NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 1 

Regulation of Inflammatory Responses: 

Influence of Sex and Gender 

Bethesda, Maryland 

September 19–20, 2006 

WORKSHOP SUMMARY 

Office of Research on Women’s Health, Office of the Director 

National Institute of Allergy and Infectious Diseases 

National Institutes of Health 

Department of Health and Human Services 

Workshop Co-Chairs 

Lisa Begg, Dr.P.H., R.N., Office of Research on Women’s Health, Office of the Director 

Christopher E. Taylor, Sc.D., National Institute of Allergy and Infectious Diseases 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 2 

Seated (Left to Right): Esther M. Sternberg, Section on Neuroendocrine Immunology and Behavior, NIMH; DeLisa 

Fairweather, Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 

Baltimore, MD; Edwin Deitch, Department of Surgery, New Jersey Medical School, Newark, NJ; Susan Kovats, 

Arthritis and Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK; Michelle Petri, 

School of Medicine, Johns Hopkins University, Baltimore, MD; Daniel E. Furst, David Geffen School of Medicine, 

University of California, Los Angeles, Los Angeles, CA. 

Standing (Left to Right): Rhonda Voskuhl, Multiple Sclerosis Program, University of California, Los Angeles, Los 

Angeles, CA; Lisa Begg, ORWH; Chris Taylor, NIAID, Lester Kkobzik, Harvard School of Public Halth, Boston, MA; 

Steven Offenbacher, Peridontology Dental Research School of Dentistry, University of North Carolina at Chapel Hill, 

Chapel Hill, NC; Elizabeth J. Kovacs, Department of Surgery, Loyola Unive4rsity, Maywood, IL; Samuel N. Breit, 

Centre for Immunology, St. Vincent’s Hospital and University of New South Wales, Sydney, NSW, Australia ; Virginia 

M. Miller, Physiology and Surgery, Mayo Clinic, Rochester, MN; Godfrey S. Getz, Division of Pathology, University of 

Chicago, Chicago, IL; Greet van den Berghe, University of Leuven, Department of Intensive Care Medicine, University 

Hospital Gasthuisberg, Leuven, Belgium; Darryl C. Zeldin, Laboratory of Respiratory Biology, National Institute of 

Environmental Health Sciences, NIH, Research Triangle Park, NC; Jeff Bender, School of Medicine Yale University, 

New Haven, CT; [Not Shown: Peter Libby, Cardiovascular Medicine, Brigham and Women’s Hospital, Harvard 

Medical School, Boston, MA]. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 3 

Table of Contents 

EXECUTIVE SUMMARY ................................................................................................ 4 

Future Research .............................................................................................................. 5 

Research Considerations................................................................................................. 5 

LIST OF ACRONYMS AND ABBREVIATIONS ........................................................... 7 

INTRODUCTION .............................................................................................................. 1 

SESSION I: INFLAMMATION: WHAT IS IT? ............................................................... 1 

An Overview of Inflammatory Mechanisms in Disease................................................. 2 

Inflammatory Modulators of Cardiovascular Risk: Cause or Effect .............................. 4 

Hormones and Inflammation: Host-Pathogen-Hormone Interactions ............................ 6 

SESSION II: SEX-RELATED INFLAMMATORY DISORDERS................................... 8 

Estrogen Receptors and Autoimmune Disease............................................................... 8 

Sex Differences in Inflammatory Responses to Shock/Trauma ..................................... 9 

Treatment of Burn Injury: Sex Effects.......................................................................... 10 

Sex Differences in Lung Function and Response to Environmental Agents................ 12 

Periodontal Disease Inflammation and Obstetric Complications ................................. 13 

SESSION III: CELLULAR AND MOLECULAR MECHANISMS IN 

INFLAMMATION RESEARCH ..................................................................................... 15 

Vascular Cell Signaling by Membrane Estrogen Receptors......................................... 15 

Sex Differences in Host Defense Against Pneumonia.................................................. 17 

Estrogen Modulation of Dendritic Cells....................................................................... 18 

Estrogen, Inflammation, and Thrombotic Risk............................................................. 19 

Use of Insulin Therapy in Treatment of Inflammatory Diseases.................................. 21 

SESSION IV: EMERGING STRATEGIES IN TREATMENT OF INFLAMMATORY 

DISEASES........................................................................................................................ 22 

TNF-Blocking Agents: Raising the Bar for Effective Treatment................................. 22 

MIC-1, a Poorly Understood TGF-β Superfamily Cytokine with Roles in Inflammation 

and Cancer .................................................................................................................... 24 

Current Lupus Treatments Neither Control Disease Activity or Prevent Organ Damage: 

The Case for New Approaches ..................................................................................... 25 

IL-12 and Tim-3 Protect Against Coxsackievirus B3-induced Myocarditis ................ 27 

SESSION V: PANEL DISCUSSIONS AND RECOMMENDATIONS ......................... 28 

Session I Discussion and Recommendations................................................................ 28 

Session II Discussion and Recommendations............................................................... 28 

Session III Discussion and Recommendations ............................................................. 29 

Session IV Discussion and Recommendations............................................................. 30 

Research Models and Other Research Considerations ................................................. 31 

CONTACT INFORMATION FOR CHAIRS AND SPEAKERS.................................... 33 

Chairs ............................................................................................................................ 33 

Speakers and Session Moderators................................................................................. 33 

LIST OF WORKSHOP ATTENDEES ............................................................................ 36 

REFERENCES ................................................................................................................. 38 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 4 

Regulation of Inflammatory Responses: Influence of Sex and 

Gender 

Office of Research on Women’s Health, Office of the Director 

National Institute of Allergy and Infectious Diseases 

September 19–20, 2006 

EXECUTIVE SUMMARY 

Several studies have shown that inflammation, which involves a constellation of 

symptoms and comprises a series of activities, underlies chronic diseases such as 

cardiovascular disease, as well as autoimmune diseases such as rheumatoid arthritis and 

lupus. Because of this increasing evidence, inflammation has become a hot topic in the 

public arena, as shown by articles in Time, Newsweek, and Wall Street Journal. The 

molecular and cellular details of inflammatory processes are well understood and are 

similar among several diseases, but initiating events for inflammation are still unknown. 

Oxidative stress, tissue damage, and age have all been implicated in the process, but no 

common specific stimuli have been identified. 

Sex-related differences have been observed in several diseases or injuries, including 

lupus, rheumatoid arthritis, heart disease, asthma, periodontal disease, and response to 

major injury, shock, or trauma. These studies further hint that sex hormones such as 

estrogen modulate both inflammation and immune response, and that these effects could 

be classified based on systemic hormone levels, the role of hormone receptor isoforms 

and variants, and the stage of disease. However, more work is needed to increase 

understanding of hormonal effects on inflammation, immune response, and disease. For 

example, much work has focused on how estrogens modulate cellular responses, but little 

information is available about other hormones, such as testosterone, progesterone, 

glucocorticoids, and insulin, and the effects of interactions among hormones on 

inflammatory processes. Moreover, existing treatments for inflammatory and 

autoimmune disease offer only limited efficacy and induce severe side effects. New 

treatment strategies will not only have to restore immune tolerance, prevent sensitization 

or neutralizing antibody production, and limit unintended biological effects, but also 

address sex differences and hormonal effects in the context of these diseases. 

On September 19–20, 2006, the National Institutes of Health Office of Research on 

Women’s Health and the National Institute of Allergy and Infectious Diseases convened 

a workshop on the influence of sex and gender on the regulation of inflammatory 

responses. Lisa Begg, Dr.P.H., R.N., and Christopher Taylor, Sc.D., served as workshop 

chairs. During this workshop, researchers from across the United States and the world 

presented their research to provide participants with a state of the science. 

Representatives of Institutes across the NIH attended the workshop, including 

representatives of the National Cancer Institute; the National Heart, Lung, and Blood 

Institute; National Institute on Aging; the National Institute on Alcohol Abuse and 

Alcoholism; the National Institute of Arthritis and Musculoskeletal and Skin Diseases; 

the National Institute of Child Health and Human Development; the National Institute of 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 5 

Dental and Craniofacial Research; the National Institute on Drug Abuse; the National 

Institute of General Medical Sciences; the National Institute of Mental Health; and the 

National Institute of Neurological Disorders and Stroke. The first session provided a 

basic foundation of what inflammation is, and subsequent sessions explored sex 

differences in the burden of disease and response to insult, cellular and molecular 

mechanisms in inflammation, and emerging strategies to treat inflammatory diseases. 

These presentations were followed by discussions in which workshop participants 

identified gaps in knowledge, research needs, and opportunities for future research. 

Recommendations for research opportunities will be incorporated into a trans-NIH 

initiative on inflammation. 

Future Research 

Session moderators and workshop participants identified the following gaps and research 

questions: 

• What is the exact stimulus or trigger for inflammatory processes? 

• How is lipid metabolism regulated by hormones? 

• How do regional differences in adiposity or energetics contribute to the differing 

burden of the metabolic syndrome? 

• What are the anatomical differences in vascular lesion formation? 

• What is the role of sex hormones in regulating HDL production, metabolism, and 

modification? 

• What is the role of hormone receptor activation in inflammatory responses? 

• Are there epigenetic effects of gender that influence sex bias? 

• What are the effects of hormones on innate immunity in healthy subjects? 

• How do sex and/or gonadal steroid hormones alter inflammatory and immune 

responses after injury? 

• How do aging, menopause, and andropause affect these responses? 

• What are the mechanisms by which hormones influence cellular responses to insult? 

• What is the temporal response of sex hormones to shock and trauma? 

• Can hormonal effects on immune or other cells account for sex biases in 

autoimmunity, infection, and recovery from trauma? 

• What is the mechanism of action of estrogen or the ER in particular cell types? 

• Which ER isoforms or variants are specific to which cell types? 

• Which hormones mediate sex-specific effects? 

• How do sex hormones interact with pathogen responses? 

• How do symptoms and responses differ by sex in humans? 

• How can new strategies employ the concept of “outflammation,” using 

chemorepellants to remove cells from places where they normally should not appear? 

• How do resolvins and the lipoxin pathway, which enhance the resolution of 

inflammation, be used? 

• Can non-steroidal anti-inflammatory drugs provide a benefit? 

Research Considerations 

• Development of new models to further explore sex differences. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 6 

• Maintaining a focus on physiology, even while conducting experiments in vitro in 

cell systems and on particular systems of interest. 

• Sorting out direct causality of chronic disease, when the initiating event has long 

passed and tissue response to that event is well established. 

• Activation and function of pro- and anti-inflammatory proteins. 

• Effects of hormone receptor resistance and hormone metabolism. 

• Influence of tissue specificity in inflammatory response. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 7 

LIST OF ACRONYMS AND ABBREVIATIONS 

ACR American College of Rheumatology 

AR androgen receptor 

ARDS acute respiratory distress syndrome 

BAL bronchoalveolar lavage 

CETP cholesterol ester transfer protein 

CHD coronary heart disease 

CVD cardiovascular disease 

CNS central nervous system 

C. rectus Campylobacter rectus 

CRP C-reactive protein 

CVB3 coxsackievirus B3 

DAS Disease Activity Score 

DC dendritic cell 

DHEA dehydroepiandrosterone 

DHT dehydrotestosterone 

DTH delayed type hypersensitivity 

EAE experimental autoimmune encephalomyelitis 

E. coli Escherichia coli

eNOS endothelial nitric oxide synthase 

ER estrogen receptor 

ER-α estrogen receptor alpha 

ER-β estrogen receptor beta 

EULAR European League Against Rheumatism 

GR glucocorticoid receptor 

HDL high-density lipoprotein (cholesterol) 

HPA hypothalamic-pituitary-adrenal 

HPT hypothalamic-pituitary-thyroid 

HRT hormone replacement therapy 

HUVEC human umbilical vein endothelial cells 

ICAM intracellular adhesion molecule 

ICU intensive care unit 

IFN interferon 

IIT intensive insulin therapy 

IκB inhibitor of nuclear factor kappa B 

IL interleukin 

iNOS inducible nitric oxide synthase 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 8 

LDL low-density lipoprotein (cholesterol) 

LeTx anthrax lethal toxin 

15-LO 15-lipoxygenase 

LPS lipopolysaccharide 

MIC-1 macrophage inhibitory cytokine 

MODS multiple organ dysfunction syndrome 

MCP-1 monocyte chemoattractant protein 1 

M-CSF monocyte colony stimulating factor 

MHC major histocompatibility complex 

MMP matrix metalloproteinase 

MS multiple sclerosis 

NFκB nuclear factor kappa B 

NHLBI National Heart, Lung, and Blood Institute 

NIA National Institute on Aging 

NIAID National Institute of Allergy and Infectious Diseases 

NIH National Institutes of Health 

NK natural killer 

NKT natural killer T cell 

NO nitric oxide 

ORWH Office of Research on Women’s Diseases 

PDGF platelet-derived growth factor 

PGE2 prostaglandin E2 

PGI prostacyclin 

PI3K phosphatidylinositol 3-kinase 

PPT propylpyrazole triol 

PR progesterone receptor 

PR-A progesterone receptor A 

PR-B progesterone receptor B 

RA rheumatoid arthritis 

RRMS relapse-remitting course of MS 

SAA serum amyloid A 

SERM selective estrogen receptor modulator 

SIRS systemic inflammatory response syndrome 

SLE systemic lupus erythromatosus 

T/HS trauma and hemorrhagic shock 

TLR Toll-like receptor 

TNF-α tumor necrosis factor alpha 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 9 

VCAM-1 vascular cell adhesion molecule 1 

VEGF vascular endothelial growth factor 

WBC white blood cells 

WHI Women’s Health Initiative 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 1 

Regulation of Inflammatory Responses: Influence of Sex and Gender 

Office of Research on Women’s Health, Office of the Director 

National Institute of Allergy and Infectious Diseases 

WORKSHOP SUMMARY 

INTRODUCTION 

The classic definition of inflammation was first proposed in the first-century description by 

Celsus of redness, swelling, heat, and pain. More recently, the study of inflammation has focused 

more and more on the cellular and molecular players involved, and recent work has revealed the 

roles of these players in several diseases and disorders, such as cardiovascular disease (CVD), 

diabetes, arthritis, and cancer. For example, atherosclerosis was once viewed as a bland, noninflammatory process resulting in a benign tumor of the smooth muscle cells. With the 

identification of various cytokines as modulators of cell proliferation in fibrotic diseases,1 this 

view of the disease process has now evolved to describe atherosclerosis as an inflammatory 

process.2,3 The idea that inflammation plays a role in CVD and pulmonary diseases represented a 

“frontier” in research, even as Pasceri and Yeh published an editorial noting the similarities in 

pathology between atherosclerosis and rheumatoid arthritis (RA) and between atherosclerosis 

and tuberculosis.4 Yet now, following a tremendous shift in thinking, the role of inflammation in 

atherosclerosis, along with other diseases, is a matter of public interest, having appeared in Time, 

Newsweek, and the New York Times. 

Although the understanding of inflammatory processes and the role they play in disease has 

increased, this field is still evolving, particularly in the area of sex and gender differences in the 

regulation of inflammatory responses. For example, recent work has shown that varying sex 

hormone levels can influence immune function and the type of immune response, but the effects 

of hormone receptor resistance are poorly understood. On September 19–20, 2006, the National 

Institutes of Health (NIH) Office of Research on Women’s Health (ORWH) and the National 

Institute of Allergy and Infectious Diseases (NIAID) co-sponsored a workshop on the influence 

of sex and gender in the regulation of inflammatory responses. The goals of this workshop were 

to assess and advance the state of the science in inflammation research and to generate ideas for 

research opportunities. Research presentations focused on the biology of inflammation, sexrelated inflammatory disorders, cellular and molecular mechanisms in inflammation research, 

and emerging strategies in treating inflammatory diseases. Following these presentations, 

moderators presented themes from each session, and workshop participants discussed research 

needs and opportunities. It is hoped that these ideas will be incorporated into a NIH Roadmap 

initiative focused on inflammation. 

SESSION I: INFLAMMATION: WHAT IS IT? 

Moderator: Virginia Miller, Ph.D., Mayo Clinic 

The presentations during this session provided a foundation for the rest of the workshop by 

discussing what inflammation is, whether biomarkers are the cause or the effects of disease, and 

how hormones affect host-pathogen interactions and inflammatory/immune responses. CVD was 

discussed as an example of the involvement of inflammatory processes in chronic disease. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 2 

An Overview of Inflammatory Mechanisms in Disease 

Peter Libby, M.D., Harvard University 

The role of inflammation in disease pathogenesis involves an interplay between innate and 

adaptive responses and a balance between pro- and anti-inflammatory stimuli.5 Because these 

factors appear in several diseases and disorders, it could be said that regardless of specialty, 

researchers most likely all study the same generic disease, in which monocyte attachment 

facilitates leukocyte adhesion to epithelial or mesenchymal cells, which in turn promote 

migration and penetration of the monocytes and their differentiation into tissue macrophages. 

The types of epithelial and mesenchymal cells, as well as the type of tissue macrophage, are 

specific to the disease of interest. 

Atherosclerosis begins when risk factors, such as high levels of low-density lipoprotein (LDL) 

cholesterol, induce monocytes to attach to the artery wall and secrete adhesion molecules, such 

as vascular cell adhesion molecule 1 (VCAM-1), that facilitate the adhesion of leukocytes to the 

endothelium. VCAM-1 specifically binds monocytes and lymphocytes found in atherosclerotic 

plaques and can be induced by cytokines. Within 1 week after rabbits are fed a diet rich in 

cholesterol and saturated fat, patches of aortic endothelial cells express VCAM-1, then bind 

leukocytes.6 Endothelial cells can modulate the type of adhesion molecules expressed and thus 

the recruitment of innate immune, adaptive immune, or acute inflammatory effector cells. 

Following leukocyte adhesion, endothelial and smooth muscle cells express chemoattractants or 

chemokines, such as monocyte chemoattractant protein 1 (MCP-1), that induce monocyte 

migration and penetration into the intima. Inactivation of MCP-1 in mutant mice rendered 

susceptible to atherosclerosis show fewer atherosclerotic plaques and less atherosclerosis 

overall.7 Following migration and penetration, monocytes in the atherosclerotic plaque 

differentiate into tissue macrophages, or foam cells, which then secrete factors promoting 

oxidative stress, coagulation, and apoptosis, resulting in the formation of a necrotic core in the 

plaque. Among the signals that stimulate the metamorphosis from monocyte to foam cell is 

monocyte colony stimulating factor (M-CSF). Inactivation of M-CSF decreases atherosclerosis, 

but it also causes problems in the bone.8

Following the initiation of atherosclerosis, lesions and plaques remain clinically stable for 

decades. Inflammation also plays a role in progression. Libby and colleagues have shown that 

cells other than leukocytes can secrete interleukin 1-beta (IL-1β). Vascular endothelial cells 

produce other cytokines, including tumor necrosis factor alpha (TNF-α), TNF-β, IL-1α, IL-1β, 

IL-6, M-CSF, MCP-1, and IL-18. Smooth muscle cells also express CD40 ligand, which is 

important for B-cell activation. In addition, CD40 ligand is expressed on vascular endothelial 

cells, smooth muscle cells, and macrophages in atherosclerotic plaques, and expression of COX2, which is induced by CD40 ligation, is augmented. Experiments also have shown that mutant 

mice susceptible to atherosclerosis and fed a high-fat diet show arrest of atherosclerosis 

progression when CD40 signaling is disrupted.9

 Thus, vascular cells actively participate in 

inflammation, rather than simply respond to it. 

Inflammation also contributes to the thrombotic complications that bring the patient most 

dramatically to the clinic. An atherosclerotic plaque consists of a lipid core surrounded by a 

fibrous cap that is formed by extracellular matrix made by smooth muscle cells. The integrity of 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 3 

the fibrous cap depends on type I and type III interstitial collagen. Matrix metalloproteinases 

(MMP) cleave collagen between glycine 775 and leucine or isoleucine 776, breaking the triple 

helix into fragments. The persistence of risk factors result in the accumulation of inflammatory 

cells and instructions to the macrophages to overexpress the collagenases MMP1, MMP8, and 

MMP13, which degrade the fibrous cap and render it susceptible to rupture at the same time 

production of interferon gamma (IFN-γ) halts new collagen synthesis by the smooth muscle 

cells.10 Other inflammatory mediators, including CD40 ligand, IL-1, TNF-α, MCP-1, and MCSF, also contribute to acute thrombotic complications,11 and inflammation induces the 

expression of the pro-coagulant tissue factor. Reducing risk factors such as lipid levels can limit 

inflammation, stabilize the fibrous cap, and facilitate the reversal or resorption of the 

atherosclerotic plaque. 

Several pro-inflammatory molecules have been proposed as independent predictors of 

myocardial infarction and stroke, two complications of atherosclerosis. Among these are the 

primary pro-inflammatory cytokines such as TNF-α; the messenger cytokine IL-6; endothelial 

adhesion molecules; and acute-phase reactants such as fibrinogen, plasminogen inhibitor, Creactive protein (CRP), and serum amyloid A (SAA). Among these molecules, CRP is most 

attractive because it is chemically stable; can be measured by a reliable, standardized, 

convenient, and inexpensive assay; has a long half-life; and shows no diurnal variation. Serum 

concentrations of CRP add prognostic value beyond the Framingham risk score. Activities that 

decrease the risk for CVD, such as exercise, weight loss, and smoking cessation, also decrease 

serum CRP concentrations. Oral estrogen and estrogen/progestin combinations, which have been 

associated with increased thrombotic liability in the populations studied, increase CRP. 

Discussion 

Although both sexes are at risk for CVD, the face of this disease has changed. In the 1950s, the 

typical CVD patient was white, middle-aged, male, hypercholesterolemic, and hypertensive. 

Now, the risk profile is shifting from smoking and hypertension to nutritional factors such as 

diabetes and the metabolic syndrome. Coronary disease has become more prevalent in women 

and in disadvantaged, minority populations, partly because of the shift in risk profiles toward 

metabolic disorders, including low high-density lipoprotein (HDL) levels, high triglyceride 

levels, and glucose intolerance or insulin resistance. In addition, although death rates associated 

with CVD have declined, these rates are age adjusted, and obesity is fast becoming a pediatric 

disease. More data is needed about diet, behavior, and exercise, and ways to encourage people to 

maintain a healthy body weight and choose a diet to forestall obesity should be explored. In 

addition, in some studies, hormone replacement therapy (HRT) has been demonstrated to be 

somewhat protective in premenopausal women, in contrast to large clinical trials that suggest that 

HRT increases the risk for heart disease in postmenopausal women. More work is needed to sort 

out the protective and detrimental effects of estrogen on the cardiovascular system, as well as the 

increased association of CVD with diabetes and the metabolic syndrome. Clinicians are only 

starting to realize that women with CVD may present with symptoms different from those 

traditionally associated with the disease. In addition, women often serve as caregivers and are 

less likely to seek care for themselves, especially for symptoms not traditionally associated with 

CVD. Because of these factors, CVD may be underdiagnosed in women. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 4 

Overall, CRP levels are higher in women than in men, but only slightly, and they vary by race 

and ethnicity, although how much of this difference is associated with dietary differences is not 

known. Estrogen affects CRP levels, although the effects of oral estrogens are more pronounced 

than those of endogenous estrogen. Despite these differences, reference values for both men and 

women are the same. 

The specific effects of foam cell production on major vessels may depend on several factors. One 

is pressure; pulmonary hypertension promotes atherosclerosis of the pulmonary arteries. In 

addition, inflammation and high cholesterol results in an accumulation of foam cells in other 

tissues, such as tendinous xanthomata. Orange tonsils are found when cholesterol cannot be 

transported out of macrophages, as in Tangiers disease. 

Inflammatory Modulators of Cardiovascular Risk: Cause or Effect 

Godfrey Getz, M.D., Ph.D., University of Chicago 

As discussed by Dr. Libby, atherosclerosis takes several decades to develop, and for many of 

those decades, patients are not aware they have it. Most risk factors have been identified through 

their relationship with clinical endpoints, even though most CVD involves underlying 

atherosclerotic processes. In a component of the PDAY Study,12 topographic distributions of 

atherosclerotic lesions were examined in autopsied persons aged 15 to 34 years. Within this 

population, males had more fatty streaks and raised lesions in the right coronary artery, 

compared with females; thus the development of atherosclerosis was more prevalent among men. 

In the future, imaging may be used to identify the presence of atherosclerosis long before a 

patient complains of symptoms. Although several modalities are under development, however, 

they are at various stages of maturity and offer differing degrees of invasiveness, and none are at 

the point of precisely determining the nature of a patient’s atherosclerotic lesion. 

Elevated LDL most likely is a cause of CVD, based on the positive correlation between total 

plasma cholesterol and LDL cholesterol with the incidence of CVD, the evidence of genetic 

hypercholesterolemia in cases of premature atherosclerosis, the reduced progression of 

atherosclerosis when total cholesterol and LDL are lowered, and the requirement for 

dyslipidemia for atherosclerosis to develop in experimental animals. In particular, oxidized LDL 

most likely plays a causal role in atherogenesis. LDL is a globular particle with a core of 

cholesterol ester and a surface consisting of cholesterol, phospholipid, and proteins, primarily 

apoB-100. Most circulating LDL is native, but as it transits through endothelial cells, the surface 

components of several LDL molecules become oxidized through the action of 15-lipoxygenase 

(15-LO) and inducible nitric oxide synthase (iNOS).13 The oxidation of phospholipids in LDL is 

highly pro-inflammatory.13 The appearance of modified LDL molecules in the subendothelial 

space activates signaling pathways, including those involving VCAM-1 and several chemokines, 

which promote the recruitment of circulating monocytes in areas where oxidized LDL has 

accumulated. Under the influence of M-CSF, these monocytes differentiate into macrophages 

and express scavenger receptors that facilitate the uptake of modified LDL and the formation of 

foam cells. Once activated, these macrophages secrete factors that promote further monocyte and 

T-cell recruitment and differentiation. Oxidized LDL inhibits the production of nitric oxide (NO) 

by endothelial NOS (eNOS), resulting in a less-pliant blood vessel. Results from several studies 

have correlated the increase in the levels of antibodies against oxidized phospholipids with 

several aspects of CVD. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 5 

High levels of HDL, on the other hand are atherprotective, as shown by an inverse relationship 

between CVD and HDL levels and studies, mostly in animals, of the influence of apoA-1, the 

major HDL protein, on atherosclerosis. Like LDL, HDL is a globular particle, but it is more 

heterogeneous. It is smaller, with many more proteins and fewer steryl esters associated with it.14

Thus, HDL is more readily remodeled in the circulation. HDL participates in reverse cholesterol 

transport, where the cell surface molecules ABCA1 and ABCG1 promote the transfer of 

cholesterol from foam cells to apoA-1. Ultimately, as remodeled HDL, apoA-1 is transported 

through the plasma to the liver for secretion as bile acid.15 Persons lacking the ABCA1 protein 

are unable to carry out reverse cholesterol transport and exhibit orange or yellow tonsils. The 

presence of apoA-1 is the primary protective property of HDL. Mice deficient in both the LDL 

receptor and apoA-1 show increased development of atherosclerotic lesions.16 The 

administration of an apoA-1 mimetic to apoE-deficient mice, even in low concentrations, 

minimizes the number of atherosclerotic lesions.17 These studies point to apoA-1 as a potential 

therapeutic target, but studies assessing the effect of apoA-1 on human atherosclerosis are still in 

preliminary stages. 

HDL is an anti-inflammatory agent.18 A culture system consisting of a synthetic artery composed 

of human endothelial cells overlying cultivated smooth muscle cells has been used to assess 

effects on monocyte recruitment.19 Oxidized LDL promotes monocyte recruitment, while the 

addition of HDL attenuates the effects of oxidized LDL. The anti-oxidant paraoxonase plays a 

role in this inhibition. Navab and colleagues19 plotted an inflammatory index based on their cell 

culture system and found that HDL reduces inflammation by about 60%. However, when an 

index was plotted for the same amount of HDL taken from patients who had experienced a 

myocardial infarction, the anti-inflammatory properties of the HDL molecules had been lost. 

Thus the quality and composition of HDL is probably just as important as the quantity, if not 

more so. 

Elevated levels of the acute inflammatory markers CRP and SAA have also been cited as risk 

factors for CVD. The relative risk for future CVD events is higher for these markers than it is for 

elevated LDL.20,21 CRP has been used as a biomarker in several CVD studies, although at the 

low limit of the acute phase reaction. SAA is transported in the bloodstream in association with 

HDL. However, it is not clear whether CRP and SAA are simply biomarkers or whether they 

have any pathogenetic influence on atherosclerotic progression. Mice do not produce CRP, and 

work with SAA has not advanced to the point of decisive genetic manipulation. 

Several inflammatory cell types are present in an atherosclerotic lesion. Genetic manipulation of 

mouse models and in vitro cell systems has shown that macrophages are central to inflammatory 

processes underlying atherosclerosis. Yet natural killer (NK) cells, CD4+

 T cells, NK T (NKT) 

cells, and CD8+ T cells all serve pro-inflammatory functions and increase atherogenesis in mouse 

models, whereas CD4+ regulatory T cells and B cells perform anti-inflammatory functions and 

decrease atherogenesis. Although few B cells are observed in atherosclerotic lesions, work with 

mouse models suggests they are protective. The pro-inflammatory cytokines IFN-γ, IL-12, and 

IL-18 and the anti-inflammatory cytokines IL-10 and transforming growth factor beta (TGF-β) 

are also present in these lesions. Work exploring the role of mature T and B cells in 

atherosclerosis is under way. However, it is clear that robust atherosclerosis may develop in the 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 6 

absence of both T and B cells, and these responses may be gender biased in some vascular beds. 

Results from the human PDAY study showed that unlike males, young females have relatively 

more abdominal aortic atherosclerosis than coronary artery atherosclerosis. Thus vascular beds 

differ in relation to gender influences in humans, as well. 

Discussion 

Studies examining expression of the estrogen receptor (ER) in the vasculature have focused 

primarily on the alpha (ER-α) isoform. However, ER-α expression in various regions of the 

vasculature has not been examined. ER studies using vascular models are discussed further in Dr. 

Bender’s section in Session III. 

Recent data has been published showing an increased fraction of pro-inflammatory HDL in 

autoimmune diseases such as systemic lupus erythromatosus (SLE) and RA. These studies also 

examined the effects of oxidation. It could be that in these diseases, the level of oxidative stress 

is high enough to overwhelm anti-oxidant enzymes. Administration of the apoA-1 mimetic 

results in the transport of paraoxonase from HDL to the mimetic. Thus, oxidative stress could 

substantially modify the composition of HDL so that it no longer functions as an antiinflammatory agent. 

It is not clear whether patients with a family history of elevated cholesterol based on high HDL 

levels are protected from atherosclerosis. Some genetic abnormalities of HDL homeostasis have 

been identified that influence their atheroprotective properties. In Japan, families with high HDL 

resulting from partial deficiency of cholesterol ester transfer protein (CETP) seem to be 

protected from atherosclerosis. CETP transfers cholesterol esters between HDL and other 

lipoproteins. Trials are in process to determine the therapeutic benefit of CETP inhibition. In 

Italy families with the apoA-1 mutation apoA-1 Milano also seem to be protected from 

atherosclerosis, though their HDL level is not increased. In addition premenopausal women show 

different distributions of HDL particle size. HDL2, the larger HDL component, is higher in 

females than in males, which may afford some premenopausal protection. More study is needed 

about the relationship between the composition of HDL and protection against atherosclerosis. 

Future research also should focus on the role of sex hormones in regulating HDL production, 

metabolism, and modification. 

Hormones and Inflammation: Host-Pathogen-Hormone Interactions 

Esther Sternberg, M.D., National Institute of Mental Health, NIH 

Steroid hormones affect host inflammatory/immune responses in a variety of ways, through the 

central nervous system (CNS), the neuroendocrine system, the hypothalamic-pituitary-adrenal 

(HPA) axis, and the hypothalamic-pituitary-gonadal (HPG) axis.22,23 The effects of estrogen, 

glucocorticoids, or progesterone on host inflammatory responses depend on its concentration. 

Low levels of estrogen tend to shift responses toward the Th1 or cellular phenotype, whereas 

high levels shift them toward the Th2 or humoral phenotype.24,25 Glucocorticoids and 

progesterone generally drive anti-inflammatory responses and tend toward immune suppressive 

states. Thus, immune function changes during the menstrual cycle, where the levels of estrogen 

and progesterone vary, and during periods of stress. During the luteal phase, when estrogen 

levels are low and progesterone levels high, the expression of T-lymphocyte receptors decrease, 

antibody production increases, and susceptibility to infection increases. Likewise, immune 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 7 

function also varies during pregnancy, with strong immunosuppression occurring in the third 

trimester as hormone levels (estrogen, progesterone, and glucocorticoids) increase dramatically: 

the number of suppressor T cells increases, the number of cytotoxic T cells decrease, and 

susceptibility to infections increase. Work is under way in Dr. Sternberg’s laboratory to assess 

the effects of sex hormones on dendritic cells, which are important in pathogen recognition and 

serve as a bridge between adaptive and innate immunity. 

Recent work has shown that bacterial toxins can interact with steroid hormone receptors and the 

associated host hormone responses. Anthrax lethal toxin (LeTx) comprises two proteins: lethal 

factor (LF), which must be transported into the cell to exert its effects, and protective antigen 

(PA), which facilitates entry of LF into the cell. Using a transient transfection system, Dr. 

Sternberg’s group showed that at extremely low concentrations, LeTx is able to repress 

activation of the glucocorticoid receptor (GR), primarily by interfering with its ability to bind 

DNA and promote downstream histone acetylation, as shown by chromatin 

immunoprecipitation.26 LeTx does not, however, affect GR ligand binding, the number of GR 

expressed by cells, GR translocation to the nucleus, or GR transrepression. The toxin also 

represses specific forms of the ER (ER-α) and progesterone receptor (PR-B) in a manner 

dependent on both the receptor and the promoter.27 Work is under way to determine whether 

repression of sex steroid receptor activity is a general phenomenon among bacterial toxins. 

Resistance to glucocorticoids, which can result from mutations or polymorphisms in the GR or in 

associated proteins, elevated levels of the antagonistic GR-β, decreased numbers of GR, or 

bacterial toxins, which results in the interruptions of the HPA axis at the tissue level that have 

been implicated in autoimmune or inflammatory diseases such as RA, SLE, Crohn’s disease, 

asthma, and multiple sclerosis (MS).22,28-36 Inhibition of the HPA axis predisposes a host to 

increased inflammation. For example, anthrax-resistant mice show increased mortality in 

response to LeTx when the adrenal glands of these animals were removed or when the 

glucocorticoid response was blocked with the GR antagonist mifepristone (RU-486).37 This 

increased mortality cannot be rescued by treatment with dexamethasone. The effects of 

mifepristone treatment on LeTx-associated mortality suggest that adrenal factors other than 

glucocorticoid activity also may be involved. Thus, treating autoimmune or inflammatory 

diseases with high concentrations of glucocorticoid might not be effective. 

Steroid hormones may play a critical role in the first line of host defense, and more attention 

should be paid to the effects of hormone receptor resistance. In addition to studies of on the 

effects of GR, other studies have shown that estrogen and ER activity influence response to 

lipopolysaccharide (LPS).38 In addition to focusing on how much hormone is present, future 

research should consider the role of steroid hormone receptor activity, including the sex hormone 

receptors, in susceptibility to inflammation. 

Discussion 

More work should be done to assess whether receptors are uniformly downregulated across all 

cell types, as receptor isoforms vary across tissue. It should be noted that the studies of LeTx 

effects on GR activation also assessed the induction of GR-induced enzymes and found this 

induction blocked. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 8 

SESSION II: SEX-RELATED INFLAMMATORY DISORDERS 

Moderator: Elizabeth Kovacs, Ph.D., Loyola University 

Presentations in this session focused on the use of epidemiological studies and animal models to 

explore sex differences in MS, shock and trauma, environmental insult, and periodontal disease. 

Estrogen Receptors and Autoimmune Disease 

Rhonda Voskuhl, M.D., University of California, Los Angeles 

The relapse-remitting course of MS (RRMS), which involves a cycle of symptomatic relapses 

followed by return to baseline, gradually progresses to a point where patients are unable to 

recover. As shown by magnetic resonance imaging, active, relapses involve gadolinium-positive 

inflammatory lesions in the white matter, but permanent disability goes beyond these lesions, 

involving atrophy in the gray matter. Existing treatment options, which are anti-inflammatory 

and only indirectly neuroprotective, are expensive and only partially effective. They reduce 

relapses by a third, but disability continues to progress. Women with RRMS are somewhat 

protected from relapse during late pregnancy, when progesterone and estrogen levels increase, 

but they become prone to relapse following delivery.39 Further understanding of the effects of 

sex hormones on RRMS may provide insight into new, more effective treatment options. 

Experimental autoimmune encephalomyelitis (EAE), a classic Th1-mediated disease with a cell 

infiltrate at the core, is a mouse model corresponding to MS. Several groups have observed that 

estriol and estradiol are protective against EAE in several genetic backgrounds. Expression of 

Th1 cytokines decreases, and in some cases, expression of Th2 cytokines such as IL-10 

increases. Estrogen treatment also reduces the function of dendritic cells and macrophages and 

expression of inflammatory chemokines, and it increases the number of CD4+CD25+ regulatory 

T cells. These mechanisms, as well as others that have yet to be identified, decrease the number 

of inflammatory cells in the CNS. Yet estrogens also appear to be neuroprotective in this model. 

Microglial activation, neuronal cell death, and oligodendrocyte cell death are all reduced, 

neurotrophic factors are induced, and the number of dendritic spines and synapses are increased 

in response to estrogen. Expression of TNF, IFN, and IL-2 decreases, and expression of IL-5 

increases. Work by two groups using knockout mice has shown that the overall protective effect 

of estrogen is mediated by ER-α.

40,41 However, this does not rule out the influence of other ER 

isoforms. 

Dr. Voskuhl’s group has further examined the anti-inflammatory and neuroprotective effects of 

the ER using the ER-α agonist propyl pyrazole triol (PPT).42 PPT works as well as estradiol in 

protecting against EAE in wild-type mice and mice deficient in ER-β, but not in mice deficient 

in ER-α, indicating a high degree of specificity for this agonist in a disease setting. Treatment 

with PPT, like that with estradiol, reduces the number of white-matter lesions and the amount of 

axonal transaction, consistent with an anti-inflammatory effect mediated through ER-α. 

Treatment with PPT also decreases the amount of permanent disability-associated neuronal 

abnormalities in the gray matter. Thus, both estradiol and the ER-α agonist PPT exert antiinflammatory and neuroprotective effects. However, whether these effects are direct and whether 

gray matter atrophy can be prevented is not yet clear. Work is under way to assess possible 

effects mediated by ER-β. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 9 

Discussion 

The effects of oral contraceptives or non-pregnancy concentrations of endogenous estrogen on 

MS or EAE are not known. No differences have been observed in ovariectomized mice, patients 

who have undergone hysterectomies, or postmenopausal women. However, these concentrations 

of estrogen are much lower than those associated with pregnancy. Clinical trials are under way. 

Sex Differences in Inflammatory Responses to Shock/Trauma 

Edwin Deitch, M.D., New Jersey Medical School 

Systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), 

and multiple organ dysfunction (MODS) are common occurrences following major shock or 

trauma. Gut barrier function plays a critical role in these syndromes. Several studies have shown 

that gut ischemia, which occurs through a shunting of blood from the intestinal circulation to the 

central circulation, is a better predictor of ARDS and MODS than are systemic hemodynamics. 

This is consistent with prospective clinical trials in which increased gut permeability predicts the 

development of ARDS and MODS and early enteral feeding, which improves the gut barrier, 

decreases the likelihood for infections and the duration of hospital stays for trauma patients. In a 

review of nine registry-based clinical studies and one prospective cohort study, Ananthakrishnan 

and colleagues found that seven of the ten studies showed improved outcomes in premenopausal 

women,43 which suggests that sex might influence outcomes following major shock or trauma. 

There are more bacteria in one person’s gut than there are people on earth, and the amount of 

endotoxin and other bacterial products in the gut is sufficient to kill the host a thousand times 

over. In the early 1980s, Dr. Deitch’s group proposed a paradigm in which loss of gut barrier 

function allows bacteria to translocate and spread to the lymphatics, resulting in bacteremia and 

systemic infection. However, this paradigm proved difficult to apply. In recent work, Dr. 

Deitch’s group subjected male Sprague-Dawley rats to trauma and hemorrhagic shock (T/HS) or 

trauma and sham shock, then collected mesenteric lymph and tested it in vitro on endothelial 

cells and neutrophils or in vivo by injecting it into normal rats.44-47 T/HS and lymph and plasma 

from T/HS rats induced lung and red-blood-cell injury, neutrophil activation, and bone marrow 

growth suppression. This was consistent with what was observed in human trauma patients, and 

the fraction of plasma with biologic activity was the same between rats and humans. These 

experiments have been repeated in a pig model, where the lymph collected from T/HS males 

increased the permeability of a monolayer of human umbilical vein endothelial cells and 

activated rat and human neutrophils.48 These results support a model of gut-origin SIRS, ARDS, 

and MODS, in which mesenteric lymph serves as the link between the gut and distant organ 

injury or inflammation. In this model, gut ischemia results in the production and release of 

biologically active factors into the lymph vein, and gut-derived factors in lymph lead to distant 

organ injuries through interactions between neutrophils and endothelial cells. The lymph factors 

responsible for biologic activity are not bacterial or cytokines, but rather large proteins and lipids 

that have yet to be identified. 

The extent of T/HS-induced organ injury and cellular dysfunction is influenced by sex.49-52 In the 

rat experiments, females appeared relatively resistant to T/HS, and unlike T/HS lymph from 

male rats, T/HS lymph from proestrus female rats did not have a biologic effect, except for mild 

neutrophil activation. Lymph from castrated males appeared somewhat protective against injury 

associated with T/HS, whereas lymph from ovariectomized females had the same biologic 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 10 

activity as that from normal T/HS males. Moreover, the inflammatory response to hypoxia and 

reoxygenation was reduced in the gut in female rats, compared with that in male rats,53 and 

intestinal iNOS activity and plasma NO concentrations were increased in males subjected to 

T/HS, but not in females.50 Similar sex differences were observed in pig48 and baboon models.54

Prospective clinical trials are under way. 

There are some caveats associated with the gut lymph model of gut-origin SIRS, ARDS, and 

MODS. Sex hormone effects might only be relevant during the early, acute post-trauma period 

after T/HS, and sex hormone levels change rapidly, both in males and females, following T/HS. 

Moreover, the effect of sex on clinical outcome following burn injury appears to differ from that 

following shock and trauma (see Dr. Kovacs’ presentation, below), and females become more 

susceptible to injury as hormone levels change. Future work should examine the temporal 

response of sex hormones to T/HS, the mechanisms by which these hormones influence cellular 

response to insult, and the potential role for sex hormone modulation in acute clinical conditions. 

Discussion 

The biologic factors in mesenteric lymph from male rats subjected to T/HS have yet to be 

identified. They are not traditional cytokines, polysaccharides, or endotoxins. Because work on 

the gut lymph model is still evolving, it is not clear whether inflammatory bowel disease or other 

chronic disease affects the ability of gut lymph to induce injury. 

Treatment of Burn Injury: Sex Effects 

Elizabeth J. Kovacs, Ph.D., Loyola University 

Of the more than 100,000 patients admitted to hospitals for burns, 80% of them are male and 

50% have ethanol in their blood at the time of admission. In most cases, ethanol exposure is 

acute rather than chronic. Patients with ethanol in their systems face increased morbidity and 

mortality associated with these injuries. They are twice as likely to experience serious 

complications and 60% more likely to undergo surgical procedures, require more rigorous 

antibiotic therapy, stay in the hospital longer, and succumb to their injuries. Yet regardless of 

alcohol exposure and sex, a similar cascade of inflammatory and immune events occurs in 

response to a burn. This cascade is initiated by an increase in the expression of pro-inflammatory 

mediators, including IL-6, followed by a decrease in T-lymphocyte activation and IL-2 

expression and a shift in the phenotype of cytokines produced from Th1 to Th2, as assessed by 

decreased expression of IFN-γ and increased expression of IL-4. Because of this, burn patients 

become more susceptible to infection. Evidence suggests that high levels of the proinflammatory mediator IL-6 for a prolonged period is associated with poor prognosis. 

Mice given ethanol before a burn injury experience immunosuppression to a greater degree than 

do those experiencing ethanol exposure or burn injury alone. These mice show decreased 

survival following bacterial challenge, decreased leukocyte chemotaxis to the skin, increased 

leukocyte chemotaxis to the lung, and diminished type hypersensitivity (DTH) response. 

Lymphocyte proliferation and IL-2 production are decreased, and the levels of several proinflammatory mediators, including IL-1, TNF-α, IL-6, and prostaglandin E2 (PGE2), are 

increased. This mouse model is therefore a useful one in teasing out sex differences in the 

response to burn injury. Dr. Kovacs’ group has investigated the use of hormonal approaches to 

reduce the amount of aberrant pro-inflammatory cytokine production following burn injury and 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 11 

to restore immunity in a mouse model. One of the hormones under investigation is estrogen, 

which controls the expression of many cytokines, up- or downregulating them at varying 

concentrations. Cycling females exhibit a more vigorous cellular and humoral immune response 

than males do, depending on the stage in the cycle. Proestrus levels of estrogen are protective, 

whereas testosterone and pregnancy levels of estrogen are immunosuppressive. 

In Dr. Kovacs’ experiments, male mice were given ethanol and subjected to a scald injury, then 

resuscitated and treated with 17β-estradiol. Immune responses were measured at various time 

points after injury. Male mice treated with proestrus levels of 17β-estradiol exhibited a marked 

recovery in DTH response and splenocyte proliferation. Additionally, in splenocyte proliferation 

assays, if macrophages were removed or antibodies against CD3 were used to stimulate 

proliferation, the administration of 17β-estradiol had no effect. When splenic macrophages were 

removed from mice given ethanol and burn injury, given estrogen in vivo, and cultured in vitro

with LPS, estrogen treatment reduced the increase in IL-6 production by 50%. The observed 

changes did not result from differences in ER expression; ER levels were the same across all 

treatment groups. Although treatment with 17β-estradiol did not affect expression of nuclear 

factor kappa B (NFκB) or inhibitor of NFκB (IκB) or phosphorylation of IκB overall, it did 

decrease the level of NFκB activation in macrophages from mice exposed to ethanol and burn 

injury. In addition, mice given the combined insult of ethanol exposure and burn injury that were 

treated with 17β-estradiol experienced improved survival following bacterial challenge, to the 

level observed in mice subjected to burn injury alone. Thus, treatment with 17β-estradiol 

improved immunity in male mice exposed to ethanol and burn injury. Hormonal approaches that 

failed to improve immunity in these mice included the testosterone derivative 

dehydrotestosterone (DHT), the androgen receptor (AR) antagonist flutamide, gonadectomy, the 

testosterone and estrogen precursor dehydroepiandrosterone (DHEA), and the ER antagonist ICI 

182,780. 

On the basis of these results, one would expect that female mice exposed to burn injury would 

have a better prognosis than males. However, the 10-day mortality following burn injury is 

similar between males and females, and the mortality following a combination of ethanol 

exposure and burn injury is more marked among females. Moreover, clinical evidence indicates 

that women do worse after burn injury than men do (Loyola Burn Registry).55-58 Although these 

studies were limited to moderate-sized burn injury—mortality following large burn injuries was 

similar between men and women and for postmenopausal women—the increased mortality 

among female burn victims suggests decreased immune response. Dr. Kovacs’ group explored 

potential differences in immune response using the same injury model described above. At 48 

hours following exposure to ethanol and burn injury, both males and females were 

immunosuppressed, as indicated by a decrease in the DTH response. At 7 days, however, female 

mice exposed to ethanol and burned remained immunosuppressed, whereas males did not. The 

DTH response was attenuated, splenocyte proliferation was suppressed, and IL-6 was elevated 

more dramatically among these females. Ovariectomy improved immunity, as shown by restored 

DTH response, greater mitogen-induced splenocyte proliferation, and reduced aberrant 

expression of IL-6. 

On the basis of these results, treatment with low, proestrus levels of estrogen can improve 

cellular immunity in male mice following ethanol exposure and burn injury, via control of IL-6 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 12 

production. Although the IL-6 promoter has no estrogen responsive elements, estrogen 

modulates its production through interaction of the ER with transcription factors such as NF-κB 

and Ap-1. Thus, sex-specific treatment regimens should be considered for burn patients. 

Alterations in estrogen levels are somewhat confusing. Ethanol exposure decreases endogenous 

estrogen levels, whereas burn injury increases it. Both males and females experience an increase 

in estrogen following burns. In males this increase occurs at a moderate level and is transient, but 

in females the level of estrogen reaches pregnancy levels and is maintained for a much longer 

period of time: weeks to months, depending on the extent of the injuries. It is not clear what the 

combined effects of ethanol exposure and burn injury would be. More work is needed to 

determine the effects of hormones on innate immunity in healthy participants; how sex and/or 

gonadal steroid hormones alter inflammatory and immune responses after injury; and how aging, 

menopause or andropause, and conditions associated with aging, such as diabetes, affect these 

responses. 

Discussion 

The relationship between IL-6 and estrogen is somewhat complex. In vitro, small but highly 

concentrated doses of estrogen can affect IL-6 activation and coactivator binding to NFκB, yet in 

vivo, as shown by Dr. Kovacs’ group, IL-6 increases in response to estrogen treatment. This 

bimodal activation of IL-6 may result from differences in mechanisms and related receptors, or it 

may represent indirect effects. 

The data indicating early protection from injury in females is consistent with other work showing 

that females are protected from injury to the gut and lung during the first 24 hours following 

burn injury. The mechanism for these effects is not clear. These sex differences might reflect 

differing cell populations activated early and late, differences in bone marrow response, or a 

combination of hormonal effects and differing cell populations. 

Sex Differences in Lung Function and Response to Environmental Agents 

Darryl C. Zeldin, M.D., National Institute of Environmental Health Sciences, NIH 

Asthma is a chronic lung disease characterized by intermittent bouts of wheezing, coughing, 

reversible airflow obstruction, airway inflammation, and bronchial hyperresponsiveness. It is of 

enormous public health importance because of its prevalence of about 11%, its mortality rate of 

more than 5,000 deaths per year, and its associations with hospital admissions, emergency-room 

visits, poorer quality of life, and high costs. On the basis of data from the Third National Health 

and Nutrition Examination Survey, clear sex differences in asthma prevalence exist. Among 

children, prevalence is higher among males than females, but around the age of 20 years and in 

most of adult life, prevalence is higher among females. At older ages, the trends change again, 

and prevalence is higher among males. 

Several studies have suggested that sex hormones influence asthma. Severity and bronchial 

hyperresponsiveness vary during the menstrual cycle;59 and half of all female patients admitted 

to the hospital for asthma are admitted during the premenstrual phase.60 Use of oral 

contraceptives can attenuate the effects of the menstrual cycle on lung function,61 and 

postmenopausal women receiving HRT experience less airway obstruction than those not taking 

HRT.62 In addition, females with Turner’s syndrome, which is characterized by low circulating 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 13 

levels of estrogen, experience increased airway obstruction that can be alleviated with estrogen 

treatment.63

Dr. Zeldin’s group has used mouse models to further explore these differences. Using an 

invasive measure of lung function, they found that response to methacholine, an airwayrestrictive agent, is more enhanced in males than in females, as shown by decreased Newtonian 

and tissue resistance and decreased elastance.64 This group also found that bacterial LPS, which 

triggers an inflammatory response in the lung, induced an exaggerated hypothermic response in 

males, compared with that seen in females. Inflammatory response in males was also 

exaggerated, as shown by the presence of neutrophils in bronchoalveolar lavage (BAL) fluid, 

increased histopathological scores, and increased cytokine response. Ovariectomy had no effect 

on hypothermic response in females, but castration abolished the inflammatory response in 

males, as demonstrated by attenuated cytokine response. Females treated with the androgen DHT 

exhibited exaggerated response to LPS, as shown by increased production of TNF-α, increased 

numbers of cells and neutrophils in BAL fluid, and enhanced hypothermic response. Thus, 

airway responsiveness is influenced by sex, and the exaggerated response seen in males is 

mediated in part by androgens. 

Despite the apparent sex differences in this mouse model, the physiologic roles of sex hormone 

receptors in the adult lung are not known, although knockout mouse models for both ER-α and 

ER-β are available.65,66 Both ER-α and ER-β are expressed in the lung,67 and although ER-β is a 

critical regulator of alveoli formation and surfactant homeostasis,68 both receptors are required 

for alveoli formation in mice.69 How the expression and function of these receptors correlate 

with the exaggerated airway response observed in male mice exposed to methacholine remains to 

be determined. 

Discussion 

No work has been done to compare estrus cycles and differences in responsiveness in females. 

Periodontal Disease Inflammation and Obstetric Complications 

Steven Offenbacher, D.D.S., Ph.D., University of North Carolina, Chapel Hill 

Periodontal problems are common in pregnancy and are more prevalent than all other obstetric 

and sexually transmitted infections combined. Several studies have shown that maternal 

periodontal disease significantly increases the risk for preterm births and preeclampsia,70 and 

active periodontal progression increases the risk independently of other factors such as 

socioeconomic status, smoking, or drug abuse. Organisms living in the oral biofilm, a slime layer 

that forms on the teeth and includes clusters of several different organisms,71 create an ulceration 

of the epithelium, gain access to the system circulation, and are disseminated through the liver 

and to the fetus, resulting in placental and fetal exposure to microbes, poor placental perfusion, 

and inflammation. These events ultimately give rise to fetal growth restriction, preterm delivery, 

and neonatal morbidity. Exposure to oral organisms is highly associated with admission to and 

lengthy stays in neonatal intensive care units, and because low birth weight has been associated 

with several adverse outcomes, including neurological deficits, intrauterine exposure to oral 

organisms can contribute to lifetime disabilities and impairments. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 14 

Most common among these organisms is Campylobacter rectus (C. rectus), a commensal 

inhabitant of the oral cavity in humans. Campylobacter species have been associated with 

abortion, premature labor, and severe perinatal infection in humans and induce abortions in 

horses, cattle, and sheep. In mice, exposure to C. fetus or C. jejuni during pregnancy results in 

adverse pregnancy outcomes and fetal complications.72 Fetal exposure C. rectus is found in 

approximately 28% of all pregnant women and in more than half of infants at 32 weeks of 

gestation.73 Although exposure to C. rectus increases the risk of premature delivery, high levels 

of CRP, the oxidative stress marker 8-iso, TNF-α, and PGE2 also increase this risk 

exponentially. 

Dr. Offenbacher’s group has studied the effects of infection by C. rectus in a mouse model.74,75

In this model, a sterile chamber was implanted in the dorsolumbar subcutaneous tissue and 

inoculated with C. rectus, then mice were bred and subjected to a timed challenge. Exposure to 

C. rectus increased the frequency of resorption and fetal growth restriction and decreased 

neonatal survival. The weight of growth-restricted fetuses was reduced by 27%, compared with 

controls. Mild inflammation was observed in the decidual zone of the placenta, and development 

of the labyrinth zone decreased in response to challenge with C. rectus. Expression of Th1 

cytokines increased, and that of Th2 cytokines and TGF-β decreased. To further explore the 

relationship between inflammation and growth restriction, Dr. Offenbacher’s group conducted 

microarray analyses on placenta from challenged mice. These analyses revealed that 202 genes 

were differentially expressed. The majority of these genes were downregulated, including genes 

encoding placental growth factors, suggesting that placental development is suppressed in 

response to C. rectus exposure. Among the genes significantly downregulated was Igf2, an 

imprinted gene that is critical for somatic growth of the fetus. 

Imprinting is a process in which DNA methylation at CpG sites converts biallelic expression to 

monoallelic expression. CpG doublets, which are often present in high-density regions of 

selected genes, are highly prevalent among master control genes that regulate transcription, cell 

growth and differentiation, morphogenesis, and neoplasm development. DNA methylation is 

often associated with gene silencing through alterations in chromatin structure, and it is 

conserved following DNA replication, resulting in a stable, somatic, heritable DNA modification 

that does not involve a sequence change. This process generally increases throughout the life 

span and controls the differentiation of CD4+

 T cells. 

A comparison of murine imprinted genes and array signals showed that among 21 shared genes, 

9 were significantly downregulated, indicating that in addition to suppressing growth and 

differentiation factors in the placenta, exposure to oral organisms also enhances imprinting. 

Bisulfite sequencing of DNA extracted from the placenta of organism-challenged mice revealed 

some sequences in Igf2 that were hypermethylated as a result of C. rectus challenge (in press). 

These results are consistent with work by McMinn and colleagues,76 who reported suppressed 

placental expression of Igf2 mRNA in human intrauterine growth restriction. The functional 

significance of Igf2 hypermethylation is not known, but it might block an Sp-1 binding element. 

Moreover, because methylation patterns are heritable in somatic cells, infection-mediated 

epigenetic alterations could affect the well-being of the offspring. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 15 

Discussion 

Other placental growth factor genes that were downregulated in response to C. rectus challenge 

have CpG doublets available for DNA methylation, and other bacterial infections, such as 

Helicobacter pylori, have also been linked with increased DNA methylation. Many intrauterine 

methylations are thought to persist throughout a person’s lifetime. 

Methylation analyses using human placenta have not yet been done. 

SESSION III: CELLULAR AND MOLECULAR MECHANISMS IN 

INFLAMMATION RESEARCH 

Moderator: Susan Kovats, Ph.D., Beckman Research Institute 

As shown by the work presented in Session II, sex differences exist in the host response to 

injury, environmental insult, and chronic disease. The presentations in Session III focused on the 

cellular and molecular mechanisms by which sex hormones influence inflammatory processes 

and immune response. 

Vascular Cell Signaling by Membrane Estrogen Receptors 

Jeff Bender, M.D., Yale University 

Although the endothelium has historically been considered a static barrier, recent work has 

shown it to be highly dynamic. In addition to its barrier function, other roles for the endothelium 

include regulation of vasomotor tone, anticoagulant and prothrombotic functions, and a role in 

inflammatory events. Endothelial dysfunction is prothrombic, present in atherosclerosis and 

hypercholesterolemic states, involved in impairment of vasomotor tone, permissive of leukocyte 

adhesion and transmigration, and involved in the alteration of barrier properties. Under normal 

conditions, the endothelium produces large quantities of NO, which inhibits platelet aggregation, 

monocyte adhesion, and smooth muscle cell proliferation and migration and promotes the 

relaxation of smooth muscle cells. These functions are also carried out by prostacyclin (PGI2), 

which is also released by the endothelium. Much less NO and PGI2 are produced by a 

dysfunctional endothelium, and macrophages and foam cells produce large amounts of 

superoxides, which quench NO and form peroxynitrite, a toxic agent that damages the vascular 

wall. 

Estrogen can have a protective effect against CVD, as suggested by low rates of coronary heart 

disease (CHD) in premenopausal women, the narrowing gender gap of CHD after menopause, 

and the increased risk for CHD in oophorectomized young women not taking estrogen. Estrogen 

might diminish the degree of monocyte or T-cell adhesion to the arterial intima, an early 

component of atherogenesis. It also might enhance the release of NO, thus impairing monocyte 

adhesion, neointima formation, and platelet aggregation. Dr. Bender’s group has shown that 

estrogen treatment of the endothelium prevents cytokine-mediated induction of adhesion 

molecules and that this protective effect is mediated by ER-α.

77

Yet there is a conundrum associated with estrogen. Data from in vitro and in vivo animal studies 

show a favorable effect in terms of CVD, but clinical trials, such as the Women’s Health 

Initiative (WHI), demonstrate an unfavorable effect. A review by Grodstein and colleagues 

found a somewhat protective effect in postmenopausal users of estrogen and HRT,78 which 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 16 

contradicted earlier studies showing no protective effect as well as the increased incidence of 

cardiovascular events observed in the WHI. Part of the explanation for this conundrum may lie in 

the conduct of these studies. Mikkola and colleagues found a large burden of atherosclerosis in 

ovariectomized primates fed an atherogenic diet.79 If these primates had been treated with 

conjugate equine estrogen at the time of ovariectomy, the burden of atherosclerosis was reduced 

by 70%. However, if the initiation of estrogen treatment was delayed by 2 years, there was no 

protective effect. Thus, timing may be critical, and the results from the WHI should be 

considered in this light. In the WHI, the average age at enrollment was 63 years, participants had 

been postmenopausal an average of 12 years, and many had established, significant vascular 

disease. 

Dr. Bender’s group has focused on estrogen effects triggered by the activation of membranebound ER. Unlike the traditional nuclear receptor pathway, which is usually delayed, the 

membrane ER pathway is rapid and induces eNOS through the phosphatidylinositol 3-kinase 

(PI3K)/Akt pathway. The resulting release of NO induces the production of cyclic GMP in 

smooth muscle cells, thereby interfering with proliferation and migration and promoting 

relaxation. Membrane-bound ER is distributed in a punctate pattern in a subset of endothelial 

cells and is found in two forms: the full-length 66 kDa ER-α, and a shorter, 46 kDa form that 

arises from a translation initiation site found in the second exon of the ER-α gene.80 No fulllength ER-α is observed in estrogen-deprived HUVEC. Treatment with estrogen induces some 

expression of the full-length ER-α in these cells, but the shorter form is predominant. 

Cardiovascular studies in ER-knockout mice have been carried out by several groups, using the 

wire loop carotid injury remodeling model, where smooth muscle cell proliferation and matrix 

deposition is inhibited by 17β-estradiol.81 Elimination of ER-α or ER-β has no effect, and 

elimination of both has only a modest effect. The shorter form of membrane ER is present in 

knockout mice in which exon 1 h as been targeted, but in knockout mice in which exon 2 has 

been targeted, there is no ER-α protein and vascular response to estrogen is eliminated. Thus, the 

shorter form of membrane ER plays an important role in vascular tissue. 

Work is under way in Dr. Bender’s laboratory to further characterize the shorter, membranebound form and its role in mediating the vascular effects of estrogen. In addition, new clinical 

trials are in progress to further clarify the effects of estrogen on CVD. Future work should 

determine how selective estrogen receptor modulators (SERMs) affect the estrogen responses 

mediated by membrane ER versus nuclear ER, how the membrane-bound ER is structured, and 

what the possible roles are for associated proteins in vascular remodeling responses. 

Discussion 

Work discussed by Dr. Bender has focused only on 17β-estradiol. The effects of pregnancy 

levels of estrogen are not known. The short, 46 kDa form of the ER most likely is not unique to 

endothelial cells, although there may be some tissue specificity with regard to splicing and any 

complexes the ER forms with coregulators. The shorter form of the ER is more efficient at 

transducing rapid responses and less so at mediating traditional nuclear receptor responses, 

although it does have that capability. It is not known whether the shorter ER is active in the 

absence of ligand. The ectodomain of this receptor is also unknown, although its sequence 

predicts a C-terminal ectodomain. Studies of the PR and GR have also shown receptors of 

varying sizes specific to the tissue in which they are expressed. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 17 

It is not clear whether work has been done to examine the ability of ER-α splice variants to 

dimerize with ER-β. 

Sex Differences in Host Defense Against Pneumonia 

Lester Kobzik, M.D., Harvard University 

Pneumococcal pneumonia is the most common form of community-acquired pneumonia and is 

associated with morbidity and mortality in hospital patients.82,83 This is partly caused by 

frequent, low-dose infectious challenges to the lungs through nocturnal aspiration.84,85 Alveolar 

macrophages play a critical role in lung defense. They are avidly phagocytic and use scavenger 

receptors to interact with unopsonized inhaled particles and pathogens.86 Depletion or 

dysfunction of alveolar macrophages has been linked to the decreased clearance of bacteria and 

particles. 

Using murine pneumococcal pneumonia as a model, Dr. Kobzik’s group has found that 

resistance to pneumonia is greater among females than among males. The percentage of 

neutrophils and bacteria found in the lung is lower in females, and survival rates are higher. 

Although these sex differences are only observed at low doses of pathogen, these doses might be 

relevant to early defense events. Sex differences in susceptibility to Pneumococcus have been 

demonstrated by several groups for many decades, beginning with Weiss and colleagues87 in the 

1970s. Yet further exploration of host defense has yielded confusing results.88

In experiments conducted by Dr. Kobzik’s group, the number of bacterial cells ingested by 

alveolar macrophages was identical between male and female mice, but the kill rate was higher 

among females. This was true also for human alveolar macrophages. The difference in kill rates 

could not be explained by differences in NADPH oxidase activity, but it was abolished by nonselective NOS inhibition, indicating involvement of the NO pathway in sex differences in host 

defense. The female advantage could be explained by a role for NOS2, which is constitutively 

expressed in both murine and human alveolar macrophages. Further examination revealed 

divergent pathways of host defense, with males relying more on iNOS/NOS2 and females 

relying more on eNOS/NOS3. NOS3 is activated by the PI3K pathway, which is triggered by 

macrophage phagocytosis and by estrogen.89 In vitro studies by Dr. Kobzik’s group using 

estrogen treatment of macrophages reproduced the female advantage observed in vivo, as well as 

the abolition of that advantage by treatment with a NOS inhibitor. Consistent with other studies 

of the endothelium, estrogen-mediated phosphorylation of Akt induced phosphorylation of 

NOS3, and inhibition of Akt blocked the estrogen-mediated advantage. 

Although in vitro studies, studies in animal models, and some epidemiological studies indicate 

sex differences in susceptibility to community-acquired pneumoccoccal pneumonia, no clear 

data has been generated to date in humans. In addition, it is not clear whether the estrogenmediated resistance to pneumoccoccal pneumonia will have therapeutic relevance. Models of 

other problems with innate immunity are needed, as well as studies exploring neonatal host 

defense susceptibility. More epidemiological studies are needed to further clarify the relevance 

of the murine pneumococcal pneumonia model. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 18 

Discussion 

The differences in activity between alveolar macrophages from females and those from males is 

likely entirely hormone driven. In vitro, an advantage is slowly conferred by estrogen treatment 

on macrophages from male humans or mice. If adoptive transfer experiments were conducted, 

alveolar macrophages most likely would assume the characteristics of their environment. 

Although eNOS is expressed in murine alveolar macrophages, the use of eNOS knockout models 

does not necessarily prove that eNOS is responsible for the observed sex differences in 

susceptibility. The use of knockout models in a macrophage-specific fashion could further 

determine whether eNOS function is important in the lungs, as opposed to the sex differences 

being related to a vascular issue. However, in the lung, macrophages encounter bacteria first, and 

it is unlikely that other cells are necessary in the time window studied. Moreover, macrophagedepletion studies support a role for eNOS in the sex differences in alveolar macrophage function. 

eNOS levels are identical between males and females within 2 hours of bacterial challenge, and 

no iNOS is observed. By 24 hours, however, iNOS has been induced. The enzymatic activities of 

eNOS and iNOS have not been compared between males and females. However, differences in 

activity may not correlate with differences in macrophage activity. In a different setting, Dr. 

Deitch’s group has demonstrated increased activity for iNOS, but this did not explain the ability 

of macrophages to kill pathogens. 

Estrogen Modulation of Dendritic Cells 

Susan Kovats, Ph.D., Beckman Research Institute 

Most lymphoid and myeloid cells express ER and AR, pointing to a role for sex hormone 

regulation in autoimmunity and response to infection. Systemic manipulation of sex hormones or 

the response to them is an attractive therapeutic approach, but it requires further understanding of 

the regulation of immune function by sex hormones, both in normal biology and in disease 

states. Dendritic cells (DC) serve as a bridge between innate and adaptive immunity. They 

facilitate contact with the antigen and pathogen products; activate antigen-presenting cells 

through antigen processing and presentation, costimulation, and cytokine production; and 

activate naïve T cells and CD4+

 T helper cell polarization. Although several studies have 

provided evidence of estrogen action at these different steps, little is known about the effects of 

estrogen on the DC themselves. 

In an ex vivo model for DC differentiation, Dr. Kovats’ group added granulocyte M-CSF (GMCSF) to total bone marrow, then monitored cellular development using flow cytometry. In some 

experiments they removed estrogen from the culture medium and added back lower physiologic 

doses, and in others they added ER antagonists to standard culture medium. The addition of 

estrogen promoted the differentiation of DC from bone marrow cells, and at the end of the time 

period studied, these cells expressed ER but not AR.90 The addition of testosterone or DHT had 

no effect. Estrogen-dependent DC expressed CD11c and intermediate levels of CD11b but were 

negative for Ly6C. These cells also expressed langerin, suggesting that estrogen stimulates the 

development of Langerhans cells, which are more capable of responding to LPS than the 

estrogen-dependent DC in these cultures, have a reduced endocytic capacity, and express lower 

levels of Toll-like receptor 4 (TLR4) but higher levels of CD86 and major histocompatibility 

complex (MHC) I and II.91 Efforts are under way to determine the mechanism of estrogen action 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 19 

on differentiating DC, the role of the ER in DC differentiation and antigen-presenting function in 

vivo, and the role of ER-mediated events in DC function during autoimmunity. 

Dr. Kovats’ group has also explored the effects of the SERMs tamoxifen and raloxifene on DC 

differentiation. Both drugs have been compared in breast cancer prevention trials. Tamoxifen 

exerts antagonist activity in breast tissue but agonist activity in endometrial tissue and bone. 

Raloxifene, which was developed as a drug targeted toward osteoporosis, exerts agonist activity 

in bone but antagonist activity in breast and endometrial tissue. Using their ex vivo culture 

system, Dr. Kovats’ group found that exposure to either drug inhibits estrogen-mediated DC 

differentiation.92 The DC that differentiated were hyporesponsive to TLR4 and TLR9 ligands 

and exhibited decreased production of MHC-II, CD86, and IL-12. However, tamoxifen exposure 

did not affect the expression of TLR4 or TLR9. These cells were therefore more likely to exist in 

a resting state than in an activated one. Future work will explore how SERMs modulate tissue 

DC differentiation in vivo, during infection, and during autoimmunity and how they modulate 

DC function in innate and adaptive immunity. 

In general, more optimal models are needed to further our understanding of sex hormone 

regulation of immune function. Development of these models should address sex and age, as 

well as systemic exogenous manipulation of hormones versus endogenous variations, ways to 

measure those variations, ER deficiency versus systemic or cell-type–specific blockade, disease 

models with sex-based differences in immune function, other hormones such as progesterone, 

and the HPA axis. 

Discussion 

The role of estrogen in the regulation of DC differentiation or function in vivo during 

autoimmunity is still unknown. In young mice prone to SLE, DC are already increased, and in 

older mice that already have SLE, more DC are observed in females than in males. More work is 

needed in a gender-divergent lupus model to determine whether this increase is a cause or effect 

of SLE and whether early stages of pathogenesis involve changes in the number or function of 

DC, or both. 

It is not clear whether the number of Langerhans cells differs between males and females. 

Estrogen, Inflammation, and Thrombotic Risk 

Virginia Miller, Ph.D., Mayo Clinic 

For thrombosis to occur, a mechanical or biochemical lesion must be present in the vascular wall 

and blood components must interact with that lesion. Components in the blood include soluble 

factors such as coagulation proteins and cellular factors such as platelets and leukocytes. 

Platelets represent a dynamic cell system, as their population turns over in about 12 days, and 

they therefore represent a target for phenotypic modulation. When activated, platelets express 

membrane adhesion proteins and secrete vasoactive, inflammatory and mitogenic cytokines from 

dense and alpha granules. Thus, when activated, they can interact with leukocytes and the 

vascular wall and facilitate the generation of thrombin. Platelet aggregation therefore plays a role 

in thrombotic risk. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 20 

Platelets are produced in the bone marrow from megakaryocytes, which express ER and AR, and 

they turn over every few days. Dr. Miller’s group has examined platelet aggregation in plateletrich plasma in pigs. As these pigs reached sexual maturity, aggregation decreased in females and 

increased in males. Ovariectomy in females caused an increase in platelet aggregation, which 

was reversed by treatment with estrogen or a SERM. Estrogen also modulated the secretion of 

TGF-β and platelet-derived growth factor (PDGF) by activated platelets.93,94 Thus, hormones can 

modulate platelet phenotypes and regulate their aggregation. However, to date, no studies have 

examined platelet function across the lifespan, and no longitiudinal studies have been done to 

relate platelet phenotype with vascular lesions in humans. 

In another set of experiments, mice were injected with Escherichia coli LPS, and platelets were 

examined before injection and at 7 days afterward, when platelets usually turn over. Platelet 

aggregation and secretion of alpha and dense granules increased following LPS injection, but 

fibrinogen binding did not.95 Thus, a single inflammatory exposure, LPS, can affect systemic 

inflammatory processes days after the initial event. In preliminary experiments, expression of 

fibrinogen receptors was examined in early menopausal women in whom platelets had been 

stimulated with ADP or sTRAP. Although the collective response, illustrated as the percentage 

of fibrinogen-positive platelets, showed large variability, individual responses to both agonists 

were consistent, suggesting that a platelet phenotype might be characteristic for an individual. 

These results, along with those in mice, point to difficulties in using any one assay to define 

changes in procoagulative phenotypes. However, individuals could be monitored to determine 

whether their responses to interventions increase or decrease, and using a panel of assays can 

provide insight into factors that contribute to procoagulative activity. In addition, the effects of 

cytokines, hormones, and pathogens can be added to a model examining changes in platelet 

phenotype and function. Dr. Miller’s group has employed this approach by conducting array 

analysis on some of their samples, and they have found increased expression of PDGF and 

RANTES expression in females, compared with males. More assays are available to determine 

what constitutes risk, but more work is needed to clarify the relationships among serology, 

hormones, cytokines, and platelet characteristics. 

A review by Herrington and colleagues found no predictive differences in allele or genotype 

frequencies to establish thrombotic risk in an analysis of proteins involved in the coagulation 

cascade.96 Thus, efforts to characterize thrombotic risk must move beyond these proteins. Dr. 

Miller’s group has examined relationships between the ER and platelet activity. In mice deficient 

for ER-β, no differences were observed in the phenotype of platelets or reticulated platelets in 

young mice. However, the percentage of reticulated platelets was increased in aged mice 

deficient in ER-β. Under these conditions, the platelets observed were positive for P-selectin, 

fibrinogen, and annexin V expression97. In addition the number of mice who died within 24 

hours of LPS injection increased slightly for aged mice deficient in ER-β, suggesting that 

hormonal status influences phenotypic expression in animals with genotypic variation in ERs. 

These results provide some insight into thrombotic risk, and they point to a paradigm shift in the 

way risk is defined. New models should further assess coagulability of the blood, the role of 

genetics and environment in platelet phenotypes, and new assays to determine individual risk. A 

randomized trial is under way to translate findings from animal models to humans. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 21 

Discussion 

At present, the location of ER-β in platelet membranes (alpha, dense granules) is not known, and 

much remains to be learned about platelet energetics with changes in hormonal status. 

Phosphorylated antibodies and assays to assess coagulative risk can be added to a model defining 

thrombotic risk. 

Use of Insulin Therapy in Treatment of Inflammatory Diseases 

Greet van den Berghe, M.D., Ph.D., University of Leuven, Belgium 

Activation of the insulin receptor results in the transduction of metabolic signals, through the 

action of PI3K, as well as transduction of a mitogenic signal.98 Type II diabetes occurs when 

excess insulin bypasses the receptor and directly influences mitogenic events. Under these 

conditions, the metabolic signal is abolished, and the number of mitogenic complications 

increases. Critical illnesses such as sepsis, burn injury, and renal failure involve a high degree of 

inflammation, but they also involve hyperglycemia arising from insulin resistance in the liver 

and muscle. High levels of insulin-like growth factor binding protein 1 can predict survival 

during critical illness.99,100

Dr. van den Berghe’s group has studied the effects of intensive insulin therapy (IIT) on critical 

illness. They found that IIT controlled blood glucose and improved survival among patients in a 

surgical intensive care unit (ICU).101,102 They also found that improvement was maintained long 

term among patients in a cardiac ICU.103 In addition, IIT alleviated morbidity associated with 

brain injury and improved recovery among neurosurgery and neurology survivors.104 Further 

analyses revealed that IIT was highly cost effective, saving a median of 2,683 euro per patient.105

Illness is more severe among patients admitted to the medical ICU, and more comorbidities, such 

as HIV/AIDS, cancer, and end-stage diseases, are present. Thus the hospital mortality rate is 

higher among patients who stay in the medical ICU for a few days. In a study similar to the 

surgical ICU study, Dr. van den Berghe and colleagues explored whether IIT would prove 

beneficial to patients in the medical ICU for at least 3 days.106 IIT significantly reduced mortality 

among patients in this group, to a degree comparable to the reduction in mortality observed 

among surgical ICU patients.101,106 Patients were weaned from the ventilator, moved from ICU, 

and discharged from the hospital at an earlier time than patients who received conventional 

treatment. The risk for kidney injury, hyperbilirubinemia, and critical illness polyneuropathy was 

also reduced. However, IIT did not affect bacteremia or infection. 

The IIT-associated cumulative reduction in risk for hospital mortality was 4% among intent-totreat patients and 8% among patients staying at least 3 days (van den Berghe et al., Diabetes

2006, in press). Multivariate logistic regression analysis revealed no sex differences in the 

efficacy of IIT. Although a recent editorial suggested that IIT only works to offset deleterious 

effects of feeding, analysis of efficacy among three tertiles of intravenous glucose showed a 

benefit even among patients who received little glucose. 

Although IIT is effective among surgical and medical ICU patients, potential harms exist, such 

as hypoglycemia and dangers associated with treatment for fewer than 3 days. Brief 

hypoglycemia increased among patients receiving IIT in the surgical and medical ICUs,102,106 but 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 22 

this hypoglycemia did not appear to affect mortality in these studies or in a nested case control 

study.107 Nor was mortality increased when IIT was given to surgical ICU patients for fewer than 

3 days. Mortality did increase among medical ICU patients, but multivariate logistic regression 

revealed history of cancer, diabetes, or kidney infection as the reasons for increased risk.106

Elevated levels of CRP, creatinine, and alanine aminotransferase also were shown as reasons to 

restrict IIT. Potential harms associated with the high insulin dose used in IIT are under 

investigation, but thus far, the clinical benefit of IIT in the ICU outweighs the potential harm. 

IIT reduces the levels of CRP and mannose-binding lectin.102 In a rabbit model of acute and 

prolonged critical illness,108 neuropathy, kidney function, and mortality increased when glucose 

levels were high, regardless of insulin.109 IIT counteracted the effects of glucose toxicity, thereby 

protecting against mitochondrial damage; increasing activity of the respiratory chain; and 

suppressing adhesion molecule expression, iNOS expression and circulating NO levels.98,110,111

Thus, IIT improves outcome by preventing glucose toxicity and its associated effects. This does 

not rule out direct effects of insulin, but these effects most likely are not independent of glucose 

control. 

Discussion 

Further work is under way to determine the role, if any, of glycated proteins in the outcomes 

associated with critical illness. Although the liver is important in estrogen metabolism, no studies 

have examined the relationship between this and illness-associated hyperglycemia. 

SESSION IV: EMERGING STRATEGIES IN TREATMENT OF 

INFLAMMATORY DISEASES 

Moderator: DeLisa Fairweather, Ph.D., Johns Hopkins University 

Presentations in this session explored flaws in current treatments, agents designed to interfere 

with the activity of inflammatory mediators, and new players in or markers of inflammation. 

TNF-Blocking Agents: Raising the Bar for Effective Treatment 

Daniel Furst, M.D., University of California, Los Angeles 

The autoimmune disease RA is more prevalent among women than men. TNF-blocking agents 

and other biologics have the potential to radically change the symptoms, quality of life, and 

outcomes associated with RA, but the mechanisms underlying sex differences in predisposition 

to RA are not known, and sex differences in response to treatment have not been tested formally. 

Raising the bar for effective treatment means improving methods to measure response and 

improving medications for RA, and expectations for successfully treating this disease are high. 

Historically, response to RA treatments was measured by counting the number of tender or 

painful joints or conducting a patient global assessment. In 1993, a panel determined that the 

number of swollen joints were most important in determining response, followed by physical 

disability and pain.112 This made it harder to compare drugs and determine efficacy, because two 

drugs, for example leflunomide and methotrexate, might look similar in terms of tender joint 

count, but not in other efficacy end points. In addition, sex differences in response were not 

tested. van der Heijde and colleagues proposed a system that examined changes in the underlying 

pathology, with an aim toward preventing structural damage.113 This scheme involved the 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 23 

systematic measurement of joints and scoring them based on the amount of erosion observed. On 

the basis of these scores, an inverse longitudinal relationship between radiographic damage and 

physical function could be observed.114,115

Comparison among RA treatments was also hampered by multiple outcome measures across 

clinical trials. To address this problem, a group of committed rheumatologists sought to develop 

a specific set of outcome measures and provide uniformity and standardization. In a consensus 

statement,116 the ACR issued criteria that include a decrease in both tender and swollen joints 

and an improvement in at least three of five measures: patient global assessment, pain, 

physician’s global assessment, validated functional measures, and acute phase reactants. These 

criteria could be applied to a 20%, 50%, or 70% response, where a 20% response equated to a 

patient feeling better and a 70% response equated to a patient being close to remission. This 

approach has allowed comparisons among studies and moved the development of anti-rheumatic 

drugs forward. On the basis of these criteria, for example, results from a trial of the TNF-blocker 

adalimumab showed a dose response at 26 weeks.117-119 In this trial, 50% of patients showed a 

20% response and only 10 to 20% showed a 70% response. ACR response criteria were also 

applied to the ASPIRE study,120,121 which compared infliximab and methotrexate, and to studies 

comparing etanercept, infliximab, methotrexate, and leflunomide. Yet although the criteria 

facilitated comparisons, they were dichotomous and did not relay information about disease 

activity at baseline. 

Investigators in the Netherlands devised the Disease Activity Score (DAS) to assess 

improvement or response.122,123 Although this was not an intuitive index,124 the DAS provided a 

continuous measure and was incorporated into the European League Against Rheumatism’s 

(EULAR) definition of response.125,126 This measurement corroborated the ACR Response 

criteria and moved the field forward as well as allowing better, quantitative comparisons. For 

example, in a trial comparing methotrexate, sulfasalzine, and a combination of the two, no 

differences were observed among these regimens, based on the DAS.127 In addition, on the basis 

of the area under the curve of improvement in DAS, researchers were able to determine that TNF 

blockers worked more quickly than methotrexate. DAS could also be correlated with ACR 

response criteria, as shown in trials of TNF blockers, methotrexate, and other therapeutic 

agents.128,129 On the basis of these indices, these drugs inhibited radiographic damage, even 

though patients did not report feeling better. For the first time, investigators could see that these 

regimens or combinations caused radiographic healing. Although none of these response 

definitions incorporated testing for sex differences, multivariate regression analyses showed no 

obvious differences. 

Thus, the development of improved outcome measures improved the ability to develop effective 

therapeutic agents and, with the development of the biologic anti-rheumatic agents, have “raised 

the bar,” in terms of both expectations and results. The DAS most likely improves the ability to 

separate drugs, but the ACR criteria form a more intuitive measure of response. Trials are in 

progress, using these indices to assess sequential monotherapy and varying combinations. Early 

results show that, on the basis of X-ray progression, DAS, and Health Assessment Questionnaire 

indices, treatment with a TNF blocker or an aggressive combination with prednisone induces a 

rapid response at an early time, and patients go into remission more quickly.130-132 Future work 

should formally test sex differences in disease activity and response to treatment in the studies 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 24 

already done and assess the causes of these differences, including genetic, hormonal, and 

environmental effects. 

Discussion 

According to some anecdotal reports, some women who have taken TNF blockers for RA and 

become pregnant reported improvement after delivery. Existing data indicate that the effects of 

pregnancy on RA vary. TNF blockers are used increasingly in pregnant patients. 

Biomarkers that can predict responsiveness to RA treatments are not yet known. 

MIC-1, a Poorly Understood TGF-β Superfamily Cytokine with Roles in 

Inflammation and Cancer 

Samuel Breit, M.D., Ph.D., University of New South Wales, Australia 

Macrophage inhibitory cytokine 1 (MIC-1), a distantly related member of the TGF-β

superfamily of cytokines, is induced in most cells by injury, inflammation, and malignancy, 

primarily through the action of p53 and EGR-1. It is variably processed, secreted cytokine; 

unprocessed MIC-1 remains tissue bound through interactions with the extracellular matrix. The 

H6D polymorphism, which changes a histidine residue to aspartic acid at position 6 in the mature 

protein, has been associated with altered risk for cancer development and outcome. 

Although MIC-1 is not generally expressed in normal tissue, serum levels increase dramatically 

and rapidly during pregnancy. The MIC-1 protein is highly expressed in the placenta and 

involved in interactions between the placenta and maternal tissue, and it inhibits trophoblast 

outgrowth and proliferation. Serum concentrations of MIC-1 have not been associated with 

preeclampsia or Down syndrome pregnancies, but they are much lower in women who miscarry 

than those who carry to term.133 In a large cohort of patients monitored through blood samples 

and imaging, MIC-1 levels decrease as early as 3 weeks before miscarriage occurs. In a smaller 

cohort of patients whose pregnancies were proven viable by ultrasound at the time of sample 

collection, MIC-1 levels were lower in women who later miscarried. No diagnostic test is 

available to predict the likelihood of miscarriage. Although more prospective studies are needed, 

serum MIC-1 levels might form the basis for such a test, predicting miscarriage early enough to 

apply therapeutic interventions. Further study should address the possible involvement of MIC-1 

in the immunoregulatory processes associated with placental implantation, growth, and 

development and roles for MIC-1 in placental health. The high expression of MIC-1 in the 

placenta, as well as its overexpression by many sex-specific cancers, suggests that this cytokine 

is influenced by sex and gender and may be regulated hormonally. 

Studies have associated MIC-1 serum levels with chronic inflammatory diseases. In a 

prospective case-control study of women aged 45 years or older with no signs of CVD or cancer, 

MIC-1 serum levels predicted cardiovascular risk, independently of CRP and other inflammatory 

factors.134 Other studies found that MIC-1 expression was upregulated in the hypertrophic 

myocardium and that MIC-1 protected against cardiac failure in animal models. MIC-1 also has 

been implicated in RA. In a pilot cross-sectional study in 60 patients, MIC-1 levels were elevated 

in unselected RA but were much more so in patients whose disease was so severe as to require 

therapy using autologous bone marrow transplant (Brown et al., Arthritis Rheum 2006, in press). 

At 3 months following transplantation, however, MIC-1 levels had returned to normal. In 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 25 

addition, the H6D allele of MIC-1 was associated with early-onset erosive disease and severe, 

treatment-resistant RA, and an algorithm incorporating serum MIC-1 levels and the H6D allele 

could predict response to hematopoietic stem cell transplantation and the presence of severe 

disease and erosions. 

Discussion 

The protective effect of overexpressed MIC-1 against cardiac failure appears to contradict study 

results showing MIC-1 as a risk factor for CVD events. It could be that MIC-1 is produced to 

mitigate injury but offers only partial protection. Higher concentrations of MIC-1 could result in 

improved outcomes, but removal of MIC-1 could be deleterious. Such effects have been found 

with IL-10 in other chronic inflammatory processes. 

Current Lupus Treatments Neither Control Disease Activity or Prevent Organ 

Damage: The Case for New Approaches 

Michelle Petri, M.D., M.P.H., Johns Hopkins University 

SLE is a prototypical autoimmune disease characterized by rashes, arthritis, and hair loss. SLE 

autoantibodies, which appear years before clinical lupus presents,135 mediate an inflammatory 

process leading to complement activation and ultimately, to fibrosis. The disease affects about 

1.5 million Americans, more commonly among women, African Americans, and Asians. The 

manifestation of SLE is similar to that for MS, with a relapse-remission pattern that progresses in 

spite of modern therapy. The most common cause of death among patients with SLE is CVD. 

Although SLE is more common among women, it is more severe among men. Production of 

antibodies against double-stranded DNA is similar between men and women, as are low levels of 

C3 and C4, but men are more likely to experience proteinuria, renal insufficience, renal failure, 

and myocardial infarction. 

The incidence of SLE has tripled since the 1950s to 1970s.136

Although the prognosis for patients with SLE has improved since the 1950s, it has remained 

about the same since the 1980s. In addition, 50% of patients with SLE have permanent organ 

damage, and for 75% of these patients, organ damage results from treatment with corticosteroids. 

In a study 498 women with SLE and 2,208 women in the Framingham Offspring Study, 

myocardial infarction was 50 times more likely among female SLE patients aged 35 to 44 

years.137 Other risk factors included older age at the time of SLE diagnosis, longer SLE disease 

duration, longer duration of corticosteroid use, and hypercholesterolemia. In Canadian studies, 

once data were adjusted for traditional CVD risk factors, hypertension was the only risk factor 

associated with increased atherosclerosis in SLE patients.138 Elevated LDL was also a factor, but 

less so. Thus existing therapies do not appear to interrupt the processes leading to accelerated 

atherosclerosis. 

The immunopathogenesis of SLE represents an interlocking circle between innate and adaptive 

immunity.139 Plasmacytoid DC produce IFN-α, which activates myeloid DC to stimulate T cells 

and B cells. Activated B cells produce autoantibodies, which form immune complexes that 

stimulate plasmacytoid DC and present antigen to T cells. Several biological approaches have 

been devised to interrupt this circle, but interruption increases SLE patients’ susceptibility to 

bacterial and fungal infections. Thus, how successful these therapies will be is not clear. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 26 

Estrogen most likely has a role in SLE. It enhances B-cell differentiation,140 autoantibody 

production,141 and cytokine production. Peripheral blood mononuclear cells are more responsive 

to estrogen, as shown by increased production of IL-10 and antibodies against double-stranded 

DNA and decreased apoptosis.142 In addition, treatment with 17β-estradiol increases production 

of T-cell activation markers,143 including calcineurin and CD154. Levels of DHEA are low in 

women with SLE and further reduced by prednisone treatment. In double-blind, randomized 

clinical trials, Dr. Petri’s group found that treatment with DHEA resulted in sustained prednisone 

reduction in patients with SLE, although effects were better at higher doses of DHEA.144 This 

effect was generalizable, as shown by studies conducted in Taiwan.145 Results from these studies 

are consistent with DHEA functioning as an immunomodulator. Other studies have shown that 

DHEA treatment reduces IL-6 production from bone marrow stromal cells through 

downregulation of NFκB,146 increases expression of peroxisome proliferator activated receptor 

beta,146 and decreases IL-10 synthesis in patients with SLE.147

Dr. Petri’s group has carried out clinical trials to determine whether exogenous estrogen is 

harmful to patients with SLE. In one randomized trial, they found that women receiving HRT 

experienced an increase in the probability of mild to severe flares, indicating that HRT increases 

the relapse-remission pattern.148 Previous studies in both animal models and humans had yielded 

contradictory data with regard to oral contraceptives,149-152 although one study found that 43% of 

women taking oral contraceptive experienced renal flares, suggesting that exogenous estrogen 

exacerbated SLE. However, most premenopausal women with SLE want to preserve ovarian 

function, and oral contraceptives alleviate their increased risk for benign gynecologic diseases. In 

a clinical trial carried out by Dr. Petri’s group, no differences in severe, moderate, or mild flares 

were observed.153 Thus oral contraceptives do not appear to exacerbate SLE, as long as the 

women receiving them are not hypercoagulative. 

Discussion 

Estrogen metabolism in patients with SLE is more likely to use the hydroxylation pathway, 

although polymorphisms predisposing to this pathway also can be found in relatives who do not 

have lupus. This predisposition might be exploited with targeted therapies, but so far no 

beneficial approaches have been identified. For example, certain foods such as broccoli can 

reverse the effects of the hydroxylation pathway, but they do not provide a clinical benefit. It 

may be that hydroxylation is important in early pathogenesis but not in clinical disease. 

Existing mouse models are not useful in studying SLE, because the disease progresses so quickly 

to death in mice. Relapse is detected in these mice based on proteinuria and end-stage renal 

disease. Other murine models are needed in which lupus can be detected based on rash or 

arthritis. 

The literature on the ER appears to conflict with what has been observed in these lupus studies. It 

might be that genetic predisposition plays a key role in ER effects. Studies of GR in lupus have 

been done, though they are not reviewed here. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 27 

IL-12 and Tim-3 Protect Against Coxsackievirus B3-induced Myocarditis 

DeLisa Fairweather, Ph.D., Johns Hopkins University 

Chronic disease involves both genetic susceptibility and environmental factors, and once a 

patient presents clinically, he or she is well past the initiating event. Dr. Fairweather’s group 

studies each stage of chronic disease pathogenesis, using an animal model of coxsackievirus B3 

(CVB3)-induced myocarditis.154 Mice are inoculated with virus, which has been passaged 

through the heart and is cleared within 16 days. Death is not induced in this model. Both 

BALB/c and BL/6 mice develop acute inflammation of the heart, but only BALB/c mice 

progress to a chronic stage of autoimmunity. Necrosis is not observed during the acute stage, but 

it is seen at the chronic stage. 

In this model at acute stages, IFN-γ deficiency results in increased viral replication and decreased 

production of neutrophils and macrophages, but it does not increase inflammation overall in the 

heart.155 Thus, IL-12–induced IFN-γ is needed to reduce viral replication. However, Th1 

responses are responsible for increasing acute inflammation of the heart in CVB3-induced 

myocarditis. Thus treatments to reduce inflammation might increase susceptibility to infection by 

CVB3 or other organisms. At chronic stages, IFN-γ deficiency results in increased myocarditis 

and fibrosis, as well as increased production of the profibrotic cytokines TGF-β1, IL-1β, and IL4.156 Dilated cardiomyopathy is observed, in addition to increased calcified fibrotic pericarditis 

and increased immune complex deposition, particularly along the pericardium. Increased 

pericarditis is associated with increased degranulating mast cells. Thus, the Th2-mediated 

response observed in IFN-γ–deficient mice increases the likelihood of chronic heart disease, and 

treatments to reduce acute inflammation might increase susceptibility to Th2-mediated heart 

disease. 

Males experience both acute and chronic myocarditis, but their response is more inflammatory. 

The number of macrophages, neutrophils, mast cells, NK cells, and CD8+ T cells is larger in 

males, and the Th1 response is enhanced. Production of IL-1 and IL-18 increases in the heart, 

and macrophages and mast cells, which behave as antigen-presenting cells following CVB3 

infection,157 express TLR4, as shown by flow cytometry.158 In mice deficient in TLR4 and 

infected with CVB3, the immune cell types in the heart resembled those observed in female mice 

infected with the virus. In TLR4-deficient mice expression of the inhibitory receptor Tim-3, 

which prevents apoptosis of Th1 cells during adaptive immune response, was increased on mast 

cells and on B and T lymphocytes, and apoptosis of CD11b+

 cells was observed. On the other 

hand, if Tim-3 function was inhibited in male mice infected with CVB3 during the innate 

immune response, inflammation and TLR4 expression increased, and the number of T regulatory 

cells decreased.159 The female response to CVB3 infection appears to be more Th2 mediated and 

fibrotic during chronic myocarditis, although the amount of viral replication is similar to that 

seen in males during the innate and acute response. In females the number of T regulatory cells 

increases, and mast cells and B and T lymphocytes express Tim-3. Thus cross-talk by TLR4 and 

Tim-3 signaling regulate inflammation in the heart: Tim-3 increases CD80, CTLA-4 expression, 

and regulatory T-cell populations, whereas TLR4 increases production of IL-1, IL-18, and 

inflammation. 

Many chronic diseases involve progression from an acute disease state to a chronic one, but as 

the above results suggest, differences between acute and chronic disease must be dissected out 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 28 

for treatments to be effective. The presence of infection also will affect efficacy. Studies focused 

on estrogens, IL-4, anti-inflammatory therapies, and therapies that block TNF, IL-1, or IFN-γ are 

under way. 

Discussion 

Early studies showed that TNF-blocking agents increased mortality in humans with congestive 

heart failure. However, this was in the case of advanced heart failure; studies have not examined 

the use of these agents during primary inflammatory myocarditis. More recent data show that 

TNF-blocking agents might not be as detrimental as once feared. 

SESSION V: PANEL DISCUSSIONS AND RECOMMENDATIONS 

Moderator: Christopher E. Taylor, National Institute of Allergy and Infectious Diseases, NIH 

Session I Discussion and Recommendations 

Session Moderator’s Summary 

The classic definition of inflammation describes a constellation of symptoms. Inflammation itself 

has been described in a large amount of cellular and molecular detail, but it could be defined 

differently by different people, depending on the diseases they study. Dr. Libby’s presentation 

highlighted cellular events that may be common among various diseases or disorders, including 

changes in lipids and cytokines and genetic influences. One must be careful when assessing a 

potential biomarker, however, as clinically useful biomarkers should be both specific and 

sensitive. Measuring hs-CRP as a biomarker of CVD relies on a highly sensitive assay, but the 

cytokine itself is not specifically regulated by a single stimulus and thus lacks specificity. 

Knowledge Gaps and Research Needs 

• What is the exact stimulus or trigger for inflammatory processes?

Tissue damage, oxidative stress, and aging have been discussed, but no clear-cut trigger has 

been identified. 

• How is lipid metabolism regulated by hormones?

• How do regional differences in adiposity or energetics contribute to the differing 

burden of the metabolic syndrome?

• What are the anatomical differences in vascular lesion formation?

• What is the role of sex hormones in regulating HDL production, metabolism, and 

modification?

• What is the role of hormone receptor activation in inflammatory responses?

• Are there epigenetic effects of gender that influence sex biases?

Session II Discussion and Recommendations 

Session Moderator’s Summary 

Presentations during Session II discussed models of sex-related differences in autoimmune 

disease and responses to injury, environmental insult, and chronic periodontal infection. They 

also hinted toward the role of varying levels of estrogens in inflammatory processes. The 

estrogen story is particularly confusing. Results from some studies suggest that estrogen exerts 

anti-inflammatory effects, but several diseases affect women more than men, and some studies 

suggest that this increased susceptibility might arise from divergent inflammatory responses. In 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 29 

addition, varying levels of estrogen, for example levels associated with pregnancy or menopause, 

can also modulate effects on inflammatory processes. 

Knowledge Gaps and Research Needs 

• What are the effects of hormones on innate immunity in healthy subjects?

Future studies should address baseline sex differences in the function of innate immune cells 

and how sex hormones modulate activation of these cells. In addition, the interactions among 

inflammatory cells and between these and non-inflammatory cells should be explored further. 

When considering the effects of sex hormones, it should be acknowledged that both estrogen 

and testosterone circulate in both sexes. How these hormones are balanced throughout the 

lifespan also should be considered. In addition, differences in the way sex hormones are used 

by animal models, compared with humans, should be kept in mind.

• How do sex and/or gonadal steroid hormones alter inflammatory and immune 

responses after injury?

More attention should be paid to the organ systems involved in inflammatory and immune 

responses, and systemic effects versus organ-specific effects should be teased out. In 

addition, to estrogen, other hormones such as progesterone, glucocorticoids, and insulin 

should be studied in this context.

Efforts to understand sex differences among these diseases might focus too much on sex 

hormones and not enough on processes that may be encoded by sex chromosomes. It could 

be that the hormonal differences discussed in these presentations are indirect effects. Future 

studies should clarify that these effects are directly caused by sex hormones and do not arise 

from the sex chromosomes themselves. However, it should be noted that the gene encoding 

the AR lies on the X chromosome, and efforts to rule out sex chromosome effects might be 

difficult because of imprinting and X-chromosome silencing in women. 

Most studies so far have focused only on specific pathways. Future studies should employ a 

systems biology approach. For example, expression of the ER is widespread and most likely 

encompasses a complex network in vivo. New tools should be developed, including tissuespecific mouse knockouts in which individual receptors are under temporal control. Some 

models with inducible ER have already been devised.

• How do aging, menopause, and andropause affect these responses?

Differences among children, adults of reproductive age, and older adults, should be studied, 

as well as the effects of comorbidities associated with aging, such as diabetes, CVD, and 

cancer.

• What are the mechanisms by which hormones influence cellular responses to insult? 

• What is the temporal response of sex hormones to shock and trauma? 

Session III Discussion and Recommendations 

Session Moderator’s Summary 

Key issues discussed during this session included the roles of ER isoforms and rapid versus 

nuclear ER signaling; the effects of variable systemic estrogen levels, for example male versus 

female, non-pregnancy versus pregnancy, and hormone replacement therapy; the effects of ER 

levels or function and hormone resistance; hormone effects on cellular differentiation, activation, 

and function; and consideration of all sex hormones and their intersection with the HPA axis. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 30 

Several ligands exist for the ER, including estrogens, phytoestrogens, and SERMs. Thus work 

with animal models must take into account other sources of estrogen or ER ligands. In addition, 

several ER heterodimers exist, and the differential outcome of ER-α and ER-β ligation depends 

on ligand concentration, ligand and receptor conformation, and relative expression of the ER and 

its coregulators. 

The development of new treatment strategies must overcome several roadblocks, including 

hormone interactions, effects during the acute stage of disease versus those during the chronic 

stage, and ubiquitous expression of ER-α. Clinical observations in genetically diverse 

populations must be reconciled with the results of laboratory experiments in model systems. 

Moreover, appropriate doses of estrogen must be determined for effects on a particular tissue, 

and specificity and potential molecular targets within ER-mediated signaling pathways should be 

addressed. Future work should also employ global approaches such as microarray and serial 

analysis of gene expression to generate a more complete picture of the effects of environmental 

modulation. 

Knowledge Gaps and Research Needs 

• Can hormonal effects on immune or other cells account for sex biases in autoimmunity, 

infection, and recovery from trauma?

• What is the mechanism of action of estrogen or the ER in particular cell types?

• Which ER isoforms or variants are specific to which cell types?

• Which hormones mediate sex-specific effects?

• How do sex hormones interact with pathogen responses?

• How do symptoms and responses differ by sex in humans?

Session IV Discussion and Recommendations 

Session Moderator’s Summary 

Current strategies in the treatment of inflammatory diseases include palliative or substitutive 

approaches such as insulin or thyroid hormone therapy, anti-inflammatory approaches such as 

the use of corticosteroids, and immunosuppressive approaches such as the use of 

cyclophosphamide. However, these strategies offer limited efficacy and severe side effects. New 

approaches should restore immune tolerance and medical benefit, prevent sensitization or 

neutralizing antibody production, and limit unintended biological effects. 

Emerging strategies include: 

• Monoclonal antibodies. To overcome the problems of sensitization and cytokine release, one 

could use humanized monoclonal antibodies and immunosuppressive agents. Engineered Fc 

regions could be used to avoid side effects and prolong half-life, and engineered variable 

regions could be used to increase affinity. 

• Approaches targeting proteins involved in inflammation and immune response, such as 

adhesion molecules, cytokines, or cell differentiation antigens. However, at present, these 

approaches induce severe side effects. 

• Soluble autoantigens, delivered nasally or orally to induce tolerance. These treatments are 

effective if given early, but lag times might increase the risk for disease acceleration or 

anaphylaxis. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 31 

• Soluble receptors and protein fusion conjugates, such as TNF, CTLA-4–immunoglobulin, or 

IL-1R. 

• Cell therapy, such as the use of tolerogenic DC or cultured T regulatory cells. 

• Gene therapy, such as the administration of immunoregulatory cytokines such as IL-10. 

Knowledge Gaps and Research Needs 

• How can new strategies employ the concept of “outflammation,” using chemorepellants 

to remove cells from places where they normally should not appear?

• How do resolvins and the lipoxin pathway, which enhance the resolution of 

inflammation, be used? 

Several derivatives have been shown to be enhanced in through aspirin therapy, and some 

industry-supported work is exploring this further. 

• Can nonsteroidal anti-inflammatory drugs provide a benefit?

These drugs might prove problematic because they inhibit side pathways, but recent papers 

suggest that these drugs block atherosclerosis. 

Research Models and Other Research Considerations 

Future research exploring sex differences in the regulation of inflammatory processes will 

require the development of new models and more completely exploit existing ones. For example, 

periodontal disease in pregnancy, in which a natural biofilm is immediately adjacent to tissue, is 

a model that offers easy access to tissue and a wealth of information, including microarray 

profiles, effects of hormonal changes throughout pregnancy, effects of constant infectious 

challenge, and ex vivo responses. The use of LPS as a stimulant in several model systems is 

advantageous because the pathways involved have been well defined, but it also might be 

problematic. 

The development of new models should account for different sources of estrogens as well as 

factors involved in the differential outcomes of ER isoforms. Several Institutes within the NIH, 

including the National Institute on Aging (NIA), are involved in creating mouse models. A series 

of discussions are under way across several agencies, including the NIH and the U.S. 

Department of Agriculture, to explore the issue of soy, which contains phytoestrogens. 

Differences among animal models and humans should also be considered. For example, studying 

the effects of hormone resistance might prove difficult, as animal models might not fully 

recapitulate it, and simply measuring hormone levels might not be useful. Protocols to measure 

GR resistance functionally in human whole blood ex vivo, rather than molecularly, could be 

adapted for use in studying other hormone receptors. However, it should be kept in mind that 

hormone receptor levels modulate when cells are kept in culture and that some phenomena might 

be observed that are not relevant to biologic processes. 

In addition, a “Rosetta stone” of effects should be established. For example, what happens in the 

whole animal following ovariectomy, including effects on various systems, is not known. This 

problem points to the need for a systems biology approach to more carefully define models and 

model systems. In addition, investigators can share with other investigators the tissues they do 

not use, facilitating the beginning of a more complete picture of what happens in a particular 

model. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 32 

Other research considerations include: 

• Maintaining a focus on physiology, even while conducting experiments in vitro in cell 

systems and on particular systems of interest. 

• Sorting out direct causality of chronic disease, when the initiating event has long passed and 

tissue response to that event is well established. 

• Activation and function of pro- and anti-inflammatory proteins. 

• Effects of hormone receptor resistance and hormone metabolism. 

• Influence of tissue specificity in inflammatory response. 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 33 

CONTACT INFORMATION FOR CHAIRS AND SPEAKERS 

Chairs 

Lisa Begg, Dr.P.H., R.N. 

Director of Research Programs 

ORWH 

Office of the Director (OD) 

NIH 

6707 Democracy Boulevard 

Suite 400 

MSC 5484 

Bethesda, MD 20892 

Phone: (301) 496-7853 

Fax: (301) 402-1798 

beggl@mail.nih.gov

Christopher E. Taylor, Sc.D. 

Program Officer/Microbiologist, Bacterial 

Respiratory Diseases 

Division of Microbiology and Infectious 

Diseases 

NIH 

6610 Rockledge Drive 

Room 5045 

Bethesda, MD 20852 

Phone: (301) 496-5305 

Fax: (301) 496-8030 

ctaylor@niaid.nih.gov

Speakers and Session Moderators 

Jeff Bender, M.D. 

Chief and Associate Professor 

New Haven Hospital 

School of Medicine 

Yale University 

15 York Street 

New Haven, CT 06510 

Phone: (203) 737-2223 

Fax: (203) 785-3588 

jeffrey.bender@yale.edu

Samuel N. Breit, M.D., Ph.D., 

Professor of Medicine 

Head of Inflammation Research Group 

Centre for Immunology 

St. Vincent’s Hospital and University of 

New South Wales 

Sydney, NSW 2010 

Australia 

Phone: +612-8382-3700 

Fax: +612-8382-2830 

s.breit@cfi.unsw.edu.au

Edwin Deitch, M.D. 

Professor of Surgery 

Department of Surgery 

New Jersey Medical School 

185 S. Orange Avenue 

Medical Sciences Building G-506 

Newark, NJ 07103 

Phone: (973) 972-5045 

Fax: (973) 972-6803 

edeitch@umdnj.edu

DeLisa Fairweather 

Assistant Professor 

Division of Toxicology 

Department of Environmental Health 

Sciences 

Johns Hopkins Bloomberg School of Public 

Health 

615 N. Wolfe Street 

Room E7628 

Baltimore, MD 21205 

Phone: (410) 502-3644 

Fax: (410) 955-0116 

dfairwea@jhsph.edu
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 34 

Daniel E. Furst, M.D. 

Carl M. Pearson Professor of Medicine 

Rheumatology 

David Geffen School of Medicine 

University of California, Los Angeles 

1000 Veteran Avenue 

Rehabilitation Center 32-59 

Los Angeles, CA 90095 

Phone: (310) 206-5366 

Fax: (310) 206-8606 

defurst@mednet.ucla.edu

Godfrey S. Getz, M.D., Ph.D. 

Professor 

Pathology 

Biological Sciences 

University of Chicago 

950 E. 59th Street 

Chicago, IL 60637 

Phone: (773) 834-4856 

Fax: (773) 834-5251 

g-getz@uchicago.edu

Lester Kobzik, M.D. 

Professor of Pathology 

Environmental Health 

Harvard School of Public Health 

665 Huntington Avenue 

Boston, MA 02115 

Phone: (617) 432-2247 

Fax: (617) 432-0014 

lkobzik@hsph.harvard.edu

Elizabeth J. Kovacs, Ph.D. 

Professor and Vice Chair for Research 

Department of Surgery 

Associate Director, Burn and Shock Trauma 

Institute 

Loyola University 

2160 S. First Avenue 

Building 110, Room 4232 

Maywood, IL 60153 

Phone: (708) 327-2477 

Fax: (708) 327-2813 

ekovacs@lumc.edu

Susan Kovats, Ph.D. 

Assistant Professor 

Arthritis and Immunology Program 

Oklahoma Medical Research Foundation 

825 NE 13th Street 

Oklahoma City, OK 73104 

Phone: (405) 271-8583 

Fax: (405) 271-7063 

susan-kovats@omrf.ouhsc.edu

Peter Libby, M.D. 

Chief 

Cardiovascular Medicine 

Brigham and Women’s Hospital 

Harvard Medical School 

77 Avenue Louis Pasteur 

New Research Building 741 

Boston, MA 02115 

Phone: (617) 525-4351 

Fax: (617) 525-4400 

plibby@rics.bwh.harvard.edu

Virginia M. Miller, Ph.D. 

Professor of Physiology and Surgery 

Mayo Clinic 

200 First Street, S.W. 

Rochester, MN 55905 

Phone: (507) 284-2290 

Fax: (507) 266-2333 

miller.virginia@mayo.edu

Steven Offenbacher, D.D.S., Ph.D. 

Distinguished Professor 

Periodontology Dental Research 

School of Dentistry 

University of North Carolina at Chapel Hill 

222 Dental Research Center 

Classroom Building 7455 

Chapel Hill, NC 27599 

Phone: (919) 962-7081 

Fax: (919) 966-7537 

steve_offenbacher@dentistry.unc.edu
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 35 

Michelle Petri, M.D., M.P.H. 

Professor of Medicine 

Rheumatology 

School of Medicine 

Johns Hopkins University 

1830 E. Monument Street 

Room 7500 

Baltimore, MD 21205 

Phone: (410) 955-3823 

Fax: (410) 614-0498 

mpetri@jhmi.edu

Esther M. Sternberg, M.D. 

Senior Investigator 

Section on Neuroendocrine Immunology 

and Behavior 

National Institute of Mental Health (NIMH) 

National Institutes of Health 

5625 Fishers Lane 

Room 4N-13 

Rockville, MD 20892 

Phone: (301) 402-2773 

Fax: (301) 496-6095 

sternbee@mail.nih.gov

Greet van den Berghe, M.D., Ph.D. 

Professor 

University of Leuven 

Department of Intensive Care Medicine 

University Hospital Gasthuisberg 

Herestraat 49 

Leuven, B-3000 

Belgium 

Phone: +32-16-34-40-21 

Fax: +32-16-34-40-15 

greta.vandenberghe@med.kuleuven.be

Rhonda Voskuhl, M.D. 

Professor and Program Director 

Neurology 

Multiple Sclerosis Program 

University of California, Los Angeles 

635 Charles E. Young Drive South 

Los Angeles, CA 90095 

Phone: (310) 206-4636 

Fax: (310)-206-9801 

rvoskuhl@ucla.edu

Darryl C. Zeldin, Ph.D. 

Senior Investigator 

Laboratory of Respiratory Biology 

National Institute of Environmental Health 

Sciences 

National Institutes of Health 

111 T.W. Alexander Drive 

Rall Building, Room D236 

Research Triangle Park, NC 27709 

Phone: (919) 541-1169 

Fax: (919) 541-4133 

zeldin@niehs.nih.gov
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 36 

LIST OF WORKSHOP ATTENDEES 

David B. Abrams, Ph.D., Office of Behavioral and Social Sciences Research, OD, NIH 

Diane Adger-Johnson, NIAID, NIH 

Sangeeta Bhargava, Ph.D., National Institute on Dental and Craniofacial Research, NIH 

Cherie L. Butts, Ph.D., National Institute of Mental Health (NIMH), NIH 

Nancy H. Colburn, Ph.D., National Cancer Institute (NCI), NIH 

Elaine S. Collier, M.D., Ph.D., National Center for Research Resources, NIH 

Anthony J. Coyle, Ph.D., MedImmune 

Alison M. Deckhut, Ph.D., NIAID, NIH 

Scott K. Durum, Ph.D., NCI, NIH 

Laurie L. Foudin, Ph.D., National Institute on Alcohol Abuse and Alcoholism (NIAAA), NIH 

Rebecca Fuldner, Ph.D., NIA, NIH 

Michael S. Gold, Ph.D., Dental School, University of Maryland 

Elizabeth T. Golub, Ph.D., Johns Hopkins Bloomberg School of Public Health 

Timothy Gondre-Lewis, Ph.D., NIAID, NIH 

Kenneth W. Hance, Ph.D., M.P.H., NCI, NIH 

Eleanor Z. Hanna, Ph.D., ORWH, OD, NIH 

Carole A. Heilman, Ph.D., NIAID, NIH 

O.M. Zack Howard, Ph.D., NCI, NIH 

Aurora C. Hutchinson, National Institute on Drug Abuse, NIH 

Danuta Krotoski, Ph.D., National Institute of Child Health and Human Development (NICHD), 

NIH 

Karen Lacouciere, Ph.D., NIAID, NIH 

Linda Lambert, Ph.D., NIAID, NIH 

Elaine Lanza, Ph.D., NCI, NIH 

Tamara Lewis-Johnson, NIAID, NIH 

Martha Lundberg, Ph.D., NHLBI, NIH 

Mamodikoe K. Makhene, M.D., NIAID, NIH 

Susan A. McCarthy, Ph.D., NCI, NIH 

Susan Meikle, M.D., M.S.P.H., Agency for Healthcare Research and Quality 

Melody Mills, Ph.D., NIAID, NIH 

Suresh Mohla, Ph.D., NCI, NIH 

Peter Moy, Ph.D., National Institute of Biomedical Imaging and Bioengineering, NIH 

Gwen Murphy, Ph.D., M.P.H., NCI, NIH 

Joseph J. Pancrazio, Ph.D., National Institute of Neurological Disorders and Stroke, NIH 

Vivian W. Pinn, M.D., ORWH, OD, NIH 

Susan F. Plaeger, Ph.D., NIAID, NIH 

Carol H. Pontzer, Ph.D., National Center for Complementary and Alternative Medicine, NIH 

Vishnudutt Purohit, Ph.D., NIAAA, NIH 

Connie Rogers, Ph.D., M.P.H., NCI, NIH 

Michael E. Rogers, Ph.D., National Institute of General Medical Sciences (NIGMS), NIH 

Neeraja Sathyamoorthy, Ph.D., NCI, NIH 

Susana A. Serrate-Sztein, M.D., National Institute of Arthritis and Musculoskeletal and Skin 

Diseases, NIH 

Grace L. Shen, Ph.D., National Eye Institute, NIH 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 37 

Christine F. Sizemore, Ph.D., NIAID, NIH 

Farida Sohrabji, Ph.D., College of Medicine, Texas A&M Science Center 

Scott D. Somers, Ph.D., NIGMS, NIH 

Pamela Stratton, M.D., NICHD, NIH 

Katherine A. Taylor, Ph.D., NIAID, NIH 

Maria L. Turner, M.D., NCI, NIH 

Nabila M. Wassef, Ph.D., NIAID, NIH 

David B. Winter, Ph.D., NIAID, NIH 
NIH Workshop: Regulation of Inflammatory Responses: Influence of Sex and Gender 38 

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