bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of Cutaneous Inflammation by Angiotensin-Converting Enzyme
Authors: Scholzen Thomas E. , Ständer Sonja , Riemann Helge , Brzoska Thomas , Luger Thomas A.
Published: April 2014

Cutaneous neurogenic inflammation is a complex biological response of the host immune system to noxious stimuli. Present evidence suggests that zinc metalloproteases may play an important role in the regulation of neurogenic inflammation by controlling the local availability of neuropeptides, such as substance P (SP), that are capable of initiating or amplifying cutaneous inflammation after release from sensory nerves. To address the hypothesis that the dipeptidyl carboxypeptidase angiotensin-converting enzyme (ACE) is capable of modulating skin inflammation, we have analyzed murine allergic contact dermatitis (ACD) and irritant contact dermatitis (ICD) using wild-type C57BL/6J (ACE+/+) or genetically engineered mice with a heterozygous deletion of somatic ACE (ACE+/−). In 2,4-dinitro-1-fluorobenzene-sensitized ACE+/− mice, ACD was significantly augmented in comparison to ACE+/+ controls as determined by the degree of ear swelling after exposure to hapten. Likewise, systemic treatment of ACE+/+ mice with the ACE inhibitor captopril before sensitization or elicitation of ACD significantly augmented the ACD response. In contrast, local damage and neuropeptide depletion of sensory nerves following capsaicin, injection of a bradykinin B2, or a SP receptor antagonist before sensitization significantly inhibited the augmented effector phase of ACD in mice with functionally absent ACE. However, in contrast to ACD, the response to the irritant croton oil was not significantly altered in ACE+/− compared with ACE+/+ mice. Thus, ACE by degrading bradykinin and SP significantly controls cutaneous inflammatory responses to allergens but not to irritants, which may explain the frequently observed exacerbation of inflammatory skin disease in patients under medication with ACE inhibitors.
Sodium Phenylacetate Inhibits Adoptive Transfer of Experimental Allergic Encephalomyelitis in SJL/J Mice at Multiple Steps
Authors: Dasgupta Subhajit , Zhou You , Jana Malabendu , Banik Naren L. , Pahan Kalipada
Published: April 2014

Experimental allergic encephalomyelitis (EAE) is the animal model for multiple sclerosis. The present study underlines the importance of sodium phenylacetate (NaPA), a drug approved for urea cycle disorders, in inhibiting the disease process of adoptively transferred EAE in female SJL/J mice at multiple steps. Myelin basic protein (MBP)-primed T cells alone induced the expression of NO synthase (iNOS) and the activation of NF-κB in mouse microglial cells through cell-cell contact. However, pretreatment of MBP-primed T cells with NaPA markedly inhibited its ability to induce microglial expression of iNOS and activation of NF-κB. Consistently, adoptive transfer of MBP-primed T cells, but not that of NaPA-pretreated MBP-primed T cells, induced the clinical symptoms of EAE in female SJL/J mice. Furthermore, MBP-primed T cells isolated from NaPA-treated donor mice were also less efficient than MBP-primed T cells isolated from normal donor mice in inducing iNOS in microglial cells and transferring EAE to recipient mice. Interestingly, clinical symptoms of EAE were much less in mice receiving NaPA through drinking water than those without NaPA. Similar to NaPA, sodium phenylbutyrate, a chemically synthesized precursor of NaPA, also inhibited the disease process of EAE. Histological and immunocytochemical analysis showed that NaPA inhibited EAE-induced spinal cord mononuclear cell invasion and normalized iNOS, nitrotyrosine, and p65 (the RelA subunit of NF-κB) expression within the spinal cord. Taken together, our results raise the possibility that NaPA or sodium phenylbutyrate taken through drinking water or milk may reduce the observed neuroinflammation and disease process in multiple sclerosis patients.
Complement Factor C5a Mediates Renal Ischemia-Reperfusion Injury Independent from Neutrophils
Authors: de Vries Bart , Köhl Jörg , Leclercq Wouter K. G. , Wolfs Tim G. A. M. , van Bijnen Annemarie A. J. H. M. , Heeringa Peter , Buurman Wim A.
Published: April 2014

The complement system has been shown to mediate renal ischemia-reperfusion (I/R) injury. However, the contribution of complement factor C5a to I/R injury, in particular in the kidney, remains to be established. In this study, we investigated the impact of blocking the C5aR pathway on the inflammatory response and on the renal function in a murine model of I/R injury. First, we analyzed C5aR expression in kidneys of healthy mice. Intriguingly, we found expression on mesangial, as well as on tubular epithelial, cells. After I/R injury, C5aR expression was up-regulated in tubular epithelial cells. In addition, mRNA levels of CXC chemokines and TNF-α increased significantly and kidneys were heavily infiltrated by neutrophils. Blocking the C5aR pathway by a specific C5a receptor antagonist (C5aRA) abrogated up-regulation of CXC chemokines but not of TNF-α and reduced neutrophil infiltration by >50%. Moreover, application of the C5aRA significantly reduced loss of renal function. This improvement of function was independent of the presence of neutrophils because neutrophil depletion by mAb NIMP-R14 did not affect the protective effect of C5aRA treatment. Furthermore, blocking of the C5aR pathway had no influence on renal apoptosis. These data provide evidence that C5a is crucially involved in the pathogenesis of renal I/R injury by modulation of neutrophil-dependent as well as neutrophil-independent pathways, which include the regulation of CXC chemokines but not TNF-α or apoptotic pathways.
IFN-γ Induces High Mobility Group Box 1 Protein Release Partly Through a TNF-Dependent Mechanism
Authors: Rendon-Mitchell Beatriz , Ochani Mahendar , Li Jianhua , Han Jialian , Wang Hong , Yang Huan , Susarla Seenu , Czura Christopher , Mitchell Robert A. , Chen Guoqian , Sama Andrew E. , Tracey Kevin J. , Wang Haichao
Published: April 2014

We recently discovered that a ubiquitous protein, high mobility group box 1 protein (HMGB1), is released by activated macrophages, and functions as a late mediator of lethal systemic inflammation. To elucidate mechanisms underlying the regulation of HMGB1 release, we examined the roles of other cytokines in induction of HMGB1 release in macrophage cell cultures. Macrophage migration inhibitory factor, macrophage-inflammatory protein 1β, and IL-6 each failed to significantly induce the release of HMGB1 even at supraphysiological levels (up to 200 ng/ml). IFN-γ, an immunoregulatory cytokine known to mediate the innate immune response, dose-dependently induced the release of HMGB1, TNF, and NO, but not other cytokines such as IL-1α, IL-1β, or IL-6. Pharmacological suppression of TNF activity with neutralizing Abs, or genetic disruption of TNF expression (TNF knockout) partially (50–60%) inhibited IFN-γ-mediated HMGB1 release. AG490, a specific inhibitor for Janus kinase 2 of the IFN-γ signaling pathway, dose-dependently attenuated IFN-γ-induced HMGB1 release. These data suggest that IFN-γ plays an important role in the regulation of HMGB1 release through a TNF- and Janus kinase 2-dependent mechanism.
Intranasal Immunotherapy Is More Effective Than Intradermal Immunotherapy for the Induction of Airway Allergen Tolerance in Th2-Sensitized Mice
Authors: Takabayashi Kenji , Libet Lev , Chisholm Dugald , Zubeldia Jose , Horner Anthony A.
Published: April 2014

Immunotherapy (IT) by injection more readily induces clinical tolerance to stinging insects than to respiratory allergens. However, while systemic immunization induces adaptive responses systemically, the induction of mucosal immunity generally requires local Ag exposure. Taken together, these observations suggest that the poor success rate of systemic IT for asthma could be a consequence of inadequate immune modulation in the airways. In support of this position, investigations presented in this report demonstrate that allergen IT more effectively induces airway allergen tolerance in Th2-sensitized mice, when delivered by the intranasal (i.n.) vs the intradermal (i.d.) route. Moreover, compared with native allergen, allergen immunostimulatory sequence oligodeoxynucleotide conjugate proved to be a more effective i.n. IT reagent for protecting allergic mice from airway hypersensitivity responses. Furthermore, for both native allergen and allergen immunostimulatory sequence oligodeoxynucleotide conjugate, i.n. and i.d. IT delivery were similarly effective in modulating systemic immune profiles in Th2-sensitized mice, while only i.n. IT had significant immunomodulatory activity on B and T cell responses in the airways. The present investigations may be the first to suggest that i.n. IT is more effective than i.d. IT for the treatment of asthma. Furthermore, our results suggest that modulating airway rather than systemic immunity may be the more important therapeutic target for the induction of clinical tolerance to respiratory allergens.
Viremia Control Despite Escape from a Rapid and Potent Autologous Neutralizing Antibody Response After Therapy Cessation in an HIV-1-Infected Individual
Authors: Montefiori David C. , Altfeld Marcus , Lee Paul K. , Bilska Miroslawa , Zhou Jintao , Johnston Mary N. , Gao Feng , Walker Bruce D. , Rosenberg Eric S.
Published: April 2014

The neutralizing Ab response after primary HIV-1 infection is delayed relative to the virus-specific CD8+ T cell response and the initial decline in plasma viremia. Because nearly all HIV-1 infections result in AIDS, it would be instructive to study cases where neutralizing Ab production commenced sooner. This was done in subject AC10, an individual treated during early infection and in whom a rapid autologous neutralizing Ab response was detected after therapy cessation as rebound viremia declined and remained below 1000 RNA copies/ml of blood for over 2.5 years. This subject’s Abs were capable of reducing the infectivity of his rebound virus by >4 logs in vitro at a time when rebound viremia was down-regulated and virus-specific CD8+ T cells were minimal, suggesting that neutralizing Abs played an important role in the early control of viremia. The rebound virus did not exhibit an unusual phenotype that might explain its high sensitivity to neutralization by autologous sera. Neutralization escape occurred within 75 days and was proceeded by neutralizing Ab production to the escape variant and subsequent escape. Notably, escape was not associated with a significant rise in plasma viremia, perhaps due to increasing CD8+ T cell responses. Sequence analysis of gp160 revealed a growing number of mutations over time, suggesting ongoing viral evolution in the face of potent antiviral immune responses. We postulate that an early effective neutralizing Ab response can provide long-term clinical benefits despite neutralization escape.
IL-12 Deficiency in MRL-FaslprMice Delays Nephritis and Intrarenal IFN-γ Expression, and Diminishes Systemic Pathology
Authors: Kikawada Eriya , Lenda Deborah M. , Kelley Vicki R.
Published: April 2014

Autoimmune disease in MRL-Faslpr mice is characterized by fatal nephritis, systemic pathology, and autoantibodies, mimicking human lupus. We previously reported that 1) intrarenal IL-12 elicits nephritis by fostering the accumulation of intrarenal IFN-γ-secreting T cells, and 2) MRL-Faslpr mice deficient in the IFN-γ receptor were spared from nephritis. Therefore, we hypothesized that eliminating IL-12 in MRL-Faslpr mice reduces IFN-γ-secreting cells and thereby prevents systemic pathology. For this purpose, we constructed an IL-12p40-deficient MRL-Faslpr(IL-12−/−) strain. We determined that glomerular and interstitial, but not perivascular, renal pathology were decreased in IL-12−/− mice vs the wild-type (WT) strain (5 mo of age). Similarly, systemic pathology (lung, lacrimal and salivary glands, skin, and lymphadenopathy) was diminished. The intrarenal accumulation of T cells (CD4+, CD8+, CD4−CD8−B220+) and macrophages was dramatically reduced in IL-12−/− MRL-Faslpr kidneys. We determined that there were fewer IFN-γ transcripts (>70%) in the IL-12−/− protected kidneys compared with the WT kidneys. Similarly, cells propagated from IL-12−/− MRL-Faslpr kidneys generated substantially less IFN-γ when stimulated with IL-12 and IL-18 compared with those from WT kidneys, and we detected fewer CD8 and B220 T cells producing IFN-γ in these IL-12−/− MRL-Faslpr kidneys. Of note, survival was modestly extended in the IL-12−/− MRL-Faslpr mice. While lung and lacrimal and salivary gland pathology remained reduced in moribund IL-12−/− MRL-Faslpr mice, renal pathology and IFN-γ expression were equivalent to those in the WT strain. Thus, we suggest that IL-12 is a therapeutic target for multiple tissues in lupus; however blocking IL-12 alone is not sufficient to confer enduring protection from lupus nephritis.
Cutting Edge: CD8+ Effector T Cells Reject Tumors by Direct Antigen Recognition but Indirect Action on Host Cells
Authors: Schüler Thomas , Blankenstein Thomas
Published: April 2014

CD8+ effector T cells recognize malignant cells by monitoring their surface for the presence of tumor-derived peptides bound to MHC class I molecules. In addition, tumor-derived Ags can be cross-presented to CD8+ effector T cells by APCs. IFN-γ production by CD8+ T cells is often critical for tumor rejection. However, it remained unclear whether 1) CD8+ T cells secrete IFN-γ in response to Ag recognition on tumor cells or APCs and 2) whether IFN-γ mediates its antitumor effect by acting on host or tumor cells. We show in this study that CD8+ effector T cells can reject tumors in bone marrow-chimeric mice incapable of cross-presenting Ag by bone marrow-derived APCs and that tumor rejection required host cells to express IFN-γR. Together, CD8+ effector T cells recognize Ag directly on tumor cells, and this recognition is sufficient to reject tumors by IFN-γ acting on host cells.
Cutting Edge: Roles of Toll-Like Receptor 4 and IL-23 in IL-17 Expression in Response toKlebsiella pneumoniaeInfection
Authors: Happel Kyle I. , Zheng Mingquan , Young Erana , Quinton Lee J. , Lockhart Euan , Ramsay Alistair J. , Shellito Judd E. , Schurr Jill R. , Bagby Gregory J. , Nelson Steve , Kolls Jay K.
Published: April 2014

Local production of IL-17 is a significant factor in effective host defense against Gram-negative bacteria. However, the proximal events mediating IL-17 elaboration by T cells remain unclear. In this study, we show in vivo that intact Toll-like receptor 4 signaling in the lung is required for induction of both the p19 transcript of IL-23 and IL-17 protein elaboration in response to Klebsiella pneumoniae. Although IL-17 is widely considered a CD4+ T cell product, we also demonstrate significant in vitro IL-17 production by CD8+ T cells after culture in medium from dendritic cells exposed to these bacteria. The dominant portion of this IL-17-inducing activity for both CD4+ and CD8+ T cells is IL-23. These data demonstrate the critical signaling pathway for IL-17 induction in the host response to Gram-negative pulmonary infection and suggest a direct role for IL-23 in CD8+ T cell IL-17 production.
Cutting Edge: Conventional CD8α+ Dendritic Cells Are Preferentially Involved in CTL Priming After Footpad Infection with Herpes Simplex Virus-1
Authors: Smith Christopher M. , Belz Gabrielle T. , Wilson Nicholas S. , Villadangos Jose A. , Shortman Ken , Carbone Francis R. , Heath William R.
Published: April 2014

CTL play a major role in immunity to HSV type 1, but little is known about the priming process. In this study, we have examined the class I-restricted presentation of an immunodominant determinant from HSV-1 glycoprotein B after footpad infection. We have found that the only cell types capable of presenting this determinant in draining popliteal lymph nodes within the first 3 days after infection are the CD11c+CD8α+CD45RA− dendritic cells. Given that such class I-restricted presentation is essential for CTL priming, this implies that these conventional CD8α+ dendritic cells are the key subset involved in CTL immunity to this virus.
A Pyrazole Derivative, YM-58483, Potently Inhibits Store-Operated Sustained Ca2+ Influx and IL-2 Production in T Lymphocytes
Authors: Ishikawa Jun , Ohga Keiko , Yoshino Taiji , Takezawa Ryuichi , Ichikawa Atsushi , Kubota Hirokazu , Yamada Toshimitsu
Published: April 2014

In nonexcitable cells, Ca2+ entry is mediated predominantly through the store depletion-dependent Ca2+ channels called store-operated Ca2+ (SOC) or Ca2+ release-activated Ca2+ channels. YM-58483, a pyrazole derivative, inhibited an anti-CD3 mAb-induced sustained Ca2+ influx in acute T cell leukemia, Jurkat cells. But it did not affect an anti-CD3 mAb-induced transient intracellular Ca2+ increase in Ca2+-free medium, nor anti-CD3 mAb-induced phosphorylation of phospholipase Cγ1. It was suggested that YM-58483 inhibited Ca2+ influx through SOC channels without affecting the TCR signal transduction cascade. Furthermore, YM-58483 inhibited thapsigargin-induced sustained Ca2+ influx with an IC50 value of 100 nM without affecting membrane potential. YM-58483 inhibited by 30-fold the Ca2+ influx through SOC channels compared with voltage-operated Ca2+ channels, while econazole inhibited both SOC channels and voltage-operated Ca2+ channels with an equivalent range of IC50 values. YM-58483 potently inhibited IL-2 production and NF-AT-driven promoter activity, but not AP-1-driven promoter activity in Jurkat cells. Moreover, this compound inhibited delayed-type hypersensitivity in mice with an ED50 of 1.1 mg/kg. Therefore, we concluded that YM-58483 was a novel store-operated Ca2+ entry blocker and a potent immunomodulator, and could be useful for the treatment of autoimmune diseases and chronic inflammation. Furthermore, YM-58483 would be a candidate for the study of capacitative Ca2+ entry mechanisms through SOC/CRAC channels and for identification of putative Ca2+ channel genes.
Decorin Reverses the Repressive Effect of Autocrine-Produced TGF-β on Mouse Macrophage Activation
Authors: Comalada Mònica , Cardó Marina , Xaus Jordi , Valledor Annabel F. , Lloberas Jorge , Ventura Francesc , Celada Antonio
Published: April 2014

Several cytokines or growth factors induce macrophages to proliferate, become activated, differentiate, or die through apoptosis. Like the major macrophage activator IFN-γ, the extracellular matrix protein decorin inhibits proliferation and protects macrophages from the induction of apoptosis. Decorin enhances the IFN-γ-induced expression of the IAα and IAβ MHC class II genes. Moreover, it increases the IFN-γ- or LPS-induced expression of inducible NO synthase, TNF-α, IL-1β, and IL-6 genes and the secretion of these cytokines. Using a number of extracellular matrix proteins, we found a negative correlation between adhesion and proliferation. However, the effects of decorin on macrophage activation do not seem to be mediated through its effect on adhesion or proliferation. Instead, this proteoglycan abolishes the binding of TGF-β to macrophages, as shown by Scatchard analysis of 125I-labeled TGF-β, which, in the absence of decorin, showed a Kd of 0.11 ± 0.03 nM and ∼5000 receptors/cell. This was confirmed when we treated macrophages with Abs to block the endogenously produced TGF-β, which enhanced macrophage activation in a way similar to decorin. The increase in activation mediated by decorin demonstrates that macrophages are under negative regulation that can be reversed by proteins of the extracellular matrix.
IL-6 Production by Pulmonary Dendritic Cells Impedes Th1 Immune Responses
Authors: Dodge Ingrid L. , Carr Michelle Woldemar , Cernadas Manuela , Brenner Michael B.
Published: April 2014

Mucosal tissues, such as the lung, are continually exposed to both foreign and environmental Ags. To counter the potential inflammatory tissue injury of chronic Th1-mediated responses against these Ags, mucosal sites may skew toward Th2 immune responses. However, the mechanism by which this occurs is unknown. Dendritic cells (DC), as orchestrators of the immune response, skew Th1/Th2 differentiation by cytokine secretion and expression of specific cell surface markers. We compared DC from mucosal and systemic locations. In this study, we show that the lung lacks a CD8α+ DC subpopulation and contains DC that appear less mature than splenic DC. Furthermore, we demonstrate that pulmonary DC produce significant levels of IL-6 and fail to produce the Th1-polarizing cytokine IL-12. Importantly, we demonstrate that IL-6 negatively regulates IL-12 production, as pulmonary DC from IL-6−/− mice produce significant levels of IL-12 and induce Th1 polarization of naive CD4+ T cells. Furthermore, we demonstrate that IL-6 is sufficient to explain the differential polarizing abilities of pulmonary and splenic DC, as splenic DC cocultures supplemented with IL-6 polarize naive T cells toward Th2, and pulmonary DC cultures in which IL-6 was removed with neutralizing Ab resulted in more Th1 polarization, pointing to IL-6 as the mechanism of Th2 polarization in the lung. We propose that the Th2 response seen in the lung is due to DC-mediated inhibition of Th1 responses via IL-6 production, rather than enhanced Th2 responses, and that this regulation decreases the likelihood of chronic inflammatory pathology in the lung.
Activation with CpG-A and CpG-B Oligonucleotides Reveals Two Distinct Regulatory Pathways of Type I IFN Synthesis in Human Plasmacytoid Dendritic Cells
Authors: Kerkmann Miren , Rothenfusser Simon , Hornung Veit , Towarowski Andreas , Wagner Moritz , Sarris Anja , Giese Thomas , Endres Stefan , Hartmann Gunther
Published: April 2014

Two different CpG oligonucleotides (ODN) were used to study the regulation of type I IFN in human plasmacytoid dendritic cells (PDC): ODN 2216, a CpG-A ODN, known to induce high amounts of IFN-α in PDC, and ODN 2006, a CpG-B ODN, which is potent at stimulating B cells. CpG-A ODN showed higher and prolonged kinetics of type I IFN production compared with that of CpG-B ODN. In contrast, CpG-B ODN was more active than CpG-A ODN in stimulating IL-8 production and increasing costimulatory and Ag-presenting molecules, suggesting that CpG-A and CpG-B trigger distinct regulatory pathways in PDC. Indeed, CpG-A ODN, but not CpG-B ODN, activated the type I IFNR-mediated autocrine feedback loop. PDC were found to express high constitutive levels of IFN regulatory factor (IRF)7. IRF7 and STAT1, but not IRF3, were equally up-regulated by both CpG-A and CpG-B. CD40 ligand synergistically increased CpG-B-induced IFN-α independent of the IFNR but did not affect CpG-B-induced IFN-β. In conclusion, our studies provide evidence for the existence of two distinct regulatory pathways of type I IFN synthesis in human PDC, one dependent on and one independent of the IFNR-mediated feedback loop. The alternate use of these pathways is based on the type of stimulus rather than the quantity of IFN-αβ available to trigger the IFNR. Constitutive expression of IRF7 and the ability to produce considerable amounts of IFN-α independent of the IFNR seem to represent characteristic features of PDC.
Phosphatidylinositol 3-Kinase Regulates the CD4/CD8 T Cell Differentiation Ratio
Authors: Rodríguez-Borlado Luis , Barber Domingo F. , Hernández Carmen , Rodríguez-Marcos Miguel A. , Sánchez Arsenio , Hirsch Emilio , Wymann Matthias , Martínez-A. Carlos , Carrera Ana C.
Published: April 2014

The signaling pathways that control T cell differentiation have only begun to be elucidated. Using T cell lines, it has been shown that class IA phosphatidylinositol 3-kinase (PI3K), a heterodimer composed of a p85 regulatory and a p110 catalytic subunit, is activated after TCR stimulation. Nonetheless, the contribution of p85/p110 PI3K isoforms in T cell development has not been described. Mice deficient in the other family of class I PI3K, p110γ, which is regulated by G protein-coupled receptors, exhibit reduced thymus size. Here we examine T cell development in p110γ-deficient mice and in mice expressing an activating mutation of the p85 regulatory subunit, p65PI3K, in T cells. We show that p110γ-deficient mice have a partial defect in pre-TCR-dependent differentiation, which is restored after expression of the p65PI3K activating mutation. Genetic alteration of both PI3K isoforms also affects positive selection; p110γ deletion decreased and p65PI3K expression augmented the CD4+/CD8+ differentiation ratio. Finally, data are presented showing that both PI3K isoforms influenced mature thymocyte migration to the periphery. These observations underscore the contribution of PI3K in T cell development, as well as its implication in determining the CD4+/CD8+ T cell differentiation ratio in vivo.
Glatiramer Acetate (Copolymer-1, Copaxone) Promotes Th2 Cell Development and Increased IL-10 Production Through Modulation of Dendritic Cells
Authors: Vieira Pedro L. , Heystek Heleen C. , Wormmeester Jan , Wierenga Eddy A. , Kapsenberg Martien L.
Published: April 2014

Glatiramer acetate (GA; copolymer-1, Copaxone) suppresses the induction of experimental autoimmune encephalomyelitis and reduces the relapse frequency in relapsing-remitting multiple sclerosis. Although it has become clear that GA induces protective degenerate Th2/IL-10 responses, its precise mode of action remains elusive. Because the cytokine profile of Th cells is often regulated by dendritic cells (DC), we studied the modulatory effects of GA on the T cell regulatory function of human DC. This study shows the novel selective inhibitory effect of GA on the production of DC-derived inflammatory mediators without affecting DC maturation or DC immunostimulatory potential. DC exposed to GA have an impaired capacity to secrete the major Th1 polarizing factor IL-12p70 in response to LPS and CD40 ligand triggering. DC exposed to GA induce effector IL-4-secreting Th2 cells and enhanced levels of the anti-inflammatory cytokine IL-10. The anti-inflammatory effect of GA is mediated via DC as GA does not affect the polarization patterns of naive Th cells activated in an APC-free system. Together, these results reveal that APC are essential for the GA-mediated shift in the Th cell profiles and indicate that DC are a prime target for the immunomodulatory effects of GA.
Distinct Pathways for NF-κB Regulation Are Associated with Aberrant Macrophage IL-12 Production in Lupus- and Diabetes-Prone Mouse Strains
Authors: Liu Jiajian , Beller David I.
Published: April 2014

One characteristic of mice prone to a variety of autoimmune diseases is the aberrant regulation of cytokine production by macrophages (Mφ), noted in cells isolated well before the onset of disease. Strikingly, the pattern of IL-12 dysregulation, in particular, is consistent with the nature of the autoimmune disease that will develop in each strain, i.e., elevated in mice prone to Th1-mediated organ-specific disease (nonobese diabetic (NOD) and SJL mice) and reduced in lupus-prone strains (MRL/+ and NZB/W). Mechanistically, the abnormal regulation of IL-12 in these strains was found to be strictly associated with novel patterns of Rel binding in vitro to the unique NF-κB site in the IL-12 p40 promoter. In this study, we report several new findings related to these Rel-κB interactions. Evaluation of the p40 NF-κB site in vivo, assessed by chromatin immunoprecipitation, revealed Rel usage patterns similar to those found in vitro using EMSA, with preferential association of the p40 κB site with c-Rel in NOD Mφ but with p50 in NZB/W Mφ. Moreover, blocking c-Rel in primary Mφ, using short interfering RNA, selectively blocked IL-12 production and normalized the minimal, residual IL-12 levels. Nuclear extracts from NOD Mφ were characterized by c-Rel hyperphosphorylation, and dephosphorylation of nuclear proteins completely blocked binding to the κB site. In contrast, elevated IκB appears to be a likely mechanism accounting for the reduced nuclear c-Rel levels noted in NZB/W Mφ. Alterations in NF-κB metabolism thus appear to define a pathway regulating intrinsic IL-12 defects in both diabetes- and lupus-prone strains.
Determinants of Human B Cell Migration Across Brain Endothelial Cells
Authors: Alter Andrea , Duddy Martin , Hebert Sherry , Biernacki Katarzyna , Prat Alexandre , Antel Jack P. , Yong Voon Wee , Nuttall Robert K. , Pennington Caroline J. , Edwards Dylan R. , Bar-Or Amit
Published: April 2014

Circulating B cells enter the CNS as part of normal immune surveillance and in pathologic states, including the common and disabling illness multiple sclerosis. However, little is known about the molecular mechanisms that mediate human B cell interaction with the specialized brain endothelial cells comprising the blood-brain barrier (BBB). We studied the molecular mechanisms that regulate the migration of normal human B cells purified ex vivo, across human adult brain-derived endothelial cells (HBECs). We found that B cells migrated across HBECs more efficiently than T cells from the same individuals. B cell migration was significantly inhibited by blocking Abs to the adhesion molecules ICAM-1 and VLA-4, but not VCAM-1, similar to the results previously reported for T cells. Blockade of the chemokines monocyte chemoattractant protein-1 and IL-8, but not RANTES or IFN-γ-inducible protein-10, significantly inhibited B cell migration, and these results were correlated with the chemokine receptor expression of B cells measured by flow cytometry and by RNase protection assay. Tissue inhibitor of metalloproteinase-1, a natural inhibitor of matrix metalloproteinases, significantly decreased B cell migration across the HBECs. A comprehensive RT-PCR comparative analysis of all known matrix metalloproteinases and tissue inhibitors of metalloproteinases in human B and T cells revealed distinct profiles of expression of these molecules in the different cell subsets. Our results provide insights into the molecular mechanisms that underlie human B cell migration across the BBB. Furthermore, they identify potential common, and unique, therapeutic targets for limiting CNS B cell infiltration and predict how therapies currently developed to target T cell migration, such as anti-VLA-4 Abs, may impact on B cell trafficking.
Homology Between an Alloantigen and a Self MHC Allele Calibrates the Avidity of the Alloreactive T Cell Repertoire Independent of TCR Affinity
Authors: Hornell Tara M. C. , Myers Nancy , Hansen Ted H. , Connolly Janet M.
Published: April 2014

The self-restricted T cell repertoire exhibits a high frequency of alloreactivity. Because these alloreactive T cells are derived from the pool of cells selected on several different self MHC alleles, it is unknown how development of the alloantigenic repertoire is influenced by homology between a self MHC allele and an alloantigen. To address this, we used the 2C transgenic TCR that is selected by Kb, is alloreactive for Ld, and cross-reacts with Lq. Lq is highly homologous to Ld and binds several of the same peptide ligands, including p2Ca, the peptide recognized by 2C. We find that Ld/p2Ca is a high avidity agonist ligand, whereas Lq/p2Ca is a low avidity agonist ligand for 2C T cells. When mice transgenic for the 2C TCR are bred to Lq-expressing mice, 2C+ T cells develop; however, they express lower levels of either the 2C TCR or CD8 and require a higher Ld/p2Ca ligand density to be activated than 2C+ T cells selected by Kb. Furthermore, the 2C T cells selected in the presence of Lq fail to detect Lq/p2Ca complexes even at high ligand density. Thus, despite possessing the identical TCR, there is a functional avidity difference between 2C+ T cells selected in the presence of Lq vs Kb. These data provide evidence that homology between the selecting ligand and an alloantigen can influence the avidity of the T cell repertoire for the alloantigen, and suggest that thymic selection can fine tune T cell avidity independent of intrinsic TCR affinity.
Correction of Defects Responsible for Impaired Qa-2 Class Ib MHC Expression on Melanoma Cells Protects Mice from Tumor Growth
Authors: Chiang Eugene Y. , Henson Maile , Stroynowski Iwona
Published: April 2014

One of the principal mechanisms of tumor immune evasion is alteration of class I MHC expression. We have identified defects contributing to down-regulation of class I MHC expression in the widely studied murine B16 melanoma and its variants B16F1, B16F10, BL6-2, BL6-8 and B78H1. Transcription of the nonclassical class I MHC genes Q8 and Q9 (Qa-2 Ags) has been switched off in the entire panel of melanoma lines, suggesting that this event occurred early during tumor progression. B78H1, unlike B16F1 and B16F10 sublines, is also selectively devoid of TAP2 and low molecular weight protein 7 as well as classical class I MHC Kb and Db transcripts. Cotransfection of B78H1 with TAP2 and class I H chain genes is sufficient to reconstitute surface expression of exogenously delivered class I MHC without concomitant re-expression of endogenous β2-microglobulin-associated class I. The serological absence of endogenous class Ia and Ib at the surface of TAP2-negative as well as TAP2-transfected B78H1 makes this system a suitable model for studying the properties of isolated class I proteins in tumors. We used this system to demonstrate that B78H1 cells genetically manipulated to re-express Q9 Ag have reduced tumor potential in syngeneic B6 mice compared with TAP2-transfected parental melanoma. Both NK cells and CTLs appear to collaborate in restraining growth of Q9-positive tumors. The results implicate Qa-2 in antitumor responses and illustrate the utility of the B78H1 system for identifying in vivo interactions between class I MHC molecules of interest and immune cells of innate and/or adaptive immunity.
IL-7-Regulated Homeostatic Maintenance of Recent Thymic Emigrants in Association with Caspase-Mediated Cell Proliferation and Apoptotic Cell Death
Authors: O’Neill Rose M. , Hassan Jaythoon , Reen Denis J.
Published: April 2014

Homeostasis of T cells is essential to the maintenance of the T cell pool and TCR diversity. In this study, mechanisms involved in the regulation of cytokine-mediated expansion of naive T cells in the absence of Ag, in particular the role of caspase activation and susceptibility to apoptosis of recent thymic emigrants (RTEs), were examined. Low level caspase-8 and caspase-3 activation was detected in proliferating IL-7-treated cells in the absence of cell death during the first days of culture. Caspase inhibitors suppressed IL-7-induced expansion of RTEs. Low level expression of CD95 and blocking Ab experiments indicated that this early caspase activation was CD95 independent. However, CD95 levels subsequently became dramatically up-regulated on proliferating naive T cells, and these cells became susceptible to CD95 ligation, resulting in high level caspase activation and apoptotic cell death. These results show a dual role for caspases in proliferation and in CD95-induced cell death during Ag-independent expansion of RTEs. This method of cell death in IL-7-expanded RTEs is a previously unrecognized mechanism for the homeostatic control of expanded T cells.
Study of the Mechanism of TCR Antagonism Using Dual-TCR-Expressing T Cells
Authors: Yang Wen , Grey Howard M.
Published: April 2014

The mechanism of action of TCR antagonists is incompletely understood. T cells expressing two distinct TCRs have been used to test competition for TCR occupancy as a potential mechanism. Previous studies with CD4 T cells showed that an antagonist for one TCR inhibited the response to the other TCR (cross-antagonism), whereas studies with CD8 cells failed to demonstrate cross-antagonism. To determine whether CD4 and CD8 cells were intrinsically different or whether the differences were the result of the use of different effector assays, we studied both CD4 and CD8 dual-TCR-expressing T cells. In the CD4 system, consistent with previous reports, cross-antagonism of proliferation was observed. In the CD8 system, cross-antagonism was observed using proliferation as readout but not when target cell cytolysis was used. These results suggest that different mechanisms may be involved in the inhibition of proliferation and inhibition of cytotoxic effector function, the latter only involving competition for TCR occupancy. Inhibition of proliferation appears to be more complex and other mechanisms such as sequestration of signaling molecules or negative signaling may be involved. The fact that 10- to 20-fold more antagonist was needed to achieve cross-antagonism compared with inhibition of the cognate TCR is consistent with the hypothesis that competition for TCR occupancy is also a major, albeit not sole, mechanism of antagonism of the proliferative responses of CD4 and CD8 cells.
Src Homology Region 2-Containing Protein Tyrosine Phosphatase-2 (SHP-2) Can Play a Direct Role in the Inhibitory Function of Killer Cell Ig-Like Receptors in Human NK Cells
Authors: Yusa Sei-ichi , Campbell Kerry S.
Published: April 2014

The inhibitory forms of killer cell Ig-like receptors (KIR) are MHC class I-binding receptors that are expressed by human NK cells and prevent their attack of normal cells. Substantial evidence indicates that the mechanism of KIR-mediated inhibition involves recruitment of the protein tyrosine phosphatase, Src homology region 2-containing protein tyrosine phosphatase (SHP)-1, to phosphorylated immunoreceptor tyrosine-based inhibitory motifs (ITIMs). However, the functional significance of parallel recruitment of a SHP-1-related phosphatase, SHP-2, to KIR ITIMs has not been addressed. In the present study, our results with mutant forms of a classical KIR, KIR3DL1, show a direct correlation between SHP-2 recruitment and functional inhibition of target cell conjugation and cytotoxicity. In addition, KIR3DL1 inhibition of target cell cytotoxicity is blocked by overexpression of a dominant-negative form of SHP-2. Finally, KIR3DL1 fused directly with the catalytic domain of SHP-2 inhibits both target cell conjugation and cytotoxicity responses. These results strongly indicate that SHP-2 catalytic activity plays a direct role in inhibitory KIR functions, and SHP-2 inhibits NK cell activation in concert with SHP-1.
Murine Neonatal Lymphocytes Show Rapid Early Cell Cycle Entry and Cell Division
Authors: Adkins Becky , Williamson Tonya , Guevara Patricia , Bu Yurong
Published: April 2014

Neonatal animals are highly susceptible to infectious agents. At least part of this susceptibility is due to the virtual absence of immunological memory in newborns. One of the hallmarks of memory is the rapidity of the response. We show in this study that neonates may make up for their lack of memory, at least in part, by the rapid entry of large proportions of naive lymphocytes into the cell cycle. Following activation, greater percentages of both CD4+ and CD8+ neonatal, as compared with adult, lymph node cells showed early cell cycle entry; this was assessed by propidium iodide staining, CFSE labeling profiles, [3H]thymidine uptake, and up-regulation of early activation markers. This rapid cycle entry was observed following polyclonal activation with anti-CD3 or with PMA and ionomycin and in both C57BL/6 and BALB/c mice. Stimulation with specific peptide also elicited more rapid proliferative responses from neonatal vs adult TCR transgenic CD4+ cells. In addition, more rapid cycle entry was observed in vivo, in lymphopenic RAG2−/− hosts. For both CD4+ and CD8+ cells, this phenomenon was observed out to 3 wk of life, although the differences between neonatal and adult cells became smaller with increasing time postbirth. These properties of peripheral neonatal T cells appeared to be inherited from their thymic precursors, because CD4+8− single-positive cells in the neonatal thymus also showed more rapid cycle entry, compared with their counterparts in the adult thymus. Interestingly, rapid early cycling was also observed among activated neonatal B cells, compared with adult B cells. Thus, early cell cycle entry by large proportions of cells may allow the naive lymphocyte population to efficiently mobilize responses against the broad range of pathogens first encountered in neonatal life.
Allelic Exclusion of the TCR α-Chain Is an Active Process Requiring TCR-Mediated Signaling and c-Cbl
Authors: Niederberger Nathalie , Holmberg Kaisa , Alam S. Munir , Sakati Wayne , Naramura Mayumi , Gu Hua , Gascoigne Nicholas R. J.
Published: April 2014

Phenotypic allelic exclusion at the TCRα locus is developmentally regulated in thymocytes. Many immature thymocytes express two cell surface α-chain species. Following positive selection, the vast majority of mature thymocytes and peripheral T cells display a single cell surface α-chain. A posttranslational mechanism occurring at the same time as positive selection and TCR up-regulation leads to this phenotypic allelic exclusion. Different models have been proposed to explain the posttranslational regulation of the α-chain allelic exclusion. In this study, we report that allelic exclusion is not regulated by competition between distinct α-chains for a single β-chain, as proposed by the dueling α-chain model, nor by limiting CD3 ζ-chain in mature TCRhigh thymocytes. Our data instead favor the selective retention model where the positive selection signal through the TCR leads to phenotypic allelic exclusion by specifically maintaining cell surface expression of the selected α-chain while the nonselected α-chain is internalized. The use of inhibitors specific for Lck and/or other Src kinases indicates a role for these protein tyrosine kinases in the signaling events leading to the down-regulation of the nonselectable α-chain. Loss of the ubiquitin ligase/TCR signaling adapter molecule c-Cbl, which is important in TCR down-modulation and is a negative regulator of T cell signaling, leads to increased dual α-chain expression on the cell surface of double-positive thymocytes. Thus, not only is there an important role for TCR signaling in causing α-chain allelic exclusion, but differential ubiquitination by c-Cbl may be an important factor in causing only the nonselected α-chain to be down-modulated.
B1 Cells Contribute to Serum IgM, But Not to Intestinal IgA, Production in Gnotobiotic Ig Allotype Chimeric Mice
Authors: Thurnheer M. Christine , Zuercher Adrian W. , Cebra John J. , Bos Nicolaas A.
Published: April 2014

B1 cells are a significant source of natural serum IgM, thereby serving as a first line of defense against systemic bacterial and viral infections. They can migrate to the intestinal lamina propria and differentiate into IgA-producing plasma cells and thus might play a similar role in mucosal immunity. To investigate the contribution of B1 cells to the intestinal IgA response induced by the commensal flora in immunocompetent animals, we generated gnotobiotic and conventionally reared Ig allotype chimeric mice. In this system B1- and B2-derived Abs can be distinguished based on different allotypes. FACS analysis of peritoneal cavity cells and analysis of B1- and B2-derived serum IgM indicated stable B1/B2 chimerism and the establishment of a functional B1 population. Monoassociation with either Morganella morganii, Bacteroides distasonis, or segmented filamentous bacteria induced germinal center reactions in Peyer’s patches and led to the production of intestinal IgA, partially reactive with bacterial Ag. A considerable amount of serum IgM was B1 cell derived in both monoassociated and conventionally reared mice. However, most of the total as well as bacteria-specific intestinal IgA was produced by B2 cells. These data suggest that intestinal IgA production induced by commensal bacteria is mainly performed by B2, not B1, cells.
TGF-β1 Suppresses Myeloid Fcγ Receptor Function by Regulating the Expression and Function of the Common γ-Subunit
Authors: Tridandapani Susheela , Wardrop Richard , Baran Christopher P. , Wang Yijie , Opalek Judy M. , Caligiuri Michael A. , Marsh Clay B.
Published: April 2014

We have previously reported that FcγR-mediated function in myeloid cells is a tightly regulated event that is influenced by the cytokines present in the milieu. TGF-β1 is an immunosuppressive cytokine with pleiotropic effects on immune responses; however, the molecular mechanism by which TGF-β suppresses immune responses is poorly understood. In this study, we have analyzed the effect of TGF-β on FcγR-mediated activation of myeloid cells. We report that TGF-β1-treated THP-1 human myeloid cells displayed reduced ability to phagocytose IgG-coated particles. Because FcγR expression is modulated by cytokines, we analyzed expression levels of FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa in cells cultured with or without TGF-β1 and found while total protein levels of the FcγR were not reduced, surface expression of FcγRI and FcγRIII was lower in cells cultured with TGF-β1. Concomitantly, there was a dose-dependent reduction in the expression of the FcγR-associated γ-subunit. This suppressive effect of TGF-β was likewise observed in bone marrow-derived murine myeloid cells and human monocytes. Importantly, TGF-β1 also significantly reduced the production of monocyte chemoattractant protein-1 induced by immobilized IgG, which would further reduce monocyte recruitment to the site of inflammation. In contrast, human alveolar macrophages were refractory to this effect, expressing low levels of TGF-β type II receptors compared with peripheral blood monocytes from the same donor. These data provide insight into the regulation of immune responses by TGF-β1 and demonstrate the selectivity of these effects.
The Cyclopentenone-Type Prostaglandin 15-Deoxy-Δ12,14-Prostaglandin J2 Inhibits CD95 Ligand Gene Expression in T Lymphocytes: Interference with Promoter Activation Via Peroxisome Proliferator-Activated Receptor-γ-Independent Mechanisms
Authors: Cippitelli Marco , Fionda Cinzia , Di Bona Danilo , Lupo Aldo , Piccoli Mario , Frati Luigi , Santoni Angela
Published: April 2014

15-Deoxy-Δ12,14-PGJ2 (15d-PGJ2) is a cyclopentenone-type PG endowed with anti-inflammatory properties and produced by different cells, including those of the immune system. 15d-PGJ2 is a natural ligand of the peroxisome proliferator-activated receptor (PPAR)-γ nuclear receptor, but relevant PPARγ-independent actions mediated by this prostanoid have been described. Fas (APO-1/CD95) and its ligand (Fas-L) are cell surface proteins whose interaction activates apoptosis of Fas-expressing targets. In T cells, the Fas-Fas-L system regulates activation-induced cell death and has been implicated in diseases in which lymphocyte homeostasis is compromised. Moreover, several studies have described the pathogenic functions of Fas and Fas-L in vivo, particularly in the induction-progression of organ-specific autoimmune diseases. In this study we describe the effect of 15d-PGJ2 on the activation of the fas-L gene in T lymphocytes. We show that 15d-PGJ2 inhibits fas-L mRNA expression, activation-induced cell death, and fas-L promoter activity by mechanisms independent of PPARγ and mediated by its chemically reactive cyclopentenone moiety. Our data indicate that 15d-PGJ2 may repress fas-L activation by interfering with the expression and/or transcriptional activity of different transcription factors (early growth response types 3 and 1, NF-κB, AP-1, c-Myc, Nur77) whose altered balancing and transactivation may contribute for overall repression of this gene. In addition, the activation/expression of the heat shock response genes HSF-1 and HSP70 is not directly involved in the repression, and the electrophilic molecule cyclopentenone (2-cyclopenten-1-one) may reproduce the effects mediated by 15d-PGJ2. These results suggest that modulation of Fas-L by 15d-PGJ2 in T cells may represent an additional tool to consider for treatment of specific autoimmune and inflammatory disorders.