bluuebunny/crossref_metadata_2025 huggingface dataset:
Intestinal and Splenic T Cell Responses to EntericListeria monocytogenesInfection: Distinct Repertoires of Responding CD8 T Lymphocytes
Authors: Huleatt James W. , Pilip Ingrid , Kerksiek Kristen , Pamer Eric G.
Published: April 2014

Listeria monocytogenes is an intracellular bacterium that causes systemic infections after traversing the intestinal mucosa. Clearance of infection and long term protective immunity are mediated by L. monocytogenes-specific CD8 T lymphocytes. In this report, we characterize the murine CD8 T cell response in the lamina propria and intestinal epithelium after enteric L. monocytogenes infection. We find that the frequency of MHC class Ia-restricted, L. monocytogenes-specific T cells is ∼4- to 5-fold greater in the lamina propria than in the spleen of mice after oral or i.v. infection. Although the kinetics of T cell expansion and contraction are similar in spleen, lamina propria, and intestinal epithelium, high frequencies of Ag-specific T cells are detected only in the lamina propria 1 mo after infection. In contrast to MHC class Ia-restricted T cells, the frequency of H2-M3-restricted, L. monocytogenes-specific T cells is decreased in the intestinal mucosa relative to that found in the spleen. In addition to this disparity, we find that MHC class Ia-restricted CD8 T cells specific for a dominant L. monocytogenes epitope have different TCR Vβ repertoires in the spleen and intestinal mucosa of individual mice. These findings indicate that the intestinal mucosa is a depot where L. monocytogenes-specific effector CD8 T cells accumulate during and after infection irrespective of immunization route. Furthermore, our results demonstrate that CD8 T cell populations in these two sites, although overlapping in Ag specificity, are distinct in terms of their repertoire.
Differential Effects of a Toll-Like Receptor Antagonist on Mycobacterium tuberculosis-Induced Macrophage Responses
Authors: Means Terry K. , Jones Bryan W. , Schromm Andra B. , Shurtleff Beth A. , Smith Jason A. , Keane Joseph , Golenbock Douglas T. , Vogel Stefanie N. , Fenton Matthew J.
Published: April 2014

We previously showed that viable Mycobacterium tuberculosis (Mtb) bacilli contain distinct ligands that activate cells via the mammalian Toll-like receptor (TLR) proteins TLR2 and TLR4. We now demonstrate that expression of a dominant negative TLR2 or TLR4 proteins in RAW 264.7 macrophages partially blocked Mtb-induced NF-κB activation. Coexpression of both dominant negative proteins blocked virtually all Mtb-induced NF-κB activation. The role of the TLR4 coreceptor MD-2 was also examined. Unlike LPS, Mtb-induced macrophage activation was not augmented by overexpression of ectopic MD-2. Moreover, cells expressing an LPS-unresponsive MD-2 mutant responded normally to Mtb. We also observed that the lipid A-like antagonist E5531 specifically inhibited TLR4-dependent Mtb-induced cellular responses. E5531 could substantially block LPS- and Mtb-induced TNF-α production in both RAW 264.7 cells and primary human alveolar macrophages (AMφ). E5531 inhibited Mtb-induced AMφ apoptosis in vitro, an effect that was a consequence of the inhibition of TNF-α production by E5531. In contrast, E5531 did not inhibit Mtb-induced NO production in RAW 264.7 cells and AMφ. Mtb-stimulated peritoneal macrophages from TLR2- and TLR4-deficient animals produced similar amounts of NO compared with control animals, demonstrating that these TLR proteins are not required for Mtb-induced NO production. Lastly, we demonstrated that a dominant negative MyD88 mutant could block Mtb-induced activation of the TNF-α promoter, but not the inducible NO synthase promoter, in murine macrophages. Together, these data suggest that Mtb-induced TNF-α production is largely dependent on TLR signaling. In contrast, Mtb-induced NO production may be either TLR independent or mediated by TLR proteins in a MyD88-independent manner.
Histamine Induces Exocytosis and IL-6 Production from Human Lung Macrophages Through Interaction with H1 Receptors
Authors: Triggiani Massimo , Gentile Marco , Secondo Agnese , Granata Francescopaolo , Oriente Alfonso , Taglialatela Maurizio , Annunziato Lucio , Marone Gianni
Published: April 2014

Increasing evidence suggests that a continuous release of histamine from mast cells occurs in the airways of asthmatic patients and that histamine may modulate functions of other inflammatory cells such as macrophages. In the present study histamine (10−9–10−6 M) increased in a concentration-dependent fashion the basal release of β-glucuronidase (EC50 = 8.2 ± 3.5 × 10−9 M) and IL-6 (EC50 = 9.3 ± 2.9 × 10−8 M) from human lung macrophages. Enhancement of β-glucuronidase release induced by histamine was evident after 30 min and peaked at 90 min, whereas that of IL-6 required 2–6 h of incubation. These effects were reproduced by the H1 agonist (6-[2-(4-imidazolyl)ethylamino]-N-(4-trifluoromethylphenyl)heptane carboxamide but not by the H2 agonist dimaprit. Furthermore, histamine induced a concentration-dependent increase of intracellular Ca2+ concentrations ([Ca2+]i) that followed three types of response, one characterized by a rapid increase, a second in which [Ca2+]i displays a slow but progressive increase, and a third characterized by an oscillatory pattern. Histamine-induced β-glucuronidase and IL-6 release and [Ca2+]i elevation were inhibited by the selective H1 antagonist fexofenadine (10−7–10−4 M), but not by the H2 antagonist ranitidine. Inhibition of histamine-induced β-glucuronidase and IL-6 release by fexofenadine was concentration dependent and displayed the characteristics of a competitive antagonism (Kd = 89 nM). These data demonstrate that histamine induces exocytosis and IL-6 production from human macrophages by activating H1 receptor and by increasing [Ca2+]i and they suggest that histamine may play a relevant role in the long-term sustainment of allergic inflammation in the airways.
Differential Regulation of Formyl Peptide Receptor-Like 1 Expression During the Differentiation of Monocytes to Dendritic Cells and Macrophages
Authors: Yang De , Chen Qian , Le Yingying , Wang Ji Ming , Oppenheim Joost J.
Published: April 2014

Monocytes are the common precursors for myeloid dendritic cells (DC) and macrophages. Identification of chemotactic receptors expressed by myeloid DC, macrophages, and their precursors in the course of differentiation and maturation is important not only for elucidation of their in vivo trafficking, but also for understanding of the functional distinction between DC and macrophages. We chose to study formyl peptide receptor like-1 (FPRL1), a chemotactic receptor known to interact with several endogenous agonists that are involved in inflammatory and host defense responses. Here we show that FPRL1 is down-regulated as monocytes differentiate into DC. This down-regulation occurs at both mRNA and functional levels. Therefore, the interaction of FPRL1 with its agonists is more likely to regulate the in vivo trafficking of DC precursors than DC. In contrast, FPRL1 expression is maintained at both mRNA and functional levels as monocytes differentiate into macrophages. Thus, our results demonstrate further distinctions between myeloid DC and macrophages, albeit they share a common precursor. The fact that macrophages rather than myeloid DC express functional FPRL1 suggests that this chemotactic receptor may be more involved in inflammatory reactions and innate host defense than in adaptive immune responses.
Differential Roles of the NPXXY Motif in Formyl Peptide Receptor Signaling
Authors: He Rong , Browning Darren D. , Ye Richard D.
Published: April 2014

The NPXXY motif (X represents any amino acid) in the seventh transmembrane domain of the chemotactic formyl peptide receptor (FPR) is highly conserved among G protein-coupled receptors. Recent work suggested that this motif contributes to G protein-coupled receptor internalization and signal transduction; however, its role in FPR signaling remains unclear. In this study we replaced Asn297 and Tyr301 in the NPXXY motif of the human FPR with Ala (N297A) and Ala/Phe (Y301A/Y301F), respectively, and determined the effects of the substitutions on FPR functions in transfected rat basophilic leukemia cells. Whereas all the mutant receptors were expressed on the cell surface, the N297A receptor exhibited reduced binding affinity and was unable to mediate activation of phospholipase C-β and the p42/44 mitogen-activated protein kinase (MAP kinase). The Y301F receptor displayed significantly decreased ligand-stimulated internalization and MAP kinase activation, suggesting that the hydrogen bonding at Tyr301 is critical for these functions. The Y301F receptor showed a chemotactic response similar to that of wild-type FPR, indicating that cell chemotaxis does not require receptor internalization and hydrogen bonding at the Tyr301 position. In contrast, the Y301A receptor displayed a left-shifted, but overall reduced, chemotaxis response that peaked at 0.1–1 nM. Finally, using a specific MAP kinase kinase inhibitor, we found that activation of MAP kinase is required for efficient FPR internalization, but is not essential for chemotaxis. These findings demonstrate that residues within the NPXXY motif differentially regulate the functions of FPR.
Effect of Redox Modulation on Xenogeneic Target Cells: The Combination of Nitric Oxide and Thiol Deprivation Protects Porcine Endothelial Cells from Lysis by IL-2-Activated Human NK Cells
Authors: Tsuyuki Shigeru , Horvath-Arcidiacono Judith A. , Bloom Eda T.
Published: April 2014

Evidence suggests that NK cells contribute to the pathogenesis of delayed rejection of vascularized xenografts, and NK cells have been suggested to participate in hyperacute xenograft rejection. Endothelial cells have been shown to be the primary target of the recipient’s immune responses that mediate both hyperacute and delayed xenograft rejection. Under conditions of oxidative stress induced by thiol deprivation, but not under normal conditions, pretreatment of porcine aortic endothelial cells (PAECs) with the NO donor, S-nitroso-N-acetyl-penicillamine, dramatically inhibited killing of PAEC target cells by IL-2-activated human NK cells. This same combined treatment reduced both surface expression and mRNA levels of E-selectin. Moreover, anti-E-selectin mAb, but not Ab to VCAM-1, protected PAEC from lysis by human IL-2-activated NK cells in a dose-dependent manner. These findings suggest that expression of porcine E-selectin is important for the cytotoxicity of PAEC mediated by activated human NK cells and may be involved in the redox-mediated modulation of that cytotoxicity. It is known that NF-κB activation is required for transcription of E-selectin, and the current data show that the suppression of E-selectin expression by S-nitroso-N-acetyl-penicillamine pretreatment and thiol deprivation was associated with reduced NF-κB DNA-binding activity in PAEC. These data suggest that the regulation of porcine E-selectin may be important for modulating delayed xenograft rejection and that manipulation of cellular redox systems may provide a means to protect xenogeneic endothelial cells from NK cell-mediated cytotoxicity.
Resting Murine Neutrophils Express Functional α4 Integrins that Signal Through Src Family Kinases
Authors: Pereira Shalini , Zhou Meijuan , Mócsai Attila , Lowell Clifford
Published: April 2014

There is mounting evidence that α4 (CD49d) integrins are involved in neutrophil recruitment and function during inflammatory responses. We report that all resting murine neutrophils derived from bone marrow or peripheral blood express easily detectable levels of α4 integrins on their surface. These α4 integrins were functional, as demonstrated by stimulation of respiratory burst when neutrophils adhered to surfaces coated with the murine vascular cell adhesion molecule-1 (mVCAM-1). Adhesion occurred via α4 integrins, as preincubation of neutrophils with an anti-α4-specific Ab inhibited attachment to mVCAM-1. Direct cross-linking of the α4 integrin subunit by surface-bound mAbs also elicited superoxide release and release of the secondary granule marker, lactoferrin. The functional responses that occurred downstream of α4 integrin cross-linking required signaling by Src family kinases. Neutrophils derived from hck−/−fgr−/−lyn−/− triple-knockout or hck−/−fgr−/− double-knockout mice failed to undergo respiratory burst when plated on mVCAM-1. Triple mutant neutrophils were also defective in release of both superoxide and lactoferrin when plated on surfaces coated with mAbs directed against α4. Correlated with impaired α4-induced functional responses, triple-mutant neutrophils also failed to spread and tightly adhere to anti-α4 mAb-coated surfaces. This is the first direct evidence that functional α4 integrins are expressed by murine PMNs, and that these surface molecules can mediate cellular responses such as tight adhesion, spreading, sustained respiratory burst, and specific granule release in vitro. Moreover the α4 integrins, like all other integrins tested, use the Src family kinases to transduce intracellular signals.
Different Therapeutic Outcomes in Experimental Allergic Encephalomyelitis Dependant Upon the Mode of Delivery of IL-10: A Comparison of the Effects of Protein, Adenoviral or Retroviral IL-10 Delivery into the Central Nervous System
Authors: Croxford J. Ludovic , Feldmann Marc , Chernajovsky Yuti , Baker David
Published: April 2014

Experimental allergic encephalomyelitis (EAE) is a CNS autoimmune disease mediated by the action of CD4+ T cells, macrophages, and proinflammatory cytokines. IL-10 is a cytokine shown to have many anti-inflammatory properties. Studies have shown both inhibition and exacerbation of EAE after systemic IL-10 protein administration. We have compared the inhibitory effect in EAE of Il10 gene delivery in the CNS. Fibroblasts transduced with retroviral vectors expressing IL-10 could inhibit EAE. This was not associated with a prevention of cellular recruitment but an alteration in their phenotype, notably an increase in the numbers of CD8+ T and B cells. In marked contrast, CNS delivery of adenovirus coding for mouse IL-10 or IL-10 protein performed over a wide dose range failed to inhibit disease, despite producing similar or greater amounts of IL-10 protein. Thus the action of IL-10 may differ depending on the local cytokine microenvironment produced by the gene-secreting cell types.
The Cutaneous Response in Humans toTreponema pallidumLipoprotein Analogues Involves Cellular Elements of Both Innate and Adaptive Immunity
Authors: Sellati Timothy J. , Waldrop Shar L. , Salazar Juan C. , Bergstresser Paul R. , Picker Louis J. , Radolf Justin D.
Published: April 2014

To extend prior studies implicating treponemal lipoproteins as major proinflammatory agonists of syphilitic infection, we examined the responses induced by intradermal injection of human subjects with synthetic lipoprotein analogues (lipopeptides) corresponding to the N termini of the 17- and 47-kDa lipoproteins of Treponema pallidum. Responses were assessed visually and by flow cytometric analysis of dermal leukocyte populations within fluids aspirated from suction blisters raised over the injection sites. Lipopeptides elicited dose-dependent increases in erythema/induration and cellular infiltrates. Compared with peripheral blood, blister fluids were highly enriched for monocytes/macrophages, cutaneous lymphocyte Ag-positive memory T cells, and dendritic cells. PB and blister fluids contained highly similar ratios of CD123−/CD11c+ (DC1) and CD123+/CD11c− (DC2) dendritic cells. Staining for maturation/differentiation markers (CD83, CD1a) and costimulatory molecules (CD80/CD86) revealed that blister fluid DC1, but not DC2, cells were more developmentally advanced than their peripheral blood counterparts. Of particular relevance to the ability of syphilitic lesions to facilitate the transmission of M-tropic strains of HIV-1 was a marked enhancement of CCR5 positivity among mononuclear cells in the blister fluids. Treponemal lipopeptides have the capacity to induce an inflammatory milieu reminiscent of that found in early syphilis lesions. In contrast with in vitro studies, which have focused upon the ability of these agonists to stimulate isolated innate immune effector cells, in this study we show that in a complex tissue environment these molecules have the capacity to recruit cellular elements representing the adaptive as well as the innate arm of the cellular immune response.
Functional IL-2 Receptor β (CD122) and γ (CD132) Chains Are Expressed by Fibroblast-Like Synoviocytes: Activation by IL-2 Stimulates Monocyte Chemoattractant Protein-1 Production
Authors: Corrigall Valerie M. , Arastu Mateen , Khan Shazia , Shah Chandrabala , Fife Mark , Smeets Tom , Tak Paul-Peter , Panayi Gabriel S.
Published: April 2014

The expression of the IL-2R α-, β-, and γ-chains, CD25, CD122, and CD132, respectively, was investigated on fibroblast-like synoviocytes (FLS) and dermal fibroblasts (DF). Both protein and mRNA for CD122 and CD132 were observed but there was no evidence of CD25 expression. Quantification of the Ag binding sites for CD122 showed that FLS expressed 4 times more receptor molecules than DF. The functional capability of these receptors was confirmed by the production of monocyte chemoattractant protein-1 (MCP-1) in direct response to stimulation by IL-2, which could be inhibited by neutralizing anti-CD122 mAb. Both rheumatoid arthritis (RA) and osteoarthritis (OA) FLS and DF spontaneously produced MCP-1 in culture over a similar range of concentrations. However, RA and OA FLS produced significantly greater levels of MCP-1 following stimulation by IL-2 and IL-1β; RA FLS produced significantly more MCP-1 than OA FLS. Addition of exogenous IL-2 caused a slight, but significant, decrease in MCP-1 production by DF. The addition of neutralizing anti-CD122 mAb to FLS cultures partially, but significantly, reduced the IL-2-induced MCP-1 secretion, but did not effect either the spontaneous or IL-1β-induced secretion of MCP-1. Increased tyrosine phosphorylation was observed in FLS lysates following 30-min incubation with IL-2. In conclusion, in the inflamed synovium, as activated T cells migrate through the sublining and lining layer, T cell-derived IL-2 may activate FLS to secrete MCP-1, thus recruiting macrophages into the rheumatoid synovium and perpetuating inflammation.
A Keratin Peptide Inhibits Mannose-Binding Lectin
Authors: Montalto Michael C. , Collard Charles D. , Buras Jon A. , Reenstra Wende R. , McClaine Rebecca , Gies David R. , Rother Russell P. , Stahl Gregory L.
Published: April 2014

Complement plays a significant role in mediating endothelial injury following oxidative stress. We have previously demonstrated that the lectin complement pathway (LCP), which is initiated by deposition of the mannose-binding lectin (MBL), is largely responsible for activating complement on endothelial cells following periods of oxidative stress. Identifying functional inhibitors that block MBL binding will be useful in characterizing the role of the LCP in disease models. The human cytokeratin peptide SFGSGFGGGY has been identified as a molecular mimic of N-acetyl-d-glucosamine (GlcNAc), a known ligand of MBL. Thus, we hypothesized that this peptide would specifically bind to MBL and functionally inhibit the LCP on endothelial cells following oxidative stress. Using a BIAcore 3000 optical biosensor, competition experiments were performed to demonstrate that the peptide SFGSGFGGGY inhibits binding of purified recombinant human MBL to GlcNAc in a concentration-dependent manner. Solution affinity data generated by BIAcore indicate this peptide binds to MBL with an affinity (KD) of 5 × 10−5 mol/L. Pretreatment of human serum (30%) with the GlcNAc-mimicking peptide (10–50 μg/ml) significantly attenuated MBL and C3 deposition on human endothelial cells subjected to oxidative stress in a dose-dependent manner, as demonstrated by cell surface ELISA and confocal microscopy. Additionally, this decapeptide sequence attenuated complement-dependent VCAM-1 expression following oxidative stress. These data indicate that a short peptide sequence that mimics GlcNAc can specifically bind to MBL and functionally inhibit the proinflammatory action of the LCP on oxidatively stressed endothelial cells.
Neuronal Expression of a Functional Receptor for the C5a Complement Activation Fragment
Authors: O’Barr Stephen A. , Caguioa Jody , Gruol Donna , Perkins Guy , Ember Julia A. , Hugli Tony , Cooper Neil R.
Published: April 2014

The present studies were undertaken to determine whether neuronal subsets in normal brains constitutively express functionally competent C5a receptors. In situ hybridization studies coupled with immunohistochemical approaches revealed that most neurons in the hippocampal formation, many pyramidal cortical neurons, and cerebellar Purkinje neurons in normal human and murine brains constitutively express C5a receptors. Neuronal C5a receptors bound C5a-coated fluorescent microspheres, and primary rodent hippocampal neurons responded to C5a with increased calcium fluxes via a pertussis-sensitive, presumably Gi-coupled protein. Additional studies with human neuroblastoma cells conducted to address the functional role of C5a receptors revealed that C5a triggered rapid activation of protein kinase C and activation and nuclear translocation of the NF-κB transcription factor. In addition, C5a was found to be mitogenic for undifferentiated human neuroblastoma cells, a novel action for the C5aR. In contrast, C5a protected terminally differentiated human neuroblastoma cells from toxicity mediated by the amyloid Aβ peptide. Thus, normal rodent hippocampal neurons as well as undifferentiated and differentiated human neuroblastoma cells express functional C5a receptors. These results have implications for understanding the role of neuronal C5aR receptors in normal neuronal development, neuronal homeostasis, and neuroinflammatory conditions such as Alzheimer’s disease.
Redirection of T Cell Effector Function In Vivo and Enhanced Collagen-Induced Arthritis Mediated by an IL-2Rβ/IL-4Rα Chimeric Cytokine Receptor Transgene
Authors: Chen Ying , Rosloniec Ed , Goral Mehmet I. , Boothby Mark , Chen Jin
Published: April 2014

Chronic inflammatory autoimmune diseases such as diabetes, experimental autoimmune encephalomyelitis, and collagen-induced arthritis (CIA) are associated with type 1 (Th1, Tc1) T cell-dependent responses against autoantigens. Immune deviation toward type 2 (Th2, Tc2) response has been proposed as a potential means of gene therapy or immunomodulation to treat autoimmune diseases based on evidence that type 2 cytokines can prevent or alleviate these conditions. In this report we assessed the effects of elevated type 2 responses on CIA using transgenic mice expressing an IL-2Rβ/IL-4Rα chimeric cytokine receptor transgene specifically in T cells. In response to IL-2 binding, this chimeric receptor transduces IL-4-specific signals and dramatically enhances type 2 responses. In contrast to published reports of Th2-mediated protection, CIA was exacerbated in IL-2Rβ/IL-4Rα chimeric receptor transgenic mice, with increased disease incidence, severity, and earlier disease onset. The aggravated disease in transgenic mice was associated with an increase in type 2 cytokines (IL-4, IL-5, IL-10) and an increase in collagen-specific IgG1 levels. However, IFN-γ production is not affected significantly in the induction phase of the disease. There is also an extensive eosinophilic infiltration in the arthritic joints of the transgenic animal, suggesting a direct contribution of type 2 response to joint inflammation. Taken together, our findings provide novel evidence that enhancement of a polyclonal type 2 response in immunocompetent hosts may exacerbate an autoimmune disease such as CIA, rather than serving a protective role. This finding raises significant caution with regard to the potential use of therapeutic approaches based on immune deviation toward type 2 responses.
Molecular Dissection of the Tissue Transglutaminase Autoantibody Response in Celiac Disease
Authors: Marzari Roberto , Sblattero Daniele , Florian Fiorella , Tongiorgi Enrico , Not Tarcisio , Tommasini Alberto , Ventura Alessandro , Bradbury Andrew
Published: April 2014

Celiac disease (CD) is an intestinal malabsorption characterized by intolerance to cereal proteins accompanied by immunological responses to dietary gliadins and tissue transglutaminase, an autoantigen located in the endomysium. Tissue transglutaminase belongs to the family of enzymes that catalyze protein cross-linking reactions and is constitutively expressed in many tissues as well as being activated during apoptosis. The role of gliadins in eliciting the immune response in CD and how transglutaminase is linked to the primary reaction are still unclear. In this work, we report the production and analysis of six phage Ab libraries from the peripheral and intestinal lymphocytes of three CD patients. We were able to isolate Abs to transglutaminase from all intestinal lymphocytes libraries but not from those obtained from peripheral lymphocytes. This is in contrast to Abs against gliadin, which could be obtained from all libraries, indicating that the humoral response against transglutaminase occurs at the local level, whereas that against gliadin occurs both peripherally and centrally. Abs from all three patients recognized the same transglutaminase epitopes with a bias toward the use of the VH5 Ab variable region family. The possible role of these anti-transglutaminase Abs in the onset of CD and associated autoimmune pathologies is discussed.
The Major Synovial Targets of the Rheumatoid Arthritis-Specific Antifilaggrin Autoantibodies Are Deiminated Forms of the α- and β-Chains of Fibrin
Authors: Masson-Bessière Christine , Sebbag Mireille , Girbal-Neuhauser Elisabeth , Nogueira Leonor , Vincent Christian , Senshu Tatsuo , Serre Guy
Published: April 2014

IgG antifilaggrin autoantibodies (AFA) are the most specific serological markers of rheumatoid arthritis. In epithelial tissues, they recognize citrulline-bearing epitopes present on various molecular forms of (pro)filaggrin. Histological analysis of rheumatoid synovial membranes with an Ab to citrulline showed labeling of interstitial amorphous deposits and mononuclear cells of various types. Immunochemical analysis of exhaustive sequential extracts of the same tissues showed that they contain several deiminated (citrulline containing) proteins. Among them, two proteins, p64–78 and p55–61, present in urea-DTT and guanidine extracts, were shown by immunoblotting to be specifically targeted by AFA. By amino-terminal sequencing the proteins were identified as deiminated forms of the α- and β-chains of fibrin, respectively. Their identity was confirmed using several Abs specific for the Aα- and/or to the Bβ-chain of fibrin(ogen). Moreover, AFA-positive rheumatoid arthritis (RA) sera and purified AFA were highly reactive to the Aα- and Bβ-chains of human fibrinogen only after deimination of the molecules by a peptidylarginine deiminase. Autoantibodies affinity purified from a pool of RA sera onto deiminated fibrinogen were reactive toward all of the epithelial and synovial targets of AFA. This confirmed that the autoantibodies to the deiminated Aα-and Bβ-chains of fibrinogen, the autoantibodies to the synovial proteins p64–78 and p55–61, and, lastly, AFA, constitute largely overlapping autoantibody populations. These results show that deiminated forms of fibrin deposited in the rheumatoid synovial membranes are the major target of AFA. They suggest that autoimmunization against deiminated fibrin is a critical step in RA pathogenesis.
Carbon Monoxide Generated by Heme Oxygenase-1 Suppresses the Rejection of Mouse-to-Rat Cardiac Transplants
Authors: Sato Koichiro , Balla Jozsef , Otterbein Leo , Smith R. Neal , Brouard Sophie , Lin Yuan , Csizmadia Eva , Sevigny Jean , Robson Simon C. , Vercellotti Gregory , Choi Augustine M. , Bach Fritz H. , Soares Miguel P.
Published: April 2014

Mouse-to-rat cardiac transplants survive long term after transient complement depletion by cobra venom factor and T cell immunosuppression by cyclosporin A. Expression of heme oxygenase-1 (HO-1) by the graft vasculature is critical to achieve graft survival. In the present study, we asked whether this protective effect was attributable to the generation of one of the catabolic products of HO-1, carbon monoxide (CO). Our present data suggests that this is the case. Under the same immunosuppressive regimen that allows mouse-to-rat cardiac transplants to survive long term (i.e., cobra venom factor plus cyclosporin A), inhibition of HO-1 activity by tin protoporphyrin, caused graft rejection in 3–7 days. Rejection was associated with widespread platelet sequestration, thrombosis of coronary arterioles, myocardial infarction, and apoptosis of endothelial cells as well as cardiac myocytes. Under inhibition of HO-1 activity by tin protoporphyrin, exogenous CO suppressed graft rejection and restored long-term graft survival. This effect of CO was associated with inhibition of platelet aggregation, thrombosis, myocardial infarction, and apoptosis. We also found that expression of HO-1 by endothelial cells in vitro inhibits platelet aggregation and protects endothelial cells from apoptosis. Both these actions of HO-1 are mediated through the generation of CO. These data suggests that HO-1 suppresses the rejection of mouse-to-rat cardiac transplants through a mechanism that involves the generation of CO. Presumably CO suppresses graft rejection by inhibiting platelet aggregation that facilitates vascular thrombosis and myocardial infarction. Additional mechanisms by which CO overcomes graft rejection may involve its ability to suppress endothelial cell apoptosis.
Immune Enhancing Effect of a Growth Hormone Secretagogue
Authors: Koo Gloria C. , Huang Christopher , Camacho Ramon , Trainor Charlotte , Blake J. Tom , Sirotina-Meisher Anna , Schleim Klaus D. , Wu Tsuei-Ju , Cheng Kang , Nargund Ravi , McKissick Gaylord
Published: April 2014

Growth hormone (GH) has been known to enhance immune responses, whether directly or through the insulin like growth factor-1, induced by GH. Recently a nonpeptidyl small m.w. compound, a GH secretagogue (GHS), was found to induce the production of GH by the pituitary gland. In this study, we examined the effect of GHS in immunological functions of 5- to 6-wk-old and 16- to 24-month-old mice. In young mice, we observed a significant increase in PBLs, but T and B cell-proliferative responses were not consistently enhanced. The old mice, treated with GHS for 3 wk, did not show increases in peripheral lymphocytes, but they exhibited a statistically significant increase in thymic cellularity and differentiation. When inoculated with a transplantable lymphoma cell line, EL4, the treated old mice showed statistically significant resistance to the initiation of tumors and the subsequent metastases. Generation of CTL to EL4 cells was also enhanced in the treated mice, suggesting that GHS has a considerable immune enhancing effect, particularly in the old mice. We have also found that GHS promoted better thymic engraftment in bone marrow transplant of SCID mice. We found more cycling cells in the spleens of treated mice, suggesting that GHS may exert its immune enhancing effect by promoting cell division in lymphoid cells. These observations ascribe to GHS a novel therapy possible for aging, AIDS, and transplant individuals, whose immune functions are compromised.
Human Monoclonal Rheumatoid Synovial B Lymphocyte Hybridoma with a New Disease-Related Specificity for Cartilage Oligomeric Matrix Protein
Authors: Souto-Carneiro Maria M. , Burkhardt Harald , Müller Ewa C. , Hermann Ralph , Otto Albrecht , Kraetsch Hans-Georg , Sack Ulrich , König Achim , Heinegård Dick , Müller-Hermelink Hans Konrad , Krenn Veit
Published: April 2014

Joint-specific self-Ags are considered to play an important role in the induction of synovial T and B cell expansion in human rheumatoid arthritis (RA). However, the nature of these autoantigens is still enigmatic. In this study a somatically mutated IgG2λ B cell hybridoma was established from the synovial membrane of an RA patient and analyzed for its Ag specificity. A heptameric peptide of cartilage oligomeric matrix protein (COMP) could be characterized as the target structure recognized by the human synovial B cell hybridoma. The clonotypic VH sequences of the COMP-specific hybridoma could also be detected in synovectomy material derived from five different RA patients but in none of the investigated osteoarthritis cases (n = 5), indicating a preferential usage of VH genes closely related to those coding for a COMP-specific Ag receptor in RA synovial B cells. Moreover, the COMP heptamer was preferentially recognized by circulating IgG in RA (n = 22) compared with osteoarthritis patients (n = 24) or age-matched healthy controls (n = 20; both p < 0.0001). Hence, the COMP-specific serum IgG is likely to reflect local immune responses toward a cartilage- and tendon-restricted Ag that might be crucial to the induction of tissue damage in RA.
Regulation of Experimental Autoimmune Encephalomyelitis in the C57BL/6J Mouse by NK1.1+, DX5+, αβ+ T Cells
Authors: Fritz Robert B. , Zhao Ming-Lang
Published: April 2014

C57BL/6 (B6) mice with targeted mutations of immune function genes were used to investigate the mechanism of recovery from experimental autoimmune encephalomyelitis (EAE). The acute phase of passive EAE in the B6 mouse is normally resolved by partial recovery followed by mild sporadic relapses. B6 TCR β-chain knockout (KO) recipients of a myelin oligodendrocyte glycoprotein p35–55 encephalitogenic T cell line failed to recover from the acute phase of passive EAE. In comparison with wild-type mice, active disease was more severe in β2-microglobulin KO mice. Reconstitution of TCR β-chain KO mice with wild-type spleen cells halted progression of disease and favored recovery. Spleen cells from T cell-deficient mice, IL-7R KO mice, or IFN-γ KO mice were ineffective in this regard. Irradiation or treatment of wild-type spleen cell population with anti-NK1.1 mAb before transfer abrogated the protective effect. Removal of DX5+ cells from wild-type spleen cells by anti-DX5 Ab-coated magnetic beads before reconstitution abrogated the suppressive properties of the spleen cells. TCR-deficient recipients of the enriched DX5+ cell population recovered normally from passively induced acute disease. DX5+ cells were sorted by FACS into DX5+ αβTCR+ and DX5+ αβTCR− populations. Only recipients of the former recovered normally from clinical disease. These results indicate that recovery from acute EAE is an active process that requires NK1.1+, DX5+ αβ+ TCR spleen cells and IFN-γ.
Molecular Basis of Deficient IL-2 Production in T Cells from Patients with Systemic Lupus Erythematosus
Authors: Solomou Elena E. , Juang Yuang-Taung , Gourley Mark F. , Kammer Gary M. , Tsokos George C.
Published: April 2014

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease characterized by diverse cellular and biochemical aberrations, including decreased production of IL-2. Here we show that nuclear extracts from unstimulated SLE T cells, unlike extracts from normal T cells, express increased amounts of phosphorylated cAMP-responsive element modulator (p-CREM) that binds the −180 site of the IL-2 promoter. Nuclear extracts from stimulated normal T cells display increased binding of phosphorylated cAMP-responsive element binding protein (p-CREB) to the −180 site of the IL-2 promoter, whereas nuclear extracts from stimulated SLE T cells display primarily p-CREM and decreased p-CREB binding. In SLE T cells, p-CREM bound to the transcriptional coactivators, CREB binding protein and p300. Increased expression of p-CREM correlated with decreased production of IL-2. The transcription of a reporter gene driven by the −180 site was enhanced in normal T cells, but was suppressed in SLE T cells. These experiments demonstrate that transcriptional repression is responsible for the decreased production of IL-2 by SLE T cells.
Identification of a Matrix-Degrading Phenotype in Human Tuberculosis In Vitro and In Vivo
Authors: Price Nicholas M. , Farrar Jeremy , Chau Tran Thi Hong , Mai Nguyen Thi Hoang , Hien Tran Tinh , Friedland Jon S.
Published: April 2014

Tuberculous meningitis is characterized by cerebral tissue destruction. Monocytes, pivotal in immune responses to Mycobacterium tuberculosis, secrete matrix metalloproteinase-9 (MMP-9), which facilitates leukocyte migration across the blood-brain barrier, but may cause cerebral injury. In vitro, human monocytic (THP-1) cells infected by live, virulent M. tuberculosis secreted MMP-9 in a dose-dependent manner. At 24 h, MMP-9 concentrations increased 10-fold to 239 ± 75 ng/ml (p = 0.001 vs controls). MMP-9 mRNA became detectable at 24–48 h. In contrast, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) gene expression and secretion were similar to constitutive levels from controls at 24 h and increased just 5-fold by 48 h. In vivo investigation revealed MMP-9 concentration per leukocyte in cerebrospinal fluid (CSF) from tuberculous meningitis patients (n = 23; median (range), 3.19 (0.19–31.00) ng/ml/cell) to be higher than that in bacterial (n = 12; 0.23 (0.01–18.37) ng/ml/cell) or viral meningitis (n = 20; 0.20 (0.04–31.00) ng/ml/cell; p < 0.01). TIMP-1, which was constitutively secreted into CSF, was not elevated in tuberculous compared with bacterial meningitis or controls. Thus, a phenotype in which MMP-9 activity is relatively unrestricted by TIMP-1 developed both in vitro and in vivo. This is functionally significant, since MMP-9 concentrations per CSF leukocyte (but not TIMP-1 concentrations) were elevated in fatal tuberculous meningitis and in patients with signs of cerebral tissue damage (unconsciousness, confusion, or neurological deficit; p < 0.05). However, MMP-9 activity was unrelated to the severity of systemic illness. In summary, M. tuberculosis-infected monocytic cells develop a matrix-degrading phenotype, which was observed in vivo and relates to clinical signs reflecting cerebral injury in tuberculous meningitis.
A Milk Protein Lactoferrin Enhances Human T Cell Leukemia Virus Type I and Suppresses HIV-1 Infection
Authors: Moriuchi Masako , Moriuchi Hiroyuki
Published: April 2014

Human T cell leukemia virus type I (HTLV-I) and HIV-1, causative agents of adult T cell leukemia/lymphoma and AIDS, respectively, are transmitted vertically via breast milk. Here we demonstrate that lactoferrin, a milk protein that has a variety of antimicrobial and immunomodulatory activities, facilitates replication of HTLV-I in lymphocytes derived from asymptomatic HTLV-I carriers and transmission to cord blood lymphocytes in vitro. Transient expression assays revealed that lactoferrin can transactivate HTLV-I long terminal repeat promoter. In contrast, lactoferrin inhibits HIV-1 replication, at least in part, at the level of viral fusion/entry. These results suggest that lactoferrin may have different effects on vertical transmission of the two milk-borne retroviruses.
Environmental Modulation of Autoimmune Arthritis Involves the Spontaneous Microbial Induction of T Cell Responses to Regulatory Determinants Within Heat Shock Protein 65
Authors: Moudgil Kamal D. , Kim Eugene , Yun Oliver J. , Chi Howard H. , Brahn Ernest , Sercarz Eli E.
Published: April 2014

Both genetic and environmental factors are believed to be involved in the induction of autoimmune diseases. Adjuvant arthritis (AA) is inducible in susceptible rat strains by injection of Mycobacterium tuberculosis, and arthritic rats raise T cell responses to the 65-kDa mycobacterial heat-shock protein (Bhsp65). We observed that Fischer 344 (F344) rats raised in a barrier facility (BF-F344) are susceptible to AA, whereas F344 rats maintained in a conventional facility (CV-F344) show significantly reduced incidence and severity of AA, despite responding well to the arthritogenic determinant within Bhsp65. The acquisition of protection from AA can be circumvented if rats are maintained on neomycin/acidified water. Strikingly, naive unimmunized CV-F344 rats but not BF-F344 rats raised T cell responses to Bhsp65 C-terminal determinants (BCTD) (we have previously shown that BCTD are involved in regulation of acute AA in the Lewis rat); however, T cells of naive CV-F344 and BF-F344 gave a comparable level of proliferative response to a mitogen, but no response at all to an irrelevant Ag. Furthermore, adoptive transfer into naive BF-F344 rats of splenic cells of naive CV-F344 rats (restimulated with BCTD in vitro) before induction of AA resulted in a considerably reduced severity of AA. These results suggest that spontaneous (inadvertent) priming of BCTD-reactive T cells, owing to determinant mimicry between Bhsp65 and its homologues in microbial agents in the conventional environment, is involved in modulating the severity of AA in CV-F344 rats. These results have important implications in broadening understanding of the host-microbe interaction in human autoimmune diseases.
Opposite Effects of IL-10 on the Ability of Dendritic Cells and Macrophages to Replicate Primary CXCR4-Dependent HIV-1 Strains
Authors: Ancuta Petronela , Bakri Youssef , Chomont Nicolas , Hocini Hakim , Gabuzda Dana , Haeffner-Cavaillon Nicole
Published: April 2014

We investigated the effect of IL-10 on replication of primary CXCR4-dependent (X4) HIV-1 strains by monocyte-derived dendritic cells (DCs) and macrophages (MΦs). MΦs efficiently replicated CXCR4-dependent HIV-1 (X4 HIV-1) strains NDK and VN44, whereas low levels of p24 were detected in supernatants of infected DCs. IL-10 significantly increased X4 HIV-1 replication by DCs but blocked viral production by MΦs as determined by p24 levels and semiquantitative nested PCR. IL-10 up-regulated CXCR4 mRNA and protein expression on DCs and MΦs, suggesting that IL-10 enhances virus entry in DCs but blocks an entry and/or postentry step in MΦs. The effect of IL-10 on the ability of DCs and MΦs to transmit virus to autologous CD4+ T lymphocytes was investigated in coculture experiments. DCs exhibited a greater ability than did MΦs to transmit a vigorous infection to CD4+ T cells despite their very low replication capacity. IL-10 had no effect on HIV-1 replication in DC:T cell cocultures but markedly decreased viral production in MΦ:T cell cocultures. These results demonstrate that IL-10 has opposite effects on the replication of primary X4 HIV-1 strains by DCs and MΦs. IL-10 increases X4-HIV-1 replication in DCs but does not alter their capacity to transmit virus to CD4+ T lymphocytes. These findings suggest that increased levels of IL-10 observed in HIV-1-infected patients with disease progression may favor the replication of X4 HIV-1 strains in vivo.
Dendritic Cells Injected Via Different Routes Induce Immunity in Cancer Patients
Authors: Fong Lawrence , Brockstedt Dirk , Benike Claudia , Wu Lijun , Engleman Edgar G.
Published: April 2014

Dendritic cells (DC) represent potent APCs that are capable of generating tumor-specific immunity. We performed a pilot clinical trial using Ag-pulsed DC as a tumor vaccine. Twenty-one patients with metastatic prostate cancer received two monthly injections of DC enriched and activated from their PBMC. DC were cocultured ex vivo with recombinant mouse prostatic acid phosphatase as the target neoantigen. Following enrichment, DC developed an activated phenotype with up-regulation of CD80, CD86, and CD83 expression. During culture, the DC maintained their levels of various adhesion molecules, including CD44, LFA-1, cutaneous lymphocyte-associated Ag, and CD49d, up-regulated CCR7, but lost CD62 ligand and CCR5. In the absence of CD62 ligand, such cells would not be expected to prime T cells efficiently if administered i.v. due to their inability to access lymphoid tissue via high endothelial venules. To assess this possibility, three patient cohorts were immunized with Ag-pulsed DC by i.v., intradermal (i.d.), or intralymphatic (i.l.) injection. All patients developed Ag-specific T cell immune responses following immunization, regardless of route. Induction of IFN-γ production, however, was seen only with i.d. and i.l. routes of administration, and no IL-4 responses were seen regardless of route, consistent with the induction of Th1-type immunity. Five of nine patients who were immunized by the i.v. route developed Ag-specific Abs compared with one of six for i.d. and two of six for i.l. routes. These results suggest that while activated DC can prime T cell immunity regardless of route, the quality of this response and induction of Ag-specific Abs may be affected by the route of administration.
Cutting Edge: Intravenous Soluble Antigen Is Presented to CD4 T Cells by CD8− Dendritic Cells, but Cross-Presented to CD8 T Cells by CD8+ Dendritic Cells
Authors: Pooley Joanne L. , Heath William R. , Shortman Ken
Published: April 2014

Mouse spleen contains three distinct mature dendritic cell (DC) populations (CD4+8−, CD4−8−, and CD4−8+) which retain a capacity to take up particulate and soluble Ags. Although the three splenic DC subtypes showed similar uptake of injected soluble OVA, they differed markedly in their capacity to present this Ag and activate proliferation in OVA-specific CD4 or CD8 T cells. For class II MHC-restricted presentation to CD4 T cells, the CD8− DC subtypes were more efficient, but for class I MHC-restricted presentation to CD8 T cells, the CD8+ DC subtype was far more effective. This differential persisted when the DC were activated with LPS. The CD8+ DC are therefore specialized for in vivo cross-presentation of exogenous soluble Ags into the class I MHC presentation pathway.
Cutting Edge: Distinct Motifs Within CD28 Regulate T Cell Proliferation and Induction of Bcl-XL
Authors: Burr John S. , Savage Nigel D. L. , Messah Grace E. , Kimzey Stephanie L. , Shaw Andrey S. , Arch Robert H. , Green Jonathan M.
Published: April 2014

CD28 provides an important costimulatory signal in T cell activation that regulates multiple cellular processes including proliferation and survival. Several signal transduction pathways are activated by CD28; however, the precise biochemical mechanism by which CD28 regulates T cell function remains controversial. Retroviral gene transfer into primary T cells from TCR-transgenic, CD28-deficient mice was used to determine the specific sequences within CD28 that determine function. Discrete regions of the cytoplasmic domain of CD28 were identified that differentially regulate T cell proliferation and induction of the anti-apoptotic protein Bcl-XL. Mutation of C-terminal proline residues abrogated the proliferative and cytokine regulatory features of CD28 costimulation while preserving Bcl-XL induction. Conversely, mutation of residues important in phosphatidylinositol 3-kinase activation partially inhibited proliferation but prevented induction of Bcl-XL. Thus the ability of CD28 to regulate proliferation and induction of Bcl-XL map to distinct motifs, suggesting independent signaling cascades modulate these biologic effects.