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Adipose-derived stem cells (ASCs) are highly attractive for cell-based therapies in tissue repair and regeneration because they have multilineage differentiation capacity and are immunosuppressive. However, the detailed epigenetic mechanisms of their immunoregulatory capacity are not fully defined. In this study, we found that Mysm1 was induced in ASCs treated with inflammatory cytokines. Adipose-derived stem cells with Mysm1 knockdown exhibited attenuated immunosuppressive capacity, evidenced by less inhibition of T cell proliferation, more pro-inflammatory factor secretion and less nitric oxide (NO) production in vitro. Mysm1-deficient ASCs exacerbated inflammatory bowel diseases but inhibited tumour growth in vivo. Mysm1-deficient ASCs also showed depressed miR-150 expression. When transduced with Mysm1 overexpression lentivirus, ASCs exhibited enhanced miR-150 expression. Furthermore, Mysm1-deficient cells transduced with lentivirus containing miR-150 mimics produced less pro-inflammatory factors and more NO. Our study reveals a new role of Mysm1 in regulating the immunomodulatory activities of ASCs by targeting miR-150. These novel insights into the mechanisms through which ASCs regulate immune reactions may lead to better clinical utility of these cells., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Spontaneous tumour regression after high-dose therapy and autologous stem cell transplantation is associated with the aplastic anaemia-like syndrome and the presence of polyclonal autoantibodies against carbonic anhydrase I (CA I). When tumour cells were grown in vitro in the presence of patients' sera positive for anti-CA I autoantibodies, their morphological pattern was altered. These changes were accompanied by modifications in the gene expression profile. We observed downregulation of genes of the basal lamina assembly (collagen type IV alpha 4, the laminin subunit gamma 2), the extracellular matrix (collagen type I alpha 1), the cytoskeleton (keratin 14 type I), the collagen triple helix repeat containing 1 and the proto-oncogene WNT7B. On the other hand, the expression of the CA 1 gene was increased in the tumour cells. It was also noticed that the presence of anti-CA I autoantibodies did not impair tumour cell proliferation and cell viability in vitro. These findings were observed only in the presence of patients' sera positive for anti-CA I autoantibodies., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Objective: CTLA-4 is a negative regulator of the immune response expressed by regulatory T (Treg) cells and activated T cells. Polymorphisms in the CTLA4 gene have been associated with autoimmune diseases, including systemic lupus erythematosus. Disease-associated polymorphisms have been shown to affect the production of the different CTLA-4 variants through an effect on alternative splicing. This study was undertaken to evaluate the role of the 1/4 CTLA-4 isoform in lupus-prone mice., Methods: We generated an MRL/lpr mouse strain that transgenically overexpresses a short isoform of CTLA-4 (1/4 CTLA-4) by backcrossing C57BL/6.1/4CTLA-4-transgenic mice to the MRL/lpr strain for 9 generations. A new antibody was generated to detect the expression of the 1/4 CTLA-4 isoform. Routine methods were used to evaluate kidney damage, humoral immunity, and cellular immunity., Results: Expression of the 1/4 CTLA-4 isoform accelerated autoimmune disease. Transgenic mice died earlier, had more severe renal disease, and had higher titers of anti-double-stranded DNA antibodies than wild-type MRL/lpr mice. The acceleration of autoimmunity and disease pathology associated with the presence of the short (1/4) isoform of CTLA-4 was linked to increased numbers of activated T cells and B cells and heightened interferon-γ production, but not to altered expression of the full-length CTLA-4 molecule or Treg cell numbers., Conclusion: Our results indicate that the presence of the alternatively spliced 1/4 CTLA-4 isoform can further promote autoimmunity and autoimmune pathology in lupus-prone mice and suggest that altered splicing of CTLA4 contributes to the expression of autoimmune disease., (Copyright © 2013 by the American College of Rheumatology.)

Objective: In vitro activation of the receptor EphB4 positively affects human osteoarthritis (OA) articular cell metabolism. However, the specific in vivo role of this ephrin receptor in OA remains unknown. We investigated in mice the in vivo effect of bone-specific EphB4 overexpression on OA pathophysiology., Methods: Morphometric, morphologic, and radiologic evaluations were performed on postnatal day 5 (P5) mice and on 10-week-old mice. Knee OA was induced surgically by destabilization of the medial meniscus (DMM) in 10-week-old male EphB4 homozygous transgenic (EphB4-Tg) and wild-type (WT) mice. Medial compartment evaluations of cartilage were performed using histology and immunohistochemistry, and evaluations of subchondral bone using histomorphometry, osteoclast staining, and micro-computed tomography., Results: There was no obvious phenotype difference in skeletal development between EphB4-Tg mice and WT mice at P5 or at 10 weeks. At 8 and 12 weeks post-DMM, the EphB4-Tg mice demonstrated significantly less cartilage alteration in the medial tibial plateau and the femoral condyle than did the WT mice. This was associated with a significant reduction of aggrecan and type II collagen degradation products, type X collagen, and collagen fibril disorganization in the operated EphB4-Tg mice. The medial tibial subchondral bone demonstrated a significant reduction in sclerosis, bone volume, trabecular thickness, and number of tartrate-resistant acid phosphatase-positive osteoclasts at both times assessed post-DMM in the EphB4-Tg mice than in the WT mice., Conclusion: This is the first in vivo evidence that bone-specific EphB4 overexpression exerts a protective effect on OA joint structural changes. The findings of this study stress the in vivo importance of subchondral bone biology in cartilage integrity., (Copyright © 2012 by the American College of Rheumatology.)

Objective: Systemic juvenile idiopathic arthritis (JIA) is an autoinflammatory syndrome in which the myelomonocytic lineage appears to play a pivotal role. Inflammatory macrophages are driven by interferon-γ (IFNγ), but studies have failed to demonstrate an IFN- induced gene signature in active systemic JIA. This study sought to characterize the status of an IFN-induced signature within affected tissue and to gauge the integrity of IFN signaling pathways within peripheral monocytes from patients with systemic JIA., Methods: Synovial tissue from 12 patients with active systemic JIA and 9 with active extended oligoarticular JIA was assessed by real-time polymerase chain reaction to quantify IFN-induced chemokine gene expression. Peripheral monocytes from 3 patients with inactive systemic JIA receiving anti-interleukin-1β (anti-IL-1β) therapy, 5 patients with active systemic JIA, and 8 healthy controls were incubated with or without IFNγ to gauge changes in gene expression and to measure phosphorylated STAT-1 (pSTAT-1) levels., Results: IFN-induced chemokine gene expression in synovium was constrained in active systemic JIA compared to the known IFN-mediated extended oligoarticular subtype. In unstimulated peripheral monocytes, IFN-induced gene expression was similar between the groups, except that lower levels of STAT1, MIG, and PIAS were observed in patients with active disease, while higher levels of PIAS1 were observed in patients with inactive disease. Basal pSTAT-1 levels in monocytes tended to be higher in systemic JIA patients compared to healthy controls, with the highest levels seen in those with inactive disease. Upon stimulation of monocytes, the fold increase in gene expression was roughly equal between groups, except for a greater increase in STAT1 in patients with inactive systemic JIA compared to controls, and a greater increase in IRF1 in those with active compared to inactive disease. Upon stimulation, the fold increase in pSTAT-1 was highest in monocytes from patients with inactive systemic JIA., Conclusion: Monocytes in patients with active systemic JIA retain the ability to respond to IFNγ, suggesting that the lack of an IFN-induced gene signature in patients with active disease reflects a limited in vivo exposure to IFNγ. In patients with inactive systemic JIA who received treatment with anti-IL-1β, hyperresponsiveness to IFNγ was observed., (Copyright © 2012 by the American College of Rheumatology.)

The thymus is the most rapidly aging tissue in the body, with progressive atrophy beginning as early as birth and not later than adolescence. Latent regenerative potential exists in the atrophic thymus, because certain stimuli can induce quantitative regrowth, but qualitative function of T lymphocytes produced by the regenerated organ has not been fully assessed. Using a genome-wide computational approach, we show that accelerated thymic aging is primarily a function of stromal cells, and that while overall cellularity of the thymus can be restored, many other aspects of thymic function cannot. Medullary islet complexity and tissue-restricted antigen expression decrease with age, representing potential mechanisms for age-related increases in autoimmune disease, but neither of these is restored by induced regrowth, suggesting that new T cells produced by the regrown thymus will probably include more autoreactive cells. Global analysis of stromal gene expression profiles implicates widespread changes in Wnt signaling as the most significant hallmark of degeneration, changes that once again persist even at peak regrowth. Consistent with the permanent nature of age-related molecular changes in stromal cells, induced thymic regrowth is not durable, with the regrown organ returning to an atrophic state within 2 weeks of reaching peak size. Our findings indicate that while quantitative regrowth of the thymus is achievable, the changes associated with aging persist, including potential negative implications for autoimmunity., (© 2011 The Authors. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

Objective: Rituximab displays therapeutic benefits in the treatment of patients with rheumatoid arthritis (RA) resistant to tumor necrosis factor (TNF) blockade. However, the precise role of B cells in the pathogenesis of RA is still unknown. We undertook this study to investigate the global molecular effects of rituximab in synovial biopsy samples obtained from anti-TNF-resistant RA patients before and after administration of the drug., Methods: Paired synovial biopsy samples were obtained from the affected knee of anti-TNF-resistant RA patients before (time 0) and 12 weeks after (time 12) initiation of rituximab therapy. Total RNA was extracted, labeled according to standard Affymetrix procedures, and hybridized on GeneChip HGU133 Plus 2.0 slides. Immunohistochemistry and quantitative real-time reverse transcriptase-polymerase chain reaction experiments were performed to confirm the differential expression of selected transcripts., Results: According to Student's paired t-tests, 549 of 54,675 investigated probe sets were differentially expressed between time 0 and time 12. Pathway analysis revealed that genes down-regulated between time 0 and time 12 were significantly enriched in immunoglobulin genes and genes involved in chemotaxis, leukocyte activation, and immune responses (Gene Ontology annotations). In contrast, genes up-regulated between time 0 and time 12 were significantly enriched in transcripts involved in cell development (Gene Ontology annotation) and wound healing (Gene Set Enrichment Analysis). At baseline, higher synovial expression of immunoglobulin genes was associated with response to therapy., Conclusion: Rituximab displays unique effects on global gene expression profiles in the synovial tissue of RA patients. These observations open new perspectives in the understanding of the biologic effects of the drug and in the selection of patients likely to benefit from this therapy., (Copyright © 2011 by the American College of Rheumatology.)

Background: Clobetasol propionate shampoo is effective and safe in treatment of scalp psoriasis (SP). Gene expression profiling of psoriatic skin biopsies led to the identification of numerous disease-related genes. However, it remained unknown whether the gene expression profile of hair follicles of SP patients was also affected., Objectives: To determine whether psoriasis-related genes are differentially regulated in the hair follicles of SP patients and whether the modulation of these genes can be correlated with clinical severity scores., Methods: A single arm, open study was conducted in three centres. SP patients received daily treatment with clobetasol propionate shampoo. At Baseline, Weeks 2 and 4, investigators assessed clinical severity parameters and collected scalp hair follicles in anagen phase. Total RNA extracted from hair follicles was used to determine the expression level of 44 genes, which were reported previously to be upregulated in the skin of psoriasis patients., Results: RNA of good quality and sufficient quantity was obtained from hair follicles of psoriasis patients and healthy volunteers (HV). The expression level of 10 inflammation-related genes was significantly increased in psoriatic hair follicles. The patient's exploratory transcriptomic score, defined as the mean fold modulation of these 10 genes compared with HV, correlated with clinical severity scores. Clobetasol propionate shampoo was effective in decreasing both the exploratory transcriptomics and the clinical severity scores., Conclusion: Hair follicles of SP patients are affected by the inflammatory process. The change in the expression level of inflammation-related genes correlates with the severity of the disease., (© 2010 Galderma R&D. Journal of the European Academy of Dermatology and Venereology © 2010 European Academy of Dermatology and Venereology.)

Objective: To quantify the expression of CD44 and variant isoforms CD44v3 and CD44v6 on T cells from patients with systemic lupus erythematosus (SLE), and to assess correlations of the level of expression of these molecules with disease manifestations., Methods: Information on clinical and demographic characteristics was collected, and blood samples were obtained from 72 patients with SLE and 32 healthy control subjects matched to the patients by sex, race, and age. Expression of CD44 and variants CD44v3 and v6 on T cell subsets was determined by flow cytometry, and Pearson's correlations of their expression levels with clinical variables, SLE Disease Activity Index (SLEDAI) scores, and presence of lupus nephritis were determined. Wilcoxon's rank sum tests and conditional multivariable regression analyses were applied to identify differences in the expression of CD44 between patients with SLE and healthy controls., Results: Expression of CD44 was higher on CD4+ and CD8+ T cells from SLE patients compared with controls (P

Objective: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) plays a role in blastocyst implantation and is down-regulated in preeclampsia and in hypertensive pregnancy disorders associated with defective extravillous trophoblast invasion. Defective placentation and severe preeclampsia are also features of the antiphospholipid syndrome (APS). The purpose of this study was to investigate whether abnormal HB-EGF expression plays a pathogenic role in antiphospholipid antibody (aPL)-mediated defective placentation., Methods: HB-EGF expression in placental tissue was evaluated by Western blotting and messenger RNA analysis in normal and APS placentae. Polyclonal IgG fractions or monoclonal beta(2)-glycoprotein I-dependent aPL and their respective controls were investigated for the following 4 features: their binding to human trophoblast monolayers, as determined by cell enzyme-linked immunosorbent assay (ELISA); their effect on HB-EGF expression by Western blotting in trophoblast cell extracts as well as by ELISA as a protein secreted in the culture supernatants; their inhibitory effect on in vitro trophoblast invasiveness, as evaluated by Matrigel assay; and their inhibitory effect on matrix metalloproteinase (MMP) levels, as measured by gelatin zymography. Experiments were also performed in the presence of serial concentrations of heparin or recombinant HB-EGF., Results: Placental APS tissue displayed reduced expression of HB-EGF. Polyclonal and monoclonal aPL bound to trophoblast monolayers and significantly reduced the in vitro synthesis and secretion of HB-EGF. Heparin inhibited aPL binding and restored HB-EGF expression in a dose-dependent manner. Addition of recombinant HB-EGF reduced the in vitro aPL-induced inhibition of Matrigel invasiveness as well as MMP-2 levels., Conclusion: These preliminary findings suggest that the reduction of aPL-mediated HB-EGF represents an additional mechanism that is responsible for the defective placentation associated with APS and that heparin protects from aPL-induced damage by inhibiting antibody binding.

Objective: Patients with psoriasis and psoriatic arthritis respond well to tumor necrosis factor alpha (TNFalpha) blockers in general; however, there is now mounting evidence that a small cohort of patients with rheumatoid arthritis who receive TNFalpha blockers develop psoriasis. This study was undertaken to explore the mechanisms underlying TNFalpha blockade-induced exacerbation of skin inflammation in murine psoriasis-like skin disease., Methods: Skin inflammation was induced in BALB/c scid/scid mice after they received CD4+CD45RB(high)CD25- (naive CD4) T cells from donor mice. These mice were treated with either anti-interleukin-12 (anti-IL-12)/23p40 antibody or murine TNFRII-Fc fusion protein and were examined for signs of disease, including histologic features, various cytokine levels in the serum, and cytokine or FoxP3 transcripts in the affected skin and draining lymph node (LN) cells. In a separate study, naive CD4+ T cells were differentiated into Th1 or Th17 lineages with anti-CD3/28 magnetic beads and appropriate cytokines in the presence or absence of TNFalpha. Cytokine gene expression from these differentiated cells was also determined., Results: Neutralization of TNFalpha exacerbated skin inflammation and markedly enhanced the expression of the proinflammatory cytokines IL-1beta, IL-6, IL-17, IL-21, and IL-22 but suppressed FoxP3 expression in the skin and reduced the number of FoxP3-positive Treg cells in the draining LNs. TNFalpha also demonstrated a divergent role during priming and reactivation of naive T cells., Conclusion: These results reveal a novel immunoregulatory role of TNFalpha on Th17 and Treg cells in some individuals, which may account for the exacerbation of skin inflammation in some patients who receive anti-TNF treatments.

Objective: Subclinical gut inflammation is common in spondylarthritis, but the immunologic abnormalities underlying this process are undefined. Perturbation of the interleukin-23 (IL-23)/Th17 axis has emerged as a fundamental trigger of chronic inflammation. This study was undertaken to investigate the expression and tissue distribution of IL-23/Th17-related molecules in Crohn's disease (CD) and in subclinical gut inflammation in ankylosing spondylitis (AS)., Methods: Quantitative gene expression analysis of Th1/Th2 and IL-23/Th17 responses was performed in intestinal biopsy samples obtained from 12 patients with CD, 15 patients with AS, and 13 controls. IL-23 tissue distribution and identification of IL-23-producing cells were evaluated by immunohistochemistry., Results: We demonstrated a strong and significant up-regulation of IL-23p19 transcripts in the terminal ileum in patients with AS and patients with CD. IL-23 was abundantly produced by infiltrating monocyte-like cells in inflamed mucosa from AS and CD patients. Notably, we also identified Paneth cells as a major source of IL-23 in patients with AS, patients with CD, and normal controls. Unlike CD, in AS patients, IL-23 was not associated with up-regulation of IL-17 and the IL-17-inducing cytokines IL-6 and IL-1beta. Finally, while the Th1-related cytokines interferon-gamma, IL-12p35, and IL-27p28 were overexpressed only in CD patients, IL-4, IL-5, and STAT-6 were also significantly increased in AS patients., Conclusion: Our findings indicate that overexpression of IL-23, but not IL-17, is a pivotal feature of subclinical gut inflammation in AS. Identification of resident Paneth cells as a pivotal source of IL-23 in physiologic and pathologic conditions strongly suggests that IL-23 is a master regulator of gut mucosal immunity, providing a pathophysiologic significance to the reported association between IL-23 receptor polymorphisms and intestinal inflammation.

Objective: Members of the suppressor of cytokine signaling (SOCS) family are key negative intracellular regulators of cytokine and growth factor responses, including those that regulate immune responses in autoimmune disorders, such as rheumatoid arthritis (RA). The aim of this study was to investigate modulation of T cell immunity for the treatment of experimental arthritis, via enhanced expression of SOCS-3 in splenic antigen-presenting cells (APCs) obtained after intravenous injection of adenovirus encoding SOCS-3., Methods: DBA/1 mice were immunized with type II collagen, and adenovirus vectors were administered by intravenous injection before the clinical onset of collagen-induced arthritis (CIA). Splenic cellular responses were analyzed by measuring cytokine production, using Luminex multi-analyte technology. Th cell populations were analyzed by flow cytometry., Results: Systemic delivery of adenovirus encoding SOCS-3 resulted in enhanced transgene expression in splenic APCs, which led to decreased production of interleukin-23 (IL-23), IL-6, and tumor necrosis factor alpha, but significantly higher production of antiinflammatory IL-10, by these cells. Fluorescence-activated cell sorting analysis showed increased numbers of splenic CD4+ T cells after SOCS-3 treatment. In the presence of SOCS-3-transduced APCs, however, purified splenic CD3+ T cells showed reduced antigen-specific proliferation and a significant reduction in the production of interferon-gamma (-43%), IL-4 (-41%), and IL-17 (-70%). Interestingly, the altered splenic cellular responses were accompanied by a protective effect on CIA development, and histologic analysis of knee joints showed reduced joint inflammation and connective tissue destruction., Conclusion: This study demonstrates effective prevention of CIA after intravenously induced overexpression of SOCS-3; this is probably caused by the generation of tolerogenic APCs, which have an inhibitory effect on Th1, Th2, and especially, Th17 cell activity.

Objective: CD4+CD25+FoxP3+ regulatory T (Treg) cells are critical regulators of autoimmunity. Yet the number of Treg cells is paradoxically increased in rheumatoid arthritis (RA) patients, and Treg cells show variable activity in human studies. The objective of this study was to characterize the expansion and function of Treg cells during the initiation and progression of experimental arthritis., Methods: To unequivocally identify Treg cells, we crossed FoxP3gfp mice with K/BxN mice to generate arthritic mice in which Treg cells express green fluorescence protein. We examined the expansion and function of Treg cells and effector T (Teff) cells during different stages of arthritis, using flow cytometry and cell proliferation analyses., Results: In K/BxN mice, thymic selection of KRN T cells resulted in an enrichment of forkhead box P3 (FoxP3)-positive Treg cells. Treg cell numbers increased during arthritis, with significant increases in spleens and draining lymph nodes, indicating selective tropism to sites of disease. In contrast to the in vitro unresponsiveness of Treg cells when cultured alone, substantial proportions of Treg cells proliferated in both nonarthritic and arthritic mice. However, they also underwent greater apoptosis, thereby maintaining equilibrium with Teff cells. Similarly, enhanced Treg cell-suppressive activity during arthritis was offset by greater resistance by their Teff cell counterparts and antigen-presenting cells., Conclusion: In this well-established model of RA, the interplay of Teff cells and Treg cells in K/BxN mice recapitulated many features of the human disease. We demonstrated an ordered expansion of Treg cells during arthritis and dynamic changes in Treg cell and Teff cell functions. By elucidating factors that govern Treg cell and Teff cell development in K/BxN(gfp) mice, we will gain insight into the pathophysiology of and develop novel therapeutics for human RA.

Associations between the AC150887.4:c.-1768T>A SNP (rs41255711), which is located in the 5' upstream region of the IL8RA gene (also known as CXCR1), and the estimated breeding values for somatic cell score in the first (P = 0.019) and second (P = 0.035) lactations were previously reported in a population of Canadian Holstein bulls. In the present study, we evaluated the impact of this SNP on the expression of IL8RA by qRT-PCR. Neutrophils were isolated from whole blood samples from a group of cows with genotypes c.-1768AA (n = 4), c.-1768AT (n = 5) and c.-1768TT (n = 5) after the cows had been challenged in vitro with lipopolysaccharide (LPS). This study demonstrated that LPS-induced expression of IL8RA in cows with the c.-1768AA genotype was significantly greater when compared with the c.-1768AT and c.-1768TT genotypes (P < 0.05) before as well as after in vitro LPS challenge.

Objective: We previously reported that the Val58Ile polymorphism of the leukocyte cell-derived chemotaxin 2 gene (LECT2) is associated with the severity of rheumatoid arthritis (RA). To define the role of LECT2 in inflammatory arthritides, we investigated the development of collagen antibody-induced arthritis (CAIA) in LECT2-deficient (LECT2(-/-)) mice., Methods: CAIA was induced in mice by administering anti-type II collagen antibodies followed by lipopolysaccharide. Daily assessment of hind paw swelling was used to monitor the development of arthritis. The histopathologic features and expression of inflammatory cytokines were also analyzed. We confirmed the role of LECT2 by introducing a LECT2 expression vector into LECT2(-/-) mice, using a hydrodynamic gene transfer method., Results: Arthritis in LECT2(-/-) mice was significantly exacerbated compared with that in wild-type (WT) controls. Histopathologic assessment of the tarsal joints showed that inflammation and erosion of cartilage and bone in LECT2(-/-) mice were more severe than that in controls. Interleukin-1beta (IL-1beta), IL-6, and certain chemokines were present at significantly higher levels in the arthritic hind paws of LECT2(-/-) mice. In contrast, the amount of LECT2 in the serum and locally in the hind paws was higher in arthritic WT mice. Finally, hydrodynamic gene transfer experiments revealed that the severity of arthritis was reduced by the systemic expression of exogenous mouse LECT2 protein in LECT2(-/-) mice., Conclusion: These results strongly suggest that LECT2 directly suppresses the development of CAIA. Manipulation of LECT2 might provide a rationale for novel therapeutic approaches to the treatment of inflammatory arthritides such as RA.

Objective: To define the roles of transforming growth factor alpha (TGFalpha) in cartilage degradation., Methods: Primary rat articular chondrocytes and articular osteochondral explants were cultured with TGFalpha to assess the effects of TGFalpha on chondrocyte physiology and phenotype., Results: TGFalpha altered chondrocyte morphology through reorganization of the actin cytoskeleton and formation of stress fibers. Expression of anabolic genes, including aggrecan, type II collagen, and cartilage link protein, was reduced in response to TGFalpha. Proliferation of chondrocytes and formation of articular chondrocyte clusters was stimulated by TGFalpha. Expression of matrix metalloproteinase 13 and cathepsin C was increased by TGFalpha. We demonstrated the down-regulation of Sox9 messenger RNA and protein levels by TGFalpha. This was associated with reduced levels of phosphorylated and total SOX9 in cartilage explants upon TGFalpha treatment. In contrast, another growth factor identified in our microarrays, Kitl, had no effects on the chondrocyte parameters tested. To examine correlations between the increased levels of TGFalpha in experimental knee osteoarthritis (OA) with the levels of TGFalpha in humans with knee OA, a microarray analysis of mRNA from 13 normal and 12 late-stage OA cartilage samples was performed. Seven OA samples showed TGFA mRNA levels similar to those in the normal controls, but expression was markedly increased in the other 5 OA samples. These data confirm that TGFA transcript levels are increased in a subset of patients with OA., Conclusion: This study adds TGFalpha to the list of dysregulated cytokines present in degrading cartilage in OA. Since TGFalpha inhibits articular chondrocyte anabolic capacity, increases catabolic factors, and contributes to the development of chondrocyte clusters, TGFalpha may be a potential target for therapeutic strategies in the treatment of OA.

Objective: Systemic lupus erythematosus (SLE) is diagnosed according to a spectrum of clinical manifestations and autoantibodies associated with abnormal expression of type I interferon (IFN-I)-stimulated genes (ISGs). The role of IFN-I in the pathogenesis of SLE remains uncertain, partly due to the lack of suitable animal models. The objective of this study was to examine the role of IFN-I signaling in the pathogenesis of murine lupus induced by 2,6,10,14-tetramethylpentadecane (TMPD)., Methods: IFN-I receptor-deficient (IFNAR(-/-)) 129Sv mice and wild-type (WT) 129Sv control mice were treated intraperitoneally with TMPD. The expression of ISGs was measured by real-time polymerase chain reaction. Autoantibody production was evaluated by immunofluorescence and enzyme-linked immunosorbent assay. Proteinuria and renal glomerular cellularity were measured and renal immune complexes were examined by immunofluorescence., Results: Increased ISG expression was observed in the peripheral blood of TMPD-treated WT mice, but not in the peripheral blood of TMPD-treated IFNAR(-/-) mice. TMPD did not induce lupus-specific autoantibodies (anti-RNP, anti-Sm, anti-double-stranded DNA) in IFNAR(-/-) mice, whereas 129Sv controls developed these specificities. Although glomerular immune complexes were present in IFNAR(-/-) mice, proteinuria and glomerular hypercellularity did not develop, whereas these features of glomerulonephritis were found in the TMPD-treated WT controls. The clinical and serologic manifestations observed in TMPD-treated mice were strongly dependent on IFNAR signaling, which is consistent with the association of increased expression of ISGs with lupus-specific autoantibodies and nephritis in humans., Conclusion: Similar to its proposed role in human SLE, signaling via the IFNAR is central to the pathogenesis of autoantibodies and glomerulonephritis in TMPD-induced lupus. This lupus model is the first animal model shown to recapitulate the "interferon signature" in peripheral blood.

Background: Recent studies point to the pathophysiological role of the peroxisome proliferators-activated receptor gamma (PPARgamma) in the inflammatory immune response. We have showed that activation of PPARgamma by specific ligands attenuates the allergic immune response via monocytes and lymphocytes. The objective of this study was to analyse the PPARgamma expression and its regulation via inflammatory cytokines., Methods: We examined the PPARgamma expression in the lesional and nonlesional skin of atopic patients by immunohistochemistry. The expression patterns of PPARgamma mRNA and its isoforms were investigated in peripheral mononuclear blood cells of atopic and nonatopic donors and in cytokine-stimulated populations by quantitative real-time RT-PCR., Results: Our data show an increased PPARgamma expression in lesional skin from atopic dermatitis patients. The analysis of PPARgamma mRNA reveals a significantly up-regulated expression of PPARgamma1 but not of PPARgamma2 in monocytes and CD4(+) T-cells from atopic dermatitis patients. Furthermore, we demonstrate that Th-cytokines, like IL-4, IL-13 and IFNgamma, which regulate the biphasic atopic immune response, directly regulate the expression of PPARgamma1., Conclusion: Taken together, these data demonstrate that PPARgamma isoforms are differently expressed and regulated by the local cytokine-milieu. Whether the increased expression of the PPARgamma1 receptor may be beneficial or not for a PPARgamma ligand-based treatment of atopic dermatitis, is currently under investigation.

Background: Signalling lymphocytic activation molecule (SLAM) and interleukin (IL)-18 induce interferon (IFN)-gamma production from Th1 cells. The allergen-induced SLAM and IL-18 mRNA expressions are increased during subcutaneous immunotherapy (SCIT), but nothing is known about their role during sublingual immunotherapy (SLIT). Transcription factor GATA-3 is associated with Th2 cells but its role in SCIT and SLIT is yet unexplored. This study was undertaken to analyse the allergen induced in vitro mRNA expression of IL-18, SLAM and GATA-3 in peripheral blood mononuclear cells (PBMC) of children with allergic rhinitis (AR) during SLIT., Methods: Ten patients with AR undergoing pollen SLIT with a weekly dose of 200,000 SQ-U, 10 with 24,000 SQ-U of mixture of Betula verrucosa, Corylus avellana and Alnus glutinosa and 10 with placebo were included. Peripheral blood mononuclear cell were stimulated with birch extract prior to, after 1 and 2 years of the treatment. The mRNA expression was assessed using kinetic real-time RT-PCR (TaqMan); Applied Biosystems, Foster City, CA, USA)., Results: The expression of IL-18 mRNA was increased in the high-dose group in comparison to the placebo group after 1 year of therapy (P = 0.028) and had an inverse correlation with the late phase skin reaction after the second study year (r = -0.41, P = 0.041). SLAM mRNA expression increased in the high-dose group from baseline to 1 year (P = 0.028) and correlated with IL-10 (r = 0.96, P < 0.0001) and transforming growth factor-beta (r = 0.80, P = 0.0037) mRNA expression. No significant changes were seen in GATA-3 mRNA expression., Conclusions: During SLIT, IL-18 and SLAM are upregulated, suggesting that the Th2 type inflammatory response is downregulated during SLIT by increased Th1 type response.

Objective: IK cytokine has been isolated as a factor that inhibits interferon-gamma (IFNgamma)-induced expression of class II major histocompatibility complex (MHC) antigens. Aberrant expression of class II MHC antigens has reportedly been recognized in the target organs of autoimmune diseases and been associated with disease activity. In this study, we investigated whether IK cytokine can ameliorate the progression of lupus nephritis in MRL/lpr mice., Methods: A truncated IK analog was prepared and transfected into a nonmetastatic fibroblastoid cell line, and then injected subcutaneously into MRL/lpr mice at ages 8 weeks (before the onset of lupus nephritis) and 12 weeks (at the early stage of the disease)., Results: An IK cytokine, when it was translated from methionine at position 316, acted as a secretory protein. This truncated IK cytokine (tIK) reduced IFNgamma-induced class II MHC expression in various cells through decreased expression of class II MHC transcription activator. Treatment of MRL/lpr mice with tIK significantly reduced renal damage as compared with control mice. A significant decrease in macrophage and T cell infiltration was found in the kidneys of tIK-treated mice, resulting in decreased production of IFNgamma and interleukin-2. Mice treated with tIK also showed significant reduction of anti-DNA antibodies and circulating immune complexes. A specific reduction of class II MHC expression was observed on B cells and monocytes as well as in the kidney., Conclusion: We prepared a potent IK analog and demonstrated its ability to ameliorate the progression of lupus nephritis. This agent may therefore provide a new therapeutic approach for lupus nephritis.

In the present study, we investigated the expression of Mcl-1 and Bcl-2 by immunohistochemistry in 85 patients of grades 1-3 and transformed follicular lymphoma (FL). In lymphoma tissue, centroblasts uniformly expressed high levels of Mcl-1 (Mcl-1(hi)) whereas centrocytes demonstrated low Mcl-1 expression (Mcl-1(lo)). Bcl-2 expression in centroblasts/centrocytes was reciprocal to Mcl-1 staining in most cases. A high number of Mcl-1(hi) centroblasts in tissue sections (> or =200/high-power field) correlated with poor overall survival (P < 0.001), independent of the International Prognostic Index and FL grade. This suggests that the number of centroblasts with strong Mcl-1 staining is associated with clinical outcome in FL patients.

Objective: To investigate whether intraarticular (IA) glucocorticoid (GC) therapy diminishes synovial cell infiltration, vascularity, expression of proinflammatory cytokines, and adhesion molecule levels in patients with chronic arthritides., Methods: Thirty-one patients with chronic arthritides received a single IA injection of triamcinolone hexacetonide to treat active large-joint inflammation. Synovial biopsy specimens were obtained with arthroscopic guidance before and 9-15 days after injection. The presence of T lymphocytes, macrophages, intercellular adhesion molecule 1 (ICAM-1), vascular endothelial growth factor (VEGF), the pan-endothelial marker CD31, and the proinflammatory cytokines interleukin-1alpha (IL-1alpha), IL-1beta, tumor necrosis factor (TNF), and high mobility group box chromosomal protein 1 (HMGB-1) was studied by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction., Results: IA GC treatment resulted in good clinical response in 29 of 31 joints. After therapeutic intervention, the number of synovial T lymphocytes declined, whereas the number of macrophages remained unchanged. Overall synovial protein expression of TNF, IL-1beta, extranuclear HMGB-1, VEGF, and ICAM-1 was reduced at followup tissue sampling, while no significant effects were observed regarding vascularity. In contrast, expression of IL-1alpha, VEGF, and cytoplasmic HMGB-1 protein in vascular endothelial cells was not affected. GC therapy down-regulated levels of messenger RNA encoding IL-1alpha and IL-1beta, but not TNF or HMGB-1., Conclusion: Synovial cell infiltration and proinflammatory cytokine expression were affected in a multifaceted manner by IA GC treatment. Marked reduction of synovial T lymphocytes, TNF, IL-1beta, extranuclear HMGB-1, ICAM-1, and VEGF occurred in association with beneficial clinical effects. Unexpectedly, macrophage infiltration and proinflammatory endothelial cytokine expression remained unchanged. These findings may reflect mechanisms controlling the transiency of clinical improvement frequently observed after IA GC injection.

Objective: To investigate the relationship between the vasculopathy of juvenile dermatomyositis (juvenile DM) and the balance between the angiostatic ELR- and angiogenic ELR+ CXC chemokines in the muscle of patients with the disease., Methods: The expression of 3 ELR- CXC chemokines (interferon-inducible protein 10 [IP-10], monokine induced by interferon-gamma, and interferon-inducible T cell alpha-chemoattractant) and 2 ELR+ CXC chemokines was quantitated in muscle biopsy samples from 7 patients with juvenile DM and 7 healthy children, by real-time polymerase chain reaction. The findings were correlated with various histopathologic features, with particular emphasis on the degree of vasculopathy. Synovial biopsy specimens from patients with juvenile rheumatoid arthritis (JRA) were used for additional comparison., Results: The angiostatic ELR- chemokines were expressed at high levels, while the angiogenic ELR+ chemokines were barely detectable, in most juvenile DM samples. This contrasted sharply with the findings in both normal muscle biopsy specimens and JRA synovial tissue specimens. The expression of the ELR- chemokines in juvenile DM samples correlated with the intensity of mononuclear cell infiltration. Furthermore, the juvenile DM samples with the highest degree of capillary loss had the highest levels of ELR- CXC chemokines. The presence of IP-10 in juvenile DM muscle specimens was confirmed by immunohistochemistry analysis. In addition, immunohistochemical staining of muscle tissue revealed that CXCR3, a receptor utilized by ELR- CXC chemokines, was expressed in vascular endothelial cells., Conclusion: Increased expression of the interferon-induced angiostatic ELR- CXC chemokines is a feature of juvenile DM that parallels the degree of vasculopathy in patients with the disease. Collectively, these findings are consistent with a model in which a subset of inflammatory cells secrete angiostatic ligands, which then contribute to a local atrophying effect on the muscle's vasculature via a receptor-mediated process.

Background: Macrophage migration inhibitory factor (MIF) has emerged to be a pivotal cytokine in immune-mediated diseases., Patients and Methods: To investigate the role of MIF in chronic hepatitis B infection, we studied two groups of hepatitis B surface antigen positive patients: group 1 (immune tolerant, n = 16) and group 2 (immune clearance, n = 16). Serum level of MIF was measured by enzyme-linked immunosorbent assay and intrahepatic expression of MIF, macrophage and T-cell localisation were detected by double immunohistochemistry., Results: An increased serum MIF correlated significantly with increased serum alanine aminotransferase activity (r = 0.73, P < 0.001) and the severity of necroinflammatory injury (r = 0.642, P < 0.001). In group 2, there was marked MIF mRNA expression in all KP-1+ macrophages and CD45RO+ activated T cells and, to a lesser extent, in hepatocytes within inflammatory areas. In contrast to its mRNA expression, the cytoplasmic MIF protein level in hepatocytes, infiltrating macrophages and T cells within the inflammatory area was reduced, which probably contributed to the increased serum MIF level., Conclusions: Our data suggested that MIF played a role in sustaining cell-mediated hepatic injury during the immune-clearance phase of chronic hepatitis B infection.

Induction of intranasal tolerance prevents the body from eliciting unwanted immune responses against harmless antigens that enter the body through the nasal mucosa. To study the intrinsic capacities of the cervical, nose draining lymph nodes (CLN), which are essential for tolerance induction, genes that are differentially expressed in CLN and not in peripheral lymph nodes (PLN) were characterized. The gene that is predominantly overexpressed in CLN codes for IgG2b. This is confirmed by a higher percentage of IgG2b+ B220+ cells in CLN compared with any PLN. However, this predominance of IgG2b-positive B cells in the CLN is not specific for the lymph node itself but rather determined by the region drained by lymph nodes at the cervical site, as transplanted PLN at these locations show a comparable predominance. It was demonstrated that IgG2b, when compared with IgG1, led to differential activation of dendritic cells (DC) through Fc receptor signalling. The results point to a unique local combination of cells and factors in the nose draining CLN leading to highly specialized immune reactivity. The results point out that predominance of a distinct IgG isotype in a lymphoid environment may lead to highly specialized immune reactivity., (Copyright 2004 Blackwell Munksgaard)

Objective: Macrophages release cytokines, such as tumor necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), and IL-6, which modulate the symptoms of rheumatoid arthritis (RA). Macrophage release of these cytokines can be modulated by estrogen. Fc gamma receptor type IIIA (CD16a) is a receptor expressed on macrophages that selectively binds IgG molecules, an important rheumatoid factor in RA. Binding of CD16 by anti-CD16 monoclonal antibodies stimulates macrophage cytokine release. We undertook this study to test the hypothesis that decreased concentrations of estrogen (17 beta-estradiol) directly cause an increase in CD16 expression, resulting in increased release of proinflammatory cytokines from monocytes and/or macrophages upon receptor binding., Methods: THP-1 cells and female human primary monocytes and monocyte-derived macrophages were treated with no 17 beta-estradiol, physiologic levels (1 x 10(-8)M) of 17 beta-estradiol, or 1 x 10(-8)M 17 beta-estradiol followed by withdrawal of 17 beta-estradiol. Surface expression of CD16 and CD16 messenger RNA was measured using fluorescence-activated cell sorting (FACS) and semiquantitative reverse transcription-polymerase chain reaction, respectively. Cytokine release from 17 beta-estradiol-treated or untreated monocytes was then quantitated by enzyme-linked immunosorbent assay and FACS after crosslinking the receptor with anti-CD16 antibodies., Results: CD16 transcript significantly increased in macrophage-like THP-1 cells and in primary, peripheral blood macrophages in the absence of 17 beta-estradiol, and the observed increase in message was dependent on transcription. CD16 receptor levels on CD14+, transforming growth factor beta-treated primary monocytes also increased in cells deprived of 17 beta-estradiol. Analysis of the cytokines released showed that CD16 crosslinking stimulated significant increases in TNF alpha, IL-1 beta, and IL-6 due to the absence of estrogen., Conclusion: Estrogen can modulate proinflammatory cytokine release from activated monocytes and/or macrophages, in part through modulation of CD16 expression.

Objective: Generalized DNA hypomethylation contributes to altered T cell function and gene expression in systemic lupus erythematosus (SLE). Some of the overexpressed genes participate in the disease process, but the full repertoire of genes affected is unknown. Methylation-sensitive T cell genes were identified by treating T cells with the DNA methyltransferase inhibitor 5-azacytidine and comparing gene expression with oligonucleotide arrays. CD70, a costimulatory ligand for B cell CD27, was one gene that reproducibly increased. We then determined whether CD70 is overexpressed on T cells treated with other DNA methylation inhibitors and on SLE T cells, and determined its functional significance., Methods: Oligonucleotide arrays, real-time reverse transcription-polymerase chain reaction, and flow cytometry were used to compare CD70 expression in T cells treated with 2 DNA methyltransferase inhibitors (5-azacytidine and procainamide) and 3 ERK pathway inhibitors known to decrease DNA methyltransferase expression (U0126, PD98059, and hydralazine). The consequences of CD70 overexpression were tested by coculture of autologous T and B cells with and without anti-CD70 and measuring IgG production by enzyme-linked immunosorbent assay. The results were compared with those of T cells from lupus patients., Results: SLE T cells and T cells treated with DNA methylation inhibitors overexpressed CD70 and overstimulated B cell IgG production. The increase in IgG synthesis was abrogated by anti-CD70., Conclusion: SLE T cells and T cells treated with DNA methyltransferase inhibitors and ERK pathway inhibitors overexpress CD70. This increased B cell costimulation and subsequent immunoglobulin overproduction may contribute to drug-induced and idiopathic lupus.

Objective: To determine the T cell receptor (TCR) structure recognizing type II collagen (CII) in HLA-DR-transgenic mice, and to examine the role of T cells with certain V(beta)-chains in collagen-induced arthritis (CIA)., Methods: T cell hybridomas were established from DR1- and DR4-transgenic mice and selected for their responses to CII and CII peptide containing the T cell determinants. RNA was extracted and reverse transcribed into complementary DNA, which was then amplified using appropriate V(beta)- and V(alpha)-subfamily-specific primers. The polymerase chain reaction products were purified and directly sequenced. To determine the role of T cells with certain V(beta)-chains in CIA, V(beta)-subfamily-specific antibodies were administered and the development and characteristics of arthritis were determined., Results: TCRs of 23 clonally distinct T cell hybridomas that were derived from DR1-transgenic mice and that were reactive to the CII peptide containing the immunodominant determinant were analyzed. These hybridomas predominantly used the TCR V(beta)14 and V(beta)8 gene segments (70% and 30%, respectively). The same restriction in V(beta) usage was also found in CII-reactive T cell hybridomas from DR4-transgenic mice. There was also restricted use of V(alpha) genes, although this was less marked than that of V(beta). In contrast, the hybridomas expressed a diverse third complementarity-determining region. Deletion of both V(beta)14-bearing and V(beta)8-bearing T cells significantly reduced the incidence and severity of CIA., Conclusion: These data demonstrate that DR1 and DR4 not only bind and present the same CII immunodominant peptide, but also stimulate a highly restricted subset of T cells.

Objective: Interleukin-8 (IL-8) plays an important role in the migration of inflammatory cells into the synovium and joint fluids in rheumatoid arthritis (RA). This study was undertaken to investigate the IL-8 inductive activity of the macrophage migration inhibitory factor (MIF) in RA synovial fibroblasts. The regulatory mechanism of IL-8 was compared with that of IL-1beta., Methods: MIF-induced IL-8 and IL-1beta transcriptional activation was studied in RA synovial fibroblasts by Northern blot analysis, enzyme-linked immunosorbent assay, and electromobility shift assay. The effect of anti-MIF antibody administration on murine passive collagen-induced arthritis (CIA) was also evaluated by histologic examination and reverse transcriptase-polymerase chain reaction., Results: MIF up-regulated the IL-8 messenger RNA (mRNA) and protein levels in a dose-dependent manner. The IL-8 mRNA up-regulation started 1 hour poststimulation by MIF, and reached a maximum level at 6 hours. IL-1beta mRNA was also up-regulated by MIF. The mRNA up-regulation of IL-8 and IL-1beta by MIF was inhibited by 2 tyrosine kinase inhibitors, a protein kinase C (PKC) inhibitor, an activator protein 1 (AP-1) inhibitor, and by an NF-kappaB inhibitor. A cAMP-dependent kinase inhibitor did not inhibit it. MIF enhanced AP-1 and NF-kappaB binding activities in a dose-dependent manner. Passive CIA enhanced mRNA levels of macrophage inflammatory protein 2 and cytokine-induced neutrophil chemoattractants and, moreover, migration and proliferation of inflammatory cells within the synovium, which were suppressed by administration of an anti-MIF antibody., Conclusion: MIF may play an important role in the migration of inflammatory cells into the synovium of rheumatoid joints via induction of IL-8. MIF up-regulates IL-8 and IL-1beta mRNA via tyrosine kinase-, PKC-, AP-1-, and NF-kappaB-dependent pathways.

Objective: To determine the role and expression of the cytokine/receptor pair interleukin-21 (IL-21)/IL-21 receptor (IL-21R) in rheumatoid arthritis (RA)., Methods: The expression of IL-21R and IL-21 was analyzed by TaqMan real-time polymerase chain reaction (PCR) and in situ hybridization of synovial biopsy samples from patients with RA and osteoarthritis (OA). Double labeling by immunohistochemistry after in situ hybridization was performed with anti-CD68 antibodies. The expression of IL-21R at the protein level was confirmed by Western blotting. Stimulation experiments were performed with recombinant IL-1beta, tumor necrosis factor alpha (TNFalpha), platelet-derived growth factor (PDGF), and transforming growth factor beta (TGFbeta). The role of IL-21R in cartilage destruction was analyzed in the SCID mouse coimplantation model of RA., Results: IL-21R was found in total RNA extracts and in synovial biopsy samples from RA patients, whereas no expression or only minimal expression was seen in samples from OA patients. Double labeling indicated that both synovial macrophages and synovial fibroblasts expressed IL-21R. Western blotting with anti-IL-21R antibodies confirmed the expression of IL-21R protein in RA synovial fibroblasts (RASFs). Of note, IL-21 was not detectable by real-time PCR and in situ hybridization in the same samples in vivo as in vitro. The level of expression of IL-21R messenger RNA (mRNA) was not altered by stimulation with IL-1beta, TNFalpha, PDGF, or TGFbeta. Interestingly, in the SCID mouse coimplantation model, RASFs did not maintain their expression of IL-21R at sites of invasion into the cartilage. Similarly, IL-21R mRNA was not expressed at sites of invasion into cartilage and bone in RA synovium., Conclusion: Our data demonstrate that IL-21R is expressed in RA synovium by RASFs and synovial macrophages. IL-21R is associated with the activated phenotype of RASFs independently of the major proinflammatory cytokines IL-1beta and TNFalpha, but correlates negatively with the destruction of articular cartilage and bone.

Objective: This study was undertaken to characterize the role of CC chemokines and their receptors in rat adjuvant-induced arthritis (AIA), a model for rheumatoid arthritis (RA). Furthermore, we investigated the signaling pathways associated with CC receptors as well as the cell type distribution of the receptors., Methods: Using TaqMan real-time reverse transcription-polymerase chain reaction, Western blot analysis, and immunohistochemistry, we defined chemokine and chemokine receptor messenger RNA (mRNA) expression, CC chemokine receptor (CCR) protein activation during the disease course, CCR-associated signaling pathways, and immunopositive CCR5, phosphorylated signal transducer and activator of transcription 1 (p-STAT-1), and p-STAT-3 cells in rat AIA versus control joints., Results: We showed significant up-regulation of CCR1, CCR2, CCR5, and macrophage inflammatory protein 1beta/CCL4 mRNA in AIA on post-adjuvant injection day 18, coincident with peak inflammation. Additionally, increases in tyrosine phosphorylation of CCR1 (days 14, 18, 21, and 24), CCR2 (days 14 and 18), and CCR5 (days 14, 18, and 21) were detected in AIA rats compared with control (nonarthritic) rats. JAK-1, STAT-1, and STAT-3 were associated with CCR1 and were highly tyrosine phosphorylated on days 14 and 18. Moreover, CCR2 was associated with JAK-2, STAT-1, and STAT-3 on day 18. The association of STAT-1 and STAT-3 with CCR5 on days 18 and 21 correlated with JAK-1 phosphorylation and binding on day 18. However, the activation of JNK was not associated with CCR5 activation in rat AIA. Immunohistochemical analysis demonstrated that the expression of CCR5, p-STAT-1, and p-STAT-3 was detected on synovial lining cells, macrophages, and endothelial cells in arthritic rat ankles on post-adjuvant injection day 18. While the majority of the CCR5 and p-STAT-1 immunostaining was on synovial lining cells and macrophages, p-STAT-3 was predominantly expressed on endothelial cells., Conclusion: CCR1, CCR2, and CCR5 mRNA expression and tyrosine phosphorylation increased with peak inflammation in the AIA model. CCR1, CCR2, and CCR5 tyrosine phosphorylation are associated with the JAK/STAT-1/STAT-3 pathway at different stages of rat AIA, as well as with macrophage and endothelial cell infiltration. However, their signaling activation overlaps with peak inflammation. Up-regulation and activation of CCRs may play a role in macrophage and endothelial cell infiltration in rat AIA joints in addition to activating the associated signaling pathways. The downstream intermediate signaling proteins associated with CC receptors may be used as potential tools to control inflammation in RA.

Objective: To assess the overexpression of B lymphocyte stimulator (BLyS) over time in patients with systemic lupus erythematosus (SLE)., Methods: Sixty-eight SLE patients were followed up longitudinally for a median 369 days. At each physician encounter, disease activity was assessed by the Systemic Lupus Erythematosus Disease Activity Index, and blood was collected for determination of the serum BLyS level, blood BLyS messenger RNA (mRNA) level, and cell surface BLyS expression. Twenty normal control subjects underwent similar laboratory evaluations., Results: In contrast to the uniformly normal serum BLyS and blood BLyS mRNA phenotypes in control subjects, SLE patients displayed marked heterogeneity, with 50% and 61% of patients manifesting persistently or intermittently elevated serum BLyS and blood BLyS mRNA phenotypes, respectively. Surface BLyS expression by SLE peripheral blood mononuclear cells was also often increased. Treatment of patients who had elevated serum BLyS levels with intensive courses of high-dose corticosteroids resulted in marked reductions in serum BLyS levels, and tapering of the corticosteroid dosage often resulted in increases in serum BLyS levels. Serum BLyS levels generally correlated with anti-double-stranded DNA (anti-dsDNA) titers (in those with detectable anti-dsDNA titers), but changes in serum BLyS levels did not correlate with changes in disease activity in individual patients. Serum BLyS phenotype did not associate with specific organ system involvement., Conclusion: Dysregulation of BLyS over extended periods of time is common in patients with SLE. Neutralization of BLyS activity with an appropriate BLyS antagonist may be therapeutically beneficial.

Objective: To examine the expression, regulation, and potential roles of bone morphogenetic proteins (BMPs) in arthritic synovium., Methods: Expression of BMPs in arthritic synovium from patients with rheumatoid arthritis (RA) or spondylarthropathy (SpA) and in noninflamed synovium from patients undergoing diagnostic or therapeutic arthroscopies was studied by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, immunohistochemistry, and 2-color immunofluorescence. In vitro regulation of gene expression in fibroblast-like synoviocytes (FLS) was determined by real-time quantitative RT-PCR and immunohistochemistry. We used (3)H-thymidine incorporation after serum deprivation-induced growth arrest to examine effects on FLS proliferation. FLS apoptosis was evaluated by flow cytometry cell cycle analysis. Apoptotic cells in synovial tissue were detected by TUNEL staining., Results: Transcripts of different BMPs, most strikingly BMP-2 and BMP-6, were detected in synovial tissues. By Western blot, BMP-2 and BMP-6 precursor protein was found in RA and SpA synovial tissue extracts, but not in extracts of noninflamed synovial tissue. By immunohistochemistry, BMP-2 and BMP-6 were detected in the hyperplastic lining and the sublining layer of synovium from RA and SpA patients, both in CD90+ cells (FLS) and in some CD68+ cells (macrophages). Proinflammatory cytokines, such as interleukin-1beta and tumor necrosis factor alpha, but not interferon-gamma, enhanced the expression of BMP-2 and BMP-6 transcripts in FLS in vitro. Neither BMP-2 nor BMP-6 affected FLS proliferation. BMP-2 promoted FLS apoptosis, whereas BMP-6 protected against nitric oxide-induced FLS apoptosis. BMP-2-positive apoptotic cells were found in arthritic synovium., Conclusion: BMP-2 and BMP-6 are expressed in arthritic synovium and are strongly up-regulated by proinflammatory cytokines. Although BMP signaling has been proposed to be involved in cartilage and bone repair in arthritis, this pathway may be equally important in modulating FLS cell populations in inflamed synovium.

Objective: In systemic sclerosis (SSc; scleroderma), T cells infiltrate organs undergoing fibrotic changes and may participate in dysregulated production of collagen by fibroblasts. The objective of this study was to functionally characterize T cells infiltrating skin lesions in early SSc and investigate their capacity to affect production of type I collagen and interstitial collagenase (matrix metalloproteinase 1 [MMP-1]) by dermal fibroblasts., Methods: Four-color cytometric analysis was used to characterize subset distribution and production of interferon-gamma (IFN gamma) and interleukin-4 (IL-4) in T cell lines generated from the skin of patients with SSc. T cell clones were generated, and their capacity to modulate collagen and MMP-1 production by fibroblasts derived from patients with SSc and from normal individuals was assessed. Neutralizing reagents were used to identify T cell mediators involved in fibroblast modulation., Results: The skin of individuals with early-stage SSc contained T cells preferentially producing high levels of IL-4. Cloned CD4+ Th2-like cells inhibited collagen production by normal fibroblasts. Th2 cell-dependent inhibition was, at least in part, contact-dependent, was essentially mediated by tumor necrosis factor alpha (TNF alpha), and was dominant over the enhancement induced by profibrotic IL-4 and transforming growth factor beta cytokines. The simultaneous induction of MMP-1 production confirmed the specificity of these observations. To be inhibitory, Th2 cells required activation by CD3 ligation. Th2 cells were less potent than were Th1 cells in inhibiting collagen production by normal fibroblasts via cell-to-cell interaction, and SSc fibroblasts were resistant to inhibition., Conclusion: These findings indicate that, despite their production of IL-4, Th2 cells reduce type I collagen synthesis by dermal fibroblasts because of the dominant effect of TNF alpha, and suggest that strategies based on TNF alpha blockade aimed at controlling fibrosis in SSc may be unwise.

Objective: A characteristic feature of the inflammatory infiltrate in rheumatoid arthritis is the segregation of CD4 and CD8 T lymphocyte subsets into distinct microdomains within the inflamed synovium. The aim of this study was to test the hypothesis that chemokines in general and stromal cell-derived factor 1 (SDF-1; CXCL12) in particular are responsible for generating this distinctive microcompartmentalization., Methods: We examined how synovial CD4/CD8 T cell subsets interacted in coculture assays with fibroblasts derived from chronic inflammatory synovial lesions and normal synovial tissue as well as from fetal lung and adult skin. We used the ability of T cells to migrate beneath fibroblasts (a process called pseudoemperipolesis) as an in vitro marker of T cell accumulation within synovial tissue., Results: Rheumatoid fibroblast-like synoviocytes (FLS) displayed a unique ability to support high levels of CD4 and CD8 T cell pseudoemperipolesis. Nonrheumatoid FLS as well as fetal lung fibroblasts supported low levels of pseudoemperipolesis, while skin-derived fibroblasts were unable to do so. CD8 T cells migrated under fibroblasts more efficiently and at a higher velocity than CD4 T cells, a feature that was intrinsic to CD8 T cells. Rheumatoid fibroblasts constitutively produced high levels of SDF-1 (CXCL12), which was functionally important, since blocking studies showed reductions in T cell pseudoemperipolesis to levels seen in nonrheumatoid FLS. Rheumatoid fibroblasts also constitutively produced high levels of vascular cell adhesion molecule 1 (VCAM-1; CD106), but this did not contribute to T cell pseudoemperipolesis, unlike the case for B cells, which require SDF-1 (CXCL12)-CXCR4 and CD49d-VCAM-1 (CD106) interactions. Importantly, only combinations of rheumatoid FLS and rheumatoid-derived synovial fluid T cells supported pseudoemperipolesis when examined ex vivo, confirming the in vivo relevance of these findings., Conclusion: These studies demonstrate that features intrinsic to both fibroblasts (the production of SDF-1) and CD8/CD4 T cells (the expression of CXCR4) are responsible for the characteristic pattern of T lymphocyte accumulation seen in the rheumatoid synovium. These findings suggest that the SDF-1/CXCR4 ligand/receptor pair is likely to play an important functional role in T lymphocyte accumulation and positioning within the rheumatoid synovium.

Objective: Treatment with anti-CD154 antibody is known to ameliorate murine lupus nephritis when given early in the disease. The aims of this study were to identify the mechanism of this early effect, to determine whether late anti-CD154 treatment could halt established nephritis, and, if so, to examine potential mechanisms of late efficacy., Methods: We studied the effects of anti-CD154 treatment on autoantibody production and immune complex deposition, renal pathology, survival, and renal cytokine and chemokine messenger RNA (mRNA) expression both in (NZB x NZW)F(1) mice (BW mice) and in NZM.2410 mice., Results: Early treatment with anti-CD154 produced long-term survival in BW mice, with abrogation of renal immune complex deposition for months after treatment was stopped. Late anti-CD154 treatment, started after development of nephritis, could halt disease in approximately 40% of mice. In some mice, proteinuria could be reversed repeatedly with sequential courses of anti-CD154 antibody. The remissions induced by late treatment with anti-CD154 occurred despite ongoing renal immune complex deposition. In preliminary studies, responding mice had rapid reductions in renal mRNA for transforming growth factor beta, interleukin-10, and tumor necrosis factor alpha., Conclusion: Amelioration of murine lupus by anti-CD154 therapy is mediated by distinct mechanisms in early versus late intervention. We postulate that anti-CD154 therapy prevents autoantibody production and renal immune complex deposition in the early, induction phase and limits secondary tissue damage in situ in the late, effector phase. These data demonstrate that CD40-CD154 interactions are critical for the maintenance of autoimmunity and suggest a potential role for anti-CD154 as a therapeutic agent in established human lupus.

Objective: Protein deimination, a process to modify arginine residues to citrulline by the addition of a neutral oxygen group, is associated with apoptosis. The presence of autoantibodies recognizing citrullinated peptides is highly specific to rheumatoid arthritis (RA) and is therefore a useful marker for the early diagnosis of RA. In this study, we explored whether anti-cyclic citrullinated peptide (anti-CCP) autoantibodies are produced in several experimental models of autoimmune diseases in mice., Methods: The levels of anti-CCP autoantibodies were analyzed by enzyme-linked immunosorbent assay in several lupus-prone strains of mice, in animals with type II collagen (CII)-induced arthritis, and after induction of neonatal tolerance to alloantigens., Results: We observed the production of these autoantibodies in 2 different lupus-prone mice, MRL-lpr/lpr and (NZW x B6)F(1)-hbcl-2 transgenic mice, characterized by the presence of abnormalities in the regulation of B cell apoptosis. Other genetic defects, determining autoimmune susceptibility, present in MRL and NZW mice were additionally required for anti-CCP autoantibody production. The induction of autoantibodies in normal BALB/c mice injected at birth with semiallogeneic spleen cells from (BALB/c x B6)F(1)-hbcl-2 transgenic mice suggested that these additional autoimmune defects may be related, at least in part, to the establishment of abnormal interactions between T cells and B cells. In addition, anti-CCP autoantibodies were not produced in the course of CII-induced arthritis, an experimental model of RA in mice., Conclusion: Our study provides evidence for the association between defects in the regulatory cell death machinery of B lymphocytes and the production of certain autoantibody specificities.

Objective: To evaluate the possible role of activation-induced, T cell-derived, and chemokine-related cytokine (ATAC)/lymphotactin (Lptn) in the pathogenesis of rheumatoid arthritis (RA)., Methods: ATAC/Lptn levels in serum and synovial fluid samples were measured by sandwich enzyme-linked immunosorbent assay. Expression of messenger RNA for ATAC/Lptn in synovial tissues was analyzed by reverse transcription-polymerase chain reaction (PCR) and by in situ hybridization, and was quantitated by real-time PCR. The phenotype of peripheral blood mononuclear cells (PBMCs) expressing ATAC/Lptn was analyzed by intracellular cytokine staining and flow cytometry., Results: Levels of ATAC/Lptn were similar in sera and synovial fluids from RA patients (n = 20) and osteoarthritis controls (n = 15). In phorbol myristate acetate/ionomycin-stimulated PBMCs, ATAC/Lptn expression was detected in CD8+ T cells and in a significantly increased proportion of CD4+,CD28- T cells from RA patients as compared with healthy controls. In synovial tissues, ATAC/Lptn was predominantly localized in CD3+ T cells in the sublining layer. Lymphocytes, synovial macrophages, and, unexpectedly, fibroblast-like synoviocytes (FLS) were identified as major target cells for ATAC/Lptn in RA synovium, as determined by analysis of the ATAC/Lptn receptor XCR1. In vitro, ATAC/Lptn stimulation of FLS resulted in a marked down-regulation of matrix metalloproteinase 2 production., Conclusion: These data indicate that in RA synovium, ATAC/Lptn is mainly produced by T cells. Considering its function as a lymphocyte-specific chemoattractant, ATAC/Lptn might be a key modulator for T cell trafficking in the pathogenesis of RA. In addition, functional studies suggest that ATAC/Lptn may exert additional immunomodulatory effects in RA.

Objective: T cells from a majority of patients with systemic lupus erythematosus (SLE) display antigen receptor-mediated signaling aberrations associated with defective T cell receptor (TCR) zeta chain, a subunit of the TCR/CD3 complex. This study was undertaken to explore the possibility that forced expression of TCR zeta chain may reverse the known signaling abnormalities and defective interleukin-2 (IL-2) production in SLE T cells., Methods: Freshly isolated SLE T cells were transfected with TCR zeta chain construct in a eukaryotic expression vector at high efficiency, by a recently developed nucleoporation technique. Restoration of TCR/CD3-mediated signaling was studied in the zeta chain-transfected cells., Results: In SLE T cells transfected with TCR zeta chain, surface expression of TCR chain was increased and the TCR/CD3-induced increased free intracytoplasmic calcium concentration response was normalized, as was hyperphosphorylation of cellular substrates. Simultaneously, the previously noted increased expression of the Fc receptor gamma chain was diminished in SLE T cells transfected with the zeta chain expression vector, and the surface membrane clusters of cell signaling molecules were redistributed to a more continuous pattern. TCR zeta chain replacement also augmented the expression of diminished TCR/CD3-mediated IL-2 production in SLE T cells, associated with increased expression of the p65 subunit of nuclear factor kappaB in the nuclear fractions of these T cells., Conclusion: These results suggest that reconstitution of deficient TCR zeta chain can reverse the TCR/CD3-mediated signaling abnormalities as well as the defective IL-2 production in T cells of patients with SLE.

Objective: Heat-shock protein 90 (Hsp90) is critical in the intracellular signaling pathways that promote inflammatory cytokine production. Geldanamycin (GD) is a benzoquinone ansamycin that inhibits the function of Hsp90. GD inhibits the production of tumor necrosis factor alpha (TNFalpha) in activated macrophages and suppresses the progression of adjuvant-induced arthritis and experimental allergic encephalomyelitis in rodents. GD has been used to investigate the mechanisms by which Hsp90 regulates inflammatory cytokine production., Methods: The macrophage cell line RAW264.7 (or primary peritoneal macrophages) was activated with lipopolysaccharide in the absence or presence of GD. The effect of GD on the transcription, stability, and translation of inflammatory cytokine messenger RNA (mRNA) was determined using nuclear run-on assays, mRNA decay assays, and sucrose gradient polysome profiles, respectively., Results: Our data revealed that GD potently inhibits the production of TNFalpha, interleukin-6 (IL-6), and IL-1beta in activated macrophages. Although GD did not significantly reduce the transcription of inflammatory cytokine mRNA, it significantly decreased the stability of these transcripts. Polysome profiles indicated that GD also inhibited the translation of TNFalpha and IL-6 transcripts. These effects may be due, in part, to inhibition of p38 mitogen-activated protein kinase, a kinase known to regulate the stability and translation of inflammatory cytokine transcripts., Conclusion: These results indicate that the function of Hsp90 is important in the posttranscriptional control of inflammatory cytokine production.

Objective: To determine whether matrix metalloproteinase (MMP)-producing inflammatory macrophages in the synovium of rheumatoid arthritis (RA) patients express the novel dendritic cell (DC)-specific C-type lectin DC-SIGN and whether this expression is associated with the presence of naive T cells expressing the DC-SIGN ligand, intercellular adhesion molecule 3 (ICAM-3)., Methods: Reverse transcription-polymerase chain reaction was performed to study the expression of DC-SIGN in synovium samples from RA, osteoarthritis (OA), and trauma patients. DC-SIGN expression on RA monocytes or on monocytes stimulated with granulocyte-macrophage colony-stimulating factor and interleukin-4 was further investigated by flow cytometry. To localize DC-SIGN in the synovium, the DC markers ICAM-3 and MMP-1 were analyzed by immunohistochemistry (single and double labeling) on serial cryostat sections., Results: Seventy percent of the inflammatory cells in the synovium of RA patients showed high expression of DC-SIGN. DC-SIGN was expressed by 80% of CD68-positive macrophages, but not by CD83-positive, DC-LAMP-positive, or Fascin-positive cells. Normal numbers of DC-SIGN-positive cells were found in the peripheral blood of RA patients, suggesting that DC-SIGN is up-regulated locally in the joint. In RA synovium, ICAM-3-positive resting T cells were found in close proximity to DC-SIGN cells. Unexpectedly, a lower percentage of DC-SIGN-expressing cells was found in OA synovium compared with RA synovium. Furthermore, ICAM-3-expressing T cells, which are known to bind DC-SIGN, were almost absent within the synovium of OA and trauma patients. DC-SIGN-positive macrophages adjacent to these T cells were located in close proximity to the cartilage-degrading proteins extracellular MMP inducer (EMMPRIN) and MMP-1., Conclusion: The C-type lectin DC-SIGN is almost absent in the synovium of trauma patients but is highly expressed by most CD68-positive macrophages in the synovium of RA patients. The lack of correlation between DC-SIGN expression and the expression of CD83, DC-LAMP, or Fascin indicates that multiple DC/macrophage subsets are present in RA synovium. Expression of DC-SIGN and its ligand, ICAM-3, is found in substantial amounts only in RA synovium, suggesting that their interaction is implicated in the additional activation of synovial macrophages that leads to the production of EMMPRIN and MMP-1.

Objective: To define the cytokine profile within rheumatoid subcutaneous nodules, and to determine whether the destructive inflammatory process in this lesion displays features of a lymphocyte-driven Th1 or Th2 granuloma., Methods: Subcutaneous nodules excised from 10 patients with rheumatoid arthritis were examined. Transcripts for interleukin 1beta (IL-1beta) IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-15, IL-18, and for tumor necrosis factor alpha (TNFalpha) and interferon-gamma (IFNgamma) were detected by reverse transcription-polymerase chain reaction of extracted RNA., Results: Nine of 10 nodules contained transcripts for IFNgamma. We observed no evidence for the expression of IL-2, IL-4, or IL-5 among the lymphokine genes analyzed. Transcripts for TNFalpha, IL-1beta, IL-10, IL-15, and IL-18 were present in all 10 nodules. Transcripts for IL-12 were present in all but one nodule. Expression of IL-13 messenger RNA was observed in only 5 nodules., Conclusion: The cytokine profile within the rheumatoid nodule (i.e., presence of IFNgamma but not IL-2, and prominent expression of IL-1beta and TNFalpha together with IL-12, IL-18, IL-15, and IL-10) is similar to the profile of cytokines in the synovial lesion of rheumatoid arthritis, which is generally accepted as being attributable to a Th1-mediated inflammatory mechanism. Our results suggest that damage to affected synovial membrane or subcutaneous tissue is caused by the same inflammatory mechanisms, and that the nodule is a Th1 granuloma.

Objective: To study the role of the activating Fc gamma receptor types I and III (Fc gamma RI and Fc gamma RIII, respectively) and the inhibiting Fc gamma receptor II (Fc gamma RII) in inflammation and in various aspects of cartilage destruction during arthritis that is solely induced by immune complexes., Methods: Immune complex-mediated arthritis (ICA) was passively induced by lysozyme-antilysozyme complexes in Fc gamma RI-, Fc gamma RIII-, and Fc gamma RII-knockout mice and their wild-type controls. Total knee joints were isolated to study inflammation and cartilage destruction (loss of proteoglycans [PGs], chondrocyte death, matrix metalloproteinase [MMP]-mediated neoepitope [VDIPEN] expression, and erosion). The presence of an active phenotype of macrophages was studied by detection of myeloid-related proteins 8 and 14 (MRP8 and MRP14, respectively)., Results: Influx and activation of inflammatory cells (MRP expression) during ICA was decreased in Fc gamma RIII-deficient mice and enhanced in mice lacking Fc gamma RII. Mild cartilage destruction reflected by loss of PGs was consistent with the degree of inflammation. Mice lacking Fc gamma RIII showed almost no PG depletion, whereas in Fc gamma RII(-/-) mice, PG depletion was increased 3-7-fold in various cartilage areas. Initiation of erosive cartilage destruction, as reflected by MMP-mediated VDIPEN expression, was reduced in Fc gamma RIII(-/-) and Fc gamma RI(-/-) mice, directing the two different critical steps of cellular influx and subsequent activation. These aspects were enhanced in Fc gamma RII(-/-) mice. In Fc gamma RI(-/-) and Fc gamma RIII(-/-) mice, VDIPEN expression was 90-99% lower, whereas in Fc gamma RII(-/-) mice, VDIPEN expression was increased 4-fold. Chondrocyte death was reduced in Fc gamma RIII(-/-) mice (68% lower) and enhanced in Fc gamma RII(-/-) mice (6-12-fold higher). Progression of arthritis and erosion of the cartilage surface were markedly elevated in Fc gamma RII(-/-) arthritic joints., Conclusion: During ICA, Fc gamma RIII is the dominant activating receptor mediating joint inflammation, whereas both Fc gamma RI and Fc gamma RIII are involved in cartilage destruction. Fc gamma RII inhibits both joint inflammation and severe cartilage destruction during ICA.

Objective: To assess the capacities of the cytokine inhibitors interleukin-1 receptor antagonist (IL-1Ra; anakinra) and PEGylated soluble tumor necrosis factor receptor I (PEG sTNFRI; pegsunercept) to suppress neovascularization., Methods: A corneal angiogenesis assay was performed by implanting nylon discs impregnated with an angiogenic stimulator (basic fibroblast growth factor or vascular endothelial growth factor) into one cornea of female Sprague-Dawley rats. Animals were treated with IL-1Ra or PEG sTNFRI for 7 days, after which new vessels were quantified. In a parallel study, male Lewis rats with mycobacteria-induced adjuvant-induced arthritis were treated with IL-1Ra or PEG sTNFRI for 7 days beginning at disease onset, after which scores for inflammation and bone erosion as well as capillary counts were acquired from sections of arthritic hind paws., Results: Treatment with IL-1Ra yielded a dose-dependent reduction in growth factor-induced corneal angiogenesis, while PEG sTNFRI did not. IL-1Ra, but not PEG sTNFRI, significantly reduced the number of capillaries in arthritic paws, even though both anticytokines reduced inflammation and bone erosion to a similar degree., Conclusion: These data support a major role for IL-1, but not TNFalpha, in angiogenesis and suggest that an additional antiarthritic mechanism afforded by IL-1 inhibitors, but not anti-TNF agents, is the suppression of the angiogenic component of pannus.

Objective: Fibroblasts play a major role in the development of systemic sclerosis (SSc), and the occurrence of serum autoantibodies reacting with fibroblast plasma membrane antigens in SSc has been reported. This study was undertaken to investigate whether IgG from SSc sera that react with human fibroblasts modulates the fibroblasts' function., Methods: Sera from 69 patients with SSc (28 with limited cutaneous SSc [lcSSc] and 41 with diffuse cutaneous SSc [dcSSc]), 30 patients with sarcoidosis, and 50 matched healthy controls were examined. We evaluated antibody binding to human skin and lung fibroblasts by cell-based enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence, and flow cytometry. We further investigated the ability of purified IgG to modulate 1) intercellular adhesion molecule 1 (ICAM-1) expression, 2) U937 cell adhesion to fibroblasts, and 3) fibroblast steady-state messenger RNA (mRNA) levels of interleukin-1alpha (IL-1alpha), IL-beta, and IL-6, and IL-6 protein production., Results: Of 69 SSc sera tested by cell-based ELISA, 58% bound to normal skin and lung fibroblasts. The prevalence of binding was significantly higher in dcSSc than in lcSSc (P < 0.05). Only IgG from SSc sera that were positive for antifibroblast antibody (AFA) induced a dose-dependent up-regulation of ICAM-1 expression and IL-6 production, enhancement of U937 cell adhesion, and increased levels of IL-1alpha, IL-1beta, and IL-6 mRNA in fibroblasts. Up-regulation of ICAM-1 mediated by AFA IgG was inhibited by the addition of IL-1 receptor antagonist, indicating an autocrine activation loop., Conclusion: Our findings confirm the presence of AFAs in SSc sera and demonstrate, for the first time, that autoantibodies reacting with fibroblast surface molecules act as an extrinsic stimulus inducing fibroblast activation in vitro.

Objective: Inhibition of T cell DNA methylation causes autoreactivity in vitro and a lupus-like disease in vivo, suggesting that T cell DNA hypomethylation may contribute to autoimmunity. The hypomethylation effects are due, in part, to overexpression of lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18). Importantly, T cells from patients with active lupus have hypomethylated DNA and overexpress LFA-1 on an autoreactive subset, suggesting that the same mechanism could contribute to human lupus. The present study investigated the nature of the methylation change that affects LFA-1 expression in vitro and in human lupus., Methods: Bisulfite sequencing was used to determine the methylation status of the ITGAL promoter and flanking regions in T cells from lupus patients and healthy subjects, and in T cells treated with DNA methylation inhibitors. "Patch" methylation of promoter sequences in reporter constructs was used to determine the functional significance of the methylation changes., Results: Hypomethylation of specific sequences flanking the ITGAL promoter was seen in T cells from patients with active lupus and in T cells treated with 5-azacytidine and procainamide. Patch methylation of this region suppressed ITGAL promoter function., Conclusion: DNA methylation changes occur in specific sequences that regulate LFA-1 expression in lupus T cells and in the hypomethylation model, indicating that altered methylation of specific genes may play a role in the pathogenesis of lupus.

Objective: To determine whether local overexpression of interleukin-12 (IL-12), a pleiotropic cytokine that promotes the development of naive T cells into Th1 cells, could aggravate murine streptococcal cell wall (SCW)-induced arthritis, a model of acute arthritis., Methods: C57BL/6 mice were injected intraarticularly with saline or with 10(7) plaque-forming units of control vector (Ad5del70-3) or IL-12 vector (AdmIL-12.1) into the right knee joint 1 day before intraarticular injection of 25 microg of SCW fragments. The development of joint swelling, changes in chondrocyte proteoglycan (PG) synthesis, and joint destruction were examined thereafter., Results: In normal joints, high levels of IL-12 (20 ng/ml on day 1) could be detected after application of the AdmIL-12.1 vector. After 14 days, expression of IL-12 was still found locally, but IL-12 alone did not induce protracted inflammation. Local expression of IL-12, in combination with SCW, markedly aggravated SCW-induced arthritis, as determined by enhanced joint swelling and prolonged inhibition of chondrocyte PG synthesis. Histologic examination on day 21 showed a chronic inflammatory process, with persistent cartilage PG depletion, cartilage erosion, and VDIPEN neoepitope expression (indicative of metalloproteinase activation). The mixture of IL-12 with SCW fragments did not lead to a chronic destructive process in mice deficient for recombination-activating gene 2, indicating the involvement of lymphocytes. In addition, systemic flare of smoldering SCW arthritis, produced by intravenous injection of SCW fragments, was only seen in the AdmIL-12/SCW group., Conclusion: These results indicate that local overexpression of IL-12 promotes conversion of an acute arthritis to a chronic destructive immune-mediated process, which is more susceptible to flares.

Objective: To analyze immunoregulatory abnormalities in patients with systemic lupus erythematosus (SLE) by assessing the expression of messenger RNA (mRNA) for types 1 and 2 recombination activating genes (RAG) in the peripheral blood of patients with active SLE., Methods: We examined B cell populations and also individual B cells from patients with SLE for the expression of RAG mRNA., Results: Analysis of bulk mRNA indicated that RAG1 and RAG2 mRNA were found routinely in peripheral B cells of patients with active SLE, but not in healthy subjects. When assessed on a single-cell basis, there was a 3-fold increase in the frequency of RAG1- and RAG2-expressing B cells in SLE patients compared with healthy subjects. Notably, B cells expressing both RAG1 and RAG2 mRNA expressed only IgD mRNA, but not IgG mRNA. Fifty percent of RAG-expressing B cells also expressed VpreB mRNA, whereas all expressed CD154 mRNA. Phenotypic analysis indicated that RAG-expressing B cells were activated, mature B cells., Conclusion: These results indicate that RAG expression is up-regulated in peripheral IgD+ and VpreB+ B cells of patients with active SLE. These cells may contribute to the immunoregulatory abnormalities in patients with SLE.