Inflammatory disease status and response to TNF blockade are associated with mechanisms of endotoxin tolerance.

The mechanisms of endotoxin tolerance (ET), which down-regulate inflammation, are well described in response to exogenous toll-like receptor ligands, but few studies have focused on ET-associated mechanisms in inflammatory disease. As blocking TNF can attenuate the development of ET, the effect of anti-TNF on the expression of key ET-associated molecules in inflammatory auto-immune disease was measured; changes in inflammatory gene expression were confirmed using an ET bioassay. The expression of immunomodulatory molecules was measured in a murine model of arthritis treated with anti-TNF and the expression of ET-associated molecules was measured in whole blood in rheumatoid arthritis (RA) and ankylosing spondylitis (AS) patients, before and after therapy. The expression of ET-associated genes was also measured in RA patient monocytes before and after therapy, in anti-TNF responders and non-responders. Tnfaip3, Ptpn6 and Irak3 were differentially expressed in affected paws, spleens, lymph nodes and circulating leucocytes in experimental murine arthritis treated with anti-TNF. Prior to therapy, the expression of TNFAIP3, INPP5D, PTPN6, CD38 and SIGIRR in whole blood differed between human healthy controls and RA or AS patients. In blood monocytes from RA patients, the expression of TNFAIP3 was significantly reduced by anti-TNF therapy in non-responders. Prior to therapy, anti-TNF non-responders had higher expression of TNFAIP3 and SLPI, compared to responders. Although the expression of TNFAIP3 was significantly higher in RA non-responders prior to treatment, the post-treatment reduction to a level similar to responders did not coincide with a clinical response to therapy.

Disease status in human and experimental arthritis, and response to TNF blockade, is associated with MHC class II invariant chain (CD74) isoform expression.

Splice variants of CD74 differentially modulate the activity of cathepsin L (CTSL). As CD74 and CTSL participate in the pathogenesis of inflammatory diseases such as rheumatoid arthritis (RA), we determined whether splice variants of CD74 could be biomarkers of disease activity. Gene expression was measured in mice with collagen-induced arthritis using quantitative PCR (qPCR). In vitro studies using murine macrophage/DC-lineage cells determined the relative influence of macrophage phenotype on isoform expression and the potential to produce CTSL in response to TNF. CD74 splice variants were measured in human RA synovium and RA patients' monocytes. In arthritic mice, the expression of the p41 CD74 isoform was significantly higher in severely affected paws compared with unaffected paws or the paws of naïve mice; the p41 isoform significantly correlated with the expression of TNF in arthritic paws. Compared with M2-like macrophages, M1-like macrophages expressed increased levels of CD74 and had higher expression, secretion and activity of CTSL. RA patients that responded to TNF blockade had significantly higher expression levels of CD74 in circulating monocytes after treatment, compared with non-responders. The expression of the human CD74 isoform a was significantly higher in RA synovia, compared with osteoarthritis synovia, and was associated with CSTL enzymatic activity. This study is the first to demonstrate differential expression of the CD74 p41 isoform in an auto-immune disorder and in response to therapy. The differential expression of CD74 splice variants indicates an association, and potentially a mechanistic role, in the pathogenesis of RA.

TLR expression profiles are a function of disease status in rheumatoid arthritis and experimental arthritis.

The role of the innate immune system has been established in the initiation and perpetuation of inflammatory disease, but less attention has been paid to its role in the resolution of inflammation and return to homeostasis. Toll-like receptor (TLR) expression profiles were analysed in tissues with differing disease status in rheumatoid arthritis (RA), ankylosing spondylitis (AS), and in experimental arthritis. TLR gene expression was measured in whole blood and monocytes, before and after TNF blockade. In RA and osteoarthritis synovia, the expression of TLRs was quantified by standard curve qPCR. In addition, four distinct stages of disease were defined and validated in collagen-induced arthritis (CIA), the gold standard animal model for RA - pre-onset, early disease, late disease and immunised mice that were resistant to the development of disease. TLR expression was measured in spleens, lymph nodes, blood cells, liver and the paws (inflamed and unaffected). In RA whole blood, the expression of TLR1, 4 and 6 was significantly reduced by TNF blockade but the differences in TLR expression profiles between responders and non-responders were less pronounced than the differences between RA and AS patients. In RA non-responders, monocytes had greater TLR2 expression prior to therapy compared to responders. The expression of TLR1, 2, 4 and 8 was higher in RA synovium compared to control OA synovium. Circulating cytokine levels in CIA resistant mice were similar to naïve mice, but anti-collagen antibodies were similar to arthritic mice. Distinct profiles of inflammatory gene expression were mapped in paws and organs with differing disease status. TLR expression in arthritic paws tended to be similar in early and late disease, with TLR1 and 2 moderately higher in late disease. TLR expression in unaffected paws varied according to gene and disease status but was generally lower in resistant paws. Disease status-specific profiles of TLR expression were observed in spleens, lymph nodes, blood cells and the liver. Notably, TLR2 expression rose then fell in the transition from naïve to pre-onset to early arthritis. TLR gene expression profiles are strongly associated with disease status. In particular, increased expression in the blood precedes clinical manifestation.

TNFα inhibitors reduce bone loss in rheumatoid arthritis independent of clinical response by reducing osteoclast precursors and IL-20.

OBJECTIVES: About half of RA patients treated with TNFα inhibitors either do not respond or lose their initial therapeutic response over time. The clinical response is measured by reduction in DAS28, which primarily reflects inflammation. However, other effects of TNFα inhibitors, such as impact on bone erosion, are not assessed by DAS28. We aimed to examine the effect of TNFα inhibitors on bone density, bone biomarkers and cytokine production in responder and non-responder patients and assessed mechanisms of action. METHODS: BMD in the lumbar spine and femur neck of 117 RA patients was measured by DEXA scan. Bone turnover biomarkers CTX, osteoprotegerin (OPG), osteocalcin and RANKL were measured by ELISA. Levels of 16 cytokines in plasma and in tissue culture supernatants of ex vivo T cells were measured by multiplex assays and ELISA. The effect of treatment with TNFα inhibitors on blood mononuclear cell (MNC) differentiation to osteoclast precursors (OCP) was measured flow cytometry and microscopy. RESULTS: TNFα inhibitors improved lumbar spine BMD but had modest effects on blood bone biomarkers, irrespective of patients' clinical response. Blood OCP numbers and the ability of monocytes to differentiate to OCP in vitro declined after treatment. Treatment also reduced RANK expression and IL-20 production. BMD improvement correlated with reduced levels of IL-20 in responder patients. CONCLUSION: This study reveals that TNFα inhibitors reduce lumbar spine bone loss in RA patients irrespective of changes in DAS28. The reduction in bone loss is associated with reduction in IL-20 levels in responder patients.

Functional and phenotypic heterogeneity of Th17 cells in health and disease.

BACKGROUND: Th17 cells have nonredundant roles in maintaining immunity, particularly at mucosal surfaces. These roles are achieved principally through the production of cytokines and the recruitment of other immune cells to maintain the integrity of mucosal barriers and prevent the dissemination of microorganisms. Th17 cells are heterogeneous and exhibit a considerable degree of plasticity. This allows these cells to respond to changing environmental challenges. However, Th17 cells also play pro-inflammatory roles in chronic autoimmune diseases. The trigger(s) that initiate these Th17 responses in chronic autoimmune diseases remain unclear. DESIGN: In this report, we provide an overview of studies involving animal models, patient data, genome wide association studies and clinical trials targeting IL-17 for treatment of patients to gain a better understanding of the pathogenic roles of Th17 cells play in a range of autoimmune diseases. RESULTS: The report sheds light on likely triggers that initiate or perpetuate Th17 responses that promote chronic inflammation and autoimmunity. The divergent effects of tumour necrosis factor alpha blockade on Th17 cells in patients, is explored. Furthermore, we highlight the role of Th17 cells in inducing autoreactive B cells, leading to autoantibody production. Pathogenic bacterial species can change Th17 cell phenotype and responses. These findings provide insights into how Th17 cells could be induced to promoting autoimmune disease pathogenesis. CONCLUSION: This article provides an overview of the distinct roles Th17 cells play in maintaining immunity at mucosal surfaces and in skin mucosa and how their functional flexibility could be linked with chronic inflammation in autoimmune rheumatic diseases.

TNFα in the regulation of Treg and Th17 cells in rheumatoid arthritis and other autoimmune inflammatory diseases.

TNFα is a principal pro-inflammatory cytokine vital for immunity to infections. However, its excessive production is involved in chronic inflammation and disease pathology in autoimmune diseases. Evidence for its pathogenic role is validated by the fact that its neutralisation by therapeutic agents in vivo is beneficial in ameliorating disease and controlling symptoms. Paradoxically, however, treatment with TNFα inhibitors can either have no clinical effects, or even exacerbate disease in some patients. The explanation for such contradictory outcomes may lay in how and which downstream signalling pathways are activated and drive disease. TNFα causes its effects by binding to either or both of two membrane-bound receptors, TNFR1 and TNFR2. Engagement of the receptors can induce cell death or cell proliferation. T cells both produce and respond to TNFα and depending on whether the cytokine is membrane-bound or soluble and the level of expression of its two receptors, the biological outcome can be distinct. In addition, polymorphisms in genes encoding TNFα and T cell signalling proteins can significantly impact the outcome of TNFα receptor engagement. Early studies revealed that effector T cells in patients with rheumatoid arthritis (RA) are hyporesponsive due to chronic exposure to TNFα. However, recent evidence indicates that the relationship between TNFα and T cell responses is complex and, at times, can be paradoxical. In addition, there is controversy as to the specific effects of TNFα on different T cell subsets. This review will summarise knowledge on how TNFα modulates T cell responses and the effect of engaging either of its two receptors. Furthermore, we discuss how such interactions can dictate the outcome of treatment with TNFα inhibitors.

In-depth characterization of CD24(high)CD38(high) transitional human B cells reveals different regulatory profiles.

BACKGROUND: CD24(high)CD38(high) transitional B cells represent cells at a key stage in their developmental pathway. In addition, these B cells have been widely ascribed regulatory functions and involvement in the control of chronic inflammatory diseases. However, the phenotypic and functional overlap between these cells and regulatory B cells remains controversial. OBJECTIVE: In this study we wanted to explore the regulatory properties of CD24(high)CD38(high) human B cells. METHODS: We used multicolor flow cytometry in combination with bioinformatics and functional studies to show that CD24(high)CD38(high) B cells can be distinguished into multiple subsets with different regulatory functions. RESULTS: For the first time, the study reveals that human transitional B cells encompass not only transitional type 1 and type 2 B cells, as previously suggested, but also distinct anergic type 3 B cells, as well as IL-10-producing CD27(+) transitional B cells. Interestingly, the latter 2 subsets differentially regulate CD4(+) T-cell proliferation and polarization toward TH1 effector cells. Additional analyses reveal that the percentage of type 3 B cells is reduced and the frequency of CD27(+) transitional B cells is increased in patients with autoimmune diseases compared with those in matched healthy subjects. CONCLUSION: This study provides evidence for the existence of different transitional B-cell subsets, each displaying unique phenotypic and regulatory functional profiles. Furthermore, the study indicates that altered distribution of transitional B-cell subsets highlights different regulatory defects in patients with different autoimmune diseases.

Harnessing the Therapeutic Potential of Th17 Cells.

Th17 cells provide protective immunity to infections by fungi and extracellular bacteria as well as cancer but are also involved in chronic inflammation. The cells were first identified by their ability to produce interleukin 17A (IL-17A) and, subsequently, associated with chronic inflammation and autoimmunity. Th17 cells have some gene profile similarity with stem cells and can remain dormant in mucosal tissues for long periods. Indeed, recent studies suggest that functionally distinct subsets of pro- and anti-inflammatory Th17 cells can interchange phenotype and functions. For development, Th17 cells require activation of the transcription factors STAT3 and RORγt while RUNX1, c-Maf, and Aiolos are involved in changes of phenotype/functions. Attempts to harness Th17 cells against pathogens and cancer using vaccination strategies are being explored. The cells gain protective abilities when induced to produce interferon γ (IFNγ). In addition, treatment with antibodies to IL-17 is effective in treating patients with psoriasis, psoriatic arthritis, and refectory rheumatoid arthritis. Moreover, since RORγt is a nuclear receptor, it is likely to be a potential future drug target for modulating Th17 functions. This review explores pathways through which Th17 subsets are induced, the molecular basis of their plasticity, and potential therapeutic strategies for their modulation in diseases.

Aberrant B-lymphocyte responses in lupus: inherent or induced and potential therapeutic targets.

BACKGROUND: Lupus is a prototype autoimmune disease of unknown aetiology. The disease is complex; manifest diverse clinical symptoms and disease mechanisms. This complexity has provided many leads to explore: from disease mechanisms to approaches for therapy. B-lymphocytes play a central role in the pathogenesis of the disease. However, the cause of aberrant B-lymphocyte responses in patients and, indeed, its causal relationship with the disease remain unclear. DESIGN: This article provides a synopsis of current knowledge of immunological abnormalities in lupus with an emphasis on abnormalities in the B-lymphocyte compartment. RESULTS: There is evidence for abnormalities in most compartments of the immune system in animal models and patients with lupus including an ever expanding list of abnormalities within the B-lymphocyte compartment. In addition, recent genome-wide linkage analyses in large cohorts of patients have identified new sets of genetic association factors some with potential links with defective B-lymphocyte responses although their full pathophysiological effects remain to be determined. The accumulating knowledge may help in the identification and application of new targeted therapies for treating lupus disease. CONCLUSIONS: Cellular, molecular and genetic studies have provided significant insights into potential causes of immunological defects associated with lupus. Most of this insight relate to defects in B- and T-lymphocyte tolerance, signalling and responses. For B-lymphocytes, there is evidence for altered regulation of inter and intracellular signalling pathways at multiple levels. Some of these abnormalities will be discussed within the context of potential implications for disease pathogenesis and targeted therapies.

CD5 promotes IL-10 production in chronic lymphocytic leukemia B cells through STAT3 and NFAT2 activation.

B lymphocytes from chronic lymphocytic leukemia (CLL) display some CD5 transcripts for CD5 containing the known exon 1 (E1A) and other CD5 transcripts containing the new exon 1 (E1B). These malignant B cells, as well as B cell lines transfected with cDNA for E1A-cd5 or with cDNA for E1B-cd5 produce IL-10, raising the possibility that CD5 participates in the secretion of IL-10. We identified transcription factors involved in this production in CD5(+) B lymphocytes from CLL patients and in E1A-cd5-transfected or E1B-cd5-transfected Jok cells. STAT3 is activated via phosphorylation of serine 727 but also NFAT2 through its translocation into the nucleus. Chromatin immunoprecipitation experiments confirmed the role of STAT3 and allowed the discovery of a role for NFAT2 in IL-10 production. Both transcription factors bind not only to the enhancer of the Il-10 gene but also to the promoter of the Il-5 and Il-13 genes. Furthermore, transfection of B cell lines with E1A-cd5 or E1B-cd5 established that activation of STAT3 and NFAT2 is regulated by CD5. The same holds true for the production of IL-10, IL-5, and IL-13 and the expression of the receptors for these cytokines. This interpretation was confirmed by two experiments. In the first, downregulation of CD5 by small interfering RNAs lowered the production of IL-10. In the second experiment, transfection of the GFP-NFAT2 gene into B lymphocytes induced nuclear translocation of NFAT2 in CD5(+) but not in CD5(-) B cells. Thus, CD5 expression is associated with NFAT2 activity (and mildly STAT3 activity), indicating that CD5 controls IL-10 secretion.

Altered patterns of epigenetic changes in systemic lupus erythematosus and auto-antibody production: is there a link?

The prominent feature of immunological defects in systemic lupus erythematosus (SLE) is the production of autoantibodies (auto-Abs) to nuclear antigens including DNA, histones and RNP. In addition, there is growing evidence that epigenetic changes play a key role in the pathogenesis of SLE. Autoreactive CD4(+) T cells and B cells in patients with SLE have evidence of altered patterns of DNA methylation as well as post-translational modifications of histones and ribonucleoproteins (RNP). A key question that has emerged from these two characteristic features of SLE is whether the two processes are linked. New data provide support for such a link. For example, there is evidence that hypomethylated DNA is immunogenic, that anti-histone auto-Abs in patients with SLE bind epigenetic-sensitive hot spots and that epigenetically-modified RNP-derived peptides can modulate lupus disease. All in all, the available evidence indicates that a better understanding of dysregulation in epigenetics in SLE may offer opportunities to develop new biomarkers and novel therapeutic strategies.

IVIg modulates BCR signaling through CD22 and promotes apoptosis in mature human B lymphocytes.

Among various mechanisms for interactions with B cells, intravenous immunoglobulin (IVIg) may operate through the insertion of its Fc part into the Fc-γ receptor, or the binding of its sialic acid (SA)-bearing glycans to the negatively regulating CD22 lectin. It appeared that IVIg reduces B lymphocyte viability in a dose- and time-dependent manner. Furthermore, we show by confocal microscopy that SA-positive IgG, but not SA-negative IgG bind to CD22. This interaction reduces the strength of B-cell receptor-mediated signaling trough down-regulating tyrosine phosphorylation of Lyn and the B-cell linker proteins, and up-regulating phospholipase Cγ2 activation. This cascade resulted in a sustained activation of Erk 1/2 and arrest of the cell cycle at the G(1) phase. These changes may be accounted for the efficacy of IVIg in autoimmune diseases.

Metabolic requirements of Th17 cells and of B cells: Regulation and defects in health and in inflammatory diseases.

The immune system protects from infections and cancer through complex cellular networks. For this purpose, immune cells require well-developed mechanisms of energy generation. However, the immune system itself can also cause diseases when defective regulation results in the emergence of autoreactive lymphocytes. Recent studies provide insights into how differential patterns of immune cell responses are associated with selective metabolic pathways. This review will examine the changing metabolic requirements of Th17 cells and of B cells at different stages of their development and activation. Both cells provide protection but can also mediate diseases through the production of autoantibodies and the production of proinflammatory mediators. In health, B cells produce antibodies and cytokines and present antigens to T cells to mount specific immunity. Th17 cells, on the other hand, provide protection against extra cellular pathogens at mucosal surfaces but can also drive chronic inflammation. The latter cells can also promote the differentiation of B cells to plasma cells to produce more autoantibodies. Metabolism-regulated checkpoints at different stages of their development ensure the that self-reactive B cells clones and needless production of interleukin (IL-)17 are limited. The metabolic regulation of the two cell types has some similarities, e.g. the utility of hypoxia induced factor (HIF)1α during low oxygen tension, to prevent autoimmunity and regulate inflammation. There are also clear differences, as Th17 cells only are vulnerable to the lack of certain amino acids. B cells, unlike Th17 cells, are also dependent of mechanistic target of rapamycin 2 (mTORC2) to function. Significant knowledge has recently been gained, particularly on Th17 cells, on how metabolism regulates these cells through influencing their epigenome. Metabolic dysregulation of Th17 cells and B cells can lead to chronic inflammation. Disease associated alterations in the genome can, in addition, cause dysregulation to metabolism and, thereby, result in epigenetic alterations in these cells. Recent studies highlight how pathology can result from the cooperation between the two cell types but only few have so far addressed the key metabolic alterations in such settings. Knowledge of the impact of metabolic dysfunction on chronic inflammation and pathology can reveal novel therapeutic targets to treat such diseases.

Dysregulated B cell function and disease pathogenesis in systemic sclerosis.

Systemic sclerosis (SSc) is a complex, immune-mediated rheumatic disease characterised by excessive extracellular matrix deposition in the skin and internal organs. B cell infiltration into lesional sites such as the alveolar interstitium and small blood vessels, alongside the production of defined clinically relevant autoantibodies indicates that B cells play a fundamental role in the pathogenesis and development of SSc. This is supported by B cell and fibroblast coculture experiments revealing that B cells directly enhance collagen and extracellular matrix synthesis in fibroblasts. In addition, B cells from SSc patients produce large amounts of profibrotic cytokines such as IL-6 and TGF-ß, which interact with other immune and endothelial cells, promoting the profibrotic loop. Furthermore, total B cell counts are increased in SSc patients compared with healthy donors and specific differences can be found in the content of naïve, memory, transitional and regulatory B cell compartments. B cells from SSc patients also show differential expression of activation markers such as CD19 which may shape interactions with other immune mediators such as T follicular helper cells and dendritic cells. The key role of B cells in SSc is further supported by the therapeutic benefit of B cell depletion with rituximab in some patients. It is notable also that B cell signaling is impaired in SSc patients, and this could underpin the failure to induce tolerance in B cells as has been shown in murine models of scleroderma.

Protein phosphorylation and kinome profiling reveal altered regulation of multiple signaling pathways in B lymphocytes from patients with systemic lupus erythematosus.

OBJECTIVE: The cause of B lymphocyte hyperactivity and autoantibody production in systemic lupus erythematosus (SLE) remains unclear. Previously, we identified abnormalities in the level and translocation of signaling molecules in B cells in SLE patients. The present study was undertaken to examine the extent of signaling abnormalities that relate to altered B cell responses in SLE. METHODS: B lymphocytes from 88 SLE patients and 72 healthy controls were isolated from blood by negative selection. Protein tyrosine phosphorylation and cellular kinase levels were analyzed by Western blotting, flow cytometry, and a kinome array protocol. Changes in protein phosphorylation were determined in ex vivo B cells and following B cell receptor engagement. RESULTS: Differences in tyrosine phosphorylation in B cells from patients with SLE, compared with matched controls, were demonstrated. Further, the kinome array analysis identified changes in the activation of key kinases, i.e., the activity of phosphatidylinositol 3-kinase, which regulates survival and differentiation, was up-regulated and the activity of Rac and Rho kinases, which regulate the cytoskeleton and migration, was increased. In contrast, the activity of ATR, which regulates the cell cycle, was down-regulated in SLE patients compared with controls. Differences in signaling pathways were seen in all SLE B lymphocyte subsets that manifested phenotypic features of immature, mature, and memory cells. CONCLUSION: This study revealed dysregulation in multiple signaling pathways that control key responses in B cells of SLE patients. Data generated in this study provide a molecular basis for further analysis of the altered B lymphocyte responses in SLE.

IL-6 modulates CD5 expression in B cells from patients with lupus by regulating DNA methylation.

B lymphocytes from patients with systemic lupus erythematosus (SLE) are characterized by reduced expression levels of membrane CD5. Recent studies from our laboratory have revealed that the level of membrane CD5 is determined by the relative level of two alternative CD5 isoforms; CD5-E1A, which is expressed on the membrane, and CD5-E1B, which is retained in the cytoplasm. Using bisulfite sequencing and methylation-sensitive endonuclease assays we show that the promoter for the alternative CD5-E1B isoform is demethylated in B cells from patients with SLE but not in healthy controls. We go on to show that differential methylation is more pronounced following BCR engagement. As a result of this demethylation, CD5-E1B mRNA is transcribed at the expense of CD5-E1A mRNA transcription. We provide further evidence that production of high IL-6 levels by SLE B cells abrogates the ability of SLE B cells to induce DNA methyl transferase (DNMT1) and then to methylate DNA, an effect that is reversed in the presence of a blocking Ab to the IL-6 receptor. The pattern of demethylation of CpG islands in the CD5-E1B promoter in SLE B cells is similar to those in B cells from healthy controls stimulated in the presence of IL-6, or treated with the methylation inhibitor PD98059. The study reveals that engagement of the BCR with constitutive IL-6 down-regulates the level of membrane CD5, which negatively regulates BCR signaling, in SLE B cells. This altered signaling could, in turn, promote the activation and expansion of autoreactive B cells in SLE patients.

Mitochondrial heat shock protein (HSP) 70 synergizes with HSP60 in transducing endothelial cell apoptosis induced by anti-HSP60 autoantibody.

Heat shock protein (HSP) 60, up-regulated by endothelial cells (ECs) to resist stress, is the target of a subgroup of apoptosis-inducing anti-EC autoantibodies (Abs) in human vasculitides. Given that HSP60 is not a transmembrane protein, the mechanism by which these auto-Abs induces apoptosis is unclear. EC membrane proteins were analyzed using bidimensional electrophoresis and Far Western blot, and the HSP60 receptor was identified by mass spectrometry. Heat stress-dependent synthesis of HSP60 and receptor was examined by semiquantitative RT-PCR, and expression was examined by flow cytometry and indirect immunofluorescence. Interaction was demonstrated by coimmunoprecipitations. Lipid rafts were purified to evaluate specific localization, and the apoptotic response was investigated by blocking monoclonal Ab. Mitochondrial HSP70 (mtHSP70) was identified as an HSP60 receptor. Stress was required for ECs to up-regulate mRNA and express mtHSP70 on their surface. HSP60 and mtHSP70 colocalized and interacted within lipid rafts. They were associated with chemokine CC motif receptor 5 (CCR5), also induced at the mRNA and protein levels in stressed ECs. CCR5 was involved in the anti-HSP60-triggered apoptosis of ECs. These results provide new insights into the mechanism by which anti-EC auto-Abs from vasculitides induce apoptosis of ECs.

Association of Defective Regulation of Autoreactive Interleukin-6-Producing Transitional B Lymphocytes With Disease in Patients With Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) has the highest case-specific mortality of any rheumatic disease, and no effective therapy is available. A clear manifestation of SSc is the presence of autoantibodies. However, the origin of autoantibody-producing B lymphocytes, their mechanisms of activation and autoantibody production, and their role remain unclear. This study was undertaken to identify mechanisms that contribute to pathogenic B cell generation and involvement in SSc and to assess the altered distribution and function of B cells in SSc patients. METHODS: Multicolor flow cytometry was performed to determine B cell subset distribution, cytokine production, and tolerance induction in SSc patients and healthy controls. Cytokine production following stimulation of the cells ex vivo was determined by multiplex assay. RESULTS: A range of defects in B lymphocyte tolerance and cytokine production in SSc were noted. There was evidence of altered distribution of transitional B cell subsets, increased production of interleukin-6 (IL-6) and IL-8, and defective tolerance induction in SSc B cells. In addition, B cells from SSc patients had a reduced ability to produce IL-10 when stimulated through innate immune pathways. In contrast to healthy individuals, tolerance checkpoints in SSc patients failed to suppress the emergence of B cells that produce autoantibodies with specificity to the Scl-70 antigen, which is strongly associated with SSc. These defects were paralleled by altered intracellular signaling and apoptosis following B cell receptor engagement. CONCLUSION: Our findings provide new insights into mechanisms underlying defective B lymphocyte responses in patients with SSc and their contribution to disease.

CD5 expression promotes IL-10 production through activation of the MAPK/Erk pathway and upregulation of TRPC1 channels in B lymphocytes.

CD5 is constitutively expressed on T cells and a subset of mature normal and leukemic B cells in patients with chronic lymphocytic leukemia (CLL). Important functional properties are associated with CD5 expression in B cells, including signal transducer and activator of transcription 3 activation, IL-10 production and the promotion of B-lymphocyte survival and transformation. However, the pathway(s) by which CD5 influences the biology of B cells and its dependence on B-cell receptor (BCR) co-signaling remain unknown. In this study, we show that CD5 expression activates a number of important signaling pathways, including Erk1/2, leading to IL-10 production through a novel pathway independent of BCR engagement. This pathway is dependent on extracellular calcium (Ca2+) entry facilitated by upregulation of the transient receptor potential channel 1 (TRPC1) protein. We also show that Erk1/2 activation in a subgroup of CLL patients is associated with TRPC1 overexpression. In this subgroup of CLL patients, small inhibitory RNA (siRNA) for CD5 reduces TRPC1 expression. Furthermore, siRNAs for CD5 or for TRPC1 inhibit IL-10 production. These findings provide new insights into the role of CD5 in B-cell biology in health and disease and could pave the way for new treatment strategies for patients with B-CLL.

Altered lipid raft-associated signaling and ganglioside expression in T lymphocytes from patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by abnormalities in T lymphocyte receptor-mediated signal transduction pathways. Our previous studies have established that lymphocyte-specific protein tyrosine kinase (LCK) is reduced in T lymphocytes from patients with SLE and that this reduction is associated with disease activity and parallels an increase in LCK ubiquitination independent of T cell activation. This study investigated the expression of molecules that regulate LCK homeostasis, such as CD45, C-terminal Src kinase (CSK), and c-Cbl, in lipid raft domains from SLE T cells and investigated the localization of these proteins during T cell receptor (TCR) triggering. Our results indicate that the expression of raft-associated ganglioside, GM1, is increased in T cells from SLE patients and LCK may be differentially regulated due to an alteration in the association of CD45 with lipid raft domains. CD45 tyrosine phosphatase, which regulates LCK activity, was differentially expressed and its localization into lipid rafts was increased in T cells from patients with SLE. Furthermore, T cells allowed to "rest" in vitro showed a reversal of the changes in LCK, CD45, and GM1 expression. The results also revealed that alterations in the level of GM1 expression and lipid raft occupancy cannot be induced by serum factors from patients with SLE but indicated that cell-cell contact, activating aberrant proximal signaling pathways, may be important in influencing abnormalities in T cell signaling and, therefore, function in patients with SLE.