Abstract: OBJECTIVE: We attempted to evaluate endothelial dysfunction and the role of AECAs in systemic lupus (SL) with low disease activity. We quantified endothelial microparticles (EMps) and attempted to find the best flow cytometry method for that purpose. METHODS: CD105, CD144 and CD146 were tested, individually or in combination, on EMp-enriched plasma. Twenty-three healthy blood donors and 27 SL patients were evaluated. SL patients with a SLEDAI <10 (median 2.6) were evaluated in our outpatient clinic. For each patient, EMps (CD105-CD146(+), CD45(-)) and AECAs were quantified and characterized. RESULTS: The monochrome composite marker CD105-CD146 appeared to be the most efficient in detecting EMps. SL patients had more circulating EMps than healthy donors: respective median values were 2575 and 130 EMps/microl (P < 0.001). SL patients had more CD54(+) and CD54(-) EMps than healthy donors (496 vs 34 EMps CD54(+)/microl, P < 0.0001; 1875 vs 89 EMps CD54(-)/microl, P < 0.0001). The ratio CD54(+) EMps/total EMps was lower for lupus patients than for healthy individuals (20.3 vs 33.7%, P = 0.03). Twenty-four patients (89%) were positive for AECAs. EMp counts were not significantly higher for patients with AECAs. CONCLUSION: Monochrome composite marker is efficient in detecting the whole population of EMps using flow cytometry. SL patients with low disease activity have a marked endothelial dysfunction. EMps released from the endothelium originate from activated and non-activated cells. AECAs do not seem to be the main cause of endothelial dysfunction in this population.
Abstract: Lupus nephritis (LN) remains the most common severe manifestation of systemic lupus erythematosus (SLE) characterized by the presence of autoantibodies (Abs) that are believed to play a central role in the pathogenesis of LN. Among more than 100 Abs reported in SLE, only a few display a direct glomerular binding capacity. Such antiglomerular Abs are detected at the onset of the disease before antinuclear Abs detection and proteinuria, this detection is associated with the related autoantigen overexpression. Antiglomerular Abs are able to interfere with cell metabolism, to penetrate living cells, and to induce glomerular cell proliferation. In addition, antiglomerular Abs could be nephritogenic causing proteinuria, particularly when they cross-react with anti-dsDNA Abs. Antiglomerular Abs encompass anti-alpha-actinin, anti-laminin-1, antifibronectin, antimyosin, and anticollagen Abs. The pathogenic activity of anti-alpha-actinin Abs has been demonstrated in non-autoimmune mice after immunization with alpha-actinin, but not with dsDNA, leading to a SLE-like disease with proteinuria and glomerular immune complex deposition. Similarly, extracorporeal immunoabsorption to remove anti-laminin-1 Abs reduces kidney-Abs deposition and proteinuria in mice and humans proving their pathogenic effect. Altogether this suggests that antiglomerular Abs participate, at least at the beginning, in the glomerular immune complex deposition and in the kidney damage.
Abstract: OBJECTIVE:: To determine if the FMS-like tyrosine kinase 3 ligand (FL), a cytokine implicated in B-cell ontogenesis and proliferation in hematological malignancies, might be responsible for the increased numbers of circulating Bm2 and Bm2' B-cell subsets in patients with primary Sjögren's syndrome (pSS). METHODS:: Serum levels of FL were measured in 64 pSS patients and 20 matched healthy controls. FL and its receptor Flt3 were quantified in circulating B cells and in salivary gland (SG) biopsies by immunofluorescence. The effect of FL on circulating B lymphocytes was then determined by coculture with human SG (HSG) cell line cells. RESULTS:: Serum levels of FL were increased in patients with pSS, compared to controls (135.8 +/- 5.5 versus 64.4 +/- 4.5 pg/mL, P < 0.001). The serum levels of FL in pSS patients correlated with the numbers of Bm2 and Bm2' (r=0.459, P<0.0006), and Flt3 was selectively expressed in Bm2 and Bm2' cells. B cell culture experiments showed that FL potentiates the proliferative effect of anti-IgM stimulation. In SGs, we found that infiltrating B cells express Flt3, and that epithelial cells produce FL. Finally, FL levels were associated with high scores of disease activity and increased risk of developing lymphoma. CONCLUSION:: Serum levels of FL are elevated in pSS and correlate with abnormal B-cell distribution. Flt3 is mainly express by Bm2 and Bm2' cells. Serum levels of FL might explain the clinical evolution of pSS to B cell lymphoma observed in some patients, thus opening the possibility of new avenues for therapy.
Abstract: Among various mechanisms for interactions with B cells, intravenous IgG (IVIg) may operate through the insertion of its Fc part into the Fc-gamma receptor, or the binding of its sialic acid (SA)-bearing glycans to the negatively-regulating CD22 lectin. It appeared that IVIg reduces B lymphocytes viability in a dose-and time dependent manner. Further, 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 signalling trough down-regulating tyrosine phosphorylation of Lyn and the B-cell linker proteins, and up-regulating phospholipase Cgamma2 activation. This cascade resulted in a sustained activation of Erk 1/2 and arrest of the cell cycle at the G1 phase. These changes may be accounted for the efficacy of IVIg in autoimmune diseases.
Abstract: During the 10th International Symposium on Sjögren's Syndrome held in Brest, France, from October 1-3, 2009 (http://www.sjogrensymposium-brest2009.org), the creation of an international epigenetic autoimmune group has been proposed to establish gold standards and to launch collaborative studies. During this "epigenetics session", leading experts in the field presented and discussed the most recent developments of this topic in Sjögren's Syndrome research. The "Brest epigenetic task force" was born and has scheduled a meeting in Ljubljana, Slovenia during the 7th Autoimmunity congress in May 2010.The following is a report of that session.
Abstract: Advances in genetics, such as sequencing of the human genome, have contributed to identification of susceptible genetic patterns in autoimmune diseases (AID). However, genetics is only one aspect of the diseases that does not reflect the influence of environment, sex or aging. Epigenetics, the control of gene packaging and expression independent of alterations in the DNA sequence, is providing new directions linking genetics and environmental factors. Recent findings have contributed to our understanding of how epigenetic modifications could influence AID development, showing differences between AID patients and healthy controls but also showing how one disease differs from another. With regards to epigenetic abnormalities, DNA methylation and histone modifications could be affected leading to large spatial and temporal changes in gene regulation. Other epigenetic processes, such as the influence of the ionic milieu around chromatin and DNA supercoiling stresses may be suspected also. The newly described role of microRNAs in control of gene expression is important by promoting or suppressing autoreactivity in AID. As a consequence control of cellular processes is affected becoming conducive, for example, to the development of autoreactive lymphocytes in systemic lupus erythematosus, synoviocyte proliferation in rheumatoid arthritis, or neural demyelination in multiple sclerosis. Application of epigenetics to AID is in its infancy and requires new hypotheses, techniques, tools, and collaborations between basic epigenetic researchers and autoimmune researchers in order to improve our comprehension of AID. From this will arise new therapeutics, means for early intervention, and perhaps prevention.
Abstract: In primary biliary cirrhosis (PBC) serum markers other than anti-mitochondrial antibodies (AMA) are promising in terms of disease severity and comorbidities, as well represented by anti-nuclear antibodies (ANA). The aim of the present study was thus to evaluate the prevalence and clinical significance of a large profile of serum autoantibodies in PBC sera. We utilized 69 sera from European patients with PBC (including 20 AMA-negative) and 297 sera from geographically and sex-matched healthy controls. All sera were tested for the presence of ANA and autoantibodies associated with thrombophilia, vasculitis, and gastrointestinal disease. Autoantibodies other than AMA were detected in 53/69 (76%) PBC sera vs. 105/297 (35%) among controls. The prevalence of ANA (targeting dsDNA, Sm, chromatin, ribosomal-P, RNP, SmRNP, SSA, SSB, and centromere) and thrombophilia-associated autoantibodies (i.e. anti-beta2GPI, phosphatydilserine, prothrombin) was common among patients with PBC. When clinical features were compared, the presence of anti-prothrombin IgM was associated with a worse prognosis as represented by a higher Mayo score. We demonstrate an increased prevalence of ANA and thrombophilia-associated autoantibodies in PBC sera and an association between the latter autoantibodies and PBC stage. The role of thrombophilia-associated antibodies will warrant further studies, based in particular on the incidence of portal hypertension at early stages of PBC.
Abstract: Autoreactive B cells are central in the pathogenesis of autoimmune diseases (AID) not only by producing autoantibodies but also by secreting cytokines and by presenting autoantigens. Changes in DNA methylation, histone modifications, and miRNA expression, the hallmarks of epigenetic failure, characterize B cells isolated from patients with AID, highlighting the contribution of epigenetic processes to autoreactivity. Additional evidence of epigenetic involvement in the development of B cell autoreactivity comes from in vivo and in vitro studies using DNA demethylating agents as accelerating factors or histone deacetylase inhibitors as repressing factors. As a result, a better understanding of the altered epigenetic processes in AID and in particular in B cells opens perspectives for the development of new therapeutics.
Abstract: Autoimmunity is believed to develop when genetically predisposed individuals encounter epigenetic modifications in response to environmental factors. Recent advances in the understanding of epigenetic mechanisms, their contribution to the immune function, and to the development of autoimmunity are presented in this special issue of Clinical Reviews in Allergy and Immunology. Potential new therapeutic strategies and biomarkers are also addressed.
Abstract: Rituximab is a monoclonal antibody to CD20, an antigen found on B cells. It was developed for the treatment of B cell lymphomas but was subsequently found useful in a number of autoimmune disorders. The efficacy of rituximab varies with the maturity and location of the target B cells, nature of the effector cells, pharmacokinetic considerations, time to B cell depletion and time to B cell restoration. As a result, substantial variability occurs across patients and between courses in a given patient. Laboratory tests can be used to monitor the effects of rituximab. Tests that predict the treatment response include CD20 density, Fc gamma IIIa receptor genotype and complement efficiency. To monitor the effect of rituximab, pharmacokinetic tests, B cell counts and xenoantibody levels are useful. Finally, to determine whether retreatment is in order, specific B cell subsets or autoantibodies can be looked for. None of the available tests is entirely reliable.
Abstract: BACKGROUND: The B cell-activating factor of the TNF family (BAFF) is upregulated in autoimmune diseases, but a number of conflicting results have cast doubts on the reliability of the ELISA protocols currently used for its quantification. This situation led us to develop a new ELISA for the measurement of BAFF. METHODS: BAFF was purified for use alongside nonglycosylated recombinant BAFF. Two monoclonal antibodies (mAbs) and two polyclonal antibodies (pAbs) to BAFF were used. RESULTS: The optimization process showed that the pAb format was preferable to the mAb format as capture antibody, because the pAbs recognized the glycosylated as well as the nonglycosylated forms of BAFF. The most efficient pair of Abs involved using the unconjugated form of a goat pAb to capture BAFF and the same biotinylated goat pAb to detect bound BAFF. This ELISA was not influenced by the presence of rheumatoid factor. CONCLUSIONS: This new ELISA helped provide insights into why serum concentrations of BAFF vary between studies for a given population of patients. It is a reliable tool for the management of the diseases in which BAFF is an indication of response to therapy.
Abstract: 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.
Abstract: B lymphocytes are divided into two subpopulations, B1 and B2 cells based on expression of the T cell-associated protein CD5. Natural B1 cells are further divided into B1a cells that express CD5 on their membrane and B1b cells that do not but share most other biological characteristics of B1a cells. Recent studies from our laboratory have revealed, in humans, the existence of two alternative isoforms of the CD5 protein. A cell surface CD5 isoform which uses exon 1A (E1A) of the gene in B1a cells, and an intracellular isoform which uses exon 1B (E1B) mainly in human B1b cells. Indeed, the protein isoform encoded by transcripts containing E1B lack the leader peptide and is, thus, retained in the cytoplasm of B cells. The restriction of interleukin (IL)-10 to B1 lymphocytes in the mouse raises the possibility that the human CD5-E1B-expressing B cells produce IL-10. This prediction was confirmed in the CD5 negative Jok-1 B cells transfected with cDNA for either isoforms resulted in high level IL-10 production. Our data indicate that E1B-CD5-expressing B cells have the capacity to interfere with the immune response through their ability to produce high levels of IL-10.
Abstract: The term pauci-immune glomerulonephritis with vasculitis encompasses a group of auto-immune disorders, which includes Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, and renal-limited vasculitis. Over the past few years, progress has been made in understanding the epidemiology and environmental and genetic risk factors of the role of antineutrophil cytoplasmic antibodies (ANCA) in kidney pathogenesis and the utilization of ANCA in diagnosis. However, certain aspects are still subject to debate including the classification and the place of ANCA in treatment.
Abstract: The anti-CD20 monoclonal antibody rituximab has been less successful in treating chronic lymphocytic leukemia (CLL) than lymphoma, possibly due to the lower density of CD20 on B lymphocytes from CLL patients than on those from lymphoma patients. This lowering may result from insufficiency of one of the transcription factors of cd20. Of these, purine-rich box-1 (PU.1) is poorly expressed in CLL. To estimate its weight in CD20 expression, pu.1 cDNA was transfected into CLL B cells and shown to raise the membrane expression of CD20 and to improve the rituximab-induced lysis of transfected cells. Granulocyte macrophage colony-stimulating factor and all-trans-retinoic acids were not involved in the defective expression of PU.1 or the excessive methylation of the pu.1 gene, because 6 of 14 CLL samples tested were normally methylated. This was confirmed by the failure of DNA methyltransferase inhibitors to restore pu.1 transcription in hypermethylated CLL, and, in fact, the expression of PU.1 was down-regulated by excessive expression of the FMS proto-oncogene-like tyrosine kinase 3 (Flt3) receptor. This abnormality is consistent with our finding of elevated levels of Flt3 ligand (FL) in 20 of 23 CLL sera tested. We propose that FL-dependent increased Flt3 signaling prevents the expression of PU.1, which down-regulates that of CD20, and accounts for resistance of leukemic B cells to rituximab-induced lysis.
Abstract: The human cd5 gene has two alternative exons 1: exon 1A (E1A) which encodes the full-length (FL) CD5 protein and exon 1B (E1B) which encodes a truncated (TR) isoform. The FL variant of CD5 protein is translocated to the plasma membrane, while its TR variant is retained in the cytoplasm. Because there is an inverse relationship between the levels of FL-CD5 and TR-CD5 in B cells, we have addressed the issue of how the selection of exon 1 is determined. In leukemic B cells, DNA methyltransferase (DNMT)1-induced methylation of E1B prevents its transcription. Furthermore, the level of mRNA for DNMT1 correlates inversely with that of mRNA for CD5-E1B. However, suppression of E1B transcription is incomplete, and some molecules of TR-CD5 continue to be synthesized. Bortezomid-induced inhibition of the proteasome establishes that these TR-CD5 molecules are cleared through the ubiquitin-proteasome pathway. Transfection of CD5 mutants into COS-1 cells locates the ubiquitin-binding site at the second destruction box of the extracellular region of CD5. Activation of the B cells by anti-IgM, Staphylococcus aureus Cowan I (SAC), or PMA up-regulates DNMT1, and thereby CD5-E1A mRNA at the expense of CD5-E1B mRNA. Aberrant synthesis of TR-CD5 is thus offset by balanced degradation of excessive protein. Dysregulation of these mechanisms reduces the expression level of membrane CD5, and thereby diminishes the threshold of the response by cells expressing CD5.
Abstract: OBJECTIVE: There is evidence to support a dominant role for B cells in the pathophysiology of primary Sjögren's syndrome (SS). Therefore, we evaluated the safety and efficacy of anti-CD20 monoclonal antibody. METHODS: Sixteen patients who met the new American-European Consensus Group criteria for primary SS and scored >50 on at least 2 of 4 visual analog scales (VAS; 100 mm) evaluating global disease, pain, fatigue, and global dryness received infusions of low-dose rituximab (375 mg/m(2)) at weeks 0 and 1 without steroid premedication. RESULTS: Slow rituximab infusions (100 mg/hour) were well tolerated, with only 1 patient experiencing serum sickness-like disease. There was a dramatic reduction in B cells of the blood and salivary gland (SG). At week 12, VAS scores with respect to fatigue and dryness (P < 0.05), tender point count (P < 0.035), and quality of life as evaluated by the Short Form 36 questionnaire (SF-36; P < 0.001) were significantly improved. At week 36, significant improvements were noted in the 4 VAS scores (P < 0.05), tender joint count (P = 0.017), tender point count (P = 0.027), and SF-36 (P < 0.03). Pulmonary manifestations were ameliorated in 1 patient. Patients with improvements on at least 3 of the 4 VAS scores at any visit (n = 11) had a shorter disease duration than the other patients (n = 5; mean +/- SD duration 3.8 +/- 5.4 versus 30.1 +/- 29.5 years; P = 0.02). CONCLUSION: Low-dose rituximab infusions were well tolerated without the benefit of steroids. Infusions induced a rapid depletion of B cells in the blood and SG and could improve primary SS. Controlled studies are needed.
Abstract: The exact role of anti-ds (double stranded) DNA antibodies in the pathogenesis of kidney injury in lupus nephritis remains a focus of continuing investigation. One theory explaining the pathogenicity of anti-dsDNA antibodies in lupus nephritis is direct cross-reactivity with renal antigens. Several years ago, alpha-actinin was identified as a major cross-reactive target for pathogenic anti-dsDNA antibodies in murine SLE. Indeed, binding of a nephritogenic murine anti-dsDNA antibody was stronger to the alpha-actinin derived from a lupus prone mouse mesangial cell line as compared to alpha-actinin in a non-autoimmune mouse mesangial cell line. Furthermore, we recently showed that immunization of non-autoimmune mice with alpha-actinin induces anti-chromatin antibodies, glomerular IgG deposition and proteinuria. In humans, anti-alpha-actinin autoantibodies (Ab) were associated with anti-dsDNA Ab in SLE. In those patients, anti-alpha-actinin rather than anti-dsDNA Ab were significantly associated with glomerulonephritis and disease activity. The anti-alpha-actinin reactivity was associated with high avidity anti-dsDNA Ab. Moreover, the anti-alpha-actinin response was related to the actin-binding site of alpha-actinin. Taken together, these studies indicate that detection of anti-alpha-actinin Ab, in association with anti-dsDNA Ab, may constitute a new marker in lupus nephritis.
Abstract: OBJECTIVES: Noninvasive objective tests are needed to diagnose primary Sjogren's syndrome (pSS) and to evaluate treatment responses. Ultrasound imaging of the salivary glands is rapid and noninvasive. Recent open-label studies suggested that anti-CD20 (rituximab) may be effective in pSS. The purpose of this study was to look for ultrasound evidence of the effects of rituximab in pSS. METHODS: We compared 16 patients fulfilling the new American-European consensus group criteria for pSS to 9 controls, using B-mode ultrasound features (parenchymal homogeneity and gland size) and Doppler waveform analysis of the transverse facial artery of parotid glands. We compared the same parameters in the patients before and after 12 weeks of intravenous rituximab therapy. RESULTS: Compared to controls, untreated patients had significant abnormalities in salivary gland structure (p < 0.0001) and parotid size (2.05 +/- 0.33 cm versus 1.70 +/- 0.28 cm; p = 0.001). Doppler waveform analysis showed significant differences before, but not after, lemon stimulation between untreated patients and controls. After rituximab treatment, significant size reductions were noted in the parotids (2.05 +/- 0.3 cm at baseline and 1.86 +/- 0.27 cm at week 12; p = 0.002) and submandibular glands (2.02 +/- 0.54 cm at baseline and 1.66 +/- 0.34 cm at week 12; p = 0.001). Doppler resistive indices after lemon stimulation were significantly increased after rituximab treatment. CONCLUSION: Salivary gland measurements and blood inflow responses to salivary stimulation as assessed by ultrasound hold promise as objective noninvasive tools for evaluating rituximab effects in patients with pSS.
Abstract: The pathophysiological relevance of B cells for systemic lupus erythematosus (SLE), particularly those expressing the T-cell marker CD5, raises the question as to how they operate upon autoimmune processes. Based on their production of low-affinity multispecific antibodies (Abs), CD5(+) B lymphocytes, also referred to as B1 cells, have originally been endowed with the autoAb making. It has since been established that high-affinity Abs to double-stranded DNA are not generated by these cells, but rather by B2 cells. It does not appear that they have the exclusive rights to the production of pathogenic autoAbs. In the light of recent findings, CD5 plays a paradoxical role in preventing autoimmunity. Hence, misguided signaling through CD5 could lead to autoimmunity. This provocative view differs from the naïve interpretation that the increased levels of B1 cells in SLE represent a direct source of autoAbs responsible for damaging organs.
Abstract: OBJECTIVE: Treatment with rituximab depletes B cells from the peripheral blood (PB) and salivary glands (SGs) of patients with primary Sjögren's syndrome (SS). The purpose of this study was to track the repopulation of B cell subsets in PB as well as their subsequent homing into SGs in patients with primary SS treated with rituximab. METHODS: A series of 4-color flow cytometry experiments delineated B cell subsets in 15 patients with primary SS. All were tested on days 8 and 15 of treatment. Nine of the patients were followed up monthly for 10 months, and the remaining 6 patients were followed up monthly for 24 months. Enzyme-linked immunosorbent assays were developed to measure serum levels of BAFF and rituximab. SGs were biopsied at the start of the study and 4 months after treatment in 15 patients, 12 months after treatment in 3 patients, and 24 months after treatment in 2 patients. RESULTS: Baseline serum levels of BAFF correlated inversely (r = -0.92, P < 5 x 10(-4)) with the duration of B cell depletion: the higher the BAFF levels, the shorter the duration of B cell depletion. Four B cell subsets repopulated the PB: plasmablasts (CD19+, CD5-,IgD-,CD38++), transitional type 1 (T1) B cells (CD19+,CD5+,IgD+,CD38++), mature Bm2 cells (CD19+,CD5+/-,IgD+,CD38+/-), and memory B cells (CD19+,CD5-,IgD-,CD38-). Increased numbers of Bm2 cells and decreased memory B cells reappeared with time. Sequential SG biopsies revealed that B cells were absent in these glands for 12 months: they were detected 24 months after rituximab treatment. Memory and T1 B cells were the first B cells identified locally. CONCLUSION: The timing of B cell repopulation is modulated by BAFF and is followed by reconstitution of the preexisting abnormalities.
Abstract: Chronic lymphocytic leukemia (CLL) does not result from accumulation of CD5(+) B cells but, presumably, represents an antigen-driven dynamic process. Autoimmune diseases (AIDs) include hemolytic anemia, thrombocytopenic purpura and immune neutropenia. In turn, CLL and B-cell lymphoma develop in the frame of an AID. Such reciprocal interactions suggest that similar B cells are involved in both disorders. Phenotypic features (i.e., several membrane markers) and functional characteristics of CD5(+) B cells (i.e., Epstein-Barr virus transformation and expression of apoptotic proteins) distinguish CLL and AID CD5(+) B cells from their normal counterparts. These cells produce often nonpathogenic polyspecific low-affinity autoantibodies, whereas they present the antigen to antiself B or T cells to feature pathogenic monospecific high-affinity autoantibody. The CD5 molecule itself plays a part by translocating phosphatases to the vicinity of the B-cell antigen receptor, thereby precluding transduction from the B-cell receptor. Such might be the link between CLL and AID, both prevented by the CD5 machinery.
Abstract: Evidence is lacking that antibodies (Ab) to endothelial cells (AECA) are pathogenic. They are frequently associated with antiphospholipid Ab (aPL), binding to complexes of phosphatidylserine (PS) with beta2GPI. Recent studies have, however, kindled a new debate on their pathogenicity of AECA. A group is responsible for PS reaching the surface of a cell, a feature of commitment to apoptosis. Defective clearance by macrophages of AECA-induced apoptotic cells might display beta2GPI on their surface, and challenge T cell tolerance, until aPL production. Some AECA are thus induced by cell membrane structures, while others recognize "planted" antigens and possibly ligand-receptor complexes. A second group promotes procoagulant factor, and a third has the capacity to trigger apoptosis. Clearly, the most direct demonstration of the pathogenicity of AECA is the autoAb-induced murine model of vasculiltis.
Abstract: OBJECTIVE: Anti-double-stranded DNA (anti-dsDNA) antibodies may contribute to the pathogenesis of glomerulonephritis (GN) by cross-reacting with alpha-actinin in murine models and in some patients with systemic lupus erythematosus (SLE). We therefore sought to determine possible disease associations with serologic and clinical features and to characterize this new autoantibody specificity. METHODS: One hundred patients with SLE were recruited into this multicenter study, as well as 100 rheumatic disease controls and 2,100 healthy blood donors. Clinical disease was evaluated by the SLE Disease Activity Index (SLEDAI; excluding the anti-DNA component). Anti-dsDNA antibodies were detected by conventional enzyme-linked immunosorbent assay (ELISA) and by a commercial enzyme immunoassay (EIA). Anti-alpha-actinin antibodies were detected by ELISA, and their specificity was confirmed by Western blotting and by indirect immunofluorescence using rat kidney sections and mesangial cells as substrates. Highly positive sera were selected for absorption experiments and were affinity-purified for cross-reactivity studies and measurement of antibody avidity. RESULTS: Sera from 62 of the SLE patients had anti-dsDNA antibodies; 21 of these sera also had anti-alpha-actinin antibodies, as compared with 1 of the 38 sera without anti-dsDNA antibodies. Of the 22 patients with anti-alpha-actinin antibodies, 10 had GN, as compared with 14 of the 78 without anti-alpha-actinin antibodies (P < 0.01). In patients with GN, anti-alpha-actinin, but not anti-dsDNA, antibodies correlated with the SLEDAI score (minus the anti-DNA component) and with treatment. The fraction of serum anti-dsDNA antibodies that cross-reacted with alpha-actinin exhibited high avidity for dsDNA, as determined using a commercial EIA for high-avidity anti-dsDNA antibodies and an in-house conventional ELISA. CONCLUSION: The alpha-actinin-binding antibodies are significantly associated with GN in SLE. Whether such autoantibodies may anticipate the development of this complication of SLE remains to be verified.
Abstract: Anti-filamentous actin antibodies characterize autoimmune hepatitis type 1 (AIH-1). Recently, the binding domain of alpha-actinin on actin was shown to be a predominant epitope. To test this reactivity, an anti-alpha-actinin enzyme-linked immunosorbent assay was developed, and positivity confirmed by Western blot. Anti-alpha-actinin antibody was found in 21/50 (42%) of AIH-1 patients, compared with 52/401 (12.9%) of liver disease control patients, and with 6/200 (6%) of blood donors. Anti-filamentous and anti-alpha-actinin activities were found specifically together in 66% of anti-filamentous-positive AIH-1 patients. This combination of specificities reflected clinical and histological disease activity, short duration and absence of treatment. Finally, using an actin-alpha-actinin complex assay, the binding of anti-filamentous actin to alpha-actinin-binding domain on actin was demonstrated, as well as that of anti-alpha-actinin on the actin-binding domain of alpha-actinin. Thus, the frequent combination of anti-filamentous and anti-alpha-actinin antibodies seems to be the hallmark of activity in AIH-1.
Abstract: The paradigm that B cell response to self antigens (Ag) is promoted by antibodies (Ab) has become unsatisfactory. Studies over the last decade have indeed revealed that B cells serve extraordinarily diverse functions within the immune system other than Ab production. They normally play a role in the development in the regulation, as well as the activation of lymphoid architecture, regulating dentritic cells and T cell subsets function through cytokine production. Receptor editing is also essential in B cells and aids in preventing autoimmunity. Both abnormalities in the distribution of B cells subsets and clinical benefit response to B cell depletion in autoimmune states illustrate their importance. Transgenic animal models have demonstrated that sensitivity of B cells to Ag receptor cross-linking correlates to autoimmunity: negative signaling by CD5 and CD22 in maintaining tolerance through recruitment of phosphatase has thus been documented. In short, a new area has been reached, whereby B lymphocytes return as a significant contributor to autoimmune disorders.
Abstract: All T lymphocytes and some B lymphocytes express CD5. This coreceptor is encoded by one gene that consists of 11 exons. We have previously described a B cell-specific alternative exon 1, leading to the synthesis of a protein, devoid of leader peptide, and, therefore, retained in the cytoplasm. The novel exon 1 originates from a human endogenous retrovirus (HERV) at a time interval between the divergence of New World monkeys from Old World monkeys, and prior to the divergence of humans from Old World monkeys. Based on sequence similarity to gamma-retroviruses, it was categorized as class I: based on the specificity of its primer binding site, it was allotted to the subclass E, and based on its location within the cd5 gene, named HERV-E.CD5. Alternative transcripts were detected in lymphoid organs including fetal liver (not adult liver), more particularly in CD5-negative cell surface B-1b than in CD5-positive cell surface B-1a, and not at all in B-2 cells. By alignment of 5' long terminal repeats, HERV-E.CD5 was distinguished from similar proviruses. This could be central to the regulation of membrane expression of CD5 in human B lymphocytes, and, thereby, to the strength of the B-cell antigen receptor signaling.
Abstract: Rheumatoid factors (RF), which are antibodies (Ab) with specificity directed against gamma (?) globulins, are the commonest auto-Ab ever described in man. Some of them are referred to as agglutinating RF, others designated non-agglutinating RF. Not only do these characterize rheumatoid arthritis (RA), but they are also encountered in a variety of disease conditions, as well as a proportion of healthy controls. Although non-specific for RA, the measurement of agglutinating IgM-RF remains the most useful serological test for the diagnosis of this disease. Demonstration of abnormal amount of serum RF by any method for which the result has been positive in less than 5% of normal subjects has indeed become one of the seven revised criteria, listed by the American College of Rheumatology (Arnett, FC, Edworthy, SM, Bloch, DA, McShane, DJ, Fries, JF, Cooper, NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum, 1988: 31: 315-24), for the classification of RA. Over the course of years, the relative importance of genetic (Carson, DA, Chen, PP, Kipps, TJ, Radoux, V, Jirik, FR, Goldfien, RD, et al. Idiotypic and genetic studies of human rheumatoid factors. Arthritis Rheum, 1987: 30: 1321-1325) and environmental (Nemazee, DA, Sato, VL. Enhancing antibody, a novel component of the immune response. Proc Natl Acad Sci USA, 1982: 79: 3828-3832) factors in the production of such intriguing auto-Ab has been delineated.
Abstract: T lymphocytes and a subpopulation of B lymphocytes express the CD5 coreceptor. Its functional importance is evident from the multiple levels and developmental stages of the regulation of its expression. We here report the discovery of a novel regulatory exon upstream of the noncoding region of the CD5 gene in humans. This alternate exon 1 is designated E1B (with the conventional exon 1 renamed E1A) and was shown to regulate the expression of CD5. E1B-containing transcripts existed exclusively in B lymphocytes and encoded a protein that was truncated and retained intracellularly. As a consequence, the amount of E1A-containing transcripts was down-regulated and the membrane CD5 expression was diminished in the presence of E1B-containing transcripts. High levels of E1A transcripts were found in chronic lymphocytic leukemia, and there were no E1A transcripts in 697 pre-B cells, which have no membrane CD5. Introduction of E1B into Jurkat cells reduced their membrane expression of CD5, and sequence analysis revealed that the E1B motif is a defective human endogenous retrovirus. A balance between the 2 alternative exons 1 might be central to the regulation of membrane CD5 in human B cells, and, through CD5-associated SH2-containing phosphatase 1, to the modulation of B-cell antigen receptor-transduced signals.
Abstract: Recent studies have revealed that B cells serve extraordinarily diverse functions within the immune system in addition to antibody production. These functions contribute to autoimmunity. They initiate the development of lymphoid architecture and regulate dendritic and T-cell function through cytokine production. Receptor editing is also essential to prevent autoimmunity. Both abnormalities in the distribution of B-cell subsets and the benefits of ablative B-cell therapy of autoimmune states confirm their importance. Results from transgenic models have demonstrated that the sensitivity of B cells to antigen receptor cross-linking correlates to autoimmunity, with particular reference to negative signaling by CD5 and CD22. These mechanisms maintain tolerance by recruiting src-homology 2 domain-containing protein tyrosine phosphatase-1. These findings open new prospects for immunotherapy of autoimmune diseases.
Abstract: In murine systemic lupus erythematosus (SLE) models, nephritogenic anti-dsDNA IgG has been shown to cross-react with a kidney antigen, alpha-actinin, and to be critical in renal pathogenesis. In humans, studies of anti-alpha-actinin antibodies (Abs) are scarce, and these antibodies remain to be evaluated. We have thus far tested sera from patients with SLE (n = 103), rheumatoid arthritis (RA, n = 93), and primary Sjögren syndrome (pSS, n = 34), and from healthy subjects (n = 160), for the presence of anti-alpha-actinin and anti-DNA Abs. The latter were tested using several methods [IIF on Crithidia luciliae (Crit) and ELISA using dsDNA]. Anti-alpha-actinin Abs were confirmed by Western blot. Sera from 23 of 103 SLE patients, 3 of 93 RA patients, 1 of 33 pSS patients, and 1 of 160 controls scored positive for anti-alpha-actinin Abs. In SLE, the positivity was significantly associated with anti-dsDNA reactivity (22 of 23): 19 of 23 sera were alpha-actinin-positive/dsDNA-positive and 13 were alpha-actinin-positive/Crit-positive. Few cases were alpha-actinin-positive/dsDNA-negative: 1 SLE, 3 RA, and 1 control. Furthermore, anti-alpha-actinin Abs have been detected at high level before or at the early stage of lupus nephritis when compared with active and inactive SLE without kidney manifestations.
Abstract: On the basis of responses to surface IgM (sIgM) cross-linking, B cells from 41 patients with B-chronic lymphocytic leukemia were categorized as 15 nonresponders (group I) and 26 responders (group II). The latter cases were subclassified as those seven where proliferation was induced (subgroup IIa) and the remaining 19 in whom apoptosis occurred (subgroup IIa). Signal disruption in group I was confirmed by the absence of Ca2+ mobilization. Activation of PI3K was constitutive in subgroup IIa, but not in subgroup IIb, and that of Akt induced by anti-mu in subgroup IIa, but not in subgroup IIb. Among the MAPK, ERK was more highly activated relative to p38 in subgroup IIa, whereas activation of p38 predominated over that of ERK in subgroup IIb. For subgroup IIb cells, based on tyrosine phosphorylation and translocation into lipid rafts, sIgM signaling was shown to be enhanced by Zap70. The different consequences of signaling through sIgM were associated with biological prognosis indicators. These included high levels of CD38, lack of mutations in the IgVH chain genes, preferential usage of full-length CD79b, and severe clinical stage. Thus, modification of sIgM-induced signaling could be a therapeutic approach.
Abstract: A total of 40 patients with B-CLL were investigated for CD5-triggered apoptosis and categorized as 20 resistant (group I) and 20 sensitive patients (group II). The densities of surface IgM (sIgM) and CD5 were lower in group I than group II, as were the percentages of CD79b+, CD38+, and Zap70-expressing B cells. CD5 signaling was mediated through the BCR in group II B cells, as established by coimmunoprecipitation of CD5 and CD79a and tyrosine phosphorylation of CD79a. Following colocalization of CD5 and sIgM in membrane lipid rafts (LRs), Syk became associated with these molecules, whereas SHP-1 was uncoupled from CD5. Nonresponsiveness to CD5 cross-linking in group I was ascribed to three possible abnormalities, and defined as three subgroups of patients. In subgroups Ia and Ib, CD5 and sIgM colocalized within the LRs. SHP-1 remained attached to the BCR in subgroup Ia, but not in subgroup Ib, where signal transduction was associated with an excess of truncated CD79b. In subgroup Ic, CD5 and sIgM segregated into different LRs, resulting in no signaling of apoptosis.
Abstract: Growing awareness of the pathophysiological importance of B cells for antiphospholipid antibody syndrome (APS), particularly those expressing the T cell marker CD5, has recently led to the proposal that their tolerance may be used as a method to reduce specific antibody (Ab) production. B cell tolerance has indeed become one of the most exciting developments in the treatment of this disease. Based on their production of multispecific Ab, these CD5+ B lymphocytes, also referred to as B-1 cells, are thought to account for most of the AutoAb in autoimmune murine models. Raised numbers of circulating CD5+ B cells correlate with high levels of anti-phospholipid (PL) Ab in some APS patients, and participate in altered immunity of women with recurrent spontaneous abortion. These findings are not surprising in view of the cross-reaction with PL of anti-bromelain-treated erythrocyte Ab secreted by these cells. Transgenic animals have, however, shown that B lymphocytes contribute to such disorders through a variety of characteristics other than Ab production. Indeed, owing to the role of the CD5 molecule in the maintenance of clonal anergy, increased proportions of B-1 cells may merely reflect their defective regulation through CD5 itself. Various B cell receptor (BCR)-associated transmembrane glycoproteins are also involved in the behavior of the cells. These include CD19 which amplifies the message, and CD22 which dampens down the BCR signaling. In addition, B lymphocytes may act as potent antigen-presenting cells for autoantigens, all the more because they secrete an excess of autocrine-acting interleukin-10 in autoimmune states. Furthermore, by modifying the specificity of their BCR, not only in the bone-marrow, but also in the secondary lymphoid organs, autoreactive B cells may initiate new immunoglobulin rearrangements. It is interesting that self-reactive Ab-making cells present with such rearrangements. Finally, B cells have the capacity to polarize into B effector (Be)-1 and Be-2, with different cytokine patterns that regulate the levels of T helper (Th)-1 and Th-2, respectively. Such a cytokine might be defective in nonorgan-specific autoimmune diseases. In conclusion, B lymphocytes are required for the initiation of anti-self Ab-associated disorders, such as APS. Their classical view in the biology of immune responses to self as autoAb secreting cells turns out to be rather naive, and an essential role for B lymphocytes may not be producing autoAb.
Abstract: The classical view of B cells in the biology of autoimmune responses to infectious and self-antigens (Ag) that they promote immunity primarily by producing antibodies (Ab) is far from being complete. Indeed, studies over the last decade suggest that B cells have extraordinarily diverse functions within the immune system other than Ab production, which could contribute to autoimmunity. They normally play a role in the development of lymphoid architecture, regulating dentritic cells (DC) and T cell subsets function through cytokine production, and in activation of T cells. Receptor editing is also important in B cells which aids in immunity to infection and, possibly, prevention of autoimmunity. Both abnormalities in the distribution of B cells subsets and clinical benefit response to B cell depletion in autoimmune diseases, including systemic lupus erythematosus (SLE), highlight their pivotal function. Transgenic (Tg) animal models have shown that sensitivity of B cells to B cell Ag receptor (BCR) cross-linking is correlated to autoimmunity. Indeed, negative signaling by CD5 and other molecules, such as CD22, in maintaining tolerance through recruitment of src-homology two domain-containing protein tyrosine phosphatase-1 (SHP-1) has also been documented. In fact, we have now reached a newer area whereby B cells returned as an important contributor to autoimmune disorders.
Abstract: As a result of damaging endothelial cells (ECs), Mycobacterium leprae triggers the production of antibodies (Abs). These anti-EC Abs (AECAs) can be divided into two types. The first type nonspecifically reacts with components of the cytosol (CY) and can be detected by enzyme-linked immunosorbent assay (ELISA). The second specifically reacts with the EC membrane (MB) and requires fluorescence-activated cell sorter (FACS) analysis to be detected. The presence of both types of AECAs was determined in 68 leprosy patients. The ELISA was positive for 35 of them but also for 30 of 34 malaria patients and 17 of 50 healthy African controls. However, whereas FACS analysis showed MB reactivity in only three malaria patients and four controls, this reactivity was found in 27 leprosy patients, more of those having the lepromatous than the tuberculoid form. Specificity for MB, which we failed to absorb by incubation with CY lysates, predominated over that for CY in leprosy, unlike malaria, where the EC reactivity was restricted to the CY. Western blot analysis and two-dimensional electrophoresis revealed that calreticulin, vimentin, tubulin, and heat shock protein 70 were targeted by AECAs from leprosy patients, but other proteins remained unidentified. These auto-Abs, but not those from malaria patients, did activate ECs, as indicated by the E-selectin and intercellular adhesion molecule 1 upregulation, and/or induced them into apoptosis, as documented by four different methods. Our findings suggest that, in some but not all leprosy patients, AECAs may play a role in pathogenesis.
Abstract: Sera from patients with systemic lupus erythematosus have been reported to contain IgM and/or IgG binding to endothelial cells (EC), i.e. anti-EC antibodies (AECA). Similar autoantibodies have been claimed to occur in a number of conditions associated with vasculitis. The original cyto-enzyme-linked immunosorbent assay (ELISA) remains the most widely used method for the detection of AECAs, although numerous pitfalls have been identified since then. These difficulties may explain why a consensus on the prevalence of AECAs has not been reached thus far. It is therefore desirable to confirm a positive result in the cyto-ELISA using other methods, such as flow cytometry, immunoprecipitation or Western blot. Yet, these methods appear to be difficult to use on a routine basis. With regard to the AECA effects, their binding induces activation of ECs, as substantiated by up-regulation of adhesion molecules, and synthesis of cytokines and chemokines, followed by their secretion. Some of these autoantibodies encourage the local production of tissue factor, and thereby favour coagulation. Other AECAs trigger apoptosis of ECs, although the Fas receptor does not seem to be involved in this process. In fact, since the target antigens are not well defined, the current challenge is to identify EC target molecules, and thus to gain further insights into the pathogenesis of diseases with vasculitis.
Abstract: The receptors FcgammaRIIIb and FcgammaRIIa for the Fc portion of IgG are naturally expressed in polymorphonuclear neutrophils (PMN). Autoantibodies (Ab) against FcgammaRIIIb exist in patients with non-organ-specific disease. These may be categorized, based on the results of an indirect immunofluorescence (IIF) test for the detection of anti-membrane-bound FcgammaRIIIb autoAbs, and an enzyme-linked immunosorbent assay for that of soluble FcgammaRIIIb-recognizing autoAbs. The IIF+ autoAbs are not cytotoxic, and prolong the survival of the cells. The autoAb-triggered anti-apoptotic signal may be transduced through FcgammaRIIa and/or CD11b, the beta-chain of the neighboring complement receptor type 3. However, FcgammaRIIIb appears to be as competent as FcgammaRIIa, because the results obtained using the respective monoclonal Abs are additive. Soluble FcgammaRIIIb binds to CD11b and produces similar effects, suggesting that autoAb-stimulated FcgammaRIIIb can work in concert with CD11b. Anti-FcgammaRIIIb-conditioned supernatant of PMNs induces the transcription of messenger RNA for granulocyte colony-stimulating factor (CSF) and granulocyte-macrophage CSF, followed by the release of these anti-apoptotic factors. The delay in apoptosis is accompanied by a down-regulated expression of Bax. Thus, apoptosis of aged PMNs can be modulated by signaling through FcgammaRIIIb, which may occur in patients with PMN-binding anti-FcgammaRIIIb autoAbs.
Abstract: Polymorphonuclear neutrophils (PMNs) express constitutively the Fc gamma receptors (Fc gamma Rs) Fc gamma RIIIb and Fc gamma RIIa for the Fc part of IgG, and soluble Fc gamma RIIIb (sFc gamma RIIIb) has been identified. Elevated levels of sFc gamma RIIIb were detected in non-organ-specific autoimmune conditions, and there was a considerably decreased number of PMNs undergoing apoptosis in the presence of sFc gamma RIIIb. Anti-Fc gamma RIIIb autoantibodies (autoAbs) have also been described in such patients. They are not cytotoxic to these cells, but they extend their survival. The anti-apoptotic signal can be transduced through Fc gamma RIIa and/or CD11b, the beta-chain of the complement receptor 3. However, Fc gamma RIIIb appears to be also competent. Anti-Fc gamma RIIIb-conditioned supernatant from cultured PMNs induces the transcription of messenger RNA for granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor, followed by protein synthesis. The delay in apoptosis may be generated by a downregulation of the death promoter Bax. Inflammation might thus be modulated by sFc gamma RIIIb and anti-Fc gamma RIIIb autoAbs in systemic diseases.
Abstract: Anticardiolipin (ACA), anti-beta2 glycoprotein I (beta2GPI), and antiprothrombin antibodies of IgG and IgM classes were quantitated by enzyme-linked immunosorbent assays in 176 untreated leprosy patients across the histopathological spectrum. Positivity rates ranged from 21% (IgG ACA) to 30% (IgM anti-prothrombin) versus 4% in healthy controls (p <10(-2) to 10(-3)). Levels of IgM anti-beta2GPI and IgG ACA were significantly higher in lepromatous leprosy and multibacillary patient subgroups. IgG3 was the most common subclass reactive to both beta2GPI and prothrombin in selected high-titer leprosy sera, unlike antibodies from patients with the antiphospholipid syndrome (APS) largely restricted to IgG2. In leprosy patients, but not in the APS control group, there was no statistical correlation between ACA and anti-beta2GPI antibody levels. Likewise, a large fraction of anti-beta2GPI positive sera (36/45 and 28/44 for IgG and IgM, respectively) were unreactive in the standard ACA assay. Most assayed anti-beta2GPI antibodies from leprosy patients showed (i) ability to recognize both human and bovine beta2GPI immobilized on non-irradiated polystyrene plates, (ii) concentration-dependent inhibition of binding by cardiolipin, and (iii) relatively high avidity binding to fluid-phase beta2GPI, thereby differing from those found in APS. Finally, the location of the major epitopic region on the beta2GPI molecule targeted by autoantibodies was different in leprosy and APS, as assessed by direct binding to domain I- and V-deleted mutants and competition with the mouse monoclonal antibody 8C3, directed at domain I. Thus, leprosy-related antiphospholipid antibodies comprise persistent IgG and IgM anti-beta2GPI that differ from APS-related ones with respect to IgG subclass, avidity and epitope specificity, possibly reflecting distinct pathophysiological significance.
Abstract: We have established that polymorphonuclear neutrophil (PMN)-binding anti-Fcgamma receptor IIIb (FcgammaRIIIb) autoantibodies (autoAb) inhibit the function of these cells but extend their survival. Here, we show that recombinant FcgammaRIIIb (rFcgammaRIIIb), as well as purified FcgammaRIIIb (pFcgammaRIIIb), deteriorated the PMN adherence and respiratory burst in a dose-dependent manner. Furthermore, rFcgammaRIIIb and pFcgammaRIIIb reduced the level of annexin V-binding PMN from 23.6 +/- 1.6 % to 6.3 +/- 1.0 and 11.0 +/- 1.0 %, respectively, while human serum albumin exerted no effects. Incubation of rFcgammaRIIIb with those autoAb binding to soluble FcgammaRIIIb resulted in the attachment of such immune complexes (IC) to the cells, thereby also delaying apoptosis (44.9 +/- 5.9 versus 18.0 +/- 2.0 % annexin V-binding PMN after 16 hours). Soluble FcgammaRIIIb, in concert with FcgammaRIIIb / anti-FcgammaRIIIb IC, produced similar effects in that the percentage of annexin V-binding PMN declined to 16.0 +/-1.9 %. It was thus suggested that FcgammaRIIIb / anti-FcgammaRIIIb IC inserted the Fc region of their IgG into the membrane FcgammaRIIIb. Such an interpretation is consistent with our finding that, whereas aggregated IgG and anti-FcgammaRIIIb monoclonal Ab prevented membrane FcgammaRIIIb / IC interaction, neither soluble FcgammaRIIIb, nor anti-cgammaRII did so. We conclude that the function and the life span of PMN are influenced synergistically by soluble FcgammaRIIIb and anti-FcgammaRIIIb autoAb.
Abstract: Our early concepts of the normal role of B cells in immunity focused on their ability to produce antibodies (Ab) and in the case of autoimmune diseases autoAbs, some of which were pathogenic. Over the past 10 years, it has became apparent that B cells display a variety of characteristics, other than Ab production, which could contribute to autoimmunity. They normally play a role in the development of lymphoid architecture, regulating T-cell subsets and dendritic cell (DC) function through cytokine production, and in activation of T cells. Receptors editing is also important in B cells which aids in immunity to infection and, possibly, prevention of autoimmunity. Transgenic animal models have now shown that B cells are necessary for many autoimmune diseases although their Ab products are not required in some cases. Negative signalling by CD5 and other molecules, such as CD22, in maintaining tolerance through recruitment of src-homology two domain-containing protein tyrosine phosphatase-1 has also been documented. In fact, we have now reached a new era whereby the B cell has returned as an important contributor to autoimmune disorders, so that the race is on to characterize signalling regulation via the B-cell receptor and coreceptors. Identification of such molecules and their potential defects should lead to effective ways of controlling the immune response and in particular preventing the development of autoimmune states. The classical view of B cells in the biology of immune responses to infectious and self-antigens (Ag) that they promote immunity primarily by producing Ab turns out to be rather naïve. Indeed, studies over the last few years indicate that this view is far from complete, and suggest that B lymphocytes have extraordinarily diverse functions within the immune system. Furthermore, it is becoming increasingly clear that the pathogenesis of autoimmune diseases cannot solely be accounted for by T cells, and intrinsic abnormalities of B cells have been described in such conditions. In this brief review we highlight some recent observations in the context of B lymphocyte in pathophysiology, and focus on their revival as pivotal players the pathophysiology in autoimmune diseases. Yet, it remains difficult to provide a model of how important B cells are in immunity and autoimmunity.
Abstract: While it has been claimed that some anti-endothelial cell antibodies (AECA) activate EC, there is also evidence that others trigger apoptosis. To address the issue of whether activation is a prerequisite for AECA-mediated apoptosis of EC, 23 AECA-positive sera were evaluated for their ability to induce activation and/or apoptosis. Activation was defined as an over-expression of E-selectin and intercellular adhesion molecule 1. Optical microscopy, annexin V binding, hypoploid cell enumeration, and determination of poly (ADP-ribose) polymerase cleavage-related products were used to assess apoptosis. Four functional profiles were defined: 10 sera promoted activation and apoptosis (act+/apo+), one was act+/apo-, six act-/apo+, and the remaining six act-/apo-. The reduced membrane expression of thrombomodulin was associated with apoptosis, rather than activation. Caspase-3 was implicated in the two models of apoptosis, the ratios of several survival proteins to Bax decreased, regardless of the ability of apo+ AECA to activate the cells, while radical oxygen species did not appear to be involved. Furthermore, it occurred that macrophages engulfed EC treated with apoptosis-promoting AECA, but not those incubated with AECA that did not induce apoptosis. Hence, AECA represent an extremely heterogeneous family of autoantibodies, not only because of the variety of their target antigens, but also the subsequent diversity of their effects.
Abstract: We have reported that human autoantibodies reacting with the polymorphonuclear neutrophil (PMN)-anchored FcgammaRIIIb (CD16) protect these cells from spontaneous apoptosis. In this study, we used anti-CD16 F(ab')(2) to delineate the mechanism(s) whereby the PMN life span is extended. As documented using four methods, CD16 cross-linking impeded spontaneous apoptosis, whereas anti-CD18 F(ab')(2) exerted no effect. Incubation of PMNs with anti-CD16 prevented the up-regulation of beta(2) integrins, particularly CD11b, which is the alpha-chain of complement receptor type 3, but also CD18, which is its beta-chain, as well as CD11a and CD11c. Anti-CD16-conditioned supernatant of PMNs diminished the percentage of annexin V-binding fresh PMNs after another 18 h in culture, whereas the negative control anti-CD18 had no effect. The expression of mRNA for G-CSF and GM-CSF was induced by anti-CD16, followed by the release of G-CSF and GM-CSF in a dose-dependent manner. Anti-G-CSF and anti-GM-CSF mAbs abrogated the antiapoptotic effect of the related growth factors. The delay in apoptosis was accompanied by a down-regulated expression of Bax, and a partial reduction of caspase-3 activity. These data suggest an autocrine involvement of anti-CD16-induced survival factors in the rescue of PMNs from spontaneous apoptosis. Thus, apoptosis of aged PMNs can be modulated by signaling through FcgammaRIIIb, which may occur in patients with PMN-binding anti-FcgammaRIIIb autoantibodies.
Abstract: OBJECTIVE: One of the main features of systemic sclerosis (SSc) is vascular damage, the mechanism of which is not understood. In the present study we examined whether screening of SSc patients for different anti-endothelial cells antibodies (AECA) of various origins increase the sensitivity of AECA detection in SSc patients. Secondary aim was an attempt to correlate AECA with other common autoantibodies. MATERIALS & METHODS: 478 SSc patients were studied for the presence AECA, anti-cardiolipin (aCL), anti-dsDNA, anti-heparin (AHA), anti-pyruvate dehydrogenase (PDH) and anti-PDC-E2 autoantibodies. AECA levels were detemined using human umbilical vein EC (HUVEC), bone marrow EC (BMEC), EC hybridoma (EA.hy 926) and Kaposi sarcoma EC (KS). RESULTS: Positive AECA were found in 49.5% of SSc patients (27.1% HUVEC; 34.3% BMEC; 26.3% EaHy 926 and 22.7% KS). The highest percent reactivity of AECA was obtained using microvascular BMEC. When combining BMEC and either other cell lines the reactivity ranged from 41.4% to 46%. A significant association between AECA on the one hand and AHA (p<0.001)) and anti-PDH (p<0.05) on the other was secn. Cross-reactivity with anti-PDC-E2 was excluded by inhibition tests, but AHA and anti-PDH may be part of the spectrum of AECA. CONCLUSIONS: Since false-negative AECA may result from lack of expression of various antigens on a specific EC, analysis of AECA in SSc patients requires using several EC types, including microvascular EC.
Abstract: Not only are some anti-endothelial cell antibodies (AECA) directed to thus far unidentified cell membrane structures, but some ot them recognize "planted" antigens and possibly ligand-receptor complexes. The functional heterogeneity of AECA is widely acknowledged: part of them activate the complement, mediate antibody-dependent cell cytotoxicity of trigger the production of tissue procoagulant factor. It has also recently been established that a proportion of AECA have the capacity to induce apoptosis of their target cells. In fact, the most direct demonstration of the pathogenicity of AECA is the autoantibody-induced murine model of vasculitis.
Abstract: Anti-Fc gamma receptor IIIb (Fc gammaRIIIb) human autoantibodies (Ab) have been classified previously into three groups, based on the results of an indirect immunofluorescence (IIF) test and an enzyme-linked immunosorbent assay (ELISA): IIF+/ELISA+ (group A), IIF+/ELISA- (group B), and IIF-/ELISA+ (group C) sera. In this study, differential effects between IIF+ autoAb, recognizing cell-bound Fc gammaR, and those ELISA+, recognizing only cell-free Fc gammaR, were studied on polymorphonuclear neutrophils (PMN). Neither group A nor B autoAb was cytotoxic, although both prolonged the survival of PMN by delaying spontaneous apoptosis. By the same extent, the PMN-binding antisera stimulated the appearance of a CD11b(dim) population, following a 12-h incubation. This event was associated with a lowered expression of beta2 integrin molecules, resulting in altered PMN function. Treatment with groups A and B autoAb reduced adhesiveness and respiratory burst. This impairment of the responses was more pronounced when the cells originated from donors NA1+ NA1+ rather than donors NA2+ NA2+. From our observations, the influences of anti-Fc gammaRIIIb autoAb on PMN survival, as well as function and subsequent dysregulation of the inflammatory response, have proven somewhat dependent on their target antigens, as determined by IIF coupled with ELISA and Fc gammaRIIIb polymorphism.
Abstract: In Black Africa, the role of hepatitis C virus (HCV) in the onset of chronic hepatic disease is unclear. This is particularly true in Senegal where the prevalence of HCV is moderate. To gain insight into this question, a case-control study including 73 patients and 73 controls was carried out at Principal Hospital in Dakar in 1995. The patients included in this study presented chronic hepatitis in 2 cases cirrhosis in 25 and hepatocellular carcinoma in 46. Patients and controls underwent serologic testing for HCV (ELISA screening test followed by 3rd generation ELISA test in case of positive results and confirmation by immunoblot) with determination of HCV serotype using the immunoenzymatic method. Testing also included research for infection by hepatitis B virus and for anti-delta antibodies. Anti-HCV antibodies were detected in two patients (3 p. 100) and serology was suspicious in two others. Serotype 2 was detected in one of these patients. No positive results were recorded in controls. Fifty-four patients (74 p. 100) and 15 controls (21 p. 100) presented the HBs antigen including 13 patients (24 p. 100) and I control (7 p. 100) with anti-delta antibodies. This study shows that HCV currently plays a minor role in the onset of hepatic disease in hospitalized patients in Senegal. It also confirms the predominant role of hepatitis B and complicating effect of the delta hepatitis virus. These findings are compared with reported data for Black African countries. The impact of HCV appears to be lower in Senegal than in central Africa.
Abstract: Anti-beta 2-glycoprotein I antibodies bind to endothelial cells through beta 2-GPI. The antibodies are present in patients with systemic lupus erythematosus and antiphospholipid syndrome and are associated with the pathogenesis of the disease. Anti-endothelial cell antibodies that react with constitutive antigens on ECs are present in patients with vasculiditis and other diseases. Both types of antibodies can activate ECs. Frequent findings in APLS and vasculitis are fibrin deposits and thromboembolic phenomena. These indicate that the coagulation system is activated. However, the mechanism of activation is not clear. ECs generate tissue factor upon stimulation with various substances. In the present study we report that monoclonal anti-beta 2-GPI antibodies and AECAs, derived from a patient with primary APLS and a patient with Takayasu's arteritis, respectively, induce a potent tissue factor in ECs. The production of TF activity, TF antigen and TF mRNA is dose and time dependent. The TF activity was induced also by F(ab)2 but not by Fc fragments and was abolished completely by pre-incubation with ant-TF antibodies. The TF that is induced in ECs by AECAs with and without beta 2-GPI specificity may activate the coagulation and thereby play a major role in the pathogenesis of fibrin deposition and thrombus formation in diseases that are associated with the presence of these antibodies.
Abstract: Patients with chronic renal failure requiring regular hemodialysis are known to be prone to thromboembolic events due to a hypercoagulable state. Vascular access thrombosis (VAT; including thrombosis of the vascular shunt or graft) represents a serious complication and jeopardizes life in these patients. In the current study, conducted on 81 consecutive patients from the Hemodialysis Unit, we have employed ELISA for the estimation of various autoantibody levels (anti-endothelial cell antibodies, anti-cardiolipin, anti-beta 2GPI and anti-modified LDL antibodies) and correlated them with the occurrence of thromboembolic events in general, and VAT in particular. We have found that the levels of antibodies reactive with human umbilical vein endothelial cells (HUVEC) but not with other EC lines (microvascular or EaHy 929) were significantly higher in hemodialysed patients with VAT in comparison with patients with no VAT (p = 0.001). A weaker but yet positive correlation was observed between the levels of anti-HUVEC and anti-cardiolipin antibodies and the occurrence of thromboembolic events including deep vein thrombosis, pulmonary infarction, cerbrovascular events and VAT (both p-values equal 0.02). Anticardiolipin antibodies (aCL) were not cross reactive with beta 2GPI or with HUVEC. Antibodies to modified LDL, although higher in hemodialyzed patients, did not correlate with thromboembolic events. The results of this study suggest that antibodies to HUVEC may prove as a fairly good marker of VAT in hemodialysis. High levels of aCL are weakly associated with thromboembolic events and antibodies to modified LDL do not correlate with a prothrombotic state.
Abstract: The IgG fraction of human anti-endothelial cell antibodies (AECA) obtained from a patient with Wegener's granulomatosis was used as immunogen to raise AECA mAb in mice selected among those which developed vasculitis-like lesions after immunization. Three mAb (BGM, 3C8 and 7G2), selected by cyto-ELISA and flow cytometry analyses, featured a specific reactivity with human umbilical vein endothelial cells (HUVEC) and the mouse endothelial cell line H5V; on the contrary, HEp2 cells, the murine melanoma B16 cell line, the extracellular matrix as well as several other antigens tested were not recognized. BGM mAb, an IgG3 precipitating a 70 kDa structure from HUVEC, was able to induce endothelial cells to secrete amounts of IL-6 significantly higher than irrelevant controls or mAb binding different endothelial antigens (i.e. CD31, CD29, ICAM-1 and HLA class I). BGM mAb induced significant levels of antibody-dependent cell cytotoxicity (13 +/- 2.5 versus 0.6 +/- 0.03%). To the best of our knowledge, BGM is the first murine mAb specific for human endothelial cells generated by idiotypic manipulation; secondly, its biological properties further support the notion of a pathogenic role for AECA in autoimmune-mediated diseases.
Abstract: An outbreak of yellow fever (YF) occurred in the central part of Senegal during October 1995. Thirty-one probable cases were detected and 79 cases were confirmed either by IgM ELISA or by virus isolation (30 strains isolated). The case fatality rate was 18.9%. Incidence of the infection was evaluated by a serosurvey in the area. Males 10-29 years old belonging to the Peul ethnic group were more affected. Moreover, 28 YF virus strains were isolated from mosquitoes and larvae pools and vertical transmission of YF virus by Aedes aegypti was also demonstrated for the first time in the field. This outbreak occurred after the major amplification of the wild cycle of YF virus in 1993 in West Africa. This epidemic represented a typical example of intermediate transmission of YF: both humans and wild vertebrates are involved in the virus cycle through wild mosquitoes with semidomestic habits, mainly Ae. furcifer, Ae. luteocephalus, and domestic vector Ae. aegypti. It was controlled by a prompt immunization campaign. The impact of inclusion of YF vaccine in the Expanded Program of Immunization, which has been conducted in Senegal for eight years, is discussed.
Abstract: As endothelial cells (EC) express heparin-like glycosaminoglycans, such as heparan sulfate, it was essential to investigate the relation of anti-EC antibody (AECA) to heparin reactivity. AECA were detected in 43 of 131 autoimmune sera and anti-heparin antibodies (AHA) in 25. These autoimmune reactivities were significantly associated (P corrected < 0.0005). Seven AECA-positive/AHA-positive and three AECA-negative/AHA-positive sera were affinity-purified using protein G column followed by a heparin-Sepharose column. Two populations of AECA were recovered from the second column. One was eluted with 0.4 M NaCl which bound to EC and to solid-phase heparin with low affinity, but not to soluble heparin. The second population of AECA, which was eluted with 4 M guanidine HCl/2 M NaCl, recognized EC and solid-phase heparin with high affinity, but also soluble heparin. The latter population of AECA might thus be an important cause of autoimmune vascular thrombosis in systemic diseases.
Abstract: IgG comprises of four subclasses which differ from each other with respect to their biological properties. Fc gamma receptor shedding as well as a variety of T cell cytokines are influential in the distribution of these subclasses, but the route the antigen is introduced into the body is also important. With regard to nonorgan-specific autoimmune conditions, such as rheumatoid arthritis and systemic lupus erythematosus, IgG1 and IgG3 autoantibodies predominate, whereas IgG4 antibodies are regularly encountered in organ-specific autoimmune diseases. This suggests that the target organ may be continuously stimulating the immune system.
