Expression of B cell receptor-associated signaling molecules in human lupus.

B cell receptor (BcR) signaling requires a tight regulation of several protein tyrosine kinases and phosphatases, and associated co-receptors. Mounting evidence indicates that abnormal BcR signaling, such as occurs in SHP-1 and Lyn-deficient mice, results in production of pathogenic autoantibodies and lupus-like glomerulonephritis, suggesting that altered signaling thresholds could underlie the development of systemic autoimmunity. To test this hypothesis, we investigated expression of BcR-associated signaling molecules in lymphocytes from patients with systemic lupus erythematosus (SLE) during inactive phases of the disease. We found that the transmembrane regulatory protein tyrosine phosphatase CD45 is expressed at abnormal levels. Strikingly, this reduction persisted during four months of follow-up. By contrast, despite its potent role as a regulator of thymus-independent immune responses and of B cell life span, the CD22 co-receptor is expressed at normal levels in B lymphocytes isolated ex vivo from SLE patients. We also noted unusual levels of the cytosolic protein tyrosine kinase Lyn and the protein tyrosine phosphatase SHP-1 in the lymphocytes of the patients. Since in normal B cells Lyn and SHP-1 act in concert within a common negative pathway in which CD45 counteracts SHP-I regulatory role, we propose that this feedback regulatory pathway is crippled to different degrees in human SLE B cells. Break of the balance between positive and negative signaling molecules likely modifies the BcR signaling thresholds. Such alterations, together with other factors, may contribute to the disruption of self-tolerance in this disease.

Abnormal distribution of CD45 isoforms expressed by CD4+ and CD8+ T cells in rheumatoid arthritis.

CD45RO+ T cells are referred to as memory or helper-inducer while CD45RA+ T cells are regarded as naive or suppressor-inducer T cells. The former population predominates in the peripheral blood and even more in the synovial fluid of patients with rheumatoid arthritis, to the expense of the latter population. Within the CD45RB+ compartment, there appears to be more of the fully-differentiated than of the early-differentiated CD4+ T cells. In spite of the fact that these lymphocytes are close to undergoing apoptosis, this programmed cell death is inhibited in the rheumatoid synovium.

Autoimmune diseases and monoclonal gammopathies.

Rheumatoid arthritis, systemic lupus erythematosus and primary Sjögren's syndrome are nonorgan-specific autoimmune diseases in which serum monoclonal immunoglobulins (MIg) have been identified repeatedly. Conversely, autoimmune traits have been detected in a number of patients with lymphoproliferative disorders such as multiple myeloma, Waldenström's macroglobulinemia and chronic lymphocytic leukemia. The latter cells have even shown to produce multispecific autoantibodies. One connection between connective tissue diseases and Iymphoid malignancies might be established by a limitedfraction of B Iymphocytes expressing the CD5 marker.

Impaired binding capacity of asialyl and agalactosyl IgG to Fc gamma receptors.

OBJECTIVE: Autoimmunity in rheumatoid arthritis has been associated with deficient glycosylation of serum and synovial IgG which could potentially be mediated through the binding of the Fc portion of the molecule to its cognate receptor. METHODS: Normal IgG1 and IgG3 were affinity-purified, and sialic acid and galactose were clipped off using neuraminidase and beta-galactosidase, respectively. The binding of these sugar-depleted IgG to the Fc gamma receptors (Fc gamma R)IIIb on polymorphonuclear leukocytes (PMN) was assessed by flow cytometry. RESULTS: The binding of both asialyl and agactosyl IgG1 and IgG3 to PMN was significantly lower than that of the native IgG1 and IgG3. CONCLUSION: These data indicate that agalactosyl and, to a lesser degree, asialyl IgG, do not bind as efficiently as native IgG to Fc gamma R. Such a reduction in the perspective of the heterogeneity of the PMN Fc gamma RIIIb.

IgG subclasses of human autoantibodies.

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.

Anti-Fc gamma receptor autoantibodies from patients with Sjögren's syndrome do not react with native receptor on human polymorphonuclear leukocytes.

Sera from patients with primary Sjögren's syndrome (pSS) have been examined for the presence of cell-free Fc-gamma receptor (Fc gamma R) IIIb, which is expressed in polymorphonuclear leukocytes (PMN), and the production of related autoantibody. Sera from 66 patients with pSS were evaluated by an ELISA using recombinant human Fc gamma RIIIb as the substrate and by flow cytometry. Cell-free Fc gamma RIIIb was also detected by an ELISA. The fine specificity of autoantibodies was established by inhibition with a preparation of Fc gamma RI plus Fc gamma RII, and two ELISAs using Fc gamma RI and Fc gamma RII as the substrates respectively. Anti-Fc gamma RIIIb activity was found in 30 patients (45%), but 25 of them did not react with autologous PMN, whereas they bound to Fc gamma RIIIb eluted from the same PMN in ELISA and Western blotting. Autoantibodies from one serum recognized the three receptors, six with Fc gamma RII in addition to Fc gamma RIII, and three sera were specific for the latter receptor. None of these reacted with Fc gamma RI- and Fc gamma RII-carrying cells. Cell-free Fc gamma RIIIb, but negligible amounts of Fc gamma RIIIa, were detectable in the patient sera. The membrane expression of CD15, an early activation marker, was diminished, while that of three PMN late activation markers was markedly enhanced. Taken together, these results suggest that autoantibodies are produced following the shedding of Fc gamma RIIIb upon PMN activation. A credible candidate for this activation is IgG-containing immune complexes.

Two populations of endothelial cell antibodies cross-react with heparin.

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.

Regulation of the expression on mouse T lymphocytes of the epitope identified by monoclonal antibody 3A35.

Monoclonal antibody 3A35 (MA 3A35) has previously been shown to be an activation marker of macrophages and T lymphocytes. It immunoprecipitated from macrophages a 200-kDa molecule belonging to the T200 family and from T cells a 85-kDa antigen. In the present work, the factors controlling the expression of the epitope identified by MA 3A35 on polyclonal activated T cells and T-cell clones, as well as the ability of 3A35 alone or together with complement to interfere with T-cell functions, were investigated. Corticoresistant thymocytes unreactive with MA 3A35 became fully reactive after 2 days of in vitro stimulation by PMA and IL-2 and the level of reactivity per cell declined to a low level thereafter. In helper and cytolytic T-cell clones, the expression of the epitope defined by MA 3A35 was also maximal soon after antigenic stimulation then declined. In helper-T-cell clones, the epitope remained detectable during the entire culture period, whereas in cytolytic clones its expression was markedly reduced at the end of the culture. The lineage of cytotoxic T lymphocytes (CTL) as studied in a bulk culture of spleen cells primed in vivo against a syngeneic tumor exhibited similar regulation by antigenic stimulation. The CTL precursors were resistant to lysis by MA 3A35 plus complement; after 3 days of culture with the stimulatory antigen, they became highly sensitive but their sensitivity then diminished and mature CTL were completely resistant. MA 3A35 plus complement also killed the activated T cells which responded to macrophage-presented antigens and were thought to be mainly Lyt-1+. Therefore, the epitope identified by MA 3A35 was expressed predominantly at an early stage of T-cell activation. At a late stage, it persisted almost exclusively on helper and Lyt-1+ cells. In addition, MA 3A35 plus complement lysed NK cells, AK cells, and their precursors present in normal spleen. In the absence of complement, MA 3A35 had no detectable effect on T-cell functions.

Ligation of CD5 on resting B cells, but not on resting T cells, results in apoptosis.

The CD5 molecule is expressed by a B cell subset. We have demonstrated that resting B cells do not proliferate in response to CD5 ligation, whereas cells preactivated with anti-IgM and IL-2 do so. Here, we specifically studied the effects of anti-CD5 and anti-IgM on apoptosis of CD5+ B cells. Both ligation of CD5 or of surface IgM (sIgM) resulted in apoptosis. This started earlier following ligation of CD5 than with sIgM, and both responses were time dependent. CD5-induced apoptosis was independent of the epitope recognized or the way the antibody was presented to the B cells. CD5+ B cells were more sensitive to IgM-induced apoptosis than CD5 B cells. Engagement of CD5 or CD3 expressed by T cells failed to induce apoptosis. Our data indicate differences in the function of CD5 molecules on tonsillar B cells, compared with blood T cells and suggest that cross-linking CD5 on B cell activates specific pathways responsible for apoptosis.

Heterogeneity of neutrophil antibodies in patients with primary Sjögren's syndrome.

Our aims were to determine the prevalence of neutrophil antibodies in patients with primary Sjögren's syndrome (pSS), identify their target antigen(s), and evaluate their functional significance. Neutrophil antibodies were detected using an indirect immunofluorescence (IIIF) test and an enzyme-linked immunosorbent assay (ELISA), using recombinant human Fc-gamma receptor (Fc gamma RIIIb) as a capture agent. Luminol-dependent chemiluminescence was then measured by an established technique. Antibodies to neutrophils were detected in 30 of 66 patients (45%) and categorized on the basis of positivity for the two assays: IIF+/ELISA+ (group A: five patients), IIF+/ELISA- (group B: five patients), and IFF-/ELISA+ (group C: 20 patients). All positive sera contained antibodies directed to the neutrophil specific Fc gamma RIIIb, and none of them bound to NAnull neutrophils. The titer of neutrophil-reactive antibodies (groups A and B) showed no correlation with the neutrophil count, but these autoantibodies did reduce the cell ability to generate a respiratory burst. Thus, neutrophil antibodies are common in patients with pSS. Their main target appears to be Fc gamma RIII, and this may partly account for the dysfunction in Fc gamma R-mediated clearance by the reticuloendothelial system reported in these patients.

Establishment and characterization of permanent human endothelial cell clones.

Though vasculitic diseases have been claimed to be associated with anti-endothelial cells antibodies (AECA), there is a widespread awareness of the limitations of the tests currently in use. Our objective was therefore to establish clones, in the hope that some of them would express disease-specific membrane autoantigens. Two EC lines and 7 clones were established by fusing human umbilical vein EC with epithelial A549/8 cells, and cloning by limiting dilution. An additional clone was derived from the EA.hy 926 cell line. All clones carried EC markers, such as thrombomoduline (TM) and platelet-EC adhesion molecule 1 but differed from each other, depending on whether they expressed HLA class II antigen, LFA-1, thrombospondin receptor or von Willebrand factor (vWf) antigen. Clones were also characterized by their ability to release tissue plasminogen activator, interleukin 6, TM and vWf. This panel is meant to distinguish reactivities of AECA.

Superantigens in autoimmune disease.

The recent identification of superantigens (of retroviral origin in mice and bacterial origin in humans) has given rise to several hypotheses linking superantigens to autoimmune responses. The most compelling argument in support of such a link is restricted V beta gene usage by lymphocytes in rheumatoid joint fluid and Sjögren's syndrome salivary tissue. However, a substantial body of evidence from mouse models militates against a link between the presence of self-superantigen-reactive T-cells and the development of autoimmune disease. Nevertheless, superantigens are powerful instruments for investigating tolerance.

The presence of anti-Fc gamma receptor autoantibodies is related to the clinical presentation of primary Sjögren's syndrome.

OBJECTIVE: Fc gamma receptor III (Fc gamma RIII) is one of the 3 structurally distinct families of receptors for the Fc domain of IgG, and its Fc gamma RIIIb isoform is exclusively expressed in polymorphonuclear (PMN) cells. We sought to detect anti-Fc gamma RIII autoantibodies in serum from patients with primary Sjögren's syndrome (SS). METHODS: Sixty-six patients with SS and 44 healthy controls were enrolled in the study. ELISA were developed. RESULTS: IgG and IgM autoantibodies were found in 16 (10 IgG+ IgM+ and 6 IgG+ IgM-) and 24 patients (10 IgG+ IgM+ and 14 IgG- IgM+) respectively. Their frequency was higher in patients with nonerosive arthritis (p < 0.02), Raynaud's phenomenon (p < 0.003), and lung involvement (p < 0.02) than in patients without such complications. The levels of IgM and IgG antibody (p < 0.05) correlated with the content of IgA without the circulating immune complex (IC), while there was no relationship between anti-Fc gamma RIII activity and the PMN count. CONCLUSION: Anti-Fc gamma RIII autoantibodies may act as an acquired additional factor further compromising IC handling in individuals who share HLA-DR3 alloantigen.

CD5+B cells: differential capping and modulation of IgM and CD5.

CD5 is associated with the B-cell antigen receptor (BcR) complex. As an approach to understanding its role in B-cell function, the authors investigated the capping and modulation of CD5 and surface IgM (sIgM). Tonsillar B cells were treated with anti-IgM or anti-CD5 antibodies, capping examined after 1 h (by fluorescence microscopy) and modulation after 24 h (by flow cytometry). CD5 co-capped and co-modulated with sIgM. Of various drugs tested, only the protein tyrosine kinase inhibitor (genistein) had any effect on capping and co-capping. Capping of sIgM (and co-capping of CD5) but not capping of CD5 (or co-capping of sIgM) was inhibited by genistein. None of the other drugs affecting PKC or cytoskeletal structures (colchicine and cytochalasin D) had any effect. However, the PKC inhibitors, staurosporine and H-7, inhibited the modulation of sIgM by anti-IgM but not CD5 by anti-CD5. In contrast, PKC activators, PMA and mezerein, inhibited modulation of CD5 by anti-CD5 but not sIgM by anti-IgM. This suggests that direct ligation of CD5 utilizes different signalling pathways compared with sIgM. It seems likely that in CD5+ cells, interaction of CD5 with its ligand CD72 modulates signals transmitted through the BcR.

Anti-CD5 extends the proliferative response of human CD5+ B cells activated with anti-IgM and interleukin-2.

The CD5 T cell glycoprotein which is expressed by a subset of B cells has been shown to be involved in T cell activation and proliferation. No similar studies, to date, have addressed the role of CD5 on the B cell subset. CD5+ and CD5- B cells were sorted and stimulated with anti-CD5 monoclonal antibody (mAb) in vitro. The activation and proliferative responses of these two populations, as measured by analysis of proliferation marker, did not differ following anti-mu and interleukin (IL)-2 stimulation. The addition of anti-CD5 did not change the responsiveness of such activated CD5+ B cells but resulted in a decrease in CD25 expression. Pre-activation of B cells with phorbol 12-myristate 13-acetate, which increased CD5 expression, failed to alter the proliferative response of CD5+ B cells to anti-mu and IL-2 with or without addition of anti-CD5 mAb. Anti-mu and IL-2 treatment of CD5+ cells resulted in optimal proliferation measured at day 3 which decreased by day 6. However, addition of anti-CD5 mAb at day 3 prevented this decline in proliferative response. This dose-dependent effect was observed only when the anti-CD5 mAb was presented to the B cells in cross-linked form. Co-stimulation of CD5 did not lower the threshold of antigen to which the B cells responded. Taken together, these data support a functional role for CD5 on B cells acting as an accessory signal, following their primary activation through the B cell receptor complex and highlight differences in the role of CD5 associated with the T cell receptor complex.

A new epitope of the T200 molecule family defined by the 3A35 monoclonal antibody and expressed by macrophages and activated T lymphocytes.

A monoclonal antibody (mAb), 3A35, produced against mouse macrophages (M phi) was found to react against certain activated T cells. This mAb, a rat IgM, resulted from a cell fusion between a mouse plasmacytoma and rat lymphocytes immunized against mouse M phi. It bound more avidly to activated than to resident M phi. It did not react against B cells and resting T lymphocytes but recognized certain dividing T cells like EL4 lymphoma, concanavalin A-activated and interleukin 2-expanded spleen cells, and helper T cell hybridomas. By contrast, other T lymphocyte-derived cell lines such as YAC-1 and CTLL2 were unreactive. No clear relationship was found between the binding of 3A35 to cells and the expression of L3T4 and Lyt-2 antigens. The specific stimulation of T cell clones with antigen rapidly induced a strong reactivity with 3A35 mAb which declined thereafter to a low (helper clones) or non-reactivity (cytotoxic clones) after 10 days of culture. Immunoprecipitation experiments, performed with M phi derived from bone marrow cell cultures, surface iodinated with 125I or metabolically labeled with [35S]methionine, showed that 3A35 bound to a 200-kDa molecule, shifting to 175 kDa under reducing conditions. In peritoneal M phi activated in vivo, in addition to the 175-kDa band, new bands migrating at 140, 120 and 85 kDa were identified by 3A35 and could be absorbed on a commercial anti-T200 mAb bound to Sepharose beads. After strengthening the cell binding of 3A35 to EL4 lymphoma cells by a cross-linking agent, only a 85-kDa molecule was immunoprecipitated. Thus, 3A35 identifies a new epitope of the T200 molecule family which is expressed on M phi and activated T cells.

Modulation of CD72 by ligation of B cell receptor complex molecules on CD5+ B cells.

B cells expressing CD5 also carry its ligand, CD72. As an approach to understanding the role of CD5 and CD72 on B cells, we have examined the association of CD72 with CD5 and slgM by modulation/co-modulation and capping/co-capping following ligation of these surface molecules with specific antibodies. Modulation and co-modulation were measured after 24 h, whilst capping was measured after 1 h. CD5 and slgM co-modulated each other, CD72 co-modulated with slgM and CD5, but anti-CD72 did not affect either slgM or CD5. CD5 and slgM co-capped each other, whilst CD72 failed to co-cap with either slgM or CD5. The CD5-induced co-modulation of CD72 was partially blocked by specific protein tyrosine kinase inhibitor, but not the slgM-induced co-modulation, Protein kinase C (PKC) inhibitors abrogated the anti-mu- but not the anti-CD5-triggered modulation of CD72, whereas PKC activators prevented the CD5- but not the slgM-induced 24 h modulation of CD72. None of these drugs was able to modify the anti-CD72-induced modulation of CD72. Our data suggest that CD5 is physically associated with slgM in the B cell receptor complex but not with CD72. Furthermore, from the effect of drugs on modulation, there appears to be different associations of CD72 with slgM and CD5. These two pathways differed in some respects, consistent with a co-stimulatory function of CD72 and CD5 in B cell activation.

A monoclonal antibody (3A33) that reacts with a mouse-specific epitope of Mac-1 antigen.

The 3A33 monoclonal antibody, obtained by fusing rat immune lymphocytes with mouse plasmacytoma cells, was directed against mouse macrophages. Antibody 3A33, a rat IgG2a, reacted with macrophages from all the mouse strains tested, with mouse blood monocytes and with 56% of bone marrow cells, but not with T lymphocytes. It immunoprecipitated an antigen with alpha and beta subunits, found to be identical to Mac-1 antigen after cross-absorption experiments with M1/70 monoclonal antibody. The two antigenic determinants of the Mac-1 molecule identified by the 3A33 and M1/70 antibodies both displayed reduced expression on inflammatory macrophages and comparable resistance to trypsin digestion. The sites of the determinants on this molecule seemed close together judging from the ability of both the 3A33 and M1/70 antibodies to block C3bi receptor sites and compete for cell binding. However, unlike antibody M1/70, 3A33 never reacted with human cells bearing Mac-1 antigen. Therefore, two closely related epitopes of the Mac-1 molecule - one specific for mouse and one common to mouse and man, were recognized by these monoclonal antibodies.

Autoantibodies to CD45 in systemic lupus erythematosus.

Western blotting with U937 cell extracts as the substrate, and enzyme-linked immunosorbent assays (ELISA) with U937-, Jurkat- and Daudi cell-purified CD45 molecules were used to detect anti-CD45 reactivity in patients with systemic lupus erythematosus (SLE). By immunoblotting, 16 of 64 SLE sera were shown to be positive (25.0%). In the ELISAs, 13 out of 18 SLE sera reacted with the target CD45. Of these, three were not detectable on the blot. Importantly, 12 of these ELISA-positive sera contained IgM and IgG auto-antibodies. Neuraminidase-treatment of U937-precipitated CD45 molecules enhanced the reactivity to most of the isoforms, indicating that the antibodies may bind to asialylated polysaccharides.