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Serum from normal individuals contains substantial amounts of natural antibodies (NA) capable of recognizing self antigens. However, the physiological implications of this autoreactivity remain unclear. We have examined the role of self-reactive NA and complement in mediating the uptake of human thyroglobulin (Tg) by human peripheral B cells in reconstituted whole blood. Significant binding of fluorescein isothiocyanate-conjugated-Tg to B cells was observed, and absorption of Tg-reactive antibodies from serum markedly reduced this uptake, as did inactivation of serum complement or blockade of complement receptor types 1 (CR1, CD35) and 2 (CR2, CD21). T cell responsiveness to Tg was examined in a preparation of peripheral blood mononuclear cells (PBMC) cultured in the presence of autologous serum. A subset of CD4(+) T cells exhibited a dose-dependent proliferative response to Tg, which was strongly inhibited by complement inactivation and by immunoabsorption of Tg-reactive antibodies. Furthermore, this T cell response was abrogated by depletion of B cells from the PBMC culture. These data imply that uptake of complement-opsonized Tg / anti-Tg complexes and subsequent presentation of Tg by B cells are prerequisites for the proliferation of Tg-reactive CD4(+) T cells, suggesting a novel role for natural autoantibodies and complement in the regulation of autoreactivity under physiological conditions.

Intravenous immunoglobulin (IVIg) was shown to decrease the severity of acute graft-versus-host disease (aGVHD) in recipients of allogeneic bone marrow transplants. To investigate the mechanisms involved in the protective effect of IVIg, we have used the parent-into-F1 model in which parental lymphocytes are transferred into semi-syngeneic non-irradiated F1 rats. Here we report that IVIg, as well as F(ab')2 fragments of IVIg, protected (Lewis × Brown-Norway) F1 rats against aGVHD induced by a single injection of Lewis lymphocytes. IVIg was given as five consecutive daily injections, starting on the day preceding that of the transfer of Lewis cells. Protection was associated with a decreased ability of lymphocytes to spontaneously proliferate and to produce NO and IFN-γ, in the absence of an increased production of IL-10. We further demonstrate that protection was associated with a decrease in CD4+ T cells bearing the activation marker CD134 in vivo, and with an enhanced apoptosis of activated CD4+ T cells by IVIg, in vitro. Our observations suggest that the prevention of aGVHD by IVIg in this model is mediated by the induction of apoptosis of activated alloreactive CD4+CD134+ donor T cells. The results further emphasize the role of normal immunoglobulin in modulating alloantigen immune responsiveness.

Serum IgM has been shown to participate in the control of IgG autoreactivity in healthy sub- jects. We have recently shown that an immunoglobulin preparation of pooled normal human IgM (IVIgM) contains anti-idiotypic antibodies against disease-associated IgG autoanti- bodies in autoimmune patients and protects rats from experimental autoimmunity. The aim of the present study was to asses the in vitro and in vivo immunomodulatory effects of IVIgM in comparison with IgG, in SCID mice reconstituted with thymic cells from a myasthenia gra- vis patient. Non-leaky SCID mice were injected i.p. with 60 ◊ 10 6 thymic cells from a patient with myasthenia gravis and 1 day later boosted with 10 6 irradiated acetylcholine receptor (AchR)-expressing TE671 cells. On days 14, 21 and 28, mice were treated with IVIgM or with equimolar amounts of human serum albumin. The level of anti-AchR antibodies in the sera of three out of four IgM-treated animals was less than 1 nM. Further, there was a significant decrease in the loss of endplate AchR on the diaphragms of IgM-treated SCID mice. These findings indicate that pooled normal IgM exerts an immunoregulatory role in experimental myasthenia gravis, and suggests that IgM may be considered as an alternative approach in the therapy of autommune diseases.

We analyzed the antibody (Ab) repertoires of IgM and IgG of patients with seropositive and patients with seronegative myasthenia gravis (MG) toward self antigens by means of a quantitative immunoblotting technique using normal human tissue extracts as sources of self antigens. Repertoires of reactivities of IgG and IgM with liver, kidney and stomach antigens were conserved between myasthenic patients and controls. IgG and IgM Ab repertoires toward muscle antigens differed significantly between patients with seropositive MG and healthy donors, as assessed by multiparametric statistical analysis. Patterns of Ab reactivities to muscle antigens were similar in patients with seronegative MG and healthy controls. Antibody repertoires of IgG and IgM toward thymus antigens of both seropositive and seronegative MG patients, differed significantly from those of healthy individuals. Our results indicate that MG is characterized by a selective impairment of self-reactive Ab repertoires toward muscle and thymus antigens. The observation that self-reactive Ab repertoires toward thymus antigens are similar in patients with seropositive and seronegative MG suggests that both forms of MG share common immunopathological features.

Normal human IgG for intravenous use (IVIg), administered intraperitoneally, protected Lewis rats against experimental allergic encephalomyelitis (EAE) induced by immunization with myelin basic protein (MBP). We demonstrate that protection was associated with an acquired unresponsiveness of lymphocytes to MBP and a decreased ability of the cells to produce IL-2, IFN-gamma and TNF-alpha and, to a lesser degree, IL-4 and IL-10, in the presence of the antigen. Lymph node (LN) cells of protected rats failed to passively transfer EAE to naive syngeneic animals. Our observations indicate that, rather than inducing selective immune deviation, IVIg induces preferential MBP unresponsiveness of Th1 cells. Whereas LN and splenic cells of IVIg-treated rats did not proliferate nor secrete IL-2 in the presence of the antigen, proliferation was restored by adding exogeneous recombinant IL-2. In contrast, LN cells of IVIg-treated rats proliferated normally and produced IL-2 in the presence of concanavalin A, indicating the selectivity for MBP of the anergy induced by IVIg when given at the time of immunization with the antigen. Treatment with IVIg also allowed a resistance to the secondary induction of EAE, indicating that IVIg protects from EAE but does not interfere with the processes that eventually lead to resistance to re-challenge. These data document the immunomodulatory effects of IVIg in T cell-dependent experimental autoimmune disease and further suggest a role for normal Ig in the selection of functional T cell repertoires.

We report on a soluble (s) form of CD21 (the C3dg/Epstein-Barr virus receptor, CR2) that is spontaneously released by B and T lymphocytes. Immunoprecipitation with anti-CD21 mAb of culture supernatants of surface and biosynthetically labeled B and T cell lines revealed a single band with an apparent molecular mass of 135 kDa. The molecule exhibited a molecular mass 10 kDa lower than that of membrane CD21. The release of soluble CD21 (sCD21) was time dependent and correlated with a parallel decrease in the expression of the membrane-associated molecule. The protein was also found in culture supernatants of tonsillar B cells and normal human thymocytes. Epitopic analysis using combinations of anti-CD21 monoclonal antibodies (mAb) indicated that sCD21 and membrane CD21 were similarly recognized by mAb directed against short consensus repeats (SCR) 1-2, SCR 4-5 and SCR 9-11. Affinity-purified sCD21 was capable of binding to purified human iC3b and to human recombinant CD23, as assessed by enzyme-linked immunosorbent assay and by using the BIAcore technology. In addition, normal human serum was found to contain a soluble form of CD21 that exhibited a similar molecular mass to that of the molecule shed by B and T cells in culture. The serum form of CD21 was recognized by all anti-CD21 mAb that we tested and showed a high reactivity with mAb directed against SCR 1-2. Our observations suggest that B and T cells shed the extracellular portion of CD21 and release a soluble molecule that retains the ligand-binding properties of CD21, thus having a potential role in immunoregulation.

We have investigated the effects of intravenous immunoglobulin (IVIg), a therapeutic preparation of normal human polyspecific IgG, on the synthesis and release of cytokines by peripheral blood monocytes. IVIg was found to selectively induce gene transcription and secretion of interleukin-1 receptor antagonist (IL-1ra) and IL-8 in cultures of normal human monocytes. The addition of IVIg to cultures of purified monocytes induced a dose-dependent secretion of IL-1ra and IL-8 without stimulating the production of IL-1 alpha, IL-1 beta, tumor necrosis factor-alpha or IL-6. The effects of IVIg required both the Fc and F(ab')2 portions of IgG. IVIg-induced production of IL-8 by monocytes was enhanced by lipopolysaccharide (LPS), although LPS inhibited the secretion of IL-1ra, suggesting that IVIg and LPS stimulate distinct intracellular pathways in monocytes. Induction of IL-1ra and IL-8 by IVIg was enhanced in the presence of autologous T lymphocytes. Our observations document the selectivity of the effects of IVIg on the synthesis of cytokines and cytokine antagonists by human monocytes. Induction of IL-1ra and IL-8 by IVIg may contribute to the anti-inflammatory effects of immunoglobulin therapy in patients with autoimmune and systemic inflammatory disorders.

The present study demonstrates that the C3b receptor CR1 (CD35) and the C3dg/Epstein-Barr virus receptor CR2 (CD21) are expressed by 25% and 70% of normal human thymocytes, respectively. The expression of CR2 extends to both CD1+ and CD1- cells in the thymus. Two subsets of CR2+ thymocytes were defined expressing low and high density of the receptor. The CR2++ subset represented 20% of CR2+ thymocytes and co-expressed the CR1 receptor. CR2++ thymocytes expressed an immature CD1dull, CD3-, CD4dull, CD8-, CD7++ phenotype and included a subpopulation of large cells expressing CD34. Twenty percent of thymocytes expressed the CD21 epitope defined by monoclonal antibody BU32, which is involved in the binding of CD23 to CD21. These observations provide a basis for a role for CD21 in the proliferation and differentiation of thymocytes at early stages of maturation. The functionality of CR1 and CR2 on thymocytes was evidenced by the ability of the receptors to mediate infection of cells with complement-opsonized human immunodeficiency virus (HIV). The results may be relevant to the immunopathogenesis of HIV infection.

In the present study we have analyzed the changes in the expressed antibody repertoire and in temporal fluctuations of antibody levels in serum that followed infusion of normal IgG (IVIg) in a patient with autoimmune thyroiditis. Administration of IVIg resulted in the stimulation of IgM production, in alterations of expressed antibody activity in serum that could not merely be accounted for by the passive transfer of antibody specificities contained in IVIg, in transient down-regulation of B cells clones expressing a specific disease-related idiotype and in the increase in serum in recipient's autoantibodies specifically reactive with F(ab')2 fragments of IVIg. In addition, infusion of IVIg shifted the pattern of spontaneous fluctuations of autoantibody activities in the patient's serum from a pattern indicative of disconnected events in the immune network to a pattern similar to that which is consistently observed in healthy controls. These results suggest that normal IgG may modulate autoreactivity by selecting expressed antibody repertoire through V region-dependent interactions with antibodies.

We have investigated the autoreactive repertoire expressed by serum IgG of healthy individuals of various age groups using a large panel of self antigens. Natural IgG autoantibodies against all self antigens of the panel were found in the purified IgG fraction of the serum of all donors that were tested. The mean binding activity to self antigens of IgG of pregnant women was higher than that of IgG purified from the serum of infants, young adults and aged individuals. No increase in IgG autoreactivity was observed with aging neither in the purified IgG fraction of serum nor in whole serum. Whereas autoantibody activity was easily detectable in purified IgG, it was low in serum. No difference was observed, however, between the binding activity of purified IgG and of IgG in serum in the case of foreign antigens nor in the case of anti-thyroglobulin autoantibodies of patients with hashimoto's thyroiditis. Purified IgM from normal serum bound to F(ab')2 fragments of autologous IgG in a dose-dependent fashion and inhibited the binding of autologous IgG to self antigens. Our results thus indicate that autologous IgM contributes to regulate expression of the natural IgG autoreactive repertoire through V region-dependent interactions, resulting in low levels of IgG autoreactivity in serum under physiological conditions.

Mouse and human natural IgM autoantibodies have been shown to be polyreactive and "connected" through V region-dependent interactions. In the present study, we have identified a connected subfraction of normal human serum IgG by using affinity chromatography of F(ab')2 fragments of pooled IgG (IVIg) or of IgG from a single donor on Sepharose-bound F(ab')2 fragments of the same source of IgG. The connected fraction of IgG exhibited a high content of autoantibodies directed against a wide panel of evolutionarily conserved self antigens and of self antigens that may be targets of autoantibodies in autoimmune diseases. Connected IgG also contained higher amounts of antibodies directed against commonly encountered microbial antigens than unfractionated IgG. The connected fraction did not, however, differ from unchromatographed IgG nor from non-connected IgG in its content of antibodies to vaccinal antigens and to distant foreign antigens. Thus, in humans as in mice, connectivity is a prominent feature of autoantibodies. Our observations are suggestive of a tight control by IgG of the expressed autoreactive repertoire in healthy individuals and strengthen the concept that the therapeutic infusion of pooled normal IgG (IVIg) may be effective in autoimmune diseases by bringing to patients normal regulatory components of the immunoglobulin network.

We have investigated the role of the membrane molecules CD11/CD18 and CD14 which may mediate the binding of lipopolysaccharide (LPS) to human monocytes, in the induction of the production and release of interleukin (IL)-1 and tumor necrosis factor-alpha (TNF-alpha) by LPS-stimulated cells. Blockade of CD11a, CD11b and CD18 with saturating concentrations of specific mAb did not inhibit the release of cytokines from LPS-stimulated monocytes. In contrast, inhibition of the release of IL-1 beta and TNF-alpha occurred in monocytes cultures that had been pretreated with either of two monoclonal antibodies (mAb) recognizing different epitopes on the CD14 molecule. The binding of LPS to CD14 has been previously shown to require serum factors. In the present study, we found that serum had an enhancing effect on the release of IL-1 and TNF-alpha from LPS-stimulated cultures of normal human monocytes. The inhibitory effect of anti-CD14 mAb was, however, observed in cultures performed in the presence or in the absence of serum, suggesting that triggering of IL-1/TNF-alpha release by CD14 is independent of LPS-binding proteins or other serum proteins. IL-1 beta and TNF-alpha were also released from LPS-stimulated cultures of monocytes from patients with paroxysmal nocturnal hemoglobinuria lacking expression of CD14. Thus, CD14 but not CD11/CD18 can trigger serum-dependent and independent cytokine release from endotoxin-stimulated normal human monocytes; CD14 is not, however, the only LPS receptor that is involved in the secretory response of endotoxin-stimulated cells.

Serum-free culture of human monocytes in the presence of monoclonal antibodies to the LFA-1 alpha chain (CD11a), CR3 alpha chain (CD11b) or beta chain (CD18) bound to Sepharose induced the dose-dependent production of cell-associated interleukin (IL) 1 activity and of IL 1 alpha and IL 1 beta antigens, but no release of extracellular IL 1 activity or antigen in the culture medium. Triggering of IL 1 production was also observed with insolubilized anti-CD11/CD18 F(ab')2 antibodies. Two cross-linked antibodies recognizing distinct epitopes on the CD11b molecule induced cell-associated IL 1. Soluble antibodies did not induce IL 1 production. The kinetics of induction of IL 1 by stimulation of adhesion-promoting proteins differed from those of IL 1 induction by adhesion to plastic. The lack of induction of IL 1 release by stimulation of the CD11/CD18 molecules resembled the intracellular accumulation of IL 1 induced by lipid A. Induction of IL 1 by adhesive processes may be a mechanism by which T cells trigger IL 1 production by monocytes during antigen presentation.

We used a quantitative immunoblotting technique to analyze the repertoires of IgG antibody reactivities in the serum of healthy young children, young adult males and aged males with self and non-self antigens. Densitometric patterns of reactivity of purified IgG with self antigens were highly conserved between individuals within a given age group and across age groups. Inter-individual differences were observed, however, upon analysis of self reactivities of IgG in whole serum. A striking heterogeneity between individuals within a given age group and across age groups characterized the reactivity of purified IgG and of IgG in whole serum with bacterial antigens. Inter-individual differences were more marked among aged individuals than among individuals of other age groups. Analysis of variances of reactivities of IgG with bacterial antigens further demonstrated an increased diversity of repertoires of aged donors compared with those of young adults and children. Our results document the stability of the self-reactive repertoires of IgG throughout life, which contrasts with the diversification of the repertoire of IgG antibody reactivities directed toward foreign antigens with aging. These findings support the concept that self-reactive antibody repertoires are positively selected throughout life by a restricted set of self antigens shared by all individuals.

Anti-thyroglobulin (TG) autoantibodies from patients with autoimmune thyroiditis express a cross-reactive alpha idiotype (Id) termed T44 which is not expressed by IgG from normal individuals. The present study demonstrates that the expression of the T44 Id is strongly associated with the recognition by anti-TG autoantibodies of a specific epitopic cluster on human TG. The epitopic reactivity of anti-TG autoantibodies was determined using a competitive inhibition assay with a panel of 15 monoclonal antibodies that define six antigenic clusters on TG. All T44+ autoantibodies from patients recognized cluster II, whereas no anti-TG IgG from healthy individuals reacted with this region. Affinity columns of Sepharose-bound intravenous therapeutic immunoglobulins which contain anti-T44 activity, retained both T44 Id-expressing antibodies and a subset of region II-specific anti-TG autoantibodies from patients with Hashimoto's disease. Restricted idiotypic and epitopic specificity may demarcate disease-associated from natural anti-TG autoantibodies, suggesting that qualitative rather than quantitative criteria should be used to identify pathological autoantibodies.

Stimulation with lipopolysaccharide (LPS) of unfractionated peripheral blood mononuclear cells in serum-free cultures resulted in the release of lower amounts of interleukin (IL) 1 as compared with those induced in cultures of purified monocytes. In addition, a dose-dependent decrease in IL 1 production was observed when purified autologous T cells were added to cultures of LPS-stimulated monocytes. The inhibitory effect of T cells on IL 1 production was reproduced by adding human recombinant IL 4 to monocyte cultures. IL 4 optimally inhibited the generation of cell-associated IL 1 and the extracellular release of IL 1 in monocyte cultures performed in the presence of low doses of LPS. IL 4 inhibited the generation of IL 1 activity and that of IL 1 alpha and IL 1 beta antigens indicating that IL 4 interfered with IL 1 transcription and/or processing rather than induced the synthesis of an IL 1 inhibitor. IL 2 and IL 3 enhanced IL 1 production by LPS-stimulated cells whereas granulocyte-monocyte colony-stimulating factor had no effect. IL 4 inhibited the production and the release of tumor necrosis factor alpha activity by monocytes stimulated with LPS. Thus, T cells and IL 4 may down-regulate the production of monokines which induce their proliferation and exert a suppressive effect on the generation of potent proinflammatory mediators.

Sephadex [alpha(1----6) cross-linked dextran] activates the human alternative pathway of complement. Substitution of hydroxyl groups of Sephadex with carboxymethyl groups (CM) results in a dose-dependent decrease of the activating capacity of the polymer in normal human serum. Sephadex bearing one CM group/glycosyl unit (CM-Seph 0.95) exhibited no activating capacity. CM groups did not interfere with the ability of the polymer to covalently bind C3b in the presence of purified alternative pathway proteins nor with the capacity of bound-C3b to form a C3 convertase in the absence of regulatory proteins. C3b that was bound to CM-Seph 0.95 was more susceptible to inactivation by factors H and I in serum than C3b bound to Sephadex. Binding studies using 125I-labeled H demonstrated that H bound with a similar affinity to the activating particle Sephadex, to Sephadex bearing C3b and to the nonactivating particle CM-Seph 0.95. However, factor H bound with a 5- to 7-fold higher affinity to CM-Seph 0.95 bearing C3b. These results demonstrate a requirement for both CM groups and C3b molecules in order for H to bind with high affinity to C3b on the non-activating surface, and indicate that H formed a ternary complex with surface-bound C3b and CM groups on CM-Seph 0.95. Using a chemically defined model system, the present study provides a molecular basis for the enhanced interaction between surface-bound C3b and factor H on nonactivators of the human alternative pathway.

Formation and function of the classical (C4b, 2a) and alternative (C3b, Bb) complement pathway C3 convertases are regulated by the intrinsic lability of the enzymes, extrinsic decay by C4bp and H, cleavage of C4b and C3b by I, and by the inhibitory action of the C3b receptor molecule (CR1). Binding of C4 nephritic factor (C4Nef) to C4b and of C3 nephritic factor (C3Nef) to C3b stabilizes the C3 convertases and bypasses inactivation by C4bp, H and/or I. In the present study, binding of C4Nef to the classical C3 convertase was found to prevent decay of C4b, 2a by inputs of CR1 that were at least 15 times the amount of CR1 which inactivated 50% unstabilized classical pathway C3 convertase sites in 2.5 min. CR1 could however inhibit lysis of C4b, 2a(C4Nef)-bearing cells in a dose-dependent manner. The latter inhibitory effect was directed at the interaction of C5 with the C5 convertase, most likely at C5 binding to cell-bound C3b. In an analogous manner to C4Nef in the classical pathway, stabilization of alternative pathway C3b, Bb convertase sites by C3Nef resulted in a relative protection of C3 convertase sites from decay by CR1. Thus, C4Nef and C3Nef can bypass all mechanisms susceptible to regulate function of the classical and alternative pathway C3 convertases. Because CR1 is essential for degradation of C3b bound to immune complexes in whole blood, stabilization of C4b,2a and C3b, Bb by C4Nef and C3Nef may alter in vivo processing of immune complexes in patients with nephritic factors.