Identification of mu-opioid receptor epitopes recognized by agonistic IgG.

We have previously reported the presence of IgG antibodies with a morphine-like activity in the serum of healthy individuals. The agonistic activity of IgG was dependent on their binding to the first and the third extracellular loops of the human mu opioid receptor. In this study we show that IgG antibodies obtained by immunizing rats with peptides corresponding to these two loops exhibited a similar morphine-like activity. Residues corresponding to Y(130), M(132), G(133), T(134) within the first and F(315) within the third extracellular segment were required for antibody binding and conferred to IgG a high mu-opioid selectivity.

Mercuric chloride-induced autoimmunity.

This unit describes methods for inducing autoimmune disease in Brown Norway rats through HgCl(2) injections as well for assessing parameters that characterize the disease by serum IgE concentration assays, anti-laminin antibody measurement, and renal immunofluorescence studies to detect autoantibodies. Also covered are disease induction using autoreactive CD4(+) T(H)2 anti-self MHC class II molecules and preparation of T cell lines. IL-4 is produced very early after the first HgCl(2) injection (beginning at day 3, peaking at day 14, and continuing up to day 30). Thus, IL-4 mRNA expression may be detected in spleen and lymph nodes from HgCl(2)-injected BN rats. The fact that HgCl(2) induces in vitro mRNA IL-4 gene expression in normal BN T cells but not in LEW T cells is probably crucial to susceptibility to the development of autoimmunity in the sense that it may condition the development of autoreactive T cells into pathogenic T(H)2 cells; a test for this condition is therefore also included.

Non-immunoglobulin serum proteins prevent the binding of IgG from normal rats and from rats with Th2-mediated autoimmune glomerulonephritis to various autoantigens including glomerular antigens.

It is now well established in normal humans and mice that purification of IgG from serum unmasks their autoantibody activity. Mercuric chloride (HgCl2) induces in Brown-Norway (BN) rats a Th2-dependent polyclonal B cell activation, a huge increase in serum IgE and IgG1 concentrations, the production of numerous autoantibodies and an autoantibody-mediated glomerulonephritis. In the present study we have compared the IgG autoantibody activity in the serum and in the purified IgG fraction from normal and HgCl2-injected BN rats. IgG autoantibodies were found to be masked in normal serum by non-immunoglobulin (nonIg) serum proteins and, provided these IgG did not encounter normal serum proteins, they could bind to glomerular antigens as assessed by immunofluorescence in a unilateral perfused kidney model. As a consequence of HgCl2-induced polyclonal activation of B cells, IgG autoantibodies were no longer complexed to non-Ig serum proteins, they were easily detected in the serum and could therefore reach their glomerular target. However, these autoantibodies could still be blocked by normal non-Ig serum proteins not only in vitro but also in a unilateral perfused kidney model so that their binding to glomerular antigens could be prevented. These findings indicate that the ratio between autoantibody level and the amount of non-Ig serum proteins may be crucial in autoantibody-mediated autoimmune diseases.

Transforming growth factor beta (TGF-beta)-dependent inhibition of T helper cell 2 (Th2)-induced autoimmunity by self-major histocompatibility complex (MHC) class II-specific, regulatory CD4(+) T cell lines.

Autoreactive anti-MHC class II T cells are found in Brown Norway (BN) and Lewis (LEW) rats that receive either HgCl2 or gold salts. These T cells have a T helper cell 2 (Th2) phenotype in the former strain and are responsible for Th2-mediated autoimmunity. In contrast, T cells that expand in LEW rats produce IL-2 and prevent experimental autoimmune encephalomyelitis, a cell-mediated autoimmune disease. The aim of this work was to investigate, using T cell lines derived from HgCl2-injected LEW rats (LEWHg), the effect of these autoreactive T cells on the development of Th2-mediated autoimmunity. The five LEWHg T cell lines obtained protect against Th2-mediated autoimmunity induced by HgCl2 in (LEW x BN)F1 hybrids. The lines produce, in addition to IL-2, IFN-gamma and TGF-beta, and the protective effect is TGF-beta dependent since protection is abrogated by anti-TGF-beta treatment. These results identify regulatory, TGF-beta-producing, autoreactive T cells that are distinct from classical Th1 or Th2 and inhibit both Th1- and Th2-mediated autoimmune diseases.

Characteristics of polyreactive and monospecific IgG anti-laminin autoantibodies in the rat mercury model.

Brown-Norway (BN) rats injected with HgCl2 produce anti-laminin antibodies responsible for an autoimmune glomerulonephritis. The properties of three IgG1 monoclonal antibodies (mAb) previously obtained in this model, and of immunoglobulins eluted from kidneys of diseased rats, were compared in the present study. Two mAb (Hg15 and Hg16) recognized laminin only, while the third one (Hg17) was polyreactive, as were some of the kidney-eluted immunoglobulins; they reacted with laminin and with several other antigens including 2,4,6-trinitrophenyl (TNP). The Hg17 mAb and kidney-eluted polyreactive antibodies were affinity purified using a TNP-bovine serum albumin (BSA) column; their affinity for TNP was high (2 x 10(-8)M, and 1 x 10(-8)M, respectively) but less than that of a TNP-specific (LO-DNP-2) mAb (2 x 10(-11) M). The Hg17 mAb and kidney-eluted antibodies reacted more effectively with TNP28-BSA than with TNP8.5-BSA, while the TNP-specific mAb reacted equally well with both conjugates. The Hg17 mAb was the most cationic (pI: 7) of the anti-laminin mAb and this was even more evident when F(ab')2 fragments were studied (pI: 8.2). The polyreactive kidney-eluted immunoglobulins that bound TNP were also more cationic (pI: 7.4-9.3) than the fraction that did not recognize TNP (pI: 5.8-8.6). The anti-laminin mAb bound in vivo to the glomerular basement membrane, but only the Hg17 mAb could be eluted with DNP alone. This study shows that polyreactive anti-laminin antibodies are produced during this autoimmune disease, and indicates that they may have pathogenic potential.

Effect of the thiol group on experimental gold-induced autoimmunity.

Brown Norway rats injected with aurothiopropanolsulfonate sodium salt develop systemic autoimmunity. The aim of this study was to assess the influence of the sulfur-containing group in this experimental model of gold-induced autoimmunity. It was shown that the sulfur-containing group does not induce autoimmunity of itself, but potentiates the immunotoxic effects of gold.

IgA polyspecific autoantibodies in IgA nephropathy.

The specificity of circulating and kidney-bound IgA during IgA nephropathy is still a matter of discussion. In the present study, high levels of IgA antibodies directed against a panel of self and non-self antigens were found in the serum from patients with IgA nephropathy and were eluted from four out of the seven kidney biopsies studied. After immunoadsorption of pooled selected serum samples on TNP and actin-coated columns, polyspecific IgA antibodies were eluted. This supports the hypothesis that IgA-bearing B cells clones most probably producing polyspecific antibodies are a major feature of human IgA nephropathy. These findings also suggest that it may be hazardous to draw conclusions from the finding of apparently monospecific IgA antibodies in this condition.

Rat anti-glomerular basement membrane antibodies in toxin-induced autoimmunity and in chronic graft-vs.-host reaction share recurrent idiotypes.

Cross-reactive idiotypes (CRId) borne on autoanti-glomerular basement membrane antibodies of Brown-Norway (BN) rats with mercury-induced glomerulonephritis have been described in the preceding study (Guéry, J.-C. et al., Eur. J. Immunol. 1990. 20:93). BN rats treated with sodium aurothiopropanol sulfonate or D-penicillamine, as well as (LEW X BN)F1 hybrids transferred with BN rat spleen cells, developed quite similar autoimmune abnormalities. In the present study, it is shown that immunoglobulins bearing such "public" idiotypes are also produced and deposited in the kidney in these three models. The CRId here described may, therefore, be considered as a marker of sets of recurrently expressed V region genes during the course of these autoimmune disorders. Anti-self class II T cells are present in the three models of toxin-induced autoimmunity and anti-allo class II T cells are responsible for the chronic graft-vs.-host reaction. The same B cell clones are probably triggered during these processes as a consequence of a polyclonal B cell activation mediated by anti-class II T cells.

Specificity and cross-reactive idiotypes of anti-glomerular basement membrane autoantibodies in HgCl2-induced autoimmune glomerulonephritis.

Mercury-induced autoimmune glomerulonephritis in the Brown-Norway (BN) rat is characterized by the successive appearance of linear and granular glomerular IgG deposits. Anti-laminin autoantibodies represent the major part of the anti-glomerular basement membrane (GBM) antibodies produced in this model. Fusions were performed in this model and four anti-GBM monoclonal antibodies (mAb) were obtained. Three of them were laminin specific. Using rabbit anti-idiotype antibodies, cross-reactive idiotypes (CRId) were characterized on anti-laminin antibodies. They were expressed on the three anti-laminin mAb, on kidney-eluted and circulating anti-laminin antibodies. CRId-bearing immunoglobulins were detected transiently in the circulation and paralleled the anti-laminin antibody activity. By immunofluorescence studies on kidney cryostat sections two different CRId were defined. One was localized close to the antigen-combining site since it was not revealed on kidney-bound antibodies, in contrast with the second CRId. This latter CRId was also found deposited in a typical linear pattern in the early phase of the disease and in a granular pattern in the late phase, demonstrating that these CRId are components of immune deposits. Taken together, these results suggest that in this model of T-dependent polyclonal B cell activation, restricted sets of V genes encode for at least a part of the anti-GBM autoantibodies.

Production of monoclonal anti-glomerular basement membrane antibodies during autoimmune glomerulonephritis.

Monoclonal anti-glomerular basement membrane (GBM) antibodies were obtained by fusing spleen cells from Brown-Norway (BN) rats injected with mercuric chloride with IR 983 F, a nonsecreting rat myeloma cell line. These antibodies showed the same pattern of fixation on renal basement membranes by indirect immunofluorescence. One of them was developed. It reacted both in vivo and in vitro with GBM but failed to react with collagenase-digested GBM, laminin, and collagen IV. This monoclonal antibody which resembles the kidney acid eluate obtained from BN rats injected with mercuric chloride induced a weak and transient proteinuria when intravenously injected into normal BN rats.

Glomerular and vascular IgG deposits in HgCl2 nephritis: role of circulating antibodies and of immune complexes.

The respective roles of circulating anti-glomerular basement membrane antibodies and of circulating immune complexes in the appearance of glomerular linear and granular IgG deposition during HgCl2-induced glomerulonephritis in the Brown-Norway rat has been studied. Syngeneic kidney transplantations have been performed at various phases of the disease. Results show that circulating antibodies are responsible for linear IgG deposition which did not change to granular deposits during the course of the disease. Electron-dense subepithelial deposits occurred only when circulating immune complexes were detected. These experiments strongly suggest that, in the mercury model, circulating immune complexes are responsible for granular IgG deposits observed in arteries and in the subepithelial space of glomeruli.

Mercuric chloride nephritis depends on host rather than kidney strain.

Brown-Norway (BN) rats are susceptible to the induction of an autoimmune glomerulonephritis (GN) by HgCl2 while Lewis (LEW) rats are resistant. When a kidney from a LEW rat (nonsusceptible) is transplanted into a binephrectomized (LEW X BN)F1 hybrid (susceptible) then HgCl2 injections into the recipient result in GN developing in the donor kidney. When a kidney from a BN or (LEW X BN)F1 hybrid (susceptible) is transplanted into a nonsusceptible rat, injections of HgCl2 into the recipient do not result in GN in the donor kidney. These observations show that kidneys from nonsusceptible rats function as susceptible targets and that induction of the disease depends more on the host immune system than on modification of kidney determinants by HgCl2.

Immune complex type disease induced by HgCl2 in Brown-Norway rats: genetic control of susceptibility.

Mercuric chloride induces a biphasic autoimmune glomerulonephritis in Brown-Norway (BN) rats but not in Lewis (LEW) rats. The genetic control of susceptibility to both phases was investigated by testing the response of segregants between BN and LEW rats and of congenic LEW.1N rats. It was confirmed that susceptibility to the first phase, characterized by the appearance of anti-glomerular basement membrane antibodies, depends on several genes one of which is RT1 linked. Susceptibility to the second phase, which is an immune complex type glomerulonephritis, was found to depend on one major RT1 linked gene or cluster of genes with a role for other(s) non-RT1 linked gene(s) controlling the magnitude of the response. However, congenic LEW.1N rats were found to be resistant. This suggests that the disease gene has been lost during the strain derivation. The question of whether both phases are two different diseases or expression of the same process cannot be definitely answered; data however indicate a dissociation of both disease processes.