Mercury-induced autoreactive anti-class II T cell line protects from experimental autoimmune encephalomyelitis by the bias of CD8+ antiergotypic cells in Lewis rats.

Brown-Norway (BN) rats injected with HgCl2 develop a systemic autoimmune disease associated with a polyclonal B cell activation, due to autoreactive T cells specific for self-class II molecules, while Lewis (LEW) rats injected with HgCl2 do not exhibit autoimmunity and develop a non-antigen-specific, CD8-mediated immunosuppression assessed by a depression of T cell functions, and a protection against experimental autoimmune encephalomyelitis (EAE). Resistance to HgCl2-induced autoimmunity is not due to these suppressor cells since treatment with an anti-CD8 monoclonal antibody (mAb) did not allow autoimmunity to appear. The absence of autoimmunity in this strain could result from the absence of autoreactive T cells, or from quantitative or qualitative differences of these cells between susceptible and resistant strains. In the present study, we show that CD4+ anti-class II T cells are present in HgCl2-injected LEW rats and are as frequent as in BN rats when assessed by limiting dilution analysis. LEW CD4+ autoreactive T cell lines were derived. They proliferated in the presence of normal class II-bearing cells, secreted interleukin 2, and did not induce B cells to produce immunoglobulins. Transfer of one of these lines, LEW Hg A, into normal LEW rats led to the appearance of CD8+ cells responsible for a non-antigen-specific immunosuppression that induced complete protection from EAE. Immunosuppression was abrogated after treatment with an anti-CD8 mAb. In vitro, CD8+ cells from rats injected with the LEW Hg A T cell line proliferated in the presence of activated T cells whatever their origin. We conclude that HgCl2 induces CD4+ autoreactive T cells that proliferate in the presence of class II+ cells in susceptible BN as well as in resistant LEW rats. But while these cells collaborate with B cells to produce autoantibodies in BN rats, they initiate in LEW rats a suppressor circuit involving antiergotypic CD8+ suppressor cells.

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.

Lethal host-versus-graft disease and hypereosinophilia in the absence of MHC I-T-cell interactions.

Neonatal injection of semiallogeneic spleen cells in BALB/c mice induces a self-limited state of chimerism that promotes the differentiation of donor-specific CD4 T cells toward the Th2 phenotype. Here we show that injection of spleen cells from beta2-microglobulin-deficient (BALB/c x C57BL/6) F1 mice into BALB/c newborns with a disrupted beta2-microglobulin (beta2m) gene results in a lethal lymphoproliferative disorder associated with uncontrolled Th2 response, long-term persistence of donor B cells, and sustained blood eosinophilia. Autoimmune manifestations are also enhanced and characterized by a severe autoantibody-mediated glomerulonephritis. Histological examination of the spleen shows a hyperplasia of periarteriolar lymphoid sheaths, with accumulation of eosinophils and basophils, and variable degree of fibrosis. Perivascular lymphoid infiltrates with eosinophils are also found in the lung and are correlated with disease severity. Such abnormalities are almost absent using beta2m-sufficient mice. These data demonstrate that induction of lymphoid chimerism in the absence of MHC class I-T-cell interactions results in a lethal form of host-versus-graft disease that represents a unique model of Th2-dependent chronic inflammatory disease associated with an hypereosinophilic syndrome in mice.

Mercuric chloride, a chemical responsible for T helper cell (Th)2-mediated autoimmunity in brown Norway rats, directly triggers T cells to produce interleukin-4.

Mercurials may induce immune manifestations in susceptible individuals. Mercuric chloride (HgCl2) induced autoimmunity in the Brown Norway (BN) strain but an immuno-suppression in the Lewis strain with, however, autoreactive anti-class II T cells present in both strains. In the present study we looked at modifications of cytokine production by PCR and cytofluorometric analyses in normal BN and Lewis rat splenocytes, cultured with or without HgCl2. Unfractionated BN rat splenocytes and purified T cells exposed to HgCl2 expressed high levels of IL-4 mRNA. Increase in class II and CD23 molecule expression on B cells was partly inhibited by anti-IL-4 mAb showing that IL-4 was produced. By contrast, no overexpression of IL-4 mRNA could be seen in Lewis rats. Although an increase in class II molecule expression was observed suggesting that other T helper cell 2 cytokines were produced, there was also a concomitant decrease in CD23 molecule expression that was abrogated after addition of an anti-IFN-gamma mAb to the culture. IFN-gamma mRNA production was induced in unfractionated spleen cells and T cells from both strains after HgCl2 exposure. Altogether these findings demonstrate that HgCl2 has very early direct effects on cytokine production and that these effects differ depending on the strain. The early effect on IL-4 production observed on BN rat spleen cells and T cells may explain that the autoreactive anti-class II T cells that are found in HgCl2-injected BN rats have a Th2 phenotype.

Nephrotic syndrome, linear glomerular IgG deposits, and minimal glomerular changes. Report of a case.

A young adult patient had an unusual acute idiopathic nephrotic syndrome. This nephrotic syndrome was remarkable for (1) association with acute renal failure and hypertension, (2) finding of minimal glomerular changes with a linear fixation of the anti-human IgG conjugate along the glomerular capillary wall without demonstrable antiglomerular basement membrane antibodies, and (3) complete recovery, including disappearance of the linear staining, after treatment with prednisone, cyclophosphamide, and plasmapheresis.

Self major histocompatibility complex class-II-specific regulatory CD4 T cells prevent both Th1- and Th2-mediated autoimmune diseases in the rat.

It is clear that functional heterogeneity of T cells may be explained by differential cytokine production. The aim of this paper was to review evidence for regulatory cells, generated after HgCl(2)-exposure. They differ from classical Th1 and Th2 cells, produce transforming growth factor-beta and interleukin-10 and exert their regulatory functions in a Th1/Th2-unrestricted fashion.

Isolation of rat and rabbit IgM from normal serum using anti-human mu antibody-polyacrylamide beads immunosorbents.

The antigenic relationship between human IgM and IgM from other species has been utilized in isolating rat and rabbit IgM from normal sera by means of an anti-human mu-polyacrylamide beads immunoadsorbent. The isolated IgM was found to be pure by immunoelectrophoretic analysis and Ouchterlony technique. The antisera obtained were monospecific for mu-chains after absorption with IgG. Lastly, the IgM isolated always retained its antibody activity.

Isolation of normal human IgA, IgM and IgG fragments by polyacrylamide beads immunoadsorbents.

Polyacrylamide beads antibody immunoabsorbents were used in order to isolate human IgA, IgM and fragments of papain-digested human IgG. The proteins obtained were pure as judged by immunochemical techniques. The antisera raised with these purified proteins were monospecific. The binding capacity and the yield were satisfactory. These antibody immunoadsorbents offer several advantages; a) highly purified antigens can be quickly obtained in a one-step procedure from body fluids; b) their handling is easy especially when using a column; c) they can be used for a long period of time and for many experiments without any noticeable loss in their binding capacity.

Complement receptors in human renal glomeruli. Further evidence by immunofluorescence.

A new method is described for demonstrating the presence of glomerular receptors for the third component of complement in human kidney. Frozen sections are incubated together with normal human serum and inulin. Activated C3 is detected by a fluoresceinated anti-C3 antiserum. Glomeruli are labelled by C3-coated inulin particles while there is no labelling in tubules and interstitium. This method is easy and rapid and it allows the exact localization of C3 glomerular receptors.

Rat monoclonal antibodies. III. A simple method for facilitation of hybridoma cell growth in vivo.

Hybridoma cells that did not grow when injected subcutaneously or intraperitoneally in histocompatible or Rnu/Rnu rats were injected intravenously into histocompatible recipients. Eight of the 9 cell lines injected in this way grew in several organs of the recipient 3-7 weeks later. Hybridoma cells proliferated mainly in the liver. When the liver homogenate of these animals was injected intraperitoneally into histocompatible recipients, hybridoma cells grew readily giving rise to ascites containing the expected monoclonal antibody for 6 of the 9 cell lines.

Magnetic solid-phase enzyme immunoassay for the detection of anti-glomerular basement membrane antibodies.

A new enzyme immunoassay has been developed for the demonstration of antiglomerular basement membrane antibodies. Magnetically responsive polyacrylamide-agarose beads (Magnogel) activated with glutaraldehyde were used to bind sonicated insoluble rat glomerular basement membranes. Both the collagenous and the non-collagenous moieties were demonstrated to be fixed on the beads. Sera from brown Norway rats with anti-glomerular basement membrane antibodies induced by HgCl2 injections were incubated with the beads. After washing, the fixed rat IgG were revealed using alkaline phosphatase labelled Fab fragments from anti-rat IgG sheep, IgGs. Comparison with a radioimmunoassay showed that results were reliable. This enzyme immunoassay has several advantages which may render this assay of considerable clinical usefulness.

Suppression of HLA-specific alloantibodies by high-dose intravenous immunoglobulins (IVIg). A potential tool for transplantation of immunized patients.

Renal transplantation in patients presenting end-stage renal failure can be hampered by the presence of alloantibodies against HLA antigens. In 4 out of 5 patients with HLA-specific alloantibodies waiting for a renal allograft, treatment with high-dose i.v. Ig resulted in a prolonged suppression (over 3 months) of most of the panel-reactive anti-HLA antibodies (PRA). Intravenous polyclonal human Ig (IVIg) and F(ab')2 fragments from IVIg inhibited the binding of patients' plasma and IgG fractions to peripheral blood lymphocytes from normal donors as well as their cytotoxicity, suggesting that the in vivo effect of IVIg was mediated by the presence, in the IVIg preparation, of anti-idiotypes directed against idiotypes borne on the anti-HLA antibodies. Thus, treatment with IVIg can be a valuable tool toward the transplantation of immunized patients.

Circulating immune complexes in the general hospital population and in patients who have renal disease. A preliminary evaluation of the platelet aggregation test for use in the hospital laboratory.

The platelet aggregation test was used to measure circulating immune complexes in patients who had glomerulonephritis and autoimmune disease, and in unselected hospitalized patients. Titers higher than those for the control group were found for groups with acute glomerulonephritis, Berger's disease, renal transplants, lupus nephritis, and polyarteritis nodosa. Hospitalized patients had titers that were higher than normal in more than one out of five cases. This preliminary study showed that the platelet aggregation test detects immune complexes in many conditions previously reported to give positive results with other assays. The test seemed suitable for use in most hospital laboratories, since it was simple, reliable, and inexpensive. The observation of detectable circulating immune complexes in 23% of the general hospital population suggests that this phenomenon may be more common than was previously suspected.

Involvement of hemostasis during an autoimmune glomerulonephritis induced by mercuric chloride in brown Norway rats.

Mercuric chloride (HgCl2) induces in Brown Norway (BN) rats an autoimmune disease characterized by a biphasic glomerulonephritis (GN). A transient nephrotic syndrome occurs during the third and fourth weeks after the first HgCl2 injection. Related to nephrotic syndrome, an hypercoagulable state develops with decreased factor XII and anti-thrombin III (AT III) levels and increased factor V activity and fibrinogen concentration. Moreover, during the same period, most of the rats were found thrombocytopenic. The presence of soluble fibrin monomer complexes and of fibrin degradation products (FDP) in the plasma of these rats associated with fibrin thrombi in glomerular capillary lumen proved the occurrence of disseminated intravascular coagulation (DIC). DIC was responsible for the death of several rats but most of these survived and clotting abnormalities were no longer found. Numerous factors can explain the occurrence of DIC in this model: anti glomerular basement membrane antibodies, circulating immune complexes, complement activation and/or glomerular endothelial cell detachment. The HgCl2 induced autoimmune disease appears as a good experimental model to study the relation between coagulation process and glomerulonephritis.

Flow cytometric analysis of intracellular interferon-gamma synthesis in rat CD4 T cells.

To date the techniques used to analyse cytokine expression by rat T cells do not give information about the simultaneous production of different cytokines from individual cells. Recently, a method for analysing the intracellular production of cytokines at the single cell level using flow cytometry has been developed. It is well established that the most critical requirement for successful intracellular cytokine staining is the availability of appropriate antibodies. In rat, it is possible to stain for intracellular IL-4 and IL-10 (Th2 cytokines) using the commercially available antibodies but not for Th1 cytokines. In the present work, we show that DB1, a mouse anti-rat IFN-gamma monoclonal antibody, could be used for intracytoplasmic staining of IFN-gamma producing rat CD4 T cells. The specificity of the staining was confirmed using a molar excess of unlabelled antibodies or recombinant cytokine. Finally, intracellular staining for IFN-gamma correlates with cytokine production in culture supernatant as evaluated by ELISA analysis.

Autoimmune glomerulonephritis induced by mercury vapour exposure in the Brown Norway rat.

Subcutaneous injections of mercuric chloride induce an autoimmune glomerulonephritis with both granular and linear IgG deposits along the glomerular capillary wall and proteinuria. This disease is due to a T cell dependent polyclonal B cell activation responsible for production of antibodies against self (glomerular basement membrane, immunoglobulins, DNA, myeloperoxydase) and non self (sheep red blood cells, trinitrophenol (TNP)) components. Increase in serum IgE concentration is the hallmark of this disease. To determine if mercury vapours have pathogenic effects is an important problem of public health. The aim of this study was, first to compare the effects of mercury vapour exposure to those of mercury injections and, second, to compare the effects of high doses to those of low doses of mercury. Two exposure levels were studied corresponding to a mercury absorption of 13.1 mumol/week per kg body wt. and 1.7 mumol/week per kg body wt. during a 5-week period. It will be shown that, whereas the mercury concentration in the kidneys was similar in injected--and vapour exposed--rats, the mercury concentration in blood at the end of the exposure was about twice as high in the injected animals. Blood concentration of mercury was related to dose level but kidney content of mercury was similar in all groups, in spite of a dose difference by a factor of seven between low and high exposure. Mercury vapour and HgCl2 injections both trigger autoimmunity to the same extent and, in both cases the extent of autoimmune manifestations was dose-dependent.

Anti-laminin antibodies in Sprague-Dawley and brown Norway rats chronically exposed to cadmium.

Sprague-Dawley and Brown-Norway female rats were chronically exposed to cadmium. The metal was administered either in drinking water at a concentration of 20 or 100 ppm for 13 months or intraperitoneally (i.p.) at the dose of 1 mg/kg, 5 times a week for 4 months. Anti-laminin and anti-type IV procollagen antibodies (laminin and type IV procollagen are components of the glomerular basement membranes) were sequentially assessed by radioimmunoassay in serial serum samples. Anti-type IV procollagen antibodies were never detectable whereas anti-laminin antibodies were transiently found in the serum of Sprague-Dawley rats only. In the i.p. group, the antibodies were detected between week 4 and 8, whereas in the oral treatment group, they were detected between month 6 and 8. At the same time, the concentration of cadmium in kidney cortex averaged about 100 ppm in the 20 ppm cadmium group and about 200 ppm in the 2 other treatment groups. The pathogenic significance of these antibodies in chronic cadmium intoxication of Sprague-Dawley rats remains to be assessed since their occurrence was not associated with concomitant immunoglobulin deposits in the kidneys.

Contribution of immunological reactions to nephrotoxicity.

Several toxic agents, such as mercurials and agents with a sulphydryl group (e.g., gold salts, D-penicillamine and captopril), are associated with the occurrence of membranous glomerulopathy. DR3 antigen-positive subjects and poor sulphoxidators are at higher risk than other patients when treated with gold salts or D-penicillamine. Other drugs, mainly nonsteroidal anti-inflammatory agents and lithium salts, are responsible for the nephrotic syndrome with minimal glomerular changes, a T-cell-mediated disease. Several models of drug-induced membranous glomerulonephritis have been developed. These have been used to confirm the role of most of the above-mentioned agents and have made it possible to shed some light on the possible mechanisms involved. Mercurials induce a polyclonal activation of B cells in rats which is related to the appearance of autoreactive T cells. Finally, numerous agents may induce immunologically mediated acute interstitial nephritis. Although cell-mediated immunity appears to be involved in most cases, experimental models are lacking to substantiate this hypothesis.

Association of overt glomerulonephritis and liver disease: a study of 34 patients.

Thirty-four patients with overt glomerulonephritis and chronic liver disease were studied. Kidney specimens were examined by light, electron and immunofluorescence microscopy. Plasma C3 levels were measured and a search for cryoglobulinemia was carried out in all patients. Twenty-six out of the thirty-four patients had an immune complex type glomerulonephritis (membrano-proliferative glomerulonephritis or glomerulosclerosis with mesangial deposits) suggestive of hepatic glomerulonephritis. The glomerular deposits almost always contained IgA and very frequently other immunoglobulins as well as C3. The membrano-proliferative glomerulonephritis was characterized by severe renal symptoms, mixed cryoglobulinemia and the frequent finding of low C3 levels. These data suggest that there is a linkage between liver disease and glomerulonephritis. The immunomorphological type of glomerulonephritis and the cryoglobulinemia are both suggestive of an immune complex disease. The lowering of the C3 levels could be due to activation of complement components by immune complexes, to hepatic hyposynthesis, or to a combination of the two.

Effect of cyclosporine A on mercury-induced autoimmune glomerulonephritis in the Brown Norway rat.

To test the effect of cyclosporine A (CsA) in mercuric chloride (HgCl2)-induced nephritis in the Brown-Norway (BN) rat, we treated groups of intoxicated rats with varying doses of CsA for a period of 2 months. All manifestations of HgCl2-induced disease were prevented in rats treated concurrently with CsA at either 7 or 10 mg/kg/day. Partial suppression was evident at lower daily doses, but not with bi-weekly CsA administration. The initial phase of HgCl2-induced nephritis could be completely suppressed with a short, 15 day course of CsA. The later phase of the disease could be tempered by CsA administration starting on day 10 after the first HgCl2 injection. The optimal regimen of 7 mg/kg/day for 60 days was not associated with any evidence of CsA toxicity. CsA appears to interfere with the polyclonal activation of B cells observed in HgCl2-induced autoimmune disease, accounting for its striking preventive and curative effect in this model.