Androgens indirectly accelerate thymocyte apoptosis.

Apoptotic processes, or the disturbance of the natural regulation of these processes, may be involved in the pathogenesis of autoimmune diseases (AID). Women are, in general, more susceptible than men to develop AID like rheumatoid arthritis. Androgens and glucocorticoids, in contrast to oestrogens, have favourable effects in AID models as well as in human AID. It is known that glucocorticoids (GC), used for treatment of AID, increase apoptosis in the thymus resulting in decreased numbers of CD4+ CD8+ thymocytes. It was asked whether androgens, in contrast to oestrogens, exert their favourable effects in the treatment of AID by a mechanism comparable to that described for GC by eliminating the apoptosis prone CD4+ CD8+ population in the thymus. Although both androgens and oestrogens proved thymolytic, a significantly decreased percentage of CD4+ CD8+ thymocytes was observed by flow cytometry after treatment of mice with the androgen methyltestosterone, but not with the oestrogen ethinylestradiol. To investigate whether the observed thymolytic effects were due to the presence of hormone receptors on thymocytes, cells were isolated from the thymus and incubated with androgens or oestrogens to measure apoptosis. Several techniques were used to determine thymocyte apoptosis in vitro, but no enhanced apoptotic signal was observed. Using the very sensitive TUNEL assay, no direct effect of androgens on thymocytes in vitro could be observed. This is in sharp contrast to the high signal observed with GC. Therefore, upon in vivo androgen treatment, other cells containing androgen receptors than thymocytes are probably involved in inducing the increase in thymic apoptosis. To study the role of the androgen receptor on thymocyte apoptosis, androgen receptor mutant (Tfm/Y) mice were treated with androgens. No alterations of thymocyte subpopulations were seen, suggesting that changes in the percentage of CD4+ CD8+ thymocytes after administration of androgens depend on the presence of functional androgen receptors. Thus, it is concluded that androgens indirectly accelerate thymocyte apoptosis in vivo.

The steroid hormone dehydroepiandrosterone (DHEA) breaks intranasally induced tolerance, when administered at time of systemic immunization.

The induction of intranasal tolerance might be dependent on specific characteristics of mucosal, nose-draining lymph nodes. Such a specific characteristic might lie in the metabolism of the steroid hormone DHEA. Conversion of the prohormone DHEAS into DHEA is dependent on DHEAS-sulphatase activity in lymph nodes. This activity is low in mucosa-draining lymph nodes compared to peripheral lymph nodes, leading to differences in microenvironment. However, administration of DHEA before the induction of intranasal tolerance, could not change tolerance induction. We next determined the effect of DHEA after the induction of intranasally induced tolerance and demonstrated that the steroid hormone and some of its derivatives are able to break tolerance, when administered at time of systemic immunization. These findings might have implications for the regulation of intranasal tolerance and the use of DHEA.

Effect of mesangiolysis on autoregulation of renal blood flow and glomerular filtration rate in rats.

Interlobular arteries and afferent arterioles are involved in autoregulation of renal blood flow (RBF) and glomerular filtration rate (GFR). The question of whether the contractile mesangial cells are also involved in autoregulation was investigated in Wistar rats. Autoregulation of RBF was examined before and 1 h after infusion of antithymocyte (anti-Thy 1-1) antibodies, and both RBF and GFR autoregulation were examined 30 h after the infusion of antibodies. Mesangial cell destruction was present 30 h after the infusion of antibodies. The angiotensin II-induced contraction of isolated glomeruli (70% of control volume, P less than 0.001) was abolished after the glomeruli had been exposed to anti-Thy 1-1 in vitro. RBF, as well as the lower limit of RBF autoregulation, were not different from control 30 h after the infusion (82 +/- 5 vs. 79 +/- 4 mmHg, P greater than 0.10). Autoregulation of GFR was maintained in the control group but was restricted in the experimental group (autoregulatory index: 0.71 +/- 0.42 for left kidney, 0.02 +/- 0.35 for control; P less than 0.05). The afferent arteriolar diameter was unchanged 30 h after the infusion of antibodies (17.8 +/- 0.8 vs. 17.6 +/- 0.4 microns, P greater than 0.10). One hour after infusion of the antibodies, RBF autoregulation was normal. It is concluded that mesangial cells do not seem to be involved in RBF autoregulation, but may in part influence autoregulation of GFR during pressure reduction.

Mesangial cell immune injury: effects of thromboxane receptor antagonism.

We assessed the renal hemodynamic changes occurring acutely after glomerular mesangial cell immune injury and the effects of thromboxane receptor antagonism on these changes. A single intravenous proteinuric dose of a monoclonal antibody raised against the rat thymocyte antigen Thy 1.1 (ER4), which is also expressed in rat mesangial cells, induced acute decrements in glomerular filtration rate and in renal blood flow in male Munich-Wistar rats. One hour after administration of 4 to 6 mg/kg of ER4 antibody, glomerular filtration rate and renal blood flow decreased by 80 and 36%, respectively. These decrements were associated with significant increments in basal thromboxane B2 synthesis in isolated glomeruli and no changes in glomerular prostaglandin E2 synthesis. Pretreatment of animals with the thromboxane receptor antagonist SQ-29,548 (2 mg/kg) significantly ameliorated decrements in glomerular filtration rate and renal blood flow. Pretreatment with a structurally dissimilar thromboxane receptor antagonist, L-670,596 (3 mg/kg) had no effect. Both antagonists at the doses employed abolished the decrements in renal blood flow induced by systemic administration of the thromboxane mimetic U-46619. Whereas the SQ-29,548 antagonist had no effect on glomerular leukotriene B4 and 12-hydroxyeicosatetraenoic acid synthesis, the L-670,596 thromboxane receptor antagonist significantly inhibited glomerular synthesis of these eicosanoids in immunologically injured glomeruli. These observations indicate that in mesangial cell immune injury the protective effect of thromboxane A2 receptor antagonism on glomerular filtration rate and renal blood flow is not solely due to inhibition of the vasoconstrictor effects of thromboxane A2. An effect on the synthesis of arachidonate lipoxygenation products may also play a role.

The mesangium in anti-Thy-1 nephritis. Influx of macrophages, mesangial cell hypercellularity, and macromolecular accumulation.

Mesangial pathology is a hallmark of focal and segmental glomerulosclerosis (FGS). In an immunologically mediated mesangial cell (MC) injury model, we analyzed the relationship between mesangial hypercellularity, increased macromolecular uptake by the mesangium, and long-term pathologic sequelae. A single injection of monoclonal anti-Thy-1 (AT) antibody induces MC apoptosis, extensive mesangiolysis, proteinuria, MC proliferation, and hypercellularity. Immunohistologic analysis indicated influx of ED 1-positive macrophages after 24 hours, which gradually subsiding thereafter. At day 12, hypercellularity was due to smooth musclelike MCs, and macrophagelike MCs were absent. Injection of iron dextran in nephritic rats indicated that mesangial uptake of iron correlated with mesangial hypercellularity, but was independent of proteinuria. Long-term studies showed no difference after 19 weeks in FGS between nephritic and control rats. In conclusion, although mesangiolysis is accompanied by influx of macrophages, a phase of smooth musclelike MC proliferation and increased macromolecular uptake, these pathologic events do not result in chronic mesangial pathology and FGS.

Thymocytes reacting with heterologous antibodies against GP 330 in autologous immune complex glomerulopathy (AICG) in the rat. The relation between susceptibility for AICG and anti-GP 330-binding thymocytes.

Autologous immune complex glomerulopathy (AICG) is induced by immunizing rats with a crude brushborder fraction of rat kidney tubules (Fx1A) or with the purified GP 330 antigen. In these animals, anti-brushborder antibodies develop, leading to subepithelial immune complexes along the glomerular capillary wall. In rats with AICG, thymocytes sensitized against Fx1A as well as thymocytes recognizing anti-Fx1A are present. These latter cells might play a role in the specific tolerance against the pathogenetic antigen GP 330. To substantiate this notion, immunofluorescence studies were performed in which the number of anti-GP 330 binding cells was quantified in thymus cell suspensions of rats with AICG, in control rats and in naive rats with different genetic background. It is shown that increased numbers of anti-GP 330-binding thymocytes in rats with AICG are associated with a decline in the serum anti-brushborder titer. Furthermore, it appears that the number of anti-GP 330-binding thymocytes in naive rats of the non-responder Brown Norway strain is significantly higher as compared to the PVG/c and Lewis strains, which are susceptible for AICG. The correlation between the numbers of anti-GP 330-binding thymocytes and the susceptibility for AICG suggests a role for these cells in maintaining the tolerance against the Fx1A (GP 330) antigen.

A monoclonal antibody against rat platelets. I. Tissue distribution in vitro and in vivo.

In this study we describe a new monoclonal antibody (MoAb PL.1) against rat platelets. Immunohistology of various rat tissues showed staining of platelets, especially in the spleen, and staining of megakaryocytes in bone marrow and spleen red pulp. In the liver small platelet aggregates and endothelial cells were stained. After in-vivo administration of MoAb PL.1 an acute severe thrombocytopenia was observed. In general the distribution of the antibody and/or antibody-coated platelet aggregates showed the same pattern as after in-vitro incubation, i.e. staining of rat platelets and platelet aggregates in spleen red pulp, and staining of megakaryocytes in spleen and bone marrow. Platelet aggregates were observed in the liver and electron microscopy indicated that they were associated with Kupffer cells. Furthermore, liver endothelial cells were positively stained. Comparison of the molecular weight of the antigens recognized by this MoAb and by human anti-platelet MoAbs, as well as comparison of staining patterns of megakaryocytes indicated that MoAb PL.1 is probably directed to a GPIIb/IIIa complex analogue. Since MoAb PL.1 is of the non-complement-binding mouse IgG1 isotype, it can be used for studying clearance of platelet aggregates by Fc-receptors of the MPS. It also promises to be a useful tool in the study of platelet involvement in rats with experimental nephritis.

The specificity of nephritogenic antibodies. V. Glomerular localization of anti-GP 330 and anti-GP 90 antibodies present in passive Heymann serum.

Administration in the rat of rabbit antibodies directed against rat tubular brushborder antigens (FXIA) leads to membranous glomerulopathy (HICN) associated with glomerular immune complexes (ICXS). These anti-FXIA antibodies (Abs) contain two major specificities, anti-GP 330 and anti-GP 90. The contribution of each specificity to the localization of anti-FXIA Abs was studied by direct immunofluorescence. Perfusion studies, under conditions which avoid ischaemia, confirmed that in this system glomerular localization of anti-FXIA Abs depends only on anti-GP 90 Abs, and the failure of anti-GP 330 Abs to localize after perfusion could not be attributed to ischaemia. In contrast, intravenous (i.v.) injection of anti-GP 330 Abs results in granular deposits of rabbit IgG, similar to those observed using anti-FXIA Abs. Injection i.v. of anti-GP 90 Abs results only in (faint) linear deposits. Using alternating and combined perfusion studies with these Abs, it is shown that anti-GP 90 Abs do not influence localization of anti-GP 330 Abs. In addition, systemic administration of anti-GP 90 Abs combined with perfusion of anti-GP 330 Abs does not facilitate localization of anti-GP 330 Abs. Although directly after i.v. injection of anti-FXIA Abs some anti-GP 90 Abs probably localize in the glomerular capillary wall, our results exclude a dominant or ancillary role for anti-GP 90 Abs in the formation of glomerular Icxs in HICN.

Glomerulonephritis induced by monoclonal anti-Thy 1.1 antibodies. A sequential histological and ultrastructural study in the rat.

The present report describes the natural history of an experimental acute glomerulonephritis with massive proteinuria induced by a single intravenous injection of a (mouse) monoclonal anti-rat Thy 1.1 antibody into the rat. The disease is characterized by direct although transient binding of this monoclonal antibody to glomerular basement membrane and mesangium after injection as demonstrated by immunofluorescence microscopy. Immediate activation of complement occurs as shown by glomerular deposition of C3 and C9. Concomitant activation of the coagulation cascade is reflected by the presence of fibrinogen deposits in the affected glomeruli. One hour after injection mesangial alterations are prominent including condensation of mesangial cell chromatin, and lysis of mesangial cells as shown by light- and electron- microscopy, leading to the formation of aneurysms in the capillary tuft. Commencing on day 4 mesangial cell proliferation can be observed, accompanied by glomerular crescent formation at day 14, which decreases gradually 3 weeks after antibody administration, whereas mesangial hypercellularity can be observed up week 10 after intravenous injection of the antibody. The disease is clinically characterized by a massive transient proteinuria starting immediately after antibody injection, reaching mean values of 300 mg/24 hours at days 2 to 4, gradually decreasing to normal levels after 3 weeks. It is concluded that in this unique model of glomerulonephritis induced by a monoclonal antibody, recognition of Thy 1.1 epitopes as well as activation of complement including the C5-C9 membrane attack complex may play a major role in the pathogenesis of this experimental disease.

The specificity of nephritogenic antibodies. IV. Binding of monoclonal antithymocyte antibodies to rat kidney.

Polyclonal rabbit antirat thymocyte serum (ATS) has been shown to form in situ glomerular immune aggregates following perfusion into normal rat kidney ex vivo. This may be due to the presence of T-cell-like epitopes in the rat kidney, or it may be a result of contaminating anti-connective-tissue antibodies in ATS. To exclude the latter possibility we investigated binding to the rat kidney of three different (mouse) monoclonal antirat thymocyte antibodies (anti-T-cell MoAbs), directed to Thy 1.1 antigen, as well as (control) anti-B-cell MoAbs. The MoAbs were incubated in vitro with kidney sections or perfused into the blood-free rat kidney ex vivo. It was shown using immunofluorescence (IF) and immunoelectron microscopy (IEM) (peroxidase technique) that the anti-T-cell MoAbs used, in contrast to anti-B-cell MoAbs, are able to bind with glomerular capillary walls, and with mesangial structures after incubation in vitro or perfusion ex vivo. Although staining patterns are not completely identical, the reaction product is clearly demonstrated throughout the glomerular basement membrane (GBM) and along the plasma membranes of endothelial and epithelial cells, after contact with either of the three anti-T-cell MoAbs used. It is concluded that the presence of T-cell-like epitopes in the rat kidney may lead to immune complex formation following contact with anti-T-cell MoAbs. The nephritogenicity of rabbit ATS, as well as of some batches of clinically used ATS, may also be explained by this mechanism rather than by the usually assumed presence of contaminating antibodies in these polyclonal antisera.

The specificity of nephritogenic antibodies. III. Binding of anti-Fx1A antibodies in glomeruli is dependent on dual specificity.

Rabbit antibodies against rat tubular brushborder antigens (Fx1A) give rise to in situ formation of immune aggregates along the glomerular capillary walls after intravenous injection into rats. These antibodies (anti-Fx1A), able to produce heterologous immune complex glomerulopathy (HIC) in the rat, have previously been shown to bind with brushborders (anti-BB) as well as with rat thymocytes (anti-T). In the present communication, this dual specificity was also demonstrated in antibodies eluted from kidneys of rats with HIC. It further appeared that, when the anti-thymocyte binding activity was selectively removed from these antibodies, using immunoabsorption with rat tissue extracts, these anti-Fx1A antibodies were no longer able to stain glomerular basement membranes (GBM) as demonstrated at the ultrastructural level using the peroxidase technique. Following perfusion of these antibodies in the normal rat kidney ex vivo, binding along the capillary walls was also below the detection level, in contrast to non anti-T depleted anti-Fx1A IgG. Biochemical analysis (including immunoblotting) showed that the anti-T moiety of anti-Fx1A was directed to a 90 kD component of Fx1A, since selective absorption of this specificity prevented staining of this 90 kD component. It is concluded, that this anti-T specificity within rabbit anti-Fx1A plays a crucial role in local immune complex formation in the rat kidney ex vivo. Whether this holds also for its role in the pathogenesis of HIC in vivo awaits further confirmation.

Acute glomerulonephritis after intravenous injection of monoclonal anti-thymocyte antibodies in the rat.

Female Wistar rats were injected with (mouse) monoclonal antibodies (Moabs) of different IgG subclasses directed to rat thymocytes or rat tumor cells. Following intravenous injection of antithymocyte Moabs, glomerular binding of mouse IgG was observed during the first 4 days along the GBM and in the mesangium. No staining for mouse IgG was detected in anti-tumor Moab injected rats. Animals injected with IgG 2a anti-thymocyte Moab developed glomerulonephritis and a massive proteinuria in contrast to rats injected with IgG 1 Moab which is non-complement fixing. The glomerulonephritis lesion consisted of microaneurysms and focal and segmental proliferation. Deposits of complement and fibrin could be detected exclusively in rats injected with IgG 2a anti-thymocyte Moab during the whole observation period of 14 days. This is the first demonstration of overt glomerulonephritis lesions on the injection of monoclonal antibodies.

The specificity of nephritogenic antibodies. II. Immune complex glomerulopathy in rats induced by heterologous antithymocyte serum.

Injection of rabbit anti-rat thymocyte serum (ATS) i.v into rats induces a transient glomerulopathy characterized by immune aggregates localized in the mesangium and along the glomerular capillary wall, as detected by immunofluorescence (IF) techniques. Neither light microscopical alterations in the kidney, nor proteinuria could be detected in these animals and no autologous IgG could be observed in the glomeruli during the observation period (45 days). Results from ex vivo perfusion studies showed identical localization of immune aggregates--which, in electron microscopy, appear to be localized subepithelially. The ATS used, was monospecific in that no other specificities could be detected after thorough absorption with rat tissue extracts including tubular brushborder antigens, so it is concluded from these data that ATS is able to participate in the formation of immune complexes in situ by recognizing epitopes both in the mesangium and at the epithelial side of the glomerular basement membrane.

The specificity of nephritogenic antibodies: I. Evidence on anti-T-cell specificity in nephritogenic antibodies detected by cytotoxicity and MIF assays.

Heterologous antibodies directed to brushborder antigens of rat kidney tubules are nephritogenic in that an immediate immune complex glomerulopathy occurs after injection ot these antibodies into normal rats. Since previous observations suggested the presence of anti-T-cell specificity within these antibodies, we now compared the specificities of these antibodies with those of heterologous anti-rat thymocyte antibodies, using immunofluorescence (IF), cytotoxicity and migration inhibition (MIF) assays. The results suggest that anti-brushborder anti-serum contains anti-thymocyte specificities, while antithymocyte antiserum contains anti-brushborder specificity. These specificities could be removed selectively from both of the antisera by absorption with insoluble tissue extracts containing the appropriate antigens, i.e. thymocyte antigens or brushborder antigens respectively, indicating that two specificities are involved rather than only cross-reacting antibodies. Since it is unlikely for several other reasons that this feature is simply the result of an immunologic cross-reaction due to antibodies raised by immunization with purified kidney tissue antigens, this dual specificity of anti-brushborder antibody might play a role in the pathogenesis of experimental glomerulopathy.