The effect of SRI 63-675, a competitive platelet-activating factor receptor-antagonist, in the generalized Shwartzman reaction.

Evidence indicates a role for platelet-activating factor (PAF) in endotoxin (LPS)-induced shock. To determine its involvement in LPS-induced intravascular coagulation, we assessed the efficacy of SRI 63-675, a specific PAF receptor antagonist, to prevent fibrin deposition in the various organs of New Zealand White rabbits 4 h after two intravenous doses of LPS (100 micrograms/kg), spaced 24 h apart. SRI 63-675 significantly lowered peak tumor necrosis factor levels after LPS challenge. Administration of SRI 63-675 around either the first (150 mg/kg) or second LPS dose (120 mg/kg), however, did not prevent coagulation. The unexpected high mortality after combined administration of SRI 63-675 and LPS precluded assessment of PAF inhibition around both LPS doses on coagulation. Sole administration of SRI 63-675 induced rapid and transient changes in peripheral blood cell counts, and blood pressure and heart rate suggestive of intrinsic PAF-like activity. Although some other intrinsic toxic effect of SRI 63-675 cannot be ruled out, it is suggested that endotoxin may have primed the rabbit to the lethal, PAF-like, effects of SRI 63-675.

Von Willebrand factor in the rat kidney: its localization and the effects of its in vivo interaction with specific antibodies.

Antiendothelial cell antibodies and increased von Willebrand factor (vWF) levels are observed in many renal diseases. We studied renal morphology after administration of rabbit anti-human factor VIII-von Willebrand factor (FVIII-vWFc) gamma-globulin (IgG) in rats. Isolated perfusion of normal rat kidney with the antibodies disclosed rabbit IgG along glomerular endothelium and in mesangial areas. Systemic administration resulted in an identical antibody distribution. C3 deposits and few electron dense deposits were transiently noted. After 1 week the mesangial deposition of rabbit IgG had increased significantly, most likely reflecting increased release of vWF by the endothelium. Thereafter, the deposits gradually relocated into mesangial stalk and into the glomerular vascular pole, and they had vanished after 6 weeks. Except for a mild influx of leukocytes, no renal injury occurred. In conclusion, although FVIII-vWFc antibodies cause mesangial immune deposits, antibodies against other endothelial antigens are apparently needed to inflict renal damage.

Antibody-mediated redistribution and shedding of endothelial antigens in the rabbit.

We report the results of studies performed in vitro and in vivo that were designed to explore individual, sequential, and concurrent Ag-antibody interactions at the surface of rabbit endothelial cells. Divalent heterologous antibodies to rabbit lung angiotensin-converting enzyme and to rabbit lung thrombomodulin were employed. In cultured monolayers, both antibodies redistributed the specific Ag and co-redistributed the immunologically unrelated Ag inducing partial or complete disappearance of the Ag from the cell surface (antigenic modulation) in 15 to 60 min. When injected into living rabbits, each antibody induced a rapid (1 to 3 min) redistribution and subsequent modulation of the specific and of the unrelated Ag at the surface of alveolar endothelial cells. Immune complexes, and the unrelated Ag, were shed in the circulation, attaining peak levels 3 to 4 min after the injection; were rapidly bound by platelets, E, and polymorphonuclear leukocytes; and were subsequently found in phagocytic cells in the spleen and in the liver. Thrombomodulin co-shed by angiotensin-converting enzyme antibody and, to a lesser degree, angiotensin-converting enzyme co-shed by thrombomodulin antibody, crossed the glomerular capillary walls and were reabsorbed by the epithelial cells of the proximal tubules within 2 to 3 min. The results show that immunologically unrelated Ag can be passively entrapped during formation of immune complexes at the cell surface, and provide new information on the kinetics of clearance of immune complexes containing endogenous, structural Ag.

Role of the membrane attack complex of complement in lung injury mediated by antibodies to endothelium.

The potential pathogenic role of the membrane attack complex (MAC) of the complement system was investigated in two models of lung injury mediated by antibodies to angiotensin-converting enzyme (ACE), an endothelial cell enzyme. In the first model, acute and fatal lung edema was induced in rabbits by intravenous administration of divalent anti-ACE antibodies. These animals died acutely. C6-deficient rabbits tolerated anti-ACE antibodies without apparent ill effects. On the other hand, C6-deficient rabbits reconstituted with C6 and then receiving anti-ACE antibodies developed acute pulmonary edema and died. These results indicate that the MAC is required for the pathogenesis of this lung injury. In the second model, intravenous administration of monovalent anti-ACE Fab fragments over 4 consecutive days induced fatal interstitial pneumonitis in normal rabbits. For C6-deficient rabbits there was a reduced inflammatory response, and no animals died, implicating a mediator function for the MAC in this model as well. These results demonstrate that MAC is an important mediator of acute pulmonary edema induced by divalent antibodies to an endothelial antigen. Moreover, the complement system was also, to some extent, involved in the recruitment of inflammatory cells leading to the development of interstitial pneumonitis in the experimental lung injury induced by monovalent anti-ACE Fab fragments that 'per se' do not activate complement.

The occurrence of IgA-nephropathy in patients with diabetes mellitus may not be coincidental: a report of five cases.

We describe five patients with IgA-nephropathy complicating diabetes mellitus. In four cases, diabetic glomerulosclerosis was present at the same time. One patient suffered from dermatitis herpetiformis. The observation of the present five cases together with the notion of an increased prevalence in diabetes mellitus of celiac disease and dermatitis herpetiformis suggests that the occurrence of IgA-nephropathy in diabetic patients is not mere coincidence.

Morphological basis of pulmonary edema in mice with cytokine-induced vascular leak syndrome.

Patients injected systemically with recombinant human interleukin-2 (rhIL-2) for treatment of solid tumor develop a vascular leak syndrome (VLS), characterized mainly by pulmonary edema whose pathogenesis is unknown. We have examined the structure of pulmonary vessels in mice with severe VLS induced by systemic injections of rhIL-2 and recombinant human interferon-alpha-A/D (rhIFN-alpha), which has a synergistic effect with IL-2. The pulmonary edema was associated with lesions of venous and capillary endothelia, alveolar basement membrane, and type I epithelial cells. These changes were more severe and diffuse than those seen in mice systemically injected with rhIL-2 alone, and in beige mice (deficient in NK cells and certain enzymes of polymorphonuclear leukocytes) injected with rhIL-2 and rhIFN-alpha. The endothelial lesions were comparable to those seen when leukocytes activated by cytokines react with activated endothelial cells in vitro, or at the site of injection of cytokines in vivo. The observations are in agreement with the interpretation that the severe lesions occurring in mice systemically injected with rhIL-2 with rhIFN-alpha result from the interaction of leukocytes with the endothelium. The results confirm the validity of previous studies performed in vitro or in animals injected intradermally with cytokines and extend their significance.

Histochemical and immunohistochemical studies of diseased human glomeruli.

In order to study the localization of Lentil lectin (LCH)-binding glycoresidues in glomeruli from patients with a variety of glomerulopathies, and to elucidate the relationship between LCH-binding sugars and the components of the extracellular matrix, laminin and type IV collagen, investigations of formalin-fixed, paraffin-embedded kidney tissues digested with trypsin were carried out by the direct and indirect immunofluorescence microscopy techniques. The glomerular basement membrane (GBM) and the mesangium reacted well with LCH, whereas areas with sclerotic lesions exhibited a decreased reactivity. The pattern of LCH binding to the GBM in various glomerulopathies was similar to that of laminin but different from that of type IV collagen. The pattern of localization of LCH-reacting sites and of laminin in the GBM included the double linear lines in diabetic nephropathy, inner linear line with outer projections (spikes) in membranous nephropathy, and reduplicated basement membrane in membranoproliferative glomerulonephritis. The results obtained by enzyme-linked immunoadsorbent assay showed that LCH had a stronger reactivity for laminin than for type IV collagen or fibronectin. These findings suggest that LCH is more reactive with laminin than with other components of the glomerular extracellular matrix.

Characterization of a tubular basement membrane component reactive with autoantibodies associated with tubulointerstitial nephritis.

A kidney tubular basement membrane (TBM) component that is bound by antibodies from individuals with anti-TBM antibody-associated tubulointerstitial nephritis (TIN) was purified and characterized (TIN antigen). TIN antigen was prepared from rabbit TBM by extraction with guanidine and purified by ion-exchange, gel filtration, and reversed-phase chromatography. Based upon yields of protein and antibody reactivity, TIN antigen accounts for about 9% of the mass of TBM and thus is a major component of this basement membrane. A predominant 58-kDa form comprises about 90% of purified TIN antigen, and a 50-kDa form accounts for the remainder. The two forms share the amino-terminal sequence Ser-Ile-Phe-Gln-Gly-Gln-Tyr-X-Arg-Ser-Phe-Gly- and give similar tryptic peptide maps, indicating that they are structurally related. Their amino acid compositions overall are similar to laminin and entactin/nidogen. The absence of hydroxyproline and hydroxylysine and the low levels of glycine in TIN antigen indicate that it is noncollagenous. No similarities were found between other known proteins and sequences of tryptic peptides and the amino terminus of TIN antigen, suggesting that it is distinct from other characterized basement membrane components. A goat polyclonal antibody toward rabbit TIN antigen showed the same kidney distribution as human antibodies and was completely inhibited in enzyme-linked immunosorbent assay by purified TIN antigen. These data further support the idea that TIN antigen is the primary target for anti-TBM antibodies associated with TIN. This research presents methods to prepare TIN antigen for biochemical studies and investigations of its role in anti-TBM autoimmune TIN.

Lung injury mediated by antibodies to endothelium. III. Effect of chlorpromazine in rabbits.

In rabbits intravenous administration of antibodies to lung angiotensin converting enzyme (ACE) results in a rapid redistribution of ACE on the plasma membrane of pulmonary endothelium with fixation of complement and development of fatal pulmonary edema. In survivors given daily injections of antibodies, ACE disappears from the lung ("antigenic modulation") and the rabbits become resistant to further immune injury. To test the hypothesis that these events depend on a functionally intact mechanism of cell activation, rabbits received, in addition to anti-ACE antibodies, chlorpromazine, a drug that inhibits calmodulin and protein kinase C and decreases plasma membrane fluidity. Initially, chlorpromazine inhibited antigen redistribution, fixation of complement, and development of pulmonary edema. In rabbits maintained on chlorpromazine and receiving daily anti-ACE antibodies this effect became attenuated and the rabbits eventually developed ACE redistribution, complement fixation, and pulmonary edema. We conclude that chlorpromazine temporarily inhibits antigenic modulation in vivo, presumably through its action on calcium-mediated antibody-cell surface antigen interaction.

Pathogenesis of an experimental model of Goodpasture's hemorrhagic pneumonitis.

The mechanisms that allow circulating basement membrane antibodies (Ab) to interact with the alveolar basement membrane (ABM) inducing Goodpasture's hemorrhagic pneumonitis are unknown. In laboratory animals the ABM is inaccessible to phlogogenic amounts of ABM Ab unless the permeability of the unfenestrated alveolar endothelium is increased. This study was designed to test the hypothesis that in the mouse polypeptide mediators, generated by activated lymphoid cells or cells infected by viruses, contribute to the pathogenesis of passive Goodpasture's hemorrhagic pneumonitis. In naive mice that received rabbit ABM Ab, these bound to the glomerular basement membrane but not to the ABM and their lungs were normal. In the lungs of mice injected with human recombinant IL-2 and IFN-alpha specific binding of ABM IgG, C3, and fibrinogen to the ABM, diffuse and severe erythrocyte extravasation, and accumulation of mononuclear and polymorphonuclear leukocytes were constantly observed. ABM Ab and IL-2 or ABM Ab and IFN-alpha did not produce comparable effects. Mice injected only with IL-2 and IFN-alpha had enlarged, edematous lungs without pulmonary hemorrhages. The results show that the synergism of IL-2 and IFN-alpha convert the lung into a preferential target for AMB Ab, suggesting that cytokines may have a role in the pathogenesis of human Goodpasture's pneumonitis.

Lung injury in rabbits induced by intravenous administration of heterologous polyclonal antibodies to angiotensin converting enzyme (kininase II).

Antibody interactions with the endothelial cell membrane glycoprotein angiotensin converting enzyme (Kininase II) in vivo exhibit features of aggregation and capping with resultant shedding similar to those events described in several in vitro isolated cell systems. Requirements for divalent ligand binding, deposition of complement and participation of cytoskeletal elements are demonstrated in vivo. Persistence of antigen in immune complexes with complement interaction appear to be necessary to induce an inflammatory response. Abrogation of this response occurs when circumstances permit antigenic modulation with removal of the immune complex from the endothelial surface.

Local formation of immune deposits in rabbit renal proximal tubules.

Rabbits passively immunized with goat antibodies to rabbit angiotensin converting enzyme (ACE), an enzyme synthesized in the endoplasmic reticulum and mainly expressed on the apical membranes of the cells of proximal tubules, developed mild and transient immune deposits in this segment of the nephron. Granular deposits of goat IgG, rabbit ACE and C3 were found in the basolateral compartment and were maximal during the first week of immunization when the highest titers of anti-ACE antibodies were present. As the antibody titer fell to an undetectable level, the immune deposits were rapidly cleared and were virtually absent 21 days after the injections. Artificial increase of glomerular permeability allowed focal binding of ACE antibodies to the brush border of some tubules, but did not significantly alter the pattern of immune injury at the base of tubular cells. The data are consistent with the interpretation that the immune deposits result from in situ formation of immune complexes. This mechanism would involve passage of circulating antibodies across the tubular basement membrane and their combination with ACE associated with tubular cell surface membranes.

Studies on cell proliferation and tracer localization in the kidneys of guinea pigs with experimental autoimmune anti-tubular basement membrane nephritis.

The mitotic activity in kidneys of guinea pigs with experimental autoimmune anti-tubular basement membrane (TBM) nephritis was investigated using autoradiographic techniques to determine the uptake of [3H]thymidine by actively dividing cells. It was observed in these animals that cells of proximal tubules, distal tubules, cortical and medullary interstitium, medullary collecting ducts, and loops of Henle took up significantly greater amounts of [3H]thymidine when compared with normal animals. In addition, the behaviour of horseradish peroxidase (HRP) and goat anti-HRP IgG in extraglomerular sites in the kidneys of these animals was studied. Contrary to what was expected, these tracers appeared to be less concentrated in the tubules and interstitium of animals with anti-TBM disease, with tracer movement restricted in areas of disrupted TBM. The significance of these observations is discussed.

The glomerular distribution of type IV collagen and laminin in human membranous glomerulonephritis.

The glomerular distribution of type IV collagen and laminin, the major collagenous and noncollagenous components of the glomerular basement membrane, was studied by immunofluorescence microscopy in idiopathic and lupus membranous glomerulonephritis. Affinity-purified antibodies against type IV collagen reacted preferentially with the inner aspect and irregularly with the adjacent outer area of the thickened basement membrane. In contrast, laminin was detected along the inner aspect of the glomerular basement membrane, in subepithelial basement membrane protrusions ("spikes"), and in the newly formed basement membrane layer above the immune deposits. We conclude that type IV collagen and laminin do not codistribute in the newly formed matrix. This aberrant antigenic distribution may reflect a loss of coordinate biosynthesis or degradation of these matrix components by visceral epithelial cells.

Studies on the formation of glomerular immune deposits in brown Norway rats injected with mercuric chloride.

Brown Norway rats injected with mercuric chloride (HgCl2) develop autoantibodies which immunolocalize along the glomerular basement membrane at first in a linear pattern and then in a granular pattern. The aim of this study was to characterize the specificity of these antibodies and to investigate the mechanisms responsible for the formation of granular immune deposits in the subepithelial zone of the glomerular basement membrane. The rats were found to develop circulating anti-laminin, anti-type IV collagen, anti-heparan sulfate proteoglycan, and anti-entactin antibodies. Antibodies against laminin and type IV collagen were found in relatively high titers in the sera and were specifically concentrated in the nephritic kidneys. Antibodies eluted from the nephritic kidneys with either linear or granular deposits reacted with basement membrane antigens synthesized and secreted by cultured rat glomerular visceral epithelial cells. Thus, in this model, the interaction of anti-laminin and type IV collagen antibodies with antigens secreted by glomerular visceral epithelial cells might, together with other mechanisms, contribute to the formation of granular immune deposits in the subepithelial part of the glomerular basement membrane.