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.

The cytomegalovirus-antigenemia assay in the diagnosis of posttransplant cytomegalovirus infection.

Cytomegalovirus (CMV) infection continues to be a major cause of morbidity and mortality in transplant recipients, yet prompt diagnosis remains a problem. A new assay has been developed that detects CMV antigens in peripheral blood leukocytes (CMV-AG). A retrospective analysis of the experience with this assay was performed, and its usefulness in the diagnosis of CMV infection in renal transplant recipients with unexplained fever was compared with that of conventional modalities (buffy coat culture, detection of circulating anti-CMV immunoglobulin M). The results suggest that the CMV-AG assay is a more rapid and sensitive test than existing modalities in the early diagnosis of CMV infection. When expressed quantitatively, it can discriminate between CMV infection and CMV disease, and it is useful in monitoring the course of infection and the response to therapy.

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.

The effect of pregnancy on the response to the TxA2/PGH2 analogue U-46619 in rabbits.

We compared the hemodynamic actions of U-46619, a stable thromboxane A2 (TxA2) prostaglandin H2 (PGH2) analogue, in nonpregnant (NP) rabbits with those observed in late pregnant (P) rabbits. An intravenous injection of U-46619 (10 micrograms) to each of eight NP chronically instrumented rabbits (mean body weight 3.4 kg) induced an immediate (1 min) and reversible fall of cardiac output (CO, 66%) and mean arterial pressure (MAP, 41%, both P < 0.01). P rabbits (n = 6, mean body weight 3.8 kg), however, responded with an elevation of MAP (5%, P < 0.02) upon intravenous injection of the drug (10 micrograms), while CO remained unchanged. The fall of CO in NP rabbits was associated with the temporary disappearance of a fraction of circulating platelets between the superior vena cava and the aortic arch. The number of platelets at 30 and 60 s after U-46619 was reduced (P < 0.05) by 14 and 20% respectively in the aortic blood, whereas caval platelet counts were unchanged until 90 s (-6%, P < 0.05). In contrast, intraaortic administration of this drug (10 micrograms) to NP rabbits resulted in neither thrombocytopenia nor hypotension. U-46619 (10-30 micrograms i.v.) caused no decrease in platelet count in the aorta of P rabbits. In vitro, U-46619-induced aggregation of platelets harvested from P rabbits was also blunted (P < 0.001). This could not be attributed to reduced affinity or number of platelet thromboxane receptors. The data indicate that U-46619 induces a fall of arterial pressure simultaneous with intravascular platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Glomerular lesions induced in the rabbit by physicochemically altered homologous IgG.

Immunization of rabbits with physicochemically altered homologous or even autologous IgG induces formation of antibodies combining with IgG of rabbit and of foreign species. Cardiac but not renal lesions were reported in such animals. This study examined the nephritogenic potential of the immune response to cationized or heat-aggregated homologous IgG of b9 or b4 allotype in rabbits of the b4 allotype. Rabbits injected with either b9 or b4 cationized IgG produced antibodies reactive with rabbit and human IgG and with histones; they also developed abnormal glomerular deposits of IgG b4 and C3 corresponding to alterations of the glomerular basement membranes (GBM). Rabbits injected with either b9 or b4 aggregated IgG developed antibodies reactive with rabbit and human IgG and abnormal glomerular deposits of IgG b4 and C3 in the GBM and in the mesangium with subendothelial and mesangial electron-dense deposits. Some rabbits in both groups had proliferative and exudative glomerulonephritis and proteinuria. The results showed that immunization of rabbits with physicochemically altered homologous IgG induces an immune response to rabbit and human IgG and to histones as well as glomerular deposits of autologous IgG and C3 and other glomerular lesions.

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.

Glomerular macrophage proliferation in experimental immune complex nephritis.

In immune complex nephritis, glomerular hypercellularity is known to result from the proliferation of intrinsic cells and from the infiltration of mononuclear cells, primarily macrophages. An immunohistochemical double-labeling procedure was used to determine whether macrophages were among the cells which may undergo mitosis within the glomerular tuft. The monoclonal antibody ED1 served as a macrophage marker; cells in the S-phase of mitosis were recognized by uptake of bromodeoxyuridine. Glomerular proliferation was studied in chronic serum sickness of LEW rats, an animal model of immune complex nephritis for which the relationship between immunopathology and pathophysiology has been well described. In normal glomeruli, resident mesangial macrophages accounted for an unexpectedly large proportion (greater than or equal to one-third) of the total mitotic activity. In immune complex glomerulonephritis, the rate of glomerular macrophage proliferation increased rapidly just at the onset of proteinuria and remained high throughout the remaining course of disease. Glomerular macrophages from rats with proliferative nephritis also divided more vigorously than normal in short term culture in vitro, while persistently expressing abnormal surface marker phenotypes. The proliferation of mesangial macrophages appears to be a prominent feature of the normal process of glomerular cell renewal. In hypercellular glomeruli, vigorous local proliferation could greatly amplify the potential of macrophages to cause damage.

Receptor antagonists of platelet-activating factor do not influence the development of passive Heymann nephritis.

Heymann nephritis in the rat is a proteinuric condition caused by glomerular lesions similar to those found in human membranous nephritis. In the present study the effect of two different receptor antagonists of platelet-activating factor (PAF) on the course of passive Heymann nephritis was assessed. It was found that rats treated with either antagonist showed the same degree of proteinuria and the same glomerular immunopathological features as untreated rats. Furthermore, at sacrifice, 7 days after the initiation of the disease, the concentration of circulating PAF in treated as well as untreated rats was normal. These results indicate that PAF is not crucial in the pathogenesis of Heymann nephritis.

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.

Effect of leukocyte stimulation on rabbit immune complex glomerulonephritis.

Phytohemagglutinin (PHA), a leukocyte mitogen, induces a lymphocyte and blast cell glomerulonephritis in rat renal allografts (Cell Immunol 13:146, 1974). The aim of this study was to assess whether PHA similarly enhances rabbit monocyte-dependent experimental, acute immune complex glomerulonephritis, and whether this effect is associated with local release of interleukin-1 (IL-1) and tumor necrosis factor (TNF). Rabbits with experimental acute serum sickness (AcSS: Group I) had focal proliferative and exudative glomerulonephritis with immune deposits, scattered subepithelial electron-dense deposits (humps), mild and transient proteinuria, normal creatinine clearance and slightly increased production of IL-1 and TNF from isolated glomeruli. Rabbits with AcSS and injected with PHA (Group II) developed severe lymphocyte and blast cell glomerulonephritis with diffuse endothelial damage; immune deposits were significantly reduced, focal subepithelial electron-dense deposits were absent, proteinuria was increased, creatinine clearance was decreased and production of IL-1 and TNF was markedly augmented as compared to rabbits in Group I. Rabbits with AcSS and injected with IL-1 beta and TNF alpha (Group V) had lesions comparable to those seen in Group II. These results show that PHA, IL-1 and TNF enhance the severity of acute immune complex glomerulonephritis, presumably by activating glomerular endothelial and mesangial cells and resident or infiltrated leukocytes.

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.

Expression and modulation of surface antigens in cultured rat glomerular visceral epithelial cells.

This study, using immunocytochemical light and electron microscopy techniques, characterizes the distribution of three antibodies bound to the surface of rat glomerular visceral epithelial cells (GEC) in culture, and tests their ability to redistribute corresponding antigens under conditions appropriate for antigenic modulation (antigen disappearance). At 4 degrees C or after fixation, anti-renal tubular brush border vesicle (BBV) IgG bound diffusely to the surface of GEC and to coated pits. Anti-gp330 IgG had a discrete distribution on the surface of GEC and reacted with coated pits. Anti-podocalyxin IgG was bound diffusely to the surface of GEC but not to coated pits. At 37 degrees C, anti-BBV IgG induced marked redistribution of immune complexes with both shedding and internalization. Anti-gp330 IgG induced weaker redistribution, with internalization of immune complexes predominating. Anti-podocalyxin IgG induced rapid redistribution of immune complexes and antigenic modulation but minimal internalization. Experiments of differential redistribution indicated that anti-BBV IgG modulated the expression of both gp330 and podocalyxin; anti-gp330 IgG had a weaker effect on BBV antigens and podocalyxin; and anti-podocalyxin failed to redistribute BBV antigens or gp330. The relevance of these immunocytochemical studies of antibody-cell surface antigen interaction in cultured GEC to understanding the pathogenesis of Heymann glomerulonephritis (HG) is discussed.