Curtis B. Wilson

The Raji cell radioimmune assay for detecting immune complexes in human sera.

A sensitivie and simple procedure for the detection and quantitation of soluble complement (C)- fixing immune complexes in sera of patients with various disease states has been developed by utilizing C receptors on Raji cells. These cells lack membrane-bound immunoglobulin but have receptors for IgG Fc, C3b, C3d, and possibly with other C proteins. Uptake experiments showed that both aggregated human gamma globulin (AHG) and 7S IgG bound to receptors for IgG Fc; however, AHG reacted with C bound to cells only via receptors for C and this binding was much more efficient than via IgG Fc receptors. AHG was used as an in vitro model of human immune complexes and its uptake by Raji cells was quantitated by 125I-radiolabeled antihuman IgG. The limit of sensitivity of this test was 6 mug AHG/ml serum. The ability of Raji cells to detect AHG in serum depended on the amount of radioactive antibody used and the size of aggregates. The presence of an excess of C somewhat inhibited binding of AHG containing C to Raji cells. The efficient binding of AHG by receptors for C on Raji cells was used for the detection and quantitation of immune complexes in human sera. Raji cells were incubated with sera to be tested and then reacted with excess radiolabeled antihuman IgG; the amount of radioactivity bound to the washed cells was determined and referred to a standard curve of radioactive antibody uptake by cells previously incubated with increasing amounts of AHG in serum. Thereby immune complexes were detected and quantitated in serum hepatitis, systemic lupus erythematosus, vasculitis, subacute sclerosing panencephalitis, dengue hemorrhagic fever, and malignancies.

Involvement of reactive oxygen intermediates in cyclooxygenase-2 expression induced by interleukin-1, tumor necrosis factor-alpha, and lipopolysaccharide.

Reactive oxygen intermediates (ROIs) play an important role in inflammatory processes as mediators of injury and potentially in signal transduction leading to gene expression. Cyclooxygenase (COX) is a rate-limiting enzyme in prostanoid biosynthesis, and its recently cloned inducible form, COX-2, is induced by proinflammatory cytokines. This study linked ROIs to the signaling pathways that induce COX-2 expression. The hydroxyl radical scavengers DMSO (1%), as well as di- and tetramethylthiourea, inhibited IL-1-, TNF alpha-, and LPS-induced COX-2 expression in rat mesangial cells. The suppression of COX-2 mRNA expression correlated with the COX-2 protein level. In comparison with the prolonged induction of the inducible gene encoding protein-tyrosine phosphatase by hydrogen peroxide, the COX-2 gene was only transiently induced. Protein-tyrosine phosphatase is also induced by heat shock and chemical stress, whereas COX-2 is not. Superoxide was a more potent inducer for COX-2 than hydrogen peroxide. In addition, NADPH stimulated COX-2 expression, and an inhibitor of NADPH oxidase blocked COX-2 expression induced by TNF alpha. COX-2 and KC gene expression costimulated by IL-1 were inhibited differentially by the scavengers. These studies demonstrate that oxidant stress is a specific and important inducer of COX-2 gene expression. This induction may contribute to the deleterious amplification of prostanoids in inflammation and compound the direct effects of ROI production.

Inhibition of constitutive nitric oxide synthase (NOS) by nitric oxide generated by inducible NOS after lipopolysaccharide administration provokes renal dysfunction in rats.

Excess NO generation plays a major role in the hypotension and systemic vasodilatation characteristic of sepsis. Yet the kidney response to sepsis is characterized by vasoconstriction resulting in renal dysfunction. We have examined the roles of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) on the renal effects of lipopolysaccharide administration by comparing the effects of specific iNOS inhibition, -N6-(1-iminoethyl)lysine (L-NIL), and 2,4-diamino6-hydroxy-pyrimidine vs. nonspecific NOS inhibitors (nitro- -arginine-methylester). cGMP responses to carbamylcholine (CCh) (stimulated, basal) and sodium nitroprusside in isolated glomeruli were used as indices of eNOS and guanylate cyclase (GC) activity, respectively. LPS significantly decreased blood pressure and GFR (112+/-4 vs. 83+/-4 mmHg; 2.66+/-0.29 vs. 0. 96+/-0.22 ml/min, P < 0.05) and inhibited the cGMP response to CCh. GC activity was reciprocally increased. L-NIL and 2, 4-diamino-6-hydroxy-pyrimidine administration prevented the decrease in GFR (2.71+/-0.28 and 3.16+/-0.18 ml/min, respectively), restored the normal response to CCh, and GC activity was normalized. In vitro application of L-NIL also restored CCh responses in LPS glomeruli. Neuronal NOS inhibitors verified that CCh responses reflected eNOS activity. L-NAME, a nonspecific inhibitor, worsened GFR (0.41+/-0.15 ml/min), a reduction that was functional and not related to glomerular thrombosis, and eliminated the CCh response. No differences were observed in eNOS mRNA expression among the experimental groups. Selective iNOS inhibition prevents reductions in GFR, whereas nonselective inhibition of NOS further decreases GFR. These findings suggest that the decrease in GFR after LPS is due to local inhibition of eNOS by iNOS, possibly via NO autoinhibition.

Abrogation of Macrophage-dependent Injury in Experimental Glomerulonephritis in the Rabbit: USE OF AN ANTIMACROPHAGE SERUM

Macrophages were shown by the use of glomerular cell culture and morphologic techniques to be present in large numbers within the glomeruli of rabbits with acute serum sickness (AcSS) and in a passive model of the autologous phase of antiglomerular basement membrane (GBM) antibody-induced glomerulonephritis (PAGBMN). To determine the part played by these cells in the glomerular injury, animals were treated with a sheep anti-rabbit macrophage serum (AMS) or normal sheep serum (NSS). NSS administration had no effect on the development of either model of glomerulonephritis. The use of AMS reduced the number of circulating monocytes and prevented the accumulation of macrophages within glomeruli in both models (AcSS/NSS, mean 126/glomerulus, range 40-251; AcSS/AMS, mean 8, range 1-44; PAGBMN/NSS, mean 52, range 27-69; PAGBMN/AMS, mean 5, range 2-7). The AMS-treated rabbits had only minor histologic lesion and profound reduction in proteinuria (AcSS/NSS, mean 516 mg/24 h, range 200-991; AcSS/AMS, mean 41, range 3-161; PAGBMN/NSS, mean 335, range 55-975; PAGBMN/AMS, mean 10, range 2-24). Similar studies in the heterologous phase of glomerular injury induced by the same anti-GBM antibody revealed no effect of the AMS on this polymorphonuclear leukocyte-related phase of injury, demonstrating the selectivity of the antisera. Complement depletion, with cobra venom factor, did not affect the development of glomerulonephritis nor the accumulation of macrophages in either model. Inhibition of macrophage accumulation can largely prevent these forms of experimental glomerulonephritis, thereby implicating macrophages as mediators of glomerular injury and consequent proteinuria.

Therapy of Anti-glomerular Basement Membrane Antibody Disease: Analysis of Prognostic Significance of Clinical, Pathologic and Treatment Factors

We have compared the effect of therapy with immunosuppression alone to immunosuppression plus plasma exchange on the clinical course and rate of disappearance of antibody in 17 patients with anti-glomerular basement membrane (anti-GBM) antibody-induced renal disease. Patients receiving immunosuppression (n = 9) and those receiving plasma exchange (n = 8) were similar in terms of entry clinical characteristics, pulmonary manifestations and complications associated with therapy. Rate of disappearance of anti-GBM antibody as estimated from serial estimates of antibody binding was significantly more rapid in patients receiving plasma exchange, and mean serum creatinine in these patients at end of therapy was half that of the patients receiving immunosuppression alone. Analysis of clinical and pathologic values at study entry, however, indicated that the percent of crescents on initial renal biopsy and entry serum creatinine correlated better with outcome than did therapeutic modality. Thus, though plasma exchange may offer some advantage over immunosuppression alone in the treatment of this disease, degree of pathologic involvement appears to be the major factor affecting outcome. Patients with low cresents (less than 30%) and well preserved function did well with either treatment, while patients with severe crescentic involvement and impaired glomerular filtration rate did poorly.

Modulation of neutrophil influx in glomerulonephritis in the rat with anti-macrophage inflammatory protein-2 (MIP-2) antibody.

The role of the chemokine, macrophage inflammatory protein-2 (MIP-2), during anti-glomerular basement membrane (GBM) antibody (Ab) glomerulonephritis (GN) was studied. Rat MIP-2 cDNA had been cloned previously. Recombinant rat MIP-2 (rMIP-2) from Escherichia coli exhibited neutrophil chemotactic activity and produced neutrophil influx when injected into the rat bladder wall. By using a riboprobe derived from the cDNA and an anti-rMIP-2 polyclonal Ab, MIP-2 was found to be induced in glomeruli with anti-GBM Ab GN as mRNA by 30 min and protein by 4 h, with both disappearing by 24 h. The expression of MIP-2 correlated with glomerular neutrophil influx. A single dose of the anti-MIP-2 Ab 30 min before anti-GBM Ab was effective in reducing neutrophil influx (40% at 4 h, P < 0.01) and periodic acid-Schiff deposits containing fibrin (54% at 24 h, P < 0.01). The anti-rMIP-2 Ab had no effect on anti-GBM Ab binding (paired-label isotope study). Functional improvement in the glomerular damage was evidenced by a reduction of abnormal proteinuria (P < 0.05). These results suggest that MIP-2 is a major neutrophil chemoattractant contributing to influx of neutrophils in Ab-induced glomerular inflammation in the rat.

Pathophysiology of Experimental Glomerulonephritis in Rats

Micropuncture, clearance, immunofluorescence and light microscopy techniques were used to study kidney structure and single nephron function in rats with autologous immune complex nephritis (AICN), a membranous glomerulonephritis developing over 5 to 20 mo, in the more acute and proliferative glomerular basement membrane (GBM) nephritis and in controls. Both models are known to have clinical counterparts in human disease. Kidney functional abnormalities correlated with the degree of architectural derangement. In both AICN and anti-GBM nephritis filtration fraction fell in direct proportion to the fall in glomerular filtration rate (GFR), renal plasma flow being unchanged. Fractional electrolyte excretion increased as the GFR fell. Despite marked heterogeneity of single nephron filtration rate (SNGFR) (AICN, 5-93 nl/min; anti-GBM, 0-50 nl/min) and of proximal tubular hydrostatic pressure (4-48 mm Hg), each nephron showed almost complete glomerulotubular balance, absolute reabsorption to the late proximal convolution varying directly with filtration rate. In addition SNGFR could be related both to proximal intratubular hydrostatic pressure and to calculated glomerular capillary pressure (Pg), being lowest in those nephrons with the highest intratubular pressure. Nephrons with very high filtration rates did not apparently reach filtration equilibrium. Mean SNGFR was significantly lower in the anti-GBM group, while calculated Pg was the same in both. This probably reflects the acute and diffuse involvement of the anti-GBM lesion with different filtration characteristics from the more chronic AICN disease. Tubular damage was more marked in AICN, and extraction of p-aminohippurate was reduced in this group.

Ischemic acute renal failure and antioxidant therapy in the rat. The relation between glomerular and tubular dysfunction.

The effects of antioxidant therapy with probucol were evaluated in rats subjected to 1 h renal ischemia and to 24 h reperfusion. Probucol exerted significant antioxidant effects in renal cortical tubules in vitro when exposed to a catalase-resistant oxidant. At 24 h probucol treatment (IP) improved single nephron glomerular filtration rate (SNGFR) (28.1 +/- 3.3 nl/min) in comparison to untreated ischemic (I) rats (15.2 +/- 3.0), primarily as a result of improving SNGFR in a population of low SNGFR, low flow and/or obstructed nephrons. However, absolute proximal reabsorption remained abnormally low in IP rats at 24 h (5.9 +/- 0.8 nl/min), and cell necrosis was greater than in I rats. Kidney GFR remained low in IP rats due to extensive tubular backleak of inulin measured by microinjection studies. Evaluations after 2 h of reperfusion revealed a higher SNGFR in IP (36 +/- 3.1 nl/min) than I rats (20.8 +/- 2.7 nl/min). Absolute proximal reabsorption was essentially normal (11.6 +/- 1.3 nl/min) in IP rats, which was higher than IP rats at 24 h and the concurrent I rats. Administration of the lipophilic antioxidant, probucol, increased SNGFR and proximal tubular reabsorption within 2 h after ischemic renal failure. Although SNGFR remained higher than I rats at 24 h, absolute reabsorption fell below normal levels and tubular necrosis was more extensive in IP rats. Early improvement in nephron filtration with antioxidants may increase load dependent metabolic demand upon tubules and increase the extent of damage and transport dysfunction.

Extraglomerular immunoglobulin deposits in human nephritis.

In order to evaluate the incidence of extraglomerular immunoglobulin deposits and to correlate their presence with histopathologic abnormalities, we performed both prospective and retrospective immunofluorescence studies of renal biopsy specimens. Of 200 diagnostic biopsy specimens examined prospectively, 21 had extraglomerular deposits, 19 in association with presumed immunologically-mediated glomerulonephritis. Nine had linear immunoglobulin deposits on tubular basement membrane (antitubular basement membrane antibodies), in one case coexisting with granular deposits, and 13 had granular deposits on tubular basement membrane, in peritubular capillaries and/or arteries, or in tubular cytoplasm (probably immune complexes). Linear deposits on tubular basement membrane were usually associated with antiglomerular basement membrane nephritis or methicillin-associated interstitial nephritis; granular extraflomerular deposits were seen primarily in systemic lupus erythematosus, cryoglobulinemia or membranoproliferative glomerulonephritis.. The incidence of immunoglobulin deposits was high in the three groups of patients examined retrospectively being present in 37 or 47 patients with antiglomerular basement membrane mephritis, 22 or 32 patients with systemic lupus erythematosus of 24 of 130 renal allograft recipients. The contribution of antitubular basement membrane antibodies to renal damage was difficult to assess, although studies in experimental animals attest to their pathogenicity. The occurrence of granular extraglomerular deposits appeared to correlate roughly with the degree of tubulointerstitial injury in the patients with systemic lupus erythematosus.

Clotting Changes, Including Disseminated Intravascular Coagulation, during Rapid Renal-Homograft Rejection

Abstract One of two patients in whom early homograft rejection developed after renal transplantation had many antidonor antibodies before operation. By the measurement of gradients across intracorp...