SUMMARYThe effector mechanisms of T cell-dependent acute glomerular injury were studied in autologous phase anti-GBM glomerulonephritis (GN) in rats. Acute proliferative GN was induced in sensitized rats by a subnephritogenic dose of sheep anti-rat GBM antibody. Injury was manifested by proteinuria and glomerular leucocyte infiltration composed predominantly of macrophages but also CD4 þ and CD8 þ T cells. T cell depletion, using an anti-CD5 MoAb, demonstrated that glomerular leucocyte infiltration and proteinuria were T cell-dependent. Inhibition of T helper cell function using an anti-CD4 MoAb prevented proteinuria and glomerular macrophage and CD4 þ T cell influx, but not accumulation of CD8 þ T cells. Depletion of CD8 þ T cells also prevented proteinuria and the influx of macrophages and CD8 þ T cells, but not accumulation of CD4 þ T cells. Macrophage depletion, using micro-encapsulated clodronate, prevented proteinuria and glomerular macrophage infiltration, but not the accumulation of CD4 þ or CD8 þ T cells, indicating that macrophages are the common cellular effectors for both CD4 and CD8 T cell-dependent injury. Evidence for cytotoxic mechanisms of injury (increased numbers of apoptotic cells or accumulation of natural killer (NK) cells in glomeruli) could not be demonstrated. These data suggest that acute glomerular injury in anti-GBM GN is the result of macrophage recruitment, which is dependent on both CD4 and CD8 T cells, and that direct T cell-mediated injury (cellular cytotoxicity) is not involved.

Macrophages release proinflammatory cytokines in response to infection that play a critical role in the pathophysiology of septic shock. We propose that targeting cytokine-neutralizing antibodies using albumin microspheres to macrophages will be more beneficial than the soluble form in reducing mortality related to peritonitis. In this study, we compared the distribution pattern of microspheres in infected and noninfected animals, evaluated the amount of microsphere taken up by peritoneal macrophages in vitro, and compared the efficacy of soluble and microsphere forms of cytokine-neutralizing antibodies in preventing lethality caused by Escherichia coli-induced peritonitis. The results indicate that twice the amount of microspheres accumulates near the site of infection (the peritoneal cavity), and 70% of the microspheres exposed to peritoneal macrophages were phagocytosed in 1 h. Treatment with the microsphere form of cytokine-neutralizing antibodies was more efficacious than using the soluble form in preventing lethality induced by E. coli. Immediate treatment was more efficacious than delayed treatment in the absence of gentamicin, whereas immediate and delayed treatment were equally efficacious in the presence of gentamicin. The combination of microspheres containing neutralizing antibodies to tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) protected 100% of the animals, whereas either one alone protected only 60%-90% of the animals from lethality caused by E. coli-induced peritonitis. In conclusion, the microsphere form of neutralizing antibodies to TNF-alpha IL-1beta may be an effective therapeutic agent in the treatment of septic shock caused by peritonitis.

A macrophage plays an important role in mediating the inflammatory response. Cytokines released by activated macrophages contribute to inflammation in glomerulonephritis (GN). Clodronate, a biphosphonate, causes macrophage depletion when administered in an encapsulated form in liposomes. We used albumin as the polymer matrix to microencapsulate clodronate to the microspheres (MS) in the 1-micron size range. The purpose of this study was to (a) determine macrophage depletion by clodronate MS, (b) determine the effect of clodronate MS on endotoxin-induced cytokine release in vitro, and (c) assess the effect of clodronate MS on macrophage infiltration in experimental antiglomerular basement membrane nephritis. Macrophage depletion by clodronate MS was assessed by staining for the EDI marker. The results indicate greater than 95% depletion of macrophages from the spleen, liver, kidney, and blood. In the whole blood model, clodronate MS attenuated endotoxin-induced tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) release, and the attenuation by the microencapsulated form of clodronate was also more effective than the free (solution) form of clodronate. Macrophage infiltration into the glomerulus in experimental GN was also blocked very effectively by pretreatment with clodronate MS. In conclusion, macrophage depletion by clodronate MS may be beneficial in reducing cytokine release and renal damage in GN.

Efficient delivery of therapeutic agents to a critical microenvironment may increase the efficacy of drugs used to modulate the allograft rejection response. This study demonstrates the ability of the combination of microspheres containing neutralizing anti-TNF alpha and anti-IL1-beta antibodies to significantly prolong murine cardiac allograft survival. These results suggest that the microsphere technique is an efficacious method to target antibody delivery to prolong allograft survival.

CNI-1493, a newly developed, water-soluble tetravalent guanylhydrazone, is a powerful inhibitor of tumor necrosis factor (TNF) and interleukin-1 (IL-1) synthesis. Microencapsulation of drugs into microcapsules that target macrophages has improved the effectiveness of both TNF and IL-1 neutralizing antibodies in experimental models of septic shock. It is the purpose of this study to determine if microencapsulation of CNI-1493 will improve cytokine inhibition. CNI-1493 was microencapsulated using albumin into 1 microm spheres. Comparable amounts of CNI-1493 in solution and in microencapsulated form were added to 1 ml aliquots of whole blood along with 100 ng of endotoxin. TNF and IL-1 were measured by ELISA. Microencapsulated CNI-1493 was also given to rats with endotoxic shock (15 mg/kg Escherichia coli endotoxin) and rats with peritonitis induced by peritoneally injecting 10(10) CFU E. coli. Equivalent amounts of encapsulated and solution CN I-493 were given intravenously. Endotoxin 15 mg/kg was also given to rats 6 and 24 h after a dose of encapsulated CNI-1493 to determine the duration of action of encapsulated drug. The microencapsulated CNI-1493 produced significantly greater inhibition of TNF and IL-1 at all doses in the whole blood model. There was significantly improved survival and cytokine inhibition in the endotoxic shock model as well as the peritonitis model in rats treated with microencapsulated CNI-1493. There was also 83% survival in rats given endotoxin 24 h after a dose of encapsulated CNI-1493. From these data, we conclude that CNI-1493 is a potent inhibitor of cytokine production and is greatly potentiated by microencapsulation, which transports the drug to macrophages.