Calcium-sensitive modulation of Ig capping: evidence supporting a cytoplasmic control of ligand-receptor complexes.

Capping of anti-Ig-Ig complexes was studied in murine B lymphocytes. Morphological studies indicated that caps formed rapidly on cells before any changes in shape. The first changes in cell shape were evident as a contraction right under the cap area. The removal of extracellular calcium had no effect on cap formation. Furthermore, the introduction of calcium by the ionophore A-23187 stopped capping. The ionophere by itself in the absence of extracellular calcium had no effect. Caps were found to be disrupted, the complexes scattering over the entire cell surface if the cells were treated by A-23187 after the caps had formed. The disruptive effect of A-23187 as dependent on extracellular calcium and could be stopped by drugs that affected energy metabolism. The cytochalasins also disrupted the formed caps. Drugs that affect energy metabolism by themselves did not disrupt the caps. We interpret the effects of the ionophore as resulting from a systemic hypercontractility of microfilaments. A theory for explaining the formation and disruption of capping is discussed.

Spontaneous redistribution of surface immunoglobulin in the motile B lymphocyte.

This paper reports that B cells undergoing translatory motion spontaneously segregate their surface Ig to one portion of their plasma membrane. The spontaneous redistribution of surface Ig was found to be: (a) selective, concanavalin A-dependent on translatory motion and energy metabolism. Abundant B cells undergoing motility were found after cultures in lipopolysaccharide or trypsin, or after brief exposure to cholinergic drugs.

A mononuclear cell component in experimental immunological glomerulonephritis.

An accelerated form of nephrotoxic serum nephritis in the rat was examined. The experimental model consisted of preimmunization of the rat with rabbit IgG 5 days before injection of subnephrotoxic doses of rabbit anti-rat kidney serum. The immunized rats developed proteinuria during the first 24 h, increasing by 48-96 h. The early 24-h proteinuria correlated with a neutrophilic infiltration of glomeruli and with deposition of rat Ig and C. The 48- to 96-h proteinuria was associated with a glomerular infiltration by mononuclear cells and proliferation of intrinsic glomerular cells. Many of the mononuclear cells were morphologically identical to monocytes and macrophages. [3H]thymidine labeling experiments indicated that the mononuclear cells originated from dividing precursors localized outside the kidney. Preimmunized rats given systemic irradiation (the kidney being protected by a shield) showed loss of the mononuclear cell infiltrate and absence of 48- to 96-h proteinuria. We conclude that mononuclear phagocytes can infiltrate the kidney in immunological glomerular disease and might contribute to the functional abnormalities.

Redistribution of myosin accompanying capping of surface Ig.

The capping of surface Ig on B cells occurs with a striking redistribution of cytoplasmic myosin. Our results suggest a close association between surface Ig and myosin which could be the basis for Ig redistribution and stimulated motility.

Prevention of diabetes in the BB rat by essential fatty acid deficiency. Relationship between physiological and biochemical changes.

Essential fatty acid (EFA) deficiency exerts a striking protective effect in several animal models of autoimmune disease. We now report that EFA deprivation prevents diabetes in the BB rat, an animal model of human insulin-dependent diabetes mellitus. In diabetes-prone (DP)-BB rats, the incidences of spontaneous diabetes and insulitis (the pathological substrate of autoimmune diabetes) were greatly reduced by EFA deficiency. This beneficial effect of the deficiency state was also seen in diabetes-resistant (DR)-BB rats that, after treatment with antibody to eliminate RT6+ T cells, would otherwise have become diabetic. The susceptibility of EFA-deprived DP-BB rats to spontaneous diabetes was restored when they were given dietary supplements of linoleate at 70 d of age (during the usual period of susceptibility), but not when they were repleted beginning at 120 d (after the peak incidence of diabetes). EFA deficiency did lead to growth retardation, but calorically restricted control rats demonstrated that the protective effect of the deficiency state was not a function of decreased weight. To examine the relationship between the biochemical changes of EFA deficiency and its physiological effects in this system, we compared the fatty acid changes that occurred in EFA-deficient animals that did and did not develop diabetes. Nondiabetic animals had significantly lower levels of (n-6) fatty acids (i.e., linoleate and arachidonate) and higher levels of oleate, an (n-9) fatty acid, than did diabetic animals. Levels of 20:3(n-9), the fatty acid that uniquely characterizes EFA deficiency, were similar in both groups, however. Among diabetic EFA-deficient rats, the age at onset of diabetes was found to correlate inversely with the level of (n-6) fatty acids, the least depleted animals becoming diabetic earliest, whereas there was no correlation with levels of 20:3(n-9). Among animals repleted with linoleate beginning at 70 d, restoration of susceptibility to diabetes correlated with normalization of the level of arachidonate. In summary, EFA deprivation reduced the frequency of diabetes in both DP and RT6-depleted DR-BB rats. This protective effect was strongly associated with depletion of (n-6) fatty acids, particularly arachidonate, but not with accumulation of the abnormal 20:3(n-9). Conjecturally, arachidonate and/or a metabolite may play a key role in mediating inflammatory injury in this animal model of autoimmune diabetes.

Localization of an Ia-bearing glomerular cell in the mesangium.

Using trypsin to render intact, isolated rat glomeruli permeable to antibody, and using an electron microscopic immunoperoxidase technique, we have localized a phagocytic immunologically-relevant cell bearing Ia determinants to the renal mesangium. Thus there are at least two functionally distinct cell types in the renal mesangium: one is a contractile smooth musclelike cell, and the other a phagocytic cell that bears immunologically-relevant surface determinants.

Essential fatty acid depletion of renal allografts and prevention of rejection.

A central hypothesis in transplantation biology is that resident leukocytes expressing class II histocompatibility antigens may determine the immunogenicity of an organ. By means of a novel method to deplete the kidney of resident leukocytes, essential fatty acid deficiency (EFAD), this hypothesis was tested in an intact, vascular organ. Kidneys subjected to EFAD and thus depleted of resident Ia-positive macrophages survived and functioned when transplanted across a major histocompatibility antigen barrier in the absence of immunosuppression of the recipient. Control allografts were rejected promptly. Allografts from donors subjected to EFAD normalized their lipid composition and were repopulated with host macrophages by 5 days. Administration of Ia-positive cells at the time of transplantation established that the resident leukocyte depletion induced by EFAD was responsible for the protective effect. These observations may provide insights into the mechanisms underlying tissue immunogenicity and the population of normal tissues with resident leukocytes.

Essential fatty acid deficiency depletes rat glomeruli of resident macrophages and inhibits angiotensin II-induced eicosanoid synthesis.

Essential fatty acid (EFA) deficiency exerts a beneficial effect on immune-mediated glomerulonephritis, preventing both the tissue injury and consequent mortality. Because both macrophages and eicosanoids are thought to play pathogenic roles in glomerulonephritis, and because macrophages play an important role in modulating arachidonate metabolism at sites of renal injury, the effects of EFA deficiency on the population of resident glomerular macrophages and on glomerular eicosanoid generation were examined. EFA deficiency led to a striking reduction in the number of resident glomerular macrophages and a corresponding reduction in the number of resident glomerular Ia+ cells. This phenomenon was not strain-specific, was not due to a decrease in circulating monocytes, was not a function of changes in cell surface labeling characteristics, and was not restricted to a specific subset of glomeruli. In addition, EFA deficiency affected other areas of the renal cortex: a comparable depletion of interstitial macrophages and Ia+ cells was also observed. In conjunction with the decrease in glomerular macrophages seen with the deficiency state, a marked decrease in both basal and angiotensin II-stimulated glomerular eicosanoid production was noted. In contrast to angiotensin II, platelet-activating factor-induced eicosanoid production was not significantly affected by the deficiency state. These changes in glomerular eicosanoid production could not be attributed to changes in glomerular cyclooxygenase or reacylation capacity. Dietary (n-6) fatty acid supplementation, but not (n-3) fatty acid supplementation, reversed both the decrease in glomerular macrophages and the diminished eicosanoid metabolism seen with the deficiency state. Understanding the mechanisms behind the changes in the glomerular microenvironment induced by EFA deficiency may provide a basis for elucidating the protective effect of dietary fatty acid manipulation on immune-mediated glomerulonephritis.

Murine glomerular leukotriene B4 synthesis. Manipulation by (n-6) fatty acid deprivation and cellular origin.

Leukotriene (LT) B4 is an important pro-inflammatory autocoid. In order to investigate the potential role of this eicosanoid in renal inflammation, in this study we determined the capability of glomeruli to synthesize this mediator. Glomeruli were able to synthesize LTB4 when provided with exogenous substrate in a dose-dependent fashion in the presence of ionophore A23187. Ionophore, although by itself a weak agonist for LTB4 formation, was required for LTB4 production from exogenous arachidonate. The identity of LTB4 was confirmed by specific radioimmunoassay, high pressure liquid chromatography, and gas chromatography/mass spectrometry. The synthesis of LTB4 was inhibited by BW755C (a lipoxygenase/cyclooxygenase inhibitor) but not indomethacin. Essential fatty acid (EFA) deficiency, obtained by the deprivation of (n-6) fatty acids, is known to exert a protective effect in renal inflammatory states. This dietary manipulation markedly attenuated the ability of glomeruli to synthesize LTB4. In contrast, the synthesis of cyclooxygenase products from exogenous arachidonate was increased by EFA deficiency. Because EFA deficiency has been shown to deplete glomeruli of resident mesangial macrophages, it was hypothesized that this effect accounted for the diminished LTB4 synthesis. To test this hypothesis, glomeruli were depleted of macrophages using x-irradiation. Glomeruli from these animals exhibited a marked decrease in LTB4 synthesis. Glomerular synthesis of cyclooxygenase products was unaffected by irradiation. In sum, glomeruli have the capability to synthesize LTB4, and this capacity is correlated with the presence of glomerular macrophages. EFA deficiency attenuates the ability of glomeruli to synthesize LTB4 by depleting them of macrophages.

Characterization of resident glomerular cells in the rat expressing Ia determinants and manifesting genetically restricted interactions with lymphocytes.

The existence of a subpopulation of rat glomerular cells bearing Ia determinants has been demonstrated with the aid of techniques for the enzymatic isolation and culture of glomerular cells. The Ia-positive cell is normally resident in the uninflamed glomerulus. It resembles a mononuclear phagocyte and consists of a functionally heterogeneous cell population with the capacity of Fc receptor display and phagocytosis, both in vivo and in vitro. A new technique for labeling these cells in situ in intact glomeruli has indicated that Ia-positive cells make up approximately 2% of the total glomerular cell population. The isolated glomerular cells can take up antigen and stimulate immune lymphocytes in an I-region-restricted interaction. They are strongly stimulatory in an allogeneic primary mixed lymphocyte culture. Characterization of this cell type suggests potential new insights into the pathogenesis of renal allograft rejection and immunologically mediated glomerulonephritis.

Local extrahepatic expression of complement genes C3, factor B, C2, and C4 is increased in murine lupus nephritis.

Systemic lupus erythematosus (SLE) is associated with the presence of complement proteins and immune complexes in affected organs. Since complement proteins are synthesized in hepatic and extrahepatic sites, we studied a murine model of SLE to ascertain the relative importance of local and humoral (liver) synthesis of complement. C3, C4, and C2 mRNA increase in kidney coincident with the development of nephritis in the MRL lpr/lpr mouse, a strain that spontaneously develops SLE. Two factor B messenger RNA transcripts are expressed in kidney and intestine; SLE nephritis is associated with decrease in the long factor B mRNA and increase in the short form. Increased local synthesis of C3 and B protein and a concomitant glomerular and renal interstitial macrophage infiltrate paralleled the increase in mRNA content in the (lpr/lpr) mice. In addition to kidney, an increase in C3, C4, C2 and factor B mRNA was noted in the lung, heart and intestine and to a lesser extent in liver of (lpr/lpr) in comparison to the MRL (+/+) animals. These results suggest that in SLE local expression of complement genes plays a role in the pathogenesis of chronic glomerulonephritis and in the autoimmune arteritis of other organs.

Essential fatty acid deficiency ameliorates acute renal dysfunction in the rat after the administration of the aminonucleoside of puromycin.

The administration of the aminonucleoside of puromycin (PAN) to rats causes the nephrotic syndrome that is associated with an acute decline in renal function, and an interstitial infiltrate. We examined whether essential fatty acid deficiency (EFAD), which inhibits macrophage infiltration in glomerulonephritis, affects PAN-induced renal dysfunction. Both control and EFAD rats developed proteinuria that resolved over 28 d. After PAN administration, there was a prominent infiltration of macrophages in rats fed a normal diet. The infiltrate was prevented by the EFAD diet. The absence of a macrophage interstitial infiltrate was associated with a significantly higher Cin in the EFAD rats than in controls at 7 d (5.21 +/- 1.19 versus 0.39 +/- 0.08, P less than 0.002 ml/min/kg BW). In addition, CPAH fell to less than 10 ml/min/kg BW by day 7 in controls, but remained the same as normal in the EFAD. After administration of PAN to control rats, there was no increase in urinary thromboxane excretion or an increase in glomerular thromboxane production. Furthermore, the effect of EFAD could not be mimicked by the administration of a thromboxane synthase inhibitor. Irradiation-induced leukopenia in rats on a normal diet markedly improved glomerular filtration and renal blood flow in acutely nephrotic rats. EFAD prevents the interstitial cellular infiltrate and the renal ischemia associated with experimental nephrosis. The recruitment of mononuclear cells into the kidney following PAN directly contributes to the decline in renal function.

Preservation of the glomerular capillary ultrafiltration coefficient during rat nephrotoxic serum nephritis by a specific leukotriene D4 receptor antagonist.

Leukotriene D4, a potent biologically active lipoxygenase derivative of arachidonic acid in activated leukocytes, depresses the glomerular capillary ultrafiltration coefficient (Kf) and contracts mesangial cells in culture. We therefore investigated its potential role in mediating the reduction in nephron filtration rate seen after induction of experimental nephrotoxic serum (NTS)-induced glomerulonephritis in the rat. Micropuncture measurements were performed in euvolemic Munich-Wistar rats 2 h after i.v. administration of 0.8 ml of rabbit serum (group 1, n = 6), 0.8 ml of rabbit anti-rat glomerular basement membrane antibody in the absence (group 2, n = 8), or presence (group 3, n = 7) of the new highly specific LTD4 receptor antagonist SK&F 104353. Quantitation of antibody binding and neutrophil infiltration revealed no differences between groups 2 and 3. Antagonism of endogenous LTD4 actions, however, was associated with prevention of the NTS-induced fall in SNGFR because of the abrogation of the fall in Kf which characterizes this form of experimental glomerulonephritis. Antagonism of endogenous LTD4 had no effect on the NTS-induced increases in pre- and postglomerular arteriolar resistances, and did not affect nephron plasma flow rate or net transcapillary hydraulic pressure difference. The observed highly localized protective action of the LTD4 antagonist on the glomerular capillary points to a possibly major functional role for intraglomerularly released LTD4, likely originating from infiltrating leukocytes, in the pathophysiology of this form of glomerulonephritis.

Predominant functional roles for thromboxane A2 and prostaglandin E2 during late nephrotoxic serum glomerulonephritis in the rat.

While much is known regarding acute nephrotoxic serum (NTS)-induced glomerular injury, the glomerular dynamics and pathophysiologic mediators of the more relevant chronic autologous phase remain poorly defined. Studies were performed in rats 14 d after injection of rabbit serum (n = 6), NTS in the absence (n = 6), or presence, of a cyclooxygenase inhibitor, ibuprofen (n = 6) or a thromboxane A2 (TxA2) receptor antagonist, L-670,596 (n = 5). A mesangial macrophage/monocyte infiltrate was noted with equal intensity in all NTS-treated rats. Glomerular generation rates of prostaglandin (PG) E2, PGF2a, and TxA2 in nephritic kidneys were dramatically increased as compared to controls. 2 wk after NTS, there was an increase in glomerular plasma flow rate (SNPF), attainment of filtration pressure disequilibrium, and augmentation of net transcapillary hydraulic pressure difference (delta P). Glomerular filtration rate (GFR), however, was reduced, due to a marked fall in the glomerular capillary ultrafiltration coefficient (Kf). Cyclooxygenase inhibition resulted in normalization of glomerular eicosanoid generation rates, amelioration of proteinuria, afferent vasoconstriction, and normalization of SNPF, delta P, Kf, and GFR. Selective antagonism of TxA2 also led to preservation of Kf, but was without effect on SNPF, thereby leading to elevated values for GFR. Thus, in contrast to the pathophysiologic role of arachidonate-lipoxygenase products in the early heterologous phase, PG-mediated vasodilatation and TxA2-induced reductions in Kf and GFR underlie glomerular functional changes during autologous mesangioproliferative glomerulonephritis.

Inhibition of p38alpha MAPK enhances proteasome inhibitor-induced apoptosis of myeloma cells by modulating Hsp27, Bcl-X(L), Mcl-1 and p53 levels in vitro and inhibits tumor growth in vivo.

Inhibition of p38 kinase blocks the production of tumor-promoting factors in the multiple myeloma (MM) bone marrow microenvironment. Proteasome inhibitors MG132 and bortezomib have been shown to have direct cytotoxic effects on MM cells. We show that a selective inhibitor of p38alpha, SCIO-469, enhances the ability of MG132 and bortezomib to induce the apoptosis of MM cells. Previously, we showed that p38 inhibition with SCIO-469 enhances MM cytotoxicity of bortezomib by inhibiting the transient expression and phosphorylation of Hsp27, a downstream target of p38. Here we show that continued treatment of MM cells with bortezomib leads to a SCIO-469-enhanced downregulation of Hsp27 and to increased MM apoptosis. Furthermore, we show that p38 inhibition enhances the bortezomib-induced MM apoptosis by upregulation of p53 and downregulation of Bcl-X(L) and Mcl-1. In a mouse xenograft plasmacytoma model of MM, we found that inhibiting p38 augments the effects of bortezomib in decreasing MM tumor growth in vivo. Thus, in addition to its role in suppressing an activated MM microenvironment, co-treatment with a p38 inhibitor, such as SCIO-469, may enhance the cytotoxicity of bortezomib by modulating pro-apoptotic and anti-apoptotic factors in MM cells, suggesting great potential for co-therapy.

Altered patterns of gene expression in response to myocardial infarction.

The use of cDNA microarrays has made it possible to simultaneously analyze gene expression for thousands of genes. Microarray technology was used to evaluate the expression of >4000 genes in a rat model of myocardial infarction. More than 200 genes were identified that showed differential expression in response to myocardial infarction. Gene expression changes were monitored from 2 to 16 weeks after infarction in 2 regions of the heart, the left ventricle free wall and interventricular septum. A novel clustering program was used to identify patterns of expression within this large set of data. Unique patterns were revealed within the transcriptional responses that illuminate changes in biological processes associated with myocardial infarction.

Macrophage-specific chemotactic lipid release by in vivo streptozocin-administered mouse islets.

The development of insulitis in animal models of immune-mediated diabetes mellitus is preceded by an influx of macrophages into the islets. In this study, ICR mice were given four 30-mg/kg doses of streptozocin over 36 h to induce insulitis. The islets were subsequently isolated and cultured in suspension. A macrophage-specific islet infiltration was observed within 48 h of the first drug injection. Concurrent with this leukocyte influx, enhanced in vitro release of a macrophage-specific chemotactic lipid by islets from streptozotocin-administered animals was observed. By chromatographic analysis, the islet-derived chemotactic factor appears to resemble a moderately polar complex lipid that is distinct from previously characterized lipid chemoattractants. Secretion of the factor is not dependent on cyclooxygenase activity. Identification of this lipid may provide important insights into the etiology of insulin-dependent diabetes mellitus and other autoimmune diseases.

Inhibition of p38 mitogen-activated protein kinase protects human islets from cryoinjury and improves the yield, viability, and quality of frozen-thawed islets.

The development of an optimal islet cryopreservation method will permit transplantation of islets from multiple donors in a single procedure and contribute to alleviation of the islet shortage. In this study, we have improved human islet cryopreservation methods under serum-free conditions using an intracellular-based islet cryopreservation solution (ICS), especially supplemented with a p38 pathway inhibitor (p38IH) to suppress p38 mitogen-activated protein kinase (MAPK) activation. Three different solutions were compared for freezing and thawing of human islets (1) conventional RPMI1640 medium, (2) ICS, and (3) ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Islet cryopreservation with ICS-p38IH significantly improved islet recovery, viability, and quality after thawing of cryopreserved islets. This improvement may allow the use of cryopreserved islets in clinical islet transplantation.

Hypersensitivity to salicylazosulfapyridine: renal and hepatic toxic reactions.

Salicylazosulfapyridine has been used for a number of years as therapy for ulcerative colitis. Reported toxicities are usually minor. This case report represents an acute allergic reaction to the drug. Characterized by fever, rash, eosinophilia, nephritis, and hepatitis. Resolution occurred with discontinuation of salicylazosulfapyridine. Although similar reactions have been reported with the antimicrobial sulfonamides, none has been fully described with salicylazosulfapyridine, a combination of a sulfonamide and salicylate.

Clinical analgesic nephropathy.

Analgesic nephropathy is recognized worldwide, but the differences in incidence in various countries, or regions, remain unexplained. Analgesic compounds may cause both functional and structural renal damage. This damage may be related to depletion of glutathione and renal vasoconstriction (probably mediated through prostaglandin depletion) and to the fact that the concentrations of glutathione and prostaglandins and their metabolites in the kidneys are manyfold their concentrations in plasma. Most patients with analgesic nephropathy are middle-aged women with histories of peptic ulcer, anemia, psychiatric disorders, headaches, and arthralgias. Investigations often show pyuria, some bacteriuria, and impaired concentrating ability, as well as other abnormalities of tubular function; caliceal abnormalities on intravenous pyelography are also frequent. It is important to discover these patients; evidence exists that with cessation of drug ingestion, renal function may stabilize and, in some cases, may improve.