James B. Lefkowith

Glucose-induced phospholipid hydrolysis in isolated pancreatic islets: quantitative effects on the phospholipid content of arachidonate and other fatty acids

Our recent findings indicate that glucose-induced insulin secretion from isolated pancreatic islets is temporally associated with accumulation of substantial amounts of free arachidonic acid and that arachidonate may serve as a second messenger for intracellular calcium mobilization in islets. In an effort to determine the source of this released arachidonate, the endogenous fatty acid composition of phospholipids from islets has been determined by thin-layer chromatographic separation of the phospholipids, methanolysis to the fatty acid methyl esters, and quantitative gas chromatographic analyses. The relative abundance of phospholipids in islets as judged by their fatty acid content was phosphatidylcholine (PC), 0.63; phosphatidylethanolamine (PE), 0.23; phosphatidylinositol (PI), 0.067; phosphatidylserine (PS), 0.049. Arachidonate constituted 17% of the total islet fatty acid content, and PC contained 43% of total islet arachidonate. Islets incubated with [3H]arachidonate in the presence of 28 mM D-glucose incorporated radiolabel into PC with a considerably higher specific activity than that of PE, PS or PI. The total fatty acid content of PC from islets incubated with 28 mM glucose for 30 min was significantly lower than that of islets incubated with 3 mM glucose, and smaller effects were observed with PE, PS and PI. The molar decrement in PC arachidonate was 3.2 pmol/islet under these conditions, which is sufficient to account for the previously observed accumulation of free arachidonate (2 pmol/islet). A sensitive method involving negative ion-chemical ionization-mass spectrometric analyses of the pentafluorobenzyl esters of fatty acids derived from trace amounts of lysophosphatidylcholine (lyso-PC) was developed, and glucose-stimulation was found to reduce islet lyso-PC content by about 10-fold. These findings indicate that the insulin secretagogue D-glucose induces phospholipid hydrolysis in islets and suggest that PC may be the major source of free arachidonate which accumulates in glucose-stimulated islets.

Fibrinogen mediates platelet-polymorphonuclear leukocyte cooperation during immune-complex glomerulonephritis in rats.

The metabolic and functional alterations which occur during the acute phase of nephrotoxic nephritis (NTN) in rats, a model of immune-mediated glomerulonephritis, result from a cooperative interaction between PMNs and platelets (PLTs). In consequence, we hypothesized that fibrinogen (Fg) might play a critical role in this process and, accordingly, we found that defibrination of animals decreased both the acute phase proteinuria in NTN (approximately 70%) as well as the influx of PLTs and PMNs into the glomerulus (approximately 40-50%). In contrast, blockade of the PLT Fg receptor, alpha IIb beta 3, with the RGD peptidomimetic SC-49992 decreased proteinuria (approximately 90%) without substantially altering the influx of PMNs or PLTs. Immunocytochemistry showed a marked increase in beta 3 integrin expression in inflamed glomeruli which was prevented either by PMN or PLT depletion before disease induction. FACS and immunocytochemical analysis of glomerular cell dissociates demonstrated that beta 3 integrin expression was predominantly on intraglomerular PLTs. In vitro, activated PLTs stimulated the PMN respiratory burst, an interaction which could be inhibited by Fg receptor blockade. In sum, acute NTN is accompanied by a marked increase in glomerular beta 3 integrin expression predominantly due to the influx of PLTs which localize to the glomerulus in a PMN-dependent fashion. Fg appears to serve a major role as a coactivating stimulus for PLT-PMNs in situ via alpha IIb beta 3, potentially mediating the PMN respiratory burst which contributes to proteinuria. Fg may also play a subsidiary role in PMN/PLT comigration.

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.

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.

Manipulation of rat brain fatty acid composition alters volatile anesthetic potency.

The molecular mechanism of volatile anesthetic action remains unknown. Attempts to elucidate this mechanism have been complicated by the absence of models in which changes in neuronal cellular properties can be correlated with changes in whole animal anesthetic effect. In this study we describe a model where diet-induced alterations in rat brain fatty acid composition are correlated with alterations in volatile anesthetic potency. Rats maintained on a fat-free diet showed significant depletion of arachidonic acid (20:4 omega 6; 5,8,11,14-eicosatetraenoic acid) and docosahexaenoic acid (22:6 omega 3; 4,7,10,13,16,19,-docosahexaenoic acid) in brain, and a corresponding increase in Mead acid (20: 3 omega 9; 5,8,11-eicosatrienoic acid). These fat-deprived rats were significantly more sensitive to all volatile anesthetics tested than were age-controlled rats on a normal diet. Parenteral supplementation of the fat-deprived animals with linolenic acid (18: 3 omega 3, 9,12,15-octadecatrienoic acid) completely reconstituted the docosahexaenoic acid content of brain without affecting anesthetic sensitivity. In contrast, supplementation of the fat-deprived rats with linoleic acid (18: omega 6; 9,12-octadecadienoic acid) caused a dramatic decrease in anesthetic sensitivity, but only a small change in whole brain arachidonate content. Further analysis revealed that linoleate supplementation of fat-deprived animals resulted in a preferential normalization of the arachidonate content of brain phosphatidylinositol as compared with other brain phosphoglycerides. These results demonstrate for the first time a correlation between changes in membrane composition and anesthetic effect, and indicate that the precise fatty acid composition (perhaps in specific phospholipids) of brain is important in the mechanism of volatile anesthetic action.

Macrophage arachidonate release via both the cytosolic Ca2+-dependent and -independent phospholipases is necessary for cell spreading

We have observed that phospholipase A2 (PLA2) activation and arachidonate (AA) release are essential for monocyte/macrophage adherence and spreading. In this study, we addressed the relationship between AA release and cell adherence/spreading in murine resident peritoneal macrophages, and the roles of specific PLA2S in these processes. The PLA2-specific inhibitors, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL, specific for the Ca(2+)-independent PLA2 (iPLA2)) and methyl arachidonoyl fluorophosphonate (MAFP, specific for the Ca(2+)-dependent phospholipase (cPLA2)) inhibited AA release and cell spreading in a correlated fashion but only modestly decreased cell adherence. Cell spreading was normalized by the addition of AA to PLA2-inhibited cells. AA release during spreading was also inhibited by Ca2+ depletion or protein kinase C (PKC) inhibition, and was accompanied by increased (but transient) phosphorylation of cPLA2-Inhibition of macrophage spreading, however, only partially inhibited AA release. Moreover, constitutive AA release was seen in fully spread macrophages which was inhibited by BEL, but not MAFP or Ca2+ depletion. BEL also reversed the phenotype of fully spread cells. These data suggest that macrophage spreading requires the release of AA by the iPLA2 (which appears to be constitutively active) and cPLA2 (which appears to be stimulated by adherence/spreading). Maintenance of macrophage spreading, in contrast, appears to be principally dependent on the iPLA2.

Serologic markers of lupus nephritis in patients: use of a tissue‐based ELISA and evidence for immunopathogenic heterogeneity

In order to assess the ability of various serologic assays to correlate with lupus nephritis, we analysed sera obtained from 60 patients with systemic lupus erythematosus (SLE), Patients were categorized as having active nephritis (group 1), active lupus without nephritis (group 2), inactive lupus with prior nephritis (group 3), or inactive lupus without prior nephritis (group 4), Three parameters were assessed including anti‐dsDNA antibodies (Farr assay), immune complexes (C1q binding), and anti‐C1q antibodies (salt‐stable C1q binding). Additionally, glomerular binding activity (GBA) was measured using a new solid‐phase immunoassay that detects immune elements by their ability to bind glomerular tissue. We found that patients with nephritis (group 1) exhibited higher mean values for each assay than patients in each of the other three groups (P= 0·001,0·009, 0·14, and 0·23 in the GBA, C1q, anti‐dsDNA, and anti‐C1q assays, respectively). The only assay which distinguished patients with nephritis (group 1) from patients having active disease without nephritis (group 2) was the GBA (mean 0·48 ± 0·09 versus 0·15 ± 0·04, (P 0·05), In terms of utility, all tests were specific for diagnosing nephritis among patients with lupus; however, only the GBA was reasonably sensitive. The information provided by the anti‐dsDNA and C1q assays were not correlated with one another, nor additive to the GBA, Patients with false negative GBA tended to have received more intensive immunosuppression. The qualitative characteristics of GBA varied among patients with nephritis. These data suggest the pathogenesis of lupus nephritis is complex, and may be mediated by an array of immune elements. Moreover, the data indicate the potential utility for a broad tissue‐based approach to detection of pathogenic immune elements over other, specific immunologic markers.

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.

Detection of glomerular-binding immune elements in murine lupus using a tissue-based ELISA

The glomerulonephritis in systemic lupus erythemalosus (SLE) is presumably triggered by ihe binding of circulating immune elements (autoantibodies and immune complexes) to the glomerulus; however, the nature of these elements is unclear. In order to detect and characterize such elements, we developed an ELISA using whole intact glomeruli as the substrate. With this assay, glomerular binding activity (GBA) was detected in the serum of MRLlpr, NZB × W, and B × SB mice, but not in non‐autoimmune BALB/c mice. Less activity was present in the serum of C3H lpr, C57B1/6J Ipr and AKR lpr animals which develop signs of autoimmunity but only modest renal disease. The GBA in MRLlpr mice contained IgG (subclasses 1, 2a and 2b), but not IgG3. IgM. igA, orC3. GBA was not significantly decreased by preadsorption of MRL lpr serum by DNA‐agarose (although anti‐DNA antibodies were). Binding activity in serum, however, was diminished by DNAase treatment. Fractionation of MRL lpr serum over a molecular sizing column showed that GBA eluled in a broad peak. GBA bound to the glomerulus ex vivo in a tissue‐specific fashion and was enriched in renal eluates relative to serum in vivo. In sum, the binding activity detected by this assay appeared to be a heterogeneous entity (possibly in part immune complexes containing DNA) which bound specifically to the glomerulus and which appeared to parallel the presence of renal disease. This novel assay system may help elucidate the pathogenesis of SLE nephritis and have utility as a disease marker.

Accelerated essential fatty acid deficiency by Δ9 desaturase induction: dissociation between the effects on liver and other tissues

Essential fatty acid (EFA) deficiency is an important tool in probing the role of arachidonic acid (20:4(n-6] in pathophysiologic processes, but requires stringent and prolonged deprivation of (n-6) fatty acids. The present study investigated whether induction of the delta 9 desaturase, which is responsible for the synthesis of oleate, the precursor of 20:3(n-9) which uniquely accumulates in the deficiency state, might serve to accelerate the biochemical and biological effects of EFA deficiency. By alternately fasting and feeding animals a fat-free diet, it was possible to induce markedly the delta 9 desaturase selectively in liver. This dietary manipulation in consequence led to dramatic and rapid changes in hepatic phospholipid fatty acid composition. Within 2 weeks, 20:3(n-9) to 20:4(n-6) ratios in liver phospholipids were several fold greater than those seen in animals fed a fat-free diet alone. These changes, however, contrasted with those seen in the serum and other tissues. The mol% of 20:3(n-9) in serum was not increased by delta 9 desaturase induction and the 20:3(n-9) to 20:4(n-6) ratio was only modestly increased. The effects of delta 9 desaturase induction were even more attenuated in tissues other than the liver. Desaturase induction led to a doubling in the 20:3(n-9) to 20:4(n-6) ratio in phosphatidylcholine in renal cortex and heart, although the ratio in the other phospholipids was unaffected. The 20:3(n-9) to 20:4(n-6) ratio in peritoneal macrophage phospholipids was unaffected by desaturase induction. Thus, delta 9 desaturase induction greatly augments the synthesis of (n-9) fatty acids within the liver and leads to the rapid and substantial accumulation of the abnormal fatty acid, 20:3(n-9). This markedly augmented synthesis of hepatic 20:3(n-9), however, is not reflected in increased plasma levels of 20:3(n-9), and thus the effects of delta 9 desaturase induction are attenuated in tissues other than the liver. These data underscore the notable ability of the liver to maintain polyunsaturated fatty acid homeostasis.