C. Schalkwijk

Cytokine-stimulated secretion of group II phospholipase A2 by rat mesangial cells. Its contribution to arachidonic acid release and prostaglandin synthesis by cultured rat glomerular cells.

Potent pro-inflammatory cytokines, such as interleukin 1 (IL-1) or tumor necrosis factor (TNF) alpha have been found to increase group II phospholipase A2 (PLA2) synthesis and secretion by mesangial cells. In all cases 85-90% of the enzyme is secreted from the cells and a parallel increase in prostaglandin (PG)E2 synthesis is observed. We report here that co-incubation with a monoclonal antibody that specifically binds and neutralizes rat group II PLA2 attenuates IL-1 beta and TNF alpha-stimulated PGE2 production by 45% and 52%, respectively. CGP43182, a specific inhibitor of group II PLA2, potently blocks mesangial cell group II PLA2 in vitro with a half-maximal inhibitory concentration (IC50) of 1.5 microM, while only slightly affecting mesangial cell high molecular weight PLA2. CGP 43182 markedly attenuates IL-1 beta- and TNF alpha-stimulated PGE2 synthesis in intact mesangial cells with IC50s of 1.3 and 1.0 microM, respectively. PLA2 secreted from cytokine-stimulated mesangial cells was purified to homogeneity. Addition of the purified enzyme to unstimulated mesangial cells causes a marked release of arachidonic acid and a subsequent increased synthesis of PGE2. Moreover, addition of purified PLA2 to a cloned rat glomerular epithelial cell line and cultured bovine glomerular endothelial cells augmented both arachidonic acid release and PGE2 synthesis, with the endothelial cells being especially sensitive. Thus, cytokine-triggered synthesis and secretion of group II PLA2 by mesangial cells contributes, at least in part, to the observed synthesis of PGE2 that occurs in parallel to the enzyme secretion. Furthermore, extracellular PLA2 secreted by mesangial cells is able to stimulate arachidonic acid release and PGE2 synthesis by the adjacent endothelial and epithelial cells. These data suggest that expression and secretion of group II PLA2 triggered by pro-inflammatory cytokines may crucially participate in the pathogenesis of inflammatory processes within the glomerulus.

A role for secretory phospholipase A2 and C-reactive protein in the removal of injured cells

The acute phase response is initiated in response to infection or physical trauma and is characterized by an increase in the levels of some plasma proteins. Here, Erik Hack and colleagues suggest that the combined actions of two of these acute phase proteins, secretory phospholipase A2 and C-reactive protein, may serve to promote phagocytosis of injured cells and tissue debris, thereby enhancing inflammation and tissue damage.

Studies on the acyl-chain selectivity of cellular phospholipases A2

The selective release of arachidonate, as opposed to monoenoic and dienoic fatty acids, after stimulation of cells has suggested the involvement of arachidonate-selective phospholipases A2. Supportive evidence for the existence of such enzymes has also come from in vitro experiments. We have studied the acyl-chain selectivity of phospholipase A2 preparations obtained from human polymorphonuclear leukocytes, human platelets and rat platelets using sn-2-[14C]oleoylphosphatidylcholine and sn-2-[3H]arachidonoylphosphatidylcholine either as single substrates or in doubly labeled mixtures. In either case, no evidence for acyl-chain selectivity was observed for human PMN and rat platelet phospholipase A2. Additional experiments with human PMN homogenates and derived extracts yielded no indication for the selective loss of an arachidonate-selective phospholipase A2. Results with human platelet cytosol were highly suggestive for the presence of an arachidonoyl-selective phospholipase A2 when separate phosphatidylcholine species were assayed. This apparent selectivity was progressively lost when the substrates were mixed or embedded in a membrane of 1-palmitoyl-2-linoleoylphosphatidylcholine. The implications for occurrence of arachidonate-selective phospholipase A2 are discussed.

X-linked adrenoleukodystrophy: defective peroxisomal oxidation of very long chain fatty acids but not of very long chain fatty acyl-CoA esters

We investigated the peroxisomal fatty acid beta-oxidation system in liver and cultured skin fibroblasts from patients with X-linked adrenoleukodystrophy known to accumulate very long chain fatty acids. In order to examine whether the deficient peroxisomal oxidation of very long chain fatty acids in these patients results from a deficiency in one of the peroxisomal beta-oxidation enzyme proteins (acyl-CoA oxidase, bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase) we carried out immunoblotting experiments using antibodies directed against the peroxisomal beta-oxidation enzyme proteins from rat liver. Furthermore, we studied the oxidation of palmitoyl-CoA and lignoceroyl-CoA in homogenates of fibroblasts from the patients. The results indicate that the peroxisomal beta-oxidation enzyme proteins are not only present immunologically but also functionally active which suggests that the defect in X-linked adrenoleukodystrophy is, indeed, as recently suggested by Hashmi and coworkers (FEBS Lett 1986;196:247-250) at the level of a deficient peroxisomal activation of very long chain fatty acids.

Therapy with interleukin-2 induces the systemic release of phospholipase-A2

Therapy with interleukin-2 (IL-2) induces remissions in some forms of cancer. This treatment however, is accompanied by side-effects which, in part, may be mediated by the formation of eicosanoids and plateletactivating factor. We investigated the systemic release of phospholipase A2 (PLA2), a rate-limiting enzyme in the formation of these lipid mediators, in patients receiving IL-2. In a pilot study of 4 patients we observed an increase in PLA2 activity in serial plasma samples obtained during the first day after a bolus infusion of IL-2, which increase closely correlated with that of antigen levels of secretory phospholipase A2 (sPLA2) as measured by enzyme-linked immunosorbent assay (r=0.92;P<0.001). In 20 patients, receiving 12×106−18×106 IU IL-2/m2, we then investigated the course of antigenic levels of sPLA2 in relation to those of the cytokines tumour necrosis factor α (TNF) and interleukin-6 (IL-6) (both cytokines may induce sPLA2 in vivo). From 4 h on, sPLA2 levels significantly increased, reaching a peak 24 h after the IL-2 infusion. Subsequent IL-2 infusions even induced a further increase of sPLA2. This increase of sPLA2 was presumably not due to a direct effect of IL-2 on, for example, hepatocytes, since this cytokine, in contrast to IL-1, IL-6, TNF and interferon γ, was not able to induce the synthesis of sPLA2 by Hep G2 cells in vitro. Consistent with this, plasma levels of TNF and IL-6 in the patients rose, reaching peak levels before a zenith of sPLA2 occurred, i.e at 2 h and 4 h after the start of the IL-2 infusion respectively. sPLA2 levels significantly correlated with the development of the side-effects increase in body weight (r=0.49;P<0.0001) and decrease in mean arterial blood pressure (r=0.40;P<0.0001). Moreover, maximum sPLA2 levels induced by IL-2 were higher in patients who had progressive disease after therapy than in patients who had stable disease or a partial response.

X-Linked Adrenoleukodystrophy: Identification of the Primary Defect at the Level of a Deficient Peroxisomal Very Long Chain Fatty Acyl-CoA Synthetase Using a Newly Developed Method for the Isolation of Peroxisomes from Skin Fibroblasts

Several types of adrenoleukodystrophy (ALD) differing in age of onset, mode of inheritance and clinical presentation have been described in the literature. The X-linked form of adrenoleukodystrophy, which is the most common, is characterized by demyelination, adrenal sufficiency and the accumulation of very long chain fatty acids, particularly hexacosanoic acid (C26:0), in tissues and body fluids (Moser et al., 1984). It is now generally accepted that the accumulation of very long chain fatty acids in X-linked ALD is caused by their impaired degradation via the peroxisomal β-oxidation system as first shown by Singh and co-workers in 1981 (see also Rizzo et al., 1984; Singh et al., 1984; Tsuji et al., 1985). Recent studies have shown that the deficient peroxisomal oxidation of very long chain fatty acids in X- linked ALD is not caused by a deficiency of one of the peroxisomal β-oxidation enzymes (acyl-CoA oxidase, bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase) since all three enzyme proteins were not only found to be immunologically present as shown by immunoblotting (Wanders et al., 1987) but also functionally active (Hashmi et al., 1986; Wanders et al., 1987). The latter finding led Hashmi and co-workers to suggest that the defect in X-linked ALD is at the level of a deficient activation of very long chain fatty acids to their CoA-esters. However, measurement of this activity in X-linked ALD fibroblasts using tetracosanoic acid (C24:0) and hexacosanoic acid (C26:0) as substrates revealed only a partial (about 25%) very long chain acyl-CoA synthetase deficiency (Wanders et al., 1987a).

The Induction of Cellular Group II Phospholipase A2 by Cytokines and its Prevention by Dexamethasone

Treatment of rat glomerular mesangial cells with interleukin-1 beta, tumor necrosis factor or forskolin resulted in the secretion of phospholipase A2 activity into the culture medium. Essentially all of this secreted phospholipase A2 activity was recognized by monoclonal antibodies elicited against rat liver mitochondrial 14 kDa group II phospholipase A2. Immunoblot analysis and gel filtration confirmed the presence of only 14 kDa phospholipase A2 in the culture supernatant. This enzyme could hardly be detected in unstimulated mesangial cells and after a lag period of 6 to 8 hours becomes detectable in both cells and culture medium. The results indicate that the increased phospholipase A2 activity upon treatment of the cells with cytokines is not due to activation of an existing cellular pool of enzyme but is caused by induced synthesis of group II phospholipase A2. Pretreatment of the cells with dexamethasone, a known inhibitor of prostaglandin synthesis, dose-dependently inhibits cytokine-induced phospholipase A2 activity. Western immunoblot analysis of cells and culture medium demonstrates that this is not due to inhibition of existing phospholipase A2 but because dexamethasone prevents the cytokine-induced synthesis of phospholipase A2 protein.

Contribution of group II phospholipase A2 to arachidonic acid release and prostaglandin synthesis by mesangial cells

Interleukin 1 (IL-1) and tumour necrosis factor (TNF)α have been found to increase group II phospholipase A2 (PLA2) synthesis and secretion by mesangial cells. In all cases 85%–90% of the enzyme is secreted from the cells and a parallel increase in prostaglandin (PGE2) synthesis is observed. We report here that co-incubation with a monoclonal antibody that specifically binds and neutralizes rat group II PLA2 attenuates IL-1β and TNFα-stimulated PGE2 production by 45% and 52%, respectively. CGP43182, a specific inhibitor of group II PLA2, potently blocks IL-1β- and TNFα-stimulated PGE2 synthesis in intact mesangial cells with 1C50s of 1.3 and 1.0 μM, respectively.