Werner Scheuer

Suppression of cytokine synthesis, integrin expression and chronic inflammation by inhibitors of cytosolic phospholipase A2

To define the isoform of phospholipases A2 active in inflammation we evaluated the effects of low-molecular-weight inhibitors of secretory and cytosolic phospholipases A2. We found that inhibitors of cytosolic phospholipase A2 had therapeutic efficacy in an in vivo model of chronic inflammation (rat adjuvant arthritis), whereas inhibitors of secretory phospholipase A2 had no beneficial effect. In vitro, inhibitors of cytosolic phospholipase A2 diminished surface expression of Mac-1 (CD11b/CD18) beta2-integrin on calcium ionophore-stimulated human blood granulocytes and suppressed synthesis of interleukin-1beta in lipopolysaccharide-stimulated human blood monocytes and U937 cells by reducing mRNA levels. Lipid mediators promote Mac-1 exocytosis and transcription of interleukin-1beta, which further enhances cytosolic phospholipase A2 activity and expression. Thus, superinduction of cytosolic phospholipase A2 may establish a positive feedback loop, converting acute inflammation into chronic inflammation. Consequently, inhibitors of cytosolic phospholipase A2 may prevent inflammation in vivo by interfering with cellular activation and infiltration. We conclude that cytosolic phospholipase A2 but not secretory phospholipase A2 is the predominant enzyme in inflammatory signalling.

Combinatorial functions of two chimeric antibodies directed to human CD4 and one directed to the α-chain of the human interleukin-2 receptor

The general feasibility of chimerization of monoclonal antibodies (mAbs) has already been shown for a large number of them. In order to evaluate in vitro parameters relevant to immunosuppressive therapy, we have chimerized and synthesized two anti-CD4 mAbs recognizing two different epitopes on the human T-lymphocyte antigen, CD4. The chimerized mAbs are produced at levels corresponding to those of the original hybridoma cell lines. With respect to activation of human complement, the individual Abs are negative; however, when used in combination, complement activation was performed. When applied in combination, they were found to modulate the CD4 antigen, whereas the individual mAb do not display this property. Individually they mediate an up to 60% inhibition of the mixed lymphocyte reaction (MLR). However, by combination of an anti-CD4 mAb with one directed against the alpha-chain of the human IL2 receptor, nearly 100% inhibition of the MLR was achieved, even with reduced dosage of the mAbs. Our data suggest that the combination of an anti-CD4 mAb and an anti-IL2R alpha chain mAb is more effective with respect to immunosuppression than each mAb by itself, indicating that this mAb cocktail could be a new strategy for immunosuppressive therapy.

Phospholipase A2 is Involved in Chemotaxis Of Human Leukocytes

Recently, it was shown that phospholipase A2 (PLA2) is essential for exposing Mac-1 (CDllb/CD18) on the cell surface of leukocytes1,2. Mac-1 is mandatory for leukocyte migration in vitro, and lack of Mac-1 on human leukocytes is associated with failure of in vitro chemotaxis3. To further substantiate the contribution of PLA2 in cell migration we investigated the effects of PLA2 inhibition on chemotaxis of mixed human leukocytes (MHL) induced by fMLP and the G-protein activator NaF7–9. Migration was measured in a modified 48-well micro-chemotaxis Boyden-chamber6. As PLA2 inhibitors manoalide4, mepacrine5, and BM 16.2115, a new PLA2 inhibitor, were employed.

Supression of Acute Experimental Inflammation by Antisense Oligonucleotides Targeting Secretory Phospholipase A2 (sPLA2)IN VITRO and IN VIVO Experiments

In HepG2 cells phosphorothioate modified antisense oligonucleotides against a sequence in the Ca2+ binding domain (AS-Ca2+) of type II sPLA2 mRNA restrained IL-6-induced synthesis of sPLA2 protein, sPLA2 mRNA (northern blot), and abolished IL-6 stimulated PGE2 release. An antisense oligonucleotide corresponding to a sequence in the catalytic domain (AS-Cat) of sPLA2 was less effective. The antisense oligonucleotides did not affect albumin synthesis in HepG2 cells, additionally demonstrating their specificity. The corresponding AS-Ca2+ against a homologous part of the rat sPLA2 mRNA depressed rat carrageenin oedema for 60–70%. Identical suppression was achieved by specific low molecular weight inhibitors of sPLA2. Since cyclo- and 5-lipoxygenase inhibitors exerted similar reductions of carrageenin oedema type II sPLA2 dependent eicosanoid formation seems to be a key cascade in this type of inflammation.

Evidence for Activation of Cyclooxygenase-1/-2 by Endogenous Nitric Oxide in Adjuvant Arthritic Lewis Rats

Increased enzyme activities of cytosolic phospholipase A2 (cPLA2) and nitric oxide synthase (NOS) have been implicated in the pathophysiology of rheumatoid arthritis(RA)1,2,3,4 and the alteration of immunological responses5,6. As determined on messenger RNA (mRNA) and protein level, in human rheumatoid synoviocytes, both, cPLA2 and growth factor-responsive prostaglandin H synthase-2 (PGHS-2 or cyclooxygenase-2, COX-2) activity were induced by interleukin-lβ, while secretory PLA2 (sPLA2) and constitutive prostaglandin H synthase-1 (PGHS-1 or cyclooxygenase-1, COX-1) remained unaffected. Reflecting the enhanced activity of this pathway, its induction was associated with high levels of prostaglandin-2 (PGE2), a mediator of pain and inflammation in the joints of patients with RA2. In addition, patients with RA showed high levels of nitrite (NO− 2, breakdown product of nitric oxide) and 3-nitrotyrosine, indicating elevated NOS activity and nitric oxide (NO•) dependent oxidative damage3,7. In order to evaluate a physiological link between cPLA2, COX-1/-2 and NOS pathway in vivo, we investigated the effect of L-nitro-arginine-methyl-ester (L-NAME), an inhibitor of NOS, in adjuvant-induced arthritis in the rat, while dexamethasone served as control drug, This model exhibits several pathological features similar to those occurring in autoimmune reactive RA in humans, characterized by chronic inflammation of the joints. In our studies following parameters have been determined: i) PGE2 as a product of the PLA2 and COX-1/-2 pathway, ii) platelet-activating-factor (PAF, PAF-acether) as product of the PLA2 and acetyltransferase pathway, iii) NO− 2 as product of the NOS pathway, iv) paw swelling as an indicator of in vivo response.

5-Hydroxy-3-vinyl-2(5H)-furanone -a New Inhibitor of Human Synovial Phospholipase A2 and Platelet Aggregation from Fermentations of a Calyptella Species (Basidiomycetes)

Abstract 5-Hydroxy-3-vinyl-2(5H)-furanone, a potent and selective inhibitor of human synovial phospholipase A2 was isolated from fermentations of a Calyptella species. Its structure as identified by spectroscopic methods is identical to PA 147, an antibiotic previously isolated from a streptomycete. 5-hydroxy-3-vinyl-2(5H)-furanone inhibits the aggregation of human and bovine platelets stimulated by different inducers and exhibits weak antimicrobial activities.

(+)-10α-Hydroxy-4-muurolen-3-one, a New Inhibitor of Leukotriene Biosynthesis from a Favolaschia Species. Comparison with Other Sesquiterpenes

Abstract A new inhibitor of leukotriene biosynthesis, (+)-10α-hydroxy-4-muurolen-3-one (1), was isolated from fermentations of Favolaschia sp. 87129. Its structure was established by spectroscopic methods. The compound exhibited no antifungal or antibacterial activities. The effects of 1 on leukotriene biosynthesis were compared with (+)-T-cadinol, (-)-3-oxo-T-cadinol, and (+)-3a-hydroxy-T-cadinol, three related sesquiterpenes.