J. Van Wauwe

Determination of T lymphocyte subpopulations by monoclonal antibodies in rheumatoid arthritis. Influence of immunomodulating agents

The pathogenesis of rheumatoid arthritis is unknown, but clear abnormalities of the immune system are well documented in this disease. We therefore evaluated T cell subpopulations in patients with rheumatoid arthritis using monoclonal antibodies previously shown to react with all T cells (OKT3), with inducer/helper T cells (OKT4) and with suppressor/cytotoxic T cells (OKT8). These investigations disclosed evidence of a significant decrease in the number of OKT8+ cells/mm3 and a high inducer-helper/suppressor-cytotoxic (OKT4+/OKT8+) ratio in active rheumatoid arthritis. A modest number of patients with active arthritis were treated wit levamisole or with synthetic thymopoietin 32-36 (thymopoietin pentapeptide or TP-5). These individuals responded with ratio decreases to more normal levels. Our data support the hypothesis that monoclonal T cell antibodies may offer an important tool for the further evaluation of patients with rheumatoid arthritis and their individual response to treatment.

Effects of antioxidants on cyclooxygenase and lipoxygenase activities in intact human platelets : Comparison with indomethacin and ETYA

Five antioxidative agents (BW755C, 1-naphtol, NDGA, propylgallate and quercetin) were compared with indomethacin and ETYA for their effects on (14C) arachidonic acid metabolism by cyclooxygenase (CO) and lipoxygenase (LPO) enzymes in intact human platelets. All tested compounds inhibited CO activity in a concentration-dependent manner. LPO activity was suppressed by NDGA, propylgallate, quercetin and ETYA but strongly enhanced by BW755C, 1-naphtol and indomethacin. Whereas NDGA and ETYA showed almost equi-potent inhibitory effects towards both fatty acid oxygenases, propylgallate and quercetin were found to be respectively 6.5 and 4 times better inhibitors of LPO than of CO activities. These data indicate that antioxidants affect arachidonic acid metabolism in intact human platelets in different ways: BW755C and 1-naphtol exerted the same activity as indomethacin, a selective CO blocker, whereas NDGA, propylgallate and quercetin behaved as ETYA, a dual CO-LPO inhibitor. Considering their inhibition selectivity, propylgallate and quercetin may serve as prototypes for more specific blockers of LPO activity.

Mitogenic actions of orthoclone OKT3 on human peripheral blood lymphocytes: Effects of monocytes and serum components

The Orthoclone monoclonal antihuman T lymphocyte antibody, OKT3, induced maximal DNA, RNA and protein synthesis in peripheral mononuclear blood cells (PMBC) at concentrations as low as 10 ng ml-1. This pronounced mitogenic activity was highly dependent on the presence of monocytes: removal of these cells from PMBC suspensions by complement (C)-dependent lysis with the antimonocyte antibody OKM1, completely abrogated the proliferative responsiveness of the remaining lymphocytes. The addition of adherent cells to OKM1-treated PMBC demonstrated the strict monocyte requirement for the mitogenic activity of OKT3. Mitogenic responses to OKT3 were most marked when PMBC were cultured in media containing heat-inactivated fetal calf serum (FCS) but they were considerably weaker in cultures supplemented with heat-inactivated human serum (HS). Moreover, aggregated human IgG and its Fc fragments (but not monomeric IgG and its Fab fragments) inhibited the mitogenicity of OKT3: their inhibition could be explained by stimulation of monocytes, resulting in increased prostaglandin E release, since (a) prostaglandin E2 itself strongly suppressed OKT3 activity and (b) indomethacin blocked the inhibitory effects of aggregated HuIgG. The present data demonstrate that OKT3 shows a particular pattern of mitogenicity: the strict monocyte requirement, the inhibitory effects of HS, aggregated human IgG and prostaglandin E2 were not observed for the phytomitogen PHA.

Cytokine production by phytohemagglutinin-stimulated human blood cells: Effects of corticosteroids, T cell immunosuppressants and phosphodiesterase IV inhibitors

The ability of dexamethasone and prednisolone (corticosteroids), FK506 and cyclosporin A (T cell immunosuppressants), and of nitraquazone and rolipram (phosphodiesterase IV inhibitors) to inhibit cytokine production by stimulated human blood was investigated. Heparinized human blood obtained from normal healthy volunteers was stimulated with phytohemagglutinin (PHA) in the presence or absence of drug. After different incubation times, supernatant levels of interleukin (IL)-2, IL-5, granulocyte-macrophage colony stimulating factor (GM-CSF) and interferonγ (IFN-γ) were quantified by ELISA. Dexamethasone strongly inhibited the production of IL-5 (IC50 = 0.004 µM), was less potent against IL-2 and IFN-γ (IC50 = 0.02–0.05 µM) and showed a relatively weak effect against GM-CSF (IC50 = 0.6 µM). Similarly prednisolone potently suppressed IL-5 generation (IC50=0.05 µM), displayed a more modest activity on IL-2 and IFN-γ (IC50 = 0.2–0.3 µM) and exerted only partial effects (43% inhibition at 1 µM) on GM-CSF. FK506 strongly suppressed the production of IL-2 (IC50 = 0.01 µM) and GM-CSF (IC50 = 0.03 µM), but was inactive (<30% inhibition at 1 µM) against IL-5 and IFN-γ. Similarly, cyclosporin A reduced the generation of IL-2 (IC50 = 0.4 µM) and GM-CSF (IC50 = 0.6 µM) while barely affecting the other two cytokines. Nitraquazone and rolipram were most active in reducing the production of IL-5 (IC50 = 0.8 and 1.3 µM, respectively), while their potency against IL-2, GM-CSF and IFN-γ was 3–6 times lower, with IC50s between 2.4 and 8.0 µM. These data indicate that corticosteroids, T cell immunosuppressants and phosphodiesterase IV inhibitors affect cytokine production by PHA-stimulated human blood cells in a differential and “pharmacotypical” manner.

Liarozole fumarate inhibits the metabolism of 4-keto-all-trans-retinoic acid

The metabolism of 4-keto-all-trans-retinoic-acid (4-keto-RA), a biologically active oxygenated metabolite of all-trans-retinoic (RA), has been examined. In vitro, incubation of [14C]4-keto-RA with hamster liver microsomes in the presence of NADPH produced two major radioactive metabolites which were more polar than the parent compound. Following isolation, appropriate derivatization and analysis by GC-MS, these compounds were tentatively identified as 2-hydroxy- and 3-hydroxy-4-ketoretinoic acid. Formation of both hydroxy-keto derivatives was suppressed by the imidazole-containing P450 inhibitor liarozole fumarate (IC50, 1.3 microM). In vitro, an i.v. injection of 4-keto-RA (20 micrograms) into rats was followed by rapid disappearance of the retinoid from plasma with a half-life of 7 min. Pretreatment with liarozole fumarate (40 mg/kg, -60 min) reduced the elimination rate of 4-keto-RA: it prolonged the plasma half-life of the retinoid to 12 min, without affecting its distribution volume. These results indicate the important role of the P450 enzyme system in the metabolism of 4-keto-RA both in vitro and in vivo. The inhibitory effect of liarozole fumarate on this metabolic process may contribute to the reported retinoid-mimetic activity of this drug.

Arabinogalactan- and dextran-induced ear inflammation in mice: differential inhibition by H1-antihistamines, 5-HT-serotonin antagonists and lipoxygenase blockers.

Intravenous injection of arabinogalactan or dextran together with pontamine sky-blue dye into mice increased vascular permeability and led to marked blueing of the ears. Arabinogalactan caused a rapidly progressing ear blueing (maximal coloration 20–30 min after injection). This response was suppressed by pretreating the animals with the histamine H1-antihistamines levocabastine and loratadine. In contrast, dextran induced a slowly evolving ear inflammation (maximal coloration 60–90 min after injection), which was blocked by the 5-HT-serotonin antagonists cinanserin, metergoline and ritanserin. Furthermore, the dextran reaction was inhibited by the lipoxygenase (LO)/cyclooxygenase (CO) inhibitors BW540C, BW755C and phenidone and by the specific 5-LO inhibitor AA-861. Both arabinogalactan and dextran responses were inhibited by aprotinin, a kallikrein inhibitor, and the mixed H1/5-HT antagonists astemizole and azatadine. The inflammogenic activity of the polysaccharides was not affected by administration of the CO inhibitors indomethacin and suprofen, the thromboxane synthetase inhibitor dazoxiben, the H2-antihistamines cimetidine and ranitidine, the anticholinergics isopropamide or the PAF-antagonist L-652,731.These data indicate the existence of distinctive endogenous molecules that mediate the pinnal extravasation reaction to both polysaccharides: histamine for arabinogalactan, serotonin and lipoxygenase-derived arachidonic acid metabolites for dextran.

The inhibitory effect of pentamidine on the production of chemotactic cytokines by in vitro stimulated human blood cells.

Pentamidine is an antiprotozoal drug with additional antiinflammatory activities that are not well understood. We now report that pentamidine inhibited the human whole blood production of the chemotactic cytokines (chemokines) interleukin (IL)-8, growth related gene α (GROα) and monocyte chemotactic protein-1 (MCP-1). The title compound dose-dependently suppressed the lipopolysaccharide (LPS)- and phytohemagglutinin (PHA)-stimulated whole blood generation of these chemokines with IC50-values of 2.1 and 2.2 μM (IL-8), 2.4 and 1.8 μM (GROα) and 2.8 and 2.4 μM (MCP-1). The inhibition was specific: when tested at 10 μM, pentamidine had no significant inhibitory effect on the PHA-induced generation of the non-chemotactic cytokines tumor necrosis factor-α (TNF-α), IL-1β, IL-2, IL-4, IL-5, IL-10 and interferon-ψ (IFN-ψ), except for a partial inhibition on IL-6. Time course experiments indicated that pentamidine (10 μM) retained its ability to inhibit PHA-stimulated IL-8 production even when its addition was delayed for up to 24 h after mitogen stimulation. Furthermore, reverse transcription PCR studies showed that pentamidine had no effect on IL-8 mRNA expression. These finding indicate that pentamidine is a posttranscription acting inhibitor of human chemokine production. This activity may contribute to the antiinflammatory action ascribed to the title compound.

Synthesis of LIAZALTM, a retinoic acid metabolism blocking agent (ramba) with potential clinical applications in oncology and dermatology

The synthesis of LIAZAL (compound 9, R085246) is described. LIAZAL inhibits all-trans-retinoic acid metabolism and thereby exerts retinoid-like effects in vivo.

Measurement of mitogen stimulation of lymphocytes with a glucose consumption test.

The increase of glycolysis in mitogen-stimulated lymphocytes was used to quantify lymphoblast transformation. Results correlated well with those by common methods, i.e. morphological evaluation and [3H]thymidine incorporation. The advantage of the glucose consumption test is that it registers the entire event of lymphocyte proliferation whereas other methods evaluate only viable transformed cells at the end of culture.

Cytoprotective effects of disodium cromoglycate on rat stomach mucosa

1 The cytoprotective effects of the anti‐asthmatic drug, disodium cromoglycate (DSCG), on gastric mucosal necrosis induced by ethanol in rats were studied. Subcutaneous, but not oral, DSCG prevented the formation of gastric lesions and this effect was dose‐dependent between 1.25 and 40 mg kg−1, with an ED50 value of 6.8 mg kg−1. Maximal cytoprotection occurred 15–30 min after DSCG treatment. 2 Histological examination revealed that DSCG effectively protected the gastric mucosa against ethanol‐induced vascular congestion, haemorrhage, epithelial desquamation and mucosal oedema. 3 Enhanced production of endogenous prostaglandins, which are known cytoprotective compounds, could not explain the mucosal protection. At a dose of 40 mg kg−1, DSCG did not change prostaglandin E2 or 6‐keto‐prostaglandin F1α concentrations in gastric mucosal tissue, although its cytoprotective activity was partially inhibited by prior treatment of the animals with indomethacin.