Studies on in vitro colony formation by mouse bone marrow cells using different inflammatory pleural exudates. Relation to colony stimulating factor (CSF).

An inflammatory exudate obtained in Swiss mice 3 hours after intrapleural injection of dextran was able to increase the number of cultivated peritoneal macrophages in S phase. This exudate was also able to stimulate the formation of colonies from mice bone marrow progenitors of macrophages and granulocytes in methylcellulose culture. The stimulating activity of this acute inflammatory exudate was compared with that of colony stimulating factor (GM.CSF). The qualitative and quantitative results showed that the biological activity of the mitogenic factor of this inflammatory exudate, inflammatory mitogenic factor (IMF) was very close to GM.CSF at optimal concentration, but when used at the same concentration, the exudate was less active than GM.CSF. A stimulating activity was also found with another inflammatory pleural exudate induced by calcium pyrophosphate. The comparative kinetics of the action of the two exudates on CFUc appeared alike but the rise was earlier with calcium pyrophosphate. These results suggest the release of a growth factor for monocyte-macrophage in different acute inflammatory exudates.

Effect of in vitro treatment with indomethacin on mouse granulocyte-macrophage colony-forming cells in culture (CFUC). Possible role of prostaglandins.

The effect of indomethacin (6 mg/kg daily orally for 2, 3 or 4 days) on myelopoiesis was studied in mice by estimating 1) differential cell counts of bone marrow, 2) proliferation of the CFUC in presence of GM-CSF, 3) proliferative state of CFUC after tritiated thymidine suicide. After 4 days of treatment, a rise in the recognizable myeloid precursors, an increase of both the colony forming capacity and the number of CFUC in S-phase were observed. These data suggest that indomethacin produced an hyperplasia of the committed stem cell compartment. The decrease of PGE2 amounts in bone marrow cells following treatment with indomethacin could explain the hyperplasia observed. These in vivo results are in accordance with the in vitro data which show that PG could control the proliferation and differentiation of myeloid progenitor cells.

Effects of donor's age on growth kinetics of rabbit articular chondrocytes in culture.

The in vitro proliferative capacity of articular chondrocytes derived from young and old rabbits was investigated to examine if the modifications incurred can be related to the in vivo aging. Determinations were made of the cartilage cell density, cell volume, cell number at confluency, plating efficiency, growth curve and DNA content distributions. The old donor cells were characterized by a decline in all the parameters of cartilage growth studied: cell number at confluency, cell replication rate (from 20 h to 45 h) as well as an increase in cell volume. The mean cycle time in vitro increased from 17.5 h compared to 27 h during in vivo aging, essentially because of an elongation of the G1 phase. Chondrocytes derived from young and old donors may be an appropriate model system for studying the in vitro effects of drugs on rheumatoid diseases as a function of in vivo aging.

Immunomodulatory effects of ornithine alpha-ketoglutarate in rats with burn injuries.

OBJECTIVE: To investigate the influence of enterally administered ornithine alpha-ketoglutarate (OKG) on muscular amino acid content, eicosanoid release, and polymorphonuclear leukocyte responsiveness after induction of burn injury in rats. DESIGN: Experimental trial. MATERIALS AND METHODS: Four groups of rats were considered: (1) healthy rats that received a standard diet supplemented with 5 g/kg per day of OKG; (2) rats with burn injuries that received the same nutrition as group 1; (3) healthy rats that received standard diet supplemented with glycine in an isonitrogenous amount relative to OKG; and (4) rats with burn injuries that received the same nutrition as group 3. The thymus and 1 skeletal muscle were weighed. The oxidative metabolism of pleural polymorphonuclear leukocytes was measured by means of superoxide generation (O2-) and the chemiluminescent response to opsonized zymosan. Prostaglandin E2 and 6-keto-prostaglandin F1 alpha were measured in the supernatants of pleural and peritoneal cells. RESULTS: The weights of the thymus and the muscle from healthy rats were similar. Those of rats from group 4 were significantly lower (P < .05), whereas those of rats from group 2 were not. Metabolism of OKG led to enhanced amounts of arginine and glutamine in skeletal muscle. The metabolic bursts of polymorphonuclear leukocytes from healthy rats were similar. Those of glycine-treated rats with burn injuries were significantly depressed (P < .05), whereas those of the OKG-treated group were not. Pleural and peritoneal cells from the rats with burn injuries that received OKG generated significantly more prostaglandins (P < .01) than did cells from the other groups of rats. CONCLUSION: Ornithine alpha-ketoglutarate administered to rats with burn injuries displays immunomodulatory properties that can enhance host-defense mechanisms in animals that are affected by a severe injury.

Effects of some antimalarial drugs on rat inflammatory polymorphonuclear leukocyte function.

In vitro concentrations of chloroquine, amodiaquine, quinine, and mefloquine were assessed with respect to functional responses (chemotaxis, anion superoxide generation, and lysosomal enzyme release) of rat polymorphonuclear leukocytes (PMN) collected after induction of acute pleural inflammation. Chloroquine, amodiaquine, and mefloquine exhibited dose-dependent inhibition of: (1) random migration and oriented migration of PMN; and (2) opsonized zymosan- or phorbol myristate acetate-stimulated PMN O2- release. These effects were not stimulus-specific and were largely reversed by washing the cells before stimulation Mefloquine was the most effective drug. Quinine had no effect on PMN migration. Apart from quinine, the drugs induced similar dose-dependent increases in beta-glucuronidase release from unstimulated PMN. Only quinine inhibited the enzyme release from stimulated PMN. Our data show that the antimalarial derivatives affect PMN functions in various ways and suggest that their effects reflect nonspecific modifications of cellular membrane.

In vivo effects of indomethacin and cyclophosphamide on CFU-GM proliferation and cyclic nucleotide levels in bone marrow of mice.

The effects of indomethacin (6 mg/kg daily, orally, for 4 days) or cyclophosphamide (150 mg/kg, a single dose, intraperitoneally) on myelopoiesis were studied in mice. A hyperplasia of the committed stem cell compartment (rise in the recognizable myeloid precursors, increase of both colony forming capacity and number of CFU-GM in S-phase) associated to an increase of cyclic AMP and GMP amounts in bone marrow cells of treated mice were observed. These results suggest a possible relationship between the in vitro enhancement of proliferative activity of CFU-GM and the in vivo increase of both cyclic nucleotide levels in medullary environment.

Ornithine alpha-ketoglutarate counteracts the decrease of liver cytochrome P-450 content in burned rats.

The effect of ornithine alpha-ketoglutarate (OKG) on cytochrome P-450 enzyme activities was studied in a well-defined model of injury (burn followed by fasting then subsequent hypocaloric diet) administered to young rats for 3 d. Hepatic microsomes were prepared by ultracentrifugation and levels of cytochromes P-450 were determined spectrophotometrically. The activities of ethoxy-resorufin-O-deethylase (EROD), benzyloxy-resorufin-O-dealkylase (BROD), and erythromycin demethylase were measured as markers of P-450 1A, 2A, and 3A isotypes respectively. The level of total hepatic microsomal proteins (8 mg/mL) remained constant. The level of cytochrome P-450 (1.14+/-0.08 nmol/mg microsomal proteins) was decreased by a hypocaloric diet (23%, P = 0.003) and burn further enhanced this phenomenon (15%, P = 0.03). Both healthy and burned rats receiving OKG showed the same level of cytochrome P-450 as the rats fed ad libitum. OKG supplementation counteracted the enhancement (40%) of EROD activity induced by hypocaloric diet but did not influence BROD and erythromycin demethylase activities. OKG sustained cytochrome P-450 levels in rats fed a hypocaloric diet, even after burning. These findings indicate that OKG may favor drug metabolism in this injured population.

Effects of niflumic acid on polyphosphoinositide and oxidative metabolism in polymorphonuclear leukocytes from healthy and thermally injured rats.

Thermal injury in rats leads to an impairment of polymorphonuclear leukocyte (PMN) functions, particularly oxidative metabolism and phosphoinositide turnover. As prostaglandin E2, which has immunosuppressive properties, is released in high levels after burn trauma, we investigated the in vitro and in vivo effects of a nonsteroidal antiinflammatory drug, niflumic acid, on oxidative and phosphoinositide metabolism in PMNs from healthy and burned rats. Given the role of fluoride ions on PMN, the influence of niflumic acid was compared with that of sodium fluoride (NaF) at equivalent doses of F-. In vitro, niflumic acid and sodium fluoride had no effect on oxidative metabolism in stimulated by formyl methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ) or nonstimulated PMNs from healthy and burned rats. Niflumic acid slightly increased the production of inositol phosphate by nonstimulated PMNs from healthy and burned rats. Niflumic acid and NaF partly restored the stimulating effect of FMLP on inositol phosphate production by PMNs from burned rats. In vivo treatment with niflumic acid and NaF increased the oxidative metabolism of PMNs from burned rats but not healthy rats. Niflumic acid, more than NaF, restored the activity of both stimulants on phosphoinositide metabolism in PMNs from burned rats. In conclusion, at non-antiinflammatory doses, while inhibiting cyclooxygenase activity, niflumic acid exerts a complex effect on the burn-induced depression of PMN functions. The fluoride anion induces similar but generally weaker effects and seems to be involved in the restoring effects of niflumic acid on PMN functions in burned rats.

Effect of an immunomodulating agent, RU 414740, on polymorphonuclear responsiveness after burn injury.

An impairment of polymorphonuclear leukocyte (PMN) functions has been described following burn trauma. It was thus of interest to investigate the effect of RU 41740, an agent known to stimulate these cells, on rat PMN functions after burn injury. In the present study the responsiveness to classical stimuli of PMN from untreated burned rats was approximately 40% lower than healthy control values. In vitro treatment with RU 41740 increased oxidative metabolism of PMNs from burned and healthy rats. The effect was dose-related but was most striking in the case of PMNs from healthy rats. Significant differences were obtained with concentrations higher than 1 micrograms/ml for healthy rats but only 10 micrograms/ml for burned rats. In vivo treatment with RU 41740 also led to an enhancement of PMN oxidative metabolism on both burned and healthy rats. The maximal effective dose was 10 mg/kg/day in both cases. In contrast, 25 and 50 mg/kg/day doses inhibited PMN oxidative metabolism.

Effect of indomethacin on collagen biosynthesis by rabbit articular chondrocytes in monolayer cultures.

The influence of indomethacin on collagen biosynthesis in rabbit articular chondrocyte monolayer cultures was studied. Two applications within the space of three days of therapeutic doses (10(-5) or 10(-6)M), as well as repeated applications four days running of lower doses (10(-8) or 10(-10)M), increased the biosynthesis of both collagen and non-collagen proteins. Two applications of higher doses (10(-3) or 10(-4M) decreased DNA synthesis and inhibited both collagen and non-collagen protein biosynthesis. These results might well be considered in connection with the adverse reactions observed in some patients with long-term use of indomethacin.

Effects of long-term treatment with D-penicillamine on antigen-induced arthritis in rabbits: studies on in vivo articular chondrocyte damage and in vitro collagen biosynthesis by cultured chondrocytes.

The effects of a long-term (120 days) treatment with D-penicillamine (DP) (50 mg/kg/day; i.v.) on antigen-induced arthritis were studied in rabbit. They were investigated by the terminal histological examination of the joints of different groups of rabbits (unimmunized treated or untreated, immunized treated or untreated) and the study of collagen and non-collagen protein biosynthesis by cultured chondrocytes obtained from articular cartilage of the same groups of animals. Treatment with D-penicillamine diminished the intensity of the erosions of cartilage and subchondral bone, the severity of the inflammatory synovitis, and the loss of chondrocyte clusters found in cartilage sections. In cultures of chondrocytes obtained from immunized treated rabbits, a partial or complete inhibition of the decreased biosynthesis of collagen and non-collagen proteins seen in culture of chondrocytes obtained from immunized untreated animals was observed. These results show that DP could be effective in preventing damage of chondrocytes and inhibition of collagen biosynthesis in them, phenomena important in cartilage destruction induced by a chronic immunological inflammation.