Faculty Bibliography

Methylprednisolone sodium succinate is an injectable form of the corticosteroid methylprednisolone. Because it is extremely soluble in water, intravenous methylprednisolone can be administered in a small volume of diluent. This characteristic is particularly beneficial when high blood levels of methylprednisolone must be achieved rapidly. Methylprednisolone sodium succinate therapy is useful in the management of many conditions seen by primary care and emergency department physicians, including allergic states, collagen disease, dermatologic conditions, cerebral and spinal edema, gastrointestinal disorders, neoplastic disease, nervous system disorders, rejection of transplanted organs, respiratory tract disease, and acute spinal cord injuries. Although intravenous methylprednisolone has widespread therapeutic uses, long-term therapy is associated with potentially serious adverse effects. Therefore, whenever methylprednisolone sodium succinate is prescribed, the clinician must individualize the dosage to ensure that the patient receives the lowest effective dose

Methotrexate, a folate antagonist, is a potent antiinflammatory agent when used weekly in low concentrations. We examined the hypothesis that the antiphlogistic effects of methotrexate result from its capacity to promote intracellular accumulation of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) that, under conditions of cell injury, increases local adenosine release. We now present the first evidence to establish this mechanism of action in an in vivo model of inflammation, the murine air pouch model. Mice were injected intraperitoneally with either methotrexate or saline for 3-4 wk during induction of air pouches. Pharmacologically relevant doses of methotrexate increased splenocyte AICAR content, raised adenosine concentrations in exudates from carrageenan-inflamed air pouches, and markedly inhibited leukocyte accumulation in inflamed air pouches. The methotrexate-mediated reduction in leukocyte accumulation was partially reversed by injection of adenosine deaminase (ADA) into the air pouch, completely reversed by a specific adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), but not affected by an adenosine A1 receptor antagonist, 8-cyclopentyl-dipropylxanthine. Neither ADA nor DMPX affected leukocyte accumulation in the inflamed pouches of animals treated with either saline or the potent antiinflammatory steroid dexamethasone. These results indicate that methotrexate is a nonsteroidal antiinflammatory agent, the antiphlogistic action of which is due to increased adenosine release at inflamed sites

Corticosteroids are the preeminent antiinflammatory agents although the molecular mechanisms that impart their efficacy have not been defined. The endothelium plays a critical role in inflammation by directing circulating leukocytes into extravascular tissues by expressing adhesive molecules for leukocytes [e.g., endothelial-leukocyte adhesion molecule 1 (ELAM-1) and intercellular adhesion molecule 1 (ICAM-1)]. We therefore determined whether corticosteroids suppress inflammation by inhibiting endothelial expression of adhesion molecules for neutrophils (polymorphonuclear leukocytes). Preincubation of endothelial cells with endotoxin [lipopolysaccharide (LPS), 1 microgram/ml] led to a 4-fold increase in subsequent adherence of polymorphonuclear leukocytes (P < 0.0001, n = 10) to endothelial cells, an increase that was markedly attenuated when endothelial cells were treated with dexamethasone (IC50 < 1 nM, P < 0.0001, n = 6 or 7) during preincubation with LPS. Moreover, the steroid receptor agonist cortisol (10 microM), but not its inactive metabolite tetrahydrocortisol (10 microM), diminished LPS-induced endothelial cell adhesiveness. Further evidence that the action of dexamethasone was mediated through ligation of corticosteroid receptors [human glucocorticoid receptors (hGRs)] was provided by experiments utilizing the steroid antagonist RU-486. RU-486 (10 microM), which prevents translocation of ligated hGR to the nucleus by inhibiting dissociation of hGR from heat shock protein 90, completely aborted the effect of dexamethasone on adhesiveness of endothelial cells (P < 0.0005, n = 3). Treatment of endothelial cells with LPS (1 microgram/ml) stimulated transcription of ELAM-1, as shown by Northern blot analysis, and expression of membrane-associated ELAM-1 and ICAM-1, as shown by quantitative immunofluorescence (both P < 0.001, n = 9). Dexamethasone markedly inhibited LPS-stimulated accumulation of mRNA for ELAM-1 and expression of ELAM-1 and ICAM-1 (IC50 < 10 nM, both P < 0.001, n = 4-9); inhibition of expression by dexamethasone was reversed by RU-486 (both P < 0.005, n = 4-6). As in the adhesion studies, cortisol but not tetrahydrocortisol inhibited expression of ELAM-1 and ICAM-1 (both P < 0.005, n = 3 or 4). In contrast, sodium salicylate (1 mM) inhibited neither adhesion nor expression of these adhesion molecules. These studies suggest that antagonism by dexamethasone of endotoxin-induced inflammation is a specific instance of the general biological principle that the glucocorticoid receptor is a hormone-dependent regulator of transcription

Peptide T, from the human immunodeficiency virus (HIV), whose sequence is Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr, has been shown to inhibit attachment of this virus to T cells and neural cells bearing the CD4 receptor. This peptide shares extensive homology with the 19-26 segment of ribonuclease A (RNase A), whose sequence is Ala-Ala-Ser-Ser-Ser-Asn-Tyr-Cys. Based on comparison of the structures of peptides occurring in proteins of known structure that are homologous to peptide T, viz, RNase A and endothiapepsin and on conformational energy calculations, we predicted that peptide T adopts a structure much like that for residues 19-26 in RNase A. A critical feature is a bend involving residues Thr 4-Asn 7 in peptide T corresponding to Ser 22-Tyr 25 in the RNase A peptide. Our proposed structure for peptide T has recently been confirmed by Cotelle et al. (Biochem. Biophys. Res. Commun. 171, 596-602). We now show directly that the RNase A peptide, with Met replacing Cys 26 to prevent disulfide exchange reactions, strongly induces monocyte-chemotaxis that is blocked by anti-CD4 monoclonal antibody. Both peptide T and RNase A fail to induce chemotaxis, however, in neutrophils which do not express surface CD4 receptors. These results suggest that both peptides interact with the CD4 receptor in inducing monocyte chemotaxis. We have also prepared cyclo-RNase A peptide with Met 26. Using molecular dynamics and conformational energy calculations, we find that the cyclic peptide cannot form a bend structure involving Ser 22-Tyr 25 that is superimposable on the RNase A bend.(ABSTRACT TRUNCATED AT 250 WORDS)

Although commonly used to control a variety of inflammatory diseases, the mechanism of action of a low dose of methotrexate remains a mystery. Methotrexate accumulates intracellularly where it may interfere with purine metabolism. Therefore, we determined whether a 48-hr pretreatment with methotrexate affected adenosine release from [14C]adenine-labeled human fibroblasts and umbilical vein endothelial cells. Methotrexate significantly increased adenosine release by fibroblasts from 4 +/- 1% to 31 +/- 6% of total purine released (EC50, 1 nM) and by endothelial cells from 24 +/- 4% to 42 +/- 7%. Methotrexate-enhanced adenosine release from fibroblasts was further increased to 51 +/- 4% (EC50, 6 nM) and from endothelial cells was increased to 58 +/- 5% of total purine released by exposure to stimulated (fMet-Leu-Phe at 0.1 microM) neutrophils. The effect of methotrexate on adenosine release was not due to cytotoxicity since cells treated with maximal concentrations of methotrexate took up [14C]adenine and released 14C-labeled purine (a measure of cell injury) in a manner identical to control cells. Methotrexate treatment of fibroblasts dramatically inhibited adherence to fibroblasts by both unstimulated neutrophils (IC50, 9 nM) and stimulated neutrophils (IC50, 13 nM). Methotrexate treatment inhibited neutrophil adherence by enhancing adenosine release from fibroblasts since digestion of extracellular adenosine by added adenosine deaminase completely abrogated the effect of methotrexate on neutrophil adherence without, itself, affecting adherence. One hypothesis that explains the effect of methotrexate on adenosine release is that, by inhibition of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase, methotrexate induces the accumulation of AICAR, the nucleoside precursor of which (5-aminoimidazole-4-carboxamide ribonucleoside referred to hereafter as acadesine) has previously been shown to cause adenosine release from ischemic cardiac tissue. We found that acadesine also promotes adenosine release from and inhibits neutrophil adherence to connective tissue cells. The observation that the antiinflammatory actions of methotrexate are due to the capacity of methotrexate to induce adenosine release may form the basis for the development of an additional class of antiinflammatory drugs

The effects of adenosine were studied on human neutrophils with respect to their generation of superoxide anion, degranulation, and aggregation in response to soluble stimuli. Adenosine markedly inhibited superoxide anion generation by neutrophils stimulated with N-formyl methionyl leucyl phenylalanine (FMLP), concanavalin A (Con A), calcium ionophore A23187, and zymosan-treated serum; it inhibited this response to PMA to a far lesser extent. The effects of adenosine were evident at concentrations ranging from 1 to 1,000 microM with maximal inhibition at 100 microM. Cellular uptake of adenosine was not required for adenosine-induced inhibition since inhibition was maintained despite the addition of dipyridamole, which blocks nucleoside uptake. Nor was metabolism of adenosine required, since both deoxycoformycin (DCF) and erythro-9-(2-hydroxy-3-nonyl) adenine did not interfere with adenosine inhibition of superoxide anion generation. The finding that 2-chloroadenosine, which is not metabolized, resembled adenosine in its ability to inhibit superoxide anion generation added further evidence that adenosine metabolism was not required for inhibition of superoxide anion generation by neutrophils. Unexpectedly, endogenously generated adenosine was present in supernatants of neutrophil suspensions at 0.14-0.28 microM. Removal of endogenous adenosine by incubation of neutrophils with exogenous adenosine deaminase (ADA) led to marked enhancement of superoxide anion generation in response to FMLP. Inactivation of ADA with DCF abrogated the enhancement of superoxide anion generation. Thus, the enhancement was not due to a nonspecific effect of added protein. Nor was the enhancement due to the generation of hypoxanthine or inosine by deamination of adenosine, since addition of these compounds did not affect neutrophil function. Adenosine did not significantly affect either aggregation or lysozyme release and only modestly affected beta-glucuronidase release by neutrophils stimulated with FMLP. These data indicate that adenosine (at concentrations that are present in plasma) acting via cell surface receptors is a specific modulator of superoxide anion generation by neutrophils.