Arthur L. Sagone

Effects of Corticosteroid Therapy on Human Monocyte Function

Since high-dose corticosteroid therapy appears to impair cellular defense mechanisms, this study examined its effect on human monocyte function. Fifteen normal volunteers were studied before and after a three-day course of prednisone therapy (50 mg every 12 hours for six doses). A transient period of monocytopenia occurred during the first few hours of therapy. Monocyte killing of Staphylococcus aureus was reduced in nine subjects from 5.6 plus or minus 0.2 (plus or minus S.E.) X 10-6 organisms before to 1.3 plus or minus 0.4 x 10-6 organisms at completion of therapy (p less than 0.01). Similary, killing of Candida tropicalis four subjects fell from 9.3 plus or minus 0.6 to 0.6 plus or minus 0.3 x 10-6 organisma (p less than 0.01). Bactericidal activity returned to normal levels 48 hours after the last dose of prednisone. These same monocyte preparations had normal or increased chemotactic response, phagocytic rate of cryptococci, hexosemonophosphate-shunt response to phagocytosis and ultrastructural characteristics. This impairment of bactericidal and fungicidal activity during prednisone therapy may contribute to the infectious complications seen in patients receiving comparable doses of corticosteroids.

Effects of Corticosteroids on Human Monocyte Function

This report examined the effect of corticosteroids in vitro on human peripheral blood monocytes, essential cells in both immune and nonimmune cellular defense mechanisms. Monocyte chemotaxis in response to sera, Escherichia coli filtrate, and lymphokine chemotactic factor was markedly reduced (P < 0.01) by hydrocortisone succinate (HCS) at 16 mug/ml. Methylprednisolone succinate and unesterified hydrocortisone produced similar impairment of monocyte chemotaxis while two drugs which unmodified do not enter cells, hydrocortisone phosphate (HCP) and cortisone acetate, had no effect on chemotaxis. HCS also significantly impaired monocyte random migration at 16 mug/ml. Monocyte bactericidal activity was reduced by HCS at 16 mug/ml (P < 0.01)) but was not affected by HCP even at 120 mug/ml. In comparison, HCS did not alter granulocyte chemotaxis even at 500 mug/ml, and bactericidal activity was reduced at 16 mug/ml (P < 0.01). Monocyte phagocytosis of cryptococci was reduced only 20% (P < 0.05) at 120 mug/ml. HCS at 120 mug/ml did not alter monocyte base-line or postphagocytic hexosemonophosphate shunt activity, viability by trypan blue exclusion, adherence to tissue culture flasks, or surface binding of IgG globulin. These corticosteroid-induced defects in monocyte function may contribute to reduced cellular defense during corticosteroid therapy.

A comparison of the metabolic response to phagocytosis in human granulocytes and monocytes.

Recent studies indicate that oxygen radicals such as superoxide or singlet oxygen may be important in the functional activity of human granulocytes. We have examined the possible importance of these radicals in the functional capacity of human blood monocytes. Monocytes, like granulocytes, generate chemiluminescence during phagocytosis. Chemiluminescence is impaired 50-90% by superoxide dismutase, an enzyme which enhances the dismutation of superoxide to hydrogen peroxide. These results indicate that superoxide is related to the chemiluminescence generated by monocytes. Superoxide dismutase in a concentration which impaired chemiluminescence also impaired the staphylococcal killing by monocytes. Hexose monophosphate shunt activity and hydrogen peroxide production by granulocytes and monocytes were also evaluated. The oxidation of [1-14C]glucose was used as a measure of hexose monophosphate shunt activity and the oxidation of [14C]formate as an estimation of hydrogen peroxide production. The oxidation of both substrates by monocytes was increased during phagocytosis but, in contrast to results in granulocytes, was not further increased by the addition of superoxide dismutase. These data indicate that superoxide may be important in bactericidal activity of human monocytes. Our results also suggest that the metabolism of oxygen radicals in monocytes and granulocytes may be different.

Leukemic reticuloendotheliosis: A study of the origin of the malignant cell

A highly pure preparation of neoplastic cells from the spleen of a patient with leukemic reticuloendotheliosis was studied for function, membrane characteristics and glucose metabolism. Glass adherence and phagocytosis of small particles (latex and carbon black) were demonstrated with phase contrast microscopy. Staphylocidal activity was similar to that of normal monocytes. Immunofluorescent assays revealed nonspecific uptake of antiserums to immunoglobulins G (IgG), M (IgM), A (IgA) and kappa and kappa and lambda light chains. Rosette assays indicated the presence of receptors for IgG on the surface of all cells but no receptors for complement (C3) or sheep red blood cells. Glucose metabolic studies revealed a pattern that differed from that of normal monocytes or lymphocytes with intermediate values for glycolysis, low hexose monophosphate shunt activity and high Krebs cycle activity. Increments in tritiated (3H)-thymidine uptake and glucose metabolism in response to phytohemagglutinin stimulation were minimal (5 per cent of normal lymphocyte values) and no response was noted with pokeweed mitogen stimulation. These findings suggest that the leukemic reticuloendotheliosis cell most closely resembles cells of the monocyte-histiocyte series.

Smoking as a cause of erythrocytosis.

Five smokers had erythrocyte masses sufficiently larger than normal to pose a problem in the differential diagnosis of polycythemia. Evaluation excluded lung disease, shunt physiology, hemoglobin with increased oxygen affinity, erythropoietin-producing tumor, renal disease, or polycythemia rubra vera as the primary cause of erythrocytosis in these patients. All were found to have levels of carboxyhemoglobin sufficient to cause clinically significant hypoxemia and to account for the increased erythrocyte masses. In two patients the erythrocytosis improved when they stopped smoking. Heavy smoking is a reversible cause of polycythemia and should be considered in the differential diagnosis of this problem.

Pulmonary embolism as a result of Hickman catheter-related thrombosis

S ince their introduction in the 197Os, Hickman catheters and similar venous access devices have become widely used in the management of patients with cancer [l-5] because of the ease they confer on blood drawing and administration of intravenous medications, nutrition, and hydration. As with other intravascular foreign objects, the complications of infection and thrombosis have been encountered but have in general been readily managed [6]. Adequate management requires prompt diagnosis for which a high index of suspicion is needed. Two cases of pulmonary embolism from superior vena caval thrombosis due to a Hickman catheter seen by the authors during a recent two-month period emphasize that this complication is not rare. These cases are presented with a review of the literature reporting thrombosis and pulmonary embolism due to Hickman catheters and similar venous access devices.

Hydroxylation of salicylate by activated neutrophils

Salicylates are metabolized in vivo to hydroxylated compounds, including 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid (gentisic acid). The present study hypothesized that activated neutrophils represent one pathway for salicylate hydroxylation. Human neutrophils were incubated in medium containing 10 mM salicylate and stimulated with phorbol myristate acetate (PMA) for 1 hr. The cell-free supernatant fractions were analyzed by HPLC. Neutrophils (1 x 10(6) cells) produced 55 +/- 11 ng of gentisic acid. Neutrophils also produced smaller quantities of 2,3-dihydroxybenzoic acid. Antioxidant inhibitor experiments indicated that superoxide dismutase (SOD), heme protein inhibitors, and glutathione blocked gentisic acid formation, whereas catalase, mannitol, and deferoxamine failed to inhibit. Experiments with the reagent hypochlorous acid (HOCl) and the model myeloperoxidase (MPO) enzyme system did not support a role for the MPO pathway in gentisic acid formation. These findings demonstrate that activated neutrophils can hydroxylate salicylate by an unknown pathway. This pathway may contribute to the increased recovery of hydroxylated salicylates in patients with inflammatory disorders.

THE EFFECT OF OXIDANT INJURY ON THE LYMPHOBLASTIC TRANSFORMATION OF HUMAN LYMPHOCYTES

Abstract— We have studied the effect of oxidant injury on the lymphoblastic transformation and glucose metabolism of human lymphocytes. Lymphocyte cultures were incubated with xanthine oxidase and xanthine, an enzyme system known to generate several highly reactive oxygen compounds. Lymphoblastic transformation to phytohemagglutinin was quantitated by the stimulation of DNA synthesis as indicated by the 3H‐thymidine uptake of the cultures at 3 days.

Evidence that OH· production by human PMNs is related to prostaglandin metabolism

Recent studies indicate that human granulocytes generate OH· during the phagocytosis of zymosan particles. Several theoretical considerations suggested to us that this OH· production might be related to prostaglandin metabolism, particularly the observation that OH· is generated by the reduction of hydroperoxides in microsomal systems. In our studies, we tested the importance of prostaglandin metabolism in the production of OH· by human granulocytes (PMNs). Indomethacin and aspirin at concentrations known to impair cyclooxygenase activity decreased OH· production by PMNs during the phagocytosis of zymosan particles. Phenol, which is known to alter prostaglandin metabolism, ablated OH· completely. None of these drugs at the concentrations used impaired the generation of O2- or H2O2 by PMNs, as indicated by their failure to diminish significantly the generation of chemiluminescence. Thus, the decrement in OH· production by these drugs could not be attributed to a nonspecific effect on the production of O2- or H2O2. These experiments therefore, indicate that the model for OH· production observed during prostaglandin synthesis with microsomal systems applies to human granulocytes.

Metabolism of phenytoin and covalent binding of reactive intermediates in activated human neutrophils

ontivation of neutrophils by phorbol-12-myristate-13-acetate (PMA) causes rapid production of superoxide radical (O2-), leading to the formation of additional reactive oxygen species, including hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and possibly hydroxyl radical (.OH). These reactive oxygen species have been associated with the oxidation of some drugs. We investigated the metabolism of phenytoin (5,5-diphenylhydantoin) and the covalent binding of reactive intermediates to cellular macromolecules in activated neutrophils. In incubations with 100 microM phenytoin, PMA-stimulated neutrophils from six human subjects produced p-, m-, and o-isomers of 5-(hydroxyphenyl)-5-phenylhydantoin (HPPH) in a ratio of 1.0:2.1:2.8, respectively, as well as unidentified polar products. Analysis of cell pellets demonstrated that phenytoin was bioactivated to reactive intermediates that bound irreversibly to macromolecules in neutrophils. Glutathione, catalase, superoxide dismutase, azide, and indomethacin all diminished the metabolism of phenytoin and the covalent binding of its reactive intermediates. The iron-inactivating chelators desferrioxamine and diethylenetriaminepentaacetic acid had little or no effect on the metabolism of phenytoin by neutrophils, demonstrating that adventitious iron was not contributing via Fenton chemistry. In an .OH-generating system containing H2O2 and Fe2+ chelated with ADP, phenytoin was oxidized rapidly to unidentified polar products and to p-, m-, and o-HPPH (ratio 1.0:1.7:1.5, respectively). Reagent HOCl and human myeloperoxidase (MPO), in the presence of Cl- and H2O2, both formed the reactive dichlorophenytoin but no HPPH. However, no chlorinated phenytoin was detected in activated neutrophils, possibly because of its high reactivity. These findings, which demonstrated that activated neutrophils biotransform phenytoin in vitro to hydroxylated products and reactive intermediates that bind irreversibly to tissue macromolecules, are consistent with phenytoin hydroxylation by .OH generated by a transition metal-independent process, chlorination by HOCl generated by MPO, and possibly cooxidation by neutrophil hydroperoxidases. Neutrophils activated in vivo may similarly convert phenytoin to reactive intermediates, which could contribute to some of the previously unexplained adverse effects of the drug.