Ira M. Goldstein

Protected environments and prophylactic antibiotics. A prospective controlled study of their utility in the therapy of acute leukemia.

Abstract To reduce the frequency of infection in acute leukemia, we employed isolation and air-filtration facilities (protected environment) and a prophylactic regimen that included oral nonabsorbable antibiotics. Eighty-eight randomized patients received identical remission-induction chemotherapy within one of three groups: protected environment combined with the prophylactic regimen (Group 1); oral nonabsorbable antibiotics alone (Group 2); and neither isolation nor prophylaxis (Group 3). The groups were comparable in factors that might influence the course of leukemia and susceptibility to infection. Environmental maneuvers. were effective in reducing the potential inoculum of ambient micro-organisms. Patients in Group 1 had 1/2 as many severe infections as those in Groups 2 and 3. Whereas approximately 1/4 of the patients in Groups 2 and 3 died of infection while on study, none in Group 1 died for that reason. Despite fewer infections in Group 1, no intergroup differences were found in remission rat...

Evidence that the superoxide-generating system of human leukocytes is associated with the cell surface.

Superoxide anion (O-2-) generation by human peripheral blood polymorphonuclear leukocytes is enhanced when these cells encounter appropriate soluble or particulate stimuli. O-2- generation requires intact, viable cells and proceeds independently of phagocytosis. To investigate the possibility that the O-2--generating system is associated with the outer surface of the polymorphonuclear leukocyte plasma membrane, we have examined the effects upon O-2- production of p-diazobenzenesulfonic acid, a reagent which can react predominantly with proteins of the external cell membrane. When normal human polymorphonuclear leukocytes were preincubated with cytochalasin B (to minimize endocytosis) and then exposed to the surface-active lectin, concanavalin A, the cells were stimulated to generate O-2- in a concentration- and time-dependent fashion and selectively to discharge the granule-associated enzyme, lysozyme, into the surrounding medium. These responses, as well as cellular binding of [H] concanavalin A, could be blocked by alpha-methyl-D-mannoside. Brief treatment (less than 5 min at 4 degrees C) of the cells with p-diazobenzenesulfonic acid (1.0-5.0 mM) significantly interfered with concanavalin A-mediated O-2- generation but had no influence upon lysozyme release or upon binding of [3H] concanavalin A. The diazonium salt did not alter cell viability or the specific activity of the cytoplasmic enzyme, lactate dehydrogenase (inhibitable under conditions which allowed entry of this reagent into the cytosol). p-Diazobenzenesulfonic acid, therefore, very likely exerted its effects at the cell surface of the intact polymorphonuclear leukocyte, selectively inhibiting O-2- production (either directly or indirectly) without influencing another response to lectin-cell contact: release of lysozyme. These results support the possibility that a polymorphonuclear leukocyte ectoenzyme is responsible for O-2- production.

Generation of a chemotactic lipid from arachidonic acid by exposure to a superoxide-generating system

Certain products of arachidonic acid have been demonstrated recently to possess chemotactic activity for human polymorphonuclear leukocytes (PMN). Enzymatic (lipoxygenase, cyclooxygenase) generation of these lipid chemotaxins proceeds through the formation of intermediate lipid peroxides. Since lipid peroxidation can be mediated by oxygen-derived free radicals, we have examined whether chemotactically active products of arachidonic acid could be produced by exposing this unsaturated fatty acid to a superoxidegenerating system. A lipid with potent chemotactic activity for human PMN was produced by incubating arachidonic acid with xanthine oxidase and acetaldehyde. Generation of chemotactic activity was time-dependent and could be inhibited to the greatest extent by scavengers of singlet oxygen (i.e., histidine, uric acid, and 2,5-dimethylfuran). Inhibition was also observed with scavengers of superoxide anion radicals (i.e., superoxide dismutase), hydrogen peroxide (i.e., catalase), and hydroxyl radicals (i.e., mannitol). Silica gel thin-layer radiochromatography demonstrated a single peak with chemotactic activity (Rf = 0.33–0.38) distinct from unaltered arachidonic acid. The product of arachidonic acid was chemotactic at a concentration of 3.0 ng/ml and chemokinetic at concentrations of 0.75–1.5 ng/ml. Since PMN produce oxygen-derived free radicals and singlet oxygen upon stimulation of their plasma membrane, and since arachidonic acid is widely distributed in human tissues, free radical-mediated generations of chemotactic activity from arachidonic acid may play an important role in amplifying inflammatory responses.

Calcium-induced lysozyme secretion from human polymorphonuclear leukocytes

Abstract Calcium ions, in the absence of other stimuli, are capable of provoking the release by exocytosis of the granule-associated enzyme, lysozyme, from human polymorphonuclear leukocytes. Calcium-induced extrusion of lysozyme occurs in a concentration, time and temperature-dependent fashion. It is enhanced in the presence of extracellular inorganic phosphate and the ionophore, A-23187, and is not accompanied by the release from cells of cytoplasmic or lysosomal marker enzymes.

Influence of corticosteroids on human polymorphonuclear leukocyte function in vitro : Reduction of lysosomal enzyme release and superoxide production.

In an experimental system in which phagocytosis or adherence of cells to surfaces were excluded as variables, we have investigated the possibility that corticosteroids may inhibit release of lysosomal constituents from viable human polymorphonuclear leukocytes into the extracellular environment. Release ofΒ-glucuronidase and lysozyme from cytochalasin B-treated cells exposed to serum-treated zymosan, heat-aggregated human IgG, and the complement component C5a was significantly reduced by pretreatment with hydrocortisone sodium succinate and methylprednisolone sodium succinate (5×10−4 M). Both steroids also reduced superoxide production by these cells. These in vitro studies provide evidence that corticosteroids influence membrane-dependent responses of intact, viable polymorphonuclear leukocytes to immune reactants. Inhibition of these responses (lysosomal enzyme release, superoxide production) may explain, in part, both the antiinflammatory actions of steroids and their deleterious effects on host defenses.

Effects of the generation of superoxide anion on permeability of liposomes

Abstract Multilamellar liposomes released previously sequestered chromate ions when these artificial membrane structures were suspended in reaction mixtures containing hypoxanthine and xanthine oxidase. Release of chromate was reduced significantly if active superoxide dismutase and/or catalase were added to these reaction mixtures. These findings suggest that superoxide anion and/or related reactive molecules are capable of perturbing lipid bilayers sufficiently to cause leakage of relatively impermeant anions.

YIN/YANG MODULATION OF LYSOSOMAL ENZYME RELEASE FROM POLYMORPHONUCLEAR LEUKOCYTES BY CYCLIC NUCLEOTIDES*

Since 1970 it has been clear that polymorphonuclear leukocytes of man and other species selectively release lysosomal, but not cytoplasmic, constituents when exposed to particulate challenges.] :i Originally demonstrated after the uptake of zymosan particles,’, this phenomenon also includes the release of lysosomal constituents when white cells are exposed to immune complexes either in the bulk phase or dispersed upon The former process, release of lysosomal hydrolases during phagocytosis of discrete particles by white cells, has been termed “regurgitation during feeding,” whereas the latter, extrusion of lysosomal enzymes after the exposure of white cells to immune complexes on a supporting surface, has been termed “reverse endocytosis” 1 or “frustrated phagocytosis.” i . l1 In these two circumstances, the white cells release such enzymes as /

A Specific Inhibitor of Complement (C5)-Derived Chemotactic Activity in Serum from Patients with Systemic Lupus Erythematosus

glucuronidase, acid phosphatase, aryl sulfatase, and myeloperoxidase (constituents of azurophil granules), in addition to lysozyme and alkaline phosphatase (constituents of specific granules) .2-11 Viability is maintained during release, and it has become appreciated that the stimuli to release of lysosomal hydrolases act via Fc receptors on the polymorphonuclear leukocytes that recognize altered immunoglobulins, such as those present in the form of immune complexes, and via C3 receptors that recognize C3b fragments present upon such particles as serum-treated zymosan.lo,

Prostaglandins, thromboxanes, and polymorphonuclear leukocytes: mediation and modulation of inflammation.

In the course of examining polymorphonuclear leukocyte (PMN) chemotaxis in patients with systemic lupus erythematosus (SLE), we have found a previously undescribed serum inhibitor of complement (C5)-derived chemotactic activity. Serum from a 25-yr-old Black female with untreated SLE, when activated with zymosan, failed completely to attract either her own or normal PMN. Incubation of normal PMN with the patients serum did not affect their subsequent random motility or chemotactic response toward normal zymosan-treated serum (ZTS). The patients serum, however, did inhibit the chemotactic activity of normal ZTS and of column-purified C5-derived peptide(s), but had no effect on the chemotactic activity of either the synthetic peptide, N-formylmethionyl leucyl-phenylalanine or a filtrate prepared from a culture of Escherichia coli (bacterial chemotactic factor). The inhibitory activity in the patients serum resisted heating at 56 degrees C for 30 min and could be separated from C5-derived chemotactic activity in the patients ZTS (or normal ZTS that had been incubated with the patients serum) by chromatography on Sephadex G-75. Despite its effect on C5-derived chemotactic activity, the patients serum did not influence two other C5-derived biologic activities: PMN lysosomal enzyme-releasing activity and PMN-aggregating activity. Chromatography of the patients serum (65% ammonium sulfate pellet) on Sephadex G-200 yielded three distinct peaks of inhibitory activity. Two were heat labile and exhibited other properties of the previously described chemotactic factor inactivators of normal human serum. The third and most active peak, however, resisted heating at 56 degrees C for 30 min, eluted with an apparent mol wt of 50,000-60,000, and acted specifically on C5-derived chemotactic activity. This uniquely specific, heat-stable inhibitor of C5-derived chemotactic activity has been found thus far in serum from 4 of 11 patients with active SLE and may account, in part, for altered host defenses against infections caused by pyogenic microorganisms.

Generation of Chemotactic Activity in Serum from Patients with Erythropoietic Protoporphyria and Porphyria Cutanea Tarda

When appropriately stimulated (even in the absence of phagocytosis), human polymorphonuclear leukocytes release and/or generate proinflammatory materials and substances capable of provoking tissue injury. These include hydrolases and nonenzymatic substances ordinarily contained within lysosomes, as well as oxygen-derived free radicals. It is now possible to add prostaglandins and thromboxanes to this list. Whereas prostaglandins are capable of eliciting many phenomena associated with inflammation, their effects on cyclic nucleotide metabolism may render these compounds antiinflammatory. Thus, the very cells that release mediators of inflammation provide a mechanism for modulating the inflammatory response.