The roles of degranulation and superoxide anion generation in neutrophil aggregation.

Human neutrophils when exposed to appropriate stimuli aggregate, generate O(2) and secrete lysosomal constituents. To determine whether a causal relationship may exist between these responses neutrophils were exposed to either N-formyl-methionyl-leucyl-phenylalanine, phorbol myristate acetate, or the two calcium ionophores, A23187 and prostaglandin Bx. Each agent elicited all of the above responses. The concentrations required to elicit the aggregation of 30 . 10(6) neutrophils/ml were comparable to that required for O(2) generation or lysozyme release. In a series of experiments designed to dissociate these responses, cells were suspended in a concentration too dilute (3 . 10(6) neutrophils/ml) to permit aggregation to occur. O(2) generation and lysozyme release was measurable and varied in a dose-dependent fashion to the concentration of stimulus. In a second series of experiments, neutrophils were treated with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid to inhibit degranulation without affecting O(2) generation. Aggregation was inhibited in a parallel fashion with lysozyme release. When detectable O(2) was removed from the medium by superoxide dismutase and catalase, aggregation and lysozyme release unaffected showing that aggregation can not be due to the presence of O(2) or its products in the extracellular medium. Neither aggregation of resting cells nor augmentation of fMet-Leu-Phe-induced aggregation was observed when cells were exposed to either supernatants of degranulated neutrophils or constituents of specific granules (lysozyme, lactoferrin). Kinetic analysis showed that in the absence of cytochalasin B degranulation preceded aggregation, while in its presence aggregation preceded degranulation.

Effects of non-steroidal anti-inflammatory agents on human neutrophil functions in vitro and in vivo.

Human blood neutrophils exposed to appropriate stimuli aggregate, degranulate and generate superoxide anion (O2-). These responses are anteceded by mobilization of membrane-associated calcium, monitored as a decrease in fluorescence of cells preloaded with chlortetracycline (CTC). We studied the effects, both in vitro and in vivo, of non-steroidal anti-inflammatory agents (aspirin, indomethacin, ibuprofen and piroxicam) on these neutrophil responses to three stimuli: a chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (FMLP); a tumor promotor, phorbol myristate acetate (PMA); and a lectin, concanavalin A (Con A). The effects of these drugs were compared with those of two polyenoic inhibitors of arachidonate metabolism: eicosatrienoic acid (ETI) and eicosatetraynoic acid (ETYA). The pattern of inhibition of neutrophil functions varied both with inhibitor and the nature of the stimulus. Thus, aspirin, piroxicam, ETYA and ETI inhibited neutrophil aggregation, degranulation, and O2- generation in response to FMLP, whereas ibuprofen inhibited only aggregation and degranulation and indomethacin only inhibited aggregation. None of the agents inhibited aggregation or degranulation induced by PMA or Con A: only piroxicam inhibited O2- generation in response to PMA or Con A. ETI and ibuprofen inhibited decrements of CTC fluorescence induced by FMLP, but whereas ETI inhibited the CTC response to PMA or Con A, ibuprofen was without effect. The agents had varying effects on binding of the stimulus [( 3H]FMLP, [3H]Con A), but these did not correlate with neutrophil responses to the ligands. Neutrophils from subjects taking therapeutic doses of ibuprofen, indomethacin, or piroxicam showed profiles of inhibited responses to FMLP similar to those observed with these agents in vitro. These data suggest that, although non-steroidal anti-inflammatory agents may inhibit discrete neutrophil functions both in vitro and in vivo, their effects do not duplicate those of polyenoic inhibitors of arachidonate metabolism. Moreover, since the susceptibility of neutrophils differed not only with respect to each inhibitor, but also to the stimulus, it is unlikely that all neutrophil responses are necessarily linked by a common pathway that is blocked by inhibitors of arachidonic acid metabolism.

Ceruloplasmin: an acute phase reactant that scavenges oxygen-derived free radicals.

Superoxide anion radicals have been implicated recently as mediators of inflammation and tissue injury. Protection from superoxide anion radicals is provided primarily by a copper-containing, intracellular enzyme (superoxide dismutase) (SOD) that catalyzes the dismutation of superoxide to hydrogen peroxide and oxygen. We have found that the action of cytoplasmic SOD to scavenge superoxide and thereby to inhibit superoxide-mediated reactions can be mimicked by the copper-containing plasma protein and acute-phase reactant, ceruloplasmin. Ceruloplasmin, at concentrations present in normal plasma, inhibited reduction of both cytochrome c and nitroblue tetrazolium (NBT) mediated by the aerobic action of xanthine oxidase on hypoxanthine (a superoxide-generating system). Ceruloplasmin neither inhibited formation of uric acid by xanthine oxidase nor accelerated autooxidation of cytochrome c. Furthermore, in an experimental system in which contact between ceruloplasmin and indicator was prevented by a relatively impermeable lipid membrane barrier, ceruloplasmin inhibited reduction of NBT trapped within liposomes exposed to xanthine oxidase and hypoxanthine. Ceruloplasmin also inhibited reduction of cytochrome c and NBT mediated by the aerobic action of xanthine oxidase on acetaldehyde (another superoxide-generating system) and mimicked the activity of purified human erythrocyte SOD by inhibiting photoreduction of NBT and by accelerating aerobic photooxidation of dianisidine. Ceruloplasmin could be separated from purified human erythrocyte SOD by electrophoresis on alkaline 12% polyacrylamide gels and identified by its superoxide-scavenging activity. These results suggest that ceruloplasmin may function as a circulating scavenger of oxygen-derived free radicals.

Neutrophil aggregation induced by sera from patients with active systemic lupus erythematosus.

Activated complement components and immune complexes cause neutrophil (PMN) aggregation in vitro and in vivo, as in dialysis-induced neutropenia and adult respiratory distress syndrome. To investigate the possible role of PMN aggregation in systemic lupus erythematosus (SLE), we studied the capacity of 59 sera from 53 patients to induce aggregation of normal PMN in vitro. Neutrophil aggregating activity (NAA) was present in the sera of 26 of 28 patients with active SLE. The mean NAA in this group was significantly greater than that found in 13 patients with inactive SLE, 20 patients with rheumatoid arthritis, and 17 normal controls. In patients with SLE there was a positive correlation between disease severity and the quantitative measure of NAA. NAA did not correlate with serum C3 or C4 nor with the presence or absence of circulating immune complexes. High levels of NAA were particularly characteristic of central nervous system lupus. These data suggest that the formation of intravascular leukoaggregates may contribute to morbidity in SLE.

A new function for ceruloplasmin as an acute-phase reactant in inflammation: a scavenger of superoxide anion radicals.

In summary, purified human ceruloplasmin inhibits several reactions mediated by superoxide anion in a fashion consistent with an ability to scavenge this free radical. It must be pointed out, however, that on a weight basis, the superoxide-scavenging activity of ceruloplasmin is substantially less than that of purified human erythrocyte superoxide dismutase. Nevertheless, since superoxide dismutase is almost exclusively an intracellular enzyme, ceruloplasmin probably represents the major circulating scavenger of superoxide anion radicals. The level of superoxide dismutase in human plasma has been reported to be 0.7 microgram/ml. It is not clear, however, how this was measured. We have found that concentrations of plasma exceeding 10% (v/v) interfere significantly with the assays routinely employed for detecting superoxide-scavenging activity. Consequently, we have not yet been able to quantify the superoxide-scavenging activity of either ceruloplasmin or superoxide dismutase in whole human plasma. Thus, we can only speculate that under conditions where levels of ceruloplasmin are markedly elevated, as during pregnancy, during acute infections, or in association with inflammatory diseases (such as rheumatoid arthritis), this acute-phase reactant may play a major role as a circulating scavenger of oxygen-derived free radicals.

Complement activation during systemic lupus erythematosus. C3a and C5a anaphylatoxins circulate during exacerbations of disease.

To determine whether activated complement components appear in the circulation of patients with systemic lupus erythematosus (SLE), we measured C5a and C3a by radioimmunoassay. Mean C5a concentration in the plasma of acutely ill SLE patients was 46.0 ng/ml, compared with 17.1 ng/ml in normal controls (P less than 0.01). Mean C3a concentration in patients with severe disease was 526 ng/ml, compared with 134 ng/ml in controls (P less than 0.01). In patients with moderately active SLE, the mean C3a concentration, but not the mean C5a concentration, was also elevated. In addition, C3a was elevated in 15 or 21 patients with active SLE, whereas low levels of C3 or C4 were noted in only 7 of these 21 patients. We conclude that the measurement of complement-derived anaphylatoxins may be useful in the management of patients with SLE. In addition, we suggest that these circulating mediators may contribute to the pathogenesis of vascular injury in patients with the disease.

Generation of C5-derived peptides and other immune reactants in the sera of patients with systemic lupus erythematosus.

Activated complement components and immune complexes cause neutrophil aggregation in vitro and in vivo. We have previously demonstrated that sera of patients with active systemic lupus erythematosus (SLE) provoke the aggregation of normal neutrophils in vitro. In this study the serum or plasma of 4 such patients was fractionated on Sephadex G-75. In 3 patients neutrophil aggregating activity (NAA) was detectable in fractions which coeluted with reference C5-derived peptides (estimated molecular radius of 17,000). The activity of these fractions was inhibitable by antibodies to human C5. All patients also had activity that coeluted with reference immune complexes. In addition, material of apparent molecular radius under 12,000 that contributed to the neutrophil aggregating activity of SLE sera was detected. In separate experiments increased levels of C5a desarg were demonstrated during active disease by means of radioimmunoassay. These findings suggest that multiple neutrophil aggregants circulate during the course of active SLE. The formation of intravascular leukoaggregates may contribute to endothelial injury in this disease.