Incubation Of PMN Research Articles

Improvement of polymorphonuclear leucocyte oxidative and bactericidal functions in chronic granulomatous disease with 4-amino-4'-hydroxylaminodiphenyl sulphone.

In an effort to restore oxidant-dependent capabilities to chronic granulomatous disease (CGD) polymorphonuclear leucocytes (PMN), we studied a dapsone derivative, 4-amino-4'-hydroxylaminodiphenyl sulphone (DDS-NOH), known to generate H2O2. After incubation of CGD PMN with 0.2 and 1.0 mM DDS-NOH for 30 min, the rate of glucose-1-14C oxidation via hexose monophosphate (HMP) shunt increased 2--4-fold and that of iodination of ingested zymosan particles 1.5--2.7-fold. Both effects could be further enhanced by superoxide dismutase (SOD) but inhibited by catalase. In three patients, 0.2 mM DDS-NOH improved in vitro killing of Staph. aureus. DDS-NOH 0.02 mM induced capping of Concanavalin A (Con A) receptor complexes suggesting interference by the drug with microtubule-associated function. Thus, optimal concentrations of DDS-NOH may be employed as an oxidant to improve metabolic and bactericidal functions of PMN from patients with CGD.

A specific inhibitor of complement (C5)-derived chemotactic activity in serum from patients with systemic lupus erythematosus.

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 patient's serum did not affect their subsequent random motility or chemotactic response toward normal zymosan-treated serum (ZTS). The patient's 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 patient's serum resisted heating at 56 degrees C for 30 min and could be separated from C5-derived chemotactic activity in the patient's ZTS (or normal ZTS that had been incubated with the patient's serum) by chromatography on Sephadex G-75. Despite its effect on C5-derived chemotactic activity, the patient's serum did not influence two other C5-derived biologic activities: PMN lysosomal enzyme-releasing activity and PMN-aggregating activity. Chromatography of the patient's 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.

591 BACTERIAL KILLING AND OXIDATIVE METABOLISM IN CHLORPRDMAZINE (CPZ) TREATED POLYMORPHONUCLEAR LEUKOCYTES (PMN): A MODEL FOR CHRONIC GRANULOMATOUS DISEASE

PMN from patients with CGD ingest and degranulate normally but do not produce superoxide ≥ or H2O2 in response to phagocytic stimuli. They kill streptococci but not staphylococci. CPZ mimics both the metabolic and bactericidal defects found in CGD PMN. Addition of 10-50 μM CPZ results in increasing inhibition of both ≥ and H2O2 production by PMN treated with opsonized zymosan or digitonin (DIG). CPZ both prolongs the lag time for maximal ≥ production and decreases the final linear rate. These effects cannot be reversed by washing the PMN in CPZ-free buffer. CPZ inhibits ZYM or DIG induced hexosemonophosphate shunt (HMP) activity but not resting on methylene blue induced HMP activity. Ingestion of opsonized particles and release of granular enzymes are unaffected by up to 100 μM CPZ. Incubation of PMN with DIG and CPZ results in undetectable NAD(P)H-dependent ≥ production in membrane fragments. CPZ inhibits NAD(P)H-dependent ≥ production in membrane fragments. 50 μM CPZ prevents the killing of staphylococci but not streptococci by human PMN. At 25 μM CPZ there is a delay in the killing of staphylococci. Thus, the metabolic, enzymatic, and bactericidal defects seen in PMN treated in vitro with CPZ are similar to those found in CGD PMN, and determination of site of CPZ attachment to PMN may aid in understanding the molecular basis of this disorder.

EFFECTS OF SURFACE ACTIVE AGENTS ON NEUTROPHIL RECEPTORS

An easily performed assay to identify the C3b and Fc receptor on human polymorphonuclear leukocytes (PMN) was developed. Salmonella typhi were directly fluoresceinated and then incubated in non-immune fresh human serum which led to C3b fixation via activation of the alternative pathway. Similarly, Type II pneumococci were fluoresceinated and opsonized with type specific rabbit anti-serum. PMN bearing C3b and Fc receptors formed rosettes with the respective bacteria which were easily readable because of their bright fluorescence. Studies employing various surface agents were employed to provide information on the physical properties of PMN receptors. Incubation of PMN at 37° C with C3-coated bacteria generated 54±5% C3b rosettes whereas PMN incubated with IgG coated bacteria yielded 78±8% rosettes. Heat inactivation of the fresh human serum at 56° C for 30 minutes completely inhibited the formation of the C3b and addition of goat anti-rabbit IgG inhibited the formation of the Fc rosettes. Pre-incubation of PMN with 1 mg/ml Trypsin, 1 mM N-ethyl maleimide, xanthine-xanthine oxidase to generate 20 nm superoxide anion and hydrogen peroxide (H2O2), 10 mM H2O2, and 8×10−4 M hydrocortisone reduced the number of C3b rosettes to values of 25±6%, 7±6%, 22±4%, 36±2%, and 24±5%, respectively, but had no significant effect on the number of Fc rosettes. Thus, this study provides evidence for selective receptors for C3b and Fc on PMN membranes.

Mechanisms of release of leucocytic pyrogen

Mechanisms of the production and release of leucocytic pyrogen (LP) from peritoneal polymorphonuclear leucocytes (PMN) from rabbits were studied in vitro. The following results were obtained. 1) The fever curves of the microsomal and the lysosomal fractions had a later onset and were longer lasting than those from the extracellular fluid. The fever curves of the supernatant of 105,000 g showed the typical response of endogenous pyrogen characterized by a rapid onset and short lasting fever, as shown by the extracellular fluid. 2) The pyrogenicity of LP was the most potent at 60 min while the 105,000 g supernatant was most potent at 30 min after PMN incubation at 37 degrees C. 3) The extracellular protein attained the maximum level at 30 min after incubation of PMN, while the protein content in the supernatant of 105,000 g of PMN decreased gradually to the constant level at 30 min. 4) It was observed that the lysosomal degradation was stimulated with bacterial pyrogen (LPS) at 37 degrees C but LPS did not directly affect the lysosomal fraction of PMN.