Microbial killing by human neutrophil cytokineplasts: similar suppressive effects of reversible and irreversible inhibitors of nitric oxide synthase.

Employing anucleate, granule-poor, motile fragments from human blood neutrophils (cytokineplasts; CKP), we previously provided evidence for a new staphylococcal killing pathway for human neutrophils involving reactive nitrogen intermediates: the NO synthase inhibitor N(omega)-monomethyl-L-arginine (NMMA), an analogue of L-arginine (L-Arg), substantially decreased the killing capacity of CKP for Staphylococcus aureus (Staph), an effect reversible by excess L-Arg but not D-Arg. We have extended these findings to two irreversible NO synthase inhibitors: the first, N-iminoethyl-L-ornithine (L-NIO), is an L-Arg analogue; the other, diphenyleneiodonium (DPI), is not. After 60 min of incubation with bacteria, despite having taken up somewhat fewer staphylococci than did controls, cytoplasts treated with NO synthase inhibitors had many more live, CKP-associated bacteria: for NMMA, 6.9 times more (40.0% of the inoculum vs. 5.8%; n = 8, P = 0.003); for L-NIO, 3.6 times more (25.5 vs. 7%; n = 4, P = 0.004); for DPI, 5.8 times more (37.4 vs. 6.4%; n = 7, P = 0.002). Results were similar after only 20 min of incubation. In two experiments in which the Gram-negative bacterium, Serratia marcescens, was employed instead of Staph, the results were again similar. In contrast, killing of either bacterium by intact neutrophils (PMN) was not inhibited by NMMA, by L-NIO, or by DPI, a failure most likely attributable to their granule content. The irreversible inhibitors of NO synthase will be especially useful in analyzing particular effects on CKP employed in multicellular systems.

Cytoplasts generate oxidants but require added neutrophil granule constituents for fungicidal activity against Candida albicans hyphae.

Killing of Candida albicans hyphae requires oxidant generation by neutrophils (PMNL), but it is uncertain whether hyphal killing is mediated by PMNL oxidants alone or requires contributions by granule constituents. This was assessed using U-cytoplasts (U-CYT), anucleate PMNL fragments depleted of cytoplasmic granules but retaining motility and respiratory burst activity. Granule-depleted U-CYT killed Staphylococcus aureus, but hyphae remained viable despite targeted generation of putatively fungicidal oxidants by avidly adherent U-CYT. Hyphal killing occurred by combining U-CYT with sublethal concentrations of purified PMNL granule extracts approximating those present in equivalent numbers of intact PMNL. Contributions of granule constituents were not entirely attributable to purified granule constituents with known antimicrobial activity (lactoferrin, cathepsin G, myeloperoxidase, and human neutrophil peptide defensins 1-3) individually or combined. Thus, oxidant generation by intact PMNL may be necessary but not always sufficient to mediate hyphal killing without complementary nonoxidative mechanisms.

Chronic granulomatous disease and glutathione peroxidase deficiency, revisited.

We have restudied two kindreds that formed the basis of the original report of autosomal recessive chronic granulomatous disease (CGD) associated with leukocyte glutathione peroxidase deficiency. Case 1 from the original study and the surviving brother of the originally reported case 2 both have severe CGD, with no detectable respiratory burst activity in purified intact neutrophils. However, their leukocytes exhibit normal glutathione peroxidase enzyme activity and gene expression. Examination of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase components known to be defective in CGD reveals no detectable cytochrome b558 nor any membrane activity in a cell-free NADPH oxidase assay system. Molecular analysis of the genes encoding cytochrome b558 subunits shows, in case 1, a C-->T substitution at nucleotide 688 of the gene encoding the gp91-phox subunit of cytochrome b558, resulting in a termination signal in place of Arginine-226. Levels of gp91-phox mRNA are markedly decreased despite normal levels of gene transcription, indicating a post-transcriptional effect of the nonsense mutation on mRNA processing or stability. The X-linked form of CGD developed in this cytogenetically normal female due to the uniform inactivation of the normal X chromosome in her granulocytes, indicated by the expression in her granulocyte mRNA of only one allele of a glucose-6-phosphate dehydrogenase polymorphisms for which she is heterozygous in genomic DNA. Case 2 (of the present study) has distinct mutations in each allele of the p22-phox gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytokineplasts and U-cytoplasts from human polymorphonuclear leukocytes: role of granule-poor motile fragments in the analysis of cell physiology.

We have been using granule-poor anucleate fragments--cytokineplasts and U-cytoplasts--released from human peripheral blood polymorphonuclear leukocytes (granulocytes; PMN) to study cell functions that bear on aspects of the inflammatory response. The work is particularly aimed at the relationships among specific but overlapping areas of leukocyte activity: adherence, locomotion, target recognition, chemotaxis, penetration of endothelial monolayers, ingestion, the increased metabolic activity that ordinarily accompanies phagocytosis or other cell activation processes, degranulation of lysosomal structures, and intracellular killing. The ways in which these activities can be separated from one another may distinguish obligate interactions from mere concomitance, and may reveal the specific pathways by which cell function is altered. We have found that cytoplasts provide a unique way of looking at the composition and function of the cell's motile and killing machinery, in greatly simplified systems.

Evidence for reactive nitrogen intermediates in killing of staphylococci by human neutrophil cytoplasts. A new microbicidal pathway for polymorphonuclear leukocytes.

In anucleate, granule-poor, motile fragments from human blood neutrophils (cytokineplasts; CKP), the nitric oxide synthase inhibitor N omega-monomethyl-L-arginine (NMMA) produced a modest decrease in uptake of staphylococci from supernatants (P less than 0.02, n = 7), and a marked decrease in the killing of cytoplast-associated bacteria (P less than 0.001, n = 7). After 60 min of incubation with bacteria, NMMA-treated cytoplasts had a mean of over 3.5 times as many live, CKP-associated staphylococci as did controls (51% of the inocula versus 14%), despite having taken up fewer. Effects on both uptake and killing were reversible by L-arginine but not by D-arginine. Results were the same with other granule-poor cytoplasts (U-cytoplasts, U-CYT), which, unlike CKP, retain activatable oxidase activity. Killing by intact PMN, including those from a patient with chronic granulomatous disease, was not inhibited by NMMA. Thus, the ability to discern effects of NMMA correlated with the paucity of granules, without regard to the presence or absence of activatable oxidase. We propose that the generation of reactive nitrogen intermediates serves as an additional microbial killing pathway in PMN, and that cytoplasts can be used to help delineate the spectrum of susceptible targets.

Identification of a thermolabile component of the human neutrophil NADPH oxidase. A model for chronic granulomatous disease caused by deficiency of the p67-phox cytosolic component.

Mild heating of human neutrophils inactivates the respiratory burst oxidase, producing a defect in superoxide production and bacterial killing comparable to that seen in patients afflicted with chronic granulomatous disease (CGD). We have now investigated the mechanism and specificity of this inactivation by examining the effect of mild heating on the known oxidase components: the membrane-bound subunits of the cytochrome b558 (gp91-phox and p22-phox) and the two cytosolic oxidase factors (p47-phox and p67-phox). Heating (46 degrees C for 7.5 min) caused intact neutrophils to lose greater than 85% of their capacity to produce superoxide, a defect which was localized to the cytosolic, but not the membrane, fraction. Complementation studies with CGD cytosols deficient in either p47-phox or p67-phox suggested that the defective component of heat-inactivated cytosol was p67-phox. This was confirmed by experiments showing that recombinant p67-phox, but not p47-phox, exhibited lability at 46 degrees C and completely reconstituted oxidase activity of heat-treated cytosol. These studies indicate that mild heating of either intact neutrophils or normal neutrophil cytosol results in a selective inactivation of p67-phox, providing a model oxidase system for the extremely rare p67-phox-deficient form of CGD.

Cryopreservable neutrophil surrogates. Stored cytoplasts from human polymorphonuclear leukocytes retain chemotactic, phagocytic, and microbicidal function.

Cryopreservation of polymorphonuclear leukocytes (PMN) has largely failed, probably because of their rich content of granular (lysosomal) enzymes. We have been developing granule-poor cytoplasts (anucleate fragments) from PMN which retain motile functions of the parent cell. The two types studied here were induced either by brief heating on surfaces (cytokineplasts) or by discontinuous gradient centrifugation (Ficoll) without heat or drugs (U-cytoplasts). Freshly made, these cytoplasts respond chemotactically to formyl peptide (fMet-Leu-Phe), and they take up and kill roughly half as many Staphylococcus aureus as their (larger, granular) parent PMN. Unlike their parent cells, after cryopreservation both cytoplasts remain chemotactic, and in matched experiments they take up and kill staphylococci with undiminished avidity. These findings are the first indications that PMN cytoplasts suitable for clinical use may be feasible.

Cytoplasts made from human blood polymorphonuclear leukocytes with or without heat: preservation of both motile function and respiratory burst oxidase activity.

Anucleate fragments (cytoplasts) from polymorphonuclear leukocytes (PMN) are simplified systems that can be used to elucidate specific pathways by which cell function is altered. PMN cytoplasts in current use are defective either in activatable respiratory burst oxidase activity or in motile function. By centrifugation of PMN on discontinuous gradients of Ficoll without cytochalasin B, we have created granule-poor cytoplasts in which both these capacities are preserved. Specifically, they generate superoxide anion (O2-.) and reduce nitroblue tetrazolium dye on appropriate stimulation; they respond chemotactically to erythrocytes destroyed by laser microirradiation or to the specific chemoattractants fMet-Leu-Phe (10 nM) and C5a (zymosan-activated serum); and they ingest and kill staphylococci. We can improve the yield of these fragments progressively by preheating (45 degrees C) the cells in suspension for increasing periods of time, but those treatments are attended by a decreasing percentage of cytoplasts with activatable oxidase activity, and a progressive inability of the cytoplasts to ingest and to kill staphylococci. These easily made and multipotent cytoplasts readily lend themselves to studies of PMN physiology.

Phagocytic capacity of cytokineplasts from human blood polymorphonuclear leukocytes.

Cytokineplasts (CKP) are membrane-bounded anucleate cytoplasmic fragments, induced from polymorphonuclear leukocytes (PMN) by the brief application of heat; derived from the cortical cytoplasm that gathers at the leading front of migrating PMN; and endowed with many of the motile properties of the parent cell. In this study we examine their phagocytic capacity by quantitative methods. CKP ingest Staphylococcus aureus and Serratia marcescens somewhat less avidly than do the corresponding intact PMN, yet rather impressively when one considers how restricted a portion of the parent cell they represent. Under the conditions employed, CKP killed about half as many of the bacteria presented to them as did their parent PMN. Thus, despite a heat-associated loss of demonstrable respiratory burst oxidase activity and a paucity of cytoplasmic granules, the organelle-depleted CKP deals with bacteria in a way that mimics its parent PMN.

Functional integrity of cytokineplasts: specific chemotactic and capping responses.

Cytokineplasts (CKP) are motile, membrane-bound, anucleate, granule-poor cytoplasmic fragments that are induced from human blood polymorphonuclear leukocytes (PMN) by the brief application of heat. We examined CKP with respect to specific chemotactic and capping responses, the presence of the N-formyl-peptide chemotactide receptor, and evidence of respiratory burst activity and compared them with CB-cytoplasts, which are fragments created by the centrifugation of cytochalasin B (CB)-treated PMN at high speeds. Under agarose, CKP responded chemotactically to both N-formyl-methionyl-leucyl-phenylalanine (fmlp) and zymosan-activated serum; CB-cytoplasts responded to neither chemoattractant. Despite the functional differences, both fragments retained N-formyl-peptide receptors as measured by affinity labeling with N-formyl-norleu-leu-phe-norleu-125I-tyr-lys and autoradiography of dried SDS-PAGE gels. For studies of capping we used a murine monoclonal antibody, PMN7C3, which binds a specific, widely distributed membrane component of intact PMN, and on warming, promptly induces capping of ligand-receptor complexes. Rhodamine-conjugated PMN7C3 at 4 degrees C labeled the surface of CKP homogeneously. As the CKP warmed to 37 degrees C, label became concentrated in small fluorescent caps at the rear of migrating fragments. Although CB-cytoplasts also bound the fluorochromed antibody homogeneously in the cold, on warming they were unable to concentrate the label normally. With respect to respiratory burst activity, the situation in the two fragments was reversed: CKP did not generate superoxide anion when stimulated either with phorbol myristate acetate or with fmlp after pretreatment with CB; CB-cytoplasts, as noted earlier by other investigators, did. These two types of cytoplasts with markedly different capabilities have complementary roles in the analysis of PMN function.

Cytokineplasts from human blood polymorphonuclear leukocytes. Lack of oxidase activity and extended functional longevity.

Cytokineplasts (CKPs) are membrane-bounded, anucleate, granule-poor cytoplasmic fragments, induced from PMNs by brief heat (45 degrees C, 9 min), which retain motile function including chemotaxis and phagocytosis. CKPs can respond to repeated chemotactic stimuli even after having been held overnight at room temperature, and hence "outlive" control PMNs. We now report that adherent CKPs lack significant oxidase activity, as measured by reduction of nitroblue tetrazolium (NBT) dye, (1) 5 min after heat, when they are often still attached to their parent PMNs (which generally do not reduce NBT either); (2) later on, when they are free; and (3) when cells have been pretreated on endotoxin-coated substrata or with phorbol myristate acetate (PMA); both pretreatments cause the large majority of adherent control PMNs to reduce NBT. Moreover, cells harvested from glass just after heat lack the normal increase in oxygen consumption seen on stimulation with PMA or with heat-killed staphylococci. PMA-stimulated respiratory burst activity was not restored to heated cells by exogenous NADPH. Thus, heat applied to normal PMNs can dissociate motile function from oxidase activity; in this respect CKPs resemble PMNs in chronic granulomatous disease. The apparent increased functional stability of CKPs may indicate that normal PMNs are not immune to their own oxidative killing mechanism.