Microtubule-granule relationships in motile human polymorphonuclear leukocytes

We examined the relationship of microtubules to the granule organization in stimulated human polymorphonuclear leukocytes (PMNs). Electron microscopic (EM) observations of critical-point-dried PMNs revealed that only a portion of the granules appeared in close association to microtubules. These closely associated granules appeared to be attached to the microtubule via smaller-diameter filaments. The remaining granules appeared either attached to microtubules at a further distance, via smaller-diameter filaments such as actin, or unassociated with microtubules. EM observations of PMNs treated with either the microtubule promoter drug taxol or the microtubule depolymerization drugs nocodozole and colchicine revealed a redistribution of granules towards the nucleus. Granule clustering at the periphery of the cell was also noted with nocodozole and colchicine. With cytochalasin B, a uniform distribution of granules was noted. However, granule clustering was noted when PMNs were coincubated with cytochalasin B and colchicine. These results indicate that microtubules may have both a direct and indirect role (through other cytoskeletal elements) in the organization of PMN granules.     

Cytokineplasts from human blood polymorphonuclear leukocytes

Cytokineplasts (CKPs) are membrane-bounded, anucleate, granule-poor cytoplasmic fragments, induced from PMNs by brief heat (45°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 outiive 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.This work, part of which has appeared in abstract (26), was supported in part by the U.S. Public Health Service (AM-10493, AM-19742, AM-07107) and by the Arthritis Foundation and its Connecticut Chapter.     

Immunofluorescence localization of calmodulin in human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMNs) were purified (approximately equal to 99%) from peripheral blood of normal, adult volunteers. The indirect immunofluorescence technique was used to investigate the presence and the localization of calmodulin in human PMNs. The cellular distribution of calmodulin has been evaluated using an affinity chromatography-purified sheep IgG anti-calmodulin and fluorescein-conjugated rabbit anti-sheep IgG. The anti-calmodulin immunofluorescence pattern suggests that calmodulin is evenly distributed throughout the cytoplasm of human PMNs.     

Effects of N-acetylcysteine on human polymorphonuclear leukocytes

N-acetylcysteine (NAC) at concentrations from 0.39 micrograms/ml to 100 micrograms/ml did not affect chemotaxis under agarose of human polymorphonuclear leukocytes (PMNLs). No reduction of phagocytic or bactericidal capacity was found in PMNLs exposed to NAC at the same concentrations. At high concentrations of NAC (25-100 micrograms/ml) a distinct inhibition of the chemiluminescent response to formylmetionyl-leucyl-phenylalanine (fMLP) known to be associated with mainly extracellular metabolic processes, was observed, consistent with the well known scavenger effects of the drug. The response to opsonized zymosan, which reflects mainly intracellular metabolic activity, was less marked. At a still higher concentration of NAC (500 micrograms/ml), a distinct effect on both intra- and extracellularly generated chemiluminescence could be demonstrated. The lack of inhibitory effects on phagocytosis and intracellular killing in spite of the effects on chemiluminescence indicates that NAC has no negative influence on the antimicrobial activity of PMNLs.     

Release of prostaglandins from human polymorphonuclear leukocytes

Human PMNs release prostaglandins E and F to the surrounding medium when these cells are exposed to zymosan. PGE₁ is the prostaglandin compound found in highest concentration in the medium, and the PGE/PGF balance is approximately 3∶1. Release of prostaglandins is not due to platelet contamination. Agents which inhibit prostaglandin synthesis (indomethacin, aspirin) prevent release of prostaglandins from phagocytic cells. Addition to cells of dibutyryl cyclic 3′,5′-adenosine monophosphate produces striking increases in concentrations of prostaglandins released during ingestion of zymosan. Prostaglandins appear to be synthesized by human PMN during phagocytosis, and their release from cells may help regulate the inflammatory response.     

Oxidation of glucosamine by human polymorphonuclear leukocytes

When exposed to a phagocytic stimulus (opsonized zymosan), human polymorphonuclear leukocytes (PMNs) produced¹⁴CO₂ from [1-¹⁴C]glucosamine at a rate 10–25% of that produced from glucose under the same conditions. The production of CO₂ from glucosamine by intact PMNs was inhibited by glucose and dependent upon activation of the hexosemonophosphate shunt (HMPS). However, the metabolic pathways for the oxidation of glucose and glucosamine by PMNs are not identical. This is suggested by the fact that glucose-6-phosphate dehydrogenase, the initiating enzyme for the HMPS, did not utilize glucosamine-6-phosphate as a substrate. In addition, glucosamine was not oxidized by sonically disrupted PMNs whereas oxidation of glucose by the same preparation was increased sevenfold over intact cells. Taken together, the data suggest that PMNs oxidize glucosamine by converting it to a compound compatible with the enzymes of the HMPS. This conversion requires intact PMNs and/or an as yet unidentified cofactor.     

Interleukin-12 production by human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMN) stimulated by lipopolysaccharide (LPS) produce interleukin-12 (IL-12). Both the free IL-12 p40 chain and minute amounts of the biologically active IL-12 p70 heterodimers are produced by PMN. Interferon-γ (IFN-γ) enhanced the LPS-induced secretion of both the free IL-12 p40 chain and the p70 heterodimer by approximately fivefold. As observed for other IL-12-producing cell types, the ratio of free p40 chain to p70 heterodimer secreted by LPS-stimulated PMN was approximately 20:1. LPS induced a 100-fold increase of IL-12 p40 mRNA, but had minimal effect on p35 mRNA accumulation, IFN-γ enhanced the LPS-induced accumulation of p40 mRNA and directly induced a several-fold increase in the accumulation of p35 mRNA. Therefore, the combined effect of LPS and IFN-γ induced sufficient expression of both p40 and p35 to attain production of the biologically active p70 heterodimer at physiologically relevant concentrations. The ratio between p40 and p35 mRNA abundance in PMN stimulated with both LPS and IFN-γ was approximately 200:1, explaining the secretion of the free p40 chain in much higher concentrations than the p70 heterodimer. IL-10, an inhibitor of the production of various cytokines in PMN, also suppressed IL-12 mRNA accumulation and secretion by PMN. Because of the important immunoregulatory function of IL-12, in particular induction of IFN-γ production and facilitation of T helper cell type 1 response, the ability of PMN to produce IL-12 suggests that neutrophils may play an active role in the regulatory interaction between innate resistance and adaptive immunity.     

Chemiluminescent response to pathogenic organisms: normal human polymorphonuclear leukocytes

Chemiluminescence (CL) is a sensitive indicator of phagocytosis and intracellular killing; however, little is known of the normal CL response by human polymorphonuclear leukocytes to different pathogenic microorganisms. We investigated the luminol-enhanced CL response of normal polymorphonuclear leukocytes to a number of common bacterial pathogens and two yeasts. We analyzed the CL response to viable and heat-killed microorganisms at 25 and 37 degrees C. The CL response to all microorganisms was greater and more rapid at 37 degrees C. Variable responses were observed with viable and heat-killed microorganisms; some were unaffected, whereas other demonstrated reduced CL. Each microorganism caused a reproducible response pattern, which could be placed into two general categories. In the first category were those which caused a rapid exponential rise and decay in CL: Enterobacter cloacae, Salmonella typhimurium, Shigella flexneri, Staphylococcus aureus, Candida albicans, and zymosan. In the second category were those which rose slowly over a longer time course to a poorly defined peak: Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, and Streptococcus pyogenes. The CL response also reflected serum opsonic activity. The effect of inactivated complement, factor B, and removal of specific antibody were investigated. Increasing the concentration of zymosan gave a proportional rise in peak CL; however, a strain of E. coli caused a variation in peak time rather than peak height. Different CL kinetics were shown for three strains of K. pneumoniae, possibly a result of each having different membrane or cell wall characteristics. This study defines the nature and factors affecting the normal CL response to a variety of common pathogenic microorganisms.     

The cytoskeleton of human polymorphonuclear leukocytes: phagocytosis and degranulation

Current evidence indicates that polymorphonuclear leukocyte (PMN) chemotaxis and phagocytosis are effected by an actin-myosin contractile system. However, the structural relationship of the contractile cytoskeleton to cell motility is still in question. In addition, while evidence suggests that microtubules are responsible for orientation during chemotaxis, the role of microtubules in degranulation is unresolved. To determine the organizational relationship between these cytoskeletal elements and phagocytosis, we examined whole-mount preparations of PMNs engulfing bacteria. These preparations were examined in the transmission electron microscope (EM) and photographed as stereo pairs. Two important observations were made. First, there was an increased density of cytoskeletal elements in the pseudopod surrounding bacteria. Second, microtubule elements were intimately associated with lysosomal granules, vesicles, and phagosomes. Lysosomal granules and vesicles aligned along microtubules and clustered around phagosomes. This suggests that the microtubules may provide a tracking mechanism whereby lysosomes are specifically parceled out to phagocytic vacuoles. These results also suggest that phagocytosis and degranulation may involve different effector mechanisms.     

The organization of actin filaments in human polymorphonuclear leukocytes

Actin constitutes a major component of the cytoskeleton of human polymorphonuclear leukocytes (PMNs). In this study, we present a comprehensive view of the organization of actin in various PMN regions and functional states. Transmission electron microscopic observations were made on whole mount, migrating, and phagocytizing PMNs. Positive identification of actin filaments was made through S-1 myosin subfragment labeling. In all PMNs studied, actin filaments were primarily organized as a three-dimensional meshwork. The density of this meshwork was greatest within the cell cortex. At peripheral regions of nonpolarized (viz., no distinct head or tail region) and polarized PMNs, actin filaments organized into parallel bundles or overlapping arcs. These bundles or arcs were oriented either perpendicular or parallel to the cell periphery. At the base of the PMN, actin filaments converged upon dense, plaquelike condensations. This latter pattern of actin organization was also observed in some pseudopods at the cell front and in phagocytic processes engulfing bacteria. In areas of internalized bacteria, the surrounding actin appeared as a loose meshwork. Treatment of PMNs with the antiactin drug, cytochalasin B, revealed shearing of the peripheral actin meshwork, condensation of the meshwork around the nuclear region, and dissolution of the basal plaquelike condensations.     

Entamoeba histolytica depresses chemiluminescence in stimulated human polymorphonuclear leukocytes

Effect of Entamoeba histolytica proteinase/toxin (Ehp/t) on the luminol-dependent chemiluminescence (CL) in stimulated human polymorphonuclear leukocytes (PMN) was studied. The role of superoxide (SO) and hydroxyl (OH) anions in the Ehp/t-associated enhancement/inhibition of CL was also studied using specific scavengers and a biological response modifier, muramyldipeptide (MDP). Ehp/t was isolated from axenic trophozoites of the HM-1:IMSS strain of virulent strain of E. histolytica. Proteinase activity was assayed on a synthetic substrate, Z-arg-arg-AFC and cytotoxicity was tested on HeLa cell monolayers. PMN isolated from blood were incubated with Ehp/t prior to stimulation by phorbol myristateacetate (PMA, 2 micrograms/ml), serum-treated zymosan (2.5 mg/ml) and glucan (2 mg/ml). CL was monitored in an LKB (Wallac) Luminometer. Ehp/t was found to depress up to 90% of CL induced by PMA, glucan and zymosan. Such a depression was Ehp/t concentration-dependent. A 150 micrograms/ml concentration of Ehp/t, obtained from a 0.015-1.5 mg/ml concentration range tested at different incubation times and temperatures, was used in most of our experiments. Incubation time and temperature optima were 15 min and 37 degrees C, respectively. Ehp/t partially inhibited the CL associated with SO and OH. MDP, in the presence of Ehp/t, enhanced CL response in human PMN to about 67% with reference to normal CL without inhibitor. PMN were confirmed to play a vital role in amebic tissue invasion mechanisms.     

Identification of calcium-dependent proteolytic activity in human polymorphonuclear leukocytes

Calcium-dependent proteolytic activity has been identified in extracts of human polymorphonuclear leukocytes. The activity is most pronounced in the neutral pH range with a pH optimum of 7.3. Maximal activation of the protease occurs at a free calcium concentration of 190 microM; it is half maximal at 91 microM. This protease activity is strongly inhibited by aprotinin and phenylmethylsulfonyl fluoride (PMSF) and more weakly inhibited by antipain, leupeptin, and o-phenanthroline. The protease is not activated by calmodulin nor is it inhibited by the calmodulin antagonist trifluoperazine. Gel filtration suggests a molecular weight of 74,100 daltons.     

Histamine,dithiaden and human polymorphonuclear leukocytes in vitro

Inflammation Research -     

Chemotaxis of human polymorphonuclear leukocytes toward Neisseria gonorrhoeae

Chemoattractive properties of Neisseria gonorrhoeae were studied by measuring leukocyte migration in agarose gel. Human serum albumin (0.5%) was present in the gel and normal human serum was excluded from all components of the assay. Viable cell populations and lysates of colonial types F62T1, F62T2 and F62T3 induced migration of polymorphonuclear leukocytes. Chemotactic activity of the lysate was not altered by heating at 100 degrees C for 10 min and was retained in the 12 100 g supernatant fraction of the heated lysate. Fractionation of the supernate by Sephadex G-100 chromatography showed that the chemotactic activity was associated primarily with an absorbance peak at 280 nm of relatively low mol. wt. The chemotactic activity of this fraction was lost after dialysis and the peak was no longer present in the Sephadex G-100 elution profile of the dialysed supernate. The gonococcal leukotaxins were sensitive to digestion by trypsin, pronase and amyloglucosidase, but insensitive to treatment with RNAase, DNAase or lipase at pH 5.7-7.1.     

Oxidation of 2-deoxyglucose by human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMNL) can metabolize [l-¹⁴C]2-deoxyglucose to¹⁴CO₂ when stimulated by either phorbol myristate acetate (PMA) or opsonized zymosan. Oxidation of 2-deoxyglucose is about 10–20% as efficient as that of glucose in normal PMNL; it does not occur in defective cells obtained from patients with chronic granulomatous disease. The increased oxidation of [ l-¹⁴C]2-deoxyglucose induced by PMA is not sufficient to explain the inhibition of transport induced by that compound; conversely increased transport of 2-deoxyglucose induced by zymosan-activated serum does not result in a significant increase in oxidation of the hexose. Oxidation of [l-¹⁴C]2-deoxyglucose appears to be mediated by the hexose monophosphate shunt as indicated by the following (1) oxidation of [1-¹⁴C]2-deoxyglucose in intermediate in activity between that of [l-¹⁴C]glucose and [6-¹⁴C]glucose; (2) the reaction is insensitive to cyanide or azide; and (3) shunt enzymes measured in a cell-free extract from human PMNL can react with 2-deoxyglucose compounds with approximately 10% the efficiency shown towards the corresponding glucose derivatives.     

Receptor-mediated internalization of tuftsin by human polymorphonuclear leukocytes

A high-performance liquid chromatography (HPLC) purified fluorescein-labeled analogue of tuftsin was prepared, which retains the full biological activity of the native molecule. Characterization of the derivatization site by amino acid analysis, N-terminal cleavage, and dansylation revealed a monofluorescinated derivative at the alpha-amino terminus. Binding of the fluorescent tuftsin to living polymorphonuclear leukocytes (PMN) was observed by means of video intensification microscopy. At 37 degrees C, diffuse membrane fluorescence was seen initially, followed by rapid aggregation and internalization. The latter was demonstrated by saltation of intracellular fluorescent aggregates. These processes are temperature-dependent and rely on specific binding to the tuftsin receptor.     

Controlling human polymorphonuclear leukocytes motility using microfabrication technology

We describe a new approach for controlling cell motility on a material surface. Transparent, photosensitive polyimide materials were used to fabricate physical structures on glass; cell motility was then followed over time using optical microscopy. Arrays of pillars and holes with 2 micron square, 4-microm height (or depth) separated by 10 microm were successfully patterned using photolithography. Neutrophils attached and spread on the smooth glass surface and surfaces with pillars. In contrast, cells were rounded and did not adhere to either smooth polyimide film or films with holes. The migration of neutrophils was much faster on holes than on polyimide surface, but it was significantly slower on pillars than on glass. These results suggest that physical patterning may be an effective tool to manipulate cell migration in the design of biomaterials for tissue engineering.