Chemiluminescent response of polymorphonuclear leukocytes to Streptococcus pneumoniae and Haemophilus influenzae in suspension and adhered to glass.

We measured the luminol- and lucigenin-enhanced chemiluminescent response of human polymorphonuclear leukocytes (PMN) stimulated by various strains of Streptococcus pneumoniae and Haemophilus influenzae. In the absence of opsonin, phagocytosis of either bacterial species elicited good PMN response when the bacteria were adhered to a surface but minimal PMN response when they were in suspension. When 10% pooled human serum was used as a source of opsonin, a moderate to excellent chemiluminescent PMN response was elicited during phagocytosis of opsonized bacteria both in suspension and adhered to surface. We conclude that opsonin significantly enhances PMN chemiluminescence when a suspension-type assay is used and that opsonin-independent mechanisms play a significant role in the chemiluminescent response of PMN during phagocytosis of adherent bacteria.     

Concurrent lipopolysaccharide enhances chemotactic response of human polymorphonuclear leukocytes to bacterial chemotaxin

Polymorphonuclear neutrophil (PMN) function is thought to be critical in resistance to infectious agents and this implies that the PMN must be able to migrate into, and to function in, environments that may have high levels of bacterial lipopolysaccharide (LPS). Therefore, we have evaluated the effect of LPS on the in vitro migration of PMNs. Our data reveal that the human PMN is resistant to the deleterious effects of high levels of LPS, that in high concentrations LPS is, itself, a direct chemoattractant for PMNs, and that PMN migration toward a bacterial chemotaxin is enhanced if LPS is also present. Such capabilities suggest that the PMN may be uniquely qualified to migrate into microenvironments that are rich in LPS.     

Immunoglobulin enhances the bioactive-glass-induced chemiluminescence response of human polymorphonuclear leukocytes

Bioactive glasses are bone substitutes that chemically bind to bone. Implanted materials always elicit a response from surrounding tissues and thereby can activate inflammatory cells, with subsequent release of biomaterial and tissue-damaging agents. Bioactive glasses can activate polymorphonuclear leukocytes (PMNL) and induce a release of reactive oxygen metabolites (ROM). Adsorption of proteins on the surface of the implanted material may influence the subsequent inflammatory cell response. The effect of Sandoglobulin(R) (SG) and albumin on the ROM release by PMNLs induced by a bioactive glass was studied by a chemiluminescence (CL) assay. An enhanced effect for SG and inhibitory effect for albumin on the CL response of PMNLs was observable. The CL response of the PMNLs was dependent on the incubation time of the glass in solution.     

Bactericidal activity in fractionated granule contents from human polymorphonuclear leukocytes: studies with leukocytes from normal individuals.

Sephadex G-100 chromatographic fractions of granule extracts from normal human polymorphonuclear leukocytes, exhibiting differences from fractions previously obtained from leukemic polymorphonuclear leukocytes, possessed cationic proteins with distinct bactericidal activity against cell wall mutants of Salmonella typhimurium LT2.     

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.     

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.     

Human polymorphonuclear leukocytes produce cytokines in response to Leishmania major promastigotes

Polymorphonuclear leukocytes (PMN) release cytokines that may influence the development of the subsequent adaptive immune response. Little is known about cytokines produced by human PMN in response to Leishmania (L.). In this study, mRNA expression of Interleukin (IL)‐12p40, IL‐12p35, Interferon (IFN)‐γ, transforming growth factor (TGF)‐β, IL‐1, and IL‐4 in PMN of volunteers stimulated with L. major promastigotes has been investigated by real‐time PCR and the results were confirmed by flow cytometer. The results showed that L. major did not induce mRNA expression of IL12p40, IL12p35, IFN‐γ, and TGF‐β in PMN, while IL‐1 and IL‐4 mRNA were induced. Flow cytometry results confirmed no IFN‐γ production by PMN with or without stimulation. IL‐12p70 was present in untreated and L. major‐treated PMN, and these cells release IL‐12 following incubation with L. major. Significant amount of IL‐1 even without treatment with promastigotes was detected in PMN. Moreover, the proportion of PMN, which produce IL‐1 in response to L. major, was increased compared with the percent of unstimulated IL‐1‐producing PMN. The results showed the accumulation of small amounts of IL‐4 in PMN after stimulation. In conclusion, our results indicate that IL‐12 and IL‐1 are pre‐stored in human PMN, nor L. major induces IL‐1 and IL‐4, but not IL‐12, IFN‐γ, nor TGF‐β expression in these cells.     

Chemiluminescence response of polymorphonuclear leukocytes in atopic dermatitis

Atopic dermatitis (AD) is characterized by many signs of immunodeficiency. Our interest was to investigate if there are also alterations of the chemiluminescence (CL) response of polymorphonuclear leukocytes (PMN) as a measure of the release of toxic oxygen radicals. Isolated PMN of 13 patients with AD with mild to moderate disease activity were stimulated with a chemotactic peptide (f-met-phe), zymosan-activated serum (ZAS), zymosan particles and phorbolmyristate acetate. In the AD group, we found a significantly decreased response after stimulation with ZAS in comparison to the controls. With the other stimuli tested no significant difference was detected. The decreased response of PMN to stimulation with ZAS from patients with AD associated with a normal reactivity to the other stimuli could be due to specific desensitization of the PMN by C5a in vivo.     

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.     

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.     

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.     

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.     

Modulation of the inflammatory response by products released from human polymorphonuclear leukocytes during phagocytosis

During phagocytosis of latex particles human polymorphonuclear leukocytes (PMNs) release a product that generates chemotactic activity from fresh human serum. Release of this product is maximal at 20–30 min of phagocytosis. It is present in resting PMNs, may be recovered from granule fractions, and functions at physiologic pH. Gel-filtration chromatography of the activated serum indicates that C5a accounts for most of the chemotactic activity generated. Studies utilizing preparations of C5, EDTA, magnesium-EGTA, CS-deficient serum, and serum heated for 20 min at 50°C demonstrate that generation of C5a results from activation of the complement system as well as from direct cleavage of C5. Activation of the complement system by this PMN-derived serum activator appears to proceed through both the alternate and classical pathways. In addition to this serum activator, an inactivator of C5a chemotactic activity is also released by the PMNs under certain conditions of phagocytosis. These studies suggest that phagocytizing PMNs have secretory functions that contribute to the localization and amplification of inflammatory responses.     

Potential of human polymorphonuclear leukocytes to synthesize and secrete sulfated proteoglycans

To analyze the function of proteoglycans (PG) in different types of leukocytes, both the relative amounts and specific types of proteoglycans produced by cultured human peripheral blood polymorphonuclear leukocytes (PMN) were were determined and compared to mononuclear leukocytes (PBMC). Media from 3-day cultured PMN contained significantly less (less than 10%) 35SO2-4-labeled PG than media from PBMC cultures. Incorporation of 35SO2-4 into cell-associated material was comparable for both types of white blood cells. In contrast to PBMC, PMN could not increase their synthesis or secretion of PG after exposure to concanavalin A or phorbol-12-myristate-13-acetate. Various inducers of leukocyte chemotaxis also failed to enhance PG production by PMNs. Release of prelabeled PG from PMNs could be induced by exposure to either opsonized or unopsonized zymosan (yeast) as well as the bacteria S. aureus, suggesting that particle ingestion may be accompanied by PG exocytosis. Both chondroitinase ABC and AC digested greater than 90% of PMN 35S-labeled material in media and 75% in cell lysates; HNO3 treatment removed less than 5% of N-linked 35SO4 from radiolabeled media and 25% from cells. Treatment with 0.5 N NaOH released shortened glycosaminoglycan chains from 35S-labeled PMN cell lysates. beta-D-xylosides did not stimulate an increase in polysaccharide chain production by cultured PMNs. These data suggest that PMNs can produce chondroitin 4-sulfate PG whose synthesis is not affected by treatments that alter PMN functions; in contrast to PBMCs, PMNs will actively release these molecules when exposed to micro-organisms that stimulate phagocytosis.     

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.     

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.