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Polymorphonuclear neutrophils (PMNs) can contribute to the regulation of the host immune response by crosstalk with innate and adaptive leukocytes, including NK cells. Mechanisms by which this immunoregulation process occurs remain incompletely understood. Here, we focused on the effect of human neutrophil-derived serine proteases on NKp46, a crucial activating receptor expressed on NK cells. We used flow cytometry, Western blotting, and mass spectrometry (MS) analysis to reveal that cathepsin G [CG; and not elastase or proteinase 3 (PR3)] induces a time- and concentration-dependent, down-regulatory effect on NKp46 expression through a restricted proteolytic mechanism. We also used a functional assay to demonstrate that NKp46 cleavage by CG severely impairs NKp46-mediated responses of NK cells, including IFN-γ production and cell degranulation. Importantly, sputa of cystic fibrosis (CF) patients, which have high concentrations of CG, also alter NKp46 on NK cells. Hence, we have identified a new immunoregulatory mechanism of neutrophils that proteolytically disarms NK cell responses.

Because of uncertainties regarding the comparability of granulocyte-macrophage and granulocyte colony-stimulating factors with regard to their effects on mature neutrophils (PMNs), we compared the actions of the two cytokines on reactive oxidant production and granular secretion by these cells. We found that chemiluminescence (CL) stimulated by formylmethionyl-leucylphenylalanine (fMLP) was not influenced by G-CSF (0.1–100 ng/ml), whereas GM-CSF priming (10 ng/ml) caused a nearly twofold increase in this PMN response. Moreover, the reactivity of PMNs treated with GM-CSF and G-CSF in combination was not different from that of PMNs treated with GM-CSF alone. GM-CSF (10 ng/ml) increased the rate of O2- production by 79%, caused a fivefold increase in fMLF-induced myeloperoxidase (MPO) secretion, and strongly enhanced CD11b expression. In contrast, G-CSF (50 ng/ml) only slightly increased O2- production (by 15%), and MPO secretion and CD11b expression remained unchanged. Both cytokines together gave results similar to those obtained with GM-CSF alone. In the presence of platelets (which by themselves enhanced PMN reactivity), the differences in the effects of the two cytokines persisted. We conclude that the priming effect of G-CSF on mature PMNs is negligible compared with that of GM-CSF. Our results are in conflict with previous reports of much more pronounced G-CSF effects but in accord with recent work showing the failure of this cytokine to induce a range of effects produced by GM-CSF. We therefore suggest that the primary role of G-CSF in mature PMN function is still unclear but may be related to the control of PMN distribution in view of the mobilizing and marginating effects of the cytokine in viva J. Leukoc. Biol. 55: 612–616; 1994.

The effects of leukotriene (LT) B4 and D4 on the motility of human peripheral blood neutrophils were investigated employing a novel analytical method. Using the under-agarose technique, migration distance and vectorial orientation of neutrophils in response to selected LT concentrations were determined with the aid of digital image processing. Neutrophil polarization induced by a chemotactic gradient was very apparent even at fields taken adjacent to the cell seeding well where little directional cell motility had occurred. Thus, cell polarization appeared to be the earliest response to chemoattractive LTs. Cell motility occurred in a dose-dependent manner to LTB4 according to determination of the leading edge. LTD4 produced similar effects on neutrophil polarization and motility, but these occurred only at very high concentrations. These data support the view that vectorial orientation is a prerequisite for directional migration of cells and it is also feasible that these are separately regulated events. Furthermore, our studies confirm that LTB4 and, to a much lesser extent, LTD4 are chemotactic for human peripheral blood neutrophils. J. Leukoc. Biol. 55: 201–208; 1994.

We have compared and quantitated transen-dothelial migration of neonatal neutrophils (N-PMNs) and adult bovine peripheral-blood PMNs (A-PMNs) in vitro using monolayers of endothelium and a two-chamber apparatus. Bovine aortic endothelial cells were cultured to confluence on polycarbonate filters perforated with 3.0-μm-diameter pores. 51Cr-labeled PMNs were added to the upper chamber, with or without an anti-CD18 antibody (monoclonal antibody 60.3). Chemotactic stimuli in the lower chambers included recombinant human interleukin-8 (rhIL-8; 75 ng/ml), rhC5a (10-7 M), and zymosan-activated bovine serum (ZAS; 10%). At 60 min incubation with rhIL-8, greater numbers (P < .01) of N-PMNs (24.70 ± 5.95%) than of A-PMNs (15.77 ± 3.66%) had migrated across the endothelial barrier, and a similar difference was present at 90 min. Migration rates of N-PMNs and A-PMNs were similar (P > .05) at all time points when using rhC5a and ZAS as stimuli. Anti-CD18 monoclonal antibody significantly decreased migration (P < .01) of both N-PMNs and A-PMNs to low levels when IL-8 and ZAS were used as stimuli. Because leukocyte integrin expression on PMNs affects transendothelial migration, we also compared surface expression of CD18, CD11a, and CD11c on PMNs from the two age groups. We found no significant quantitative differences in integrin expression between PMNs from the two age groups, regardless of whether the PMNs were incubated with buffer alone or with chemotaxins (rhIL-8, rhC5a, ZAS). J. Leukoc. Biol. 55: 43–49; 1994.

We examined the hypothesis that neutrophil (PMN)-mediated injury of the vascular endothelium is dependent on adhesion of PMNs to endothelial cells via the leukocyte adhesion glycoprotein CD11/CD18. We compared the PMN activation responses [i.e. adhesion to cultured endothelial cells, superoxide (O2-) production, degranulation, and cytosolic [Ca2+] ([Ca2+]i)] and endothelial injury elicited by opsonized zymosan (OZ, which is phagocytosed by PMNs) or phorbol 12-myristate 13-acetate (PMA, a protein kinase C activator). The basal adherence of nonstimulated PMNs to bovine pulmonary artery endothelial cells (BPAEC) was 9.0 ± 1.1 PMN/field. PMA and OZ increased PMN adherence to BPAEC (to 31.1 ± 1.4 and 39.8 ± 3.8 PMN/field, respectively), which in both cases was inhibited by anti-GDI 8 monoclonal antibody (mAb) IB4. Stimulation of PMNs with PMA or OZ produced injury to 73% and 53% of BPAEC examined, respectively, which corresponded to 6.8-fold and 3.5-fold increases in transendothelial 125I-albumin permeability from baseline. Pretreatment of PMNs with mAb IB4 prevented endothelial injury in both cases. Both PMA and OZ increased the production of O2- (by 7.6-fold and 3.1-fold over control, respectively) and promoted the release of myeloperoxidase (5.2-fold and 9.1-fold over control, respectively) (P < .01). IB4 did not inhibit the PMA- or OZ-induced increases in O2-. IB4 did not inhibit the PMA-induced myeloperoxidase release but reduced by 29% the OZ-induced myeloperoxidase release. Stimulation of PMNs layered on BPAEC with OZ (0.5 mg/ml) caused an 7-fold increase in PMN [Ca2+]i over baseline, which decayed to a steady-state level above baseline at 10 min. IB4 (10 μg/ml) alone did not alter baseline [Ca2+]i and did not inhibit the OZ-induced increase in [Ca2+]i. In contrast to OZ, stimulation of PMNs with PMA did not increase [Ca2+]i. The results indicate that the protective effects of the anti-CD18 mAb IB4 were associated predominantly with its antiadherence property. Therefore, CD18 integrin-mediated PMN adhesion to the endothelium is a critical determinant of endothelial injury irrespective of the PMN-activating stimulus. J. Leukoc. Biol. 55: 58–63; 1994.

Human milk neutrophils and macrophages were examined by flow cytometry to determine whether they displayed phenotypic markers of activation. The markers were CDllb and L-selectin, which are increased or shed, respectively, from the surface of activated neutrophils. Phenotypic features of milk neutrophils and macrophages were similar to blood neutrophils stimulated with fMLP: plasma membrane expression of CDllb was increased and L-selectin was decreased. After blood neutrophils were incubated in acellular milk, their expression of CDllb increased and L-selectin decreased. The activation was not affected by trypsin but was significantly decreased by treating acellular milk with chloroform or ether. Sedimentation studies suggested that particulate fractions of milk were active. Further, the activation was partly blocked by treating target blood neutrophils with cytochalasin B. Thus, human milk neutrophils are activated, and the activation may be due partly to phagocytosis of membranous structures in milk.

Medium-chain triglyceride (MCT) and long- chain triglyceride (LCT) emulsions currently used in nutritional therapy were evaluated for their in vitro effect on neutrophil oxidative metabolism, phagocytosis, and bacterial killing activities. Neutrophils from healthy adult male volunteers were assessed after blood incubation with commercially available fat emulsions containing LCT, MCT, or a mixture of 50% MCT and 50% LCT at a final triglyceride concentration of 20 mg/ml. It was observed that MCT-containing emulsions stimulated nitroblue tetrazolium (NBT) dye reduction by neutrophils as determined by a cytochemical NBT test performed directly on whole blood. This effect was dose dependent. However, after lipid removal by cell washing, the MCT-treated neutrophils showed decreased production of hydrogen peroxide (H2O2) and NBT reduction in response to bacterial lipopolysaccharide or phorbol my- ristate acetate stimuli as well as impaired phagocytosis and killing of Staphylococcus aureus. In contrast, the LCT emulsion did not alter any of the neutrophil functions evaluated. The present data suggest that MCTs elicit the oxidative metabolism of neutrophils, probably by phagocytosis of fat particles and, depending on the lipid concentration, this effect may not be reversible, leading to impairment of the cellular response to subsequent membrane stimuli. J. Leukoc. Biol. 53: 404–410; 1993.

Recruitment of cells to an inflammatory site is a process that is selectively regulated. At an inflammatory site caused by bacterial infection, predominantly neutrophil accumulation is observed. This is in contrast to air lergic inflammation, where predominantly eosinophil accumulation occurs. Mac-1 is an inducible adhesion molecule for both neutrophils and eosinophils. We examined the mobilization of this receptor on neutrophils and eosinophils after exposure to factors related to bacterial infections and allergic inflammation. We found more pronounced mobilization of Mac-1 on neutrophils than eosinophils after exposure to N-formylmethionyl- leucyl-phenylalanine, lipopolysaccharides, and activated sera (C5a). There was no significant difference in Mac-1 expression after exposure to aggregated immunoglobulin G. Incubation with interleukin-5 (IL-5) caused a significant increase of Mac-1 expression on eosinophils but not on neutrophils. Neutrophils seem to respond to a greater extent than eosinophils to factors related to bacterial infections, whereas eosinophils respond better to IL-5 associated with allergic inflammation. We measured the total pool of Mac-1 to evaluate whether these differences could depend on the size of the intracellular pool. Eosinophils had a larger total pool of Mac-1 than neutrophils. This finding increases the difference between eosinophils and neutrophils when relating the mobilized pool to the total pool. Stimulation with receptor- independent stimuli such as phorbol myristate acetate and ionomycin induced more pronounced mobilization of Mac-1 on eosinophils, but no differences were obtained if the mobilized pool was related to their total pool. These results indicate that the difference in responsiveness depends on different receptor-mediated signaling, since receptor-independent stimulation resulted in relatively similar mobilization of the intracellular pool of Mac-1. J. Leukoc. Biol. 53: 336–341; 1993.

The relationship between activated neutro- phils and end-organ injury in endotoxemia was studied. The function of peripheral blood neutrophils (PMNs) in rabbits with the generalized Shwartzman reaction (GSR) was compared to that of PMNs rabbits receiving a single injection of endotoxin. The following results were ob- tained: (I) PMNs from rabbits with the GSR demon- strated enhanced adherence to endothelial cells and in- creased mitochondrial ATP production; (2) the GSR did not enhance chemotaxis and oxygen radical production of PMNs; (3) a single injection of endotoxin did not cause necrosis of visceral organs; (4) in vitro detachment of endothelial cells by PMNs was increased in rabbits with the GSR; (5) in vivo administration of monoclonal antibody (mAb) against CD11b/CD18 (Mac-i) sup- pressed the increase in PMN adherence; and (6) hemor- rhagic necrosis did not occur when mAb to Mac-i was in- jected. Thus, enhanced adherence of PMNs to endothelial cells appears to play a key role in endotoxin- induced end-organ injuries in this animal model. J. Leukoc. Biol. 53: 256-263; i993. In this study, the functional characteristics of PMNs were studied and compared with those of PMNs from a group of animals injected only once with endotoxin. A single injection did not cause histologic necrosis. Through comparison of the PMN activation state in the two groups, we sought to clarify the functional attributes crucial to end-organ injury in the GSR. The results help to differentiate between useful activation of host defenses by endotoxin and overactivation leading to organ injury.

In this study, we used the fluorescent probe Fluo-3 to show that an increase in cytosolic free calcium, [Ca2+]i, occurred when suspensions of bovine neutrophils were incubated with sublethal concentrations of P. haemolytica leukotoxin. This increase in [Ca2+]i was dependent on the concentration of leukotoxin present in the medium and, at a given concentration of leukotoxin, dependent on the external calcium concentration. The calcium channel blocker verapamil and the β-adrenergic antagonist propranolol inhibited leukotoxin-stimulated Ca2+ gain, as did a neutralizing antileukotoxin monoclonal antibody. As reported previously, incubation of bovine neutrophils with partially purified leukotoxin stimulated a vigorous luminol-dependent chemiluminescence response (LDCL). The present study shows that LDCL stimulation was dependent on the presence of extracellular calcium and was inhibited by the addition of verapamil and propranolol. These data indicate that bovine neutrophils exhibit a considerable increase in cytoplasmic free calcium when they are incubated with P. haemolytica leukotoxin in the presence of external calcium. They also provide evidence that an increased [Ca2+]i is required for functional activation of the bovine neutrophil oxidative burst by P. haemolytica leukotoxin.

The agent(s) and mechanism(s) responsible for suppression of neutrophil chemotaxis in association with major thermal injury have not been identified. We have proposed that the reduced random motility characterizing patients' cells may contribute to their generalized chemotactic dysfunction. Here we report that actin polymerization may be responsible for the loss of neutrophil motility associated with major thermal injury. Using a fluorescent ligand specific for polymerized or filamentous actin (NBD-phallacidin) in conjunction with flow cytometry, we have discovered that peripheral blood and exudate neutrophils from patients with major thermal injury contain increased levels of actin in a stably polymerized form. Because cyclic polymerization and depolymerization of actin is essential to cell motility, we suggest that actin polymerization may contribute in a major way to the attenuation of neutrophil random and chemotactic functions induced by major thermal injury.

We have previously demonstrated that bactericidal activity and superoxide anion (O2-) production are depressed concomitantly in polymorphonuclear leukocytes (PMNs) following thermal injury in a guinea pig model, and the bactericidal defect is related to elevation of intracellular cyclic-3',5'-adenosine monophosphate (cAMP). The purpose of the present investigation was to determine the relationship between elevation of intracellular cAMP and depression of O2- production in PMNs following thermal injury and determine the involvement of circulating factors in the development of these alterations. The kinetics of O2- production and dose responses to formylmethionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA) were depressed in peripheral PMNs following thermal injury in this experimental model. Sera obtained during the period of PMN dysfunction induced depression of O2- production in response to fMLP and elevation of intracellular cAMP in normal PMNs. Pretreatment of normal PMNs with nonsteroidal anti-inflammatory drugs (NSAID; indomethacin or piroxicam) inhibited the elevation of intracellular cAMP mediated by sera from the injured animals but had no effect on the depression of O2- production observed under similar conditions. Treatment of PMNs from injured animals with NSAID under conditions known to reduce the cAMP content of the cells and correct the bactericidal defect did not normalize O2- production. Studies utilizing sera from two thermally injured patients confirmed findings in the guinea pig model of serum-mediated elevation of intracellular cAMP and depression of O2- production in normal PMNs and effects observed with NSAID. These results suggest that circulating factors contribute to the elevation of intracellular cAMP and depression of O2- production in PMNs following thermal injury. Whereas the increase in intracellular cAMP may be involved in the depression of O2- production, our results suggest that there is not a direct link between these alterations.

A simple, precise method has been developed for assessing neutrophil secretory responses (release of vitamin B12 binding protein from specific granules) to challenge of aliquots of whole blood with the bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). Dose-response studies performed on blood from normal healthy volunteers showed higher maximal secretory responses in males than females (33.3 ± SEM 2.2 vs. 27.4 ± 2.5, P

The endothelial adhesion protein P-selectin binds to a ligand present on the surface of leukocytes. We have characterized the binding interaction between P-selectin and polymorphonuclear leukocytes (PMNs) in an in vitro assay. These studies have utilized a soluble chimeric protein termed receptor globulin (Rg), which consists of the lectin-EGF-CR-CR extracellular domains of P-selectin fused to a human immunoglobulin G Fc domain. The PMNs bound to immobilized Rg in a saturable and concentration-dependent manner. The binding was specific for the Rg, as preincubation of the cells with soluble Rg inhibited binding to immobilized Rg, and binding was dependent on the presence of free divalent cations. The PMNs expressed a ligand for both P-selectin and E-selectin but not for L-selectin. Previously it was shown that sulfatide is a ligand for P-selectin binding in transformed cells. We have demonstrated that the presence of sulfatide in the P-selectin–PMN adhesion assay inhibits binding in a dose-dependent manner.

Membrane lipid fluidity of peripheral blood polymorphonuclear cells (PMNs) of 24 newborn infants, 2–4 days after birth, was determined by steady-state fluorescence polarization with 1,6-diphenyl 1,3,5-hexatriene (DPH) as a probe and compared with that of PMNs from 23 adults. Measurements with intact cells, which correspond to all cellular lipid domains, did not display any statistically significant difference between PMNs of the two groups. However, application of bixinoyl glucosamine, a membrane-impermeable fluorescence quencher, revealed that the PMN plasma membrane of the newborn is about 23% more fluid than that of the adult. Total cholesterol-to-phospholipid ratio of newborn PMNs was found to be lower by about 10% than that of the adult, which could account for the difference in their plasma membrane fluidity. The possible implication of this finding for the deficit in chemotactic ability of leukocytes from newborns was tested with neonatal PMNs that have incorporated cholesteryl hemisuccinate (CHS), an efficient plasma membrane rigidifier. In all neonatal PMNs tested a mild incorporation of CHS (0.5–1 min incubation in 50 μg/ml dispersion) caused a significant improvement in their net chemotaxis, from an average value of 28 ± 7 to 43 ± 11. Longer incubations with CHS caused a gradual decrease in chemotactic ability that approached the basal level after about 5 min incubation. The net chemotaxis in adult PMNs was significantly higher than that of neonatal PMNs (72 ± 13) and was gradually inhibited by incorporation of CHS without any initial augmentation. Based on these results it was estimated that about 27% of the chemotactic deficit of neonatal PMNs is mediated by their immature fluid membrane.

Metabolism of platelet-activating factor (PAF) was studied in cultured polymorphonuclear neutrophils (PMNs) obtained from Pichinde virus–infected strain 13 guinea pigs. Neutrophils obtained from control and infected guinea pigs on postinoculation days 10 and 14 were incubated with [3H]lyso-PAF, [3H]PAF, or [3H]acetate. After incubation for 1 h at 37°C, formation of [3H]acyl-PAF from either [3H]lyso-PAF or [3H]PAF increased significantly in PMNs from infected guinea pigs compared to control PMNs. Furthermore, total radioactivity from [3H]lyso-PAF or [3H]PAF was higher in PMNs from infected animals than in those from controls. However, compared to PAF production by control PMNs, production of PAF by infected PMNs was unchanged. These results suggest that PMNs may not be the major source of increased blood PAF levels during Pichinde viral disease.

We have measured the capacity of two types of granule-poor anucleate cytoplasmic fragments (cytoplasts) from human polymorphonuclear leukocytes (PMN) to migrate across the barrier imposed by monolayers of human umbilical vein endothelial cells (EC) with and without chemotactic stimulation by fMLP. Cytoplasts were made by brief heating of PMN attached to surfaces (CKP) or by discontinuous gradient centrifugation (U-CYT). In the absence of chemoattractant, both types of cytoplast adhered poorly to endothelial cell monolayers, as did unstimulated intact PMN from which the cytoplasts were derived. In the presence of a transendothelial chemoattractant gradient both types of cytoplast exhibited a marked increase in adherence to, and migration across, endothelial monolayers; CKP did so to the same extent as chemoattractant-stimulated intact PMN. Since these motile cytoplasts are markedly deficient in most cytoplasmic organelles they may serve as useful tools for the dissection of cellular mechanisms that mediate PMN migration across endothelia.

Burn trauma is associated with alterations of various components of host defenses, including impaired neutrophil functions. In an animal model of experimental thermal injury, we studied if the modifications of cellular reactivity result from alterations in signalling systems by comparing polyphosphoinositide breakdown, particularly the production of inositol phosphates (IP, IP2 IP3), in healthy and burned rat polymorphonuclear neutrophil leukocytes (PMNs). Neutrophil activators such as N-formyl-methionyl-leucyl-phenylalanine (fMLP) and serum-opsonized zymosan increased in vitro production of inositol phosphates in PMNs from healthy rats. The immunomodulator RU 41740 had no effect by itself, but decreased the stimulating effect of fMLP and zymosan. In PMNs from burned rats, the stimulating effects of fMLP and zymosan were decreased, while RU 41740 stimulated inositol phosphate generation. In vivo treatment with RU 41740 inhibited the activation of phosphoinositide metabolism by fMLP or zymosan in healthy rat PMNs. Similar treatment of burned rats after injury restored the stimulating effect of fMLP and zymosan on inositol phosphate accumulation in PMNs. Thus, RU 41740 can modulate fMLP and zymosan receptor-mediated signal transduction, inducing an attenuation of the phosphatidylinositol hydrolysis response. After burn injury, when the activating effects of fMLP and zymosan are inhibited, RU 41740 can, on the contrary, stimulate phospholipase C-mediated polyphosphoinositide turnover and the formation of intracellular messengers such as IP3. These data show that RU 41740 has different effects on polyphosphoinositide metabolism in rat PMNs, according to the physiological and pathological state of the animals. Interestingly, it has a beneficial action on the post-burn decrease in PMN reactivity.

Skin test anergy, the failure to produce a delayed type hypersensitivity (DTH) response, is associated with an increase in infection-related complications and death usually due to multiple organ failure (MOF). Refractory intravascular activation of polymorphonuclear neutrophils (PMNs) has been implicated in the development of MOF. We studied 20 critically ill surgical patients with life threatening infections to determine if PMN intravascular activation was present and how this affected essential PMN functions such as exudation. The 11 anergic patients had a more intense inflammatory response to their infection. Plasma lactoferrin was 6.1 ± 0.3 μg/ml in anergic patients compared to 3.9 ± 1.5 in reactives P < 0.05, accompanied by reduced total primary (3.3 ± 1.9 vs 4.7 ± 2.1 μg/106 PMN P < 0.01) and secondary (2.8 ± 0.4 vs 5.0 ± 0.9 μg/106 PMN P < 0.01) granule content, respectively. In vitro superoxide production following 100 ng/ml PMA stimulation was 0.44 ± 0.1 in anergics vs 0.36 ± 0.1 nmol/μg PMN protein in reactivities, P < 0.05. PMN chemotaxis was 8.2 ± 0.6 PMNs/HPF in anergics compared to 10.2 ± 1.6 PMNs/HPF in reactives P < 0.05, accompanied by decreased PMN delivery to skin blister windows (3.2 ± 1.4 vs 4.5 ± 1.9 × 107 PMN/ml, respectively, P < 0.05). We conclude that critically ill anergic surgical patients have increased intravascular PMN activation, which may contribute to oxygen-derived tissue damage in the vascular space, as well as a deficient delivery of effector cells in areas of bacterial invasion. This may lead to inability to clear the inflammatory signals which set up the vicious circle of MOF leading to death.

Human neutrophils adherent to simulated biologic surfaces undergo significant activation of the respiratory burst over prolonged periods of time in response to stimulation with the cytokines tumor necrosis factor-alpha (TNF alpha) or tumor necrosis factor-beta (TNF beta) or with the chemotactic peptide N-formyl-methionylleucylphenylalanine (FMLP). In this study, neutrophils were examined for their ability to generate the highly reactive and powerful oxidant hypochlorous acid (HOCl) and the longer-lived, less reactive endogenous nitrogen-chlorine (N-Cl) derivatives in response to these stimuli either alone or when exposed to recombinant human TNF alpha (rTNF alpha) or beta (rTNF beta) prior to addition of FMLP. Neutrophils adherent to fetal bovine serum-coated polystyrene tissue culture wells were able to generate only small quantities of HOCl when incubated with rTNF alpha, rTNF beta, or FMLP individually. However, when neutrophils were first incubated with either rTNF alpha or rTNF beta prior to addition of FMLP, there was a marked increase in HOCl generation. Neutrophils stimulated in such a manner consumed approximately 18% of the HOCl generated in the formation of N-Cl derivatives. Further scrutiny of the response to the combination of rTNF alpha and FMLP revealed that HOCl release was rapid, with 80% of total HOCl accumulation occurring within 15 min after FMLP addition. The amount of HOCl generated was dependent on the number of cells added and on the concentration of both rTNF alpha and FMLP. Comparison of HOCl generation with superoxide anion and myeloperoxidase release showed that the amount of HOCl generated was limited primarily by the amount of myeloperoxidase released rather than by the degree of respiratory burst activation. These results demonstrate that human neutrophils stimulated with FMLP after a brief incubation with rTNF alpha or rTNF beta can generate cytotoxic and microbicidal concentrations of chlorinated oxidants.

Antibody-dependent cellular cytotoxicity (ADCC) activity against pseudorabies virus-infected target cells has been found to be lower in young pig peripheral blood leukocytes (PBLs) than in adults. Experiments were designed to investigate the reason(s) for low activity in the young, which are more at risk of fatal infection than adults. The percentage of polymorphonuclear leukocytes (PMNs), the major ADCC effector cell, in the whole leukocyte population did not have a bearing on the deficiency. Enrichment for PMNs did not alleviate differences in activity between young and adult pigs. Additionally, no suppressor cell(s) or factor(s) could be demonstrated to account for the ADCC deficiency. The source of the ADCC deficiency in the young was found to be related to the decreased ability of young pig effector cells to bind antibody-sensitized targets. This deficiency relative to adults was associated with decreased antibody binding to high affinity Fc receptors on young pig neutrophils.

Platelets are currently thought to play a role in tissue injury and inflammatory states both directly and indirectly through their action on neutrophils (PMNs). Both stimulation and inhibition of PMN superoxide anion (O2-) production by platelets has been reported. To clarify these discrepant observations, we investigated the effects of wide ranges of platelet to PMN ratios as well as concentrations of ATP and ADP on human PMN O2- production. Platelets, at low platelet-to-PMN ratios (1:1 and 5:1), primed PMNs which were stimulated with either FMLP or PMA. However, at higher platelet-to-PMN ratios (25:1, 50:1, and 100:1), inhibition of O2- production was seen. ATP also had a biphasic effect on O2- production: low concentrations of ATP (1 x 10-6 to 3.2 x 10-4 M) increased O2 production and high concentrations of ATP (6.4 x 10-4 M and above) inhibited O2 production. ADP, when added to stimulatory concentrations of ATP, also caused inhibition of O2- production. The incubation time for platelet-neutrophil interactions in vitro was also crucial. Short incubation periods lead to priming, whereas longer periods (> 5 min) lead to inhibition. We believe that these studies help to resolve the controversy over the effects of platelets upon the production of O2- by human PMNs and lend further support to the notion that platelets may modulate injury resulting from neutrophil activation.

We investigated the role of neutrophils in increased vascular permeability by a selective reduction of neutrophils using a monoclonal antibody, RP-3. An intraperitoneal injection of RP-3 not only selectively depleted peripheral blood neutrophils, but prevented the neutrophil infiltration to the tissues. Proteose peptone, zymosan, and BCG induced three different types of inflammatory edema, showing the early phase only, early plus late phase, and the late phase only, respectively. Only the late phase response of zymosan and BCG was inhibited by a depletion of neutrophils by RP-3, though the early phase response induced by proteose peptone and zymosan was not affected. Reconstitution of neutrophil-depleted rats by in situ infection of these cells restored the inflammatory edema induced by BCG, depending upon the number of neutrophils injected.

Neutrophil accumulation and plasma leakage induced in rabbit skin by neutrophil-activating peptide-1 (NAP-1, a 72 amino acid peptide produced by monocytes and a variety of tissue cells), E. coli endotoxin, and interleukin-1 (IL-1) were compared. Neutrophil accumulation at sites injected with NAP-1 was intense, rapid, and long-lasting; it reached a maximum rate during the first 30 min, continued at constant rate for 4–6 h, and remained detectable up to at least 8 h. In contrast, the neutrophil-attracting effect of endotoxin and IL-1 was slower in onset and more transient; it peaked in the first 2 h and declined to a very low level after 4 h. Plasma leakage induced by NAP-1 had a shorter time course than neutrophil accumulation and ceased after 6 h. Depletion of blood neutrophils by treatment with hydroxyurea prevented the plasma leakage induced by NAP-1 or endotoxin but not by histamine. Desensitization to NAP-1 was studied by restimulation of lesions. Following restimulation with NAP-1 after intervals from 6–10 h, there was diminished infiltration of neutrophils, while nearly normal responses were obtained after an interval of 24 h. Desensitization was dose dependent and affected both plasma leakage and neutrophil accumulation. In lesions initiated with NAP-1 there were normal responses following restimulation with endotoxin but marked desensitization to IL-1, suggesting that NAP-1 may contribute to inflammation induced by IL-1 but not by endotoxin. This study indicates that NAP-1 is a potent mediator of neutrophil accumulation in vivo, with characteristics similar to those reported for C5 fragments, but with a more protracted action.

Association of platelets and neutrophils is frequently observed within thrombi or inflammatory sites. Interactions between these two cell populations have been reported for the production of several mediators of inflammation such as hydrogen peroxides or leukotrienes. Another potential mediator of thrombosis and inflammation is paf-acether, which is synthesized by activated platelets and neutrophils. Since platelets form and release large amounts of the paf-acether precursor lyso paf-acether, platelet and neutrophil cooperation for paf-acether biosynthesis was investigated. Purified human neutrophils (4 x 106/ml) stimulated by opsonized zymosan (ZC, 1 mg/ml) formed 4.5 ± 2.5 ng/ml paf-acether. Human washed platelets (3 x 108/ml) stimulated with thrombin (1 lU/ml) formed 0.60 ± 0.43 ng/ml paf-acether. Platelets and neutrophils, incubated together and both stimulated by their specific agonist, formed more than twice as much paf-acether as did platelets and neutrophils separately (10.90 ± 4.25 ng/ml, n = 6, P < .001). The formation of lyso paf-acether and the release of lysozyme and LDH were unchanged under the cooperation conditions. The formation of paf-acether almost doubled (10.24 ± 3.81 ng/ml paf-acether vs. 5.30 ± 2.23, P < .05, n = 4) when ZC-stimulated neutrophils were incubated with supernatants from thrombin-stimulated platelets as well as with synthetic lyso paf-acether. Extracted and purified lyso paf-acether from thrombin-stimulated platelets led to an increase of biosynthesis of paf-acether by neutrophils (13.86 ± 2.26 ng/ml paf-acether vs. 5.76 ± 0.38, P < .05, n = 3). These results indicate that a cooperation between platelets and neutrophils exists for paf-acether formation. The phenomenon depends on a platelet-derived soluble factor, possibly lyso paf-acether. This cell-to-cell interaction is of interest since paf-acether is formed by and acting on platelets and neutrophils and represents a molecular basis for potent amplification of inflammatory reactions.

Polymorphonuclear leukocytes (PMN) were stimulated with phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl phenylalanine (FMLP) to clarify the role of excited oxygen species in inducing changes of membrane fluidity. Membrane fluidity was assessed by the excimer-forming lipid technique using pyrenedecanoic acid and flow cytometry. Membrane fluidity of PMN decreased following stimulation with PMA, and the extent of decrease was both time- and dose-dependent. FMLP at 10-5 M induced a decrease, while FMLP at 10-7 M induced a rapid increase. On stimulation with 10-7 M FMLP as well as in a resting condition, the change of membrane fluidity of PMN from patients with chronic granulomatous disease (CGD) was similar to that of normal PMN. However, on stimulation with PMA or 10-5 M FMLP, CGD PMN did not show a significant decrease. In addition, normal PMN incubated with catalase inhibited the decrease. These findings suggest that the generation of excited oxygen species, particularly of H2O2, is important in inducing a decrease of PMN membrane fluidity.

To study the effect of endotoxin (LPS) on the basal and chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (fMLP) -induced alterations in neutrophil cytoskeleton, we purified (>98%) LPS-free neutrophils (LPS- < 10 pg/ml LPS), compared their cytoskeletal organization to that of circulating neutrophils, and examined the effect of LPS exposure on the basal and fMLP-induced change in the cytoskeleton as reflected by F-actin content and distribution. Shape, F-actin content and distribution were monitored by FACS analysis and fluorescence microscopy of NBDphallicidin-stained cells. The F-actin content of basal and fMLP-activated, purified LPS- cells is similar to that of circulating neutrophils (defined as cells drawn in LPS- buffers at 37°C and analyzed after

Chemotactic factors and phorbol esters may act synergistically to evoke neutrophil responses, but the mechanism of such interaction is not entirely clear. We investigated the combined effects of the chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA) on protein kinase C (PKC) activity in human neutrophils. FMLP had little effect on the sharp decrease in cytosolic PKC activity induced by PMA. However, cells exposed to FMLP and PMA exhibited a synergistic increase in particulate PKC activity (1 ± 1 pmol 32P/107 PMNs/min in unstimulated cells, 53 ± 12 pmol 32P with 20 ng/ml PMA, 6 ± 3 pmol 32P with 10-7 M FMLP, and 191 ± 17 pmol 32P with FMLP and PMA). FMLP also markedly increased calcium/phospholipid-independent protein kinase activity in particulate fractions of control and PMA-treated cells. Enhancement of PKC activity required the presence of cytochalasin B during cell stimulation. Cellular calcium was crucial to the FMLP effect since enhancement was decreased in cells incubated with EGTA or Quin2. These results suggest that chemotactic factors and phorbol esters may mediate synergistic effects on neutrophil responses through enhancement of particulate PKC activity. The enhancing effect is probably mediated through chemoattractant-mediated increases in intracellular calcium.

Cytokineplasts (CKP) are granule-poor cytoplasts from human polymorphonuclear leukocytes (PMN) that retain motile function, even (unlike the parent PMN) after cryopreservation. Employing time-lapse videomicroscopy, we examined the chemotactic properties of CKP after cryopreservation toward erythrocytes lysed by laser microirradiation. Paths of locomotion were plotted for six CKP in the field, and velocities were calculated at 10-sec intervals. Mean velocities of the six fragments, ranging from 9.3 to 20.8 μm/min, are of the order of fresh, intact PMN, the fastest of locomoting cells.

We studied the effect of the linoleic acid metabolite 13-hydroxyoctadecadienoic acid (13-HODE) on the increase of the intracellular calcium concentration, [Ca2+]i, induced by platelet-activating factor (PAF), leukotriene B4 (LTB4), and formyl-Met-Leu-Phe (fMLP) in human polymorphonuclear leukocytes (PMNs). 13-HODE by itself did not change the [Ca2+]i of PMNs. However, when PMNs were preincubated with 13-HODE and subsequently stimulated with PAF, LTB4, or fMLP, an inhibition of the increase of the [Ca2+]i was observed. The PAF-induced calcium response was concentration-dependently inhibited between 0.1 and 5 μM 13-HODE. These results suggest that 13-HODE can affect the increase of the [Ca2+]i and hence can modulate activation of PMNs. J. Leukoc. Biol. 56: 200–202; 1994.

Neutrophil accumulation in tissues is a hallmark of inflammation and is associated with a variety of pathological conditions. In bacterial infection neutrophils are selectively attracted in large numbers to phagocytose and kill invading microorganisms. However, activated neutrophils can also cause injury to tissues. To investigate functional alterations in liver recruited neutrophils (PMNs), we studied the functional characteristics of circulating blood and liver sequestered PMNs in terms of host defense mechanisms, such as nitric oxide (NO) and superoxide (SO) generation, β 2 integrin expression, phagocytosis, and eicosanoid profile. Cells were isolated from rats infused with a nonlethal dose (320 μg/kg) of E. coli endotoxin (ET) or pyrogen-free saline for 90 min. Liver PMNs produced significantly more NO both in the absence and in the presence of an in vitro endotoxin challenge than did blood PMNs. No significant difference was observed in phorbol myristate acetate-stimulated SO generation. Endotoxin infusion significantly up-regulated the expression of CD11b/c in circulating and even more so in liver PMNs. Phagocytosis was significantly enhanced by in vivo ET treatment in blood PMNs, and liver PMNs showed even greater phagocytic activity than blood PMNs or Kupffer cells. The percent distribution of prostaglandins D2 and E2 of total 14C-eicosanoids was significantly higher and that of thromboxane B2 and 5-, 12-, and 15-HETEs was significantly lower in liver than in blood PMNs. Our study demonstrates several functional differences between liver-recruited and circulating PMNs in an acute endotoxic model. The implications of altered neutrophil function may extend to mechanisms of host defense and hepatotoxicity associated with sepsis and endotoxemia. J. Leukoc. Biol. 56: 166–173; 1994.

A short-term bone marrow culture was established to determine the functional and proliferative responses of neutrophils sampled from sheep exposed to endotoxin in vivo. Iliac crest bone marrow was drawn from surgical control animals and from sheep given a 12-h infusion of endotoxin. In these bone marrow aspirates, neutrophils from endotoxin-treated sheep showed primed superoxide anion release compared with controls when stimulated with phorbol myristate acetate. Priming was no longer present in neutrophils tested after 2 days in culture. The neutrophil cell count, however, was higher after 2 days in culture than in controls. Increased neutrophil production/viability was reproduced in naive bone marrow cultures by replacing part of the normal serum medium with plasma taken from sheep several hours after endotoxin infusion. These data suggest that a dissociation exists between priming and increased neutrophil production/viability in response to endotoxin. J. Leukoc. Biol. 56: 145–150; 1994.

The effect of A1F4- on the degranulation process in human neutrophils was investigated. In intact neutrophils, A1F4- induced degranulation, whereas in electropermeabilized neutrophils A1F4- did not stimulate degranulation. In electropermeabilized neutrophils, fluoride ions proved to be inhibitory for the degranulation induced by addition of Ca2+ and/or GTP-γ-S. Another phosphatase inhibitor, okadaic acid, inhibited degranulation induced by Ca2+ or by GTP-γ-S but not degranulation induced by the combination of Ca2+ plus GTP-γ-S. It is concluded that under suboptimal conditions of stimulation with Ca2+ or GTP-γ-S, protein dephosphorylation plays an important role in the degranulation response in human neutrophils. J. Leukoc. Biol. 55: 489–495; 1994.

We previously found that platelet-activating factor (PAF) stimulated greater lactoferrin secretion by neutrophils of atopies than nonatopics. To help understand underlying mechanisms, we compared agonist- stimulated lactoferrin secretion with another response, superoxide generation, to determine whether there is a global alteration in the reactivity of atopic neutrophils to these stimuli. We also determined Ca2+ mobilization in such neutrophils because such mobilization is a signaling pathway for both superoxide generation and granule content exocytosis. Although PAF again stimulated greater lactoferrin secretion in atopic than in nonatopic neutrophils, the peak superoxide secretion and cytosolic Ca2+ mobilization were not significantly different in atopies and nonatopics. Leukotriene B4-induced superoxide secretion and cytosolic Ca2+ shifts were also similar in atopies and nonatopics. These findings suggest that (1) the enhanced PAF-induced lactoferrin secretion in atopic neutrophils is not a reflection of greater PAF binding or broad-based cell hyperreactivity and (2) a selective signaling pathway in atopic neutrophils may be responsible for the enhanced lactoferrin secretion. These findings may be relevant to in vivo events because we have found increased PAF and lactoferrin release in skin chambers overlying immunoglobulin E-mediated human skin reactions. J. Leukoc. Biol. 53: 727–731; 1993.

To examine whether neutrophils are involved in the pathogenesis of experimental liver dysfunction, we observed the effect of selective in vivo neutrophil depletion by a monoclonal antibody (RP-3) on the pathogenesis of acute experimental hepatic necrosis in rats induced by a preparative injection of heat-killed Corynebacterium parvum and a challenging dose of lipopolysaccharide (LPS) (positive control group). The serum transaminase titer 8 h after LPS injection was reduced by selective depletion of peripheral blood neutrophils as a result of RP-3 treatment (RP-3 group). A kinetic study showed that the serum transaminase titer of the RP-3 group was significantly lower than that of the positive control group from 4 to 24 h after LPS injection. The transaminase level was significantly lower in the group with less than 400/mm3 peripheral blood neutrophils than in the group with a greater number. The effect of RP-3 on the transaminase level was due neither to the injection of cancer ascites nor to RP-3 injection with the accompanying decrease in complement titer. These results suggest that neutrophils play an important role in the induction of liver dysfunction in this system. J. Leukoc. Biol. 53: 144–150; 1993.

Tumor necrosis factor-alpha (TNF-α), a cytokine produced by mononuclear cells in response to endotoxin, inhibits neutrophil chemotaxis. We analyzed the effects of TNF-α on the orientation and movement of individual neutrophils in a chemoattractant gradient. Neutrophils, treated or untreated with TNF-α, were observed migrating in a gradient of the chemotactic peptide N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP) on a specially constructed chamber (Zigmond bridge). The movement of these cells was videotaped, digitized, and then tracked using a newly designed computer algorithm. The data obtained from this algorithm were then utilized to calculate distance traveled, speed and ability to polarize and migrate in a directed manner for each individual cell. TNF-a-treated cells behaved like cells not exposed to fMLP in that they failed to orient in a chemotactic gradient and moved in a manner similar to randomly migrating cells. This study provides unique observations of the effect of TNF-α on multiple parameters of PMN migration.

Interleukin 1 (IL-1) has been shown to modulate various functional activities of neutrophils, presumably by first binding to cell surface IL-1 receptors. In this study, we characterized and identified IL-1 receptors on bovine neutrophils using direct and competitive receptor binding studies and affinity cross-linking analysis. The results of direct binding studies demonstrated that bovine neutrophils bound 125I-labeled bovine IL-1 by a single affinity binding site (Kd = 4.6 nM, 5600 binding sites per cell). Competitive receptor binding studies demonstrated that unlabeled recombinant bovine IL-1β, murine IL-1α, and human IL-1β competitively blocked neutrophil binding of 125I-labeled bovine IL-1β. In contrast, the IL-1 receptor antagonist (IL-1ra) demonstrated no detectable binding to bovine neutrophils as judged by direct and competitive receptor binding assays. Affinity cross-linking of 125I-labeled bovine IL-1β to neutrophils was used to identify cell surface IL-1 receptors. Two specific cross-linked products were observed with molecular sizes of 89 kd (a deduced receptor size of 71.5 kd) and more than 200 kd. The latter may indicate a complex of IL-1 receptor-associated signal-transducing components, IL-1 receptors, and IL-1. The presence of 100 nM unlabeled bovine, murine, or human IL-1 during the receptor binding and cross-linking reactions prevented the formation of cross-linked complexes of 125I-labeled bovine IL-1β and its receptor. In contrast, the IL-1ra failed to inhibit the formation of cross-linked complexes of 125I-labeled bovine IL-1β and its receptor.

The capacity of phorbol myristate acetate (PMA) to prime neutrophil cytotoxic responses induced by a second stimulus was investigated. Treatment of neutrophils with low concentrations of PMA (0.2–0.5 ng/ml) for 18 hr at 37°C markedly enhanced cytotoxicity triggered by Ca2+ ionophore A23187, N-formyl-methionyl-leucyl-phenylalanine (FMLP) and PMA. Pretreatment with PMA also enabled neutrophils to mediate significant cytotoxicity when triggered by platelet-activating factor (PAF), a stimulus unable to induce untreated cells to display cytotoxicity. Conversely, neutrophil cytotoxicity triggered by immune complexes (IC) was not modified by PMA treatment, whereas cytolytic activity of neutrophils against antibody-sensitized target cells was significantly increased. Treatment with PMA concentrations higher than 1 ng/ml directly triggered neutrophil cytotoxicity. Interestingly, we found that PMA-triggered neutrophils were able to sustain maximal levels of cytotoxicity for at least 8 hr after stimulation. With regard to the mechanisms involved in neutrophil activation by PMA, we found that catalase but not superoxide dismutase (SOD) prevented neutrophil activation measured as 1) induction of cytotoxic responses, 2) increase of neutrophil adhesiveness to cell-free surfaces, and 3) inhibition of chemotactic responses to FMLP. These findings suggest that H2O2 may play a major role in neutrophil activation induced by PMA.

Infiltration of polymorphonuclear neutrophils (PMN) in the rat liver 3 hr after an intravenous (IV) injection of a sublethal dose of Escherichia coli lipopolysaccharide (LPS) was observed without any significant alteration in the total number of Kupffer and endothelial cells. Since previous studies have demonstrated that phagocytic cells in the liver were in a state of metabolic activation under similar experimental conditions, we investigated the in vitro generation of superoxide anion (O2-) by this cell type following the administration of LPS. Kupffer cells from normal rats did not release O2-, in contrast to those obtained from LPS-treated rats. The generation of O2- by Kupffer cells from endotoxic rats was elevated from 3.0 ± 1.9 nmol/106 cells/60 min (mean ± SD) in the absence of macrophage (Mφ) activators, to 5.0 ± 2.36, 11.33 ± 5.40, and 4.33 ± 0.90 in the presence of opsonized zymosan, phorbol myristate acetate (PMA), and the calcium ionophore A23187, respectively. Hepatocytes from normal or endotoxic rats did not produce detectable O2-. Endothelial cells from LPS-treated rats generated < 0.8 nmol/106 cells in the presence of zymosan. PMN that accumulated in the livers of endotoxic rats released O2- only in the presence of zymosan (8.12 ± 5.40), PMA (15.43 ± 5.84), or A23187 (1.70 ± 0.12). The O2- generation by blood monocytes and PMN increased significantly after endotoxin administration and in the presence of activators. These results suggest that the hypermetabolic state of phagocytic cells in the liver shortly after LPS treatment may be correlated with the increased generation of O2-. The latter may subsequently contribute to the induction of hepatic injury in endotoxemia.

Neutrophils (PMNs) may be exposed to high concentrations of biliary products during cholestasis and other hepatic disorders. We have previously reported that bile and certain bile salts enhance superoxide (O2-) release from neutrophils activated with phorbol myristate acetate (PMA) (Dahm et al.: Toxicol. Appl. Pharmacol. 95,82,1988), suggesting that PMN oxidative metabolism might be altered in toxicoses or disease states characterized by elevations in serum bile salts and other biliary products. In the present study, we characterized the priming effect of lithocholate for O2- release and also examined the effects of lithocholate on enzyme release from PMNs. PMNs preincubated with lithocholate at concentrations which did not directly stimulate O2- release (3–100 μM) and activated with PMA released greater amounts of O2- than controls exposed to PMA alone, illustrating a priming effect O2- release from lithocholate-primed PMNs rose sharply between 5 and 10 min after PMA addition and then ceased between 10 and 30 min. The priming effect of lithocholate toward PMA-activated PMNs was reduced approximately 50% by washing PMNs after lithocholate addition and was not dependent on extracellular Ca2+, although removal of Ca2+ from the incubation buffer enhanced the cytotoxicity of lithocholate toward PMNs. In Ca2+-supplemented medium, lithocholate primed PMNs for O2- release when formyl-methionyl-leucyl-phenylalanine (FMLP, 10-8-10-6 M) or calcium ionophore, A23187 (10-7 or 10-6 M), was used to activate PMNs. Lithocholate (100 μM) by Itself had only marginal effects on release of lysozyme or β-glucuronidase from PMNs. However, lithocholate (100 μM) inhibited β-glucuronidase release from FMLP-stimulated PMNs to near-baseline levels. When FMLP was added to PMNs prior to lithocholate, β-glucuronidase release was not reduced as it was when the order of addition was the reverse. Lithocholate had no effect on PMA-stimulated lysozyme release. These results indicate that lithocholate has different actions on PMN O2- release and enzyme release and suggest that lithocholate might exert its action on the PMN plasma membrane.

Recombinant human Interleukin-1 (rhlL-1) β was found to enhance stimulus-induced granule exocytosis from human polymorphonuclear leukocytes (PMNs). PMNs were incubated with rhlL-1β and then stimulated with either heat-aggregated IgG (Hagg) or N-formyhmethionyl leucylphenylalanine (FMLP). The release of the azurophil enzyme myeloperoxidase (MPO) was measured. Low concentrations of stimuli (10 μg/ml Hagg, 2.5 x 10-9 M FMLP) did not stimulate degranulation in the absence of rhlL-1β. However, such concentrations elicited marked degranulation from PMNs preincubated with rhlL-1β (0.2-100 ng/ml). The enhancement of degranulation was dependent on the concentration of rhIL-1β employed and on the period of incubation. In other experiments, the effect of rhlL-1β on the PMN oxidative response was determined. rhlL-1β did not directly stimulate the production of superoxide anions or enhance the oxidative response to Hagg or FMLP. It is suggested that in rheumatoid joints, IL-1β may potentiate PMN degranulation, but not their oxidative response.

Human mononuclear cells exposed to staphylococcal peptidoglycan in serum-free culture rapidly produce an inhibitor of neutrophil chemotaxis which we have previously described. We found that this inhibitor of chemotaxis has its most potent effect on the inhibition of neutrophil shape change from a spherical to a polarized configuration. In order to quantify this shape change inhibition, we developed an assay using flow cytometric techniques. Neutrophils exposed to a chemoattractant simultaneously change their shape and decrease their forward angle light scattering intensity (δFLS) with a correlation coefficient of 0.886 (p < 0.001). In 51 experiments, neutrophils pretreated with the inhibitor of chemotaxis decreased their FLS by only 6.8 ±1.3 channels, while neutrophils pretreated with medium or control culture supernatants decreased theirs by 26.4 ±1.9 and 20.5 ± 3.0 channels respectively (p < 0.001). The factor which causes inhibition of shape change was indistinguishable from the inhibitor of chemotaxis by physical properties and chromatography. We conclude that this inhibitor of chemotaxis may act by inhibiting a physiologic step at or before shape change.

Both purified human monocyte interleukin-1 and recombinant interleukin-1 (beta) primed neutrophils for increased superoxide production and chemiluminescence in response to f-met-leu-phe. In addition, purified human monocyte interleukin-1 and recombinant interleukin-1 (beta) altered neutrophil shape. Recombinant interleukin-1 (alpha) used at the same concentration of interleukin-1 (beta) did not prime neutrophils for increased superoxide production after stimulation with f-met-leu-phe. interleukin-1 expressed by monocytes in response to endotoxin stimulation could act as a modulator of neutrophil function.

Interferon-γ (IFN-γ) treatment of polymorphonuclear leukocytes (PMNs) results in an activation of their functions. Studying the IFN responsiveness of PMNs and using antibodies to the IFN-induced proteins, we have observed the ability of IFN-α to stimulate the production of the IFN-induced 67,000 and 56,000 dalton proteins and the ability of IFN-γ to induce the synthesis of the 67,000, 56,000, and 42,000 dalton proteins. The induction of these proteins is dependent on de novo RNA synthesis, as its induction is inhibited if the IFNs and actinomycin D are added to the cells simultaneously. The results of this study confirm the ability of PMNs to carry out gene activation and demonstrate the ability of PMNs to respond to both IFN-α and IFN-γ.

Chemotactic substances confer a migratory pattern for neutrohil granulocytes under agarose that is characteristic for each agent. To analyse the cause of such differences, we have studied neutrophil migration and orientation with fMet-Leu-Phe (fMLP), leukotriene B4 (LTB4), and serum as chemoattractants. When these agents were used at optimal concentrations, it was observed that cells stimulated by LTB4 did not start migration as fast and did not migrate as far as those exposed to fMLP, but they maintained a higher degree of orientation. This delay in initiation of migration and maximal degree of orientation was even more marked when serum was the chemoattractant. These migration variables were related to the generation of gradients in the agarose of fML[3H]P, arachidonic-[3H]acid (AA, of which LTB4 is a metabolite), and fluorescein. The curvilinear AA gradient was flatter and more stable than those of fML[3H]P and fluorescein, which were linear. Thus, differences in the development and shape of the gradient of chemoattractant may contribute to differences in migration kinetics.

The migration of polymorphonuclear leukocytes (PMN) in response to recombinant interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF), C5a, and f-met-leu-phe-lys (FMLPL) in vivo was studied using a mouse subcutaneous sponge implantation model. In this model sponges were implanted in C3H/OUJ mice, and 2 days later they were injected with the test sample. After varying times, sponges were removed and digested with collagenase, and total cell counts and differentials were enumerated. IL-1 was found to stimulate a significant influx of PMN, which peaked at 6 hr and declined to near baseline levels by 24 hr. This response was dose-dependent, with the greatest response observed when 5 units of IL-1 were injected. When the IL-1 concentration was increased to 10 U, the total number of PMN migrating into the sponge was decreased, compared with that observed with 5 U of IL-1. The overall number of PMN migrating into the sponge 6 hr after injecting 5 U of IL-1 averaged 269% of the number of PMN migrating randomly into the sponge. No difference in the total number of macrophages or lymphocytes in control or IL-1-injected sponges was observed in this time frame. Heat treatment of the IL-1 at 90°C for 30 min ablated the response. Similar studies with TNF and C5a showed that both of these agents also stimulated an influx of PMN that peaked 6 hr postinjection. In contrast, FMLPL did not stimulate a PMN response. When IL-1 and TNF were injected simultaneously, an additive response was observed. These data indicate that IL-1, TNF, and C5a can all stimulate a PMN response in vivo and support the hypothesis that these substances are actively involved in the mobilization of PMN to inflammatory sites in vivo.

The in vitro generation and release of 5-lipoxygenase metabolites of arachidonic acid by bovine peripheral blood neutrophils and alveolar neutrophils elicited with either a heat-killed bacterium, Haemophilus somnus, or platelet-activating factor, were compared. After stimulation with calcium lonophore A23187 for 2.5-60 min, up to 4.5 ± 0.7 (mean ± SEM) ng of LTB4 per 106 cells was released into the media by circulating neutrophils. LTB4 release by alveolar neutrophils was significantly less (P

Serum from a patient with inactive systemic lupus erythematosus (SLE) and ibuprofen-induced transient neutropenia was used as a probe to define further the control of human polymorphonuclear leukocyte (PMN) exocytosis and superoxide (O-2) generation. Thirty-minute preincubation of normal PMNs with 10-50H v/v of this serum, followed by washing, produced a specific dose-related suppression of leukotriene B4 (LTB4)-elicited β-glucuronidase and lysozyme release of up to 45% and 30% respectively. If cells were not washed, the inhibition increased to 60% and 40%. Superoxide production stimulated by LTB4 was unaffected. The serum had no effect on formyl-met-leu-phe (FMLP) or phorbol myristate acetate-stimulated O2- or exocytosis. O2- and β-glucuronidase release elicited by zymosan-treated serum (ZTS) were both decreased by 15%, but there was no increased inhibition seen if cells were not washed, or if the time of preincubation was increased from 7 to 30 min. In contradistinction, the serum inhibition of LTB4 exocytosis did show time dependence. Serum obtained when the patient was not leukopenic and sera from 6 normal controls, 2 patients with inactive SLE, 1 patient with SLE and chronic leukopenia, and 2 controls taking Ibuprofen did not influence any PMN function. The serum inhibition of ZTS-induced functions was qualitatively similar to that observed when PMNs were preincubated and desensitized with ZTS in vitro. Selective inhibition of LTB4 exocytosis was not seen when PMNs were desensitized with LTB4 in vitro. These observations indicate that LTB4-elicfted O2- and exocytosis can be independently and specifically regulated. The cellular site at which this serum factor acts is not clear, but the current studies strongly suggest that this inhibition is not due to in vitro deactivation by LTB4 activity.

This study examines the effect of epithelial permeability on 1) the passage of the chemoattractant tritiated formyl methionyl-leucyl-phenylalanine (3H-fMLP; m.w. 437) and 2) the migration of leukocytes. In addition, it also compares the kinetics of neutrophil and monocyte transepithelial migration. As we had demonstrated with neutrophils (Milks et al.: Journal of Cell Biology 96:1241, 1983), when the permeability of the epithelium decreased, the accumulation of 3H-fMLP and the emigration of monocytes also decreased. When neutrophils and monocytes traversed epithelia with similar permeability, neutrophil accumulation was at least ninefold greater than that of monocytes at 30, 60, and 90 min. During the same time intervals, the number of neutrophil invasion sites/mm epithelium exceeded the number of monocyte invasion sites by at least fivefold, with approximately twice as many neutrophils as monocytes traversing each invasion site. These studies demonstrate that epithelial permeability affects the passage of the chemoattractant and the emigration of leukocytes. In addition, the enhanced ability with which neutrophils traverse occluding junctions compared to monocytes helps to explain, at least in part, the more rapid accumulation of neutrophils at inflammatory lesions.

When sterile gelatin sponges are implanted under the skin of a mouse and retrieved 6 hr later, approximately 106 neutrophils per mouse, in a 98–99% pure population, may be retrieved by simply squeezing and rinsing the sponges. These neutrophils behave similarly to peritoneal exudate neutrophils in chemotaxis and phagocytosis assays, but are bacteriostatic rather than bactericidal. The sponge method yields substantially more neutrophils than could be obtained by exsanguinating the mouse and isolating blood neutrophils, and a 5-fold purer population than is normally obtained by passage of peritoneal exudate cells over Ficoll. In addition, sponge-elicited neutrophils may be ready for use within a half hour after removal from the mouse, without being exposed to osmotic shock or Ficoll.