Oxygen radicals, inflammation, and tissue injury

Inflammatory reactions often result in the activation and recruitment of phagocytic cells (e.g., neutrophils and/or tissue macrophages) whose products result in injury to the tissue. In killing of endothelial cells by activated neutrophils as well as in lung injury produced by either activated neutrophils or activated macrophages there is evidence that H2O2 and iron play a role. HO. may be a key oxygen product related to the process of injury. Endothelial cells in some vascular compartments may be susceptible to neutrophil mediated injury in a manner that is independent of oxygen radicals. On the basis of in vitro observations, a synergy exits between platelets and neutrophils, resulting in enhanced oxygen radical formation by the latter. Finally, the cytokines, interleukin 1 and tumor necrosis factor, released from macrophages have both direct stimulatory effects on oxygen radical formation in neutrophils and can "prime" macrophages for enhanced oxygen radical responses to other agonists. Cytokines may also alter endothelial cells rendering them more susceptible to oxygen radical mediated injury by neutrophils. This suggests a complex network of interactions between phagocytic cells and peptide mediators, the result of which is acute, oxygen radical mediated tissue injury.     

Exogenous nitric oxide elicits chemotaxis of neutrophils in vitro

Nitric oxide (NO) has been shown to be both an intercellular and intracellular messenger. We propose here that exogenous NO induces chemotactic locomotion of human neutrophils. Indeed, when human neutrophils were placed in a gradient of a nitric oxide donor (S-nitroso-N-acetylpenicillamine; SNAP), a directed locomotion was induced, as evidenced by experiments of chemotaxis under agarose. Degraded SNAP (i.e., SNAP solution which had previously released NO) did not induce directed locomotion. Moreover, oxyhemoglobin, a scavenger of free NO, suppressed the chemotactic effect of SNAP, whereas LY-83583, a soluble guanylate cyclase inhibitor, inhibited the SNAP-mediated chemotaxis in a dose-response manner. Other unrelated NO donors, SIN-1 and S-nitroso-cysteine—a natural S-nitroso-compound, also induced a directed locomotion of neutrophils. Taken together, these in vitro experiments indicate that exogenous NO could mediate the chemotaxis of neutrophils and thus suggest that NO could contribute to neutrophil recruitment in vivo. © 1995 Wiley-Liss Inc.     

Hepatocyte growth factor stimulates neutrophil degranulation but not respiratory burst

Neutrophil function is regulated in part by cytokines with growth factor activities for different cell types. Hepatocyte growth factor (HGF) is a cytokine produced during injury to the liver and other organs. Neutrophils are numerous in such tissue injury sites and may be influenced by HGF. In the present study the effect of HGF on neutrophils was investigated. The data show that HGF at 1-10 ng/ml increased lysosomal enzyme release from both specific and azurophilic granules of cytochalasin-B treated neutrophils. The release of specific granule contents in response to N-formyl-methionyl-leucylphenylalanine was also increased by HGF. In contrast there were no significant effects of HGF on neutrophil respiratory burst, adherence or locomotion. It is concluded that HGF modulates neutrophil granule exocytosis.     

Physical association of beta 2 integrin with GPI-80, a novel glycosylphosphatidylinositol-anchored protein with potential for regulating adhesion and migration

We recently found a novel GPI-anchored protein, GPI-80, on human phagocytes that may regulate leukocyte adherence, locomotion, and extravasation. To clarify the mechanisms by which GPI-80 functions on leukocytes, we explored the possibility of its physical association with beta 2 integrin which is important for leukocyte adherence, locomotion, and extravasation. beta 2 integrin, detected by anti-CD18 mAb, was coprecipitated with GPI-80 from human neutrophil lysates by a mAb to GPI-80. In addition, GPI-80 was immunoprecipitated from human neutrophil lysates by anti-human CD18 mAb. These results clearly show that GPI-80 is physically associated with beta 2 integrin in human neutrophils.     

High mannose type N-linked oligosaccharides on endothelial cells may influence β2 integrin mediated neutrophil adherence in vitro

We report herein on the role of N-linked oligosaccharide processing of endothelial cell surface proteins on the adhesion of neutrophils. Monolayers of human umbilical vein endothelial cells were treated for 24 h with deoxymannojirimycin (DMJ), an inhibitor of golgi mannosidase I, which results in changes in glycoprotein processing, and then incubated with neutrophils to examine their ability to adhere to the treated endothelial cells. Treatment with DMJ, which leads to accumulation of high mannose type oligosaccharides, resulted in a twofold increase in adherence of phorbol ester (PMA) activated neutrophils compared to attachment to untreated endothelial cells. This adherence was likely mediated by the β2 integrin, Mac-1, and could be specifically inhibited with monoclonal antibodies to ICAM-1 and to the integrin β2 subunit. Similarly, IL-1 treatment resulted in a β2 integrin mediated increase in neutrophil adherence to the DMJ treated endothelial cells in a dose dependent manner. However, the IL-1 induced adherence was not significantly inhibited by the anti-ICAM-1 antibody, thus, suggesting the presence of other inducible components on the endothelial cell surface. Our results demonstrate that alterations in glycosylation of N-linked oligosaccharides, resulting in the synthesis of high mannose type sugars on molecules that may interact with the β2 integrins, leads to an increased adherence of PMA activated neutrophils to endothelial cells. © 1993 Wiley-Liss, Inc.     

Escape of Mycobacterium tuberculosis from oxidative killing by neutrophils

Neutrophils enter sites of infection, where they can eliminate pathogenic bacteria in an oxidative manner. Despite their predominance in active tuberculosis lesions, the function of neutrophils in this important human infection is still highly controversial. We observed that virulent Mycobacterium tuberculosis survived inside human neutrophils despite prompt activation of these defence cells' microbicidal effectors. Survival of M. tuberculosis was accompanied by necrotic cell death of infected neutrophils. Necrotic cell death entirely depended on radical oxygen species production since chronic granulomatous disease neutrophils were protected from M. tuberculosis-triggered necrosis. More, importantly, the M. tuberculosis ΔRD1 mutant failed to induce neutrophil necrosis rendering this strain susceptible to radical oxygen species-mediated killing. We conclude that this virulence function is instrumental for M. tuberculosis to escape killing by neutrophils and contributes to pathogenesis in tuberculosis.     

Lysophosphatidic acid increases phosphatidic acid formation, phospholipase D activity and degranulation by human neutrophils

I-oleoyl-sn-glycero-3-phosphate, a lysophosphatidic acid (LPA), in serum is a biologically active lipid and has multiple functions depending on the cell types. Several studies have shown that LPA stimulates phospholipase D (PLD) activity in fibroblasts and prostate cancer cells in culture. PLD plays a central role in regulating neutrophil functions. One of the functions of the lipid product, phosphatidic acid (PA), of PLD action in neutrophils is to promote degranulation. In the present study, we examined the effect of LPA on PLD activity and degranulation by human neutrophils. The results show that exogenous LPA increased PA formation, PLD activity and degranulation by human neutrophils in a time and concentration dependent manner. These findings suggest that LPA released from activated platelets during blood clotting may participate in bacterial killing and wound healing process. On the other hand, augmented LPA production might be involved in inflammation, causing damage of the host tissues.     

Induction of neutrophil apoptosis by the Pseudomonas aeruginosa exotoxin pyocyanin: a potential mechanism of persistent infection

Pseudomonas aeruginosa colonizes and infects human tissues, although the mechanisms by which the organism evades the normal, predominantly neutrophilic, host defenses are unclear. Phenazine products of P. aeruginosa can induce death in Caenorhabditis elegans. We hypothesized that phenazines induce death of human neutrophils, and thus impair neutrophil-mediated bacterial killing. We investigated the effects of two phenazines, pyocyanin and 1-hydroxyphenazine, upon apoptosis of neutrophils in vitro. Pyocyanin induced a concentration- and time-dependent acceleration of neutrophil apoptosis, with 50 microM pyocyanin causing a 10-fold induction of apoptosis at 5 h (p < 0.001), a concentration that has been documented in sputum from patients colonized with P. aeruginosa. 1-hydroxyphenazine was without effect. In contrast to its rapid induction of neutrophil apoptosis, pyocyanin did not induce significant apoptosis of monocyte-derived macrophages or airway epithelial cells at time points up to 24 h. Comparison of wild-type and phenazine-deleted strains of P. aeruginosa showed a highly significant reduction in neutrophil killing by the phenazine-deleted strain. In clinical isolates of P. aeruginosa pyocyanin production was associated with a proapoptotic effect upon neutrophils in culture. Pyocyanin-induced neutrophil apoptosis was not delayed either by treatment with LPS, a powerfully antiapoptotic bacterial product, or in neutrophils from cystic fibrosis patients. Pyocyanin-induced apoptosis was associated with rapid and sustained generation of reactive oxygen intermediates and subsequent reduction of intracellular cAMP. Treatment of neutrophils with either antioxidants or synthetic cAMP analogues significantly abrogated pyocyanin-induced apoptosis. We conclude that pyocyanin-induced neutrophil apoptosis may be a clinically important mechanism of persistence of P. aeruginosa in human tissue.     

Effects of D-Glucose on Chemokinesis and Resting Production of Reactive Oxygen Species in Neutrophil Granulocytes of Lean or Obese-Hyperglycemic Mouse

The response to D-glucose (0–21 mM) was studied in neutrophil granulocytes from obese, hyperglycemic and hyperinsulinemic Umeå ob/ob mice and their lean, littermate controls in order to further elucidate the effects of in vivo and in vitro hyperglycemia on neutrophil function. Neutrophil random locomotion on glass and neutrophil resting luminol-enhanced chemiluminescence in cell suspension were studied. Random locomotion was stimulated by D-glucose in neutrophils from both Umeå ob/ob and control mice but the locomotive activity in Umeå ob/ob mouse neutrophils was significantly higher than that found in the controls at 4–21 mM glucose. In both types of mice, the stimulatory effect of D-glucose on random locomotion was diminished at 21 mM glucose (not significantly different from that at 0 mM glucose). Resting chemiluminescence from mouse neutrophils was also stimulated by glucose but here the magnitude of response was similar in neutrophils from both types of mice. These results indicate that chronic hyperglycemia and hyperinsulinemia in the Umeå ob/ob mouse may be associated with an increased neutrophil random locomotive activity but a similar resting production of reactive oxygen species, as compared with neutrophils from control mice at physiological and hyperglycemic glucose concentrations in vitro.     

Characterization of f-Met-Leu-Phe-stimulated fluid pinocytosis in human polymorphonuclear leukocytes by flow cytometry

N-formylated chemotactic peptide stimulation of human neutrophils initiates a number of cellular processes, such as lysosomal enzyme release and superoxide anion production, that are indicative of the events of neutrophil activation during the acute inflammatory response in disease. This study characterizes a newly recognized neutrophil activation event, N-formylated chemotactic peptide-stimulated fluid pinocytosis in human neutrophils, using a novel flow cytometric assay for this activity. Fluid pinocytosis was found to be inhibited by acidic pH and low temperature but could be enhanced by cytochalasin B treatment or surface adherence by neutrophils. The activity measured by this new assay of fluid pinocytosis appears to be separate and distinct from lysosomal enzyme release and receptor-mediated adsorptive endocytosis in neutrophils. The physiologic significance of N-formylated chemotactic peptide-stimulated fluid pinocytosis is not known, but a possible relationship to neutrophil locomotion is discussed.     

Intracellular signaling in neutrophil priming and activation

In order for neutrophils to function effectively in host defense, they have evolved specific attributes including the ability to migrate to the site of inflammation and release an array of toxic products including proteolytic enzymes, reactive oxygen species, and cationic proteins. While these compounds are intended for killing invading pathogens, if released inappropriately, they may also contribute to tissue damage. Such inflammatory tissue injury may be important in the pathogenesis of a variety of clinical disorders including arthritis, ischemia-reperfusion tissue injury, the systemic inflammatory response syndrome (SIRS), and the acute respiratory distress syndrome (ARDS). Despite the importance of neutrophil function in host defense and dysfunction in disease states, much remains unknown about the intracellular signaling pathways regulating neutrophil activity. This review will focus on the signaling molecules regulating leukocyte ‘effector’ functions including receptors, GTP-binding proteins, phospholipases, polyphosphoinositide metabolism, and protein kinases and phosphatases.     

Selective inhibition of neutrophil activation by the subendothelial extracellular matrix: Possible role in protection of the vessel wall during diapedesis

Mobilization of circulating neutrophils toward an inflamed area involves adherence of the cells to the vascular endothelium and subsequent penetration through the endothelial cell layer without causing significant damage. To investigate the nature of a possible protective mechanism, granulocytes were incubated with the extracellular matrix (ECM) produced by cultured endothelial cells and tested for release of enzymes, chemoattractants, and free oxygen radicals. In the absence of exogenously added stimuli, the neutrophils adhered to the ECM but there was no detectable release of lysozyme, chemotactic activity, or production of O2-. In contrast, the cells readily released a heparan sulfate-degrading endoglycosidase (heparanase) to an extent comparable with that released in contact with polystyrene surfaces. Neutrophils treated with the calcium ionophore A23187 or with the peptide FMLP produced O2- to a much lesser degree when incubated in contact with ECM-coated surfaces than did those incubated in contact with uncoated polystyrene culture dishes. The ECM itself was devoid of superoxide dismutase activity. Stimulation with opsonized zymosan was not inhibited by the ECM. Experiments with isolated constituents of the ECM revealed that fibronectin but not collagen type IV or laminin could partially inhibit O2- production by Ca2+ ionophore-stimulated neutrophils. Treatment of the ECM with proteolytic enzymes, but not with heparanase, abolished its inhibitory effect on neutrophil activation. These results indicate that the subendothelial basement membrane has the capacity to inhibit release of potentially noxious agents excluding heparanase, suggesting a preferential involvement of this enzyme in neutrophil diapedesis.     

Neutrophil-induced changes in the biomechanical properties of endothelial cells: roles of ICAM-1 and reactive oxygen species

This study evaluated the changes in the biomechanical properties of endothelial cells (ECs) induced by neutrophil adhesion and the roles of ICAM-1 and reactive oxygen species (ROS) in modulating these changes. Neutrophil adherence to 24-h TNF-alpha-activated pulmonary microvascular ECs induced an increase in the apparent stiffness of ECs within 2 min, measured with magnetic twisting cytometry. An anti-ICAM-1 Ab blocked the EC stiffening response without inhibiting neutrophil adherence. Moreover, cross-linking ICAM-1 mimicked the stiffening response induced by neutrophils. The neutrophil-induced increase in the apparent stiffness of ECs was inhibited with 1% DMSO (a hydroxyl radical scavenger), allopurinol (a xanthine oxidase inhibitor), or deferoxamine (an iron chelator), suggesting that ROS may be involved in mediating the EC stiffening response. The cellular sources of ROS were determined by measuring the oxidation of dichlorofluorescein. Neutrophil adherence to TNF-alpha-activated ECs induced ROS production only in ECs, and not in neutrophils. This ROS production in ECs was completely prevented by the anti-ICAM-1 Ab and partially inhibited by allopurinol. These results suggest that ICAM-1-mediated signaling events during neutrophil adherence may activate xanthine oxidase, which in turn mediates the ROS production in ECs that leads to stiffening. ROS generated in ECs on neutrophil adherence appear to mediate cytoskeletal remodeling, which may modulate subsequent inflammatory responses.     

Human neutrophils kill Bacillus anthracis

Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified alpha-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that alpha-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.     

Activation of AMPK Enhances Neutrophil Chemotaxis and Bacterial Killing

An inability of neutrophils to eliminate invading microorganisms is frequently associated with severe infection and may contribute to the high mortality rates associated with sepsis. In the present studies, we examined whether metformin and other 5′ adenosine monophosphate-activated protein kinase (AMPK) activators affect neutrophil motility, phagocytosis and bacterial killing. We found that activation of AMPK enhanced neutrophil chemotaxis in vitro and in vivo, and also counteracted the inhibition of chemotaxis induced by exposure of neutrophils to lipopolysaccharide (LPS). In contrast, small interfering RNA (siRNA)-mediated knockdown of AMPKα1 or blockade of AMPK activation through treatment of neutrophils with the AMPK inhibitor compound C diminished neutrophil chemotaxis. In addition to their effects on chemotaxis, treatment of neutrophils with metformin or aminoimidazole carboxamide ribonucleotide (AICAR) improved phagocytosis and bacterial killing, including more efficient eradication of bacteria in a mouse model of peritonitis-induced sepsis. Immunocytochemistry showed that, in contrast to LPS, metformin or AICAR induced robust actin polymerization and distinct formation of neutrophil leading edges. Although LPS diminished AMPK phosphorylation, metformin or AICAR was able to partially decrease the effects of LPS/toll-like receptor 4 (TLR4) engagement on downstream signaling events, particularly LPS-induced IκBα degradation. The IκB kinase (IKK) inhibitor PS-1145 diminished IκBα degradation and also prevented LPS-induced inhibition of chemotaxis. These results suggest that AMPK activation with clinically approved agents, such as metformin, may facilitate bacterial eradication in sepsis and other inflammatory conditions associated with inhibition of neutrophil activation and chemotaxis.     

Inflammatory response of tracheobronchial epithelial cells to endotoxin

Respiratory epithelial cells play a crucial role in the inflammatory response in endotoxin-induced lung injury, an experimental model for acute lung injury. To determine the role of epithelial cells in the upper respiratory compartment in the inflammatory response to endotoxin, we exposed tracheobronchial epithelial cells (TBEC) to lipopolysaccharide (LPS). Expression of inflammatory mediators was analyzed, and the biological implications were assessed using chemotaxis and adherence assays. Epithelial cell necrosis and apoptosis were determined to identify LPS-induced cell damage. Treatment of TBEC with LPS induced enhanced protein expression of cytokines and chemokines (increases of 235-654%, P < 0.05), with increased chemotactic activity regarding neutrophil recruitment. Expression of the intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was enhanced by 52-101% (P < 0.0001). This upregulation led to increased adhesion of neutrophils, with >95% adherence to TBEC after LPS stimulation, which could be blocked by either ICAM-1 (69%) or VCAM-1 antibodies (55%) (P < 0.05). Enhanced neutrophil-induced necrosis of TBEC was observed when TBEC were exposed to LPS. Reduced neutrophil adherence by ICAM-1 or VCAM-1 antibodies resulted in significantly lower TBEC death (52 and 34%, respectively, P < 0.05). Therefore, tight adherence of neutrophils to TBEC appears to promote epithelial cell killing. In addition to indirect effector cell-induced TBEC death, direct LPS-induced cell damage was seen with increased apoptosis rate in LPS-stimulated TBEC (36% increase of caspase-3, P < 0.01). These data provide evidence that LPS induces TBEC killing in a necrosis- and apoptosis-dependent manner.     

Structural divergence of GPI-80 in activated human neutrophils

GPI-80 is a glycosylphosphatidylinositol (GPI)-anchored protein that is mainly expressed in human neutrophils. Previous studies using 3H9, a monoclonal antibody (mAb) against GPI-80, suggested that GPI-80 regulates leukocyte adherence and migration through Mac-1. GPI-80, which is anchored at the plasma membrane in resting neutrophils, moves into the pseudopodia and is released from activated human neutrophils. Here, we demonstrate that neutrophil activation affects GPI-80 dynamics using a new anti-GPI-80 mAb, designated 4D4, which is directed against the form of GPI-80 found on resting human neutrophils. Similar to 3H9, 4D4 influences Mac-1-dependent neutrophil adhesion. Treatment of purified GPI-80 with periodic acid and trypsin indicated that 3H9 and 4D4 recognize peptide and carbohydrate moieties, respectively. Stimulation with fMLP decreased the binding of 4D4 to GPI-80 on the neutrophil surface but increased the overall expression of GPI-80, as visualized by the 3H9 signal. Confocal laser microscopy revealed the 4D4 signal mainly on cell bodies and at a low level on pseudopodia during migration toward increasing concentrations of fMLP, whereas the 3H9 signal was observed in both areas. In addition, soluble GPI-80 released from activated neutrophils did not bind 4D4. These results suggest that there are two populations of GPI-80 that differ in the ability to bind 4D4. The 4D4-recognized form may regulate Mac-1-dependent neutrophil adhesion, and may subsequently be converted to a 4D4-unrecognized form during neutrophil activation.     

The lipooligosaccharide‐modifying enzyme LptA enhances gonococcal defence against human neutrophils

Infection with Neisseria gonorrhoeae (Gc) is marked by an influx of neutrophils to the site of infection. Despite a robust immune response, viable Gc can be recovered from neutrophil‐rich gonorrhoeal secretions. Gc enzymatically modifies the lipid A portion of lipooligosaccharide by the addition of phosphoethanolamine to the phosphate group at the 4′ position. Loss of lipooligosaccharide phosphoethanolamine transferase A (LptA), the enzyme catalysing this reaction, increases bacterial sensitivity to killing by human complement and cationic antimicrobial peptides. Here, we investigated the importance of LptA for interactions between Gc and human neutrophils. We found that lptA mutant Gc was significantly more sensitive to killing by human neutrophils. Three mechanisms underlie the increased sensitivity of lptA mutant Gc to neutrophils. (i) lptA mutant Gc is more likely to reside in mature phagolysosomes than LptA‐expressing bacteria. (ii) lptA mutant Gc is more sensitive to killing by components found in neutrophil granules, including CAP37/azurocidin, human neutrophil peptide 1 and the serine protease cathepsin G. (iii) lptA mutant Gc is more susceptible to killing by antimicrobial components that are exocytosed from neutrophils, including those decorating neutrophil extracellular traps. By increasing the resistance of Gc to the bactericidal activity of neutrophils, LptA‐catalysed modification of lipooligosaccharide enhances survival of Gc from the human inflammatory response during acute gonorrhoea.     

Inactivation of a subpopulation of human neutrophils by exposure to ultrahigh-molecular-weight polyethylene wear debris

Polymorphonuclear neutrophils, a first line of defence against invading microbial pathogens, may be attracted by inflammatory mediators triggered by ultrahigh-molecular-weight polyethylene (UHMWPE) wear particles released from orthopaedic prostheses. Phagocytosis of UHMWPE particles by neutrophils may indirectly compromise their phagocytic-bactericidal mechanisms, thus enhancing host susceptibility to microbial infections. In an in vitro assay, pre-exposure of purified human neutrophils to UHMWPE micrometre- and submicrometre-sized wear particles interfered with subsequent Staphylococcos aureus uptake in a heterogeneous way, as assessed by a dual label fluorescence microscopic assay that discriminated intracellular rhodamine-labelled UHMWPE particles from fluorescein isothiocyanate-labelled S. aureus. Indeed, a higher percentage (44%) of neutrophils having engulfed UHMWPE particles lost the ability to phagocytize S. aureus, compared with UHMWPE-free neutrophils (<3%). Pre-exposure of neutrophils to UHMWPE wear particles did not impair but rather stimulated their oxidative burst response in a chemoluminescence assay. The presence of UHMWPE wear particles did not lead to significant overall consumption of complement-mediated opsonic factors nor decreased surface membrane display of neutrophil complement receptors. In conclusion, engulfment of UHMWPE wear particles led to inactivation of S. aureus uptake in nearly half of the neutrophil population, which may potentially impair host clearance mechanisms against pyogenic infections.     

Mechanism of priming of human neutrophils by a soluble lymphoblastoid cell factor

Treatment of human neutrophils (PMN) with a cytokine-like factor in the supernatants of human lymphoblastoid cells (Raji) increased the random mobility and enhanced the migration of treated cells in response to other chemoattractants nearly 21/2-fold, although the supernatant itself was not a chemoattractant. Supernatant treatment also increased the adherence of bacteria threefold and the bacterial killing fourfold compared with PMN treated with control media. In examining the metabolic basis for the enhanced bactericidal ability, we observed a significant increase in spontaneous hexose monophosphate shunt activity of Raji cell supernatant (RS)-treated neutrophils even in the absence of additional stimuli. RS-treated PMN also had significantly enhanced production of superoxide anion and chemiluminescence response upon subsequent stimulation with a variety of soluble and particulate stimuli. Unlike other agents that prime neutrophil activation, however, the factor(s) in RS did not cause degranulation. It also differed in its ability to progressively enhance PMN functions with a longer period of preincubation (up to 3 hr). These data suggest that the RS factor(s) primes neutrophils by a unique mechanism. The neutrophil-enhancing activities of RS, which are the opposite of those activities described for leukocyte inhibitory factor, eluted off a Sephacryl S-200 column at approximately 30,000 m.w. This factor expands the relationship between neutrophils and lymphocytes, and may be a useful agent to provide valuable insights into the mechanism of respiratory burst activation and regulation.