CCR9(+) macrophages are required for eradication of peritoneal bacterial infections and prevention of polymicrobial sepsis

Sepsis is a systemic inflammatory response to infection associated with multiple organ dysfunction syndrome and a high mortality rate. In septic shock induced by severe peritonitis, early response of peritoneal macrophages against infected microbes is vital in preventing the spread of infection. We found that the mucosal homing receptor CCR9, is induced in peritoneal macrophages in response to inflammatory stimulation. We used a cecal ligation and puncture (CLP) model of sepsis to determine the role of CCR9 with respect to peritoneal macrophages, and controlling peritoneal infection and systemic inflammation. CCR9(-/-) mice showed aggravated septic shock with higher mortality rates compared with wild-type (WT) mice. Six hours after CLP, CCR9(-/-) mice demonstrated a greater inflammatory response. This was associated with higher production of inflammatory cytokines, such as IL-6, TNF and IP-10 in peritoneal lavage compared with WT mice. Although the numbers of peritoneal bacteria were elevated in CCR9(-/-) mice subjected to CLP compared with WT mice, this was normalized in CCR9(-/-) mice subjected to CLP through the adoptive transfer of WT peritoneal macrophages. We conclude that CCR9 is required for recruitment of peritoneal macrophages in the steady state to control systemic sepsis during early phases of peritoneal infection.     

Systemic mast cell degranulation increases mortality during polymicrobial septic peritonitis in mice

MCs are required for an effective host response during septic peritonitis. Local MC degranulation facilitates neutrophil recruitment, activation, and bacterial killing. However, the role of MCs located distant from the site of infection is unknown. We studied the temporal and spacial degranulation of MCs following CLP-induced septic peritonitis. The functional importance of systemic MC degranulation during infection was evaluated by compartment-specific MC reconstitution. Serum histamine, reflecting MC degranulation, was elevated 4 h after onset of septic peritonitis. Histologic examination revealed progressive MC degranulation in select tissues during the first 24 h of infection. MC-deficient Wsh mice, reconstituted only in the peritoneal compartment, had improved survival after CLP compared with controls. However, reconstitution in peritoneal plus systemic compartments worsened survival after CLP. IL-6 contributed to the detrimental effects of systemic MCs on survival, as mice systemically reconstituted with IL-6(-/-) MCs were more likely to survive than control mice. These results indicate that in contrast to the benefits of local MC activation during infection, systemic MC activation worsens survival during CLP-induced sepsis.     

Neutrophil Sequestration in Liver and Lung is Differentially Regulated by C-X-C Chemokines During Experimental Peritonitis

C-X-C chemokines play an important role in the migration and activation of neutrophils (PMNs) during an inflammatory event. We measured mRNA and protein expression of the murine C-X-C chemokines macrophage inflammatory protein-2 (MIP-2) and KC in the lungs, liver, blood, and peritoneal cavity of Swiss Webster mice after cecal ligation and puncture (CLP). Neutralizing antibodies to MIP-2 and KC were also used to determine the biological effects of these chemokines on neutrophil sequestration and organ injury in the CLP model. The data showed that early after CLP, MIP-2 mRNA and protein were expressed predominantly by the lung, whereas KC mRNA and protein were expressed by the liver. Inhibition of MIP-2 reduced both lung neutrophil sequestration and peritoneal neutrophil migration. Inhibition of KC had no effect on overall neutrophil sequestration in liver but reduced injury as measured by serum transaminases. An early survival benefit was found with anti-KC treatment, although overall survival was not different. Our study showed a differential expression by organs of C-X-C chemokines during sepsis and suggested that such chemokine effects are tissue-specific.     

Chemokine C10 promotes disease resolution and survival in an experimental model of bacterial sepsis

Previous studies have suggested that the C-C chemokine C10 is involved in the chronic stages of host defense reactions. The present study addressed the role of C10 in a murine model of septic peritonitis, induced by cecal ligation and puncture (CLP). Unlike other C-C chemokines, C10 levels in the peritoneal wash were increased approximately 30-fold above baseline levels at 48 h after CLP surgery. Immunoneutralization of peritoneal C10 levels with polyclonal anti-C10 antiserum during CLP-induced peritonitis negatively impacted mouse survival over 4 days. In contrast, when 500 ng of recombinant murine C10 was administered immediately after CLP surgery, the 4-day survival rate increased from 20% to over 60%. The C10 therapy appeared to facilitate a rapid and significant enhancement of the levels of tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein-1 (MCP-1) and a later increase in interleukin-13 (IL-13) levels in the peritoneal cavity. In vitro studies showed that the combination of IL-1beta and C10 markedly augmented TNF-alpha synthesis by peritoneal macrophages and that C10 synthesis was induced in these cells following their exposure to IL-13. At 24 h after CLP surgery, only 25% of C10-treated mice were bacteremic versus 85% of the control group that exhibited dissemination of bacteria into the circulation. The lack of bacteremia in C10-treated mice appeared to be related, in part, to in vitro evidence that C10 significantly enhanced the bacterial phagocytic activity of peritoneal macrophages. In addition, in vivo evidence suggested that C10 therapy significantly reduced the amount of material that leaked from the damaged gut. Taken together, the results of this study demonstrate that the C10 chemokine rapidly promotes disease resolution in the CLP model through its direct effects on the cellular events critically involved in host defense during septic peritonitis.     

BTLA expression contributes to septic morbidity and mortality by inducing innate inflammatory cell dysfunction

A proper innate inflammatory response is essential for prevention of the systemic inflammation associated with sepsis. BTLA is an immune-regulatory receptor demonstrated to be expressed not only on adaptive immune populations and have potent inhibitory effects on CD4(+) T cells but is also expressed on innate cell populations (CD11c(+) and CD11b(+) cells) and has been shown to diminish pathogen clearance following bacterial and parasite infection. The role of BTLA in sepsis and the mechanisms by which BTLA alters pathogen clearance, however, have not been addressed clearly. Here, we show that following acute experimental sepsis induction in mice (CLP), the number of infiltrating BTLA- and HVEM (the ligand for BTLA)-expressing macrophages, inflammatory monocytes, mature and immature DCs, and neutrophils increased in the peritoneum compared with sham surgery, suggesting that a high level of HVEM:BTLA interactions occurs between these cells at the site of septic insult. Given this, we evaluated BTLA(-/-) mice, 24 h post-CLP, and observed a marked increase in the degree of activation on these cell populations, as well as a reduction in peritoneal bacterial burden and IL-10 induction, and most importantly, BTLA(-/-) mice exhibited a higher rate of survival and protection from organ injury when compared with WT mice. Such changes were not restricted to experimental mice, as circulating BTLA+ and HVEM+ monocytes and HVEM+ granulocytes were increased in septic ICU patients, supporting a role for BTLA and/or HVEM as potential, novel diagnostic markers of innate immune response/status and as therapeutic targets of sepsis.     

CC chemokine receptor 2 regulates leukocyte recruitment and IL-10 production during acute polymicrobial sepsis

Chemokine receptors are important for recruiting leukocytes to sites of infection and may contribute to immune cell activation. The present study investigated the role of the chemokine receptor CCR2 in polymicrobial septic peritonitis. The results showed that peritoneal production of the CCR2 ligands CCL2 and CCL12 in septic mice was largely independent of the common Toll-like receptor signaling adaptor MyD88. Antibody blockade of CCR2 reduced the recruitment of macrophages and neutrophils to the infected peritoneal cavities of both wild-type and MyD88-deficient mice, suggesting that CCR2 engagement contributes to the MyD88-independent cellular response against polymicrobial septic peritonitis. Notably, administration of blocking CCR2 antibodies markedly increased local and systemic IL-10 levels in septic wild-type mice, whereas IL-10 was not detected in MyD88-deficient mice irrespective of whether CCR2 was blocked or not. Inhibition of CCR2 directly augmented Toll-like receptor-induced IL-10, but not TNF and IL-6, production of macrophages in vitro. Concomitant with enhanced IL-10 production, CCR2 blockade caused impaired bacterial clearance and aggravated kidney injury in wild-type, but not MyD88-null mice. These results indicate that CCR2 engagement modulates the innate immune response to polymicrobial septic peritonitis by both MyD88-dependent and -independent processes and suggest that a major function of CCR2 in sepsis is to attenuate IL-10 production and IL-10-mediated suppression of host defense.     

Pulmonary and Hepatic Gene Expression following Cecal Ligation and Puncture: Monophosphoryl Lipid A Prophylaxis Attenuates Sepsis-Induced Cytokine and Chemokine Expression and Neutrophil Infiltration

Polymicrobial sepsis induced by cecal ligation and puncture (CLP) reproduces many of the pathophysiologic features of septic shock. In this study, we demonstrate that mRNA for a broad range of pro- and anti-inflammatory cytokine and chemokine genes are temporally regulated after CLP in the lung and liver. We also assessed whether prophylactic administration of monophosphoryl lipid A (MPL), a nontoxic derivative of lipopolysaccharide (LPS) that induces endotoxin tolerance and attenuates the sepsis syndrome in mice after CLP, would alter tissue-specific gene expression post-CLP. Levels of pulmonary interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), IL-1 receptor antagonist (IL-1ra), and IL-10 mRNA, as well as hepatic IL-1β, IL-6, gamma interferon (IFN-γ), G-CSF, inducible nitric oxide synthase, and IL-10 mRNA, were reduced in MPL-pretreated mice after CLP compared to control mice. Chemokine mRNA expression was also profoundly mitigated in MPL-pretreated mice after CLP. Specifically, levels of pulmonary and hepatic macrophage inflammatory protein 1α (MIP-1α), MIP-1β, MIP-2, and monocyte chemoattractant protein-1 (MCP-1) mRNA, as well as hepatic IFN-γ-inducible protein 10 and KC mRNA, were attenuated in MPL-pretreated mice after CLP. Attenuated levels of IL-6, TNF-α, MCP-1, MIP-1α, and MIP-2 in serum also were observed in MPL-pretreated mice after CLP. Diminished pulmonary chemokine mRNA production was associated with reduced neutrophil margination and pulmonary myeloperoxidase activity. These data suggest that prophylactic administration of MPL mitigates the sepsis syndrome by reducing chemokine production and the recruitment of inflammatory cells into tissues, thereby attenuating the production of proinflammatory cytokines.     

Elevated levels of macrophage inflammatory protein 2 in severe murine peritonitis increase neutrophil recruitment and mortality.

We hypothesized that chemokines may play important roles in a cecal ligation and puncture (CLP) model of septic peritonitis in CD-1 mice. Concentrations of C-X-C (macrophage inflammatory protein 2 [MIP-2] and ENA-78) and C-C (MIP-1alpha and JE) chemokines were measured (by enzyme-linked immunosorbent assay) in serum, peritoneal lavage fluid, lung, and liver at 4, 8, 24, 48, and 96 h after CLP. Significant elevations in all measured chemokines occurred in peritoneal fluid after CLP (P < 0.05). MIP-2, in particular, increased dramatically (>400-fold, P < 0.001) in peritoneal fluid, serum, and to a lesser extent lung and liver (P < 0.05). Increased MIP-2 was correlated with severity of sepsis (P < 0.001). To determine the significance of this finding, mice were passively immunized prior to CLP with polyclonal antibody to MIP-2, which decreased mortality from 85 to 38% at 96 h (P < 0.01). To further understand the mechanism of the effect of MIP-2, additional measurements demonstrated that anti-MIP-2 prior to CLP decreased the percent neutrophils in peritoneal fluid (55% +/- 12%, compared with 82% +/- 10% in controls), but no significant changes in tumor necrosis factor alpha, interleukin-6, or interleukin-10 occurred. MIP-2 contributes to the inflammatory response and overall mortality in this model of severe septic peritonitis, possibly by increasing recruitment of neutrophils, which clear bacteria but may also injure the host.     

Interleukin-18 facilitates the early antimicrobial host response to Escherichia coli peritonitis

To determine the role of endogenous interleukin-18 (IL-18) during peritonitis, IL-18 gene-deficient (IL-18 KO) mice and wild-type mice were intraperitoneally (i.p.) infected with Escherichia coli, the most common causative agent found in septic peritonitis. Peritonitis was associated with a bacterial dose-dependent increase in IL-18 concentrations in peritoneal fluid and plasma. After infection, IL-18 KO mice had significantly more bacteria in the peritoneal lavage fluid and were more susceptible for progression to systemic infection at 6 and 20 h postinoculation than wild-type mice. The relative inability of IL-18 KO mice to clear E. coli from the abdominal cavity was not due to an intrinsic defect in the phagocytosing capacity of their peritoneal macrophages or neutrophils. IL-18 KO mice displayed an increased neutrophil influx into the peritoneal cavity, but these migratory neutrophils were less activate, as reflected by a reduced CD11b surface expression. These data suggest that endogenous IL-18 plays an important role in the early antibacterial host response during E. coli-induced peritonitis.     

Divergence of protection induced by bacterial products and sepsis-induced immune suppression

Susceptibility to bacterial infections after a primary immune stimulation differs drastically depending on the presensitization of the innate immune system. To determine the conditions that either induce protection or enhanced susceptibility to infection with Salmonella enterica serovar Typhimurium, we pretreated mice either with tumor necrosis factor (TNF), whole killed bacteria, or sublethal cecal ligation and puncture (CLP) as a mouse model for septic peritonitis. Impaired production of the cytokines TNF, interleukin-6 (IL-6), and IL-10 was induced by these pretreatment schedules, with TNF-signaling not being essential for this effect. Injection of TNF or killed bacteria enhanced survival of mice infected subsequently with serovar Typhimurium. In contrast, sepsis such as that induced by CLP only protected from shock induced by d-galactosamine and lipopolysaccharide or by a high dose of bacteria but sensitized to a secondary bacterial infection. Such sepsis-induced enhanced susceptibility to infection was critically dependent on TNF function.     

Deficiency of the CGRP receptor component RAMP1 attenuates immunosuppression during the early phase of septic peritonitis

The neuropeptide CGRP contributes to the control of excessive cytokine production in endotoxemia models. However, the function of CGRP in sepsis caused by infection with viable pathogens is unknown. Here, we show that mice deficient for the CGRP receptor component RAMP1 have an improved anti-bacterial defense during the early, but not late, phase of polymicrobial septic peritonitis. The protective effect of Ramp1-deficiency was associated with reduced levels of IL-10 in plasma and peritoneal lavage fluid. Consistent with these findings, CGRP markedly increased IL-10 production of peritoneal and bone marrow-derived macrophages in response to short term stimulation with LPS in vitro. In addition, the lack of an intact CGRP receptor resulted in an increased recruitment and activation of neutrophils and caused an enhanced release of defensin-α1 in the peritoneal cavity. Considered together, our results identify the neuropeptide CGRP as a crucial immunosuppressive mediator impairing host defense during the early, but not late, phase of septic peritonitis.     

Simvastatin antagonizes CD40L secretion, CXC chemokine formation, and pulmonary infiltration of neutrophils in abdominal sepsis

Statins have been reported to exert anti-inflammatory actions and protect against septic organ dysfunction. Herein, we hypothesized that simvastatin may attenuate neutrophil activation and lung damage in abdominal sepsis. Male C57BL/6 mice were pretreated with simvastatin (0.5 or 10 mg/kg) before CLP. In separate groups, mice received an anti-CD40L antibody or a CXCR2 antagonist (SB225002) prior to CLP. BALF and lung tissue were harvested for analysis of neutrophil infiltration, as well as edema and CXC chemokine formation. Blood was collected for analysis of Mac-1 and CD40L expression on neutrophils and platelets, as well as soluble CD40L in plasma. Simvastatin decreased CLP-induced neutrophil infiltration and edema formation in the lung. Moreover, Mac-1 expression increased on septic neutrophils, which was significantly attenuated by simvastatin. Inhibition of CD40L reduced CLP-induced up-regulation of Mac-1 on neutrophils. Simvastatin prevented CD40L shedding from the surface of platelets and reduced circulating levels of CD40L in septic mice. CXC chemokine-induced migration of neutrophils in vitro was decreased greatly by simvastatin. Moreover, simvastatin abolished CLP-evoked formation of CXC chemokines in the lung, and a CXCR2 antagonist attenuated pulmonary accumulation of neutrophils. Our data suggest that the inhibitory effect of simvastatin on pulmonary accumulation of neutrophils may be related to a reduction of CD40L secretion into the circulation, as well as a decrease in CXC chemokine formation in the lung. Thus, these protective mechanisms help to explain the beneficial actions exerted by statins, such as simvastatin, in sepsis.     

Critical role of CD14 for production of proinflammatory cytokines and cytokine inhibitors during sepsis with failure to alter morbidity or mortality

We investigated the immunopathophysiologic responses during sepsis induced by cecal ligation and puncture (CLP) in CD4-deficient (CD14 knockout [CD14KO]) mice. Our studies were designed to specifically test the role of CD14 in the inflammatory response to sepsis and to ascertain if alterations would improve morbidity or mortality. Sepsis was induced using the CLP model with appropriate antibiotic treatment. The severity of sepsis increased in the CD14KO mice with increasing puncture size (18 gauge [18G], 21G, and 25G). Following CLP, body temperature (at 12 h) and gross motor activity levels of the sham and 25G CLP groups recovered to normal, while the 21G and 18G CLP groups exhibited severe hypothermia coupled with decreased gross motor activity and body weight. There were no significant differences in survival, temperature, body weight, or activity levels between CD14KO and control mice after 21G CLP. However, CD14KO mice expressed two- to fourfold less pro-inflammatory (interleukin-1beta [IL-1beta], tumor necrosis factor [TNF], and IL-6) and anti-inflammatory (IL-10, IL-1 receptor antagonist, and TNF receptors I and II) cytokines in the blood after 21G CLP. Plasma levels of the chemokines macrophage inflammatory protein 2alpha and KC were similarly reduced in CD14KO mice. A similar trend of decreased cytokine and cytokine inhibitor levels was observed in the peritoneal cavity of CD14KO mice. Our results indicate that the CD14 pathway of activation plays a critical role in the production of both pro-inflammatory cytokines and cytokine inhibitors but has minimal impact on the morbidity or mortality induced by the CLP model of sepsis.     

Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome

Severe acute respiratory syndrome (SARS) is a recently emerged infectious disease caused by a novel coronavirus, but its immunopathological mechanisms have not yet been fully elucidated. We investigated changes in plasma T helper (Th) cell cytokines, inflammatory cytokines and chemokines in 20 patients diagnosed with SARS. Cytokine profile of SARS patients showed marked elevation of Th1 cytokine interferon (IFN)-gamma, inflammatory cytokines interleukin (IL)-1, IL-6 and IL-12 for at least 2 weeks after disease onset, but there was no significant elevation of inflammatory cytokine tumour necrosis factor (TNF)-alpha, anti-inflammatory cytokine IL-10, Th1 cytokine IL-2 and Th2 cytokine IL-4. The chemokine profile demonstrated significant elevation of neutrophil chemokine IL-8, monocyte chemoattractant protein-1 (MCP-1), and Th1 chemokine IFN-gamma-inducible protein-10 (IP-10). Corticosteroid reduced significantly IL-8, MCP-1 and IP-10 concentrations from 5 to 8 days after treatment (all P < 0.001). Together, the elevation of Th1 cytokine IFN-gamma, inflammatory cytokines IL-1, IL-6 and IL-12 and chemokines IL-8, MCP-1 and IP-10 confirmed the activation of Th1 cell-mediated immunity and hyperinnate inflammatory response in SARS through the accumulation of monocytes/macrophages and neutrophils.     

An essential role of macrophage inflammatory protein 1alpha/CCL3 on the expression of host's innate immunities against infectious complications

Sepsis was induced by well-controlled cecal ligation and puncture (CLP) in macrophage inflammatory protein 1alpha (MIP-1alpha)/CCL3 knockout (CCL3(-/-)) and severe combined immunodeficiency (SCID) mice. CCL3(-/-) mice and their littermates (CCL3(+/+) mice) treated with anti-CCL3 monoclonal antibodies were susceptible (0-20% survival) to CLP-induced sepsis, and CCL3(-/-) mice supplemented with recombinant (r)CCL3 (250 ng/mouse) and CCL3(+/+) mice were resistant (70-80% survival). The resistance of SCID mice to CLP was markedly improved by the rCCL3 administration (88% survival), and SCID mice treated with saline were shown to be middling resistant to the same CLP (45% survival). However, the resistance of SCID-M mice (SCID mice depleted of the macrophage function) to CLP was not improved by the rCCL3 administration (11% survival), and 41% of SCID-M mice reconstituted with normal peritoneal macrophages and 79% of SCID-M mice inoculated with CCL3-treated peritoneal macrophages survived. In addition, the resistance of SCID-MN mice (SCID mice depleted of functional macrophages and neutrophils) to CLP was improved by the inoculation of CCL3-treated macrophages (78% survival), and all of SCID-MN mice inoculated with CCL3-treated neutrophils died. CCL3 is shown to be essential to the host resistance against bacterial sepsis. Macrophages but not neutrophils are highlighted as the major effector cells when protective innate immunities against sepsis are improved by CCL3.     

Mechanisms of acute inflammatory lung injury induced by abdominal sepsis

Sequestration of neutrophils and release of histotoxic mediators are considered important for the development of pathologic alterations of the lung defined as adult respiratory distress syndrome. Mechanisms of inflammatory lung injury caused by abdominal sepsis were investigated using the colon ascendens stent peritonitis (CASP) model that closely mimics the human disease. In the CASP model, a continuous leakage of intraluminal bacteria into the peritoneal cavity is induced by implantation of a stent in the ascending colon, generating a septic focus. In contrast to the cecal ligation and puncture model of peritonitis, survival of mice following CASP surgery is dependent on IFN-gamma, but independent of tumor necrosis factor (TNF). Here we show that the systemic inflammation induced by CASP surgery results in a rapid and profound increase of lung vascular permeability that was associated with the activation and recruitment of neutrophils to the lung. Activation of circulating granulocytes was characterized by increased production of serine proteinases and reactive oxygen metabolites, as well as elevated expression of cell surface Mac-1. Expression of MIP-2, KC, MIP-1alpha and E-selectin mRNA in lung was strongly increased within 3 h following CASP surgery, whereas up-regulation of IP-10, MCP-1 and P-selectin was delayed. In contrast, induction of RANTES, LIX, ICAM-1 and VCAM-1 mRNA was weak or not detectable after CASP surgery. Importantly, recruitment of leukocytes to the lung was normal in lipopolysaccharide-resistant mice, and was not affected by antibody neutralization of TNF or the chemokines MIP-2 and KC.     

Cholecystokinin protects rats against sepsis induced by Staphylococcus aureus

Staphylococcus aureus is a Gram-positive bacteria described as an important causative agent of sepsis. The contact between host leukocytes and bacteria activates the innate immune response. Nitric oxide, tumor necrosis factor (TNF)-α and interleukin (IL)-1β play a key role in increasing microbicidal activity and controlling cell influx into infectious focus. Contrarily, IL-10 acts as an anti-inflammatory cytokine and bacterial killing suppressor. Immunoregulatory properties have also been attributed to hormones, including cholecystokinin (CCK). CCK protects cardiovascular function and inhibits the inflammatory response induced by lipopolysaccharide, product derived from Gram-negative bacteria. Nevertheless, the role of CCK during Gram-positive infection remains a literature gap. Our aims were to investigate whether CCK protects rats against bacterial dissemination during sepsis induced by S. aureus. We determined whether CCK modulates local and systemic inflammatory response, as well as the cell migration into the infectious focus and the bactericidal capacity of leukocytes. Our results revealed that proglumide (nonselective CCK receptor antagonist) pretreated rats showed higher bacterial counts in blood and peritoneal lavage fluid (PLF) and reduced TNF-α and IL-10 levels in PLF. Moreover, the dissemination of S. aureus may be related to the failure of neutrophil and macrophage migration into the peritoneal cavity. Also, CCK improved the phagocytic and bactericidal ability of these inflammatory cells. Noteworthy is that the adoptive transfer of CCK-treated neutrophils and macrophages in septic rats improved immune defense, reducing bacterial number in blood and PLF. All together, our study clearly demonstrates an important protective role of CCK against sepsis induced by S. aureus.     

Modulation of human neutrophil and monocyte chemotaxis and superoxide responses by recombinant TNF-alpha and GM-CSF

The effects of recombinant TNF and GM-CSF on human peripheral blood neutrophil and monocyte chemotaxis and the superoxide response were studied. TNF exhibited a slight chemotactic activity for both cell types. Preincubation of neutrophils with as little as 40 units/ml strongly inhibited the neutrophil chemotaxis towards f-Met-Leu-Phe. The inhibition of monocyte chemotaxis required higher concentrations of TNF (greater than 400 units/ml). TNF at concentrations higher than 500 units/ml enhanced the generation of superoxide anions by neutrophils stimulated with f-Met-Leu-Phe. In contrast, TNF even at 2,000 units/ml did not prime monocytes for enhanced superoxide response. GM-CSF alone did not exhibit any chemotactic activity for any of the cell types tested. Preincubation of cells with GM-CSF inhibited chemotaxis of neutrophils but not of monocytes. GM-CSF was as potent as TNF in enhancing the generation of superoxide response by neutrophils. However, GM-CSF did not have any effect on monocyte superoxide response. The priming ability of TNF and GM-CSF on neutrophils was heat-sensitive. We conclude that TNF and GM-CSF play a more pronounced regulatory role on neutrophils than on monocytes. Inhibition of neutrophil chemotaxis followed by enhancement of the superoxide response by TNF and GM-CSF may provide an attractive mechanism by which these cytokines assist in fighting invading microorganisms.