Neutrophil leukocyte emigration induced by endotoxin. Mediator roles of interleukin 1 and tumor necrosis factor alpha 1

The hypothesis that cytokines mediate neutrophil emigration induced by endotoxin (LPS) was studied by examining the potency, the kinetics of neutrophil emigration, and the tachyphylaxis of intradermal sites with IL-1, TNF-alpha and LPS. Human rIL-1 alpha and IL-1 beta, synthetic lipid A, and LPS were several orders of magnitude more potent than human rTNF. The kinetic profiles of neutrophil emigration induced by IL-1 alpha, TNF, and LPS were characterized by minimal emigration in the first 30 min, followed by rapid and transient emigration. After the injection of LPS, the onset and the time at which the rate of emigration was maximal consistently appeared 30 min later than IL-alpha or TNF, suggesting that neutrophil emigration in response to LPS was mediated by a locally generated cytokine. IL-1 and TNF were then examined as potential secondary mediators of LPS-induced emigration by comparing the patterns of tachyphylaxis between LPS and IL-1 alpha or TNF; i.e., the magnitude of neutrophil emigration into inflammatory sites was compared with sites injected 6 h previously (desensitizing injections) with a cytokine or with LPS. Tachyphylaxis was dose dependent with each and also between the IL-1 species; therefore, when tachyphylaxis between the cytokines and LPS was examined, relatively higher doses were selected for the desensitizing injections than for the test injections. With this approach, desensitizing injections of IL-1 alpha diminished the neutrophil accumulation after LPS, and LPS also desensitized sites to IL-1 alpha. However, tachyphylaxis was not observed between TNF and LPS, or between TNF and IL-1 alpha. These data suggest that IL-1, but not TNF, is a potential mediator of LPS-induced neutrophil emigration.     

Early response cytokines and innate immunity: essential roles for TNF receptor 1 and type I IL-1 receptor during Escherichia coli pneumonia in mice

The early response cytokines, TNF and IL-1, have overlapping biologic effects that may function to propagate, amplify, and coordinate host responses to microbial challenges. To determine whether signaling from these early response cytokines is essential to orchestrating innate immune responses to intrapulmonary bacteria, the early inflammatory events induced by instillation of Escherichia coli into the lungs were compared in wild-type (WT) mice and mice deficient in both TNF receptor 1 (TNFR1) and the type I IL-1 receptor (IL1R1). Neutrophil emigration and edema accumulation induced by E. coli were significantly compromised by TNFR1/IL1R1 deficiency. Neutrophil numbers in the circulation and within alveolar septae did not differ between WT and TNFR1/IL1R1 mice, suggesting that decreased neutrophil emigration did not result from decreased sequestration or delivery of intravascular neutrophils. The nuclear translocation of NF-kappa B and the expression of the chemokine macrophage inflammatory protein-2 did not differ between WT and TNFR1/IL1R1 lungs. However, the concentration of the chemokine KC was significantly decreased in the bronchoalveolar lavage fluids of TNFR1/IL1R1 mice compared with that in WT mice. Thus, while many of the molecular and cellular responses to E. coli in the lungs did not require signaling by either TNFR1 or IL1R1, early response cytokine signaling was critical to KC expression in the pulmonary air spaces and neutrophil emigration from the alveolar septae.     

Evidence that a receptor-operated event on the neutrophil mediates neutrophil accumulation in vivo. Pretreatment of 111In-neutrophils with pertussis toxin in vitro inhibits their accumulation in vivo

The role of neutrophil chemoattractant receptors in neutrophil stimulation in vitro is well established, however, the precise mechanisms underlying local neutrophil accumulation at inflammatory sites in vivo have not been defined. A fundamental question that remains open is whether chemoattractants act on the endothelial cell or the neutrophil to initiate the process of neutrophil migration in vivo. To address this question we have investigated whether neutrophil accumulation in vivo can occur if chemoattractant receptor occupancy is uncoupled from neutrophil stimulation. For this purpose we have used pertussis toxin (PT) as the pharmacologic tool. We have investigated the effect of in vitro pretreatment of rabbit neutrophils with PT on their responses in vitro and on their accumulation in vivo. Pretreatment of rabbit neutrophils with PT inhibited FMLP- and C5a-, but not PMA- induced increases in CD18 expression, neutrophil adherence, and degranulation in vitro. This pretreatment procedure with PT inhibited the accumulation of radiolabeled neutrophils in vivo in response to intradermally injected FMLP, C5a, C5a des Arg, leukotriene B4, IL-8, and zymosan in rabbit skin. Further, in contrast to the in vitro results, PT inhibited the PMA-induced 111In-neutrophil accumulation in vivo. Interestingly, pretreatment of neutrophils with PT also inhibited accumulation in response to intradermally injected IL-1, despite the reports that IL-1 lacks neutrophil chemoattractant activity in vitro. Although the experimental techniques used cannot distinguish the different stages of neutrophil migration involved, these results suggest that the accumulation of neutrophils induced by local extravascular chemoattractants in vivo depends on a pertussis toxin-sensitive receptor operated event on the neutrophil itself. Further, PMA and IL-1 may release secondary chemoattractants in vivo.     

Vascular hyperpermeability induced by tumor necrosis factor and its augmentation by IL-1 and IFN-gamma is inhibited by selective depletion of neutrophils with a monoclonal antibody

We investigated whether various recombinant cytokines induce vascular hyperpermeability when intradermally injected into rats. Only TNF did so. Of the other cytokines examined (IL-1, IL-2, granulocyte-CSF, IFN-alpha, IFN-beta, IFN-gamma) none had this effect. The increase in vascular permeability was dose dependent, and the peak response was observed at 90 min after TNF injection. When mixtures of TNF and various other cytokines (IL-1, IL-2, granulocyte-CSF, IFN-alpha, IFN-beta, IFN-gamma) were injected, only IL-1 and IFN-gamma augmented TNF-induced vascular hyperpermeability, the increase occurring in a dose-dependent manner. The induction of vascular hyperpermeability by TNF and its enhancement by IL-1 and IFN-gamma were inhibited by selective depletion of peripheral blood neutrophils with i.p. administration of an anti-rat neutrophil mAb, RP-3. Reconstitution of neutrophils to the depleted rats by in situ injection of these cells, restored TNF increased vascular permeability.     

Mediators and regulation of neutrophil accumulation in inflammatory responses in lung: insights from the IgG immune complex model

Neutrophil trafficking in lung involves transendothelial migration, migration in tissue interstitium, and transepithelial migration. In a rat model of IgG immune complex-induced lung injury, it was demonstrated that neutrophil emigration involves regulatory mechanisms including complement activation, cytokine regulation, chemokine production, activation of adhesion molecules, and their respective counter receptors. The process is presumably initiated and modulated by the production of early response cytokines and chemokines from lung cells, especially from alveolar macrophages. TNF-alpha and IL-1 up-regulate intracellular adhesion molecule-1 (ICAM-1) and E-selectin, setting the stage for neutrophil migration through endothelium. The CXC chemokines, such as macrophage inflammatory protein (MIP)-2 and cytokine-inducible neutrophil chemoattractant (CINC), constitute chemokine gradient to orchestrate neutrophil migration in lung. Complement activation induced by IgG immune complex deposition is another important event leading to neutrophil accumulation in lung. Complement activation product C5a not only plays an important role in chemoattracting neutrophils into lung, but regulates adhesion molecules, chemokines, and cytokines expression. In addition, oxidative stress may regulate neutrophil accumulation in lung by modulation of adhesion molecule activation and chemokine production. In this review, we focus on the current knowledge of the mechanisms leading to accumulation of neutrophils during acute lung injury.     

Effect of cytokines on polymorphonuclear neutrophil infiltration in the mouse. Prostaglandin- and leukotriene-independent induction of infiltration by IL-1 and tumor necrosis factor

The i.p. injection of mice with highly purified recombinant human rIL-1 alpha or beta resulted in the rapid influx of a large number of polymorphonuclear neutrophils (PMN) into the peritoneal cavity. Significant increases in the number of PMN were induced by doses of IL-1 which ranged from 0.005 to 5 ng/injection. Interestingly the dose response for PMN influx was bell-shaped because 50 ng of IL-1 did not result in a significant increase in peritoneal PMN. IL-1 induced PMN infiltration was detectable by 1 h with peak levels of PMN obtained by about 2 h, followed by a subsequent decline by 24 h. Other cytokines, IL-2, IFN-gamma, IFN alpha beta, granulocyte-CSF, granulocyte-macrophage-CSF, IL-3, TNF-alpha, and TNF-beta were compared to IL-1 for their ability to induce a PMN influx into the peritoneum. Only TNF-alpha or TNF-beta (lymphotoxin) were able to induce a significant influx of PMN within 2 h. However, based on total protein administered, about 100 times more TNF than IL-1 was required to produce a comparable PMN infiltration. Intraperitoneal injection of inhibitors of the cyclooxygenase or lipoxygenase pathways did not inhibit the IL-1-induced influx of PMN. Also, neither IL-1 nor TNF triggered an increase in PG or leukotriene release from peritoneal cells in vitro. Furthermore, direct peritoneal injection of leukotriene B4, a potent PMN chemoattractant in vitro, did not induce any significant increase in PMN in the peritoneal cavity indicating that chemotactic activity alone is insufficient for inducing peritoneal infiltration. These results suggest that the local production of very low levels of IL-1 in vivo would be sufficient to initiate a sequence of events that results in a rapid accumulation of PMN. Because IL-1 was not chemotactic for PMN in vitro, our data suggest that IL-1 induces production of factors that are chemotactic for PMN. Alternatively, IL-1 may act on other stages of the complex sequence of events that regulates the emigration of PMN into tissue sites in vivo. The synergy apparent in PMN influx when suboptimal concentrations of IL-1 and TNF were injected suggests that the local production of very low concentrations of these cytokines in situ could play a critical role in the emigration of PMN during infection.     

Neurokinin-1 receptor agonists are involved in mediating neutrophil accumulation in the inflamed, but not normal, cutaneous microvasculature: an in vivo study using neurokinin-1 receptor knockout mice

We have used tachykinin neurokinin-1 receptor (NK1 receptor) knockout mice to learn of the link between NK1 receptors and neutrophil accumulation in normal naive skin, as compared with inflamed skin. Intradermal substance P (300 pmol) induced edema formation in wild-type mice, but not in NK1 knockout mice, as expected. However, in contrast to IL-1beta (0.3 pmol), substance P did not induce neutrophil accumulation in wild-type mice. IL-1beta-induced neutrophil accumulation was similar in wild-type and knockout mice, but a significant (p < 0.05) contributory effect of added NK1 agonists, which by themselves have no effect on neutrophil accumulation in normal skin, was observed. The results support the concept that NK1 agonists such as substance P cannot act on their own to mediate neutrophil accumulation in naive skin and provide direct evidence that in inflamed skin, under certain circumstances, the NK1 receptor can play a pivotal role in modulating neutrophil accumulation during the ongoing inflammatory process. We investigated responses to two inflammatory stimuli (carrageenin and zymosan). Neutrophil accumulation was significantly attenuated (p < 0.001) in carrageenin- but not zymosan-induced inflammation in NK1 knockout mice. The carrageenin (500 microg)-induced response was inhibited (p < 0.05) by a NK1 receptor antagonist, SR140333 (480 nmol/kg i.v. at -5 min), in the wild-type group. The bradykinin B1 and B2 receptor antagonists (desArg9[Leu8]bradykinin and HOE 140) each reduced neutrophil accumulation to carrageenin in wild-type animals (p < 0.05), but did not cause further reduction of the suppressed response of knockout mice. The results provide evidence that kinin receptors participate in NK1 receptor-dependent neutrophil accumulation in inflamed mouse skin.     

Tumor Necrosis Factor,but Not Neutrophils,Alters the Metabolic Profile in Acute Experimental Arthritis

Metabolic alterations are associated with arthritis apart from obesity. However, it is still unclear which is the underlying process behind these metabolic changes. Here, we investigate the role of tumor necrosis factor (TNF) in this process in an acute model of antigen-induced arthritis (AIA). Immunized male BALB/c mice received an intra-articular injection of PBS (control) or methylated bovine serum albumin (mBSA) into their knees, and were also pre-treated with different drugs: Etanercept, an anti-TNF drug, DF2156A, a CXCR1/2 receptor antagonist, or a monoclonal antibody RB6-8C5 to deplete neutrophils. Local challenge with mBSA evoked an acute neutrophil influx into the knee joint, and enhanced the joint nociception, along with a transient systemic metabolic alteration (higher levels of glucose and lipids, and altered adipocytokines). Pre-treatment with the conventional biological Etanercept, an inhibitor of TNF action, ameliorated the nociception and the acute joint inflammation dominated by neutrophils, and markedly improved many of the altered systemic metabolites (glucose and lipids), adipocytokines and PTX3. However, the lessening of metabolic changes was not due to diminished accumulation of neutrophils in the joint by Etanercept. Reduction of neutrophil recruitment by pre-treating AIA mice with DF2156A, or even the depletion of these cells by using RB6-8C5 reduced all of the inflammatory parameters and hypernociception developed after AIA challenge, but could not prevent the metabolic changes. Therefore, the induction of joint inflammation provoked acute metabolic alterations which were involved with TNF. We suggest that the role of TNF in arthritis-associated metabolic changes is not due to local neutrophils, which are the major cells present in this model, but rather due to cytokines.     

The role of histamine and other mediators in microvascular changes in acute inflammation

In addition to bronchial smooth muscle, histamine and other mediators act in bronchial asthma on the microcirculation of pulmonary connective tissue. The mediators induce enhanced blood flow, and by acting on the blood-tissue barrier, they induce increase in vasopermeability with edema and in more severe injury microhemorrhage and microthrombosis and infiltration of the connective tissue by leukocytes, predominantly neutrophils. The scheme proposed 10 years ago by K. F. Austen and co-workers in 1976 still holds true: a short-acting humoral-cellular phase is followed by a longer-acting pathopharmacologic or inflammatory phase. Some mediators, including histamine, serotonin, bradykinin, the sulfido leukotrienes and platelet activating factor, have a direct effect on endothelium and smooth muscle cells and more severe injury is due to mediators that exert their effect via neutrophils, which release lysosomal constituents. The intact complement-derived fragments C3a and C5a act directly and for a short period, as do histamine and the other direct-action mediators. The accumulation of neutrophils is brought about by C5a and its stable derivative C5ades Arg, leukotriene B4, PAF, and interleukin-1. Unlike the direct-action mediators, whose effect on the microcirculation does not extend beyond 20-25 min, those mediating via neutrophils have a delayed effect (peak 1-2 h), which parallels the neutrophil influx. The direct action mediators exert their effects on the microcirculation also in neutropenic animals. The most potent agents causing enhanced blood flow are the prostaglandins of the E class. Through the enhanced blood flow increase in vasopermeability, neutrophil emigration and hemorrhage are enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proinflammatory effects of copper deficiency on neutrophils and lung endothelial cells

Dietary copper deficiency increases the accumulation of circulating neutrophils in the rat lung microcirculation. This process includes neutrophil adhesion to, migration along, and emigration though the vascular endothelium. The current study was designed to examine the role of copper in each of these steps. Neutrophils were isolated from rats fed either a copper-adequate (CuA, 6.1 microg Cu/g diet) or copper-deficient diet (CuD, 0.3 microg Cu/g diet) for 4 weeks. First, transient and firm adhesion of neutrophils to P-selectin in a flow chamber showed there were more adhered CuD neutrophils than CuA ones. This effect is probably caused by the increased expression of CD11b that was observed in the current study. Second, the evaluation of neutrophil migration under agarose showed that the CuD neutrophils moved farther than the CuA group in response to IL-8 but not fMLP; this suggests an increased sensitivity to a CD11/CD18-independent signalling pathway. Third, the contractile mechanism of endothelial cells was studied. Elevated F-actin formation in Cu-chelated lung microvascular endothelial cells suggests that neutrophil emigration may be promoted by enhanced cytoskeletal reorganization of the endothelium during copper deficiency. Combined, these results support the theory that dietary copper deficiency has proinflammatory effects on both neutrophils and the microvascular endothelium that promote neutrophil-endothelial interactions.     

A critical role for sphingosine kinase in anaphylatoxin-induced neutropenia, peritonitis, and cytokine production in vivo

The aim of our study was to investigate the roles played by sphingosine kinase (SPHK) in the anaphylatoxin C5a-triggered responses in vivo. Our data show that i.v. administration of C5a triggers a rapid neutropenic response, but pretreating mice with the SPHK inhibitor, N,N-dimethylsphingosine (DMS), 10 min before the C5a i.v. administration substantially inhibited the C5a-triggered neutropenia. Similarly the i.v. administration of C5a caused a rapid increase in the serum levels of TNF-alpha and IL-6, and this increase in cytokine levels was blocked by DMS. We then induced acute peritonitis with C5a. The C5a i.p. injection triggered a fast recruitment of neutrophils, later followed by monocytes, into the peritoneal cavity. Vascular permeability was also observed: when we i.v. injected Evans blue before C5a i.p. injection, we could observe a continued influx of the dye into the peritoneum. In mice pretreated with DMS, there was a significant reduction on the C5a-triggered neutrophil and monocyte infiltration, as well as a marked reduction on the Evans blue influx. Our data also show that the i.p. administration of C5a caused a rapid increase in TNF-alpha and IL-6 levels in the peritoneal cavity, and this increase in cytokine levels was substantially inhibited in mice pretreated with the SPHK inhibitor. Taken together, these observations suggest a potential role for SPHK in the C5a-triggered inflammatory responses in vivo.     

Comparison of the effects of IL-1 alpha and TNF-alpha on phagocyte accumulation and murine antibacterial immunity

IL-1 and TNF both are reported to increase host antibacterial resistance. To directly compare their effects on tissue phagocyte accumulation and antibacterial activity, we infused recombinant human IL-1 alpha and TNF-alpha into C3H/HeJ mice. Although IL-1, at a dose of 1 microgram/day, did not significantly elevate blood neutrophil concentrations, it increased the number of PMNs within the spleen three to fourfold within 2 days. Similar neutrophil accumulation also occurred in the lungs, bone marrow, and liver of treated animals without detectable changes in macrophage numbers. IL-1 also increased myelopoiesis in the spleen by Days 3-4 of infusions. The capacity of splenocytes from IL-1-treated animals to kill Listeria monocytogenes in vitro and to suppress listeria proliferation in vivo after the intravenous infusion of bacteria both rose in parallel with PMN accumulation. Comparable doses of TNF also enhanced listeria killing in vivo but in contrast to IL-1, it significantly depressed peripheral blood neutrophil counts, and inhibited splenic neutrophil accumulation and in vitro listericidal activity in listeria-infected mice. Our results suggest that IL-1 enhances host resistance to infection by increasing tissue neutrophil accumulation while TNF protects by a different mechanism, despite a net inhibitory effect on neutrophil accumulation.     

Neutrophil influx in response to a peritoneal infection with Salmonella is delayed in lipopolysaccharide-binding protein or CD14-deficient mice

The induction of an adaptive immune response to a previously unencountered pathogen is a time-consuming process and initially the infection must be held in check by the innate immune system. In the case of an i.p. infection with Salmonella typhimurium, survival requires both CD14 and LPS-binding protein (LBP) which, together with Toll-like receptor 4 and myeloid differentiation protein 2, provide a sensitive means to detect bacterial LPS. In this study, we show that in the first hours after i.p. infection with Salmonella a local inflammatory response is evident and that concomitantly neutrophils flood into the peritoneum. This rapid neutrophil influx is dependent on TNF since it is 1) abolished in TNF KO mice and 2) can be induced by i.p. injection of TNF in uninfected animals. Neutrophil influx is not strictly dependent on the presence of either LBP or CD14. However, in their absence, no local inflammatory response is evident, neutrophil migration is delayed, and the mice succumb to the infection. Using confocal microscopy, we show that the neutrophils which accumulate in CD14 and LBP null mice, albeit with delayed kinetics, are nevertheless fully capable of ingesting the bacteria. We suggest that the short delay in neutrophil influx gives the pathogen a decisive advantage in this infection model.     

The sequential generation of neutrophil chemoattractant proteins in acute inflammation in the rabbit in vivo. Relationship between C5a and proteins with the characteristics of IL-8/neutrophil-activating protein 1

An in vivo experimental peritonitis model was investigated in the rabbit using zymosan as the inflammatory stimulus. After an i.p. injection of zymosan, exudate was removed at intervals and tested in the back skin of assay rabbits. Assay rabbits received i.v. injections of 125I-albumin and 111In-neutrophils, and the local accumulation of each label was measured in response to intradermal injections of exudate samples mixed with a potentiating dose of PGE2. When peritoneal exudate samples were tested in the presence of a specific anti-C5a antibody, virtually all the edema-inducing and neutrophil chemoattractant activity was abolished in samples taken up to 2 h after the zymosan injection. Later samples, however, contained increasing levels of a non-C5a component. In C5a-depleted 6-h exudate two peaks of inflammatory activity were separated using cation exchange HPLC. Evidence is presented that C5a itself is unable to stimulate the production of these activities. Both peaks of activity appear related to IL-8/NAP-1 as they inhibited the binding of 125I-IL-8/NAP-1 to human neutrophils.     

Human neutrophil Fc gamma RIIIB and formyl peptide receptors are functionally linked during formyl-methionyl-leucyl-phenylalanine-induced chemotaxis.

The formyl peptide receptor (FPR) and the glycosyl-phosphatidylinositol-linked type III receptor for the Fc portion of IgG (Fc gamma RIIIB; CD16) play important roles in various inflammatory responses in human neutrophils. The mechanisms of signaling by the glycosyl phosphatidylinositol-anchored Fc gamma RIIIB are not known. Therefore, we investigated the possibility that Fc gamma RIIIB and FPR may act in concert to mediate neutrophil functions. We observed that pretreatment of normal human neutrophils with Fab fragments of a mAb to the Fc gamma RIII (3G8) specifically inhibited their chemotaxis into micropore filters in response to the formylated peptides FMLP or formyl-norleucyl-leucyl-phenylalanine. Pretreatment of neutrophils with a saturating concentration of 3G8 Fab (100 nM or 5 micrograms/ml) followed by exposure to FMLP (0.5 to 500 nM) indicated that significant inhibition of chemotaxis was observed at peptide concentrations greater than 5 nM. However, 3G8 Fab had no effect on the neutrophil response to a wide range (0.05 to 500 nM) of other chemotactic factors, including C5a, leukotriene B4, IL-8 (neutrophil-activating peptide-1), and platelet-activating factor. Moreover, pretreatment of neutrophils with mAb to other cell surface molecules (decay-accelerating factor, Fc gamma RII, and HLA class I) did not affect chemotaxis to FMLP. Inhibition of movement was not due to degradation of FMLP by the cell surface endopeptidase 24.11 (CD10), because neutrophils pretreated with the CD10 inhibitor phosphoramidone and 3G8 Fab displayed the same altered response to FMLP as cells pretreated with 3G8 Fab alone. Ligation of the Fc binding site of Fc gamma RIIIB appears to be essential for altering the FMLP-induced response, since soluble aggregated IgG and other anti-Fc gamma RIII antibodies, all of which recognize the ligand binding site, mimic the inhibitory effect of the 3G8 Fab on FMLP-induced chemotaxis. In contrast, a mAb (214.1) that does not recognize the Fc binding site of Fc gamma RIIIB had no effect on FMLP-induced chemotaxis. Not only did anti-Fc gamma RIII inhibit neutrophil chemotaxis to FMLP in a filter-based migration assay, but 3G8 Fab also inhibited FMLP-induced neutrophil transendothelial migration. Scatchard plot analysis of radioligand binding experiments indicated that 3G8 Fab did not significantly alter the number of FMLP binding sites on neutrophils but significantly increased the affinity of the FPR for [3H]FMLP. Removal of greater than 80% of cell surface Fc gamma RIIIB by phospholipase C abolished the neutrophil chemotactic response to FMLP but did not affect movement toward C5a, IL-8, or leukotriene B4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Macrophages induce neutrophil apoptosis through membrane TNF, a process amplified by Leishmania major

Neutrophils are recruited to the site of parasite inoculation within a few hours of infection with the protozoan parasite Leishmania major. In C57BL/6 mice, which are resistant to infection, neutrophils are cleared from the site of s.c. infection within 3 days, whereas they persist for at least 10 days in susceptible BALB/c mice. In the present study, we investigated the role of macrophages (MPhi) in regulating neutrophil number. Inflammatory cells were recruited by i.p. injection of either 2% starch or L. major promastigotes. Neutrophils were isolated and cultured in the presence of increasing numbers of MPhi. Extent of neutrophil apoptosis positively correlated with the number of MPhi added. This process was strictly dependent on TNF because MPhi from TNF-deficient mice failed to induce neutrophil apoptosis. Assays using MPhi derived from membrane TNF knock-in mice or cultures in Transwell chambers revealed that contact with MPhi was necessary to induce neutrophil apoptosis, a process requiring expression of membrane TNF. L. major was shown to exacerbate MPhi-induced apoptosis of neutrophils, but BALB/c MPhi were not as potent as C57BL/6 MPhi in this induction. Our results emphasize the importance of MPhi-induced neutrophil apoptosis, and membrane TNF in the early control of inflammation.     

Increased susceptibility of TNF-alpha lymphotoxin-alpha double knockout mice to systemic candidiasis through impaired recruitment of neutrophils and phagocytosis of Candida albicans.

TNF-alpha and lymphotoxin-alpha (LT) are members of the TNF family, and these cytokines play crucial roles in the defense against infection with Candida albicans. The aim of the present study was to investigate the role of endogenous TNF and LT during disseminated candidiasis in TNF-/-LT-/- knockout mice. The TNF- and LT-deficient animals had a significantly increased mortality following C. albicans infection compared with control mice, and this was due to a 10- to 1000-fold increased outgrowth of the yeast in their organs. No differences between TNF-/-LT-/- mice and TNF+/+LT+/+ were observed when mice were rendered neutropenic, suggesting that activation of neutrophils mediates the beneficial effects of endogenous TNF and LT. Histopathology of the organs, combined with neutrophil recruitment experiments, showed a dramatic delay in the neutrophil recruitment at the sites of Candida infection in the TNF-/-LT-/- mice. Moreover, the neutrophils of deficient animals were less potent to phagocytize Candida blastospores than control neutrophils. In contrast, the killing of Candida and the oxygen radical production did not differ between neutrophils of TNF-/-LT-/- and TNF+/+LT+/+ mice. Peak circulating IL-6 was significantly higher in TNF-/-LT-/- mice during infection. Peritoneal macrophages of TNF-/-LT-/- mice did not produce TNF, and synthesized significantly lower amounts of IL-1alpha, IL-1beta, IL-6, and macrophage-inflammatory protein-1alpha than macrophages of TNF+/+LT+/+ animals did. In conclusion, endogenous TNF and/or LT contribute to host resistance to disseminated candidiasis, and their absence in TNF-/-LT-/- mice renders the animals susceptible through impaired recruitment of neutrophils and impaired phagocytosis of C. albicans.     

Divergent signaling pathways in phagocytic cells during sepsis

Neutrophil accumulation in the lung plays a pivotal role in the pathogenesis of acute lung injury during sepsis. Directed movement of neutrophils is mediated by a group of chemoattractants, especially CXC chemokines. Local lung production of CXC chemokines is intensified during experimental sepsis induced by cecal ligation and puncture (CLP), as reflected by rising levels of MIP-2 and cytokine-induced neutrophil chemoattractant-1 in bronchoalveolar lavage fluids. Alveolar macrophages are primed and blood neutrophils are down-regulated for production of MIP-2 and cytokine-induced neutrophil chemoattractant production in response to LPS and C5a. Under these conditions of stimulation, activation of MAPKs (p38, p42/p44) occurs in sham neutrophils but not in CLP neutrophils, while under the same conditions phosphorylation of p38 and p42/p44 occurs in both sham and CLP alveolar macrophages. These data indicate that, under septic conditions, there is impaired signaling in neutrophils and enhanced signaling in alveolar macrophages, resulting in CXC chemokine production, and C5a appears to play a pivotal role in this process. As a result, CXC chemokines increase in lung, setting the stage for neutrophil accumulation in lung during sepsis.     

Accumulation of 111In-neutrophils in rabbit skin in allergic and non-allergic inflammatory reactions in vivo. Inhibition by neutrophil pretreatment in vitro with a monoclonal antibody recognizing the CD18 antigen

The mAb 60.3 recognizes the neutrophil CD18 Ag. We have investigated the effect of in vitro pretreatment of radiolabeled neutrophils with mAb 60.3 on their accumulation in vivo. Further, we have compared the in vivo effects of mAb 60.3 with its effects on neutrophil adherence in vitro. Neutrophil accumulation in vivo was measured in response to: 1) exogenous mediators FMLP, C5a des Arg, LTB4 and IL-1; 2) endogenous mediators generated in a non-allergic inflammatory reaction induced by zymosan; and 3) endogenous mediators generated in two allergic inflammatory reactions, a passive cutaneous anaphylactic reaction and a reversed passive Arthus reaction in rabbit skin. Pretreatment of neutrophils with mAb 60.3 inhibited their accumulation in all the responses. The results demonstrate that there is a common mechanism mediating neutrophil accumulation in these inflammatory reactions. Neutrophils pretreated with mAb 60.3 were also unresponsive to chemoattractants in in vitro adherence assays. However, the antibody-treated neutrophils responded normally to FMLP and C5a with respect to granular enzyme release. These results suggest that the basal expression of CD18 Ag is important for the adherence of neutrophils to microvascular endothelial cells stimulated by the local generation, or administration, of chemical mediators in vivo. Despite the fact that mediators such as FMLP can increase CD18 expression in vitro, it appears more likely that such mediators act in vivo by inducing a conformational change in the basally expressed neutrophil adhesive molecules.