Circulating cytokines and endotoxin are not necessary for the activation of the sickness or corticosterone response produced by peripheral E. coli challenge.

Peripheral administration of a variety of inflammatory stimuli, such as endotoxin or cytokines, induces an orchestrated set of brain-mediated events referred to as the sickness response. The mechanism for how immune products signal the brain is not clear, but accumulating evidence supports the existence of neural as well as blood-borne pathways. Although endotoxin or cytokine administration results in sickness responses, few data exist regarding the role of circulating endotoxin or cytokines in the induction of sickness during a real bacterial infection. Thus the present studies examined whether subcutaneously administered Escherichia coli can activate sickness responses and whether circulating endotoxin and/or proinflammatory cytokines are a prerequisite for these responses. Male Sprague-Dawley rats were injected subcutaneously with one of three doses (2.5 x 10(7), 2.5 x 10(8), 2.5 x 10(9) colony-forming units) of replicating E. coli, a ubiquitous bacterial strain, or vehicle. Core body temperature (Tc) and activity were measured for 3 days after the injection. A second set of groups of animals were killed 3, 6, 12, 18, 24, and 48 h after the injection, and blood samples and brains were collected. Injections dose dependently and consistently increased Tc and decreased activity, with increases in Tc beginning 4 h after the injection. In addition, E. coli significantly increased serum interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha and brain IL-1beta levels beginning at the 6-h time point. Corticosterone and endotoxin were first elevated in the circulation at 3 and 18 h after the injection, respectively. Because fever onset preceded brain cytokine induction, we also examined cytokine levels in the serum, brain, and inflammation site 2 and 4 h after injection. Cytokines were elevated at the inflammation site but were not detectable in the serum or brain at 2 and 4 h. We conclude that subcutaneous injection of replicating E. coli induces a consistent and naturalistic infection that includes features of the sickness response as well as increases in circulating, brain, and inflammation site tissue cytokines. In addition, injection of replicating E. coli produces a robust fever and corticosterone response at a time when there are no detectable increases in circulating cytokines or endotoxin. These results suggest that elevated levels of circulating cytokines and endotoxin are not necessary for the activation of the sickness or corticosterone response. Therefore, fever, activity reduction, and corticosterone elevation induced by E. coli infection may have been evoked by a neural, rather than a humoral, pathway from the periphery to the brain.     

Leukocyte antibacterial functions are not impaired by perfluorocarbon exposure in vitro

Application of liquid, aerosolized, and vaporized perfluorocarbons (PFC) in acute lung injury has shown anti-inflammatory effects. Although this may be beneficial in states of pulmonary hyperinflammation, it also could increase susceptibility to nosocomial lung infection. We hypothesized that PFC impair cellular host defense and therefore investigated in an in vitro model the influence of perfluorohexane (PFH) on crucial mechanisms of bacterial elimination in human neutrophils and monocytes. Using scanning and transmission electron microscopy, we could show membrane-bound and ingested PFH particles that morphologically did not alter adherence and phagocytosis of Escherichia coli or leukocyte viability. The amount of adherent and phagocytosed bacteria as determined by flow cytometry was not influenced in cells only pretreated with PFH for 1 and 4 h. When PFH was present during E. coli challenge, bacterial adherence was decreased in polymorphonuclear neutrophils, but respective intracellular uptake was not impaired and was even significantly promoted in monocytes. Overall, E. coli-induced respiratory burst capacity was not reduced by PFH. Our findings provide evidence that key functions of innate host defense are not compromised by PFH treatment in vitro.     

Differential activation of RAGE by HMGB1 modulates neutrophil-associated NADPH oxidase activity and bacterial killing

The receptor for advanced glycation end products (RAGE) plays an important role in host defense against bacterial infection. In the present experiments, we investigated the mechanisms by which RAGE contributes to the ability of neutrophils to eradicate bacteria. Wild-type (RAGE(+/+)) neutrophils demonstrated significantly greater ability to kill Escherichia coli compared with RAGE(-/-) neutrophils. After intraperitoneal injection of E. coli, increased numbers of bacteria were found in the peritoneal fluid from RAGE(-/-) as compared with RAGE(+/+) mice. Exposure of neutrophils to the protypical RAGE ligand AGE resulted in activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and enhanced killing of E. coli, and intraperitoneal injection of AGE enhanced bacterial clearance during peritonitis. However, incubation of neutrophils with high mobility group box 1 protein (HMGB1), which also binds to RAGE, diminished E. coli-induced activation of NADPH oxidase in neutrophils and bacterial killing both in vitro and in vivo. Deletion of the COOH-terminal tail of HMGB1, a region necessary for binding to RAGE, abrogated the ability of HMGB1 to inhibit bacterial killing. Incubation of neutrophils with HMGB1 diminished bacterial or AGE-dependent activation of NADPH oxidase. The increase in phosphorylation of the p40(phox) subunit of NADPH oxidase that occurred after culture of neutrophils with E. coli was inhibited by exposure of the cells to HMGB1. These results showing that HMGB1, through RAGE-dependent mechanisms, diminishes bacterial killing by neutrophils as well as NADPH oxidase activation provide a novel mechanism by which HMGB1 can potentiate sepsis-associated organ dysfunction and mortality.     

Naturally occurring hypothermia is more advantageous than fever in severe forms of lipopolysaccharide- and Escherichia coli-induced systemic inflammation

The natural switch from fever to hypothermia observed in the most severe cases of systemic inflammation is a phenomenon that continues to puzzle clinicians and scientists. The present study was the first to evaluate in direct experiments how the development of hypothermia vs. fever during severe forms of systemic inflammation impacts the pathophysiology of this malady and mortality rates in rats. Following administration of bacterial lipopolysaccharide (LPS; 5 or 18 mg/kg) or of a clinical Escherichia coli isolate (5 × 10(9) or 1 × 10(10) CFU/kg), hypothermia developed in rats exposed to a mildly cool environment, but not in rats exposed to a warm environment; only fever was revealed in the warm environment. Development of hypothermia instead of fever suppressed endotoxemia in E. coli-infected rats, but not in LPS-injected rats. The infiltration of the lungs by neutrophils was similarly suppressed in E. coli-infected rats of the hypothermic group. These potentially beneficial effects came with costs, as hypothermia increased bacterial burden in the liver. Furthermore, the hypotensive responses to LPS or E. coli were exaggerated in rats of the hypothermic group. This exaggeration, however, occurred independently of changes in inflammatory cytokines and prostaglandins. Despite possible costs, development of hypothermia lessened abdominal organ dysfunction and reduced overall mortality rates in both the E. coli and LPS models. By demonstrating that naturally occurring hypothermia is more advantageous than fever in severe forms of aseptic (LPS-induced) or septic (E. coli-induced) systemic inflammation, this study provides new grounds for the management of this deadly condition.     

Tumor necrosis factor-alpha activation of the c-Jun N-terminal kinase pathway in human neutrophils. Integrin involvement in a pathway leading from cytoplasmic tyrosine kinases apoptosis

The intensity and duration of an inflammatory response depends on the balance of factors that favor perpetuation versus resolution. At sites of inflammation, neutrophils adherent to other cells or matrix components are exposed to tumor necrosis factor-alpha (TNFalpha). Although TNFalpha has been implicated in induction of pro-inflammatory responses, it may also inhibit the intensity of neutrophilic inflammation by promoting apoptosis. Since TNFalpha is not only an important activator of the stress-induced pathways leading to p38 MAPk and c-Jun N-terminal kinase (JNK) but also a potent effector of apoptosis, we investigated the effects of TNFalpha on the JNK pathway in adherent human neutrophils and the potential involvement of this pathway in neutrophil apoptosis. Stimulation with TNFalpha was found to result in beta2 integrin-mediated activation of the cytoplasmic tyrosine kinases Pyk2 and Syk, and activation of a three-part MAPk module composed of MEKK1, MKK7, and/or MKK4 and JNK1. JNK activation was attenuated by blocking antibodies to beta2 integrins, the tyrosine kinase inhibitors, genistein, and tyrphostin A9, a Pyk2-specific inhibitor, and piceatannol, a Syk-specific inhibitor. Exposure of adherent neutrophils to TNFalpha led to the rapid onset of apoptosis that was demonstrated by augmented annexin V binding and caspase-3 cleavage. TNFalpha-induced increases in annexin V binding to neutrophils were attenuated by blocking antibodies to beta2 integrins, and the caspase-3 cleavage was attenuated by tyrphostin A9. Hence, exposure of adherent neutrophils to TNFalpha leads to utilization of the JNK-signaling pathways that may contribute to diverse functional responses including induction of apoptosis and subsequent resolution of the inflammatory response.     

Escherichia coli-specific T lymphocytes in experimental pyelonephritis

We have assessed the phenotype and specificity of infiltrating mononuclear cells in a model of unilateral ascending acute pyelonephritis induced in rats with nephritogenic Escherichia coli or Pseudomonas aeruginosa. Histologic examination showed a predominance of mononuclear cells in the interstitium at all periods examined (4, 8, 15, 21, and 25 days), although at 4 and 8 days neutrophils were also abundant. Most of the mononuclear cells had the morphologic appearance of large lymphocytes. Immunoperoxidase studies with mAb showed that most of the mononuclear cells were W3/25+; many were W3/13+ and a small proportion were OX8+. Many of the mononuclear cells were Ia+. T cells were propagated in IL-2-containing media from small fragments of renal tissue with pyelonephritic lesions. Most of the propagated cells were W3/25+; fewer than (10%) were OX8+ or Ia+. T cells propagated from kidneys infected with E. coli responded, in proliferation assays, to the infecting strain or other E. coli strains, but not to P. aeruginosa or enterococci. The response to non-p-pilus-bearing E. coli was as great or greater than to E. coli with adhesins. T cells derived from lesions induced by P. aeruginosa responded to the infecting organisms, but not to E. coli. The response to the infecting organism (E. coli or P. aeruginosa) was MHC restricted, as indicated by the requirement for syngeneic APC. The results show that large numbers of T lymphocytes, especially with the "helper/inducer" phenotype, accumulate in the lesions of acute pyelonephritis in rats. Among the infiltrating T lymphocytes are activated cells and cells with specific reactivity to the infecting bacteria (or related strains). The findings indicate that T lymphocytes play a role within the kidney in response to the invading bacteria.     

Effects of endotoxin and tumor necrosis factor alpha on regional brain neurotransmitters in mice

Alterations in regional brain concentration of dopamine (DA), norepinephrine (NE), serotonin (5-HT) and their metabolites were investigated in male BALB/c mice injected intraperitoneally with bacterial lipopolysaccharide (LPS, 2 mg kg(-1)) or recombinant murine tumor necrosis factor alpha (TNFalpha, 0.1 mg kg(-1)) at 2, 6, 12 and 24 h after the injection. At 2 h post-injection the LPS administration resulted in hypothermia, which was not apparent at later time points. No consistent effects were observed by either LPS or TNFalpha on peripheral leukocyte counts or plasma transaminase levels. Both LPS and TNFalpha slightly elevated NE metabolism in the striatum at 2-12 h. Concentrations of DA and its metabolites were significantly elevated only in the hypothalamus following TNFalpha at 24 h. Tumor necrosis factor alpha exerted pronounced effects on 5-HT metabolism in most brain regions at 2 h. Results suggest that the effect of LPS is more complex compared with TNFalpha because of the endogenous production of other cytokines including the TNFalpha.     

Production of granulocyte colony-stimulating factor in the nonspecific acute phase response enhances host resistance to bacterial infection

Mice mounting an acute phase response, induced by sterile inflammation after a single s.c. injection of casein 24 h beforehand, were remarkably protected against lethal infection with Gram-positive or Gram-negative bacteria. This was associated with enhanced early clearance of bacteremia, greater phagocytosis and oxidative burst responses by neutrophils, and enhanced recruitment of neutrophils into tissues compared with control, nonacute phase mice. Casein-induced inflammation was also associated with increased concentrations of G-CSF in serum, and administration of neutralizing Ab to this cytokine completely abrogated protection against Escherichia coli infection after casein pretreatment. Injection of recombinant murine G-CSF between 3 and 24 h before infection conferred the same protection as casein injection. In contrast, the casein-induced acute phase response affected neither serum values of TNF-alpha, IL-1 beta, or IL-6 after E. coli infection nor susceptibility to LPS toxicity. Furthermore, protection against infection was unaffected in IL-1R knockout mice, which have deficient acute phase plasma protein responses, or after nonspecific inhibition of acute phase protein synthesis by D-galactosamine or specific depletion of complement C3 by cobra venom factor. Increased production of G-CSF in the acute phase response is thus a key physiological component of host defense, and pretreatment with G-CSF to prevent bacterial infection in at-risk patients now merits further study, especially in view of increasing bacterial resistance to antibiotics.     

A novel Mutein of TNFalpha Containing the Arg-Gly-Asp Sequence Shows Reduced Toxicity in Intestine

The effects of human tumour necrosis factor-alpha (TNFalpha), or its mutein (F4168) having the cell adhesive Arg-Gly-Asp sequence at the N-terminus, on intestinal injury, were examined. Histopathological examination revealed that an intravenous injection of TNFalpha resulted in marked haemorrhage or oedema in the caecum of rats, whereas F4168 showed no such effects even at the same therapeutic dose. Moreover, the number of neutrophils that adhered to endothelial cells or infiltrated the mucosal tissue was much higher after TNFalpha injection compared with F4168 in vivo. The enhanced adhesion of neutrophils on to human umbilical vein endothelial cells also occurred when the latter were pre-stimulated with TNFalpha but not with F4168 in vitro. The expression of the cell adhesion molecules including endothelial leukocyte adhesion molecule-1 or intercellular adhesion molecule-1 on F4168- stimulated human umbilical vein endothelial ceils was significantly lower than that stimulated with TNFalpha. These results suggest that the Arg-Gly-Asp sequence introduced into the TNFalpha molecule abrogates the side effect of this cytokine such as tissue injury or shock, and that F4168 could be useful for systemic therapy.     

Involvement of KC, MIP-2, and MCP-1 in leukocyte infiltration following injection of necrotic cells into the peritoneal cavity

Although necrotic cells are known to induce inflammation in vivo, the underlying mechanism remains largely unexplored. In order to examine the mechanism, we used an inflammation model induced by injection of necrotic leukemic P388 cells into the peritoneal cavity in this study. The injection of necrotic cells induced the infiltration of neutrophils and subsequently that of monocytes/macrophages. In agreement with this, the injection also induced the production of KC and MIP-2, and subsequently that of MCP-1. Although the level of KC was higher than that of MIP-2, both anti-KC Ab and anti-MIP-2 Ab significantly inhibited the infiltration of neutrophils. Antibodies against CXCR2, a sole receptor for KC and MIP-2, almost completely inhibited the infiltration of neutrophils and monocytes/macrophages. Anti-MCP-1 Ab, on the other hand, inhibited the infiltration of monocytes/macrophages but not neutrophils. These results indicate that KC and MIP-2 play important roles in the infiltration of neutrophils into the site of injection of necrotic cells and that neutrophils may regulate monocyte/macrophage infiltration in our model.     

Catalase and superoxide dismutase in Escherichia coli

We assessed the roles of intrabacterial catalase and superoxide dismutase in the resistance of Escherichia coli to killing by neutrophils. E. coli in which the synthesis of superoxide dismutase and catalase were induced by paraquat 10-fold and 5-fold, respectively, did not resist killing by neutrophils. When bacteria were allowed to recover from the toxicity of paraquat for 1 h on ice and for 30 min at 37 degrees C, they still failed to resist killing by neutrophils. Induction of the synthesis of catalase 9-fold by growth in the presence of phenazine methosulfate did not render E. coli resistant to killing by either neutrophils or by H2O2 itself. The lack of protection by intrabacterial catalase from killing by neutrophils could not be attributed to an impermeable bacterial membrane; the evolution of O2 from H2O2 was no less rapid in suspensions of E. coli than in lysates. The failure of intrabacterial catalase or superoxide dismutase to protect bacteria from killing by neutrophils might indicate either that the flux of O-2 and H2O2 in the phagosome is too great for the intrabacterial enzymes to alter or that the site of injury is at the bacterial surface.     

Escherichia coli induces apoptosis and proliferation of mammary cells

Mammary cell apoptosis and proliferation were assessed after injection of Escherichia coli into the left mammary quarters of six cows. Bacteriological analysis of foremilk samples revealed coliform infection in the injected quarters of four cows. Milk somatic cell counts increased in these quarters and peaked at 24 h after bacterial injection. Body temperature also increased, peaking at 12 h postinjection. The number of apoptotic cells was significantly higher in the mastitic tissue than in the uninfected control. Expression of Bax and interleukin-1beta converting enzyme increased in the mastitic tissue at 24 h and 72 h postinfection, whereas Bcl-2 expression decreased at 24 h but did not differ significantly from the control at 72 h postinfection. Induction of matrix metalloproteinase-9, stromelysin-1 and urokinase-type plasminogen activator was also observed in the mastitic tissue. Moreover, cell proliferation increased in the infected tissue. These results demonstrate that Escherichia coli-induced mastitis promotes apoptosis and cell proliferation.     

Neutrophils and the calcium-binding protein MRP-14 mediate carrageenan-induced antinociception in mice

BACKGROUND: We have previously shown that the calcium-binding protein MRP-14 secreted by neutrophils mediates the antinociceptive response in an acute inflammatory model induced by the intraperitoneal injection of glycogen in mice. AIM: In an attempt to broaden the concept that neutrophils and MRP-14 controls inflammatory pain induced by different type of irritants, in the present study, after demonstrating that carrageenan (Cg) also induces atinociception in mice, we investigated the participation of both neutrophils and MRP-14 in the phenomenon. METHODS: Male Swiss mice were injected intraperitoneally with Cg and after different time intervals, the pattern of cell migration of the peritoneal exudate and the nociceptive response of animals submitted to the writhing test were evaluated. The participation of neutrophils and of the MRP-14 on the Cg effect was evaluated by systemic inoculation of monoclonal antibodies anti-granulocyte and anti-MRP-14. RESULTS: Our results demonstrate that the acute neutrophilic peritonitis evoked by Cg induced antinociception 2, 4 and 8 h after inoculation of the irritant. Monoclonal antibodies anti-granulocyte or anti-MRP-14 reverts the antinociceptive response only 2 and 8 h after Cg injection. The antibody anti-MRP-14 partially reverts the antinociception observed after 4 h of Cg injection while the anti-granulocyte antibody enhances this effect. This effect is reverted by simultaneous treatment of the animals with both antibodies. After 4 h of Cg injection in neutrophil-depleted mice a significant expression of the calcium-binding protein MRP-14 was detected in the cytoplasm of peritoneal macrophages. This suggests that the enhancement of the effect observed after treatment with the anti-neutrophil antibody may be due to secretion of MRP-14 by macrophages. It has also been demonstrated that endogenous opioids and glucocorticoids are not involved in the antinociception observed at the 4th hour after Cg injection. CONCLUSION: These data support the hypothesis that neutrophils and the calcium-binding protein MRP-14 are participants of the endogenous control of inflammatory pain in mice despite the model of acute inflammation used.     

Dendritic cells express CCR7 and migrate in response to CCL19 (MIP-3beta) after exposure to Helicobacter pylori

Helicobacter pylori infection induces chronic inflammation in the gastric mucosa with a marked increase in the number of lymphoid follicles consisting of infiltrating B and T cells, neutrophils, dendritic cells (DC) and macrophages. It has been suggested that an accumulation of mature DC in the tissue, resulting from a failure of DC to migrate to lymph nodes, may contribute to this chronic inflammation. Migration of DC to lymph nodes is regulated by chemokine receptor CCR7, expressed on mature DC, and the CCR7 ligands CCL19 and CCL21. In this study we analysed the maturation, in vitro migration and cytokine production of human DC after stimulation with live H. pylori. For comparison, DC responses to non-pathogenic Escherichia coli bacteria were also evaluated. Stimulation with H. pylori induced maturation of DC, i.e. up-regulation of the chemokine receptors CCR7 and CXCR4 and the maturation markers HLA-DR, CD80 and CD86. The H. pylori-stimulated DC also induced CD4(+) T-cell proliferation. DC stimulated with H. pylori secreted significantly more interleukin (IL)-12 compared to DC stimulated with E. coli, while E. coli-stimulated DC secreted more IL-10. Despite low surface expression of CCR7 protein following stimulation with H. pylori compared to E. coli, the DC migrated equally well towards CCL19 after stimulation with both bacteria. Thus, we could not detect any failure in the migration of H. pylori stimulated DC in vitro that may contribute to chronic gastritis in vivo, and our results suggest that H. pylori induces maturation and migration of DC to lymph nodes where they promote T cell responses.     

Outer membrane protein A of Escherichia coli K1 selectively enhances the expression of intercellular adhesion molecule-1 in brain microvascular endothelial cells

Escherichia coli K1 meningitis is a serious central nervous system disease with unchanged mortality and morbidity rates for last few decades. Intercellular adhesion molecule 1 (ICAM-1) is a cell adhesion molecule involved in leukocyte trafficking toward inflammatory stimuli at the vascular endothelium; however, the effect of E. coli invasion of endothelial cells on the expression of ICAM-1 is not known. We demonstrate here that E. coli K1 invasion of human brain microvascular endothelial cells (HBMEC) selectively up-regulates the expression of ICAM-1, which occurs only in HBMEC invaded by the bacteria. The interaction of outer membrane protein A (OmpA) of E. coli with its receptor, Ecgp, on HBMEC was critical for the up-regulation of ICAM-1 and was depend on PKC-alpha and PI3-kinase signaling. Of note, the E. coli-induced up-regulation of ICAM-1 was not due to the cytokines secreted by HBMEC upon bacterial infection. Activation of NF-kappaB was required for E. coli mediated expression of ICAM-1, which was significantly inhibited by over-expressing the dominant negative forms of PKC-alpha and p85 subunit of PI3-kinase. The increased expression of ICAM-1 also enhanced the binding of THP-1 cells to HBMEC. Taken together, these data suggest that localized increase in ICAM-1 expression in HBMEC invaded by E. coli requires a novel interaction between OmpA and its receptor, Ecgp.     

Induction of a novel mechanism of accelerated bacterial clearance by lipopolysaccharide in CD14-deficient and Toll-like receptor 4-deficient mice

Despite the lack of a proinflammatory response to LPS, CD14-deficient mice clear Gram-negative bacteria (Escherichia coli 0111) at least 10 times more efficiently than normal mice. In this study, we show that this is due to an early and intense recruitment of neutrophils following the injection of Gram-negative bacteria or LPS in CD14-deficient mice; in contrast, neutrophil infiltration is delayed by 24 h in normal mice. Similar results of early LPS-induced PMN infiltration and enhanced clearance of E. coli were seen in Toll-like receptor (TLR) 4-deficient mice. Furthermore, the lipid A moiety of LPS induced early neutrophil infiltration not only in CD14-deficient and TLR-4-deficient mice, but also in normal mice. In conclusion, the lipid A component of LPS stimulates a unique and critical pathway of innate immune responses that is independent of CD14 and TLR4 and results in early neutrophil infiltration and enhanced bacterial clearance.     

Neurogenic inflammation of the rat trachea: fate of neutrophils that adhere to venules

The goal of this study was to determine whether neutrophils that adhere to the vascular endothelium in association with neurogenic inflammation in the respiratory tract migrate out of the blood vessels or whether they detach and reenter the circulation. We also sought to determine whether the fate of the neutrophils is influenced by neutral endopeptidase (NEP), an enzyme that degrades the tachykinins that produce neurogenic inflammation. Neutrophils in the tracheal mucosa of anesthetized pathogen-free rats were examined 5 min or 4 h after neurogenic inflammation was produced by an injection of capsaicin (100 or 200 micrograms/kg iv). In whole mounts of these tracheae stained histochemically for myeloperoxidase, adherent intravascular neutrophils had a spherical or teardrop (regular) shape and migrating neutrophils had a polarized amoeboid (irregular) shape. The number of regular neutrophils in the tracheae was increased at both times, but the increase at 4 h was only half that present at 5 min. The reduction between 5 min and 4 h was not offset by an appreciable increase in the number of irregular neutrophils, unless NEP was inhibited by phosphoramidon. We interpret these results as indicating that the rapid adherence of neutrophils to the vascular endothelium after an injection of capsaicin is followed by a gradual reentry of the neutrophils into the circulation and comparatively little neutrophil migration. However, when the effect of the stimulus is increased and/or prolonged by inhibition of NEP, some of the adherent neutrophils migrate out of the vessels. Thus the activity of NEP can regulate both the magnitude of the neutrophil adherence and the fate of the adherent cells.     

Time-dependent reversal of sepsis-induced PMN uptake and lung vascular injury by expression of CD18 antagonist

We determined the time-dependent effects of conditional expression of neutrophil inhibitory factor (NIF), a specific 41-kDa CD18 integrin antagonist, on the time course of NIF expression and lung PMN (polymorphonuclear leukocyte) infiltration and vascular injury in a model of Escherichia coli-induced sepsis in mice. Studies were made in mice transduced with the E-selectin (ES) promoter-NIF construct (using liposomes) in which the NIF cDNA was driven by the inflammation- and endothelial cell-specific ES promoter. We observed time-dependent expression of NIF in pulmonary vascular endothelium that paralleled the ES expression. Expression of both was evident at 1 h after E. coli challenge, peaked at 3-6 h, and returned to basal level within 48 h. We observed that increases in PMN uptake and transalveolar PMN migration induced by E. coli challenge were reversed in a time-dependent manner following NIF expression in mice. NIF expression also prevented the progression of lung vascular injury and edema formation following E. coli challenge. Thus the conditional expression of NIF using the ES promoter can reverse, in a time-dependent manner, lung PMN infiltration and vascular injury induced by gram-negative sepsis. The results support the model that initial engagement of CD18 integrins enables the further recruitment of additional PMN into lung tissues such that PMN continue to sequester and migrate after E. coli challenge.