Reactive nitrogen intermediates and monokines induce caspase-3 mediated macrophage apoptosis by anaerobically stressed Salmonella typhi.

A successful pathogen manipulates its host for its own benefit. After ingestion, on reaching the intestine Salmonella encounters the resident tissue macrophages. Rather than being destroyed by these professional phagocytes after internalization, Salmonella survives intracellularly. Invasive Salmonella has been reported to induce apoptosis of macrophages as a part of its infection process, which may allow it to avoid detection by the innate immune system. However, the induction of apoptosis under different host environments, including the anaerobic stress encountered by the pathogen in the gut, remains to be examined. The present study is aimed at investigating the apoptotic potential of S. enterica serovar Typhi (S. typhi) grown under anaerobic conditions simulating the in vivo situation encountered by the pathogen. Apoptotic cell death was determined by assessment of nucleosomal DNA and flow cytometric analysis. Evaluation of the data revealed that anaerobically grown S. typhi could induce apoptosis in significantly more number of macrophages compared to the bacterial cells grown under aerobic conditions. A significantly enhanced generation of reactive nitrogen intermediates and caspase-3 activity during macrophage apoptosis induced by anaerobic S. typhi correlated with the increased generation of tumour necrosis factor-alpha, interleukin (IL)-1alpha and IL-6. The results indicate that reactive nitrogen intermediates and monokines induce caspase-3 mediated apoptosis of macrophages by S. typhi under anaerobic conditions. These findings may be relevant for clearer understanding of the Salmonella-macrophage interactions and may be of clinical importance in the development of preventive intervention against the infection.

Chemoattractants, extracellular proteases, and the integrated host defense response.

The host response to tissue injury and/or infection is dependent on the action of numerous extracellular proteases. Proteolytic cascades trigger blood clotting, fibrinolysis, and complement activation, while proteases released upon leukocyte degranulation are integral to the processes of inflammation and immunity. Modulation of effector protein activity by proteases provides a critical layer of posttranslational control that enables rapid enzymatic regulation of target proteins. This report reviews the emerging literature describing a novel class of proteolytic targets, leukocyte chemoattractants, and, in particular, chemerin, a dendritic cell and macrophage chemoattractant activated by serine proteases of the coagulation, fibrinolytic, and inflammatory cascades. As chemoattractants are critical for both systemic leukocyte positioning by triggering integrin activation and subsequent recruitment from circulation, and local intratissue leukocyte positioning via chemotaxis, modulation of attractant activities by proteases may have profound effects on the immune response.

Macrophage inflammatory protein-2 contributes to liver resection-induced acceleration of hepatic metastatic tumor growth.

AIM: To study the role of macrophage inflammatory protein (MIP)-2 in liver resection-induced acceleration of tumor growth in a mouse model of hepatic metastasis. METHODS: After a 50% hepatectomy, 1x10(5) CT26.WT cells were implanted into the left liver lobe of syngeneic balb/c mice (PHx). Additional animals were treated with a monoclonal antibody (MAB452) neutralizing MIP-2 (PHx+mAB). Non-resected and non-mAB-treated mice (Con) served as controls. After 7 d, tumor angiogenesis and microcirculation as well as cell proliferation, tumor growth, and CXCR-2 expression were analyzed using intravital fluorescence microscopy, histology, immunohistochemistry, and flow cytometry. RESULTS: Partial hepatectomy increased (P<0.05) the expression of the MIP-2 receptor CXCR-2 on tumor cells when compared with non-resected controls, and markedly accelerated (P<0.05) angiogenesis and metastatic tumor growth. Neutralization of MIP-2 by MAB452 treatment significantly (P<0.05) depressed CXCR-2 expression. Further, the blockade of MIP-2 reduced the angiogenic response (P<0.05) and inhibited tumor growth (P<0.05). Of interest, liver resection-induced hepatocyte proliferation was not effected by anti-MIP-2 treatment. CONCLUSION: MIP-2 significantly contributes to liver resection-induced acceleration of colorectal CT26.WT hepatic metastasis growth.

TLR-2 is upregulated and mobilized to the hepatocyte plasma membrane in the space of Disse and to the Kupffer cells TLR-4 dependently during acute endotoxemia in mice.

Membrane components of bacteria and fungi are recognized by Toll-like receptors (TLRs) which, when activated, induce several inflammatory mediators important in the host defense. As the liver is constantly exposed to ingested bacteria, hepatic TLRs must be broadly responsive and highly regulated to prevent uncontrolled inflammatory activation. Although several hepatic cells express microbe recognition molecules and inflammatory mediators in vitro, the regulation and cellular localization of these proteins in vivo remain uncertain. The expression and regulation of TLR-2 and TLR-4, and the cytokine expression patterns were evaluated in mouse tissues using a model of acute inflammation induced by intraperitoneal injection of LPS. Five hours after intraperitoneal LPS, induction of TLR-4 was evident in lung, while the low hepatic TLR-4 expression was non-inducible. TLR-2 mRNA and protein were induced both in lung and liver TLR-4 dependently. However, IL-1alpha also contributed to this induction, and IL-1R1 antibody attenuated the TLR-2 increase. Immunoelectron microscopy showed accumulation of cytoplasmic TLR-2 to vesicles near the hepatocyte plasma membrane in the space of Disse, to the sinusoidal endothelium and to the Kupffer cells. NF-kappaB activation was clear in Kupffer cells and hepatocytes during LPS-challenge, suggesting these cells to be the main source of in vivo cytokine production. Hepatic cytokine response to LPS was remarkably rapid in liver, whereas lung responded less acutely. Secondary inflammatory challenge attenuated the TLR-2 response. The innate immune system of the liver is rapidly and transiently activated during endotoxemia by mechanism involving both TLR-4 and TLR-2.

Divergent roles of murine neutrophil chemokines in hemorrhage induced priming for acute lung injury.

Neutrophil associated lung injury is identified with a variety of local and systemic priming insults. In vitro studies have shown that TNF-alpha mediated suppression of neutrophil apoptosis is due to the secretion of interleukin-8 (IL-8), a human chemokine shown to alter neutrophil chemotaxis. Our initial in vitro antibody neutralization studies with neutrophil chemotactic proteins, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2alpha (MIP-2alpha), mouse IL-8 homologues, indicate that MIP-2alpha but not KC appears to mediate TNF-alpha suppression of mouse neutrophil apoptosis. Therefore, we hypothesized that in vivo neutralization of KC or MIP-2alpha during an initial priming insult would produce differential effects on the extent of lung injury by restoring normal neutrophil apoptotic function. To assess this, mice were hemorrhaged followed with septic challenge at 24 h. Antibody against KC or MIP-2alpha or a nonspecific IgG was given during resuscitation immediately following hemorrhage. Anti-MIP-2alpha treatment resulted in a significant reduction in lung tissue IL-6 and myeloperoxidase levels. Percentage of neutrophil apoptosis increased significantly in the anti-KC group. Tissue and plasma KC and MIP-2alpha were reduced in their respective treatment groups. These data suggest that KC and MIP-2alpha differ in their mediation of neutrophil function (apoptosis and chemotaxis) and contribution to the pathogenesis of lung injury following hemorrhage subsequent to sepsis.

Smoking promotes pathogenesis of aortic aneurysm through the 5-lipoxygenase pathway.

Smoking has been known as a risk factor for aortic aneurysm. 5-Lipoxygenase is the key enzyme in leukotriene biosynthesis and catalyzes initial steps in the conversion of arachidonic acid to these biologically active lipid mediators, which are known to exert proinflammatory effects in vivo. Smoking can induce 5-lipoxygenase expression in colon neoplasm, and may activate the 5-lipoxygenase pathway also in aortic tissue. 5-Lipoxygenase has a role in promoting the formation of aneurysms through potential plasma macrophage inflammatory protein-1alpha and -2 chemokine-dependent inflammatory circuits involving both myeloid and endothelial cells. Therefore, smoking may promote pathogenesis of aortic aneurysm through the 5-lipoxygenase pathway.

Role of endogenous macrophage inflammatory protein-2 in regulating fever induced by bacterial endotoxin in normal and immunosuppressed rats.

During myelosuppressive chemotherapy, Gram-negative bacterial infection with consequent exposure to lipopolysaccharide (LPS) is one of the most important causes of persistent fever. The classical model of the pathogenesis of fever suggests that pro-inflammatory cytokines, produced by leucocytes in the bloodstream in response to exogenous pyrogens such as bacterial LPS, represent the distal mediators of the febrile response. Neutrophils are the first effectors cells and the most prominent leucocyte population involved in acute bacterial infection. Macrophage inflammatory protein (MIP)-2 plays a crucial role in influencing early cell trafficking and neutrophil activation during pathophysiological processes and serves the same chemotactic function as human interleukin-8. In the present study, we investigated the role of MIP-2 in the development of a febrile response induced by LPS in immunocompetent and leukopenic rats. Intraperitoneal injection of LPS in leukopenic rats elicited a biphasic febrile response of rapid onset, the magnitude and duration of which were significantly greater than in immunocompetent animals. The febrile responses to LPS were accompanied by a pronounced induction of serum MIP-2 levels at 1, 2 and 4 h compared with their respective controls. In both normal and leukopenic rats, neutralization of endogenous MIP-2 bioactivity by systemic administration of antirat MIP-2 antibody caused a significant attenuation of the early phase of LPS fever. However, in contrast with normal rats, the second phase of fever was unimpaired by anti-MIP-2 in leukopenic rats. These findings suggest that circulating MIP-2 is involved in the generation of the early phase of LPS fever that contributes to the maintenance of the later phase of fever in immunocompetent, but not leukopenic, rats. Our data support a regulatory role for endogenous MIP-2 in initiating the fever responses to LPS. Furthermore, these results provide evidence that different cellular and humoral mechanisms are implicated in the development of a febrile response triggered by Gram-negative bacterial infections in leukopenic hosts.

Essential role for the p40 subunit of interleukin-12 in neutrophil-mediated early host defense against pulmonary infection with Streptococcus pneumoniae: involvement of interferon-gamma.

Interleukin (IL)-12 is a critical cytokine in the T helper (Th)1 response and host defense against intracellular microorganisms, while its role in host resistance to extracellular bacteria remains elusive. In the present study, we elucidated the role of IL-12 in the early-phase host defense against acute pulmonary infection with Streptococcus pneumoniae, a typical extracellular bacterium, using IL-12p40 gene-disrupted (IL-12p40KO) mice. IL-12p40KO mice were highly susceptible to S. pneumoniae infection, as indicated by the shortened survival time, which was completely restored by the replacement therapy with recombinant (r) IL-12, and increased bacterial counts in the lung. In these mice, recruitment of neutrophils in the lung was significantly attenuated when compared to that in wild-type (WT) mice, which correlated well with the reduced production of macrophage inflammatory protein (MIP-2) and tumor necrosis factor (TNF)-alpha in the infected tissues at the early phase of infection. In vitro synthesis of both cytokines by S. pneumoniae-stimulated lung leukocytes was significantly lower in IL-12p40KO mice than in WT mice, and addition of rIL-12 or interferon (IFN)-gamma restored the reduced production of MIP-2 and TNF-alpha in IL-12p40KO mice. Neutralizing anti-IFN-gamma monoclonal antibody (mAb) significantly decreased the effect of rIL-12. Anti-IFN-gamma mAb shortened the survival time of infected mice and reduced the recruitment of neutrophils and production of MIP-2 and TNF-alpha in the lungs. Our results indicated that IL-12p40 plays a critical role in the early-phase host defense against S. pneumoniae infection by promoting the recruitment of neutrophils to the infected tissues.

Angiogenic activity of human CC chemokine CCL15 in vitro and in vivo.

CCL15 is a novel human CC chemokine and exerts its biological activities on immune cells through CCR1 and CCR3. Because a number of chemokines induce angiogenesis and endothelial cells express CCR1 and CCR3, we investigated the angiogenic activity of CCL15. Both CCL15(1-92) and N-terminal truncated CCL15(25-92) stimulate the chemotactic endothelial cell migration and differentiation, but CCL15(25-92) is at least 100-fold more potent than CCL15(1-92). Treatment with pertussis toxin (PTX), with anti-CCR1, or with anti-CCR3 antibody inhibits the CCL15(25-92)-induced endothelial cell migration. CCL15(25-92) also stimulates sprouting of vessels from aortic rings and mediates angiogenesis in the chick chorioallantoic membrane assay. Our findings demonstrate that CCL15(25-92) has in vitro and in vivo angiogenic activity, and suggest roles of the chemokine in angiogenesis.

Immunolocalization of CXC chemokine and recruitment of polymorphonuclear leukocytes in the rat molar periodontal tissue after topical application of lipopolysaccharide.

This study investigated the recruitment of polymorphonuclear leukocytes (PMNs) and the immunolocalization of CXC chemokines, including macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant-2 (CINC-2) in rat periodontal tissue after topical application of lipopolysaccharide (LPS; 5 mg/ml) from Escherichia coli into the rat molar gingival sulcus. In normal periodontal tissues, a small number of MIP-2- and CINC-2-positive cells were seen in junctional epithelium (JE), especially in its coronal half. After topical application of LPS, a prominent increase of MIP-2- and CINC-2-positive JE cells was observed. Almost all JE cells strongly expressed them at day 1 and day 2, and then the number of chemokine-positive cells returned to normal at day 7. Corresponding to these chemokine expressions, LPS application induced a significant increase in the number of PMNs in the sub-JE area from 1 h to 2 days and a significant increase in JE area from 3 h to 5 days, indicating a dynamic flow of PMNs from the sub-JE area into JE. These findings indicated that JE cells produced MIP-2 and CINC-2 in response to LPS stimulation and suggested that MIP-2 and CINC-2 may be responsible for PMN migration toward the periodontal pathogen and may play an important role in the initiation of inflammation and subsequent periodontal tissue destruction.

NK cells regulate CD8+ T cell effector function in response to an intracellular pathogen.

We studied the role of NK cells in regulating human CD8+ T cell effector function against mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Depletion of NK cells from PBMC of healthy tuberculin reactors reduced the frequency of M. tuberculosis-responsive CD8+IFN-gamma+ cells and decreased their capacity to lyse M. tuberculosis-infected monocytes. The frequency of CD8+ IFN-gamma+ cells was restored by soluble factors produced by activated NK cells and was dependent on IFN-gamma, IL-15, and IL-18. M. tuberculosis-activated NK cells produced IFN-gamma, activated NK cells stimulated infected monocytes to produce IL-15 and IL-18, and production of IL-15 and IL-18 were inhibited by anti-IFN-gamma. These findings suggest that NK cells maintain the frequency of M. tuberculosis-responsive CD8+IFN-gamma+ T cells by producing IFN-gamma, which elicits secretion of IL-15 and IL-18 by monocytes. These monokines in turn favor expansion of Tc1 CD8+ T cells. The capacity of NK cells to prime CD8+ T cells to lyse M. tuberculosis-infected target cells required cell-cell contact between NK cells and infected monocytes and depended on interactions between the CD40 ligand on NK cells and CD40 on infected monocytes. NK cells link the innate and the adaptive immune responses by optimizing the capacity of CD8+ T cells to produce IFN-gamma and to lyse infected cells, functions that are critical for protective immunity against M. tuberculosis and other intracellular pathogens.

Critical role of CXC chemokines in endotoxemic liver injury in mice.

Tissue accumulation of leukocytes constitutes a rate-limiting step in endotoxin-induced tissue injury. Chemokines have the capacity to regulate leukocyte trafficking. However, the role of CXC chemokines, i.e., macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC), in leukocyte recruitment, microvascular perfusion failure, cellular injury, and apoptosis in the liver remains elusive. Herein, mice were challenged with lipopolysaccharide (LPS) in combination with D-galactosamine, and intravital microscopy of the liver microcirculation was conducted 6 h later. It was found that immunoneutralization of MIP-2 and KC did not reduce LPS-induced leukocyte rolling and adhesion in postsinusoidal venules. In contrast, pretreatment with monoclonal antibodies against MIP-2 and KC abolished (83% reduction) extravascular recruitment of leukocytes in the livers of endotoxemic mice. Notably, endotoxin challenge increased the expression of CXC chemokines, which was mainly confined to hepatocytes. Moreover, endotoxin-induced increases of liver enzymes and hepatocellular apoptosis were decreased by more than 82% and 68%, respectively, and sinusoidal perfusion was restored in mice passively immunized against MIP-2 and KC. In conclusion, this study indicates that intravascular accumulation of leukocytes in the liver is independent of CXC chemokines in endotoxemic mice. Instead, our novel data suggest that CXC chemokines are instrumental in regulating endotoxin-induced transmigration and extravascular tissue accumulation of leukocytes. Indeed, these findings demonstrate that interference with MIP-2 and KC functions protects against septic liver damage and may constitute a potential therapeutic strategy to control pathological inflammation in endotoxemia.

Adhesion molecules and CXC chemokines in endotoxin-induced liver injury.

Interactions between leukocytes and sinusoidal endothelial cells are known to be involved in the pathogenesis of acute liver injury. Various adhesion molecules and chemokines play key roles in these cell-to-cell interactions, and the expression of these adhesion molecules and the production of chemokines are regulated by inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and interferon-gamma (IFN-gamma). We have shown that the expression of intercellular adhesion molecule-1 (ICAM-1) on cultured rat sinusoidal endothelial cells stimulated with TNF-alpha increases in a dose-dependent manner. The number of neutrophils that adhered to sinusoidal endothelial cells pretreated with TNF-alpha also increased in a dose-dependent manner and significantly decreased upon incubation with an anti-ICAM-1 antibody. In endotoxin-induced rat liver injury, the number of neutrophils infiltrating the sinusoids increased after serum TNF-alpha, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) reached their peak levels. In addition, the level of ICAM-1 expression on sinusoidal endothelial cells greatly increased from 8 h after exposure to endotoxin, and these cells were adhered to neutrophils that expressed both LFA-1 and Mac-1. Moreover, lipo-prostaglandin E1 (PGE1) reduced the extent of liver injury, and also reduced the number of neutrophils that infiltrated the liver, was reduced the production of MIP-2 and CINC, but not that of TNF-alpha, in rats injected with endotoxin.

Chemokines accumulate in the lungs of rats with severe pulmonary embolism induced by polystyrene microspheres.

Pulmonary thromboembolism (PEm) is a serious and life threatening disease and the most common cause of acute pulmonary vascular occlusion. Even following successful treatment of PEm, many patients experience long-term disability due to diminished heart and lung function. Considerable damage to the lungs presumably occurs due to reperfusion injury following anti-occlusive treatments for PEm and the resulting chronic inflammatory state in the lung vasculature. We have used a rat model of irreversible PEm to ask whether pulmonary vascular occlusion in the absence of reperfusion is itself sufficient to induce an inflammatory response in lungs. By adjusting the severity of the vascular occlusion, we were able to generate hypertensive and nonhypertensive PEm, and then examine lung tissue for expression of CXC and C-C chemokine genes and bronchoalveolar lavage (BAL) fluid for the presence of chemokine proteins. Hypertensive and nonhypertensive PEm resulted in increased expression of both CXC and C-C chemokines genes in lung tissues. Hypertensive PEm was also associated with a 50-100-fold increase in protein content in lung BAL fluid, which included the CXC chemokines cytokine-induced neutrophil chemoattractant and macrophage-inflammatory protein 2. The presence of chemokines in BALs was reflected by a potent neutrophil chemotactic activity in in vitro chemotaxis assays. Abs to cytokine-induced neutrophil chemoattractant blocked the in vitro neutrophil chemotactic activity of BAL by 44%. Our results indicate that the ischemia and hypertension associated with PEm are sufficient to induce expression of proinflammatory mediators such as chemokines, and establish a proinflammatory environment in the ischemic lung even before reperfusion.

Staphylococcal enterotoxin B-induced acute inflammation is inhibited by dexamethasone: important role of CXC chemokines KC and macrophage inflammatory protein 2.

This study was conducted to examine the anti-inflammatory mechanisms of dexamethasone during leukocyte recruitment and expression of the CXC chemokines macrophage inflammatory protein 2 (MIP-2) (CXCL2) and cytokine-induced neutrophil chemoattractant (KC) (CXCL1) in staphylococcal enterotoxin B (SEB)-induced acute inflammation. To do this, SEB was injected into murine air pouches with or without dexamethasone pretreatment for 2 h. SEB induced infiltration of leukocytes in a dose- and time-dependent manner, with the maximal response observed after 4 h of treatment with 10 microg of SEB. The recruited leukocytes comprised more than 77% neutrophils. Moreover, SEB challenge (10 microg) provoked time-dependent secretion of CXC chemokines, which peaked after 1 h. Local administration of antibodies against MIP-2 and KC significantly reduced SEB-triggered neutrophil accumulation by 38 and 59%, respectively. Dexamethasone (10 mg kg(-1)) significantly decreased neutrophil recruitment by 82% and reduced secretion of MIP-2 and KC by 89 and 85%, respectively, in response to SEB challenge. Our data demonstrate that dexamethasone potently inhibits neutrophil recruitment in SEB-induced inflammation. Moreover, we provide evidence that MIP-2 and KC are key mediators in the neutrophil response to SEB. Furthermore, our findings demonstrate that dexamethasone attenuates SEB-induced expression of MIP-2 and KC. Thus, this study elucidates important signaling pathways of SEB-induced neutrophil recruitment and anti-inflammatory mechanisms of action of dexamethasone.

Role of macrophage inflammatory peptide-2 in cerulein-induced acute pancreatitis and pancreatitis-associated lung injury.

Acute pancreatitis is an inflammatory process of variable severity, and leukocytes are thought to play a key role in the development of pancreatitis and pancreatitis-associated lung injury. The effects of mediators released by these inflammatory cells may induce tissue damage. The aim of our study was to evaluate the role of the chemokine, macrophage inflammatory protein-2 (MIP-2), in the pathogenesis of cerulein-induced pancreatitis and pancreatitis-associated lung injury. The severity of pancreatitis was measured by serum amylase, pancreatic edema, acinar cell necrosis, and myeloperoxidase activity. Lung injury was quantitated by evaluating lung microvascular permeability and lung myeloperoxidase activity. To determine the role of MIP-2 in the pathophysiology of the disease, anti-MIP-2 antibody was administered either 1 hour before or 2 hours after the start of cerulein administration. MIP-2 concentrations increased in serum, pancreas, and lung tissues in mice treated with cerulein. Anti-MIP-2 antibody administrated either before or after cerulein partially protected against pancreas and lung injury. These results show that MIP-2 plays a key role in the pathophysiology of acute pancreatitis and that MIP-2 blockade may improve the outcome of the disease.

CD40 ligand trimer enhances the response of CD8+ T cells to Mycobacterium tuberculosis.

We investigated the effect of recombinant CD40 ligand trimer (CD40LT) on the functional capacity of peripheral blood CD8(+) T cells from healthy tuberculin reactors that were cultured with Mycobacterium tuberculosis-infected autologous monocytes. CD40LT enhanced the capacity of M. tuberculosis-responsive CD8(+) T cells to produce IFN-gamma by increasing the number of IFN-gamma-producing CD8(+) T cells and the amount of IFN-gamma produced per cell. CD40LT-induced IFN-gamma production was dependent on production of IL-12 and IL-18, but did not require IL-15. CD40LT up-regulated expression of the transcription factors phosphorylated CREB and c-Jun, both of which have been previously shown to stimulate IFN-gamma mRNA transcription by binding to the IFN-gamma promoter. CD40LT also enhanced the capacity of CD8(+) T cells to lyse M. tuberculosis-infected monocytes, and increased CTL activity was associated with higher expression of perforin and granulysin, but not of Fas ligand. We conclude that CD40LT can enhance CD8(+) T cell effector function in response to M. tuberculosis.

Role and regulation of CXC-chemokines in acute experimental keratitis.

The aim of this study was to elucidate the expression of chemokines, their role and regulation in bacterial corneal infection using three bacterial strains (Pseudomonas. aeruginosa- invasive, cytotoxic and contact lens induced acute red eye strains) which have been shown to produce three distinct patterns of corneal disease in the mouse. The predominant chemokine expressed in response to all three strains was MIP-2. Prolonged expression of high levels of MIP-2 was associated with increased severity of corneal inflammation. Significantly reduced disease severity upon administration of anti-MIP-2 antibodies suggested that MIP-2 may play an important role in the pathogenesis of Pseudomonas keratitis at least in part by being a major chemoattractant for polymorphonuclear leukocytes (PMN) recruitment. Interestingly, the numbers of bacteria in eyes with neutralized MIP-2 activity did not decrease even though the severity of the disease was decreased. This implies PMNs as the major destructive factor in microbial keratitis. Further, neutralization of IL-1beta activity alone using monoclonal antibodies resulted in significant reduction of both MIP-2 and KC activity indicating that chemokine levels were regulated by IL-1beta. These studies demonstrate that the regulation of MIP-2 activity may be beneficial in reducing corneal damage during microbial keratitis in rodents and perhaps that regulation of the human homologue of MIP-2, IL-8, may be useful for controlling keratitis in humans.

[Stimulating the expression of macrophage inflammatory protein-2gamma by lipopolysaccharide in-vitro].

AIM: To study the expression level of macrophage inflammatory protein-2gamma(MIP-2gamma)stimulated by lipopolysaccharide(LPS)in-vitro. METHODS: Murine monocyte macrophage like cell line RAW264.7 and BALB/c mice primary kidney cell culture were stimulated with LPS. MIP-2gamma mRNA expression level in these cells was detected by real time fluorescence quantitative RT-PCR. RESULTS: MIP-2gamma mRNA expression level of RAW264.7 cell line increased quickly and reached the peak (7 times the normal level) within 2 hours after LPS stimulation and dropped to the base level within 16 hours, while that of cultured promary kidney cells of BALB/c mice elevated about 50 times compared with base level 12 hours following LPS stimulation. CONCLUSION: LPS can significantly stimulate the expression of MIP-2gamma mRNA, suggesting that MIP-2gamma may play some role in inflammatory process.