Macrophage Inflammatory Protein-2 Levels Research Articles

Leukotriene B4 receptor (BLT-1) modulates neutrophil influx into the peritoneum but not the lung and liver during surgically induced bacterial peritonitis in mice.

Leukotriene B4 (LTB4) is a rapidly synthesized, early neutrophil chemoattractant that signals via its cell surface receptor, BLT-1, to attract and activate neutrophils during peritonitis. BLT-1-deficient (BLT-1(-/-)) mice were used to determine the effects of LTB4 on neutrophil migration and activation, bacterial levels, and survival after cecal ligation and puncture (CLP). Male BLT-1(-/-) or wild-type (WT) BALB/c mice underwent CLP. Tissues were harvested for determination of levels of bacteria, myeloperoxidase (MPO), LTB4, macrophage inflammatory protein 2 (MIP-2), and neutrophil (polymorphonuclear leukocyte [PMN]) numbers at 4 and 18 h after CLP. PMN activation was determined by an assessment of phagocytosis ability and CD11b expression. Survival was also determined. BLT-1(-/-) mice had decreased numbers of PMNs in the peritoneum at both 4 and 18 h after CLP but increased numbers of PMNs in the blood at 18 h compared with WT mice. Liver and lung MPO levels were significantly higher in BLT-1(-/-) mice at both 4 and 18 h after CLP, with increased bacterial levels in the blood, the liver, and peritoneal fluid at 4 h. Bacterial levels remained higher in peritoneal fluid at 18 h, but blood and liver bacterial levels at 18 h were not different from levels at 4 h. PMN phagocytosis and CD11b levels were decreased in BLT-1(-/-) mice. LTB4 levels were similar between the groups before and after CLP, but MIP-2 levels were decreased both locally and systemically in BLT-1(-/-) mice. Survival was significantly improved in BLT-1(-/-) mice (71%) compared with WT mice (14%) at 48 h post-CLP. Thus, LTB4 modulates neutrophil migration into the mouse peritoneum, but not the lung or liver, after CLP. Despite higher bacterial and PMN levels at remote sites, there was increased survival in BLT-1(-/-) mice compared to WT mice. Decreased PMN activation may result in less remote organ dysfunction and improved survival.

Protein Kinase Cδ Mediates Lysophosphatidic Acid-induced NF-κB Activation and Interleukin-8 Secretion in Human Bronchial Epithelial Cells

Lysophosphatidic acid (LPA), a potent bioactive lipid, elicits many of its biological actions via the specific G-protein-coupled receptors LPA1, LPA2, LPA3, and LPA4. Recently, we have shown that LPA-induced transactivation of platelet-derived growth factor receptor-beta is regulated by phospholipase D2 in human bronchial epithelial cells (HBEpCs) (Wang, L., Cummings, R. J., Zhao, Y., Kazlauskas, A., Sham, J., Morris, A., Brindley, D. N., Georas, S., and Natarajan, V. (2003) J. Biol. Chem. 278, 39931-39940). Here, we report that protein kinase Cdelta (PKCdelta) mediates LPA-induced NF-kappaB transcription and interleukin-8 (IL-8) secretion in HBEpCs. Treatment of HBEpCs with LPA increased both IL-8 gene and protein expression, which was coupled to Gi and G(12/13) proteins. LPA caused a marked activation of NF-kappaB in HBEpCs as determined by IkappaB phosphorylation and of NF-kappaB nuclear translocation and a strong induction of NF-kappaB promoter-mediated luciferase activity. Furthermore, LPA-activated PKCdelta and the LPA-mediated activation of NF-kappaB and IL-8 production were attenuated by overexpression of dominant-negative PKCdelta and rottlerin. Intratracheal administration of LPA in mice resulted in elevated levels of macrophage inflammatory protein-2, a murine homolog of IL-8, and an influx of neutrophils in the bronchoalveolar lavage fluid. These results demonstrate for the first time that LPA is a potent stimulator of IL-8 production in HBEpCs, which involves PKCdelta/NF-kappaB signaling pathways.

Stimulation of toll-like receptor 2 in mononuclear cells from HIV-infected patients induces chemokine responses: possible pathogenic consequences.

Toll-like receptor 2 (TLR2) stimulation in monocytes may contribute to enhanced inflammation and viral replication in HIV infection. In the present study we examined if TLR2 stimulation could modulate chemokine responses in peripheral blood mononuclear cells (PBMC) from HIV-infected patients and healthy controls. Our main findings were, with similar qualitative patterns in both healthy controls and HIV-infected patients: (1) TLR2 stimulation induced up-regulation of several chemokines at the mRNA level as well as increased protein levels of macrophage inflammatory protein (MIP)-1alpha, interleukin (IL)-8 and regulated on activation, normal T cell expressed and secreted (RANTES); (2) TLR2 stimulation induced enhanced protein expression of CCR5 (a receptor for MIP-1alpha and RANTES) on monocytes; (3) In vitro stimulation with RANTES induced release of MIP-1alpha, MCP-1, IL-8 and interferon-gamma from PBMC. While increased levels of beta-chemokines possibly have antiviral effects, TLR2 stimulation may also promote a chemokine-driven inflammatory loop, potentially contributing to the immunopathogenesis of HIV infection.

Role of Inducible Nitric Oxide Synthase-Derived Nitric Oxide in Silica-Induced Pulmonary Inflammation and Fibrosis

Inhalation of crystalline silica can produce lung inflammation and fibrosis. Inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) is believed to be involved in silica-induced lung disease. To investigate the role of iNOS-derived NO in this disease, the responses of iNOS knockout (KO) versus C57Bl/6J wild-type (WT) mice to silica were compared. Male mice (8–10 wk old, mean body weight 24.0 g) were anesthetized and exposed, by aspiration, to silica (40 mg/kg) or saline. At 24 h and 42 d postexposure, lungs were lavaged with saline. The first bronchoalveolar lavage (BAL) fluid supernatant was analyzed for lactate dehydrogenase (LDH) activity, levels of albumin, tumor necrosis factor-α (TNF-α), and macrophage inflammatory protein-2 (MIP-2), as well as total antioxidant capacity (TAC). The cellular fraction of the total BAL was used to determine alveolar macrophage (AM) and polymorphonuclear leukocyte (PMN) counts, and zymosan-stimulated AM chemiluminescence (AM-CL). In separate mice, lung histopathological changes were evaluated 42 d postexposure. Acute (24-h) silica exposure decreased AMs, increased PMNs, increased LDH activity and levels of albumin, TNF-α, and MIP-2 in BAL fluid, and enhanced AM-CL in both iNOS KO and WT mice. However, iNOS KO mice exhibited less AM activation (defined as increased AM-CL and decreased AM yield) than WT. Furthermore, TAC following acute silica decreased in WT but was maintained in iNOS KO mice. Pulmonary reactions to subchronic (42 d) silica exposure were similar to acute. However, histopathological and BAL fluid indices of lung damage and inflammation, AM activation, and lung hydroxyproline levels were significantly less in iNOS KO compared to WT mice. These results suggest that iNOS-derived NO contributes to the pathogenesis of silica-induced lung disease in this mouse model.

CXCR2 inhibition suppresses hemorrhage-induced priming for acute lung injury in mice.

Polymorphonuclear neutrophil (PMN) extravasation/sequestration in the lung and a dysregulated inflammatory response characterize the pathogenesis of acute lung injury (ALI). Previously, we have shown that hemorrhage (Hem) serves to prime PMN such that subsequent septic challenge [cecal ligation and puncture (CLP)] produces a pathological, inflammatory response and consequent lung injury in mice. Keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2) are murine CXC chemokines found elevated in the lungs and plasma following Hem/CLP and have been reported by others to share a common receptor (CXCR2). Based on these data, we hypothesize that blockade of CXCR2 immediately following Hem would suppress KC and MIP-2 priming of PMN, thereby reducing the inflammatory injury observed following CLP. To assess this, Hem mice (90 min at 35+/-5 mmHg) were randomized to receive 0, 0.4, or 1 mg antileukinate (a hexapeptide inhibitor of CXCRs) in 100 microl phosphate-bufferd saline (PBS)/mouse subcutaneously, immediately following resuscitation (Ringer's lactate-4x drawn blood volume). Twenty-four hours post-Hem, mice were subjected to CLP and killed 24 h later. The results show that blockade of CXCR2 significantly (P<0.05, Tukey's test) reduced PMN influx, lung protein leak, and lung-tissue content of interleukin (IL)-6, KC, and MIP-2 and increased tissue IL-10 levels. Plasma IL-6 was significantly decreased, and IL-10 levels increased in a dose-dependent manner compared with PBS-treated mice. A differential effect was observed in plasma levels of KC and MIP-2. KC showed a significant reduction at the 0.4 mg antileukinate dose. In contrast, plasma MIP-2 was significantly elevated at both doses compared with the PBS-treated controls. Together, these data demonstrate that blockade of CXCR2 signaling attenuates shock-induced priming and ALI observed following Hem and subsequent septic challenge in mice.

Macrophage migration inhibitory factor up-regulates the expression of interleukin-8 messenger RNA in synovial fibroblasts of rheumatoid arthritis patients: common transcriptional regulatory mechanism between interleukin-8 and interleukin-1beta.

Interleukin-8 (IL-8) plays an important role in the migration of inflammatory cells into the synovium and joint fluids in rheumatoid arthritis (RA). This study was undertaken to investigate the IL-8 inductive activity of the macrophage migration inhibitory factor (MIF) in RA synovial fibroblasts. The regulatory mechanism of IL-8 was compared with that of IL-1beta. MIF-induced IL-8 and IL-1beta transcriptional activation was studied in RA synovial fibroblasts by Northern blot analysis, enzyme-linked immunosorbent assay, and electromobility shift assay. The effect of anti-MIF antibody administration on murine passive collagen-induced arthritis (CIA) was also evaluated by histologic examination and reverse transcriptase-polymerase chain reaction. MIF up-regulated the IL-8 messenger RNA (mRNA) and protein levels in a dose-dependent manner. The IL-8 mRNA up-regulation started 1 hour poststimulation by MIF, and reached a maximum level at 6 hours. IL-1beta mRNA was also up-regulated by MIF. The mRNA up-regulation of IL-8 and IL-1beta by MIF was inhibited by 2 tyrosine kinase inhibitors, a protein kinase C (PKC) inhibitor, an activator protein 1 (AP-1) inhibitor, and by an NF-kappaB inhibitor. A cAMP-dependent kinase inhibitor did not inhibit it. MIF enhanced AP-1 and NF-kappaB binding activities in a dose-dependent manner. Passive CIA enhanced mRNA levels of macrophage inflammatory protein 2 and cytokine-induced neutrophil chemoattractants and, moreover, migration and proliferation of inflammatory cells within the synovium, which were suppressed by administration of an anti-MIF antibody. MIF may play an important role in the migration of inflammatory cells into the synovium of rheumatoid joints via induction of IL-8. MIF up-regulates IL-8 and IL-1beta mRNA via tyrosine kinase-, PKC-, AP-1-, and NF-kappaB-dependent pathways.

Treatment with alpha-galactosylceramide attenuates the development of bleomycin-induced pulmonary fibrosis.

Pulmonary fibrosis is an end-stage disorder for which efficacious therapeutic options are not readily available. Although its pathogenesis is poorly understood, pulmonary fibrosis occurs as a result of various inflammations. NKT cells modulate inflammation because of their ability to produce large amounts of cytokines by stimulation with their glycolipid ligand. In the present study, we investigated the effects of alpha-galactosylceramide (alpha-GalCer), a selective NKT cell ligand, on the development of bleomycin-induced pulmonary fibrosis. Treatment of mice with alpha-GalCer prolonged their survival under bleomycin administration by attenuating the development of pulmonary fibrosis. The protective effects of alpha-GalCer were associated with an increase in the pulmonary level of IFN-gamma and a decrease in the pulmonary level of fibrogenic cytokines such as TGF-beta and connective tissue growth factor. The initial pulmonary inflammation caused by bleomycin was also attenuated by alpha-GalCer with the reduction of the macrophage inflammatory protein-2 level. The protective effects of alpha-GalCer were markedly reduced in mice lacking NKT cells or as a result of treatment with anti-IFN-gamma Ab. These results suggest that alpha-GalCer suppresses bleomycin-induced acute pulmonary inflammation and thus attenuates the development of pulmonary fibrosis possibly by regulating several cytokine levels.

The CepIR quorum-sensing system contributes to the virulence of Burkholderia cenocepacia respiratory infections.

The cepIR genes encode an N-acyl homoserine lactone (AHL)-dependent quorum-sensing system consisting of an AHL synthase that directs the synthesis of N-octanoyl-L-homoserine lactone (ohl) and n-hexanoyl-L-homoserine lactone and a transcriptional regulator. The virulence of cepIR mutants was examined in two animal models. Rats were infected with agar beads containing Burkholderia cenocepacia K56-2, K56-I2 (cepI : : Tp(r)) or K56-R2 (cepR : : Tn5-OT182). At 10 days post-infection, the extent of lung histopathological changes was significantly lower in lungs infected with K56-I2 or K56-R2 compared to the parent strain. Intranasal infections were performed in Cftr((-/-)) mice and their wild-type siblings. K56-2 was more virulent in both groups of mice. K56-I2 was the least virulent strain and was not invasive in the Cftr((-/-)) mice. OHL was readily detected in lung homogenates from Cftr((-/-)) mice infected with K56-2 but was only detected at levels slightly above background in a few mice infected with K56-I2. Lung homogenates from mice infected with K56-2 had significantly higher levels of the inflammatory mediators murine macrophage inflammatory protein-2, KC/N51, interleukin-1beta and interleukin-6 than those from K56-I2-infected animals. These studies indicate that a functional CepIR quorum-sensing system contributes to the severity of B. cenocepacia infections. A zinc metalloprotease gene (zmpA) was shown to be regulated by CepR and may be one of the factors that accounts for the difference in virulence between the cepI mutant and the parent strain.

Selective transport of cytokine-induced neutrophil chemoattractant from the lung to the blood facilitates pulmonary neutrophil recruitment.

The CXC chemokines cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 (MIP-2) are potent neutrophil chemoattractants in rats. We have previously shown that CINC, unlike MIP-2 and most other proinflammatory cytokines, is elevated in the systemic circulation in response to an intratracheal (IT) challenge. Therefore, we hypothesized that CINC generated within the lung selectively enters the vascular compartment to facilitate pulmonary neutrophil recruitment. Rats were administered IT LPS, and plasma CINC and MIP-2 levels were measured 90 min and 4 h after injection, along with mRNA expression in lung, spleen, liver, and kidney. Ninety minutes and 4 h after IT LPS, CINC and MIP-2 mRNA expression were largely confined to lung homogenate, but of the two chemokines, only CINC was present in plasma. In separate experiments, rats received IT injections of recombinant CINC and/or MIP-2. Here, plasma levels of CINC, but not MIP-2, were significantly increased throughout the 4-h observation period. This finding was verified by individually administering (125)I-labeled forms of each chemokine. Instillation of recombinant MIP-2 or CINC into the lung increased the number of neutrophils recovered in bronchoalveolar lavage fluid at 4 h, and this effect was enhanced when both chemokines were administered together. In addition, intravenous (IV) CINC, but not IV MIP-2, increased pulmonary neutrophil recruitment in response to IT MIP-2. Our results show that CINC, in contrast to MIP-2, is selectively transported from the lung to the systemic circulation, where it promotes neutrophil migration into the lung in response to a chemotactic stimulus.

Fas ligand-induced murine pulmonary inflammation is reduced by a stable decoy receptor 3 analogue.

Fas ligand (FasL)-induced lung inflammation has recently been suggested to play an important role in the pathogenesis of acute respiratory disease syndrome (ARDS). In order to further explore this connection, we established a FasL-induced murine model of pulmonary inflammation. Instillation of recombinant FasL (rFasL) into the lung induced neutrophil infiltration and increased pulmonary permeability, as evidenced by increased total protein in the airspace; both occur in patients with ARDS. These effects were accompanied with a rapid induction of proinflammatory mediators: cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) and the chemokines macrophage inflammatory protein-2 (MIP-2) and KC. Pretreatment with a FasL antagonist, a decoy receptor 3 analogue (DcR3 analogue), reduced neutrophil infiltration into the airspace and resulted in a highly significant reduction in the levels of GM-CSF, MIP-2 and KC in bronchoalveolar lavage (BAL) fluid. We postulate that rFasL may be responsible for induction of proinflammatory chemokines and cytokines in the lung, which in turn attract neutrophil infiltration into the airspace. This proinflammatory process and the associated pulmonary permeability may, in part, explain the association of FasL with severe pulmonary inflammation, such as ARDS, and shed new light on FasL and its role in lung injury.

Rapamycin causes down-regulation of CCR5 and accumulation of anti-HIV beta-chemokines: an approach to suppress R5 strains of HIV-1.

Propagation of R5 strains of HIV-1 on CD4 lymphocytes and macrophages requires expression of the CCR5 coreceptor on the cell surface. Individuals lacking CCR5 (CCR5 Delta 32 homozygous genotype) are phenotypically normal and resistant to infection with HIV-1. CCR5 expression on lymphocytes depends on signaling through the IL-2 receptor. By FACS analysis we demonstrate that rapamycin (RAPA), a drug that disrupts IL-2 receptor signaling, reduces CCR5 surface expression on T cells at concentrations as low as 1 nM. In addition, lower concentrations of RAPA (0.01 nM) were sufficient to reduce CCR5 surface expression on maturing monocytes. PCR analysis on peripheral blood mononuclear cells (PBMCs) showed that RAPA interfered with CCR5 expression at the transcriptional level. Reduced expression of CCR5 on PBMCs cultured in the presence of RAPA was associated with increased extracellular levels of macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta. In infectivity assays, RAPA suppressed the replication of R5 strains of HIV-1 both in PBMC and macrophage cultures. In total PBMC cultures, RAPA-mediated inhibition of CCR5-using strains of HIV-1 occurred at 0.01 nM, a concentration of drug that is approximately 103 times lower than therapeutic through levels of drug in renal transplant recipients. In addition, RAPA enhanced the antiviral activity of the CCR5 antagonist TAK-779. These results suggest that low concentrations of RAPA may have a role in both the treatment and prevention of HIV-1 infection.

Increased production of chemotactic cytokines and elevated proliferation and expression of intercellular adhesion molecule-1 in rat mesangial cells treated with erythrogenic toxin type B and its precursor isolated from nephritogenic streptococci.

Previous reports have demonstrated the presence of streptococcal erythrogenic toxin type B (ETB) as well as proliferation and expression of adhesion molecules along with leukocyte infiltrations in biopsies from patients with acute post-streptococcal glomerulonephritis (APSGN). The purpose of the present study was to correlate infiltrative and proliferative events with interactions between ETB or its precursor (ETBP) and intrinsic mesangial cells. Rat mesangial cells were cultured with ETB or ETBP (50 micro g/ml) while measuring production of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) and while examining proliferation and expression of intercellular adhesion molecule-1 (ICAM-1). After 24, 48 and 96 h of incubation, MCP-1 and MIP-2 in culture supernatants were assessed by enzyme-linked immunosorbent assay (ELISA). Cells were assessed for proliferation by incorporation of radioactive thymidine and expression of ICAM-1 was measured by indirect immunofluorescence and by cellular ELISA. Compared with controls, treatment with either ETBP or ETB significantly increased MCP-1 and MIP-2 levels in mesangial cell cultures. Mesangial cells also showed elevated proliferation at 96 h of culture when treated with streptococcal proteins. Although production of MCP-1 and MIP-2 was not correlated with proliferation, treatment with ETBP resulted in a significant correlation between MCP-1 production and proliferation. Immunofluorescence studies revealed an increased expression of ICAM-1 in ETBP/ETB-treated mesangial cells. In addition, cellular ELISA studies showed increased absorbance in cultures treated with ETBP/ETB. Finally, low serum concentrations in the culture medium potentiated the stimulatory effect of ETB on MCP-1 production. Our findings, by demonstrating a role for cationic streptococcal ETB or ETBP in the induction of chemotactic molecules as well as the proliferation and expression of adhesion molecules, delineate an additional possible pathway for the pathogenesis of APSGN.

Impact of human interleukin-10 on vector-induced inflammation and early graft function in rat lung transplantation.

This study was undertaken to examine the time course of human interleukin (hIL)-10 gene expression after transtracheal administration of adenoviral (Ad)hIL-10 and its effect on the early adenoviral proinflammatory cytokine response and on post-transplant lung function. Using a rat lung transplant model, we observed that lungs retrieved 12 h after the administration of AdhIL-10 were associated with significant improvement in post-transplant lung function. Shorter periods of transfection were associated with significantly elevated levels of tumor necrosis factor-alpha and macrophage inflammatory protein-2 in lung tissue, leading to an increased degree of injury. The release of proinflammatory cytokines secondary to the adenoviral vector was reduced by high-dose methylprednisolone (30 mg/kg) administered 3 h before transfection. Reduction in the early adenoviral inflammatory response was associated with significant improvement in post-transplant lung function when lungs were retrieved 6 or 12 h after transtracheal administration of AdhIL-10. Transtracheal administration of adenoviral-mediated hIL-10 to donor lungs is associated with a significant early inflammatory response that may enhance ischemia-reperfusion injury if insufficient hIL-10 is expressed in lung tissue before retrieval. The period between delivery of AdhIL-10 and lung retrieval can be reduced if the early inflammatory response is suppressed with methylprednisolone.

Interleukin-18 impairs the pulmonary host response to Pseudomonas aeruginosa.

Interleukin-18 (IL-18) is a potent cytokine with many different proinflammatory activities. To study the role of IL-18 in the pathogenesis of Pseudomonas pneumonia, IL-18-deficient (IL-18(-/-)) and wild-type mice were intranasally inoculated with Pseudomonas aeruginosa. IL-18 deficiency was associated with reduced outgrowth of Pseudomonas in the lungs and diminished dissemination of the infection. In addition, pulmonary inflammation (histopathology) and levels of tumor necrosis factor alpha, IL-6, and macrophage inflammatory protein-2 in lungs and plasma were lower in IL-18(-/-) mice. Consistent with results obtained for IL-18(-/-) mice, treatment of wild-type mice with a neutralizing IL-18 binding protein-immunoglobulin G Fc fusion construct also attenuated outgrowth of Pseudomonas compared with that for mice treated with a control protein. These results demonstrate that the presence of endogenous IL-18 activity facilitates inflammatory responses in the lung during Pseudomonas pneumonia, concurrently impairing bacterial clearance.

Macrophage Inflammatory Protein-2 Levels Are Associated With Changes in Serum Leptin Concentrations Following Ozone-Induced Airway Inflammation

A positive correlation between obesity and the risk of acquiring asthma has been suggested by epidemiologic studies, but the mechanism underlying this relationship has not been established. The levels of leptin, a proinflammatory, satiety-controlling cytokine, are increased in the serum of obese individuals; therefore, we hypothesized that obesity promotes the development of asthma through leptin-induced augmentation of airway inflammation. The purpose of this study was to test this hypothesis by experimentally manipulating endogenous serum leptin levels and assessing their effects on markers of ozone-induced airway inflammation.

IL-17, produced by lymphocytes and neutrophils, is necessary for lipopolysaccharide-induced airway neutrophilia: IL-15 as a possible trigger.

IL-17 is a cytokine implicated in the regulation of inflammation. We investigated the role of this cytokine in neutrophil recruitment using a model of LPS-induced lung inflammation in mice. In the bronchoalveolar lavage, LPS induced a first influx of neutrophils peaking at day 1, followed by a second wave, peaking at day 2. IL-17 levels were increased during the late phase neutrophilia (day 2), and this was concomitant with an increased number of T cells and macrophages, together with an increase of KC and macrophage-inflammatory protein-2 levels in the lung tissue. Intranasal treatment with a neutralizing murine anti-IL-17 Ab inhibited the late phase neutrophilia. In the bronchoalveolar lavage cells, IL-17 mRNA was detected at days 1, 2, and 3 postchallenge, with a strong expression at day 2. This expression was associated with CD4(+) and CD8(+) cells, but also with neutrophils. When challenged with LPS, despite the absence of T cells, SCID mice also developed a neutrophilic response associated with IL-17 production. In BALB/c mice, IL-15 mRNA, associated mainly with neutrophils, was evidenced 1 day after LPS challenge. In vitro, IL-15 was able to induce IL-17 release from purified spleen CD4(+) cells, but not spleen CD8(+) or airway neutrophils. We have shown that IL-17, produced mainly by CD4(+) cells, but also by neutrophils, plays a role in the mobilization of lung neutrophils following bacterial challenge. In addition, our results suggest that IL-15 could represent a physiological trigger that leads to IL-17 production following bacterial infection.

Up-regulation of macrophage inflammatory protein-2 and complement 3A receptor by the trichothecenes deoxynivalenol and satratoxin G

The trichothecenes are a group of mycotoxins that target leukocytes and have a wide range of immunomodulatory effects. Differential display analysis was applied to assess the effects of the trichothecenes deoxynivalenol (vomitoxin, DON) and satratoxin G (SG), on mRNA in the RAW 264.7 macrophage cell line. Cells were incubated with DON (1 μg/ml) or SG (5 ng/ml) for 2 h and total RNA then subjected to RT-PCR with a set of oligo(dT) primers. Resultant cDNA was amplified using an oligo (dT) downstream primer and an arbitrary decanucleotide upstream primer to make 35S-labeled PCR products. After separation of the products in denaturing polyacrylamide gel, 23 differentially expressed cDNA fragments were isolated and sequenced. Two of these were identified as known genes, namely, macrophage inflammatory protein-2 (MIP-2), a potent neutrophil chemoattractant involved in tissue injury and inflammation, and complement 3a receptor (C3aR), a proinflammatory mediator. Both MIP-2 and C3aR mRNAs were up-regulated by DON while only MIP-2 mRNA was induced by SG. Using commercially available antibodies, MIP-2 protein was also found to be induced by both DON and SG in RAW 264.7 cell cultures. When mice were treated with DON (12.5 mg/kg), splenic MIP-2 mRNA and serum MIP-2 levels were increased. MIP-2 mRNA and serum MIP-2 levels were synergistically increased when mice were co-treated with DON and LPS. Up-regulation of MIP-2 and C3aR are consistent with previous reports of trichothecene-induced inflammatory gene up-regulation and suggest that the specific genes affected may depend on trichothecene structures.

Mast cells modulate the inflammatory but not the proliferative response in healing wounds.

Upon stimulation, mast cells release a heterogeneous group of factors that promote inflammation and influence cell proliferation. Mast cells accumulate at sites of injury, further suggesting a critical role in wound healing. To assess the importance of mast cells in tissue repair, we compared wound healing in mast cell-deficient WBB6F1/J-KitW/KitW-v (KitW/KitW-v) and wild type WBB6F1/++ (WT) mice. During the inflammatory phase, neutrophil infiltration into wounds of the KitW/KitW-v mice was significantly less than that of WT mice (84.6 +/- 10.3 vs. 218 +/- 26.0 cells/10 high-power fields at day 3, p < 0.001), while wound macrophage and T cell infiltration were similar in both strains. The decrease in neutrophils could not be explained by changes in tumor necrosis factor-alpha or macrophage inflammatory protein-2 levels, because the amounts of these two neutrophil chemoattractants were similar in both KitW/KitW-v and WT mice. Surprisingly, the absence of mast cells had no effect on the proliferative aspects of wound healing, including reepithelialization, collagen synthesis, and angiogenesis. Although mast cells are known to release proangiogenic mediators, vascular endothelial growth factor levels were similar in WT and KitW/KitW-v mice. Moreover, levels of fibroblast growth factor-2 were increased in KitW/KitW-v mice (4206 +/- 107 vs. 1865 +/- 249 pg/ml, p < 0.01). These results suggest that mast cells modulate the recruitment of neutrophils into sites of injury, yet indicate that mast cells are unlikely to exert a major influence on the proliferative response within healing wounds.

Modulation Of Sulfur Mustard-induced Inflammation And Gene Expression By Olvanil In The Hairless Mouse Vesicant Model

Cutaneous exposure to sulfur mustard [bis(2-chloroethyl) sulfide (SM)] produces a delayed inflammatory skin response that is followed by severe dermal injury. Assessment of anti-inflammatory therapies against SM-induced skin injury has mainly relied on qualitative histopathological evaluation. The goal of this study was to identify proinflammatory biomarkers in the hairless mouse vesicant model that could be used as additional indicators of SM-induced skin injury for evaluating anti-inflammatory treatment. SM-induced inflammation was determined at 2, 6, and 24 hr postexposure by changes in edema. Ribonuclease protection assay (RPA) was used to determine changes in gene expression of inflammatory mediators. At 2, 6, and 24 hr postexposure, a time-dependent increase in edema was observed in SM-exposed skin, which was significant at 6 and 24 hr when compared to unexposed controls. Ribonuclease protection assay analysis revealed a two-fold or greater increase in monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), MIP-1α, tumor necrosis factor-α, and interleukin (IL)-1β following exposure to SM when compared to unexposed controls. A significant time-dependent increase was observed in MCP-1, MIP-1α, and IL-1β over the 24 hr time period. At 24 hr postexposure, skin treated with the anti-inflammatory drug olvanil showed a significant decrease in SM-induced edema. Additionally, mRNA levels of MCP-1, MIP-2, and IL-1β were decreased when compared to skin exposed to SM alone. In this study, we identified molecular biomarkers at the mRNA level, up-regulated in skin exposed to SM, which can be partially suppressed by olvanil. Further characterization of the mRNA and protein expression patterns of proinflammatory biomarkers may enable the use of other classes of anti-inflammatory drugs or therapeutic treatments against SM dermal injury.

The Role of Free Radicals in the Toxic and Inflammatory Effects of Four Different Ultrafine Particle Types

PM 10 contains an ultrafine component, which is generally derived from combustion processes. This ultrafine fraction may be a factor in the increases in exacerbations of respiratory disease and deaths from cardiorespiratory causes associated with transient increases in levels of PM 10. By using four different ultrafine particles (carbon black, cobalt, nickel, and titanium dioxide), we set out to determine the attributes of the ultrafine particle (surface area, chemical composition, particle number, or surface reactivity) that contribute most to its toxicity and proinflammatory effects both in vivo and in vitro. Instillation of 125 µg ultrafine carbon black (UFCB) and ultrafine cobalt (UFCo) particles induced a significant influx of neutrophils at both 4 and 18 h postinstillation. Accompanying the influx of neutrophils was an increase in macrophage inflammatory protein-2 (MIP-2) (at 4 h) and an increase in γ-glutamyl transpeptidase (at 18 h) in bronchoalveolar lavage fluid (BAL). Ultrafine nickel (UFNi) did not induce a significant increase in neutrophil influx until 18 h postinstillation. The increase in neutrophils induced by UFNi at this timepoint was comparable to that induced by UFCo and UFCB. UFTi did not induce a significant increase in neutrophils following instillation into the rat lung. The levels of MIP-2 observed at 4 h and neutrophil influx at 18 h induced by the particle samples were consistent with the pattern of surface free radical generation (as measured by the plasmid scission assay) whereby UFCo, UFCB, and UFNi all cause significant increases in inflammatory markers, as well as inducing a significant depletion of supercoiled plasmid DNA, indicative of hydroxyl radical generation. A role for free radicals and reactive oxygen species (ROS) in mediating ultrafine inflammation is further strengthened by the ability of the antioxidants N-acetylcysteine (NAC) and glutathione monoethyl ester (GSHme) to block the particle induced release of tumour necrosis factor-α (TNF-α) from alveolar macrophages in vitro. The ultrafine particles in PM 10 may cause adverse effects via oxidative stress, and this could have implications for susceptible individuals. Susceptible individuals, such as those with COPD or asthma, already exhibit preexisting oxidative stress and hence are in a primed state for further oxidative stress induced by occupational or environmental particles.