Chlorine (Cl₂) is a highly irritating and reactive gas with potential occupational and environmental hazards. Acute exposure to Cl₂ induces severe epithelial damage, airway hyperreactivity, impaired alveolar fluid clearance, and pulmonary edema in the presence of heightened inflammation and significant neutrophil accumulation in the lungs. Herein, we investigated whether Cl₂ exposure affected the lung antimicrobial immune response leading to increased susceptibility to opportunistic infections. Mice exposed to Cl₂ and challenged intratracheally 24 h thereafter with the opportunistic mold Aspergillus fumigatus demonstrated an >500-fold increase in A. fumigatus lung burden 72 h postchallenge compared with A. fumigatus mice exposed to room air. Cl₂-exposed A. fumigatus challenged mice also demonstrated significantly higher lung resistance following methacholine challenge and increased levels of plasma proteins (albumin and IgG) in the bronchoalveolar lavage fluid. Despite enhanced recruitment of inflammatory cells to the lungs of Cl₂-exposed A. fumigatus challenged mice, these cells (>60% of which were neutrophils) demonstrated a profound impairment in generating superoxide. Significantly higher A. fumigatus burden in the lungs of Cl₂ exposed mice correlated with enhanced production of IL-6, TNF-α, CXCL1, CCL2, and CCL3. Surprisingly, however, Cl₂-exposed A. fumigatus challenged mice had a specific impairment in the production of IL-17A and IL-22 in the lungs compared with mice exposed to room air and challenged with A. fumigatus. In summary, our results indicate that Cl₂ exposure markedly impairs the antimicrobial activity and inflammatory reactivity of myeloid cells in the lung leading to increased susceptibility to opportunistic pathogens.

BACKGROUND: Respiratory viral infections are characterized by the infiltration of leukocytes, including activated neutrophils into the lung that can lead to sustained lung injury and potentially contribute to chronic lung disease. Specific mechanisms recruiting neutrophils to the lung during virus-induced lung inflammation and injury have not been fully elucidated. Since CXCL1 and CXCL2/3, acting through CXCR2, are potent neutrophil chemoattractants, we investigated their role in dsRNA-induced lung injury, where dsRNA (Poly IC) is a well-described synthetic agent mimicking acute viral infection. METHODS: We used 6-8 week old female BALB/c mice to intratracheally inject either single-stranded (ssRNA) or double-stranded RNA (dsRNA) into the airways. The lungs were then harvested at designated timepoints to characterize the elicited chemokine response and resultant lung injury following dsRNA exposure as demonstrated qualititatively by histopathologic analysis, and quantitatively by FACS, protein, and mRNA analysis of BAL fluid and tissue samples. We then repeated the experiments by first pretreating mice with an anti-PMN or corresponding control antibody, and then subsequently pretreating a separate cohort of mice with an anti-CXCR2 or corresponding control antibody prior to dsRNA exposure. RESULTS: Intratracheal dsRNA led to significant increases in neutrophil infiltration and lung injury in BALB/c mice at 72 h following dsRNA, but not in response to ssRNA (Poly C; control) treatment. Expression of CXCR2 ligands and CXCR2 paralleled neutrophil recruitment to the lung. Neutrophil depletion studies significantly reduced neutrophil infiltration and lung injury in response to dsRNA when mice were pretreated with an anti-PMN monoclonal Ab. Furthermore, inhibition of CXCR2 ligands/CXCR2 interaction by pretreating dsRNA-exposed mice with an anti-CXCR2 neutralizing Ab also significantly attenuated neutrophil sequestration and lung injury. CONCLUSION: These findings demonstrate that CXC chemokine ligand/CXCR2 biological axis is critical during the pathogenesis of dsRNA-induced lung injury relevant to acute viral infections.

The foreign body reaction (FBR) differs between subcutaneously and supra-epicardially implanted materials. We hypothesize that this is a result of differences in cytokine, chemokine and matrix metalloproteinase (MMP) dynamics. Therefore we applied collagen disks subcutaneously and on the epicardium in mice and analyzed the FBR from day 1 to 21. Both the influx of leukocytes and implant degradation were higher in supra-epicardially implanted collagen than in subcutaneously implanted material. This correlated with a higher gene expression of pro-inflammatory cytokines such as IL-1 and IL-6, and a lower expression of the anti-inflammatory cytokine IL-10. Furthermore, the higher supra-epicardial expression of PMN attractants CXCL1/KC and CXCL2/MIP2 correlated with a higher and prolonged PMN influx. The gene expression levels of collagen degrading MMPs, i.e. MMP8, MMP13 and MMP14 were similar in subcutaneous and supra-epicardial disks. However, the activity of these enzymes was markedly higher supra-epicardially. In addition, the MMP9 expression was higher supra-epicardially, suggesting a role for this enzyme in the degradation process. In conclusion, a strong pro-inflammatory milieu is generated after supra-epicardial implantation that enables prolonged PMN presence and activation. This, together with the high supra-epicardial MMP9 level, could explain the observed difference in Col-I degradation between locations.

Background. Lymphatic filariasis is a chronic human parasitic disease in which the parasites repeatedly provoke acute and chronic inflammatory reactions in the host bloodstream and lymphatics. Excretory-secretory products derived from filariae are believed to play an important role in the development of associated immunologic conditions; however, the specific mechanisms involved in these changes are not well understood. Recently, human cytoplasmic aminoacyl-transfer (t) RNA synthetases, which are autoantigens in idiopathic inflammatory myopathies, were shown to activate chemokine receptors on T lymphocytes, monocytes, and immature dendritic cells by recruiting immune cells that could induce innate and adaptive immune responses. Filarial (Brugia malayi) asparaginyl-tRNA synthetase (AsnRS) is known to be an immunodominant antigen that induces strong human immunoglobulin G3 responses.Methods. Recombinant B. malayi AsnRS was used to perform cellular function assays--for example, chemotaxis and kinase activation assays.Results. Unlike human AsnRS, parasite AsnRS is chemotactic for neutrophils and eosinophils. Recombinant B. malayi AsnRS but not recombinant human AsnRS induced chemotaxis of CXCR1 and CXCR2 single-receptor-transfected HEK-293 cell lines, blocked CXCL1-induced calcium flux, and induced mitogen-activated protein kinase.Conclusions. Our findings suggest that a filarial parasite chemoattractant protein may contribute to the development of chronic inflammatory disease and that chemokine receptors may be therapeutic targets to ameliorate parasite-induced pathology.

OBJECTIVE

Our previous studies showed that NF-kappaB2/p52 is involved in the castration-resistant growth of the androgen-sensitive LNCaP prostate cancer cells. The role of NF-kappaB2/p52 in lymphomagenesis has been studied extensively, but its target genes in other cancers remain unknown. In order to identify genes potentially regulated by p52 in prostate cancer cells, we performed a genome-wide microarray analysis of genes differentially up- or down-regulated by the overexpression of p52 by adenoviral-mediated gene delivery in LNCaP cells.

METHODS

Total RNAs from vector control-infected and Adeno-p52-infected LNCaP cells were used to prepare cDNAs, which were hybridized to the Whole Genome Human 44k Microarray chips (Agilent Technologies). Data analysis was performed using GeneSpring and Ingenuity Pathway Analysis software. Validation of microarray results was performed by real-time quantitative RT-PCR and Western blot analyses.

RESULTS

Expression of approximately 130 genes was differentially upregulated by >5-fold, whereas approximately 60 genes were differentially downregulated by >2-fold in p52-expressing LNCaP cells. Pathway analysis revealed that the upregulated genes belong to functional categories like cell growth and proliferation, cellular movement, cell-to-cell signaling and interaction, cancer, cell cycle, etc., whereas the downregulated genes were represented by functional categories like cell movement, antigen presentation, and cell death. Six of the top upregulated genes including annexin A2, PLAU, RND3, Twist2, VEGFC, and CXCL1 were validated by real-time PCR and Western blot analysis.

CONCLUSIONS

This study provides a comprehensive analysis of genes potentially regulated by NF-kappaB2/p52 in the LNCaP prostate cancer cell line and provides a rationale for the induction of castration-resistant growth by p52 in LNCaP cells.

We demonstrated recently that Francisella tularensis profoundly impairs human neutrophil apoptosis, but how this is achieved is largely unknown. Herein we used human oligonucleotide microarrays to test the hypothesis that changes in neutrophil gene expression contribute to this phenotype, and now demonstrate that F. tularensis live vaccine strain (LVS) caused significant changes in neutrophil gene expression over a 24-hour time period relative to the uninfected controls. Of approximately 47,000 genes analyzed, 3,435 were significantly up- or downregulated by LVS, including 365 unique genes associated with apoptosis and cell survival. Specific targets in this category included genes asso-ciated with the intrinsic and extrinsic apoptotic pathways (CFLAR, TNFAIP3, TNFRSF10D, SOD2, BCL2A1, BIRC4, PIM2, TNFSF10, TNFRSF10C, CASP2 and CASP8) and genes that act via the NFĸB pathway and other mechanisms to prolong cell viability (NFKB1, NFKB2 and RELA, IL1B, CAST, CDK2,GADD45B, BCL3, BIRC3, CDK2, IL1A, PBEF1, IL6, CXCL1, CCL4 and VEGF). The microarray data were confirmed by qPCR and pathway analysis. Moreover, we demonstrate that the X-linked inhibitor of apoptosis protein remained abundant in polymorphonuclear leukocytes over 48 h of LVS infection, whereas BAX mRNA and protein were progressively downregulated. These data strongly suggest that antiapoptotic and prosurvival mechanisms collaborate to sustain the viability of F. tularensis--infected neutrophils.

The incidence of cancers that metastasize to the peritoneum increases with age. Intraperitoneal cancer dissemination depends largely on angiogenesis and interactions with the peritoneal mesothelium. We assessed the proangiogenic potential of human peritoneal mesothelial cells. Conditioned media collected from these cells at senescence stimulated proliferation of endothelial cells to a significantly greater extent compared to media from early-passage cells. The effect was accompanied by a significantly increased release of proangiogenic mediators -- VEGF, CXCL1/GROalpha, CXCL8/IL-8, and CCL2/MCP-1. These results indicate that the senescent mesothelium exhibits increased angiogenic activity, which may contribute to accelerated intraperitoneal cancer progression in the aged.

Extracts from the edible mushroom Agaricus blazei Murill (AbM) are used extensively as a non-prescription remedy against cancer, infections, and immune related diseases. The presumed effect is to activate certain parts of the immune system. In order to investigate the effect, we examined the changes of gene expression caused by the extract on a human monocyte cell line (THP-1). Changes in the levels of mRNA transcripts were measured using 35 k microarrays, and the changes in select cytokine gene products by immuno assays. Lipopolysaccharide (LPS) was included for comparison. Both AbM and LPS had drastic effects on gene expression. Genes related to immune function were selectively up-regulated, particularly proinflammatoric genes such as the interleukins IL1B and IL8. Although most genes induced by AbM were also induced by LPS, AbM produced a unique profile, e.g., as to a particular increase in mRNA for the cytokines IL1A, CXCL1, CXCL2 and CXCL3, as well as PTGS2 (cyclooxygenase2).

The existence of multipotent cardiac stromal cells expressing stem cell antigen (Sca)-1 has been reported, and their proangiogenic properties have been demonstrated in myocardial infarction models. In this study, we tested the hypothesis that stimulation of adenosine receptors on cardiac Sca-1(+) cells up-regulates their secretion of proangiogenic factors. We found that Sca-1 is expressed in subsets of mouse cardiac stromal CD31(-) and endothelial CD31(+) cells. The population of Sca-1(+)CD31(+) endothelial cells was significantly reduced, whereas the population of Sca-1(+)CD31(-) stromal cells was increased 1 week after myocardial infarction, indicating their relative functional importance in this pathophysiological process. An increase in adenosine levels in adenosine deaminase-deficient mice in vivo significantly augmented vascular endothelial growth factor (VEGF) production in cardiac Sca-1(+)CD31(-) stromal cells but not in Sca-1(+)CD31(+) endothelial cells. We found that mouse cardiac Sca-1(+)CD31(-) stromal cells predominantly express mRNA encoding A(2B) adenosine receptors. Stimulation of adenosine receptors significantly increased interleukin (IL)-6, CXCL1 (a mouse ortholog of human IL-8), and VEGF release from these cells. Using conditionally immortalized Sca-1(+)CD31(-) stromal cells obtained from wild-type and A(2B) receptor knockout mouse hearts, we demonstrated that A(2B) receptors are essential for adenosine-dependent up-regulation of their paracrine functions. We found that the human heart also harbors a population of stromal cells similar to the mouse cardiac Sca-1(+)CD31(-) stromal cells that increase release of IL-6, IL-8, and VEGF in response to A(2B) receptor stimulation. Thus, our study identified A(2B) adenosine receptors on cardiac stromal cells as potential targets for up-regulation of proangiogenic factors in the ischemic heart.

Dendritic cells (DCs), the most potent and specialized antigen-presenting cells, play a key role in the regulation of the adaptive immunity. Immature DCs were generated by in vitro culturing of peripheral blood monocytes and functionally activated with the classical pathogen-associated molecular pattern lipopolysaccharide (LPS). Alternative activation resulting in Th-2 polarization was induced with lipid oxidation products derived from 1-palmitoyl-2-arachidoyl-sn-glycerol-3-phosphorylcholin (OxPAPC). Tolerogenic cells were obtained by treating DCs with human rhinovirus (HRV). The aim of this study was the identification of proteome profiles related to the functionally different dendritic cell phenotypes. Cytoplasmic proteins were analyzed by shotgun proteomics resulting in the identification of 1690 proteins. While mature and alternatively activated DCs displayed highly distinct protein expression profiles, HRV-treated DCs showed minor proteome alterations. As DCs exert many specific functions via secretion, we investigated the secretomes by a combination of 2D-PAGE and shotgun proteomics. We successfully identified a broad variety of cytokines (e.g., GM-CSF, TNF-alpha, interleukin-1beta, 6, 12 beta, 28B and 29), chemokines (e.g., CCL3, 5, 8, 17, 18, 19, 24, CXCL1, 2, 9 and 10) and growth factors (growth/differentiation factor 8, C-type lectin domain family 11 member A). The relative composition of secretome profiles, although comprising much less proteins, was found to be much more affected by functional alteration of cells than the cytoplasmic protein composition. In conclusion, we demonstrate that functional distinct subsets of DCs display distinct proteome profiles which comprise biomarker candidates. These proteins may prove useful for the interpretation of complex clinical proteomics data.

As the current staging system is imprecise for estimating prognosis of early stage non-small cell lung cancer (NSCLC), it is important to identify other methods for selecting high-risk patients after failed surgical treatment. The aim of the study was to evaluate the expression of 23 genes as putative prognostic markers in early stage NSCLC. The study was performed on 109 pairs of tumor and matched unaffected lung tissue surgical specimens taken from stage I and II NSCLC patients. We evaluated the mRNA level of 23 genes using the real-time PCR method. The difference in the expression between the tumor and normal tissue for each gene was analyzed using a general linear model. The influence of gene expression on survival was analyzed by using the proportional hazards model. Eighteen out of the 23 genes showed statistically significant differences in expression between the tumor and non-tumor tissue. For 12 genes (ITGB1, ITGB3, <em>CXCL1</em>, CXCL8, CXCL9, <em>CXCL1</em>0, <em>CXCL1</em>1, CXCR3, CXCR4, TNF, CHKA, AGFG1, and CTC1), the expression was lower, and for six genes (ITGA5, IL8, IL6, CXCL2, CXCL3, and <em>CXCL1</em>2), it was higher in the tumor tissue as compared to the matched normal tissue. Expression changes were more pronounced in squamous cell carcinomas than in adenocarcinomas or large cell carcinomas. Of all the analyzed genes, only CXCL5 was found to statistically significantly (p = 0.04) influence both overall and disease-free survival. Among the 23 genes previously suggested to be relevant for early staged NSCLC patients' postoperative outcome, only CXCL5 showed a statistically significant prognostic effect.

The inability of epithelial cells from the cornea and other tissues to respond to LPS is reportedly due to low expression of the TLR4 co-receptor MD-2. We generated MD-2(-/-) bone marrow chimeras, and showed that MD-2 expression on non-myeloid cells was sufficient to mediate LPS-induced corneal inflammation. As IFN-γ is produced during Pseudomonas aeruginosa corneal infection, we examined the role of this cytokine on MD-2 expression by primary human corneal epithelial (HCE) cells and HCE cell lines. Exogenous IFN-γ was found to induce MD-2 mRNA, MD-2 cell surface expression, and LPS responsiveness as determined by p65 translocation to the nucleus and production of IL-6, CXCL1, and CXCL8/IL-8. Incubation with either the AG490 JAK2 inhibitor or with STAT1 siRNA blocked STAT1 phosphorylation and MD-2 transcription. Furthermore, EMSA analysis demonstrated that STAT1 binds to the MD-2 promoter, indicating that STAT1 is an MD-2 transcription factor. Together, these findings demonstrate that IFN-γ induces MD-2 expression and LPS responsiveness in HCE cells by JAK-2-dependent STAT1 activation and direct binding to the MD-2 promoter. Furthermore, given our findings on LPS-induced corneal inflammation, it is likely that IFN-γ-induced MD-2 expression by corneal epithelial cells contributes to the host response in vivo, determining the extent of tissue damage and bacterial clearance.

Inflammatory bowel disease (IBD) affects millions of people worldwide but its pathophysiology remains unclear. Therefore, experimental models of colitis have contributed crucially for the understanding of IBD, and also in the investigations for effective therapies. Herein we investigated the kinetics of inflammatory mediator production and cell infiltration during acute and chronic dextran sodium sulfate (DSS)-induced colitis. The induction phases with DSS were characterized by severe disease activity with massive colonic polymorphonuclear infiltration and increased levels of tumor necrosis factor-α (TNF-α), keratinocyte-derived chemokine (CXCL1/KC), interleukin (IL)-17 and vascular adhesion molecule-1 (VCAM-1). Interestingly, in the recovery periods, we found marked increase of anti-inflammatory mediators IL-10, IL-4, transforming growth factor-β (TGF-β) and cyclooxygenase 2 (COX-2) that seems be essential for the resolution of intestinal inflammation. Furthermore, nuclear factor κB (NFκB) and regulatory T cell marker forkhead box P3 (FoxP3) were increased gradually during experimental colitis, demonstrating a discrepant profile response and evident immune disbalance in the chronic phase of intestinal mucosal inflammation. Taken together, these results provide valuable information for studies on DSS-induced colitis and especially for the identification of biomarkers that predict disease course and possible therapeutic interventions.

BACKGROUND

Women have an increased prevalence of severe asthma compared with men. IL-17A is associated with severe asthma and requires IL-23 receptor (IL-23R) signaling, which is negatively regulated by let-7f microRNA.

OBJECTIVE

We sought to Determine the mechanism by which 17β-estradiol (E2) and progesterone (P4) increase IL-17A production.

METHODS

IL-17A production was determined by using flow cytometry in TH17 cells from women (n = 14) and men (n = 15) with severe asthma. Cytokine levels were measured by using ELISA, and IL-23R and let-7f expression was measured by using quantitative PCR in TH17-differentiated cells from healthy women (n = 13) and men (n = 14). In sham-operated or ovariectomized female mice, 17β-E2, P4, 17β-E2+P4, or vehicle pellets were administered for 3 weeks before ex vivo TH17 cell differentiation. Airway neutrophil infiltration and CXCL1 (KC) expression were also determined in ovalbumin (OVA)-challenged wild-type female recipient mice with an adoptive transfer of OVA-specific TH17 cells from female and male mice.

RESULTS

In patients with severe asthma and healthy control subjects, IL-17A production was increased in TH17 cells from women compared with men. IL-23R expression was increased and let-7f expression was decreased in TH17-differentiated cells from women compared with men. In ovariectomized mice IL-17A and IL-23R expression was increased and Let-7f expression was decreased in TH17 cells from mice administered 17β-E2+P4 compared with those administered vehicle. Furthermore, transfer of female OVA-specific TH17 cells increased acute neutrophil infiltration in the lungs of OVA-challenged recipient mice compared with transfer of male OVA-specific TH17 cells.

CONCLUSIONS

17β-E2+P4 increased IL-17A production from TH17 cells, providing a potential mechanism for the increased prevalence of severe asthma in women compared with men.

Despite the obvious clinical significance of post-stroke angiogenesis in aged subjects, a detailed transcriptomic analysis of post-stroke angiogenesis has not yet been undertaken in an aged experimental model. In this study, by combining stroke transcriptomics with immunohistochemistry in aged rats and post-stroke patients, we sought to identify an age-specific gene expression pattern that may characterize the angiogenic process after stroke. We found that both young and old infarcted rats initiated vigorous angiogenesis. However, the young rats had a higher vascular density by day 14 post-stroke. "New-for-stroke" genes that were linked to the increased vasculature density in young animals included Angpt2, Angptl2, Angptl4, Cib1, Ccr2, Col4a2, <em>Cxcl1</em>, Lef1, Hhex, Lamc1, Nid2, Pcam1, Plod2, Runx3, Scpep1, S100a4, Tgfbi, and Wnt4, which are required for sprouting angiogenesis, reconstruction of the basal lamina (BL), and the resolution phase. The vast majority of genes involved in sprouting angiogenesis (Angpt2, Angptl4, Cib1, Col8a1, Nrp1, Pcam1, Pttg1ip, Rac2, Runx1, Tnp4, Wnt4); reconstruction of a new BL (Col4a2, Lamc1, Plod2); or tube formation and maturation (Angpt1, Gpc3, Igfbp7, Sparc, Tie2, Tnfsf10), had however, a delayed upregulation in the aged rats. The angiogenic response in aged rats was further diminished by the persistent upregulation of "inflammatory" genes (<em>Cxcl1</em>2, Mmp8, Mmp12, Mmp14, Mpeg1, Tnfrsf1a, Tnfrsf1b) and vigorous expression of genes required for the buildup of the fibrotic scar (Cthrc1, Il6ra, Il13ar1, Il18, Mmp2, Rassf4, Tgfb1, Tgfbr2, Timp1). Beyond this barrier, angiogenesis in the aged brains was similar to that in young brains. We also found that the aged human brain is capable of mounting a vigorous angiogenic response after stroke, which most likely reflects the remaining brain plasticity of the aged brain.

BACKGROUND

Aspergillus fumigatus causes invasive aspergillosis, a potentially fatal infection in oncohematological patients. Innate immune detection of A. fumigatus involves Toll-like receptor (TLR) 4 and TLR2, which forms a heterodimer with either TLR1 or TLR6. The role of those coreceptors in Aspergillus sensing is unknown.

METHODS

Cytokine production was measured in bone marrow-derived macrophages (BMDMs) from wild-type (WT) and TLR-deficient mice after incubation with a WT and an immunogenic RodA-deficient (ΔrodA-47) strain of A. fumigatus and in lungs from these mice after intranasal mold inoculation. Aspergillus fumigatus-mediated NF-κB activation was measured in HEK293T cells transfected with plasmids expressing mouse or human TLRs.

RESULTS

Bone marrow-derived macrophages from TLR1- and TLR6-deficient mice produced lower amounts of interleukin 12p40, CXCL2, interleukin 6, and tumor necrosis factor α than BMDMs from WT mice after stimulation with A. fumigatus. Lungs from TLR1- and TLR6-deficient mice had diminished CXCL1 and CXCL2 production and increased fungal burden after intranasal inoculation of ΔrodA A. fumigatus compared with lungs from WT mice. ΔrodA strain-mediated NF-κB activation was observed in HEK293T cells expressing mouse TLR2/1, mouse TLR2/6, and human TLR2/1 but not human TLR2/6.

CONCLUSIONS

Innate immune detection of A. fumigatus is mediated by TLR4 and TLR2 together with TLR1 or TLR6 in mice and TLR1 but not TLR6 in humans.

Initial observations suggested that C-C motif chemokines exclusively mediate chemotaxis of mononuclear cells. In addition, recent studies also implicated these chemotactic cytokines in the recruitment of neutrophils. The underlying mechanisms remained largely unknown. Using in vivo microscopy on the mouse cremaster muscle, intravascular adherence and subsequent paracellular transmigration of neutrophils elicited by the chemokine (C-C motif) ligand 3 (CCL3, synonym MIP-1α) were significantly diminished in mice with a deficiency of the chemokine (C-C motif) receptor 1 (Ccr1(-/-)) or 5 (Ccr5(-/-)). Using cell-transfer techniques, neutrophil responses required leukocyte CCR1 and nonleukocyte CCR5. Furthermore, neutrophil extravasation elicited by CCL3 was almost completely abolished on inhibition of G protein-receptor coupling and PI3Kγ-dependent signaling, while neutrophil recruitment induced by the canonical neutrophil attractants chemokine (C-X-C motif) ligand 1 (CXCL1, synonym KC) or the lipid mediator platetelet-activating factor (PAF) was only partially reduced. Moreover, Ab blockade of β(2) integrins, of α(4) integrins, or of their putative counter receptors ICAM-1 and VCAM-1 significantly attenuated CCL3-, CXCL1-, or PAF-elicited intravascular adherence and paracellular transmigration of neutrophils. These data indicate that the C-C motif chemokine CCL3 and canonical neutrophil attractants exhibit both common and distinct mechanisms for the regulation of intravascular adherence and transmigration of neutrophils.

Recruitment of mesenchymal stem cells (MSC) to tissue damages is a promising approach for in situ tissue regeneration. The physiological mechanisms and regulatory processes of MSC trafficking to injured tissue remain poorly understood. However, the pivotal role of chemokines in MSC recruitment has already been shown. The aim of this study was to determine the migratory potential and the gene expression profile of MSC stimulated with the CC chemokine CCL25 (TECK). Bone marrow derived human MSC were exposed to different doses of CCL25 in a standardized chemotaxis assay. Microarray gene expression profiling and pathway analysis were performed for CCL25 stimulated MSC. Maximum migration of MSC towards CCL25 was observed at 10(3) nM. Microarray analysis revealed an induction of molecules directly involved in chemotaxis and homing of bone marrow cells (CXCL1-3, CXCL8, PDE4B), cytoskeletal and membrane reorganisation (CXCL8, PLD1, IGFBP1), cellular polarity (PLD1), and cell movement (CXCL1-3, CXCL6, CXCL8, PTGS2, PDE4B, TGM2). Respective chemokine secretion was confirmed by protein membrane-array analysis. The activation of CXCR2 ligands (CXCL1-3, CXCL5-6, CXCL8) and a LIF-receptor/gp130 ligand (LIF) indicated an involvement of the respective signaling pathways during initiation of chemotaxis and migration. These results suggest CCL25 as a new potential candidate for further in situ regeneration approaches.

Aqueous humor (AH) samples from patients with Behçet's disease (BD) (n=29), Vogt-Koyanagi-Harada (VKH) disease (n=21), and HLA-B27-associated uveitis (n=8), and 42 control patients were assayed for the neutrophil chemoattractants <em>CXCL1</em>/GRO-α and CXCL8/IL-8 and the lymphocyte chemoattractants CXCL9/MIG, <em>CXCL1</em>0/IP-10 and <em>CXCL1</em>2/SDF-1 with the use of a multiplex chemokine assay. Chemokine levels except SDF-1 were significantly higher in the 3 disease groups than in normal controls. Considering all patients, mean GRO-α levels were 15-fold higher than IL-8 levels and mean IP-10 levels were 22-fold higher than MIG levels. In patients with the same disease activity, AH levels of GRO-α and IP-10 were significantly higher in patients with BD than in patients with VKH disease and HLA-B27-associated uveitis (p=0.0474; p<0.001, respectively). These data suggest that GRO-α and IP-10 are the predominant CXC chemokines involved in neutrophil and activated T lymphocyte chemoattraction in endogenous uveitis, particularly in BD.

Interleukin-17 (IL-17)-secreting T helper 17 cells were recently identified as a CD4(+) T helper subset and implicated in various inflammatory and autoimmune diseases. The issues of whether and by what mechanism hyperlipidemic stress induces IL-17A to activate aortic endothelial cells (ECs) and enhance monocyte adhesion remained largely unknown. Using biochemical, immunological, microarray, experimental data mining analysis, and pathological approaches focused on primary human and mouse aortic ECs (HAECs and MAECs) and our newly generated apolipoprotein E (ApoE)(-/-)/IL-17A(-/-) mice, we report the following new findings. 1) The hyperlipidemia stimulus oxidized low density lipoprotein up-regulated IL-17 receptor(s) in HAECs and MAECs. 2) IL-17A activated HAECs and increased human monocyte adhesion in vitro. 3) A deficiency of IL-17A reduced leukocyte adhesion to endothelium in vivo. 3) IL-17A activated HAECs and MAECs via up-regulation of proinflammatory cytokines IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), chemokine CXC motif ligand 1 (CXCL1), and CXCL2. 4) IL-17A activated ECs specifically via the p38 mitogen-activated protein kinases (MAPK) pathway; the inhibition of p38 MAPK in ECs attenuated IL-17A-mediated activation by ameliorating the expression of the aforementioned proinflammatory cytokines, chemokines, and EC adhesion molecules including intercellular adhesion molecule 1. Taken together, our results demonstrate for the first time that IL-17A activates aortic ECs specifically via p38 MAPK pathway.

Heme oxygenase-1 (HO-1) has been shown to display anti-inflammatory properties in models of acute pulmonary inflammation. For the first time, we investigated the role of leukocytic HO-1 using a model of HO-1(flox/flox) mice lacking leukocytic HO-1 that were subjected to lipopolysaccharide (LPS)-induced acute pulmonary inflammation. Immunohistology and flow cytometry demonstrated that activation of HO-1 using hemin decreased migration of polymorphonuclear leukocytes (PMNs) to the lung interstitium and bronchoalveolar lavage (BAL) in the wild-type and, surprisingly, also in HO-1(flox/flox) mice, emphasizing the anti-inflammatory potential of nonmyeloid HO-1. Nevertheless, hemin reduced the CXCL1, CXCL2/3, tumor necrosis factor-α (TNFα), and interleukin 6 (IL6) levels in both animal strains. Microvascular permeability was attenuated by hemin in wild-type and HO-1(flox/flox) mice, indicating a crucial role of non-myeloid HO-1 in endothelial integrity. The determination of the activity of HO-1 in mouse lungs revealed no compensatory increase in the HO-1(flox/flox) mice. Topical administration of hemin via inhalation reduced the dose required to attenuate PMN migration and microvascular permeability by a factor of 40, emphasizing its clinical potential. In addition, HO-1 stimulation was protective against pulmonary inflammation when initiated after the inflammatory stimulus. In conclusion, nonmyeloid HO-1 is crucial for the anti-inflammatory effect of this enzyme on PMN migration to different compartments of the lung and on microvascular permeability.

BACKGROUND

Acute lung injury (ALI) is a common disease in critically ill patients with a high morbidity and mortality. 12/15-lipoxygenase (12/15-LO) is an enzyme generating 12-hydroxy-eicosatetraenoic acid (12-HETE) and 15-HETE from arachidonic acid. It has been shown that 12/15-LO is involved in the regulation of vascular permeability during ALI.

METHODS

To test whether 12/15-LO participates in leukocyte recruitment into the lung, we investigated the role of 12/15-LO in mouse models of lipopolysaccharide (LPS)-induced pulmonary inflammation and acid-induced ALI, a clinically relevant model of acute lung injury.

RESULTS

The increase in neutrophil recruitment following LPS inhalation was reduced in 12/15-LO-deficient (Alox15(-/-)) mice and in wild-type (WT) mice after the blocking of 12/15-LO with a pharmacological inhibitor. Bone marrow chimeras revealed that 12/15-LO in hematopoietic cells regulates neutrophil accumulation in the interstitial and alveolar compartments, whereas the accumulation of neutrophils in the intravascular compartment is regulated by 12/15-LO in non-hematopoietic and hematopoietic cells. Mechanistically, the increased plasma levels of the chemokine CXCL1 in Alox15(-/-) mice led to a reduced response of the neutrophil chemokine receptor CXCR2 to stimulation with CXCL1, which in turn abrogated neutrophil recruitment. Alox15(-/-) mice also showed decreased edema formation, reduced neutrophil recruitment and improved gas exchange in an acid-induced ALI model.

CONCLUSIONS

Our findings suggest that 12/15-LO modulates neutrophil recruitment into the lung by regulating chemokine/chemokine receptor homeostasis.

BACKGROUND

Serum and bronchoalveolar fluid IL-6 are increased in patients with acute respiratory distress syndrome (ARDS) and predict prolonged mechanical ventilation and poor outcomes, although the role of intra-alveolar IL-6 in indirect lung injury is unknown. We investigated the role of endogenous and exogenous intra-alveolar IL-6 in AKI-mediated lung injury (indirect lung injury), intraperitoneal (IP) endotoxin administration (indirect lung injury) and, for comparison, intratracheal (IT) endotoxin administration (direct lung injury) with the hypothesis that IL-6 would exert a pro-inflammatory effect in these causes of acute lung inflammation.

METHODS

Bronchoalveolar cytokines (IL-6, CXCL1, TNF-α, IL-1β, and IL-10), BAL fluid neutrophils, lung inflammation (lung cytokines, MPO activity [a biochemical marker of neutrophil infiltration]), and serum cytokines were determined in adult male C57Bl/6 mice with no intervention or 4 hours after ischemic AKI (22 minutes of renal pedicle clamping), IP endotoxin (10 µg), or IT endotoxin (80 µg) with and without intratracheal (IT) IL-6 (25 ng or 200 ng) treatment.

RESULTS

Lung inflammation was similar after AKI, IP endotoxin, and IT endotoxin. BAL fluid IL-6 was markedly increased after IT endotoxin, and not increased after AKI or IP endotoxin. Unexpectedly, IT IL-6 exerted an anti-inflammatory effect in healthy mice characterized by reduced BAL fluid cytokines. IT IL-6 also exerted an anti-inflammatory effect in IT endotoxin characterized by reduced BAL fluid cytokines and lung inflammation; IT IL-6 had no effect on lung inflammation in AKI or IP endotoxin.

CONCLUSIONS

IL-6 exerts an anti-inflammatory effect in direct lung injury from IT endotoxin, yet has no role in the pathogenesis or treatment of indirect lung injury from AKI or IP endotoxin. Since intra-alveolar inflammation is important in the pathogenesis of direct, but not indirect, causes of lung inflammation, IT anti-inflammatory treatments may have a role in direct, but not indirect, causes of ARDS.

Endothelial inflammation is regulated by a complex molecular mechanism. TC1(C8orf4) is a novel regulator implicated in cancer and inflammation. It is a small protein conserved well among vertebrates. In zebrafish embryos, it is mostly expressed in angio-hematopoietic system and the overexpression induces edema. In human aortic endothelial cells and umbilical vein endothelial cells, TC1 transfection up-regulates key inflammatory cytokines, enzymes, and adhesion proteins including IL-6, IL-1alpha, COX-2, CXCL1, CCL5, CCL2, IL-8, ICAM1, VCAM1, and E-selectin, while TC1 knockdown down-regulates them. TC1 also enhances inflammatory parameters such as monocyte-endothelial adhesion and endothelial monolayer permeability. TC1 is up-regulated by IL-1beta, TNF-alpha, LPS, and phorbol ester, and the up-regulation is inhibited by I-kappaB-kinase inhibitors. TC1, in turn, enhances the nuclear translocation of RelA and the DNA binding activity, suggesting a biological role of amplifying NF-kappaB signaling via a positive feedback. Our findings suggest that TC1 is a novel endothelial inflammatory regulator that might be implicated in inflammatory vascular diseases.