Ursolic Acid Ameliorates the Injury of H9c2 Cells Caused by Hypoxia and Reoxygenation Through Mediating CXCL2/NF-κB Pathway.

Ursolic acid (UA) has been reported to possess several biological benefits, such as anti-cancer, anti-inflammation, antibacterial, and neuroprotective functions. This study detects the function and molecular mechanism of UA in H9c2 cells under hypoxia and reoxygenation (H/R) conditions.Under H/R stimulation, the effects of UA on H9c2 cells were examined using ELISA and western blot assays. The Comparative Toxicogenomics Database was employed to analyze the target molecule of UA. Small interfering RNA was used to knock down CXCL2 expression, further exploring the function of CXCL2 in H/R-induced H9c2 cells. The genes related to the nuclear factor-kappa B (NF-κB) pathway were assessed using western blot analysis.Significant effects of UA on H/R-induced H9c2 cell damage were observed, accompanied by reduced inflammation and oxidative stress injury. Additionally, the increased level of CXCL2 in H/R-induced H9c2 cells was reduced after UA stimulation. Moreover, CXCL2 knockdown strengthened the beneficial effect of UA on H/R-induced H9c2 cells. HY-18739, an activator of the NF-κB pathway, can increase CXCL2 expression. Moreover, the increased levels of p-P65 NF-κB and p-IκBα in H/R-induced H9c2 cells were remarkably attenuated by UA treatment.In summary, the results indicated that UA may alleviate the damage of H9c2 cells by targeting the CXCL2/NF-κB pathway under H/R conditions.

MyD88 in Macrophages Enhances Liver Fibrosis by Activation of NLRP3 Inflammasome in HSCs.

Chronic liver disease mediated by the activation of hepatic stellate cells (HSCs) leads to liver fibrosis. The signal adaptor MyD88 of Toll-like receptor (TLR) signaling is involved during the progression of liver fibrosis. However, the specific role of MyD88 in myeloid cells in liver fibrosis has not been thoroughly investigated. In this study, we used a carbon tetrachloride (CCl4)-induced mouse fibrosis model in which MyD88 was selectively depleted in myeloid cells. MyD88 deficiency in myeloid cells attenuated liver fibrosis in mice and decreased inflammatory cell infiltration. Furthermore, deficiency of MyD88 in macrophages inhibits the secretion of CXC motif chemokine 2 (CXCL2), which restrains the activation of HSCs characterized by NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome activation. Moreover, targeting CXCL2 by CXCR2 inhibitors attenuated the activation of HSCs and reduced liver fibrosis. Thus, MyD88 may represent a potential candidate target for the prevention and treatment of liver fibrosis.

Mechanically stimulated osteocytes promote the proliferation and migration of breast cancer cells via a potential CXCL1/2 mechanism.

Bone represents the most common site for breast cancer metastasis. Bone is a highly dynamic organ that is constantly adapting to its biophysical environment, orchestrated largely by the resident osteocyte network. Osteocytes subjected to physiologically relevant biophysical conditions may therefore represent a source of key factors mediating breast cancer cell metastasis to bone. Therefore, we investigated the potential proliferative and migratory capacity of soluble factors released by mechanically stimulated osteocytes on breast cancer cell behaviour. Interestingly the secretome of mechanically stimulated osteocytes enhanced both the proliferation and migration of cancer cells when compared to the secretome of statically cultured osteocytes, demonstrating that mechanical stimuli is an important physiological stimulus that should be considered when identifying potential targets. Using a cytokine array, we further identified a group of mechanically activated cytokines in the osteocyte secretome, which potentially drive breast cancer metastasis. In particular, CXCL1 and CXCL2 cytokines are highly expressed, mechanically regulated, and are known to interact with one another. Lastly, we demonstrate that these specific factors enhance breast cancer cell migration independently and in a synergistic manner, identifying potential osteocyte derived factors mediating breast cancer metastasis to bone.

CXCL1 and CXCL2 Inhibit the Axon Outgrowth in a Time- and Cell-Type-Dependent Manner in Adult Rat Dorsal Root Ganglia Neurons.

The ability to regrow their axons after an injury is a hallmark of neurons in peripheral nervous system which distinguish them from central nervous system neurons. This ability is influenced by their intrinsic capacity to regrow and by the extracellular environment which needs to be supportive of regrowth. CXCL1 [Chemokine (C-X-C motif) Ligand 1] and CXCL2 [Chemokine (C-X-C motif) Ligand 2] are two low-molecular-weight chemokines which can influence neuronal proliferation, differentiation and neurogenesis, but which are also upregulated by injury or inflammation. In this study we investigated the effects of long-term incubation (24, 48 and 72 h) with different concentrations of CXCL1 (0.4, 4 or 40 nM) or CXCL2 (0.36, 3.6 or 36 nM) on the axon outgrowth of adult rat dorsal root ganglia neurons in culture. The results showed that both chemokines significantly inhibited the axon outgrowth, with large and medium NF200 (NeuroFilament 200) (+) dorsal root ganglia neurons affected quicker, compared to small IB4 (Isolectin B4) (+) dorsal root ganglia neurons which were affected after longer exposure. Blocking CXCR2 (C-X-C motif chemokine receptor 2) which mediates the effects of CXCL1 and CXCL2 prevented these effects, suggesting that CXCR2 may represent a new therapeutic target for promoting the axon outgrowth after a peripheral nerve injury.

Re-education of Tumor-Associated Macrophages by CXCR2 Blockade Drives Senescence and Tumor Inhibition in Advanced Prostate Cancer.

Tumor-associated macrophages (TAMs) represent a major component of the tumor microenvironment supporting tumorigenesis. TAMs re-education has been proposed as a strategy to promote tumor inhibition. However, whether this approach may work in prostate cancer is unknown. Here we find that Pten-null prostate tumors are strongly infiltrated by TAMs expressing C-X-C chemokine receptor type 2 (CXCR2), and activation of this receptor through CXCL2 polarizes macrophages toward an anti-inflammatory phenotype. Notably, pharmacological blockade of CXCR2 receptor by a selective antagonist promoted the re-education of TAMs toward a pro-inflammatory phenotype. Strikingly, CXCR2 knockout monocytes infused in Ptenpc-/-; Trp53pc-/- mice differentiated in tumor necrosis factor alpha (TNF-α)-releasing pro-inflammatory macrophages, leading to senescence and tumor inhibition. Mechanistically, PTEN-deficient tumor cells are vulnerable to TNF-α-induced senescence, because of an increase of TNFR1. Our results identify TAMs as targets in prostate cancer and describe a therapeutic strategy based on CXCR2 blockade to harness anti-tumorigenic potential of macrophages against this disease.

Transcriptome profiling analysis reveals that CXCL2 is involved in anlotinib resistance in human lung cancer cells.

BACKGROUND: Anlotinib has been demonstrated its anti-tumor efficacy on non-small cell lung cancer (NSCLC) in clinical trials at 3rd line. However, anlotinib resistance occurs during its administration, and the underlying mechanism is still unclear. METHODS: Anlotinib resistant lung cancer cell line NCI-H1975 was established in vitro. Toxicologic effects undergoing anlotinib stress were observed upon NCI-H1975 cells and anlotinib resistant NCI-H1975 cells, respectively. Transcriptome profiling was performed to screen anlotinib resistance-associated genes between NCI-H1975 cells and anlotinib resistant NCI-H1975 cells. Functional assays were performed to examine the correlations between CXCL2 gene expression and anlotinib resistance. RESULTS: We found anlotinib inhibits cell proliferation and cell viability in NCI-1975 cells, whereas it attenuates these activities in anlotinib resistant NCI-H1975 cells. Transcriptome profiling analysis identified 769 anlotinib-responsive genes enriched in the biological processes of microtubule-based process, cytoskeleton organization, and wound healing. Furthermore, we found 127 genes are associated with anlotinib resistance. In particular, we demonstrated that CXCL2 contributes to anlotinib resistance in NCI-H1975 cells. CONCLUSIONS: This study suggested that CXCL2 is involved in anlotinib resistance in NCI-H1975 cells and provided an insight for understanding the resistant mechanism of anlotinib.

Rapid Mobilization Reveals a Highly Engraftable Hematopoietic Stem Cell.

Hematopoietic stem cell transplantation is a potential curative therapy for malignant and nonmalignant diseases. Improving the efficiency of stem cell collection and the quality of the cells acquired can broaden the donor pool and improve patient outcomes. We developed a rapid stem cell mobilization regimen utilizing a unique CXCR2 agonist, GROß, and the CXCR4 antagonist AMD3100. A single injection of both agents resulted in stem cell mobilization peaking within 15 min that was equivalent in magnitude to a standard multi-day regimen of granulocyte colony-stimulating factor (G-CSF). Mechanistic studies determined that rapid mobilization results from synergistic signaling on neutrophils, resulting in enhanced MMP-9 release, and unexpectedly revealed genetic polymorphisms in MMP-9 that alter activity. This mobilization regimen results in preferential trafficking of stem cells that demonstrate a higher engraftment efficiency than those mobilized by G-CSF. Our studies suggest a potential new strategy for the rapid collection of an improved hematopoietic graft.

Clinical relevance of circulating anti-ENA and anti-dsDNA secreting cells from SLE patients and their dependence on STAT-3 activation.

Disturbances of plasma cell homeostasis and auto-antibody production are hallmarks of systemic lupus erythematosus. The aim of this study was to explore the presence of circulating anti-ENA and anti-dsDNA antibody-secreting cells, to determine their dependence on plasma cell-niche cytokines and to analyze their clinical value. The study was performed in SLE patients with serum anti-ENA and/or anti-dsDNA antibodies (n = 57). Enriched B-cell fractions and sorted antibody-secreting cells (CD19low CD38high ) were obtained from blood. dsDNA- and ENA-specific antibody-secreting cells were identified as cells capable of active auto-antibody production in culture. The addition of a combination of IL-6, IL-21, BAFF, APRIL, and CXCL12 to the cultures significantly augmented auto-antibody production and antibody-secreting cell proliferation, whereas it diminished apoptosis. The effect on auto-antibody production was dependent on STAT-3 activation as it was abrogated in the presence of the JAK/STAT-3 pathway inhibitors ruxolitinib and stattic. Among patients with serum anti-dsDNA antibodies, the detection of circulating anti-dsDNA-antibody-secreting cells was associated with higher disease activity markers. In conclusion, auto-antibody production in response to plasma cell-niche cytokines that are usually at high levels in SLE patients is dependent on JAK/STAT-3 activation. Thus, patients with circulating anti-dsDNA antibody-secreting cells and active disease could potentially benefit from therapies targeting the JAK/STAT3 pathway.

Antitumour activity of the recombination polypeptide GST-NT21MP is mediated by inhibition of CXCR4 pathway in breast cancer.

BACKGROUND: CXC chemokine receptor 4 (CXCR4) and its ligand stromal cell-derived factor-1α (SDF-1α, also known as CXCL12) have important roles in promoting tumour growth and metastasis. Therefore, targeting CXCR4 could be a promising strategy for treatment of human cancer. METHODS: To achieve this goal, we developed a highly purified recombination polypeptide (GST-NT21MP), which is a synthetic 21-mer peptide antagonist of CXCR4 (NT21MP) derived from the viral macrophage inflammatory protein II by fermentation technology, affinity chromatography and fast protein liquid chromatography. In this study, we used multiple methods such as MTT assay, FACS, invasion assay, RT-PCR and western blot to explore the efficacy and mechanism by which GST-NT21MP inhibits cell growth, migration and invasion of breast cancer in vitro and in vivo. RESULTS: We found that blockade of CXCR4 pathway by GST-NT21MP decreased SDF-1-induced cell growth, adhesion and migration capacities in breast cancer cells. Moreover, GST-NT21MP significantly retarded pulmonary metastasis in vivo. Furthermore, GST-NT21MP-mediated antitumour activity was found to be associated with reduced phosphorylated Src, Akt, FAK and ERK1/2 as well as decreased Bcl-2. CONCLUSIONS: Our results suggest that GST-NT21MP could be a potential anticancer agent for the treatment of breast cancer.

Mesenchymal stem cells tune the development of monocyte-derived dendritic cells toward a myeloid-derived suppressive phenotype through growth-regulated oncogene chemokines.

Mesenchymal stem/stromal cells (MSCs) are promising potential candidates for the treatment of immunological diseases because of their immunosuppressive functions. However, the molecular mechanisms that mediate MSCs' immunosuppressive activity remain elusive. In this article, we report for the first time, to our knowledge, that secreted growth-regulated oncogene (GRO) chemokines, specifically GRO-γ, in human MSC-conditioned media have an effect on the differentiation and the function of human monocyte-derived dendritic cells. The monocyte-derived dendritic cells were driven toward a myeloid-derived suppressor cell (MDSC)-like phenotype by the GRO chemokines. GRO-γ-treated MDSCs had a tolerogenic phenotype that was characterized by an increase in the secretion of IL-10 and IL-4, and a reduction in the production of IL-12 and IFN-γ. We have also shown that the mRNA expression levels of the arginase-1 and inducible NO synthase genes, which characterize MDSCs, were upregulated by GRO-γ-primed mouse bone marrow cells. In addition, the ability of GRO-γ-treated bone marrow-derived dendritic cells to stimulate the OVA-specific CD8(+) T (OT-1) cell proliferation and the cytokine production of IFN-γ and TNF-α were significantly decreased in vivo. Our findings allow a greater understanding of how MDSCs can be generated and offer new perspectives to exploit the potential of MDSCs for alternative approaches to treat chronic inflammation and autoimmunity, as well as for the prevention of transplant rejection.

Human chemokines as antimicrobial peptides with direct parasiticidal effect on Leishmania mexicana in vitro.

Chemokines and chemokine receptor-mediated effects are important mediators of the immunological response and cure in human leishmaniasis. However, in addition to their signalling properties for leukocytes, many chemokines have also been shown to act directly as antimicrobial peptides on bacteria and fungi. We screened ten human chemokines (CXCL2, CXCL6, CXCL8, CXCL9, CXCL10, CCL2, CCL3, CCL20, CCL27, CCL28) for antimicrobial effects on the promastigote form of the protozoan parasite Leishmania mexicana, and observed direct parasiticidal effects of several, CCL28 being the most potent. Damage to the plasma membrane integrity could be visualised by entrance of propidium iodide, as measured with flow cytometry, and by scanning electron microscopy, which showed morphological changes and aggregation of cells. The findings were in concordance with parasiticidal activity, measured by decreased mitochondrial activity in an MTT-assay. This is the first report of direct antimicrobial activity by chemokines on parasites. This component of immunity against Leishmania parasites identified here warrants further investigation that might lead to new insight in the mechanisms of human infection and/or new therapeutic approaches.

Inefficient antimicrobial functions of innate phagocytes render infant mice more susceptible to bacterial infection.

Neonates and infants, due to the immaturity in their adaptive immunity, are thought to depend largely on the innate immune system for protection against bacterial infection. However, the innate immunity-mediated antimicrobial response in neonates and infants is incompletely characterized. Here, we report that infant mice were more susceptible to microbial sepsis than adult mice, with significantly reduced bacterial clearance from the circulation and visceral organs. Infant PMNs exhibited less constitutive expression of the chemokine receptor CXCR2, and bacterial infection caused further reduction of PMN CXCR2 in infant mice compared with adult mice. This correlates with diminished in vitro chemotaxis of infant PMNs toward the chemoattractant CXCL2 and impaired in vivo recruitment of infant PMNs into the infectious site. Furthermore, consistent with the reduced antimicrobial response in vivo, infant macrophages displayed an impaired bactericidal activity with a defect in phagosome maturation after ingestion of either gram-positive or gram-negative bacteria. Thus, infant mice exhibit an increased vulnerability to microbial infection with delayed bacterial clearance, which is associated with the inefficiency in their innate phagocyte-associated antimicrobial functions characterized by defects in PMN recruitment and macrophage phagosome maturation during microbial sepsis.

Fer kinase limits neutrophil chemotaxis toward end target chemoattractants.

Neutrophil recruitment and directional movement toward chemotactic stimuli are important processes in innate immune responses. This study examines the role of Fer kinase in neutrophil recruitment and chemotaxis to various chemoattractants in vitro and in vivo. Mice targeted with a kinase-inactivating mutation (Fer(DR/DR)) or wild type (WT) were studied using time-lapse intravital microscopy to examine leukocyte recruitment and chemotaxis in vivo. In response to keratinocyte-derived cytokine, no difference in leukocyte chemotaxis was observed between WT and Fer(DR/DR) mice. However, in response to the chemotactic peptide WKYMVm, a selective agonist of the formyl peptide receptor, a 2-fold increase in leukocyte emigration was noted in Fer(DR/DR) mice (p < 0.05). To determine whether these defects were due to Fer signaling in the endothelium or other nonhematopoietic cells, bone marrow chimeras were generated. WKYMVm-induced leukocyte recruitment in chimeric mice (WT bone marrow to Fer(DR/DR) recipients or vice versa) was similar to WT mice, suggesting that Fer kinase signaling in both leukocytes and endothelial cells serves to limit chemotaxis. Purified Fer(DR/DR) neutrophils demonstrated enhanced chemotaxis toward end target chemoattractants (WKYMVm and C5a) compared with WT using an under-agarose gel chemotaxis assay. These defects were not observed in response to intermediate chemoattractants (keratinocyte-derived cytokine, MIP-2, or LTB(4)). Increased WKYMVm-induced chemotaxis of Fer(DR/DR) neutrophils correlated with sustained PI3K activity and reduced reliance on the p38 MAPK pathway compared with WT neutrophils. Together, these data identify Fer as a novel inhibitory kinase for neutrophil chemotaxis toward end target chemoattractants through modulation of PI3K activity.

Different microvascular permeability responses elicited by the CXC chemokines MIP-2 and KC during leukocyte recruitment: role of LSP1.

The CXC chemokines keratinocyte-derived chemokine (KC, CXCL1) and macrophage inflammatory protein-2 (MIP-2, CXCL2) activate G-protein coupled receptor CXCR2 and are believed to have similar inflammatory effects in mice. Their specific signaling mechanisms remain elusive. A wide variety of cellular events, mediators and signaling pathways are known to regulate microvascular permeability. Leukocyte-specific protein 1 (LSP1), a Ca2+- and F-actin binding protein, is one of the major downstream substrates of p38 MAPK. LSP1 was previously shown to play a pivotal role in leukocyte transmigration and microvascular permeability. Using intravital microscopy visualizing microvasculature of murine cremaster muscle, we demonstrate that KC and MIP-2 triggered increased leukocyte recruitment which was significantly reduced in LSP1-deficient mice compared to the wild-type control mice. Fluorescence imaging revealed that KC induced more substantial increases of microvascular permeability to FITC-labeled albumin than MIP-2. We found that LSP1 had a more prominent role in microvascular hyperpermeability induced by KC than that triggered by MIP-2. Moreover, Western blotting showed enhanced phosphorylation of p38 MAPK in the cremasteric tissue after stimulation with KC but not with MIP-2 and KC-induced but not MIP-2-induced hyperpermeability was blunted by pharmacological inhibition of p38 MAPK. In conclusion, LSP1 plays an important role in leukocyte recruitment induced by both KC and MIP-2. KC elicits more profoundly increased microvascular permeability than MIP-2. KC is at least partially effective through LSP1 and the phosphorylation of p38 MAPK.

CXCR2 mediates NADPH oxidase-independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation.

Upon activation, neutrophils release DNA fibers decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). Although NETs are bactericidal and contribute to innate host defense, excessive NET formation has been linked to the pathogenesis of autoinflammatory diseases. However, the mechanisms regulating NET formation, particularly during chronic inflammation, are poorly understood. Here we show that the G protein-coupled receptor (GPCR) CXCR2 mediates NET formation. Downstream analyses showed that CXCR2-mediated NET formation was independent of NADPH oxidase and involved Src family kinases. We show the pathophysiological relevance of this mechanism in cystic fibrosis lung disease, characterized by chronic neutrophilic inflammation. We found abundant NETs in airway fluids of individuals with cystic fibrosis and mouse cystic fibrosis lung disease, and NET amounts correlated with impaired obstructive lung function. Pulmonary blockade of CXCR2 by intra-airway delivery of small-molecule antagonists inhibited NET formation and improved lung function in vivo without affecting neutrophil recruitment, proteolytic activity or antibacterial host defense. These studies establish CXCR2 as a receptor mediating NADPH oxidase-independent NET formation and provide evidence that this GPCR pathway is operative and druggable in cystic fibrosis lung disease.

Differential activity of pro-angiogenic CXC chemokines.

We showed previously in a mouse model of lung ischemia-induced angiogenesis, enhanced expression of the three ELR+ CXC chemokines (KC, LIX, and MIP-2) and that blockade of the ligand receptor CXCR(2) limited neovascularization. The present study was undertaken to determine the relative abundance and angiogenic potential of the three CXC chemokines and whether RhoA activation explained the measured differences in potencies. We found that LIX showed the greatest absolute amount in the in vivo model 4 h after left pulmonary artery obstruction (LIX>KC>MIP-2; p<0.05). In vitro, LIX induced the greatest degree of arterial endothelial cell chemotaxis and KC was without effect. A significant increase (approximately 40%) in active RhoA was observed with both LIX and MIP-2 compared with vehicle control (p<0.05). On average, LIX induced the greatest amount of tube formation within pleural tissue in culture. Thus, the results of the present study suggest that among the three ELR+ CXC chemokines, LIX predominates in eliciting a pro-angiogenic phenotype.

Platelet-derived CD40L (CD154) mediates neutrophil upregulation of Mac-1 and recruitment in septic lung injury.

OBJECTIVE: To define the role of CD40L in abdominal sepsis. BACKGROUND: Platelets promote sepsis-induced pulmonary recruitment of neutrophils. However, the identity of the platelet-derived molecule regulating neutrophil infiltration is not known. The hypothesis of the present study was that platelet-derived CD40L might be responsible for platelet-mediated activation and accumulation of neutrophils in sepsis. METHODS: Wild-type C57BL/6 mice and CD40L gene-deficient mice were exposed to cecal ligation and puncture (CLP). Lung edema, bronchoalveolar neutrophils, CD40L and macrophage inflammatory protein-2 (MIP-2) plasma levels, myeloperoxidase activity and Mac-1 expression were determined up to 24 hours after CLP induction. For platelet depletion was an anti-GP1balpha antibody administered before CLP. RESULTS: Plasma levels of soluble CD40L increased and surface expression of CD40L on platelets decreased in CLP mice. Platelet depletion reduced CLP-induced CD40L levels by 90%. CLP-provoked Mac-1 expression on neutrophils was abolished in CD40L-deficient mice. Interestingly, CLP-induced edema and myeloperoxidase activity in the lung as well as neutrophil infiltration in the broncoalveolar space were markedly reduced in mice lacking CD40L. In vitro experiments showed that CD40L was not capable of directly increasing Mac-1 levels on neutrophils. Instead, CLP-induced plasma levels of MIP-2 were significantly reduced in CD40L-deficient mice and inhibition of the MIP-2 receptor (CXCR2) decreased Mac-1 expression on neutrophils in septic animals. CONCLUSIONS: CD40L derived from platelets is a potent activator of neutrophils and mediates sepsis-induced neutrophil recruitment and lung edema. The neutrophil activating mechanism of CD40L is indirect and mediated via MIP-2 formation and CXCR2 signaling. Targeting CD40L may be an effective approach to limit pulmonary damage in abdominal sepsis.

Myeloid-specific deletion of tumor suppressor PTEN augments neutrophil transendothelial migration during inflammation.

Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) is a second messenger that is involved in a number of cell activities including cell growth, proliferation, and motility. PIP(3) is produced by PI3K and regulated by PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP lipid phosphatases. Evidence from our experiments shows that enhanced PIP(3) production results in elevated neutrophil recruitment under inflammatory conditions. However, the mechanism of this elevation is not well understood. We used intravital video microscopy to investigate neutrophil recruitment in the cremaster venules of wild-type and PTEN knockout (KO) mice. Neutrophil transmigration was augmented in PTEN KO mice 4 h after TNF-alpha intrascrotal injection. PTEN KO neutrophils also showed significantly enhanced transmigration 2 h after MIP-2 intrascrotal injection, an effect that dramatically decreased when PI3K or Src kinase inhibitor treatments preceded MIP-2 stimulation. Similarly, fMLP superfusion of the cremaster muscle lead to enhanced emigration in PTEN KO mice. The observed elevation in neutrophil emigration was likely caused by increased speed of crawling, crossing the venular wall, and migrating through the muscular tissue in PTEN KO mice because the effect of PTEN depletion on neutrophil rolling or adhesion was minimal. Interestingly, chemoattractant-induced release of gelatinase and elastase was also elevated in PTEN null neutrophils, providing a potential mechanism for the enhanced neutrophil migration in the PTEN KO mice. Collectively, these results demonstrate that PTEN deletion in neutrophils enhances their invasivity and recruitment to inflamed sites more likely by raising the cell physical capability to cross the vascular and tissue barriers.

Hepatocyte signaling through CXC chemokine receptor-2 is detrimental to liver recovery after ischemia/reperfusion in mice.

UNLABELLED: CXC chemokines and their receptor, CXC chemokine receptor-2 (CXCR2), are important components of the hepatic inflammatory response to ischemia/reperfusion (I/R). However, direct effects of CXC chemokines on hepatocytes during this response have not been studied. Wild-type and CXCR2(-/-) mice were subjected to 90 minutes of partial hepatic ischemia followed by up to 96 hours of reperfusion. CXCR2(-/-) mice had significantly less liver injury at all reperfusion times compared with wild-type mice. Early neutrophil recruitment (12 hours) was diminished in CXCR2(-/-) mice, but within 24 hours it was the same as that of wild-type mice. Hepatocyte proliferation and regeneration was accelerated in CXCR2(-/-) mice compared with wild-type mice. These effects were associated with increased activation of nuclear factor kappaB and signal transducers and activators of transcription-3, despite there being no difference in the expression of proliferative factors such as tumor necrosis factor alpha, interleukin-6, and hepatocyte growth factor. To establish whether the accelerated proliferation and regeneration observed in CXCR2(-/-) mice was due to effects on hepatocytes rather than just a generalized decrease in acute inflammatory injury, mice were treated with the CXCR2 antagonist, SB225002, after neutrophil recruitment and injury were maximal (24 hours after reperfusion). SB225002 treatment increased hepatocyte proliferation and regeneration in a manner identical to that observed in CXCR2(-/-) mice. Treatment of primary wild-type hepatocytes with macrophage inflammatory protein-2 revealed that low concentrations protected against cell death, whereas high concentrations induced cell death. These effects were absent in hepatocytes from CXCR2(-/-) mice. CONCLUSION: Our data suggest that hepatocyte CXCR2 regulates proliferation and regeneration after I/R injury and reveal important differences in the role of this receptor in liver regeneration and repair induced under different conditions that may be related to ligand concentration.

Migration across the sinusoidal endothelium regulates neutrophil mobilization in response to ELR + CXC chemokines.

An increase in circulating neutrophils is a characteristic feature of many inflammatory reactions and is a result of the rapid mobilization of neutrophils from the bone marrow, driven by inflammatory mediators, including the ELR + CXC chemokines. In this paper, using a combination of light and electron microscopy and an in situ perfusion system of the rat femoral bone marrow, we examined this mobilization process in detail. We show that mobilization of neutrophils stimulated by the CXC chemokine, rat MIP-2, involves neutrophil migration from the haematopoietic compartment of the bone marrow across the bone marrow sinusoidal endothelium via a transcellular route. The critical role of the bone marrow sinusoidal endothelium in regulating neutrophil mobilization was demonstrated by artificially disrupting the bone marrow endothelial barrier by treatment with cytochalasin D, which results in the non-selective release of leucocytes from the bone marrow. In contrast, inhibiting the activity of p38 mitogen-activated protein kinase, inhibited both MIP-2 stimulated chemotaxis of bone marrow neutrophils in vitro and neutrophil mobilization in situ while, a broad spectrum matrix metalloproteinase inhibitor, BB94, had no effect on neutrophil mobilization. These results support the hypothesis that neutrophil migration drives their mobilization and highlights the function of the sinusoidal endothelium in regulating this process.