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.

Endothelial LSP1 Modulates Extravascular Neutrophil Chemotaxis by Regulating Nonhematopoietic Vascular PECAM-1 Expression.

During inflammation, leukocyte-endothelial cell interactions generate molecular signals that regulate cell functions. The Ca(2+)- and F-actin-binding leukocyte-specific protein 1 (LSP1) expressed in leukocytes and nonhematopoietic endothelial cells is pivotal in regulating microvascular permeability and leukocyte recruitment. However, cell-specific function of LSP1 during leukocyte recruitment remains elusive. Using intravital microscopy of cremasteric microvasculature of chimeric LSP1-deficient mice, we show that not neutrophil but endothelial LSP1 regulates neutrophil transendothelial migration and extravascular directionality without affecting the speed of neutrophil migration in tissue in response to CXCL2 chemokine gradient. The expression of PECAM-1-sensitive α6ß1 integrins on the surface of transmigrated neutrophils was blunted in mice deficient in endothelial LSP1. Functional blocking studies in vivo and in vitro elucidated that α6ß1 integrins orchestrated extravascular directionality but not the speed of neutrophil migration. In LSP1-deficient mice, PECAM-1 expression was reduced in endothelial cells, but not in neutrophils. Similarly, LSP1-targeted small interfering RNA silencing in murine endothelial cells mitigated mRNA and protein expression of PECAM-1, but not ICAM-1 or VCAM-1. Overexpression of LSP1 in endothelial cells upregulated PECAM-1 expression. Furthermore, the expression of transcription factor GATA-2 that regulates endothelial PECAM-1 expression was blunted in LSP1-deficient or LSP1-silenced endothelial cells. The present study unravels endothelial LSP1 as a novel cell-specific regulator of integrin α6ß1-dependent neutrophil extravascular chemotactic function in vivo, effective through GATA-2-dependent transcriptional regulation of endothelial PECAM-1 expression.

CXC chemokines function as a rheostat for hepatocyte proliferation and liver regeneration.

BACKGROUND: Our previous in vitro studies have demonstrated dose-dependent effects of CXCR2 ligands on hepatocyte cell death and proliferation. In the current study, we sought to determine if CXCR2 ligand concentration is responsible for the divergent effects of these mediators on liver regeneration after ischemia/reperfusion injury and partial hepatectomy. METHODS: Murine models of partial ischemia/reperfusion injury and hepatectomy were used to study the effect of CXCR2 ligands on liver regeneration. RESULTS: We found that hepatic expression of the CXCR2 ligands, macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC), was significantly increased after both I/R injury and partial hepatectomy. However, expression of these ligands after I/R injury was 30-100-fold greater than after hepatectomy. Interestingly, the same pattern of expression was found in ischemic versus non-ischemic liver lobes following I/R injury with expression significantly greater in the ischemic liver lobes. In both systems, lower ligand expression was associated with increased hepatocyte proliferation and liver regeneration in a CXCR2-dependent fashion. To confirm that these effects were related to ligand concentration, we administered exogenous MIP-2 and KC to mice undergoing partial hepatectomy. Mice received a "high" dose that replicated serum levels found after I/R injury and a "low" dose that was similar to that found after hepatectomy. Mice receiving the "high" dose had reduced levels of hepatocyte proliferation and regeneration whereas the "low" dose promoted hepatocyte proliferation and regeneration. CONCLUSIONS: Together, these data demonstrate that concentrations of CXC chemokines regulate the hepatic proliferative response and subsequent liver regeneration.

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.

Rho-kinase regulates adhesive and mechanical mechanisms of pulmonary recruitment of neutrophils in abdominal sepsis.

We hypothesized that Rho-kinase signaling plays a role in mechanical and adhesive mechanisms of neutrophil accumulation in lung. Male C57BL/6 mice were treated with the Rho-kinase inhibitor Y-27632 prior to cecal ligation and puncture (CLP). Lung levels of myeloperoxidase (MPO) and histological tissue damage were determined 6h and 24h after CLP. Expression of Mac-1 and F-actin formation in neutrophils were quantified by using flow cytometry 6h after CLP. Mac-1 expression and F-actin formation were also determined in isolated neutrophils up to 3h after stimulation with CXCL2. Labeled and activated neutrophils co-incubated with Y-27632, an anti-Mac-1 antibody and cytochalasin B were adoptively transferred to CLP mice. Y-27632 reduced the CLP-induced pulmonary injury and MPO activity as well as Mac-1 on neutrophils. Neutrophil F-actin formation peaked at 6h and returned to baseline levels 24h after CLP induction. Rho-kinase inhibition decreased CLP-provoked F-actin formation in neutrophils. CXCL2 rapidly increased Mac-1 expression and F-actin formation in neutrophils. Co-incubation with Y-27632 abolished CXCL2-induced Mac-1 up-regulation and formation of F-actin in neutrophils. Notably, co-incubation with cytochalasin B inhibited formation of F-actin but did not reduce Mac-1 expression on activated neutrophils. Adoptive transfer experiments revealed that co-incubation of neutrophils with the anti-Mac-1 antibody or cytochalasin B significantly decreased pulmonary accumulation of neutrophils in septic mice. Our data show that targeting Rho-kinase effectively reduces neutrophil recruitment and tissue damage in abdominal sepsis. Moreover, these findings demonstrate that Rho-kinase-dependent neutrophil accumulation in septic lung injury is regulated by both adhesive and mechanical mechanisms.

Tracking neutrophil intraluminal crawling, transendothelial migration and chemotaxis in tissue by intravital video microscopy.

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation(1,2). In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling(3). Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)(4). Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment(2,5). In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm(3) size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

Increased recruitment but impaired function of leukocytes during inflammation in mouse models of type 1 and type 2 diabetes.

BACKGROUND: Patients suffering from diabetes show defective bacterial clearance. This study investigates the effects of elevated plasma glucose levels during diabetes on leukocyte recruitment and function in established models of inflammation. METHODOLOGY/PRINCIPAL FINDINGS: Diabetes was induced in C57Bl/6 mice by intravenous alloxan (causing severe hyperglycemia), or by high fat diet (moderate hyperglycemia). Leukocyte recruitment was studied in anaesthetized mice using intravital microscopy of exposed cremaster muscles, where numbers of rolling, adherent and emigrated leukocytes were quantified before and during exposure to the inflammatory chemokine MIP-2 (0.5 nM). During basal conditions, prior to addition of chemokine, the adherent and emigrated leukocytes were increased in both alloxan- (62±18% and 85±21%, respectively) and high fat diet-induced (77±25% and 86±17%, respectively) diabetes compared to control mice. MIP-2 induced leukocyte emigration in all groups, albeit significantly more cells emigrated in alloxan-treated mice (15.3±1.0) compared to control (8.0±1.1) mice. Bacterial clearance was followed for 10 days after subcutaneous injection of bioluminescent S. aureus using non-invasive IVIS imaging, and the inflammatory response was assessed by Myeloperoxidase-ELISA and confocal imaging. The phagocytic ability of leukocytes was assessed using LPS-coated fluorescent beads and flow cytometry. Despite efficient leukocyte recruitment, alloxan-treated mice demonstrated an impaired ability to clear bacterial infection, which we found correlated to a 50% decreased phagocytic ability of leukocytes in diabetic mice. CONCLUSIONS/SIGNIFICANCE: These results indicate that reduced ability to clear bacterial infections observed during experimentally induced diabetes is not due to reduced leukocyte recruitment since sustained hyperglycemia results in increased levels of adherent and emigrated leukocytes in mouse models of type 1 and type 2 diabetes. Instead, decreased phagocytic ability observed for leukocytes isolated from diabetic mice might account for the impaired bacterial clearance.

Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1α, suppress amyloid ß-induced neurotoxicity.

Alzheimer's disease (AD) is characterized by a progressive cognitive decline and accumulation of neurotoxic oligomeric peptides amyloid-ß (Aß). Although the molecular events are not entirely known, it has become evident that inflammation, environmental and other risk factors may play a causal, disruptive and/or protective role in the development of AD. The present study investigated the ability of the chemokines, macrophage inflammatory protein-2 (MIP-2) and stromal cell-derived factor-1α (SDF-1α), the respective ligands for chemokine receptors CXCR2 and CXCR4, to suppress Aß-induced neurotoxicity in vitro and in vivo. Pretreatment with MIP-2 or SDF-1α significantly protected neurons from Aß-induced dendritic regression and apoptosis in vitro through activation of Akt, ERK1/2 and maintenance of metalloproteinase ADAM17 especially with SDF-1α. Intra-cerebroventricular (ICV) injection of Aß led to reduction in dendritic length and spine density of pyramidal neurons in the CA1 area of the hippocampus and increased oxidative damage 24h following the exposure. The Aß-induced morphometric changes of neurons and increase in biomarkers of oxidative damage, F(2)-isoprostanes, were significantly inhibited by pretreatment with the chemokines MIP-2 or SDF-1α. Additionally, MIP-2 or SDF-1α was able to suppress the aberrant mislocalization of p21-activated kinase (PAK), one of the proteins involved in the maintenance of dendritic spines. Furthermore, MIP-2 also protected neurons against Aß neurotoxicity in CXCR2-/- mice, potentially through observed up regulation of CXCR1 mRNA. Understanding the neuroprotective potential of chemokines is crucial in defining the role for their employment during the early stages of neurodegeneration.