The chemokine CXCL14 is negatively associated with obesity and concomitant type-2 diabetes in humans.

Chemokine (C-X-C motif) ligand-14 (CXCL14) levels are downregulated in experimental rodent models of obesity. Moreover, CXCL14 reportedly favors insulin sensitization in obese mice. Here we examined, for the first time, the role of CXCL14 in human obesity. We found that circulating levels of CXCL14 were decreased in patients with obesity and, especially, those with concomitant type-2 diabetes. CXCL14 levels were negatively associated with BMI and with indices of impaired glucose/insulin homeostasis. CXCL14 expression was decreased in subcutaneous adipose tissue from patients with obesity and type-2 diabetes. In adipose tissue, CXCL14 expression was negatively correlated with the expression of genes encoding pro-inflammatory molecules, and positively correlated with GLUT4 and adiponectin expression. In conclusion, obesity, and especially, concomitant type-2 diabetes are associated with abnormally decreased levels of CXCL14 in blood and impaired CXCL14 expression in adipose tissue. CXCL14 downregulation may be a novel biomarker of altered metabolism in obesity. CXCL14 also deserves further research as a therapeutic candidate.

Improved Host Cell Protein Analysis in Monoclonal Antibody Products through Molecular Weight Cutoff Enrichment.

Host cell proteins (HCPs) are process-related impurities that are generated by the host organism and are typically present at low levels in recombinant biopharmaceutical products, such as therapeutic antibodies. While overall HCP levels are usually monitored by enzyme-linked immunosorbent assay (ELISA), liquid chromatography coupled to mass spectrometry (LC-MS) is emerging as a powerful tool that can provide both qualitative and quantitative information about HCP levels during purification process development. However, a major challenge for LC-MS-based methods is that there can be a more than 5 orders of magnitude difference in the concentration between HCPs and therapeutic antibody in solution, which precludes the effective identification of low abundance HCPs in antibody product. This work reports a simple and powerful strategy to identify HCPs in antibody drug substance by applying molecular weight cutoff (MWCO) filtration step followed by shotgun proteomic analysis. After dissociating the interaction between HCPs and antibody with an anionic detergent, the depletion of antibody from HCPs can be easily achieved with the MWCO filtration step. Using this method, we observed that the dynamic range across proteins in the HCP samples was significantly decreased up to 1000-fold. In addition, by spiking in known amounts of HCPs to purified antibody drug substance with low levels of HCPs, we demonstrated that our method could detect HCP with low molecular weight (11 kDa and 17 kDa) at a concentration as low as 1 ppm. When applying this methodology to the study of HCPs in NIST monoclonal antibody (NISTmAb), more than 150 HCPs were confidently identified, which doubles the number of identified HCPs that have been previously reported. Parallel reaction monitoring (PRM) results confirmed that the novel HCPs found using this method were present in very low abundance (0.01-8 ppm), highlighting that our method reduces the dynamic range by removing antibody interference and improving the sensitivity of HCP identification and quantification.

CXCL14 downregulation in human keratinocytes is a potential biomarker for a novel in vitro skin sensitization test.

To elucidate the roles of epidermal keratinocytes in the toxicological outcomes of chemically induced contact dermatitis, genome-scale transcriptional analyses were performed using normal human keratinocytes (NHKCs) treated with 10 µM sodium lauryl sulfate (SLS) or 5 µM urushiol. In Gene Ontology (GO) enrichment analyses, SLS- and urushiol-induced upregulated DEGs are commonly associated with the regulation of pro-inflammatory responses and epidermal differentiation processes in NHKCs whereas cellular protein metabolic process was also identified as a commonly downregulated DEG signature. Among the downregulated DEGs, CXCL14 was investigated as a potential biomarker for a new in vitro skin sensitization test using OECD TG429 reference chemicals. CXCL14 was significantly downregulated in NHKCs in response to 62.5% of the OECD TG429 sensitizers in a concentration-dependent manner. When the sensitizer-induced upregulation of chemokine CXCL8 was included in the analysis, 87.5% of the OECD TG429 reference sensitizing chemicals significantly induced either CXCL8 upregulation or CXCL14 downregulation in NHKCs. Only one OECD TG429 reference non-sensitizer changed the constitutive CXCL14 expression in NHKCs whereas five out of six non-sensitizers altered CXCL8 production. The reference irritating non-sensitizer SLS caused a false-positive outcome. The downregulation of constitutively expressed CXCL14 was regulated by both the MAPK/ERK and JAK3/STAT6 pathways in NHKCs. CXCL14 can be used as a mechanism-based biomarker in the development of in vitro skin sensitization tests and may help improve the distinction between allergenic sensitizers and non-sensitizers.

Proteomic signatures of angiogenesis in androgen-independent prostate cancer.

INTRODUCTION: The observation that angiogenesis, the process of new blood vessel formation, in healthy prostate and early prostate cancer is androgen-dependent gave rise to significant questions on how hypervascularization and increased angiogenesis is also achieved at the molecular level in advanced androgen-independent prostate cancer. The exact paracrine molecular network that is hardwired into the proteome of the endothelial and cancer subpopulations participating in this process remains partially understood. METHODS: Here, we interrogated the signaling pathways and the molecular functional signatures across the proteome of endothelial cells after interacting with various secretomes produced by androgen-dependent and -independent prostate cancer cells. RESULTS: We found the significant overexpression (P < 0.05) of prominent markers of angiogenesis, such as vonWillebrand factor (vWF) (∼ 2.5-fold) and CD31 (∼ 2-fold) in HUVECs stimulated with conditioned media from the androgen-independent prostate cancer cell line PC3. By mining the proteome of PC3 conditioned media, we discovered a signature of chemokine CXC motif ligands (i.e., CXCL3, CXCL5, CXCL6 and CXCL8) that could potentially coordinate increased angiogenesis in androgen-independent prostate cancer and verified their increased expression (P < 0.05) in both in vitro and xenograft models of androgen-independence. DISCUSSION: Our findings form the basis for understanding the regulation of crucial metastatic phenomena during the transition of androgen-dependent prostate cancer into the highly aggressive, androgen-independent state and provide further insight on potential therapeutic targets of cancer-related angiogenesis.

Comprehensive analysis of the clinical and biological significances for chemokine CXCL3 in cholangiocarcinoma.

Cholangiocarcinoma (CHOL) is a race malignant cancer arising from bile duct epithelial cells in clinical practice. C-X-C motif chemokine ligand 3 (CXCL3) is a member of chemokines family, which participates in the pathogenesis of various tumors. However, the association between CXCL3 and CHOL is unclear. This present study was to assess the role of CXCL3 expression in the progress of CHOL. TIMER, GEPIA, UALCAN, GSCA, LinkedOmics, Metascape and STRING databases were performed to evaluate the clinical and biological significances for CXCL3 with CHOL patients including expression, clinicopathological factors, immune cell infiltration, GO enrichment and KEGG pathway analyses, as well as PPI network analysis. The immunohistochemistry analysis of tissue microarray was conducted to detect the protein expression level, subcellular localization, clinicopathological factors and prognosis of CXCL3 in CHOL. The mRNA and protein expression levels of CXCL3 were markedly increased in CHOL tissues. The overexpression of CXCL3 was strongly associated with maximum tumor diameter of patients with CHOL. Additionally, there were negative correlations between the expression of CXCL3 and monocyte as well as Th17. Low infiltration of neutrophil indicated significantly shorter cumulative survival in CHOL patients. And CXCL3 was significantly associated with arm-level deletion of CD8+ T cell. Furthermore, functional network analysis suggested that CXCL3 and its associated genes were mainly enriched for chemotaxis, secretory granule membrane, cytokine activity and IL-17 signaling pathway. CXCL3 might potentially participate in the carcinogenesis of CHOL, which provided a direction for future research on the mechanism of CXCL3 in CHOL.

Effect of bacterial peptidoglycan on erythrocyte death and adhesion to endothelial cells.

Peptidoglycans, bacterial wall components, have previously been shown to trigger eryptosis, the suicidal erythrocyte death, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Phosphatidylserine exposing erythrocytes adhere to the vascular wall at least partially by interaction of erythrocytic phosphatidylserine with endothelial CXC chemokine ligand 16 (CXCL16). The present study explored whether peptidoglycan exposure fosters the adhesion of erythrocytes to human umbilical vein endothelial cells (HUVEC). To this end, HUVEC were treated for 48 h with peptidoglycan (10 µg/ml) and CXCL16 abundance determined by confocal microscopy and FACS analysis. Moreover, human erythrocytes were exposed for 48 h to peptidoglycan (10 µg/ml) and phosphatidylserine exposure estimated from binding of fluorescent annexin-V, cell volume from forward scatter in FACS analysis and erythrocyte adhesion to human umbilical vein endothelial cells (HUVEC) from trapping of labeled erythrocytes in a flow chamber. As a result, bacterial peptidoglycan exposure was followed by increased CXCL16 expression in HUVEC as well as erythrocyte shrinkage, phosphatidylserine exposure and adhesion to HUVEC under flow conditions at arterial shear rates. The adhesion was significantly attenuated but not abrogated in the presence of either, erythrocyte phosphatidylserine-coating annexin-V (5 µl/ml) or CXCL16 neutralizing antibody directed against endothelial CXCL16 (4 µg/ml). In conclusion, exposure to peptidoglycan increases endothelial CXCL16 expression and leads to eryptosis followed by phosphatidylserine- and CXCL16-mediated adhesion of eryptotic erythrocytes to vascular endothelial cells.

Activation of innate immunity accelerates sialoadenitis in a mouse model for Sjögren's syndrome-like disease.

OBJECTIVE: Sjögren's syndrome is a chronic autoimmune disorder characterized by progressive lymphocytic infiltration within the salivary and lacrimal glands. This study was undertaken to investigate the effects of innate immunity activation on sialoadenitis in a mouse strain genetically susceptible for development of SS-like disease. METHODS: Female New Zealand Black X New Zealand White F1 mice were repeatedly treated with toll-like 3 receptor agonist poly(I:C). Submandibular glands were investigated at different time points for sialoadenitis by immunohistochemistry and for gene expression of different chemokines by quantitative PCR. Submandibular gland-infiltrating cells were characterized by flow cytometry. RESULTS: Poly(I:C) treatment significantly upregulated the expression of multiple chemokines within the submandibular glands. The severity and incidence of sialoadenitis was considerably higher in poly(I:C)-treated mice. There was a preponderance of dendritic cells and NK cells in the initial inflammatory cell infiltrates, and these were followed by CD4+ T cells. CONCLUSIONS: Our data clearly demonstrate that systemic activation of innate immunity accelerates sialoadenitis in a mouse model for SS-like disease. These findings suggest that chronic activation of innate immunity can influence certain features of SS.

Bioinformatics analysis of the role of CXC ligands in the microenvironment of head and neck tumor.

Chemokines play a significant role in cancer. CXC-motif chemokine ligands (CXCLs) are associated with the tumorigenesis and progression of head and neck squamous cell carcinoma (HNSC); however, their specific functions in the tumor microenvironment remain unclear. Here, we analyzed the molecular networks and transcriptional data of HNSC patients from the Oncomine, GEPIA, String, cBioPortal, Metascape, TISCH, and TIMER databases. To verify immune functions of CXCLs, their expression was analyzed in different immune cell types. To our knowledge, this is the first report on the correlation between CXCL9-12 and 14 expression and advanced tumor stage. CXCL2, 3, 8, 10, 13, and 16 were remarkably related to tumor immunity. Kaplan-Meier and TIMER survival analyses revealed that high expression of CXCL1, 2, 4, and 6-8 is correlated with low survival in HNSC patients, whereas high expression of CXCL9, 10, 13, 14, and 17 predicts high survival. Only CXCL13 and 14 were associated with overall survival in human papilloma virus (HPV)-negative patients. Single-cell datasets confirmed that CXCLs are associated with HNSC-related immune cells. Thus, CXCL1-6, 8-10, 12-14, and 17 could be prognostic targets for HNSC, and CXCL13 and 14 could be novel biomarkers of HPV-negative HNSC.

Comprehensive analysis of the prognosis and immune infiltration for CXC chemokines in colorectal cancer.

The C-X-C motif (CXC) chemokines are a family of chemotactic molecules that have been identified as potential prognostic markers and prospective therapeutic targets for many kinds of cancer types. Increasing evidence shows that CXC chemokines are associated with the progression of colorectal cancer (CRC); however, the correlations of CXC chemokines with prognostic and immune infiltrates in CRC remain to be clarified. In this study, we analyzed the mRNA expression level, prognostic data and immune infiltrates of CXC chemokines in CRC patients from the Gene Expression Profiling Interactive Analysis, Oncomine, cBioPortal and databases using GeneMANIA, STRING, DAVID 6.8, and TIMER. Our results showed that CXCL1/2/3/4/5/8/9/10/11/13/14/16 were significantly overexpressed in CRC tissues. Furthermore, expression of CXCL1/2/3/9/10/11 was associated with tumor stage in CRC. A significant association was also identified between the co-expression of CXCL16 with EGFR, KRAS and NRAS. In addition, the survival analysis suggested that high CXCL2/3/8/9/10/11/14 expression is correlated with clinical outcomes of CRC patients. Moreover, a significant association was observed between the CXCL8/9/10/11 expression and immune infiltration in colonic and rectal adenocarcinoma. Overall, CXC chemokines are not only implicated as prognostic biomarkers for CRC patients, but may also influence the immune status of CRC tissues.

Chemokine CXCL14; a double-edged sword in cancer development.

Cancer is a leading cause of death worldwide and imposes a substantial financial burden. Therefore, it is essential to develop cost-effective approaches to inhibit tumor growth and development. The imbalance of cytokines and chemokines play an important role among different mechanisms involved in cancer development. One of the strongly conserved chemokines that is constitutively expressed in skin epithelia is the chemokine CXCL14. As a member of the CXC subfamily of chemokines, CXCL14 is responsible for the infiltration of immune cells, maturation of dendritic cells, upregulation of major histocompatibility complex (MHC)-I expression, and cell mobilization. Moreover, dysregulation of CXCL14 in several cancers has been identified by several studies. Depending on the type or origin of the tumor and components of the tumor microenvironment, CXCL14 plays a conflicting role in cancer. Although fibroblast-derived CXCL14 has a tumor-supportive role, epithelial-derived CXCL14 mainly inhibits tumor progression. Hence, this review will elucidate what is known on the mechanisms of CXCL14 and its therapeutic approaches in tumor treatment. CXCL14 is a promising approach for cancer immunotherapy.

Aberrant chemokine receptor expression and chemokine production by Langerhans cells underlies the pathogenesis of Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is characterized by a clonal proliferation and retention of cells with a Langerhans cell (LC)-like phenotype at various sites within the body. The present study set out to elucidate whether aberrant expression of chemokine receptors or dysregulation of chemokine production in LCH lesions could explain abnormal retention of these cells. Immunohistochemical analysis on 13 LCH biopsies of bone, skin, and lymph node all expressed the immature dendritic cell (DC) marker CCR6 on the lesional LCs and absence of the mature DC marker CCR7. Furthermore, regardless of the tissue site, LCH lesions markedly overexpressed CCL20/MIP-3alpha, the ligand for CCR6. The lesional LCs appeared to be the source of this CCL20/MIP-3alpha production as well as other inflammatory chemokines such as CCL5/RANTES and CXCL11/I-TAC. These may explain the recruitment of eosinophils and CD4+CD45RO+ T cells commonly found in LCH lesions. The findings of this study emphasize that, despite abundant TNF-alpha, lesional LCs remain in an immature state and are induced to produce chemokines, which via autocrine and paracrine mechanisms cause not only the retention of the lesional LCs but also the recruitment and retention of other lesional cells. We postulate that the lesional LCs themselves control the persistence and progression of LCH.

Interferon gamma-independent rejection of interleukin 12-transduced carcinoma cells requires CD4+ T cells and Granulocyte/Macrophage colony-stimulating factor.

We analyzed the ability of interferon (IFN)-gamma knockout mice (GKO) to reject a colon carcinoma transduced with interleukin (IL)-12 genes (C26/IL-12). Although the absence of IFN-gamma impaired the early response and reduced the time to tumor onset in GKO mice, the overall tumor take rate was similar to that of BALB/c mice. In GKO mice, C26/IL-12 tumors had a reduced number of infiltrating leukocytes, especially CD8 and natural killer cells. Analysis of the tumor site, draining nodes, and spleens of GKO mice revealed reduced expression of IFN- inducible protein 10 and monokine induced by gamma-IFN. Despite these defects, GKO mice that rejected C26/IL-12 tumor, and mice that were primed in vivo with irradiated C26/IL-12 cells, showed the same cytotoxic T lymphocyte activity but higher production of granulocyte/macrophage colony-stimulating factor (GM-CSF) as compared with control BALB/c mice. Treatment with monoclonal antibodies against GM-CSF abrogated tumor regression in GKO but not in BALB/c mice. CD4 T lymphocytes, which proved unnecessary or suppressive during rejection of C26/IL-12 cells in BALB/c mice, were required for tumor rejection in GKO mice. CD4 T cell depletion was coupled with a decline in GM-CSF expression by lymphocytes infiltrating the tumors or in the draining nodes, and with the reduction and disappearance of granulocytes and CD8 T cells, respectively, in tumor nodules. These results suggest that GM-CSF can substitute for IFN-gamma in maintaining the CD8-polymorphonuclear leukocyte cross-talk that is a hallmark of tumor rejection.

Inflammogenic effect of well-characterized fullerenes in inhalation and intratracheal instillation studies.

BACKGROUND: We used fullerenes, whose dispersion at the nano-level was stabilized by grinding in nitrogen gas in an agitation mill, to conduct an intratracheal instillation study and an inhalation exposure study. Fullerenes were individually dispersed in distilled water including 0.1% Tween 80, and the diameter of the fullerenes was 33 nm. These suspensions were directly injected as a solution in the intratracheal instillation study. The reference material was nickel oxide in distilled water. Wistar male rats intratracheally received a dose of 0.1 mg, 0.2 mg, or 1 mg of fullerenes and were sacrificed after 3 days, 1 week, 1 month, 3 months, and 6 months. In the inhalation study, Wistar rats were exposed to fullerene agglomerates (diameter: 96 +/- 5 nm; 0.12 +/- 0.03 mg/m3; 6 hours/days for 5 days/week) for 4 weeks and were sacrificed at 3 days, 1 month, and 3 months after the end of exposure. The inflammatory responses and gene expression of cytokine-induced neutrophil chemoattractants (CINCs) were examined in rat lungs in both studies. RESULTS: In the intratracheal instillation study, both the 0.1 mg and 0.2 mg fullerene groups did not show a significant increase of the total cell and neutrophil count in BALF or in the expression of CINC-1,-2alphabeta and-3 in the lung, while the high-dose, 1 mg group only showed a transient significant increase of neutrophils and expression of CINC-1,-2alphabeta and -3. In the inhalation study, there were no increases of total cell and neutrophil count in BALF, CINC-1,-2alphabeta and-3 in the fullerene group. CONCLUSION: These data in intratracheal instillation and inhalation studies suggested that well-dispersed fullerenes do not have strong potential of neutrophil inflammation.

CXCL17-mediated downregulation of type I collagen via MMP1 and miR-29 in skin fibroblasts possibly contributes to the fibrosis in systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is characterized by excessive deposition of collagen in the skin and internal organs. Recent studies have shown that chemokine (C-X-C motif) ligands (CXCLs) are involved in the pathogenesis of SSc. OBJECTIVE: Our aim was to examine the anti-fibrotic potential of CXCL17, a newly discovered chemokine, in cultured skin fibroblasts and in a bleomycin-induced SSc mouse model. Moreover, we examined serum level of CXCL17 in patients with SSc. METHODS: Type I collagen expression was evaluated in SSc skin and cultured fibroblasts treated with CXCL17 using immunoblotting and quantitative reverse transcription-PCR. Serum CXCL17 levels were determined using enzyme-linked immunosorbent assay in 63 patients with SSc and 17 healthy subjects. A bleomycin-induced SSc mouse model was used to evaluate the effect of CXCL17 on skin fibrosis. RESULTS: CXCL17 reduced the expression of type I collagen in healthy control fibroblasts. CXCL17 also induced matrix metalloproteinase 1 (MMP1) and miR-29 expression in fibroblasts, indicating that CXCL17 regulates type I collagen expression in part via post-transcriptional mechanisms through MMP1 and miR-29. We found that local injection of CXCL17 attenuated bleomycin-induced skin fibrosis in mice. CXCL17 levels in SSc skin were lower than those in healthy controls, in contrast to the high serum CXCL17 levels in patients with SSc. The low expression of CXCL17 in SSc skin possibly affects type I collagen accumulation in this disease. CONCLUSION: Our data indicate that understanding CXCL17 signaling may lead to a better therapeutic approach for SSc.

Regulation of CXCL16 expression and secretion by myeloid cells is not altered in rheumatoid arthritis.

OBJECTIVE: Chemokine (C-X-C motif) ligand 16 (CXCL16) is secreted by macrophages and dendritic cells (DCs) to attract memory type T cells. CXCL16 expression is increased in arthritic joints of patients with rheumatoid arthritis (RA) and a role for CXCL16 has been suggested in the pathogenesis of RA. To date, little is known about the regulation of CXCL16 on monocytes/macrophages and DCs. The aim of this study was to elucidate how CXCL16 expression is regulated in healthy donors and patients with RA. METHODS: CD14+cells were isolated from the peripheral blood or synovial fluid of patients with RA and healthy controls, differentiated into different types of dendritic cells or macrophages and stimulated with various cytokines or lipopolysaccharide (LPS). Cell surface proteins, including surface CXCL16, were measured by flow cytometry and soluble CXCL16 was measured by ELISA. RESULTS: Distinct types of dendritic cells constitutively express and secrete CXCL16, which is not affected by maturation. Monocytes rapidly upregulate membrane-bound CXCL16 expression and release soluble CXCL16 upon culture. CXCL16 expression by monocytes is transiently inhibited by the Toll-like receptor (TLR)4 ligand LPS. Th2 type cytokines inhibit soluble CXCL16, whereas T helper (Th)1 cell stimulus enhances its release. In RA monocytes/macrophages, neither CXCL16 expression, nor CXCL16 regulation is different from healthy controls. CONCLUSIONS: Culture of monocytes is the main trigger for CXCL16 surface expression in vitro, which is not altered in RA. Together our data suggest that the increased CXCL16 expression in patients with RA is likely to be caused by increased influx of monocytes rather than intrinsic differences in CXCL16 regulation.

Carcinoma cell-derived chemokines and their presence in oral fluid.

Chemokines are important in inflammation and in carcinogenesis. We hypothesized that besides oro-laryngeal cancer, oral inflammatory states, such as periodontitis, may also influence the chemokine profile of oral fluid. The aim of this study was to characterize the chemokine isoforms in the oral fluid of patients with periodontitis and in the oral fluid of patients with head and neck cancer. Using enzyme-linked immunosorbent assays (ELISA), it was found that the concentrations of CXCL8, CXCL10, and CCL14 were significantly elevated in the oral fluids of the cancer patients. However, periodontitis did not significantly alter the chemokine levels in oral fluid. Identification of chemokine isoforms by a proteomic approach using a newly developed three-step purification procedure was applied on the oral fluid of head and neck cancer and periodontitis patients and on the conditioned medium from carcinoma cells. Carcinoma cells produced predominantly intact CXCL1, CXCL2, CXCL8, and CCL2, whereas CXCL8 also appeared in a truncated, more active, form. Unfortunately, the chemokine concentrations in oral fluids were too low to allow full biochemical identification of the modified isoforms. However, the chemokine profile of head and neck cancer significantly changed after therapy, indicating that it is a useful parameter in clinical practice.

Anti-mycobacterial treatment reduces high plasma levels of CXC-chemokines detected in active tuberculosis by cytometric bead array.

Chemokines recruit and activate leukocytes, assisting granuloma formation. Herein, we evaluated plasma chemokines in patients with active tuberculosis (ATB) and after completing treatment (TTB) and compared them to BCG-vaccinated healthy controls (HC). Levels of chemokines were measured by cytometric bead array. Levels of CXCL8, CXCL9 and CXCL10 were higher in ATB patients compared to HC, but they decreased in TTB. Levels of CCL2 and CCL5 in ATB patients were similar to those observed in HC. Thus, the high levels of CXC-chemokines detected during ATB, which can modulate the trafficking of immune cells from the periphery to the site of infection, were reversed by anti-mycobacterial treatment.

CXCR6 is expressed in human prostate cancer in vivo and is involved in the in vitro invasion of PC3 and LNCap cells.

In spite of the clinical importance of prostate cancer (PCa) bone metastasis, the precise mechanisms for the directed migration of malignant cells remain unclear. In the present study, the expression of CXCR6 in human PCa and benign prostatic hyperplasia samples, and the expression of CXCL16 in human osseous tissues were determined by immunohistochemistry. It was found that the level of CXCR6 protein expression was elevated in human malignant prostate tumors, and CXCL16 was expressed positively by human osteocytes in vivo. The in vitro experiments further confirmed that the PCa cell lines PC3 and LNCap expressed CXCR6 at both the mRNA and protein levels, and exogenous CXCL16 has the potential to stimulate the invasion of PC3 and LNCap. To further elucidate the role of the CXCL16-CXCR6 axis in PCa progression, we compared the expression of CXCR6 and CXCR4 in human PCa tissues and the effects of CXCL16 and CXCL12 on the in vitro invasion of PC3 and LNCap cells. It was shown that CXCR6 and CXCR4 proteins were coexpressed and elevated in human PCa samples, and CXCL16 and CXCL12 promoted the invasion of PC3 and LNCap via their respective receptors. Furthermore, in contrast to CXCL12, which enhanced the activity of matrix metalloproteinase (MMP) 9 and MMP2 in PC3 and LNCap, CXCL16 ligation resulted in stronger MMP9 and MMP2 activity in LNCap but not in PC3. Our results suggest that besides CXCL12/CXCR4, CXCL16/CXCR6 might be another important factor involved in PCa bone metastasis.

CXCR6-CXCL16 interaction in the pathogenesis of Juvenile Idiopathic Arthritis.

In order to evaluate the role of CXCR6/CXCL16 in driving lymphocyte migration into inflamed joints of children with oligoarticular Juvenile Idiopathic Arthritis (JIA) we analysed CXCR6 expression and functional capability in lymphocytes from synovial fluid (SF) by flow cytometry, by real-time polymerase chain reaction (RT-PCR) and migration assays. Furthermore, CXCR6 and CXCL16 expression in synovial tissue (ST) was analysed by immunohistochemistry. T cells isolated from SF of patients with JIA expressed CXCR6 which was functionally active as shown by chemotactic assays. The same cells expressed CXCR3 and it exerted a migratory activity in response to CXCL10. CXCL16 and CXCR6 were intensively expressed on the synovium cells, respectively on macrophages, synoviocytes and endothelial cells and on lymphocytes, synoviocytes and endothelial cells. Taken together, these data suggest that CXCR6 and CXCR3 act coordinately with respective ligands and are involved in the pathophysiology of JIA-associated inflammatory processes.

The expression of E-selectin and chemokines in the cultured human lymphatic endothelium with lipopolysaccharides.

This study investigated the expression of selectins and chemokines in cultured human lymphatic endothelial cells stimulated with lipopolysaccharides. In microarray, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 gene expressions in the lymphatic endothelium with lipopolysaccharides did not change at 0.5 h but increased two- to three-fold at 12 h, whereas E-selectin increased 10-fold at 0.5 h and 68-fold at 12 h compared with untreated cells. The E-selectin mRNA and protein increased in the lymphatic endothelial cells with lipopolysaccharides at more than two-fold levels compared with human umbilical vein endothelial cells. Induction of Cys-Cys chemokine ligand 2, 3, 5, 7, 8 and 20 mRNAs in the lymphatic endothelial cells with lipopolysaccharides was detected in microarray and real-time PCR. The Cys-Cys chemokine ligand 2, 5 and 20 mRNA amounts in cells with high concentration lipopolysaccharides were larger in the lymphatic endothelial cells than in human umbilical vein endothelial cells. The Cys-Cys chemokine ligand 3 and 8 mRNAs were not detected in human umbilical vein endothelial cells. Induction of Cys-X-Cys chemokine ligand 1, 3, 5, 6 and 8 mRNAs was detected in the lymphatic endothelial cells with lipopolysaccharides. The Cys-X-Cys chemokine ligand 3, 5 and 8 mRNA amounts in cells with high concentration lipopolysaccharides were larger in the lymphatic endothelial cells than in human umbilical vein endothelial cells. In conclusion, it was demonstrated that the cultured human lymphatic endothelial cells express E-selectin and phagocyte-attractive chemokine genes.