The development of clinical signs of rheumatoid synovial inflammation is associated with increased synthesis of the chemokine CXCL8 (interleukin-8)

Paired synovial tissue samples were obtained from both clinically uninvolved (CU) and clinically involved (CI) knee joints of eight rheumatoid arthritis (RA) patients. In addition, biopsies were taken from five control subjects. We observed the expression of the chemokines CXCL8, CXCL9, CXCL10, CCL2 and CCL4 in CI and CU joints of RA patients. In particular, CXCL8 protein levels were specifically increased in CI joints compared with CU joints, which was confirmed by immunohistochemistry and in situ hybridization.     

Chemokines regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by proliferation of synoviocytes that produce inflammatory cytokines and chemokines. The expressed chemokines are thought to be involved in the migration of inflammatory cells into the synovium. In this study we show that CCL2/monocyte chemotactic protein-1, CCL5/RANTES, and CXCL12/stromal cell-derived factor-1 enhanced IL-6 and IL-8 production by fibroblast-like synoviocytes (FLS) from patients with RA, and their corresponding receptors, CCR2, CCR5, and CXCR4, respectively, were expressed by RA FLS. The chemokines stimulated RA FLS more effectively than skin fibroblasts. Culture with CCL2 enhanced phosphorylation of extracellular signal-related kinase 1 (ERK1) and ERK2, but not phosphorylation of p38 or Src. Moreover, activation of ERK1/2 was inhibited by pertussis toxin, a G(i)-coupled protein inhibitor, and RS-504393, CCR2 antagonist, suggesting that ERK1/2 was activated by CCL2 via CCR2 and G(i)-coupled protein. On the other hand, CCL2, CCL5, and CXCL12 were expressed on RA FLS, and their production was regulated by TNF-alpha, IL-1beta, and TGF-beta1. Our results indicate that the chemokines not only play a role in inflammatory cell migration, but are also involved in the activation of FLS in RA synovium, possibly in an autocrine or paracrine manner.     

Molecular aspects of rheumatoid arthritis: chemokines in the joints of patients

Rheumatoid arthritis (RA) is a chronic symmetric polyarticular joint disease that primarily affects the small joints of the hands and feet. The inflammatory process is characterized by infiltration of inflammatory cells into the joints, leading to proliferation of synoviocytes and destruction of cartilage and bone. In RA synovial tissue, the infiltrating cells such as macrophages, T cells, B cells and dendritic cells play important role in the pathogenesis of RA. Migration of leukocytes into the synovium is a regulated multi-step process, involving interactions between leukocytes and endothelial cells, cellular adhesion molecules, as well as chemokines and chemokine receptors. Chemokines are small, chemoattractant cytokines which play key roles in the accumulation of inflammatory cells at the site of inflammation. It is known that synovial tissue and synovial fluid from RA patients contain increased concentrations of several chemokines, such as monocyte chemoattractant protein-4 (MCP-4)/CCL13, pulmonary and activation-regulated chemokine (PARC)/CCL18, monokine induced by interferon-gamma (Mig)/CXCL9, stromal cell-derived factor 1 (SDF-1)/CXCL12, monocyte chemotactic protein 1 (MCP-1)/CCL2, macrophage inflammatory protein 1alpha (MIP-1alpha)/CCL3, and Fractalkine/CXC3CL1. Therefore, chemokines and chemokine-receptors are considered to be important molecules in RA pathology.     

IL-6 Amplifies TLR Mediated Cytokine and Chemokine Production: Implications for the Pathogenesis of Rheumatic Inflammatory Diseases

The role of Interleukin(IL)-6 in the pathogenesis of joint and systemic inflammation in rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis (s-JIA) has been clearly demonstrated. However, the mechanisms by which IL-6 contributes to the pathogenesis are not completely understood. This study investigates whether IL-6 affects, alone or upon toll like receptor (TLR) ligand stimulation, the production of inflammatory cytokines and chemokines in human peripheral blood mononuclear cells (PBMCs), synovial fluid mononuclear cells from JIA patients (SFMCs) and fibroblast-like synoviocytes from rheumatoid arthritis patients (RA synoviocytes) and signalling pathways involved. PBMCs were pre-treated with IL-6 and soluble IL-6 Receptor (sIL-6R). SFMCs and RA synoviocytes were pre-treated with IL-6/sIL-6R or sIL-6R, alone or in combination with Tocilizumab (TCZ). Cells were stimulated with LPS, S100A8-9, poly(I-C), CpG, Pam2CSK4, MDP, IL-1β. Treatment of PBMCs with IL-6 induced production of TNF-α, CXCL8, and CCL2, but not IL-1β. Addition of IL-6 to the same cells after stimulation with poly(I-C), CpG, Pam2CSK4, and MDP induced a significant increase in IL-1β and CXCL8, but not TNF-α production compared with TLR ligands alone. This enhanced production of IL-1β and CXCL8 paralleled increased p65 NF-κB activation. In contrast, addition of IL-6 to PBMCs stimulated with LPS or S100A8-9 (TLR-4 ligands) led to reduction of IL-1β, TNF-α and CXCL8 with reduced p65 NF-κB activation. IL-6/IL-1β co-stimulation increased CXCL8, CCL2 and IL-6 production. Addition of IL-6 to SFMCs stimulated with LPS or S100A8 increased CXCL8, CCL2 and IL-1β production. Treatment of RA synoviocytes with sIL-6R increased IL-6, CXCL8 and CCL2 production, with increased STAT3 and p65 NF-κB phosphorylation. Our results suggest that IL-6 amplifies TLR-induced inflammatory response. This effect may be relevant in the presence of high IL-6 and sIL-6R levels, such as in arthritic joints in the context of stimulation by endogenous TLR ligands.     

CXCL8 and CCL20 Enhance Osteoclastogenesis via Modulation of Cytokine Production by Human Primary Osteoblasts

Generalized osteoporosis is common in patients with inflammatory diseases, possibly because of circulating inflammatory factors that affect osteoblast and osteoclast formation and activity. Serum levels of the inflammatory factors CXCL8 and CCL20 are elevated in rheumatoid arthritis, but whether these factors affect bone metabolism is unknown. We hypothesized that CXCL8 and CCL20 decrease osteoblast proliferation and differentiation, and enhance osteoblast-mediated osteoclast formation and activity. Human primary osteoblasts were cultured with or without CXCL8 (2–200 pg/ml) or CCL20 (5–500 pg/ml) for 14 days. Osteoblast proliferation and gene expression of matrix proteins and cytokines were analyzed. Osteoclast precursors were cultured with CXCL8 (200 pg/ml) and CCL20 (500 pg/ml), or with conditioned medium (CM) from CXCL8 and CCL20-treated osteoblasts with or without IL-6 inhibitor. After 3 weeks osteoclast formation and activity were determined. CXCL8 (200 pg/ml) and CCL20 (500 pg/ml) enhanced mRNA expression of KI67 (2.5–2.7-fold), ALP (1.6–1.7-fold), and IL-6 protein production (1.3–1.6-fold) by osteoblasts. CXCL8-CM enhanced the number of osteoclasts with 3–5 nuclei (1.7-fold), and with >5 nuclei (3-fold). CCL20-CM enhanced the number of osteoclasts with 3–5 nuclei (1.3-fold), and with >5 nuclei (2.8-fold). IL-6 inhibition reduced the stimulatory effect of CXCL8-CM and CCL20-CM on formation of osteoclasts. In conclusion, CXCL8 and CCL20 did not decrease osteoblast proliferation or gene expression of matrix proteins. CXCL8 and CCL20 did not directly affect osteoclastogenesis. However, CXCL8 and CCL20 enhanced osteoblast-mediated osteoclastogenesis, partly via IL-6 production, suggesting that CXCL8 and CCL20 may contribute to osteoporosis in rheumatoid arthritis by affecting bone cell communication.     

Identification of biologically active chemokine isoforms from ascitic fluid and elevated levels of CCL18/pulmonary and activation-regulated chemokine in ovarian carcinoma

Chemokines are important in leukocyte homeostasis, inflammation, angiogenesis, and metastasis. Here, the molecular diversity of chemokines present in ovarian carcinoma was studied by purifying the proteins to homogeneity from ascitic fluid. Biologically active intact CCL2 and processed CXCL8, CCL3, and CCL18 isoforms were recovered. CCL7 and CCL20 were also purified, but their levels were 10-fold lower compared with CXCL8, CCL2, and CCL3 and even 100-fold lower than the amounts of CCL18 isolated. In ascitic fluids from patients with ovarian carcinoma (n = 12), significantly higher levels of CXCL8 and CCL18 (2.0 versus 0.7 ng/ml (p = 0.01) and 120 versus 44 ng/ml (p = 0.0002), respectively) were detected compared with those in nonovarian carcinoma patients (n = 12). In contrast to CXCL8, CCL18 was not inducible in carcinoma cell lines. Immunostaining showed CCL18 expression in tumor-infiltrating cells with monocyte/macrophage morphology but not in the ovarian carcinoma cells. Our data demonstrate that biochemically heterogenous but biologically active forms of several chemokines are present at different concentrations in ovarian carcinoma ascitic fluid. This points to a delicate balance of chemokines in epithelial ovarian cancer and to a potentially major role for CXCL8 and CCL18 in this tumor.     

Eotaxin/CCL11 suppresses IL-8/CXCL8 secretion from human dermal microvascular endothelial cells

The CC chemokine eotaxin/CCL11 is known to bind to the receptor CCR3 on eosinophils and Th2-type lymphocytes. In this study, we demonstrate that CCR3 is expressed on a subpopulation of primary human dermal microvascular endothelial cells and is up-regulated by TNF-alpha. We found that incubation of human dermal microvascular endothelial cells with recombinant eotaxin/CCL11 suppresses TNF-alpha-induced production of the neutrophil-specific chemokine IL-8/CXCL8. The eotaxin/CCL11-suppressive effect on endothelial cells was not seen on IL-1beta-induced IL-8/CXCL8 release. Eotaxin/CCL11 showed no effect on TNF-alpha-induced up-regulation of growth-related oncogene-alpha or IFN-gamma-inducible protein-10, two other CXC chemokines tested, and did not affect production of the CC chemokines monocyte chemoattractant protein-1/CCL2 and RANTES/CCL5, or the adhesion molecules ICAM-1 and E-selectin. These results suggest that eotaxin/CXCL11 is not effecting a general suppression of TNF-alphaR levels or signal transduction. Suppression of IL-8/CXCL8 was abrogated in the presence of anti-CCR3 mAb, pertussis toxin, and wortmannin, indicating it was mediated by the CCR3 receptor, G(i) proteins, and phosphatidylinositol 3-kinase signaling. Eotaxin/CCL11 decreased steady state levels of IL-8/CXCL8 mRNA in TNF-alpha-stimulated cells, an effect mediated in part by an acceleration of IL-8 mRNA decay. Eotaxin/CCL11 may down-regulate production of the neutrophil chemoattractant IL-8/CXCL8 by endothelial cells in vivo, acting as a negative regulator of neutrophil recruitment. This may play an important biological role in the prevention of overzealous inflammatory responses, aiding in the resolution of acute inflammation or transition from neutrophilic to mononuclear/eosinophilic inflammation.     

Membrane type 1-matrix metalloproteinase is regulated by chemokines monocyte-chemoattractant protein-1/ccl2 and interleukin-8/CXCL8 in endothelial cells during angiogenesis

We have investigated the putative role and regulation of membrane type 1-matrix metalloproteinase (MT1-MMP) in angiogenesis induced by inflammatory factors of the chemokine family. The absence of MT1-MMP from null mice or derived mouse lung endothelial cells or the blockade of its activity with inhibitory antibodies resulted in the specific decrease of in vivo and in vitro angiogenesis induced by CCL2 but not CXCL12. Similarly, CCL2- and CXCL8-induced tube formation by human endothelial cells (ECs) was highly dependent on MT1-MMP activity. CCL2 and CXCL8 significantly increased MT1-MMP surface expression, clustering, activity, and function in human ECs. Investigation of the signaling pathways involved in chemokine-induced MT1-MMP activity in ECs revealed that CCL2 and CXCL8 induced cortical actin polymerization and sustained activation of phosphatidylinositol 3-kinase (PI3K) and the small GTPase Rac. Inhibition of PI3K or actin polymerization impaired CCL2-induced MT1-MMP activity. Finally, dimerization of MT1-MMP was found to be enhanced by CCL2 in ECs in a PI3K- and actin polymerization-dependent manner. In summary, we identify MT1-MMP as a molecular target preferentially involved in angiogenesis mediated by CCL2 and CXCL8, but not CXCL12, and suggest that MT1-MMP dimerization might be an important mechanism of its regulation during angiogenesis.     

Expression of MDC/CCL22 and its receptor CCR4 in rheumatoid arthritis,psoriatic arthritis and osteoarthritis

The pathogenesis of rheumatoid arthritis (RA) and psoriatic arthritis (PsA) involves an abnormal chemokine regulation. The chemokine receptor CCR4 is necessary for T cell migration to the skin. We, therefore, studied if CCR4 and its ligand macrophage-derived chemokine (MDC/CCL22) could participate in spreading the disease between skin and joints by examining RA, PsA and osteoarthritis (OA) patients. In synovial fluid from RA and PsA patients we observed a significantly higher MDC/CCL22 level compared to OA patients. Additionally, the MDC/CCL22 protein was found to be elevated in RA and PsA plasma compared to OA and healthy volunteers. Flow cytometry revealed that most CD4⁺CCR4⁺ lymphocytes also co-expressed CD45RO. Neither the MDC/CCL22 level nor the expression of CCR4 correlated to CRP. Immunohistochemistry of the RA and OA synovial membrane demonstrated CCR4 to be expressed by mononuclear cells and endothelial cells. Our results show that MDC/CCL22 is present within the synovial membrane of RA and OA patients and in high amount in the synovial fluid of patients with RA and PsA. This will enable migration of CCR4 expressing memory cells supporting that MDC/CCR4 could play a role in attracting skin specific memory T cells to the joints.     

Proinflammatory chemokine gene expression influences survival of patients with non-Hodgkin's lymphoma

The migration, survival and proliferation of cells is the basis for all physiologic and pathologic processes in the human body. All these reactions are regulated by a complex chemokine network that guides lymphocytes homing, chemotaxis, adhesion and interplay between immunologic system response cells. Chemokines are also responsible for metastatic dissemination of cancers, including Hodgkin's and non-Hodgkin's lymphomas. The purpose of this study was to determine chemokine gene expression (CXCL8, CXCL10, CCL2, CCL3, CCL4 and CCL5) in lymphoma lymph nodes compared to their expression in reactive lymph nodes. We also analyzed the influence of chemokine gene expression on the survival of lymphoma patients. Chemokine gene expression was evaluated in 37 lymphoma lymph nodes and in 25 samples of reactive lymph nodes. Gene expression of chemokines CXCL8, CXCL10, CCL2, CCL3, CCL4 and CCL5 was measured using the PCR method. Statistical analysis was performed using CSS Statistica for Windows (version 7.0) software. Probability values 〈 〈 0.05 were considered statistically significant and those between 0.05 and 0.1 as indicative of a trend. We found lower CXCL8 and CXCL10 gene expression in lymphoma lymph nodes compared to reactive lymph nodes. In the cases of CCL2 and CCL3, expression in lymphomas was higher than in reactive lymph nodes. Patients with high expression of CCL2 and CXCL10 had shorter survival.     

Expression of the inflammatory chemokines CCL5, CCL3 and CXCL10 in juvenile idiopathic arthritis, and demonstration of CCL5 production by an atypical subset of CD8+ T cells

This study focuses upon three chemokines, namely CCL5, CXCL10 and CCL3, which are potential novel therapeutic targets in arthritis. The aim of the study was to analyse the expression and production of these three chemokines within the joints of children with juvenile idiopathic arthritis (JIA) of the oligoarticular and polyarticular subtypes. All three of these chemokines are highly expressed at the level of mRNA, with the most significant increase in mRNA levels being demonstrated for CCL5 when compared with matched peripheral blood samples and controls. We show that high levels of all three chemokines are present in synovial fluid of children with JIA. We investigate the major source of CCL5 from inflammatory synovial cells, which we show to be CD8+ T cells. This CD8+ synovial T cell population has an unexpected phenotype that has not been described previously, being CCR7- yet predominantly CD28+ and CD45RA-. These cells contain high levels of stored intracellular CCL5, and rapid release of CCL5 takes place on T cell stimulation, without requiring new protein synthesis. In addition, we demonstrate that CCL5 is present in synovial biopsies from these patients, in particular on the endothelium of small and medium sized vessels. We believe this to be the first in depth analysis of these mediators of inflammation in JIA.     

Identification of differentially expressed genes in synovial tissue of rheumatoid arthritis and osteoarthritis in patients

Rheumatoid arthritis (RA) and osteoarthritis (OA) are the common joints disorder in the world. Although they have showed the analogous clinical manifestation and overlapping cellular and molecular foundation, the pathogenesis of RA and OA were different. The pathophysiologic mechanisms of arthritis in RA and OA have not been investigated thoroughly. Thus, the aim of study is to identify the potential crucial genes and pathways associated with RA and OA and further analyze the molecular mechanisms implicated in genesis. First, we compared gene expression profiles in synovial tissue between RA and OA from the National Center of Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database. Gene Expression Series (GSE) 1919, GSE55235, and GSE36700 were downloaded from the GEO database, including 20 patients of OA and 21 patients of RA. Differentially expressed genes (DEGs) including “CXCL13,” “CD247,” “CCL5,” “GZMB,” “IGKC,” “IL7R,” “UBD///GABBR1,” “ADAMDEC1,” “BTC,” “AIM2,” “SHANK2,” “CCL18,” “LAMP3,” “CR1,” and “IL32.” Second, Gene Ontology analyses revealed that DEGs were significantly enriched in integral component of extracellular space, extracellular region, and plasma membrane in the molecular function group. Signaling pathway analyses indicated that DEGs had common pathways in chemokine signaling pathway, cytokine-cytokine receptor interaction, and cytosolic DNA-sensing pathway. Third, DEGs showed the complex DEGs protein-protein interaction network with the Coexpression of 83.22%, Shared protein domains of 8.40%, Colocalization of 4.76%, Predicted of 2.87%, and Genetic interactions of 0.75%. In conclusion, the novel DEGs and pathways between RA and OA identified in this study may provide new insight into the underlying molecular mechanisms of RA.     

Escherichia coli K1 induces IL-8 expression in human brain microvascular endothelial cells

Microbial penetration of the blood-brain barrier (BBB) into the central nervous system is essential for the development of meningitis. Considerable progress has been achieved in understanding the pathophysiology of meningitis, however, relatively little is known about the early inflammatory events occurring at the time of bacterial crossing of the BBB. We investigated, using real-time quantitative PCR, the expression of the neutrophil chemoattractants alpha-chemokines CXCL1 (Groalpha) and CXCL8 (IL-8), and of the monocyte chemoattractant beta-chemokine CCL2 (MCP-1) by human brain microvascular endothelial cells (HBMEC) in response to the meningitis-causing E. coli K1 strain RS218 or its isogenic mutants lacking the ability to bind to and invade HBMEC. A nonpathogenic, laboratory E. coli strain HB101 was used as a negative control. CXCL8 was shown to be significantly expressed in HBMEC 4 hours after infection with E. coli K1, while no significant alterations were noted for CXCL1 and CCL2 expression. This upregulation of CXCL8 was induced by E. coli K1 strain RS218 and its derivatives lacking the ability to bind and invade HBMEC, but was not induced by the laboratory strain HB101. In contrast, no upregulation of CXCL8 was observed in human umbilical vein endothelial cells (HUVEC) after stimulation with E. coli RS218. These findings indicate that the CXCL8 expression is the result of the specific response of HBMEC to meningitis-causing E. coli K1.     

Immune-related chemotactic factors were found in acute coronary syndromes by bioinformatics

DNA microarray data for thrombus-related leukocyte from patients with acute coronary syndrome (ACS) was analyzed to acquire key genes associated with ACS. Microarray data set GSE19339, including four ACS patients’ samples and four normal samples, were downloaded from Gene Expression Omnibus database. Raw data was pre-processed and differentially expressed genes (DEGs) were identified by Affy packages of R. The interaction network was established with STRING. DrugBank was retrieved to obtain relevant small molecules. A total of 487 differentially expressed genes were identified as DEGs between normal and disease samples. Among which, ten up-regulated genes belonging to chemokine family (CCL2, CCR1, CXCL3, CXCL2, CCL8, CXCL11, CCL7, IL10, CCL22 and CCL20) were related to inflammatory response. In addition, two inhibitors of CCL2 (L-Mimosine) were retrieved from the DrugBank database. Considering the roles of inflammatory response in the progression of ACS and the functions of the ten up-regulated genes, we speculated that these genes might be related to ACS. Moreover, the inhibitors could provide guidelines for future drug design acting on these genes.     

Variability in chemokine-induced adhesion of human mesenchymal stromal cells

BACKGROUND AIMS. Intravenously applied mesenchymal stromal cells (MSC) are under investigation for numerous clinical indications. However, their capacity to activate shear stress-dependent adhesion to endothelial ligands is incompletely characterized. METHODS. Parallel-plate flow chambers were used to induce firm adhesion of MSC to integrin ligand vascular cell adhesion molecule (VCAM)-1. Human MSC were stimulated by chemokine (C-C motif) ligand (CCL15)/macrophage inflammatory protein (MIP-5), CCL19/MIP-3β chemokine (C-X-C motif) ligand (CXCL8)/interleukin (IL)-8, CXCL12/ stromal derived factor (SDF-1) or CXCL13/B lymphocyte chemoattractant (BLC). RESULTS. Two MSC isolates responded to three chemokines (either to CCL15, CCL19 and CXCL13, or to CCL19, CXCL12 and CXCL13), two isolates responded to two chemokines (to CCL15 and CCL19, or to CCL19 and CXCL13), and one isolate responded to CCL19 only. In contrast, all tested MSC isolates responded to selectins (P-selectin and E-selectin) or integrin ligand VCAM-1, as visualized by a velocity reduction under flow. CONCLUSIONS. Inter-individual variability of chemokine-induced integrin activation should be considered when evaluating human MSC as cellular therapies.     

Association of inflammatory and other immune markers with gallbladder cancer: Results from two independent case-control studies

Most gallbladder cancer (GBC) cases arise in the context of gallstones, which cause inflammation, but few gallstone patients develop GBC. We explored inflammation/immune-related markers measured in bile and serum in GBC cases compared to gallstone patients to better understand how inflammatory patterns in these two conditions differ. We measured 65 immune-related markers in serum and bile from 41 GBC cases and 127 gallstone patients from Shanghai, China, and calculated age- and sex-adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs) for GBC versus gallstones. We then focused on the markers that were significantly elevated in bile and serum to replicate the findings in serum from 35 GBC cases and 31 gallstone controls from Chile. Comparing the highest versus lowest quantile, 15 markers (23%) were elevated in both serum and bile from GBC versus gallstone patients in the Shanghai study (p < 0.05). The strongest OR was for CXCL8 (interleukin-8) in serum (96.8, 95% CI: 11.9–790.2). Of these 15 markers, 6 were also significantly elevated in serum from Chile (CCL20, C-reactive protein, CXCL8, CXCL10, resistin, serum amyloid A). Pooled ORs from Shanghai and Chile for these 6 markers ranged from 7.2 (95% CI: 2.8–18.4) for CXCL10 to 58.2 (95% CI: 12.4–273.0) for CXCL8. GBC is associated with inflammation above and beyond that generated by gallstones alone. This local inflammatory process is reflected systemically. Future longitudinal studies are needed to identify the key players in cancer development, which may guide translational efforts to identify individuals at high risk of developing GBC.     

Towards in situ tissue repair: human mesenchymal stem cells express chemokine receptors CXCR1, CXCR2 and CCR2, and migrate upon stimulation with CXCL8 but not CCL2

The recruitment of bone marrow CD34- mesenchymal stem- and progenitor cells (MSC) and their subsequent differentiation into distinct tissues is the precondition for in situ tissue engineering. The objective of this study was to determine the entire chemokine receptor expression profile of human MSC and to investigate their chemotactic response to the selected chemokines CCL2, CXCL8 and CXCL12. Human MSC were isolated from iliac crest bone marrow aspirates and showed a homogeneous population presenting a typical MSC-related cell surface antigen profile (CD14-, CD34-, CD44+, CD45-, CD166+, SH-2+). The expression profile of all 18 chemokine receptors was determined by real-time PCR and immunohistochemistry. Both methods consistently demonstrated that MSC express CC, CXC, C and CX(3)C receptors. Gene expression and immunohistochemical analysis documented that MSC express chemokine receptors CCR2, CCR8, CXCR1, CXCR2 and CXCR3. A dose-dependent chemotactic activity of CXCR4 and CXCR1/CXCR2 ligands CXCL12 and CXCL8 (interleukin-8) was demonstrated using a 96-well chemotaxis assay. In contrast, the CCR2 ligand CCL2 (monocyte chemoattractant protein-1, MCP-1) did not recruited human MSC. In conclusion, we report that the chemokine receptor expression profile of human MSC is much broader than known before. Furthermore, for the first time, we demonstrate that human MSC migrate upon stimulation with CXCL8 but not CCL2. In combination with already known data on MSC recruitment and differentiation these are promising results towards in situ regenerative medicine approaches based on guiding of MSC to sites of degenerated tissues.     

Assessment of chemokine profiles in human skin biopsies by an immunoaffinity capillary electrophoresis chip

Atopic dermatitis is a skin condition resulting in a skin rash from exposure to environmental factors. Skin biopsies taken from patients suffering from atopic dermatitis were micro-dissected and analyzed using a microchip-based immunoaffinity CE system for the presence of CXCL1, CXCL5 and CXCL8 and CCL1, CCL3 and CCL5 chemokines. Disposable immunoaffinity disks with immobilized antibodies were used to capture the CXC and CC chemokines from the homogenized skin samples. The captured analytes were then labeled with AlexaFluor 633, eluted from the disk and separated by CE. The labeled chemokines were identified and quantified by laser induced fluorescence. The total analysis time was less than 40min, including the biopsy microdissection, pre-analysis preparation of the sample and the ICE-CHIP analysis, which took less than 10min with inter- and intra-assay CV's below 6.4%. Microchip-based immunoaffinity CE could distinguish between normal skin biopsies and those with inflammation. Patients with neutrophil cellular infiltrates by histopathology showed increased concentrations of CXCL1, CXCL5 and CXCL8 while increases of CCL1, CCL3 and CCL5 corresponded to the patient group demonstrating monocytic and T-lymphocyte infiltration by histopathology. This system demonstrates the ability to identify and quantify immunochemical analytes in frozen sections taken from clinical histopathology samples.     

Human periosteum-derived progenitor cells express distinct chemokine receptors and migrate upon stimulation with CCL2, CCL25, CXCL8, CXCL12, and CXCL13

For bone repair, transplantation of periosteal progenitor cells (PCs), which had been amplified within supportive scaffolds, is applied clinically. More innovative bone tissue engineering approaches focus on the in situ recruitment of stem and progenitor cells to defective sites and their subsequent use for guided tissue repair. Chemokines are known to induce the directed migration of bone marrow CD34(-) mesenchymal stem cells (MSCs). The aim of our study was to determine the chemokine receptor expression profile of human CD34(-) PCs and to demonstrate that these cells migrate upon stimulation with selected chemokines. PCs were isolated from periosteum of the mastoid bone and displayed a homogenous cell population presenting an MSC-related cell-surface antigen profile (ALCAM(+), SH2(+), SH3(+), CD14(-), CD34(-), CD44(+), CD45(-), CD90(+)). The expression profile of chemokine receptors was determined by real-time PCR and immunohistochemistry. Both methods consistently demonstrated that PCs express receptors of all four chemokine subfamilies CC, CXC, CX(3)C, and C. Migration of PCs and a dose-dependent migratory effect of the chemokines CCL2 (MCP1), CCL25 (TECK), CXCL8 (IL8), CXCL12 (SDF1alpha), and CXCL13 (BCA1), but not CCL22 (MDC) were demonstrated using a 96-multiwell chemotaxis assay. In conclusion, for the first time, here we report that human PCs express chemokine receptors, present their profile, and demonstrate a dose-dependent migratory effect of distinct chemokines on these cells. These results are promising towards in situ bone repair therapies based on guiding PCs to bone defects, and encourage further in vivo studies.