CXCL8, a ligand for the chemokine receptor CXCR2, was recently reported to be a transcriptional target of Ras signaling, but its role in Ras-induced tumorigenesis has not been fully defined. Here, we investigated the role of KC and MIP-2, the murine homologues of CXCL8, in Kras(LA1) mice, which develop lung adenocarcinoma owing to somatic activation of the KRAS oncogene. We first investigated biological evidence of CXCR2 ligands in Kras(LA1) mice. Malignant progression of normal alveolar epithelial cells to adenocarcinoma in Kras(LA1) mice was associated with enhanced intralesional vascularity and neutrophilic inflammation, which are hallmarks of chemoattraction by CXCR2 ligands. In in vitro migration assays, supernatants of bronchoalveolar lavage samples from Kras(LA1) mice chemoattracted murine endothelial cells, alveolar inflammatory cells, and the LKR-13 lung adenocarcinoma cell line derived from Kras(LA1) mice, an effect that was abrogated by pretreatment of the cells with a CXCR2-neutralizing antibody. CXCR2 and its ligands were highly expressed in LKR-13 cells and premalignant alveolar lesions in Kras(LA1) mice. Treatment of Kras(LA1) mice with a CXCR2-neutralizing antibody inhibited the progression of premalignant alveolar lesions and induced apoptosis of vascular endothelial cells within alveolar lesions. Whereas the proliferation of LKR-13 cells in vitro was resistant to treatment with the antibody, LKR-13 cells established as syngeneic tumors were sensitive, supporting a role for the tumor microenvironment in the activity of CXCR2. Thus, high expression of CXCR2 ligands may contribute to the expansion of early alveolar neoplastic lesions induced by oncogenic KRAS.

Interleukin-8 (IL-8; CXCL8) is a cytokine of the CXC chemokine family that is involved in neutrophil recruitment and activation. In addition, IL-8 has been implicated in a wide variety of other processes, including angiogenesis and metastasis in lung cancer. Lung adenocarcinoma and muco-epidermoid carcinoma cells produce substantial amounts of IL-8, and express both CXCR1 and CXCR2 IL-8 receptors. We hypothesized that IL-8 stimulates proliferation of non-small cell lung cancer cells, involving transactivation of the epidermal growth factor receptor (EGFR). The EGFR plays a central role in regulating cell proliferation and it has been therefore implicated in lung cancer. Both EGFR ligands and transactivation of the receptor may lead to downstream signalling events, including mitogen-activated protein kinase (MAPK) activation. Transactivation of the EGFR has been shown to occur in response to ligands of various G-protein coupled receptors (GPCRs) and involves metalloproteinase-mediated release of membrane bound EGFR ligands. The aim of the present study was to investigate the effect of IL-8 on proliferation of lung adenocarcinoma and muco-epidermoid carcinoma cells, and to explore the mechanisms leading to this proliferation in two different non-small cell lung cancer cell lines (A549 and NCI-H292). In both NSCLC cell lines, we observed that IL-8 stimulates epithelial cell proliferation in a dose-dependent manner. The ability of IL-8 to increase cell proliferation was blocked both by an inhibitor of EGFR tyrosine kinase, by a specific anti-EGFR blocking antibody and by a panmetalloproteinase inhibitor. Similar results were obtained using the GPCR inhibitor pertussis toxin. Inhibition of the MAPK p42/44 (ERK1/2) also blocked the mitogenic effect of IL-8, while a p38 MAPK inhibitor did not affect IL-8-induced cell proliferation. These results suggest that IL-8 increases cell proliferation in NSCLC cell lines via transactivation of the EGFR and that this mechanism involves metalloproteinase activity.

BACKGROUND

Chronic obstructive pulmonary disease (COPD) is often associated with peripheral muscle weakness, which is caused by several factors. Acute exacerbations may contribute, but their impact on muscle force remains unclear. Correlations between peripheral muscle force and inflammatory and anabolic markers have never been studied in COPD. The effect of an acute exacerbation on quadriceps peak torque (QPT) was therefore studied in hospitalised patients, and the aforementioned correlations were examined in hospitalised and in stable patients.

METHODS

Lung function, respiratory and peripheral muscle force, and inflammatory and anabolic markers were assessed in hospitalised patients on days 3 and 8 of the hospital admission and 90 days later. The results on day 3 (n=34) were compared with those in clinically stable outpatients (n=13) and sedentary healthy elderly subjects (n=10).

RESULTS

Hospitalised patients had lowest mean (SD) QPT (66 (22)% predicted) and highest median (IQR) levels of systemic interleukin-8 (CXCL8, 6.1 (4.5 to 8.3) pg/ml). Insulin-like growth factor I (IGF-I) tended to be higher in healthy elderly subjects (p=0.09). QPT declined between days 3 and 8 in hospital (mean -5% predicted (95% CI -22 to 8)) and partially recovered 90 days after admission to hospital (mean 6% predicted (95% CI -1 to 23)). QPT was negatively correlated with CXCL8 and positively correlated with IGF-I and lung transfer factor in hospitalised and in stable patients.

CONCLUSIONS

Peripheral muscle weakness is enhanced during an acute exacerbation of COPD. CXCL8 and IGF-I may be involved in the development of peripheral muscle weakness in hospitalised and in stable patients with COPD.

CXCL10 was recently shown to exert antimalignancy functions by influencing the tumor microenvironment. Here, we have taken a different approach, investigating the effects of CXCL10 directly on tumor-promoting functions in colorectal carcinoma (CRC) cells. CXCL10 expression was detected in preferred metastatic sites of CRC (liver, lungs, and lymph nodes), and its CXCR3 receptor was expressed by eight CRC cell lines (detected: reverse transcription-PCR and/or flow cytometry). Detailed analysis was done on two cell lines derived from primary CRC tumors (SW480, KM12C) and their metastatic descendents (SW620 and KM12SM). The three known variants of CXCR3 (CXCR3-A, CXCR3-B, and CXCR3-alt) were detected in all four cell lines. CXCR3 expression was also observed on colorectal tumor cells in biopsies of CRC patients (immunohistochemistry). CXCL10 and CXCR3 expression were potently induced in CRC cells by Interferon gamma and all four CRC cell lines responded to CXCL10 by extracellular signal-regulated kinase 1/2 dephosphorylation. The chemokine did not affect tumor cell growth or angiogenesis-related functions in the tumor cells, such as CXCL8 and vascular endothelial growth factor secretion. Importantly, CXCL10 significantly up-regulated invasion-related properties in CRC cells: It promoted matrix metalloproteinase 9 expression and induced CRC cell migration. Of note, CXCL10-induced migration was detected only in the two metastatic cells and not in their primary counterparts. Also, CXCL10 promoted the adhesion of metastatic cells to laminin. These results suggest that CXCL10 can be exploited by CRC cells toward their progression, thus possibly antagonizing the antimalignancy effects of the chemokine on the tumor microenvironment. Therefore, care should be taken when considering CXCL10 as a therapeutic antitumor modality for CRC treatment.

Chemokines are small proteins that control several tissue functions, including cell recruitment and activation under homeostatic and inflammatory conditions. CXCL8 (interleukin-8) is a member of the chemokine family that acts on CXCR1 and CXCR2 receptors. CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL7 are also ELR+ chemokine members that bind to these receptors, especially CXCR2. The majority of studies on the biology of CXCL8 and their receptors have been performed in polymorphonuclear leukocytes. However, many other cells express CXCR1/CXCR2, including epithelial, endothelial, fibroblasts and neurons, contributing to the biological effects of CXCL8. There is substantial amount of experimental data suggesting that CXCL8 and receptors contribute to elimination of pathogens, but may also contribute significantly to disease-associated processes, including tissue injury, fibrosis, angiogenesis and tumorigenesis. Here, we discuss the biology of CXCL8 family and the potential therapeutic use of antagonists or blockers of these molecules in the context of organ-specific diseases.

Platelets are a rich source of different chemokines and express chemokine receptors. CXCL4 is highly abundant in platelets and involved in promoting monocyte arrest from rolling and monocyte differentiation to macrophages. CXCL4 can also associate with CCL5 and amplify its effect on monocytes. The megakaryocyte CXCL7 gene product is proteolytically cleaved into the strong neutrophil chemoattractant, NAP-2, which has also been implicated in repair cell homing to vascular lesions. Platelet adhesion can induce release of CCL2 and CXCL8 from endothelial cells. Conversely, the chemokines CCL17, CCL22, and CXCL12 made by other cells amplify platelet activation. Platelet chemokines enhance recruitment of various hematopoietic cells to the vascular wall, fostering processes such as neointima formation, atherosclerosis, and thrombosis, but also vessel repair and regeneration after vascular injury.

Astrocytes play a key role in maintenance of neuronal functions in the central nervous system by producing various cytokines, chemokines, and growth factors, which act as a molecular coordinator of neuron-glia communication. At the site of neuroinflammation, astrocyte-derived cytokines and chemokines play both neuroprotective and neurotoxic roles in brain lesions of human neurological diseases. At present, the comprehensive profile of human astrocyte-derived cytokines and chemokines during inflammation remains to be fully characterized. We investigated the cytokine secretome profile of highly purified human astrocytes by using a protein microarray. Non-stimulated human astrocytes in culture expressed eight cytokines, including G-CSF, GM-CSF, GROα (CXCL1), IL-6, IL-8 (CXCL8), MCP-1 (CCL2), MIF and Serpin E1. Following stimulation with IL-1β and TNF-α, activated astrocytes newly produced IL-1β, IL-1ra, TNF-α, IP-10 (CXCL10), MIP-1α (CCL3) and RANTES (CCL5), in addition to the induction of sICAM-1 and complement component 5. Database search indicated that most of cytokines and chemokines produced by non-stimulated and activated astrocytes are direct targets of the transcription factor NF-kB. These results indicated that cultured human astrocytes express a distinct set of NF-kB-target cytokines and chemokines in resting and activated conditions, suggesting that the NF-kB signaling pathway differentially regulates gene expression of cytokines and chemokines in human astrocytes under physiological and inflammatory conditions.

Cells of the tumor microenvironment play active roles in determining the malignancy phenotype. The host cells and the cancer cells cross-talk via a large variety of soluble factors, whose effects on both partners determine the final outcome of the tumorigenic process. In this review, we focus on the interactions between cancer cells and fibroblasts that are found in their proximity in the growing and progressing tumor and describe the roles of chemokines in mediating such cross-talks. Cancer-associated fibroblasts (CAFs, also termed tumor-associated fibroblasts) were found recently to acquire properties that promote tumor development and metastasis formation, as is also the case for specific members of the chemokine family. In this review, we suggest that there is a bidirectional cross-talk between tumor cells and CAFs, which leads via chemokine activities to increased malignancy. This cross-talk is manifested by the fact that cancer cells release factors that enhance the ability of the fibroblasts to secrete a variety of tumor-promoting chemokines, which then act back on the malignant cells to promote their proliferative, migratory, and invasive properties. The CAF-released chemokines also affect the tumor microenvironment, leading to increased angiogenesis and possibly to an elevated presence of cancer-supporting macrophages in tumors. Here, we describe these bidirectional interactions and the chemokines that are involved in these processes: mainly the CXCL12-CXCR4 pair but also other chemokines, including CCL2, CCL5, CCL7, CXCL8, and CXCL14. The overall findings suggest that chemokines stand at the crossroads of tumor-CAF interactions that lead to increased malignancy in many cancer diseases.

The progression of epithelial cancer is associated with an intense immunological interaction between the tumor cells and immune cells of the host. However, little is known about the interaction between tumor cells and polymorphonuclear granulocytes (PMNs) in patients with head and neck squamous cell carcinoma (HNSCC). In our study, we investigated systemic PMN-related alterations in HNSCC, the role of tumor-infiltrating PMNs and their modulation by the tumor microenvironment. We assessed the infiltration of HNSCC tissue by PMNs (retrospectively) and systemic PMN-related alterations in blood values (prospectively) in HNSCC patients (n = 99 and 114, respectively) and control subjects (n = 41). PMN recruitment, apoptosis and inflammatory activity were investigated in an in vitro system of peripheral blood PMNs and a human HNSCC cell line (FaDu). HNSCC tissue exhibited considerable infiltration by PMNs, and strong infiltration was associated with poorer survival in advanced disease. PMN count, neutrophil-to-lymphocyte ratio and serum concentrations of CXCL8 (interleukin-8), CCL4 (MIP-1β) and CCL5 (RANTES) were significantly higher in the peripheral blood of HNSCC patients than in that of controls. In vitro, HNSCC-conditioned medium inhibited apoptosis of PMNs, increased chemokinesis and chemotaxis of PMNs, induced release of lactoferrin and matrix metalloproteinase 9 by PMNs and enhanced the secretion of CCL4 by PMN. Our findings demonstrate alterations in PMN biology in HNSCC patients. In vitro, tumor-derived factors modulate cellular functions of PMNs and increase their inflammatory activity. Thus, the interaction between HNSCC and PMNs may contribute to host-mediated changes in the tumor microenvironment.

Neutrophilic inflammation plays an important role in lung tissue destruction occurring in many chronic pulmonary diseases. Neutrophils can be recruited to sites of inflammation via the action of the cytokine IL-17. In this study, we report that IL-17RA and IL-17RC mRNA expression is significantly increased in asthmatic bronchoscopic biopsies and that these receptors are not only expressed on epithelial and inflammatory cells but also on endothelial cells. IL-17 potently stimulates lung microvascular endothelial cells to produce chemoattractants (CXCL8 and derivatives of the 5-lipoxygenase pathway) that selectively drive neutrophil but not lymphocyte chemotaxis. Moreover, IL-17 promotes endothelial activation by inducing the expression of endothelial adhesion markers (E-selectin, VCAM-1, and ICAM-1) in a p38 MAPK-dependent manner. This increased expression of adhesion molecules stimulates the trans-endothelial migration of neutrophils, as well as the transmigration of HT-29 colon carcinoma cells, suggesting a further role in promoting lung metastasis. Finally, IL-17 increased neutrophil adhesion to the endothelium in vivo as determined by intravital microscopy of mice cremaster muscle. Overall, our results demonstrate that IL-17 is a potent activator of the endothelium in vivo leading to neutrophil infiltration. Therefore, preventing neutrophil recruitment by blocking the action of IL-17 on endothelial cells may prove to be highly beneficial in diseases in which neutrophilic inflammation plays a key role.

The chemokine receptors CXCR1/2 and their ligand CXCL8 are essential for the activation and trafficking of inflammatory mediators as well as tumor progression and metastasis. The CXCL8-CXCR1/2 signaling axis is involved in the pathogenesis of several diseases including chronic obstructive pulmonary diseases (COPD), asthma, cystic fibrosis and cancer. Interaction between CXCL8 secreted by select cancer cells and CXCR1/2 in the tumor microenvironment is critical for cancer progression and metastasis. The CXCL8-CXCR1/2 axis may play an important role in tumor progression and metastasis by regulating cancer stem cell (CSC) proliferation and self-renewal. During the past two decades, several small-molecule CXCR1/2 inhibitors, CXCL8 releasing inhibitors, and neutralizing antibodies against CXCL8 and CXCR1/2 have been reported. As single agents, such inhibitors are expected to be efficacious in various inflammatory diseases. Several preclinical studies suggest that combination of CXCR1/2 inhibitors along with other targeted therapies, chemotherapies, and immunotherapy may be effective in treating select cancers. Currently, several of these inhibitors are in advanced clinical trials for COPD, asthma, and metastatic breast cancer. In this review, we provide a comprehensive analysis of the role of the CXCL8-CXCR1/2 axis and select genes co-expressed in this pathway in disease progression. We also discuss the latest progress in developing small-molecule drugs targeting this pathway.

Interleukin-8 (IL-8/CXCL8), a paracrine angiogenic factor, modulates multiple biologic functions in CXCR1 and CXCR2 expressing endothelial cells. Several reports suggest that inflammation, infection, cellular stress and tumor presence regulate IL-8 production in endothelial cells. In the present study, we test the hypothesis that IL-8 regulates multiple biological effects in endothelial cells in an autocrine manner. We examined the autocrine role of IL-8 in regulating angiogenesis by using a neutralizing antibody to IL-8, CXCR1 or CXCR2 in human vein umbilical endothelial cell (HUVEC) and human dermal microvascular endothelial cell (HMEC). Neutralizing antibody to IL-8, CXCR1 or CXCR2 inhibited endothelial cell proliferation, and MMP-2 production as compared to cells cultured with medium alone or control antibody. In addition, we observed that the number of apoptotic cells was significantly higher in anti-IL-8, anti-CXCR1 and anti-CXCR2 treated endothelial cells, which coincided with decreased survival-associated gene expression. We observed reduced migration of endothelial cells treated with anti-IL-8 and anti-CXCR2 antibody, but not anti-CXCR1 antibody as compared to controls. Further, we observed an inhibition of capillary tube formation and neovascularization following treatment with anti-IL-8, anti-CXCR1 and anti-CXCR2 antibodies. Together these data suggest that IL-8 functions as an important autocrine growth and angiogenic factor in regulating multiple biological activities in endothelial cells.

The chemokine system controls leukocyte trafficking during homeostasis as well as during inflammation and is necessary for the linkage between innate and adaptive immunity. Tissue regulation outside the hematopoietic compartment, for instance, angiogenesis, organogenesis and tumor development, growth and metastasis, is another important function of the chemokine system. The chemokine-mediated regulation of angiogenesis is highly sophisticated and fine tuned, and involves pro-angiogenic chemokines, for instance, CXCL8/IL8 interacting with the CXCR2 receptor, and anti-angiogenic (i.e. angiostatic) chemokines, for instance, CXCL10/IP10 interacting with the CXCR3 receptor. Chemokines also regulate angiogenesis in a receptor-independent manner by means of a perturbation of bFGF and VEGF function. The current review focuses on the influence of the chemokine system in angiogenesis. Examples of the delicate angiogenesis regulation by the chemokine system in, for instance, wound healing and of the dysregulation in, for instance, tumor development are provided along with the interesting phenomenon of molecular piracy of host-encoded genes within the chemokine system. This phenomenon is a general strategy to circumvent and exploit the immune system -- and thereby improve survival -- for many viruses. Yet, a certain group of herpesviruses -- the gamma2-herpesviruses -- encode a functional CXCR2 receptor homolog that is activated by angiogenic chemokines and antagonized by angiostatic chemokines, and this particular gene seems to cause the development of a vascular tumor -- Kaposi's sarcoma -- in the host.

Chemokines are attractants and regulators of cell activation. Several CXC family chemokine members induce angiogenesis and promote tumor growth. In contrast, the only CC chemokine, reported to play a direct role in angiogenesis is monocyte-chemotactic protein-1. Here we report that another CC chemokine, eotaxin (also known as CCL11), also induced chemotaxis of human microvascular endothelial cells. CCL11-induced chemotactic responses were comparable with those induced by monocyte-chemotactic protein-1 (CCL2), but lower than those induced by stroma-derived factor-1alpha (CXCL12) and IL-8 (CXCL8). The chemotactic activity was consistent with the expression of CCR3, the receptor for CCL11, on human microvascular endothelial cells and was inhibited by mAbs to either human CCL11 or human CCR3. CCL11 also induced the formation of blood vessels in vivo as assessed by the chick chorioallantoic membrane and Matrigel plug assays. The angiogenic response induced by CCL11 was about one-half of that induced by basic fibroblast factor, and it was accompanied by an inflammatory infiltrate, which consisted predominantly of eosinophils. Because the rat aortic sprouting assay, which is not infiltrated by eosinophils, yielded a positive response to CCL11, this angiogenic response appears to be direct and is not mediated by eosinophil products. This suggests that CCL11 may contribute to angiogenesis in conditions characterized by increased CCL11 production and eosinophil infiltration such as Hodgkin's lymphoma, nasal polyposis, endometriosis, and allergic diathesis.

Bloodsucking parasites such as ticks have evolved a wide variety of immunomodulatory proteins that are secreted in their saliva, allowing them to feed for long periods of time without being detected by the host immune system. One possible strategy used by ticks to evade the host immune response is to produce proteins that selectively bind and neutralize the chemokines that normally recruit cells of the innate immune system that protect the host from parasites. We have identified distinct cDNAs encoding novel chemokine binding proteins (CHPBs), which we have termed Evasins, using an expression cloning approach. These CHBPs have unusually stringent chemokine selectivity, differentiating them from broader spectrum viral CHBPs. Evasin-1 binds to CCL3, CCL4, and CCL18; Evasin-3 binds to CXCL8 and CXCL1; and Evasin-4 binds to CCL5 and CCL11. We report the characterization of Evasin-1 and -3, which are unrelated in primary sequence and tertiary structure, and reveal novel folds. Administration of recombinant Evasin-1 and -3 in animal models of disease demonstrates that they have potent antiinflammatory properties. These novel CHBPs designed by nature are even smaller than the recently described single-domain antibodies (Hollinger, P., and P.J. Hudson. 2005. Nat. Biotechnol. 23:1126-1136), and may be therapeutically useful as novel antiinflammatory agents in the future.

BACKGROUND

Interleukin-8 (IL-8, CXCL8) is a potent chemoattractant for neutrophils and contributes to acute liver inflammation. Much less is known about IL-8 in chronic liver diseases (CLD), but elevated levels were reported from alcoholic and hepatitis C-related CLD. We investigated the regulation of IL-8, its receptors CXCR1 and CXCR2 and possible IL-8 responding cells in CLD patients.

METHODS

Serum IL-8 levels were measured in CLD patients (n = 200) and healthy controls (n = 141). Intrahepatic IL-8, CXCR1 and CXCR2 gene expression was quantified from liver samples (n = 41), alongside immunohistochemical neutrophil (MPO) and macrophage (CD68) stainings. CXCR1 and CXCR2 expression was analyzed on purified monocytes from patients (n = 111) and controls (n = 31). In vitro analyses explored IL-8 secretion by different leukocyte subsets.

RESULTS

IL-8 serum levels were significantly increased in CLD patients, especially in end-stage cirrhosis. Interestingly, patients with cholestatic diseases exhibited highest IL-8 serum concentrations. IL-8 correlated with liver function, inflammatory cytokines and non-invasive fibrosis markers. Intrahepatically, IL-8 and CXCR1 expression were strongly up-regulated. However, intrahepatic IL-8 could only be associated to neutrophil infiltration in patients with primary biliary cirrhosis (PBC). In non-cholestatic cirrhosis, increased IL-8 and CXCR1 levels were associated with hepatic macrophage accumulation. In line, CXCR1, but not CXCR2 or CXCR3, expression was increased on circulating monocytes from cirrhotic patients. Moreover, monocyte-derived macrophages from CLD patients, especially the non-classical CD16⁺ subtype, displayed enhanced IL-8 secretion in vitro.

CONCLUSIONS

IL-8 is strongly activated in CLD, thus likely contributing to hepatic inflammation. Our study suggests a novel role of IL-8 for recruitment and activation of hepatic macrophages via CXCR1 in human liver cirrhosis.

Inflammatory mediators present in the tumor milieu may promote cancer progression and are considered promising targets of novel biological therapies. We previously reported that the marine antitumor agent trabectedin, approved in Europe in 2007 for soft tissue sarcomas and in 2009 for ovarian cancer, was able to downmodulate the production of selected cytokines/chemokines in immune cells. Patients with myxoid liposarcoma (MLS), a subtype characterized by the expression of the oncogenic transcript FUS-CHOP, are highly responsive to trabectedin. The drug had marked antiproliferative effects on MLS cell lines at low nanomolar concentrations. We tested the hypothesis that trabectedin could also affect the inflammatory mediators produced by cancer cells. Here, we show that MLS express several cytokines, chemokines, and growth factors (CCL2, CCL3, CCL5, CXCL8, CXCL12, MIF, VEGF, SPARC) and the inflammatory and matrix-binder protein pentraxin 3 (PTX3), which build up a prominent inflammatory environment. In vitro treatment with noncytotoxic concentrations of trabectedin selectively inhibited the production of CCL2, CXCL8, IL-6, VEGF, and PTX3 by MLS primary tumor cultures and/or cell lines. A xenograft mouse model of human MLS showed marked reduction of CCL2, CXCL8, CD68+ infiltrating macrophages, CD31+ tumor vessels, and partial decrease of PTX3 after trabectedin treatment. Similar findings were observed in a patient tumor sample excised after several cycles of therapy, indicating that the results observed in vitro might have in vivo relevance. In conclusion, trabectedin has dual effects in liposarcoma: in addition to direct growth inhibition, it affects the tumor microenvironment by reducing the production of key inflammatory mediators.

Keratinocytes play a crucial role in the regulation of skin inflammation, responding to environmental and immune cells stimuli. They produce soluble factors that can act in an autocrine or paracrine manner on immune cells or directly on aggressors. A screening of the activities of 36 cytokines on keratinocyte gene expression identified IL-17A, IL-22, oncostatin M, TNF-alpha, and IL-1alpha as potent cytokines in inducing cutaneous inflammation. These five proinflammatory cytokines synergistically increased production of CXCL8 and beta-defensin 2 (BD2). In addition, ex vivo studies on human skin explants demonstrated upregulation of BD2, S100A7, and CXCL8 expression in response to the same combination of cytokines. In vivo intradermal injection of these five cytokines in mouse increased CXCL1, CXCL2, CXCL3, S100A9, and BD3 expression, associated with neutrophil infiltration. We confirmed and extended this synergistic effect using quantitative real-time PCR analysis and observed increased expression of nine chemokines and 12 antimicrobial peptides. Production of CXCL, CXCL5, and CXCL8 by keratinocytes stimulated in the presence of this cytokine combination was associated with increased neutrophil chemotactic activity. Similarly, high production of BD2, BD3, and S100A7 was associated with an increased antimicrobial activity. Finally, the transcriptional profile observed in this in vitro model of inflammatory keratinocytes correlated with the one of lesional psoriatic skin. Our results demonstrate the important potentiating activities of IL-17A, IL-22, oncostatin M, TNF-alpha, and IL-1alpha on keratinocytes. This is particularly interesting in the context of psoriasis where these cytokines are overexpressed and could synergize to play an important role in upregulation of chemokines and antimicrobial peptides production.

Although mast cells (MCs) often are abundant in the synovial tissues of patients with rheumatoid arthritis, the contribution of MCs to joint inflammation and cartilage loss remains poorly understood. MC-restricted tryptase/heparin complexes have proinflammatory activity, and significant amounts of human tryptase beta (hTryptase-beta) are present in rheumatoid arthritis synovial fluid. Mouse MC protease-6 (mMCP-6) is the ortholog of hTryptase-beta, and this serine protease is abundant in the synovium of arthritic mice. We now report that C57BL/6 (B6) mice lacking their tryptase/heparin complexes have attenuated arthritic responses, with mMCP-6 as the dominant tryptase responsible for augmenting neutrophil infiltration in the K/BxN mouse serum-transfer arthritis model. While inflammation in this experimental arthritis model was not dependent on protease-activated receptor-2, it was dependent on the chemokine receptor CXCR2. In support of the latter data, exposure of synovial fibroblasts to hTryptase-beta/heparin or mMCP-6/heparin complexes resulted in expression of the neutrophil chemotactic factors CXCL1/KC, CXCL5/LIX, and CXCL8/IL-8. Our proteomics, histochemistry, and immunohistochemistry data also revealed substantial loss of cartilage-derived aggrecan proteoglycans in the arthritic joints of wild-type B6 mice but not mMCP-6-null B6 mice. These observations demonstrate the functional contribution of MC-restricted tryptase/heparin complexes in the K/BxN mouse arthritis model and connect our mouse findings with rheumatoid arthritis pathophysiology.

Functional interleuin-8 (IL-8) receptors (IL-8RA and IL-8RB: CXCR1 and CXCR2, respectively) have been described in human, monkey, dog, rabbit, and guinea pig. Although three IL-8R homologues have been found in rat, only one of these, rat CXCR2, appears to be functional based on responsiveness to ligands. Similarly, CXC chemokines induce biological responses through the murine homolog of CXCR2, but the identification of functional rodent CXCR1 homologues has remained elusive. We have identified and characterized the mouse CXCR1 homologue (mCXCR1). Murine CXCR1 shares 68 and 88% amino acid identity with its human and rat counterparts, respectively. Similar to the tissue distribution pattern of rat CXCR1, we found murine CXCR1 mRNA expression predominantly in lung, stomach, bone marrow, and leukocyte-rich tissues. In contrast to previous reports, we determined that mCXCR1 is a functional receptor. We show predominant engagement of this receptor by mouse GCP-2/CXCL6, human GCP-2, and IL-8/CXCL8 by binding, stimulation of GTPgammaS exchange, and chemotaxis of mCXCR1-transfected cells. Furthermore, murine CXCR1 is not responsive to the human CXCR2 ligands ENA-78/CXCL5, NAP-2/CXCL7, GRO-alpha, -beta, -gamma/CXCL1-3, or rat CINC-1-3. In addition, we show concomitant elevation of mCXCR1 and its proposed major ligand, GCP-2, positively correlated with paw swelling in murine collagen-induced arthritis. This report represents the first description of a functional CXCR1-like receptor in rodents.

The first chemokine structure, that of IL-8/CXCL8, was determined in 1990. Since then, many chemokine structures have emerged. To the initial disappointment of structural biologists, the tertiary structures of these small proteins were found to be highly conserved. However, they have since proven to be much more interesting and diverse than originally expected. Somewhat like lego blocks, many chemokines oligomerize and there is significant diversity in their oligomeric forms and propensity to oligomerize. Chemokines not only interact with receptors where different oligomeric forms can induce different signaling responses, they also interact with glycosaminoglycans which can stabilize oligomers and other structures that would not otherwise form in solution. Although chemokine monomers and dimers yielded quickly to structure determination, structural information about larger chemokine oligomers, chemokines receptors, and complexes of chemokines with glycosaminoglycans and receptors has been more difficult to obtain, but recent breakthroughs suggest that this information will be forthcoming, especially with receptor structures. Equally important and challenging, will be efforts to correlate the structural information with function.

The pathogenic mechanisms that contribute to multiple sclerosis (MS) include leukocyte chemotaxis into the central nervous system (CNS) and the production of inflammatory mediators, resulting in oligodendrocyte damage, demyelination, and neuronal injury. Thus, factors that regulate leukocyte entry may contribute to early events in MS, as well as to later stages of lesion pathogenesis. CXCL12 (SDF-1alpha), a chemokine essential in CNS development and a chemoattractant for resting and activated T cells, as well as monocytes, is constitutively expressed at low levels in the CNS and has been implicated in T cell and monocyte baseline trafficking. To determine whether CXCL12 is increased in MS, immunohistochemical analyses of lesions of chronic active and chronic silent MS were performed. CXCL12 protein was detected on endothelial cells (EC) in blood vessels within normal human brain sections and on a small number of astrocytes within the brain parenchyma. In active MS lesions, CXCL12 levels were high on astrocytes throughout lesion areas and on some monocytes/macrophages within vessels and perivascular cuffs, with lesser staining on EC. In silent MS lesions, CXCL12 staining was less than that observed in active MS lesions, and also was detected on EC and astrocytes, particularly hypertrophic astrocytes near the lesion edge. Experiments in vitro demonstrated that IL-1beta and myelin basic protein (MBP) induced CXCL12 in astrocytes by signaling pathways involving ERK and PI3-K. Human umbilical vein EC did not produce CXCL12 after treatment with MBP or IL-1beta. However, these EC cultures expressed CXCR4, the receptor for CXCL12, suggesting that this chemokine may activate EC to produce other mediators involved in MS. In agreement, EC treatment with CXCL12 was found to upregulate CCL2 (MCP-1) and CXCL8 (IL-8) by PI3-K and p38-dependent mechanisms. Our findings suggest that increased CXCL12 may initiate and augment the inflammatory response during MS.

BACKGROUND

Chronic Fatigue Syndrome (CFS) studies from our laboratory and others have described cytokine abnormalities. Other studies reported no difference between CFS and controls. However, methodologies varied widely and few studies measured more than 4 or 5 cytokines. Multiplex technology permits the determination of cytokines for a large panel of cytokines simultaneously with high sensitivity and with only 30 ul of plasma per sample. No widely accepted laboratory test or marker is available for the diagnosis or prognosis of CFS. This study screened plasma factors to identify circulating biomarkers associated with CFS.

METHODS

Cytokines were measured in plasma from female CFS cases and female healthy controls. Multiplex technology provided profiles of 16 plasma factors including the pro -inflammatory cytokines: tumor necrosis factor alpha (TNFalpha), lymphotoxin alpha (LTalpha), interleukin (IL) - IL-Ialpha, IL-1beta, IL-6; TH1 cytokines: interferon gamma (IFNgamma), IL-12p70, IL-2, IL-15; TH2: IL-4, IL-5; TH17 cytokines, IL-17 and IL-23; anti-inflammatory cytokines IL-10, IL-13; the inflammatory mediator and neutrophil attracting chemokine IL-8 (CXCL8). Analysis by receiver operating characteristic (ROC) curve assessed the biomarker potential of each cytokine.

RESULTS

The following cytokines were elevated in CFS compared to controls: LTalpha, IL-1alpha, IL-1beta, IL-4, IL-5, IL-6 and IL-12. The following cytokines were decreased in CFS: IL-8, IL-13 and IL-15. The following cytokines were not different: TNFalpha, IFNgamma, IL-2, IL-10, IL-23 and IL-17. Applying (ROC) curve analyses, areas under the curves (AUC) for IL-5 (0. 84), LTalpha (0.77), IL-4 (0.77), IL-12 (0.76) indicated good biomarker potential. The AUC of IL-6 (0.73), IL-15 (0.73), IL-8 (0.69), IL-13 (0.68) IL-1alpha (0.62), IL-1beta (0.62) showed fair potential as biomarkers.

CONCLUSIONS

Cytokine abnormalities are common in CFS. In this study, 10 of 16 cytokines examined showed good to fair promise as biomarkers. However, the cytokine changes observed are likely to more indicative of immune activation and inflammation, rather than specific for CFS. As such, they are targets for herapeutic strategies. Newer techniques allow evaluation of large panels of cytokines in a cost effective fashion.

Previous studies in the K14-HPV/E(2) mouse model of cervical carcinogenesis demonstrated that infiltrating macrophages are the major source of matrix metalloproteinase 9 (MMP-9), a metalloprotease important for tumor angiogenesis and progression. We observed increased expression of the macrophage chemoattractant, CCL2, and its receptor, CCR2, concomitant with macrophage influx and MMP-9 expression. To study the role of CCL2-CCR2 signaling in cervical tumorigenesis, we generated CCR2-deficient K14-HPV/E(2) mice. Cervixes of CCR2-null mice contained significantly fewer macrophages. Surprisingly, there was only a modest delay in time to progression from dysplasia to carcinoma in the CCR2-deficient mice, and no difference in end-stage tumor incidence or burden. Moreover, there was an unexpected persistence of MMP-9 activity, associated with increased abundance of MMP-9(+) neutrophils in tumors from CCR2-null mice. In vitro bioassays revealed that macrophages produce soluble factor(s) that can suppress neutrophil dynamics, as evidenced by reduced chemotaxis in response to CXCL8, and impaired invasion into three-dimensional tumor masses grown in vitro. Our data suggest a mechanism whereby CCL2 attracts proangiogenic CCR2(+) macrophages with the ancillary capability to limit infiltration by neutrophils. If such tumor-promoting macrophages are suppressed, MMP-9(+) neutrophils are then recruited, providing alternative paracrine support for tumor angiogenesis and progression.