Chemokines are key mediators of the selective migration of leukocytes that occurs in neurodegenerative diseases and related inflammatory processes. Astrocytes, the most abundant cell type in the CNS, have an active role in brain inflammation. To ascertain the role of astrocytes during neuropathological processes, we have investigated in two models of primary cells (human fetal and simian adult astrocytes) the repertoire of chemokines and their receptors expressed in response to inflammatory stimuli. We demonstrated that, in the absence of any stimulation, human fetal and simian adult astrocytes express mRNA for receptors APJ, BOB/GPR15, Bonzo/CXCR6, CCR2, CCR3, CCR5, CCR8, ChemR23, CXCR3/GPR9, CXCR4, GPR1, and V28/CX3CR1. Moreover, TNFalpha and IL-1beta significantly increase BOB/GPR15, CCR2, and V28/CX3CR1 mRNA levels in both models. Furthermore, TNFalpha and IFNgamma act synergistically to induce expression of the major coreceptors for HIV infection, CXCR4 and CCR5, at both the mRNA and protein levels in human and simian astrocytes, whereas CCR3 expression was not affected by cytokine treatment. Finally, TNFalpha/IFNgamma was the most significant cytokine combination in leading to a pronounced upregulation in a comparable, time-dependent manner of the production of chemokines IP-10/CXCL10, RANTES/CCL5, MIG/CXCL9, MCP-1/CCL2, and IL-8/CXCL8. In summary, these data suggest that astrocytes serve as an important source of chemokines under the dependence of a complex cytokine regulation, and TNFalpha and IFNgamma are important modulators of chemokines and chemokine receptor expression in human as well as simian astrocytes. Finally, with the conditions we used, there was no difference between species or age of tissue.

Thymic stromal lymphopoietin (TSLP) is highly expressed by bronchial epithelial cells and skin keratinocytes in allergic diseases. TSLP acts as a master switch for allergic inflammation through the activation of dendritic cells and mast cells for initiating inflammatory type 2 T-helper lymphocyte responses. To elucidate the immunological cascades of epithelium/keratinocyte-eosinophil-mediated allergic inflammation, we examined the modulating effects of TSLP on human eosinophils. Expression of TSLP receptor complex was detected by RT-PCR, flow cytometry, and Western blot. Adhesion molecules, cytokine, and chemokines were quantitated by flow cytometry or ELISA. Intracellular signal transduction molecules were measured by Western blot and flow cytometry. We observed that human eosinophils constitutively expressed functional heterodimeric TSLP receptor complex comprising TSLP-binding chain TSLPR and IL-7Ralpha chain. TSLP could significantly delay eosinophil apoptosis, up-regulate cell surface expression of adhesion molecule CD18 and intercellular adhesion molecule-1, but down-regulate L-selectin, enhance eosinophil adhesion onto fibronectin, and induce the release of inflammatory cytokine IL-6 and chemokines CXCL8, CXCL1, and CCL2 (all P < 0.05). All these effects were concentration dependent and TSLP specific. TSLP regulated the above effects through the activation of extracellular signal-regulated protein kinase, p38 mitogen-activated protein kinase, and NF-kappaB signaling pathway, but not signal transducer and activator of transcription 5 and 3, which were usually activated in other effector cells upon TSLP stimulation. Collectively, the above findings elucidate the proallergic mechanisms of TSLP via the activation of distinct intracellular signaling pathways in eosinophils.

Rapid translocation of P-selectin from WPB to the surface of endothelial cells is crucial for early neutrophil recruitment to acute inflammatory lesions. Likewise, the chemokine CXCL8/IL-8 is sorted to WPB in human endothelial cells, but little is known about its functional importance in lack of a suitable animal model. Here, we explored the distribution of the functional IL-8 homologues CXCL1/KC, CXCL2/MIP-2, and CXCL5-6/LIX in resting and inflamed murine vessels by confocal microscopy and paired immunostaining with markers of WPB, discovering that these chemokines did not localize to WPB but displayed a granular pattern in a subset of vessels in healthy skin compatible with sorting to the type 2 endothelial compartment for regulated secretion. Moreover, all chemokines colocalized with VWF and P-selectin in platelets, suggesting that their storage in platelet alpha-granules might represent an alternative source of rapidly available, neutrophil-recruiting chemokines. In conclusion, WPB appear not to be involved in regulated secretion of chemokines in the mouse, and instead, the possible existence of type 2 granules and the role of platelets in rapid leukocyte adhesion deserve further attention.

IL-17A and IL-17F are members of the IL-17 family that play crucial roles in allergic inflammation. Recent studies reported that IL-17A and IL-17F production from a distinct Th lymphocyte subset, Th17, was specifically induced by IL-23, which was produced by dendritic cells and macrophages in response to microbial stimuli. The IL-23-IL-17 axis might therefore provide a link between infections and allergic diseases. In the present study, we investigated the effects of IL-17A, IL-17F, and IL-23, alone or in combination, on cytokine and chemokine release from eosinophils and the underlying intracellular mechanisms. Human eosinophils were found to constitutively express receptors for IL-17A, IL-17F, and IL-23 at the protein level. IL-17A, IL-17F, and IL-23 could induce the release of chemokines GRO-alpha/CXCL1, IL-8/CXCL8, and MIP-1beta/CCL4 from eosinophils, while IL-17F and IL-23 could also increase the production of proinflammatory cytokines IL-1beta and IL-6. Synergistic effects were observed in the combined treatment of IL-17F and IL-23 on the release of proinflammatory cytokines, and the effects were dose-dependently enhanced by IL-23, but not IL-17F. Further investigations showed that IL-17A, IL-17F, and IL-23 differentially activated the ERK, p38 MAPK, and NF-kappaB pathways. Moreover, inhibition of these pathways using selective inhibitors could significantly abolish the chemokine release induced by IL-17A, IL-17F, and IL-23 and the synergistic increases on IL-1beta and IL-6 production mediated by combined treatment of IL-17F and IL-23. Taken together, our findings provide insight for the Th17 lymphocyte-mediated activation of eosinophils via differential intracellular signaling cascades in allergic inflammation.

Triple negative breast cancer (TNBC) is a type of aggressive breast cancer lacking the expression of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor-2 (HER-2). TNBC patients account for approximately 15% of total breast cancer patients and are more prevalent among young African, African-American and Latino women patients. The currently available ER-targeted and Her-2-based therapies are not effective for treating TNBC. Recent studies have revealed a number of novel features of TNBC. In the present work, we comprehensively addressed these features and discussed potential therapeutic approaches based on these features for TNBC, with particular focus on: 1) the pathological features of TNBC/basal-like breast cancer; 2) E(2)/ERbeta-mediated signaling pathways; 3) G-protein coupling receptor-30/epithelial growth factor receptor (GPCR-30/EGFR) signaling pathway; 4) interactions of ERbeta with breast cancer 1/2 (BRCA1/2); 5) chemokine CXCL8 and related chemokines; 6) altered microRNA signatures and suppression of ERalpha expression/ERalpha-signaling by micro-RNAs; 7) altered expression of several pro-oncongenic and tumor suppressor proteins; and 8) genotoxic effects caused by oxidative estrogen metabolites. Gaining better insights into these molecular pathways in TNBC may lead to identification of novel biomarkers and targets for development of diagnostic and therapeutic approaches for prevention and treatment of TNBC.

BACKGROUND

Inflammation and matrix degradation are the hallmarks of high-risk atherosclerosis that leads to myocardial infarction and stroke. Toll-like receptors (TLRs), key players in innate immunity, are upregulated in atherosclerotic lesions, but their functional role in human atherosclerosis is unknown. We explored the effects of blocking TLR-2, TLR-4, and myeloid differentiation primary response gene 88 (MyD88), a signaling adaptor shared by most TLRs and interleukin-1 receptor (IL-1R), in an in vitro model of human atherosclerosis.

RESULTS

Carotid endarterectomies were obtained from patients with symptomatic carotid disease. Cells were isolated via enzymatic tissue dissociation and cultured in the presence or absence of TLR signaling blockers. A dominant-negative form of MyD88 (MyD88(DN)) decreased the production of monocyte chemotactic protein-1/CCL2 (P=0.000), IL-8/CXCL8 (P=0.006), IL-6 (P=0.002), matrix metalloproteinase-1 (MMP-1; P=0.002), and MMP-3 (P=0.000), as well as nuclear factor-kappaB activation (P<0.05) in atheroma cell cultures. IL-1R antagonist, TLR-4 blocking antibodies, or overexpression of a dominant-negative form of the TLR-4 signaling adaptor TRIF-related adaptor molecule reduced nuclear factor-kappaB activity but did not have a broad impact on the production of the mediators studied. In contrast, TLR-2 neutralizing antibodies inhibited nuclear factor-kappaB activation (P<0.05) and significantly reduced monocyte chemotactic protein-1/CCL2 (P=0.000), IL-8/CXCL8 (P=0.009), IL-6 (P=0.000), and MMP-1 (P=0.000), MMP-2 (P=0.004), MMP-3 (P=0.000), and MMP-9 (P=0.006) production.

CONCLUSIONS

Our data indicate that TLR-2 signaling through MyD88 plays a predominant role in inflammation and matrix degradation in human atherosclerosis. TLR-2 blockade may represent a therapeutic strategy for atherosclerosis and its complications.

Astrocytes play key roles in CNS development, inflammation, and repair by producing a wide variety of cytokines, chemokines, and growth factors. Understanding the regulation of this network is important for a full understanding of astrocyte functioning. In this study, expression levels of 268 genes encoding cytokines, chemokines, growth factors, and their receptors were established in cultured human adult astrocytes using cDNA arrays. Also, changes in this gene profile were determined following stimulation with TNFalpha, IL-1beta, and IFNgamma. The data obtained reveal a highly reproducible pattern of gene expression not only between different astrocyte cultures from a single source, but also between astrocytes from different donors. They also identify several gene products not previously described for human astrocytes, including a.o. IL-17, CD70, CD147, and BIGH3. When stimulated with TNFalpha astrocytes respond with increased expression of several genes, notably including those encoding the chemokines CCL2 (MCP-1), CCL5 (RANTES), and CXCL8 (IL-8), growth factors including BMP-2A, BMP-3, neuromodulin (GAP43), BDNF, and G-CSF, and receptors such as the CRF receptor, the calcitonin receptor (CTR), and TKT. The response to IL-1beta involves largely the same range of genes, but responses were blunted in comparison to the TNFalpha response. Treatment with IFNgamma had no or only marginal effects on expression of any of the 268 genes analyzed. Astrocytes treated with a mixture of all three stimuli together displayed responses that are largely similar to those found in response to TNFalpha or IL-1beta alone, with only few additional synergistic effects.

BACKGROUND

Vitamin D deficiency has been shown to be independently associated with increased risk of viral acute respiratory infection (ARI) in a number of observational studies, and meta-analysis of clinical trials of vitamin D supplementation for prevention of ARI has demonstrated protective effects. Several cellular studies have investigated the effects of vitamin D metabolites on immune responses to respiratory viruses, but syntheses of these reports are lacking.

METHODS

In this article, we review the literature reporting results of in vitro experiments investigating immunomodulatory actions of vitamin D metabolites in human respiratory epithelial cells infected with respiratory viruses.

RESULTS

Vitamin D metabolites do not consistently influence replication or clearance of rhinovirus, respiratory syncytial virus (RSV) or influenza A virus in human respiratory epithelial cell culture, although they do modulate expression and secretion of type 1 interferon, chemokines including CXCL8 and CXCL10 and pro-inflammatory cytokines, such as TNF and IL-6.

CONCLUSIONS

More studies are needed to clarify the effects of vitamin D metabolites on respiratory virus-induced expression of cell surface markers mediating viral entry and bacterial adhesion to respiratory epithelial cells.

OBJECTIVE

One of the most important rate-limiting steps in adoptive cell transfer (ACT) is the inefficient migration of T cells to tumors. Because melanomas specifically express the chemokines CXCL1 and CXCL8 that are known to facilitate the CXCR2-dependent migration by monocytes, our aim is to evaluate whether introduction of the CXCR2 gene into tumor-specific T cells could further improve the effectiveness of ACT by enhancing T-cell migration to tumor.

METHODS

In this study, we used transgenic pmel-1 T cells, which recognize gp100 in the context of H-2Db, that were transduced with luciferase gene to monitor the migration of transferred T cells in vivo. To visualize luciferase-expressing T cells within a tumor, a nonpigmented tumor is required. Therefore, we used the MC38 tumor model, which naturally expresses CXCL1.

RESULTS

Mice bearing MC38/gp100 tumor cells treated with CXCR2/luciferase-transduced pmel-1 T cells showed enhanced tumor regression and survival compared with mice receiving control luciferase-transduced pmel-1 T cells. We also observed preferential accumulation of CXCR2-expressing pmel-1 T cells in the tumor sites of these mice using bioluminescence imaging. A similar enhancement in tumor regression and survival was observed when CXCR2-transduced pmel-1 T cells were transferred into mice bearing CXCL1-transduced B16 tumors compared with mice treated with control pmel-1 T cells.

CONCLUSIONS

These results implicate that the introduction of the CXCR2 gene into tumor-specific T cells can enhance their localization to tumors and improve antitumor immune responses. This strategy may ultimately enable personalization of cancer therapies based on chemokine expression by tumors.

Biological functions of proteins are influenced by posttranslational modifications such as on/off switching by phosphorylation and modulation by glycosylation. Proteolytic processing regulates cytokine and chemokine activities. In this study, we report that natural posttranslational citrullination or deimination alters the biological activities of the neutrophil chemoattractant and angiogenic cytokine CXCL8/interleukin-8 (IL-8). Citrullination of arginine in position 5 was discovered on 14% of natural leukocyte-derived CXCL8(1-77), generating CXCL8(1-77)Cit(5). Peptidylarginine deiminase (PAD) is known to citrullinate structural proteins, and it may initiate autoimmune diseases. PAD efficiently and site-specifically citrullinated CXCL5, CXCL8, CCL17, CCL26, but not IL-1beta. In comparison with CXCL8(1-77), CXCL8(1-77)Cit(5) had reduced affinity for glycosaminoglycans and induced less CXCR2-dependent calcium signaling and extracellular signal-regulated kinase 1/2 phosphorylation. In contrast to CXCL8(1-77), CXCL8(1-77)Cit(5) was resistant to thrombin- or plasmin-dependent potentiation into CXCL8(6-77). Upon intraperitoneal injection, CXCL8(6-77) was a more potent inducer of neutrophil extravasation compared with CXCL8(1-77). Despite its retained chemotactic activity in vitro, CXCL8(1-77)Cit(5) was unable to attract neutrophils to the peritoneum. Finally, in the rabbit cornea angiogenesis assay, the equally potent CXCL8(1-77) and CXCL8(1-77)Cit(5) were less efficient angiogenic molecules than CXCL8(6-77). This study shows that PAD citrullinates the chemokine CXCL8, and thus may dampen neutrophil extravasation during acute or chronic inflammation.

Renal cell carcinoma (RCC) accounts for 3% of new cancer incidence and mortality in the United States. Studies in RCC have predominantly focused on VEGF in promoting tumor-associated angiogenesis. However, other angiogenic factors may contribute to the overall angiogenic milieu of RCC. We hypothesized that the CXCR2/CXCR2 ligand biological axis represents a mechanism by which RCC cells promote angiogenesis and facilitate tumor growth and metastasis. Therefore, we first examined tumor biopsies and plasma of patients with metastatic RCC for levels of CXCR2 ligands, and RCC tumor biopsies for the expression of CXCR2. The proangiogenic CXCR2 ligands CXCL1, CXCL3, CXCL5, and CXCL8, as well as VEGF were elevated in the plasma of these patients and found to be expressed within the tumors. CXCR2 was found to be expressed on endothelial cells within the tumors. To assess the role of ELR(+) CXC chemokines in RCC, we next used a model of syngeneic RCC (i.e., RENCA) in BALB/c mice. CXCR2 ligand and VEGF expression temporally increased in direct correlation with RENCA growth in CXCR2(+/+) mice. However, there was a marked reduction of RENCA tumor growth in CXCR2(-/-) mice, which correlated with decreased angiogenesis and increased tumor necrosis. Furthermore, in the absence of CXCR2, orthotopic RENCA tumors demonstrated a reduced potential to metastasize to the lungs of CXCR2(-/-) mice. These data support the notion that CXCR2/CXCR2 ligand biology is an important component of RCC tumor-associated angiogenesis and tumorigenesis.

Chemokines promote tumour progression by enhancing proliferation and modifying the immune response. The purpose of this study was to test the hypothesis that CCL2 monocyte chemotactic protein-1 (MCP-1) contributes to the progression of colorectal cancer by influencing the number and distribution of tumour associated macrophages (TAMs). Chemokine expression was assessed in human colorectal adenocarcinomas by ribonuclease protection assay (RPA). Colonic adenocarcinoma cell lines were used to assess chemokine production by enzyme linked immunosorbant assay (ELISA), and Boyden microchemotaxis assays were performed to determine cell line supernatant monocyte chemotactic activity. CCL2 production was assessed in paraffin embedded tumour samples by immunohistochemistry. Finally, the number of macrophages and their distribution was determined in the same colorectal adenocarcinomas and compared with CCL2 expression and tumour stage. Results showed that CCL2 produced by cell lines induced monocyte chemoattraction, the expression of this chemokine in solid cancers increased with tumour stage (P < 0.05) and immunohistochemistry localized production to tumour cells. Analysis of the macrophage infiltrate showed that the accumulation was significantly greater in tumours than controls (P < 0.005) and within tumours it was greatest in necrotic regions (median 44,600 per mm(3)). Macrophage accumulation increased with tumour stage and correlated with CCL2 expression (r(s) = 0.8). CXCL8 interleukin 8 (IL-8), a potent angiogenic factor and growth factor, was expressed in all tumours and cell lines. It is concluded that CCL2 induces the accumulation of tumour promoting TAMs in human colorectal cancer and represents a therapeutic target to modify the macrophage response and direct immune mediated therapy.

During inflammatory skin disorders such as psoriasis, atopic dermatitis, and allergic contact dermatitis, epidermal keratinocytes overexpress large amounts of soluble epidermal growth factor receptor ligands in response to tumor necrosis factor alpha and interferon gamma. These cytokines also promote de novo synthesis of numerous chemokines, including CCL2/MCP-1, CCL5/RANTES, CXCL10/IP-10, and CXCL8/IL-8, in turn responsible for the recruitment of different leukocyte populations. This study demonstrates that stimulation of EGFR down-regulates CCL2, CCL5, and CXCL10, while it increases CXCL8 expression in keratinocytes. Conversely, EGFR signaling blockade produces opposite effects, with increased CCL2, CCL5, and CXCL10, and reduced CXCL8 expression. In a mouse model of contact hypersensitivity, a single topical administration of a selective EGFR kinase blocker before antigen challenge results in a markedly enhanced immune response with increased chemokine expression and heavier inflammatory cell infiltrate. Targeting EGFR on epithelial cells may thus have profound impact on inflammatory and immune responses.

Malaria during pregnancy is associated with poor birth outcomes, particularly low birth weight. Recently, monocyte infiltration into the placental intervillous space has been identified as a key risk factor for low birth weight. However, the malaria-induced chemokines involved in recruiting and activating placental monocytes have not been identified. In this study, we determined which chemokines are elevated during placental malaria infection and the association between chemokine expression and placental monocyte infiltration. Placental malaria infection was associated with elevations in mRNA expression of three beta chemokines, macrophage-inflammatory protein 1 (MIP-1) alpha (CCL3), monocyte chemoattractant protein 1 (MCP-1; CCL2), and I-309 (CCL1), and one alpha chemokine, IL-8 (CXCL8); all correlated with monocyte density in the placental intervillous space. Placental plasma concentrations of MIP-1 alpha and IL-8 were increased in women with placental malaria and were associated with placental monocyte infiltration. By immunohistochemistry, we localized placental chemokine production in malaria-infected placentas: some but not all hemozoin-laden maternal macrophages produced MIP-1 beta and MCP-1, and fetal stromal cells produced MCP-1. In sum, local placental production of chemokines is increased in malaria, and may be an important trigger for monocyte accumulation in the placenta.

Neutrophils play a pivotal role in the innate immune response. The small cytokine CXCL8 (also known as IL-8) is known to be one of the most potent chemoattractant molecules that, among several other functions, is responsible for guiding neutrophils through the tissue matrix until they reach sites of injury. Unlike mice and rats that lack a CXCL8 homolog, zebrafish has two distinct CXCL8 homologs: Cxcl8-l1 and Cxcl8-l2. Cxcl8-l1 is known to be upregulated under inflammatory conditions caused by bacterial or chemical insult but until now the role of Cxcl8s in neutrophil recruitment has not been studied. In this study we show that both Cxcl8 genes are upregulated in response to an acute inflammatory stimulus, and that both are crucial for normal neutrophil recruitment to the wound and normal resolution of inflammation. Additionally, we have analyzed neutrophil migratory behavior through tissues to the site of injury in vivo, using open-access phagocyte tracking software PhagoSight. Surprisingly, we observed that in the absence of these chemokines, the speed of the neutrophils migrating to the wound was significantly increased in comparison with control neutrophils, although the directionality was not affected. Our analysis suggests that zebrafish may possess a subpopulation of neutrophils whose recruitment to inflamed areas occurs independently of Cxcl8 chemokines. Moreover, we report that Cxcl8-l2 signaled through Cxcr2 for inducing neutrophil recruitment. Our study, therefore, confirms the zebrafish as an excellent in vivo model to shed light on the roles of CXCL8 in neutrophil biology.

OBJECTIVE

To examine the effect of different sources of good manufacturing practice clinical grade adipose-derived mesenchymal stem cells (AD-MSCs) on inflammatory factors in osteoarthritic (OA) chondrocytes and synoviocytes.

METHODS

AD-MSCs from infrapatellar Hoffa fat, subcutaneous (SC) hip fat, and SC abdominal fat were cocultured in Transwells with chondrocytes or synoviocytes. Inflammatory factors (interleukin-1β [IL-1β], tumor necrosis factor α, IL-6, CXCL1/growth-related oncogene α, CXCL8/IL-8, CCL2/monocyte chemotactic protein 1, CCL3/macrophage inflammatory protein 1α, and CCL5/RANTES) were evaluated by quantitative reverse transcription-polymerase chain reaction or multiplex bead-based immunoassay. The role of different immunomodulators was analyzed.

RESULTS

All the inflammatory factors analyzed were down-modulated at the messenger RNA or protein level independently by all 3 AD-MSC sources or by allogeneic AD-MSCs used in coculture with chondrocytes or synoviocytes. Inflammatory factor down-modulation was observed only when AD-MSCs were cocultured with chondrocytes or synoviocytes that produced high levels of inflammatory factors, but no effect was observed in cells that produced low levels of those factors, thus highlighting a dependence of the AD-MSC effect on existing inflammation. The immunomodulators IL-10, IL-1 receptor antagonist, fibroblast growth factor 2, indoleamine 2,3-dioxygenase 1, and galectin 1 were not involved in AD-MSC effects, whereas the cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2 ) pathway exerted a role in the mechanism of antiinflammatory AD-MSC action.

CONCLUSIONS

The antiinflammatory effects of AD-MSCs are probably not dependent on AD-MSC adipose tissue sources and donors but rather on the inflammatory status of OA chondrocytes and synoviocytes. AD-MSCs seem to be able to sense and respond to the local environment. Even though a combination of different molecules may be involved in AD-MSC effects, the COX-2/PGE2 pathway may play a role, suggesting that AD-MSCs may be useful for therapies in osteoarticular diseases.

Foci of chondrocyte hypertrophy that commonly develop in osteoarthritic (OA) cartilage can promote dysregulated matrix repair and pathologic calcification in OA. The closely related chemokines IL-8/CXCL8 and growth-related oncogene alpha (GROalpha)/CXCL1 and their receptors are up-regulated in OA cartilage chondrocytes. Because these chemokines regulate leukocyte activation through p38 mitogen-activated protein kinase signaling, a pathway implicated in chondrocyte hypertrophic differentiation, we tested whether IL-8 and GROalpha promote chondrocyte hypertrophy. We observed that normal human and bovine primary articular chondrocytes expressed both IL-8Rs (CXCR1, CXCR2). IL-8 and the selective CXCR2 ligand GROalpha (10 ng/ml) induced tissue inhibitor of metalloproteinase-3 expression, markers of hypertrophy (type X collagen and MMP-13 expression, alkaline phosphatase activity), as well as matrix calcification. IL-8 and the selective CXCR2 ligand GROalpha also induced increased transamidation activity of chondrocyte transglutaminases (TGs), enzymes up-regulated in chondrocyte hypertrophy that have the potential to modulate differentiation and calcification. Under these conditions, p38 mitogen-activated protein kinase pathway signaling mediated induction of both type X collagen and TG activity. Studies using mouse knee chondrocytes lacking one of the two known articular chondrocyte-expressed TG isoenzymes (TG2) demonstrated that TG2 was essential for murine GROalpha homologue KC-induced TG activity and critically mediated induction by KC of type X collagen, matrix metalloproteinase-13, alkaline phosphatase, and calcification. In conclusion, IL-8 and GROalpha induce articular chondrocyte hypertrophy and calcification through p38 and TG2. Our results suggest a novel linkage between inflammation and altered differentiation of articular chondrocytes. Furthermore, CXCR2 and TG2 may be sites for intervention in the pathogenesis of OA.

We examined the expression of <em>CXCL8</em> (interleukin-8), its receptors, CXCR1 and CXCR2, and vessel density in human melanoma by immunohistochemical analysis of tumors from different Clark levels, depths, and thicknesses. Expression of <em>CXCL8</em> and CXCR2 was lower in Clark level I and II specimens than in level III through V specimens and metastases. CXCR1 expression was observed ubiquitously in the majority of human melanoma tumor specimens irrespective of disease state, with the highest intensity in Clark level III specimens. We observed a significant difference in <em>CXCL8</em> and CXCR2 expression between thin (<or=0.75 mm) and thick (>0.75 mm) melanomas and between thin and metastatic lesions. Positive correlations were observed between Clark level and <em>CXCL8</em> or CXCR2 and between thickness and CXCR2 expression. We found no correlation between vessel density and Clark level or thickness. Our data suggest that expression of <em>CXCL8</em> and CXCR2 contributes to aggressive growth and metastasis in human malignant melanoma. Consistent with the transition from radial to vertical growth phase melanoma, expression of <em>CXCL8</em> and its receptor, CXCR2, may be key in the switch to an aggressive, more metastatic phenotype.

Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in Cftr(F508del) homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation.

Myocardial infarction is associated with an inflammatory response leading to leukocyte recruitment, healing and formation of a scar. Members of the chemokine superfamily are rapidly induced in the infarcted myocardium and may critically regulate the post-infarction inflammatory response. CXCL8/Interleukin (IL)-8 is upregulated in the infarcted area and may induce neutrophil infiltration. In addition, mononuclear cell chemoattractants, such as the CC chemokines CCL2/Monocyte Chemoattractant Protein (MCP)-1, CCL3/Macrophage Inflammatory Protein (MIP)1alpha, and CCL4/MIP-1beta are expressed in the ischemic area, and may regulate monocyte and lymphocyte recruitment. However, chemokines may have additional effects on healing infarcts beyond their leukotactic properties. The CXC chemokine CXCL10/Interferon-y inducible Protein (IP)-10, a potent angiostatic factor with antifibrotic properties, is induced in the infarct and may prevent premature angiogenesis and fibrous tissue deposition, until the infarct is debrided and provisional matrix necessary to support granulation tissue ingrowth is formed. Chemokine induction in the infarct is transient, suggesting that inhibitory mediators (such as transforming growth Factor (TGF)-beta) may be activated suppressing chemokine synthesis and leading to resolution of inflammation and transition to fibrosis. Brief repetitive ischemia in mice also results in chemokine upregulation followed by suppression of chemokine synthesis and interstitial fibrosis, in the absence of myocardial infarction. Chemokine expression may play a role in the pathogenesis of non-infarctive ischemic cardiomyopathy, where early ischemia-induced chemokine expression may be followed by activation of inhibitory mediators that suppress inflammation, but induce fibrosis.

Epidemiologic studies implicate inflammatory stimuli in the development of ovarian cancer. The proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) and both its receptors (TNFRI and TNFRII) are expressed in biopsies of this malignancy. Here, we tested the hypothesis that TNF-alpha is a regulator of the proinflammatory microenvironment of ovarian cancer. A cancer profiling array showed higher expression of TNF-alpha in ovarian tumors compared with normal ovarian tissue, and cultured ovarian cancer cells expressed up to 1,000 times more TNF-alpha mRNA than cultured normal ovarian surface epithelial cells; TNF-alpha protein was only detected in the supernatant of tumor cell cultures. Treatment with TNF-alpha induced TNF-alpha mRNA via TNFRI in both malignant and normal cells with evidence for enhanced TNF-alpha mRNA stability in tumor cells. TNF-alpha induced TNF-alpha protein in an autocrine fashion in tumor but not in normal ovarian surface epithelial cells. The TNF-alpha neutralizing antibody infliximab reduced the constitutive levels of TNF-alpha mRNA in tumor cell lines capable of autocrine TNF-alpha production. Apart from TNF-alpha mRNA expression, several other proinflammatory cytokines were constitutively expressed in malignant and normal ovarian surface epithelial cells, including interleukin (IL)-1alpha, IL-6, CCL2, CXCL8, and M-CSF. TNF-alpha treatment further induced these cytokines with de novo transcription of IL-6 mRNA contrasting with the increased stability of CCL2 mRNA. RNA interference directed against TNF-alpha was highly effective in abolishing constitutive IL-6 production by ovarian tumor cells. In summary, we show that TNF-alpha is differentially regulated in ovarian cancer cells compared with untransformed cells and modulates production of several cytokines that may promote ovarian tumorigenesis. Infliximab treatment may have a role in suppressing the TNF-alpha-driven inflammatory response associated with ovarian cancer.

Interleukin-8 (IL8/CXCL8) is present in decidua and trophoblast, which also expresses the IL8 receptors, CXCR1 and CXCR2. IL8 was shown to stimulate trophoblast migration. Matrix metalloproteinase (MMP)2, MMP9, and integrins alpha(5)beta(1) and alpha(1)beta(1) were found to play important roles in trophoblast invasion. We hypothesized that IL8 would increase this cell migration and invasion by HTR-8/SVneo cells through the activity of MMPs and integrins. Isolated first trimester of pregnancy cytotrophoblast (CT) and HTR-8/SVneo cell line were used. Migration was studied by monolayer wounding test, and invasion by Matrigel invasion test. The effects of IL8 on MMPs and integrin subunit expression were determined in HTR-8/SVneo cells by gelatin zymography and western blot respectively. The results that were obtained showed that exogenous IL8 stimulated HTR-8/SVneo cell migration and invasion. MMP2 and MMP9 levels were stimulated to 182% (P<0.01) and 134% (P<0.01) respectively. Integrin alpha(5) expression was increased to 119% (P<0.05) and integrin beta(1) expression to 173% (P<0.001) of the control values. The data that were obtained show for the first time the sensitivity of the HTR-8/SVneo cells, in addition to isolated first trimester CT, to IL8. Exogenous IL8/CXCL8 increased trophoblast cell migration and invasion, which may be partly attributable to stimulation of MMP2 and MMP9 levels and an increase in integrins. HTR-8/SVneo cell viability and proliferation were also increased.

A central feature of all solid tumor growth is the presence of neovascularization. The CXC chemokines GRO-gamma/CXCL3, ENA-78/CXCL5, and IL-8/CXCL8 have profound angiogenic potential mediated through the CXCR2 receptor. The aim of the present study was to evaluate the expression of the angiogenic chemokines in three human pancreatic cancer cell lines and to determine the role of these proteins in pancreatic cancer angiogenesis. Secreted CXC protein levels in the supernatant of the cell lines were analyzed by ELISA. A rat corneal micropocket model was used to determine the angiogenic potential of these secreted CXC chemokines in vivo. ELISA confirmed expression of all three tested CXC chemokines in the supernatant of two cell lines. In the corneal micropocket assay, neovascularization was induced using pelleted supernatant of all three-cell lines. Using an anti-CXCR2 antibody, neovascularization was significantly inhibited in the high expressing BxPC-3 cell line samples. In addition, the expression of ENA-78/CXCL5 and IL-8/CXCL8 has been evaluated in human pancreatic cancer tissue samples by using immunohistochemistry in order to further investigate the potential role of CXC chemokines in pancreatic cancer angiogenesis and tumorigenesis.

The Duffy Ag expressed on RBCs, capillaries, and postcapillary venular endothelial cells binds selective CXC and CC chemokines with high affinity. Cells transfected with the Duffy Ag internalize but do not degrade chemokine ligand. It has been proposed that Duffy Ag transports chemokines across the endothelium. We hypothesized that Duffy Ag participates in the movement of chemokines across the endothelium and, by doing so, modifies neutrophil transmigration. We found that the Duffy Ag transfected into human endothelial cells facilitates movement of the radiolabeled CXC chemokine, growth related oncogene-alpha/CXC chemokine ligand 1 (GRO-alpha/CXCL1), across an endothelial monolayer. In addition, neutrophil migration toward GRO-alpha/CXCL1 and IL-8 (IL-8/CXCL8) was enhanced across an endothelial monolayer expressing the Duffy Ag. Furthermore, GRO-alpha/CXCL1 stimulation of endothelial cells expressing the Duffy Ag did not affect gene expression by oligonucleotide microarray analysis. These in vitro observations are supported by the finding that IL-8/CXCL8-driven neutrophil recruitment into the lungs was markedly attenuated in transgenic mice lacking the Duffy Ag. We conclude that Duffy Ag has a role in enhancing leukocyte recruitment to sites of inflammation by facilitating movement of chemokines across the endothelium.