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.

CXCR3 ligands were secreted by tissue fibroblasts and peripheral blood-derived mononuclear leukocytes in response to interferon-gamma (IFN-gamma) and Toll-like receptor (TLR) ligands. Subsequent purification and identification revealed the presence of truncated CXCL11 variants missing up to 6 amino acids. In combination with CD26/dipeptidyl peptidase IV, the metalloprotease aminopeptidase N (APN), identical to the myeloid cell marker CD13, rapidly processed CXCL11, but not CXCL8, to generate truncated CXCL11 forms. Truncated CXCL11 had reduced binding, signaling, and chemotactic properties for lymphocytes and CXCR3- or CXCR7-transfected cells. CD13/APN-truncated CXCL11 failed to induce an intracellular calcium increase but was still able to bind and desensitize CXCR3 for intact CXCL11 signaling. CXCL11 efficiently bound to CXCR7, but CXCL11 was not able to induce calcium signaling or ERK1/2 or Akt phosphorylation through CXCR7. CD26-truncated CXCL11 failed to attract lymphocytes but still inhibited microvascular endothelial cell (HMVEC) migration. However, further processing of CXCL11 by CD13 resulted in significant reduction of inhibition of HMVEC migration. Taken together, during inflammation or cancer, CXCL11 processing by CD13 may lead to a reduced number of tumor-infiltrating lymphocytes and in a more angiogenic environment.

Tuberculosis is characterized by granuloma formation and caseous necrosis, but the factors causing tissue destruction are poorly understood. Matrix metalloproteinase (MMP)-9 (92-kDa gelatinase) secretion from monocytes is stimulated by Mycobacterium tuberculosis (M. tb) and associated with local tissue injury in tuberculosis patients. We demonstrate strong immunohistochemical MMP-9 staining in monocytic cells at the center of granuloma and adjacent to caseous necrosis in M. tb-infected patient lymph nodes. Minimal tissue inhibitor of MMPs-1 staining indicated that MMP-9 activity is unopposed. Because granulomas characteristically contain few mycobacteria, we investigated whether monocyte-monocyte cytokine networks amplify MMP-9 secretion. Conditioned medium from M. tb-infected primary human monocytes or THP-1 cells (CoMTB) stimulated MMP-9 gene expression and a >10-fold increase in MMP-9 secretion by monocytes at 3-4 days (p < 0.009, vs controls). Although CoMTB stimulated dose-dependent MMP-9 secretion, MMP-1 (52-kDa collagenase) was not induced. Anti-TNF-alpha Ab but not IL-1R antagonist pretreatment decreased CoMTB-induced MMP-9 secretion by 50% (p = 0.0001). Anti-TNF-alpha Ab also inhibited MMP-9 secretion from monocytic cells by 50%, 24 h after direct M. tb infection (p = 0.0002). Conversely, TNF-alpha directly stimulated dose-dependent MMP-9 secretion. Pertussis toxin inhibited CoMTB-induced MMP-9 secretion and enhanced the inhibitory effect of anti-TNF-alpha Ab (p = 0.05). Although chemokines bind to G protein-linked receptors, CXCL8, CXCL10, CCL2, and CCL5 did not stimulate monocyte MMP-9 secretion. However, the response to cholera toxin confirmed that G protein signaling pathways were intact. In summary, MMP-9 within tuberculous granuloma is associated with tissue destruction, and TNF-alpha, critical for antimycobacterial granuloma formation, is a key autocrine and paracrine regulator of MMP-9 secretion.

The IL-23/IL-17 pathway plays an important role in chronic inflammatory diseases, including inflammatory bowel disease. In inflammatory bowel disease, intestinal epithelial cells are an important source of chemokines that recruit inflammatory cells. We examined the effect of IL-17 on chemokine expression of HT-29 colonic epithelial cells. IL-17 strongly repressed TNF-alpha-stimulated expression of CXCL10, CXCL11, and CCL5, but synergized with TNF-alpha for induction of CXCL8, CXCL1, and CCL20 mRNAs. For CXCL10, IL-17 strongly inhibited promoter activity but had no effect on mRNA stability. In contrast, for CXCL8, IL-17 slightly decreased promoter activity but stabilized its normally unstable mRNA, leading to a net increase in steady-state mRNA abundance. IL-17 synergized with TNF-alpha in transactivating the epidermal growth factor receptor (EGFR) and in activating ERK and p38 MAPK. The p38 and ERK pathway inhibitors SB203580 and U0126 reversed the repressive effect of IL-17 on CXCL10 mRNA abundance and promoter activity and also reversed the inductive effect of IL-17 on CXCL8 mRNA, indicating that MAPK signaling mediates both the transcriptional repression of CXCL10 and the stabilization of CXCL8 mRNA by IL-17. The EGFR kinase inhibitor AG1478 partially reversed the effects of IL-17 on CXCL8 and CXCL10 mRNA, demonstrating a role for EGFR in downstream IL-17 signaling. The overall results indicate a positive effect of IL-17 on chemokines that recruit neutrophils (CXCL8 and CXCL1), and Th17 cells (CCL20). In contrast, IL-17 represses expression of CXCL10, CXCL11, and CCR5, three chemokines that selectively recruit Th1 but not other effector T cells.

Cytokine-induced inflammation is involved in the pathogenesis of type 2 diabetes mellitus (DM). We investigated plasma concentrations and ex vivo production of cytokines and chemokines, and intracellular signalling molecules, mitogen-activated protein kinases (MAPK) in T helper (Th) cells and monocytes in 94 type 2 diabetic patients with or without nephropathy and 20 healthy controls. Plasma concentrations of inflammatory cytokines tumour necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-18 and chemokine CCL2 in patients with diabetic nephropathy (DN) were significantly higher than control subjects, while IL-10, CXCL8, CXCL9, CXCL10 and adiponectin concentrations of DN were significantly higher than patients without diabetic nephropathy (NDN) and control subjects (all P < 0.05). Plasma concentrations of TNF-alpha, IL-6, IL-10, IL-18, CCL2, CXCL8, CXCL9, CXCL10 and adiponectin exhibited significant positive correlation with urine albumin : creatinine ratio in DN patients. The percentage increases of ex vivo production of IL-6, CXCL8, CXCL10, CCL2 and CCL5 upon TNF-alpha activation were significantly higher in both NDN and DN patients than controls (all P < 0.05). The percentage increases in IL-18-induced phosphorylation of extracellular signal-regulated kinase (ERK) in Th cells of NDN and DN were significantly higher than controls (P < 0.05), while the percentage increase in TNF-alpha-induced phosphorylation of p38 MAPK in monocytes and IL-18-induced phosphorylation of p38 MAPK in Th cells and monocytes were significantly higher in NDN patients than controls. These results confirmed that the aberrant production of inflammatory cytokines and chemokines and differential activation of MAPK in different leucocytes are the underlying immunopathological mechanisms of type 2 DM patients with DN.

BACKGROUND

Monocytes and macrophages are key innate immune effector cells that produce cytokines and chemokines upon activation. We and others have shown that 17beta-estradiol (E2) has a direct role in the modulation of monocyte and macrophage immune function. However, relatively little is known about the ability of E2 to regulate isoform expression of estrogen receptors (ERs) in these cells.

RESULTS

In this study, we quantify expression of ERalpha and ERbeta in human monocytes and macrophages. We also show for the first time that the N-terminal truncated ERalpha variant, ERalpha46, is expressed in both cell types. Promoter utilization studies reveal that transcription of ERalpha in both cell types occurs from upstream promoters E and F. Treatment with E2 induces ERalpha expression in macrophages but has no effect on ERbeta levels in either cell type. During monocyte-to-macrophage differentiation, ERalpha is upregulated in a time-dependent manner. Previous studies by our group demonstrated that E2 treatment attenuates production of the chemokine CXCL8 in an ER-dependent manner. We now show that ERalpha expression levels parallel the ability of E2 to suppress CXCL8 production.

CONCLUSIONS

This work demonstrates for the first time that human macrophages predominantly express the truncated ER variant ERalphap46, which is estradiol-inducible. This is mediated through usage of the ERalpha F promoter. Alternative promoter usage may account for tissue and cell type-specific differences in estradiol-induced effects on gene expression. These studies signify the importance of ERalpha expression and regulation in the ability of E2 to modulate innate immune responses.

Human cytomegalovirus (HCMV) is linked to the acceleration of vascular diseases such as atherosclerosis and transplant vasculopathy. One of the hallmarks of these diseases is angiogenesis (AG) and neovessel formation. Endothelial cells (ECs) are an integral part of AG and are sites of HCMV persistence. AG requires multiple synchronous processes that include EC proliferation, migration, and vessel stabilization. Virus-free supernatant (secretome) from HCMV-infected ECs induces AG. To identify factor(s) involved in this process, we performed a human cytokine array. Several cytokines were significantly induced in the HCMV secretomes including interleukin-6 (IL-6), granulocyte macrophage colony-stimulating factor, and IL-8/CXCL8. Using in vitro AG assays, neutralization of IL-6 significantly reduced neovessel formation. Addition of the HCMV secretome to preformed vessels extended neovessel survival, but this effect was blocked by neutralization of IL-6. In these cells, IL-6 prevented apoptosis by blocking caspase-3 and -7 activation through the induction of survivin. Neutralization of IL-6 receptor on ECs abolished the ability of HCMV secretome to increase survivin expression and activated effector caspases. Moreover, survivin shRNA expression induced rapid regression of tubule capillary networks in ECs stimulated with HCMV secretome and activated effector caspases. These observations may explain how CMV accelerates vascular disease despite limited infection in tissues.

Full-skin substitutes, epidermal substitutes, and dermal substitutes are currently being used to heal deep burns and chronic ulcers. In this study, we investigated which wound-healing mediators are released from these constructs and whether keratinocyte-fibroblast interactions are involved. Autologous skin substitutes were constructed from human keratinocytes, fibroblasts, and acellular donor dermis. Full-thickness skin was used to represent an autograft. Secretion of wound-healing mediators was investigated by means of protein array, enzyme-linked immunosorbent assay, neutralizing antibodies, and conditioned culture supernatants. Full-skin substitutes and autografts produce high amounts of inflammatory/angiogenic mediators (IL-6, CCL2, CXCL1, CXCL8, and sST2). Epidermal and dermal substitutes produced less of these proteins. Epidermal-derived proinflammatory cytokines interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) were found to mediate synergistically the secretion of these wound-healing mediators (with the exception of sST2) from fibroblasts in dermal substitutes. The secretion of proinflammatory cytokines (IL-1alpha, TNF-alpha), chemokine/mitogen (CCL5) and angiogenic factor (vascular endothelial growth factor) by epidermal substitutes and tissue remodeling factors (tissue inhibitor of metalloproteinase-2, hepatocyte growth factor) by dermal substitutes was not influenced by keratinocyte-fibroblast interactions. The full-skin substitute has a greater potential to stimulate wound healing than epidermal or dermal substitutes. Both epidermal-derived IL-1alpha and TNF-alpha are required to trigger the release of dermal-derived inflammatory/angiogenic mediators from skin substitutes.

Strong evidence suggests that neutrophils may play an active role in acute and chronic inflammatory disorders, such as rheumatoid arthritis and atherosclerosis. Given the role of pro-inflammatory cytokine TNF-alpha in these inflammatory processes, we planned the present study to investigate the effect of short term incubation with TNF-alpha on neutrophil migration to CCL3, a chemokine produced in inflammatory sites and normally devoid of neutrophil chemotactic properties. We found that TNF-alpha primed neutrophils for migration to CCL3 via CCR5. TNF-alpha-induced migration was a consequence of the TNF-alpha-induced up-regulation of integrin CD11b/CD18 (Mac-1) on neutrophil surface. Furthermore, TNF-alpha activity was found to be strictly dependent on the activation of ERK 1/2 p44, cooperating with the intracellular pathways involving Src kinases, PI3K/Akt, p38 MAPK, well known as activated in response to classical chemoattractants (CXCL8) or priming agents (GM-CSF). On the contrary, the effect of TNF-alpha on neutrophil migration to CCL3 was not dependent on JNK 1/2. In conclusion, the present report shows that TNF-alpha unveils a previously unknown capacity of neutrophils to migrate to CCL3 through the intervention of Mac-1. TNF-alpha regulates Mac-1 up-regulation through signalling pathways, involving various kinases, but not JNK 1/2. Although highly speculative, ERK 1/2 p44 may represent a selective target for the pharmacologic manipulation of neutrophil-mediated adverse activities in TNF-alpha-mediated inflammatory states.

Field cancerization involves the lateral spread of premalignant or malignant disease and contributes to the recurrence of head and neck tumors. The overall hypothesis underlying this work is that endothelial cells actively participate in tumor cell invasion by secreting chemokines and creating a chemotactic gradient for tumor cells. Here we demonstrate that conditioned medium from head and neck tumor cells enhance Bcl-2 expression in neovascular endothelial cells. Oral squamous cell carcinoma-3 (OSCC3) and Kaposi's sarcoma (SLK) show enhanced invasiveness when cocultured with pools of human dermal microvascular endothelial cells stably expressing Bcl-2 (HDMEC-Bcl-2), compared to cocultures with empty vector controls (HDMEC-LXSN). Xenografted OSCC3 tumors vascularized with HDMEC-Bcl-2 presented higher local invasion than OSCC3 tumors vascularized with control HDMEC-LXSN. CXCL1 and CXCL8 were upregulated in primary endothelial cells exposed to vascular endothelial growth factor (VEGF), as well as in HDMEC-Bcl-2. Notably, blockade of CXCR2 signaling, but not CXCR1, inhibited OSCC3 and SLK invasion toward endothelial cells. These data demonstrate that CXC chemokines secreted by endothelial cells induce tumor cell invasion and suggest that the process of lateral spread of tumor cells observed in field cancerization is guided by chemotactic signals that originated from endothelial cells.

Chemokine expression is markedly upregulated in healing myocardial infarcts and may play an important role in regulating leukocyte infiltration and activity and in modulating infarct angiogenesis as well as fibrous tissue deposition. The CC chemokine monocyte chemoattractant protein-1/CCL2 has important effects in infarct healing. Monocyte chemoattractant protein-1 -/- mice exhibit reduced macrophage infiltration and activation, suppressed cytokine synthesis, delayed phagocytotic removal of dead cardiomyocytes, diminished myofibroblast accumulation, and decreased ventricular remodeling after myocardial infarction. Monocyte chemoattractant protein-1 may also play an important role in the development of interstitial fibrosis in ischemic noninfarctive cardiomyopathy. CXC chemokines are also induced in healing infarcts. Interleukin-8/CXCL8 may mediate neutrophil recruitment and activation and may promote neovessel formation, whereas induction of the angiostatic and antifibrotic chemokine interferon-gamma-inducible protein-10/CXCL10 may serve to prevent premature wound angiogenesis and fibrous tissue deposition in the infarct, until the injured myocardium has been cleared from dead cells and debris and a fibrin-rich provisional matrix is formed. Understanding of the role of chemokines in myocardial ischemia may result in novel strategies in the treatment of patients with ischemic heart disease.

Human mast cells are found in skin and mucosal surfaces and next to blood vessels. They play a sentinel cell role in immunity, recognizing invading pathogens and producing proinflammatory mediators. Mast cells can recruit granulocytes, and monocytes in allergic disease and bacterial infection, but their ability to recruit antiviral effector cells such as natural killer (NK) cells and T cells has not been fully elucidated. To investigate the role of human mast cells in response to virus-associated stimuli, human cord blood-derived mast cells (CBMCs) were stimulated with polyinosinic.polycytidylic acid, a double-stranded RNA analog, or infected with the double-stranded RNA virus, reovirus serotype 3 Dearing for 24 hours. CBMCs responded to stimulation with polyinosinic.polycytidylic acid by producing a distinct chemokine profile, including CCL4, CXCL8, and CXCL10. CBMCs produced significant amounts of CXCL8 in response to low levels of reovirus infection, while both skin- and lung-derived fibroblasts were unresponsive unless higher doses of reovirus were used. Supernatants from CBMCs infected with reovirus induced substantial NK cell chemotaxis that was highly dependent on CXCL8 and CXCR1. These results suggest a novel role for mast cells in the recruitment of human NK cells to sites of early viral infection via CXCL8.

BACKGROUND

Obesity is a significant risk factor for psoriasis and body mass index (BMI) correlates with disease severity. Objectives To investigate the relationship between obesity and psoriasis, focusing on the role of adipokines such as leptin and resistin.

METHODS

Patients with psoriasis (n = 30) were recruited and their BMI, waist circumference and disease severity [Psoriasis Area and Severity Index (PASI)] were recorded. Fasting serum samples were obtained on enrolment and after a course of ultraviolet (UV) B treatment. Age-, sex- and BMI-matched healthy controls were also recruited.

RESULTS

On enrolment, serum leptin and soluble leptin receptor levels were not raised compared with the controls. However, resistin, interleukin (IL)-1beta, IL-6, and chemokines CCL2, CXCL8 and CXCL9 were all significantly elevated in the patient group and serum resistin correlated with disease severity (r = 0.372, P = 0.043). Improvement after UVB treatment was accompanied by decreased serum CXCL8. In vitro, both leptin and resistin could induce CXCL8 and tumour necrosis factor-alpha production by blood monocytes, and leptin could additionally induce IL-1beta and IL-1 receptor antagonist production. Leptin also dose dependently increased secretion of the growth factor amphiregulin by ex vivo-cultured lesional psoriasis skin.

CONCLUSIONS

These data support the view that leptin and resistin may be involved in the pathogenesis of psoriasis in overweight individuals, possibly by augmenting the cytokine expression by the inflammatory infiltrate.

c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) family and controls essential processes such as inflammation, cell differentiation, and apoptosis. JNK signalling is triggered by extracellular signals such as cytokines and environmental stresses. Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory cytokine with chemokine-like functions in leukocyte recruitment and atherosclerosis. MIF promotes MAPK signalling through ERK1/2, while it can either activate or inhibit JNK phosphorylation, depending on the cell type and underlying stimulation context. MIF activities are mediated by non-cognate interactions with the CXC chemokine receptors CXCR2 and CXCR4 or by ligation of CD74, which is the cell surface expressed form of the class II invariant chain. ERK1/2 signalling stimulated by MIF is dependent on CD74, but the receptor pathway involved in MIF activation of the JNK pathway is unknown. Here we comprehensively characterize the stimulatory effect of MIF on the canonical JNK/c-Jun/AP-1 pathway in fibroblasts and T cell lines and identify the upstream signalling components. Physiological concentrations of recombinant MIF triggered the phosphorylation of JNK and c-Jun and rapidly activated AP-1. In T cells, MIF-mediated activation of the JNK pathway led to upregulated gene expression of the inflammatory chemokine CXCL8. Activation of JNK signalling by MIF involved the upstream kinases PI3K and SRC and was found to be dependent on CXCR4 and CD74. Together, these data show that the CXCR4/CD74/SRC/PI3K axis mediates a rapid and transient activation of the JNK pathway as triggered by the inflammatory cytokine MIF in T cells and fibroblasts.

OBJECTIVE

High expression of galectin 3 at sites of joint destruction in rheumatoid arthritis (RA) suggests that galectin 3 plays a role in RA pathogenesis. Previous studies have demonstrated the effects of galectins on immune cells, such as lymphocytes and macrophages. This study was undertaken to investigate the hypothesis that galectin 3 induces proinflammatory effects in RA by modulating the pattern of cytokine and chemokine production in synovial fibroblasts.

METHODS

Matched samples of RA synovial and skin fibroblasts were pretreated with galectin 3 or tumor necrosis factor alpha (TNFalpha), and the levels of a panel of cytokines, chemokines, and matrix metalloproteinases (MMPs) were determined using enzyme-linked immunosorbent assays and multiplex assays. Specific inhibitors were used to dissect signaling pathways, which were confirmed by Western blotting and NF-kappaB activation assay.

RESULTS

Galectin 3 induced secretion of interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor, CXCL8, and MMP-3 in both synovial and skin fibroblasts. By contrast, galectin 3-induced secretion of TNFalpha, CCL2, CCL3, and CCL5 was significantly greater in synovial fibroblasts than in skin fibroblasts. TNFalpha blockade ruled out autocrine TNFalpha-stimulated induction of chemokines. The MAPKs p38, JNK, and ERK were necessary for IL-6 production, but phosphatidylinositol 3-kinase (PI 3-kinase) was required for selective CCL5 induction. NF-kappaB activation was required for production of both IL-6 and CCL5.

CONCLUSIONS

Our findings indicate that galectin 3 promotes proinflammatory cytokine secretion by tissue fibroblasts. However, galectin 3 induces the production of mononuclear cell-recruiting chemokines uniquely from synovial fibroblasts, but not matched skin fibroblasts, via a PI 3-kinase signaling pathway. These data provide further evidence of the role of synovial fibroblasts in regulating the pattern and persistence of the inflammatory infiltrate in RA and suggest a new and important functional consequence of the observed high expression of galectin 3 in the rheumatoid synovium.

Elevated tumor cyclooxygenase (COX)-2 activity plays a multifaceted role in non-small cell lung cancer (NSCLC). To elucidate the role of COX-2 in the in vitro and in vivo expression of two known NSCLC angiogenic peptides, CXC ligand (CXCL) 8 and CXCL5, we studied two COX-2 gene-modified NSCLC cell lines, A549 and H157. COX-2 overexpression enhanced the in vitro expression of both CXCL8 and CXCL5. In contrast, specific COX-2 inhibition decreased the production of both peptides as well as nuclear translocation of nuclear factor kappaB. In a severe combined immunodeficient mouse model of human NSCLC, the enhanced tumor growth of COX-2-overexpressing tumors was inhibited by neutralizing anti-CXCL5 and anti-CXCL8 antisera. We conclude that COX-2 contributes to the progression of NSCLC tumorigenesis by enhancing the expression of angiogenic chemokines CXCL8 and CXCL5.

OBJECTIVE

Chemokines initiate the immune response by controlling leukocyte migration and lymphocyte development. Macrophage infiltration of the decidua has been implicated in the genesis of recurrent miscarriage and preeclampsia. Therefore, we determined whether cultured human decidual cells produce monocyte/macrophage-recruiting chemokines in response to a potent pro-inflammatory cytokine, interleukin-1beta (IL-1beta), and whether decidual cell-conditioned medium contains monocyte- and macrophage-chemoattractant activity.

METHODS

Leukocyte-free first trimester decidual cells were treated for 6h with estradiol (E(2)) and medroxyprogesterone acetate (MPA) to mimic the steroidal milieu of pregnancy, or E(2) and MPA and IL-1beta (1 ng/ml) to mimic inflamed decidua. Total RNA was used for cDNA synthesis. Biotinylated cRNAs were generated and chemically fragmented for hybridization on Affymetrix HG_U133 Plus 2.0 chips followed by fluorescence labeling and optical scanning. Raw data generated from Affymetrix GCOS 1.2 (GeneChip Operating Software) were analyzed by GeneSpring 7.2 software. Subsequently microarray results were validated by real time RT-PCR and Western blotting. A functional study of monocyte migration was carried out also using conditioned media from culture.

RESULTS

Five chemokines responsible for monocyte/macrophage chemoattraction and activation, including C-C motif ligand 2 (CCL2), CCL5, C-X-C motif ligand 2 (CXCL2), CXCL3 and CXCL8, were markedly elevated from 29- to 975-fold after exposure to IL-1beta in cultured first trimester decidual cells. The results of real-time RT-PCR (up-regulation from 43- to 3069-fold) and Western blotting (up-regulation from 15- to 300-fold) confirmed the microarray findings. Monocyte migration was significantly induced by the conditioned medium from IL-1beta-treated decidual cells.

CONCLUSIONS

Treatment of first trimester decidual cells with IL-1beta induces secretion of monocyte/macrophage recruiting-chemokines and promotes monocyte migration. Extrapolation of these in vitro results to the milieu of implantation site suggests a mechanism whereby IL-1beta could mediate excessive macrophage infiltration of the decidua.

Lung epithelial cells are the primary cellular targets for respiratory virus pathogens such as influenza and parainfluenza viruses. Here, we have analyzed influenza A, influenza B and Sendai virus-induced chemokine response in human A549 lung epithelial cells. Influenza virus infection resulted in low CCL2/MCP-1, CCL5/RANTES, CXCL8/IL-8 and CXCL10/IP-10 production at late times of infection. However, when cells were pretreated with TNF-alpha or IFN-alpha, influenza-A-virus-induced chemokine production was greatly enhanced. Cytokine pretreatment resulted in enhanced expression of RIG-I, IKKepsilon, interferon regulatory factor (IRF)1, IRF7 and p50 proteins. Most importantly, influenza-A-virus-induced DNA binding of IRF1, IRF3, IRF7 and NF-kappaB onto CXCL10 ISRE and NF-kappaB elements, respectively, was markedly enhanced in cytokine-pretreated cells. Our results suggest that IFN-alpha and TNF-alpha have a significant role in priming epithelial cells for higher cytokine and chemokine production in influenza A virus infection.

BACKGROUND

Breast cancer progression is promoted by stromal cells that populate the tumors, including cancer-associated fibroblasts (CAFs) and mesenchymal stem/stromal cells (MSCs). The activities of CAFs and MSCs in breast cancer are integrated within an intimate inflammatory tumor microenvironment (TME) that includes high levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β). Here, we identified the impact of TNF-α and IL-1β on the inflammatory phenotype of CAFs and MSCs by determining the expression of inflammatory chemokines that are well-characterized as pro-tumorigenic in breast cancer: CCL2 (MCP-1), CXCL8 (IL-8) and CCL5 (RANTES).

METHODS

Chemokine expression was determined in breast cancer patient-derived CAFs by ELISA and in patient biopsies by immunohistochemistry. Chemokine levels were determined by ELISA in (1) human bone marrow-derived MSCs stimulated by tumor conditioned media (Tumor CM) of breast tumor cells (MDA-MB-231 and MCF-7) at the end of MSC-to-CAF-conversion process; (2) Tumor CM-derived CAFs, patient CAFs and MSCs stimulated by TNF-α (and IL-1β). The roles of AP-1 and NF-κB in chemokine secretion were analyzed by Western blotting and by siRNAs to c-Jun and p65, respectively. Migration of monocytic cells was determined in modified Boyden chambers.

RESULTS

TNF-α (and IL-1β) induced the release of CCL2, CXCL8 and CCL5 by MSCs and CAFs generated by prolonged stimulation of MSCs with Tumor CM of MDA-MB-231 and MCF-7 cells. Patient-derived CAFs expressed CCL2 and CXCL8, and secreted CCL5 following TNF-α (and IL-1β) stimulation. CCL2 was expressed in CAFs residing in proximity to breast tumor cells in biopsies of patients diagnosed with invasive ductal carcinoma. CCL2 release by TNF-α-stimulated MSCs was mediated by TNF-RI and TNF-RII, through the NF-κB but not via the AP-1 pathway. Exposure of MSCs to TNF-α led to potent CCL2-induced migration of monocytic cells, a process that may yield pro-cancerous myeloid infiltrates in breast tumors.

CONCLUSIONS

Our novel results emphasize the important roles of inflammation-stroma interactions in breast cancer, and suggest that NF-κB may be a potential target for inhibition in tumor-adjacent stromal cells, enabling improved tumor control in inflammation-driven malignancies.

The expression of chemokine receptors and chemokine production by adult human non-transformed astrocytes, grade III astrocytoma and grade IV glioblastoma tumour cell lines were determined. Here, we show an increased expression of CXCR3 and CXCR4, and a decreased expression of CXCR1 and CCR4 by glioma cells compared to adult human astrocytes. Glioma cells showed increased production of CXCL10, whereas production of other chemokines was decreased (CXCL8, CCL2, CCL5, and CCL22). CXCL10 induced an ERK1/2-dependent increase in [(3)H] thymidine uptake. These results suggest that expression of chemokine receptor/ligand pairs such as CXCR3/CXCL10 have an important role in the proliferation of glioma cells.

BACKGROUND

Ulcerative colitis is characterized by relapsing mucosal inflammation where the lesions include tissue-damaging granulocytes. In addition, T cells and natural killer (NK) cells play important pathophysiologic roles. Chemokines are a large family of peptides that play key roles in the regulation of inflammation. The CXC-chemokines, growth-related oncogene (GRO)-alpha/CXCL1 and interleukin (IL)-8/CXCL8, both recruit neutrophils and possess mitogenic properties, whereas the interferon-dependent CXC-chemokines monokine induced by gamma-interferon (MIG)/CXCL9, interferon-gamma inducible protein of 10 kD/CXCL10, and IFN-inducible T cell alpha chemoattractant/CXCL11 recruit and activate T cells and NK cells.

METHODS

The expression of CXC-chemokines was studied in eight controls and in 11 patients suffering from ulcerative colitis in the distal part of the colon, before and during topical treatment with corticosteroids. Perfusates (obtained before, after 7 days, and after 28 days of treatment) and pinch biopsies (obtained before and after 28 days of treatment) were collected by colonoscopy. The rectal release of GRO-alpha and MIG was determined by enzyme-linked immunosorbent assay (ELISA), and tissue expression of the chemokines was detected in colonic tissue by immunohistochemistry.

RESULTS

In perfusates, high levels of GRO-alpha, IL-8, and MIG were detected compared with controls (p=0.02, 0.005, and p=0.03, respectively). During treatment with corticosteroids, both GRO-alpha and MIG decreased. In clinical nonresponders, characterized by sustained inflammation, the levels of GRO-alpha and MIG remained elevated. Both epithelial cells and granulocytes, present in the submucosa, expressed GRO-alpha and MIG as detected by immunohistochemistry.

CONCLUSIONS

CXC-chemokines are likely to be important in the pathophysiology of ulcerative colitis and may become targets for novel treatment strategies. In addition, GRO-alpha may serve as a marker of disease activity.

BACKGROUND

Rhinovirus infections are frequent causes of asthma exacerbations.

OBJECTIVE

This study was conducted to test whether subjects with and without allergic asthma have different responses to infection and to identify baseline patient risk factors that predict cold outcomes.

METHODS

Twenty subjects with mild persistent allergic asthma and 18 healthy subjects were experimentally inoculated with rhinovirus-16. Subjects were evaluated at baseline, during the acute infection, and during recovery for asthma and cold symptoms by using a validated questionnaire. Sputum and nasal lavage fluid were evaluated for viral shedding, cytokines, and cellular inflammation.

RESULTS

There were no group-specific significant differences in peak cold symptom scores (10.0 +/- 5.8 vs 11.1 +/- 6.2, asthmatic vs healthy subjects), peak nasal viral titers (log(10) 4.3 +/- 0.8 vs 3.7 +/- 1.4 50% tissue culture infective dose/mL, respectively), or changes in peak flow during the study (10% +/- 10% vs 8% +/- 6%, respectively). Rhinovirus-16 infection increased peak asthma index values in the asthmatic group (median, 6 ->> 13; P = .003) but only marginally in the healthy group (median, 4 ->> 7; P = .09). More asthmatic subjects had detectable eosinophils in nasal lavage and sputum samples at baseline and during infection, but otherwise, cellular and cytokine responses were similar. Baseline sputum eosinophilia and CXCL8 (IL-8) levels were positively associated with cold symptoms, whereas CCL2 (monocyte chemotactic protein 1) levels were inversely associated with nasal viral shedding.

CONCLUSIONS

These findings suggest that subjects with mild allergic asthma and healthy subjects have similar cold symptoms and inflammatory and antiviral responses. In addition, eosinophilia and other selective baseline measures of airway inflammation in subjects with or without asthma might predict respiratory outcomes with rhinovirus infection.

BACKGROUND

The Duffy antigen receptor for chemokines (DARC) is known to be a promiscuous chemokine receptor that binds a variety of CXC and CC chemokines in the absence of any detectable signal transduction events. Within the CXC group of chemokines, DARC binds the angiogenic CXC chemokines including IL-8 (CXCL8), GROalpha (CXCL1) and ENA-78 (CXCL5), all of which have previously been shown to be important in non-small cell lung carcinoma (NSCLC) tumor growth. We hypothesized that overexpression of DARC by a NSCLC tumor cell line would result in the binding of the angiogenic ELR+ CXC chemokines by the tumor cells themselves, and thus interfere with the stimulation of endothelial cells and induction of angiogenesis by the tumor cell-derived angiogenic chemokines.

RESULTS

NSCLC tumor cells that constitutively expressed DARC were generated and their growth characteristics were compared to control transfected cells in vitro and in vivo in SCID animals. We found that tumors derived from DARC-expressing cells were significantly larger in size than tumors derived from control-transfected cells. However, upon histological examination we found that DARC-expressing tumors had significantly more necrosis and decreased tumor cellularity, as compared to control tumors. Expression of DARC by NSCLC cells was also associated with a decrease in tumor-associated vasculature and a reduction in metastatic potential.

CONCLUSIONS

The expression of DARC in the context of NSCLC tumors may act as a chemokine decoy receptor and interferes with normal tumor growth and chemokine-induced tumor neovascularization.

Macroautophagy, a catabolic process of cellular self-digestion, is an important tumor cell survival mechanism and a potential target in antineoplastic therapies. Recent discoveries have implicated autophagy in the cellular secretory process, but potential roles of autophagy-mediated secretion in modifying the tumor microenvironment are poorly understood. Furthermore, efforts to inhibit autophagy in clinical trials have been hampered by suboptimal methods to quantitatively measure tumor autophagy levels. Here, we leveraged the autophagy-based involvement in cellular secretion to identify shed proteins associated with autophagy levels in melanoma. The secretome of low-autophagy WM793 melanoma cells was compared to its highly autophagic metastatic derivative, 1205Lu in physiological 3-dimensional cell culture using quantitative proteomics. These comparisons identified candidate autophagy biomarkers IL1B (interleukin 1, β), CXCL8 (chemokine (C-X-C motif) ligand 8), LIF (leukemia inhibitory factor), FAM3C (family with sequence similarity 3, member C), and DKK3 (dickkopf WNT signaling pathway inhibitor 3) with known roles in inflammation and tumorigenesis, and these proteins were subsequently shown to be elevated in supernatants of an independent panel of high-autophagy melanoma cell lines. Secretion levels of these proteins increased when low-autophagy melanoma cells were treated with the autophagy-inducing tat-BECN1 (Beclin 1) peptide and decreased when ATG7 (autophagy-related 7) was silenced in high-autophagy cells, thereby supporting a mechanistic link between these secreted proteins and autophagy. In addition, serum from metastatic melanoma patients with high tumor autophagy levels exhibited higher levels of these proteins than serum from patients with low-autophagy tumors. These results suggest that autophagy-related secretion affects the tumor microenvironment and measurement of autophagy-associated secreted proteins in plasma and possibly in tumors can serve as surrogates for intracellular autophagy dynamics in tumor cells.