Aberrant TGFbeta signaling is common in human cancers and contributes to tumor metastasis. Here, we demonstrate that Gr-1+CD11b+ myeloid cells are recruited into mammary carcinomas with type II TGF beta receptor gene (Tgfbr2) deletion and directly promote tumor metastasis. Gr-1+CD11b+ cells infiltrate into the invasive front of tumor tissues and facilitate tumor cell invasion and metastasis through a process involving metalloproteinase activity. This infiltration of Gr-1+CD11b+ cells also results in increased abundance of TGF beta 1 in tumors with Tgfbr2 deletion. The recruitment of Gr-1+CD11b+ cells into tumors with Tgfbr2 deletion involves two chemokine receptor axes, the SDF-1/CXCR4 and CXCL5/CXCR2 axes. Together, these data indicate that Gr-1+CD11b+ cells contribute to TGFbeta-mediated metastasis through enhancing tumor cell invasion and metastasis.

IL-22 belongs to a family of cytokines structurally related to IL-10, including IL-19, IL-20, IL-24, and IL-26. In contrast to IL-10, IL-22 has proinflammatory activities. IL-22 signals through a class II cytokine receptor composed of an IL-22-binding chain, IL-22RA1, and the IL-10RB subunit, which is shared with the IL-10R. In the present study, we show that short-term cultured human epidermal keratinocytes express a functional IL-22R but no IL-10R. Accordingly, IL-22 but not IL-10 induces STAT3 activation in keratinocytes. Using a cDNA array screening approach, real-time RT-PCR, and Western blot analysis, we demonstrate that IL-22 up-regulates, in a dose-dependent manner, the expression of S100A7, S100A8, S100A9, a group of proinflammatory molecules belonging to the S100 family of calcium-binding proteins, as well as the matrix metalloproteinase 3, the platelet-derived growth factor A, and the CXCL5 chemokine. In addition, IL-22 induces keratinocyte migration in an in vitro injury model and down-regulates the expression of at least seven genes associated with keratinocyte differentiation. Finally, we show that IL-22 strongly induces hyperplasia of reconstituted human epidermis. Taken together, these results suggest that IL-22 plays an important role in skin inflammatory processes and wound healing.

Tumor-derived exosomes containing the tetraspanin Tspan8 can efficiently induce angiogenesis in tumors and tumor-free tissues. However, little information exists on exosome-endothelial cell (EC) interactions or the proangiogenic role of tetraspanins, which are a constitutive component of exosomes. In this study, we used a rat adenocarcinoma model (AS-Tspan8) to explore the effects of exosomal Tspan8 on angiogenesis. Tspan8 contributed to a selective recruitment of proteins and mRNA into exosomes, including CD106 and CD49d, which were implicated in exosome-EC binding and EC internalization. We found that EC internalized Tspan8-CD49d complex-containing exosomes. Exosome uptake induced vascular endothelial growth factor (VEGF)-independent regulation of several angiogenesis-related genes, including von Willebrand factor, Tspan8, chemokines CXCL5 and MIF, chemokine receptor CCR1, and, together with VEGF, VEGF receptor 2. EC uptake of Tspan8-CD49d complex-containing exosomes was accompanied by enhanced EC proliferation, migration, sprouting, and maturation of EC progenitors. Unraveling these new pathways of exosome-initiated EC regulation could provide new options for therapeutic interference with tumor-induced angiogenesis.

In this study, we examined the biological action of IL-17 on human non-small cell lung cancer (NSCLC). Although IL-17 had no direct effect on the in vitro growth rate of NSCLC, IL-17 selectively augmented the secretion of an array of angiogenic CXC chemokines, including CXCL1, CXCL5, CXCL6, and CXCL8 but not angiostatic chemokines, by three different NSCLC lines. Endothelial cell chemotactic activity (as a measure of net angiogenic potential) was increased in response to conditioned medium from NSCLC stimulated with IL-17 compared with those from unstimulated NSCLC. Enhanced chemotactic activity was suppressed by neutralizing mAb(s) to CXCL1, CXCL5, and CXCL8 or to CXCR-2 but not to vascular endothelial growth factor-A. Transfection with IL-17 into NSCLC had no effect on the in vitro growth, whereas IL-17 transfectants grew more rapidly compared with controls when transplanted in SCID mice. This IL-17-elicited enhancement of NSCLC growth was associated with increased tumor vascularity. Moreover, treatment with anti-mouse CXCR-2-neutralizing Ab significantly attenuated the growth of both neomycin phosphotransferase gene-transfected and IL-17-transfected NSCLC tumors in SCID mice. A potential role for IL-17 in modulation of the human NSCLC phenotype was supported by the findings that, in primary NSCLC tissues, IL-17 expression was frequently detected in accumulating and infiltrating inflammatory cells and that high levels of IL-17 expression were associated with increased tumor vascularity. These results demonstrate that IL-17 increases the net angiogenic activity and in vivo growth of NSCLC via promoting CXCR-2-dependent angiogenesis and suggest that targeting CXCR-2 signaling may be a novel promising strategy to treat patients with NSCLC.

In order to metastasize, cancer cells need to acquire a motile phenotype. Previously, development of this phenotype was thought to rely on the acquisition of selected, random mutations and thus would occur late in cancer progression. However, recent studies show that cancer cells disseminate early, implying the existence of a different, faster route to the metastatic motile phenotype. Using a spontaneous murine model of melanoma, we show that a subset of bone marrow-derived immune cells (myeloid-derived suppressor cells or MDSC) preferentially infiltrates the primary tumor and actively promotes cancer cell dissemination by inducing epithelial-mesenchymal transition (EMT). CXCL5 is the main chemokine attracting MDSC to the primary tumor. In vitro assay using purified MDSC showed that TGF-β, EGF, and HGF signaling pathways are all used by MDSC to induce EMT in cancer cells. These findings explain how cancer cells acquire a motile phenotype so early and provide a mechanistic explanation for the long recognized link between inflammation and cancer progression.

Neutrophils can either promote or inhibit tumor progression, depending on the tumor microenvironment, via release of cytokines. Neither the factors produced by tumor-associated neutrophils (TANs) nor their effects on tumor progression have been characterized. We investigated the roles of TANs in progression of hepatocellular carcinoma (HCC) using cell lines and immune cells isolated from patients.

We performed studies with HepG2, PLC/PRF/5, MHCC97H, and HCCLM3 human and Hepa1-6 and H22 mouse HCC cell lines; expression of chemokines and cytokines were knocked down with small hairpin RNAs. Cells were analyzed in chemotaxis assays and as growth as tumors in mice. HCC tissues and peripheral blood were collected from 20 patients undergoing curative resection or 20 healthy individuals (controls) in 2012 at Zhongshan Hospital in China. TANs and peripheral blood neutrophils (PBNs) were isolated and exposed to conditioned media from HCC cell lines; reverse-transcription polymerase chain reaction was used to quantify the expression of cytokines and chemokines. We collected neutrophils from another 60 patients undergoing curative resection for HCC in 2012 to measure the production of C-C motif chemokine ligand 2(CCL2) and CCL17. Patients were followed up until March 15, 2014. For immunohistochemical analyses, we collected HCC tissues and paired, adjacent, nontumor cirrhotic liver tissues from 832 HCC patients undergoing curative resection from 2006 through 2008. All patients were followed up until March 15, 2013. To study the effects of sorafenib, we collected clinical and pathology data from 46 patients who underwent curative resection in 2010.

CCL2 and CCL17 were the cytokines most highly expressed by TANs and HCC cell-activated PBNs. Levels of CCL2 and CCL17 messenger RNAs and proteins were significantly higher in TANs than in PBNs, and increased in patients with HCC recurrence. CCL2 and CCL17 messenger RNA and proteins also increased when PBNs were exposed to conditioned media from HCC cell lines. Immunohistochemical analysis of a tissue microarray showed that CCL2+ and CCL17+ cells, which also expressed the neutrophil marker CD66b, were distributed throughout the HCC stroma, but not in tumor cells or the adjacent nontumor liver cells. The number of CCL2+ or CCL17+ TANs correlated with tumor size, microvascular invasion, tumor encapsulation, tumor differentiation, and stage. Patients whose tumors had lower levels of CCL2+ or CCL17+ cells had longer survival times than those with higher numbers of these cells. TAN-conditioned media, as well as recombinant CCL2 and CCL17, increased the migratory activity of the macrophages and T-regulatory (Treg) cells from patients or mice with HCC to a greater extent that PBN-conditioned media. Neutralizing antibodies against CCL2 and CCL17, or their receptors C-C chemokine receptor 2 and C-C chemokine receptor 4, reduced the migratory activities of macrophage and Treg cells. HCC cell lines injected into mice formed larger tumors when they were co-injected with TANs and formed more pulmonary metastases; these tumors were infiltrated by Ly6G+ cells, F4/80+ macrophages, and Foxp3+ Treg cells. In a phosphokinase array of human PBNs, levels of phosphorylated AKT and P38 increased after exposure to conditioned media from all 4 HCC cell types. Pharmacologic inhibitors of AKT and P38 inhibited secretion of CCL2 and CCL17 by these PBNs. In tumor-bearing mice, sorafenib increased the numbers of TANs and levels of CCL2 and CCL17 in tumors. HCC tissues from patients who received sorafenib before surgery contained more TANs than tissues from patients who did not receive sorafenib. In knockdown cells, HCC cell-derived CXCL5 was the strongest effector of neutrophil migration under hypoxic conditions. In mice, the combination of sorafenib and TAN depletion inhibited tumor growth and neovascularization to a greater extent than sorafenib alone.

TANs recruit macrophages and Treg cells to HCCs to promote their growth, progression, and resistance to sorafenib.

IL-17A and IL-17F are related homodimeric proteins of the IL-17 family produced by Th17 cells. In this study, we show that mouse Th17 cells also produce an IL-17F/A heterodimeric protein. Whereas naive CD4(+) T cells differentiating toward the Th17 cell lineage expressed IL-17F/A in higher amounts than IL-17A/A homodimer and in lower amounts than IL-17F/F homodimer, differentiated Th17 cells expressed IL-17F/A in higher amounts than either homodimer. In vitro, IL-17F/A was more potent than IL-17F/F and less potent than IL-17A/A in regulating CXCL1 expression. Neutralization of IL-17F/A with an IL-17A-specific Ab, and not with an IL-17F-specific Ab, reduced the majority of IL-17F/A-induced CXCL1 expression. To study these cytokines in vivo, we established a Th17 cell adoptive transfer model characterized by increased neutrophilia in the airways. An IL-17A-specific Ab completely prevented Th17 cell-induced neutrophilia and CXCL5 expression, whereas Abs specific for IL-17F or IL-22, a cytokine also produced by Th17 cells, had no effects. Direct administration of mouse IL-17A/A or IL-17F/A, and not IL-17F/F or IL-22, into the airways significantly increased neutrophil and chemokine expression. Taken together, our data elucidate the regulation of IL-17F/A heterodimer expression by Th17 cells and demonstrate an in vivo function for this cytokine in airway neutrophilia.

Oncolytic viruses (OVs) are selected or designed to eliminate malignancies by direct infection and lysis of cancer cells. In contrast to this concept of direct tumor lysis by viral infection, we observed that a significant portion of the in vivo tumor killing activity of two OVs, vesicular stomatitis virus (VSV) and vaccinia virus is caused by indirect killing of uninfected tumor cells. Shortly after administering the oncolytic virus we observed limited virus infection, coincident with a loss of blood flow to the interior of the tumor that correlated with induction of apoptosis in tumor cells. Transcript profiling of tumors showed that virus infection resulted in a dramatic transcriptional activation of pro-inflammatory genes including the neutrophil chemoattractants CXCL1 and CXCL5. Immunohistochemical examination of infected tumors revealed infiltration by neutrophils correlating with chemokine induction. Depletion of neutrophils in animals prior to VSV administration eliminated uninfected tumor cell apoptosis and permitted more extensive replication and spreading of the virus throughout the tumor. Taken all together, these results indicate that targeted recruitment of neutrophils to infected tumor beds enhances the killing of malignant cells. We propose that activation of inflammatory cells can be used for enhancing the effectiveness of oncolytic virus therapeutics, and that this approach should influence the planning of therapeutic doses.

Cancer metastases arise following extravasation of circulating tumor cells with certain tumors exhibiting high organ specificity. Here, we developed a 3D microfluidic model to analyze the specificity of human breast cancer metastases to bone, recreating a vascularized osteo-cell conditioned microenvironment with human osteo-differentiated bone marrow-derived mesenchymal stem cells and endothelial cells. The tri-culture system allowed us to study the transendothelial migration of highly metastatic breast cancer cells and to monitor their behavior within the bone-like matrix. Extravasation, quantified 24 h after cancer cell injection, was significantly higher in the osteo-cell conditioned microenvironment compared to collagen gel-only matrices (77.5 ± 3.7% vs. 37.6 ± 7.3%), and the migration distance was also significantly greater (50.8 ± 6.2 μm vs. 31.8 ± 5.0 μm). Extravasated cells proliferated to form micrometastases of various sizes containing 4 to more than 60 cells by day 5. We demonstrated that the breast cancer cell receptor CXCR2 and the bone-secreted chemokine CXCL5 play a major role in the extravasation process, influencing extravasation rate and traveled distance. Our study provides novel 3D in vitro quantitative data on extravasation and micrometastasis generation of breast cancer cells within a bone-like microenvironment and demonstrates the potential value of microfluidic systems to better understand cancer biology and screen for new therapeutics.

IL-17A (IL-17) is the signature cytokine produced by Th17 cells and has been implicated in host defense against infection and the pathophysiology of autoimmunity and cardiovascular disease. Little is known, however, about the influence of IL-17 on endothelial activation and leukocyte influx to sites of inflammation. We hypothesized that IL-17 would induce a distinct pattern of endothelial activation and leukocyte recruitment when compared with the Th1 cytokine IFN-γ. We found that IL-17 alone had minimal activating effects on cultured endothelium, whereas the combination of TNF-α and IL-17 produced a synergistic increase in the expression of both P-selectin and E-selectin. Using intravital microscopy of the mouse cremaster muscle, we found that TNF-α and IL-17 also led to a synergistic increase in E-selectin-dependent leukocyte rolling on microvascular endothelium in vivo. In addition, TNF-α and IL-17 enhanced endothelial expression of the neutrophilic chemokines CXCL1, CXCL2, and CXCL5 and led to a functional increase in leukocyte transmigration in vivo and CXCR2-dependent neutrophil but not T cell transmigration in a parallel-plate flow chamber system. By contrast, endothelial activation with TNF-α and IFN-γ preferentially induced the expression of the integrin ligands ICAM-1 and VCAM-1, as well as the T cell chemokines CXCL9, CXCL10, and CCL5. These effects were further associated with a functional increase in T cell but not neutrophil transmigration under laminar shear flow. Overall, these data show that IL-17 and TNF-α act in a synergistic manner to induce a distinct pattern of endothelial activation that sustains and enhances neutrophil influx to sites of inflammation.

We have applied immunohistology and in situ hybridization to bronchial biopsies of patients with chronic obstructive pulmonary disease (COPD) to examine neutrophil recruitment and to determine neutrophil chemoattractant and CXC receptor (CXCR) 1 and CXCR2 gene expression associated with acute severe exacerbations. Cells were counted in endobronchial biopsies of (1) patients with COPD intubated for exacerbations (E-COPD; n = 15), (2) those with COPD in a stable phase of their disease (S-COPD; n = 7), and (3) nonsmoker surgical control subjects intubated for a nonrespiratory surgical procedure (n = 15). In comparison with the nonrespiratory surgical procedure and S-COPD groups, neutrophilia and gene expression for epithelial-derived neutrophil attractant-78 (CXCL5), interleukin-8 (CXCL8), CXCR1, and CXCR2 were each upregulated in the E-COPD group (p < 0.01); compared with the S-COPD group, by 97-, 6-, 6-, 3-, and 7-fold, respectively (p < 0.01). In E-COPD, there was a significant positive association between the number of neutrophils and CXCR2 mRNA-positive cells (r = 0.79; p < 0.01) but not between the number of neutrophils and CXCR1 mRNA-positive cells. At the time of sampling of the mucosa, there was no association between neutrophil number and either the length of intubation or viral infection. Thus, in COPD, in addition to CXCL8 and CXCR1, CXCL5 and CXCR2 appear to play important roles in the airway neutrophilia characteristic of severe exacerbations.

Blood platelets play critical roles in hemostasis, providing rapid essential protection against bleeding and catalyzing the important slower formation of stable blood clots via the coagulation cascade. They are also involved in protection from infection by phagocytosis of pathogens and by secreting chemokines that attract leukocytes. Platelet function usually is activated by primary agonists such as adenosine diphosphate (ADP), thrombin, and collagen, whereas secondary agonists like adrenalin do not induce aggregation on their own but become highly effective in the presence of low levels of primary agonists. Current research has revealed that chemokines represent an important additional class of agonists capable of causing significant activation of platelet function. Early work on platelet alpha-granule proteins suggested that platelet factor 4, now known as CXCL4, modulated aggregation and secretion induced by low agonist levels. Subsequent reports revealed the presence in platelets of messenger RNA for several additional chemokines and chemokine receptors. Three chemokines in particular, CXCL12 (SDF-1), CCL17 (TARC), and CCL22 (MDC), recently have been shown to be strong and rapid activators of platelet aggregation and adhesion after their binding to platelet CXCR4 or CCR4, when acting in combination with low levels of primary agonists. CXCL12 can be secreted by endothelial cells and is present in atherosclerotic plaques, whereas CCL17 and CCL22 are secreted by monocytes and macrophages. Platelet activation leads to the release of alpha-granule chemokines, including CCL3 (MIP-1alpha), CCL5 (RANTES), CCL7 (MCP-3), CCL17, CXCL1 (growth-regulated oncogene-alpha), CXCL5 (ENA-78), and CXCL8 (IL-8), which attract leukocytes and further activate other platelets. These findings help to provide a direct linkage between hemostasis, infection, and inflammation and the development of atherosclerosis.

During metastasis, host cells are recruited to disseminated tumor cells to form specialized microenvironments ("niches") that promote metastatic progression, but the mechanisms guiding the assembly of these niches are largely unknown. Tumor cells may autonomously recruit host cells or, alternatively, host cell-to-host cell interactions may guide the formation of these prometastatic microenvironments. Here, we show that platelet-derived rather than tumor cell-derived signals are required for the rapid recruitment of granulocytes to tumor cells to form "early metastatic niches." Granulocyte recruitment relies on the secretion of CXCL5 and CXCL7 chemokines by platelets upon contact with tumor cells. Blockade of the CXCL5/7 receptor CXCR2, or transient depletion of either platelets or granulocytes prevents the formation of early metastatic niches and significantly reduces metastatic seeding and progression. Thus, platelets recruit granulocytes and guide the formation of early metastatic niches, which are crucial for metastasis.

On chemokine stimulation, leucocytes produce and secrete proteolytic enzymes for innate immune defence mechanisms. Some of these proteases modify the biological activity of the chemokines. For instance, neutrophils secrete gelatinase B (matrix metalloproteinase-9, MMP-9) and neutrophil collagenase (MMP-8) after stimulation with interleukin-8/CXCL8 (IL-8). Gelatinase B cleaves and potentiates IL-8, generating a positive feedback. Here, we extend these findings and compare the processing of the CXC chemokines human and mouse granulocyte chemotactic protein-2/CXCL6 (GCP-2) and the closely related human epithelial-cell derived neutrophil activating peptide-78/CXCL5 (ENA-78) with that of human IL-8. Human GCP-2 and ENA-78 are cleaved by gelatinase B at similar rates to IL-8. In addition, GCP-2 is cleaved by neutrophil collagenase, but at a lower rate. The cleavage of GCP-2 is exclusively N-terminal and does not result in any change in biological activity. In contrast, ENA-78 is cleaved by gelatinase B at eight positions at various rates, finally generating inactive fragments. Physiologically, sequential cleavage of ENA-78 may result in early potentiation and later in inactivation of the chemokine. Remarkably, in the mouse, which lacks IL-8 which is replaced by GCP-2/LIX as the most potent neutrophil activating chemokine, N-terminal clipping and twofold potentiation by gelatinase B was also observed. In addition to the similarities in the potentiation of IL-8 in humans and GCP-2 in mice, the conversion of mouse GCP-2/LIX by mouse gelatinase B is the fastest for any combination of chemokines and MMPs so far reported. This rapid conversion was also performed by crude neutrophil granule secretion under physiological conditions, extending the relevance of this proteolytic cleavage to the in vivo situation.

Recent studies indicate that TLRs are critical in generating innate immune responses during infection with HSV-1. In this study, we investigated the role of TLR2 signaling in regulating the production of neuroimmune mediators by examining cytokine and chemokine expression using primary microglial cells obtained from TLR2-/- as well as wild-type mice. Data presented here demonstrate that TLR2 signaling is required for the production of proinflammatory cytokines and chemokines: TNF-alpha, IL-1beta, IL-6, IL-12, CCL7, CCL8, CCL9, CXCL1, CXCL2, CXCL4, and CXCL5. CXCL9 and CXCL10 were also induced by HSV, but their production was not dependent upon TLR2 signaling. Because TLR2-/- mice display significantly reduced mortality and diminished neuroinflammation in response to brain infection with HSV, the TLR2-dependent cytokines identified here might function as key players influencing viral neuropathogenesis.

OBJECTIVE

To evaluate the association of serum factors with the severity of diabetic retinopathy and to assess their presence in retinal tissue obtained at autopsy.

METHODS

The following serum factors of 93 subjects were examined at the National Eye Institute (NEI) clinical center: the chemokines regulated on activation, normal T-cell expressed and presumably secreted (RANTES)/CCL5, epithelial neutrophil activator (ENA)-78/CXCL5, interferon-induced protein (IP)-10/CXCL10, stromal cell-derived factor (SDF)-1alpha/CXCLl2, monocyte chemoattractant protein (MCP)-1/CCL2, macrophage inflammatory protein (MIP)-1alpha/CCL3, interleukin (IL)-8/CXCL8; the cytokine IL-6; the cell adhesion molecules intercellular adhesion molecule (ICAM-1/CD54) and vascular cell adhesion molecule (VCAM/CD106); and the growth factor vascular endothelial growth factor (VEGF). Logistic regression was performed to assess the association of these factors with age, sex, severity of retinopathy, hemoglobin A(1C), total cholesterol, creatinine, duration of diabetes, and presence of macular edema. The outcome assessed was severity of retinopathy. Frozen sections of two donor eyes obtained at autopsy from a donor with documented severe nonproliferative diabetic retinopathy and diabetic macular edema and of a normal nondiabetic eye were processed by immunoperoxidase staining with primary antibodies against RANTES, MCP-1, ICAM-1, and LFA-1alpha/CD11a.

RESULTS

The levels of RANTES and SDF-1alpha were significantly elevated in patients with at least severe nonproliferative diabetic retinopathy compared with those with less severe diabetic retinopathy (P < 0.001 and 0.007, respectively). Positive immunostaining was observed in the inner retina for MCP-1 and RANTES of the patient with diabetes. Staining was strongly positive throughout the diabetic retina for ICAM-1. Normal retinal tissues showed little reactivity.

CONCLUSIONS

Serum chemokines were significantly elevated in patients with at least severe nonproliferative diabetic retinopathy compared with those who had less severe retinopathy. Elevated levels of the chemokines and cell adhesion molecules were also identified in eyes of a donor with ischemic diabetic retinopathy. These findings provide evidence to support the role of inflammation in the pathogenesis of diabetic retinopathy.

We identify matrix metalloproteinase (MMP)-8, the polymorphonuclear (PMN) leukocyte collagenase, as a critical mediator initiating lipopolysaccharide (LPS)-responsiveness in vivo. PMN infiltration towards LPS is abrogated in Mmp8-null mice. MMP-8 cleaves LPS-induced CXC chemokine (LIX) at Ser(4)-Val(5) and Lys(79)-Arg(80). LIX bioactivity is increased upon N-terminal cleavage, enhancing intracellular calcium mobilization and chemotaxis upon binding its cognate receptor, CXCR2. As there is no difference in PMN chemotaxis in Mmp8-null mice compared with wild-type mice towards synthetic analogues of MMP-8-cleaved LIX, MMP-8 is not essential for extravasation or cell migration in collagenous matrices in vivo. However, with biochemical redundancy between MMPs 1, 2, 9, and 13, which also cleave LIX at position 4 approximately 5, it was surprising to observe such a markedly reduced PMN infiltration towards LPS and LIX in Mmp8-/- mice. This lack of physiological redundancy in vivo identifies MMP-8 as a key mediator in the regulation of innate immunity. Comparable results were found with CXCL8/IL-8 and CXCL5/ENA-78, the human orthologues of LIX. MMP-8 cleaves CXCL8 at Arg(5)-Ser(6) and at Val(7)-Leu(8) in CXCL5 to activate respective chemokines. Hence, rather than collagen, these PMN chemoattractants are important MMP-8 substrates in vivo; PMN-derived MMP-8 cleaves and activates LIX to execute an in cis PMN-controlled feed-forward mechanism to orchestrate the initial inflammatory response and promote LPS responsiveness in tissue.

The circadian system is an important regulator of immune function. Human inflammatory lung diseases frequently show time-of-day variation in symptom severity and lung function, but the mechanisms and cell types underlying these effects remain unclear. We show that pulmonary antibacterial responses are modulated by a circadian clock within epithelial club (Clara) cells. These drive circadian neutrophil recruitment to the lung via the chemokine CXCL5. Genetic ablation of the clock gene Bmal1 (also called Arntl or MOP3) in bronchiolar cells disrupts rhythmic Cxcl5 expression, resulting in exaggerated inflammatory responses to lipopolysaccharide and an impaired host response to Streptococcus pneumoniae infection. Adrenalectomy blocks rhythmic inflammatory responses and the circadian regulation of CXCL5, suggesting a key role for the adrenal axis in driving CXCL5 expression and pulmonary neutrophil recruitment. Glucocorticoid receptor occupancy at the Cxcl5 locus shows circadian oscillations, but this is disrupted in mice with bronchiole-specific ablation of Bmal1, leading to enhanced CXCL5 expression despite normal corticosteroid secretion. The therapeutic effects of the synthetic glucocorticoid dexamethasone depend on intact clock function in the airway. We now define a regulatory mechanism that links the circadian clock and glucocorticoid hormones to control both time-of-day variation and the magnitude of pulmonary inflammation and responses to bacterial infection.

OBJECTIVE

Alcoholic hepatitis (AH) is characterized by hepatocellular damage, inflammation, and fibrosis. We performed a prospective study to associate hepatic expression of the CXC subfamily of chemokines with histology findings and prognosis of patients with AH.

METHODS

Liver biopsy samples from 105 patients with AH and 5 normal liver samples (controls) were evaluated for steatosis, inflammation, fibrosis, and cholestasis. Computer-based morphometric analysis assessed the numbers of infiltrating CD3+ T cells and CD15+ cells (neutrophils); terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining was used to quantify apoptosis. Expression of CXC and CC chemokines and selected signaling components were assessed by quantitative reverse-transcription polymerase chain reaction; protein levels of interleukin (IL)-8 and Gro-alpha also were determined by immunohistochemistry. Serum levels of IL-8 and Gro-alpha were measured by enzyme-linked immunosorbent assay. The Cox regression model identified variables associated with mortality.

RESULTS

Most patients (75%) had severe AH; their 90-day mortality rate was 21.9%. In AH liver samples, expression of the CXC subfamily members IL-8, Gro-alpha, CXCL5, CXCL6, CXCL10, and platelet factor 4 was up-regulated and compared with controls. The CC chemokine CCL2, but not CCL5, also was up-regulated. Higher expression levels of IL-8, CXCL5, Gro-gamma, and CXCL6 were associated with worse prognosis. Expression of CXC components correlated with neutrophil infiltration and the severity of portal hypertension. In the multivariate analysis, IL-8 protein levels were an independent predictor of 90-day mortality. IL-8 and Gro-alpha serum levels did not correlate with prognosis.

CONCLUSIONS

Hepatic expression of CXC components correlates with prognosis of patients with AH. Reagents that target CXC chemokines might be developed as therapeutics.

BACKGROUND

Despite growing recognition of an etiologic role for inflammation in lung carcinogenesis, few prospective epidemiologic studies have comprehensively investigated the association of circulating inflammation markers with lung cancer.

METHODS

We conducted a nested case-control study (n = 526 lung cancer patients and n = 592 control subjects) within the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Control subjects were matched to lung cancer case patients on age, sex, follow-up time (median = 2.9 years), randomization year, and smoking (pack-years and time since quitting). Serum levels of 77 inflammation markers were measured using a Luminex bead-based assay. Conditional logistic regression and weighted Cox models were used to estimate odds ratios (ORs) and cumulative risks, respectively.

RESULTS

Of 68 evaluable markers, 11 were statistically significantly associated with lung cancer risk (P trend across marker categories < .05), including acute-phase proteins (C-reactive protein [CRP], serum amyloid A [SAA]), proinflammatory cytokines (soluble tumor necrosis factor receptor 2 [sTNFRII]), anti-inflammatory cytokines (interleukin 1 receptor antagonist [IL-1RA]), lymphoid differentiation cytokines (interleukin 7 [IL-7]), growth factors (transforming growth factor alpha [TGF-A]), and chemokines (epithelial neutrophil-activating peptide 78 [ENA 78/CXCL5], monokine induced by gamma interferon [MIG/CXCL9], B cell-attracting chemokine 1 [BCA-1/CXCL13], thymus activation regulated chemokine [TARC/CCL17], macrophage-derived chemokine [MDC/CCL22]). Elevated marker levels were associated with increased lung cancer risk, with odds ratios comparing the highest vs the lowest group ranging from 1.47 (IL-7) to 2.27 (CRP). For IL-1RA, elevated levels were associated with decreased lung cancer risk (OR = 0.71; 95% confidence interval = 0.51 to 1.00). Associations did not differ by smoking, lung cancer histology, or latency. A cross-validated inflammation score using four independent markers (CRP, BCA-1/CXCL13, MDC/CCL22, and IL-1RA) provided good separation in 10-year lung cancer cumulative risks among former smokers (quartile [Q] 1 = 1.1% vs Q4 = 3.1%) and current smokers (Q1 = 2.3% vs Q4 = 7.9%) even after adjustment for smoking.

CONCLUSIONS

Some circulating inflammation marker levels are associated with prospective lung cancer risk.

Interleukin (IL)-17 is the founding member of an emerging family of inflammatory cytokines whose functions remain poorly defined. IL-17 has been linked to the pathogenesis of rheumatoid arthritis, and numerous studies implicate this cytokine in inflammation-induced bone loss. It is clear that a major function of IL-17 is to amplify the immune response by triggering production of chemokines, cytokines, and cell-surface markers, ultimately leading to neutrophil chemotaxis and inflammation. As an IL-17 signaling deficiency in mice causes a dramatic reduction in neutrophil chemotaxis and a consequent increased susceptibility to bacterial infection, it is important to define gene targets involved in IL-17-mediated neutrophil trafficking. Here, we demonstrate that IL-17 and tumor necrosis factor alpha (TNF-alpha) cooperatively induce the lipopolysaccharide-inducible CXC chemokine (LIX; a.k.a., CXC chemokine ligand 5, Scya5, or murine granulocyte chemotactic protein-2) in the preosteoblast cell line MC3T3. LIX is induced rapidly at the mRNA and protein levels, likely through the activation of new gene transcription. Conditioned media from MC3T3 cells treated with IL-17 and/or TNF-alpha stimulates neutrophil mobility potently, and LIX is a significant contributing factor to this process. In addition, IL-17 cooperates with bacterial components involved in periodontal disease to up-regulate LIX expression. This study is the first demonstration of LIX expression in bone cells and has implications for inflammatory bone diseases such as arthritis and periodontal disease.

The signaling mechanisms between prostate cancer cells and infiltrating immune cells may illuminate novel therapeutic approaches. Here, utilizing a prostate adenocarcinoma model driven by loss of Pten and Smad4, we identify polymorphonuclear myeloid-derived suppressor cells (MDSC) as the major infiltrating immune cell type, and depletion of MDSCs blocks progression. Employing a novel dual reporter prostate cancer model, epithelial and stromal transcriptomic profiling identified CXCL5 as a cancer-secreted chemokine to attract CXCR2-expressing MDSCs, and, correspondingly, pharmacologic inhibition of CXCR2 impeded tumor progression. Integrated analyses identified hyperactivated Hippo-YAP signaling in driving CXCL5 upregulation in cancer cells through the YAP-TEAD complex and promoting MDSC recruitment. Clinicopathologic studies reveal upregulation and activation of YAP1 in a subset of human prostate tumors, and the YAP1 signature is enriched in primary prostate tumor samples with stronger expression of MDSC-relevant genes. Together, YAP-driven MDSC recruitment via heterotypic CXCL5-CXCR2 signaling reveals an effective therapeutic strategy for advanced prostate cancer.

CONCLUSIONS

We demonstrate a critical role of MDSCs in prostate tumor progression and discover a cancer cell nonautonomous function of the Hippo-YAP pathway in regulation of CXCL5, a ligand for CXCR2-expressing MDSCs. Pharmacologic elimination of MDSCs or blocking the heterotypic CXCL5-CXCR2 signaling circuit elicits robust antitumor responses and prolongs survival.

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.

CXCL5 (epithelial neutrophil-activating peptide-78) is a member of a proangiogenic subgroup of the CXC-type chemokine family of small, secreted proteins. Recently, evidence that CXCL5 is involved in carcinogenesis and cancer progression has emerged. To investigate the role of CXCL5 in tumor growth, invasion, and prognosis of hepatocellular carcinoma (HCC), we examined CXCL5 messenger RNA (mRNA) and protein levels in HCC cell lines with various metastatic potentials and in three independent cohorts of 919 HCC patients. We found that CXCL5 expression was increased in the highly metastatic HCC cell lines and in tumor tissues from patients with recurrent HCC compared to controls. CXCL5 activated the PI3K-Akt and ERK1/2 signaling pathways in HCC cells and promoted proliferation, migration, and invasion. Furthermore, we found that CXCL5 had a direct chemoattractant effect on neutrophils in vitro. In animal studies, the up-regulation of CXCL5 in HCC cells promoted tumor growth, lung metastasis, and intratumoral neutrophil infiltration. Conversely, down-regulation of CXCL5 in HCC cells reduced tumor growth, metastasis, and intratumoral neutrophil infiltration. Immunohistochemical analysis in HCC samples showed that overexpression of CXCL5 was well correlated with intratumoral neutrophil infiltration, shorter overall survival, and tumor recurrence. Multivariate analysis revealed that CXCL5 overexpression alone, or combined with the presence of intratumoral neutrophils, was an independent prognostic indicator for overall survival and cumulative recurrence.

CONCLUSIONS

CXCL5 promotes HCC cell proliferation, invasion, and intratumoral neutrophil infiltration. CXCL5 overexpression, alone or combined with intratumoral neutrophil presence, is a novel prognostic predictor, and CXCL5 is a potential therapeutic target for HCC.