Plasma endothelial cell-derived exosomes (EDEs) and platelet-derived exosomes (PDEs) were precipitated and enriched separately by immunospecific absorption procedures for analyses of cargo proteins relevant to atherosclerosis. EDEs had usual exosome size and marker protein content, and significantly higher levels than PDEs of the endothelial proteins vascular cell adhesion molecule-1 (VCAM-1) and endothelial nitric oxide synthase, whereas PDEs had significantly higher levels of platelet glycoprotein VI. EDE levels of VCAM-1, von Willebrand factor, platelet-derived growth factor (PDGF)-BB, angiopoietin-1, and lysyl oxidase-2 and the cerebrovascular-selective proteins glucose transporter 1, permeability-glycoprotein, and large neutral amino acid transporter 1 were significantly higher for 18 patients with cerebrovascular disease (CeVD) than for 18 age- and gender-matched control subjects. PDE levels of PDGF-AA, platelet glycoprotein VI, integrin-linked kinase-1, high mobility group box-1 protein, chemokine CXCL4, and thrombospondin-1 were significantly higher in patients with CeVD than in control subjects, but differences were less with greater overlaps than for EDE proteins. EDE levels of Yes-associated protein (YAP) were higher and of P(S127)-YAP lower in patients with CeVD than in control subjects, consistent with heightened activity of this mechanical force-sensitive system in atherosclerosis. Elevated EDE and PDE levels of atherosclerosis-promoting proteins in CeVD justify clinical studies of their potential value as biomarkers.-Goetzl, E. J., Schwartz, J. B., Mustapic, M., Lobach, I. V., Daneman, R., Abner, E. L., Jicha, G. A. Altered cargo proteins of human plasma endothelial cell-derived exosomes in atherosclerotic cerebrovascular disease.

Coagulation and the immune system interact in several physiological and pathological conditions, including tissue repair, host defense, and homeostatic maintenance. This network plays a key role in diseases of the central nervous system (CNS) by involving several cells (CNS resident cells, platelets, endothelium, and leukocytes) and molecular pathways (protease activity, complement factors, platelet granule content). Endothelial damage prompts platelet activation and the coagulation cascade as the first physiological step to support the rescue of damaged tissues, a flawed rescuing system ultimately producing neuroinflammation. Leukocytes, platelets, and endothelial cells are sensitive to the damage and indeed can release or respond to chemokines and cytokines (platelet factor 4, CXCL4, TNF, interleukins), and growth factors (including platelet-derived growth factor, vascular endothelial growth factor, and brain-derived neurotrophic factor) with platelet activation, change in capillary permeability, migration or differentiation of leukocytes. Thrombin, plasmin, activated complement factors and matrix metalloproteinase-1 (MMP-1), furthermore, activate intracellular transduction through complement or protease-activated receptors. Impairment of the neuro-immune hemostasis network induces acute or chronic CNS pathologies related to the neurovascular unit, either directly or by the systemic activation of its main steps. Neurons, glial cells (astrocytes and microglia) and the extracellular matrix play a crucial function in a "tetrapartite" synaptic model. Taking into account the neurovascular unit, in this review we thoroughly analyzed the influence of neuro-immune hemostasis on these five elements acting as a functional unit ("pentapartite" synapse) in the adaptive and maladaptive plasticity and discuss the relevance of these events in inflammatory, cerebrovascular, Alzheimer, neoplastic and psychiatric diseases. Finally, based on the solid reviewed data, we hypothesize a model of neuro-immune hemostatic network based on protein-protein interactions. In addition, we propose that, to better understand and favor the maintenance of adaptive plasticity, it would be useful to construct predictive molecular models, able to enlighten the regulating logic of the complex molecular network, which belongs to different cellular domains. A modeling approach would help to define how nodes of the network interact with basic cellular functions, such as mitochondrial metabolism, autophagy or apoptosis. It is expected that dynamic systems biology models might help to elucidate the fine structure of molecular events generated by blood coagulation and neuro-immune responses in several CNS diseases, thereby opening the way to more effective treatments.

GNA13 has been found overexpressed in various types of cancer, which is related to tumor metastasis and progression. However, the biological functions of GNA13 in colorectal cancer (CRC) progression remain unclear. This study aimed to explore the role of GNA13 in CRC and investigate the mechanism of how GNA13 promotes tumor growth. Interestingly, our findings showed that GNA13 is commonly upregulated in CRC, where these events are associated with a worse histologic grade and poor survival. Increased expression levels of GNA13 promoted cell growth, migration, invasion, and epithelial-mesenchymal transition, whereas GNA13 silencing abrogated these malignant phenotypes. In addition, overexpressing GNA13 in cancer cells increased the levels of the chemokines CXCL1, CXCL2, and CXCL4, which contributed to CRC proliferation and colony formation. Moreover, our mechanistic investigations suggest that the NF-κB/p65 signaling pathway was activated by the increase in GNA13 levels. Inhibiting the NF-κB/p65 pathway with an inhibitor decreased GNA13-induced migration, invasion and CXCL chemokine level increases, indicating the critical role of NF-κB/p65 signaling in mediating the effects of GNA13 in CRC. Together, these results demonstrate a key role of GNA13 overexpression in CRC that contributes to malignant behavior in cancer cells, at least in part through stimulating angiogenesis and increasing the levels of the NF-κB-dependent chemokines CXCL1, CXCL2, and CXCL4.

BACKGROUND

Osteosarcoma is the most common malignant bone tumor in children. Despite the advent of chemotherapy, the survival of osteosarcoma patients has not been significantly improved recently. Chemokines are a group of signaling molecules that have been implicated in tumorigenesis and metastasis.

METHODS

The authors used an antibody microarray to identify chemokines that were elevated in the plasma samples of osteosarcoma patients. The results were validated using enzyme-linked immunosorbent assays on an independent set of samples. The tumor expressions of 3 chemokines were examined in 2 sets of osteosarcoma tissue arrays. The authors also evaluated the proliferative effect of the chemokines in 4 osteosarcoma cell lines.

RESULTS

The authors found that the plasma levels of CXCL4, CXCL6, and CXCL12 in the osteosarcoma patients were significantly higher than those in the controls, and the results were validated by an independent osteosarcoma cohort (P < .05). However, CXCL4 (100%) and CXCL6 (91%) were frequently expressed in osteosarcoma, whereas CXCL12 was only expressed in 4%. Survival analysis further showed that higher circulating levels of CXCL4 and CXCL6, but not CXCL12, were associated with a poorer outcome of osteosarcoma patients. Addition of exogenous chemokines significantly promoted the growth of different osteosarcoma cells (P < .05).

CONCLUSIONS

The results demonstrate that CXCL4 and CXCL6 are frequently expressed in osteosarcoma, and that the plasma levels of these 2 chemokines are associated with patient outcomes. Further study of these circulating chemokines may provide a promising approach for prognostication of osteosarcoma. Targeting these chemokines or their receptors may also lead to a novel therapeutic invention.

Dendritic cells (DCs) are professional antigen-presenting cells and members of the adoptive immunity. In addition, they play an important role in innate immunity within the systemic inflammatory response to trauma and sepsis. In this study, gene expression patterns of DC in patients with multiple trauma were studied. Total RNA was isolated from highly purified DCs (purity>95%) that were enriched from peripheral blood mononuclear cells and whole blood, respectively. Samples were obtained from 10 multiple trauma patients (injury severity score, 35.4+/-10.6 on day of admission) and 5 healthy volunteers (control). Aliquots of target cDNAs and reference samples (cDNA derived from the monocytic cell line SIGM5) were cohybridized on a thematic medium-density microarray assessing 780 inflammation-related transcripts. Twenty transcripts were up-regulated in DCs of multiple trauma patients compared with healthy volunteers, whereas these differences were missed when RNA from whole blood was subjected to transcriptomic profiling. This cluster included central effector molecules of DC such as transcripts encoding for 5-lipoxygenase and the corresponding leukotriene 4 receptor, which regulate DC migration, adoptive immune responses, and airway inflammation, as well as CD74, CXCL4, or platelet factor 4, a chemokine not implicated as a product of DCs to date. In addition, genes involved in antiapoptosis (BCL2), intracellular signal transduction (mitogen-activated protein kinase), and secretion of mediators (VAMP2) were found to be up-regulated. The up-regulated transcripts suggest that life span and signaling function of DCs are altered by trauma. Furthermore, these data confirm and expand the central role of chemokines and lipid mediators as effector molecules of DC-mediated immune responses in systemic inflammation associated with severe trauma.

OBJECTIVE

Over the last decade there has been mounting experimental data demonstrating that platelets contribute to acute vascular inflammation and atherosclerosis. This review focuses on recent findings that link platelets to inflammatory responses of relevance to transplants.

RESULTS

Although it has been known that platelets modify vascular inflammation by secretion of soluble mediators and release of microparticles, new aspects of these mechanisms are being defined. For example, platelet-derived CCL5 not only functions in homomers, but also forms more potent heteromers with platelet factor 4 (PF4; CXCL4). This heteromer formation can be inhibited with small molecules. New findings also demonstrate heterologous interactions of platelet microparticles with leukocytes that may increase their range of impact. By attaching to neutrophils, platelet microparticles appear to migrate out of blood vessels and into other compartments where they stimulate secretion of cytokines. Contact of platelets with extracellular matrix also can result in cleavage of hyaluronan into fragments that serve as an endogenous danger signal.

CONCLUSIONS

Recent findings have expanded the range of interactions by which platelets can modify innate and adaptive immune responses to transplants.

Feeder cells of irradiated mouse fibroblasts are commonly used for, and are generally necessary for, the in vitro maintenance and growth of many fastidious cell types, particularly embryonic stem cells or induced pluripotent stem cells. Quantitative and semiquantitative immunoassays of conditioned media were performed to identify some of the soluble cytokines, chemokines, protein hormones, and cell matrix/adhesion molecules that are elaborated from two commonly used feeder cells, STO and CF-1. Among those quantitatively assayed, the most abundant cytokine proteins expressed by the feeder cells were activin A, hepatocyte growth factor (HGF), insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding protein (IGFBP)-6, macrophage colony-stimulating factor (a.k.a. CSF-1), and pigment epithelium-derived factor (a.k.a. serine protease inhibitor, clade F, member 1). CF-1 cells expressed ten times more activin A than STO cells and also produced larger amounts of interleukin-6 and IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5. Conversely, STO cell produced almost ten times more HGF and five times more stem cell factor (a.k.a. c-kit ligand) than CF-1 cells. Assayed semiquantitatively, relatively large amounts of chemokines were produced by both feeder cells including fractalkine (CX3CL1), interferon-inducible protein 10 (a.k.a. CXCL10 and cytokine-responsive gene-2, CRG-2), monocyte chemotactic protein (MCP)-1 (a.k.a. CCL2 and junctional epithelium chemokine (JE), MCP-5/CCL12), keratinocyte-derived chemokine (a.k.a. CXCL1 and growth-related oncogene alpha, GROα), nephroblastoma overexpressed gene (CCN3, IGFBP-9), stromal cell-derived factor 1 (CXCL12), and serpin E1 (PAI-1). In contrast to one another, STO produced more CXCL16 than CF-1 cells, and CF-1 cell produced more MCP-5 (CCL12), macrophage inflammatory protein (MIP)-1α (CCL3), MIP-1β (CCL4), pentraxin-3 (TSG-14), and platelet factor-4 (CXCL4) than STO cells. Soluble adhesion molecule, sICAM (ICAM-1, CD54), was expressed by CF-1 cells, but not STO cells, and similarly, the cell matrix-associated molecules endocan (endothelial cell-specific molecule 1), endostatin (collagen XVIII), and matrix metalloproteinase 3 were expressed more by CF-1 cells. Tissue inhibitor of metalloproteinases 1 was robustly expressed by both feeder cells. Other proteins primarily detected from CF-1 cells included retinol-binding protein 4 and FGF21, while STO cells secreted more interferon gamma. Both feeder cells produced no or low amounts of LIF, tumor necrosis factor alpha, vascular endothelial growth factor (VEGF), VEGF-B, prolactin, various interleukins, fibroblast growth factor (FGF)-1, FGF-2, FGF-7, EGF, HB-EGF, and amphiregulin. The results may explain some of the cell growth and maintenance responses by various types of cells co-cultured on STO or CF-1 feeder cells.

The CXC chemokines, CXCL4, -9, -10, -11, CXCL4L1, and the CC chemokine CCL21, activate CXC chemokine receptor 3 (CXCR3), a cell-surface G protein-coupled receptor expressed mainly by Th1 cells, cytotoxic T (Tc) cells and NK cells that have a key role in immunity and inflammation. However, CXCR3 is also expressed by vascular smooth muscle and endothelial cells, and appears to be important in controlling physiological vascular function. In the last decade, evidence from pre-clinical and clinical studies has revealed the participation of CXCR3 and its ligands in multiple cardiovascular diseases (CVDs) of different aetiologies including atherosclerosis, hypertension, cardiac hypertrophy and heart failure, as well as in heart transplant rejection and transplant coronary artery disease (CAD). CXCR3 ligands have also proven to be valid biomarkers for the development of heart failure and left ventricular dysfunction, suggesting an underlining pathophysiological relation between levels of these chemokines and the development of adverse cardiac remodelling. The observation that several of the above-mentioned chemokines exert biological actions independent of CXCR3 provides both opportunities and challenges for developing effective drug strategies. In this review, we provide evidence to support our contention that CXCR3 and its ligands actively participate in the development and progression of CVDs, and may additionally have utility as diagnostic and prognostic biomarkers.

Platelet factor 4 (CXCL4-PF4) is a chemokine that binds to and neutralizes heparin and other negatively charged proteoglycans, but is also involved in angiogenesis and cancer development. In some patients exposed to heparin, antibodies are generated against the CXCL-PF4/heparin complex that may activate platelets and coagulation and lead to thrombocytopenia and arterial or venous thrombosis, a condition commonly named heparin induced thrombocytopenia (HIT). HIT has been investigated in numerous clinical settings, but there is limited data on the epidemiology and phenotype of HIT in cancer patients. The present review describes the role of CXCL4-PF4 in cancer, the immunobiology, clinical presentation and diagnosis of HIT, and the specific problems faced in cancer patients.

Platelets functions in hemostasis have been widely studied. Currently, growing evidence shows that platelets have also a role in the immune innate response. Recently, protein expression of Toll-like receptors (TLR's) 2, 4, 7, 8, and 9, and the presence of TLRs 1 and 6 mRNA in human platelets was described. Up to now the functionality of TLR-2, 4 and 9 in human platelets has been demonstrated. Due to the relevance of TLRs functions to PAMPS (pathogen-associated molecular patterns) recognizing, we evaluated the presence of TLR3 in human platelets founding low percentages of platelets expressing surface or intracellular TLR3 protein. The activation with thrombin induced an increase in the percentage of platelets expressing surface TLR3 and higher levels of TLR3 expression in the whole population. Human platelets responded to poly I:C by increasing [Ca(2+)]i, the percentages of cells expressing TLR4 and CD62P, and by releasing CXCL4 and IL-1β in comparison to unstimulated platelets. These results demonstrate that human platelets express TLR3 and are capable of responding to poly I:C, suggesting that these cells might influence the immune innate response when detecting viral dsRNA.

The regulation of hematopoietic stem cell (HSC) survival and self-renewal within the bone marrow (BM) niche is not well understood. We therefore investigated global transcriptomic profiling of normal human HSC/hematopoietic progenitor cells [HPCs], revealing that several chemokine ligands (CXCL1-4, CXCL6, CXCL10, CXCL11, and CXCL13) were upregulated in human quiescent CD34(+)Hoescht(-)Pyronin Y(-) and primitive CD34(+)38(-), as compared with proliferating CD34(+)Hoechst(+)Pyronin Y(+) and CD34(+)38(+) stem/progenitor cells. This suggested that chemokines might play an important role in the homeostasis of HSCs. In human CD34(+) hematopoietic cells, knockdown of CXCL4 or pharmacologic inhibition of the chemokine receptor CXCR2, significantly decreased cell viability and colony forming cell (CFC) potential. Studies on Cxcr2(-/-) mice demonstrated enhanced BM and spleen cellularity, with significantly increased numbers of HSCs, hematopoietic progenitor cell-1 (HPC-1), HPC-2, and Lin(-)Sca-1(+)c-Kit(+) subpopulations. Cxcr2(-/-) stem/progenitor cells showed reduced self-renewal capacity as measured in serial transplantation assays. Parallel studies on Cxcl4 demonstrated reduced numbers of CFC in primary and secondary assays following knockdown in murine c-Kit(+) cells, and Cxcl4(-/-) mice showed a decrease in HSC and reduced self-renewal capacity after secondary transplantation. These data demonstrate that the CXCR2 network and CXCL4 play a role in the maintenance of normal HSC/HPC cell fates, including survival and self-renewal.

Activated platelets release micromolar concentrations of the chemokine CXCL4/Platelet Factor-4. Deposition of CXCL4 onto the vascular endothelium is involved in atherosclerosis, facilitating monocyte arrest and recruitment by an as yet, unidentified receptor. Here, we demonstrate that CXCL4 drives chemotaxis of the monocytic cell line THP-1. Migration and intracellular calcium responses induced by CXCL4 were pertussis toxin-sensitive, implicating a GPCR in signal transduction. Cell treatment with chondroitinase ABC ablated migration, suggesting that cis presentation of CXCL4 by cell surface glycosaminoglycans to a GPCR is required. Although CXCR3 has been previously described as a CXCL4 receptor, THP-1 cells were unresponsive to CXCR3 ligands and CXCL4-induced migration was insensitive to a CXCR3 antagonist, suggesting that an alternative receptor is involved. Interrogating CC-class chemokine receptor transfectants, we unexpectedly found that CXCL4 could induce the migration of CCR1-expressing cells and also induce CCR1 endocytosis. Extending our findings to primary human monocytes, we observed that CXCL4 induced CCR1 endocytosis and could induce monocyte chemotaxis in a CCR1 antagonist-sensitive manner. Collectively, our data identify CCR1 as a previously elusive monocyte CXCL4 receptor and suggest that CCR1 may play a role in inflammation where the release of CXCL4 is implicated.

First thought to orchestrate exclusively leukocyte trafficking, chemokines are now acknowledged for their multiple roles in the regulation of cell proliferation, differentiation, and survival. Dysregulation of their normal functions contributes to various pathologies, including inflammatory diseases and cancer. The two chemokine receptor 3 variants CXCR3-A and CXCR3-B, together with their cognate chemokines (CXCL11, CXCL10, CXCL9, CXCL4, and CXCL4L1), are involved in the control but also in the development of many tumors. CXCR3-A drives the infiltration of leukocytes to the tumor bed to modulate tumor progression (paracrine axis). Conversely, tumor-driven changes in the expression of the CXCR3 variants and their ligands promote cancer progression (autocrine axis). This review summarizes the anti- and pro-tumoral activities of the CXCR3 variants and their associated chemokines with a focus on the understanding of their distinct biological roles in the tumor microenvironment.

BACKGROUND

Photochemical treatment (PCT) for pathogen reduction of platelet concentrates (PCs) affects all cells containing DNA and/or RNA. Soluble mediators, which may cause transfusion reactions, are determined by the balance between secretion and/or cell destruction and binding and/or degradation.

METHODS

Ten double-dose single-donor leukoreduced PCs were split in two identical units. Two study arms were created: Study Arm A consisting of five PCT PCs with corresponding untreated control PCs and Study Arm B consisting of five PCT PCs with corresponding gamma-irradiated control PCs. PCs that had added PAS-III (Intersol) were treated with amotosalen and ultraviolet A light. Corresponding controls PCs, to which PAS-II (T-sol) were added, received no treatment or were gamma-irradiated before storage. Platelet (PLT)-derived (CCL5/RANTES, CXCL4/PF4, CCL3/MIP-1alpha, transforming growth factor [TGF]-beta, CXCL8/interleukin [IL]-8, IL-1beta) as well as white blood cell (WBC)-associated (IL-6, IL-10, IL-11, IL-12, tumor necrosis factor, interferon-gamma) cytokines were investigated by enzyme-linked immunosorbent assay and cytometric bead array during storage for up to 12 days.

RESULTS

Independent of previous treatment we observed that all concentrates showed low levels of WBC-associated cytokines. PLT-derived cytokines were detected at higher levels and showed significant increase during storage. Statistical analysis showed lower PLT content per unit in PCT PCs, higher levels of activation variables in PCT PCs, and higher levels and accumulation rate of CCL5, CXCL4, TGF-beta, and CXCL8 in PCT PCs.

CONCLUSIONS

PLTs are the main source of released cytokines during storage of untreated, gamma-irradiated, and PCT PCs. PCT may affect the level of PLT-derived cytokines in PCs. No additional reduction of WBC-associated cytokines were observed after PCT in prestorage leukoreduced PCs.

Signal transduction mechanisms associated with neutrophil activation by platelet factor 4 (PF4; CXCL4) are as yet poorly characterized. In a recent report, we showed that PF4-induced neutrophil functions (such as adhesion and secondary granule exocytosis) involve the activation of Src-kinases. By analyzing intracellular signals leading to adherence, we here demonstrate by several lines of evidence that in addition to Src-kinases, PF4 signaling involves the monomeric GTPase Ras, the tyrosine kinase Syk, and the MAP kinase JNK. Furthermore, on stimulation, GTPases Rac2 and RhoA were activated, and each was translocated to a different membrane compartment. As shown by inhibitor studies, Rac2 and JNK are located downstream of Syk and Ras. Most intriguingly, the latter 2 elements appear to control the activity of Rac2 and JNK independently of each other at different phases of the activation process. Although a first phase of Rac2 and JNK activation of up to 5 minutes is initiated by Ras, the second phase (5-30 minutes) depends predominantly on the activity of Syk. In summary, we describe that coordinated activity of Syk, Ras, and JNK mediates neutrophil adhesion to endothelial cells and that PF4 induces sequential activation of these elements.

Platelet factor-4 (CXCL4/PF-4) is the first chemokine identified to have several biological functions. Notably, CXCL4/PF-4 inhibits endothelial cell proliferation and migration, leading to suppression of angiogenesis. Since angiogenesis is essential for the growth of most primary tumors and their subsequent metastases, it is a target for cancer therapy; due to its multiple functions, CXCL4/PF-4 is a potential clinical anti-tumor agent. This report reviews the mechanisms of CXCL4/PF-4 angiostatic activity, including interference with angiogenic growth factors bFGF-2 and VEGF165, activation of CXCR3B, interactions with integrins, interference with cell cycle, interactions with factors such as VEGF121 and CXCL8/IL-8, and derived molecules of CXCL4/PF-4 with angiostatic and anti-tumoral activities in different models in vivo or in vitro.

Inflammatory cells play important roles in progression of solid neoplasms including ovarian cancers. Tumor-associated macrophages (TAMs) contribute to angiogenesis and immune suppression by modulating microenvironment. Ovarian cancer develops occasionally on the bases of endometriosis, a chronic inflammatory disease. We have recently demonstrated differential expressions of CXCR3 variants in endometriosis and ovarian cancers. In this study, we showed impaired CXCL4 expression in TAMs of ovarian cancers arising in endometriosis. The expressions of CXCL4 and its variant CXCL4L1 were investigated among normal ovaries (n = 26), endometriosis (n = 18) and endometriosis-associated ovarian cancers (EAOCs) composed of clear cell (n = 13) and endometrioid (n = 11) types. In addition, four cases of EAOCs that contained both benign and cancer lesions contiguously in single cysts were investigated in the study. Western blot and quantitative RT-PCR analyses revealed significant downregulation of CXCL4 and CXCL4L1 in EAOCs compared with those in endometriosis. In all EAOCs coexisting with endometriosis in the single cyst, the expression levels of CXCL4 and CXCL4L1 were significantly lower in cancer lesions than in corresponding endometriosis. Histopathological study revealed that CXCL4 was strongly expressed in CD68 (+) infiltrating macrophages of endometriosis. In microscopically transitional zone between endometriosis and EAOC, CD68 (+) macrophages often demonstrated CXCL4 (-) pattern. The majority of CD68 (+) TAMs in overt cancer lesions were negative for CXCL4. Collective data indicate that that CXCL4 insufficiency may be involved in specific inflammatory microenvironment of ovarian cancers arising in endometriosis. Suppression of CXCL4 in cancer lesions is likely to be attributable to TAMs in part.

MKEY, a synthetic cyclic peptide inhibitor of CXCL4-CCL5 heterodimer formation, has been shown to protect against atherosclerosis and aortic aneurysm formation by mediating inflammation, but whether it modulates neuroinflammation and brain injury has not been studied. We therefore studied the role of MKEY in stroke-induced brain injury in mice.

MKEY was injected into mice after stroke with 60 minutes of middle cerebral artery occlusion. Infarct volume and neurological deficit scores were measured. Protein levels of CCL5 and its receptor CCR5 were detected by Western blot and fluorescence-activated cell sorting (FACS), respectively. Numbers of microglia-derived macrophages (MiMΦs) and monocyte-derived MΦs (MoMΦs) in the brain, and their subsets, based on the surface markers CD45, CD11b, CCR2, CX3CR1, and Ly6C, were analyzed by FACS. MΦs and neutrophil infiltration in the ischemic brain were stained with CD68 and myeloperoxidase (MPO), respectively, and assessed by immunofluorescent confocal microscopy. The results showed that expressions of CCL5 and its receptor CCR5, were increased in the ischemic brain after stroke. MKEY injection significantly reduced infarct sizes and improved neurological deficit scores measured 72 hours after stroke. In addition, MKEY injection inhibited the number of MoMΦs, but not MiMΦs, in the ischemic brain. Furthermore, MKEY inhibited protein expression levels of Ly6C,CCR2, and CX3CR1 on MoMΦs. Lastly, the confocal study also suggests that the number of CD68-positive MΦs and MPO-positive neutrophils was inhibited by MKEY injection.

MKEY injection protects against stroke-induced brain injury, probably by inhibiting MoMΦ-mediated neuroinflammation.

Bone marrow-derived myeloid cells can form a premetastatic niche and provide a tumor-promoting microenvironment. However, subsets of myeloid cells have also been reported to have anti-tumor properties. It is not clear whether there is a transition between anti- and pro- tumor function of these myeloid cells, and if so, what are the underlying molecular mechanisms. Here we report platelet factor 4 (PF4), or CXCL4, but not the other family members CXCL9, 10, and 11, was produced at higher levels in the normal lung and early stage premetastatic lungs but decreased in later stage lungs. PF4 was mostly produced by Ly6G+CD11b+ myeloid cell subset. Although the number of Ly6G+CD11b+ cells was increased in the premetastatic lungs, the expression level of PF4 in these cells was decreased during the metastatic progression. Deletion of PF4 (PF4 knockout or KO mice) led an increased metastasis suggesting an inhibitory function of PF4. There were two underlying mechanisms: decreased blood vessel integrity in the premetastatic lungs and increased production of hematopoietic stem/progenitor cells (HSCs) and myeloid derived suppressor cells (MDSCs) in tumor-bearing PF4 KO mice. In cancer patients, PF4 expression levels were negatively correlated with tumor stage and positively correlated with patient survival. Our studies suggest that PF4 is a critical anti-tumor factor in the premetastatic site. Our finding of PF4 function in the tumor host provides new insight to the mechanistic understanding of tumor metastasis.

During acute inflammation, monocytes are essential in abolishing invading micro-organisms and encouraging wound healing. Recruitment by CC chemokines is an important step in targeting monocytes to the inflamed tissue. However, cell surface expression of the corresponding chemokine receptors is subject to regulation by various endogenous stimuli which so far have not been comprehensively identified. We report that the platelet-derived CXC chemokine ligand 4 (CXCL4), a known activator of human monocytes, induces down-regulation of CC chemokine receptors (CCR) 1, -2, and -5, resulting in drastic impairment of monocyte chemotactic migration towards cognate CC chemokine ligands (CCL) for these receptors. Interestingly, CXCL4-mediated down-regulation of CCR1, CCR2 and CCR5 was strongly dependent on the chemokine's ability to stimulate autocrine/paracrine release of TNF-α. In turn, TNF-α induced the secretion CCL3 and CCL4, two chemokines selective for CCR1 and CCR5, while the secretion of CCR2-ligand CCL2 was TNF-α-independent. Culture supernatants of CXCL4-stimulated monocytes as well as chemokine-enriched preparations thereof reproduced CXCL4-induced CCR down-regulation. In conclusion, CXCL4 may act as a selective regulator of monocyte migration by stimulating the release of autocrine, receptor-desensitizing chemokine ligands. Our results stress a co-ordinating role for CXCL4 in the cross-talk between platelets and monocytes during early inflammation.

The CXCL4 paralog CXCL4L1 is a less studied chemokine that has been suggested to exert an antiangiogenic function. However, CXCL4L1 is also expressed in patient tumors, tumor cell lines, and murine xenografts, prompting a more detailed analysis of its role in cancer pathogenesis. We used genetic and antibody-based approaches to attenuate CXCL4L1 in models of pancreatic ductal adenocarcinoma (PDAC). Mechanisms of expression were assessed in cell coculture experiments, murine, and avian xenotransplants, including through an evaluation of CpG methylation and mutation of critical CpG residues. CXCL4L1 gene expression was increased greatly in primary and metastatic PDAC. We found that myofibroblasts triggered cues in the tumor microenvironment, which led to induction of CXCL4L1 in tumor cells. CXCL4L1 expression was also controlled by epigenetic modifications at critical CpG islands, which were mapped. CXCL4L1 inhibited angiogenesis but also affected tumor development more directly, depending on the tumor cell type. In vivo administration of an mAb against CXCL4L1 demonstrated a blockade in the growth of tumors positive for CXCR3, a critical receptor for CXCL4 ligands. Our findings define a protumorigenic role in PDAC development for endogenous CXCL4L1, which is independent of its antiangiogenic function. Cancer Res; 76(22); 6507-19. ©2016 AACR.

Chemokines affect inflammation and cancer through leukocyte attraction and angiogenesis. Here, we demonstrate that CXCL4L1/platelet factor-4 variant (PF-4var), a highly angiostatic chemokine, is poorly chemotactic for phagocytes and is inducible in monocytes by inflammatory mediators but remained undetectable in macrophages and neutrophils. In addition, CXCL4L1/PF-4var production by mesenchymal tumor cells was evidenced in vitro and in vivo by specific ELISA and immunohistochemistry. CXCL4L1/PF-4var, but not CXCL4/PF-4, was coinduced with the angiogenic chemokine CXCL6/granulocyte chemotactic protein-2 (GCP-2) by cytokines, e.g., IL-1beta and IL-17, in sarcoma cells, but not in diploid fibroblasts. Furthermore, the induction of CXCL6/GCP-2 in endothelial cells by IL-1beta was enhanced synergistically by TNF-alpha but inhibited by IFN-gamma, which synergized with IL-1beta to produce the angiostatic CXCL10/IFN-gamma-induced protein-10. These findings indicate that the equilibrium between angiostatic and angiogenic factors during inflammation and tumor progression is rather complex and differs depending on the chemokine, cell type, and stimulus. Selective intervention in the chemokine network may drastically disturb this delicate balance of angiogenesis and tissue repair. Application of angiostatic CXCL4L1/PF-4var without attraction of protumoral phagocytes may be beneficial in cancer therapy.

The G protein-coupled receptor (GPCR) C-X-C chemokine receptor 3 (CXCR3) is a potential drug target that mediates signaling involved in cancer metastasis and inflammatory diseases. The CXCR3 primary transcript has three potential alternative splice variants and cell-type specific expression results in receptor variants that are believed to have different functional characteristics. However, the molecular pharmacology of ligand binding to CXCR3 alternative splice variants and their downstream signaling pathways remain poorly explored. To better understand the role of the functional consequences of alternative splicing of CXCR3, we measured signaling in response to four different chemokine ligands (CXCL4, CXCL9, CXCL10, and CXCL11) with agonist activity at CXCR3. Both CXCL10 and CXCL11 activated splice variant CXCR3A. Whereas CXCL10 displayed full agonistic activity for Gαi activation and extracellular signal regulated kinase (ERK) 1/2 phosphorylation and partial agonist activity for β-arrestin recruitment, CXCL9 triggered only modest ERK1/2 phosphorylation. CXCL11 induced CXCR3B-mediated β-arrestin recruitment and little ERK phosphorylation. CXCR3Alt signaling was limited to modest ligand-induced receptor internalization and ERK1/2 phosphorylation in response to chemokines CXCL11, CXCL10, and CXCL9. These results show that CXCR3 splice variants activate different signaling pathways and that CXCR3 variant function is not redundant, suggesting a mechanism for tissue specific biased agonism. Our data show an additional layer of complexity for chemokine receptor signaling that might be exploited to target specific CXCR3 splice variants.

Activated platelets and neutrophils exacerbate atherosclerosis. Platelets release the chemokines CXCL4, CXCL4L1 and CCL5, whereas myeloperoxidase (MPO) and azurocidin are neutrophil-derived. We investigated whether plasma levels of these platelet and neutrophil mediators are affected by the acute coronary syndrome (ACS), its medical treatment, concomitant clinical or laboratory parameters, and predictive for the progression of coronary artery disease (CAD). In an observational study, the association of various factors with plasma concentrations of platelet chemokines and neutrophil mediators in 204 patients, either upon admission with ACS and 6 hours later or without ACS or CAD, was determined by multiple linear regression. Mediator release was further analysed after activation of blood with ACS-associated triggers such as plaque material. CXCL4, CXCL4L1, CCL5, MPO and azurocidin levels were elevated in ACS. CXCL4 and CCL5 but not CXCL4L1 or MPO were associated with platelet counts and CRP. CXCL4 (in association with heparin treatment) and MPO declined over 6 hours during ACS. Elevated CCL5 was associated with a progression of CAD. Incubating blood with plaque material, PAR1 and PAR4 activation induced a marked release of CXCL4 and CCL5, whereas CXCL4L1 and MPO were hardly or not altered. Platelet chemokines and neutrophil products are concomitantly elevated in ACS and differentially modulated by heparin treatment. CCL5 levels during ACS predict a progression of preexisting CAD. Platelet-derived products appear to dominate the inflammatory response during ACS, adding to the emerging evidence that ACS per se may promote vascular inflammation.