CXC chemokines are involved in chemotaxis, regulation of cell growth, induction of apoptosis and modulation of angiostatic effects. CXCL9, CXCL10, CXCL11, CXCL4 and its variant CXCL4L1 are members of the CXC chemokine family, which bind to the CXCR3 receptor to exert their biological effects. These chemokines are associated with a variety of human diseases including chronic inflammation, immune dysfunction, cancer and metastasis. In this review, we focus on accumulating evidence demonstrating the pivotal role of CXCR3 in tumor progression. Its effects are mediated directly in tumor cells or indirectly through the regulation of angiogenesis and tumor immunity. Understanding the emerging role of CXCR3 and its signaling mechanisms further validates this receptor as a biomarker and therapeutic target for tumor progression and tumor angiogenesis.

Chemokines are a family of small proteins that have significant roles in inflammation, angiogenesis and cellular homing. Since inflammation and hemostasis/thrombosis have multiple overlapping roles and pathways, one could expect that some chemokines would also have biologically significant roles in hemostasis/thrombosis as well. This would especially be true for chemokines that are localized solely or predominantly within platelets and released in large amounts at sites of platelet activation such as platelet factor 4 (PF4, CXCL4) and its closely related chemokine, platelet basic protein (PBP, CXCL7). Our group and others have clearly demonstrated an in vivo role for PF4 in hemostasis/thrombosis, but not for PBP, which in contrast has clear proinflammatory properties. This review will focus on PF4 and its potential roles in hemostasis/thrombosis and the underlying pathways by which PF4 may be especially important in such pathologic thrombotic states as heparin-induced thrombocytopenia (HIT) and septic shock.

Atherosclerosis is a chronic inflammatory disease of the arterial wall and an increasing body of evidence suggests that the immune system actively participates in the initiation, progression and persistence of atherosclerosis. Different types of leukocytes such as T and B lymphocytes, natural killer cells (NK) and NKT cells, macrophages, dendritic cells and mast cells have been found within atherosclerosis-prone aortas. The mechanisms of monocyte recruitment have been partially characterized and involve P-selectin, E-selectin, VCAM-1, ICAM-1 and JAM-A. CXCL1, CCL5, CXCL4, CXCL7 and MIF are also implicated in monocyte trafficking into aortas. Recently it has been reported that Ly6C(high) and Ly6C(low) monocyte subsets differently use CCL2, CX3CL1 and CCL5 for their homing into atherosclerotic aortas. T and B lymphocytes constitutively migrate into the normal and atherosclerotic aortic wall in an L-selectin-dependent manner. Recent studies suggest an important role of CCL5, CXCL10, CXCL16, CXCR6 and MIF in T cell influx into the atherosclerotic wall. However, there is little information available on the mechanisms of recruitment of other types of the immune cells such as NK, NKT and mast cells. In this review we shall summarize what is known about leukocyte recruitment into the aortic wall during atherosclerosis with a focus on mouse model systems.

During atherogenesis, blood monocytes transmigrate into the subendothelial space and differentiate toward macrophages and foam cells. The major driver of monocyte-macrophage differentiation is macrophage colony-stimulating factor (M-CSF). M-CSF-induced macrophages are important promoters of atherogenesis as demonstrated in M-CSF and M-CSF receptor knock out mice. However, M-CSF is not the only relevant promoter of macrophage differentiation. The platelet chemokine CXCL4 also prevents monocyte apoptosis and promotes macrophage differentiation in vitro. It is secreted from activated platelets and has effects on various cell types relevant in atherogenesis. Knocking out the Pf4 gene coding for CXCL4 in Apoe(-/-) mice leads to reduced atherogenesis. Thus, it seems likely that CXC4-induced macrophages may have specific pro-atherogenic capacities. We have studied CXC4-induced differentiation of human macrophages using gene chips, systems biology, and functional in vitro and ex vivo experiments. Our data indicate that CXCL4-induced macrophages are distinct from both their M-CSF-induced counterparts and other known macrophage polarizations like M1 macrophages (induced by lipopolysaccharide and interferon-gamma) or M2 macrophages (induced by interleukin-4). CXCL4-induced macrophages have distinct phenotypic and functional characteristics, e.g., the complete loss of the hemoglobin-haptoglobin (Hb-Hp) scavenger receptor CD163 which is necessary for effective hemoglobin clearance after plaque hemorrhage. Lack of CD163 is accompanied by the inability to upregulate the atheroprotective enzyme heme oxygenase-1 in response to Hb-Hp complexes. This review covers the current knowledge about CXCL4-induced macrophages. Based on their unique properties, we have suggested to call these macrophages "M4." CXCL4 may represent an important orchestrator of macrophage heterogeneity within atherosclerotic lesions. Further dissecting its effects on macrophage differentiation may help to identify novel therapeutic targets in atherogenesis.

BACKGROUND

Carcinoma cells must circumvent the normally suppressive signals to disseminate. While often considered 'stop' signals for adherent cells, CXCR3-binding chemokines have recently been correlated positively with cancer progression though the molecular basis remains unclear.

RESULTS

Here, we examined the expression and function of two CXCR3 variants in human prostate cancer biopsies and cell lines. Globally, both CXCR3 mRNA and protein were elevated in localized and metastatic human cancer biopsies compared to normal. Additionally, CXCR3A mRNA level was upregulated while CXCR3B mRNA was downregulated in these prostate cancer specimens. In contrast to normal prostate epithelial cells (RWPE-1), CXCR3A was up to half the receptor in the invasive and metastatic DU-145 and PC-3 prostate cancer cells, but not in the localized LNCaP cells. Instead of inhibiting cell migration as in RWPE-1 cells, the CXCR3 ligands CXCL4/PF4 and CXCL10/IP10 promoted cell motility and invasiveness in both DU-145 and PC-3 cells via PLCβ3 and μ-calpain activation. CXCR3-mediated diminution of cell motility in RWPE-1 cells is likely a result of cAMP upregulation and m-calpain inhibition via CXCR3B signal transduction. Interestingly, overexpression of CXCR3B in DU-145 cells decreased cell movement and invasion.

CONCLUSIONS

These data suggest that the aberrant expression of CXCR3A and down-regulation of CXCR3B may switch a progression "stop" to a "go" signal to promote prostate tumor metastasis via stimulating cell migration and invasion.

Murine CD4 and CD8 T cells express predominantly types 1 and 4 sphingosine 1-phosphate (S1P) G protein-coupled receptors (designated S1P1 and S1P4 or previously endothelial differentiation gene-encoded 1 and 6) for S1P, which has a normal plasma concentration of 0.1-1 microM. S1P now is shown to enhance chemotaxis of CD4 T cells to CCL-21 and CCL-5 by up to 2.5-fold at 10 nM to 0.1 microM, whereas 0.3-3 microM S1P inhibits this chemotaxis by up to 70%. Chemotaxis of S1P(1), but not S1P(4), transfectants to CXCL1 and CXCL4 was similarly affected by S1P. Activation of CD4 T cells, which decreases S1P receptor expression, suppressed effects of S1P on chemotaxis. Pretreatment of labeled CD4 T cells with S1P before reintroduction into mice inhibited by a maximum of 75% their migration into chemokine-challenged s.c. air pouches. The S1P-S1P(1) receptor axis thus controls recruitment of naive T cells by maintaining their response threshold to diverse lymphotactic factors.

BACKGROUND

CXCL4 is a platelet-derived chemokine that promotes macrophage differentiation from monocytes. Deletion of the PF4 gene that encodes CXCL4 reduces atherosclerotic lesions in ApoE(-/-) mice.

OBJECTIVE

We sought to study effects of CXCL4 on macrophage differentiation with possible relevance for atherogenesis.

RESULTS

Flow cytometry for expression of surface markers in macrophage colony-stimulating factor (M-CSF)- and CXCL4-induced macrophages demonstrated virtually complete absence of the hemoglobin scavenger receptor CD163 in CXCL4-induced macrophages. mRNA for CD163 was downregulated as early as 2 hours after CXCL4. CD163 protein reached a minimum after 3 days, which was not reversed by treatment of cells with M-CSF. The CXCL4 effect was entirely neutralized by heparin, which bound CXCL4 and prevented CXCL4 surface binding to monocytes. Pretreatment of cells with chlorate, which inhibits glycosaminoglycan synthesis, strongly inhibited CXCL4-dependent downregulation of CD163. Similar to recombinant CXCL4, releasate from human platelets also reduced CD163 expression. CXCL4-differentiated macrophages were unable to upregulate the atheroprotective enzyme heme oxygenase-1 at the RNA and protein level in response to hemoglobin-haptoglobin complexes. Immunofluorescence of human atherosclerotic plaques demonstrated presence of both CD68+CD163+ and CD68+CD163- macrophages. PF4 and CD163 gene expression within human atherosclerotic lesions were inversely correlated, supporting the in vivo relevance of CXCL4-induced downregulation of CD163.

CONCLUSIONS

CXCL4 may promote atherogenesis by suppressing CD163 in macrophages, which are then unable to upregulate the atheroprotective enzyme heme oxygenase-1 in response to hemoglobin.

Undoubtedly, platelets are key elements in the regulation of thrombosis and haemostasis. Along with their primary task to prevent blood loss from injured vessels, platelets have emerged as regulators of a variety of processes in the vasculature. Multiple challenges, from the contact and adhesion to subendothelial matrix after injury of the vessel wall, to interactions with blood cells in inflammatory conditions, result in platelet activation with concomitant shape change and release of numerous substances. Among these, chemokines have been found to modulate several processes in the vasculature, such as atherosclerosis and angiogenesis. In particular, the chemokines connective tissue activating protein III (CTAP-III) and its precursors, or truncation products (CXCL7), platelet factor 4, (PF4, CXCL4) and its variant PF4alt (CXCL4L1) or regulated upon activation and normal T cell expressed and secreted (RANTES, CCL5), have been investigated thoroughly. Defined common properties as their aptitude to bind glycosaminoglycans or their predisposition to associate and form homooligomers are pre-requisites for their role in the vasculature and function in vivo. The current review summarizes the development of these single chemokines, and their cooperative effects that may in part be dependent on their physical interactions.

The present study characterized platelet secretion and surface expression of proangiogenic stromal cell-derived factor-1α (SDF-1α) and vascular endothelial growth factor (VEGF) and antiangiogenic PF4 and endostatin on activation. The angiogenic factors presented in randomly distributed granules in resting platelets, which were peripherized on activation. Confocal and immunogold electron microscopy demonstrated that SDF-1α/CXCL12 and PF4/CXCL4 mostly present in different granules. Platelet activation induced marked SDF-1α and endostatin but mild PF4 or no VEGF surface expression. PAR1-activating peptide (PAR1-AP), adenosine diphosphate (via P2Y1/P2Y12), and glycoprotein VI-targeting collagen-related peptide induced massive SDF-1α and VEGF but modest PF4 or no endostatin release. In contrast, PAR4-AP triggered marked PF4 and sole endostatin release but limited SDF-1α or VEGF secretion. Distinct platelet release of SDF-1α and endostatin involved different engagements of intracellular signaling pathways. In conclusion, different platelet stimuli evoke distinct secretion and surface expression of proangiogenic and antiangiogenic factors. PAR1, adenosine diphosphate, and glycoprotein VI stimulation favors proangiogenic, whereas PAR4 promotes antiangiogenic, factor release.

Platelets restrict the growth of intraerythrocytic malaria parasites by binding to parasitized cells and killing the parasite within. Here, we show that the platelet molecule platelet factor 4 (PF4 or CXCL4) and the erythrocyte Duffy-antigen receptor (Fy) are necessary for platelet-mediated killing of Plasmodium falciparum parasites. PF4 is released by platelets on contact with parasitized red cells, and the protein directly kills intraerythrocytic parasites. This function for PF4 is critically dependent on Fy, which binds PF4. Genetic disruption of Fy expression inhibits binding of PF4 to parasitized cells and concomitantly prevents parasite killing by both human platelets and recombinant human PF4. The protective function afforded by platelets during a malarial infection may therefore be compromised in Duffy-negative individuals, who do not express Fy.

The chemokine CXCL4/platelet factor-4 is released by activated platelets in micromolar concentrations and is a chemoattractant for leukocytes via an unidentified receptor. Recently, a variant of the human chemokine receptor CXCR3 (CXCR3-B) was described, which transduced apoptotic but not chemotactic signals in microvascular endothelial cells following exposure to high concentrations of CXCL4. Here, we show that CXCL4 can induce intracellular calcium release and the migration of activated human T lymphocytes. CXCL4-induced chemotaxis of T lymphocytes was inhibited by a CXCR3 antagonist and pretreatment of cells with pertussis toxin (PTX), suggestive of CXCR3-mediated G-protein signaling via Galphai-sensitive subunits. Specific binding by T lymphocytes of the CXCR3 ligand CXCL10 was not effectively competed by CXCL4, suggesting that the two are allotopic ligands. We subsequently used expression systems to dissect the potential roles of each CXCR3 isoform in mediating CXCL4 function. Transient expression of the CXCR3-A and CXCR3-B isoforms in the murine pre-B cell L1.2 produced cells that migrated in response to CXCL4 in a manner sensitive to PTX and a CXCR3 antagonist. Binding of radiolabeled CXCL4 to L1.2 CXCR3 transfectants was of low affinity and appeared to be mediated chiefly by glycosaminoglycans (GAGs), as no specific CXCL4 binding was observed in GAG-deficient 745-Chinese hamster ovary cells stably expressing CXCR3. We suggest that following platelet activation, the CXCR3/CXCL4 axis may play a role in T lymphocyte recruitment and the subsequent amplification of inflammation observed in diseases such as atherosclerosis. In such a setting, antagonism of the CXCR3/CXCL4 axis may represent a useful, therapeutic intervention.

Plasmodium falciparum in a subset of patients can lead to a diffuse encephalopathy known as cerebral malaria (CM). Despite treatment, mortality caused by CM can be as high as 30% while 10% of survivors of the disease may experience short- and long-term neurological complications. The pathogenesis of CM involves alterations in cytokine and chemokine expression, local inflammation, vascular injury and repair processes. These diverse factors have limited the rate of discovery of prognostic predictors of fatal CM. Identification of reliable early predictors of CM severity will enable clinicians to adjust this risk with appropriate management of CM. Recent studies revealed that elevated levels of CXCL10 expression in cerebrospinal fluid and peripheral blood plasma independently predicted severe and fatal CM. CXCR3, a promiscuous receptor of CXCL10, plays an important role in pathogenesis of mouse model of CM. In this study the role of corresponding CXCR3 ligands (CXCL11, CXCL10, CXCL9 & CXCL4) in fatal or severe CM was evaluated by comparing their levels in 16 healthy control (HC), 26 mild malaria (MM), 26 cerebral malaria survivors (CMS) and 12 non-survivors (CMNS) using enzyme linked immunosorbent assay (ELISA). Levels of CXCL4 and CXCL10 were significantly elevated in CMNS patients (p < 0.05) when compared with HC, MM and CMS. Elevated plasma levels of CXCL10 and CXCL4 were tightly associated with CM mortality. Receiver Operating Characteristic (ROC) curve analysis revealed that CXCL4 and CXCL10 can discriminate CMNS from MM (p < 0.0001) and CMS (p <0.0001) with an area under the curve (AUC)=1. These results suggest that CXCL4 and CXCL10 play a prominent role in pathogenesis of CM associated death and may be used as functional or surrogate biomarkers for predicting CM severity.

Many persistent pain states (pain lasting for hours, days, or longer) are poorly treated because of the limitations of existing therapies. Analgesics such as nonsteroidal anti-inflammatory drugs and opioids often provide incomplete pain relief and prolonged use results in the development of severe side effects. Identification of the key mediators of various types of pain could improve such therapies. Here, we tested the hypothesis that hitherto unrecognized cytokines and chemokines might act as mediators in inflammatory pain. We used ultraviolet B (UVB) irradiation to induce persistent, abnormal sensitivity to pain in humans and rats. The expression of more than 90 different inflammatory mediators was measured in treated skin at the peak of UVB-induced hypersensitivity with custom-made polymerase chain reaction arrays. There was a significant positive correlation in the overall expression profiles between the two species. The expression of several genes [interleukin-1β (IL-1β), IL-6, and cyclooxygenase-2 (COX-2)], previously shown to contribute to pain hypersensitivity, was significantly increased after UVB exposure, and there was dysregulation of several chemokines (CCL2, CCL3, CCL4, CCL7, CCL11, CXCL1, CXCL2, CXCL4, CXCL7, and CXCL8). Among the genes measured, CXCL5 was induced to the greatest extent by UVB treatment in human skin; when injected into the skin of rats, CXCL5 recapitulated the mechanical hypersensitivity caused by UVB irradiation. This hypersensitivity was associated with the infiltration of neutrophils and macrophages into the dermis, and neutralizing the effects of CXCL5 attenuated the abnormal pain-like behavior. Our findings demonstrate that the chemokine CXCL5 is a peripheral mediator of UVB-induced inflammatory pain, likely in humans as well as rats.

In skin wounds, the chemokine CXCR3 receptor appears to play a key role in coordinating the switch from regeneration of the ontogenically distinct mesenchymal and epithelial compartments toward maturation. However, because CXCR3 equivalently binds four different ELR-devoid CXC chemokines (ie, PF4/CXCL4, IP-10/CXCL10, MIG/CXCL9, and IP-9/CXCL11), we sought to identify the ligand that coordinates epidermal coverage with the maturation of the underlying superficial dermis. Because CXCL11 (IP-9 or I-TAC) is produced by redifferentiating keratinocytes late in the regenerative phase when re-epithelialization is completed and matrix maturation ensues, we generated mice in which an antisense construct (IP-9AS) eliminated IP-9 expression during the wound-healing process. Both full and partial thickness excisional wounds were created and analyzed histologically throughout a 2-month period. Wound healing was impaired in the IP-9AS mice, with a hypercellular and immature dermis noted even after 60 days. Re-epithelialization was delayed with a deficient delineating basement membrane persisting in mice expressing the IP-9AS construct. Provisional matrix components persisted in the dermis, and the mature basement membrane components laminin V and collagen IV were severely diminished. Interestingly, the inflammatory response was not diminished despite IP-9/I-TAC being chemotactic for such cells. We conclude that IP-9 is a key ligand in the CXCR3 signaling system for wound repair, promoting re-epithelialization and modulating the maturation of the superficial dermis.

We investigated possible cellular receptors for the human CXC chemokine platelet factor-4 variant/CXCL4L1, a potent inhibitor of angiogenesis. We found that CXCL4L1 has lower affinity for heparin and chondroitin sulfate-E than platelet factor-4 (CXCL4) and showed that CXCL10 and CXCL4L1 could displace each other on microvascular endothelial cells. Labeled CXCL4L1 also bound to CXCR3A- and CXCR3B-transfectants and was displaced by CXCL4L1, CXCL4, and CXCL10. The CXCL4L1 anti-angiogenic activity was blocked by anti-CXCR3 antibodies (Abs) in the Matrigel and cornea micropocket assays. CXCL4L1 application in CXCR3(-/-) or in wild-type mice treated with neutralizing anti-CXCR3 Abs, resulted in reduced inhibitory activity of CXCL4L1 on tumor growth and vascularization of Lewis lung carcinoma. Furthermore, CXCL4L1 and CXCL4 chemoattracted activated T cells, human natural killer cells, and human immature dendritic cells (DCs). Migration of DCs toward CXCL4 and CXCL4L1 was desensitized by preincubation with CXCL10 and CXCL11, inhibited by pertussis toxin, and neutralized by anti-CXCR3 Abs. Chemotaxis of T cells, natural killer cells, and DCs is likely to contribute to the antitumoral action. However, the in vivo data indicate that the angiostatic property of CXCL4L1 is equally important in retarding tumor growth. Thus, both CXCR3A and CXCR3B are implicated in the chemotactic and vascular effects of CXCL4L1.

Myelodysplastic syndromes and chronic myelomonocytic leukemia (CMML) are characterized by mutations in genes encoding epigenetic modifiers and aberrant DNA methylation. DNA methyltransferase inhibitors (DMTis) are used to treat these disorders, but response is highly variable, with few means to predict which patients will benefit. Here, we examined baseline differences in mutations, DNA methylation, and gene expression in 40 CMML patients who were responsive or resistant to decitabine (DAC) in order to develop a molecular means of predicting response at diagnosis. While somatic mutations did not differentiate responders from nonresponders, we identified 167 differentially methylated regions (DMRs) of DNA at baseline that distinguished responders from nonresponders using next-generation sequencing. These DMRs were primarily localized to nonpromoter regions and overlapped with distal regulatory enhancers. Using the methylation profiles, we developed an epigenetic classifier that accurately predicted DAC response at the time of diagnosis. Transcriptional analysis revealed differences in gene expression at diagnosis between responders and nonresponders. In responders, the upregulated genes included those that are associated with the cell cycle, potentially contributing to effective DAC incorporation. Treatment with CXCL4 and CXCL7, which were overexpressed in nonresponders, blocked DAC effects in isolated normal CD34+ and primary CMML cells, suggesting that their upregulation contributes to primary DAC resistance.

The platelet factor-4 variant, designated PF-4var/CXCL4L1, is a recently described natural non-allelic gene variant of the CXC chemokine platelet factor-4/CXCL4. PF-4var/CXCL4L1 was cloned, and the purified recombinant protein strongly inhibited angiogenesis. Recombinant PF-4var/CXCL4L1 was angiostatically more active (at nanomolar concentration) than PF-4/CXCL4 in various test systems, including wound-healing and migration assays for microvascular endothelial cells and the rat cornea micropocket assay for angiogenesis. Furthermore, PF-4var/CXCL4L1 more efficiently inhibited tumor growth in animal models of melanoma and lung carcinoma than PF-4/CXCL4 at an equimolar concentration. For B16 melanoma in nude mice, a significant reduction in tumor size and the number of small i.t. blood vessels was obtained with i.t. applied PF-4var/CXCL4L1. For A549 adenocarcinoma in severe combined immunodeficient mice, i.t. PF-4var/CXCL4L1 reduced tumor growth and microvasculature more efficiently than PF-4/CXCL4 and prevented metastasis to various organs better than the angiostatic IFN-inducible protein 10/CXCL10. Finally, in the syngeneic model of Lewis lung carcinoma, PF-4var/CXCL4L1 inhibited tumor growth equally well as monokine induced by IFN-gamma (Mig)/CXCL9, also known to attract effector T lymphocytes. Taken together, PF-4var/CXCL4L1 is a highly potent antitumoral chemokine preventing development and metastasis of various tumors by inhibition of angiogenesis. These data confirm the clinical potential of locally released chemokines in cancer therapy.

The goal to attenuate inflammation without inducing generalized immunosuppression has focused the attention on chemokines, a family of chemotactic peptides that regulate the leukocyte traffick into tissues. However, the development of drugs that block ckemokine activity may be hampered by the observation that some chemokines display pleiotropic biologic functions. For example, the chemokines CXCL9/Mig, CXCL10/IP-10, and CXCL11/I-TAC exhibit the ability to recruit different leukocytes subsets, the capacity to induce the proliferation of vascular pericytes as well as powerful anti-tumor effects, which are mediated by a common receptor, named CXCR3. Because of their pleiotropic biologic effects, these chemokines have been proposed as possible therapeutic targets in cancer, allograft rejection, glomerulonephritis, diabetes, multiple sclerosis, and autoimmune disorders of the thyroid. The chemokine CXCL4/PF4 shares several activities with CXCL9, CXCL10, and CXCL11, including angiostatic effects, although its specific receptor has remained unknown for a long time. Recently, we provided evidence that the different functions of CXCL9, CXCL10, and CXCL11 on distinct cell types can be at least partly explained by the interaction of these chemokines with two distinct receptors. Indeed, in addition to the classic form of CXCR3 receptor, which we have renamed as CXCR3-A, a novel CXCR3 receptor variant (CXCR3-B) was identified, that not only mediates the angiostatic activity of CXCR3 ligands, but also acts as functional receptor for CXCL4. In this review, we focus on the accumulating evidence demonstrating the pivotal role of CXCR3-binding chemokines in several human diseases. Studies based on CXCR3 targeting have shown its importance in different pathologic conditions and orally active small molecules capable of inhibiting this receptor are now being developed in order to be tested for their activity in humans.

Chemokines binding the CXCR3 receptor have been shown to inhibit angiogenesis via the CXCR3-B isoform, but the underlying molecular mechanisms are unknown. Aim of this study was to elucidate the effects of CXCR3-B on activation of members of the mitogen-activated protein kinase family, and to explore the relevance of defined signaling pathways to the angiostatic effects of CXCR3-B ligands. Human embryonic kidney (HEK) 293 cells were transfected with expression vectors encoding for CXCR3-A or CXCR3-B. In cells expressing CXCR3-A, CXCL10 (IP-10) at nanomolar concentrations induced activation of ERK, Akt, and Src, as previously described in human vascular pericytes. In HEK-293 cells expressing CXCR3-B, exposure to CXCL10 in the micromolar concentration range led to activation of the p38(MAPK) pathway, as indicated by phosphorylation of p38(MAPK) itself, and of MKK3/6 and MAPKAPK-2, that lie upstream and downstream of p38(MAPK), respectively. Similar results were obtained in cells stimulated with CXCL4 (PF4), a specific ligand of CXCR3-B. In contrast, CXCL4 was unable to activate p38(MAPK) in mock-transfected HEK-293 cells. Only a modest induction of ERK or JNK was observed upon CXCR3-B activation. In human microvascular endothelial cells, which selectively express CXCR3-B, in a cell cycle-dependent fashion, CXCL10 and CXCL4 increased the enzymatic activity of p38(MAPK). Pharmacologic inhibition of p38(MAPK) by SB302580 resulted in a significant increase in DNA synthesis and in reversal of the inhibitory action of CXCL10. In conclusion, the p38(MAPK) pathway is a downstream effector of CXCR3-B implicated in the angiostatic action of this chemokine receptor.

OBJECTIVE

For our understanding of the pathogenesis of rheumatoid arthritis (RA), it is important to elucidate the mechanisms underlying early stages of synovitis. Here, synovial cytokine production was investigated in patients with very early arthritis.

METHODS

Synovial biopsies were obtained from patients with at least one clinically swollen joint within 12 weeks of symptom onset. At an 18-month follow-up visit, patients who went on to develop RA, or whose arthritis spontaneously resolved, were identified. Biopsies were also obtained from patients with RA with longer symptom duration (>12 weeks) and individuals with no clinically apparent inflammation. Synovial mRNA expression of 117 cytokines was quantified using PCR techniques and analysed using standard and novel methods of data analysis. Synovial tissue sections were stained for CXCL4, CXCL7, CD41, CD68 and von Willebrand factor.

RESULTS

A machine learning approach identified expression of mRNA for CXCL4 and CXCL7 as potentially important in the classification of early RA versus resolving arthritis. mRNA levels for these chemokines were significantly elevated in patients with early RA compared with uninflamed controls. Significantly increased CXCL4 and CXCL7 protein expression was observed in patients with early RA compared with those with resolving arthritis or longer established disease. CXCL4 and CXCL7 co-localised with blood vessels, platelets and CD68(+) macrophages. Extravascular CXCL7 expression was significantly higher in patients with very early RA compared with longer duration RA or resolving arthritis

CONCLUSIONS

Taken together, these observations suggest a transient increase in synovial CXCL4 and CXCL7 levels in early RA.

Interferon-gamma-inducible protein-10 (IP-10)/CXCL10 is a CXC chemokine that attracts T lymphocytes and NK cells through activation of CXCR3, the only chemokine receptor identified to date that binds IP-10/CXCL10. We have found that several nonhemopoietic cell types, including epithelial and endothelial cells, have abundant levels of a receptor that binds IP-10/CXCL10 with a Kd of 1-6 nM. Surprisingly, these cells expressed no detectable CXCR3 mRNA. Furthermore, no cell surface expression of CXCR3 was detectable by flow cytometry, and the binding of 125I-labeled IP-10/CXCL10 to these cells was not competed by the other high affinity ligands for CXCR3, monokine induced by IFN-gamma/CXCL9, and I-TAC/CXCL11. Although IP-10/CXCL10 binds to cell surface heparan sulfate glycosaminoglycan (GAG), the receptor expressed by these cells is not GAG, since the affinity of IP-10/CXCL10 for this receptor is much higher than it is for GAG, its binding is not competed by platelet factor 4/CXCL4, and it is present on cells that are genetically incapable of synthesizing GAG. Furthermore, in contrast to IP-10/CXCL10 binding to GAG, IP-10/CXCL10 binding to these cells induces new gene expression and chemotaxis, indicating the ability of this receptor to transduce a signal. These high affinity IP-10/CXCL10-specific receptors on epithelial cells may be involved in cell migration and, perhaps, in the spread of metastatic cells as they exit from the vasculature. (All of the lung cancer cells we examined also expressed CXCR4, which has been shown to play a role in breast cancer metastasis.) CXCR3-negative endothelial cells may also use this receptor to mediate the angiostatic activity of IP-10/CXCL10, which is also expressed by these cells in an autocrine manner.

Both chemokine oligomerization and binding to glycosaminoglycans (GAGs) are required for their function in cell recruitment. Interactions with GAGs facilitate the formation of chemokine gradients, which provide directional cues for migrating cells. In contrast, chemokine oligomerization is thought to contribute to the affinity of GAG interactions by providing a more extensive binding surface than single subunits alone. However, the importance of chemokine oligomerization to GAG binding has not been extensively quantified. Additionally, the ability of chemokines to form different oligomers has been suggested to impart specificity to GAG interactions, but most studies have been limited to heparin. In this study, several differentially oligomerizing chemokines (CCL2, CCL3, CCL5, CCL7, CXCL4, CXCL8, CXCL11 and CXCL12) and select oligomerization-deficient mutants were systematically characterized by surface plasmon resonance to determine their relative affinities for heparin, heparan sulfate (HS) and chondroitin sulfate-A (CS-A). Wild-type chemokines demonstrated a hierarchy of binding affinities for heparin and HS that was markedly dependent on oligomerization. These results were corroborated by their relative propensity to accumulate on cells and the critical role of oligomerization in cell presentation. CS-A was found to exhibit greater chemokine selectivity than heparin or HS, as it only bound a subset of chemokines; moreover, binding to CS-A was ablated with oligomerization-deficient mutants. Overall, this study definitively demonstrates the importance of oligomerization for chemokine-GAG interactions, and demonstrates diversity in the affinity and specificity of different chemokines for GAGs. These data support the idea that GAG interactions provide a mechanism for fine-tuning chemokine function.

Surgical repair of the rotator cuff repair is one of the most common procedures in orthopedic surgery. Despite it being the focus of much research, the physiological tendon-bone insertion is not recreated following repair and there is an anatomic non-healing rate of up to 94%. During the healing phase, several growth factors are upregulated that induce cellular proliferation and matrix deposition. Subsequently, this provisional matrix is replaced by the definitive matrix. Leukocyte- and platelet-rich fibrin (L-PRF) contain growth factors and has a stable dense fibrin matrix. Therefore, use of LPRF in rotator cuff repair is theoretically attractive. The aim of the present study was to determine 1) the optimal protocol to achieve the highest leukocyte content; 2) whether L-PRF releases growth factors in a sustained manner over 28 days; 3) whether standard/gelatinous or dry/compressed matrix preparation methods result in higher growth factor concentrations. 1) The standard L-PRF centrifugation protocol with 400 x g showed the highest concentration of platelets and leukocytes. 2) The L-PRF clots cultured in medium showed a continuous slow release with an increase in the absolute release of growth factors TGF-β1, VEGF and MPO in the first 7 days, and for IGF1, PDGF-AB and platelet activity (PF4=CXCL4) in the first 8 hours, followed by a decrease to close to zero at 28 days. Significantly higher levels of growth factor were expressed relative to the control values of normal blood at each culture time point. 3) Except for MPO and the TGFβ-1, there was always a tendency towards higher release of growth factors (i.e., CXCL4, IGF-1, PDGF-AB, and VEGF) in the standard/gelatinous- compared to the dry/compressed group. L-PRF in its optimal standard/gelatinous-type matrix can store and deliver locally specific healing growth factors for up to 28 days and may be a useful adjunct in rotator cuff repair.

The concept of platelets as important players in the process of atherogenesis has become increasingly accepted due to accumulating experimental and clinical evidence. Despite the progress in understanding the molecular details of atherosclerosis, particularly by using animal models, the inflammatory and thrombotic roles of activated platelet s especially in the human system remain difficult to dissect, as often only the complications of atherosclerosis, i.e., stroke and myocardial infarction are definable but not the plaque burden. Platelet indices including platelet count and mean platelet volume (MPV) and soluble mediators released by activated platelets are associated with atherosclerosis. The chemokine CXCL4 has multiple atherogenic activities, e.g., altering the differentiation of T cells and macrophages by inhibiting neutrophil and monocyte apoptosis and by increasing the uptake of oxLDL and synergizing with CCL5. CCL5 is released and deposited on endothelium by activated platelets thereby triggering atherogenic monocyte recruitment, which can be attenuated by blocking the corresponding chemokine receptor CCR5. Atheroprotective and plaque stabilizing properties are attributed to CXCL12, which plays an important role in regenerative processes by attracting progenitor cells. Its release from luminal attached platelets accelerates endothelial healing after injury. Platelet surface molecules GPIIb/IIIa, GP1bα, P-selectin, JAM-A and the CD40/CD40L dyade are crucially involved in the interaction with endothelial cells, leukocytes and matrix molecules affecting atherogenesis. Beyond the effects on the arterial inflammatory infiltrate, platelets affect cholesterol metabolism by binding, modifying and endocytosing LDL particles via their scavenger receptors and contribute to the formation of lipid laden macrophages. Current medical therapies for the prevention of atherosclerotic therapies enable the elucidation of mechanisms linking platelets to inflammation and atherosclerosis.