Wound healing is a complex, orchestrated series of biological events that is controlled by extracellular components that communicate between cell types to re-establish lost tissue. We have found that signaling by ELR-negative CXC chemokines through their common CXCR3 receptor is critical for dermal maturation during the resolving phase. In addition there needs to be complete maturation of the epidermis and regeneration of a delineating basement membrane for proper functioning. The role of this ligand-receptor system appears confounding as one ligand, CXCL4/(PF4), is present during the initial dissolution and two others, CXCL10/(IP-10) and CXCL11/(IP-9/I-TAC), are expressed by keratinocytes in the later regenerative and resolving phases during which the basement membrane is re-established. We examined CXCR3 signaling role in healing using a mouse lacking this receptor, as all three ligands act solely via the common receptor. Reepithelialization was delayed in CXCR3-deficient mice in both full and partial-thickness excisional wounds. Even at 90 days postwounding, the epidermis of these mice appeared less mature with lower levels of E-cadherin and cytokeratin 18. The underlying basement membrane, a product of both dermal fibroblasts and epidermal keratinocytes, was not fully established with persistent diffuse expression of the matrix components laminin 5, collagen IV, and collagen VII throughout the wound bed. These results suggest that CXCR3 and its ligands play an important role in the re-establishment of the basement membrane and epidermis. These studies further establish the emerging signaling network that involves the CXCR3 chemokine receptor and its ligands as a key regulator of wound repair.

Effective ex vivo expansion of hematopoietic stem cells (HSCs) is a prerequisite for HSC transplantation. Growth and maintenance of HSC is dependent on cytokine and niche factors. We investigated whether mesenchymal stem cells (MSCs) or osteogenic cytokine-differentiated MSCs play a role in HSC expansion. We used the human HM3.B10 (B10) MSC cell line and the osteoblast-differentiated B10 (Ost-B10) as a feeder layer and examined ex vivo expansion of CD34(+)CD38(-) HSCs obtained from peripheral blood (PB) and cord blood (CB) with or without several growth cytokines. Both undifferentiated B10 and Ost-B10 cells exhibited similar effects on total HSC expansion; however, Ost-B10 demonstrated a higher potency in CD34(+)CD38(-) cell-specific proliferation in the presence of cytokines compared to undifferentiated B10 HSCs. Colony-forming cell assay and long-term culture initiating cell assay revealed that Ost-B10 displayed multipotent differentiation and enabled long-term ex vivo culture of HSCs. We next examined the relationship between HSC expansion and the presence of various chemokines. CXCL4 and CXCL12 expression were increased in Ost-B10 cells compared with the B10 cells. CD34(+)CD38(-) cells were significantly increased with CXCL12, but not CXCL4 treatment. siRNA inhibition of CXCL12 decreased CXCL12 secretion in both B10 and Ost-B10, whereas expansion of CD34(+)CD38(-) cells was decreased in Ost-B10 alone. These results demonstrated that ex vivo expansion of HSCs may be highly effective through osteoblast-differentiated MSCs acting as a feeder layer, and likely operates through the CXCL12 chemokines signaling pathway.

In this study, we have examined the major platelet-derived CXC chemokines connective tissue-activating peptide III (CTAP-III), its truncation product neutrophil-activating peptide 2 (CXC chemokine ligand 7 (CXCL7)), as well as the structurally related platelet factor 4 (CXCL4) for their impact on neutrophil adhesion to and transmigration through unstimulated vascular endothelium. Using monolayers of cultured HUVEC, we found all three chemokines to promote neutrophil adhesion, while only CXCL7 induced transmigration. Induction of cell adhesion following exposure to CTAP-III, a molecule to date described to lack neutrophil-stimulating capacity, depended on proteolytical conversion of the inactive chemokine into CXCL7 by neutrophils. This was evident from experiments in which inhibition of the CTAP-III-processing protease and simultaneous blockade of the CXCL7 high affinity receptor CXCR-2 led to complete abrogation of CTAP-III-mediated neutrophil adhesion. CXCL4 at substimulatory dosages modulated CTAP-III- as well as CXCL7-induced adhesion. Although cell adhesion following exposure to CTAP-III was drastically reduced, CXCL7-mediated adhesion underwent significant enhancement. Transendothelial migration of neutrophils in response to CXCL7 or IL-8 (CXCL8) was subject to modulation by CTAP-III, but not CXCL4, as seen by drastic desensitization of the migratory response of neutrophils pre-exposed to CTAP-III, which was paralleled by selective down-modulation of CXCR-2. Altogether our results demonstrate that there exist multiple interactions between platelet-derived chemokines in the regulation of neutrophil adhesion and transendothelial migration.

Platelet factor-4 (PF4), a platelet-derived chemokine, has two important functions in the vasculature. It has a pro-atherogenic role while also having anti-angiogenic effects. The activity of platelet factor-4 (PF4), unlike other chemokines that bind to specific receptors, depends on its unusually high affinity for proteoglycans and other negatively charged molecules. High affinity for heparan sulfates was thought to be central to all of PF4's biological functions. However, other mechanisms have been described such as direct growth factor binding, activation of the CXCR3B chemokine receptor isoform that is present in some vascular cells or binding to lipoprotein-related protein-1 (LRP1). Furthermore, PF4 also binds to integrins with affinities similar to matrix molecules. These interactions may explain the effects of PF4 in healthy and pathological tissues. However, the mechanisms involved in PF4's activity are complex and may depend on a given tissue or localisation. Overall, while much is already known about PF4, its specific role in atherosclerosis and angiogenesis remains still to be clarified.

The natural history of HIV-1 infection is highly variable in different individuals, spanning from a rapidly progressive course to a long-term asymptomatic infection. A major determinant of the pace of disease progression is the in vivo level of HIV-1 replication, which is regulated by a complex network of cytokines and chemokines expressed by immune and inflammatory cells. The chemokine system is critically involved in the control of HIV-1 replication by virtue of the role played by specific chemokine receptors, most notably CCR5 and CXCR4, as cell-surface coreceptors for HIV-1 entry; hence, the chemokines that naturally bind such coreceptors act as endogenous inhibitors of HIV-1. Here, we show that the CXC chemokine CXCL4 (PF-4), the most abundant protein contained within the α-granules of platelets, is a broad-spectrum inhibitor of HIV-1 infection. Unlike other known HIV-suppressive chemokines, CXCL4 inhibits infection by the majority of primary HIV-1 isolates regardless of their coreceptor-usage phenotype or genetic subtype. Consistent with the lack of viral phenotype specificity, blockade of HIV-1 infection occurs at the level of virus attachment and entry via a unique mechanism that involves direct interaction of CXCL4 with the major viral envelope glycoprotein, gp120. The binding site for CXCL4 was mapped to a region of the gp120 outer domain proximal to the CD4-binding site. The identification of a platelet-derived chemokine as an endogenous antiviral factor may have relevance for the pathogenesis and treatment of HIV-1 infection.

Platelets are inflammatory anuclear cells with a well-established role in the development and manifestation of atherosclerosis. Activated platelets secrete a plethora of chemokines including CXCL4 or platelet factor 4 (PF4), CCL5, CXCL12 or stromal cell derived factor-1α (SDF-1α), CXCL16 and others, which initiate or promote local inflammatory processes at sites of vascular injury. These processes are mainly mediated by the recruitment of circulating haematopoietic stem cells, neutrophils, monocytes or lymphocytes on vascular wall. Under acute ischemic conditions platelet-derived chemokines may promote the mobilization of bone marrow-derived progenitor cells and their homing at lesion sites. This review focuses on the role of platelet-derived chemokines in inflammation and atherosclerosis. Further, we discuss the clinical value of plasma levels of chemokines in the prognosis of atherosclerotic heart disease.

This study aimed at examining the presence and role of chemokines (angiogenic CCL2/MCP-1 and angiostatic CXCL4/PF-4, CXCL9/Mig, CXCL10/IP-10) in proliferative diabetic retinopathy (PDR). Regulated chemokine production in human retinal microvascular cells (HRMEC) and chemokine levels in vitreous samples from 40 PDR and 29 non-diabetic patients were analyzed. MCP-1, PF-4, Mig, IP-10 and VEGF levels in vitreous fluid from PDR patients were significantly higher than in controls. Except for IP-10, cytokine levels were significantly higher in PDR with active neovascularization and PDR without traction retinal detachment (TRD) than those in inactive PDR, PDR with TRD and control subjects. Exploratory regression analysis identified associations between higher levels of IP-10 and inactive PDR and PDR with TRD. VEGF levels correlated positively with MCP-1 and IP-10. Significant positive correlations were observed between MCP-1 and IP-10 levels. In line with these clinical findings Western blot analysis revealed increased PF-4 expression in diabetic rat retinas. HRMEC produced MCP-1, Mig and IP-10 after stimulation with IFN-γ, IL-1β or lipopolysaccharide. IFN-γ synergistically enhanced Mig and IP-10 production in response to IL-1β or lipopolysaccharide. MCP-1 was produced by HRMEC in response to VEGF treatment and activated HRMEC via the ERK and Akt/PKB pathway. On the other hand, phosphorylation of ERK induced by VEGF and MCP-1 was inhibited by PF-4, Mig and IP-10. In accordance with inhibition of angiogenic signal transduction pathways, PF-4 inhibited in vitro migration of HRMEC. Thus, regulatory roles for chemokines in PDR were demonstrated. In particular, IP-10 might be associated with the resolution of active PDR and the development of TRD.

Myelodysplastic syndromes (MDS) are among the most frequent hematologic malignancies. Patients have a short survival and often progress to acute myeloid leukemia. The diagnosis of MDS can be difficult; there is a paucity of molecular markers, and the pathophysiology is largely unknown. Therefore, we conducted a multicenter study investigating whether serum proteome profiling may serve as a noninvasive platform to discover novel molecular markers for MDS. We generated serum proteome profiles from 218 individuals by MS and identified a profile that distinguishes MDS from non-MDS cytopenias in a learning sample set. This profile was validated by testing its ability to predict MDS in a first independent validation set and a second, prospectively collected, independent validation set run 5 months apart. Accuracy was 80.5% in the first and 79.0% in the second validation set. Peptide mass fingerprinting and quadrupole TOF MS identified two differential proteins: CXC chemokine ligands 4 (CXCL4) and 7 (CXCL7), both of which had significantly decreased serum levels in MDS, as confirmed with independent antibody assays. Western blot analyses of platelet lysates for these two platelet-derived molecules revealed a lack of CXCL4 and CXCL7 in MDS. Subtype analyses revealed that these two proteins have decreased serum levels in advanced MDS, suggesting the possibility of a concerted disturbance of transcription or translation of these chemokines in advanced MDS.

In this study, we investigated cytokine expression during experimental pneumococcal meningitis. Mice were intracisternally infected with Streptococcus pneumoniae and treated with ceftriaxone starting at 24 h after infection. At different time points before and after antibiotic therapy, the cytokine expression pattern was determined in mouse brains using protein arrays. Underlining the power of this method, the meningitis-relevant cytokines interleukin-1beta (IL-1beta), IL-6, KC, macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractant protein-1 (MCP-1/CCL2) were markedly elevated in infected animals. Newly identified proteins during the acute stage of the disease (until 30 h after infection) included lymphotactin (XCL-1), MIP-1gamma (CCL9) and MCP-5 (CCL12), cytokine responsive gene- 2 (CRG-2/CXCL10) and CXCL16, and insulin-like growth factor binding protein 3 (IGFBP3). During later stages, an induction of T-cell activation-3 (TCA-3/CCL1), platelet factor-4 (PF-4/CXCL4) and stromal derived factor-1alpha (SDF-1alpha/CXCL13), and IL-4 was observed. The validity of this method was supported by an additional ELISA analysis of the expression profile of CXCL16 and IGFBP3, which was identical to that observed by protein array. In conclusion, the use of protein array technology led to an extension of the current picture of protein expression in pneumococcal meningitis. Most important, new factors that might play a role in pneumococcal meningitis were identified.

CXC and CC chemokines are involved in numerous biological processes, and their function in situ may be significantly influenced by heterodimer formation, as was recently reported, for example, for CXC chemokines CXCL4/PF4 and CXCL8/IL8 that interact to form heterodimers that modulate chemotactic and cell proliferation activities. Here we used molecular dynamics simulations to determine relative association free energies (overall average and per residue) for homo- and heterodimer pairs of CXC (CXCL4/PF4, CXCL8/IL8, CXCL1/Gro-alpha, and CXCL7/NAP-2) and CC (CCL5/RANTES, CCL2/MCP-1, and CCL8/MCP-2) chemokines. Even though structural homology among monomer folds of all CXC and CC chemokines permits heterodimer assembly, our calculated association free energies depend upon the particular pair of chemokines in terms of the net electrostatic and nonelectrostatic forces involved, as well as (for CC/CXC mixed chemokines) the selection of dimer type (CC or CXC). These relative free energies indicate that association of some pairs of chemokines is more favorable than others. Our approach is validated by correlation of calculated and experimentally determined free energies. Results are discussed in terms of CXC and CC chemokine function and have significant biological implications.

Platelet-derived chemokines, such as regulated on activation, normal T expressed and secreted (RANTES; CC chemokine ligand 5), platelet factor 4 [PF4; CXC chemokine ligand 4 (CXCL4)], and epithelial neutrophil-activating protein 78 (ENA-78; CXCL5), or precursors, such as beta-thromboglobulin, which can be processed to neutrophil-activating protein-2 (NAP-2; CXCL7), may play an important role in monocyte recruitment during atherogenesis. Platelets can deposit chemokines on inflamed endothelium; however, little is known about differential or additive effects of platelet chemokines on monocyte arrest. Here, we demonstrate that preincubation of activated human microvascular endothelial cells (HMVECs) with RANTES, PF4, or NAP-2 but not ENA-78 dose-dependently increased surface immobilization and subsequent monocyte arrest in flow. RANTES was the most potent and efficient arrest chemokine. Pretreatment of HMVECs with beta-thromboglobulin enhanced monocyte arrest in the presence of cathepsin G generating NAP-2. Combined pretreatment of HMVECs with RANTES and PF4 at suboptimal concentrations synergistically increased arrest, and preincubation with chondroitinase ABC abrogated RANTES- and PF4-induced monocyte arrest. This was associated with reduced expression of chondroitin sulfate, RANTES, and PF4 on the HMVEC surface. Perfusion of HMVECs with platelets known to deposit RANTES and PF4 on the endothelial surface enhanced monocyte arrest, which was inhibited by Met-RANTES, chondroitinase, or a blocking antibody to PF4 but not to ENA-78. The relevance of platelet-derived chemokines was confirmed in adhesion assays with activated whole blood, where Met-RANTES and to a lesser extent, antibodies to PF4 and NAP-2 inhibited arrest of CD14-positive monocytes. Thus, multiple platelet-derived chemokines and processable precursors, which can be presented by specific endothelial proteoglycans, may contribute and cooperate differentially to induce monocyte recruitment.

Platelet-derived exosomes mediate platelet atherogenic interactions with endothelial cells and monocytes. A new method for isolation of plasma platelet-derived exosomes is described and used to examine effects of aging and aspirin on exosome cargo proteins. Exosome secretion by purified platelets in vitro did not increase after exposure to thrombin or collagen, as assessed by exosome counts and quantification of the CD81 exosome marker. Thrombin and collagen increased exosome content of α-granule chemokines CXCL4 and CXCL7 and cytoplasmic high-mobility group box 1 (HMGB1) protein, but not membrane platelet glycoprotein VI (GPVI), with dependence on extracellular calcium. Aspirin consumption significantly blocked thrombin- and collagen-induced increases in exosome cargo levels of chemokines and HMGB1, without altering total exosome secretion or GPVI cargo. Plasma platelet-derived exosomes, enriched by absorption with mouse antihuman CD42b [platelet glycoprotein Ib (GPIb)] mAb, had sizes and cargo protein contents similar to those of exosomes from purified platelets. The plasma platelet-derived exosome number is lower and its chemokine and HMGB1 levels higher after age 65 yr. Aspirin consumption significantly suppressed cargo protein levels of plasma platelet-derived exosomes without altering total levels of exosomes. Cargo proteins of human plasma platelet-derived exosomes may biomark platelet abnormalities and in vivo effects of drugs.- Goetzl, E. J., Goetzl, L., Karliner, J. S., Tang, N., Pulliam, L. Human plasma platelet-derived exosomes: effects of aspirin.

The chemokine receptor CXCR3 and its ligands CXCL9, CXCL10 and CXCL11 are involved in variety of inflammatory disorders including multiple sclerosis, rheumatoid arthritis, psoriasis and sarcoidosis. Two alternatively spliced variants of the human CXCR3-A receptor have been described, termed CXCR3-B and CXCR3-alt. Human CXCR3-B binds CXCL9, CXCL10, CXCL11 as well as an additional ligand CXCL4. In contrast, CXCR3-alt only binds CXCL11. We report that CXCL4 induces intracellular calcium mobilization as well as Akt and p44/p42 extracellular signal-regulated kinase phosphorylation, in activated human T lymphocytes. These responses have similar concentration dependence and time-courses to those induced by established CXCR3 agonists. Moreover, phosphorylation of Akt and p44/p42 is inhibited by pertussis toxin, suggesting coupling to Gα(i) protein. Surprisingly, and in contrast with the other CXCR3 agonists, stimulation of T lymphocytes with CXCL4 failed to elicit migratory responses and did not lead to loss of surface CXCR3 expression. Taken together, our findings show that, although CXCL4 is coupled to downstream biochemical machinery, its role in T cells is probably distinct from that of CXCR3-A agonists.

Systemic sclerosis (SSc) is a multisystem life-threatening fibrosing disorder that lacks effective treatment. The link between the inflammation observed in organs such as the skin and profibrotic mechanisms is not well understood. The plasmacytoid dendritic cell (pDC) is a key cell type mediating Toll-like receptor (TLR)-induced inflammation in autoimmune disease patients, including lupus and skin diseases with interface dermatitis. However, the role of pDCs in fibrosis is less clear. We show that pDCs infiltrate the skin of SSc patients and are chronically activated, leading to secretion of interferon-α (IFN-α) and CXCL4, which are both hallmarks of the disease. We demonstrate that the secretion of CXCL4 is under the control of phosphatidylinositol 3-kinase δ and is due to the aberrant presence of TLR8 on pDCs of SSc patients, which is not seen in healthy donors or in lupus pDCs, and that CXCL4 primarily acts by potentiating TLR8- but also TLR9-induced IFN production by pDCs. Depleting pDCs prevented disease in a mouse model of scleroderma and could revert fibrosis in mice with established disease. In contrast, the disease was exacerbated in mice transgenic for TLR8 with recruitment of pDCs to the fibrotic skin, whereas TLR7 only partially contributed to the inflammatory response, indicating that TLR8 is the key RNA-sensing TLR involved in the establishment of fibrosis. We conclude that the pDC is an essential cell type involved in the pathogenesis of SSc and its removal using depleting antibodies or attenuating pDC function could be a novel approach to treat SSc patients.

The development of a megakaryocyte lineage specific Cre deleter, using the Pf4 (CXCL4) promoter (Pf4-Cre), was a significant step forward in the specific analysis of platelet and megakaryocyte cell biology. However, in the present study we have employed a sensitive reporter-based approach to demonstrate that Pf4-Cre also recombines in a significant proportion of both fetal liver and bone marrow hematopoietic stem cells (HSCs), including the most primitive fraction containing the long-term repopulating HSCs. Consequently, we demonstrate that Pf4-Cre activity is not megakaryocyte lineage-specific but extends to other myeloid and lymphoid lineages at significant levels between 15-60%. Finally, we show for the first time that Pf4 transcripts are present in adult HSCs and primitive hematopoietic progenitor cells. These results have fundamental implications for the use of the Pf4-Cre mouse model and for our understanding of a possible role for Pf4 in the development of the hematopoietic lineage.

The CXCL12/CXCR4 axis has been posited widely to have significant roles in many primary tumors and metastases. It is known that CXCR7 can also be engaged by CXCL12, but the exact function of CXCR7 is controversial. This prompted us to investigate the expression, specific function and signal transduction of CXCR7 in hepatocellular carcinoma (HCC). In this study, CXCR7 and CXCR4 were differentially expressed in nine cell lines of HCC, and that elevated expression of both CXCR7 and CXCL4 were correlated with highly metastatic ability of HCC cells. Moreover, CXCR7 expression was significantly upregulated in metastatic HCC samples compared with the non-metastatic ones by staining of high-density tissue microarrays constructed from a cohort of 48 human HCC specimens. CXCR7 overexpression enhanced cell growth and invasiveness in vitro, and tumorigenicity and lung metastasis in vivo. By contrast, CXCR7 stable knockdown markedly reduced these malignant behaviors. In addition, it was observed that alterations in CXCR7 expression were positively correlated with the phosphorylation levels of mitogen-activated protein kinase (MAPK) pathway proteins. Targeting extracellular regulated kinase pathway by using U0126 inhibitor or using CCX771, a selective CXCR7 antagonist, drastically reduced CXCR7-mediated cell proliferation. Importantly, by using human biotin-based antibody arrays, several differentially expressed proteins were identified in CXCR7-overexpression and depletion groups. Comparative analysis indicated that upstream regulators including TP53 and IL-6 were involved in CXCR7 signal transduction. CXCR7 expression was further proved to regulate expression of vascular endothelial growth factor A and galectin-3, which may contribute to tumor angiogenesis and invasiveness. Consequently, elevated expression of CXCR7 contributes to HCC growth and invasiveness via activation of MAPK and angiogenesis signaling pathways. Targeting CXCR7 may prevent metastasis and provide a potential therapeutic strategy for HCC.

OBJECTIVE

Leukocyte invasion in the arterial wall is critical in the development of atherosclerotic lesions. This review describes recent advances in the understanding of leukocyte recruitment in atherogenesis and in the development of vulnerable plaque. It also discusses limitations in the current knowledge of this process and how these limitations may be addressed.

RESULTS

The adhesive function of platelets has recently been highlighted as an important recruitment mechanism in atherosclerosis. For example, targeted deficiency of P-selectin in platelets reduces atherosclerosis in mice. Platelets also increase monocyte recruitment in atherosclerosis by secreting chemokines such as platelet factor 4 (CXCL4) or RANTES (CCL5), which trigger monocyte arrest in atherosclerotic arteries. A causal role for RANTES in atherosclerosis was shown by a protective effect of the blockage of RANTES receptors in apolipoprotein E-deficient mice. A similar effect was also demonstrated for the fractalkine receptor CX3CR1. Moreover, the classic chemoattractant LTB4 plays important roles in atherosclerosis, inasmuch as the absence of the principal LTB4 receptor (BLT1) reduces early atherosclerosis in mice. Novel data have also shown that many types of cells in lesions express 5-lipoxygenase, which indicates a rich source of leukotrienes in plaque.

CONCLUSIONS

Recent data provide evidence for the involvement of several adhesive and signalling mechanisms in leukocyte recruitment in atherosclerosis. However, the specific mechanisms that are responsible for the accumulation of proatherogenic leukocytes in lesions are unclear. Detailed study of certain subclasses of leukocytes in the recruitment process will be important in future studies in this field.

BACKGROUND

Two variants of the CXCR3 receptor exist, one (CXCR3-A) reactive with CXCL9, CXCL10, and CXCL11 and the other (CXCR3-B) also reactive with CXCL4. Both variants are contemporarily expressed by human T cells.

OBJECTIVE

We sought to investigate the in vitro effects of CXCL10 and CXCL4 on the production of TH1 or TH2 cytokines.

METHODS

The cytokine profile of antigen-specific human CD4+ T-cell lines obtained in the absence or presence of CXCL10 or CXCL4 was evaluated by means of quantitative RT-PCR, flow cytometry, and ELISA.

RESULTS

CXCL10 upregulated IFN-gamma and downregulated IL-4, IL-5, and IL-13 production, whereas CXCL4 downregulated IFN-gamma and upregulated TH2 cytokines. Similar effects were also observed on polyclonally activated pure naive CD4+ T cells. The opposite effects of CXCL10 and CXCL4 on TH1 and TH2 cytokine production were inhibited by an anti-CXCR3 antibody able to neutralize both CXCR3-A and CXCR3-B and were apparently related to the activation of distinct signal transduction pathways. Moreover, CXCL10 upregulated mRNA levels of T-box expressed in T cells and downregulated GATA-3 expression, whereas CXCL4 downregulated T-box expressed in T cells and upregulated GATA-3. Finally, CXCL4, but not CXCL10, induced direct activation of IL-5 and IL-13 promoters.

CONCLUSIONS

CXCL10 and CXCL4 exert opposite effects on the production of human TH1 and TH2 cytokines, likely through their respective interaction with CXCR3-A or CXCR3-B and the consequent activation of different signal transduction pathways. This might represent an internal regulatory pathway of TH cell responses and might contribute to the modulation of chronic inflammatory reactions, including allergy.

Inflammatory illness is associated with depression. Preclinical work has shown that chemokines are linked with peripheral-central crosstalk and may be important in mediating depressive behaviours. We sought to establish what evidence exists that differences in blood or cerebrospinal fluid chemokine concentration discriminate between individuals with depression and those without. Following PRISMA guidelines, we systematically searched Embase, PsycINFO and Medline databases. We included participants with physical illness for subgroup analysis, and excluded participants with comorbid psychiatric diagnoses. Seventy-three studies met the inclusion criteria for the meta-analysis. Individuals with depression had higher levels of blood CXCL4 and CXCL7 and lower levels of blood CCL4. Sensitivity analysis of studies with only physically healthy participants identified higher blood levels of CCL2, CCL3, CCL11, CXCL7 and CXCL8 and lower blood levels of CCL4. All other chemokines examined did not reveal significant differences (blood CCL5, CCL7, CXCL9, CXCL10 and cerebrospinal fluid CXCL8 and CXCL10). Analysis of the clinical utility of the effect size of plasma CXCL8 in healthy individuals found a negative predictive value 93.5%, given the population prevalence of depression of 10%. Overall, our meta-analysis finds evidence linking abnormalities of blood chemokines with depression in humans. Furthermore, we have demonstrated the possibility of classifying individuals with depression based on their inflammatory biomarker profile. Future research should explore putative mechanisms underlying this association, attempt to replicate existing findings in larger populations and aim to develop new diagnostic and therapeutic strategies.

Data from animal studies indicate that platelets play a key role in tumor dissemination and metastasis. We therefore hypothesized that metastastic cancer patients may display a specific platelet phenotype. Percentage of activated, p-selectin positive platelets as well as platelet contents (i.e., plasma and platelet count-corrected serum levels of VEGF-A, CXCL12, CXCL4, and thrombospondin-1) were analyzed in 43 patients with newly diagnosed metastatic disease prior to treatment. Tumor patients had increased platelet counts and significantly elevated percentages of activated platelets. Moreover, the platelet content of VEGF-A in cancer patients was significantly increased compared to healthy controls, while thrombospondin-1, CXCL12 and CXCL4 were significantly decreased. Our data contain several unexpected results: firstly, CXCL12 was found in minute quantities in the serum as compared with murine studies. Secondly, CXCL4, which was found by mass spectrometry to be the single massively upregulated intraplatelet chemokine in mice after tumor xenotransplantation, was decreased in tumor patient platelets. While increased contents of VEGF-A have been attributed to platelet scavenger activity, the differential decrease of specific platelet contents may be due to differential secretion or altered megakaryopoiesis in metastatic cancer patients.

We tested the possibility that immune complexes formed following platelet factor 4 (PF4/CXCL4) binding to anti-PF4 antibody can stimulate neutrophil activation, similar to previous reports with platelets. Monoclonal Abs against PF4 and IgG from a heparin-induced thrombocytopenia (HIT) patient were applied. We observed that although PF4 or anti-PF4 antibody alone did not alter neutrophil function, costimulation with both reagents resulted in approximately 3-fold increase in cell surface Mac-1 expression, enhanced cell adhesion via L-selectin and CD18 integrins, and degranulation of secondary and tertiary granules. The level of Mac-1 up-regulation peaked at an intermediate PF4 dose, suggesting that functional response varies with antigen-antibody stoichiometry. PF4 binding to neutrophils was blocked by chondroitinase ABC. Cell activation was inhibited by both chondroitinase ABC and anti-CD32/FcgammaRII blocking mAb, IV.3. Confocal microscopy demonstrated that immune complexes colocalize with CD32a. Studies with HIT IgG demonstrated that neutrophils could be activated in the absence of exogenous heparin. These data, together, show that leukocyte surface chondroitin sulfates promote neutrophil activation by enhancing immune-complex binding to CD32a. Studies with recombinant PF4 suggest a role for arginine 49 in stabilizing PF4-chondroitin binding. Neutrophils activated via this mechanism may contribute to thrombosis and inflammation in patients mounting an immune response to PF4-heparin.

The generation of reactive oxygen species (ROS) represents a pivotal element of phagocyte defense against microbial invaders. However, oxidative stress also participates in pathophysiological processes of vascular damage leading to cell death of endothelial cells (EC). Currently, ROS-producing cells involved in this process as well as the corresponding extracellular signals required for their activation are ill-defined. In this study, we investigate the impact of the platelet-derived CXC chemokine platelet factor 4 (PF4/CXCL4) on the interaction of human monocytes and EC. We can show for the first time that PF4-activated monocytes become cytotoxic for EC but not epithelial cells. Cytotoxicity was time- and dose-dependent, and earliest effects were seen after 15 h of culture and at a concentration from 0.125 microM PF4 up. By performing transwell experiments and by using specific inhibitory antibodies, we could show that direct cell contact between effector and target cells, mediated by beta(2)integrins as well as their corresponding ligand ICAM-1, is essential for the cytotoxic effect. Investigations of the cellular mechanisms of cytotoxicity revealed that in the presence of EC, PF4-activated monocytes are capable of releasing high amounts of ROS for more than 2 h following stimulation. This causes programmed cell death in EC, as inhibitors of the NADPH oxidase (diphenyleneiodonium and apocynin) effectively blocked PF4-induced monocyte oxidative burst and protected EC from undergoing apoptosis. Taken together, our data suggest a role for platelet-derived PF4 in oxidative stress-mediated vascular disorders, as observed during atherosclerosis or ischemia/reperfusion injury.

Inflammation represents a fundamental aspect of the healing process. Besides their primary role in hemostasis, platelets play an active role in the immunological and inflammatory aspect of tissue healing. Indeed , they can be directly involved in the inflammatory response by the production and release of several inflammatory mediators, including a variety of cytokines, such as TGF-beta, IL-1 beta, CD40L, and chemokines, such as CXCL7, CXCL4, CXCL4L1, CCl5, CXCL1, CXCL8, CXCL5, CXCL12, CCL2, CCL3. Platelet are not only a source of several chemokine involved in the inflammatory response and tissue healing, but they also express chemokine receptors, in particular CCR1 CCR3 CCR4 and CXCR4, thus being able to being able to regulate the inflammatory response associated to the healing process. However, this local inflammation must be taken under control, and platelets can prevent the excess of leukocytes recruitment by anti-inflammatory cytokines, such as TGF-beta. For this biological properties of platelets, platelet rich plasma therapy (PRP) is considered an innovative and promising approach that has been extended to many field of medicine, ranging from non-union defects, bone fractures, spinal fusion, bone implant and osteointegration, joint arthroplasty, to the treatment of several traumatic or degenerative pathologies of tendons, cartilage and ligaments.

OBJECTIVE

Macrophages are critical contributors to abdominal aortic aneurysm (AAA) disease. We examined the ability of MKEY, a peptide inhibitor of CXCL4-CCL5 interaction, to influence AAA progression in murine models.

RESULTS

AAAs were created in 10-week-old male C57BL/6J mice by transient infrarenal aortic porcine pancreatic elastase infusion. Mice were treated with MKEY via intravenous injection either (1) before porcine pancreatic elastase infusion or (2) after aneurysm initiation. Immunostaining demonstrated CCL5 and CCR5 expression on aneurysmal aortae and mural monocytes/macrophages, respectively. MKEY treatment partially inhibited migration of adaptively transferred leukocytes into aneurysmal aortae in recipient mice. Although all vehicle-pretreated mice developed AAAs, aneurysms formed in only 60% (3/5) and 14% (1/7) of mice pretreated with MKEY at 10 and 20 mg/kg, respectively. MKEY pretreatment reduced aortic diameter enlargement, preserved medial elastin fibers and smooth muscle cells, and attenuated mural macrophage infiltration, angiogenesis, and aortic metalloproteinase 2 and 9 expression after porcine pancreatic elastase infusion. MKEY initiated after porcine pancreatic elastase infusion also stabilized or reduced enlargement of existing AAAs. Finally, MKEY treatment was effective in limiting AAA formation after angiotensin II infusion in apolipoprotein E-deficient mice.

CONCLUSIONS

MKEY suppresses AAA formation and progression in 2 complementary experimental models. Peptide inhibition of CXCL4-CCL5 interactions may represent a viable translational strategy to limit progression of human AAA disease.