The heterodimerization of platelet-derived chemokines

Chemokines encompass a large family of proteins that act as chemoattractants and are involved in many biological processes. In particular, chemokines guide the migration of leukocytes during normal and inflammatory conditions. Recent studies reveal that the heterophilic interactions between chemokines significantly affect their biological activity, possibly representing a novel regulatory mechanism of the chemokine activities. The co-localization of platelet-derived chemokines in vivo allows them to interact. Here, we used nano-spray ionization mass spectrometry to screen eleven different CXC and CC platelet-derived chemokines for possible interactions with the two most abundant chemokines present in platelets, CXCL4 and CXCL7. Results indicate that many screened chemokines, although not all of them, form heterodimers with CXCL4 and/or CXCL7. In particular, a strong heterodimerization was observed between CXCL12 and CXCL4 or CXCL7. Compared to other chemokines, the main structural difference of CXCL12 is in the orientation and packing of the C-terminal alpha-helix in relation to the beta-sheet. The analysis of one possible structure of the CXCL4/CXCL12 heterodimer, CXC-type structure, using molecular dynamics (MD) trajectory reveals that CXCL4 may undergo a conformational transition to alter the alpha helix orientation. In this new orientation, the alpha-helix of CXCL4 aligns in parallel with the CXCL12 alpha-helix, an energetically more favorable conformation. Further, we determined that CXCL4 and CXCL12 physically interact to form heterodimers by co-immunoprecipitations from human platelets. Overall, our results highlight that many platelet-derived chemokines are capable of heterophilic interactions and strongly support future studies of the biological impact of these interactions.     

Interference with Glycosaminoglycan-Chemokine Interactions with a Probe to Alter Leukocyte Recruitment and Inflammation In Vivo

In vivo leukocyte recruitment is not fully understood and may result from interactions of chemokines with glycosaminoglycans/GAGs. We previously showed that chlorite-oxidized oxyamylose/COAM binds the neutrophil chemokine GCP-2/CXCL6. Here, mouse chemokine binding by COAM was studied systematically and binding affinities of chemokines to COAM versus GAGs were compared. COAM and heparan sulphate bound the mouse CXC chemokines KC/CXCL1, MIP-2/CXCL2, IP-10/CXCL10 and I-TAC/CXCL11 and the CC chemokine RANTES/CCL5 with affinities in the nanomolar range, whereas no binding interactions were observed for mouse MCP-1/CCL2, MIP-1α/CCL3 and MIP-1β/CCL4. The affinities of COAM-interacting chemokines were similar to or higher than those observed for heparan sulphate. Although COAM did not display chemotactic activity by itself, its co-administration with mouse GCP-2/CXCL6 and MIP-2/CXCL2 or its binding of endogenous chemokines resulted in fast and cooperative peritoneal neutrophil recruitment and in extravasation into the cremaster muscle in vivo. These local GAG mimetic features by COAM within tissues superseded systemic effects and were sufficient and applicable to reduce LPS-induced liver-specific neutrophil recruitment and activation. COAM mimics glycosaminoglycans and is a nontoxic probe for the study of leukocyte recruitment and inflammation in vivo.     

Echinococcus multilocularis: inflammatory and regulatory chemokine responses in patients with progressive, stable and cured alveolar echinococcosis

The progressive growth of Echinococcus multilocularis metacestodes and their tissue infiltration will cause organ malfunction and finally failure. In few patients, E. multilocularis metacestode proliferation will spontaneously regress, but little is known about the determinants which may restrain metacestode survival and growth. In this study, chemokine responses were investigated in E. multilocularis patients at different states of infection, i.e. with progressive, stable and cured alveolar echinococcosis (AE). Characteristic chemokine profiles and changes in their production were observed in AE patients and infection-free controls when their peripheral blood cells were cultured with E. multilocularis antigens. The production of CC and CXC chemokines which associate with inflammation (MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5 and GRO-α/CXCL1) was constitutively larger in AE patients than in controls; and the elevated chemokine releases were equal in patients with progressive, stable or cured AE. Cluster analyses identified three distinct chemokine response profiles; chemokines were enhanced, depressed or produced in similar quantities in AE patients and controls. A disparate cellular responsiveness was observed in AE patients to viable E. multilocularis vesicles; cluster 1 (GRO-α/CXCL1, MCP-3/CCL7, MCP-4/CCL13, TARC/CCL17, LARC/CCL20) and cluster 2 chemokines (PARC/CCL18, MDC/CCL22, MIG/CXCL9) were clearly diminished, while cluster 3 chemokines (MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5) augmented. The increased production of inflammatory chemokines in patients even with cured AE could be induced by residual E. multilocularis metacestode lesions which continuously stimulate production of inflammatory chemokines. E. multilocularis metacestodes also suppressed cellular chemokine production in AE patients, and this may constitute an immune escape mechanism which reduces inflammatory host responses, prevents tissue destruction and organ damage, but may also facilitate parasite persistence.     

Eotaxin/CCL11 suppresses IL-8/CXCL8 secretion from human dermal microvascular endothelial cells

The CC chemokine eotaxin/CCL11 is known to bind to the receptor CCR3 on eosinophils and Th2-type lymphocytes. In this study, we demonstrate that CCR3 is expressed on a subpopulation of primary human dermal microvascular endothelial cells and is up-regulated by TNF-alpha. We found that incubation of human dermal microvascular endothelial cells with recombinant eotaxin/CCL11 suppresses TNF-alpha-induced production of the neutrophil-specific chemokine IL-8/CXCL8. The eotaxin/CCL11-suppressive effect on endothelial cells was not seen on IL-1beta-induced IL-8/CXCL8 release. Eotaxin/CCL11 showed no effect on TNF-alpha-induced up-regulation of growth-related oncogene-alpha or IFN-gamma-inducible protein-10, two other CXC chemokines tested, and did not affect production of the CC chemokines monocyte chemoattractant protein-1/CCL2 and RANTES/CCL5, or the adhesion molecules ICAM-1 and E-selectin. These results suggest that eotaxin/CXCL11 is not effecting a general suppression of TNF-alphaR levels or signal transduction. Suppression of IL-8/CXCL8 was abrogated in the presence of anti-CCR3 mAb, pertussis toxin, and wortmannin, indicating it was mediated by the CCR3 receptor, G(i) proteins, and phosphatidylinositol 3-kinase signaling. Eotaxin/CCL11 decreased steady state levels of IL-8/CXCL8 mRNA in TNF-alpha-stimulated cells, an effect mediated in part by an acceleration of IL-8 mRNA decay. Eotaxin/CCL11 may down-regulate production of the neutrophil chemoattractant IL-8/CXCL8 by endothelial cells in vivo, acting as a negative regulator of neutrophil recruitment. This may play an important biological role in the prevention of overzealous inflammatory responses, aiding in the resolution of acute inflammation or transition from neutrophilic to mononuclear/eosinophilic inflammation.     

Towards in situ tissue repair: human mesenchymal stem cells express chemokine receptors CXCR1, CXCR2 and CCR2, and migrate upon stimulation with CXCL8 but not CCL2

The recruitment of bone marrow CD34- mesenchymal stem- and progenitor cells (MSC) and their subsequent differentiation into distinct tissues is the precondition for in situ tissue engineering. The objective of this study was to determine the entire chemokine receptor expression profile of human MSC and to investigate their chemotactic response to the selected chemokines CCL2, CXCL8 and CXCL12. Human MSC were isolated from iliac crest bone marrow aspirates and showed a homogeneous population presenting a typical MSC-related cell surface antigen profile (CD14-, CD34-, CD44+, CD45-, CD166+, SH-2+). The expression profile of all 18 chemokine receptors was determined by real-time PCR and immunohistochemistry. Both methods consistently demonstrated that MSC express CC, CXC, C and CX(3)C receptors. Gene expression and immunohistochemical analysis documented that MSC express chemokine receptors CCR2, CCR8, CXCR1, CXCR2 and CXCR3. A dose-dependent chemotactic activity of CXCR4 and CXCR1/CXCR2 ligands CXCL12 and CXCL8 (interleukin-8) was demonstrated using a 96-well chemotaxis assay. In contrast, the CCR2 ligand CCL2 (monocyte chemoattractant protein-1, MCP-1) did not recruited human MSC. In conclusion, we report that the chemokine receptor expression profile of human MSC is much broader than known before. Furthermore, for the first time, we demonstrate that human MSC migrate upon stimulation with CXCL8 but not CCL2. In combination with already known data on MSC recruitment and differentiation these are promising results towards in situ regenerative medicine approaches based on guiding of MSC to sites of degenerated tissues.     

Heterodimerization of CCR2 chemokines and regulation by glycosaminoglycan binding

Despite the wide range of sequence diversity among chemokines, their tertiary structures are remarkably similar. Furthermore, many chemokines form dimers or higher order oligomers, but all characterized oligomeric structures are based primarily on two dimerization motifs represented by CC-chemokine or CXC-chemokine dimer interfaces. These observations raise the possibility that some chemokines could form unique hetero-oligomers using the same oligomerization motifs. Such interactions could modulate the overall signaling response of the receptors, thereby providing a general mechanism for regulating chemokine function. For some chemokines, homo-oligomerization has also been shown to be coupled to glycosaminoglycan (GAG)-binding. However, the effect of GAG binding on chemokine hetero-oligomerization has not yet been demonstrated. In this report, we characterized the heterodimerization of the CCR2 ligands MCP-1 (CCL2), MCP-2 (CCL8), MCP-3 (CCL7), MCP-4 (CCL13), and eotaxin (CCL11), as well as the effects of GAG binding, using electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry. Strong heterodimerization was observed between CCL2 and CCL8 at the expense of homodimer formation. Using NMR, we showed that the heterodimer is predominant in solution and forms a specific CC chemokine-like dimer. By contrast, only moderate heterodimer formation was observed between CCL2.CCL13, CCL2.CCL11 and CCL8.CCL13, and no heterodimerization was observed when any other CCR2 ligand was added to CCL7. To investigate the effect of a highly sulfated GAG on the formation of heterodimers, each chemokine pair was mixed with the heparin pentasaccharide, Arixtra, and assayed by ESI-FTICR mass spectrometry. Although no CCL8.CCL11 heterodimer was observed in the absence of GAG, abundant ions corresponding to the ternary complex, CCL8.CCL11.Arixtra, were observed upon addition of Arixtra. Heterodimerization between CCL2 and CCL11 was also enhanced in the presence of Arixtra. In summary, these results indicate that some CCR2 ligands can form stable heterodimers in preference to homodimers and that these interactions, like those of homo-oligomers, can be influenced by some GAGs.     

Stimulation of oral fibroblast chemokine receptors identifies CCR3 and CCR4 as potential wound healing targets

The focus of this study was to determine which chemokine receptors are present on oral fibroblasts and whether these receptors influence proliferation, migration, and/or the release of wound healing mediators. This information may provide insight into the superior wound healing characteristics of the oral mucosa. The gingiva fibroblasts expressed 12 different chemokine receptors (CCR3, CCR4, CCR6, CCR9, CCR10, CXCR1, CXCR2, CXCR4, CXCR5, CXCR7, CX3CR1, and XCR1), as analyzed by flow cytometry. Fourteen corresponding chemokines (CCL5, CCL15, CCL20, CCL22, CCL25, CCL27, CCL28, CXCL1, CXCL8, CXCL11, CXCL12, CXCL13, CX3CL1, and XCL1) were used to study the activation of these receptors on gingiva fibroblasts. Twelve of these fourteen chemokines stimulated gingiva fibroblast migration (all except for CXCL8 and CXCL12). Five of the chemokines stimulated proliferation (CCL5/CCR3, CCL15/CCR3, CCL22/CCR4, CCL28/CCR3/CCR10, and XCL1/XCR1). Furthermore, CCL28/CCR3/CCR10 and CCL22/CCR4 stimulation increased IL‐6 secretion and CCL28/CCR3/CCR10 together with CCL27/CCR10 upregulated HGF secretion. Moreover, TIMP‐1 secretion was reduced by CCL15/CCR3. In conclusion, this in‐vitro study identifies chemokine receptor‐ligand pairs which may be used in future targeted wound healing strategies. In particular, we identified the chemokine receptors CCR3 and CCR4, and the mucosa specific chemokine CCL28, as having an predominant role in oral wound healing by increasing human gingiva fibroblast proliferation, migration, and the secretion of IL‐6 and HGF and reducing the secretion of TIMP‐1.     

CXC chemokine ligand 4 (CXCL4) down-regulates CC chemokine receptor expression on human monocytes

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.     

Chemokines and cytokines in patients with an occult Onchocerca volvulus infection

Repeated ivermectin treatment will clear microfilaria (Mf) of Onchocerca volvulus from skin and eyes of onchocerciasis patients while adult filaria remains alive and reproductive, and such occult O. volvulus infection may persist for years. To investigate the effect of residual adult filaria on the immune response profile, chemokines and cytokines were quantified 1) in onchocerciasis patients who developed an occult O. volvulus infection (Mf-negative) due to repeated ivermectin treatments, 2) patients who became Mf-negative without ivermectin treatments due to missing re-infection, and 3) endemic and non-endemic O. volvulus Mf-negative controls. With occult O. volvulus infection, serum levels of pro-inflammatory chemokines MCP-1/CCL2, MIP-1α/CCL3, MIP-1β/CCL4, MPIF-1/CCL23 and CXCL8/IL-8 enhanced and approached higher concentrations as determined in infection-free controls, whilst regulatory and Th2-type cytokines and chemokines MCP-4/CCL13, MIP-1δ/CCL15, TARC/CCL17 and IL-13 lessened. Levels of Eotaxin-2/CCL24, MCP-3/CCL7 and BCA-1/CXCL13 remained unchanged. At 3 days post-initial ivermectin treatment, MCP-1/CCL2, MCP-4/CCL13, MPIF-1/CCL23 and Eotaxin-2/CCL24 were strongly enhanced, suggesting that monocytes and eosinophil granulocytes have mediated Mf clearance. In summary, with occult and expiring O. volvulus infections the serum levels of inflammatory chemokines enhanced over time while regulatory and Th2-type-promoting cytokines and chemokines lessened; these changes may reflect a decreasing effector cell activation against Mf of O. volvulus, and in parallel, an enhancing inflammatory immune responsiveness.     

Platelet factor 4 and interleukin-8 CXC chemokine heterodimer formation modulates function at the quaternary structural level

The apparent complexity of biology increases as more biomolecular interactions that mediate function become known. We have used NMR spectroscopy and molecular modeling to provide direct evidence that tetrameric platelet factor-4 (PF4) and dimeric interleukin-8 (IL8), two members of the CXC chemokine family, readily interact by exchanging subunits and forming heterodimers via extension of their antiparallel beta-sheet domains. We further demonstrate using functional assays that PF4/IL8 heterodimerization has a direct and significant consequence on the biological activity of both chemokines. Formation of heterodimers enhances the anti-proliferative effect of PF4 on endothelial cells in culture, as well as the IL8-induced migration of CXCR2 vector-transfected Baf3 cells. These results suggest that CXC chemokine biology, and perhaps cytokine biology in general, may be functionally modulated at the molecular level by formation of heterodimers. This concept, in turn, has implications for designing chemokine/cytokine variants with modified biological properties.     

人CXCL4蛋白原核表达与纯化

CXCL4又名血小板因子4(PF4),属于CXC趋化因子亚家族,能够广谱的作用于多种细胞,在炎症,凝血等众多生理及病理反应中发挥重要作用。将CXCL4全长基因构建到原核表达载体pET43.1a(+),利用大肠杆菌系统表达,继而纯化获得高纯度的目的蛋白。实验表明重组人CXCL4蛋白可以有效抑制人肾癌细胞增殖,具有生物学活性。重组人CXCL4原核表达与纯化方法的建立将有助于其生物学结构与功能的研究,并且对趋化因子家族其它蛋白质的表达与纯化具有参考价值。     

Effect of vitamin D(3) on chemokine expression in pulmonary tuberculosis

1,25 Dihydroxy vitamin D(3) (vitamin D(3)) is an immunomodulator and its deficiency has been associated with susceptibility to tuberculosis. We have studied the immunoregulatory role of vitamin D(3) on various chemokine expression in pulmonary tuberculosis. Peripheral blood mononuclear cells obtained from 21 pulmonary tuberculosis (PTB) patients and 24 healthy controls (HCs) were cultured for 48h with culture filtrate antigen (CFA) of Mycobacterium tuberculosis with or without vitamin D(3) at a concentration 1×10(-7)M. The relative mRNA expression of monocyte chemoattractant protein-1 (MCP-1, CCL2), macrophage inflammatory protein-1α (MIP-1α, CCL3), macrophage inflammatory protein-1β (MIP-1β, CCL4), and regulated upon-activation, normal T cell-expressed and secreted (RANTES, CCL5) and IFN-γ inducible protein-10 (IP-10, CXCL10) chemokines were estimated from 48h old macrophages using real-time polymerase chain reaction (RT-PCR). The culture supernatants were used to estimate the various chemokines including monokine induced by IFN-γ (MIG, CXCL9) levels using cytometric bead array. In HCs, vitamin D(3) significantly suppressed the MCP-1 mRNA expression of CFA stimulated cells (p=0.0027), while no such effect was observed in PTB patients. Vitamin D(3) showed no significant effect on MIP-1α, MIP-1β and RANTES in both the study groups. The CFA induced IP-10 mRNA and protein expression was significantly suppressed by vitamin D(3) in both the study groups (p<0.05). A similar suppressive effect of vitamin D(3) was observed with MIG protein in healthy controls (p=0.0029) and a trend towards a suppression was observed in PTB patients. The suppressive effect of vitamin D(3) is more prominent in CXC chemokines rather than CC chemokines. This suggests that vitamin D(3) may down regulate the recruitment and activation of T-cells through CXC chemokines at the site of infection and may act as a potential anti-inflammatory agent.     

ELR+ CXC chemokines in human milk

CXC chemokines bearing the glutamic acid-leucine-arginine (ELR) motif are crucial mediators in neutrophil-dependent acute inflammation. Interestingly, however, Interleukin (IL)-8/CXC ligand (CXCL) 8 is expressed in human milk in biologically significant concentrations, and may play a local maturational role in the developing human intestine. In this chemokine subfamily, there are six other known peptides beside IL-8/CXCL8, all sharing similar effects on neutrophil chemotaxis and angiogenesis. In this study, we measured the concentrations of these chemokines in human milk, sought their presence in human mammary tissue by immunohistochemistry, and confirmed chemokine expression in cultured human mammary epithelial cells (HMECs). Each of the seven ELR(+) CXC chemokines was measurable in milk, and except for NAP-2/CXCL7, these concentrations were higher than serum. The concentrations were higher in colostrum (except for GRO-beta/CXCL2 and NAP-2/CXCL7), and correlated negatively with time elapsed postpartum. IL-8/CXCL8, GRO-gamma/CXCL3, and ENA-78/CXCL5 concentrations were higher in preterm milk. There was intense immunoreactivity in mammary epithelial cells for all ELR(+) CXC chemokines, and the intensity of staining was higher in breast tissue with lactational changes. The supernatants from confluent HMEC cultures also contained measurable concentrations of all the seven ELR(+) CXC chemokines. These results confirm that all ELR(+) CXC chemokines are actively secreted by the mammary epithelial cells into human milk. Further studies are needed to determine if these chemokines share with IL-8/CXCL8 the protective effects on intestinal epithelial cells.     

Molecular aspects of rheumatoid arthritis: chemokines in the joints of patients

Rheumatoid arthritis (RA) is a chronic symmetric polyarticular joint disease that primarily affects the small joints of the hands and feet. The inflammatory process is characterized by infiltration of inflammatory cells into the joints, leading to proliferation of synoviocytes and destruction of cartilage and bone. In RA synovial tissue, the infiltrating cells such as macrophages, T cells, B cells and dendritic cells play important role in the pathogenesis of RA. Migration of leukocytes into the synovium is a regulated multi-step process, involving interactions between leukocytes and endothelial cells, cellular adhesion molecules, as well as chemokines and chemokine receptors. Chemokines are small, chemoattractant cytokines which play key roles in the accumulation of inflammatory cells at the site of inflammation. It is known that synovial tissue and synovial fluid from RA patients contain increased concentrations of several chemokines, such as monocyte chemoattractant protein-4 (MCP-4)/CCL13, pulmonary and activation-regulated chemokine (PARC)/CCL18, monokine induced by interferon-gamma (Mig)/CXCL9, stromal cell-derived factor 1 (SDF-1)/CXCL12, monocyte chemotactic protein 1 (MCP-1)/CCL2, macrophage inflammatory protein 1alpha (MIP-1alpha)/CCL3, and Fractalkine/CXC3CL1. Therefore, chemokines and chemokine-receptors are considered to be important molecules in RA pathology.     

Assessment of chemokine profiles in human skin biopsies by an immunoaffinity capillary electrophoresis chip

Atopic dermatitis is a skin condition resulting in a skin rash from exposure to environmental factors. Skin biopsies taken from patients suffering from atopic dermatitis were micro-dissected and analyzed using a microchip-based immunoaffinity CE system for the presence of CXCL1, CXCL5 and CXCL8 and CCL1, CCL3 and CCL5 chemokines. Disposable immunoaffinity disks with immobilized antibodies were used to capture the CXC and CC chemokines from the homogenized skin samples. The captured analytes were then labeled with AlexaFluor 633, eluted from the disk and separated by CE. The labeled chemokines were identified and quantified by laser induced fluorescence. The total analysis time was less than 40min, including the biopsy microdissection, pre-analysis preparation of the sample and the ICE-CHIP analysis, which took less than 10min with inter- and intra-assay CV's below 6.4%. Microchip-based immunoaffinity CE could distinguish between normal skin biopsies and those with inflammation. Patients with neutrophil cellular infiltrates by histopathology showed increased concentrations of CXCL1, CXCL5 and CXCL8 while increases of CCL1, CCL3 and CCL5 corresponded to the patient group demonstrating monocytic and T-lymphocyte infiltration by histopathology. This system demonstrates the ability to identify and quantify immunochemical analytes in frozen sections taken from clinical histopathology samples.     

Chemokines in proliferative diabetic retinopathy and proliferative vitreoretinopathy

PURPOSE: To determine levels of the chemokines CCL1/I-309, CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL7/MCP-3, CCL8/MCP-2, CXCL5/ENA-78, CXCL6/GCP-2, CXCL10/IP-10, and CXCL11/I-TAC in the vitreous humor and serum, from patients with proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), and rhegmatogenous retinal detachment with no PVR (RD), and to investigate the expression of MCP-1, CXCL12/SDF-1, and the chemokine receptor CXCR3 in epiretinal membranes. METHODS: Paired vitreous humor and serum samples were obtained from patients undergoing vitrectomy for the treatment of RD (57 specimens), PVR (32 specimens), and PDR (88 specimens). The levels of chemokines were measured by enzyme-linked immunosorbent assays. Eighteen PDR and 5 PVR membranes were studied by immunohistochemical techniques. RESULTS: Of all the chemokines studied, only MCP-1 and IP-10 were detected in vitreous humor samples. MCP-1 levels in vitreous humor samples were significantly higher than in serum samples (p < 0.001). MCP-1 levels were significantly higher in vitreous humor samples from patients with PVR and PDR compared with RD (p = 0.0002). MCP-1 levels in vitreous humor samples from patients with active PDR were significantly higher than in inactive PDR cases (p = 0.0224). IP-10 levels in vitreous humor samples were significantly higher than in serum samples (p = 0.0035). IP-10 levels were significantly higher in vitreous humor samples from patients with PVR and PDR compared with RD (p = 0.0083). The incidence of IP-10 detection in vitreous humor samples was significantly higher in active PDR cases compared with inactive cases (p = 0.0214). There was a significant association between the incidence of IP-10 detection and increased levels of MCP-1 in vitreous humor samples from all patients, and patients with RD and PDR (p < 0.001 for all comparisons). MCP-1, and SDF-1 were localized in myofibroblasts in PVR and PDR membranes and in vascular endothelial cells in PDR membranes. CXCR3 was expressed by vascular endothelial cells in PDR membranes. CONCLUSION: MCP-1, IP-10 and SDF-1 may participate in pathogenesis of PVR and PDR. Myofibroblasts and vascular endothelial cells are the major cell types expressing MCP-1, SDF-1, and CXCR3 in epiretinal membranes.     

G-protein-coupled receptor independent, immunomodulatory properties of chemokine CXCL9

Certain chemokines possess anti-angiogenic and antibacterial activity, in addition to their ability to recruit leukocytes. Herein, we demonstrate that CXCL9/MIG induces the expression, by a monocytic cell line and peripheral blood mononuclear cells, of a variety of chemokines including CXCL8/IL-8, CCL3/MIP-1α, CCL4/MIP-1β, CCL2/MCP-1 in a pertussis toxin insensitive manner. Similarly, another cationic chemokine CCL20/MIP-3α, but not the non-cationic chemokines CCL2 or CCL3, stimulated monocytic cells to produce substantial amounts of CXCL8 and CCL3. Microarray experiments demonstrated that CXCL9, but not CCL2, induced the expression of hundreds of genes, many of which have known or proposed immunomodulatory functions. Induction of CXCL8 required the p38 and ERK1/2 mitogen-activated protein kinases but not NFκB, JAK-STAT or JNK signaling pathways. These results collectively demonstrate that CXCL9 has immunomodulatory functions that are not mediated through a G-protein coupled receptor and may possess additional roles in host defenses against infection.     

Expression of CXC and CC type of chemokines and its receptors in tuberculous and non-tuberculous effusions

Chemokines mediate their biological functions by transmigration of various immune cells to the site of infection. Tuberculous pleurisy provides an effective model to study the role of chemokines in the recruitment of immune cells to the pleura. Our aim was to understand the cumulative effect of chemokines (IP-10, MIG, IL-8, MCP-1, MIP-1α and RANTES) and its receptors (CXCR2, CXCR3, CCR1, CCR2, CCR5 and CCR7) in the recruitment of CD4⁺ T cells obtained from blood (BL) and pleural fluid (PF) of tuberculous (TB) and non-tuberculous (NTB) patients. We observed significant increase in CD4⁺ T cells in TB PF indicating lymphocytic rich effusion. All chemokines except RANTES were significantly high in PF compared to BL in TB group, whereas IL-8 and MCP-1 showed significant increase only in NTB PF. The significantly high levels of IFN-γ and ΤΝF-α in TB PF and their positive correlation with IP-10 and MIP-1α indicated their synergistic action to elicit a strong protective Th1 response. In spite of high levels of Th1 cytokines and chemokines in TB PF, significantly lower levels of RANTES indicated its limited role at the site. The CXC receptors in PF of both the groups and CC receptors except CCR5 in TB PF were significantly high compared to BL. Only CXCR2, CCR5 and CCR7 showed significant increase in TB compared to NTB. Thus a selective concentration of chemokines, cytokines and abundant expression of chemokine receptors confirm the accumulation of activated and memory T cells at the site of infection and help in polarizing Th1 immune response.     

Statins affect the presentation of endothelial chemokines by targeting to multivesicular bodies

BACKGROUND: In addition to lowering cholesterol, statins are thought to beneficially modulate inflammation. Several chemokines including CXCL1/growth-related oncogene (GRO)-α, CXCL8/interleukin (IL)-8 and CCL2/monocyte chemoattractant protein (MCP)-1 are important in the pathogenesis of atherosclerosis and can be influenced by statin-treatment. Recently, we observed that atorvastatin-treatment alters the intracellular content and subcellular distribution of GRO-α in cultured human umbilical vein endothelial cells (HUVECs). The objective of this study was to investigate the mechanisms involved in this phenomenon. METHODOLOGY/ PRINCIPAL FINDINGS: The effect of atorvastatin on secretion levels and subcellular distribution of GRO-α, IL-8 and MCP-1 in HUVECs activated by interleukin (IL)-1β were evaluated by ELISA, confocal microscopy and immunoelectron microscopy. Atorvastatin increased the intracellular contents of GRO-α, IL-8, and MCP-1 and induced colocalization with E-selectin in multivesicular bodies. This effect was prevented by adding the isoprenylation substrate GGPP, but not the cholesterol precursor squalene, indicating that atorvastatin exerts these effects by inhibiting isoprenylation rather than depleting the cells of cholesterol. CONCLUSIONS/ SIGNIFICANCE: Atorvastatin targets inflammatory chemokines to the endocytic pathway and multivesicular bodies and may contribute to explain the anti-inflammatory effect of statins at the level of endothelial cell function.