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PubMed2007

Chemokine responses are increased in HIV-infected Malawian children with invasive pneumococcal disease.

BACKGROUND: Chemokines play an important role in the recruitment and regulation of leukocyte traffic during bacterial infection. The aims of this study were to investigate the chemokine response to invasive pneumococcal disease (IPD) and to examine the influence of HIV infection on the chemokine response, pneumococcal bacterial loads, and outcome. METHODS: We prospectively studied 95 children with IPD, and blood and cerebrospinal fluid (CSF) samples were taken at admission for the determination of chemokines, interferon-gamma (IFNgamma), and pneumococcal bacterial loads. RESULTS: Plasma CXCL8 and CCL2, CSF CXCL8 and CCL4, and IFNgamma were significantly higher in HIV-infected children than in HIV-uninfected children. Blood and CSF pneumococcal bacterial loads correlated with plasma and CSF chemokines, respectively, and were higher in HIV-infected children compared with HIV-uninfected children. Among HIV-infected children, plasma concentrations of CXCL8 and CCL2 were significantly higher in nonsurvivors than in survivors, but CCL5 was significantly lower. HIV-infected and HIV-uninfected children with IPD had higher concentrations of chemokines (except CCL5) than acutely ill HIV-infected and HIV-uninfected children with no detectable bacterial infection. Male gender and low plasma CCL2 concentrations were shown to be independently associated with survival. CONCLUSIONS: Chemokines, in particular CCL2, are associated with survival in IPD and correlate with pneumococcal bacterial loads, disease presentation, and outcome.

Adolescent↗

PubMed2005

Chemokine response to febrile urinary tract infection.

BACKGROUND: Mucosal CXC chemokines recruit inflammatory cells to the infected urinary tract. The chemokine response repertoire of the urinary tract and the relationship to disease severity have not been examined, however. METHODS: This study quantified CXC (CXCL1, CXCL3, CXCL5, CXCL8, CXCL9, and CXCL10) and CC (CCL2, CCL4, and CCL5) chemokines in sequential urine samples obtained from 50 patients with febrile urinary tract infections during 24 hours after diagnosis. RESULTS: All patients had elevated chemokine levels, but bacteremic infections caused higher CXCL1, CXCL3, CXCL5, CXCL8, and CCL2 responses. CCL2 and CXCL8 levels were higher in patients with acute pyelonephritis symptoms and CCL2, CXCL3, CCL4, CXCL5, and CXCL10 were significantly correlated to C-reactive protein (CRP) and temperature. Women and men showed different chemokine responses. CONCLUSION: Febrile urinary tract infections are accompanied by a complex chemokine response. The response magnitude reflects disease severity, and the repertoire is influenced by gender and underlying disease.

Adolescent↗

PubMed2004

Collateral artery growth (arteriogenesis) after experimental arterial occlusion is impaired in mice lacking CC-chemokine receptor-2.

Arteriogenesis has been associated with the presence of monocytes/macrophages within the collateral vessel wall. Induced macrophage migration in vivo is driven by the binding of monocyte chemoattractant protein-1 (MCP-1, or CCL2 in the new nomenclature) to the CCR2-chemokine receptor on macrophages. To determine whether the CCL2-CCR2 signaling pathway is involved in the accumulation of macrophages in growing collateral vessels, we used mice that are deficient in CCR2 in a model of experimental arterial occlusion and collateral vessel growth. In an in vitro CCL2-driven chemotaxis assay, mononuclear cells isolated from wild-type BALB/c mice exhibited CCL2 concentration-dependent migration, whereas this migration was abolished in cells from CCR2(-/-) mice on a BALB/c genetic background. In vivo, blood flow recovery as measured by laser Doppler (LDI) and MRI (MRI) was impaired in CCR2(-/-) mice on either the BALB/c or C57BL/6 genetic backgrounds. Three weeks after femoral artery ligation, LDI perfusion ratio of operated versus nonoperated distal hindlimb in BALB/c wild-type mice increased to 0.45+/-0.06 and in CCR2(-/-) animals only to 0.21+/-0.03 (P<0.01). In C57BL/6 mice, ratio increased to 0.96+/-0.09 and 0.85+/-0.08 (P<0.05), respectively. MRI at 3 weeks (0.76+/-0.06 versus 0.62+/-0.01; P<0.05) and hemoglobin oxygen saturation measurements confirmed these findings. Active foot movement score significantly decreased and gastrocnemius muscle atrophy was significantly greater in CCR2(-/-) mice. Morphometric analysis showed a lesser increase in collateral vessel diameters in CCR2(-/-) mice. Importantly, the number of invaded monocytes/macrophages in the perivascular space of collateral arteries of CCR2(-/-) animals was dramatically reduced in comparison to wild-type mice. In conclusion, our results demonstrate that the CCR2 signaling pathway is essential for efficient collateral artery growth.

Animals↗

PubMed2006

Lipopolysaccharide regulated protein expression is only partly impaired in monocytes from patients with type I diabetes.

BACKGROUND: Monocytes play an important role in innate immunity and atherosclerosis. A disturbed secretion of cytokines in lipopolysaccharide (LPS) activated monocytes from type 1 diabetes (T1D) patients has been described and may contribute to the impaired inflammatory response in these individuals. In the present study the influence of LPS on five different proteins with a function in immunity and atherosclerosis was analyzed in monocytes from controls and T1D patients. METHODS: Monocytes were isolated from controls and T1D patients and the LPS-stimulated increase of IL-6, CXCL8, monocyte chemotactic protein 1 (CCL2, MCP-1) and superoxide dismutase (SOD 2), as well as the LPS-mediated decrease of apolipoprotein E (Apo E) in primary human monocytes from controls and T1D patients was determined. RESULTS: CCL2 and IL-6 secretion in response to LPS was found significantly reduced in monocytes from T1D patients when compared to controls whereas basal CCL2 release was similar in control and T1D cells. In contrast, CXCL8 and apolipoprotein E secretion and SOD 2 expression upon LPS stimulation is similar from T1D and control monocytes. CONCLUSION: These data indicate that LPS-mediated protein expression is only partly disturbed in monocytes from T1D patients. Reduced secretion of IL-6 and CCL2 in activated monocytes of these patients may contribute to an impaired inflammatory response and vascular disease.

Adult↗

PubMed2025

Rare damaging CCR2 variants are associated with lower lifetime cardiovascular risk.

BACKGROUND: Previous work has shown a role of CCL2, a key chemokine governing monocyte trafficking, in atherosclerosis. However, it remains unknown whether targeting CCR2, the cognate receptor of CCL2, provides protection against human atherosclerotic cardiovascular disease. METHODS: Computationally predicted damaging or loss-of-function (REVEL > 0.5) variants within CCR2 were detected in whole-exome-sequencing data from 454,775 UK Biobank participants and tested for association with cardiovascular endpoints in gene-burden tests. Given the key role of CCR2 in monocyte mobilization, variants associated with lower monocyte count were prioritized for experimental validation. The response to CCL2 of human cells transfected with these variants was tested in migration and cAMP assays. Validated damaging variants were tested for association with cardiovascular endpoints, atherosclerosis burden, and vascular risk factors. Significant associations were replicated in six independent datasets (n = 1,062,595). RESULTS: Carriers of 45 predicted damaging or loss-of-function CCR2 variants (n = 787 individuals) were at lower risk of myocardial infarction and coronary artery disease. One of these variants (M249K, n = 585, 0.15% of European ancestry individuals) was associated with lower monocyte count and with both decreased downstream signaling and chemoattraction in response to CCL2. While M249K showed no association with conventional vascular risk factors, it was consistently associated with a lower risk of myocardial infarction (odds ratio [OR]: 0.66, 95% confidence interval [CI]: 0.54-0.81, p = 6.1 × 10-5) and coronary artery disease (OR: 0.74, 95%CI: 0.63-0.87, p = 2.9 × 10-4) in the UK Biobank and in six replication cohorts. In a phenome-wide association study, there was no evidence of a higher risk of infections among M249K carriers. CONCLUSIONS: Carriers of an experimentally confirmed damaging CCR2 variant are at a lower lifetime risk of myocardial infarction and coronary artery disease without carrying a higher risk of infections. Our findings provide genetic support for the translational potential of CCR2-targeting as an atheroprotective approach.

Humans↗

PubMed2001

Cutting edge: Differential regulation of chemoattractant receptor-induced degranulation and chemokine production by receptor phosphorylation.

Phosphorylation of G protein-coupled receptors and the subsequent recruitment of beta-arrestin play an important role in desensitization of receptor-mediated responses, including degranulation in leukocytes. In this study, we report that receptor phosphorylation also provides a stimulatory signal for CCR ligand 2 (CCL2) production. C3a stimulated degranulation in a basophilic leukemia RBL-2H3 cell expressing wild-type C3aR or a phosphorylation-deficient mutant (DeltaST-C3aR). In contrast, C3a caused CCL2 production only in C3aR but not DeltaST-C3aR cells. Furthermore, overexpression of G protein-coupled receptor kinase 2 resulted in enhancement of both ligand-induced receptor phosphorylation and CCL2 production but inhibition of degranulation. Agonist activation of C3aR, but not DeltaST-C3aR, led to the translocation of green fluorescent protein tagged beta-arrestin 2 from the cytoplasm to the plasma membrane. These data demonstrate that receptor phosphorylation, which provides a turn off signal for degranulation, is essential for CCL2 production.

Amino Acid Sequence↗

PubMed2006

Macrophage migration inhibitory factor induces macrophage recruitment via CC chemokine ligand 2.

Macrophage migration inhibitory factor (MIF) was originally identified for its ability to inhibit the random migration of macrophages in vitro. MIF is now recognized as an important mediator in a range of inflammatory disorders. We recently observed that the absence of MIF is associated with a reduction in leukocyte-endothelial cell interactions induced by a range of inflammatory mediators, suggesting that one mechanism whereby MIF acts during inflammatory responses is by promoting leukocyte recruitment. However, it is unknown whether MIF is capable of inducing leukocyte recruitment independently of additional inflammatory stimuli. In this study, we report that MIF is capable of inducing leukocyte adhesion and transmigration in postcapillary venules in vivo. Moreover, leukocytes recruited in response to MIF were predominantly CD68(+) cells of the monocyte/macrophage lineage. Abs against the monocyte-selective chemokine CCL2 (JE/MCP-1) and its receptor CCR2, but not CCL3 and CXCL2, significantly inhibited MIF-induced monocyte adhesion and transmigration. CCL2(-/-) mice displayed a similar reduction in MIF-induced recruitment indicating a critical role of CCL2 in the MIF-induced response. This hypothesis was supported by findings that MIF induced CCL2 release from primary microvascular endothelial cells. These data demonstrate a previously unrecognized function of this pleiotropic cytokine: induction of monocyte migration into tissues. This function may be critical to the ability of MIF to promote diseases such as atherosclerosis and rheumatoid arthritis, in which macrophages are key participants.

Animals↗

PubMed2004

Induction of glial L-CCR mRNA expression in spinal cord and brain in experimental autoimmune encephalomyelitis.

Chemokines and chemokine receptors are important regulators of leukocyte trafficking and immune response. It is well established that chemokines and their receptors are also expressed in the central nervous system (CNS), where their expression has been associated with various neuroinflammatory diseases, such as multiple sclerosis (MS). One of the most important chemokines involved in MS pathology is CCL2 (previously known as MCP-1). CCL2, released by glial cells, activates the chemokine receptor CCR2, causing the infiltration of blood monocytes in tissues affected by MS. There is evidence, however, that CCL2 also has local effects on CNS cells, including induction or modulation of cytokine release and synthesis of matrix metalloproteinases, that might contribute to CNS pathology. These effects are most likely independent of CCR2, since CCR2 expression in glial cells is rarely observed. We have recently provided evidence for the presence of an alternative CCL2 receptor in glial cells called L-CCR and have investigated the expression of L-CCR mRNA in a murine EAE model. It is shown that L-CCR mRNA is expressed in infiltrating macrophages during EAE, but not in infiltrating T cells. Prominent expression of L-CCR mRNA was detected in astrocytes and microglia already at early time points throughout the brain and spinal cord supporting the hypothesis that L-CCR expression in glial cells is related to CNS inflammation.

Animals↗

PubMed2006

Attenuated liver tumor formation in the absence of CCR2 with a concomitant reduction in the accumulation of hepatic stellate cells, macrophages and neovascularization.

The liver parenchyma is populated by hepatocytes and several nonparenchymal cell types, including Kupffer cells and hepatic stellate cells. Both Kupffer cells and hepatic stellate cells are responsive to the chemokine CCL2, but the precise roles of CCL2 and these cells in liver tumor formation remain undefined. Hence, we investigated the effects of the lack of the major CCL2 receptor, CCR2, on liver tumor formation induced by intraportal injection of the murine colon adenocarcinoma cell line, colon 26. Wild-type mice showed macroscopic tumor foci in the liver 10 days after injection of colon 26 cells. After 10 days, CCL2 proteins were detected predominantly in tumor cells, coincident with increased intratumoral macrophage and hepatic stellate cell numbers. Although tumor formation occurred at similar rates in wild-type and CCR2-deficient mice up to 10 days after tumor cell injection, the number and size of tumor foci were significantly attenuated in CCR2-deficient mice relative to wild-type mice thereafter. Moreover, neovascularization and matrix metalloproteinase 2 expression were diminished in CCR2-deficient mice with a concomitant reduction in the accumulation of macrophages and hepatic stellate cells. Furthermore, matrix metalloproteinase 2 was detected predominantly in hepatic stellate cells but not in macrophages. We provided the first definitive evidence that the absence of CCR2-mediated signals can reduce the trafficking of hepatic stellate cells, a main source of matrix metalloproteinase 2, and consequently can diminish neovascularization during liver tumor formation.

Adenocarcinoma↗

PubMed1988

The mechanism of the suicidal, reductive inactivation of microsomal cytochrome P-450 by carbon tetrachloride.

1. Stoichiometric losses of microsomal haem and cytochrome P-450 were observed when carbon tetrachloride (CCl4) was incubated anaerobically with rat liver microsomes using NADPH or sodium dithionite as a reducing agent. A rapid destruction of haem was also observed during the non-enzymatic reductive incubation of CCl4 with soluble haem preparations (methaemalbumin) in presence of sodium dithionite. The results indicate that haem is both the site and the target of the suicidal activation of CCl4 by cytochrome P-450. 2. When an additional, fluorimetric assay for haem determination was used, an equimolar loss of protoporphyrin IX fluorescence was also observed in both the enzymatic and non-enzymatic system, indicating that the haem moiety of cytochrome P-450 has undergone a structural change, involving either loss or labilization of the porphyrin tetrapyrrolic structure. In both systems the loss of porphyrin was prevented by carbon monoxide (CO). 3. A dichlorocarbene-cytochrome P-450 ligand complex is partially responsible for the difference spectrum obtained on addition of CCl4 to anaerobically reduced rat liver microsomes. A molar extinction coefficient for this complex has been calculated. The carbene trapping agent 2,3-dimethyl-2-butene (DMB) strongly inhibited (greater than 95%) the formation of this spectrum but did not modify the loss of haem in reduced CCl4-supplemented microsomal incubations. The results suggest that dichlorocarbene (:CCl2) is not significantly involved in CCl4-dependent haem destruction. 4. Pretreatment of rats with different microsomal enzyme inducers was responsible for similar but not identical patterns of :CCl2 and CO formation and haem loss during incubation of CCl4 with reduced microsomes. This indicates a critical role of CCl4 metabolism in the suicidal destruction of cytochrome P-450 haem and suggests that the apoprotein of cytochrome P-450 is capable of modulating not only the metabolism of CCl4 to :CCl2 but also the hydrolysis of :CCl2 to CO. 5. Inactivation of cytochrome P-450 by CCl4 with reduced microsomes from Aroclor-pretreated rats was saturable and followed pseudo first-order kinetics. This provides further evidence to conclude that CCl4 activation is a suicidal process where the reactive metabolite(s) formed bind to haem, we predict, in a one to one stoichiometry. 6. The partition ratio between loss of cytochrome P-450 haem and CCl4 metabolism by liver microsomes from Aroclor pretreated rats has been investigated using limiting concentrations of CCl4. It was calculated that approximately 26 molecules of CCl4 had to be metabolised to achieve the loss of one molecule of haem.

Animals↗

PubMed2002

Chemokines in health and disease.

Chemokines belong to a large family of structurally related proteins that play a pivotal role in immune system development and deployment. While a large number of chemokines (approximately 50) and their receptors (approximately 20) have been identified from humans or mice, only a few are known in domestic veterinary species. Recent data implicate CXCL8 (old name, IL-8), CXCL10 (old name, IP-10) (both CXC chemokines) and CCL2 (old name, MCP-1) (a CC chemokine) in veterinary infections, inflammatory diseases or reproduction. There is compelling evidence for neutrophil targeting chemokines such as CXCL8, in ovine bacterial mastitis, bovine pneumonic pasturellosis and equine chronic obstructive pulmonary disease (COPD). Monocyte and lymphocyte targeting chemokines appear to play a role in caprine arthritis encephalitis (CCL2) and canine endotoxemia (CXCL10). Interestingly CCL2 is considered a missing link between hormonal and cellular control of luteolysis. On the other hand, canine cardiovascular conditions are associated with overexpression of CCL2 and CXCL8. Furthermore, a number of veterinary viral pathogens encode chemokine/chemokine receptor like molecules or chemokine binding proteins that may help viruses to evade the immune system. Here, we provide an overview of the chemokine system and critically evaluate the current literature implicating chemokines in veterinary pathophysiology. Furthermore, we highlight promising areas for further research and discuss how and why chemokine antagonists are viewed as next generation anti-inflammatory drugs for the 21st century.

Animals↗

PubMed2004

Vibrational frequencies and structural determinations of tetrachlorobutatriene.

The normal mode frequencies and corresponding vibrational assignments of tetrachlorobutatriene in D2h symmetry are examined theoretically using the Gaussian 98 set of quantum chemistry codes. All normal modes were successfully assigned to one of the six types of motion (C=C stretch, CCl2 scissors, CCl2 twist, CCl2 wag, CCl2 rock, and C=C=C bend) predicted by a group theoretical analysis. By comparing the vibrational frequencies with IR and Raman spectra available in the literature, a set of scaling factors is derived.

Chlorine↗

PubMed2005

Accurate ab initio determination of spectroscopic and thermochemical properties of mono- and dichlorocarbenes.

The best technically feasible values for the triplet-singlet energy gap and the enthalpies of formation of the HCCl and CCl2 radicals have been determined through the focal-point approach. The electronic structure computations were based on high-level coupled cluster (CC) methods, up to quadruple excitations (CCSDTQ), and large-size correlation-consistent basis sets, ranging from aug-cc-pVDZ to aug-cc-pV6Z, followed by extrapolation to the complete basis set limit. Small corrections due to core correlation, relativistic effects, diagonal Born-Oppenheimer correction, as well as harmonic and anharmonic zero-point vibrational energy corrections have been taken into account. The final estimates for the triplet-singlet energy gap, T0(ã), are 2170+/-40 cm-1 for HCCl and 7045+/-60 cm-1 for CCl2, favoring the singlet states in both cases. Complete quartic force fields in internal coordinates have been computed for both the X and ã states of both radicals at the frozen-core CCSD(T)/aug-cc-pVQZ level. Using these force fields vibrational energy levels of {HCCl, DCCl, CCl2} up to {6000, 5000, 7000} cm-1 were calculated both by second-order vibrational perturbation theory (VPT2) and variationally. These results, especially the variational ones, show excellent agreement with the experimentally determined energy levels. The enthalpies of formation of HCCl (X1A') and CCl2(X1A1), at 0 K, are 76.28+/-0.20 and 54.54+/-0.20 kcal mol-1, respectively.

Algorithms↗

PubMed2002

Overexpression of ccl1-2 can bypass the need for the putative apocytochrome chaperone CycH during the biogenesis of c-type cytochromes.

In Gram-negative bacteria, including Rhodobacter capsulatus, the membrane protein CycH acts as a putative apocytochrome chaperone during the biogenesis of c-type cytochromes. CycH-null mutants are unable to produce various c-type cytochromes and sustain photosynthetic (Ps) growth that requires the cytochromes c1 and c2 or cy. However, Ps+ revertants are readily obtained only on minimal, but not on enriched, medium. To obtain further information about the biogenesis of c-type cytochromes, these suppressor mutants were studied. Complementation of a CycH-null mutant for Ps+ growth by a genomic library constructed using DNA from a Ps+ suppressor yielded a plasmid carrying the ccl1-2 operon, the products of which, Ccl1 and Ccl2, are also involved in the biogenesis of c-type cytochromes. DNA sequence analysis revealed that the complementing activity resulted from a single point mutation, G488A, located upstream of the coding region of ccl1-2. This mutation changed the -35 region of the ccl1-2 promoter from TTGGCC to TTGACC, improving its similarity to the consensus sequence of Escherichia colisigma 70-dependent promoters. That the G488A mutation indeed enhanced transcription of ccl1-2 was demonstrated by the use of reporter gene fusions. An appropriate ccl1-2::lacZ transcriptional-translational fusion carrying the G488A mutation produced in R. capsulatus over 30-fold higher beta-galactosidase activity than a wild-type construct. Immunoblot analyses confirmed that Ccl1 and Ccl2 were overproduced in the Ps+ suppressors. Deletion of either ccl1 or ccl2, from the ccl1-2 cluster carrying the G488A mutation abolished the complementing ability, indicating that overexpression of both ccl1 and ccl2 was required to confer the Ps+ phenotype on a CycH-null mutant. These findings therefore demonstrate that, during R. capsulatus growth on minimal medium, the requirement for CycH in c-type cytochrome biogenesis could be bypassed by overexpressing the ccl1-2 operon.

Amino Acid Substitution↗

PubMed2003

A highly selective CCR2 chemokine agonist encoded by human herpesvirus 6.

The chemokine-like, secreted protein product of the U83 gene from human herpesvirus 6, here named vCCL4, was chemically synthesized to be characterized in a complete library of the 18 known human chemokine receptors expressed individually in stably transfected cell lines. vCCL4 was found to cause calcium mobilization as efficiently as the endogenous chemokine ligand CCL2 through the CCR2 receptor, whereas the virally encoded chemokine did not affect any of the other 17 human chemokine receptors tested. Mutual cross-desensitization between CCL2 and vCCL4 was demonstrated in the CCR2-transfected cells. The affinity of vCCL4 for the CCR2 receptor was 79 nm as determined in competition binding against radioactively labeled CCL2. In the murine pre-B lymphocyte cell line L1.2 stably transfected with the CCR2 receptor, vCCL4 acted as a relatively low potency but highly efficacious chemoattractant being equally or more efficacious in causing cell migration than CCL2 and CCL7 and considerably more efficacious than CCL8 and CCL13. It is concluded that human herpesvirus 6 encodes a highly selective and efficacious CCR2 agonist, which will attract CCR2 expressing cells, for example macrophages and monocytes, conceivably for the virus to infect and to establish latency in. It is suggested that vCCL4 during reactivation of the virus in for example monocyte-derived microglia could perhaps be involved in the pathogenesis of the CCR2-dependent disease, multiple sclerosis.

Amino Acid Sequence↗

PubMed1997

Differential levels of specific cytochrome c biogenesis proteins in response to oxygen: analysis of the ccl operon in Rhodobacter capsulatus.

The photosynthetic bacterium Rhodobacter capsulatus synthesizes c-type cytochromes under a variety of growth conditions. For example, under aerobic growth, c-type cytochromes are synthesized as part of an electron transport pathway, using oxygen as the terminal electron acceptor. Anaerobically in the light, R. capsulatus requires cytochrome bc1 and other c-type cytochromes for the photosynthetic electron transport pathway. It is shown here that the ccl1 and ccl2 genes of R. capsulatus are required for the synthesis of all c-type cytochromes, including the cytochrome c' protein of unknown function but of structural similarity to cytochrome b562. Polar and nonpolar mutations constructed in each gene demonstrated that the ccl12 genes form an operon. Expression of the ccl12 genes was examined by using lacZ and phoA fusions as translational reporters. Primer extension analysis was used to determine transcriptional control and the start site of the ccl12 promoter. Finally, antiserum to the Ccl2 protein was used to quantitate levels of Ccl2 under six different growth conditions. The Ccl2 protein is present at 20-fold-higher levels under conditions where oxygen is present. In contrast, other cytochromes c biogenesis proteins, HelA and HelX, previously shown to be part of an helABCDX operon, are at relatively similar levels under these six growth conditions. This discovery is discussed in terms of the physiology and evolution of cytochromes c biogenesis, with particular attention to oxidative environments.

Aerobiosis↗

PubMed2002

Chemokine stimulation of monocyte matrix metalloproteinase-9 requires endogenous TNF-alpha.

Leukocyte extravasation into tissues is a multi-step process culminating in the migration of cells through the basement membrane. This requires the production of matrix-degrading enzymes, in particular matrix metalloproteinases (MMP). We investigated the role of chemokines in regulating MMP production in the monocytic cell line THP-1 and in peripheral blood monocytes (PBM). The CC chemokines CCL2 (MCP-1), CCL3 (MIP-1alpha), and CCL5 (RANTES) stimulated the release of monocyte MMP-9 protein in a bell-shaped dose-dependent manner. The increase in MMP-9 protein detected at 24 h was due to de novo synthesis, confirmed by Northern blotting, with MMP-9 mRNA detectable at 6-8 h. Autocrine TNF-alpha was necessary for chemokine stimulation of MMP-9. Chemokines increased TNF-alpha mRNA levels and protein release in monocytes and THP-1 cells, and neutralizing anti-TNF-alpha antibodies inhibited CCL2-induced MMP-9 release. Furthermore, the broad spectrum MMP inhibitor BB 2516, which inhibits TNF-alpha release, abrogated CCL2- and CCL5-induced MMP-9 release in both THP-1 cells and freshly isolated monocytes. Monocyte production of MMP is of major importance in the pathology of cancer, asthma, and rheumatoid arthritis. An understanding of the mechanisms by which these MMP are produced may lead to novel therapies to modulate extravasation of leukocytes in disease.

Cell Line↗

PubMed2005

Chemokine receptor CCR2 expression by systemic sclerosis fibroblasts: evidence for autocrine regulation of myofibroblast differentiation.

OBJECTIVE: To investigate expression of the chemokine receptor CCR2 on key cell types involved in the pathogenesis of systemic sclerosis (SSc) and to assess the potential for autocrine activation of SSc dermal fibroblasts via CCL2/CCR2. METHODS: Chemokine receptor expression in skin biopsy tissues and explanted dermal fibroblasts from a well-characterized cohort of SSc patients was examined using immunohistochemistry and flow cytometry techniques. Autocrine regulation of the expression of fibrotic markers in CCR2+ SSc fibroblast cell lines was assessed using specific ligand or receptor antagonists. RESULTS: We identified strong CCR2 expression in skin biopsy samples of early-stage diffuse cutaneous SSc (dcSSc), but not late-stage dcSSc or limited cutaneous SSc. Double labeling confirmed up-regulation of CCL2/CCR2 on myofibroblasts, pericytes, lymphocytes, macrophages, and endothelial cells. Explanted dermal fibroblasts from early dcSSc tissues expressed CCR2 and CXCR2 in 55% and 66% of cell strains, respectively. There was no expression in control fibroblasts. CCR2+ fibroblasts demonstrated a profibrotic phenotype, with overexpression of alpha-smooth muscle actin (alpha-SMA), connective tissue growth factor (CTGF), and CCL2. Flow cytometric analysis identified a subset of CCR2+ SSc fibroblasts expressing the myofibroblast marker alpha-SMA. In these cultures, specific inhibition of CCL2 or CCR2 attenuated the overexpression of alpha-SMA, but not CTGF or plasminogen activator inhibitor 1. CONCLUSION: Our results show that CCR2 is up-regulated in early dcSSc on cell types known to be activated in the disease, which is consistent with a key role in SSc pathogenesis. CCR2 expression on SSc fibroblasts appears to regulate the expression of CCL2 and alpha-SMA. Our findings suggest potential autocrine regulation of key profibrotic properties via a CCL2/CCR2 loop in early-stage dcSSc.

Actins↗