MAP-Kinase Activated Protein Kinase 2 Links Endothelial Activation and Monocyte/macrophage Recruitment in Arteriogenesis

Arteriogenesis, the growth of natural bypass arteries, is triggered by hemodynamic forces within vessels and requires a balanced inflammatory response, involving induction of the chemokine MCP-1 and recruitment of leukocytes. However, little is known how these processes are coordinated. The MAP-kinase-activated-proteinkinase-2 (MK2) is a critical regulator of inflammatory processes and might represent an important link between cytokine production and cell recruitment during postnatal arteriogenesis. Therefore, the present study investigated the functional role of MK2 during postnatal arteriogenesis. In a mouse model of hindlimb ischemia (HLI) MK2-deficiency (MK2KO) significantly impaired ischemic blood flow recovery and growth of collateral arteries as well as perivascular recruitment of mononuclear cells and macrophages. This was accompanied by induction of endothelial MCP-1 expression in wildtype (WT) but not in MK2KO collateral arteries. Following HLI, MK2 activation rapidly occured in the endothelium of growing WT arteries in vivo. In vitro, inflammatory cytokines and cyclic stretch activated MK2 in endothelial cells, which was required for stretch- and cytokine-induced release of MCP-1. In addition, a monocyte cell autonomous function of MK2 was uncovered potentially regulating MCP-1-dependent monocyte recruitment to vessels: MCP-1 stimulation of WT monocytes induced MK2 activation and monocyte migration in vitro. The latter was reduced in MK2KO monocytes, while in vivo MK2 was activated in monocytes recruited to collateral arteries. In conclusion, MK2 regulates postnatal arteriogenesis by controlling vascular recruitment of monocytes/macrophages in a dual manner: regulation of endothelial MCP-1 expression in response to hemodynamic and inflammatory forces as well as MCP-1 dependent monocyte migration.     

Alveolar macrophage-derived cytokines induce monocyte chemoattractant protein-1 expression from human pulmonary type II-like epithelial cells

Many acute and chronic lung diseases are characterized by the presence of increased numbers of activated macrophages. These macrophages are derived predominantly from newly recruited peripheral blood monocytes and may play a role in the amplification and perpetuation of an initial lung insult. The process of inflammatory cell recruitment is poorly understood, although the expression of inflammatory cell-specific chemoattractants and subsequent generation of chemotactic gradients is likely involved. Although immune cells such as macrophages and lymphocytes are known to generate several inflammatory cell chemoattractants, parenchymal cells can also synthesize and secrete a number of bioactive factors. We now demonstrate the generation of significant monocyte chemotactic activity from tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta-treated pulmonary type II-like epithelial cells (A549). The predominant inducible monocyte chemotaxin had an estimated molecular mass of approximately 14-15 kDa and was neutralized by specific antibody to human monocyte chemotactic protein-1 (MCP-1). Induction of activity was accompanied by increases in steady-state mRNA level for MCP-1. These data are consistent with the induction of MCP-1 expression from A549 cells by TNF and IL-1. MCP-1 production from A549 cells could be induced by lipopolysaccharide (LPS)-stimulated alveolar macrophage (AM)-conditioned media, but not by LPS alone. The inducing activity in AM-conditioned media was neutralized with specific antibodies to IL-1 beta, but not TNF-alpha. Our findings suggest that the alveolar epithelium can participate in inflammatory cell recruitment via the production of MCP-1 and that cytokine networking between contiguous alveolar macrophages and the pulmonary epithelium may be essential for parenchymal cell MCP-1 expression.     

Impact of the anti-inflammatory agent bindarit on the chemokinome: selective inhibition of the monocyte chemotactic proteins

Bindarit is an indazolic derivative that is devoid of any immunosuppressive effects and has no effect on arachidonic acid metabolism. However, it has been proved to have anti-inflammatory activity in a number of experimental diseases, including pancreatitis, arthritis, and lupus nephritis. This therapeutic effect has been associated with its ability to interfere selectively with monocyte recruitment, although the underlying molecular mechanisms are unknown. Here we comprehensively examine the effect of bindarit on the chemokine system, and report that in activated monocytes and endothelial cells, it selectively inhibits the production of the monocyte chemotactic protein subfamily of CC inflammatory chemokines (MCP-1/CCL2, MCP-3/CCL7, MCP-2/CCL8). The capacity of bindarit to inhibit the production of a defined set of related CC chemokines by monocytes and endothelial cells likely underlies the anti-inflammatory activity of this agent in disease. The exploitation of the chemokine system as drug target in inflammatory disease has relied mainly on the development of receptor antagonists and blocking antibodies. Here we report on the use of inhibition of synthesis as a potentially viable and selective approach to modify the chemokine system.     

Attenuation of the pulmonary inflammatory response following butylated hydroxytoluene treatment of cytosolic phospholipase A2 null mice

Administration of butylated hydroxytoluene (BHT) to mice causes lung damage characterized by the death of alveolar type I pneumocytes and the proliferation and subsequent differentiation of type II cells to replace them. Herein, we demonstrate this injury elicits an inflammatory response marked by chemokine secretion, alveolar macrophage recruitment, and elevated expression of enzymes in the eicosanoid pathway. Cytosolic phospholipase A(2) (cPLA(2)) catalyzes release of arachidonic acid from membrane phospholipids to initiate the synthesis of prostaglandins and other inflammatory mediators. A role for cPLA(2) in this response was examined by determining cPLA(2) expression and enzymatic activity in distal respiratory epithelia and macrophages and by assessing the consequences of cPLA(2) genetic ablation. BHT-induced lung inflammation, particularly monocyte infiltration, was depressed in cPLA(2) null mice. Monocyte chemotactic protein-1 (MCP-1) content in bronchoalveolar lavage fluid increases after BHT treatment but before monocyte influx, suggesting a causative role. Bronchiolar Clara cells isolated from cPLA(2) null mice secrete less MCP-1 than Clara cells from wild-type mice, consistent with the hypothesis that cPLA(2) is required to secrete sufficient MCP-1 to induce an inflammatory monocytic response.     

Rho-kinase mediates lysophosphatidic acid-induced IL-8 and MCP-1 production via p38 and JNK pathways in human endothelial cells

Lysophosphatidic acid (LPA), an inflammatory mediator that is elevated in multiple inflammatory diseases, is a potent activator of Rho kinase (ROCK) signaling and of chemokine production in endothelial cells. In this study, LPA activated ROCK, p38, JNK and NF-κB pathways and induced interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) mRNA and protein expression in human endothelial cells. We mapped signaling events downstream of ROCK, driving chemokine production. In summary, MCP-1 production was partly regulated by ROCK acting upstream of p38 and JNK and mediated downstream by NF-κB. IL-8 production was largely driven by ROCK through p38 and JNK activation, but with no involvement of NF-κB.     

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.     

Phosphorothioate oligodeoxynucleotides inhibit basic fibroblast growth factor-induced angiogenesis in vitro and in vivo.

Angiogenesis is regulated by heparin-binding growth factors, such as basic fibroblast growth factor (bFGF). We investigated the effects of phosphorothioate-mediated oligodeoxynucleotides (PS-ODN) on bFGF-induced angiogenesis. Because PS-ODN are polyanions, they can also bind many heparin-binding proteins. On a basement matrix using a Matrigel matrix, we observed <50% tube formation by human umbilical endothelial cells with 10 microM bFGF, vascular endothelial growth factor, or nuclear factor-kappaB (NF-kappaB) antisense and sense PS-ODN, while phosphodiester oligodeoxynucleotides (PO-ODNs) were not affected. The PS-ODN, but not the PO-ODN, inhibited the bFGF-induced rabbit corneal neovascularization. In albino rats, the NF-kappaB antisense PS-ODN showed a low rescue score for bFGF-dependent photoreceptor rescue because of their degradation by constant light exposure. However, antisense PS-ODN active against bFGF inhibited angiogenesis more strongly than did the antisense NF-kappaB PS-ODN. Because of the important role bFGF plays in angiogenesis, some PS-ODN may serve as potent antiangiogenic compounds that act through a combination of polyanionic phosphorothioate effects and a sequence-specific antisense mechanism.     

Effects of antioxidant and nitric oxide on chemokine production in TNF-alpha-stimulated human dermal microvascular endothelial cells

Chemokines have been implicated convincingly in the driving of leukocyte emigration in different inflammatory reactions. Multiple signaling mechanisms are reported to be involved in intracellular activation of chemokine expression in vascular endothelial cells by various stimuli. Nevertheless, redox-regulated mechanisms of chemokine expression in human dermal microvascular endothelial cells (HDMEC) remain unclear. This study examined the effects of pyrrolidine dithiocarbamate (PDTC, 0.1 mM) and spermine NONOate (Sper-NO, 1 mM) on the secretion and gene expression of chemokines, interleukin (IL)-8, monocyte chemotactic protein (MCP)-1, regulated upon activation normal T cell expressed and secreted (RANTES), and eotaxin. This study also addresses PDTC and Sper-NO effects on activation of nuclear factor kappa B (NF-kappaB) induced by TNF-alpha (10 ng/ml). Treatment with TNF-alpha for 8 h significantly increased secretion of IL-8, MCP-1, and RANTES, but not of eotaxin, in cultured HDMEC. Up-regulation of these chemokines was suppressed significantly by pretreatment with PDTC or Sper-NO for 1 h, but not by 1 mM 8-bromo-cyclic GMP. The mRNA accumulation of IL-8, MCP-1, RANTES, and eotaxin, and activation of NF-kappaB were induced by TNF-alpha for 2 h; all were suppressed significantly by the above two pretreatments. These findings indicate that both secretion and mRNA accumulation of IL-8, MCP-1, and RANTES in HDMEC induced by TNF-alpha are inhibited significantly by pretreatment with PDTC or Sper-NO, possibly via blocking redox-regulated NF-kappaB activation. These results suggest that restoration of the redox balance using antioxidant agents or nitric oxide pathway modulators may offer new opportunities for therapeutic interventions in inflammatory skin diseases.     

Role of chemokines and formyl peptides in pneumococcal pneumonia-induced monocyte/macrophage recruitment

Host-derived chemoattractant factors are suggested to play crucial roles in leukocyte recruitment elicited by inflammatory stimuli in vitro and in vivo. However, in the case of acute bacterial infections, pathogen-derived chemoattractant factors are also present, and it has not yet been clarified how cross-talk between chemoattractant receptors orchestrates diapedesis of leukocytes in this context of complex chemoattractant arrays. To investigate the role of chemokine (host-derived) and formyl peptide (pathogen-derived) chemoattractants in leukocyte extravasation in life-threatening infectious diseases, we used a mouse model of pneumococcal pneumonia. We found an increase in mRNA expression of eight chemokines (RANTES, macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, IP-10, monocyte chemoattractant protein (MCP)-1, T cell activation 3, and KC) within the lungs during the course of infection. KC and MIP-2 protein expression closely preceded pulmonary neutrophil recruitment, whereas MCP-1 protein production coincided more closely than MIP-1alpha with the kinetics of macrophage infiltration. In situ hybridization of MCP-1 mRNA suggested that MCP-1 expression started at peribronchovascular regions and expanded to alveoli-facing epithelial cells and infiltrated macrophages. Interestingly, administration of a neutralizing Ab against MCP-1, RANTES, or MIP-1alpha alone did not prevent macrophage infiltration into infected alveoli, whereas combination of the three Abs significantly reduced macrophage infiltration without affecting neutrophil recruitment. The use of an antagonist to N-formyl peptides, N-t-Boc-Phe-D-Leu-Phe-D-Leu-Phe, reduced both macrophages and neutrophils significantly. These data demonstrate that a complex chemokine network is activated in response to pulmonary pneumococcal infection, and also suggest an important role for fMLP receptor in monocyte/macrophage recruitment in that model.     

Low-level monocyte chemoattractant protein-1 stimulation of monocytes leads to tumor formation in nontumorigenic melanoma cells

Tumors commonly produce chemokines for recruitment of host cells, but the biological significance of tumor-infiltrating inflammatory cells, such as monocytes/macrophages, for disease outcome is not clear. Here, we show that all of 30 melanoma cell lines secreted monocyte chemoattractant protein-1 (MCP-1), whereas normal melanocytes did not. When low MCP-1-producing melanoma cells from a biologically early, nontumorigenic stage were transduced to overexpress the MCP-1 gene, tumor formation depended on the level of chemokine secretion and monocyte infiltration; low-level MCP-1 secretion with modest monocyte infiltration resulted in tumor formation, whereas high secretion was associated with massive monocyte/macrophage infiltration into the tumor mass, leading to its destruction within a few days after injection into mice. Tumor growth stimulated by monocytes/macrophages was due to increased angiogenesis. Vessel formation in vitro was inhibited with mAbs against TNF-alpha, which, when secreted by cocultures of melanoma cells with human monocytes, induced endothelial cells under collagen gels to form branching, tubular structures. These studies demonstrate that the biological effects of tumor-derived MCP-1 are biphasic, depending on the level of secretion. This correlates with the degree of monocytic cell infiltration, which results in increased tumor vascularization and TNF-alpha production.     

Monocyte chemoattractant protein-1--a major contributor to the inflammatory process associated with diabetes

There is evidence that strongly suggests that inflammation plays an important role in diabetes and cardiovascular diseases. The high glucose-induced inflammatory process is characterised by the cooperation of a complex network of inflammatory molecules such as cytokines, adhesion molecules, growth factors, and chemokines. Among the chemokine family, monocyte chemoattractant protein (MCP-1) is a potent chemotactic factor, which is upregulated at sites of inflammation being in control of leukocytes trafficking. Here, we review the current knowledge on MCP-1 and its regulation by high glucose level in vascular cells involved in diabetes-induced accelerated atherosclerosis. The signalling pathways involved in MCP-1 modulation by high glucose, the proximal signalling events that stimulate downstream effects and the role of this chemokine in the pathophysiology of diabetes and its complications, are discussed.     

Effects of Antioxidant and Nitric Oxide on Chemokine Production in TNF-α-stimulated Human Dermal Microvascular Endothelial Cells

Chemokines have been implicated convincingly in the driving of leukocyte emigration in different inflammatory reactions. Multiple signaling mechanisms are reported to be involved in intracellular activation of chemokine expression in vascular endothelial cells by various stimuli. Nevertheless, redox-regulated mechanisms of chemokine expression in human dermal microvascular endothelial cells (HDMEC) remain unclear. This study examined the effects of pyrrolidine dithiocarbamate (PDTC, 0.1?mM) and spermine NONOate (Sper-NO, 1?mM) on the secretion and gene expression of chemokines, interleukin (IL)-8, monocyte chemotactic protein (MCP)-1, regulated upon activation normal T cell expressed and secreted (RANTES), and eotaxin. This study also addresses PDTC and Sper-NO effects on activation of nuclear factor kappa B (NF-κB) induced by TNF-α (10?ng/ml). Treatment with TNF-α for 8?h significantly increased secretion of IL-8, MCP-1, and RANTES, but not of eotaxin, in cultured HDMEC. Up-regulation of these chemokines was suppressed significantly by pretreatment with PDTC or Sper-NO for 1?h, but not by 1?mM 8-bromo-cyclic GMP. The mRNA accumulation of IL-8, MCP-1, RANTES, and eotaxin, and activation of NF-κB were induced by TNF-α for 2?h; all were suppressed significantly by the above two pretreatments. These findings indicate that both secretion and mRNA accumulation of IL-8, MCP-1, and RANTES in HDMEC induced by TNF-α are inhibited significantly by pretreatment with PDTC or Sper-NO, possibly via blocking redox-regulated NF-κB activation. These results suggest that restoration of the redox balance using antioxidant agents or nitric oxide pathway modulators may offer new opportunities for therapeutic interventions in inflammatory skin diseases.     

Additive roles for MCP-1 and MCP-3 in CCR2-mediated recruitment of inflammatory monocytes during Listeria monocytogenes infection

Chemokine receptor-mediated recruitment of inflammatory cells is essential for innate immune defense against microbial infection. Recruitment of Ly6C(high) inflammatory monocytes from bone marrow to sites of microbial infection is dependent on CCR2, a chemokine receptor that responds to MCP-1 and MCP-3. Although CCR2(-/-) mice are markedly more susceptible to Listeria monocytogenes infection than are wild-type mice, MCP-1(-/-) mice have an intermediate phenotype, suggesting that other CCR2 ligands contribute to antimicrobial defense. Herein, we show that L. monocytogenes infection rapidly induces MCP-3 in tissue culture macrophages and in serum, spleen, liver, and kidney following in vivo infection. Only cytosol invasive L. monocytogenes induce MCP-3, suggesting that cytosolic innate immune detection mechanisms trigger chemokine production. MCP-3(-/-) mice clear bacteria less effectively from the spleen than do wild-type mice, a defect that correlates with diminished inflammatory monocyte recruitment. MCP-3(-/-) mice have significantly fewer Ly6C(high) monocytes in the spleen and bloodstream, and increased monocyte numbers in bone marrow. MCP-3(-/-) mice, like MCP-1(-/-) mice, have fewer TNF- and inducible NO synthase-producing dendritic cells (Tip-DCs) in the spleen following L. monocytogenes infection. Our data demonstrate that MCP-3 and MCP-1 provide parallel contributions to CCR2-mediated inflammatory monocyte recruitment and that both chemokines are required for optimal innate immune defense against L. monocytogenes infection.     

Differences in hyaluronic acid-mediated functions and signaling in arterial, microvessel, and vein-derived human endothelial cells

Hyaluronic acid (HA), a nonsulfated glycosaminoglycan, regulates cell adhesion and migration. Small HA fragments (3-25 disaccharide units) induce neovascularization. We investigated the effect of HA and a HA fragment (10-15 disaccharide units, F1) on primary human endothelial cells (ECs). Human pulmonary ECs (HPAEC) and lung microvessel ECs (HMVEC-L) bound HA (K(d) approximately 1 and 2.3 nm, respectively) and expressed 17,780 and 16,690 HA binding sites, respectively. Both ECs showed HA-mediated cell adhesion; however, HMVEC-L was 1.5-fold better. Human umbilical vein ECs neither bound HA nor showed HA-mediated adhesion. All three ECs expressed CD44 ( approximately 110 kDa). The expression of receptor for HA-mediated motility (RHAMM) (approximately 80 kDa) was the highest in HMVEC-L, followed by HPAEC and human umbilical vein ECs. RHAMM, not CD44, bound HA in all three ECs. F1 was better than HA and stimulated a 2. 5- and 1.8-fold mitogenic response in HMVEC-L and HPAEC, respectively. Both HA and F1 induced tyrosine phosphorylation of p125(FAK), paxillin, and p42/44 ERK in HMVEC-L and HPAEC, which was blocked by an anti-RHAMM antibody. These results demonstrate that RHAMM is the functional HA receptor in primary human ECs. Heterogeneity exists among primary human ECs of different vascular origins, with respect to functional HA receptor expression and function.     

Mitogen-activated protein kinase phosphatase 1 activity is necessary for oxidized phospholipids to induce monocyte chemotactic activity in human aortic endothelial cells

Entrapment and oxidation of low density lipoproteins (LDL) in the sub-endothelial space is a key process in the initiation of atherosclerotic lesion development. Functional changes induced by oxidized lipids in endothelial cells are early events in the pathogenesis of atherosclerosis. Oxidized-l-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (ox-PAPC), a major component of minimally modified/oxidized-LDL (MM-LDL) mimics the biological activities assigned to MM-LDL both in vitro in a co-culture model as well as in vivo in mice. We hypothesized that ox-PAPC initiates gene expression changes in endothelial cells that result in enhanced endothelial/monocyte interactions. To analyze the gene expression changes that oxidized lipids induce in endothelial cells, we used a suppression subtractive hybridization procedure to compare mRNA from PAPC-treated human aortic endothelial cells (HAEC) with that of ox-PAPC-treated cells. We report here the identification of a gene, mitogen-activated protein kinase phosphatase 1 (MKP-1), that is rapidly and transiently induced in ox-PAPC-treated HAEC. Inhibition of MKP-1 using either the phosphatase inhibitor sodium orthovanadate or antisense oligonucleotides prevents the accumulation of monocyte chemotactic activity in ox-PAPC-treated HAEC supernatants. Furthermore, we show that decreased monocyte chemotactic activity in HAEC treated with sodium orthovanadate or MKP-1 antisense oligonucleotides is due to decreased MCP-1 protein. Our results implicate a direct role for MKP-1 in ox-PAPC-induced signaling pathways that result in the production of MCP-1 protein by ox-PAPC-treated HAEC.     

Role of monocyte chemotactic protein-1/CC chemokine ligand 2 on gamma delta T lymphocyte trafficking during inflammation induced by lipopolysaccharide or Mycobacterium bovis bacille Calmette-Guérin

Gammadelta T lymphocytes are involved in a great variety of inflammatory and infectious responses. However, the mechanisms by which gammadelta T lymphocytes migrate to inflamed sites are poorly understood. In this study we investigate the role of monocyte chemotactic protein (MCP)-1 in regulating gammadelta T cell migration after LPS or Mycobacterium bovis bacille Calmette-Guérin (BCG) challenge. LPS-induced gammadelta T cell influx was significantly inhibited by either pretreatment with dexamethasone or vaccinia virus Lister 35-kDa chemokine binding protein, vCKBP, a CC chemokine neutralizing protein, suggesting a role for CC chemokines in this phenomenon. LPS stimulation increased the expression of MCP-1 mRNA and protein at the inflammation site within 6 h. It is noteworthy that LPS was unable to increase MCP-1 production or gammadelta T cell recruitment in C3H/HeJ, indicative of the involvement of Toll-like receptor 4. Gammadelta T cells express MCP-1 receptor CCR2. Pretreatment with anti-MCP-1 mAb drastically inhibited LPS-induced in vivo gammadelta T cell mobilization. Indeed, MCP-1 knockout mice were unable to recruit gammadelta T cells to the pleural cavity after LPS stimulation, effect that could be restored by coadministration of MCP-1. In addition, BCG-induced gammadelta lymphocyte accumulation was significantly reduced in MCP-1 knockout mice when compared with wild-type mice. In conclusion, our results indicate that LPS-induced gammadelta T lymphocyte migration is dependent on Toll-like receptor 4 and sensitive to both dexamethasone and CC chemokine-binding protein inhibition. Moreover, by using MCP-1 neutralizing Abs and genetically deficient mice we show that LPS- and BCG-induced gammadelta T lymphocyte influx to the pleural cavity of mice is mainly orchestrated by the CC chemokine MCP-1.