Monocyte chemoattractant protein-1 and macrophage inflammatory protein-2 are involved in both excitotoxin-induced neurodegeneration and regeneration

Intrahippocamal injections of kainic acid (KA) significantly increase the expression of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) in the ipsilateral hippocampus at 2-4 h and 21-45 days post-administration, suggesting the possible involvement of these chemokines in both neurodegenerative and regenerative processes. To examine the possible role of these chemokines on neuronal cell death, hippocampal neurons were incubated with either MCP-1 or MIP-2 in vitro and examined to assess the effects on neuronal cell viability. These treatments resulted in significant neuronal apoptosis that could be abrogated by prior treatment with the caspase-1 inhibitor, Z-VAD-FMK, the caspase-3 inhibitor, Z-DEVD-FMK, the Galphai inhibitor, pertussis toxin, or the MAO-B inhibitor, (-)deprenyl. Furthermore, this chemokine apoptotic effect could also be observed in vivo as intrahippocampal injections of MCP-1 or MIP-2 resulted in the apoptosis of hippocampal neurons, thus supporting a direct role of these chemokines in neuronal death. In contrast, immunohistological analysis of kainic acid lesions on days 21-45 revealed significant expression of MCP-1 and MIP-2 associated with reactive astrocytes and macrophages, respectively, with no apoptotic populations being observed. These results suggested that these chemokines might also mediate distinct biological effects on local microenvironmental cell populations at various stages post truama and during cellular repair. To address this possibility, astrocyte were cultured in the presence or absence of these chemokines and examined by microarray analysis for effects on astrocytes gene expression. A number of genes encoding proteins associated with inflammation, cellular signaling, differentiation, and repair were directly modulated by chemokine treatment. More specifically, the RNA and protein expression of the neurotrophic factor, basic fibroblast growth factor (bFGF), was found to be significantly increased upon culture with MCP-1 and MIP-2. Conditioned media derived from chemokine-stimulated astrocytes also facilitated bFGF-dependent neuronal cell differentiation and promoted survival of H19-7 neurons in vitro, suggesting a possible role for chemokine-activated astrocytes as a source of trophic support. Taken together, these data support possible autocrine and paracrine roles for MCP-1 and MIP-2 in both the "death and life" of hippocampal neurons following CNS injury.     

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.     

Monocyte chemoattractant protein-1 functions as a neuromodulator in dorsal root ganglia neurons

It has previously been observed that expression of chemokine monocyte chemoattractant protein-1 (MCP-1/CC chemokine ligand 2 (CCL2)) and its receptor CC chemokine receptor 2 (CCR2) is up-regulated by dorsal root ganglion (DRG) neurons in association with rodent models of neuropathic pain. MCP-1 increases the excitability of nociceptive neurons after a peripheral nerve injury, while disruption of MCP-1/CCR2 signaling blocks the development of neuropathic pain, suggesting MCP-1 signaling is responsible for heightened pain sensitivity. To define the mechanisms of MCP-1 signaling in DRG, we studied intracellular processing, release, and receptor-mediated signaling of MCP-1 in DRG neurons. We found that in a focal demyelination model of neuropathic pain both MCP-1 and CCR2 were up-regulated by the same neurons including transient receptor potential vanilloid receptor subtype 1 (TRPV1) expressing nociceptors. MCP-1 expressed by DRG neurons was packaged into large dense-core vesicles whose release could be induced from the soma by depolarization in a Ca²⁺-dependent manner. Activation of CCR2 by MCP-1 could sensitize nociceptors via transactivation of transient receptor potential channels. Our results suggest that MCP-1 and CCR2, up-regulated by sensory neurons following peripheral nerve injury, might participate in neural signal processing which contributes to sustained excitability of primary afferent neurons.     

Monocyte chemoattractant protein-1 and CCR2 interactions are required for IFN-alpha/beta-induced inflammatory responses and antiviral defense in liver

IFN-alpha/beta-mediated functions promote production of MIP-1alpha (or CCL3) by mediating the recruitment of MIP-1alpha-producing macrophages to the liver during early infection with murine CMV. These responses are essential for induction of NK cell inflammation and IFN-gamma delivery to support effective control of local infection. Nevertheless, it remains to be established if additional chemokine functions are regulated by IFN-alpha/beta and/or play intermediary roles in supporting macrophage trafficking. The chemokine MCP-1 (or CCL2) plays a distinctive role in the recruitment of macrophages by predominantly stimulating the CCR2 chemokine receptor. Here, we examine the roles of MCP-1 and CCR2 during murine CMV infection in liver. MCP-1 production preceded that of MIP-1alpha during infection and was dependent on IFN-alpha/beta effects for induction. Resident F4/80(+) liver leukocytes were identified as primary IFN-alpha/beta responders and major producers of MCP-1. Moreover, MCP-1 deficiency was associated with a dramatic reduction in the accumulation of macrophages and NK cells, as well as decreased production of MIP-1alpha and IFN-gamma in liver. These responses were also markedly impaired in mice with a targeted disruption of CCR2. Furthermore, MCP-1- and CCR2-deficient mice exhibited increased viral titers and elevated expression of the liver enzyme alanine aminotransferase in serum. These mice also had widespread virus-induced liver pathology and succumbed to infection. Collectively, these results establish MCP-1 and CCR2 interactions as factors promoting early liver inflammatory responses and define a mechanism for innate cytokines in regulation of chemokine functions critical for effective localized antiviral defenses.     

Monocyte chemoattractant protein-1 induces endothelial cell apoptosis in vitro through a p53-dependent mitochondrial pathway

The cystine-cystine (CC) chemokine monocyte chemoattractant protein-1 (MCP-1) has been established playing a pathogenic role in the development of atherosclerosis due to its chemotactic ability of leading monocytes to locate to subendothelia. Recent studies have revealed more MCP-1 functions other than chemotaxis. Here we reported that various concentrations (0.1-100 ng/ml) of MCP-1 induced human umbilical vein endothelial cell (HUVEC) strain CRL-1730 apoptosis, caspase-9 activation, and a couple of mitochondrial alterations. Moreover, MCP-1 upregulated p53 expression of HUVECs and the p53-specific inhibitor pifithrin-α (PFTα) rescued the MCP-1-induced apoptosis of HUVECs. Furthermore, PKC (protein kinase C) activation or inhibition might also affect HUVECs apoptosis induced by MCP-1. These findings together demonstrate that MCP-1 exerts direct proapoptotic effects on HUVECs in vitro via a p53-dependent mitochondrial pathway.     

Monocyte chemoattractant protein-1 induces scavenger receptor expression and monocyte differentiation into foam cells

Accumulation of monocytes and the entrapment of oxidized-low-density lipoprotein (ox-LDL) in monocytes are important in the differentiation into "foam" macrophages and the pathogenesis of atherosclerosis. We investigated the role of monocyte chemoattractant protein-1 (MCP-1) in the expression of scavenger receptor (SCR) by using resting monocytes prepared by counterflow centrifugal elutriation. Our results showed that: (1) MCP-1 increased the expression of CD36 SCR by flow cytometric analysis. (2) MCP-1 increased incorporation of 125I-labeled ox-LDL and oil red O staining. (3) MCP-1 and ox-LDL enhanced in vitro transendothelial monocyte migration. (4) These functions were mediated at least in part via extracellular signal-regulated kinase (ERK) pathway. (5) MCP-1 and ox-LDL did not induce monocyte proliferation. Our results imply that MCP-1 is involved in the inflammatory process of atherosclerosis through the induction of SCR expression via the ERK pathway and differentiation of monocytes into foam macrophages, as well as induction of monocyte migration.     

Monocyte chemoattractant protein-1 regulates adhesion molecule expression and cytokine production in human monocytes.

Monocytes play a critical role in defending the host against foreign organisms and in regulating the behavior of other cells. Monocytes circulate as nonadherent cells in the blood and migrate as adherent cells through tissues. Adhesion molecules mediate not only cell adhesion, but also migration, phagocytosis, and many other adhesion-dependent functions. Monocyte chemoattractant protein-1 (MCP-1) is thought to be responsible for monocyte recruitment in acute inflammatory conditions and may be an important mediator in chronic inflammation. In this study, immunofluorescence flow cytometry was used to determine whether MCP-1 can regulate the cell surface expression of adhesion molecules, particularly beta-2 and alpha-4 integrins and the leukocyte adhesion molecule-1. We found that MCP-1 induced expression of CD11c (p150,95 alpha-subunit) and CD11b (Mac-1 alpha-subunit), and caused little or no change of CD11a (lymphocyte function-associated Ag-1 alpha-subunit), very late activation Ag-4, or leukocyte adhesion molecule-1. We demonstrated that antibodies to beta-2 and alpha-4 integrins inhibited MCP-1-induced monocyte chemotaxis. We also showed that MCP-1 is capable of inducing IL-1 and IL-6, but not TNF production of monocytes. These results indicate that MCP-1 is not only a chemoattractant but also a novel cytokine with the capacity to regulate several parameters of monocyte function.     

Monocyte chemoattractant protein-1 production by intestinal myofibroblasts in response to staphylococcal enterotoxin a: relevance to staphylococcal enterotoxigenic disease

Food poisoning due to staphylococcal enterotoxins A and B (SEA and SEB) affects hundreds of thousands of people annually. SEA and SEB induce massive intestinal cytokine production, which is believed to be the key factor in staphylococcal enterotoxin enteropathy. MHC class II molecules are the major receptors for staphylococcal enterotoxins. We recently demonstrated that normal human subepithelial intestinal myofibroblasts (IMFs) express MHC class II molecules. We hypothesized that IMFs are among the first cells to respond to staphylococcal enterotoxins and contribute to the cytokine production associated with staphylococcal enterotoxin pathogenesis. We demonstrated here that primary cultured IMFs bind staphylococcal enterotoxins in a MHC class II-dependent fashion in vitro. We also demonstrated that staphylococcal enterotoxins can cross a CaCo-2 epithelial monolayer in coculture with IMFs and bind to the MHC class II on IMFs. IMFs responded to SEA, but not SEB, exposure with 3- to 20-fold increases in the production of proinflammatory chemokines (MCP-1, IL-8), cytokines (IL-6), and growth factors (GM-CSF and G-CSF). The SEA induction of the proinflammatory mediators by IMFs resulted from the efficient cross-linking of MHC class II molecules because cross-linking of class II MHC by biotinylated anti-HLA-DR Abs induced similar cytokine patterns. The studies presented here show that MCP-1 is central to the production of other cytokines elicited by SEA in IMFs because its neutralization with specific Abs prevented the expression of IL-6 and IL-8 by IMFs. Thus, MCP-1 may play a leading role in initiation of inflammatory injury associated with staphylococcal enterotoxigenic disease.     

Monocyte chemoattractant protein-1 influences trauma-hemorrhage-induced distal organ damage via regulation of keratinocyte-derived chemokine production

Leukocyte infiltration, mediated by chemokines, is a key step in the development of organ dysfunction. Lung and liver neutrophil infiltration following trauma-hemorrhage is associated with upregulation of monocyte chemoattractant protein-1 (MCP-1). Because MCP-1 is not a major attractant for neutrophils, we hypothesized that MCP-1 influences neutrophil infiltration via regulation of keratinocyte-derived chemokines (KC). To study this, male C3H/HeN mice were pretreated with MCP-1 antiserum or control serum and subjected to trauma-hemorrhage or sham operation. Animals were killed 4 h after resuscitation. One group of trauma-hemorrhage mice receiving MCP-1 antiserum was also treated with murine KC during resuscitation. Plasma levels and tissue content of MCP-1 and KC were determined by cytometric bead arrays. Immunohistochemistry was performed to determine neutrophil infiltration; organ damage was assessed by edema formation. Treatment with MCP-1 antiserum significantly decreased systemic, lung, and liver levels of MCP-1 and KC following trauma-hemorrhage. This decrease in MCP-1 levels was associated with decreased neutrophil infiltration and edema formation in lung and liver following trauma-hemorrhage. Restitution of KC in mice treated with MCP-1 antiserum restored tissue neutrophil infiltration and edema. These results lead us to conclude that increased levels of MCP-1 cause neutrophil accumulation and distant organ damage by regulating KC production during the postinjury inflammatory response.     

Monocyte chemoattractant protein-1 (MCP-1) gene transduction: an effective tumor vaccine strategy for non-intracranial tumors

Recently, there has been renewed interest in the concept of tumor vaccines using genetically engineered tumor cells expressing a variety of cytokines to increase their immunogenicity. Human MCP-1 (JE) is a potent chemoattractant and activator of monocytes and T lymphocytes and thus a good candidate gene for a tumor vaccine. We therefore evaluated the efficacy of vaccines consisting of irradiated tumor cells transduced with the murine MCP-1 gene in the syngeneic 9L gliosarcoma brain tumor model. 9L cell lines stably expressing murine MCP-1 (9L-JE) and control cell lines expressing neomycin 3 phosphotransferase (9L-Neo) were generated by infection with a Moloney murine leukemia retroviral vector. Fisher 344 rats were immunized with intradermal injections of 5×10⁵ or 2×10⁶ irradiated (5000 cGy) 9L-JE, 9L-Neo, and wild-type 9L (9L-WT) cells. Two weeks later immunized an non-immunized animals were challenged with varyious doses of intradermal (5×10⁶–5×10⁷) or intracerebral (2×10⁴–5×10⁵) 9L-WT cells. Intradermal tumors grew in all non-immunized animals. No tumors grew in animals immunized with irradiated 9L-JE or 9L-Neo cells and challenged with inocula of fewer than 5×10⁵ 9L-WT cells. With higher inocula up to 10⁷ cells, tumors appeared in all the animals. Tumors in animals immunized with 9L-JE were always smaller than tumors in the other groups. In addition, only the 9L-JE vaccine protected against tumor inocula of 5×10⁷ cells. Thus vaccination with MCP-1-expressing cells was able to protect animals against at least a 100-fold larger number of challenge tumor cells than vaccination with control cells. In contrast to studies with intradermal tumors, immunization with 9L-JE and 9L-Neo produced only minimal protection against intracerebral tumors. There was no significant difference between the 9L-JE and 9L-Neo vaccines in intracerebral challenge. This study suggests that tumor vaccines expressing cytokine genes such as MCP-1 can increase the antitumor response. However, the protective effect of these vaccines appears to be largely limited to intradermal tumors rather than intracerebral tumors.     

Monocyte chemoattractant protein-1 in schizophrenia: -2518A/G genetic variant and protein levels in Armenian population

Monocyte chemoattractant protein-1 (MCP-1) has been proposed as a contributory factor in pathophysiology of schizophrenia. The aim of the current study was to explore the possible association of the MCP-1-2518A/G genetic polymorphism and plasma levels of MCP-1 in patients with paranoid schizophrenia. The MCP-1-2518A/G (rs1024611) polymorphism and blood levels of MCP-1 in patients with paranoid schizophrenia and healthy subjects were evaluated and compared. One hundred and three chronic patients with paranoid schizophrenia treated with neuroleptics and 105 healthy subjects were genotyped using polymerase chain reaction with sequence-specific primers (PCR-SSP) and their MCP-1 plasma levels were measured by a solid-phase enzyme-linked immunosorbent assay (ELISA). When comparisons were made between patients and controls, the frequency of the MCP-1-2518*G minor allele (35% vs 23%, p=0.009, OR=1.77, 95% CI: 1.1-2.04) and also of the MCP-1-2518*G carriers (60% vs 40%, p=0.003, OR=2.27, 95% CI: 1.13-2.01) were higher in patients. The mean value of the MCP-1 plasma level in patients with schizophrenia was significantly higher than in controls. Interestingly, the patients with the GG genotype had the highest MCP-1 level (711.4 ± 211.4 pg/ml), followed by those with the AG genotype (472.1 ± 135.8 pg/ml) and AA (372.4 ± 180.2 pg/ml) homozygotes. In conclusion, we report here the association of the -2518A/G genetic polymorphism and increased plasma levels of MCP-1 with schizophrenia and nominate -2518*G minor allele as a risk factor for schizophrenia in Armenian population.     

Monocyte chemoattractant protein-1/CC chemokine ligand 2 enhances apoptotic cell removal by macrophages through Rac1 activation

Apoptotic cell removal (efferocytosis) is an essential process in the regulation of inflammation and tissue repair. We have shown that monocyte chemoattractant protein-1/CC chemokine ligand 2 (MCP-1/CCL2) enhances efferocytosis by alveolar macrophages in murine bacterial pneumonia. However, the mechanism by which MCP-1 exerts this effect remains to be determined. Here we explored that hypothesis that MCP-1 enhances efferocytosis through a Rac1/phosphatidylinositol 3-kinase (PI3-kinase)-dependent mechanism.We assessed phagocytosis of apoptotic cells by MCP-1 treated murine macrophages in vitro and in vivo. Rac activity in macrophages was measured using a Rac pull down assay and an ELISA based assay (GLISA). The downstream Rac1 activation pathway was studied using a specific Rac1 inhibitor and PI3-kinase inhibitor in in vitro assays.MCP-1 enhanced efferocytosis of apoptotic cells by murine alveolar macrophages (AMs), peritoneal macrophages (PMs), the J774 macrophage cell line (J774s) in vitro, and murine AMs in vivo. Rac1 activation was demonstrated in these cell lines. The effect of MCP-1 on efferocytosis was completely negated by the Rac1 inhibitor and PI3-kinase inhibitor.We demonstrated that MCP-1 enhances efferocytosis in a Rac1-PI3 kinase-dependent manner. Therefore, MCP-1-Rac1-PI3K interaction plays a critical role in resolution of acute lung inflammation.     

Human monocyte chemoattractant protein-1 expressed in a baculovirus system

Human monocyte chemoattractant protein-1 (hMCP-1) was produced using a baculovirus system. The hMCP-1 cDNA was inserted into the genomic DNA of Autographa californica nuclear polyhedrosis virus (AcNPV) using a transfer vector, pJVP10Z. Spodoptera frugiperda insect cells, which were infected with this recombinant virus, secreted recombinant hMCP-1 (re-hMCP-1) at the level of 10–20 μg/ml of culture medium. This product was shown to chemoattract monocytes. Three distinct bands of 11,11.5 and 12 kDa were revealed by immunoblotting analysis, and this heterogeneity was assigned to differences in carbohydrate processing. N-terminal amino-acid sequence analysis of the purified product revealed identity with hMCP-1. Thus, in this system, re-hMCP-1 was produced in large quantities and modified in a manner similar to native hMCP-1.     

Role of CC chemokines (macrophage inflammatory protein-1 beta, monocyte chemoattractant protein-1, RANTES) in acute lung injury in rats

The role of the CC chemokines, macrophage inflammatory protein-1 beta (MIP-1 beta), monocyte chemotactic peptide-1 (MCP-1), and RANTES, in acute lung inflammatory injury induced by intrapulmonary deposition of IgG immune complexes injury in rats was determined. Rat MIP-1 beta, MCP-1, and RANTES were cloned, the proteins were expressed, and neutralizing Abs were developed. mRNA and protein expression for MIP-1 beta and MCP-1 were up-regulated during the inflammatory response, while mRNA and protein expression for RANTES were constitutive and unchanged during the inflammatory response. Treatment of rats with anti-MIP-1 beta Ab significantly decreased vascular permeability by 37% (p = 0.012), reduced neutrophil recruitment into lung by 65% (p = 0.047), and suppressed levels of TNF-alpha in bronchoalveolar lavage fluids by 61% (p = 0.008). Treatment of rats with anti-rat MCP-1 or anti-rat RANTES had no effect on the development of lung injury. In animals pretreated intratracheally with blocking Abs to MCP-1, RANTES, or MIP-1 beta, significant reductions in the bronchoalveolar lavage content of these chemokines occurred, suggesting that these Abs had reached their targets. Conversely, exogenously MIP-1 beta, but not RANTES or MCP-1, caused enhancement of the lung vascular leak. These data indicate that MIP-1 beta, but not MCP-1 or RANTES, plays an important role in intrapulmonary recruitment of neutrophils and development of lung injury in the model employed. The findings suggest that in chemokine-dependent inflammatory responses in lung CC chemokines do not necessarily demonstrate redundant function.     

Monocyte chemoattractant protein-1 (CCL-2) integrates mechanical and endocrine signals that mediate term and preterm labor

Recent evidence suggests that leukocytes infiltrate uterine tissues at or around the time of parturition, implicating inflammation as a key mechanism of human labor. MCP-1 (also known as C-C chemokine motif ligand 2, CCL-2) is a proinflammatory cytokine that is up-regulated in human myometrium during labor. Myometrium was collected from pregnant rats across gestation and at labor. Total RNA and proteins were subjected to real-time PCR and ELISA, respectively. Ccl-2 gene and protein expression was significantly up-regulated in the gravid rat myometrium before and during labor, which might suggest that it is regulated positively by mechanical stretch of the uterus imposed by the growing fetus and negatively by physiological withdrawal of progesterone (P4). We confirmed in vivo that: 1) administration of P4 receptor antagonist RU486 induced an increase in Ccl-2 mRNA and preterm labor, whereas 2) artificial maintenance of elevated P4 levels at late gestation caused a significant decrease in gene expression and blocked labor; 3) Ccl-2 was elevated specifically in the gravid horn of unilaterally pregnant rats suggesting that mechanical strain imposed by the growing fetus controls its expression in the myometrium; 4) in vitro static mechanical stretch of primary rat myometrial smooth muscle cells (25% elongation) induced a release of Ccl-2 protein, which was repressed by pretreatment with P4 (1 microM); and 5) stretch enhanced their monocyte chemoattractant activity. These data indicate that Ccl-2 protein serves to integrate mechanical and endocrine signals contributing to uterine inflammation and the induction of labor and thus may represent a novel target for therapeutic prevention of preterm labor in humans.     

Monocyte chemoattractant protein-2 can exert its effects through the MCP-1 receptor (CC CKR2B)

We studied the activities of the monocyte chemoattractant proteins MCP-1, MCP-2 and MCP-3 on human embryonic kidney 293-EBNA cells transfected with the MCP-1 receptor (CC CKR2B). At 4 nM, MCP-2 induced a Ca²⁺ influx which was as potent as that with MCP-1 at 4 nM, although the increase by MCP-2 became saturated at higher concentrations. In addition, all three MCPs showed dose-dependent inhibition of adenylyl cyclase activity stimulated by forskolin (IC₅₀ values: 0.3 nM for MCP-1, 7 nM for MCP-2, and 1.5 nM for MCP-3). In conclusion, our data indicate that MCP-2 can exert its effects through the MCP-1 receptor, CC CKR2B.     

Macrophage inflammatory protein-1

Macrophage inflammatory protein-1 (MIP-1) and MIP-1β are highly related members of the CC chemokine subfamily. Despite their structural similarities, MIP-1 and MIP-1β show diverging signaling capacities. Depending on the MIP-1 subtype and its NH₂-terminal processing, one or more of the CC chemokine receptors CCR1, CCR2, CCR3 and CCR5 are recognized. Since both human MIP-1 subtypes (LD78 and LD78β) and MIP-1β signal through CCR5, the major co-receptor for M-tropic HIV-1 strains, these chemokines are capable of inhibiting HIV-1 infection in susceptible cells. In this review, different aspects of human and mouse MIP-1 and MIP-1β are discussed, including their protein and gene structures, their regulated production, their receptor usage and biological activities and their role in several pathologies including HIV-1 infection.