In vivo properties of monocyte chemoattractant protein-1

BACKGROUND AND OBJECTIVE: In adult patients with acute myeloid leukemia (AML), central nervous system (CNS) involvement is a rare event and treatment has not yet been defined. Because there are no definitive data as to the most appropriate therapeutic approach to CNS leukemia in AML, we retrospectively analyzed a cohort of AML patients with meningeal leukemia in order to increase our knowledge on this particular matter. METHODS: Out of 410 patients with de novo AML observed at our Institute from 1986 to 1995, 9 (2.2%) showed CNS leukemia (CNSL) during the follow-up. CNSL was treated as follows: in a first group of 4 patients we combined systemic HD Ara-C 3 g/m2 (every 12 hours by 3-hour infusion, for 6 doses), cranial radiation therapy and intrathecal (IT) methotrexate (MTX); a second group of 4 patients was treated with HD Ara-C, IT MTX without cranial irradiation; HD Ara-C alone was administered in one patient. RESULTS: All patients of the first group and 2 patients of the second who achieved a complete remission (CR) had a median survival of 10 months (range 5-25+) after CNS involvement, while for the non-remitters it was 2 months (range 1-5). The only patient still living underwent allogeneic bone marrow transplantation. INTERPRETATION AND CONCLUSIONS: The combination treatment of HD Ara-C, IT MTX and cranial irradiation is well tolerated and seems to be an effective therapy for CNSL, presenting a high incidence of neurologic CR that correlates with a longer survival. As expected, the number of AML patients with CNSL was small, due to the fact that CNS in those patients is a rare complication. However, this study provides further information about the therapeutic possibilities in such restricted subsets of AML patients.     

Alleviation of monocrotaline-induced pulmonary hypertension by antibodies to monocyte chemotactic and activating factor/monocyte chemoattractant protein-1

Administration of monocrotaline (MCT) causes pulmonary vascular lesions consisting of monocyte/macrophage infiltration in the early phase and medial thickening in pulmonary arteries and arterioles associated with pulmonary hypertension (PH) in the later phase. However, the molecular mechanism of monocyte/macrophage infiltration and its roles remain elusive. Herein, we have evaluated the role of a potent monocyte chemotactic and activating chemokine/monocyte chemoattractant protein-1 (MCAF/MCP-1) in MCT-induced PH in rats. A single injection of MCT induced PH at Day 21, as evidenced by increases in the ratio of right ventricular to left ventricular and septum weights (RV/LV+S) and right ventricular systolic pressure (RVSP). A significant increase in macrophage number in lungs started at Day 14, reaching a maximum at Day 21. MCAF/MCP-1 levels in bronchoalveolar lavage fluids were elevated significantly at Day 14 and remained high until Day 28, whereas plasma MCAF/MCP-1 levels increased at Day 7, returning to normal levels by Day 21. Immunoreactive MCAF/MCP-1 proteins were mainly detected in macrophages in alveoli and in perivascular regions of pulmonary arterioles and venules. Intravenous administration of anti-MCAF/MCP-1 antibodies with MCT significantly decreased macrophage infiltration and eventually reduced the increases in RV/LV+S and RVSP, as well as medial thickening of pulmonary arterioles. Thus, MCAF/MCP-1 is essentially involved in MCT-induced PH by recruiting and activating macrophages.     

Hyaluronan induces monocyte chemoattractant protein-1 expression in renal tubular epithelial cells

Hyaluronan (HA) is a nonsulfated glycosaminoglycan that accumulates in the renal interstitium in immune-mediated kidney diseases. The functional significance of such HA deposition in the kidney has not been elucidated. Several studies have suggested that HA may exhibit proinflammatory effects. Since chemokines such as monocyte chemoattractant protein-1 (MCP-1) play an important role in the recruitment of leukocytes in renal injury, this study tested whether HA and its fragments could promote MCP-1 production by renal parenchymal cells. Mouse cortical tubular cells were stimulated with fragmented HA or with high molecular weight HA (Healon) in vitro and were examined for MCP-1 expression. Fragmented HA, but not Healon, increased MCP-1 mRNA within 30 min with a peak after 2 h. In addition, a 10-fold increase of MCP-1 protein in the supernatant was found after a 6-h stimulation with fragmented HA. The enhanced MCP-1 mRNA and protein expression in response to HA was dose-dependent between 1 and 100 microg/ml. Upregulation of MCP-1 protein production could be blocked by preincubation with actinomycin D or cycloheximide, suggesting that MCP-1 mRNA and protein expression in response to HA are based on de novo synthesis. The HA-stimulated MCP-1 production was also inhibited with anti-CD44 antibodies, suggesting that MCP-1 is upregulated at least in part by signaling through CD44. In summary, fragmented HA markedly stimulates renal tubular MCP-1 production by mechanisms that involve binding to the HA receptor CD44. It is hypothesized that the accumulation of HA in immune renal injury could participate in the recruitment and activation of inflammatory cells in vivo through production of MCP-1.     

Identification of an inhibitor targeting macrophage production of monocyte chemoattractant protein-1 as TGF-beta 1

Monocyte chemoattractant protein-1 (MCP-1) is expressed in a diverse range of cells in response to various pathologic stimuli, whereas little is known about endogenous inhibitors of MCP-1 expression. I sought negative regulators of MCP-1 in culture medium conditioned by several cell lines and found that glomerular mesangial cells exclusively secrete a factor that inhibits expression of MCP-1 by activated macrophages. Treatment of J774.2 macrophages with conditioned medium from mesangial cells blunted the induction of MCP-1 by LPS. Even after the induction, subsequent treatment of macrophages with the conditioned medium down-regulated the MCP-1 expression. Medium conditioned by normal rat glomeruli contained a similar inhibitory activity that was enhanced in regenerating glomeruli, where mesangial cells are activated. The activity of the conditioned medium was not diminished, but enhanced by heat treatment, which was consistent with the unique property of TGF-beta family of molecules. Indeed, the mesangial cell-derived medium contained active TGF-beta 1. An anti-TGF-beta 1 neutralizing Ab abolished the inhibitory effect exerted by the mesangial cell medium, and externally added TGF-beta 1 suppressed the MCP-1 expression by macrophages at both mRNA and protein levels. The inhibitory effect of TGF-beta 1 on MCP-1 was observed in other macrophage cell lines, RAW264.7 and NR8383, and peritoneal macrophages, but not in fibroblastic cell line NRK49F. Treatment of J774.2 macrophages with TGF-beta 1 inhibited LPS induction of c-jun that was found to be crucial for the MCP-1 expression. These data demonstrate that TGF-beta 1 functions as an inhibitor of MCP-1 expression in macrophages possibly via down-regulation of c-Jun/activator protein-1.     

Regulation of monocyte chemoattractant protein-1 expression in adult human non-neoplastic astrocytes is sensitive to tumor necrosis factor (TNF) or antibody to the 55-kDa TNF receptor

A trifluoroacetamide analogue of siastatin B, (3S,4S,5R,6R)-6-(trifluoroacetamido)-4,5-dihydroxy-3-piperidine carboxylic acid has been chemically synthesized. This compound, as well as the previously synthesized analogue, (3R,4R,5R,6R)-6-(trifluoroacetamido)-3,4,5-trihydroxy-3-piperid inecarboxylic acid, showed marked inhibitory activity against beta-glucuronidase and significant inhibition of experimental pulmonary metastasis of the highly metastatic melanoma B16.     

Monomeric monocyte chemoattractant protein-1 (MCP-1) binds and activates the MCP-1 receptor CCR2B

To address the role of dimerization in the function of the monocyte chemoattractant protein-1, MCP-1, we mutated residues that comprise the core of the dimerization interface and characterized the ability of these mutants to dimerize and to bind and activate the MCP-1 receptor, CCR2b. One mutant, P8A*, does not dimerize. However, it has wild type binding affinity, stimulates chemotaxis, inhibits adenylate cyclase, and stimulates calcium influx with wild type potency and efficacy. These data suggest that MCP-1 binds and activates its receptor as a monomer. In contrast, Y13A*, another monomeric mutant, has a 100-fold weaker binding affinity, is a much less potent inhibitor of adenylate cyclase and stimulator of calcium influx, and is unable to stimulate chemotaxis. Thus Tyr13 may make important contacts with the receptor that are required for high affinity binding and signal transduction. We also explored whether a mutant, [1+9-76]MCP-1 (MCP-1 lacking residues 2-8), antagonizes wild type MCP-1 by competitive inhibition, or by a dominant negative mechanism wherein heterodimers of MCP-1 and [1+9-76]MCP-1 bind to the receptor but are signaling incompetent. Consistent with the finding that MCP-1 can bind and activate the receptor as a monomer, we demonstrate that binding of MCP-1 in the presence of [1+9-76]MCP-1 over a range of concentrations of both ligands fits well to a simple model in which monomeric [1+9-76]MCP-1 functions as a competitive inhibitor of monomeric MCP-1. These results are crucial for elucidating the molecular details of receptor binding and activation, for interpreting mutagenesis data, for understanding how antagonistic chemokine variants function, and for the design of receptor antagonists.     

IL-1beta-induced monocyte chemoattractant protein-1 gene expression in endothelial cells is blocked by proteasome inhibitors

Human monocyte chemoattractant protein-1 (MCP-1) is expressed by a variety of cell types in response to various stimuli. MCP-1 expressed by the endothelium plays an important role in cell migration and activation. MCP-1 is a major chemoattractant for monocytes, T lymphocytes, and basophils. In the present study, we present evidence that the proteasome complex is involved in mediating the interleukin (IL)-1beta induction of MCP-1 in endothelial cells. We present evidence that a proteasome inhibitor, N-acetyl-leucinyl-leucinyl-norleucinal (norLeu), and the protease inhibitor tosyl-Phe-chloromethylketone (TPCK) block IL-1beta induction of MCP-1 protein expression. norLeu and TPCK also blocked IL-1beta-induced MCP-1 promoter-driven reporter gene expression as well as nuclear factor (NF)-kappaB-mediated reporter gene expression. The effects of norLeu were due to its inhibition of the proteasome rather than calpain, because other calpain inhibitors had no effect on MCP-1 expression. In contrast to TPCK, which blocked NF-kappaB translocation to the nucleus, norLeu had no effect on NF-kappaB nuclear translocation or IL-1beta-induced phosphorylation of p65. This study demonstrates that the proteasome pathway is involved in IL-1beta-induced MCP-1 gene expression in human endothelial cells.     

Production and regulation of monocyte chemoattractant protein-1 in lipopolysaccharide- or monosodium urate crystal-induced arthritis in rabbits: roles of tumor necrosis factor alpha, interleukin-1, and interleukin-8

The production of monocyte chemoattractant protein-1 (MCP-1) and its regulation by TNFalpha, IL-1, and IL-8 were investigated in two rabbit models of arthritis induced by intra-articular injection of lipopolysaccharide (LPS) or monosodium urate (MSU) crystals. We first prepared recombinant rabbit MCP-1 and antibodies and then developed an immunoassay. The immunoassay detected 3 pg/ml rabbit MCP-1 and did not cross-react with other rabbit chemokines such as IL-8 or GRO. MCP-1 was first detected in synovial fluid (SF) at 1 hour, and peaked at 4 or 2 hours after the injection of LPS or MSU crystals, respectively. Immunohistochemically, MCP-1 was detected in synovial lining cells and infiltrating neutrophils. The amounts of MCP-1 detected in SF from neutrophil-depleted rabbits were similar to those in normal rabbits, suggesting that synovial lining cells were the main source of MCP-1 detected in SF. The peak level of MCP-1 in SF after LPS-injection was inhibited by 57% with anti-TNFalpha mAb and by 41% with IL-1 receptor antagonist (IL-1Ra). Coadministration of anti-TNFalpha mAb and IL-1Ra inhibited 90% of MCP-1 production. In contrast, the peak level of MCP-1 in SF after MSU crystal-injection was not affected by any cytokine inhibitor, but was reduced by 52% with coadministration of anti-TNFalpha mAb and IL-1Ra. Anti-IL-8 IgG had no effect on the production of MCP-1 in either model. Thus, the production of MCP-1 in LPS-induced arthritis was mostly regulated by TNFalpha and IL-1, whereas half the extent of MCP-1 production in MSU crystal-induced arthritis was independent of TNFalpha or IL-1. IL-8 does not seem to regulate the production of MCP-1 in SF either directly or indirectly. Finally, administration of neutralizing anti-MCP-1 antibody inhibited LPS- and MSU crystal-induced monocyte infiltration by 58.4% and 44.9%, respectively, suggesting that synovial production of MCP-1 plays an important role in the recruitment of monocytes in these arthritis models.     

Expression of monocyte chemoattractant protein-1 in monocytes and effects of native and oxidized very low density lipoproteins

Rheumatoid neutrophilic dermatitis (RND) is a rare cutaneous finding in patients with severe rheumatoid arthritis or with high-titer rheumatoid factors. Most commonly, these lesions are erythematous papules or plaques distributed symmetrically on extensor surfaces. On histologic examination a dense dermal neutrophilic infiltrate without vasculitis is apparent. The pathogenesis of RND is unclear, and few treatments are known. With careful clinical and histologic examination, RND may be differentiated from the wide array of other cutaneous findings in rheumatoid arthritis.     

Expression of monocyte chemoattractant protein-1 and other beta-chemokines by resident glia and inflammatory cells in multiple sclerosis lesions

Beta-chemokines induce the directional migration of monocytes and T lymphocytes and are thus associated with chronic inflammation. Using immunocytochemistry and in situ hybridisation (ISH) techniques, we have examined the expression of the beta-chemokines monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated upon activation, normal T cell expressed and secreted) in post-mortem human brain from multiple sclerosis (MS) cases, at different stages of lesion development. In actively demyelinating MS plaques RANTES expression was restricted to the blood vessel endothelium, perivascular cells and surrounding astrocytes, suggesting a role in the recruitment of inflammatory cells from the circulation. MCP-1 was expressed by astrocytes and macrophages within acute MS lesions, but was restricted to reactive astrocytes in the parenchyma surrounding the lesion. MIP-1alpha was expressed by astrocytes and macrophages within the plaque, while MIP-1beta was expressed by macrophages and microglia within the lesion, and by microglia in surrounding white matter. Glial cells may be stimulated to produce chemokines and continue the local inflammatory response by forming chemotactic gradients to attract T cells and mononuclear phagocytes from the circulation and surrounding tissue.     

Neutrophil attractant protein-1 and monocyte chemoattractant protein-1 in human serum. Effects of intravenous lipopolysaccharide on free attractants, specific IgG autoantibodies and immune complexes

We recently found that normal human sera contain IgG antibodies against two chemoattractants, neutrophil attractant protein-1 (NAP-1/IL-8) and monocyte chemoattractant protein-1 (MCP-1), as well as immune complexes of these proteins. Intravenously administered LPS was reported to cause a sharp rise in serum NAP-1 concentration. Our study was designed to determine if LPS also caused an increase in MCP-1 and to measure associated changes in concentrations of antibody and immune complex. LPS caused a rise to peak within 2 to 3 h in serum concentrations of free NAP-1 and MCP-1, followed by an almost equally rapid fall toward base-line levels by about 5 h postinjection. MCP-1 concentration in sera from the 11 subjects rose to a peak of 330 +/- 52 pM. The peak value for NAP-1 was 80 +/- 11 pM. In 10 of the 11 subjects, free IgG autoantibody to MCP-1 decreased from a mean pre-LPS value of 1820 +/- 660 pM to a mean low of 53% of the respective initial values. Corresponding data for IgG anti-NAP-1 were a pre-LPS concentration of 216 +/- 7 pM, which decreased to a mean low of 44% of the respective initial values. The finding in some subjects of a rapid rise in free antibody after the nadir suggests the possibility of acute regulation of autoantibody secretion rates. Although the results suggested that LPS-induced chemoattractant combined with free antibody, serum concentrations of MCP-1-IgG or NAP-1-IgG did not increase, which points to an as yet unknown mechanism for trapping and elimination of the immune complexes.     

Lysine 58 and histidine 66 at the C-terminal alpha-helix of monocyte chemoattractant protein-1 are essential for glycosaminoglycan binding

Monocytes rolling on the endothelial cell layer interact with monocyte chemoattractant protein-1 (MCP-1) that is tethered to the proteoglycans on the luminal side of the endothelial cells and consequently initiate adhesion of monocytes in the early phase of immune response. The amino acid residues in MCP-1 involved in tethering to the proteoglycans have not been elucidated. MCP-1 showed binding to [3H]heparin with a KD of 1.5 microM. We substituted lysine or histidine residues at the C-terminal end of MCP-1 with alanine residues and tested these mutants for their ability to bind heparin, heparan sulfate, hyaluronic acid, and chondroitin sulfate-C. Substitution of Lys-58 or His-66 drastically reduced glycosaminoglycan binding. Substitution of Lys-56 or deletion of the five amino acid residues at the C terminus, including Lys-75, did not alter the heparin binding ability, suggesting that the other lysine residues at the C terminus are not involved in glycosaminoglycan binding. MCP-1 and its mutants did not bind hyaluronic acid as strongly as the other subunits of the GAGs. Substitution of Lys-58 or His-66 by alanine that prevented glycosaminoglycan binding did not affect Ca2+ influx, receptor binding, or chemotactic activity elicited by the chemokine on monocytic THP-1 cells. Therefore, we conclude that the Lys-58 and His-66 residues in the C-terminal alpha-helix of MCP-1 are essential for glycosaminoglycan binding and probably for the binding to the endothelial surface proteoglycans.     

Plasma levels of monocyte chemoattractant protein-1 but not those of macrophage inhibitory protein-1alpha and RANTES correlate with virus load in human immunodeficiency virus infection

Plasma levels of proinflammatory cytokines, cytokine inhibitors, and the beta chemokines RANTES, macrophage inhibitory protein (MIP)-1alpha, and monocyte chemoattractant protein (MCP)-1 were studied in relationship with virus load in 40 patients exhibiting plasma levels of HIV RNA ranging between undetectable and levels >10(6) copies/mL. Mean plasma levels of MCP-1 were increased in patients with high virus load compared with HIV-seropositive subjects with undetectable plasma viral RNA and healthy controls. MCP-1 levels were directly correlated with plasma levels of HIV RNA. No correlation was observed between virus load and plasma concentrations of MIP-1alpha and RANTES. The results suggest that low rates of viral replication in vivo are not dependent on increased production of the suppressive chemokines RANTES and MIP-1alpha. Since MCP-1 upregulates viral replication in vitro, the results may suggest a role for MCP-1 in triggering viral replication in HIV disease.     

Chemokine receptor CCR2 expression and monocyte chemoattractant protein-1-mediated chemotaxis in human monocytes. A regulatory role for plasma LDL

The subendothelial accumulation of macrophage-derived foam cells is one of the hallmarks of atherosclerosis. The recruitment of monocytes to the intima requires the interaction of locally produced chemokines with specific cell surface receptors, including the receptor (CCR2) for monocyte chemoattractant protein-1 (MCP-1). We have previously reported that monocyte CCR2 gene expression and function are effectively downregulated by proinflammatory cytokines. In this study we identified low density lipoprotein (LDL) as a positive regulator of CCR2 expression. Monocyte CCR2 expression was dramatically increased in hypercholesterolemic patients compared with normocholesterolemic controls. Similarly, incubation of human THP-1 monocytes with LDL induced a rapid increase in CCR2 mRNA and protein. By 24 hours the number of cell surface receptors was doubled, causing a 3-fold increase in the chemotactic response to MCP-1. The increase in CCR2 expression and chemotaxis was promoted by native LDL but not by oxidized LDL. Oxidized LDL rapidly downregulated CCR2 expression, whereas reductively methylated LDL, which does not bind to the LDL receptor, had only modest effects on CCR2 expression. A neutralizing anti-LDL receptor antibody prevented the effect of LDL, suggesting that binding and internalization of LDL were essential for CCR2 upregulation. The induction of CCR2 expression appeared to be mediated by LDL-derived cholesterol, because cells treated with free cholesterol also showed increased CCR2 expression. These data suggest that elevated plasma LDL levels in conditions such as hypercholesterolemia enhance monocyte CCR2 expression and chemotactic response and potentially contribute to increased monocyte recruitment to the vessel wall in chronic inflammation and atherogenesis.     

Interleukin 8 and monocyte chemoattractant protein-1 in patients with juvenile rheumatoid arthritis. Relation to onset types, disease activity, and synovial fluid leukocytes

OBJECTIVE: To measure serum and synovial fluid (SF) levels of interleukin 8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in patients with juvenile rheumatoid arthritis (JRA) and to compare them with adult rheumatoid factor-positive rheumatoid arthritis (RA). METHODS: IL-8 and MCP-1 were measured by immunoassay (1) in sera obtained from 55 children with JRA and from 16 adults with RA, and (2) in SF obtained from 30 children with JRA and 11 adults with RA. RESULTS: Patients with active systemic JRA had serum levels of IL-8 and MCP-1 higher than in controls (p<0.01) and in patients with active polyarticular or pauciarticular JRA (p<0.05). In patients with RA serum MCP-1 levels were higher than in patients with the 3 JRA onset types, while no difference was found for IL-8 levels. Patients with systemic JRA and with current systemic features had serum levels of IL-8 and MCP-1 higher (p = 0.03 and p = 0.04, respectively) than patients in which systemic features had subsided. No significant differences in SF IL-8 or MCP-1 levels were found among the 3 JRA onset types or adults with RA. In patients with JRA SF leukocyte counts were correlated with SF IL-8 levels (p = 0.002), but not with MCP-1 levels. Moreover, SF levels of both IL-8 and MCP-1 were correlated with those of IL-1beta (p<0.001) and IL-6 (p<0.01), but not with those of TNF-alpha. CONCLUSION: Elevated serum levels of IL-8 and MCP-1 in patients with systemic JRA with current systemic features at sampling suggest systemic production of the 2 chemokines during systemic phases of the disease. Similar SF levels of IL-8 and MCP-1 among the 3 JRA onset-types and RA suggest comparable local production of the 2 chemokines.     

MaMi, a human endometrial stromal sarcoma cell line that constitutively produces interleukin-6, interleukin-8, and monocyte chemoattractant protein-1

BACKGROUND: Uterine endometrial stromal sarcoma is a rare neoplasm with a morphology that closely resembles that of the proliferative endometrial stroma. To understand its pathologic characteristics, we established a novel cell line, MaMi, from a primary culture of an endometrial stromal sarcoma obtained from a 65-year-old Japanese woman. METHODS: We observed the morphology of MaMi cells and performed immunohistochemical analysis on the primary tumor and transplants in nude mice. Prolactin, insulin-like growth factor-binding protein-1, interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, E-selectin, vascular cell adhesion molecule-1, and fibronectin production in the culture medium of MaMi cells were also examined. RESULTS: MaMi cells were shown to exhibit a fibroblast-like morphology in vitro, and they adopted a more elongated appearance in response to 12-O-tetradecanoyl phorbol-13-acetate (TPA). On injection into nude mice, the cells gave rise to subcutaneous tumors. Immunohistologically, both the primary tumor and MaMi cell-induced tumors stained positively with antibodies to neuron-specific enolase or vimentin. MaMi cells constitutively produced IL-6, IL-8, and monocyte chemoattractant protein-1 in vitro. Interleukin-1beta, (100 pmol/L), tumor necrosis factor-alpha (1 nmol/L), and lipopolysaccharide (1 microg/mL) each increased the release of IL-6, IL-8, and monocyte chemoattractant protein-1 by MaMi cells. TPA (10 nmol/L) also stimulated the production of IL-6 and IL-8 by these cells, but inhibited that of monocyte chemoattractant protein-1. CONCLUSIONS: We demonstrated that MaMi cells closely resemble proliferative endometrial stromal cells not only morphologically, but also functionally. This cell line may prove valuable in understanding the role of cytokines produced by tumor cells in the pathogenesis of endometrial stromal sarcoma and may also be useful as an in vitro model of functioning endometrial stromal cells.