Teizo Yoshimura

Detection of monocyte chemoattractant protein-1 in human atherosclerotic lesions by an anti-monocyte chemoattractant protein-1 monoclonal antibody

The infiltration of blood monocytes into the subendothelial space is thought to be one of the most important pathologic events in early atherogenesis. To examine the mechanism of monocyte migration in early atherosclerotic lesions we investigated immunohistochemically the production of monocyte chemoattractant protein-1 (MCP-1) in various atherosclerotic lesions, including diffuse intimal thickening, fatty streaks, and atheromatous plaques, obtained during autopsies of patients of various ages. A highly specific anti-MCP-1 monoclonal antibody that does not cross-react with neutrophil-activating, attractant protein-1/interleukin-8 or platelet proteins that have an amino acid sequence similar to MCP-1 was used to localize MCP-1 in situ. To characterize the cells constituting the atherosclerotic lesions a panel of monoclonal and polyclonal antibodies that are specific to smooth muscle cells (HHF-35), monocyte/macrophages (HAM56, Leu-M3, Leu-M5, EBM11, and PM-2K), and endothelial cells (anti-von Willebrand factor) was used. Double immunohistochemical staining with anti-MCP-1 and one of the cell type-specific antibodies was performed to identify the nature of MCP-1-positive cells. Endothelial cells stained positively for MCP-1 in nine of 14 diffuse intimal thickening lesions. Scattered macrophages in thickened intima also were positive for MCP-1. Endothelial staining of MCP-1 was observed in 14 of 21 fatty streak lesions. Subendothelial macrophages were strongly stained for MCP-1 in all fatty streak lesions examined. Subendothelial macrophages were stained for MCP-1 in atherosclerotic plaques; however, endothelial cells were only slightly positive for MCP-1. A few smooth muscle cells in the intima were positive for MCP-1 in atheromatous plaques. From these results it is concluded that the cell populations positive for MCP-1 are different in early and advanced atherosclerotic lesions, and that the endothelial cells and subendothelial macrophages are considered to be the major sources of MCP-1 in early atherosclerotic lesions.

The role of monocyte chemoattractant protein-1 (MCP-1) in the pathogenesis of collagen-induced arthritis in rats

Collagen‐induced arthritis was produced in rats by intradermal immunization with type II collagen and the expression and production of monocyte chemoattractant protein‐1 (MCP‐1) were examined by immunohistochemistry, enzyme‐linked immunosorbent assay (ELISA), and Northern blot analysis. Two to three weeks after the immunization, the hindfeet showed swelling and redness, followed by the development of severe arthritis, particularly in the ankle joints. During this period, prominent infiltration of neutrophils and macrophages was observed. Sandwich ELISA and Northern blot analysis revealed that MCP‐1 concentrations in the joint lavages and MCP‐1 mRNA levels in the joint tissues both peaked at 2 weeks after the immunization. By immunohistochemistry, various types of cells, particularly neutrophils, macrophages, synovial cells, and vascular endothelial cells, stained positively for MCP‐1. Finally, injection of a neutralizing monoclonal antibody against rat MCP‐1 significantly decreased the number of exudate macrophages in the lesions and reduced the ankle swelling by about 30 per cent compared with controls. These results suggest that MCP‐1 plays a critical role in this model in the recruitment of monocytes and in the development of arthritis.

Important Role of Local Angiotensin II Activity Mediated via Type 1 Receptor in the Pathogenesis of Cardiovascular Inflammatory Changes Induced by Chronic Blockade of Nitric Oxide Synthesis in Rats

BACKGROUND The chronic inhibition of NO synthesis by N(omega)-nitro-L-arginine methyl ester (L-NAME) upregulates the cardiovascular tissue angiotensin II (Ang II)-generating system and induces cardiovascular inflammatory changes in rats. METHODS AND RESULTS We used a rat model to investigate the role of local Ang II activity in the pathogenesis of such inflammatory changes. Marked increases in monocyte infiltration into coronary vessels and myocardial interstitial areas, monocyte chemoattractant protein-1 (MCP-1) expression, and nuclear factor-kappaB (NF-kappaB, an important redox-sensitive transcriptional factor that induces MCP-1) activity were observed on day 3 of L-NAME administration. Along with these changes, vascular superoxide anion production was also increased. Treatment with an Ang II type 1 receptor antagonist or with a thiol-containing antioxidant, N-acetylcysteine, prevented all of these changes. CONCLUSIONS Increased Ang II activity mediated via the type 1 receptor may thus be important in the pathogenesis of early cardiovascular inflammatory changes in this model. Endothelium-derived NO may decrease MCP-1 production and oxidative stress-sensitive signals by suppressing localized activity of Ang II.

Rho-Kinase Mediates Angiotensin II-Induced Monocyte Chemoattractant Protein-1 Expression in Rat Vascular Smooth Muscle Cells

Recently, it was shown that Rho-kinase plays an important role in blood pressure regulation. However, it is not known whether Rho-kinase is involved in atherogenesis. Monocyte chemoattractant protein-1 (MCP-1) is an important chemokine that regulates monocyte recruitment and atherogenesis. Therefore, we examined the role of Rho and Rho-kinase in the angiotensin (Ang) II-induced expression of MCP-1. Ang II dose- and time-dependently enhanced the expression of MCP-1 mRNA and the protein production in vascular smooth muscle cells. CV11974, an Ang II type 1 receptor (AT1-R) specific antagonist inhibited the enhancement of MCP-1 expression by Ang II, suggesting that the effect of Ang II is mediated by the AT1-R. Botulinum C3 exotoxin, a specific inhibitor of Rho, suppressed Ang II-induced MCP-1 production. To examine the role of Rho-kinase in Ang II-induced MCP-1 expression, we used adenovirus-mediated overexpression of the dominant negative mutant of Rho-kinase (AdDNRhoK) or Y-27632, a specific inhibitor of Rho-kinase. Both AdDNRhoK and Y-27632 strongly inhibited Ang II-induced MCP-1 expression. Although inhibition of extracellular signal-regulated protein kinase (ERK) by PD 098,059 also inhibited Ang II-induced MCP-1 expression, Y-27632 did not affect Ang II-induced activation of ERK. These results indicate that Rho-kinase plays a critical role in Ang II-induced MCP-1 production independent of ERK. The Rho-Rho-kinase pathway may be a novel target for the inhibition of Ang II signaling and the treatment of atherosclerosis.

Bacterial c-di-GMP is an immunostimulatory molecule.

Cyclic diguanylate (c-di-GMP) is a bacterial intracellular signaling molecule. We have shown that treatment with exogenous c-di-GMP inhibits Staphylococcus aureus infection in a mouse model. We now report that c-di-GMP is an immodulator and immunostimulatory molecule. Intramammary treatment of mice with c-di-GMP 12 and 6 h before S. aureus challenge gave a protective effect and a 10,000-fold reduction in CFUs in tissues (p < 0.001). Intramuscular vaccination of mice with c-di-GMP coinjected with S. aureus clumping factor A (ClfA) Ag produced serum with significantly higher anti-ClfA IgG Ab titers (p < 0.001) compared with ClfA alone. Intraperitoneal injection of mice with c-di-GMP activated monocyte and granulocyte recruitment. Human immature dendritic cells (DCs) cultured in the presence of c-di-GMP showed increased expression of costimulatory molecules CD80/CD86 and maturation marker CD83, increased MHC class II and cytokines and chemokines such as IL-12, IFN-γ, IL-8, MCP-1, IFN-γ-inducible protein 10, and RANTES, and altered expression of chemokine receptors including CCR1, CCR7, and CXCR4. c-di-GMP-matured DCs demonstrated enhanced T cell stimulatory activity. c-di-GMP activated p38 MAPK in human DCs and ERK phosphorylation in human macrophages. c-di-GMP is stable in human serum. We propose that cyclic dinucleotides like c-di-GMP can be used clinically in humans and animals as an immunomodulator, immune enhancer, immunotherapeutic, immunoprophylactic, or vaccine adjuvant.

Molecular cloning of rat monocyte chemoattractant protein-1 (MCP-1) and its expression in rat spleen cells and tumor cell lines

Rat monocyte chemoattractant protein-1 (MCP-1) cDNA was cloned. A cDNA library was constructed from mRNA extracted from Con A-stimulated rat spleen cells. After 2 rounds of screening with a radiolabeled oligonucleotide probe and DNA sequencing from plasmid DNAs, one clone which encoded for MCP-1 was obtained. The cDNA clone comprises 665 base pairs with an open reading frame which encodes for 148 amino acid protein. Spleen cells expressed a significant amount of MCP-1 mRNA without stimulus. But higher expression was observed when cells were stimulated with Con A. MCP-1 mRNA was also expressed in tumor cell lines, although the amount of expressed MCP-1 mRNA was different among cell lines.

Inhibition of NO Synthesis Induces Inflammatory Changes and Monocyte Chemoattractant Protein-1 Expression in Rat Hearts and Vessels

We recently showed that chronic inhibition of NO synthesis by N(omega)-nitro-L-arginine methyl ester (L-NAME) causes coronary vascular remodeling (ie, vascular fibrosis and medial thickening) in rats. To test the hypothesis that the inhibition of NO synthesis induces inflammatory changes in the heart, we characterized the inflammatory lesions that occurred during L-NAME administration and determined whether inflammation involved the induction of monocyte chemoattractant protein-1 (MCP-1) in vivo. During the first week of L-NAME administration to Wistar-Kyoto rats, we observed a marked infiltration of mononuclear leukocytes (ED1-positive macrophages) and fibroblast-like cells (alpha-smooth muscle actin-positive myofibroblasts) into the coronary vessels and myocardial interstitial areas. These inflammatory changes were associated with the expression of proliferating cell nuclear antigen and MCP-1 (both mRNA and protein). The areas affected by inflammatory changes, as well as the expression of MCP-1 mRNA, declined after longer (28 days) treatment with L-NAME and were replaced by vascular and myocardial remodeling. Our results support the hypothesis that the inhibition of NO synthesis induces inflammatory changes in coronary vascular and myocardial tissues and involves MCP-1 expression. Results also suggest that the early stages of inflammatory changes are important in the development of later-stage structural changes observed in rat hearts.

Macrophages cultured in vitro release leukotriene B4 and neutrophil attractant/activation protein (interleukin 8) sequentially in response to stimulation with lipopolysaccharide and zymosan.

The capacity of lipopolysaccharide (LPS), zymosan, and calcium ionophore A23187 to induce neutrophil chemotactic activity (NCA), leukotriene B4 (LTB4), and neutrophil attractant/activation protein (NAP-1) release from human alveolar macrophages (AM) retrieved from normal nonsmokers was evaluated. LPS induced a dose-dependent release of LTB4 that began by 1 h, 4.0 +/- 3.2 ng/10(6) viable AM; peaked at 3 h, 24.7 +/- 13.5 ng/10(6) viable AM; and decreased by 24 h, 1.2 +/- 1.0 ng/10(6) viable AM (n = 8). Quantities of LTB4 in cell-free supernatants of AM stimulated with LPS were determined by reverse-phase high-performance liquid chromatography and corresponded well with results obtained by radioimmunoassay. By contrast, NAP-1 release began approximately 3-5 h after stimulation of AM with LPS, 197 +/- 192 ng/ml, and peaked at 24 h, 790 +/- 124 ng/ml. Release of NAP-1 was stimulus specific because A23187 evoked the release of LTB4 but not NAP-1, whereas LPS and zymosan induced the release of both LTB4 and NAP-1. The appearance of neutrophil chemotactic activity in supernatants of AM challenged with LPS for 3 h could be explained completely by the quantities of LTB4 present. After stimulation with LPS or zymosan for 24 h, AM had metabolized almost all generated LTB4. Preincubation of AM with nordihydroguiaretic acid (10(-4) M) completely abolished the appearance of NCA, LTB4, and NAP-1 in supernatants of AM challenged with LPS. Therefore, LPS and zymosan particles were potent stimuli of the sequential release of LTB4 and NAP-1 from AM.

Role of Monocyte Chemoattractant Protein-1 in Cardiovascular Remodeling Induced by Chronic Blockade of Nitric Oxide Synthesis

BackgroundChronic inhibition of endothelial nitric oxide (NO) synthesis by the administration of N&ohgr;-nitro-l-arginine methyl ester (L-NAME) to rats induces early vascular inflammatory changes (monocyte infiltration into coronary vessels and monocyte chemoattractant protein-1 [MCP-1] expression) as well as subsequent arteriosclerosis (medial thickening and perivascular fibrosis) and cardiac fibrosis. However, the role of MCP-1 in this process is not known. Methods and ResultsWe investigated the effect of a specific monoclonal anti–MCP-1 neutralizing antibody in rats treated with L-NAME to determine the role of monocytes in the regulation of cardiovascular remodeling. We found increased expression of MCP-1 mRNA in vascular endothelial cells and monocytes in inflammatory lesions. Cotreatment with an anti–MCP-1 antibody, but not with control IgG, prevented the L-NAME–induced early inflammation and reduced late coronary vascular medial thickening. In contrast, the anti–MCP-1 antibody did not decrease the development of perivascular fibrosis, the expression of transforming growth factor (TGF)-&bgr;1 mRNA, or systolic pressure overload induced by L-NAME administration. ConclusionsThese results suggest that MCP-1 is necessary for the development of medial thickening as well as monocyte recruitment. In contrast, the pathogenesis of fibrosis may involve other factors, such as TGF-&bgr;1.

Monocyte chemoattractant protein-1 in idiopathic pulmonary fibrosis and other interstitial lung diseases.

Macrophages play a crucial role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). To examine the mechanisms for increased monocyte/macrophage recruitment in IPF and nonIPF interstitial lung diseases (nonIPF) the localization of monocyte chemoattractant protein-1 (MCP-1) was investigated in 14 cases of IPF, seven cases of nonIPF, and seven normal control lungs (CTRL) by immunohistochemistry using a specific anti-MCP-1 monoclonal antibody, F9. By double immunohistochemical staining using F9 and one of the cell type specific antibodies significant differences in the staining pattern of MCP-1 were observed between IPF and nonIPF. In IPF MCP-1 was observed in cuboidal and flattened metaplastic epithelial cells, alveolar macrophages, and vascular endothelial cells. In contrast, no epithelial cells were stained for MCP-1 in nonIPF cases, although alveolar macrophages and vascular endothelial cells were labeled. Northern hybridization analysis of selected cases showed marked expression of MCP-1 messenger RNA (mRNA) in IPF and nonIPF compared with CTRL. These findings suggest that the MCP-1 production in IPF and nonIPF plays an important role in the recruitment of monocyte/macrophages. Monocyte chemoattractant protein-1 production by epithelial cells in IPF may be caused by the metaplastic nature of the epithelial cells and may be one of the key factors inducing the irreversible progression of IPF.