Tadashi Kasahara

Induction of Monocyte Chemoattractant Protein-1 Synthesis in Human Monocytes During Transendothelial Migration In Vitro

Monocyte chemoattractant protein-1 (MCP-1, or monocyte chemotactic and activating factor) plays important roles in the recruitment of monocytes and thus in the development of atherosclerosis. In this study, we determined whether MCP-1 synthesis was induced by the cellular interaction between monocytes and endothelial cells during the process of transendothelial migration. We found that when human peripheral blood monocytes (2.5 x 10(6) cells) and umbilical vein endothelial cells (HUVECs; 5.0 x 10(5) cells) were cocultured for 5 hours, 7.9 ng/mL MCP-1 was secreted into the medium, whereas when the two were cultured separately, MCP-1 levels were 1.0 and 0.9 ng/mL, respectively. Furthermore, the use of interleukin-1 beta (IL-1 beta)-pretreated HUVECs in cocultures induced twice the levels of MCP-1 as in unstimulated HUVEC culture. Conditioned medium had transendothelial chemotactic activity for monocytes, and this activity was completely abolished by addition of anti-MCP-1 antibody. Although MCP-1 mRNA levels were very low or undetectable in HUVECs or monocytes alone, message could be detected after 2 hours of coculture in total mRNA preparations from both monocytes and HUVECs. mRNA levels increased by 4 hours and had declined slightly by 24 hours. The rapid induction of message suggests that cell contact between monocytes and HUVECs induces the de novo synthesis of MCP-1 protein. Anti-interleukin (IL)-1 alpha/beta and anti-tumor necrosis factor-alpha antibodies, or anti-lymphocyte function-associated antigen-1 and very late antigen-4 antibodies, had little or no inhibitory effects on MCP-1 secretion by cocultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation of specific antibodies against murine IL-1ra and the establishment of IL-1ra as an endogenous regulator of bacteria-induced fulminant hepatitis in mice.

Blocking monoclonal antibodies (mAbs) specific to mouse interleukin‐1 receptor antagonist (IL‐1ra) were prepared by immunizing Armenian hamsters with recombinant mouse IL‐1ra. A sensitive and specific ELISA against mouse IL‐1ra was also established. In Propionibacterium αcnes‐induced liver injury, P. acnes induced transient increase of serum tumor necrosis factor‐α levels but not those of IL‐1ra, IL‐1, and IL‐6. However, subsequent lipopolysaccharide (LPS) challenge induced the increase of serum levels of all these cytokines and the peak serum IL‐1ra level was more than 20 times as high as serum IL‐1 levels. Immunohistochemical analysis demonstrated that IL‐1ra was predominantly produced by hepatocytes during the course of the priming phase by P. acnes and eliciting phase by LPS challenge. Furthermore, the administration of a mAb to mouse IL‐1ra exacerbates the liver injury induced by P. acnes and sublethal dose of LPS, suggesting a protective role of endogenous IL‐1ra in this liver injury model. J. Leukoc. Biol. 58: 90–98; 1995.

Interleukin-8 levels and granulocyte counts in cervical mucus during pregnancy

PROBLEM: We measured interleukin‐8 (IL‐8) and granulocyte counts in cervical mucus to assess local immunity and parturition. METHOD OF STUDY: We detected IL‐8 by enzyme linked immunosorbent assay (ELISA) and Western blotting. Granulocytes were counted on a slide glass containing mucus from the external cervical os. RESULTS: ELISA and Western blotting revealed IL‐8 in cervical mucus from both nonpregnant and pregnant women. There were no significant differences in cervical mucus IL‐8 levels or granulocyte counts between follicular, ovulatory, and luteal phases. However, IL‐8 and granulocyte counts were significantly decreased after menopause. IL‐8 and granulocyte counts were increased significantly during pregnancy, and were further increased after 38 weeks of gestation and at labor. IL‐8 levels were significantly correlated with granulocyte counts, based on the study of 678 samples (P<0.0001). CONCLUSION: IL‐8 is involved in the increase of cervical granulocytes and in the process of parturition.

Detection and localization of interleukin-8 mRNA and protein in human placenta and decidual tissues

It has been reported that interleukin-8 (IL-8) is secreted from the placental and decidual tissues and that IL-8 levels in the amniotic fluids are significantly elevated by chorioamnionitis or labor pain. The present study was aimed at defining the localization of IL-8 mRNA as well as IL-8 protein at the feto-maternal interface using in situ hybridization and immunohistochemical staining. Both IL-8 mRNA and protein were localized in cytotrophoblast, syncytiotrophoblast and Hofbauer cells of the placenta, decidual stromal cells, decidual lymphocytes and endometrial gland cells. IL-8 secretion from glandular cells has not previously been reported. In addition, we confirmed IL-8 mRNA expression and secretion of IL-8 by an endometrial cancer cell line (Ishikawa) using the reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) methods, respectively.

Nitric oxide synthesis in rat cardiac myocytes and fibroblasts

We investigated nitric oxide (NO) synthase activity in cultured neonatal rat cardiac myocytes and fibroblasts upon treatment with interleukin 1 beta (IL-1 beta) and lipopolysaccharide (LPS). Incubation of cardiac myocytes for 24 h with IL-1 beta or LPS caused a significant increase in NO and cGMP production. Simultaneous incubation of IL-1 beta with NG-monomethyl-L-arginine or transforming growth factor beta (TGF-beta) completely inhibited the IL-1 beta-induced NO and cGMP production in cardiac myocytes. In contrast, incubation of cardiac fibroblasts for 24 h with IL-1 beta or LPS showed no significant effect on NO or cGMP production. Addition of IL-1 beta decreased the beating rate of cardiac myocytes, but TGF-beta overcame that inhibition. These observations suggest the presence of iNOS in cardiac myocytes, which is an important regulator of contractile function of the heart.

Induction of Metallothionein in Astrocytes by Cytokines and Heavy Metals

Immunohistochemical staining of human and rat brains for metallothionein (MT) using a monoclonal anti-MT antibody (MT45) revealed that protoplasmic astrocytes, which are densely localized in the gray matter, contain high levels of MT. Human U373MG astrocytoma cells were stimulated with interleukin (IL)-1 or heavy metals to produce MT. When expression of MT in U373MG cells was analyzed by Northern blotting or indirect immunofluorescence using the MT45 antibody, it was found that IL-1 (> or = 10 U/ml), CdCl2 (50 microM) and high concentrations of ZnCl2 (500 microM) induced marked biosynthesis of MT.IL-6 (up to 3,000 U/ml) and lower concentrations of ZnCl2 (10-50 microM), however, showed little inducing activity. Hippocampal astrocytes in primary culture produced a relatively high basal level of MT. The MT level increased in response to addition of IL-1 (> or = 10 U/ml), ZnCl2 (50 microM) and CdCl2 (5 microM). However, the increase induced by IL-6 (1,000 U/ml) was not very marked. The in vivo induction of MT in the brain by cytokines is not fully understood. However, our data and other indirect evidence suggest that IL-1 may be a potent stimulator of MT induction in astrocytes. Furthermore, the astrocytoma cell line, U373MG, is a suitable in vitro system to analyze the expression of MT in astrocytes.

Induction of metallothionein in a human astrocytoma cell line by interleukin-1 and heavy metals

The effects of cytokines and heavy metals on the expression and localization of metallothioneins (MTs) within U373MG astrocytoma cells were analyzed by using indirect immunofluorescence using a monoclonal anti‐MT antibody (MT45). IL‐1, CdCl2 (50μM) or ZnCI2, (500μM) remarkably augmented intracellular MT levels, whereas IL‐6 or 10 μM of ZnCl2 showed no inducing activity. From 24 to 48 h after the addition of CdCl2 or IL‐1, immunoreactive MTs were found in the cytoplasm and the nucleus. After 72 h, immunoreactive MTs accumulated in a granular form near the cell surfaces in the presence of CdCl2 (50 μM) or IL‐1 plus ZnCI2 (10 μM). However, this accumulation was not observed when only IL‐1 was added. Thus, Zn2+ facilitated the appearance of the granular form of immunoreactive MTs at a concentration where they do not induce MTs by themselves.

Induction of inflammatory cytokines in the pleural effusion of cancer patients after the administration of an immunomodulator, OK-432 : role of IL-8 for neutrophil infiltration

When OK-432, a well-known streptococcal preparation for an anti-tumour drug, was administered into the pleural cavity of patients with malignant pleurisy, a rapid and prominent leukocytosis, predominantly consisting of neutrophils, was observed in the cavity. Neutrophil infiltration usually peaked 6-9 h after OK-432 administration, and levelled down after 24 h. Prior to the neutrophil accumulation, transient but marked elevation of various inflammatory cytokine levels including IL-1 beta, TNF-alpha, IL-8 and G-CSF was observed. In particular, IL-8 levels increased more than 10-fold, while GM-CSF did not change significantly. A good correlation between IL-8 levels and neutrophil chemotactic response was observed particularly during 0-3 h. Specific neutralization or removal of IL-8 by antibody column abrogated half of the neutrophil chemotaxis, while neutralization of C5a removed around 40%. Sequential removal of IL-8 and C5a abrogated totally 80% of chemotaxis, confirming that these two factors are mostly responsible for the neutrophil chemotaxis in the pleural fluids. These results have suggested that rapid neutrophil infiltration induced by OK-432 in vivo is ascribable largely to IL-8 and in part to C5a.

Suppressive Role of Endogenous Endothelial Monocyte Chemoattractant Protein–1 on Monocyte Transendothelial Migration In Vitro

Monocyte chemoattractant protein-1 (MCP-1, or monocyte chemotactic and activating factor) is thought to play an important role in monocyte infiltration into tissue, but little is known about its effect on monocyte-endothelium interaction. We examined the effect of MCP-1 produced by cytokine-activated endothelial cells (ECs) on monocyte-endothelium adhesion and subsequent transendothelial migration by using a double-chamber vessel model. Unstimulated ECs showed no MCP-1 expression, but exposure to interleukin-1 beta (IL-1 beta, 25 U/mL) induced marked MCP-1 mRNA expression and protein synthesis. When placed in the lower compartment, recombinant human (rh) MCP-1 (100 ng/mL) produced a 1.9-fold and a 2.7-fold increase in adhesion and migration, respectively, compared with a corresponding 51% and 59% decrease when placed in the upper compartment. Migration of monocytes was dependent on a gradient of rh-MCP-1 from the apical to basilar side of the EC layer. Furthermore, a forward gradient of MCP-1 induced adherent cells to increase their subsequent migration, whereas a reverse gradient induced the cells to detach and completely inhibited their subsequent migration. Pretreatment with IL-1 beta for 4 and 24 hours produced a 20% and 63% increase in monocyte migration, respectively. In the presence of anti-MCP-1 antibody, the increase was further enhanced by 52% and 152%, respectively. These results suggest that endogenous endothelial MCP-1, when secreted by IL-1-stimulated ECs, suppresses monocyte migration in the presence of MCP-1 on the basilar side of the EC layer.(ABSTRACT TRUNCATED AT 250 WORDS)