Kenichi Norioka

Production of B cell stimulatory factor-2/interleukin-6 activity by human endothelial cells

The effect of culture supernatants of endothelial cell (EC) lines on the immunoglobulin-M(IgM) synthesis by human B cell line, SKW6-CL4 cells, was investigated. Supernatants of human EC stimulated IgM synthesis, as high as 6-fold, but supernatants of bovine EC did not. This enhancing activity was completely blocked by addition of anti-human B cell stimulatory factor-2/interleukin-6 (BSF-2/IL-6) antibody. These data suggest that human EC might participate in the human antibody production system by producing soluble factor, BSF-2/IL-6.

Inhibitory effect of human recombinant interleukin-1 α and β on growth of human vascular endothelial cells

Abstract Endothelial cell growth factor(ECGF) is a potent polypeptyde mitogen which stimulates the growth of endothelial cells. The mitogenic effect of ECGF was inhibited by addition of recombinant interleukin-1 (rIL-1) α or β in a concentration dependent manner. The morphological change was not observed distinctly. In the condition without ECGF, both types of rIL-1 enhanced [3H] -thymidine uptake slightly, but failed to increase cell numbers. These data suggest the possibility that the effect of rIL-1 on EC is modulated by the presence of ECGF.

Characterization of human monocytic cell line, U937, in taking up acetylated low-density lipoprotein and cholesteryl ester accumulation. A flow cytometric and HPLC study

The uptake of LDL and acetylated LDL and the ability of cholesteryl ester accumulation by cells of a human monocytic cell line, U937, has been characterized by flow cytometric assay using a fluorescent probe, DiI, and by high-performance liquid chromatography (HPLC). The increase of mean fluorescence intensity of U937 incubated with DiI-labeled lipoproteins demonstrates that this cell line could incorporate DiI-AcLDL, as well as DiI-labeled LDL. Competition and saturation studies indicate that the manner of taking up DiI-AcLDL is receptor-mediated. While differentiated U937 incubated with 16 nM phorbol myristate acetate for 24 h took up little DiI-AcLDL, HPLC analysis confirmed that intracellular free and esterified cholesterols significantly increase in the U937 cells incubated with AcLDL or LDL. The ability of mouse peritoneal macrophage to abundantly accumulate at least five kinds of cholesteryl ester were also shown in this analysis. In contrast, in U937 cells, free fatty acids are incorporated into various substances rather than into cholesteryl esters (as revealed by HPLC analysis), so that the cholesterol in AcLDL taken up by U937 cells is not synthesized into cholesteryl esters to any great extent.

Augmentation of LDL receptor activities on lymphocytes by interleukin-2 and anti-CD3 antibody: a flow cytometric analysis

Dormant lymphocytes are known to show little LDL receptor (LDL-R) activities. The present study was designed to determine whether or not LDL-R activities of lymphocytes from normal subjects were high enough to be measured by flow cytometry after the cells had been stimulated with recombinant interleukin-2 (IL-2) and anti-CD3 monoclonal antibody (mAb). IL-2 or anti-CD3 mAb individually provokes proliferation of lymphocytes in a serum-free medium. Proliferation rate was accelerated when the two reagents were used in combination. Stimulated cells cultured for 5 days expressed more than 85% CD3 positive, less than 0.5% CD14 positive, and less than 1.5% CD20 positive. The LDL-R activities of the cells were examined by the uptake of a fluorescence probe, DiI-labeled LDL (DiI-LDL) and analyzed by flow cytometry. Stimulated cells showed increased uptake of DiI-LDL and 84 +/- 9% were positive, whereas only 3.0 +/- 2.5% of the cells without stimulation were positive (P less than 0.001). Under the same conditions stimulated lymphocytes from a homozygous familial hypercholesterolemia (FH) patient showed little LDL-R activities; 14% of the cells were positive. Displacement assays reveal that the uptake of LDL by these cells is occurring by way of its specific pathway. These data imply the lymphocytes stimulated with the reagents used in the study might be used for detecting defects in LDL-R, perhaps defects in other genomic systems as well.

Pretreatment of Human Vascular Smooth Muscle Cells with Interleukin-1 Enhances Interleukin-6 Production and Cell Proliferation (Action of IL-1 on Vascular Smooth Muscle Cells)

Systemic vasculitis is an inflammatory disorder of blood vessels characterized by a perivascular mononuclear cell infiltration around the vessel and fibrinoid necrosis within vessel walls. Interleukin-1 (IL-1) is a multipotent inflammatory mediator and affects several properties of vascular cells. To determine whether IL-1 could contribute to the pathogenesis of vascular diseases, we examined the effect of IL-1 on B cell stimulatory factor-2/interleukin-6 (IL-6) production by cultured human vascular smooth muscle cells (SMC) and the proliferation of these cells. Supernatants of SMC stimulated IgM synthesis of human B cell line. SKW6-CL4 cells. This activity was increased (1.7 to 2.6-fold) when SMC were pretreated with IL-1 or calcium ionophore A23187 for 48 h, and was completely blocked by rabbit anti-human IL-6 antibodies. These IL-6 activities of the SMC supernatants were also assessed by using an IL-6 dependent murine hybridoma cell line. MH-60. BSF-2. In addition, we observed that pretreatment of SMC with IL-1 for 48 h stimulated growth of SMC during the 96 h incubations, as assessed by cell number (p less than 0.05). These results suggest that IL-1 may contribute to the pathogenesis of inflammatory and immunological vasculitis by the augmentation of IL-6 release and growth of SMC.

Expression of TLiSA1 on T cells from patients with rheumatoid arthritis and systemic lupus erythematosus

The expression of a new activation antigen, T cell lineage specific activation antigen (TLiSA1) on peripheral blood T cells from 16 rheumatoid arthritis (RA) and 8 systemic lupus erythematosus (SLE) patients and synovial fluid T cells from RA patients was determined in the context of T cell activation. The percentages of TLiSA1 positive T cells from inactive (4.6 +/- 5.2, mean +/- SE) or active RA (19.3 +/- 8.6) or inactive (1.7 +/- 2.1) or active SLE (8.7 +/- 2.7) were significantly increased compared with that of normal controls (0.7 +/- 0.4) (P less than 0.01). All patients with vasculitis showed relatively high positive percentages. The mean fluorocytometric intensity of TLiSA1 positive T cells from RA and SLE patients was significantly higher than that from normals. Percentages of TLiSA1 positive T cells from synovial fluids (21.8 +/- 4.9%) were significantly increased compared with those from peripheral blood of the same patients, indicating the local activation of T cells in patients with RA. An increase in the expression of TLiSA1 with no increase in the expression of the very late activating antigen 1 (VLA-1) was found in peripheral blood from RA, suggesting a difference in the stage of T cell activation in RA. In RA, there was a clinical correlation with levels of TLiSA1 expression on peripheral T cells. After stimulation with PHA, TLiSA1 positive percentages were increased on Day 2 and continued to increase through 5 days of culture. The maximum expression was obtained on Day 5. An increased number of TLiSA1 positive T cells belonged to OKT8. These results suggest that there is the systemic and the local activation of T cells in RA, following antigen stimulation, or a generalized nonspecific activation of immune system that could provide a means to monitor the abnormal immunologic activity in RA.

Stimulatory effect of CD5 antibody on B cells from patients with rheumatoid arthritis.

In order to clarify the role of CD5 antigen on B cell in autoimmunity, we examined B cells from patients with rheumatoid arthritis (RA). The percentages of CD5 positive B cells were increased in peripheral blood from RA compared with normal. Normal and RA B cells were stimulated with two kinds of monoclonal antibodies to CD5 (Leu-1, SL-1) which recognize different epitopes. RA B cells proliferated and secreted IgM by CD5 antibody stimulation in combination with IL-1. Our observations imply that CD5 positive B cells in RA are in their differentiation stage and that CD5 antigen might be one of the triggers to activate CD5 positive B cells in vivo to produce autoantibody.

Differential abnormality in cell-cycle stage of peripheral B cells from patients with systemic lupus erythematosus.

SummaryIn order to clarify the stage of abnormal activation of systemic lupus erythematosus (SLE) B cells, we investigated the cell-cycle phase of SLE B cells by flow-cytometric analysis. This study uses the simultaneous flow-cytometric analysis of cellular DNA content and incorporated bromodeoxyuridine, an analogue of thymidine, as indicators for DNA synthesis to detect activated B cells in peripheral blood of patients with SLE. In active SLE, the percentage of B cells increased in the S and G2M phase and decreased in the G0G1 phase as compared with normal control subjects. In inactive SLE, the percentage of B cells in the S phase also increased. Active SLE B cells did not progress through the cell cycle with the stimulation of anti-IgM plus PMA or SAC plus BSF, although normal B cells transit into S and G2M phase with such stimulation. These data suggest that SLE B cells are already activated and shifted to a matured state and that for this reason the B cells were poorly responsive to mitogen.

Measurement of spontaneous and stimulated anti-microsomal antibody synthesis in vitro by avidin-biotin enzyme immunoassay

The spontaneous and stimulated anti-microsomal (anti-Mic) antibody synthesis in vitro by peripheral blood lymphocytes (PBL) from patients with Hashimotos thyroiditis (HT) was studied by a highly sensitive and thyroid microsome-specific enzyme immunoassay using an avidin-biotin system (A-B EIA). Since the amount of the synthesized anti-Mic antibody by PBL in vitro is very small, it is difficult to study its kinetics and response to mitogens or the specific antigen by conventional assay systems. We applied the avidin-biotin system to conventional indirect EIA and established an assay system which was about four times as sensitive as indirect EIA. PBL from patients with HT synthesized significant amount of IgG anti-Mic antibody spontaneously but those from normal individuals and patients with rheumatoid arthritis did not. IgG anti-Mic antibody synthesis with pokeweed mitogen stimulation was increased in all HT patients and that with thyroid microsome stimulation was increased in three out of five patients. These results indicate that A-B EIA is a useful system to study the mechanism of anti-Mic antibody synthesis in vitro.