Yoshinori Goto

An essential role of myosin light-chain kinase in the regulation of agonist- and fluid flow-stimulated Ca2+ influx in endothelial cells

Cytosolic Ca2+ ([Ca2+];) plays an important role in endothelial cell signaling. Although it has been suggested that the influx of Ca2+ can be triggered by depletion of intracellular Ca2+ stores, the mechanism (or mechanisms) underlying this phenomenon needs further elaboration. In the present study, involvement of myosin light‐chain kinase (MLCK) in the regulation of Ca2+ signaling was investigated in agonist‐ and fluid flow‐stimulated endothelial cells loaded with Ca2+‐sensitive dyes. Bradykinin (BK) and thapsigargin caused an increase in [Ca2+]i followed by a sustained rise due to Ca2+ influx from extracellular space and shifted total myosin light‐chain (MLC) from the unphosphorylated to the diphosphorylated form. ML‐9 (100 pM), an inhibitor of MLCK, abolished Ca2+ influx and prevented MLC diphosphorylation in BK‐ and thapsigargin‐treated cells, but did not affect Ca2+ mobilization from internal stores. Fluid flow stimulation (shear stress=5 dynes/cm2) increased [Ca2+]i and enhanced MLC phosphorylation. ML‐9 also inhibited Ca2+ response and MLC phosphorylation in fluid flow‐stimulated cells. The Ca2+ influx in response to BK was linearly correlated with the diphosphorylation of MLC in ML‐9 treated cells. Effects of ML‐5 and ML‐7, analogs of ML‐9, to inhibit Ca2+ influx paralleled their potencies to inhibit MLCK activity. These findings demonstrate that MLCK plays an essential role in regulating the plasmalemmal Ca2+ influx in agonist‐ and fluid flow‐stimulated endothelial cells. This study is the first to report the close relationship between Ca2+ influx and MLC diphosphorylation.—Watanabe, H., Takahashi, R., Zhang, X.‐X., Goto, Y., Hayashi, H., Ando, J., Isshiki, M., Seto, M., Hidaka, H., Niki, I., Ohno, R. An essential role of myosin light‐chain kinase in the regulation of agonist‐ and fluid flow‐stimulated Ca2+ influx in endothelial cells. FASEB J. 12, 341–348 (1998)

Abnormal production of B cell growth factor in patients with systemic lupus erythematosus.

In order to clarify the role of B cell growth factor (BCGF) in the pathogenesis of systemic lupus erythematosus(SLE), BCGF production by peripheral blood mononuclear cells(PBMC) and T cells was studied using a new bioassay for BCGF activity. For this purpose, we established an Epstein‐Barr (EB) virus‐transformed B cell line KS‐3.F10 that proliferates only in response to two B cell‐specific BCGF, low‐mol. wt BCGF (LMW‐BCGF) and high‐mol. wt BCGF (HMW‐BCGF). PBMC from active SLE patients produced less BCGF when stimulated with phytohaemagglutinin (PHA) compared with controls. The decreased BCGF production by PHA‐stimulated PBMC from active SLE reverted to control values when SLE became inactive. However, PHA‐stimulated T cells from active SLE patients produced more BCGF compared with controls, whereas those from inactive SLE showed normal BCGF production. Spontaneous BCGF production by T cells was not observed in active SLE patients. These findings suggest that decreased BCGF production by SLE PBMC is due to excessive BCGF consumption by B cells in vitro and that SLE T cells produce large amounts of BCGF with appropriate immune stimuli in vitro to promote polyclonal B cell activation.

Relations between surface expression of the interleukin-2 receptor and release of the soluble form of the receptor in cultured mononuclear cells from patients with rheumatoid arthritis or systemic lupus erythematosus

The relationship between surface expression of the interleukin-2 receptor (IL-2R) and release of the soluble form of the receptor (sIL-2Rα or sCD25) was investigated with peripheral blood mononuclear cells (PBMCs) from individuals with rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). The spontaneous release of sCD25 was significantly increased in PBMCs from RA patients and decreased in cells from SLE patients, compared with normal controls. However, the extent of sCD25 release from phytohaemagglutinin (PHA)-stimulated PBMCs did not differ between RA or SLE patients and normal controls. The serum concentration of sCD25 was significantly increased in SLE or RA patients compared with the normal controls. Whereas the surface expression of CD25 by unstimulated PBMCs did not differ among the three groups of subjects, this parameter was significantly reduced for PHA-stimulated PBMCs from RA patients relative to those from normal controls. The surface expression of CD25 showed a positive correlation with sCD25 release for PBMMCs from SLE patients under either basal or stimulated conditions. No such relation was apparent for cells from RA patients. The surface expression of IL-2Rβ (CD122) under basal or stimulated conditions was significantly reduced in PBMCs from RA or SLE patients, compared with cells from normal controls. Thus, the increased concentration of sCD25 in the serum of individuals with these autoimmune rheumatic diseases may result from two different mechanisms: an increase in the spontaneous release of sCD25 in RA, and reduced clearance of this protein in SLE.

Activation-induced cell death of peripheral blood T lymphocytes in patients with primary Sjögren’s syndrome

Activation-induced cell death (AICD) in T lymphocytes is important for the maintenance of peripheral tolerance. We studied AICD of peripheral blood T cells from patients with primary Sjögren’s syndrome (SS). AICD was induced in mitogen-activated T cellsin vitro using mAb to CD3 or Fas (CD95). Cell death and proliferation, Fas and Fas ligand (FasL) expression, and soluble Fas and soluble FasL production were measured. Surface phenotypes and cytokine production of AICD-surviving cells and effects of cytokines on AICD were examined. Anti-CD3 mAb induced cell death is SS and normal T cells in the presence of exogenous interleukin (IL)-2. In the absence of IL-2 anti-CD3 mAb induced cell proliferation in SS and normal T cells. There was no significant difference in Fas/FasL expression and sFas/sFasL production between SS patients and normals. AICD-surviving cells consisted of more CD4+ T cells and less CD8+ T cells in SS compared to normals. AICD-surviving cells produced abundant interferon-γ and little IL-4. There was no significant difference in the effects of cytokines on AICD between SS patients and normals. These findings suggest that IL-2 is a critical factor for AICD. AICD works almost normally in SS T cells when sufficient IL-2 is present prior to T cell receptor re-stimulation.

Pulmonary thromboembolism in a patient with idiopathic hypereosinophilic syndrome

We report a successful treatment in a patient with idiopathic hypereosinophilic syndrome (HES) presenting with left pulmonary truncal thromboembolism and right pleural effusion. A treatment with urokinase infusion by a Swan-Gants catheter near a left pulmonary thrombus was performed and left pulmonary arterial occlusions were recanalized completely. After corticosteroid therapy was started, right pleural effusion disappeared. Both anticoagulant and corticosteroid therapy was necessary for treatment in HES patients who had thromboembolic episodes.