Kazuhiko Takehara

Ligand-dependent inhibition of B16 melanoma cell migration and invasion via endogenous S1P2 G protein-coupled receptor. Requirement of inhibition of cellular RAC activity.

We investigated mechanisms for inhibition of B16 melanoma cell migration and invasion by sphingosine-1-phosphate (S1P), which is the ligand for the Edg family G protein-coupled receptors and also implicated as an intracellular second messenger. S1P, dihydro-S1P, and sphingosylphosphorylcholine inhibited B16 cell migration and invasion with the relative potencies expected as S1P2 receptor agonists. The S1P2-selective antagonist JTE013 completely abolished the responses to these agonists. In addition, JTE013 abrogated the inhibition by sphingosine, which is the S1P precursor but not an agonist for S1P receptors, indicating that the sphingosine effects were mediated via S1P2 stimulation, most likely by S1P that was converted from sphingosine. S1P induced inhibition and activation, respectively, of Rac and RhoA in B16 cells, which were abrogated by JTE013. Adenovirus-mediated expression of N17Rac mimicked S1P inhibition of migration, whereas C3 toxin pretreatment, but not Rho kinase inhibitors, reversed the S1P inhibition. Overexpression of S1P2 sensitized, and that of either S1P1 or S1P3 desensitized, B16 cells to S1P inhibition of Rac and migration. In JTE013-pretreated, S1P3-overexpressing B16 cells, S1P stimulated cellular RhoA but failed to inhibit either Rac or migration, indicating that RhoA stimulation itself is not sufficient for inhibition of migration. These results provide compelling evidence that endogenously expressed S1P2 negatively regulates cell motility and invasion through ligand-dependent reciprocal regulation of cellular Rac and RhoA activities. In the presence of JTE013, S1P instead stimulated Rac and migration in B16 cells that overexpress either S1P1 or S1P3, unveiling counteractions between S1P2 and S1P1 or S1P3 chemotactic receptor.

Epidemiological study of patients with systemic sclerosis in Tokyo

SummaryWe conducted an epidemiological study of systemic sclerosis in the city of Tokyo using the records of patients who had been registered to receive free medical service for intractable diseases. A total of 636 patients were registered as having systemic sclerosis in 1987, and we sent questionnaires to the doctor of each patient. The contents of the questionnaires included the patients name, sex, age, occupation, major symptoms, therapy and laboratory findings. We received 357 completed replies, and were able to analyse them. Our study estimated that at 1 January 1988 the prevalence rate in Japan was between 2.1 and 5.3 per 100000. The male/female ratio was 14:1. The ages of the patients when surveyed ranged from 17 to 82 years, with a mean age of 51 years, peaking with the most numerous group being 50–59 years. The characteristic signs of systemic sclerosis were as follows: proximal scleroderma, 75%; sclerodactyly, 91%; pitting scars, 49%; short sublingual frenulum, 49%; pulmonary fibrosis, 45%; diffuse pigmentation, 45%; and phalangeal contracture, 35%. Raynauds phenomenon was present in 93% of patients, and was the initial symptom in 59% of cases. With respect to specific antinuclear antibodies, anticentromere antibody was present in 19% and antitopoisomerase I antibody was present in 27%.

Demonstration of interleukin-2, interleukin-4 and interleukin-6 in sera from patients with localized scleroderma

Localized scleroderma has been reported to be accompanied by immunological abnormalities related to B cells, but little is known about T-cell activation in this disease. In this study, serum levels of interleukin-2 (IL-2), interleukin-4 (IL-4) and interleukin-6 (IL-6), which are known to be released by activated T cells, were determined using a sensitive enzyme-linked immunosorbent assay in 48 patients with localized scleroderma and 20 with systemic sclerosis, and in 20 healthy control subjects. IL-2, IL-4 and IL-6 were detected in serum from patients with localized scleroderma but not in that from healthy controls. The presence of antihistone antibodies correlated significantly with elevated IL-4 and IL-6 levels. Decreased serum levels of IL-2, IL-4 and IL-6 paralleled improvement in cutaneous sclerosis. Frequent detection of these lymphokines in serum from patients with localized scleroderma reflects T-cell activation in this disorder.

Clinical characteristics associated with antihistone antibodies in patients with localized scleroderma

BACKGROUNDnRecently we demonstrated the presence of antihistone antibodies (AHA) in localized scleroderma.nnnOBJECTIVEnOur purpose was to determine clinical characteristics associated with AHA in patients with localized scleroderma.nnnMETHODSnWe examined 57 serum samples by an enzyme-linked immunosorbent assay in the following three subgroups: 15 patients with generalized morphea, 27 with linear scleroderma, and 15 with morphea. We classified the patients as having generalized morphea when they had four or more lesions on at least two areas of the body, irrespective of whether the lesions were of morphea or linear type.nnnRESULTSnAHA were detected in 42% of patients with localized scleroderma (24 of 57), and in 87% of patients with generalized morphea (13 of 15). The presence of AHA strongly correlated with the number of morphea lesions, the total number of lesions, and the number of involved areas of the body. However, AHA did not correlate with the presence or number of linear lesions. The presence of AHA showed a 87% sensitivity (13 of 15 patients) and a 74% specificity (31 of 42 patients) for generalized morphea.nnnCONCLUSIONnOur data suggest that AHA are a serologic marker for generalized morphea and that the validity of our new classification for generalized morphea is supported by the high frequency of AHA detection.

Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis

In order to determine the prevalence and clinical significance of β2‐GPI‐dependent anticardiolipin antibodies (β2‐GPI/aCL) in patients with systemic sclerosis (SSc), serum samples from 80 patients with SSc, 20 patients with systemic lupus erythematosus (SLE), and 120 healthy control subjects were examined by ELISA using purified β2‐GPI. IgG isotype β2‐GPI/aCL was present in eight of 80 patients with SSc (10%), and the presence of β2‐GPI/aCL IgG was significantly correlated with the presence of isolated pulmonary hypertension (PH). Furthermore, levels of β2‐GPI/aCL IgG were significantly correlated with levels of mean pulmonary arterial pressure. These data suggest that IgG isotype β2‐GPI/aCL might be a serological indicator of the severity of PH in patients with SSc.

Effects of various growth factors and histamine on cultured keloid fibroblasts.

OBJECTIVE AND METHODSnWe investigated the effects of several growth factors on [3H]thymidine incorporation and procollagen type I carboxyterminal propeptide (P1CP) production, which reflects type I collagen metabolism, in keloid and normal fibroblasts.nnnRESULTSnSix fibroblast cell strains, derived from keloid or normal skin, exhibited similar growth responses to platelet-derived growth factor, transforming growth factor beta 1 (TGF-beta 1), gamma-interferon (gamma-IFN) and histamine. In contrast, keloid fibroblasts showed significantly greater growth response to epidermal growth factor (EGF) than normal fibroblasts. P1CP production was 4.4 times higher in 6 strains of keloid fibroblasts than in 6 controls. Treatment with gamma-IFN (100 U/ml) decreased P1CP production in both groups; the effect was significantly greater in keloid fibroblasts. TGF-beta 1 treatment upregulated P1CP production in both groups. Treatment with histamine increased P1CP production in keloid fibroblasts, although it did not change that in the controls.nnnCONCLUSIONnEGF and histamine may play some role in the development of keloids.

Abnormal expression of intracellular cytokines and chemokine receptors in peripheral blood T lymphocytes from patients with systemic sclerosis

In patients with systemic sclerosis (SSc), there are conflicting findings regarding which is predominant between type 1 and type 2 immune responses. To determine the balance between type 1 and type 2 T lymphocytes in peripheral blood from SSc patients, we investigated the expression of intracellular cytokines, such as interferon‐γ (IFN‐γ), interleukin‐2 (IL‐2), IL‐4, and IL‐13, and chemokine receptors such as CXCR3 and CCR4 by flow cytometry. The frequency of IFN‐γ‐producing cells among CD8+ cells was significantly increased in patients with diffuse cutaneous SSc (nu2003=u200311, Pu2003<u2003 0·0001) and limited cutaneous SSc (lSSc; n= 16, Pu2003<u20030·0001) compared with normal controls (nu2003=u200317) while there was no significant difference in the frequency of IL‐4‐ or IL‐13‐producing cells. In contrast, the frequency of IFN‐γ‐ or IL‐4‐producing cells among CD4+ cells was similar between the three groups. Similar results were obtained when absolute numbers were assessed. The frequency of IFN‐γ‐producing cells among CD8+ cells inversely correlated with percentage DLco in SSc patients (ru2003=u2003−u20030·650, Pu2003<u20030·005). CXCR3+u2003CD8+ cells selectively produced IFN‐γ, and the frequency of CXCR3+u2003CD45RO+ cells among CD8+ cells was higher in lSSc patients (nu2003=u200314, Pu2003<u20030·01) than in normal controls (nu2003=u200322). In contrast, there was no significant difference in the frequencies of CXCR3‐ or CCR4‐expressing CD45RO+ cells among CD4+ cells. These results demonstrate the predominance of type 1 cytokine‐producing cells (Tc1 cells) in peripheral blood CD8+ T cells from SSc patients, but no definite Th1/Th2 imbalance in CD4+ T cells. Tc1 cells may be associated with pulmonary vascular damage in SSc.

Elevated plasma endothelin levels in systemic sclerosis.

Endothelin is a novel potent vasoconstrictor peptide produced mainly by endothelial cells. Thrombomodulin is a high-affinity thrombin receptor on vascular endothelial cells that plays an important role as a natural anticoagulant. In this study, we measured plasma levels of endothelin and thrombomodulin in patients with systemic sclerosis or Raynauds disease. Plasma levels of endothelin and the ratio of thrombomodulin to creatinine were significantly increased in patients with systemic sclerosis compared with normal controls, and there was a positive correlation between these two indicators (r=0.615, P=0.004). Moreover, plasma levels of endothelin were significantly higher in patients with diffuse systemic sclerosis than in patients with limited systemic sclerosis. In contrast, plasma levels of endothelin in patients with Raynauds disease were not significantly increased. These results suggest that increased plasma levels of endothelin and thrombomodulin may reflect microvascular damage in systemic sclerosis.

Clinical evaluation of scleroderma spectrum disorders using a points system

We have established a new diagnostic method using a points system to evaluate patients with early scleroderma and those with scleroderma spectrum disorders (SSD). To examine the clinical usefulness of this method, it was applied to a total of 215 cases including 97 patients with scleroderma, 32 with SSD, 28 with presumed primary Raynauds phenomenon (RP) and 58 with other connective tissue disorders (CTD). A total score was obtained for each patient as the sum of the following five factors: (1) extent of skin sclerosis (maximum, 10 points); (2) pulmonary changes (maximum, 4 points); (3) antinuclear antibodies (maximum, 5 points); (4) pattern of Raynauds phenomenon (maximum, 3 points); and (5) nailfold bleeding (maximum, 2 points). Of the 97 scleroderma patients, 86 (89%) had 9 or more points, and of the 32 SSD patients, 28 (88%) had 5 to 8 points. In contrast, all patients with presumed primary RP and 54 of 58 (93%) patients with other CTD had 0 to 4 points. These data suggest that this diagnostic method is very useful not only for clinical evaluation of SSD, but also for the differentiation of scleroderma and SSD from other CTD and primary RP.

Increased levels of circulating intercellular adhesion molecule-1 in patients with localized scleroderma

BACKGROUNDnIntercellular adhesion molecule-1 (ICAM-1) is important in immune-mediated mechanisms, and its circulating form (cICAM-1) may be an indicator of immune activation. Localized scleroderma is accompanied by various immunologic abnormalities.nnnOBJECTIVEnWe investigated whether the serum level of cICAM-1 in patients with localized scleroderma was elevated and was correlated with the clinical or serologic features of this disease.nnnMETHODSnSerum cICAM-1 levels were determined by an enzyme-linked immunosorbent assay in 48 patients with localized scleroderma, in 20 patients with systemic sclerosis, and in 20 healthy control subjects.nnnRESULTSnSerum levels of cICAM-1 were significantly higher in patients with localized scleroderma than in the healthy control subjects. These levels correlated with the number of lesions, the number of involved areas, levels of antihistone antibody IgM, and levels of soluble interleukin 2 receptor.nnnCONCLUSIONnThe results suggest that immune activation may be a factor in localized scleroderma.