Increase of RP105-lacking activated B cells in the peripheral blood and salivary glands in patients with Sjögren's syndrome.

OBJECTIVE: To quantify the activated B cells in the peripheral blood and salivary glands of patients with Sjögren's syndrome (SS) by analyzing the expression of RP105 molecule on the B cells. METHODS: The expression of RP105 on the peripheral blood B cells of patients with SS (19 cases) was analyzed by flow cytometry. RP105-positive and negative B cells were sorted and cultured in vitro and the amount of immunoglobulins (IgG and IgM) produced in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA). Salivary gland biopsy samples from 9 SS patients were histologically evaluated and the sequential frozen sections were separately immunostained by anti-RP105 and anti-CD20 monoclonal antibodies. RESULTS: A significantly higher proportion of peripheral blood RP105-negative B cells was found in SS patients than in healthy individuals. RP105-negative, but not positive, B cells from SS patients were capable of producing IgG and IgM spontaneously in vitro, which was enhanced by the addition of Staphylococcus aureus Cowan I strain (SAC) or IL-6. Salivary glands from 2 of 9 SS patients were found to have lymphoid follicles whose germinal centers consisted of RP105-negative B cells. Moreover, a larger proportion of B cells extensively infiltrating the area other than lymphoid follicles was also RP105-negative. CONCLUSION: RP105-negative B cells, a subset of highly activated and well differentiated B cells, which are increased in number in the peripheral blood and extensively infiltrate salivary glands, may be responsible for the production of class-switched immunoglobulin in SS. In addition, those cells might be associated with the inflammation and tissue damage of the salivary glands.

Synthesis and secretion of protein C inhibitor by the human hepatoma-derived cell line, Hep G2.

The site of synthesis of protein C inhibitor, a recently identified human plasma inhibitor against activated protein C, is not known. We have studied the production and secretion of protein C inhibitor by an established human liver cell line derived from hepatocellular carcinoma (Hep G2). The concentration of protein C inhibitor, as measured by a specific radioimmunoassay, increased in the medium of Hep G2 cells with time. There was no evidence for a significant intracellular pool of this protein. Protein C inhibitor secreted from Hep G2 cells (G2 protein C inhibitor) inhibited the activity of purified activated protein C in a functional assay. De novo synthesis of protein C inhibitor was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 h of labeling of the cells with [35S]methionine. Analysis of the immunoprecipitates by SDS-polyacrylamide gel electrophoresis showed a peak of radioactivity corresponding to Mr 57 000. These results indicate that the liver is a site of protein C inhibitor production.

Prominent increase in the amount of a cytosol protein in transformed fibroblasts and ascites hepatoma cells.

Analysis of cytosol proteins by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis revealed a prominent increase in the amount of a cytosol protein with molecular weight of 88 000 in transformed human adult, human embryo, mouse adult, and hamster embryo fibroblasts as compared with normal fibroblasts. The cytosol protein with Mr 88 000 is also increased in the cytosol of four kinds of rat ascites hepatoma cell as compared to normal and regenerating liver. The protein with Mr 88 000 exists as one of the major cytosol proteins in transformed fibroblasts hepatoma cells and HeLa cells, constituting 7--10% of total cytosol proteins. The data suggest that the cytosol protein with Mr 88 000 is associated with certain growth characteristics of cells.

Effects of simvastatin, a cholesterol synthesis inhibitor, on phosphatidylcholine synthesis in HepG2 cells.

The effects of the addition of a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, simvastatin, to the medium on sterol synthesis and phosphatidylcholine (PC) synthesis were studied in HepG2 cells. The cells were cultured with simvastatin at concentrations of 10(-7) and 10(-6) mol/L for 6 hours, and radioactive lipid precursors were added 1 hour before harvesting. Simvastatin inhibited cholesterol synthesis from [14C]acetate in a dose-dependent manner. It also decreased the incorporation of [14C]choline into PC by 30%; this decrease was accompanied by a decrease in phosphocholine cytidylyltransferase activity in cell homogenates. Simvastatin had no significant effects on the incorporation of [3H]glycerol into phospholipids. These data indicate that simvastatin has two different functions: inhibition of HMG-CoA reductase and depression of de novo synthesis of PC via the cytidine diphosphate-choline pathway, which, in turn, may result in a decrease in plasma lipid levels.

Effect of forskolin on platelet deaggregation and cyclic AMP generation.

The diterpene, forskolin, induced a partial deaggregation of ADP- or collagen-aggregated human platelets in vitro. An increase in platelet cyclic AMP by forskolin was assumed to mediate the platelet deaggregation. PGE1 also deaggregated these platelets, and a combination of forskolin and PGE1 produced deaggregation greater than the maximum which could be obtained with each agent alone. A greater than additive effect was observed on the platelet cyclic AMP level in the presence of both forskolin and PGE1. No additive effect was observed in the phosphorylation of molecular weight (Mr) 21K polypeptide using forskolin (0.1 mmol/l) and PGE1 (5 mumol/l) suggesting that although cyclic AMP is responsible for the deaggregation process a mechanism other than phosphorylation through cyclic AMP-dependent protein kinase may be responsible for the effect of forskolin on platelet deaggregation.

Genetic analysis of systemic lupus erythematosus: association with a T cell receptor restriction fragment length polymorphism in Japanese patients.

Many studies have suggested the involvement of multiple genetic loci in the development of systemic lupus erythematosus (SLE). We have analyzed the correlation between various genetic markers and the susceptibility to SLE. In this study, the association of SLE and restriction fragment length polymorphism (RFLP) of T cell receptor gene was evaluated. The cDNA for constant regions of alpha, beta and gamma chain were used as probes and RFLPs were analyzed after digestion with Eco RI, Bam HI, Pst I, Pvu II, Hind III and Bgl II. Among them, polymorphisms were detected using Bgl II- and Hind III-digested DNA and C beta as a probe. Association with SLE in Japanese patients was found only after digestion with Hind III. The absence of the 13 kb polymorphic band appeared to be correlated with the development of SLE (relative risk = 4.78).

Pharmacological effects of dai-saiko-to on lipid biosynthesis in cultured human hepatocyte HepG2 cells.

The pharmacological effects of Dai-saiko-to, a Japanese and Chinese traditional medicinal mixture (Kampohozai), on lipid biosynthesis were investigated in cultured human hepatocyte HepG2 cells. The addition of Dai-saiko-to (0.5 mg/ml), which had no significant effect on cell proliferation, caused a marked decrease in the intracellular triglyceride content with no significant changes in the other lipid fraction. At the same time, the incorporation of 14C-acetate or 3H-glycerol into the triglyceride or diglyceride fractions also decreased significantly. These results suggest that Dai-saiko-to decreases hepatic triglyceride biosynthesis, which might contribute to a reduction in plasma VLDL levels.

Ex vivo CD(+) T-cell cytokine expression from patients with Sjögren's syndrome following in vitro stimulation to induce proliferation.

OBJECTIVE: To assess ex vivo CD4(+) T-cell cytokine expression from patients with primary Sjögren's syndrome (SS) following in vitro stimulation to induce proliferation, as proliferation is closely related to differentiation of cytokine-producing cells. METHODS: Peripheral blood mononuclear cells (PBMCs) separated from primary SS patients (n = 28) and controls (n = 25) were analysed. PBMCs were stimulated with concanavalin A followed by phorbol 12-myristate 13-acetate and ionomycin. Intracellular interferon-gamma (IFN-gamma) and interleukin-4 (IL)-4 in proliferating CD4(+) T cells were assessed by flow cytometry. The proportion of cytokine-producing cells and proliferating cells in each division cycle was assessed using [5(and 6)-carboxyfluorescein diacetate, succinimidyl ester]-labelled CD4(+/-) T cells. RESULTS: The proportion of IFN-gamma+ proliferating CD4(+) T cells in each cell division cycle from extraglandular SS was increased in glandular SS patients compared glandular SS patients with controls (P<0.05 approximately 0.01). The percentage of IFN-gamma single positive proliferating CD4(+) T cells was greater in extraglandular SS patients (26.7+/-14.1%) compared with glandular SS (9.9 +/- 9.1%) (P<0.01) and controls (9.4 +/- 5.8%) (P<0.001). There was no significant difference in the percentages of IL-4(+) proliferating CD4(+) T cells among the groups. However, the proliferating response of CD4(+) T cells was significantly decreased in extraglandular SS patients (percentage of proliferating cells 38.4 +/- 18.6%) compared with that in glandular SS patients (64.2 +/- 17.2%) (P<0.05) and controls (63.1+/-10.6%) (P<0.01). CONCLUSIONS: CD4(+) T cells from extraglandular SS patients may have a predisposition for entry into the IFN-gamma-producing effector pathway as a result of the stimulations. These results are helpful for understanding the immunological difference between glandular and extraglandular SS and the mechanisms of disease progression.

Testosterone eliminates the attenuating effect of castration on the progressive glomerular injury in hypercholesterolemic male Imai rats.

Hypercholesterolemic Imai rats, especially males, spontaneously develop proteinuria and glomerulosclerosis. We have shown that castration attenuated the progression of glomerular injury in male Imai rats. The present study was designed to investigate whether the attenuating effect of castration on glomerulosclerosis is eliminated by administration of testosterone. Testosterone propionate (TP) eliminated the attenuating effect of castration on the progression of glomerular injury by significantly and dose dependently increasing proteinuria and enhancing glomerular sclerosis. TP at 1 mg failed to reverse glomerulosclerosis to the control levels, despite a dose of TP sufficient to restore urinary protein excretion, serom testosterone level and glomerular growth. The results indicated that besides testosterone other sex-related hormones may be involved in the development of glomerulosclerosis in male Imai rats.

Low responsiveness to the anti Leu 4 antibody by T cells from patients with active systemic lupus erythematosus.

It has been widely accepted that murine monoclonal antibodies against the CD 3 antigen have mitogenic effects on human T cells which mimic antigen binding via the T cell receptors. We report that T cells from patients with active systemic lupus erythematosus cannot respond to anti Leu 4 antibody, an anti CD 3 murine monoclonal antibody of the subclass IgG1. It was observed that T cells from patients with the active disease expressed as much of the Leu 4 and OKT 3 epitopes as compared to high responders, and sera from active patients could not change the expression of the Leu 4 epitope. Similarly, the low responsiveness could not be attributed to suppressor T cells, poor production of IL 2 or less expression of the IL 2 receptor. The accessory nonrosetting mononuclear leucocytes from high responders could convert the low responsiveness of these patients to high, whereas the accessory cells from active patients failed to help the responses of T cells from high responders. Likewise, exogenous IL 1 and anti Leu 4 antibody bound to Sepharose beads independently or cooperatively failed to propagate T cells from low responders. We suggest that the low response to anti Leu 4 antibody as demonstrated in the T cells of patients with active SLE may be due in part to the impairment of the secondary signals, distinct from IL 1, between helping accessory cells and responding T cells, or to the dysfunction of intracellular transmission of stimulation, and may play significant roles in the pathogenesis of SLE.

Testosterone does not eliminate the attenuating effect of estrogen on progressive glomerular injury in estrogen-treated hypercholesterolemic male Imai rats.

Hypercholesterolemic Imai rats spontaneously develop proteinuria and glomerulosclerosis, especially males. Estrogen attenuates the progressive glomerular injury in these male rats. To clarify whether this attenuating effect of estrogen depends on a reduction of testosterone and/or a reduction of the sex-related factors, we investigated whether testosterone administration eliminates the attenuating effect of estrogen on the development of glomerular injury in estrogen-treated male Imai rats. Estrogen significantly reduced sex-related low molecular weight protein excretion to undetectable levels; and treatment with estrogen and testosterone failed to increase these levels. Unexpectedly, treatment with estrogen and testosterone attenuated glomerular injury more than treatment with estrogen only. Estrogen significantly increased both levels of estrogen and growth hormone (GH), whereas it suppressed testosterone levels. Testosterone administration resulted in an increase in serum testosterone levels of about fivefold above the control levels, but reduced the elevated serum GH to the levels of the controls. These results suggest that estrogen appears to play a protective role by itself or in association with sex-related factors, independent of the levels of serum testosterone, and that testosterone does not exert its effect on augmenting glomerular injury and rather may act to attenuate glomerular injury associated with a reduction of GH levels.

The ovaries attenuate the aggravating effect of testosterone on glomerular injury in Adriamycin-induced nephropathy of female rats.

To clarify whether the ovaries have a potential to attenuate the aggravating effect of testosterone (T) on glomerular injury, we investigated the effect of T in female rats with or without ovaries, using Adriamycin (ADR)-induced nephropathy in female Sprague-Dawley rats. Group 1 consisted of female control rats, group 2 received T, groups 3 and 4 were subjected to ovariectomy (OVX) at 5 weeks of age, and group 4 received further T treatment. Group 5 consisted of male control rats. T was injected subcutaneously every 4 weeks from 5 weeks of age through the end of the experiment. ADR 2 mg/kg was administered intravenously to all rats twice, at 8 weeks of age and 20 days later. Body weight, blood pressure, urinary protein and serum constituents were investigated every 4 weeks from 4 through 24 weeks after the second ADR injection. Each group was studied morphologically 24 weeks after the second ADR injection. Treatment with T or with OVX and T significantly increased the urinary protein excretion. OVX had no significant effect on the urinary protein excretion. Treatment with either T or OVX did not induce any significant effects on the renal function with regard to blood urea nitrogen (BUN), serum creatinine (Cr) and Cr clearance (Ccr) levels, but a combined treatment with OVX and T significantly lowered the serum albumin levels, increased the levels of BUN and Cr and lowered the Ccr values. The glomerulosclerosis index was significantly and markedly higher in control male rats than in control females. Treatment with T resulted in a slight but significant increase in glomerular injury to levels similar to those seen in ovariectomized rats. Combined treatment with OVX and T significantly aggravated glomerular injury in a somewhat accelerated manner, associated with a significant increase in glomerular tuft volume. Our results suggested that the ovaries could not completely suppress glomerular injury worsened by T administered at serum levels similar to those of male rats, but they had a potential to attenuate glomerular injury induced by T, and the protective effect of the ovaries on glomerular injury may be related to their attenuating effect on glomerular growth.

Attenuating effect of castration on glomerular injury is age-dependent in unilaterally nephrectomized male Sprague-Dawley rats.

To clarify a role of sex hormones in greater susceptibility of young rats than adults to the development of focal and segmental glomerulosclerosis (FSGS), we castrated animals at different ages and investigated whether the attenuating effect of castration on FSGS is age-dependent in unilaterally nephrectomized male Sprague-Dawley (SD) rats. At 6 weeks of age, all groups received unilateral right nephrectomy (Nx) and group 2 was simultaneously castrated, while group 1 received a sham operation. Group 3 was castrated at 3 months of age, and group 4 at 6 months of age. Body weight, blood pressure, urinary protein and serum constituents were investigated every 2 months from 4 to 14 months of age. At 6 and 14 months of age, rats were studied morphologically. Castration at 6 weeks of age or at 3 months of age significantly inhibited the compensatory glomerular hypertrophy and hyperfunction with regard to the creatinine clearance as seen in Nx rats at 6 months of age and significantly reduced glomerular injury at the end of the experiment, while castration at 6 months produced neither an inhibitory effect on glomerular hypertrophy nor an attenuating effect on glomerular injury. Serum levels of growth hormone (GH) and somatomedin-C (SmC) were decreased by castration to a greater extent when castrated at younger age. These findings indicated that GH and SmC influenced by male sex hormone seem to play a more important role at younger age than in adults in exerting its effect on glomerular growth, leading somehow to glomerular injury in aging, unilaterally nephrectomized male SD rats.

Effect of ovariectomy on glomerular injury in hypercholesterolemic female Imai rats.

To clarify the pathogenesis of focal and segmental glomerulosclerosis, we investigated the effect of ovariectomy in hypercholesterolemic female Imai rats. At 5 weeks of age, control female (group 1) and control male rats (group 3) were sham-operated, female rats (group 2) were ovariectomized and male rats (group 4) were castrated. Body weight, blood pressure, urinary protein and serum constituents were checked every 2 months from 2 through 12 months of age. All groups were studied morphologically at 6 months of age and further female groups (1 and 2) studied at 12 months. Both control female and control male rats developed marked proteinuria, to a significantly greater extent in the male rats. Castration reduced proteinuria, while ovariectomy did not influence it and there were no significant differences in proteinuria among the control females, the ovariectomized females and the castrated males. Control male rats had significantly lower serum albumin levels, higher cholesterol levels and a significantly greater impairment of renal function in blood urea nitrogen (BUN) levels than did the control female rats at 6 months. Castration significantly increased serum albumin levels and lowered BUN levels, while ovariectomy did not basically influence these values in the female rats. The glomerulosclerosis index at 6 months of age was significantly higher in the control males than in the control females. Castration attenuated glomerular injury, while ovariectomy aggravated glomerular injury to the same levels as found in the castrated males. This aggravating effect of ovariectomy observed at 6 months, however, disappeared at 12 months. These results suggested that sex-related factors regulated by the ovaries may play an inhibitory role in the development of glomerulosclerosis before 6 months of age, but not thereafter, in hypercholesterolemic female Imai rats.

Ovariectomy attenuates proteinuria and glomerular injury in unilaterally nephrectomized female Sprague-Dawley rats.

To determine the contribution of the ovary to the development of glomerulo-sclerosis, we investigated the effect of ovariectomy on glomerulosclerosis, using the unilaterally nephrectomized (Nx) female Sprague-Dawley rat. At 6 weeks of age, groups 2 and 3 underwent unilateral right nephrectomy and group 3 was simultaneously ovariectomized, while group 1 underwent a sham operation. Body weight, blood pressure, urinary protein, serum albumin, cholesterol, blood urea nitrogen and serum creatinine were checked every 2 months from 2 to 12 months after right nephrectomy. Control group 1, the Nx group 2 and the ovariectomized (Nx + ovariectomized) group 3 were studied morphologically at 6 and 12 months after nephrectomy. Body weight significantly increased in ovariectomized rats as compared with control and Nx rats. Nx rats became proteinuric with age. Ovariectomy significantly reduced proteinuria to the same levels in the controls. The glomerulosclerosis index was significantly higher in Nx rats than in either controls or ovariectomized rats. Ovariectomy attenuated glomerular injury in Nx rats, though not to the same levels in the control rats. Three groups showed no significant differences in either blood pressure or plasma somatomedin C. Growth hormone (GH) was significantly decreased by ovariectomy. The severity of glomerular injury and the glomerular tuft volume correlated with GH levels. Our results suggested that a decrease in plasma GH may contribute to the attenuating effect of ovariectomy on the development of glomerular injury in aging unilaterally Nx female Sprague-Dawley rats.

Sex difference in progression of adriamycin-induced nephropathy in rats.

To clarify the pathogenesis of focal-segmental glomerulosclerosis, we investigated the sex-related difference and the effect of castration in Adriamycin (ADR) induced nephropathy of Sprague-Dawley rats. At 5 weeks of age, group 1 female and group 2 male rats were sham operated, and group 3 male rats were castrated. ADR 2 mg/kg was intravenously administered to all rats at 8 weeks of age twice at a 20-day interval. Body weight, blood pressure, urinary protein, and serum constituents were investigated every 4 weeks, 4-20 weeks after the second ADR injection. Each group was studied morphologically 12 and 20 weeks after the second ADR injection. ADR induced massive proteinuria in male rats, whereas it induced significantly lower proteinuria in female rats, and castration significantly reduced proteinuria of male rats to an extent equal to the levels seen in female rats. Control male rats had significantly lower serum albumin levels and a significantly greater impairment of renal function (blood urea nitrogen and creatinine levels) than the female rats or the castrated male rats at 20 weeks. The glomerulosclerosis index was significantly higher in control male rats than in female rats, and castration attenuated glomerular injury of male rats to an extent close to the levels seen in female rats, though there was a significant difference in the glomerulosclerosis index between female rats and castrated male rats. The three groups did not differ in blood pressure and plasma somatomedin C and serum growth hormone levels, whereas the plasma testosterone levels were decreased to undetectable in female and castrated male rats, resulting in a reduction of sex-related low molecular weight protein in urine. These observations suggest that sex hormones such as testosterone and estrogen and/or sex-related low molecular weight protein regulated by testosterone and estrogen may play a contributory role in sex differences in the progression of glomerulosclerosis in ADR-treated rats.