Interleukin-1beta induces elevation of spermidine/spermine N1-acetyltransferase activity and an increase in the amount of putrescine in synovial adherent cells from patients with rheumatoid arthritis.

OBJECTIVE: To investigate polyamine metabolism in rheumatoid synovial adherent cells stimulated by interleukin- 1beta (IL-1beta). METHODS: Synovial adherent cells obtained from patients with rheumatoid arthritis (RA) were cultured and incubated in the presence or absence of human recombinant IL-1beta at a concentration of 10 ng/ml for 24 h. The cellular contents of polyamines as well as the activities of spermidine/spermine N1-acetyltransferase (SAT) and ornithine decarboxylase (ODC) were measured. RESULTS: Polyamines in synovial adherent cells decreased significantly after 24 h incubation in the absence of IL-1beta. However, in the presence of IL-Ibeta, putrescine and N'-acetylspermidine increased significantly. No significant difference was observed between the amount of spermidine in synovial adherent cells incubated with and without IL-1beta. Spermine and N8-acetylspermidine in synovial adherent cells incubated with IL-1beta decreased significantly more than in synovial adherent cells incubated without. SAT activity reached a peak 12 h after the addition of IL-1beta and then decreased, while the ODC activity did not increase. SAT activity was elevated by the addition of IL-1beta in a dose dependent manner. CONCLUSION: An increase in the putrescine level in rheumatoid synovial adherent cells as a result of the elevation of SAT activity induced by IL-1beta may play a role in RA.

Levels of serum and synovial fluid pyridinium crosslinks in patients with rheumatoid arthritis.

OBJECTIVE: To elucidate the major source of pyridinium crosslinks in rheumatoid arthritis (RA). METHODS: Serum samples were collected from 75 patients with RA and 41 healthy controls, and synovial fluid (SF) samples were collected from 20 patients with RA and 13 with osteoarthritis (OA). Paired samples of serum and SF were collected at the same time from 26 patients with RA. Levels of pyridinium crosslinks were determined by a recently developed high sensitivity assay method using high pressure liquid chromatography. RESULTS: The levels of serum pyridinoline (PYD) and serum deoxypyridinoline (DPD) were significantly higher in patients with RA than in healthy controls, and significantly correlated with laboratory variables indicating disease activity and severity. The levels of SF DPD, but not SF PYD, were significantly higher in patients with RA than in patients with OA. The levels of SF PYD and SF DPD both showed a significantly positive correlation with those of either SF interleukin 1beta or SF interleukin 6 in patients with RA. Finally, the levels of PYD, but not DPD, were higher in SF than in serum in all paired RA samples collected at the same time, with significant correlation between the members of each pair. CONCLUSION: These observations suggest than an increase of PYD in RA serum may originate mostly from affected joints and that an increase of DPD in RA serum may be influenced more by systemic bone resorption.

Positive correlation between levels of IL-1 or IL-2 and 1,25(OH)2D/25-OH-D ratio in synovial fluid of patients with rheumatoid arthritis.

The present study determined the levels in synovial fluid (SF) of vitamin D metabolites (1,25-dihydroxyvitamin D (1,25(OH)2D), 24,25-dihydroxyvitamin D (24,25(OH)2D) and 25-hydroxyvitamin D (25-OH-D)), and of the cytokines. We evaluated SF from 21 patients with rheumatoid arthritis (RA) and 6 patients with osteoarthritis (OA). The levels of vitamin D metabolites in SF, as determined by two different extraction methods, were significantly correlated (p < 0.05, n=7). The levels of 3 vitamin D metabolites were significantly higher in the RA SF than in OA SF (p < 0.05). The ratio of 1,25(OH)2D/25-OH-D in RA SF, which is presumed to reflect the activity of 25-OH-D-1-hydroxylase (1-OH-ase), was positively correlated with the levels of interleukin-1alpha (IL-1alpha), IL-1beta, and IL-2 in such SF, and was significantly higher than that in sera from RA patients. This suggests an important role for these cytokines in the activation of 1-OH-ase in RA synovium. The ratio of 24,25(OH)2D/25-OH-D, which is presumed to reflect 25-OH-D-24-hydroxylase (24-OH-ase) activity, was significantly correlated with 1,25(OH)2D levels only in RA SF, but not in sera from RA patients, suggesting a local regulation of vitamin D metabolism that 1,25-(OH)2D induces 24-OH-ase as in other target cells. Our observations suggested that 1,25(OH)2D and 24,25(OH)2D are produced locally from 25-OH-D in RA synovium, and that the syntheses of 1,25(OH)2D and 24,25(OH)2D may be affected by IL-1/IL-2 and 1,25(OH)2D in RA SF, respectively.

Levels of hepatocyte growth factor in synovial fluid and serum of patients with rheumatoid arthritis and release of hepatocyte growth factor by rheumatoid synovial fluid cells.

OBJECTIVE: To determine the levels of hepatocyte growth factor (HGF) in synovial fluids (SF) and sera from patients with rheumatoid arthritis (RA); to examine how these correlate with several disease variables in patients with RA and with levels of interleukin-6 (IL-6) in SF of these patients; and to examine whether HGF is released from adherent synovial cells (ASC) and synovial fluid cells (SF cells). METHODS: An enzyme linked immunosorbent assay was used to measure levels of HGF and IL-6. SF samples were obtained from 22 patients with RA, 12 with osteoarthritis (OA), and one with septic arthritis. Serum samples were collected from 40 patients with RA. HGF levels in culture supernatants from ASC and SF cells were measured. RESULTS: The mean values of HGF in SF were 1.21 ng/ml for patients with RA, 0.19 ng/ml for those with OA and 0.18 ng/ml for the one with septic arthritis. HGF levels in SF of patients with RA were significantly higher than of those with OA (p < 0.01). The levels for patients with RA correlated with the serum C-reactive protein concentrations (r = 0.626, p < 0.01) and IL-6 levels in SF (r = 0.476, p < 0.05). The mean value of HGF in sera from patients with RA was 0.28 ng/ml. HGF levels in SF were higher than those in sera drawn simultaneously from the same patients with RA. In vitro, release of HGF from rheumatoid ASC was not detected. However, SF cells from patients with RA released HGF spontaneously. CONCLUSION: Our observations suggest that HGF in SF of patients with RA is produced by SF cells and is related to disease activity of RA, and thus that HGF may play a role in RA.

Levels of urinary polyamines in patients with rheumatoid arthritis.

OBJECTIVE: To compare urinary polyamine levels in patients with rheumatoid arthritis (RA), with osteoarthritis (OA), and in healthy controls and examine the relationship between urinary polyamine levels and several disease variables in patients with RA. METHODS: We determined the concentrations of urinary polyamines in 33 patients with RA, 24 with OA, and 20 healthy controls, using the enzymatic assay method. For patients with RA relevant clinical and laboratory variables were obtained and functional and radiologic scores determined for the joints. RESULTS: Urinary polyamine levels were significantly higher in patients with RA versus those with OA and healthy controls. In patients with RA the levels of urinary polyamines correlated significantly with the concentrations of serum C-reactive protein (CRP); there was also a statistically significant negative correlation between their urinary polyamine levels and average grip strength in either hand. Moreover, the levels of urinary polyamines in patients with RA showed an increase in proportion to the degree of joint functional damage and radiologic progression. CONCLUSION: Our results confirm our previous report of an increase in the amount of free putrescine in synovial fluids and a significant correlation between the putrescine contents of synovial tissues and the serum CRP concentrations in patients with RA; they also suggest that urinary polyamine levels may be related to the activity and progression of RA, indicating that polyamine may play an important role in RA.

Polyamine levels in synovial tissues and synovial fluids of patients with rheumatoid arthritis.

We determined the polyamine contents of the synovial tissues from 11 patients with rheumatoid arthritis (RA), and the free putrescine levels in the synovial fluids (SF) from 10 patients with RA, 7 with osteoarthritis (OA), 5 with posttraumatic arthritis, and 3 with infectious arthritis. Putrescine levels in the synovial tissues correlated with serum C reactive protein concentration in patients with RA. Free putrescine levels in SF were significantly elevated in patients with infectious arthritis, compared with those found in RA, OA, and posttraumatic arthritis. Free putrescine levels in SF from patients with RA were significantly higher than in those with OA. Our findings suggest that polyamines may play an important role in RA.

Two mechanisms of spermidine/spermine N1-acetyltransferase-induction.

The changes in activity of spermidine/spermine N1-acetyltransferase (SAT), a rate-limiting enzyme in polyamine degradation, were investigated to understand the mechanism of the induction of this enzyme in bovine lymphocytes. The activity of SAT was induced by stimulation with phytohemagglutinin (PHA), calcium ionophore A23187, sodium n-butyrate, or methylglyoxal bis(guanylhydrazone) (MGBG). When the cells were treated with a combination of PHA with either MGBG or butyrate, the increase in SAT was synergistic. However, the treatment of cells with both PHA and A23187 did not cause more induction of the enzyme activity than the stimulatory effects of each agent alone. The elevation in SAT caused by PHA or A23187 was inhibited by the simultaneous addition of 25 microM H-7, a protein kinase C inhibitor; the induction of the enzyme activity by MGBG or butyrate was slightly enhanced in the presence of H-7. In cells treated with a high concentration of O-tetradecanoylphorbol 13-acetate, which results in the breakdown of protein kinase C, PHA and A23187 did not give the maximum response, and MGBG slightly enhanced the enzyme activity. Dibutyryl cyclic AMP inhibited PHA-induced enzyme activity, but it stimulated MGBG- or butyrate-induced activity. Exposure to PHA or A23187 but not to MGBG or butyrate significantly increased the ornithine decarboxylase activity and DNA synthesis. These results showed that there were two different mechanisms of SAT induction. One is dependent on protein kinase C. The other one is independent of protein kinase C and is enhanced by cyclic AMP.

Effect of 1 alpha,25-dihydroxyvitamin D3 on polyamine metabolism in human monocyte cell line-U937.

1. Pretreatment with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] caused an increase in ornithine decarboxylase (EC 4.1.1.17) (ODC) activity in lipopolysaccharide (LPS)-stimulated human monocyte cell line, U937. 2. The increase in ODC activity was dose-dependent and preincubation-time dependent. 3. 26,26,26,27,27,27-Hexafluoro-1,25-dihydroxyvitamin D3 was ca 7 times more potent than 1,25-(OH)2D3 in increasing ODC activity. 4. Pretreatment with 1,25-(OH)2D3 also potentiated the activity of spermidine/spermine-N1-acetyltransferase in LPS-stimulated U937 cells. 5. Putrescine levels in cells pretreated with 1,25-(OH)2D3 increased ca 2-fold 4-8 hr after LPS addition. 6. However, pretreatment with 1,25-(OH)2D3 did not cause any increase in ODC mRNA level, suggesting that 1,25-(OH)2D3 may modulate polyamine metabolism at the posttranscriptional level rather than the transcriptional step.

Calcium/calmodulin-mediated action of calcitonin on lipid metabolism in rats.

The effects of calcitonin on lipid metabolism were investigated in three kinds of rats, one strain of rabbits, and a primary culture of rat hepatocytes. In a short-term experiment, calcitonin decreased serum cholesterol and triglycerides after injection in rats on either an ordinary or high-fat diet. In a long-term experiment, calcitonin decreased the serum cholesterol and triglycerides in uremic rats, hypothalamic obese rats, and Watanabe-heritable hyperlipidemic rabbits. In cultured hepatocytes, calcitonin reduced the incorporation of [14C]acetate into cholesterol and triglycerides in a dose-dependent way. Treatment with W7, a calmodulin inhibitor, overcame the decrease caused by calcitonin in serum lipids in rats and in the synthesis of triglycerides from acetate or palmitate in the hepatocytes, but did not alter the intracellular cAMP level or incorporation of [32P]Pi into PI in the cells. The results suggest that calcitonin lowers serum lipid levels and lipogenesis in hepatocytes in a calcium/calmodulin-dependent way.

Biological activity of 26,26,26,27,27,27-hexafluorinated analogs of vitamin D3 in inhibiting interleukin-2 production by peripheral blood mononuclear cells stimulated by phytohemagglutinin.

Vitamin D compounds suppress the production of interleukin-2 (IL-2) by peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin in a dose-dependent manner. We used this suppression to test 26,26,26,27,27,27-hexafluorinated analogs of vitamin D3 for their immunosuppressive activity in PBMCs. 26,26,26,27,27,27-Hexafluoro-1,25-dihydroxyvitamin D3 and 26,26,26,27,27,27-hexafluoro-1,24-dihydroxyvitamin D3 were approximately 10 times more potent than 1,25-dihydroxyvitamin D3 in suppressing IL-2 production. 26,26,26,27,27,27-Hexafluoro-1-hydroxyvitamin D3 was 20 to 30 times less potent than 1,25-dihydroxyvitamin D3 in causing this effect. The relative biopotency of each vitamin D3 analog toward PBMC proliferation was roughly similar to that toward IL-2 production by PBMCs. Suppression of PBMC proliferation by vitamin D3 analogs seemed to be a secondary effect of their inhibition of IL-2 production.

Polyamine biosynthesis is necessary for interleukin-2-dependent proliferation but not for interleukin-2 production or high-affinity interleukin-2 receptor expression.

DL-alpha-Difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase [EC 4.1.1.17] (ODC), inhibited concanavalin A-induced proliferation of splenic mononuclear cells (SMNC). The inhibition was not reversed by interleukin-2 (IL-2) addition. Although DFMO did not affect the production of IL-2 or the expression of high-affinity IL-2 receptor, IL-2-dependent proliferation of SMNC was inhibited by DFMO, and the inhibition was reversed by exogenous putrescine. The inhibition of IL-2-dependent DNA synthesis appeared to be related to the decrease in intracellular polyamines. When the proliferation of SMNC was induced by IL-2, ODC activity was also increased. A similar result was obtained in the proliferation of an IL-2-dependent T cell line, CTLL. The time course of ODC induction was similar to that of IL-2 production by concanavalin A-stimulated SMNC. These results indicate that polyamine biosynthesis is necessary for IL-2-dependent proliferation, but not for IL-2 production or IL-2 receptor expression.

Biological activity of fluorinated vitamin D analogs at C-26 and C-27 on human promyelocytic leukemia cells, HL-60.

Vitamin D compounds added to the culture medium induce HL-60 cells to differentiate into macrophage/monocytes via a receptor mechanism. This system provides a biologically relevant assay for the study of biopotency of vitamin D analogs. Using this system, the biological activity of various fluorinated derivatives of vitamin D3 was compared with that of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). As assessed by cell morphology, nitroblue tetrazolium reduction and nonspecific esterase activity, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 (26,27-F6-1,25-(OH)2D3) and 26,26,26,27,27,27-hexafluoro-1,24-dihydroxyvitamin D3 (26,27-F6-1,24-(OH)2D3) were about 10 times as potent as 1,25-(OH)2D3 in suppressing HL-60 cell proliferation and inducing cell differentiation. The biological activity of 26,26,26,27,27,27-hexafluoro-1-hydroxyvitamin D3 (26,27-F6-1-OH-D3) was equal to that of 1,25-(OH)2D3 in this system. 1,25-(OH)2D3 and its fluorinated analogs exerted their effects on HL-60 cells in a dose-dependent manner. HL-60 cells have a specific receptor for 1,25-(OH)2D3 with an apparent Kd of 0.25 nM, identical with that of chick intestinal receptor. While the binding affinities of 26,27-F6-1,25-(OH)2D3 and 26,27-F6-1,24-(OH)2D3 for chick intestinal receptor were lower than that of 1,25-(OH)2D3 by factors of 3 and 1.5, respectively, they were as competent as 1,25-(OH)2D3 in binding to HL-60 cell receptor. The ability of 26,27-F6-1-OH-D3 to compete for receptor protein from HL-60 cells and chick intestine was about 1/70 that of 1,25-(OH)2D3. These results indicate that trifluorination of carbons 26 and 27 of vitamin D3 can markedly enhance the effect on HL-60 cells.

Polyamines in 1 alpha, 25-dihydroxycholecalciferol-induced differentiation of human promyelocytic leukemia cells, HL-60.

The human promyelocytic leukemia cell line, HL-60, differentiated into macrophage/monocytes in the presence of 1 alpha,25-dihydroxycholecalciferol [1 alpha,25(OH)2D3], as assessed by the percentage of morphologically mature cells and their ability to reduce nitroblue tetrazolium. In this study of the mechanism involved, the activities of ornithine decarboxylase and spermidine/spermine-N1-acetyltransferase (SAT), the rate-limiting enzymes of polyamine metabolism, as well as the cellular levels of polyamine were measured. ODC activity reached a peak 24 h after the addition of 1 alpha,25(OH)2D3 and then decreased, while SAT activity gradually increased as differentiation commenced. An increase in putrescine and decreases in spermidine and spermine were also observed. Addition of alpha-difluoromethylornithine, an irreversible inhibitor of ODC, with or without methylglyoxalbis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, caused no effect on 1 alpha,25(OH)2D3-induced cell differentiation, although the cellular levels of putrescine and spermidine decreased markedly. Addition of alpha-difluoromethylornithine markedly suppressed cell proliferation; this effect was reversed by the addition of exogenous putrescine. Addition of exogenous spermidine or spermine to overcome activation of SAT also had no effect on 1 alpha,25(OH)2D3-induced cell differentiation. These results suggest both that polyamine metabolism is not important in 1 alpha,25(OH)2D3-induced differentiation of HL-60 cells, but that it is intimately involved in the proliferation of these cells.

Nutrition and renal osteodystrophy.

Nutritional factors, especially calcium, calorie and fat intakes may be important in the treatment with active vitamin D, so that the effect appears more efficient. Incidence of bone changes, due to hyperparathyroidism in diabetic nephropathy, was less than that in non-diabetic patients under hemodialysis. No effect of control status of diabetes mellitus was demonstrated, regarding incidence of subperiosteal resorption of finger bones. Bone mass was decreased in diabetic patients in whom the control of blood glucose was inadequate.