Serum factors from patients with systemic lupus erythematosus enhancing superoxide generation by normal neutrophils.

It has been suggested that human neutrophils exposed to performed immune complexes or activated complement fragments generate O2- anions in extracellular medium. In vivo studies have revealed that oxygen intermediates produced by immune complex-activated neutrophils play an important role in subsequent tissue damage. Since it is difficult to obtain direct evidence that O2- is released into plasma in patients with systemic lupus erythematosus (SLE), we studied the capacities of their sera to stimulate O2- release by human neutrophils in vitro. Sera from patients with SLE significantly enhanced O2- generation by neutrophils compared to normal sera. The enhancing activity of serum in the induction of increased O2- generation correlated positively with the presence of serum immune complexes and negatively with serum complement levels. The enhancing factors were analyzed by serum fractionation on Sephadex G-200 gel filtration, and were concluded to be immune complexes of intermediate size containing an activated complement fragment.

Human plasma proopiomelanocortin N-terminal peptide and adrenocorticotropin: circadian rhythm, dexamethasone suppression, and corticotropin-releasing hormone stimulation.

The circadian rhythm, suppression with dexamethasone, and stimulation by corticotropin-releasing hormone (CRH) of plasma immunoreactive (IR) proopiomelanocortin N-terminal (NT) and IR-ACTH were studied in nine normal subjects and two patients with Addison's disease. The RIA for human NT (hNT) used was specific for NT except for partial cross-reactivity with gamma 2MSH. In normal subjects, plasma IR-hNT and IR-ACTH had almost parallel circadian rhythms and were suppressed by dexamethasone. The mean plasma levels of IR-hNT and IR-ACTH at 0800 h were 140 +/- 23 (SD) and 23 +/- 5 pg/ml, respectively. Plasma IR-hNT increased in parallel with IR-ACTH 15 to 30 min after iv injection of 100 micrograms ovine CRH. Maximum percent increases in plasma IR-hNT and IR-ACTH were 185 +/- 47 and 235 +/- 10%, respectively. In Addison's disease, on the other hand, plasma levels of IR-hNT and IR-ACTH were markedly elevated and the circadian rhythms were parallel. The mean plasma IR-hNT and IR-ACTH levels at 0900 h were 4363 and 1750 pg/ml, respectively. These results suggest that plasma hNT and ACTH are produced from a common precursor in the pituitary gland and secreted concomitantly under various physiological conditions such as stimulation by CRH and inhibition by glucocorticoid.

NSAID-induced gastrointestinal damage. Epidemiology, risk and prevention, with an evaluation of the role of misoprostol. An Asia-Pacific perspective and consensus.

The problem of nonsteroidal anti-inflammatory drug (NSAID)-induced gastrointestinal toxicity was reviewed by members of the Asia Pacific League of Associations for Rheumatology (APLAR) in a consensus conference in September 1992. This paper by the participants presents the consensus conclusions incorporating knowledge from recent publications. There had been a high level of concern that much of the toxicity had resulted from extensive and indiscriminate prescribing of NSAIDs. The implementation of evidence-based guidelines was considered likely to be able to effect a substantial reduction in toxicity without significant loss of overall therapeutic benefit. The evidence from which such guidelines could be developed is critically appraised.

The possible role of endothelial cells in hypertensive disorders during pregnancy.

OBJECTIVE: To clarify the role of endothelial cells in pregnancy-related hypertensive disorders, we studied the cytotoxic effect of sera from normal pregnant women and from gravidas with various hypertensive complications of pregnancy. METHODS: We obtained serum samples from 84 Japanese women: 17 with preeclampsia, ten with gestational hypertension, six with chronic hypertension, five with chronic hypertension with superimposed preeclampsia, 21 normal gravidas, and 25 healthy nonpregnant women. Endothelial cell injury was measured by the release of radiolabeled chromium from the cells into the culture medium. RESULTS: The mean (+/- standard error of the mean) values of chromium 51 release in preeclampsia, gestational hypertension, chronic hypertension, chronic hypertension with superimposed preeclampsia, normal pregnancy, and healthy nonpregnant women were: 21.9 +/- 2.1, 10.0 +/- 2.0, 9.2 +/- 2.3, 12.9 +/- 0.8, 8.4 +/- 1.4, and 7.3 +/- 1.6%, respectively. Normal pregnant and nonpregnant subjects did not differ with respect to endothelial cell injury. Sera from women with preeclampsia demonstrated significantly greater endothelial cell injury than did sera from normal gravidas. Subjects with the three other categories of hypertensive disorders did not differ significantly from normal gravidas. CONCLUSION: Preeclampsia is characterized by the presence of a serum factor cytotoxic to endothelial cells. Therefore, the mechanism responsible for the increase in blood pressure differs between women with preeclampsia and those with other hypertensive disorders in pregnancy.

Serum Antibodies to Cytoskeletal Systems in Patients with Adult T-Cell Leukemia and Healthy HTLV-1 Carriers.

Serum antibodies to cytoskeletal systems were detected, using indirect immunofluorescence in patients with adult T-cell leukemia (ATL), healthy carriers of human T cell lymphotropic virus 1 (HTLV-1), patients with infectious mononucleosis and healthy adults. Healthy carriers of HTLV-1 had IgG antibodies to cytoskeletal systems as evidenced by an increased incidence of IgG antibodies to actin and vimentin. Decreased IgG antibody levels to Epstein-Barr virus nucleic acid (EBNA) were also evident. In patients with ATL, the titers of IgM antibodies to vimentin and cytokeratin showed a positive correlation with decreased serum levels of IgM, despite the fact that serum concentrations of IgM were significantly decreased in patients with ATL. The IgM antibody titer divided by the IgM concentration (the antibody ratio) was nigher than that of healthy carriers and healthy adults, suggesting that the IgM antibody response to cytoskeletal systems was preferentially preserved in these cases. There was also a suggestion of the presence of polyclonal as well as specific antibody responses to cytoskeletal systems in patients with infectious mononucleosis. As a result of these findings we suggest that there is some difference in the mechanisms responsible for the production of autoantibodies to cytoskeletal systems following HTLV-1 infection and Epstein-Barr virus infection.

Trilineage response to rhG-CSF with subsequent clonal hematopoiesis in a patient with severe bone marrow aplasia.

We treated a patient with severe aplastic anemia with long-term administration of recombinant human granulocyte-colony stimulating factor (rhG-CSF). When a trilineage response of hematopoiesis was obtained after the first treatment, a chromosomal change [45XX, -7] was observed in 20 of the 20 metaphases examined. Later, we were able to show a monoclonal X inactivation pattern in the phosphoglycerate kinase (PGK) gene in the peripheral blood polymorphonuclear leukocytes and mononuclear cells, indicating the presence of clonal hematopoiesis regardless of the disappearance of the karyotype abnormality. We suggest that it is important to pay close attention to the appearance of clonal hematopoiesis during the administration of G-CSF to patients with idiopathic severe bone marrow aplasia.

Possible mechanism of vascular damage in pre-eclampsia.

To clarify the pathophysiological changes of pre-eclampsia, we investigated the injurious effect of sera from women with pre-eclampsia on cultured endothelial cells, vascular smooth muscle cells and fibroblasts. We obtained serum samples from 32 Japanese women, including 10 healthy nonpregnant women, 12 normal pregnant women and 10 women with pre-eclampsia. Cell injury was measured by the release of radiolabelled chromium from the cells into the culture medium. The mean values +/- SD of percentage chromium release from endothelial cells in normal nonpregnant, normal pregnant and pre-eclamptic subjects were 8.5 +/- 2.4, 8.8 +/- 2.1 and 19.7 +/- 3.6%, respectively. Sera from women with pre-eclampsia demonstrated significantly greater endothelial cell injury than did sera from normal pregnant and nonpregnant subjects. However, normal pregnant and pre-eclamptic subjects did not differ with respect to both vascular smooth muscle cell injury and fibroblast injury. These results indicate that a serum cytotoxic to endothelial cells is present in pre-eclampsia and that this activity has a cellular specificity for endothelial cells.

Recombinant human IL-1 beta and TNF-alpha stimulate production of IL-1 alpha and IL-1 beta by vascular smooth muscle cells and IL-1 alpha by vascular endothelial cells.

Vascular endothelial cells (EC) produced IL-1 alpha but not IL-1 beta into extracellular fluids. Vascular smooth muscle cells (SMC), on the other hand, produced both IL-1 alpha and IL-1 beta, and IL-1 beta produced was much higher than IL-1 alpha. The addition of recombinant human IL-1 beta or recombinant human TNF-alpha significantly enhanced IL-1 alpha production in EC, and IL-1 alpha and IL-1 beta production in SMC. IL-1 beta release was not observed even when EC were stimulated with TNF-alpha. These results suggest that the species of released form of IL-1 are different in different cell types and that cytokines enhance IL-1 alpha and IL-1 beta production in SMC and IL-1 alpha production in EC.

The effect of recombinant tumor necrosis factor-alpha on superoxide and metalloproteinase production by synovial cells and chondrocytes.

We studied the effect of recombinant tumor necrosis factor-alpha (rTNF-alpha) on the production of superoxide and metalloproteinase by rheumatoid synovial cells or osteoarthritis chondrocytes. rTNF-alpha significantly inhibited superoxide generation by osteoarthritis chondrocytes and rheumatoid synovial cells at a concentration of 23 U/ml. On the other hand, rTNF-alpha at a concentration of 1500 U/ml significantly enhanced superoxide production by rheumatoid synovial cells, osteoarthritis synovial cells and osteoarthritis chondrocytes, respectively. Metalloproteinase released by rheumatoid synovial cells and chondrocytes derived from osteoarthritis patients were stimulated by rTNF-alpha at a concentration of 94 U/ml. rTNF-alpha at the highest concentration (15000 U/ml) significantly inhibited metalloproteinase release by rheumatoid synovial cells. The enhancing effect of rTNF-alpha at higher concentrations on superoxide production by rheumatoid synovial cells and osteoarthritis chondrocytes was time dependent. These results suggest that rTNF-alpha has a biphasic effect on superoxide and metalloproteinase production, and hence may play an important role in the pathogenesis of inflammatory joint diseases.

Correlation of metal-binding proteins and proteinase inhibitors with immunological parameters in rheumatoid synovial fluids.

Metal-binding proteins (ceruloplasmin, transferrin, ferritin, and lactoferrin), proteinase inhibitors (alpha 1-antitrypsin, alpha 2-macroglobulin and inter-alpha-trypsin inhibitors), and albumin were assayed in synovial fluid obtained from 20 patients with rheumatoid arthritis (RA) and 15 with osteoarthritis (OA). The levels of proteinase inhibitors and metal-binding proteins, except transferrin, were significantly increased in synovial fluid from RA patients as compared with synovial fluid from OA patients. Metal-binding proteins significantly correlated with rheumatoid factor and immune complexes in synovial fluid from RA patients. Proteinase inhibitor levels also significantly correlated with C-reactive protein, and complement components. These results suggest that the raised level of metal-binding proteins and proteinase inhibitors in synovial fluid from RA patients reflect inflammatory activity, and hence may play an important role in the pathogenesis of inflammatory joint diseases.

Sjögren's syndrome and mixed connective tissue disease.

We investigated the clinical significance of the close association of Sjögren's syndrome (SS) with mixed connective tissue disease (MCTD) by analyzing the clinical manifestations, sialographic findings and immunological parameters of MCTD and primary SS. The prevalence of sialectasia or SS in MCTD was significantly higher than in any other connective tissue diseases. The prevalence of Raynaud's phenomenon, swollen fingers, arthralgias, lymphadenopathy, sclerodactyly, muscle weakness, fever and erythema was significantly higher in MCTD than in primary SS. There were no significant differences between these manifestations in MCTD patients with sialectasia or SS, and those in MCTD patients without sialectasia or SS. Although the levels or prevalence of the erythrocyte sedimentation rate, CRP, antinuclear factor, anti-DNA antibody and anti-RNP antibody were significantly greater in MCTD than in primary SS, there were no significant differences in the levels or the prevalence of laboratory abnormalities between MCTD with sialectasia or SS, and MCTD without sialectasia or SS. Moreover, there was a strict dissociation between the occurrence of anti-RNP antibody and anti-SS-B antibody both in MCTD and primary SS. These results suggest that the association of secondary SS or sialectasia in MCTD, although more common than in other connective tissue diseases, is merely a consequence of MCTD and does not influence the clinical course of MCTD.

Influences of water deprivation and fasting on hypothalamic, pituitary and plasma opioid peptides and prolactin in rats.

To study the physiological roles of endogenous opioid peptides in drinking and feeding behaviors, the effects of water deprivation and fasting on plasma immunoreactive (IR) beta-endorphin (beta-end), IR-Antidiuretic hormone (ADH) and IR-Prolactin (Prl), pituitary IR-beta-end and IR-methionine-enkephalin (IR-Met-enk) and IR-ADH, and hypothalamic IR-beta-end and IR-Met-enk were observed in rats. The effects of water deprivation on hypothalamic dopaminergic system was also studied. In water deprived rats, plasma IR-beta-end and Prl were decreased significantly. In the neurointermediate lobe, IR-Met-enk, but not IR-beta-end, was decreased, although these peptides did not change in the anterior lobe and hypothalamus. Intraperitoneal injection of haloperidol reversed the decrease in plasma IR-beta-end in water deprived rats but did not change it in control rats. Subcutaneous injection of CB-154, on the other hand, decreased the plasma IR-beta-end in control rats but not in water deprived rats. The dopamine (DA) turnover rate in hypothalamus, in addition, was increased in water deprived rats as compared with controls. In fasted rats, IR-beta-end in plasma, but not in pituitary lobes and hypothalamus, was increased. The present results suggest that the increase of hypothalamic dopaminergic activity, in part, is related to the suppressed secretions of pituitary IR-beta-end and Prl in water deprivation, and plasma IR-beta-end play some roles in feeding behavior in rats.

Influences of feeding and drinking on circadian rhythms of opioid peptides in plasma, hypothalamus and pituitary gland in rats.

Changes of plasma, hypothalamic and pituitary immunoreactive beta-endorphin (IR-beta-end), methionine-enkephalin (IR-Met-enk) and ACTH (IR-ACTH) were studied under various conditions of feeding and watering in rats. When rats were fed from 17:00 to 09:00 hr and water was given ad lib, plasma IR-beta-end and IR-ACTH had parallel circadian rhythms with a peak before feeding and drinking. In the hypothalamus, IR-beta-end and IR-Met-enk showed parallel circadian rhythms with a decrease before these behaviors. When rats were fed from 09:00 to 17:00 hr, the peaks of plasma IR-beta-end and IR-ACTH shifted to one hour before the onset of feeding and drinking. When feeding and watering were restricted to 17:00-09:00 hr and 09:00-12:00 hr respectively, plasma IR-beta-end and IR-ACTH exhibited parallel circadian rhythms with two separate peaks at one hour before drinking and feeding, respectively. In the hypothalamus, IR-beta-end, IR-Met-enk and IR-ACTH showed parallel circadian rhythms with a decrease before feeding but not before drinking. When rats were fed from 17:00 to 20:00 hr, plasma IR-beta-end increased and neurohypophysial IR-beta-end and IR-Met-enk decreased at 16:00 hr, one hour before feeding. It was observed that locomotor activities increased at the time of transition from light to dark and at one hour before the onset of feeding and drinking. The present results suggest that endogenous opioid peptides may have some physiological roles in feeding and drinking behaviors.

Generation of superoxide by immunologically stimulated normal human neutrophils and possible modulation by intracellular and extracellular SOD and rheumatoid factors.

Rheumatoid synovial fluids generated significantly greater amounts of superoxide, lysosomal enzymes, and superoxide dismutase from neutrophils into extracellular fluid than osteoarthritic synovial fluids. Rheumatoid factors isolated from serum suppressed superoxide-generating activity of performed immune complexes, but did not suppress that of intermediate-sized immune complexes isolated from RA serum. Synovial fluid neutrophils has a greater capacity to generate superoxide and lower intracellular superoxide dismutase activity, compared with peripheral neutrophils of the corresponding patients. These results suggest that neutrophil superoxide release may be modulated, both by rheumatoid factor and by intracellular and extracellular superoxide dismutase.

Effects of recombinant human IL-1 beta on production of prostaglandin E2, leukotriene B4, NAG, and superoxide by human synovial cells and chondrocytes.

The effects of recombinant human IL-1 beta on the production of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), N-acetyl-beta-D-glucosaminidase (NAG), and superoxide by synovial cells and chondrocytes derived from osteoarthritis patients were determined. IL-1 beta markedly enhanced PGE2 production in chondrocytes and, to the lesser extent, in synovial cells. Synovial cells and chondrocytes spontaneously released LTB4 into culture medium and IL-1 beta significantly inhibited LTB4 production by these cells. IL-1 beta significantly suppressed the release of NAG and superoxide by synovial cells, whereas it significantly enhanced the production of NAG and superoxide by chondrocytes. Production of intracellular superoxide dismutase by synovial cells was significantly enhanced on incubation with IL-1 beta, but that of chondrocytes was not altered. IL-6, unlike IL-1 beta, significantly suppressed the production of NAG and superoxide by synovial cells and chondrocytes. These results suggest that IL-1 has differing effects on the release of mediators by synovial cells and chondrocytes and that these cells also vary in their responses to IL-1 beta and IL-6.

Relationship between leukotriene B4 and immunological parameters in rheumatoid synovial fluids.

Leukotriene B4 (LTB4) was measured in synovial fluid from 20 patients with rheumatoid arthritis and 15 patients with osteoarthritis. The level of LTB4 was significantly higher in synovial fluid from rheumatoid arthritis patients as compared with synovial fluid from osteoarthritis patients. LTB4 levels also significantly correlated with cell numbers, rheumatoid factor, and immune complexes in synovial fluid from rheumatoid arthritis patients. There was an inverse correlation between LTB4 levels and complement components. The high-pressure liquid chromatography peak of immunoreactivity extracted from the synovial fluid occurred at a retention volume identical to that of authentic LTB4. These results suggest that the increased level of this mediator in synovial fluid may contribute to perpetuation of inflammation and tissue destruction in rheumatoid arthritis.

Human vascular smooth muscle cells and endothelial cells lack catalase activity and are susceptible to hydrogen peroxide.

51Cr release as lytic and cell detachment as nonlytic injury were employed to estimate neutrophil-mediated injury of cultured human vascular smooth muscle cells and endothelial cells. The reagents hydrogen peroxide or hypoxanthine-xanthine oxidase produced dose-dependent killing and nonlytic cell detachment, which were specifically inhibited by catalase but not by superoxide dismutase. The concentration of hydrogen peroxide or xanthine oxidase to induce cell detachment was less than lytic dose, suggesting that cell detachment was a much more sensitive assay of injury. Neutrophil-mediated cell lysis averaged 15% at most and was mostly dependent on hydrogen peroxide, while neutrophil-mediated cell detachment was nearly 100% and its dependency on hydrogen peroxide varied from 46% to 60%. These results suggest that vascular smooth muscle cells and endothelial cells in neutrophil-mediated events are destroyed by a hydrogen peroxide-dependent process, mainly via a nonlytic cell detachment mechanism. There was no striking difference of sensitivity to hydrogen peroxide between vascular smooth muscle cells and endothelial cells. Vascular smooth muscle cells and endothelial cells contained fairly high concentrations of superoxide dismutase, but not catalase, activity. The sensitivity of these cells to hydrogen peroxide but not to superoxide may arise from the fact that these cells lack intracellular catalase activity. The injury of vascular cells, which constitute important components of blood vessels, may lead to vascular injury and subsequent tissue damage.

C1q binding to human vascular smooth muscle cells mediates immune complex deposition and superoxide generation.

Evidence was obtained for the binding of C1q to the membrane of cultured vascular smooth muscle cells derived from human umbilical cord veins. C1q was fixed to the cell membrane at 4 degrees C, whereas it was ingested into the cytoplasm, as a cytoplasmic inclusion, when tested at 37 degrees C. The addition of C1q in advance inhibited the subsequent binding of C1q. Neither fibronectin nor laminin was detected on the cell membrane. Aggregated IgG bound to vascular smooth muscle cells in the case of preincubation with C1q at 4 degrees C, whereas aggregated IgG did not bind to the cells in the absence of C1q. The addition of C1q molecules to the cells in suspension enhanced superoxide generation by vascular smooth muscle cells. There was no effect of C1q on superoxide generation by the cells in monolayer. These results suggest that C1q binds on the membrane of vascular smooth muscle cells via its specific receptor that mediates immune complex binding to the cells and superoxide generation. These properties elucidate the mechanisms by which circulating immune complexes deposit in the vascular wall, and subsequent degradation of tissue components surrounding vascular smooth muscle cells occurs through oxidative burst of the cells.

Role of oxygen radicals and IL-6 in IL-1-dependent cartilage matrix degradation.

It has been suggested that IL-1 produces cartilage matrix degradation by metalloproteinases such as collagenase and that such degradation is regulated by metalloproteinase inhibitors. In the present study, the effects of IL-6 and oxygen radical scavengers on cartilage matrix degradation were studied. Superoxide dismutase, catalase, or methionine all significantly inhibited cartilage matrix degradation both in IL-1 beta-stimulated and unstimulated experimental conditions. Both 10 mM EDTA and 100 nM tissue inhibitor of metalloproteinase (TIMP) significantly inhibited cartilage matrix degradation. The addition of methionine significantly inhibited collagenase activity produced in the culture supernatants of chondrocytes stimulated with IL-1 beta. IL-6 significantly suppressed cartilage matrix degradation produced spontaneously or by IL-1 beta stimulation in chondrocytes. IL-6 inhibited superoxide production by chondrocytes both in IL-1 beta-stimulated or unstimulated conditions. These results suggest that oxygen radicals are involved in cartilage matrix degradation mediated by both paracrine and autocrine IL-1 mechanisms and that oxygen radical-mediated activation of collagenase in chondrocytes may explain the mechanisms of how oxygen radicals are involved in cartilage matrix degradation. IL-6 inhibited superoxide production in chondrocytes and thus inhibited cartilage matrix degradation.

Production of soluble ICAM-1 from human endothelial cells induced by IL-1 beta and TNF-alpha.

The present study was designed to establish the effects of cytokines on soluble ICAM-1 (sICAM-1) production by human endothelial cells (EC) and ICAM-1 expression on these cells and the effects of purified sICAM-1 on lymphocyte-EC adhesion. Expression of ICAM-1 and production of sICAM-1 were measured by a specific ELISA method. ICAM-1 expression was enhanced by IL-1 beta, TNF-alpha, and most effectively by IFN-gamma. IL-4, IL-6, M-CSF, or GM-CSF showed no effects on ICAM-1 expression. IL-4 (100 units/ml) or IL-6(100 units/ml) abolished the enhancing effect of IL-1 beta, while TNF-alpha (1, 10, 100 units/ml) synergized with IL-1 beta to promote ICAM-1 expression in EC. In contrast with the transient increase of cell-associated ICAM-1 expression after activation by IL-1 beta, which peaked 40 h poststimulation and declined thereafter, sICAM-1 continued to accumulate in culture supernatants even after 48 h poststimulation in IL-1 beta-stimulated EC. IL-1 beta treatment resulted in an increase in adhesion. sICAM-1, purified from cell-free supernatants obtained after a 48-h culture of EC in IL-1 beta by affinity chromatography using monoclonal ICAM-1 antibody coupled to Sepharose beads, significantly inhibited lymphocyte EC adhesion. Preincubation of lymphocytes with conditioned medium of EC cultured with 100 units/ml IL-1 beta for 48 h, which contained a considerable amount of sICAM-1, resulted in a significant inhibition of lymphocyte adhesion to IL-1 beta-stimulated EC. These results suggest that there is a cumulative increase in sICAM-1 concentration in the vicinity of cytokine-stimulated EC and that this sICAM-1 modulates ICAM-1-mediated cell to cell interaction.