Leukotriene B4 receptor levels and intracellular calcium signalling in polymorphonuclear leukocytes from patients with Crohn's disease.

OBJECTIVE: To investigate the leukotriene B4 (LTB4) signal transducing mechanism in polymorphonuclear neutrophils (PMNs) from patients with Crohn's disease. METHODS: Cytosolic free calcium ([Ca2+]i), inositol (1,4,5)-trisphosphate [(1,4,5)-IP3] chemotaxis, LTB4 receptor number and affinity were investigated in peripheral PMNs from 11 patients with Crohn's disease and 11 healthy controls. RESULTS: There was a slight reduction (P = 0.31) in the number of LTB4 receptor sites per cell expressed on PMNs (mean Bmax 931) from nine of the 11 patients studied compared with the healthy controls (mean Bmax 1095). LTB4-mediated (1,4,5)-IP3 formation and the increase in [Ca2+]i were markedly decreased in PMNs from the 11 patients with Crohn's disease [(1,4,5)-IP3, mean +/- SEM 12 +/- 0.84 and 27.4 +/- 1.4 pmol/l/tube for patients and controls, respectively; [Ca2+]i, mean +/- SEM 295 +/- 2.75 and 598 +/- 4.7 nmol/l for patients and controls, respectively]. The decrease in calcium might be related to the decrease in Bmax (P < 0.05). Ionomycin, a calcium ionophore which bypasses the initial steps of LTB4 receptor activation, showed only a minor difference in peak [Ca2+]i between PMNs from patients and controls. LTB4-directed chemotaxis showed that the sensitivity to suboptimal concentrations of LTB4 (1.0 nmol/l) was significantly depressed in PMNs from patients (P < 0.05). CONCLUSION: Peripheral PMNs from patients with Crohn's disease had a small deficit in the expression of LTB4 receptors. This deficiency was paralleled by marked alterations in cellular signalling. Whether these results are specific to Crohn's disease or simply result from the exposure of circulating PMNs to elevated levels of LTB4 remains to be established.

Cyclosporine-induced renal morphologic and immunohistologic changes in patients with chronic uveitis.

Ten long-term cyclosporine-treated patients with chronic uveitis underwent percutaneous renal biopsy in order to evaluate a) abnormalities of renal morphology and b) the nature of lymphocytic infiltrates by immunohistochemistry. Pretransplant renal biopsies from eleven cadaveric donors served as controls. Eight of the ten patients had lymphocytic infiltrates consisting predominantly of T lymphocytes with a CD4+/CD8+ ratio of 1.46, which is identical to that of peripheral blood in healthy donors. Evidence of immune activation as estimated by the presence of interleukin-2 receptors, transferrin receptors or by the expression of MHC class II antigens was not demonstrated in any of the patients. The severity of morphologic alterations did not correlate with any of the clinical or paraclinical data. Percutaneous renal biopsy should be performed within 18 months of treatment to identify patients susceptible to renal side effects of cyclosporine.

Arachidonic acid metabolism in neutrophil granulocytes obtained from synovial fluid in rheumatoid arthritis.

Circulating human neutrophil granulocytes (PMNs) from patients with rheumatoid arthritis (RA) have earlier been described to possess an enhanced capacity for production of certain 5-lipoxygenase-derived metabolites of arachidonic acid (AA), 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4). In the present investigation the endogenous AA metabolism of synovial fluid PMNs of RA patients was studied and compared with that of the corresponding circulating PMNs. Synovial fluid PMNs prelabelled with 14C-AA released significantly less radioactivity than circulating PMNs when stimulated with calcium ionophore. Furthermore, synovial fluid PMNs produced significantly smaller amounts of both 5-HETE and LTB4 than circulating PMNs from the same patients, whereas no such difference was observed in the LTB4 catabolites or the cyclo-oxygenase products. More information dealing with the complex way in which arachidonic acid is metabolized in diseased RA joints may provide future rational approaches in the treatment of this chronic inflammatory disease.

Elemental diet: a therapeutic approach in chronic inflammatory bowel disease.

The therapeutic effect of an elemental diet (Pepti 2000) and blended normal diet (placebo) was investigated in 43 out-patients with chronic inflammatory bowel disease (IBD); 24 with ulcerative colitis (UC) and 19 with Crohn's disease (CD), in a mild to moderate state of disease activity. A pilot study on healthy volunteers was executed to investigate palatability of the two diets. The patients were randomized in a double-blind study to the two diet regimes for 14 d. A simultaneous determination of laboratory data including plasma C3c split product and urinary excretion of 51Cr-EDTA was carried out together with a careful registration of the clinical symptoms and signs. No significant effect on the stage of clinical activity was seen in CD. A significant effect on clinical activity was obtained in both UC groups. The clinical improvement was primarily due to a decrease in number of bowel movements both in the elemental diet group and in the group of patients on the blended normal diet. The gross appearance of rectal mucosa did not improve during the study period in the Pepti 2000 or in the placebo group. The concentration of complement split products in plasma remained unchanged. 51Cr-EDTA excretion, as an expression of a leaky bowel mucosa, also remained unchanged. It was concluded that an effect on inflammation could not be demonstrated even if both diets seem to have a beneficial effect on the stage of clinical activity, especially diarrhoea, in patients with UC.

Inhibition of human neutrophils by auranofin: chemotaxis and metabolism of arachidonate via the 5-lipoxygenase pathway.

The effect of auranofin on human neutrophil (PMN) 5-lipoxygenase activity and leucotriene B4 (LTB4) chemotaxis was investigated. [1-14C]Arachidonic acid was incorporated into the purified cells until steady state conditions were obtained. After preincubations with serial dilutions of auranofin arachidonic acid release and metabolism were stimulated with calcium ionophore A23187. The radioactive eicosanoids released were extracted and separated by thin layer chromatography, followed by autoradiography and quantitative laser densitometry. Chemotaxis of PMNs towards LTB4 was measured in a modified Boyden chamber. Auranofin showed dose dependent inhibition of both the 5-lipoxygenase pathway (IC50 17.4 X 10(-6) mol/l) and of chemotaxis (IC50 45 X 10(-6) mol/l). The release of arachidonic acid from phospholipids was unaffected in the concentration range tested (1-1000 mumol/l). Inhibition of both neutrophil motility and cellular synthesis of proinflammatory eicosanoids may thus contribute to the beneficial clinical effects of auranofin in rheumatoid arthritis.

Inhibition of intestinal macrophage chemotaxis to leukotriene B4 by sulphasalazine, olsalazine, and 5-aminosalicylic acid.

Purified intestinal macrophages obtained at resections for colonic neoplasms were investigated for chemotaxis to leukotriene B4 (LTB4) by the Millipore filter assay and leading front technique. Possible inhibition by drugs effective in the treatment of chronic inflammatory bowel disease (sulphasalazine, olsalazine, its active moiety 5-aminosalicylic acid (5-ASA), and the 5-ASA metabolite N-acetylated-5-ASA (ac-5-ASA)) was tested at therapeutic colonic concentrations of 0.01-10 mM. Leukotriene B4 at a dose of 10 nM was equipotent with casein (5 g litre-1) as regards chemoattraction of macrophages. Sulphasalazine, olsalazine and 5-ASA were potent inhibitors of macrophages chemotaxis to LTB4 with IC50 values of 0.43, 0.39 and 0.24 mM, respectively. These concentrations are below the lowest concentration of 5-ASA (2 mM) in the colonic lumen during conventional sulphasalazine treatment of patients with chronic inflammatory bowel disease. The inhibition of macrophage chemotaxis by these drugs may be important for this limitation of the local inflammatory process in chronic inflammatory bowel disease, and may in part explain the beneficial effect of systemic and local treatment with sulphasalazine. Leukotriene B4 appears to be an important inflammatory mediator for the activation of macrophages in colonic inflammation.

A comparison of the effect of timegadine, levamisole, and D-penicillamine on human neutrophil metabolism of endogenous arachidonic acid and chemotaxis.

The effect of timegadine, a novel experimental antirheumatic drug, on human neutrophil (PMN) 5-lipoxygenase activity and leukotriene B4 (LTB4) chemotaxis was compared with that of two second-line antiinflammatory drugs, D-penicillamine and levamisole. 1-14C-Arachidonic acid (AA) was incorporated into the purified cells until steady state conditions were obtained. After preincubation with serial dilutions of the three drugs, AA release and metabolism was stimulated with calcium ionophore A23187. The radioactive eicosanoids released were extracted and separated by thin-layer chromatography, followed by autoradiography and quantitative laser densitometry. Chemotaxis of PMNs towards LTB4 was measured in a modified Boyden chamber. Timegadine showed dose-dependent inhibition of both the 5-lipoxygenase pathway (IC50 3.4 x 10(-5) M), and of chemotaxis (IC50 3 x 10(-4) M). Inhibition of the release of AA from phospholipids, however, occurred only at therapeutically irrelevant doses (millimolar concentrations). Levamisole and D-penicillamine did not inhibit any of the cell functions investigated. Inhibition of both neutrophil motility and cellular synthesis of pro-inflammatory eicosanoids, may thus contribute to the clinical effects of timegadine in rheumatoid arthritis.

Serum interferon activity in inflammatory bowel disease. Arachidonic acid release and lipoxygenation activated by alpha-class interferon in human neutrophils.

Serum interferon (IFN) of alpha-class was studied in 64 consecutive patients, 26 with Crohn's disease, 38 with ulcerative colitis, and in 34 healthy volunteers. Detectable IFN-alpha in 10 patients was associated with a moderate to severe activity of chronic inflammatory bowel disease (CIBD). However, 19 of 28 patients (68%) with activity in their disease did not have elevated IFN-alpha levels. The three groups, ulcerative colitis, Crohn's disease, and healthy volunteers did not reveal any statistically significant difference in serum IFN-alpha, as four of 34 healthy controls without intercurrent infections had elevated levels as well. Possible effects of alpha, beta, and gamma classes of IFN on endogenous arachidonic acid (AA) release and metabolism in human neutrophils was investigated in a substudy. IFN-alpha caused a dose-dependent release of AA from phospholipids and metabolism of a modest fraction of leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) at doses reaching a maximum of 100 IU/ml. IFN of the beta and gamma classes did not exert such effects. Addition of complement 5a to cells activated by IFN-alpha caused induction of increased 5-lipoxygenase activity with unchanged release of AA. As only 16% of all CIBD patients had elevated IFN-alpha levels as compared to 12% among the group of healthy volunteers, IFN-alpha does not seem to be of importance for the perpetuation of the inflammatory reaction in ulcerative colitis or Crohn's disease, and other factors may therefore be responsible for activation of the inflammatory cells to production of LTB4 and 5-HETE.

Activation of classical pathway complement in chronic inflammation. Elevated levels of circulating C3d and C4d split products in rheumatoid arthritis and Crohn's disease.

Split products of complement component 3 (C3) and complement component 4 (C4) derived from activation of the alternative and classical complement pathways were measured in untreated outpatients, 20 with Crohn's disease and 19 with rheumatoid arthritis. Elevated levels of the d split product of C4 (C4d) were observed in 12 of 19 patients with rheumatoid arthritis and in 9 of 20 patients with Crohn's disease. Levels of the d split product of C3 (C3d) were increased in 14 of 19 patients with rheumatoid arthritis and in 6 of 20 Crohn's disease patients. The median values of C4d and C3d were significantly increased in both groups of patients. C3d concentrations correlated positively with C4d levels (rs = 0.51-0.56, p less than 0.005). The complement activation was not reflected in reduced plasma levels of native C3 and C4. The data indicate activation of the classical complement pathway in both rheumatoid arthritis and Crohn's disease.

Activation of neutrophil chemotaxis by leukotriene B4 and 5-hydroxyeicosatetraenoic acid in chronic inflammatory bowel disease.

Circulating neutrophils were investigated in 15 patients with Crohn's disease (CD), 15 with ulcerative colitis (UC), and 15 healthy volunteers. Dose-response curves for chemotaxis in Boyden chambers were analysed for sensitivity to leukotriene B4 (LTB4), its 20-hydroxy-LTB4 (20-OH-LTB4) and 20-carboxy-LTB4 (20-COOH-LTB4) catabolites, and 5- and 15-hydroxyeicosatetraenoic acids (HETEs). Positive controls included: complement 5a (C5a), formy-L-methionyl-L-leucyl-L-phenylalanine (f-Met-Leu-Phe), and casein. Control chemotaxis test were performed at concentrations yielding optimal responses in leucocytes of healthy volunteers. Chemotaxis to suboptimal concentrations of LTB4 1.0 and 3.2 nmol/l, and 5-HETE 316 nmol/l, was markedly depressed in patients with chronic inflammatory bowel disease (CIBD). Analyses of individual dose-response curves revealed an underlying decreased sensitivity to LTB4 in 11 out of 30 patients, to 5-HETE in 10 out of 30 patients with a corresponding decrease of median sensitivity to LTB4 and 5-HETE in both CD and UC. Peak responses to LTB4, 5-HETE, f-Met-Leu-Phe, and casein were identical in the three groups tested, whereas the C5a values were significantly depressed in both groups of patients (p less than 0.05). The potency of LTB4 exceeded that of 5-HETE by a factor of approximately 100 whereas 20-OH-LTB4 was nearly as potent as LTB4. 20-COOH-LTB4 and 15-HETE did not activate chemotaxis of human neutrophils. These findings are suggestive of a competitive inhibition of receptors with heterogeneity for LTB4 and 5-HETE.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced capacity for release of leucotriene B4 by neutrophils in rheumatoid arthritis.

The calcium dependent metabolism of endogenous arachidonic acid (AA) was investigated in 17 patients with rheumatoid arthritis during treatment with dextropropoxyphene alone and in 25 healthy volunteers. Incorporation of [1-14C]AA into intracellular phospholipids of purified neutrophils was achieved by incubation until steady state before activation with ionophore A23187. Analysis of extracellular metabolites was performed by extraction, thin layer chromatography, autoradiography, and laser densitometry. The patients showed a twofold increase in the total capacity for oxidation of AA. Release of leucotriene B4 (LTB4) and its omega oxidation products, 20-OH LTB4 and 20-COOH LTB4, was 29%, range 11-48%, in patients compared with 8%, range 4-12%, in healthy volunteers. Total amounts of radioactivity released and the specific activity of LTB4, as assessed by high pressure liquid chromatography, were equal in experimental and control groups. The demonstrated increased capacity for metabolism of AA to the major proinflammatory metabolite, LTB4, via the 5-lipoxygenase pathway may contribute to perpetuation of inflammation and to tissue destruction in rheumatoid arthritis.

Inhibition of 5-lipoxygenase pathway of arachidonic acid metabolism in human neutrophils by sulfasalazine and 5-aminosalicylic acid.

The possible effect of sulfasalazine, 5-aminosalicylic acid, and acetyl-5-aminosalicylic acid on endogenous arachidonic acid release and metabolism was studied in human polymorphonuclear leukocytes (PMNs). A new in vitro assay was used by which [1-14C]arachidonic acid is incorporated by purified peripheral PMNs until steady state was obtained (5 hr). After preincubation with the test drugs prior to activation with calcium ionophore A23187, the released eicosanoids were isolated by extraction and thin-layer chromatography (TLC) and quantitated by autoradiography and laser densitometry. Median drug concentrations needed for 50% inhibition of leukotriene B4 and 5-hydroxyeicosatetraenoic acid (5-HETE) release was 4-5 mM (range 1-9 mM) for both sulfasalazine and 5-aminosalicylic acid. The acetylated derivative of 5-aminosalicylic acid was ineffective. The present data suggest that inhibition of arachidonic acid lipoxygenation may be an essential action of sulfasalazine and its active metabolite, 5-aminosalicylic acid. Interference with lipoxygenase enzymes, rather than a steroid-like inhibition of arachidonic acid release from intracellular phospholipids, seems to be the mode of action.

Abnormal metabolism of arachidonic acid in chronic inflammatory bowel disease: enhanced release of leucotriene B4 from activated neutrophils.

The metabolism of endogenous arachidonic acid P(AA) was investigated in activated neutrophils from 20 patients with Crohn's disease, 20 with ulcerative colitis, and 25 healthy volunteers. 1-14C-P(AA) was incorporated into intracellular pools of phospholipids prior to activation of the cells with ionophore A23187 and analyses of released arachidonic acid metabolites by thin layer chromatography. Total release of radioactivity expressing the release of arachidonic acid and its metabolites, was equal in the experimental and control groups, which suggests a normal substrate availability. In contrast, there was a marked increase in the relative release of leucotriene B4 (LTB4) and its omega-oxidation products, 20-hydroxy-LTB4 (20-OH-LTB4) and 20-carboxy-LTB4 (20-COOH-LTB4), with LTB4 values exceeding the reference interval in seven of 20 patients with Crohn's disease, median 8.7%, and in six of 20 patients with ulcerative colitis, median 7.7%, as compared with a median of 5.3% in healthy volunteers. Furthermore, a decreased release of unmetabolised arachidonic acid, correlating inversely with the release of LTB4 in all experimental and control groups, and normal values for the production of other metabolites of arachidonic acid--for example, 5-hydroxyeicosatetraenoic acid (5-HETE) and 12-hydroxyheptadecatrienoic acid (HHT), point to an enzymatic abnormality such as increased activity of leucotriene B synthetase. An increased capacity for release of LTB4, the major pro-inflammatory metabolite of arachidonic acid lipoxygenation by polymorphonuclear leucocytes, may contribute to perpetuation of the inflammation and to tissue destruction in chronic inflammatory bowel disease. Our findings agree with previous reports of an increased release of LTB4 by the colonic mucosa in this condition.

Impaired activation of the neutrophil oxidative metabolism in chronic inflammatory bowel disease.

The oxidative metabolism of circulating neutrophils was studied in patients with Crohn's disease and ulcerative colitis. The production of superoxide anion (02-.) in patients with ulcerative colitis was markedly decreased irrespective of whether soluble or particulate, non-chemotactic or chemotactic stimuli were used. Crohn's disease neutrophils showed a just marginally diminished 02-. production. Disease activity, as defined by the Crohn's disease activity index, was negatively correlated with the neutrophil O2-. production in both diseases. In both Crohn's disease and in ulcerative colitis neutrophil hydrogen peroxide (H2O2) production was normal. It is concluded that the neutrophil function, as-assessed by superoxide anion production, is impaired in patients with inflammatory bowel disease. It is therefore suggested that the suboptimal microbicidal function of these cells, as demonstrated in the present study, may contribute to the disease process.

Release of leukotriene B4 and 5-hydroxyeicosatetraenoic acid during phagocytosis of artificial immune complexes by peripheral neutrophils in chronic inflammatory bowel disease.

The capacity of peripheral neutrophils for activation of the arachidonic acid (AA) metabolism was studied during phagocytosis of IgG containing immune complexes (ICs) binding to Fc-receptors. Release of approximately 9% of the intracellular pool of radiolabelled AA in phospholipids, and release of the pro-inflammatory mediators, leukotriene B4 (LTB4), constituting 1.8%, and 5-hydroxyeicosatetraenoic acid (5-HETE), constituting 2.9% of the total radioactivity released, were demonstrated in 15 patients with untreated Crohn's disease, 15 patients with ulcerative colitis, and in 15 healthy volunteers. The concentrations of LTB4 and 5-HETE released were within the range of chemotactic activity for the two lipoxygenase products. Multiple large IgG containing ICs were revealed in neutrophils after phagocytosis by immunofluorescence. A minor defect in the IC uptake in patients with Crohn's disease observed in the absence of complement only, did not result in a subnormal activation of arachidonic acid release or metabolism. The study suggests that complexes of the IgG-class previously demonstrated in chronic inflammatory bowel disease, particularly in Crohn's disease, may activate inflammatory neutrophils leading to release of significant amounts of the pro-inflammatory lipoxygenase metabolites, LTB4 and 5-HETE.

Arachidonic acid metabolism in human neutrophils: lack of effect of cyclosporine A.

The metabolism of arachidonic acid (AA) in human neutrophils was studied by incorporation of 1-14C-AA, removal of excess 1-14C-AA, and stimulation of radiolabelled cells with A23187. Radiolabelled lipids were quantitated by extraction, thin-layer chromatography, autoradiography, and laser densitometry. Following 5 h of incubation with 1-14C-AA, the maximum amount of radioactivity was located in triglycerides, 70%, and phospholipids, 30%. Activation of the cells with calcium ionophore A23187 led to release of free AA, 58%, whereas AA-metabolites revealed mainly lipoxygenase (arachidonate 5 lipoxygenase, E.C. 1. 13. 11. 34) activity, 5-HETE 13%, LTB4 5%, with only small amounts of cyclooxygenase (prostaglandin synthase, E.C. 1. 14. 99. 1) metabolites, HHT 2%. Intra-assay coefficient of variation for release of metabolites was approximately 15%. A potent immunosuppressive agent, cyclosporine A (CS-A) was shown to be without any effect in AA-release and metabolism. This method is applicable to studies of both basic cell function in human disease and to further immunopharmacological investigations.

Progressive muscular dystrophy in Denmark. Natural history, prevalence and incidence.

Prevalence of progressive muscular dystrophy in 1965 and incidence in Denmark for the period 1965 to 1975 was studied by collection of data from hospital departments, nursery homes and general practitioners. This material was supplemented with information from death certificates and disablement pension records. Total patient group included 445 patients with progressive muscular dystrophy alive January 1st 1965. Prevalence was 69.4 per 10(6) male inhabitants in the Duchenne type, whereas the prevalences in the limb-girdle and facioscapulohumeral types were 36.5 and 18.6 per 10(6) inhabitants, respectively. Incidence was 222 per 10(6) male newborns in the Duchenne type, 66 and 26 per 10(6) newborns for the limb-girdle and the facioscapulohumeral types, respectively. Figures for the Duchenne type are in agreement with previous results. Both prevalence and incidence rates for the limb-girdle and the facioscapulohumeral types exceed published figures by a factor 3 to 6. These high Danish rates seem to reflect the true prevalence and incidence in the less serious types of progressive muscular dystrophy, probably because the Danish health system with free medical care and easy access to specialized hospital departments makes it possible to identify all cases of progressive muscular dystrophy.