Jens Elmgreen

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 newin 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.

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.

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 Crohns 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 Crohns 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.

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.

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

Circulating neutrophils were investigated in 15 patients with Crohns 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)

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.

Defective release of C5a related chemo-attractant activity from complement in Crohn's disease.

Complement was studied in 20 untreated cases of Crohns disease and in 20 healthy volunteers by an in vitro activation of the cascade reaction. Total haemolytic complement was normal in all patients. In contrast, activation of the alternative pathway lead to a decreased release of C5a related chemo-attractant activity together with a subnormal utilisation of the main complement component C3. This abnormality of complement function was not related to the activity of the disease, site of involvement or to disease duration. The results suggest that an inadequate stimulation of important neutrophil functions may result when bacterial lipopolysaccharides and other macromolecules activating the alternative pathway penetrate the gut mucosa. A delayed clearance from the tissue of such foreign material could be a further pathogenic factor in Crohns disease leading to granulomatous inflammation by a foreign body reaction.

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.

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

Serum interferon (IFN) ofα-class was studied in 64 consecutive patients, 26 with Crohns disease, 38 with ulcerative colitis, and in 34 healthy volunteers. Detectable IFN-α 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-α levels. The three groups, ulcerative colitis, Crohns disease, and healthy volunteers did not reveal any statistically significant difference in serum IFN-α, as four of 34 healthy controls without intercurrent infections had elevated levels as well. Possible effects ofα, β, andγ classes of IFN on endogenous arachidonic acid (AA) release and metabolism in human neutrophils was investigated in a substudy. IFN-α 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 theβ andγ classes did not exert such effects. Addition of complement 5a to cells activated by IFN-α caused induction of increased 5-li-poxygenase activity with unchanged release of AA. As only 16% of all CIBD patients had elevated IFN-α levels as compared to 12% among the group of healthy volunteers, IFN-α does not seem to be of importance for the perpetuation of the inflammatory reaction in ulcerative colitis or Crohns disease, and other factors may therefore be responsible for activation of the inflammatory cells to production of LTB4 and 5-HETE.

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.