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Fatty Acids (1990) 39,227-229

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Induction of Endogenous Arachidonic Acid Metabolism in Human Neutrophils with Snake Venom Phospholipase AZ, Immune Complexes, and A23187 E. Langholz and 0. H. Nielsen* Depurtment of Medical Gastroenterology C, Herlev Hospital, University of Copenhagen, and *Department of Medicine C, Glostrup Hospital, University of Copenhagen, Ndr. Ringvej, DK-2600 Golstrup, Denmark

The stimuli responsible for eicosanoid secretion of phagocytes in chronic inflammatory disorders like rheumatoid arthritis and chronic inflammatory bowel disease are unknown. Phospholipase AZ (PLA3, found in Russelli vipera snake venom, has been proposed to be more than 100 times more potent on a molar basis than A23187 in releasing leukotriene B4 (LTB4) from porcine neutrophils. Therefore, this enzyme was investigated as a challenger of human neutrophils (PMNs) and compared with immune complexes and A23187. l-14C-Arachidonic acid (AA) was incorporated into purified human PMNs until steady state conditions were obtained. AA release and metabolism were stimulated with either PLAz isoenzyme of Russelli vipera, immune complexes, or A23187. The radioactive eicosanoids released were extracted and separated by thin layer chromatography, followed by autoradiography and quantitative laser densitometry. Stimulation with PLAz, immune complexes, or A23187 resulted in LT& formation of 0%, 1.8%, and 5.3%, respectively, of total released radioactivity. In conclusion, Russelli vipera PLAz does not stimulate AA-release and metabolism in human PMNs, and immune complexes are weak as compared to the unphysiologic challenger A23187 in this respect.

ABSTRACT.

INTRODUCTION

metabolism in human PMNs which possibly could be used as potent physiological challengers in future experimental models for leukotriene research. Recently this PLA2 isoenzyme has been described to be a useful probe for the study of AA-metabolism in porcine PMNs (4).

Neutrophil granulocytes (PMNs) are important modulators of inflammatory processes (1). When activated, these cells may release and metabolize arachidonic acid (AA) from their phospholipid membrane structures (2). Further, these cells may secrete various enzymes and produce oxygen free radicals (3). The physiological stimuli evoking an activation of these cells, as seen in various autoimmune disorders, are unknown, but bacterial derived peptides, phagocytosis, interferons, and kinins have been implicated (1). The aim of this study was to compare the activatof the Russelli vipera ing effect venom phospholipase AZ (PLA_3 isoenzyme with immune complexes and calcium ionophore A23187 on AA-

MATERIALS AND METHODS Blood from ten healthy volunteers was drawn in EDTA (10 mM). None of these persons had received any drug treatment within the last four weeks apart from peroral anticonception. All patients gave informed consent before the blood samples were drawn, and approval was given by the Scientific Ethical Committee of the Copenhagen County. The PMNs were isolated from peripheral blood by a modification of Boyurn’s method (5) resulting in a purity of more than 95%, and a recovery of 45%. In short, this method was: 1) methylcellulose

‘Address correspondence to Ole Haagen Nielsen 227

228

Prostaglandins Leukotrienes and Essential Fatty Acids

sedimentation of erythrocytes; 2) gradient centrifugation of ‘buffy coat’ leukocytes; and 3) hypotonic lysis of residual red cells. Finally these cells were resuspended in Gey’s solution (5 x 106 PMNs/ml) with albumin (2%), glucose (0.2%), streptomycin (50 pg/ml), and penicillin (200 IU/ml) (7). PLAzisoenzyme (10 -4 - 10m8M) (Russelli vipera venom) was obtained from Sigma Chemicals, St. Louis, MO, USA, A23187 (10e5M) (Calbiochem, La Jolla, CA, USA), and immune complexes consisting of purified tetanus toxoid with human anti-tetanus IgG without complement, at two times antibody excess (5 pg/ml) (Dr. U. E. Nydegger, WHO, Geneva, Switzerland). AA metabolism in purified PMNs was investigated by incorporation of l-14C-AA (37 x lo3 Bq/5x106 cells, 2.2 x lo9 Bq/mmol) (Amersham International, Buckinghamshire, UK) for 5 hours at 37 “C to obtain a steady-state for labelling of the intracellular pools of AA (7). After removal of excess extracellular AA by washing, the cells were challenged with either PLAz, immune complexes, or A23187 for 0 - 30 min. Extracellular fluid containing radiolabelled metabolites was prepared by instantaneous removal of the cells by centrifugation (8000 g, 1 minute) through dibutyl phthalate: dinonyl phthalate, 3:1 (density 1.033 g/ml) before extraction of eicosanoids (7,‘8). The radioactive metabolites were then separated by thin-layer chromatography and measured by autoradiography and laser densitometry as previously described (7). Fractions more lipophilic than arachidonic acid were not included in these calculations. Identification of AA and metabolites was carried out with co-chromatography using pure

% LTB, 7

t

6

I

standards (Paesel GMBH, Frankfurt am Main, FRG), and evaluation of specific activities was performed by high pressure liquid chromatography (7). The coefficient of variation of duplicate determinations of LTB4 and 5-hydroxyeicosatetraenoic acid (5-HETE) was less than 0.16 (t statistics). Phagocytosis of immune complexes for 15 minutes - a time period found in a preliminary series of experiments - was evaluated by an immunofluorescence technique as the end point. Immune complexes as described above were labelled with fluorescine isothiocyanate (green stain) or rhodamine (red stain) conjugated rabbit IgG specific for human IgG. The cell specimens were investigated with a Leitz Ortoplan fluorescence microscope (Leitz, Wetzlar, FRG) equipped with incident light illumination (9), and 400 PMNs were counted from each person.

RESULTS The median total radioactivity released, i.e. AA and metabolites, was 680 Bq/5 x lo6 cells with A23187 (range 270-890), 315 Ba/5 x lo6 cells with immune complexes (range 260-370), whereas PLA2 did not increase the AA-release and metabolism over unstimulated cells (150 Bq/ 5x 106 cells (range 90-230)). Stimulation with A23187, immune complexes and PLA:! resulted in a median maximal formation of 5.4% (range 2.6-9.3), 1.8% (range 0.7-5.1) and 0% (range 0.0-0.1) LTB4, respectively, and of 15.1% (range 9.3-20.1), 3.3% (range 2.0-6.8), and 0.1% (range 0.0-0.3) 5-HETE, respectively of total released radioactivity in the time range investigated (Figure). Assessed by immunofluorescence a median of 96% (range 93-98%) of PMNs were IgG positively staining indicating a phagocytic uptake of immune complexes. The median specific activity of LTB4 was equal in the groups of cells stimulated with immune complexes and A23187 (6.2 x lo8 Bq/mmol, range 4.8-7.4).

DISCUSSION PLAz and immune

z PLAz_isoenzyme 1 0

Figure

5

10

20

30 Time (min)

Release of leukotriene B, (LTBJ from human neutrophils in per cent of total released radioactivity and length of stimulation (min). Challengers: PLArisoenzyme. immune complexes, or A23187. Detection limit: 0.2%. Median values with Q, ranges are given (n = 10).

complexes were compared to A23187 in the present study. PLA2 from Russelli vipera venom has recently been described to be lOOfold more potent than A23187 on a molar basis in releasing LTB4 from porcine neutrophils (4), and immune complexes may cause aggregation and degranulation of PMNs (2). Further, local excessive amounts of IgG immune complexes have been described in chronic inflammatory conditions like

Induction of Endogenous

rheumatoid arthritis and chronic inflammatory bowel disease (lo), and phagocytosis of such immune complexes as seen in the present investigation may possibly perpetuate the inflammatory reaction (II). PLATisoenzymes released from macrophages and other leukocytes during cell activation and immune complexes are present at sites of inflammation. We were unable to confirm the report by Lam et al (4) of an eicosanoid formation activating action of snake venom PLAz apart from an extremely weak stimulation of 5-HETE production. Species differences between porcine and human PMNs may explain this discrepancy. Furthermore, the challenge with immune complexes of the IgG class was weak as compared to A23187. However, phagocytosis of immune complexes of IgG class may thus lead to secretion of important eicosanoid metabolites like LTB4 and 5-HETE. If validated on the basis of LTB4 and 5-HETE release, immune complexes were respectively approximately 10 and 15 times less potent than A23187. Other, so far unknown, mediators may therefore be of relevance in inflammatory diseases characterized by an excessive eicosanoid release, e.g. rheumatoid arthritis and chronic inflammatory bowel disease (1, 12). The possibility exists that PLAZ-isoenzymes of a different origin than snake venom and immune complexes may take part in a general stimulus-secretion coupling mechanism which, in combination with other stimuli, may perpetuate and amplify the complex inflammatory process. Acknowledgements The authors wish to thank Helma Furhauge for skilfull technical assistance. This paper was supported by grants from handelsgartner Ove Villiam Buhl Olesen’s and aegtefiel-le Edith Buhl Olesen’s Foundation, direckw Jacob Madsen’s and Hustru Olga Madsen’s Foundation, and the Danish Medical Research Council.

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in Human Neutrophils 229

References 1. Nielsen 0 H. In vitro studies on the significance of arachidonate metabolism and other oxidative processes in the inflammatory response of human neutrophils and macrophages with special reference to chronic inflammatory bowel disease, Stand J Gastroenterol 23 (suppl. 150): 1-21, 1988. 2. Weissmann G, Smolen J E, Korchak H M. Release of inflammatory mediators from stimulated neutroohils. N Enel J Med 303: 27-34. 1980. 3. Weiss ‘S J. Tissui destruction by neutrophils. N Engl J Med 320: 365-76, I989. 4. Lam B K, Lee C-Y and Wong P Y-K. Phospholipase AZ releases lipoxins and leukotrienes from endogenous sources. Ann N Y Acad Sci 524: 27-34: 1988. 5. BGyum A. Isolation of leukocytes from human blood. Stand J Clin Lab Invest 21 (suuol. \ 97): 9-29, 1968. 6. Nielsen 0 H. Bukhave K, Elmgreen J and Ahnfelt-R6nne J. Inhibition of 5-lipoxygenase pathway of arachidonic acid metabolism in human neutrophils by sulfasalazine, and 5-aminosalicvlic acid. Dig Dis- Sci 32: 577-82, 1987. 7. Nielsen 0 H. Bukhave K. Ahnfelt-R@nne I. Elmgreen J. Arachidonic’acid metabbrism in human neutrophils: lack of effect of cyclosporin A. Int J Immunopharmacol 8: 419-26, i986.. 8. Folch J. Lees M. Sloane-Stanlev G H. A simole method for the isolation and pirification of total lipids from animal tissues. J Biol Chem 226: 497-509,1957. 9. Wiik A, Jensen E, Friis J. Granulocyte-specific antinuclear factors in synovial fluids and sera from patients with rheumatoid arthritis. Ann Rheum Dis 33: 515-22,1974. 10. Baklien K. Brandtzaeg P. Immunohistochemical characterization of local immunoglobulin formation in Crohn’s disease of the ileum. Stand J Gastroenterol 11: 447-57, 1976. 11. Mall J M H. Iriflammatory bowel disease. Clin Rheum Dis 11: 87-111, 1985. 12. Weissmann G. Pathogenesis of inflammation: effects of the pharmacological manipulation of arachidonic acid metabolism of the cytological response to inflammatory stimuli. Drugs 33 (suppl. 1): 28-37, 1987. II

Induction of endogenous arachidonic acid metabolism in human neutrophils with snake venom phospholipase A2, immune complexes, and A23187.

The stimuli responsible for eicosanoid secretion of phagocytes in chronic inflammatory disorders like rheumatoid arthritis and chronic inflammatory bo...
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