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Effect of imidazole salicylate on the respiratory burst of polymorphonuclear leukocytes
CURRENT THERAPEUTIC RESEARCH VOL. 54, NO. 2, AUGUST1993
EFFECT OF IMIDAZOLE SALICYLATE ON THE RESPIRATORY BURST OF POLYMORPHONUCLEAR LEUKOCYTES A. KANTAR,1 N. OGGIANO, 1 R. GABBIANELLI, 1 P. L. GIORGI, 1 AND M. BIRAGHI2 IPediatric Clinic, University of Ancona, Ancona, and 2Medical Department, VALEAS, Milan, Italy
ABSTRACT The effect of imidazole salicylate (IS), a n organic salt of imidazole a n d salicylic acid, on the respiratory burst of p o l y m o r p h o n u c l e a r leukocytes (PMNs) was investigated using a c h e m i l u m i n e s c e n c e (CL) assay. L u m i n o l - a n d lucigenin-amplified CL of PMNs s t i m u l a t e d with N - f o r m y l - m e t h i o n y l - l e u c y l - p h e n y l a l a n i n e ( F M L P ) or phorbol m y r i s t a t e acetate (PMA) were evaluated in the absence or presence of 1 to 4 ~tg/ml of IS. L u m i n o l - a m p l i f i e d CL of PMNs activated with F M L P was s i g n i f i c a n t l y reduced in the presence of IS, whereas no s i g n i f i c a n t effect was observed on P M A - s t i m u l a t e d PMNs. L u c i g e n i n amplified CL of PMA-stimulated PMNs was s i g n i f i c a n t l y inhibited by IS. CL of the x a n t h i n e / x a n t h i n e oxidase system was abolished in the presence of IS. These results suggest t h a t IS is a scavenger of the superoxide a n i o n a n d that it can influence the respiratory burst of PMNs, probably by i n t e r a c t i n g with the p l a s m a m e m b r a n e . INTRODUCTION
There is little argument that the products of activated polymorphonuclear leukocytes (PMNs) damage tissue. 1 This is especially true in the case of inflammatory reactions that feature an infiltration of PMNs. Activated PMNs injure tissue by releasing products stored in granules and generating new factors, including oxygen species. The latter process, known as the respiratory burst, is initiated when, after a suitable stimulus, molecular oxygen undergoes a one-electron reduction to superoxide anion at the expense of nicotinamide adenine dinucleotide phosphate (NADPH). 2 This reaction is mediated by a multicomponent enzyme system, known as respiratory burst oxidase. 3 In studies of the inflammatory injury of tissue, oxidants, which are generated by stimulated PMNs, appear to participate significantly in the attack on cells and supporting tissue. 4 Nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibiting the synthesis and release of prostaglandinsS'6; however, this may not account for all the effects of NSAIDs at all Address correspondence to: Dr. A. Kantar, Pediatric Clinic, University of Ancona, via Corridoni 11, 1-60123 Ancona,Italy. Received for publication on June 1, 1993. Printed in the U.S.A. Reproduction in whole or part is not permitted. 241
0011-393X/93/$3.50
IMIDAZOLESALICYLATEAND PMNs
doses. Alternatively, NSAIDs m a y modulate the response of PMNs to various stimuli. 7,s Imidazole salicylate* (IS) is an organic NSAID consisting of salt of imidazole and salicylic acid. In this study, we investigated the effect of IS on the oxidative metabolism of PMNs using luminol- and lucigeninamplified chemiluminescence (CL) assays. The ability of IS to act as a radical scavenger was investigated by using CL in the xanthine/xanthine oxidase system. M A T E R I A L S AND M E T H O D S
PMNs were isolated using Mono-Poly Resolving Medium (ICN, Biomedicals, Milan, Italy) as previously described, 9 and the cells were resuspended in a Krebs-Ringer phosphate solution containing 1 mg/ml of glucose, A reaction mixture of 106 cells and 10 btmol/L of luminol (Sigma Chemical Co., St. Louis, Missouri) in 1 ml of Krebs-Ringer phosphate solution was prepared with or without 1, 2, 3, or 4 ~g/ml of IS. The CL was measured immediately in an AutoLumat LB 953 (Berthold Co., Wilbad, Germany) after activation with 10-7 mol/L of N-formyl-methionyl-leucylphenylalanine (FMLP) (Sigma Chemical Co.) or 3 × 10 -4 mol/L phorbol myristate acetate (PMA) (Sigma Chemical Co.) as previously described. 1°'11 A reaction mixture of 106 cells and 150 ~mol/L of lucigenin (Sigma Chemical Co.) in 1 ml of Krebs-Ringer phosphate solution was prepared with or without IS using the same concentration as used for the luminolamplified CL assay. PMNs were later activated with PMA, and the CL was observed for 60 minutes as previously described. 11'12 A 1-ml reaction mixture containing 0.9 U of xanthine oxidase and 150 ~mol/L of lucigenin in Krebs-Ringer phosphate solution plus glucose (pH 7.4) in the absence or presence of 1, 2, 3, or 4 ~tg/ml of IS was prepared. The reaction was initiated by injecting 0.05 ml ofxanthine (final concentration, 50 ~Lmol/L). The CL measurement was observed for 40 seconds. The significance of the values obtained was calculated using Student's t test. RESULTS
When F M L P was added to PMNs, a bimodal CL response was obtained. The first peak appeared approximately 1 minute after injection of FMLP, and the second peak occurred after 3 to 5 minutes (Figure 1). When the cells were exposed to FMLP in the presence of IS, a dose-dependent inhibition of both peaks was obtained (Table I). When PMNs were activated with PMA, a peak CL value was obtained 15 to 17 minutes after stimulation. No significant inhibition was observed in the presence of IS. In lucigenin-amplified CL of PMNs activated with PMA, a peak value was obtained 18 to 20 minutes after stimulation. A dose-dependent inhi* Trademark: Flogozen ® (VALEAS S.p.A., Milan, Italy). 242
A. K A N T A R E T AL.
3.0 c p m x 10 7
2.5
, 2.0
i
1.5
1.0
0.5
0.0
o.oo
2. o
6. o
4.oo
I
8.()0
10.00
min
Figure 1. Time course of luminol-amplified chemiluminescence of polymorphonuclear leukocytes stimulated with N-formyl-methionyl-leucyl-phenylalanine. Chemiluminescence was measured as counts per minute (cpm).
bition of CL by IS was observed (Table II). CL produced by superoxide generated from the xanthine/xanthine oxidase reaction was completely abolished in the presence of IS (Figure 2). DISCUSSION
In response to certain stimuli, PMNs undergo an oxidative burst during which a series Of reactive oxygen metabolites are generated. 2 This process, Table I. Extracellular (first peak) and intracellular (second peak) luminol-amplified chemiluminescence (CL) obtained from polymorphonuclear leukocytes after activation with 10 -7 mol/L of N-formyl-methionyl-leucyl-phenylalanine and the effect of different concentrations of imidazole salicylate.
Imidazoie Salicylate None 1 p.g/ml 2 ~g/ml 3 i~g/ml 4 i~g/ml
Extracellular CL (First Peak) 100 76.5 65.6 61.1 51.6
-+ 5.2 +- 2.9 +- 2.0 -+ 2.8 -+ 5.2
Intracellular CL (Second Peak) 100 85.8 75.2 74.1 68.6
-+ 4.6 -+ 4.0 +- 2.2 +- 3.0 -+ 2.0
Data are mean _+ SD of ten measurements and are expressed as a percentage of the value obtained without imidazole salicylate.
243
IMIDAZOLE SALICYLATE AND PMNs
Table II. Lucigenin-amplified chemiluminescence (CL) obtained from polymorphonuclear leukocytes after activation with 3 × 10-4 mol/L of phorbol myristate acetate and the effect of different concentration of imidazole salicylate.
Imidazole Salicylate
Lucigenin CL
None 1 #g/ml 2 r~g/ml 3 i~g/ml 4 ~g/ml
100 83.8 72.7 64.3 57.1
-+ 3.8 -+ 4.0 _+ 2.4 _+ 2.5 _+ 2.8
Data are mean _+ SD of ten measurements and are expressed as a percentage of the values obtained without imidazole salicylate.
which accompanies phagocytosis, can be triggered by a variety of inflammatory mediators that are capable of activating the multicomponent NADPH oxidase system. 13'14 This complex enzyme is responsible for generating the superoxide anion (02) at the expense of NADPH. Superoxide anion dismutes rapidly to produce H 2 0 2 and oxygen. 02 and H 2 0 2 a r e used to generate various reactive oxygen species, including hypochlorite produced by the myeloperoxidase-hydrogen peroxide-halide reaction, chloramines, and hydroxyl radicals. 1'2 2.0 cps x 104
1.5.
1.0.
0.5-
0.0 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 sec Figure 2. Time course of lucigenin-amplified chemiluminescence of the xanthine/xanthine oxidase reaction. Chemiluminescence was measured as counts per second (cps).
244
A. KANTARET AL.
The CL assay provides a simple and sensitive method of assessing the overall oxidative metabolic potential of PMNs by introducing substrates (ie, chemiluminigenic probes) that are susceptible to oxygenation and high CL yields. 15 Lucigenin-amplified CL is dependent on 0 2 production, whereas luminol-amplified CL is dependent on myeloperoxidase release and hydrogen peroxide production. 16-1s Our results demonstrated that the CL response of PMNs activated by FMLP is bimodal. Recent studies by Dahlgren is have demonstrated that the first peak reflects the extracellular release of H202 and the second peak reflects the intracellular activity of the myeloperoxidase system. Imidazole salicylate inhibited both responses in a dose-dependent manner. No significant inhibition was observed in PMA-stimulated PMNs. A difference between an FMLP- and PMA-induced cellular response could be expected, because these substances use different routes to activate the cells. FMLP acts through a specific receptor on the membranes of PMNs, 19 whereas PMA acts on protein kinase C and does not need a membrane receptor. 2° Imidazole salicylate inhibited the second peak of FMLPstimulated PMNs but did not significantly influence PMA-stimulated PMNs. These results suggest that IS inhibits the respiratory burst of FMLP-activated PMNs, probably by interfering with the plasma membrane. The fact that IS significantly inhibited the first peak of the CL response of FMLP-stimulated PMNs may be attributed to an antioxidant effect or to an inhibitory effect on the respiratory burst activation mechanisms. To examine whether IS possesses a scavenging effect on superoxide anions, we used the xanthine/xanthine oxidase system. The xanthine/ xanthine oxidase system produced a superoxide anion 21 that was inhibited by various concentrations of IS. Similar results were obtained by using lucigenin-amplified CL, which reflects the release of superoxide anion by PMNs. 16 These results indicate that IS is a scavenger of superoxide anion. In conclusion, our data demonstrate that IS is a scavenger of superoxide anion that can interfere with the oxidative metabolism of PMNs, probably by influencing the plasma membrane of PMNs. Recent studies 9'22 have suggested an interaction between some NSAIDs and the plasma membrane of PMNs. We are conducting further studies to investigate the effect of IS on plasma membrane using fluorescence techniques. References:
1. Haliwell B. Reactive oxygen species in living systems: Source, biochemistry, and role in human disease. A m J Med 1991; 91:14S-22S. 2. Clark RA. Molecular dissection of the leukocyte superoxide generating system. In: Je-
245
IMIDAZOLESALICYLATEANDPMNs
saitis AJ, Dratz ES, eds. Molecular basis of oxidative damage by leukocytes. Boca Raton, FL: CRC Press, 1992:25-35. 3. Babior BM. The respiratory burst oxidase and the molecular basis of chronic granulomatous disease. A m J Hematol 1991; 37:263-266. 4. Cochrane CG. Mechanism of cell damage by oxidants. In: Jesaitis AJ, Dratz ES, eds. Molecular basis of oxidative damage by leukocytes. Boca Raton, FL: CRC Press, 1992: 149-162. 5. Abramson SB, Weissmann G. The mechanisms of action of nonsteroidal antiinflammatory drugs. Arthritis Rheum 1989; 32:1-9. 6. Vane JR, Botting RM. The mode of action of anti-inflammatory drugs. Postgrad Med J 1990; 66(Suppl 4):$2-S17. 7. Halliwell B, Houllt JR, Blake DR. Oxidants, inflammation, and anti-inflammatory drugs. FASEB J 1988; 2:2867-2873. 8. Weissmann G. Aspirin. Sci A m 1991; 264:84-89. 9. Kantar A, Wilkins G, Swoboda B, et al. Alterations of the respiratory burst of polymorphonuclear leukocytes from diabetic children. Acta Paediatr Scand 1990; 79:535-541. 10. Kantar A, Oggiano N, Romagnoni GG, Giorgi PL. Effect of oral administration of bacterial extracts on the bactericidal capacity of polymorphonuclear leukocytes in children with recurrent respiratory infections. J Int Med Res 1991; 19:451-456. 11. Fiorini R, Curatola G, Bertoli E, et al. Changes of fluorescence anisotropy in plasma membrane of human polymorphonuclear leukocytes during the respiratory burst phenomenon. FEBS Lett 1990; 273:122-126. 12. Kantar A, Bruni S, Oggiano N, et al. Influence of ketone bodies on the oxidative burst of polymorphonuclear leukocytes: Implications for the use of ketogenic diets in obese children. Pediatr Adolesc Med 1992; 2:230-234. 13. Lambeth JD. Activation of the respiratory burst oxidase in neutrophils: On the role of membrane-derived second messengers, Ca2+, and protein kinase C. J Bioenerg B iomembranes 1988; 20:709-733. 14. Baggiolini M, Wymann MP. Turning on the respiratory burst. Trends Biochem Sci 1990; 15:69-72. 15. Murphy ME, Sies H. Visible-range low-level chemiluminescence in biological systems. Methods Enzymol 1990; 186:595-610. 16. Gyllenhammar H. Lucigenin chemiluminescence in the assessment of neutrophil superoxide production. J Immunol Methods 1987; 97:209-213. 17. Briheim G, Stendahl O, Dahlgren C. Intra- and extracellular events in luminoldependent chemiluminescence of polymorphonuclear leukocytes. Infect Immun 1984; 45: 1-5. 18. Dahlgren C. Effect of different inhibitors on the intracellularly and extracellularly generated chemiluminescence induced by formylmethionyl-leucyl-phenylalaninein polymorphonuclear leukocytes. Cellular response in the presence of mannitol, benzoate, taurine, indomethacin and NDGA. J Biolumin Chemilumin 1991; 6:29-34. 19. Jesaitis AJ, Allen RA. Activation of the neutrophil respiratory burst by chemoattractants: Regulation of the N-formyl peptide receptor in plasma membrane. J Bioenerg Biomembranes 1988; 20:679-707. 246
A. KANTAR
ET AL.
20. Heyworth PG, Badwey JA. Protein phosphorylation associated with the stimulation of neutrophils. Modulation of superoxide production by protein kinase C and calcium. J Bioenerg Biomembranes 1990; 22:1-26. 21. Cotelle N, Berneir JL, Henichart JP, et al. Scavenger and antioxidant properties of ten synthetic flavones. Free Radic Biol Med 1992; 13:211-219. 22. Abramson SB, Leszczynska-Piziak J, Haines K, Reibman J. Non-steroidal antiinflammatory drugs: Effects on a GPT binding protein within the neutrophil plasma membrane. Biochem Pharmacol 1991; 41:1567-1573.
247
EFFECT OF IMIDAZOLE SALICYLATE ON THE RESPIRATORY BURST OF POLYMORPHONUCLEAR LEUKOCYTES A. KANTAR,1 N. OGGIANO, 1 R. GABBIANELLI, 1 P. L. GIORGI, 1 AND M. BIRAGHI2 IPediatric Clinic, University of Ancona, Ancona, and 2Medical Department, VALEAS, Milan, Italy
ABSTRACT The effect of imidazole salicylate (IS), a n organic salt of imidazole a n d salicylic acid, on the respiratory burst of p o l y m o r p h o n u c l e a r leukocytes (PMNs) was investigated using a c h e m i l u m i n e s c e n c e (CL) assay. L u m i n o l - a n d lucigenin-amplified CL of PMNs s t i m u l a t e d with N - f o r m y l - m e t h i o n y l - l e u c y l - p h e n y l a l a n i n e ( F M L P ) or phorbol m y r i s t a t e acetate (PMA) were evaluated in the absence or presence of 1 to 4 ~tg/ml of IS. L u m i n o l - a m p l i f i e d CL of PMNs activated with F M L P was s i g n i f i c a n t l y reduced in the presence of IS, whereas no s i g n i f i c a n t effect was observed on P M A - s t i m u l a t e d PMNs. L u c i g e n i n amplified CL of PMA-stimulated PMNs was s i g n i f i c a n t l y inhibited by IS. CL of the x a n t h i n e / x a n t h i n e oxidase system was abolished in the presence of IS. These results suggest t h a t IS is a scavenger of the superoxide a n i o n a n d that it can influence the respiratory burst of PMNs, probably by i n t e r a c t i n g with the p l a s m a m e m b r a n e . INTRODUCTION
There is little argument that the products of activated polymorphonuclear leukocytes (PMNs) damage tissue. 1 This is especially true in the case of inflammatory reactions that feature an infiltration of PMNs. Activated PMNs injure tissue by releasing products stored in granules and generating new factors, including oxygen species. The latter process, known as the respiratory burst, is initiated when, after a suitable stimulus, molecular oxygen undergoes a one-electron reduction to superoxide anion at the expense of nicotinamide adenine dinucleotide phosphate (NADPH). 2 This reaction is mediated by a multicomponent enzyme system, known as respiratory burst oxidase. 3 In studies of the inflammatory injury of tissue, oxidants, which are generated by stimulated PMNs, appear to participate significantly in the attack on cells and supporting tissue. 4 Nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibiting the synthesis and release of prostaglandinsS'6; however, this may not account for all the effects of NSAIDs at all Address correspondence to: Dr. A. Kantar, Pediatric Clinic, University of Ancona, via Corridoni 11, 1-60123 Ancona,Italy. Received for publication on June 1, 1993. Printed in the U.S.A. Reproduction in whole or part is not permitted. 241
0011-393X/93/$3.50
IMIDAZOLESALICYLATEAND PMNs
doses. Alternatively, NSAIDs m a y modulate the response of PMNs to various stimuli. 7,s Imidazole salicylate* (IS) is an organic NSAID consisting of salt of imidazole and salicylic acid. In this study, we investigated the effect of IS on the oxidative metabolism of PMNs using luminol- and lucigeninamplified chemiluminescence (CL) assays. The ability of IS to act as a radical scavenger was investigated by using CL in the xanthine/xanthine oxidase system. M A T E R I A L S AND M E T H O D S
PMNs were isolated using Mono-Poly Resolving Medium (ICN, Biomedicals, Milan, Italy) as previously described, 9 and the cells were resuspended in a Krebs-Ringer phosphate solution containing 1 mg/ml of glucose, A reaction mixture of 106 cells and 10 btmol/L of luminol (Sigma Chemical Co., St. Louis, Missouri) in 1 ml of Krebs-Ringer phosphate solution was prepared with or without 1, 2, 3, or 4 ~g/ml of IS. The CL was measured immediately in an AutoLumat LB 953 (Berthold Co., Wilbad, Germany) after activation with 10-7 mol/L of N-formyl-methionyl-leucylphenylalanine (FMLP) (Sigma Chemical Co.) or 3 × 10 -4 mol/L phorbol myristate acetate (PMA) (Sigma Chemical Co.) as previously described. 1°'11 A reaction mixture of 106 cells and 150 ~mol/L of lucigenin (Sigma Chemical Co.) in 1 ml of Krebs-Ringer phosphate solution was prepared with or without IS using the same concentration as used for the luminolamplified CL assay. PMNs were later activated with PMA, and the CL was observed for 60 minutes as previously described. 11'12 A 1-ml reaction mixture containing 0.9 U of xanthine oxidase and 150 ~mol/L of lucigenin in Krebs-Ringer phosphate solution plus glucose (pH 7.4) in the absence or presence of 1, 2, 3, or 4 ~tg/ml of IS was prepared. The reaction was initiated by injecting 0.05 ml ofxanthine (final concentration, 50 ~Lmol/L). The CL measurement was observed for 40 seconds. The significance of the values obtained was calculated using Student's t test. RESULTS
When F M L P was added to PMNs, a bimodal CL response was obtained. The first peak appeared approximately 1 minute after injection of FMLP, and the second peak occurred after 3 to 5 minutes (Figure 1). When the cells were exposed to FMLP in the presence of IS, a dose-dependent inhibition of both peaks was obtained (Table I). When PMNs were activated with PMA, a peak CL value was obtained 15 to 17 minutes after stimulation. No significant inhibition was observed in the presence of IS. In lucigenin-amplified CL of PMNs activated with PMA, a peak value was obtained 18 to 20 minutes after stimulation. A dose-dependent inhi* Trademark: Flogozen ® (VALEAS S.p.A., Milan, Italy). 242
A. K A N T A R E T AL.
3.0 c p m x 10 7
2.5
, 2.0
i
1.5
1.0
0.5
0.0
o.oo
2. o
6. o
4.oo
I
8.()0
10.00
min
Figure 1. Time course of luminol-amplified chemiluminescence of polymorphonuclear leukocytes stimulated with N-formyl-methionyl-leucyl-phenylalanine. Chemiluminescence was measured as counts per minute (cpm).
bition of CL by IS was observed (Table II). CL produced by superoxide generated from the xanthine/xanthine oxidase reaction was completely abolished in the presence of IS (Figure 2). DISCUSSION
In response to certain stimuli, PMNs undergo an oxidative burst during which a series Of reactive oxygen metabolites are generated. 2 This process, Table I. Extracellular (first peak) and intracellular (second peak) luminol-amplified chemiluminescence (CL) obtained from polymorphonuclear leukocytes after activation with 10 -7 mol/L of N-formyl-methionyl-leucyl-phenylalanine and the effect of different concentrations of imidazole salicylate.
Imidazoie Salicylate None 1 p.g/ml 2 ~g/ml 3 i~g/ml 4 i~g/ml
Extracellular CL (First Peak) 100 76.5 65.6 61.1 51.6
-+ 5.2 +- 2.9 +- 2.0 -+ 2.8 -+ 5.2
Intracellular CL (Second Peak) 100 85.8 75.2 74.1 68.6
-+ 4.6 -+ 4.0 +- 2.2 +- 3.0 -+ 2.0
Data are mean _+ SD of ten measurements and are expressed as a percentage of the value obtained without imidazole salicylate.
243
IMIDAZOLE SALICYLATE AND PMNs
Table II. Lucigenin-amplified chemiluminescence (CL) obtained from polymorphonuclear leukocytes after activation with 3 × 10-4 mol/L of phorbol myristate acetate and the effect of different concentration of imidazole salicylate.
Imidazole Salicylate
Lucigenin CL
None 1 #g/ml 2 r~g/ml 3 i~g/ml 4 ~g/ml
100 83.8 72.7 64.3 57.1
-+ 3.8 -+ 4.0 _+ 2.4 _+ 2.5 _+ 2.8
Data are mean _+ SD of ten measurements and are expressed as a percentage of the values obtained without imidazole salicylate.
which accompanies phagocytosis, can be triggered by a variety of inflammatory mediators that are capable of activating the multicomponent NADPH oxidase system. 13'14 This complex enzyme is responsible for generating the superoxide anion (02) at the expense of NADPH. Superoxide anion dismutes rapidly to produce H 2 0 2 and oxygen. 02 and H 2 0 2 a r e used to generate various reactive oxygen species, including hypochlorite produced by the myeloperoxidase-hydrogen peroxide-halide reaction, chloramines, and hydroxyl radicals. 1'2 2.0 cps x 104
1.5.
1.0.
0.5-
0.0 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 sec Figure 2. Time course of lucigenin-amplified chemiluminescence of the xanthine/xanthine oxidase reaction. Chemiluminescence was measured as counts per second (cps).
244
A. KANTARET AL.
The CL assay provides a simple and sensitive method of assessing the overall oxidative metabolic potential of PMNs by introducing substrates (ie, chemiluminigenic probes) that are susceptible to oxygenation and high CL yields. 15 Lucigenin-amplified CL is dependent on 0 2 production, whereas luminol-amplified CL is dependent on myeloperoxidase release and hydrogen peroxide production. 16-1s Our results demonstrated that the CL response of PMNs activated by FMLP is bimodal. Recent studies by Dahlgren is have demonstrated that the first peak reflects the extracellular release of H202 and the second peak reflects the intracellular activity of the myeloperoxidase system. Imidazole salicylate inhibited both responses in a dose-dependent manner. No significant inhibition was observed in PMA-stimulated PMNs. A difference between an FMLP- and PMA-induced cellular response could be expected, because these substances use different routes to activate the cells. FMLP acts through a specific receptor on the membranes of PMNs, 19 whereas PMA acts on protein kinase C and does not need a membrane receptor. 2° Imidazole salicylate inhibited the second peak of FMLPstimulated PMNs but did not significantly influence PMA-stimulated PMNs. These results suggest that IS inhibits the respiratory burst of FMLP-activated PMNs, probably by interfering with the plasma membrane. The fact that IS significantly inhibited the first peak of the CL response of FMLP-stimulated PMNs may be attributed to an antioxidant effect or to an inhibitory effect on the respiratory burst activation mechanisms. To examine whether IS possesses a scavenging effect on superoxide anions, we used the xanthine/xanthine oxidase system. The xanthine/ xanthine oxidase system produced a superoxide anion 21 that was inhibited by various concentrations of IS. Similar results were obtained by using lucigenin-amplified CL, which reflects the release of superoxide anion by PMNs. 16 These results indicate that IS is a scavenger of superoxide anion. In conclusion, our data demonstrate that IS is a scavenger of superoxide anion that can interfere with the oxidative metabolism of PMNs, probably by influencing the plasma membrane of PMNs. Recent studies 9'22 have suggested an interaction between some NSAIDs and the plasma membrane of PMNs. We are conducting further studies to investigate the effect of IS on plasma membrane using fluorescence techniques. References:
1. Haliwell B. Reactive oxygen species in living systems: Source, biochemistry, and role in human disease. A m J Med 1991; 91:14S-22S. 2. Clark RA. Molecular dissection of the leukocyte superoxide generating system. In: Je-
245
IMIDAZOLESALICYLATEANDPMNs
saitis AJ, Dratz ES, eds. Molecular basis of oxidative damage by leukocytes. Boca Raton, FL: CRC Press, 1992:25-35. 3. Babior BM. The respiratory burst oxidase and the molecular basis of chronic granulomatous disease. A m J Hematol 1991; 37:263-266. 4. Cochrane CG. Mechanism of cell damage by oxidants. In: Jesaitis AJ, Dratz ES, eds. Molecular basis of oxidative damage by leukocytes. Boca Raton, FL: CRC Press, 1992: 149-162. 5. Abramson SB, Weissmann G. The mechanisms of action of nonsteroidal antiinflammatory drugs. Arthritis Rheum 1989; 32:1-9. 6. Vane JR, Botting RM. The mode of action of anti-inflammatory drugs. Postgrad Med J 1990; 66(Suppl 4):$2-S17. 7. Halliwell B, Houllt JR, Blake DR. Oxidants, inflammation, and anti-inflammatory drugs. FASEB J 1988; 2:2867-2873. 8. Weissmann G. Aspirin. Sci A m 1991; 264:84-89. 9. Kantar A, Wilkins G, Swoboda B, et al. Alterations of the respiratory burst of polymorphonuclear leukocytes from diabetic children. Acta Paediatr Scand 1990; 79:535-541. 10. Kantar A, Oggiano N, Romagnoni GG, Giorgi PL. Effect of oral administration of bacterial extracts on the bactericidal capacity of polymorphonuclear leukocytes in children with recurrent respiratory infections. J Int Med Res 1991; 19:451-456. 11. Fiorini R, Curatola G, Bertoli E, et al. Changes of fluorescence anisotropy in plasma membrane of human polymorphonuclear leukocytes during the respiratory burst phenomenon. FEBS Lett 1990; 273:122-126. 12. Kantar A, Bruni S, Oggiano N, et al. Influence of ketone bodies on the oxidative burst of polymorphonuclear leukocytes: Implications for the use of ketogenic diets in obese children. Pediatr Adolesc Med 1992; 2:230-234. 13. Lambeth JD. Activation of the respiratory burst oxidase in neutrophils: On the role of membrane-derived second messengers, Ca2+, and protein kinase C. J Bioenerg B iomembranes 1988; 20:709-733. 14. Baggiolini M, Wymann MP. Turning on the respiratory burst. Trends Biochem Sci 1990; 15:69-72. 15. Murphy ME, Sies H. Visible-range low-level chemiluminescence in biological systems. Methods Enzymol 1990; 186:595-610. 16. Gyllenhammar H. Lucigenin chemiluminescence in the assessment of neutrophil superoxide production. J Immunol Methods 1987; 97:209-213. 17. Briheim G, Stendahl O, Dahlgren C. Intra- and extracellular events in luminoldependent chemiluminescence of polymorphonuclear leukocytes. Infect Immun 1984; 45: 1-5. 18. Dahlgren C. Effect of different inhibitors on the intracellularly and extracellularly generated chemiluminescence induced by formylmethionyl-leucyl-phenylalaninein polymorphonuclear leukocytes. Cellular response in the presence of mannitol, benzoate, taurine, indomethacin and NDGA. J Biolumin Chemilumin 1991; 6:29-34. 19. Jesaitis AJ, Allen RA. Activation of the neutrophil respiratory burst by chemoattractants: Regulation of the N-formyl peptide receptor in plasma membrane. J Bioenerg Biomembranes 1988; 20:679-707. 246
A. KANTAR
ET AL.
20. Heyworth PG, Badwey JA. Protein phosphorylation associated with the stimulation of neutrophils. Modulation of superoxide production by protein kinase C and calcium. J Bioenerg Biomembranes 1990; 22:1-26. 21. Cotelle N, Berneir JL, Henichart JP, et al. Scavenger and antioxidant properties of ten synthetic flavones. Free Radic Biol Med 1992; 13:211-219. 22. Abramson SB, Leszczynska-Piziak J, Haines K, Reibman J. Non-steroidal antiinflammatory drugs: Effects on a GPT binding protein within the neutrophil plasma membrane. Biochem Pharmacol 1991; 41:1567-1573.
247