Inhibition of neutrophil degranulation and superoxide production by sulfasalazine (original) (raw)
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Gut, 1989
The possibility that the mode of action of sulphasalazine and its active metabolite 5-amino-salicylic acid (5ASA) involves modification of toxic oxygen metabolite production by neutrophils has been investigated by measuring the effect of these drugs on luminol-dependent chemiluminescence, superoxide release and oxygen consumption by stimulated neutrophils in vitro. 5ASA, and to a lesser extent sulphasalazine, had profound inhibitory effects on the luminol dependent chemiluminescent response of neutrophils stimulated with formyl-methionyl-leucylphenylalanine (1 [tM)+cytochalasin B (5 [igIml). A concentration of 50 ,tM 5ASA or sulphasalazine produced 93.8 (2-3)% and 65.7 (3.7)% inhibition of control responses respectively. The concentration of 5ASA and sulphasalazine producing 50% inhibition of chemiluminescence were 3-6 (1.8) [1M and 16.5 (6) FM respectively. Both drugs had little effect on the chemiluminescent response of neutrophils stimulated with phorbol myristate acetate (1 [ig/ml), producing only 11.4 (3.9)% and 34 (7)% inhibition respectively, at a concentration of 50 FM. Superoxide release from fMLP+CB stimulated neutrophils was also inhibited slightly by 5ASA (50 [tM) by 35-6% and by sulphasalazine (50 FM) by 7.9%. Similarly, there was little inhibition in the rate of oxygen consumption by fMLP+ CB stimulated neutrophils by either 5ASA or sulphasalazine at concentrations which produced near total abolition of luminol dependent chemiluminescence. These results show that sulphasalazine and 5ASA inhibit the reaction of toxic metabolites produced by stimulated neutrophils with luminol, without inhibition of the oxidase system producing these metabolites. The site of action of these drugs on neutrophils in vitro is thus extracellular, by scavenging a released metabolite, probably hypochlorite. This has important implications for their mode of action in vivo in inflammatory bowel disease. The beneficial effects of sulphasalazine on ulcerative colitis were first noted in the 1940s. ' Sulphasalazine is broken down in the colon by enteric bacteria into sulphapyridine and 5-amino-salicylic acid (5ASA).2 The active metabolite of SAL is 5ASA4 which has recently been used in the treatment of ulcerative colitis, being delivered to the colon in a pH sensitive coated capsule.' The mode of action of sulphasalazine and its active metabolite, 5ASA, in inflammatory bowel disease
Digestive Diseases and Sciences, 1991
The in vitro antioxidant capacity of sulfasalazine (SASP), its metabolites (SP, 5-ASA), and olsalazine (OAZ), was studied by evaluating their effects on superoxide (02-') production. Assay systems were the xanthine-xanthine oxidase (X/XOD) reaction and phorbol myristate acetate (PMA)-activated polymorphonuclear leukocytes (PMNs), using the cytochrome c (cyt-c) reduction assay and a luminot-dependent chemiluminescence method. 5-ASA, SASP, and OAZ showed a dose-dependent scavenger effect in both 02-. generating systems, 5-ASA being the most powerful (>50% of inhibition in the PMNs system and >70% in the X/XOD system at 10 izM concentration). SP had an inhibitor.." effect only in the PMNs system but did not modify the activity of xanthine oxidase, thus excluding a scavenger action. These data suggest that the scavenger effect of 5-ASA, SASP, and OAZ may be an important mechanism of action.
Digestive Diseases and Sciences, 1991
The in vitro antioxidant capacity of sulfasalazine (SASP), its metabolites (SP,, and olsalazine (OAZ), was studied by evaluating their effects on superoxide (02-') production. Assay systems were the xanthine-xanthine oxidase (X/XOD) reaction and phorbol myristate acetate (PMA) -activated polymorphonuclear leukocytes (PMNs), using the cytochrome c (cyt-c) reduction assay and a luminot-dependent chemiluminescence method. 5-ASA, SASP, and OAZ showed a dose-dependent scavenger effect in both 02-. generating systems, 5-ASA being the most powerful (>50% of inhibition in the PMNs system and >70% in the X/XOD system at 10 izM concentration). SP had an inhibitor.." effect only in the PMNs system but did not modify the activity of xanthine oxidase, thus excluding a scavenger action. These data suggest that the scavenger effect of 5-ASA, SASP, and OAZ may be an important mechanism of action.
Redox Report, 2010
Sulfasalazine is a prodrug composed by a molecule of 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP), linked by an azo bond, which has been shown to be effective in the therapy of inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease, as well as of rheumatic diseases, such as rheumatoid arthritis and ankylosing spondylitis. The precise mechanism of action of sulfasalazine and/or its metabolites has not been completely elucidated, though its antioxidant effects are well established and are probably due to its scavenging effects against reactive oxygen and nitrogen species (ROS and RNS), as well as metal chelating properties, in association to its inhibitory effects over neutrophil oxidative burst. The present work was focused on screening and comparing the potential scavenging activity for an array of ROS (O 2 • •-, H 2 O 2 , 1 O 2 , ROO • • and HOCl) and RNS ( • • NO and ONOO -), mediated by sulfasalazine and its metabolites 5-ASA and SP, using validated in vitro screening systems. The results showed that both 5-ASA and sulfasalazine were able to scavenge all the tested ROS while SP was practically ineffective in all the assays. For HOCl, 1 O 2 , and ROO • • , 5-ASA showed the best scavenging effects. A new and important finding of the present study was the strong scavenging effect of 5-ASA against 1 O 2 . 5-ASA was shown to be a strong scavenger of • • NO and ONOO -. Sulfasalazine was also able to scavenge these RNS, although with a much lower potency than 5-ASA. SP was unable to scavenge • • NO in the tested concentrations but was shown to scavenge ONOO -, with a higher strength when the assay was performed in the presence of 25 mM bicarbonate, suggesting further scavenging of oxidizing carbonate radical. In conclusion, the ROS-and RNS-scavenging effects of sulfasalazine and its metabolites shown in this study may contribute to the anti-inflammatory effects mediated by sulfasalazine through the prevention of the oxidative/nitrative/nitrosative damages caused by these species.
European Journal of Pharmacology, 1989
A sulfasalazine analogue, 5'-(2,4-dichlorobenzoyl)2'-hydroxyphenylacetic acid (CL 42A), potently inhibited the formation of 5-1ipoxygenase products (leukotrienes B 4 and C 4 and 5-hydroxyeicosatetraenoic acid) by human leukocytes. Half-maximal inhibition of leukotriene production was obtained with 5 and 10 ~tM CL 42A after stimulation with serum-treated zymosan or ionophore A23187, respectively. CL 42A was equipotent to nordihydroguaiaretic acid and about 50 times more potent than sulfasalazine and benoxaprofen in studies on the inhibition of LTB 4 formation in leukocyte suspensions stimulated with serum-treated zymosan. Furthermore, CL 42A had no inhibitory effect on the production of 15-hydroxyeicosatetraenoic acid after incubation of human leukocytes with ionophore A23187 in the presence of exogenous arachidonic acid. Sulfasalazine inhibited the synthesis of 5-1ipoxygenase products (5-hydroxyeicosatetraenoic acid and leukotriene B4:IC50 250 ~tM, leukotriene C4:IC50 100/~M) in a concentration-dependent manner but had no effect on 15-hydroxyeicosatetraenoic acid formation. The metabolites of sulfasalazine, sulfapyridine and 5-aminosalicylic acid, and the isomer, 4-aminosalicylic acid, were all less potent than sulfasalazine as inhibitors of leukotriene formation. Both CL 42A (ICs0 20 /~M) and sulfasalazine (ICs0 500 /~M) inhibited the synthesis of thromboxane B 2 and hydroxyheptadecatrienoic acid in human platelet suspensions after arachidonic acid stimulation. However, while CL 42A inhibited cyclooxygenase, the inhibitory effect of sulfasalazine was exerted mainly on thromboxane synthase. The platelet formation of 12-hydroxyeicosatetraenoic acid was not inhibited by CL 42A whereas sulfasalazine had a weak inhibitory effect.
Clinical Science, 1995
1. It is well known that neutrophils act as mediators of tissue injury in a variety of inflammatory diseases. Their histotoxic activity is presently thought to involve proteinases and oxidants, primarily hypochlorous acid (HOCl). This oxidant is also capable of inactivating the specific inhibitor of neutrophil elastase (α1-antitrypsin), thereby favouring digestion of the connective matrix. 2. In the present work, we found that sulphanilamide and some sulphanilamide-related anti-inflammatory drugs such as dapsone, nimesulide and sulphapyridine reduce the availability of HOCl in the extracellular microenvironment of activated neutrophils and prevent the inactivation of α1-antitrypsin by these cells in a dose-dependent manner. The ability of each drug to prevent α1-antitrypsin from inactivation by neutrophils correlates significantly with its capacity to reduce the recovery of HOCl from neutrophils. Five other non-steroidal anti-inflammatory drugs were completely ineffective. 3. Theref...
Clinical and Experimental Pharmacology and Physiology, 2009
1. Neutrophils release several histotoxic molecules that cause tissue injury. Neutrophil apoptosis is a crucial process that governs the persistence of inflammatory disorders and tissue damage. Thus, in the present study, we investigated whether the anti-inflammatory drug sulphasalazine (SSZ) affects neutrophil apoptosis in the presence of insoluble immune complex (IC). 2. Neutrophils were obtained from healthy donors. Neutrophils were resuspended in incubation medium and incubated for 2-12 h with or without 10, 30 or 100 lmol/L SSZ and 25 lg/mL IC. In some experiments, cells were co-incubated with 20 lmol/L Z-IETD-fmk (a caspase 8 inhibitor) or 20 lmol/L Z-LEHD-fmk (a caspase 9 inhibitor). Apoptosis was evaluated morphologically on cytological preparations stained with May-Grü nwald-Giemsa as well as by flow cytometry analysis of annexin V and propidium iodide staining. Caspase 3 activity was determined spectrophotometrically. 3. At 100 lmol/L, SSZ significantly accelerated IC-induced neutrophil apoptosis. Treatment of neutrophils with 20 lmol/L of the caspase 8 or 9 inhibitors Z-IETD-fmk or Z-LEHD-fmk, respectively, demonstrated that the SSZ-induced pro-apoptotic effect was mediated by a caspase 8-but not caspase 9-dependent pathway. The caspase 3 activity assay showed that treatment with 100 lmol/L SSZ increased caspase 3 activation. 4. In conlusion, the results of the present study indicate that it is possible that the molecular mechanism underlying SSZ protection against neutrophil-mediated tissue injury inflammatory disorders, such as rheumatoid arthritis and inflammatory bowel diseases, involves a caspase 8-dependent pathway.
International Journal of Molecular Sciences
Sulforaphane has several effects on the human body, including anti-inflammation, antioxidation, antimicrobial and anti-obesity effects. In this study, we examined the effect of sulforaphane on several neutrophil functions: reactive oxygen species (ROS) production, degranulation, phagocytosis, and neutrophil extracellular trap (NET) formation. We also examined the direct antioxidant effect of sulforaphane. First, we measured neutrophil ROS production induced by zymosan in whole blood in the presence of 0 to 560 µM sulforaphane. Second, we examined the direct antioxidant activity of sulforaphane using a HOCl removal test. In addition, inflammation-related proteins, including an azurophilic granule component, were measured by collecting supernatants following ROS measurements. Finally, neutrophils were isolated from blood, and phagocytosis and NET formation were measured. Sulforaphane reduced neutrophil ROS production in a concentration-dependent manner. The ability of sulforaphane to ...