,4cta Med Scand 206: 45 1-457, 1979

The Effect of Sulfasalazine and Its Active Components on Human Polymorphonuclear Leukocyte Function in Relation to Ulcerative Colitis Lars Molin and Olle Stendahl From the Departments of Dermatology and Medical Microbiology, University Hospital, Linktiping, Sweden

ABSTRACT. Sulfasalazine and its active components, 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP), are potent modulators of inflammatory reactions but with somewhat different modes of action. Investigating the effect of these compounds on normal human polymorphonuclear leukocytes. in vitro, we show inhibition of different stages in the phagocytic process, such as migration (sulfasalazine and SP), superoxide production (sulfasalazine and SP), myeloperoxidase-mediated iodination and cytotoxicity (5-ASA and SP). It is thus suggested that sulfasalazine is not just a vehicle for delivering its active components in the colon, but that its therapeutic effect in ulcerative colitis and other inflammatory reactions is a result of the concurrent action of the three compounds. Key Mjords: polymorphonuclear leukocyte, sulfasalazine, 5-aminosalicylic acid, sulfapyridine, myeloperoxidase, ulcerative colitis.

Acta Med Scand 206: 45 1, 1979.

Sulfasalazine, formerly known as salicylazosulfapyridine (Salazopyrin@,Azulfidine@),consists of 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP) linked together by an azo bound. The combination of a sulfonamide with a salicylate radicle was introduced by Svartz for the treatment of rheumatoid arthritis and ulcerative colitis (29). The therapeutic effect in ulcerative colitis has been verified by several clinical investigations (4, 11, 12). In rheumatoid arthritis, however, the preparation is not effective enough. Although sulfasalazine has been used successfully in clinical practice for more than 30 years, its mode of action is still mainly unknown. It is often regarded as a vehicle for delivering its possible active components to the colon in higher concentrations than could be achieved by oral administration

of either one alone (10, 24). About 70% of the sulfasalazine reaches the colon intact, where it undergoes reductive cleavage at the azo linkage, releasing 5-ASA and SP (27). The split is induced mainly by the colonic bacteria (1). All SP is virtually absorbed from the colon, metabolized and then excreted in the urine. Some part of 5-ASA is also absorbed but the main unabsorbed part is excreted in the faeces (24, 27). Recent investigations have regarded 5-ASA as the active moiety during sulfasalazine treatment. Azad Khan et al. ( 2 ) showed that local administration of 5-ASA resulted in a significant improvement in patients with ulcerative colitis, whereas SP had little effect. Nevertheless, the beneficial action of sulfasalazine is far from elucidated. The present investigation of the effect of sulfasalazine on the function of polymorphonuclear leukocytes (PMNL) was initiated from our earlier observation that dapsone, a sulfone analogue to SP, has a specific effect on the function of PMNL (28), and has been used as a substitute for sulfasalazine, e.g. in Mb. Crohn (30). Since ulcerative colitis is characterized by an acute mucosal inflammation dominated by PMNL accumulation, the effect of sulfasalazine on PMNL activity was relevant to analyse. Our results reveal that sulfasalazine, as well as 5-ASA and SP, inhibits different stages in the phagocytic process, such as random migration, Abbreviations: 5-ASA=aminosalicylic acid, SP=sulfapyridine, PMNL=polyrnorphonuclear leukocytes, KRG= Krehs-Ringer phosphate buffer containing glucose, FITC=fluorescein isothiocyanate, PBS=phosphate-buffered saline, TCA=trichloroacetic acid, STZ=serumtreated zymosan, SOD=superoxide disrnutase, MPO= myeloperoxidase. Reprint requests: Dr L. Molin, Departmknt of Dermatology, University Hospital, S-58185 Linkoping, Sweden. Actir Med Sctrnd206

452

L . Moliti and 0. Strnduhl

phagocytosis, myeloperoxidase (MPO)-mediated iodination, oxidative metabolism and cytotoxicity. W e thus show that both sulfasalazine and its components are potent modulators of inflammatory reactions but with somewhat different modes of action, thereby complementing each other's qualities. These findings are discussed in relation t o the therapeutic effect of sulfasalazine in ulcerative colitis as well as in other inflammatory processes. M A T E R I A L A N D METHODS L 4 m y t e preparcition. Blood was obtained from apparently healthy adult volunteers (age 18-35) and the leukocytes were isolated according to Beyum ( 5 ) , using Hypaque-dextran sedimentation for the separation of cells from EDTA blood. After separation, washing and hypotonic lysis of contaminating erythrocytes, the leukocytes were suspended to 1 X lo7 cells/ml in Krebs-Ringer phosphate buffer containing 5 mM glucose (KRG), pH 7.2. A differential count was performed to determine the number of PMNL. Trypane blue exclusion was used to assay viability. Motiliry measurement system. Leukocyte motility was studied by a modification of the method described by Nelson et al. (22). Briefly, agarose was dissolved (1.5 %) in sterile water by heating. After cooling to SOT, the agarose was mixed with an equal volume of prewarmed Gey's solution in twice its usual concentration. Of the agarose medium, 8 ml were poured into 60x 15 mm tissue culture dishes (Flow Laboratories, Irvine, Scotland). Six sets of three wells were cut in the agarose. The wells had a diameter of 2.4 mm and in each set they were placed 2.4 m m apart. The drug tested was mixed with the Gey's solution to obtain the same concentration of the drug throughout the agarose. In each set of wells were placed 10 p1of the cell suspension (5x lo7 PMNL/ml) in the middle well, 10 pI of Gey's solution in the inner well, and as an attractant 10 p1 of human normal serum in the outer well. When normal serum is incubated in the agar, chemotactic factors are generated (26). To determine random locomotion of the PMNL population, both the inner and the outer well was filled with Gey's solution. The dishes were incubated for 2 hours at 37°C. After fixation in methanol for 30 min, the agarose was removed, and the cells stained with Giemsa for 15 min. The distances of migration were measured with an ocular micrometer. Phagocytic uptake. The ability of isolated PMNL to ingest heat-killed, fluorescein isothiocyanate (FITC)labelled yeast cells (Saccharomyces cerevisiae) was assayed as described by Hed (15). Briefly, isolated PMNL were allowed to adhere to glass slides and non-adhering cells were removed by washing. To the monolayers were added 0. I ml of FITC-labelled yeast cells ( 2 . 5 10" ~ cells/ ml) in KRG. The yeast particles were preopsonized with I0 pg/ml of anti-yeast IgG. The reaction mixtures were then incubated at 37°C in a humidified chamber. After 30 min, the slides were washed and kept in cold (4°C) phosphate-buffered saline (PBS) until examined under the mi-

croscope. To separate ingested yeast cells from those attached, a drop of crystal violet (0.5 mg/ml in 0.15 M NaC1) was added. The dye stains and thereby extinguishes the fluorescence of the attached particles, but does not reach the intracellular ones, which still fluoresce. By combining fluorescence and phase-contrast microscopy, the number of attached and ingested particles is determined. One hundred cells were counted and the phagocytic uptake was expressed as the number of yeast particles ingested per PMNL. Measurement of leukocyte iodination was carried out essentially as described by Olsson et al. (23). The reaction mixture contained 1 x log leukocytes, 10% pooled human serum, 30 nmoles of sodium iodide (0.5 pCi of lz5I), 1 X 10' yeast particles and KRG to a final volume of 0.5 ml. The tubes were incubated at 37°C and the reaction was terminated after 30 min with 0.1 ml of 0.1 M sodium thiosulphate. Five ml of cold 10% trichloroacetic acid (TCA) was then added. After centrifugation, the precipitates were washed three times with 5 ml of TCA. The iodination was expressed as nmoles I- precipitated per 1x 108 PMLN per 30 min. Superoxide generation was assayed essentially as described by Curnutte and Babior (9). The reaction mixtures contained 5 x 10" PMNL, 75 p M horse heart ferricytochrome c (Cyt c , Type 111, Sigma Chemical Co.), 5 my serum-treated zymosan (STZ) and KRG to a final volume of 3 ml. The reaction was initiated by incubating 1.5 ml of the reaction mixture at 37°C in a water bath, 1.5 ml being kept at 0°C in melting ice and used as a blank. After 30 min the test tubes were placed in melting ice and then centrifuged at 4°C (750 g , 10 rnin). The supernatant was collected and assayed for the amount of reduced Cyt c with a Beckman DU-2 spectrophotometer at 550 nm. The amount of reduced Cyt c was calculated using an absorbance coefficient of 15.5 mM-', cm-', at 550 nm. The superoxidedependent Cyt c reduction was expressed as the difference in Cyt c reduction between reaction mixtures containing no superoxide dismutase (SOD) (Sigma Chemical Co.) and those containing 200 U/ml of SOD. These controls were run with each set of experiments. Lysosomal enzyme release. The extracellular release of the PMNL granule-associated enzyme P-N-acetylglucosaminidase (E.C. 3.21.30) in the presence of the drugs was assayed from reaction mixtures similar to those employed for the determination of superoxide generation, but in the absence of Cyt c and using the fluorochrome substrate 4-methyl umbelliferyl 2-deoxy-2-acetamidoP-D-glucopyranoside (25). Maximum enzyme release was determined as the amount released by 0.2% Triton X-100 (Rohm and Haas Co., Philadelphia, Pa). Cell-free MPO-mediated iodination. MPO isolated from human PMNL was supplied by I. Olsson, Lund, Sweden. The reaction mixtures contained varying concentrations (0.2-10 pg/ml) of MPO. 1x lo8 yeast cells, 120 nmoles of NaI (1.0 pCi of '"I) and 0.05 M Tris-HC1 buffer, pH 7.0, to a final volume of 2.0 ml. The reaction was initiated by addition of 0.2 ml of 1 mM H2OZ.After 15 min the reaction was terminated by addition of 0.1 ml of 0.1 M sodium thiosulphate, and 5 ml of 10% TCA was added. The precipitate was washed three times with 5 ml of TCA and assayed for radioactivity.

Sulfasalazine effect on leukocytes

453

RESULTS

0

0.1

0.2 0.3 0.L

Concentration

0.5

(mM)

Fig. I . Effect of different concentrations of sulfasalazine (0),5-ASA ( O ) , S P (0) or 5-ASA+SP (W) on random

migration (top) and chemotaxis (bottom). Mean of three experiments.

To determine the type of inhibition on MPO-mediated iodination, the kinetics of the reaction was studied using varying concentrations of iodide (0.05-1 .O mM). The incubation mixture contained 5 pg MPO, iodide (0.5 pCi T), 1 X lo8 yeast cells and Tris-HCI to a final volume of 2.0 ml. The reaction was initiated by addition of 0.2 ml of 1 mM H,O,. MPO-H,O,-halide-mediated cytotoxicity . The cytotoxic effect of the MPO-H,O,-halide system was assessed essentially as described by Clark et al. (8), assaying the W r release from prelabelled mammalian target cells. Human embryonic lung fibroblasts (W138, Flow Lab., Irwine, Scotland) were used as target cells. The tumour cells were grown in vitro in plastic Petri dishes (60x13 mm, Flow Lab.) in Eagle's modified medium supplemented with 10% fetal calf serum and antibiotics. The cells were labelled by incubating them with 25 pCi of Na, 51Cr04in KRG at 37°C for 1 h. After washing the cells four times in PBS, a reaction mixture was added containing 10 pg MPO, a peroxide-generating system of 0.5 ml glucose (20 mglml) and 0.2 ml glucose oxidase (100 Fglml; E.C. 1.1.3.4. Sigma Chemical Co.) and 0.03 mM sodium phosphate buffer, pH 7.0, supplemented with 1.5xlOP M KHZP04, 1.5x M MgSOl and 0.1 NaCl in 3 ml. After 240 min incubation at 37"C, 0.5 ml samples were drawn to test tubes containing 2 ml PBS, kept on melting ice and centrifuged at 4°C (250 g , 5 min). The supernatants were collected and assayed for radioactivity. Maximum ''Cr release was determined by counting the supernatants from dishes where 0.2% Triton X-100 had been added. The cytotoxicity was expressed as per cent of maximum releasable activity in duplicate samples. Drugs. Sulfasalazine, 5-aminosalicylic acid and sulfapyridine (supplied by Pharmacia, Uppsala, Sweden) were solubilized to 1 mM in PBS and kept in the dark at 4°C.

Effect of sulfasalazine and its components 5-ASA and SP on PMNL locomotion The influence of the compounds on PMNL locomotion was assayed with the agarose method according to Nelson et al. (22), using agarose-activated serum as chemotactic stimulus. Sulfasalazine exhibits a pronounced dose-dependent inhibition of random migration-0.1 mM decreases the migration to 30% and 0.5 mM abolishes it completely (Fig. 1). The drug has, however, no effect on the chemotactic response per se since the relative increase in migration in the presence of activated serum was not changed in the presence of 0.1 mM-the decreased motility observed in the chemotactic assay was primarily due to decreased random migration. 5-ASA shows no effect in either random migration or chemotaxis. Neither does SP show any effect at the lower concentration (0.1 mM), most compatible with the concentration reached in vivo. At the higher concentrations (0.25 and 0.5 mM) we observed a moderate but significant inhibition on random migration, but no effect on chemotaxis. To evaluate whether the pronounced effect of sulfasalazine is due to the sulfasalazine molecule or to the synergistic effect of 5-ASA and SP, the later compounds were mixed and incubated with the cells. No synergistic effect was, however, observed-the inhibition was the same as of SP alone (Fig. 1). Effect on phagocytosis and metabolic activity The effects of sulfasalazine, 5-ASA and SP on the phagocytic activity were assayed by counting the number of FITC-labelled yeast cells phagocytozed by PMNL attached to glass slides. This method permits an accurate quantitation of the number of ingested and attached particles. Table I shows that only sulfasalazine at a high non-physiological concentration (0.5 mM) reduced the uptake moderately, by 25% @