Inhibition of cytotoxicity by sulfasalazine. I. Sulfasalazine inhibits spontaneous cell-mediated cytotoxicity by peripheral blood and intestinal mononuclear cells from control and inflammatory bowel disease patients

Immunopharmacology, 11 (1986) 101-109 Elsevier

101

IMO 00316

Inhibition of Cytotoxicity by Sulfasalazine. I. Sulfasalazine Inhibits Spontaneous Cell-Mediated Cytotoxicity by Peripheral Blood and Intestinal Mononuclear Cells from Control and Inflammatory Bowel Disease Patients Richard P. M a c D e r m o t t , M a r y G. Kane, Loren L. Steele and William F. Stenson Division qf Gastroenterology, Department of Medicine, Barnes and Jewish Hospitals Washington University Medical Center, 660 South Euclid Avenue, St. Louis, MO 63110, U.S.A. (Received 29 July 1985: accepted 2 December 1985)

Abstract: We have studied the effects of sulfasalazine and its metabolites on cell-mediated cytotoxicity by peripheral blood and intestinal mononuclear cells from both control and inflammatory bowel disease (IBD) patients. Sulfasalazine and sulfapyridine, as well as hydrocortisone and nordihydroguaiaretic acid inhibited spontaneous cell-mediated cytotoxicity by control and IBD peripheral blood cells. Sulfasalazine and nordihydroguaiaretic acid inhibited spontaneous cell-mediated cytotoxicity by control and IBD intestinal mononuclear cells cultured for 72 h in media alone. In contrast, 5-aminosalicylate, indomethacin and benzylimidazole had no effect on cytotoxicity by any cell population. Lectin-induced, antibody-dependent and interleukin-2-induced cell-mediated cytotoxicity, as well as lymphokine-activated killing were not inhibited by the drugs: inhibitory effects in these assays were primarily upon the underlying spontaneous cell-mediated cytotoxicity. The inhibition induced by sulfasalazine, sulfapyridine and nordihydroguaiaretic acid could not be reversed by adding the lipoxygenase metabolites leukotrine B4 or 12-hydroxyeicosatetraenoic acid. These findings demonstrate that spontaneous cell-mediated cytotoxicity by control and IBD mononuclear cells can be inhibited by sulfasalazine. Key words:

Sulfasalazine: Cytotoxicity; Inflammatory bowel disease; Intestinal mononuclear cells

Introduction

Cell-mediated cytotoxicity has been suggested as a possible mechanism of injury to the intestine in inflammatory bowel disease (IBD) (Broberger and Perlmann, 1963; Perlmann and Broberger, 1963; Watson et al., 1966; Shorter et al., 1968, 1969, 1970, 1973; Stobo et al., 1976). Perlmann and Broberger (Broberger and Perlmann, 1963; Perlmann and Broberger, 1963) demonstrated that peripheral blood mononuclear cells from patients with ulcerAbbreviations: IBD, inflammatory bowel disease: 5-ASA, 5aminosalicylic acid; HETE, hydroxyeicosatetraenoic acid; LT; leukotriene; NDGA, nordihydroguaiaretic acid: SCMC, spontaneous cell-mediated cytotoxicity; MNC, mononuclcar cell.

ative colitis could kill human fetal colonic epithelial cells in vitro. These studies were then expanded (Watson et al., 1966; Shorter et al., 1968, 1969, 1970, 1973; Stobo et al., 1976) with the proposal that a possible mechanism in the immunopathogenesis of IBD is the destruction of colonic targets by Fc receptor-bearing cells, in conjunction with IgM anti-colon antibodies. Auer and co-workers (Auer et al., 1980; Auer and Ziemer, 1980) demonstrated that peripheral blood mononuclear cells from Crohn's disease patients also exhibit decreased cytotoxic capabilities toward a variety of cell line targets. In comparison with peripheral blood mononuclear cells, intestinal mononuclear cells are poor mediators of cell-mediated cytotoxicity (Bookman

0162-3109/86/$03.50 ~;; 1986 Elsevier Science Publishers B.V. (Biomedical Division)

102 and Bull, 1979; MacDermott et al., 1980). However, lectins can induce cellular cytotoxicity by intestinal mononuclear cells (MacDermott et al., 1980, 1986; Falchuk et al., 1981), interferon can induce cytotoxic function by control and IBD intestinal mononuclear cells (MacDermott et al., 1986), and it has recently been demonstrated that interleukin-2 induces cytotoxocity by both control and IBD intestinal mononuclear cells after a 48 72-h preincubation (Fiocchi et al., 1985). Sulfasalazine is effective in the therapy of inflammatory bowel disease and is degraded in vivo into its two components, sulfapyridine and 5-aminosalicylate (5-ASA). Sulfapyridine is absorbed, but 5ASA remains in the lumen and is thought to be the active compound (Klotz et al., 1980). The mechanism of action of these drugs in IBD is not known, but it has recently been demonstrated that sulfasalazine and 5-ASA block the synthesis of arachidonic acid metabolites by human peripheral blood neutrophils and colonic mucosa (Stenson and Lobos, 1982; Sharon and Stenson, 1984). Arachidonic acid can be metabolized via the cyclooxygenase pathway to prostaglandins and thromboxane, or via the lipoxygenase pathway to the hydroxyeicosatetraenoic acids (HETEs). Among these products are 5-HETE, 12-HETE and leukotrine B4 (LTB4). Both 5-HETE and LTB4 are potent chemotactic agents, and could play a role in the initiation or propagation of inflammation in the gut. It has recently been found that synthesis of LTB4 is enhanced in the colonic mucosa of IBD patients relative to controls (Sharon and Stenson, 1984), and that LTB4 in the mucosa of patients with ulcerative colitis is the major chemotactic factor (Lobos et al., 1984). Several inhibitors of arachidonic acid metabolism vary in their selectivity for different pathways. Steroids inhibit the release of fatty acids from phospholipids and therefore the generation of arachidonic acid. Nordihydroguaiaretic acid (NDGA) is an inhibitor of the lipoxygenase pathway. In this study we have examined the effect of sulfasalazine and its metabolites, as well as other inhibitors and products of arachidonic acid metabolism, on cellular cytotoxicity by control and IBD peripheral blood and intestinal mononuclear cells.

Methods

Isolation of peripheral blood lymphocytes Peripheral blood mononuclear cells (MNC) were obtained from freshly drawn blood of healthy volunteers by fractionation over Ficoll-Hypaque gradients according to the method of Boyum (1968). After isolation, the peripheral blood MNC were washed in M199 assay medium: M199 (Grand Island Biologicals (GIBCO), Grand Island, NY) with 2 mM L-glutamine (Sigma, St. Louis, MO), 1% Hepes buffer (Sigma), 10% heat-inactivated pooled human serum (GIBCO), 1% pen-strep-fungizone solution (GIBCO: 10000 units/ml-10000 #g/ml10000 #g/ml), gentamicin 50 #g/ml (Sigma), and NaOH to bring the pH to 7.4. Cells were counted and viability determined by trypan blue exclusion.

Isolation q]" human intestinal mononuclear cells Intestinal tissue (large or small bowel) was obtained as previously described (MacDermott et al., 1980, 1981a, 1981b) at the time of surgery from patients with ulcerative colitis, Crohn's disease, and adenocarcinoma (for control samples). MNC from intestinal mucosa were isolated as described by Bookman and Bull (1979) and modified by MacDermott et al. (1980, 1981a, 1981b). The supernatant from the overnight collagenase digestion was collected and the resultant intestinal MNC, red blood cells, and debris were layered over a Ficoll-Hypaque gradient. Interface cells were harvested and washed with calcium-magnesium-free Hanks' balanced salt solution with 5 mM EDTA. The pellet was resuspended in 10 ml Percoll solution (specific gravity 1.040; Pharmacia, Piscataway, N J), centrifuged at 500 x g for 15 rain to remove epithelial cells, then washed and resuspended in M199 assay medium. Sephadex G-10 columns were used to deplete macrophages from MNC populations as previously described (MacDermott and Stacey, 1981). The non-adherent cells (purified lymphocytes) were eluted using 50 ml warm (37°C) RPMI 1640 containing 20% fetal bovine serum (GIBCO).

103

Drugs, arachidonic acid metabolites and lymphokines Sulfasalazine and sulfapyridine (Pharmacia, Uppsala, Sweden) were dissolved in 1 part 0.1 N N a O H and 9 parts media, and brought to a pH of 7.4 with 0.1 N HC1. 5-ASA (Pharmacia) was dissolved in media, and brought to pH 7.4 with 0.l N NaOH. The final concentrations in the cytotoxicity assays were 0.5 × 10 3 M sulfasalazine, 1 × 10 -3 M sulfapyridine and 2 x 10 -3 M 5-ASA. N D G A (Sigma) was dissolved in ethanol, diluted 1:300 (v/v) in media, and serially diluted in medium to concent r a t i o n s o f 5 x 10-6,1 x 1 0 - S a n d 5 × 1 0 - S M . Hydrocortisone (Elkins-Sinn, Cherry Hill, N J) was diluted in medium to final assay concentrations of 1 x i 0 - 4 and 1 x I0-3 M. Indomethacin (Sigma) was dissolved in ethanol, diluted 1:1000 (v/v) in medium, and serially diluted to 1 x 10 -6 and 1 x 10 -5 M. 1-Benzylimidazole (Aldrich, Milwaukee, WI) was dissolved in medium, pH adjusted to 7.4 and diluted to concentrations of 3 x 10 -5, 1 x 10 4 a n d 3 x 10 - 4 M . L T B a ( a g i f t o f J . Rokach, Merck-Frosst, Quebec) was dissolved in medium and serially diluted to final assay concentration of 1 x 10 - 1 2 to 1 X 10 7 M. Interleukin-2 (Cellular Products, Buffalo, NY) was diluted to 5%, 10% or 20% in M199 assay media. 12-HETE was biosynthesized by incubating arachidonic acid with human platelets and was purified by high performance liquid chromatography. The 12-HETE was diluted to final concentrations of 1 x 10 -8 to 1 × 10 - 6 M. Diluents used above had no effect on any of the cytotoxicity assays.

final concentration of 0.05 × l06 cells/ml. Assays were performed in triplicate in round-bottom microtiter plates as previously described (MacDermott et al., 1980, 1986). For spontaneous cellmediated cytotoxicity (SCMC) assays, 50/zl of the final suspension of 51Cr-labelled target cells were employed. Appropriate dilutions of effector MNC in M199 assay medium were added in 50 #l aliquots, drugs and inhibitors were added in 50 pl aliquots, and 10 pl of M199 assay medium was also added. For antibody-dependent cellular cytotoxicity assays, 10 pl per well of rabbit anti-K562 or anti-colon cell antibody was added instead of medium. In lectin-induced cellular cytotoxicity assays, pokeweed mitogen at a dilution of 1:100 was added directly into the assay instead of antibody. After incubation at 37°C for 18 h, the plates were harvested using a Titertek harvesting system (Flow Labs, McLean, VA). The amount of s~Cr label in each supernatant (counts per minute, CPM) was determined in a Beckman BioGamma gamma counter (Beckman Instruments, Palo Alto, CA). Parallel incubation mixtures containing radiolabelled cell lines in media alone defined spontaneous 5~Cr release. Maximal 5aCr release (determined by the addition of 100 pl Triton X-100 (Sigma) to the targets alone) averaged 93%, and spontaneous release averaged approximately 11% of the incorporated chromium. The percent cytotoxicity was determined from the experimental (Cexp), spontaneous (c~) and maximal (cm) CPM using the formula: Percent cvtotoxicitv

-

Cexp

--

Crn - -

cs

×

100.

Cs

Cell-mediated cytotoxicity assays Statistical analyses Human colon tumor cells (RPMI 4788) were harvested after treatment with trypsin-EDTA (GIBCO), and the myeloid cell line K-562 (CCL 243; American Type Culture Collection, Rockville, MD) was cultured in suspension. For radioactive labelling, 0.5% Ci of NaS~CrO4 (10 mCi/ml, specific activity 500 Ci/g chromium; ICN Radiochemicals, Irvine, CA) were added to the cells and incubated for 2 h. The target cells were washed three times in medium and diluted in M199 assay medium to a

Statistical significance was determined by normalizing the values of each assay and using the paired Student's t-test.

Results

Sulfasalazine and sulfapyridine but not 5-ASA inhibited SCMC by control peripheral blood M N C

104 ss!

a g a i n s t c o l o n t u m o r cell line t a r g e t s a t all t h r e e c o n c e n t r a t i o n s s t u d i e d ( F i g . 1; p < 0.05). T h e I D s 0 f o r both sulfasalazine and sulfapyridine was approxix"..

O

., '-. T...........~ [ ....... ....... 2o|£11"~,". ",~,

mately 1 mM. Similar results were obtained using

.,."

K - 5 6 2 cell line t a r g e t s . A p p a r e n t s u p p r e s s i o n o f a n tibody-dependent

b

~.

cellular cytotoxicity and lectin-

induced cellular cytotoxicity was actually due to the effects o f t h e d r u g s o n S C M C

}

0.05 m M

inhibited SCMC

( T a b l e I). N D G A

( F i g . 2; p <

control peripheral blood MNC 05

0

50

20

'5

at

0.05) b y

as p r e v i o u s l y de-

s c r i b e d b y S e a m a n (1983). T h e I D s 0 w a s 0.03 m M

DRUG CONCENTRATION (raM)

for both targets. Although suppression was apparFig. 1. Effect of sulfasalazine (A), sulfapyridine ( 0 ) and 5-ASA (O) on SCMC by control peripheral blood MNC. Colon cell line target cells were incubated with effector cells at an effector to target ratio of 50:1. Inhibitors were added at concentrations of 0.5 mM, 1 mM and 2 mM at the beginning of an 18-h s~Cr release assay with cytotoxicity determined by percentage of total releasable 5'Cr with 10% Triton-X target dissolution, as described in the methods. Sulfasalazine and sulfapyridine significantly suppressed at all concentrations (p < 0.05). Similar results were seen at 25:1 and 10:1 effector to target cell ratios with colon cell line targets and with K-562 cell line cells as targets at all 3 effector to target cell ratios. Values represent the mean + SEM for 8 experiments.

ently produced by NDGA

in b o t h t h e a n t i b o d y - d e -

p e n d e n t a n d l e c t i n - i n d u c e d c e l l u l a r c y t o t o x i c i t y assays, t h i s a g a i n w a s a c t u a l l y d u e t o t h e effects o f NDGA

on SCMC

( T a b l e I). S i m i l a r l y , h y d r o c o r -

tisone inhibited spontaneous

but not antibody-de-

p e n d e n t k i l l i n g ( T a b l e I). H y d r o c o r t i s o n e , h o w e v e r , d i d i n h i b i t l e c t i n - i n d u c e d k i l l i n g ( T a b l e I). A cyclooxygenase inhibitor, indomethacin M, l x

10-5 M), and a thromboxane

i n h i b i t o r , b e n z y l i m i d a z o l e (3 x M),

were

without

effect o n

10 - 5 -

(1

x

10 6

synthetase 3 x

spontaneous,

10 4 anti-

TABLE 1 Effects of inhibitors on cytotoxicity by control peripheral blood MNC against colon target cells" Inhibitor

Concn. (M)

25 ± 7c

No Drug (8) Sulfasalazine (8)

I x

2 Sulfapyridine(8)

Nordihydroguaiaretic acid (7)

SCMC

10 3 M

I I ± 3*

4 ± 4**

× 10 . 3 M

l x 10 3 M 2 x 10 3 M

13 ± 3* 6 ± 2*

I x 10 - s M

16 ± 3

5 x I0 5 M Hydrocortisone(8)

6 ± 2*

I x 10 ~ M I x I0 3 M

-I2

ADCC b

ADCC-SCMC a LICC ~

LICC-SCMC a

62 ± 4

37 ± 10

53 ± 5

28 ± 8

53 ± 5*

39 ±

9

42 -k 3*

28 ± 5

43 ± 8**

39 ±

9

24 i 4**

20 ± 5

59 ± 5 56 ± 4

46 ± 50 ±

6 5

35 + 4** 29 ± 4**

22 ± 4 23 ± 4

54 ± 5*

39 ±

6

46 i

30 ± 6

41 ± 5**

35 ±

5

27 i 5**

5 ± 2**

58 ± 3

53 ±

2

± 4**

33 ± 6

45 ±

4

6

11 ± 3** -2

± 5**

21 ± 4 6 ± 2* 2 ± 4**

" Percent cytotoxicity: 50:1 killer:target ratio; 18-h assay. Data represent mean ± SEM. Numbers in parenthesis indicated the number of experiments, each performed in triplicate. b Antibody-dependent cellular cytotoxicity. c Lectin-induced cellular cytotxocity. a SCMC subtracted from ADCC or LICC in each individual experiment prior to calculating the mean + SEM. Similar results were obtained with K-562 cells as targets, and with lBD peripheral blood mononuclear cells as cffectors. * p < 0.05: ** p < 0.01. Statistically significant using correlated Student's t-test.

105 body-dependent, 3O )25

~"

20

k)

15

cell-mediated

s i g n i f i c a n t l y i n h i b i t e d S C M C by I B D p e r i p h e r a l b l o o d M N C a g a i n s t c o l o n t u m o r cell line t a r g e t s ""........

IT\\.\

(Fig. 3). S i m i l a r results w e r e o b t a i n e d w i t h K - 5 6 2

?iI

~z l0 k.) c~ m Q_

lectin-induced

0.01) a n d s u l f a p y r i d i n e (p < 0.005), b u t n o t 5 - A S A ,

U

o

or

c y t o t o x i c i t y ( d a t a n o t s h o w n ) . S u l f a s a l a z i n e (p <

5

0.005

I

I

0.01

0.05

NDGA CONCENTRATION(raM)

Fig. 2. Effect of NDGA on SCMC by control peripheral blood MNC. K-562 ( 0 ) and colon (O) cell line target cells were incubated with effector control peripheral blood MNC at an effector to target ratio of 50:1. NDGA was added at concentrationsof5 x 10-6,1 x 10-5, and5 x 10-5 M at the beginning of an 18-h s~Cr release assay with cytotoxicity determined by percentage of total releasable 51Cr with 10% Triton-X dissolution. NDGA (0.05 mM) significantly suppressed spontaneous cytotoxicity (p < 0.05). Similar results were seen at 25:1 and 10:1 effector to target cell ratios for both target cell types. Values represent the mean ± SEM for 7 experiments.

targets. In p r e i n c u b a t i o n studies, p e r i p h e r a l b l o o d M N C w e r e t r e a t e d w i t h s u l f a s a l a z i n e o r N D G A for 30 rain a n d t h e n i n t e r l e u k i n - 2 was a d d e d at t h e b e g i n n i n g o f the c y t o t o x i c i t y a s s a y (Fig. 4). S u l f a s a l a z i n e and NDGA

i n h i b i t e d S C M C (p < 0.05). H o w e v e r ,

apparent inhibition of interleukin-2-induced cytot o x i c i t y was d u e to i n h i b i t i o n o f S C M C . T h e r e f o r e , s i m i l a r to a n t i b o d y - d e p e n d e n t

and lectin-induced

c y t o t o x i c i t y ( T a b l e I), i n t e r l e u k i n - 2 - i n d u c e d killing is n o t i n h i b i t e d by s u l f a s a l a z i n e o r N D G A . Intestinal MNC

exhibited cytotoxicity against

c o l o n cell line t a r g e t s a f t e r i n c u b a t i o n

in m e d i a

a l o n e for 3 d a y s (Fig. 5), w h i l e fresh u n i n c u b a t e d i n t e s t i n a l cells e x h i b i t little o r n o lytic c a p a b i l i t y ( M a c D e r m o t t et al., 1980, 1986). A f t e r 72 h o f cult u r e in m e d i a a l o n e , c o n t r o l i n t e s t i n a l M N C

(Fig.

5A) e x h i b i t e d g r e a t e r S C M C (p < 0.05) t h a n t h o s e f r o m I B D p a t i e n t s (Fig. 5B). I n c u b a t i o n w i t h 5, 10 L o

25 80

70

~

No D,~gs

~o~

50

I

0

0)5

i

10

i

15

T0

DRUG CONCENTRATION (raM)

Fig. 3. Effect of sulfasalazine (A), sulfapyridine ( 0 ) and 5-ASA (O) on SCMC by IBD peripheral blood MNC. K-562 target cells were used at an effector to target ratio of 50:1. Inhibitors were added at concentrations of 0.5, 1 and 2 mM at the beginning of an 18-h 51Cr release assay with cytotoxicity determined by percentage of total releasable s lCr with 10% Triton-X target dissolution, as described in the Methods section. The values represent the mean 4- SEM for 6 experiments. Sulfasalazine caused significant suppression (p < 0.01) at I and 2 mM; while sulfapyridine significantly suppressed SCMC (p < 0.005) at all concentrations; 5-ASA did not significantly suppress SCMC at any concentration. Similar results were obtained with colon tumor cell line cells as targets at all effector to target cell ratios and at 25:1 and 10:1 killer to target cell ratios for K-562 cell line targets.

eL

I '

I

CONCENTRATION IL-2 (VOLUME :VOLUME)

Fig. 4. Effect of 2 m M sulfasalazine ( A ) and 5 x 10 .5 M N D G A (Fq) preincubation on interleukin-2-induced cell-mediated cytotoxicity toward K-562 cell line targets. Control per-

ipheral blood MNC were incubated for 30 minutes with or without nordihydroguaiaretic acid or sulfasalazine. Interleukin-2 was added in concentrations of 0%, 5%, 10% and 20% at the beginning of an 18-h slCr release assay (50:I effector to target cell ratio) with cytotoxicity determined by percentage of total releasable 51Cr with Triton-X target dissolution. The results shown are the mean ± SEM for 4 experiments.

106 NO DRUG A Control

2o © 8~

o

m l, l l l

0%

U > k#

5%

10%

20% iL-2

B Inflammatory Bowel Disease

}

5%

0%

I0%

20% [L-2

Fig. 5. Effect of 2 mM sulfasalazine (black) and 5 x 10 5 M N D G A (shaded) on SCMC and interleukin-2-induced cell-mediated cytotoxicity by intestinal MNC. Control (panel A) or IBD (panel B) intestinal MNC were incubated with media alone or interleukin-2 (IL-2) at concentrations of 0%, 5%, 10% and 20% (v/v) for 72 h. The cells were washed and resuspended in media. Sulfasalazine or N D G A was added at the beginning of an 18-h 51Cr release assay with colon tumor cell line targets at a killer to target ratio of 50:1. Cytotoxicity was determined as the percent of total releasable 51Cr. Bars represent the mean ± SEM for 4 experiments.

TABLE II The effect of B4 and 12-HETE on cytotoxicity by control peripheral blood MNC in the presence and absence of sulfasalazine" Buffer

LTB4(3)

0 10 12 M 1 x 10 9 M 1 x 10-TM 1 x

12-HETE(4) 0 10-aM 1 x 10-VM 1 x 10 6 M

1 x

Sulfasalazine (2 raM)

36 ± 9 32 ± 12 28± 7 32 ± 4 17 14 19 13

I ± 1 0 ± 0

1 ± 4 1 ±

1

±

3

±

2

0 ± 1 1 ±

± ±

2 1

4 ± 1 1 ± 1

1

a K-562 cell line targets; 50:1 killer:target ratio; 18-h assay. Values represent the mean ± SEM percent cytotoxicity. Numbers in parenthesis indicate the number of experiments, each performed in triplicate.

or 20% (v/v) interleukin-2 resulted in increased levels of cytotoxicity as shown by Fiocchi et al. (1985). Both sulfasalazine and N D G A significantly (p < 0.05) inhibited control intestinal MNC-mediated cytotoxicity due to inhibition of the SCMC recovered by the intestinal MNC during the 72-h culture period (Fig. 5A). Using IBD intestinal MNC as effectors (Fig. 5B), with the lower level of SCMC recovered during 72 h of culture, lymphokine-activated killing was inhibited by sulfasalazine and N D G A . Neither sulfapyradine nor 5-ASA inhibited SCMC or lymphokine-activated killing by control or IBD intestinal MNC. Because inhibition of the lipoxygenase pathway may be a mechanism by which sulfasalazine inhibits SCMC, reversal of this process was attempted with products of the lipoxygenase pathway. LTB4 or 12-HETE was added in the presence and absence of sulfasalazine (Table II), but neither had any effect on SCMC or the suppression of cytotoxicity induced by sulfasalazine.

Discussion

In this study we have demonstrated that sulfasalazine inhibits SCMC by control and IBD peripheral blood and intestinal MNC. The IDso for sulfasalazine ( ~ 1 mM) is well within the range of concentrations found in the colonic lumens of treated patients (Stenson, 1984). The IDso for inhibition of SCMC by control and IBD peripheral blood MNC by sulfapyridine (1 raM) is higher than either the serum or lumenal concentrations. In contrast, 5ASA had no effect on cytotoxicity by any cell population. Sulfasalazine, sulfapyridine and N D G A inhibited SCMC by control and IBD peripheral blood MNC, but did not have any effect on the additional cytotoxicity induced by the addition of antibody, lectin or interleukin-2. The potential interrelationship between arachidonic acid metabolism and cell-mediated cytotox±city has recently begun to be explored. In studies by Rola-Pleszczynski and coworkers (Rola-Pleszczynski et al., 1983, 1984; Gagnon et al., 1984), LTB4 enhanced cytotoxicity. Seaman (1983) found

107 that N D G A reversibly inhibits cytotoxicity by peripheral blood M N C in a dose-dependent manner. Cyclooxygenase inhibitors enhance cytotoxicity, although N D G A and inhibitors of thromboxane synthetase inhibit cytotoxicity (Seaman, 1983; Rola-Pleszczynski et al., 1983, 1984; Gagnon et al., 1984). Leung et al. also have found that N D G A inhibited natural killer activity in a dose-dependent fashion in rat spleen cells (Leung and Ip, 1984). Recent evidence suggests, therefore, that arachidonic acid metabolites may be involved in regulating cell-mediated cytotoxic processes. The data presented here demonstrate that sulfasalazine, sylfapyridine, hydrocortisone and N D G A inhibit SCMC. Whether or not this relates to their effects on the lipoxygenase pathway is far less clear. The IDs0 for sulfasalazine suppression of SCMC is 1 mM, which is the same as its IDso for lipoxygenase (Stenson and Lobos, 1982). The IDso for N D G A suppression of SCMC (30 /aM) is higher than its IDso for lipoxygenase (1 /~M) (Palmer and Salmon, 1985). This suggests that the effect of N D G A on cytotoxicity is not mediated by its effects on lipoxygenase. The inhibition of cytotoxicity by sulfapyridine is certainly not mediated by its effects on lipoxygenase since sulfapyridine is not a lipoxygenase inhibitor (Stenson and Lobos, 1982). This in turn suggests that the effects of sulfasalazine on cytotoxicity may not be mediated through the lipoxygenase pathway, if one is to assume that the suppression of cytotoxicity by both sulfasalazine and its cleavage product operate by similar mechanisms. Further evidence against a role for involvement of the lipoxygenase pathway is the failure of the lipoxygenase products LTB4 and 12-HETE to reverse the inhibition of cytotoxicity caused by sulfasalazine. The inability of these compounds to alter cytotoxicity does not prove that the lipoxygenase pathway is uninvolved in SCMC because it is possible that other lipoxygenase products would have effects. These results are remarkably similar to those of a recent study on the effects of eicosatetraynoic acid on cytotoxic T lymphocyte-mediated lysis in which eicosatetraynoic acid, a lipoxygenase inhibitor, was found to inhibit cytotoxic T lymphocyte-mediated lysis but this appeared to be unrelat-

ed to its effects on the lipoxygenase pathway (Taylor et al., 1985). Sulfasalazine is cleaved in the colon to sulfapyrdine and 5-ASA. The preponderance of evidence suggests that 5-ASA is the therapeutic agent and that sulfapyridine is not therapeutic (Klotz et al., 1980). The data presented here are not inconsistent with that evidence. As shown in the present study, sulfapyridine suppresses cytotoxicity only at concentrations of 1 raM, a concentration not achieved in man. Sulfasalazine also blocks cytotoxicity at a concentration of 1 mM, but this is well within the range of concentrations of sulfasalazine found in the colons of treated individuals (Stenson, 1984). Although there is evidence that 5-ASA is therapeutic, there is also evidence that the parent molecule, , sulfasalazine, has pharmacologic properties distinct from those of 5-ASA, and it is possible that sulfasalazine has therapeutic properties in its own right (Stenson, 1984). Thus sulfasalazine may work in part through inhibition of SCMC, whereas 5-ASA may work through separate mechanisms. The inhibition of cytotoxic function by sulfasalazine or sulfapyridine was not merely due to nonspecific toxicity toward the effector cells, in that sulfasalazine and sulfapyridine had no effect on lectin-induced, lymphokine-activated or antibody-dependent cellular cytotoxicity. The present study adds to the mechanisms (MacDermott et al., 1980, 1986; Falchuk et al., 1981; Fiocchi et al., 1985) of inducing intestinal MNC to exhibit cytotoxicity by demonstrating that 72 h of culture in media alone will result in potent cytotoxic effector cells. However, similar to recent data (MacDermott et al., 1986), IBD intestinal MNC are hyporesponsive compared to control intestinal MNC even after 72 h of incubation in media alone. There are two ways one might envisage a primary role for altered cytotoxicity in IBD. Excessive cytotoxicity which fails to distinguish normal from abnormal cells could result in damage to the intestine. On the other hand, impaired cytotoxicity could prevent an appropriate response to abnormal cells or to those infected with a causative agent. Our data provide support for a theory of spontaneous cell-mediated cytotoxic damage playing a role in the intestine in

108 IBD: drugs that are useful in treating IBD also appear to be able to inhibit SCMC by both peripheral blood and intestinal MNC. If cytotoxic effector cells are induced in the intestine in IBD, one of the therapeutic effects of drugs useful in the treatment of IBD (sulfasalazine and hydrocortisone) may be inhibition of SCMC.

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