Inhibition of Antibody Secretion by 5-Aminosalicylic Acid

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1 GASTROENTEROLOGY 1989;96:442-8 Inhibition of Antibody Secretion by 5-Aminosalicylic Acid RICHARD P. MACDERMOTT, SUANNE R. SCHLOEMANN, MICHAEL J. BERTOVICH, GEOFFREY S. NASH, MARION PETERS, and WILLIAM F. STENSON Division of Gastroenterology, Department of Medicine, Barnes and Jeish Hospitals, Washington University Medical Center, St. Louis, Missouri We have examined the effects of sulfasalazine and its metabolites sulfapyridine and 5-aminosalicylic acid on antibody secretion by normal peripheral blood and intestinal mononuclear cells. Sulfasalazine and 5-aminosalicylic acid both inhibited pokeeed mitogen-stimulated secretion of immunoglobulins (Igs) A, G, and M by peripheral blood mononuclear cells in a dose-dependent manner, hereas sulfapyridine had little effect. Sulfasalazine and 5-aminosalicylic acid also inhibited spontaneous secretion of IgA by intestinal mononuclear cells, but sulfapyridine did not. Sulfasalazine inhibited pokeeed mitogen-stimulated lymphocyte proliferation, hile 5-aminosalicylic acid and sulfapyridine exhibited minimal inhibition. Sulfasalazine as toxic for peripheral blood mononuclear cells, hereas 5-aminosalicylic acid and sulfapyridine ere not toxic. Thus, the inhibition of antibody secretion by sulfasalazine as due to direct toxicity. On the other hand, 5-aminosalicylic acid, the therapeutically active component of sulfasalazine, as neither toxic nor anti proliferative, and appeared to exert its effects on metabolic pathays directly related to antibody synthesis. The calculated ID5 values of 5-aminosalicylic acid for antibody secretion ere 1.35 mm for IgA and 1.5 mm for IgG, concentrations that are achieved in the colons of treated individuals. Indomethacin did not inhibit antibody secretion at pharmacologically relevant concentrations. 5-Aminosalicylic acid mediated inhibition of antibody secretion may play a role in inflammatory boel disease by stopping antibodymediated memory events involved in the induction or perpetuation of the disease process. There is increasing evidence that antibody-mediated processes could play an important role in the pathogenesis of inflammatory boel disease (IBD). Immunopathologic studies of the intestines of patients ith IBD have demonstrated that immunoglobulin (Ig) G-containing cells increase 3 times, IgA-containing cells to times, and IgM-containing cells five times above control values (1-5). Increased spontaneous secretion of immunoglobulins occurs from peripheral blood (PB) and intestinal mononuclear cells (MNC) from patients ith IBD (6-9). Sulfasalazine has been used in the therapy of IBD for many years. It is effective in the treatment of ulcerative colitis and Crohn's colitis as ell as in the maintenance of remissions of ulcerative colitis. Sulfasalazine is split by bacteria in the colon into sulfapyridine and 5-aminosalicylic acid (5-ASA). Whereas sulfapyridine is absorbed and metabolized in the liver, 5-ASA remains in the intestinal lumen and is the active compound in the treatment of IBD (1). Although the mechanism of action of sulfasalazine and 5-ASA is not knon, a number of potential mechanisms of action have been put forth. Both compounds inhibit cyclooxygenase (11) and 5-lipoxygenase (12), mechanisms that ould reduce the elevated levels of prostaglandin E z and leukotriene B4 in IBD mucosa. Sulfasalazine and 5-ASA are also free radical scavengers (13), Hoever, specific antibodies directed against colonic antigens could playa role in the induction of antigen-specific effector mechanisms in IBD. If so, an additional mechanism by hich sulfasalazine, or its therapeutically active metabolite 5-ASA, may exert its pharmacologic effects could be through inhibition of antibody secretion. Abbreviations used in this paper: 5-ASA, 5-aminosalicylic acid; IBD, inflammatory boel disease; MNC, mononuclear cells; PH, peripheral blood; PWM, pokeeed mitogen by the American Gastroenterological Association /89/$ 3.5

2 February ASA INHIBITS ANTIBODY SECRETION 443 Materials and Methods Isolation of Peripheral Blood Mononuclear Cells Peripheral blood MNC ere obtained from heparinized blood of healthy volunteers by fractionation over Ficoll-Hypaque gradients according to the method of Boyum (14). After isolation, the PB MNC ere ashed in the folloing assay medium: RPMI-164 (GIBCO, Grand Island, N.Y.), 2 mm L-glutamine (Sigma, St. Louis, Mo.), 2% penicillin-streptomycin (GIBCO), and 1% heat-inactivated fetal calf serum (GIBCO). Cells ere counted and viability as determined by trypan blue dye exclusion. Isolation of Human Intestinal Mononuclear Cells Intestinal tissue (large boel) as obtained as previously described (6,15,16) at the time of surgery from patients ith adenocarcinoma. (Uninvolved tissue, at least 1 cm from the site of the lesion, as used for control samples.) Mononuclear cells ere isolated from intestinal mucosa as described by Bookman and Bull (17) and modified by MacDermott et al. (6,8,15,16). After overnight digestion, the supernatant as collected, layered over a Ficoll-Hypaque gradient, and centrifuged at 4 g for 4 min. The interface cells ere harvested, ashed, resuspended in 1 ml of Percoll solution (Percoll diluted ith calcium-magnesium-free Hanks' balanced salt solution to a specific gravity of 1.4; Pharmacia, Piscataay, N.J.), and centrifuged at 5 g for 15 min. The cell pellet as resuspended in assay medium. Drugs Used in the Studies Sulfasalazine and sulfapyridine (Pharmacia, Uppsala, Seden) ere dissolved in.1 N NaOH, then media as added to bring the concentration to 6 mm, and the ph as adjusted to 7.4 ith.1 N HCl. 5-Aminosalicylic acid (Pharmacia) as dissolved in media, and brought to ph 7.4 ith.1 N NaOH. In Vitro Antibody Secretion Peripheral blood or intestinal MNC, cultured in vitro in assay medium alone or in the presence of pokeeed mitogen (PWM) (1:1 final dilution) at 2 x 1 6 MNC/ml, ere incubated at 37 C in a humidified 5% CO 2-95% air incubator in flat-bottom microtiter plates in.2 ml of assay medium for 14 days. The amount of IgA, IgG, and IgM secreted as determined by solid-phase radioimmunoassay (8,18). The results in terms of micrograms of antibody per milliliter of media in each assay ell (2 x 1 6 cells/ml) ere determined by linear regression analyses of the points on the standard curve and the geometric means and arithmetic means obtained. Mitogenesis and Cell Viability Studies Pokeeed mitogen-induced proliferation as carried out utilizing 2 x 1 5 MNC ith and ithout PWM (1: 1 i= 75 '" ~ '" <{ 5,9,y1 25 i=." I iii :I:... ~ -- ~ Sulfosolozine 5-ASA 1 Sulfopyridine,if om.1 1 DRUG CONCENTRATION (mm) Figure 1. Inhibition of 14-day PWM-stimulated IgA secretion by sulfasalazine and its component molecules 5-ASA and sulfapyridine. Varying dosages of the drugs ere incubated ith PB MNC from 6 normal donors for 14 days. The data are presented as arithmetic mean ± SEM percentage of inhibition of IgA secretion. 1, vol/vol) in 96-ell microtiter plates. Tritiated thymidine incorporation as measured during the last 4 h of a 72-h culture. Cell viability as determined by incubating PB MNC ith drugs at varying concentrations for 1, 2, or 3 days and the percentage of viable cells as determined using trypan blue exclusion. Statistical analyses ere performed by the Student's t-test. Results The effect of sulfasalazine, sulfapyridine, and 5-ASA on PWM-stimulated IgA secretion by PB MNC from six different normal donors during 14 days of in vitro culture is shon in Figure 1. Sulfasalazine had a marked inhibitory effect on PWMstimulated IgA secretion at concentrations >.1 mm, ith a 96.4% reduction of IgA secretion from 4.67 don to.17 f,tg/ml in cultures containing 2 mm sulfasalazine (p <.1). To minimize variability in results due to heterogeneity beteen different donors, the data are presented as mean percentage of inhibition of PWM-stimulated control cultures for each donor (Figure 1). The mean percentage of inhibition of IgA secretion induced by sulfasalazine as dose-dependent ith.1,.1, and 2 mm sulfasalazine concentrations resulting in -2.3%, 35.6%, and 89.2% inhibition of IgA secretion, respectively (Figure 1). Sulfapyridine, hich does not have a therapeutic role in the treatment of patients ith IBD, had little or no effect on IgA secretion (Figure 1). 5-Aminosalicylic acid, hich is the therapeutically active component of sulfasalazine, exhibited a

3 444 MACDERMOTT ET AL. GASTROENTEROLOGY Vol. 96, No.2, Part 1 dose-dependent inhibition of IgA secretion ith inhibition from the PWM-stimuJated control value of 4.6 to 2.82 JLg/ml in the presence of.5 mm 5-ASA (29.4% inhibition, p <.1), 2.5 JLg/ml in the presence of 1 mm 5-ASA (46.9% inhibition, p <.1), and.9 JLg/ml in the presence of 2 mm 5-ASA (62.2% inhibition, p <.1). Similar results ere observed for IgG and IgM secretion by PB MNC (data not shon). The ID5 for sulfasalazine inhibition of IgA secretion as.16 mm, of IgG secretion.2 mm, and of IgM secretion.36 mm. The ID5 for 5-ASA inhibition of IgA secretion as 1.35 mm and of IgG secretion 1.5 mm. Thus, both sulfasalazine and 5-ASA inhibit PWM-stimulated immunoglobulin secretion by PB MNC. We next examined the effects of sulfasalazine, sulfapyridine; and 5-ASA on the spontaneous synthesis and secretion of IgA by control intestinal MNC (Figure 2). Although PB MNC require stimulation (PWM) for antibody secretion, normal intestinal MNC spontaneously secrete large amounts of IgA ithout stimulation (6-8). Sulfasalazine markedly inhibited (p <.5) IgA synthesis and secretion by intestinal MNC ith an ID5 of.6 mm, hereas 5-ASA also produced a dose-dependent inhibition of IgA secretion by intestinal MNC ith an ID5 of 2. mm (Figure 2). Once again sulfapyridine had no effect on secretion of IgA. To determine if the inhibition caused by the drugs as related to an anti proliferative or toxic effect, e examined the effect of the drugs on lymphocyte proliferation and cellular viability. During a 14-day z 1 i= 75.: U W V'l <{ 5-ASA CI... LL. i= iii r... u.: W Q" Sullosalazine ~ Sulfapyridine t I I I I I III I I I I I II I, 1 DRUG CONCENTRATION (mm) Figure 2. Inhibition of spontaneous IgA secretion by control intestinal MNC cultured in vitro for 14 days ith sulfasalazine and its component molecules 5-ASA and sulfapyridine. The data are presented as arithmetic mean ± SEM percentage of inhibition of IgA secretion for four separate experiments. 1 ~ _ - Q - - Sulfasalazine - i. 5 - '1'---+" O ~ ~ = ~ - - ~ r - o L 5-ASA Sulfapyridine -25 l ~ t ~ ~ - - ' ~ ' ~ ' ~ ' u ' ~ U l' ~ llu -l ~ L - - L ' DRUG CONCENTRATION (mm) Figure 3. Percentage of suppression of PWM-induced mitogenesis after incubation ith sulfasalazine and its component molecules 5-ASA and sulfapyridine. The mean ± SEM percentage of suppression as calculated by comparing tritiated thymidine incorporation by PB MNC from 4 normal donors in the presence of drugs ith the PWM-stimulated control value. antibody secretion assay, the proliferation in response to PWM occurs ithin the first 72 h, after hich the stimulated cells then undergo differentiation into antibody-secreting plasma cells. As seen in Figure 3, varying concentrations of 5-ASA and sulfapyridine had little effect on suppression of PWM-stimulated lymphocyte proliferation of normal PB MNC during a 3-day culture period. In contrast, the parent compound, sulfasalazine, caused a marked inhibition of mitogenesis ith concentrations of sulfasalazine of >.5 mm resulting in 9.8%-93.5% inhibition of mitogenesis (p <.1, Figure 3). Similar effects of sulfasalazine, 5-ASA, and sulfapyridine ere observed at 7 and 1 days of culture (data not shon), ith 5-ASA exhibiting no inhibition of cell proliferation in comparison ith media controls. Therefore, the inhibition of IgA secretion by sulfasalazine as most likely related to a direct inhibitory effect on cellular proliferation, hereas the effect of 5-ASA as not due to inhibition of lymphocyte proliferation. One of the potential reasons for the suppression of proliferation by sulfasalazine could be direct cellular toxicity. As shon in Figure 4, varying concentrations of sulfapyridine and 5-ASA had minimal effects on viability during 3 days of culture. Sulfasalazine, in contrast, as markedly toxic to normal PWM-stimulated PB MNC, ith an 8.3% reduction in cell viability after 3 days in culture ith 3 mm sulfasalazine (p <.1). Similar results due to sulfasalazine, 5-ASA, and sulfapyridine ere ob-

4 February ASA INHIBITS ANTIBODY SECRETION 445 >!:: al «1 ;; crl 5 V... ~ ~ Sulfapyridine ASA Sulfasalazine.1 1 DRUG CONCENTRATION (mm ) Figure 4. Cell viability (%) of PE MNC cultured for 3 days ith sulfasalazine and its component molecules 5-ASA and sulfapyridine, as determined by trypan blue dye exclusion. Mean ± SEM for three experiments, calculated by comparing the number of viable cells in the presence of drug ith the PWM-stimulated control values. served at 7 and 1 days of culture (data not shon), ith 5-ASA exhibiting no direct toxic effects in comparison ith media controls. Therefore, the in vitro effects of sulfasalazine appear to be due to direct cell death, hereas the inhibition of IgA secretion by 5-ASA is not due to direct toxicity. We next examined the effect of pretreatment of PB MNC on the secretion of IgA (Table 1). Varying concentrations of sulfasalazine, sulfapyridine, and 5-ASA ere added to normal PB MNC for 1, 2, or 3 days. The cells ere ashed extensively to remove the drugs and then resuspended in media before a 14-day culture period ith PWM. The amounts of secreted immunoglobulins ere determined in the supernatants. Sulfasalazine had a dose- and timedependent inhibitory effect on subsequent IgA secretion most likely due to direct toxicity. On the other hand, neither sulfapyridine nor 5-ASA preincubation had any significant effect on subsequent immunoglobulin secretion, even at very high drug concentrations, indicating that the specific inhibitory effects of 5-ASA require the continued presence of the drug after the first 3 days of the culture period (Table 1). The lack of inhibitory effects after 5-ASA preincubation reinforces the conclusion that 5-ASA is not directly toxic, but instead exerts its effects through metabolic pathays directly related to antibody synthesis and secretion. To determine hether or not the in vitro effects of 5-ASA might have clinical relevance, e next examined the effects of indomethacin, a cyclooxygenase inhibitor that does not have beneficial effects in the treatment of patients ith IBD. As seen in Figure 5A, at pharmacologically relevant concentrations (1-3 J-LM) indomethacin had no effect on IgA secretion by either PWM-stimulated PB MNC or unstimulated intestinal MNC. Inhibition of antibody secretion by indomethacin as observed only at very high concentrations (5-1 J-LM) and indomethacin as more inhibitory for PWM-stimulated PB MNC than spontaneously secreting intestinal MNC. In addition, the mechanism of inhibition of antibody secretion by indomethacin at these high drug concentrations ould appear to be through an anti proliferative action, as it does not exhibit direct toxic effects (Figure 5B). Discussion There is evidence that antibody-mediated mechanisms may be of relevance and importance in Table 1. Effect of Drug Pretreatment of Peripheral Blood Mononuclear Cells on Immunoglobulin A Secretion by Normal Donors Q Pretreatment Percentage of control IgA secretion at No. of period Drug experiments (days) 3.mM 1. mm.5mm 5-ASA ± 47 b 12 ± ± ± ± ± ± ± ± 74 Sulfasalazine ± 3" 143 ± ± ± 8 e 3 ± ± ± 1" 4 ± 1 7 ± 6 d Sulfapyridine ± 3 11 ± ± ± ± 7 d 118 ± ± ± ± 42 IgA, immunoglobulin A. a Peripheral blood mononuclear cells from normal donors ere incubated at 37 C ith drugs and pokeeed mitogen (1:1 concentration) for 1, 2, or 3 days, ashed free of drugs, and incubated for 14 days at 37 C ith pokeeed mitogen. b Percentage of pokeeed mitogen-stimulated IgA secretion by peripheral blood mononuclear cells from normal donors (mean ± SEM). c p <.1. d P <.1.

5 446 MACDERMOTT ET AL. GASTROENTEROLOGY Vol. 96, No.2, Part 1 z Oz 1 75 ~ 5 O iili= I ~ 25 U... I-V) «WCI U... ~ ~ ~ 1 :::; iil z«5 75 i=-i ~ u : ; :J: U 5 ~ O I- «25 WV) ~ V i - 5 L L ~ ~ I U I ~ I! LI I~ W I" IW, L, I l~ l ~ I!-~ I ~ L-! I L1 ~ I 1 PWM-STIMULATED PERIPHERAL BLOOD MNC UNSTIMULATED It. (Spontaneous Secr.lion) / INTESTINAL MNC / MITOGENESIS W W CELL VIABILITY ~ z W O ~ ~ ~ - - ~ - - ~ ~ ~ ~ ~ g -25L1 - L ~ U W ~ l u l ~ I! I ~! I- ~ 1 -I UL I I ' ~ " ~ " I ~ 1 ID m DRUG CONCENTRATION (IJM) B Figure 5. Effect of indomethacin on antibody secretion, mitogenesis, and cell viability. The top panel (A) shos the percentage of inhibition (mean ± SEM) of IgA secretion due to indomethacin in culture for 14 days ith PWMstimulated PB MNC or unstimulated (spontaneously secreting) intestinal MNC. The bottom panel (B) shos the effect of indomethacin on mitogenesis and cell viability of PB MNC. the immunopathogenesis of IBD (19). In this study e have demonstrated that 5-ASA, the therapeutically active component of sulfasalazine, specifically inhibits the secretion of IgA, IgG, and IgM by normal PB MNC and IgA secretion by normal intestinal MNC during a 14-day in vitro culture period. The effects of 5-ASA ere not due to impairment of cell division or cell viability, as measured over a 14-day culture period. Sulfasalazine, the parent compound, as also able to inhibit antibody secretion, but this effect as due to direct toxicity, hich resulted in the blocking of cell division and decreased cell viability. Sulfapyridine, hich is the therapeutically ineffective component of sulfasalazine, had no effect on antibody secretion, proliferation, or cell viability. Indomethacin did not inhibit antibody secretion at pharmacologically relevant drug concentrations. Previous studies in humans have shon that sulfasalazine does not have an effect on the number of circulating T cells, immunoglobulin-bearing B cells, A or complement receptor-bearing lymphocytes (2). Additional studies demonstrated that sulfasalazine does not alter PB immune function in normal human controls (21). In contrast, immunologic alterations seen in patients ith IBD, including decreased T-cell percentages in PB, decreased lymphocyte proliferation, and the increased percentage of activated monocytes, ere reversed after treatment of patients ith sulfasalazine (21). Patients ith IBD ere found to have increased numbers of indomethacin-sensitive, prostaglandin-producing suppressor cells that ere not affected by sulfasalazine or corticosteroids (22). Prior studies have not examined the effects of sulfasalazine and its metabolites on in vitro antibody secretion. We have previously demonstrated that sulfapyridine but not 5-ASA inhibits spontaneous cell-mediated cytotoxicity by control and IBD PB cells (23,24). Other types of cytotoxicity, such as lectin-induced, antibody-dependent, and interleukin 2-induced cell-mediated cytotoxicity, ere not inhibited by sulfapyridine, but instead inhibitory effects in those assays ere due to the inhibition of underlying spontaneous cell-mediated cytotoxicity (23,24). Our previous studies have demonstrated that alterations occur in the spontaneous synthesis and secretion of IgA and IgG and their subclasses, hich could relate to the initiation or perpetuation of memory and effector immune events in IBD (6-9,19). We have observed that (a) spontaneous secretion of immunoglobulins in general, and IgA in particular, is increased from IBD PB MNC; (b) normal intestinal MNC spontaneously secrete large amounts of dimeric IgA, hereas IBD intestinal MNC exhibit decreased total IgA secretion; (c) there are increases in the percentage of monomeric IgA as ell as IgA subclass 1 secretion from both ulcerative colitis and Crohn's disease intestinal MNC; (d) intestinal MNC from ulcerative colitis colonic specimens secrete markedly increased amounts of IgG, hich is due to greatly increased production of IgG subclass 1; and (e) in contrast, Crohn's disease intestinal MNC secrete moderately increased amounts of IgG, hich is due in large part to increased IgG subclass 2 production (6-9,19). Therefore, major alterations in the synthesis and secretion patterns of immunoglobulins in general, and IgA and IgG and their subclasses in particular, occur ith IBD peripheral blood and intestinal mononuclear cells. The IgG subclasses represent specific responses to different types of antigens in both mice and humans, and are capable of inducing and mediating complementinduced as ell as cell-mediated processes that result in the destruction of target cells (25). The data presented here come from in vitro experiments performed ith PB MNC from normal indi-

6 February ASA INHIBITS ANTIBODY SECRETION 447 viduals. Previous studies (6-9) have demonstrated alterations in immunoglobulin secretion by intestinal MNC from patients ith led, and in some studies, these cells came from patients on sulfasalazine. The in vitro data presented here raise the question of ho these MNC exposed to 5-ASA in vivo ere capable of secreting immunoglobulin in vitro. There are several factors that may contribute to this finding. First, these cells came from surgically resected intestinal specimens; thus the patients ere selected for not having responded to medical therapy including sulfasalazine. It is thus possible that in vivo effects of 5-ASA on immunoglobulin secretion may have been present if the cells studied had come from patients ith led ho had responded clinically to sulfasalazine. Also, the patients had received preoperative antibiotics, hich ould have killed the bacteria required to cleave sulfasalazine and release 5-ASA. Finally, at pharmacologically relevant concentrations 5-ASA causes partial and not complete inhibition of antibody secretion. The present data do not define the mechanism by hich 5-ASA blocks antibody synthesis other than to establish that the effect is not just toxic or antiproliferative. The combination of the indomethacin data and the 5-ASA data suggests that the effects of 5-ASA on antibody synthesis do not relate to its effects on cyclooxygenase. At lo concentrations (1-3 /LM) the only pharmacologic effect of indomethacin is cyclooxygenase inhibition, hereas at higher concentrations indomethacin has numerous pharmacologic effects including phosphodiesterase inhibition (26). Indomethacin at very high concentrations (5-1 /LM) blocks antibody synthesis, apparently by blocking proliferation, but at concentrations that totally inhibit cyclooxygenase (1-3 /LM) indomethacin has no effect on antibody synthesis. Thus, it appears that cyclooxygenase inhibition does not block antibody synthesis in this system and that 5-ASA has its effect by some other mechanism. We have no demonstrated that 5-ASA, the therapeutically active metabolite of sulfasalazine, is capable of inhibiting antibody secretion, hereas sulfapyridine, hich is not therapeutically active, does not inhibit antibody secretion. The ability of 5-ASA to inhibit antibody secretion could be of importance ith regard to its ability to decrease the recurrence rate in patients ho take sulfasalazine long term. Because ulcerative colitis and Crohn's disease are recurrent relapsing disorders, inhibition of antibodymediated memory events (19,25) involved in directing destructive effector processes to the intestine could be a mechanism of 5-ASA action. Previous studies have delineated possible roles for 5-ASA in the inhibition of pro inflammatory events mediated by prostaglandins (11), leukotrienes (12), and oxygen radicals (13), hich may explain therapeutic mechanisms of action related to the acute inflammatory processes that occur in led. Although these proposed mechanisms of action, particularly lip oxygenase inhibition and free radical scavenging, are reasonable in terms of hat is knon about the pathogenesis of led, none of the drug effects has been firmly established as the mechanism of therapeutic efficacy for these drugs. The present study describes a possible role of 5-ASA in the inhibition of cellular recognition and effector pathays (19), hich may explain therapeutic mechanisms of action related to interruption of chronic cell-mediated autoimmune processes in led. References 1. Brandtzaeg p, Baklien K, Fausa, Hoel PS. Immunohistochemical characterization of local immunoglobulin formation in ulcerative colitis. Gastroenterology 1974;66: Baklien K, Brandtzaeg P. Comparative mapping of the local distribution of immunoglobulin-containing cells in ulcerative colitis and Crohn's disease of the colon. Clin Exp Immunol 1975;22: Rosekrans PCM, Meijer CJLM, Van der Wal AM, Cornelisse CJ, Lindeman J. Immunoglobulin containing cells in inflammatory boel disease of the colon: a morphometric and immunohistochemical study. Gut 198;21: Scott BB, Goodall A, Stephenson P, Jenkins D. Rectal mucosal plasma cells in inflammatory boel disease. Gut 1983;24: Keren DF, Appelman HD, Dobbins WO, et al. Correlation of histopathologic evidence of disease activity ith the presence of immunoglobulin-containing cells in the colons of patients ith inflammatory boel disease. Hum Pathol 1984;15: MacDermott RP, Nash GS, Bertovich MJ, Seiden MV, Bragdon MJ, Beale MG. Alterations of IgM, IgG, and IgA synthesis and secretion by peripheral blood and intestinal mononuclear cells from patients ith ulcerative colitis and Crohn's disease. Gastroenterology 1981:81: MacDermott RP, Beale MG, Alley CD, Nash GS, Bertovich MJ, Bragdon MJ. Synthesis and secretion of IgA, IgM, and IgG by peripheral blood mononuclear cells in human disease states, by isolated human intestinal mononuclear cells, and by human bone marro mononuclear cells from ribs. In: Mc Ghee JR, Mestecky J, eds. The secretory immune system. Annals of the Ne York Academy of Sciences, Volume 49. Ne York: Ne York Academy of Sciences, 1983: MacDermott RP, Nash GS, Bertovich MJ, et al. Altered patterns of secretion of monomeric IgA and IgA subclass 1 by intestinal mononuclear cells in inflammatory boel disease. Gastroenterology 1986;91: Scott MG, Nahm MH, Macke K, Nash GS, Bertovich MJ, MacDermott RP. Spontaneous secretion of IgG subclasses by intestinal mononuclear cells: differences beteen ulcerative colitis, Crohn's disease, and controls. Clin Exp Immunol 1986;66: Klotz U, Maier K, Fischer C, Heinkel K. Therapeutic efficacy of sulfasalazine and its metabolites in patients ith ulcerative colitis and Crohn's disease. N Engl J Med 198;33: Hakey CJ, Truelove SC. Inhibition of prostaglandin synthetase in human rectal mucosa. Gut 1983;24:213-7.

7 448 MACDERMOTT ET AL. GASTROENTEROLOGY Vol. 96, No.2, Part Stenson WF, Lobos E. Sulfasalazine inhibits the synthesis of chemotactic lipids by neutrophils. J Clin Invest 1982;69: , Miyachi Y, Yoshioka A, Imamura S, Nia Y. Effect of sulfasalazine and its metabolites on the generation of reactive oxygen species. Gut 1987;28: Boyum A. Separation of leucocytes from blood and bone marro. Scand J Clin Lab Invest 1968;21: MacDermott RP, Franklin GO, Jenkins KM, Kodner KJ. Nash GS, Weinrieb IJ. Human intestinal mononuclear cells: I. Investigation of antibody-dependent, lectin-induced, and spontaneous cell-mediated cytotoxic capabilities. Gastroenterology 198;78: MacDermott RP, Bragdon M1. Jenkins KM, Franklin GO, Shedlofsky S, Kodner KJ. Human intestinal mononuclear cells: II. Demonstration of a naturally occurring subclass of T cells hich respond in the allogeneic mixed leukocyte reaction but do not effect cell-mediated lympholysis. Gastroenterology 1981;8: Bookman MA, Bull DM. Characteristics of isolated intestinal mucosal lymphoid cells in inflammatory boel disease. Gastroenterology 1979;77: Nash GS, Seiden MV, Beale MG. MacDermott RP. A rapid miniaturized solid-phase radioimmunoassay technique for secreted immunoglobulins, employing microtiter plates, antibody bound to polyacrylamide beads, and filter strip harvesting. J Immunol Methods 1982;49: MacDermott RP, Stenson WF. Alterations of the immune system in ulcerative colitis and Crohn's disease. Adv ImmunoI1988;42: Thayer WR, Charland C, Field CE. Effects of sulfasalazine on selected lymphocyte subpopulations in vivo and in vitro. Dig Dis Sci 1979;24: Rubinstein A, Das KM, Melamed 1. Murphy RA. Comparative analysis of systemic immunological parameters in ulcerative colitis and idiopathic proctitis: effects of sulfasalazine in vivo and in vitro. Clin Exp ImmunoI1978;33: Holdstock G, Chastenay BF, Kraitt EL. Increased suppressor cell activity in inflammatory boel disease. Gut 1981;22: MacDermott RP, Kane MG, Steele LL, Stenson WF. Inhibition of cytotoxicity by sulfasalazine. I. Sulfasalazine inhibits spontaneous cell-mediated cytotoxicity by peripheral blood and intestinal mononuclear cells from control and inflammatory boel disease patients. Immunopharmacology 1986;11: Shanahan F, Niederlehner A, MacDermott RP, Stenson WF, Kane MG, Targan S. Inhibition of cytotoxicity by sulfasalazine. II. Sulfasalazine and sulfapyridine inhibit different stages of the NK and NKCF lytic processes. Immunopharmacology 1986;11: MacDermott RP, Nahm MH. Expression of human immunoglobulin G subclasses in inflammatory boel disease. Gastroenterology 1987;93: Stenson WF, Parker CWo Prostaglandins. In: Sirois P, Rola Pleszczynski M, eds. Immunopharmacology. Amsterdam: Elsevier. 1982: Received January 7, Accepted September 26, Address requests for reprints to: Richard P. MacDermott, M.D., Division of Gastroenterology, Box 8124, Washington University Medical Center, 66 South Euclid Avenue, St. Louis, Missouri This ork as supported in part by U.S. Public Health grants DK DK33487, and DK33165; and by grants from the National Foundation for Ileitis and Colitis and from Marion Laboratories. This ork as presented at the 89th annual meeting of the American Gastroenterological Association, May 18, 1988, and as published in abstract form (Gastroenterology 1988;94:A275).