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Prostanoid synthesis by cultured peripheral blood mononuclear cells in inflammatory diseases of the bowel
GASTROENTEROLOGY
1982:82:673-g
Prostanoid Synthesis by Cultured Peripheral Blood Mononuclear Cells in Inflammatory Diseases of the Bowel DANIEL RACHMILEWITZ, MOSHE LIGUMSKY, ADRIANA HAIMOVITZ, and ABRAHAM J. TREVES Departments of Gastroenterology, Radiation Hospital, The Hebrew University-Hadassah
and Clinical Oncology, Hadassah University Medical School, Jerusalem, Israel
Prostanoid synthesis by cultured peripheral blood mononuclear cells and monocytes in inflammatory bowel disease patients was determined because monocytosis was reported in inflammatory bowel diseases and prostanoids are synthesized by peripheral blood mononuclear cells in response to inflammatory stimuli. Prostaglandin EZ and thromboxane Bz accumulation in the medium of cultured peripheral blood mononuclear cells isolated from patients with active Crohn’s disease was two and three times higher than their respective accumulation by peripheral blood mononuclear cells isolated from normal subjects or patients in remission. In ulcerative colitis, prostaglandin Ez and thromboxane Bz accumulation was not enhanced. 6-Keto-prostaglandin F1, was not detected in any of the cultured medium. The absolute number of monocytes was determined according to their adherence to plastic surfaces, and the percent of phagocytic cells was significantly higher among peripheral blood mononuclear cells in patients with Crohn’s disease and ulcerative colitis as compared with peripheral blood mononuclear cells isolated from normal subjects. However, prostaglandin E2 secretion, by the same number of cultured monocytes isolated from all groups of patients, was similar. Flufenamic acid, methylprednisolone, and 5aminosalicylic acid significantly inhibited prostaglandin E, and thromboxane Bz accumula-
Received April 23, 1981. Accepted November 12, 1981. Address requests for reprints to: Daniel Rachmilewitz, M.D., Department of Gastroenterology, Hadassah Hospital, P. 0. BOX 12000. Jerusalem, Israel 91120. This study was supported in part by a grant from the National Foundation for Ileitis and Colitis, Inc. (U.S.A.), and by a grant from the Ministry of Health, State of Israel, to Daniel Rachmilewitz. The authors thank Ms. G. Halevi for her excellent technical help. 0 1982 by the American Gastroenterological 0016-5085/82/040673-07$02.50
Association
tion. These results suggest that in Crohn’s disease enhanced prostanoid synthesis is probably due to the monocytosis, whereas in ulcerative colitis the monocytosis is not accompanied by a significant increase in prostanoid synthesis in vitro. Ulcerative colitis and Crohn’s disease are two chronic inflammatory diseases of the bowel whose etiologies are still not known. In both diseases, monocytosis (1,2) and overproduction of monocytes (3) were reported. We have previously used transcobalamin II content in peripheral blood mononuclear (PBM) cells as an indicator of the monocyte response and found it also to be elevated in patients with active ulcerative colitis and Crohn’s disease (4). Irrespective of the specific etiology, prostanoids were suggested by us to mediate the inflammatory response in ulcerative colitis: enhanced prostaglandin Ez (PGE& thromboxane AZ (TXA2), and prostacyclin I2 (PGIJ syntheses by cultured rectal mucosa was observed during the active stage of the disease (5,6). Moreover, it was also suggested that tissue inflammatory cells are those responsible for the enhanced prostanoid synthesis (7). The aim of the present study was to determine and characterize prostanoid production by cultured PBM cells and monocytes in inflammatory bowel disease (IBD) during active and remission periods because: (a) tissue macrophages are recruited from peripheral blood monocytes and their precursors in the bone marrow (8), and (b) both monocytes and macrophages are among the cells reported to synthesize prostanoids in response to inflammatory stimuli (9).
Materials and Methods Patients Thirty milliliter of venous blood was drawn and mixed with ethylenediaminetetraacetate (EDTA) to a final
674
Table
GASTROENTEROLOGY Vol. 82, No. 4
RACHMILEWITZ ET AL.
1.
Sex and Age of Patients Bowel Disease
with Inflammatory Age
Sex W/F1
Range
Mean t SE
18
1216
17-69
35.9 t 4.2
17 12
1215 715
16-59 20-58
32.8 2 3.5 41.6 f 6.3
15 11
916 615 213
26-64 27-63 20-75
40.3 k 5.4 44.2 L 4.7 45.3 t 12.1
n
Normal subjects Ulcerative colitis Active Remission Crohn’s disease Active Remission Shigellosis
5
concentration of 0.1% from the following groups of patients: normal subjects without any signs or symptoms of gastrointestinal or other diseases; patients with active Crohn’s disease of the large or small intestine before therapy; patients suffering from Crohn’s disease during remission maintained by sulfasalazine; patients suffering from active ulcerative colitis before treatment; and patients with ulcerative colitis in remission maintained by sulfasalazine. Patients with shigellosis served as an additional control. The sex and age distribution of patients in the various groups is listed in Table 1. In all patients suffering from ulcerative colitis, the disease was diagnosed by sigmoidoscopy and confirmed histologically. Crohn’s disease was diagnosed radiologically and histologically. The clinical factors taken into consideration in assessing disease activity were: fever, abdominal pain, frequency of defecation, and increase in sedimentation rate. Shigellosis was confirmed by culturing the stools. Isolation Cells
of Peripheral
Blood
Mononuclear
Peripheral blood mononuclear cells were isolated as previously described by us in detail (10). The blood was centrifuged for 15 min at 250 g, and the supernatant, which contained most of the platelets, was discarded. The buffy coat was mixed with two volumes of phosphatebuffered saline (PBS) without Ca2+ and Mg2+ (pH 7.2) and separated by a standard Ficoll-Hypaque sedimentation for 20 min at 700 g. The cells were washed once with PBS and suspended in RPM1 1640 medium (Grand Island Biological Co., Santa Clara, Calif.) containing 2% heat-inactivated human AB+ serum (HS); the medium was supplemented with MEM vitamin solution (1:loo),penicillin (50 cLglm1) and streptomycin (50 Fg/ml), L-glutamine (2 mM), sodium pyruvate (1 mM), and HEPES buffer (10 mM). All media ingredients, except for the HS, were purchased from GIBCO. The PBM cells recovered by this procedure contained between 0% and 10% of the original number of platelets. Monocyte
Preparation
and Enumeration
Monolayers of monocytes were prepared by incubating 4 X lo6 PBM cells in 1 ml of medium in multiwell
plates (Linbro, Hamden, Conn.) for 1 h. The nonadherent cells were then removed by repeated pipeting, and fresh medium was added to the remaining adherent cells. The adherent cells consisted of >95% monocytes and OS-4% of lymphocytes as previously described by us (10). No granulocytes or platelets could be identified morphologically among the adherent cell population. After 24 h of incubation the cultured supernatants were removed, and the number of adherent cells in each well was evaluated by counting the number of nuclei collected from the well after incubation for 8 min with Zap-Oglobin (Coulter Electronics Ltd., Harpenden, England). The mean recovery of monocytes in different experiments was 82% of the phagocytic cells in the original sample of PBM cells. The percentage of monocytes in the PBM-cell samples was determined by counting the proportion of cells that had phagocytized more than five particles of latex (0.8 pm diameter, Sigma Chemical, Co., St. Louis, MO.). Latex phagocytosis was tested by incubation of 1 x 10” PBM cells in 1 ml of medium containing 0.02% of latex particles for 24 h in 12 x 75-mm silicon-coated tissue-culture tubes [Falcon, Oxnard, Calif.). Some of the differences between the number of phagocytic and adherent cells may be ascribed to the presence of an additional population of nonadherent phagocytic monocytes as previously described (10). Prostanoid
Secretion
by Cultured
Cells
Peripheral blood mononuclear cells ( 1 X 10' cells) or adherent cells derived from 4 x lo6 PBM cells were incubated for 24 h in 1 ml of medium containing 2% HS. Control cultures were incubated without cells. At the end of the incubation the media were collected, centrifuged for 5 min at 800 g, and frozen immediately at -20°C. Each experimental group consisted of media from two wells or tubes. In some experiments, PBM cells isolated from the same normal subjects were cultured in medium containing either sulfasalazine (50-200 pg/ml), sulfapyridine (5-500 pg/ml), or 5-aminosalicylic acid (5-ASA) (5-500 &ml), all obtained from Pharmacia, Uppsala, Sweden. In other experiments, methyl prednisolone (0.1-50.0 pg/ml) (Upjohn, Belgium) or flufenamic acid (0.1-10.0 pg/ml) (RAFA Laboratories, Jerusalem, Israel] were added to the medium. In these series of experiments, prostanoid accumulation in the medium of PBM cells cultured in drug-free medium served as control. Solutions of all drugs were prepared in 0.02 N NaOH, 25-100 ~1 of which were added to each well. The addition of these agents did not change the pH of the culture medium. At the end of the culture, phagocytic activity of the cells cultured in the presence of all drugs was determined by their phagocytosis of latex particles. All incubation procedures were performed in humidified 5% COz in an air incubator at 3 7°C. Viable cell counts were performed by the exclusion of 0.05% trypan blue. Prostaglandin Ez, 6-Keto-Prostaglandin and Thromboxane B, Determination
Flo,
Prostaglandin Ez was determined by radioimmunoassay (11). Antibodies were obtained from Miles
PROSTANOID SYNTHESIS BY MONONUCLEAR CELLS IN IBD
April 1982
675
were expressed as nanograms per prostanoid accumulated in the medium during 24-h culture of 1 x lo6PBM cells or 1 X lo5 monocytes. Statistical evaluation of the data was performed according to the Student’s t-test for unpaired data.
Results
licerative TFiFqGz
Coliti
Figure 1. Prostaglandin E, accumulation in the medium of tured PBM cells isolated from normal subjects, patients suffering from Crohn’s disease, ulcerative tis, and shigellosis. Shown are individual values mean ‘- SE.
culand coland
Laboratories, Rehovot, Israel. 6-Keto-prostaglandin F1, and TXBz were determined by radioimmunoassay as previously described by us in detail (6). In brief, rats were immunized with prostaglandin-bovine serum albumin conjugate and were bled lo-14 days after booster injections. The sera were characterized by a radioimmunoassay procedure, and a titer of 1:100,000was obtained after the third booster injection. The antisera was found to be 100% specific against either 6-keto-PGF1, or TXBz with a cross reaction of
During 24 h of culture, PBM cells isolated from all groups of patients were found to synthesize and secrete PGEz and TXBp to the culture medium. 6-Keto-prostaglandin F1, did not accumulate in the medium during the culture period. Prostaglandin E, accumulation in the medium of cultured PBM cells isolated from 15 patients with active Crohn’s disease and from 5 patients with shigellosis was two and three times higher than its respective accumulation by PBM cells isolated from 18 normal subjects (p < 0.01). Prostaglandin Ez accumulation by cultured PBM cells isolated from patients with Crohn’s disease in remission was significantly lower when compared with its accumulation by PBM cells isolated during the active state of the diseases (p < 0.01) and similar to its accumulation by PBM cells isolated from normal subjects. No significant difference in PGEz accumulation was noted between cultured PBM cells isolated from normal subjects or from patients with ulcerative colitis both during active and remission periods (Figure 1). Thromboxane B2 accumulation in the medium of cultured PBM cells isolated from normal subjects was similar to its accumulation by cultured PBM cells isolated from patients with Crohn’s disease in remission, patients with ulcerative colitis both during active and remission periods, and patients with shigellosis. Thromboxane B2 secretion by cultured PBM cells isolated from patients with active Crohn’s disease was twice as high as its secretion by PBM cells isolated from normal subjects (p < 0.05) (Figure 2). Thomboxane B2 accumulation by cultured PBM cells isolated from patients with shigellosis was higher but not significantly different from its accumulation by PBM cells isolated from normal subjects (Figure 2). In 5 patients with active Crohn’s disease, consecutively selected, PBM cells were isolated both at time of clinical activity and after improvement. In 3 of them, improvement was achieved by surgical resection of the diseased section of bowel. In all of them, both PGEz and TXBz accumulation in the medium was found to decrease with clinical improvement (Figure 3). In these patients the number of monocytes, measured as the percent of phagocytic cells in PBM cells, was reduced from 26.3 ? 8.0 (mean + SE) during clinical ment. The
activity number
to 14.3 of adherent
-+ 3.0 cells
at time obtained
of improvefrom
4 X
676
RACHMILEWITZ ET AL.
GASTROENTEROLOGY Vol. 82, No.
6.0-
50 CI ; LO\ ul = 2
3.0-
$& .p
0
2.0 - CzJ
V
0
m” c-”
1.0 O%ZO 0 0 00
O-
Normal Subject!
llcerative TTiiqzG -I_
Figure
(kohn’s
Colitis I
i
disease
higellosir
Ic(irr
i
2. Thromboxane B2 accumulation in the medium of cultured PBM cells isolated from normal subjects, and patients suffering from Crohn’s disease, ulcerative colitis, and shigellosis. Shown are individual values and mean t SE.
lo6 PBM cells and the percent of phagocytic cells among PBM cells isolated from the various groups of patients is given in Table 2. The percentage of monocytes, as indicated by those two parameters, was significantly higher among PBM cells isolated from patients with active ulcerative colitis and Crohn’s disease as compared with the percentage among PBM cells isolated from controls. The number of adherent cells was also increased in PBM cells isolated from patients in remission whereas in shigellosis only the percent of phagocytic cells was increased.
No significant difference in PGEz secretion by the same number of cultured monocytes isolated from all groups of patients and normal subjects was observed: 4.9 + 0.8, 3.7 + 0.8, 5.3 k 1.1, and 8.3 k 3.9 (mean k SE) rig/l x lo5 cells in monocytes isolated from normal subjects, patients with active ulcerative colitis, patients with Crohn’s disease, and patients with shigellosis, respectively. Thromboxane Bz secretion by cultured monocytes isolated from patients with active Crohn’s disease and shigellosis was significantly (p < 0.05) higher: 31.3 k 4.4 and 31.7 + 7.2 rig/l X lo5 cells, respectively, than its secretion by cultured monocytes isolated from normal subjects: 16.1 5 2.5 rig/l X lo5 cells. The effect of various drugs on PGE:! and TXB, accumulation by cultured PBM cells is detailed in Table 3. The addition of flufenamic acid (0.1-10.0 pglml) to the medium resulted in a dose response inhibition of PGE, and TXBz accumulation by cultured PBM cells isolated from normal subjects. Methyl prednisolone (0.1 pg/ml) did not affect PGE2 or TXB, accumulation. Methyl prednisolone (1 .O kg/ ml) induced a 45% inhibition of TXBz accumulation, whereas a higher dose (50 &ml) further inhibited TXBz accumulation and also induced a 60% inhibition of PGE2 accumulation. Salazopyrine (50-200 PgIml) did not affect significantly PGE2 accumulation, whereas only the higher dose inhibited TBX, accumulation. Sulfapyridine (5-500 pg/ml) also did not affect PGEz whereas 250 and 500 pug/ml inhibited TXBz accumulation by 38% and 57% respectively. 5Aminosalicylic acid (5 pg/ml) did not affect the accumulation of both prostanoids. A 30% inhibition of PGEz accumulation was noted after the addition of 50 pg/ml to the medium. Higher doses further inhibited PGE2, and they also induced a significant inhibition of TXBz accumulation. The phagocytic activity of PBM cells was also determined at the end of the culture with each of the above-mentioned drugs. No significant difference in the percent of phagocytic cells was noted among
Figure
months
4
3. Prostaglandin Ez (left panel) and TXBz (right panel) accumulation in the medium of cultured PBM cells isolated from patients suffering from Crohn’s disease during active and remission periods.
PROSTANOID
April1982
Table 2.
Number of Adherent Cells and Percent of Phagocytic Cells Among Peripheral Blood Mononuclear Cells
Normal subjects Ulcerative colitis Active Remission Crohn’s disease Active Remission Shigellosis
n
No. of adherent cells (x105)
18
4.7 2 0.5
12.9" 1.7
17 12
8.62 0.6" 7.22 0.6"
18.72 2.0' 14.0* 1.7
15 11 5
8.4t 0.8" 7.1k 0.7b 5.9r 1.3
23.52 3.6b 15.1+ 2.3 23.32 2.7"
Phagocytic cells (%)
Four million PBM cells recovered after Ficoll-Hypaque gradients were plated in multiwell plates for 90 min. Nonadherent cells were removed by repeated pipetting and those remaining attached to the plastic surface were counted after the addition of zapoglobin as described in Materials and Methods. Latex phagocytosis was determined by incubating 1 x 10” PBM cells in 1.0 ml of medium containing O.OZ"~of latex particles for 24 h. Results are mean * SE. Significantly different from normal subjects: a p < 0.01; b p < 0.02.
cells cultured in the absence or presence of the various concentrations of all drugs used, indicating that the viability of the cultured PBM cells was not affected.
SYNTHESIS
BY MONONUCLEAR
Prostanoids are among the major secretory products of monocytes and macrophages. Prostaglandin E mediates the inflammatory response (12) and the immune precursor cell proliferation (131, and regulates several mature immune cell functions (14). Thromboxane can oppose PGE actions in certain biologic systems (15). We have previously reported enhanced production of three prostanoids, PGE2, PG12, and TXA2, by the inflamed rectal mucosa in ulcerative colitis (5,6). Experiments recently performed in our laboratory confirmed enhanced colonic prostanoid synthesis also in active Crohn’s disease of the large bowel. These observations suggest that prostanoids may mediate the inflammatory response in ulcerative colitis and Crohn’s disease. Moreover, we have also established that intestinal inflammatory cells are those responsible for most of the enhanced colonic PGE, and TXA, synthesis (7). Tissue macrophages are recruited from peripheral blood mononuclear cells and their precursors in the bone marrow. Counting peripheral blood monocytes according to their morphologic identifications revealed monocytosis both in ulcerative colitis and in Crohn’s disease (1,2). We have confirmed this obseration by counting isolated adherent monocytes as
IN IBD
677
well as by counting the number of phagocytic cells in the peripheral blood. Plastic adherence and phagocytic activity are among the most useful criteria used to identify and isolate peripheral blood monocytes (10). Cultured PBM cells isolated from both normal subjects and patients with inflammatory diseases of the bowel were found to synthesize PGEB and TXB2, the stable metabolite of TXA2, both of which accumulated in the medium during the culture. During 24 h of culture, 6-keto-PGF,,, the stable metabolite of PGIZ, did not accumulate in the medium. It was previously reported that PBM cells do not synthesize this prostanoid (16)and that PGEz and TXBz are the major products released by human monocytes in day 1 of the culture after cell adherence (17). Prostaglandin Ez and TXB2 secretion by PBM cells isolated from patients with active Crohn’s disease was found to be two times higher than their secretion by PBM cells isolated from normal subjects. Prostaglandin Ez secretion by PBM cells isolated from patients with shigellosis was also increased. Prostanoid secretion by PBM cells was not enhanced in Crohn’s disease
Table 3.
Effect of Drugs on Prostanoid Accumulation by Cultured Peripheral Blood Mononuclear Cells
Drug
Discussion
CELLS
Flufenamic acidb 0.1 1.0 10.0 Methyl prednisoloneb 0.1
1.0 50.0
No.
PGE,”
TXB,”
4 5 4
85.02 32.0 31.9-t9.6' 13.6+ 8.4"
33.6-+2.7' 9.32 2.5' 3.12 1.8'
2 4 4
61.72 22.9 45.7T 6.5 39.6k 8.6"
75.85 12.7 55.22 6.0' 42.42 6.5c
5 5 4
153.0? 50.0 170.0? 68.8 257.0+ 107.0
104.02 20.0 67.52 15.6 22.02 8.0c
Salazopyrineb
50 100 200 Sulfapyridineb
71.4k 77.0+ 126.02 100.0k
5 50 250 500 5-Aminosalycilic 5 50 75 500
11.4 11.6 16.1 28.0
80.82 9.7 82.42 10.1 62.3+.7.3e 43.0" 4.8c
108.02 9.0 71.4rf7.9d 48.4t 9.4d 24.0-+6.4'
102.0? 18.4 74.42 16.2 41.0+-6.0' 3.8f 2.2'
acidb
Peripheral blood mononuclear cells were separated from normal subjects and cultured in medium containing each of the drugs for 24 h. Prostanoid accumulation in the medium was determined by radioimmunoassay as described in Materials and Methods. Prostanoid accumulation by PBM cells cultured in drug-free medium served as control and was regarded as 100%. Results are mean * SE. a Percent of accumulation in drug-free medium. b &ml. Significantly different from prostanoid accumulation in drug-free medium:” p < 0.01; d p < 0.02;e p -=c 0.05.
676
RACHMILEWITZ
ET AL.
patients in remission and in patients with active ulcerative colitis. When tested in the same individuals with Crohn’s disease, prostanoid synthesis by PBM cells was found to decrease with clinical improvement achieved either by medical therapy or following surgical resection of the diseased bowel. Enhanced prostanoid secretion by PBM cells in active Crohn’s disease may be either due to quantitative difference in their secretion by individual monocytes, or due to an absolute increase in the number of monocytes among PBM cells. The results reported indicate that the same number of monocytes isolated from Crohn’s disease patients and normal subjects secreted similar amounts of PGE2. It is therefore suggested that there is no qualitative difference and that the enhanced PGE;! secretion by PBM cells is due to an increase in the absolute number of monocytes in Crohn’s disease. In view of the similar increase in the number of monocytes observed in active ulcerative colitis and Crohn’s disease, the difference in their PBM-cell prostanoid production may be explained by other mechanisms such as either the presence of a prostanoid inhibitor in cells isolated in ulcerative colitis, or the presence of a separate subset of cells. A slight increase in TXBz secretion by monocytes was observed in active Crohn’s disease and shigellosis, indicating that in these two diseases monocytes differ in their capacity to secrete this prostanoid. Prostaglandin Ez and TXB, secretion by cultured PBM cells reflects their recent synthesis, as flufenamic acid, a cyclooxygenase inhibitor, induced about 90% inhibition of their accumulation. Methyl prednisolone also inhibited their synthesis although sulfasalazine in a high pharmacologic dose of 200 pg/ ml only inhibited TXBz and did not affect PGEz synthesis. Sulfapyridine in high concentrations 5(>250 pg/ml) inhibited only TXBz synthesis. Aminosalicylic acid, the other component of sulfasalazine, also inhibited PGEz accumulation only when added in a high dose of 50 pg/ml. These results are similar to our observations about sulfasalazine effects on prostanoid production by cultured rectal mucosa (6). The inhibition of PGEz and TXB2 accumulation induced by all drugs is not due to induction of irreversible cell damage, as latex phagocytosis regarded as a parameter of cell viability was not affected by any of them even when used in high concentrations. In active ulcerative colitis, circulating complement receptor-positive lymphocytes are increased, T-cell percentages and lymphocyte functions are decreased, and erythrocyte antibody component phagocytozing esterase-positive cells-indicative of activated monocytes-are demonstrated. Whereas successful treatment with sulfasalazine reversed
GASTROENTEROLOGY
Vol. 82, No. 4
these immunologic changes, in vitro incubation of sulfasalzine and its metabolites with leukocytes did not alter these immunologic changes (18). Similar results were obtained in the present study; in vitro incubation of cultured PBM cells obtained from normal subjects with therapeutic plasma concentrations of sulfasalazine or its constituents failed to inhibit PGEz or TXB2 synthesis whereas in Crohn’s disease patients in remission maintained by sulfasalzine the enhanced prostanoid production by cultured PBM cells was reversed to normal. It is still not clear which is the active moiety in sulfasalazine and what is the mechanism responsible for its therapeutic efficacy in IBD. The discrepancy between the in vitro and in vivo drug effects may suggest an indirect effect of the drug, but more work and probably other approaches are needed to clarify this issue.
References 1. Auer IO, Wechsler W, Ziemer E, et al. Immune status in Crohn’s disease. I. Leukocyte and lymphocyte subpopulations in peripheral blood. Stand J Gastroenterol 1978;13:56171. 2. Thayer WR, Charland C, Field CA. The subpopulations of circulating white blood cells in inflammatory bowel disease. Gastroenterology 1976;71:379-84. 3. Meuret G, Bitzi A, Hammer B. Macrophage turnover in Crohn’s disease and ulcerative colitis. Gastroenterology 1978;74:501-3. 4. Rachmilewitz D, Ligumsky M, Rachmilewitz B, et al. Transcobalamin II level in peripheral blood monocytes-a biochemical marker in inflammatory diseases of the bowel. Gastroenterology 1980;78:43-6. 5. Sharon P, Ligumsky M, Rachmilewitz D, et al. Role of prostaglandins in ulcerative coltis. Enhanced production during active disease and inhibition by sulfasalazine. Gastroenterology 1978;75:638-40. 6 Ligumsky M, Karmeli F, Sharon P, et al. Enhanced thromboxane Az and prostacyclin production by cultured rectal mucosa in ulcerative colitis and its inhibition by steroids and sulfasalazine. Gasteroenterology 1981;81:444-9. 7. Rachmilewitz D, Zifroni A, Ligumsky M, et al. Enhanced prostanoids synthesis by cultured peripheral blood mononuclears (PBM) and intestinal lymphoid cells in Crohn’s disease (abstr). Gastroenterology 196i;80:1256. 8. Spector WG. The macrophage in inflammation. Ser Haematol 1970;3:132-44. 9. Humes JL, Bonney RJ, Pelus L, et al. Macrophages synthesize and release prostaglandins in response to inflammatory stimuli. Nature 1977;269:149. 10. Treves AJ, Yagoda D, Haimovitz A, e“1 al. The isolation and purification of human peripheral blood monocytes in cell suspension. J Immunol Methods 1980;39:71-80. 11. Bauminger S, Zor U, Lindner HR. Radioimmunological assay of prostaglandin synthetase activity. Prostanglandins 1973; 4:313-24. 12. Ferreira SH, Moncada S, Vane JR. Prostaglandins and signs and symptoms of inflammation. In: Robinson HJ, Vane JR, eds. Prostaglandin synthetase inhibitors. New York: Raven Press, 1974:175-87. 13. Bockman RS, Rothschild M. Prostaglandin E inhibition of Tlymphocyte colony formation: a possible mechanism of
PROSTANOID
April 1982
monocyte
modulation
of clonal
expansion.
J Clin Invest
1979:64:812-Q. 14. Droller
MJ, Schneider MU, Perlman P. A possible role of prostaglandins in the inhibition of natural and antibody dependent cell-mediated cytotoxicity against tumor cells. Cell Immunol 1978;39:165-77. 15. Miller OU, Johnson RA, Gorman RR. Inhibition of PGE, stimulated CAMP accumulation in human platelets by thromboxane AZ. Prostaglandins 1977;13:599-609. 16. Kenney MS, Stobo JD, Goldyne ME. In vitro synthesis of
SYNTHESIS
BY MONONUCLEAR
CELLS IN IBD
679
prostaglandins and related lipids by populations of peripheral blood mononuclear cells. Prostaglandins 1980;20:135-45. 17. Bockman RS. Prostaglandin production by human blood monocytes and mouse peritoneal macrophages: synthesis dependent on in vitro culture conditions. Prostaglandins 1981;21:9-31. 18. Rubinstein A, Das KM, Melamed J, et al. Comparative analysis of systemic immunological parameters in ulcerative colitis and idiopathic proctitis: effects of sulfasalazine in vivo and in vitro. Clin Exp Immunol 1978:33:217-224.
1982:82:673-g
Prostanoid Synthesis by Cultured Peripheral Blood Mononuclear Cells in Inflammatory Diseases of the Bowel DANIEL RACHMILEWITZ, MOSHE LIGUMSKY, ADRIANA HAIMOVITZ, and ABRAHAM J. TREVES Departments of Gastroenterology, Radiation Hospital, The Hebrew University-Hadassah
and Clinical Oncology, Hadassah University Medical School, Jerusalem, Israel
Prostanoid synthesis by cultured peripheral blood mononuclear cells and monocytes in inflammatory bowel disease patients was determined because monocytosis was reported in inflammatory bowel diseases and prostanoids are synthesized by peripheral blood mononuclear cells in response to inflammatory stimuli. Prostaglandin EZ and thromboxane Bz accumulation in the medium of cultured peripheral blood mononuclear cells isolated from patients with active Crohn’s disease was two and three times higher than their respective accumulation by peripheral blood mononuclear cells isolated from normal subjects or patients in remission. In ulcerative colitis, prostaglandin Ez and thromboxane Bz accumulation was not enhanced. 6-Keto-prostaglandin F1, was not detected in any of the cultured medium. The absolute number of monocytes was determined according to their adherence to plastic surfaces, and the percent of phagocytic cells was significantly higher among peripheral blood mononuclear cells in patients with Crohn’s disease and ulcerative colitis as compared with peripheral blood mononuclear cells isolated from normal subjects. However, prostaglandin E2 secretion, by the same number of cultured monocytes isolated from all groups of patients, was similar. Flufenamic acid, methylprednisolone, and 5aminosalicylic acid significantly inhibited prostaglandin E, and thromboxane Bz accumula-
Received April 23, 1981. Accepted November 12, 1981. Address requests for reprints to: Daniel Rachmilewitz, M.D., Department of Gastroenterology, Hadassah Hospital, P. 0. BOX 12000. Jerusalem, Israel 91120. This study was supported in part by a grant from the National Foundation for Ileitis and Colitis, Inc. (U.S.A.), and by a grant from the Ministry of Health, State of Israel, to Daniel Rachmilewitz. The authors thank Ms. G. Halevi for her excellent technical help. 0 1982 by the American Gastroenterological 0016-5085/82/040673-07$02.50
Association
tion. These results suggest that in Crohn’s disease enhanced prostanoid synthesis is probably due to the monocytosis, whereas in ulcerative colitis the monocytosis is not accompanied by a significant increase in prostanoid synthesis in vitro. Ulcerative colitis and Crohn’s disease are two chronic inflammatory diseases of the bowel whose etiologies are still not known. In both diseases, monocytosis (1,2) and overproduction of monocytes (3) were reported. We have previously used transcobalamin II content in peripheral blood mononuclear (PBM) cells as an indicator of the monocyte response and found it also to be elevated in patients with active ulcerative colitis and Crohn’s disease (4). Irrespective of the specific etiology, prostanoids were suggested by us to mediate the inflammatory response in ulcerative colitis: enhanced prostaglandin Ez (PGE& thromboxane AZ (TXA2), and prostacyclin I2 (PGIJ syntheses by cultured rectal mucosa was observed during the active stage of the disease (5,6). Moreover, it was also suggested that tissue inflammatory cells are those responsible for the enhanced prostanoid synthesis (7). The aim of the present study was to determine and characterize prostanoid production by cultured PBM cells and monocytes in inflammatory bowel disease (IBD) during active and remission periods because: (a) tissue macrophages are recruited from peripheral blood monocytes and their precursors in the bone marrow (8), and (b) both monocytes and macrophages are among the cells reported to synthesize prostanoids in response to inflammatory stimuli (9).
Materials and Methods Patients Thirty milliliter of venous blood was drawn and mixed with ethylenediaminetetraacetate (EDTA) to a final
674
Table
GASTROENTEROLOGY Vol. 82, No. 4
RACHMILEWITZ ET AL.
1.
Sex and Age of Patients Bowel Disease
with Inflammatory Age
Sex W/F1
Range
Mean t SE
18
1216
17-69
35.9 t 4.2
17 12
1215 715
16-59 20-58
32.8 2 3.5 41.6 f 6.3
15 11
916 615 213
26-64 27-63 20-75
40.3 k 5.4 44.2 L 4.7 45.3 t 12.1
n
Normal subjects Ulcerative colitis Active Remission Crohn’s disease Active Remission Shigellosis
5
concentration of 0.1% from the following groups of patients: normal subjects without any signs or symptoms of gastrointestinal or other diseases; patients with active Crohn’s disease of the large or small intestine before therapy; patients suffering from Crohn’s disease during remission maintained by sulfasalazine; patients suffering from active ulcerative colitis before treatment; and patients with ulcerative colitis in remission maintained by sulfasalazine. Patients with shigellosis served as an additional control. The sex and age distribution of patients in the various groups is listed in Table 1. In all patients suffering from ulcerative colitis, the disease was diagnosed by sigmoidoscopy and confirmed histologically. Crohn’s disease was diagnosed radiologically and histologically. The clinical factors taken into consideration in assessing disease activity were: fever, abdominal pain, frequency of defecation, and increase in sedimentation rate. Shigellosis was confirmed by culturing the stools. Isolation Cells
of Peripheral
Blood
Mononuclear
Peripheral blood mononuclear cells were isolated as previously described by us in detail (10). The blood was centrifuged for 15 min at 250 g, and the supernatant, which contained most of the platelets, was discarded. The buffy coat was mixed with two volumes of phosphatebuffered saline (PBS) without Ca2+ and Mg2+ (pH 7.2) and separated by a standard Ficoll-Hypaque sedimentation for 20 min at 700 g. The cells were washed once with PBS and suspended in RPM1 1640 medium (Grand Island Biological Co., Santa Clara, Calif.) containing 2% heat-inactivated human AB+ serum (HS); the medium was supplemented with MEM vitamin solution (1:loo),penicillin (50 cLglm1) and streptomycin (50 Fg/ml), L-glutamine (2 mM), sodium pyruvate (1 mM), and HEPES buffer (10 mM). All media ingredients, except for the HS, were purchased from GIBCO. The PBM cells recovered by this procedure contained between 0% and 10% of the original number of platelets. Monocyte
Preparation
and Enumeration
Monolayers of monocytes were prepared by incubating 4 X lo6 PBM cells in 1 ml of medium in multiwell
plates (Linbro, Hamden, Conn.) for 1 h. The nonadherent cells were then removed by repeated pipeting, and fresh medium was added to the remaining adherent cells. The adherent cells consisted of >95% monocytes and OS-4% of lymphocytes as previously described by us (10). No granulocytes or platelets could be identified morphologically among the adherent cell population. After 24 h of incubation the cultured supernatants were removed, and the number of adherent cells in each well was evaluated by counting the number of nuclei collected from the well after incubation for 8 min with Zap-Oglobin (Coulter Electronics Ltd., Harpenden, England). The mean recovery of monocytes in different experiments was 82% of the phagocytic cells in the original sample of PBM cells. The percentage of monocytes in the PBM-cell samples was determined by counting the proportion of cells that had phagocytized more than five particles of latex (0.8 pm diameter, Sigma Chemical, Co., St. Louis, MO.). Latex phagocytosis was tested by incubation of 1 x 10” PBM cells in 1 ml of medium containing 0.02% of latex particles for 24 h in 12 x 75-mm silicon-coated tissue-culture tubes [Falcon, Oxnard, Calif.). Some of the differences between the number of phagocytic and adherent cells may be ascribed to the presence of an additional population of nonadherent phagocytic monocytes as previously described (10). Prostanoid
Secretion
by Cultured
Cells
Peripheral blood mononuclear cells ( 1 X 10' cells) or adherent cells derived from 4 x lo6 PBM cells were incubated for 24 h in 1 ml of medium containing 2% HS. Control cultures were incubated without cells. At the end of the incubation the media were collected, centrifuged for 5 min at 800 g, and frozen immediately at -20°C. Each experimental group consisted of media from two wells or tubes. In some experiments, PBM cells isolated from the same normal subjects were cultured in medium containing either sulfasalazine (50-200 pg/ml), sulfapyridine (5-500 pg/ml), or 5-aminosalicylic acid (5-ASA) (5-500 &ml), all obtained from Pharmacia, Uppsala, Sweden. In other experiments, methyl prednisolone (0.1-50.0 pg/ml) (Upjohn, Belgium) or flufenamic acid (0.1-10.0 pg/ml) (RAFA Laboratories, Jerusalem, Israel] were added to the medium. In these series of experiments, prostanoid accumulation in the medium of PBM cells cultured in drug-free medium served as control. Solutions of all drugs were prepared in 0.02 N NaOH, 25-100 ~1 of which were added to each well. The addition of these agents did not change the pH of the culture medium. At the end of the culture, phagocytic activity of the cells cultured in the presence of all drugs was determined by their phagocytosis of latex particles. All incubation procedures were performed in humidified 5% COz in an air incubator at 3 7°C. Viable cell counts were performed by the exclusion of 0.05% trypan blue. Prostaglandin Ez, 6-Keto-Prostaglandin and Thromboxane B, Determination
Flo,
Prostaglandin Ez was determined by radioimmunoassay (11). Antibodies were obtained from Miles
PROSTANOID SYNTHESIS BY MONONUCLEAR CELLS IN IBD
April 1982
675
were expressed as nanograms per prostanoid accumulated in the medium during 24-h culture of 1 x lo6PBM cells or 1 X lo5 monocytes. Statistical evaluation of the data was performed according to the Student’s t-test for unpaired data.
Results
licerative TFiFqGz
Coliti
Figure 1. Prostaglandin E, accumulation in the medium of tured PBM cells isolated from normal subjects, patients suffering from Crohn’s disease, ulcerative tis, and shigellosis. Shown are individual values mean ‘- SE.
culand coland
Laboratories, Rehovot, Israel. 6-Keto-prostaglandin F1, and TXBz were determined by radioimmunoassay as previously described by us in detail (6). In brief, rats were immunized with prostaglandin-bovine serum albumin conjugate and were bled lo-14 days after booster injections. The sera were characterized by a radioimmunoassay procedure, and a titer of 1:100,000was obtained after the third booster injection. The antisera was found to be 100% specific against either 6-keto-PGF1, or TXBz with a cross reaction of
During 24 h of culture, PBM cells isolated from all groups of patients were found to synthesize and secrete PGEz and TXBp to the culture medium. 6-Keto-prostaglandin F1, did not accumulate in the medium during the culture period. Prostaglandin E, accumulation in the medium of cultured PBM cells isolated from 15 patients with active Crohn’s disease and from 5 patients with shigellosis was two and three times higher than its respective accumulation by PBM cells isolated from 18 normal subjects (p < 0.01). Prostaglandin Ez accumulation by cultured PBM cells isolated from patients with Crohn’s disease in remission was significantly lower when compared with its accumulation by PBM cells isolated during the active state of the diseases (p < 0.01) and similar to its accumulation by PBM cells isolated from normal subjects. No significant difference in PGEz accumulation was noted between cultured PBM cells isolated from normal subjects or from patients with ulcerative colitis both during active and remission periods (Figure 1). Thromboxane B2 accumulation in the medium of cultured PBM cells isolated from normal subjects was similar to its accumulation by cultured PBM cells isolated from patients with Crohn’s disease in remission, patients with ulcerative colitis both during active and remission periods, and patients with shigellosis. Thromboxane B2 secretion by cultured PBM cells isolated from patients with active Crohn’s disease was twice as high as its secretion by PBM cells isolated from normal subjects (p < 0.05) (Figure 2). Thomboxane B2 accumulation by cultured PBM cells isolated from patients with shigellosis was higher but not significantly different from its accumulation by PBM cells isolated from normal subjects (Figure 2). In 5 patients with active Crohn’s disease, consecutively selected, PBM cells were isolated both at time of clinical activity and after improvement. In 3 of them, improvement was achieved by surgical resection of the diseased section of bowel. In all of them, both PGEz and TXBz accumulation in the medium was found to decrease with clinical improvement (Figure 3). In these patients the number of monocytes, measured as the percent of phagocytic cells in PBM cells, was reduced from 26.3 ? 8.0 (mean + SE) during clinical ment. The
activity number
to 14.3 of adherent
-+ 3.0 cells
at time obtained
of improvefrom
4 X
676
RACHMILEWITZ ET AL.
GASTROENTEROLOGY Vol. 82, No.
6.0-
50 CI ; LO\ ul = 2
3.0-
$& .p
0
2.0 - CzJ
V
0
m” c-”
1.0 O%ZO 0 0 00
O-
Normal Subject!
llcerative TTiiqzG -I_
Figure
(kohn’s
Colitis I
i
disease
higellosir
Ic(irr
i
2. Thromboxane B2 accumulation in the medium of cultured PBM cells isolated from normal subjects, and patients suffering from Crohn’s disease, ulcerative colitis, and shigellosis. Shown are individual values and mean t SE.
lo6 PBM cells and the percent of phagocytic cells among PBM cells isolated from the various groups of patients is given in Table 2. The percentage of monocytes, as indicated by those two parameters, was significantly higher among PBM cells isolated from patients with active ulcerative colitis and Crohn’s disease as compared with the percentage among PBM cells isolated from controls. The number of adherent cells was also increased in PBM cells isolated from patients in remission whereas in shigellosis only the percent of phagocytic cells was increased.
No significant difference in PGEz secretion by the same number of cultured monocytes isolated from all groups of patients and normal subjects was observed: 4.9 + 0.8, 3.7 + 0.8, 5.3 k 1.1, and 8.3 k 3.9 (mean k SE) rig/l x lo5 cells in monocytes isolated from normal subjects, patients with active ulcerative colitis, patients with Crohn’s disease, and patients with shigellosis, respectively. Thromboxane Bz secretion by cultured monocytes isolated from patients with active Crohn’s disease and shigellosis was significantly (p < 0.05) higher: 31.3 k 4.4 and 31.7 + 7.2 rig/l X lo5 cells, respectively, than its secretion by cultured monocytes isolated from normal subjects: 16.1 5 2.5 rig/l X lo5 cells. The effect of various drugs on PGE:! and TXB, accumulation by cultured PBM cells is detailed in Table 3. The addition of flufenamic acid (0.1-10.0 pglml) to the medium resulted in a dose response inhibition of PGE, and TXBz accumulation by cultured PBM cells isolated from normal subjects. Methyl prednisolone (0.1 pg/ml) did not affect PGE2 or TXB, accumulation. Methyl prednisolone (1 .O kg/ ml) induced a 45% inhibition of TXBz accumulation, whereas a higher dose (50 &ml) further inhibited TXBz accumulation and also induced a 60% inhibition of PGE2 accumulation. Salazopyrine (50-200 PgIml) did not affect significantly PGE2 accumulation, whereas only the higher dose inhibited TBX, accumulation. Sulfapyridine (5-500 pg/ml) also did not affect PGEz whereas 250 and 500 pug/ml inhibited TXBz accumulation by 38% and 57% respectively. 5Aminosalicylic acid (5 pg/ml) did not affect the accumulation of both prostanoids. A 30% inhibition of PGEz accumulation was noted after the addition of 50 pg/ml to the medium. Higher doses further inhibited PGE2, and they also induced a significant inhibition of TXBz accumulation. The phagocytic activity of PBM cells was also determined at the end of the culture with each of the above-mentioned drugs. No significant difference in the percent of phagocytic cells was noted among
Figure
months
4
3. Prostaglandin Ez (left panel) and TXBz (right panel) accumulation in the medium of cultured PBM cells isolated from patients suffering from Crohn’s disease during active and remission periods.
PROSTANOID
April1982
Table 2.
Number of Adherent Cells and Percent of Phagocytic Cells Among Peripheral Blood Mononuclear Cells
Normal subjects Ulcerative colitis Active Remission Crohn’s disease Active Remission Shigellosis
n
No. of adherent cells (x105)
18
4.7 2 0.5
12.9" 1.7
17 12
8.62 0.6" 7.22 0.6"
18.72 2.0' 14.0* 1.7
15 11 5
8.4t 0.8" 7.1k 0.7b 5.9r 1.3
23.52 3.6b 15.1+ 2.3 23.32 2.7"
Phagocytic cells (%)
Four million PBM cells recovered after Ficoll-Hypaque gradients were plated in multiwell plates for 90 min. Nonadherent cells were removed by repeated pipetting and those remaining attached to the plastic surface were counted after the addition of zapoglobin as described in Materials and Methods. Latex phagocytosis was determined by incubating 1 x 10” PBM cells in 1.0 ml of medium containing O.OZ"~of latex particles for 24 h. Results are mean * SE. Significantly different from normal subjects: a p < 0.01; b p < 0.02.
cells cultured in the absence or presence of the various concentrations of all drugs used, indicating that the viability of the cultured PBM cells was not affected.
SYNTHESIS
BY MONONUCLEAR
Prostanoids are among the major secretory products of monocytes and macrophages. Prostaglandin E mediates the inflammatory response (12) and the immune precursor cell proliferation (131, and regulates several mature immune cell functions (14). Thromboxane can oppose PGE actions in certain biologic systems (15). We have previously reported enhanced production of three prostanoids, PGE2, PG12, and TXA2, by the inflamed rectal mucosa in ulcerative colitis (5,6). Experiments recently performed in our laboratory confirmed enhanced colonic prostanoid synthesis also in active Crohn’s disease of the large bowel. These observations suggest that prostanoids may mediate the inflammatory response in ulcerative colitis and Crohn’s disease. Moreover, we have also established that intestinal inflammatory cells are those responsible for most of the enhanced colonic PGE, and TXA, synthesis (7). Tissue macrophages are recruited from peripheral blood mononuclear cells and their precursors in the bone marrow. Counting peripheral blood monocytes according to their morphologic identifications revealed monocytosis both in ulcerative colitis and in Crohn’s disease (1,2). We have confirmed this obseration by counting isolated adherent monocytes as
IN IBD
677
well as by counting the number of phagocytic cells in the peripheral blood. Plastic adherence and phagocytic activity are among the most useful criteria used to identify and isolate peripheral blood monocytes (10). Cultured PBM cells isolated from both normal subjects and patients with inflammatory diseases of the bowel were found to synthesize PGEB and TXB2, the stable metabolite of TXA2, both of which accumulated in the medium during the culture. During 24 h of culture, 6-keto-PGF,,, the stable metabolite of PGIZ, did not accumulate in the medium. It was previously reported that PBM cells do not synthesize this prostanoid (16)and that PGEz and TXBz are the major products released by human monocytes in day 1 of the culture after cell adherence (17). Prostaglandin Ez and TXB2 secretion by PBM cells isolated from patients with active Crohn’s disease was found to be two times higher than their secretion by PBM cells isolated from normal subjects. Prostaglandin Ez secretion by PBM cells isolated from patients with shigellosis was also increased. Prostanoid secretion by PBM cells was not enhanced in Crohn’s disease
Table 3.
Effect of Drugs on Prostanoid Accumulation by Cultured Peripheral Blood Mononuclear Cells
Drug
Discussion
CELLS
Flufenamic acidb 0.1 1.0 10.0 Methyl prednisoloneb 0.1
1.0 50.0
No.
PGE,”
TXB,”
4 5 4
85.02 32.0 31.9-t9.6' 13.6+ 8.4"
33.6-+2.7' 9.32 2.5' 3.12 1.8'
2 4 4
61.72 22.9 45.7T 6.5 39.6k 8.6"
75.85 12.7 55.22 6.0' 42.42 6.5c
5 5 4
153.0? 50.0 170.0? 68.8 257.0+ 107.0
104.02 20.0 67.52 15.6 22.02 8.0c
Salazopyrineb
50 100 200 Sulfapyridineb
71.4k 77.0+ 126.02 100.0k
5 50 250 500 5-Aminosalycilic 5 50 75 500
11.4 11.6 16.1 28.0
80.82 9.7 82.42 10.1 62.3+.7.3e 43.0" 4.8c
108.02 9.0 71.4rf7.9d 48.4t 9.4d 24.0-+6.4'
102.0? 18.4 74.42 16.2 41.0+-6.0' 3.8f 2.2'
acidb
Peripheral blood mononuclear cells were separated from normal subjects and cultured in medium containing each of the drugs for 24 h. Prostanoid accumulation in the medium was determined by radioimmunoassay as described in Materials and Methods. Prostanoid accumulation by PBM cells cultured in drug-free medium served as control and was regarded as 100%. Results are mean * SE. a Percent of accumulation in drug-free medium. b &ml. Significantly different from prostanoid accumulation in drug-free medium:” p < 0.01; d p < 0.02;e p -=c 0.05.
676
RACHMILEWITZ
ET AL.
patients in remission and in patients with active ulcerative colitis. When tested in the same individuals with Crohn’s disease, prostanoid synthesis by PBM cells was found to decrease with clinical improvement achieved either by medical therapy or following surgical resection of the diseased bowel. Enhanced prostanoid secretion by PBM cells in active Crohn’s disease may be either due to quantitative difference in their secretion by individual monocytes, or due to an absolute increase in the number of monocytes among PBM cells. The results reported indicate that the same number of monocytes isolated from Crohn’s disease patients and normal subjects secreted similar amounts of PGE2. It is therefore suggested that there is no qualitative difference and that the enhanced PGE;! secretion by PBM cells is due to an increase in the absolute number of monocytes in Crohn’s disease. In view of the similar increase in the number of monocytes observed in active ulcerative colitis and Crohn’s disease, the difference in their PBM-cell prostanoid production may be explained by other mechanisms such as either the presence of a prostanoid inhibitor in cells isolated in ulcerative colitis, or the presence of a separate subset of cells. A slight increase in TXBz secretion by monocytes was observed in active Crohn’s disease and shigellosis, indicating that in these two diseases monocytes differ in their capacity to secrete this prostanoid. Prostaglandin Ez and TXB, secretion by cultured PBM cells reflects their recent synthesis, as flufenamic acid, a cyclooxygenase inhibitor, induced about 90% inhibition of their accumulation. Methyl prednisolone also inhibited their synthesis although sulfasalazine in a high pharmacologic dose of 200 pg/ ml only inhibited TXBz and did not affect PGEz synthesis. Sulfapyridine in high concentrations 5(>250 pg/ml) inhibited only TXBz synthesis. Aminosalicylic acid, the other component of sulfasalazine, also inhibited PGEz accumulation only when added in a high dose of 50 pg/ml. These results are similar to our observations about sulfasalazine effects on prostanoid production by cultured rectal mucosa (6). The inhibition of PGEz and TXB2 accumulation induced by all drugs is not due to induction of irreversible cell damage, as latex phagocytosis regarded as a parameter of cell viability was not affected by any of them even when used in high concentrations. In active ulcerative colitis, circulating complement receptor-positive lymphocytes are increased, T-cell percentages and lymphocyte functions are decreased, and erythrocyte antibody component phagocytozing esterase-positive cells-indicative of activated monocytes-are demonstrated. Whereas successful treatment with sulfasalazine reversed
GASTROENTEROLOGY
Vol. 82, No. 4
these immunologic changes, in vitro incubation of sulfasalzine and its metabolites with leukocytes did not alter these immunologic changes (18). Similar results were obtained in the present study; in vitro incubation of cultured PBM cells obtained from normal subjects with therapeutic plasma concentrations of sulfasalazine or its constituents failed to inhibit PGEz or TXB2 synthesis whereas in Crohn’s disease patients in remission maintained by sulfasalzine the enhanced prostanoid production by cultured PBM cells was reversed to normal. It is still not clear which is the active moiety in sulfasalazine and what is the mechanism responsible for its therapeutic efficacy in IBD. The discrepancy between the in vitro and in vivo drug effects may suggest an indirect effect of the drug, but more work and probably other approaches are needed to clarify this issue.
References 1. Auer IO, Wechsler W, Ziemer E, et al. Immune status in Crohn’s disease. I. Leukocyte and lymphocyte subpopulations in peripheral blood. Stand J Gastroenterol 1978;13:56171. 2. Thayer WR, Charland C, Field CA. The subpopulations of circulating white blood cells in inflammatory bowel disease. Gastroenterology 1976;71:379-84. 3. Meuret G, Bitzi A, Hammer B. Macrophage turnover in Crohn’s disease and ulcerative colitis. Gastroenterology 1978;74:501-3. 4. Rachmilewitz D, Ligumsky M, Rachmilewitz B, et al. Transcobalamin II level in peripheral blood monocytes-a biochemical marker in inflammatory diseases of the bowel. Gastroenterology 1980;78:43-6. 5. Sharon P, Ligumsky M, Rachmilewitz D, et al. Role of prostaglandins in ulcerative coltis. Enhanced production during active disease and inhibition by sulfasalazine. Gastroenterology 1978;75:638-40. 6 Ligumsky M, Karmeli F, Sharon P, et al. Enhanced thromboxane Az and prostacyclin production by cultured rectal mucosa in ulcerative colitis and its inhibition by steroids and sulfasalazine. Gasteroenterology 1981;81:444-9. 7. Rachmilewitz D, Zifroni A, Ligumsky M, et al. Enhanced prostanoids synthesis by cultured peripheral blood mononuclears (PBM) and intestinal lymphoid cells in Crohn’s disease (abstr). Gastroenterology 196i;80:1256. 8. Spector WG. The macrophage in inflammation. Ser Haematol 1970;3:132-44. 9. Humes JL, Bonney RJ, Pelus L, et al. Macrophages synthesize and release prostaglandins in response to inflammatory stimuli. Nature 1977;269:149. 10. Treves AJ, Yagoda D, Haimovitz A, e“1 al. The isolation and purification of human peripheral blood monocytes in cell suspension. J Immunol Methods 1980;39:71-80. 11. Bauminger S, Zor U, Lindner HR. Radioimmunological assay of prostaglandin synthetase activity. Prostanglandins 1973; 4:313-24. 12. Ferreira SH, Moncada S, Vane JR. Prostaglandins and signs and symptoms of inflammation. In: Robinson HJ, Vane JR, eds. Prostaglandin synthetase inhibitors. New York: Raven Press, 1974:175-87. 13. Bockman RS, Rothschild M. Prostaglandin E inhibition of Tlymphocyte colony formation: a possible mechanism of
PROSTANOID
April 1982
monocyte
modulation
of clonal
expansion.
J Clin Invest
1979:64:812-Q. 14. Droller
MJ, Schneider MU, Perlman P. A possible role of prostaglandins in the inhibition of natural and antibody dependent cell-mediated cytotoxicity against tumor cells. Cell Immunol 1978;39:165-77. 15. Miller OU, Johnson RA, Gorman RR. Inhibition of PGE, stimulated CAMP accumulation in human platelets by thromboxane AZ. Prostaglandins 1977;13:599-609. 16. Kenney MS, Stobo JD, Goldyne ME. In vitro synthesis of
SYNTHESIS
BY MONONUCLEAR
CELLS IN IBD
679
prostaglandins and related lipids by populations of peripheral blood mononuclear cells. Prostaglandins 1980;20:135-45. 17. Bockman RS. Prostaglandin production by human blood monocytes and mouse peritoneal macrophages: synthesis dependent on in vitro culture conditions. Prostaglandins 1981;21:9-31. 18. Rubinstein A, Das KM, Melamed J, et al. Comparative analysis of systemic immunological parameters in ulcerative colitis and idiopathic proctitis: effects of sulfasalazine in vivo and in vitro. Clin Exp Immunol 1978:33:217-224.