Clin. exp. Immunol. (1978) 33, 217-224.
Comparative analysis of systemic immunological parameters in ulcerative colitis and idiopathic proctitis: effects of sulfasalazine in vivo and in vitro A. RUBINSTEIN, K. M. DAS, J. MELAMED & RAE ANN MURPHY Departments of Medicine (Division of Gastrointestinal and Liver Disease), Cell Biology and Pediatrics (Division ofImmunology), Albert Einstein College ofMedicine, Bronx, New York 10461, USA
(Received 20 February 1978) SUMMARY
Comparative analysis of the systemic immunity revealed similarities between ulcerative colitis and idiopathic proctitis. In the active stage of both diseases, circulating complement receptor positive cells were increased whereas T-cell percentages and lymphocyte functions were decreased. In severe forms of ulcerative colitis and idiopathic proctitis circulating EAC-phagocytosing esterase positive cells, indicative of activated monocytes, were demonstrated. Successful treatment with salicylazosulfapyridine (SASP) reversed these immunological changes. Incubation of SASP and its metabolites with leucocytes from patients and control subjects, in concentrations similar to those demonstrated in sera from patients treated with SASP, did not alter the immunological changes. INTRODUCTION There is a large body of evidence to demonstrate that both systemic and local immunological abnormalities are present in inflammatory bowel disease (Kraft & Kirsner, 1971; Brandtzaeg et al., 1974; Skinner & Whitehead, 1974; Thayer, Charland & Field, 1976), but their pathogenetic roles are still obscure. In Crohn's disease, a major feature appears to be a deficient cell mediated immunity (Sachar et al., 1973; Asquith, Kraft & Rothberg, 1973). Patients with active ulcerative colitis (UC), with a few exceptions (Thayer et al., 1976), were reported to exhibit increased numbers of circulating B-cells (Sachar et al., 1973; Strickland et al., 1974), often accompanied by the presence of circulating anti-colon antibodies (Wright & Truelove, 1966). In some patients with UC, a decrease in the absolute number of peripheral blood T-cells and abnormalities of delayed type hypersensitivity reactions have been documented (Sachar et al., 1973). Idiopathic proctitis (IP), a localized form of inflammatory bowel disease, is considered clinically to be a forerunner of ulcerative colitis (Lennard-Jones et al., 1962; Sparberg, Fennessy & Kirsner, 1966). Disturbed local immunity has been observed in histologically diseased, as well as in histologically normal, colonic mucosal biopsy specimens from patients with idiopathic proctitis (Das, Erber & Rubinstein, 1977a). However, the sequential analysis of systemic immunological parameters in such patients and their possible alteration by salicylazosulfapyridine (SASP), the drug most extensively used in the treatment of UC and IP, has not yet been determined. The present study was undertaken firstly to compare the systemic immunity in untreated patients with IP and UC, secondly to explore the in vivo effects of SASP by the temporal analysis of the immunological parameters in treated patients as correlated with the blood levels of the drug and its metabolites, and thirdly to investigate the in vitro effects of the drug on the peripheral blood lymphocytes. Correspondence: Dr A. Rubinstein, Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA. 0099-9104/78/0800-0217$02.00 (C 1978 Blackwell Scientific Publications
217
218
A. Rubinstein et al. MATERIALS AND METHODS
Patients. Thirteen patients (eight females and five males) with mild to moderate active ulcerative colitis and thirteen patients (ten females) with active idiopathic proctitis were studied as out-patients. Their ages ranged between 22-62 years. The diagnosis was confirmed on the basis of symptoms, sigmoidoscopic and/or colonoscopic examinations and barium enema studies. The clinical state of the disease was evaluated as active or in remission on the basis of the number of bowel movements, presence of blood and mucus in the stool, general sense of well being and sigmoidoscopic findings. All patients were treated with 3-4g ofSASP per day. Only one patient with extensive UC received 15 mg prednisone daily, and one patient with IP was treated with hydrocortisone enema in addition to SASP. All twenty-six patients with active disease were studied immunologically prior to treatment. Of these, nine patients with UC and six with IP were re-evaluated 6 to 16 weeks after treatment. All these fifteen patients, except for one with UC and one with IP, were in remission during the follow-up studies. Serum immunoglobulins. IgA, IgD, IgG, IgM and the third complement component (C3), were measured by radial immunodiffusion, using specific antisera (Mancini, Carbonara & Heremans, 1965). Serum IgE levels were determined by radioimmunoassay using the Phadebas Kit, Pharmacia, New Jersey. Serum concentrations of SASP and its metabolites were estimated spectrophotometrically (Das et al., 1973). Peripheral blood lymphocytes (PBL). These were isolated by Ficoll-Hypaque density gradients. Blast transformation of PBL in vitro was measured by the incorporation of tritiated thymidine after stimulation with 0-01% phytohaemagglutinin (PHA-P), 0-01% pokeweed mitogen (PWM), and 10 [ug/ml Concanavalin A (Con A). Throughout the study the same batches of foetal calf serum and of mitogens were employed for the same patient. 200,000 lymphocytes were cultured in Linbro tissue culture multidishes in triplicate in RPMI 1640, supplemented with 15% foetal calf serum. Cells were harvested after 72 hr (for PHA, Con A, PWM), or after 120 hr (for specific antigens) with the automatic cell harvester Mash 2. Mixed lymphocyte cultures were performed according to Bach & Voynow (1969). Thymic derived lymphocytes (T-cells). These were detected by rosetting with sheep red blood cells (SRBC) treated with sulfhydryl compound 2-amino ethylisothiouronium bromide (EAET) (Pellegrino et al., 1975). Rosettes were examined in a mixture of regular and ultraviolet light with a fluorescent microscope after staining the cell suspension with euchrysin (Allison, 1977). Complement receptor positive cells (CR +cells). These were counted after rosetting with sheep red blood cells coated with Forssman rabbit anti-SRBC serum and mouse complement (EAC) (Bianco, Patrick & Nussenzweig, 1970). Serum from C5 deficient mice (B10 D2-OSN, Jackson Labs., Bar Harbor, Maine) was used as complement source. Trypsinized SRBC were employed in the preparation of EAC to avoid cross-reactivity with T-cells (Weiner, Bianco & Nussenzweig, 1973). Early in the course of these studies, we noted abundant phagocytosed EAC and rosette formation by monocytes. Three techniques were employed to avoid reading such rosettes as CR+B-cells. Firstly, rosettes were quantified by the euchrysine technique, which permits discrimination of monocytes from lymphocytes; secondly, EAC rosetted PBL smears were stained by esterase (Yam, Li, & Crosby, 1971). Esterase positive rosetted cells were subtracted from the total CR+cells. Thirdly, rosetted PBL were pelleted by a Ficoll-Hypaque density gradient. The erythrocytes in the pellet were lysed by 0-87% NH4C1, and a differential count of mononuclear cells was obtained by esterase stain or by the Technicon Hemalog D 10,000-cell automated differential analyser. All three techniques yielded comparable results. The net CR+ lymphocytes were found to correlate best with surface immunoglobulin positive cells, and were thus termed B-cells (Ehlenberger et al., 1976). Activated monocytes. These were identified by their capacity to phagocytose EAC after 60 min of incubation at 370C. IgM fraction of EAC was purified on a Sephadex G-200 column to remove most residual IgG. The presence of intracellular erythrocytes was verified by euchrystine fluorescence and by esterase stains of PBL smears. Particularly with the euchrysine fluorescence-stain, intracellular EAC could easily be distinguished from extracellular rosetted erythrocytes. In some experiments, the validity of the euchrysin quantification of EAC-phagocytosis by monocytes was confirmed by re--valuation following lysis ofextracellular EAC with 0-87% NH4CI. Fcy receptor positive PBL. These were identified by rosetting with trypsinized SRBC coated with the IgG fraction of a rabbit anti-SRBC serum (EA-IgG). EA-IgG indicator cells were mixed with PBL, pelleted and incubated at room temperature for 30 min. Rosettes were ready by the euchrysine technique. All immunological parameters were studied in patients with UC and IP, before and after treatment with SASP, in a double blind protocol. The code was broken at the end ofthe protocol. In vitro effects of salicylazosulfapyridine (SASP), and its metabolites, 5-aminosalicylate (5-ASA), and sulfapyridine (SP) on PBL. Toxicity of SASP, 5-ASA and SP in vitro was evaluated by incubation of normal PBL with the drugs at various concentrations. Following incubation, residual cell numbers, cell viability (trypan blue exclusion test), and spontaneous cellular DNA synthesis rates (3H-thymidine uptake) were determined. The drug concentrations used in the evaluation of the in vitro drug effect on the various immunological parameters (for SASP and SP, above 100 ,ug/ml and for 5-ASA, above 10 ,Lg/ml) were below their cell toxic range and higher than serum concentrations attained in treated patients (5-32, 0-1 and 15-42 ug/ml of SASP, 5-ASA and total SP, respectively). Studies with the three drugs were performed in vitro on cultured PBL obtained from six normal controls, two patients with UC, and three patients with IP. PBL were incubated with the drug for 2 hr or throughout the culture period (3-5 days). Cell numbers, cell viability, differential white blood cell count, B-cells, T-cells, Fcy-positive cells and 3H-thymidine uptake were determined on completion of the incubation and simultaneously compared to drug free controls. The in vitro drug effects were studied on PBL culture with or without mitogenic stimulation (with PHA, Con A, PWM, PPD, Candida antigen, or with mitomycin C treated allogeneic cells).
Reversal of altered immunity by salicylazosulfapyridine
219
RESULTS Serum immunoglobulins These revealed no characteristic pattern. Serum IgA levels were in the upper limits of normal or slightly elevated in five out of the eleven UC patients (340, 350, 465, 650 and 560 mg%), and in four of the thirteen IP patients (305, 430, 460 and 580 mg%), as compared to 64-407 mg% in controls. Serum IgE levels were increased in one IP patient (500 u/ml) and in one UC patient (950 u/ml), as compared with the upper limit of normal controls (300 u/ml). Serum IgG, C3 and total complement levels were normal in all patients. Following treatment, there was no change in serum IgD, IgE, IgG and IgM levels. In three patients with UC there was a marked increase in serum IgA level as compared to their pretreatment levels (50, 71 and 1250% increase). In vitro blast transformation ofPBL To PHA and Con A, this was significantly decreased in eleven out of the twenty-six patients with UC and IP (Table 1); responses to PWM being less frequently affected, as compared to twenty controls (Student's t-test for PHA, Con A, P< 0.05). Nine patients with UC and six patients with IP could be
TABLE 1. 3H-thymidine uptake by peripheral blood lymphocytes in patients with UC or with IP before and after treatment with SASP (cpm-minus background, mean value and + 1 SE) Normal controls
Before SASP
PHA Con A PWM
After SASP
PHA Con A PWM
*
N
=
Ulcerative Colitis
Idiopathic Proctitis
N*
RI±1 SE
N* NPt
X ±1 s.e.
N* NPt
20 20 20
41,840±3962
13 13 13
51 64 44
16,182±2470 18,369±3097 18,412±4097
13 13 13
8 8 8
24 14
35,608±6630
33,447±3757
14
25,650±3152
4 4 4
37,982±4332
29,170±2611
64
54 31
34 0 0
X +1 s.e. 13,882±3600 18,369±3935 15,887±2512
19,467±3840 18,959±3591 18,429±3866
Total number of subjects studied for the specified parameter.
t NP= Number of patients with immunological disturbances as compared to the complete range of twenty controls. (Shaded areas in Figs. 1-3). 4 = Abnormally decreased value.
studied for this parameter after therapy (Fig. 1). The mean values for PHA, Con A and PWM lymphocyte mitogenic responses (LMT) were calculated before and after SASP treatment (Table 1). The Student's t-test did not show a significant difference before and after SASP treatment, (except for PHA and Con A LMT in UC), probably because of the wide variation of results. In individual IP patients there was, however, a clear trend towards improvement (Fig. 1). The same data were therefore analysed by comparing each patient to controls before and after SASP treatment. Patients were divided into two groups according to their LMT to PHA: group 1 = below X-ISE of twenty normal controls, group 2 = above X-ISE. The statistical significance of the difference in proportions of patients in each group before and after SASP treatment was obtained by the 2 x 2 contingency test. The results proved that after SASP treatment there were significantly more patients in group 2 (P < 0.05).
220
A. Rubinstein et al.
c
0
~cr .To c
BE E
-e
R CI We
6
Weeks
FIG. 1. 3H-thymidine uptake by peripheral blood lymphocytes following mitogenic stimulation with PHA in patients with ulcerative colitis and idiopathic proctitis before and after SASP therapy. Shaded areas represent the complete range in twenty controls. Before treatment with salicylazosulfapyridine; 0 after treatment with ulterative colitis and 0 normal range. idiopathic proctitis; salicylazosulfapyridine; A
- - -
600[
600g A
400 --._ 0
2O~0~~~~~~~
3000
2000
1000
i-' ', AI
2
4
6 Weeks
8
'Af
*-
12
16
2
4
6
8
12
16
Weeks
FIG. 2. Peripheral blood T cells and B cells in patients with ulcerative colitis before and after treatment with SASP. Shaded areas represent the complete range in twenty controls. A Before treatment with salicylazosulfapyridine; * after treatment with salicylazosulfapyridine and 0 normal range. FIG. 3. Peripheral blood T cells and B cells in patients with idiopathic proctitis before and after treatment with SASP. Shaded areas represent the complete range in twenty controls. A Before treatment with salicylazosulfapyridine; 0 after treatment with salicylazosulfapyridine and 0 normal range.
T-cells Absolute numbers were decreased in five out of the twelve UC patients and in one out of the twelve IP patients as compared to the complete range in twenty controls (Fig. 2, Table 2). The percentage of T-cells was decreased by the same criteria in ten out of twenty-four patients with UC and IP. Following
221
Reversal ofaltered immunity by salicylazosulfapyridine
TABLE 2. Peripheral blood lymphocyte subpopulations in patients with UC or with IP before and after treatment with SASP (mean value i 1 s.e.)
Ulcerative colitis
Normal controls
Before SASP
41
63+5
12
64
66-6±2-8
51
917+120 75±4
12 6
14 It 1450+162 14 73+2-6
1162±66 22-1±4-5
6
81
12
14 2T 1566±330 18±14 81
13 9
7T IT
307+37 10-6±1-7
12 10T 6
2T
9 9
21 31
166±51
X= 25
6 6
3T
20
77+2-3 % of PBL
12
12 9
9
9 13
77±2-3
20
After SASP
Before SASP
X ± Is.e.
N* NPt
11±2-5
CR+ cells
Fcy+ cells
N* NPt
X ±ls.e.
After SASP
Before SASP
± I s.e.
N*
T-cells
11±2-5
20
17±4-2
above 850 per cmm % of PBL above 850 per cmm %of PBL below 284 per cmm %of PBL below 284 per cmm % of PBL
Idiopathic proctitis
I1
(14-38)
21
330±48 13-6±1-1
251+44 X-21
(15-26)
* N= Total number of patients studied for the specified parameter. NP= Number of patients with immunological disturbances as compared to the complete range of twenty controls (shaded areas in Figs. 1-3). 4=Abnormally decreased value. T=Abnormally increased value.
treatment with SASP, T-cells increased beyond the lowest level of the normal range in their percentage and absolute numbers in all but two patients (one IP, one UC) (Table 2, Figs. 2, 3). Calculation of T-cell numbers and percentages before and after SASP treatment by the 2 x 2 contingency test (as used for the analysis of LMT changes by SASP) revealed a significant increase (P < 0.02) in UC patients.
CR+ B-cells These were initially evaluated by light microscopy of EAC-rosettes. Their percentage was markedly higher than the number of B-cells found by simultaneous surface immunoglobulin fluorescence in some patients. This discrepancy alerted us to the possibility of increased numbers of monocytes binding EAC, or of phagocytosis of EAC by monocytes. By employing the euchrysine technique, and esterase stains of smears of rosetted PBL, the net CR+ B-cells were found elevated in both UC and IP patients (Table 2, Figs. 2, 3). After treatment with SASP, the CR+ B-cell number decreased significantly (Table 2, Figs. 2, 3) in most UC and IP patients (2 x 2 contingency test P< 0.05). Esterase positive monocytes The total number per cmm did not deviate significantly from normal controls and was unchanged by treatment. The mean value +1 s.e. of the total monocyte count was higher in thirteen UC patients
222
A. Rubinstein et A
(392±32), as compared to thirteen IP patients (281±13). Both were, however, still in the normal range. Monocytes were abnormally high (700, 760 and 880 monocytes per cmm) in only three of the thirteen patients with UC. Phagocytosis ofEAC This was noted by peripheral blood monocytes in six out of twenty-four patients. These six patients were found to have moderately severe active disease on the basis of symptomatology and sigmoidoscopic findings. In five of these patients (four UC, one IP), more than 24% of all monocytes phagocytosed EAC. In twenty normal controls at most 8% of the monocytes phagocytosed EAC. Following SASP treatment, the percentage of EAC phagocytosing monocytes decreased in all patients, in four to the normal, and in two (UC) to 12% and 14%, respectively. The mean of the percentage of the Fcy positive lymphocytes was elevated in both UC and IP patients, but more so in the UC patients (Table 2). In vitro effects ofSASP and its metabolites While treatment with SASP in vivo affected LMTs, T and CR+ B-cell numbers, and EAC phagocytosis by monocytes, no such effects were detected by in vitro incubation of normal controls' or patients' leucocytes with the drugs in two folds of the 'therapeutic concentration ranges' (Das et al., 1973).
DISCUSSION Idiopathic proctitis is an inflammatory bowel disease localized to the distal 15-20 cm of the large intestine, and is clinically considered to be a forerunner of UC (Lennard-Jones et al., 1962; Sparberg et al., 1966). We have demonstrated previously abnormalities in secretory IgA in histologically normal proximal colonic mucosa of patients with IP (Das et al., 1977a). While the pathogenetic significance of this finding is still obscure, it suggests that the disease process is more extensive than the macroscopic and light microscopic abnormalities (Das et al., 1977a; Das et al., 1977b) and probably reflects various spectra of this process. The present analysis of systemic immunological functions in UC and IP also indicates similar alterations. In both diseases, increased circulating B-cells and an in vitro decreased lymphocyte mitogenic responses to PHA and Con A as well as low T-cell numbers were noted. Most intriguingly, however, the peripheral blood monocytes were found in the active stages of UC and IP to phagocytose sheep red blood cells coated by IgM antibody and complement (EAC). The SRBC-IgM-C3 complex (EAC) is routinely used to detect CR+ lymphocytes (Bianco et al., 1970). The employment of the euchrysin fluorescence (Allison, 1977) and the esterase stain (Yam et al., 1971) in this study permitted the discrimination between bona fide EAC rosetted CR+ lymphocytes and EAC attached to monocytes or phagocytosed by them. In a few instances more than a quarter of the peripheral blood monocytes obtained from patients with active UC and IP phagocytosed EAC. Normal monocytes carry both C3b and C3d receptors and phagocytose only SRBC coated by IgG and C3. Activated monocytes alone phagocytose SRBC coated by the IgM-C3 complex too (Ehlenberger & Nussenzweig, 1977). They are frequently highly secretory (Gordon, 1976; Vassalli & Reich, 1977) and their proteolytic enzymes may contribute to the mucosal damage as found in patients with IP and UC. Hypothetically, such an effect could be most injurious in IP and UC in view of the additional abnormality of the colonic secretory IgA system (Das et al., 1977a; Simada, 1976). It has been suggested that secretory IgA may regulate tissue autoproteolysis (Tomasi & Hauptman, 1974) by its association with alpha-l-antitrypsin. The local IgA deficiency may thus render the colonic mucosa more vulnerable to the proteolytic enzymes secreted by activated monocytes. The isolation of a highly active monocyte/macrophage population from normal human intestinal mucosa has recently been reported (Bull & Bookman, 1977). This population was absent in normal peripheral blood and was defined by its increased cell size, granularity, motility, glass adherence and latex particle phagocytic activity. It has yet to be determined whether that population finds access into the peripheral blood in inflammatory bowel disease. Our observation that a successful SASP treatment invariably diminished the number of circulating EAC phagocytosing esterase positive cells, would suggest the inter-relationship of this cell subset with the disease process. The decrease in
Reversal of altered immunity by salicylazosulfapyridine
223
EAC phagocytosing monocytes was, however, not accompanied by a diminution of the total monocyte number in the peripheral blood. There was also evidence to suggest a drug related correction of other immunological abnormalities such as the decrease in T-cell numbers and the elevation of CR+ B-cells, as well as the abnormalities of in vitro LMTs (Table 1, 2, Figs. 1, 2 & 3). However, a complete dissociation between the in vivo and in vitro effects of SASP was noted. Studies with patients' or controls' PBL, in tissue culture medium containing SASP or its derivatives in up to two folds of the therapeutic serum levels, failed to demonstrate any influence on immunocytes. Currently, little is known about the mechanism of the action of SASP. In view of the dichotomy between the in vivo and in vitro drug effects, we may conjecture that SASP exerts its immunological influence indirectly. It may, for example by its antimicrobial activity (West et al., 1974), eliminate bacterial antigens from the colonic tissue. We have recently extracted from colectomy specimens an antibody that cross-reacts with an as yet undefined antigen(s) present in the colonic mucosa of patients with UC (Das & Dubin, 1977). This finding may be indicative of local immune complexes that in turn could activate monocytes. Further studies related to quantification and functional characterization of local monocytes, before and after treatment with SASP, may yield further information concerning their pathogenetic role and a possible mode of action of the drug. The authors wish to acknowledge the invaluable help of Dr N. A. Kowatch, Supervisor of the Hematology Laboratory, the Hospital of the Albert Einstein College of Medicine, in analysing white blood cell differentials on the Technicon Hemalog D counter. We are also grateful to Dr V. Krishnaswamy for the statistical analysis of the data and Dr B. Zapp for his assistance. Supported in part by a grant from the National Institutes of Health Al 10702-05 and by the National Foundation for Ileitis and Colitis, Inc., New York. Dr Das is the recipient of a Clinical Investigator Award from the National Institutes of Health grant # IK08 AM00237.
REFERENCES ALLISON, A.C. (1977). Fluorescence microscopy of lymphocytes and mononuclear phagocytes and the use of silica to eliminate the latter. In Vitro Methods in Cell Mediated and Tumor Immunity (ed. by B.R. Bloom, and J.R. David), p. 395. Academic Press, New York. ASQUITH, P., KRAFT, S.C. & TOTHBERG, R.M., (1973). Lymphocyte responses to nonspecific mitogens in inflammatory bowel disease. Gastroenterology, 65, 1. BACH, F.H. & VOYNOW, N.K., (1969). One way stimulation in mixed leukocyte cultures. Science, 153, 545. BIANCO, C., PATRICK, R. & NUSSENZEIG, V., (1970). A population of lymphocytes bearing a membrane receptor for antigen antibody complement complexes. I. Separation and characterization.]. exp. Med. 132, 702. BRANDTZAEG, P., BAKLIEN, K., FAUSA, 0. & HOEL, P.S., (1974). Immunohistochemical characterization of local immunoglobulin formation in ulcerative colitis. Gastroenterology, 66, 1123. BULL, D.M. & BOOKMAN, M.A., (1977). Isolation and functional characterization of human intestinal mucosal lymphoid cells.]. clin. Invest. 59, 966. DAS, K.M. & DUBIN, R., (1977). Isolation and characterization of colonic tissue bound antibodies from patients with ulcerative colitis. Gastroenterology, 72, 1043 (abstr.). DAS, K.M., EASTWOOD, M.A., MCMANUS, J.P.A. & SIRCUS, W., (1973). The metabolism of salicylazosulphapyridine in ulcerative colitis. I. The relationship between metabolites and the response to treatment in patients. II. The relationship between metabolites and the progress of the disease studied in out-patients. Gut, 14, 631. DAS, K.M., ERBER, W.F. & RUBINSTEIN, A., (1977a). Immunohistochemical changes in morphologically involved and uninvolved colonic mucosa of patients with idiopathic proctitis.]. clin. Invest. 59, 379. DAS, K.M., MORECKi, R., NAIR, P. & BERKOWITZ, J.M., (1977b). Idiopathic proctitis: I. The morphology of
proximal colonic mucosa and its clinical significance.
Am.J. Dig. Dis. 22, 524. EHLENBERGER, A.G., MCWILLIAMS, M., PHILLIPS-QUAGLIATA, J.M, LAMM, M.E., & NUSSENZWEIG, V. (1976). Immunoglobulin-bearing and complement-receptor lymphocytes constitute the same population in human peripheral blood._T. Clin. Invest. 57, 53. EHLENBERGER, A.G. & NUSSENZWEIG, V., (1977). The role of membrane receptors for C3b and C3d in phagocytosis. J. exp. Med. 145, 537. GORDON, S., (1976). Macrophage neutral proteinases and chronic inflammation. Ann. N.Y. Acad. Sci. 278, 176. KRAFT, S.C. & KIRSNER, J.B., (1971). Immunological apparatus of the gut and inflammatory bowel disease. Gastroenterology, 60, 922. LENNARD-JONES, J.E., COOPER, G.W., NEWELL, A.C., WILSON, C.W.E. & JONES, F.A., (1962). Observations on idiopathic proctitis. Gut, 3, 201. MANCINI, G., CARBONARA, A.W. and HEREMANS, J.F., (1965). Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry. 2, 235. PELLEGRINO, M.A., FERRONE, S., DIERICH, M.P. & REISFELD, R.A., (1975). Enhancement of sheep red blood cell human lymphocyte rosette formation by the sulfhydryl compound 2-amino ethylisothiourodium bromide. Clin. Immunol. Immunopathol. 3, 324. SACHAR, D., TAUB, R.N., BROWN, S.M., PRESENT, D.H., KORELTZ, B.I. & JANOWITZ, H.D., (1973). Impaired lymphocyte responsiveness in inflammatory bowel disease. Gastroenterology, 64, 203. SIMADA, H., (1976). Immunohistochemical study of ulcerative colitis with special reference to local immunoglobulin. Jap. J. Dig. Dis. 73, 832. SKINNER, J.M. & WHITEHEAD, R., (1974). The plasma cells in inflammatory disease of the colon: A quantitative study.]. Clin. Pathol. 27, 643.
224
A. Rubinstein et al.
SPARBERG, M., FENNESSY, J. and KIRSNER, J.B., (1966). Ulcerative proctitis and mild ulcerative colitis: A study of 220 patients. Medicine (Baltimore), 45, 391. STRICKLAND, R.G., KORSMEYER, S., SOLTis, R.D., WILSON, I.D. & WILLIAMS, JR., R.C. (1974). Peripheral blood T and B cells in chronic inflammatory bowel disease. Gastroenterology, 67, 569. THAYER, JR., W.R., CHARLAND, C. & FIELD, C.E., (1976). The subpopulations of circulating white blood cells in inflammatory bowel disease. Gastroenterology, 71, 379. TomASI, JR., T.B. & HAUPTMAN, S., (1974). Modulation of the assembly of immunoglobulin subunits by J chain. In the immunoglobulin A system (ed. by J. Mestecky, and A.R. Lawton,) p. 111. Pleum Press, New York.
VASSALLI, T.D. & REICH, E., (1977). Macrophage plasminogen activator: induction by products of activated lymphoid cells.]. exp. Med. 145,429. WEST, B., LENDRUM, R., HILL, M.S. & WALKER, G., (1974). Effects of sulfasalazine (salazopyrin) on fecal flora in patients with inflammatory bowel disease. Gut, 15, 960. WEINER, M.S., BIANCO, C., & NUSSENZWEIG, V. (1973). Enhanced binding of Neuraminidase treated sheep erythrocytes to human T-lymphocytes. Blood, 42, 939. WRIGHT, R. & TRUELOVE, S.C., (1966). Autoimmune reactions in ulcerative colitis. Gut, 7, 32. YAM, L.T., LI, C.Y. & CROSBY, W.H., (1971). Cytochemical identification of monocytes and granulocytes. Amer. J. Chem. Pathol. 55, 283.
Comparative analysis of systemic immunological parameters in ulcerative colitis and idiopathic proctitis: effects of sulfasalazine in vivo and in vitro A. RUBINSTEIN, K. M. DAS, J. MELAMED & RAE ANN MURPHY Departments of Medicine (Division of Gastrointestinal and Liver Disease), Cell Biology and Pediatrics (Division ofImmunology), Albert Einstein College ofMedicine, Bronx, New York 10461, USA
(Received 20 February 1978) SUMMARY
Comparative analysis of the systemic immunity revealed similarities between ulcerative colitis and idiopathic proctitis. In the active stage of both diseases, circulating complement receptor positive cells were increased whereas T-cell percentages and lymphocyte functions were decreased. In severe forms of ulcerative colitis and idiopathic proctitis circulating EAC-phagocytosing esterase positive cells, indicative of activated monocytes, were demonstrated. Successful treatment with salicylazosulfapyridine (SASP) reversed these immunological changes. Incubation of SASP and its metabolites with leucocytes from patients and control subjects, in concentrations similar to those demonstrated in sera from patients treated with SASP, did not alter the immunological changes. INTRODUCTION There is a large body of evidence to demonstrate that both systemic and local immunological abnormalities are present in inflammatory bowel disease (Kraft & Kirsner, 1971; Brandtzaeg et al., 1974; Skinner & Whitehead, 1974; Thayer, Charland & Field, 1976), but their pathogenetic roles are still obscure. In Crohn's disease, a major feature appears to be a deficient cell mediated immunity (Sachar et al., 1973; Asquith, Kraft & Rothberg, 1973). Patients with active ulcerative colitis (UC), with a few exceptions (Thayer et al., 1976), were reported to exhibit increased numbers of circulating B-cells (Sachar et al., 1973; Strickland et al., 1974), often accompanied by the presence of circulating anti-colon antibodies (Wright & Truelove, 1966). In some patients with UC, a decrease in the absolute number of peripheral blood T-cells and abnormalities of delayed type hypersensitivity reactions have been documented (Sachar et al., 1973). Idiopathic proctitis (IP), a localized form of inflammatory bowel disease, is considered clinically to be a forerunner of ulcerative colitis (Lennard-Jones et al., 1962; Sparberg, Fennessy & Kirsner, 1966). Disturbed local immunity has been observed in histologically diseased, as well as in histologically normal, colonic mucosal biopsy specimens from patients with idiopathic proctitis (Das, Erber & Rubinstein, 1977a). However, the sequential analysis of systemic immunological parameters in such patients and their possible alteration by salicylazosulfapyridine (SASP), the drug most extensively used in the treatment of UC and IP, has not yet been determined. The present study was undertaken firstly to compare the systemic immunity in untreated patients with IP and UC, secondly to explore the in vivo effects of SASP by the temporal analysis of the immunological parameters in treated patients as correlated with the blood levels of the drug and its metabolites, and thirdly to investigate the in vitro effects of the drug on the peripheral blood lymphocytes. Correspondence: Dr A. Rubinstein, Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA. 0099-9104/78/0800-0217$02.00 (C 1978 Blackwell Scientific Publications
217
218
A. Rubinstein et al. MATERIALS AND METHODS
Patients. Thirteen patients (eight females and five males) with mild to moderate active ulcerative colitis and thirteen patients (ten females) with active idiopathic proctitis were studied as out-patients. Their ages ranged between 22-62 years. The diagnosis was confirmed on the basis of symptoms, sigmoidoscopic and/or colonoscopic examinations and barium enema studies. The clinical state of the disease was evaluated as active or in remission on the basis of the number of bowel movements, presence of blood and mucus in the stool, general sense of well being and sigmoidoscopic findings. All patients were treated with 3-4g ofSASP per day. Only one patient with extensive UC received 15 mg prednisone daily, and one patient with IP was treated with hydrocortisone enema in addition to SASP. All twenty-six patients with active disease were studied immunologically prior to treatment. Of these, nine patients with UC and six with IP were re-evaluated 6 to 16 weeks after treatment. All these fifteen patients, except for one with UC and one with IP, were in remission during the follow-up studies. Serum immunoglobulins. IgA, IgD, IgG, IgM and the third complement component (C3), were measured by radial immunodiffusion, using specific antisera (Mancini, Carbonara & Heremans, 1965). Serum IgE levels were determined by radioimmunoassay using the Phadebas Kit, Pharmacia, New Jersey. Serum concentrations of SASP and its metabolites were estimated spectrophotometrically (Das et al., 1973). Peripheral blood lymphocytes (PBL). These were isolated by Ficoll-Hypaque density gradients. Blast transformation of PBL in vitro was measured by the incorporation of tritiated thymidine after stimulation with 0-01% phytohaemagglutinin (PHA-P), 0-01% pokeweed mitogen (PWM), and 10 [ug/ml Concanavalin A (Con A). Throughout the study the same batches of foetal calf serum and of mitogens were employed for the same patient. 200,000 lymphocytes were cultured in Linbro tissue culture multidishes in triplicate in RPMI 1640, supplemented with 15% foetal calf serum. Cells were harvested after 72 hr (for PHA, Con A, PWM), or after 120 hr (for specific antigens) with the automatic cell harvester Mash 2. Mixed lymphocyte cultures were performed according to Bach & Voynow (1969). Thymic derived lymphocytes (T-cells). These were detected by rosetting with sheep red blood cells (SRBC) treated with sulfhydryl compound 2-amino ethylisothiouronium bromide (EAET) (Pellegrino et al., 1975). Rosettes were examined in a mixture of regular and ultraviolet light with a fluorescent microscope after staining the cell suspension with euchrysin (Allison, 1977). Complement receptor positive cells (CR +cells). These were counted after rosetting with sheep red blood cells coated with Forssman rabbit anti-SRBC serum and mouse complement (EAC) (Bianco, Patrick & Nussenzweig, 1970). Serum from C5 deficient mice (B10 D2-OSN, Jackson Labs., Bar Harbor, Maine) was used as complement source. Trypsinized SRBC were employed in the preparation of EAC to avoid cross-reactivity with T-cells (Weiner, Bianco & Nussenzweig, 1973). Early in the course of these studies, we noted abundant phagocytosed EAC and rosette formation by monocytes. Three techniques were employed to avoid reading such rosettes as CR+B-cells. Firstly, rosettes were quantified by the euchrysine technique, which permits discrimination of monocytes from lymphocytes; secondly, EAC rosetted PBL smears were stained by esterase (Yam, Li, & Crosby, 1971). Esterase positive rosetted cells were subtracted from the total CR+cells. Thirdly, rosetted PBL were pelleted by a Ficoll-Hypaque density gradient. The erythrocytes in the pellet were lysed by 0-87% NH4C1, and a differential count of mononuclear cells was obtained by esterase stain or by the Technicon Hemalog D 10,000-cell automated differential analyser. All three techniques yielded comparable results. The net CR+ lymphocytes were found to correlate best with surface immunoglobulin positive cells, and were thus termed B-cells (Ehlenberger et al., 1976). Activated monocytes. These were identified by their capacity to phagocytose EAC after 60 min of incubation at 370C. IgM fraction of EAC was purified on a Sephadex G-200 column to remove most residual IgG. The presence of intracellular erythrocytes was verified by euchrystine fluorescence and by esterase stains of PBL smears. Particularly with the euchrysine fluorescence-stain, intracellular EAC could easily be distinguished from extracellular rosetted erythrocytes. In some experiments, the validity of the euchrysin quantification of EAC-phagocytosis by monocytes was confirmed by re--valuation following lysis ofextracellular EAC with 0-87% NH4CI. Fcy receptor positive PBL. These were identified by rosetting with trypsinized SRBC coated with the IgG fraction of a rabbit anti-SRBC serum (EA-IgG). EA-IgG indicator cells were mixed with PBL, pelleted and incubated at room temperature for 30 min. Rosettes were ready by the euchrysine technique. All immunological parameters were studied in patients with UC and IP, before and after treatment with SASP, in a double blind protocol. The code was broken at the end ofthe protocol. In vitro effects of salicylazosulfapyridine (SASP), and its metabolites, 5-aminosalicylate (5-ASA), and sulfapyridine (SP) on PBL. Toxicity of SASP, 5-ASA and SP in vitro was evaluated by incubation of normal PBL with the drugs at various concentrations. Following incubation, residual cell numbers, cell viability (trypan blue exclusion test), and spontaneous cellular DNA synthesis rates (3H-thymidine uptake) were determined. The drug concentrations used in the evaluation of the in vitro drug effect on the various immunological parameters (for SASP and SP, above 100 ,ug/ml and for 5-ASA, above 10 ,Lg/ml) were below their cell toxic range and higher than serum concentrations attained in treated patients (5-32, 0-1 and 15-42 ug/ml of SASP, 5-ASA and total SP, respectively). Studies with the three drugs were performed in vitro on cultured PBL obtained from six normal controls, two patients with UC, and three patients with IP. PBL were incubated with the drug for 2 hr or throughout the culture period (3-5 days). Cell numbers, cell viability, differential white blood cell count, B-cells, T-cells, Fcy-positive cells and 3H-thymidine uptake were determined on completion of the incubation and simultaneously compared to drug free controls. The in vitro drug effects were studied on PBL culture with or without mitogenic stimulation (with PHA, Con A, PWM, PPD, Candida antigen, or with mitomycin C treated allogeneic cells).
Reversal of altered immunity by salicylazosulfapyridine
219
RESULTS Serum immunoglobulins These revealed no characteristic pattern. Serum IgA levels were in the upper limits of normal or slightly elevated in five out of the eleven UC patients (340, 350, 465, 650 and 560 mg%), and in four of the thirteen IP patients (305, 430, 460 and 580 mg%), as compared to 64-407 mg% in controls. Serum IgE levels were increased in one IP patient (500 u/ml) and in one UC patient (950 u/ml), as compared with the upper limit of normal controls (300 u/ml). Serum IgG, C3 and total complement levels were normal in all patients. Following treatment, there was no change in serum IgD, IgE, IgG and IgM levels. In three patients with UC there was a marked increase in serum IgA level as compared to their pretreatment levels (50, 71 and 1250% increase). In vitro blast transformation ofPBL To PHA and Con A, this was significantly decreased in eleven out of the twenty-six patients with UC and IP (Table 1); responses to PWM being less frequently affected, as compared to twenty controls (Student's t-test for PHA, Con A, P< 0.05). Nine patients with UC and six patients with IP could be
TABLE 1. 3H-thymidine uptake by peripheral blood lymphocytes in patients with UC or with IP before and after treatment with SASP (cpm-minus background, mean value and + 1 SE) Normal controls
Before SASP
PHA Con A PWM
After SASP
PHA Con A PWM
*
N
=
Ulcerative Colitis
Idiopathic Proctitis
N*
RI±1 SE
N* NPt
X ±1 s.e.
N* NPt
20 20 20
41,840±3962
13 13 13
51 64 44
16,182±2470 18,369±3097 18,412±4097
13 13 13
8 8 8
24 14
35,608±6630
33,447±3757
14
25,650±3152
4 4 4
37,982±4332
29,170±2611
64
54 31
34 0 0
X +1 s.e. 13,882±3600 18,369±3935 15,887±2512
19,467±3840 18,959±3591 18,429±3866
Total number of subjects studied for the specified parameter.
t NP= Number of patients with immunological disturbances as compared to the complete range of twenty controls. (Shaded areas in Figs. 1-3). 4 = Abnormally decreased value.
studied for this parameter after therapy (Fig. 1). The mean values for PHA, Con A and PWM lymphocyte mitogenic responses (LMT) were calculated before and after SASP treatment (Table 1). The Student's t-test did not show a significant difference before and after SASP treatment, (except for PHA and Con A LMT in UC), probably because of the wide variation of results. In individual IP patients there was, however, a clear trend towards improvement (Fig. 1). The same data were therefore analysed by comparing each patient to controls before and after SASP treatment. Patients were divided into two groups according to their LMT to PHA: group 1 = below X-ISE of twenty normal controls, group 2 = above X-ISE. The statistical significance of the difference in proportions of patients in each group before and after SASP treatment was obtained by the 2 x 2 contingency test. The results proved that after SASP treatment there were significantly more patients in group 2 (P < 0.05).
220
A. Rubinstein et al.
c
0
~cr .To c
BE E
-e
R CI We
6
Weeks
FIG. 1. 3H-thymidine uptake by peripheral blood lymphocytes following mitogenic stimulation with PHA in patients with ulcerative colitis and idiopathic proctitis before and after SASP therapy. Shaded areas represent the complete range in twenty controls. Before treatment with salicylazosulfapyridine; 0 after treatment with ulterative colitis and 0 normal range. idiopathic proctitis; salicylazosulfapyridine; A
- - -
600[
600g A
400 --._ 0
2O~0~~~~~~~
3000
2000
1000
i-' ', AI
2
4
6 Weeks
8
'Af
*-
12
16
2
4
6
8
12
16
Weeks
FIG. 2. Peripheral blood T cells and B cells in patients with ulcerative colitis before and after treatment with SASP. Shaded areas represent the complete range in twenty controls. A Before treatment with salicylazosulfapyridine; * after treatment with salicylazosulfapyridine and 0 normal range. FIG. 3. Peripheral blood T cells and B cells in patients with idiopathic proctitis before and after treatment with SASP. Shaded areas represent the complete range in twenty controls. A Before treatment with salicylazosulfapyridine; 0 after treatment with salicylazosulfapyridine and 0 normal range.
T-cells Absolute numbers were decreased in five out of the twelve UC patients and in one out of the twelve IP patients as compared to the complete range in twenty controls (Fig. 2, Table 2). The percentage of T-cells was decreased by the same criteria in ten out of twenty-four patients with UC and IP. Following
221
Reversal ofaltered immunity by salicylazosulfapyridine
TABLE 2. Peripheral blood lymphocyte subpopulations in patients with UC or with IP before and after treatment with SASP (mean value i 1 s.e.)
Ulcerative colitis
Normal controls
Before SASP
41
63+5
12
64
66-6±2-8
51
917+120 75±4
12 6
14 It 1450+162 14 73+2-6
1162±66 22-1±4-5
6
81
12
14 2T 1566±330 18±14 81
13 9
7T IT
307+37 10-6±1-7
12 10T 6
2T
9 9
21 31
166±51
X= 25
6 6
3T
20
77+2-3 % of PBL
12
12 9
9
9 13
77±2-3
20
After SASP
Before SASP
X ± Is.e.
N* NPt
11±2-5
CR+ cells
Fcy+ cells
N* NPt
X ±ls.e.
After SASP
Before SASP
± I s.e.
N*
T-cells
11±2-5
20
17±4-2
above 850 per cmm % of PBL above 850 per cmm %of PBL below 284 per cmm %of PBL below 284 per cmm % of PBL
Idiopathic proctitis
I1
(14-38)
21
330±48 13-6±1-1
251+44 X-21
(15-26)
* N= Total number of patients studied for the specified parameter. NP= Number of patients with immunological disturbances as compared to the complete range of twenty controls (shaded areas in Figs. 1-3). 4=Abnormally decreased value. T=Abnormally increased value.
treatment with SASP, T-cells increased beyond the lowest level of the normal range in their percentage and absolute numbers in all but two patients (one IP, one UC) (Table 2, Figs. 2, 3). Calculation of T-cell numbers and percentages before and after SASP treatment by the 2 x 2 contingency test (as used for the analysis of LMT changes by SASP) revealed a significant increase (P < 0.02) in UC patients.
CR+ B-cells These were initially evaluated by light microscopy of EAC-rosettes. Their percentage was markedly higher than the number of B-cells found by simultaneous surface immunoglobulin fluorescence in some patients. This discrepancy alerted us to the possibility of increased numbers of monocytes binding EAC, or of phagocytosis of EAC by monocytes. By employing the euchrysine technique, and esterase stains of smears of rosetted PBL, the net CR+ B-cells were found elevated in both UC and IP patients (Table 2, Figs. 2, 3). After treatment with SASP, the CR+ B-cell number decreased significantly (Table 2, Figs. 2, 3) in most UC and IP patients (2 x 2 contingency test P< 0.05). Esterase positive monocytes The total number per cmm did not deviate significantly from normal controls and was unchanged by treatment. The mean value +1 s.e. of the total monocyte count was higher in thirteen UC patients
222
A. Rubinstein et A
(392±32), as compared to thirteen IP patients (281±13). Both were, however, still in the normal range. Monocytes were abnormally high (700, 760 and 880 monocytes per cmm) in only three of the thirteen patients with UC. Phagocytosis ofEAC This was noted by peripheral blood monocytes in six out of twenty-four patients. These six patients were found to have moderately severe active disease on the basis of symptomatology and sigmoidoscopic findings. In five of these patients (four UC, one IP), more than 24% of all monocytes phagocytosed EAC. In twenty normal controls at most 8% of the monocytes phagocytosed EAC. Following SASP treatment, the percentage of EAC phagocytosing monocytes decreased in all patients, in four to the normal, and in two (UC) to 12% and 14%, respectively. The mean of the percentage of the Fcy positive lymphocytes was elevated in both UC and IP patients, but more so in the UC patients (Table 2). In vitro effects ofSASP and its metabolites While treatment with SASP in vivo affected LMTs, T and CR+ B-cell numbers, and EAC phagocytosis by monocytes, no such effects were detected by in vitro incubation of normal controls' or patients' leucocytes with the drugs in two folds of the 'therapeutic concentration ranges' (Das et al., 1973).
DISCUSSION Idiopathic proctitis is an inflammatory bowel disease localized to the distal 15-20 cm of the large intestine, and is clinically considered to be a forerunner of UC (Lennard-Jones et al., 1962; Sparberg et al., 1966). We have demonstrated previously abnormalities in secretory IgA in histologically normal proximal colonic mucosa of patients with IP (Das et al., 1977a). While the pathogenetic significance of this finding is still obscure, it suggests that the disease process is more extensive than the macroscopic and light microscopic abnormalities (Das et al., 1977a; Das et al., 1977b) and probably reflects various spectra of this process. The present analysis of systemic immunological functions in UC and IP also indicates similar alterations. In both diseases, increased circulating B-cells and an in vitro decreased lymphocyte mitogenic responses to PHA and Con A as well as low T-cell numbers were noted. Most intriguingly, however, the peripheral blood monocytes were found in the active stages of UC and IP to phagocytose sheep red blood cells coated by IgM antibody and complement (EAC). The SRBC-IgM-C3 complex (EAC) is routinely used to detect CR+ lymphocytes (Bianco et al., 1970). The employment of the euchrysin fluorescence (Allison, 1977) and the esterase stain (Yam et al., 1971) in this study permitted the discrimination between bona fide EAC rosetted CR+ lymphocytes and EAC attached to monocytes or phagocytosed by them. In a few instances more than a quarter of the peripheral blood monocytes obtained from patients with active UC and IP phagocytosed EAC. Normal monocytes carry both C3b and C3d receptors and phagocytose only SRBC coated by IgG and C3. Activated monocytes alone phagocytose SRBC coated by the IgM-C3 complex too (Ehlenberger & Nussenzweig, 1977). They are frequently highly secretory (Gordon, 1976; Vassalli & Reich, 1977) and their proteolytic enzymes may contribute to the mucosal damage as found in patients with IP and UC. Hypothetically, such an effect could be most injurious in IP and UC in view of the additional abnormality of the colonic secretory IgA system (Das et al., 1977a; Simada, 1976). It has been suggested that secretory IgA may regulate tissue autoproteolysis (Tomasi & Hauptman, 1974) by its association with alpha-l-antitrypsin. The local IgA deficiency may thus render the colonic mucosa more vulnerable to the proteolytic enzymes secreted by activated monocytes. The isolation of a highly active monocyte/macrophage population from normal human intestinal mucosa has recently been reported (Bull & Bookman, 1977). This population was absent in normal peripheral blood and was defined by its increased cell size, granularity, motility, glass adherence and latex particle phagocytic activity. It has yet to be determined whether that population finds access into the peripheral blood in inflammatory bowel disease. Our observation that a successful SASP treatment invariably diminished the number of circulating EAC phagocytosing esterase positive cells, would suggest the inter-relationship of this cell subset with the disease process. The decrease in
Reversal of altered immunity by salicylazosulfapyridine
223
EAC phagocytosing monocytes was, however, not accompanied by a diminution of the total monocyte number in the peripheral blood. There was also evidence to suggest a drug related correction of other immunological abnormalities such as the decrease in T-cell numbers and the elevation of CR+ B-cells, as well as the abnormalities of in vitro LMTs (Table 1, 2, Figs. 1, 2 & 3). However, a complete dissociation between the in vivo and in vitro effects of SASP was noted. Studies with patients' or controls' PBL, in tissue culture medium containing SASP or its derivatives in up to two folds of the therapeutic serum levels, failed to demonstrate any influence on immunocytes. Currently, little is known about the mechanism of the action of SASP. In view of the dichotomy between the in vivo and in vitro drug effects, we may conjecture that SASP exerts its immunological influence indirectly. It may, for example by its antimicrobial activity (West et al., 1974), eliminate bacterial antigens from the colonic tissue. We have recently extracted from colectomy specimens an antibody that cross-reacts with an as yet undefined antigen(s) present in the colonic mucosa of patients with UC (Das & Dubin, 1977). This finding may be indicative of local immune complexes that in turn could activate monocytes. Further studies related to quantification and functional characterization of local monocytes, before and after treatment with SASP, may yield further information concerning their pathogenetic role and a possible mode of action of the drug. The authors wish to acknowledge the invaluable help of Dr N. A. Kowatch, Supervisor of the Hematology Laboratory, the Hospital of the Albert Einstein College of Medicine, in analysing white blood cell differentials on the Technicon Hemalog D counter. We are also grateful to Dr V. Krishnaswamy for the statistical analysis of the data and Dr B. Zapp for his assistance. Supported in part by a grant from the National Institutes of Health Al 10702-05 and by the National Foundation for Ileitis and Colitis, Inc., New York. Dr Das is the recipient of a Clinical Investigator Award from the National Institutes of Health grant # IK08 AM00237.
REFERENCES ALLISON, A.C. (1977). Fluorescence microscopy of lymphocytes and mononuclear phagocytes and the use of silica to eliminate the latter. In Vitro Methods in Cell Mediated and Tumor Immunity (ed. by B.R. Bloom, and J.R. David), p. 395. Academic Press, New York. ASQUITH, P., KRAFT, S.C. & TOTHBERG, R.M., (1973). Lymphocyte responses to nonspecific mitogens in inflammatory bowel disease. Gastroenterology, 65, 1. BACH, F.H. & VOYNOW, N.K., (1969). One way stimulation in mixed leukocyte cultures. Science, 153, 545. BIANCO, C., PATRICK, R. & NUSSENZEIG, V., (1970). A population of lymphocytes bearing a membrane receptor for antigen antibody complement complexes. I. Separation and characterization.]. exp. Med. 132, 702. BRANDTZAEG, P., BAKLIEN, K., FAUSA, 0. & HOEL, P.S., (1974). Immunohistochemical characterization of local immunoglobulin formation in ulcerative colitis. Gastroenterology, 66, 1123. BULL, D.M. & BOOKMAN, M.A., (1977). Isolation and functional characterization of human intestinal mucosal lymphoid cells.]. clin. Invest. 59, 966. DAS, K.M. & DUBIN, R., (1977). Isolation and characterization of colonic tissue bound antibodies from patients with ulcerative colitis. Gastroenterology, 72, 1043 (abstr.). DAS, K.M., EASTWOOD, M.A., MCMANUS, J.P.A. & SIRCUS, W., (1973). The metabolism of salicylazosulphapyridine in ulcerative colitis. I. The relationship between metabolites and the response to treatment in patients. II. The relationship between metabolites and the progress of the disease studied in out-patients. Gut, 14, 631. DAS, K.M., ERBER, W.F. & RUBINSTEIN, A., (1977a). Immunohistochemical changes in morphologically involved and uninvolved colonic mucosa of patients with idiopathic proctitis.]. clin. Invest. 59, 379. DAS, K.M., MORECKi, R., NAIR, P. & BERKOWITZ, J.M., (1977b). Idiopathic proctitis: I. The morphology of
proximal colonic mucosa and its clinical significance.
Am.J. Dig. Dis. 22, 524. EHLENBERGER, A.G., MCWILLIAMS, M., PHILLIPS-QUAGLIATA, J.M, LAMM, M.E., & NUSSENZWEIG, V. (1976). Immunoglobulin-bearing and complement-receptor lymphocytes constitute the same population in human peripheral blood._T. Clin. Invest. 57, 53. EHLENBERGER, A.G. & NUSSENZWEIG, V., (1977). The role of membrane receptors for C3b and C3d in phagocytosis. J. exp. Med. 145, 537. GORDON, S., (1976). Macrophage neutral proteinases and chronic inflammation. Ann. N.Y. Acad. Sci. 278, 176. KRAFT, S.C. & KIRSNER, J.B., (1971). Immunological apparatus of the gut and inflammatory bowel disease. Gastroenterology, 60, 922. LENNARD-JONES, J.E., COOPER, G.W., NEWELL, A.C., WILSON, C.W.E. & JONES, F.A., (1962). Observations on idiopathic proctitis. Gut, 3, 201. MANCINI, G., CARBONARA, A.W. and HEREMANS, J.F., (1965). Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry. 2, 235. PELLEGRINO, M.A., FERRONE, S., DIERICH, M.P. & REISFELD, R.A., (1975). Enhancement of sheep red blood cell human lymphocyte rosette formation by the sulfhydryl compound 2-amino ethylisothiourodium bromide. Clin. Immunol. Immunopathol. 3, 324. SACHAR, D., TAUB, R.N., BROWN, S.M., PRESENT, D.H., KORELTZ, B.I. & JANOWITZ, H.D., (1973). Impaired lymphocyte responsiveness in inflammatory bowel disease. Gastroenterology, 64, 203. SIMADA, H., (1976). Immunohistochemical study of ulcerative colitis with special reference to local immunoglobulin. Jap. J. Dig. Dis. 73, 832. SKINNER, J.M. & WHITEHEAD, R., (1974). The plasma cells in inflammatory disease of the colon: A quantitative study.]. Clin. Pathol. 27, 643.
224
A. Rubinstein et al.
SPARBERG, M., FENNESSY, J. and KIRSNER, J.B., (1966). Ulcerative proctitis and mild ulcerative colitis: A study of 220 patients. Medicine (Baltimore), 45, 391. STRICKLAND, R.G., KORSMEYER, S., SOLTis, R.D., WILSON, I.D. & WILLIAMS, JR., R.C. (1974). Peripheral blood T and B cells in chronic inflammatory bowel disease. Gastroenterology, 67, 569. THAYER, JR., W.R., CHARLAND, C. & FIELD, C.E., (1976). The subpopulations of circulating white blood cells in inflammatory bowel disease. Gastroenterology, 71, 379. TomASI, JR., T.B. & HAUPTMAN, S., (1974). Modulation of the assembly of immunoglobulin subunits by J chain. In the immunoglobulin A system (ed. by J. Mestecky, and A.R. Lawton,) p. 111. Pleum Press, New York.
VASSALLI, T.D. & REICH, E., (1977). Macrophage plasminogen activator: induction by products of activated lymphoid cells.]. exp. Med. 145,429. WEST, B., LENDRUM, R., HILL, M.S. & WALKER, G., (1974). Effects of sulfasalazine (salazopyrin) on fecal flora in patients with inflammatory bowel disease. Gut, 15, 960. WEINER, M.S., BIANCO, C., & NUSSENZWEIG, V. (1973). Enhanced binding of Neuraminidase treated sheep erythrocytes to human T-lymphocytes. Blood, 42, 939. WRIGHT, R. & TRUELOVE, S.C., (1966). Autoimmune reactions in ulcerative colitis. Gut, 7, 32. YAM, L.T., LI, C.Y. & CROSBY, W.H., (1971). Cytochemical identification of monocytes and granulocytes. Amer. J. Chem. Pathol. 55, 283.
