Clin. exp. Immunol. (1978) 32,451-458.
Decreased suppressor cell activity in inflammatory bowel disease H. J. F. HODGSON, J. R. WANDS & K. J. ISSELBACHER Department of Medicine, Harvard Medical School, and the Massachusetts General Hospital (Gastrointestinal Unit), Boston, Massachusetts, USA
(Received 10January 1978) S UMMARY
Studies were performed on eleven patients with inflammatory bowel disease to determine if there was an alteration in concanavalin A (Con A) induced suppressor cell activity. Similar investigations were also performed on twenty-one control subjects and five patients with other inflammatory conditions. Suppressor cells were generated by pre-incubation of peripheral blood mononuclear cells with a mitogenic concentration of Con A, followed by treatment with mitomycin C and a-methyl mannoside. Under these conditions, cells obtained from normal individuals are then capable of suppressing the Con A-stimulated blast transformation responses of fresh allogeneic lymphocytes in new cultures. We found that in twenty out of twenty-one control subjects, and all five patients with other inflammatory disorders, Con A-stimulated suppressor cell activity was demonstrable. Four patients with inflammatory bowel disease, whose disease was mildly active or was in clinical remission, had elicitable suppressor cell activity which fell within the normal range. In contrast, suppressor cell activity was markedly diminished or absent in seven patients with severe and active inflammatory bowel disease. These studies suggest that an alteration in Con A-stimulated suppressor cells exists in patients with active inflammatory bowel disease, which may contribute, in part, to the persistent inflammation in the gastrointestinal tract. INTRODUCTION Patients with ulcerative colitis and Crohn's disease show cellular immune responses to gut and bacterial antigens. This hypersensitivity has been demonstrated by a variety of techniques. Enterobacterial common antigens and colonic epithelial cells elicit the production of macrophage and leucocyte migration inhibition factor from lymphocytes of patients with inflammatory bowel disease (Bull & Ignaczack, 1973; Bendixen, 1969; Bartnik, Swarbrick & Williams, 1974). In vitro studies with autologous colonic epithelial cells induce a blast transformation response in peripheral blood mononuclear cells (Hunt & Trotter, 1973). Delayed-type hypersensitivity skin responses to colonic antigens incubated with E. coli are present in such patients (Fink & Mais, 1968). Peripheral blood lymphocytes from patients with inflammatory bowel diseases are also cytotoxic for colonic epithelial cells (Perlmann & Broberger, 1963; Watson, Quigley & Bolt, 1966; Shorter et al., 1969), although this is probably not a manifestation of classical cell-mediated immunity, as humoral factors are also involved (Stobo et al., 1976a). There is only a poor correlation between the various aspects of cellular immunity observed and the activity of the inflammatory bowel disease. Lymphocyte responses to bacterial antigens have been reported as enhanced, unchanged or diminished during the active disease (Bartnik et al., 1974; Stefani & Fink, 1967; Hinz, Perlmann & Hammarstrom, 1967). The degree of lymphocyte cytotoxicity observed has been very similar in all patients studied, and is apparently unaffected by inflammatory disease activity (Watson et al., 1966). More importantly, most of these cellular immune responses have only been demonstrated in vitro and their role in vivo is unknown. Correspondence: Dr H. J. F. Hodgson, Department of Medicine, Royal Postgraduate Medical School, Du Cane Road, London W12 OHS. 0099-9104/78/0600-0451$02.00 (©1978 Blackwell Scientific Publications
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H. J. F. Hodgson, J. R. Wands & K. 9. Isselbacher
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Recent animal studies have suggested that the expression of both cellular and humoral immunity by antigen-sensitized lymphocytes is regulated by helper and suppressor T cells (Gershon, 1974; Dutton, 1975). It has become apparent that similar cells, not exclusively confined to the T-cell population, with an immunoregulatory function, may be present in man. Thus it has been postulated that abnormalities in the function of suppressor cells may contribute, in part, to variable hypogammaglobulinaemia (Waldman et al., 1974; Siegal, Siegal & Good, 1976), multiple myeloma (Broder et al., 1975), Hodgkin's disease (Twomey et al., 1975), systemic lupus erythematosus (Abdou et al., 1976; Bresnihan & Jasin, 1977) and severe fungal infections (Stobo et al., 1976b). It is therefore possible that in inflammatory bowel disease, increased helper or decreased suppressor cell activity may influence the expression of the immune response. In the present investigation, we have explored this possibility by measuring the Con A-stimulated suppressor cell activity elicited from peripheral blood mononuclear cells in patients with ulcerative colitis and Crohn's disease. The suppressor cell assay was based on the demonstration that peripheral blood mononuclear cells from normal individuals, when incubated in vitro with Con A, develop the ability to suppress the blast transformation responses of fresh allogeneic lymphocytes (Shou, Schwartz & Good, 1976; Hubert, Delespesse & Govaerts, 1976). We report here an alteration in Con A-stimulated suppressor cell activity in patients with inflammatory bowel disease.
MATERIALS AND METHODS Patients. Eleven patients with inflammatory bowel disease, aged from 16 to 66 (mean 36 years), were studied. Some clinical data are shown in Table 1. Diagnoses were made on standard clinical, histological and radiological grounds, and the disease extent defined radiologically. Disease severity was classified according to the criteria of Truelove & Witts (1955) for ulcerative colitis and De Dombal et al. (1974) for Crohn's disease. Twenty-one subjects, comprising six healthy individuals and fifteen hospitalized patients with benign disorders (hiatus hernia, peptic ulceration and cardiac arrhythmias), were studied as a control group (mean age 39 years, range 19-72 years). Five additional patients with acute inflammatory conditions (three with lobar pneumonia, one with gonococcal septicaemia and one with cellulitis) were studied as an inflammatory disease control group (mean age 57 years, range 26-81 years). TABLE 1. Clinical details of patients with inflammatory bowel disease
Prednisone Patient number (1) (2) (3) (4) (5) (6) (7) (8) (9) (10)
(11)
Sex
Age
M F M M M F M F M M M
19 35 66 30 41 62 24 46 16 39 20
Diagnosis Ulcerative colitis Ulcerative colitis Ulcerative colitis Ulcerative colitis Ulcerative colitis Ulcerative colitis Crohn's disease Crohn's disease Crohn's disease Crohn's disease Crohn's disease
Region affected Total Total Transverse colon rectum Descending colon rectum Total Total Ileal
Ileo-colonic Ileo-colonic Ileo-caecal Total
Severity Severe Remission Mild Mild
Moderately severe
Moderately severe Severe Remission Severe Severe Severe
dosage (mg daily)
Suppression (%)
0 0 0 0 0 60 20 30 0 10 30
-35 22
21-9 30 7 -108 -69
-18-9 14-8 4.3 -27-8 1-5
Suppressor cell assay. 40 ml heparinized venous blood were obtained from each subject. In each assay, cells from both normal subjects and patients with inflammatory bowel disease were studied simultaneously for comparison. The mononuclear cells were separated by Ficoll-Hypaque centrifugation (Boyum, 1968), washed in RPMI 1640 medium (Microbiological Associates, Bethesda, Maryland) and resuspended in 10 ml RPMI 1640 enriched with 10% inactivated foetal calf serum, 4mM glutamine, 100 u/ml penicillin and 100 jg/ml streptomycin (complete medium). Cells were then divided into
Suppressor cells in inflammatory bowel disease
453
two groups containing 3 to 5 x 106 cells per ml, which were incubated (first culture) with or without 6 j±g/ml Con A (MilesYeda Inc, Indiana) in sterile Universal Containers (Sterilin Ltd, U.K.), generating 'suppressor' and 'control' populations respectively. 48 hr later, both cell suspensions were treated with 50 jig/ml mitomycin C (Sigma Chemicals) for 30 min at 370C to block DNA synthesis, and then washed three times with 30 mm a-methyl mannoside in RPMI 1640 medium to remove residual cell-bound Con A. Finally, cells were resuspended in complete medium, counted in a haemocytometer and adjusted to a concentration of 106 per ml. An aliquot was tested for viability by trypan blue exclusion; under these circumstances, viability was greater than 85%. Fresh mononuclear cells (responder cells) were prepared from a normal donor in a similar manner and adjusted to a concentration of 106 cells per ml in complete medium. One donor served as the source of responder cells in all experiments (unless otherwise specified) to ensure a reproducible Con A-reacfive cell population. These responder cells were stimulated to blast transformation by Con A in the presence of either the 'suppressor' or 'control' cells (second culture). Incubations were performed in quadruplicate using microtitre plates (Cooke Engineering, Alexandria, Virginia), to which were added 0-1 ml responder cells, 0- 1 ml of the suppressor or control cell suspension and 0-025 ml of 60 jig/ml solution of Con A. After an 80 hr incubation, 2 jLCi of [3H]thymidine (sp. act. 20-40 Ci/mM, New England Nuclear, Boston Massachusetts) were added to each well. The cells were harvested 18 hr later in an automatic harvester (MASH II, Microbiological Associates). The filters were placed in 3-0 ml of toluene scintillation fluid (New England Nuclear) and their radioactivity measured for 10 min in a Beckman liquid scintillation counter. The results of the suppressor assay were expressed in the following manner: [3H]thymidine incorporation in presence of suppressor cells) 100. (1 [3H]thymidine incorporation in presence of control cells 9 Comparison between groups were made using the Student's t-test. Dose-response characteristics of suppressor cell activity generated by Con A. In another series of experiments we studied the effectiveness of varying doses of Con A in inducing both suppressor cell activity and blast transformation in the same population of peripheral blood mononuclear cells from normal individuals. Cells were incubated for 48 hr at doses of Con A varying from 0-60 [Lg/ml, as described above. In 0-1 ml aliquots from each cell suspension in triplicate, the blast transformation response was determined by incubation for a further 6 hr with 2-0 [tCi [3H]thymidine before harvesting. The remainder of each cell suspension was treated with mitomycin C and the suppressor function assayed as described above. X
RESULTS
Characteristics ofstppressor cell assay Peripheral blood mononuclear cells from normal donors, after pre-incubation at doses of Con A from 0-1 to 20 gg/ml, suppressed the Con A-stimulated blast transformation responses in fresh allogeneic lymphocytes. Suppressor activity was thus generated by concentrations of Con A that only stimulated minor increases in the [3H]thymidine incorporation during pre-incubation as well as by concentrations which induced maximal blast transformation (see Table 2). When higher doses of Con A (60 jg/ml) were TABLE 2. Comparison between blast transformation induced in peripheral blood mononuclear cells by pre-incubation with various doses of Con A (first culture), and the effect of those pre-incubated cells in suppressing Con A-stimulated blast transformation in fresh allogeneic responder cells (second culture)
[3H]thymidine uptake of Concentration of Con A (jig/ml) (1st culture)
[3H]thymidine uptake (54 hr)
0 0-5 1 2 5 10 20
1152±120 1614+100 4124±208 10312±386 57519±589 132307±2919 163604±4176
(ct/min±s.e.m.)*
Stimulation Index -
1-4 3-6 90
49.9 114-8 142-0
responder cells (2nd culture)
(ct/min~s.e.m.) 82759+2327 74874±3986 64076± 1742 57304±642 60305±1786 29490± 1124
9-6 22-6 30-8 27-1 64-4
49244±2859
40.5
Concentration of Con A employed in second culture was 6-00,g/ml. * 48 hr incubation with Con A followed by 2 ,uCi [3H]thymidine for 6 hr. ct/min error ofthe mean (s.e.m.).
Suppression (%)
=
-
Mean count per minute± standard
H. J. F. Hodgson, J. R. Wands & K. J. Isselbacher
454
employed, leucoagglutination occured, and the replicate measurements of the blast transformation response of responder cells in the presence of the suppressor population showed tremendous variability. The suppressor cell activity in control subjects and patients with inflammatory bowel diseases in the present investigation were generated by a mitogenic concentration of Con A (6 gg/ml). We also studied the reproducibility of the suppressor cell assay in serial studies, using both allogeneic and autologous responder cell populations. Serial weekly suppressor cell assays in one normal subject, when the responder cells were autologous to the Con A pre-treated and control cells, showed suppression of 48-2, 66-3 and 32%. In two other individuals using allogeneic responder cells, less variation was found (68.3 and 634%, 45X3 and 46.8%). Suppressor cellfunction in patients with inflammatory bowel disease and control subjects The results of a typical suppressor assay on peripheral blood mononuclear cells from two patients and two control subjects are shown in Table 3, in order to illustrate the magnitude of the changes observed in the cultures. TABLE 3. Representative data on the suppressor cell assay in two patients and two control subjects.
Control Control PatientNo. 3 Patient No. 7
(A) Responder cells plus Con A pre-treated cells plus Con A
(B) Responder cells plus control cells plus Con A
54081±3846 76026±3709
114341±3368 102742±3858 109850±2346 102661+5408
85839±3279 122131±5682
Suppression (%) 52-8 26 21-9 -18-9
Mean ct/min (+ s.e.m.) of responder cells from one donor in presence of Con A pre-treated cells (column A) or control cells (column B) and Con A. For patient No. refer to Table 1. 80 6040
0
-6 60
-80 _ -100
_
Control
Inflammatory
(n = 21)
bowel disease
(n- I1) FIG. 1. Results of suppressor cell measurement in control subjects and patients with inflammatory bowel disease. The ordinate shows the percentage suppression. Points below the dashed line indicate enhancement rather than suppression of 3H-thymidine incorporation. Mean ± s.e.m. for each group is indicated by shaded area. Difference between the two groups is significant: P < 0 005.
In twenty out of twenty-one control subjects (96%), the blast transformation responses of fresh allogeneic lymphocytes were suppressed by Con A pre-treated cells as compared to control cells. The mean suppressor cell activity for this group was 2744% (Fig. 1). Furthermore, there was no difference between the mean suppressor cell activity obtained from the healthy controls when compared to the hospitalized control subjects. In contrast, Con A-generated suppressor cells were found in only six out of eleven patients (54%) with inflammatory bowel disease. Moreover, in the other five patients with
Suppressor cells in inflammatory bowel disease
455
inflammatory bowel disease, the blast transformation of fresh lymphocytes was greater in the presence of the Con A pre-treated cells than the controls, suggesting a helper rather than a suppressor effect. The mean level of suppressor activity elicitable from peripheral blood mononuclear cells in the inflammatory bowel disease group was - 9 2% (+ 11.8 % s.e.m.). These results differ significantly from the control group (P < 0005). The loss of mononuclear cell suppressor activity was closely related to disease activity. There were four patients, none of whom had been admitted to the hospital, whose disease was classified as mildly active or in remission (see Table 1). Each of these patients had a normal suppressor cell response (22-4 +3 3%), and this group did not differ from the control population. In contrast, seven patients had their disease classified as severe or moderately severe; these had a prolonged hospital course for treatment of 40
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Mild
Severe Other inflammatory conditions (0:-4) (n =7) Inflammatory bowel (0=5)
diseose FIG. 2. Results of suppressor cell measurement in patients with inflammatory bowel disease, classified according to disease severity, and in patients with other inflammatory conditions. The ordinate show the percentage suppression. Mean + s.e.m. and dashed line as in Fig. 1. The differences between mild vs severe inflammatory bowel disease are significant: P< 0 05; as are differences between severe inflammatory bowel disease vs other inflammatory conditions: P< 0-025. 40
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.
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63/ 10 _% s_ -
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-20 _ -30 40 ~ -
atDa 0
_
2
4
6 8 10 12 Time (weeks)
14
16
Admitted Discharged FIG. 3. Serial study of suppressor cell activity in a patient with severe ulcerative colitis. Prednisone therapy commenced after first assay performed. The abscissa shows time. The ordinate shows the percentage suppression. Shaded area show the mean + s.e.m. ofcontrol subjects.
456
H. 3. F. Hodgson, 3. R. Wands & K. 3. Isselbacher
their disease, and five underwent bowel resection (the suppressor cell assay had been performed prior to surgery). Only two out of the seven patients exhibited a suppressor cell response; the observed mean suppressor cell activity for all these severely affected patients was - 272± 14-5 %, which was significantly less than both the control population and the patients with mild inflammatory bowel disease (Fig. 2). Furthermore, this result was significantly different from the patients with other inflammatory disorders
(21'84±5.5%). One patient (Table 1, patient 1), with severe ulcerative colitis, was studied serially after admission with toxic megacolon. The clinical course is illustrated in Fig. 3. Suppressor activity could not be elicited from his cells during repeated testing over 3 months, despite a slow clinical improvement with medical management, including high-dose corticosteroids (60 mg prednisone daily) and sulphasalazine. The first measurement after hospital discharge, at which time the patient was symptom-free, still showed altered suppressor cell activity; however, it returned to the normal range 6 weeks later. At that time there was no evidence of disease activity, as judged by symptoms, sigmoidoscopic examination and rectal biopsy. Of the eleven patients studied, five were on treatment with corticosteroids at the time the assay was performed (Table 1). The serial study detailed above indicated little change in the activity of the Con Astimulated suppressor cells at the time of initiation of treatment with prednisone, 60 mg daily (-35%), or after 1 week of therapy (- 28%). However, it is quite possible that corticosteroids affect Con Astimulated suppressor cell activity after prolonged administration. Additional studies are needed before a firm conclusion can be reached on the role of corticosteroids on Con A-generated suppressor cell activity. DISCUSSION The suppressor cell assay employed in the present investigation measures suppressor cell function generated by pre-incubation of cells with Con A. While this assay does not measure the spontaneous suppressor activity present in the mononuclear cell population, it indicates the presence of a population or subpopulation ofcells which can develop in vitro suppressor function after Con A activation. A number of studies (Shou et al., 1976; Hubert et at., 1976; Hallgren & Yunis, 1977) have confirmed that a Con Ainduced suppressor cell population is present in normal subjects and the putative effector cell has been identified as a high-density T cell (Sakane & Green, 1977). We have shown altered suppressor cell activity in patients with severe or moderately severe inflammatory bowel disease. There are a number of possible explanations for the altered suppressor cell activity in these patients. A generalized lymphocyte hyporesponsiveness has been reported by some authors in inflammatory bowel disease, particularly Crohn's disease (Walter & Greaves, 1969; Brown et al., 1970), although Asquith, Kraft & Rothberg (1973) and Aas et al. (1972) both found the blast transformation response induced by Con A to be normal in Crohn's disease. However, it seems unlikely that a failure to generate suppressor cells during pre-incubation with Con A is the same phenomenon as a generalized lymphocyte hyporesponsiveness, as the subpopulation of lymphocytes responsible for Con A-induced suppression in normal human subjects has been shown to possess a poor blastogenic response to this lectin (Sakane & Green, 1977). Also relevant in this regard is our finding that doses of Con A that caused only minor increases in thymidine uptake induced considerable suppressor activity in the same mononuclear cell population. Corticosteroids affect lymphocyte responsiveness in vitro and alter lymphocyte subpopulations in vivo (Fauci & Dale, 1974). Although a majority of the patients with severe disease were on such treatment, there was little difference in the results of the suppressor assay performed before and after initiation of this therapy, as illustrated by the serial study. Abnormal suppressor responses were also seen in those patients with active disease not on such therapy. Additional studies are necessary, however, to clarify the role of corticosteroids on Con A-stimulated suppressor cells. It is possible that our findings could be explained by an alteration in the lymphocyte subpopulations during active disease. Strickland et al. (1975) found reduced numbers of T cells in patients with inflammatory bowel disease, although Bird & Britton (1974) and Thayer, Charland & Field (1976) found
Suppressor cells in inflammatory bowel disease
457
normal T-cell numbers. In the present investigation, we cannot distinguish between the loss of a Con A-reactive T-cell subpopulation with suppressor activity, hyporesponsiveness of suppressor cells to Con A, or an abnormal helper cell population which could mask the suppressor cell effect. A comment is also required concerning the mode of calculation of the suppressor cell activity. Suppressor cell activity was expressed as a reduction in the ratio of the blast transformation response of fresh lymphocytes in the presence of Con A pre-treated cells compared to their response in the presence of control cells; if the control cells retain spontaneous suppressor activity, this ratio could increase even if the Con A-induced suppressor activity was unchanged. However, this seems unlikely, since spontaneous suppressor cells are apparently short-lived in culture, and their activity is markedly diminished after 24 hr in vitro (Bresnihan & Jasin, 1977). With our present knowledge we cannot judge whether the demonstrated defect in Con A-induced suppressor cell activity plays a role in maintaining the disease process in inflammatory bowel disease. While the close relationship between disease activity and loss of suppressor function might suggest that the defect is merely a secondary phenomenon, the normal suppressor cell responses among patients with other severe inflammatory conditions indicates that inflammation per se is not associated with abnormal suppressor cell responses. It was also noteworthy that the return to normal suppressor function in the patient studied serially lagged behind the clinical remission of the disease. It is indeed conceivable that a loss of suppressor function might contribute, in part, to the pathogenesis of inflammatory bowel disease. In another chronic inflammatory disease, systemic lupus erythematosus, a suppressor cell defect has been described (Bresnihan & Jasin 1977) and it has been proposed that a lack of suppression may contribute to the development of autoimmunity and resulting tissue damage in that disease. Serial measurements of suppressor cell activity in patients with systemic lupus erythematosus have shown a return to normal with remission (Bresnihan & Jasin, 1977). In inflammatory bowel disease, while the initiating factor remains unknown, e.g. a transmissible agent (Cave et al., 1973) or an initial mucosal breach allowing sensitization to gut and bacterial antigens (Shorter, Huizenga & Spencer, 1973), a loss of suppressor cell activity, permitting the expression of damaging immune responses, might contribute to the persistence of inflammation in the gastrointestinal tract. This work was supported in part by grants from the National Institutes of Health, Bethesda, Maryland, U.S.A. (AA-02666 and AM-2030). J. R. Wands is a recipient of a Clinical Investigator Award: AM-00127. H. J. F. Hodgson held the Radcliffe Travelling Fellowship of University College, Oxford.
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Decreased suppressor cell activity in inflammatory bowel disease H. J. F. HODGSON, J. R. WANDS & K. J. ISSELBACHER Department of Medicine, Harvard Medical School, and the Massachusetts General Hospital (Gastrointestinal Unit), Boston, Massachusetts, USA
(Received 10January 1978) S UMMARY
Studies were performed on eleven patients with inflammatory bowel disease to determine if there was an alteration in concanavalin A (Con A) induced suppressor cell activity. Similar investigations were also performed on twenty-one control subjects and five patients with other inflammatory conditions. Suppressor cells were generated by pre-incubation of peripheral blood mononuclear cells with a mitogenic concentration of Con A, followed by treatment with mitomycin C and a-methyl mannoside. Under these conditions, cells obtained from normal individuals are then capable of suppressing the Con A-stimulated blast transformation responses of fresh allogeneic lymphocytes in new cultures. We found that in twenty out of twenty-one control subjects, and all five patients with other inflammatory disorders, Con A-stimulated suppressor cell activity was demonstrable. Four patients with inflammatory bowel disease, whose disease was mildly active or was in clinical remission, had elicitable suppressor cell activity which fell within the normal range. In contrast, suppressor cell activity was markedly diminished or absent in seven patients with severe and active inflammatory bowel disease. These studies suggest that an alteration in Con A-stimulated suppressor cells exists in patients with active inflammatory bowel disease, which may contribute, in part, to the persistent inflammation in the gastrointestinal tract. INTRODUCTION Patients with ulcerative colitis and Crohn's disease show cellular immune responses to gut and bacterial antigens. This hypersensitivity has been demonstrated by a variety of techniques. Enterobacterial common antigens and colonic epithelial cells elicit the production of macrophage and leucocyte migration inhibition factor from lymphocytes of patients with inflammatory bowel disease (Bull & Ignaczack, 1973; Bendixen, 1969; Bartnik, Swarbrick & Williams, 1974). In vitro studies with autologous colonic epithelial cells induce a blast transformation response in peripheral blood mononuclear cells (Hunt & Trotter, 1973). Delayed-type hypersensitivity skin responses to colonic antigens incubated with E. coli are present in such patients (Fink & Mais, 1968). Peripheral blood lymphocytes from patients with inflammatory bowel diseases are also cytotoxic for colonic epithelial cells (Perlmann & Broberger, 1963; Watson, Quigley & Bolt, 1966; Shorter et al., 1969), although this is probably not a manifestation of classical cell-mediated immunity, as humoral factors are also involved (Stobo et al., 1976a). There is only a poor correlation between the various aspects of cellular immunity observed and the activity of the inflammatory bowel disease. Lymphocyte responses to bacterial antigens have been reported as enhanced, unchanged or diminished during the active disease (Bartnik et al., 1974; Stefani & Fink, 1967; Hinz, Perlmann & Hammarstrom, 1967). The degree of lymphocyte cytotoxicity observed has been very similar in all patients studied, and is apparently unaffected by inflammatory disease activity (Watson et al., 1966). More importantly, most of these cellular immune responses have only been demonstrated in vitro and their role in vivo is unknown. Correspondence: Dr H. J. F. Hodgson, Department of Medicine, Royal Postgraduate Medical School, Du Cane Road, London W12 OHS. 0099-9104/78/0600-0451$02.00 (©1978 Blackwell Scientific Publications
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Recent animal studies have suggested that the expression of both cellular and humoral immunity by antigen-sensitized lymphocytes is regulated by helper and suppressor T cells (Gershon, 1974; Dutton, 1975). It has become apparent that similar cells, not exclusively confined to the T-cell population, with an immunoregulatory function, may be present in man. Thus it has been postulated that abnormalities in the function of suppressor cells may contribute, in part, to variable hypogammaglobulinaemia (Waldman et al., 1974; Siegal, Siegal & Good, 1976), multiple myeloma (Broder et al., 1975), Hodgkin's disease (Twomey et al., 1975), systemic lupus erythematosus (Abdou et al., 1976; Bresnihan & Jasin, 1977) and severe fungal infections (Stobo et al., 1976b). It is therefore possible that in inflammatory bowel disease, increased helper or decreased suppressor cell activity may influence the expression of the immune response. In the present investigation, we have explored this possibility by measuring the Con A-stimulated suppressor cell activity elicited from peripheral blood mononuclear cells in patients with ulcerative colitis and Crohn's disease. The suppressor cell assay was based on the demonstration that peripheral blood mononuclear cells from normal individuals, when incubated in vitro with Con A, develop the ability to suppress the blast transformation responses of fresh allogeneic lymphocytes (Shou, Schwartz & Good, 1976; Hubert, Delespesse & Govaerts, 1976). We report here an alteration in Con A-stimulated suppressor cell activity in patients with inflammatory bowel disease.
MATERIALS AND METHODS Patients. Eleven patients with inflammatory bowel disease, aged from 16 to 66 (mean 36 years), were studied. Some clinical data are shown in Table 1. Diagnoses were made on standard clinical, histological and radiological grounds, and the disease extent defined radiologically. Disease severity was classified according to the criteria of Truelove & Witts (1955) for ulcerative colitis and De Dombal et al. (1974) for Crohn's disease. Twenty-one subjects, comprising six healthy individuals and fifteen hospitalized patients with benign disorders (hiatus hernia, peptic ulceration and cardiac arrhythmias), were studied as a control group (mean age 39 years, range 19-72 years). Five additional patients with acute inflammatory conditions (three with lobar pneumonia, one with gonococcal septicaemia and one with cellulitis) were studied as an inflammatory disease control group (mean age 57 years, range 26-81 years). TABLE 1. Clinical details of patients with inflammatory bowel disease
Prednisone Patient number (1) (2) (3) (4) (5) (6) (7) (8) (9) (10)
(11)
Sex
Age
M F M M M F M F M M M
19 35 66 30 41 62 24 46 16 39 20
Diagnosis Ulcerative colitis Ulcerative colitis Ulcerative colitis Ulcerative colitis Ulcerative colitis Ulcerative colitis Crohn's disease Crohn's disease Crohn's disease Crohn's disease Crohn's disease
Region affected Total Total Transverse colon rectum Descending colon rectum Total Total Ileal
Ileo-colonic Ileo-colonic Ileo-caecal Total
Severity Severe Remission Mild Mild
Moderately severe
Moderately severe Severe Remission Severe Severe Severe
dosage (mg daily)
Suppression (%)
0 0 0 0 0 60 20 30 0 10 30
-35 22
21-9 30 7 -108 -69
-18-9 14-8 4.3 -27-8 1-5
Suppressor cell assay. 40 ml heparinized venous blood were obtained from each subject. In each assay, cells from both normal subjects and patients with inflammatory bowel disease were studied simultaneously for comparison. The mononuclear cells were separated by Ficoll-Hypaque centrifugation (Boyum, 1968), washed in RPMI 1640 medium (Microbiological Associates, Bethesda, Maryland) and resuspended in 10 ml RPMI 1640 enriched with 10% inactivated foetal calf serum, 4mM glutamine, 100 u/ml penicillin and 100 jg/ml streptomycin (complete medium). Cells were then divided into
Suppressor cells in inflammatory bowel disease
453
two groups containing 3 to 5 x 106 cells per ml, which were incubated (first culture) with or without 6 j±g/ml Con A (MilesYeda Inc, Indiana) in sterile Universal Containers (Sterilin Ltd, U.K.), generating 'suppressor' and 'control' populations respectively. 48 hr later, both cell suspensions were treated with 50 jig/ml mitomycin C (Sigma Chemicals) for 30 min at 370C to block DNA synthesis, and then washed three times with 30 mm a-methyl mannoside in RPMI 1640 medium to remove residual cell-bound Con A. Finally, cells were resuspended in complete medium, counted in a haemocytometer and adjusted to a concentration of 106 per ml. An aliquot was tested for viability by trypan blue exclusion; under these circumstances, viability was greater than 85%. Fresh mononuclear cells (responder cells) were prepared from a normal donor in a similar manner and adjusted to a concentration of 106 cells per ml in complete medium. One donor served as the source of responder cells in all experiments (unless otherwise specified) to ensure a reproducible Con A-reacfive cell population. These responder cells were stimulated to blast transformation by Con A in the presence of either the 'suppressor' or 'control' cells (second culture). Incubations were performed in quadruplicate using microtitre plates (Cooke Engineering, Alexandria, Virginia), to which were added 0-1 ml responder cells, 0- 1 ml of the suppressor or control cell suspension and 0-025 ml of 60 jig/ml solution of Con A. After an 80 hr incubation, 2 jLCi of [3H]thymidine (sp. act. 20-40 Ci/mM, New England Nuclear, Boston Massachusetts) were added to each well. The cells were harvested 18 hr later in an automatic harvester (MASH II, Microbiological Associates). The filters were placed in 3-0 ml of toluene scintillation fluid (New England Nuclear) and their radioactivity measured for 10 min in a Beckman liquid scintillation counter. The results of the suppressor assay were expressed in the following manner: [3H]thymidine incorporation in presence of suppressor cells) 100. (1 [3H]thymidine incorporation in presence of control cells 9 Comparison between groups were made using the Student's t-test. Dose-response characteristics of suppressor cell activity generated by Con A. In another series of experiments we studied the effectiveness of varying doses of Con A in inducing both suppressor cell activity and blast transformation in the same population of peripheral blood mononuclear cells from normal individuals. Cells were incubated for 48 hr at doses of Con A varying from 0-60 [Lg/ml, as described above. In 0-1 ml aliquots from each cell suspension in triplicate, the blast transformation response was determined by incubation for a further 6 hr with 2-0 [tCi [3H]thymidine before harvesting. The remainder of each cell suspension was treated with mitomycin C and the suppressor function assayed as described above. X
RESULTS
Characteristics ofstppressor cell assay Peripheral blood mononuclear cells from normal donors, after pre-incubation at doses of Con A from 0-1 to 20 gg/ml, suppressed the Con A-stimulated blast transformation responses in fresh allogeneic lymphocytes. Suppressor activity was thus generated by concentrations of Con A that only stimulated minor increases in the [3H]thymidine incorporation during pre-incubation as well as by concentrations which induced maximal blast transformation (see Table 2). When higher doses of Con A (60 jg/ml) were TABLE 2. Comparison between blast transformation induced in peripheral blood mononuclear cells by pre-incubation with various doses of Con A (first culture), and the effect of those pre-incubated cells in suppressing Con A-stimulated blast transformation in fresh allogeneic responder cells (second culture)
[3H]thymidine uptake of Concentration of Con A (jig/ml) (1st culture)
[3H]thymidine uptake (54 hr)
0 0-5 1 2 5 10 20
1152±120 1614+100 4124±208 10312±386 57519±589 132307±2919 163604±4176
(ct/min±s.e.m.)*
Stimulation Index -
1-4 3-6 90
49.9 114-8 142-0
responder cells (2nd culture)
(ct/min~s.e.m.) 82759+2327 74874±3986 64076± 1742 57304±642 60305±1786 29490± 1124
9-6 22-6 30-8 27-1 64-4
49244±2859
40.5
Concentration of Con A employed in second culture was 6-00,g/ml. * 48 hr incubation with Con A followed by 2 ,uCi [3H]thymidine for 6 hr. ct/min error ofthe mean (s.e.m.).
Suppression (%)
=
-
Mean count per minute± standard
H. J. F. Hodgson, J. R. Wands & K. J. Isselbacher
454
employed, leucoagglutination occured, and the replicate measurements of the blast transformation response of responder cells in the presence of the suppressor population showed tremendous variability. The suppressor cell activity in control subjects and patients with inflammatory bowel diseases in the present investigation were generated by a mitogenic concentration of Con A (6 gg/ml). We also studied the reproducibility of the suppressor cell assay in serial studies, using both allogeneic and autologous responder cell populations. Serial weekly suppressor cell assays in one normal subject, when the responder cells were autologous to the Con A pre-treated and control cells, showed suppression of 48-2, 66-3 and 32%. In two other individuals using allogeneic responder cells, less variation was found (68.3 and 634%, 45X3 and 46.8%). Suppressor cellfunction in patients with inflammatory bowel disease and control subjects The results of a typical suppressor assay on peripheral blood mononuclear cells from two patients and two control subjects are shown in Table 3, in order to illustrate the magnitude of the changes observed in the cultures. TABLE 3. Representative data on the suppressor cell assay in two patients and two control subjects.
Control Control PatientNo. 3 Patient No. 7
(A) Responder cells plus Con A pre-treated cells plus Con A
(B) Responder cells plus control cells plus Con A
54081±3846 76026±3709
114341±3368 102742±3858 109850±2346 102661+5408
85839±3279 122131±5682
Suppression (%) 52-8 26 21-9 -18-9
Mean ct/min (+ s.e.m.) of responder cells from one donor in presence of Con A pre-treated cells (column A) or control cells (column B) and Con A. For patient No. refer to Table 1. 80 6040
0
-6 60
-80 _ -100
_
Control
Inflammatory
(n = 21)
bowel disease
(n- I1) FIG. 1. Results of suppressor cell measurement in control subjects and patients with inflammatory bowel disease. The ordinate shows the percentage suppression. Points below the dashed line indicate enhancement rather than suppression of 3H-thymidine incorporation. Mean ± s.e.m. for each group is indicated by shaded area. Difference between the two groups is significant: P < 0 005.
In twenty out of twenty-one control subjects (96%), the blast transformation responses of fresh allogeneic lymphocytes were suppressed by Con A pre-treated cells as compared to control cells. The mean suppressor cell activity for this group was 2744% (Fig. 1). Furthermore, there was no difference between the mean suppressor cell activity obtained from the healthy controls when compared to the hospitalized control subjects. In contrast, Con A-generated suppressor cells were found in only six out of eleven patients (54%) with inflammatory bowel disease. Moreover, in the other five patients with
Suppressor cells in inflammatory bowel disease
455
inflammatory bowel disease, the blast transformation of fresh lymphocytes was greater in the presence of the Con A pre-treated cells than the controls, suggesting a helper rather than a suppressor effect. The mean level of suppressor activity elicitable from peripheral blood mononuclear cells in the inflammatory bowel disease group was - 9 2% (+ 11.8 % s.e.m.). These results differ significantly from the control group (P < 0005). The loss of mononuclear cell suppressor activity was closely related to disease activity. There were four patients, none of whom had been admitted to the hospital, whose disease was classified as mildly active or in remission (see Table 1). Each of these patients had a normal suppressor cell response (22-4 +3 3%), and this group did not differ from the control population. In contrast, seven patients had their disease classified as severe or moderately severe; these had a prolonged hospital course for treatment of 40
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Severe Other inflammatory conditions (0:-4) (n =7) Inflammatory bowel (0=5)
diseose FIG. 2. Results of suppressor cell measurement in patients with inflammatory bowel disease, classified according to disease severity, and in patients with other inflammatory conditions. The ordinate show the percentage suppression. Mean + s.e.m. and dashed line as in Fig. 1. The differences between mild vs severe inflammatory bowel disease are significant: P< 0 05; as are differences between severe inflammatory bowel disease vs other inflammatory conditions: P< 0-025. 40
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6 8 10 12 Time (weeks)
14
16
Admitted Discharged FIG. 3. Serial study of suppressor cell activity in a patient with severe ulcerative colitis. Prednisone therapy commenced after first assay performed. The abscissa shows time. The ordinate shows the percentage suppression. Shaded area show the mean + s.e.m. ofcontrol subjects.
456
H. 3. F. Hodgson, 3. R. Wands & K. 3. Isselbacher
their disease, and five underwent bowel resection (the suppressor cell assay had been performed prior to surgery). Only two out of the seven patients exhibited a suppressor cell response; the observed mean suppressor cell activity for all these severely affected patients was - 272± 14-5 %, which was significantly less than both the control population and the patients with mild inflammatory bowel disease (Fig. 2). Furthermore, this result was significantly different from the patients with other inflammatory disorders
(21'84±5.5%). One patient (Table 1, patient 1), with severe ulcerative colitis, was studied serially after admission with toxic megacolon. The clinical course is illustrated in Fig. 3. Suppressor activity could not be elicited from his cells during repeated testing over 3 months, despite a slow clinical improvement with medical management, including high-dose corticosteroids (60 mg prednisone daily) and sulphasalazine. The first measurement after hospital discharge, at which time the patient was symptom-free, still showed altered suppressor cell activity; however, it returned to the normal range 6 weeks later. At that time there was no evidence of disease activity, as judged by symptoms, sigmoidoscopic examination and rectal biopsy. Of the eleven patients studied, five were on treatment with corticosteroids at the time the assay was performed (Table 1). The serial study detailed above indicated little change in the activity of the Con Astimulated suppressor cells at the time of initiation of treatment with prednisone, 60 mg daily (-35%), or after 1 week of therapy (- 28%). However, it is quite possible that corticosteroids affect Con Astimulated suppressor cell activity after prolonged administration. Additional studies are needed before a firm conclusion can be reached on the role of corticosteroids on Con A-generated suppressor cell activity. DISCUSSION The suppressor cell assay employed in the present investigation measures suppressor cell function generated by pre-incubation of cells with Con A. While this assay does not measure the spontaneous suppressor activity present in the mononuclear cell population, it indicates the presence of a population or subpopulation ofcells which can develop in vitro suppressor function after Con A activation. A number of studies (Shou et al., 1976; Hubert et at., 1976; Hallgren & Yunis, 1977) have confirmed that a Con Ainduced suppressor cell population is present in normal subjects and the putative effector cell has been identified as a high-density T cell (Sakane & Green, 1977). We have shown altered suppressor cell activity in patients with severe or moderately severe inflammatory bowel disease. There are a number of possible explanations for the altered suppressor cell activity in these patients. A generalized lymphocyte hyporesponsiveness has been reported by some authors in inflammatory bowel disease, particularly Crohn's disease (Walter & Greaves, 1969; Brown et al., 1970), although Asquith, Kraft & Rothberg (1973) and Aas et al. (1972) both found the blast transformation response induced by Con A to be normal in Crohn's disease. However, it seems unlikely that a failure to generate suppressor cells during pre-incubation with Con A is the same phenomenon as a generalized lymphocyte hyporesponsiveness, as the subpopulation of lymphocytes responsible for Con A-induced suppression in normal human subjects has been shown to possess a poor blastogenic response to this lectin (Sakane & Green, 1977). Also relevant in this regard is our finding that doses of Con A that caused only minor increases in thymidine uptake induced considerable suppressor activity in the same mononuclear cell population. Corticosteroids affect lymphocyte responsiveness in vitro and alter lymphocyte subpopulations in vivo (Fauci & Dale, 1974). Although a majority of the patients with severe disease were on such treatment, there was little difference in the results of the suppressor assay performed before and after initiation of this therapy, as illustrated by the serial study. Abnormal suppressor responses were also seen in those patients with active disease not on such therapy. Additional studies are necessary, however, to clarify the role of corticosteroids on Con A-stimulated suppressor cells. It is possible that our findings could be explained by an alteration in the lymphocyte subpopulations during active disease. Strickland et al. (1975) found reduced numbers of T cells in patients with inflammatory bowel disease, although Bird & Britton (1974) and Thayer, Charland & Field (1976) found
Suppressor cells in inflammatory bowel disease
457
normal T-cell numbers. In the present investigation, we cannot distinguish between the loss of a Con A-reactive T-cell subpopulation with suppressor activity, hyporesponsiveness of suppressor cells to Con A, or an abnormal helper cell population which could mask the suppressor cell effect. A comment is also required concerning the mode of calculation of the suppressor cell activity. Suppressor cell activity was expressed as a reduction in the ratio of the blast transformation response of fresh lymphocytes in the presence of Con A pre-treated cells compared to their response in the presence of control cells; if the control cells retain spontaneous suppressor activity, this ratio could increase even if the Con A-induced suppressor activity was unchanged. However, this seems unlikely, since spontaneous suppressor cells are apparently short-lived in culture, and their activity is markedly diminished after 24 hr in vitro (Bresnihan & Jasin, 1977). With our present knowledge we cannot judge whether the demonstrated defect in Con A-induced suppressor cell activity plays a role in maintaining the disease process in inflammatory bowel disease. While the close relationship between disease activity and loss of suppressor function might suggest that the defect is merely a secondary phenomenon, the normal suppressor cell responses among patients with other severe inflammatory conditions indicates that inflammation per se is not associated with abnormal suppressor cell responses. It was also noteworthy that the return to normal suppressor function in the patient studied serially lagged behind the clinical remission of the disease. It is indeed conceivable that a loss of suppressor function might contribute, in part, to the pathogenesis of inflammatory bowel disease. In another chronic inflammatory disease, systemic lupus erythematosus, a suppressor cell defect has been described (Bresnihan & Jasin 1977) and it has been proposed that a lack of suppression may contribute to the development of autoimmunity and resulting tissue damage in that disease. Serial measurements of suppressor cell activity in patients with systemic lupus erythematosus have shown a return to normal with remission (Bresnihan & Jasin, 1977). In inflammatory bowel disease, while the initiating factor remains unknown, e.g. a transmissible agent (Cave et al., 1973) or an initial mucosal breach allowing sensitization to gut and bacterial antigens (Shorter, Huizenga & Spencer, 1973), a loss of suppressor cell activity, permitting the expression of damaging immune responses, might contribute to the persistence of inflammation in the gastrointestinal tract. This work was supported in part by grants from the National Institutes of Health, Bethesda, Maryland, U.S.A. (AA-02666 and AM-2030). J. R. Wands is a recipient of a Clinical Investigator Award: AM-00127. H. J. F. Hodgson held the Radcliffe Travelling Fellowship of University College, Oxford.
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