Lymphocyte Responses to Nonspecific Mitogens in Inflammatory Bowel Disease

GASTROENTEROLOGY® Official Publication of the American Gastroenterological Association @ CoPVRIGtrr

VoLUME

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1973

THE WILLIAMS

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W1LXINS Co.

July 1973

Number 1

LYMPHOCYTE RESPONSES TO NONSPECIFIC MITOGENS IN INFLAMMATORY BOWEL DISEASE PETER AsQUITH, M.D., SUMNER C. KRAFT, M.D. , AND RICHARD M. RoTHBERG , M.D.

Departments of Medicine and Pediatrics, University of Chicago, Chicago, Illinois

To assess cell-mediated immunity, circulating lymphocytes from 13 patients with Crohn's disease, 14 patients with ulcerative colitis, and 24 healthy control subjects of comparable ages were cultured in the presence of multiple doses of phytohemagglutinin, concanavalin A, and pokeweed mitogen. There was considerable variation of the results with marked overlap among the three groups of subjects, and much variability existed as to the optimal dose of a mitogen needed to elicit a maximal response in a given subject. Several of the patients with either type of inflammatory bowel disease had poor lymphocyte responses, but no significant differences were demonstrated among the three groups. There was a trend towards poorer lymphocyte responsiveness in patients with Crohn's disease of moderate to severe clinical activity. The fact that equally low values also were found in healthy individuals suggests that such isolated in vitro observations are in themselves inadequate bases upon which to draw etiological or pathogenetic conclusions. Continuing emphasis has been placed on the possible involvement of immunological mechanisms in the etiology and pathogenesis of ulcerative colitis and Crohn's disReceived December 13, 1972. Accepted March 6, 1973. Address requests for reprints to: Dr . Sumner C. Kraft, Box 400, University of Chicago Hospitals, 950 East 59th Street, Chicago, Illinois 60637. This work was supported in part by Research Grants AM-02133 and AI-07854 from the National Institutes of Health, United States Public Health Service, and by the Gastro-Intestinal Research Foundation, Chicago, Illinois. Dr. Asquith performed these studies during the tenure of a Sir Henry Wellcome Travelling Fellowship. His present address is: Department of Experi-

ease, 1• 2 with much recent interest in studying the integrity and reactivity of the cellular immune system. 3 In Crohn's disease, for example, some have described a variable impairment of the responses of mental Pathology, University of Birmingham, Birmingham, England. Dr. Kraft was the recipient of United States Public Health Service Research Career Development Award 5-K3-AI-13,936. Dr. Rothberg is the recipient of United States Public Health Service Research Career Development Award 5-K04-AI-38,899. The expert technical assistance of Slavko T. Stanis and Harold E. Ford is gratefully acknowledged. Dr. Howard Schachter performed the dinitrochlorobenzene skin tests.

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circulating thymus-dependent lymphocytes to phytohemagglutinin (PHA) 4 - 7 and concanavalin A (Con A) . 5 However, not all workers have been able to confirm these observations 8 • " ; and sharply conflicting results have been reported using pokeweed mitogen (PWM), 5 • 8 a more selective stimulus of B lymphocytes. While circulating lymphocytes from patients with ulcerative colitis have been reported to have normal responses to such nonspecific mitogens, 4 • 6 • 8 • 10 • --. 1 some of these individuals have shown diminished lymphocyte responses to PHA. 6 • 7 To assess the integrity of the cellular immune system in patients with inflammatory bowel disease , circulating lymphocytes from these patients and healthy control subjects were cultured in the presence of PHA, Con A, and PWM. In view of the importance of dose-response measurements in studies of this type, 6 • 12- 14 multiple doses of each mitogen were employed for each individual studied .

Materials and Methods Subjec ts. There were 24 control subjects, consisting of 22 healthy hospital physicians and laboratory personnel and 2 patients with a well documented irritable colon syndrome. The mean age was 36 years (range, 21 to 85) . There were 13 patients with Crohn 's disease, of whom 7 had ileocolitis, 3 had regional enteritis, and 3 had evidence of recurrent disease after bowel resection and ileotransverse colostomy. The mean age of this group was 34 years (range, 16 to 63). There were 14 patients with ulcerative colitis, of whom 8 had involvement of the entire colon, 5 had evidence of only left-sided disease, and 1 had ulcerative proctitis. The mean age of this group was 44 years (range, 16 to 71). The diagnosis of inflammatory bowel disease depended upon the clinical course, laboratory findings, X-ray studies of the small and large intestine , proctoscopic examination, and the results of biopsy or surgery when performed . The current clinical status was determined on the basis of the number of stools per day, their character, the presence of macroscopic blood in the stool, abdominal pain, fever , and recent loss of weight. Using combined clinical and laboratory data, disease activity was classified on the basis of the parameters described by Cooke 15 (excluding seromucoid levels) for the patients with Crohn's disease, and the criteria of True-

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love and Witts 16 for the patients with ulcerative colitis. No patient was receiving corticosteroids or cytotoxic agents such as azathioprine. The following laboratory investigations were performed on the day of the lymphocyte study: complete blood count, erythrocyte sedimentation rate, total serum protein, quantitative protein electrophoresis, and serum concentrations of IgG, IgA, and IgM. Lymphocyte separation. With the patients' consent, approximately 75 ml of peripheral venous blood were obtained aseptically, transferred to sterile disposal containers (Falcon Plastics, Oxnard , Calif.) and defibrinated by gently stirring with sterile wooden sticks. The clot was discarded and a sterile 0.1% solution of methylcellulose was added in a concentration of 1 ml per 10 ml of blood. The plastic containers were stoppered and inverted several times to produce mixing and then kept upright at 37 C. After 1 hr, the huffy coat and supernatant plasma were aspirated and transferred to an identical container. The leukocytes were concentrated by centrifugation at 750 rpm for 5 min at room temperature. The supernatant again was aspirated with a sterile pipette. The cell button was agitated gently and washed twice with 5 mi of culture medium consisting of Eagle's Minimal Essential Medium (Grand Island Biological Co., Grand Island, N. Y.) containing penicillin (140 U per ml), streptomycin (140 llg per ml), and glutamine (0.16 mg per ml). A drop of leukocyte-rich solution then was removed and a smear made to determine the proportion of lymphocytes. Lymphocyte culture and thymidine incorporation. The remaining leukocytes were resuspended in 4 ml of the culture medium and the concentration of lymphocytes determined. The culture medium was supplemented with 15% normal human serum and lymphocytes were added to a final concentration of 1 x 10 6 per ml. To minimize the possible effects of isoagglutinins, 17 the serum used throughout the whole series of experiments was from a single pool donated by four healthy volunteers of blood group AB rhesus-positive.· Two milliliters of the final culture medium , containing 2 x 10" lymphocytes, were then added to sterile disposal snap-top plastic tubes (Stayne Laboratories, London, England) . The cultures were performed in duplicate and each contained either 0.1 ml of sterile phosphate-buffered saline pH 7.2 or 0.1 ml of sterile preparations of either reagent-grade PHA (Burroughs-Wellcome and Co., Tuckahoe, N. Y.). Con A (Miles Laboratories, Elkhart, Ind .) or PWM (Grand Island Biological Co., Grand Island, N . Y.). Four

different concentrations of these mitogens were used, i.e., PHA 250, 100, 50, and 10 /lg; Con A 200, 100, 50, and 10 11g; and PWM 12.0, 6.0, 0.6, and 0.06 /lg. These tubes were stoppered and placed in a vertical position and incubated for 3 days at 37 C in an atmosphere of 5% C0 2 and 95% air. Sixteen hours before termination, 0.1 ml of phosphate-buffered saline containing 1.0 11c tritiated ("H) thymidine (specific activity 6. 7 11c per mole as 3 H-methyl thymidine; New England Nuclear Corp., Boston, Mass.) was added to each culture. At the end of the 3 days, the plastic culture tubes were gently agitated to resuspend the cultured lymphocytes and the latter were poured into disposable glass centrifuge tubes. The plastic culture tubes then were washed free of remaining lymphocytes with isotonic saline. The cells then were processed for deoxyribonucleic acid synthesis by the method of Ling and Holt'" except that the final step involved dissolving the cell precipitate in 0.3 ml of 1 N sodium hydroxide . The processed cultures were transferred into disposable plastic scintillation bottles by two successive 5-ml washes of scintillation fluid made up by adding 160 ml of Liquifluor (New England Nuclear Corp., Boston, Mass.) to 1 gallon of reagent-grade toluene. The scintillation bottles remained at 4 C for a minimum of 4 hr prior to counting in a Packard Tri-Carb liquid scintillation spectrometer. Scintillation counts were corrected to 100% efficiency by correcting for quenching. The data were recorded as mean absolute scintillation counts per minute . As shown in table 1, the unstimulated scintillation counts were very similar in the patient and control groups, and the degree of stimulation caused by each mitogen therefore was expressed as the ratio of the mean scintillation counts in disintegrations per minute in the duplicate mitogencontaining cultures to the mean of the disintegrations per minute in the duplicate phosphatebuffered saline control cultures. The mean and standard deviations of the ratios were calculated for each group and the significance of TABLE

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LYMPHOCYTE RESPONSES IN BOWEL DISEASE

July 1973

1. Unstimulated scintillation counts

Group

Control .. . .. Ulcerative colitis .. Crohn's disease . . ..

Mean No. of counts subper jects minute

24 14 13

3491 3569 3415

so

Range

2002 1401- 10564 1916 1658- 8551 1345 1950-5613

the differences between groups was determined by the Student's t -test. Data analysis using mean stimulated scintillation counts minus mean unstimulated scintillation counts gave comparable results.

Results Figure 1 depicts the lymphocyte response ratios using PHA at each of the four doses. There was a wide range of values at each dose with considerable overlap among the three groups of subjects. Several individuals with either Crohn's disease or ulcerative colitis had lymphocyte responses of 1 so or more below the mean of the control group, but the mean responses were not significantly different from those of the control group. For example, the mean responses of the patients with Crohn's disease at doses of 100 and 50 1-lg did not differ statistically from the control group (0.05 < p < 0.10). The lymphocytes from these three groups of subjects were tested using multiple doses of Con A and PWM. Figure 2 shows the ratios obtained using one of the concentrations of Con A (100 /olg) and PWM (6.0 1-lg). These relationships are representative of those obtained with the other doses of these two mitogens. Again, there was PHYTOHEMAGGLUTININ

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FIG. 1. Responses of circulating lymphocytes to phytohemagglutinin at doses of 250, 100, 50, and 10 llg. Each solid circle represents the ratio of duplicate stimulated to unstimulated lymphocyte responses for a given subject. Solid horizontal lines represent the mean ratios of a group; interrupted horizontal lines depict 1 so. C, control group; C.D ., Crohn's disease group ; U.C., ulcerative colitis group .

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FIG. 2. Responses of circulating lymphocytes to 100 p.g of concanavalin A and 6.0 p.g of pokeweed mitogen. Each solid circle represents the ratio of duplicate stimulated to unstimulated lymphocyte responses for a given subject. Solid horizontal lines represent the mean rat ios of a group ; interrupted horizontal lines depict 1 so. C, control group; C.D. , Crohn 's disease group ; U. C., ulcerative colitis group .

included . The mean response of lymphocytes from patients with Crohn's disease tended to be lower than those from the control or ulcerative colitis groups, but this difference was not statistically significant. A similar analysis showed no significant differences between the mean maximum responses to Con A or PWM in patients with either of the inflammatory bowel diseases in comparison with the healthy control subjects. Table 3 compares the clinical activity of the inflammatory bowel disease with lymphocyte responses to PHA using doses of either 100 or 50 J.Lg. Although the numbers in these subgroups are small, there is a trend towards poorer lymphocyte responsiveness in those patients with Crohn's disease of moderate to severe clinical activity. In patients with ulcerative colitis, on the other hand, the more severely active cases tended to have normal or even somewhat augmented lymphocyte responsiveness to PHA. We observed no relationships between lymphocyte reactivity and the concentrations of the three major serum TABLE

2. Max imum ly mphocyte resp onses to

phy tohemagglutinin

considerable variability of the results and marked overlap among the three groups of subjects. The responses of lymphocytes from all subjects tended to be greatest at doses of Con A of 100 and 50 J.Lg and at doses of PWM of 6.0 and 0.6 J.Lg. The mean responses of the two groups of patients did not differ significantly from the mean responses of the healthy control subjects using either of these mitogens. Since the results using PHA suggested a partial impairment of the responses of lymphocytes obtained from certain patients, and since individuals vary with respect to the dose of mitogen which evokes a maximum response, the data also were analyzed using the maximum response of the lymphocytes to each mitogen, irrespective of dose. Table 2 depicts the maximum lymphocyte response ratios using PHA. The number of individuals shown is less than the total number of subjects studied because only patients with sufficient lymphocyte yields to study all doses were

Mean No. of maxisu b- m al jects• response

Group

Con trol . Ul cerative colit is . Crohn 's disease . .. .

17 9 8

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Range

142. 1 76.2 78. 1- 378.8 167.0 82.6 34 .2- 285.6 98.6 41.8 39.8- 166.8

• See text for criteria for inclusion in t his an alysis.

3. Correlation between clinical activity of inflammatory bowel diease and ly mphocyte responses to 100 or 50 p.g of phy tohemagg lut inin (PHA )

TABLE

Group

Clinical activity

No. Mean ratio of sub- P HA- PHA· jects 100 50

Cont rol 24 130.9 129 .8 Ulcerative colitis Quiescent-mild" 9 106.2 102.2 Modera te-severe 5 178.5 151.5 Crohn 's disease Quiescent-mild" 8 116.7 105.2 Moderate-severe 5 73.2 78.1 • Based on criteri a of Truelove and Witts. 16 • Based on parameters of Cooke ."

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immunoglobulin classes in the patients with inflammatory bowel disease.

Discussion Peripheral lymphocytes from healthy control subjects and from patients with nonspecific ulcerative colitis or Crohn's disease were tested for their responsiveness in short term tissue cultures to three nonspecific plant mitogens, i.e., PHA, Con A, and PWM. The dose responsiveness of the lymphocytes to these mitogens was examined in view of observations 6 • 12- 14 of differences in the reactivity of lymphocytes when tested in this manner. Our data support earlier studies showing that considerable variability exists among so-called healthy control subjects, and that variability exists as to the optimal dose of a mitogen needed to elicit a maximal response in a given subject. 6 • 12- 14 The mean responses of lymphocytes from healthy subjects in this study using doses of 100 or 50 J.Lg of PHA were quite comparable to those in other studies utilizing this in vitro system and 3-day lymphocyte cultures. While some studies have suggested impaired lymphocyte responses to PHA in patients with Crohn's disease, 4- 7 not all workers have agreed. 8 • 9 In this study, impaired responsiveness has not been a uniform finding in Crohn's disease; while some patients with ulcerative colitis have had comparably low responses to PHA. Utilizing either mean or maximum responses to PHA, no statistically significant differences were noted in comparing patients with Crohn's disease and healthy control subjects. Furthermore, no differences existed in studying the same patients' maximum responses to Con A and PWM. The diminished lymphocyte responses noted in certain individuals would appear to represent an alteration in the lymphocyte populations rather than the result of a serum factor since (a) we attempted to remove autologous serum from the lymphocytes by washing, (b) we suspended the lymphocytes in human serum rather than in fetal calf serum which is known to have a varying stimulatory effect upon human lymphocytes, (c) the human serum used

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was a single batch of pooled serum so that any other potential serum factor would be constant throughout the whole series of experiments, and (d) serum from group AB rhesus-positive blood was used because some authors have noted that human blood group isoagglutinins may stimulate lymphocytes. 17 The heterogeneity of the patients studied in this and other series might be considered as an important factor in differing results. Although generally it has not been possible to correlate diminished lymphocyte responses with disease activity, site, and extent, diminished lymphocyte reactivity to PHA among patients with long-standing inactive Crohn's disease and a trend towards increased responsiveness during exacerbations have recently been reported. 19 It is interesting that we noted the latter findings among patients with active ulcerative colitis (table 3). Although the nature and amounts of current and previous medications may be other variables, negative in vitro studies of the influence of Azulfidine therapy on lymphocyte responses have also been described in patients with inflammatory bowel disease. 7 On the other hand, corticosteroid administration has been shown to have a suppressive effect upon lymphocyte responsiveness to mitogens in this and other diseases. 20 Although none of these patients were receiving steroids, we have had occasion to study 4 additional patients with Crohn's disease who were taking low doses of prednisone at the time of lymphocyte testing. Three of these 4 patients showed depressed lymphocyte responses to PHA, i.e., their maximum PHA response ratios were 50, 40, and 20. It is impossible to tell whether such low values represented the suppressive effect of the low doses of prednisone on lymphocyte reactivity or whether the poor in vitro responses were in some way related to the fact that these patients tended to be sicker and thus were treated with this drug. What is the clinical significance of the observation of diminished lymphocyte responses to nonspecific plant mitogens in limited numbers of patients with Crohn's

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disease or ulcerative colitis? Clearly, impaired lymphocyte transformation has been observed in a number of diseases, 3 as well as in subjects with iron-deficiency anemia. 21 In patients with inflammatory bowel disease, a lymphocyte defect might be either qualitative or quantitative. Lymphopenia does not appear to explain our in vitro results since a constant number of lymphocytes was used, but specific subpopulations of lymphocytes may have been lost, as has been shown in patients with intestinal lymphangiectasia. 22 In that disease, a cellular immune deficiency exists secondary to the gastrointestinal loss and consequent depletion of a recirculating, long-lived lymphocyte population from the peripheral lymphocyte pool. 22 Since Crohn's disease also may result in a secondary lymphangiectasia, the loss of lymphocyte-rich lymph could explain in part the occasionally depressed lymphocyte responses observed in this and earlier studies, and account for diminished tuberculin skin reactivity 23 and an impaired ability to be sensitized to dinitrochlorobenzene 24 in some patients with Crohn's disease. Dinitrochlorobenzene skin tests were performed in a few of the patients with Crohn's disease in this study. Both negative and positive results were obtained, without correlation with the in vitro lymphocyte responsiveness of the same subjects. The relevance of very low lymphocyte responses to nonspecific mitogens to the etiology or pathogenesis of these inflammatory bowel diseases remains unclear. The occurrence of this finding in a limited number of these individuals, as well as the fact that equally low values also were found in apparently healthy subjects, might suggest that it is not of great significance. On the other hand, if these nonspecific inflammatory bowel diseases are looked upon as part of a spectrum of similar conditions with many potentially different etiologies, additional studies might be directed at subgroups with very low in vitro lymphocyte responses. Such studies might include the assessment of the reactivity of both circulating and intestinal lymphocytes, using multiple in vivo and in

vitro parameters of cell-mediated immunity. REFERENCES 1. Kraft SC, Kirsner JB: Immunological apparatus of the gut and inflammatory bowel disease. Gastroenterology 60:922-951, 1971 2. Dykes PW: Immunology of Crohn's disease . Clin Gastroenterol 1:349-366, 1972 3. Kraft SC: Cellular immunity in Crohn 's disease. Gastroenterology 61:545- 548, 1971 4. Walker JG , Greaves MF: Delayed hypersensitivity and lymphocyte transformation in Crohn's disease and proctocolitis (abstr). Gut 10:414, 1969 5. Brown SM, Taub RN, Present DH, et al : Shortterm lymphocyte cultures in regional enteritis. Lancet 1:1112, 1970 6. Parent K, Barrett J , Wilson ID : Investigation of the pathogenic mechanisms in regional enteritis with in vitro lymphocyte cultures. Gastroenterology 61:431- 439, 1971 7. Sachar DB, Taub RN, Brown SM, et al: Lymphocyte responsiveness in inflammatory bowel disease (abstr). Gastroenterology 62:804, 1972 8. McHattie J, Magi! A, Jeejeebhoy K, et al: Immunoresponsiveness of lymphocytes from patients with regional ileocolitis (Crohn's disease) by in vitro testing (abstr). Clin Res 19:779, 1971 9. Aas J, Huizenga KA, Newcomer AD, et al: Inflammatory bowel disease: lymphocytic responses to nonspecific stimulation in vitro. Scand J Gastroenterol 7:299-303, 1972 10. Hinz CF Jr, Perlmann P, Hammarstrom S: Reactivity in vitro of lymphocytes from patients with ulcerative colitis. J Lab Clin Med 70:752- 759, 1967 11. Stefani S, Fink S: Effect of E. coli antigens, tuberculin, and phytohaemagglutinin upon ulcerative colitis lymphocytes. Gut 8:249- 252, 1967 12. Richter M , Naspitz CK: The variation in response of human peripheral lymphocytes to phytohemagglutinin in vitro. Int Arch Allerg 32:288- 293, 1967 13. Fitzgerald MG: The establishment of a normal human population dose-response curve for lymphocytes cultured with PHA (phytohaemagglutinin) . Clin Exp Immunol 8:421-425, 1971 14. Carr MC, Stites DP, Fudenberg HH: Cellular immune aspects of the human fetal-maternal relationship. I. In vitro response of cord blood lymphocytes to phytohemagglutinin. Cell Immunol 5:21-29, 1972 15. Cooke WT: Survey of results of treatment of Crohn 's disease. Clin Gastroenterol 1:521-531, 1972

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16. Truelove SC, Witts LJ : Cortisone in ulcerative colitis: final report on a therapeutic trial. Br Med J 2:1267-1272, 1956 17. Ling NR: Lymphocyte stimulation. New York, J Wiley and Sons Inc, 1968, p 123 18. Ling NR, Holt PJ : The activation and reactivation of peripheral lymphocytes in culture. J Cell Sci 2:57-70, 1967 19. Maclaurin BP, Cooke WT, Ling NR: Impaired lymphocyte reactivity against tumour cells in patients with Crohn's disease. Gut 13:614- 620 1972 20. Claman HN: Corticosteroids and lymphoid cells. N Eng! J Med 287:388- 397, 1972 21. Joynson DHM, Jacobs A, Walker DM, et al:

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Defect of cell-mediated immunity in patients with iron-deficiency anaemia . Lancet 2:1058-1059, 1972 22. Weiden PL, Blaese RM, Strober W, et al: Impaired lymphocyte transformation in intestinal lymphangiectasia: evidence for at least two functionally distinct lymphocyte populations in man. J Clin Invest 51:1319- 1325, 1972 23. Jones Williams W: A study of Crohn's syndrome using tissue extracts and the Kveim and Mantoux tests . Gut 6:503-505, 1965 24. Jones JV, Housley J, Ashurst PM, et al: Development of delayed hypersensitivity to dinitrochlorobenzene in patients with Crohn's disease. Gut 10:52-56, 1969