Clin. exp. Immunol. (1991) 83, 320-325

ADONIS

000991049100058Z

Induction of suppressor cells by Mycobacterium paratuberculosis antigen in inflammatory bowel disease E. C. EBERT, B. D. BHATT, S. LIU & K. M. DAS Department of Medicine, University of Medicine and Dentistry New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ, USA

of

(Acceptedfor publication 5 September 1990)

SUMMARY We studied the M. paratuberculosis-induced proliferation and suppressor cell generation by peripheral blood lymphocytes from patients with inflammatory bowel disease. Peripheral blood lymphocytes were separated from 33 patients with Crohn's disease, 18 with ulcerative colitis, nine with other intestinal diseases, and five with autoimmune disorders. Proliferation of peripheral blood lymphocytes from normal individuals in response to 10 yg/ml of M. paratuberculosis antigen was reduced by depletion of CD4+ T cells. The ability of M. paratuberculosis antigen to suppress concanavalin A-induced proliferation (expressed as a percentage suppression) was reduced by depletion of CD8+ T cells. This suppression was the same whether peripheral blood lymphocytes were from normal individuals, patients with intestinal diseases other than inflammatory bowel disease, or patients with autoimmune disorders (47 + 14%, 44 + 24%, and 30 + 26%, respectively). In contrast, the suppression induced by M. paratuberculosis for patients with Crohn's disease and ulcerative colitis (66+22% and 67+22%) was much greater than that for normal individuals (P < 0-00 1). In particular, lymphocytes from patients with active Crohn's disease demonstrated little proliferation in response to this antigen but marked suppressor activity (79+13%). How the immunomodulatory effects of this antigen relate to the pathogenesis of the inflammatory bowel diseases remains to be determined. Keywords Mycobacterium paratuberculosis Inflammatory Bowel Disease Ulcerative colitis Crohn's disease Suppressor lymphocytes

antibodies in their serum or intestine against mycobacteria (Thayer et al., 1987; Chao et al., 1988). These findings are compatible with several possibilities: (i) mycobacterial organisms may cause IBD; (ii) these organisms may invade intestinal ulcers and set up local colonies in these immunosuppressed hosts without altering the disease process; or (iii) invasion by these organisms may represent a secondary event that perpetuates some destructive immune response. In support of the last possibility is evidence that mycobacteria may evoke autoimmune diseases of various sorts by molecular mimicry (Shoenfeld & Isenberg, 1988). In other words, antigenic similarities between mycobacterial constituents and tissue antigens result in humoral and cellular immune responses that destroy not only the organism, but also normal tissue. It would be difficult, if not impossible, to prove that mycobacteria are the primary cause of IBD by studying humans rather than animal models. However, one can determine whether these organisms alter the immune response of the host, and so affect the activity of the disease. This is the first study to evaluate the possible suppressive effects of mycobacterial antigens on the immune response of patients with IBD, using an approach similar to that applied to leprosy. The M. leprae antigens have certain components that

INTRODUCTION There has been a recent resurgence of interest in the possibility that inflammatory bowel disease (IBD), particularly Crohn's disease, might be caused by a mycobacterial organism. Ever since Crohn's disease was described as an entity distinct from intestinal tuberculosis, the clinical similarity of these two disorders has been noted. The possible benefit of anti-tuberculosis drugs in the treatment of Crohn's disease was reported in several small studies (Warren, Rees & Cox, 1986; Schultz et al., 1987) and has led to more extensive trials. Particular interest has centered around M. paratuberculosis, the pathogen in Johne's disease, a granulomatous enteritis in cattle that resembles Crohn's disease. Recently, this organism has been found to affect primates in a similar manner (McClure et al., 1987). Mycobacteria have been isolated from diseased tissue obtained from patients with or without IBD (Chiodini et al., 1984; Graham, Markesich & Yoshimura, 1987). In addition, a large proportion of patients with Crohn's disease have Correspondence: Ellen C. Ebert, MD, UMDNJ-Robert Wood Johnson Medical School, One Robert Wood Johnson Place, CN 19, New Brunswick, NJ 08903-0019, USA.

320

321

M. paratuberculosis and inflammatory bowel disease suppressor

or if they were taking equivalent.

Study population Heparinized blood was obtained from 23 normal individuals, 33 patients with Crohn's disease, 18 patients with ulcerative colitis, nine acutely ill patients with other inflammatory diseases of the intestine, and five with autoimmune disorders (Table 1). Patients were not included in the study if they were HIV positive

Isolation and culture of lymphocytes PBL were isolated from heparinized blood by density gradient centrifugation with Ficoll (Bionetics Laboratory Products, Kensington, MD). In some experiments, PBL were either depleted of macrophages by adherence to a Petri dish, or depleted of CD4+ or CD8+ T cells by antibody and complement lysis (Ebert, 1989). Lymphocytes (I x 105/0-2 ml) were cultured for 3 or 6 days at 37°C in 95% air/5% CO2 in RPMI 1640 medium (GIBCO, Grand Island, NY) containing 10% heatinactivated fetal calf serum (Microbiological Associates Bioproducts, Walkersville, MD), 1% antibiotic-antimycotic solution (GIBCO), and 10 mM N-2 hydroxyethylpiperazine-N'2 ethanesulphonic acid (Sigma). Lymphocytes (I x 106/0-2 ml) were cultured with medium alone, with antigen (10 yg/ml of sonicated whole M. paratuberculosis, M. tuberculosis, or M. leprae antigens or a 1/100 dilution of Candida antigen), with 2 5 pug/ml of concanavalin A (Con A, ICN Pharmaceuticals, Cleveland, OH), or with both antigen and mitogen. The M. paratuberculosis antigen was a generous gift from Dr Harold Yoshimura, Baylor College of Medicine, Texas, and the M. tuberculosis, M. keprae and Candida antigens were generous gifts from Dr Barry Bloom, Albert Einstein College of Medicine, Bronx, NY. Lymphocyte proliferation was measured by 3H-thymidine incorporation as described previously (Ebert, 1989). These concentrations of the antigens were chosen since they resulted in the

stimulate lymphocyte proliferation and others that induce activity. The peripheral blood lymphocytes (PBL) from patients with the lepromatous form of the disease proliferate minimally in response to the M. leprae antigens, but demonstrate more M. keprae-induced suppression of mitogenic responses than do PBL from patients with the tuberculoid form of this disease (Mehra et al., 1979). When T cells were cloned from the lesions of lepromatous leprosy, the CD8 + T cell clones were found to inhibit the M. leprae-specific CD4+ T cell proliferation (Bloom, 1986). Such findings have clinical correlations. Patients with the lepromatous form exhibit depressed cellmediated immunity and retain acid-fast bacilli in their tissues, sometimes in large numbers, whereas those with the tuberculous form have an active cell-mediated immune response that clears the bacilli from the tissues (Bloom, 1986). Such studies have helped to understand the pathogenesis of leprosy. In IBD, the antigens that trigger the disease are unknown. Studying antigenspecific immune responses will help to determine the importance of that antigen in the disease process. SUBJECTS AND METHODS

more

than 15 mg of prednisone or the

Table 1. Clinical data of patients

Disease

Mean age (range)

Location/type of disease*

Disease activityt

Medicationst

Crohn's disease (n = 33)

35 (9-77)

Small bowel (12) Colon (11) Both (10)

Active (13) Inactive (20)

None (1 1) (4 active disease) Sulphasalazine (16) Metronidazole (1) Corticosteroids (7)

Ulcerative colitis (n= 18)

43 (8-73)

Left colon only (11) Left and transverse colon (1) Total colon (6)

Active (6) Inactive (13)

Diseased controls (n =9)

35 (18-63)

Active (9) Inactive (0)

Autoimmune disease (n = 5)

37 (23-51)

Infectious diarrhoea (3) Radiation enteritis (2) Pseudomembranous colitis (1) Coeliac sprue (2) Appendiceal abscess (1) Rheumatoid arthritis (2) Systemic lupus erythematosus (2) Hashimoto's thyroiditis (1)

None (3) (2 active disease) Sulphasalazine (9) Olsalazine (2) Corticosteroids (4) 5-ASA enemas (3) Cortenemas (2) None (8) Metronidazole (1) Corticosteroids (1)

None (3) Corticosteroids (2) Metronidazole (1)

* The extent of inflammatory bowel disease in the colon was usually determined by colonoscopy and biopsies while the extent in the small bowel was determined by radiologic studies (n of patients). t Disease was considered active if the patient had diarrhoea (at least three bowel movements daily), rectal bleeding due to inflammation, fever, and/or moderate abdominal pain, using the criteria outlined for ulcerative colitis (Truelove & Witts, 1955) and the criteria modified for Crohn's disease (de Dombal et al., 1974) (n of patients). t No patient took more than 15 mg of prednisone daily or the equivalent (n of patients).

322

E. C. Ebert et al. 180-

250 H

0

0

200 H

170

150 H S 0

100_

80

0

0 x

x

a) c

0

60 _

.L_-0 60 V 0

-5 E

40 _

0

0 0

-I

E

0

-.

en

S 0

40 _

0

0

0

0

0

S

20 _

201

0

-I--

0 -

0

*~~~~ * ifML MTA

0

-0-

0

emo ofit-N

Healthy

0

8

I

Crohn's disease

Ulcerative

colitis

6dr(

Diseased control

l

u.

F

MPT

Autoimrnmune disorders

0

0

-C-

A

-

r-

--

MPT

C

Healthy

ML MT Crohn's disease

.b

C

Fig. 1. Proliferation of peripheral blood lymphocytes (PBL) in response paratuberculosis. PBL (1 x 105/0 2 ml) from five patient groups were cultured with M. paratuberculosis (10 pg/ml) or with medium alone for 3 days. Proliferative responses were measured by 3H-thymidine incorporation and a stimulation index was calculated as described in Subjects and Methods. 0, Active disease; 0, inactive disease.

Fig. 2. Proliferation of peripheral blood lymphocytes (PBL) in response x 105/0 2 ml) from healthy individuals or from patients with Crohn's disease were cultured with M. paraluberculosis (MPT), M. leprae (ML), M. tuberculosis (MT), or Candida (C) antigens and the proliferative responses determined after a 6-day incubation. 0, Active disease; 0, inactive disease.

best proliferative responses by PBL from normal individuals and from IBD patients. The stimulation index (SI) for antigen was calculated as follows:

although a high value from a sixth patient caused the average to be the same as the SI for control PBL (Fig. 1). The low SI to M. paratuberculosis demonstrated by PBL from patients with active Crohn's disease was not due to tolerance resulting from high doses of antigen since the proliferative responses did not

to M.

S

ct/min (antigen) ct/min (medium)

to various antigens. PBL (1

The percentage of suppression of Con A-induced proliferation of PBL due to the presence of antigen was calculated as:

r-P

Induction of suppressor cells by Mycobacterium paratuberculosis antigen in inflammatory bowel disease.

We studied the M. paratuberculosis-induced proliferation and suppressor cell generation by peripheral blood lymphocytes from patients with inflammator...
873KB Sizes 0 Downloads 0 Views