Leucocyte Migration Test with Autologous Colonic Mucosa as Antigen in Patients with Ulcerative Colitis LONE ASTRUP, S. N6RBY RASMUSSEN & VIBEKE BINDER Dept. of Internal Medicine B (Gastroenterology), Gentofte Hospital, University of Copenhagen, Copenhagen, Denmark

Astrup, L., Rasmussen, S. N. & Binder, V. Leucocyte migration test with autologous colonic mucosa as antigen in patients with ulcerative colitis. Scand. J. Gasfroenr. 1977, Scand J Gastroenterol Downloaded from informahealthcare.com by Queen's University on 12/31/14 For personal use only.

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The leucocyte migration agarose technique (LMAT) was applied in a study of the migration of peripheral leucocytes in 16 patients with ulcerative colitis using three different autologous types of tissue as antigen: rectal mucosa, skin and buccal mucosa. In all cases the migration indices were within normal limits, and they did not differ from a group of control patients suffering from peptic ulcer, irritable colon, or haemorrhoidal tumours. The present study does not support the theory of cellular hypersensitivity against colonic mucosa in patients with ulcerative colitis. Keywords: Cell-mediatedhypersensitivity;leucocytemigration test; ulcerativecolitis Lone Asfrup, M.D., Dept. of Internal Medicine E . Frederiksberg Hospifal, Ndr. Fasanvej 59, 2000 Copenhagen F, Denmark

The inhibition of the migration of peripheral leucocytes induced by specific antigens has been widely accepted as a parameter of cellular hypersensitivity. The leucocyte migration test seems to be an appropriate technique for the in vitro examination of cell-mediated immunity. It has been used for the investigation of a number of diseases of a possibly immunological nature, e.g. idiopathic Addison’s Disease ( IO),thyroiditis (13). and pernicious anaemia (3). In a number of studies the technique has been used for the investigation of ulcerative colitis and Crohn’s disease (1, 9, 11, 14). Bendixen ( 1 ) and Marcussen & Bendixen (9), using extract of foetal colonic and jejunal mucosa as antigen, demonstrated cellular hypersensitivity in some patients with ulcerative colitis in contrast to patients with Crohn’s disease. Richens et al. ( 1 1) used homologous colonic mucosa from patients with Crohn’s disease as an antigen and demonstrated cellular hypersensitivity with this antigen in 16 out of 32 patients with Crohn’s disease. Williams et al. (14) reproduced Richens’s results in Crohn’s disease, but found no significant hypersensitivity against colonic mucosa in ulcerative colitis patients.

The purpose of the present investigation was to further evaluate the immunological parameters of ulcerative colitis by examining the patient’s own colonic mucosa as a possible antigen. The procedure was intended to bypass the tissue incompatibility that may exist against another individual’s colonic mucosa. A second purpose of this investigation was to evaluate whether a possible autohypersensitivity was specific to colonic mucosa by comparing the results obtained by this antigen to those obtained by using skin and buccal mucosa. MATERIAL Sixteen patients with ulcerative colitis were examined. The patients fulfilled at least 3 of 4 sets of criteria for the diagnosis according to Riis & Anthonisen (12). The material was classified with respect to clinical activity of the disease in accordance with Binder (2). Five patients were inactive, seven moderately active, and four very active. The patients were untreated at the time of examination. If they had been given salazosulfapyridine or steroid, these drugs were withdrawn at least one and four weeks respectively beforehand. There were 12

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females and 4 males. Their ages ranged from 2 1 to 66 years, median 41 years. The control series consisted of 12 patients, 6 females and 6 males, suffering from peptic ulcer, irritable colon, and haemorrhoidal tumours. Their ages ranged from 30 to 67 years, median 49 years. In order to evaluate the technique 10 tuberculinpositive individuals were tested with a tuberculin solution as antigen, containing 1 mg purified protein derivative (PPD) per ml phosphate buffer from the State Serum Institute, Copenhagen, Denmark. METHODS Preparation of leucocj-te suspension Venous blood was collected in 10 ml polysterole tubes (Nunc, Roskilde. Denmark) each containing 250 i.u. heparin and 1.5ml 5% dextran (dextran 250 with mol wt 250,000). After sedimentation for one hour at 37OC the leucocyte-rich plasma was removed. centrifuged for 5 min at 220 g, and the cell pellet thereafter washed 3 times in Hanks balanced salt solution. After the third wash the leucocytes were resuspended in a solution containing TC- 199 medium and 10% horse serum. The added volume was calculated so that the final content of 7p1 was 2 mill. leucocytes. Preparation of antigen The biopsy from the rectal mucosa was taken 8 to 10 cm from the anus. The mucosa was inflamed in the biopsy specimens from active ulcerative colitis patients, normal in those from inactive patients.The skin biopsy was taken on the ulnar region of the right upper arm by means of a skin drill. In advance the skin was washed with propyl alcohol, and ethylene chloride was applied for local anaesthesia. The buccal mucosa biopsy was taken in the lower right vestibulum at the height of the first molar after the application of leostesine gel. The biopsy specimens were thoroughly washed in 0.9%saline solution and immediately frozen in liquid nitrogen. Homogenization was made a.m. Goudie ( 7 ) by cutting the biopsy specimen in 6 pm slices on a freezing microtome and then shaking in 0.9% saline solution by means of a Vortex Genie for 10 min. The homogenate was centrifuged for 15 min at 3000 rev./min and

filtered sterile with a pore size of 0.6pm. The concentration of protein was measured in accordance with Lowry's method (8). The extract was immediately frozen in a sterile glass ampulla at -18OC until the test was performed within one week. Leucocyte migration test The leucocyte migration agarose technique (LMAT) described by Clausen (4) was used. A 2 per cent agarose solution (Litex, Glostrup, Denmark) was prepared on the day of examination. The medium contained 9% ten times concentrated tissue culture medium (Difco TC-medium 199 x lo), 10% horse serum (the Serum Institute, Copenhagen, Denmark), 50% sterile water, and the final concentration of agarose was 1%. Penicillin and streptomycin (TC-penicillin-streptomycin) was added to a concentration of 66 units per ml and 66 pg per ml medium respectively. Sodium bicarbonate (Difco-Sodium bicarbonate solution 10%) was added to a final pH in the agarose medium of 7.2-7.4. Five ml medium was thereafter poured into plastic petri dishes (Millipore). After a gel had formed, 6 holes, 2.3mm in diameter, were cut in each agarose plate, using a stainless steel punch. The leucocyte suspension was now divided into 2 portions, and for half an hour one portion was incubated with antigen solution and the other with equivalent amounts of 0.9% saline. The concentration of protein in the three different types of antigen solution was standardized to 100 pg/ml medium. Solutions of antigen and control were now placed in the agarose medium, 7 pI in each hole. The final concentration of leucocytes in the solutions was 2.8 x 108/ml. The agarose plates were incubated at 37 "C for 20 hours in 2% CO, in atmospherical air saturated with water. Thereafter the plates were placed in methanol for 4 hours. This procedure allowed an easy removal of the agarose. Thereafter the migration circle was estimated after measurement of 2 diameters by means of a magnifying glass. The area of the hole in the agarose plate was withdrawn. Migration index was calculated according to the formula M.I.: area with antigen/area without antigen. The nominator as well as the denominator were calculated as the mean of six migration areas.

Leucocyte Migration Test

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RESULTS Primarily a series of double LMATs with PPD as antigen was performed in 10 volunteers who were Mantoux positive. There was migration inhibition in all with a range from 0.40-0.69 and a mean index of 0.52. The difference between the double determinations varied from 0.07 to 0.13, mean 0.0 1, S.D.= 0.07. These results are equivalent to the results of Clausen (5). The results of LMAT carried out in patients with ulcerative colitis and control persons are shown in Table I. and further illustrated in Fig. 1. The migration indices were within normal limits in all patient groups whether colonic tissue, skin or oral mucosa was used as antigen. Analysis of variance did not

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disclose significant differences between the means (F= 1.25, p > 0.05). When patients and controls were compared by means of Student's t-test, no significant difference between the M.1.s could be demonstrated either. On the control series all measurements except 4 were carried out as double determinations on 2 different days. With rectal mucosa the differences varied from -0.12 to 0.24, mean 0.02, S.D.= 0.12; with skin from -0.18 to 0.17. mean 0.00, S.D.= 0.10; with buccal mucosa from -0.24 to 0.07, mean -0.02, S.D. 0.08. In ulcerative colitis patients double determinations were only performed in a few cases, because treatment had to be started in most cases after the first investigation. However, the variation of the

Migration index in ulcerative colitis and normal controls RECTAL MUCOSA

MI

uc

SKIN

BUCCAL MUCOSA

uc

1.4-

1.2----,---. $ A

1.0-

0.8-

$ .u+

-+'.

0.60.4-

0.2Fig. 1. Migration indices of leucocyte migration test in patients with ulcerative colitis (UC) and a group of controls ( C ) .Straight lines indicate mean values: broken lines indicate intervals corresponding to ? 2 S.D. + are very active and A moderately active UC patients, 0 are inactive UC patients and controls.

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L . Astrup. S . N . Rasmussen & V. Binder

Table I. Leucocyte migration indices obtained with 3 different autologous tissue antigens in patients with ulcerative colitis and normal subjects

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ANTIGEN Rectal mucosa

Skin

Buccal mucosa

Ulcerative colitis patients

1.02 (S.D.: 0.08) n = 16

1.04 (S.D.: 0.08) n = 16

0.98 (S.D.: 0.10) n = 13

Normal controls

1.03 (S.D.: 0.13) n = 12

1.07 (S.D.: 0.14) n = 12

1.02 (S.D.: 0.99) n= I1

double determinations obtained did not point towards any difference when compared to the results obtained in the group of controls. No correlation between M.I. and the clinical activity of ulcerative colitis could be demonstrated. DISCUSSION In several studies concerning the pathogenesis of ulcerative colitis the results especially of the different immunological tests have been difficult to interpret. Bendixen ( 1) investigated cell-mediated immunity by means of the leucocyte migration test with the capillary tube method and using foreign colon tissues (homologous, foetal) as an antigen. He found inhibition of leucocyte migration in 1 1 of 19 patients with ulcerative colitis. In a later publication, however, with the same method, Marcussen & Bendixen (9) found inhibition of leucocyte migration in only 6 of 26 patients with ulcerative colitis. A recent study by Fixa et al. (6) of LMAT with foetal colon antigen supports the findings of Bendixen (1) and shows a similar inhibition of migration when E. coli 014 is used as an antigen. The tissue used as antigen was taken at autopsy up to 12 hours after death, which gives the risk of postmortem alterations of tisue antigen. The use of autologous colonic tissue in the present series eliminates the possibility of genetic incompatibility but does not eliminate the possibility of bacterial antigens completely, even when the homogenate was filtered through a 0.6 pm filter, since split products of lesser size could possibly pass. The negative results of the study, however, Received 2 May 1977 Accepted 1 June 1977

make this hypothetical possibility less interesting. On the other hand, the negative results of the present study raise the question whether the concentration of antigen used is sufficient. The homogenization procedure used is similar to that used by Bendixen et al., and the final protein concentration in their antigen preparation was 5080 pg/ml-in ours 100pg/ml. We must therefore conclude that no antigen-activity of autologous colon mucosa, buccal mucosa, and skin was demonstrable in ulcerative colitis by means of the leucocyte migration test. REFERENCES 1. Bendixen,G. Scand. J . Gastroent. 1967.2.2 14-22 1 2. Binder, V. Scand. J. Gastroent. 1970, 5, 627-632 3. Brostoff, J. Proc. Roy. Soc. Med. 1970,63.905-906 4. Clausen, J. E. Acta Allerg. (Kbh.) 1971, 56-80 5. Clausen, J. E. The agarose migration inhibition technique for in vitro demonstration of cell-mediated immunity in man. Thesis 1974. Skaunic, V., Nerad, M., 6. Fixa, B., Komarkova, 0.. Kojecky, Z. & Benysek, L. Scand. J. Gastroent. 1975, 5, 490-492 7. Goudie, R. B. et al. Lancet 1957, 273, 976-979 8. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L. & Randall, R. J.J. Biol. Chem. 195 1,193,265-275 9. Marcussen, H. 8t Bendixen, G. Scand. J. Gastroenr. 1974, 9, 149-152 10. Nerup, J.. Andersen, V. & Bendixen, G. Clin. Exp. Immunol. 1969, 4, 355-363 1 1. Richens, Elizabeth R., Williams. M. J.. Gough, K.R. & Ancill, R. J. Gut 1974, 15, 19-23 12. Riis, P. & Anthonisen. P. The differential diagnosis

between ulcerative colitis and Crohn’s disease of the colon. Modern Gastroenterology. VIIIth International Congress of Gastroenterology,Prague, 1968 13. Soborg, M. & Halberg, P. Acta Med. Scand. 1968, 183, 101-105 14. Williams. M. J., Richens, E. R., Gough, K. R. & Ancill, R. J. Dig. Dis. 1975, 20, 5

Leucocyte migration test with autologous colonic mucosa as antigen in patients with ulcerative colitis.

Leucocyte Migration Test with Autologous Colonic Mucosa as Antigen in Patients with Ulcerative Colitis LONE ASTRUP, S. N6RBY RASMUSSEN & VIBEKE BINDER...
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