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

Scand J Gastroenterol Downloaded from informahealthcare.com by McMaster University on 12/28/14 For personal use only.

Astrup, L., Rasmussen, S. N. & Binder, V. Leucocyte migration test with autologous colonic mucosa as antigen in patients with ulcerative colitis. Scand. J. Gastroent. 1977, 12, 95 1-955. 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-mediated hypersensitivity; colitis, ulcerative; leucocyte migration test Lone Astrup, M.D., Dept. of Internal Medicine E , Frederiksberg Hospital, Ndr. Fasanvej 59, 2000 Copenhagen F , Denmark

The inhibition of the migration of peripheral leucccytes 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 cellmediated immunity. It has been used for the investigation of a number of diseases of a possibly immunological nature, e.g. idiopathic Addison’s Disease ( lo), 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. I 1. 14). Bendixen ( I ) 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. (11) 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 the results of Richens in Crohn’s disease, but found no significant hypersensitivity against colonic mucosa in ulcerative colitis patients. The purpose of the present investigation was t o

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 t o 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 females and 4 males. The age range was from 2 1 to 66 years, median 4 I years. The control series consisted

952

L . Astrup, S . N . Rasrnussen & V . Binder

Scand J Gastroenterol Downloaded from informahealthcare.com by McMaster University on 12/28/14 For personal use only.

of 12 patients, 6 females and 6 males, suffering from peptic ulcer, irritable colon, and haemorrhoidal tumows. The age range was from 30 to 67 years, median 49 years. In order to evaluate the technique 10 tuberculinpositive individuals were treated with a tuberculin solution as antigen, containing 1mg purified protein derivative (PPD) per ml phosphate buffer from the State Serum Institute, Copenhagen. Denmark.

Lowry's method (8). The extract was immediately frozen in a sterile glass ampulla at - 18 "C until the test was performed within one week.

Leucocyte migration tees1

The leucocyte migration agarose technique (LMAT) described by Clausen (4) was used. A 2% 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 METHODS serum (the Serum Institute, Copenhagen, Denmark), 50% sterile water, and the final concentraPreparation of leucocyte suspension tion of agarose was one per cent. Penicillin and Venous blood was collected in 10 ml polysterol streptomycine (TC-penicillin-streptomycine) were tubes (Nunc, Roskilde, Denmark) each containing added t o a concentration of 6 6 units per ml and 250i.u. Heparin and 1.5ml 5% dextran (dextran 66pg per ml medium respectively. Sodium bicar250 with mol wt 250,000). After sedimentation for bonate (Difco-Sodium bicarbonate solution 10%) one hour at 37OC the leucocyte-rich plasma was was added to a final pH in the agarose medium of removed and centrifuged for 5 min at 220g and the 7.2-7.4. Five ml medium was thereafter poured into cell pellet was thereafter washed 3 times in Hanks plastic petri dishes (Millipore). After a gel had balanced salt solution. formed, 6 holes, 2.3mm in diameter, were cut in After the third wash the leucocytes were resuseach agarose plate, using a stainless steel punch. pended in a solution containing TC- 199 and 10% The leucocyte suspension was now divided into 2 horse serum. The added volumen was calculated so portions and for half an hour one portion was that the final content of 7p1 was 2mi11. leucocytes. incubated with antigen solution and the other with equivalent amounts of 0.9% saline. Preparation of antigen The concentration of protein in the three different The biopsy from the rectal mucosa was taken 8 to types of antigen solution was standardized t o 10 cm from the anus. The mucosa was inflamed in 100 pg/ml medium. Solutions of antigen and control the biopsy specimens from active ulcerative colitis were now placed in the agarose medium, 7 PI in each patients, normal from inactive patients. The skin hole. The final concentration of leucocytes in the biopsy was taken on the u l n a region of the right solutions was 2.8 x 1O8/ml. The agarose plates were upper arm by means of a skin drill. In advance the incubated at 37°C for 20 hours in 2% CO, in skin was washed with propyl alcohol and ethylene atmospherical air saturated with water. Thereafter chloride was applied for local anaesthesia. The bucthe plates were placed in methanol for 4 hours. This cal mucosa biopsy was taken in the lower right procedure allowed an easy removal of the agarose. vestibulum at the height of the first molar after the Thereafter the migration circle was estimated application of leostesine gel. The biopsy specimens after measurement of 2 diameters by means of a were thoroughly washed in 0.9% saline solution and magnifying glass. The area of the hole in the agarose immediately frozen in liquid nitrogen. Homogenizaplate was withdrawn. Migration index was calcution was made a.m. Goudie (7) by cutting the biopsy lated according to the formula specimen in 6 pm slices on a freezing microtome and then shaking in 0.9% saline solution by means of a area with antigen M.I. = Vortex Genie for 10 minutes. The homogenate was area without antigen centrifuged for 15 min at 3000 rev/min and filtered sterile with a pore size of 0.6 pm. The concentration The nominator as well as the denominator were of protein was measured in accordance with calculated as the mean of six migration areas.

Leucocyte Migration Test in Ulcerative Colitis

953

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

Scand J Gastroenterol Downloaded from informahealthcare.com by McMaster University on 12/28/14 For personal use only.

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.: 0.09)

R E C T A L MUCOSA

MI

1.4

BUCCAL MUCOSA

SKIN

uc

n=Il

C

-8.

1.2. 1.08

0.8,

0.6 0.4-

0.2. Fig. 1. Migration indicesofLMT inpatients withulcerativecolitis (UC)andagroupofcontrols (C).Straightlinesindicate mean values; broken lines indicate intervals corresponding to ? 2 S.D. + are very activeand Amoderately active ulcerative colitis patients, 0 are inactive ulcerative colitis patients and controls.

954

L.Astrup, S. N . Rasmussen & Y . Binder

inhibition of leucocyte migration in 1 I of 19 Primarily a series of double LMATs with P P D as patients with ulcerative colitis. In a later publication, antigen was performed in 10 volunteers who were however, with the same method Marcussen & BenMantoux positive. There was migration inhibition in dixen (9) found inhibition of leucocyte migration in all, with a range from 0.40-0.69 and a mean index only 6 of 26 patients with ulcerative colitis. A recent of 0.52. The difference between the double determi- study by Fixa et al. (6) of LMAT with foetal colon nations varied from -0.07 to 0.13, mean 0.01, antigen supports the findings of Bendixen ( I ) and S.D.zO.07. These results are equivalent t o the re- shows a similar inhibition of migration when E . coli 0 14 is used as an antigen. The tissue used as antigen sults of Clausen (5). was taken at autopsy up to 12 hours after death, The results of LMAT carried out in patients with which introduces the risk of postmortem alterations ulcerative colitis and control persons are shown in of tissue antigen. Table I, and further illustrated in Fig. 1. The migraof autologous colonic tissue in the presThe use tion indices were within normal limits in all patient ent series eliminates the possibility of genetic groups whatever colonic tissue, skin or oral mucosa incompability but does not eliminate the possibility was used as antigen. Analysis of variance did not of bacterial antigens completely, even when the disclose significant differences between the means homogenate was filtered through a 0.6prn filter, (F= 1.25, p > 0.05).. of lesser size could possibly pass since split products When patients and controls were compared by through. The negative results of the study, however. means of Student's t-test, no significant difference make this hypothetical possibility less interesting. between the M.1.s could be demonstrated either. On the other hand, the negative results of the present On the control series all measurements except 4 were carried out as double determinations on 2 study raise the question whether the concentration different days. With rectal mucosa the differences of antigen used is sufficient. The homogenization varied from -0.12 t o 0 . 2 4 , mean 0.02, S.D. 0.12; procedure used is similar to that used by Bendixen et with skin from -0.18 to 0.17, mean 0.00, a]., and the final protein concentration in their antiS.D. 0.10; with buccal mucosa from -0.24 to 0.07, gen preparation was 50-80 pg/ml-in ours 100pg/ ml. We must therefore conclude that no antigenmean -0.02, S.D. 0.08. In ulcerative colitis patients double determina- activity of autologous colon-mucosa, buccal-mutions were only performed in a few cases, because cosa and skin was demonstrable in ulcerative colitis, treatment had to be started in most cases after the by means of the leucocyte migration test. first investigation. However, the variation of the double determinations obtained did not point towards any difference when compared t o the results REFERENCES obtained in the group of controls. 1. Bendixen, G. Scand. J . Gastraent. 1967,2,2 14-22 1 2. Binder, V. Scand. J. Gastraent. 1970, 5, 621-632 No correlation between M.I. and the clinical 3. Brmtoff, J. Proc. Roy. Soc. Med. 1970,63,905-906 activity of ulcerative colitis could be demonstrated. 4. Clausen, J. E. Acfu 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 6. Fixa, B., Komarkova, O., Skaunic, V., Nerad, M., Kojecky, Z. & Benysek, L. Scand. J . Gastraent. DISCUSSION

Scand J Gastroenterol Downloaded from informahealthcare.com by McMaster University on 12/28/14 For personal use only.

RESULTS

1975, 5, 49&492 7. Goudie, R. B. et al. Luncet 1957, 273, 976-979 A. L. 8c 8. Lowry, O. H . t Rosenbrough,N. J., Randall, R. J. J. Biol. Chem. 1951, 193, 265-275 9, Marcussen, H. & G , stand. J . G ~ 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., Cough, K. R. & Ancill, R. J. Gut 1974. 15, 19-23 I

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 leucwyte migration test with the capi'Iary tube method and using foreign "ion tissues (homologous,foetal)as an antigen. He found

~

Leucocyte Migration Test in Ulcerative Colitis 12. Riis, P. & Anthonisen, P. Modern Gastroenterology. VIIIth International Congress of Gastroenterology, July 1968, Prague

Scand J Gastroenterol Downloaded from informahealthcare.com by McMaster University on 12/28/14 For personal use only.

Received 2 May 1977 Accepted July 19 1977

955

13. Soborg, M. & Halberg, P. Acra Med. Scand. 1968, 183, 10 1- 105 14. Williams, M. J.. Richens, E. R., Gough, K. R. & Ancill, R. J. Dig. Dis. 1975, 20, No. 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. NORBY RASMUSSEN & VIBEKE BINDER...
296KB Sizes 0 Downloads 0 Views