The Autologous Leukocyte Skin Test in Patients with Ulcerative Colitis

Vol. 49, No.6 Printed in U.S.A.

GASTROENTEROLOGY

Copyright © 1965 by The Williams & Wilkins Co.

THE AUTOLOGOUS LEUKOCYTE SKIN TEST IN PATIENTS WITH ULCERATIVE COLITIS DAviD W. WATSON, M.D ., HAROLD J . STYLER, M.D ., AND RoBERT J. BoLT, M .D.

Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan

There is much about ulcerative colitis to suggest that immunological mechanisms may play a role in the pathogenesis of the disease itself or at least in some of its manifestations. The chronic, intermittent and progessive nature of the disease; the many extracolonic manifestations; the infiltration of the colon with lymphocytes and plasma cells; the response, however temporary, to adrenal steroids, which are known to modify or interfere with certain immunological mechanisms ;1 the demonstration of antinuclear factors in a significant number of patients with ulcerative colitis and their relatives ;2 • 3 and finally, the demonstration of a cytotoxic effect of ulcerative colitis leukocytes for human fetal colon epithelial cells, 4 all serve to create this impression. For these reasons, it occurred to us that the leukocyte, important in delayed hypersensitivity, might have some special role in ulcerative colitis. Since a positive delayed cutaneous response to autologous leukocytes has been reported in systemic lupus erythematosus 5 and hemorrhagic cutaneous anaphylaxis due to autosensitization to DNA, 6 - 8 we decided to perform this test on our patients with ulcerative colitis. Materials and Methods

A total of 37 unselected in-patients and out-patients with ulcerative colitis were Received May 10, 1965. Accepted July 28, 1965. Address requests for reprints to: Dr. David W. Watson, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48104. This investigation was supported in part by United States Public Health Service Support to Graduate Education #6.

studied. The diagnosis of ulcerative colitis was established by barium enema and sigmoidoscopy in all cases. A group of 37 control patients without ulcerative colitis or suspected connective tissue disease was also studied. They were either healthy volunteers or patients hospitalized for a variety of other reasons. The leukocytes were obtained in a manner similar to that used by Friedman et al. 5 Into a 30-ml syringe was placed 1 ml of heparin (1000 U.S.P. units per ml) and 2 ml of 20% dextran solution (clinical grade H with a molecular weight of 186,000) as an agglomerant. Then 27 ml of blood was drawn into the syringe and thoroughly mixed by gentle rotation. The syringe was inverted with the capped disposable needle uppermost and the red cells allowed to sediment at 4 C for 1 hr. The plasma containing the leukocytes and platelets was then expressed through a 19-G scalp vein infusion set into a sterile, graduated centrifuge tube and centrifuged for 10 min at 1000 rpm. One-tenth milliliter of plasma was placed into a tuberculin syringe to be used as a control. The leukocytes were resuspended in 1 ml of plasma and a 0.1-ml aliquot of this used for the skin test. A leukocyte count was performed on the white blood cell suspension so that the approximate number of cells injected could be calculated. Each patient was tested with autologous leukocytes, red cells, plasma and dextran. One-tenth milliliter volumes were injected intradermally on either the forearm or the back. The skin tests were read at 24 and 48 hr. Biopsies were also taken at these times. Frequently two leukocyte skin tests were placed in order to biopsy at both 24and 48-hr intervals in the same patient.

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A test was considered positive if by 24 or 48 hr there was 10 mm or more of induration with or without erythema. I n addition to t he skin tests, each patient had a serum protein electrophoresis according to the method of Briere and Mull 9 and a detailed clinical history by one of t he investigators. Results

The results of the leukocyte skin tests are recorded in table 1. Of the 37 patients

Age, sex

Patient

M.F ... .... K.M .. . . .... .. R.C ... . . . K. S ... . . . . . . . .

c.w ... . . . .

D . J .. ..... .. D. F .. . ... . . L . H ... . . . . . . . . B. D .. . ... . . . Z.M . . J . G . .... . . R. S . . . . . ... . . . M.A .. . . .. . . .. . R. V .. . . . . . . R.W .. . . . . . s. s ... .. . . . C. S .... .. .. . . . . S. V .. . . ... .. . . D.R .. E. P . . . . . . . . . . R. B .. . . . . . . . . . .. .. .. J. M .. D. A .. . . . . . .. .. W.A .. .. .... . . . S. B ..... . . . . . . . .... . S.M ... C. C . . .. .. .. . R . S. . N.C .. . . . . . . . . W. H .. . . . ... . . . . .. . . J.D .. R . R . .. . . . . . .. . •

•

0

0

0

with ulcerative colitis, 12 or 32% gave a positive response. If the patients receiving · steroids at the t ime of the test are excluded, the figure for positive responses is 40%. No response was evident to the injections of red cells, plasma or dextran. Similarly, no positive responses were elicited in the 37 control subjects. I n all cases, responses were maximal by 24 hr. Table 2 compares the reactors, nonreactors and controls in respect to sex, average

1 Results of the autologous leukocyte skin test in 37 patients with ulcerative colitis

TABLE

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Vol . 49, No.6

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•

•

•

•

•

•

•

•

0

.

•

•

•

•

•

•

•

0

•

c. v .. . ... .. . . .

J. H . . . . . . . . . A.W .. .. . . . . . . . J. B .. . . . . . . . . . L.M .. .

20, F 26, M 39,M 30, M 19, F 19,M 20, F 68, F 21, M 32, M 34, M 48, F 37, F 14, F 63, M 17, F 19, F 40, F 34, M 40, F 14, M 16, M 25, M 34, M 14, F 32, F 39, M 24, M 64, M 38, M 30, M 28, M 17, M 60, M 15, M 36,M 17, F

No. of cells injected

Induration

months

millions

mm

84 62 24 96 3 52 5 6 5 120 84 13 36 7 180 48 15 96 84 168 48 30 24 12 17 1 18 17 66 48 96 72 12 480 12

qns• 9.2 5, 10, 20 8.0 17.1 18 .0 14 .6 7.6 9.2 12 .5 9.4 5.5 8 .0 9.7 18.0 11.2 6 .7 10.5 9.2 6.2 4.5 12.0 8.5 7.0 7.0 6.5 3.5 10.2 9.4 12.0 10.0 9.6 12.2 10.0 10 . 2 10.2 9.6

13 8 6 7 4 13 9 5 12 12 6 8 0 8 10 6 9 10 13 6 6 10 5 6 8 4 4 12 5 10 6 16 10 5 5 4 5

Duration of colitis

60

18

ExtracoJonic manifestations

Hepatitis Arthritis

Arthritis Arthritis, erythema multiforme

Arthritis, erythema multiforme Arthritis Pyoderma gangrenosum

Arthritis, episcleritis

Iritis, ankylosing spondylitis

Erythema nodosum Erythema nodosum, iritis, spondylitis

a This patient was leukopenic and there were insufficient cells to provide both for a reasonable inoculum and a cell count.

December 1965 TABLE

651

AUTOLOGOUS LEUKOCYTE SKIN TEST . 2. Relationship of leukocyte skin test results to age, sex, duration of the colitis and number of cells injected Positive (12)

. . . . . . ...... . . . Mean induration (mm) .. . . . . . . Mean number of cells injected (millions) . . . . .. . .. .

12 12.1 (range, 9.2-18) 29 .3 Mean age (years). ...... . .... . . ......... . . . . . . ... Sex 10 (83%) Male . ..... . . . . . . . . . . . . . . . . .. . . .. .. . . . . .. . .. . . . . 2 (17%) Female . .. . . . . . . . . . . . .. . . ........ . . . . 66.6 Mean duration of ulcerative colitis (months) . .. . . . •

•

•

•

•

•

•

•

0

age, amount of induration, number of cells injected and duration of the colitis. It is possible that the higher average number of cells injected in the reactors might account for the difference in response. This seems unlikely, however, since the range for number of cells injected in patients giving a positive response is 9.2 to 18.0 million and the same figure for those giving a negative response is 5 to 20 million. Thus it can be seen that similar numbers of cells are capable of either a positive or negative response. Indeed, in one patient the injection of 5, 10 and 20 million cells produced a uniformly negative response. No correlation was apparent between a positive skin test, age of the patient or duration of the disease. Neither was it possible to discern a relationship to the severity of the colitis at the time of the test. Table 3 shows the relationship between the skin tests, serum protein electrophoresis and the presence or absence of clinical allergies. No undue occurrence of allergy was found in the group as a whole or in those giving a positive skin test. A relationship was evident, however, between a positive skin test and the presence of an elevated serum yglobulin (P = 0.01). The y-globulin was taken to be elevated if it amounted to 1.7 g per 100 ml or more and comprised at least 20% of the total serum protein. No statistically significant relationship was found between a positive skin test and extracolonic manifestations. The results of the biopsies were generally unrewarding. In two patients with positive tests, minor degrees of perivascular cuffing

•

N egative (25)

Control (37 )

6 8.8 (range, 5-20) 31.8

5 8.5 (range, 4. 6-21. 8) 30.3

13 (52%) 12 (48%) 56.8

14 (38% ) 23 (62% )

with leukocytes was noted, as well as minimal collagen necrosis and slightly increased numbers of eosinophils. By and large, however, no significant differences from negative or control tests could be seen. In all of our cases whether reactors, nonreactors or controls, large numbers of polymorphonuclear neutrophiles were seen in the dermis, occasionally infiltrating between collagen bundles but more often in the form of a microabscess. Perhaps the cellular nature of the inoculum readily masks any subtle change that might have taken place, thus rendering correlation with macroscopic events difficult. Figure 1 shows the histopathology of a positive skin test at 24 hr and figure 2 shows a negative control test at the same interval. Discussion

A positive delayed response to autologous leukocytes has been described in systemic lupus erythematosus (SLE) ,5 hemorrhagic cutaneous anaphylaxis due to autosensitization to DNA 6 - 8 and two cases of pyoderma gangrenosum,l 0 at least one of which had ulcerative colitis. A few patients with rheumatoid arthritis have also been found to have positive tests but since from 12 to 20% of randomly selected patients with rheumatoid arthritis have a positive LE cell prep 11 it is difficult to separate such patients from those with SLE. Our results would place ulcerative colitis in the group of disorders which exhibit a delayed response to autologous leukocytes, although the magnitude of response is not of the order seen in SLE or autosensitization to DNA.

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WATSON ET AL.

3. Correlation of skin test results, serum -y-globulin levels and occurrence of clinical allergies Patient

Skin test

M . F .. K. MeA . ... . .. ... .. .. .. R . C .. .. . K. S ...

+

D. J . .. D. F ... . . . ... .. . .... L. H ..... . .... . . . . . . B. D ... ... .. . . ... . .. .... Z.M ....... . . J. G .. ..... . . . ... . . .... . R . S ....... . ... ... . ..... M.A ... .. . ... . . . . ... ... R . V ..... ......... . . R.W . . . ... . . . ... . . . . . .

+

-y-Globulin

% Tpa

g/100 tnl

65 14 21 12 8 25

5.8 1.0 1.4 0.7 0.4 1.7 0.6

c. w ...

s. s .. c. s ... ... .. . . . . .. . .. ... v. s .. ....... . .

D. R . . .... ... . . .... . . . . E.P ... ...... . . ........ R.L . B ...... .. . . ... . . . . J . MeG .. . .. . .... ..... D. A ............ . . . .. .. . W.W .... S. B ... . . . ... .. .... .. .. S.M ..... . . . . . . . . . . .

Clinical allergies

11

+ +

36

+

30

11

10 13 9 24

15

+ +

c. v ... .

.. ...... . J. H .. A.W ... J. B. .. . .. . . .... . . . . . L . McK .. . a b

0 0 0 0 0 0 0 0 0 0 0 0 0

3.3 0.6 0.7 0.9 0.6 2.3 1.7 1.0

Asthmab Eczema

0 Drug allergy-eczema

+

19

1.1

19 19

1.5 1.4

0 0 0 0 0 Allergic rhinitis

0 Allergic rhinitis

c. c .. ........ ...... ....

... . . ... . ... R. s .. N. c .... .. .. .... ...... .. W.H ... ... J.D . . ... ...... ..... .. .. R . R .... ....

Drug rash

0

+ + + +

19 13 22 19 25 17 19 13

1.5 0.9 2.0 1.4 1.9 0.8 1.6 0.7

Food allergy

0 Allergic rhinitis Drug allergy

0 0 0 Allergic rhinitis

0 0

TP = total protein. Clinically active at time of test.

Friedman reported histological differences between patients with SLE and controls. In those with positive skin tests, he described a marked cellular infiltration of the deeper dermis with polymorphonuclear neutrophiles and limited numbers of mononuclear cells. In the controls no such cellular infiltration was evident; only scattered cellular debris was seen. We were unable to demonstrate such a difference. In all cases the histological appearance was similar to

Friedman's reactors. In no case did we observe a picture similar to that in his controls. The reason for this discrepancy is not apparent. What is implied by such a response is not known. There are two possible interpretations of such a reaction. On the one hand, it may imply that the leukocyte is in some way a mediator of the pathological changes in ulcerative colitis and other so-called autoimmune diseases. Certainly lympho-

December 1965

AUTOLOGOUS LEUKOCYTE SKIN TEST

Fw . 1 (top) . Histopathology of a positive skin test at 24 hr. Note the infiltration of collagen by the neutrophiles with microabscess formation. FIG. 2 (bottom). Histopathology of a negative skin test at 24 hr. Note the similarity to figure 1.

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cytic infiltration is a prominent feature of ulcerative colitis and one could readily visualize their participation in local tissue damage. Lymphocytes are also known to be the bearers of transfer factor/ 2 important in delayed hypersensitivity. That leukocytes are capable of cytotoxicity is also known from the work of Perlmann and Broberger with fetal colon 4 and the work of Weaver et al. with millipore filters. 13 Of perhaps more importance is the fact that experimentally induced autoimmune diseases such as encephalomyelitis and thyroiditis depend on cell-bound mechanisms for their development. 14 • 15 It is not too difficult to visualize such a role for these cells in ulcerative colitis or at least in some of its manifestations. If the lymphocytes do effect tissue changes one would have to postulate the presence of antigens in various tissues, such as skin, colon, liver, etc., capable of interacting with such sensitized cells. The parallel elevation in serum yglobulin would be difficult to reconcile with such a view, however, since this finding would imply an increase in circulating antibodies. It has been suggested that the autologous leukocyte skin test is in reality a test for immediate hypersensitivity with the time required to achieve a positive response representing the time required for the appropriate antigen to be liberated. If this were true it would be easier to explain a positive skin test in association with an elevated serum y-globulin. One could then postulate that the leukocytes contained a substance which upon liberation reacted with circulating antibody to produce the local lesion. This leads us to a consideration of the second possible explanation for a positive skin test. In this view the leukocyte might simply be a convenient agent useful in demonstrating a hypersensitivity to some cellular constituent such as nucleoprotein. Thus it is possible that the antinuclear factors, present in some patients with ulcerative colitis, might combine with the leukocytes to cause a visible skin reaction. Such cellular constituents might be antigenically similar to antigenic determinants arising within the colon as a result of the stimulus responsible for initiating the disease. This possibility is

suggested by the reported cases of autosensitization to DNA, for it is in this condition that the most pronounced reactions to leukocytes and DNA are elicited. This might also explain why antinuclear factors are so prominent in conditions such as SLE and ulcerative colitis. It is, however, possible that the responsible constituent of the leukocyte is merely an antigen or other noxious material coating the cell which is derived from absorbed products of bacteria or foodstuffs. That leukocytes can adsorb antigens is apparent from the work of Boyden with tuberculoprotein. 16 It is also possible that both views are correct. Perhaps an alteration of nucleoprotein or other cell constituent initiates a state of delayed hypersensitivity in which the leukocyte not only becomes the instrument of tissue damage by virtue of its fundamental role in such reactions but also at the same time shares the hypothetical alteration with other cells or at least contains material antigenically related to it. Whatever the explanation, the leukocyte, especially the lymphocyte, is deserving of further investigation to ascertain what part, if any, it plays in ulcerative colitis. Summary

The autologous leukocyte skin test was studied in 37 patients with ulcerative colitis. Twelve of the 37 exhibited positive delayed cutaneous reactions to autologous leukocytes. Five of the nonreactors were on steroids at the time of the test. No reactions were observed to red blood cells, plasma or dextran. None of the 37 control subjects gave a positive response. A positive response could not be correlated with the age and sex of the patient, duration of the disease, clinical activity, presence of clinical allergy or the presence of extracolonic manifestations. A correlation was found between a positive skin test and the presence of an elevated serum y-globulin. REFERENCES 1. Stoerck, H. C. 1959. Cortisone and lymphoid tissue in relation to hypersensitivity, p. 623-631. In J. H. Shaffer, G. A. LoGrippo, and M. W. Chase [ed.] Mechanisms of hypersensitivity. Little, Brown & Company, Boston.

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2. Calabresi, P., W. R. Thayer, Jr., and H. M. Spiro. 1961. Demonstration of circulating antinuclear globulins in ulcerative colitis. J. Clin. Invest. 40: 2126. 3. Thayer, W. R., Jr., and J. M. Spiro. 1963. Protein abnormalities in ulcerative colitis patients and their families. Gastroenterology 44: 444. 4. Perlmann, P ., and 0. Broberger. 1963. In vitro studies of ulcerative colitis. II. Cytotoxic action of white blood cells from patients on human fetal colon cells. J. Exp. Med. 117:

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5. Friedman, E. A., W. A. Bardawil, J. P. Merrill, and C. Hanau. 1960. "Delayed" cutaneous hypersensitivity to leukocytes in disseminated lupus erythematosus. New Eng. J. Med. 262: 486. 6. Levin, M. B., and H. Pinkus. 1961. Autosensitivity to desoxyribonucleic acid (DNA): Report of a case with inflammatory skin lesions controlled by Chloroquine. New Eng. J. Med. 264: 533. 7. Schwartz, R. S., F. B. Lewis, and W. Dameshek. 1962. Hemorrhagic cutaneous anaphylaxis due to autosensitization to deoxyribonucleic acid. New Eng. J. Me d. 267: 1105. 8. Little, A. S., and H . E. Bell. 1964. Painful subcutaneous hemorrhages of the extremities with unusual reaction to injected deoxyribonucleic acid. Ann. Intern. Med. 60: 886. 9. Briere, R. 0., and J. D. Mull. 1964. Electrophoresis of serum protein with cellulose

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acetate. A method for quantitation. Amer. J. Clin. Path. 42: 547. Long, P. I., and C. T. Uesu. 1964. Pyoderma gangraenosum. J. A. M.A. 187: 336. McEwen, C. 1960. The diagnosis and differential diagnosis of rheumatoid arthritis, p. 237. In J. L. Hollander [ed.] Arthritis. Lea & Febiger, Philadelphia. Lawrence, H. S., F. T. Rapaport, J. M. Converse, and W. S. Tillett. 1960. Transfer of delayed hypersensitivity to skin homografts with leukocyte extracts in man. J . Clin. Invest. 39: 185. Weaver, J. M., G. H. Algire, and R. T. Prehn. 1955. The growth of cells in vivo in diffusion chambers. II. The role of cells in the destruction of homografts in mice. J. Nat. Cancer Inst. 15: 1737. Paterson, P. Y. 1962. Cellular and humoral immune factors in allergic encephalomyelitis, p. 184-192. In P. Grabar and P. Miescher [ed.] Mechanism of cell and tissue damage produced by immune reaction. Schwabe & Co., Basel. Roitt, I. M., H. E. H. Jones, and D. Doniach. 1962. Mechanisms of tissue damage in human and experimental autoimmune thyroiditis, p. 174-183. In P. Grabar and P. Miescher [ed.] M echanism of cell and tissue damage produced by immune reactions. Schwabe & Co., Basel. Boyden, S. V. 1953. Fixation of bacterial products by erythrocytes in vivo and by leucocytes. Nature (London) 171: 402.