J. Endocrinol. Invest. 2: 375, 1979

Luekocyte adherence inhibition in response to human orbital and lacrimal extracts in patients with Graves' ophthalmopathy

J.R. Wall, B.A.J. Walters, and C. Grant Department of Medicine, Repatriation General Hospital, Greenslopes, Queensland, Australia

ABSTRACT. Leukocyte adherence inhibition (LAI) may be a test for cell-mediated immunity or cy­tophilic antibodies. LAI tests were carried out on patients with Graves' ophthalmopathy, using normal human lacrimal gland, orbital fat, and eye muscle extracts as antigens. The results were ex­pressed as % LA!. The percentage leukocyte adherence inhibition in tests from patients and nor­mal subjects were not significantly different for any preparation (Mann-Whitney tests). Taking the upper limit of normal as the mean % LAI +2S0 for normal subjects, tests were positive, to eye mus­cle extract, in only one patient whilst borderline inhibition, in response to eye muscle and fat ex­tracts respectively, was demonstrated in two patients. All three had active disease. LAI tests may be positive only during the acute stage of Graves' ophthalmopathy. The significance of these find­ings in respect to the pathogenesis of Graves' ophthalmopathy is discussed.

INTRODUCTION Graves' ophthalmopathy is likely to be an autoimmune disorder (1-3). Although serum antibodies against orbi­tal antigens have not been convincingly demonstrated there is evidence for cell-mediated immunity to orbital fractions, as determined by macrophage migration in­hibition tests (4-7). In addition, the weJl known close as­sociation with Graves' hyperthyroidism and other auto­immune disorders, the response to immunosuppres­sive therapy (3, 8) and the orbital infiltration with lym­phocytes and plasma cells (9) suggests an auto­immune pathogenesis'. Although it is uncertain whether leukocyte adherence inhibition (LAI) is a test for cell-mediated immunity (10-11) or cytophilic antibodies (12, 13), it is generaJly ac­cepted to have an immunological basis. In this study, LAI tests were carried out on patients with Graves' oph­thalmopathy using preparations of human orbital and lacrimal gland tissues as antigen, in an attempt to de­fine another parameter of orbital immunoreactivity in this disorder.

MATERIALS AND METHODS Patients Fifteen patients with Graves' ophthalmopathy, 8 fe­males and 7 males aged 47-49 yr (mean age 59 yr) were studied. Eleven patients had associated hyper-

Key-words.- Graves' ophthalmopathy, leukocyte adherence Inhibition. cell­mediated immunity, lymphocytes, orbital antigens

Correspondence J.R. Wall Department of Medicine, Hotel D,eu Hospital, King­ston. Ontario, Canada, K7L 3H6

Received October 3, 1978: accepted May 23. 1979

375

thyroidism which was untreated in 3 patients and treat­ed in 8 patients. Four patients had no associated hyper­thyroidism ("euthyroid Graves' disease"). The eye dis­ease was active in 11 patients, after treatment with cyclophosphamide in 3 and in the absence of specific treatment in 8. This was considered severe in 6 pa­tients and mild in 5. The other 4 patients had "burnt out" disease (3 patients) or inactive disease after cyclo­phosphamide treatment (1 patient). No patient was be­ing treated with immunosuppressive drugs or steroids at the time of study. Also studied were 15 normal sub­jects, 9 females and 6 males aged 22-55 yr (mean age 53 yr) as controls, Patients and normals were tested concurrently.

Peripheral blood leukocytes Peripheral blood leukocytes (PBL) were harvested from 25 ml of venous blood using a Hypaque-FicoJl gra­dient (14). The ceJl population consisted of from 80-90% lymphocytes, 9-19% monocytes, and 0-1 % neu­trophils,

Leukocyte adherence inhibition The method used was based on that of Holan et at. (15) and Grosser and Thomson (16), as modified by Walters et at. (17) and Rutherford et al. (18). Equal volumes (0.1 ml) of mononuclear ceJl suspen­sion (4 x 106 cells) and lacrimal, eye muscle or fat ex­tract were mixed, and medium was added to make the final volume 0.4 ml. Duplicate mixtures were made of each cell suspension with the three extracts and con­trols (without extract) in small plastic-capped tubes. The tubes were incubated at 37 C for 30 min. with peri­odic shaking. After incubation the contents of the tubes were removed into plastic cups a nd cells were counted

JR. Wall, BAJ Walters, and C. Grant

in subsamples of 0,02 mL The remaining cell mixtures were aspirated with Pasteur pipettes and placed at the bottom of Kimax screw-capped test tubes (Owens­Illinois Glass Company. Toledo. Ohio) These tubes were then incubated at 37C for 2 h in an almost horiz­ontal position so that the contents ran along the top three-quarters of the tube, After incubation. the tubes were individually raised to the vertical position. the me­dium and nonadherent cells at the bottom mixed by re­peated pipetting. and the cells recounted, The percentage adherence of cells was determined for each mixture by the formula

final count Percentage adherence = 100 x 100

initial count

An arithmetic mean was calculated for each pair of,du­plicate mixtures; the individual values did not differ from th'e mean by more than 6%. The percentage LAI was calculated for each combination of leukocytes and ex­tract by the formula :

Percentage LAI

Mean % adherence with no extract­Mean % adherence with extract

-------------------------xl00 Mean % adherence with no extract

The upper limit of normal for each preparation was tak­en as the mean % LAI for normals + 2S0, and levels above this considered significant. Percentaqe LAI for patients and normal subjects were compared statisti­cally for each fraction using the Mann-Whitney test. The method used for preparation of tissue extracts was based on that of Schnieder and Hogeboom (19), Fresh normal human lacrimal gland. orbital fat. and eye mus­cle were obtained at autopsy, Approximately 109 of tissue were homogenized in 1 ml 0.25 M sucrose. fil­tered through two layers of gauze and made up to 5 ml in phosphate buffered saline (PBS) (pH 7.2), The solu­ble proteins were extracted by incubating overnight at 4 C, The mixture was centrifuged at 2.000 rpm and the pellet discarded. The protein concentration of the su­pernatant was calculated using the optical density me­thod and adjusted to 2 mg/ml in PBS, One ml aliquots were stored at -20 C until used, All procedures were carried out at 4 C, Fresh normal human lung and peri­pheral nerve extracts were prepared in the same way as control extracts, Single batches of tissues were used for the study, The final concentration of extracts used in LAI tests was 100 Jlg Iml for all preparations, Concentrations of up to 200 Jlg/ml were found. in preli­minary tests. to be nontoxic to normal lymphocytes, In some tests 50 and 200 Jlg/ml were also used, Control tests consisted of (i) saline instead of extract (unstimu­lated tests), and (ii) tests with nerve or lung extracts (tissue controls)

RESULTS The results are summarIZed In Figure 1 There was no significant difference between % LAI in tests from pa-

376

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Fig, 1 - Percentage teukocyte adherence mhibltlOn m re ­sponse to human tacrrmat gtand. orbital lal. and eye muscle extracts in patients with Graves' ophthalmopathy and normat subjects, Horizontat Imes represent mean values, The upper limit of normat (not shown) = mean + 2SD for each prepara­tion Percentage LAI above these were considered posJllve Concentrations of extracts used were 50. 1 DO. and ?o.o. pg m l. Maximum values onty are shown

tients and normal subjects for any preparation tested (Mann-Whitney tests, NS) The mean percentage LAI for normal subjects was + 0,23 (± 1,1 SE) for lacrimal extract, -0.11 (± 1,3 SE) for orbital muscle extract and + 0,62 (± 1.27 SE) for orbital fat extract. The upper limit of normaJ (mean + 2S0) was taken as 7,6 for lacrimal. 9,5 for eye muscle, and 92 for fat. respectively. and % LAI greater than these considered significant. Signifi­cant LAI (+ 39%) in response to eye muscle was dem­onstrated in only one patient. although borderline lev­els in tests with eye muscle and fat, respectively. were found in two other patients All three patients had active disease which was of recent onset in two, There was no close correlation between % LAI and the severity ac­tivity, or duration of the eye disease for any preparation Similarly. there was no relationship between prevIous treatment with cyclophosphamide and percentage LAI for any preparation

DISCUSSION In this study we have failed to convinCingly demon-

strate significant LAI to lacrimal or orbital extracts in patients with Graves' ophthalmopathy, Tests were, however, positive to eye muscle extract in one patient with active disease of recent onset whilst borderline re­sponses to eye muscle and fat respetively were dem­onstrated in 2 other patients with active disease, sug­gesting that tests may be positive for a short time during the active phase of the disease, There is now considerable evidence that leukocyte ad­herence inhibition represents a specific immunological reaction, Significant LAI has been demonstrated in re e

sponse to fungi in patients with chronic dermatophytic infections (17), tumor antigens in patients with cancer (10, 16, 18), and testis extracts in patients with leprom­atous leprosy (20), The mechanism of the reaction is, however, unclear, Although some workers consider that LAI is a test for cell-mediated immunity (10, 11), others have shown evidence that the test measures cytophilic antibodies attached to the surface of mono­cytes (12, 13) It is likely that ophthalmopathy and hyperthyroidism are separate autoimmune disorders and that thyroid­stimulating antibodies, whether measured as LATS, LATS-protector (LA TS-P) or TSH-displacing activity play no role in the pathogenesis of the eye disease (2,3, 21, 22), Although it is likely that Graves' ophthalmo­pathy is an autoimmune disease, the evidence for this is not so strong as for Graves' hyperthyroidism, Whilst macrophage migration inhibitory factor (MIF) produc­tion in response to orbital antigens has been demon­strated by several workers (4-7), PBLtransformation in response to these antigens has not been found, al­though significant transformation in response to la­crimal gland extracts has recently been demonstrated in one third of patients With Graves' ophthalmopathy (23), Serum orbital antibodies have not been detected although an IgG from the serum of patients with Graves' ophthalmopathy produced eye disease exper­imentally in one study (24) Fakhri and Hobbs (25) have shown evidence that a specific IgG and normal (un­sensitized) lymphocytes may cooperate to produce ophthalmopathy, Killer cell activity against orbital mus­cle cells has not been reported, AlthoughTSH subfrag­ments have been found to cause experimental exoph­thalmos (26), this cannot explain the development of eye disease inassociation with hyperthyroidism, where TSH is suppressed, Kriss and colleagues (27) have shown that eye muscle has an affinity for thyroglobulin and thyroglobulin­antithyroglobulln complexes, which they claim, have been transported via communicating lymphatics from the thyroid Thyroglobulin has even been found in nor­mal eye muscle (7), Moreover, these workers have re­cently shown evidence for specific cytotoxicity against a thyroglobulin-eye muscle complex In patients with eye disease and associated hyperthyrOidism (28) Whilst such reaclions cannot explain the development of eye disease in the absence of thyroid autoimmunity,

377

Leukocyte adherence inhibition in ophthalmopathy

it is likely that investigation of the mechanisms for the association of Graves' hyperthyroidism and ophthal­mopathy will be rewarding, A prerequisite for such stu­dies would appear to be the identification and charac­terization of the orbital autoantigens, partly achieved by Mullin and colleagues (7), We have recently demonstrated that thyroid hormones influence lymphocyte function (29), One possible cause of the development of eye disease in associa­tion with hyperthyroidism, therefore, is an effect of T 4

on suppressor cells leading to the appearance of a se­cond autoimmune disorder, We are currently testing this hypothesis in hyperthyroid, hypothyroid and euthy­roid guinea pigs immunized with orbital antigens,

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Symposium on Autoimmune Aspects of EndOCrine Disorders. Academic Press. New York. In press