|Year : 1985 | Volume
| Issue : 3 | Page : 145-149
Ocular immunity in uveitis : A study of cellular response
Madan Mohan, RV Azad, RK Batta, KS Ratnakar
Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India
Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Mohan M, Azad R V, Batta R K, Ratnakar K S. Ocular immunity in uveitis : A study of cellular response. Indian J Ophthalmol 1985;33:145-9
Of the various techniques for the evaluation of cell mediated immunity (CMI) in clinical and experimental situation, leukocyte migration test has been found to be a good correlate of the delayed hypersensitivity. The capillary tube migration systems has been described by George and Vaughan and since then this test has been carried out in many occular disorders and antigen induced inhibition has been observed in chronic keratrtis uveits after intraocular surgery Sjogren disease; chorioretinitis sympathetic ophthalmitis and Harada's disease senile macular degeneration, Bests Vitelliform degeneration and optic neuropathy using corneal, uveal and retinal antigen. Findings of these tests suggest that the CMI may be helpful in detecting cellular hypersensitivity in ocular disease but their diagnostic and prognostic value is yet to be proved. The present study attempts at under-standing of cell mediated response in different types of uveitis and also uveitis following surgical intervention using leukocyte migration test.
| Materials and methods|| |
Antigen preparation: Human eyes were obtained from the eye bank service, were stored in a container at -140°C from 4 to 16 weeks. Uvea and retina were dissected out separately after bringing down the temperature to 37° C. Uvea was minced, under sterile conditions in phosphate buffered saline at pH 8.00. It was homogenised at the speed of 2000 rpm and the protein concentration of supernatant was adjusted to 4-6 mg/100cc. The antigen thus prepared was stored at 140° C for use.
Standardisation of antigen was done by viability test and cell migration test. An optimal concentration of 0.15 mg% was obtained and used throughout the study. Patients in the study were taken from uvea clinic of this Centre and were divided into following groups
i) Controls having no ocular or systemic disease 10 cases
a) Immediate uveal reaction 5 cases
b) Extra capsular 5 cases
iii) Acute uveitis 10 cases
iv) Chronic uveitis 10 cases
| Methods|| |
Modified method of George and Vaughan was employed. About 12 to 15 ml of blood from cubital vein of patients was drawn and was defibrinated in a conical flask containing 4 to 5 glass beads. To this defibrinated blood equal amount of gelatin 2% (freshly prepared) was mixed thoroughly by inverting the tubes 3-4 times. This was allowed to sediment for 30 to 45 minutes. The supernatant rich in leukocytes with few R. B. Cs and plasma consitituents was transferred to another test tube. This was centrifuged for 5 minutes at a speed of 1200 rpm. The supernatant was discarded and the leukocyte rich button was mixed with Hanks Basal salt solution thoroughly and centrifuged at 1200 rpm for 5 minutes. The supernatant was again discarded and the cells were twice washed in Hanks solution and centrifuged to obtain a R. B. C. free washed leukocytes. The supernatant of the last centrifuge was discarded off as usual and the leukocyte rich pellet was mixed with minimum essential medium (MEM) 10°x, (with penicillin streptomycin) and 5% fetal calf serum. The total WBC count was done to adjust the final concentration of cells upto 31-40 X 10 -5/cumin. These cells enriched with MEM were sucked in capillary tubes (German Hawksely No. -184). The capillary tubes were sealed at one end with plasticene and again centrifuged at 1500 rpm for 6 minutes. The capillaaries were cut at cell fluid interphase. The cut end of capillaries containing leukocytes was ploced into the perspex sheet chamber (LMI chamber). The chamber was sealed from both sides with paraffin wax and through the side opening, MEM and 5% fetal calf serum with antigen were syringed inside the chamber. The chamber which contained antigen was marked `A'. These chambers were kept in incubator at room temperature (at 37°C) for 20 hours. After 20 hours the chambers were taken out and the migration fans were outlined on an inch graph paper by projection microscope and were photographed [Figure - 1]. Area of migration in chambers `A', i.e., containing antigen and area of migration in chamber 'S', i. e., without antigen was calculated (by inch graph paper method) and migration index, percentage migration and percentage inhibition was determined,
In each group mean percentage migration mean migration index and mean percentage inhibition were statistically evaluated for `p' value, mean standard deviation and also relationship between various groups was found out.
| Observations|| |
The migration index in cases of control having no ocular or systemic disease was 0.81 + 0.08 [Table - 1]. The results in the iimmediate uveal reaction and extracapular subgroups are shown in [Table 2][Table 3]. The mean migration index in immediate uveal reaction following intracapsular lens extraction was 0.73±0.23 while it was 0.53+0.25 after extracapsular lens extraction. There was no statistical difference in the mean migration index of intracapsular lens extraction, i.e., 0.75 ± 0.25 when compared to that of control 0.81 ± 0.08. When the mean migration index of extracapsular group, i. e., 0.53 ±0.25 were compared to that of control of 0.81 ± 0.08 the difference in the results were statistically significant (P/0.01). The results obtained in acute uveities group are as shown in the Table. The mean migration index in this group was 0.59 +0.13 and when these values were compared with the control migration index the difference was statistically highly significant (P<0.001).
In chronic uveitis a mean migration index of 0.43 ± 0.19 was obtained as shown in the [Table - 1] and this was highly significant when the results of control were compared (P<0.001).
No statistically significant difference was observed when mean migration indices of chronic uveitis vs. acute uveitis, immediate postoperative uveitis following intracapsular lens extraction vs. postoperative extracapsular lens extraction were compared.
| Discussions|| |
In the present study the postoperative group had two types of uveitis. Immediate uveal reaction occurred in those patients who had undergone uneventful intracapsular lens extraction and had very mild type of iritis and LMI was done on 1st, 2nd and 3rd postoperative days and was merely due to operative intervention. The second type of uveitis followed extracapsular lens extraction and in some cases it manifested in severe form. LMI in these cases was done on the day of appearance of even minimal uveal reaction unto full blow picture of uveitis in these cases.
The findings in immediate postoperative uveal reaction group (mean migration index 0.73+0.23) using uveal antigen shown no significant inhibition when compared to that of control (mean migration index 0.81±0.08) Hence this study indicates that no cell mediated response to uveal antigen exists in such cases. This confirms the finding of Henley et al (1974). In postoperative extracapsular lens extraction group (mean migration index 0.53:1 0.25) a statistically significant (PL0.01) difference was found when compared to the mean migration index of control. The significant inhibition in extracapsular group does not give much lead to arrive at a definite conclusion owing to the following facts
i) Mild uveitis in this group may have been caused by mere operative intervention.
ii) Not all the types of uveitis following extracapsular lens extraction are mediated by cellular mechanism.
iii) Even those mediated by cellular mechanism lack substantial proof in support of their being cell mediated. For this, separate work is required to know the immunological status of uveitis following extracapsular extraction which would include phacotoxic and phacoanaphylactic uveitis.
In the two groups of pidiopathic uveitis (acute uveitis MMI 0.59±0.13; chronic uveitis MMI 0.43 ±0.19) statistically highly significant inhibition was noted (P < 0.001). However, no correlation of migration index with the duration of the disease could be found in these cases which indicates that duration of the disease has no bearing on the induction of CMI. Hence no statistical difference in the result between these two groups was observed. Occurrence of delayed reaction in idiopathic acute uveitis can be explained because of failure of humoral mechanism to overcome the offending bacteria. It can also occur in those acute cases where the antigen is being released slowly. Causing tissue damages. The antigen thus acts in the way of depot and reactivate lymphocyte. It is believed that recurrent and chronic "1166of obscure aetiology causes delayed hypersensitivity by memory cells. The memory celiare retained in the uvea, after an initial episode of uveitis and result in reactivation on further exposure to the original antigen. These memory cells are sensitised lymphocy of T type which are the mediators of cellular response. The induction of cell mediated immunity occurs after initial episode of inflammation either in uvea or anywhere in eye caused by various antigenic stimulus like Bacterial, viral toxicins etc. Antigenecity of uvea being low it is believed that altered inflammed uvea and concurrent infection form stronger antigenic stimulus to perpetuate the uveal inflammatory process in the form of recurrent uveitis.
Occurrence of delayed immunity in indiopathic uveitis as evident from this work has bearing on its therapy. Many of recurrent uveitis cases are recalcitrant to treatment and one has to look for, other modes of therapy than corticosteroid. Though steroid has immunosuppressive effect cytopathic effect on B & T lymphocyte and inhibitory effect on Prostaglandin and anybody synthesis, yet it's therapeutic effect is far from being satisfactory. Folic Acid and purine analogues act by compectetive inhibition and suppress lymphocyte proliferation. They have been tried in recurrent uveitis especially in Bechets desease but not with much success. Anti lymphocyte serum containing IgG is most effective against T & B lymphocytes by the process of immune adherence and phagocytosis. At present clinical role of ALS is not fully established. We have tried ALS in experimental corneal heterograft and achieved success Mohan et al 1978. However, its role in recurrent uveitis remains speculative.
More over prolong use of ALS has side effects due to difficult procurement of pure ALS IgG from the serum.
| References|| |
George M and Vaughan J. M., 1972, Proc. Soc. Exp. Biol, 3 : 574.
Shore, B., Leopold, I., and Henley, W. L., 1972, Amer J. Ophthalmol., 73 :63.
Henley, W. L., Okas S. and Leopold H. I, 1972 Amer. J. Ophthalmol 73 : 67.
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Berry, H., Bacon, P. A. and Davis, J. D. Ann. Rheum. Dis., 31 :298.
Leopold, I. H., 1973, Amer. J. Ophthalmol 76: 619.
Hammer, H., 1974, Brit. J. Ophthalmol 58 773.
Henley, W. L., Okas, S. and Leopold.. H. 1. 1974, Anger J. Ophthalmol 77: 59,
[Figure - 1]
[Table - 1]