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| ARTICLES |
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| Year : 1972 | Volume
: 20
| Issue : 2 | Page : 84-87 |
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Corneal immune reactions
BR Shukla, NC Gupta, OP Ahuja
Aligarh Muslim University Institute of Ophthalmology, Aligarh, India
Correspondence Address:
B R Shukla
Aligarh Muslim University Institute of Ophthalmology, Aligarh
India
 Source of Support: None, Conflict of Interest: None  | Check |
PMID: 4668482 
How to cite this article:
Shukla B R, Gupta N C, Ahuja O P. Corneal immune reactions. Indian J Ophthalmol 1972;20:84-7 |
In the science of keratoplasty, immunological rejection of the grafted cornea is a well recognised fact. To enhance the chance of a successful corneal transplant, attempts are being made to suppress this phenomenon. Several procedures like irradiation (Sterzl Marchioro and Woddel [8] ), Corticosteriod therapy (Leibowitz and Elloitt [2] , Smolin and Keates [7] and Olson [3] ) and certain immuno suppressive drugs have been evaluated in this respect. In the present study, attempts were made to investigate the effect of Purinethol,* on experimentally produced corneal hypersensitivity reactions. The drug is a known nucleic acid anti-metabolite and a suppressive of haemopoietic system.
| Materials and Methods | |  |
The present study was undertaken on 24 albino rabbits of either sex and of average weight of 3 pounds (varying between 2.5 to 4 pounds). They were labelled 1 through 24. These rabbits were anaesthetised with 2% sodium seconal injected into the marginal vein of pinna. It was supplemented with topical instillation of 1 % anaethaine and 1 cc of 2% novocain retrobuibarly.
Each eye was proptosed and fixed with fixation forceps. 0.06 cc of sterile bovine serum albumin solution (100 mg./cc) was injected intralamellarly at the centre of the cornea with a 1 cc tuberculin syringe fitted with a 27 gauge needle. The injection was repeated on the 20th day.
The animals were then divided into three groups for observation
Group I :- This consisted of rabbits 1 through 9. No post inoculation medication was given. They were carefully examined daily by oblique illumination and biomicroscopy. Blood was withdrawn by intracardiac puncture on 5th, 14th, 22nd and 30th day after inoculation for estimation of antibody titre and total white cell count. Double agar diffusion technique was employed for antibody estimation.
One eye at similar intervals was enuclated under general anaesthesia. The histological examination was made of serial sections stained with haematoxyline - eosine and a methyl green-pyronine stain.
Group II :- This consisted of rabbits 10 through 18. They were given a daily intramuscular injection of purinethol (2 mg./lb body weight) for 20 days starting from the day of inoculation. Observations, similar to group I were made but in addition a daily record of weight was kept for any evidence of loss of weight. The animals were watched for any diarrhoea or loss of appetite.
Group III:- Consisted of rabbits 19 through 24. A biweekly subconjunctival injection of 0.25 cc of 5% purinethol solution was injected in both eyes of each animal. The medication was instituted on the day of inoculation and continued for 30 days. This group was studied on the lines laid down for groups I and II.
| Results | | |
Group 1 (Control)
Within 4-7 days following the inoculation, there appeared corneal opacification circum limbal congestion and corneal vascularization These signs persisted for about a week and then all the signs disappeared except for a mild injection opacity in the centre of the cornea. The eyes remained so upto the 20th. day of inocuatlon when a second inoculation was made. 24 hours after the second injection there appeared circum corneal congestion, corneal vascularization and a white ring of opacity in the cornea around the injection site.
Histological examination showed a mononuclear infiltration of limbus during the first phase of reaction but in the second phase there were also present many plasma cells - mature and immature.
The precipitin reactions for circulating antibodies gave uniformly negative results in all the samples of blood when tested by double agar diffusion technique.
Group II (I/M Purinethol)
Only 7 out of 19 eyes developed the first reaction 4-7 days after inoculation. Only a mild central opacity or ciliary congestion was present in the other 11.
None of the 11 eyes reinoculated on 20th day developed any reaction in the form of corneal vascularisation or white immune ring around injection site. Only two eyes showed some limbal vascular dilatation.
Histological examination confirmed the suppression of clinical response as only a few mononuclear cells with insignificant number of plasma cells were seen in the limbal infiltrates.
Immunological tests gave negative results like the control group
Group III (SIC Purinethol)
There was no effect of therapy in this group. Clinical, histological and immunological results were similar to those in control group.
Drug Toxicity :
No toxic side effects of the drug were seen when given by subconjunctival route. However on systemic therapy, there was a significant leucopenia and diarrhoea in 3 of the animals. One of them died on 14th day of therapy.
| Discussion | | |
The first reaction following inoculation developed after 4-7 days. This was characterised by corneal opacification, circum corneal congestion and a lymphocytic infiltratration of limbus. The picture was suggestive of delayed hypersensitivity reaction. No circulating antibodies were demonstrable then. 24 hours following the second inoculation, there developed the second reaction characterised by circum corneal congestion, a white opaque ring around the injection site and a mononuclear limbal infiltration with many plasma cells. This was against the observations of Germuth [1] who observed this reaction on the 14th day following a single inoculation. This was the immediate type of hypersensitivity reaction. As we failed to get the reaction following a single inoculation, and could get a similar reaction only after a second inoculation it is suggested that although a reactive level of circulating antibodies was reached, the antigen depot in the cornea had exhausted. This antigen was then provided by a second injection. This early exhaustion of antigen is difficult to explain. It may be that the breed or strain of animals that we employed was different from those used by other workers. The limbal infiltrate of delayed hypersensitivity reaction consisted of mononuclear cells, predominantly the lymphocytes. During the immediate response, large number of mature and immature plasma cells were also seen among the mononuclear cells. The appearance of plasma cells at this stage is suggestive of their role in the formation of antibodies locally or extra ocularly. Though the antigenantibody reaction was observed clinically and confirmed histologically yet in our study no circulating antibodies could be demonstrated. As the standard double agar diffusion technique using the standard material was employed, the negative immunological tests only suggest that although a reactive level of antibodies was present in the serum, the titre was probably not high enough to be demonstrated on agar plates.
Intramuscular purinethol therapy markedly suppressed both the types of hypersensitivity reactions. Only 7 eyes out of 18 eyes developed typical delayed type of reaction. Immediate reaction developed in none of those 11 eyes which were reinjected on 20th day.
Purinethol, a synthetic purine analogue interferes with nucleic acid biosynthesis. It suppresses the formation of lymphocytes and thereby of antibodies. It is interesting to note that although systemic purinethol suppressed immediate hypersensitivity reaction in almost all the 11 eyes leucopenia was observed in only three animals. This suggests that the immuno-suppressive effect of the drug is not directly related to its lymphocyte suppressing effect on the haemopoietic system. It may however be possible that the drug inhibits antibody formation even in the presence of normal lymphocytic count probably by inhibiting the conversion of lymphocytes into antibody forming plasma cells.
Suppression of delayed hypersensitivity by systemic purinethol suggests that it has some inhibitory effect on the sensitization of tissues. Thus the drug has both central and peripheral action.
Subconjunctival route failed to suppress the hypersensitivity reaction. It is understandable that this route, which allows a comparatively small amount of the agent to be introduced, does not produce effective levels of drug in general circulation. As very little antibody if at all is formed locally, this route of medication did not suppress the reactions.
Systemic therapy is toxic as it produced diarrhoea and leucopenia in some of the animals.
| Summary | | |
Both eyes of all the 24 animals were inoculated by an intra corneal injection of B.S.A. The injection was repeated on 20th. day.
The control group which received no medication showed a delayed hypersensitivity reaction 4-7 days after first injection and an immediate hypersensitivity reaction 24 hours after second inoculation.
Among the treated groups, intra muscular purinethol therapy markedly suppressed both the delayed and immediate types of hypersensitivity. The sub conjunctival route of purinethol therapy did not have a significant effect.
Systemic purinethol therapy produced toxic side effects like diarrohoea and leucopenia but the subconjunctival route was non-toxic.
| References | | |
| 1. | Germuth. F.G. et al (1962) J. Expt. Med.; 115, 919.  |
| 2. | Leibowitz, H.M. Elliott, J.H. (1965). Arch. Ophth.; 74, 835. |
| 3. | Olson, C.L. (1966) Arch Ophthal; 75, 651. |
| 4. | Parks, J.J.: Leibowitz, H.M. and Maumenee. A.E. (1962) J. Exp. Med.: 113, 867. |
| 5. | Schwartz, R.; Stack, J. and Dameshek, W. (1958); Proc. Soc. Exper. Biol. and Med.: 99, 164. |
| 6. | Sellyei, L.F.; and Ellis P.P. (1966) Amen. J. Ophth.; 61, 702. |
| 7. | Smolin, G,; and Kaetes. R. H. (1967): Am. J. Ophth., 63, 335. |
| 8. | Sterzl, T.E. Marchiors, T.L.; and Woddell, W.R. (1963) Surg. Gynae. and Obstet 117, 385. |
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