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Year : 1982  |  Volume : 30  |  Issue : 1  |  Page : 29-35

T-Lymphocyte mediated response in conjunctiva and cornea (an experimental study)

Institute of Ophthalmology, A.M.U., Aligarh, India

Correspondence Address:
R Gogi
Institute of Ophthalmology, A.M.U., Aligarh
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Source of Support: None, Conflict of Interest: None

PMID: 6982853

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How to cite this article:
Gogi R, Kulkarni P, Nath K. T-Lymphocyte mediated response in conjunctiva and cornea (an experimental study). Indian J Ophthalmol 1982;30:29-35

How to cite this URL:
Gogi R, Kulkarni P, Nath K. T-Lymphocyte mediated response in conjunctiva and cornea (an experimental study). Indian J Ophthalmol [serial online] 1982 [cited 2021 Jun 18];30:29-35. Available from: https://www.ijo.in/text.asp?1982/30/1/29/27913

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Table 1

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Table 1

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Delayed hypersensitivity reactions are involved in the eye in a numb--r of circumstance such as corneal graft rejections, viral keratitis helminth infestation of the retina, fungal infection of the choroid and conjunctivitis provoked by sensitization to cosmetic eye pre­parations[1]. Apart from this, delayed hyper­sensitivity reactions in ocular structures are found in various inflammatory processes dur­ing bacterial[2], mycotic[3] and viral infestations[4]. The experimental model of delayed hypersen­sitivity in ocular structures has been demon­strated repeated by using tuberculoproteins as antigens[5],[6],[7],[8],[9],[10].

One basic problem with tuberculoproteins is that besides eliciting a T-cell response they also stimulate B-lymphocyte population[11]. Therefore the emergent picture is not necessa­rily one of a pure T-cell mediated cellular immunity but rather results in a pleomorphic histologic appearances.

In the present study a pure T-cell response in the conjunctiva and cornea of rabbits shall be studied with the help of 2,4-dinitrochloro­benzane (DNCB). This chemical acts as hapten. It forms covalent bonds with lysin group of epithelium[12],[13], has high sensitizing properties and can produce a pure delayed hypersensitivity reaction by stimulating the T-cells exclusively[14]

  Material and methods Top

The study was conducted on 18 male rabbits. Their average weight was 500 gms (range 450 to 550 gms). D.N.C B. was used as hapten. 2% solution (Solution A) and 0.2% (Solution B) of DNCB was prepared in acetone and were used for sensitization and challenge respectively. Haptic contact lenses with a central fenestration (2 to 5 mm in dia­meter) were fabricated after taking a mould. The rabbits were divided into group A (10 rabbits) and group B (8 rabbits).

Group A

Sensitization :
Ten microliters of solution A was applied on the outer and inner surface of the contact lens and acetone was allowed to evaporate. The lens was fitted on anaesthe­tized eye and retained for 24 hours and remo­ved thereafter.

Challenge : The animals were challenged after 14-18 days of sensitization with ten microliters of solution B with the help of contact lens. The lenses were retained for 2 to 24 hours depending on the experimental proce­dure and removed thereafter.

The rabbits were observed for any swelling and hyperaemia of lids, amount and nature of discharge, conjunctival congestion and chemosis, corneal haze, neovascularization and for any evidence of ulceration following sensitization and challenge. Antibiotics drops were instilled to ward off any secondary infec­tion.

Enucleations were carried out 2, 6, 18, 24, 48 72 and 96 hours after the challenge dose. Cornea, upper and lower palpebral and bulbar conjunctiva were subjected to histopathological study. 3 to 6 micron sections were stained by haematoxylin and eosin and with Mallory's phosphotungstic acid haematoxylin and Lendrum's acid picro-Mallory stain to study the distribution of fibrin.

Group B

This group served as control for group A and was divided into 4 subgroups.

Subgroup 131 : 0.2 mg DNCB was applied in the virgin eyes of 2 rabbits in order to study the direct phlogistic impact of sensitizing dose. Eyes were enucleated after 24 hours.

Subgroup B2 : 0.02 mg DNCB was applied and biopsied after 24 two rabbits.

Subgroup B3 : Two rabbits were sensiti­zed as in group A and biopsies were taken on 14th day to study the preexisting histological changes before the challenge dose.

Subgroup B4 : In two rabbits, contact lens painted with acetone only was fitted and tissues were biopsied after 24 hours.

  Observations Top

Group A : (1) Macroscopic changes

A) After sensitization :
The grading of various clinical findings are summarised in Table 1. An arbitrary index was designed denoting the sum total of the grades of the various clinical signs. It was 13 +, 24 hours after the sensitization and declined rapidly thereafter and was 1 +, after one week.

B) After Challenge : The animals res­ponded rapidly and more intensely to the challenge dose and grading of the various clinical signs is summarised in Table II. The translucency of cornea was maximum (+++) between 18 and 72 hours [Figure - 1]. Peripheral blood vessels were also markedly congested between 18 and 48 hours and the cornea showed superficial and deep neovascularization [Figure - 1]. The inflammatory response started appearing 2 hours (4+) increased in severity upto 24 hours (21+) and started declining thereafter as measured by inflammatory index.

(2) Microscopic changes

Vasodilation in the conjunctiva was noted after 2 hours and in 6 to 18 hours, the lumen of the blood vessels showed stuffing by lym­phocytes, polymorphs and. eosinophils [Figure - 2]. Endothelial proliferation at certain places was noted by 48 to 72 hours and a fair degree of vascular changes were noted even after 96 hours. The stroma was oedematosus and fibrin was present in maximum amount bet­ween 24 and 48 hours after the callenge. The cellular infiltrates were chiefly neutrophils and a few lymphocytes after 2 hours and by 6 hours a considerable number of eosinophils were also present and maximum infiltration was noted after 24 and 48 hours [Figure - 3]. In 48 to 72 hours, few epithelioid cells were observed without any giant cell reaction [Figure - 4] Cellular infiltration started decreasing after 48 hours. All these changes were more prominent in the bulbar conjunctiva as com­pared to that of palpebral.

Cornea also shared the immunological response. It was more intense at the limbus and the peripheral part of cornea. Vascular changes were present in 2 and 6 hour speci­mens. Corneal stroma was oedematosus result­ing in the lack of compactness of the corneal lamellae. This was associated with fair amount of fibrin content. There was evidence of neovascularization in superficial and midstromal by 24 to 48 hours after the challenge [Figure - 5]. Cellular infiltration by lymphocytes, neutrophils, eosinophils, and basophils was the highlight of the microscopic pathology. Epitheloid cells or giant cells reaction could not be seen in any of the cases. These inflam­matory cells started appearing 2 hours after the challenge and could be seen in a fair num­ber after 96 hours and the maximum number of cells were present in the superficial stroma. Corneal epithelium was oedematosus with the formation of micro bullae. There was also a variable amount of epithelial necrosis.

An arbitrary inflammatory index was devised. It shows that changes were detectable even at 2 hour intervals and it gradually increased being highest between 24 and 48 hours after challenge and thereafter it showed a steady decline [Figure - 6].

Group B (Control group)

In the subgroup B I a response similar to that obtained after sensitization in group A was seen. In subgroup B2 eyes showed a mild grade of inflammation as manifested by mild conjunctival congestion (-I-) and slight peripheral corneal haze (+). In subgroup B3, there was no inflammatory reaction except the presence of mild peripheral corneal haze (+) in 2 eyes. In subgroup B4, there was no evidence of inflammation. Histo­pathology did not show any evidence of immunological response except in group B3, where the residual changes 14 days after the sensitization consisted of isolated foci of lymphocytes. Phlogistic effect of DNCB (subgroup B1 and B2) excited only a polymor­phonuclear response and epithelial oedema.

  Discussion Top

During the process of sensitization of skin by dinitrochlorobenzene some of the protein bound hapten is picked up by the Langerhan's cells which are present in the basal layer of the epidermis of skin[15]. These cells are known to carry the antigens to the regional lymph follicles where sensitization takes place[16]. Similar types of Langerhan's cells have been demonstrated in the human cornea[17] and limbal conjunctiva[18]. We feel that a similar mechanism is operative in rabbits where pro­tein bound hapten is carried by these cells to the corresponding lymph follicles which are located in the fornix and nictitating mem­brane in rabbits[19]. DNCB induced immuno­logical reaction has been demonstrated by us for the first time in the rabbit conjunctiva and cornea. The reaction is set in gear when sensitized lymphocytes come in contact with protein bound DNCB at the time of challenge.

Sensitization with 0.2 mg DNCB brings inflammatory response. However with lower amount (0.02 mg) inflammatory changes were short lived and it failed to evoke a true inflammatory response. The sensitized animals of group A when challenged responded rapidly and vigorously and it appeared as early as 2 hours and increasing in intensity rapidly. It was present even at the end of 96 hours. In the conjunctiva there was generalised hypera­emia with chemosis whereas neovasculariza­tion was well marked in the cornea. It is now accepted that delayed hypersensitivity reaction do not occur in the cornea in the absence of neovascularization[20]

The change at the microscopic level was vasodilation and variable degree of endothelial proliferation. These vascular changes were associated with perivascular infiltration which was predominantly lymphocytic. There was a variable amount of fibrin in the tissue. The presence of lymphocyte at the reaction site is a specific indication of delayed hypersensi­tivity reactions. It was earlier considered that majority of the lymphocyte present at the site of reaction were sensitized lymphocytes. Wilson[21] showed that 1-2% of such lymphocytes are immunologically active. Such lymphocytes on coming in contact with antigen liberate biologically active substances known as lym­phokines[22]. The lymphokines can damage the tissue at the site and tissue some distance away from the active site[23]

The delayed hypersensitivity response in the cornea and conjunctiva can best be seen in phlyctenular keratoconjunctivitis where the lesion is dominated by the mononuclear cells alongwith the polymorphs[24]. In the corneal allografts in the human the failure to take up graft is due to the lack of histocompatibi­lity. The graft is technically successful and clear during the first three weeks. This is the period when the sensitization is taking place. The moment these sensitized T-lymphocytes appear at the site of allograft an immunologi­cal reaction is generated resulting in the for­mation of an opaque line at the site of the junction of the donor's and recipient's cornea. This line represents the interface between the rejected endothelium and the advancing infla­mmatory infiltrates[25]. Such rejections are more common if the cornea is vascular prior to surgery because it provides an easy approach for the sensitized lymphocytes to reach the site of allograft[26],[27]

The term `delayed hypersensitivity' is a misnomer. The classic time course for skin is 48 to 72 hours whereas in conjunctiva and cornea it appears as early as 2 hours in the present study. This class of immunologic reactions are not always dependent upon the T-lymphocytes cells as an effector but is largely mediated by its soluble products known as lymphokines. Hence it is not really cellular hypersensitivity. The better name for this group of reactions could simply be Type IV hypersensitivity reactions. In the present study a pure T-lymphocyte response was pro­duced. The present model can act as a base­line for testing efficacy of different therapeutic agents such as immuno-suppressants.

  Summary Top

Dinitrochlorobenzene can be used to elicit a pure T-lymphocyte mediated hypersensitivity response in the cornea and conjunctiva of rabbit. Various clinical and histopathological changes have been detailed. Microscopic changes so produced correspond with the histological changes associated with "type IV hypersensitivity reactions", elsewhere in many ways.

  References Top

Rahi, A.H.S. and Garner, A.. 1976, In Immuno. pathology of the Eye P. 90. Blackwell, Oxford, pp. 90.  Back to cited text no. 1
Spink, W.W., 1956 Biucellosis. P. 131. University of Minnesota Press, Minneapolis.  Back to cited text no. 2
Salvin, S.B., 1963 : Progr. Allergy, 7: 213.  Back to cited text no. 3
Turk, J.L., Allison, A.C. and Oxman, M.N., 1962, Lancet. I : 405.  Back to cited text no. 4
Wolff-Eisner, A., 1909, Munch. Med. Wschr. 44 : 2266.  Back to cited text no. 5
Silverstein, A.M. and Zimmerman, L.E., 1959 Amer. J. Ophthalmol 48 : 435.  Back to cited text no. 6
Leikowitz, S. and Waksman, B.H., 1960 : J. Immunol. 84: 58.  Back to cited text no. 7
Salvin, S.B, and Gregg, M.B.. 1961 : Proc. Soc. Exp. Biol. Med. 107 : 478.  Back to cited text no. 8
Parks, J.J., Leibowitz. H.M.I. and Maumenee. A.E, 1962: J. Exp. Med. 115: 867.  Back to cited text no. 9
Elliott, J.H , Flax, M.H. & Letbowitz, H.M.I., 1966: Arch. Ophthalmol 76 : 104.  Back to cited text no. 10
Schwartz, H.J. and Zimmerman, T.S., 1971 J. Immunol. 106: 450.  Back to cited text no. 11
Eisen, H.N.. Orris]. and Belman, S., 1952 J. Exp. Med. 95, 473.  Back to cited text no. 12
Eisen, H.N., and Tabachnick, M., 1958: J. Exp. Med. 108 : 773.  Back to cited text no. 13
Waksman, B.H , 1960: J. Allergy. 31 :468.  Back to cited text no. 14
Maximov, A.A. and Bloom, W., 1954 : In `A Text book of Histology' Saunders, Philadelphia. pp. 315.  Back to cited text no. 15
Frey, J.R. and Wenk, P., 1957 : Int. Arch. Allergy. 11 :81.  Back to cited text no. 16
Henkind, P., 1965 : Eye Res. 4 : 42.  Back to cited text no. 17
Sugiura, S. and Matsuda, H., 1968 : Trans. Soc. Ophihalmol. Jap. 72 : 2435.  Back to cited text no. 18
Prince, J.H., 1964 : In `The rabbit in eye research' Thomas C. Charles, Springfield, Illinois (U.S.A.). p.p. 93.  Back to cited text no. 19
Schlossman, S. and Stetson, C.A., 1957 : J. Immunol. 79 :208.  Back to cited text no. 20
Wilson. D.B , 1965: J. Exp. Med. 122: 143.  Back to cited text no. 21
Dumonde, D.C., 1970 : Proc. ror. Soc. Med. 63 : 899.  Back to cited text no. 22
Johnson, R,L., Fink, C.W. and Ziff, M., 1972 J. Clin, Invest. 51 :2435.  Back to cited text no. 23
Duke Elder S., 1965 : In `System of Ophthal­mology' Vol. VIII Part I, Henry Kimpton, London. pp. 465.  Back to cited text no. 24
Khodadoust, A.A., 1973 : In Corneal Graft Failure Ciba Foundation Symposium 15, Excerpta Medica. Amsterdam. p.p. 151-164.  Back to cited text no. 25
Maumenee, A.E., 1962 . Invest. Ophthalmol. 1 :244.  Back to cited text no. 26
Polak, F.M., 1973 : In `Corneal Graft Failure' Ciba Foundation Symposium 15, Excerpta Medica, Amsterdam. P. 18.  Back to cited text no. 27


  [Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6]

  [Table - 1], [Table - 2]


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