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ARTICLE
Year : 1960  |  Volume : 8  |  Issue : 1  |  Page : 1-10

Immunological approach to etiology of cataract


King Edward Memorial Hospital, Bombay, India

Date of Web Publication6-May-2008

Correspondence Address:
S N Cooper
King Edward Memorial Hospital, Bombay
India
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Source of Support: None, Conflict of Interest: None


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How to cite this article:
Cooper S N, Padukone K V, Bhatia P R. Immunological approach to etiology of cataract. Indian J Ophthalmol 1960;8:1-10

How to cite this URL:
Cooper S N, Padukone K V, Bhatia P R. Immunological approach to etiology of cataract. Indian J Ophthalmol [serial online] 1960 [cited 2020 Jul 7];8:1-10. Available from: http://www.ijo.in/text.asp?1960/8/1/1/40689

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The paper to-day is a continuation of the work which I presented before this Society while giving the Aden­walla oration in 1957. By six experi­ments we are showing how we have drifted into an immunological ap­proach to etiology of cataract.

I shall start by representing the experiment (Experiment I) and the figure [Figure - 1] I had shown then, which is of the result of Ouchter­loney's technique applied for detection of lens proteins in the aqueous, vitre­ous and serum of rabbits. It shows clearly that in the aqueous and vitre­ous of rabbits there are present pro­teins which are immunologically simi­lar to lens-proteins but in the serum they are not to be found. Dr. Rao confirmed the same by showing elec­trophoretic similarity between the pro­teins of the aqueous and lens on one hand and of the vitreous and serum on the other. However electrophoretic­ally it was not possible to demonstrate the presence of traces of lens pro­teins in the vitreous which could be done by the agar-precipitin technique of Ouchterloney. The conclusion we (Rao, Kulkarni, Cooper and Radha­krishna-1954) had drawn was that lens proteins do not remain encaged within the lens capsule, as it is general­ly supposed, but they keep on escap­ing continuously in the aqueous and vitreous. At that time we thought that lens proteins do not reach the blood stream because they undergo degrada­tion by enzymes in the aqueous and vitreous, and so cannot be traced in the blood stream in an undegraded form.

As our original study was for pha­coanaphylaxis we had interested our­selves in the demonstration of lens antibodies in the serum. We were looking out for some quantitative method of determining lens-antibodies. We interested ourselves in the haemag­glutination technique, as described by Stavitzky (1954).


  Method Top


In this method sheep's red blood cells are ­treated with a 1 in 20,000 solution of tan­nic acid so that they get agglutinated in the presence of corresponding antigen and antibody.

Preparation of tannic acid R.B.C.'s: This was done in exactly the same way ass described by Stavitzky (1956).

Diluent. From horse serum which was pre­served at-1 ºC, with merthiolate as preser­vative, 2 ml. were inactivated in a water bath at 56ºC for half an hour and ab­sorbed with an equal amount of packed R.B.C. 's and kept at room temperature for halt an hour to remove all non-specific antibodies in the diluent. one ml. of re­absorbed horse serum was diluted 1 in 100 with normal saline which was used as the diluent.

Antigens. Under aseptic precautions and working under ultra-violet light, twenty five bovine lenses, removed from eyes of buffaloes which had been slaughtered four hours previously were crushed and stocked in lots of five in 25 ml. sterile normal saline -at-4ºC. Only those lots which were found .sterile two days later, as shown by sterility tests in glucose-broth, were made use of. The sterile lots were pooled and centrifug­ed at 7,000 r.p.m. for twenty minutes twice and the supernate was collected in a sterile bottle. After dialysing, estimations of the total solids, and total proteins were re­corded and the concentration of total solids was brought to 1 in 100 by diluting with sterile normal salin. The pH was then ad­justed to 6.8 and the preparation stored in aliquotes of 10 ml in sterilized bottles in the freezing chamber of a refrigerator. This was our stock solution of bovine­soluble lens antigen.

Similarly stock solutions of rabbits' lenses and human cataractous lenses were pre­pared and stored.

From these stock-solutions, preparations of alpha-and beta-crystallin fractions were then prepared. By adding in acetic acid to the solution of bovine-soluble lens anti­gen, the pH was adjusted to 4.8 with a pH meter, which being the iso-electric point of alpha--it precipitated. It was re­dissolved in saline and reprecipitated simi­larly three times and finally centrifuged to remove all acetic acid. A t in too solution in normal saline was prepared and stocked as above. After removal of the alpha-frac­tion, to the supernate was added in Na OH to bring the solution to pH 6 at which point beta-precipitates. This fraction was purified by repeating the process three times finally centrifuging and washing with distilled water. This was lyophilised and stored in sterile ampoules and used freshly in the required concentration, be­cause a solution of pur beta-crystallin pre­cipitates readily.

Sensitization of tannic-acid R.B.C.'s: To 8 ml. Of pH 6.4 buffered saline and 2 ml. of the required antigen were added 2 ml. Of tannic acid R.B.C., mixed and kept at room temperature for to minutes .and then centrifuged. After discarding the supernate, the cells were washed with 4 ml. of the horse serum diluent and resus­pended in 2 ml. of the same. Antigens thus prepared show agglutination when they come in contact with traces of their cor­responding antibodies.

Preparation of test-sera. All test sera after separation from the clotted blood were inactivated at 56º C for half an hour in a water bath. One ml. of this was absorbed - with an equal amount of washed packed R.B.C.'s (untreated by tannic acid) and kept at room temperature for half an hour, then centrifugd. Sera thus absorbed were ready for the test.

Tests were carried out in 10 x 100 test ­tubes. For each test 10 tubes were taken, in each of which was placed 0.5 ml, of the diluent. To the first tube was added 0.5 ml. of the test serum. After mixing, 0.5 ml. from this was removed with a pipette and put into the next tube and this was repeated upto the tenth tube discarding the last 0.5 ml, in the pipette.

Addition of the antigen. To the above tubes 0.5 ml. of the prepared antigen (described above) was added, the tubes were shaken gently then plugged and kept at room temperature. The tests were read after 6 and 12 hours. The intensity of the reaction was recorded as described by Stavitzky (1956).

Controls.

1. To each of three dilutions of the test serum were added 0.5 ml. of the un­treated tannic acid R.B.C. prepa­ration.

2. To 0.5 ml. of the diluent was added 0.5 ml. of sensitized tannic acid R.B.C. preparation.

Experiment 2. We tested the sera of normal (ages 15-29-64) and cata­ractous (ages 35-54.5-70) patients for this test. We were surprised to find the presence of anti-bodies to lens­proteins not only in cataractous but even in normal individuals [Table - 1] because Halbert et al (1957) were not able to find antibodies to lens in cata­ractous subjects. They had used the agar precipitin technic. They quote several references where anti-lens anti­bodies have not been recovered from cataractous patients although certain cataractous patients show dermal hy­persensitivity to lens proteins. We also found that the anti-lens antibodies were present in much larger quanti­ties in cataractous patients. Another interesting fact we found was, for the same sera the haemagglutination test was stronger with weaker antigen, to a limit, than with stronger.

Comparing the haemagglutination test with different dilutions of the same antigen we found that maximum haemagglutination took place with a in 10,000 dilution of the extract as shown in the table [Table - 1] whereas for in 1,000 dilution the reactions were weaker and for a in 1oo dilu­tion there were even weaker reactions. In cataractous subjects the tests were much more strongly positive.

A better idea can be had from the curves depicted by a graph of the test in normal and cataractous subjects with different dilutions of the serum and antigen. It distinctly shows what is called a zone phenomenon, that is the maximum reaction is not with the undiluted serum and antigen but with a critical dilution which in this inst­ance is in the neighbourhood of in 20 - 3o dilution of the serum and in 10,000 of our antigen [Figure - 2]. After the critical dilution, further dilutions cause a precipitous drop in the reac­tion capacity.

[Table - 2] shows the results of the haemagglutination test in unsensitized rabbits and those sensitized to the frac­tions of lens proteins.

It was thus possible for us to de­monstrate for the first time auto-lens-antibodies even in non-cataractous human beings and unsensitized rabbits.

The two facts we believe we have thus brought to light are (i) lens proteins can be found in normal aque­ous and vitreous but not in normal serum, (z) auto-lens antibodies can be found in normal serum.

This raises a bogey: if antibodies arc circulating in the blood of even normal individuals, every person then runs the risk of getting cataract be­cause the ultimate nutrition of the lens comes from blood serum, and if these antibodies to lens proteins can get in­side the lens capsule, an antigen anti­body precipitation may occur and that could be the beginning of a cataract­ous process.

So we took upon ourselves a study of production of cataract on immuno­logical lines.

Experiment 3 :
One eye of each of three normal rabbits was enucleated. The aqueous of these eyes was aspi­rated and pooled. The vitreous was also aspirated and pooled. The idea of enucleating the eye and then aspi­rating was to avoid any chance of puncturing a vessel of the choroid and contaminating the vitreous with serum, which incidentally contains antibodies.

To half ml. of the pooled aqueous and vitreous the h=agglutination test was applied with a i in 10,000 dilution of the antigen.

Result . No antibodies to lens-pro­teins could be demonstrated either in the aqueous or vitreous. The situation can thus be visualized by the following figure. [Figure - 3]

Thus, there appears to be a kind of barrier against the passage of anti­bodies into the aqueous-vitreous.


  Study of the Barrier Top


Experiment 4: To study this we took five groups of three rabbits each and made them immune-allergic to five antigens - (1) bovine soluble-lens (2) rabbit soluble-lens. (3) bovine alpha-crystallin, (4) bovine beta- cry­stallin (5) soluble part of human cataractous lens, keeping a, sixth group as control

Technique of sensitizing. Various me­thods are available. We used a in 100 solution of the antigens for the purpose. We injected subcutaneously into the rabbits the following closes of the antigen.

1st day o.1 c.c. - 13th day 1.oo c.c.

5th day o.2 c.c. - 19th day 1.5 c.c.

9th day 0.5 c.c.- 25th day 2.o c. c.

After this, 2.0 c.c. was injected intra­muscularly every week for a total period of two months.

Within four to six weeks it was pos­sible to obtain a strong antibody res­ponse as determined by the haemag­glutination test of the serum of these animals.

We recovered the aqueous and vitreous of one rabbit of each of the groups as previously done by remov­ing one eye. We carried out the haemagglutination test with a 1/10,000 dilution of the antigen and having found that there appeared to be no difference between the aqueous and vitreous as far as this test is concern­ed, we tested the aqueous only of the other rabbits without enucleating the eyes, thus saving unnecessary enuclea­tion, assuming at this stage that vitre­ous changes will be parallel to aque­ous changes.

The results of these tests arc tabu­lated below :-

It can be seen that (1) rabbit lens (homologus lens proteins) produces fewer lens antibodies than heterologus lens proteins. (2) The antibody res­ponse is almost similar to all the variet­ies of the extract except beta-crystallin, which appears to be comparatively less antibodygenic. (3) Presence of antibodies in the aqueous/vitreous of rabbits sensitized to lens proteins can be demonstrated.

It can thus be concluded that the barrier we found in Experiment 3 is a relative barrier and that it can be overcome if there is an excess of anti­bodies in the serum, experimentally produced.


  Nature of the Barrier Top


[Figure - 4] helps to visualize the situa­tion in immune-allergic rabbits with excess antibodies in the serum, aque­ous and vitreous. It may therefore be visualized from [Figure - 5] that in eyes of rabbits unsensitized to lens-proteins, escape of antibodies from the serum into the aqueous-vitreous is taking place constantly just as much as es­cape of lens-proteins is taking place constantly through the capsule as de­termined in Experiment 1 (Rao, Kul­karni and Cooper, Radhakrishna - r954), but there being excess of anti­gen over antibody in the aqueous-vitreous of unsensitized animals, [Figure - 3], the haemagglutination tests for lens anti-bodies in the aqueous/vitreous came negative. In sensitized animals on the other hand, [Figure - 6] there being excess of antibody over antigen in the acqueous-vitreous, the haemagglutina­tion test came positive for acqueous/vitreous. Thus we may assume, that the usual escape of lens-antigen into the acqueous (ex.1) wards off the few antibodies that may be escaping into the equcous/vitreous which otherwise hold a threat of entering through the lens capsule. [Figure - 6]. Thus the barrier is a immunochemical barrier and not a biological barrier as we at first thought (sec page 1) nor a mechanical or vital­ly selective one as in the case with other substances having a blood-acque­ous barrier. Incidentally this type of a barrier acts as a line of defence against cataract formation, as otherwise there are greater chances of antibodies en­tering through the lens capsule and causing an antigen-antibody precipita­tion in the lens, setting the pace for cataract formation.


  Permeability of the Capsule Top


Arguing from the fact that there are excess antibodies in the aqueous-vitreous of animals sensitized to lens proteins, there should be some catar­actous changes in the lenses of animals sensitized to lens-proteins.

On observing with a slit-lamp, not one animal showed any sign of catar­act formation. Halbert et al (1957) got the same result. Evidently some bar must be there to prevent the pass­age of the antibodies to the lens sub­stance. We investigated the role of the capsule in this direction.

Experiment 5 : [Figure - 7] shows the scheme of this experiment. In the bottom of a sterilised Kahn's tube is put o.5 cc of a 1 in 100 sterile solu­tion of soluble lens-protein. Over it is placed 0.5 cc. of sterile 1% agar, ice-box in a sterile condition ready for use.

At the time of a cataract operation, the lens removed intracapsularly is placed in a sterile petri-dish. Its cap­sule is incised with a cataract knife and a jet of sterile normal saline, col­oured with 1 in 1,000 acriflavin from a McKewan's irrigator is played upon it. It is possible to separate the cap­sule completely without furthur dam­age. The slightly straw-coloured solu­tion stains the lens-matter a pale yel­low and so a lens capsule can be obtained, visibly free of all lens mat­ter, in an almost undamaged state. This could be spread out over the agar layer evenly in the test-tube pre­pared for the experiment, with the help of a sterile camel-hair brush wetted in sterlie saline.

Now a 0.5 cc layer of 1% agar is placed over the capsule and over it a layer of 0.5 cc. antiserum. The tubes were left at room temperature for one month. During that time a faint cloudiness appeared in the part of the agar above the capsule with a tend­ency to arrange itself in strata not parallel with the surface, but the por­tion of agar below the capsule remain­ed clear. [Figure - 6]

In the control tube where we had placed a strip of filter-paper in place of the capsule, no cloudiness took place either above or below the paper strip.

This experiment is open to the criticism that a little lens matter may have remained adherent to the lens capsule giving cloudiness in the direc­tion facing the anti-serum. The ex­periment needs to be repeated with better controls and although we have not proved conclusively, we suspect there is a one-way traffic through the capsule, that is lens-antigen can pass out through the lens capsule, but lens-antibodies cannot pass through it on their way to the lens substance.


  Effect of Traumatisation Top


If the capsule may be offering resist­ance to the passage of antibodies and thus acting as a line of defence against cataract formation, then there should be no difficulty in producing a cataract by traumatising the capsule in sensitiz­ed animals.

Experiment 6 : The capsules of two of each group of the animals were traumatised, the anterior part of the capsule by a cataract knife through a puncture at the limbus and the post­erior capsule by a Ziggler's knife through the sclera under direct vision through the pupil; it can easily be done. We confirmed the traumatisation by seeing with a slit-lamp and corneal microscope.

To our disappointment, no opacities developed in the lens even after one month proving that mere traumatisa­tion of the capsule even of sensitized animals was not sufficient to produce a cataract. There should be something more.


  Effect of Diet Top


As we were despairing, our atten­tion was drawn to a paper by Kalyan Bagchi of Calcutta Hygiene Research Institute (1957). He was experiment­ing with rats and was determining the sulphydril content of their lenses after putting them on a protein poor diet, since in cataract formation as well as in a diet deficient in protein the sul­phydril content gets diminished. There are two fractions of sulphydril in the lens, - glutathion aril a protein-bound substance (PBS for short) which con­tains the different enzymes. The sulphydrill content suffers at the ex­pense of the glutathion content, the PBS not suffering much. Bagchi noticed that although the sulphydrill content was lowered and there were differences in the staining properties of excised lenses from such animals he found no cataractous changes in these animals as could be determined by bio-microscopy.

All that was now left for us to do was to put our animals on a protein poor diet.

Bagchi had a diet which was defi­cient in the amino-acid methionin. He used the diet called Godwin A, which is rich in starch (Maize starch 92, Casein 3, salt-mixture 3, yeast 2, fortified by arachis oil given bi-weekly which supplied the vitamins A,D,E,K and essential fatty acids).

Experiment : 7 The usual diet of all our rabbits was milk, vegetables, green grass and carrots, the later two supplying the required vitamins in sufficient quantity.

In this experiment we kept the same diet but witheld milk completely and instead fed them on bread. The animals had not lost any appreciable weight during the three weeks that the diet was changed. The results were as shown in [Table - 4].

Only the two rabbits that were sen­sitized to alpha-crystallin and whose lenses were traumatised developed cataracts. The opacities did not show any familiar pattern, but were irregu­larly placed and irregular in density.


  Comments and Conclusions Top


It will be seen that production of cataract on immunological lines is pos­sible but very difficult, although the physiological conditions as they exist are very favourable for the production of cataract, viz. the presence of auto­antibodies to lens in normal sera of animals. Nature has provided at least 5 safe-guards.

1. Continuous leakage of lens­proteins into the aqueous and vitreous warding off the onslaught of anti­bodies.

2. Difficulty in production of sen­sitization to autologus lens as against heterologus lens, [Table - 3] although there is a certain minimum concentra­tion of autolens-antibodies always pre­sent.

3. One-way traffic through the lens capsule which presumably allows the passage of lens-proteins out through the capsule and prevents the passage of lens-antibodies inwards. A question may be asked at this stage how it is that we can get traumatic cataracts. It must be remembered that every form of lens trauma does not produce a cataract. In the 1920's when artificial ripening of immature cataracts used to be attempted by us, we remember, not in all cases could we succeed in bringing about an increase in the opa­city. Secondly it falls to the experi­ence of every ophthalmologist, to come across a case of intraocular foreign body in the lens with no cataract. In cases of traumatic cataracts, either the capsular damage is very gross or there may be other contributing causes. The important thing in our experiment is to realise that an injury to the capsule did not produce a cataract, until it was placed on a protein-poor diet, without any further traumatisation, and that also in the rabbit sensitised to alpha--crystallin.

4.A balance between alpha- and. beta-crystallin. Allan Woods has shown that alpha- and beta-crystallin have a mutual anti-precipitating property over each other.

Selective-sensitization by alpha-crystallin alone was responsible for catar­act formation. This would suggest that the balance between alhpha-and beta­crystallin must be disturbed before a cataract can form. - Woods (1933).

5.A balance between glutathion and protein-bound substance, the sul­phydril components of the lens, which. can be disturbed by nutritional distur­bances.

As can be seen, only when all the­ five safeguards against this form of cataract formation are broken, then alone a cataract may form. So if I gave you any cause for alarm after experi­ment II, it will now be softened be­cause all the five safeguards together cannot be broken easily, at the same time breaking through all the five safe-­guards is not an improbability in every­day life.

Frequency of cataracts in tropical countries is established. The tropical heat of the sun can easily cause minute points of thermal trauma to the capsule.

Frequency of cataracts in diabetics may suggest a chemical or metabolic trauma to the capsule.

Nutritional and dietetic (protein) deficiencies are the rule in India.

The missing link is selective sensiti­zation to alpha-crystallin in clinical practice the study of which will be the aim in our future studies on the subject.


  Summary Top


By a series of seven experiments, cataract formation was attempted in rabbits on immunological lines. Suc­cess could be obtained only in the case of those rabbits which were sensitized to alpha-crystallin, whose capsules were traumatised and who were kept on a protein substituted starchy diet.

In control animals that were either not sensitized or sensitized to whole ­lens, similar trauma to lens capsule and/or diet deficiency failed to produce cataract.[6]

From this, five natural safeguards to production of cataract on immuno­logical lines have been theorized.

 
  References Top

1.
Stavitzky A.B. (1954) J of Immun. 72, 360.  Back to cited text no. 1
    
2.
Rao S. S. Kulkarni, M.E. and Cooper S. N., Radhakrishnan M. R., (1955) Brit. J. Ophthal. 39, 165-167.  Back to cited text no. 2
    
3.
Ouchterlony, O. (1953), Sixth Inter­nal. Cong Microbiol. 2, 140.  Back to cited text no. 3
    
4.
Oudin (1957) Bull. Soc. Chim. Biol. (Paris), 29, 140.  Back to cited text no. 4
    
5.
Bagchi K. (1958). Ind. J. Med. Re­search, 47, 184.  Back to cited text no. 5
    
6.
Woods, A.C. (1933), Allergy and Im­munity in Ophthalmology, The Johns Hopkins Press, Baltimore. p. 61.  Back to cited text no. 6
    


    Figures

  [Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6], [Figure - 7]
 
 
    Tables

  [Table - 1], [Table - 2], [Table - 3], [Table - 4]



 

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