|Year : 1960 | Volume
| Issue : 1 | Page : 1-10
Immunological approach to etiology of cataract
SN Cooper, KV Padukone, PR Bhatia
King Edward Memorial Hospital, Bombay, India
|Date of Web Publication||6-May-2008|
S N Cooper
King Edward Memorial Hospital, Bombay
Source of Support: None, Conflict of Interest: None
|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
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|| |
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).
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|| |
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|| |
[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|| |
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|| |
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|| |
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|| |
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|| |
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.
From this, five natural safeguards to production of cataract on immunological lines have been theorized.
| References|| |
Stavitzky A.B. (1954) J of Immun. 72, 360.
Rao S. S. Kulkarni, M.E. and Cooper S. N., Radhakrishnan M. R., (1955) Brit. J. Ophthal. 39, 165-167.
Ouchterlony, O. (1953), Sixth Internal. Cong Microbiol. 2, 140.
Oudin (1957) Bull. Soc. Chim. Biol. (Paris), 29, 140.
Bagchi K. (1958). Ind. J. Med. Research, 47, 184.
Woods, A.C. (1933), Allergy and Immunity in Ophthalmology, The Johns Hopkins Press, Baltimore. p. 61.
[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6], [Figure - 7]
[Table - 1], [Table - 2], [Table - 3], [Table - 4]