Specificity of antibody formation after intravitreal immunization with bovine gamma globulin and ovalbumin

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1 Specificity of antibody formation after intravitreal immunization with bovine gamma globulin and ovalbumin I. Primary response Joan M. Hall Rabbits were immunized intravitreally with bovine gamma globulin in one eye and ovalbumin in the other. Uveal tract cells of a given eye produced antibody only to the antigen with which the eye had been injected. Cells from the draining lymph node produced antibody almost exclusively to the antigen injected into the eye drained by that node. When both antigens were injected into the same eye, uveal tract and lymph node cells produced antibody to both antigens. No antibodies were produced by cells from the uninjected eye. Circulating s and hemagglutinins to both antigens were demonstrated in all rabbits. Antibody was also detected in the aqueous humor. The response in the lymph nodes of rabbits that received the two antigens via the footpad was not specific. Key words: hemolytic antibody, plaque-forming cells, intravitreal injection, bovine gamma globulin, ovalbumin, uveal tract, lymphoid cells, specificity, aqueous antibody,ur previous investigations 1 showed that uveal tract cells from eyes of rabbits immunized intravitreally with a single antigen, bovine gamma globulin (), produced specific antibody. Cells from the uninjected contralateral eyes did not produce antibody nor did the spleen cells and preauricular lymph nodes that we tested. The From the Francis I. Proctor Foundation for Research in Ophthalmology, University of California San Francisco, San Francisco, Calif. Supported in part by United States Public Health Service Program Research Grant EY-31. Manuscript submitted July 14, 1971; manuscript accepted Aug. 6, Reprint requests: Dr. Joan M. Hall, Francis I. Proctor Foundation, University of California, San Francisco, Calif experiments of Silverstein 2 showed that the anamnestic response that follows challenge of rabbits initially injected intravitreally with protein antigens is a specific response. Only the eye originally injected with the challenge antigen responded with rapid development of inflammation. The present experiments were undertaken to determine whether specificity could be demonstrated at the level of the antibody-forming cell; that is, whether cells from the uveal tract produce antibody only to the particular antigen with which the eye has been injected, regardless of the rabbit's systemic immunity to another simultaneously injected antigen. For this purpose we used two antigens, and ovalbumin (), in the following three sets of experiments: (1) Rabbits were in- Downloaded From: on 1/28/216

2 776 Hall Investigative Ophthalmology October 1971 jected with in one eye and in the other. (2) Both antigens were injected into the same eye. (3) Both antigens were injected into the footpads, one in the right foot and the other in the left foot. The ability of uveal tract, lymph node, and spleen cells to produce antibody against each of these antigens under these various conditions was determined by the hemo- Jytic plaque technique. Materials and methods Animals. The experimental animals were New Zealand white rabbits weighing approximately 2.5 kilograms. Antigens and injection procedure. The was obtained from Miles Laboratories, Chicago, 111., and the from Schwarz/Mann, Orangeburg, N. Y. The antigens were dissolved in normal saline in concentrations of 3 mg. per milliliter and were sterilized by filtration through a.45 micron Millipore filter. The procedure for making the intravitreal injections has been described previously. 3 Intravitreally injected animals received: (1) 1.5 mg. of in the right eye and 1.5 mg. of in the left eye, or (2) both antigens in the right eye. Other rabbits were injected with 15 mg. of in the right hind footpad and 15 mg. of in the left hind footpad. With one exception, adjuvants were not used for the footpad injections. Antibody determinations. Serum was examined for antibody by passive hemagglutination and passive hemolysis tests. The latter test was adapted from the procedure described by Hiibner and Gengozian. 4 Sheep erythrocytes were coated with the antigens, with carbodiimide as the coupling agent. A mixture of.1 ml. of the coated cells and 1. ml. of.7 per cent agarose in Eagles' minimal essential medium (MEM) (Hyland Laboratories) was poured onto the surface of a 6 mm. plastic Petri dish containing 2. ml. of the agarose. Serial twofold dilutions of serum or aqueous humor were made with the Microtiter apparatus (Cooke Engineering Co., Alexandria, Va.). A drop of each dilution was placed on the surface of the prepared Petri dish with a finely drawn Pasteur pipette. Each serum was tested for its ability to lyse cells coated with and cells coated with. Uncoated erythrocytes served as controls. After the serum was absorbed, the plates were rinsed with MEM, and 1. ml. of guinea pig complement (1:1 dilution) was added. The plates were incubated for one hour. The highest dilution showing a spot of lysis was considered the end point. Titers were expressed as log:. Plaque assay. The hemolytic plaque assay adapted for use with protein antigens was carried out as previously described, 1 but in this instance uveal tract cells from each eye were treated separately. In some experiments cervical and preauricular lymph nodes were also tested for their ability to produce antibody. When the rabbits' footpads were injected, popliteal lymph nodes and spleen cells were assayed. The results of the plaque assays were adjusted to express the number of plaque-forming cells (PFC) per million cells in the original suspension. Results Inflammatory response. The inflammatory reaction that developed after intravitreal injection of or was essentially the same as that previously described. 3 Most rabbits showed grossly apparent uveitis by postinjection Day 6 or 7. The degree of inflammation varied, but there were no apparent differences between the responses to and. Plaque assay and antibody determinations. Each cell suspension was tested for the presence of cells producing antibody to both and. Table I gives the results of the plaque assays on the uveal tract cells of seven rabbits injected with in the right eye and in the left eye. Table I also gives the peak titers in the sera of these rabbits. Table II gives the results of the plaque assays on uveal tract and lymph node cells of six additional rabbits injected in the same manner. Peak serum titers, and titers in aqueous on the day of sacrifice, are also given. These results indicate that the response as measured by the activity of the PFC is indeed specific. Uveal tract cells produced antibody only against the antigen with which the eye was injected. The response in the cervical lymph nodes draining the injected eye also showed a high degree of specificity; very little cross-reactivity was noted. Antibody was detected in the sera of all rabbits. Antibody was also present in all of the aqueous humor specimens tested. Some aqueous samples showed antibody to both antigen, even though the PFC response in the uveal tract was specific. Downloaded From: on 1/28/216

3 Volume 1 Number 1 Antibody formation after intraditreal immunization 111 Table I. Plaque formation by uveal tract cells from rabbits injected with bovine gamma globulin in the right eye and ovalbumin in the left eye Rabbit No. Cells assayed Antigen Plaques per 1 e cells Peak serum titer logt S 434, 2, BCC , 5, BCG 2, BGC, 5, BCG BGC, 2, BGC , 3, BGC, 3, 2 31 BCC , 5, 7 Table III gives the results of plaque assays and antibody determinations in seven rabbits injected with 1.5 mg. of and 1.5 mg. of in the right eye. Uveal tissue from the right eye contained cells that produced antibodies to both antigens. Cells from the uninjected left eyes did not produce antibody. The cervical lymph nodes contained small numbers of cells that produced both antibodies. Some of the left cervical lymph nodes contained a very small number of PFC. Six rabbits were injected with in the right hind footpad and in the left hind footpad. Popliteal lymph nodes and spleens were tested for the presence of antibody-forming cells. The results of these experiments are presented in Table IV. Four of the six rabbits had cells that produced anti- antibodies almost exclusively in both the right and left lymph nodes and in the spleen. The response in the lymph nodes of the rabbit that received antigen in complete Freund's adjuvant appeared to be more specific than the response in rabbits receiving antigen in saline. Some rabbits did not produce serum antibody because they were killed too soon (on postinjection Day 4 or 6) for antibody to have been synthesized in amounts detectable by our tests. All serum samples were tested for s and hemagglutinins. Because of the small amount of material obtained, the aqueous humor samples were tested for s only. Since the s correspond more closely than the hemag- Downloaded From: on 1/28/216

4 778 Hall Investigative Ophthalmology October 1971 Table II. Plaque formation by uveal tract and lymph node cells from rabbits injected with in the right eye and ovalbumin in the left eye Rabbit No Cells assayed Antigen Plaques per 1 s cells 1, , , , Peak serum titer logi, 6, 5, 5, 5, 3, 11, 4, 4, 3, 5, 7, 6 Aqueous titer log., 7, 2, 1,,, 12, 8, 6,, 9, 7, 5, 5, 8 = right uveal tract; = left uveal tract; = right cervical lymph node; = left cervical lymph node. glutinins to the hemolytic antibodies produced by the PFC, only the titers are given in the tables. The and hemagglutinin titers correlated well in most rabbits, however. Fig. 1, A and B, shows a typical antibody curve for a rabbit injected with in the right eye and in the left eye. The serum antibody response varied from rabbit to rabbit, but the general pattern of the response was the same in all rabbits injected. The aqueous antibody titer was usually as high or higher than the serum antibody titer. Animals with the highest antibody titers appeared to have the most PFC in their uveal tracts. Downloaded From: on 1/28/216

5 Volume 1 Number 1 Antibody formation after intravitreal immunization 779 Table III. Plaque formation by uveal tract and lymph node cells from rabbits injected with and ovalbumin in right eye only Rabbit No Cells assayed Vitreous exudate Antigen BCG BCG Plaques per 1 e cells Peak serum titer logt, 8, 8, 6, 6, 4, 11, 4, 6, 4, 8, 5, 7, 2, 4 Aqueous titer logt, 8, 11,,, 4, 2,, = right uveal tract; = left uveal tract; = right cervical lymph node; = left cervical lymph node. Discussion The present experiments confirm our earlier observations 3 and those of Parks and associates 5 that intravitreal injection of soluble protein antigens is an effective method of stimulating antibody production in rabbits. Intravenous injection of the same amounts of BSA,, and HSA 7 has not had this effect. The plaque assay was carried out routinely 12 days after intravitreal injection because preliminary experiments had shown that the maximal number of PFC were detected several days after the onset Downloaded From: on 1/28/216

6 78 Hall Investigative Ophthalmology October 1971 Table IV. Antibody production by lymphoid cells from rabbits injected with bovine gamma globulin in right hind footpad and ovalbumin in left hind footpad Rabbit No. 262 Cells assayed Antigen 263* f \ = right lymph node; = left lymph node. "Rabbit received two injections of antigen in each footpad instead of one. f Killed on Day 4. tantigen given in Freund's complete adjuvant. Killed on Day 13. of ocular inflammation. In most systems in which erythrocyte antigens are used, the maximal PFC response has been detected 4 days after antigen injection. But Daniels and Weigle 6 detected the maximal number of PFC in rabbit lymphoid tissue nine or ten days after an intravenous injection of soluble BSA. The number of PFC found in uveal tract tissue after intravitreal injection did not differ from the number found in our previous experiments 1 but was significantly Plaques per 1 6 cells Peak serum titer log;, 7,, 7, 8,,,,, 4,, 9, 8 higher than the number found in lymph nodes after injection into the foot pad. The results in the latter instance are comparable to those of Daniels and Weigle 6 who injected BSA intravenously. These results lend support to our supposition that intravitreally injected antigen is released slowly into the circulation, an effect comparable to that obtained when antigen emulsified in Freund's adjuvant is injected into a peripheral site. Preliminary evidence indicates that antigen is present in detect- Downloaded From: on 1/28/216

7 Volume 1 Number 1 Antibody formation after intravitreal iinmunization 781 able quantities for at least 14 days after a single intravitreal injection of 1.5 mg. of or. The investigations reported here definitely indicate that antibody production by uveal tract cells is specific, and that this specificity extends to some extent to the draining lymph nodes. Cells from the right eyes produced plaques only when plated with -coated erythrocytes, and cells from the left eyes produced plaques only when plated with -coated erythrocytes. The apparent cross-reaction with cells of rabbit No. 268 was probably due to technical error during manipulation of the cell suspensions. Cells from the draining lymph nodes produced a predominant amount of antibodv to the antigen injected into the eye drained by that particular node. Very little cross-reactivity between the two sides was observed. In all cases, more PFC were found in the uveal tract than in the draining nodes. Although the specificity of the response shown in Tables I and II was not entirely unexpected, it is an unusual phenomenon. Several investigators have studied the antibody response in lymphoid tissue following injection of two unrelated antigens. The high degree of specificity demonstrated in the present experiments has not been shown in other systems. White s used a fluorescent antibody technique to detect antibody-containing cells in lymph nodes of rabbits injected via the footpad with diphtheria toxoid and. Cells of both specificities were found in the draining popliteal node. Green 9 injected guinea pigs with both and, varying the physical nature of the antigen and the sites of injection. He injected the antigens either in Freund's adjuvant or in pertussis vaccine and detected the antibody-producing cells by a fluorescent antibody procedure. Antibodyproducing cells were randomly mixed in draining lymph nodes whether the antigens had been mixed before injection or were injected into separate sites. No nodes con- CM 2._ Heinolysin titer T T Hemagglutinin titer Days after injection Hemolysin titer T T Hemagglutinin titer Days after injection Fig. 1. A, Hemagglutinating and hemolytic antibody in serum of a rabbit injected with in the right eye and ovalbumin in the left eye. Response to. B, Hemagglutinating and hemolytic antibody in serum of a rabbit injected with in the right eye and ovalbumin in the left eye. Response to ovalbumin. taining cells productive of only one type of antibody were found. Nakano and Braun 1 injected mice simultaneously with sheep and chicken erythrocytes. cells of these mice contained PFC for both antigens. Similar responses to simultaneous injections of heterologous erythrocytes were noted by Moiler and Sjoberg, 11 Gershon and associates, 12 Radovich and Talmage, 13 and Waterson. 14 A possible explanation for the specificity found in our system is that antigen is released slowly after intravitreal injection. In a previous publication of ours and in one of Silverstein's, 2 it was postulated that after an initial stimulation of peripheral Downloaded From: on 1/28/216

8 782 Hall Investigative Ophthalmology October 1971 lymphoid tissue, most of the antibodyforming cells or their precursors are recruited to the eye where most of the antibody is produced. Antibody-forming cells are apparently not recruited to the uninjected contralateral eye. There is no reason to suppose that a different mechanism operates when a different antigen is injected into each eye. The antibody-forming cells corresponding to a particular antigen are apparently recruited only to the eye injected with that antigen. Draining lymph nodes may be minimally stimulated. The antigen probably does not reach the nodes draining the opposite eye, and so the response in the nodes of a given side of the body remains quite specific. It seems that very little antigen reaches the spleen since few PFC are found in spleen cell suspensions. The results of experiments in which both antigens were injected into the same eye would tend to corroborate the above explanation. Cells from the uveal tracts and draining lymph nodes of rabbits so injected contained cells that produced antibody to both antigens. The uninjected eyes of such rabbits did not contain antibody-forming cells. When rabbits were injected via the footpad, the situation was less clear. Four of the rabbit injected with and in saline appeared to produce antibody almost exclusively to the. The phenomenon of antigenic competition has been adequately reviewed elsewhere ie Some evidence indicates that competition is not always manifest when two antigens are injected simultaneously, and this was certainly the case after the intravitreal immunization which we have described. Adler reported that competition was in evidence after simultaneous injection of rabbits with BSA and serum globulins. The fact that in our study popliteal lymph nodes from the left side produced antibody to was not surprising. Green 9 showed that antibody production can occur in lymph nodes other than those draining the site of injection. Askonas and White 17 also showed that contralateral lymph nodes, as well as the draining flank and popliteal nodes, produced antibody in guinea pigs after footpad injection of. Kamarytova ls injected rabbits with diphtheria toxoid and found gamma globulincontaining cells in both draining and contralateral nodes. In our experiments, however, it seemed logical to expect the lymph nodes draining the left foot to produce antibody to. A possible explanation for the fact that they did not do so is that the coating procedure failed in this case and that PFC for were present but not detected. In at least one instance, however, the efficacy of the coating procedure was tested by dropping known positive antiserum onto -coated erythrocytes suspended in agarose. The possibility remains that antigenic competition did take place in the rabbits described and that this may have been related to the relative degree of antigenicity of the two antigens. Fauci and Johnson 1 have demonstrated competition at the cellular level in rabbit lymph nodes following footpad injection of two hapten-protein conjugates in Freund's adjuvant. Another interesting aspect of the study was the presence of antibody in the aqueous humor. Antibody was expected in the aqueous in fairly high concentrations if the cells in the vitreous of that eye were known to be producing the majority of the antibody detected. However, some rabbits (Nos. 38, 312, and 313) manifested antibody to both antigens in the aqueous humor despite the fact that cells in the vitreous of that eye were producing antibody only to the antigen with which the eye was injected. Since the introduction of antigen into the vitreous alters vascular permeability, serum antibody could have entered the anterior chamber from the circulation. This would account for the presence of antibodies of both specificities. This aspect of the study is being investigated further. Downloaded From: on 1/28/216

9 Volume 1 Number 1 Antibody formation after intravitreal immunization 783 We are planning to make additional experiments on the specificity of the secondary response after intravitreal or systemic changes with the antigens concerned. FENCES 1. Hall, J. M., and O'Connor, G. R.: Correlation between ocular inflammation and antibody production. II. Hemolytic plaque formation by cells of the uveal tract, J. Immunol. 14: 44, Silverstein, A. M.: Ectopic antibody formation in the eye: Pathologic implications, in Maumenee, A. E., and Silverstein, A. M., editors: Immunopathology of uveitis, Baltimore, 1964, The Williams & Wilkins Company, p Hall, J. M., and O'Connor, G. R.: Correlation between ocular inflammation and antibody production. I. Serum antibody response following intravitreal immunization with protein antigens, J. Immunol. 15: 432, Hiibner, K. F., and Gengozian, N.: Critical variables of the Jerne plaque technique as applied to rodent antibody forming systems responding to heterologous red cell antigens, J. Immunol. 12: 155, Parks, J. J., Leibowitz, H. M. T., and Maumenee, A. E.: The effect of route of inoculation upon development of antibody in rabbits, J. Immunol. 87: 199, Daniels, J. C, and Weigle, W. O.: Antibodyproducing cells in rabbits injected with soluble BSA. II. Kinetics and dose response, J. Immunol. 11: 123, Naspitz, C. K., Singhal, S. K., and Richter, M.: The effect of phytohemagglutinin in rabbits. III. Antibody production in vivo by immune rabbit lymphoid cells incubated with phytohemagglutinin in vitro, Int. Arch. Allergy Appl. Immunol. 34: 53, White, R. G.: Antibody production by single cells, Nature 182: 1383, Green, L.: Distribution of antibody-forming cells of different specificities in the lymph nodes and spleens of guinea pigs, J. Exp. Med. 128: 729, Nakano, M., and Braun, W.: Fluctuation tests with antibody-forming spleen cell populations, Science 151: 338, Moller, G., and Sjoberg, O.: Effect of antigenic competition on antigen-sensitive cells and on adoptively transferred immunocompetent cells, Cell. Immunol. 1: 11, Gershon, H., Bauminger, S., Sela, M., and Feldman, M.: Studies on the competence of single cells to produce antibodies of two specificities, J. Exp. Med. 128: 223, Radovich, J., and Talmage, D. W.: Antigenic competition, cellular or humoral, Science 158: 512, Waterson, R. H.: Antigenic competition: A paradox, Science 17: 118, Adler, F. L.: Competition of antigens, in Shaffer, J. H., Lo Grippo, G. A., and Chase, M. W., editors: Mechanisms of hypersensitivity, Boston, 1959, Little, Brown & Company, p Adler, F. L.: Competition of antigens, Progr. Allergy 8: 41, Askonas, B. A., and White, R. G.: Sites of antibody production in the guinea pig. The relation between in vitro synthesis of antiovalbumin and gamma globulin and distribution of antibody containing plasma cells, Br. J. Exp. Pathol. 37: 61, Kamarytova, V.: Number of gamma globulin containing cells after the single local administration of antigen, Folia Microbiol. (Praha) 14: 345, Fauci, A. S., and Johnson, J. S.: Antigenic competition at the cellular level: Response of rabbit lymph node cells to the trinitrophenyl and p-arsanilic haptens, J. Immunol. 16: , Downloaded From: on 1/28/216