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An Immunologic Study of Rabbits Sensitized with Homologous Uveal Tissue*
AN IMMUNOLOGIC STUDY
377
4. Eames, T. H. : Comparison of eye conditions among 1,000 reading failures, 500 ophthalmic patients, and ISO unselected children. Am. J. Ophth., 31:713-717 (June) 1948. 5. Farris, L. P. : Visual defects as factors influencing achievement in reading. J. Exper. Education, 5:59 (Sept.) 1936. 6. Taylor, E. A. : Controlled Reading. Chicago, Univ. Chicago Press, 1937.
AN IMMUNOLOGIC STUDY O F RABBITS SENSITIZED W I T H HOMOLOGOUS U V E A L TISSUE* TED SUIE, P H . D . , AND MATT C. DODD, P H . D .
Columbus, Ohio INTRODUCTION
Generally, it is agreed that uveitis fre quently demonstrates allergic manifestations. This ocular sensitivity may be classified into the granulomatous and the nongranulomatous types. Woods 1 states that in the granulo matous type the tissues are actually in vaded by micro-organisms such as Mycobacterium tuberculosis and Treponema pallidum and the hypersensitivity results when the organism or its products again reaches the eye. On the other hand, the nongranu lomatous type is believed by some to be a sterile allergic reaction resulting from intoxi cation with specific allergens.1 In 1903, Elschnig 2 ' 3 ' 4 postulated that sym pathetic ophthalmia, a type of uveitis which is characterized chiefly by its tendency to involve the uveal tissue of both eyes, was an isoallergy to modified uveal tissue. Later, the recognition that certain tissues such as brain and lens protein possessed a greater degree of organ specificity than species specificity became perhaps one of the earliest demonstrations of the feasibility of autoantibody for normal tissues. Attempts to produce isoallergic conditions in experimental animals are numerous but until recently results were difficult to obtain ♦From the Departments of Ophthalmology and Bacteriology, College of Medicine, The Ohio State University. Grateful acknowledgement is made to W. D. Rummel, M.D., and H. Farmer, M.D., for their aid in biomicroscopic studies and dissections of the uveal tracts.
except by a long process involving many injections. Even then, the success was rela tively poor. However, with the introduction of the adjuvant technique by Freund, 5 a method was devised which greatly facilitated the production of experimental isoallergic encephalitis apparently by enhancing the pro duction of antibodies to homologous tissue extract. In fact, a single injection sufficed to produce this condition in some rabbits. The present work was undertaken to study the serology and hypersensitive reac tions which might occur in rabbits sensitized with homologous uveal tissue suspension with and without the aid of Freund's ad juvant. METHODS AND MATERIALS
Unless otherwise noted, albino rabbits of both sexes and varying weights were used throughout these experiments. None of the animals had a history of infection. All antigens used in this study were pre pared from rabbit uveal tissue. All materials were stored, without preservatives, at —23°C. Thioglycollate broth was used to determine the sterility of all the antigens. The uveal tracts were washed several times in saline. The tissue was then ground in a mortar and enough sterile saline was added to make dilutions of 1:10 and 1:20. The emulsion was placed in sterile vials. Each vial contained uveal tracts pooled from four or five rabbits. The 1:10 dilution of uveal suspension was used to sensitize the eyes,
378
TED SUIE AND MATT C. DODD
and the 1:20 dilution was employed for in travenous injections and for skin testing. Albino, as well as pigmented, rabbits were used for the preparation of uveal-adjuvant emulsion. The inoculated mixture consisted of 0.5 gm. of uveal tissue, 3.5 ml. of mineral oil, and 10.0 mg. of killed and dried tubercle bacilli. The bacteria were ground in a sterile mortar and oil was gradually added. This mixture was then introduced slowly into an homogenate of uveal tissue and again ground in a mortar. This emulsion was stored at - 2 0 ° C . until used. The tubercle bacilli suspension for skin testing was prepared as follows : Previously killed and dried tubercle bacilli were re duced to a fine powder in a mortar and enough sterile saline was added to make a final concentration of 0.2 mg. of tubercle bacilli per ml. of saline. The rabbits were divided into two groups : GROUP I
Nine rabbits were inoculated intraocularly with 0.1 ml. of a 1:10 dilution of uveal ex tract. One-half percent pontocain was used as the local anesthetic. Only one eye was inoculated; the fellow eye served as a con trol. Normal nonimmunized rabbits also served as controls. A 27-gauge hypodermic needle attached to a 0.25-ml. tuberculin syringe was inserted tangentially into the anterior chamber at the limbus. Then 0.2 ml. of aqueous humor was removed. The needle was left in position and a new syringe con taining 0.1 ml. of uveal suspension was ex changed for the one containing the aqueous humor. This material was then injected into the anterior chamber slowly. Extreme pre cautions were taken in order to prevent in jury to the lenses. After the nonspecific inflammation sub sided, the rabbits were given one ml. of a 1:20 dilution of homologous antigen intra venously. The rabbits were observed with the slitlamp and macroscopically immediately be
fore the injection, one hour later, and after ward at indefinite intervals. GROUP II
This group consisted of 18 rabbits inocu lated into the footpads of both hind legs with uveal-adjuvant emulsion. The hair on the footpads was removed with an electric clipper and the pads were cleaned carefully with 70-percent alcohol. Using a 27-gauge needle and a 0.25-ml. tuberculin syringe, 0.05 ml. of this emulsion was injected intradermally into each of the footpads. All animals in this group were skin tested with 0.05 ml. of tubercle bacilli suspension and 0.1 ml. of uveal suspension. The right side and left side of the rabbits were clipped and cleaned thoroughly with 70-percent al cohol prior to the injections. Separate syringes and needles were used. Animals from both groups were bled by cardiac puncture at weekly intervals and the sera were stored at —23°C. All the rab bits were observed daily either macroscopi cally or with the slitlamp for evidence of eye inflammation. Three rabbits from Group II were later inoculated intravenously as described above. Controls consisted of two normal, nonimmunized animals which were injected in travenously with 1.0 ml. of uveal tissue and two immunized rabbits which received 1.0 ml. albumin by the same route. Complement fixation tests were performed as follows : The anticomplementary value of the an tigens was determined before each series of tests. Commercially prepared, lyophilized complement was used throughout. Twentyfive hundredths ml. of 1:40 saline suspen sion of uveal tissue was added to a series of tubes containing the following amounts of antisera : 0.5, 0.2, 0.1, 0.05, 0.02 ml. Then, 0.1 ml. of complement, containing two units, was added to each. The tubes were incu bated at refrigerator temperature overnight and then the indicator system was added. Preliminary readings were made after the
AN IMMUNOLOGIC STUDY tubes were incubated at 37°C. for 30 min utes, and final readings after storing over night at refrigerator temperature. Serum, antigen, hemolytic system, and cell controls were included. The results were recorded ac cording to the degree of fixation of comple ment. Gradations in fixation ranged from 4 + for complete fixation to — (minus) for absence of fixation. Several methods were used in an attempt to transfer the hypersensitivity passively. The first method consisted of injecting each of two normal albino rabbits intradermally with 0.2 ml. of uveal antiserum into three sites, one of which served as a control. Eighteen hours later 0.1 ml. of antigen (homologous uveal extract) was injected into one of the sites and 0.02 ml. of the anti gen was injected into another. An addi tional injection of 0.1 ml. of antigen was used as a control. The rabbits were then ob served for anaphylactic and tuberculinlike reactions. The second method was carried out as follows : Three rabbits which had been immunized with uveal-adjuvant emulsion one and a half months prior to the test and two nor mal rabbits were given 0.2 ml. of rabbit uveal antisera into the anterior chamber of the right eye. Normal rabbit sera was in troduced into the fellow eye of each animal as a control. The sera were obtained by car diac puncture and precautions were taken to insure sterility. The eyes of the rabbits were examined daily macroscopically and periodi cally with the slitlamp. The titer of the sera was determined by complement fixation tests prior to the injections. FINDINGS
Animals in Group I demonstrated inflam matory reactions in the anterior chamber after the homologous antigen was given in travenously. In some animals the changes in the anterior chamber could be observed dur ing the first 24 hours after the shock dose was administered.
379
These changes were manifested by lacrimation, injection of the vessels of the an terior segment of the eye, and the presence of flocculent material in the anterior cham ber of the injected eyes. Within three days, one could generally observe keratic precipitates on the endo thelium of the cornea, aqueous rays, fibrinous exudates in the anterior chamber, as well as hyperemia of the circumcorneal, iris, and conjunctival vessels. Only one animal in this group demonstrated a sympathetic ophthal mialike reaction. Periodic observations of rabbits in Group II with the slitlamp for several months failed to reveal evidence of ocular inflam mation. Two rabbits (CI and CII) which had been inoculated with the uveal-adjuvant emulsion six months previously and one rabbit ( D I ) which had been sensitized with the same material for a period of three months were again observed with the slitlamp and appeared normal. Immediately afterward, each animal re ceived 1.0 ml. of a 1:20 dilution of aqueous uveal suspension intravenously. No evidence of inflammation of the eyes appeared after one hour. However, after 24 hours both eyes showed varying degrees of diffuse, fine, al bumin-type precipitation on the endothelium of the cornea. No aqueous ray was noted and there was a minimal hyperemia of the iris vessels. The eyes appeared normal in four days. The rabbits immunized with the uvealadjuvant emulsion did not develop inflam mation within four days after the intra venous injection of albumin. Likewise, nonimmunized rabbits did not demonstrate in flammation after the injection of aqueous uveal suspension intravenously. In contrast, rabbits in Group I, which had previously demonstrated a local organ hy persensitivity 84 days before, did not show evidence of uveitis before or after the in travenous injection of aqueous uveal extract. Complement fixing antibiodies were dem onstrated in the sera of rabbits in Group I
T E D S U I E A N D M A T T C. D O D D
380
TABLE 1 COMPLEMENT FIXATION TITERS OF SELECTED RABBITS IN G R O U P II WHICH W E R E INOCULATED WITH THE UVEAL-ADJUVANT EMULSION
Rabbit Number Reciprocal of Serum Dilution
12.5 25 50 125 250
cm
CI Normal —. —· — — —
Weeks 2 1+ 1+ 2+ 2+ 1+
3
5
4+ 2+ 2+ 1+ 1+
Normal — — — — —
3+ 1+ 1+ + +
(table 1). Significant rise in titer occurred during the third week after the inoculation. Normal antibodies were detected in titers of 1:12.5 and 1:25. The complement fixa tion titers were greater in the animals in Group II as compared with those in Group I (tables 2 and 3). The highest titers in the former were detected during the third week after the injections. No complement fixing antibodies were demonstrable in the aqueous humor of the injected eyes or in the con trols. As indicated in Tables 3 and 4, rabbits in Group II which were inoculated with uvealadjuvant emulsion demonstrated a delayed type of skin hypersensitivity to the aqueous suspension of uveal extract. The skin reac tivity, both to the uveal suspension and tubercle bacilli suspension, developed as early as four days after the inoculation and was still present two weeks later. There was very little change in the de
Weeks 2
3
5
2+ 4+ 3+ 2+ 1+
1+ 2+ 3+ 2+ 1+
1+ 2+ 2+ + —
gree of skin reactions to uveal suspension in the same rabbits tested four days and 14 days after the inoculations. However, the acid-fast bacilli suspension induced a greater area of hyperemia and induration after 14 days. The control rabbit, which did not re ceive uveal-adjuvant emulsion previously, was not sensitive to the uveal or tubercle bacilli suspensions. Passive transfer of such skin sensitivity was not accomplished. The serum control was negative 24 hours after the inoculation. Ap proximately the same amount of erythema and induration appeared in the control site (antigen alone) as in the site containing the antigen-serum mixture. In 24 and 48 hours the antigen control and test areas showed the same amount of induration and erythema. The attempts to transfer sensitivity pas sively by means of anti-uveal sera into the eye also were unsuccessful. An iritis and
TABLE 2 COMPLEMENT FIXATION TITERS O F SELECTED RABBITS IN G R O U P I WHICH WERE INOCULATED INTRAOCULARLY AND SUBSEQUENTLY GIVEN AN INTRAVENOUS INJECTION OF HOMOLOGOUS ANTIGEN
Rabbit Number of Serum 12.5 25 50 125 250
AI
All
Normal
20 Days
Normal
5 Days
20 Days
2+ 1+ + + +
2+ 2+ 3+ 2+ 1+
2+ +
2+ 2+ 1+ 2+ 1+
2+ 2+ 2+ 2+ 1+
±
— —
AN IMMUNOLOGIC STUDY
381
TABLE 3 RESULTS OF SKIN TESTS WITH UVEAL SUSPENSION AND TUBERCLE BACILLI SUSPENSION FOUR DAYS AFTER INOCULATION
Rabbit Number
T.B.
48 Hours
24 Hours
3 Hours
Uvea
T.B.
++ +++ +++ ++ ++
++ +++ ++ ++
'+ + + ++
+
+ ++ +++ ++ ++
+ ++
±
+++ ++
+ ++
++
T.B.
Uvea
Cl C2 C3 C4 CS C6 C7 C8 C9 (control)*
+
Uvea
±
+
+
±
++ ++
* Control did not receive uveal-adjuvant emulsion + — No reaction ± Barely perceptible erythema or induration (neither exceeding 5 mm. in diameter) + Erythema and induration between 5 to 10 mm. in diameter + + Erythema and induration between 10 to 15 mm. in diameter + + + Erythema and induration exceeding IS mm. in diameter + + + + Very marked reactions with necrosis
circumcorneal injection developed within sev eral days. However, the control eyes which were injected with normal rabbit sera pro duced a similar manifestation. This inflam mation was considered nonspecific and was probably due to the intraocular injections. After a period of two weeks both eyes ap peared normal and no evidence of uveitis developed during the three months of obser vation. DISCUSSION
The experimental findings in the present work indicate that rabbits, which had been previously sensitized to uveal tissue intra-
ocularly, develop changes in the anterior chamber as a result of a shock dose of homologous antigen given intravenously two weeks later. These manifestations varied in degree from one animal to another. Generally, one could observe keratic pre cipitates on the endothelium of the cornea, aqueous ray, fibrinous exudates in the ante rior chamber, as well as hyperemia of the circumcorneal, iris, and conjunctival vessels. Macroscopic evidence of inflammation oc curred in a few animals and consisted of lacrimation and of hyperemia of the vessels of the anterior segment of the eye. Only one rabbit out of seven in Group I
TABLE 4 R E S U L T S OF SKIN TESTS WITH UVEAL SUSPENSION AND TUBERCLE BACILLI SUSPENSION FOURTEEN DAYS AFTER INOCULATION
Rabbit Number
3 Hours T.B.
Cl C2 C3 C4 C5 C6 C7 C8 C9 (control) See Table 3 for legend.
24 Hours Uvea
T.B.
++ +++ +++ +++ +++ ++ ++ ++ +
48 Hours
Uvea
++ +++ ++ ++ +++ + ±
++
T.B.
+++ ++++ +++ +++ ++ + ++ ++
Uvea
++
+++ ++ ++ + + +
++
382
TED SUIE AND MATT C. DODD
demonstrated a sympathetic ophthalmialike effect. Other workers 6 ' 7 have also noted a sympathizing effect in animals sensitized with foreign proteins. The white precipitate which was seen in the anterior chamber in certain of the ani mals might have been the result of an anti body-antigen reaction in vivo. Foss e noted that if one injected 0.1 ml. of horse serum into the vitreous of rabbits and the same amount of antihorse serum was injected into the anterior chamber, precipitation occurred in the anterior chamber. The precipitates were partly deposited as a ring around the pupillary brim. Rabbits in Group II which had been in oculated into the footpads with uvealadjuvant emulsion did not show evidence of ocular inflammation during the several months of observation with the slitlamp. Since trauma and infection will change the permeability of the surface membrane of the eye, various substances injected into the blood and which ordinarily would not get into the aqueous humor because of the normal blood-aqueous barriers, may now be found in the fluid of the anterior chamber. This phenomenon may explain the devel opment of eye inflammations which occurred in the rabbits in Group I and the failure of this reaction to appear in the eyes of the animals in Group II. The former group received antigenic stim ulation directly into the anterior chamber and as a result of the injection and paracentesis the normal blood-aqueous barriers were modified. Since residual inflammation was still present at the time of the shock dose, it is highly doubtful that these barriers were restored during the period of sensitization. Subsequent injection of the homologous antigen intravenously might permit the anti gen to react with the preformed antibodies present in the aqueous humor. In the rabbits of Group II, the barriers probably played an important role in pre venting localization of the antigen in the anterior chamber. However, other possibili
ties for the failure to incite ocular inflam matory reactions by this method may exist. Perhaps the antigenic dosage which was in corporated in the adjuvant was too small to elicit an ocular manifestation. Three animals which had been sensitized with uveal-adjuvant emulsion developed slight changes in the anterior chamber when large amounts of homologous antigen was inoculated intravenously even after an elapse of time of six months from the primary injections. This would indicate that larger amounts of uveal tissue added to the adju vant might suffice to produce local organ hypersensitivity. Furthermore, it is conceivable that the local organ must be directly sensi tized in order to produce an ocular in flammation. The rabbits in Group I which demon strated an ocular anaphylactic-type of reac tion also had circulating antibodies two weeks after the initial inoculation. At the same time, the group of animals which did not develop ocular inflammation did possess com plement-fixing antibodies. The highest titers were obtained after the third week of im munization with a slight decrease during the fifth week. The complement fixing titers generally correlated with the skin reactivity observed in the animals of Group II. The demonstration of circulating anti bodies, the presence of an Arthuslike phe nomenon at the site of inoculation of the uveal-adjuvant emulsion, and the immediate response of the sensitized animals to a shock dose of homologous antigen would suggest an allergic reaction of the immediate type. However, the majority of the animals in the second group which were skin tested demonstrated a delayed rather than an ana phylactic-type of skin reactivity. This could be explained by the report of Dienes and Schoenheit8 who found that pro teins which induce anaphylactic sensitivity may give rise to a delayed type of sensi tivity if such antigens are injected into a tuberculous focus. Later, Freund and McDermott9 obtained the same effect when horse
383
AN IMMUNOLOGIC STUDY serum, which normally gives rise to an im mediate sensitivity was incorporated in an adjuvant. Whether or not this phenomenon is due to the presence of more than one anti body is not clear. The fact that a mild ocular sensitivity in the animals in Group II was detected after a period of three and six months and no sensitivity could be demonstrated in ani mals in Group I after 84 days would indi cate that Freund's adjuvant sustains sensi tization for longer periods of time. This finding is in agreement with the results of other investigators.9 The work presented thus far would in^ dicate that uveal tissue is antigenic since iso-antibodies can be demonstrated. Further more, this homologous tissue may cause an ocular inflammation and is capable of pro ducing skin sensitivity. In part, these find ings confirm the investigations of other authors 2 ' 10 · 11 and further support Elschnig's theory of the possible immunologie mech anism of sympathetic ophthalmia. Passive transfers by several methods were unsuccessful. However, this does not pre clude the presence of sensitizing antibodies in the sera since complement fixation titer of the sera used for passive transfer was 1:250. Perhaps the antibodies are removed from the circulation and neutralized as quickly as they are introduced artificially into the animals or perhaps the complementfixing antibodies are not responsible for the sensitization. Furthermore, the possibility that the antibody seemingly responsible for the inflammation of the eyes is of the sessile type should not be overlooked. Similarly, the many attempts by various investigators to produce encephalomyelitis passively by injecting large amounts of antibrain sera into animals have failed. The possibility exists that sensitization might occur, not only to uveal tissue but also to a bacterial-uveal complex similar to the immunologie mechanism postulated for rheumatic fever and glomerulonephritis. As mentioned previously, Cavelti12 was
able to demonstrate specific antibodies to kid ney by immunizing animals with Group A streptococci and homologous kidney tissue. This same investigator also was able to dem onstrate auto-antibodies to human heart ex tract in the sera of patients with rheumatic fever. With such a mechanism, one can postulate an immunologie process in human uveitis involving three methods : ( 1 ) Sensitivity to a bacterial-uveal tissue complex, (2) sensi tivity to uveal tissue, (3) sensitivity to bac teria and their products. Diagram 1 shows the different pathways which might be involved in the production of hypersensitivity resulting in clinical uve itis. The paths on the right hand side of the bar indicate the generally accepted route in certain cases of uveitis, while the hypo thetic mechanism, which is strengthened by the experimental results in this paper, is indicated on the right hand side. Modifica tion of the uveal tract could result as a direct injury to the eye either mechanically or biologically. Likewise, in the experimental work, the removed uveâl tracts undoubtedly were modified by mechanical grinding of the tissue. The correlations of sérologie, hypersensi tive, and clinical manifestations in experi mental animals immunized with homologous uveal tissue could lend support to an isoimmunologic mechanism which might occur in humans as a result of autosensitization to modified and normal uveal tissue. SUMMARY
Rabbits which were sensitized intraocularly and subsequently inoculated intrave nously with homologous uveal tissue devel oped inflammatory reactions in the anterior chamber. This sensitivity could not be dem onstrated 84 days after the intraocular in jections. During the period of sensitization, complement-fixation tests demonstrated the presence of antibodies. Neither the skin nor eyes could be sensitized by passive transfer of anti-uveal sera.
TED SUIE AND MATT C. DODD
384
VIHUSES, BACTEHIA ITERIAL .PRODUCTS -
I
TRAUMA
DIBECT INJURY TO UVEAL CELLS
PHODÜCTS OF UVEAL T I S S U E DAMA8B UVEAL TISSUE
REACTION WITH SENSITIZED UVEAL CULLS
AUTOAOTI BODIES, .CTERIAL-UVEAL* TISSUE COMPLEX
REACTION WITH MODIFIED AND NORMAL UVEAL CELLS
/
HTPERSENSITIVITT
UVEITIS
Diagram 1 (Suie and Dodd). Pathways which may be involved in the production of hypersensitivity resulting in clinical uveitis.
The rabbits which were inoculated into the footpads with uveal suspension mixed with Freund's adjuvant demonstrated com plement-fixing antibodies and a delayed-type of skin reactivity. Ocular inflammation could not be elicited by this method of immuniza tion. Sensitivity to homologous uveal tissue was demonstrated six months later by the appear ance of a fine precipitate in the anterior chamber following intravenous injection of the antigen. Passive transfer of this sensitization could not be demonstrated. Failure to produce an experimental uveitis
in these animals may be due to insufficient dosage of antigen or the presence of the blood-aqueous barriers in the anterior cham ber. Furthermore, it is conceivable that the local organ must be directly sensitized in order to produce this manifestation. The correlations of the sérologie, hyper sensitive, and clinical manifestations of ex perimental animals which were immunized with homologous uveal tissue are discussed. Various suggestions are made concerning the possible roles of this type of isoallergic phenomena in human uveitis. College of Medicine (10).
REFERENCES
1. Woods, A. C. : Endogenous Uveitis: A Manual. Am. Acad. Ophth., 1949. 2. Elschnig, A. : Studien zur sympthischin Ophthalmic Arch. f. Ophth., 75 :507-S46, 1910. 3. : Studien zur sympthischin Ophthalmie. Arch, f. Ophth., 76 :4S9-473, 1910. 4. : Studien zur sympthischin Ophthalmie. Arch. f. Ophth., 78:549-585, 1911. 5. Freund, J., Casals, J., and Hesmer, L. : Sensitization and antibody formation after injection of tubercle bacilli and paraffin oil. Proc. Soc. Exper. Biol. & Med., 37 :509-513, 1937. 6. Foss, F.: Experimental anaphylactic iridocyclitis. Acta path, et microbiol. scandinav. 8 1 : (Mono graph), 1949. 7. Seegal, D., and Seegal, B. C. : Local organ hypersensitiveness. J. Exper. Med., 54:249-263, 1931. 8. Dienes, L., and Schoenheit, E. W. : The reproduction of tuberculin hypersensitiveness in guinea pigs with various protein substances. Am. Rev. Tuberc, 20 :92, 1929. 9. Freund, J., and McDermott, D. : Sensitization to horse serum by means of adjuvants. Proc. Soc. Exper. Biol. & Med., 49 :548-553, 1942. 10. Henton, H. O. : Opsonic index for uveal pigment in treated patients. Arch. Ophth., 17:113-116, 1937. 11. Woods, A. C. : Immune reactions following injuries to the uveal tract. J.A.M.A., 77 :1317-1322, 1921. 12. Cavelti, P. A.: Autoantibodies in rheumatic fever. Proc. Soc. Exper. Biol. & Med., 78:59; 379-381, 1943.
377
4. Eames, T. H. : Comparison of eye conditions among 1,000 reading failures, 500 ophthalmic patients, and ISO unselected children. Am. J. Ophth., 31:713-717 (June) 1948. 5. Farris, L. P. : Visual defects as factors influencing achievement in reading. J. Exper. Education, 5:59 (Sept.) 1936. 6. Taylor, E. A. : Controlled Reading. Chicago, Univ. Chicago Press, 1937.
AN IMMUNOLOGIC STUDY O F RABBITS SENSITIZED W I T H HOMOLOGOUS U V E A L TISSUE* TED SUIE, P H . D . , AND MATT C. DODD, P H . D .
Columbus, Ohio INTRODUCTION
Generally, it is agreed that uveitis fre quently demonstrates allergic manifestations. This ocular sensitivity may be classified into the granulomatous and the nongranulomatous types. Woods 1 states that in the granulo matous type the tissues are actually in vaded by micro-organisms such as Mycobacterium tuberculosis and Treponema pallidum and the hypersensitivity results when the organism or its products again reaches the eye. On the other hand, the nongranu lomatous type is believed by some to be a sterile allergic reaction resulting from intoxi cation with specific allergens.1 In 1903, Elschnig 2 ' 3 ' 4 postulated that sym pathetic ophthalmia, a type of uveitis which is characterized chiefly by its tendency to involve the uveal tissue of both eyes, was an isoallergy to modified uveal tissue. Later, the recognition that certain tissues such as brain and lens protein possessed a greater degree of organ specificity than species specificity became perhaps one of the earliest demonstrations of the feasibility of autoantibody for normal tissues. Attempts to produce isoallergic conditions in experimental animals are numerous but until recently results were difficult to obtain ♦From the Departments of Ophthalmology and Bacteriology, College of Medicine, The Ohio State University. Grateful acknowledgement is made to W. D. Rummel, M.D., and H. Farmer, M.D., for their aid in biomicroscopic studies and dissections of the uveal tracts.
except by a long process involving many injections. Even then, the success was rela tively poor. However, with the introduction of the adjuvant technique by Freund, 5 a method was devised which greatly facilitated the production of experimental isoallergic encephalitis apparently by enhancing the pro duction of antibodies to homologous tissue extract. In fact, a single injection sufficed to produce this condition in some rabbits. The present work was undertaken to study the serology and hypersensitive reac tions which might occur in rabbits sensitized with homologous uveal tissue suspension with and without the aid of Freund's ad juvant. METHODS AND MATERIALS
Unless otherwise noted, albino rabbits of both sexes and varying weights were used throughout these experiments. None of the animals had a history of infection. All antigens used in this study were pre pared from rabbit uveal tissue. All materials were stored, without preservatives, at —23°C. Thioglycollate broth was used to determine the sterility of all the antigens. The uveal tracts were washed several times in saline. The tissue was then ground in a mortar and enough sterile saline was added to make dilutions of 1:10 and 1:20. The emulsion was placed in sterile vials. Each vial contained uveal tracts pooled from four or five rabbits. The 1:10 dilution of uveal suspension was used to sensitize the eyes,
378
TED SUIE AND MATT C. DODD
and the 1:20 dilution was employed for in travenous injections and for skin testing. Albino, as well as pigmented, rabbits were used for the preparation of uveal-adjuvant emulsion. The inoculated mixture consisted of 0.5 gm. of uveal tissue, 3.5 ml. of mineral oil, and 10.0 mg. of killed and dried tubercle bacilli. The bacteria were ground in a sterile mortar and oil was gradually added. This mixture was then introduced slowly into an homogenate of uveal tissue and again ground in a mortar. This emulsion was stored at - 2 0 ° C . until used. The tubercle bacilli suspension for skin testing was prepared as follows : Previously killed and dried tubercle bacilli were re duced to a fine powder in a mortar and enough sterile saline was added to make a final concentration of 0.2 mg. of tubercle bacilli per ml. of saline. The rabbits were divided into two groups : GROUP I
Nine rabbits were inoculated intraocularly with 0.1 ml. of a 1:10 dilution of uveal ex tract. One-half percent pontocain was used as the local anesthetic. Only one eye was inoculated; the fellow eye served as a con trol. Normal nonimmunized rabbits also served as controls. A 27-gauge hypodermic needle attached to a 0.25-ml. tuberculin syringe was inserted tangentially into the anterior chamber at the limbus. Then 0.2 ml. of aqueous humor was removed. The needle was left in position and a new syringe con taining 0.1 ml. of uveal suspension was ex changed for the one containing the aqueous humor. This material was then injected into the anterior chamber slowly. Extreme pre cautions were taken in order to prevent in jury to the lenses. After the nonspecific inflammation sub sided, the rabbits were given one ml. of a 1:20 dilution of homologous antigen intra venously. The rabbits were observed with the slitlamp and macroscopically immediately be
fore the injection, one hour later, and after ward at indefinite intervals. GROUP II
This group consisted of 18 rabbits inocu lated into the footpads of both hind legs with uveal-adjuvant emulsion. The hair on the footpads was removed with an electric clipper and the pads were cleaned carefully with 70-percent alcohol. Using a 27-gauge needle and a 0.25-ml. tuberculin syringe, 0.05 ml. of this emulsion was injected intradermally into each of the footpads. All animals in this group were skin tested with 0.05 ml. of tubercle bacilli suspension and 0.1 ml. of uveal suspension. The right side and left side of the rabbits were clipped and cleaned thoroughly with 70-percent al cohol prior to the injections. Separate syringes and needles were used. Animals from both groups were bled by cardiac puncture at weekly intervals and the sera were stored at —23°C. All the rab bits were observed daily either macroscopi cally or with the slitlamp for evidence of eye inflammation. Three rabbits from Group II were later inoculated intravenously as described above. Controls consisted of two normal, nonimmunized animals which were injected in travenously with 1.0 ml. of uveal tissue and two immunized rabbits which received 1.0 ml. albumin by the same route. Complement fixation tests were performed as follows : The anticomplementary value of the an tigens was determined before each series of tests. Commercially prepared, lyophilized complement was used throughout. Twentyfive hundredths ml. of 1:40 saline suspen sion of uveal tissue was added to a series of tubes containing the following amounts of antisera : 0.5, 0.2, 0.1, 0.05, 0.02 ml. Then, 0.1 ml. of complement, containing two units, was added to each. The tubes were incu bated at refrigerator temperature overnight and then the indicator system was added. Preliminary readings were made after the
AN IMMUNOLOGIC STUDY tubes were incubated at 37°C. for 30 min utes, and final readings after storing over night at refrigerator temperature. Serum, antigen, hemolytic system, and cell controls were included. The results were recorded ac cording to the degree of fixation of comple ment. Gradations in fixation ranged from 4 + for complete fixation to — (minus) for absence of fixation. Several methods were used in an attempt to transfer the hypersensitivity passively. The first method consisted of injecting each of two normal albino rabbits intradermally with 0.2 ml. of uveal antiserum into three sites, one of which served as a control. Eighteen hours later 0.1 ml. of antigen (homologous uveal extract) was injected into one of the sites and 0.02 ml. of the anti gen was injected into another. An addi tional injection of 0.1 ml. of antigen was used as a control. The rabbits were then ob served for anaphylactic and tuberculinlike reactions. The second method was carried out as follows : Three rabbits which had been immunized with uveal-adjuvant emulsion one and a half months prior to the test and two nor mal rabbits were given 0.2 ml. of rabbit uveal antisera into the anterior chamber of the right eye. Normal rabbit sera was in troduced into the fellow eye of each animal as a control. The sera were obtained by car diac puncture and precautions were taken to insure sterility. The eyes of the rabbits were examined daily macroscopically and periodi cally with the slitlamp. The titer of the sera was determined by complement fixation tests prior to the injections. FINDINGS
Animals in Group I demonstrated inflam matory reactions in the anterior chamber after the homologous antigen was given in travenously. In some animals the changes in the anterior chamber could be observed dur ing the first 24 hours after the shock dose was administered.
379
These changes were manifested by lacrimation, injection of the vessels of the an terior segment of the eye, and the presence of flocculent material in the anterior cham ber of the injected eyes. Within three days, one could generally observe keratic precipitates on the endo thelium of the cornea, aqueous rays, fibrinous exudates in the anterior chamber, as well as hyperemia of the circumcorneal, iris, and conjunctival vessels. Only one animal in this group demonstrated a sympathetic ophthal mialike reaction. Periodic observations of rabbits in Group II with the slitlamp for several months failed to reveal evidence of ocular inflam mation. Two rabbits (CI and CII) which had been inoculated with the uveal-adjuvant emulsion six months previously and one rabbit ( D I ) which had been sensitized with the same material for a period of three months were again observed with the slitlamp and appeared normal. Immediately afterward, each animal re ceived 1.0 ml. of a 1:20 dilution of aqueous uveal suspension intravenously. No evidence of inflammation of the eyes appeared after one hour. However, after 24 hours both eyes showed varying degrees of diffuse, fine, al bumin-type precipitation on the endothelium of the cornea. No aqueous ray was noted and there was a minimal hyperemia of the iris vessels. The eyes appeared normal in four days. The rabbits immunized with the uvealadjuvant emulsion did not develop inflam mation within four days after the intra venous injection of albumin. Likewise, nonimmunized rabbits did not demonstrate in flammation after the injection of aqueous uveal suspension intravenously. In contrast, rabbits in Group I, which had previously demonstrated a local organ hy persensitivity 84 days before, did not show evidence of uveitis before or after the in travenous injection of aqueous uveal extract. Complement fixing antibiodies were dem onstrated in the sera of rabbits in Group I
T E D S U I E A N D M A T T C. D O D D
380
TABLE 1 COMPLEMENT FIXATION TITERS OF SELECTED RABBITS IN G R O U P II WHICH W E R E INOCULATED WITH THE UVEAL-ADJUVANT EMULSION
Rabbit Number Reciprocal of Serum Dilution
12.5 25 50 125 250
cm
CI Normal —. —· — — —
Weeks 2 1+ 1+ 2+ 2+ 1+
3
5
4+ 2+ 2+ 1+ 1+
Normal — — — — —
3+ 1+ 1+ + +
(table 1). Significant rise in titer occurred during the third week after the inoculation. Normal antibodies were detected in titers of 1:12.5 and 1:25. The complement fixa tion titers were greater in the animals in Group II as compared with those in Group I (tables 2 and 3). The highest titers in the former were detected during the third week after the injections. No complement fixing antibodies were demonstrable in the aqueous humor of the injected eyes or in the con trols. As indicated in Tables 3 and 4, rabbits in Group II which were inoculated with uvealadjuvant emulsion demonstrated a delayed type of skin hypersensitivity to the aqueous suspension of uveal extract. The skin reac tivity, both to the uveal suspension and tubercle bacilli suspension, developed as early as four days after the inoculation and was still present two weeks later. There was very little change in the de
Weeks 2
3
5
2+ 4+ 3+ 2+ 1+
1+ 2+ 3+ 2+ 1+
1+ 2+ 2+ + —
gree of skin reactions to uveal suspension in the same rabbits tested four days and 14 days after the inoculations. However, the acid-fast bacilli suspension induced a greater area of hyperemia and induration after 14 days. The control rabbit, which did not re ceive uveal-adjuvant emulsion previously, was not sensitive to the uveal or tubercle bacilli suspensions. Passive transfer of such skin sensitivity was not accomplished. The serum control was negative 24 hours after the inoculation. Ap proximately the same amount of erythema and induration appeared in the control site (antigen alone) as in the site containing the antigen-serum mixture. In 24 and 48 hours the antigen control and test areas showed the same amount of induration and erythema. The attempts to transfer sensitivity pas sively by means of anti-uveal sera into the eye also were unsuccessful. An iritis and
TABLE 2 COMPLEMENT FIXATION TITERS O F SELECTED RABBITS IN G R O U P I WHICH WERE INOCULATED INTRAOCULARLY AND SUBSEQUENTLY GIVEN AN INTRAVENOUS INJECTION OF HOMOLOGOUS ANTIGEN
Rabbit Number of Serum 12.5 25 50 125 250
AI
All
Normal
20 Days
Normal
5 Days
20 Days
2+ 1+ + + +
2+ 2+ 3+ 2+ 1+
2+ +
2+ 2+ 1+ 2+ 1+
2+ 2+ 2+ 2+ 1+
±
— —
AN IMMUNOLOGIC STUDY
381
TABLE 3 RESULTS OF SKIN TESTS WITH UVEAL SUSPENSION AND TUBERCLE BACILLI SUSPENSION FOUR DAYS AFTER INOCULATION
Rabbit Number
T.B.
48 Hours
24 Hours
3 Hours
Uvea
T.B.
++ +++ +++ ++ ++
++ +++ ++ ++
'+ + + ++
+
+ ++ +++ ++ ++
+ ++
±
+++ ++
+ ++
++
T.B.
Uvea
Cl C2 C3 C4 CS C6 C7 C8 C9 (control)*
+
Uvea
±
+
+
±
++ ++
* Control did not receive uveal-adjuvant emulsion + — No reaction ± Barely perceptible erythema or induration (neither exceeding 5 mm. in diameter) + Erythema and induration between 5 to 10 mm. in diameter + + Erythema and induration between 10 to 15 mm. in diameter + + + Erythema and induration exceeding IS mm. in diameter + + + + Very marked reactions with necrosis
circumcorneal injection developed within sev eral days. However, the control eyes which were injected with normal rabbit sera pro duced a similar manifestation. This inflam mation was considered nonspecific and was probably due to the intraocular injections. After a period of two weeks both eyes ap peared normal and no evidence of uveitis developed during the three months of obser vation. DISCUSSION
The experimental findings in the present work indicate that rabbits, which had been previously sensitized to uveal tissue intra-
ocularly, develop changes in the anterior chamber as a result of a shock dose of homologous antigen given intravenously two weeks later. These manifestations varied in degree from one animal to another. Generally, one could observe keratic pre cipitates on the endothelium of the cornea, aqueous ray, fibrinous exudates in the ante rior chamber, as well as hyperemia of the circumcorneal, iris, and conjunctival vessels. Macroscopic evidence of inflammation oc curred in a few animals and consisted of lacrimation and of hyperemia of the vessels of the anterior segment of the eye. Only one rabbit out of seven in Group I
TABLE 4 R E S U L T S OF SKIN TESTS WITH UVEAL SUSPENSION AND TUBERCLE BACILLI SUSPENSION FOURTEEN DAYS AFTER INOCULATION
Rabbit Number
3 Hours T.B.
Cl C2 C3 C4 C5 C6 C7 C8 C9 (control) See Table 3 for legend.
24 Hours Uvea
T.B.
++ +++ +++ +++ +++ ++ ++ ++ +
48 Hours
Uvea
++ +++ ++ ++ +++ + ±
++
T.B.
+++ ++++ +++ +++ ++ + ++ ++
Uvea
++
+++ ++ ++ + + +
++
382
TED SUIE AND MATT C. DODD
demonstrated a sympathetic ophthalmialike effect. Other workers 6 ' 7 have also noted a sympathizing effect in animals sensitized with foreign proteins. The white precipitate which was seen in the anterior chamber in certain of the ani mals might have been the result of an anti body-antigen reaction in vivo. Foss e noted that if one injected 0.1 ml. of horse serum into the vitreous of rabbits and the same amount of antihorse serum was injected into the anterior chamber, precipitation occurred in the anterior chamber. The precipitates were partly deposited as a ring around the pupillary brim. Rabbits in Group II which had been in oculated into the footpads with uvealadjuvant emulsion did not show evidence of ocular inflammation during the several months of observation with the slitlamp. Since trauma and infection will change the permeability of the surface membrane of the eye, various substances injected into the blood and which ordinarily would not get into the aqueous humor because of the normal blood-aqueous barriers, may now be found in the fluid of the anterior chamber. This phenomenon may explain the devel opment of eye inflammations which occurred in the rabbits in Group I and the failure of this reaction to appear in the eyes of the animals in Group II. The former group received antigenic stim ulation directly into the anterior chamber and as a result of the injection and paracentesis the normal blood-aqueous barriers were modified. Since residual inflammation was still present at the time of the shock dose, it is highly doubtful that these barriers were restored during the period of sensitization. Subsequent injection of the homologous antigen intravenously might permit the anti gen to react with the preformed antibodies present in the aqueous humor. In the rabbits of Group II, the barriers probably played an important role in pre venting localization of the antigen in the anterior chamber. However, other possibili
ties for the failure to incite ocular inflam matory reactions by this method may exist. Perhaps the antigenic dosage which was in corporated in the adjuvant was too small to elicit an ocular manifestation. Three animals which had been sensitized with uveal-adjuvant emulsion developed slight changes in the anterior chamber when large amounts of homologous antigen was inoculated intravenously even after an elapse of time of six months from the primary injections. This would indicate that larger amounts of uveal tissue added to the adju vant might suffice to produce local organ hypersensitivity. Furthermore, it is conceivable that the local organ must be directly sensi tized in order to produce an ocular in flammation. The rabbits in Group I which demon strated an ocular anaphylactic-type of reac tion also had circulating antibodies two weeks after the initial inoculation. At the same time, the group of animals which did not develop ocular inflammation did possess com plement-fixing antibodies. The highest titers were obtained after the third week of im munization with a slight decrease during the fifth week. The complement fixing titers generally correlated with the skin reactivity observed in the animals of Group II. The demonstration of circulating anti bodies, the presence of an Arthuslike phe nomenon at the site of inoculation of the uveal-adjuvant emulsion, and the immediate response of the sensitized animals to a shock dose of homologous antigen would suggest an allergic reaction of the immediate type. However, the majority of the animals in the second group which were skin tested demonstrated a delayed rather than an ana phylactic-type of skin reactivity. This could be explained by the report of Dienes and Schoenheit8 who found that pro teins which induce anaphylactic sensitivity may give rise to a delayed type of sensi tivity if such antigens are injected into a tuberculous focus. Later, Freund and McDermott9 obtained the same effect when horse
383
AN IMMUNOLOGIC STUDY serum, which normally gives rise to an im mediate sensitivity was incorporated in an adjuvant. Whether or not this phenomenon is due to the presence of more than one anti body is not clear. The fact that a mild ocular sensitivity in the animals in Group II was detected after a period of three and six months and no sensitivity could be demonstrated in ani mals in Group I after 84 days would indi cate that Freund's adjuvant sustains sensi tization for longer periods of time. This finding is in agreement with the results of other investigators.9 The work presented thus far would in^ dicate that uveal tissue is antigenic since iso-antibodies can be demonstrated. Further more, this homologous tissue may cause an ocular inflammation and is capable of pro ducing skin sensitivity. In part, these find ings confirm the investigations of other authors 2 ' 10 · 11 and further support Elschnig's theory of the possible immunologie mech anism of sympathetic ophthalmia. Passive transfers by several methods were unsuccessful. However, this does not pre clude the presence of sensitizing antibodies in the sera since complement fixation titer of the sera used for passive transfer was 1:250. Perhaps the antibodies are removed from the circulation and neutralized as quickly as they are introduced artificially into the animals or perhaps the complementfixing antibodies are not responsible for the sensitization. Furthermore, the possibility that the antibody seemingly responsible for the inflammation of the eyes is of the sessile type should not be overlooked. Similarly, the many attempts by various investigators to produce encephalomyelitis passively by injecting large amounts of antibrain sera into animals have failed. The possibility exists that sensitization might occur, not only to uveal tissue but also to a bacterial-uveal complex similar to the immunologie mechanism postulated for rheumatic fever and glomerulonephritis. As mentioned previously, Cavelti12 was
able to demonstrate specific antibodies to kid ney by immunizing animals with Group A streptococci and homologous kidney tissue. This same investigator also was able to dem onstrate auto-antibodies to human heart ex tract in the sera of patients with rheumatic fever. With such a mechanism, one can postulate an immunologie process in human uveitis involving three methods : ( 1 ) Sensitivity to a bacterial-uveal tissue complex, (2) sensi tivity to uveal tissue, (3) sensitivity to bac teria and their products. Diagram 1 shows the different pathways which might be involved in the production of hypersensitivity resulting in clinical uve itis. The paths on the right hand side of the bar indicate the generally accepted route in certain cases of uveitis, while the hypo thetic mechanism, which is strengthened by the experimental results in this paper, is indicated on the right hand side. Modifica tion of the uveal tract could result as a direct injury to the eye either mechanically or biologically. Likewise, in the experimental work, the removed uveâl tracts undoubtedly were modified by mechanical grinding of the tissue. The correlations of sérologie, hypersensi tive, and clinical manifestations in experi mental animals immunized with homologous uveal tissue could lend support to an isoimmunologic mechanism which might occur in humans as a result of autosensitization to modified and normal uveal tissue. SUMMARY
Rabbits which were sensitized intraocularly and subsequently inoculated intrave nously with homologous uveal tissue devel oped inflammatory reactions in the anterior chamber. This sensitivity could not be dem onstrated 84 days after the intraocular in jections. During the period of sensitization, complement-fixation tests demonstrated the presence of antibodies. Neither the skin nor eyes could be sensitized by passive transfer of anti-uveal sera.
TED SUIE AND MATT C. DODD
384
VIHUSES, BACTEHIA ITERIAL .PRODUCTS -
I
TRAUMA
DIBECT INJURY TO UVEAL CELLS
PHODÜCTS OF UVEAL T I S S U E DAMA8B UVEAL TISSUE
REACTION WITH SENSITIZED UVEAL CULLS
AUTOAOTI BODIES, .CTERIAL-UVEAL* TISSUE COMPLEX
REACTION WITH MODIFIED AND NORMAL UVEAL CELLS
/
HTPERSENSITIVITT
UVEITIS
Diagram 1 (Suie and Dodd). Pathways which may be involved in the production of hypersensitivity resulting in clinical uveitis.
The rabbits which were inoculated into the footpads with uveal suspension mixed with Freund's adjuvant demonstrated com plement-fixing antibodies and a delayed-type of skin reactivity. Ocular inflammation could not be elicited by this method of immuniza tion. Sensitivity to homologous uveal tissue was demonstrated six months later by the appear ance of a fine precipitate in the anterior chamber following intravenous injection of the antigen. Passive transfer of this sensitization could not be demonstrated. Failure to produce an experimental uveitis
in these animals may be due to insufficient dosage of antigen or the presence of the blood-aqueous barriers in the anterior cham ber. Furthermore, it is conceivable that the local organ must be directly sensitized in order to produce this manifestation. The correlations of the sérologie, hyper sensitive, and clinical manifestations of ex perimental animals which were immunized with homologous uveal tissue are discussed. Various suggestions are made concerning the possible roles of this type of isoallergic phenomena in human uveitis. College of Medicine (10).
REFERENCES
1. Woods, A. C. : Endogenous Uveitis: A Manual. Am. Acad. Ophth., 1949. 2. Elschnig, A. : Studien zur sympthischin Ophthalmic Arch. f. Ophth., 75 :507-S46, 1910. 3. : Studien zur sympthischin Ophthalmie. Arch, f. Ophth., 76 :4S9-473, 1910. 4. : Studien zur sympthischin Ophthalmie. Arch. f. Ophth., 78:549-585, 1911. 5. Freund, J., Casals, J., and Hesmer, L. : Sensitization and antibody formation after injection of tubercle bacilli and paraffin oil. Proc. Soc. Exper. Biol. & Med., 37 :509-513, 1937. 6. Foss, F.: Experimental anaphylactic iridocyclitis. Acta path, et microbiol. scandinav. 8 1 : (Mono graph), 1949. 7. Seegal, D., and Seegal, B. C. : Local organ hypersensitiveness. J. Exper. Med., 54:249-263, 1931. 8. Dienes, L., and Schoenheit, E. W. : The reproduction of tuberculin hypersensitiveness in guinea pigs with various protein substances. Am. Rev. Tuberc, 20 :92, 1929. 9. Freund, J., and McDermott, D. : Sensitization to horse serum by means of adjuvants. Proc. Soc. Exper. Biol. & Med., 49 :548-553, 1942. 10. Henton, H. O. : Opsonic index for uveal pigment in treated patients. Arch. Ophth., 17:113-116, 1937. 11. Woods, A. C. : Immune reactions following injuries to the uveal tract. J.A.M.A., 77 :1317-1322, 1921. 12. Cavelti, P. A.: Autoantibodies in rheumatic fever. Proc. Soc. Exper. Biol. & Med., 78:59; 379-381, 1943.