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Specificity of antibody formation after intravitreal immunization with bovine gamma globulin and ovalbumin
CELLULAR
IMMUNOLOGY
Specificity
11,
64-73 (19%)
of Antibody with Bovine
Formation After Gamma Globulin
ft. Nonspecific
Enhancement
of the
Intravitreal Immunization and Ovalbumin Secondary
Response 1
JOAN M. HALL Francis
I. Proctor
Foundation
Research
for
in
San Fruncisco,
Ophthalmology,
California
University
of California,
University
of
94143
AND JAMES Naval Biomedical
Research
Laboratory, Berkeley, Received
F. PRIBNOW of Public Health, California 94720
School
August
California,
16, 1973
Rabbits immunized intravitreally with bovine gamma globulin and ovalbumin were challenged intravitreally with both bovine gamma globulin and ovalbumin, with bovine gamma globulin and human serum albumin, or with bovine gamma globulin alone. The specificity demonstrated in the primary response did not appear in rabbits challenged with both antigens ; antibody-producing cells of both specificities were found in the uveal tracts of both eyes. Anti-ovalbumin-producing cells were found in the uveal tracts of the rabbits challenged with bovine gamma globulin alone, or with bovine gamma globulin and human serum albumin. Anti-bovine gamma globulin producing cells were also present. No antibody-producing cells were found in the unchallenged eyes of rabbits challenged with bovine gamma globulin aIone. The action of a nonspecific soluble enhancing factor is postulated to explain the enhanced response to ovalbumin that resulted when rabbits were challenged with bovine gamma globulin alone.
INTRODUCTION Our previous publications have described the cellular responses that follow intravitreal injection of protein antigens into the eyes of rabbits (1, 2). Although the majority of the antibody producing cells were found in the uveal tracts of these animals, plaque forming cells (PFC) were first demonstrated in the extraocular lymphoid tissues (1) . Rabbits immunized intravitreally with small amounts of antigen developed high serum antibody titers (2). The titers in the aqueous and vitreous humors were higher than the serum titers. Antigen was present in the vitreous for at least two weeks after its injection (1). A second intravitreal 1 This work was supported in part by United States Public Health Service Program Grant EY-00310, and by the Office of Naval Research under a contract between the Office of Naval Research and the Regents of the University of California. 64 Copyright All rights
0 1974 by Academic Press, of reproduction ,in any form
Inc. reserved.
SPECIFICITY
OF
ANTIBODY
FOXMATJOIG
II
6.5
injection in the opposite eye resulted in the accelerated protluction of serunl antibody. In other experiments (3) rabbits were injected with bovine gamma globulin ( RGG) in the right eye and with ovalbumin (0-A) in the left eye. Uveal tract cells from the right eye produced only anti-BGG. and uveal tract cells from the left eye produced only anti-OA. although antibody specific for both antigens was present in the serum and in the aqueous humor from both eyes. .-\tltlitional investigations showed that the specificity of antihotly formation by uveal tract cells persisted for at least 23 days after a primary intravitreal injection. The initial purpose of the present investigations was to determine whether the specificity of the antibody response would be retained after intravitreal challenge with the same protein antigens used in the sensitizing injections. This reljort also clescrihes nonspecific enhancement of the secondar\- ocular immune response. 14ATERIAT5
AKD
11 ETHODS
Immunization
The experimental animals were 48 New Zealand white rabbits weighing G.5 11). In a series of experiments, they were injected intravitreally with approximatel! 1.5 mg of various antigens according to a method previously described (2). Group 1 was injected with BGG only and in the right eye only, Groups 2-7 were injected with BGG in the right eye and OA in the left eye, and a final group of six rabbits was injected with BGG in the right eye and HSA in the left eve. Thr animals were bled at stated intervals, and the hemolvtic antibodv titers wf’rc cletermined. The rabbits were challenged approximately five months after the sensitizing injections. No hemolytic antibody was demonstrable at the time of challenge. Rabbits initially injected with BGG, or with EGG and OA or HSA in comhination, were challenged according to the following protocol : Group Group (;roup Group Group Group Group Groul)
1. 2. 3. 4. 5. 6. 7. 8.
BGG in the left eye only (14 rabbits). BGG in the right eye and OA in the left eye (10 rabbits). BGG in the left eye and OA in the right el-e (7 rabbits). BGG in the right eye only. Left eye unchallenged (4 rabbits) BGG in the left eye only. Right eye unchallenged (3 rabbits 1. BGG in the right eye and HSA in the left eye (2 rabbits). BGG in the left eye and HSA in the right eye (2 rabbits‘). Tn the final group of six rabbits whose left eyes \vere sensitized to TTSA instead of to OA, the same challenges wet-c made as in tllr :il)cn~ groups escept that nonr of the animals received 3 h~lcrolog~~~s antigen.
1n all instances
the dose of the challenge antigen 1~;~s 1 ..q mg.
The rabbits were killed on the sixth postchallengr day. The ryes were enucleated and the aqueous and vitreous humors aspirated. Cell suspensions were made fro111 the uveal tracts of both eyes, from the cer\.icxl lymph nodes from both sides, and in some cases from the spleen. The procedure for enumerating the plaque-forming
66
SECONDARY
HALL
AND
PRIBNOW
TABLE
1
RESPONSE OF RABBITS INJECTED CHALLENGED WITH INTRAVITREAL Source of cells assayed
Right eyes Left eyes
INTRAVITREALLY WITH BGG INJECTION OF BGG IN LEFT
Average no. PFC per million cells 0 1482
Average
IN RIGHT EYE ONLY. EYE ONLY
peak hemagglutinin titer (Logz)
Primary response Secondary response
3.2 1 9.84
cells (PFC) has previously been described (1). The number of PFC are expressed as the number per log nucleated cells. In the case of the uveal tract cells, the plaque numbers may actually have been higher than stated in the tables since many of the nucleated cells were not lymphoid cells. Antibody
determination
Antibody titers in the serum and in the aqueous and vitreous humors were determined separately by a hemolytic assay previously described (1). We inactivated the serum samples by heating them at 56°C for 30 min, and we removed nonspecific hemolytic antibody by absorbing the samples with washed sheep erythrocytes. Since the aqueous and vitreous samples usually showed little or no nonspecific lysis of the control sheep erythrocytes, these samples were not inactivated. As described in a previous publication (3), we determined the presence of precipitating antibody by double-diffusion in agarose, and the presence of residual antigen in vitreous samples by immunoelectrophoresis. RESULTS Table 1 shows the results of plaque assays on 14 rabbits whose right eyes were initially injected with BGG and that were challenged approximately five months later by intravitreal injections of 1.5 mg BGG into the previously uninjected left eyes. All rabbits developed uveitis in the left eye within two days of the challenge injection and were killed on postchallenge Day 6. There were no PFC in any of the unchallenged right eyes but seven of the 14 rabbits had more than 2000 plaques in the uveal tract cells of the challenged left eyes. In a previous study (3) there were few or no plaques present in any uveal tract cells on the sixth day after primary injections of BGG. The results of the plaque assays and antibody determinations in the rabbits in Groups 2 and 3 are presented in Tables 2 and 3. The responses of the two groups were similar. PFC for both BGG and OA were found in the uveal tract cells of both right and left eyes. This was true whether a given eye had been challenged with the antigen used for initial sensitization, or with the antigen initially injected into the contralateral eye. In most cases there were more PFC for the antigen used to sensitize the challenged eye than for the antigen used to sensitize the opposite eye. Few PFC were found in the lymph nodes, and no specific plaques were found in the spleens tested. Hemolytic antibody specific for both BGG and OA was demonstrated in the serum and in the aqueous humor from both the right and left
SPECIFICITY
Y\I~KR.~GK
OF
PFC ASI) ANTIBODY RIBPONSE IN T,EFT EYE. CHALLENGED
0.4
ANTIRODY
I:ORRfATION
OF RABBITS WITH BGG IN
INJECTLI) WITH RIGHT Eye AM)
11
BGG OA
IS RIGHT I
.iverage hemolyt antibody titer (I,ogL’)
Right Left Right
eyes”
10 9 6
eyes* lymph
Ideft lymph
nodes nodes
6 4
BGG 0‘4 BGG OA BGG OA BGG 0‘1
671 97 113 147 58 21 3 6
(46-1287) (O-617) (O-32.5) (3-581) (O-101)
.\NI)
ic
h11pk
BGG
o:\
Serum
6..37
7.87
1.0 5.i
5.62 6.87
4..13 6.85
.5.88 0.0
Right Vitreous Right hclueolki I
‘l All right eyes contained PFC for BGG; 7 of 10 right eyes contained PITC for OA. ‘) .\I1 left eyes contained PFC for OX ; 6 of 7 left eyes contained PFC for 13GG. r Number varies because (1) in some cases the sheep erythrocytes did not coat with Or\, and the I’FC could not be determined; and (2) many eyes injected with OrI developed lenh olxtcities beveral months after initial injection. In some rabbits the cornea was alho opaque.
eyes. When no antibody was demonstrable in the vitreous humor, immunoelectrophoresis showed the presence of residual antigen. The precipitating antihotly determinations correlated well with the hemolytic antibody determinations.
Right
eyes”
I>eft eyes” Right Left
lymph lymph
nodes nodes
7 6 6 5 6 6 5 5
BGG OA BGG OA BGG 0‘4 BGG 0‘4
781 42.5 801 314 21 4 59 6
(36-2308) (74-923) (65-2666) (O-1166) (O-65) (O-21) (O-129) (O-30)
Serum Right \Ytreous Right Aqueous I.eft \“itreous I xft .\qu’ou~
6.2 1 I .o 3.8 1l.i
7.2 0 75 .i .6 11.7
(I All right eyes contained PFC for BGG; 5 of 6 right eyes contained PFC for 0.X. (III OIW VSperiment the sheep erythrocytes did not coat with 0.4). * Five of six left eyes contained PFC for both BGG and OA; 4 of 5 left eyes contained I’t;C for OA. (One microphthalmic left eye was not used; lens was opaque and only a few cells were obtainable from the minced uvea).
68
HALL
AND
PRIBNOW
The responses of the rabbits in Groups 2 and 3 suggested that the specificity of the primary response was lost when the eyes were challenged with the sensitizing antigens. Two possible explanations were (i) that a sufficient amount of antigen reached the opposite eye to initiate antibody production, or (ii) that circulating memory cells were attracted to both eyes because both were undergoing an inflammatory response. The next two experiments were designed to test the effect of challenge with only one of the sensitizing antigens. Would ‘the previously injected contralateral eye react under these conditions ? As shown in Table 1, rabbits injected with a single antigen and challenged in the opposite eye had antibody-producing cells only in the challenged eye. The seven rabbits in Groups 4 and 5 were challenged with BGG only. The opposite eyes were left unchallenged. The results of the plaque assays and antibody determinations on the rabbits in these groups are presented in Tables 4 and 5. With the exception of rabbits 401 and 422, the unchallenged eyes of these rabbits did not have PFC for either antigen. The number of PFC in the unchallenged eyes of rabbits 401 and 422 was much smaller than the number in the challenged TABLE PFC
Rabbit no.
400
AND
ANTIBODY RESPONSE IN RABBITS INJECTED IN LEFT EYE. CHALLENGED WITH BGG
Source of cells assayed RE
LE RLN 401
RE
LE RLN 416
RE
LE 421
4
RE
LE LLN
PFC per million cells
WITH BGG IN RIGHT IN RIGHT EYE ONLY
Hemolytic Sample
BGG OA
2,000 239
BGG OA BGG OA BGG OA
0 0 0 0 11,000 1,900
BGG OA BGG OA BGG OA
100 33 8 0 10,000 740
BGG OA BGG OA
0 0 200 0
BGG OA BGG OA
0 0 0 0
EYE
antibody
titer
Anti-BGG
AND
OA
(Log*) Anti-OA
8+
8+ 8+
0 3
0 6
Serum R. aqueous R. vitreous L. aqueous L. vitreous
12+ 8+
12+ 8+ 8+
Serum R. aqueous R. vitreous L. aqueous L. vitreous
8 12 0 6 5
Serum R. aqueous R. vitreous L. aqueous L. vitreous
12
Serum R. aqueous R. vitreous L. aqueous
4
0 4 2
8 8
8 12 0 7 4 8
12+
12+
0 0 1
0 0 4
SPECIFICITY
OF
ANTIBODY
FORMATION
00
II
Inillion .\trti-HGG
cells BGG
0 0
OA
BGG OA RGG 413
OA BGG OX
BGG 0-l 422
BGG OA I3GG 0.4
BGG OAl BGG OA
9,000 1,000
900 30 0 0
5,000 39 0 0 50 0
1,876
I2 0
\nti-0.4 10
0
XS 8+
8+ (I
8+ 0
11
0
I 7
0 (I
12
10
.(
(1
12+ 3
II+ (I
0 12+ 0
II I!+ 0
11
23 4
eyes. As expected, the challenged eyes contained many BGG-specific PFC. Ilowever, OA-specific PFC were found in six of the challenged eyes, even though no OA was reinjected. Anti-OA antibody was demonstrated in the serum and aqueous llumor of the challenged eyes and in some of the aqueous and vitreous samples from the contralateral eyes. Titers for OA were often as high as those for Ti(X;. but the number of anti-OA-producing cells was smaller. Similar results were obtained with one rabbit challenged with OrZ in tile right eye only. The uveal tract cells from the right eye and the lymph node cells from the right side contained anti-BGG-producing cells. and anti-RGG-alltihod~ WIS found in the aqueous and vitreous humors from the right eye. The results of the plaque assays on the rabbits in (;rnups 3 nlltl 5 indicatc~tl that. as in the case of the rabbits sensitized in one eye only and challenged in the other -eye only, PFC were found only in the challenged eyes. 111 order to demonstrate loss of specificity, it is apparently necessary to induce inflammation in both eyes. The next two experiments were designed to test the effect of nonspecific inflammation on an eye by challenging it with an antigen unrelated to either of the sensitizing antigens. If one eye were challenged with IHSA ant1 the other with BGG. would the eye challenged with HSA have anti-BGG-producing cells, or would thy inflammation have to be induced by one of the initially injected (sensitizing) antigens ?
70
HALL
AND
PRIBNOW
Tables 6 and 7 present the results of the plaque assays and antibody determinations on the rabbits in Groups 6 and 7. The eye challenged with BGG developed inflammation within two days after challenge, while the eye injected with HSA showed only minimal inflammation at the time of sacrifice six days later. The two rabbits in Group 6 had anti-BGG-producing cells in both the right and left eyes. Both of the BGG-challenged left eyes and one of the HSA-challenged right eyes of the rabbits of Group 7 also contained BGG-specific-PFC. Both rabbits in this group had anti-OA-producing cells in the BGG-challenged left eyes (previously sensitized to OA). All rabbits tested for hemolytic antibody titers in both groups had antibody to OA in their sera and in some of the aqueous and vitreous samples. To confirm the observations reported above, similar experiments were performed with rabbits initially given BGG and HSA. Rabbits challenged with both antigens had responses similar to those obtained in Groups 2 and 3. Technical problems have precluded the presentation of results of assays on rabbits challenged with only BGG or HSA. DISCUSSION Our previous experiments uveal tract was the primary onstrated, as did Silverstein
showed that after intravitreal injection of BGG the site of antibody production (1). However, we dem(4), that extraocular lymphoid cells were stimulated. TABLE
PFC
Rabbit ll0.
418
AND ANTIBODY 1~ LEFT EYE.
Source of cells assayed RE
LE
LLN
RLN
420
RE
LE
RLN
RESPONSE CHALLENGED
IN
6
RABBITS INJECTED WITH WITH BGG IN RIGHT EYE
PFC per million cells
Hemolytic Sample
BGG OA HSA
192 0 0
BGG OA HSA BGG OA HSA BGG OA HSA BGG OA HSA
341 0 0 8 4 0 6 12 0 1,300 0 0
BGG OA HSA BGG OA HSA
13 0 13 3 2 0
Serum R. aqueous R. vitreous L. aqueous L. vitreous
Serum R. aqueous R. vitreous L. aqueous L. vitreous
BGG AND
IN RIGHT EYE AND IN LEFT Eye
OA
HSA
antibody Anti-BGG
titer
(Logz)
Anti-OA
ND
ND
12+
12+
0 0 0
0 0 0
8
Anti-HSA ND 0 0 0 0
11
0
12+
12+
0
0 3 0
0 3 0
0
0 0
l
71
BGG OX HSA
0 0 0
BGG 0‘4 HSA BGG 0‘4 HSA BGG 0‘4 HSA BGG o=\ HS,\
448 60 0 0 0 0 71 7 3 23 0 0
BBG 0‘4 HS‘\ BGG Oi\ HSA BGG OA HS.4
6,000 42 0 0 0 0 47 13 0
12+
12+
0
11+
12-t 0 h 5
0 I) 0 II
0 5 i
l;urther evidence for systemic stimulation was providetl by Silverstein’s tlemonatration that such rabbits responded to intravenous challenge by the antigen originally injected with a rapid onset of uleitis in the previously injected eye (4). In our model, when one eye was challenged with one antigen alone, only the cells of the challenged eye produced antibody (Groups 1, 4, and 5). Antibody was present in the serum and in the aqueous humor of both eyes. When the rabbits were challenged in both previously injected eyes (Groups 2 and 3) the uveal tract cells of both eyes produced anti-EGG and anti-OA, although each eye was challenged with only one antigen. An accelerated response to both antigens ~vas exljected, since the rabbits would have had immunologic memory for both BGG autl OA. The lack of specificity of the antibody producing cells would be explained if antigen reached the contralateral eye in quantities sufficient to stimulate a secondary response, or if antigen-stimulated circulating “memory cells” were attracted to both eyes. Because of the eye’s isolation from the lymphoid tissues, it seemed necessary to produce inflammation in both eyes in order to facilitate passage of antigen or of memory cells to the contralateral eyes. The results of PFC and antibody determinations in the rabbits of Groups 6 and i indicate that this inf-lammation can be nonspecifically induced. Jacobsen and Thorbecke (5 ) and
72
HALL
AND
PRIBNOW
Stavitsky and Folds (6) demonstrated immunologic memory in contralateral nodes of rabbits immunized by foot-pad injections. An unexpected but more significant finding was the demonstration of anti-OA producing PFC in the uveal tracts and lymph nodes of rabbits challenged with BGG only (Groups 4 and 5) and in some of the rabbits challenged with BGG and HSA (Group 7). These PFC probably had not persisted from the initial injection since one rabbit killed months after initial sensitization showed no PFC among its uveal tract cells and no antibody in its serum, aqueous, or vitreous humors. No anti-OA producing cells would have been present initially in the right eyes (challenged eyes) of the rabbits in Group 4, since the initial injection was with BGG, and the primary PFC response has been shown to be specific. Gel diffusion and immunoelectrophoresis tests indicated that there was no cross reaction between the BGG and OA preparations we used. The same two antigens in the hands of other investigators also failed to show evidence of cross reactivity (4). Repeated contamination of one antigen with the other seemed highly unlikely. Several reports of nonspecific or heterologous enhancement of immune responses have recently appeared. Allogenic stimulation of the antibody response was reported by Katz et al. (7, S), by Dutton (9)) and by Shimpl and Wecker (10). Katz postulated that a nonspecific factor, elaborated by the T cells as a result of a graft-versus-host reaction acted on B cells to enhance antibody production to the priming hapten. Van and Galloway ( 11) used histocompatibility antigens as a nonspecific stimulus, and Stavitsky and Self (12)) Mond (13)) and Ivanyi (14) also described nonspecific stimulation of secondary responses, possibly mediated by a soluble factor. Rubin and Coons (15) observed an accelerated primary response to sheep erythrocytes when the priming antigen was added to spleen cultures containing SRBC. The effect was attributed to the action of a soluble enhancing factor (16-18). Feldman and Basten (19) postulated the existence of two soluble factors. The antigen-specific factor was thought to act early in the response, and the allogeneic factor was thought to augment the antibody response by increasing the proliferation of previously immunized B cells. In our BGG challenged rabbits, memory cells specific for OA could have been nonspecifically attracted to the challenged eye along with the BGG-specific memory cells. A nonspecific factor, produced by the interaction of the BGG-specific cells and the injected BGG, could have stimulated these OA primed cells to proliferate and to produce antibody in the presence of small amounts of OA. Since both T and B cells can be memory cells (20, 21) it is not necessary to define the nature of the memory cells involved. The requirement for OA can be satisfied for the rabbits in group 5 if one postulates that antigen reached the contralateral eye after the first injection and remained there. Antigen can reach the contralateral eye, (22-24) although in our experiments it did not induce a primary response and could not be demonstrated by immunoelectrophoresis. Ovalbumin remaining in the eye after the first injection would satisfy this requirement in the rabbits of groups 5 and 7. OA could have been present as an antigen-antibody complex, as has been described for antigen persisting in lymphoid tissues of systemically immunized animals (25-28). Complex formation in the vitreous is possible, since antigen is retained for long periods, and antibody appears in the vitreous by the 16th postimmunization day (1). A
SPECIFICITY
OF
ANTIBODY
I;OR’LIATION
II
;\z
nonspecific factor could also have augmented the response to the heterologtts antigen in the rabbits of Groups 2 and 3. The nonspecific factor proposed by Feldman and Basten might also explain the lack of response to HSA, since no HSA-primed cells would have been present ill the eye or in the circulation to react with the nonspecific factor. In these rabbits. HSA was not present in the eyes undergoing accelerated inflammation. l’ro!)al)l~ no solul)le factor would ha\:e been producet! in the niininiallv inflanird txvt* cl1:11lcnget! with HSA. ‘f’hr existence of a nonspecific enhancing factor would cs!j!ain niali~ (Ii 0t1r results. Iqurther experiments to tlemonstrate such a factor are in progrrss. ‘1’11t unique characteristic of the eye as an “isolated lymph node” makes it !)articularl! useful for in Z&I studies of this nature, and such studies may more closely paralel natural situations than in vitro experiments \\,hich utilize cells removed frc)nl their natural environment. Nonspecific enhancement of ocular immune response5 could be important in the consideration of such problems as recurrent anteriol uveitis, in which the recurrences might be attributed to antigens unrelated tco those. responsible for the original inflammation. REFERENCES 1. 2. 3. 3. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 2.5. 26. 27. 28.
Hall, J. RI., and Pribnow, J. F., Cell. Iwurtano~. 5, 419, 1972. Hall, J. M., and O’Connor, G. R., 1. Immunol. 104, 432, 1970. Hall, J. M., Iwest. Ophfhal. 10, 775, 1971. Silverstein, A. M., In “Immunopathology of Uveitis” (-4. E. Maumenee and A. 11. Silvcrstein, Eds.), pp. 83-97. Williams and Wilkins, Baltimore, 1964. Jacobsen, E. B., and Thorbecke, G. J., J. Exp. Med. 130, 287, 1969. Stavitsky, A. B., and Folds, J. D., J. Inzmu~ol. 108, 152, 1972. Katz, D. H., Paul, W., Goidl, E. A., and Benacrrraf, B., J. E.rp. .Ilrd. 133, 109, 1071. Katz, D. H., Transplant. Rev. 12, 141, 1972. Dutton, R. W., Falkoff, R., Hurst, J. A., Hoffman, M., Kappler, J. W., Kettman. J. Ii.. Lesley, J. F., and Vann, D., Progr. Imnzunol. 1, 355, 1971. Shimpl, A., and Wecker, E., Nature New Biol. 237, 15, 1972. \‘ann, D. C., and Galloway, P. C., J. Zmnzu~ol. 110, 1542, 1973. Stavitsky, A. B., and Self, C. H., Iwznm14nol. Comm. 1, 491, 1972. Mond, J. J., Takahashi, T., and Thorbecke, G. J., J. E.zp. Med. 136, 721, 1972. Ivanyi, J., Eur. J. Inlmunol. 2, 360, 1973. Rubin, A. S., and Coons, A. H., Proc. Nut. Arad Sci. USA 68, 1665, 1971. Rubin, A. S., and Coons, A. H., J. Exfi. Med. 135, 437, 1972. Rubin, -4. S., and Coons, A. H., J. In~m~~nol. 108, 1597, 1972. Rubin, A. S., and Coons, A. H., J. Exfi. Med. 136, 1501, 1972. Feldmann, M., and Basten, .A., J. Exp. Tiled. 136, 722, 1972. Miller, H. C., and Cudkowicz, G., J. Exfi. Med. 135, 1028, 1972. Jehn, U. W., and Karlin, L., J. Imrtl~nol. 106, 946, 1971. Jensen, I’. R.. Aronson, S. B., Pollyco~q M., and Yamamoto, E., .-lvclt. Oplzthrr/. 77, 814, 1967. Schnellman, 2). C., and Aronson, S. B., Arck. Ophthnl. 75, 213, 1966. Larsen, G., ilrta Ophthal. 39, 737, 1961. Lang, R. G., and Ada, G. L., In~~vzz~noZ. 13, 523, 1967. Hanna, M. S., and Peters, L. C., Immuvzol. 20, 707, 1971. Hanna, M. S., and Szakal, A. K.. J. Zmmz4nol. 101, 949. 1968. Nossal. G. J. IT., Abbot, A., Mitchell, J., and Lummis, A. J., .I. Enp. .Iird. 127. 277, 19r1X.
IMMUNOLOGY
Specificity
11,
64-73 (19%)
of Antibody with Bovine
Formation After Gamma Globulin
ft. Nonspecific
Enhancement
of the
Intravitreal Immunization and Ovalbumin Secondary
Response 1
JOAN M. HALL Francis
I. Proctor
Foundation
Research
for
in
San Fruncisco,
Ophthalmology,
California
University
of California,
University
of
94143
AND JAMES Naval Biomedical
Research
Laboratory, Berkeley, Received
F. PRIBNOW of Public Health, California 94720
School
August
California,
16, 1973
Rabbits immunized intravitreally with bovine gamma globulin and ovalbumin were challenged intravitreally with both bovine gamma globulin and ovalbumin, with bovine gamma globulin and human serum albumin, or with bovine gamma globulin alone. The specificity demonstrated in the primary response did not appear in rabbits challenged with both antigens ; antibody-producing cells of both specificities were found in the uveal tracts of both eyes. Anti-ovalbumin-producing cells were found in the uveal tracts of the rabbits challenged with bovine gamma globulin alone, or with bovine gamma globulin and human serum albumin. Anti-bovine gamma globulin producing cells were also present. No antibody-producing cells were found in the unchallenged eyes of rabbits challenged with bovine gamma globulin aIone. The action of a nonspecific soluble enhancing factor is postulated to explain the enhanced response to ovalbumin that resulted when rabbits were challenged with bovine gamma globulin alone.
INTRODUCTION Our previous publications have described the cellular responses that follow intravitreal injection of protein antigens into the eyes of rabbits (1, 2). Although the majority of the antibody producing cells were found in the uveal tracts of these animals, plaque forming cells (PFC) were first demonstrated in the extraocular lymphoid tissues (1) . Rabbits immunized intravitreally with small amounts of antigen developed high serum antibody titers (2). The titers in the aqueous and vitreous humors were higher than the serum titers. Antigen was present in the vitreous for at least two weeks after its injection (1). A second intravitreal 1 This work was supported in part by United States Public Health Service Program Grant EY-00310, and by the Office of Naval Research under a contract between the Office of Naval Research and the Regents of the University of California. 64 Copyright All rights
0 1974 by Academic Press, of reproduction ,in any form
Inc. reserved.
SPECIFICITY
OF
ANTIBODY
FOXMATJOIG
II
6.5
injection in the opposite eye resulted in the accelerated protluction of serunl antibody. In other experiments (3) rabbits were injected with bovine gamma globulin ( RGG) in the right eye and with ovalbumin (0-A) in the left eye. Uveal tract cells from the right eye produced only anti-BGG. and uveal tract cells from the left eye produced only anti-OA. although antibody specific for both antigens was present in the serum and in the aqueous humor from both eyes. .-\tltlitional investigations showed that the specificity of antihotly formation by uveal tract cells persisted for at least 23 days after a primary intravitreal injection. The initial purpose of the present investigations was to determine whether the specificity of the antibody response would be retained after intravitreal challenge with the same protein antigens used in the sensitizing injections. This reljort also clescrihes nonspecific enhancement of the secondar\- ocular immune response. 14ATERIAT5
AKD
11 ETHODS
Immunization
The experimental animals were 48 New Zealand white rabbits weighing G.5 11). In a series of experiments, they were injected intravitreally with approximatel! 1.5 mg of various antigens according to a method previously described (2). Group 1 was injected with BGG only and in the right eye only, Groups 2-7 were injected with BGG in the right eye and OA in the left eye, and a final group of six rabbits was injected with BGG in the right eye and HSA in the left eve. Thr animals were bled at stated intervals, and the hemolvtic antibodv titers wf’rc cletermined. The rabbits were challenged approximately five months after the sensitizing injections. No hemolytic antibody was demonstrable at the time of challenge. Rabbits initially injected with BGG, or with EGG and OA or HSA in comhination, were challenged according to the following protocol : Group Group (;roup Group Group Group Group Groul)
1. 2. 3. 4. 5. 6. 7. 8.
BGG in the left eye only (14 rabbits). BGG in the right eye and OA in the left eye (10 rabbits). BGG in the left eye and OA in the right el-e (7 rabbits). BGG in the right eye only. Left eye unchallenged (4 rabbits) BGG in the left eye only. Right eye unchallenged (3 rabbits 1. BGG in the right eye and HSA in the left eye (2 rabbits). BGG in the left eye and HSA in the right eye (2 rabbits‘). Tn the final group of six rabbits whose left eyes \vere sensitized to TTSA instead of to OA, the same challenges wet-c made as in tllr :il)cn~ groups escept that nonr of the animals received 3 h~lcrolog~~~s antigen.
1n all instances
the dose of the challenge antigen 1~;~s 1 ..q mg.
The rabbits were killed on the sixth postchallengr day. The ryes were enucleated and the aqueous and vitreous humors aspirated. Cell suspensions were made fro111 the uveal tracts of both eyes, from the cer\.icxl lymph nodes from both sides, and in some cases from the spleen. The procedure for enumerating the plaque-forming
66
SECONDARY
HALL
AND
PRIBNOW
TABLE
1
RESPONSE OF RABBITS INJECTED CHALLENGED WITH INTRAVITREAL Source of cells assayed
Right eyes Left eyes
INTRAVITREALLY WITH BGG INJECTION OF BGG IN LEFT
Average no. PFC per million cells 0 1482
Average
IN RIGHT EYE ONLY. EYE ONLY
peak hemagglutinin titer (Logz)
Primary response Secondary response
3.2 1 9.84
cells (PFC) has previously been described (1). The number of PFC are expressed as the number per log nucleated cells. In the case of the uveal tract cells, the plaque numbers may actually have been higher than stated in the tables since many of the nucleated cells were not lymphoid cells. Antibody
determination
Antibody titers in the serum and in the aqueous and vitreous humors were determined separately by a hemolytic assay previously described (1). We inactivated the serum samples by heating them at 56°C for 30 min, and we removed nonspecific hemolytic antibody by absorbing the samples with washed sheep erythrocytes. Since the aqueous and vitreous samples usually showed little or no nonspecific lysis of the control sheep erythrocytes, these samples were not inactivated. As described in a previous publication (3), we determined the presence of precipitating antibody by double-diffusion in agarose, and the presence of residual antigen in vitreous samples by immunoelectrophoresis. RESULTS Table 1 shows the results of plaque assays on 14 rabbits whose right eyes were initially injected with BGG and that were challenged approximately five months later by intravitreal injections of 1.5 mg BGG into the previously uninjected left eyes. All rabbits developed uveitis in the left eye within two days of the challenge injection and were killed on postchallenge Day 6. There were no PFC in any of the unchallenged right eyes but seven of the 14 rabbits had more than 2000 plaques in the uveal tract cells of the challenged left eyes. In a previous study (3) there were few or no plaques present in any uveal tract cells on the sixth day after primary injections of BGG. The results of the plaque assays and antibody determinations in the rabbits in Groups 2 and 3 are presented in Tables 2 and 3. The responses of the two groups were similar. PFC for both BGG and OA were found in the uveal tract cells of both right and left eyes. This was true whether a given eye had been challenged with the antigen used for initial sensitization, or with the antigen initially injected into the contralateral eye. In most cases there were more PFC for the antigen used to sensitize the challenged eye than for the antigen used to sensitize the opposite eye. Few PFC were found in the lymph nodes, and no specific plaques were found in the spleens tested. Hemolytic antibody specific for both BGG and OA was demonstrated in the serum and in the aqueous humor from both the right and left
SPECIFICITY
Y\I~KR.~GK
OF
PFC ASI) ANTIBODY RIBPONSE IN T,EFT EYE. CHALLENGED
0.4
ANTIRODY
I:ORRfATION
OF RABBITS WITH BGG IN
INJECTLI) WITH RIGHT Eye AM)
11
BGG OA
IS RIGHT I
.iverage hemolyt antibody titer (I,ogL’)
Right Left Right
eyes”
10 9 6
eyes* lymph
Ideft lymph
nodes nodes
6 4
BGG 0‘4 BGG OA BGG OA BGG 0‘1
671 97 113 147 58 21 3 6
(46-1287) (O-617) (O-32.5) (3-581) (O-101)
.\NI)
ic
h11pk
BGG
o:\
Serum
6..37
7.87
1.0 5.i
5.62 6.87
4..13 6.85
.5.88 0.0
Right Vitreous Right hclueolki I
‘l All right eyes contained PFC for BGG; 7 of 10 right eyes contained PITC for OA. ‘) .\I1 left eyes contained PFC for OX ; 6 of 7 left eyes contained PFC for 13GG. r Number varies because (1) in some cases the sheep erythrocytes did not coat with Or\, and the I’FC could not be determined; and (2) many eyes injected with OrI developed lenh olxtcities beveral months after initial injection. In some rabbits the cornea was alho opaque.
eyes. When no antibody was demonstrable in the vitreous humor, immunoelectrophoresis showed the presence of residual antigen. The precipitating antihotly determinations correlated well with the hemolytic antibody determinations.
Right
eyes”
I>eft eyes” Right Left
lymph lymph
nodes nodes
7 6 6 5 6 6 5 5
BGG OA BGG OA BGG 0‘4 BGG 0‘4
781 42.5 801 314 21 4 59 6
(36-2308) (74-923) (65-2666) (O-1166) (O-65) (O-21) (O-129) (O-30)
Serum Right \Ytreous Right Aqueous I.eft \“itreous I xft .\qu’ou~
6.2 1 I .o 3.8 1l.i
7.2 0 75 .i .6 11.7
(I All right eyes contained PFC for BGG; 5 of 6 right eyes contained PFC for 0.X. (III OIW VSperiment the sheep erythrocytes did not coat with 0.4). * Five of six left eyes contained PFC for both BGG and OA; 4 of 5 left eyes contained I’t;C for OA. (One microphthalmic left eye was not used; lens was opaque and only a few cells were obtainable from the minced uvea).
68
HALL
AND
PRIBNOW
The responses of the rabbits in Groups 2 and 3 suggested that the specificity of the primary response was lost when the eyes were challenged with the sensitizing antigens. Two possible explanations were (i) that a sufficient amount of antigen reached the opposite eye to initiate antibody production, or (ii) that circulating memory cells were attracted to both eyes because both were undergoing an inflammatory response. The next two experiments were designed to test the effect of challenge with only one of the sensitizing antigens. Would ‘the previously injected contralateral eye react under these conditions ? As shown in Table 1, rabbits injected with a single antigen and challenged in the opposite eye had antibody-producing cells only in the challenged eye. The seven rabbits in Groups 4 and 5 were challenged with BGG only. The opposite eyes were left unchallenged. The results of the plaque assays and antibody determinations on the rabbits in these groups are presented in Tables 4 and 5. With the exception of rabbits 401 and 422, the unchallenged eyes of these rabbits did not have PFC for either antigen. The number of PFC in the unchallenged eyes of rabbits 401 and 422 was much smaller than the number in the challenged TABLE PFC
Rabbit no.
400
AND
ANTIBODY RESPONSE IN RABBITS INJECTED IN LEFT EYE. CHALLENGED WITH BGG
Source of cells assayed RE
LE RLN 401
RE
LE RLN 416
RE
LE 421
4
RE
LE LLN
PFC per million cells
WITH BGG IN RIGHT IN RIGHT EYE ONLY
Hemolytic Sample
BGG OA
2,000 239
BGG OA BGG OA BGG OA
0 0 0 0 11,000 1,900
BGG OA BGG OA BGG OA
100 33 8 0 10,000 740
BGG OA BGG OA
0 0 200 0
BGG OA BGG OA
0 0 0 0
EYE
antibody
titer
Anti-BGG
AND
OA
(Log*) Anti-OA
8+
8+ 8+
0 3
0 6
Serum R. aqueous R. vitreous L. aqueous L. vitreous
12+ 8+
12+ 8+ 8+
Serum R. aqueous R. vitreous L. aqueous L. vitreous
8 12 0 6 5
Serum R. aqueous R. vitreous L. aqueous L. vitreous
12
Serum R. aqueous R. vitreous L. aqueous
4
0 4 2
8 8
8 12 0 7 4 8
12+
12+
0 0 1
0 0 4
SPECIFICITY
OF
ANTIBODY
FORMATION
00
II
Inillion .\trti-HGG
cells BGG
0 0
OA
BGG OA RGG 413
OA BGG OX
BGG 0-l 422
BGG OA I3GG 0.4
BGG OAl BGG OA
9,000 1,000
900 30 0 0
5,000 39 0 0 50 0
1,876
I2 0
\nti-0.4 10
0
XS 8+
8+ (I
8+ 0
11
0
I 7
0 (I
12
10
.(
(1
12+ 3
II+ (I
0 12+ 0
II I!+ 0
11
23 4
eyes. As expected, the challenged eyes contained many BGG-specific PFC. Ilowever, OA-specific PFC were found in six of the challenged eyes, even though no OA was reinjected. Anti-OA antibody was demonstrated in the serum and aqueous llumor of the challenged eyes and in some of the aqueous and vitreous samples from the contralateral eyes. Titers for OA were often as high as those for Ti(X;. but the number of anti-OA-producing cells was smaller. Similar results were obtained with one rabbit challenged with OrZ in tile right eye only. The uveal tract cells from the right eye and the lymph node cells from the right side contained anti-BGG-producing cells. and anti-RGG-alltihod~ WIS found in the aqueous and vitreous humors from the right eye. The results of the plaque assays on the rabbits in (;rnups 3 nlltl 5 indicatc~tl that. as in the case of the rabbits sensitized in one eye only and challenged in the other -eye only, PFC were found only in the challenged eyes. 111 order to demonstrate loss of specificity, it is apparently necessary to induce inflammation in both eyes. The next two experiments were designed to test the effect of nonspecific inflammation on an eye by challenging it with an antigen unrelated to either of the sensitizing antigens. If one eye were challenged with IHSA ant1 the other with BGG. would the eye challenged with HSA have anti-BGG-producing cells, or would thy inflammation have to be induced by one of the initially injected (sensitizing) antigens ?
70
HALL
AND
PRIBNOW
Tables 6 and 7 present the results of the plaque assays and antibody determinations on the rabbits in Groups 6 and 7. The eye challenged with BGG developed inflammation within two days after challenge, while the eye injected with HSA showed only minimal inflammation at the time of sacrifice six days later. The two rabbits in Group 6 had anti-BGG-producing cells in both the right and left eyes. Both of the BGG-challenged left eyes and one of the HSA-challenged right eyes of the rabbits of Group 7 also contained BGG-specific-PFC. Both rabbits in this group had anti-OA-producing cells in the BGG-challenged left eyes (previously sensitized to OA). All rabbits tested for hemolytic antibody titers in both groups had antibody to OA in their sera and in some of the aqueous and vitreous samples. To confirm the observations reported above, similar experiments were performed with rabbits initially given BGG and HSA. Rabbits challenged with both antigens had responses similar to those obtained in Groups 2 and 3. Technical problems have precluded the presentation of results of assays on rabbits challenged with only BGG or HSA. DISCUSSION Our previous experiments uveal tract was the primary onstrated, as did Silverstein
showed that after intravitreal injection of BGG the site of antibody production (1). However, we dem(4), that extraocular lymphoid cells were stimulated. TABLE
PFC
Rabbit ll0.
418
AND ANTIBODY 1~ LEFT EYE.
Source of cells assayed RE
LE
LLN
RLN
420
RE
LE
RLN
RESPONSE CHALLENGED
IN
6
RABBITS INJECTED WITH WITH BGG IN RIGHT EYE
PFC per million cells
Hemolytic Sample
BGG OA HSA
192 0 0
BGG OA HSA BGG OA HSA BGG OA HSA BGG OA HSA
341 0 0 8 4 0 6 12 0 1,300 0 0
BGG OA HSA BGG OA HSA
13 0 13 3 2 0
Serum R. aqueous R. vitreous L. aqueous L. vitreous
Serum R. aqueous R. vitreous L. aqueous L. vitreous
BGG AND
IN RIGHT EYE AND IN LEFT Eye
OA
HSA
antibody Anti-BGG
titer
(Logz)
Anti-OA
ND
ND
12+
12+
0 0 0
0 0 0
8
Anti-HSA ND 0 0 0 0
11
0
12+
12+
0
0 3 0
0 3 0
0
0 0
l
71
BGG OX HSA
0 0 0
BGG 0‘4 HSA BGG 0‘4 HSA BGG 0‘4 HSA BGG o=\ HS,\
448 60 0 0 0 0 71 7 3 23 0 0
BBG 0‘4 HS‘\ BGG Oi\ HSA BGG OA HS.4
6,000 42 0 0 0 0 47 13 0
12+
12+
0
11+
12-t 0 h 5
0 I) 0 II
0 5 i
l;urther evidence for systemic stimulation was providetl by Silverstein’s tlemonatration that such rabbits responded to intravenous challenge by the antigen originally injected with a rapid onset of uleitis in the previously injected eye (4). In our model, when one eye was challenged with one antigen alone, only the cells of the challenged eye produced antibody (Groups 1, 4, and 5). Antibody was present in the serum and in the aqueous humor of both eyes. When the rabbits were challenged in both previously injected eyes (Groups 2 and 3) the uveal tract cells of both eyes produced anti-EGG and anti-OA, although each eye was challenged with only one antigen. An accelerated response to both antigens ~vas exljected, since the rabbits would have had immunologic memory for both BGG autl OA. The lack of specificity of the antibody producing cells would be explained if antigen reached the contralateral eye in quantities sufficient to stimulate a secondary response, or if antigen-stimulated circulating “memory cells” were attracted to both eyes. Because of the eye’s isolation from the lymphoid tissues, it seemed necessary to produce inflammation in both eyes in order to facilitate passage of antigen or of memory cells to the contralateral eyes. The results of PFC and antibody determinations in the rabbits of Groups 6 and i indicate that this inf-lammation can be nonspecifically induced. Jacobsen and Thorbecke (5 ) and
72
HALL
AND
PRIBNOW
Stavitsky and Folds (6) demonstrated immunologic memory in contralateral nodes of rabbits immunized by foot-pad injections. An unexpected but more significant finding was the demonstration of anti-OA producing PFC in the uveal tracts and lymph nodes of rabbits challenged with BGG only (Groups 4 and 5) and in some of the rabbits challenged with BGG and HSA (Group 7). These PFC probably had not persisted from the initial injection since one rabbit killed months after initial sensitization showed no PFC among its uveal tract cells and no antibody in its serum, aqueous, or vitreous humors. No anti-OA producing cells would have been present initially in the right eyes (challenged eyes) of the rabbits in Group 4, since the initial injection was with BGG, and the primary PFC response has been shown to be specific. Gel diffusion and immunoelectrophoresis tests indicated that there was no cross reaction between the BGG and OA preparations we used. The same two antigens in the hands of other investigators also failed to show evidence of cross reactivity (4). Repeated contamination of one antigen with the other seemed highly unlikely. Several reports of nonspecific or heterologous enhancement of immune responses have recently appeared. Allogenic stimulation of the antibody response was reported by Katz et al. (7, S), by Dutton (9)) and by Shimpl and Wecker (10). Katz postulated that a nonspecific factor, elaborated by the T cells as a result of a graft-versus-host reaction acted on B cells to enhance antibody production to the priming hapten. Van and Galloway ( 11) used histocompatibility antigens as a nonspecific stimulus, and Stavitsky and Self (12)) Mond (13)) and Ivanyi (14) also described nonspecific stimulation of secondary responses, possibly mediated by a soluble factor. Rubin and Coons (15) observed an accelerated primary response to sheep erythrocytes when the priming antigen was added to spleen cultures containing SRBC. The effect was attributed to the action of a soluble enhancing factor (16-18). Feldman and Basten (19) postulated the existence of two soluble factors. The antigen-specific factor was thought to act early in the response, and the allogeneic factor was thought to augment the antibody response by increasing the proliferation of previously immunized B cells. In our BGG challenged rabbits, memory cells specific for OA could have been nonspecifically attracted to the challenged eye along with the BGG-specific memory cells. A nonspecific factor, produced by the interaction of the BGG-specific cells and the injected BGG, could have stimulated these OA primed cells to proliferate and to produce antibody in the presence of small amounts of OA. Since both T and B cells can be memory cells (20, 21) it is not necessary to define the nature of the memory cells involved. The requirement for OA can be satisfied for the rabbits in group 5 if one postulates that antigen reached the contralateral eye after the first injection and remained there. Antigen can reach the contralateral eye, (22-24) although in our experiments it did not induce a primary response and could not be demonstrated by immunoelectrophoresis. Ovalbumin remaining in the eye after the first injection would satisfy this requirement in the rabbits of groups 5 and 7. OA could have been present as an antigen-antibody complex, as has been described for antigen persisting in lymphoid tissues of systemically immunized animals (25-28). Complex formation in the vitreous is possible, since antigen is retained for long periods, and antibody appears in the vitreous by the 16th postimmunization day (1). A
SPECIFICITY
OF
ANTIBODY
I;OR’LIATION
II
;\z
nonspecific factor could also have augmented the response to the heterologtts antigen in the rabbits of Groups 2 and 3. The nonspecific factor proposed by Feldman and Basten might also explain the lack of response to HSA, since no HSA-primed cells would have been present ill the eye or in the circulation to react with the nonspecific factor. In these rabbits. HSA was not present in the eyes undergoing accelerated inflammation. l’ro!)al)l~ no solul)le factor would ha\:e been producet! in the niininiallv inflanird txvt* cl1:11lcnget! with HSA. ‘f’hr existence of a nonspecific enhancing factor would cs!j!ain niali~ (Ii 0t1r results. Iqurther experiments to tlemonstrate such a factor are in progrrss. ‘1’11t unique characteristic of the eye as an “isolated lymph node” makes it !)articularl! useful for in Z&I studies of this nature, and such studies may more closely paralel natural situations than in vitro experiments \\,hich utilize cells removed frc)nl their natural environment. Nonspecific enhancement of ocular immune response5 could be important in the consideration of such problems as recurrent anteriol uveitis, in which the recurrences might be attributed to antigens unrelated tco those. responsible for the original inflammation. REFERENCES 1. 2. 3. 3. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 2.5. 26. 27. 28.
Hall, J. RI., and Pribnow, J. F., Cell. Iwurtano~. 5, 419, 1972. Hall, J. M., and O’Connor, G. R., 1. Immunol. 104, 432, 1970. Hall, J. M., Iwest. Ophfhal. 10, 775, 1971. Silverstein, A. M., In “Immunopathology of Uveitis” (-4. E. Maumenee and A. 11. Silvcrstein, Eds.), pp. 83-97. Williams and Wilkins, Baltimore, 1964. Jacobsen, E. B., and Thorbecke, G. J., J. Exp. Med. 130, 287, 1969. Stavitsky, A. B., and Folds, J. D., J. Inzmu~ol. 108, 152, 1972. Katz, D. H., Paul, W., Goidl, E. A., and Benacrrraf, B., J. E.rp. .Ilrd. 133, 109, 1071. Katz, D. H., Transplant. Rev. 12, 141, 1972. Dutton, R. W., Falkoff, R., Hurst, J. A., Hoffman, M., Kappler, J. W., Kettman. J. Ii.. Lesley, J. F., and Vann, D., Progr. Imnzunol. 1, 355, 1971. Shimpl, A., and Wecker, E., Nature New Biol. 237, 15, 1972. \‘ann, D. C., and Galloway, P. C., J. Zmnzu~ol. 110, 1542, 1973. Stavitsky, A. B., and Self, C. H., Iwznm14nol. Comm. 1, 491, 1972. Mond, J. J., Takahashi, T., and Thorbecke, G. J., J. E.zp. Med. 136, 721, 1972. Ivanyi, J., Eur. J. Inlmunol. 2, 360, 1973. Rubin, A. S., and Coons, A. H., Proc. Nut. Arad Sci. USA 68, 1665, 1971. Rubin, A. S., and Coons, A. H., J. Exfi. Med. 135, 437, 1972. Rubin, -4. S., and Coons, A. H., J. In~m~~nol. 108, 1597, 1972. Rubin, A. S., and Coons, A. H., J. Exfi. Med. 136, 1501, 1972. Feldmann, M., and Basten, .A., J. Exp. Tiled. 136, 722, 1972. Miller, H. C., and Cudkowicz, G., J. Exfi. Med. 135, 1028, 1972. Jehn, U. W., and Karlin, L., J. Imrtl~nol. 106, 946, 1971. Jensen, I’. R.. Aronson, S. B., Pollyco~q M., and Yamamoto, E., .-lvclt. Oplzthrr/. 77, 814, 1967. Schnellman, 2). C., and Aronson, S. B., Arck. Ophthnl. 75, 213, 1966. Larsen, G., ilrta Ophthal. 39, 737, 1961. Lang, R. G., and Ada, G. L., In~~vzz~noZ. 13, 523, 1967. Hanna, M. S., and Peters, L. C., Immuvzol. 20, 707, 1971. Hanna, M. S., and Szakal, A. K.. J. Zmmz4nol. 101, 949. 1968. Nossal. G. J. IT., Abbot, A., Mitchell, J., and Lummis, A. J., .I. Enp. .Iird. 127. 277, 19r1X.