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Local production of Ia-inducing activity in experimental immunogenic uveitis
CELLULAR
86, 557-56 1 (1984)
IMMUNOLOGY
Local Production of la-Inducing Activity in Experimental Immunogenic Uveitis’ JOHN J. DONNELLY*'~ AND ROBERTA.PRENDERGAST Department
of Ophthalmology, Johns Hopkins Baltimore, Maryland Received
December
University 21205
7, 1983; accepted
March
School
of Medicine,
12, 1984
The production of soluble lymphokine products of T lymphocytes was studied in the aqueous of rabbits with uveitis induced by intravitreal injection of 2.0 mg of ovalbumin. Aqueous from uveitic eyes demonstrated Ia-inducing activity at a dilution of 1:50 which diluted out at 1:250. The control aqueous and sera from normal or uveitic animals were negative. While production of interleukin 2 (K-2) in uveitic eyescould not be demonstrated in vitro, control rabbit concanavalin A-induced supematants of nylon-wool-purified T cells contained both Ia-inducing and IL-2 activity. These data demonstrate antigendriven production of a lymphokine activity in the aqueous of the uveitic eye. Local production of lymphokine may be a potent mechanism for amplification of the inflammatory process in uveitis.
INTRODUCTION Intravitreal injection of antigen in the rabbit eye induces an intraocular inflammatory response which culminates in the local production of antibody. In this instance, the eye simulates organized lymphoid tissue since the response is immunologically specific and can be restimulated in that eye only with the same antigen (1, 2). The role of lymphokines, produced locally by antigen-specific T lymphocytes, in the induction of immunopathologic responses in specialized sites such as the eye is incompletely understood. It has been demonstrated that ocular antibody responses can be induced in systemically sensitized rabbits if lymphokines are injected intravitreally (3). Thus, lymphokines may fulfill key roles in the initiation and amplification of intraocular immune responses. The present studies investigate local production of a particular lymphokine activity during primary immunogenic uveitis induced by intravitreal injection of ovalbumin. The induction of Ia (class II histocompatibility) antigen expression on rabbit macrophages was selected as the lymphokine activity for study because of the requirement of Ia-bearing accessory cells in the activation of antigen-specific T lymphocytes and the importance of both cell types in the amplification of the immune response (4, 5). In human and murine systems, induction of class II antigen expression can ’ Supported * Recipient Health 3 To whom Eye Institute,
in part by Grant EY-03521 from the National Institutes of Health. of Individual National Research Service Award EY-05622 from the National Institutes of correspondence should be sent at the present address: Department of Ophthalmology, Scheie University of Pennsylvania School of Medicine, 51 N. 39th Street, Philadelphia, Pa. 19104. 557 OOOS-8749/84 $3.00 Copyright Q 1984 by Academic Pres, Inc. All rights of repreduction in any form reserved.
558
SHORT COMMUNICATIONS
be mediated by interferon-y, a T-cell product (6, 7). In this communication we demonstrate that Ia-inducing activity is present in aqueous humor of eyes during experimental immunogenic uveitis. MATERIALS
AND
METHODS
Primary immunogenic uveitis was induced in three groups of eight each of New Zealand white rabbits by intravitreal injection of 2 mg of ovalbumin (Sigma Chemical Co., St. Louis, MO.) in sterile Hanks’ balanced salt solution (HBSS)4 in the left eyes. Sterile HBSS alone was injected intravitreally in the right eyes. Eyes were examined on alternate days by Zeiss slitlamp microscopy for the development of uveitis. In all cases ovalbumin-injected eyes became inflamed while the contralateral eyes remained clear. Anterior chamber paracentesis of both eyes (0.1-0.25 ml/eye) with a 27-gauge needle and a l-ml disposable syringe and bleeding, by cardiac puncture, were carried out under ketamine-xylazine anesthesia. Taps were performed on Days 10 and 12 after intravitreal injection and just before and 24 hr after intravenous challenge on Day 56. Cell surface membrane Ia antigen expression was assayed on rabbit macrophages obtained by lavage 3 days after an intraperitoneal injection of 100 ml of 5% thioglycollate (Difco, Detroit, Mich.). Macrophages were washed in HBSS and resuspended in RPM1 1640 plus 15% fetal bovine serum (PBS) and 5 mM Hepes buffer at 2 X lo6 cells/ml. These cells were cultured on 12 mm round coverslips (Bellco, Vineland, N.J.) in 24-well plates (Linbro, Boston, Mass.) at 1 ml/well for 90 min at 37’C, and the nonadherent cells were removed by washing once with HBSS. One milliliter of fresh medium and 0.1 ml of fluid to be assayed for Ia-inducing activity were added to 3-6 replicate cultures and the cultures were incubated at 37°C in 5% CO* for 5 days. Aqueous and serum from groups of six animals were pooled and dialyzed against 1000 vol of phosphate-buffered saline (PBS), pH 7.4, centrifuged at lO,OOOg, and sterile filtered. The final dilutions of the aqueous in the assays for Iainduction were 1:50, 1:250, and 1: 1250. Serum samples were diluted to an absorbance at 280 nm equivalent to that of the aqueous from inflamed eyes, and then diluted 1:50, 1:250, and 1: 1250. Supematants were prepared from nylon-wool-purified rabbit lymph node T lymphocytes, cocultured with a minimal number of macrophages, stimulated by insoluble concanavalin A (Con A) or unstimulated (3), and used at a 1: 10 final dilution as positive and negative controls. Monolayers of cultured macrophages were fixed for 15 min in freshly prepared 1% paraformaldehyde, and stained with 15 ~1 of monoclonal anti-Ia (first step) at 5 pg/ml and 15 ~1 of FITC anti-mouse IgG at 50 pg/ml (second step) (Tago, Inc., Burlingame, Calif.), as described by Beller et al. (8). (Monoclonal anti-rabbit Ia was a kind gift of Dr. K. Knight and Dr. S. Lobel, University of Chicago (9).) The antibody was prepared by immunization of Balb/c mice with spleen cells from homozygous Ia’/Ia’ and Ia2/Ia2 rabbits (9). The antibody stained predominantly Ig+ lymphocytes in lymph node and spleen, and inhibited the one-way MLR by 80% (9). Polyacrylamide gel electrophoresis of immunoprecipitates of spleen cell homogenates prepared with the monoclonal antibody and with anti-Ia alloantisera gave identical patterns. Both ‘Abbreviations used: Con A, concanavalin A, FITC-IgG, fluorescein isothiocyanate conjugated immunoglobulin G, HBSS, Hanks’ balanced salt solution; Hepes, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, IG2, interleukin 2; M&J, macrophages; PBS, phosphate-buffered saline; RPM1 1640, culture medium 1640 from Roswell Park Memorial Institute.
559
SHORT COMMUNICATIONS
precipitated three polypeptides with apparent molecular weights of 35,000, 3 1,000, and 28,000 after extensive reduction and alkylation (9). Cover-slips stained with antiIa or control mouse IgG and FITC-goat anti-mouse IgG were examined using a Zeiss microscope with mercury lamp, darkfield condenser, BG 12 exciting, and GG 14 barrier filter. Assays for Ia-inducing activity in the murine system were carried out in parallel with the studies employing rabbit macrophages. Interleukin 2 (IL-2) assays were carried out using the IL2dependent mouse T-cell line, CT6 (gift of Dr. J. Farrar, NIH), maintained in rat IG2-containing medium, as described by Gillis et al. (10). Data were analyzed statistically using two-way analysis of variance. RESULTS Aqueous obtained at the peak of ocular inflammation 10 days after intravitreal injection of 2 mg of ovalbumin induced Ia expression on 24% (range 23-25; P < 0.025) of macrophages after 5 days in culture (Table 1). Aqueous obtained from contralateral control eyes, and serum obtained at the same time, did not induce Ia expression above the background levels of 2- 12%. By 12 days after intravitreal antigen injection, the difference in Ia-inducing activity between the aqueous from inflamed and control eyes was less marked (P > 0.05, Table 1). By 8 weeks after injection, only barely detectable Ia-inducing activity was present in aqueous or serum. Intravenous challenge injection of 20 mg ovalbumin on Day 56 induced a recall uveitis in the sensitized eye on the following day. Only aqueous obtained on Day 57 from inflamed eyes contained significant Ia-inducing activity (range, 18-2 1; P < 0.0 1). Dilutions of 1:250 of aqueous from inflamed eyes were less than two standard deviations above the corresponding values for control aqueous, while 1: 1250 dilutions were identical to the controls. The effect of rabbit Con A supernatant on Ia expression of cultured rabbit macrophages is shown in Fig. 1A. Thioglycollate-induced rabbit peritoneal macrophages averaged 38% Ia positive (SEM, 3.3; range, 30-58, five repetitions) after 24 hr in culture. Addition of control supematant at this time was followed by a decline of Ia expression to 15% after 3 days and 9% or less (SEM, 2.3; range, 2-17, five repetitions) TABLE
1
Ia-Inducing Activity in Aqueous Humor Aqueous humor Day after intravitreal injection
Inflamed eyes”
Control eyes”
Serum”
10 12 56d 57
24 f 2.2b 16 f 2.3’ <2 20 + 1.56
9+ 1.6 11 f 1.9 5 + 2.9 <2
9+ 1.7 10 + 1.7 <2 12
n Percent Ia+ macrophages + SEM after 5 days culture with aqueous or serum added at a final concentration of 1:50; mean of three experiments. b Significantly different (P < 0.025) from control aqueous. ’ Not significantly different (P > 0.05) from control aqueous. dEighteen rabbits were given 20 mg ovalbumin intravenously immediately after aqueous taps on Day 56.
SHORT COMMUNICATIONS
560 6o
A
50 I” A 5 8 a 2
30
20 IO
+
400 i
i DAYS
IN CULTURE
FIG. 1. Effect of Con A-stimulated and control lymphocyte supematants on macrophage (Mg) expression of Ia. Arrows indicate the time of addition of supematants to a final concentration of 1:10 in all cultures. (A) Rabbit peritoneal exudate adherent cells exposed to Con A-stimulated q compared to control lB rabbit lymphocyte culture supematants. (B) Effect of rabbit- and mouse-lymphocytederived supematants on expression of Ia on mouse peritoneal exudate adherent cells exposed to Con A-stimulated q and control n mouse supematants, compared to stimulated •4and control c3 rabbit supematants (SEM ranged from 0.8 to 3.3% in all cultures).
alter 5 days of culture. Addition of active Con A-stimulated supematant after 24 hr of culture maintained the proportion of Ia positive macrophages at 38% (SEM, 3.0; range 25-47; five repetitions) on Days 3 and 5. The percentage of Ia+ macrophages after 5 days culture with Con A supematant was significantly different from that of macrophages cultured with control supematant (P 4 O.Ol), but was not significantly different (P B 0.05) from that of freshly obtained macrophages. In order to determine whether rabbit and mouse Ia-inducing activities were comparable, rabbit or mouse Con A-stimulated or control supematants were added to mouse peritoneal macrophages after 1 day of culture (Fig. 1B). Mouse Con A supematant induced Ia expression on 55-60% of mouse macrophages after 5 days, while rabbit Con A supematant induced Ia on 25% of mouse macrophages. Cultures given control supematants of mouse or rabbit origin showed background Ia expression of 5-7% after 5 days (Fig. 1B). Samples of pooled aqueous from inflamed eyes containing significant Ia-inducing activity, and of pooled aqueous from contralateral control eyes with essentially no Ia-inducing properties, were tested for IL-2 activity by the CT6 cell assay technique. No IL-2 activity was detected in aqueous humor from inflamed or control eyes. Pooled rabbit serum also did not show IL2 activity. Supematants from rabbit lymph node cells stimulated with insoluble Con A, however, did show IL-2 activity. Peak [3H]thymidine incorporation by CT6 cells in response to rabbit Con A supematant was 80% of that obtained in response to an equivalent dilution of the standard rat Con A supemate used for maintaining the CT6 cell line. DISCUSSION The presence of Ia-inducing activity in aqueous from eyes undergoing immunogenic uveitis, in the absence of detectable activity in contralateral control aqueous or in serum, indicates that an Ia-inducing lymphokine activity may be produced locally. This effect was demonstrated in primary uveitis or 24 hr after secondary uveitis induced by intravenous antigenic challenge. The degree of biologically active material recoverable from the aqueous was found to correspond to the clinical extent of the
SHORT COMMUNICATIONS
561
ocular inflammatory reaction. The marked difference in Ia-inducing activity between aqueous samples obtained 10 and 12 days after intraocular antigen injection also supports the hypothesis that peak local lymphokine production may occur during a relatively short interval. The presence of Ia-inducing activity in supematants of Con A-stimulated, nylon-wool-purified T cells, and not in supematants of nonstimulated T cells, suggests that this factor is of T-cell origin. Corresponding activities in mice and humans have been identified as interferon-y, a T-cell product (6,7). The absence of demonstrable IL-2 activity from aqueous containing Ia-inducing lymphokine may be due to the rapid consumption of IL-2 by dividing T cells (lo), or to the presence of inhibitors of the action of IL2 on its target cells (11). The local production of Ia-inducing lymphokine activity indicates that antigenspecific T cells are being triggered in the eye in the immunogenic inflammation induced by intravitreal injection of antigen (3,5). The presence of Ia-inducing activity in high local concentrations would greatly increase the efficiency of accessory cell presentation of antigen to specific T cells and help to amplify antigen-specific intraocular immune responses. Vascular endothelial cells also may be induced to express Ia by treatment with lymphokines (12). These la-bearing endothelial cells may serve as antigen-presenting cells as well as stimulator cells in the mixed leukocyte reaction ( 13, 14). Induction of Ia expression on choroidal vascular endothelium would greatly increase the intraocular capability for antigen presentation. Further, induction of Ia expression on comeal endothelial cells by locally produced lymphokines might play a significant role in comeal allograft rejection. The site-specific regulation of Ia expression, as demonstrated in the experiments reported in this communication, broadens our understanding of ocular immune reactions and may be of considerable importance in efforts to suppress such immunopathological phenomena in vivo. ACKNOWLEDGMENTS The authors are grateful to Dr. K. Knight and Dr. S. Lobe1 for their gift of the monoclonal anti-rabbit Ia, and to Dr. J. Farrar for his gift of the CT6 cells.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Shimada, K., and Silverstein, A. M., Invest. Ophthalmol. 14, 513, 1975. Prendergast, R. A., and Franklin, R. M., Invest. Ophthalmol. 10, 695, 1971. Liu, S. H., Prendergast, R. A., and Silverstein, A. M., Invest. Ophthalmol. Visual Sci. 24, 361, 1983. Farrar, J. J., Benjamin, W. R., Hilfiker, M. L., Howard, M., Farrar, W. L., and Fuller-Farrar, J., Immunol. Rev. 63, 130, 1982. Unanue, E. R., Adv. Immunol. 31, 1, 1981. Steeg, P. S., Johnson, H. M., and Oppenheim, J. J., J. Immunol. 129, 2402, 1982. Basham, T. Y., and Merigan, T. C., J. Immunol. 130, 1492, 1983. Beller, D. I., Kiely, J.-M., and Unanue, E. R., J. Immunol. 124, 1426, 1980. Lobel, S., and Knight, K., Immunology 51, 35, 1984. Gillii, S., Mochizuki, D. Y., Conlon, P. J., Hefeneider, S. H., Ramthun, C. A., Gillis, A. E., Frank, M. B., Henney, C. S., and Watson, J. D., Immunol. Rev. 63, 167, 1982. Hard& C., Rollinghoff, M., Phzenmaier, K., Mosmann, H., and Wagner, H., J. Exp. Med. 154, 262, 1981. Pober, J. S., Gimbrone, M. A., Cotran, R. S., Reiss, C. S., Burakoff, S. J., Fiers, W., and Ault, K. A., J. Exp. Med. 157, 1339, 1983. Hirschberg, H., Braathen, L. R., and Thorsby, E., Immunol. Rev. 65, 57, 1982. Hirschberg, H., Evensen, S. A., Hemiksen, T., and Thorsby, E., Transplantation 19, 495, 1975.
86, 557-56 1 (1984)
IMMUNOLOGY
Local Production of la-Inducing Activity in Experimental Immunogenic Uveitis’ JOHN J. DONNELLY*'~ AND ROBERTA.PRENDERGAST Department
of Ophthalmology, Johns Hopkins Baltimore, Maryland Received
December
University 21205
7, 1983; accepted
March
School
of Medicine,
12, 1984
The production of soluble lymphokine products of T lymphocytes was studied in the aqueous of rabbits with uveitis induced by intravitreal injection of 2.0 mg of ovalbumin. Aqueous from uveitic eyes demonstrated Ia-inducing activity at a dilution of 1:50 which diluted out at 1:250. The control aqueous and sera from normal or uveitic animals were negative. While production of interleukin 2 (K-2) in uveitic eyescould not be demonstrated in vitro, control rabbit concanavalin A-induced supematants of nylon-wool-purified T cells contained both Ia-inducing and IL-2 activity. These data demonstrate antigendriven production of a lymphokine activity in the aqueous of the uveitic eye. Local production of lymphokine may be a potent mechanism for amplification of the inflammatory process in uveitis.
INTRODUCTION Intravitreal injection of antigen in the rabbit eye induces an intraocular inflammatory response which culminates in the local production of antibody. In this instance, the eye simulates organized lymphoid tissue since the response is immunologically specific and can be restimulated in that eye only with the same antigen (1, 2). The role of lymphokines, produced locally by antigen-specific T lymphocytes, in the induction of immunopathologic responses in specialized sites such as the eye is incompletely understood. It has been demonstrated that ocular antibody responses can be induced in systemically sensitized rabbits if lymphokines are injected intravitreally (3). Thus, lymphokines may fulfill key roles in the initiation and amplification of intraocular immune responses. The present studies investigate local production of a particular lymphokine activity during primary immunogenic uveitis induced by intravitreal injection of ovalbumin. The induction of Ia (class II histocompatibility) antigen expression on rabbit macrophages was selected as the lymphokine activity for study because of the requirement of Ia-bearing accessory cells in the activation of antigen-specific T lymphocytes and the importance of both cell types in the amplification of the immune response (4, 5). In human and murine systems, induction of class II antigen expression can ’ Supported * Recipient Health 3 To whom Eye Institute,
in part by Grant EY-03521 from the National Institutes of Health. of Individual National Research Service Award EY-05622 from the National Institutes of correspondence should be sent at the present address: Department of Ophthalmology, Scheie University of Pennsylvania School of Medicine, 51 N. 39th Street, Philadelphia, Pa. 19104. 557 OOOS-8749/84 $3.00 Copyright Q 1984 by Academic Pres, Inc. All rights of repreduction in any form reserved.
558
SHORT COMMUNICATIONS
be mediated by interferon-y, a T-cell product (6, 7). In this communication we demonstrate that Ia-inducing activity is present in aqueous humor of eyes during experimental immunogenic uveitis. MATERIALS
AND
METHODS
Primary immunogenic uveitis was induced in three groups of eight each of New Zealand white rabbits by intravitreal injection of 2 mg of ovalbumin (Sigma Chemical Co., St. Louis, MO.) in sterile Hanks’ balanced salt solution (HBSS)4 in the left eyes. Sterile HBSS alone was injected intravitreally in the right eyes. Eyes were examined on alternate days by Zeiss slitlamp microscopy for the development of uveitis. In all cases ovalbumin-injected eyes became inflamed while the contralateral eyes remained clear. Anterior chamber paracentesis of both eyes (0.1-0.25 ml/eye) with a 27-gauge needle and a l-ml disposable syringe and bleeding, by cardiac puncture, were carried out under ketamine-xylazine anesthesia. Taps were performed on Days 10 and 12 after intravitreal injection and just before and 24 hr after intravenous challenge on Day 56. Cell surface membrane Ia antigen expression was assayed on rabbit macrophages obtained by lavage 3 days after an intraperitoneal injection of 100 ml of 5% thioglycollate (Difco, Detroit, Mich.). Macrophages were washed in HBSS and resuspended in RPM1 1640 plus 15% fetal bovine serum (PBS) and 5 mM Hepes buffer at 2 X lo6 cells/ml. These cells were cultured on 12 mm round coverslips (Bellco, Vineland, N.J.) in 24-well plates (Linbro, Boston, Mass.) at 1 ml/well for 90 min at 37’C, and the nonadherent cells were removed by washing once with HBSS. One milliliter of fresh medium and 0.1 ml of fluid to be assayed for Ia-inducing activity were added to 3-6 replicate cultures and the cultures were incubated at 37°C in 5% CO* for 5 days. Aqueous and serum from groups of six animals were pooled and dialyzed against 1000 vol of phosphate-buffered saline (PBS), pH 7.4, centrifuged at lO,OOOg, and sterile filtered. The final dilutions of the aqueous in the assays for Iainduction were 1:50, 1:250, and 1: 1250. Serum samples were diluted to an absorbance at 280 nm equivalent to that of the aqueous from inflamed eyes, and then diluted 1:50, 1:250, and 1: 1250. Supematants were prepared from nylon-wool-purified rabbit lymph node T lymphocytes, cocultured with a minimal number of macrophages, stimulated by insoluble concanavalin A (Con A) or unstimulated (3), and used at a 1: 10 final dilution as positive and negative controls. Monolayers of cultured macrophages were fixed for 15 min in freshly prepared 1% paraformaldehyde, and stained with 15 ~1 of monoclonal anti-Ia (first step) at 5 pg/ml and 15 ~1 of FITC anti-mouse IgG at 50 pg/ml (second step) (Tago, Inc., Burlingame, Calif.), as described by Beller et al. (8). (Monoclonal anti-rabbit Ia was a kind gift of Dr. K. Knight and Dr. S. Lobel, University of Chicago (9).) The antibody was prepared by immunization of Balb/c mice with spleen cells from homozygous Ia’/Ia’ and Ia2/Ia2 rabbits (9). The antibody stained predominantly Ig+ lymphocytes in lymph node and spleen, and inhibited the one-way MLR by 80% (9). Polyacrylamide gel electrophoresis of immunoprecipitates of spleen cell homogenates prepared with the monoclonal antibody and with anti-Ia alloantisera gave identical patterns. Both ‘Abbreviations used: Con A, concanavalin A, FITC-IgG, fluorescein isothiocyanate conjugated immunoglobulin G, HBSS, Hanks’ balanced salt solution; Hepes, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, IG2, interleukin 2; M&J, macrophages; PBS, phosphate-buffered saline; RPM1 1640, culture medium 1640 from Roswell Park Memorial Institute.
559
SHORT COMMUNICATIONS
precipitated three polypeptides with apparent molecular weights of 35,000, 3 1,000, and 28,000 after extensive reduction and alkylation (9). Cover-slips stained with antiIa or control mouse IgG and FITC-goat anti-mouse IgG were examined using a Zeiss microscope with mercury lamp, darkfield condenser, BG 12 exciting, and GG 14 barrier filter. Assays for Ia-inducing activity in the murine system were carried out in parallel with the studies employing rabbit macrophages. Interleukin 2 (IL-2) assays were carried out using the IL2dependent mouse T-cell line, CT6 (gift of Dr. J. Farrar, NIH), maintained in rat IG2-containing medium, as described by Gillis et al. (10). Data were analyzed statistically using two-way analysis of variance. RESULTS Aqueous obtained at the peak of ocular inflammation 10 days after intravitreal injection of 2 mg of ovalbumin induced Ia expression on 24% (range 23-25; P < 0.025) of macrophages after 5 days in culture (Table 1). Aqueous obtained from contralateral control eyes, and serum obtained at the same time, did not induce Ia expression above the background levels of 2- 12%. By 12 days after intravitreal antigen injection, the difference in Ia-inducing activity between the aqueous from inflamed and control eyes was less marked (P > 0.05, Table 1). By 8 weeks after injection, only barely detectable Ia-inducing activity was present in aqueous or serum. Intravenous challenge injection of 20 mg ovalbumin on Day 56 induced a recall uveitis in the sensitized eye on the following day. Only aqueous obtained on Day 57 from inflamed eyes contained significant Ia-inducing activity (range, 18-2 1; P < 0.0 1). Dilutions of 1:250 of aqueous from inflamed eyes were less than two standard deviations above the corresponding values for control aqueous, while 1: 1250 dilutions were identical to the controls. The effect of rabbit Con A supernatant on Ia expression of cultured rabbit macrophages is shown in Fig. 1A. Thioglycollate-induced rabbit peritoneal macrophages averaged 38% Ia positive (SEM, 3.3; range, 30-58, five repetitions) after 24 hr in culture. Addition of control supematant at this time was followed by a decline of Ia expression to 15% after 3 days and 9% or less (SEM, 2.3; range, 2-17, five repetitions) TABLE
1
Ia-Inducing Activity in Aqueous Humor Aqueous humor Day after intravitreal injection
Inflamed eyes”
Control eyes”
Serum”
10 12 56d 57
24 f 2.2b 16 f 2.3’ <2 20 + 1.56
9+ 1.6 11 f 1.9 5 + 2.9 <2
9+ 1.7 10 + 1.7 <2 12
n Percent Ia+ macrophages + SEM after 5 days culture with aqueous or serum added at a final concentration of 1:50; mean of three experiments. b Significantly different (P < 0.025) from control aqueous. ’ Not significantly different (P > 0.05) from control aqueous. dEighteen rabbits were given 20 mg ovalbumin intravenously immediately after aqueous taps on Day 56.
SHORT COMMUNICATIONS
560 6o
A
50 I” A 5 8 a 2
30
20 IO
+
400 i
i DAYS
IN CULTURE
FIG. 1. Effect of Con A-stimulated and control lymphocyte supematants on macrophage (Mg) expression of Ia. Arrows indicate the time of addition of supematants to a final concentration of 1:10 in all cultures. (A) Rabbit peritoneal exudate adherent cells exposed to Con A-stimulated q compared to control lB rabbit lymphocyte culture supematants. (B) Effect of rabbit- and mouse-lymphocytederived supematants on expression of Ia on mouse peritoneal exudate adherent cells exposed to Con A-stimulated q and control n mouse supematants, compared to stimulated •4and control c3 rabbit supematants (SEM ranged from 0.8 to 3.3% in all cultures).
alter 5 days of culture. Addition of active Con A-stimulated supematant after 24 hr of culture maintained the proportion of Ia positive macrophages at 38% (SEM, 3.0; range 25-47; five repetitions) on Days 3 and 5. The percentage of Ia+ macrophages after 5 days culture with Con A supematant was significantly different from that of macrophages cultured with control supematant (P 4 O.Ol), but was not significantly different (P B 0.05) from that of freshly obtained macrophages. In order to determine whether rabbit and mouse Ia-inducing activities were comparable, rabbit or mouse Con A-stimulated or control supematants were added to mouse peritoneal macrophages after 1 day of culture (Fig. 1B). Mouse Con A supematant induced Ia expression on 55-60% of mouse macrophages after 5 days, while rabbit Con A supematant induced Ia on 25% of mouse macrophages. Cultures given control supematants of mouse or rabbit origin showed background Ia expression of 5-7% after 5 days (Fig. 1B). Samples of pooled aqueous from inflamed eyes containing significant Ia-inducing activity, and of pooled aqueous from contralateral control eyes with essentially no Ia-inducing properties, were tested for IL-2 activity by the CT6 cell assay technique. No IL-2 activity was detected in aqueous humor from inflamed or control eyes. Pooled rabbit serum also did not show IL2 activity. Supematants from rabbit lymph node cells stimulated with insoluble Con A, however, did show IL-2 activity. Peak [3H]thymidine incorporation by CT6 cells in response to rabbit Con A supematant was 80% of that obtained in response to an equivalent dilution of the standard rat Con A supemate used for maintaining the CT6 cell line. DISCUSSION The presence of Ia-inducing activity in aqueous from eyes undergoing immunogenic uveitis, in the absence of detectable activity in contralateral control aqueous or in serum, indicates that an Ia-inducing lymphokine activity may be produced locally. This effect was demonstrated in primary uveitis or 24 hr after secondary uveitis induced by intravenous antigenic challenge. The degree of biologically active material recoverable from the aqueous was found to correspond to the clinical extent of the
SHORT COMMUNICATIONS
561
ocular inflammatory reaction. The marked difference in Ia-inducing activity between aqueous samples obtained 10 and 12 days after intraocular antigen injection also supports the hypothesis that peak local lymphokine production may occur during a relatively short interval. The presence of Ia-inducing activity in supematants of Con A-stimulated, nylon-wool-purified T cells, and not in supematants of nonstimulated T cells, suggests that this factor is of T-cell origin. Corresponding activities in mice and humans have been identified as interferon-y, a T-cell product (6,7). The absence of demonstrable IL-2 activity from aqueous containing Ia-inducing lymphokine may be due to the rapid consumption of IL-2 by dividing T cells (lo), or to the presence of inhibitors of the action of IL2 on its target cells (11). The local production of Ia-inducing lymphokine activity indicates that antigenspecific T cells are being triggered in the eye in the immunogenic inflammation induced by intravitreal injection of antigen (3,5). The presence of Ia-inducing activity in high local concentrations would greatly increase the efficiency of accessory cell presentation of antigen to specific T cells and help to amplify antigen-specific intraocular immune responses. Vascular endothelial cells also may be induced to express Ia by treatment with lymphokines (12). These la-bearing endothelial cells may serve as antigen-presenting cells as well as stimulator cells in the mixed leukocyte reaction ( 13, 14). Induction of Ia expression on choroidal vascular endothelium would greatly increase the intraocular capability for antigen presentation. Further, induction of Ia expression on comeal endothelial cells by locally produced lymphokines might play a significant role in comeal allograft rejection. The site-specific regulation of Ia expression, as demonstrated in the experiments reported in this communication, broadens our understanding of ocular immune reactions and may be of considerable importance in efforts to suppress such immunopathological phenomena in vivo. ACKNOWLEDGMENTS The authors are grateful to Dr. K. Knight and Dr. S. Lobe1 for their gift of the monoclonal anti-rabbit Ia, and to Dr. J. Farrar for his gift of the CT6 cells.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Shimada, K., and Silverstein, A. M., Invest. Ophthalmol. 14, 513, 1975. Prendergast, R. A., and Franklin, R. M., Invest. Ophthalmol. 10, 695, 1971. Liu, S. H., Prendergast, R. A., and Silverstein, A. M., Invest. Ophthalmol. Visual Sci. 24, 361, 1983. Farrar, J. J., Benjamin, W. R., Hilfiker, M. L., Howard, M., Farrar, W. L., and Fuller-Farrar, J., Immunol. Rev. 63, 130, 1982. Unanue, E. R., Adv. Immunol. 31, 1, 1981. Steeg, P. S., Johnson, H. M., and Oppenheim, J. J., J. Immunol. 129, 2402, 1982. Basham, T. Y., and Merigan, T. C., J. Immunol. 130, 1492, 1983. Beller, D. I., Kiely, J.-M., and Unanue, E. R., J. Immunol. 124, 1426, 1980. Lobel, S., and Knight, K., Immunology 51, 35, 1984. Gillii, S., Mochizuki, D. Y., Conlon, P. J., Hefeneider, S. H., Ramthun, C. A., Gillis, A. E., Frank, M. B., Henney, C. S., and Watson, J. D., Immunol. Rev. 63, 167, 1982. Hard& C., Rollinghoff, M., Phzenmaier, K., Mosmann, H., and Wagner, H., J. Exp. Med. 154, 262, 1981. Pober, J. S., Gimbrone, M. A., Cotran, R. S., Reiss, C. S., Burakoff, S. J., Fiers, W., and Ault, K. A., J. Exp. Med. 157, 1339, 1983. Hirschberg, H., Braathen, L. R., and Thorsby, E., Immunol. Rev. 65, 57, 1982. Hirschberg, H., Evensen, S. A., Hemiksen, T., and Thorsby, E., Transplantation 19, 495, 1975.