Current Eye Research
ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20
A new effective and non-harmful chemical adjuvant for the induction of experimental autoimmune uveoretinitis Francois G. Roberge, Dasheng Xu & Chi-Chao Chan To cite this article: Francois G. Roberge, Dasheng Xu & Chi-Chao Chan (1992) A new effective and non-harmful chemical adjuvant for the induction of experimental autoimmune uveoretinitis, Current Eye Research, 11:4, 371-376, DOI: 10.3109/02713689209001790 To link to this article: http://dx.doi.org/10.3109/02713689209001790
Published online: 02 Jul 2009.
Submit your article to this journal
Article views: 2
View related articles
Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=icey20 Download by: [University of Saskatchewan Library]
Date: 25 March 2016, At: 19:58
Current Eye Research
Volume I1 number 4 1092. 371 -376
A new effective and non-harmful chemical adjuvant for the induction of experimental autoimmune uveoretinitis Francois G.Roberge, Dasheng Xu and Chi-Chao C h a d
Current Eye Research 1992.11:371-376.
Department of Ophthalmology, Notre-Dame Hospital, University of Montreal, Montreal, QC, Canada and 'National Eye Institute, Bethesda, MD, USA
ABSTRACT The induction of T cell mediated disease models in animals is usually dependent on the use of complete Freund's adjuvant (CFA). In order to avoid the painful side effects of CFA on the animals, we tested the capacity to induce experimental autoimmune uveoretinitis (EAU) with Hunter's adjuvant (HA). This new adjuvant makes use of nonionic copolymer surfactants, and does not cause deleterious effects to animals. We have found that EAU could be efficiently induced in rats with low doses of S-Ag (10 pg) in very small quantity of HA (10 pl). The biologic parameters of EAU induction showed a potent stimulation of lymphocytes proliferation maximal 11 days after immunization, as well as high levels of antibody production. INTRODUCTION A variety of organ specific autoimmune disease models are induced in animal by immunization with tissue specific proteins. Some of the best studied models are experimental autoimmune encephalomyelitis (EAE), experimental autoimmune thyroiditis (EAT), collagen induced arthritis (CIA), and experimental autoimmune uveoretinitis (1-5). These models are all dependent on the use of complete Freund's adjuvant in their immunization protocol. However the need to use CFA poses many problems. One is that most of the immune response is directed against non-relevant proteins from the mycobacterial component of the adjuvant. In addition the mycobacteria causes the formation of a painful granuloma at
Rcccived
oil
the site of injection as well as a non-specific arthritis in the animal ( 6 ) . These side effects have motivated restrictions on the use of CFA by animal care councils in many countries and generated a search for alternatives. In an attempt to find an alternate more ethically acceptable method of inducing autoimmune disease models with the same reliability as with CFA, we tested a new adjuvant developed by Robert L. Hunter, and recently made available under the trade mark TiterMax. The main components of Hunter's adjuvant are block copolymers of polyoxyethylene (POE) and polyoxypropylene (POP) linked to microparticles of silica and suspended in squalene (7, 8). The adjuvant activity of the POE-POP-POE polymer chains appears to stem from the formation of hydrophilic adhesive surfaces (9, 10). Proteins are bound on these surfaces and made available to cells of the immune system. Additional properties of the polymers consist in the activation of macrophages and complement, and in the stimulation of leukocytes chemotaxis (11-13). From the standpoint of animal welfare, the adjuvant activity was reported to be dissociated from the level of inflammation produced at the site of injection (11). High levels of antibody production in the serum of animals immunized with antigen in Hunter's adjuvant have been obtained (7, 11, 14). However no study has been reported on the capacity of immunization with HA to induce T
January 10. 1992; accepted o n March 16. 1992
0 Oxford University Press
37 1
Current Eye Research
Current Eye Research 1992.11:371-376.
cell mediated disease in animals. We have conducted experiments designed to evaluate that capacity by using the EAU model. EAU is a T cell mediated disease inducible in genetically susceptible species with purified retinal proteins (4,5,15-19). It serves as a model for several intraocular inflammatory diseases in humans, generally grouped under the term non-infectious uveitis. It is most commonly induced in rats by subcutaneous injection of the antigen emulsified in CFA containing 2.0 to 2.5 mg/ml of Mycobacteriurn tuberculosis. Histologically the disease is characterized by a monocytic and lymphocytic infiltration of uveal and retinal tissues leading to the destruction of the retina. MATERIALS AND METHODS
.
.
Animal i m m m z a h a Male Lewis rats (Charles-River, Montreal, Qc.) between 8 and 12 weeks were used for all experiments. Animal care conformed to the ARVO Resolution on the Use of Animals for Research. The animals were immunized in one hind footpad with various doses of S-Antigen (S-Ag) solubilized in PBS pH 7.4, and suspended 1:l (water in oil) in the indicated volume of HA (TiterMaxm, CytRx Corp., Norcross, GA) by syringe injection mixing. Controls received PBS in HA. The rats were examined daily for clinical sign of EAU. After 16 days the eyes were collected, processed for histopathology and the severity of the disease scored on a scale of 0.0 to 4.0 as previously described (19). In some experiments rats were immunized with 30 pg S-Ag suspended 1:l v:v in 50 pl CFA containing 2.0 mg/ml Mycobacteriurn tuberculosis strain H37 RA (Difco, Detroit, MI). S-Ag was prepared from bovine retinae following the method described by Dorey et al (20), and the concentration determined by U.V. absorption. Droliferation assav. Popliteal draining lymph node (DLN) cell suspensions were prepared 6, 9, 1 1 , 13, and 15
372
days after immunization. Four rats were evaluated individually at each time point. The cells were cultured in quadruplicate in flat bottom microplate at 2 x 105 cells/we11/200 p1 of RPMl 1640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 5x1 0-5 M 2-mercaptoethanol, 50 pg/ml gentarnycin, and containing 10% FBS (Hyclone, Logan, UT). S-Ag was added at 5 pg/ml. The cultures were pulsed with 0.5 pCi/well of [3H]thyrnidine (Amersham, Oakville, Ont) for the last 16 h of the indicated culture periods, and the incorporated radioactivity measured by I iqu id scintillation. The results are given in Acpm representing (cpm of Ag stimulated culture) (cpm of nonstimulated culture), and as stimulation index (S.I.) representing (cpm of Ag stimulated culture) / (cpm of nonstimulated culture). odv Droduction measurement Serum Ab levels were measured by solid phase ELISA as described by Tuyen et al. (21). The secondary antibodies were peroxydase-labelled goat anti rat IgM and IgG (Kierkegaard and Perry, Gaithersburg, MD). Levels were compared by unpaired Student’s t-test. RESULTS Lewis rats immunized with graded doses of S-Ag in HA developed severe EAU in a consistent and reproducible fashion, even at doses as low as 10 pg of S-Ag in 10 pI of HA. One of repeated experiments is shown in Table 1. The kinetic for clinical disease appearance was comparable to that usually observed with CFA (16). On histology the disease consisted in a predominant monocytic and lymphocytic infiltration of the uvea and the retina, with few polymorphonuclear leukocytes. The pathology was identical to earlier description of EAU, induced with S-Ag in CFA, at a similar advanced stage (16). During the 2 weeks following immunization, the injected footpad swelling was minimal. The rats
Current Eye Research
--
TABLE 1 INDUCTION OF EAU IN RATS WITH S-Ag IN HUNTER'S ADJUVANT IMMUNI7ATION DOSF
FAU POSlTlVF RATS
QAYAZ
!aYXiw
+STD
S-Ag (50 pg) in HA (50 pl)
313
S-Ag (30 pg) in HA (30 pl)
213
313
not done
S-Ag (20 pg) in HA (20 pl)
213
313
4.0 kO.0
S-Ag (10 pg) in HA (10 pl)
517
617
not done
3.5 k0.5
717
Lewis rats were immunized in one hind footpad with S-Ag (lpglpl in PBS) emulsified 1:1 in the indicated adjuvant volume. Eyes were removed for histopathology 16 days after immunization. Disease was bilateral in all rats. Current Eye Research 1992.11:371-376.
-
appeared not to suffer from pain because they walked normally on the injected foot. The cell proliferative response of the DLN to S-Ag was found to be maximal 11 days after immunization (Fig.1). A distinctive feature of DLN cell culture from antigen in HA immunized rats was the very high level of proliferation observed in the non-stimulated cultures. In fact when the culture period was prolonged from 2 to 4 days, the levels in the nonstimulated cultures increased to reach those of the antigen driven cultures (Fig. 2 ) . The absence of proliferation of DLN cells from rats immunized with PBS in HA alone indicated that this high background response was not due to a direct effect of the adjuvant (data not shown). Likewise, increasing the number of washes from 2 to 4 did not affect the background proliferation. The levels of anti S-Ag antibody production were compared between rats immunized with the respective uveitogenic doses of 10 pg S-Ag in 10 pl HA or 30 pg S-Ag in 50 1.11 CFA. Figure 3 illustrates that, at these comparable disease inducing injections, the titer of serum antibody level was approximately double in the rats immunized in HA compared to those immunized in CFA.
LNC PROLIFERATIVE RESPONSE TO S-Ag 110
-
loo
-
T
m m -
m w 50-
4030-
1 1
20 5
7
9
11
13
,
15
DAY
Fiaure 1. Lewis rats DLN were collected 6, 9, 11, 13, and 15 days after immunization with S-Ag 20 pg in 20 pl HA. The in vitro cell proliferation to SAg was measured by [3H]t hy mid i ne incorporation during the last 16 h of a 64 h culture period. Results represent the average Acpm kSEM of 4 rats cultured individually for each time point.
Statistical analysis showed values of p< 0.001 for dilutions of 1/50 tO 11400 and p l 0.01 for the other dilutions.
373
Current Eye Research S.I.
2.56
S.I.
1.8 I
1.01
T - r
E
Q8 3
1
x
h 0
m
1.4
1.2 1 0.8 0.6 0.4
40
0.2 20
0
0
40
7 2
96
Current Eye Research 1992.11:371-376.
TIME (h)
Fiaure 2. High level of in vitro cell proliferation in nonstimulated cultures after immunization with SAg in HA. DLN were collected 10 days after immunization . The cells were cultured for the indicated periods of time in the presence of SAg (5 pg/ml) (H) or in medium only (H). The results are the average from 6 rats, and represent the incorporation of [sHJthymidine in cpm kSEM during the last 16 h of each culture period, and the calculated S.I. ~
DISCUSSION There is a pressing need to develop new adjuvants for research that have a high degree of efficacy while minimizing discomfort to animals. The pain caused to rodents by the injection of CFA in the footpad is leading many research organizations to disapprove of this type of immunization. This situation is creating difficulties for the study of autoimmune disease models. We have shown here with the EAU model that Hunter’s Adjuvant could efficiently substitute for CFA in a T cell mediated disease. There was minimal swelling at the injection site and the animals did not avoid walking on the injected foot as they usually do when CFA is employed. Another advantage was that EAU could be reproducibly induced with less S-Ag and with a much smaller injection volume. We now routinely use
374
1IDILUTION
Fiaure 3. Serum anti S-Ag antibody levels in Lewis rats immunized 16 days before with S-Ag in HA or CFA at the doses indicated in Materials and Methods. Results of solid phase ELISA measurements represent the average kSEM of groups of 7 rats.
an injection of 20 pg S-Ag in 20 p1 HA for all our experiments in rats, and consistently achieve adequate immunization. The antibody levels that were obtained suggest that it could also be useful in antibody mediated disease models. The serum Ab levels were actually higher with HA than with CFA despite a lower immunizing dose of S-Ag. Concerning the in vitro DLN cell proliferation, the response curve to S-Ag was similar to the one previously observed after immunization in CFA (22). However there was a strong proliferation level in the cultures in which no antigen was added. It suggests that antigen is carried over with the cells possibly associated with the silica microparticles and slowly released for processing. This phenomenon also raised the possibility that EAU could be transferred directly with DLN cells without having to undergo the usual in vitro stimulation culture period. Preliminary assays have shown this direct transfer to be possible. In conclusion Hunter’s Adjuvant appears to offer a
Current Eye Research simple, reliable, and ethically acceptable alternative for the induction of T cell-mediated disease models in animals. ACKNOWLEDGMENTS We thank Mr FranCois Boisvert and Serge Boucher, and Mrs Nicole Newman for excellent technical assistance. This work was supported by a grant from Fondation OCULUS pour les maladies de I'oeil, and Fond de Recherche de I'Universite de Montreal.
Current Eye Research 1992.11:371-376.
CORRESPONDING AUTHOR Dr Francois G. Roberge, National Institutes of Health, Bldg. 10, Room 10N 202, Bethesda, MD 20892. USA.
7.
s,
8.
9.
10. REFERENCES Cohen, I.R. (1985) Experimental 1. autoimmune encephalomyelitis: Pathogenesis and prevention. In "I rnm uno reg uIat o r y Processes i n Experimental Allergic Encephalomyelitis and Multiple Sclerosis", (Eds. Vandenbark, A.A., and Raus, J.C.M.). P. 91. Elsevier Press, Amsterdam. Romagnani, S., Ricci, M., Passaleva, A. and 2. Biliotti, G. (1970) Cell-mediated immune responses to heterologous and homologous thyroglobulin in guinea pigs immunized with heterologous thyroid extracts. Immunology, 19,599-61 2. Holmdahl, R., Mo, J., Nordling, C., Larsson, 3. P., Jansson, L., Goldschmidt, T., Andersson, M. and Klareskog, L. (1989) Collagen induced arthritis: an experimental model for rheumatoid arthritis with involvement of both DTH and immune complex mediated mechanisms. Clin. Exp. Rheumatol. ISUDDI. 31, S41-46. Faure, J.-P. (1980) Autoimmunity and the 4. retina. Curr. Topics Eye Res. 2, 215-302. Gery, I., Mochizuki, M. and Nussenblatt, 5. R.B. (1986) Retinal specific antigens and immunopathogenic processes they provoke. Prog. Retinal Res. 5, 75-109. Allison, A.C. (1989) Antigens and 6. adjuvants for a new generation of v accines . In I mm uno Iog ic a I Adj uv a nt s and Vaccines", (Eds. Gregoriadie, G., "
Allison, A.C. and Poste, G.) Pp. 1-12. Plenum, New York, USA. Snippe, H., De Reuver, M.J., Strickland F., Willers, J.M.N. and Hunter, R.L. (1981) Adjuvant effect of nonionic block polymer surfactants in humoral and cellular immunity. Int. Archs. Allergy appl. Immun. 390-398. Hunter, R.L., Bennett, B., Howerton, D., Buynitzky, S. and Check, I.J. (1989) In "Immunological Adjuvants and Vaccines", (Eds. Gregoriadie, G., Allison, A.C. and Poste, G.) Pp. 133-144. Plenum, New York, USA. Hunter, R. Strickland, F. and Kezdy, F. (1981) The adjuvant activity of nonionic block polymer surfactants. I . The role of hydrophile-lipophile balance. J. Immunol. 122, 1244-1250. Hunter, R.L. and Bennett, B. (1986) The adjuvant activity of nonionic block polymer surfactants. 1II.Characterization of selected biologically active surfaces. Scand. J. Immunol. 287-300. Hunter, R.L. and Bennett, B. (1984) The adjuvant activity of nonionic block polymer surfactants. It. Antibody formation and inflammation related to the structure of triblock and octablock 31 67-31 75. copolymers. J. Immunol. Howerton, D., Check, I.J. and Hunter, R.L. (1987) Induction of macrophages class II MHC antigen expression by synthetic copolymer surfactants: Relation to physicochemical structure and adjuvant activity. Fed. Proc., 46,939. Howerton, D.A., Hunter,R.L., Ziegler, H.K. and Check, I.J. (1990) Induction of macrophage la expression in vivo by a synthetic block copolymer, L81. J. Immunol. 144,1578-1584. Hunter, R., Olsen, M. and Buynitzky, S. (1991) Adjuvant activity of non-ionic block copolymers. IV. Effect of molecular weight and formulation on titre and isotype of antibody. Vaccine, 9, 250-256. Wacker, W.B. and Lipton, M.M. (1968) Experimental allergic uveitis. I. Production in the guinea-pig and rabbit by immunization with retina in adjuvant. J. Immunol. 101,151-156. de Kozak, Y., Sakai, J., Thillaye, B. and Faure, J.P. (1981) S antigen-induced experimental autoimmune uveo-retinitis
a,
11.
m,
12.
13.
14.
15.
16.
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Current Eye Research 17.
18.
Current Eye Research 1992.11:371-376.
19.
in rats. Curr. Eye Res. 1,327-337. Salinas-Carmona, M.C., Nussenblatt, R.B. and Gery, I. (1982) Experimental autoimmune uveitis in the athymic nude rat. Eur. J. Immunol. 12,480-484. Mochizuki, M.,Kuwabara,T., McAllister,C., Nussenblatt, R.B. and Gery, I. (1985) Adoptive transfer of experimental autoimmune uveoretinitis in rats: lmmunopathogenic mechanisms and histologic features. Invest. Ophthalmol. Vis. Sci. 26. 1-9. Roberge, F.G., Lorberboum-Galski, H., Le Hoang, P., De Smet, M., Chan, C.-C., Fitzgerald, D. and Pastan, I. (1989) Selective immunosuppression of activated T cells with the chimeric toxin IL-2PE40. Inhibition of Experimental Autoimmune Uveoretinitis. J. Immunol. 3498-3502. Dorey, C., Cozette, J. and Faure, J.P. (1982) A simple and rapid method for isolation of retinal S antigen. Ophthal. Res. 14,249-255. Tuyen, V.V., Faure, J.P., Thillaye,B., de Kozak,Y. and Fortier, B. (1982) Antibody determination by ELlSA in rats with retinal S antigen-induced uveoretinitis. Curr. Eye Res. 2,7-12. Nussenblatt, R.B., Salinas-Carmona, M., Waksman, B.H. and Gery, I. (1983) Cyclosporin A: alteration of the cellular immune response in S-antigen-induced experimental uveitis. Int. Arch. Allergy Appl. Immunol. Zp, 289-294.
m,
20.
21.
22.
376
ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20
A new effective and non-harmful chemical adjuvant for the induction of experimental autoimmune uveoretinitis Francois G. Roberge, Dasheng Xu & Chi-Chao Chan To cite this article: Francois G. Roberge, Dasheng Xu & Chi-Chao Chan (1992) A new effective and non-harmful chemical adjuvant for the induction of experimental autoimmune uveoretinitis, Current Eye Research, 11:4, 371-376, DOI: 10.3109/02713689209001790 To link to this article: http://dx.doi.org/10.3109/02713689209001790
Published online: 02 Jul 2009.
Submit your article to this journal
Article views: 2
View related articles
Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=icey20 Download by: [University of Saskatchewan Library]
Date: 25 March 2016, At: 19:58
Current Eye Research
Volume I1 number 4 1092. 371 -376
A new effective and non-harmful chemical adjuvant for the induction of experimental autoimmune uveoretinitis Francois G.Roberge, Dasheng Xu and Chi-Chao C h a d
Current Eye Research 1992.11:371-376.
Department of Ophthalmology, Notre-Dame Hospital, University of Montreal, Montreal, QC, Canada and 'National Eye Institute, Bethesda, MD, USA
ABSTRACT The induction of T cell mediated disease models in animals is usually dependent on the use of complete Freund's adjuvant (CFA). In order to avoid the painful side effects of CFA on the animals, we tested the capacity to induce experimental autoimmune uveoretinitis (EAU) with Hunter's adjuvant (HA). This new adjuvant makes use of nonionic copolymer surfactants, and does not cause deleterious effects to animals. We have found that EAU could be efficiently induced in rats with low doses of S-Ag (10 pg) in very small quantity of HA (10 pl). The biologic parameters of EAU induction showed a potent stimulation of lymphocytes proliferation maximal 11 days after immunization, as well as high levels of antibody production. INTRODUCTION A variety of organ specific autoimmune disease models are induced in animal by immunization with tissue specific proteins. Some of the best studied models are experimental autoimmune encephalomyelitis (EAE), experimental autoimmune thyroiditis (EAT), collagen induced arthritis (CIA), and experimental autoimmune uveoretinitis (1-5). These models are all dependent on the use of complete Freund's adjuvant in their immunization protocol. However the need to use CFA poses many problems. One is that most of the immune response is directed against non-relevant proteins from the mycobacterial component of the adjuvant. In addition the mycobacteria causes the formation of a painful granuloma at
Rcccived
oil
the site of injection as well as a non-specific arthritis in the animal ( 6 ) . These side effects have motivated restrictions on the use of CFA by animal care councils in many countries and generated a search for alternatives. In an attempt to find an alternate more ethically acceptable method of inducing autoimmune disease models with the same reliability as with CFA, we tested a new adjuvant developed by Robert L. Hunter, and recently made available under the trade mark TiterMax. The main components of Hunter's adjuvant are block copolymers of polyoxyethylene (POE) and polyoxypropylene (POP) linked to microparticles of silica and suspended in squalene (7, 8). The adjuvant activity of the POE-POP-POE polymer chains appears to stem from the formation of hydrophilic adhesive surfaces (9, 10). Proteins are bound on these surfaces and made available to cells of the immune system. Additional properties of the polymers consist in the activation of macrophages and complement, and in the stimulation of leukocytes chemotaxis (11-13). From the standpoint of animal welfare, the adjuvant activity was reported to be dissociated from the level of inflammation produced at the site of injection (11). High levels of antibody production in the serum of animals immunized with antigen in Hunter's adjuvant have been obtained (7, 11, 14). However no study has been reported on the capacity of immunization with HA to induce T
January 10. 1992; accepted o n March 16. 1992
0 Oxford University Press
37 1
Current Eye Research
Current Eye Research 1992.11:371-376.
cell mediated disease in animals. We have conducted experiments designed to evaluate that capacity by using the EAU model. EAU is a T cell mediated disease inducible in genetically susceptible species with purified retinal proteins (4,5,15-19). It serves as a model for several intraocular inflammatory diseases in humans, generally grouped under the term non-infectious uveitis. It is most commonly induced in rats by subcutaneous injection of the antigen emulsified in CFA containing 2.0 to 2.5 mg/ml of Mycobacteriurn tuberculosis. Histologically the disease is characterized by a monocytic and lymphocytic infiltration of uveal and retinal tissues leading to the destruction of the retina. MATERIALS AND METHODS
.
.
Animal i m m m z a h a Male Lewis rats (Charles-River, Montreal, Qc.) between 8 and 12 weeks were used for all experiments. Animal care conformed to the ARVO Resolution on the Use of Animals for Research. The animals were immunized in one hind footpad with various doses of S-Antigen (S-Ag) solubilized in PBS pH 7.4, and suspended 1:l (water in oil) in the indicated volume of HA (TiterMaxm, CytRx Corp., Norcross, GA) by syringe injection mixing. Controls received PBS in HA. The rats were examined daily for clinical sign of EAU. After 16 days the eyes were collected, processed for histopathology and the severity of the disease scored on a scale of 0.0 to 4.0 as previously described (19). In some experiments rats were immunized with 30 pg S-Ag suspended 1:l v:v in 50 pl CFA containing 2.0 mg/ml Mycobacteriurn tuberculosis strain H37 RA (Difco, Detroit, MI). S-Ag was prepared from bovine retinae following the method described by Dorey et al (20), and the concentration determined by U.V. absorption. Droliferation assav. Popliteal draining lymph node (DLN) cell suspensions were prepared 6, 9, 1 1 , 13, and 15
372
days after immunization. Four rats were evaluated individually at each time point. The cells were cultured in quadruplicate in flat bottom microplate at 2 x 105 cells/we11/200 p1 of RPMl 1640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 5x1 0-5 M 2-mercaptoethanol, 50 pg/ml gentarnycin, and containing 10% FBS (Hyclone, Logan, UT). S-Ag was added at 5 pg/ml. The cultures were pulsed with 0.5 pCi/well of [3H]thyrnidine (Amersham, Oakville, Ont) for the last 16 h of the indicated culture periods, and the incorporated radioactivity measured by I iqu id scintillation. The results are given in Acpm representing (cpm of Ag stimulated culture) (cpm of nonstimulated culture), and as stimulation index (S.I.) representing (cpm of Ag stimulated culture) / (cpm of nonstimulated culture). odv Droduction measurement Serum Ab levels were measured by solid phase ELISA as described by Tuyen et al. (21). The secondary antibodies were peroxydase-labelled goat anti rat IgM and IgG (Kierkegaard and Perry, Gaithersburg, MD). Levels were compared by unpaired Student’s t-test. RESULTS Lewis rats immunized with graded doses of S-Ag in HA developed severe EAU in a consistent and reproducible fashion, even at doses as low as 10 pg of S-Ag in 10 pI of HA. One of repeated experiments is shown in Table 1. The kinetic for clinical disease appearance was comparable to that usually observed with CFA (16). On histology the disease consisted in a predominant monocytic and lymphocytic infiltration of the uvea and the retina, with few polymorphonuclear leukocytes. The pathology was identical to earlier description of EAU, induced with S-Ag in CFA, at a similar advanced stage (16). During the 2 weeks following immunization, the injected footpad swelling was minimal. The rats
Current Eye Research
--
TABLE 1 INDUCTION OF EAU IN RATS WITH S-Ag IN HUNTER'S ADJUVANT IMMUNI7ATION DOSF
FAU POSlTlVF RATS
QAYAZ
!aYXiw
+STD
S-Ag (50 pg) in HA (50 pl)
313
S-Ag (30 pg) in HA (30 pl)
213
313
not done
S-Ag (20 pg) in HA (20 pl)
213
313
4.0 kO.0
S-Ag (10 pg) in HA (10 pl)
517
617
not done
3.5 k0.5
717
Lewis rats were immunized in one hind footpad with S-Ag (lpglpl in PBS) emulsified 1:1 in the indicated adjuvant volume. Eyes were removed for histopathology 16 days after immunization. Disease was bilateral in all rats. Current Eye Research 1992.11:371-376.
-
appeared not to suffer from pain because they walked normally on the injected foot. The cell proliferative response of the DLN to S-Ag was found to be maximal 11 days after immunization (Fig.1). A distinctive feature of DLN cell culture from antigen in HA immunized rats was the very high level of proliferation observed in the non-stimulated cultures. In fact when the culture period was prolonged from 2 to 4 days, the levels in the nonstimulated cultures increased to reach those of the antigen driven cultures (Fig. 2 ) . The absence of proliferation of DLN cells from rats immunized with PBS in HA alone indicated that this high background response was not due to a direct effect of the adjuvant (data not shown). Likewise, increasing the number of washes from 2 to 4 did not affect the background proliferation. The levels of anti S-Ag antibody production were compared between rats immunized with the respective uveitogenic doses of 10 pg S-Ag in 10 pl HA or 30 pg S-Ag in 50 1.11 CFA. Figure 3 illustrates that, at these comparable disease inducing injections, the titer of serum antibody level was approximately double in the rats immunized in HA compared to those immunized in CFA.
LNC PROLIFERATIVE RESPONSE TO S-Ag 110
-
loo
-
T
m m -
m w 50-
4030-
1 1
20 5
7
9
11
13
,
15
DAY
Fiaure 1. Lewis rats DLN were collected 6, 9, 11, 13, and 15 days after immunization with S-Ag 20 pg in 20 pl HA. The in vitro cell proliferation to SAg was measured by [3H]t hy mid i ne incorporation during the last 16 h of a 64 h culture period. Results represent the average Acpm kSEM of 4 rats cultured individually for each time point.
Statistical analysis showed values of p< 0.001 for dilutions of 1/50 tO 11400 and p l 0.01 for the other dilutions.
373
Current Eye Research S.I.
2.56
S.I.
1.8 I
1.01
T - r
E
Q8 3
1
x
h 0
m
1.4
1.2 1 0.8 0.6 0.4
40
0.2 20
0
0
40
7 2
96
Current Eye Research 1992.11:371-376.
TIME (h)
Fiaure 2. High level of in vitro cell proliferation in nonstimulated cultures after immunization with SAg in HA. DLN were collected 10 days after immunization . The cells were cultured for the indicated periods of time in the presence of SAg (5 pg/ml) (H) or in medium only (H). The results are the average from 6 rats, and represent the incorporation of [sHJthymidine in cpm kSEM during the last 16 h of each culture period, and the calculated S.I. ~
DISCUSSION There is a pressing need to develop new adjuvants for research that have a high degree of efficacy while minimizing discomfort to animals. The pain caused to rodents by the injection of CFA in the footpad is leading many research organizations to disapprove of this type of immunization. This situation is creating difficulties for the study of autoimmune disease models. We have shown here with the EAU model that Hunter’s Adjuvant could efficiently substitute for CFA in a T cell mediated disease. There was minimal swelling at the injection site and the animals did not avoid walking on the injected foot as they usually do when CFA is employed. Another advantage was that EAU could be reproducibly induced with less S-Ag and with a much smaller injection volume. We now routinely use
374
1IDILUTION
Fiaure 3. Serum anti S-Ag antibody levels in Lewis rats immunized 16 days before with S-Ag in HA or CFA at the doses indicated in Materials and Methods. Results of solid phase ELISA measurements represent the average kSEM of groups of 7 rats.
an injection of 20 pg S-Ag in 20 p1 HA for all our experiments in rats, and consistently achieve adequate immunization. The antibody levels that were obtained suggest that it could also be useful in antibody mediated disease models. The serum Ab levels were actually higher with HA than with CFA despite a lower immunizing dose of S-Ag. Concerning the in vitro DLN cell proliferation, the response curve to S-Ag was similar to the one previously observed after immunization in CFA (22). However there was a strong proliferation level in the cultures in which no antigen was added. It suggests that antigen is carried over with the cells possibly associated with the silica microparticles and slowly released for processing. This phenomenon also raised the possibility that EAU could be transferred directly with DLN cells without having to undergo the usual in vitro stimulation culture period. Preliminary assays have shown this direct transfer to be possible. In conclusion Hunter’s Adjuvant appears to offer a
Current Eye Research simple, reliable, and ethically acceptable alternative for the induction of T cell-mediated disease models in animals. ACKNOWLEDGMENTS We thank Mr FranCois Boisvert and Serge Boucher, and Mrs Nicole Newman for excellent technical assistance. This work was supported by a grant from Fondation OCULUS pour les maladies de I'oeil, and Fond de Recherche de I'Universite de Montreal.
Current Eye Research 1992.11:371-376.
CORRESPONDING AUTHOR Dr Francois G. Roberge, National Institutes of Health, Bldg. 10, Room 10N 202, Bethesda, MD 20892. USA.
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