Aiovimmunity, 1992, Vol. 12, pp. 175-184 Reprints available directly from the publisher Photocopying permitted by license only
0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
INHIBITION OF EXPERIMENTAL AUTOIMMUNE UVEORETINITIS BY ORAL ADMINISTRATION OF S-ANTIGEN AND SYNTHETIC PEPTIDES TAMARA R. VRABEC', DALE S. GREGERSON', H.S. DUA and LARRY A. DONOSO' 'Thc Retina Service and the Henry and Corinne Bower Laboratory for Macular Degeneration, Wills Eye Hospital, Philadelphia, P A 19107; and the 'Department of'Ophthalmology, University of Minnesota, Minneapolis, MN 55455
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/29/14 For personal use only.
(Received September 9, 1991; infina1,for.m January 23, 1992) S-Antigen, a photoreceptor cell protein, is highly efficient in inducing experimental autoimmune uveoretinitis (EAU), a severe inflammation of the weal tract and retina of the eye. S-Antigen and six synthetic peptides. all of which correspond to known T-cell or B-cell recognition sites, were tested for their abiity to induce oral tolerance to EAU in LEW rats. Feeding three l-mg doses o f native S-Antigen or three doses of one synthetic peptide, designated BSA(343-362) (200 pg per dose), reduced the incidence and severity of EAU induced by immunization with 5Opg of S-Antigen. Another peptide, BSA(270-289), was able to inhibit EAU only when a low dose (I0 p g ) of the uveitogenic S-Antigen was used to induce EAU. Animals which received 200 pg doses of four other immunologically active peptides, BSA(3 1-5 I), BSA( 143-162). BSA(303-327) and BSA(333-352). were not significantly protected. Furthermore, animals fed BSA(343-362) were significantly less susceptible to EAU induced by adoptive transfer (tEAU) of the uveitogenic R9 T-cell lines. Con A-activated lymphocytes purified from spleens of rats fed peptide BSA(343-362) transferred partial resistance to tEAU induced by adoptive transfer of R9 line cells. The resistance of orally tolerized rats to induction of EAU by adoptive transfer of an activated, pathogenic T-cell line, and the ability of lymphocytes from orallytolerized animals to transfer resistance to tEAU shows that effector mechanisms can be inhibited by oral feeding as well as the afferent mechanisms reported here and elsewhere. Circulating levels of IgG specific for S-Antigen were not affected by feeding any of the peptides. KEY WORDS: Adoptive transfer, autoimmunity, oral tolerance, S-Antigen, synthetic peptides, uveitis. ABBREVIATIONS: EAU, experimental autoimmune uveoretinitis: aEAU, actively induced EAU; tEAU, adoptive transfer EAU; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; APC, antigen-presenting cells.
INTRODUCTION Immunologic tolerance can be induced by oral administration of antigenic proteins. This phenomenon has been demonstrated for T-cell dependent, but not T-cell independent antigens','. Higgins and Weiner' demonstrated dose dependent inhibition of acute experimental autoimmune encephalomyelitis (EAE), a T-cell mediated inflammation of the central nervous system, by orally administered myelin basic protein (MBP). Although tolerization was more complete in animals fed before the induction of EAE, a protective eflect was demonstrable in animals with established disease. T-cell proliferative responses to MBP were also suppressed in these animals. Recently, these findings were expanded to show that oral feeding of MBP reduces the severity of relapsing EAE, demonstrating that ongoing, established responses can also be suppressed4. Correspondence to: Larry A. Donoso, M.D., Ph.D., Wills Eye Hospital, 900 Walnut Street, Philadelphia, PA 19107. TEL: (215) 928-3274; FAX: (215) 592-4628,
Experimental autoimmune uveoretinitis (EAU), a CD4' T-cell mediated inflammatory disease involving the uvea and retina of the eye, can be induced by several well-characterized photoreceptor cell proteins, including S-Antigen'. The principal role of T-cells in the pathogenesis of EAU is indicated by the finding that disease can be elicited by the adoptive transfer of T-cell lines or sensitized lymph node cells specific for S-Antigen"' of pathogenic peptides8-I0. Like EAE, EAU can also be prevented by oral administration of the intact autoantigen prior to immunization. In this regard, native bovine S-Antigen has been shown to be an effective agent for the induction of oral tolerance against EAU". Less is known about the role of orally administered synthetic peptides of S-Antigen in preventing EAU, since, in one study, synthetic peptides containing the pathogenic sites in residues 286-297 and 303-314 did not protect against EAU induction by intact bovine S-Antigen". In this study, bovine S-Antigen and synthetic peptides corresponding to several known T-cell and Bcell recognition sites were used to investigate the oral induction of immunologic tolerance in LEW rats. One I75
T.R. VRABEC ET AL.
176
synthetic peptide, BSA(343-362), which corresponds to a recently reported highly pathogenic sequence', was found to inhibit both active induction of EAU by S-Antigen (aEAU), and the adoptive transfer of EAU by T-cell lines (tEAU). MATERIALS AND METHODS
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/29/14 For personal use only.
S-Antigen and synthetic peptides
S-Antigen was purified from retinal homogenates as described previously5. The amino acid sequence of bovine S-Antigen'' and the synthesis of S-Antigen peptides have 'been rep~rted''.'~.Each peptide has been designated according to its amino acid sequence. For example, BSA(343-362) refers to the peptide comprised of amino acid residues 343-362 in bovine S-Antigen. Induction of tolerance was attempted with six synthetic peptides corresponding to regions of bovine S-Antigen identified previously as potentially important functional domains in the immune response to bovine S-Antigen in LEW rats, and designated as . The peptides representing sites A, B, C, and D9*10s13-'s these sites and their immunologic activities are described in Table 1. Peptide BSA(3 1-5 1) contains an antibody epitope reported to be involved in modulating EAU via an anti-idiotypic networki6. Animals
Female LEW rats age 6-8 weeks weighing 150-250 g were obtained from Charles River (Wilmington, MA). Specific pathogen-free animals were used in the adoptive transfer study. Induction of oral tolerance
Two hundred pg of peptide or 1 mg of bovine SAntigen in 0.5 ml water were fed by oral-gastric intubation to rats under light ketamine/chlorpromazine sedation on days 1, 3, and 5 of the study. Feeding was done through a 25-gauge polyethylene tube attached
to a 27-gauge needle on a tuberculin syringe. The tubing was passed from the mouth to the stomach of the animals for each feeding to insure that each dose was ingested completely. In a separate group of animals, 5 , 50 and lOOpg doses of peptide BSA(343-362) were fed in a similar manner on days 1, 3, and 5 . Where indicated, some animals were fed four times on days 1, 3, 5 and 7 prior to immunization. Animals which received saline only or were not treated served as controls. Prior to adoptive transfer of activated lymphocytes, the rats were fed three 200pg doses of BSA(343-362) in a similar fashion on days 1, 3, and 5 of the study. Immunization f o r active EAU induction
On day 7, following intravenous injection of 0.4 ml 6. pertussis ( 1 ~ 1 0 'killed ~ organisms, Mass. Public Health Biologics Laboratory), animals were immunized with 50 pg of native bovine S-Antigen in 100 pI of complete Freund's adjuvant (CFA) emulsion supplemented with 2.5 mg/ml M . tuberculosis H37Ra. After one week, the animals were examined daily for biomicroscopic evidence of uveitis. Twenty-one days after immunization, the animals were sacrificed. All eyes without biomicroscopic evidence of uveitis and diseased eyes were removed for routine histopathologic examination. Animals which were immunized with the reduced dose of bovine S-Antigen received IOpg of antigen in the above adjuvant, and were not given the 6. pertussis. Adoptive transfer of EAU by T-cell lines
Fifteen animals were fed 200pg doses of peptides BSA(343-362) on days 1 , 3 and 5 as described above. On day 10; a group of eight animals was injected with the LEW rat R9 T-cell line (bovine S-Antigen specific) and seven animals received R610 line cells (BSA(352-364)-specific). Both of these T-cell lines have been described elsewhere','. The T-cell lines were activated with Con A for two days prior to intra-
Table 1 Sequences and properties of the bovine S-Antigen peptides.
Sequences und properties of the bovine S-Antigen peptides Pepfide
Amino acid scquence
Pathogenic"
In vitro proliferation'
Antibody ei)ifoue'
BSA(31-51) BSA( 143-162) BSA(270-289) BSA( 303-327) BSA(333-352) BS A( 343-362)
Y IDHVERVEPVDGVVLVDPEL CGVDFEIKAFATHSTDVEED PNSSLTKTLTLVPLLANNRE
No No Yes Yes Yes Yes
No Yes Yes No Weak Weak
Yes No No Yes No NO
DTNLASSTIIKEGlDKTVMGILVS LTVSGLLGELTSSEVATEVP TSSEVATEVPFRLMHPQPED
~
"Pathogenicityin LEW ruts (aec RefsY. 19. 13. 14. IS. 16). "As determined by lheir ubilily lo slimulale iit virro proliferalive response.; in lymph node cells and T-cell lines from bovine S-Anligen immunized LEW unpublisheddata). 'Determined using antisera from bovine S-Anligcn-inimuiiiced LEW ruts ( w e Refs 18. 19).
*TI:
(are Refs 10. IS. 17 arid
PREVENTION OF UVEORETINITIS
venous tail vein transfer of 7x106 line cells to each animal. Fifteen unfed animals that received identical numbers and types of T-cells served as controls. Beginning three days after transfer (day 13), the animals were examined daily for clinical evidence of EAU. The experiment was terminated on day 31, 21 days after transfer. All eyes of unaffected and affected animals were submitted for histopathologic examination.
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/29/14 For personal use only.
Adoptive transfer of tolerance Spleen cells were taken from animals fed BSA(343-362) as described above, or unfed controls, and cultured with Con A for two days. The two groups of lymphocytes were purified by density gradient centrifugation on Lympholyte M (Cedarlane Laboratories, Ltd, Hornby, Ont.) and 80x lo6 lymphocytes were given i.v. to each of 6 naive animals in two groups. After I day, the animals were challenged by tail vein inoculation of 2 ~ 1 0Con ~ A-activated R9 T-cells and observed for evidence of EAU. Assessment 0fEAl.J For the clinical evaluation of EAU, animals were observed daily for clinical signs of EAU by biomicroscopy. The clinical grading system has previously been described'". Onset is defined as the day post immunization or transfer, on which the first definitive signs of EAU are clinically present. For histological examination, both eyes from each animal were fixed in 10% buffered formalin, paraffin-embedded, sectioned (five-micron), and stained with hematoxylin and eosin. The eyes were examined by conventional light microscopy in a masked fashion, and the severity of EAU in each eye was graded from 0 (no EAU) to 4+ (severe) according to criteria described in detail e Ise where I".
171
The slides were decoded and the eyes from each animal were averaged to give a single score per animal. In those instances where the severity observed by clinical examination appears not to correspond to the histopathology, the discrepancy between the clinical scores and the histopathology is easily explained by considering the time when the eyes were taken for histology. All eyes were taken at 21 days post immunization or transfer. This allowed the full clinical course to be observed prior to sacrifice. Consequently, the eyes were becoming "quiet" when they were taken for histology, and the pathology shows a narrower range of features. Conversely, clinical observations made at the peak of disease expression reveal the maximal severity observed in any given eye.
RESULTS Inhibition of actively induced EAU The tolerogenic effect of oral administration of bovine S-Antigen and each of the six synthetic peptides on the active induction of EAU by bovine S-Antigen is shown in Table 2. Naive rats were fed peptides of bovine S-Antigen as indicated three times, once each on days 1, 3, and 5, and then immunized with a highly uveitogenic 50 p g dose of bovine S-Antigen on day 7. As shown, 200 p g doses of BSA(343-362) were also effective in conferring a state of resistance to aEAU following immunization with bovine S-Antigen in CFA. Only one of six animals that received three oral 2 0 0 p g doses of BSA(343-362) showed clinical or histopathologic evidence of retinal or uveal inflammation (Figure 1 ) during the course of the experiment (2 1 days following immunization). Similarly, only one of three animals fed three 1-mg doses of SAntigen developed uveitis, and it was only mild, unilateral clinical disease (5% hypopyon), with a delayed
178
T.R. VRABEC ET AL.
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/29/14 For personal use only.
onset as compared to the control animals (average onset at day 16 versus day 12). All control animals exhibited severe (4+), bilateral EAU with early onset. The dose dependencey of protection conferred by feeding BSA(343-362) was assessed by feeding 5 , 50 and 100 p g doses of the peptide (Table 3). All animals fed l00,ug doses developed EAU, but with a reduced mean clinical severity of 2+ and an average onset of 16 days. The 50 pg dose was minimally protective in
Figure 1 (A) Absence of ocular inflammatory response in a LEW rat fed peptide BSA(343-362). (B) Severe inflammatory response in control animal. Note the diffuse inflammatory response throughout all retinal layers with complete destruction of the photoreceptor cells. There is an associated vitritis and subretinal exudate.
that onset was delayed from 12 to 16 days, while animals fed 5 p g doses of BSA(343-362) were clearly not protected. By comparison, all control animals developed severe (4+) clinical EAU by day 12 (Table 3). The minimal dose clearly capable of inhibiting EAU induction was 200 pg of BSA(343-362) given three times. With regards to the other peptides, BSA(3 1-5 l), BSA( 143-162) and BSA(333-352) did not reduce the
PREVENTION OF UVEORETINITIS
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/29/14 For personal use only.
incidence of EAU in repeated 200pg doses; all animals in both groups developed bilateral uveitis with at least 50% hypopyon as early as 14 days after immunization with 5 0 p g of bovine S-Antigen (Table 2). Only two of three animals in each of the remaining groups fed either BSA(303-327) or BSA(270-289) developed uveitis with onset delayed from 12 to 14 days, suggestive of protection from EAU. To determine if a milder dose of bovine S-Antigen might permit expression of a reduced ability to inhibit EAU, peptide BSA(270-289) was fed four times on days 1, 3, 5 and 7, to a group of four rats which were then immunized with 1Opg of bovine S-Antigen rather than 50 pg; also, the B . pertussis was omitted. Under
179
these conditions, peptide BSA(270-289) was able to inhibit EAU induction (Table 3). Suppression of EAU induced by adoptive transfer of uveitogenic T-cell lines
The effect of BSA(343-362) feeding on tEAU induced by adoptive transfer of the uveitogenic R9 and R610 T-cell lines was examined. The R9 T-cell line, isolated from a bovine S-Antigen immunized animal and maintained in vitro with bovine S-Antigen6, was tested first. The R9 line has little proliferative response to the BSA(343-362) region (note Table 6), but is highly uveitogenic. Unlike the R610 line, which
Table 3 Inhibition of aEAU by oral administration of synthetic peptides. Orul
tolet.i)pn
Dose of S-Antigen
Dose ner feeding
aEAU Incidence Affictedl animalsd
AJffcredl eyesh
Aiwage sever;ty'
A vei'uge onsetd 16 16
12 12 16 15
Table 4
Inhibition of tEAU by oral feeding peptide BSA(343-362). tEA U
Anintul
Linc c,ell.r
R9 R9 R6lO R6 10
Pretreatnrent
NONE BSA(343-362) none BSA(343-362)
Inc,idence
his to pa tho log.^^
Onset'
Animals"
Eyes'
MeankSD
818
16/16 12/16 7/14 5/14
5.9+ I .s 8.8f I .9 7.7+ I .5 I I .81t4.8
718 517 4/7
frobd
MeankSD
2.6k0.4 I Bf0.9 I .o+I . I 0.9f I.3
0.0002 0.04
Proh''
0.009 0.38
"Numhcr 0 1 aflecled aniiiials/nuniher of aninials ininiuniied. 'Numlw 01 nflecled cye~holalnuniher ol'cyc\ ' A l l ~ ~ i canimal* d only. day, poht tramfer "C.mi1pmwii of fed versu\ unfed animal\: I-ICCI. unpdircd. onc-t;iilcd. 'All c \ L . \ 01 ,111 iiiilnlnl\.
Table 5
5 6 7 8 9 I0 II
Clinical course of tEAU following oral feeding with peptide BSA(343-362)
16
33 43 53 60 57 42
I 6 10 16
25 27 30
0.06 0.001
0.007 0.002 0.0005 0.0004 0.2
'M;tnii Whiiney 11.conrclcd I : compariwn 01 unlreiited i t n d pcplldc-led m m a l r
0 2 8 13
0 0 I
I1
4 5
II
4
8
4
0.32 0.12 0.29 0.22 0.12 0.24
Ixo
T.R. VRABEC ET AL.
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/29/14 For personal use only.
is specific for epitopes contained in the BSA( 343-362) peptide used to induce tolerance, the response of the R9 line is multi-specific, responding to several sitesh.". In animals which received the R9 line cells, 8 of 8 untreated animals developed EAU (Table 4). However, in contrast to controls, the disease in animals fed BSA(343-362) was delayed in average onset from 5.9 days to 8.6 days (P=0.002; Table 4, and Fig. 2A). In addition, there was a marked reduction in clinical severity in this group of animals as compared to untreated controls on all days from day 6 to 1 1 post transfer (P value ranged from < 0.0004 to
0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
INHIBITION OF EXPERIMENTAL AUTOIMMUNE UVEORETINITIS BY ORAL ADMINISTRATION OF S-ANTIGEN AND SYNTHETIC PEPTIDES TAMARA R. VRABEC', DALE S. GREGERSON', H.S. DUA and LARRY A. DONOSO' 'Thc Retina Service and the Henry and Corinne Bower Laboratory for Macular Degeneration, Wills Eye Hospital, Philadelphia, P A 19107; and the 'Department of'Ophthalmology, University of Minnesota, Minneapolis, MN 55455
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/29/14 For personal use only.
(Received September 9, 1991; infina1,for.m January 23, 1992) S-Antigen, a photoreceptor cell protein, is highly efficient in inducing experimental autoimmune uveoretinitis (EAU), a severe inflammation of the weal tract and retina of the eye. S-Antigen and six synthetic peptides. all of which correspond to known T-cell or B-cell recognition sites, were tested for their abiity to induce oral tolerance to EAU in LEW rats. Feeding three l-mg doses o f native S-Antigen or three doses of one synthetic peptide, designated BSA(343-362) (200 pg per dose), reduced the incidence and severity of EAU induced by immunization with 5Opg of S-Antigen. Another peptide, BSA(270-289), was able to inhibit EAU only when a low dose (I0 p g ) of the uveitogenic S-Antigen was used to induce EAU. Animals which received 200 pg doses of four other immunologically active peptides, BSA(3 1-5 I), BSA( 143-162). BSA(303-327) and BSA(333-352). were not significantly protected. Furthermore, animals fed BSA(343-362) were significantly less susceptible to EAU induced by adoptive transfer (tEAU) of the uveitogenic R9 T-cell lines. Con A-activated lymphocytes purified from spleens of rats fed peptide BSA(343-362) transferred partial resistance to tEAU induced by adoptive transfer of R9 line cells. The resistance of orally tolerized rats to induction of EAU by adoptive transfer of an activated, pathogenic T-cell line, and the ability of lymphocytes from orallytolerized animals to transfer resistance to tEAU shows that effector mechanisms can be inhibited by oral feeding as well as the afferent mechanisms reported here and elsewhere. Circulating levels of IgG specific for S-Antigen were not affected by feeding any of the peptides. KEY WORDS: Adoptive transfer, autoimmunity, oral tolerance, S-Antigen, synthetic peptides, uveitis. ABBREVIATIONS: EAU, experimental autoimmune uveoretinitis: aEAU, actively induced EAU; tEAU, adoptive transfer EAU; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; APC, antigen-presenting cells.
INTRODUCTION Immunologic tolerance can be induced by oral administration of antigenic proteins. This phenomenon has been demonstrated for T-cell dependent, but not T-cell independent antigens','. Higgins and Weiner' demonstrated dose dependent inhibition of acute experimental autoimmune encephalomyelitis (EAE), a T-cell mediated inflammation of the central nervous system, by orally administered myelin basic protein (MBP). Although tolerization was more complete in animals fed before the induction of EAE, a protective eflect was demonstrable in animals with established disease. T-cell proliferative responses to MBP were also suppressed in these animals. Recently, these findings were expanded to show that oral feeding of MBP reduces the severity of relapsing EAE, demonstrating that ongoing, established responses can also be suppressed4. Correspondence to: Larry A. Donoso, M.D., Ph.D., Wills Eye Hospital, 900 Walnut Street, Philadelphia, PA 19107. TEL: (215) 928-3274; FAX: (215) 592-4628,
Experimental autoimmune uveoretinitis (EAU), a CD4' T-cell mediated inflammatory disease involving the uvea and retina of the eye, can be induced by several well-characterized photoreceptor cell proteins, including S-Antigen'. The principal role of T-cells in the pathogenesis of EAU is indicated by the finding that disease can be elicited by the adoptive transfer of T-cell lines or sensitized lymph node cells specific for S-Antigen"' of pathogenic peptides8-I0. Like EAE, EAU can also be prevented by oral administration of the intact autoantigen prior to immunization. In this regard, native bovine S-Antigen has been shown to be an effective agent for the induction of oral tolerance against EAU". Less is known about the role of orally administered synthetic peptides of S-Antigen in preventing EAU, since, in one study, synthetic peptides containing the pathogenic sites in residues 286-297 and 303-314 did not protect against EAU induction by intact bovine S-Antigen". In this study, bovine S-Antigen and synthetic peptides corresponding to several known T-cell and Bcell recognition sites were used to investigate the oral induction of immunologic tolerance in LEW rats. One I75
T.R. VRABEC ET AL.
176
synthetic peptide, BSA(343-362), which corresponds to a recently reported highly pathogenic sequence', was found to inhibit both active induction of EAU by S-Antigen (aEAU), and the adoptive transfer of EAU by T-cell lines (tEAU). MATERIALS AND METHODS
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/29/14 For personal use only.
S-Antigen and synthetic peptides
S-Antigen was purified from retinal homogenates as described previously5. The amino acid sequence of bovine S-Antigen'' and the synthesis of S-Antigen peptides have 'been rep~rted''.'~.Each peptide has been designated according to its amino acid sequence. For example, BSA(343-362) refers to the peptide comprised of amino acid residues 343-362 in bovine S-Antigen. Induction of tolerance was attempted with six synthetic peptides corresponding to regions of bovine S-Antigen identified previously as potentially important functional domains in the immune response to bovine S-Antigen in LEW rats, and designated as . The peptides representing sites A, B, C, and D9*10s13-'s these sites and their immunologic activities are described in Table 1. Peptide BSA(3 1-5 1) contains an antibody epitope reported to be involved in modulating EAU via an anti-idiotypic networki6. Animals
Female LEW rats age 6-8 weeks weighing 150-250 g were obtained from Charles River (Wilmington, MA). Specific pathogen-free animals were used in the adoptive transfer study. Induction of oral tolerance
Two hundred pg of peptide or 1 mg of bovine SAntigen in 0.5 ml water were fed by oral-gastric intubation to rats under light ketamine/chlorpromazine sedation on days 1, 3, and 5 of the study. Feeding was done through a 25-gauge polyethylene tube attached
to a 27-gauge needle on a tuberculin syringe. The tubing was passed from the mouth to the stomach of the animals for each feeding to insure that each dose was ingested completely. In a separate group of animals, 5 , 50 and lOOpg doses of peptide BSA(343-362) were fed in a similar manner on days 1, 3, and 5 . Where indicated, some animals were fed four times on days 1, 3, 5 and 7 prior to immunization. Animals which received saline only or were not treated served as controls. Prior to adoptive transfer of activated lymphocytes, the rats were fed three 200pg doses of BSA(343-362) in a similar fashion on days 1, 3, and 5 of the study. Immunization f o r active EAU induction
On day 7, following intravenous injection of 0.4 ml 6. pertussis ( 1 ~ 1 0 'killed ~ organisms, Mass. Public Health Biologics Laboratory), animals were immunized with 50 pg of native bovine S-Antigen in 100 pI of complete Freund's adjuvant (CFA) emulsion supplemented with 2.5 mg/ml M . tuberculosis H37Ra. After one week, the animals were examined daily for biomicroscopic evidence of uveitis. Twenty-one days after immunization, the animals were sacrificed. All eyes without biomicroscopic evidence of uveitis and diseased eyes were removed for routine histopathologic examination. Animals which were immunized with the reduced dose of bovine S-Antigen received IOpg of antigen in the above adjuvant, and were not given the 6. pertussis. Adoptive transfer of EAU by T-cell lines
Fifteen animals were fed 200pg doses of peptides BSA(343-362) on days 1 , 3 and 5 as described above. On day 10; a group of eight animals was injected with the LEW rat R9 T-cell line (bovine S-Antigen specific) and seven animals received R610 line cells (BSA(352-364)-specific). Both of these T-cell lines have been described elsewhere','. The T-cell lines were activated with Con A for two days prior to intra-
Table 1 Sequences and properties of the bovine S-Antigen peptides.
Sequences und properties of the bovine S-Antigen peptides Pepfide
Amino acid scquence
Pathogenic"
In vitro proliferation'
Antibody ei)ifoue'
BSA(31-51) BSA( 143-162) BSA(270-289) BSA( 303-327) BSA(333-352) BS A( 343-362)
Y IDHVERVEPVDGVVLVDPEL CGVDFEIKAFATHSTDVEED PNSSLTKTLTLVPLLANNRE
No No Yes Yes Yes Yes
No Yes Yes No Weak Weak
Yes No No Yes No NO
DTNLASSTIIKEGlDKTVMGILVS LTVSGLLGELTSSEVATEVP TSSEVATEVPFRLMHPQPED
~
"Pathogenicityin LEW ruts (aec RefsY. 19. 13. 14. IS. 16). "As determined by lheir ubilily lo slimulale iit virro proliferalive response.; in lymph node cells and T-cell lines from bovine S-Anligen immunized LEW unpublisheddata). 'Determined using antisera from bovine S-Anligcn-inimuiiiced LEW ruts ( w e Refs 18. 19).
*TI:
(are Refs 10. IS. 17 arid
PREVENTION OF UVEORETINITIS
venous tail vein transfer of 7x106 line cells to each animal. Fifteen unfed animals that received identical numbers and types of T-cells served as controls. Beginning three days after transfer (day 13), the animals were examined daily for clinical evidence of EAU. The experiment was terminated on day 31, 21 days after transfer. All eyes of unaffected and affected animals were submitted for histopathologic examination.
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/29/14 For personal use only.
Adoptive transfer of tolerance Spleen cells were taken from animals fed BSA(343-362) as described above, or unfed controls, and cultured with Con A for two days. The two groups of lymphocytes were purified by density gradient centrifugation on Lympholyte M (Cedarlane Laboratories, Ltd, Hornby, Ont.) and 80x lo6 lymphocytes were given i.v. to each of 6 naive animals in two groups. After I day, the animals were challenged by tail vein inoculation of 2 ~ 1 0Con ~ A-activated R9 T-cells and observed for evidence of EAU. Assessment 0fEAl.J For the clinical evaluation of EAU, animals were observed daily for clinical signs of EAU by biomicroscopy. The clinical grading system has previously been described'". Onset is defined as the day post immunization or transfer, on which the first definitive signs of EAU are clinically present. For histological examination, both eyes from each animal were fixed in 10% buffered formalin, paraffin-embedded, sectioned (five-micron), and stained with hematoxylin and eosin. The eyes were examined by conventional light microscopy in a masked fashion, and the severity of EAU in each eye was graded from 0 (no EAU) to 4+ (severe) according to criteria described in detail e Ise where I".
171
The slides were decoded and the eyes from each animal were averaged to give a single score per animal. In those instances where the severity observed by clinical examination appears not to correspond to the histopathology, the discrepancy between the clinical scores and the histopathology is easily explained by considering the time when the eyes were taken for histology. All eyes were taken at 21 days post immunization or transfer. This allowed the full clinical course to be observed prior to sacrifice. Consequently, the eyes were becoming "quiet" when they were taken for histology, and the pathology shows a narrower range of features. Conversely, clinical observations made at the peak of disease expression reveal the maximal severity observed in any given eye.
RESULTS Inhibition of actively induced EAU The tolerogenic effect of oral administration of bovine S-Antigen and each of the six synthetic peptides on the active induction of EAU by bovine S-Antigen is shown in Table 2. Naive rats were fed peptides of bovine S-Antigen as indicated three times, once each on days 1, 3, and 5, and then immunized with a highly uveitogenic 50 p g dose of bovine S-Antigen on day 7. As shown, 200 p g doses of BSA(343-362) were also effective in conferring a state of resistance to aEAU following immunization with bovine S-Antigen in CFA. Only one of six animals that received three oral 2 0 0 p g doses of BSA(343-362) showed clinical or histopathologic evidence of retinal or uveal inflammation (Figure 1 ) during the course of the experiment (2 1 days following immunization). Similarly, only one of three animals fed three 1-mg doses of SAntigen developed uveitis, and it was only mild, unilateral clinical disease (5% hypopyon), with a delayed
178
T.R. VRABEC ET AL.
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/29/14 For personal use only.
onset as compared to the control animals (average onset at day 16 versus day 12). All control animals exhibited severe (4+), bilateral EAU with early onset. The dose dependencey of protection conferred by feeding BSA(343-362) was assessed by feeding 5 , 50 and 100 p g doses of the peptide (Table 3). All animals fed l00,ug doses developed EAU, but with a reduced mean clinical severity of 2+ and an average onset of 16 days. The 50 pg dose was minimally protective in
Figure 1 (A) Absence of ocular inflammatory response in a LEW rat fed peptide BSA(343-362). (B) Severe inflammatory response in control animal. Note the diffuse inflammatory response throughout all retinal layers with complete destruction of the photoreceptor cells. There is an associated vitritis and subretinal exudate.
that onset was delayed from 12 to 16 days, while animals fed 5 p g doses of BSA(343-362) were clearly not protected. By comparison, all control animals developed severe (4+) clinical EAU by day 12 (Table 3). The minimal dose clearly capable of inhibiting EAU induction was 200 pg of BSA(343-362) given three times. With regards to the other peptides, BSA(3 1-5 l), BSA( 143-162) and BSA(333-352) did not reduce the
PREVENTION OF UVEORETINITIS
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/29/14 For personal use only.
incidence of EAU in repeated 200pg doses; all animals in both groups developed bilateral uveitis with at least 50% hypopyon as early as 14 days after immunization with 5 0 p g of bovine S-Antigen (Table 2). Only two of three animals in each of the remaining groups fed either BSA(303-327) or BSA(270-289) developed uveitis with onset delayed from 12 to 14 days, suggestive of protection from EAU. To determine if a milder dose of bovine S-Antigen might permit expression of a reduced ability to inhibit EAU, peptide BSA(270-289) was fed four times on days 1, 3, 5 and 7, to a group of four rats which were then immunized with 1Opg of bovine S-Antigen rather than 50 pg; also, the B . pertussis was omitted. Under
179
these conditions, peptide BSA(270-289) was able to inhibit EAU induction (Table 3). Suppression of EAU induced by adoptive transfer of uveitogenic T-cell lines
The effect of BSA(343-362) feeding on tEAU induced by adoptive transfer of the uveitogenic R9 and R610 T-cell lines was examined. The R9 T-cell line, isolated from a bovine S-Antigen immunized animal and maintained in vitro with bovine S-Antigen6, was tested first. The R9 line has little proliferative response to the BSA(343-362) region (note Table 6), but is highly uveitogenic. Unlike the R610 line, which
Table 3 Inhibition of aEAU by oral administration of synthetic peptides. Orul
tolet.i)pn
Dose of S-Antigen
Dose ner feeding
aEAU Incidence Affictedl animalsd
AJffcredl eyesh
Aiwage sever;ty'
A vei'uge onsetd 16 16
12 12 16 15
Table 4
Inhibition of tEAU by oral feeding peptide BSA(343-362). tEA U
Anintul
Linc c,ell.r
R9 R9 R6lO R6 10
Pretreatnrent
NONE BSA(343-362) none BSA(343-362)
Inc,idence
his to pa tho log.^^
Onset'
Animals"
Eyes'
MeankSD
818
16/16 12/16 7/14 5/14
5.9+ I .s 8.8f I .9 7.7+ I .5 I I .81t4.8
718 517 4/7
frobd
MeankSD
2.6k0.4 I Bf0.9 I .o+I . I 0.9f I.3
0.0002 0.04
Proh''
0.009 0.38
"Numhcr 0 1 aflecled aniiiials/nuniher of aninials ininiuniied. 'Numlw 01 nflecled cye~holalnuniher ol'cyc\ ' A l l ~ ~ i canimal* d only. day, poht tramfer "C.mi1pmwii of fed versu\ unfed animal\: I-ICCI. unpdircd. onc-t;iilcd. 'All c \ L . \ 01 ,111 iiiilnlnl\.
Table 5
5 6 7 8 9 I0 II
Clinical course of tEAU following oral feeding with peptide BSA(343-362)
16
33 43 53 60 57 42
I 6 10 16
25 27 30
0.06 0.001
0.007 0.002 0.0005 0.0004 0.2
'M;tnii Whiiney 11.conrclcd I : compariwn 01 unlreiited i t n d pcplldc-led m m a l r
0 2 8 13
0 0 I
I1
4 5
II
4
8
4
0.32 0.12 0.29 0.22 0.12 0.24
Ixo
T.R. VRABEC ET AL.
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/29/14 For personal use only.
is specific for epitopes contained in the BSA( 343-362) peptide used to induce tolerance, the response of the R9 line is multi-specific, responding to several sitesh.". In animals which received the R9 line cells, 8 of 8 untreated animals developed EAU (Table 4). However, in contrast to controls, the disease in animals fed BSA(343-362) was delayed in average onset from 5.9 days to 8.6 days (P=0.002; Table 4, and Fig. 2A). In addition, there was a marked reduction in clinical severity in this group of animals as compared to untreated controls on all days from day 6 to 1 1 post transfer (P value ranged from < 0.0004 to
