Immunology 1991 74 348-354
Prevention of experimental autoimmune uveoretinitis and experimental autoimmune pinealitis in (Lewis x Brown-Norway) F1 rats by HgCl2 injections A. SAOUDI, B. BELLON, Y. DE KOZAK*, J. KUHN, M.-C. VIAL, B. THILLAYE* & PH. DRUET INSERM U28, H6pital Broussais and *INSERM U86, Institut des Cordeliers, Paris, France Acceptedfor publication 4 June 1991
SUMMARY Mercuric chloride (HgCI2) induces in Brown-Norway (BN) and (Lewis x Brown-Norway) F1 hybrid rats a transient autoimmune disease characterized by the production of various antibodies to self and non-self antigens and by a dramatic increase of serum IgE. Experimental autoimmune uveoretinitis (EAU) can be induced in Lewis (LEW) and (LEW x BN) F. hybrid rats by a single immunization with retinal S-antigen (S-Ag). Besides uveoretinitis, animals immunized with S-Ag develop an autoimmune pinealitis (EAP). We demonstrate in this study that (LEW x BN) F. hybrid rats, injected with HgCl2 7 days before S-Ag immunization, are quite efficiently protected against EAU and EAP. We also show that HgCl2-induced protection is neither due to a cytotoxic effect of HgCl2 nor to CD8 + Tcell dependent mechanisms nor to the HgCI2-induced increase of serum IgE concentration. The role of other hypothetical mechanisms, such as anti-S-Ag anti-idiotypic antibodies and/or HgCI2-induced unbalance between T-helper cell subsets, is discussed.
highly susceptible to mercury-induced autoimmunity'7"'8 and resistant to EAU.4'i'l (LEW x BN) F, hybrid rats are susceptible to both diseases.' '71'8 The aim of the present study was to evaluate in (LEW x BN)F1 hybrid rats the effect of mercury-induced systemic autoimmunity on EAU, an organ-specific autoimmune disease. Our results demonstrate that HgCl2 injections, started 1 week before S-Ag immunization, prevent clinical and histological manifestations of EAU and, moreover, protect against EAP. This protective phenomenon is not due to a cytotoxic effect of HgCl2 and is not CD8 + T-cell dependent. The possible implications of anti-S-Ag anti-idiotypic antibodies and of an unbalance between T-helper I (Th- 1)- and Th-2-like subsets during HgCl2induced autoimmunity are discussed.
INTRODUCTION Experimental autoimmune uveoretinitis (EAU) is an ocular autoimmune disease that can be induced in various species by a single immunization with a few micrograms of the soluble retinal S-antigen (S-Ag). 1'4Besides uveoretinitis, animals immunized with S-Ag develop an autoimmune pinealitis (EAP).5 6 It is now well established that EAU is a T-cell mediated autoimmune disease.7 9 In addition, there is evidence that mast cells play an accessory role by facilitating the egress of T cells'°0" due to the release of mediators.'2 Mercuric chloride (HgCI2) induces in Brown-Norway (BN) rats an autoimmune disease due to a T-dependent polyclonal activation of B cells,'3 leading to the production of various antibodies (Ab) to self and non-self Ag'3 1' and to a tremendous increase in total serum IgE.'6 These autoimmune phenomena are transient and no longer observed after the second month, even if HgCl2 injections are continued.'31'41'6 Mercury-induced autoimmunity and EAU are genetically controlled. Lewis (LEW) rats are highly susceptible to EAU4." and resistant to mercury-induced autoimmunity.'7 BN rats are
MATERIALS AND METHODS Animals
(Lewis x Brown-Norway) F. hybrid rats were bred in our animal house from LEW female rats and BN male rats, initially obtained from the CSEAL (CNRS, Orleans, La Source, France). Eight- to 13-week-old (male or female) rats were used in the following experiments.
Abbreviations: Ab, antibody; BN, Brown-Norway; EAE, experimental allergic encephalomyelitis; EAU, experimental autoimmune uveoretinitis; EAP, experimental autoimmune pinealitis; LEW, Lewis, PPD, purified protein derivative of tuberculin; S-Ag, retinal S-antigen; SI. stimulation index. Correspondence: Dr A. Saoudi, INSERM U28, H6pital Broussais 96, rue Didot, 75674 Paris, Cedex 14, France.
Monoclonal antibodies and antisera Fluoresceinated (FITC) goat F(ab')2 anti-mouse IgG antibodies (Janssen Biochimica, Beerse, Belgium) were adsorbed with pooled decomplemented normal rat sera (v/v) prior to use. The
348
Prevention of EAU and EAP by HgCl2 Table 1. Experimental protocol
Weeks Group*
n
H20 S-Ago
10 10
HgCl2 S-Ago H20 S-Ag7
HgCI2 S-Ag7 H20
HgCl2
0
1
2
3
4
5
9 13 7 11
*(LEW x BN)FI rats were immunized in the footpads with S-Ag and CFA supplemented with M. tuberculosis. At the same time they received an i.p. injection of H. pertussis. HgCl2 or H20 was subcutaneously injected three times a week. Horizontal arrows indicate the duration of treatment with HgCl2 or H20. Vertical arrows indicate the time of S-Ag immunization.
OX8 mouse monoclonal antibody (mAb) was produced in our laboratory from the corresponding antibody-producing hybridoma, kindly provided by Dr A. Williams (MRC, Oxford, U.K.). This IgGI antibody recognizes the rat CD8 molecule.'9 Hybridoma cells were grown in Dulbecco's medium (Biochrom, KG, Berlin, Germany) supplemented with 2 mM L-glutamine, 1 % non-essential amino acids (Biochrom), 2-5 mm sodium pyruvate, 100 IU/ml penicillin, 100 yg/ml streptomycin (Gibco, Paisley, Renfrewshire, U.K.) and 15% foetal calf serum (FCS; Gibco). Cells were then injected intraperitoneally (i.p.) into pristane-primed BALB/c mice and mAb was purified on a protein A column (Pharmacia, Uppsala, Sweden). Mouse mAb B1OH2 (thyroglobulin specific) was provided by Dr D. Glotz (H6pital Broussais, Paris, France) and used as an irrelevant IgGl mAb. Induction of autoimmune diseases EA Uand EAP. (LEW x BN) F, rats were immunized once in the hind footpads with 50 jg of purified bovine S-Ag314 in 0-1 ml of phosphate-buffered saline (PBS), emulsified in 0-1 ml of complete Freund's adjuvant (CFA; Difco laboratories, Detroit, MI) and supplemented with 250 jig of Mycobacterium tuberculosis H 37 Ra (Difco). At the same time the animals were i.p. injected with killed Hemophilus pertussis (Institut Merieux, Lyon, France). Mercury-induced autoimmune disease. Mercuric chloride, 1 mg/ml in distilled water, was injected subcutaneously (s.c.) three times a week, at a dose of 100 pg per 100 g body weight, as described previously.'5 Control rats received the same volume of distilled water, adjusted to the same pH (3 8) as the HgC12 solution. '
E.xperimental protocols (Table 1) Twenty-three (LEW x BN) F, rats were injected with HgCl2 and immunized with bovine S-Ag, either the same day as the first HgCI2 injection (HgCI2 S-Ago; n= 10), or 7 days later (HgCI2 S-Ag7; n= 13). Nineteen control rats received the control solution and were immunized with S-Ag either the same day as
349
the first H20 injection (H20 S-Ago; n = 10) or 7 days later (H20 S-Ag7; n = 9). Rats were killed 28 days after S-Ag immunization. Injections of H20 or HgCl2 were continued throughout the experiments. Seven rats received H20 alone and 11 rats HgCI2 alone; they were killed at Day 35 of H20 or HgCI2 administration. Ten other (LEW x BN)F, rats were treated as HgCl2 S-Ag7 rats and 10 others as H20 S-Ag7 rats (not shown in Table 1). Five rats from each group were i.p. injected with 0 5 mg of OX8 mAb as ascites once a week on Days - 1, 6, 13, 20 and 27 of HgCl2 or H20 injections. To determine the effect of OX8 mAb, these rats were bled just before the second (Day 6) and the third (Day 13) injection of the mAb. Mononuclear cells were then isolated from heparinized venous blood by centrifugation on a FicollHypaque gradient (Pharmacia, Uppsala, Sweden), incubated with either B1OH2 or OX8 mAb and stained with FITCconjugated goat F(ab')2 anti-mouse IgG antibodies. The cells were analysed by flow cytometry with a FACScan (BectonDickinson, Synnvale, CA). Popliteal and mesenteric lymph nodes (LN) were removed from animals killed on Day 35 of HgCl2 or H20 administration, i.e. I week after the last injection of OX8 mAb. The percentage of CD8 + cells was determined by FACScan analysis, as above.
Clinico-pathological assessment of EA U and EA P Clinical assessment of EA U. The eyes were examined daily after S-Ag immunization in order to note the day of onset and the severity of uveitis. The severity of clinical EAU was graded from 0 to 4 as follows: 0, normal iris dilation after instillation with a mydriatic drug, no cells in the aqueous humour and the vitreous body; 1, anterior uveitis with cell deposits in the pupil (hypopion); 2, total invasion of the pupil by the cellular infiltrate, no dilation under mydriatic drug; 3, severe inflammation, associated with a corneal oedema; 4, ocular protrusion, haemorrhages in the anterior chamber. Histological assessment oJ EA U and EA P. Histology was performed on Day 28, following S-Ag immunization. Both eyes and pineal gland were removed, dissected, fixed in Bouin's solution, embedded in paraffin and sections were stained with haematoxylin & eosin. The histological lesions were graded from 0 to 7 as follows: 0, no destruction, no cell infiltration; 1-6, limited or total destruction of the various layers-of rods and cones, 1-2, of the outer nuclear layer, 3-4, of the inner nuclear layer, 5-6; 7, destruction of the ganglion cell layer. The pineal gland was examined thoroughly for the presence of mononuclear cells on 16 serial sections for each rat. The presence of mononuclear cells on 1-5 sections was scored as 1, on 6-10 sections as 2, and on 1 1-16 sections as 3. Only cells present in the pineal gland were taken into account. Cells infiltrating the parenchymal stalk were not considered in the scoring of the pinealitis induced by S-Ag immunization.
Antibody determination Rats were bled at the tail artery under ether anaesthesia once a week. Individual serum IgE concentration was measured using previously.'" To detect anti-S-Ag antibodies, the following ELISA was designed: microtitre plates were coated with 100 pl per well of bovine S-Ag at 1 pg/ml in 0-05 M carbonate buffer, pH 9-6, for 1 hr at 37 and then overnight at 4 . After three washes with 0-1 %'Y Tween-20 in PBS, plates were incubated for 90 min at 37- with individual sera an ELISA as described
A. Saoudi et al.
350
Table 2. Effect of HgC12 on EAU and EAP in (LEW x BN)F, rats immunized with S-Ag EAU
Clinical data
Histological data
n positive
Groupt H2O S-Ago HgCl2 S-Ago
H20 S-Ag7 HgCl2 S-Ag7 H2O
HgCl2
eyes/total
EAP
Histological data
n positive
Onset
timet
n positive
eyes/total
Score¶4
rats/total
Scorett
18/20 12/20
16.2+2 3*
3-7+0-5 3-2+1-0
18/20 20/20
5 3+0 5 4 2+1 3**
7/8 9/9
2-6+0-8 2 4+0 5
18/18 6/26
12-3+ 1-1 16-8+2-6***
3-7+0-5 3 7+0 5
18/18 6/26
6 2+0-7 5 0+0 0**
6/9 3/13
2 5+0 8 2 0+ 1 0
0 0
0/14 0/22
0/7 0/11
0 0
0/14 0/22
14-1 + 1-8
Score§
0 0
t Groups have been detailed in Table 1. T Mean number of days between S-Ag immunization and onset of EAU + SD. § Mean grade of clinical EAU at the time of maximal intensity of inflammation + SD. Mean grade of histological lesions of ¶ retina + SD or of ttpineal gland + SD. * P
Prevention of experimental autoimmune uveoretinitis and experimental autoimmune pinealitis in (Lewis x Brown-Norway) F1 rats by HgCl2 injections A. SAOUDI, B. BELLON, Y. DE KOZAK*, J. KUHN, M.-C. VIAL, B. THILLAYE* & PH. DRUET INSERM U28, H6pital Broussais and *INSERM U86, Institut des Cordeliers, Paris, France Acceptedfor publication 4 June 1991
SUMMARY Mercuric chloride (HgCI2) induces in Brown-Norway (BN) and (Lewis x Brown-Norway) F1 hybrid rats a transient autoimmune disease characterized by the production of various antibodies to self and non-self antigens and by a dramatic increase of serum IgE. Experimental autoimmune uveoretinitis (EAU) can be induced in Lewis (LEW) and (LEW x BN) F. hybrid rats by a single immunization with retinal S-antigen (S-Ag). Besides uveoretinitis, animals immunized with S-Ag develop an autoimmune pinealitis (EAP). We demonstrate in this study that (LEW x BN) F. hybrid rats, injected with HgCl2 7 days before S-Ag immunization, are quite efficiently protected against EAU and EAP. We also show that HgCl2-induced protection is neither due to a cytotoxic effect of HgCl2 nor to CD8 + Tcell dependent mechanisms nor to the HgCI2-induced increase of serum IgE concentration. The role of other hypothetical mechanisms, such as anti-S-Ag anti-idiotypic antibodies and/or HgCI2-induced unbalance between T-helper cell subsets, is discussed.
highly susceptible to mercury-induced autoimmunity'7"'8 and resistant to EAU.4'i'l (LEW x BN) F, hybrid rats are susceptible to both diseases.' '71'8 The aim of the present study was to evaluate in (LEW x BN)F1 hybrid rats the effect of mercury-induced systemic autoimmunity on EAU, an organ-specific autoimmune disease. Our results demonstrate that HgCl2 injections, started 1 week before S-Ag immunization, prevent clinical and histological manifestations of EAU and, moreover, protect against EAP. This protective phenomenon is not due to a cytotoxic effect of HgCl2 and is not CD8 + T-cell dependent. The possible implications of anti-S-Ag anti-idiotypic antibodies and of an unbalance between T-helper I (Th- 1)- and Th-2-like subsets during HgCl2induced autoimmunity are discussed.
INTRODUCTION Experimental autoimmune uveoretinitis (EAU) is an ocular autoimmune disease that can be induced in various species by a single immunization with a few micrograms of the soluble retinal S-antigen (S-Ag). 1'4Besides uveoretinitis, animals immunized with S-Ag develop an autoimmune pinealitis (EAP).5 6 It is now well established that EAU is a T-cell mediated autoimmune disease.7 9 In addition, there is evidence that mast cells play an accessory role by facilitating the egress of T cells'°0" due to the release of mediators.'2 Mercuric chloride (HgCI2) induces in Brown-Norway (BN) rats an autoimmune disease due to a T-dependent polyclonal activation of B cells,'3 leading to the production of various antibodies (Ab) to self and non-self Ag'3 1' and to a tremendous increase in total serum IgE.'6 These autoimmune phenomena are transient and no longer observed after the second month, even if HgCl2 injections are continued.'31'41'6 Mercury-induced autoimmunity and EAU are genetically controlled. Lewis (LEW) rats are highly susceptible to EAU4." and resistant to mercury-induced autoimmunity.'7 BN rats are
MATERIALS AND METHODS Animals
(Lewis x Brown-Norway) F. hybrid rats were bred in our animal house from LEW female rats and BN male rats, initially obtained from the CSEAL (CNRS, Orleans, La Source, France). Eight- to 13-week-old (male or female) rats were used in the following experiments.
Abbreviations: Ab, antibody; BN, Brown-Norway; EAE, experimental allergic encephalomyelitis; EAU, experimental autoimmune uveoretinitis; EAP, experimental autoimmune pinealitis; LEW, Lewis, PPD, purified protein derivative of tuberculin; S-Ag, retinal S-antigen; SI. stimulation index. Correspondence: Dr A. Saoudi, INSERM U28, H6pital Broussais 96, rue Didot, 75674 Paris, Cedex 14, France.
Monoclonal antibodies and antisera Fluoresceinated (FITC) goat F(ab')2 anti-mouse IgG antibodies (Janssen Biochimica, Beerse, Belgium) were adsorbed with pooled decomplemented normal rat sera (v/v) prior to use. The
348
Prevention of EAU and EAP by HgCl2 Table 1. Experimental protocol
Weeks Group*
n
H20 S-Ago
10 10
HgCl2 S-Ago H20 S-Ag7
HgCI2 S-Ag7 H20
HgCl2
0
1
2
3
4
5
9 13 7 11
*(LEW x BN)FI rats were immunized in the footpads with S-Ag and CFA supplemented with M. tuberculosis. At the same time they received an i.p. injection of H. pertussis. HgCl2 or H20 was subcutaneously injected three times a week. Horizontal arrows indicate the duration of treatment with HgCl2 or H20. Vertical arrows indicate the time of S-Ag immunization.
OX8 mouse monoclonal antibody (mAb) was produced in our laboratory from the corresponding antibody-producing hybridoma, kindly provided by Dr A. Williams (MRC, Oxford, U.K.). This IgGI antibody recognizes the rat CD8 molecule.'9 Hybridoma cells were grown in Dulbecco's medium (Biochrom, KG, Berlin, Germany) supplemented with 2 mM L-glutamine, 1 % non-essential amino acids (Biochrom), 2-5 mm sodium pyruvate, 100 IU/ml penicillin, 100 yg/ml streptomycin (Gibco, Paisley, Renfrewshire, U.K.) and 15% foetal calf serum (FCS; Gibco). Cells were then injected intraperitoneally (i.p.) into pristane-primed BALB/c mice and mAb was purified on a protein A column (Pharmacia, Uppsala, Sweden). Mouse mAb B1OH2 (thyroglobulin specific) was provided by Dr D. Glotz (H6pital Broussais, Paris, France) and used as an irrelevant IgGl mAb. Induction of autoimmune diseases EA Uand EAP. (LEW x BN) F, rats were immunized once in the hind footpads with 50 jg of purified bovine S-Ag314 in 0-1 ml of phosphate-buffered saline (PBS), emulsified in 0-1 ml of complete Freund's adjuvant (CFA; Difco laboratories, Detroit, MI) and supplemented with 250 jig of Mycobacterium tuberculosis H 37 Ra (Difco). At the same time the animals were i.p. injected with killed Hemophilus pertussis (Institut Merieux, Lyon, France). Mercury-induced autoimmune disease. Mercuric chloride, 1 mg/ml in distilled water, was injected subcutaneously (s.c.) three times a week, at a dose of 100 pg per 100 g body weight, as described previously.'5 Control rats received the same volume of distilled water, adjusted to the same pH (3 8) as the HgC12 solution. '
E.xperimental protocols (Table 1) Twenty-three (LEW x BN) F, rats were injected with HgCl2 and immunized with bovine S-Ag, either the same day as the first HgCI2 injection (HgCI2 S-Ago; n= 10), or 7 days later (HgCI2 S-Ag7; n= 13). Nineteen control rats received the control solution and were immunized with S-Ag either the same day as
349
the first H20 injection (H20 S-Ago; n = 10) or 7 days later (H20 S-Ag7; n = 9). Rats were killed 28 days after S-Ag immunization. Injections of H20 or HgCl2 were continued throughout the experiments. Seven rats received H20 alone and 11 rats HgCI2 alone; they were killed at Day 35 of H20 or HgCI2 administration. Ten other (LEW x BN)F, rats were treated as HgCl2 S-Ag7 rats and 10 others as H20 S-Ag7 rats (not shown in Table 1). Five rats from each group were i.p. injected with 0 5 mg of OX8 mAb as ascites once a week on Days - 1, 6, 13, 20 and 27 of HgCl2 or H20 injections. To determine the effect of OX8 mAb, these rats were bled just before the second (Day 6) and the third (Day 13) injection of the mAb. Mononuclear cells were then isolated from heparinized venous blood by centrifugation on a FicollHypaque gradient (Pharmacia, Uppsala, Sweden), incubated with either B1OH2 or OX8 mAb and stained with FITCconjugated goat F(ab')2 anti-mouse IgG antibodies. The cells were analysed by flow cytometry with a FACScan (BectonDickinson, Synnvale, CA). Popliteal and mesenteric lymph nodes (LN) were removed from animals killed on Day 35 of HgCl2 or H20 administration, i.e. I week after the last injection of OX8 mAb. The percentage of CD8 + cells was determined by FACScan analysis, as above.
Clinico-pathological assessment of EA U and EA P Clinical assessment of EA U. The eyes were examined daily after S-Ag immunization in order to note the day of onset and the severity of uveitis. The severity of clinical EAU was graded from 0 to 4 as follows: 0, normal iris dilation after instillation with a mydriatic drug, no cells in the aqueous humour and the vitreous body; 1, anterior uveitis with cell deposits in the pupil (hypopion); 2, total invasion of the pupil by the cellular infiltrate, no dilation under mydriatic drug; 3, severe inflammation, associated with a corneal oedema; 4, ocular protrusion, haemorrhages in the anterior chamber. Histological assessment oJ EA U and EA P. Histology was performed on Day 28, following S-Ag immunization. Both eyes and pineal gland were removed, dissected, fixed in Bouin's solution, embedded in paraffin and sections were stained with haematoxylin & eosin. The histological lesions were graded from 0 to 7 as follows: 0, no destruction, no cell infiltration; 1-6, limited or total destruction of the various layers-of rods and cones, 1-2, of the outer nuclear layer, 3-4, of the inner nuclear layer, 5-6; 7, destruction of the ganglion cell layer. The pineal gland was examined thoroughly for the presence of mononuclear cells on 16 serial sections for each rat. The presence of mononuclear cells on 1-5 sections was scored as 1, on 6-10 sections as 2, and on 1 1-16 sections as 3. Only cells present in the pineal gland were taken into account. Cells infiltrating the parenchymal stalk were not considered in the scoring of the pinealitis induced by S-Ag immunization.
Antibody determination Rats were bled at the tail artery under ether anaesthesia once a week. Individual serum IgE concentration was measured using previously.'" To detect anti-S-Ag antibodies, the following ELISA was designed: microtitre plates were coated with 100 pl per well of bovine S-Ag at 1 pg/ml in 0-05 M carbonate buffer, pH 9-6, for 1 hr at 37 and then overnight at 4 . After three washes with 0-1 %'Y Tween-20 in PBS, plates were incubated for 90 min at 37- with individual sera an ELISA as described
A. Saoudi et al.
350
Table 2. Effect of HgC12 on EAU and EAP in (LEW x BN)F, rats immunized with S-Ag EAU
Clinical data
Histological data
n positive
Groupt H2O S-Ago HgCl2 S-Ago
H20 S-Ag7 HgCl2 S-Ag7 H2O
HgCl2
eyes/total
EAP
Histological data
n positive
Onset
timet
n positive
eyes/total
Score¶4
rats/total
Scorett
18/20 12/20
16.2+2 3*
3-7+0-5 3-2+1-0
18/20 20/20
5 3+0 5 4 2+1 3**
7/8 9/9
2-6+0-8 2 4+0 5
18/18 6/26
12-3+ 1-1 16-8+2-6***
3-7+0-5 3 7+0 5
18/18 6/26
6 2+0-7 5 0+0 0**
6/9 3/13
2 5+0 8 2 0+ 1 0
0 0
0/14 0/22
0/7 0/11
0 0
0/14 0/22
14-1 + 1-8
Score§
0 0
t Groups have been detailed in Table 1. T Mean number of days between S-Ag immunization and onset of EAU + SD. § Mean grade of clinical EAU at the time of maximal intensity of inflammation + SD. Mean grade of histological lesions of ¶ retina + SD or of ttpineal gland + SD. * P
