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Actively induced experimental allergic orchitis in lewis-resistant (Le-R) Rats: Reversibility of disease resistance by immunization with Bordetella pertussis
CELLULARIMMUNOLOGY
119,233-238
(1989)
SHORT COMMUNICATION Actively Induced Experimental Allergic Orchitis in Lewis-Resistant (Le-R) Rats: Reversibility of Disease Resistance by Immunization with Bordetella pertussis C. TEUSCHER,
Z-Z.
ZHOU,
Y. ZHENG, AND W. F. HICKEY*
Division of Reproductive Biology, Department of Obstetrics and Gynecology, and *Department of Pathology and Laboratory Medicine, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Received August 15.1988; accepted October 25, 1988 Differential susceptibility to the induction of experimental allergic orchitis (EAO) was examined in Lewis/NCr and Le-R subline rats. Lewis/NCr rats were found to be fully susceptible to the induction of EAO whereas Le-R subline rats were not. Disease resistance exhibited by Le-R rats could be overcome by including Bordetella pertussis in the immunization protocol. However, reversal of resistance with B. pertussis was dependent on the dose of rat testicular homogenate in the inoculum and found to be effective only at lower doses of antigen ( 10 m&at). Disease resistance in Le-R rats as well as B. petit&s-induced reversal of resistance did not appear to be associated with either (1) a significant difference in the number of mast cells in the ductus efferentes, the anatomic location of the earliest inflammatory infiltrates, or (2) an alteration in the phenotypic expression of either innate or B. pertussis-induced sensitivity to vasoactive amines. The results are discussed in the context of the role of B. pertussis in other animal models of organ-specific autoimmune diseases. It is proposed that the phenotypic expression of resistance to EAO in Le-R rats is a result of a mutation in a common regulatory locus affecting susceptibility to multiple autoimmune diseases and whose immunoreguiatory action is normally exerted during the sensitization phase of the immune response. 0 1989 Academic press, IIIC.
INTRODUCTION Experimental allergic orchitis (EAO) is an organ-specific autoimmune disease which can be elicited in a genetically susceptible animal by immunization with either homologous or autologous testicular and/or sperm antigens in conjunction with the appropriate adjuvants (reviewed in (l-3)). EAO is manifest as inflammation of the testis (orchitis), aspermatogenesis, epididymitis, and/or vasitis. Susceptibility to the induction of EAO has been shown to be controlled by both major histocompatibility complex (MHC)-linked and non-MHC-linked genes (4,5). The identity and mechanism of action of non-MHC-linked genes in the regulation of organ-specific autoimmune diseases are, for the most part, ill defined and poorly understood. One approach which is potentially useful in identifying and elucidating their function(s) is to utilize experimental animals which have a very high degree of genetic similarity, but differ in the phenotypic expression of certain traits, i.e., coisogenie strains and/or mutant sublines of inbred animals. With regard to organ-specific autoimmune diseases, one such example is the Lewis-resistant @e-R) strain of rat. 233 0008-8749189
$3.00
Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
234
SHORT
COMMUNICATION
The Le-R subline was derived from a Lewis colony which was originally susceptible to the induction of experimental allergic encephalomyelitis (EAE) (6). Serologically, antigenic differences between the two substrains could not be detected using antisera from reciprocal immunization (6) nor has it been possible to generate monoclonal antibodies which will discriminate between the two substrains (7). However, the two will eventually reject reciprocal skin grafts (W. F. Hickey, unpublished observation). Nevertheless, Le-R rats possess sufficient genetic similarity to Lewis rats to allow for adoptive transfer of EAE from Lewis to Le-R animals (6). Gasser et al. (7) suggested that the Le-R subline developed as the result of a simple mutational event since the gene or genes for nonsusceptibility developed in the Le-R subline while it was a closed colony. Furthermore, they demonstrated that the nonsusceptibility of Le-R rats is not linked to RT 1 and is in fact attributable to non-RT 1-linked genes. Le-R rats are also resistant to experimental allergic uveitis (EAU) while Lewis rats are susceptible to EAU (8). The mechanism underlying the loss of susceptibility to EAE and EAU in the Le-R rat is unknown. However, Mochizuki et al. (8) reported that Le-R rats have about f the number of mast cells in the choroid of the eye as compared to Lewis rats. They proposed that the mutation in the Le-R subline resulted in a decrease in the prevalence of choroidal mast cells and, consequently, in a reduced susceptibility for the induction of EAU. In addition, resistance to EAU and EAE was reported to be completely reversible when Bordetellapertussis was used as an additional adjuvant during immunization (8). In this study, we examined the differential susceptibility of Lewis/ NCr and Le-R rats to the induction of EAO using immunization protocols which did and did not include B. pertussis. Accordingly, susceptibility to B. pertussis-induced sensitivity to histamine, a phenotype which has been shown to be associated with susceptibility to EAE and EAO in mice (5,9), was also scored in all test animals. We also examined the potential relationship between the number of local mast cells at the site of the earliest lesions of rat EAO, i.e., the ductus efferentes (lo), and disease susceptibility. Our results demonstrate that Le-R rats are resistant to the induction of EAO and that resistance is reversed by B. pertussis when the inoculum contains lower doses of testicular autoantigens. However, unlike EAU, there does not appear to be an association between the number of local mast cells at the site of the earliest inflammatory infiltrates and susceptibility to the induction of EAO. Lewis and Le-R rats were found not to differ significantly from each other in the number of mast cells in the ductus efferentes. Furthermore, disease resistance in Le-R rats was not associated with an alteration in the phenotypic expression of vasoactive amine sensitization. MATERIALS
AND METHODS
Animals. Male Lewis/NCr rats weighing 250-300 g were purchased from the Animal Genetics and Production Branch of the National Cancer Institute. Male Le-R rats of similar age and weight were generated in our closed colony at the University of Pennsylvania School of Medicine (Philadelphia, PA). All animals were fed Purina rat pellets and water ad Zibitum and were in good health. Induction ofEA0. Groups of animals were immunized with varying doses of syngeneic rat testicular homogenate (RTH) emulsified in complete Freund’s adjuvant (CFA) containing 450 pg of Mycobacterium tuberculosis H37Ra. Each animal re-
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235
FIG.1. Photomicrographs (H&E) of the inflammatory lesion of rat EAO: (A) mild orchitis accompanied by aspermatogenesis (200x), (B) severe orchitis (200x), (C) mild epididymitis (360x), (D) severe epididymitis (200X), and(E) mild vasitis (360x).
ceived 1.O ml of the emulsion distributed in both hind footpads and in four sites on animals received 20 X lo9 organisms in 1 ml of the back. B. pertussis-immunized sterile saline iv at the time of immunization. Histology. All animals were killed and studied at 30 days postimmunization. The testes were fixed in Bouin’s fixative and embedded in paraffin. Five-micrometer-thick sections were obtained and stained with hematoxylin-eosin. Multiple sections were examined in a double-blind fashion for the histopathologic lesions of EAO, i.e., orchitis, aspermatogenesis, epididymitis, and vasitis (Fig. 1). Histochemistry and mast cell quantitation. Frozen sections of the ductus efferentes (6 pm) were cut, air-dried for 2-5 hr, fixed in acetone for 10 min at 4°C and washed
236
SHORT COMMUNICATION TABLE I Susceptibility of Lewis/NCr and Le-R Subline Rats to the Induction of EAO” Dose
Strain Lewis/NCr Le-R Lewis/NCr Le-R Lewis/NCr Le-R
B. pertussis
RTH (mg)
20 x lo9 organisms
100 100 100 100 50 50 50 50 10 10 10 10
+ + + + + +
-
Orchitis
Aspermatogenesis
Epididymitis
515 (100) 414 (100) O/5 (0) O/4 (0) 2/4 (50) 4/4 (loo) o/4 (0) 114 (25) 213 (66) 4/4 (100) O/4 (0) 5110 (50)
515 (100) 414 (100) 215 (50) O/4 (0) 214 (50) 4/4 (100) O/4 (0) 114 (25) 213 (66) 4/4 (1W O/4 (0) 7110 (70)
515 (100) 414 (100) O/5 (0) O/4 (0) 214 (50) O/3 (0) O/4 (0) l/4 (25) 213 (66) 4/4 (1W O/4 (0) 6110 (60)
Vasitis O/4 O/4 O/5 O/4 O/4 O/3 O/4 O/4
(0) (0) (0) (0) (0) (0) (0) (0) 012 (0) O/4 (0) O/4 (0) 4110 (40)
’ Animals were immunized with varying doses of RTH-CFA emulsion in both bind footpads and in four sites on the back. Animals receiving B. pertussis got 20 X lo9 organisms in 1.0 ml sterile saline iv at the time of immunization, whereas non-B. pertussis-immunized rats received 1.Oml sterile saline. All animals were killed at 30 days after immunization and examined histologically in a double-blind fashion for histopathologic lesions.
in phosphate-buffered saline (PBS). 3-Amino-9-ethylcarbazole (AEC) staining of mast cells was detected by direct staining (11). Tissue sections were counterstained with Delafield’s hematoxylin and mounted and the number of morphologically identifiable mast cells ( 12) in 1 to 10 high power fields was counted per specimen. Histamine sensitivity determination (13). Histamine sensitivity of Lewis and Le-R rats following immunization with and without B. pertussis was determined at the time of sacrifice. Rats were challenged ip with 1000 mg/kg of histamine-free base in PBS. Deaths were recorded after 2 hr and the results expressed as the number of deaths over the total number of animals studied. RESULTS
AND DISCUSSION
Lewis/NCr rats were found to be susceptible to the induction of EAO when immunized with and without B. pertussis at all doses of RTH studied (Table 1). However, Lewis/NCr rats immunized with B. pertussisexhibited more extensive inflammatory infiltrates as compared to non-B. pertussis-immunized animals. This was observed at all doses of RTH used. In contrast, Le-R rats immunized without B. pertussis did not exhibit any of the characteristic inflammatory lesions of EAO at any of the doses of RTH used (Table 1). Furthermore, Le-R rats immunized with B. pertussis at doses of 100 and 50 mg of RTH/rat did not develop significant disease (O/4 and l/4, respectively). However, a significant number (6/10) of Le-R rats immunized with B. pertussis at a dose of 10 mg RTH exhibited extensive inflammatory infiltrates (Table 1). These results suggest that the reversibility of resistance to EAO by B. pertussis in LeR rats is not only a result of the inclusion of B. pertussis at the time of immunization, but is also a function of the dose of sensitizing antigen since the reversal of disease is
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237
seen only at lower doses of RTH (10 mg). Such a response is indicative of an active immunoregulatory mechanism in the control of the phenotypic expression of disease susceptibility. The results of the analysis of the number of mast cells in the ductus efferentes, the anatomic location of the earliest lesions of EAO in the rat (lo), indicate that Lewis/ NCr and Le-R rats do not differ significantly from each other (4.4 f 2.3 and 4.2 f 1.3, respectively). It has been postulated that the difference in mast cell density is the salient difference distinguishing Lewis rats from Le-R rats as regards the development of EAU (8). With respect to EAO, we were unable to find such a difference in the ductus efferentes. This must raise the possibility that the differential expression of susceptibility to experimental autoimmune diseases between these two substrains is not dependent upon mast cell number. A comparison of the phenotypic expression of B. pertussis-induced sensitivity to histamine between the two substrains indicates that both possess the susceptible phenotype and that the two sublines appear to be essentially equivalent in both their innate sensitivity as well as their B. pertussis-induced sensitivity to histamine challenge (Lewis/NCr: l/ 12 without B. pertussis and 10112 with B. pertussis; Le-R: 2/ 13 without B. pertussis and 15118 with B. pertussis). The role of histamine and differential susceptibility to B. pertussis-induced histamine sensitivity has been examined in murine EAE (9) and EAO (5). The differential susceptibility and resistance exhibited by BALB/c substrain mice to both diseases appears to be independent of histamine sensitivity ( 14). Similarly, in the Lewis and Le-R subline rat model, EAO development is not overtly related to differential susceptibility to histamine sensitization. The role of B. pertussis in eliciting acute EAE is in part believed to be due to its ability to increase the permeability of the blood-brain barrier via the hypersensitivity of the vascular endothelium to vasoactive amines. It is postulated that this provides a means by which sensitized T-cells more easily gain access to the target tissue (9). A similar mechanism has been suggested for B. pertussis in the reversal of resistance to EAU in Le-R rats (8). However, in view of our findings it seems unlikely that such a mechanism(s) can fully account for either the differential susceptibility to EAO or the reversal of disease resistance in Le-R rats. B. pertussis is maximally effective at eliciting both actively induced murine EAE and EAO when administered from Day 2 before sensitization to Day 6 after sensitization with antigen emulsified in CFA (5, 15). Similarly, in passively induced murine EAE, B. pertussis is essential in the adoptive transfer of disease in that antigen-specific effector T-cells capable of passively inducing EAE can only be generated from donors which have been immunized with antigen plus B. pertussis, whereas it is not necessary to treat the donors with B. pertussis in order to elicit disease ( 16). Thus it appears that B. pertussis plays a more crucial role during the sensitization phase of the induction of EAE and EAO than during the inflammatory or effector phase (5, 15, 16). We believe that these observations taken together with our data indicate that the mutation which has rendered Le-R resistant to the induction of EAE, EAU, and EAO involves an alteration in an immunoregulatory locus which is normally operative in Lewis/NCr rats during the sensitization phase of disease induction rather than during the effector phase. Although the nature of the gene(s) controlling resistance to the induction of these three autoimmune diseases in Le-R rats remains unknown, the possibility exists that a single mutation in the regulatory locus affecting susceptibility to EAE also affects susceptibility to EAU and EAO. In this vein, it has been reported that the Le-
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R subline may have an immunologically significant mutation in the CBi region of the T-cell receptor gene ( 17). A mutation in such a T-cell receptor gene potentially has important ramifications for T-cell-mediated autoimmune diseases ( 18). Alternatively, Le-R rats may possess multiple mutations in independent regulatory loci controlling susceptibility to each disease. However, this possibility seems less likely since all such mutations would have to be overcome by this inclusion of B. pertussis. The identification and characterization of non-MHC-linked genes involved in controlling organ-specific T-cell-mediated autoimmune diseases is a focus of ongoing research in these laboratories. Whether or not this goal is achieved, Le-R subline rats nevertheless present a novel opportunity for the immunogenetic and pathogenetic analysis of mechanisms involved in organ-specific autoimmune disease. ACKNOWLEDGMENTS The authors thank Ms. Debbie Coffin for typing the manuscript. This research was supported by NIH Grants HD-21100, HD-21926, HD-06274 (C.T.), and NS-25533 (W.F.H.). Z.-Z. Zhou is the recipient of a postdoctoral fellowship from the Mellon Foundation and Y. Zheng is the recipient of a Rockefeller Foundation postdoctoral fellowship.
REFERENCES 1. Tung, K. S. K., In “Immunological Aspects of Infertility and Fertility Regulation” (D. H. Dhindsa and G. F. B. Schumaker, Eds.), pp. 33-9 1. Elsevier/North-Holland, New York, 1980. 2. Tung, K. S. K., and Menge, A. C., In “The Autoimmune Diseases” (N. R. Rose and I. R. Mackay, Eds.), pp. 537-584. Academic Press, New York, 1985. 3. Kosuda, L. L., and Bigazzi, P. E., In “Immunology of the Male Reproductive System” (P. E. Bigazzi, Ed.), pp. 253-352. Dekker, New York, 1987. 4. Teuscher, C., Smith, S. M., Goldberg, E. H., Shearer, G. M., and Tung, K. S. K., Immunogenetics 22, 323, 1985. 5. Teuscher, C., Immunogenetics 22,417, 1985. 6. Waxman, F. J., Perryman, L. E., Hinrichs, D. J., and Coe, J. E., J. Exp. Med. 153,61, 198 1. 7. Gasser, D. L., Hickey, W. F., and Gonatas, N. K., Immunogenetics 17,441, 1983. 8. Mochizuki, M., Kuwabara, T., Chart, C.-C., Nussenblatt, R. B., Metcalfe, D. D., and Gery, I., .I. Immunol. 133,1699, 1984. 9. Linthicum, D. S., and Frelinger, J. A., J. Exp. Med. 156,3 1, 1982. 10. Zhou, Z.-Z., Zheng, Y., Steenstra, R., Hickey, W. F., and Teuscher, C., Autoimmunity, in press. 11. Graham, R. C., Lundholm, U., and Kamovsky, M. J., J. Histochem. Cytochem. 13, 150, 1965. 12. Metcalf, D. D., Kaliner, M., and Donlon, M. A., CRC Crit. Rev. Immunol. 3,23, I98 1. 13. Malkiel, S., and Hargis, B. J., Proc. Sot. Exp. Biol. Med. 81,689, 1952. 14. Teuscher, C., Blankenhom, E. P., and Hickey, W. F., Cell. Immunol. 110,294, 1987. 15. Munoz, J. J., Bernard, C. C. A., and Mackay, I. R., Cell. Immunol. 83,92, 1984. 16. Lando, Z., and Ben-Nun, A., Clin. Immunol. Immunopathol. 30,290, 1984. 17. Hickey, W. F., and Blankenhom, E. P., J. Cell. Biochem. (Suppl.) llD, 297, 1987. 18. Zamvil, S. S., Mitchell, D. J., Lee, N. E., Moore, A. C., Waldor, M. K., Sakai, K., Rothbard, J. B., McDevitt, H. O., Steinman, L., and Acha-Orbea, H., J. Exp. Med. 167, 1586, 1988.
119,233-238
(1989)
SHORT COMMUNICATION Actively Induced Experimental Allergic Orchitis in Lewis-Resistant (Le-R) Rats: Reversibility of Disease Resistance by Immunization with Bordetella pertussis C. TEUSCHER,
Z-Z.
ZHOU,
Y. ZHENG, AND W. F. HICKEY*
Division of Reproductive Biology, Department of Obstetrics and Gynecology, and *Department of Pathology and Laboratory Medicine, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Received August 15.1988; accepted October 25, 1988 Differential susceptibility to the induction of experimental allergic orchitis (EAO) was examined in Lewis/NCr and Le-R subline rats. Lewis/NCr rats were found to be fully susceptible to the induction of EAO whereas Le-R subline rats were not. Disease resistance exhibited by Le-R rats could be overcome by including Bordetella pertussis in the immunization protocol. However, reversal of resistance with B. pertussis was dependent on the dose of rat testicular homogenate in the inoculum and found to be effective only at lower doses of antigen ( 10 m&at). Disease resistance in Le-R rats as well as B. petit&s-induced reversal of resistance did not appear to be associated with either (1) a significant difference in the number of mast cells in the ductus efferentes, the anatomic location of the earliest inflammatory infiltrates, or (2) an alteration in the phenotypic expression of either innate or B. pertussis-induced sensitivity to vasoactive amines. The results are discussed in the context of the role of B. pertussis in other animal models of organ-specific autoimmune diseases. It is proposed that the phenotypic expression of resistance to EAO in Le-R rats is a result of a mutation in a common regulatory locus affecting susceptibility to multiple autoimmune diseases and whose immunoreguiatory action is normally exerted during the sensitization phase of the immune response. 0 1989 Academic press, IIIC.
INTRODUCTION Experimental allergic orchitis (EAO) is an organ-specific autoimmune disease which can be elicited in a genetically susceptible animal by immunization with either homologous or autologous testicular and/or sperm antigens in conjunction with the appropriate adjuvants (reviewed in (l-3)). EAO is manifest as inflammation of the testis (orchitis), aspermatogenesis, epididymitis, and/or vasitis. Susceptibility to the induction of EAO has been shown to be controlled by both major histocompatibility complex (MHC)-linked and non-MHC-linked genes (4,5). The identity and mechanism of action of non-MHC-linked genes in the regulation of organ-specific autoimmune diseases are, for the most part, ill defined and poorly understood. One approach which is potentially useful in identifying and elucidating their function(s) is to utilize experimental animals which have a very high degree of genetic similarity, but differ in the phenotypic expression of certain traits, i.e., coisogenie strains and/or mutant sublines of inbred animals. With regard to organ-specific autoimmune diseases, one such example is the Lewis-resistant @e-R) strain of rat. 233 0008-8749189
$3.00
Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
234
SHORT
COMMUNICATION
The Le-R subline was derived from a Lewis colony which was originally susceptible to the induction of experimental allergic encephalomyelitis (EAE) (6). Serologically, antigenic differences between the two substrains could not be detected using antisera from reciprocal immunization (6) nor has it been possible to generate monoclonal antibodies which will discriminate between the two substrains (7). However, the two will eventually reject reciprocal skin grafts (W. F. Hickey, unpublished observation). Nevertheless, Le-R rats possess sufficient genetic similarity to Lewis rats to allow for adoptive transfer of EAE from Lewis to Le-R animals (6). Gasser et al. (7) suggested that the Le-R subline developed as the result of a simple mutational event since the gene or genes for nonsusceptibility developed in the Le-R subline while it was a closed colony. Furthermore, they demonstrated that the nonsusceptibility of Le-R rats is not linked to RT 1 and is in fact attributable to non-RT 1-linked genes. Le-R rats are also resistant to experimental allergic uveitis (EAU) while Lewis rats are susceptible to EAU (8). The mechanism underlying the loss of susceptibility to EAE and EAU in the Le-R rat is unknown. However, Mochizuki et al. (8) reported that Le-R rats have about f the number of mast cells in the choroid of the eye as compared to Lewis rats. They proposed that the mutation in the Le-R subline resulted in a decrease in the prevalence of choroidal mast cells and, consequently, in a reduced susceptibility for the induction of EAU. In addition, resistance to EAU and EAE was reported to be completely reversible when Bordetellapertussis was used as an additional adjuvant during immunization (8). In this study, we examined the differential susceptibility of Lewis/ NCr and Le-R rats to the induction of EAO using immunization protocols which did and did not include B. pertussis. Accordingly, susceptibility to B. pertussis-induced sensitivity to histamine, a phenotype which has been shown to be associated with susceptibility to EAE and EAO in mice (5,9), was also scored in all test animals. We also examined the potential relationship between the number of local mast cells at the site of the earliest lesions of rat EAO, i.e., the ductus efferentes (lo), and disease susceptibility. Our results demonstrate that Le-R rats are resistant to the induction of EAO and that resistance is reversed by B. pertussis when the inoculum contains lower doses of testicular autoantigens. However, unlike EAU, there does not appear to be an association between the number of local mast cells at the site of the earliest inflammatory infiltrates and susceptibility to the induction of EAO. Lewis and Le-R rats were found not to differ significantly from each other in the number of mast cells in the ductus efferentes. Furthermore, disease resistance in Le-R rats was not associated with an alteration in the phenotypic expression of vasoactive amine sensitization. MATERIALS
AND METHODS
Animals. Male Lewis/NCr rats weighing 250-300 g were purchased from the Animal Genetics and Production Branch of the National Cancer Institute. Male Le-R rats of similar age and weight were generated in our closed colony at the University of Pennsylvania School of Medicine (Philadelphia, PA). All animals were fed Purina rat pellets and water ad Zibitum and were in good health. Induction ofEA0. Groups of animals were immunized with varying doses of syngeneic rat testicular homogenate (RTH) emulsified in complete Freund’s adjuvant (CFA) containing 450 pg of Mycobacterium tuberculosis H37Ra. Each animal re-
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235
FIG.1. Photomicrographs (H&E) of the inflammatory lesion of rat EAO: (A) mild orchitis accompanied by aspermatogenesis (200x), (B) severe orchitis (200x), (C) mild epididymitis (360x), (D) severe epididymitis (200X), and(E) mild vasitis (360x).
ceived 1.O ml of the emulsion distributed in both hind footpads and in four sites on animals received 20 X lo9 organisms in 1 ml of the back. B. pertussis-immunized sterile saline iv at the time of immunization. Histology. All animals were killed and studied at 30 days postimmunization. The testes were fixed in Bouin’s fixative and embedded in paraffin. Five-micrometer-thick sections were obtained and stained with hematoxylin-eosin. Multiple sections were examined in a double-blind fashion for the histopathologic lesions of EAO, i.e., orchitis, aspermatogenesis, epididymitis, and vasitis (Fig. 1). Histochemistry and mast cell quantitation. Frozen sections of the ductus efferentes (6 pm) were cut, air-dried for 2-5 hr, fixed in acetone for 10 min at 4°C and washed
236
SHORT COMMUNICATION TABLE I Susceptibility of Lewis/NCr and Le-R Subline Rats to the Induction of EAO” Dose
Strain Lewis/NCr Le-R Lewis/NCr Le-R Lewis/NCr Le-R
B. pertussis
RTH (mg)
20 x lo9 organisms
100 100 100 100 50 50 50 50 10 10 10 10
+ + + + + +
-
Orchitis
Aspermatogenesis
Epididymitis
515 (100) 414 (100) O/5 (0) O/4 (0) 2/4 (50) 4/4 (loo) o/4 (0) 114 (25) 213 (66) 4/4 (100) O/4 (0) 5110 (50)
515 (100) 414 (100) 215 (50) O/4 (0) 214 (50) 4/4 (100) O/4 (0) 114 (25) 213 (66) 4/4 (1W O/4 (0) 7110 (70)
515 (100) 414 (100) O/5 (0) O/4 (0) 214 (50) O/3 (0) O/4 (0) l/4 (25) 213 (66) 4/4 (1W O/4 (0) 6110 (60)
Vasitis O/4 O/4 O/5 O/4 O/4 O/3 O/4 O/4
(0) (0) (0) (0) (0) (0) (0) (0) 012 (0) O/4 (0) O/4 (0) 4110 (40)
’ Animals were immunized with varying doses of RTH-CFA emulsion in both bind footpads and in four sites on the back. Animals receiving B. pertussis got 20 X lo9 organisms in 1.0 ml sterile saline iv at the time of immunization, whereas non-B. pertussis-immunized rats received 1.Oml sterile saline. All animals were killed at 30 days after immunization and examined histologically in a double-blind fashion for histopathologic lesions.
in phosphate-buffered saline (PBS). 3-Amino-9-ethylcarbazole (AEC) staining of mast cells was detected by direct staining (11). Tissue sections were counterstained with Delafield’s hematoxylin and mounted and the number of morphologically identifiable mast cells ( 12) in 1 to 10 high power fields was counted per specimen. Histamine sensitivity determination (13). Histamine sensitivity of Lewis and Le-R rats following immunization with and without B. pertussis was determined at the time of sacrifice. Rats were challenged ip with 1000 mg/kg of histamine-free base in PBS. Deaths were recorded after 2 hr and the results expressed as the number of deaths over the total number of animals studied. RESULTS
AND DISCUSSION
Lewis/NCr rats were found to be susceptible to the induction of EAO when immunized with and without B. pertussis at all doses of RTH studied (Table 1). However, Lewis/NCr rats immunized with B. pertussisexhibited more extensive inflammatory infiltrates as compared to non-B. pertussis-immunized animals. This was observed at all doses of RTH used. In contrast, Le-R rats immunized without B. pertussis did not exhibit any of the characteristic inflammatory lesions of EAO at any of the doses of RTH used (Table 1). Furthermore, Le-R rats immunized with B. pertussis at doses of 100 and 50 mg of RTH/rat did not develop significant disease (O/4 and l/4, respectively). However, a significant number (6/10) of Le-R rats immunized with B. pertussis at a dose of 10 mg RTH exhibited extensive inflammatory infiltrates (Table 1). These results suggest that the reversibility of resistance to EAO by B. pertussis in LeR rats is not only a result of the inclusion of B. pertussis at the time of immunization, but is also a function of the dose of sensitizing antigen since the reversal of disease is
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seen only at lower doses of RTH (10 mg). Such a response is indicative of an active immunoregulatory mechanism in the control of the phenotypic expression of disease susceptibility. The results of the analysis of the number of mast cells in the ductus efferentes, the anatomic location of the earliest lesions of EAO in the rat (lo), indicate that Lewis/ NCr and Le-R rats do not differ significantly from each other (4.4 f 2.3 and 4.2 f 1.3, respectively). It has been postulated that the difference in mast cell density is the salient difference distinguishing Lewis rats from Le-R rats as regards the development of EAU (8). With respect to EAO, we were unable to find such a difference in the ductus efferentes. This must raise the possibility that the differential expression of susceptibility to experimental autoimmune diseases between these two substrains is not dependent upon mast cell number. A comparison of the phenotypic expression of B. pertussis-induced sensitivity to histamine between the two substrains indicates that both possess the susceptible phenotype and that the two sublines appear to be essentially equivalent in both their innate sensitivity as well as their B. pertussis-induced sensitivity to histamine challenge (Lewis/NCr: l/ 12 without B. pertussis and 10112 with B. pertussis; Le-R: 2/ 13 without B. pertussis and 15118 with B. pertussis). The role of histamine and differential susceptibility to B. pertussis-induced histamine sensitivity has been examined in murine EAE (9) and EAO (5). The differential susceptibility and resistance exhibited by BALB/c substrain mice to both diseases appears to be independent of histamine sensitivity ( 14). Similarly, in the Lewis and Le-R subline rat model, EAO development is not overtly related to differential susceptibility to histamine sensitization. The role of B. pertussis in eliciting acute EAE is in part believed to be due to its ability to increase the permeability of the blood-brain barrier via the hypersensitivity of the vascular endothelium to vasoactive amines. It is postulated that this provides a means by which sensitized T-cells more easily gain access to the target tissue (9). A similar mechanism has been suggested for B. pertussis in the reversal of resistance to EAU in Le-R rats (8). However, in view of our findings it seems unlikely that such a mechanism(s) can fully account for either the differential susceptibility to EAO or the reversal of disease resistance in Le-R rats. B. pertussis is maximally effective at eliciting both actively induced murine EAE and EAO when administered from Day 2 before sensitization to Day 6 after sensitization with antigen emulsified in CFA (5, 15). Similarly, in passively induced murine EAE, B. pertussis is essential in the adoptive transfer of disease in that antigen-specific effector T-cells capable of passively inducing EAE can only be generated from donors which have been immunized with antigen plus B. pertussis, whereas it is not necessary to treat the donors with B. pertussis in order to elicit disease ( 16). Thus it appears that B. pertussis plays a more crucial role during the sensitization phase of the induction of EAE and EAO than during the inflammatory or effector phase (5, 15, 16). We believe that these observations taken together with our data indicate that the mutation which has rendered Le-R resistant to the induction of EAE, EAU, and EAO involves an alteration in an immunoregulatory locus which is normally operative in Lewis/NCr rats during the sensitization phase of disease induction rather than during the effector phase. Although the nature of the gene(s) controlling resistance to the induction of these three autoimmune diseases in Le-R rats remains unknown, the possibility exists that a single mutation in the regulatory locus affecting susceptibility to EAE also affects susceptibility to EAU and EAO. In this vein, it has been reported that the Le-
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R subline may have an immunologically significant mutation in the CBi region of the T-cell receptor gene ( 17). A mutation in such a T-cell receptor gene potentially has important ramifications for T-cell-mediated autoimmune diseases ( 18). Alternatively, Le-R rats may possess multiple mutations in independent regulatory loci controlling susceptibility to each disease. However, this possibility seems less likely since all such mutations would have to be overcome by this inclusion of B. pertussis. The identification and characterization of non-MHC-linked genes involved in controlling organ-specific T-cell-mediated autoimmune diseases is a focus of ongoing research in these laboratories. Whether or not this goal is achieved, Le-R subline rats nevertheless present a novel opportunity for the immunogenetic and pathogenetic analysis of mechanisms involved in organ-specific autoimmune disease. ACKNOWLEDGMENTS The authors thank Ms. Debbie Coffin for typing the manuscript. This research was supported by NIH Grants HD-21100, HD-21926, HD-06274 (C.T.), and NS-25533 (W.F.H.). Z.-Z. Zhou is the recipient of a postdoctoral fellowship from the Mellon Foundation and Y. Zheng is the recipient of a Rockefeller Foundation postdoctoral fellowship.
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