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Delayed-type hypersensitivity to elastase-soluble lung peptides in the tight-skin (Tsk) mouse
CELLULAR
IMMUNOLOGY
81, 175-179
(1983)
Delayed-Type Hypersensitivity to Elastase-Soluble Peptides in the Tight-Skin (Tsk) Mouse’ F. A. DELUSTRO,* A. M. MACKEL, Division
qf Rheumatoiogy
Received
and Immunology, Charleston, South April
AND E. C. LEROY
Medical University Carolina 29425
13, 1983; accepted
Lung
of South
Carolina,
June 14, 1983
The development of immunity to homologous connective tissue antigens was studied with respect to aging in the tight-skin (Tsk) mouse mutant. A delayed-type hypersensitivity (DTH) response to elastase-solubilized lung peptides in Tsk/+ mice, which became evident at 10 weeks of age and increased in intensity until 22 weeks, was observed. Tsk mice did not demonstrate significant DTH responseswhen challenged with type I or IV collagen, and normal (+/+) littermates of all ages did not respond to any of the antigens under study. DTH responses could be adoptively transferred to normal +/+ and C57BL/6 mice with spleen cells from 30-week-old Tsk/+ mice; treatment with anti-Thy I .2 antibodies plus complement significantly reduced the ability of these Tsk/+ cells to transfer DTH reactivity. No antibody activity to the antigens under study could be detected in the sera of Tsk/+ or +/+ mice at any age. These results are discussed with regard to the pathological manifestations observed in the Tsk/+ mutant mouse. INTRODUCTION
In 1976, Green et al. (I) described a dominant gene mutation, tight skin (Tsk),3 which is located on chromosome 2 of the mouse. While homozygous (Tsk/Tsk) mice die in utero, they demonstrated that heterozygous (Tsk/+) mice display excessively taut skin due to hyperplasia of the loose connective tissue, as well as of the cartilage and bone. Menton et al. (2) further characterized the defects in collagen deposition and fiber organization in the Tsk/+ mouse; they noted the common features of cutaneous abnormalities found in this mouse mutant and in patients with scleroderma. In addition to these aspects of the Tsk/+ mouse, Szapiel et al. (3) found that the lungs undergo emphysematous changes with elastin degradation, enlargement of the air spaces, and increased total lung compliance and capacity. More recently, several groups (4, 5) have reported that the collagen content of the skin from Tsk/+ mice is significantly increased when compared to normal (+/+) littermates. These data have clearly demonstrated the abnormal spontaneous accumulation of excessive
’ These studies were supported by NIH Grant AM-3043 1. ’ To whom correspondence should be addressed: Connective Tissue Research Laboratories, Collagen Corporation, 2500 Faber PI., Palo Alto, Cahf. 94303. ’ Abbreviations used: DTH, delayed-type hypersensitivity; ELISA, enzyme-linked immunosorbent assay; ip. intraperitoneal; PBS, phosphate-buffered saline; SE, standard error; Tsk, tight-skin gene mutation in mice. 175 0008-8749183 Copyright All n&s
$3.00
0 1983 by Academic Press. Inc. of reprcducrion ,n any form reserved
176
SHORT COMMUNICATIONS
quantities of loose connective tissue in the Tsk/+ mouse and its similarity to the manifestations of scleroderma in man. We have previously published that mice can be sensitized to murine basement membrane (type IV collagen and laminin) components and to type I collagen at the level of cell-mediated (6, 7) and humoral immunity (7, 8). Furthermore, patients with scleroderma were found to have antibodies to type I and IV collagen, and the presence of these antibodies was associated with pulmonary abnormalities as measured by diminished diffusion capacity (9). Because of the connective tissue abnormalities associated with the Tsk/+ mouse, we examined these mice for immunity to connective tissue antigens during the development of the disease process and found the occurrence of cell-mediated immunity to elastase-soluble lung peptides. MATERIALS AND METHODS Animals. C57BL/6 Tsk/+ and +/+ mice were obtained at 4-5 weeks of age from Dr. S. Lane (Jackson Laboratories, Bar Harbor, Maine) and C57BL/6 mice were purchased at 6-8 weeks of age from the Department of Laboratory Animal Medicine (Medical University of South Carolina). Antigens. Type I and IV collagen were prepared as previously described (6-10). Type I collagen was isolated from mouse tail tendon by acid solubilization and type IV collagen was extracted from the matrix of the Engelbroth-Helm/Swarm sarcoma following pepsinization and DEAE-cellulose chromatography. Elastase-soluble lung peptides (elastin peptides) were prepared according to the technique of Damule et al. (11). After removal of the bronchi and major blood vessels from lungs of normal C57BL/6 mice, lung parenchyma was homogenized in 1 M NaCl and stirred for 3 hr at 4°C. After centrifugation, the residue was homogenized in 5 M guanidine-HCl (pH 7.0) at 5% w/v and stirred at room temperature for 24 hr. The suspension was centrifuged and the residue treated with guanidine-HCl twice more as described above. The final residue was washed free of guanidine-HCl and resuspended in 0.2 M Tris (pH 7.4). Purified bacterial collagenase (6, 12) was added at 1: 150 and the mixture incubated at 37°C for 48 hr with rocking. After centrifugation, the residue was washed and incubated with trypsin (1:50) for 24 hr at 37°C with mixing. The residue was washed extensively with phosphate-buffered saline (PBS) and resuspended with elastase ( lo3 U/g wet wt; porcine pancreas, EC 134; Elastin Products, Pacific, Missouri). After mixing for 24 hr at 37°C the suspension was centrifuged and the supernatant dialyzed against PBS for 48 hr. The soluble protein concentration was determined (13) and aliquots were frozen at -70°C. Delayed-type hypersensitivity (DTH) response.The footpad swelling response was utilized to measure DTH to antigenic challenge as previously described (6, 7, 10). Mice were injected intradermally with 5 pg antigen in 30 ~1 into the plantar surface of the hind foot. Footpad diameters were measured at 0,4, and 24 hr with a micrometer. The data are expressed as the mean percentage footpad swelling + standard error (SE): mm after challenge - mm before challenge x 100. % footpad swelling = mm before challenge Adoptive transfer. Spleens were removed from Tsk/+ mice at 30 weeks of age. A single-cell suspension was left untreated or treated with monoclonal murine anti-Thy 1.2 antibody (New England Nuclear, Boston, Mass.) plus guinea pig complement
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(M. A. Bioproducts, Wallace, Md.) as previously described (6, 7, 10). C57BL/6 and +/+ mice were injected intraperitoneally (ip) with 25 X lo6 viable cells in 0.5 ml PBS, and recipient mice were challenged 48 hr after cell transfer for DTH as described above. RESULTS
AND
DISCUSSION
Because of the spontaneous abnormalities of the interstitial and pulmonary connective tissue which have been described in Tsk/+ mutant mice (l-5), we examined immunity to murine connective tissue components (type I and IV collagen and elastin peptides) in Tsk/+ and +/+ littermates as a function of age. While no DTH was 6
T
A
7
10
14 Age
18
22
lweeksl
FIG. 1. Tsk/+ (a) and +/+ (m) mice were challenged in the footpad at 7 to 22 weeks of age with elastin peptides (A), type I collagen (B), or type IV collagen (C). The DTH response was measured after 24 hr and the results are expressed as percentage footpad swelling f SE. Data from several experiments were pooled and the number of mice per group is indicated above each bar.
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COMMUNICATIONS
observed at 7 weeks of age, Tsk/+ mice displayed a significant DTH response to elastase-soluble lung peptides by 10 weeks of age compared to +/+ littermates, and this response increased markedly by 14 weeks (Fig. 1A). In contrast, DTH responses to type I and IV collagen in Tsk/+ mice remained low at all times (~5% mean footpad swelling) and did not show a pattern consistent with autoimmune sensitization (Figs. 1B and C). When footpad swelling was monitored at 4 and 24 hr postchallenge in 18-week-old Tsk/+ mice (n = 6) challenged with elastin peptides, no significant swelling was detected at 4 hr (3.65 + 0.39%) while DTH was clearly evident 24 hr after challenge (16.3 + 0.71%). In this last experiment, the 18-week-old Tsk/+ mice had been untreated until this time indicating that the observed DTH response was not due to sensitization from repeated challenge with antigen. The kinetics of the response to antigenic challenge in the footpad is consistent with a T-cell-mediated DTH response as we have previously described for other connective tissue antigens (6, 7, 10). To determine if T lymphocytes from old Tsk/+ mice were responsible for this reactivity to elastin peptides, 30-week-old +/+ mice were injected ip with untreated or anti-Thy 1.2 antibody plus complement (anti-Thy 1.2)-treated spleen cells from 30-week-old Tsk/+ mice. As indicated in Fig. 2, untreated spleen cells from Tsk/+ mice can adoptively transfer DTH responsiveness to elastase-soluble lung peptides to +/+ littermates or normal C57BL/6 mice. Anti-Thy 1.2~treated (T-cell-depleted) spleen cells were significantly less effective in adoptively transferring immunity (Fig. 2). These data indicate that Tsk/+ mice generate a cell-mediated autoimmune response to elastin peptides which is evident by 10 weeks of age and increases with time. Using the ELISA (7-lo), we have been unable to detect antibodies to the antigens under study at any age in Tsk/+ or +/+ mice. Additional work is needed to determine
TT
Untreated
Anti
-Thy
1.2
FIG. 2. DTH responsiveness to elastase-soluble lung peptides was adoptively transferred to normal C57BL/6 (m) and +/+ (W) mice using spleen cells from 30-week-old Tsk/+ mice. Spleen cell suspensions were left untreated or treated with anti-Thy 1.2 antibody plus complement prior to injection as described under Materials and Methods. Data are expressed as in Fig 1.
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179
if adoptive transfer of T cells from Tsk/+ mice can initiate connective tissue disease in +/+ littermates or if the observed autoimmune response is secondary to pulmonary disease (3) and unrelated to the initiation or propagation of the connective tissue abnormalities associated with the Tsk/+ mouse mutant (l-5). The dominant features of scleroderma in man are the microvascular abnormalities and the excessive deposition of newly synthesized collagen (14). However, several investigators have noted marked damage to existing elastin and collagen early in the disease process. Getzowa (15) described cystic areas of elastin and microvascular dissolution associated with pulmonary sclerosis among patients with scleroderma as similar to those found in emphysema. Furthermore, both Lange et al. ( 16) and Markowitz et al. (17) detected fragmentation of the elastic fibers in the skin of polyvinyl chloride workers with scleroderma. These data indicate that the skin and pulmonary features of the Tsk/+ mouse closely resemble those found in patients with scleroderma. Using the Tsk/+ mouse as a model for spontaneous connective tissue disease, we examined humoral and cell-mediated immunity to the major components of the involved skin and lungs. Darnule et al. (18) demonstrated that guinea pigs could mount an antigen-specific, cell-mediated immune response to human elastin peptides. In the Tsk/+ mouse, cell-mediated immunity to elastase-soluble murine lung peptides became evident with age while DTH responses to type I or IV collagen were not detected. Studies are in progress to determine the relationship between age and the pathological changes in the connective tissue of Tsk/+ mice, as well as the ability of immunocompetent cells from Tsk/+ mice to transfer disease to healthy +/+ littermates. ACKNOWLEDGMENTS The authors wish to thank Barbara DeLustro for technical assistance and Judy Anderson for secretarial assistance.
REFERENCES 1. Green, M. C., Sweet, H. O., and Bunker, L. E., Amer. J. P&ho/. 82, 493, 1976. 2. Menton, D. N., Hess, R. A., Lichtenstein, J. R., and Eisen, A. Z., J. Invest. Dermatol. 70, 4, 1978. 3. Szapiel, S. V., Fulmer, J. D., Hunninghake, G. W., Elson, N. A., Kawanami, O., Ferrans, V. J., and Crystal, R. G., Amer. Rev. Respir. Dis. 123, 680, 1981. 4. Osbom, T. G., Bauer, N. E., Moore, T. L., Zuckner, J., and Domer, R. W., Arthritis Rheum. 25, S14, 1982. 5. Jimenez, S. A., Bashey, R. I., and Millan, A., Arthritis Rheum. 25, S14, 1982. 6. Mackel, A. M., DeLustro, F., and LeRoy, E. C., Clin. Immunol. Immunopathol. 21, 204, 1981. 7. Mackel, A. M., DeLustro, F., and LeRoy, E. C., Proc. Sot. Exp. Biol. Med. 171, 98, 1982. 8. Mackel, A. M., DeLustro, F., DeLustro, B., Fudenberg, H. H., and LeRoy, E. C., Connect. Tissue Rex 10, 333, 1982. 9. Mackel, A. M., DeLustro, F., Harper, F. E., and LeRoy, E. C., Arfhrifis Rheum. 25, 522, 1982. 10. Mackel, A. M., DeLustro, F., and LeRoy, E. C., J. Exp. Med. 156, 1042, 1982. 11. Damule, T. V., Likhite, V., Turino, G. M., and Mandel, I., Connect. Tissue Res. 5, 67, 1977. 12. Peterkofsky, B., and Diegelmann, R., Biochemistry IO, 988, 1971. 13. Bradford, M. M., Anal. B&hem. 72, 248, 1976. 14. LeRoy, E. C., J. Invest. Dermatol. 79, 87S, 1982. 15. Getzowa, S., Arch. Pathol. 40, 99, 1945. 16. Lange, C. E., Juhe, S., Stein, G., and Veltman, G., Int. Arch. Arbeitsmed. 32, 1, 1974. 17. Markowitz, S. S., McDonald, C. J., Fethiere, W., and Kerzner, M. S., Arch. Dermatol. 106,2 19, 1972. 18. Darnule, T. V., Likhite, V., Turino, G. M., and Mandl, I., Proc. Sot. Exp. Biol. Med. 165, 413, 1980.
IMMUNOLOGY
81, 175-179
(1983)
Delayed-Type Hypersensitivity to Elastase-Soluble Peptides in the Tight-Skin (Tsk) Mouse’ F. A. DELUSTRO,* A. M. MACKEL, Division
qf Rheumatoiogy
Received
and Immunology, Charleston, South April
AND E. C. LEROY
Medical University Carolina 29425
13, 1983; accepted
Lung
of South
Carolina,
June 14, 1983
The development of immunity to homologous connective tissue antigens was studied with respect to aging in the tight-skin (Tsk) mouse mutant. A delayed-type hypersensitivity (DTH) response to elastase-solubilized lung peptides in Tsk/+ mice, which became evident at 10 weeks of age and increased in intensity until 22 weeks, was observed. Tsk mice did not demonstrate significant DTH responseswhen challenged with type I or IV collagen, and normal (+/+) littermates of all ages did not respond to any of the antigens under study. DTH responses could be adoptively transferred to normal +/+ and C57BL/6 mice with spleen cells from 30-week-old Tsk/+ mice; treatment with anti-Thy I .2 antibodies plus complement significantly reduced the ability of these Tsk/+ cells to transfer DTH reactivity. No antibody activity to the antigens under study could be detected in the sera of Tsk/+ or +/+ mice at any age. These results are discussed with regard to the pathological manifestations observed in the Tsk/+ mutant mouse. INTRODUCTION
In 1976, Green et al. (I) described a dominant gene mutation, tight skin (Tsk),3 which is located on chromosome 2 of the mouse. While homozygous (Tsk/Tsk) mice die in utero, they demonstrated that heterozygous (Tsk/+) mice display excessively taut skin due to hyperplasia of the loose connective tissue, as well as of the cartilage and bone. Menton et al. (2) further characterized the defects in collagen deposition and fiber organization in the Tsk/+ mouse; they noted the common features of cutaneous abnormalities found in this mouse mutant and in patients with scleroderma. In addition to these aspects of the Tsk/+ mouse, Szapiel et al. (3) found that the lungs undergo emphysematous changes with elastin degradation, enlargement of the air spaces, and increased total lung compliance and capacity. More recently, several groups (4, 5) have reported that the collagen content of the skin from Tsk/+ mice is significantly increased when compared to normal (+/+) littermates. These data have clearly demonstrated the abnormal spontaneous accumulation of excessive
’ These studies were supported by NIH Grant AM-3043 1. ’ To whom correspondence should be addressed: Connective Tissue Research Laboratories, Collagen Corporation, 2500 Faber PI., Palo Alto, Cahf. 94303. ’ Abbreviations used: DTH, delayed-type hypersensitivity; ELISA, enzyme-linked immunosorbent assay; ip. intraperitoneal; PBS, phosphate-buffered saline; SE, standard error; Tsk, tight-skin gene mutation in mice. 175 0008-8749183 Copyright All n&s
$3.00
0 1983 by Academic Press. Inc. of reprcducrion ,n any form reserved
176
SHORT COMMUNICATIONS
quantities of loose connective tissue in the Tsk/+ mouse and its similarity to the manifestations of scleroderma in man. We have previously published that mice can be sensitized to murine basement membrane (type IV collagen and laminin) components and to type I collagen at the level of cell-mediated (6, 7) and humoral immunity (7, 8). Furthermore, patients with scleroderma were found to have antibodies to type I and IV collagen, and the presence of these antibodies was associated with pulmonary abnormalities as measured by diminished diffusion capacity (9). Because of the connective tissue abnormalities associated with the Tsk/+ mouse, we examined these mice for immunity to connective tissue antigens during the development of the disease process and found the occurrence of cell-mediated immunity to elastase-soluble lung peptides. MATERIALS AND METHODS Animals. C57BL/6 Tsk/+ and +/+ mice were obtained at 4-5 weeks of age from Dr. S. Lane (Jackson Laboratories, Bar Harbor, Maine) and C57BL/6 mice were purchased at 6-8 weeks of age from the Department of Laboratory Animal Medicine (Medical University of South Carolina). Antigens. Type I and IV collagen were prepared as previously described (6-10). Type I collagen was isolated from mouse tail tendon by acid solubilization and type IV collagen was extracted from the matrix of the Engelbroth-Helm/Swarm sarcoma following pepsinization and DEAE-cellulose chromatography. Elastase-soluble lung peptides (elastin peptides) were prepared according to the technique of Damule et al. (11). After removal of the bronchi and major blood vessels from lungs of normal C57BL/6 mice, lung parenchyma was homogenized in 1 M NaCl and stirred for 3 hr at 4°C. After centrifugation, the residue was homogenized in 5 M guanidine-HCl (pH 7.0) at 5% w/v and stirred at room temperature for 24 hr. The suspension was centrifuged and the residue treated with guanidine-HCl twice more as described above. The final residue was washed free of guanidine-HCl and resuspended in 0.2 M Tris (pH 7.4). Purified bacterial collagenase (6, 12) was added at 1: 150 and the mixture incubated at 37°C for 48 hr with rocking. After centrifugation, the residue was washed and incubated with trypsin (1:50) for 24 hr at 37°C with mixing. The residue was washed extensively with phosphate-buffered saline (PBS) and resuspended with elastase ( lo3 U/g wet wt; porcine pancreas, EC 134; Elastin Products, Pacific, Missouri). After mixing for 24 hr at 37°C the suspension was centrifuged and the supernatant dialyzed against PBS for 48 hr. The soluble protein concentration was determined (13) and aliquots were frozen at -70°C. Delayed-type hypersensitivity (DTH) response.The footpad swelling response was utilized to measure DTH to antigenic challenge as previously described (6, 7, 10). Mice were injected intradermally with 5 pg antigen in 30 ~1 into the plantar surface of the hind foot. Footpad diameters were measured at 0,4, and 24 hr with a micrometer. The data are expressed as the mean percentage footpad swelling + standard error (SE): mm after challenge - mm before challenge x 100. % footpad swelling = mm before challenge Adoptive transfer. Spleens were removed from Tsk/+ mice at 30 weeks of age. A single-cell suspension was left untreated or treated with monoclonal murine anti-Thy 1.2 antibody (New England Nuclear, Boston, Mass.) plus guinea pig complement
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COMMUNICATIONS
(M. A. Bioproducts, Wallace, Md.) as previously described (6, 7, 10). C57BL/6 and +/+ mice were injected intraperitoneally (ip) with 25 X lo6 viable cells in 0.5 ml PBS, and recipient mice were challenged 48 hr after cell transfer for DTH as described above. RESULTS
AND
DISCUSSION
Because of the spontaneous abnormalities of the interstitial and pulmonary connective tissue which have been described in Tsk/+ mutant mice (l-5), we examined immunity to murine connective tissue components (type I and IV collagen and elastin peptides) in Tsk/+ and +/+ littermates as a function of age. While no DTH was 6
T
A
7
10
14 Age
18
22
lweeksl
FIG. 1. Tsk/+ (a) and +/+ (m) mice were challenged in the footpad at 7 to 22 weeks of age with elastin peptides (A), type I collagen (B), or type IV collagen (C). The DTH response was measured after 24 hr and the results are expressed as percentage footpad swelling f SE. Data from several experiments were pooled and the number of mice per group is indicated above each bar.
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COMMUNICATIONS
observed at 7 weeks of age, Tsk/+ mice displayed a significant DTH response to elastase-soluble lung peptides by 10 weeks of age compared to +/+ littermates, and this response increased markedly by 14 weeks (Fig. 1A). In contrast, DTH responses to type I and IV collagen in Tsk/+ mice remained low at all times (~5% mean footpad swelling) and did not show a pattern consistent with autoimmune sensitization (Figs. 1B and C). When footpad swelling was monitored at 4 and 24 hr postchallenge in 18-week-old Tsk/+ mice (n = 6) challenged with elastin peptides, no significant swelling was detected at 4 hr (3.65 + 0.39%) while DTH was clearly evident 24 hr after challenge (16.3 + 0.71%). In this last experiment, the 18-week-old Tsk/+ mice had been untreated until this time indicating that the observed DTH response was not due to sensitization from repeated challenge with antigen. The kinetics of the response to antigenic challenge in the footpad is consistent with a T-cell-mediated DTH response as we have previously described for other connective tissue antigens (6, 7, 10). To determine if T lymphocytes from old Tsk/+ mice were responsible for this reactivity to elastin peptides, 30-week-old +/+ mice were injected ip with untreated or anti-Thy 1.2 antibody plus complement (anti-Thy 1.2)-treated spleen cells from 30-week-old Tsk/+ mice. As indicated in Fig. 2, untreated spleen cells from Tsk/+ mice can adoptively transfer DTH responsiveness to elastase-soluble lung peptides to +/+ littermates or normal C57BL/6 mice. Anti-Thy 1.2~treated (T-cell-depleted) spleen cells were significantly less effective in adoptively transferring immunity (Fig. 2). These data indicate that Tsk/+ mice generate a cell-mediated autoimmune response to elastin peptides which is evident by 10 weeks of age and increases with time. Using the ELISA (7-lo), we have been unable to detect antibodies to the antigens under study at any age in Tsk/+ or +/+ mice. Additional work is needed to determine
TT
Untreated
Anti
-Thy
1.2
FIG. 2. DTH responsiveness to elastase-soluble lung peptides was adoptively transferred to normal C57BL/6 (m) and +/+ (W) mice using spleen cells from 30-week-old Tsk/+ mice. Spleen cell suspensions were left untreated or treated with anti-Thy 1.2 antibody plus complement prior to injection as described under Materials and Methods. Data are expressed as in Fig 1.
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179
if adoptive transfer of T cells from Tsk/+ mice can initiate connective tissue disease in +/+ littermates or if the observed autoimmune response is secondary to pulmonary disease (3) and unrelated to the initiation or propagation of the connective tissue abnormalities associated with the Tsk/+ mouse mutant (l-5). The dominant features of scleroderma in man are the microvascular abnormalities and the excessive deposition of newly synthesized collagen (14). However, several investigators have noted marked damage to existing elastin and collagen early in the disease process. Getzowa (15) described cystic areas of elastin and microvascular dissolution associated with pulmonary sclerosis among patients with scleroderma as similar to those found in emphysema. Furthermore, both Lange et al. ( 16) and Markowitz et al. (17) detected fragmentation of the elastic fibers in the skin of polyvinyl chloride workers with scleroderma. These data indicate that the skin and pulmonary features of the Tsk/+ mouse closely resemble those found in patients with scleroderma. Using the Tsk/+ mouse as a model for spontaneous connective tissue disease, we examined humoral and cell-mediated immunity to the major components of the involved skin and lungs. Darnule et al. (18) demonstrated that guinea pigs could mount an antigen-specific, cell-mediated immune response to human elastin peptides. In the Tsk/+ mouse, cell-mediated immunity to elastase-soluble murine lung peptides became evident with age while DTH responses to type I or IV collagen were not detected. Studies are in progress to determine the relationship between age and the pathological changes in the connective tissue of Tsk/+ mice, as well as the ability of immunocompetent cells from Tsk/+ mice to transfer disease to healthy +/+ littermates. ACKNOWLEDGMENTS The authors wish to thank Barbara DeLustro for technical assistance and Judy Anderson for secretarial assistance.
REFERENCES 1. Green, M. C., Sweet, H. O., and Bunker, L. E., Amer. J. P&ho/. 82, 493, 1976. 2. Menton, D. N., Hess, R. A., Lichtenstein, J. R., and Eisen, A. Z., J. Invest. Dermatol. 70, 4, 1978. 3. Szapiel, S. V., Fulmer, J. D., Hunninghake, G. W., Elson, N. A., Kawanami, O., Ferrans, V. J., and Crystal, R. G., Amer. Rev. Respir. Dis. 123, 680, 1981. 4. Osbom, T. G., Bauer, N. E., Moore, T. L., Zuckner, J., and Domer, R. W., Arthritis Rheum. 25, S14, 1982. 5. Jimenez, S. A., Bashey, R. I., and Millan, A., Arthritis Rheum. 25, S14, 1982. 6. Mackel, A. M., DeLustro, F., and LeRoy, E. C., Clin. Immunol. Immunopathol. 21, 204, 1981. 7. Mackel, A. M., DeLustro, F., and LeRoy, E. C., Proc. Sot. Exp. Biol. Med. 171, 98, 1982. 8. Mackel, A. M., DeLustro, F., DeLustro, B., Fudenberg, H. H., and LeRoy, E. C., Connect. Tissue Rex 10, 333, 1982. 9. Mackel, A. M., DeLustro, F., Harper, F. E., and LeRoy, E. C., Arfhrifis Rheum. 25, 522, 1982. 10. Mackel, A. M., DeLustro, F., and LeRoy, E. C., J. Exp. Med. 156, 1042, 1982. 11. Damule, T. V., Likhite, V., Turino, G. M., and Mandel, I., Connect. Tissue Res. 5, 67, 1977. 12. Peterkofsky, B., and Diegelmann, R., Biochemistry IO, 988, 1971. 13. Bradford, M. M., Anal. B&hem. 72, 248, 1976. 14. LeRoy, E. C., J. Invest. Dermatol. 79, 87S, 1982. 15. Getzowa, S., Arch. Pathol. 40, 99, 1945. 16. Lange, C. E., Juhe, S., Stein, G., and Veltman, G., Int. Arch. Arbeitsmed. 32, 1, 1974. 17. Markowitz, S. S., McDonald, C. J., Fethiere, W., and Kerzner, M. S., Arch. Dermatol. 106,2 19, 1972. 18. Darnule, T. V., Likhite, V., Turino, G. M., and Mandl, I., Proc. Sot. Exp. Biol. Med. 165, 413, 1980.