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A role for CD4+ T cells in the pathogenesis of skin fibrosis in tight skin mice

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A role for CD4+ T cells in the pathogenesis of skin fibrosis in tight skin mice
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  Eur. J. Immunol. 1994. 24: 1463-1466 CD4+ T cells and skin fibrosis in the tight skin mouse 1463 Short paper Valerie A. Wallace., Seiji KondoA, Takeshi KonoA, Zhou Xing., Emma Timms., Caren FurlongerO, Edward Keystoneo, Jack Gauldie., Daniel N. SanderA, Tak W. Mak. and Christopher J. PaigeO Amgen Institute, Ontario Cancer Institute, Departments of Immunology and Medical Biophysics., University of Toronto, Toronto, Sunnybrook Health Science CentreA, University of Toronto, Toronto, Department of Pathology., McMaster University, Hamilton and Wellesley Hospital Research Instituteo, Toronto A role for CD4 T cells in the pathogenesis of skin fibrosis in tight skin mice The tight skin (TsWf) mouse represents a murine model of heritable fibrosis with some similarities to the skin fibrosis seen in human scleroderma. TsW+ animals display alterations in connective tissue in some internal organs. Skin fibrosis can be adoptively transferred to normal recipients withTsW+ bone marrow or spleen cells and older TsW+ animals develop autoantibodies against topoisomerase suggesting that some of the pathogenesis in theTsW+ mouse may be mediated by autoimmunity. To determine the role of T cell subsets in the pathogenesis of fibrotic disease,TsW+ mice were bred with CD4- and CD8-deficient CD4-’- and CD8-/-) mice.TsW+ CD4-’- mice showed a marked reduction in skin fibrosis as well as decreased cellularity and only mild collagen disorganization as compared toTsW+ CD4+ CD8+ control mice yet did not differ fromTsk controls in the level of serum anti-topoisomerase activity. In contrast, TsW+ CD8-/- mice exhibited the same histology in the skin as TsW+ controls yet had significantly reduced levels of serum anti-topoisomerase activity. Lung pathology, zy   zyxwv   emphysema, was unaffected by both the CD4 or CD8 mutations.These data show that only some of the pathological effects of the Tsk mutation are Tcell dependent and that different T cell subsets affect different parameters in this multi-organ model of fibrotic disease. 1 Introduction The tight skin TsW+) mouse strain represents a model of heritable fibrosis that bears some similarity with human scleroderma [l]. The Tsk mutation maps to chromosome 2 and is inherited in an autosomal dominant fashion zyxwvu   11. Mice that are homozygous for the Tsk defect die n utero [l]. TsW+ heterozygous mice have excessive accumulation of collagen in skin and some internal organs (heart, lung and retroperitoneum) characteristic of scleroderma [2, 31. Although there are differences between the Tsk mouse and human scleroderma, zyxwvutsrq .e. a paucity of inflammatory changes and an absence of vascular lesions in the animal model, there is evidence for an autoimmune component to the disease in the TsW+ mouse. Antinuclear antibodies directed against topoisomerase have been detected in TsW+ mice and the disease can be adoptively transferred with TsW+ bone marrow or spleen cells [4, 51. It has been demonstrated that T cells are involved in most experimentally induced autoimmune diseases [6]. CD4+ T cells are normally required for the initiation of disease while CD8+ T cells may play a role in regulating the severity [I 127301 * This work was supported by grants from the Medical Research Council and National Cancer Institute of Canada. Correspondence: Christopher J. Paige, Wellesley Hospital Research Institute, 160 Wellesley St. E., Toronto, Ontario, M4Y 153, Canada Abbreviations: zyxwvutsr sk Tight skin CD4-/- and zyxwvut D8-k CD4- and CD8-deficient mice, respectively CD4+/- and CD8+/-: Mice heterozygous for the targeted CD4 or CD8 alleles, respectively Key words: ight skin mouse zyxwvutsr   Skin fibrosis /Autoimmune disease I CD4+ T cells / CD8+ T cells of the disease [6,7].To nvestigate the role of CD4 and CD8 T cell subsets in the pathogenesis of theTsk fibrosis model, the Tsk mutation was bred onto a CD4 or CD8 gene- deficient background [8, 91. Skin and lung samples fom control and TsW+ mice were examined histologically and sera were analyzed for the titer of anti-topoisomerase antibodies. There was no difference in the presence of pulmonary changes (consistent with emphysema) exhibited by TsW+ CD4+ CD8+ control, Tsk/+ CD4-’- or TsW+ CD8-/- mice. However, TsW+ CD4-’- mice exhibited a significant reduction in skin pathology as compared to TsW+ control or TsW+ CD8-l- mice. In contrast TsW+ CD8-’- but not TsW+ CD4-I- animals exhibited signifi- cantly lower levels of serum anti-topoisomerase activity as compared toTsW+ controls.These results demonstrate that the pathogenesis of skin fibrosis in the TsW+ mouse is influenced by alterations in peripheral T cell populations. Since alterations in T cell subsets did not affect the lung pathology, these results also emphasize the pleiotropic nature of this murine model of fibrosis. 2 Materials and methods 2.1 Mice TsW+ mice on a C57BL/6 background were obtained from the Jackson Laboratory (Bar Harbor, ME). Mice homozy- gous for the CD4 or CD8 gene mutations CD4-’- or CD8-/-) have been previously described [S, 91 and were bred in the animal facility of the Ontario Cancer Institute. In order to generate mice with the Tsk mutation on a CD4-/- background, C57BL/6 TsW+ mice were mated with CD4-’- animals. The TsM+ CD4+’- progeny were then crossed with CD4-/- mice to generate normal or TsW+ offspring on both CD4+/- and CD4-/- backgrounds. An identical breeding strategy was followed to obtainTsW+ VCH Verlagsgeseilschaft mbH, D-69451 Weinheim, 1994 0014-2980194lO606-1463 10.00 + 25/0  1464 V. A. Wallace, S. Kondo, T. Kono et al. Eur. J. Immunol. 1994.24: 1463-1466 CD8-’- animals. Tsk mice were distinguished from their normal littermates by tightness of the skin in the intrasca- pular region which was unaffected by the absence of either CD4 or CD8 Tcells. CD4 and CD8 expression was determined by immunofluorescence staining of blood cells as previously described [7, 81. Mice ranging in age from 4 to 10months (CD8-I- and CD8+/- littermates), 8 to 11 months (CD4-’- and CD4+/- littermates) and older than lyear (Tsk/+ C57BL/6 mice) were used for the analysis.The mutant mice used in this study were H-2b/b nd were not on a C57BL/6 congenic background. For this reason, CD4-I- TsW+ and CD8-’- TsW+ mice were always compared to CD4+’- and CD8+/- TsW+ littermates in all of the analyses. The phenotype of CD4+/- TsW+, CD8+/- TsW+ did not differ from C57BL/6 TsW+ mice in any of the parameters that were examined suggesting that the non- congenic background had little influence on the TsW+ phenotype. In addition, the TsW+ mutation has been examined on three different genetic backgrounds, BlO.D2(58N), C3H/Di and C57BL/6, and there is no evidence to date which demonstrates that background genes influence the TsW+ phenotype [l]. 2.2 Serum anti-topoisomerase ELISA The level of serum antibodies directed against topoisomer- ase was determined by an ELISA. Plates coated with topoisomerase were purchased from Advanced Biological Products Lim. ( SCL-100) (Brampton, Ontario). Sera were obtained from tail vein blood. Serially diluted samples were plated in SO-pl aliquots and incubated at 37°C for 1 h. Plates were washed eight times with cold tap water and incubated for an additional 2 h at 37°C with goat anti- mouse immunoglobulin (Ig) coupled to peroxidase (Sigma Chemical Company, St. Louis, MO). Plates were washed and developed for 1 h with zyxwvut ,2’-azino-bis 3-ethylbenzthia- zoline-6-sulfonic acid) (ABTS) substrate (Sigma). Dilu- tions of purified mouse IgG standards (used as controls) were incubated on plates coated with goat anti-mouse Ig and subsequently developed using the same protocol as described for the topoisomerase plates. Absorbance was read with a microplate reader at 405 nm. In order to estimate the amount of antibody retained on the topoisom- erase plates the zyxwvuts 5 values obtained for the test sera were compared to standard titrations made with purified mouse IgG of known concentrations. 2.3 Histology Lungs were fixed by intratracheal perfusion with 4 zyxwv   paraformaldehyde and further fixed overnight in the same material and transferred to 70 ethanol. The lungs were mounted in paraffin and serial sections were stained for light microscopy with hematoxylin and eosin. Full thick- ness biopsies of shaved skin were excised deep enough to include underlying fascia from the dorsal surface of the trunk. The specimens were fixed with 10% buffered formalin and processed for light microscopy. Slides were stained with hematoxylin and eosin H & E) and Penta- chrome (Movats Pentachrome-Carolga modification). All sections were coded and read independently by two observers. Dermal and hypodermal thickness were mea- sured by an ocular micrometer. Cellularity of the dermis and hypodermis was evaluated by counting nuclei under light microscopy in ten areas randomly selected, and expressed as the mean number of cells per high-power field (HPF, magnification x lO00). At least four mice per group were included in the skin analysis. Differences between groups were analyzed using an unpaired, two-tailed Stu- dent’s t-test or a Mann Whitney rank sum test as indi- cated. 3 Results 3.1 Generation of TsW CD4-’- and TsW+ CD8-/- mice The Tsk mutation maps to chromosome 2 and the Tsk phenotype is inherited in an autosomal dominant fashion l] In crosses between TsW+ CD4+’- and normal CD4-I- mice the expected numbers of TsW+ CD4+’- and TsW+ CD4-I- mice were obtained (110 mice total: 31 TsW+ CD4+/- and 27 TsW+ CD4-’-). The crosses onto the CD8 background yielded lower than the expected number of TsW+ CD8-/- mice (120 mice total: 16 TsW CD8-’- versus 30 TsW+ CD8-I- expected). However, this reduc- tion was accompanied by a decrease in the number of CD8-/- mice obtained (120 mice total: 32 CD8-’- observed versus 60 CD8-/- expected). The reduction in the frequen- cy of CD8-/- mice was not due to the genetic background or the Tsk mutation as it was also observed on other genetic backgrounds J. Penninger, personal communication). The presence of either the CD4 or CD8 mutations did not mask or exacerbate on a gross morphological level the TsW+ phenotype. 3.2 Autoantibody production in TsW+ CD4-’- and CD8-I- mice It has been previously demonstrated that older TsW+ mice > 8 months) develop antinuclear antibodies [4]. The spe- cificity of these antibodies is directed against topoisomerase similar to what is observed in patients with scleroderma [4]. The effect of the CD4 or CD8 mutations on the production of autoantibodies by TsW+ mice was examined. Fig. 1 shows the level of anti-topoisomerase antibody detected in the serum of individual mice 8 months of age and older.The levels of serum anti-topoisomerase activity in TsW+ CD4+/- and TsW+ CD8+/- mice were not significantly different from the levels measured in TsW+ C57BL/6 control mice (data not shown) and therefore, the data from these groups of mice comprise the TsW+ control sample (Fig. 1). Although there are individual variations, TsW+ mice had higher levels of anti-topoisomerase antibodies than did normal controls (p < 0.06 in the Mann-Whitney rank sum two-sample test). Anti-topoisomerase levels in TsW+ CD8-I- mice were significantly lower than those from TsW+ mice (p < 0.02) while there was no significant difference between theTsW+ CD4-/- and theTsW+ control group (p > 0.1). 3.3 Lung and skin histology of TsW+ mice When lung histology of the TsW+ mice was compared to that of normal mice, the presence of emphysema-like  Eur. J. Immunol. 1994.24: 1463-1466 CD4+ T cells and skin fibrosis in the tight skin mouse 1465 Tsk/+ CD4+/- n = 4) and Tsk/+ CDW- n = 1) litter- mate controls were analyzed. In all mice there was marked evidence of emphysema data not shown). Lung sections from non-Tsk CD4-’- and CD8-/- mice were normal with no evidence of inflammation or changes in the alveolar spaces. Overall, there were no differences observed in the lung pathology and there was no consistent presence or absence of inflammatory cells between the various groups of TsW+ mice. Therefore the absence of either CD4+ or CD8+ T cells had no apparent effect on the lung pathology in TsM+ mice. changes inTsW+ mice was consistent with previous reports 131 and data not shown). Lung sections fromTsk/+ CD4-’- mice n = 7) and TsW+ CD8-I- n = 2) mice and their zyxwvutsrqpo  -/-) zyxwvutsrqpo  -/-) + 4 -/-) zyxwvutsrq (+) tsk normal zyxwvut igure 1 Levels of anti-topoisomerase antibodies detected by ELISA in the serum of individual mice (X 10 ng/ml). Figure 2. Histological findings of normal zyxwvuts sW+, TsW+ CD4-/- and TsW+ CD8-/- mice. A) normal skin; B) TsW+; C) TsM+ CD4-/-; D) TsW+ CD8-/-. d, dermal layer; f, adipocyte layer; h, hypodermal layer; p, panniculum carnosum. Note the increased hypodermal thickness in €3) and D). In contrast to lung pathology,TsW+ CD4-’- mice exhibited a marked decrease in the thickness mean +_ SD) of hypodermis 246.2 pm 60.0 pm, n = 5 compared to TsW+ CD4+ CD8+ 652.5 pm zy   00.4 pm, n = 10) (p < 0.05, Student’s t-test) and TsW+ CD8-/- mice 549.7 pm + 91.8 pm, n = 4) (p < 0.05 Figs. 2 and 3a). No significant differences were found in the dermal thick- ness between all groups of mice Fig. 3a). TsW+ CD4-’- mice also demonstrated a marked decrease n the cellularity in the dermis 16.9 k 2.1/HPF) and hypodermis (10.5 f .6/HPF) compared to Tsk/+ CD4+ CD8+ mice 34.1 k .9/HPF in dermis and 28.2 .3/HPF in hypo- dermis) (p < 0.001, Student’s t-test) and TsW+ CD8-’- mice 31.5 .3/HPF in dermis and 29.9 C_ 1.5/HPF in hypodermis) (p < 0.001) Fig. 3b). It should be noted that the skin histology inTsW+ CD4+/- and CD8+’- littermates of TsW+ CD4-I- and CD8-/- mice did not differ signifi- cantly from that in TsW+ C57BL/6 mice and these groups comprise the TsW+ sample Fig. 3). These data show that t T 8 600 a v1 400 .- zoo 1 a 23456 ** 123456 Figure 3 a) Skin thickness was measured by an ocular microm- eter. Open bars represent dermal thickness and solid bars represent hypodermal thickness in pm. * p < 0.05. Error bars represent SD. I) normal CD4+ CD8+; 2 normal CD4-/-; 3) normal CD8-/-; 4) TsW+ CD4+ CD8+; 5) TsW+ CD4-/-; 6) TsW+ CD8-/-. b) Cellularity of the dermis open bars) and hypodermis solid bars) was evaluated by counting nuclei in ten randomly selected high-power fields HPF) and expressed as the mean number of cells per HPF. ** p < 0.001. Error bars represent SEM 1 normal CD4+ CDW; 2 normal CD4-’-; 3) normal zy D8 ’ ; TsW+ CD4 CD8+; 5) TsW CD4-/-; 6) TsW+ CD8-/-.  1466 V. A. Wallace, S. Kondo, T. Kono et al. Eur. J. Immunol. 1994. 24: 1463-1466 TsW+ CD8-/- and TsW+ CD4+ CD8+ control mice did not differ significantly from each other in any parameter analyzed, whereas TsM+ CD4-/- mice displayed marked reduction in skin pathology. 4 Discussion The aim of this study was to investigate whether the absence of CD4 or CD8 T cells would affect the pathogen- esis of skin, pulmonary disease or the production of auto-antibodies in TsM+ mice. Neither the CD4 or CD8 mutations altered the development of emphysema inTsW+ mice. This finding is consistent with the observation that adoptive transfer of TsW+ splenocytes or bone marrow cells to normal recipients did not reproduce the lung pathology observed in TsM+ mice [5]. The levels of serum anti- topoisomerase antibodies were significantly reduced in TsW+ CD8-’- mice as compared to TsW+ control animals. However, this reduction in auto-antibodies did not corre- late with disease severity since there was no difference in the skin or the lung pathology betweenTsW+ CD8-/- mice and TsW+ controls. The way in which CD8+ T cells might influence the level of serum anti-topoisomerase activity in TsW+ mice is unclear. However, it has been demonstrated that CD8+ Tcells do play a regulatory role in other autoimmune disease models [7, 101. We observed a striking reduction in the skin pathology in Tsk CD4-/- mice as compared to their Tsk CD4+’- littermates which suggests that T helper cell function is involved in the skin fibrosis observed in TsW+ mice. Previous studies have shown that in CD4-I- mice T helper activity can be detected and the level of T help depends on the type antigenic challenge [ll] and Rahemutalla, A., Kuendig,T. M., Paige, C. J., Zinkernagel, R. M. and Mak, T. W.; manuscript submitted]. One explanation for the reduction of skin pathology observed in CD4-’- TsW+ mice is that the remaining level of T help is too low to influence skin pathology in this model.This is the first direct demonstration that the skin fibrosis in TsW+ mice is influenced by CD4+ T cells. These results are consistent with the findings in human scleroderma in which CD4+ T cell involvement in skin fibrosis has been documented [12, 131. How CD4 cells could be involved in the progres- sive skin fibrosis observed inTsk/+ mice is not clear since an inflammatory infiltrate is not observed in the skin of TsW+ mice. Although an inflammatory infiltrate is observed in the skin during the early stages of human scleroderma, the latter stages of the disease are characterized by progressive skin fibrosis which occurs in the absence of inflammation [14]. The occurrence of skin fibrosis in the absence of an inflammatory infiltrate in the TsW+ mouse has been proposed to be cytokine mediated [15]. Our findings are consistant with a model in which CD4+ Tcell-derived cytokines mediate skin fibrosis in the TsW+ mouse. Taken together, the results of the present study clearly demon- strate that CD4+ T cells are involved in the pathogenesis of skin fibrosis in the TsW+ mouse and that different T cell subsets may be involved in different aspects of this multi-organ fibrosis model. In this context, the TsW+ mouse may be helpful in dissecting the role of T cells in the pathogenesis of skin fibrosis in scleroderma. We would like to thank Dr. J. Penninger for helpful discussion. Received January 18, 1994; in revised form March 1, 1994; accepted March 10, 1994. 5 References 1 Green, M. C., Sweet, H. 0. andBunker, L. E., Am. J. Puthol. 1976. 82: 493. 2 Jimenez, S. A., Millan, A. and Bashey, R. I. Arthritis Rheum. 1984. 27: 180. 3 Szapiel, S. V., Fulmer, J. D., Hunninghake, G. W., Elson, N. A., Kawanami, O., Ferrans,V. J. and Crystal, R. G., Am. Rev. Respir. Dis. 1981. 123: 680. 4 Muryoi,T., Kasturi, K. N., Kafina, M. J., Saitoh,Y., Usuba, O., Perlich, J. S., Heishmayer, R. and Bopna, C., Autoimmunity 1991. 9: 109. 5 Walker, M. A., Harley, R. A., DeLustro, F. A. and LeRoy, E. C., Proc. SOC. Exp. Biol. Med. 1989. 192: 196. 6 Sinha, A. A., Lopez, M. T. and McDevitt, H. O., Science 1990. 248: 1380. 7 Koh, D.-R., Fung-Leung, W.-I?, Ho, A., Gray, D., Acha- Orbea, H. and Mak, T. W., Science 1992. 256: 1210. 8 Rahemtulla, A., Fung-Leung,W. zy ? Schilham, M. W., Kundig, T. M., Sambhara, S. R., Narendran, A., Arabian, A., Wake- ham, A., Paige, C. J., Zinkernagel, R. M., Miller, R. G. and Mak, T. W., Nature 1991. 353: 180. 9 Fung-Leung,W.-I?, chilham, M. W., Rahemtulla, A., Kundig, T. M., Vollenweider, M., Potter, J., van Ewijk, W. and Mak, T. W., Cell 1991. 65: 443. 10 Penninger, J. M., Neu, N., Timms, E., Wallace, V. A., Koh, D.-R., Kishihara, K., Pummerer, C. and Mak, T. W., J. Exp. Med. 1993. 178: 1837. 11 Locksley, R. M., Reiner, S. L., Hatam, F., Littmen, D. R. and Killeen, N., Science 1993. 261: 1448. 12 Korn, J., Curr. Opinion Rheumatol. 1990. 2: 922. 13 Fiocco, U., Rosada, M., Cozzi, L., Ortolani, C., De Silvestro, G., Ruffatti, A., Cozzi, E., Gallo, C. and Todesco, S., Ann. Rheum. Dis. 1993. 52: 272. 14 Roumm, A. D., Whiteside, T. L., Medsger, Jr., T. A., and Rodnan, G. I? Arthritis Rheum. 1984. 27: 645. 15 Walker, M., Harley, R. and LeRoy, E. C., zyx   Rheum. 1989.17: 57.
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