Cathepsin-L Influences the Expression of Extracellular Matrix in Lymphoid Organs and Plays a Role in the Regulation of Thymic Output and of
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免疫学杂志 2005年第11期
Abstract
Nackt mice, which are deficient in cathepsin-L (CTSL), show an early impairment during positive selection in the context of class II MHC molecules and as a consequence, the percentage and absolute number of CD4+ thymocytes are significantly decreased. In this study, we show that lymph nodes from nackt mice are hypertrophied, showing normal absolute numbers of CD4+ T cells and marked increases in the number of CD8+ T lymphocytes. Basal proliferative levels are increased in the CD4+ but not in the CD8+ population. Lymph node T cells show increases in the expression of 5, 6, and 1 integrin chains. These alterations correlate with increases in the expression of extracellular matrix (ECM) components in lymph nodes. Interestingly, laminin, fibronectin, and collagen I and IV are markedly decreased in nackt thymus which shows an augmented output of CD8+ cells. These results demonstrate that a mutation in the Ctsl gene influences the levels of ECM components in lymphoid organs, the thymic output, and the number of T cells in the periphery. They further raise the possibility that, by regulating the level of expression of ECM components in lymphoid organs, CTSL is able to broadly affect the immune system.
Introduction
Cells in lymphoid tissues are surrounded by a web of extracellular matrix (ECM) 3 glycoproteins, which contribute to the determination of the particular tissue microenvironment and its functional characteristics. During intrathymic maturation, thymocytes interact with cellular and noncellular stromal thymic components. Thymic ECM is not restricted to the basal membranes, but is also heterogeneously distributed in the thymic interstitium forming a thick network in the medullary region of the thymic lobules, whereas very thin ECM fibers are found within the cortex (1, 2). In lymph nodes, ECM components are abundant in the interfollicular compartments but they are poorly expressed in both primary and secondary follicles (3). In addition to their mechanistical role, ECM glycoproteins are important signaling molecules with the ability to strongly influence thymocyte and lymphocyte programs, promoting differentiation, migration, proliferation, and activation (4, 5, 6, 7, 8, 9). Moreover, different growth factors, chemokines, and hormones are associated with ECM, this association being an important factor in regulating cellular responsiveness (10, 11, 12, 13, 14).
The lysosomal cysteine peptidase cathepsin-L (CTSL), an abundant and ubiquitously expressed member of the papain family that contributes to the terminal degradation of proteins in the lysosome, can also be secreted (15). There is accumulating evidence for specific functions of CTSL and other lysosomal proteases in health and disease (16, 17, 18, 19, 20, 21, 22). The use of "knockout" (KO) or mutant mice deficient in CTSL has provided a valuable tool for gaining new insights into the in vivo functions of this protease (21, 22, 23, 24, 25, 26, 27).
In mice, CTSL has been shown to be critical for epidermal homeostasis, the regulation of the hair cycle (18, 23), and the maintenance of the heart structure and function (24). A role for CTSL in trophoblast invasion (17), tumor metastasis (16), thyroid function (25), and mammary gland involution (19) has also been demonstrated. CTSL plays an important role in the MHC class II-mediated peptide presentation in thymic epithelial cells, acting both in Ii degradation (21) and in the generation of MHC class II-bound peptide ligands presented by cortical thymic epithelial cells (22). Nakagawa et al. (21) reported that CTSL is critical for degradation of Ii in cortical epithelial cells and as a consequence, CTSL KO mice exhibit a marked reduction in the percentage of CD4+ cells in the thymus and the periphery. We have shown that nackt (nkt/nkt) mice–which carry a mutation in the Ctsl gene (26)–have an early impairment during positive selection of CD4 thymocytes (27). In this study, we report that nkt/nkt mice show hypertrophied lymph nodes, which have normal absolute numbers of CD4+ cells and marked increases in the number of CD8+ lymphocytes. Whereas the normalization in the number of lymph node CD4+ cells correlates with increases in basal proliferative levels, increases in CD8+ cells correlate with a significant increase in thymic export of these T cells. Interestingly, the thymus and lymph nodes of nkt/nkt mice show opposite alterations in the expression levels of ECM glycoproteins.
Materials and Methods
Statistics
The two-tailed Student t test or the Mann-Whitney U test were used to assess the statistical significance of the results. A value of p < 0.05 was considered indicative of a significant difference.
Results
Alterations in T cell populations in peripheral lymphoid organs from nkt/nkt mice
We have previously reported that the positive selection of CD4+ cells is impaired in the thymus of nkt/nkt mice, thus showing a considerable decrease in the percentage and absolute number of CD4+ single-positive (SP) thymocytes (27). Herein we investigated whether these mice showed alterations in peripheral lymphoid organ T cells. The spleen of nkt/nkt mice showed normal cellularity. The percentage and the absolute number of CD4+ cells were significantly decreased whereas the number of CD8+ cells was increased (Fig. 1 and Table I). Lymph nodes were hypertrophied (17.1 x 106 ± 5.1 (n = 22) total lymphocytes per lymph node in nkt/nkt mice vs 6.0 x 106 ± 3.2 (n = 15) in nkt/+ mice, p < 0.001 (mean ± SD)). The histological examination of lymph nodes showed that the overall structure was modified by: 1) the presence in the upper cortex of compressed subcapsular follicle-like structures without germinal centers, mostly composed of small, dark-stained lymphoid cells; 2) an enlarged paracortical area that seems to compress the cortical areas and projects deeply into the medulla; 3) a medullary area with dilated sinuses with macrophages and medullary cords variable in size and shape, composed by darkly stained and loosely arranged lymphoid cells (Fig. 2).
As can be observed in Fig. 1 and Table I, although the percentage of CD4+ T cells was decreased in nkt/nkt lymph nodes, their absolute number was not significantly different from that of nkt/+ mice. The percentage and absolute number of CD8+ T cells in nkt/nkt lymph nodes was markedly increased. The absolute number of B220+ lymph node cells was also found to be increased in nkt/nkt mice (Table I).
Concerning the expression of activation/adhesion molecules, the percentage of CD44high and CD69+ cells within CD4+ lymph node cells was found to be significantly increased in nkt/nkt mice. A 3-fold increase in the percentage of CD4+ cells expressing high levels of CD44 and a 2-fold increase in the percentage of cells expressing CD69 was consistently recorded in mutant mice (Fig. 3 and Table II). In the spleen, CD4+ cells showed a significant increase in the percentage of CD44high cells whereas the percentage of CD69+ cells remained unchanged (Fig. 3). CD8+ cells did not show significant differences in the expression of CD44 or CD69 molecules either in lymph nodes or in the spleen (Table II).
The thymus of nkt/nkt mice shows decreases in ECM components
In a conventional histological study, the thymus from nkt/nkt mice showed a loss of the basic lobular pattern mainly due to a diminished septa thickness (Fig. 9). A normally vascularized corticomedullar junction showed an ill-defined boundary. The corticomedullar relation was altered with clear predominance of the cortex. Less packed thymocytes were found in the outer cortex. To investigate the expression of thymic ECM glycoproteins, immunohistochemical studies were conducted. A significant decrease in the level of expression of fibronectin, laminin, and collagen I and IV was observed not only in the medulla but also in the cortex in mutant mice. As can be observed in Fig. 5, I and J, the decrease of fibronectin expression was observed in epithelial cells, basal membranes, and perivascular spaces. Fibronectin was minimally expressed in the capsula/septa. A general decrease in laminin expression was also observed in the thymus from nkt/nkt mice. A very marked decrease was observed in the medullar area and in epithelial cells. (Fig. 5, K and L). The expression of collagen I (Fig. 5, M and N) and collagen VI (Fig. 5, O and P) was also diminished. A weak stain for collagen IV was observed in both the cortical and the medullar network and also in some scattered epithelial cells as well as in capsule/septa and perivascular space. The expression of collagen I showed a general diminution. Thymocytes were negative for all the ECM components studied. Staining for ECM components was analyzed by image analysis both in the cortex and medulla. A significant decrease in the cortical staining was observed in nkt/nkt mice (laminin: 8.1 ± 0.5 vs 5.5 ± 0.6; fibronectin: 8.2 ± 0.9 vs 1.9 ± 0.7; collagen IV: 2.2 ± 0.3 vs 0.23 ± 0.22; collagen I: 5.2 ± 1.0 vs 1.2 ± 0.5, expressed as mean percentage of the stained area ± SEM (n = 5). In the medulla, levels of ECM components were also shown to be significantly lower in nkt/nkt mice (laminin 11.6 ± 0.9 vs 4.2 ± 0.6; fibronectin: 11.4 ± 1.1 vs 2.6 ± 0.9; collagen IV: 2.24 ± 0.3 vs 1.2 ± 0.16; collagen I: 4.9 ± 1.3 vs 0.78 ± 0.3). Overall, these data indicate a clear decrease in the main components of ECM in the thymus of nkt/nkt mice.
Discussion
We had previously described that nkt/nkt mice, which have a deletion in the Ctsl gene (26), showed an early impairment during positive selection in the context of major class II MHC molecules (27). Consequently, the percentage and absolute number of CD4+ SP thymocytes were significantly decreased. No differences in the percentage and absolute number of SP CD8+ thymocytes could be registered.
In this study, we show that peripheral lymph nodes of nkt/nkt mice are enlarged, showing a 2- to 3-fold increase in cell number. Histologically, the most remarkable features were compressed subcapsular follicle-like structures without germinal centers and an enlarged paracortical area that projects deeply into the medulla. The overall structure seems to correspond to a hyperplastic nonreactive lymph node. Accordingly, the numbers of B and T cells in nkt/nkt lymph nodes were significantly increased. The number of CD4+ cells was similar to that of normal littermates. This subpopulation of T cells displayed a significant overrepresentation of cells with an activated/memory phenotype–based on the expression of CD44 and CD69 molecules–and an increased basal proliferative level. The percentage and absolute number of CD8+ cells were markedly increased. Differently from results registered in the CD4+ population, the CD8+ subset showed a normal proportion of cells with an activated/memory phenotype and a normal basal proliferative level. In the spleen, which shows normal cellularity, the number of CD4+ cells was decreased whereas CD8+ cells were found to be highly increased. The same phenotypic characteristics (i.e., increases in lymph node cellularity, in the number of CD8+ lymph node cells and in CD4+ proliferative levels along with higher numbers of CD4+ cells expressing high levels of CD44) were observed in nkt/nkt mice backcrossed on C57BL/6J.
The number of CD4+ cells leaving the thymus daily was found to be diminished in nkt/nkt mice and their thymic export rate–as suggested in a previous report (27)–was not significantly altered when compared with nkt/+ littermates. On the contrary, both the number of CD8+ cells leaving the thymus daily and the CD8+ thymic export rate were significantly increased in mutant mice.
These data show that the marked increment in the number of CD8+ peripheral cells correlates with increases in CD8+ thymic output. The low number of CD4+ T cells in the spleen correlates with the decrease in CD4 thymic output. In contrast, in lymph nodes, the mechanisms by which nkt/nkt mice reach normal numbers of CD4+ cells involve increases in cell proliferation.
Lysosomal cystein peptidases of the papain family exhibit high collagenolytic (34) and elastinolytic (35) activity. Although CTSL is located mainly in the endosomal/lysosomal compartment, 10% is physiologically secreted and can be extracellularly activated (15). There, it is capable of processing ECM proteins such as fibronectin, laminin, elastin, and collagen types I, IV, and XVII (15, 34, 36, 37, 38). In turn, CTSL activity induces further ECM degradation by other proteases (35, 37). In vivo, the level of CTSL mRNA is related to tumor progression and metastatic potential, and this is thought to be related to the ability of CTSL to degrade ECM and basement membranes (16). In addition, it has been recently proposed that CTSL plays a role in ECM degradation during early mammary gland involution (19). Thus, the absence of CTSL would generate the accumulation of components of the ECM as it has been suggested for myocardium in CTSL KO mice (24) and observed in nkt/nkt heart (our unpublished results). In accordance, ECM components such as laminin, fibronectin, collagen I and IV were found to be increased in lymph nodes from nkt/nkt mice. Concomitantly, the percentage of lymph node T cells expressing high levels of 5, 6, and 1 integrin chains was increased. However, in the spleen, no alterations in the level of ECM components or changes in the level of expression of 5, 6, and 1 integrin chains in T cells could be detected.
ECM components have been shown to transduce survival signals through integrins (6, 7, 39, 40). It has also been demonstrated that ECM components promote the proliferation of T cells by signaling through integrins (9). Besides, different growth factors, chemokines and hormones have been shown to be associated with ECM (10, 11, 12, 13), this association being an important factor in regulating not only their availability but also the cellular responsiveness (10, 12, 13, 14). Thus, it can be considered that increases in ECM components may increase the number of niches allowing the survival of an increased number of T (and B) cells in lymph nodes and also may favor cell proliferation. Increases in cell proliferation were detected in CD4+ but not in CD8+ lymph node T cells. The mechanisms underlying selective CD4+ proliferation remain unknown. However several hypotheses can be proposed. Although homeostatic proliferation cannot be discarded it is difficult to reconcile with the presence of high numbers of CD8+ cells in lymph nodes. Moreover, a significant percentage of CD4+ cells express CD69, an early marker of T cell activation which has not been observed in cells undergoing homeostatic proliferation (41). Neither can alterations in the recognition of self Ags in the periphery be discarded since it has been reported that CTSL KO mice display an altered repertoire in CD4+ (22). Contrary to lymph nodes, the spleen–which show no alterations in ECM components and integrin chains levels–showed no changes in the total number or in the basal proliferative level of CD4+ cells. Thus, it is possible that, in a situation of low thymic output of CD4+ cells, increased ECM components in lymph nodes could lead to their proliferation.
Overall, these results suggest that the mutation in the Ctsl gene present in nkt/nkt mice leads to alterations in lymph node cell counts probably by causing increases in ECM components.
Immunohistochemical studies showed that nkt/nkt mice had important decreases in several components of thymic ECM such as laminin, fibronectin, and type I and IV collagens, both in the medulla and in the cortex. The regulatory mechanisms involved in the control of thymic ECM production and degradation are poorly understood (reviewed in Ref. 42). Although some metalloproteinases have been shown to be constitutively expressed in human thymus (43) and transcripts for metalloproteinase-9 have also been detected in fetal mouse thymocyte precursors (44), their role in the physiological breakdown of thymic ECM remains unclear. The possibility exists that in nkt/nkt mice, the lack of CTSL leads to increases in other proteases–as it has been recently shown for procathepsin D in the thyroid gland of CTSL–/– mice (25)–which could be responsible for ECM degradation. In contrast, different factors have been involved in the control of ECM production (45, 46, 47). In human thymic epithelial cells, insulin growth factor-1 (IGF-1) stimulates the expression of laminin and fibronectin, as well as their receptors VLA-5 and VLA-6 (46). It has been recently reported that CTSL-deficient fibroblasts show a decreased degradation of IGF binding protein-3 (IGFBP-3) (48). Limited proteolysis of IGFBP from IGF/IGFBP complexes constitutes a central mechanism for the release of IGF from these complexes (49). Thus, a putative decrease in IGFBP degradation in the thymus could play a role in the decreased thymic ECM composition of nkt/nkt mice. However, we were not able to detect alterations in the levels of laminin 1, 2, and 5 and fibronectin mRNAs in the thymus of nkt/nkt mice (data not shown) nor in thymocyte expression of 6, 5, 4, and 1 integrin chains. Therefore, these results do not support a central role for alterations in the proteolysis of IGF/IGFBP complexes in the thymic decrease of ECM components. In contrast, in vitro studies have shown that thyroid hormones are able to enhance laminin and fibronectin as well as VLA-5 and VLA-6 expression in mouse and human thymic epithelial cells. CTSL has been proposed to be involved in the processing and solubilization of the prohormone thyroglobulin (47). In this sense, alterations in thyroid function could play a role in the thymic decrease of ECM in nkt/nkt mice.
Whatever the mechanisms involved, decreases in thymic ECM glycoproteins not only could greatly alter the availability and activity of different chemokines (13, 14), hormones, and growth factors (10, 11), but also could have effects on integrins/ligands interactions, thus influencing thymocyte development. Interactions between integrins displayed by thymocytes and ECM components, especially fibronectin and laminin, have been shown to strongly influence T cell differentiation/maturation possibly by stabilizing interactions between thymocytes and the epithelial components of the thymus (4, 5, 6, 50). The interaction of thymocyte integrins with their ECM ligands also leads to intracellular signaling (50). It has also been proposed that thymic ECM glycoproteins would constitute a macromolecular arrangement allowing differentiating thymocytes to migrate (51). Dysregulated ECM production/distribution correlates with alterations in intrathymic cell maturation and trafficking as recently shown in laminin 2-chain mutant dy3k/dy3k mice (52) and in experimental Chagas’ disease (53). In the case of dy3k/dy3k mice, the lack of laminin 2 (merosin) is associated with a severe thymic atrophy resulting from apoptosis of double-positive (DP) cells. In vitro experiments suggested that the interaction of merosin with integrin 61 in the thymus is able to block the apoptosis of DP thymocytes. In contrast, during the course of Trypanosoma cruzi infection, a progressive increase in fibronectin and laminin expression correlates with increases in thymocyte migration ability; as a result, immature DP cells are found in the periphery of infected animals.
Herein, we show that in nkt/nkt mice, the decrease in thymic ECM components correlates with an increase both in the daily output of CD8+ cells and in their thymic export rate. Taking into account that the total number of hemopoietic bone marrow cells is increased in mutant mice (our unpublished results), the high output of mature CD8+ T cells could be related to increases in the entry of T cell precursors into the thymus. Alterations in thymocyte traffic and maturation could also be involved. Changes in thymocyte traffic could be recorded at least in the rate of emigration of CD8+ cells. No differences in the percentage of CD8+ SP thymocytes expressing CD69 high, intermediate, and negative levels were detected in the CD8 thymic compartment when nkt/nkt and control mice were compared. Although the mechanisms underlying the selective increased export of CD8+ SP thymocytes remain unknown, our data suggest that changes in the speed of maturation and exit would include all the stages of CD8+ SP thymocytes since neither diminution nor accumulation could be detected along all stages of their maturation. Finally, it is tempting to speculate that alterations in thymic ECM could also affect lineage commitment. The intracellular signaling pathways stimulated by integrin-ligand binding are shared by a number of other surface receptors expressed on thymocytes, including growth factor receptors, the TCR-CD3 complex, CD4 and CD8 (54). It has been reported that the intensity of signals appears to be crucial for positive selection and CD4/CD8 lineage commitment (55, 56, 57, 58, 59). Stronger and/or longer TCR signals may be required for CD4 than for CD8 lineage commitment (56, 57, 58, 59, 60). In addition, it has been shown that a strong MAPK signal is required for CD4+ differentiation (61), and that blocking this signal favors CD8+ production (61, 62, 63). Schmeissner et al. (64), using a mouse system that transgenically expresses a chimeric molecule shown to have dominant negative effects on integrin functions, suggested that integrin signaling is required for the generation of CD4+ cells but is not essential for the production of CD8+ cells. Moreover, the authors suggested that the disruption of integrin function leads to a shift from the CD4 to the CD8 lineage that correlates with an increased CD8:CD4 ratio in the spleen (64). Although integrin activation was not investigated in nkt/nkt mice, no alterations in the expression of 4, 5, 6, and 1 molecules during the different stages of thymocyte development could be detected. However, mutant mice showed a marked decrease in laminin, fibronectin, collagen I and IV thymic expression. Thus, in a scenario in which CD4+-positive selection is impaired, the possibility exists that thymocyte signaling through integrin-ECM interactions is decreased and that this could influence lineage commitment favoring a shift from the CD4+ to the CD8+ fate.
Benavides et al. (26) were not able to detect CTSL in the skin of nkt/nkt mice using the Ab M-19 from Santa Cruz Biotechnology, raised against a peptide mapping at the C terminus of the L chain of CTSL. The 118-bp deletion they described for CTSL in nkt/nkt mice involves the end of exon 6 and almost all of exon 7, sequences partially coding for the protein H and L chains. In accordance with the sequence reported (26), no C terminus of the L chain could be expected to be produced and therefore the amino acid sequence recognized by the M-19 Ab should be absent from a putative mutated form of CTSL. Results reported herein show that, although at a lower level, it has been possible to detect CTSL in thymus and skin from nkt/nkt mice using the AF1515 Ab (R&D Systems) directed to aa 18–334 in Western blot assays. Moreover, the immunohistochemical analysis of nkt/nkt tissues exhibited a very strong staining for CTSL (data not shown). Chauhan et al. (65) suggested that human CTSL lacking the 16 C-terminal amino acids is retained in the endoplasmic reticulum presumably because of its improper folding. Our results suggest that nkt/nkt mice express a mutated form of CTSL. The possibility exists that the presence of a mutated CTSL in nkt/nkt mice could lead to the appearance of phenotypic differences between KO and nkt/nkt mice. Therefore, it would be of interest to investigate which of the nkt/nkt phenotypes described herein are also shared by CTSL KO mice, to determine whether the presence of a mutated form of CTSL plays a role in some of the alterations observed.
To our knowledge, these results demonstrate by the first time that a mutation in the Ctsl gene is able to affect the levels of ECM components in lymphoid organs. Moreover, the opposite effects registered in thymic and lymph node ECM in CTSL mutant mice further underline the variability of CTSL functions among different organs. Results reported herein also show that the nkt mutation broadly affects the immune system, influencing the thymic output of CD8+ cells and the regulation of the number of T cells in the periphery. We postulate that these influences would be a consequence of alterations in ECM expression.
Acknowledgments
We thank Dr. C. D. Pasqualini for helpful discussions. We are grateful to Antonio Morales, Julio Tejeda, and Juan Portaluppi for efficient technical assistance.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by grants from CONICET, L-Agencia para la Promoción Cientifíca y Tecnológica (ANPCYT)-Fundación para Combatir la Leucemia (FUNDALEU).
2 Address correspondence and reprint requests to Dr. Irene Nepomnaschy, División Medicina Experimental, Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Pacheco de Melo 3081(1425) Buenos Aires, Argentina. E-mail address: irenepo{at}hematologia.anm.edu.ar
3 Abbreviations used in this paper: ECM, extracellular matrix; CTSL, cathepsin-L; KO, knockout; Cy, CyChrome; RTE, recent thymic emigrant; PI, propidium iodide; SP, single positive; IGF, insulin growth factor; IGFBP, IGF binding protein; DP, double positive.
Received for publication April 19, 2004. Accepted for publication March 25, 2005.
References
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Lannes-Veira, J., M. Dardenne, W. Savino. 1991. Extracellular matrix components of the mouse thymus microenvironment. Ontogenetic studies and modulation by glucocorticoid hormones. J. Histochem. Cytochem. 39: 1539-1546.
Casta?os-Velez, E., P. Biberfeld, M. Patarroyo. 1995. Extracellular matrix proteins and integrin receptors in reactive and non-reactive lymph nodes. Immunology 86: 270-278.
Utsumi, K., M. Sawada, S. Narumiya, J. Nagamine, T. Sakata, S. Iwagami, Y. Kita, H. Teraoka, H. Hirano, M. Ogata, et al 1991. Adhesion of immature thymocytes to thymic stromal cells through fibronectin molecule and its significance for the induction of thymocyte differentiation. Proc. Natl. Acad. Sci. USA 88: 5685-5689.
Salomon, D. R., C. F. Mojcik, A. C. Chang, S. Wadsworth, D. H. Adams, J. E. Coligan, E. M. Shevach. 1994. Constitutive activation of integrin 41 defines a unique stage of human thymocytes development. J. Exp. Med. 179: 1573-1584.(Gabriela Lombardi*, Dalia)
Nackt mice, which are deficient in cathepsin-L (CTSL), show an early impairment during positive selection in the context of class II MHC molecules and as a consequence, the percentage and absolute number of CD4+ thymocytes are significantly decreased. In this study, we show that lymph nodes from nackt mice are hypertrophied, showing normal absolute numbers of CD4+ T cells and marked increases in the number of CD8+ T lymphocytes. Basal proliferative levels are increased in the CD4+ but not in the CD8+ population. Lymph node T cells show increases in the expression of 5, 6, and 1 integrin chains. These alterations correlate with increases in the expression of extracellular matrix (ECM) components in lymph nodes. Interestingly, laminin, fibronectin, and collagen I and IV are markedly decreased in nackt thymus which shows an augmented output of CD8+ cells. These results demonstrate that a mutation in the Ctsl gene influences the levels of ECM components in lymphoid organs, the thymic output, and the number of T cells in the periphery. They further raise the possibility that, by regulating the level of expression of ECM components in lymphoid organs, CTSL is able to broadly affect the immune system.
Introduction
Cells in lymphoid tissues are surrounded by a web of extracellular matrix (ECM) 3 glycoproteins, which contribute to the determination of the particular tissue microenvironment and its functional characteristics. During intrathymic maturation, thymocytes interact with cellular and noncellular stromal thymic components. Thymic ECM is not restricted to the basal membranes, but is also heterogeneously distributed in the thymic interstitium forming a thick network in the medullary region of the thymic lobules, whereas very thin ECM fibers are found within the cortex (1, 2). In lymph nodes, ECM components are abundant in the interfollicular compartments but they are poorly expressed in both primary and secondary follicles (3). In addition to their mechanistical role, ECM glycoproteins are important signaling molecules with the ability to strongly influence thymocyte and lymphocyte programs, promoting differentiation, migration, proliferation, and activation (4, 5, 6, 7, 8, 9). Moreover, different growth factors, chemokines, and hormones are associated with ECM, this association being an important factor in regulating cellular responsiveness (10, 11, 12, 13, 14).
The lysosomal cysteine peptidase cathepsin-L (CTSL), an abundant and ubiquitously expressed member of the papain family that contributes to the terminal degradation of proteins in the lysosome, can also be secreted (15). There is accumulating evidence for specific functions of CTSL and other lysosomal proteases in health and disease (16, 17, 18, 19, 20, 21, 22). The use of "knockout" (KO) or mutant mice deficient in CTSL has provided a valuable tool for gaining new insights into the in vivo functions of this protease (21, 22, 23, 24, 25, 26, 27).
In mice, CTSL has been shown to be critical for epidermal homeostasis, the regulation of the hair cycle (18, 23), and the maintenance of the heart structure and function (24). A role for CTSL in trophoblast invasion (17), tumor metastasis (16), thyroid function (25), and mammary gland involution (19) has also been demonstrated. CTSL plays an important role in the MHC class II-mediated peptide presentation in thymic epithelial cells, acting both in Ii degradation (21) and in the generation of MHC class II-bound peptide ligands presented by cortical thymic epithelial cells (22). Nakagawa et al. (21) reported that CTSL is critical for degradation of Ii in cortical epithelial cells and as a consequence, CTSL KO mice exhibit a marked reduction in the percentage of CD4+ cells in the thymus and the periphery. We have shown that nackt (nkt/nkt) mice–which carry a mutation in the Ctsl gene (26)–have an early impairment during positive selection of CD4 thymocytes (27). In this study, we report that nkt/nkt mice show hypertrophied lymph nodes, which have normal absolute numbers of CD4+ cells and marked increases in the number of CD8+ lymphocytes. Whereas the normalization in the number of lymph node CD4+ cells correlates with increases in basal proliferative levels, increases in CD8+ cells correlate with a significant increase in thymic export of these T cells. Interestingly, the thymus and lymph nodes of nkt/nkt mice show opposite alterations in the expression levels of ECM glycoproteins.
Materials and Methods
Statistics
The two-tailed Student t test or the Mann-Whitney U test were used to assess the statistical significance of the results. A value of p < 0.05 was considered indicative of a significant difference.
Results
Alterations in T cell populations in peripheral lymphoid organs from nkt/nkt mice
We have previously reported that the positive selection of CD4+ cells is impaired in the thymus of nkt/nkt mice, thus showing a considerable decrease in the percentage and absolute number of CD4+ single-positive (SP) thymocytes (27). Herein we investigated whether these mice showed alterations in peripheral lymphoid organ T cells. The spleen of nkt/nkt mice showed normal cellularity. The percentage and the absolute number of CD4+ cells were significantly decreased whereas the number of CD8+ cells was increased (Fig. 1 and Table I). Lymph nodes were hypertrophied (17.1 x 106 ± 5.1 (n = 22) total lymphocytes per lymph node in nkt/nkt mice vs 6.0 x 106 ± 3.2 (n = 15) in nkt/+ mice, p < 0.001 (mean ± SD)). The histological examination of lymph nodes showed that the overall structure was modified by: 1) the presence in the upper cortex of compressed subcapsular follicle-like structures without germinal centers, mostly composed of small, dark-stained lymphoid cells; 2) an enlarged paracortical area that seems to compress the cortical areas and projects deeply into the medulla; 3) a medullary area with dilated sinuses with macrophages and medullary cords variable in size and shape, composed by darkly stained and loosely arranged lymphoid cells (Fig. 2).
As can be observed in Fig. 1 and Table I, although the percentage of CD4+ T cells was decreased in nkt/nkt lymph nodes, their absolute number was not significantly different from that of nkt/+ mice. The percentage and absolute number of CD8+ T cells in nkt/nkt lymph nodes was markedly increased. The absolute number of B220+ lymph node cells was also found to be increased in nkt/nkt mice (Table I).
Concerning the expression of activation/adhesion molecules, the percentage of CD44high and CD69+ cells within CD4+ lymph node cells was found to be significantly increased in nkt/nkt mice. A 3-fold increase in the percentage of CD4+ cells expressing high levels of CD44 and a 2-fold increase in the percentage of cells expressing CD69 was consistently recorded in mutant mice (Fig. 3 and Table II). In the spleen, CD4+ cells showed a significant increase in the percentage of CD44high cells whereas the percentage of CD69+ cells remained unchanged (Fig. 3). CD8+ cells did not show significant differences in the expression of CD44 or CD69 molecules either in lymph nodes or in the spleen (Table II).
The thymus of nkt/nkt mice shows decreases in ECM components
In a conventional histological study, the thymus from nkt/nkt mice showed a loss of the basic lobular pattern mainly due to a diminished septa thickness (Fig. 9). A normally vascularized corticomedullar junction showed an ill-defined boundary. The corticomedullar relation was altered with clear predominance of the cortex. Less packed thymocytes were found in the outer cortex. To investigate the expression of thymic ECM glycoproteins, immunohistochemical studies were conducted. A significant decrease in the level of expression of fibronectin, laminin, and collagen I and IV was observed not only in the medulla but also in the cortex in mutant mice. As can be observed in Fig. 5, I and J, the decrease of fibronectin expression was observed in epithelial cells, basal membranes, and perivascular spaces. Fibronectin was minimally expressed in the capsula/septa. A general decrease in laminin expression was also observed in the thymus from nkt/nkt mice. A very marked decrease was observed in the medullar area and in epithelial cells. (Fig. 5, K and L). The expression of collagen I (Fig. 5, M and N) and collagen VI (Fig. 5, O and P) was also diminished. A weak stain for collagen IV was observed in both the cortical and the medullar network and also in some scattered epithelial cells as well as in capsule/septa and perivascular space. The expression of collagen I showed a general diminution. Thymocytes were negative for all the ECM components studied. Staining for ECM components was analyzed by image analysis both in the cortex and medulla. A significant decrease in the cortical staining was observed in nkt/nkt mice (laminin: 8.1 ± 0.5 vs 5.5 ± 0.6; fibronectin: 8.2 ± 0.9 vs 1.9 ± 0.7; collagen IV: 2.2 ± 0.3 vs 0.23 ± 0.22; collagen I: 5.2 ± 1.0 vs 1.2 ± 0.5, expressed as mean percentage of the stained area ± SEM (n = 5). In the medulla, levels of ECM components were also shown to be significantly lower in nkt/nkt mice (laminin 11.6 ± 0.9 vs 4.2 ± 0.6; fibronectin: 11.4 ± 1.1 vs 2.6 ± 0.9; collagen IV: 2.24 ± 0.3 vs 1.2 ± 0.16; collagen I: 4.9 ± 1.3 vs 0.78 ± 0.3). Overall, these data indicate a clear decrease in the main components of ECM in the thymus of nkt/nkt mice.
Discussion
We had previously described that nkt/nkt mice, which have a deletion in the Ctsl gene (26), showed an early impairment during positive selection in the context of major class II MHC molecules (27). Consequently, the percentage and absolute number of CD4+ SP thymocytes were significantly decreased. No differences in the percentage and absolute number of SP CD8+ thymocytes could be registered.
In this study, we show that peripheral lymph nodes of nkt/nkt mice are enlarged, showing a 2- to 3-fold increase in cell number. Histologically, the most remarkable features were compressed subcapsular follicle-like structures without germinal centers and an enlarged paracortical area that projects deeply into the medulla. The overall structure seems to correspond to a hyperplastic nonreactive lymph node. Accordingly, the numbers of B and T cells in nkt/nkt lymph nodes were significantly increased. The number of CD4+ cells was similar to that of normal littermates. This subpopulation of T cells displayed a significant overrepresentation of cells with an activated/memory phenotype–based on the expression of CD44 and CD69 molecules–and an increased basal proliferative level. The percentage and absolute number of CD8+ cells were markedly increased. Differently from results registered in the CD4+ population, the CD8+ subset showed a normal proportion of cells with an activated/memory phenotype and a normal basal proliferative level. In the spleen, which shows normal cellularity, the number of CD4+ cells was decreased whereas CD8+ cells were found to be highly increased. The same phenotypic characteristics (i.e., increases in lymph node cellularity, in the number of CD8+ lymph node cells and in CD4+ proliferative levels along with higher numbers of CD4+ cells expressing high levels of CD44) were observed in nkt/nkt mice backcrossed on C57BL/6J.
The number of CD4+ cells leaving the thymus daily was found to be diminished in nkt/nkt mice and their thymic export rate–as suggested in a previous report (27)–was not significantly altered when compared with nkt/+ littermates. On the contrary, both the number of CD8+ cells leaving the thymus daily and the CD8+ thymic export rate were significantly increased in mutant mice.
These data show that the marked increment in the number of CD8+ peripheral cells correlates with increases in CD8+ thymic output. The low number of CD4+ T cells in the spleen correlates with the decrease in CD4 thymic output. In contrast, in lymph nodes, the mechanisms by which nkt/nkt mice reach normal numbers of CD4+ cells involve increases in cell proliferation.
Lysosomal cystein peptidases of the papain family exhibit high collagenolytic (34) and elastinolytic (35) activity. Although CTSL is located mainly in the endosomal/lysosomal compartment, 10% is physiologically secreted and can be extracellularly activated (15). There, it is capable of processing ECM proteins such as fibronectin, laminin, elastin, and collagen types I, IV, and XVII (15, 34, 36, 37, 38). In turn, CTSL activity induces further ECM degradation by other proteases (35, 37). In vivo, the level of CTSL mRNA is related to tumor progression and metastatic potential, and this is thought to be related to the ability of CTSL to degrade ECM and basement membranes (16). In addition, it has been recently proposed that CTSL plays a role in ECM degradation during early mammary gland involution (19). Thus, the absence of CTSL would generate the accumulation of components of the ECM as it has been suggested for myocardium in CTSL KO mice (24) and observed in nkt/nkt heart (our unpublished results). In accordance, ECM components such as laminin, fibronectin, collagen I and IV were found to be increased in lymph nodes from nkt/nkt mice. Concomitantly, the percentage of lymph node T cells expressing high levels of 5, 6, and 1 integrin chains was increased. However, in the spleen, no alterations in the level of ECM components or changes in the level of expression of 5, 6, and 1 integrin chains in T cells could be detected.
ECM components have been shown to transduce survival signals through integrins (6, 7, 39, 40). It has also been demonstrated that ECM components promote the proliferation of T cells by signaling through integrins (9). Besides, different growth factors, chemokines and hormones have been shown to be associated with ECM (10, 11, 12, 13), this association being an important factor in regulating not only their availability but also the cellular responsiveness (10, 12, 13, 14). Thus, it can be considered that increases in ECM components may increase the number of niches allowing the survival of an increased number of T (and B) cells in lymph nodes and also may favor cell proliferation. Increases in cell proliferation were detected in CD4+ but not in CD8+ lymph node T cells. The mechanisms underlying selective CD4+ proliferation remain unknown. However several hypotheses can be proposed. Although homeostatic proliferation cannot be discarded it is difficult to reconcile with the presence of high numbers of CD8+ cells in lymph nodes. Moreover, a significant percentage of CD4+ cells express CD69, an early marker of T cell activation which has not been observed in cells undergoing homeostatic proliferation (41). Neither can alterations in the recognition of self Ags in the periphery be discarded since it has been reported that CTSL KO mice display an altered repertoire in CD4+ (22). Contrary to lymph nodes, the spleen–which show no alterations in ECM components and integrin chains levels–showed no changes in the total number or in the basal proliferative level of CD4+ cells. Thus, it is possible that, in a situation of low thymic output of CD4+ cells, increased ECM components in lymph nodes could lead to their proliferation.
Overall, these results suggest that the mutation in the Ctsl gene present in nkt/nkt mice leads to alterations in lymph node cell counts probably by causing increases in ECM components.
Immunohistochemical studies showed that nkt/nkt mice had important decreases in several components of thymic ECM such as laminin, fibronectin, and type I and IV collagens, both in the medulla and in the cortex. The regulatory mechanisms involved in the control of thymic ECM production and degradation are poorly understood (reviewed in Ref. 42). Although some metalloproteinases have been shown to be constitutively expressed in human thymus (43) and transcripts for metalloproteinase-9 have also been detected in fetal mouse thymocyte precursors (44), their role in the physiological breakdown of thymic ECM remains unclear. The possibility exists that in nkt/nkt mice, the lack of CTSL leads to increases in other proteases–as it has been recently shown for procathepsin D in the thyroid gland of CTSL–/– mice (25)–which could be responsible for ECM degradation. In contrast, different factors have been involved in the control of ECM production (45, 46, 47). In human thymic epithelial cells, insulin growth factor-1 (IGF-1) stimulates the expression of laminin and fibronectin, as well as their receptors VLA-5 and VLA-6 (46). It has been recently reported that CTSL-deficient fibroblasts show a decreased degradation of IGF binding protein-3 (IGFBP-3) (48). Limited proteolysis of IGFBP from IGF/IGFBP complexes constitutes a central mechanism for the release of IGF from these complexes (49). Thus, a putative decrease in IGFBP degradation in the thymus could play a role in the decreased thymic ECM composition of nkt/nkt mice. However, we were not able to detect alterations in the levels of laminin 1, 2, and 5 and fibronectin mRNAs in the thymus of nkt/nkt mice (data not shown) nor in thymocyte expression of 6, 5, 4, and 1 integrin chains. Therefore, these results do not support a central role for alterations in the proteolysis of IGF/IGFBP complexes in the thymic decrease of ECM components. In contrast, in vitro studies have shown that thyroid hormones are able to enhance laminin and fibronectin as well as VLA-5 and VLA-6 expression in mouse and human thymic epithelial cells. CTSL has been proposed to be involved in the processing and solubilization of the prohormone thyroglobulin (47). In this sense, alterations in thyroid function could play a role in the thymic decrease of ECM in nkt/nkt mice.
Whatever the mechanisms involved, decreases in thymic ECM glycoproteins not only could greatly alter the availability and activity of different chemokines (13, 14), hormones, and growth factors (10, 11), but also could have effects on integrins/ligands interactions, thus influencing thymocyte development. Interactions between integrins displayed by thymocytes and ECM components, especially fibronectin and laminin, have been shown to strongly influence T cell differentiation/maturation possibly by stabilizing interactions between thymocytes and the epithelial components of the thymus (4, 5, 6, 50). The interaction of thymocyte integrins with their ECM ligands also leads to intracellular signaling (50). It has also been proposed that thymic ECM glycoproteins would constitute a macromolecular arrangement allowing differentiating thymocytes to migrate (51). Dysregulated ECM production/distribution correlates with alterations in intrathymic cell maturation and trafficking as recently shown in laminin 2-chain mutant dy3k/dy3k mice (52) and in experimental Chagas’ disease (53). In the case of dy3k/dy3k mice, the lack of laminin 2 (merosin) is associated with a severe thymic atrophy resulting from apoptosis of double-positive (DP) cells. In vitro experiments suggested that the interaction of merosin with integrin 61 in the thymus is able to block the apoptosis of DP thymocytes. In contrast, during the course of Trypanosoma cruzi infection, a progressive increase in fibronectin and laminin expression correlates with increases in thymocyte migration ability; as a result, immature DP cells are found in the periphery of infected animals.
Herein, we show that in nkt/nkt mice, the decrease in thymic ECM components correlates with an increase both in the daily output of CD8+ cells and in their thymic export rate. Taking into account that the total number of hemopoietic bone marrow cells is increased in mutant mice (our unpublished results), the high output of mature CD8+ T cells could be related to increases in the entry of T cell precursors into the thymus. Alterations in thymocyte traffic and maturation could also be involved. Changes in thymocyte traffic could be recorded at least in the rate of emigration of CD8+ cells. No differences in the percentage of CD8+ SP thymocytes expressing CD69 high, intermediate, and negative levels were detected in the CD8 thymic compartment when nkt/nkt and control mice were compared. Although the mechanisms underlying the selective increased export of CD8+ SP thymocytes remain unknown, our data suggest that changes in the speed of maturation and exit would include all the stages of CD8+ SP thymocytes since neither diminution nor accumulation could be detected along all stages of their maturation. Finally, it is tempting to speculate that alterations in thymic ECM could also affect lineage commitment. The intracellular signaling pathways stimulated by integrin-ligand binding are shared by a number of other surface receptors expressed on thymocytes, including growth factor receptors, the TCR-CD3 complex, CD4 and CD8 (54). It has been reported that the intensity of signals appears to be crucial for positive selection and CD4/CD8 lineage commitment (55, 56, 57, 58, 59). Stronger and/or longer TCR signals may be required for CD4 than for CD8 lineage commitment (56, 57, 58, 59, 60). In addition, it has been shown that a strong MAPK signal is required for CD4+ differentiation (61), and that blocking this signal favors CD8+ production (61, 62, 63). Schmeissner et al. (64), using a mouse system that transgenically expresses a chimeric molecule shown to have dominant negative effects on integrin functions, suggested that integrin signaling is required for the generation of CD4+ cells but is not essential for the production of CD8+ cells. Moreover, the authors suggested that the disruption of integrin function leads to a shift from the CD4 to the CD8 lineage that correlates with an increased CD8:CD4 ratio in the spleen (64). Although integrin activation was not investigated in nkt/nkt mice, no alterations in the expression of 4, 5, 6, and 1 molecules during the different stages of thymocyte development could be detected. However, mutant mice showed a marked decrease in laminin, fibronectin, collagen I and IV thymic expression. Thus, in a scenario in which CD4+-positive selection is impaired, the possibility exists that thymocyte signaling through integrin-ECM interactions is decreased and that this could influence lineage commitment favoring a shift from the CD4+ to the CD8+ fate.
Benavides et al. (26) were not able to detect CTSL in the skin of nkt/nkt mice using the Ab M-19 from Santa Cruz Biotechnology, raised against a peptide mapping at the C terminus of the L chain of CTSL. The 118-bp deletion they described for CTSL in nkt/nkt mice involves the end of exon 6 and almost all of exon 7, sequences partially coding for the protein H and L chains. In accordance with the sequence reported (26), no C terminus of the L chain could be expected to be produced and therefore the amino acid sequence recognized by the M-19 Ab should be absent from a putative mutated form of CTSL. Results reported herein show that, although at a lower level, it has been possible to detect CTSL in thymus and skin from nkt/nkt mice using the AF1515 Ab (R&D Systems) directed to aa 18–334 in Western blot assays. Moreover, the immunohistochemical analysis of nkt/nkt tissues exhibited a very strong staining for CTSL (data not shown). Chauhan et al. (65) suggested that human CTSL lacking the 16 C-terminal amino acids is retained in the endoplasmic reticulum presumably because of its improper folding. Our results suggest that nkt/nkt mice express a mutated form of CTSL. The possibility exists that the presence of a mutated CTSL in nkt/nkt mice could lead to the appearance of phenotypic differences between KO and nkt/nkt mice. Therefore, it would be of interest to investigate which of the nkt/nkt phenotypes described herein are also shared by CTSL KO mice, to determine whether the presence of a mutated form of CTSL plays a role in some of the alterations observed.
To our knowledge, these results demonstrate by the first time that a mutation in the Ctsl gene is able to affect the levels of ECM components in lymphoid organs. Moreover, the opposite effects registered in thymic and lymph node ECM in CTSL mutant mice further underline the variability of CTSL functions among different organs. Results reported herein also show that the nkt mutation broadly affects the immune system, influencing the thymic output of CD8+ cells and the regulation of the number of T cells in the periphery. We postulate that these influences would be a consequence of alterations in ECM expression.
Acknowledgments
We thank Dr. C. D. Pasqualini for helpful discussions. We are grateful to Antonio Morales, Julio Tejeda, and Juan Portaluppi for efficient technical assistance.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by grants from CONICET, L-Agencia para la Promoción Cientifíca y Tecnológica (ANPCYT)-Fundación para Combatir la Leucemia (FUNDALEU).
2 Address correspondence and reprint requests to Dr. Irene Nepomnaschy, División Medicina Experimental, Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Pacheco de Melo 3081(1425) Buenos Aires, Argentina. E-mail address: irenepo{at}hematologia.anm.edu.ar
3 Abbreviations used in this paper: ECM, extracellular matrix; CTSL, cathepsin-L; KO, knockout; Cy, CyChrome; RTE, recent thymic emigrant; PI, propidium iodide; SP, single positive; IGF, insulin growth factor; IGFBP, IGF binding protein; DP, double positive.
Received for publication April 19, 2004. Accepted for publication March 25, 2005.
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