ARTICLE |
Correspondence to: Andrew G. Farr, Dept. of Biological Structure, Box 357420, U. of Washington, Seattle, WA 98195-7420.
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Summary |
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We characterized the distribution of CD40 and CD40 ligand (CD40-L) in the adult and developing murine thymus. Before birth, CD40 was almost exclusively localized to scattered foci of medullary cells. By birth there was a dramatic upregulation of CD40 expression by cortical epithelial cells, which was accompanied by a consolidation of medullary epithelial foci. CD40-L+ thymocytes displayed a medullary location. Analysis of mice deficient in CD40-L expression indicated that CD40-L/CD40 interactions were not required for development of the medullary compartment. Overexpression of CD40-L targeted to thymocytes altered thymic architecture, as reflected by a dramatic loss of cortical epithelial cells, expansion of the medullary compartment, and extensive infiltration of the capsule with a mixture of CD3+ cells, B-cells, and macrophages/dendritic cells. Reconstitution of lethally irradiated normal mice with lck CD40-L bone marrow cells also resulted in loss of cortical epithelium and expansion of the medullary compartment. Disruption of the normal pattern of thymic architecture and epithelial differentiation as a consequence of increased intrathymic levels of CD40-L expression points to a role for CD40-L/CD40 interactions in the normal pattern of epithelial compartmentalization/differentiation within the thymic environment. (J Histochem Cytochem 45:129-141, 1996)
Key Words: Thymic epithelium, CD40-L, CD40, gp39
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Introduction |
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The CD40 molecule is a 50-kd glycoprotein belonging to the TNF/NGF receptor superfamily (
The role of CD40 interactions in T-cell development is less clear. The absence of significant alterations of T-cell development in CD40null or CD40-Lnull mice suggests that CD40/CD40-L interactions are not absolutely required for T-cell development to occur (
In this study we characterized the expression of CD40 and CD40-L within the developing and adult normal murine thymus and assessed the effects of either the lack of CD40-L expression or elevated levels of CD40-L expression on the development and organization of the thymic environment. Although thymic tissue from mice lacking CD40-L displayed reduced expression of Class II MHC antigens and accessory molecules within the medullary compartment, the over all thymic architecture and phenotype was essentially normal. In contrast, thymic tissue from mice overexpressing CD40-L was profoundly affected, with B-cell infiltration/expansion, disorganization and loss of cortical epithelium, and dramatic increases of the medullary compartment.
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Materials and Methods |
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Mice.
CD40-L-deficient mice produced by -irradiated (1000 rads) 6-week-old female C57Bl/6 mice with 107 bone marrow cells from female lckgp39 transgenic mice or syngenic female donors. The donor bone marrow cells were depleted of T-cells by treatment with anti-Thy1.2 MAb (clone 30-H12; American Type Culture Collection, Rockville, MD) and rabbit complement (Lo-Tox; Accurate Chemical and Scientific, Westbury, NY). Chimeric mice were analyzed 4-6 weeks after reconstitution.
Antibodies.
The following MAbs were obtained from the American Type Culture Collection: anti-CD4 (clone GK 1.5); anti-CD45 (clone RA3-3A1/6.1); anti-B7.2 (clone GL-1); anti-Class II MHC (clone M5/114.15.2); and anti-mouse CD11c (clone N418). The following MAbs were also used: anti-E-cadherin MAb [clone ECCD-2 (-aminocaproic acid N-hydrosuccinimide ester (Boehringer Mannheim) or d-biotinoyl-
-aminocaproic acid N-hydroxy-succinimide ester (Pierce) was performed according to the manufacturer's instructions.
Light Microscopic Immunohistochemistry.
For light microscopy, frozen sections of thymic tissue embedded in OCT compound (Miles; Elkhart, IN) were mounted on aminoalkylsilane-subbed slides and allowed to air-dry for 2 hr before fixation in cold (-20°C) acetone for 20 min. After washing twice for 5 min in PBS, antigens were detected by either two- or three-step enzyme immunohistochemistry. For the two-step procedure, digoxigenin-conjugated MAbs were diluted in PBS containing 5% w/v nonfat dry milk and incubated with the fixed and hydrated tissue sections for 60 min at room temperature (RT). After washing three times for 5 min in PBS to remove unbound MAbs, peroxidase-conjugated Fab fragments of goat anti-digoxigenin were applied. The diluent for this conjugate was PBS containing 10% normal mouse serum (NMS). Enzyme activity was detected with a mixture of 3,3'-diaminobenzidine (DAB) and H2O2. The three-step procedure was similar, except that the primary MAbs were in the form of hybridoma supernatants or purified protein and the secondary MAb was digoxigenin-modified polyclonal goat anti-rat IgG antibody, which was detected with goat anti-digoxigenin antibody/enzyme conjugates. In some instances it was necessary to inhibit endogenous peroxidase before application of antibodies. Acetone-fixed tissue was incubated for 1 hr at 37°C in PBS containing 1 mM NaN3, 10 mM glucose, and 1 U/ml of glucose oxidase (US Biochemical; Cleveland, OH).
Electron Microscopic Immunohistochemistry.
Under ketamine/xylazine anesthesia, mice were fixed by cardiac perfusion with 5 ml of HBSS, followed by
30 ml of 0.1 M cacodylate buffer, pH 7.4, containing 4% paraformaldehyde and 1 mM CaCl2 (all at RT). Thymic tissue was then excised and cut into 50-µm-thick sections with a vibratome (Ted Pella; Tustin, CA). Tissue sections were incubated overnight at 4°C with primary antibodies diluted to 50 µg/ml in PBS containing 1% BSA and 5% sucrose. Sections were then washed three to five times with PBS/sucrose and incubated for about 6 hr with peroxidase-conjugated goat anti-rat IgG antibodies diluted to 30 µg/ml in PBS/BSA/sucrose. After repeated washing in PBS/sucrose, the sections were immersed for 15 min in PBS containing 1% glutaraldehyde, washed extensively with PBS/sucrose, and then immersed for 15-20 min in 0.05 M Tris buffer, pH 7.6, containing 0.0058% H2O2, 0.2 mg/ml DAB, and 5% sucrose. After additional washing in PBS/sucrose, the sections were immersed in 0.1 M cacodylate buffer, pH 7.4, containing 1% OsO4. After additional washing in distilled water, the sections were then prepared for conventional transmission electron microscopy.
Cell Culture.
Thymocytes at 107 cells /ml were cultured in RPMI 1640 containing 10% FBS, 5 x 10-5 M ß2-mercaptoethanol, penicillin (100 U/ml), and streptomycin (100 µg/ml) (CM). CM in some cultures also contained ionomycin (120 ng/ml) and PMA (12 ng/ml). After 1 hr at 37°C in a humidified environment containing 5% CO2, the cells were recovered and washed by centrifugation, then processed for flow cyometry. Aliquots of the starting cell population were also taken for flow cytometric analyses.
Flow Cytometry.
One million cells were incubated for 1 hr at 4°C with antibodies used at concentrations previously d etermined to yield optimal labeling. With direct fluorochrome-conjugated antibodies or fluorochrome-labeled anti-digoxigenin antibodies used in conjunction with digoxigenin-conjugated primary antibodies, the diluent contained 2.4G2 antibodies (
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Results |
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Thymic CD40 Expression
In adult thymic tissue, staining with anti-CD40 MAb revealed a reticular pattern in the cortex and a more heavily labeled confluent pattern in the medulla (Figure 1a). Adjacent sections labeled with ER-TR5 and ER-TR4, which stain medullary and cortical TE, respectively (
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Ultrastructural immunohistochemistry confirmed that the CD40 expression in the thymic cortex was associated with epithelial cells (Figure 1d). For the most part, there was a delicate linear distribution of the CD40 reaction product along the epithelial cell processes, which was consistent with the relatively light labeling of cortical CD40 at the light microscopic level. Interestingly, there were small segments of the epithelial cell processes that bore larger amounts of the reaction product, resulting in a patchy labeling pattern. In the medullary compartment, dendritic cells and epithelial cells, identified by ultrastructural features, were labeled with anti-CD40 MAb (Figure 1e and Figure 1f). In contrast to dendritic cells, which appeared to uniformly label well with anti-CD40 MAb, some medullary epithelial cells displayed very little reaction product (Figure 1e), whereas the labeling of others approached that displayed by dendritic cells (Figure 1f).
Thymocyte Expression of CD40-L
Previous flow cytometric analyses of normal young adult Balb/c thymocytes demonstrated low levels of CD40-L expressed by a small population of thymocytes, which increased following exposure to ConA or anti-CD3 MAbs (60%) and CD4+8+ (
25%) thymocyte populations (Figure 2b). Within the CD4+8+ thymocyte subset, CD40-L was preferentially expressed by cells expressing higher levels of CD4 and CD8 (Figure 2b).
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Exposure of thymocytes to PMA and ionophore for 1 hr resulted in a rapid upregulation of CD40-L (Figure 2c). The distribution of CD40-L+ cells among thymocyte populations defined by CD4/CD8 expression was similar to that of untreated thymocytes, although there was further skewing of MR1+ cells to the CD4+8- population (Figure 2d). Although this treatment had little effect on the representation of thymocyte subsets defined by CD4 and CD8 expression, marked downregulation of CD4 labeling was observed after PMA/ionophore treatment.
Immunohistochemical analysis of gp 39 expression in frozen sections of adult thymus tissue detected rare, scattered CD40-L+ cells in the cortex and more labeling in areas corresponding to the medulla (Figure 3a). A punctate anti-CD40-L labeling pattern predominated among medullary thymocytes, although profiles of partial circumferential labeling were also observed (Figure 3b). Demonstration of CD3 expression on adjacent thymic sections (Figure 3c) confirmed that the location of the majority of the CD40-L+ thymocytes coincided with the distribution of strongly labeled CD3+ cells in the medulla.
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Ontogeny of Thymic CD40 and CD40-L Expression
At Day 14 of gestation, the earliest time point examined, expression of CD40 was preferentially associated with aggregates of cells in the central portion of the thymus (Figure 4a). With advancing gestational age (Days 16 and 18), increased levels of CD40 labeling were associated with cords of medullary cells (Figure 4b and Figure 4c, respectively). Levels of cortical CD40 expression observed in adult thymic tissue were not observed in fetal thymic tissue through Day 18 of gestation, but were dramatically increased by birth (Figure 4d). As shown in Figure 4e and Figure 4g, CD40-L+ thymocytes were not detectable in Day 18 fetal thymic tissue. By birth (Figure 4f and Figure 4h), CD40-L+ thymocytes were readily detectable and were preferentially associated with the medullary compartment, although isolated CD40-L+ thymocytes were scattered throughout the cortex. The upregulation of cortical CD40 expression and the appearance of CD40-L+ thymocytes coincided temporally with consolidation of the medullary thymic compartment. Scattered foci of ER-TR5+ and CD40+ epithelial cells present at Day 18 of gestation (Figure 4i) had coalesced into fewer but larger foci by birth (Figure 4j). The distribution of ER-TR5+ and medullary CD40+ cells was largely overlapping, suggesting that many of the ER-TR5+ stromal cells also expressed CD40 at this stage of development.
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Targeted Overexpression of CD40-L to the Thymus Results in Altered Cortical, Medullary, and Subcapsular Compartments
The temporal coincidence of the appearance of CD40-L+ thymocytes and the organization of the medulla raised the possibility that CD40-L/CD40 interactions might contribute to the differentiation or expansion of this tissue compartment. Therefore, we examined the organization and phenotype of thymic tissue in which the thymocyte expression of CD40-L was abrogated (
Thymic tissue from mice bearing a disruption of the CD40-L gene displayed fairly normal histology, with well-defined cortical and medullary compartments and no obvious defect in thymocyte development (
Thymic tissue from transgenic mice expressing different levels of CD40-L transgene under control of the proximal lck promoter were examined to assess the consequences of CD40-L overexpression on the organization and development of thymic stromal compartments. The extent to which overexpression of CD40-L affected the thymic environment was correlated with the number of CD40-L transgene copies, which in turn correlated with levels of transgene expression. In thymic tissue from transgenic mice expressing 40 copies of the transgene (Figure 5), the thymic epithelial component identified by E-cadherin expression (
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In addition to the disrupted segregation/representation of cortical and medullary epithelial cells, the expression patterns of several putative cell interaction molecules were elevated in the lckCD40-L transgenic thymus tissue. Labeling of B7-1 and B7-2 was widespread throughout the epithelial and extraepithelial thymic compartments (data not shown). Dendritic cells and macrophages defined by CD11c expression were also widely distributed throughout both thymic compartments (data not shown).
The expanded extraepithelial subcapsular space defined by the absence of E-cadherin+ epithelium (Figure 6a) contained accumulations of B220+ (Figure 6b) and CD40+ (Figure 6c) cells. Widespread CD40 expression was also evident within the epithelial compartment of the thymus (Figure 6c). The extraepithelial compartment also contained substantial numbers of CD3+ (Figure 6d) cells expressing CD40-L (Figure 6e). Although the level of CD40-L expression was dramatically elevated in the transgenic thymic tissue, the punctate labeling pattern was similar to that observed in normal mice (inset to Figure 6e). In addition to B-cells and CD3+ cells, these areas also contained significant numbers of CD11c+ cells (Figure 6f) and thus resembled diffuse peripheral lymphoid tissue. Endogenous peroxidase activity was predominantly associated with the epithelial component of the transgenic thymic tissue (Figure 6g). As in normal thymic tissue, this activity was due largely to eosinophils, as determined by ultrastructural cytochemistry (data not shown).
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Elevated intrathymic CD40-L expression could be affecting the initial expansion of different stromal cell populations or the differentiation program of TE subpopulations. If promiscuous CD40-L/CD40 interactions were primarily altering the expansion potential of different TE populations, thymocytes overexpressing CD40-L would not be expected to significantly alter the representation of relatively radio-resistant cortical and medullary elements in an established thymic environment that had developed normally. On the other hand, if the loss of cortical TE and expansion of the medullary compartment within the lckCD40-L thymus reflected disruption of the normal developmental relationship between these two epithelial populations, then elevated levels of CD40-L expression should cause similar epithelial alterations in thymic tissue that had developed normally. We generated radiation bone marrow chimeric mice by lethally irradiating C57Bl/6 mice and reconstituting their hematopoietic compartment with T-cell-depleted bone marrow from syngenic normal mice or from lckCD40-L mice. As shown in Figure 7, reconstitution of irradiated but otherwise normal adult thymic tissue with syngenic bone marrow cells resulted in normal thymic architecture. In contrast, repopulation of normal thymic tissue with thymocytes overexpressing CD40-L resulted in a thymic phenotype virtually indistinguishable from that displayed by lckCD40-L transgenic mice. In addition to loss of cortical TE and expansion of medullary TE, lckCD40-L &Aelig; C57Bl/6 thymic tissue also displayed accumulations of N418+ cells, elevated levels of CD40 expression, and focal subcapsular accumulations of T-cells, B-cells, and dendritic cells.
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Discussion |
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The pattern of CD40 expression in the murine thymus was similar to that described for the human thymus (
Flow cytometric and immunohistochemical analyses indicated that CD40-L is expressed at low levels by thymocytes, but preferentially by medullary CD4+8- and, to a lesser extent, CD4+8+ subsets. The distinctive punctate nature of much of the MR1 labeling may represent CD40-L sequestered in a cytoplasmic compartment within thymocytes or redistribution of CD40-L on the thymocyte cell surface as a consequence of crosslinking induced by interactions with CD40+ cells in the thymus. Dynamic regulation of CD40-L expression by CD4+ thymocytes is suggested by reports that CD40-L/CD40 interactions lead to downregulation of CD40-L expression by CD4+ T-cells (Yellin et al., 1992), and that human CD4+ memory T-cells contain preformed CD40-L that is rapidly brought to the cell surface after TCR-mediated activation (
Previous studies of SCID or RAG-deficient mice demonstrated that normal development and organization of medullary, but not cortical, thymic epithelium was dependent on the participation of mature CD3+ thymocytes (ß TCR begin to accumulate (
or combinations of IFN
and other pro-in flammatory cytokines (Farr, unpublished observations). If the cytokine requirements for CD40 expression by TE cells in vitro reflect physiological requirements for this process in vivo, the neonatal upregulation of CD40 expression may be related to the accumulation of CD4 and CD8 single positive thymocytes, which can produce high titers of IFN
and TNF
in vitro after activation with calcium ionophore and PMA (
Targeted overexpression of CD40-L in thymocytes resulted in reduced thymic cellularity, disrupted thymocyte development, and profound alterations in the phenotype and organization of the thymic environment, the severity of which increased with increased transgene expression. At low levels of transgene expression (<12 copies) thymic cellularity was reduced two- to 10-fold, with fairly normal representation of thymocyte subsets defined by CD4 and CD8 expression. With high copy number there was a preferential loss of CD4+8+ thymocytes (up to 4000-fold reduction) and skewing of the mature thymocyte representation to the CD8 single positive subset. Thymocyte development in radiation bone marrow chimeras repopulated with transgenic bone marrow yielded similar results (Clegg CH et al., submitted for publication). Based on the work of
A potential role for CD40-L/CD40 interactions in regulating epithelial cell growth/differentiation is suggested by the progressively increased representation of medullary TE cells and the decline in the frequency of cortical TE cells as levels of transgene-derived CD40-L expression increased. It is presently not known if cortical and medullary epithelia derive from a common 3rd pharyngeal pouch endodermal epithelial progenitor population (
The observed alterations in the thymic environment may not represent a direct sequelae of CD40/CD40-L interactions. Alterations of thymocyte development as a consequence of dysregulated expression of CD40L could modify other stimuli that in turn regulate the differentiation of thymic epithelial cells. A loss of cortical epithelium has been reported in transgenic mice that are homozygous for a human CD3 transgene and display a block in thymocyte differentiation at the CD44-CD25- stage (
transgenic mice does not appear to be accompanied by expansion of the medullary compartment or capsular involvement, suggesting that the requirements for expansion and maintenance of cortical TE may be different. Interestingly, thymic tissue from transgenic mice with thymic overexpression of oncostatin M (Clegg and Farr, manuscript in preparation) or LIF (
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health (AI-24137 and AG-04360) and by the Bristol-Myers Squibb Pharmaceutical Research Institute.
We thank Dr Roger Perlmutter for critical review of the manuscript.
Received for publication May 15, 1996; accepted August 5, 1996.
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