In vitro evidence for participation of DEC-205 expressed by thymic cortical epithelial cells in clearance of apoptotic thymocytes

Myra Small1 and Georg Kraal2

1 Department of Cell Biology and Histology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Israel 2 Department of Cell Biology and Immunology, Vrije Universiteit Medical Center, 1081 BT Amsterdam, The Netherlands

Correspondence to: M. Small; E-mail: msmall{at}post.tau.ac.il
Transmitting editor: D. Wallach


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Binding of apoptotic cells was compared after incubation of thymocytes with two clones of murine thymic stromal cells to which CD4+/CD8+ thymocytes attach. With the BA/10, but not the BA/2, clone, thymocytes with apoptotic morphology were bound irreversibly. These tightly bound thymocytes were further identified as apoptotic in terms of active caspase-3 and DNA fragmentation assayed in situ. FACS analysis indicated that the apoptotic thymocytes are at an early double-positive stage and results with mice mutant for the Fas gene showed that the Fas–Fas ligand system is not involved. Comparison of BA/10 and BA/2 cells showed that the former, but not the latter, can be induced to express CDR-1 antigen which is characteristic of cortical epithelial thymic stroma and constitutively express DEC-205, a surface protein common to cortical thymic epithelium and dendritic cells. Antibody NLDC-145 that is specific for the DEC-205 protein strongly reduced the number of stromal cells with bound apoptotic thymocytes. Preincubation of thymocytes in dexamethasone dramatically increased the number of bound apoptotic cells, indicating that the thymic cortical epithelial cells can participate in clearance of apoptotic thymocytes through involvement of DEC-205.

Keywords: apoptotic thymocyte, DEC-205/NLDC-145 antigen, thymic stroma, thymocyte clearance


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In recent years it has become increasingly apparent that apoptosis plays a central role in the differentiation of T cells in the thymus (1,2). Random rearrangement of DNA encoding the receptor for foreign antigens is followed by two types of thymocyte selection. Positive selection, apparently of thymocytes with moderate affinity for thymic stromal elements, rescues them from death by apoptosis and yields a population that is self-MHC restricted (25). Negative selection is thought to occur in thymocytes with high affinity for the thymic stroma and eliminates, by apoptosis, cells with the potential to react against their host (2,3,6,7). Apoptosis by negative selection and apoptosis by lack of positive selection, both in double-positive (DP) thymocytes, have been detected in separate sites in the thymus (2,3). Negative selection has been detected most often (but not exclusively) in association with thymic antigen-presenting cells in the medulla or at the cortical/medullary border (2,7) and the role of accessory signals in this process has also been investigated (1,2,8). Elegant organ culture experiments point to cortical epithelial cells as responsible for positive selection (9). In vivo, apoptotic thymocytes found in the cortex of normal mice [usually engulfed by macrophages (3)] have been classified as primarily related to the default for positive selection (13).

While information on the apoptotic process has been increasing exponentially, we still have much to learn about the thymic environment in which apoptosis occurs. The final stage of apoptosis, the clearance mechanism by which dead cells are removed rapidly, has only recently come under attention. The importance of this process has been recognized, for example, with the suggestion that autoimmune disease could result from impaired clearance of apoptotic cells (10) or that tolerance could be induced (extrathymically) by presentation of antigens from internalized apoptotic cells (11,12). The current investigation was conducted to assess activity of epithelial cells in thymocyte apoptosis. The results of these in vitro studies indicate that clearance of apoptotic thymocytes can occur in cortical epithelial cells and involves DEC-205, for the first time pointing to a role for this receptor in thymic epithelial cells.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells
The BA/10 line and the BA/2 line, cloned independently from C57BL fetal thymus (13,14), were maintained in Iscove’s DMEM with 10% FCS (Biological Industries, Kibbutz Beit Haemek) as described previously (13). Thymocyte suspensions were prepared from 1- to 2-month-old C57BL/6 mice which were maintained (as approved) in the animal colony of the Sackler Faculty of Medicine, by pressing thymus tissue through a sterile wire mesh. In a few cases thymocytes were taken from Fas-deficient B6. MRL-Faslpr mice obtained from Jackson Laboratories (Bar Harbor, ME). Approximately 1.5 x 106 BA/10 or BA/2 stromal cells in 35-mm Falcon dishes were overlayed with thymocytes (usually 100 x 106) in Iscove’s DMEM + 10% FCS and incubated in a 37°C incubator with 10% CO2. Of the times tested, optimum incubation was 6 h, but bound apoptotic thymocytes were also seen after overnight incubation. Each dish was then rinsed 10 times with 1-ml aliquots of warm PBS (containing calcium and magnesium). Calcium and magnesium-free balanced salt solution with 1% BSA was added for 1 h, and the majority of the thymocytes detached easily with gentle agitation. Fresh balanced salt solution/BSA was added, the stromal cells detached from the dish by pipettation and four drops were centrifuged in a Shandon cytospin. After brief drying, the slides were fixed in methanol, stained with Giemsa stain (BDH Chemicals, Poole, UK) and 100 stromal cells scored microscopically on each of two slides. Results are presented as the percentage of stromal cells with bound apoptotic thymocytes both in order to focus on the stromal cells responding and because the number of thymocytes bound per stromal cell did not vary in experiments with total thymocytes. A stromal cell was considered positive when one or more thymocytes were bound.

Reagents
NLDC-145/anti-DEC-205 antibody was prepared as described (15) and used as culture supernatant or Protein G purified from Ig-free supernatant. 6D2 antibody binds a different epitope on the NLDC-145 antigen (16) and was used as culture supernatant. The FITC–F(ab')2 fragment of goat anti-rat IgG was obtained from Jackson ImmunoResearch (West Grove, PA). CDR-1 ammonium sulfate-purified antibody was provided by Robert Rouse (Stanford University Medical Center) (17,18). Biotinylated anti-rat antibody, Vector ABC peroxidase reagents and 3-amino-9-ethylcarbazole (AEC) were from Vector (Burlingame, CA). Anti-active caspase-3 (clone C92-605), FITC–anti-mouse Thy-1.2 (CD 90.2/53-2.1), FITC–anti-mouse TCR ß chain (H57-597), FITC–anti-mouse CD8a (53-6.7) and phycoerythrin-conjugated anti-mouse CD4 (GK 1.5) were obtained from PharMingen (San Diego CA). Peroxidase-labeled anti-rabbit serum was from Dako (Santa Barbara, CA). Recombinant IFN was from Schering (Bloomfield, NJ) and used at 104 U/ml. Dexamethasone was from Sigma (St Louis, MO). To check for DNA fragmentation by in situ staining (19), we used the ApopTag detection kit from Oncor (Gaithersburg, MD). The procedure as outlined in the kit included fixation of thymocytes and BA/10 cells (on cytospin slides) with neutral buffered formalin, addition of digoxigenin nucleotide to fragmented DNA by means of terminal deoxynucleotide transferase, followed by treatment with peroxidase-labeled anti-digoxigenin, which was then revealed with diaminobenzidine and peroxide.

Histochemical staining
Active caspase-3
Cytospin slides were fixed with cold acetone, treated briefly with H2O2 and 10% normal rabbit serum before anti-active caspase-3 or an irrelevant rabbit antibody. This was followed by peroxidase-labeled anti-rabbit serum and diaminobenzidine.

CDR-1
Cytospin slides fixed with cold acetone were stained with CDR-1 antibody followed by biotinylated anti-rat antibody, the Vector ABC peroxidase reagents and AEC.

FACS analysis
The FACS of the Sackler Faculty of Medicine was used as described previously (13). This is a three-channel FACSort from Becton Dickinson (Sunnyvale CA), equipped with CellQuest software. The FACS was run at standard settings and fine tuning of compensation was done daily with control samples. Viable cells excluded 1 µg/ml propidium iodide. Occasionally cells were fixed with 0.37% formaldehyde and 1.25 x PBS. WinMDI software was used for data presentation.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
When freshly isolated thymocytes were incubated for 6 h with BA/10 or BA/2 stromal cells, CD4+/CD8+ thymocytes were bound to both types of stromal cells. In the absence of calcium and magnesium most thymocytes readily detached. In the case of the BA/10 clone, condensed cells with the classical morphology of apoptotic cells remained tightly bound (Fig. 1A). At higher magnification (Fig. 1F), in addition to cell shrinkage, blebbing was sometimes seen. Typically, one to 15 condensed thymocytes were bound to a stromal cell (Fig. 1A) and, as is shown in Fig. 2, condensed thymocytes were usually found on 60–70% of the BA/10 stromal cells. In comparison, with the BA/2 clone apoptotic thymocytes were tightly bound to only 10–14% of the stroma (Fig. 2). In order to characterize this apoptotic process we proceeded to check (i) additional evidence that the condensed cells are apoptotic, (ii) characteristics of the thymocytes involved and (iii) characteristics of the BA/10 stroma in comparison to the BA/2 clone



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Fig. 1. Apoptotic thymocytes bound to BA/10 stromal cells. (A and F) Giemsa staining of cytospin slides after 6 h of incubation. The bound apoptotic cells (enclosed in boxes) are smaller and stain darker than viable thymocytes (which are seen at the edge of the stromal cell). (B and C) Detection of bound apoptotic thymocytes by staining of anti-active caspase-3 (after overnight incubation with BA/10 stroma): (B) shows peroxidase-labeled thymocytes treated with anti-active caspase-3 (arrows), while (C) shows no staining when an irrelevant primary antibody was used. (D and E) Detection of bound apoptotic thymocytes with an in situ assay of DNA fragmentation which was detected by addition of digoxigenin nucleotide, peroxidase-labeled anti-digoxigenin and diaminobenzidine plus peroxide: (D) shows peroxidase-labeled thymocytes, while (E) shows thymocytes stained in parallel. Magnification: (A)–(C), x40; (D)–(F), x100.

 


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Fig. 2. Percent stromal cells binding morphologically apoptotic thymocytes. Mean values and SD shown. Treatment with antibodies diluted 1:20 in culture medium was for 1 or 2 h at 4°C. Antibodies were decanted and the thymocytes added in fresh culture medium for 6 h at 37°C. pNLDC-145 was purified antibody diluted to 50 µg/ml. *P < 0.01 in comparison to untreated controls by t-test.

 
Although apoptosis was originally defined morphologically (20), additional evidence for apoptosis was obtained from immunohistochemical demonstration of active caspase-3 and from the presence of fragmented DNA. Caspase-3 is activated in cells undergoing apoptosis (21). Active caspase-3 was demonstrated in many of the condensed thymocytes and Fig. 1(B) shows two of these darkly stained cells on a background of BA/10 cells. Another characteristic of apoptosis which can be detected in situ (19) is DNA fragmentation. When this indicator of apoptosis was checked, positive cells were found, although in smaller numbers (Fig. 1D). These two additional criteria confirm our identification of the condensed thymocytes as apoptotic.

The possibility that this type of apoptosis involves Fas–Fas ligand was checked by testing thymocytes from B6.MRL-Faslpr mice. The results did not differ from those obtained with normal mice (not shown), thus indicating lack of involvement of Fas–Fas ligand in the process under study.

Which thymocytes are involved in the apoptosis observed here? As an order of magnitude, 1–5% of total thymocytes were tightly bound to the BA/10 clone. Since they were tightly bound and dead as well, an indirect approach was taken to determine their phenotype. Because apoptotic thymocytes were not bound to the majority of the BA/2 stroma (Fig. 2), we incubated thymocytes on BA/2 overnight and transferred reversibly bound cells to BA/10 to see whether this population includes the cells that bind tightly to BA/10. Table 1 shows that this was the case and we used this selection event to delineate the phenotype of the thymocytes by FACS analysis. To verify that the apoptotic cells are in fact thymocytes, we first checked the cells released from BA/2 with anti-Thy-1 antibody. In the curve shown in Fig. 3(A), 99% of the released cells stained with anti-Thy-1. High levels of Thy-1 are characteristic of immature thymocytes (22,23). Staining for CD4 and CD8 is shown in Fig. 3(B). Thymocytes released from BA/2 were 96% CD4/CD8 DP. This profile may be compared to results published previously (13) showing 98% DP released from BA/10 stroma, while total thymocytes have 70–80% DP. In Fig. 3(C), staining for {alpha}ß TCR in thymocytes released from BA/2 is compared to staining of total thymocytes. The released thymocytes were TCRlo/med (i.e. immature). These profiles indicate that the apoptotic thymocytes are early DP thymocytes.


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Table 1. Transfer to BA/10 stromal cells of thymocytes bound reversibly during overnight incubation with BA/2 or BA/10
 


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Fig. 3. Antigenic characteristics of thymocytes released from BA/2 stroma after overnight incubation. These profiles were gated on FSC/SSC of thymocytes alone. (A) FITC–Thy-1 staining of released thymocytes (heavy line, right hand peak) is shown on a background of unstained thymocytes (dashed line, left hand peak) and total thymocytes stained with anti-Thy-1 (dotted line, striped double peak). (B) The CD4/CD8 profile of released thymocytes is shown with quadrants determined by staining of total thymocytes. (C) FITC–{alpha}ß TCR staining of released thymocytes (heavy line, open peak) is shown on a background of {alpha}ß TCR staining of total thymocytes (filled, triple peak).

 
How do these two stromal clones differ? The BA/10 stromal cells can be classified as cortical epithelial cells because they were induced to express the cortical epithelial antigen CDR-1 (17) after treatment with IFN-{gamma} and IL-2 (18), as shown in Fig 4(A), while such staining was not seen with the BA/2 clone. In addition, BA/10 cells constitutively express the DEC-205 receptor (Fig. 5), which is also expressed on cortical thymic epithelial cells in vivo (15), while it was at background levels on the BA/2 cell line (Fig. 5). This prompted us to look for a role for DEC-205 in the interaction between apoptotic thymocytes and BA/10 cells. DEC-205 is involved in endocytosis and has been characterized as a pattern recognition molecule (24,25). We analyzed its activity in the BA/10 cells by pre-incubating BA/10 cells with either hybridoma supernatant or purified NLDC-145 antibody against DEC-205. A strong reduction in the number of stromal cells with bound apoptotic thymocytes was found (Fig. 2). Parallel treatment with antibody 6D2, prepared to a different epitope on the NLDC-145 antigen (16), had no effect (Fig. 2). Thus DEC-205, through its epitope recognized by NLDC-145, is involved at some stage in this apoptotic process. While the stromal cells that bind apoptotic thymocytes were found to be cortical epithelial cells which express DEC-205, these characteristics were not found with the BA/2 cells (Table 2).



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Fig. 4. Characterization of BA/10 cells as cortical epithelium. (A) After growth with IFN-{gamma} and IL-2, cytospin slides of BA/10 cells fixed with cold acetone were stained with purified CDR-1, biotinylated anti-rat IgG, Vector ABC reagents for peroxidase staining and AEC as substrate. (B) The same cells were treated without the primary antibody.

 


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Fig. 5. FACS profile of NLDC-145 staining. Viable BA/10 cells were stained with NLDC-145 supernatant and FITC-conjugated goat anti-rat IgG (gray right-hand peak), while control cells were stained with the second antibody only (white left-hand peak). Viable BA/2 cells were stained with NLDC-145 supernatant and FITC–goat anti-rat IgG (heavy line gray-left hand peak).

 

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Table 2. Differential characteristics of the stromal lines studied
 
In an attempt to delineate whether killing or clearance of apoptotic cells occurred here, thymocytes were treated for 4 h with 10–6 M dexamethasone to induce apoptosis and compared to untreated thymocytes. After dexamethasone treatment, instead of <=15 thymocytes bound per BA/10 cell as in all the experiments described so far, >=40 bound cells could be seen as shown in Fig. 6. Attachment of increased numbers of dying cells to the BA/10 stroma is evidence both for preferential attachment of dead cells and for clearance of these cells by the stromal epithelium. Additional evidence compatible with dead cell clearance by the BA/10 stroma is found in Table 1 where primary incubation of total thymocytes on the BA/10 cells appeared to remove most of the apoptotic cells.



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Fig. 6. Binding of apoptotic dexamethasone-treated thymocytes to BA/10 cells. Giemsa stained cytospin slides show (A) binding to BA/10 cells of thymocytes pretreated for 4 h with 10–6 M dexamethasone, (B) viable thymocytes at the same magnification for comparison of size and (C) binding to BA/10 cells of untreated thymocytes for comparison of number.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In these experiments, thymic cortical epithelial cells were found to participate in thymocyte apoptosis with the involvement of the DEC-205 receptor. Previous evidence for activity of cortical epithelial cells in thymocyte apoptosis was scant. While apoptotic thymocytes had been found scattered throughout the cortex, these cells seemed to be within macrophages (3). At least three groups have reported that apoptosis of thymocytes in suspension could be induced by thymic epithelial cells in vitro (2628). However, this contact was generally lethal, while with the BA/10 clone thymocytes that bind reversibly can mature to CD4+ or CD8+ cells after 2 days of culture on primary thymic stroma (M. Small, unpublished results). Apoptotic thymocytes have also been found within thymic nurse cells (2931). Two of these investigations reported that the apoptosis was induced elsewhere and the apoptotic cells engulfed for clearance by the nurse cells (29,30). In the third of these reports (31), viable thymocytes entered into thymic nurse cells and apoptotic thymocytes remained internalized. One question remaining open for further experimentation is a more defined identity for the BA/10 cells, which awaits the availability of appropriate antibodies. Such antibodies will also allow us to determine the relative frequency of BA/10-like cells in the fetal and adult thymus.

Staining profiles of the cells studied here indicate that they are early DP thymocytes in terms of Thy-1, CD4, CD8 and {alpha}ß TCR levels, and they represent 1–5% of total thymocytes.

The DEC-205 surface protein on the epithelial cells seems to participate in the binding of apoptotic (but not viable) thymocytes. (i) The NLDC-145 antibody was able to interfere with this binding and (ii) the BA/2 line, which shows only marginal binding of apoptotic cells, does not express the DEC-205 protein. The DEC-205 antigen is abundant in the thymic cortex as well as a characteristic of one class of dendritic cells (15) and we might ask if these cell types have some common function. In recent years there have been considerable advances in clarifying the pathways of interaction between dendritic cells and mature T lymphocytes. Indeed a recent publication implicates dendritic cells in induction of unresponsiveness in peripheral T cells (32). DEC-205 has been shown to function in endocytosis from coated pits to a late endosomal compartment (24,33). This receptor was shown to be highly efficient in antigen presentation for both reactivity and unresponsiveness (24,32,33). Endocytosis and antigen presentation of apoptotic cells within dendritic cells have also been described with the involvement of other receptors (34,35). Recently, the ability of dendritic cells to endocytose dying cells has been pinpointed to the CD8+ subset (11). Interestingly, such dendritic cells are also known to express DEC-205, although dendritic cells from DEC-205 knockout mice were still able to ingest dead splenocytes (11). However, clearance systems are known for redundancy of receptors (10). Here we show for the first time a role for DEC-205 in thymic cortical epithelial cells. Since DEC-205 is a surface protein (Fig. 5) and in dendritic cells has been linked with endocytosis (24,33), we hypothesize that this receptor is the first site of interaction between the BA/10 cells and the thymocytes which will eventually be tightly bound. The considerable increase in numbers of apoptotic thymocytes bound per stromal cell after dexamethasone treatment of the thymocytes implicates the BA/10 cells in ingestion and clearance of dead or dying cells. Recent investigations have begun to show the importance and the complexities of this clearance phase (1012,2931,34). When the stromal clones were checked with an antibody against corticosterone there was strong staining of this steroid in the BA/10 cells, but not the BA/2 cells, and both an inhibitor of glucocorticoid synthesis and a blocker of the glucocorticoid receptor clearly reduced the number of stromal cells with bound apoptotic thymocytes. These results (not shown) suggest a role for locally synthesized corticosterone, but at the present time the precise role is unclear.

It does seem clear, however, that the final outcome of this process is clearance of apoptotic thymocytes by cortical epithelial cells with the participation of DEC-205 expressed on the cortical epithelial cells. Sixteen years after publication of the finding that NLDC-145 antigen is found on cortical epithelial cells of the thymus (15), we present in vitro evidence of a function for the protein in this location. Further work will show to what extent this also occurs in vivo.


    Acknowledgements
 
We thank Itzhak Oschry for skilled FACS operation, Reina Mebius for assistance with antibody production and Eli Golumb for generously sharing ApopTag reagents.


    Abbreviations
 
AEC—3-amino-9-ethylcarbazole

DP—double positive


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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