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Thymus-derived glucocorticoids regulate antigen-specific positive selection.

Vacchio MS, Ashwell JD - J. Exp. Med. (1997)

Bottom Line: We have previously demonstrated that glucocorticoids are produced in the thymus, and that they antagonize deletion caused by TCR cross-linking.Inhibition of glucocorticoid production in thymi from alpha/beta-TCR transgenic mice resulted in the antigen- and MHC-specific loss of thymocytes that normally recognize self antigen/MHC with sufficient avidity to result in positive selection.These results indicate that the balance of TCR and glucocorticoid receptor signaling influences the antigen-specific thymocyte development by allowing cells with low-to-moderate avidity for self antigen/MHC to survive.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology, Division of Hematologic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20852, USA.

ABSTRACT
While it is generally believed that the avidity of the T cell antigen receptor (TCR) for self antigen/major histocompatibility complex (MHC) determines a thymocyte's fate, how the cell discriminates between a stimulus that causes positive selection (survival) and one that causes negative selection (death) is unknown. We have previously demonstrated that glucocorticoids are produced in the thymus, and that they antagonize deletion caused by TCR cross-linking. To examine the role of glucocorticoids during MHC-dependent selection, we examined thymocyte development in organ cultures in which corticosteroid biosynthesis was inhibited. Inhibition of glucocorticoid production in thymi from alpha/beta-TCR transgenic mice resulted in the antigen- and MHC-specific loss of thymocytes that normally recognize self antigen/MHC with sufficient avidity to result in positive selection. Furthermore, inhibition of glucocorticoid production caused an increase in apoptosis only in CD+CD8(+) thymocytes bearing transgenic TCRs that recognized self antigen/MHC. These results indicate that the balance of TCR and glucocorticoid receptor signaling influences the antigen-specific thymocyte development by allowing cells with low-to-moderate avidity for self antigen/MHC to survive.

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Modified TUNEL  assay for thymocyte apoptosis.  Thymi from 1-2-d-old female  RAG-2−/− H-Y/Db-specific  TCR transgenic mice homozygous for either H-2d or H-2b  were separated into individual  lobes and cultured in medium  alone or with 200 μg/ml of  freshly diluted metyrapone. After  18 h, thymocytes were analyzed  for expression of CD4 and CD8  and evaluated for apoptosis on  the FACScan® using a modified  TUNEL assay to detect nicked  DNA. To determine the amount  of apoptosis in the DP population, thymocytes were gated for  expression of both CD4 and  CD8 and analyzed for dUTPFITC incorporation.
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Figure 3: Modified TUNEL assay for thymocyte apoptosis. Thymi from 1-2-d-old female RAG-2−/− H-Y/Db-specific TCR transgenic mice homozygous for either H-2d or H-2b were separated into individual lobes and cultured in medium alone or with 200 μg/ml of freshly diluted metyrapone. After 18 h, thymocytes were analyzed for expression of CD4 and CD8 and evaluated for apoptosis on the FACScan® using a modified TUNEL assay to detect nicked DNA. To determine the amount of apoptosis in the DP population, thymocytes were gated for expression of both CD4 and CD8 and analyzed for dUTPFITC incorporation.

Mentions: Antigen-specific thymocyte deletion is due to the induction of apoptotic cell death (17, 18). Therefore, experiments were performed to determine if the decrease in thymocyte number when corticosteroid production was inhibited was due to apoptosis of DP thymocytes. Cells undergoing apoptotic death acquire single- and double-strand DNA breaks, which can be detected by techniques in which labeled oligonucleotides are incorporated into the damaged DNA. We have used a modified version of the original TUNEL assay in which harvested thymocytes are stained with antibodies to cell surface molecules such as CD4 and CD8, permeabilized, and incubated with DNA polymerase I, allowing the incorporation of fluoresceinated dUTP into nicked DNA. The cells are analyzed by flow cytometry and the number of TUNELpositive cells quantitated. A representative example of profiles of thymocytes from a RAG-2−/− H-2b or a RAG2−/− H-2d thymus cultured in the absence or presence of metyrapone for 24 h is shown in Fig. 3. In the nonselecting H-2d haplotype, the background level of spontaneous cell death was unaffected by the addition of metyrapone. In contrast, metyrapone caused a substantial increase in DP TUNEL-positive cells compared to medium alone in DP thymocytes of the H-2b haplotype. When the data from multiple experiments were averaged (Fig. 4), it was found that there was little if any increase in spontaneous apoptosis in DP cells when thymi from RAG-2−/− H-2d mice expressing the H-Y/H-Db-specific TCR were cultured with the corticosteroid-synthesis inhibitor. In contrast, in thymi of H-2b littermates there was an induction of almost 20% specific apoptosis. Therefore, inhibition of local corticosteroid production in the thymus specifically causes the apoptotic death and deletion of DP cells only when their TCRs recognize self.


Thymus-derived glucocorticoids regulate antigen-specific positive selection.

Vacchio MS, Ashwell JD - J. Exp. Med. (1997)

Modified TUNEL  assay for thymocyte apoptosis.  Thymi from 1-2-d-old female  RAG-2−/− H-Y/Db-specific  TCR transgenic mice homozygous for either H-2d or H-2b  were separated into individual  lobes and cultured in medium  alone or with 200 μg/ml of  freshly diluted metyrapone. After  18 h, thymocytes were analyzed  for expression of CD4 and CD8  and evaluated for apoptosis on  the FACScan® using a modified  TUNEL assay to detect nicked  DNA. To determine the amount  of apoptosis in the DP population, thymocytes were gated for  expression of both CD4 and  CD8 and analyzed for dUTPFITC incorporation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196338&req=5

Figure 3: Modified TUNEL assay for thymocyte apoptosis. Thymi from 1-2-d-old female RAG-2−/− H-Y/Db-specific TCR transgenic mice homozygous for either H-2d or H-2b were separated into individual lobes and cultured in medium alone or with 200 μg/ml of freshly diluted metyrapone. After 18 h, thymocytes were analyzed for expression of CD4 and CD8 and evaluated for apoptosis on the FACScan® using a modified TUNEL assay to detect nicked DNA. To determine the amount of apoptosis in the DP population, thymocytes were gated for expression of both CD4 and CD8 and analyzed for dUTPFITC incorporation.
Mentions: Antigen-specific thymocyte deletion is due to the induction of apoptotic cell death (17, 18). Therefore, experiments were performed to determine if the decrease in thymocyte number when corticosteroid production was inhibited was due to apoptosis of DP thymocytes. Cells undergoing apoptotic death acquire single- and double-strand DNA breaks, which can be detected by techniques in which labeled oligonucleotides are incorporated into the damaged DNA. We have used a modified version of the original TUNEL assay in which harvested thymocytes are stained with antibodies to cell surface molecules such as CD4 and CD8, permeabilized, and incubated with DNA polymerase I, allowing the incorporation of fluoresceinated dUTP into nicked DNA. The cells are analyzed by flow cytometry and the number of TUNELpositive cells quantitated. A representative example of profiles of thymocytes from a RAG-2−/− H-2b or a RAG2−/− H-2d thymus cultured in the absence or presence of metyrapone for 24 h is shown in Fig. 3. In the nonselecting H-2d haplotype, the background level of spontaneous cell death was unaffected by the addition of metyrapone. In contrast, metyrapone caused a substantial increase in DP TUNEL-positive cells compared to medium alone in DP thymocytes of the H-2b haplotype. When the data from multiple experiments were averaged (Fig. 4), it was found that there was little if any increase in spontaneous apoptosis in DP cells when thymi from RAG-2−/− H-2d mice expressing the H-Y/H-Db-specific TCR were cultured with the corticosteroid-synthesis inhibitor. In contrast, in thymi of H-2b littermates there was an induction of almost 20% specific apoptosis. Therefore, inhibition of local corticosteroid production in the thymus specifically causes the apoptotic death and deletion of DP cells only when their TCRs recognize self.

Bottom Line: We have previously demonstrated that glucocorticoids are produced in the thymus, and that they antagonize deletion caused by TCR cross-linking.Inhibition of glucocorticoid production in thymi from alpha/beta-TCR transgenic mice resulted in the antigen- and MHC-specific loss of thymocytes that normally recognize self antigen/MHC with sufficient avidity to result in positive selection.These results indicate that the balance of TCR and glucocorticoid receptor signaling influences the antigen-specific thymocyte development by allowing cells with low-to-moderate avidity for self antigen/MHC to survive.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology, Division of Hematologic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20852, USA.

ABSTRACT
While it is generally believed that the avidity of the T cell antigen receptor (TCR) for self antigen/major histocompatibility complex (MHC) determines a thymocyte's fate, how the cell discriminates between a stimulus that causes positive selection (survival) and one that causes negative selection (death) is unknown. We have previously demonstrated that glucocorticoids are produced in the thymus, and that they antagonize deletion caused by TCR cross-linking. To examine the role of glucocorticoids during MHC-dependent selection, we examined thymocyte development in organ cultures in which corticosteroid biosynthesis was inhibited. Inhibition of glucocorticoid production in thymi from alpha/beta-TCR transgenic mice resulted in the antigen- and MHC-specific loss of thymocytes that normally recognize self antigen/MHC with sufficient avidity to result in positive selection. Furthermore, inhibition of glucocorticoid production caused an increase in apoptosis only in CD+CD8(+) thymocytes bearing transgenic TCRs that recognized self antigen/MHC. These results indicate that the balance of TCR and glucocorticoid receptor signaling influences the antigen-specific thymocyte development by allowing cells with low-to-moderate avidity for self antigen/MHC to survive.

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