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T cell-specific ablation of Fas leads to Fas ligand-mediated lymphocyte depletion and inflammatory pulmonary fibrosis.

Hao Z, Hampel B, Yagita H, Rajewsky K - J. Exp. Med. (2004)

Bottom Line: Experiments in which Fas was ablated in T cells, B cells, T and B cells, or in a more generalized manner demonstrated that the development of lymphoproliferative disease as seen in Fas-deficient mice requires Fas ablation in lymphoid and nonlymphoid tissues.In addition, the mutant animals developed a fatal wasting syndrome caused by massive leukocyte infiltration in the lungs together with increased inflammatory cytokine production and pulmonary fibrosis.Thus, FasL-mediated interaction of activated, Fas-deficient T cells with Fas-expressing cells in their environment leads to break down of lymphocyte homeostasis and development of a lung disease strikingly resembling idiopathic pulmonary fibrosis in humans, a common and severe disease for which the mutant mice may serve as a first animal model.

View Article: PubMed Central - PubMed

Affiliation: 620 University Ave., Toronto, ON, M5G 2C1, Canada. zyhao@uhnres.utoronto.ca

ABSTRACT
To study the role of Fas-Fas ligand (FasL) interaction-mediated apoptosis in lymphocyte homeostasis, we generated a mutant fas allele allowing conditional inactivation of the fas gene through Cre-mediated recombination. Experiments in which Fas was ablated in T cells, B cells, T and B cells, or in a more generalized manner demonstrated that the development of lymphoproliferative disease as seen in Fas-deficient mice requires Fas ablation in lymphoid and nonlymphoid tissues. Selective inactivation of Fas in T cells led to a severe lymphopenia over time, accompanied by up-regulation of FasL on activated T cells and apoptosis of peripheral lymphocytes. In addition, the mutant animals developed a fatal wasting syndrome caused by massive leukocyte infiltration in the lungs together with increased inflammatory cytokine production and pulmonary fibrosis. Inhibition of Fas-FasL interaction in vivo completely prevented the loss of lymphocytes and initial lymphocyte infiltration in the lungs. Thus, FasL-mediated interaction of activated, Fas-deficient T cells with Fas-expressing cells in their environment leads to break down of lymphocyte homeostasis and development of a lung disease strikingly resembling idiopathic pulmonary fibrosis in humans, a common and severe disease for which the mutant mice may serve as a first animal model.

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Loss of peripheral lymphocytes upon T cell–specific Fas inactivation. (a) Cellularity in the spleen and inguinal LNs. Each circle represents an individual mouse. Lines connect mean values of each group. (b) Immunohistochemical staining of spleens of 5–6-mo-old mice (four mice per group analyzed) with anti-CD3 (red) and anti-CD19 (blue) antibody. (c) Annexin V staining of T and B cells in LNs (four mice per group analyzed) at the age of 16 wk. (d) TUNEL staining of the splenic sections from animals (four mice per group analyzed) at the age of 16 wk. Green and red staining shows apoptotic cells and nuclei, respectively. (b and d) Bars, 200 μm.
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fig3: Loss of peripheral lymphocytes upon T cell–specific Fas inactivation. (a) Cellularity in the spleen and inguinal LNs. Each circle represents an individual mouse. Lines connect mean values of each group. (b) Immunohistochemical staining of spleens of 5–6-mo-old mice (four mice per group analyzed) with anti-CD3 (red) and anti-CD19 (blue) antibody. (c) Annexin V staining of T and B cells in LNs (four mice per group analyzed) at the age of 16 wk. (d) TUNEL staining of the splenic sections from animals (four mice per group analyzed) at the age of 16 wk. Green and red staining shows apoptotic cells and nuclei, respectively. (b and d) Bars, 200 μm.

Mentions: Rather than leading to lymphoproliferation, T cell–specific Fas inactivation resulted in a decline of both T and B cell numbers in the secondary lymphoid organs over time, and finally led to an almost complete lymphopenia in the LNs of the animals (Fig. 3, a and b). The numbers of total cells, T cells, and B cells in the spleens of T cell–specific Fas KO mice were ∼160% of the controls at the age of 2 mo. However, T cell and B cell counts in the mutants declined to ∼60% of the controls at the age of 5 mo, and further to 23% for T cells and 44% for B cells at the age of 7 mo (Fig. 3 a). As with the spleen, cell numbers in the inguinal LNs of the mutants were comparable to control values at the age of 2 mo (Fig. 3 a). However, at 5 mo of age, the inguinal LNs in 6 out of 8 mutant animals analyzed were atrophic and contained <0.1 million cells. At 7 mo, the inguinal LNs in all of four mutants analyzed were almost empty. Similar to the inguinal LNs, age-dependent cell loss was also observed in other LNs, including mesenteric, axillary, and cervical LNs (unpublished data). Enlargement of the spleen (157 ± 27 mg against 85 ± 25 mg in the controls; n = 4) and disruption of splenic architecture characterized by loss of white pulp, sclerosis, and hyalinization became evident in the mutants at the age of 5 mo (Fig. 3 b and not depicted). Flow cytometric analysis showed that 65% of T cells and 64% of B cells in the LNs of 16-wk-old mutants were positive for annexin V, a marker for cells undergoing apoptosis (Fig. 3 c). Similarly, widespread cellular apoptosis was observed in histological sections of the spleens of the mutants, whereas, in the controls, apoptotic (TUNEL positive) cells were rare and exclusively located in the splenic red pulp (Fig. 3 d). These results indicate that cellular apoptosis is the main cause for lymphocyte depletion upon T cell–specific Fas inactivation. A subset of DCs called “myeloid” DCs in mouse spleen is CD4 positive (27) and, thus, it is possible that the fas gene was also deleted in these cells. To clarify this point, we bred the fasfl/fl mice to lck-cre transgenic mice in which Cre recombinase is only expressed in T cells but not DCs (20). Similar to fasfl/fl, CD4-cre mice, the LNs of fasfl/fl, lck-cre mice at the age of 7 mo contained <0.1 million cells, confirming that loss of T and B cells is indeed due to Fas deletion in T cells (unpublished data). The possibility that Cre expression as such is toxic for T cells was excluded by two pieces of evidence as follows: T cell numbers in aged fasfl/+, CD4-cre mice were comparable to those in fasfl/fl controls; and aged fasdel/del; CD4-cre mice had the expected splenomegaly and lymphadenopathy (unpublished data).


T cell-specific ablation of Fas leads to Fas ligand-mediated lymphocyte depletion and inflammatory pulmonary fibrosis.

Hao Z, Hampel B, Yagita H, Rajewsky K - J. Exp. Med. (2004)

Loss of peripheral lymphocytes upon T cell–specific Fas inactivation. (a) Cellularity in the spleen and inguinal LNs. Each circle represents an individual mouse. Lines connect mean values of each group. (b) Immunohistochemical staining of spleens of 5–6-mo-old mice (four mice per group analyzed) with anti-CD3 (red) and anti-CD19 (blue) antibody. (c) Annexin V staining of T and B cells in LNs (four mice per group analyzed) at the age of 16 wk. (d) TUNEL staining of the splenic sections from animals (four mice per group analyzed) at the age of 16 wk. Green and red staining shows apoptotic cells and nuclei, respectively. (b and d) Bars, 200 μm.
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fig3: Loss of peripheral lymphocytes upon T cell–specific Fas inactivation. (a) Cellularity in the spleen and inguinal LNs. Each circle represents an individual mouse. Lines connect mean values of each group. (b) Immunohistochemical staining of spleens of 5–6-mo-old mice (four mice per group analyzed) with anti-CD3 (red) and anti-CD19 (blue) antibody. (c) Annexin V staining of T and B cells in LNs (four mice per group analyzed) at the age of 16 wk. (d) TUNEL staining of the splenic sections from animals (four mice per group analyzed) at the age of 16 wk. Green and red staining shows apoptotic cells and nuclei, respectively. (b and d) Bars, 200 μm.
Mentions: Rather than leading to lymphoproliferation, T cell–specific Fas inactivation resulted in a decline of both T and B cell numbers in the secondary lymphoid organs over time, and finally led to an almost complete lymphopenia in the LNs of the animals (Fig. 3, a and b). The numbers of total cells, T cells, and B cells in the spleens of T cell–specific Fas KO mice were ∼160% of the controls at the age of 2 mo. However, T cell and B cell counts in the mutants declined to ∼60% of the controls at the age of 5 mo, and further to 23% for T cells and 44% for B cells at the age of 7 mo (Fig. 3 a). As with the spleen, cell numbers in the inguinal LNs of the mutants were comparable to control values at the age of 2 mo (Fig. 3 a). However, at 5 mo of age, the inguinal LNs in 6 out of 8 mutant animals analyzed were atrophic and contained <0.1 million cells. At 7 mo, the inguinal LNs in all of four mutants analyzed were almost empty. Similar to the inguinal LNs, age-dependent cell loss was also observed in other LNs, including mesenteric, axillary, and cervical LNs (unpublished data). Enlargement of the spleen (157 ± 27 mg against 85 ± 25 mg in the controls; n = 4) and disruption of splenic architecture characterized by loss of white pulp, sclerosis, and hyalinization became evident in the mutants at the age of 5 mo (Fig. 3 b and not depicted). Flow cytometric analysis showed that 65% of T cells and 64% of B cells in the LNs of 16-wk-old mutants were positive for annexin V, a marker for cells undergoing apoptosis (Fig. 3 c). Similarly, widespread cellular apoptosis was observed in histological sections of the spleens of the mutants, whereas, in the controls, apoptotic (TUNEL positive) cells were rare and exclusively located in the splenic red pulp (Fig. 3 d). These results indicate that cellular apoptosis is the main cause for lymphocyte depletion upon T cell–specific Fas inactivation. A subset of DCs called “myeloid” DCs in mouse spleen is CD4 positive (27) and, thus, it is possible that the fas gene was also deleted in these cells. To clarify this point, we bred the fasfl/fl mice to lck-cre transgenic mice in which Cre recombinase is only expressed in T cells but not DCs (20). Similar to fasfl/fl, CD4-cre mice, the LNs of fasfl/fl, lck-cre mice at the age of 7 mo contained <0.1 million cells, confirming that loss of T and B cells is indeed due to Fas deletion in T cells (unpublished data). The possibility that Cre expression as such is toxic for T cells was excluded by two pieces of evidence as follows: T cell numbers in aged fasfl/+, CD4-cre mice were comparable to those in fasfl/fl controls; and aged fasdel/del; CD4-cre mice had the expected splenomegaly and lymphadenopathy (unpublished data).

Bottom Line: Experiments in which Fas was ablated in T cells, B cells, T and B cells, or in a more generalized manner demonstrated that the development of lymphoproliferative disease as seen in Fas-deficient mice requires Fas ablation in lymphoid and nonlymphoid tissues.In addition, the mutant animals developed a fatal wasting syndrome caused by massive leukocyte infiltration in the lungs together with increased inflammatory cytokine production and pulmonary fibrosis.Thus, FasL-mediated interaction of activated, Fas-deficient T cells with Fas-expressing cells in their environment leads to break down of lymphocyte homeostasis and development of a lung disease strikingly resembling idiopathic pulmonary fibrosis in humans, a common and severe disease for which the mutant mice may serve as a first animal model.

View Article: PubMed Central - PubMed

Affiliation: 620 University Ave., Toronto, ON, M5G 2C1, Canada. zyhao@uhnres.utoronto.ca

ABSTRACT
To study the role of Fas-Fas ligand (FasL) interaction-mediated apoptosis in lymphocyte homeostasis, we generated a mutant fas allele allowing conditional inactivation of the fas gene through Cre-mediated recombination. Experiments in which Fas was ablated in T cells, B cells, T and B cells, or in a more generalized manner demonstrated that the development of lymphoproliferative disease as seen in Fas-deficient mice requires Fas ablation in lymphoid and nonlymphoid tissues. Selective inactivation of Fas in T cells led to a severe lymphopenia over time, accompanied by up-regulation of FasL on activated T cells and apoptosis of peripheral lymphocytes. In addition, the mutant animals developed a fatal wasting syndrome caused by massive leukocyte infiltration in the lungs together with increased inflammatory cytokine production and pulmonary fibrosis. Inhibition of Fas-FasL interaction in vivo completely prevented the loss of lymphocytes and initial lymphocyte infiltration in the lungs. Thus, FasL-mediated interaction of activated, Fas-deficient T cells with Fas-expressing cells in their environment leads to break down of lymphocyte homeostasis and development of a lung disease strikingly resembling idiopathic pulmonary fibrosis in humans, a common and severe disease for which the mutant mice may serve as a first animal model.

Show MeSH
Related in: MedlinePlus