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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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T cell–specific KO of Fas. (a) Targeting scheme. (1) Genomic structure of the fas locus surrounding exon IX. (2) Targeting vector construction. The chromosomal locus after homologous recombination is shown in 3. Cre-mediated deletion produces the fas floxed (4, fasfl) and deleted alleles (5, fasdel). (triangles) loxP sites. Ev, EcoRV; S, SphI; C, ClaI restriction site. (b) Southern blot analysis on DNA from wild-type embryonic stem cells (Bruce-4) and homologous recombinants (clones 117 and 173 and neor deleted clones 117#5 and 173#5). (c) T cell–specific deletion of Fas. T, thymocytes; B, purified splenic B cells (CD19+); XTB, purified nonlymphocyte population. Deletion efficiencies are indicated. (d) FACS® analysis showing loss of Fas expression in thymocytes of fasfl/fl, CD4-cre mice.
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fig1: T cell–specific KO of Fas. (a) Targeting scheme. (1) Genomic structure of the fas locus surrounding exon IX. (2) Targeting vector construction. The chromosomal locus after homologous recombination is shown in 3. Cre-mediated deletion produces the fas floxed (4, fasfl) and deleted alleles (5, fasdel). (triangles) loxP sites. Ev, EcoRV; S, SphI; C, ClaI restriction site. (b) Southern blot analysis on DNA from wild-type embryonic stem cells (Bruce-4) and homologous recombinants (clones 117 and 173 and neor deleted clones 117#5 and 173#5). (c) T cell–specific deletion of Fas. T, thymocytes; B, purified splenic B cells (CD19+); XTB, purified nonlymphocyte population. Deletion efficiencies are indicated. (d) FACS® analysis showing loss of Fas expression in thymocytes of fasfl/fl, CD4-cre mice.

Mentions: A conditional fas allele (fasfl) in which the death domain–encoding exon IX flanked by lox/P-sites (Fig. 1, a and b) was generated to allow conditional inactivation of Fas through cell type–specific expression of Cre recombinase. Fasfl/fl mice showed normal lymphocyte development and subset distribution as determined by FACS® analysis (unpublished data). Deletion of the loxP-flanked (floxed) fas sequence through crossing to the deleter strain (24) produced mice carrying an inactive fas allele (fasdel) in all cells of the body. As expected, fasdel/del mice developed the typical lpr phenotype (unpublished data). To delete Fas selectively in T cells, fasfl/fl mice were crossed to CD4-cre transgenic mice in which the cre coding sequence is under the control of a CD4 minigene (19). Southern blot analysis of DNA isolated from thymus and various cell populations in the spleen of fasfl/fl, CD4-cre mice demonstrated efficient Cre-mediated recombination in T cells (Fig. 1 c). In the nonlymphocyte population of the spleen, a band characteristic for the fasdel allele was also detectable, but its intensity (16% of that representing the fasfl allele) corresponded to the fraction of contaminating T cells (17%). The absence of Fas expression in thymocytes of the compound mutants was also confirmed by FACS® analysis at the protein level (Fig. 1 d). Similarly, efficient Cre-mediated recombination in B cells, and both T and B cells was observed in fasfl/fl, CD19-cre and fasfl/fl, CD4-cre/CD19-cre mice, respectively (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)

T cell–specific KO of Fas. (a) Targeting scheme. (1) Genomic structure of the fas locus surrounding exon IX. (2) Targeting vector construction. The chromosomal locus after homologous recombination is shown in 3. Cre-mediated deletion produces the fas floxed (4, fasfl) and deleted alleles (5, fasdel). (triangles) loxP sites. Ev, EcoRV; S, SphI; C, ClaI restriction site. (b) Southern blot analysis on DNA from wild-type embryonic stem cells (Bruce-4) and homologous recombinants (clones 117 and 173 and neor deleted clones 117#5 and 173#5). (c) T cell–specific deletion of Fas. T, thymocytes; B, purified splenic B cells (CD19+); XTB, purified nonlymphocyte population. Deletion efficiencies are indicated. (d) FACS® analysis showing loss of Fas expression in thymocytes of fasfl/fl, CD4-cre mice.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211818&req=5

fig1: T cell–specific KO of Fas. (a) Targeting scheme. (1) Genomic structure of the fas locus surrounding exon IX. (2) Targeting vector construction. The chromosomal locus after homologous recombination is shown in 3. Cre-mediated deletion produces the fas floxed (4, fasfl) and deleted alleles (5, fasdel). (triangles) loxP sites. Ev, EcoRV; S, SphI; C, ClaI restriction site. (b) Southern blot analysis on DNA from wild-type embryonic stem cells (Bruce-4) and homologous recombinants (clones 117 and 173 and neor deleted clones 117#5 and 173#5). (c) T cell–specific deletion of Fas. T, thymocytes; B, purified splenic B cells (CD19+); XTB, purified nonlymphocyte population. Deletion efficiencies are indicated. (d) FACS® analysis showing loss of Fas expression in thymocytes of fasfl/fl, CD4-cre mice.
Mentions: A conditional fas allele (fasfl) in which the death domain–encoding exon IX flanked by lox/P-sites (Fig. 1, a and b) was generated to allow conditional inactivation of Fas through cell type–specific expression of Cre recombinase. Fasfl/fl mice showed normal lymphocyte development and subset distribution as determined by FACS® analysis (unpublished data). Deletion of the loxP-flanked (floxed) fas sequence through crossing to the deleter strain (24) produced mice carrying an inactive fas allele (fasdel) in all cells of the body. As expected, fasdel/del mice developed the typical lpr phenotype (unpublished data). To delete Fas selectively in T cells, fasfl/fl mice were crossed to CD4-cre transgenic mice in which the cre coding sequence is under the control of a CD4 minigene (19). Southern blot analysis of DNA isolated from thymus and various cell populations in the spleen of fasfl/fl, CD4-cre mice demonstrated efficient Cre-mediated recombination in T cells (Fig. 1 c). In the nonlymphocyte population of the spleen, a band characteristic for the fasdel allele was also detectable, but its intensity (16% of that representing the fasfl allele) corresponded to the fraction of contaminating T cells (17%). The absence of Fas expression in thymocytes of the compound mutants was also confirmed by FACS® analysis at the protein level (Fig. 1 d). Similarly, efficient Cre-mediated recombination in B cells, and both T and B cells was observed in fasfl/fl, CD19-cre and fasfl/fl, CD4-cre/CD19-cre mice, respectively (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