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Antigen receptor-induced activation and cytoskeletal rearrangement are impaired in Wiskott-Aldrich syndrome protein-deficient lymphocytes.

Zhang J, Shehabeldin A, da Cruz LA, Butler J, Somani AK, McGavin M, Kozieradzki I, dos Santos AO, Nagy A, Grinstein S, Penninger JM, Siminovitch KA - J. Exp. Med. (1999)

Bottom Line: In the thymus, this abnormality was associated with impaired progression from the CD44(-)CD25(+) to the CD44(-)CD25(-) stage of differentiation.This defect in TCR signaling was associated with a reduction in TCR-evoked upregulation of the early activation marker CD69 and in TCR-triggered apoptosis.While induction of TCR-zeta, ZAP70, and total protein tyrosine phosphorylation as well as mitogen-activated protein kinase (MAPK) and stress-activated protein/c-Jun NH(2)-terminal kinase (SAPK/JNK) activation appeared normal in TCR-stimulated WAS(-)(/)(-) cells, TCR-evoked increases in intracellular calcium concentration were decreased in WASp-deficient relative to wild-type cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Toronto, Ontario, Canada M5G 1X5.

ABSTRACT
The Wiskott-Aldrich syndrome protein (WASp) has been implicated in modulation of lymphocyte activation and cytoskeletal reorganization. To address the mechanisms whereby WASp subserves such functions, we have examined WASp roles in lymphocyte development and activation using mice carrying a WAS allele (WAS(-)(/)(-)). Enumeration of hemopoietic cells in these animals revealed total numbers of thymocytes, peripheral B and T lymphocytes, and platelets to be significantly diminished relative to wild-type mice. In the thymus, this abnormality was associated with impaired progression from the CD44(-)CD25(+) to the CD44(-)CD25(-) stage of differentiation. WASp-deficient thymocytes and T cells also exhibited impaired proliferation and interleukin (IL)-2 production in response to T cell antigen receptor (TCR) stimulation, but proliferated normally in response to phorbol ester/ionomycin. This defect in TCR signaling was associated with a reduction in TCR-evoked upregulation of the early activation marker CD69 and in TCR-triggered apoptosis. While induction of TCR-zeta, ZAP70, and total protein tyrosine phosphorylation as well as mitogen-activated protein kinase (MAPK) and stress-activated protein/c-Jun NH(2)-terminal kinase (SAPK/JNK) activation appeared normal in TCR-stimulated WAS(-)(/)(-) cells, TCR-evoked increases in intracellular calcium concentration were decreased in WASp-deficient relative to wild-type cells. WAS(-)(/)(-) lymphocytes also manifested a marked reduction in actin polymerization and both antigen receptor capping and endocytosis after TCR stimulation, whereas WAS(-)(/)(-) neutrophils exhibited reduced phagocytic activity. Together, these results provide evidence of roles for WASp in driving lymphocyte development, as well as in the translation of antigen receptor stimulation to proliferative or apoptotic responses, cytokine production, and cytoskeletal rearrangement. The data also reveal a role for WASp in modulating endocytosis and phagocytosis and, accordingly, suggest that the immune deficit conferred by WASp deficiency reflects the disruption of a broad range of cellular behaviors.

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Related in: MedlinePlus

Characterization of WAS genotypes and thymocyte populations in WAS−/− mice. (A) Long-range PCR genotyping used in screening the ES clones. The wild-type (+/+) allele appears as an 8-kb fragment, and the mutant (−/−) allele appears as a 6.3-kb fragment. A molecular mass standard (1-kb ladder) is shown on the left. (B) Southern blot analysis for detection of targeted clones. BamHI-digested DNA from the respective clones was electrophoresed through 0.8% agarose, transferred to nitrocellulose, and the filters were hybridized with a 450-bp segment from the 5′ flanking region of the WAS gene. The 10.5-kb fragment represents hybridization to homologously targeted alleles, and the 6.5-kb band represents hybridization to the wild-type allele. (C) Spleen and thymus from WAS−/− mice are  for the expression of WASp. Splenocyte and thymocyte lysates from control (+/+) and WAS−/− mice were analyzed by Western blotting as described in Materials and Methods. The blot was probed with an anti-WASp polyclonal antibody (top panel). Equal loading was assessed by reprobing the blot with an anti–β-actin antibody (bottom panel). (D) Immunofluorescence analysis of T and B cell development in WAS−/− mice. Thymocytes and lymph node T cells from 4–8-wk-old WAS−/− and age-matched control (+/+) mice were assayed for CD4 and CD8 expression (top and middle panels) or expression of CD25 and CD44 (bottom panel). To analyze the early stages of thymic development, four-color staining of thymocytes was carried out by staining the cells with a cocktail containing allophycocyanin-labeled anti-CD3, anti-CD4, anti-CD8, anti–TCR-α/β, and anti-B220 antibodies, and subsequently with FITC-Ly9.1, biotinylated anti-CD25 (detected with Texas red–conjugated streptavidin), and PE-labeled anti-CD44. Fluorescence signals were evaluated using Becton Dickson FACScan™ and CELLQuest™ software and were gated to display CD25 versus CD44 on Ly-9.1+ (ES-derived) CD4, CD8, CD3 triple-negative cells (bottom panel). Numbers in the quadrants indicate percentages of different cell populations, while numbers below each panel indicate total number of thymic cells. Data shown are representative of four independent experiments.
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Figure 1: Characterization of WAS genotypes and thymocyte populations in WAS−/− mice. (A) Long-range PCR genotyping used in screening the ES clones. The wild-type (+/+) allele appears as an 8-kb fragment, and the mutant (−/−) allele appears as a 6.3-kb fragment. A molecular mass standard (1-kb ladder) is shown on the left. (B) Southern blot analysis for detection of targeted clones. BamHI-digested DNA from the respective clones was electrophoresed through 0.8% agarose, transferred to nitrocellulose, and the filters were hybridized with a 450-bp segment from the 5′ flanking region of the WAS gene. The 10.5-kb fragment represents hybridization to homologously targeted alleles, and the 6.5-kb band represents hybridization to the wild-type allele. (C) Spleen and thymus from WAS−/− mice are for the expression of WASp. Splenocyte and thymocyte lysates from control (+/+) and WAS−/− mice were analyzed by Western blotting as described in Materials and Methods. The blot was probed with an anti-WASp polyclonal antibody (top panel). Equal loading was assessed by reprobing the blot with an anti–β-actin antibody (bottom panel). (D) Immunofluorescence analysis of T and B cell development in WAS−/− mice. Thymocytes and lymph node T cells from 4–8-wk-old WAS−/− and age-matched control (+/+) mice were assayed for CD4 and CD8 expression (top and middle panels) or expression of CD25 and CD44 (bottom panel). To analyze the early stages of thymic development, four-color staining of thymocytes was carried out by staining the cells with a cocktail containing allophycocyanin-labeled anti-CD3, anti-CD4, anti-CD8, anti–TCR-α/β, and anti-B220 antibodies, and subsequently with FITC-Ly9.1, biotinylated anti-CD25 (detected with Texas red–conjugated streptavidin), and PE-labeled anti-CD44. Fluorescence signals were evaluated using Becton Dickson FACScan™ and CELLQuest™ software and were gated to display CD25 versus CD44 on Ly-9.1+ (ES-derived) CD4, CD8, CD3 triple-negative cells (bottom panel). Numbers in the quadrants indicate percentages of different cell populations, while numbers below each panel indicate total number of thymic cells. Data shown are representative of four independent experiments.

Mentions: A WAS gene targeting vector was derived by subcloning a 3.3-kb segment of the WAS gene encompassing ∼2 kb of the 5′ flanking sequence upstream of the initiation codon through exon 4 of the gene into the Ssc8387I site of the pPNT expression cassette 46 and a 3.4-kb fragment encompassing the XbaI site in exon 11 through to a BamHI site within intron 11 into the pPNT XbaI-BamHI sites. Embryonic stem (ES) cells from the male-derived R1 ES cell line (129/Sv) were electroporated with this targeting vector and selected with neomycin and gancyclovir 47. Surviving clones were screened for homologous recombination at the WAS locus by PCR using the following primer pair: 5′-GTGAAGGATAACCCTCAGAAGTCC-3′ (forward primer, S1, derived from sequences within exon 2 of the WAS gene) and 5′-CGGAGCAGAATCTAGATGGCAGAGT-3′ (reverse primer, S2, representing sequences in the 3′ region downstream from exon 12 of the WAS gene) (see Fig. 1 A). Targeted clones were verified by Southern blotting with a probe derived from a 450-bp segment external to the 5′ region of homology (see Fig. 1 B). Two independently derived WAS−/− clones were then either aggregated with CD1 eight cell stage embryos or microinjected into 3.5-d recombinase activating gene 2–deficient (RAG-2−/−) blastocysts 48, and the aggregates or blastocysts were then implanted into pseudopregnant foster mothers.


Antigen receptor-induced activation and cytoskeletal rearrangement are impaired in Wiskott-Aldrich syndrome protein-deficient lymphocytes.

Zhang J, Shehabeldin A, da Cruz LA, Butler J, Somani AK, McGavin M, Kozieradzki I, dos Santos AO, Nagy A, Grinstein S, Penninger JM, Siminovitch KA - J. Exp. Med. (1999)

Characterization of WAS genotypes and thymocyte populations in WAS−/− mice. (A) Long-range PCR genotyping used in screening the ES clones. The wild-type (+/+) allele appears as an 8-kb fragment, and the mutant (−/−) allele appears as a 6.3-kb fragment. A molecular mass standard (1-kb ladder) is shown on the left. (B) Southern blot analysis for detection of targeted clones. BamHI-digested DNA from the respective clones was electrophoresed through 0.8% agarose, transferred to nitrocellulose, and the filters were hybridized with a 450-bp segment from the 5′ flanking region of the WAS gene. The 10.5-kb fragment represents hybridization to homologously targeted alleles, and the 6.5-kb band represents hybridization to the wild-type allele. (C) Spleen and thymus from WAS−/− mice are  for the expression of WASp. Splenocyte and thymocyte lysates from control (+/+) and WAS−/− mice were analyzed by Western blotting as described in Materials and Methods. The blot was probed with an anti-WASp polyclonal antibody (top panel). Equal loading was assessed by reprobing the blot with an anti–β-actin antibody (bottom panel). (D) Immunofluorescence analysis of T and B cell development in WAS−/− mice. Thymocytes and lymph node T cells from 4–8-wk-old WAS−/− and age-matched control (+/+) mice were assayed for CD4 and CD8 expression (top and middle panels) or expression of CD25 and CD44 (bottom panel). To analyze the early stages of thymic development, four-color staining of thymocytes was carried out by staining the cells with a cocktail containing allophycocyanin-labeled anti-CD3, anti-CD4, anti-CD8, anti–TCR-α/β, and anti-B220 antibodies, and subsequently with FITC-Ly9.1, biotinylated anti-CD25 (detected with Texas red–conjugated streptavidin), and PE-labeled anti-CD44. Fluorescence signals were evaluated using Becton Dickson FACScan™ and CELLQuest™ software and were gated to display CD25 versus CD44 on Ly-9.1+ (ES-derived) CD4, CD8, CD3 triple-negative cells (bottom panel). Numbers in the quadrants indicate percentages of different cell populations, while numbers below each panel indicate total number of thymic cells. Data shown are representative of four independent experiments.
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Related In: Results  -  Collection

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Figure 1: Characterization of WAS genotypes and thymocyte populations in WAS−/− mice. (A) Long-range PCR genotyping used in screening the ES clones. The wild-type (+/+) allele appears as an 8-kb fragment, and the mutant (−/−) allele appears as a 6.3-kb fragment. A molecular mass standard (1-kb ladder) is shown on the left. (B) Southern blot analysis for detection of targeted clones. BamHI-digested DNA from the respective clones was electrophoresed through 0.8% agarose, transferred to nitrocellulose, and the filters were hybridized with a 450-bp segment from the 5′ flanking region of the WAS gene. The 10.5-kb fragment represents hybridization to homologously targeted alleles, and the 6.5-kb band represents hybridization to the wild-type allele. (C) Spleen and thymus from WAS−/− mice are for the expression of WASp. Splenocyte and thymocyte lysates from control (+/+) and WAS−/− mice were analyzed by Western blotting as described in Materials and Methods. The blot was probed with an anti-WASp polyclonal antibody (top panel). Equal loading was assessed by reprobing the blot with an anti–β-actin antibody (bottom panel). (D) Immunofluorescence analysis of T and B cell development in WAS−/− mice. Thymocytes and lymph node T cells from 4–8-wk-old WAS−/− and age-matched control (+/+) mice were assayed for CD4 and CD8 expression (top and middle panels) or expression of CD25 and CD44 (bottom panel). To analyze the early stages of thymic development, four-color staining of thymocytes was carried out by staining the cells with a cocktail containing allophycocyanin-labeled anti-CD3, anti-CD4, anti-CD8, anti–TCR-α/β, and anti-B220 antibodies, and subsequently with FITC-Ly9.1, biotinylated anti-CD25 (detected with Texas red–conjugated streptavidin), and PE-labeled anti-CD44. Fluorescence signals were evaluated using Becton Dickson FACScan™ and CELLQuest™ software and were gated to display CD25 versus CD44 on Ly-9.1+ (ES-derived) CD4, CD8, CD3 triple-negative cells (bottom panel). Numbers in the quadrants indicate percentages of different cell populations, while numbers below each panel indicate total number of thymic cells. Data shown are representative of four independent experiments.
Mentions: A WAS gene targeting vector was derived by subcloning a 3.3-kb segment of the WAS gene encompassing ∼2 kb of the 5′ flanking sequence upstream of the initiation codon through exon 4 of the gene into the Ssc8387I site of the pPNT expression cassette 46 and a 3.4-kb fragment encompassing the XbaI site in exon 11 through to a BamHI site within intron 11 into the pPNT XbaI-BamHI sites. Embryonic stem (ES) cells from the male-derived R1 ES cell line (129/Sv) were electroporated with this targeting vector and selected with neomycin and gancyclovir 47. Surviving clones were screened for homologous recombination at the WAS locus by PCR using the following primer pair: 5′-GTGAAGGATAACCCTCAGAAGTCC-3′ (forward primer, S1, derived from sequences within exon 2 of the WAS gene) and 5′-CGGAGCAGAATCTAGATGGCAGAGT-3′ (reverse primer, S2, representing sequences in the 3′ region downstream from exon 12 of the WAS gene) (see Fig. 1 A). Targeted clones were verified by Southern blotting with a probe derived from a 450-bp segment external to the 5′ region of homology (see Fig. 1 B). Two independently derived WAS−/− clones were then either aggregated with CD1 eight cell stage embryos or microinjected into 3.5-d recombinase activating gene 2–deficient (RAG-2−/−) blastocysts 48, and the aggregates or blastocysts were then implanted into pseudopregnant foster mothers.

Bottom Line: In the thymus, this abnormality was associated with impaired progression from the CD44(-)CD25(+) to the CD44(-)CD25(-) stage of differentiation.This defect in TCR signaling was associated with a reduction in TCR-evoked upregulation of the early activation marker CD69 and in TCR-triggered apoptosis.While induction of TCR-zeta, ZAP70, and total protein tyrosine phosphorylation as well as mitogen-activated protein kinase (MAPK) and stress-activated protein/c-Jun NH(2)-terminal kinase (SAPK/JNK) activation appeared normal in TCR-stimulated WAS(-)(/)(-) cells, TCR-evoked increases in intracellular calcium concentration were decreased in WASp-deficient relative to wild-type cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Toronto, Ontario, Canada M5G 1X5.

ABSTRACT
The Wiskott-Aldrich syndrome protein (WASp) has been implicated in modulation of lymphocyte activation and cytoskeletal reorganization. To address the mechanisms whereby WASp subserves such functions, we have examined WASp roles in lymphocyte development and activation using mice carrying a WAS allele (WAS(-)(/)(-)). Enumeration of hemopoietic cells in these animals revealed total numbers of thymocytes, peripheral B and T lymphocytes, and platelets to be significantly diminished relative to wild-type mice. In the thymus, this abnormality was associated with impaired progression from the CD44(-)CD25(+) to the CD44(-)CD25(-) stage of differentiation. WASp-deficient thymocytes and T cells also exhibited impaired proliferation and interleukin (IL)-2 production in response to T cell antigen receptor (TCR) stimulation, but proliferated normally in response to phorbol ester/ionomycin. This defect in TCR signaling was associated with a reduction in TCR-evoked upregulation of the early activation marker CD69 and in TCR-triggered apoptosis. While induction of TCR-zeta, ZAP70, and total protein tyrosine phosphorylation as well as mitogen-activated protein kinase (MAPK) and stress-activated protein/c-Jun NH(2)-terminal kinase (SAPK/JNK) activation appeared normal in TCR-stimulated WAS(-)(/)(-) cells, TCR-evoked increases in intracellular calcium concentration were decreased in WASp-deficient relative to wild-type cells. WAS(-)(/)(-) lymphocytes also manifested a marked reduction in actin polymerization and both antigen receptor capping and endocytosis after TCR stimulation, whereas WAS(-)(/)(-) neutrophils exhibited reduced phagocytic activity. Together, these results provide evidence of roles for WASp in driving lymphocyte development, as well as in the translation of antigen receptor stimulation to proliferative or apoptotic responses, cytokine production, and cytoskeletal rearrangement. The data also reveal a role for WASp in modulating endocytosis and phagocytosis and, accordingly, suggest that the immune deficit conferred by WASp deficiency reflects the disruption of a broad range of cellular behaviors.

Show MeSH
Related in: MedlinePlus