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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

Analysis of lymphocyte cap formation, actin polymerization, and endocytosis and neutrophil phagocytosis in WAS−/− mice. (A) Percentages (±SD) of WAS+/+ and WAS−/− thymocytes and splenic B cells showing antigen receptor clustering after antigen receptor ligation. Peripheral lymph node T cells and splenic cells from WAS−/− and wild-type (+/+) mice were incubated with soluble biotinylated anti-TCR antibody (1 μg/ml) or with 1 μg/ml biotinylated anti–mouse IgD antibody, respectively, followed by FITC-conjugated streptavidin and then fixed with 2% paraformaldehyde as described in Materials and Methods. Capped cells were then visualized by fluorescence confocal microscopy, and the percentage of capped cells was determined by scoring cells from 10 microscope fields per sample (100–200 cells/field). (B) Fluorescence confocal micrograph (original magnification: ×126) showing TCR capping of anti-TCR–stimulated thymocytes (as above) from WAS−/− (bottom) and wild-type (top) mice. (C) Impairment of actin polymerization in WAS−/− mice. Thymocytes from WAS−/− and WAS+/+ mice were isolated and stimulated with anti-CD3∈ antibody (1 μg/ml) for 30 min on ice, followed by cross-linking with a secondary antibody (outlined histograms) or alternatively, treated with the secondary antibody alone (filled histograms). Cells were fixed with 4% paraformaldehyde, and F-actin content was quantitated by flow cytometric analysis of FITC-phalloidin–stained cells. One result representative of three independent experiments is shown. (D) TCR internalization in WAS−/− T cells. Lymph node T cells from WAS−/− and wild-type mice were incubated for 30 min on ice with anti-CD3 antibody (1 μg/ml) and then washed and incubated for an additional 30 min on ice with biotinylated goat anti–hamster antibody (2 μg/ml). Cells were then warmed to 37°C, and aliquots were removed at 5 and 60 min, mixed with 0.1% NaN3 on ice, and stained with FITC-conjugated streptavidin. Cells were then fixed for 15 min in 4% paraformaldehyde, and surface TCR expression was analyzed by flow cytometry. (E) Phagocytic activity of bone marrow neutrophils from WAS−/− mice. Neutrophils were purified from WAS−/− and wild-type bone marrow as described in Materials and Methods and then incubated for 5 min at 37°C with opsonized zymosan and lucifer yellow. Cells were then washed, and the percentage of lucifer yellow–containing phagosomes was evaluated by fluorescence microscopy. The percent phagocytosis was calculated from the total number of cells with lucifer yellow–stained phagosomes relative to the total number of cells in six to eight high-power fields. Values represent the mean ± SEM of five independent experiments.
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Figure 4: Analysis of lymphocyte cap formation, actin polymerization, and endocytosis and neutrophil phagocytosis in WAS−/− mice. (A) Percentages (±SD) of WAS+/+ and WAS−/− thymocytes and splenic B cells showing antigen receptor clustering after antigen receptor ligation. Peripheral lymph node T cells and splenic cells from WAS−/− and wild-type (+/+) mice were incubated with soluble biotinylated anti-TCR antibody (1 μg/ml) or with 1 μg/ml biotinylated anti–mouse IgD antibody, respectively, followed by FITC-conjugated streptavidin and then fixed with 2% paraformaldehyde as described in Materials and Methods. Capped cells were then visualized by fluorescence confocal microscopy, and the percentage of capped cells was determined by scoring cells from 10 microscope fields per sample (100–200 cells/field). (B) Fluorescence confocal micrograph (original magnification: ×126) showing TCR capping of anti-TCR–stimulated thymocytes (as above) from WAS−/− (bottom) and wild-type (top) mice. (C) Impairment of actin polymerization in WAS−/− mice. Thymocytes from WAS−/− and WAS+/+ mice were isolated and stimulated with anti-CD3∈ antibody (1 μg/ml) for 30 min on ice, followed by cross-linking with a secondary antibody (outlined histograms) or alternatively, treated with the secondary antibody alone (filled histograms). Cells were fixed with 4% paraformaldehyde, and F-actin content was quantitated by flow cytometric analysis of FITC-phalloidin–stained cells. One result representative of three independent experiments is shown. (D) TCR internalization in WAS−/− T cells. Lymph node T cells from WAS−/− and wild-type mice were incubated for 30 min on ice with anti-CD3 antibody (1 μg/ml) and then washed and incubated for an additional 30 min on ice with biotinylated goat anti–hamster antibody (2 μg/ml). Cells were then warmed to 37°C, and aliquots were removed at 5 and 60 min, mixed with 0.1% NaN3 on ice, and stained with FITC-conjugated streptavidin. Cells were then fixed for 15 min in 4% paraformaldehyde, and surface TCR expression was analyzed by flow cytometry. (E) Phagocytic activity of bone marrow neutrophils from WAS−/− mice. Neutrophils were purified from WAS−/− and wild-type bone marrow as described in Materials and Methods and then incubated for 5 min at 37°C with opsonized zymosan and lucifer yellow. Cells were then washed, and the percentage of lucifer yellow–containing phagosomes was evaluated by fluorescence microscopy. The percent phagocytosis was calculated from the total number of cells with lucifer yellow–stained phagosomes relative to the total number of cells in six to eight high-power fields. Values represent the mean ± SEM of five independent experiments.

Mentions: As WASp has also been implicated by many lines of evidence in the regulation of cytoskeletal architecture, the relevance of WASp to lymphocyte cytoskeletal rearrangements induced by antigen receptor engagement was also investigated. To this end, WAS−/− lymphocytes were studied with respect to the induction of antigen receptor capping, a phenomenon that requires de novo actin polymerization and microfilament rearrangement. As indicated in Fig. 4 A and illustrated by the representative micrograph (Fig. 4 B), a defect in ligand-induced capping was detected in WAS−/− thymocytes, the number of these cells showing T cell antigen receptor clustering to be ∼40% less than that of wild-type thymocytes. As is consistent with a defect, albeit mild in BCR-induced proliferation, anti-Ig–mediated capping of the BCR was also impaired in the context of WASp deficiency. In addition, immunofluorescence analysis of anti-CD3–treated phalloidin-labeled WAS−/− thymocytes, an assay that selectively detects expression of polymerized F-actin, revealed that antigen receptor–mediated actin polymerization was markedly reduced in WAS−/− thymocytes (Fig. 4 C) and lymph node T cells (data not shown). Together, these data demonstrate a requirement for WASp in the regulation of cytoskeletal rearrangement in response to antigen receptor engagement.


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)

Analysis of lymphocyte cap formation, actin polymerization, and endocytosis and neutrophil phagocytosis in WAS−/− mice. (A) Percentages (±SD) of WAS+/+ and WAS−/− thymocytes and splenic B cells showing antigen receptor clustering after antigen receptor ligation. Peripheral lymph node T cells and splenic cells from WAS−/− and wild-type (+/+) mice were incubated with soluble biotinylated anti-TCR antibody (1 μg/ml) or with 1 μg/ml biotinylated anti–mouse IgD antibody, respectively, followed by FITC-conjugated streptavidin and then fixed with 2% paraformaldehyde as described in Materials and Methods. Capped cells were then visualized by fluorescence confocal microscopy, and the percentage of capped cells was determined by scoring cells from 10 microscope fields per sample (100–200 cells/field). (B) Fluorescence confocal micrograph (original magnification: ×126) showing TCR capping of anti-TCR–stimulated thymocytes (as above) from WAS−/− (bottom) and wild-type (top) mice. (C) Impairment of actin polymerization in WAS−/− mice. Thymocytes from WAS−/− and WAS+/+ mice were isolated and stimulated with anti-CD3∈ antibody (1 μg/ml) for 30 min on ice, followed by cross-linking with a secondary antibody (outlined histograms) or alternatively, treated with the secondary antibody alone (filled histograms). Cells were fixed with 4% paraformaldehyde, and F-actin content was quantitated by flow cytometric analysis of FITC-phalloidin–stained cells. One result representative of three independent experiments is shown. (D) TCR internalization in WAS−/− T cells. Lymph node T cells from WAS−/− and wild-type mice were incubated for 30 min on ice with anti-CD3 antibody (1 μg/ml) and then washed and incubated for an additional 30 min on ice with biotinylated goat anti–hamster antibody (2 μg/ml). Cells were then warmed to 37°C, and aliquots were removed at 5 and 60 min, mixed with 0.1% NaN3 on ice, and stained with FITC-conjugated streptavidin. Cells were then fixed for 15 min in 4% paraformaldehyde, and surface TCR expression was analyzed by flow cytometry. (E) Phagocytic activity of bone marrow neutrophils from WAS−/− mice. Neutrophils were purified from WAS−/− and wild-type bone marrow as described in Materials and Methods and then incubated for 5 min at 37°C with opsonized zymosan and lucifer yellow. Cells were then washed, and the percentage of lucifer yellow–containing phagosomes was evaluated by fluorescence microscopy. The percent phagocytosis was calculated from the total number of cells with lucifer yellow–stained phagosomes relative to the total number of cells in six to eight high-power fields. Values represent the mean ± SEM of five independent experiments.
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Related In: Results  -  Collection

Show All Figures
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Figure 4: Analysis of lymphocyte cap formation, actin polymerization, and endocytosis and neutrophil phagocytosis in WAS−/− mice. (A) Percentages (±SD) of WAS+/+ and WAS−/− thymocytes and splenic B cells showing antigen receptor clustering after antigen receptor ligation. Peripheral lymph node T cells and splenic cells from WAS−/− and wild-type (+/+) mice were incubated with soluble biotinylated anti-TCR antibody (1 μg/ml) or with 1 μg/ml biotinylated anti–mouse IgD antibody, respectively, followed by FITC-conjugated streptavidin and then fixed with 2% paraformaldehyde as described in Materials and Methods. Capped cells were then visualized by fluorescence confocal microscopy, and the percentage of capped cells was determined by scoring cells from 10 microscope fields per sample (100–200 cells/field). (B) Fluorescence confocal micrograph (original magnification: ×126) showing TCR capping of anti-TCR–stimulated thymocytes (as above) from WAS−/− (bottom) and wild-type (top) mice. (C) Impairment of actin polymerization in WAS−/− mice. Thymocytes from WAS−/− and WAS+/+ mice were isolated and stimulated with anti-CD3∈ antibody (1 μg/ml) for 30 min on ice, followed by cross-linking with a secondary antibody (outlined histograms) or alternatively, treated with the secondary antibody alone (filled histograms). Cells were fixed with 4% paraformaldehyde, and F-actin content was quantitated by flow cytometric analysis of FITC-phalloidin–stained cells. One result representative of three independent experiments is shown. (D) TCR internalization in WAS−/− T cells. Lymph node T cells from WAS−/− and wild-type mice were incubated for 30 min on ice with anti-CD3 antibody (1 μg/ml) and then washed and incubated for an additional 30 min on ice with biotinylated goat anti–hamster antibody (2 μg/ml). Cells were then warmed to 37°C, and aliquots were removed at 5 and 60 min, mixed with 0.1% NaN3 on ice, and stained with FITC-conjugated streptavidin. Cells were then fixed for 15 min in 4% paraformaldehyde, and surface TCR expression was analyzed by flow cytometry. (E) Phagocytic activity of bone marrow neutrophils from WAS−/− mice. Neutrophils were purified from WAS−/− and wild-type bone marrow as described in Materials and Methods and then incubated for 5 min at 37°C with opsonized zymosan and lucifer yellow. Cells were then washed, and the percentage of lucifer yellow–containing phagosomes was evaluated by fluorescence microscopy. The percent phagocytosis was calculated from the total number of cells with lucifer yellow–stained phagosomes relative to the total number of cells in six to eight high-power fields. Values represent the mean ± SEM of five independent experiments.
Mentions: As WASp has also been implicated by many lines of evidence in the regulation of cytoskeletal architecture, the relevance of WASp to lymphocyte cytoskeletal rearrangements induced by antigen receptor engagement was also investigated. To this end, WAS−/− lymphocytes were studied with respect to the induction of antigen receptor capping, a phenomenon that requires de novo actin polymerization and microfilament rearrangement. As indicated in Fig. 4 A and illustrated by the representative micrograph (Fig. 4 B), a defect in ligand-induced capping was detected in WAS−/− thymocytes, the number of these cells showing T cell antigen receptor clustering to be ∼40% less than that of wild-type thymocytes. As is consistent with a defect, albeit mild in BCR-induced proliferation, anti-Ig–mediated capping of the BCR was also impaired in the context of WASp deficiency. In addition, immunofluorescence analysis of anti-CD3–treated phalloidin-labeled WAS−/− thymocytes, an assay that selectively detects expression of polymerized F-actin, revealed that antigen receptor–mediated actin polymerization was markedly reduced in WAS−/− thymocytes (Fig. 4 C) and lymph node T cells (data not shown). Together, these data demonstrate a requirement for WASp in the regulation of cytoskeletal rearrangement in response to antigen receptor engagement.

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