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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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Effects of WASp deficiency on TCR signaling. WAS−/− or WAS+/+ thymocytes or lymph node T cells were stimulated with biotinylated anti-TCR antibody (10 μg/ml) followed by streptavidin cross-linking (10 μg/ml) for the indicated times. Lysates were prepared as in Materials and Methods, and the lysate proteins were either (A) resolved over SDS-PAGE and subjected to antiphosphotyrosine immunoblotting analysis; (B) subjected to immunoprecipitation with anti–TCR-ζ (top panel) or anti-ZAP70 (bottom panel) antibodies as well as control rabbit IgG(c) and subsequent sequential immunoblotting with antiphosphotyrosine (anti p-Tyr) and anti–TCR-ζ or anti-ZAP70 antibodies, respectively; (C) immunoprecipitated with anti-ERK1 and anti-ERK2 antibodies as well as control IgG(c), and the immune complexes were evaluated for ability to phosphorylate MBP by SDS-PAGE and autoradiography (upper panel) and for amount of ERK1 and ERK2 by immunoblotting analysis (lower panel); or (D) resolved over SDS-PAGE and subjected to sequential immunoblotting analysis with antiphospho-SAPK/JNK (pSAPK/JNK) and anti-SAPK/JNK (JNK1 and JNK2) antibodies. (E) Flow cytometric analysis of Ca2+ influx after stimulation of thymocytes with anti–TCR-α/β antibody. Arrows indicate addition of cross-linking streptavidin.
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Figure 3: Effects of WASp deficiency on TCR signaling. WAS−/− or WAS+/+ thymocytes or lymph node T cells were stimulated with biotinylated anti-TCR antibody (10 μg/ml) followed by streptavidin cross-linking (10 μg/ml) for the indicated times. Lysates were prepared as in Materials and Methods, and the lysate proteins were either (A) resolved over SDS-PAGE and subjected to antiphosphotyrosine immunoblotting analysis; (B) subjected to immunoprecipitation with anti–TCR-ζ (top panel) or anti-ZAP70 (bottom panel) antibodies as well as control rabbit IgG(c) and subsequent sequential immunoblotting with antiphosphotyrosine (anti p-Tyr) and anti–TCR-ζ or anti-ZAP70 antibodies, respectively; (C) immunoprecipitated with anti-ERK1 and anti-ERK2 antibodies as well as control IgG(c), and the immune complexes were evaluated for ability to phosphorylate MBP by SDS-PAGE and autoradiography (upper panel) and for amount of ERK1 and ERK2 by immunoblotting analysis (lower panel); or (D) resolved over SDS-PAGE and subjected to sequential immunoblotting analysis with antiphospho-SAPK/JNK (pSAPK/JNK) and anti-SAPK/JNK (JNK1 and JNK2) antibodies. (E) Flow cytometric analysis of Ca2+ influx after stimulation of thymocytes with anti–TCR-α/β antibody. Arrows indicate addition of cross-linking streptavidin.

Mentions: To elucidate the biochemical basis for the T cell functional defects conferred by WASp deficiency, WAS−/− T cells were evaluated with respect to the signaling events elicited by TCR engagement. As indicated by the antiphosphotyrosine immunoblots shown in Fig. 3a and Fig. b, the levels of TCR-ζ, ZAP70, and total cellular protein tyrosine phosphorylation detected in resting and TCR-stimulated WAS−/− thymocytes and peripheral T cells were essentially identical to those observed in similarly treated wild-type cells. Along similar lines, TCR-evoked increases in MAPK activation and in phosphorylation, and presumably activation of SAPK/JNK were comparable (Fig. 3C and Fig. D). By contrast, the increase in intracellular calcium levels induced by TCR ligation was less sustained in the WAS−/− compared with wild-type cells, with intracellular calcium concentrations 20 and 25% reduced in the WAS−/− relative to wild-type cells at the 600- and 700-s time points, respectively (Fig. 3 E). At present, it is unclear whether this quantitative difference in intracellular calcium mobilization observed in wild-type compared with WAS−/− cells translates to a sufficiently significant perturbance of other downstream signaling events, such as NF-AT translocation to the nucleus, so as to account for the very significant impairment in TCR-induced IL-2 expression by the mutant cells.


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)

Effects of WASp deficiency on TCR signaling. WAS−/− or WAS+/+ thymocytes or lymph node T cells were stimulated with biotinylated anti-TCR antibody (10 μg/ml) followed by streptavidin cross-linking (10 μg/ml) for the indicated times. Lysates were prepared as in Materials and Methods, and the lysate proteins were either (A) resolved over SDS-PAGE and subjected to antiphosphotyrosine immunoblotting analysis; (B) subjected to immunoprecipitation with anti–TCR-ζ (top panel) or anti-ZAP70 (bottom panel) antibodies as well as control rabbit IgG(c) and subsequent sequential immunoblotting with antiphosphotyrosine (anti p-Tyr) and anti–TCR-ζ or anti-ZAP70 antibodies, respectively; (C) immunoprecipitated with anti-ERK1 and anti-ERK2 antibodies as well as control IgG(c), and the immune complexes were evaluated for ability to phosphorylate MBP by SDS-PAGE and autoradiography (upper panel) and for amount of ERK1 and ERK2 by immunoblotting analysis (lower panel); or (D) resolved over SDS-PAGE and subjected to sequential immunoblotting analysis with antiphospho-SAPK/JNK (pSAPK/JNK) and anti-SAPK/JNK (JNK1 and JNK2) antibodies. (E) Flow cytometric analysis of Ca2+ influx after stimulation of thymocytes with anti–TCR-α/β antibody. Arrows indicate addition of cross-linking streptavidin.
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Figure 3: Effects of WASp deficiency on TCR signaling. WAS−/− or WAS+/+ thymocytes or lymph node T cells were stimulated with biotinylated anti-TCR antibody (10 μg/ml) followed by streptavidin cross-linking (10 μg/ml) for the indicated times. Lysates were prepared as in Materials and Methods, and the lysate proteins were either (A) resolved over SDS-PAGE and subjected to antiphosphotyrosine immunoblotting analysis; (B) subjected to immunoprecipitation with anti–TCR-ζ (top panel) or anti-ZAP70 (bottom panel) antibodies as well as control rabbit IgG(c) and subsequent sequential immunoblotting with antiphosphotyrosine (anti p-Tyr) and anti–TCR-ζ or anti-ZAP70 antibodies, respectively; (C) immunoprecipitated with anti-ERK1 and anti-ERK2 antibodies as well as control IgG(c), and the immune complexes were evaluated for ability to phosphorylate MBP by SDS-PAGE and autoradiography (upper panel) and for amount of ERK1 and ERK2 by immunoblotting analysis (lower panel); or (D) resolved over SDS-PAGE and subjected to sequential immunoblotting analysis with antiphospho-SAPK/JNK (pSAPK/JNK) and anti-SAPK/JNK (JNK1 and JNK2) antibodies. (E) Flow cytometric analysis of Ca2+ influx after stimulation of thymocytes with anti–TCR-α/β antibody. Arrows indicate addition of cross-linking streptavidin.
Mentions: To elucidate the biochemical basis for the T cell functional defects conferred by WASp deficiency, WAS−/− T cells were evaluated with respect to the signaling events elicited by TCR engagement. As indicated by the antiphosphotyrosine immunoblots shown in Fig. 3a and Fig. b, the levels of TCR-ζ, ZAP70, and total cellular protein tyrosine phosphorylation detected in resting and TCR-stimulated WAS−/− thymocytes and peripheral T cells were essentially identical to those observed in similarly treated wild-type cells. Along similar lines, TCR-evoked increases in MAPK activation and in phosphorylation, and presumably activation of SAPK/JNK were comparable (Fig. 3C and Fig. D). By contrast, the increase in intracellular calcium levels induced by TCR ligation was less sustained in the WAS−/− compared with wild-type cells, with intracellular calcium concentrations 20 and 25% reduced in the WAS−/− relative to wild-type cells at the 600- and 700-s time points, respectively (Fig. 3 E). At present, it is unclear whether this quantitative difference in intracellular calcium mobilization observed in wild-type compared with WAS−/− cells translates to a sufficiently significant perturbance of other downstream signaling events, such as NF-AT translocation to the nucleus, so as to account for the very significant impairment in TCR-induced IL-2 expression by the mutant cells.

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