The motheaten mutation rescues B cell signaling and development in CD45-deficient mice - PubMed (original) (raw)

The motheaten mutation rescues B cell signaling and development in CD45-deficient mice

G Pani et al. J Exp Med. 1997.

Abstract

The cytosolic SHP-1 and transmembrane CD45 protein tyrosine phosphatases (PTP) play critical roles in regulating signal transduction via the B cell antigen receptor (BCR). These PTPs differ, however, in their effects on BCR function. For example, BCR-mediated mitogenesis is essentially ablated in mice lacking CD45 (CD45(-)), but is enhanced in SHP-1-deficient motheaten (me) and viable motheaten (mev) mice. To determine whether these PTPs act independently or coordinately in modulating the physiologic outcome of BCR engagement, we assessed B cell development and signaling in CD45-deficient mev (CD45-/SHP-1-) mice. Here we report that the CD45-/SHP-1-) cells undergo appropriate induction of protein kinase activity, mitogen-activated protein kinase activation, and proliferative responses after BCR aggregation. However, BCR-elicited increases in the tyrosine phosphorylation of several SHP-1-associated phosphoproteins, including CD19, were substantially enhanced in CD45-/SHP-1-, compared to wild-type and CD45- cells. In addition, we observed that the patterns of cell surface expression of mu, delta, and CD5, which distinguish the PTP-deficient from normal mice, are largely restored to normal levels in the double mutant animals. These findings indicate a critical role for the balance of SHP-1 and CD45 activities in determining the outcome of BCR stimulation and suggest that these PTPs act in a coordinate fashion to couple antigen receptor engagement to B cell activation and maturation.

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Figures

Figure 2

Figure 2

B cell proliferative responses and autoantibody production in PTP-deficient mice. (A) Effects of anti-Ig and LPS on proliferation ([3H]thymidine incorporation) of CD45−/SHP-1−, CD45−, and CD45+/ SHP-1+ (wild-type) B cells (all LPS-treated groups as well as anti-μ–treated groups (CD45+/SHP-1+) and (CD45−/SHP-1−) differed significantly from their counterparts in the No stimulation group based on analysis by a two-tailed t test (P <0.05 in all cases). (B) Comparisons of antitopoisomerase antibody titers in 4-wk-old CD45−/SHP-1−, CD45−, CD45+/ SHP-1+ and SHP-1− mice. (C) Immunostaining of renal tissues from 4-wk-old CD45+/SHP-1+, CD45−, CD45−/SHP-1−, and SHP-1− mice.

Figure 2

Figure 2

B cell proliferative responses and autoantibody production in PTP-deficient mice. (A) Effects of anti-Ig and LPS on proliferation ([3H]thymidine incorporation) of CD45−/SHP-1−, CD45−, and CD45+/ SHP-1+ (wild-type) B cells (all LPS-treated groups as well as anti-μ–treated groups (CD45+/SHP-1+) and (CD45−/SHP-1−) differed significantly from their counterparts in the No stimulation group based on analysis by a two-tailed t test (P <0.05 in all cases). (B) Comparisons of antitopoisomerase antibody titers in 4-wk-old CD45−/SHP-1−, CD45−, CD45+/ SHP-1+ and SHP-1− mice. (C) Immunostaining of renal tissues from 4-wk-old CD45+/SHP-1+, CD45−, CD45−/SHP-1−, and SHP-1− mice.

Figure 1

Figure 1

Representative flow cytometric analyses of splenic B cells from PTP-deficient mice. Expression of IgM and IgD was evaluated on splenic cells from age-matched 4-wk-old mice: CD45+/SHP-1+ (C57B1/6 wild type); CD45−/ SHP-1+ (C57B1/6 CD45 exon 6−/−; originally derived by K. Kishihara; reference ; eighth backcross generation), CD45+/SHP-1− (C57B1/6 mev/mev); CD45−/SHP-1− mice (derived by mating C57B1/6 CD45 exon 6−/− [eighth backcross generation] with C57B1/6 mev/+ mice). The percentages of cells within the boxed regions correspond to the following pattern: I, percent δ+μ− in the lymphoid gate; II, percent δhiμlo of III; III, percent δ+μ+ in the lymphoid gate; IV, percent δ−μ+ in the lymphoid gate. The lymphoid gate was set by standard procedures relying on the forward and side scatter properties of the splenic cells. The absolute numbers of various subsets of cells found in the spleens of the same mice (× 106) for CD45+/SHP-1+, CD45−/SHP-1+, CD45+/SHP-1−, CD45−/SHP-1−), total splenic cells were 79, 120, 140, and 56, respectively. Lymphoid gate: 68, 95, 95, 41; μ+δ+: 33, 76, 7.2, 9.1; μloδlo: 15, 5.2, 3.6, 4.4; μ+δ−, 1.6, 3.4, 8.2, 1.1; μ−δ+: 0.5, 0.06, 1.6, 0.05; μ+CD5+: 5.7 (representing 16% of μ+ cells), 4.5 (6%), 11 (71%), 3.6 (37%). Data are representative of four independent experiments carried out as described in Materials and Methods.

Figure 1

Figure 1

Representative flow cytometric analyses of splenic B cells from PTP-deficient mice. Expression of IgM and IgD was evaluated on splenic cells from age-matched 4-wk-old mice: CD45+/SHP-1+ (C57B1/6 wild type); CD45−/ SHP-1+ (C57B1/6 CD45 exon 6−/−; originally derived by K. Kishihara; reference ; eighth backcross generation), CD45+/SHP-1− (C57B1/6 mev/mev); CD45−/SHP-1− mice (derived by mating C57B1/6 CD45 exon 6−/− [eighth backcross generation] with C57B1/6 mev/+ mice). The percentages of cells within the boxed regions correspond to the following pattern: I, percent δ+μ− in the lymphoid gate; II, percent δhiμlo of III; III, percent δ+μ+ in the lymphoid gate; IV, percent δ−μ+ in the lymphoid gate. The lymphoid gate was set by standard procedures relying on the forward and side scatter properties of the splenic cells. The absolute numbers of various subsets of cells found in the spleens of the same mice (× 106) for CD45+/SHP-1+, CD45−/SHP-1+, CD45+/SHP-1−, CD45−/SHP-1−), total splenic cells were 79, 120, 140, and 56, respectively. Lymphoid gate: 68, 95, 95, 41; μ+δ+: 33, 76, 7.2, 9.1; μloδlo: 15, 5.2, 3.6, 4.4; μ+δ−, 1.6, 3.4, 8.2, 1.1; μ−δ+: 0.5, 0.06, 1.6, 0.05; μ+CD5+: 5.7 (representing 16% of μ+ cells), 4.5 (6%), 11 (71%), 3.6 (37%). Data are representative of four independent experiments carried out as described in Materials and Methods.

Figure 3

Figure 3

Analysis of SHP-1 phosphoprotein binding and MAP kinase activation in stimulated B cells from PTP-deficient mice. (A) Comparison of protein tyrosine phosphorylation in resting and anti-Ig–treated B cells from wild-type (CD45+/SHP-1+), CD45−CD45−/SHP-1−, and CD45+/ SHP-1− mice. Loading of equivalent amounts of lysate proteins was confirmed by reblotting with anti–mb-1 antibody (bottom). (B) Antiphosphotyrosine (anti pTyr) immunoblots (top) showing the tyrosine-phosphorylated species coprecipitated with SHP-1 from resting and anti-Ig–treated B cells from wild-type (CD45+/SHP-1+), CD45−, and CD45−/SHP-1− mice (left) and from CD45+/SHP-1− mice (right). Arrows indicate the positions of SHP-1 and three associated phosphoproteins that appear differentially phosphorylated in the CD45− compared to CD45−/SHP-1− and SHP-1− cells. Mobilities of molecular mass standards are shown on the left. Loading of equivalent amounts of lysate protein was confirmed by reblotting with anti–SHP-1 antibody (bottom). Data are representative of three independent experiments on nine mice. (C) Antiphosphotyrosine immunoblot (top) showing the tyrosine phosphorylation status of CD19 immunoprecipitates derived from biotinylated resting and anti-Ig–treated wild-type, CD45−, and CD45−/SHP-1− cells was carried out as described in Materials and Methods. The position of CD19 is indicated by the arrow on the right. Loading of equivalent amounts of CD19 was confirmed by reblotting with HRP-avidin (bottom). (D) Representative example showing the levels of MAP kinase activities before and 5 min after BCR ligation in wild-type, CD45−, CD45−/SHP-1−, and CD45+/SHP-1− cells (top). Analysis of equivalent amounts of Erk-2 was confirmed by anti-Erk2 immunoblotting of equivalent aliquots of each Erk-2 immunoprecipitate (bottom).

Figure 3

Figure 3

Analysis of SHP-1 phosphoprotein binding and MAP kinase activation in stimulated B cells from PTP-deficient mice. (A) Comparison of protein tyrosine phosphorylation in resting and anti-Ig–treated B cells from wild-type (CD45+/SHP-1+), CD45−CD45−/SHP-1−, and CD45+/ SHP-1− mice. Loading of equivalent amounts of lysate proteins was confirmed by reblotting with anti–mb-1 antibody (bottom). (B) Antiphosphotyrosine (anti pTyr) immunoblots (top) showing the tyrosine-phosphorylated species coprecipitated with SHP-1 from resting and anti-Ig–treated B cells from wild-type (CD45+/SHP-1+), CD45−, and CD45−/SHP-1− mice (left) and from CD45+/SHP-1− mice (right). Arrows indicate the positions of SHP-1 and three associated phosphoproteins that appear differentially phosphorylated in the CD45− compared to CD45−/SHP-1− and SHP-1− cells. Mobilities of molecular mass standards are shown on the left. Loading of equivalent amounts of lysate protein was confirmed by reblotting with anti–SHP-1 antibody (bottom). Data are representative of three independent experiments on nine mice. (C) Antiphosphotyrosine immunoblot (top) showing the tyrosine phosphorylation status of CD19 immunoprecipitates derived from biotinylated resting and anti-Ig–treated wild-type, CD45−, and CD45−/SHP-1− cells was carried out as described in Materials and Methods. The position of CD19 is indicated by the arrow on the right. Loading of equivalent amounts of CD19 was confirmed by reblotting with HRP-avidin (bottom). (D) Representative example showing the levels of MAP kinase activities before and 5 min after BCR ligation in wild-type, CD45−, CD45−/SHP-1−, and CD45+/SHP-1− cells (top). Analysis of equivalent amounts of Erk-2 was confirmed by anti-Erk2 immunoblotting of equivalent aliquots of each Erk-2 immunoprecipitate (bottom).

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