A dual role for Src homology 2 domain-containing inositol-5-phosphatase (SHIP) in immunity: aberrant development and enhanced function of b lymphocytes in ship -/- mice - PubMed (original) (raw)

A dual role for Src homology 2 domain-containing inositol-5-phosphatase (SHIP) in immunity: aberrant development and enhanced function of b lymphocytes in ship -/- mice

C D Helgason et al. J Exp Med. 2000.

Abstract

In this report, we demonstrate that the Src homology 2 domain-containing inositol-5-phosphatase (SHIP) plays a critical role in regulating both B cell development and responsiveness to antigen stimulation. SHIP(-/-) mice exhibit a transplantable alteration in B lymphoid development that results in reduced numbers of precursor B (fraction C) and immature B cells in the bone marrow. In vitro, purified SHIP(-/)- B cells exhibit enhanced proliferation in response to B cell receptor stimulation in both the presence and absence of Fcgamma receptor IIB coligation. This enhancement is associated with increased phosphorylation of both mitogen-activated protein kinase and Akt, as well as with increased survival and cell cycling. SHIP(-/)- mice manifest elevated serum immunoglobulin (Ig) levels and an exaggerated IgG response to the T cell-independent type 2 antigen trinitrophenyl Ficoll. However, only altered B cell development was apparent upon transplantation into nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice. The in vitro hyperresponsiveness, together with the in vivo findings, suggests that SHIP regulates B lymphoid development and antigen responsiveness by both intrinsic and extrinsic mechanisms.

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Figures

Figure 1

Figure 1

Expression of SHIP in B cell subsets. (A) Southern blots of amplified total cDNA prepared from phenotypically defined bone marrow and spleen B cell subsets, as well as total bone marrow (TBM), were sequentially hybridized with probes specific for SHIP and actin as indicated. (B) Signal intensities after hybridization were quantified by PhosphorImager® analysis, and expression levels of SHIP relative to actin were calculated. Average expression levels from two determinations are shown normalized to expression in the B220+c-kit+ population.

Figure 1

Figure 1

Expression of SHIP in B cell subsets. (A) Southern blots of amplified total cDNA prepared from phenotypically defined bone marrow and spleen B cell subsets, as well as total bone marrow (TBM), were sequentially hybridized with probes specific for SHIP and actin as indicated. (B) Signal intensities after hybridization were quantified by PhosphorImager® analysis, and expression levels of SHIP relative to actin were calculated. Average expression levels from two determinations are shown normalized to expression in the B220+c-kit+ population.

Figure 2

Figure 2

Proliferation of purified B cells in response to various stimuli. (A) Purified splenic B cells from wild-type (•) or SHIP−/− (♦) mice were incubated with intact goat anti–mouse IgM antibody in the presence (solid line) or absence (broken line) of the anti-FcR antibody 2.4G2 for 60 h before addition of [3H]thymidine for determination of specific incorporation. Results are the mean ± SEM of triplicate determinations (four experiments with cells pooled from three or four mice per experiment). Purified splenic B cells from wild-type (•, solid line) and SHIP−/− (♦, broken line) mice were incubated with various concentrations of the F(ab′)2 fragment of goat anti–mouse IgM antibody (B) or bacterial LPS (C), and proliferation was determined as above. (D) Purified splenic B cells from wild-type (white bars) and SHIP−/− (black bars) mice were incubated with the indicated concentrations of IL-4 or anti-CD40 culture supernatants. (E) Wild-type (•, solid line) and SHIP−/− (♦, broken line) B cells were incubated with 1% IL-4 supernatant and the indicated percentages of anti-CD40 supernatant for determination of proliferation as indicated above. Statistical analysis between similarly treated wild-type and SHIP−/− cells was carried out using the Student's t test. *P ≤ 0.05; **P ≤ 0.005.

Figure 3

Figure 3

Phosphorylation of Akt and MAPK after BCR stimulation of purified B cells. Western blot analysis showing the time course of Akt (Akt-P) and MAPK (p44-P and p42-P) phosphorylation in wild-type (+/+) and knockout (−/−) B cells after BCR stimulation with the F(ab′)2 (left) or intact (right) goat anti–mouse IgM. Protein levels of Akt and MAPK are shown to indicate relative expression levels.

Figure 4

Figure 4

Serum Ig levels in 4–8-wk-old SHIP wild-type (+/+), heterozygous (+/−), and knockout (−/−) mice. Serial dilutions of serum obtained from age-matched mice (n = 8–9) were used. Symbols represent levels in individual animals, and the bar represents the mean value. Statistical analysis was carried out using the Student's t test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005.

Figure 5

Figure 5

Serum Ig levels after antigenic challenge. (A) Anti-TNP–specific Ig levels before (d0) and 8 d (d8) after immunization with the TI-2 antigen TNP-Ficoll. (B) Anti-TNP–specific Ig at the indicated times after injection of the T cell–dependent antigen TNP-KLH. Symbols represent levels in individual animals, and the column represents the mean value. Statistical analysis was carried out using the Student's t test. *P ≤ 0.05.

Figure 5

Figure 5

Serum Ig levels after antigenic challenge. (A) Anti-TNP–specific Ig levels before (d0) and 8 d (d8) after immunization with the TI-2 antigen TNP-Ficoll. (B) Anti-TNP–specific Ig at the indicated times after injection of the T cell–dependent antigen TNP-KLH. Symbols represent levels in individual animals, and the column represents the mean value. Statistical analysis was carried out using the Student's t test. *P ≤ 0.05.

Figure 6

Figure 6

B lymphocyte and myeloid populations in transplanted NOD/SCID mice. FACS® analysis was carried out on peripheral blood cells isolated from NOD/SCID mice transplanted with wild-type (•) or SHIP knockout (♦) bone marrow cells to determine the percentages of cells expressing B220 or IgM plus IgD. Nontransplanted mice exhibited undetectable levels of these markers (not shown). In addition, the percentages of transplant-derived cells expressing the Mac-1 and Gr-1 surface antigens were determined (Ly5.2+Mac+Gr+). Results are shown for 4–10 mice. Numbers above the column represent the mean ± SD for the indicated animals. Statistical analysis was carried out using the Student's t test. *P ≤ 0.005.

Figure 7

Figure 7

Serum Ig and anti-TNP–specific antibody levels in NOD/SCID mice transplanted with wild-type or SHIP−/− bone marrow cells. (A) Serum Ig levels were determined by ELISA measurements on serial dilutions of serum taken from representative mice transplanted with wild-type (•) or SHIP−/− (♦) bone marrow. (B) Nonirradiated NOD/SCID mice with equivalent levels of Ly5.2+B220+ cells in the peripheral blood were injected intravenously with TNP-Ficoll, and serum samples were collected 9 d after immunization. ELISAs were carried out to determine the levels of anti-TNP–specific antibodies in recipients of wild-type (•) or SHIP−/− (♦) bone marrow. Statistical analysis was carried out using the Student's t test. **P ≤ 0.01; ***P ≤ 0.005.

Figure 7

Figure 7

Serum Ig and anti-TNP–specific antibody levels in NOD/SCID mice transplanted with wild-type or SHIP−/− bone marrow cells. (A) Serum Ig levels were determined by ELISA measurements on serial dilutions of serum taken from representative mice transplanted with wild-type (•) or SHIP−/− (♦) bone marrow. (B) Nonirradiated NOD/SCID mice with equivalent levels of Ly5.2+B220+ cells in the peripheral blood were injected intravenously with TNP-Ficoll, and serum samples were collected 9 d after immunization. ELISAs were carried out to determine the levels of anti-TNP–specific antibodies in recipients of wild-type (•) or SHIP−/− (♦) bone marrow. Statistical analysis was carried out using the Student's t test. **P ≤ 0.01; ***P ≤ 0.005.

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