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The Journal of Immunology, 2001, 167: 204-211.
Copyright © 2001 by The American Association of Immunologists

Partially Distinct Molecular Mechanisms Mediate Inhibitory Fc{gamma}RIIB Signaling in Resting and Activated B Cells1

Anne Brauweiler*,{dagger}, Idan Tamir*,{dagger}, Susanne Marschner*,{dagger}, Cheryl D. Helgason{ddagger} and John C. Cambier2,*,{dagger}

* Department of Immunology, National Jewish Medical and Research Center, Denver, CO 80206; {dagger} Department of Immunology, University of Colorado Health Science Center, Denver, CO 80206; and {ddagger} Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fc{gamma}RIIB functions as an inhibitory receptor to dampen B cell Ag receptor signals and immune responses. Accumulating evidence indicates that ex vivo B cells require the inositol 5-phosphatase, Src homology domain 2-containing inositol 5-phosphatase (SHIP), for Fc{gamma}RIIB-mediated inhibitory signaling. However, we report here that LPS-activated primary B cells do not require SHIP and thus differ from resting B cells. SHIP-deficient B cell blasts display efficient Fc{gamma}RIIB-dependent inhibition of calcium mobilization as well as Akt and extracellular signal-related protein kinase phosphorylation. Surprisingly, Fc{gamma}RIIB-dependent degradation of phosphatidylinositol 3,4,5-trisphosphate and conversion into phosphatidylinositol 3,4-bisphosphate occur in SHIP-deficient B cell blasts, demonstrating the function of an additional inositol 5-phosphatase. Further analysis reveals that while resting cells express only SHIP, B cell blasts also express the recently described inositol 5-phosphatase, SHIP-2. Finally, data suggest that both SHIP-2 and SHIP can mediate downstream biologic consequences of Fc{gamma}RIIB signaling, including inhibition of the proliferative response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
During late phases of humoral immune responses, immune complexes composed of IgG Abs and Ag may bind simultaneously to B cell Ag receptors (BCRs)3 and low-affinity IgG Fc receptors (Fc{gamma}RIIB) coexpressed on B cells. Coaggregation of the BCR with Fc{gamma}RIIB inhibits BCR signaling, blocking downstream biologic responses including activation, proliferation, and Ab production (1), and further serves to reduce the development of autoimmune disease (2). The initial event in inhibitory signaling is phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM) tyrosine found in the Fc{gamma}RIIB cytoplasmic tail (3). This modification results in recruitment of a limited number of Src homology 2 domain (SH2)-containing phosphatases (4, 5), predominantly the SH2 containing inositol 5-phosphatase (SHIP) (6, 7, 8). Recruitment and activation of SHIP causes a dramatic and immediate hydrolysis of the membrane lipid, phosphatidylinositol 3,4,5-trisphosphate (PI(3, 4, 5)P3), yielding phosphatidylinositol 3,4-bisphosphate (PI(3, 4)P2) (9, 10). PI(3, 4, 5)P3 is the docking site for Pleckstrin homology domain containing proteins, including Bruton’s tyrosine kinase, and phospholipase C{gamma} (PLC{gamma}) (2, 11, 12). Therefore, SHIP-mediated hydrolysis of PI(3, 4, 5)P3 leads to impaired membrane translocation of these signal transducing molecules. Bruton’s tyrosine kinase is required for activation of PLC{gamma} and hydrolysis of phosphatidylinositol 4,5-bisphosphate yielding inositol 1,4,5-trisphosphate (Ins(1, 4, 5)P3) and diacylglycerol. Thus SHIP inhibits the generation of second messengers that mediate calcium mobilization and protein kinase C (PKC) activation, respectively (13). Furthermore, activation of mitogen-activated protein (MAP) kinases (14, 15) and the recruitment of the antiapoptotic kinase, Akt (16, 17, 18), are suppressed by Fc{gamma}RIIB coaggregation with the BCR, leading to inhibition of cell proliferation and survival.

In addition to its catalytic activity, SHIP functions as an adaptor, binding Shc (19, 20) and p62Dok (14). Fc{gamma}RIIB binding to SHIP reportedly inhibits ras activation (21, 22). In turn, impaired downstream activation of the extracellular signal-related protein kinase (ERK) family of MAP kinases and arrest of cell cycle progression and proliferation occur. However, inhibition of ERK has also been attributed to impaired PLC{gamma}-mediated activation of PKC (23). Therefore, Fc{gamma}RIIB-mediated inhibition of ERK may occur by at least two mechanisms; SHIP linkage to Shc or Dok, and SHIP enzymatic degradation of PI(3, 4, 5)P3, preventing activation of PKC.

The role of SHIP as a crucial regulator of cell signaling has been conclusively demonstrated through studies of SHIP gene-ablated mice. Although viable, these mice display a variety of abnormalities including shortened life span, splenomegaly, increased B cell numbers, elevated levels of basal serum Ab, and elevated Ab production upon challenge with trinitrophenol-Ficoll (24, 25). SHIP-/- B cells are characterized as hypersensitive to both constitutive and Ag-induced signals (26), displaying altered patterns of development, increased survival, and increased activation (26, 27). Strikingly, ex vivo B cells from these mice are insensitive to Fc{gamma}RIIB-mediated inhibition of calcium mobilization, Akt and ERK activation, and BCR-induced proliferation (25, 27).

Although these results suggest that SHIP is the sole mediator of inhibitory signaling in primary B cells, other recent studies indicate that the 5-phosphatase, SHIP-2, also binds to the phosphorylated (p)-ITIM sequence in the cytoplasmic tail of Fc{gamma}RIIB (28, 29). Although not previously detected in primary B cells, SHIP-2 is highly expressed in nonhemopoietic cells (30, 31, 32) and in select T and B cell lines (33, 34). SHIP-2 closely resembles SHIP in both structure and enzymatic activity, but is the product of a distinct gene (35). SHIP-2-deficient mice display loss of negative regulation of insulin signaling and die shortly after birth (36). Recent results have demonstrated that enforced expression of SHIP-2 in phosphatidylinositol 3-phosphatase-deficient glioblastoma cells results in hydrolysis of PI(3, 4, 5)P3, generating PI(3, 4)P2 (37). In addition, constitutive Akt activation is abolished, and cell cycle progression is arrested in G1. These results demonstrate that SHIP-2 is a potent negative regulator of PI(3, 4, 5)P3-mediated signals.

In the studies presented here, we unexpectedly found that LPS-activated B cells from SHIP-deficient mice exhibit significant Fc{gamma}RIIB-mediated inhibition of calcium mobilization and Ins(1, 4, 5)P3 production, as well as ERK and Akt phosphorylation. This inhibition correlates with the expression of SHIP-2 in activated, but not resting, primary B cells. Fc{gamma}RIIB-dependent degradation of PI(3, 4, 5)P3 and conversion into PI(3, 4)P2 occur in SHIP-deficient B cell blasts, thus supporting a role for SHIP-2 enzymatic activity. The observed degradation of PI(3, 4, 5)P3 during Fc{gamma}RIIB signaling occurs with a concomitant reduction in B cell proliferation, suggesting that both SHIP and SHIP-2 can function in trans to block proliferative signals generated by LPS activation. Therefore, Fc{gamma}RIIB and its effectors, SHIP and SHIP-2, may play important roles in immune complex-mediated termination of ongoing immune responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and Abs

F(ab')2 and intact purified rabbit anti-mouse IgG and IgM (H+L) Abs were purchased from Zymed (South San Francisco, CA), and used for cell stimulation. Abs directed against the following molecules were used for immunoblot analysis. Polyclonal rabbit anti-Akt, p-Akt, ERK, and p-ERK were obtained from Cell Signaling. Polyclonal rabbit anti-SHIP was prepared as previously described (15) (NEB, Beverly, MA). Polyclonal rabbit anti-SHIP-2 was a generous gift from S. Swendemen and B. Clarkson (32). LPS (055:B5) and wortmannin were obtained from Sigma (St. Louis, MO).

Animals and cells

All animals used in this study were aged-matched 6- to 10-wk-old SHIP-/- mice and control SHIP+/+ littermates generated as the F1 progeny of SHIP+/- mice (24). Usually mice between 6 and 8 wk old were used to avoid the pathology associated with aged SHIP-/- mice. Splenic B cells were prepared as previously described (38). Briefly, spleens were excised from mice, cells were dispersed, and RBCs were lysed using Gey’s solution. Splenocytes were depleted of T cells by complement lysis using HO13.4 and T24 Abs, and B cells were further purified by discontinuous Percoll density gradient centrifugation (p > 1.07). For certain experiments, purified B cells were cultured with 25 µg/ml LPS in 20% FBS with 100 IU/ml penicillin and 100 µg/ml streptomycin for 48 h.

Calcium mobilization

For measurements of intracellular free calcium, 106 cells/ml in IMDM cells were loaded with Indo-AM (Molecular Probes, Eugene, OR) and stimulated with F(ab')2 or intact anti-IgM Ab. Mean intracellular free calcium was evaluated over time using a flow cytometer (model 50H; Ortho Diagnostic Systems, Raritan, NJ) with appended data acquisition system and MultiTime software (Phoenix Flow Systems, San Diego, CA) as previously described (38).

Measurement of PI(3, 4, 5)P3 and PI(3, 4)P2 generation

Splenic B cells from SHIP-/- or SHIP+/+ control littermates were depleted of RBCs and T cells and further purified by discontinuous Percoll density gradient centrifugation as described above. Cells were cultured for 48 h in 20% FBS as described above with the addition of 25 µg/ml LPS (incorporation of 32P label into the cellular ATP pool requires actively metabolizing cells). After 48 h in culture, cells were harvested, washed three times, and incubated for 90 min at 107 cells/ml in low phosphate medium with 0.5 mCi/ml [32P]orthophosphate. 32P-labeled cells were stimulated with F(ab')2 or intact anti-IgG for the indicated time and immediately lysed in 2.4 N HCl/methanol/chloroform (1:0.9:1.4 v/v). Lipids were extracted, deacylated with methanol/25% methylamine/n-butanol, and analyzed by HPLC on a SAX ion exchange column (Phenomenex, Torrance, CA) as previously described (26, 39).

Measurement of Ins(1, 4, 5)P3 generation

Ins(1, 4, 5)P3 generation was measured using a [3H] radioreceptor inhibition assay kit (DuPont-NEN, Boston, MA) according to the manufacturer’s instructions. Splenic B cells used in this study were cultured with LPS for 48 h as described above.

B cell stimulation and cell lysis

The cells were washed with IMDM three times and resuspended in IMDM. After prewarming at 37°C for 10 min, cells were stimulated with intact or F(ab')2 rabbit anti-mouse Ig (H + L) for the period indicated. After stimulation, the cells were washed three times with ice-cold PBS and lysed with solubilizing buffer (1% Triton X-100, 10 mM Tris pH 7.5, 150 mM NaCl, 0.4 mM EDTA, 10 mM NaF, 2 mM Na3VO4, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml {alpha}1-antitrypsin, and 1 mM PMSF), and cleared supernatants were retained for further processing.

Immunoblotting analysis

Cleared cell lysates were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). After blocking, polyvinylidene difluoride membranes were blotted with the indicated Ab and detected using the ECL Western blotting system (Amersham, Little Chalfont, U.K.). In some cases, the membranes were stripped to remove the Ab and subject to sequential blotting with other Abs. The membranes were incubated with the anti-p-AKT, -Akt, -SHIP, -SHIP-2, -p44/p42 ERK, or -phospho-p44/p42 ERK Ab followed by incubation with HRP-conjugated protein A.

In vitro B cell proliferation

Freshly purified splenic B cells (1 x 105/100 µl) were cultured with 20 µg/ml LPS as described above. After 24 h of LPS stimulation, 40 µg/ml of intact or F(ab')2 anti-IgM Ab was added. After 1–72 h of LPS stimulation, cells were incubated with 1 uCi/well of [3H]thymidine (Amersham) and harvested 4 h after thymidine addition. Experiments were performed in triplicate wells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fc{gamma}RIIB-mediated inhibition of calcium mobilization occurs independently of SHIP in LPS-activated B cells

Fc{gamma}RIIB coaggregation with the BCR leads to inhibition of Ins(1, 4, 5)P3 generation and calcium mobilization. A significant role for SHIP in mediating inhibitory signals has been shown through recent biochemical and genetic studies. Examination of ex vivo B cells from SHIP-deficient mice revealed ablation of Fc{gamma}RIIB-mediated inhibition of the calcium response (25), leading to substantial increases in extracellular calcium influx (Fig. 1GoA). Although ex vivo B cells from SHIP-deficient mice demonstrated virtually complete ablation of the inhibitory response through Fc{gamma}RIIB, a limited number of studies have indicated that signaling may be altered in LPS-activated B cell blasts (40, 41). To test the role of SHIP in these populations, B cells from SHIP-deficient or wild-type littermates were activated with the mitogen, LPS, for 48 h. Calcium mobilization responses were monitored after cross-linking the BCR with F(ab')2 of rabbit anti-mouse Ig or with intact Ab to coaggregate Fc{gamma}RIIB with the BCR. In marked contrast to resting B cells, LPS blasts derived from SHIP-/- mice exhibited significant Fc{gamma}RIIB-mediated inhibition of calcium influx and Ins(1, 4, 5)P3 production (Fig. 1Go, B and C). Thus, partially distinct mechanisms must mediate Fc{gamma}RIIB signaling in resting and activated B cells.



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  		FIGURE 1. Calcium mobilization and Ins(1,4,5)P3 production are inhibited by Fc{gamma}RIIB coaggregation in activated B cells from SHIP-deficient mice. B cells derived from either SHIP-/- or wild-type littermates were loaded with Indo-1, and intracellular free calcium levels were monitored by flow cytometry. Cells were stimulated with either 10 µg/ml of F(ab')2 anti-Ig to aggregate the BCR or with equal molar concentrations of intact Ab to coaggregate Fc{gamma}RIIB. The analysis was conducted under conditions of 70–100 nM buffered extracellular free calcium initially, and later CaCl2 was added to achieve a final free Ca2+ concentration of 1.3 mM. Calcium mobilization responses in freshly isolated resting B cells (A) or 48-h LPS blasts (B) are shown. C, Ins(1,4,5)P3 levels in SHIP-/- and control B cell LPS blasts after stimulation with F(ab')2 anti-Ig (12 µg/ml) or equal molar concentrations of intact Ab are shown.

 
BCR-mediated Akt phosphorylation is inhibited by Fc{gamma}RIIB coaggregation in SHIP-deficient B cell blasts

The inhibition of calcium mobilization and PLC{gamma}-induced Ins(1, 4, 5)P3 generation observed in SHIP-/- B cell blasts prompted us to examine whether additional Fc{gamma}RIIB-mediated responses were similarly attenuated. Fc{gamma}RIIB-mediated recruitment of SHIP leads to inhibition of activation of the antiapoptotic enzyme Akt by preventing its PI(3, 4, 5)P3-dependent translocation to the plasma membrane (16, 18). Subsequent serine/threonine phosphorylation is consequently reduced, resulting in attenuated Akt activation. Consistent with studies by Helgason (27), examination of Akt phosphorylation following Fc{gamma}RIIB coaggregation with the BCR revealed inhibition in wild-type but not SHIP-deficient resting primary B cells (Fig. 2GoA). In contrast, Fc{gamma}RIIB-mediated inhibition of BCR-induced Akt phosphorylation was observed in both wild-type and SHIP-deficient B cell blasts (Fig. 2GoB), particularly at later time points. Therefore, unlike resting B cells, SHIP-/- B cell blasts retain Fc{gamma}RIIB-mediated inhibition of Akt activation, supporting the hypothesis that additional inhibitory signals function in activated B cells.



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  		FIGURE 2. BCR-mediated Akt phosphorylation is inhibited by Fc{gamma}RIIB coaggregation in activated B cells from SHIP-deficient mice. Resting (A) or activated (B) B cells (5 x 106) from wild-type or SHIP-deficient mice were either left unstimulated (U) or were stimulated for the indicated times with either 10 µg/ml F(ab')2 (F) anti-mouse Ig to aggregate the BCR or with equal molar concentrations of intact Ab (I) to coaggregate Fc{gamma}RIIB. Total cell lysates were run on SDS-PAGE and blotted with anti-phospho-Akt (Ser473) Ab, then reprobed for total Akt protein.

 
BCR-mediated ERK phosphorylation is inhibited by Fc{gamma}RIIB coaggregation in SHIP-deficient B cell blasts

Two possible mechanisms have been proposed to account for SHIP-mediated inhibition of ERK activation following Fc{gamma}RIIB coaggregation with the BCR. ERK inhibition could occur through attenuation of PLC{gamma}-mediated PKC signaling (23). Alternatively, ERK inhibition could occur through attenuation of ras activation (21), because SHIP also mediates recruitment of rasGAP via p62Dok (14).

Previous reports (25, 27) and Fig. 3GoA, demonstrate that SHIP deficiency abolishes Fc{gamma}RIIB-mediated inhibition of ERK signaling in ex vivo B cells. In contrast, LPS-activated B cells from SHIP-/- mice display inhibition of ERK phosphorylation (Fig. 3GoB). It should be noted that the kinetics of inhibition were delayed compared with resting splenocytes, with at least 30 min required before a reduction in phosphorylation was apparent. Fc{gamma}RIIB-mediated inhibition of ERK has been attributed to phosphorylation and recruitment of p62Dok. However, previous studies by Tamir et al. have demonstrated that Fc{gamma}RIIB-induced phosphorylation of Dok is completely absent in SHIP-deficient B cell blasts (14). Therefore, in activated cells, Fc{gamma}RIIB-mediated inhibition of ERK occurs independently of p62Dok phosphorylation, possibly through attenuation of PKC activation.



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  		FIGURE 3. BCR-mediated ERK phosphorylation is inhibited upon Fc{gamma}RIIB coaggregation in activated B cells from SHIP-deficient mice. Resting (A) or activated (B) B cells (2 x 106) from wild-type or SHIP-deficient mice were left unstimulated (U) or were stimulated for the indicated times with either 10 µg/ml F(ab')2 (F) anti-IgM to aggregate the BCR or with intact Ab (I) to coaggregate Fc{gamma}RIIB. Total cell lysates were run on SDS-PAGE, blotted with anti-phospho-ERK Ab, and reprobed for total ERK.

 
BCR-mediated PI(3, 4, 5)P3 accumulation is attenuated by Fc{gamma}RIIB coaggregation in SHIP-/- B cell blasts

Because calcium mobilization and phosphorylation of Akt and ERK are dependent upon PI(3, 4, 5)P3 generation, we next determined whether the Fc{gamma}RIIB-mediated inhibition of signaling in SHIP-/- B cells reflected reduced PI(3, 4, 5)P3 accumulation. To determine the enzymatic activity of SHIP in B cell blasts, levels of PI(3, 4, 5)P3 and its breakdown product, PI(3, 4)P2, were measured by HPLC (26, 39, 42). B cell blasts generated from SHIP-/- or control littermates were stimulated with F(ab')2 of anti-BCR or intact Ab to coaggregate Fc{gamma}RIIB with the BCR. Fig. 4GoA shows that coaggregation of the BCR and Fc{gamma}RIIB resulted in nearly total hydrolysis of PI(3, 4, 5)P3 in wild-type cells. Approximately 99% of PI(3, 4, 5)P3 in wild-type cells was degraded to PI(3, 4)P2 even at the earliest measurable time point, 30 s. As previously described (26), there are significant increases in PI(3, 4, 5)P3 levels following BCR aggregation in the SHIP-/- B cells, suggesting that SHIP may negatively regulate signaling through the BCR. Surprisingly, enzymatic degradation of PI(3, 4, 5)P3 and resultant PI(3, 4)P2 formation were still apparent in the SHIP-deficient B cell blasts (Fig. 4GoB), suggesting the action of an additional inositol 5-phosphatase. Therefore, multiple pathways control the levels of PI(3, 4, 5)P3 in activated B cells.



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  		FIGURE 4. BCR-mediated PI(3,4,5)P3 signals are terminated by Fc{gamma}RIIB coaggregation in activated B cells from SHIP-/- mice. LPS-activated B cells from SHIP-/- or control littermates were labeled with [32P]orthophosphate for 1.5 h. The SHIP-deficient and wild-type cells were then stimulated in parallel for the indicated times with either 12 µg/ml F(ab')2 anti-Ig to aggregate the BCR or with equal molar concentrations of intact Ab to coaggregate Fc{gamma}RIIB with the BCR. After stimulation, the cells were immediately lysed with methanol/chloroform. Phospholipids were extracted, deacylated, and fractionated by HPLC. The fractions containing PI(3,4,5)P3 (A) and the SHIP breakdown product PI(3,4)P2 (B) were quantitated by liquid scintillation. Each data point represents 5 x 106 cells. Cells depicted in B were stimulated for 30 s.

 
Induction of SHIP-2 and SHIP expression upon B cell activation

Because the above results demonstrate an additional inositol 5-phosphatase activity present and operative in SHIP-/- B cell blasts, we were prompted to assess SHIP-2 expression in these cells. SHIP-2 is widely expressed (30), while SHIP is restricted to cells of hemopoietic lineage (43). Although SHIP-2 has recently been shown to become phosphorylated and associate with Fc{gamma}RIIB in the A20 B cell line (28, 29), its expression in primary B cells has not been observed. As shown in Fig. 5Go, SHIP-2 was not detected in resting B cells, but expression was induced following stimulation for 48 h with LPS. Equal levels of SHIP-2 were detected in both wild-type and SHIP-deficient LPS blasts. In addition, levels of SHIP increased 10-fold following B cell activation. In comparison, levels of total p42/44 ERK remained relatively constant throughout activation. A time course analysis revealed that increases in SHIP and SHIP-2 occur after 24 h of stimulation with LPS (data not shown). Therefore, in resting cells, SHIP is likely to be the primary mediator of inhibitory signaling, while in activated cells, both SHIP and SHIP-2 contribute. Upon B cell activation, SHIP-2 apparently also contributes to the inhibition of calcium mobilization, Ins(1, 4, 5)P3 generation, Akt/ERK phosphorylation, as well as PI(3, 4, 5)P3 degradation, seen following Fc{gamma}RIIB coaggregation with the BCR.



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  		FIGURE 5. Expression of both SHIP and SHIP-2 is induced upon B cell activation. A total cell lysate (wild-type or SHIP-/-) of 107 resting B cells or 48-h LPS blasts were subjected to SDS-PAGE. Transferred blots were probed with anti-SHIP, anti-SHIP-2, or anti-ERK Abs as indicated.

 
SHIP-/- B cell blasts are sensitive to Fc{gamma}RIIB-mediated inhibition of proliferation

Coaggregation of Fc{gamma}RIIB inhibits downstream BCR-mediated cell proliferation. Previous studies have shown that Fc{gamma}RIIB-mediated inhibition of BCR-induced proliferation is completely ablated in ex vivo B cells from SHIP-deficient mice (25, 27). To determine whether Fc{gamma}RIIB can function to block proliferation of activated B cells, ex vivo B cells were stimulated for 24 h with LPS and then treated with F(ab')2 of anti-BCR or with intact Ab to coaggregate Fc{gamma}RIIB with the BCR. As shown in Fig. 6Go, B cells continued to proliferate following aggregation of the BCR; however, coaggregation of Fc{gamma}RIIB resulted in attenuation of proliferation. Thus Fc{gamma}RIIB can function in trans to inhibit proliferation stimulated by LPS treatment. Interestingly, SHIP-/- B cell blasts were also sensitive to Fc{gamma}RIIB-mediated inhibition of ongoing proliferation, suggesting that the inhibition can be mediated by SHIP-2. It is likely that Fc{gamma}RIIB-mediated inhibition is linked to degradation of PI(3, 4, 5)P3, because the addition of wortmannin had similar antiproliferative effects (Fig. 6Go). Therefore, in B cell blasts, SHIP- and SHIP-2-mediated degradation of PI(3, 4, 5)P3 are likely to contribute to Fc{gamma}RIIB-mediated inhibition of proliferation. It should be noted that PLC{gamma} activation, calcium mobilization, Akt, PKC, and MAP kinase activation all have been implicated as regulators of cell cycle progression. Consequently, it remains to be determined which of these PI(3, 4, 5)P3-regulated pathways, singly, or in combination, contributes to inhibition of cell cycle progression induced by Fc{gamma}RIIB coaggregation.



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  		FIGURE 6. B cells preactivated by LPS are sensitive to Fc{gamma}RIIB-mediated inhibition of proliferation. Purified B cells (1.5 x 105) from SHIP-/- mice or wild-type littermates were stimulated with LPS at time 0. A, At the indicated time points, cells were pulsed with thymidine to follow the kinetics of B cell activation. Twenty-four hours after stimulation with LPS, the cells were then restimulated with either 40 µg/ml F(ab')2 anti-IgM to aggregate the BCR or with equal molar concentrations of intact Ab to coaggregate Fc{gamma}RIIB and BCR. The kinetics of proliferation were then followed for an additional 30 h. Some cells were treated with LPS for 24 h as described above, then incubated with 200 nM wortmannin for an additional 24 h as indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
SHIP activation and association with Fc{gamma}RIIB inhibits BCR-mediated signaling, resulting in a blockade of PLC{gamma} activation, calcium mobilization, ERK and Akt phosphorylation, and downstream blastogenesis (2, 8). Previous reports have shown that resting ex vivo B cells from SHIP-deficient mice are insensitive to Fc{gamma}RIIB-mediated inhibition (25, 27). In contrast, we report that LPS-activated B cells from SHIP-deficient mice are significantly inhibited by signals mediated through Fc{gamma}RIIB. Surprisingly, SHIP-deficient LPS blasts display prominent Fc{gamma}RIIB-mediated inhibition of calcium mobilization, Ins(1, 4, 5)P3 production, as well as Akt and ERK phosphorylation.

We attribute this inhibition to the expression of SHIP-2 in LPS B cell blasts. Although not detectable in resting splenocytes, we find that SHIP-2 is inducibly expressed upon B cell activation. Previous in vitro analysis of SHIP-2 enzymatic activity has revealed that SHIP and SHIP-2 have similar functions (35). Furthermore, SHIP and SHIP-2 have partially homologous SH2 domains (30) and nearly identical affinity for the p-ITIM in the cytoplasmic tail of Fc{gamma}RIIB (29). Together, this suggests that the Fc{gamma}RIIB-mediated degradation of PI(3, 4, 5)P3 observed in SHIP-deficient B cell blasts is mediated by de novo expression of SHIP-2. Concomitantly, Fc{gamma}RIIB expression is increased 10-fold upon LPS stimulation (41). Therefore, in activated B cells, the up-regulation of Fc{gamma}RIIB, SHIP, and SHIP-2 all may contribute to the inhibition of phosphatidylinositol 3-kinase (PI3-K) signaling pathways induced by immune complexes.

Although SHIP and SHIP-2 have similar phosphatase activities and affinity for Fc{gamma}RIIB, their divergent amino acid sequences suggest differing linker function. Both SHIP and SHIP-2 bind to the adapter molecule, Shc (6, 32), but SHIP-2 selectively binds the SH3 domain of abl, while SHIP binds the SH3 domain of src (32). Surprisingly, SHIP-2, unlike SHIP, does not associate with grb-2 (32). Examination of the C-terminal domain of SHIP-2, a region essential for SHIP translocation and function in vivo (44, 45, 46), reveals that SHIP-2 contains only one NPXY motif (30). Furthermore, because Fc{gamma}RIIB ligation in SHIP-/- blasts does not lead to enhanced Dok phosphorylation, we suspect that Fc{gamma}RIIB coaggregation does not induce an association between SHIP-2 and p62Dok (14). This indicates that SHIP and SHIP-2 associate with different sets of effectors, and thus may have partially distinct function, in activated B cells.

SHIP and, more recently, SHIP-2 have been strongly implicated as negative regulators in a variety of cell types. SHIP has demonstrated activity as an inhibitor of cytokine and growth factor-induced signaling in mast cells (47). Because mast cell degranulation via Fc{epsilon}RI (48) or steel factor receptor (49) is significantly enhanced in SHIP-deficient bone marrow-derived mast cells, SHIP has been described as the ’"gatekeeper" for mast cell degranulation. Furthermore, SHIP has been linked to inhibition of growth factor-mediated proliferation in these cells (50). SHIP-2 also negatively regulates signaling events induced by numerous growth factor receptors and insulin (36). Consistent with the above studies, a recent report has directly demonstrated that SHIP-2 expression in glioblastoma cells suppresses growth responses (37).

Previous studies of T cells have shown a requirement for PI3-K for cell proliferation, because PI(3, 4, 5)P3-mediated activation of Akt is sufficient to induce E2F activity (51). PI3-K is required for phosphorylation of Rb, induction of cyclin D3, and degradation of p27Kip1 (51). These results establish a crucial link between PI3-K and the cell cycle machinery. PI3-K-mediated activation of Akt also promotes cell survival signals through NF-{kappa}{beta} phosphorylation and activation of the I{kappa}-{beta} kinase (52). In addition, Akt phosphorylation inhibits the proapoptotic activities of Bad (53) and caspase-9 (54). We find that activated B cells, like T cells, are likely to have similar requirements for PI3-K activity, as wortmannin significantly inhibits both B and T cell proliferation. Consistent with studies presented here, T lymphocytes have been shown to increase expression of SHIP-2 following activation (33). These results suggest that induction of SHIP and/or SHIP-2 and subsequent degradation of PI(3, 4, 5)P3, may be a common mechanism used by hemopoietic cells to modulate the effects of stimulation.

The above results support a model in which mitogenic stimulation of quiescent B cells triggers PI(3, 4, 5)P3 generation, Akt activation, and cell cycle progression. Support for this model comes from studies that demonstrate that the addition of wortmannin inhibits this progression. In contrast, elevated basal levels of PI(3, 4, 5)P3 can trigger unregulated cell cycle progression, lymphocyte hyperproliferation, and autoimmunity (55), indicating that PI(3, 4, 5)P3 normally limits these responses. Taken together with the fact that mice deficient in SHIP or PTEN, a phosphatidylinositol 3-phosphatase tend toward lymphocyte hyperproliferation, the precise regulation of PI(3, 4, 5)P3 levels within the cell are of utmost importance. We suggest that in normal cells, immune complex-induced degradation of PI(3, 4, 5)P3 counterbalances the effects of mitogenic stimulation and causes arrest of cell cycle progression. This may explain the attenuating effects of Fc{gamma}RIIB late in the immune response.

Intriguingly, we and others have found that Fc{gamma}RIIB-mediated inhibition can function to block proliferative signals generated by LPS or IL-4 stimulation (56), even in the absence of direct coaggregation with these receptors. Thus, cross-talk between LPS- or IL-4-mediated activating signals and Fc{gamma}RIIB-mediated inhibitory signals must occur. In contrast, killer Ig-related receptors require direct coassociation with the activating receptor to attenuate signaling. This may reflect the fact that killer Ig-related receptors tether SH2 domain-containing protein tyrosine phosphatase, which can act in only a localized area. In contrast, by efficiently reducing PI(3, 4, 5)P3 levels cell-wide, SHIP can indirectly inhibit signaling by all receptors that require PI(3, 4, 5)P3 generation. Thus, the negative signal transmitted upon B cell encounter with immune complexes likely inhibits responses to many ligands.

Although activated or proliferating B cell blasts may represent an important target of immune complex-mediated inhibition of the immune response, elucidation of the inhibitory signaling mechanisms operative in this B cell subset is only beginning. Activated B cells differ from resting B cells in their significantly elevated levels of SHIP, SHIP-2, and Fc{gamma}RIIB. Thus, these cells may be more sensitive to negative feedback regulation by immune complexes.


   Acknowledgments
 
We thank Drs. Steve Swendeman and B. Clarkson for the anti-SHIP-2 Ab and Drs. Stephan Gauld and Robert Benschop for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health. J.C.C. is the Ida and Cecil Green Professor of Cell Biology. S.M. is supported by a grant from the American Foundation for AIDS Research. A.B. is supported by a grant from the Colorado Cancer League. C.D.H. is the recipient of a British Columbia Health Joint Research Scholarship. Back

2 Address correspondence and reprint requests to Dr. John C. Cambier, Integrated Department of Immunology, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: cambierj{at}njc.org Back

3 Abbreviations used in this paper: BCR, B cell Ag receptor; PI3-K, phosphatidylinositol 3-kinase; ERK, extracellular signal-related protein kinase; ITIM, immunoreceptor tyrosine-based inhibitory motif; SH2, Src homology domain 2; SHIP, SH2 containing inositol 5-phosphatase; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PI(3,4)P2, phosphatidylinositol 3,4,-bisphosphate; PLC{gamma}, phospholipase C{gamma}; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; PKC, protein kinase C; MAP, mitogen-activated protein; p, phosphorylated. Back

Received for publication January 24, 2001. Accepted for publication April 27, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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