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The Journal of Immunology, 2005, 175: 5034-5042.
Copyright © 2005 by The American Association of Immunologists

Tight Regulation of IFN-{gamma} Transcription and Secretion in Immature and Mature B cells by the Inhibitory MHC Class I Receptor, Ly49G21

Gili Hart, Liat Flaishon, Shirly Becker-Herman and Idit Shachar2

Department of Immunology, Weizmann Institute of Science, Rehovot, Israel


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
To complete their maturation and to participate in the humoral immune response, immature B cells that leave the bone marrow are targeted to specific areas in the spleen, where they differentiate into mature cells. Previously, we showed that immature B cells actively down-regulate their integrin-mediated migration to lymph nodes or sites of inflammation, enabling their targeting to the spleen to allow their final maturation. This inhibition is mediated by IFN-{gamma}, which is transcribed and secreted at low levels by these immature B cells and is down-regulated at the mature stage. The activating MHC class I receptor, Ly49D, which is expressed at high levels on immature B cells, stimulates this IFN-{gamma} secretion. In this study we show that B cells coexpress the inhibitory MHC class I receptor, Ly49G2. In addition, we demonstrate a tight regulation in the expression of the Ly49 family members on B cells that depends on their cell surface levels. High levels of Ly49G2 have a dominant inhibitory effect on Ly49D expressed at low levels on immature bone marrow and mature B cells, resulting in inhibition of IFN-{gamma} secretion. However, low levels of the inhibitory receptor, Ly49G2, coexpressed with high levels of the activating receptor, Ly49D, on the immigrating immature B cells enable the secretion of specific low levels of IFN-{gamma}. This expression pattern insures the inhibitory control of peripheral immature B cell to prevent premature encounter with an Ag while enabling entry to the lymph nodes during the mature stage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The differentiation pathway from stem cell to mature B lymphocyte can be divided into several stages, characterized by differentiation processes, proliferation phases, and control steps. Precursor B cells differentiate into immature B lymphocytes after successfully expressing a surface Ig receptor (IgM). These immature B cells emerge from the bone marrow (BM)3 to the periphery and migrate into the spleen for their final maturation step. This migration proceeds through the terminal branches of central arterioles to blood sinusoids of the marginal zone (1, 2). Before arriving at the spleen, the optimal site of their maturation, immature B cells are excluded from nonsplenic secondary lymphoid organs and from sites of infection and inflammation, where Ag encounter could lead to effective clonal elimination because of the negative selection process.

Previously we demonstrated that immature B cells can down-regulate their own integrin-mediated adhesion to the extracellular matrix and thereby suppress their migration into nonsplenic sites (3). This inhibition is mediated by IFN-{gamma}, which is transcribed and secreted at low levels by immature B cells. The inhibitory signal of IFN-{gamma} is transmitted through the IFN-{gamma}R, whose engagement leads to inhibition of cytoskeleton rearrangement, which is required for promoting integrin-mediated adhesion and migration of B cells (4, 5). We have shown that IFN-{gamma} secretion is regulated by the MHC class I receptor, Ly49D. This activating receptor is expressed on peripheral immature B cells and recognizes MHC class I on peripheral tissues. Ly49D and MHC class I interaction induces stable secretion of IFN-{gamma} by immature B cells, thereby down-regulating their homing to the lymph nodes (LN) or to sites of inflammation (6).

It was previously shown that effector cells that express the activating Ly49D receptor also coexpress, at very high levels, the inhibitory Ly49G2 or Ly49A receptors, which recognize the same MHC class I ligand and dominate over the activating function (7). Co-cross-linking with anti-Ly49G2 and anti-Ly49D blocks Ly49D mediated activation and IFN-{gamma} secretion (8). Thus, engagement of activating Ly49 NK receptors in vivo appears to be constantly balanced by inhibitory forces. The biological role of such tight regulation remains unknown.

To test whether this mechanism exists in B cells, we analyzed Ly49G2 transcription and translation in immature and mature B cell populations. We show in this study that low levels of Ly49G2 are expressed on peripheral immature B cells, whereas BM immature and peripheral mature B cells elevate its levels. We suggest that the outcome of the stimulation of both inhibitory and activating Ly49 receptors depends on their cell surface levels. Low levels of Ly49D expressed on immature BM and on mature B cells can induce IFN-{gamma} transcription; however, this process is inhibited by the dominant inhibitory effect mediated by the Ly49G2 receptor. In peripheral immature B cells, Ly49G2 levels are low, allowing transcription of low levels of IFN-{gamma}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6, Ii–/– (9), Kb–/–, and Db–/– (10) mice were used at 6–8 wk of age. The animal research committee at the Weizmann Institute approved all animal procedures.

Cells and separation of B cells

Spleen and BM cells were obtained from the various mice as previously described (11, 12). The purity of the purified cells (between 95 and 98%) was analyzed by FACS after each experiment. Ii–/– B cells were purified using CD19 beads. Control IgD– cells were separated from IgD+ B cells using the MACS system. To separate the IgD– and CD21-positive and -negative populations, IgD– cells were separated according to their CD21 expression using the MACS system as previously described (6, 12). To isolate T1, T2, and mature B cells, we followed the procedures described previously (13). Briefly, the non-B cell population was depleted using a B cell-negative selection kit (Miltenyi Biotec). The purified B cells were then sorted into various populations using anti-CD21, anti-CD24, and anti-CD23 mAb: T1 cells: CD21–CD23–CD24high; T2 cells, CD21+,CD23–CD24high ’ and mature cells, CD21+CD23+CD24low (13) BM immature B cells were purified by depletion of IgD+ B cells and isolation of IgM+ B cells using the MACS system (Miltenyi Biotec).

RNA isolation and RT

Total RNA was isolated from cells using the Tri-Reagent kit (Molecular Research Center). RT was conducted using SuperScript II RT (Invitrogen Life Technologies). The primers used included: Ly49D, 5'-CCTGGCAGCTCATTGTGATAG-3' and 5'ATTCTGGCAGCTCTGTTTACATC-3'; IFN-{gamma}, 5'-CATTGAAAGCCTAGAAAGTCTG-3' and 5'-CTCATGAATGCATCCTTTTTCG-3'; hypoxanthine phosphoribosyltransferase, 5'-GAGGGTAGGCTGGCCTATGGCT-3' and 5GTTGGATACAGGCCAGACTTTGTTG-3'; and Ly49G2, 5'-GGCAATGATCTTCYGGAATCCC-3' and 5'-CGTTGTGTTCAAGGCAAGT-3'.

Abs, immunofluorescence, and flow cytometry

Staining was performed on freshly isolated splenocytes as previously described (14). The following Abs were used: RA3-6B2 anti-CD45R/B220, AMS 9.1 anti-IgD, R6-60.2 anti-IgM, B3B4 anti-CD23, 4E5 anti-Ly49D, 4D11 anti-Ly49G2, and 7G6 anti-CD21 M1/69 anti-CD24; all mAb were obtained from BD Pharmingen.

Stimulation of primary B cells

For Ly49D or Ly49G2 stimulation, 5 x 106 primary B cells were suspended in 100 µl of DMEM containing 10% (v/v) FCS. Then 5 µg of 4E5 mAb or 4D11 mAb was added to each tube, and the tubes were immediately placed at 37°C for 2.5 min. Immediately after stimulation, the cells were washed and fast-frozen in liquid N2.

Preparation of cell extract

Stimulated cells were lysed in lysis buffer containing 25 mM Tris (pH 7.4), 2 mM vanadate, 75 mM glycophosphate (pH 7.2), 2 mM EDTA, 2 mM EGTA, 10 mM tetrasodium pyrophosphate, and 0.5% Nonidet P-40 in the presence of the following protease inhibitors: 10 mg/ml leupeptin, 10 mg/ml aprotinin, 10 mg/ml pepstatin, 10 mg/ml chymostatin (Roche), 1 mM PMSF (Sigma-Aldrich), and 20 mM N-ethylmalemide (Sigma-Aldrich).

Immunoprecipitation and Western blot analysis

Protein G-Sepharose beads (Pharmacia Biotech) were conjugated to phosphorylated tyrosine (p-Tyr) mAb (Santa Cruz Biotechnology) at a 1:20 ratio for 2 h at 4°C, followed by three washes in PBS. Beads were added to the cell lysates and p-Tyr proteins were immunoprecipitated overnight. The protein G-bound material was washed three times with PBS containing 0.1% SDS and 0.5% Nonidet P-40. Lysates or immunoprecipitates were separated by 10% (w/v) SDS-PAGE. The proteins were transferred into nitrocellulose membrane and probed with anti-p-Tyr99 (Santa Cruz Biotechnology), followed by HRP-conjugated anti-mouse or anti-rabbit IgG (Jackson ImmunoResearch Laboratories) or anti-Syk (Santa Cruz Biotechnology), followed by HRP-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories).

Syk inhibition

Immature B cells from Ii–/– were incubated in the presence of 120 µM of the Syk inhibitor, piceatannol (Sigma-Aldrich) or in the presence of DMSO. After 3 h of incubation at 37°C, the cells were immediately washed, and RNA was purified.

Detection of IFN-{gamma}

Lysates of cells were separated by 10% (w/v) SDS-PAGE. IFN-{gamma} was detected as previously described (3).

Intracellular staining

Total splenocytes (106 cells/ml) were incubated in the presence of PMA (50 ng/ml) and ionomycin (750 ng/ml) for 2 h. Cells were then incubated for 4 h with brefeldin A (10 µg/ml). The cells were blocked with anti-FcR and stained for cell surface B220, then fixed in PBS with 4% paraformaldehyde and permeabilized in PBS with 0.1% saponin, 5%FCS, and 0.1% azide. After permeabilization, cells were stained with FITC-conjugated anti-mouse IFN-{gamma} ((XMG1.2; BD Pharmingen) or an FITC-conjugated isotype control (BD Pharmingen). IFN-{gamma} expression was analyzed by FACS.

Cytoskeleton rearrangement

B cells were stimulated for 1 h in the presence or the absence of anti-Ly49D, Ly49G2, or both (BD Pharmingen) at 37°C as described above. Cytoskeleton rearrangement was evaluated as previously described (4).

ELISA

Ii–/– immature CD19+ cells were stimulated in the presence or the absence of anti-Ly49G2 as described above. Cell supernatants were collected, and IFN-{gamma} levels were determined by ELISA (BD Pharmingen) according to the manufacturer’s instructions.

Tracking of cells in vivo

Ii–/– B cells were stimulated in the presence or the absence of anti-Ly49G2 or control Ab (anti-CD8). After 1 h, the cells were labeled with 5 µM CFDA-SE (Molecular Probes) for 15 min at room temperature. The cells were then injected into control mice, and spleen and LN were analyzed 3 h later by FACS.

Calculation of ratio

The ratio in each figure was calculated as the intensity of a band divided by the intensity of hypoxanthine phosphoribosyltransferase, tubulin, or an unrelated band in untreated or lesser expressing cells, was normalized to 1. The ratio for the treatment was calculated as the intensity for that treatment relative to 1.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
B cells express active Ly49G2 receptor on their surface that is up-regulated throughout maturation

The Ly49D and Ly49G2 receptors are commonly expressed as pairs on NK cells (8). To determine whether B cells express both receptors as well, we first analyzed the transcription of Ly49G2 in immature and mature cells. IgD– peripheral immature B cells were purified from either control (C57BL/6; Fig. 1A) or invariant chain deficient (Ii–/–) mice (Fig. 1B), whose B cells are arrested at an immature stage (14). Low levels of Ly49G2 message were detected in immature B cells from both control and Ii–/– mice, which transcribe (Fig. 1B) and express (Fig. 1C) IFN-{gamma}. However, a dramatic up-regulation of Ly49G2 mRNA levels was observed in the mature (IgD+CD19+) cells (Fig. 1A), and the total control B population (CD19+), which consist mostly of mature cells, with ~10% immature B cells (Fig. 1B), expressed little, if any, IFN-{gamma} protein (Fig. 1C). To further show that Ly49G2 is transcribed in B cells, cells were separated using anti-IgM Abs, resulting in enrichment of the entire peripheral B population. In control mice, this constitutes a mixture containing mostly mature B cells, whereas in the Ii–/– mouse, it consists of mostly immature B cells. As shown in Fig. 1D, high levels of Ly49G2 mRNA were detected in control B cells, whereas low levels of this message were detected in the Ii–/– population.



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  		FIGURE 1. Ly49G2 message is expressed at low levels on immature B cells and is up-regulated throughout differentiation to mature cells. A and B, Ly49G2 expression in immature (IgD–CD19+) and mature (IgD+) B cells derived from control mice (A) and total B cells (CD19+) from Ii–/– and control mice. RT-PCR was performed as described in Materials and Methods. C, Intracellular staining of IFN-{gamma} in control and Ii–/– B220+ cells. D and E, IgM+ B cells from control or invariant chain-deficient (Ii–/–) mice (D) and control IgD+CD21–, IgD–CD21+, and IgD–CD21–B220+, Ii–/–IgD–CD21+, and IgD–CD21–B220+ (E) were purified. RT-PCR was performed as described in Materials and Methods. F, T1, T2, and mature B populations were purified as described in Materials and Methods. RT-PCR was performed as described in Materials and Methods. The results presented are representative of at least three different experiments.

 
Newly emigrant immature B cells in the spleen can be separated from marginal zone B cells by their surface expression of CD21. To determine whether the marginal zone B cell population transcribes the Ly49G2 message, we analyzed Ly49G2 mRNA in CD21+ and negative IgD– populations from control and Ii–/– mice. As shown in Fig. 1E, higher levels of the Ly49G2 message were detected in the IgD+CD21– mature population compared with the control IgD–CD21– immature B cells, and Ly49G2 message was hardly observed in the marginal zone B cells, IgD-CD21+ cells. Taken together, Ly49G2 is transcribed mostly in follicular mature B cells.

The newly arrived immature B cells enter the spleen through the central arteriole and develop into T1 cells, which are located at the outer periarteriolar lymphoid sheath. They then develop into T2 cells, which are situated within primary follicles and adjacent to mature cells (15). To determine at which specific stage of differentiation Ly49G2 transcription is up-regulated, we sorted these populations using cell surface markers attributed to each population (13) and followed their Ly49D and Ly49G2 messages by RT-PCR. As shown in Fig. 1F, although Ly49D message was down-regulated during the differentiation from T1 to T2 B cells, Ly49G2 mRNA was increased during the transition to T2 cells. Thus, Ly49D and Ly49G2 RNA levels were inversely regulated over the course of B cell maturation. Cells that expressed high levels of Ly49D showed low levels of Ly49G2 and vice versa.

To directly show cell surface expression of Ly49G2 on mature B cells, B cells derived from control or Ii–/– mice were analyzed by FACS for Ly49G2 expression. As shown in Fig. 2A, Ii–/– immature B cells expressed lower levels of Ly49G2 than the control B populations (B220+, IgM+, or CD19+). However, Ly49G2 expression levels on mature B cells were still lower than the cell surface Ly49G2 levels on the B220-negative population or NK1.1 cells (Fig. 2B). In addition, control immature B cells (IgD– or CD23–B220+ cells) expressed lower levels of Ly49G2 receptor, which was significantly elevated on the mature (IgD+ or CD23+) population (Fig. 2C). These results show that, unlike Ly49D, which is expressed on immature B cells and dramatically down-regulated during differentiation to mature cells (6), Ly49G2 expression follows an inverse pattern and is expressed mostly on mature cells.



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  		FIGURE 2. Ly49G2 receptor is enriched on the surface of mature B cells. A, Splenocytes from control and Ii–/– mice were double stained with: lane 1, anti-B220 and Ly49G2 (solid line) or an isotype control Ab (dashed line); lane 2, anti-IgM and anti-Ly49G2 Abs; or lane 3, anti-CD19 and anti-Ly49G2. Histograms show isotype control or Ly49G2 expression on B220+/IgM+ cells or on CD19+ cells. Overlay, Control (black) line over Ii–/– (gray) line. B, Splenocytes from control and Ii–/– mice were double stained with anti-B220 and anti-Ly49G2 (lane 1) or anti-NK1.1 (lane 2) and anti-Ly49G2. Histograms show Ly49G2 expression on the B220-negative or NK1.1-positive populations. C, Splenocytes from control mice were stained with anti-Ly49G2, anti-IgD (lane 1), or anti-CD23 (lane 2), and anti-B220 Abs. Histograms show Ly49G2 expression on mature B220+CD23+ or B220+IgD+ and immature B220+CD23– or B220+IgD– cells. Overlay, Mature (black) line over immature (gray) line. The results presented are representative of five different experiments.

 
To determine whether incubation of immature and mature B cells with anti-Ly49G2 Ab induces a signaling cascade, Ii–/– B cells and control B cells were stimulated with anti-Ly49G2 (4D11) Abs. As shown in Fig. 3, A and B, anti-Ly49G2 induced a specific phosphorylation of several proteins, peaking 2.5 min after stimulation, showing that Ly49G2 expressed on these cells is active. Moreover, activation of Ly49G2 on Ii–/– B cells resulted in stimulation, but to a lesser extent (Fig. 3C).



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  		FIGURE 3. Ly49G2 receptor expressed on B cells is active. A–C, Control (A and B) or Ii–/– (C) B cells were stimulated with anti-Ly49G2 (4D11; A–C) or anti-CD8 (A) Abs. Lysates were separated on SDS-PAGE and blotted with anti-p-Tyr or anti-tubulin Ab. The results presented are representative of three different experiments.

 
We have previously shown that immature BM B cells express low levels of Ly49D. After their exit from the BM compartment, Ly49D is up-regulated on the peripheral immature population (6). To determine the expression of Ly49G2 in the immature BM cells, IgM+IgD– BM B cells were separated, and their Ly49G2 mRNA levels were compared with those of peripheral immature (CD19+IgD–) and mature (IgD+) populations. As shown in Fig. 4A, high levels of Ly49G2 message were detected in immature BM B cells. After their exit from the BM, these immature B cells down-regulated their Ly49G2 transcription. Differentiation to mature B cells resulted in elevation of Ly49G2 transcription to levels similar to those in BM cells. FACS analysis of Ly49G2 revealed that high protein levels of this receptor were expressed on immature BM cells (Fig. 4B). Exit from the BM to the periphery was accompanied by down-regulation of Ly49G2 cell surface expression on the peripheral immature population and its elevated expression on mature B cells (Fig. 4B). Thus, BM and mature B cells exhibited similar Ly49 receptor expression patterns.



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  		FIGURE 4. Ly49G2 is expressed at high levels on BM immature B cells and is down-regulated during their exit to the periphery. A, Immature (IgD–IgM+) BM, immature (IgD– CD19+), and mature (IgD+) splenic B cells derived from control mice were purified. RT-PCR was performed as described in Materials and Methods. B, Lymphocytes from BM and spleen from control mice were triple stained with anti-Ly49G2, anti-B220, and anti-IgD or anti-IgM Abs. Histograms show Ly49G2 expression on the BM IgMhigh immature B cells and on peripheral immature (IgD–) and mature (IgD+) B cells. The results presented are representative of three different experiments.

 
Activation of Ly49G2 on immature B cells leads to a decrease in IFN-{gamma} transcription and translation, and restores their cytoskeleton rearrangement and migration

To determine whether IFN-{gamma} expression correlates with the expression of the Ly49 family members, we analyzed IFN-{gamma} message in the various B cell populations. As shown in Fig. 4A, BM immature and mature B cells, which expressed high levels of Ly49G2, transcribed low levels of IFN-{gamma}, whereas IFN-{gamma} transcription was increased in peripheral immature cells. In addition, during differentiation of immature T1 B cells to T2 and mature cells, there was a down-regulation in IFN-{gamma} expression levels (Fig. 1E). Thus, IFN-{gamma} expression correlates with high levels of Ly49D and low Ly49G2 expression.

We also wanted to determine whether Ly49G2 stimulation influences IFN-{gamma} expression in immature B cells. To this end, we followed IFN-{gamma} message in stimulated or unstimulated B cells derived from Ii–/– mice. Ii–/–CD19+ cells were incubated in the presence or the absence of anti-Ly49G2, anti-Ly49D, or nonspecific Abs. The anti-Ly49 Abs were previously shown to each stimulate their corresponding receptor (Fig. 3, A and B) (6). IFN-{gamma} mRNA was compared in unstimulated and stimulated cells by RT-PCR. As shown in Fig. 5, A–C, Ly49D stimulation led to a substantial increase in the levels of IFN-{gamma} message, as we previously showed (6). However, Ly49G2 stimulation resulted in a substantial reduction in IFN-{gamma} mRNA levels (Fig. 5, A–C) below its levels in unstimulated cells or in cells stimulated with a nonspecific Ab (Fig. 5B). Consistent with the dominant effect of the inhibitory Ly49G2 receptor, stimulation with both anti-Ly49D and anti-Ly49G2 resulted in the return of IFN-{gamma} message to levels similar to those in unstimulated cells (Fig. 5C), further demonstrating the tight regulation of IFN-{gamma} transcription by both receptors.



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  		FIGURE 5. Ly49G2 stimulation leads to a decrease in IFN-{gamma} transcription and translation in immature B cells. A, Immature B splenocytes from Ii–/– mice incubated in the presence or the absence of anti-Ly49D or anti-Ly49G2. RT-PCR was performed as described in Materials and Methods. B, Immature B cells from Ii–/– mice were incubated in the presence or the absence of anti-Ly49G2, anti-Ly49D, and a nonspecific Ab (CD8). RT-PCR was performed as described in Materials and Methods. C, Immature B splenocytes from Ii–/– mice were incubated in the presence or the absence of anti-Ly49D, anti-Ly49G2, or both. RT-PCR was performed as described in Materials and Methods. D, Immature B splenocytes from Ii–/– mice were incubated in the presence or the absence of Ly49G2. Cells were lysed and analyzed by Western blot with anti-IFN-{gamma} and anti-tubulin as described in Materials and Methods. E, Immature B splenocytes from Ii–/– mice were stimulated, or not, with anti-Ly49G2 Ab, and IFN-{gamma} levels were detected in the conditioned medium by ELISA. The results presented are representative of three different experiments.

 
Next, we stimulated immature B cells (Ii–/–) with anti-Ly49G2 and followed IFN-{gamma} protein levels. Ly49G2 stimulation down-regulated IFN-{gamma} protein levels, as detected in immature B cells by Western blot analysis (Fig. 5D) or by ELISA of cell supernatants (Fig. 5E). These results demonstrated down-regulation of both intracellular and secreted IFN-{gamma} after Ly49G2 stimulation. Thus, both Ly49G2 and Ly49D regulated IFN-{gamma} secretion in peripheral immature B cells. Although Ly49D stimulation resulted in augmented secretion of this cytokine (6), Ly49G2 activation inhibited IFN-{gamma} secretion.

Previously, we showed that specific low levels of IFN-{gamma} secreted by immature B cells inhibited adhesion and migration of immature and mature B cells, whereas at lower or higher levels of IFN-{gamma}, this inhibition was released (3, 5). To determine whether the decrease in IFN-{gamma} expression induced by Ly49G2 stimulation regulates the motility of the cells, and thus their migration, we followed the degree of actin polymerization in Ii–/– (immature) B cells in the presence or the absence of Ly49G2 stimulation. As shown in Fig. 6A, anti-Ly49G2 treatment caused a substantial increase in cytoskeleton rearrangement in response to CXCL12, a potent B cell chemoattractant (16). This loss of inhibition could be explained by the decrease in IFN-{gamma} secretion to below its inhibitory levels. Our previous studies demonstrated that conditioned medium derived from immature B cells negatively regulates cytoskeleton rearrangement and migration of mature B cells (3, 4). To show a down-regulation in IFN-{gamma} secretion in response to Ly49G2 stimulation, conditioned medium derived from unstimulated control or Ii–/– B cells and Ii–/– B cells stimulated in the presence of anti-Ly49G2 for 1 h was collected. Cytoskeleton rearrangement of mature B cells incubated in the various conditioned media was monitored. As shown in Fig. 6B, addition of conditioned media from immature cells exposed to Ly49G2 stimulation vs conditioned medium derived from unstimulated immature B cells resulted in a dramatic recovery (to wild-type levels) of the ability of mature B cells to polymerize their actin. This loss of inhibition could be explained by the reduction in IFN-{gamma} secretion to below its inhibitory levels.



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  		FIGURE 6. Stimulation of immature B cells with anti-Ly49G2 down-regulates their actin polymerization and homing. A, Ii–/– B splenocytes were pretreated in the presence or the absence of anti-Ly49G2 for 1 h. The cells were then stimulated with CXCL12. Next, the cells were fixed and permeabilized, and their intracellular F-actin was stained with FITC-phalloidin and analyzed by FACS. B, Ii–/– and control B splenocytes were pretreated in the presence or the absence of anti-Ly49G2 for 1 h, and their conditioned medium (Sup) was collected and incubated for 1 h with mature B splenocytes from control mice. The cells were then stimulated with CXCL12. Next, the cells were fixed and permeabilized, and their intercellular F-actin was stained with FITC-phalloidin and analyzed by FACS. C, Ii–/– B splenocytes were pretreated in the presence or the absence of anti-CD8, anti-Ly49G2, or anti-Ly49D together with anti-Ly49G2 for 1 h. The cells were then stimulated with CXCL12. Next, the cells were fixed and permeabilized, and their intracellular F-actin was stained with FITC-phalloidin and analyzed by FACS. D and E, Ii–/– cells were incubated in the presence of anti-Ly49G2 or nonspecific Ab (anti-CD8). The treated cells were injected into C57BL/6 mice. After 3 h, the spleen (D) and LN (E) were collected, and the FITC-positive population was counted by FACS. The results presented are representative of at least three different experiments.

 
To show the tight control of IFN-{gamma} secretion, cytoskeleton rearrangement was monitored in immature B cells stimulated in the presence or the absence of anti-Ly49D, anti-Ly49G2, or both. As previously shown, Ly49D stimulation resulted in elevation of IFN-{gamma} secretion above its inhibitory levels (6). As shown above, Ly49G2 stimulation down-regulated IFN-{gamma} secretion to levels below its inhibitory dose. Combination of the two signals resulted in retrieval of the inhibitory effect to levels similar to those in untreated cells (Fig. 6C). Thus, signals from the two opposite receptors resulted in the secretion of specific low levels of IFN-{gamma} that inhibited cytoskeleton rearrangement.

To demonstrate the biological effect of Ly49G2 stimulation, we next analyzed the entry of cells into LN in vivo after Ly49G2 activation. Total splenocytes from Ii–/– mice were stimulated in vitro in the presence of anti-Ly49G2 or a nonspecific (anti-CD8) Ab. The cells were washed and labeled with fluorescent dye, and an equal number of live cells was injected i.v. into C57BL/6 mice. The proportion of labeled cells recovered in the spleen and LN was determined 3 h after injection. The extent of labeled cell accumulation in the spleen was unaffected by the various pretreatments (Fig. 6D). In contrast, migration of anti-Ly49G2-stimulated Ii–/– cells to the LN was significantly increased, whereas cells incubated with nonspecific Ab exhibited low migration (Fig. 6E). Thus, Ly49G2 stimulation leads to a rapid decrease in IFN-{gamma} transcription and translation, enabling increased cytoskeletal rearrangement and homing of immature B cells to the LN.

MHC I Db recognition is required for Ly49G2 activation

We have previously shown that MHC I recognition is essential for the secretion of low dose IFN-{gamma} that results in down-regulation of immature B cell migration and homing (6). To determine whether MHC-I Db regulates Ly49G2 activation as well, we first analyzed IFN-{gamma} transcription levels in H2-Db–/–, H2-Kb–/–, and control total B cells. As shown in Fig. 7A, B cells deficient in the MHC class I H2-Db transcribed reduced levels of IFN-{gamma} compared with H2-Kb–/– or control B cells, suggesting that interaction of Ly49D with the H2-Db haplotype is essential for IFN-{gamma} transcription. To determine whether IFN-{gamma} levels are regulated by both activating and inhibitory receptors that recognize the same MHC class I molecule, we separated the activities of these two receptors. We analyzed mature B cells that express high levels of Ly49G2 and lower levels of Ly49D, deficient in MHC class I (H2-Db–/– and H2-Kb–/–). We hypothesized that because both receptors are inactive in the absence of their ligand, in the absence of an inhibitory signal from Ly49G2, artificial stimulation of Ly49D will augment IFN-{gamma} secretion. Therefore, H2-Db–/– and H2-Kb–/– B cells were incubated in the presence or the absence of anti-Ly49D Abs to induce IFN-{gamma} secretion. As shown in Fig. 7B, unstimulated H2-Db–/– B cells transcribed very low levels of IFN-{gamma} message. However, in the absence of an inhibitory signal in the H2-Db-deficient B cells, Ly49D activation led to augmented IFN-{gamma} transcription. In H2-Kb-deficient B cells, because the inhibitory signal through Ly49G2 was maintained, activation of Ly49D by Ab did not affect IFN-{gamma} levels (Fig. 7C). These results suggest that recognition of H2-Db by Ly49G2 leads to down-regulation of IFN-{gamma} secretion. When this signal is absent, and the activating receptor is stimulated, even mature B cells can transcribe IFN-{gamma} message.



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  		FIGURE 7. H-2Db is the ligand for Ly49G2. A, Total B splenocytes (CD19+) derived from Q7 (con), H-2Db–/– (Db–/–), and H2-Kb–/– (Kb–/–) mice were purified. RT-PCR for IFN-{gamma} was performed as described in Materials and Methods. B and C, Mature B splenocytes (IgD+) derived from Db–/– (B) or Kb–/– (C) mice were purified, and the cells were stimulated with anti-Ly49D for various times. RT-PCR for IFN-{gamma} was performed as described in Methods. The results presented are representative of three different experiments.

 
Finally, because Ly49G2 and Ly49D are activated by the same MHC class I ligand, we wanted to determine whether both receptors activate similar signaling cascades. To this end, we followed their downstream signaling pathways. Stimulation of NK Ly49D receptor led to recruitment of Syk and its phosphorylation (8). We therefore analyzed Syk phosphorylation in Ii–/– cells after anti-Ly49D or anti-Ly49G2 stimulation. The phosphorylation of Syk was directly assessed by precipitation of phosphotyrosine proteins, followed by anti-Syk immunoblotting. As shown in Fig. 8, although Ly49D stimulation led to a rapid phosphorylation of Syk (Fig. 8A), activation of Ly49G2 did not result in elevated phosphorylation of this protein (Fig. 8B). To determine whether Syk phosphorylation leads to IFN-{gamma} transcription, immature B cells were simulated with anti-Ly49D and incubated for 3 h in the presence or the absence of the Syk inhibitor, piceatannol (17). As shown in Fig. 8C, piceatannol inhibited IFN-{gamma} transcription, suggesting that Syk activity is essential for IFN-{gamma} transcription, which inhibits B cell homing, whereas the inhibitory effects of Ly49G2 are not mediated through the Syk pathway.



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  		FIGURE 8. Stimulation of Ly49D receptor leads to recruitment of Syk and its phosphorylation. A and B, Immature B cells from Ii–/– were stimulated with Ly49D or Ly49G2 or were left unstimulated for various time periods. The cells were lysed and p-Tyr proteins were immunoprecipitated from most of the lysate, leaving an aliquot for total proteins. The total and immunoprecipitated proteins were separated on SDS-PAGE and analyzed by Western blot with anti-Syk as described in Materials and Methods. C, Immature B cells from Ii–/– mice were incubated in the presence or the absence of 120 µM Syk inhibitor (piceatannol) or with 6 µl of DMSO for 3 h. RT-PCR for IFN-{gamma} was performed as described in Materials and Methods. The results presented are representative of three different experiments. RT-PCR for IFN-{gamma} was performed as described in Materials and Methods. The results presented are representative of three different experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The Ly49 gene family encodes MHC class I-recognizing type II transmembrane receptors primarily on murine NK cells. Inhibitory Ly49 receptors allow murine NK cells to kill cells with aberrant class I MHC expression while sparing normal cells. This is accomplished by their recognition of specific class I MHC products and inhibition of NK cell lysis of cells that present a normal repertoire of class I MHC ligands. The inhibitory receptors contain cytoplasmic ITIMs that are phosphorylated upon stimulation, leading to recruitment of Src homology region 2-containing phosphatase 1 and attenuation of intercellular signaling that inhibits NK cell activity (18, 19, 20). Ly49 receptors that lack the cytoplasmic ITIM motif, which is required for inhibition of killing, and contain a positively charged residue in their transmembrane region (21, 22, 23, 24) stimulate NK killing and are therefore referred to as activating Ly49 receptors (25).

One of the classic hallmarks of adaptive immunity is the strong synergy observed upon coreceptor activation. NK cells can express both activating and inhibitory Ly49 receptors on their cell surfaces. When cells expressing both receptors are presented with a ligand, inhibition dominates as the functional outcome (7, 26). Ly49D receptor ligation can lead to the rapid and potent secretion of IFN-{gamma}. Consistent with the dominant inhibitory function of Ly49G, NK cells coexpressing Ly49D and Ly49G show a profound reduction in IFN-{gamma} secretion after interaction with targets expressing their common ligand, H-2Dd (26).

In this study we followed the expression of the inhibitory receptor, Ly49G2, on B cells and analyzed its interaction with the activating receptor, Ly49D, that was previously shown to be expressed on immature B cells (6). We demonstrate for the first time that B cells display surface Ly49G2. Ly49G2 and Ly49D transcription, translation, and cell surface expression are coregulated during B cell differentiation. Although high levels of Ly49G2 are expressed in immature BM B cells, its expression is dramatically down-regulated in peripheral immature B cells. Differentiation to mature cells results in a strong enrichment of its expression levels. Furthermore, the Ly49G2 receptor on immature and mature B cells is functional and is able to tyrosine phosphorylate specific proteins after its stimulation. We have previously shown that Ly49D expression in B cells is regulated as well, but in an inverse pattern. Although bone marrow immature B cells express low levels of this receptor, peripheral immature cells dramatically up-regulate its levels, and differentiation to mature cells results in its reduced levels (6). Thus, there is a tight regulation in the expression of the Ly49 family members on B cells.

Previously, we demonstrated that immature B cells can down-regulate their own integrin-mediated adhesion to the extracellular matrix and thereby suppress their migration into nonsplenic sites. This inhibition is mediated by the chemokine receptor CCR2 (27) and IFN-{gamma}, which is transcribed and secreted at low levels by immature B cells (3). The inhibitory signal of IFN-{gamma} is transmitted through the IFN-{gamma}R, whose engagement leads to inhibition of cytoskeleton rearrangement, which is required for promoting integrin-mediated adhesion and migration of B cells (4, 5).

In this study we show that high Ly49G2 inhibits IFN-{gamma} expression in B cells. Although activation of Ly49D on immature B cells augments IFN-{gamma} transcription and secretion levels (6), cross-linking of Ly49G2 on these cells dramatically inhibits these processes and restores cytoskeleton rearrangement and homing of cells to LN. In addition, cross-linking of both Ly49D and Ly49G2 receptors on immature B cells results in transcription of specific low levels of IFN-{gamma}. Thus, high levels of the inhibitory receptor expressed on immature BM and mature B cells dominate, and IFN-{gamma} secretion is largely inhibited; low levels of the inhibitory receptor, Ly49G2, coexpressed with high levels of the activating receptor, Ly49D, result in regulated and specific secretion of low levels of IFN-{gamma}. This regulation insures the inhibitory control of immature B cells homing to the LN and removal of this regulation in BM and during the mature stage.

It is well established that class I MHC molecules serve as ligands for Ly49 receptors. Ly49 receptors are allele specific in their recognition of class I ligands; it is known that Ly49G binds Dd, Dr, and possibly an H-2k product (25). Our studies demonstrate that B cells that express Ly49G2 on their surface specifically recognize the H2-Db allele of MHC class I. This recognition results in a downstream pathway that prevents transcription of IFN-{gamma}. By preventing this specific recognition in H2-Db-deficient mice, stimulation of Ly49D on mature B cells results in up-regulation of IFN-{gamma} transcription. Immature and mature B cells in our studies were not incubated in the presence of any additional cells; therefore, Ly49G2 expressed on these cells could recognize only H2-Db and not any other MHC class I allele. Recent studies suggest that Ly49A inhibitory receptor may also interact weakly with H-2b molecules, but that the affinity of this interaction may be too low to observe inhibition of target cell lysis in vitro, but sufficient to induce effects on Ly49A receptor expression in vivo (28, 29, 30). We believe that Ly49G2 in B cells might have a similar interaction with H2-Db, and this interaction should be further studied.

Our current model suggests that Ly49 family members regulate IFN-{gamma} by B cells from their formation stage in the BM through their maturation in the spleen. Immature BM B cells express high levels of Ly49G2 and low levels of Ly49D, resulting in inhibition of IFN-{gamma} secretion. In preparation for their exit to the periphery, these cells down-regulate their Ly49G2 expression and up-regulate cell surface Ly49D levels. MHC class I recognition leads to Ly49D activation, enabling the secretion of constant low levels of IFN-{gamma}. We propose that this IFN-{gamma} breaks the CXCL12 retention signals in the BM, allowing the release of immature B cell from this compartment. IFN-{gamma} secretion by peripheral immature B cells prevents their arrival at Ag-enriched sites, allowing their direct targeting to the spleen. After their arrival at the spleen, immature B cells complete their maturation process. Mature B cells down-regulate their Ly49D and elevate their Ly49G2 levels. Mature cells that no longer secrete IFN-{gamma} can recirculate in the periphery and home to various compartments, such as LN or sites of inflammation.


   Acknowledgments
 
We thank Dr. Lemonnier for the gift of the MHC class I-deficient mice; Prof. Lea Eisenbach, Dr. Esther Zahovel, and Ezra Vadaai for the gift of mice; and the Shachar laboratory for helpful discussions and review of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Israel Science Foundation founded by the Academy of Sciences and Humanities, and the Migration and Targeting Cell Migration in Chronic Inflammation European Union grant. I.S. is the incumbent of the Alvin and Gertrude Levine Career Development Chair of Cancer Research. Back

2 Address correspondence and reprint requests to Dr. Idit Shachar, Department of Immunology, Weizmann Institute of Science, 76100 Rehovot, Israel. E-mail address: idit.shachar{at}weizmann.ac.il Back

3 Abbreviations used in this paper: BM, bone marrow; LN, lymph node; p-Tyr, phosphorylated tyrosine. Back

Received for publication April 6, 2005. Accepted for publication August 2, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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