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Ly49D Receptor Expressed on Immature B Cells Regulates Their IFN- Secretion, Actin Polymerization, and Homing¹

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

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Low levels of IFN- secreted by immature B cells prevent their own migration and homing to the lymph nodes and premature encounter with Ag. In this study we followed the mechanism regulating IFN- secretion by immature B cells. We show that the MHC class I receptor, Ly49D, is expressed on immature B cells and is down-regulated during maturation. Activation of this receptor leads to increase in IFN- transcription and translation and results in the altered ability of B cells to polymerize actin in response to chemokine stimulation. Moreover, we show that H2-D blockage inhibits the ability of immature B cells to transcribe the IFN- gene and results in rescue of cytoskeletal rearrangement. Thus, Ly49D that is expressed on immature B cells recognizes MHC class I on the peripheral tissues, inducing the secretion of low levels of IFN- and thereby down-regulating immature B cell homing to the lymph nodes or to sites of inflammation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion 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 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, immature B cells are excluded from nonsplenic secondary lymphoid organs, which are specialized tissues for collecting Ags (3), and from sites of infection and inflammation. In these secondary lymphoid organs, in which differentiation to a mature phenotype does not occur, Ag encounter could lead to effective clonal elimination because of a 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 (4). This inhibition is mediated by IFN-, which is transcribed and secreted at low levels by immature B cells. The inhibitory signal of IFN- is transmitted through the IFN- receptor, whose engagement leads to inhibition of cytoskeleton rearrangement, which is required for promoting integrin-mediated adhesion and migration of B cells (5, 6).

The NK-activating receptor, Ly49D, is a type II transmembrane glycoprotein of the C-type lectin superfamily that recognizes MHC class I proteins. Ly49D does not contain the cytoplasmic immune receptor tyrosine-based inhibitory motifs (ITIMs)³ that are phosphorylated upon stimulation, confirming that it is not an inhibitory receptor (7, 8, 9). This receptor was recently shown to be a potent inducer of IFN- expression. Furthermore, stimulation with mAb Ly49D on NK cells induces IFN- gene transcription and higher levels of IFN- protein following receptor ligation (10), although the function of this induction was not analyzed. The expression of Ly49 family proteins was described mostly on NK cells; however, a member of this family, Ly49A/D, was recently found on the peritoneal B1 subpopulation of B cells (11). In this study we investigated whether Ly49D may be expressed on immature B cells and may control B cell homing. Our results show that immature B cells express Ly49D, which controls their IFN- secretion and consequently their actin polymerization and migration.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

C57BL/6, Ii^-/- (12), IFN-^-/- (Jackson), K^b-/- and D^b-/- (13) 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

The murine pre-B cell-like lymphoma line, 70Z/3 (14) was grown in suspension culture at 37°C in RPMI 1640 medium containing 10% (v/v) FCS and 200 µM 2-ME in the presence or absence of LPS (5 µg/ml). Spleen cells were obtained as previously described (15). Control IgD^-, IgD⁺ CD21⁺, and CD21-B were separated as previously described (16). CD19⁺ cells were separated using anti-CD19 magnetic beads (Miltenyi Biotec, Auburn, CA). Bone marrow cells were obtained from the femurs of control and Ii^-/- mice. The marrow was rinsed with PBS, and the RBC were lysed. Pre-pro- and pro-B cells were separated by FACS sorting using anti-B220, CD19, Bp1, and IgM Abs (BD PharMingen, San Diego, CA). Bone marrow immature B cells were purified by incubating bone marrow cells with anti-IgD-PE Ab (BD PharMingen) and separation with anti-PE magnetic beads using the MACS system (Miltenyi Biotec). After depletion of the mature IgD⁺ B population, cells were incubated with anti-IgM-FITC Ab (BD PharMingen), and the positive population was separated with anti-FITC beads using the MACS system (Miltenyi Biotec). NK cells were purified using the anti-Dex5 magnetic beads (Miltenyi Biotec). 493⁺ cells were separated using anti-493-PE Abs (BD PharMingen), followed by separation with anti-PE magnetic beads (Miltenyi Biotec).

RNA isolation and RT

Total RNA was isolated using the Tri-Reagent kit (Molecular Research Center). RT was conducted using Superscript II reverse transcriptase (Life Technologies, Grand Island, NY). The primers used for IFN- and hypoxanthine phosphoribosyltransferase (HPRT) were described previously (4). For Ly49D, the primers were 5'-CCTGGCAGCTCATTGTGATAG-3' and 5'-ATTCTGGCAGCTCTGTTTACATC-3'.

Immunofluorescence and flow cytometry

Staining was performed as previously described (17, 18). The Abs used were as follows: RA3-6B2, anti-CD45R/B220 (Southern Biotechnology Associates, Birmingham, AL); AMS 9.1, anti-IgD; R6-60.2, anti-IgM; B3B4, anti-CD23; 4E5, anti-Ly49D; 493, and anti-493; and 53-7.3, anti-CD5 (BD PharMingen).

Stimulation of B cells with mAb 4E5

Primary B cells (5 x 10⁶) were suspended in 100 µl of RPMI 1640 containing 10% (v/v) FCS. Next, 5 µg of 4E5 mAb was added to each tube, and the tubes were immediately placed in 37°C for 2.5 min. Immediately after stimulation, the cells were washed and fast-frozen in liquid N₂.

Preparation of cell extract

Stimulated primary B cells were lysed as previously described (5).

Immunoprecipitation and Western blot analysis

Protein G-Sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ) were conjugated to mAb Ly49D (4E5) at a 1/20 ratio for 2 h at 4°C, followed by three PBS washes. Beads were added to the cell lysates, and Ly49D was 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 12% (w/v) SDS-PAGE. The proteins were transferred into nitrocellulose membrane and probed with anti p-tyr (pTyr99) (Santa Cruz Biotechnology, Santa Cruz, CA), followed by HRP-conjugated anti-mouse or rabbit IgG (Jackson ImmunoResearch Laboratories, WesT Grove, PA).

Detection of IFN-

Lysates of cells were separated by 10% (w/v) SDS-PAGE. IFN- was detected as previously described (4).

Radiolabeling and immunoprecipitation

B splenocytes (2 x 10⁷) were incubated in 2 ml of RPMI 1640 methionine-free medium with 10% dialyzed FCS at 37°C. After 1 h [³⁵S]methionine (200 mCi; Amersham Pharmacia Biotech) was added, and the cells were pulsed for 2.5 h. Cells were lysed, and Ly49D was immunoprecipitated and separated on 10% SDS-PAGE.

Cytoskeleton rearrangement

B splenocytes were stimulated in the presence or the absence of anti-Ly49D as described above. Cytoskeleton rearrangement was evaluated using FITC-phalloidin as previously described (5).

MHC blocking

B splenocytes (2 x 10⁷) were incubated in 1 ml of hybridoma supernatant containing 10% FCS. The hybridomas used were 20-8-4S (anti-H-2 K^b), 28-14-8S (anti-H-2 D^b), and Afg-120.1.2 (anti-I-A^b). The tubes were incubated for 2.5 h on ice and then placed at 37°C for 2 h more.

ELISA

Ii^-/- immature B220⁺ cells were incubated in the presence or the absence of anti-Ly49D as described above. After 1 h of stimulation cell supernatants were collected, and IFN- levels were determined by ELISA (BD PharMingen) according to the manufacturer’s specification.

Tracking of cells in vivo

Ii^-/- B cells were stimulated in the presence or the absence of anti-Ly49D or control Ab (anti-CD8). One hour later the cells were labeled with 5 µM carboxyfluorescein diacetate succinimidyl ester (Molecular Probes, Eugene, OR) for 15 min at room temperature. The cells were then injected into control mice, and spleen and lymph nodes (LN) were analyzed 3 h later by FACS.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Immature B cells express active Ly49D receptor on their surface

Previously we showed that immature B cells secrete low levels of IFN- that down-regulates the B cells’ own integrin-mediated adhesion to the extracellular matrix and thereby suppress their migration and homing into Ag-enriched sites. The Ly49D receptor has been shown to regulate IFN- secretion by NK cells. We therefore determined whether this receptor is expressed on immature B cells, and whether it may also regulate IFN- secretion in this cell type. To this end we first analyzed the mRNA expression of Ly49D in immature (B220⁺IgD^-) and mature (B220⁺IgD⁺) B populations. IgD^- immature B cells were purified from either control (C57BL/6) or invariant chain-deficient (Ii^-/-) mice, whose B cells are arrested at an immature stage (17, 18, 19, 20). Mature IgD⁺ cells were purified from control (C57BL/6) mice. As shown in Fig. 1A, Ly49D message, which appeared in the immature B population from both control (Ii^+/+) and Ii^-/- cells, was dramatically decreased in the mature cells. It is known that activated NK cells express on their cell surface low levels of the B cell marker B220. To directly show that Ly49D is expressed on B cells, and that the expression we detected did not result from contamination of NK cells, B cells from either control (C57BL/6) or Ii^-/- mice were separated using anti-IgM and -IgD (Fig. 1B) or anti-CD19 (Fig. 1C) Abs. Separation with anti-IgM or anti-CD19 results in enrichment of the entire peripheral B population, and in the control mice it constitutes a mixture of mostly mature cells, with 10% immature B cells. However, since maturation of B cells in the Ii^-/- mice is arrested, anti-CD19 or anti-IgM-purified Ii^-/- B cells are mainly of the immature population. As shown in Fig. 1C, high levels of Ly49D mRNA were detected in Ii^-/- CD19⁺ immature B cells, while these levels were dramatically reduced in the control CD19⁺ population that is enriched with mature cells (Fig. 1C). Furthermore, separation of control C57BL/6 B cells with anti-IgD results in the enrichment of mature B cells. As shown in Fig. 1B, Ii^-/- immature IgM⁺ B cells express high levels of Ly49D mRNA. The levels of this message are dramatically decreased in the control IgM⁺ population, which contains only a small proportion of immature B cells, and are almost absent from the control IgD⁺ mature population. To further confirm the transcription of Ly49D message in immature B cells, we purified this population using the Ab493 that was previously shown to be expressed exclusively on early B cells (21). As shown in Fig. 1D, Ly49D message was detected in 493⁺ cells. Newly emigrant immature B cells can be separated from marginal zone cells by expression of the CD21 marker. To determine which population transcribes Ly49D message, we analyzed Ly49D mRNA in the CD21-positive and -negative IgD^- populations. As shown in Fig. 1E, Ly49D mRNA can be detected in IgD^- CD21^- B cells from both control (Ii^+/+) and Ii^-/-Ii mice. After acquisition of CD21, the cells down-regulate their Ly49D expression. Thus, Ly49D is transcribed mostly in newly arrived immature B cells. Finally, to further prove that the detection of Ly49D mRNA in B cells does not result from contamination of NK cells, this message was analyzed in the 70Z/3 cell line, which is a murine pre-B lymphoma that can differentiate to immature B cells in the presence of LPS. As shown in Fig. 1F, Ly49D mRNA was detected in early B cells; following differentiation these cells down-regulate their Ly49D transcription. Thus, B cells transcribe Ly49D message that is down-regulated following differentiation.

View larger version (70K): [in this window] [in a new window]   FIGURE 1. Ly49D message and protein are expressed in B cells and are down-regulated as part of the transition between immature and mature B cells. A, Immature IgD- B cells derived from control (Ii+/+) or invariant chain-deficient (Ii-/-) mice and control mature IgD+ cells were purified. RT-PCR was performed as described in Materials and Methods. B, IgM+ B cells from control (Ii+/+) or Ii-/- mice and control mature (IgD+) B cells were purified. RT-PCR was performed as described in Materials and Methods. C, CD19+ B cells from control (Ii+/+) or Ii-/- mice were purified. RT-PCR was performed as described in Materials and Methods. D, Immature 493+ B cells derived from control mice and control mature IgD+ cells were purified. RT-PCR was performed as described in Materials and Methods. E, IgD+, IgD-CD21+, and IgD-CD21- B cells were purified. RT-PCR was performed as described in Materials and Methods. F, 70Z/3 cells were incubated in the presence or the absence of LPS for 24 h. RT-PCR was performed as described in Materials and Methods. G and H, B cells derived from Ii-/- mice (G and H), IgD+ mature (G) and total B cells from control (Ii+/+) mice (H), and NK cells from control mice (H) were labeled with [35S]methionine for 2.5 h. Immunoprecipitation was performed using anti-Ly49D or control Abs. Ly49D was immunoprecipitated from the cell lysates and separated on 10% SDS-PAGE. The results presented are representative of four different experiments.

To directly show the expression of Ly49D on the surface of immature B cells, B cells derived from control or Ii^-/- mice were analyzed by FACS. As shown in Fig. 2, B cells derived from Ii^-/- mice (B220⁺ or IgM⁺ cells) expressed higher levels of Ly49D compared with the control population (Fig. 2, B and C). The expression of Ly49D in the B1 CD5⁺ population was almost identical (Fig. 2D). Furthermore, separation of the immature (493⁺, IgD^-, or CD23^-) and mature (493^-, IgD⁺, or CD23⁺) B220⁺ populations revealed the down-regulation of Ly49D expression in the mature cells (Fig. 2, E–G). Thus, immature B cells express Ly49D, and its expression is down-regulated in the mature B population. Unlike its expression on NK cells, the staining of Ly49D on B cells did not show a discrete homogenous peak, but, rather, showed broad diffuse staining. We suggest that B cells express lower levels of Ly49D receptor on their surface, probably due to its different role in these cells.

View larger version (31K): [in this window] [in a new window]   FIGURE 2. Ly49D is expressed on immature B cells. A and B, Splenocytes from control (Ii+/+) and Ii-/- mice were double-stained with anti-Ly49D (B) or irrelevant isotype control (IgG2a ; A) Abs and anti-B220. Histograms show Ly49D and isotype control expression on B220+ cells. C and D, Splenocytes from control (Ii+/+) and Ii-/- mice were triple-stained with anti-B220, anti-Ly49D, and anti-IgM or anti-CD5 Abs. Histograms show Ly49D expression on B220+IgM+ (C) or B220+CD5+ (D) cells. E–G, Splenocytes from control and Ii-/- mice were triple-stained with anti-Ly49D, anti-B220, and anti-493 (E), anti-IgD (F), or anti-CD23 (G). Histograms show Ly49D expression on the immature and mature populations derived from the various mice. The results presented are representative of six different experiments.

View larger version (44K): [in this window] [in a new window]   FIGURE 3. Ly49D message and protein are expressed in immature B cells in low levels and are up-regulated as part of the transition to peripheral immature B cells. A, Bone marrow pre-pro- and pro-B cells and peripheral immature B cells were purified. RT-PCR was performed as described in Materials and Methods. B, Bone marrow and peripheral immature B cells were purified. RT-PCR was performed as described in Materials and Methods. C, Control bone marrow and spleen cells were triple-stained with anti-B220, anti-Ly49D, and anti-493 or anti-IgD Abs. Histograms show Ly49D expression on immature B220+493+ or B220+IgD- cells.

View larger version (60K): [in this window] [in a new window]   FIGURE 4. Ly49D stimulation leads to phosphorylation of downstream proteins. A, B cells derived from invariant chain-deficient (Ii-/-) mice were incubated in the presence or the absence of anti-Ly49D for 2.5 min. Cells were lysed and analyzed with anti-p-tyr Abs as described in Materials and Methods. B, B cells derived from Ii-/- mice were stimulated with mAb Ly49D, anti-CD4, or anti-IgM for 2.5 min. Cells were lysed and analyzed with anti-p-tyr Abs as described in Materials and Methods. C and D, B cells derived from Ii-/- or control (Ii+/+) mice were incubated in the presence (C and D) or the absence (D) of anti-Ly49D Ab for 2.5 min. Ly49D was immunoprecipitated from cell lysates. The control lane contained lysis buffer with no cells (C and D, lanes 5 and 10). Protein G-Sepharose beads conjugated to mAb Ly49D (4E5) were added to the lysates as described in Materials and Methods. The immunoprecipitates were separated under nonreducing conditions on a 12% SDS-PAGE gel and analyzed with anti-p-tyr Abs, followed by anti-mouse HRP Ab (C and D, lanes 1–5) or anti-mouse HRP alone (D, lanes 6–10).

Activation of Ly49D on immature B cells leads to an elevation of IFN- transcription and translation

Low levels of IFN- secreted by immature B cells were previously shown to down-regulate the homing of these cells to Ag-enriched sites (4). To determine whether Ly49D activation leads to elevation of IFN- transcription in immature B cells, we followed IFN- message in B cells derived from control (Ii^+/+) or Ii^-/- mice. B220⁺ cells were incubated with the anti-Ly49D stimulatory Ab (4E5), and IFN- mRNA was compared in unstimulated and stimulated cells by RT-PCR. As shown in Fig. 5A, stimulation of Ly49D Ii^-/- immature B cells led to a substantial increase in the levels of IFN- message compared with the unstimulated cells. Moreover, while IFN- message was hardly detected in control unstimulated cells (4) (Fig. 5A), stimulation of Ly49D resulted in elevation of IFN- mRNA, probably due to activation of the small immature population (10% of the cells). To directly show that mature B cells respond poorly to Ly49D stimulation, control mature IgD⁺ cells were purified and were compared with IgM⁺ immature B cells derived from Ii^-/- mice. As shown in Fig. 5B, mature B cells transcribed almost undetectable levels of IFN- message after Ly49D ligation, while this stimulation dramatically elevated the level of Ii^-/- immature B cells IFN- mRNA. In addition, 493⁺ (Fig. 5C) and IgD^- (Fig. 5D) immature B cells were shown to increase their IFN- mRNA following Ly49D stimulation, while IFN- levels were not elevated following stimulation of mature IgD⁺ B cells (Fig. 5D). To determine whether Ly49D stimulation results in increased translation of IFN-, control B220⁺ cells (which contain mostly the mature population with 10% of immature cells), control (Ii^+/+) mature IgD⁺ B cells, and B220⁺ B cells from invariant chain-deficient mice (immature B cells) were analyzed following Ly49D stimulation. As shown in Fig. 5E, stimulated mature cells did not express IFN- protein, while this protein was detected in the total control population, which contains a small population of immature cells. Stimulation of Ii^-/- immature B cells resulted in the expression of high levels of IFN- protein (Fig. 5, E and F) and its secretion (Fig. 5G). Thus, stimulation of Ly49D, which is expressed on immature B cells, results in elevation of IFN- transcription and translation.

View larger version (45K): [in this window] [in a new window]   FIGURE 5. Stimulation of immature B cells with Ly49D elevates IFN- transcription and translation. A, B220+ cells derived from invariant chain-deficient (Ii-/-; immature B cells) and control (Ii+/+) mice (mostly mature B cells) were purified. The cells were incubated in the presence or the absence of anti-Ly49D mAb for 1 h. RT-PCR with IFN- and HPRT primers was performed as described in Materials and Methods. B, IgM+ B cells from control (Ii+/+) or Ii-/- mice and control mature (IgD+) B cells were purified. The cells were stimulated in the presence or the absence of mAb Ly49D as described in Materials and Methods. RT-PCR with IFN- and HPRT primers was performed as described in Materials and Methods. C, Control immature 493+ cells were purified. The cells were stimulated in the presence or the absence of mAb Ly49D as described in Materials and Methods. RT-PCR with IFN- and HPRT primers was performed as described in Materials and Methods. D, Control immature IgD- and mature IgD+ B cells were purified. The cells were stimulated in the presence or the absence of mAb Ly49D as described in Materials and Methods. RT-PCR with IFN- and HPRT primers was performed as described in Materials and Methods. E, Steady state protein levels of IFN- in total cell lysates of B220+ cells derived from Ii-/- and the mature IgD+ population from control (Ii+/+) mice (E), which were stimulated for 1 h with anti-Ly49D mAb. Protein was detected by Western blot, showing the band representing IFN- detected by anti-IFN- Ab, followed by anti-rat Ab (E, lanes 1–3). As a control, the membrane was probed with anti-rat alone (lanes 4–6). F, Steady state protein levels of IFN- and tubulin in total cell lysates of immature IgD- from control mice that were incubated in the presence or the absence of Ly-49D stimulation. Protein was detected by Western blot, showing the bands representing IFN- and tubulin. G, B cells IFN- levels detected in stimulated and nonstimulated immature B cell-conditioned medium by ELISA.

View larger version (14K): [in this window] [in a new window]   FIGURE 6. Stimulation of immature B cells with Ly49D regulates actin polymerization and homing. A and B, Ii-/- and control (Ii+/+) B splenocytes (A) or control mature IgD+ and immature 493+ cells (B) were pretreated in the presence or the absence of anti-Ly49D for 1 h. The cells were then stimulated with SDF-1. Next, the cells were fixed and permeabilized, and their intracellular filamentous actin was stained with FITC-phalloidin and analyzed by FACS. The percent increase in actin polymerization was calculated as the: polymerization of actin in the presence of SDF-1 - polymerization of actin without SDF-1/polymerization of actin without SDF-1. C, Homing of labeled cells to the LN. Ii-/- cells that were incubated in the presence or the absence of specific (Ly49D) or nonspecific (CD8) stimulation were injected into control mice. After 3 h the spleen and LN were collected, and the FITC-positive population was analyzed by FACS.

View larger version (39K): [in this window] [in a new window]   FIGURE 7. Interaction with MHC I molecules controls immature B cell IFN- transcription, actin polymerization, and homing. A, Immature (IgD-) and mature (IgD+) B cells derived from IFN--/- mice were purified. RT-PCR was performed as described in Materials and Methods. B, IFN--/- and control B220+ splenocytes were pretreated in the presence or the absence of anti-Ly49D for 1 h. The cells were then stimulated with SDF-1. Next the cells were fixed and permeabilized, and their intracellular filamentous actin was stained with FITC-phalloidin and analyzed by FACS. The graph represents the average of four independent experiments and shows the percent change in actin polymerization in each population. C, B cells from Ii-/- mice were incubated with anti-H2Db (28148), anti-H2Kb (2084), or anti-MHC II (Iab) as described in Materials and Methods. IFN- mRNA levels was analyzed by RT-PCR. D, Immature B cells from control mice were incubated in the presence or the absence of anti-H2Db (28148) as described in Materials and Methods. IFN- mRNA levels were analyzed by RT-PCR. E, Ii-/- and control B220+ splenocytes were pretreated in the presence or the absence of anti-H2Db (28148) or anti-H2Kb (2084) or anti-MHC II (I-Ab) Abs for 1 h. The cells were then stimulated with SDF-1. Next, the cells were fixed and permeabilized, and their intracellular filamentous actin was stained with FITC-phalloidin and analyzed by FACS. F, Analysis of spleen and LN derived from control, H2D-/-, or H2K-/- mice. Histograms show the expression of IgD on IgM+ B cells. The data shown are derived from one mouse of four analyzed.

Blocking of MHC I leads to inhibition of Ly49D activity and down-regulation of IFN- expression

We have shown in vitro that cultured immature B cells secrete low levels of IFN-, which are up-regulated following stimulation of Ly49D. It is not known, however, whether this low level of IFN- secretion requires a basal level of Ly49D stimulation, or whether Ly49D is only required to further up-regulate IFN- expression. MHC class I molecules have been shown to serve as ligands for Ly-49 receptors. To determine whether MHC I recognition is essential for IFN- secretion, Ii^-/- immature B cells were incubated with MHC I blocking Abs with specificities for H2-D^b (281484) or to H2-K^b (2084) or with a control anti-MHC class II (I-A^b) Ab, and IFN- message was analyzed by RT-PCR. As shown in Fig. 7C, while blocking of MHC class II and H2-K^b had no effect on IFN- mRNA levels, incubation of the cells with anti-H2-D^b resulted in a significant decrease in IFN- mRNA levels. This indicates that even basal levels of IFN- production require Ly49D ligation. Moreover immature B cells derived from control mice were incubated with MHC class I blocking Abs for H2-D^b, and IFN- message was analyzed. As shown in Fig. 7D, H2-D^b blocking caused a significant decrease in IFN- mRNA levels.

To evaluate the effect of blocking MHC I Ags on the capacity of immature B cells to polymerize actin, we blocked MHC I or II on immature B cells from Ii^-/- mice, stimulated the cells with SDF-1, and analyzed the increase in polymerized actin. As shown in Fig. 7E, the blocking of H2-K^b had no effect on actin polymerization in the immature B cells, while blocking of H2-D^b led to a substantial increase in actin polymerization. The ability of H2-D^b blockade to reduce IFN- transcription suggests that immature B cells must bind their MHC class I ligand to enable secretion of IFN-. When this recognition is blocked, there is a dramatic inhibition in IFN- transcription and a corresponding increase in actin polymerization.

Finally, to directly show that MHC class I recognition is essential for down-regulation of immature B cell migration and homing, we analyzed the B cell population in the LN of mice lacking H2-K or H2-D. Previously we have shown that secretion of low levels of IFN- results in depletion of immature B cells from the LN, while in IFN-^-/- mice there is accumulation of immature B cell in this compartment (4). As shown in Fig. 7F, in H2-D-negative mice, there is accumulation of IgM⁺IgD^- immature B cells in the LN, while their arrival in the spleen was unchanged. Taken together, these results indicate that H2-D is recognized by Ly49D, a process that is essential to induce IFN- secretion and to inhibit the homing of immature B cells to the LN.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mouse lectin-related Ly49 receptors are type II transmembrane proteins of the C-type lectin superfamily, which mediate the ability of NK cells to bind classical MHC class I proteins and regulate the NK cell activities (24, 25). The NK Ly49 receptors are able to distinguish individual MHC class I alleles and positively or negatively regulate their function. In the case of inhibitory Ly49 receptors, each subunit contains an ITIM motif. Upon receptor engagement, the ITIM becomes phosphorylated, which, in turn, recruits Src homology 2 domain-containing tyrosine phosphatase (24). In contrast, the Ly 49 members that lack the ITIMs deliver activation signals and phosphorylate homodimers with a monomer mass of 16 kDa (pp16) (22).

Ly49D was first described as an activator of NK cell cytotoxicity, proliferation, and cytokine production and was shown to be expressed mostly on murine NK cells. Cross-linking of the Ly49D receptor was found to induce transcription of IFN-, lymphotactin, and macrophage inflammatory protein 1 and 1 (10). Thus, it was suggested that a primary role for the activating receptor in vivo might be to trigger soluble factor production and regulation of the immune response.

In this study we have shown that in addition to its expression on NK cells, T cell subsets, and B1 cells in C57BL/6 mice (11), Ly49D is specifically expressed on immature B cells. Ly49D was found to be transcribed, translated, and expressed on the surface of B cells. Ly49D expression is enriched in the peripheral immature population, and its transcription and translation are down-regulated following maturation of cells in the spleen. Furthermore, the Ly49D receptor on immature B cells was able to tyrosine phosphorylate specific proteins following its stimulation. Specifically, a phosphorylated protein of 32 kDa, which might correlate with a homodimer of pp16, was coimmunoprecipitated with Ly49D following its activation. Thus, an active Ly49D receptor is expressed on the cell surface of immature B cells.

We have previously shown that low levels of IFN- down-regulate adhesion and migration of immature and mature B cells (4). Cross-linking of the Ly49D receptor induces transcription and translation of IFN- at low levels, which results in inhibition of cytoskeletal rearrangement of the control B cell population. However, activation of Ly49D on purified immature B cells results in elevation of IFN- transcription and translation and recovery of cytoskeleton rearrangement. This lack of inhibition might result from the fact that stimulation of purified immature population could lead to accumulation of high levels of IFN-, which are above the inhibitory doses, or alternatively, the anti-Ly49D Ab might stimulate the cells more strongly than the H-2D ligand. Since stimulation of the diluted (10% of the cells) immature population results in inhibition of cell cytoskeleton rearrangement of the entire control population, we believe that stimulation of purified immature B cells results in accumulation of high levels of IFN- that no longer inhibit actin polymerization and homing to the LN. Our studies demonstrate that immature B cells that express Ly49D on their surface specifically recognize the H2-D^b allele of MHC class I. This recognition results in a downstream pathway that allows transcription of IFN-. By blocking this specific recognition, immature B cells down-regulated their IFN- transcription, and the inhibition of their actin polymerization is removed. Moreover, in the absence of H2-D, immature B cells are able to migrate more efficiently to the LN, while loss of H2-K does not change their homing capability. MHC class I molecules are expressed on almost all cell types; therefore, immature B cells can receive a constant signal from the various peripheral cell populations, leading to basal secretion of IFN-. In our studies purified immature B cells transmit a signal that results in IFN- secretion. Ly49D was found to stimulate allo-reactive killing of H2-D^d-expressing cells, but not of other alleles of mouse MHC class I Ags (H2-D^b, K^b, K^k). Moreover, activation of NK cells by Ly49D stimulates lysis of Con A blasts of H-2^d mice, but not blasts from H-2^b or H-2^k mice (26). Immature B cells in our studies were not incubated in the presence of any additional cells; therefore, Ly49D expressed on these cells could recognize only the H2-D^b allele and not any other MHC class I alleles. However, Ly49A is the best-characterized member of the Ly49 family that has been shown to recognize the class I molecules H-2Dd, Dk, and Dp. Recent studies suggest that Ly49A may also interact weakly with H-2b molecules, but that affinity may be too low to observe inhibition of target cell lysis in vitro, but sufficient to induce effects on Ly49A receptor expression in vivo (27, 28, 29). We believe that Ly49D might have the same interaction with H-2^b, and this interaction should be further studied. However, we show that anti-H-2^b block IFN- transcription. It might be suggested that this Ab directly interacts with the MHC class I molecule, a process that causes signaling which results in inhibition of IFN- transcription. Since mice lacking H-2D^b show an increase in their immature B cell population in the LN, we could argue that the lack of H-2D and not activation of its downstream pathway results in the down-regulation in IFN- transcription.

We therefore suggest that Ly49D is stimulated by H2-D^b, a process that results in activation of a signaling pathway leading to IFN- secretion. This IFN- activation cascade might be separately regulated and involve unique proteins different from the signaling pathway resulting in activation of killing.

The entrance of B cells to the LN is a process that requires interaction between L-selectin and ligands on the high endothelial venules. Subsequently, a chemokine-mediated triggering event (using chemokines such as SDF-1, secondary lymphoid tissue chemokine, or EBV-induced molecule 1 ligand chemokine) causes integrin activation and adhesion (30, 31). LFA-1, ₄₇, and ₄₁ contribute to this integrin adhesion requirement (31, 32). Despite our detailed understanding of the steps involved in lymphocyte entry to LN, relatively little is known about how the cells enter the spleen and the white pulp of this compartment. 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, outside of the follicle. These cells then further develop to T2 cells, which are situated within primary follicles, adjacent to mature cells (33). The role of integrins in homing of immature B cells to the spleen has not been fully elucidated. The entry of B cells to the spleen was regarded until recently as a process for which integrin activation was not required (34, 35, 36). Nevertheless, recent results show that integrin activation also has a role in the entry of mature B cells into the splenic white pulp. However, although integrin inhibition causes a decrease in B cell entry to the white pulp, it has no influence on the total B cell number in the spleen (37). In addition, while inhibition of a single integrin (LFA-1 or ₄₇) results in a dramatic inhibition of homing of T and B cells to the LN, it has only a minor effect on their entrance to the splenic white pulp. Only dual integrin inhibition can inhibit B cell entrance to the white pulp (37). These studies suggest that homing of immature B cells to the spleen is different from their homing to the LN and sites of inflammation. Thus, engagement of Ly49D leading to IFN- secretion and integrin-dependent homing inhibition interferes with the arrival of these cells to the LN, while their homing to the spleen is not affected.

Taken together, we demonstrate that immature B cells circulate in the periphery before their final arrival to the spleen for their maturation. Ly49D expressed on these immature cells recognizes MHC class I on various peripheral tissues, resulting in constant low level secretion of IFN-. This constant recognition and IFN- secretion prevent immature B cells arrival to Ag-enriched sites and target them to their maturation compartment in the spleen. After maturation, B cells down-regulate their Ly49D expression, and this down-regulation terminates the IFN- secretion.

	Acknowledgments

 
We thank Prof. Lea Eisenbach, Dr. Esther Zahovel, and Ezra Vadaai for the generous gift of Abs and mice, and the Shachar laboratory for helpful discussions and review of this manuscript.

	Footnotes

 
¹ This work was supported by the Israel Science Foundation founded by the Academy of Sciences and Humanities and by the Minerva Foundation. I.S. is the incumbent of the Alvin and Gertrude Levine Career Development Chair of Cancer Research.

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

³ Abbreviations used in this paper: ITIM, immune receptor tyrosine-based inhibitory motif; HPRT, hypoxanthine phosphoribosyltransferase; LN, lymph nodes; SDF-1, stromal cell-derived factor 1.

Received for publication February 7, 2003. Accepted for publication August 28, 2003.

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