Structurally distinct phosphatases CD45 and CD148 both regulate B cell and macrophage immunoreceptor signaling

Abstract

The receptor-type protein tyrosine phosphatase (RPTP) CD148 is thought to have an inhibitory function in signaling and proliferation in nonhematopoietic cells. However, its role in the immune system has not been thoroughly studied. Our analysis of CD148 loss-of-function mice showed that CD148 has a positive regulatory function in B cells and macrophages, similar to the role of CD45 as a positive regulator of Src family kinases (SFKs). Analysis of CD148 and CD45 doubly deficient B cells and macrophages revealed hyperphosphorylation of the C-terminal inhibitory tyrosine of SFKs accompanied by substantial alterations in B and myeloid lineage development and defective immunoreceptor signaling. Because these findings suggest the C-terminal tyrosine of SFKs is a common substrate for both CD148 and CD45 phosphatases and imply a level of redundancy not previously appreciated, a reassessment of the function of CD45 in the B and myeloid lineages based on prior data from the CD45-deficient mouse is warranted.

Figure 1. T and B cell phenotypes in CD148 TM-KO mice

(A) Representative FACS analyses of thymocytes from 8-week-old B6 WT and CD148 TM-KO mice. (B) Splenocytes, pre-gated on CD19+ CD1d^lo to exclude MZ cells, were further separated into T1 (IgM^hi IgD^lo) and T2 + FM (follicular mature) B cells (IgM^hi IgD^hi for T2 and IgM^lo IgD^hi for FM). (C) Splenocytes from the indicated mice were stained with mAb to CD19 and CD1d. Percentages of MZ (CD1d^hi) cells in the CD19+ gate are shown. (D) FACS analysis of peritoneal lavage from 8-week-old B6 WT and CD148 TM-KO mice, stained with mAb to CD19, CD5 and CD23. Percentages of B1b (CD5+, CD23-), B1a (CD5-, CD23-) and B2 (CD5-, CD23+) cells in the CD19+ gate are shown. (E,F,G) Quantitation of percentages and cell numbers of T1 and T2+FM (E), MZ (F), B1a and B2 (G) are indicated. Complete quantification of all subsets is provided in supplementary table 1.

Figure 2. CD45/CD148 DKO mice on the B6 background die prematurely

(A) Body weight of 4-6 week old mice of the indicated genotypes. (B) Percent survival of mice of the indicated genotypes. (C,D) Total cell numbers of spleens (C) and lymph nodes (D) from 4-6 week old mice of the indicated genotypes. (E) Liver sections (10X) from 5 week old mice of the indicated genotypes stained with H&E. Inserts are 63X. (F) Lung sections (10X) from 5 week old mice of the indicated genotypes stained with H&E.

Figure 3. B cell developmental block in CD45/CD148 DKO mice on the B6 background

(A) Percentages and cell numbers of CD19+ splenocytes from 4-6 week-old mice of the indicated genotypes. (B) Representative FACS analyses of CD19+ splenocytes stained with mAb to CD19, IgM and IgD. Percentages of CD19+ B cells, T1 (IgM^hi IgD^lo) cells and T2 (IgM^hi IgD^hi) plus follicular mature (FM) (IgM^lo IgD^hi) B cells are shown. (C) Percentages and absolute numbers of each B cell subset are shown. (D) Percentages and cell numbers of CD19+ lymphocytes in the BM from 4-6 week old mice of the indicated genotypes. (E) Representative FACS analyses of BM cells stained with mAb to CD19 and CD43. Percentages of CD19+ B cells and CD43- or CD43+ in CD19+ gate are shown. (F) Based on the FACS analysis, CD19+ BM B cells were identified as fraction B (CD43+ CD24+, BP1-), fraction C (CD43+ CD24+, BP1+), fraction D (CD43-IgM- IgD-), fraction E (CD43- IgM+ IgD-), fraction F (CD43- IgM+ IgD+) (see also Supplementary Fig. 2). Fraction A (B220+, CD19-) could not be analyzed due to the lack of the B220 marker in DKO mice (ND)(Hardy et al., 2000).

Figure 4. Impaired BCR-mediated signaling in CD45/CD148 DKO mice

(A) Purified lymph node B cells of the indicated mice were loaded with Fluo3-AM and Fura Red and intracellular free Ca²⁺ concentrations were monitored before and after addition of IgM F(ab')₂ (5μg/ml). (B) Purified lymph node B cells of all four genotypes were stimulated with F(ab')2 fragments of anti-IgM antibody (5 μg/ml). At the indicated time points, the cells were lysed in SDS-PAGE sample buffer and the lysates were analyzed by immunoblotting with phosphotyrosine antibody. (C) Samples from panel (B) were subjected to immunoblotting with phospho-specific antibodies as indicated. Staining of total Erk served as a loading control. (D) Samples from panel (B) (the 0 min and 1 min timepoints) were stained with site-specific antibody against phosphorylated activation loop of SFKs (Src-family P-Y416, this antibody crossreacts with multiple SFKs) and against C-terminal inhibitory phosphotyrosine of Lyn (Lyn P-Y507). The right hand panels are protein controls in the various genotypes. See quantification for westerns shown in (C) and (D) in supplementary Fig. 6.

Figure 5. BCR mediated signaling and B cell development in competitively reconstituted chimeras

(A) Cell autonomous defect in intracellular free Ca²⁺ increase. Lethally irradiated recipient mice were reconstituted with BM from CD45/CD148 DKO and WT control mice. The donor origins of reconstituted B cells were identified using CD45.2 as a congenic marker: WT B cells were CD45.2+ and DKO B cells were CD45.2-. Lymph node B cells were stained with CD45.2 (upper left) and IgM /IgD (upper right) antibodies. The intracellular free Ca²⁺ levels were monitored before and after addition of IgM F(ab')₂ (5μg/ml). (B) Cell autonomous defect in activation of ERK. Purified B cells as in (A) were stimulated with IgM F(ab')₂ (5μg/ml) or PMA for 2.5 minutes, fixed, permeabilized and then stained with phospho-ERK along with other surface makers. Follicular mature B cells (IgM+IgD+CD23+) were separated based on the origin of the donors (same as in (A)). Phospho-ERK levels of CD45.2+ (WT origin) and CD45.2- (DKO origin) upon indicated stimulation were overlaid in histograms. (C) B cell development in competitively reconstituted chimera. Cells from BM and spleen were isolated and stained with antibodies as indicated. For BM, CD19+ B cells were gated to separate CD43+, and CD43-. The CD43- cells were further subdivided based on expression of IgM and IgD. For the spleen, CD19+ B cells were separated based on IgM and IgD expression. The donor origin of each subset was then identified by CD45.2 staining. Percentages of WT and DKO B cells of each sunset were presented in the overlaid bar graph. Results in (C) are representive of 4 independent experiments.

Figure 6. CD148 and CD45 regulate Fc receptor mediated phagocytosis and cytokine production in BMDMs

(A) BMDMs were incubated with human red blood cells coated with mouse IgG2a mAb. BMDMs with ingested erythrocytes were fixed, labeled with Hoechst and quantified under fluorescent microscope. Phagocytic index was determined as [number of erythrocytes] / [number of nuclei]. Phagocytic index of WT macrophages was then set as 100%. * p< 0.001 (B,C) BMDMs were stimulated with plate-bound mAb to FcR II/III (2.4G2) (B) or LPS (C) in the presence of brefeldin A for 4 hours. TNFα production was detected by intracellular staining with TNFα-specific antibody. (D) BMDMs were stained with mAb to FcγR II/III (2.4G2) or mouse IgG. The expression levels on the BMDMs of the indicated genotypes over the negative control (shaded histogram) are shown.

Figure 7. Impaired FcR mediated signaling in DKO BMDMs

(A) BMDMs of all four genotypes were sensitized with purified mouse IgG (20 μg/ml) and then stimulated with anti-mouse secondary antibody (10 μg/ml). Cells were lysed in SDS-PAGE sample buffer and the lysates were analyzed by immunoblotting with phosphotyrosine antibody (4G10). Staining of total Erk served as a loading control. (B) Samples from BMDMs stimulated the same way as in (a) were subjected to immunoblotting with phospho-specific antibodies as indicated. Staining of tubulin represents a loading control. (C) Samples from panel (B) 0 min and 2 min timepoints were stained with site-specific antibody against phosphorylated activation loop of SFKs (Src-family P-Y416) and against the C-terminal inhibitory phosphotyrosine of Lyn (Lyn P-Y507). See quantification for westerns shown in (B) and (C) in supplementary Fig. 6.