CD72 negatively regulates KIT-mediated responses in human mast cells

Abstract

KIT activation, through binding of its ligand, stem cell factor, is crucial for normal mast cell growth, differentiation, and survival. Furthermore, KIT may also contribute to mast cell homing and cytokine generation. Activating mutations in KIT lead to the dysregulated mast cell growth associated with the myeloproliferative disorder, mastocytosis. We investigated the potential of downregulating such responses through mast cell inhibitory receptor activation. In this study, we report that the B cell-associated ITIM-containing inhibitory receptor, CD72, is expressed in human mast cells. Ligation of CD72 with the agonistic Ab, BU40, or with recombinant human CD100 (rCD100), its natural ligand, induced the phosphorylation of CD72 with a resulting increase in its association with the tyrosine phosphatase SH2 domain-containing phosphatase-1. This, in turn, resulted in an inhibition of KIT-induced phosphorylation of Src family kinases and extracellular-regulated kinases (ERK1/2). As a consequence of these effects, KIT-mediated mast cell proliferation, chemotaxis, and chemokine production were significantly reduced by BU40 and rCD100. Furthermore, BU40 and rCD100 also downregulated the growth of the HMC1.2 human mast cell line. Thus, targeting CD72 may provide a novel approach to the suppression of mast cell disease such as mastocytosis.

Figure 1. Human mast cells and mast cell lines express CD72

(A) RT-PCR. (B) Western blotting. (C) Flow cytometry were performed as described in Material and Methods. The Raji cells were used as a positive control and the U937 cells as a negative control.

Figure 2

The effects of BU40 or recombinant CD100 on the tyrosine phosphorylation of CD72, the association of SHP-1 with CD72 and the tyrosine phosphorylation of SHP-1 in huMCs. (A) BU40 or recombinant CD100 administration with SCF to huMCs induces the tyrosine phosphorylation of CD72 and the association with SHP-1, whereas (B) BU40 or recombinant CD100 administration in the absence of SCF did not. HuMCs were incubated for 0 or 5 min with control IgG (10 µg/ml), BU40 (10 µg/ml), or recombinant CD100 (10 µg/ml) in the presence or absence of SCF (10 ng/ml). CD72 was immunoprecipitated with anti-CD72 (H-96), and visualized with anti-phosphotyrosine 4G10, anti-SHP-1, or anti-CD72. (C) BU40 or recombinant CD100 administration with SCF (10 ng/ml) to huMCs induces the tyrosine phosphorylation of SHP-1, when incubated for 5 min. SHP-1 was immunoprecipitated with an anti-SHP-1 antibody (C-19), and visualized with anti-phosphotyrosine 4G10 or anti-SHP-1. Data are representative of three individual experiments.

Figure 3

The effects of BU40 or recombinant CD100 on KIT-mediated responses of huMCs. (A) The ligation of CD72 with BU40 or recombinant CD100 suppresses KIT-induced proliferation of huMCs (BrdU assay, n = 4). Human mast cells were incubated for 24 h with or without control IgG, BU40, or recombinant CD100 (10 µg/ml, respectively) in the absence or presence of SCF (10 ng/ml). HuMC proliferation was assessed using a BrdU cell proliferation assay kit according to the manufacture’s protocol. The relative values are indicated when the value of SCF + control IgG is 100. *; P < 0.05, when compared with SCF + control IgG. (B) Ligation of CD72 with BU40 or recombinant CD100 suppresses SCF-induced mast cell chemotaxis. HuMCs (1 × 10⁵) with or without control IgG, BU40, or recombinant CD100 (10 µg/ml, respectively) were incubated in the upper chambers to assess the migration to the lower chambers containing SCF (30 ng/ml) for 4 hours (n = 3). After the incubation, cell migration to the lower wells was assed by counting by microscopy. *; P < 0.05, when compared with SCF + control IgG. (C) Ligation of CD72 with BU40 or recombinant CD100 suppresses SCF-induced MCP-1 secretion. HuMCs were incubated for 6 h with or without control IgG, BU40, or recombinant CD100 (10 µg/ml, respectively) in the absence or presence of SCF (10 ng/ml) (n = 5). The culture supernatants were used for the ELISA assay for human MCP-1. *; P < 0.05, when compared with SCF + control IgG.

Figure 4

The effects of BU40 or recombinant CD100 on IgE-triggered degranulation of huMCs. BU40 or recombinant CD100 administration did not inhibit IgE/streptavidin- (FcεRI-mediated) induced degranulation in the absence of SCF. However, BU40 and recombinant CD100 administration suppressed SCF-induced augmentation of IgE/streptavidin-induced degranulation of huMCs. Degranulation from huMCs was evaluated by β-hexominidase release as described in Material and Methods. HuMCs were sensitized overnight in cytokine-free medium containing biotinylated-human myeloma IgE (100 ng/ml) in the presence of control IgG, BU40, or rCD100 (10 µg/ml, respectively), and activated with streptavidin (100 ng/ml) with or without SCF (1 ng/ml) for 30 min (n = 3). *; P < 0.05, when compared with control IgG in the absence or presence of SCF.

Figure 5

Ligation of CD72 with BU40 or recombinant CD100 suppresses the activation of KIT signaling in huMCs. HuMCs were incubated for the indicated times with control IgG, BU40, or recombinant CD100 (10 µg/ml, respectively) in the presence of SCF (10 ng/ml). The levels of phospho-KIT, phospho-ERK, phospho-Src family kinases (Tyr 416), and β-actin were then evaluated by immunoblot analysis. Data are representative from three individual experiments.

Figure 6

Ligation of CD72 with BU40 or recombinant CD100 suppresses mutated KIT-driven proliferation of HMC1.2 (BrdU assay, n = 3). HMC1.2 cells were incubated for 24 h with control medium, control IgG, BU40, or recombinant CD100 (10 µg/ml, respectively). Incorporation of BrdU into the HMC1.2 cells was assessed using a BrdU cell proliferation assay kit according to the manufacture’s protocol. The relative values are indicated when the value of control IgG is 100. *; P < 0.05, when compared with control IgG.

Figure 7

The effects of BU40 or recombinant CD100 on the tyrosine phosphorylation of CD72, the association with SHP-1 with CD72, and on the activation of signaling molecules in HMC1.2 cells. BU40 or recombinant CD100 administration to HMC1.2 cells up-regulates (A) the tyrosine phosphorylation of CD72, the association with SHP-1 and (B) the tyrosine phosphorylation of SHP-1. HMC1.2 cells were incubated for 0 or 5 min with control IgG, BU40, or recombinant CD100 (10 µg/ml respectively) in the absence of SCF. CD72 was immunoprecipitated with anti-CD72 (H-96) or anti-SHP-1 (C-19), and visualized with anti-phosphotyrosine 4G10, anti-SHP-1, or anti-CD72. Data are representative from three individual experiments. (C) Ligation of CD72 with BU40 or recombinant CD100 suppresses the activation of signal molecules in HMC1.2 cells. HMC1.2 cells were incubated for the indicated time with control IgG, BU40, or recombinant CD100 (10 µg/ml respectively). The levels of phospho-KIT, phospho-ERK, phospho-Src family kinases (Tyr 416), and β-actin were then assssed by immunoblot analysis. Data are representative from three individual experiments.

Figure 8

Proposed model of the effects of CD72 – CD100 system on human mast cells. SFKs; Src family kinases, ERKs; extracellular-regulated kinases.