The vitronectin receptor and its associated CD47 molecule mediates proinflammatory cytokine synthesis in human monocytes by interaction with soluble CD23

Abstract

The vitronectin receptor, alphavbeta3 integrin, plays an important role in tumor cell invasion, angiogenesis, and phagocytosis of apoptotic cells. CD47, a member of the multispan transmembrane receptor family, physically and functionally associates with vitronectin receptor (VnR). Although vitronectin (Vn) is not a ligand of CD47, anti-CD47 and beta3 mAbs suppress Vn, but not fibronectin (Fn) binding and function. Here, we show that anti-CD47, anti-beta3 mAb and Vn, but not Fn, inhibit sCD23-mediated proinflammatory function (TNF-alpha, IL-12, and IFN-gamma release). Surprisingly, anti-CD47 and beta3 mAbs do not block sCD23 binding to alphav+beta3+ T cell lines, whereas Vn and an alphav mAb (clone AMF7) do inhibit sCD23 binding, suggesting the VnR complex may be a functional receptor for sCD23. sCD23 directly binds alphav+beta3+/CD47(-) cell lines, but coexpression of CD47 increases binding. Moreover, sCD23 binds purified alphav protein and a single human alphav chain CHO transfectant. We conclude that the VnR and its associated CD47 molecule may function as a novel receptor for sCD23 to mediate its proinflammatory activity and, as such, may be involved in the inflammatory process of the immune response.

Figure 1

Clone 10G2 neutralizes sCD23 biological activities and recognizes CD47 Ag. (A) Inhibition of sCD23 costimulation of IFN-γ production in T cell/monocyte cocultures by anti-CD47 mAbs (clones 10G2, B6H12, C1Km1, and BRIC126). Mean ± SD for 10G2 P < 0.03 (ten experiments) and for other mAbs P < 0.001 (five independent experiments). (B) 10G2 mAb staining of untransfected (COS, dotted line) or CD47-transfected COS cell line (COS-47, bold line). Control mAb staining of both cell lines: (COS, thin line; COS-47, dashed line). (C) Fluorescence staining of Jurkat T cell line (left) and THP-1 monocyte cell line (right) by increasing concentrations of anti-CD47 mAbs (clones 10G2 and B6H12). One representative experiment out of three.

Figure 2

Anti-CD47 mAb inhibits CD23 costimulation of IFN-γ production. (A) Dose-dependent inhibition of IFN-γ production in IL-2 plus sCD23 stimulated coculture system by mAb clone BRIC126 and F(ab′)₂ fragments of mAb clone B6H12. One representative experiment out of two. (B) Anti-CD47 mAb (clone B6H12) inhibition of IFN-γ secretion by T cells cocultured with autologous monocytes and increasing numbers of untransfected or CD23-transfected CHO cells. Similar data were obtained using clone 10G2 in three independent experiments. (C and D) Anti-CD47 mAbs (clone B6H12) mediated inhibition of IFN-γ and IL-12 secretion in cocultures of IL-2– or IL-15–stimulated T cells and autologous monocytes in the absence or presence of sCD23. Mean ± SD of six experiments. Anti-CD47 mAb inhibition in IL-2– or IL-15–stimulated cocultures, for IFN-γ production P < 0.001, and P < 0.01 for IL-12 secretion.

Figure 3

Anti-CD47 mAb inhibition of sCD23-induced monokine release by purified monocytes. Monocytes (10⁶/ml) were cultured in the absence or presence of sCD23 or LPS with or without anti-CD47 mAb (clone 10G2). After overnight cultures, TNF-α (A), IL-1β, IL-8, or PGE₂ (B) was measured in the culture supernatant. Mean ± SD of six experiments (*P < 0.05, **P < 0.01).

Figure 4

Suppression of sCD23-induced TNF-α release by anti-β₃ (CD61) mAb and Vn. sCD23-activated monocytes were cultured with anti-β₃ (CD61) clone AP3, anti-α_vβ₃ (CD51/CD61; clone LM609), or isotype-matched mAbs (A) and with soluble Vn or Fn (20 μg/ml, B). Mean ± SD of four experiments (P < 0.01).

Figure 5

Effect of mAbs or natural ligands to VnR/CD47 complex on sCD23 binding to Jurkat and Raji cell lines. Jurkat α_v ⁺β₃ ⁺ (CD51⁺/CD61⁺) (a–e), or Raji α_v ⁺β₃ ⁻ (CD51⁺/CD61⁻) (f) cell lines were stained by B-BSA (dotted line) or B-sCD23 in the absence (solid line) or presence of the following inhibitors (plain histograms): anti-CD23 mAb (a); anti-CD47 mAbs (cocktail of four anti-CD47 mAbs; b); anti-β₃ (CD61) mAb (c); Vn (d); anti-α_v (CD51) mAb (e and f). B-sCD23 plus isotype-matched control mAb (a–c, e, and f) or Fn (d) were shown as dashed lines. One representative experiment out of four.

Figure 6

Western blot analysis of α_v ⁺β₃ ⁺ (CD51⁺/CD61⁺) melanoma cell line. Cell lysate was purified on anti-β₃ (CD61) mAb-affinity column. The eluate was separated by SDS-PAGE (5%) and transferred to membrane. Staining by anti-α_v (CD51) mAb (clone AMF7 and LM142; lane 2), anti-β₃ (CD61, clone AP3; lane 4), isotype-control matched mAbs (lanes 1 and 3), B-BSA (lane 5), and B-sCD23 (lane 6) is shown.

Figure 7

Binding of sCD23 to CHO-51 (CHO α_v) transfected cell line. (A) α_v (CD51)-transfected CHO cell line was stained with (a) anti-CD47 mAbs (clones 10G2, solid line and B6H12, dotted line) or isotype-matched control mAbs or with (b) anti-α_v (CD51) mAb (clone AMF7, dashed line) and anti-β₃ (CD61) mAb (clone AP3, solid line), or isotype-matched cont mAbs. (B) Untransfected (a), and α_v (CD51)-transfected CHO cell lines (b) were stained with B-sCD23 (plain histograms) or B-BSA (dotted line). One representative experiment out of three.

Figure 8

Binding of sCD23 to CD47⁺ and CD47⁻ cell lines. CD47⁺ and CD47⁻ cell lines were stained with two anti-CD47 mAbs (clone 10G2 and B6H12), anti-α_v (CD51) and anti-β₃ (CD61) mAbs, or B-sCD23 as described in Materials and Methods. OV10 carcinoma and Jurkat cell lines express different levels of α_v (CD51) chain which correlate with sCD23 binding.

Figure 9

sCD23 and Vn share the same functional receptor, VnR/CD47 complex. (A) sCD23 binds to α_v/CD51 and Vn to α_vβ₃ (CD51/CD61) conformational site while neither bind to CD47. Binding of Vn to α_vβ₃ (CD51/CD61) obliterates sCD23 binding site, and vice versa. (B and C) Anti-CD47 and anti-β₃ (CD61) mAbs suppress Vn binding and function (left) while they inhibit sCD23 function, but not binding (right).