OSCAR is a receptor for surfactant protein D that activates TNF-α release from human CCR2+ inflammatory monocytes

Abstract

Surfactant protein D (SP-D) is critical for maintenance of lung homeostasis and provides a first line of defense to pathogens at mucosal surfaces. Polymorphisms in the SP-D-encoding gene SFTPD have been associated with chronic obstructive pulmonary disease and ulcerative colitis. Identification of the immunoreceptors that bind SP-D is essential for understanding its contribution to lung homeostasis and mucosal defense. We located a putative binding motif for the osteoclast-associated receptor (OSCAR) within the SP-D collagenous domain. An OSCAR-Fc fusion protein specifically bound to the collagenous region of recombinant SP-D and captured native SP-D from human bronchoalveolar lavage. OSCAR localized in an intracellular compartment of alveolar macrophages together with SP-D. Moreover, we found OSCAR on the surface of interstitial lung and blood CCR2(+) inflammatory monocytes, which secreted TNF-α when exposed to SP-D in an OSCAR-dependent fashion. OSCAR and SP-D did not exclusively colocalize in lung, as they were also highly expressed in atherosclerotic plaques of human aorta, supporting a role for this interaction in atherosclerosis. Our results identify the OSCAR:SP-D interaction as a potential therapeutic target in chronic inflammatory diseases of the lung as well as other diseases involving tissue accumulation of SP-D, infiltration of inflammatory monocytes, and release of TNF-α.

FIGURE 1

OSCAR is a novel SP-D receptor. (A) OSCAR-Fc, LAIR1-Fc, ILT3-Fc and TREM1-Fc binding to different human (h) and rat (r) SP-D isoforms and collagen I (± 5mM Ca²⁺) in solid-phase (*, P <0.05). (B) Capture of Hyb-246-04 mAb reactive protein from human BAL (n=1 donor) or (C) soluble recombinant hSP-D dodecamer by plate-immobilized OSCAR-Fc (circles) or LAIR1-Fc (triangles) (*, P <0.05). (D) GFP expression from OSCAR-CD3ζ NFAT-GFP reporter cells incubated with recombinant SP-D isoforms in solid- or (E) soluble-phase. Data were performed in triplicate and are representative of three independent experiments.

FIGURE 2

OSCAR expression in lung resident immune cells. (A) Representative light microscopy sections of normal lung immunostained with: (i) rabbit (brown) and mouse IgG1 (blue; x200 objective) control antibodies; (ii) rabbit anti-SP-D (brown; x200 objective); (iii) rabbit anti-SP-D and TTF1 mAb (blue; x600 objective); or (iv) OSCAR C-terminus mAb (brown; x200 objective). (B) Representative confocal sections of normal (i–iii) or COPD (iv–vi) lungs immunostained with: (i) goat (red) and mouse IgG2a (green) control antibodies; (ii–vi) goat anti-OSCAR (red) and CD68 mAb (green; co-localization, yellow), Bar=20μM; (iii & v) Higher magnification confocal microscopy of areas demarcated by white squares in (ii) and (iv), respectively, Bars=5μM. (vi) Alveolar macrophage (arrow) and smaller interstitial lung monocyte (arrowhead), Bar=5μM. (C) Representative confocal sections of normal lungs immunostained with: (i) SP-D mAb 245-01 (green) and goat anti-OSCAR (red), Bar=20μM. (ii) Higher magnification confocal microscopy of area demarcated by white square in (i), Bar=2μM.

FIGURE 3

Lung and blood monocytes express cell surface OSCAR. (A) Representative histogram plots for lung cells triple-stained with mAbs to CD14, OSCAR and HLA-DR (open histograms) compared to isotype control mAb (gray histograms) for low (Lo) Forward Scatter (FSC)/side scatter (SSC) lung tissue monocytes (Gate R1, upper histogram panels) or high (Hi) FSC/SSC ‘alveolar macrophages’ (Gate R2, lower histogram panels). (B) Representative plots for OSCAR immunostaining of peripheral blood SSC^HiCD45⁺CD56⁻CD16⁺ granulocytes (upper panels). Within the SSC^LoCD45⁺CD56⁻HLA-DR⁺ monocytic population, CCR2⁺CD14^HiCD16⁻ ‘inflammatory’ monocytes express OSCAR (bottom panels) but not CCR2⁻CD14^LoCD16^Hi ‘non-inflammatory’ monocytes (center panels) (n=3 donors).

FIGURE 4

SP-D functionally interacts with OSCAR. (A) TNF-α release by human CCR2⁺ monocytes from two different donors treated with soluble SP-D in the presence of either IgG1 or anti-OSCAR blocking mAbs, 11.20.08 or 11.20.25. (B) Representative sections of atherosclerotic lesions (tunica intima, i–iv; tunica media, v–viii) from human aorta immunostained with: (i & v) rabbit anti-SP-D (brown; x100 objective); (ii & vi) anti-OSCAR C-terminal mAb 13-9-17 (brown; x100 objective); (iii & vii) x200 objective of areas demarcated by black rectangles in (ii) and (vi), respectively; (iv) OSCAR C-terminal mAb 13-9-17 (brown) and anti-CD163 mAb (blue, x600 objective); (viii) rabbit (brown) and mouse IgG1 (blue) control antibodies (x200 objective).