C-reactive protein-mediated phagocytosis and phospholipase D signalling through the high-affinity receptor for immunoglobulin G (FcgammaRI)

Abstract

C-reactive protein (CRP) is the prototypic acute-phase protein in man which performs innate immune functions. CRP-mediated phagocytosis may be indirect, through activation of complement and complement receptors, or direct, through receptors for the Fc portion of immunoglobulin G (IgG; FcgammaRs) or even a putative CRP-specific receptor. No strong evidence has been shown to indicate which receptors may be responsible for phagocytosis or signalling responses. Using BIAcore technology, we confirm that CRP binds directly to the extracellular portion of FcgammaRI with a threefold higher affinity than IgG (KD = 0.81 x 10-9 m). Binding is Ca2+ dependent and is inhibited by IgG1 but not by phosphorylcholine (PC). CRP opsonization (using CRP concentrations within the normal human serum range) of PC-conjugated sheep erythrocytes increased phagocytosis of these particles by COS-7 cells transfected with FcgammaRI-II chimaera or FcgammaRI/gamma-chain. Interferon-gamma-treated U937 cells, which signal through FcgammaRI to activate phospholipase D (PLD) in response to cross-linked IgG, were also activated by CRP without any requirement for further cross-linking. These studies indicate that CRP is capable of binding to and cross-linking FcgammaRI thereby resulting in PLD activation and increased phagocytosis. Uptake by FcgammaRI has been reported to promote various acquired immune responses suggesting that CRP could act in a similar way.

Figure 1

CRP binding to immobilized FcγRI on the BIAcore. (a) Sensogram showing sequential binding of FcγRI-GPI and CRP (1 mg/ml) to the GαM IgG1 and mouse anti-FcγRI (22/32) coated sensor chip surface. (b) Sensogram showing the same sequence as (a) in the absence of calcium, resulting in no binding of CRP to immobilized FcγRI. (c) The theoretical curve fit for the association of CRP for FcγRI (as determined by BIA evaluation 2·1 curve fit) directly overlays the actual curve from the sensogram in (a).

Figure 2

Phagocytosis of erythrocytes (E) opsonized with CRP or IgG1 by PBMC adherent cells. Day 6 adherent PBMCs were incubated with (a) erythrocytes, (b) BSA-coupled erythrocytes (BSAE), or (c) PC-BSA-conjugated erythrocytes (PCE) which were unopsonized (open bars) or had been preopsonized with either 2 μg/ml CRP (filled bars) or rabbit IgG1 anti-stromal antibody (hatched bars). After lysing uninternalized cells, the percentage phagocytosis was determined using light micoscopy. Each bar represents the mean ±SEM of three independent experiments. The phagocytic index was expressed as the number of erythrocytes/100 cells for PCE conjugated erythrocytes (d). Each bar represents the mean ±SEM of eight independent experiments.

Figure 3

Rosetting and phagocytosis of CRP- or IgG1-opsonized PCE by COS-7 cells transfected with FcγRI-II or FcγRI-GPI. COS-7 cells transfected with either FcγRI-II (open bars) or FcγRI-GPI (filled bars) were incubated with PCE or IgG1-opsonized PCE (a,b) and PCE or CRP-opsonized PCE (c,d). The percentage rosetted cells (a,c) and, after lysis of uninternalized erythrocytes, the percentage phagocytosis (b,d) were determined using light microscopy. In addition, the number of internalized erythrocytes per 100 cells was determined in FcγRI-II chimaera-transfected COS-7 cells after incubation with PCE, CRP-PCE and IgG1-PCE (e). The data represent the mean ±SEM of six independent experiments.

Figure 4

Rosetting and phagocytosis of CRP- or IgG1-opsonized PCE by COS-7 cells transfected with FcγRI ±γ-chain. COS-7 cells transfected with either FcγRI and γ-chain (open bars) or FcγRI alone (filled bars) were incubated with PCE or IgG1-opsonized PCE (a,b) and PCE or CRP-opsonized PCE (c,d). The percentage rosetted cells (a,c) and, after lysis of uninternalized erythrocytes, the percentage phagocytosis (b,d) were determined using light microscopy. In addition, the number of internalized erythrocytes per 100 cells was determined in FcγRI and γ-chain-transfected COS-7 cells after incubation with PCE, CRP-PCE and IgG1-PCE (e). The data represent the mean ±SEM of four independent experiments.

Figure 5

Induction of PLD activity by CRP or cross-linked IgG1 in IFN-γ treated U937 cells. IFN-γ-treated U937 cells were incubated with medium (basal), CRP (20 μg/ml) or IgG1 (40 μg/ml followed by F(ab)′₂ anti-IgG). Following the addition of [³H]butanol, incorporation of phosphatidylbutanol was measured and expressed as a percentage of total [³H]palmitate incorporation. The bars represent the mean ±SEM of four independent experiments.