Reversal of proinflammatory responses by ligating the macrophage Fcgamma receptor type I

Abstract

Macrophages can respond to a variety of infectious and/or inflammatory stimuli by secreting an array of proinflammatory cytokines, the overproduction of which can result in shock or even death. In this report, we demonstrate that ligation of macrophage Fcgamma receptors (FcgammaR) can lead to a reversal of macrophage proinflammatory responses by inducing an upregulation of interleukin (IL)-10, with a reciprocal inhibition of IL-12 production. IL-10 upregulation was specific to FcgammaR ligation, since the ligation of the Mac-1 receptor did not alter IL-10 production. The identification of the specific FcgammaR subtype responsible for IL-10 upregulation was determined in gene knockout mice. Macrophages from mice lacking the FcR gamma chain, which is required for assembly and signaling by FcgammaRI and FcgammaRIII, failed to upregulate IL-10 in response to immune complexes. However, mice lacking either the FcgammaRII or the FcgammaRIII were fully capable of upregulating IL-10 production, implicating FcgammaRI in this process. The biological consequences of FcgammaRI ligation were determined in both in vitro and in vivo models of inflammation and sepsis. In all of the models tested, the ligation of FcgammaR promoted the production of IL-10 and inhibited the secretion of IL-12. This reciprocal alteration in the pattern of macrophage cytokine production illustrates a potentially important role for FcgammaR-mediated clearance in suppressing macrophage proinflammatory responses.

Figure 1

FcγR ligation enhances LPS-induced IL-10 production. (A) BMMφ were exposed to either LPS alone or LPS in combination with either E-IgG or E-C3bi. 6 h after the addition of stimuli, total RNA was isolated and used to carry out competitive RT-PCR. Input cDNAs were adjusted to yield comparable ratios of competitor (upper band in each reaction) to wild-type (lower band in each reaction) intensities for the amplification reaction for hypoxanthine-guanine phosphoribosyltransferase (HPRT), as resolved on a 2% ethidium-stained agarose gel. The adjusted input cDNAs were then used in subsequent RT-PCR reactions using primers for IL-10. Results are representative of two separate experiments. (B) cDNA generated from BMMφ exposed to LPS or LPS in combination with E-IgG, were first normalized for HPRT levels. Constant volumes of normalized cDNAs were then amplified in the presence of increasing concentrations of competitor (PQRS), using primers for IL-10. The concentration of the experimental cDNA is represented by the equivalent intensities of competitor and wild-type bands. The fold increase in IL-10 levels between BMMφ exposed to LPS or LPS in combination with E-IgG can be determined by taking the ratio of their equivalence points. (C) BMMφ were exposed to either media, LPS, E-IgG, or E-C3bi (inset), or LPS alone or LPS in combination with either E-IgG or E-C3bi. After 24 h, the supernatant was harvested, and IL-10 levels were determined by ELISA. Values represent the mean of three independent experiments, each performed in triplicate, ±SE.

Figure 1

FcγR ligation enhances LPS-induced IL-10 production. (A) BMMφ were exposed to either LPS alone or LPS in combination with either E-IgG or E-C3bi. 6 h after the addition of stimuli, total RNA was isolated and used to carry out competitive RT-PCR. Input cDNAs were adjusted to yield comparable ratios of competitor (upper band in each reaction) to wild-type (lower band in each reaction) intensities for the amplification reaction for hypoxanthine-guanine phosphoribosyltransferase (HPRT), as resolved on a 2% ethidium-stained agarose gel. The adjusted input cDNAs were then used in subsequent RT-PCR reactions using primers for IL-10. Results are representative of two separate experiments. (B) cDNA generated from BMMφ exposed to LPS or LPS in combination with E-IgG, were first normalized for HPRT levels. Constant volumes of normalized cDNAs were then amplified in the presence of increasing concentrations of competitor (PQRS), using primers for IL-10. The concentration of the experimental cDNA is represented by the equivalent intensities of competitor and wild-type bands. The fold increase in IL-10 levels between BMMφ exposed to LPS or LPS in combination with E-IgG can be determined by taking the ratio of their equivalence points. (C) BMMφ were exposed to either media, LPS, E-IgG, or E-C3bi (inset), or LPS alone or LPS in combination with either E-IgG or E-C3bi. After 24 h, the supernatant was harvested, and IL-10 levels were determined by ELISA. Values represent the mean of three independent experiments, each performed in triplicate, ±SE.

Figure 1

FcγR ligation enhances LPS-induced IL-10 production. (A) BMMφ were exposed to either LPS alone or LPS in combination with either E-IgG or E-C3bi. 6 h after the addition of stimuli, total RNA was isolated and used to carry out competitive RT-PCR. Input cDNAs were adjusted to yield comparable ratios of competitor (upper band in each reaction) to wild-type (lower band in each reaction) intensities for the amplification reaction for hypoxanthine-guanine phosphoribosyltransferase (HPRT), as resolved on a 2% ethidium-stained agarose gel. The adjusted input cDNAs were then used in subsequent RT-PCR reactions using primers for IL-10. Results are representative of two separate experiments. (B) cDNA generated from BMMφ exposed to LPS or LPS in combination with E-IgG, were first normalized for HPRT levels. Constant volumes of normalized cDNAs were then amplified in the presence of increasing concentrations of competitor (PQRS), using primers for IL-10. The concentration of the experimental cDNA is represented by the equivalent intensities of competitor and wild-type bands. The fold increase in IL-10 levels between BMMφ exposed to LPS or LPS in combination with E-IgG can be determined by taking the ratio of their equivalence points. (C) BMMφ were exposed to either media, LPS, E-IgG, or E-C3bi (inset), or LPS alone or LPS in combination with either E-IgG or E-C3bi. After 24 h, the supernatant was harvested, and IL-10 levels were determined by ELISA. Values represent the mean of three independent experiments, each performed in triplicate, ±SE.

Figure 2

FcγRI is responsible for the FcγR-mediated enhancement of IL-10 production. BMMφ from C57BL/6, FcRγ^−/−, FcγRII^−/−, or FcγRIII^−/− mice were exposed to LPS alone or LPS in combination with either E-IgG or unopsonized erythrocytes (E). After 24 h, the supernatant was harvested, and IL-10 levels were determined by ELISA. Determinations were performed in triplicate, and values are expressed as the means ± SD. Results are representative of three separate experiments.

Figure 3

IL-10 produced by macrophages stimulated with LPS/FcγR can suppress IL-12 production. Supernatants from BMMφ exposed to either media alone or LPS in combination with E-IgG for 24 h were harvested and filtered through a 0.2-μm filter. Supernatants were diluted 1:3 with media and incubated for 15 min at 4°C in either the presence or absence of a neutralizing mAb to IL-10 (JESS-2A5; 20 μg/ml). Diluted supernatants were then added to BMMφ that had been primed with IFN-γ (100 U/ml) for 8 h, and immediately treated with LPS. After 24 h, the supernatant was harvested, and IL-12(p70) levels were determined by ELISA. Values represent the mean of three independent experiments, each performed in triplicate, ±SE.

Figure 4

The modulation of inflammatory response by FcγR ligation. BMMφ were exposed to either media, LPS, or IgG-LPS (A and B). After 24 h, the supernatant was harvested, and IL-10 (A) and IL-12(p40) (B) levels were determined by ELISA. Determinations were performed in triplicate, and values are expressed as the means ± SD. Results are representative of four separate experiments. BMMφ were incubated with media alone or with equal numbers of either unopsonized or IgG-opsonized H. influenzae (C and D). After 24 h, the supernatant was harvested, and IL-10 (C) and IL-12(p40) (D) levels were determined by ELISA. Determinations were performed in triplicate, and values are expressed as the means ± SD. Results are representative of three separate experiments.

Figure 4

The modulation of inflammatory response by FcγR ligation. BMMφ were exposed to either media, LPS, or IgG-LPS (A and B). After 24 h, the supernatant was harvested, and IL-10 (A) and IL-12(p40) (B) levels were determined by ELISA. Determinations were performed in triplicate, and values are expressed as the means ± SD. Results are representative of four separate experiments. BMMφ were incubated with media alone or with equal numbers of either unopsonized or IgG-opsonized H. influenzae (C and D). After 24 h, the supernatant was harvested, and IL-10 (C) and IL-12(p40) (D) levels were determined by ELISA. Determinations were performed in triplicate, and values are expressed as the means ± SD. Results are representative of three separate experiments.

Figure 5

Production of IL-12(p40) and IL-10 in a murine model of septic shock. RAG-1^−/− mice received either LPS or IgG-LPS intravenously at a final LPS dose of 4 μg per mouse. Serum levels of IL-12(p40) (A) and IL-10 (B) were measured at the indicated times after challenge. Data show the mean ± SD of groups of four separately handled mice. *P < 0.01, and **P < 0.08 (significant by rank–sum analysis) versus the LPS-treated group as determined by Student's t test.