CD300a contributes to the resolution of articular inflammation triggered by MSU crystals by controlling neutrophil apoptosis

Abstract

Gout is an inflammatory disease triggered by deposition of monosodium urate (MSU) crystals in the joints, resulting in high neutrophil influx and pain. Here, we studied the role of the inhibitory receptor CD300a in the resolution process in a murine model of gout. We found increased CD300a expression on neutrophils emigrated to the joint. When compared to WT mice, CD300a^-/- mice had persistent neutrophil influx till 24 hr after MSU injection. This was associated with increased concentration of IL-1β and greater tissue damage in the joints of CD300a^-/- mice. There was an increase in the percentage of apoptotic neutrophils in the synovial lavage of WT mice, as compared to CD300a^-/- mice. This difference was reflected in the decline of efferocytic events in the synovial cavity of CD300a^-/- mice 24 hr after MSU injection. A CD300a agonistic antibody was shown, for the first time, to increase apoptosis of human neutrophils, and this was associated with cleavage of caspase-8. In conclusion, our results reveal an important role of CD300a in the control of leucocyte infiltration, IL-1β production and caspase-8 cleavage in neutrophils, contributing to the resolution of inflammation triggered by MSU injection.

Keywords: CD300a; apoptosis; gout; neutrophilic inflammation; resolution of inflammation.

FIGURE 1

CD300a expression on neutrophils from blood and synovial cavity. BALB/c male mice were injected with 30 µg of MSU crystals into the tibiofemoral joint. Cells were harvested from the blood and articular cavity 12h after injection to measure CD300a expression on neutrophils by flow cytometry. Neutrophils were stained with anti‐Ly6G, and CD300a expression was evaluated on positive Ly6G cells (a). The median fluorescence intensity (MFI) of CD300a was measured on the neutrophils from the blood and synovial cavity (b). Bars show the mean ± SEM of 6 mice per group and are from one experiment representative of two independent experiments. Significance was calculated in relation to the control group (two‐tailed unpaired Student's t‐test). *p < 0·05

FIGURE 2

Time course of cell infiltrate and inflammatory mediators in acute gout inflammation. BALB/c and CD300a^−/− male mice were injected with MSU crystals into the tibiofemoral joint. Cells from the articular cavity and periarticular tissue were harvested at 12, 24 and 48 hr after injection. From the articular cavity, total cells (a), mononuclear cells (b) and polymorphonuclear cells (c) were analysed. Polymorphonuclear and mononuclear cell populations were determined in cytospin preparation count. Periarticular tissue was processed, and the inflammatory chemokine CXCL1 (d) and the inflammatory cytokine IL‐1β (e) were measured. Bars show the mean ± SEM of 6 mice per group and are from one experiment representative of two independent experiments. Significance was calculated using ANOVA followed by the Newman–Keuls test. **p < 0·005, *** p < 0·001

FIGURE 3

Histopathological analysis and articular dysfunction. BALB/c and CD300a^−/− male mice were injected with MSU crystals into the tibiofemoral joint. Histopathologic analysis (magnification of 100×) was observed 24 hr after MSU crystal injection by criteria previously described (a) (scale bar: 100 µm). Semiquantitative analysis of histological findings is also shown (b). To evaluate articular dysfunction, hypernociception was evaluated using an electronic pressure meter test at 24 hr after injection. Bars show the mean ± SEM of 6 mice per group and are from one experiment representative of two independent experiments. Significance was calculated using the ANOVA followed by the Newman–Keuls test and two‐tailed unpaired Student's t‐test in relation to the control group (the exact p‐value is shown in the figure). **p < 0·005

FIGURE 4

Difference in apoptosis among wild‐type and CD300a^−/− neutrophils. BALB/c and CD300a^−/− male mice were injected with MSU crystals into the tibiofemoral joint. Cells from the articular cavity were harvested 24 hr after injection, and cells with distinctive apoptotic morphology were evaluated on cytospin and expressed as per cent of neutrophil with apoptotic morphology (a). Representative figures (magnification of 1000×) of viable and apoptotic neutrophil (arrow) (b). BALB/c and CD300a^−/− male mice were injected with MSU crystals into the peritoneal cavity. Neutrophils were recovered and isolated 3 hr after peritoneal injection and incubated for 24 hr at 37°. After incubation, neutrophils were stained with annexin V and propidium iodide to assess early apoptosis through flow cytometry (c). The dot plot is an example of general analysis of early apoptosis. Bars show the mean ± SEM of 7 mice per group and are from one experiment representative of two independent experiments. Significance was calculated in relation to the control group (two‐tailed unpaired Student's t‐test). **p < 0·005

FIGURE 5

Effects of the deletion of CD300a on efferocytosis. The in vivo efferocytosis analysis was performed in BALB/c (WT) and CD300a^−/− male mice injected with MSU crystals into the tibiofemoral joint. Cells from the articular cavity were harvested 24 hr after injection, stained with surface marker F4/80, permeabilized and intracellularly stained with Ly6G. Efferocytosis was assessed by flow cytometry analysing F4/80⁺Ly6G⁺ (a). The ex vivo efferocytosis analysis was performed in BALB/c (WT) and CD300a^−/− male mice injected with zymosan into the peritoneal cavity. Cells were recovered, and neutrophils were separated by Histopaque gradient. Apoptotic neutrophils from WT and CD300a^−/− labelled with fluorescent CFSE were incubated with WT macrophages in a proportion of 3 neutrophils per macrophage. Efferocytosis was assessed by flow cytometry analysing F4/80⁺CFSE⁺ (b). Bars show the means ± SEM of 5 mice per group and are from one experiment representative of two independent experiments. Significance was calculated in relation to the control group (two‐tailed unpaired Student's t‐test). *p < 0·05 and **p < 0·005

FIGURE 6

Apoptosis and cleavage of caspase‐8 on human blood neutrophils stimulated with anti‐CD300a agonistic antibody. Neutrophils were purified from the blood of human donors, and 1 × 105 cells were incubated with or without human agonistic CD300a antibody (α‐CD300a) to assess early neutrophil apoptosis (a). Neutrophils were purified from the blood of human donors, and 2 × 106 cells were incubated with or without α‐CD300a. Total cell lysates were separated by SDS‐PAGE and subjected to Western blotting for cleaved caspase‐8 detection after 5 and 15 min of incubation. The image represents one of two independent experiments (b), and the densimetry of two independent experiments is shown (c). Significance was calculated using ANOVA followed by the Newman–Keuls test and two‐tailed unpaired Student's t‐test in relation to the control group. *p < 0·05 and **p < 0·005