C5aR2 Deficiency Lessens C5aR1 Distribution and Expression in Neutrophils and Macrophages

Abstract

As another receptor for complement activation product C5a, C5aR2 has been paid much attention these years. Although controversial and complex, its specific signals or roles in modulating the classic receptor C5aR1 have been investigated and gradually revealed. The hypothesis of the heterodimer of C5aR1 and C5aR2 has also been suggested and observed under extremely high C5a concentrations. In this article, we tried to investigate whether C5aR2 would affect C5aR1 expression under normal or inflammatory conditions in WT and C5ar2 ^-/- mice of C57BL/6 background. We focused on the innate immune cells-neutrophils and macrophages. The mRNA levels of C5ar1 in normal kidney, liver, and the mRNA or protein levels of naïve-bone marrow and peripheral blood leukocytes and peritoneal Mφs were comparable between WT and C5ar2 ^-/- mice, indicating the technique of C5aR2 knockout did not affect the transcription of its neighboring gene C5aR1. However, the mean fluorescence intensity of surface C5aR1 on naïve circulating C5ar2 ^-/- neutrophils detected by FACS was reduced, which might be due to the reduced internalization of C5aR1 on C5ar2 ^-/- neutrophils. In the peritonitis model induced by i.p. injection of thioglycollate, more neutrophils were raised after 10 hr in C5ar2 ^-/- peritoneal cavity, indicating the antagonism of C5aR2 on C5aR1 signal in neutrophil chemotaxis. After 3 days of thioglycollate injection, the mainly infiltrating macrophages were comparable between WT and C5ar2 ^-/- mice, but the C5ar1 mRNA and surface or total C5aR1 protein expression were both reduced in C5ar2 ^-/- macrophages, combined with our previous study of reduced chemokines and cytokines expression in C5ar2 ^-/- peritoneal macrophages, indicating that C5aR2 in macrophages may cooperate with C5aR1 inflammatory signals. Our article found C5aR2 deficiency lessened C5aR1 distribution and expression in neutrophils and macrophages with different functions, indicating C5aR2 might function differently in different cells.

Figure 1

C5aR2 deficiency does not affect total C5aR1 expression under naïve conditions. (a) Relative mRNA levels of C5ar1 in kidney and liver tissue, naïve primarily cultured RTECs, and peripheral blood leukocytes from normal WT and C5ar2^-/- mice determined by RT-qPCR (n = 4/group). (b) A typical agarose gel showing the 155-bp C5ar1 band and the 453-bp Gapdh (internal control) band of conventional RT-PCR of 35 cycles about the C5ar1 mRNA levels in normal kidneys from female WT and C5ar2^-/- mice. The 100-bp DNA markers (M) are shown alongside the gels. (c) The percentages of neutrophils and monocytes in peripheral blood CD45⁺ leukocytes from normal female WT and C5ar2^-/- mice as assessed by flow cytometry (n = 8/group). (d) Total C5aR1 expression in CD45⁺ circulating leukocytes, neutrophils, and monocytes from female WT and C5ar2^-/- mice as assessed by flow cytometry (n = 8/group). Each dot represents an individual mouse. ns, no significance, Student's t test.

Figure 2

C5aR2 deficiency lessens C5aR1 distribution on peripheral blood neutrophil surface under naïve conditions. (a) Representative FACS analysis of surface C5aR1 expression on peripheral blood neutrophils and monocytes. (b) C5aR1 expression on circulating neutrophils and monocytes from WT and C5ar2^-/- mice as assessed by flow cytometry (n = 8/group). Each dot represents an individual mouse.  ^∗P < 0.05, ns, no significance, Student's t test.

Figure 3

C5aR1 distribution and expression are similar in naïve intraperitoneal Mφs between WT and C5ar2^-/- mice. n = 8/group. Each dot represents an individual mouse. ns, no significance, Student's t test.

Figure 4

Neutrophil infiltration is higher after 10 hr of 3% TG injection. (a) The representative diagram of peritoneal cells constituent and C5aR1 expression after 10 hr of TG injection. (b) Neutrophils and Mo/Mφs infiltration of WT and C5ar2^-/- mice. (c) Representative FACS analysis of total C5aR1 expression in neutrophils and Mo/Mφs. (d and e) C5aR1 expression in CD45⁺ leukocytes, in/on neutrophils and Mo/Mφs from WT and C5ar2^-/- mice as assessed by flow cytometry (n = 8/group). Each dot represents an individual mouse.  ^∗P < 0.05,  ^∗∗P < 0.01, ns, no significance, Student's t test.

Figure 5

C5aR2 deficiency lessens C5aR1 distribution and expression in Mφs after 3 days of 3% TG injection. (a) The representative diagram and statistic graph of peritoneal cells constituent between WT and C5ar2^-/- mice after 3 days of TG injection (n = 8/group). (b) Representative FACS analysis of surface and total C5aR1 expression in Mφs. (c) Surface and total C5aR1 expression in Mφs from WT and C5ar2^-/- mice as assessed by flow cytometry (n = 8/group). (d) Relative mRNA levels of C5aR1 between WT and C5ar2^-/- peritoneal cells 3 days after TG injection determined by RT-qPCR. Each dot represents an individual mouse (n = 6/group).  ^∗P  < 0.05,  ^∗∗P  < 0.01,  ^∗∗∗P  < 0.001,  ^∗∗∗∗P  < 0.0001, ns, no significance, Student's t test.

Figure 6

C5aR2 deficiency may be in favor of C5aR1 internalization on neutrophils upon C5a stimulation. Flow cytometry analysis of surface C5aR1⁺% on WT and C5ar2^-/- bone marrow neutrophils, monocytes, and naïve peritoneal Mφs after C5a (1–50 nM) stimulation for 30 min. (n = 3/group). The surface C5aR1⁺% on these cells with no C5a stimulation was considered as 100%. Each dot represents an individual mouse.  ^∗∗P < 0.01, ns, no significance, two-way ANOVA.