Impaired inflammatory responses in the reverse arthus reaction through genetic deletion of the C5a receptor

Abstract

We recently demonstrated that gene-targeted disruption of the C5a anaphylatoxin receptor prevented lung injury in immune complex-mediated inflammation. In this study, we compare the effect of C5aR deficiency in immune complex-induced inflammation in the peritoneal cavity and skin with the results derived from our immune complex alveolitis model. C5aR- deficient mice exhibit decreased migration of neutrophils and decreased levels of TNF-alpha and interleukin 6 in the peritoneal reverse passive Arthus reaction compared to their wild-type littermates. In the reverse passive Arthus reaction in the skin the C5aR was also required for the full expression of neutrophil influx and edema formation; C5aR-deficient mice showed reduced neutrophil migration and microvascular permeability changes. In contrast to our studies in immune complex-induced lung inflammation, C5aR deficiency does not completely prevent injury in the peritoneal cavity and skin. These data indicate a dominant role for the C5aR and its ligand in the reverse passive Arthus reaction in the lung and a synergistic role together with other inflammatory mediators in immune complex-mediated peritonitis and skin injury.

Figure 1

Role of C5a in neutrophil elicitation in immune complex–induced peritonitis. The reverse passive Arthus reaction was allowed to proceed for 4 h (A) or 8 h (B) in C5aR (−/−) mice (striped bars) and their wild-type littermates (black bars). Total cell numbers (data not shown) and percentage of neutrophils in the peritoneal fluid were determined as described previously (13). Ab controls (Ab control) received Ab to chicken egg albumin i.p. without i.v. injection of chicken egg albumin. Mice treated with PBS i.p. followed by i.v. chicken egg albumin served as Ag controls (Ag control). Data are represented as mean ± SEM, n = 7–8 mice *P <0.004 (A), and n = 8–11 mice *P <0.05 (B).

Figure 1

Role of C5a in neutrophil elicitation in immune complex–induced peritonitis. The reverse passive Arthus reaction was allowed to proceed for 4 h (A) or 8 h (B) in C5aR (−/−) mice (striped bars) and their wild-type littermates (black bars). Total cell numbers (data not shown) and percentage of neutrophils in the peritoneal fluid were determined as described previously (13). Ab controls (Ab control) received Ab to chicken egg albumin i.p. without i.v. injection of chicken egg albumin. Mice treated with PBS i.p. followed by i.v. chicken egg albumin served as Ag controls (Ag control). Data are represented as mean ± SEM, n = 7–8 mice *P <0.004 (A), and n = 8–11 mice *P <0.05 (B).

Figure 2

C5a-dependent secretion of TNF-α. The reverse passive Arthus reaction (IC) was allowed to proceed for 4 or 8 h in C5aR (−/−) (dotted bars) mice and their wild-type littermates (black bars). TNF-α content in the peritoneal fluid was determined by a TNF-α–specific ELISA. Ab controls (Ab control) received Ab to chicken egg albumin i.p. without i.v. injection of chicken egg albumin. Mice treated with PBS i.p. followed by i.v. chicken egg albumin served as Ag controls (Ag control). Data are represented as mean ± SEM, n = 14–17 mice (4 h) and n = 7–9 mice (8 h). *P <0.025.

Figure 3

C5a-dependent secretion of IL-6. The reverse passive Arthus reaction (IC) was allowed to proceed for 4 or 8 h in C5aR (−/−) mice (dotted bars) and their wild-type littermates (black bars). IL-6 content in the peritoneal fluid was determined by an IL-6–specific ELISA. Ab controls (Ab control) received Ab to chicken egg albumin i.p. without i.v. injection of chicken egg albumin. Mice treated with PBS i.p. followed by i.v. chicken egg albumin served as Ag controls (Ag control). Data are represented as mean ± SEM, n = 14–17 mice (4 h) and n = 7–9 mice (8 h). *P <0.004.

Figure 4

PMN infiltration in the reverse Arthus reaction in the skin. Extraction of MPO from skin of C5aR (−/−) (striped bars) mice and their wild-type littermates (black bars) was performed 8 h after initiation of the Arthus reaction. Ab controls (Ab control) received Ab to chicken egg albumin intratracheally without i.v. injection of chicken egg albumin. Mice treated with PBS intratracheally followed by i.v. chicken egg albumin served as Ag controls (Ag control). Data are represented as mean ± SEM, n = 12–13 animals in each group. *P <0.003.

Figure 5

Edema formation in the reverse Arthus reaction in the skin. 25 μg of rabbit anti–chicken egg albumin IgG was injected i.d. followed by 20 mg/kg body weight i.v. chicken egg albumin together with 100 μl Evans blue dye (6.25 mg/ml). Dorsal skins were harvested from C5aR (−/−) mice (striped bars) and their wild-type littermates (black bars) after 3 h and edema was evaluated as weight of 1 cm² skin sections (A). In addition, edema was quantitated as enhanced microvascular permeability by extraction of extravasated Evan's blue dye with formamide from injected skin sections (B). Ab controls (Ab control) received Ab to chicken egg albumin intratracheally without i.v. injection of chicken egg albumin. Mice treated with PBS intratracheally followed by i.v. chicken egg albumin served as Ag controls (Ag control). Data are represented as mean ± SEM, n = 8–12 animals in each group. *P <0.001 (A), *P <0.0001 (B).

Figure 5

Edema formation in the reverse Arthus reaction in the skin. 25 μg of rabbit anti–chicken egg albumin IgG was injected i.d. followed by 20 mg/kg body weight i.v. chicken egg albumin together with 100 μl Evans blue dye (6.25 mg/ml). Dorsal skins were harvested from C5aR (−/−) mice (striped bars) and their wild-type littermates (black bars) after 3 h and edema was evaluated as weight of 1 cm² skin sections (A). In addition, edema was quantitated as enhanced microvascular permeability by extraction of extravasated Evan's blue dye with formamide from injected skin sections (B). Ab controls (Ab control) received Ab to chicken egg albumin intratracheally without i.v. injection of chicken egg albumin. Mice treated with PBS intratracheally followed by i.v. chicken egg albumin served as Ag controls (Ag control). Data are represented as mean ± SEM, n = 8–12 animals in each group. *P <0.001 (A), *P <0.0001 (B).

Figure 6

Effect of C5aR deficiency in immune complex–induced neutrophil influx and permeability changes. The decrease in neutrophil influx and permeability changes in C5aR (−/−) (dotted bars) is shown as percentage of wild-type controls (black bars; normalized to 100%). Results are expressed as means, n = 7–13 animals in each group.