Increased C5a receptor expression in sepsis

Abstract

Excessive production of the complement activation product C5a appears to be harmful during the development of sepsis in rodents. Little is known about the role of the C5a receptor (C5aR) and its presence in different organs during sepsis. Using the cecal ligation/puncture (CLP) model in mice, we show here that C5aR immunoreactivity was strikingly increased in lung, liver, kidney, and heart early in sepsis in both control and neutrophil-depleted mice. C5aR mRNA expression in these organs was also significantly increased during sepsis. Immunohistochemical analysis revealed patterns of increased C5aR expression in parenchymal cells in all four organs following CLP. Mice injected at the start of CLP with a blocking IgG to C5aR (alphaC5aR) showed dramatically improved survival when compared with animals receiving nonspecific IgG, as did mice injected with alphaC5a. In alphaC5aR-treated mice, serum levels of IL-6 and TNF-alpha and bacterial counts in various organs were significantly reduced during CLP when compared with control CLP animals. These studies demonstrate for the first time that C5aR is upregulated in lung, liver, kidney, and heart during the early phases of sepsis and that blockade of C5aR is highly protective from the lethal outcome of sepsis.

Figure 1

(a) In vivo binding of ¹²⁵I-αC5aR to organs 0, 3, 6, and 12 hours after CLP. Binding is expressed as the ratio of cpm per g organ to cpm in 100 μl blood from each animal, obtained 15 minutes after intravenous injection of ¹²⁵I-αC5aR. (b) In vivo binding of ¹²⁵I-labeled rabbit IgG to similar organs expressed similarly to the data in a. Data are representative of three to six animals for each time point. *Statistical significance in treated groups when compared with control animals.

Figure 2

In vivo binding of ¹²⁵I-αC5aR to various organs in neutrophil-depleted mice 0 and 6 hours after CLP. Binding is expressed as the ratio of cpm per g organ to cpm in 100 μl blood from each animal, obtained 15 minutes after intravenous injection of ¹²⁵I-αC5aR. *Statistical significance in treated groups when compared with control animals. Data are representative of four to seven animals per group. PMN, polymorphonuclear neutrophil.

Figure 3

Inhibition of binding of ¹²⁵I-labeled mouse C5a (¹²⁵I-C5a) to peritoneal mouse neutrophils in vitro (a) and various organs in vivo (b) by αC5aR. (a) Binding of ¹²⁵I-C5a is expressed as cpm. Groups were treated (as indicated) for 20 minutes prior to binding of ¹²⁵I-C5a. Data are representative of five independent experiments per condition. (b) Binding of ¹²⁵I-C5a is expressed as the ratio of cpm per g organ to cpm in 100 μl blood from each animal (n = 4 per condition) obtained 10 minutes after intravenous injection of ¹²⁵I-C5a and 6 hours after CLP. αC5aR (6 μg/mouse) or an equal amount of irrelevant rabbit IgG (control) was administered intravenously 15 minutes prior to ¹²⁵I-C5a infusion. *Statistical significance in treated groups when compared with control animals. Ctrl, control.

Figure 4

Semiquantitative RT-PCR for C5aR mRNA in lung, liver, kidney, and heart. RT-PCR was performed using RNA isolated 0, 3, 6, and 12 hours after CLP. Approximately equal loading of the DNA product is demonstrated by expression of GAPDH mRNA (lower half of each panel). Results are representative of two independent and separate experiments for each group and time point.

Figure 5

Immunohistochemical stains of organ sections for C5aR from mice, as indicated in the text. Compared are staining results from organ sections from control animals (left panels) and from animals 12 hours after CLP (right panels). Results are representative of three to six stainings from two independent experiments per condition. Magnification, ×20; insets in right panels, ×40 (hematoxylin staining).

Figure 6

Survival study in CLP mice over 7 days. Mice were treated intravenously with 20 μg αC5aR (n = 11), 20 μg αC5a (n = 10), or 20 μg rabbit IgG (n = 12), each being infused at the time of CLP. In another experiment, 20 μg αC5aR (n = 10) was injected 6 hours after CLP.

Figure 7

(a) ELISA measurements for IL-6 and TNF-α serum levels at 6 and 18 hours after CLP-induced sepsis and in healthy control mice (normal). When used, αC5aR treatment consisted of 20 μg/mouse at the start of CLP, with a companion group treated with control IgG (20 μg/mouse). (b) Content of aerobic bacteria in lung, liver, and kidney 18 hours after CLP-induced sepsis in mice. Compared groups were treated as described for a (n = 4 for all groups). *Statistically significant changes between αC5aR- and control IgG–treated CLP groups.