Macrophage-produced IL-12p70 mediates hemorrhage-induced damage in a complement-dependent manner

Abstract

Hemorrhage and hemorrhagic shock instigate intestinal damage and inflammation. Multiple components of the innate immune response, including complement and neutrophil infiltration, are implicated in this pathology. To investigate the interaction of complement activation and other components of the innate immune response during hemorrhage, we treated mice after hemorrhage with CR2-fH, a targeted inhibitor of the alternative complement pathway and assessed intestinal damage and inflammation 2 h after hemorrhage. In wild-type mice, CR2-fH attenuated hemorrhage-induced, midjejunal damage and inflammation as determined by decreased mucosal damage, macrophage infiltration, leukotriene B4, IL-12p40, and TNF-[alpha] production. The critical nature of intestinal macrophage infiltration and activation in the response to hemorrhage was further determined using mice pretreated with clodronate-containing liposomes. The absence of either macrophages or IL-12p70 attenuated intestinal damage. These data suggest that complement activation and macrophage infiltration with IL-12p70 production are critical to hemorrhage-induced midjejunal damage and inflammation.

FIGURE 1. Targeted alternative pathway inhibitor attenuates hemorrhage induced complement activation, intestinal injury andinflammation

C57Bl/6 (B6) mice subjected to Sham or hemorrhage (HS) in the presence or absence of CR2-fH. Serum C5a (A) and ex vivo intestinal LTB₄ (B), IL-12p40 (C) and TNF-α (D) concentrations were determined. Representative photomicrographs (original magnification 100X) of H&E stained mid-jejunal tissue sections from HS-treated, C57Bl/6 mice with or without administration of CR2-fH (E-F). Each bar is the average ± SEM of 4-10 mice per group. (*) Indicates p ≤ 0.05 compared to pooled Sham treatment and (ϕ) indicates p ≤ 0.05 compared to C57Bl/6 HS mice.

FIGURE 2. Macrophages infiltrate into the intestinal tissue following hemorrhage

Mid-jejunal tissue sections from C57Bl/6 (B6) (A-D), IL-12p40^-/- (E) or IL-12p35^-/- (F) mice after Sham (A) or hemorrhage (HS) (B-F) treated with liposomes containing PBS (B), CR2-fH (C) or liposomes containing clodronate (Clod) (D) were stained for F4/80. Photomicrographs original magnification of 400X (A-C) or 200X (D-F) are representative of three individual experiments with 5-7 photos per treatment group in each experiment. The number of macrophages per villus was quantitated from 3-4 mice per treatment group with 20-30 villi per mouse (G). (*) Indicates p ≤ 0.05 compared to respective pooled Sham treatment; (ϕ) indicates p ≤ 0.05 compared to C57Bl/6 HS-treated animals.

FIGURE 3. Macrophage or IL-12 depletion inhibits C3 deposition after hemorrhage

Mid-jejunal tissue sections from C57Bl/6 (B6) (A-D), IL-12p40^-/- (E) or IL-12p35^-/- (F) mice after Sham (A) or hemorrhage (HS) (B-F) treated with liposomes containing PBS (B) or liposomes containing clodronate (Clod) (C) were stained for C3 deposition. Photomicrographs (original magnification of 200X) are representative of three individual experiments with 5-7 photos per treatment group in each experiment).

FIGURE 4. Complement inhibition or macrophage depletion attenuates hemorrhage induced IL-12p35 RNA

C57Bl/6 (B6) mice were subjected to Sham or hemorrhage (HS) after inhibition of complement (FH-CR2) or macrophage depletion with liposomes containing Clodronate (Clod). Intestinal RNA transcripts from each treatment group were analyzed for IL-12p40, IL-12p35 and IL-23p19 by realtime RT-PCR. Each bar represents the average of 4-6 animals. (*) Indicates p ≤ 0.05 compared to pooled Sham treatment; (ϕ) indicates p ≤ 0.05 compared to C57Bl/6 HS-treated animals.

FIGURE 5. IL-12p40 or IL-12p35 but not IL-23p19 deficiency attenuates inflammatory cytokine production following hemorrhage

Ex vivo intestinal supernatants were used to determine LTB₄ (A), and TNF-α (B) produced by C57Bl/6 (B6), IL-12p40^-/- or IL-12p35^-/- mice after Sham or hemorrhage (HS) treatment. One hour after injection of anti-IL-12p40, IL-23p19 or isotype (Ig) control antibodies, wildtype (B6) mice were subjected to Sham or hemorrhage (HS) treatment and intestinal cytokines determined (C). All concentrations were normalized to tissue protein content and expressed as pg per mg of intestinal tissue. Each bar represents the average of 4-10 animals. (*) Indicates p ≤ 0.05 compared to pooled Sham treatment; (ϕ) indicates p ≤ 0.05 compared to C57Bl/6 HS-treated animals.