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. 2011 Jul;25(7):2500-8.
doi: 10.1096/fj.11-183236. Epub 2011 Apr 8.

Complement dependency of cardiomyocyte release of mediators during sepsis

Gelareh Atefi  1 , Firas S Zetoune, Todd J Herron, José Jalife, Markus Bosmann, Rami Al-Aref, J Vidya Sarma, Peter A Ward
Affiliations

Complement dependency of cardiomyocyte release of mediators during sepsis

Gelareh Atefi et al. FASEB J. 2011 Jul.

Abstract

We have recently shown that antibody-induced blockade of C5a, C5a receptors, or IL-17A greatly reduced the harmful outcomes of sepsis. In the current study, normal cardiomyocytes from young (300 g) male Sprague-Dawley rats responded in vitro to C5a (ED(50)=55 nM) with release of IL-6 and TNFα, peaking between 2 to 8 h. Neutralizing antibodies to mouse C5a or IL-17A (ED(50)=40 μg for each, based on improved survival) reduced spontaneous in vitro release of cardiosuppressive cytokines and chemokines in cardiomyocytes obtained from mice with polymicrobial sepsis. A non-neutralizing C5a antibody had no such effects. Cardiomyocytes from septic mice (C57Bl/6) showed increased mRNA for TNFR1, IL-6 (gp80), and C5aR at 6 h after sepsis. Cardiomyocytes from septic C5aR(-/-) or C5L2(-/-) mice did not show spontaneous in vitro release of cytokines and chemokines. These data suggest that cardiomyocytes from septic mice release suppressive cytokines in a C5a-, C5aR-, and IL-17A-dependent manner, followed by mediator reactivity with receptors on cardiomyocytes, resulting in defective contractility and relaxation. These data may be relevant to a strategy for the treatment of heart dysfunction developing during sepsis.

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Figures

Figure 1.
Figure 1.
A) Normal rat CM immunostained with primary antibody against α-actin and Texas-Red conjugated secondary antibody. B) Time course for release of IL-6 from rat CMs (2×106) exposed to 100 nM recombinant rat C5a at 37°C. C, D) Dose responses for sham rat CMs (2×106) incubated with HBSS (none) or recombinant rat C5a (40–100 nM) for 12 h at 37°C, following which supernatant fluids were evaluated by ELISA for TNFα (C) and IL-6 (D). For each bar, n = 5. E) Frozen sections of rat heart obtained before CLP (panels 1–3) or 24 h after CLP and stained for TNFα (panel 4) or IL-1β (panel 5). In panel 1, the primary antibody (to TNFα) was omitted.
Figure 2.
Figure 2.
Presence of cytokines and chemokines in plasma from sham-treated and WT mice at 12, 24, 48, and 72 h after CLP. A) IL-6. B) TNFα. C) IL-1β. D) MIP-1α. E) MIP-2. F) MCP-1. G) KC. H) IL-10. For each bar, n ≥ 5.
Figure 3.
Figure 3.
Presence of cytokines and chemokines in heart homogenates from sham-treated and WT mice at 12, 24, 48, and 72 h after CLP. A) IL-6. B) TNFα. C) IL-1β. D) MIP-1β. E) MIP-2. F) MCP-1. G) KC. H) IL-10. Data are expressed as amount of mediator (pg/ml) per gram total protein. For each bar, n ≥ 5.
Figure 4.
Figure 4.
Release of mediators from sham-treated or CLP WT CMs obtained at 8, 12, 24, and 48 h after CLP. A) IL-6. B) TNFα. C) MIP-1α. D) MIP-2. CMs were cultured for 18 h at 37°C. For each bar, n ≥ 5.
Figure 5.
Figure 5.
mRNA expression based on real-time PCR for C5a receptors (C5aR, C5L2), IL-6R (gp80) and TNFR1, in sham-treated (control) WT mouse hearts and in hearts at 6, 12, and 24 h following CLP. A) C5aR. B) C5L2. C) IL-6R. D) TNFR1. For each bar, n = 3 separate samples.
Figure 6.
Figure 6.
Release of mediators from CMs obtained from WT mice before (sham) and 24 h after CLP, as well as from CLP mice that were C5aR−/− or C5L2−/−. A) IL-6. B) TNFα. C) IL-1β. D) MIP-1α. E) MIP-2. F) MCP-1. G) KC. H) IL-10. For each bar, n = 5.
Figure 7.
Figure 7.
Cytokines (TNFα, IL-6) and chemokines (MIP-1α, MIP-2) released from sham-treated mouse CMs and from 24 h CLP CMs obtained from WT mice administered control IgG, anti-C5a (40 μg), or anti-IL-17A IgG (50 μg), all administered i.v. at time 0. A) TNFα. B) IL-6. C) MIP-1α. D) MIP-2.For each bar, n ≥ 5.
Figure 8.
Figure 8.
Proposed mechanisms of septic cardiomyopathy based on the data presented herein.
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