IL-6 is known to be an important pro- and anti-inflammatory cytokine, which is up-regulated during sepsis. Our previous work has suggested a role for IL-6 in the up-regulation of C5aR in sepsis. We reported earlier that interception of C5a or C5aR results in improved outcomes in experimental sepsis. Using the cecal ligation/puncture (CLP) model in mice, we now demonstrate that treatment with anti-IL-6 Ab (anti-IL-6) results in significantly improved survival, dependent on the amount of Ab infused. CLP animals showed significantly increased binding of 125I-labeled anti-C5aR to organs when compared to either control mice at 0 h or CLP animals infused with normal rabbit 125I-labeled IgG. Binding of 125I-labeled anti-C5aR to lung, liver, kidney, and heart was significantly decreased in anti-IL-6-treated animals 6 h after CLP. RT-PCR experiments with mRNA isolated from various organs obtained 3, 6, and 12 h after CLP demonstrated increased C5aR mRNA expression during the onset of sepsis, which was greatly suppressed in CLP mice treated with anti-IL-6. These data suggest that IL-6 plays an important role in the increased expression of C5aR in lung, liver, kidney, and heart during the development of sepsis in mice and that interception of IL-6 leads to reduced expression of C5aR and improved survival.

The current studies demonstrate protective and harmful effects of neutrophils (PMN) during experimental sepsis after cecal ligation and puncture (CLP). It is known that CLP induces signaling defects in blood PMN. When PMN were depleted 12 h after CLP, there were dramatic reductions in levels of bacteremia, evidence for reduced liver and renal dysfunction, sharp reductions in serum levels of cytokines (IL-1beta, IL-6, IL-10, TNF-alpha, and IL-2), and improved survival. In contrast, PMN depletion before CLP resulted in substantial increases in bacteremia and no evidence for attenuation of liver and renal failure dysfunction. These data suggest that at the onset of sepsis, PMN are important in regulating the levels of bacteremia, whereas after the onset of sepsis, as they lose innate immune functions, their presence is associated with higher levels of bacteremia and intensified organ dysfunction.

In sepsis, there is evidence that excessive C5a generation leads to compromised innate immune functions, being associated with poor outcome. We now report that in vitro exposure of neutrophils to C5a causes increased levels of IkappaBalpha, decreased NF-kappaB-dependent gene transcription of TNFalpha, and decreased lipopolysaccharide (LPS)-induced TNFalpha production. Similar findings were obtained with neutrophils from cecal ligation/puncture (CLP)-induced septic rats. Such changes were reversed by antibody-induced in vivo blockade of C5a. In contrast, in vitro exposure of alveolar macrophages to C5a and LPS resulted in enhanced production of TNFalpha and no increase in IkappaBalpha. These data suggest that CLP-induced sepsis causes a C5a-dependent dysfunction of neutrophils, which is characterized by altered signaling associated with NF-kappaB activation.

Complement fragment 5a (C5a)-C5a receptor (C5aR) signaling plays an essential role in neutrophil innate immunity. Blockade of either the ligand or the receptor improves survival rates in experimental sepsis. In the current study, sepsis was induced in rats by cecal ligation/puncture. Early in sepsis C5aR content on neutrophils significantly dropped, reached the nadir at 24 h after onset of sepsis, and progressively elevated thereafter. Western-blot, RT-PCR, and confocal microscopy analyses revealed that the loss and re-expression of C5aR during sepsis might be due, at least in part, to the receptor internalization and reconstitution. The reduction and reconstitution of C5aR correlate with the loss and restoration of innate immune functions of blood neutrophils (chemotaxis and reactive oxygen species production), respectively. Quantitative measurements of C5aR on blood neutrophils are highly predictive of survival or death during sepsis. These data suggest that neutrophil C5aR content represents an essential component of an efficient defense system in sepsis and may serve as a prognostic marker for the outcome.

There is mounting evidence that apoptosis plays a significant role in tissue damage during acute lung injury. To evaluate the role of the apoptosis mediators Fas and FasL in acute lung injury, Fas (lpr)- or FasL (gld)-deficient and wild-type mice were challenged with intrapulmonary deposition of IgG immune complexes. Lung injury parameters ((125)I-albumin leak, accumulation of myeloperoxidase, and wet lung weights) were measured and found to be consistently reduced in both lpr and gld mice. In wild-type mice, lung injury was associated with a marked increase in Fas protein in lung. Inflamed lungs of wild-type mice showed striking evidence of activated caspase-3, which was much diminished in inflamed lungs from lpr mice. Intratracheal administration of a monoclonal Fas-activating antibody (Jo2) in wild-type mice induced MIP-2 and KC production in bronchoalveolar lavage fluids, and a murine alveolar macrophage cell line (MH-S) showed significantly increased MIP-2 production after incubation with this antibody. Bronchoalveolar lavage fluid content of MIP-2 and KC was substantially reduced in lpr mice after lung injury when compared to levels in wild-type mice. These data suggest that the Fas/FasL system regulates the acute lung inflammatory response by positively affecting CXC-chemokine production, ultimately leading to enhanced neutrophil influx and tissue damage.