The data described here show for the first time a differentialy effect of inhibiting CD14/TLR4/MD-2 and complement in E. coli
-induced inflammation in porcine whole blood. The findings support the hypothesis that these two upstream systems are both pivotal for induction of the inflammatory response. The rationale for inhibiting both the CD14/TLR and complement systems involves blocking not only exogenous microbial ligands but various endogenous ligands as well (24
). The main exogenous ligand for the CD14/TLR4/MD-2 complex is LPS, whereas a number of endogenous ligands have recently been described (9
). Similarly, complement recognizes not only microbial ligands but also endogenous structures. Interestingly, we recently demonstrated that the inflammatory response induced by meconium, containing no bacteria and having a low LPS content, was completely attenuated by inhibition of complement and CD14 (31
). Evidence suggests that “pattern recognition” and “danger signaling” might be important both in the protection of the host against external microbes and in the maintenance of tissue homeostasis, the latter of which contributes to tissue damage during inflammation. Inhibition of complement and CD14 might therefore attenuate the combined exogenous and endogenous activation of the innate immune system in conditions like gram-negative sepsis.
The anti-CD14 clone MIL-2 was initially described as a clone that bound to a cell surface molecule which was moderately expressed on granulocytes and strongly expressed on monocytes/macrophages in porcine blood (12
). Later, the target of the monoclonal antibody was found to be porcine CD14 (39
). The Third Swine CD Workshop determined that anti-CD14 MIL-2 inhibited binding of LPS (39
). In the present study, we showed that anti-CD14 MIL-2 bound to and saturated porcine granulocyte CD14 in a dose-dependent manner. Furthermore, anti-CD14 MIL-2 completely inhibited LPS-induced proinflammatory cytokine/chemokine production in porcine whole blood. At a dose of 50 μg anti-CD14/ml whole blood the inhibition was complete. Ultrapure (phenol-extracted) E. coli
LPS was used, which is known to activate specifically through the CD14/TLR4/MD-2 complex and not through TLR2, with which crude LPS preparations are known to react through contaminants like lipoproteins (13
). Anti-CD14 inhibited E. coli
-induced release of TNF-α and IL-1β, but not IL-8, which was significantly reduced by complement inhibition. This was in contrast to the results for LPS-induced IL-8, which anti-CD14 inhibited completely.
Two complement inhibitors were used in this study. VCP is a protein that is secreted by vaccinia virus-infected mammalian cells. It is structurally related to C4b-binding protein and is functionally closely related to CR1. It binds to C3b and C4b in the rodent and human complement cascade and thereby arrests the C3 convertases (15
). Several previous studies have demonstrated the beneficial effects of VCP as a complement inhibitor in rodent disease models, such as collagen-induced arthritis (16
) and atherosclerosis (40
). Recently, we showed that VCP is an efficient inhibitor of complement activation in porcine serum by several known complement activators (41
). In the present study we first tested the ability of VCP to inhibit complement in porcine whole blood and found that VCP efficiently and in a dose-dependent manner inhibited E. coli
-induced complement activation. SPICE is a protein that is secreted by variola virus-infected human cells. Since smallpox was eradicated in 1977 and live variola virus is not available for study, only molecularly engineered SPICE is available (30
). SPICE closely resembles VCP; only 11 of 244 amino acids (approximately 4.5%) are different (30
). It was shown previously that SPICE was 100-fold more potent than VCP for inactivation of human C3b (30
). A recent study showed that just a few amino acid changes in the VCP molecule may be responsible for this functional difference (48
). Interestingly, SPICE was only fivefold more potent than VCP in inhibiting porcine complement, with the reservation that our readout was TCC and not C3b.
Interestingly, the complement inhibitor SPICE reduced bacterial clearance from porcine whole blood. This is not surprising and can be explained by reduced opsonization, phagocytosis, and bacterial killing, in which complement is known to play an essential role (5
). In contrast, anti-CD14 had no influence on bacterial clearance, indicating that complement is more important than CD14 for bacterial killing in this setting. Therefore, appropriate antibacterial therapy must be established before complement is inhibited. Since the host defense system is a double-edged sword, a combined strategy involving antimicrobial treatment and attenuation of the host defense, including complement and CD14, might be a rational strategy for treatment of sepsis.
CD11b in complex with CD18 forms the CR3 receptor in humans. CR3 is a β2 integrin and is the principal receptor involved in the phagocytosis of iC3b-coated bacteria. Upregulation of CD11b/CR3 on human granulocytes has previously been shown to be complement dependent (23
). Porcine wCD11R3, although slightly smaller than the human α chain of CR3 (155 kDa instead of 165 kDa [8
]), is also associated with CD18 and has the same cellular distribution in pigs that CD11b has in humans (8
). We found that wCD11R3 on porcine granulocytes was upregulated by E. coli
in a dose-dependent manner. Based on these findings, the ability of VCP to inhibit this upregulation was tested. E. coli
-induced wCD11R3 upregulation was inhibited by VCP in a dose-dependent manner, and at a concentration of 100 μg VCP/ml whole blood the upregulation was inhibited to background values. These findings indicate that the E. coli
-induced upregulation of porcine wCD11R3 is complement dependent, as previously described for CD11b in humans (5
In summary, inhibition of CD14 and complement was shown to differentially attenuate the E. coli-induced inflammatory response in porcine whole blood. The results obtained with the Orthopoxvirus complement inhibitors VCP and SPICE showed that the wCD11R3 expression induced on granulocytes was completely dependent on complement and that IL-8 production was markedly reduced due to complement inhibition, but this inhibition had no effect on TNF-α or IL-1β. In contrast, the inflammatory cytokines TNF-α and IL-1β were to a large extent CD14 dependent. Furthermore, anti-CD14 inhibited IL-8 induced by LPS, whereas there was no effect on the production induced by whole bacteria. This finding emphasizes the importance of including whole bacteria and not only LPS when the effect of gram-negative bacteria on innate immune responses is studied.
Altogether, these data strengthen the hypothesis that inhibition of complement and CD14/TLR4/MD-2, two main upstream initiators of gram-negative bacterium-induced inflammation, is a rational approach for attenuating inflammation. Further in vivo studies in the pig could provide further support for this hypothesis.