The Antimicrobial Antiproteinase Elafin Binds to Lipopolysaccharide and Modulates Macrophage Responses

Abstract

Lipopolysaccharides (LPS) of the outer membrane of Gram-negative bacteria represent a primary target for innate immune responses. We demonstrate here that the antimicrobial/anti-neutrophil elastase full-length elafin (FL-EL) is able to bind both smooth and rough forms of LPS. The N-terminus was shown to bind both forms of LPS more avidly. We demonstrate that the lipid A core-binding proteins polymyxin B (PB) and LPS-binding protein (LBP) compete with elafin for binding, and that LBP is able to displace prebound elafin from LPS. When PB, FL-EL, N-EL, and C-EL were pre-incubated with LPS before addition to immobilized LBP, PB was the most potent inhibitor of LPS transfer to LBP. These data prompted us to examine the biological consequences of elafin binding to LPS, using tumor necrosis factor (TNF)-alpha release by murine macrophages. In serum-containing conditions, N-EL had no effect, whereas both C-EL and FL-EL inhibited TNF-alpha production. In serum-free conditions, however, all moieties had a stimulatory activity on TNF-alpha release, with C-EL being the most potent at the highest concentration. The differential biological activity of elafin in different conditions suggests a role for this molecule in either LPS detoxification or activation of innate immune responses, depending on the external cellular environment.

Elafin, an Elastase-specific Inhibitor, Is Cleaved by Its Cognate Enzyme Neutrophil Elastase in Sputum from Individuals with Cystic Fibrosis

“…The reason for these antagonistic effects remains unknown. More intriguingly, the elafin fragment, which is less potent than trappin-2 to bind LPS, is much more effective in enhancing the secretion of TNF-␣ by macrophages exposed to LPS in serum-containing conditions (33). In our report we found that NE cleaves elafin at its N terminus, generating a fragment starting from Ser-10, and similarly, we demonstrated that elafin binds to LPS more strongly than NE-treated elafin.…”

“…Some molecules like BPI (bactericidal/permeability-increasing protein), granulysin/NK lysin, histatins, histone H2A, LL-37, and SLPI can neutralize the pro-inflammatory effects of LPS, whereas another molecule like azurocidin/HBP (heparin-binding protein) can enhance the capacity of LPS to induce the release of pro-inflammatory mediators by cells. A recent study demon-strated that trappin-2 (pre-elafin) and a fragment of elafin starting from Pro-13 were able to bind LPS and modulate the macrophage response to LPS (33). Although trappin-2 binds LPS much more strongly than the elafin fragment, both molecules can inhibit and enhance the LPS-induced activation of macrophages, respectively, in the presence and in absence of serum (33).…”

“…A recent study demon-strated that trappin-2 (pre-elafin) and a fragment of elafin starting from Pro-13 were able to bind LPS and modulate the macrophage response to LPS (33). Although trappin-2 binds LPS much more strongly than the elafin fragment, both molecules can inhibit and enhance the LPS-induced activation of macrophages, respectively, in the presence and in absence of serum (33). The reason for these antagonistic effects remains unknown.…”

“…Elafin is an antibacterial component possessing the capacity to interact with the endotoxin LPS of Gram-negative bacteria and to modulate macrophage responses after LPS stimulation (33). We, therefore, investigated the effect of NE on the ability of the inhibitor to bind to LPS.…”

Functional Characterization of Polymorphisms in the Peptidase Inhibitor 3 (Elafin) Gene and Validation of Their Contribution to Risk of Acute Respiratory Distress Syndrome

“…Contrary to what we expected, our results suggest that SNP rs2664581 (T34P) augments the proteinase inhibitor's capacity to locally protect ECM proteins from PMN-mediated degradation. It is also noteworthy that, in addition to its serine proteinase inhibitory properties, preelafin has been reported to have opposing activities in regulating the inflammatory response, limiting some inflammatory responses, but increasing others (44)(45)(46). Thus, it is possible that the increased TGase-mediated binding of MT pre-elafin to ECM proteins may increase activation of some leukocytes during lung inflammatory responses to injury.…”