IL-33 enhances lipopolysaccharide-induced inflammatory cytokine production from mouse macrophages by regulating lipopolysaccharide receptor complex.

Bacterial LPS triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. However, once exposed to LPS, they become hyporesponsive to a subsequent endotoxin challenge. This phenomenon is defined as LPS desensitization or tolerance. Previous studies have identified some components of the biochemical pathways involved in negative modulation of LPS responses. In particular, it has been shown that the IL-1R-related protein ST2 could be implicated in LPS tolerance. The natural ligand of ST2 was recently identified as IL-33, a new member of the IL-1 family. In this study, we investigated whether IL-33 triggering of ST2 was able to induce LPS desensitization of mouse macrophages. We found that IL-33 actually enhances the LPS response of macrophages and does not induce LPS desensitization. We demonstrate that this IL-33 enhancing effect of LPS response is mediated by the ST2 receptor because it is not found in ST2 knockout mice. The biochemical consequences of IL-33 pretreatment of mouse macrophages were investigated. Our results show that IL-33 increases the expression of the LPS receptor components MD2 (myeloid differentiation protein 2) and TLR-4, the soluble form of CD14 and the MyD88 adaptor molecule. In addition, IL-33 pretreatment of macrophages enhances the cytokine response to TLR-2 but not to TLR-3 ligands. Thus, IL-33 treatment preferentially affects the MyD88-dependent pathway activated by the TLR.

Synthetic peptides representing the N-terminal segment of surfactant protein C modulate LPS-stimulated TNF-alpha production by macrophages.

Surfactant protein C (SP-C) consists of a hydrophobic alpha-helix inserted in pulmonary surfactant membranes, and a more polar N-terminal palmitoylated segment exposed to the aqueous phase. Previously, we showed that SP-C inserted in lipid vesicles interacts with bacterial lipopolysaccharide (LPS) and reduces LPS-elicited responses. As the N-terminal segment of SP-C was the most likely region responsible for these effects, a set of synthetic analogs of this stretch (SPC((1-13)) ) were studied. Binding studies showed that SPC((1-13)) binds LPS to the same extent as porcine SP-C under lipid-free conditions. In the absence of serum, both, palmitoylated and non-palmitoylated analogs enhanced the binding of tritiated LPS to macrophages as well as the LPS-induced production of TNF-alpha by these cells. These effects were reversed in the presence of serum; the analogs reduced the production of TNF-alpha in LPS-stimulated macrophages, probably by interfering with the formation of LPS/CD14/LBP complexes as suggested by analysis of the fluorescence emitted by a FITC derivative of Re-LPS. Our data indicate that water-soluble analogs of the N-terminal segment of SP-C can reduce LPS effects in the presence of serum, and thus might help in the design of new derivatives to fight endotoxic shock and pro-inflammatory events.

Inactivation of endotoxin by human plasma gelsolin.

Septic shock from bacterial endotoxin, triggered by the release of lipopolysaccharide (LPS) molecules from the outer wall of Gram-negative bacteria, is a major cause of human death for which there is no effective treatment once the complex inflammatory pathways stimulated by these small amphipathic molecules are activated. Here we report that plasma gelsolin, a highly conserved human protein, binds LPS from various bacteria with high affinity. Solid-phase binding assays, fluorescence measurements, and functional assays of actin depolymerizing effects show that gelsolin binds more tightly to LPS than it does to its other known lipid ligands, phosphatidylinositol 4,5-bisphosphate and lysophosphatidic acid. Gelsolin also competes with LPS-binding protein (LBP), a high-affinity carrier for LPS. One result of gelsolin-LPS binding is inhibition of the actin binding activity of gelsolin as well as the actin depolymerizing activity of blood serum. Simultaneously, effects of LPS on cellular functions, including cytoskeletal actin remodeling, and collagen-induced platelet activation by pathways independent of toll-like receptors (TLRs) are neutralized by gelsolin and by a peptide based on gelsolin residues 160-169 (GSN160-169) which comprise part of gelsolin's phosphoinositide binding site. Additionally, TLR-dependent NF-kappaB translocation in astrocytes appears to be blocked by gelsolin. These results show a strong effect of LPS on plasma gelsolin function and suggest that some effects of endotoxin in vivo may be mediated or inhibited by plasma gelsolin.

Interactions between LPS and lung surfactant proteins.

After penetration into the lower airways, bacterial lipopolysaccharide (LPS) interacts with alveolar cells in a fluid environment consisting of pulmonary surfactant, a lipid-protein complex which prevents alveolar collapsing and participates in lung defense. The two hydrophilic surfactant components SP-A and SP-D are proteins with collagen-like and lectin domains (collectins) able to interact with carbohydrate-containing ligands present on microbial membranes, and with defined regions of LPS. This explains their capacity to damage the bacterial envelope and induce an antimicrobial effect. In addition, they modulate LPS-induced production of pro-inflammatory mediators in leukocytes by interaction with LPS or with leukocyte receptors. A third surfactant component, SP-C, is a small, highly hydrophobic lipopeptide which interacts with lipid A and reduces LPS-induced effects in macrophages and splenocyte cultures. The interaction of the different SPs with CD14 might explain their ability to modulate some LPS responses. Although the alveolar fluid contains other antiLPS and antimicrobial agents, SPs are the most abundant proteins which might contribute to protect the lung epithelium and reduce the incidence of LPS-induced lung injury. The presence of the surfactant collectins SP-A and SP-D in non-pulmonary tissues, such as the female genital tract, extends their field of action to other mucosal surfaces.

N-terminally extended surfactant protein (SP) C isolated from SP-B-deficient children has reduced surface activity and inhibited lipopolysaccharide binding.

In both humans and mice, a deficiency of surfactant protein B (SP-B) is associated with a decreased concentration of mature SP-C and accumulation of a larger SP-C peptide, denoted SP-C(i), which is not observed under normal conditions. Isolation of hydrophobic polypeptides from the lungs of children who died with two different SP-B mutations yielded pure SP-C(i) and showed only trace amounts of mature SP-C. Determination of the SP-C(i) covalent structure revealed a 12-residue N-terminal peptide segment, followed by a 35-residue segment that is identical to mature SP-C. The SP-C(i) structure determined herein is similar to that of a proposed late intermediate in the processing of proSP-C, suggesting that SP-C(i) is the immediate precursor of SP-C. In bronchoalveolar lavage fluid from transgenic mice with a focal deficiency of SP-B, SP-C(i) was detected in the biophysically active, large aggregate fraction and was associated with membrane structures that are typical for a large aggregate surfactant. However, unlike SP-C, SP-C(i) exhibited a very poor ability to promote phospholipid adsorption, gave high surface tension during cyclic film compression, and did not bind lipopolysaccharide in vitro. SP-C(i) is thus capable of associating with surfactant lipids, but its N-terminal dodecapeptide segment must be proteolytically removed to generate a biologically functional peptide. The results of this study indicate that the early postnatal fatal respiratory distress seen in SP-B-deficient children is combined with the near absence of active variants of SP-C.

Cellular antiendotoxin activities of lung surfactant protein C in lipid vesicles.

The respiratory system is continuously exposed to airborne particles containing lipopolysaccharide. Our laboratory established previously that the hydrophobic surfactant protein C (SP-C) binds to lipopolysaccharide and to one of its cellular receptors, CD14. Here we examined the influence of SP-C, and of a synthetic analog, on some cellular in vitro effects of lipopolysaccharide. When associated with vesicles of dipalmitoylphosphatidylcholine, SP-C inhibits the binding of a tritium-labeled lipopolysaccharide to the macrophage cell line RAW 264.7. Under similar conditions of presentation, SP-C inhibits the mitogenic effect of lipopolysaccharide on mouse splenocytes, and inhibits the lipopolysaccharide-induced production of tumor necrosis factor-alpha by peritoneal and alveolar macrophages, and of nitric oxide by RAW 264.7 cells. In contrast, tumor necrosis factor-alpha production induced by a lipopeptide, and nitric oxide production induced by picolinic acid, were not affected by SP-C. The lipopolysaccharide-binding capacity of SP-C is resistant to peroxynitrite, a known mediator of acute lung injury formed by reaction of nitric oxide with superoxide anions. These results indicate that SP-C may play a role in lung defense; SP-C resists degradation under inflammatory conditions and traps lipopolysaccharide, preventing it from inducing production of noxious mediators in alveolar cells.