Soluble MD-2 activity in plasma from patients with severe sepsis and septic shock

Abstract

In this paper, we show that plasma from patients with severe sepsis and septic shock but not normal plasma supports lipopolysaccharide (LPS) activation of epithelial cells expressing Toll-like receptor 4 (TLR4). Recombinant soluble myeloid differentiation protein-2 (MD-2) complemented normal plasma and allowed LPS activation of epithelial cells to levels measured with "septic" plasma, whereas soluble MD-2-depleted plasma lost its effects. The same "MD-2 activity" was found in urine from a patient with septic shock and in lung edema fluids from patients with adult respiratory distress syndrome (ARDS) . IntroductionHuman sepsis accounts for more than 200 000 deaths per year in the United States 1 and results from a dysregulation of innate responses to bacterial pathogens. 2,3 Septic shock, its most severe form, is characterized by a profound vasodilation and extravascular plasma leakage resulting from an increase in endothelial permeability. 4 Gram-negative lipopolysaccharide (LPS) triggers host responses similar to those seen during human septic shock and has served for several decades as a useful in vivo and in vitro model molecule. 5 Several host humoral and cell-surface proteins participate in the innate recognition of LPS, including the acute-phase LPS-binding protein (LBP), and the glycoproteins CD14, Toll-like receptor 4 (TLR4), and myeloid differentiation protein-2 (MD-2). 6 In myeloid cells, the LPS receptor complex is made of CD14, TLR4, and MD-2, whereas in endothelial cells only TLR4 and MD-2 are expressed. 6 LPS responses of endothelial cells require the presentation of LPS to the TLR4/MD-2 membrane receptor by LBP and soluble CD14. 7 Epithelial cells may only express TLR4, and the cofactor requirement of these cells for LPS responses remains to be defined. [8][9][10] MD-2 is produced by several cell types as a 20-to 25-kDa glycoprotein, binds to LPS, and confers LPS responsiveness to TLR4-expressing cells. [11][12][13][14][15][16][17][18][19] Some of the MD-2 molecules remain attached to TLR4, whereas the rest is secreted in the extracellular milieu. 7,20 MD-2 seems to be essential in mouse cells to bring TLR4 from the Golgi to the cell surface. 21 Cystein residues, N-glycosylation, and oligomerization are important for MD-2 function. 22-26 MD-2 knock-out mice are resistant to LPS-induced lethality, 21 a phenotype very similar to that observed in mice lacking TLR4, CD14, or LBP. [27][28][29] However, little is known about the function of soluble MD-2 with regard to LPS activation of cells and as a potential mediator of septic shock. 7,20,26 In this paper, we propose that soluble MD-2 in plasma from patients with severe sepsis and septic shock is an important cofactor for LPS activation of TLR4-expressing cells. Plasma LBP and soluble CD14 also play a role, most probably by shuttling LPS to MD-2 or the MD-2/TLR4 receptor complex. Soluble MD-2 is required for LPS activation of epithelial cells expressing TLR4, whereas it down-regulates LPS activation of monocytic and endothelial cells expressing both T...

MD-2 Mediates the Ability of Tetra-Acylated and Penta-Acylated Lipopolysaccharides to AntagonizeEscherichia coliLipopolysaccharide at the TLR4 Signaling Complex

“…It has been suggested that sMD-2 might play an important role in modulating the innate responses of epithelial cells that express TLR4 but not MD-2 (39). Relevant to this finding, a recent report has suggested that plasma-derived sMD-2 might play a major role in promoting organ inflammation during the progression of Gram-negative bacterial-induced septic shock (53). Information derived from additional investigation of msbB LPS and P. gingivalis LPS interactions with LBP, CD14, and MD-2 will be critical in further elucidating the molecular mechanism of LPS-dependent antagonism.…”

Antifibroblast antibodies in systemic sclerosis induce fibroblasts to produce profibrotic chemokines, with partial exploitation of toll‐like receptor 4

“…Dulbecco's modified Eagle's medium (DMEM), nonessential amino acids, sodium pyruvate, penicillin, and streptomycin were obtained from Gibco Invitrogen (Karlsruhe, Germany), ␣-ketoglutaric acid, ␤-amino propionitrile, and fetal calf serum (FCS) were from Sigma (St. Louis, MO), and recombinant human tumor necrosis factor ␣ (rHuTNF␣) was from R&D Systems (Minneapolis, MN). The inhibitors U-0126, SB203580, MG-132, and SP600125 were from Calbiochem (San Diego, CA), IFN␥ was from Roussel Uclaf (Paris, France), the anti-human TLR-2 mAb (T2.5), the anti-human TLR-4 mAb (15C1) (36,37), and the control isotype mAb were kind gifts from Dr. G. Elson (NovImmune, Plan-les-Ouates, Switzerland). The antagonists used, lipopolysaccharide (LPS) from Rhodobacter sphaeroides (LPS-RD) and LPS from Escherichia coli, were from InvivoGen (San Diego, CA) and Difco Laboratories (Detroit, MI), respectively.…”

Recombinant Soluble Forms of Extracellular TLR4 Domain and MD-2 Inhibit Lipopolysaccharide Binding on Cell Surface and Dampen Lipopolysaccharide-Induced Pulmonary Inflammation in Mice

“…Plasma from patients with severe sepsis and septic shock and even normal plasma appears to contain soluble MD-2 that elicits LPS activation in TLR4-transfected HEK293 cells and HUVECs (16,27), whereas the patients' plasma tends to somewhat decrease, although not significantly, LPS responsiveness in leukocytes and differentiated THP-1 cells expressing both TLR4 and MD-2 on cell surface. Previous studies (6,28) have shown that soluble MD-2 interacts with the extracellular TLR4 domain and confers LPS responsiveness on TLR4-transfected cells.…”