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

Laboratory of the Division of Intensive Care, Department of Internal Medicine, University Hospital of Geneva, 24, rue Micheli-du-Crest, 1211 Geneva 14, Switzerland.
Blood (Impact Factor: 9.78). 01/2005; 104(13):4071-9. DOI: 10.1182/blood-2003-04-1290
Source: PubMed

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). Recombinant soluble MD-2 enabled LPS-dependent activation of epithelial cells bearing TLR4. LPS-binding protein (LBP) and soluble CD14 increased the sensitivity of TLR4-expressing epithelial cells to LPS but were not able to mediate LPS activation of these cells in the absence of soluble MD-2. An anti-MD-2 monoclonal antibody blocked LPS activation of TLR4-expressing cells only in the presence of septic plasma or septic urine. These results suggest that septic plasma containing soluble MD-2 leaking into the extravascular space supports LPS activation of TLR4-expressing epithelial cells. We therefore propose that soluble MD-2 is an important mediator of organ inflammation during sepsis.

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    ABSTRACT: IntroductionThe pathophysiology of endotoxemia-induced acute kidney injury (AKI) is characterized by an intense activation of the host immune system and renal resident cells by lipopolysaccharide (LPS) and derived pro-inflammatory products. However, the occurrence of renal fibrosis in this setting has been poorly investigated. The aim of the present study was to investigate the possible association between endothelial dysfunction and acute development of tissue fibrosis in a swine model of LPS-induced AKI. Moreover we studied the possible effects of coupled plasma filtration adsorption (CPFA) in this setting.Methods After 9 h from LPS infusion and 6 h of CPFA treatment, histological and biochemical changes were analyzed in pigs. Apoptosis and endothelial dysfunction were assessed on renal biopsies. The levels of LPS binding protein (LBP) were quantified by ELISA. Endothelial cells (EC) were stimulated in vitro with LPS and cultured in the presence of swine sera and were analyzed by FACS and Real time RT-PCR.ResultsIn a swine model of LPS-induced AKI, we observed that acute tubulointerstitial fibrosis occurred within 9 h from LPS injection. Acute fibrosis was associated with dysfunctional alpha-smooth muscle actin (¿-SMA)+ EC characterized by active proliferation (Ki-67+) without apoptosis (Caspase-3-). In accordance, LPS led to EC dysfunction in vitro with significant Vimentin and N-cadherin expressions and increased collagen I mRNA synthesis. Therapeutic intervention by citrate-based CPFA significantly prevented acute fibrosis in endotoxemic animals, by preserving EC phenotype both in peritubular capillaries and renal arteries. We found that the removal of LBP from plasma was crucial to eliminate the effects of LPS on EC dysfunction, by blocking LPS-induced collagen I production.Conclusions Our data indicates that EC dysfunction might be pivotal in the acute development of tubulointerstitial fibrosis in LPS-induced AKI. Selective removal of the LPS adaptor protein LBP might represent a future therapeutic option to prevent EC dysfunction and tissue fibrosis in endotoxemia-induced AKI.
    Critical care (London, England) 09/2014; 18(5):520. · 5.04 Impact Factor
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    ABSTRACT: The innate immune system plays an important role as a first response to tissue injury. This first response is carried out via germline-encoded receptors. Toll-like receptors (TLRs) are the first identified and best studied family of pattern recognition receptors. TLRs are expressed on a variety of cell types, including epithelial cells, endothelia, dendritic cells, monocytes/macrophages, and B- and T-cells. TLRs initiate innate immune responses and concurrently shape the subsequent adaptive immune response. They are sensors of both pathogens, through the exogenous pathogen-associated molecular patterns (PAMPs), and tissue injury, through the endogenous danger-associated molecular patterns (DAMPs). TLR signaling is critical in defending against invading microorganisms; however, sustained receptor activation is also implicated in the pathogenesis of inflammatory diseases. Ischemic kidney injury involves early TLR-driven immunopathology, and the resolution of inflammation is needed for rapid regeneration of injured tubule cells. Notably, the activation of TLRs also has been implicated in epithelial repair. This review focuses on the role of TLRs and their endogenous ligands within the inflammatory response of acute kidney injury.
    International Journal of Nephrology and Renovascular Disease 01/2014; 7:241-51.
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    ABSTRACT: MD-2 is essential for lipopolysaccharide (LPS) recognition of Toll-like receptor 4 (TLR4) but not for cell surface expression. The TLR4/MD-2 complex is formed intracellularly through co-expression. Extracellular complex formation remains a matter for debate because of the aggregative nature of secreted MD-2 in the absence of TLR4 co-expression. We demonstrated extracellular complex formation using three independent monoclonal antibodies (mAbs), all of which are specific for complexed TLR4 but unreactive with free TLR4 and MD-2. These mAbs bound to TLR4-expressing Ba/F3 cells only when co-cultured with MD-2-secreting Chinese hamster ovary cells or incubated with conditioned medium from these cells. All three mAbs bound the extracellularly formed complex indistinguishably from the intracellularly formed complex in titration studies. In addition, we demonstrated that two mAbs lost their affinity for TLR4/MD-2 on LPS stimulation, suggesting that these mAbs bound to conformation-sensitive epitopes. This was also found when the extracellularly formed complex was stimulated with LPS. Additionally, we showed that cell surface TLR4 and extrinsically secreted MD-2 are capable of forming the functional complex extracellularly, indicating an additional or alternative pathway for the complex formation.
    Biochemical and Biophysical Research Communications 09/2013; · 2.28 Impact Factor

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