R‐form LPS, the master key to the activation ofTLR4/MD‐2‐positive cells

Abstract

Lipopolysaccharide (endotoxin, LPS) is a major recognition marker for the detection of gram-negative bacteria by the host and a powerful initiator of the inflammatory response to infection. Using S-and R-form LPS from wild-type and R-mutants of Salmonella and E. coli, we show that R-form LPS readily activates mouse cells expressing the signaling receptor Toll-like receptor 4/myeloid differentiation protein 2 (TLR4/MD-2), while the S-form requires further the help of the LPS-binding proteins CD14 and LBP, which limits its activating capacity. Therefore, the R-form LPS under physiological conditions recruits a larger spectrum of cells in endotoxic reactions than S-form LPS. We also show that soluble CD14 at high concentrations enables CD14-negative cells to respond to S-form LPS. The presented in vitro data are corroborated by an in vivo study measuring TNF-a levels in response to injection of R-and S-form LPS in mice. Since the R-form LPS constitutes ubiquitously part of the total LPS present in all wild-type bacteria its contribution to the innate immune response and pathophysiology of infection is much higher than anticipated during the last half century.Supporting information for this article is available at http://www.wiley-vch.de/contents/jc_2040/2006/35593_pdf IntroductionThe interaction of highly conserved microbial constituents with the innate immune system forms the basis of recognition of and reaction against intruding pathogens in mammals. One such constituent is lipopolysaccharide (endotoxin, LPS), a major recognition marker common to gram-negative bacteria [1][2][3], a large group comprising important human pathogens as well as commensals. Thus, the interaction of LPS with cells of the innate immune system leads to the formation and release of endogenous mediators initiating inflammatory and immune responses essential for an antibacterial defense [3,4]. This primarily protective mechanism may become overshadowed by an acute pathophysiological response with the typical clinical symptoms of septic shock that frequently follows the release of inflammatory mediators, such as tumor necrosis factor (TNF)-a during infection [5][6][7].Innate immunity * Both authors contributed equally to this work. [10]. Binding of allergen to its specific IgE on the surface of MC results in an immediate release of preformed pro-inflammatory mediators (e.g., histamine, TNF-a and proteases) from cytoplasmic granules (degranulation) [11] and a later release of de novo synthesized arachidonic acid metabolites and various cytokines like interleukin 6 (IL-6), TNF-a, and chemokines [10,11]. Unlike allergens, LPS induces no degranulation of MC, but like allergens, it stimulates the de novo synthesis and release of cytokines in these cells [9].Activation of cells by LPS is mediated by the Toll-like receptor 4 (TLR4), a member of the highly conserved protein family of TLR, which are specialized in the recognition of microbial components. In mice, defects in TLR4 result in LPS unresponsiveness [12]. For functional interaction with LP...

Nontransformed, GM-CSF–dependent macrophage lines are a unique model to study tissue macrophage functions

“…Two types of biologically active LPS are synthetized by Gram-negative bacteria, smooth (S) and rough (R) form LPS (S-LPS and R-LPS). LPS-binding proteins (LBP) and CD14 are required for the activation of cells by S-LPS via the TLR4/ myeloid differentiation factor-2 receptor complex, whereas R-LPS activates various target cells including BMMs in the absence of these proteins (18,19). In agreement, we found that the TNF-α response to S-LPS in MPI cells, AMs, and BMMs is highly dependent on LBP and CD14 (Fig.…”

“…According to the current consensus, LBP/CD14 help is required for S-LPSbut not R-LPS-triggered cell activation (18,19,31). Therefore, our present finding suggests the existence of an alternative, cell type-specific activation mechanism in MPI cells and AMs.…”

CREB and AP‐1 activation regulates MKP‐1 induction by LPS or M‐CSF and their kinetics correlate with macrophage activation versus proliferation

“…In several experiments, the results obtained with commercial LPS were compared with highly purified LPS from Salmonella abortus equi, kindly donated by Dr. C. Galanos (Max Planck Institute, Freiburg, Germany) [70]. No differences were found.…”

Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood

“…Recently, it has been shown that the rough (R)-form LPS can interact with TLR4 without CD14 (12,13). Re-form LPS (lipid A + two 3-deoxy-D-manno-2-octurosonic acid residues), but not the smooth (S)-form LPS, can induce tumor necrosis factor-alpha (TNF-α) responses also in the absence of CD14 (12).…”

“…Re-form LPS (lipid A + two 3-deoxy-D-manno-2-octurosonic acid residues), but not the smooth (S)-form LPS, can induce tumor necrosis factor-alpha (TNF-α) responses also in the absence of CD14 (12). R-form LPS was a potent activator of mast cells (that lack CD14), while S-form LPS was practically devoid of stimulatory activity (13). Since human neutrophils either lack or express low amounts of mCD14, it became important to study if LPS-induced neutrophil activation is different when stimulated by S-or R-form LPS.…”