Despite their "immaturity", band forms are capable of mediating crucial innate immune functions during severe infections and sepsis. Their fate and capacity to mature in vivo remain to be determined.

Innate recognition of bacteria is a key step in the activation of inflammation and coagulation, and it is dependent on pathogen-associated molecular pattern (PAMP) ligation to Toll-like receptors (TLRs) and CD14. The dominant receptors activated when cells encounter a whole bacterium, which express several PAMPs, are poorly defined. Herein, we have stimulated various human cells with prototypic Gramnegative and Gram-positive bacteria. Receptor-dependent responses to whole bacteria were assessed using both TLRtransfected cells and specific monoclonal antibodies against TLRs, MD-2, and CD14. Enterobacteria-activated leukocytes and endothelial cells in a TLR4/MD-2-dependent manner, most likely via lipopolysaccharide (LPS). TLR2 activation was observed with a high bacterial inoculum, and in epithelial cells expressing TLR2 but not TLR4. Pseudomonas aeruginosa stimulated cells by both TLR2 and TLR4/MD-2. Gram-positive bacteria activated cells only at high concentrations, in a partially TLR2-dependent but TLR4/MD-2-independent manner. Either TLR or CD14 neutralization blocked activation to all bacterial strains tested with the exception of some Gram-positive strains in whole blood in which partial inhibition was noted. This study identifies dominant TLRs involved in responses to whole bacteria. It also validates the concept that host cell activation by bacterial pathogens can be therapeutically reduced by anti-TLR4, -TLR2, and -CD14 mAbs. IntroductionThe recognition of bacteria as nonself agents by mammalian cells is key in mounting an innate response to control infection. Several bacterial antigens, known as pathogen-associated molecular patterns (PAMPs), are sensed as "nonself" molecules by host immune cells, using receptors of the innate immune system. PAMPs are, for the majority, cell-wall molecules. Some PAMPs are found in both Gram-negative and Gram-positive bacteria, (lipopeptides, peptidoglycan, flagellin, and bacterial DNA). Others are specific either for Gram-negative bacteria (LPS), Gram-positive bacteria (lipoteichoic acid), or mycobacteria (lipoarabinomannan).Toll-like receptors (TLRs) belong to a family of leucine-rich repeat (LRR) proteins that are either expressed at the cell surface or in intracellular compartments. 1 They recognize and bind a wide variety of bacterial PAMPs, including LPS (typically recognized by TLR4, although some LPS species can be recognized by TLR2), lipopeptides (TLR1, TLR2, TLR6), lipoarabinomannan and lipoteichoic acid (TLR2 and other TLRs), flagellin (TLR5), and bacterial DNA (TLR9). [2][3][4] The ligation of PAMPs to TLRs induces a profound reprogramming of immune cells and activates several innate pathways such as inflammation, coagulation, and cell death. Some of the nucleotide-binding oligomerization domain (NOD) LRR proteins, such as Nod1 and Nod2, are also intracellular sensors for bacterial molecules and mediate inflammation on recognition of specific motifs in bacterial peptidoglycan. 5 Other receptors also recognize PAMPs and are essential for the phagocytosis of b...

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...