SummaryThe Hsp100/Clp ATPases constitute a family of closely related proteins of which some members function solely as chaperones whereas others additionally can associate with the unrelated ClpP peptidase forming a Clp proteolytic complex. We have investigated the role of four Clp ATPases in the versatile pathogen, Staphylococcus aureus . Previously, we showed that ClpX is required for expression of major virulence factors and for virulence of S. aureus , but not for survival during heat shock. In the present study, we have inactivated clpC , clpB and clpL and, while none of these mutations affected toxin production, both ClpC and ClpB and to a minor extent ClpL were required for intracellular multiplication within bovine mammary epithelial cells. These defects were paralleled by an inability of the clpC mutant to grow at high temperature and of the clpB mutant to induce thermotolerance indicating that the protective functions of these proteins are required both at high temperature and during infection. By primer extension analysis and footprint studies, we show that expression of clpC and clpB is controlled by the negative heatshock regulator, CtsR, and that ClpC is required for its repressor activity. Thus, ClpC is a likely sensor of stress encountered during both environmental stress and infection. In addition to virulence factor production the ability to form biofilms is of importance to S. aureus as a nosocomial pathogen. Interestingly, biofilm formation was reduced in the absence of ClpX or ClpC whereas it was enhanced in the absence of ClpP. Thus, our data show that Clp proteolytic complexes and the Clp ATPases control several key processes of importance to the success of S. aureus as a pathogen.

The liver sinusoidal endothelial cell (LSEC) forms the fenestrated wall of the hepatic sinusoid and functions as a control post regulating and surveying the trafficking of molecules and cells between the liver parenchyma and the blood. The cell acts as a scavenger cell responsible for removal of potential dangerous macromolecules from blood, and is increasingly acknowledged as an important player in liver immunity. This review provides an update of the major functions of the LSEC, including its role in plasma ultrafiltration and regulation of the hepatic microcirculation, scavenger functions, immune functions, and role in liver aging, as well as issues that are either undercommunicated or confusingly dealt with in the literature. These include metabolic functions, including energy metabolic interplay between the LSEC and the hepatocyte, and adequate ways of identifying and distinguishing the cells.

To maintain homeostasis, the animal body is equipped with a powerful system to remove circulating waste. This review presents evidence that the scavenger endothelial cell (SEC) is responsible for the clearance of blood-borne waste macromolecules in vertebrates. SECs express pattern-recognition endocytosis receptors (mannose and scavenger receptors), and in mammals, the endocytic Fc gamma-receptor IIb2. This cell type has an endocytic machinery capable of super-efficient uptake and degradation of physiological and foreign waste material, including all major classes of biological macromolecules. In terrestrial vertebrates, most SECs line the wall of the liver sinusoid. In phylogenetically older vertebrates, SECs reside instead in heart, kidney, or gills. SECs, thus, by virtue of their efficient nonphagocytic elimination of physiological and microbial substances, play a critical role in the innate immunity of vertebrates. In major invertebrate phyla, including insects, the same function is carried out by nephrocytes. The concept of a dual-cell principle of waste clearance is introduced to emphasize that professional phagocytes (macrophages in vertebrates; hemocytes in invertebrates) eliminate larger particles (>0.5 μm) by phagocytosis, whereas soluble macromolecules and smaller particles are eliminated efficiently and preferentially by clathrin-mediated endocytosis in nonphagocytic SECs in vertebrates or nephrocytes in invertebrates. Including these cells as important players in immunology and physiology provides an additional basis for understanding host defense and tissue homeostasis.

Morphological changes in the hepatic sinusoid with old age are increasingly recognized. These include thickening and defenestration of the liver sinusoidal endothelial cell, sporadic deposition of collagen and basal lamina in the extracellular space of Disse, and increased numbers of fat engorged, nonactivated stellate cells. In addition, there is endothelial upregulation of von Willebrand factor and ICAM-1 with reduced expression of caveolin-1. These changes have been termed age-related pseudocapillarization. The effects of old age on Kupffer cells are inconsistent, but impaired responsiveness is likely. There are functional implications of these aging changes in the hepatic sinusoid. There is reduced sinusoidal perfusion, which will impair the hepatic clearance of highly extracted substrates. Blood clearance of a variety of waste macromolecules takes place in liver sinusoidal endothelial cells (LSECs). Previous studies indicated either that aging had no effect, or reduced the endocytic capacity of LSECs. However, a recent study in mice showed reduced endocytosis in pericentral regions of the liver lobules. Reduced endocytosis may increase systemic exposure to potential harmful waste macromolecules such as advanced glycation end products Loss of fenestrations leads to impaired transfer of lipoproteins from blood to hepatocytes. This provides a mechanism for impaired chylomicron remnant clearance and postprandial hyperlipidemia associated with old age. Given the extensive range of substrates metabolized by the liver, age-related changes in the hepatic sinusoid and microcirculation have important systemic implications for aging and age-related diseases. Anat Rec,

I n his pioneering work on the ultrastructure of the hepatic sinusoid 30 years ago, Wisse (1) observed numerous ''smoothwalled macropinocytic vesicles'' in the sinusoidal liver endothelial cells (LEC), suggestive of active pinocytic scavenging of blood plasma proteins. The subsequent finding that the specific activities of lysosomal enzymes in LEC were as high as in the Kupffer cells (2), and for some enzymes even higher, substantiated Wisse's notion. In a series of papers over the next 20 years, it was shown that LEC eliminate an array of soluble macromolecular physiologic and foreign waste products from the circulation by receptor-mediated endocytosis (3-7). It is now well established that the population of LEC in mammals serves as the most important site of elimination of an array of circulating soluble macromolecular waste products. To carry out this scavenger function, LEC express at least five types of specific receptors for endocytosis of major physiologic waste products: (i) the hyaluronan receptor for major matrix polysaccharides and proteoglycans such as hyaluronan and chondroitin sulfate (8); (ii) the collagen alpha-chain receptor for collagen alpha-chains of several types of collagen (9); (iii) the scavenger receptor for amino-terminal propeptides of types I and III procollagen (4), modified macromolecules such as atherogenic advanced glycation end (AGE) products (5) and oxidized LDL (6), oligodeoxynucleotides (10), and blood proteins modified by the process of blood clotting and platelet activation (11); (iv) the mannose receptor for carboxy-terminal propeptide of type I procollagen (7) and tissue plasminogen activator (12); and (v) the Fc-␥ receptor for IgG-antigen immune complexes (13). It is interesting to note that the LEC are able to clear all major categories of biological macromolecules (proteins, polysaccharides, lipids, and nucleic acids) by means of these five types of endocytosis receptors. The blood concentration of most of these substances is normally very low (ng͞ml) because of highly efficient elimination by endocytosis receptors on LEC.On the basis of several studies showing that LEC represent an important nonphagocytic scavenger cell in mammals, it has been suggested that the hepatic scavenger function is shared between Kupffer cells, which eliminate insoluble waste by phagocytosis, and LEC, which remove soluble colloidal or macromolecular waste (3). Hypothesizing that the scavenger function of LEC in mammals be represented by similar nonmacrophagic, nonphagocytic scavenger endothelia in animals of all vertebrate classes, we set out to screen animal species from the seven major vertebrate classes for the presence of scavenger cells equivalent of mammalian LEC.The findings from these studies strongly suggest the presence of a nonmacrophagic system of scavenger endothelial cells that clear the blood from an array of circulating waste macromolecules and colloids. These scavenger endothelial cells make up the backbone of the reticuloendothelial system (RES), and thus represent an import...

Aging of the liver is associated with impaired metabolism of drugs, adverse drug interactions, and susceptibility to toxins. Since reduced hepatic blood flow is suspected to contribute this impairment, we examined age-related alterations in hepatic microcirculation.. Livers of C57Bl/6 mice were examined at 0.8 (pre-pubertal), 3 (young adult), 14 (middle-aged) and 27 (senescent) months of age using in vivo and electron microscopic methods. The results demonstrated a 14% reduction in the numbers of perfused sinusoids between 0.8 and 27 month mice associated with 35% reduction in sinusoidal blood flow. This was accompanied by an inflammatory response evidenced by a 5-fold increase in leukocyte adhesion in 27 month mice, up-regulated expression of ICAM-1, and increases in intrahepatic macrophages. Sinusoidal diameter decreased 6-10%. Liver sinusoidal endothelial cell (LSEC) dysfunction was seen as early as 14 months when there was a 3-fold increase in the numbers of swollen LSEC. The endocytotic capacity of LSEC also was found to be reduced in older animals. The sinusoidal endothelium in 27 month old mice exhibited pseudocapillarization. In conclusion, the results suggest that leukocyte accumulation in the sinusoids and narrowing of sinusoidal lumens due to pseudocapillarization and dysfunction of LSEC reduce sinusoidal blood flow in aged livers.

The purpose of this study was to identify the receptor responsible for endocytosis of denatured collagen from blood. The major site of clearance of this material (at least 0.5 g/day in humans) is a receptor on liver sinusoidal endothelial cells (LSECs). We have now identified an 180-kDa endocytic receptor on LSECs, peptide mass fingerprinting of which revealed it to be the mannose receptor. Challenge of mannose-receptor knockout mice and their cultured LSECs revealed significantly reduced blood clearance and a complete absence of LSEC endocytosis of denatured collagen. Organ analysis of wild-type versus knockout mice after injection of denatured collagen revealed significantly reduced liver uptake in the knockout mice. Clearance/endocytosis of ligands for other receptors in these animals was as that for wild-type mice, and denatured collagen uptake in wild-type mice was not affected by other ligands of the mannose receptor, namely mannose and mannan. Furthermore, unlike that of mannose and mannan, endocytosis of denatured collagen by the mannose receptor is calcium independent. This suggests that the binding site for denatured collagen is distinct from that for mannose/mannan. Mannose receptors on LSECs appear to have less affinity for circulating triple helical type I collagen. Conclusion: The mannose receptor is the main candidate for being the endocytic denatured collagen receptor on LSECs. (HEPATOLOGY 2007;45:1454-1461

Staphylococcus aureus causes infections ranging from superficial wound infections to life-threatening systemic infections. Essential for S. aureus pathogenicity are a number of cell-wall-associated and secreted proteins that are controlled by a complex regulatory network involving the quorum-sensing agr locus and a large set of transcription factors belonging to the Sar family. Recently, we revealed a new layer of regulation by showing that mutants lacking the ClpXP protease produce reduced amounts of several extracellular virulence factors and that, independently of ClpP, ClpX is required for transcription of spa, encoding Protein A. Here we find that the independent effect of ClpX is not general for other cell wall proteins, as expression of fibronectin-and fibrinogen-binding proteins was increased in the absence of either ClpX or ClpP. To assess the roles of ClpX and ClpP within the sar/agr regulatory network, deletions in clpX and clpP were combined with mutations in these genes. Interestingly, the derepression of spa transcription normally observed in an agrnegative strain was abolished in cells devoid of ClpX, and apparently ClpX modulates both SarS-dependent and SarS-independent control of spa expression, perhaps through the Sar family member Rot. Examination of expression of a single secreted protein, the SspA serine protease, revealed that ClpXP, similar to agr, is required for growth phase-dependent transcriptional induction of sspa. Intriguingly, induction was restored by the concomitant inactivation of Rot. We hypothesize that RNAIII accumulating in the postexponential phase may target Rot for degradation by ClpXP, leading to derepression of sspA.