The double-radiolabeled LPS substrate can also be used to quantitate the rate and extent of LPS deacylation in vivo
. Whether the LPS is injected subcutaneously (footpad, skin site), intraperitoneally or intravenously, deacylation occurs over many hours (Lu et al., 2005
; Shao et al., 2007
). Despite this seemingly sluggish performance, AOAH-mediated deacylation completely inactivated almost 80% of a subcutaneous dose of LPS before the LPS could travel to draining lymph nodes (Lu et al., 2005
). No loss of the LPS secondary acyl chains was detected in Aoah−/−
mice, and in neither Aoah−/−
animals was there loss of primary (3-hydroxymyristoyl) acyl chains from the backbone. LPS deacylation in vivo
is thus remarkably selective and limited. Of the various LPS-catabolizing enzymes produced by D. discoideum
(Verret et al., 1982a
), which eat bacteria as a foodstuff, only AOAH has been conserved during animal evolution.
One important unresolved issue is the extent to which LPS deacylation occurs inside and outside host cells. An intracellular site was suggested by the enzyme’s acid pH optimum, its location in an intracellular vacuole (Staab et al., 1994
), and the apparent colocalization of AOAH and deacylated LPS in neutrophils (Luchi and Munford, 1993
). Both rabbit macrophages and murine dendritic cells deacylate the LPS in phagocytosed Escherichia coli
in an AOAH-dependent fashion (Katz et al., 1999
; Lu et al., 2003
). Moreover, AOAH does not act on LPS in buffered salt solutions in the absence of a nonionic detergent such as Triton X-100, suggesting that it may require an intracellular environment or factor(s) to do its job. The enzyme can be secreted by rabbit neutrophils and monocytes (Erwin and Munford, 1991
), however, and extracellular deacylation has been demonstrated in rabbit peritoneal exudate fluid (Katz et al., 1999
), a rich mixture of extravasated plasma, leukocyte products, and other molecules. The AOAH secreted into the urine by renal cortical epithelial cells can also deacylate LPS (Feulner et al., 2004
). Gioannini et al.
recently reported that CD14 and LBP can bind LPS in a way that allows AOAH to deacylate its lipid A moiety (Gioannini et al., 2007
), providing an attractive mechanism for extracellular LPS deacylation (Weinrauch et al., 1999
). The relative contributions of intra- and extracellular deacylation to LPS inactivation in vivo
Very little is known about how AOAH activity is regulated in vivo
. In part this reflects the enzyme’s low abundance, which has hindered quantitative detection of both AOAH protein and mRNA. In addition, it has not been possible to detect AOAH activity or protein in human plasma or serum. Since the enzyme is easily measured in rodent and rabbit serum, most studies of AOAH regulation have been performed in these animals. In rabbits, plasma AOAH levels rise dramatically within a few minutes of an intravenous injection of LPS and remain elevated for many hours (Erwin and Munford, 1991
). The increase in AOAH activity was significantly less in animals that had been given methchlorethamine to induce leukopenia, suggesting that much of the extracellular enzyme is produced and released by neutrophils or monocytes. Indeed, rabbit leukocytes released AOAH in response to stimulation by LPS ex vivo
(Erwin and Munford, 1991
). In studies performed in mice, Cody et al.
found that AOAH mRNA and activity in liver and lung increased several-fold following intraperitoneal treatment with LPS (Cody et al., 1997
). In addition, low concentrations of LPS and interferon-γ induced greater than 10-fold increases in AOAH mRNA in cultured murine macrophages. Neither IL-10 nor dexamethasone prevented AOAH mRNA accumulation in response to LPS, in keeping with the discovery, many years later, that AOAH participates in the anti-inflammatory (recovery) phase of local infection. Indeed, Mages et al. (2008)
found an approximately sixfold increase in AOAH mRNA abundance in LPS-primed (tolerant) murine macrophages relative to unstimulated controls. Immature murine dendritic cells also produce AOAH; cytokine-induced maturation was associated with diminished LPS-deacylating ability, whereas exposure to LPS, CpG oligonucleotides, or staphylococci was stimulatory (Lu et al., 2003
). Unfortunately, it is uncertain that mice are useful models for human AOAH regulation, since the tissue-specific expression of the enzyme differs substantially (mice produce much more AOAH in the kidney than do humans, and less in myeloid cells). DeLeo and colleagues found that AOAH mRNA abundance decreased approximately twofold in human neutrophils during the 6 h following phagocytosis of latex beads, a time when many of the neutrophils were undergoing apoptosis (Kobayashi et al., 2003
). Further study of AOAH regulation in human phagocytes is needed.