This study of LPS trafficking in vivo
yielded 3 note worthy findings. First, we found that LPS moves from a local injection site principally via lymphatics. Endotoxemia, or the presence of LPS in the circulating blood, is a much-feared complication of many Gram-negative bacterial diseases. LPS in an extravascular tissue might enter the blood directly, via venous capillaries, or indirectly, by draining first through lymphatic channels to the thoracic duct. Our results indicate that lymphatics provide an important conduit from an inflamed subcutaneous site to the circulating blood, just as lymphatic drainage is a major route of LPS clearance from infected peritoneal fluid (24
). The slow course of lymphatic drainage not only provides greater time for LPS inactivation by deacylation prior to entering the blood, but the LPS may also be exposed to inhibitory factors in lymph (25
). It is possible that some LPS moved directly from the footpad into the blood, or that it was internalized by footpad phagocytes that then left the injection site and entered the bloodstream, but these seemed to be minor modes of LPS egress from the injection site. Although almost five per cent of the LPS injectate was found in the liver on day 1 after injection, the LPS recovered from the livers of wildtype mice had lost over half of its secondary acyl chains, consistent with AOAH-mediated deacylation/inactivation either prior to, or after (12
), hepatic uptake of LPS from the circulation.
Second, most of the injected LPS passed through the DLN without entering the paracortex or B follicles. Radiolabeled LPS did not accumulate in the DLN. FITC-LPS that entered the DLN was found principally within the subcapsular sinus, where it was associated with macrophages (CD169+) and lymphatic endothelial cells (LYVE1+), and also in the medulla. With time, discrete foci of FITC-LPS and TR-LPS appeared in the paracortex, closely resembling the pattern observed for subcutaneously-injected fluorescent beads, suggesting that some of the LPS is carried to the DLN by cells. This was more prominent at later time points (1 to 2 days after injection). Although a minor fraction of the injected LPS was found in the paracortex, this may be sufficient to bring LPS into direct contact with B cells (20
), a prerequisite for B cell stimulation by LPS in mice (MF Lu, unpublished results).
Third, our results document the important role that AOAH plays in modulating the bioactivity of LPS in vivo
. Acyloxyacyl hydrolase is a lipase, found principally in myeloid cells, that removes two of the six fatty acids that are required for LPS to be sensed by its receptor on animal cells, the MD-2—TLR4 complex. In three different experimental settings – involving B cells, Kupffer cells, and peritoneal macrophages – mice that lacked AOAH were unable to restore homeostasis after they were exposed to small amounts of LPS in vivo
). In each instance, the response to LPS exposure was exaggerated and prolonged in the absence of AOAH. Here we found that over 70% of the injected LPS was deacylated by AOAH before it left the injection site in Aoah+/+
mice. There was strong evidence that phagocytes took part in deacylating LPS, but we were unable to distinguish extracellular from intracellular deacylation. AOAH has an acid pH optimum and resides within intracellular granules, yet it may be secreted and taken up by other cells in a mannose-6-phosphate-dependent fashion (27
). In addition, Gioannini et al. found that soluble CD14 and LPS binding protein (LBP) can bind LPS in a manner that makes it accessible to deacylation by AOAH (28
). Although there is evidence that LPS can be released after being processed by cultured macrophages (18
), the absence of a defined mechanism for this phenomenon makes it seem less likely than extracellular deacylation within the footpad by secreted AOAH.
To be able to follow LPS molecules quantitatively in vivo
, we injected LPS subcutaneously into a footpad or a site on the back. Although this approach was meant to mimic the LPS released by bacteria into an infected tissue site, it required relatively high doses of LPS and utilized purified LPS instead of intact bacteria. Using rough (Ra or Rc) LPS or lipooligosaccharide (N. meningitidis
) offered several advantages over smooth (long polysaccharide-containing) LPS preparations: each of the LPS preparations used here has a relatively uniform structure, with 6 fatty acyl chains attached to the lipid A moiety of most molecules, and each potently activates MD-2—TLR4 (29
). A disadvantage is the tendency of rough LPS preparations to form aggregates or micelles that, while possibly resembling the size of bacterial outer membrane fragments, are clearly artificial. LPS is not soluble in methanol, yet it is possible that cell-free FITC-LPS was washed away during tissue fixation for microscopy. Although our FITC-LPS was partially deacylated during the labeling process, there is strong evidence that partially deacylated LPS structures bind to LPS binding protein (LBP), CD14 and MD-2—TLR4 (30
). It thus seems unlikely that this degree of deacylation would alter the FITC-LPS’s ability to interact normally with cells.
To our knowledge, these are the first studies to track the fates of LPS molecules in tissues other than the bloodstream. We did not expect to find the very slow disappearance kinetics from the injection site, the prominent role played by lymphatics in removing LPS from the subcutaneous tissue, the passage of a large fraction of the LPS through DLN without entering the parenchyma of the node, or the inactivation of such a large fraction of the LPS before it entered the lymph. The immunological potency of LPS in vivo, measured here as B cell activation to produce polyclonal antibodies, was greatly influenced by the kinetics of drainage and enzymatic inactivation as well as by lymph node anatomy.