Search

During Gram-negative bacterial infections, excessive LPS induces inflammation and sepsis via action on immune cells. However, the bulk of LPS can be cleared from circulation by the liver. Liver clearance is thought to be a slow process mediated exclusively by phagocytic resident macrophages, Kupffer cells (KC). However, we discovered that LPS disappears rapidly from the circulation, with a half-life of 2-4 min in mice, and liver eliminates about three quarters of LPS from blood circulation. Using microscopic techniques, we found that ∼75% of fluor-tagged LPS in liver became associated with liver sinusoidal endothelial cells (LSEC) and only ∼25% with KC. Notably, the ratio of LSEC-KC-associated LPS remained unchanged 45 min after infusion, indicating that LSEC independently processes the LPS. Most interestingly, results of kinetic analysis of LPS bioactivity, using modified limulus amebocyte lysate assay, suggest that recombinant factor C, an LPS binding protein, competitively inhibits high-density lipoprotein (HDL)-mediated LPS association with LSEC early in the process. Supporting the previous notion, 3 min postinfusion, 75% of infused fluorescently tagged LPS-HDL complex associates with LSEC, suggesting that HDL facilitates LPS clearance. These results lead us to propose a new paradigm of LSEC and HDL in clearing LPS with a potential to avoid inflammation during sepsis., (Copyright © 2016 by The American Association of Immunologists, Inc.)

It has long been known that the ITIM-bearing IgG Fc receptor (FcγRIIb, RIIb) is expressed on liver sinusoidal endothelial cells (LSEC) and that the liver is the major site of small immune complex (SIC) clearance. Thus, we proposed that RIIb of LSEC eliminates blood-borne SIC, thereby controlling immune complex-mediated autoimmune disease. Testing this hypothesis, we found most RIIb of the mouse, fully three-quarters, to be expressed in liver. Moreover, most (90%) liver RIIb was expressed in LSEC, the remainder in Kupffer cells. An absent FcRγ in LSEC implied that RIIb is the sole FcγR expressed. Testing the capacity of liver RIIb to clear blood-borne SIC, we infused mice intravenously with radio-iodinated SIC made of OVA and rabbit IgG anti-OVA. Tracking decay of SIC from the blood, we found the RIIb knockout strain to be severely deficient in eliminating SIC compared with the wild-type strain, terminal half-lives being 6 and 1.5 h, respectively. RIIb on LSEC, a major scavenger, keeps SIC blood concentrations low and minimizes pathologic deposition of inflammatory immune complex.

In adults, the nonclassical MHC class I molecule, FcRn, binds both IgG and albumin and rescues both from a degradative fate, endowing both proteins with high plasma concentrations. FcRn also transports IgG from mother to young during gestation. Anticipating that a detailed understanding of gestational IgG transport in the mouse may give us a useful model to understand FcRn function in the human placenta, we have studied FcRn in the mouse yolk sac placenta in detail. Analyzing day 19-20 fetuses of the three FcRn genotypes resulting from matings of FcRn(+/-) parents, we found that FcRn(-/-) fetuses showed negligible IgG concentrations (1.5 microg/ml), whereas IgG concentrations in FcRn(+/-) fetuses were about a half (176 microg/ml) that of FcRn(+/+) fetuses (336 microg/ml), indicating that FcRn is responsible for virtually all IgG transport from mother to fetus. Immunofluorescence and immunoblotting studies indicated that FcRn is expressed in the endoderm of the yolk sac placenta but not in other cells of the yolk sac placenta or in the chorioallantoic placenta. IgG was found in the endoderm of both FcRn(+/+) and FcRn(-/-) yolk sac placentas and in the mesenchyme of FcRn(+/+) but was missing from the mesenchyme of FcRn(-/-) yolk sac placentas, indicating that IgG enters the endoderm constitutively but is moved out of the endoderm by FcRn. The similarities of these results to human placental FcRn expression and function are striking.

Placental transfer of IgG from maternal circulation to that of the fetus is crucial for fetal and newborn immunity. This process requires that IgG broach two cellular layers of the placenta. IgG transport across the first layer, the syncytiotrophoblast, is almost certainly mediated by the MHC-related FcR for IgG, FcRn. The second layer, the villus endothelium, was until recently thought to allow IgG movement nonspecifically by constitutive transcytosis in caveolae. However, we recently showed that villus endothelium expressed a separate FcR for IgG, the inhibitory motif-bearing Fc gammaRIIb2 seen most notably on macrophages and as a minor fraction of the Fc gammaRIIb expressed on B cells. Now, by quantitative microscopy, we find Fc gammaRIIb2 to be expressed abundantly in an unidentifiable and likely novel organelle of the villus endothelium, unassociated with caveolae. About half of these Fc gammaRIIb2 organelles contain IgG; the remainder lack IgG. The majority fraction (approximately 80%) of IgG-containing organelles is associated with Fc gammaRIIb. No IgG-containing organelles are associated with caveolin. These findings are compatible with Fc gammaRIIb-mediated transfer of IgG across the villus endothelium, independent of caveolae.

Abs of the IgG isotype are efficiently transported from mother to neonate and have an extended serum t(1/2) compared with Abs of other isotypes. Circumstantial evidence suggests that the MHC class I-related protein, the neonatal FcR (FcRn), is the FcR responsible for both in vivo functions. To understand the phenotypes imposed by FcRn, we produced and analyzed mice with a defective FcRn gene. The results provide direct evidence that perinatal IgG transport and protection of IgG from catabolism are mediated by FcRn, and that the latter function is key to IgG homeostasis, essential for generating a potent IgG response to foreign Ags, and the basis of enhanced efficacy of Fc-IgG-based therapeutics. FcRn is therefore a promising therapeutic target for enhancing protective humoral immunity, treating autoimmune disease, and improving drug efficacy.

FcgammaR-mediated phagocytosis is accompanied by the generation of tissue-damaging products such as inflammatory cytokines and reactive oxygen species. Hence, the phagocytic response must be a tightly regulated process. Recent studies have established that clustering FcgammaR on human myeloid cells causes tyrosine phosphorylation of Src homology 2 domain-containing inositol polyphosphate phosphatase (SHIP). However, it is not known how these immunoreceptor tyrosine-based activation motif (ITAM)-bearing phagocytic FcgammaR activate SHIP, or whether the activation of SHIP by ITAMs has any functional relevance. Experiments addressing the mechanism of SHIP association with ITAMs have been done in in vitro systems using phosphopeptides. In this study we undertook to dissect the molecular mechanism by which SHIP associates with the native ITAM-FcgammaR and becomes phosphorylated. In this report we provide evidence that first, SHIP is indeed phosphorylated by ITAM-FcgammaR, using cell systems that lack FcgammaRIIb expression; second, coimmunoprecipitation experiments demonstrate that SHIP associates with native ITAM-bearing FcgammaRIIa in vivo; and third, phosphorylation of SHIP by FcgammaRIIa is inhibited by overexpressing either the SHIP Src homology 2 domain or a dominant negative mutant of Shc. In contrast, SHIP phosphorylation was not inhibited by a dominant negative mutant of Grb2. We extend these observations to show that SHIP activation by ITAM-FcgammaR down-regulates NF-kappaB-induced gene transcription. These findings both provide a molecular mechanism for SHIP association with native ITAM-bearing receptors and demonstrate that SHIP association with ITAM-FcgammaR serves to regulate gene expression during the phagocytic process.