Mouse Liver Sinusoidal Endothelium Eliminates HIV-Like Particles from Blood at a Rate of 100 Million per Minute by a Second-Order Kinetic Process.

We crafted human immunodeficiency virus (HIV)-like particles of diameter about 140 nm, which expressed two major HIV-1 proteins, namely, env and gag gene products, and used this reagent to simulate the rate of decay of HIV from the blood stream of BALB/c male mice. We found that most (~90%) of the particles were eliminated (cleared) from the blood by the liver sinusoidal endothelial cells (LSECs), the remainder from Kupffer cells; suggesting that LSECs are the major liver scavengers for HIV clearance from blood. Decay was rapid with kinetics suggesting second order with respect to particles, which infers dimerization of a putative receptor on LSEC. The number of HIV-like particles required for saturating the clearance mechanism was approximated. The capacity for elimination of blood-borne HIV-like particles by the sinusoid was 112 million particles per minute. Assuming that the sinusoid endothelial cells were about the size of glass-adherent macrophages, then elimination capacity was more than 540 particles per hour per endothelial cell.

Blood-Borne Lipopolysaccharide Is Rapidly Eliminated by Liver Sinusoidal Endothelial Cells via High-Density Lipoprotein.

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.

Scavenger receptor B1, the HDL receptor, is expressed abundantly in liver sinusoidal endothelial cells.

Cholesterol from peripheral tissue, carried by HDL, is metabolized in the liver after uptake by the HDL receptor, SR-B1. Hepatocytes have long been considered the only liver cells expressing SR-B1; however, in this study we describe two disparate immunofluorescence (IF) experiments that suggest otherwise. Using high-resolution confocal microscopy employing ultrathin (120 nm) sections of mouse liver, improving z-axis resolution, we identified the liver sinusoidal endothelial cells (LSEC), marked by FcγRIIb, as the cell within the liver expressing abundant SR-B1. In contrast, the hepatocyte, identified with ß-catenin, expressed considerably weaker levels, although optical resolution of SR-B1 was inadequate. Thus, we moved to a different IF strategy, first separating dissociated liver cells by gradient centrifugation into two portions, hepatocytes (parenchymal cells) and LSEC (non-parenchymal cells). Characterizing both portions for the cellular expression of SR-B1 by flow cytometry, we found that LSEC expressed considerable amounts of SR-B1 while in hepatocytes SR-B1 expression was barely perceptible. Assessing mRNA of SR-B1 by real time PCR we found messenger expression in LSEC to be about 5 times higher than in hepatocytes.

Surmounting an impasse of FcRn structure.

FcRn, resembling a major histocompatibility complex class I molecule with a closed peptide cleft, is an intracellular molecule that binds endocytosed albumin and IgG by a pH-dependent mechanism, diverting them from degradative fates and moving them out of the cell. The turnover of both of these important plasma proteins is thus regulated, as discussed by Schmidt and colleagues in this issue of Structure.

Abundant intracellular IgG in enterocytes and endoderm lacking FcRn.

FcRn, a non-classical MHCI molecule, transports IgG from mother to young and regulates the rate of IgG degradation throughout life. Brambell proposed a mechanism that unified these two functions, saying that IgG was pinocytosed nonspecifically by the cell into an FcRn-expressing endosome, where, at low pH, it bound to FcRn and was exocytosed. This theory was immediately challenged by claims that FcRn specificity for ligand could be conferred at the cell surface in neonatal jejunum. Assessing Brambell's hypothesis we found abundant nonspecifically endocytosed IgG present in the cytoplasm of FcRn(-/-) enterocytes. Further, IgG was present in the intercellular clefts and the cores of FcRn(+/+) but not FcRn(-/-) jejunum. FcRn specificity for ligand could be determined within the cell.

FcγRIIb on liver sinusoidal endothelium clears small immune complexes.

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.

Rapid and efficient clearance of blood-borne virus by liver sinusoidal endothelium.

The liver removes quickly the great bulk of virus circulating in blood, leaving only a small fraction to infect the host, in a manner characteristic of each virus. The scavenger cells of the liver sinusoids are implicated, but the mechanism is entirely unknown. Here we show, borrowing a mouse model of adenovirus clearance, that nearly all infused adenovirus is cleared by the liver sinusoidal endothelial cell (LSEC). Using refined immunofluorescence microscopy techniques for distinguishing macrophages and endothelial cells in fixed liver, and identifying virus by two distinct physicochemical methods, we localized adenovirus 1 minute after infusion mainly to the LSEC (∼90%), finding ∼10% with Kupffer cells (KC) and none with hepatocytes. Electron microscopy confirmed our results. In contrast with much prior work claiming the main scavenger to be the KC, our results locate the clearance mechanism to the LSEC and identify this cell as a key site of antiviral activity.

IgG is transported across the mouse yolk sac independently of FcgammaRIIb.

While generally accepted that FcRn of the human syncytiotrophoblast and the mouse yolk sac endoderm is the major IgG transporter, the finding of a different Fc receptor FcgammaRIIb (RIIb) in the human placental endothelium has suggested the existence of an additional IgG transporter. Testing our hypothesis in mouse, we found that while RIIb is expressed in the yolk sac vasculature, IgG concentrations in fetuses of wild-type mice (RIIb(+/+)) and mice with a null mutation in the gene encoding RIIb (RIIb(-/-) mice) are not different, and we thus reject our hypothesis that yolk sac RIIb transports IgG in utero in the mouse. However, the capillary bed in the mouse yolk sac is structurally more complex than in human placenta, consisting of three types of cells: an RIIb-negative endothelium, a unique RIIb-bearing cell that also expresses 2 out of 4 macrophage markers but not endothelial cell or pericyte markers, and pericytes. As in the human placenta the b2 isoform of RIIb predominates in the mouse yolk sac. Remarkably only a single capillary channel rather than 2 channels with a loop is found in each yolk sac villus, which, along with intracapillary erythrocytes, suggests that blood flow is peristaltic, mediated by pericytes. It is not clear whether RIIb in the human placental villus might mediate an IgG transport function in light of the mouse yolk sac equivalent failing to do so.

FcRn in the yolk sac endoderm of mouse is required for IgG transport to fetus.

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.

Beta 2-microglobulin deficient mice catabolize IgG more rapidly than FcRn- alpha-chain deficient mice.

FcRn, a nonclassical MHC-I protein bound to beta 2-microglobulin (beta 2m), diverts IgG and albumin from an intracellular degradative fate, prolonging the half-lives of both. While knockout mouse strains lacking either FcRn-alpha-chain (AK) or beta 2m (BK) show much shorter half-lives of IgG and albumin than normal mice, the plasma IgG half-life in the BK and AK strains is different, being shorter in the BK strain. Since beta 2m does not affect the IgG production rate, we tested whether an additional beta 2m-associated mechanism protects IgG from catabolism. First, we compared the fractional disappearance rate in plasma of an intravenous dose of radioiodinated IgG in a mouse strain deficient in both FcRn-alpha-chain and beta 2m (ABK), in the two parental knockout strains (AK and BK), and in the background wild-type (WT) strain. We found that IgG survived longer in the beta 2m-expressing AK strain than in the beta 2m-lacking ABK and BK strains, whereas the IgG half-lives between the ABK and BK strains were identical. Then we compared endogenous concentrations of four typical plasma proteins among the four strains and found that steady-state plasma concentrations of both IgG and albumin were higher in the AK strain than in either the BK or the ABK strain. These results suggest that a beta 2m-associated effect other than FcRn prolongs the survival of both IgG and albumin, although leaky gene transcription in the AK strain cannot be ruled out.

Kinetics of FcRn-mediated recycling of IgG and albumin in human: pathophysiology and therapeutic implications using a simplified mechanism-based model.

The nonclassical MHC class-I molecule, FcRn, salvages both IgG and albumin from degradation. Here we introduce a mechanism-based kinetic model for human to quantify FcRn-mediated recycling of both ligands based on saturable kinetics and data from the literature using easily measurable plasma concentrations rather than unmeasurable endosomal concentrations. The FcRn-mediated fractional recycling rates of IgG and albumin were 142% and 44% of their fractional catabolic rates, respectively. Clearly, FcRn-mediated recycling is a major contributor to the high endogenous concentrations of these two important plasma proteins. While familial hypercatabolic hypoproteinemia is caused by complete FcRn deficiency, the hypercatabolic IgG deficiency of myotonic dystrophy could be explained, based on the kinetic analyses, by a normal number of FcRn with lowered affinity for IgG but normal affinity for albumin. A simulation study demonstrates that the plasma concentrations of IgG and albumin could be dynamically controlled by both FcRn-related and -unrelated parameters.

Accelerated transferrin degradation in HFE-deficient mice is associated with increased transferrin saturation.

HFE, a major histocompatibility complex class I-related protein, is implicated in the iron overload disease, hereditary hemochromatosis. Whereas patients with hereditary hemochromatosis have low serum transferrin levels, little is known about transferrin turnover in HFE deficiency states. We injected mice intravenously with radioiodinated transferrin and compared plasma transferrin decay and steady-state endogenous transferrin concentration in the plasma between HFE-deficient and wild-type C57BL/6 mouse strains. HFE-deficient mice degraded transferrin faster than normal (P < 0.001) and had lower plasma transferrin concentrations (P < 0.001). Both HFE-deficient and wild-type mice were then fed diets with 3 different iron concentrations that we designated deficient (2-5 mg/kg of iron), control (0.2 g/kg), and overload (20 g/kg) for 6 wk immediately after weaning to create a range of serum iron concentrations and resultant transferrin saturations ranging from 16 to 78%. We found an inverse correlation between transferrin saturation and transferrin half-life (P < 0.0001, r = -0.839) for both HFE-deficient and wild-type mice, which suggests that HFE does not have a direct effect on transferrin catabolism; rather, HFE may influence transferrin half-life indirectly through its effect on transferrin saturation, which in turn enhances transferrin decay in HFE-deficient mice.

Perspective-- FcRn transports albumin: relevance to immunology and medicine.

Recent evidence validates a forgotten 40-year-old hypothesis: the MHC-related Fc receptor for IgG (FcRn) protects albumin from intracellular catabolic degradation, as it does for IgG, accounting for the uniquely long half-lives of both molecules and explaining their direct concentration-catabolism relationships. Albumin and IgG bind to FcRn at low pH but not at physiological pH. These two ligands bind independently of one another by distinctive mechanisms and to different surfaces of the receptor. Kinetic studies of FcRn-deficient mice indicate that, at steady-state, FcRn salvages from the degradative pathway a similar amount of albumin as is produced by mice and almost four-times more IgG than is produced. Thirty-fivefold more albumin than IgG molecules are protected from degradation by FcRn per unit time. It can be inferred that FcRn is expressed in nearly all cells. This receptor, originally described as transporting IgG from the mother to the fetus or neonate, now has a wider role central to the homeostatic regulation and conservation of both albumin and IgG throughout life.

Albumin binding to FcRn: distinct from the FcRn-IgG interaction.

The MHC-related Fc receptor for IgG (FcRn) protects albumin and IgG from degradation by binding both proteins with high affinity at low pH in the acid endosome and diverting both from a lysosomal pathway, returning them to the extracellular compartment. Immunoblotting and surface plasmon resonance studies show that both IgG and albumin bind noncooperatively to distinct sites on FcRn, that the affinity of FcRn for albumin decreases approximately 200-fold from acidic to neutral pH, and that the FcRn-albumin interaction shows rapid association and dissociation kinetics. Isothermal titration calorimetry shows that albumin binds FcRn with a 1:1 stoichiometry and the interaction has hydrophobic features as evidenced by a large positive change in entropy upon binding. Our results suggest that the FcRn-albumin interaction has unique features distinct from FcRn-IgG binding despite the overall similarity in the pH-dependent binding mechanism by which both ligands are protected from degradation.

Familial hypercatabolic hypoproteinemia caused by deficiency of the neonatal Fc receptor, FcRn, due to a mutant beta2-microglobulin gene.

Two siblings, products of a consanguineous marriage, were markedly deficient in both albumin and IgG because of rapid degradation of these proteins, suggesting a lack of the neonatal Fc receptor, FcRn. FcRn is a heterodimeric receptor composed of a nonclassical MHC class I alpha-chain and beta(2)-microglobulin (beta(2)m) that binds two ligands, IgG and albumin, and extends the catabolic half-lives of both. Eight relatives of the siblings were moderately IgG-deficient. From sera archived for 35 years, we sequenced the two siblings' genes for the heterodimeric FcRn. We found that, although the alpha-chain gene sequences of the siblings were normal, the beta(2)m genes contained a single nucleotide transversion that would mutate a conserved alanine to proline at the midpoint of the signal sequence. Concentrations of soluble beta(2)m and HLA in the siblings' sera were <1% of normal. Transfection assays of beta(2)m-deficient cultured cells with beta(2)m cDNA indicated that the mutant beta(2)m supported <20% of normal expression of beta(2)m, MHC class I, and FcRn proteins. We concluded that a beta(2)m gene mutation underlies the hypercatabolism and reduced serum levels of albumin and IgG in the two siblings with familial hypercatabolic hypoproteinemia. This experiment of nature affirms our hypothesis that FcRn binds IgG and albumin, salvages both from a degradative fate, and maintains their physiologic concentrations.

Albumin turnover: FcRn-mediated recycling saves as much albumin from degradation as the liver produces.

It is now understood that the nonclassical major histocompatibility complex-I molecule FcRn binds albumin and retrieves it from an intracellular degradative fate. Whether FcRn in the liver modulates albumin turnover through effects on biosynthesis and production is not known. Thus we quantified the appearance of biosynthetically labeled albumin in plasma after an intravenous bolus injection of [(3)H]leucine in FcRn-deficient mice. The production rates for both albumin (FcRn substrate) and transferrin (nonsubstrate) are increased by approximately 20% in FcRn-deficient mice compared with normal mice, likely compensating for the lowered plasma oncotic pressure caused by hypoalbuminemia in FcRn-deficient mice. Determining the magnitude of FcRn-mediated effects on albumin turnover, we then measured the steady-state plasma concentrations of biosynthetically labeled albumin and transferrin during [(3)H]leucine infusion. The concentration of albumin was approximately 40% lower in FcRn-deficient mice compared with normal mice. Furthermore, the approximately 40% lower plasma albumin concentration in FcRn-deficient mice along with the approximately 20% increase in albumin production indicate, by the mass-balance equation, that albumin degradation in FcRn-deficient mice is twice that of normal mice. These studies of biosynthetically labeled, and thus native, albumin support our previous finding that FcRn protects albumin from degradation. Permitting quantification of the magnitude of FcRn-mediated recycling, they further indicate that FcRn has extraordinary capacity: the amount of albumin saved from degradation by FcRn-mediated recycling is the same as that produced by the liver.