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D H Perlmutter

Publications and source records attributed to D H Perlmutter.

At least 37 records · Page 2Linked to original sources
PubMed1998

Ligand substitution of receptor targeted DNA complexes affects gene transfer into hepatoma cells.

We have targeted the serpin enzyme complex receptor for gene transfer in human hepatoma cell lines using peptides < 30 amino acids in length which contain the five amino acid recognition sequence for this receptor, coupled to poly K of average chain length 100 K, using the heterobifunctional coupling reagent sulfo-LC SPDP. The number of sulfo-LC SPDP modified poly-L-lysine residues, as well as the degree of peptide substitution was assessed by nuclear magnetic resonance spectroscopy. Conjugates were prepared in which 3.5%, 7.8% or 26% of the lysine residues contained the sulfo-LC SPDP moiety. Each of these conjugates was then coupled with ligand peptides so that one in 370, one in 1039, or one in 5882 lysines were substituted with receptor ligand. Electron microscopy and atomic force microscopy were used to assess complex structure and size. HuH7 human hepatoma cells were transfected with complexes of these conjugates with the plasmid pGL3 and luciferase expression measured 2 to 16 days after treatment. All the protein conjugates in which 26% of the K residues were modified with sulfo-LC SPDP were poor gene transfer reagents. Complexes containing less substituted poly K, averaged 17 +/- 0.5 nm in diameter and gave peak transgene expression of 3-4 x 10(6) ILU/mg which persisted (> 7 x 10(5) ILU) at 16 days. Of these, more substituted polymers condensed DNA into complexes averaging 20 +/- 0.7 nm in diameter and gave five-fold less luciferase than complexes containing less substituted conjugates. As few as eight to 11 ligands per complex are optimal for DNA delivery via the SEC receptor. The extent of substitution of receptor-mediated gene transfer complexes affects the size of the complexes, as well as the intensity and duration of transgene expression. These observations may permit tailoring of complex construction for the usage required.

Animals↗
PubMed1998

Alpha-1-antitrypsin deficiency.

Homozygous PIZZ alpha 1-antitrypsin deficiency, which has an incident of 1 in 1600 to 1 in 2000 live births, is the most common genetic cause of liver disease in children. It is also associated with chronic liver disease and hepatocellular carcinoma in adults. It is a well-known cause of pulmonary emphysema. Although emphysema is due to uninhibited proteolytic destruction of the connective tissue backbone of the lung, liver disease is thought to result from the toxic effects of the mutant alpha 1AT molecule retained within the endoplasmic reticulum of liver cells. Screening studies done by Sveger in Sweden have shown that only 10 to 15% of the PIZZ population develop clinically significant liver disease over the first 20 years of life. Recent studies have suggested that a subgroup of PIZZ individuals are predisposed to liver injury because of an inefficient degradation of mutant alpha 1ATZ within the endoplasmic reticulum. Altered migration of the abnormal alpha 1ATZ molecule in isoelectric focussing gels is the basis of the diagnosis of alpha 1AT deficiency. Treatment of alpha 1AT deficiency-associated liver disease is mostly supportive. Liver replacement therapy has been used successfully for severe liver injury. An increasing number of patients with severe emphysema have undergone lung transplantation.

Adult↗
PubMed1997

Reconstitution of bile acid transport in a heterologous cell by cotransfection of transporters for bile acid uptake and efflux.

The rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 (CBATP) is a 110-kDa transmembrane phosphoglycoprotein that is thought to have bile acid efflux, ecto-ATPase, and cell adhesion properties. Its extracellular amino-terminal domain is highly homologous to carcinoembryonic antigen (CEA), a glycophosphatidyl inositol-anchored membrane protein with cell adhesion properties and a marker for adenocarcinoma. In the current study, we examined the possibility of more clearly defining the role of CBATP in bile acid efflux by cotransfecting a heterologous cell, the COS cell, with cDNAs for a bile acid importer, the ileal bile acid transporter (IBAT), as well as for CBATP. The results show that when IBAT mediates uptake of [3H]taurocholate to a level 20-fold higher than that achieved previously by nonspecific pinocytosis, CBATP mediates time-, temperature- and concentration-dependent efflux. Efflux of [3H]taurocholate mediated by CBATP in the cotransfected COS cells is saturable and has curvilinear kinetic characteristics (Vmax = 400 pmol/mg protein/min, Km = 70 microM). It is inhibited by 4,4'-diisothiocyanostilbene-2,2-disulfonic acid and dependent on ATP but not dependent on membrane potential. Although CEA could not mediate bile acid efflux in COS cells cotransfected with IBAT and CEA, efflux of [3H]taurocholate was detected in COS cells cotransfected with IBAT and a chimeric molecule having the carboxyl-terminal tail and membrane spanning domain of CBATP and the amino-terminal extracellular tail of CEA. Taken together, these data provide further evidence that CBATP confers bile acid efflux properties on heterologous cells and that its cytoplasmic tail and membrane spanning segment are integral to this property. The data also establish a model system for more clearly defining the molecular determinants of bile acid transport mediated by this molecule.

Adenosine Triphosphatases↗
PubMed1997

Review: alpha 1-antitrypsin deficiency associated liver disease.

alpha 1-Antitrypsin (alpha 1-AT) deficiency is the most common genetic cause of liver disease in children and genetic disease for which children undergo liver transplantation. It also causes cirrhosis and hepatocellular carcinoma in adults. Studies by Sveger in Sweden have shown that only a subgroup of the population with homozygous PiZZ alpha 1-AT deficiency develop clinically significant liver injury. Other studies have shown that the mutant alpha 1-AT Z molecule undergoes polymerization in the endoplasmic reticulum and that a subpopulation of alpha 1-AT-deficient individuals may be susceptible to liver injury because they also have a trait that reduces the efficiency by which the mutant alpha 1-AT Z molecule is degraded in the endoplasmic reticulum.

Adult↗
PubMed1997

Gene transfer into hepatoma cell lines via the serpin enzyme complex receptor.

The serpin enzyme complex receptor (SECR) expressed on hepatocytes binds to a conserved sequence in alpha 1-antitrypain (alpha 1-AT) and other serpins. A molecular conjugate consisting of a synthetic peptide (C1315) based on the SECR binding motif of human alpha 1-AT covalently coupled to poly-L-lysine was used to introduce reporter genes into hepatoma cell lines in culture. This conjugate condensed DNA into spheroidal particles 18-25 nm in diameter. When transfected with the SECR-directed complex containing pGL3, Hep G2 cells that express the receptor, but not Hep G2 cells that do not, expressed a peak luciferase activity of 538,731 +/- 144,346 integrated light units/mg protein 4 days after transfection. Free peptide inhibited uptake and expression in a dose-dependent manner. Complexes of DNA condensed with polylysine or LC-sulfo-N-succinimidyl-3-(2-pyridyldithio)propionate-substituted polylysine were ineffective. Transfection with a plasmid encoding human factor IX produced expression in Hep G2 (high) and HuH7 cells that express SECR but not Hep G2 (low) cells that lack the receptor. Fluorescein-labeled C1315 peptide labeled 9-31% of Hep G2 (high), 10-14% of HuH7, and 0.6-3.4% of Hep G2 (low) cells, and when the lac Z gene was transfected, only these cells expressed beta-galactosidase. SECR-mediated gene transfer gives efficient, specific uptake and high-level expression of three reporter genes, and the system merits further study for gene therapy.

Carcinoma, Hepatocellular↗
PubMed1996

Site-directed mutagenesis within an ectoplasmic ATPase consensus sequence abrogates the cell aggregating properties of the rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 and carcinoembryonic antigen.

Recent studies of the rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 (CBATP), a member of the carcinoembryonic antigen (CEA) supergene family, indicate that it is a multifunctional protein possessing bile acid efflux, ecto-ATPase, and intercellular aggregating properties. Cheung et al. (Cheung, P. H., Luo, W., Qiu, Y., Zhang, K. E., Millron, P., Lin, S. H. (1993) J. Biol. Chem. 268, 24303-24310) have shown that the amino-terminal Ig V-like domain of this protein is required for its aggregating properties, much like the homologous amino-terminal domain of CEA is required for its aggregating properties. The amino-terminal domains of both CBATP and CEA include a consensus ATPase sequence. Site-directed mutagenesis within this ATPase consensus sequence completely eliminates the ecto-ATPase activity of CBATP (Sippel, C. J., McCollum, M., Perlmutter, D. H. (1994) J. Biol. Chem. 269, 2820-2826). In this study we examined the possibility that it is this ATPase consensus sequence which is required for the cell aggregating properties of CBATP and CEA and whether there is a relationship between ATPase, aggregating, and bile acid efflux activities. For this we used a baculovirus vector to express in Sf9 cells wild type as well as mutant and chimeric CBATP and CEA molecules. The results indicate that Arg-98 in the ATPase consensus sequence of CBATP and the corresponding residue of CEA are essential for the aggregating properties of these molecules. Moreover Arg-98 is essential for CBATP to interact with itself, CEA to interact with itself, and CBATP to interact with CEA. However, the role of Arg-98 in aggregation is distinct from its role in ecto-ATPase activity and the aggregating properties cannot be attributed to a change in ATP metabolism in the pericellular milieu.

Adenosine Triphosphatases↗
PubMed1996

Degradation of a mutant secretory protein, alpha1-antitrypsin Z, in the endoplasmic reticulum requires proteasome activity.

Degradation of proteins that are retained in the quality control apparatus of the endoplasmic reticulum (ER) has been attributed to a third proteolytic system, distinct from the lysosomal and the cytoplasmic ubiquitin-dependent proteosomal proteolytic pathways. However, several recent studies have shown that ER degradation of a mutant membrane protein, CFTRdeltaF508, is at least in part mediated from the cytoplasmic side by the 26 S proteasome. In this study, we examined the possibility that ER degradation of mutant secretory protein alpha1-antitrypsin (alpha1-AT) Z, the mutant protein associated with infantile liver disease and adult-onset emphysema of alpha1-AT deficiency, is mediated by the proteasome. The results show that a specific proteasome inhibitor, lactacystin, inhibits ER degradation of alpha1-ATZ in transfected human fibroblast cell lines and in a cell-free microsomal translocation system. Although it is relatively easy to conceptualize how a transmembrane protein like CFTRDeltaF508 might be accessible on the cytoplasmic aspect of the ER membrane for ubiquitination and degradation by the proteasome, it is more difficult to conceptualize how this might occur for a luminal polypeptide. The results show that, once within the lumen of the ER, alpha1-ATZ interacts with the transmembrane molecular chaperone calnexin and specifically induces the polyubiquitination of calnexin. The results, therefore, provide evidence that the proteasome, from its cytoplasmic localization, induces the degradation of the luminal alpha1-ATZ molecule by first attacking the cytoplasmic tail of calnexin molecules that are associated with alpha1-ATZ.

Acetylcysteine↗
PubMed1996

The serpin-enzyme complex receptor recognizes soluble, nontoxic amyloid-beta peptide but not aggregated, cytotoxic amyloid-beta peptide.

There is now extensive evidence that amyloid-beta peptide is toxic to neurons and that its cytotoxic effects can be attributed to a domain corresponding to amyloid-beta 25-35, GSNKGAIIGLM. We have shown recently that the serine proteinase inhibitor (serpin)-enzyme complex receptor (SEC-R), a receptor initially identified for binding of alpha1-antitrypsin (alpha1-AT) and other serine protease inhibitors, also recognizes the amyloid-beta 25-35 domain. In fact, by recognizing the amyloid-beta 25-35 domain, SEC-R mediates cell surface binding, internalization, and degradation of soluble amyloid-beta peptide. In this study, we examined the possibility that SEC-R mediates the neurotoxic effect of amyloid-beta peptide. A series of peptides based on the sequences of amyloid-beta peptide and alpha1-AT was prepared soluble in dimethyl sulfoxide or insoluble in water and examined in assays for SEC-R binding, for cytotoxicity in neuronal PC12 cells and murine cortical neurons in primary culture, and for aggregation in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results show that amyloid-beta peptide 25-35 and amyloid-beta peptide 1-40 prepared soluble in dimethyl sulfoxide compete for binding to SEC-R, are nontoxic, and migrate as monomers in SDS-PAGE analysis. In contrast, the same peptides aged in water did not compete for binding to SEC-R but were toxic and migrated as aggregates in SDS-PAGE. An all-D-amyloid-beta 25-35 peptide was not recognized at all by SEC-R but retained full toxic/aggregating properties. Using a series of deleted, substituted, and chimeric ambeta/alpha1-AT peptides, toxicity correlated well with aggregation but poorly with SEC-R recognition. In a subclone of PC12 cells which developed resistance to the toxic effect of aggregated amyloid-beta 25-35 there was a 2.5-3-fold increase in the number of SEC-R molecules/cell compared with the parent PC12 cell line. These data show that SEC-R does not mediate the cytotoxic effect of aggregated amyloid-beta peptide. Rather, SEC-R could play a protective role by mediating clearance and catabolism of soluble, monomeric amyloid-beta peptide, if soluble amyloid-beta peptide proves to be an in vivo precursor of the insoluble, toxic peptide.

Amino Acid Sequence↗
PubMed1996

The endoplasmic reticulum degradation pathway for mutant secretory proteins alpha1-antitrypsin Z and S is distinct from that for an unassembled membrane protein.

We have theorized that a subset of PiZZ alpha1-antitrypsin (alpha1-AT)-deficient individuals is more susceptible to liver injury by virtue of second inherited trait(s) or environmental factor(s), which exaggerate the accumulation of mutant alpha1-AT Z within the endoplasmic reticulum (ER) of liver cells. Using a complementation approach in which cell lines from PiZZ individuals with liver disease ("susceptible" hosts) and from PiZZ individuals without liver disease ("protected" hosts) are transduced with the mutant alpha1-AT Z gene, we have recently shown that there is a delay in ER degradation of mutant alpha1-AT Z protein that is only present in cell lines from susceptible hosts and correlates with the liver disease phenotype. In the present study we examined the specificity of this ER degradation pathway to determine if it is responsible for degrading other misfolded mutants of alpha1-AT and/or for unassembled membrane proteins. The S mutant of alpha1-AT and H2a subunit of the asialoglycoprotein receptor (ASGPR H2a) were expressed in skin fibroblast cell lines from susceptible and protected hosts. The results showed in both susceptible and protected hosts that alpha1-AT S was associated with a delay in secretion as compared with wild type alpha1-AT. The alpha1-AT S mutant was retained in ER, albeit to a lesser extent than the alpha1-AT Z mutant. There was, however, a significant increase in retention of alpha1-AT S in the ER of susceptible as compared with protected host cells. The same host cell lines were transduced to express an unassembled membrane protein, ASGPR H2a. There was no difference in the kinetics of ER degradation of ASGPR H2a in susceptible as compared with protected hosts. Taken together, the results show that alpha1-AT S is associated with a defect in biogenesis, intracellular retention, which is similar to but milder than alpha1-AT Z. Like alpha1-AT Z, alpha1-AT S is degraded by a pathway in the ER, which is relatively inefficient in PiZZ individuals with the liver disease phenotype. However, this pathway appears to be different from that previously described for a model unassembled membrane protein.

Cell Line↗
PubMed1996

Regulation of sucrase-isomaltase gene expression in human intestinal epithelial cells by inflammatory cytokines.

Using metabolic labeling techniques in human intestinal epithelial cell lines in tissue culture and in situ hybridization techniques in normal and inflamed (Crohn's) intestine, recent studies have shown that there is synthesis of acute phase proteins in enterocytes. Moreover, these studies have shown that acute phase protein biosynthesis in enterocytes is regulated by inflammatory cytokines in a manner characteristic of the physiologic acute phase response. In the course of these studies it was noticed that one inflammatory cytokine, interleukin-6 (IL-6), mediated selective down-regulation of the enterocyte-specific, differentiation-dependent integral membrane protein sucrase-isomaltase (SI) in the Caco2 intestinal epithelial cell line. In the current study we examined the effect of several other inflammatory cytokines interleukin-1 (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and interferon gamma (IFN gamma) on synthesis of SI in Caco2 cells, examined the possibility that inflammatory cytokines affect the synthesis of other enterocyte integral membrane proteins using lactase as a prototype, and examined the possibility that SI gene expression was down-regulated in villous enterocytes in vivo during the local inflammatory response of Crohn's disease. The results show that IL-6 and IFN gamma each mediate a decrease and TNF alpha mediates an increase in synthesis of SI in Caco2 cells. The magnitude of down-regulation by IL-6 and IFN gamma is significantly greater than the up-regulation by TNF alpha. IL-1 beta has no effect on synthesis of SI. Synthesis of lactase is not affected by any of the cytokines. There is a marked specific decrease in SI gene expression in villous enterocytes in acutely inflamed Crohn's ileum as compared to adjacent uninflamed ileum and normal ileum. Taken together, these data show that inflammatory cytokines have specific and selective effects on the expression of the brush border hydrolase SI in tissue culture and in vivo and provide evidence for a previously unrecognized mechanism for disaccharidase deficiency in intestinal inflammation.

Cell Line↗
PubMed1996

Biliary atresia: current concepts and research directions. Summary of a symposium.

Biliary atresia (BA) is the end result of a destructive, inflammatory process that affects intra- and extrahepatic bile ducts, leading to fibrosis and obliteration of the biliary tract with the development of biliary cirrhosis. It is the commonest cause of chronic cholestasis in infants and children, and therefore is the most frequent indication for liver transplantation in this age group. The disease occurs worldwide, affecting an estimated 1 in 8,000 to 12,000 live births. At present, there is no specific therapy for BA; however, sequential surgical therapy begins with creation of a hepatoportoenterostomy (HPE); in those with end-stage liver disease, liver transplantation is indicated. Since most candidates are young children of small size, there is a shortage of size-matched donors for liver transplantation. At present, an increased awareness to ensure early diagnosis and development of methods to prevent progressive fibrosis are needed. These considerations are dependent on detailed studies of the pathogenesis of BA. Recent studies have focused on normal and altered bile duct morphogenesis and the role of various factors (infectious or toxic agents and metabolic insults) in isolation or in combination with a genetic or immunologic susceptibility in the etiology of BA.

Biliary Atresia↗
PubMed1996

Alpha-1-antitrypsin deficiency: biochemistry and clinical manifestations.

Alpha-1-antitrypsin (alpha 1-AT) deficiency is a well known cause of emphysema in adults. A subgroup of deficient individuals develops liver injury during infancy and childhood. In fact, it is the most common genetic cause of liver disease in children. Although lung injury is due to the decrease in alpha 1-AT function in the lung, allowing uninhibited elastolytic destruction of its connective tissue integrity, liver injury is probably due to retention of the mutant alpha 1-AT molecule in the endoplasmic reticulum (ER) of liver cells. Recent studies have shown that the mutant alpha 1-AT molecule polymerizes in the ER by a novel loop-sheet insertion mechanism. Other recent studies show that the subgroup of deficient individuals is susceptible to liver injury by virtue of unlinked genetic traits and/or environmental factors which interfere with degradation of the mutant alpha 1-AT molecules within the ER.

Adult↗
PubMed1995

Specificity in recognition of amyloid-beta peptide by the serpin-enzyme complex receptor in hepatoma cells and neuronal cells.

The serpin-enzyme complex (SEC) receptor was originally identified using a synthetic peptide (peptide 105Y) based on the sequence of a candidate receptor-binding domain of alpha 1-antitrypsin (1-AT) and was subsequently shown to be a receptor on the surface of hepatocytes, monocytes, and neutrophils for recognition of alpha 1-AT-elastase and several other serpin-enzyme complexes (Perlmutter, D. H., Glover, G. I., Rivetna, M., Schasteen, C. S., and Fallon, R.J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3753-3757). Studies of the minimal requirements for binding to SEC receptor (SEC-R) showed that a pentapeptide FVFLM within the carboxyl-terminal tail of alpha 1-AT was sufficient for binding to SEC-R and interacted with SEC-R in a sequence-specific manner (Joslin, G., Krause, J. E., Hershey, A. D., Adams, S. P., Fallon, R. J., and Perlmutter, D. H. (1991) J. Biol. Chem. 266, 21897-21902). Sequence motifs bearing homology with this pentapeptide domain were found in the amyloid-beta peptide, and amyloid-beta peptide 1-42 was shown to compete for binding to SEC-R on hepatoma cells (Joslin, G., Fallon, R. J., Bullock, J., Adams, S. P., and Perlmutter, D. H. (1991) J. Biol. Chem, 266, 11281-11288). In this study we examined the sequence specificity by which amyloid-beta peptide competes for binding to SEC-R and examined the possibility that SEC-R is expressed in cells of neuronal origin. The results show that amyloid-beta-(25-35) and amyloid-beta-(31-35) compete for binding to SEC-R as effectively as amyloid-beta-(1-39), amyloid-beta-(1-40), and amyloid-beta-(1-42). Amyloid-beta-(1-16) does not compete for binding to SEC-R. There is cross-competition for binding to the same site by 125I-peptide 105Y and amyloid-beta-(25-35) as well as by 125I-Y amyloid-beta-(25-35) and peptide 105Y. By deletions and substitutions within amyloid-beta-(25-35) and generation of chimeric amyloid-beta-alpha 1-AT peptides, amyloid-beta-(31-35) is shown to be critical for binding to the SEC receptor. However, the upstream region, amyloid-beta-(25-30), also contributes to recognition by SEC-R. The SEC-R is present on the surface of a neuronal cell line PC12 as well as that of murine cortical neurons in primary culture, and the specificity of neuronal SEC-R for amyloid-beta peptide is identical to that on hepatoma cells. Finally SEC-R mediates internalization and degradation of amyloid beta-peptide in PC12 cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1995

N-formylpeptide and complement C5a receptors are expressed in liver cells and mediate hepatic acute phase gene regulation.

Although the classical chemotactic receptor for complement anaphylatoxin C5a has been associated with polymorphonuclear and mononuclear phagocytes, several recent studies have indicated that this receptor is expressed on nonmyeloid cells including human endothelial cells, vascular smooth muscle cells, bronchial and alveolar epithelial cells, hepatocytes, and in the human hepatoma cell line HepG2. In this study, we examined the possibility that other members of the chemotactic receptor family are expressed in HepG2 cells and human liver, and the possibility that such receptors mediate changes in acute phase gene expression in HepG2 cells. Using polymerase chain reaction (PCR) amplification of HepG2 mRNA with primers based on highly conserved regions of the chemotactic subgroup of the G protein-coupled receptor family, we identified a PCR fragment from the formyl-methionyl-leucyl-phenylalanine (FMLP) receptor, as well as one from the C5a receptor. Immunostaining with antipeptide antisera to FMLPR confirmed the presence of this receptor in HepG2 cells. Receptor binding studies showed specific saturable binding of a radioiodinated FMLP analogue to HepG2 cells (Kd approximately 2.47 nM; R approximately 6 x 10(3) plasma membrane receptors per cell). In situ hybridization analysis showed the presence of FMLPR mRNA in parenchymal cells of the human liver in vivo. Both C5a and FMLP mediated concentration- and time-dependent changes in synthesis of acute phase proteins in HepG2 cells including increases in complement C3, factor B, and alpha 1-antichymotrypsin, as well as concomitant decreases in albumin and transferrin synthesis. The effects of C5a and FMLP on the synthesis of these acute phase proteins was evident at concentrations as low as 1 nM, and they were specifically blocked by antipeptide antisera for the corresponding receptor. In contrast to the effect of other mediators of hepatic acute phase gene regulation, such as interleukin 6, the effects of C5a and FMLP were reversed by increased concentrations well above the saturation point of the respective receptor. These results suggest that acute phase gene regulation by C5a and FMLP is desensitized at high concentrations, a property that is unique among the several known mechanisms for hepatic acute phase gene regulation.

Acute-Phase Proteins↗
PubMed1995

Cellular expression of the C5a anaphylatoxin receptor (C5aR): demonstration of C5aR on nonmyeloid cells of the liver and lung.

The small-complement C5 activation fragment, C5a, is a potent phlogistic molecule that, on binding to the C5a Receptor (C5aR), mediates contraction of smooth muscle, enhances vascular permeability, and promotes leukocyte functions such as directed chemotaxis, degranulation, mediator release, and production of superoxide anions. Although C5aR expression has traditionally been thought to be limited primarily to myeloid blood cells, including neutrophils, monocytes, macrophages, and eosinophils, we report here that C5aR is expressed by liver and lung cells as well as by cells in several other tissues. By Northern blot analysis, it was determined that mouse liver, baboon liver, human liver, and the human hepatoma-derived cell line HepG2 express a normal size (2.3 kb) C5aR mRNA; in HepG2 cells, the quantity of C5aR mRNA was comparable to that contained in dbcAMP-differentiated U937 cells. HepG2 cells were demonstrated to express the C5aR on their cell surface by flow cytometric and immunofluorescence analyses as well as by 125I-C5a binding assays. The binding data indicated that HepG2 cells express a single class of C5aR with a Kd of 1.18 nM and approximately 28,000 receptors per cell. In vivo expression of C5aR in human liver cells was demonstrated by in situ hybridization and immunohistochemistry analyses. Northern blot analysis of murine and baboon organs shows that, in addition to the liver, other tissues express C5aR mRNA in significant quantities, including the spleen, lung, heart, kidney, and intestine. Moreover, mice treated with LPS show a large increase in C5aR mRNA in all these tissues except the intestine. Immunostaining of human lung tissue demonstrated that bronchial and alveolar epithelial cells, as well as vascular smooth muscle and endothelial cells, also express the C5aR. Collectively, these data indicate that the C5aR is expressed in several different types of cells in liver and lung, and in yet undetermined cell types in spleen, heart, intestine, and kidney. Furthermore, these data suggest that the C5a anaphylatoxin mediates previously unrecognized functions by binding to tissue cells that express the C5aR.

Amino Acid Sequence↗