Expression and induction of anaphylatoxin C5a receptors in the rat liver.

The C5a-anaphylatoxin which is generated by limited proteolysis upon activation of the fifth component of complement may be induced by the classical, the alternative or the lectin pathway. C5a has been shown, under normal conditions, to induce the release of prostanoids from Kupffer cells (KC) and hepatic stellate cells (HSC) and thereby indirectly to increase glucose output from hepatocytes (HC). A direct action of C5a on HC would require the expression of the specific C5a receptor (C5aR). In studies using quantitative RT-PCR it was shown that non-stimulated HC lack C5aR, in contrast to KC, HSC and sinusoidal endothelial cells (SEC) all of which contained mRNA for the C5aR in decreasing amounts. FACS analyses, immunohisto- and immunocytochemistry as well as functional analyses confirmed the results of the RT-PCR assays. Under inflammatory situations the C5aR was found to be upregulated in various organs and tissues which included the liver. Interleukin-6 (IL-6) as a main inflammatory mediator in the liver induced a de novo expression of functional C5aR in HC in-vitro and in-vivo. In contrast, LPS failed to induce C5aR directly in cultured HC in-vitro but induced C5aR in HC in vivo and in co-cultures of HC and KC which release IL-6 upon stimulation with LPS. So far, the only known effector function of C5a on HSC was the induction of prostanoid release. In an approach to reveal new functions of C5aR in HSC, the cells responsible for liver fibrosis, it could be shown that C5a upregulated fibronectin-specific mRNA five-fold whereas entactin, collagen IV and the structure protein smooth muscle actin were not affected. In addition, C5a did not upregulate specific mRNA for the profibrotic cytokine TGF-beta1 in either isolated KC or HSC. Thus, C5a alone appears to have only a limited role in the induction of liver fibrosis.

Analysis of the tissue distribution of the rat C5a receptor and inhibition of C5a-mediated effects through the use of two MoAbs.

The C5-anaphylatoxin C5a is a protein of 74 (human) or 77 (rat) amino-acid residues, respectively, the generation of which may be induced by either the classical and/or the alternative pathways. C5a binds specifically to its receptor (C5aR/CD88) which belongs to the superfamily of G-protein-coupled receptors with seven transmembrane segments. In this study we describe the tissue distribution of the rat C5aR (rC5aR) and the blocking of its ligand by the application of two monoclonal antibodies (MoAbs). The first antibody (MoAb R63) which is directed against the amino-terminal domain Ex1 of the rat C5aR was generated in mice immunized with RBL-2H3 cells which had been stably transfected with the rat C5a receptor gene. Checking the rC5aR expression in various tissues bronchial epithelial cells stained positive only in tissue samples from animals with a mycoplasm infection indicating that the receptor may be induced in this cell type as a consequence of an inflammatory process. Using immunohistochemistry there was no evidence for nonmyeloid expression in the large or small intestine, heart, lung, kidney or liver of the normal rat. The MoAb R63 was found to be a reliable tool for the investigation of the expression of the receptor by FACS analyses or immunohistochemistry. Despite numerous attempts neutralizing antibodies could not be generated against the receptor. Therefore a C5a-ligand neutralizing MoAb was generated against the synthesized carboxyterminal 20mer peptide. This antibody (6-9F) recognized the carboxy terminus of C5a/C5a-FLUOS and prevented its binding at a three-fold molar excess as evidenced by FACS-analyses. It also blocked the C5a-mediated signal transduction as demonstrated by the inhibition of intracellular Ca2+-release (at a 16-fold molar excess) and the release of N-Acetyl-beta-D-glucosaminidase (at a 25-fold molar excess).

Rat complement factor I: molecular cloning, sequencing and expression in tissues and isolated cells.

Factor I (FI) is a regulatory serine protease of the complement system which cleaves three peptide bonds in the alpha-chain of C3b and two bonds in the alpha-chain of C4b thereby inactivating these proteins. The human protein and the recently characterized mouse factor I are heterodimers of about 88,000 MW which consist of a non-catalytic heavy chain of 50,000 MW which is linked to a catalytic light chain of 38,000 MW by a disulphide bond. For the screening of a rat liver cDNA library we used a hybridization probe produced by polymerase chain reaction (PCR) using degenerated primers which corresponded to conserved parts of the human and the murine factor I nucleotide sequences. One of the identified sequences, which had a length of 2243 base pairs (bp), contained the complete coding region and the whole 3' untranslated region. The length of the coding region in rat consisted of 1812 bp followed by a 3' untranslated region of 207 bp including the polyadenylation signal and the beginning of the poly A tail. Comparison of the rat cDNA-derived coding sequence revealed identities of 87% to the mouse and of 78% to the human FI nucleotide sequence. The translation product of rat FI mRNA was 604 amino acid residues (aa) in length with an identity of 85% to the mouse (603 aa) and 69% to the human protein (583 aa). The comparison of the molecular mass predicted by the primary structure and derived from rat FI isolated from rat serum as detected in immunoblot analyses suggested a glycosylation of more than 20% of the total mass of the FI protein. Expression studies using reverse transcription (RT)-PCR assays indicated that FI-specific mRNA could neither be identified in B cells, nor in T cells, monocytes or granulocytes from rat and human peripheral blood nor in rat peritoneal macrophages. These data were in agreement with the results of RT-PCR obtained with several human lymphoma cell lines (Jurkat, MOLT-4, HUT102, Wil 2-NS, Ramos, Raji, U937) all of which were devoid of FI-specific mRNA. In accord with our data from two rat hepatoma cell lines (FAO and H4IIE) and one from man (HepG2) only isolated rat hepatocytes (HC) but neither Kupffer cells (KC), hepatic stellate cells (HSC; Ito cells) nor sinusoidal endothelial cells (SEC) expressed FI-specific mRNA. FI mRNA was also detected in human umbilical vein endothelial cells (HUVEC) and in the uterus and small intestine of the rat. Spleen and lymph nodes did not contain any detectable FI-specific mRNA.

Differential expression of the C5a receptor on the main cell types of rat liver as demonstrated with a novel monoclonal antibody and by C5a anaphylatoxin-induced Ca2+ release.

The C5-anaphylatoxin (C5a) is a protein of 74 (human) or 77 (rat) amino acid residues, respectively, which is generated by limited proteolysis upon activation of the fifth component of complement. Its generation may be induced by both the classical and alternative pathways. C5a has been shown to indirectly increase glucose output from hepatocytes (HC) in perfused rat liver by inducing prostanoid release from Kupffer cells (KC) and hepatic stellate cells (HSC). A direct action of C5a on hepatocytes would require their expression of the specific C5a receptor (C5aR). In former studies using quantitative reverse transcription polymerase chain reaction (RT-PCR) it was shown that HC lack this receptor in contrast to KC, HSC and, probably, sinusoidal endothelial cells (SEC), all of which contained mRNA for the C5aR in decreasing amounts. Using a novel monoclonal antibody (mAb R63) against the rat receptor, expression of the rat receptor on the four cell types was investigated by FACS analysis, immunohistochemistry, and immunocytochemistry. The data obtained were confirmed by functional studies in which the Ca2+ response after stimulation of the isolated cells with recombinant rat C5a (rrC5a), the ligand for the receptor was recorded. The FACS and the immunocytochemical data presented here clearly indicate that rat HC do not express the C5aR, whereas KC have the highest expression level followed by HSC. SEC expressed the receptor only weakly. In line with these findings, a strong Ca2+ response was observed after stimulation of KC and HSC, and a weak one with SEC. However, no signal was obtained upon stimulation of HC. The results of this study support the indirect stimulation of glucose output from HC via prostanoid release from nonparenchymal liver cells and contradict the formerly proposed hypothesis of a direct action of C5 anaphylatoxin on hepatocytes.

Stimulation by anaphylatoxin C5a of glycogen phosphorylase in rat hepatocytes via prostanoid release from hepatic stellate cells but not sinusoidal endothelial cells.

In the perfused rat liver, the anaphylatoxin C5a has been shown to enhance glucose output. Since hepatocytes lack C5a receptor mRNA, the metabolic effect of C5a must be elicited indirectly via C5a receptor expressing non-parenchymal liver cells. Kupffer cells were found to be able to mediate the C5a action via release of prostanoids. However, elimination of the Kupffer cells by pretreatment of the animals with gadolinium chloride reduced the metabolic effect of C5a to only about 40%. Therefore, it was investigated whether not only Kupffer cells but in addition also hepatic stellate cells or sinusoidal endothelial cells released prostanoids in response to C5a. In isolated hepatic stellate cells but not in sinusoidal endothelial cells, recombinant rat C5a induced a time- and dose-dependent release of thromboxane B2 and prostaglandins D2, E2 and F2alpha. The rate of prostanoid release was maximal within the first two minutes and then declined again. C5a-induced prostanoid release from hepatic stellate cells was smaller than that from Kupffer cells and it differed in the prostanoid ratios (PGE2/PGD2/PGF2alpha/TXB2 = 1:1:0.1:0.6 and 1:4:1:3, respectively). RrC5a activated hepatocellular glycogen phosphorylase via prostanoid release in cocultures of hepatocytes with hepatic stellate cells but not with sinusoidal endothelial cells. Thus, the part of the rrC5a-induced glucose output in the perfused rat liver, which was not abrogated by elimination of the Kupffer cells with gadolinium chloride, most likely was mediated by prostanoids released from hepatic stellate cells.

Molecular cloning and expression of the functional rat C5a receptor.

The C5a receptor belongs to the superfamily of G-protein coupled receptors with seven transmembrane segments. In this study we report on the cloning of the rat C5a receptor (ratC5aR). We used a hybridization probe produced by PCR utilizing degenerate primers which corresponded to conserved parts of the human, canine and murine C5a receptor nucleotide sequences and to the published partial amino acid sequence of the rat C5a receptor to screen a rat macrophage cDNA library. We found two overlapping clones containing an open reading frame of 1056 bp, a 3'untranslated region of 683 bp and a 5'untranslated region of 27 bp. The overall nucleotide acid sequence identity, compared to the murine, human and canine C5a receptor sequences, was 85.8, 70.5 and 68.9%, respectively. The greatest diversity exists in the putative extracellular domains, especially in the aminoterminal domain which is assumed to be involved in ligand binding. An N-glycosylation site is present within the N-terminal sequence at residue 6. One of the cDNA containing the 5'untranslated region, the coding sequence and part of the 3'untranslated region was cloned into an eucaryotic expression vector and stably transfected into the rat basophilic leukemia cell line RBL-2H3. Expression of the rat C5a receptor on the surface of these cells could be demonstrated by flow cytometric analysis using FITC-labeled recombinant rat C5a (rrC5a). By measuring the release of calcium from intracellular stores after stimulation with rrC5a it could further be shown that the receptor is functionally coupled. Receptor binding assays showed that rrC5a specifically binds to the ratC5aR with a KD of 0.91 +/- 0.36 and to the human C5a receptor (huC5aR) with a KD of 7.19 +/- 1.56. The determined KD for binding of human C5a (huC5a) to the huCSaR was 2.16 +/- 0.65. No binding of huC5a to the ratC5aR could be observed although high concentrations of this ligand (> 60 nM) promoted chemotaxis of RBL cells transfected with the huC5aR.

Anaphylatoxin C5a receptor mRNA is strongly expressed in Kupffer and stellate cells and weakly in sinusoidal endothelial cells but not in hepatocytes of normal rat liver.

Anaphylatoxins (C5a and C3a), which are generated during complement activation, have recently been shown to increase glucose output from hepatocytes (HC) in perfused rat liver. They did not act directly on HC but indirectly by prostanoid release from non-parenchymal cells (NPC), probably Kupffer cells (KC). In order to corroborate this mechanism, the distribution of anaphylatoxin receptors in the different cell types of rat liver was determined by quantitative RT-PCR with primers specific for the rat C5a receptor (rC5aR) using RNA isolated from KC, sinusoidal endothelial cells (SEC), hepatic stellate cells (HSC) and HC. In line with functional data, C5aR mRNA was detected in freshly isolated NPC but not in HC of rat liver. Mainly KC but also HSC clearly expressed C5aR mRNA, while SEC did so only weakly. KC expressed up to 10-fold more C5aR mRNA than HSC and these in turn up to 10-fold more than SEC. These results support the proposed indirect action of anaphylatoxins on HC.

Nucleotide and corrected amino acid sequence of the functional recombinant rat anaphylatoxin C5a.

For bacterial expression of rat anaphylatoxin C5a, the cDNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using rat liver RNA and degenerate primers designed according to the published amino acid sequence [1]. Surprisingly, the amino acid sequence deduced from cDNA differed at positions 55 (N for K), 63 (K for H), 67 (E for N), 68 (S for E) and 69 (H for S) from the published sequence. The overall amino acid composition, however, was unchanged because these 5 amino acids were located at different positions compared to the published sequence. As a consequence, the proposed N-glycosylation site was absent, suggesting O-glycosylation of the mature molecule. Recombinant rat C5a with a 6 histidine tag at the N-terminus was expressed in bacteria, purified and renatured. The peptide was as potent as recombinant human C5a in eliciting lysosomal enzyme release from human granulocytes.

Inhibition of anaphylatoxin C3a- and C5a- but not nerve stimulation- or Noradrenaline-dependent increase in glucose output and reduction of flow in Kupffer cell-depleted perfused rat livers.

In isolated in situ perfused rat livers, infusion of anaphylatoxins C3a and C5a, activation peptides of the complement system, as well as stimulation of sympathetic hepatic nerves have been shown to increase hepatic glucose output and to reduce hepatic flow. These effects were mediated via an at least partially prostanoid-dependent intercellular signalling chain between nonparenchymal cells and hepatocytes. Kupffer cells have been implicated as the source of prostanoids in the anaphylatoxin-dependent signalling chain and Ito cells in the nerve stimulation-dependent signalling chain, because anaphylatoxins and noradrenaline increased prostanoid synthesis in isolated Kupffer and Ito cells, respectively. To further corroborate this hypothesis, anaphylatoxins were infused and hepatic nerves were stimulated in perfused rat livers in which Kupffer cells had been largely depleted by treatment of the animals with gadolinium chloride. Native human anaphylatoxin C3a (nhC3a) and recombinant rat anaphylatoxin C5a (rrC5a) increased prostanoid formation as well as glucose output and reduced flow in perfused rat liver. In Kupffer cell-depleted rat livers, the nhC3a- and rrC5a-mediated prostanoid formation was nearly abolished, and the increase in glucose output and the reduction of flow were reduced to between 30% and 50% (area under the curve [AUC]) of control livers. In contrast, stimulation of hepatic nerves increased glucose output and reduced flow to a similar extent in Kupffer cell-depleted livers as in control livers. These results indicate that Kupffer cells were not involved in the prostanoid-mediated nerve stimulation-dependent increase in glucose output and reduction of flow. Kupffer cells seemed, however, to be at least one major source of the anaphylatoxin-mediated prostanoid formation and, consequently, stimulation of glucose release and flow reduction in perfused liver. Because the metabolic and hemodynamic anaphylatoxin effects were not completely blocked in livers of gadolinium-treated animals, either Kupffer cells may not have been entirely eliminated, or yet another nonparenchymal cell type and mediator might be involved in the anaphylatoxin-elicited intercellular communication between nonparenchymal cells and hepatocytes.

Characterization and mapping of a highly conserved processed pseudogene and an intron-carrying gene of the heat shock cognate protein 70 (Hsc70) gene family in the rat.

A processed pseudogene of the rat Hsc70 gene, Hsc70-ps1, is described, which still presents the open reading frame of the original gene. The pseudogene does not appear to be expressed. It maps to rat Chromosome (Chr) 2. The intron-carrying Hsc70 gene localizes to Chr 8. Hsc70-specific probes detect a large number of more than 20 cross-hybridizing fragments, which show only limited length polymorphism among various inbred rat strains.

Restriction fragment length polymorphism of the major histocompatibility complex-linked heat shock protein 70 (Hsp70) genes of the rat.

DNAs from inbred rat strains representing eighteen major histocompatibility complex (RT1) haplotypes have been tested for restriction fragment length polymorphisms of the three heat shock protein 70 (Hsp70) genes mapping into this complex. The DNAs were digested with restriction enzymes BamHI, EcoRI, PstI or PvuII and assayed by probes hybridizing to all three genes or selectively to Hsp70-1, Hsp70-2 and Hsp70-3, respectively. Three to four alleles were detected with each enzyme and in total five different strain distribution patterns could be found. Neither deletions nor amplifications of genes were detected in the three-gene Hsp70 cluster.

A novel, conserved gene of the rat that is developmentally regulated in the testis.

From a rat testis library three overlapping cDNA clones were isolated that represent a novel single-copy gene, designated Tegt. Two transcripts of 2.8 and 1.0 kb were found in each organ tested. The shorter transcript was highly abundant in adult testis. A similar expression pattern was found in the mouse. Analysis of rat RNA from different stages of spermatogenesis indicated that accumulation of the short transcript occurred mainly postmeiotically. The rat Tegt gene maps to Chromosome (Chr) 7, and its mouse homolog to Chr 15.

Characterization of a class Ib gene of the rat major histocompatibility complex.

The cDNA and a partial genomic sequence of a rat class I major histocompatibility (RT1) gene, 11/3R, is reported here. The sequence contains several unique amino acid residues at certain positions, mutations in exon 7 (which is not expressed), a mutation of the canonical exon 8 stop codon to a sense codon, and includes a long 3' untranslated region (utr). The structure of exon 7 differs from that found in most rat class I genes and resembles exon 7 of most H-2K,D,L,Q genes. Parts of the 3' noncoding region are homologous to the RT1.A-4 and certain H-2 genes. Expression is detectable by northern blot analysis in mitogen-stimulated lymphocytes only, by polymerase chain reaction (PCR) in each tissue tested. After transfection into L cells 11/3R can be shown to be expressible at the cell surface. Probes derived from the 3' noncoding part crosshybridize with a number of restriction fragments which map to the RT1.C region, thus defining a subfamily of RT1.C region genes. Several members of this subfamily are deleted in the lm1 RT1 mutant. The 11/3R gene presents typical features of a class Ib gene. Aspects of evolution and the potential function of the gene are discussed.

Genetic analysis of susceptibility to diabetes mellitus in F2-hybrids between diabetes-prone BB and various MHC-recombinant congenic rat strains.

The occurrence of diabetes mellitus was analysed in F2 hybrids bred from diabetic BB male rats and females of congenic rat strains carrying different major histocompatibility (RT1) haplotypes on the common LEW strain genetic background. Permissiveness for the disease in BB rats is probably determined by class II genes of the RT1 complex, which are also present in normal rats. Class I and class III genes, notably the Hsp70 genes in the class III region, do not appear to be involved. Among the diabetic F2 hybrids, about 90% are shown by DNA analysis to be homozygous for the class II genes of the permissive RT1 haplotype. The segregation patterns are in accord with the action of two independent recessive genes, one of them being RT1-linked.