Mouse B1 Research Articles

Ceramide Synthase Inhibition by Fumonisin B1 Treatment Activates Sphingolipid-Metabolizing Systems in Mouse Liver

Sphingolipids are important components of cell structure and cell signaling. Both external and internal stimuli can alter levels of cellular sphingolipids by regulating enzyme activities associated with sphingolipid metabolism. Fumonisin B1, mycotoxin produced by Fusarium verticillioides, is a reportedly specific inhibitor of ceramide synthase. In order to test our hypothesis whether ceramide synthase inhibition by fumonisin B1 alters other sphingolipid-metabolizing enzymes, we investigated the changes in free sphingoid bases and sphingomyelin (SM) and activities of key enzymes for their metabolism, sphingomyelinase (SMase), serine palmitoyltransferase (SPT), and sphingosine kinase (SPHK) in mouse liver. The hepatic free sphingoid bases increased significantly following five daily treatments with fumonisin B1 in mice. The activity of acidic SMase was enhanced by fumonisin B1, accompanied with a decrease in liver SM content. The expression and activities of SPT and SPHK1 in liver increased significantly following fumonisin B1 treatment. Another hepatotoxicant acetaminophen caused liver regeneration similar to fumonisin B1 but did not produce similar effects on liver sphingolipid-metabolizing enzymes, suggesting that activation of sphingolipid metabolism was not a consequence of hepatocyte regeneration. Data suggest that ceramide synthase inhibition by fumonisin B1 treatment stimulates sphingolipid-metabolizing systems to maintain a balance of cellular sphingolipids.

Alu elements contain many binding sites for transcription factors and may play a role in regulation of developmental processes

BackgroundThe human genome contains over one million Alu repeat elements whose distribution is not uniform. While metabolism-related genes were shown to be enriched with Alu, in structural genes Alu elements are under-represented. Such observations led researchers to suggest that Alu elements were involved in gene regulation and were selected to be present in some genes and absent from others. This hypothesis is gaining strength due to findings that indicate involvement of Alu elements in a variety of functions; for example, Alu sequences were found to contain several functional transcription factor (TF) binding sites (BSs). We performed a search for new putative BSs on Alu elements, using a database of Position Specific Score Matrices (PSSMs). We searched consensus Alu sequences as well as specific Alu elements that appear on the 5 Kbp regions upstream to the transcription start site (TSS) of about 14000 genes.ResultsWe found that the upstream regions of the TSS are enriched with Alu elements, and the Alu consensus sequences contain dozens of putative BSs for TFs. Hence several TFs have Alu-associated BSs upstream of the TSS of many genes. For several TFs most of the putative BSs reside on Alu; a few of these were previously found and their association with Alu was also reported. In four cases the fact that the identified BSs resided on Alu went unnoticed, and we report this association for the first time. We found dozens of new putative BSs. Interestingly, many of the corresponding TFs are associated with early markers of development, even though the upstream regions of development-related genes are Alu-poor, compared with translational and protein biosynthesis related genes, which are Alu-rich. Finally, we found a correlation between the mouse B1 and human Alu densities within the corresponding upstream regions of orthologous genes.ConclusionWe propose that evolution used transposable elements to insert TF binding motifs into promoter regions. We observed enrichment of biosynthesis genes with Alu-associated BSs of developmental TFs. Since development and cell proliferation (of which biosynthesis is an essential component) were proposed to be opposing processes, these TFs possibly play inhibitory roles, suppressing proliferation during differentiation.

Antinociceptive Pharmacology ofN-[[4-(4,5-Dihydro-1H-imidazol-2-yl)phenyl]methyl]-2-[2-[[(4-methoxy-2,6-dimethylphenyl) sulfonyl]methylamino]ethoxy]-N-methylacetamide, Fumarate (LF22-0542), a Novel Nonpeptidic Bradykinin B1Receptor Antagonist

The antinociceptive pharmacology of N-[[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]methyl]-2-[2-[[(4-methoxy-2,6-dimethylphenyl) sulfonyl]methylamino]ethoxy]-N-methylacetamide fumarate (LF22-0542), a novel nonpeptidic B1 antagonist, was characterized. LF22-0542 showed high affinity for human and mouse B1 receptors with virtually no affinity for the human B2 receptor; a selectivity index of at least 4000 times was obtained when LF22-0542 was profiled throughout binding or cell biology assays on 64 other G-protein-coupled receptor, 10 ion channels, and seven enzymes. LF22-0542 was a competitive B1 receptor antagonist and elicited significant antinociceptive actions in the mouse acetic acid-induced writhing assay, as well as in the second phases of formalin-induced nociception in mice and in both the first and second phases of the formalin response in rats. LF22-0542 was active after s.c. but not p.o. administration. In B1 receptor knockout (KO) mice, acetic acid and formalin responses were significantly reduced and LF22-0542 had no additional effects in these animals. LF22-0542 alleviated thermal hypersensitivity in both acute (carrageenan) and persistent inflammatory (complete Freund's adjuvant) pain models in rats. LF22-0542 produced a full reversal of experimental neuropathic thermal hypersensitivity but was inactive in reversing nerve injury-induced tactile hypersensitivity in rats. In agreement with this observation, B1 KO mice subjected to peripheral nerve injury did not show thermal hypersensitivity but developed nerve injury-induced tactile hypersensitivity normally. The data demonstrate the antihyperalgesic actions of a selective systemically administered B1 receptor antagonist and suggest the utility of this class of agents for the treatment of inflammatory pain states and for some aspects of neuropathic pain.

Further evidence for recombination between mouse hemoglobin beta b1 and b2 genes based on the nucleotide sequences of intron, UTR, and intergenic spacer regions

Nucleotide sequences of the intron regions and UTRs (Untranslated regions) of the hemoglobin beta adult genes, b1 and b2, and of the intergenic spacer region were determined for mouse strains representing the d, p, and w1 hemoglobin haplotypes defined by protein electrophoretic analyses. The hypothesis of recombination of the b1 and b2 genes between the d and w1 haplotypes previously reported in the cDNA nucleotide sequences was confirmed by neighbor-joining analyses of the intron regions and UTRs within the b1 and b2 genes, suggesting that all of the structures of hemoglobin beta adult genes support the hypothesis that the p haplotype was established by hybridization between d and w1 haplotype mice. The resultant recombinant of the p haplotype was found to have a d-like b1 gene and a w1-like b2 gene. In addition to the possible recombination, a break point was suggested around 2-3 kb downstream of the b1 gene within the intergenic spacer region, despite the absence of clear properties that could stimulate the recombination machinery. Some large insertions or deletions (indels) specific to the p or d haplotypes were located within the intergenic spacer region, in which the 1010-bp indel specific to the p haplotype was shared by all examined strains representing the p haplotype.

Skin tumorigenic potential of aflatoxin B1 in mice

Aflatoxin B1 (AFB1) has been classified as a category I human carcinogen, which is responsible for a high incidence of hepatocellular carcinoma. Since exposure to AFB1 can occur through skin contact in addition to ingestion and inhalation, the carcinogenic potential of topically applied AFB1 on mouse skin was investigated. Our results show that single topical application of AFB1 (80 nmol) as a tumor initiator, followed by twice weekly application of 12-tetradecanoyl phorbol myristate acetate (TPA, 4 nmol), resulted in tumor formation after 13 weeks. However, no tumorigenic potential was observed when AFB1 (16 nmol) was used either as a complete carcinogen or as a tumor promoter (4 nmol). Histological analysis of skin showed squamous cell carcinoma in the AFB1/TPA treated group. The application of AFB1 as a complete carcinogen, an initiator or a promoter after 24 weeks demonstrated widespread degenerative and necrotic changes in hepatic tissue as well, suggesting liver to be the target organ following percutaneous absorption. Additionally, twice weekly topical application of AFB1 caused significant induction of cutaneous CYP IA monoxygenases without any effect on hepatic levels while glutathione-S-transferase activity was induced more in the liver than skin. The topical application of AFB1 also resulted in increased hepatic and cutaneous lipid peroxidation with concomitant depletion of glutathione content. It is likely that due to higher induction of hepatic GST activity, products of lipid peroxidation may be detoxified and therefore unable to cause DNA damage making mice resistant to hepatic tumor formation. The overall results indicate a tumor initiating potential of AFB1 in mice and suggest that continued dermal exposure of AFB1, even at low doses, might lead to degenerative changes in hepatocytes.

Analysis of the contribution of changes in mRNA stability to the changes in steady‐state levels of cyclin mRNA in the mammalian cell cycle

Cyclins are the essential regulatory subunits of cyclin-dependent protein kinases. They accumulate and disappear periodically at specific phases of the cell cycle. Here we investigated whether variations in cyclin mRNA levels in exponentially growing cells can be attributed to changes in mRNA stability. Mouse EL4 lymphoma cells and 3T3 fibroblasts were synchronized by elutriation or cell sorting. Steady-state levels and degradation of cyclin mRNAs and some other cell cycle related mRNAs were measured at early G1, late G1, S and G2/M phases. In both cell lines mRNAs of cyclins C, D1 and D3 remained unchanged throughout the cell cycle. In contrast, cyclin A2 and B1 mRNAs accumulated 3.1- and 5.7-fold between early G1 and G2/M phase, whereas cyclin E1 mRNA decreased 1.7-fold. Mouse cyclin A2 and B1 genes, by alternative polyadenylation, gave rise to more than one transcript. In both cases, the longer transcripts were the minor species but accumulated more strongly in G2/M phase. All mRNAs were rather stable with half-lives of 1.5-2 h for cyclin E1 mRNA and 3-4 h for the others. Changes in mRNA stability accounted for the accumulation in G2/M phase of the short cyclin A2 and B1 mRNAs, but contributed only partially to changes in levels of the other mRNAs.

Clustering, duplication and chromosomal distribution of mouse SINE retrotransposons

We analyzed potential mechanisms determining chromosomal distributions of the mouse B1 and B2 non-LTR retrotransposons, also known as SINE elements. We report that young B1 and B2 SINEs are underrepresented on chromosome X relative to autosomes, which is consistent with their integration in male germ lines. As the age of the SINE elements progresses, their densities on chromosome X increase relative to autosomal densities, possibly due to differences in ectopic recombination rates between chromosome X and autosomes. Furthermore, unlike young human Alus that tend to be integrated outside Alu-dense regions, young B1 and B2 elements are found mostly in SINE-rich clusters. The B1- or B2-rich clusters are more likely to contain duplicated elements than B1- or B2-poor chromosomal regions. We also present evidence indicating potential association of B1 and B2 elements with intra-chromosomal segmental duplications. No such association was found with inter-chromosomal duplications. We propose that the accumulation of mouse SINE elements observed in GC-rich regions may be due to the excess of DNA duplications over deletions in gene-rich regions that tend to be GC rich.

Antihyperalgesic activity of a novel nonpeptide bradykinin B1 receptor antagonist in transgenic mice expressing the human B1 receptor

We describe the properties of a novel nonpeptide kinin B1 receptor antagonist, NVP-SAA164, and demonstrate its in vivo activity in models of inflammatory pain in transgenic mice expressing the human B1 receptor. NVP-SAA164 showed high affinity for the human B1 receptor expressed in HEK293 cells (K(i) 8 nM), and inhibited increases in intracellular calcium induced by desArg10kallidin (desArg10KD) (IC50 33 nM). While a similar high affinity was observed in monkey fibroblasts (K(i) 7.7 nM), NVP-SAA164 showed no affinity for the rat B1 receptor expressed in Cos-7 cells. In transgenic mice in which the native B1 receptor was deleted and the gene encoding the human B1 receptor was inserted (hB1 knockin, hB1-KI), hB1 receptor mRNA was induced in tissues following LPS treatment. No mRNA encoding the mouse or human B1 receptor was detected in mouse B1 receptor knockout (mB1-KO) mice following LPS treatment. Freund's complete adjuvant-induced mechanical hyperalgesia was similar in wild-type and hB(1)-KI mice, but was significantly reduced in mB1-KO animals. Mechanical hyperalgesia induced by injection of the B1 agonist desArg10KD into the contralateral paw 24 h following FCA injection was similar in wild-type and hB1-KI mice, but was absent in mB1-KO animals. Oral administration of NVP-SAA164 produced a dose-related reversal of FCA-induced mechanical hyperalgesia and desArg10KD-induced hyperalgesia in hB1-KI mice, but was inactive against inflammatory pain in wild-type mice. These data demonstrate the use of transgenic technology to investigate the in vivo efficacy of species selective agents and show that NVP-SAA164 is a novel orally active B1 receptor antagonist, providing further support for the utility of B1 receptor antagonists in inflammatory pain conditions in man.

Effects of aflatoxin B1 in pregnant mice and rats

Effects of aflatoxin B1 in pregnant mice and rats

The SINE-encoded mouse B2 RNA represses mRNA transcription in response to heat shock.

Cells respond to changes in environmental conditions via orchestrated modifications in gene expression. For example, in response to heat shock, cells execute a program of gene-specific transcriptional activation and repression. Although the activation of genes upon heat shock has been widely studied, the mechanism of mRNA transcriptional repression upon heat shock is unexplained. Here we show that during the heat shock response in mouse cells, a small noncoding RNA polymerase III transcript, B2 RNA, associates with RNA polymerase II and represses transcription of specific mRNA genes. These studies define a unique transcriptional regulatory mechanism involving an RNA regulator and reveal how mRNA transcription is repressed upon heat shock. Moreover, we identify a function for B2 RNA, which is transcribed from short interspersed elements that are abundant in the mouse genome and historically considered to be 'junk DNA.'

Predicting mammalian SINE subfamily activity from A-tail length.

Based on previous observations that newly inserted LINEs and SINEs have particularly long 3' A-tails, which shorten rapidly during evolutionary time, we have analyzed the rat and mouse genomes for evidence of recently inserted SINEs and LINEs. We find that the youngest predicted subfamilies of rodent identifier (ID) elements, a rodent-specific SINE derived from tRNA(Ala), are preferentially associated with A-tails over 50 bases in the rat genome, as predicted. Furthermore, these studies detected a subfamily of ID elements that has made over 15,000 copies that is younger than any previously reported ID subfamily. We use PCR analysis of genomic loci to demonstrate that all subfamily members tested inserted after the divergence of Rattus norvegicus from Rattus rattus. We also found evidence that the rodent B1 family of elements is much more active currently in mouse than in rat. These data provide useful estimates of recent activity from all of the mammalian retrotransposons, as well as allowing identification of the most recent insertions for use as population and speciation markers in those species. Both the current rat ID and mouse B1 elements that are active have small, specific interruptions in their 3' A-tail sequences. We suggest that these interruptions stabilize the length of the A-tails and contribute to the activity of these subfamilies. We present a model in which the dynamics of the 3' A-tail may be a central controlling factor in SINE activity.

Morphological characterization of mouse B1 cells

At least three B cell subsets, B-1a, B-1b and B-2 are present circulating peripherally in the mouse. In these animals, B-1 cells constitute a minor fraction of B cells in spleen and are absent in lymph nodes although they represent the main B cell population in peritoneal and pleural cavities. Currently these cells are identified by a surface phenotypic repertoire; they express Mac-1, IgM(high), and B220(low). B-1a cells express CDS. The aim of this work emerged from the fact that the morphology of B-1 cells is not fully characterized. Here we identified B-1 cells using colloidal gold immunocytochemical assays and purified B-1 cells from supernatants of adherent peritoneal cell cultures by a magnetic bead technique. These techniques lead us to demonstrate that, in mice, either B-1a or B-1b cells have a unique morphology distinct from that of B-2 cells.

Selective stimulation of translational expression by Alu RNA.

Human Alu and adenovirus VA1 RNAs each stimulate the translational expression of reporter genes in co-transient transfection assays without affecting either the rate of global protein synthesis or the abundance of the reporter mRNA. This selective, post-transcriptional stimulation of expression, which is observed in human and mouse cell lines and for three reporters, acts through a PKR- independent mechanism. The activity of Alu and VA1 RNAs in this assay is transient, causing a reduction in the lag time for the translational expression of the newly synthesized reporter mRNAs. The reduction in this lag time accounts for the relative selectivity of the effect upon the expression of the reporter and suggests novel roles for Alu and VA1 RNA in cell stress recovery and viral infection. Deletion analysis demonstrates that a specific region residing within the right monomer of the dimeric Alu consensus sequence is necessary for activity. Highly abundant left Alu monomer transcripts are inactive but the right Alu monomer is fully active, although its transcripts are scarce. Mouse B1 and B2 SINE RNAs stimulate reporter gene expression in mouse cells, suggesting that this activity is a general property of eucaryotic SINEs.

Genomic structure, organization, and promoter region analysis of murine guanylyl cyclase/atrial natriuretic peptide receptor-A gene

We have determined the complete genomic nucleotide sequence and analyzed the promoter region of murine guanylyl cyclase/natriuretic peptide receptor-A gene (Npr1,coding for NPRA). The gene spans about 17.8 kb and contains 22 exons interrupted by 21 introns. All the exon–intron boundaries possess the consensus GT/AG splice junctions. Four different types of short interspersed nuclear elements (ten mouse B1 elements, seven mouse B2–B4 elements, one ID and one MIR element) and one medium reiteration frequency repeats have been found in the non-coding regions of the gene. Eleven tandem repeats, including three in the promoter region of the gene, have been identified. The transcription start site, 362 bp upstream from the start codon, was determined by 5′- rapid amplification of cDNA ends. The 1.98 kb 5′-flanking region contains three potential SP1 binding sites and one inverted CCAAT box but lacks the TATA box. This region also contains several putative cis-acting motifs known to bind kidney specific nuclear protein HFH-3, cAMP-responsive element binding protein (CREB) and AP-4. In addition, the binding sites for a variety of transcription factors: AML-1α, SRY, Nkx-2.5, LyF-1, p300, GATA-1/2, HNF-3β, c/EBP α/β and USF have been localized in the promoter region of Npr1 gene. The analyses and characterization of the genomic structure of murine Npr1 gene should yield important insights into the species-specific regulation of this important gene family.

DETECTION OF DIFFERENTIALLY EXPRESSED GENES IN MOUSE LUNG ADENOCARCINOMAS

Increasing evidence suggests that altered gene expression is associated with the induction and maintenance of malignancy in various organs including mouse lung adenocarcinomas. A competitive cDNA library screening (CCLS) was used to examine gene expression in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung adenocarcinomas from (C3H/HeJ x A/J])F1 mice. Comparisons of RNA expression in lung adenocarcinomas to those of normal surrounding lung tissue revealed altered expression in 220 clones from more than 50,000 clones screened. Fifty clones were selected for quantitative reverse transcriptase-polymerase chain reaction (PCR) analysis to verify altered expression. PCR primers were designed based on partial sequence analysis of the clones. Twenty-two clones were found to be differentially expressed in lung adenocarcinomas compared with normal lungs. GenBank database analysis showed that 14 of the 22 clones were homologous with known genes, whereas 8 clones contained novel sequences. Thirteen clones were down regulated in tumors compared to normal lung tissues, and 9 were overexpressed. The clones underexpressed or absent include adipocyte p27, carbonic anhydrase III, carbonyl reductase, cytochrome CYP2E1, skelemin, myosin, major urinary protein, and contrapsin. Overexpressed clones include Bruton's tyrosine kinase, cyclin D3, poly(A)-binding protein, alpha-fetoprotein, transferrin, and mouse B2 family repetitive sequence. Further examination of biologic implications of the differentially expressed genes in lung adenocarcinomas is necessary to understand their role(s) in mouse lung carcinogenesis.

A mechanistic approach to modelling the risk of liver tumours in mice exposed to fumonisin B1 in the diet

Data from the National Toxicology Program's carcinogenesis study of fumonisin B1 in B6C3F1 mice, conducted at the National Center for Toxicological Research, were used to fit the Moolgavkar-Venzon-Knudson (MVK) two-stage, clonal-expansion model of carcinogenesis. In addition to tumour data from the conventional 2-year bioassay, the study included data on tissue weights, cell proliferation, cell death, and sphingolipid metabolism in primary target organs. The model was used to predict 2-year liver tumour rates in female and male mice based on differences among dose groups in the effect of fumonisin B1 on the growth of normal tissue and on the proliferation of preneoplastic cells as a compensatory response to sphinganine-induced cell death. Fumonisin B1 was assumed to be non-genotoxic, i.e. the model did not include any effect of fumonisin B1 on either of the two mutation rates of the MVK model. The model was able to reproduce reasonably well the observed tumour rates in both female and male mice, predicting substantially increased rates above background only at the highest doses of fumonisin B1 in females.

Mouse B1 cell line responds to lipopolysaccharide via membrane-bound CD14

The role of membrane-bound CD14 in the response of mouse B1 cell lines to lipopolysaccharide (LPS) was studied. The surface profile of mouse TH2.52 B cells was positive for CD5, IgM, B220, CD11b and F4/80, suggesting that TH2.52 cells carried the typical phenotype of B1 cells. Furthermore, TH2.52 B1 cells were found to express membrane-bound CD14, which plays a critical role in LPS recognition. TH2.52 B1 cells responded to a very low concentration of LPS and exhibited: (i) augmentation of DNA synthesis; (ii) activation of nuclear factor (NF)-kappaB; and (iii) phosphorylation of extracellular signal regulated kinase 1/2 (Erk1/2). They were markedly inhibited by anti-CD14 antibody. Therefore, the expression of membrane-bound CD14 was suggested to provide high sensitivity to LPS for TH2.52 B1 cells.

Classification of kinin receptors.

This minireview is divided into three parts: the first part refers to the characterization and classification of kinin receptors using agonists and antagonists in isolated tissues (classical pharmacology). Two kinin receptors have been considered on the basis of their distinct pharmacology, namely the B1 receptor of the rabbit aorta (rank order of potency of agonists: LysdesArg9BK > desArg9BK > or = LysBK > BK; apparent affinities of antagonists Lys[Leu8]desArg9BK (pIC50 8.4) > [Leu8]desArg9BK (pIC50 7.4) >>> HOE 140, a B2 receptor antagonist, pIC50<5.0), and the B2 receptor of the rabbit jugular vein (potency of agonists: LysBK = BK >>> LysdesArg9BK = desArg9BK and HOE 140 (pIC50 9.0) >>> Lys[Leu8]desArg9BK, pIC50<5.0). The second part describes species-related B1 receptor subtypes, demonstrated by different pharmacological profiles of agonists and antagonists: human, rabbit and pig subtypes (LysdesArg9BK >> desArg9BK and Lys[Leu8]desArg9BK > [Leu8]desArg9BK) and dog, rat, mouse and hamster B1 receptors (desArg9BK = LysdesArg9BK and [Leus]desArg9BK = Lys[Leu8]desArg9BK). Affinities of agonists and antagonists in some species (man, rabbit, pig) are significantly increased (at least 10-fold) by the presence of a Lys at their N-terminus. The last part describes species-related B2 receptor subtypes supported by results obtained with non-peptide receptor agonists (FR 190997) and antagonists (FR 173657). While BK acts as a full agonist in man, rabbit and pig, FR 190997 behaves as a full agonist on human, as partial agonist on rabbit, and as pure antagonist on pig B2 receptors. Various hypotheses are considered to interpret these findings.

DETECTION OF DIFFERENTIALLY EXPRESSED GENES IN MOUSE LUNG ADENOCARCINOMAS

Increasing evidence suggests that altered gene expression is associated with the induction and maintenance of malignancy in various organs including mouse lung adenocarcinomas. A competitivecDNA libraryscreening (CCLS)was used to examinegene expression in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung adenocarcinomas from (C3H/HeJ A/J)F1 mice. Comparisons of RNA expression in lung adenocarcinomas to those of normal surrounding lung tissue revealed altered expression in 220 clones from more than 50,000 clones screened. Fifty clones were selected for quantitative reverse transcriptase-polymerase chain reaction (PCR) analysis to verify altered expression. PCR primers were designed based on partial sequence analysis of the clones. Twenty-two clones were found to be differentially expressed in lung adenocarcinomas compared with normal lungs. GenBank database analysis showed that 14 of the 22 clones were homologous with known genes, whereas 8 clones contained novel sequences. Thirteen clones were down regulated in tumors compared to normal lung tissues, and 9 were overexpressed. The clones underexpressed or absent include adipocyte p27, carbonic anhydrase III, carbonyl reductase, cytochrome CYP2E1, skelemin, myosin, major urinary protein, and contrapsin. Overexpressed clones include Bruton's tyrosine kinase, cyclin D3, poly(A)-binding protein, alpha-fetoprotein, transferrin, and mouse B2 family repetitive sequence. Further examination of biologic implications of the differentially expressed genes in lung adenocarcinomas is necessaryto understand their role(s)in mouse lung carcinogenesis.

B and T cell responses to the spliceosomal heterogeneous nuclear ribonucleoproteins A2 and B1 in normal and lupus mice.

Autoantibodies directed against spliceosomal heterogeneous nuclear ribonucleoproteins (hnRNPs) are a typical feature of rheumatoid arthritis, systemic lupus erythematosus, and mixed-connective tissue disease. With the aim of investigating a potential pathogenic role of these Abs, we have studied the Ab response to A2/B1 hnRNPs in different murine models of lupus. The specificity of anti-A2/B1 Abs was tested with a series of 14 overlapping synthetic peptides covering the region 1-206 of A2 that contains most of the epitopes recognized by patients' Abs. A major epitope recognized very early during the course of the disease by Abs from most of MRL lpr/lpr mice but not from other lupus mice and from mice of different MHC haplotypes immunized against B1 was identified in residues 50-70. This peptide contains a highly conserved sequence RGFGFVTF also present in other hnRNPs and small nuclear ribonucleoproteins. Abs reacting with a second A2 epitope identified in residues 35-55 were detectable several weeks later, suggesting an intramolecular B cell epitope spreading during the course of the disease. We identified several T cell epitopes within the region 35-175 that generated an effective Th cell response with IL-2 and IFN-gamma secretion in nonautoimmune CBA/J mice sharing the same MHC haplotype H-2k as MRL/lpr mice. None of the peptides stimulated T cells primed in vivo with B1. Because Abs to peptide 50-70 were detected significantly earlier than Abs reacting with other A2 peptides and the protein itself, it is possible that within the protein, this segment contains residues playing an initiator role in the induction of the anti-A2/B1 and antispliceosome Ab response.