ABSTRACT
Fas (CD95) is a cell surface protein that mediates apoptosis. lpr is a mutation of the Fas gene caused by a retroviral insertion resulting in premature termination of transcription and aberrant splicing of Fas mRNA. Mice homozygous for the lpr gene develop lymphoproliferation and produce autoantibodies closely resembling those of human systemic lupus erythematosus. While lpr mice have been reported to express low levels of normally spliced Fas mRNA, it is unknown whether they express functional Fas protein. Here we show that splenocytes from lpr mice that have been damaged by gamma-irradiation expressed Fas protein. Fas was up-regulated on irradiated B6 cells and could be detected on B6/lpr cells undergoing apoptosis following in vitro culture. Detection of Fas on live lpr cells was demonstrable when apoptosis was blocked by zinc. In a short term chimera system, Fas was shown to play a role, in vivo, in the disposition of radiation-injured cells from both normal and lpr mice. The addition of anti-Fas Ab to in vitro cultures resulted in an increase in apoptosis in both B6 and B6/lpr cells. Detection of intact Fas message and low levels of Fas protein in lpr mice has led to the consideration of lpr as a leaky mutation. This study demonstrates that lpr mice can produce functional Fas protein. This system is also appropriate for identifying the in vivo role of Fas/FasL in apoptosis following other cell manipulations.
Subject(s)
Apoptosis/radiation effects , Gamma Rays/adverse effects , Radiation Injuries, Experimental/immunology , fas Receptor/physiology , Animals , Apoptosis/drug effects , Autoimmune Diseases/immunology , Autoimmune Diseases/pathology , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Cells, Cultured , Disease Models, Animal , Fas Ligand Protein , Gene Expression Regulation/radiation effects , Lupus Erythematosus, Systemic/immunology , Lupus Erythematosus, Systemic/pathology , Membrane Glycoproteins/physiology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred MRL lpr , RNA, Messenger/biosynthesis , Radiation Chimera , Radiation Injuries, Experimental/pathology , Spleen/pathology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/pathology , Zinc/pharmacology , fas Receptor/biosynthesisABSTRACT
Antibody which reacts with phosphatidyl choline can be detected in all normal mice. Generation of this specificity requires the use of either the unmutated VH11 or VH12 genes, with constraints on the length and sequence of CDR3, and specific light chain association. Given that those parts of the antibody that are subject to somatically generated diversity are restricted, we hypothesized that the germline VH11 and VH12 genes may be evolutionarily conserved to a greater extent than other VH genes. The nucleotide sequence was determined from a panel of inbred strains and Mus species for VH11 and VH12. The results were compared to the three functional members of the S107 VH family and show that VH11 and VH12 have a total of 13 silent and three replacement mutations while the S107 genes have 11 silent and 20 replacement mutations. This implies that there has been strong selection to conserve the VH11 and VH12 gene products, which must reflect a substantial survival value to the individual. Sequence comparisons also show that the alleles present in the recently derived inbred strains arose prior to speciation. While there is polymorphism within the inbred strains, there are alleles which are shared between species which diverged 3-5 million years ago. Conservation at the amino acid and nucleotide levels argues against the idea that Ig genes evolve at a rapid rate and suggests that the rate at which mutations are incorporated may be determined by the importance of the encoded protein.
Subject(s)
Antibody Diversity/immunology , Autoantibodies/immunology , Genes, Immunoglobulin/genetics , Immunoglobulin Variable Region/genetics , Phosphatidylcholines/immunology , Alleles , Amino Acid Sequence , Animals , B-Lymphocytes/immunology , Base Sequence , Biological Evolution , DNA Primers , DNA Probes , Genes, Immunoglobulin/immunology , Immunoglobulin G/immunology , Immunoglobulin Variable Region/immunology , Mice , Mice, Inbred Strains , Molecular Sequence Data , Polymorphism, Genetic , Receptors, Antigen, B-Cell/immunologySubject(s)
Antigens, CD/analysis , Immunoglobulin Light Chains/genetics , Immunoglobulin Variable Region/genetics , Mice, Inbred Strains/growth & development , Phosphatidylcholines/immunology , Aging/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal , B-Lymphocyte Subsets/immunology , Base Sequence , CD5 Antigens , Enzyme-Linked Immunosorbent Assay , Hybridomas/immunology , Lymphoma/immunology , Mice , Mice, Inbred Strains/immunology , Molecular Sequence Data , Peritoneal Cavity/physiology , Poly A/genetics , Poly A/isolation & purification , RNA, Messenger/genetics , RNA, Messenger/isolation & purificationABSTRACT
The in vivo frequency of mutants resulting from mutation at the hprt locus in human T-lymphocytes can be determined by a cloning assay. This assay quantifies the frequency of 6-thioguanine-resistant (TGr) T-cells through growth of colonies in 96-well microtiter dishes. The reproducibility of the TGr mutant frequency values has now been assessed in a longitudinal study of six individuals (three male, three female, aged 22-33 years) employing 4-5 blood samples over a 26-37 week time period. Cloning assays were performed with both fresh and cryopreserved cell samples. No significant differences were found among the mutant frequency values for multiple samples from each individual with both fresh and cryopreserved cell samples. These results demonstrate the reproducibility of this cloning assay for in vivo mutant frequency determinations in human T-lymphocytes.
