RESUMO
OBJECTIVES: Previous studies of decreased cochlear DNA binding in autoimmune mice suggested that antibodies against a cochlear cell surface DNA receptor cause autoimmune hearing loss. However, the presence of a cochlear DNA receptor has not been determined. Therefore, immunohistochemistry with an anti-DNA receptor antibody was performed on MRL.MpJ-Fas(lpr) (MRL/lpr) autoimmune mice to determine 1) which inner ear structures contain DNA receptors and 2) whether the receptor staining pattern changes as autoimmune disease progresses and hearing thresholds increase. STUDY DESIGN: A prospective study of the progression of hearing loss in autoimmune mice and correlated alterations in immunostaining for the inner ear DNA receptor. METHODS: One group of MRL/lpr mice (n = 10) was allowed to develop autoimmune disease, and auditory brainstem response (ABR) audiometry was performed at 4, 6, and 9 months of age to measure the progression of hearing loss. A second group (n = 5) was tested for ABR thresholds at 2 months of age and immediately killed to assess receptor staining before the onset of autoimmune disease and hearing loss. The inner ears from all mice were immunohistochemically stained with an anti-DNA receptor antibody, and a qualitative analysis of the staining of cochlear structures was performed. RESULTS: Auditory brainstem response audiometry revealed a significant 20- to 30-dB elevation of thresholds as systemic disease progressed. Anti-DNA receptor staining was heaviest in the spiral ligament and less intense in the spiral ganglion and cochlear nerve. Both groups showed a similar pattern of staining in these structures. The stria vascularis and hair cells also stained in both groups. However, the stria cells of normal-hearing mice showed diffuse intracellular immunoreactivity, whereas older mice displayed less staining that was confined to the cell membranes. CONCLUSIONS: The inner ears of MRL/lpr mice contain DNA receptors. Autoimmune hearing loss was correlated with weaker overall intracellular staining in the stria vascularis and hair cells but increased staining of the cell membranes. This suggested DNA receptors have impaired endocytosis and more receptors remain on the cell membrane, possibly as a result of binding by circulating autoantibodies.
Assuntos
Doenças Autoimunes , Cóclea/metabolismo , DNA/análise , Perda Auditiva Neurossensorial/etiologia , Células Receptoras Sensoriais/metabolismo , Fatores Etários , Animais , Audiometria de Resposta Evocada , Doenças Autoimunes/complicações , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Cóclea/fisiologia , Perda Auditiva Neurossensorial/diagnóstico , Imuno-Histoquímica , Lúpus Eritematoso Sistêmico/complicações , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Camundongos Endogâmicos MRL lpr , Estudos Prospectivos , Coloração e RotulagemRESUMO
This study was designed to assess the effect of exposure to long-term extremely low-frequency electric and magnetic fields (ELF-EMF) from a 500 kV transmission line on IL-1 and IL-2 activity in sheep. The primary hypothesis was that the reduction in IL-1 activity observed in our two previous short-term studies (10 months) was due to EMF exposure from this transmission line. To repeat and expand these studies and to characterize the components of EMF responsible for the previously observed reduction in IL-1 activity, the current experiment examined not only the effect of exposure to electric and magnetic fields, but also the magnetic field component alone. In the current study, IL-2 was examined to characterize the effects of EMF exposure on an indicator of T cell responses. 45 Suffolk ewe lambs were randomized into three groups of 15 animals each. One group of animals was placed in the EMF pen, located directly beneath the transmission line. A second group was placed in the shielded MF (magnetic field only) pen, also directly beneath the transmission line. The third group of animals was placed in the control pen located several hundred meters away from the transmission line. During the 27 month exposure period, blood samples were taken from all animals monthly. When the data were analyzed collectively over time, no significant differences between the groups were found for IL-1 or IL-2 activity. In previous studies ewe lambs of 8-10 weeks of age were used as the study animals and significant differences in IL-1 activity were observed after exposure of these animals to EMF at mean magnetic fields of 3.5-3.8 microT (35-38 mG) and mean electric fields of 5.2-5.8 kV/m. At the start of the current study EMF levels were reduced as compared to previous studies. One interpretation of the current data is that magnetic field strength and age of the animals may be important variables in determining whether EMF exposure will affect IL-1 activity.
Assuntos
Eletricidade , Campos Eletromagnéticos , Interleucina-1/sangue , Interleucina-2/sangue , Linfócitos/efeitos da radiação , Ovinos/imunologia , Análise de Variância , Animais , Exposição Ambiental , Feminino , Abrigo para Animais , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Fatores de TempoRESUMO
Studies to clone a cell-surface DNA-binding protein involved in the binding and internalization of extracellular DNA have led to the isolation of a gene for a membrane-associated nucleic acid-binding protein (MNAB). The full-length cDNA is 4.3 kilobases with an open reading frame of 3576 base pairs encoding a protein of approximately 130 kDa (GenBank accession numbers and ). The MNAB gene is on human chromosome 9 with wide expression in normal tissues and tumor cells. A C3HC4 RING finger and a CCCH zinc finger have been identified in the amino-terminal half of the protein. MNAB bound DNA (K(D) approximately 4 nm) and mutagenesis of a single conserved amino acid in the zinc finger reduced DNA binding by 50%. A potential transmembrane domain exists near the carboxyl terminus. Antibodies against the amino-terminal half of the protein immunoprecipitated a protein of molecular mass approximately 150 kDa and reacted with cell surfaces. The MNAB protein is membrane-associated and primarily localized to the perinuclear space, probably to the endoplasmic reticulum or trans-Golgi network. Characterization of the MNAB protein as a cell-surface DNA-binding protein, critical in binding and internalization of extracellular DNA, awaits confirmation of its localization to cell surfaces.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Membrana/genética , Sequência de Aminoácidos , Animais , Células COS , Membrana Celular/química , Clonagem Molecular , DNA/metabolismo , Proteínas de Ligação a DNA/química , Células HeLa , Humanos , Proteínas de Membrana/química , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Dedos de ZincoRESUMO
DNA binding to cell-surfaces has been documented in several studies. The interaction of DNA with cells has been shown to have therapeutic potential as a non-viral form of gene delivery and DNA vaccination. Recently, bacterial DNA binding and internalization has been demonstrated in some cells to trigger secretion of cytokines and cell activation. Previous studies to quantify DNA binding to cells have used radiolabeled DNA. Here we report a non-radioactive assay for quantification of cell-surface DNA binding based on the isoparametric analysis of flow cytometric data as described by Chatelier et al., Embo J., 5 (1986) 1181. This assay has the advantage over previously used procedures in not employing radioactive material and being able to discriminate viable from non-viable cells that bind DNA. With the importance of understanding the interaction of DNA with cells, this assay may have application for the identification and characterization of reagents designed to either enhance or inhibit DNA binding to cells.
Assuntos
Membrana Celular/metabolismo , Citometria de Fluxo/métodos , Plasmídeos/metabolismo , Linhagem Celular Transformada , HumanosRESUMO
Corticosteroid therapy is used to reverse autoimmune sensorineural hearing loss, although little is known of the mechanism by which this occurs. This has been due to the lack of a suitable animal model with spontaneous hearing loss that is steroid responsive. The present study examined the effects of prednisolone treatment on auditory thresholds in the MRL.MpJ-Fas(lpr) autoimmune mouse to determine its suitability as such a model. Autoimmune mice at 3.5-4. 5 months of age were evaluated by pure-tone auditory brainstem response (ABR) to establish threshold elevations due to the disease. The steroid treatment group was then given prednisolone in their drinking water for 2.5 months, while untreated controls were given tap water. Significantly more steroid treated mice survived to the time of post-treatment ABR evaluation. Half of the steroid treated ears demonstrated either improvement or no change in cochlear function compared to only 25% in the untreated controls. Overall, cochlear thresholds in the untreated controls increased by 14.7 dB, whereas no significant threshold increase was seen in the steroid treated group (4.3 dB) over the treatment period. No qualitative anatomical differences were seen in the ears of those mice surviving to the end of the study. These findings establish the autoimmune mouse as a model for studies of steroid responsive mechanisms within the ear. This could apply to autoimmune sensorineural hearing loss, as well as any hearing disorder for which steroid therapy is recommended.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Glucocorticoides/uso terapêutico , Perda Auditiva Neurossensorial/tratamento farmacológico , Prednisolona/uso terapêutico , Administração Oral , Animais , Limiar Auditivo/efeitos dos fármacos , Doenças Autoimunes/patologia , Doenças Autoimunes/fisiopatologia , Modelos Animais de Doenças , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Glucocorticoides/administração & dosagem , Perda Auditiva Neurossensorial/patologia , Perda Auditiva Neurossensorial/fisiopatologia , Transporte de Íons/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos MRL lpr , Prednisolona/administração & dosagem , Sódio/metabolismoRESUMO
Corticosteroid therapy reverses clinical autoimmune sensorineural hearing loss, although little is known of how steroids restore normal auditory function. If suppression of systemic autoimmune processes underlies hearing restoration, then preventing autoimmune symptoms from developing should prevent cochlear dysfunction. MRL. MpJ-Fas(lpr) autoimmune mice were used to test this potential mechanism by initiating oral prednisolone treatment at 6 weeks of age, prior to autoimmune disease and hearing loss onset. The steroid treatment group was given prednisolone in their drinking water, while untreated controls were given tap water. Treatment continued for 7 months with periodic evaluations of cochlear function with auditory brainstem response (ABR) audiometry. Autoimmune mice given the steroid lived longer and did not develop levels of serum immune complexes seen in their untreated controls. Also, their ABR thresholds remained near normal throughout the 7 months of treatment, while untreated controls showed progressive threshold elevations typical for autoimmune disease. This correlation of suppressed systemic autoimmune activity and maintenance of normal cochlear function identifies one potential mechanism for autoimmune hearing loss and hearing restoration with steroid therapy. The autoimmune mouse should serve as a valuable model for future studies of the cochlear mechanisms responsive to steroid treatment in autoimmune hearing loss.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Cóclea/efeitos dos fármacos , Glucocorticoides/uso terapêutico , Perda Auditiva Neurossensorial/tratamento farmacológico , Prednisolona/uso terapêutico , Administração Oral , Fatores Etários , Animais , Audiometria de Tons Puros , Doenças Autoimunes/patologia , Doenças Autoimunes/fisiopatologia , Cóclea/patologia , Cóclea/fisiopatologia , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Glucocorticoides/administração & dosagem , Perda Auditiva Neurossensorial/patologia , Perda Auditiva Neurossensorial/fisiopatologia , Camundongos , Camundongos Endogâmicos MRL lpr , Prednisolona/administração & dosagemRESUMO
Heat shock protein 70 (HSP70) has been suggested as the putative cochlear antigen underlying a proposed autoimmune etiology in certain cases of Meniere's disease and idiopathic hearing loss. To determine if antibodies to this cellular protein are capable of altering cochlear function, BALB/c (N= 3) and CBA/J (N= 9) mice were inoculated with bovine HSP70 by intraperitoneal injections (10 microg in saline) every 10 days for 7 or 10 months, respectively. An equal number of control mice were injected with PBS according to the same schedule. ABR thresholds at 4, 8, 16, and 32 kHz in the HSP70-inoculated mice did not change over the 10 month period and were similar to saline controls. Furthermore, serum immune complexes and antinuclear antibodies did not increase over the inoculation period. ELISA analysis demonstrated the mice created antibodies to the foreign HSP70, but these apparently caused no abnormalities in the auditory or immune systems. It was concluded that foreign HSP70 is antigenic and inoculation with it will raise antibodies, but these antibodies were neither immunopathogenic nor cochleopathic. Therefore, these findings do not support current theories that elevated anti-HSP70 antibodies are the underlying cause of hearing loss in patients with such antibodies present.
Assuntos
Anticorpos/metabolismo , Cóclea/imunologia , Cóclea/fisiologia , Proteínas de Choque Térmico HSP70/imunologia , Animais , Complexo Antígeno-Anticorpo/metabolismo , Antígenos/metabolismo , Autoantígenos/metabolismo , Doenças Autoimunes/etiologia , Doenças Autoimunes/imunologia , Bovinos , Proteínas de Choque Térmico HSP70/fisiologia , Perda Auditiva Neurossensorial/etiologia , Perda Auditiva Neurossensorial/imunologia , Doença de Meniere/etiologia , Doença de Meniere/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBARESUMO
Inner ear function and systemic autoimmune disease were evaluated in the MRL/lpr mouse to determine their relationship with alterations in cell surface DNA receptors of 28-30 and 68-70 kDa size. Auditory brainstem response thresholds in the autoimmune disease mice were significantly elevated as early as 2 months of age when compared to MRL/++ controls. Hearing thresholds continued to rise with progression of the disease, manifested as increasing spleen weights, antinuclear (anti-DNA) antibodies, and serum immune complexes. Cochlear membranous labyrinth cells in the autoimmune mice bound less DNA, suggesting the DNA receptors were abnormally occupied by circulating antibodies. Western blots of a murine T-cell line probed with autoimmune mouse sera demonstrated reactivity to 28-30 and 68-70 kDa proteins after disease onset. It is hypothesized that cell surface DNA binding molecules could be masked or down-regulated by circulating antibodies in autoimmune disease. This interference with DNA receptor activity may be occurring within the inner ear and underlie the cochlear dysfunction seen in autoimmune sensorineural hearing loss.
Assuntos
Doenças Autoimunes/etiologia , Orelha Interna/metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Perda Auditiva Neurossensorial/etiologia , Receptores de Superfície Celular/metabolismo , Análise de Variância , Animais , Anticorpos Antinucleares/imunologia , Complexo Antígeno-Anticorpo/sangue , Limiar Auditivo/fisiologia , Doenças Autoimunes/imunologia , Ligação Competitiva , Western Blotting , Linhagem Celular , Cóclea/citologia , Cóclea/imunologia , Desoxirribonucleases/metabolismo , Orelha Interna/imunologia , Eletroforese em Gel de Poliacrilamida , Perda Auditiva Neurossensorial/imunologia , Camundongos , Camundongos Endogâmicos MRL lpr , Peso Molecular , Tamanho do Órgão/fisiologia , Receptores de Superfície Celular/imunologia , Baço/imunologia , Baço/patologia , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
Vascular endothelial cells secrete the pluripotent cytokine interleukin-6, and the induction of this secretion can be regulated by a number of other immune-related cytokines. To determine whether a cellular alloimmunologic response to vascular endothelial cells alters the expression of interleukin-6 production by endothelial cells, we cocultured peripheral blood lymphocytes with a pool of human aortic endothelial cells. In response to the pool of allogeneic human aortic endothelial cells, lymphocytes from 10 separate donors proliferated to varying degrees after 5 days of coculturing. After 20 hours, human aortic endothelial cell-derived messenger RNA coding for interleukin-6 increased an average of 96% after exposure to allogeneic lymphocytes and the amount of biologically active interleukin-6 released into the media increased 69%. The kinetics of human aortic endothelial cell interleukin-6 messenger RNA expression in response to lymphocytes from an additional three donors was determined over a 48-hour period. Human aortic endothelial cell interleukin-6 messenger RNA increased approximately threefold over control, as early as 2 hours after exposure to allogeneic lymphocytes and returned toward control levels by 48 hours. Activation of six additional isolates of lymphocytes with phorbol myristate acetate before exposure to human aortic endothelial cells resulted in an increase in human aortic endothelial cell-derived interleukin-6 bioactivity regardless of whether the cells were in direct contact with the human aortic endothelial cells, but the interleukin-6 level increase was approximately twofold higher in those cocultures where there was direct contact. These data show that allogeneic lymphocytes have the potential of regulating vascular endothelial cell-derived interleukin-6, and direct lymphocyte-endothelial cell contact appears to be required for optimal interleukin-6 induction in this in vitro system.
Assuntos
Aorta/metabolismo , Endotélio Vascular/metabolismo , Interleucina-6/biossíntese , Linfócitos/fisiologia , Comunicação Celular , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Meios de Cultivo Condicionados , Humanos , Interleucina-1/biossíntese , Interleucina-6/genética , Ativação Linfocitária , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Fatores de Tempo , Regulação para CimaRESUMO
Allograft recipients who have preformed antibodies to MHC determinants or develop these antibodies post-transplantation have a higher incidence of cellular rejection and graft loss. It is unclear whether this association is an etiologic one or whether the presence of these antibodies solely identifies individuals with a more pronounced alloimmunologic response. To determine whether antibodies to MHC determinants have a direct role in enhancing cell-mediated immunity, specifically in altering effector-target cell adhesion, the expression of endothelial cell surface intercellular adhesion molecule-1 (ICAM-1) in response to serum with high-titer anti-HLA antibodies was investigated. The target cells used were a pool of blood group O human aortic endothelial cells (HAECs) representing a wide range of HLA-A, B, C, and DR phenotypes. The test serum was serum pooled from 30 highly sensitized individuals (panel-reactive antibody 80%). Antibody binding to HAECs, and HAEC expression of class I and class II major histocompatibility (MHC) antigens and ICAM-1 were assessed by flow cytometry. General HAEC metabolic changes were assessed by 3H-uridine incorporation as a measure of RNA synthesis. Test serum resulted in almost a 14-fold increase in HAEC surface ICAM-1 expression compared with control serum, and titrations of test serum yielded a strong correlation between IgG bound to HAECs and HAEC ICAM-1 expression (r = 0.92). Test serum induced no change in expression of HAEC class I or class II MHC antigens, or 3H-uridine incorporation. The HAEC ICAM-1-inducing ability of the test serum was retained by concentrating the high molecular weight (> 100 kilodaltons) fraction of the test serum, isolation and purification of IgG from the test serum, and lost by absorbing this fraction with pooled platelets, suggesting that the activity was mediated by antibodies directed against MHC class I determinants. These data suggest that the presence of anti-HLA antibodies is more than a marker for individuals with greater alloreactive responsiveness. Anti-HLA antibodies may directly and specifically alter adhesion of effector cells to the allograft.
Assuntos
Moléculas de Adesão Celular/análise , Endotélio Vascular/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Isoanticorpos/imunologia , Aorta/imunologia , Aorta/metabolismo , Endotélio Vascular/metabolismo , Rejeição de Enxerto , Antígenos de Histocompatibilidade Classe I/análise , Antígenos de Histocompatibilidade Classe II/análise , Humanos , Molécula 1 de Adesão Intercelular , Interferon gama/farmacologia , RNA/metabolismo , Proteínas Recombinantes , Transplante HomólogoRESUMO
DNA binds to cell-surface proteins on human and murine leukocytes and induces secretion of the cytokine interleukin 6 (IL-6). Cell-surface DNA binding molecules have been shown to serve as target antigens for the production of autoantibodies in patients with systemic lupus erythematosus (SLE), and in lupus-prone mice. Recent studies have demonstrated that a subset of anti-anti-DNA antibodies, isolated from patients with SLE, are idiotypically related to antibodies reactive with a cell-surface DNA binding molecule. We now report that immunization of normal mice with a murine monoclonal anti-DNA antibody induces an anti-idiotypic response which has reactivity with a cell-surface DNA binding molecule. An anti-idiotypic anti-DNA monoclonal antibody (LB17) was isolated from the spleen of an immunized mouse. This monoclonal antibody blocked the binding of DNA to murine splenocytes and mimicked the functional effect of DNA by stimulating the secretion of IL-6. These experiments provide further evidence for an idiotypic connectivity between antibodies to cell-surface DNA binding proteins and anti-DNA antibodies. It is hypothesized that this idiotypic system is part of the network of natural autoantibodies and that its perturbation may give rise to pathogenic antibodies.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Antinucleares/imunologia , Anticorpos Monoclonais/imunologia , Proteínas de Ligação a DNA/imunologia , Animais , Ligação Competitiva , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Interleucina-1/biossíntese , Interleucina-6/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologiaRESUMO
Anti-idiotypic anti-DNA antibodies (anti-anti-DNA) have previously been described in both patients with systemic lupus erythematosus and healthy individuals. Jerne's hypothesis predicts that such antibodies would bear a paratope reactive with non-sequence specific DNA binding proteins. Here we have explored the notion of a molecular mimicry between anti-anti-DNA antibodies and antibodies to a previously described 28-29 kD cell surface DNA binding molecule. It was shown that affinity purified anti-anti-DNA antibodies inhibit the binding of DNA to cells and that MoAb to the 28-29 kD receptor react with anti-DNA antibodies. These findings indicate that a subset of anti-anti-DNA antibodies are idiotypically related to antibodies reactive with a cell surface DNA binding molecule. It is hypothesized that anti-DNA antibodies may arise when a convergence of genetic and environmental influences favours an unrestrained anti-idiotypic response to cell surface DNA binding molecule(s).
Assuntos
Anticorpos Antinucleares/metabolismo , Idiótipos de Imunoglobulinas/imunologia , Receptores de Superfície Celular/fisiologia , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais , Ligação Competitiva , Humanos , Lúpus Eritematoso Sistêmico/sangueRESUMO
The molecular basis for the cellular interaction of DNA and nucleosomes and the physiological consequences of this binding were examined. Both DNA and nucleosomes were demonstrated to bind specifically to the surface of human peripheral blood mononuclear cells and the murine T cell line S49. Western blots of S49 cell membranes, using probes of biotin-labeled DNA and nucleosomes, showed reactivity at 29 and 69 kDa. Functionally, the interaction of DNA and nucleosomes with murine spleen cells stimulated the release of significant amounts of IL-6 activity. There is evidence that nucleosomes, a product of apoptosis, are the major component of circulating DNA found in the plasma of patients with systemic lupus erythematosus (SLE). The interaction of nucleosomes with cell-surface DNA binding molecules may have physiological relevance to some of the immune aberrations observed in patients with SLE.
Assuntos
Citocinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Nucleossomos/metabolismo , Animais , Linhagem Celular , Separação Celular , Citometria de Fluxo , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Salmão , Linfócitos T/citologia , Linfócitos T/metabolismo , Testículo/química , Testículo/ultraestruturaRESUMO
Autoimmunity to a 28-29-kDa cell-surface DNA-binding molecule has previously been described in patients with systemic lupus erythematosus and related autoimmune diseases. This report describes experiments that implicate a similar antigen-antibody system in the evolution of autoimmunity in lupus-prone mice. DNA binding to murine spleen cells was found to be a saturable phenomenon that was inhibited by excess cold DNA and trypsinization. The role of autoimmunity to murine cell-surface DNA-binding molecules in lupus-prone mice (MRL lpr/lpr, MRL +/+, BXSB) was compared to normal mice (BALB/c, C3H.SW) by means of an assay that measured the inhibition of cell-surface DNA binding. Only sera from lupus strains had inhibitory activity and this component was shown to be an IgM autoantibody. Furthermore, we isolated a spontaneously occurring IgM monoclonal antibody from the spleen of an MRL/lpr mouse, which inhibited DNA binding to mouse cells. Time-course studies indicated that young female MRL/lpr mice lacked detectable activity against cell-surface DNA-binding molecules; however, by 8-10 weeks maximal inhibitory activity was observed. This response occurred prior to the development of significant antinuclear antibody activity. With the appearance of overt disease and anti-DNA antibodies, inhibition of DNA-binding activity became undetectable. These findings mirror previous studies on autoimmunity to a cell-surface DNA-binding molecule on human leucocytes, but have the added advantage of permitting the study of the temporal evolution of this inhibitory activity in relation to disease expression.
Assuntos
Autoantígenos/metabolismo , DNA/imunologia , DNA/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Animais , Anticorpos Antinucleares/biossíntese , Autoimunidade , Sítios de Ligação , Ligação Competitiva , Membrana Celular/imunologia , Membrana Celular/metabolismo , Feminino , Imunoglobulina M/isolamento & purificação , Imunoglobulina M/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Baço/imunologia , Baço/metabolismoRESUMO
Previous experiments have established the presence of a 30-kD DNA binding protein on the surface of human leukocytes. Herein we report that selected sera from patients with systemic lupus erythematosus (SLE) and MCTD are reactive with a 28-30 kD protein on immunoblots of peripheral blood mononuclear cells (PBMC) cell membrane preparations; the reactivity is abolished by prior incubation of the blot with DNA. Antibodies eluted from the 28-30 kD strip inhibited the binding of 3H. DNA to human PBMC. An immunomatrix of 28-30 kD reactive immunoglobulins was able to extract a 29-kD DNA binding protein from a PBMC cell membrane preparation. Flow cytometry experiments confirmed the cell surface IgG reactivity of sera with T lymphocytes. Additional experiments indicated that cell surface IgG binding was not due to antibodies binding to cell surface DNA, DNA anti-DNA immune complexes reacting with a DNA binding protein, anti-histone antibodies or anti-Sm antibodies. It is hypothesized that this autoimmune response could be one component of an idiotypic network involving anti-DNA antibodies.
Assuntos
Autoimunidade , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Doença Mista do Tecido Conjuntivo/imunologia , Autoanticorpos/análise , Western Blotting , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Humanos , Linfócitos T/citologiaRESUMO
We have previously reported the existence of a cell-membrane-associated molecule on human PBMC, which binds DNA and has the characteristics of a receptor. Monoclonal antibodies have been made to this receptor and have been used successfully for the purification of this cell-surface molecule. Preliminary studies have indicated a receptor for DNA on murine kidney and spleen cells which is similar in molecular weight to the human DNA receptor (30 kD). The occurrence of autoantibodies to cell-surface receptors has been described in several autoimmune diseases and we have noted that the serum of patients with lupus and similar disorders inhibit the binding of labeled DNA to human leukocytes. Using a "dot-blot" assay with affinity-purified human DNA receptor, sera from patients with various CTD and from healthy volunteers were screened for anti-receptor antibodies; anti-receptor antibodies were found in many patients with CTD and some of their first-degree relatives. The prevalence of anti-receptor antibodies in normal blood donors was less than 2%. It is hypothesized that anti-receptor antibodies represent an early immune response in lupus and kindred disorders and that anti-DNA antibodies may arise from the corresponding anti-idiotypic response.
Assuntos
Lúpus Eritematoso Sistêmico/metabolismo , Receptores de Superfície Celular/metabolismo , Anticorpos/análise , Anticorpos Monoclonais/imunologia , Autoanticorpos/imunologia , Membrana Celular/metabolismo , DNA/metabolismo , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/isolamento & purificaçãoRESUMO
Two murine mAb have been generated with a reactivity toward a 30,000 m.w. DNA binding protein found on the cell surface of human leukocytes; mAb 12A has an IgG1/k isotype, and mAb 24T has an IgG2b/k isotype. Both react with the DNA binding domain or adjacent region of the putative DNA receptor and inhibit the binding of [3H]DNA to PBMC at concentrations as low as 100 ng/ml. Stoichiometric studies indicate that both mAb react with monocytes and T cells with a kDa of 10(-7) M; about 0.5 x 10(6) molecules bind per cell at saturation. Flow cytometry indicated that 67% of lymphocytes and 98% of monocytes bore the DNA receptor. Dual labeling studies showed that 90% of B cells and 50% of T cells express the receptor; 50% of CD4+ T cells are receptor positive. Immunomatrices constructed with both mAb 12A and 24T allowed the receptor to be purified to a high degree of purity. A single protein of Mr 30,000 was readily observed after SDS-PAGE and silver staining of the gel; after electropheretic transfer of nitrocellulose this protein was shown to be a DNA binding molecule by use of a probe of biotin labeled DNA. These experiments provide further evidence to support the existence of a specific DNA receptor on human leukocytes; the availability of mAb to the receptor should be useful in its further characterization.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Superfície/imunologia , Proteínas de Ligação a DNA/imunologia , Receptores de Superfície Celular/imunologia , Sítios de Ligação , DNA/metabolismo , Epitopos , Citometria de Fluxo , Histonas/imunologia , Peso Molecular , Monócitos/imunologia , Linfócitos T/imunologiaRESUMO
Locally injected endotoxin induces potent inflammatory changes in the rabbit eye. To clarify the possible role of interleukin 1 (Il-1), an endotoxin-induced monokine, in rabbit eye inflammation, we injected rabbits with recombinant Il-1 (rIl-1). Twelve and a half to 200 U of intravitreally injected rIl-1 consistently induced inflammation, which was documented using slit-lamp biomicroscopy, histologic methods, or direct quantitation of protein in the aqueous humor. Responses including a cellular infiltrate in the anterior chamber, protein extravasation, and iris vessel dilatation became evident within six hours, peaked at 24 hours, and began to recede by 48 to 72 hours after the injection. Pathologic changes primarily occurred in the anterior chamber and included edema, hemorrhage, and cellular infiltration. Locally injected corticosteroid reduced but did not prevent rIl-1-induced changes in vascular permeability. Heat-inactivated rIl-1 induced minimal changes, as determined by histologic methods, slit-lamp examination, or direct protein measurement. These data support the conclusion that Il-1 should be considered as a potential mediator of ocular inflammation.
Assuntos
Endoftalmite/induzido quimicamente , Interleucina-1/farmacologia , Animais , Humor Aquoso/metabolismo , Betametasona/farmacologia , Endoftalmite/patologia , Proteínas do Olho/metabolismo , Feminino , Injeções , Coelhos , Proteínas Recombinantes/farmacologia , Corpo VítreoRESUMO
Consistent with the reports of others, we have demonstrated that human peripheral blood lymphocytes adhere to cultured human umbilical vein-derived endothelial cells (EC) in vitro. In our studies adherence was increased twofold to threefold by a 6-hr preincubation of the EC with IL 1. Recombinant human IL 1 alpha induced a maximal adherence response at less than 1 U per 2 X 10(4) EC. In contrast, recombinant murine IL 1 alpha was found to be 250- to 1250-fold less active in the adherence assay, based on units of IL 1 activity defined by the murine thymocyte proliferation assay. Moreover, when EC were preincubated with excess murine IL 1, no inhibition of the adherence-inducing effect of human IL 1 was noted. To characterize further this dichotomy of biological potency of murine and human IL 1 on the adherence assay, IL 1 binding studies were initiated. Recombinant human and murine IL 1 alpha were equally effective in inhibiting the binding of 125I-labeled human and murine IL 1, based on both micrograms of protein and units of IL 1 activity. The results of this study demonstrate that although human and murine IL 1 bind with equal affinity to receptors on human EC, human IL 1 is significantly more potent at inducing the increased EC adhesiveness for lymphocytes. The implications of these results for endothelial cell IL 1 receptor function are discussed.
Assuntos
Endotélio/fisiologia , Interleucina-1/fisiologia , Linfócitos/citologia , Receptores Imunológicos/fisiologia , Animais , Ligação Competitiva , Adesão Celular , Endotélio/citologia , Humanos , Camundongos , Receptores de Interleucina-1RESUMO
2A3 monoclonal antibody (gamma 1, kappa) is a novel high-affinity reagent for detecting the human interleukin 2 (IL-2) receptor. The antibody inhibits IL-2 binding to its receptor and is an antagonist of IL-2 action. Detailed analysis of the mechanism of binding of the IgG, (Fab')2 and Fab' of 2A3 antibody shows that the bivalent species cross-link on the cell surface when bound. Measurements of IL-2 receptor expression on digitonin-permeabilized cells suggest that the intracellular pool of receptors is small. The antibody will bind to IL-2 receptors on glutaraldehyde-fixed cells in the presence of Triton X-100. This property is used in designing an assay for quantitative measurements of IL-2 receptor concn in solution. This assay can be used to monitor receptor protein during purification to homogeneity.