RESUMEN
FoxP3 reporter mice expressing green fluorescence protein (GFP) have been used as a very convenient tool to investigate the impact of regulatory T (Treg) cells on pathogenesis in autoimmune diseases. Here, we found that GFP-FoxP3+ knock-in (KI) mice showed alterations in the production of anti-nuclear autoantibodies (ANAs) and nephritis with different extent, depending on the presence or absence of lupus susceptibility gene locus 1 (Sle1) and KI method: contrasting with B6.Sle1.fGFP-FoxP3 mice, expressing GFP via N-terminal insertion, B6.Sle1.iGFP-FoxP3, expressing GFP via bicistronic internal ribosome entry site-driven promotion, exhibited significantly lower penetrance of serum ANA, comparing to control B6.Sle1 mice. Moreover, B6.Sle1.GFP-FoxP3+ mice reduced the Sle1-induced splenomegaly and B-cell expansion independently of the KI method employed, mainly by reducing the numbers of transitional 1 (T1) B cells and CD21-CD23- B cells, including plasmablasts and plasma cells. The absolute numbers of both splenic CD4+ T cells and Treg cells from B6.Sle1.GFP-FoxP3 KI mice were significantly reduced but their proportion was not changed, compared to B6.Sle1 mice. Although the glomerular basement membranes were thickened in both B6.Sle1 and B6.Sle1.iGFP-FoxP3 mice, they were thinner in B6.Sle1.fGFP-FoxP3 mice. The latter mice expressed more nephrophilic autoantibodies and deposited more complement component 3 in glomeruli compared to B6.iGFP-FoxP3 mice. FoxP3+ Treg cells may modulate B-cell tolerance in lupus-prone B6.Sle1 mice, presumably by modulating pathogenic, nephrophilic autoantibody production and nephritis.
Asunto(s)
Técnicas de Sustitución del Gen , Proteínas Fluorescentes Verdes , Lupus Eritematoso Sistémico , Penetrancia , Linfocitos T Reguladores , Animales , Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Tolerancia Inmunológica , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Ratones , Ratones Transgénicos , Especificidad de la Especie , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
Bile acids (BAs) control metabolism and inflammation by interacting with several receptors. Here, we report that intravenous infusion of taurodeoxycholate (TDCA) decreases serum pro-inflammatory cytokines, normalizes hypotension, protects against renal injury, and prolongs mouse survival during sepsis. TDCA increases the number of granulocytic myeloid-derived suppressor cells (MDSCLT) distinctive from MDSCs obtained without TDCA treatment (MDSCL) in the spleen of septic mice. FACS-sorted MDSCLT cells suppress T-cell proliferation and confer protection against sepsis when adoptively transferred better than MDSCL. Proteogenomic analysis indicated that TDCA controls chromatin silencing, alternative splicing, and translation of the immune proteome of MDSCLT, which increases the expression of anti-inflammatory molecules such as oncostatin, lactoferrin and CD244. TDCA also decreases the expression of pro-inflammatory molecules such as neutrophil elastase. These findings suggest that TDCA globally edits the proteome to increase the number of MDSCLT cells and affect their immune-regulatory functions to resolve systemic inflammation during sepsis.
Asunto(s)
Células Supresoras de Origen Mieloide/inmunología , Sepsis/inmunología , Linfocitos T/inmunología , Ácido Taurodesoxicólico/metabolismo , Animales , Recuento de Células , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Tolerancia Inmunológica , Elastasa de Leucocito/genética , Elastasa de Leucocito/metabolismo , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oncostatina M/genética , Oncostatina M/metabolismoRESUMEN
INTRODUCTION: Pre-naïve B cells represent an intermediate stage in human B-cell development with some functions of mature cells, but their involvement in immune responses is unknown. The aim of this study was to determine the functional role of normal pre-naïve B cells during immune responses and possible abnormalities in systemic lupus erythematosus (SLE) that might contribute to disease pathogenesis. METHODS: Pre-naïve, naïve, and memory B cells from healthy individuals and SLE patients were stimulated through CD40 and were analyzed for interleukin-10 (IL-10) production and co-stimulatory molecule expression and their regulation of T-cell activation. Autoreactivity of antibodies produced by pre-naïve B cells was tested by measuring immunoglobulin M (IgM) autoantibodies in culture supernatants after differentiation. RESULTS: CD40-stimulated pre-naïve B cells produce larger amounts of IL-10 but did not suppress CD4(+) T-cell cytokine production. Activated pre-naïve B cells demonstrated IL-10-mediated ineffective promotion of CD4(+) T-cell proliferation and induction of CD4(+)FoxP3(+) T cells and IL-10 independent impairment of co-stimulatory molecule expression and tumor necrosis factor-alpha (TNF-α) and IL-6 production. IgM antibodies produced by differentiated pre-naïve B cells were reactive to single-stranded deoxyribonucleic acid. SLE pre-naïve B cells were defective in producing IL-10, and co-stimulatory molecule expression was enhanced, resulting in promotion of robust CD4(+) T-cell proliferation. CONCLUSIONS: There is an inherent and IL-10-mediated mechanism that limits the capacity of normal pre-naïve B cells from participating in cellular immune response, but these cells can differentiate into autoantibody-secreting plasma cells. In SLE, defects in IL-10 secretion permit pre-naïve B cells to promote CD4(+) T-cell activation and may thereby enhance the development of autoimmunity.
Asunto(s)
Linfocitos B/metabolismo , Homeostasis/fisiología , Interleucina-10/fisiología , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Adulto , Femenino , Humanos , Inmunidad Humoral/fisiología , Masculino , Persona de Mediana EdadRESUMEN
Systemic lupus erythematosus is characterized by the spontaneous production of IgG autoantibodies in patients and lupus-prone mice. In this study, we investigated the effect of the Sle1 lupus susceptibility locus on the peripheral development of 56R(+) anti-DNA transgenic B cells by tracking 56R(+) B cells in mice without (B6.56R) or with (B6.Sle1.56R) the Sle1 locus. Compared with B6.56R mice, B6.Sle1.56R mice exhibited increased class-switched IgG2a anti-DNA Abs in their serum, encoded by the transgene. Interestingly, within the spleen, Sle1 facilitated the development of these cells into clusters of IgG2a class-switched B cells juxtaposed to CD4(+) T cells within extrafollicular sites. Through sequence analysis of B cell hybridomas, we also found that B cells from B6.Sle1.56R mice are inefficient at Ig H and L chain editing. Thus, the Ig H chains in Sle1.56R(+) B cells are partnered more often with cationic L chains that facilitate DNA binding. Taken together, these findings indicate that the Sle1 lupus-susceptibility locus may facilitate the emergence of anti-DNA B cells by subduing BCR revision and possibly by shaping the extrafollicular development of effector B cells, although the precise molecular mechanisms await further study.
Asunto(s)
Anticuerpos Antinucleares/inmunología , Linfocitos B/inmunología , Sitios Genéticos/inmunología , Predisposición Genética a la Enfermedad , Inmunoglobulina G/inmunología , Lupus Eritematoso Sistémico/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Animales , Anticuerpos Antinucleares/genética , Linfocitos B/patología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Inmunoglobulina G/genética , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/patología , Ratones , Ratones Mutantes , Receptores de Antígenos de Linfocitos B/genéticaRESUMEN
When monoclonal ANAs and non-ANAs generated from a genetically simplified mouse model of lupus, B6.Sle1, were recently compared, the ANAs exhibited three sequence motifs in their immunoglobulin heavy chains, including increased cationicity in CDR3 ("motif A"), reduced anionicity in CDR2 ("motif B") and increased aspartate at H50 ("motif C"). The present study was designed to elucidate the extent to which these ANA-associated sequence motifs might be hard-wired into the primary B-cell repertoire in lupus. The immunoglobulin heavy chain sequence of total splenic B-cells, follicular B-cells and marginal zone B-cells from B6.Sle1 congenic mice and C57BL/6 controls were amplified by single-cell PCR and compared. Analysis of the primary immunoglobulin heavy chain repertoire indicated that the first two sequence motifs "A" and "B" were already encoded in the naïve repertoire of B6.Sle1(z) mice, whereas the third motif "C" was introduced in part by somatic mutation. Site-directed mutagenesis confirmed that non-anionic CDR2 and cationic CDR3 residues in the immunoglobulin heavy chain facilitated nuclear antigen binding in concert, whereas aspartate at H50 strongly vetoed DNA-binding, while preserving nucleosome reactivity. Hence, anti-nuclear antibodies appear to arise as a consequence of two distinct processes-genetically programmed selection of specific CDR charge motifs into the primary immunoglobulin repertoire, with secondary contribution from somatic mutation. Polymorphisms in the lupus susceptibility gene Ly108 that impair central B-cell tolerance may be mechanistically responsible for these early repertoire differences in lupus.
Asunto(s)
Anticuerpos Antinucleares/genética , Anticuerpos Monoclonales/genética , Antígenos Ly/genética , Linfocitos B/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Lupus Eritematoso Sistémico/genética , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/inmunología , Animales , Anticuerpos Antinucleares/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos Ly/inmunología , Antígenos Nucleares/genética , Antígenos Nucleares/inmunología , Modelos Animales de Enfermedad , Cadenas Pesadas de Inmunoglobulina/inmunología , Lupus Eritematoso Sistémico/inmunología , Ratones , Polimorfismo Genético/genética , Polimorfismo Genético/inmunologíaRESUMEN
Dendritic cells (DCs) induce innate immune responses by recognizing bacterial LPS through TLR4 receptor complexes. In this study, we compared gene expression profiles of TLR4 knockout (TLR4(neg)) DCs and wild type (TLR4(pos)) DCs after stimulating with LPS. We found that the expression of various inflammatory genes by LPS were TLR4-independent. Among them, interleukin 1 receptor antagonist (IL-1rn) was of particular interest since IL-1rn is a potent natural inhibitor of proinflammatory IL-1. Using RT-PCR, real-time PCR, immunoblotting and ELISA, we demonstrated that IL-1rn was induced by DCs stimulated with LPS in the absence of TLR4. 2-Aminopurine, a pharmacological PKR inhibitor, completely abrogated LPS-induced expression of IL-1rn in TLR4(neg) DCs, suggesting that LPS-induced TLR4-independent expression of IL-1rn might be mediated by PKR pathways. Considering that IL-1rn is a physiological inhibitor of IL-1, TLR4-independent and PKR-dependent pathways might be crucial in counter-balancing proinflammatory effector functions of DCs resulted from TLR4-dependent activation by LPS.
Asunto(s)
Citocinas/metabolismo , Células Dendríticas/inmunología , Proteína Antagonista del Receptor de Interleucina 1/biosíntesis , Lipopolisacáridos/inmunología , Receptor Toll-Like 4/metabolismo , eIF-2 Quinasa/metabolismo , 2-Aminopurina/farmacología , Animales , Citocinas/genética , Citocinas/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Perfilación de la Expresión Génica , Proteína Antagonista del Receptor de Interleucina 1/agonistas , Proteína Antagonista del Receptor de Interleucina 1/inmunología , Lectinas/genética , Lectinas/inmunología , Lectinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , eIF-2 Quinasa/antagonistas & inhibidores , eIF-2 Quinasa/inmunologíaRESUMEN
Anti-nuclear antibodies constitute the hallmark of lupus. The NZM2410-derived Sle1 lupus susceptibility interval on murine chromosome 1 breaches tolerance, leading to the emergence of anti-nuclear autoantibodies targeting nucleosomes. However, little is known about the molecular structure of the anti-nucleosome autoantibodies from this genetically simplified mouse model of lupus. In this study, the immunoglobulin heavy chain and light chain sequences of 50 anti-nuclear monoclonal antibodies derived from five B6.Sle1(z) mice were compared to non-nuclear antibody controls. Compared to two different sets of non-nuclear antibodies, anti-nucleosome antibodies derived from B6.Sle1(z) congenic mice exhibited a high degree of clonal expansion and three distinct sequence motifs in their heavy chains - cationic CDR3 stretches, non-anionic CDR2 regions, and an increased frequency of aspartate residues at H50, which together increased the likelihood of an antibody being chromatin-reactive by approximately 4-fold.
Asunto(s)
Anticuerpos Antinucleares/inmunología , Cromatina/inmunología , Lupus Eritematoso Sistémico/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Predisposición Genética a la Enfermedad , Hibridomas , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Lupus Eritematoso Sistémico/genética , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Nucleosomas/inmunologíaRESUMEN
We have identified a distinct pre-naive B cell population circulating in human peripheral blood that exhibits an intermediate phenotype between transitional and naive B cells. Like human transitional B cells, these cells express CD5 but have intermediate densities of CD38, CD10, CD9, and the ABCB1 transporter compared with transitional and naive B cells. These pre-naive B cells account for a majority of circulating human CD5(+) B cells. Importantly, CD5(+) pre-naive B cells could be induced to differentiate into cells with a naive phenotype in vitro. CD5(+) pre-naive B cells show only partial responses to BCR stimulation and CD40 ligation and undergo more spontaneous apoptosis and cell death than do naive B cells, whereas BAFF/BLyS (B cell-activating factor belonging to the TNF family) did not enhance their survival compared with naive B cells. In contrast, CD5(+) pre-naive B cells carry out certain functions comparable to naive B cells, including the capacity to differentiate into plasma cells and the ability to function as APCs. Notably, an increased proportion of CD5(+) pre-naive B cells were found in peripheral blood of patients with systemic lupus erythematosus. These results have identified a unique intermediate in human naive B cell development within the peripheral blood and derangements of its homeostasis in patients with systemic lupus erythematosus.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Antígenos CD5/inmunología , Diferenciación Celular/inmunología , Lupus Eritematoso Sistémico/inmunología , Células Precursoras de Linfocitos B/inmunología , Adulto , Anciano , Subgrupos de Linfocitos B/citología , Antígenos CD5/metabolismo , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismoRESUMEN
INTRODUCTION: The present study was devised to understand the role of systemic indoleamine 2,3-dioxygenase (IDO) in the tolerance induction for orally tolerized mice in collagen-induced arthritis (CIA). We examined whether IDO-expressing dendritic cells (DCs) are involved in the generation of CD4+CD25+ regulatory T cells during the induction of oral tolerance in a murine CIA model. METHODS: Type II collagen was fed six times to DBA/1 mice beginning 2 weeks before immunization, and the effect on arthritis was assessed. To examine the IDO expression, the DCs of messenger RNA and protein were analyzed by RT-PCR and Flow cytometry. In addition, a proliferative response assay was also carried out to determine the suppressive effects of DCs through IDO. The ability of DCs expressing IDO to induce CD4+CD25+ T regulatory cells was examined. RESULTS: CD11c+ DCs in Peyer's patches from orally tolerized mice expressed a higher level of IDO than DCs from nontolerized CIA mice. IDO-expressing CD11c+ DCs were involved in the suppression of type II collagen-specific T-cell proliferation and in the downregulation of proinflammatory T helper 1 cytokine production. The suppressive effect of IDO-expressing CD11c+ DCs was mediated by Foxp3+CD4+CD25+ regulatory T cells. CONCLUSION: Our data suggest that tolerogenic CD11c+ DCs are closely linked with the induction of oral tolerance through an IDO-dependent mechanism and that this pathway may provide a new therapeutic modality to treat autoimmune arthritis.
Asunto(s)
Artritis Experimental/enzimología , Artritis Experimental/inmunología , Células Dendríticas/inmunología , Tolerancia Inmunológica , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Ganglios Linfáticos Agregados/inmunología , Linfocitos T Reguladores/inmunología , Administración Oral , Animales , Artritis Experimental/prevención & control , Antígeno CD11c/metabolismo , Proliferación Celular , Senescencia Celular , Colágeno Tipo II/administración & dosificación , Colágeno Tipo II/inmunología , Células Dendríticas/enzimología , Ratones , Ratones Endogámicos DBA , Boca/inmunología , Ganglios Linfáticos Agregados/patología , Fenotipo , Linfocitos T Reguladores/patologíaRESUMEN
OBJECTIVE: B cell hyperactivity is a common denominator in murine and human systemic lupus erythematosus. Some susceptibility genes in lupus are associated with B cell hyperactivity, but others are clearly not. While the Sle1 lupus susceptibility locus of NZM2410/NZW origin leads to chromatin-focused autoimmunity, genetically engineered overexpression of CD19 leads to "generalized" B cell hyperactivity. We undertook this study to determine the degree to which generalized B cell hyperactivity can amplify lupus pathogenesis. METHODS: To elucidate the impact of generalized B cell hyperactivity on Sle1-triggered autoimmunity, B6 mice bearing the human CD19 transgene were rendered congenic for the Sle1(z) genetic locus and phenotyped for serologic, cellular, and pathologic evidence of lupus. RESULTS: As expected, B6.Sle1.hCD19(Tg/Tg) mice, homozygous at Sle1 and bearing the hCD19 transgene, exhibited high levels of IgM and IgG anti-DNA/antiglomerular autoantibodies, skewed B cell subsets, and profoundly activated B and T cells. Despite exhibiting glomerular IgM, IgG, and complement deposits, these mice did not exhibit accelerated mortality or any clinical evidence of renal dysfunction. CONCLUSION: Generalized B cell hyperactivity may augment humoral autoimmunity, but this may not suffice to engender end-organ disease in lupus. These findings allude to the presence of an additional distal checkpoint that dissociates pathogenic autoantibody formation and renal immunoglobulin deposition from the progression to clinical nephritis in lupus.
Asunto(s)
Formación de Anticuerpos , Antígenos CD19/biosíntesis , Autoinmunidad/inmunología , Linfocitos B/inmunología , Nefritis/genética , Nefritis/inmunología , Animales , Epistasis Genética , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , RatonesRESUMEN
Transforming growth factor (TGF)-beta1 is a pleiotropic cytokine with many functions, including those related to growth modulation, immunosuppression, and pro-inflammation, in a wide variety of cell types. In this study, we investigated the ability of TGF-beta1 to regulate RANTES production by activated rheumatoid synovial fibroblasts. Fibroblast-like synoviocytes (FLS) were cultured in the presence of TGF-beta1 and IL-1beta, IL-15, TNFalpha, or IL-17, and the secretion of RANTES into culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA). Expression of RANTES encoded mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR), and NF-kappaB binding activity for RANTES transcription was determined by electrophoretic mobility shift assay (EMSA). We found that the concentrations of RANTES in synovial fluid (SF) from rheumatoid arthritis (RA) patients were lower than in SF from osteoarthritis (OA) patients, whereas the concentrations of TGF-beta1 were higher in RA SF than in OA SF. TGF-beta1 dose-dependently inhibited TNFalpha-induced production of RANTES protein and mRNA from RA FLS. Addition of RA SF with high-level TGF-beta1 mimicked the effect of TGF-beta1 on TNFalpha-induced RANTES production, which was inhibited by treatment with anti-TGF-beta1 neutralizing antibody. TGF-beta1 blocked the degradation of cytosolic IkappaB-alpha and the translocation of activated NF-kappaB to the nucleus. EMSA showed that the inhibitory effect of TGF-beta1 was associated with decreased binding of NF-kappaB to the RANTES promoter. These results suggest that elevated TGF-beta1 in rheumatoid synovial tissue may suppress joint inflammation by inhibiting RANTES secretion from synovial fibroblasts, thus blocking the infiltration of immune cells. These findings may provide an explanation for the mechanism by which TGF-beta1 regulates immune function in RA.
Asunto(s)
Artritis Reumatoide/metabolismo , Quimiocina CCL5/biosíntesis , Regulación hacia Abajo/efectos de los fármacos , FN-kappa B/metabolismo , Membrana Sinovial/citología , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adulto , Anciano , Anciano de 80 o más Años , Artritis Reumatoide/genética , Células Cultivadas , Quimiocina CCL5/genética , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Osteoartritis/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Mensajero/genética , Líquido Sinovial/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1 , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Although a rich database of Igh a allotype mouse immunoglobulin germline genes exists, current information on Igh b allotype immunoglobulin germline genes is limited. Among the immunoglobulin VH genes, single-cell amplified from six Igh b (C57BL/6 background) spleens in this study, 602 clonally independent immunoglobulin VH sequences belonging to the VH1/J558 family were identified. Whereas 335 of these sequences could be traced to have originated from 29 different VH1/J558 germline genes deposited in the NCBI Igblast database, the remaining 267 sequences appeared to have originated from 21 novel germline genes. Of the 50 VH1/J558 germline genes utilized in the peripheral repertoire of these Igh b allotype mice, the most frequently used genes included 45.21.2, V165.1, J558.6, J558.18A, and V23. Whereas the majority of the novel genes uncovered represented allelic counterparts of previously described Balb/c (Igh a allotype) genes, some appeared to represent truly novel germline genes. Collectively, the VH1/J558 germline genes exhibited high amino acid residue usage variability at the CDR1 positions, H31, H33, and H35, and the CDR2 positions, H50, H52, H53, H54, H56, and H58. The 50 VH1/J558 germline genes expressed in the peripheral Igh b repertoire also varied widely in the net charge of their CDR regions, raising the possibility that they may be differentially utilized to encode anti-nuclear autoantibodies.
Asunto(s)
Genes de Inmunoglobulinas , Ratones Endogámicos C57BL/genética , Ratones Endogámicos C57BL/inmunología , Animales , Secuencia de Bases , Regiones Determinantes de Complementariedad , ADN/química , ADN/genética , Alotipos de Inmunoglobulinas/genética , Cadenas Pesadas de Inmunoglobulina/genética , RatonesRESUMEN
OBJECTIVE: (SWR x NZB)F(1) (or SNF(1)) hybrid mice succumb to lupus nephritis. A previous analysis of SNF(1) x NZB backcross mice revealed the existence of 4 SWR loci (H2 on chromosome 17, Swrl-1 on chromosome 1, Swrl-2 on chromosome 14, and Swrl-3 on chromosome 18) and 2 NZB loci (Nba1 and Lbw2/Sbw2, both on chromosome 4). A second study focusing on SNF(1) x SWR backcross offspring uncovered 5 suggestive loci for antinuclear antibody formation, consisting of 3 dominant NZB contributions (Nba4 on chromosome 5, Lbw4 on chromosome 6, and Nba5 on chromosome 7) and 2 recessive SWR contributions (Swrl-1 on chromosome 1 and Swrl-4 on chromosome 10). The present intercross study was executed to replicate the earlier findings, using an independent panel of (SWR x NZB)F(2) offspring. METHODS: A panel of (NZB x SWR)F(2) hybrids were phenotyped (for renal disease, early mortality, and a variety of autoantibodies) and genotyped (using 95 microsatellite primers positioned across all 19 autosomes and the X chromosome). Linkage analysis was conducted using the derived phenotype and genotype data, with the interval-mapping program MapManager. RESULTS: Four suggestive loci were mapped: Swrl-5 on chromosome 1 (peak at 106 cM), linked to hypergammaglobulinemia; an NZB locus on chromosome 5 (Nba4; peak at 15 cM), linked to IgG anti-single-stranded DNA (anti-ssDNA) antibodies, IgG anti-doubled-stranded DNA (anti-dsDNA) antibodies, and glomerulonephritis; an NZB locus on chromosome 13 (Nba6; peak at 28 cM), linked to IgG anti-dsDNA antibodies; and an SWR locus on chromosome 14 (Swrl-2; peak at 30 cM), linked to IgG anti-ssDNA antibodies. Eight additional loci revealed linkage at P < 0.01, of which 7 co-mapped with lupus susceptibility loci previously identified in other models. CONCLUSION: Considering all 3 mapping studies together, lupus in SWR/NZB hybrids appears to be the epistatic end product of several distinct loci, of which 3 SWR-derived loci (Swrl-1, Swrl-2, and Swrl-3) and 5 NZB-derived loci (Nba1, Nba3, Nba4, Nba5, and Lbw4) have been independently confirmed. The immunologic functions and molecular identities of these loci await elucidation.
Asunto(s)
Nefritis Lúpica/genética , Animales , Anticuerpos Antinucleares/sangre , Quimera , Mapeo Cromosómico , Cruzamientos Genéticos , ADN/inmunología , ADN de Cadena Simple/inmunología , Ligamiento Genético , Genotipo , Inmunoglobulina G/análisis , Ratones , Ratones Endogámicos NZBRESUMEN
OBJECTIVE: The autoantibody specificities that dominate the deposits in lupus kidneys remain unclear. Reasoning that previously utilized elution buffers such as acidic glycine and ammonium thiocyanate may not have been maximally effective in eluting all Ig deposits from the kidneys, this study was conducted to experiment with a stronger dissociating agent, urea-glycine. METHODS: Seven antinuclear antibody-positive, nephritic female (SWR x NZB)F(1) (SNF1) lupus mice were selected for the elution study. Deposited Ig was eluted from their kidneys using 3 different elution buffers: 0.15M glycine-HCl buffer, 1.3M ammonia thiocyanate/0.15M glycine-HCl buffer, and 5M urea/0.15M glycine-HCl buffer. All eluates were tested for specificity against a variety of nuclear and glomerular antigens. RESULTS: Compared with conventional elution buffers, the urea-based regimen eluted severalfold more IgG and IgM antinuclear antibodies from the kidneys of nephritic SNF1 lupus mice. IgG anti-double-stranded DNA (anti-dsDNA) antibodies were not only the most prevalent species in these renal deposits, they were also heavily enriched in the kidneys, relative to the corresponding serum levels. A substantial fraction of the anti-single-stranded DNA and antihistone/DNA (but not antihistone) reactivity in these eluates was due to cross-reactive anti-dsDNA antibodies. No reactivity with SSA, SSB, Sm, RNP, Jo-1, Scl-70, or ribosomal P antigens could be demonstrated in these eluates. Importantly, the urea-glycine eluates differed from the conventional eluates in having significantly greater reactivity to glomerular substrate and laminin. CONCLUSION: This novel urea-based elution provides further support for the dominance of antibodies in lupus kidneys, with strong polyreactivity to DNA and glomerular substrate.
Asunto(s)
Anticuerpos Antinucleares/sangre , Anticuerpos Antinucleares/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/métodos , Nefritis Lúpica/inmunología , Animales , Tampones (Química) , ADN/inmunología , ADN de Cadena Simple/inmunología , Modelos Animales de Enfermedad , Glicina , Histonas/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina M/sangre , Inmunoglobulina M/aislamiento & purificación , Ratones , Ratones Endogámicos NZB , Organismos Libres de Patógenos Específicos , UreaRESUMEN
A database of the Ig heavy chains of 143 anti-ssDNA, 103 anti-dsDNA and 23 anti-nucleosome antinuclear antibodies (ANAs) was constructed, with no clonal overlap, gleaning from published literature. In comparison to the Kabat database of antibodies (N>3600), ANAs (total=269) demonstrated several significant changes, particularly in the incidence of charged or polar residues, in their CDR regions. In particular, anti-dsDNA ANAs differed significantly from anti-ssDNA ANAs in having (a) more 'D' residues at H31 and more 'Y' residues at H33, in CDR1, (b) significantly different distributions of charged or polar residues at H53, H55 and H56 of CDR2, and (c) more 'R' residues at H95-H100 of CDR3. Whereas, the differences in CDR1 and CDR3 are likely to characterize anti-dsDNA ANAs encoded by all VH families, the sequence differences in CDR2 are likely to be VH family specific. Finally, among anti-dsDNA ANAs, there was an enrichment of VH1/J558 germline genes (notably, VH 45.21.1), which bear germline-encoded amino acid residues in their CDR regions that may potentially facilitate nuclear antigen binding. This ANA heavy chain database thus constitutes a useful resource for analyzing the molecular requirements for nuclear antigen reactivity.