RESUMO
OBJECTIVE: Sex disparities in rheumatoid arthritis (RA) are well documented despite the lack of any known major RA susceptibility genes mapped to sex chromosomes. Murine chromosome 15 carries the sex-affected Pgia8 locus that mediates proteoglycan-induced arthritis, and homologous human loci are associated with RA. This study was undertaken to identify genes/mechanisms implicated in sex disparities in arthritis. METHODS: Gene expression analysis was performed using RNA isolated from the paws of male and female Pgia8-congenic mice with collagen antibody-induced arthritis. Results were corroborated by reverse transcription-polymerase chain reaction, and mice were also studied prior to disease onset. Ingenuity Pathways Analysis of the expression patterns and gene functions was used to discover locus-specific and sex-affected signature transcripts. RESULTS: We found that the Pgia8 locus regulates antibody-mediated inflammatory arthritis differently in males and females. In Pgia8-congenic males, arthritis severity was 30% less (P < 0.005) than in wild-type males, but the antiinflammatory effect was similar in wild-type and congenic females. Transcriptome analysis indicated that 12 genes within the locus were significantly dysregulated in arthritic joints of congenic mice; expression of these genes was also sex specific. The genes that correlated most highly with arthritis severity included those for collagen triple-helix repeat-containing 1 (Cthrc1), metalloproteinase (Adamts12), R-spondin (Rspo2), and syndecan (Sdc2) (r = 0.87-0.91). The level of Cthrc1 message also correlated with that of the genes for the proinflammatory cytokines interleukin-1ß and interleukin-6. CONCLUSION: These results indicate that sex-specific disparities in RA are linked to transcriptional regulation of genes involved in cartilage degradation (Adamts12) and canonical and noncanonical Wnt signaling (Cthrc1, Rspo2, Sdc2).
Assuntos
Artrite Experimental/genética , Artrite Reumatoide/genética , Cromossomos Humanos Par 15/metabolismo , Via de Sinalização Wnt/genética , Animais , Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Modelos Animais de Doenças , Feminino , Expressão Gênica , Predisposição Genética para Doença , Humanos , Masculino , Camundongos , Índice de Gravidade de Doença , Fatores SexuaisRESUMO
We found a spontaneous autosomal mutation in a mouse leading to neutrophil infiltration with ulceration in the upper dermis of homozygous offspring. These animals had increased neutrophil numbers, associated with normal lymphocyte count, in peripheral blood and bone marrow, suggesting a myeloproliferative disorder; however, granulocyte precursor proliferation in bone marrow was actually reduced (because circulating neutrophils were less susceptible to apoptosis). Neutrophil infiltration of the skin and other organs and high serum levels of immunoglobulins and autoantibodies, cytokines, and acute-phase proteins were additional abnormalities, all of which could be reduced by high-dose corticosteroid treatment or neutrophil depletion by antibodies. Use of genome-wide screening localized the mutation within an 0.4-Mbp region on mouse chromosome 6. We identified insertion of a B2 element in exon 6 of the Ptpn6 gene (protein tyrosine phosphatase, non-receptor type 6; also known as Shp-1). This insertion involves amino acid substitutions that significantly reduced the enzyme activity in mice homozygous for the mutation. Disease onset was delayed, and the clinical phenotype was milder than the phenotypes of other Ptpn6-mutants described in motheaten (me, mev) mice; we designated this new genotype as Ptpn6(meB2/meB2) and the phenotype as meB2. This new phenotype encompasses an autoinflammatory disease showing similarities to many aspects of the so-called neutrophilic dermatoses, a heterogeneous group of skin diseases with unknown etiology in humans.
Assuntos
Doenças Hereditárias Autoinflamatórias/genética , Neutrófilos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/fisiologia , Dermatopatias/metabolismo , Corticosteroides/farmacologia , Animais , Autoanticorpos/química , Mapeamento Cromossômico , Homozigoto , Humanos , Imunoglobulinas/química , Inflamação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , MutaçãoRESUMO
Using genetic linkage analysis of proteoglycan-induced arthritis (PGIA), a murine model for rheumatoid arthritis, we identified two loci, Pgia8 and Pgia9, on chromosome 15 (chr15) that appear to be implicated in disease susceptibility. Immunization of congenic strains carrying the entire chr15 and separately each of the two loci of DBA/2 arthritis-resistant origin in susceptible BALB/c background confirmed locations of two loci on chr15: the major Pgia9 and lesser Pgia8 locus. Distal part of chr15 (Pgia9) showed a major suppressive effect on PGIA susceptibility in females (40%, p < 0.001), whereas the effect of this locus in congenic males was still significant but weaker. Proximal part of chr15 (Pgia8) demonstrated mild and transient effect upon arthritis; this effect was PGIA-promoting in males and suppressive in females. Pgia8 and Pgia9 loci demonstrated an additive mode of inheritance, since when they were both incorporated in consomic chr15 strain, the total effect was a sum of the two loci. Using F(2) population of the intercross of wild-type and chr15 consomic strain, we confirmed and refined quantitative trait locus positions and identified a strong correlation between disease susceptibility and lymphocyte-producing cytokines of TNF-alpha and IL-6. Both Pgia8 and Pgia9 loci on chr15 appear to control IL-6 production in spleen cultures of arthritic mice, providing an important link to the mechanism of autoimmune inflammation.
Assuntos
Artrite Experimental/genética , Cromossomos de Mamíferos/genética , Interleucina-6/metabolismo , Ativação Linfocitária , Locos de Características Quantitativas , Fator de Necrose Tumoral alfa/metabolismo , Animais , Artrite Experimental/imunologia , Proliferação de Células , Feminino , Predisposição Genética para Doença , Humanos , Interleucina-6/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Proteoglicanas/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Background: The purpose of this study was to determine whether plasma levels of the collagen triple helix repeat containing 1 (CTHRC1) protein can serve as a blood-based biomarker for improved diagnosis of rheumatoid arthritis (RA) patients and monitoring of RA disease activity. Methods: We measured levels of CTHRC1 in the plasma of patients diagnosed with RA, osteoarthritis (OA), reactive arthritis (ReA), as well as in healthy individuals. We then assessed the correlation between CTHRC1 protein and a range of indices including the 28-joint disease activity score (DAS28), rheumatoid factor (RF), C-reactive protein (CRP), anti-citrullinated protein antibodies (ACPA), erythrocyte sedimentation rate (ESR), as well as a panel of cytokines, including interleukin 1 beta (IL-1ß), interleukin 6 (IL-6), interleukin 8 (IL-8), and interferon gamma (IFNγ). Receiver operating characteristic (ROC) analysis was further performed to assess the diagnostic value of CTHRC1. Results: CTHRC1 plasma levels were significantly elevated in RA patients compared to healthy individuals, OA and ReA patients. ROC curve and risk score analysis suggested that plasma CTHRC1 can accurately discriminate patients with RA from healthy controls and may have practical value for RA diagnosis. CTHRC1 levels were positively associated with RF, ACPA, CRP, and disease activity based on the combined index of DAS28 with CRP (DAS28-CRP), and also strongly correlated with IL-1ß, IL-6, IL-8, and IFNγ. Conclusion: Our studies show that CTHRC1 is a sensitive and easy-to-measure plasma marker that differentiates between RA and healthy status and also distinguishes between RA and other forms of arthritis, such as OA and ReA. At the current level of understanding, plasma CTHRC1 levels may improve the diagnosis of RA and these findings warrant confirmation in a larger, more comprehensive patient population.
Assuntos
Artrite Reumatoide/diagnóstico , Biomarcadores/sangue , Proteínas da Matriz Extracelular/sangue , Osteoartrite/diagnóstico , Adulto , Idoso , Anticorpos Antiproteína Citrulinada/metabolismo , Citocinas/metabolismo , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proibitinas , Índice de Gravidade de Doença , Regulação para CimaRESUMO
Collagen triple helix repeat-containing1 (Cthrc1) has previously been implicated in osteogenic differentiation and positive regulation of bone mass, however, the underlying mechanisms remain unclear. Here we characterized the bone phenotype of a novel Cthrc1 null mouse strain using bone histomorphometry, µCT analysis and functional readouts for bone strength. In male Cthrc1 null mice both trabecular bone as well as cortical bone formation was impaired, whereas in female Cthrc1 null mice only trabecular bone parameters were altered. Novel and highly specific monoclonal antibodies revealed that CTHRC1 is expressed by osteocytes and osteoblasts, but not osteoclasts. Furthermore, Cthrc1 null mice exhibited increased bone resorption with increased number of osteoclast and increased osteoclast activity together with enhanced expression of osteoclastogenic genes such as c-Fos, Rankl, Trap, and Nfatc1. Differentiation of bone marrow-derived monocytes isolated from Cthrc1 null mice differentiated into osteoclasts as effectively as those from wildtype mice. In the presence of CTHRC1 osteoclastogenic differentiation of bone marrow-derived monocytes was dramatically inhibited as was functional bone resorption by osteoclasts. This process was accompanied by downregulation of osteoclastogenic marker genes, indicating that extrinsically derived CTHRC1 is required for such activity. In vitro, CTHRC1 had no effect on osteogenic differentiation of bone marrow stromal cells, however, calvarial osteoblasts from Cthrc1 null mice exhibited reduced osteogenic differentiation compared to osteoblasts from wildtypes. In a collagen antibody-induced arthritis model Cthrc1 null mice suffered significantly more severe inflammation and joint destruction than wildtypes, suggesting that CTHRC1 expressed by the activated synoviocytes has anti-inflammatory effects. Mechanistically, we found that CTHRC1 inhibited NFκB activation by preventing IκBα degradation while also inhibiting ERK1/2 activation. Collectively our studies demonstrate that CTHRC1 secreted from osteocytes and osteoblasts functions as an inhibitor of osteoclast differentiation via inhibition of NFκB-dependent signaling. Furthermore, our data suggest that CTHRC1 has potent anti-inflammatory properties that limit arthritic joint destruction.
Assuntos
Artrite Experimental/metabolismo , Artrite Experimental/patologia , Diferenciação Celular , Proteínas da Matriz Extracelular/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patologia , Animais , Anticorpos , Fenômenos Biomecânicos , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Reabsorção Óssea/patologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/patologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Osteócitos/patologia , Osteogênese/efeitos dos fármacos , Ligante RANK/farmacologia , Transdução de Sinais/efeitos dos fármacos , Crânio/patologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Microtomografia por Raio-XRESUMO
To explore early signature genes playing critical roles in the initial steps in an autoimmune murine model of rheumatoid arthritis (RA) (proteoglycan (PG)-induced arthritis; PGIA), we performed gene expression profiling of "arthritogenic" spleen cells stimulated with cartilage PG, and compared them to differentially expressed genes, identified in joints prior to the onset of arthritis, and then in the acute and chronic phases of the disease. A total of 280 genes were up-regulated and 226 genes were suppressed in in vitro PG-stimulated lymphocytes at a minimum of 2-fold expression change. Functional gene classification identified several major clusters of biological activity. Expression of immunoglobulin genes (66 transcripts) was downregulated by approximately 3.7-fold, whereas most of the other genes with immune/inflammation-associated functions such as interleukins (IL-1, -2, -4, -6, -10, -12, -16, -17), chemokine receptors and their ligands (Cxcl1, Ccl2, 7, 8, 9, 10, 22, Ccr2, Ccr5), and major components of the complement cascade were upregulated. Using adoptive disease transfer with stimulated lymphocytes into SCID mice, followed by gene expression profiling of SCID paws, indicated that 37 genes were differentially expressed in yet non-inflamed (pre-arthritic) paws; these genes were related mostly to chemokine, IFN-gamma and TNF-alpha signaling. However, the majority of differentially expressed immune response-related genes were silent in pre-arthritic joints, and only 12 genes were found differentially expressed both in antigen (PG)-stimulated lymphocytes and in the synovium prior to the onset of arthritis. Most of these "arthritis-initiation" genes belonged to chemokine mediated cell motility. Transcripts of chemokine receptor 5 (Ccr5), chemokine ligand 7 (Ccl7) and IFN-gamma-inducible proteins (Ifi47) and GTP-ase 1 were expressed at the highest levels in both antigen-stimulated lymphocytes and pre-inflamed synovium, which suggests a key role of these genes in both lymphocyte maturation and arthritis initiation.
Assuntos
Artrite Reumatoide/genética , Expressão Gênica/imunologia , Linfócitos/imunologia , Membrana Sinovial/metabolismo , Transferência Adotiva , Animais , Artrite Experimental/genética , Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Análise de Sequência com Séries de OligonucleotídeosRESUMO
BACKGROUND: The formation of destructive hypercellular pannus is critical to joint damage in rheumatoid arthritis (RA). The collagen triple helix repeat containing 1 (CTHRC1) protein expressed by activated stromal cells of diverse origin has previously been implicated in tissue remodeling and carcinogenesis. We recently discovered that the synovial Cthrc1 mRNA directly correlates with arthritis severity in mice. This study characterizes the role of CTHRC1 in arthritic pannus formation. METHODS: Synovial joints of mice with collagen antibody-induced arthritis (CAIA) and human RA-fibroblast-like synoviocytes (FLS) were immunostained for CTHRC1, FLS and macrophage-specific markers. CTHRC1 levels in plasma from patients with RA were measured using sandwich ELISA. The migratory response of fibroblasts was studied with a transwell migration assay and time-lapse microscopy. Velocity and directness of cell migration was analyzed by recording the trajectories of cells treated with rhCTHRC1. RESULTS: Immunohistochemical analysis of normal and inflamed synovium revealed highly inducible expression of CTHRC1 in arthritis (10.9-fold). At the tissue level, CTHRC1-expressing cells occupied the same niche as large fibroblast-like cells positive for α-smooth muscle actin (α-SMA) and cadherin 11 (CDH11). CTHRC1 was produced by activated FLS predominantly located at the synovial intimal lining and at the bone-pannus interface. Cultured RA-FLS expressed CDH11, α-SMA, and CTHRC1. Upon treatment with exogenous rhCTHRC1, embryonic fibroblasts and RA-FLS significantly increased migration velocity, directness, and cell length along the front-tail axis (1.4-fold, p < 0.01). CONCLUSION: CTHRC1 was established as a novel marker of activated synoviocytes in murine experimental arthritis and RA. The pro-migratory effect of CTHRC1 on synoviocytes is considered one of the mechanisms promoting hypercellularity of the arthritic pannus.
Assuntos
Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Biomarcadores/análise , Proteínas da Matriz Extracelular/biossíntese , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Western Blotting , Movimento Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Fibroblastos/metabolismo , Tecido de Granulação , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase em Tempo Real , Sinoviócitos/metabolismoRESUMO
Brucellosis, a frequent bacterial zoonosis, can produce debilitating chronic disease with involvement of multiple organs in human patients. Whereas acute brucellosis is well studied using the murine animal model, long-term complications of host-pathogen interaction remain largely elusive. Human brucellosis frequently results in persistent, chronic osteoarticular system involvement, with complications such as arthritis, spondylitis and sacroiliitis. Here, we focused on identifying infectious sites in the mouse that parallel Brucella melitensis foci observed in patients. In vivo imaging showed rapid bacterial dispersal to multiple sites of the murine axial skeleton. In agreement with these findings, immunohistochemistry revealed the presence of bacteria in bones and limbs, and in the lower spine vertebrae of the axial skeleton where they were preferentially located in the bone marrow. Surprisingly, some animals developed arthritis in paws and spine after infection, but without obvious bacteria in these sites. The identification of Brucella in the bones of mice corroborates the findings in humans that these osteoarticular sites are important niches for the persistence of Brucella in the host, but the mechanisms that mediate pathological manifestations in these sites remain unclear. Future studies addressing the immune responses within osteoarticular tissue foci could elucidate important tissue injury mediators and Brucella survival strategies.
Assuntos
Osso e Ossos/microbiologia , Osso e Ossos/patologia , Brucelose/microbiologia , Brucelose/patologia , Articulações/microbiologia , Articulações/patologia , Animais , Artrite/microbiologia , Artrite/patologia , Medula Óssea/microbiologia , Medula Óssea/patologia , Brucella melitensis/fisiologia , Feminino , Interações Hospedeiro-Patógeno , Humanos , Fígado/microbiologia , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/microbiologia , Baço/patologiaRESUMO
BACKGROUND: Angiotensin-converting enzyme (ACE) metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling. Elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases, including asthma. Previously, a molecular mechanism underlying a 5-fold familial increase of blood ACE was discovered: Pro1199Leu substitution enhanced the cleavage-secretion process. Carriers of this mutation were Caucasians from Europe (mostly Dutch) or had European roots. METHODOLOGY/PRINCIPAL FINDINGS: We have found a family of African-American descent whose affected members' blood ACE level was increased 13-fold over normal. In affected family members, codon TGG coding for Trp1197 was substituted in one allele by TGA (stop codon). As a result, half of ACE expressed in these individuals had a length of 1196 amino acids and lacked a transmembrane anchor. This ACE mutant is not trafficked to the cell membrane and is directly secreted out of cells; this mechanism apparently accounts for the high serum ACE level seen in affected individuals. A haplotype of the mutant ACE allele was determined based on 12 polymorphisms, which may help to identify other carriers of this mutation. Some but not all carriers of this mutation demonstrated airflow obstruction, and some but not all have hypertension. CONCLUSIONS/SIGNIFICANCE: We have identified a novel Trp1197Stop mutation that results in dramatic elevation of serum ACE. Since blood ACE elevation is often taken as a marker of disease activity (sarcoidosis and Gaucher diseases), it is important for clinicians and medical scientists to be aware of alternative genetic causes of elevated blood ACE that are not apparently linked to disease.
Assuntos
Substituição de Aminoácidos/genética , Mutação/genética , Peptidil Dipeptidase A/sangue , Peptidil Dipeptidase A/genética , Animais , Sequência de Bases , Western Blotting , Células CHO , Análise por Conglomerados , Cricetinae , Cricetulus , Análise Mutacional de DNA , Etnicidade/genética , Feminino , Dosagem de Genes/genética , Haplótipos/genética , Humanos , Masculino , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Linhagem , Peptidil Dipeptidase A/química , Conformação Proteica , Proteínas Recombinantes/genética , TransfecçãoRESUMO
Spondyloarthropathies (SpAs), including ankylosing spondylitis, are chronic inflammatory diseases of the axial skeleton. Genomic scans of SpA families revealed the overwhelming complexity of the disease, which appears to be under the control of over 20 chromosome loci, including the major SpA gene HLA-B27 within class I of the major histocompatibility complex (MHC). Animal models confirmed the primary role of MHC in SpA susceptibility and supported the hypothesis that certain enterobacterial infections can trigger SpA. Immunization of mice with proteoglycan aggrecan also can provoke SpA, thus providing the opportunity to study genetic and clinical details of the disease initiation.
Assuntos
Modelos Animais de Doenças , Complexo Principal de Histocompatibilidade/genética , Espondilite Anquilosante/genética , Espondilite Anquilosante/patologia , Agrecanas , Animais , Proteoglicanas de Sulfatos de Condroitina/imunologia , Proteínas da Matriz Extracelular/imunologia , Predisposição Genética para Doença , Genoma Humano , Humanos , Lectinas Tipo C/imunologia , Camundongos , Espondilite Anquilosante/imunologiaRESUMO
We present here an extensive study of differential gene expression in the initiation, acute and chronic phases of murine autoimmune arthritis with the use of high-density oligonucleotide arrays interrogating the entire mouse genome. Arthritis was induced in severe combined immunodeficient mice by using adoptive transfer of lymphocytes from proteoglycan-immunized arthritic BALB/c mice. In this unique system only proteoglycan-specific lymphocytes are transferred from arthritic mice into syngeneic immunodeficient recipients that lack adaptive immunity but have intact innate immunity on an identical (BALB/c) genetic background.Differential gene expression in response to donor lymphocytes that migrated into the joint can therefore be monitored in a precisely timed manner, even before the onset of inflammation. The initiation phase of adoptively transferred disease (several days before the onset of joint swelling) was characterized by differential expression of 37 genes, mostly related to chemokines, interferon-gamma and tumor necrosis factor-alpha signaling, and T cell functions. These were designated early arthritis 'signature' genes because they could distinguish between the naive and the pre-arthritic state. Acute joint inflammation was characterized by at least twofold overexpression of 256 genes and the downregulation of 21 genes, whereas in chronic arthritis a total of 418 genes with an equal proportion of upregulated and downregulated transcripts were expressed differentially. Hierarchical clustering and functional classification of inflammation-related and arthritis-related genes indicated that the most common biological activities were represented by genes encoding interleukins, chemokine receptors and ligands, and by those involved in antigen recognition and processing.
Assuntos
Artrite Experimental/genética , Doenças Autoimunes/genética , Perfilação da Expressão Gênica , Doença Aguda , Transferência Adotiva , Animais , Artrite Experimental/etiologia , Artrite Experimental/imunologia , Doenças Autoimunes/etiologia , Doenças Autoimunes/imunologia , Quimiocinas/biossíntese , Quimiocinas/genética , Doença Crônica , Progressão da Doença , Feminino , Regulação da Expressão Gênica , Humanos , Interferon gama/biossíntese , Interferon gama/genética , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Proteoglicanas/imunologia , Baço/imunologia , Subpopulações de Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
Autoimmune spondylitis was induced in BALB/c mice and their MHC-matched (BALB/c x DBA/2)F1 and F2 hybrids by systemic immunization with cartilage/intervertebral disk proteoglycan (PG). As in human ankylosing spondylitis, the MHC was the major permissive genetic locus in murine PG-induced spondylitis (PGIS). Two major non-MHC chromosome loci with highly significant linkage were found on chromosomes 2 (Pgis2) and 18 (Pgis1) accounting for 40% of the entire F2 trait variance. The dominant spondylitis-susceptibility allele for Pgis2 locus is derived from the BALB/c strain, whereas the Pgis1 recessive allele was present in the disease-resistant DBA/2 strain. The Pgis1 locus significantly affected the disease-controlling Pgis2 locus, inducing as high incidence of spondylitis in F2 hybrids as was found in the spondylitis-susceptible parent BALB/c strain. Additional disease-controlling loci with suggestive linkage were mapped to the chromosomes 12, 15, and 19. Severity of spondylitis in F2 mice positively correlated with serum levels of amyloid A, IL-6, and Pg-specific Abs, and showed negative correlation with Ag-induced T cell proliferation, IFN-gamma, IL-4, and TNF-alpha production. A major locus controlling serum IL-6 was found on chromosome 14 near osteoclast differentiation factor Tnfsf11. Locus on chromosome 11 near the Stat3 and Stat5 genes controlled serum level of the Ig IgG2a isotype. The two major genetic loci Pgis1 and Pgis2 of murine spondylitis were homologous to chromosome regions in human genome, which control ankylosing spondylitis in human patients. Thus, this animal model of experimentally induced spondylitis might facilitate the identification of spondylitis-susceptibility genes in humans.
Assuntos
Predisposição Genética para Doença , Espondiloartropatias/genética , Espondiloartropatias/imunologia , Animais , Mapeamento Cromossômico , Cruzamentos Genéticos , Modelos Animais de Doenças , Feminino , Ligação Genética , Marcadores Genéticos/imunologia , Humanos , Imunidade Inata/genética , Incidência , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Proteoglicanas/administração & dosagem , Proteoglicanas/imunologia , Locos de Características Quantitativas/genética , Locos de Características Quantitativas/imunologia , Homologia de Sequência do Ácido NucleicoRESUMO
We previously reported that the alpha-subunit of heterotrimeric G13 protein induces either mitogenesis and neoplastic transformation or apoptosis in a cell-dependent manner. Here, we analyzed which signaling pathways are required for G alpha 13-induced mitogenesis or apoptosis using a novel mutant of G alpha 13. We have identified that in human cell line LoVo, the mutation encoding substitution of Arg260 to stop codon in mRNA of G alpha 13 subunit produced a mutant protein (G alpha 13-T) that lacks a COOH terminus and is endogenously expressed in LoVo cells as a polypeptide of 30 kDa. We found that G alpha 13-T lost its ability to promote proliferation and transformation but retained its ability to induce apoptosis. We found that full-length G alpha 13 could stimulate Elk1 transcription factor, whereas truncated G alpha 13 lost this ability. G alpha 13-dependent stimulation of Elk1 was inhibited by dominant-negative extracellular signal-regulated kinase (MEK) but not by dominant-negative MEKK1. Similarly, MEK inhibitor PD-98059 blocked G alpha 13-induced Elk1 stimulation, whereas JNK inhibitor SB-203580 was ineffective. In Rat-1 fibroblasts, G alpha 13-induced cell proliferation and foci formation were also inhibited by dominant-negative MEK and PD-98059 but not by dominant-negative MEKK1 and SB-203580. Whereas G alpha 13-T alone did not induce transformation, coexpression with constitutively active MEK partially restored its ability to transform Rat-1 cells. Importantly, full-length but not G alpha 13-T could stimulate Src kinase activity. Moreover, G alpha 13-dependent stimulation of Elk1, cell proliferation, and foci formation were inhibited by tyrosine kinase inhibitor, genistein, or by dominant-negative Src kinase, suggesting the involvement of a Src-dependent pathway in the G alpha 13-mediated cell proliferation and transformation. Importantly, truncated G alpha 13 retained its ability to stimulate apoptosis signal-regulated kinase ASK1 and c-Jun terminal kinase, JNK. Interestingly, the apoptosis induced by G alpha 13-T was inhibited by dominant-negative ASK1 or by SB-203580.
Assuntos
Apoptose/fisiologia , Transformação Celular Neoplásica , Proteínas de Ligação a DNA/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Fibroblastos/fisiologia , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP , Humanos , Mitógenos/farmacologia , Conformação Molecular , Mutação , Ratos , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Collagen-induced arthritis (CIA) in DBA/1 and proteoglycan-induced arthritis (PGIA) in BALB/c mice are the most frequently used mouse models for studying clinical, immunological and genetic factors contributing to rheumatoid arthritis. DBA/1 ( H2(q)) mice are susceptible to CIA but resistant to PGIA, whereas BALB/c mice ( H2 (d)) are susceptible to PGIA and resistant to CIA. To gain insight into the mechanisms of how the major clinical (disease susceptibility, severity and onset of arthritis) and immunological traits (antigen-specific T- and B-cell responses) are influenced by the major histocompatibility complex (MHC), we have generated a unique intercross of BALB/c and DBA/1 parent strains, and the F1 and F2 hybrids were immunized for either CIA or PGIA. The major clinical and immunological traits were identified as either binary (qualitative) or quantitative traits on Chromosome 17 with a peak at MHC when the entire population was analyzed. In contrast, when only arthritic (susceptible) mice were selected and analyzed, the major clinical traits (severity and onset) 'lost' the linkage to MHC. Thus, MHC dictates disease susceptibility, but not the severity of arthritis. This was even more evident in the case of the H2(q) allele, which was clearly responsible for the dominant inheritance of arthritis in F2 hybrids (either CIA or PGIA). In conclusion, while certain MHC alleles strongly affect disease susceptibility and determine the mode of inheritance of a polygenic autoimmune disease, neither the type of inheritance (dominant vs recessive) nor other MHC components have evident effects upon the clinical symptoms of arthritis.
Assuntos
Artrite Reumatoide/genética , Predisposição Genética para Doença , Complexo Principal de Histocompatibilidade , Animais , Anticorpos/sangue , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/imunologia , Colágeno , Cruzamentos Genéticos , Genes Dominantes , Ligação Genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Proteoglicanas , Característica Quantitativa Herdável , Especificidade da Espécie , Linfócitos T/imunologiaRESUMO
Collagen-induced arthritis (CIA) and proteoglycan-induced arthritis (PGIA) are murine models for rheumatoid arthritis both in terms of their pathology and genetics. Using the F(2) hybrids of the CIA-susceptible, but PGIA-resistant DBA/1 mice, and the CIA-resistant, but PGIA-susceptible BALB/c mice, our goals were to 1) identify both model-specific and shared loci that confer disease susceptibility, 2) determine whether any pathophysiological parameters could be used as markers that distinguish between nonarthritic and arthritic mice, and 3) analyze whether any immune subtraits showed colocalization with arthritis-related loci. To identify chromosomal loci, we performed a genome scan on 939 F(2) hybrid mice. For pathophysiological analyses, we measured pro- and anti-inflammatory cytokines (IL-1, IL-6, TNF-alpha, IFN-gamma, IL-4, IL-10, IL-12), Ag-specific T cell proliferation and IL-2 production, serum IgG1 and IgG2 levels of both auto- and heteroantibodies, and soluble CD44. In addition to multiple CIA- and PGIA-related loci identified in previous studies, we have identified nine new CIA- and eight new PGIA-linked loci. Comprehensive statistical analysis demonstrated that IL-2 production, T cell proliferation, and IFN-gamma levels differed significantly between arthritic and nonarthritic animals in both CIA and PGIA populations. High levels of TNF-alpha, IFN-gamma, IL-2, and Ab production were detected in F(2) hybrids with CIA, whereas T cell proliferation, IL-2 and IFN-gamma production, and a shift to IgG2a isotype were more characteristic of PGIA. Quantitative trait loci analysis demonstrated colocalization of numerous immune subtraits with arthritis-related traits. Quantitative trait loci on chromosomes 5, 10, 17, 18, and X were found to control arthritis in both models.
Assuntos
Artrite Experimental/genética , Artrite Experimental/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Modelos Animais de Doenças , Locos de Características Quantitativas/imunologia , Doença Aguda , Animais , Artrite Experimental/patologia , Artrite Experimental/fisiopatologia , Doenças Autoimunes/patologia , Doenças Autoimunes/fisiopatologia , Biomarcadores/análise , Colágeno Tipo II/administração & dosagem , Cruzamentos Genéticos , Feminino , Ligação Genética/imunologia , Predisposição Genética para Doença , Genoma , Imunidade Inata/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Proteoglicanas/administração & dosagem , Índice de Gravidade de Doença , Especificidade da EspécieRESUMO
OBJECTIVE: To explore the effect of sex on clinical and immunologic traits in major histocompatibility complex-matched (H-2d) F(2) hybrid mice with proteoglycan (PG)-induced arthritis and to identify how the quantitative trait locus (QTL) on the X chromosome influences the onset QTL of another chromosome. METHODS: (BALB/c x DBA/2)F(2) hybrid mice were immunized with cartilage PG, and a genome-wide linkage analysis was performed using >200 simple sequence-length polymorphic markers. The major clinical traits (susceptibility, onset, and severity) were assessed, and PG-specific T and B cell responses, and the production of proinflammatory and antiinflammatory cytokines (tumor necrosis factor alpha, interleukin-1 [IL-1], IL-6, interferon-gamma, IL-4, IL-10, and IL-12) were measured in 133 arthritic and 426 nonarthritic female and male F(2) hybrid mice. The major clinical and immunologic traits were linked to genetic loci, and potential linkages among these QTLs and the effect of sex were analyzed. RESULTS: Thirteen QTLs reported in previous studies were confirmed. Binary traits (susceptibility to arthritis) and disease onset were female specific and were identified on chromosomes 3, 7, 10, 11, 13, and X. QTLs for disease severity were mostly male specific and were located on chromosomes 1, 4, 5, 8, 14, 15, and 19. In addition, we identified 4 new QTLs for the onset of arthritis on chromosomes 3, 4, and 11, and 1 new QTL for severity on chromosome 14; all showed a strong gender association. A locus on the X chromosome interacted with a QTL on chromosome 10, and these 2 loci together seemed to control disease incidence and onset. Most of the clinical traits (QTLs) shared common regions with the immunologic traits and frequently showed a locus-locus interaction. CONCLUSION: Numerous immunologic QTLs overlap with clinical QTLs, thus providing information about possible mechanisms underlying QTL function. Disease susceptibility and onset showed predominant linkage with the female sex, under the control of a QTL on the X chromosome, while the severity QTLs were more strongly linked to the male sex.