RESUMEN
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies to certain cellular macromolecules, such as the small nuclear ribonucleoprotein particles (snRNPs), which had been considered to be passive targets of the autoimmune response. SLE is also characterized by the increased expression of type I interferon (IFN), which appears to be associated with the development and severity of disease. Here, we show that specific, highly conserved RNA sequences within snRNPs can stimulate Toll-like receptors (TLRs) 7 and 8 as well as activate innate immune cells, such as plasmacytoid dendritic cells (pDCs), which respond by secreting high levels of type I IFN. SLE patient sera containing autoantibodies to snRNPs form immune complexes that are taken up through the Fc receptor gammaRII and efficiently stimulate pDCs to secrete type I IFNs. These results demonstrate that a prototype autoantigen, the snRNP, can directly stimulate innate immunity and suggest that autoantibodies against snRNP may initiate SLE by stimulating TLR7/8.
Asunto(s)
Autoantígenos/inmunología , Inmunidad Innata , Lupus Eritematoso Sistémico/inmunología , ARN Nuclear Pequeño/inmunología , Receptor Toll-Like 7/inmunología , Receptor Toll-Like 8/inmunología , Animales , Anticuerpos Antinucleares/inmunología , Línea Celular , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Humanos , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Masculino , Ratones , Células Plasmáticas/inmunología , Receptores de IgG/inmunologíaRESUMEN
STUDY OBJECTIVE: s: The impairment of cardiac contractility during endotoxemia involves induction of nitric oxide formation through a cascade of events initiated by overexpression of proinflammatory cytokines. We previously showed that hypothermia attenuates endotoxin-induced overexpression of nitric oxide in rat lungs. In the present study, we tested the hypothesis that hypothermia protects against endotoxin-induced myocardial inflammation by changing the balance of pro- and anti-inflammatory cytokines, inhibiting myeloperoxidase, an indicator of neutrophil activity, and inhibiting nitric oxide-mediated protein damage. DESIGN: Rats were randomized to treatment with either hypothermia (n = 6; 18 to 24 degrees C) or normothermia (n = 6; 36 to 38 degrees C). Endotoxin (15 mg/kg) was administered intravascularly to anesthetized animals, and heart tissue was harvested 150 min later. MEASUREMENTS AND RESULTS: Using enzyme-linked immunosorbent assays (ELISAs), we found that hypothermia induced myocardial expression of the anti-inflammatory cytokines interleukin (IL)-4 and IL-10, while decreasing concentrations of the pro-inflammatory cytokines IL-1beta and growth-related oncogene/cytokine-induced neutrophil chemoattractant (rat homolog of IL-8). Electromobility shift assay revealed that hypothermia inhibited the nuclear translocation of nuclear factor-kappaB. Reverse transcriptase-polymerase chain reaction and Western blot assays revealed that hypothermia attenuated the endotoxin-induced overexpression of both inducible nitric oxide synthase (iNOS) messenger RNA and iNOS protein, respectively. Hypothermia also attenuated nitric oxide-mediated myocardial protein damage, as determined by a nitrotyrosine ELISA. Myocardial myeloperoxidase content, an indicator of neutrophil accumulation and oxidative activity, was also inhibited by hypothermia in endotoxemic rats. CONCLUSION: These data demonstrate that hypothermia induces an anti-inflammatory cytokine profile, inhibits neutrophil aggregation, and inhibits the formation of nitric oxide during endotoxemia in the rat.
Asunto(s)
Citocinas/metabolismo , Endotoxemia/metabolismo , Endotoxemia/terapia , Hipotermia Inducida , Miocardio/metabolismo , Óxido Nítrico/biosíntesis , Peroxidasa/antagonistas & inhibidores , Animales , Ensayo de Inmunoadsorción Enzimática , Inflamación/prevención & control , Interleucina-1/metabolismo , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Interleucina-8/metabolismo , Masculino , FN-kappa B/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
Lymphoid neogenesis is associated with antibody-mediated autoimmune diseases such as Sjogren's syndrome and rheumatoid arthritis. Although systemic lupus erythematosus is the prototypical B-cell-mediated autoimmune disease, the role of lymphoid neogenesis in its pathogenesis is unknown. Intraperitoneal injection of 2,6,10,14-tetramethyl-pentadecane (TMPD, pristane) or mineral oil causes lipogranuloma formation in mice, but only TMPD-treated mice develop lupus. We report that lipogranulomas are a form of lymphoid neogenesis. Immunoperoxidase staining of lipogranulomas revealed B cells, CD4(+) T cells, and dendritic cells and in some cases organization into T- and B-cell zones. Lipogranulomas also expressed the lymphoid chemokines CCL21, CCL19, CXCL13, CXCL12, and CCL22. Expression of the type I interferon (IFN-I)-inducible genes Mx1, IRF7, IP-10, and ISG-15 was greatly increased in TMPD- versus mineral oil-induced lipogranulomas. Dendritic cells from TMPD lipogranulomas underwent activation/maturation with high CD86 and interleukin-12 expression. Magnetic bead depletion of dendritic cells markedly diminished IFN-inducible gene (Mx1) expression. We conclude that TMPD-induced lupus is associated with the formation of ectopic lymphoid tissue containing activated dendritic cells producing IFN-I and interleukin-12. In view of the increased IFN-I production in systemic lupus erythematosus, these studies suggest that IFN-I from ectopic lymphoid tissue could play a role in the pathogenesis of experimental lupus in mice.
Asunto(s)
Granuloma/metabolismo , Interferón Tipo I/biosíntesis , Tejido Linfoide/metabolismo , Terpenos , Animales , Línea Celular , Quimiocina CXCL10 , Quimiocinas/biosíntesis , Quimiocinas CXC/biosíntesis , Coristoma/inducido químicamente , Coristoma/metabolismo , Coristoma/patología , Citocinas/biosíntesis , Células Dendríticas/metabolismo , Femenino , Proteínas de Unión al GTP/biosíntesis , Granuloma/inducido químicamente , Granuloma/patología , Factor 7 Regulador del Interferón/biosíntesis , Interleucina-12/biosíntesis , Lupus Eritematoso Sistémico/inducido químicamente , Lupus Eritematoso Sistémico/patología , Tejido Linfoide/efectos de los fármacos , Tejido Linfoide/patología , Ratones , Ratones Endogámicos BALB C , Aceite Mineral , Proteínas de Resistencia a Mixovirus , Peritoneo , Ubiquitinas/biosíntesisRESUMEN
OBJECTIVE: Most lupus patients produce autoantibodies against small ribonucleoproteins such as Sm/RNP and Ro 60 (containing U1 and Y1-Y5 RNAs, respectively). We undertook this study to investigate whether the RNA components of these antigens, which contain extensive tracts of single- and double-stranded RNA, signatures of viral infection, activate innate immunity. METHODS: U1 and Y RNAs were affinity purified from K562 cells. Murine bone marrow-derived dendritic cells (DCs), human HEK 293 cells, and murine RAW264.7 cells were stimulated with U1 RNA and other known Toll-like receptor (TLR) ligands. Expression of the interferon (IFN)-inducible gene Mx1 and other genes was quantified using real-time polymerase chain reaction, and cytokine production was measured by enzyme-linked immunosorbent assay. DC maturation was assessed using flow cytometry. RESULTS: Purified U1 and Y1-Y5 RNAs and synthetic stem-loop II of U1 RNA stimulated type I IFN (IFN-I) production by cell lines and murine bone marrow-derived DCs and promoted DC maturation (CD86 expression). U1 RNA-stimulated, but not TLR-3 ligand-stimulated, IFN-I was blocked by bafilomycin A1, indicating that immunostimulation by U1 RNA requires endosomal acidification. Myeloid differentiation factor 88-deficient cells responded poorly to U1 RNA, suggesting that an endosomal TLR, probably TLR-7, mediates the stimulatory effects of U1 RNA. U1 RNA-induced IFN-I and interleukin-6 production also were protein kinase R (PKR) dependent (abrogated by 2-aminopurine and greatly reduced in PKR-/- cells). CONCLUSION: We conclude that the RNA components of the Ro 60 (Y1-Y5 RNA) and Sm/RNP (U1 RNA) small ribonucleoproteins act as endogenous adjuvants that could play a role in the pathogenesis of autoimmunity by stimulating DC maturation and IFN-I production.
Asunto(s)
Autoantígenos/inmunología , Lupus Eritematoso Sistémico/inmunología , ARN Citoplasmático Pequeño/inmunología , Ribonucleoproteínas Nucleares Pequeñas/inmunología , Ribonucleoproteínas/inmunología , Animales , Células Cultivadas , Endosomas/inmunología , Humanos , Interferón Tipo I/biosíntesis , Ratones , Receptores Toll-Like/fisiologíaRESUMEN
Statins possess anti-inflammatory and immunomodulatory properties beyond their cholesterol-lowering effects. To determine whether short-term atorvastatin treatment affects the acute phase and humoral immune responses to tetanus toxoid (TT) in normal healthy volunteers, we conducted a randomized, double blind, placebo-controlled trial. Twenty healthy volunteers were assigned a 10-day treatment with atorvastatin (40 mg) or placebo. All volunteers received a TT booster on the fifth day. Subjects in the atorvastatin group had a significant reduction in total and low-density lipoprotein cholesterol (both p<0.001). Unexpectedly, the production of anti-TT antibodies (predominately IgG1) was three-fold higher in the atorvastatin group 15 days post-vaccination (2306 +/- 468 versus 713+/-21 units, p = 0.008). Atorvastatin also suppressed the post-vaccination rise in platelet and lymphocyte counts (both p<0.05). Acute phase parameters did not change significantly in either group. This study illustrates a novel immunomodulatory effect of atorvastatin raising the possibility of using HMG-CoA reductase inhibitors to enhance humoral responses to vaccination.
Asunto(s)
Anticuerpos Antibacterianos/biosíntesis , Ácidos Heptanoicos/administración & dosificación , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Factores Inmunológicos/administración & dosificación , Pirroles/administración & dosificación , Toxoide Tetánico/administración & dosificación , Proteínas de Fase Aguda/biosíntesis , Adulto , Especificidad de Anticuerpos , Atorvastatina , Clostridium tetani/inmunología , Citocinas/biosíntesis , Método Doble Ciego , Femenino , Humanos , Inmunoglobulina G/biosíntesis , Cinética , MasculinoRESUMEN
Regulatory T cells (Tregs), including natural CD4+CD25+ Tregs and inducible IL-10 producing T regulatory type 1 (T(R)1) cells, maintain tolerance and inhibit autoimmunity. Recently, increased percentages of Tregs have been observed in the blood of septic patients, and ex vivo-activated Tregs were shown to prevent polymicrobial sepsis mortality. Whether endogenous Tregs contribute to sepsis outcome remains unclear. Polymicrobial sepsis, induced by cecal ligation and puncture, caused an increased number of splenic Tregs compared with sham-treated mice. Splenic CD4+CD25+ T cells from septic mice expressed higher levels of Foxp3 mRNA and were more efficient suppressors of CD4+CD25- T effector cell proliferation. Isolated CD4+ T cells from septic mice displayed increased intracellular IL-10 staining following stimulation, indicating that T(R)1 cells may also be elevated in sepsis. Surprisingly, Ab depletion of total CD4+ or CD4+CD25+ populations did not affect mortality. Furthermore, no difference in survival outcome was found between CD25 or IL-10 null mice and wild-type littermates, indicating that Treg or T(R)1-generated IL-10 are not required for survival. These results demonstrate that, although sepsis causes a relative increase in Treg number and increases their suppressive function, their presence does not contribute significantly to overall survival in this model.
Asunto(s)
Subunidad alfa del Receptor de Interleucina-2/inmunología , Sepsis/inmunología , Sepsis/mortalidad , Linfocitos T Reguladores/inmunología , Animales , Proliferación Celular , Femenino , Citometría de Flujo , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Interleucina-10/inmunología , Interleucina-10/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sepsis/microbiología , Bazo/citología , Bazo/inmunologíaRESUMEN
Astrocyte inflammation, reactive oxygen species (ROS) formation, and dysfunction form a common denominator shared by all the major neurodegenerative disorders. Viral infections are emerging as important events in the etiology of CNS damage involving astrocytes, but molecular understanding is incomplete. Double-stranded RNA (dsRNA) is a byproduct of viral replication and serves as the signature molecule for viral infection via Toll-like receptor 3 (TLR3) largely restricted to circulating peripheral dendritic cells. However, astrocytes are strategically located at the blood-brain barrier (BBB) and throughout brain tissues, making these cells ideal candidates as innate immunity sentinels within the CNS. We hypothesized that extracellular dsRNA, mimicked by polyinosinic-polycytidylic acid (Poly(I:C); PIC), initiates signaling of the double-edged sword of antiviral plus pathophysiological events in astrocytes. Using Western blot analysis and real-time qPCR, we determined that neonatal rat astrocyte cultures constitutively express TLR3 mRNA and protein, and that PIC dsRNA induced phosphorylation of eIF2alpha, as well as mRNA type I interferon (alpha/beta IFN)-response genes Mx1, PKR, and TLR3. Astrocyte TLR3 protein was downregulated after PIC treatment, however. PIC signaled degradation of IkappaBalpha with the consequence of upregulating iNOS, TNF-alpha, and IL-1beta mRNAs and proteins. In addition to antiviral protection events, dsRNA induced astrocyte dysfunction, evidenced by inhibiting EAAT1/GLAST transporter gene expression and attenuating L-glutamate uptake via sodium-dependent transport system X(AG)-, as well as inducing cytotoxicity. Anti-TLR3 blocking antibody attenuated PIC upregulation of TNF-alpha mRNA and iNOS activity. Extracellular PIC-induced events were prevented by 2-aminopurine, implicating PKR as an important downstream player in astrocyte dsRNA sensing pathways. The effects of plasma membrane impermeable poly(I:C) were dose-dependent (0-50 microM). In concert, these data provide evidence that dsRNA/TLR3-activated astrocytes initiate a battery of rapid innate pathogen-associated molecular pattern (PAMP) immune responses that are important for mounting antiviral defense in the CNS, yet also lead to pathophysiological events associated with the glutamate neurotoxicity of neurodegenerative diseases.
Asunto(s)
Astrocitos/patología , Astrocitos/virología , Ácido Glutámico/metabolismo , ARN Bicatenario/fisiología , Transducción de Señal/fisiología , Animales , Anticuerpos Bloqueadores/farmacología , Antivirales/farmacología , Astrocitos/metabolismo , Western Blotting , Supervivencia Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Espacio Extracelular/virología , Proteínas I-kappa B/fisiología , FN-kappa B/fisiología , Enfermedades Neurodegenerativas/patología , Nitritos/metabolismo , Poli I-C/farmacología , Sondas ARN , Ratas , Ratas Endogámicas WKY , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Lupus patients selectively produce autoantibodies against nucleoproteins. Since the RNA/DNA components of these autoantigens are endogenous TLR ligands capable of stimulating Type I interferon (IFN-I) production, we asked whether autoantibodies against the ribonucleoproteins Sm/RNP and Ro60 and double-stranded DNA are associated with high levels of IFN-I. IFN-I levels were increased in SLE (n = 88) vs. other autoimmune diseases (n = 82) and controls (n = 57) (P < 0.0001) and were associated positively with autoantibodies against Sm/RNP, Ro60/La, and dsDNA but negatively with anti-phospholipid. Low numbers of circulating plasmacytoid and myeloid dendritic cells also were associated with these autoantibodies. The IFN-I and dendritic cell abnormalities correlated with disease severity and were not therapy-related. These findings suggest that immunostimulatory nucleic acid components of autoantigens may act as endogenous adjuvants by promoting IFN-I production and dendritic cell maturation, helping to explain the high prevalence of autoantibodies against nucleoprotein antigens in SLE.