RESUMEN
AIMS: Electro-acupuncture pretreatment (EAP) plays a protective role in myocardial ischemia (MI) injury. However, the underlying mechanism remains unclear. A growing body of evidence suggests postinfarction inflammatory response directly affects the remodeling of ventricular function. The purpose of this study was to investigate whether EAP alleviates MI through NLRP3 inflammasome inhibition. MATERIALS AND METHODS: We constructed an AMI model by ligating the left anterior descending (LAD) coronary artery after 3 days of EAP with C57BL/6 mice. Echocardiography and TTC staining were employed to evaluate cardiac function and infarct size after 24 h of ischemia. HE staining and immunohistochemistry were employed to determine inflammatory level. Then, inflammasome activation was detected by western blotting, and macrophage polarization and neutrophil infiltration were observed by flow cytometry. KEY FINDINGS: Our preliminary findings showed that EAP reduced the infarct area and increased fractional shortening (FS) and ejection fraction (EF) and decreased the degree of inflammation after AMI injury. Meanwhile, EAP inhibited the expression of NLRP3, cleaved caspase-1 and IL-1ß in ischemia myocardial tissue, companied by inhibiting the expression of F4/80+, CD11b+, CD206low macrophages and activated M2 macrophage, and decreasing Ly-6G+CD11b+ neutrophils in ischemia myocardial and spleen tissue. SIGNIFICANCE: EAP inhibits the activation of NLRP3 inflammasome, promotes M2 polarization of macrophages and reduces the recruitment of neutrophils in damaged myocardium, thereby decreases the infarct size and improves the cardiac function.
Asunto(s)
Electroacupuntura/métodos , Inflamasomas/inmunología , Precondicionamiento Isquémico Miocárdico , Isquemia Miocárdica/genética , Isquemia Miocárdica/terapia , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Animales , Antígenos Ly/genética , Antígenos Ly/inmunología , Antígeno CD11b/genética , Antígeno CD11b/inmunología , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/inmunología , Caspasa 1/genética , Caspasa 1/inmunología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Inflamasomas/genética , Inflamación , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Macrófagos/inmunología , Macrófagos/patología , Masculino , Receptor de Manosa , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/inmunología , Ratones , Ratones Endogámicos C57BL , Isquemia Miocárdica/inmunología , Isquemia Miocárdica/patología , Miocardio/inmunología , Miocardio/patología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Infiltración Neutrófila , Neutrófilos/inmunología , Neutrófilos/patología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/inmunología , Transducción de SeñalRESUMEN
BACKGROUND: Brain edema as a result of secondary injury following traumatic brain injury (TBI) is a major clinical concern. Neutrophils are known to cause increased vascular permeability leading to edema formation in peripheral tissue, but their role in the pathology following TBI remains unclear. METHODS: In this study we used controlled cortical impact (CCI) as a model for TBI and investigated the role of neutrophils in the response to injury. The outcome of mice that were depleted of neutrophils using an anti-Gr-1 antibody was compared to that in mice with intact neutrophil count. The effect of neutrophil depletion on blood-brain barrier function was assessed by Evan's blue dye extravasation, and analysis of brain water content was used as a measurement of brain edema formation (24 and 48 hours after CCI). Lesion volume was measured 7 and 14 days after CCI. Immunohistochemistry was used to assess cell death, using a marker for cleaved caspase-3 at 24 hours after injury, and microglial/macrophage activation 7 days after CCI. Data were analyzed using Mann-Whitney test for non-parametric data. RESULTS: Neutrophil depletion did not significantly affect Evan's blue extravasation at any time-point after CCI. However, neutrophil-depleted mice exhibited a decreased water content both at 24 and 48 hours after CCI indicating reduced edema formation. Furthermore, brain tissue loss was attenuated in neutropenic mice at 7 and 14 days after injury. Additionally, these mice had a significantly reduced number of activated microglia/macrophages 7 days after CCI, and of cleaved caspase-3 positive cells 24 h after injury. CONCLUSION: Our results suggest that neutrophils are involved in the edema formation, but not the extravasation of large proteins, as well as contributing to cell death and tissue loss following TBI in mice.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Edema Encefálico/terapia , Lesiones Encefálicas/patología , Neutrófilos/patología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos Ly/inmunología , Antígenos Ly/metabolismo , Apoptosis/efectos de los fármacos , Barrera Hematoencefálica/efectos de los fármacos , Edema Encefálico/etiología , Edema Encefálico/patología , Lesiones Encefálicas/complicaciones , Caspasa 3/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Esquema de Medicación , Azul de Evans , Galectina 3/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Activación de Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/patología , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Factores de Tiempo , Agua/metabolismoRESUMEN
Calreticulin (CRT) plays a critical role in MHC class I antigen processing and elicits peptide-specific CD8(+) T cell responses against tumours when administered with peptides. However, how CRT contributes to class I antigen processing and the mechanism of its adjuvant effect in anti-tumour responses, remain to be elucidated. Here we show that reduced class I expression in CRT deficient cells can be restored by the direct delivery of peptides into the ER or by incubation at low temperature. CRT deficient cells exhibited a TAP-deficient phenotype in terms of class I assembly, without loss of TAP expression or functionality. Furthermore, a higher concentration of antigen in the cytosol is required for specific T cell stimulation, suggesting that CRT has a functional role in the maintenance of the low peptide concentration threshold required in the ER for efficient antigen presentation. In the absence of CRT, ERp57 is up-regulated, which indicates that they collaborate with each other in class I antigen processing.
Asunto(s)
Presentación de Antígeno/inmunología , Retículo Endoplásmico/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Péptidos/inmunología , Proteína G de Unión al Calcio S100/inmunología , Animales , Presentación de Antígeno/genética , Antígenos Ly/genética , Antígenos Ly/inmunología , Linfocitos T CD8-positivos , Calbindina 2 , Línea Celular , Retículo Endoplásmico/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Proteínas de Neoplasias/inmunología , Neoplasias/inmunología , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/inmunología , Proteína G de Unión al Calcio S100/genéticaRESUMEN
Here, we identified and characterized a Ly49 family member, designated as Ly49Q. The Ly49q gene encodes a 273-aa protein with an immunoreceptor tyrosine-based inhibitory motif (ITIM) at the N terminus of its cytoplasmic domain. We show that the ITIM of Ly49Q can recruit SHP-2 and SHP-1 in a tyrosine phosphorylation-dependent manner. In contrast to other known members of the Ly49 family, Ly49Q was found not to be expressed on NK1.1(+) cells, but instead was detectable on virtually all Gr-1(+) cells, such as myeloid precursors in bone marrow. Monocytes/macrophages also expressed low levels of Ly49Q, and the expression was enhanced by the treatment of cells with IFN-gamma. Treatment of activated macrophages with anti-Ly49Q mAb induced rapid formation of polarized actin structures, showing filopodia-like structure on one side and lamellipodial-like structure on the other side. A panel of proteins became tyrosine-phosphorylated in myeloid cells when treated with the mAb. Induction of the phosphorylation depends on the ITIM of Ly49Q. Thus, Ly49Q has unique features different from other known Ly49 family members and appears to be involved in regulation of cytoskeletal architecture of macrophages through ITIM-mediated signaling.
Asunto(s)
Antígenos Ly/metabolismo , Células de la Médula Ósea/metabolismo , Citoesqueleto/metabolismo , Regulación de la Expresión Génica , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Ly/inmunología , Secuencia de Bases , Linaje de la Célula , Clonación Molecular , Cartilla de ADN , ADN Complementario , Citometría de Flujo , Lectinas Tipo C , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Subfamilia A de Receptores Similares a Lectina de Células NK , Fosforilación , Reacción en Cadena de la Polimerasa , Receptores Similares a Lectina de Células NK , Tirosina/metabolismoRESUMEN
NK cells can migrate into sites of inflammatory responses or malignancies in response to chemokines. Target killing by rodent NK cells is restricted by opposing signals from inhibitory and activating Ly49 receptors. The rat NK leukemic cell line RNK16 constitutively expresses functional receptors for the inflammatory chemokine CXC chemokine ligand (CXCL)10 (CXCR3) and the homeostatic chemokine CXCL12 (CXCR4). RNK-16 cells transfected with either the activating Ly49D receptor or the inhibitory Ly49A receptor were used to examine the effects of NK receptor ligation on CXCL10- and CXCL12-mediated chemotaxis. Ligation of Ly49A, either with Abs or its MHC class I ligand H2-D(d), led to a decrease in chemotactic responses to either CXCL10 or CXCL12. In contrast, Ly49D ligation with Abs or H2-D(d) led to an increase in migration toward CXCL10, but a decrease in chemotaxis toward CXCL12. Ly49-dependent effects on RNK-16 chemotaxis were not the result of surface modulation of CXCR3 or CXCR4 as demonstrated by flow cytometry. A mutation of the Src homology phosphatase-1 binding motif in Ly49A completely abrogated Ly49-dependent effects on both CXCL10 and CXCL12 chemotaxis, suggesting a role for Src homology phosphatase-1 in Ly49A/chemokine receptor cross-talk. Ly49D-transfected cells were pretreated with the Syk kinase inhibitor Piceatannol before ligation, which abrogated the previously observed changes in migration toward CXCL10 and CXCL12. Piceatannol also abrogated Ly49A-dependent inhibition of chemotaxis toward CXCL10, but not CXCL12. Collectively, these data suggest that Ly49 receptors can influence NK cell chemotaxis within sites of inflammation or tumor growth upon interaction with target cells.
Asunto(s)
Antígenos Ly/fisiología , Inhibición de Migración Celular , Quimiocinas CXC/fisiología , Quimiotaxis de Leucocito/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Receptores Inmunológicos/fisiología , Adyuvantes Inmunológicos/metabolismo , Adyuvantes Inmunológicos/fisiología , Secuencias de Aminoácidos/fisiología , Animales , Antígenos Ly/inmunología , Antígenos Ly/metabolismo , Línea Celular , Línea Celular Tumoral , Membrana Celular/inmunología , Membrana Celular/metabolismo , Quimiocina CXCL10 , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Precursores Enzimáticos/fisiología , Antígenos H-2/metabolismo , Antígeno de Histocompatibilidad H-2D , Humanos , Péptidos y Proteínas de Señalización Intracelular , Células Asesinas Naturales/citología , Células Asesinas Naturales/enzimología , Lectinas Tipo C , Ligandos , Activación de Linfocitos/inmunología , Proteínas Tirosina Quinasas/fisiología , Ratas , Receptores CXCR3 , Receptores CXCR4/biosíntesis , Receptores de Quimiocina/biosíntesis , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Receptores Similares a Lectina de Células NK , Quinasa Syk , Tirosina/metabolismo , Tirosina/fisiologíaRESUMEN
The majority of the known Ly49 family members have been isolated from either C57BL/6 (B6) or BALB/c mice. Interestingly, the anti-Ly49 Ab reactivities observed in 129/J mice are different from those of B6 mice. Furthermore, immunoprecipitation of 129/J NK cell lysates with YE1/32 and YE1/48, Abs specific for the inhibitory Ly49A in B6, resulted in detection of the activation-associated DAP12 molecule. These results indicated a need for a more detailed study of this strain. Therefore, a cloning strategy was devised to isolate Ly49 cDNAs from 129/J mice. An immunoreceptor tyrosine-based inhibitory motif-containing, Ly49D-related clone was discovered that we have named Ly49O, and one immunoreceptor tyrosine-based inhibitory motif-lacking, Ly49A-related clone was discovered that we have named Ly49P. No anti-Ly49 mAb reacted with Ly49O, whereas the molecule encoded by the Ly49P cDNA was found to react with YE1/32 and YE1/48. Ly49P was found to associate with mouse DAP12, and Ab-mediated cross-linking of Ly49P resulted in mouse DAP12 phosphorylation and Ca2+ mobilization, indicating that Ly49P is a competent activation receptor. Ly49P, therefore, represents a novel member of the Ly49 activating receptor subfamily.
Asunto(s)
Antígenos Ly/genética , Proteínas Portadoras/genética , Activación de Linfocitos/inmunología , Proteínas de la Membrana/genética , Receptores Inmunológicos/genética , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Reacciones Antígeno-Anticuerpo , Antígenos Ly/química , Antígenos Ly/inmunología , Antígenos Ly/metabolismo , Secuencia de Bases , Señalización del Calcio/inmunología , Clonación Molecular , ADN Complementario/aislamiento & purificación , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Lectinas Tipo C , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Subfamilia A de Receptores Similares a Lectina de Células NK , Fosforilación , Reacción en Cadena de la Polimerasa , Receptores Inmunológicos/química , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/fisiología , Receptores Similares a Lectina de Células NKRESUMEN
A study was made of the distribution of 125I (a chloramine method of labeling) monoclonal antibodies to the surface antigen Ly 2.1 of T-lymphocytes during action of immunomodulators (tactivin, hydrocortisone, tactivin administered after hydrocortisone) on ACR mice. These antibodies were shown to retain antigen binding capacity, permitting monitoring of the redistribution of the antigen in the body exposed to immunomodulators.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Anticuerpos Monoclonales/farmacocinética , Antígenos Ly/inmunología , Radioisótopos de Yodo , Linfocitos T/inmunología , Animales , Femenino , Hidrocortisona/farmacología , Masculino , Ratones , Ratones Endogámicos AKR , Péptidos/farmacología , Linfocitos T/efectos de los fármacos , Extractos del Timo/farmacología , Factores de Tiempo , Distribución Tisular/efectos de los fármacosRESUMEN
Purified populations of B cells expressing the Ly-1 and/or Mac-1 surface Ag were isolated from normal unmanipulated mice by cell sorting. The number of lymphocytes in each population secreting antibodies reactive with DNA, bromelain-treated mouse RBC, phosphorylcholine and TNP-keyhole limpet hemocyanin was quantitated by ELISA spot assay. The proportion of B cells secreting Ig in vivo and the repertoire of antibodies they produced varied as a function of B cell phenotype and location. Among peritoneal lymphocytes, those that were Ly-1+ or Ly-1- Mac-1+ secreted Ig 10 times more frequently that Mac-1- Ly-1- B cells from the same location. In addition, the former populations expressed repertoires that were significantly skewed toward the production of antibodies reactive with bromelain-treated mouse RBC (p less than 0.001). In contrast, splenic B cells expressing the Ly-1 surface Ag did not differ significantly from splenic Ly-1- B cells in their expressed repertoire or frequency of Ig production. B cells isolated from the spleen and peritoneum tended to differ in antibody specificity from bone marrow and lymph node-derived lymphocytes. For example, B cells from the spleen secreted anti-DNA antibodies two to four times more frequently than B cells from other organs. These results demonstrate that phenotype and microenvironment influence the repertoire of antibodies expressed by B cells in vivo.
Asunto(s)
Antígenos Ly/inmunología , Linfocitos B/inmunología , Animales , Diversidad de Anticuerpos , Formación de Anticuerpos , Antígenos de Diferenciación/análisis , Antígenos de Diferenciación/inmunología , Antígenos CD5 , Citometría de Flujo , Antígeno de Macrófago-1 , Ratones , Ratones Endogámicos , Cavidad Peritoneal/citología , Receptores de Adhesión de Leucocito/análisis , Bazo/citología , Bazo/inmunologíaRESUMEN
The presence of metastases in the regional lymph nodes is the major prognostic factor in breast cancer in the absence of overt distant metastases and is also an important indicator of the need for adjuvant therapy in "early" breast cancer. Currently, the accurate assessment of axillary lymph node status requires axillary dissection which has an associated morbidity. An alternative method of identifying patients who are "node positive" has been developed by means of immunolymphoscintigraphy with s.c. administered radioiodinated monoclonal antibody. The 131I-labeled anti-breast cancer antibody (RCC-1; 400 micrograms) and cold iodine-labeled "blocking" antibody (Ly-2.1; 2 mg which is nonreactive with breast cancer) were injected s.c. into both arms and scintigraphy images were obtained 16-18 h after the injection, using the axilla contralateral to the side of the breast cancer as the control. Studies were reported as positive (and therefore indicative of lymph node metastases) if the amount of background-subtracted radioactive count in the axilla of interest exceeded the normal side by a radio equal to or greater than 1.5:1.0 as assessed by computer analysis. In 38 of 40 patients the findings on scintigraphy were correlated with operative and histopathological findings on the axillary dissection specimen or cytological findings of fine needle aspiration of axillary lymph nodes. In a prospective study of 26 patients, the method is more sensitive (86%) and specific (92%) than preoperative clinical assessment (57% sensitivity, 58% specificity) in the detection of axillary lymph node metastases; and by combining both modalities of assessment, there was an improvement in the sensitivity (100%) but a deterioration in the specificity (50%). There was no significant complication from this essentially outpatient procedure and only 1 of 40 patients developed a human anti-mouse antibody response. This novel and safe method of imaging may become a most useful adjunct in the surgical management of breast cancer.
Asunto(s)
Anticuerpos Monoclonales , Neoplasias de la Mama/diagnóstico por imagen , Ganglios Linfáticos/diagnóstico por imagen , Metástasis Linfática , Animales , Antígenos Ly/inmunología , Axila , Femenino , Humanos , Técnicas para Inmunoenzimas , Radioisótopos de Yodo , Ratones , CintigrafíaRESUMEN
RIIIS/J mice produce low antibody responses to several polysaccharide Ag of bacterial origin. They have low levels of serum IgM and IgG3 and high levels of serum IgG2a and IgG2b. Low serum IgM and IgG3 have been attributed to a low frequency of CD5 (Ly-1) B cells, which play an important role in the production of natural antibodies. Indeed, RIIIS/J mice have a low frequency of CD5 (Ly-1)+, IgM bright+, Ly-5 (B220)dull+ (i.e., CD5 (Ly-1) B) cells in their peritoneum. RIIIS/J mice treated with LPS produce a low anti-bromelain-treated mouse RBC splenic plaque-forming cell response and a normal anti-mouse transferrin splenic PFC response. Those data are compatible with the fact that CD5 (Ly-1) B cells contain the precursors of B lymphocytes secreting anti-bromelain-treated mouse RBC antibody. However, they have a higher frequency of IgM bright+, Mac-1+ cells in their peritoneum. These cells represent the CD5 (Ly-1) "sister population" of CD5 (Ly-1) B cells described by others. This suggests that characteristics usually associated with the CD5 (Ly-1) lineage are applicable only to the CD5 (Ly-1)+ Mac-1+ IgM+ population, but not the related CD5 (Ly-1)- Mac-1+ IgM+ population. RIIIS/J mice should thus prove a valuable model to study the CD5 (Ly-1) B cell lineage.
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Antígenos Ly/inmunología , Linfocitos B/inmunología , Síndromes de Inmunodeficiencia/inmunología , Animales , Autoanticuerpos/biosíntesis , Autoantígenos/inmunología , Linfocitos B/clasificación , Femenino , Citometría de Flujo , Técnica de Placa Hemolítica , Isotipos de Inmunoglobulinas/metabolismo , Síndromes de Inmunodeficiencia/sangre , Síndromes de Inmunodeficiencia/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NZB , Fenotipo , BazoAsunto(s)
Antígenos Ly/inmunología , Autoanticuerpos/biosíntesis , Autoantígenos/inmunología , Linfocitos B/inmunología , Eritrocitos/inmunología , Animales , Linfocitos B/clasificación , Bromelaínas/farmacología , Técnica de Placa Hemolítica , Inmunoglobulina M/biosíntesis , Ratones , Ratones Endogámicos NZB , Bazo/citología , Bazo/inmunologíaRESUMEN
The X-linked CBA/N immune defect was used to investigate the roles of Lyb-5- and Lyb-5+ B cells in the memory response to PC-KLH (phosphocholine-conjugated keyhole limpet hemocyanin). (CBA/N X BALB/c)F1 (CB) male mice express the xid mutation and thereby lack the Lyb-5+ B cell subset, whereas their female littermates are normal and express both Lyb-5+ and Lyb-5- B cells. After priming with PC-conjugated hemocyanin (PC-Hy) in complete Freund's adjuvant, female B cells produce three phenotypic sets of PC-KLH-specific antibody. The first set (group I) is dominated by T15+, IgM, IgA, and IgG3, PC-specific antibodies. The second subset (group II) is specific for phenylphosphocholine (PPC), and is dominated by T15-, IgG1, and IgG2 antibodies. The third set (group III) recognizes an epitope(s) composed of both the PPC hapten and carrier determinants. PC-Hy-primed B cells from immune defective CB male mice produce the same number of IgG1 and IgG2 plaque-forming cells (PFC) as do PC-Hy-primed normal female cells, and these PFC are also predominantly T15- and PPC specific (group II). In addition, a significant amount of group III IgG1 and IgG2 antibody is observed in the immune defective male response. In contrast to female B cells, immune defective male B cells produce a low IgM, IgA, and IgG3 memory response that is not composed of PC-specific (group I) antibodies; in fact, most of these antibodies arise from group III precursors and are not inhibited by either PC or PPC. PC-specific antibodies usually represent less than 25% of the anti-PC-KLH response in immune defective mice; however, these PC-specific antibodies are predominantly T15-. These data suggest that the Lyb-5-B cells in both normal and immune defective mice produce the T15-, IgG1, and IgG2 antibodies that dominate the secondary immune response to PC-KLH, and that the Lyb-5+ B cells produce the T15+, IgM, IgA, and IgG3 portion of the secondary response in normal mice. This hypothesis was confirmed by priming normal mice with the R36a strain of Streptococcus pneumoniae or with PC-Hy in saline. These forms of PC-antigen prime only the Lyb-5+ B cell subset. The adoptive transfer of these two B cell sources results in an anti-PC-KLH response that is T15 dominant and totally PC inhibitable.(ABSTRACT TRUNCATED AT 400 WORDS)