RESUMEN
Alpha1-antitrypsin (AAT) is a major circulating and tissues inhibitor of serine proteinases implicated in the regulation of inflammation and host defence. There is now increasing evidence that AAT may also exhibit anti-inflammatory activities independent of its protease inhibitor function. This study was undertaken to investigate the effects of native (inhibitory) and polymerized (non-inhibitory) forms of AAT on MID (Moraxella IgD binding protein)-induced human tonsillar B cell activation in vitro. We found that 0.5 microg/ml MID induces B cell proliferation and stimulates IL-6 release (p<0.001) relative to non-stimulated controls. Both native and polymerized AAT (0.5 mg/ml) inhibited MID-stimulated B cell proliferation in a similar manner (by 70%, p<0.001), whereas MID-induced IL-6 release was more strongly suppressed by polymerized (9.9-fold, p<0.001) as compared to native AAT (2.8-fold, p<0.01). Electrophoretic analysis of cell culture media did not indicate any interaction between AAT and MID, and flow cytometry data showed no competition for the same receptor. The effects of AATs were observed whether added together with MID or 2h after MID-addition to cell cultures. Thus, our data demonstrate that AAT inhibits MID-induced B cell activation in vitro that is unrelated to its protease inhibitory activity and is not dependent on MID binding to the cell surface.
Asunto(s)
Adhesinas Bacterianas/inmunología , Adhesinas Bacterianas/farmacología , Linfocitos B/inmunología , Proliferación Celular/efectos de los fármacos , Tonsila Palatina/inmunología , alfa 1-Antitripsina/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Interleucina-6/biosíntesis , Interleucina-6/metabolismo , Tonsila Palatina/citologíaRESUMEN
The human DUSP15 gene encodes an uncharacterized 235-amino acid member of the subfamily of small dual specificity protein phosphatases related to the Vaccinia virus VH1 phosphatase. Similar to VHR-related MKPX (VHX) (DUSP22), the predicted protein has an N-terminal myristoylation recognition sequence, and we show here that both are indeed modified by the attachment of a myristate to Gly-2. In recognition of this relatedness to VHX, we refer to the DUSP15-encoded protein as VH1-related member Y (VHY). We report that VHY is expressed at high levels in the testis and barely detectable levels in the brain, spinal cord, and thyroid. A VHY-specific antiserum detected a protein with an apparent molecular mass of 26 kDa, and histochemical analysis showed that VHY was readily detectable in pachytene spermatocytes (midstage of meiotic division I) and round spermatids and weakly in Leydig cells (somatic cells outside of the seminiferous tubules). When expressed in 293T or NIH-3T3 cells, VHY was concentrated at the plasma membrane with some staining of vesicular structures in the Golgi region. Mutation of the myristoylation site Gly-2 abrogated membrane location. Finally, we demonstrate that VHY is an active phosphatase in vitro. We conclude that VHY is a new member of a subgroup of myristoylated VH1-like small dual specificity phosphatases.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos , Fosfoproteínas Fosfatasas/química , Proteínas Tirosina Fosfatasas/química , Proteínas Represoras/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Northern Blotting , Southern Blotting , Línea Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Citoplasma/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Fosfatasas de Especificidad Dual , Embrión de Mamíferos/metabolismo , Fibroblastos/metabolismo , Glutatión Transferasa/metabolismo , Glicina/química , Aparato de Golgi/metabolismo , Humanos , Immunoblotting , Inmunohistoquímica , MAP Quinasa Quinasa 4 , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Microscopía Fluorescente , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosfatasas de la Proteína Quinasa Activada por Mitógenos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Ácidos Mirísticos/química , Células 3T3 NIH , Nitrofenoles/química , Compuestos Organofosforados/química , Monoéster Fosfórico Hidrolasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Tirosina Fosfatasas/metabolismo , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Homología de Secuencia de Aminoácido , Espermátides/metabolismo , Testículo/metabolismo , Transfección , Virus Vaccinia/metabolismoRESUMEN
Moraxella IgD binding protein (MID) is a novel bacterial outer membrane protein with IgD-binding properties. MID was purified from the respiratory pathogen Moraxella catarrhalis and is here shown to have B cell stimulatory properties. Purified MID in the range of 0.01-0.1 microg/ml was optimal to induce a proliferative response in human PBL. MID coupled to Sepharose and formalin-fixed M. catarrhalis preparations induced similar proliferative responses in PBL cultures. MID or MID-Sepharose stimulated purified human peripheral B cells as measured by proliferation. In contrast, MID or MID-Sepharose did not activate T cells. Preincubation of purified B cells with anti-IgD Abs inhibited MID-Sepharose-induced B cell proliferation. The addition of IL-4 specifically induced IL-6 production in MID-Sepharose-activated B cells. IgM secretion was detected in B cell cultures stimulated with MID or MID-Sepharose and IL-2 for 10 days. Secretion of IgG and IgA was efficiently induced in cultures from purified B cells stimulated with the combination of MID or MID-Sepharose and IL-4, IL-10, and soluble CD40 ligand, suggesting that Th2-derived cytokines were required for optimal plasma cell generation. Taken together, MID has properties that make it an important tool to study IgD-targeted activation of B cells.
Asunto(s)
Adhesinas Bacterianas , Linfocitos B/efectos de los fármacos , Proteínas Portadoras/farmacología , Activación de Linfocitos/efectos de los fármacos , Células Th2/inmunología , Linfocitos B/inmunología , Citocinas , Relación Dosis-Respuesta a Droga , Humanos , Inmunoglobulina A/biosíntesis , Inmunoglobulina D/fisiología , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Interleucina-2/farmacología , Interleucina-6/biosíntesisRESUMEN
Stroke is one of the most complex diseases, with several subtypes, as well as secondary risk factors, such as hypertension, hyperlipidemia, and diabetes, which, in turn, have genetic and environmental risk factors of their own. Here, we report the results of a genomewide search for susceptibility genes for the common forms of stroke. We cross-matched a population-based list of patients with stroke in Iceland with an extensive computerized genealogy database clustering 476 patients with stroke within 179 extended pedigrees. Linkage to 5q12 was detected, and the LOD score at this locus meets the criteria for genomewide significance (multipoint allele-sharing LOD score of 4.40, P=3.9 x 10(-6)). A 20-cM region on 5q was physically and genetically mapped to obtain accurate marker order and intermarker distances. This locus on 5q12, which we have designated as "STRK1," does not correspond to known susceptibility loci for stroke or for its risk factors and represents the first mapping of a locus for common stroke.