RESUMEN
Allergic asthma is a chronic inflammatory disease of the airways characterized by excessive eosinophilic and lymphocytic inflammation with associated changes in the extracellular matrix (ECM) resulting in airway wall remodeling. Hyaluronan (HA) is a nonsulfated glycosaminoglycan ECM component that functions as a structural cushion in its high molecular mass (HMM) but has been implicated in metastasis and other disease processes when it is degraded to smaller fragments. However, relatively little is known about the role HA in mediating inflammatory responses in allergy and asthma. In the present study, we used a murine Aspergillus fumigatus inhalational model to mimic human disease. After observing in vivo that a robust B cell recruitment followed a massive eosinophilic egress to the lumen of the allergic lung and corresponded with the detection of low molecular mass HA (LMM HA), we examined the effect of HA on B cell chemotaxis and cytokine production in the ex vivo studies. We found that LMM HA functioned through a CD44-mediated mechanism to elicit chemotaxis of B lymphocytes, while high molecular mass HA (HMM HA) had little effect. LMM HA, but not HMM HA, also elicited the production of IL-10 and TGF-ß1 in these cells. Taken together, these findings demonstrate a critical role for ECM components in mediating leukocyte migration and function which are critical to the maintenance of allergic inflammatory responses.
Asunto(s)
Aspergillus fumigatus/inmunología , Asma/inmunología , Linfocitos B/inmunología , Quimiotaxis/inmunología , Ácido Hialurónico/inmunología , Animales , Antígenos Fúngicos/inmunología , Asma/microbiología , Linfocitos B/metabolismo , Líquido del Lavado Bronquioalveolar/inmunología , Modelos Animales de Enfermedad , Matriz Extracelular/inmunología , Femenino , Receptores de Hialuranos/inmunología , Inmunoglobulina E/inmunología , Interleucina-10/biosíntesis , Recuento de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Factor de Crecimiento Transformador beta1/biosíntesisRESUMEN
Sensitization to fungi often leads to a severe form of asthma that is particularly difficult to manage clinically, resulting in increased morbidity and hospitalizations in these patients. Although B lymphocytes might exacerbate asthma symptoms through the production of IgE, these cells might also be important in the protective response against inhaled fungi. Through cytokine release and T-cell interactions, these lymphocytes might also influence the development and maintenance of airway wall fibrosis. J(H)(-/-) mice lack the JH gene for the heavy chain component of antibodies, which is critical for B-cell function and survival. These animals have facilitated the elucidation of the role of B lymphocytes in a number of immune responses; however, J(H)(-/-) mice have not been used to study fungal allergy. In this study, we examined the role of B lymphocytes using an Aspergillus fumigatus murine fungal aeroallergen model that mimics human airway disease that is triggered by environmental fungal exposure. We compared disease progression in sensitized wild-type BALB/c and J(H)(-/-) mice that were exposed to repeated fungal exposure and found no differences in airway hyperresponsiveness, overall pulmonary inflammation or collagen deposition around the large airways. However, the levels of the Th2-type cytokines IL-4 and IL-13 were significantly attenuated in the airways of J(H)(-/-) mice relative to the BALB/c controls. By contrast, levels of the inflammatory cytokines IL-17A and IL-6 were significantly elevated in the J(H)(-/-) animals, and there was significantly more robust airway eosinophilia and neutrophilia than in control animals. Taken together, these findings demonstrate that B lymphocytes help to regulate granulocytic responses to fungal exposure in the pulmonary compartment.
Asunto(s)
Asma/inmunología , Linfocitos B/inmunología , Hiperreactividad Bronquial/inmunología , Modelos Animales de Enfermedad , Granulocitos/inmunología , Pulmón/inmunología , Neumonía/inmunología , Animales , Asma/microbiología , Asma/patología , Linfocitos B/microbiología , Linfocitos B/patología , Western Blotting , Hiperreactividad Bronquial/microbiología , Hiperreactividad Bronquial/patología , Líquido del Lavado Bronquioalveolar , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Granulocitos/microbiología , Granulocitos/patología , Humanos , Inmunoglobulina E , Cadenas Pesadas de Inmunoglobulina/fisiología , Región de Unión de la Inmunoglobulina/fisiología , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neumonía/microbiología , Neumonía/patología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esporas Fúngicas/patogenicidadRESUMEN
Vasoactive intestinal peptide receptor-1 signaling in lymphocytes has been shown to regulate chemotaxis, proliferation, apoptosis and differentiation. During T cell activation, VPAC1 mRNA is downregulated, but the effect on its protein levels is less clear. A small number of studies have reported measurement of human VPAC1 by flow cytometry, but murine VPAC1 reagents are unavailable. Therefore, we set out to generate a reliable and highly specific α-mouse VPAC1 polyclonal antibody for use with flow cytometry. After successfully generating a rabbit α-VPAC1 polyclonal antibody (α-mVPAC1 pAb), we characterized its cross-reactivity and showed that it does not recognize other family receptors (mouse VPAC2 and PAC1, and human VPAC1, VPAC2 and PAC1) by flow cytometry. Partial purification of the rabbit α-VPAC1 sera increased the specific-activity of the α-mVPAC1 pAb by 20-fold, and immunofluorescence microscopy (IF) confirmed a plasma membrane subcellular localization for mouse VPAC1 protein. To test the usefulness of this specific α-mVPAC1 pAb, we showed that primary, resting mouse T cells express detectable levels of VPAC1 protein, with little detectable signal from activated T cells, or CD19 B cells. These data support our previously published data showing a downregulation of VPAC1 mRNA during T cell activation. Collectively, we have established a well-characterized, and highly species specific α-mVPAC1 pAb for VPAC1 surface measurement by IF and flow cytometry.