RESUMEN
OBJECTIVE: The objective of this study was to determine the effects of transforming growth factor (TGF)-beta1 on mast cell development. MATERIALS AND METHODS: Mast cells were cultured from mouse bone marrow in interleukin (IL)-3 + stem cell factor, in the presence or absence of TGF-beta1. We assessed mast cell development by measuring the expression of kit, T1/ST2, FcvarepsilonRI, and Fcgamma receptors. Cell morphology was determined by histochemical staining. Alterations in FcvarepsilonRI subunit expression were measured by Western blot analysis. Adoptive transfer of cultured mast cells into mast cell-deficient W/W(v) mice was used to determine if the in vivo environment could reverse the inhibitory effects of TGF-beta1. RESULTS: TGF-beta1 decreased FcvarepsilonRI, c-kit, T1/ST2, and FcgammaR expression, and inhibited granule formation in developing mast cells. Accessory cells were not required for this inhibition. Smad3 deficiency did not alter the response of bone marrow cells to TGF-beta1. TGF-beta1 inhibited expression of the FcvarepsilonRI alpha subunit protein, without decreasing beta or gamma proteins. Mast cells derived in the presence of TGF-beta1 were functionally impaired, as IgE-mediated cytokine secretion was greatly reduced. The changes in granule formation and surface antigen expression were long-standing, as they were not reversed by transfer to W/W(v) mice. CONCLUSIONS: TGF-beta1 may contribute to mast cell homeostasis by inhibiting maturation from bone marrow precursors. The effects of TGF-beta1 result in greatly diminished expression of cell surface markers, reduced granulation, and lack of responsiveness to IgE-mediated activation. Thus TGF-beta1 can serve as a potent and multifunctional regulator of mast cell maturation.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Mastocitos/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Traslado Adoptivo , Animales , Médula Ósea , Degranulación de la Célula/efectos de los fármacos , Células Cultivadas , Femenino , Homeostasis , Inmunoglobulina E/farmacología , Inmunofenotipificación , Mastocitos/citología , Mastocitos/inmunología , Mastocitos/trasplante , Ratones , Ratones Endogámicos C57BL , Receptores de Superficie Celular/análisis , Factor de Crecimiento Transformador beta1RESUMEN
To examine whether prematurity significantly changes the lung inflammatory response to oxygen, rabbit lung explant cultures were exposed to 95% or 5% oxygen for 24 hours. Interleukin (IL)-8 protein concentrations from homogenates of the premature lung rose significantly after hyperoxia (6.8 +/- 1.8 in 5% O2 to 45.7 +/- 21.3 pg/microg protein in 95% O2) but not in the term lung (15.9 +/- 6.7 to 20.4 +/- 4.3 pg/microg protein). There was no change in IL-8 mRNA after hyperoxia in either age group. Preterm lungs demonstrated higher IL-8 levels by fluorescence-activated cell sorting (FACs) analysis and immunohistochemistry. This model may help determine why premature lungs are more susceptible to oxygen-induced disease.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Hiperoxia/fisiopatología , Interleucina-8/genética , Pulmón/metabolismo , Oxígeno/farmacología , Nacimiento Prematuro/genética , Nacimiento a Término/genética , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Hiperoxia/genética , Técnicas In Vitro , Interleucina-8/análisis , Interleucina-8/biosíntesis , Pulmón/efectos de los fármacos , Embarazo , Nacimiento Prematuro/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Nacimiento a Término/metabolismoRESUMEN
Thymosin beta4 (Tbeta4), a 5 kDa polypeptide, is a member of the beta-thymosin family. It acts as the principal intracellular G-actin sequestering peptide and exhibits extracellular functions in angiogenesis and wound healing. The N-terminus of Tbeta4 contains a bioactive tetrapeptide, acSDKP, a negative regulator of hematopoietic stem-cell proliferation. Here, we show that both peptides inhibit mast-cell proliferation over the concentration range of 10(-6) to 10(-17) M with the maximum effect of both at 10(-14) M. Both Tbeta4 and acSDKP caused dysplastic mast-cell nuclei that were confirmed by DAPI fluorescent staining. Flow-cytometric analysis of ploidy revealed that the dysplastic nuclei were not multinucleated, but fragmented nuclei in G2 growth arrest. We could further demonstrate that 10(-8) or 10(-14) M Tbeta4 or acSDKP induce mast-cell degranulation. A concentration of 10(-8) M Tbeta4 or acSDKP caused 57 or 89% degranulation, respectively. A number of tryptic fragments of Tbeta4 were assayed beside intact Tbeta4 and the tetrapeptide, and found to be inactive.
Asunto(s)
Apoptosis/fisiología , Degranulación de la Célula/fisiología , Núcleo Celular/fisiología , Inhibidores de Crecimiento/fisiología , Mastocitos/citología , Mastocitos/fisiología , Oligopéptidos/fisiología , Timosina/fisiología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Proliferación Celular , Células Cultivadas , Inhibidores de Crecimiento/química , Ratones , Ratones Endogámicos BALB C , Oligopéptidos/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/fisiología , Timosina/químicaRESUMEN
To determine if the alveolar macrophage inflammatory cytokine response to oxygen differs in premature cells, macrophages were obtained from litters of premature (27 days) and term (31 days) rabbits. The majority of these cells were nonspecific esterase positive and actively phagocytosed latex particles. The cells that expressed cytokines also reacted with a monoclonal antibody against rabbit macrophages. After incubation overnight in 5 or 95% oxygen, the amount of interleukin (IL)-1beta and IL-8 mRNA was assessed by RT-PCR and the amount of cytokine protein by quantitative immunofluorescence microscopy. The preterm macrophage showed a significant increase in cytokine mRNA and protein after overnight incubation in 95% oxygen. This response was not seen in the term cells. Only premature macrophages had a significant increase in intracellular oxygen radical content, measured by 2',7'-dichlorofluorescin analysis, after incubation in 95% oxygen. This enhanced inflammatory cytokine response to oxygen may be one mechanism involved in the early development of chronic lung disease in premature infants.