RESUMEN
Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in the pathogenesis of inflammatory disorders such as infection, sepsis, and autoimmune disease. MIF exists preformed in cytoplasmic pools and exhibits an intrinsic tautomerase and oxidoreductase activity. MIF levels are elevated in the serum of animals and patients with infection or different inflammatory disorders. To elucidate how MIF actions are controlled, we searched for endogenous MIF-interacting proteins with the potential to interfere with key MIF functions. Using in vivo biotin-tagging and endogenous co-immunoprecipitation, the ribosomal protein S19 (RPS19) was identified as a novel MIF binding partner. Surface plasmon resonance and pulldown experiments with wild type and mutant MIF revealed a direct physical interaction of the two proteins (K(D) = 1.3 x 10(-6) m). As RPS19 is released in inflammatory lesions by apoptotic cells, we explored whether it affects MIF function and inhibits its binding to receptors CD74 and CXCR2. Low doses of RPS19 were found to strongly inhibit MIF-CD74 interaction. Furthermore, RPS19 significantly compromised CXCR2-dependent MIF-triggered adhesion of monocytes to endothelial cells under flow conditions. We, therefore, propose that RPS19 acts as an extracellular negative regulator of MIF.
Asunto(s)
Células Endoteliales/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Monocitos/metabolismo , Receptores de Interleucina-8B/metabolismo , Proteínas Ribosómicas/metabolismo , Animales , Apoptosis/genética , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/metabolismo , Adhesión Celular/genética , Humanos , Inflamación/genética , Inflamación/metabolismo , Factores Inhibidores de la Migración de Macrófagos/genética , Ratones , Mutación , Células 3T3 NIH , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Unión Proteica/genética , Ratas , Receptores de Interleucina-8B/agonistas , Receptores de Interleucina-8B/genética , Proteínas Ribosómicas/genética , Sepsis/genética , Sepsis/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
In ciliated airway epithelial cells endothelial nitric oxide synthase as well as several other membrane bound proteins are located in the apical cell pole. To date, mechanisms that serve to target and to keep these proteins in this region are unknown. Endothelial nitric oxide synthase is known to target to caveolae by interaction with caveolin-1 or caveolin-3. Since caveolin-1 is found only in a subpopulation of ciliated cells at the basolateral cell membrane, we examined if caveolin-3 could be responsible for the apical localization of endothelial nitric oxide synthase in ciliated cells. We used real-time RT-PCR, laser-assisted microdissection, Western blotting and double-labeling immunohistochemistry to examine the presence of caveolin-3 in the airway epithelium of the rat. Indeed, we found caveolin-3-mRNA as well as protein in ciliated cells throughout the trachea and the bronchial tree. Caveolin-3-immunoreactivity was confined to the apical region and was colocalized with endothelial nitric oxide synthase and the high affinity choline transporter in a compartment distinct from the plasma membrane at the light microscopic level. No caveolae were found in the apical plasma membrane of ciliated cells but a tubulovesicular network was present in the apical region that reached up to the basal bodies of the cilia and was in close contact with mitochondria. Co-immunoprecipitation of caveolin-3 with endothelial nitric oxide synthase verified that both proteins interact in airway ciliated cells. These findings indicate that caveolin-3 is responsible to keep endothelial nitric oxide synthase in a membrane compartment in the apical region of ciliated cells.
