RESUMEN
The rapid response of neutrophils throughout the body to a systemic challenge is a critical first step in resolution of bacterial infection such as Escherichia coli (E. coli). Here we delineated the dynamics of this response, revealing novel insights into the molecular mechanisms using lung and spleen intravital microscopy and 3D ex vivo culture of living precision cut splenic slices in combination with fluorescent labelling of endogenous leukocytes. Within seconds after challenge, intravascular marginated neutrophils and lung endothelial cells (ECs) work cooperatively to capture pathogens. Neutrophils retained on lung ECs slow their velocity and aggregate in clusters that enlarge as circulating neutrophils carrying E. coli stop within the microvasculature. The absolute number of splenic neutrophils does not change following challenge; however, neutrophils increase their velocity, migrate to the marginal zone (MZ) and form clusters. Irrespective of their location all neutrophils capturing heat-inactivated E. coli take on an activated phenotype showing increasing surface CD11b. At a molecular level we show that neutralization of ICAM-1 results in splenic neutrophil redistribution to the MZ under homeostasis. Following challenge, splenic levels of CXCL12 and ICAM-1 are reduced allowing neutrophils to migrate to the MZ in a CD29-integrin dependent manner, where the enlargement of splenic neutrophil clusters is CXCR2-CXCL2 dependent. We show directly molecular mechanisms that allow tissue resident neutrophils to provide the first lines of antimicrobial defense by capturing circulating E. coli and forming clusters both in the microvessels of the lung and in the parenchyma of the spleen.
Asunto(s)
Movimiento Celular/inmunología , Infecciones por Escherichia coli/inmunología , Escherichia coli/inmunología , Pulmón/inmunología , Neutrófilos/inmunología , Bazo/inmunología , Animales , Quimiocina CXCL12/inmunología , Células Endoteliales/inmunología , Células Endoteliales/patología , Infecciones por Escherichia coli/patología , Femenino , Molécula 1 de Adhesión Intercelular/inmunología , Pulmón/patología , Ratones , Neutrófilos/patología , Bazo/patologíaRESUMEN
Following the FDA-approval of the hematopoietic stem cell (HSC) mobilizer plerixafor, orally available and potent CXCR4 antagonists were pursued. One such proposition was AMD11070, which was orally active and had superior antagonism in vitro; however, it did not appear as effective for HSC mobilization in vivo. Here we show that while AMD11070 acts as a full antagonist, plerixafor acts biased by stimulating ß-arrestin recruitment while fully antagonizing G protein. Consequently, while AMD11070 prevents the constitutive receptor internalization, plerixafor allows it and thereby decreases receptor expression. These findings are confirmed by the successful transfer of both ligands' binding sites and action to the related CXCR3 receptor. In vivo, plerixafor exhibits superior HSC mobilization associated with a dramatic reversal of the CXCL12 gradient across the bone marrow endothelium, which is not seen for AMD11070. We propose that the biased action of plerixafor is central for its superior therapeutic effect in HSC mobilization.
Asunto(s)
Bencilaminas/farmacología , Ciclamas/farmacología , Movilización de Célula Madre Hematopoyética/métodos , Receptores CXCR4/metabolismo , Aminoquinolinas/metabolismo , Aminoquinolinas/farmacología , Animales , Bencimidazoles/metabolismo , Bencimidazoles/farmacología , Bencilaminas/metabolismo , Butilaminas/metabolismo , Butilaminas/farmacología , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Ciclamas/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Femenino , Factor Estimulante de Colonias de Granulocitos , Células HEK293 , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Preparaciones Farmacéuticas/metabolismo , Receptores CXCR3/efectos de los fármacos , Receptores CXCR3/metabolismo , Receptores CXCR4/efectos de los fármacos , beta-Arrestinas/efectos de los fármacos , beta-Arrestinas/metabolismoRESUMEN
Treatment with the CXCR4 antagonist, plerixafor (AMD3100), has been proposed for clinical use in patients with WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome and in pulmonary fibrosis. However, there is controversy with respect to the impact of plerixafor on neutrophil dynamics in the lung, which may affect its safety profile. In this study, we investigated the kinetics of endogenous neutrophils by direct imaging, using confocal intravital microscopy in mouse bone marrow, spleen, and lungs. Neutrophils are observed increasing their velocity and exiting the bone marrow following plerixafor administration, with a concomitant increase in neutrophil numbers in the blood and spleen, while the marginated pool of neutrophils in the lung microvasculature remained unchanged in terms of numbers and cell velocity. Use of autologous radiolabeled neutrophils and SPECT/CT imaging in healthy volunteers showed that plerixafor did not affect GM-CSF-primed neutrophil entrapment or release in the lungs. Taken together, these data suggest that plerixafor causes neutrophil mobilization from the bone marrow but does not impact on lung marginated neutrophil dynamics and thus is unlikely to compromise respiratory host defense both in humans and mice.