Lymphocyte chemotaxis is a complex process by which cells move within tissues and across barriers such as vascular endothelium and is usually stimulated by chemokines such as stromal cell-derived factor-1 (CXCL12) acting via G protein-coupled receptors. Because members of this receptor family are regulated (''desensitized'') by G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and ␤-arrestin binding, we examined signaling and chemotactic responses in splenocytes derived from knockout mice deficient in various ␤-arrestins and GRKs, with the expectation that these responses might be enhanced. Knockouts of ␤-arrestin2, GRK5, and GRK6 were examined because all three proteins are expressed at high levels in purified mouse CD3؉ T and B220؉ B splenocytes. CXCL12 stimulation of membrane GTPase activity was unaffected in splenocytes derived from GRK5-deficient mice but was increased in splenocytes from the ␤-arrestin2-and GRK6-deficient animals. Surprisingly, however, both T and B cells from ␤-arrestin2-deficient animals and T cells from GRK6-deficient animals were strikingly impaired in their ability to respond to CXCL12 both in transwell migration assays and in transendothelial migration assays. Chemotactic responses of lymphocytes from GRK5-deficient mice were unaffected. Thus, these results indicate that ␤-arrestin2 and GRK6 actually play positive regulatory roles in mediating the chemotactic responses of T and B lymphocytes to CXCL12.L eukocytes migrate to sites of inflammation by recognizing a gradient of chemoattractants, and moving toward the chemoattractant source. This process of ligand recognition, cell polarization and directed cell migration is complex, given that a cell needs to integrate numerous signals arising from different spatial orientations to decide in which direction to move. In lymphocytes, the signals that guide the cell in making these decisions arise from chemokine activation of heptahelical G protein-coupled receptors (GPCR) linked to G␣i proteins. Directional migration, however, requires the activity of G␤␥, but not G␣, proteins (1, 2). In addition, Rho family guanosine triphosphatases (GTPases), the phosphoinositide 3-kinases, and possibly extracellular receptor kinases each play important roles in generating the cell polarity and cytoskeletal reorganization required for directional migration (3-7).Migration to chemokine gradients is dose dependent. Chemotaxis occurs at relatively low concentrations of chemokines, but at higher chemokine concentrations, cells become paralyzed and no longer migrate toward the chemoattractant source. The mechanisms by which cells are paralyzed at high chemokine concentrations are not clear but may involve agonist-dependent desensitization and receptor endocytosis mediated by G proteincoupled receptor kinases (GRKs) and arrestins. GRKs phosphorylate serine and threonine residues in the C terminus and intracellular loops of GPCRs, allowing for the association of arrestins that act to prevent heterotrimeric G␣␤␥ protein association with a...

An increasing body of evidence suggests that bone marrow–derived myeloid cells play a critical role in the pathophysiology of pulmonary hypertension (PH). However, the true requirement for myeloid cells in PH development has not been demonstrated, and a specific disease-promoting myeloid cell population has not been identified. Using bone marrow chimeras, lineage labeling, and proliferation studies, we determined that, in murine hypoxia-induced PH, Ly6Clo nonclassical monocytes are recruited to small pulmonary arteries and differentiate into pulmonary interstitial macrophages. Accumulation of these nonclassical monocyte–derived pulmonary interstitial macrophages around pulmonary vasculature is associated with increased muscularization of small pulmonary arteries and disease severity. To determine if the sensing of hypoxia by nonclassical monocytes contributes to the development of PH, mice lacking expression of hypoxia-inducible factor-1α in the Ly6Clo monocyte lineage were exposed to hypoxia. In these mice, vascular remodeling and PH severity were significantly reduced. Transcriptome analyses suggest that the Ly6Clo monocyte lineage regulates PH through complement, phagocytosis, Ag presentation, and chemokine/cytokine pathways. Consistent with these murine findings, relative to controls, lungs from pulmonary arterial hypertension patients displayed a significant increase in the frequency of nonclassical monocytes. Taken together, these findings show that, in response to hypoxia, nonclassical monocytes in the lung sense hypoxia, infiltrate small pulmonary arteries, and promote vascular remodeling and development of PH. Our results demonstrate that myeloid cells, specifically cells of the nonclassical monocyte lineage, play a direct role in the pathogenesis of PH.

We report here senescent changes in the structure and organization of the mucociliary pseudostratified epithelium of the mouse trachea and main stem bronchi. We confirm previous reports of the gradual appearance of age-related, gland-like structures (ARGLS) in the submucosa, especially in the intercartilage regions and carina. Immunohistochemistry shows these structures contain ciliated and secretory cells and Krt5+ basal cells, but not the myoepithelial cells or ciliated ducts typical of normal submucosal glands. Data suggest they arise de novo by budding from the surface epithelium rather than by delayed growth of rudimentary or cryptic submucosal glands. In old mice the surface epithelium contains fewer cells per unit length than in young mice and the proportion of Krt5+, p63+ basal cells is reduced in both males and females. However, there appears to be no significant difference in the ability of basal stem cells isolated from individual young and old mice to form clonal tracheospheres in culture or in the ability of the epithelium to repair after damage by inhaled sulfur dioxide. Gene expression analysis by Affymetrix microarray and quantitative PCR, as well as immunohistochemistry and flow sorting studies, are consistent with low-grade chronic inflammation in the tracheas of old versus young mice and an increase in the number of immune cells. The significance of these changes for ARGL formation are not clear since several treatments that induce acute inflammation in young mice did not result in budding of the surface epithelium.