Ellen E. Millman

Neutrophil Sequestration and Pulmonary Dysfunction in a Canine Model of Open Heart Surgery With Cardiopulmonary Bypass Evidence for a CD18-Dependent Mechanism

BACKGROUND Previous studies documented an inflammatory reaction to cardiopulmonary bypass with neutrophil (PMN) sequestration in the lungs, contributing to microvascular injury and postoperative pulmonary dysfunction. This study explored the hypothesis that the beta 2 integrin CD18, a leukocyte adhesion molecule, mediates this response. METHODS AND RESULTS Fifteen adult, mixed-breed dogs underwent 90 minutes of cardiopulmonary bypass with 3 hours of subsequent recovery. Seven additional dogs were treated before cardiopulmonary bypass with a 1-mg/kg IV bolus of R15.7 IgG, a monoclonal antibody to CD18. Both groups were compared with 5 sham bypass control dogs. Bypassed dogs demonstrated an increased number of PMNs sequestered in the lungs 3 hours after bypass compared with sham bypass control dogs (1466 +/- 75 versus 516 +/- 43 PMN/mm2 alveolar surface area, mean +/- SEM, P < .001). Also, when PMNs from bypass dogs were compared with those from sham dogs, they produced more H2O2 (305 +/- 45 versus 144 +/- 48 amol H2O2 per phagocyte per 20 minutes, P < .05). Bypass dogs had significantly decreased arterial oxygenation 3 hours after the procedure compared with shams (457 +/- 20 versus 246 +/- 49 mm Hg, P < .05), and they had a significantly increased lung wet-to-dry weight ratio (5.38 +/- 0.14 versus 4.54 +/- 0.15, P = .003), demonstrating a significant increase in lung water. R15.7 markedly attenuated pulmonary PMN accumulation in bypass dogs (412 +/- 73 PMN/mm2, P < .001) and significantly inhibited PMN production of H2O2 (146 +/- 18 amol H2O2 per phagocyte per 20 minutes, P < .05) Bypass dogs pretreated with R15.7 also had improved oxygenation (445 +/- 28 mm Hg, P < .05) and tended to have less lung water accumulation after bypass (4.99 +/- 0.20). CONCLUSIONS Pulmonary dysfunction after cardiopulmonary bypass is caused, at least in part, by a neutrophil-mediated, CD18-dependent mechanism.

Rab11 regulates the recycling and lysosome targeting of β2-adrenergic receptors

The pericentriolar recycling endosome (RE) may be an alternative compartment through which some β2-adrenergic receptors (β2ARs) recycle from early endosomes to the cell surface during prolonged exposure to agonist. For the transferrin receptor, CXCR2, and the M4-muscarinic acetylcholine receptor, trafficking through the RE and receptor recycling is regulated by the small GTPase rab11. The precise role of the RE and rab11 in regulating the cellular trafficking of the β2AR is not understood. We therefore monitored trafficking of β2ARs in HEK293 cells following the modulation of rab11 activity. Expression of a rab11 mutant deficient in GTP binding (as a fusion between enhanced green fluorescent protein (EGFP) and the rab11S25N mutant) significantly slowed receptor recycling to the cell surface from dispersed transferrin-positive peripheral vesicles following a brief exposure to agonist. The agonist was applied at a time when receptors have undergone only one or two rounds of endocytosis and recycling. In cells overexpressing wild-type rab11, β2ARs localized to a rab11-positive compartment and the rate of β2AR recycling to the cell surface was reduced, but only after prolonged exposure to agonist and multiple rounds of receptor endocytosis and recycling. This effect was associated with impaired β2AR trafficking to lysosomes and receptor proteolysis, whereas the sorting of low-density lipoprotein from transferrin-positive vesicles to late endosomes and lysosomes was not affected. These data highlight a pivotal role for rab11 in regulating the traffic of a G protein-coupled receptor at the level of the RE, where modulation of rab11 activity dictates the balance between receptor recycling and downregulation during prolonged exposure to agonist.

Role of the G Protein-coupled Receptor Kinase Site Serine Cluster in β2-Adrenergic Receptor Internalization, Desensitization, and β-Arrestin Translocation

There is considerable evidence for the role of carboxyl-terminal serines 355, 356, and 364 in G protein-coupled receptor kinase (GRK)-mediated phosphorylation and desensitization of β2-adrenergic receptors (β2ARs). In this study we used receptors in which these serines were changed to alanines (SA3) or to aspartic acids (SD3) to determine the role of these sites in β-arrestin-dependent β2AR internalization and desensitization. Coupling efficiencies for epinephrine activation of adenylyl cyclase were similar in wild-type and mutant receptors, demonstrating that the SD3 mutant did not drive constitutive GRK desensitization. Treatment of wild-type and mutant receptors with 0.3 nm isoproterenol for 5 min induced ∼2-fold increases in the EC50 for agonist activation of adenylyl cyclase, consistent with protein kinase A (PKA) site-mediated desensitization. When exposed to 1 μm isoproterenol to trigger GRK site-mediated desensitization, only wild-type receptors showed significant further desensitization. Using a phospho site-specific antibody, we determined that there is no requirement for these GRK sites in PKA-mediated phosphorylation at high agonist concentration. The rates of agonist-induced internalization of the SD3 and SA3 mutants were 44 and 13%, respectively, relative to that of wild-type receptors, but the SD3 mutant recruited enhanced green fluorescent protein (EGFP)-β-arrestin 2 to the plasma membrane, whereas the SA3 mutant did not. EGFP-β-Arrestin2 overexpression triggered a significant increase in the extent of SD3 mutant desensitization but had no effect on the desensitization of wild-type receptors or the SA3 mutant. Expression of a phosphorylation-independent β-arrestin 1 mutant (R169E) significantly rescued the internalization defect of the SA3 mutant but inhibited the phosphorylation of serines 355 and 356 in wild-type receptors. Our data demonstrate that (i) the lack of GRK sites does not impair PKA site phosphorylation, (ii) the SD3 mutation inhibits GRK-mediated desensitization although it supports some agonist-induced β-arrestin binding and receptor internalization, and (iii) serines 355, 356, and 364 play a pivotal role in the GRK-mediated desensitization, β-arrestin binding, and internalization of β2ARs.

Rapid Recycling of β2‐Adrenergic Receptors is Dependent on the Actin Cytoskeleton and Myosin Vb

For the β2‐adrenergic receptor (β2AR), published evidence suggests that an intact actin cytoskeleton is required for the endocytosis of receptors and their proper sorting to the rapid recycling pathway. We have characterized the role of the actin cytoskeleton in the regulation of β2AR trafficking in human embryonic kidney 293 (HEK293) cells using two distinct actin filament disrupting compounds, cytochalasin D and latrunculin B (LB). In cells pretreated with either drug, β2AR internalization into transferrin‐positive vesicles was not altered but both agents significantly decreased the rate at which β2ARs recycled to the cell surface. In LB‐treated cells, nonrecycled β2ARs were localized to early embryonic antigen 1‐positive endosomes and also accumulated in the recycling endosome (RE), but only a small fraction of receptors localized to LAMP‐positive late endosomes and lysosomes. Treatment with LB also markedly enhanced the inhibitory effect of rab11 overexpression on receptor recycling. Dissociating receptors from actin by expression of the myosin Vb tail fragment resulted in missorting of β2ARs to the RE, while the expression of various CART fragments or the depletion of actinin‐4 had no detectable effect on β2AR sorting. These results indicate that the actin cytoskeleton is required for the efficient recycling of β2ARs, a process that likely is dependent on myosin Vb.

Endosome sorting of β2‐adrenoceptors is GRK5 independent

We have investigated the role of G protein‐coupled receptor kinase 5 (GRK5) in the regulation of endosome sorting of human β2‐adrenoceptors. Expressing GRK5 at a high level significantly increased the extent of internalization of wild‐type β2‐adrenoceptors and of an internalization‐defective mutant receptor, and increased receptor phosphorylation at serines 355 and 356 in the cytoplasmic tail. Overexpressing GRK5 did not alter β2‐adrenoceptor recycling as assessed by immunofluorescence microscopy and radioligand binding assays nor was there any change in receptor downregulation. These data indicate that GRK5 does not regulate the sorting of β2‐adrenoceptors in the endocytic pathway.

Mutating the dileucine motif of the human β2-adrenoceptor reduces the high initial rate of receptor phosphorylation by GRK without affecting postendocytic sorting

The internalization of beta(2)-adrenoceptors after agonist activation results in a desensitized and phosphorylated receptor that either resensitizes by recycling to the cell surface or becomes degraded by postendocytic sorting to lysosomes. The duration and physiological effects of agonists therefore depend on beta(2)-adrenoceptor sorting, highlighting the importance of sorting signals. Dileucine motifs within other membrane proteins act as signals for endocytosis and/or postendocytic sorting, and the beta(2)-adrenoceptor has a dileucine motif within helix 8 that might play a role in efficient receptor recycling and/or downregulation. beta(2)-adrenoceptor internalization and sorting were studied in HEK293 cells stably expressing wild type or mutant dialanine L339A,L340A beta(2)-adrenoceptors. The mutant beta(2)-adrenoceptors showed a significantly lower initial rate of phosphorylation at the prominent G-protein coupled receptor kinase (GRK) sites Ser355 and 356 compared to wild type beta(2)-adrenoceptors. Furthermore, the agonist-induced endocytic rate constant for L339A,L340A beta(2)-adrenoceptors was reduced to approximately 25% that of wild type beta(2)-adrenoceptors, which resulted in a similar reduction in agonist-induced downregulation. Internalized L339A,L340A beta(2)-adrenoceptors recycled to the surface with a rate and extent similar to that of wild type beta(2)-adrenoceptors. Therefore, although the role of L339,L340 in beta(2)-adrenoceptor recycling or postendocytic sorting seems minimal, we conclude that L339,L340 is required for the initial high rate of phosphorylation by G-protein coupled receptor kinases at Ser355,356, which in turn is required for efficient beta(2)-adrenoceptors endocytosis.