Cystic fibrosis transmembrane conductance regulator recruitment to phagosomes in neutrophils.

Optimal microbicidal activity of human polymorphonuclear leukocytes (PMN) relies on the generation of toxic agents such as hypochlorous acid (HOCl) in phagosomes. HOCl formation requires H2O2 produced by the NADPH oxidase, myeloperoxidase derived from azurophilic granules, and chloride ion. Chloride transport from cytoplasm into phagosomes requires chloride channels which include cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. However, the phagosomal targeting of CFTR in PMN has not been defined. Using human peripheral blood PMN, we determined that 95-99% of lysosomal-associated membrane protein 1 (LAMP-1)-positive mature phagosomes were CFTR positive, as judged by immunostaining and flow cytometric analysis. To establish a model cell system to evaluate CFTR phagosomal recruitment, we stably expressed enhanced green fluorescent protein (EGFP) alone, EGFP-wt-CFTR and EGFP-DF508-CFTR fusion proteins in promyelocytic PLB-985 cells, respectively. After differentiation into neutrophil-like cells, CFTR presentation to phagosomes was examined. EGFP-wt-CFTR was observed to associate with phagosomes and colocalize with LAMP-1. Flow cytometric analysis of the isolated phagosomes indicated that such a phagosomal targeting was determined by the CFTR portion of the fusion protein. In contrast, significantly less EGFP-DF508-CFTR was found in phagosomes, indicating a defective targeting of the molecule to the organelle. Importantly, the CFTR corrector compound VRT-325 facilitated the recruitment of DF508-CFTR to phagosomes. These data demonstrate the possibility of pharmacologic correction of impaired recruitment of mutant CFTR, thereby providing a potential means to augment chloride supply to the phagosomes of PMN in patients with cystic fibrosis to enhance their microbicidal function.

Chloride transport in functionally active phagosomes isolated from Human neutrophils.

Chloride anion is critical for hypochlorous acid (HOCl) production and microbial killing in neutrophil phagosomes. However, the molecular mechanism by which this anion is transported to the organelle is poorly understood. In this report, membrane-enclosed and functionally active phagosomes were isolated from human neutrophils by using opsonized paramagnetic latex microspheres and a rapid magnetic separation method. The phagosomes recovered were highly enriched for specific protein markers associated with this organelle such as lysosomal-associated membrane protein-1, myeloperoxidase (MPO), lactoferrin, and NADPH oxidase. When FITC-dextran was included in the phagocytosis medium, the majority of the isolated phagosomes retained the fluorescent label after isolation, indicative of intact membrane structure. Flow cytometric measurement of acridine orange, a fluorescent pH indicator, in the purified phagosomes demonstrated that the organelle in its isolated state was capable of transporting protons to the phagosomal lumen via the vacuolar-type ATPase proton pump (V-ATPase). When NADPH was supplied, the isolated phagosomes constitutively oxidized dihydrorhodamine 123, indicating their ability to produce hydrogen peroxide. The preparations also showed a robust production of HOCl within the phagosomal lumen when assayed with the HOCl-specific fluorescent probe R19-S by flow cytometry. MPO-mediated iodination of the proteins covalently conjugated to the phagocytosed beads was quantitatively measured. Phagosomal uptake of iodide and protein iodination were significantly blocked by chloride channel inhibitors, including CFTRinh-172 and NPPB. Further experiments determined that the V-ATPase-driving proton flux into the isolated phagosomes required chloride cotransport, and the cAMP-activated CFTR chloride channel was a major contributor to the chloride transport. Taken together, the data suggest that the phagosomal preparation described herein retains ion transport properties, and multiple chloride channels including CFTR are responsible for chloride supply to neutrophil phagosomes.

Crosstalk between PKA and Epac regulates the phenotypic maturation and function of human dendritic cells.

The cAMP-dependent signaling pathways that orchestrate dendritic cell (DC) maturation remain to be defined in detail. Although cAMP was previously thought to signal exclusively through protein kinase A (PKA), it is now clear that cAMP also activates exchange protein activated by cAMP (Epac), a second major cAMP effector. Whether cAMP signaling via PKA is sufficient to drive DC maturation or whether Epac plays a role has not been examined. In this study, we used cAMP analogs to selectively activate PKA or Epac in human monocyte-derived DCs and examined the effect of these signaling pathways on several hallmarks of DC maturation. We show that PKA activation induces DC maturation as evidenced by the increased cell-surface expression of MHC class II, costimulatory molecules, and the maturation marker CD83. PKA activation also reduces DC endocytosis and stimulates chemotaxis to the lymph node-associated chemokines CXCL12 and CCL21. Although PKA signaling largely suppresses cytokine production, the net effect of PKA activation translates to enhanced DC activation of allogeneic T cells. In contrast to the stimulatory effects of PKA, Epac signaling has no effect on DC maturation or function. Rather, Epac suppresses the effects of PKA when both pathways are activated simultaneously. These data reveal a previously unrecognized crosstalk between the PKA and Epac signaling pathways in DCs and raise the possibility that therapeutics targeting PKA may generate immunogenic DCs, whereas those that activate Epac may produce tolerogenic DCs capable of attenuating allergic or autoimmune disease.

Utility of the radioiodine whole-body retention at 48 hours for modifying empiric activity of 131-iodine for the treatment of metastatic well-differentiated thyroid carcinoma.

BACKGROUND: Dosimetry has been used to help identify when empiric dosages of 131-I treatment for suspected metastatic well-differentiated thyroid carcinoma (WDTC) may be increased or should be decreased, but dosimetry is complex, and easier approaches would be useful. The three objectives of this study were to assess the utility of the percent whole-body retention of 131-I at 48 hours (%WBR(48hr)) in identifying patients with WDTC in whom the therapeutic empiric prescribed activity of 131-I might be increased/decreased, to evaluate the thresholds proposed by Sisson et al. in 2003 for increasing or decreasing activity, and to determine the relationship between %WBR(48hr) and maximum tolerated activity (MTA). METHOD: A retrospective review was conducted of patients who had WDTC, total thyroidectomy, suspected metastatic disease, thyroid hormone withdrawal, and 131-I dosimetry. The %WBR(48hr) was determined based on the Benua-Leeper dosimetry protocol, and the four thresholds and recommendations of Sisson et al., 2003 for the use of %WBR(48hr) were evaluated relative to an empiric activity (EA) of 7.4 GBq of 131-I. A biexponential equation was determined from the %WBR(48hr) data. RESULTS: Of 142 patients, 47 patients had a %WBR(48hr) of <9%, and all could have received more than the EA of 7.4 GBq with an average of 21.0 GBq (incremental range of 6.8-23.2 GBq). Ten patients had a %WBR(48hr) < or = 5%, and all could have had their EA of 7.4 GBq safely increased by at least 250%. Conversely, if the %WBR(48hr) was >24.8%, then 7 of 14 of these patients would have exceeded the MTA by 0.37-3.18 GBq with an EA of 7.4 GBq. Finally, for patients with a %WBR(48hr) > 40%, five of six patients would have exceeded the MTA by 0.85-3.18 GBq. A biexponential regression equation is presented. CONCLUSION: We conclude that, with respect to the treatment of metastatic epithelial cell thyroid cancer, the %WBR(48hr) of 131-I helps identify those patients in whom the empiric therapeutic prescribed activity of 131-I may be increased or should be decreased so as not to exceed the MTA and that Sisson et al.'s thresholds published in 2003 are applicable. We favor a biexponential regression model using the %WBR(48hr) and a lower limit threshold as a potentially useful method for determining how much an empiric therapeutic prescribed activity of 131-I can be increased or decreased.

The utility of radioiodine scans prior to iodine 131 ablation in patients with well-differentiated thyroid cancer.

BACKGROUND: The utility of radioiodine (RAI) scans prior to (131)I ablation is controversial. The objective of this study was to evaluate the utility of RAI scans prior to (131)I ablation in patient with well-differentiated thyroid cancer. METHOD: All RAI scans performed prior to (131)I ablation from July 2000 to November 2006 at Washington Hospital Center were reviewed retrospectively. Patients were excluded who were suspected of having 1) loco-regional disease, 2) distant metastases, and/or 3) physiological uptake that might alter management prior to the pre-ablation RAI scans. RAI scans were performed either 24 hours after dosing with 37-148 MBq of (123)I or 48 hours after dosing with 37-148 MBq of (131)I with imaging of the whole body, the thyroid bed/neck with a pinhole collimator, and the neck and chest with a parallel-hole collimator. One reviewer blindly evaluated each set of scans using six criteria, and for the purpose of this study, the thresholds for each criterion for which the patient's management may have been altered prior to (131)I ablation are noted in parentheses: 1) the number of foci of RAI uptake in thyroid bed/neck (0 or > or =6), 2) the location(s) of these foci in the thyroid bed/neck (outside the thyroid bed), 3) the size of the largest foci in thyroid bed/neck (> or =1 lobe), 4) the percent uptake in the thyroid bed/neck (> or =15%), 5) uptake suggestive of distant metastases, and 6) significant altered biodistribution (e.g., any breast, marked salivary gland, or marked gastrointestinal uptake). RESULTS: Of 355 sets of scans reviewed, 53% of patients had findings on the RAI scans that might have altered the patient's management prior to their (131)I ablation. The data grouped by the criteria noted above were 1) 12% with six or more foci suggesting local metastases and 6% (22) with no focal uptake, 2) 14% with suggestion of lymph node metastases, 3) 1.1% with at least one focus > or =1 lobe, 4) 8% with > or =15% uptake, 5) 4% with distant metastases, 6) 16% demonstrating altered distribution with 6% breast, 3% salivary, 10% GI, and 0.3% urinary bladder. CONCLUSION: Pre-ablation RAI scans demonstrate a significant number of findings that may alter the management of patients with well-differentiated thyroid cancer prior to (131)I ablation.

Ca2+-dependent calmodulin binding to FcRn affects immunoglobulin G transport in the transcytotic pathway.

The Fcgamma receptor FcRn transports immunoglobulin G (IgG) so as to avoid lysosomal degradation and to carry it bidirectionally across epithelial barriers to affect mucosal immunity. Here, we identify a calmodulin-binding site within the FcRn cytoplasmic tail that affects FcRn trafficking. Calmodulin binding to the FcRn tail is direct, calcium-dependent, reversible, and specific to residues comprising a putative short amphipathic alpha-helix immediately adjacent to the membrane. FcRn mutants with single residue substitutions in this motif, or FcRn mutants lacking the cytoplasmic tail completely, exhibit a shorter half-life and attenuated transcytosis. Chemical inhibitors of calmodulin phenocopy the mutant FcRn defect in transcytosis. These results suggest a novel mechanism for regulation of IgG transport by calmodulin-dependent sorting of FcRn and its cargo away from a degradative pathway and into a bidirectional transcytotic route.