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. 2011 Nov;45(5):962-8.
doi: 10.1165/rcmb.2010-0514OC. Epub 2011 Apr 14.

Calcium-activated potassium channel KCa3.1 in lung dendritic cell migration

Zhifei Shao  1 , Toluwalope O Makinde, Devendra K Agrawal
Affiliations

Calcium-activated potassium channel KCa3.1 in lung dendritic cell migration

Zhifei Shao et al. Am J Respir Cell Mol Biol. 2011 Nov.

Abstract

Migration to draining lymph nodes is a critical requirement for dendritic cells (DCs) to control T-cell-mediated immunity. The calcium-activated potassium channel KCa3.1 has been shown to be involved in regulating cell migration in multiple cell types. In this study, KCa3.1 expression and its functional role in lung DC migration were examined. Fluorescence-labeled antigen was intranasally delivered into mouse lungs to label lung Ag-carrying DCs. Lung CD11c(high)CD11b(low) and CD11c(low)CD11b(high) DCs from PBS-treated and ovalbumin (OVA)-sensitized mice were sorted using MACS and FACS. Indo-1 and DiBAC4(3) were used to measure intracellular Ca(2+) and membrane potential, respectively. The mRNA expression of KCa3.1 was examined using real-time PCR. Expression of KCa3.1 protein and CCR7 was measured using flow cytometry. Migration of two lung DC subsets to lymphatic chemokines was examined using TransWell in the absence or presence of the KCa3.1 blocker TRAM-34. OVA sensitization up-regulated mRNA and protein expression of KCa3.1 in lung DCs, with a greater response by the CD11c(high)CD11b(low) than CD11c(low)CD11b(high) DCs. Although KCa3.1 expression in Ag-carrying DCs was higher than that in non-Ag-carrying DCs in OVA-sensitized mice, the difference was not as prominent. However, Ag-carrying lung DCs expressed significantly higher CCR7 than non-Ag-carrying DCs. CCL19, CCL21, and KCa3.1 activator 1-EBIO induced an increase in intracellular calcium in both DC subsets. In addition, 1-EBIO-induced calcium increase was suppressed by TRAM-34. In vitro blockade of KCa3.1 with TRAM-34 impaired CCL19/CCL21-induced transmigration. In conclusion, KCa3.1 expression in lung DCs is up-regulated by OVA sensitization in both lung DC subsets, and KCa3.1 is involved in lung DC migration to lymphatic chemokines.

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Figures

Figure 1.
Figure 1.
Sensitization protocol. (A [solid frame]) Ovalbumin (OVA) sensitization and PBS-treated controls. (B [dashed frame]) Identification of lung Ag-carrying cells. A total of 150 μg of OVA-AlexaFluor647 in 30 μl was intranasally delivered into lungs of OVA-sensitized mice on Day 45. Lungs were collected on Day 47 for the evaluation of antigen uptake by lung dendritic cell (DC) subsets.
Figure 2.
Figure 2.
Expression of KCa3.1 in lung DC subsets. (A) The expression of KCa3.1 in lung DC subsets measured by immunostaining (FITC) and flow cytometry as compared with isotype controls are presented as a histogram of cell frequency versus mean fluorescence intensity (MFI). Data are representative of three independent experiments. (B) Quantitative analysis of KCa3.1 protein expression in lung DC subsets. Protein expression was calculated according to the formula (MFIKCa3.1 – MFIIsotype) (n = 3; error bars, 95% confidence interval [CI]). (C) Fold change of mRNA expression of KCa3.1 in lung DC subsets. The value of fold change between OVA-treated and PBS-treated mice was calculated according to the formula 2−(ΔCTOVA− ΔCTPBS). One sample t test was used to test statistical significance with respect to value 0 (n = 4; error bars, 95% CI). (D) Localization of KCa3.1 on cell membrane. D1, KCa3.1 expression (FITC); D2, cell membrane (WGA-tetramethylrhodamine); D3, nuclear (DAPI); D4, merged image (cell was a MACS-separated–FACS-isolated lung CD11clowCD11bhigh DC, a representative example of at least three independent experiments; original magnification: ×60).
Figure 3.
Figure 3.
–KCa3.1 activator 1-EBIO–induced membrane hyperpolarization and intracellular calcium increase. AutoMACS-separated CD11c+ lung cells were loaded with membrane potential dye DiBAC4(3) (1 μM) or indo-1 (5 μM) at 37°C for 45 minutes. Signal acquisition was performed to obtain the baseline values. The KCa3.1 activator 1-EBIO was added to the CD11c+ cell suspension containing lung CD11chighCD11blow (upper panel) and CD11clowCD11bhigh (lower panel) DCs at 90 ± 5 seconds (A, B) and 45 ± 5 seconds (C, D), with a final concentration of 500 μM (A–C) and 1 mM (D). (E) TRAM-34 (1 μM) was added to 1-EBIO–treated lung DCs. Signal acquisition started 15 seconds after the addition of TRAM-34 and lasted for 300 seconds. Data were collected from three independent experiments using lung DCs pooled from four to six experimental animals for each experiment.
Figure 4.
Figure 4.
Expression of KCa3.1 in Ag-carrying and non–Ag-carrying lung DCs. (A) Ag-carrying DCs were defined based on AlexaFluor647 signal intensity in the cells isolated from the mouse lungs not receiving labeled antigen. (B) KCa3.1 expression in the Ag-carrying and non–Ag-carrying DCs. (C) KCa3.1 expression levels were quantified and indicated by mean fluorescence intensity (n = 3; data obtained from three experimental animals and one control animal).
Figure 5.
Figure 5.
Expression of CCR7 in Ag-carrying and non–Ag-carrying lung DCs. (A) Ag-carrying DCs were defined based on DQ-OVA signal intensity in the cells isolated from the mouse lungs not receiving labeled antigen. (B) CCR7 expression in the Ag-carrying and non–Ag-carrying DCs. (C) Quantification of CCR7 expression indicated by mean fluorescence intensity (n = 3, data obtained from three experimental animals and one control animal).
Figure 6.
Figure 6.
CCL19/CCL21-induced intracellular calcium increase. Cells were labeled with CD11c and CD11b antibody and incubated with Indo-1 for 45 minutes. Intracellular calcium levels were recorded by flow cytometry before and after the addition of ionomycin (1 μg/ml) (A, B), CCL19 (1 μg/ml) (C, D) and CCL21 (1 μg/ml) (E, F) in lung CD11chighCD11blow (upper panel) and CD11clowCD11bhigh (lower panel) DCs. Data were collected from three independent experiments using lung DCs pooled from four to six experimental animals for each experiment.
Figure 7.
Figure 7.
Effect of TRAM-34 on lung DC chemotaxis. Final concentration of chemokines and blocker: CCL19, 100 ng/mL; CCL21, 100 ng/mL; TRAM34, 200 nM (n = 3). Upper panel: CD11chighCD11blow DCs. Lower panel: CD11clowCD11bhigh DCs. The chemotactic index, a measure of the specificity of migration, was determined as follows: (number of cells migrating to chemokines)/(number of cells that migrated to medium alone).
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References

    1. Yanagihara S, Komura E, Nagafune J, Watarai H, Yamaguchi Y. EBI1/CCR7 is a new member of dendritic cell chemokine receptor that is up-regulated upon maturation. J Immunol 1998;161:3096–3102 - PubMed
    1. Chan VW, Kothakota S, Rohan MC, Panganiban-Lustan L, Gardner JP, Wachowicz MS, Winter JA, Williams LT. Secondary lymphoid-tissue chemokine (SLC) is chemotactic for mature dendritic cells. Blood 1999;93:3610–3616 - PubMed
    1. Berridge MJ. Inositol trisphosphate and calcium signalling mechanisms. Biochim Biophys Acta 2009;1793:933–940 - PubMed
    1. Hsu S, O'Connell PJ, Klyachko VA, Badminton MN, Thomson AW, Jackson MB, Clapham DE, Ahern GP. Fundamental Ca2+ signaling mechanisms in mouse dendritic cells: CRAC is the major Ca2+ entry pathway. J Immunol 2001;166:6126–6133 - PubMed
    1. Scandella E, Men Y, Legler DF, Gillessen S, Prikler L, Ludewig B, Groettrup M. CCL19/CCL21-triggered signal transduction and migration of dendritic cells requires prostaglandin E2. Blood 2004;103:1595–1601 - PubMed

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