|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
First published online April 16, 2004
doi: 10.1242/10.1242/jcs.01072
Research Article |
,25-dihydroxyvitamin D3–phosphoinositide 3-kinase pathway
1 Division of Infectious Diseases, Department of Medicine, The University of British Columbia and Vancouver Hospital Health Sciences Center, D452-HP, 2733 Heather Street, Vancouver, BC, V5Z 3J5 Canada
2 Laboratoire d'Immunologie, Faculté des Sciences Dhar Mahraz, Université Mohamed Ben Abdallah. BP 1796, Atlas, Fès, 30000, Morocco
3 Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA
* Author for correspondence (e-mail: hmama{at}interchange.ubc.ca)
Accepted 31 December 2003
Macrophage cell membranes were labeled with PKH26 and subsequently incubated with latex beads to generate phagosomes surrounded by a red-fluorescent membrane suitable for flow cytometry. Following cell disruption and partial purification of phagosomes, these vesicles were readily distinguished from both cell debris and free beads released from disrupted vacuoles. Flow cytometry analysis of phagosomes stained with specific mAbs and FITC-labeled secondary antibodies showed progressive acquisition of both Rab7 and LAMP-1 consistent with movement along the endocytic pathway. Alternatively, macrophages were preloaded with the lysosomal tracer FITC-dextran before membrane labeling with PKH and incubation with latex beads. Phagosome-lysosome fusion was then quantified on the basis of the colocalization of red and green signals. Using these flow cytometry-based systems, we showed that co-internalization of beads with lysates of Mycobacterium tuberculosis, but not lysates from the nonpathogenic organism Mycobacterium smegmatis, markedly decreased phagosome acquisition of Rab7 and LAMP-1 and vesicle fusion with FITC-dextran-loaded lysosomes. Inhibition of phagolysosome fusion could be attributed, at least in part, to the mycobacterial cell wall glycolipid lipoarabinomannan, and further analysis showed complete rescue of phagosome maturation when cells were pretreated with vitamin D3 before exposure to lipoarabinomannan. Moreover, the ability of vitamin D3 to reverse the phenotype of phagosomes in the presence of the glycolipid was completely abrogated by LY-294002, suggesting that vitamin D3 promotes phagolysosome fusion via a phosphoinositide 3-kinase signaling pathway.
These findings establish a robust platform technology based on labeling of phagocyte cell membranes and flow cytometry capable of supporting broad-based screens to identify microbial and other bioactive compounds that influence phagosome biology.
Key words: Flow cytometry, THP-1, PKH26, Phagosome maturation, Mycobacteria, Lipoarabinomannan
This article has been cited by other articles:
|
|
 |
|
  A. Moeenrezakhanlou, L. Shephard, L. Lam, and N. E. Reiner Myeloid cell differentiation in response to calcitriol for expression CD11b and CD14 is regulated by myeloid zinc finger-1 protein downstream of phosphatidylinositol 3-kinase J. Leukoc. Biol., August 1, 2008; 84(2): 519 - 528. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Welin, M. E. Winberg, H. Abdalla, E. Sarndahl, B. Rasmusson, O. Stendahl, and M. Lerm Incorporation of Mycobacterium tuberculosis Lipoarabinomannan into Macrophage Membrane Rafts Is a Prerequisite for the Phagosomal Maturation Block Infect. Immun., July 1, 2008; 76(7): 2882 - 2887. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Saldias, J. Lamothe, R. Wu, and M. A. Valvano Burkholderia cenocepacia Requires the RpoN Sigma Factor for Biofilm Formation and Intracellular Trafficking within Macrophages Infect. Immun., March 1, 2008; 76(3): 1059 - 1067. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Sun, A.-E. Deghmane, H. Soualhine, T. Hong, C. Bucci, A. Solodkin, and Z. Hmama Mycobacterium bovis BCG disrupts the interaction of Rab7 with RILP contributing to inhibition of phagosome maturation J. Leukoc. Biol., December 1, 2007; 82(6): 1437 - 1445. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Soualhine, A.-E. Deghmane, J. Sun, K. Mak, A. Talal, Y. Av-Gay, and Z. Hmama Mycobacterium bovis Bacillus Calmette-Guerin Secreting Active Cathepsin S Stimulates Expression of Mature MHC Class II Molecules and Antigen Presentation in Human Macrophages J. Immunol., October 15, 2007; 179(8): 5137 - 5145. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.-E. Deghmane, H. Soualhine, H. Bach, K. Sendide, S. Itoh, A. Tam, S. Noubir, A. Talal, R. Lo, S. Toyoshima, et al. Lipoamide dehydrogenase mediates retention of coronin-1 on BCG vacuoles, leading to arrest in phagosome maturation J. Cell Sci., August 15, 2007; 120(16): 2796 - 2806. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Martineau, R. J. Wilkinson, K. A. Wilkinson, S. M. Newton, B. Kampmann, B. M. Hall, G. E. Packe, R. N. Davidson, S. M. Eldridge, Z. J. Maunsell, et al. A Single Dose of Vitamin D Enhances Immunity to Mycobacteria Am. J. Respir. Crit. Care Med., July 15, 2007; 176(2): 208 - 213. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. R. Martineau, K. A. Wilkinson, S. M. Newton, R. A. Floto, A. W. Norman, K. Skolimowska, R. N. Davidson, O. E. Sorensen, B. Kampmann, C. J. Griffiths, et al. IFN-{gamma}- and TNF-Independent Vitamin D-Inducible Human Suppression of Mycobacteria: The Role of Cathelicidin LL-37 J. Immunol., June 1, 2007; 178(11): 7190 - 7198. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Moeenrezakhanlou, D. Nandan, L. Shephard, and N. E. Reiner 1{alpha},25-Dihydroxycholecalciferol activates binding of CREB to a CRE site in the CD14 promoter and drives promoter activity in a phosphatidylinositol-3 kinase-dependent manner J. Leukoc. Biol., May 1, 2007; 81(5): 1311 - 1321. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. B. Torrelles, A. K. Azad, and L. S. Schlesinger Fine Discrimination in the Recognition of Individual Species of Phosphatidyl-myo-Inositol Mannosides from Mycobacterium tuberculosis by C-Type Lectin Pattern Recognition Receptors J. Immunol., August 1, 2006; 177(3): 1805 - 1816. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. M. Chertow, S. Blumenthal, S. Turner, M. Roppolo, L. Stern, E. M. Chi, J. Reed, and on behalf of the CONTROL Investigators Cinacalcet Hydrochloride (Sensipar) in Hemodialysis Patients on Active Vitamin D Derivatives with Controlled PTH and Elevated Calcium x Phosphate Clin. J. Am. Soc. Nephrol., March 1, 2006; 1(2): 305 - 312. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. B. Kang, A. K. Azad, J. B. Torrelles, T. M. Kaufman, A. Beharka, E. Tibesar, L. E. DesJardin, and L. S. Schlesinger The human macrophage mannose receptor directs Mycobacterium tuberculosis lipoarabinomannan-mediated phagosome biogenesis J. Exp. Med., October 3, 2005; 202(7): 987 - 999. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Sendide, N. E. Reiner, J. S. I. Lee, S. Bourgoin, A. Talal, and Z. Hmama Cross-Talk between CD14 and Complement Receptor 3 Promotes Phagocytosis of Mycobacteria: Regulation by Phosphatidylinositol 3-Kinase and Cytohesin-1 J. Immunol., April 1, 2005; 174(7): 4210 - 4219. [Abstract] [Full Text] [PDF]
|
|
