CD8+ T lymphocytes and leukotriene B4: Novel interactions in the persistence and progression of asthma Erwin W. Gelfand, MD, Azzeddine Dakhama, PhD

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Presentation transcript:

CD8+ T lymphocytes and leukotriene B4: Novel interactions in the persistence and progression of asthma Erwin W. Gelfand, MD, Azzeddine Dakhama, PhD Journal of Allergy and Clinical Immunology Volume 117, Issue 3, Pages 577-582 (March 2006) DOI: 10.1016/j.jaci.2005.12.1340 Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 1 Model of the CD8+/BLT1+/IL-13+ axis in asthma pathogenesis. Mast cells, activated through the high-affinity receptor (FcεRI) after cross-linking with allergen, initiate a cascade resulting in the rapid synthesis and release of LTB4. Binding of LTB4 to its high-affinity receptor, BLT1, on a subset of CD8+ T cells results in their accumulation in the lung. This CD8+ subset is an important source of IL-13, which affects many of the cell types involved in asthma pathogenesis. PLA2, Phospholipase A2; FLAP, 5-LO activating protein; LTA4, leukotriene A4; LTA4H, LTA4 hydrolase. Journal of Allergy and Clinical Immunology 2006 117, 577-582DOI: (10.1016/j.jaci.2005.12.1340) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 2 Immunofluorescent detection of CD8+, BLT1+, and IL-13+ cells in BAL fluid (A) and tissue (B) of asthmatic subjects. Fresh BAL fluid cells were stimulated for 4 hours in culture with phorbol 12-myristate 13-acetate (5 ng/mL) and ionomycin (500 ng/mL) in the presence of brefeldin A (10 μg/mL). Cells were stained in suspension with mouse monoclonal anti-human CD8 antibody (DakoCytomation, Carpinteria, Calif) and then fixed with paraformaldehyde (4% in PBS, 10 minutes) and permeabilized with saponin (0.1% in staining buffer). Cells were then incubated with allophycocyanin (APC)–conjugated rat anti-human IL-13 (Biolegend, San Diego, Calif) and rabbit anti-human BLT1 (Cayman Chemicals, Ann Arbor, Mich), followed by fluorescein isothiocyanate (FITC)–conjugated goat anti-rabbit Ig (Jackson ImmunoResearch Laboratories, Inc, West Grove, Pa). This anti-BLT1 antibody recognizes the C-terminus of the BLT1 receptor, which is located on the intracellular side of the plasma membrane. After washing, cells were incubated with alkaline phosphatase–conjugated goat anti-mouse Ig (DakoCytomation), followed by incubation with fluorescent Permanent Red alkaline phosphatase substrate (DakoCytomation), and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, Calif). For tissue staining, 5-μm sections of formalin-fixed and paraffin-embedded lung tissue specimens were permeabilized with 0.1% Triton X-100, incubated with rabbit anti-BLT1 and mouse anti-CD8 antibodies, washed with Tris-buffered saline, and incubated with FITC-conjugated goat anti-rabbit Ig and alkaline phosphatase-conjugated goat anti-mouse Ig, followed by washing and incubation with fluorescent Permanent Red alkaline phosphatase substrate and mounting as above. Staining was examined under a Leica DMRXA fluorescent microscope (Leica, Wetzlar, Germany) by using the following filters: Texas Red for CD8 (Orange), FITC for BLT1 (green), and APC for IL-13 (blue). Arrows indicate the same cells stained for CD8, BLT1, and IL-13 (Fig 1, A). Arrowheads indicate BLT1 staining localized on the intracellular side of the membrane of CD8+ cells (Fig 1, B, inset representing the boxed area after Z-scan). Journal of Allergy and Clinical Immunology 2006 117, 577-582DOI: (10.1016/j.jaci.2005.12.1340) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions