Blood, Vol. 114, Issue 15, 3316-3324, October 8, 2009

Fc{gamma}RI ligation leads to a complex with BLT1 in lipid rafts that enhances rat lung macrophage antimicrobial functions
Blood Serezani et al. 114: 3316

Supplemental materials for: Serezani et al

Files in this Data Supplement:

  • Figure S1. BLT1 and CysLT1 redistribute to detergent-resistant LR microdomains upon FcR engagement (JPG, 197 KB) -
    AMs were challenged for 5 min without (A) or with (B) IgG-RBCs (50:1), lysed with 1% Triton X-100, and subjected to discontinuous sucrose gradient (40% and 10%) centrifugation for 18 h at 100,000 x g. Fractions were collected from the gradient and analyzed by SDS-PAGE and membranes immunoblotted for the LR protein flotillin-1, the LR-excluded protein CD45, BLT1, and CysLT1. Data shown are from a single experiment representative of 3 independent experiments.





  • Figure S2. CysLT1-induced signaling and enhancement of AM antimicrobial functions do not require functional LRs (JPG, 639 KB) -
    (A) AMs were pretreated with or without 100 nM LTD4 for 5 min followed by incubation with or without 10:1 IgG-RBCs for 10 min. LR and non-LR membrane fractions were pooled and subjected to immunoblot shown on the right. The experiment depicted is representative of 5 independent experiments. (B) AMs were incubated with (ii and iv) or without (i and iii) 10:1 IgG-RBCs for 10 min, after which LR patches were stained with CTxB-alexa455/anti-CTxB; fixed and stained with anti-CysLT1 alone (i and ii) or anti-CysLT1 and anti-FcRI primary antibodies (iii and iv), followed by a FITC-labeled (for CysLT1) or a rhodamine-labeled (for FcγRI) secondary antibody and then visualized by confocal microcopy. Images are representative of at least 3 independent experiments. (C) AMs were pretreated for 5 min with vehicle control or cyclodextrin compounds followed by the addition of 100 nM LTD4 for 5 min. They were then incubated with IgG-RBCs (50:1) and phagocytic indices determined. (D) AMs were preincubated with cyclodextrin compounds as in (C) before the addition of 50:1 serum-opsonized K. pneumoniae. Thirty min after infection, cells were incubated with 100 nM LTD4 or vehicle. Microbicidal activity (C) and phagocytosis (D) were assessed and expressed as the mean ± SEM from 3 independent experiments, each performed in quintuplicate. *, p 4 for 5 min, followed by incubation with or without 10:1 IgG-RBCs for 10 min. LR and non-LR fractions were pooled and subjected to immunoblot analysis for the proteins of interest shown on the right. The data are representative of at least 3 independent experiments.





  • Figure S3. Cholesterol depletion disrupts LR integrity and interferes with BLT1 and CysLT1 redistribution (JPG, 193 KB) -
    AMs were pretreated with (B) or without (A) 5 µM MβCD for 5 min followed by 10 min incubation with or without 10:1 IgG-RBCs. Cells were lysed by sonication and the membranes were subjected to discountinuous Optiprep™ gradient centrifugation as described in “Material and Methods.” Fractions were harvested and subjected to immunoblot analysis for the proteins of interest shown on the right. The experiment depicted is representative of 2 independent experiments.





  • Figure S4. LR integrity is not required for basal AM antimicrobial functions (JPG, 316 KB) -
    (A) AMs were pretreated for 5 min with vehicle control or 5 mM MβCD, 2-βHPCD, or MαCD followed by IgG-RBCs (50:1) and phagocytic indices determined as described in “Material and Methods.” Phagocytosis is expressed as the percentage of the control value in which no drugs were added. (B) AMs were preincubated with cyclodextrin compounds as in (A) before the addition of 50:1 serum-opsonized K. pneumoniae. Thirty min after infection, cells were incubated with cyclodextrins or vehicle. Microbicidal activity was assessed and expressed as the mean ± SEM percentage survival of ingested bacteria from 3 individual experiments, each performed in triplicate.





  • Figure S5. Protein tyrosine kinase activity is required for FcγR engagement to trigger FcγRI-BLT1 association in LRs (JPG, 462 KB) -
    (A) AMs were pretreated with or without the PTK inhibitor genistein (100 µM) or the PKC inhibitor calphostin C (100 nM) for 20 min followed by incubation with or without 30:1 IgG-RBCs for 10 min. Pooled LR and non-LR fractions were prepared and subjected to immunoblot for the proteins of interest indicated at the right. (B) Relative distribution of total BLT1 and FcγRI was determined by densitometric analysis of immunoblots from 3 different experiments as depicted in (A) and expressed as LR/Non-LR ratio of each. *, p