title: Uptake of muramyl dipeptide fluorescent congeners by normal rabbit bronchoalveolar lavage cells: a study using flow cytometry.
abstract: Muramyl dipeptide (MDP) is the minimal adjuvant-active structure of mycobacterial cell walls and is known to activate monocytes/macrophages, but mechanisms involved with uptake and activation of these cells have not been completely defined. Earlier studies addressing uptake of MDP and the question of receptors have utilized radioligands and murine peritoneal macrophages. We used fluorescent congeners of MDP and flow cytometry to explore kinetics and specificity of uptake by bronchoalveolar cells of normal rabbits. Both washed cells and cell suspensions from which the fluorescent congeners were not washed were used, and incubation was carried out primarily at 4 degrees C. Fluorescence microscopy consistently revealed intracellular but no visible membrane fluorescence of alveolar macrophages. Uptake was dose dependent but was not saturable up to concentration limits of fluoresceinated muramyl tripeptide (MTP-FITC) imposed by the system, and was partially inhibited by excess unlabeled MDP, consistent with specific inhibition. Alveolar macrophages, but not lymphocytes, demonstrated specific uptake at 4 degrees C, with rapid on- and off-times. Uptake was enhanced 7-fold at 37 degrees C. Uptake was greater by larger, more granular macrophages than by smaller, less granular macrophages, but no difference in uptake was found when cells of similar size but different densities were compared. The exact mechanism of the rapid uptake at 4 degrees C is uncertain but appears to be competed for by unlabeled MDP.
authors.affiliation: Department of Internal Medicine, College of Medicine, University of Iowa, Iowa City.
authors.forename: H B
authors.initials: HB
authors.lastname: Richerson
authors.forename: P A
authors.initials: PA
authors.lastname: Adams
authors.forename: Y
authors.initials: Y
authors.lastname: Iwai
authors.forename: C F
authors.initials: CF
authors.lastname: Barfknecht
mesh_terms: D000119:Acetylmuramyl-Alanyl-Isoglutamine / Q000031:analogs & derivatives / Q000378:metabolism*; D000276:Adjuvants, Immunologic; D000595:Amino Acid Sequence; D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology* / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins / Q000378:metabolism; D005453:Fluorescence; D006168:Guinea Pigs; D007700:Kinetics; D008214:Lymphocytes / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D008969:Molecular Sequence Data; D011650:Pulmonary Alveoli; D011817:Rabbits; D013696:Temperature
chemical_list: D000276:Adjuvants, Immunologic; D005452:Fluoresceins; C063223:N-acetylmuramyl-alanyl-isoglutamyl-lysine-fluorescein isothiocyanate; D000119:Acetylmuramyl-Alanyl-Isoglutamine; D016650:Fluorescein-5-isothiocyanate