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Abstract: Our previous results on the phagocytic activity of alveolar macrophages (Mϕs) toward poly(lactic-co-glycolic) acid microspheres (PLGA MS) loaded with the anti-tuberculosis agent rifampicin (R-PLGA MS) suggest that the phagocytosis of R-PLGA MS enhances the phagocytic activity of Mϕ cells . To confirm this possibility, we examined the effect of phagocytosis of R-PLGA MS and polystyrene latex (PSL) MS on the phagocytic uptake of fluorescent PSL (F-PSL) MS by cells of the rat alveolar macrophage cell line NR8383 at 37°C. Phagocytic activity was examined in terms of the population of Mϕ cells that had phagocytosed MS (N total) and the total number of MS phagocytosed (n total) by counting the phagocytic Mϕ cells and the MS ingested in optical microscopic fields. Phagocytosis of R-PLGA MS enhanced about 1.5 times the values of N total and n total of the phagocytosis of F-PSL MS under the conditions where the phagocytosis of F-PSL MS did not attain the saturated level. In contrast, the phagocytosis of PSL MS did not enhance the phagocytic activity of Mϕ cells toward F-PSL MS. In conclusion, R-PLGA MS are favorable for drug delivery of anti-tuberculosis agents into alveolar Mϕs due to their ability to up-regulate the phagocytosis of MS. [Copyright &y& Elsevier]

Abstract: Understanding of the association of phagocytosis of polymers with signaling of innate immunity of macrophages is the major purpose of this study. Polymer conjugates have been utilized for clinical therapy of cancer and infections, such as Mycobacterium tuberculosis, as effective vectors of drug-delivery systems. They are incorporated through phagocytosis into macrophages and activate innate immunity signaling, which plays a crucial role in its therapeutic and side effects. Macrophage phagocytosis of polystyrene latex microspheres was examined and assayed by treatment of macrophages with the cholesterol depletor methyl-β-cyclodextrin (MβCD) or the sphingolipid depletor n-octyl-β-d-glucopyranoside (OGP). Expressions of various mRNAs during phagocytosis were quantified by real-time PCR. Phagocytosis of polystyrene latex microspheres by various macrophages, such as murine monocyte-derived macrophage J774, rat alveolar macrophage NR8383, and murine Kupffer cell KC13-2, was suppressed by treatment with MβCD or OGP in a concentration-dependent manner. The expression of mRNAs of TNFα, IL-1β, IL-6 and CXCL10 genes induced by lipopolysaccharide (LPS) was not suppressed by treatment with MβCD in J774 cells. Moreover, genes that were induced by LPS were up-regulated even in the absence of LPS by the phagocytosis of polymer conjugates, but such up-regulations were not suppressed by the treatment with MβCD. It was shown that lipid rafts play a significant role in incorporation of polymer conjugates through phagocytosis of macrophages, but their association with signal transduction in innate immunity is very limited. [Copyright &y& Elsevier]

Abstract: A method for exact determination of phagocytic activity of alveolar macrophage (Mϕ) cells toward synthetic microspheres (MS) by optical microscopy was developed. We examined the effectiveness of the treatment of Mϕ samples with trypsin, acid or xylene to remove the polystyrene latex microspheres (PSL MS) attached to Mϕ cell membranes during their phagocytosis by Mϕ cells. We found that centrifugation, which was employed to collect Mϕ samples after incubation with MS, affected significantly the efficiency of the various treatments. Of the three treatments, xylene treatment without centrifugation was the most effective to determine the phagocytic activity of Mϕ cells, as xylene dissolved the PSL MS on the cell surface almost completely. This treatment was also effective in the case of poly(lactic-co-glycolic acid) MS (PLGA MS), which have been commonly used as an efficient vehicle for drug delivery system. [Copyright &y& Elsevier]

Polystyrene microspheres with diameters of 0.2, 0.5, 1.0, 6.0 and 10 μm were added to alveolar macrophages, and their uptake was determined as the amount of superoxide generated from macrophages by the usage of chemiluminescence assay with luminol. The amount of superoxide generated was apparently higher with polystyrene microspheres with a diameter of 1 μm than those with diameters smaller than 1 μm (i.e. 0.2 or 0.5 μm) and with larger than 1 μm (6 or 10 μm). The effects of the functional groups located on the microsphere surfaces upon the uptake by alveolar macrophages were studied with polystyrene microspheres of 1 μm diameter having the primary amine, sulfate, hydroxyl, or carboxyl groups on their surfaces. We found that the macrophages most effectively trapped polystyrene microspheres with primary amine groups, those with carboxyl groups to a slightly lesser extent, and other microspheres much less amounts. The surface properties of these microspheres were determined by measuring their electrophoretic mobility in phosphate buffer solution (pH 7.4) with various ionic strengths. By the analysis of data with Ohshima''s electrokinetic theory for soft particles, the surface charge density and the electrophoretic softness of the microsphere surfaces were determined. All the microsphere surfaces were found to be negatively charged, and those with primary amine groups and carboxyl groups were softer than other microspheres. From these findings, it is suggested that microspheres having soft surfaces are easily accessible to alveolar macrophages, and effectively trapped by macrophages. [Copyright &y& Elsevier]

In order to determine whether the microsphere size affects the phagocytic uptake of microspheres in vitro, polystyrene microspheres with diameters of 0.2, 0.5, 1.0, 6.0 and 10 μm were added to a HL-60 differentiated macrophage-like cell culture system. The extent of the uptake was determined by the amount of superoxide generated from activated macrophages by the usage of a chemiluminescence (CL) assay with luminol. The release amount of superoxide was higher when polystyrene microspheres with diameters between 0.5 and 6 μm (i.e. 0.5, 1, and 6 μm were added to the macrophages than that when those with a diameter 0.2 or 10 μm were added. Especially, polystyrene microspheres with an average diameter of 1 μm were most effectively recognized as foreign materials by the macrophages. The effects of the functional groups on the microsphere surfaces upon the uptake by macrophages were studied using polystyrene microspheres with an average diameter of 1 μm, having primary amine, sulfate, hydroxyl, or carboxyl groups on their surfaces. Polystyrene microspheres having primary amine groups on their surfaces were more effectively engulfed by the macrophages than polystyrene microspheres having other functional groups. Also, polystyrene microspheres coated with human IgG was more easily recognized as foreign materials than intact polystyrene microspheres. [Copyright &y& Elsevier]