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Following inhalation of Mycobacterium tuberculosis, including bacillus Calmette-Guérin (BCG), pathogens enter and grow inside macrophages by taking advantage of their phagocytic mechanisms. Macrophages often fail to eliminate intracellular M. tuberculosis, leading to the induction of host macrophage death. Despite accumulating evidence, the molecular mechanisms underlying M. tuberculosis infection-induced cell death remain controversial. In this study, we show the involvement of two distinct pathways triggered by TLR2 and β2 integrin in BCG infection-induced macrophage apoptosis. First, BCG infection induced activation of ERK1/2, which in turn caused phosphorylation/activation of the proapoptotic protein Bim in mouse macrophage-like Raw 264.7 cells. BCG-infected Raw cells treated with U0126, an MEK/ERK inhibitor, led to the suppression of Bim phosphorylation alongside a remarkable increase in the number of viable macrophages. Small interfering RNA-mediated knockdown of Bim rescued the macrophages from the apoptotic cell death induced by BCG infection. Stimulation with Pam3CSK, a TLR2 agonist, induced macrophage apoptosis with a concomitant increase in the phosphorylation/activation of MEK/ERK and Bim. These observations indicate the important role of the TLR2/MEK/ERK/Bim pathway in BCG infection-induced macrophage apoptosis. Second, we used the β2 integrin agonists C3bi and fibronectin to show that the β2 integrin-derived signal was involved in BCG infection-induced apoptosis, independent of MEK/ERK activation. Interestingly, latex beads coated with Pam3CSK and C3bi were able to induce apoptosis in macrophages to the same extent and specificity as that induced by BCG. Taken together, two distinct pattern-recognition membrane receptors, TLR2 and β2 integrin, acted as triggers in BCG infection-induced macrophage apoptosis, in which MEK/ERK activation played a crucial role following the engagement of TLR2.

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 © 2011 Elsevier B.V. All rights reserved.)

Background: Micro- and nanospheres have been used as carrier materials for effective delivery of drugs into macrophages via endocytosis including phagocytosis for the treatment of cancer and infections., Materials and Methods: To analyze the role of lipid rafts in the endocytic uptake of microspheres by macrophages, we studied the incorporation of polystyrene latex microspheres (PSL MS) by treating macrophages with reagents that affect the physicochemical and biochemical functions of their lipid rafts, such as the cholesterol depletor methyl-β-cyclodextrin (MβCD) and the inhibitor of scavenger receptors polyinosinic acid (poly I). For this study, we used 3-μm fluorescent polystyrene latex microspheres (F-PSL MS) and J774 murine macrophage., Results: We found that the endocytosis of J774 cells was remarkably reduced in a concentration-dependent manner by treatment of the cells with MβCD and that endocytosis was suppressed by approximately 50% by treatment with 100 μg/ml poly I. These results suggest that scavenger receptors are associated with phagocytosis of F-PSL MS and that their functions are regulated by lipid rafts., Conclusion: PSL MS are phagocytosed via a raft-dependent pathway.

Background and Aims: Tumor necrosis factor (TNF) production by the macrophages in intestines appears to play a critical role in the pathogenesis of Crohn's disease (CD). However, it is reported that resident intestinal macrophages (both colonic and small-bowel) do not produce TNF after lipopolysaccharide (LPS) stimulation. It has not yet been proven whether or not intestinal macrophages have an inherent potential to produce TNF. The purpose of this study is to answer this question., Materials and Methods: Colonic macrophages were isolated from lamina propria of human large intestine and stimulated with a variety of substances: LPS, a lipid A derivative (ONO-4007), killed Streptococcus bacterial body (OK-432), phorbol 12-myristate 13-acetate, and lectins (pokeweed mitogen and Sarcophaga lectin)., Results: Colonic macrophages were phenotypically negative for CD14 and positive for CD68 and produced very little TNF in response to LPS, as reported previously. Of the substances tested, only Sarcophaga lectin, which is a defense protein of fleshflies (Sarcophaga peregrina), induced TNF production by the intestinal macrophages. In addition, when the colonic macrophages were cultured on immunoglobulin-A-coated dishes, their characteristic response to LPS was altered, and they produced TNF at a level 6.6 times higher than when on collagen-coated dishes., Conclusion: Colonic macrophages have an inherent ability to produce TNF. Activation of colonic macrophages by unknown substances may contribute to the induction of TNF production, which causes the intestinal inflammation of CD.

Background: Over the last decade, tumor-specific antigens have been discovered, but so far it has not been possible to use them as part of an effective acquired immunotherapy. This failure may be due to the fact that the expression of the MHC class 1 is low and in lung cancer cells is heterogeneous. Therefore, it may be advantageous to develop techniques that activate the antitumor mechanism of the innate immune system. An experimental model was developed for testing lung cancer therapies that are based on the stimulation of macrophages, which then activate innate immunity., Materials and Methods: A549, a human lung adenocarcinoma cell line, was co-cultured with a rat macrophage cell line (NR8383), or a human macrophage cell line (THP 1) at the ratios of 1:1 or 1:5. The experiments were performed with lipopolysaccharide (LPS) or in its absence. The cytotoxicity rate to A549 cells was estimated over time using a dye-uptake method and the amount of lactate dehydrogenase released was measured. The amount of nitric oxide (NO) induced in the medium was assayed, because it may be a candidate as a useful cytotoxic factor., Results: High cytotoxicity was observed to A549 cells when co-cultured with NR8383 cells in the presence of LPS. This effect was not observed in the absence of LPS. Similar results, although to a lesser extent, were observed when A549 cells were co-cultured with THP-1 cells. A high concentration of NO was measured in the co-culture medium of A549 cells and NR8383 cells when LPS was present., Conclusion: The induction of cell death in lung cancer cells occurred after contact with macrophages that had been activated by LPS. The NO that was produced by macrophages in response to LPS was responsible for some of this effect.

Appropriate and rational modulation of innate immunity may enhance the therapeutic efficacy of emerging immune therapies for treating cancer. One of the crucial cells of innate immunity is the macrophage. The purpose of this article was to review those issues that suggest ways of exploiting macrophage local functions in immune therapy, and to discuss the suitability of low molecular-weight lipopolysaccharides as potent modulators of macrophage functions for immune therapy of cancer.

Investigates whether or not intestinal macrophages have an inherent potential to produce tumor necrosis factors. Method used to obtain human colonic macrophages from lamina propia; Details of preparing immunoglobulin-coated dishes; Isolation of human peripheral blood monocytes; Results of the study.

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: Inhalation delivery of poly(lactic-co-glycolic) acid (PLGA) microspheres (MS) loaded with the anti-tuberculosis agent rifampicin (RFP–PLGA MS) to alveolar macrophage (Mϕ) cells could be an effective drug delivery system for the treatment of tuberculosis. To examine this possibility, we studied (1) the bactericidal effect of RFP–PLGA MS on Mycobacterium bovis Bacillus Calmette–Guérin (BCG)-infected rat alveolar Mϕ NR8383 cells, and (2) changes in the biochemical events induced in these cells by the uptake of RFP–PLGA MS. The amount of intracellular RFP imported into the Mϕs by RFP–PLGA MS containing 0.25 and 2.50μg RFP/mL was more than twice and ten times, respectively, than that attained with 5.00μg/mL of RFP solution; and the MS exerted more potent bactericidal effect on BCG inside Mϕ cells than 5.00μg RFP/mL solution after incubation for 7days. RFP–PLGA MS little affected the viability of Mϕ cells, whereas the polystyrene latex (PSL) MS used as a reference decreased it significantly. RFP–PLGA MS did not stimulate the production of tumor necrosis factor-α (TNF-α), nitric oxide, interleukin-10 (IL-10), and transforming growth factor-β1 (TGF-β1) by the Mϕ cells, whereas PSL MS stimulated all of these mediators except IL-10. We conclude that RFP–PLGA MS are bio-safe microspheres due to their “silent” nature when taken into Mϕ cells and that they are promising for the treatment of tuberculosis by pulmonary inhalation. [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]

Abstract: We examined the phagocytic activities of alveolar macrophages (NR8383 cells) toward poly(lactic-co-glycolic) acid (PLGA) microspheres (MS) loaded with the anti-tuberculosis agent rifampicin (RFP), the sizes of which were between 1 μm and 10 μm. We found that 1) the phagocytosis was dependent greatly on the particle size and the number of particles added; 2) macrophages phagocytosed considerably the PLGA microspheres loaded with RFP, the diameter of which was between 1 μm and 6 μm, but took up few 10-μm particles; 3) the population of the macrophages that phagocytosed 1-μm or 3-μm particles was larger than that of those phagocytosed 6- or 10-μm particles; 4) a considerable population of macrophages were not able to phagocytose even the 1- and 3-μm particles; 5) the most efficient deliveries of RFP into each macrophage cell and a large population of macrophages were achieved by the phagocytosis of 3-μm particles; and 6) phagocytosis did not affect macrophage viability in 4 h after the start of phagocytosis. [Copyright &y& Elsevier]

Abstract: Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(dl-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9μm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP–PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4μg/1×106 cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15μg/ml) on intracellular bacilli. In vivo efficacy of RFP–PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP–PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages. [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]