Cultures Of Human Monocyte-derived Macrophages Research Articles

Some new insights into the biological activities of carboxymethylated polysaccharides from Lasiodiplodia theobromae

BackgroundCarboxymethylated Lasiodiplodan (LaEPS-C), Lasiodiplodia theobromae β-glucan exopolysaccharide derivative, has a well-known range of biological activities. Compared to LaEPS-C, its fractions, Linear (LLaEPS-C) and Branched (BLaEPS-C), have biological potentialities scarcely described in the literature. So, in this study, we investigate the immunomodulatory, antiviral, antiproliferative, and anticoagulant activities of LLaEPS-C and BLaEPS-C and compare them to the LaEPS-C.MethodsLaEPS was obtained from L. theobromae MMBJ. After carboxymethylation, LaEPS-C structural characteristics were confirmed by Elementary Composition Analysis by Energy Dispersive X-Ray Detector (EDS), Fourier Transform Infrared (FTIR), and Nuclear Magnetic Resonance (NMR). The immunomodulatory activity on cytokine secretion was evaluated in human monocyte-derived macrophage cultures. The antiviral activity was evaluated by Hep-2 cell viability in the presence or absence of hRSV (human respiratory syncytial virus). In vitro antiproliferative activity was tested by sulforhodamine B assay. The anticoagulant activity was determined by APTT (Activated Partial Thromboplastin Time) and PT (Prothrombin Time).ResultsLaEPS-C showed low macrophage cell viability only at 100 µg/mL (52.84 ± 24.06, 48 h), and LLaEPS-C presented no effect. Conversely, BLaEPS-C showed cytotoxicity from 25 to 100 µg/mL (44.36 ± 20.16, 40.64 ± 25.55, 33.87 ± 25.16; 48 h). LaEPS-C and LLaEPS-C showed anti-inflammatory activity. LaEPS-C presented this at 100 µg/mL (36.75 ± 5.53, 48 h) for IL-10, and LLaEPS-C reduces TNF-α cytokine productions at 100 µg/mL (18.27 ± 5.80, 48 h). LLaEPS-C showed an anti-hRSV activity (0.7 µg/ml) plus a low cytotoxic activity for Hep-2 cells (1.4 µg/ml). LaEPS-C presented an antiproliferative activity for NCI-ADR/RES (GI50 65.3 µg/mL). A better PT was achieved for LLaEPS-C at 5.0 µg/mL (11.85 ± 0.87s).ConclusionsThese findings demonstrated that carboxymethylation effectively improves the biological potential of the LaEPS-C and their fractions. From those polysaccharides tested, LLaEPS provided the best results with low toxicity for anti-inflammatory, antiviral, and anticoagulant activities.

Abstract 99: Single Cell RNA-Sequencing Reveals Array Of Immune Activation States In Intracerebral Hemorrhage

Introduction: Secondary inflammation is a well-established cause of injury in intracerebral hemorrhage (ICH). One potential treatment for ICH limits inflammation through targeted metabolic modulation of immune cells, but leukocyte activation states in ICH are poorly defined. Using single-cell RNA sequencing (scRNA-seq) of patient hematoma evacuates, we characterized the inflammatory and metabolic profile of over a dozen identifiable leukocyte populations. This research provides insight into the dynamics of immune activation in ICH. Methods: Evacuated hematoma and peripheral blood samples from 8 ICH patients (62 ± 2.1 years) were collected via minimally invasive surgery (n=5) or craniotomy (n=3) (Fig. 1a). Collection time ranged from 0 to 4 days after ICH onset (1.5 ± 0.5 days). scRNA-seq was done on the 10X Genomics platform and analyzed using Seurat. Human monocyte-derived macrophage cultures were stimulated with S100A9 and IL-1β, two ICH-relevant inflammatory molecules, or LPS. Echinomycin and 2-deoxy-D-glucose were used to inhibit HIF signaling and glycolysis respectively. Results: Clustering revealed over a dozen transcriptionally distinct CNS-resident and immune cell types, many of which have little prior study in ICH (Fig. 1b). 3,395 differentially expressed genes between hematoma and peripheral blood (p<0.05) were found in CD14 + monocytes and 932 in CD4 + effector T cells. Pathway analysis found upregulation of HIF signaling, glycolysis, and correlated networks of cytokine expression in the hematoma. The findings were significant across all immune populations but most prominent in myeloid cells (Fig. 1c). Inhibition of HIF and glycolysis in human macrophages in-vitro abrogated inflammatory and reparative cytokine production following inflammatory stimulation. In conclusion, this work highlights the interplay of inflammation and metabolism at a single cell level in ICH, contributing to knowledge of the immune response to brain injury.

Abstract A39: Inflammatory macrophage derived TNF alpha induces downregulation of estrogen receptor alpha in breast cancer cells by inactivation of Foxo3A

Abstract Breast cancers are divided into different subtypes based on receptor expression status: estrogen receptor (ER), progesterone receptor (PR) and Her2. The luminal A subtype (ER+PR+Her2-) is most often associated with a good prognosis while the triple-negative (TN) cancers (ER-PR-Her2-) have a poor prognosis. ER+ cancer cells depend on estrogen for their growth and can be treated with drugs to block its effects. While most human breast cancers express ER alpha, some tumors are hormone independent despite the presence of ER and others become ER negative over time. Myeloid cells are known to affect tumor development, and infiltration of tumor-associated macrophages is associated with a worse prognosis. Theoretically, however, macrophages can be both beneficial and bad for the patient. Proinflammatory M1 macrophages (CD68+ CD163-) can eliminate cancer cells and promote antitumor immune responses while anti-inflammatory M2 macrophages (CD163+CD68+) or anti-inflammatory myeloid cells (CD68-CD163+) can promote tumor progression by inhibiting proinflammatory immune responses and induce wound-healing processes. This includes angiogenesis and matrix degradation, both of which promote metastatic spread and possibly even EMT. Macrophages have also been proposed to downregulate ER in breast cancer cells through an unknown mechanism. Due to the recent reports suggesting that macrophages can induce EMT and downregulate ER, we evaluated the effect of human primary macrophages on ER expression in breast cancer cells. Immunohistochemistry analyses of breast cancer tumor xenografts, established with co-transplantation of primary human myeloid cells together with ER+ breast cancer cells in NSG mice, showed association of monocytes with downregulation of ER-receptor expression. Using primary human monocyte-derived macrophage cultures In vitro, we show that the different subtypes of macrophages have unique cytokine secretion profiles with M1 macrophages secreting significantly higher levels of TNF alpha compared to M2 macrophages. ER alpha positive MCF-7 and ER alpha negative MDA-MB-231 breast cancer cells were cultured in macrophage condition media, and both Western blot and qPCR analysis showed downregulation of ER was caused by M1 macrophage-derived TNF alpha that inhibits FoxO3A transcription of ER alpha. We propose that proinflammatory macrophages (M1), despite being tumoricidal, may have direct effects on endocrine resistance in breast cancer patients. Our findings suggest that different macrophage subtypes have various and unique impacts on breast cancer progression, and that proinflammatory macrophages, despite being tumoricidal, may have unwanted and direct effects on endocrine resistance mechanisms in breast cancer patients. Citation Format: Frida Björk Gunnarsdottir, Catharina Hagerling, Caroline Bergenfelz, Camilla M. Rydberg, Meliha Mehmeti, Roni Allaoui, Sofie Mohlin, Sven Påhlman, Daniel Bexell, Karin Leandersson. Inflammatory macrophage derived TNF alpha induces downregulation of estrogen receptor alpha in breast cancer cells by inactivation of Foxo3A [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A39.

Mitochondrial Calcium Uptake Is Instrumental to Alternative Macrophage Polarization and Phagocytic Activity.

Macrophages are highly plastic and dynamic cells that exert much of their function through phagocytosis. Phagocytosis depends on a coordinated, finely tuned, and compartmentalized regulation of calcium concentrations. We examined the role of mitochondrial calcium uptake and mitochondrial calcium uniporter (MCU) in macrophage polarization and function. In primary cultures of human monocyte-derived macrophages, calcium uptake in mitochondria was instrumental for alternative (M2) macrophage polarization. Mitochondrial calcium uniporter inhibition with KB-R7943 or MCU knockdown, which prevented mitochondrial calcium uptake, reduced M2 polarization, while not affecting classical (M1) polarization. Challenging macrophages with E. coli fragments induced spikes of mitochondrial calcium concentrations, which were prevented by MCU inhibition or silencing. In addition, mitochondria remodelled in M2 macrophages during phagocytosis, especially close to sites of E. coli internalization. Remarkably, inhibition or knockdown of MCU significantly reduced the phagocytic capacity of M2 macrophages. KB-R7943, which also inhibits the membrane sodium/calcium exchanger and Complex I, reduced mitochondria energization and cellular ATP levels, but such effects were not observed with MCU silencing. Therefore, phagocytosis inhibition by MCU knockdown depended on the impaired mitochondrial calcium buffering rather than changes in mitochondrial and cellular energy status. These data uncover a new role for MCU in alternative macrophage polarization and phagocytic activity.

Abstract B055: Optimized human monocyte-derived macrophage cultures accurately reflect macrophage immunomodulatory function

Abstract Prostate cancer tumor growth and disease progression is highly influenced by noncancerous host cells within the tumor microenvironment. Tumor-associated macrophages (TAMs) are a critical component of this microenvironment, and high TAM infiltrate correlates with poor prognosis in prostate cancer. Macrophages can inhibit or support tumor growth depending on their polarization to an M1 or M2 state. M1 macrophages, as part of the Th1 immune response that fights bacterial or viral infection, promote helper and killer T-cell recruitment and activation and induce cell death of infected cells. In the tumor, M1s and their recruited T cells target the cancer cells for cell death. Conversely, as part of the Th2 immune response that repairs tissue injury, M2 macrophages promote cell growth and proliferation, inhibit helper and killer T-cell function, and induce regulatory T-cell differentiation. Protumor M2s TAMS make up the majority of macrophage infiltrate in prostate cancer tumors and protect the cancer cells from attack by the immune system. Techniques for studying TAMs in vitro are limited, which is confounded by the lack of procedure consensus for in vitro macrophage differentiation and polarization. The Pienta Lab established an optimized protocol for generating homogenous populations of M1 and M2 macrophages from human monocytes isolated from whole blood (doi: 10.2144/000114435). While these cell populations have been well characterized by their marker expression, their functional ability to modulate T-cell activation is critical to determining their validity as an in vitro TAM model. Following our optimized protocol, monocytes isolated from human blood were differentiated and cultured as resting macrophages or polarized into M1 or M2 macrophages. Conditioned media were collected at 24, 48, and 72 hours. The immunomodulatory capacity of the macrophages was evaluated by measuring the secreted levels of Th1 cytokines IL-1B and TNF known to recruit and activate helper and killer T cells and Th2 cytokine IL-10 known to prevent helper and killer T-cell function and promote regulatory T-cell induction. The in vitro-polarized human M1s secreted high levels of Th1 cytokines IL-1B and TNF, while levels from both the resting and M2-polarized macrophages were below the limit of detection. Likewise, conditioned media from M2-polarized macrophages showed high levels of Th2 cytokine IL-10, but none was detected in resting or M1 cultures. Taken together, this indicates that M1 and M2 macrophages isolated and polarized using our optimized protocol accurately reflect anticipated macrophage immunomodulatory function. Further work will confirm these data by RT-qPCR of RNA taken from M1, M2, and resting macrophage cultures, and conditioned media from serum-free cultures will be assessed to determine the secretion of additional immunomodulatory cytokines present at abundant levels in serum (e.g., TGFB1). Citation Format: Amber E. de Groot, Kenneth J. Pienta. Optimized human monocyte-derived macrophage cultures accurately reflect macrophage immunomodulatory function [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B055.

Proteomic profiling of macrophages by 2D electrophoresis.

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages migrate in an activation-dependent manner to chemokines involved in neuroinflammation

BackgroundIn neuroinflammatory diseases, macrophages can play a dual role in the process of tissue damage, depending on their activation status (M1 / M2). M1 macrophages are considered to exert damaging effects to neurons, whereas M2 macrophages are reported to aid regeneration and repair of neurons. Their migration within the central nervous system may be of critical importance in the final outcome of neurodegeneration in neuroinflammatory diseases e.g. multiple sclerosis (MS). To provide insight into this process, we examined the migratory capacity of human monocyte-derived M1 and M2 polarised macrophages towards chemoattractants, relevant for neuroinflammatory diseases like MS.MethodsPrimary cultures of human monocyte-derived macrophages were exposed to interferon gamma and lipopolysaccharide (LPS) to evoke proinflammatory (M1) activation or IL-4 to evoke anti-inflammatory (M2) activation. In a TAXIScan assay, migration of M0, M1 and M2 towards chemoattractants was measured and quantified. Furthermore the adhesion capacity and the expression levels of integrins as well as chemokine receptors of M0, M1 and M2 were assessed. Alterations in cell morphology were analysed using fluorescent labelling of the cytoskeleton.ResultsSignificant differences were observed between M1 and M2 macrophages in the migration towards chemoattractants. We show that M2 macrophages migrated over longer distances towards CCL2, CCL5, CXCL10, CXCL12 and C1q compared to non-activated (M0) and M1 macrophages. No differences were observed in the adhesion of M0, M1 and M2 macrophages to multiple matrix components, nor in the expression of integrins and chemokine receptors. Significant changes were observed in the cytoskeleton organization upon stimulation with CCL2, M0, M1 and M2 macrophages adopt a spherical morphology and the cytoskeleton is rapidly rearranged. M0 and M2 macrophages are able to form filopodia, whereas M1 macrophages only adapt a spherical morphology.ConclusionsTogether our results indicate that the alternative activation status of macrophages promotes their migratory properties to chemoattractants relevant for neuroinflammatory diseases like MS. Conversely, classically activated, proinflammatory macrophages have reduced migratory properties. Based on our results, we postulate that the activation status of the macrophage influences the capacity of the macrophages to rearrange their cytoskeleton. This is the first step in understanding how modulation of macrophage activation affects macrophage migration in neuroinflammatory diseases like MS.

Inhaled Microparticles Containing Clofazimine Are Efficacious in Treatment of Experimental Tuberculosis in Mice

Inhalable clofazimine-containing dry powder microparticles (CFM-DPI) and native clofazimine (CFM) were evaluated for activity against Mycobacterium tuberculosis in human monocyte-derived macrophage cultures and in mice infected with a low-dose aerosol. Both formulations resulted in 99% killing at 2.5 μg/ml in vitro. In mice, 480 μg and 720 μg CFM-DPI inhaled twice per week over 4 weeks reduced numbers of CFU in the lung by as much as log(10) 2.6; 500 μg oral CFM achieved a log(10) 0.7 reduction.

Comparison of Ultrastructural Cytotoxic Effects of Carbon and Carbon/Iron Particulates on Human Monocyte-Derived Macrophages

In this study, we tested the hypothesis that the presence of iron in carbon particulates enhances ultrastructural perturbation in human monocyte-derived macrophages (MDMs) after phagocytosis. We used 1-μm synthetic carbon-based particulates, designed to simulate environmental particulates of mass median aerodynamic diameter ≤ 2.5 μm (PM2.5). Cultures of human MDMs or T-lymphocytes (as a nonphagocytic control) were exposed to carbon or carbon/iron particulates for various time periods and examined by transmission electron microscopy for ultrastructural changes. T-cells failed to internalize either of the particulates and showed no organelle or nuclear changes. Conversely, MDMs avidly phagocytized the particulates. MDMs treated with C particulates exhibited morphologic evidence of macrophage activation but no evidence of lysis of organelles. In contrast, MDMs treated with C/Fe particulates exhibited coalescence of particulate-containing lysosomes. This phenomenon was not observed in the case of C particulates. By 24 hr there was a tendency of the C/Fe particulates to agglomerate into loose or compact clusters. Surrounding the compact C/Fe agglomerates was a uniform zone of nearly total organelle lysis. The lytic changes diminished in proportion to the distance from the agglomerate. In such cells, the nucleus showed loss of chromatin. Although C particles induced no detectable oxidative burst on treated MDMs, C/Fe particles induced a nearly 5-fold increase in the extracellular oxidative burst by treated MDMs compared with untreated controls. Iron bound to C particles catalyzed the decomposition of hydrogen peroxide to generate hydroxyl radicals. Results of these studies suggest that, among particulates of similar size, biologic activity can vary profoundly as a function of particulate physicochemical properties.

Mycobacterium avium infection and modulation of human macrophage gene expression.

Mycobacterium avium is a facultative intracellular pathogen cleared rapidly via intact host defense mechanisms. In the absence of adequate T cell function, as occurs in HIV-1-induced immunodeficiency, M. avium becomes an opportunistic infection with uncontrolled replication and reinfection of macrophage hosts. How M. avium infects, survives, and replicates in macrophages without signaling an effective microbicidal counterattack is unresolved. To address whether M. avium signals the expression of molecules, which influence mycobacterial survival or clearance, human monocyte-derived macrophage cultures were exposed to M. avium. Within minutes, M. avium, or its cell wall lipoarabinomannan, binds to the adherent macrophages and induces a spectrum of gene expression. In this innate response, the most abundant genes detected within 2 h by cDNA expression array involved proinflammatory chemokines, cytokines including TNF-alpha and IL-1, and adhesion molecules. Associated with this rapid initial up-regulation of recruitment and amplification molecules was enhanced expression of transcription factors and signaling molecules. By 24 h, this proinflammatory response subsided, and after 4 days, when some bacteria were being degraded, others escaped destruction to replicate within intracellular vacuoles. Under these conditions, inducible NO synthase was not up-regulated and increased transferrin receptors may facilitate iron-dependent mycobacterial growth. Sustained adhesion molecule and chemokine expression along with the formation of multinucleated giant cells appeared consistent with in vivo events. Thus, in the absence of T lymphocyte mediators, macrophages are insufficiently microbicidal and provide a nonhostile environment in which mycobacteria not only survive and replicate, but continue to promote recruitment of new macrophages to perpetuate the infection.

Soluble gC1q-R/p33, a cell protein that binds to the globular "heads" of C1q, effectively inhibits the growth of HIV-1 strains in cell cultures.

C1q and the outer envelope protein of HIV, gp120, have several structural and functional similarities. Therefore, it is plausible to assume that proteins that are able to interact with C1q may also interact with isolated gp120 as well as the whole HIV-1 virus. Based on this hypothesis, we studied the potential ability of the recombinant form of the 33-kDa protein, which binds to the globular "heads" of C1q (gC1q-R/p33), to inhibit the growth of different HIV-1 strains in cell cultures. gC1q-R/p33 was found to effectively and dose-dependently inhibit the production of one T-lymphotropic (X4) and one macrophage-tropic (R5) strain in human T cell lines (MT-4 and H9) and human monocyte-derived macrophage cultures, respectively. At a concentration range of 5-25 microg/ml, gC1q-R caused a marked and prolonged suppression of virus production. The extent of inhibition was enhanced when gC1q-R was first incubated with and then removed from the target cell cultures before virus infection, compared to that when the cells were infected with gC1q-R-HIV mixtures. The extent of inhibition was comparable to that of the Leu3a anti-CD4 antibody. Addition of gC1q-R to the cell cultures on day 1 or 2 after infection induced markedly less inhibition of HIV-1 growth than pretreatment of the cells just before or together with the infective HIV strains. In ELISA experiments, gC1q-R did not bind to a solid-phase recombinant gp120 while strong and dose-dependent binding of gC1q-R to solid-phase CD4 was observed. Our present findings indicate that gC1q-R is an effective inhibitor of HIV-1 infection, which prevents viral entry by blocking the interaction between CD4 and gp120. Since gC1q-R is a human protein, it is most probably not antigenic in humans. It would seem logical, therefore, to consider gC1q-R or its fragments involved in the CD4 binding as potential therapeutic agents.

Nitric oxide synthesis enhances human immunodeficiency virus replication in primary human macrophages.

Macrophages are suspected to play a major role in human immunodeficiency virus (HIV) infection pathogenesis, not only by their contribution to virus dissemination and persistence in the host but also through the dysregulation of immune functions. The production of NO, a highly reactive free radical, is thought to act as an important component of the host immune response in several viral infections. The aim of this study was to evaluate the effects of HIV type 1 (HIV-1) Ba-L replication on inducible nitric oxide synthase (iNOS) mRNA expression in primary cultures of human monocyte-derived macrophages (MDM) and then examine the effects of NO production on the level of HIV-1 replication. Significant induction of the iNOS gene was observed in cultured MDM concomitantly with the peak of virus replication. However, this induction was not accompanied by a measurable production of NO, suggesting a weak synthesis of NO. Surprisingly, exposure to low concentrations of a NO-generating compound (sodium nitroprusside) and L-arginine, the natural substrate of iNOS, results in a significant increase in HIV replication. Accordingly, reduction of L-arginine bioavailability after addition of arginase to the medium significantly reduced HIV replication. The specific involvement of NO was further demonstrated by a dose-dependent inhibition of viral replication that was observed in infected macrophages exposed to N(G)-monomethyl L-arginine and N(G)-nitro-L-arginine methyl ester (L-NAME), two inhibitors of the iNOS. Moreover, an excess of L-arginine reversed the addition of L-NAME, confirming that an arginine-dependent mechanism is involved. Finally, inhibitory effects of hemoglobin which can trap free NO in culture supernatants and in biological fluids in vivo confirmed that endogenously produced NO could interfere with HIV replication in human macrophages.

Chylomicron-remnant-induced foam cell formation and cytotoxicity: a possible mechanism of cell death in atherosclerosis

The effects of chylomicron remnants on cytoplasmic lipid loading and cell viability were assessed in cultures of human monocyte-derived macrophages and rabbit arterial smooth muscle cells. At a cholesterol concentration of 150 microg/ml, chylomicron remnants induced substantial cytoplasmic lipid loading of macrophages, but not of smooth muscle cells, within 6 h of exposure. Chylomicron remnants were found to be cytotoxic to macrophages and smooth muscle cells, although the latter were generally more resistant. Chylomicron remnants contained no detectable oxysterols (>1 ng) and contained less non-esterified ('free') fatty acids than non-lipolysed nascent chylomicrons. Chylomicron-remnant-induced cytotoxicity appeared to be time- and dose-dependent. Macrophage and smooth muscle cell viability were inversely related to the production of superoxide free radicals and were significantly improved in the combined presence of superoxide dismutase and catalase. Collectively, our data suggest that, in macrophages, cell viability is compromised as a consequence of superoxide free radical production following uptake of chylomicron remnants. We would suggest that, in arterial smooth muscle cells, chylomicron-remnant-induced cell death also occurs as a consequence of superoxide free radical production. Our observations in the present study suggest that macrophage foam cells in atherosclerotic plaques might be derived from the cellular uptake of chylomicron remnants. Furthermore, arterial accumulation of chylomicron remnants might contribute to plaque destabilization as a consequence of cell death following superoxide free radical production by macrophages and smooth muscle cells.

Inhibition of phagocytosis of human macrophages induced by airborne particulates

Human monocyte-derived macrophages isolated from peripheral blood were treated with different extracts of airborne particulates collected in the highly industrialized Rhine-Ruhr area. All tested extracts showed a substantial impairment of phagocytosis by inhibition of phagocytic activity as well as phagocytic capacity, while cell viability was rather well maintained. Significant reduction of phagocytosis already appeared at a concentration equivalent to extracted particulates from 3.8 m 3 air. Having properties of alveolar macrophages, human monocyte-derived macrophage cultures may offer a reliable in vitro model for assessment of pulmonary toxicity by respirable pollutants.

Tumour Promoters/Protein‐Kinase C Activators Augment the Survival and Function of Human Monocyte‐Derived Macrophages in Long‐Term Cultures

In this study the effect of various Protein kinase C (PKC) activators/tumour promoters on the maturation and activity of human peripheral blood monocytes was examined. Monocytes were cultured in the absence or presence of various PKC activators for up to 2 weeks, and examined for the number of adherent cells, expression of myeloperoxidase enzymes, CD14 antigens, mannose/N-acetylglucosamine (Man/GlcNAc) receptors, and the production of TNF-alpha. The presence of PKC activators in cultures of monocyte-derived macrophages (HuMoDM) prevented the loss in the number of initially plated monocytes, otherwise observed in long-term tissue cultures with time of incubation. This effect of PKC activators on monocyte survival was diminished in the presence of PKC inhibitors. HuMoDM obtained in the presence of PKC activators maintained a normal differentiation pattern, as was evident by the loss of granular myeloperoxidase enzymes and CD14 antigens, and the acquisition of membrane Man/GlcNAc receptors. HuMoDM which differentiated in the presence of PKC activators also released TNF-alpha in comparable amounts to freshly harvested human monocytes. PKC activators/tumour promoters augmented the viability of long-term cultures of human monocyte-derived macrophages. Such macrophages may facilitate cell and molecular biology studies of differentiated human macrophages.

Infection of human monocyte‐derived macrophages by human immunodeficiency virus mediated by cell‐to‐cell transmission

We have infected ten-day-old primary cultures of human monocyte-derived macrophages (MDM) with HIV-1 by cocultivation with chronically infected monocytic cell lines. This work has involved the U-937 monocytoid cell line, chronically infected with the HIV-IIIB strain of HIV-1 (U-937HIV IIIB) as well as a number of cell clones, termed UHC, which were derived from U-937HIV IIIB by limiting dilution. Cell-free virus, derived from each of U-937HIV IIIB cells and the UHC1 clone were noninfectious for MDM, as determined by failure to express viral p24 antigen (Ag). In contrast, viral p24 Ag production was detected in MDM that had been cocultivated with U-937HIV IIB, and with each of three UHC clones that produced infectious virus. Infection, in each case, was confirmed by polymerase chain reaction detection via the amplification of proviral DNA. In contrast, cocultivation with the UHC15.7 clone, which fails to cleave viral gp160 to its gp120 and gp41 products or the UHC8 clone, which lacks functional reverse transcriptase, did not lead to infection of MDM. Pretreatment of MDM for 2 hr with 1 microM AZT completely prevented infection by culture fluids containing HIVada, a macrophage-tropic virus, but did not affect infection mediated by cocultivation. These results suggest that cell-to-cell transmission of HIV-1, among monocyte-derived macrophages, can be mediated by proviral DNA. Moreover, gp120 at the surface of infected cells may play an important role in this process, since cell-to-cell HIV transmission could not be demonstrated with the UHC clone that is defective in cleavage of the viral envelope glycoprotein gp160.

Human monocyte-derived macrophage cultures: An alternative test system for the detection of pulmonary toxicity induced by inhaled particulate pollutants

Alveolar macrophages are major target cells for toxicity from inhaled particulates. To investigate pulmonary toxicity induced by airborne particulates a phagocytosis assay using human monocyte-derived macrophage cultures was used. Monocytes were isolated from peripheral blood and cultured for 10–14 days. During this period the monocytes differentiated to macrophages. After treatment with various concentrations of different samples of airborne particulate matter, phagocytosis was induced by the addition of Polychromatic Fluoresbrite Microspheres at a cell/particle ratio of 1:10. Phagocytosis was assessed from the determination of phagocytic activity (% cells showing phagocytosis) and of phagocytic capacity (number of phagocytized particles/cell). A concentration-dependent reduction of phagocytic activity and capacity was observed, while cell viability was not greatly affected as compared with the control. These results were in good agreement with those obtained from in vivo inhalation experiments with rodents as well as with those using rat alveolar macrophages obtained after bronchoalveolar lavage and treated in vitro with extracts of particulate matter. Therefore, it is suggested that the use of human monocyte-derived macrophage cultures represents a promising in vitro test system for the evaluation of pulmonary toxicity induced by particulate pollutants.

Comparative binding and degradation of lipoprotein(a) and low density lipoprotein by human monocyte-derived macrophages.

The binding and degradation of equimolar concentrations of lipoprotein(a) (Lp(a)) and low density lipoprotein (LDL) isolated from the same individual were studied in primary cultures of human monocyte-derived macrophages (HMDM). At 4 degrees C, LDL receptor-mediated binding of both Lp(a) and LDL was of low affinity, being 0.8 and 0.23 microM, respectively. Competitive binding studies indicated that the binding of Lp(a) to HMDM was competed 63% by excess LDL. In contrast to the 4 degrees C binding data, the degradation of Lp(a) at 37 degrees C was mainly nonspecific because the amount of Lp(a) processed by the LDL receptor pathway in 5 h was 17% that of LDL. According to pulse-chase experiments, this phenomenon may be accounted for by the facts that less Lp(a) is bound to HMDM at 37 degrees C and that Lp(a) has a lower intrinsic degradation rate and was not due to increased intracellular accumulation or retroendocytosis of the lipoprotein. Degradation of both lipoproteins was primarily lysosomal and only modestly affected by up- or down-regulation of the LDL receptor. The rate of retroendocytosis in HMDM was approximately equal to the degradation rate and appeared to be independent of the type of lipoprotein used, up- or down-regulation of the LDL receptor, or the presence of the lysosomotropic agent chloroquine. Overall, the results indicate that HMDM degrade Lp(a) mainly via a nonspecific pathway with only 25% of total Lp(a) degradation occurring through the LDL receptor pathway. As both 37 degrees C degradation and 4 degrees C binding of LDL are mainly LDL receptor specific, the different metabolic behavior observed at 37 degrees C suggests that Lp(a) undergoes temperature-induced conformational changes on cooling to 4 degrees C that allows better recognition of Lp(a) by the LDL receptor at a temperature lower than the physiological temperature of 37 degrees C. How apo(a) affects these structural changes remains to be established.

Modification of low density lipoproteins by polymorphonuclear cell elastase leads to enhanced uptake by human monocyte-derived macrophages via the low density lipoprotein receptor pathway

In previous studies we reported that polymorphonuclear cell (PMN) elastase cleaves apoB-100 of human plasma low density lipoprotein (LDL) into seven or eight large Mr fragments (1, Polacek, D., R.E. Byrne, G.M. Fless, and A.M. Scanu. 1986. J. Biol. Chem. 261: 2057-2063). In the present studies we examined the interaction of native and elastase-digested LDL (ED-LDL) with primary cultures of human monocyte-derived macrophages (HMD-M). For this purpose LDL was digested with purified PMN elastase, re-isolated by ultracentrifugation at d 1.063 g/ml to remove the enzyme, and radiolabeled with 125I. At all LDL concentrations in the medium, the degradation of 125I-labeled ED-LDL was 1.5- to 2.5-fold greater than that of 125I-labeled native LDL, and for both lipoproteins species it was further enhanced by prior incubation of the cells in autologous lipoprotein-deficient serum (ALPDS). ED-LDL incubated with HMD-M in a medium containing [14C]oleate stimulated cholesteryl [14C]oleate formation 2- to 3-fold more than native LDL. In competitive degradation experiments, unlabeled ED-LDL did not inhibit the degradation of 125I-labeled acetylated LDL, whereas it caused a 90% inhibition of the degradation of 125I-labeled native LDL. At 4 degrees C, the binding of both 125I-labeled native and 125I-labeled ED-LDL was specific and of a high affinity. At saturation (Bmax), the binding of 125I-labeled ED-LDL was 2-fold higher (68 ng/mg cell protein) than that of 125I-labeled native LDL (31 ng/mg), with Kd values of 6.5 x 10(-8) M and 2.1 x 10(-8) M, respectively. A possible explanation of the binding data was provided by electrophoretic analyses suggesting that ED-LDL was twice the size of native LDL and thus potentially capable of delivering proportionately more cholesterol to the cells. Taken together, the results indicate that 1) digestion of LDL by purified PMN elastase results in a greater mass of ED-LDL (relative to native LDL) being degraded per unit time by HMD-M; 2) uptake of ED-LDL occurs via the LDL receptor; and 3) LDL digested by PMN elastase undergoes a physical change that may be responsible for its unique interactions with HMD-M. We speculate that if this process were to occur in vivo during an inflammatory process, macrophages could acquire excess cholesterol and be transformed into foam cells which are considered to be precursors of the atherosclerotic process.

Membrane phospholipid asymmetry as a determinant of erythrocyte recognition by macrophages.

To assess the role of transbilayer phospholipid distribution in the recognition and phagocytosis of erythrocytes by macrophages, human erythrocytes with either a symmetric or asymmetric distribution of membrane phospholipids were prepared by hypotonic hemolysis and then incubated with cultures of human monocyte-derived macrophages. Erythrocytes with an abnormal, symmetric distribution were phagocytosed 4 times more readily than their counterparts with an asymmetric distribution or than normal, asymmetric intact erythrocytes. This enhanced phagocytosis correlated with two biophysical properties of the membrane: the spacing of phospholipids, as assessed by binding of the dye merocyanine 540, and the relative hydrophobicity, as measured by aqueous two-phase polymer partitioning. These results suggest a mechanism by which loss of membrane asymmetry is translated into recognition by macrophages and provide guidelines in loading erythrocytes that may be useful in manipulating the mode of delivery when erythrocytes are used as drug carriers in vivo.