NDT Advance Access originally published online on February 14, 2008
Nephrology Dialysis Transplantation 2008 23(6):1876-1885; doi:10.1093/ndt/gfm901
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Mesangial matrix-activated monocytes express functional scavenger receptors and accumulate intracellular lipid
1 Centre for Nephrology, Department of Medicine, Hampstead Campus, Royal Free and University College Medical School, London, NW3 2PF, UK 2 Faculty of Medicine and Biological Sciences, University of Leicester, LE1 9HN, UK 3 Department of Pathology, Royal Free and University College Medical School, London, NW3 2PF, UK
Correspondence and offprint requests to: Correspondence and offprint requests to: David C. Wheeler, Centre for Nephrology, Royal Free and University College Medical School, Hampstead Campus, Rowland Hill Street, London NW3 2PF, UK. Tel: +44-20-7830-2930; Fax: +44-20-7317-8591; E-mail: d.wheeler{at}medsch.ucl.ac.uk
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Abstract |
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Background. Monocyte recruitment into the mesangium and foam cell formation are recognized features of glomerular injury. External signals encountered by infiltrating mononuclear cells may determine their behaviour and thereby potentially influence disease outcome. Having previously demonstrated that monocytes are activated by exposure to matrix secreted by mesangial cells, we set out to determine whether matrix activation of monocytes led to expression of a macrophage phenotype.
Methods. THP-1 mononuclear cells were incubated for up to 120 h (5 days) with 500 µg/ml solublized matrix extracted from cultured human mesangial cells or with phorbol myristate ester (PMA-positive control) or albumin (negative control). Expression of peroxisome proliferator activated receptor-
(PPAR-) and of scavenger receptors was used as a marker of monocyte to macrophage differentiation. The presence of functional scavenger receptors was examined by assessing cellular uptake of Dil-labelled acetylated (Ac)-LDL by flow cytometry. Matrix-mediated LDL oxidation was assessed using agarose gel electrophoresis to determine mobility shifts.
Results. Matrix activation was associated with an increase in the expression of PPAR-, scavenger receptor-B (CD36) and scavenger receptor-A mRNA with a corresponding increase in PPAR- protein. Matrix-activated cells incubated with Ac-LDL demonstrated foam cell formation, whilst incubation with Dil-labelled Ac-LDL led to an increase in mean fluorescence intensity of 373 ± 34.8% (P < 0.005) as compared to albumin (100%) and PMA (423 ± 55.8%) (P < 0.005). This could be inhibited by the addition of excess unlabelled ligand, suggesting specific involvement of scavenger receptors. Incubation of LDL with mesangial matrix in the absence of mesangial cells or monocytes led to enhanced electrophoretic mobility of the recovered lipoprotein on agarose gel, an effect that could be inhibited by the addition of anti-oxidants.
Conclusion. Exposure to mesangial cell matrix induces expression of monocyte characteristics associated with a macrophage phenotype and promotes oxidation of LDL, thereby converting this lipoprotein to a scavenger receptor ligand. These observations may help to explain foam cell formation in the mesangium in the context of glomerular disease.
Keywords: foam cell; macrophage; mesangial matrix; monocyte; scavenger receptor
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Introduction |
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Transendothelial migration of monocytes into the glomerular mesangium is a recognized early feature of glomerular injury in man and experimental models of kidney disease [1]. These cells play a central role in orchestrating tissue inflammation and may be critical in determining whether the final outcome of an acute inflammatory glomerular lesion is complete resolution or permanent scarring [2]. Interactions between monocytes and extracellular structures encountered during the process of transmigration may play a critical role in determining the phenotype and therefore the behaviour of the activated tissue macrophage. Extracellular matrix is a highly ordered network of fibrous proteins and associated glycoproteins embedded in a hydrated ground substance of glycosaminoglycans and proteoglycans. It is recognized that matrix not only provides a structural framework, but also influences cellular behaviour. For example, integrin-mediated adhesion of monocytes to extracellular matrix may regulate expression of numerous inflammatory and immune response genes [3]. The importance of this process is demonstrated by disease states thought to arise from dysregulation of matrix–integrin interactions [4].
In previous studies, we demonstrated that exposure of human monocytes to both intact glomerular matrix and its individual components enhanced the production of a range of inflammatory cytokines and matrix-degrading metalloproteinases [5]. However, these experiments did not conclusively demonstrate that such interactions induced monocyte to macrophage differentiation. The present study set out to test the hypothesis that activation by mesangial matrix converts monocytes to a macrophage phenotype. The expression of three macrophage-specific markers was studied: (a) the peroxisome proliferator activated receptor- (PPAR-), a nuclear receptor that acts as a transcriptional mediator for genes involved in lipid metabolism and adipogenesis [6], (b) CD36, a class B scavenger receptor and (c) scavenger receptor class A. Both these scavenger receptors are located in the plasma membrane of the macrophage and are involved in the cellular uptake of modified lipoproteins [7]. Since unregulated uptake of modified lipoproteins is a characteristic of the tissue macrophage, we tested the capacity of matrix-activated monocytes to accumulate intracellular lipid when exposed to acetylated low-density lipoprotein (Ac-LDL), a scavenger receptor ligand. To further examine the role of matrix in foam cell formation, we also assessed the capacity of matrix to modify LDL in the absence of cells to produce oxidized LDL (ox-LDL), a naturally occurring scavenger receptor ligand identified in diseased glomeruli. Our results demonstrate that mesangial cell matrix has the potential both to induce monocyte to macrophage maturation and to oxidize LDL, thereby indicating a likely modulatory role in glomerular inflammation and foam cell formation.
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Materials and methods |
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Cell culture
Human mesangial cells were grown from glomeruli isolated from nephrectomy specimens using standard techniques [8]. Mesangial cells were plated in RPMI medium supplemented with 20% foetal calf serum (FCS) (Gibco BRL, Paisley, UK), L-glutamine (300 µg/ml), insulin (5 µg/ml), transferrin (5 µg/ml), selenium (5 ng/ml), penicillin (100 IU/ml) and streptomycin (100 µg/ml) (Sigma, Dorset, UK) [5]. Mesangial cells were subcultured using trypsin-EDTA (Life Technologies Ltd, Paisley, Scotland) into tissue culture flasks and used between passages 5 and 10. Cells of the human leukaemic THP-1 monocytic line (ECACC, Porton Down, UK) were grown in the supplemented RPMI medium as above in 10% FCS, with the addition of 20 µM mercaptoethanol. Prior to experiments, monocytes were harvested by centrifugation at 350 x g for 5 min. The cell pellet was rinsed twice and resuspended at 1.5 x 106 cells/ml in the supplemented RPMI medium with 5% fibronectin-free homologous serum. Cell viability was determined by trypan blue exclusion and was >95% in all experiments. All reagents and materials used in the experiments including matrix and buffers were tested for endotoxin contamination using a Limulus amebocyte lysate test kit (Sigma) and proved negative.
Mesangial cell matrix isolation
Mesangial cells were grown to 
90% confluence, washed three times with the RPMI medium and then growth arrested in the serum-free RPMI medium for 48 h. The cell layer was removed by the addition of 2.5 mM NH4OH and 0.1% Triton X-100 for 3 min, leaving behind cell matrix [9]. This matrix was then washed three times with PBS, collected by mechanical scraping and sonicated. After measuring the protein concentration using a modified Lowry method [10], the matrix was resuspended in the RPMI medium at concentrations of 10, 50, 100 and 500 µg/ml. Matrix was isolated from mesangial cells derived from four different glomerular preparations and used on the day of isolation. The matrix contained very low concentrations of TGF-β (<0.05 pg/µg) and virtually undetectable amounts of TNF-
(<0.02 pg/µg), IL-1β (<0.003 pg/µg) and IL-6 (<0.003 pg/µg) as measured by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Abingdon, Oxon, UK) according to the manufacturer's instructions.
Analysis of PPAR- CD36 and scavenger receptor-A gene expression using reverse transcriptase-polymerase chain reaction
Based on previous experiments, monocytes were incubated in the presence of soluble matrix for various times up to 120 h (5 days) at 37°C in a humidified atmosphere of 5% CO2. PMA (125 nM) and cell culture grade bovine serum albumin (BSA, 500 µg/ml) (Sigma) served as positive and negative controls, respectively. Cells were trypsinized and recovered by centrifugation. Total RNA (500 ng) was extracted from the cell pellet using a published method [11]. The RNA was used as a template for the reverse transcriptase-polymerase chain reaction (RT-PCR) and the resultant cDNA was amplified for PPAR-, CD36 and scavenger receptor-A by PCR, using β-actin as a control. The following primers were used: PPAR- upper primer 5'-GGC AAT TGA ATG TCG TGT CTG TGG AGA TAA-3' and PPAR- lower primer 5'-AGC TCC AGG GCT TGT AGC AGG TTG TCT TGA-3', CD36 upper primer 5'-CAG CCT CAT TTC CAC CTT TTG TT-3 and CD36 lower primer 5'-GTT GAC CTG CAG CCG TTT TG-3, scavenger receptor-A upper primer 5'-TCG CTC AAT GAC AGC TTT GC-3' and scavenger receptor-A lower primer 5'-CCA TGT TGC TCA TGT GTT CC-3', β-actin upper primer 5'-ATG GAT GAT GAT ATC GCC GCG-3' and β-actin lower primer 5'-CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG GCC-3' (Biogenesis, Poole, UK). Twenty microlitres of the PCR product was subjected to electrophoresis on a 2% agarose gel and bands visualized by staining with ethidium bromide under UV light. Gels were subjected to densitometric analysis using a Gel Doc 2000 scanner (Bio-Rad, Herts, UK), and band intensity normalized to β-actin to control for variations in loading then normalized to the appropriate control conditions (100%).
PPAR- expression using Western analysis
Modulation of PPAR- in THP-1 monocytes in response to mesangial cell matrix was also examined by Western blot analysis. Monocytes were seeded into 24-well plates at a density of 1.5 x 106 cells/well and incubated with matrix (500 µg/ml) for various times up to 48 h. Supplemented RPMI media containing either PMA or BSA served as a positive and negative control, respectively. After stimulation, cells were collected, washed twice with cold PBS and lysed in Laemmli buffer (60 mmol/l Tris, pH 6.8, 10% glycerol, 2% sodium deoxycholate, 100 mmol/l dithiothreitol and 0.01% bromophenol blue). Cell lysates were heated at 100°C for 5 min and subjected to 10% SDS–polyacrylamide gel electrophoresis (Bio-Rad, Herts, UK). Proteins were transferred on to Protran nitrocellulose membranes (Schleicher and Schuell BioSciences GmbH, Dassel, Germany). PPAR- was detected using a monoclonal antibody (SC7273; SantaCruz Biotech, Cambridge, UK) at 1:1000 dilution and a secondary horseradish peroxidase-linked anti-mouse IgG antibody (A4416; Sigma) at 1:2000 dilution. Bound antibodies were visualized using an enhanced chemiluminescence system (Amersham Biosciences, Bucks, UK). For densitometric analysis, bands from Western blots were scanned and quantified using Scion Image version 4.0.2.
Preparation of low-density lipoprotein
Plasma was collected from healthy human volunteers and LDL isolated by sequential ultracentrifugation [12]. LDL was acetylated by incubation with a saturated sodium acetate solution at a ratio of 1:2 and stirred continuously for 30 min at 4°C. Aliquots of acetic anhydride (1.5 µl/mg LDL) were added to the mixture over 90 min. The Ac-LDL was then dialyzed against PBS containing 0.01% EDTA, pH 7.4 and purified using a PD10 Column (Amersham). Freshly isolated native LDL and Ac-LDL were then passed through a 0.2-µm filter and protein concentrations were measured using a modified Lowry method [10]. Acetylation of LDL was confirmed by assessing the changes in mobility of modified lipoprotein using agarose gel electrophoresis.
Flow cytometric analysis of scavenger receptor-A activity
Based on previous experiments, THP-1 monocytic cells (1.5 x 106 cells/ml) were resuspended in the supplemented RPMI medium and incubated with 500 µg/ml of solubilized matrix or with 500 µg/ml BSA protein (negative protein control) or 125 nm PMA for 48 h at 37°C. Cells were then exposed for a further 3 h to 10 µg/ml Ac-LDL labelled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil-labelled Ac-LDL, Biogenesis, Poole, UK) in the presence or absence of an excess of unlabelled Ac-LDL (250 µg/ml) to confirm the specificity of receptor-mediated uptake. After incubation, the cells were recovered by centrifugation at 350 x g for 5 min, washed three times with PBS and fixed in a 5% formalin solution in PBS. Ac-LDL binding and uptake was assessed by flow cytometry (EPICS XL-MCL; Beckman Coulter, Bucks, UK). Forward and side scatter gates were established to exclude dead cells and cell debris from the analysis. Fluorescence signals from the accumulated Dil associated with the cells were detected at 555–600 nm by a photomultiplier and then converted to digital format and processed. The mean fluorescence intensity (MFI) of 5 x 103 cells were analysed in each sample. Auto-fluorescence signals generated by unlabelled cells were used as negative controls in each experiment. The MFI of the Dil-labelled cells was calculated by subtracting the auto-fluorescence intensity from the observed MFI of labelled cells. Each experiment was carried out in duplicate, on four separate occasions with different preparations of cells and Ac-LDL. The average of the duplicate determinations was used for statistical analysis.
Morphological examination of functional scavenger receptors
THP-1 monocytes were incubated in chamber slides (0.5 x 106 cells/ml) with solubilized matrix (500 µg/ml). PMA (125 nM) and BSA (500 µg/ml) served as positive and negative controls, respectively. Polyinosinic acid (poly I) was also added as an inhibitor of the scavenger receptor. After 120 h incubation at 37°C, cells were further incubated with 50 µg/ml Ac-LDL for 48 h. Cells were then washed with PBS, fixed for 30 min with a 5% formalin solution in PBS, stained with Oil Red O for 30 min and counter-stained with haematoxylin for another 5 min. Lipid inclusion was assessed by observing at least eight fields under a light microscope.
LDL oxidation by mesangial cell matrix
Relative electrophoretic mobility was used to assess oxidation of LDL by matrix [8]. Matrix (500 µg/ml) and native LDL (250 µg/ml) were co-incubated in the absence of cells. As a positive control, native LDL (250 µg/ml) was incubated in the presence of CuSO4 (10 µM). Native LDL co-incubated with BSA (500 µg/ml) and native LDL alone served as negative controls. The effect of the anti-oxidants EDTA (100 µM) and butylated hydroxytoluene (BHT, 20 µM) on matrix co-incubated with LDL was also assessed. Following incubation for 24 h at 37°C, the protein fraction was adjusted to 50 µg/ml with PBS and 5 µl of each sample was loaded on to a 0.5% agarose gel (Paragon Lipogel, Beckman, Austria) and subjected to electrophoresis. Bands were visualized by staining gels according to the manufacturer's instructions.
Staining of human kidney biopsy material for macrophage activation markers
Ethical approval to use human kidney biopsy material was obtained. Sections of formalin-fixed paraffin embedded kidney tissue were dewaxed and treated with hydrogen peroxide to block endogenous peroxidase. Four kidney samples from patients with non-inflammatory conditions (two with ischaemic nephropathy, one with thin membrane nephropathy and one with myoglobinuria) and five from patients with inflammatory diseases (three with pauci-immune necrotizing/vasculitic glomerulonephritis, one with anti-glomerular basement membrane antibody disease and one with lupus nephritis) were analysed. The sections were then heated in TRIS-EDTA buffer (pH 9.0) before being stained with either a mouse monoclonal antibody to CD68 diluted 1:200 (PG-M1 antibody, Dako, Cambridge, UK), a mouse monoclonal antibody to PPAR- diluted 1:100 (Santa Cruz) or a goat antibody to scavenger receptor diluted 1:500 (Abcam, Cambridge, UK). In the case of the anti-CD68 antibody, samples were pre-treated with trypsin for 10 min. After incubation with the first stage antibody for 1 h, an Envision kit (Dako) was used for the second stage in the case of the mouse antibodies and a peroxidase-conjugated rabbit anti-sheep antibody in the case of the goat antibody. Hydrogen peroxidase and diaminobenzidine were used as substrates and sections were counter-stained with haematoxylin.
Statistical analysis
Statistical analysis was performed using a Mann–Whitney unpaired non-parametric two-tailed test. Data are expressed as mean ± SEM and P < 0.005 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAbbreviationsReferences |
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PPAR-
expression by matrix-activated monocytesWhilst no PPAR-
mRNA was detected by RT-PCR analysis of total RNA extracted from freshly isolated THP-1 monocytes, message was detectable within 24 h when cells were incubated with soluble mesangial matrix (500 µg/ml). Expression was maximal at 48 h, persisting over at least 5 days and was comparable to that observed when cells were stimulated with PMA over a similar time period under identical experimental conditions (Figure 1). Increased expression of PPAR- protein within 24 h of exposure of matrix stimulation was confirmed by Western analysis, with levels of expression being similar to those observed following PMA stimulation (Figure 2). No further increase in expression was observed when incubation was extended beyond 48 h to 7 days (data not shown).
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CD36 expression by matrix-activated monocytes
Enhanced expression of CD36 mRNA was detected by RT-PCR analysis of total RNA extracted from THP-1 monocytes exposed to soluble mesangial matrix (500 µg/ml). An increase in message was detected after 48 h of incubation and was comparable with that observed when cells were stimulated with PMA for the same time period under identical experimental conditions (Figure 3). No further increase in expression was observed when incubation was extended beyond 120 h to 7 days (data not shown).
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Scavenger receptor expression by matrix-activated monocytes
Scavenger receptor-A mRNA expression increased in a concentration-dependent manner when THP-1 monocytes were incubated with increasing concentrations of soluble matrix protein for 48 h with a maximal response at 100 µg/ml (Figure 4). A time-dependent increase was observed with the addition of 500 µg/ml matrix protein increasing up to 120-h incubation (Figure 5), with no further change up to 7 days (data not shown). No expression was observed under baseline conditions prior to stimulation, neither did equivalent concentrations of BSA induce detectable scavenger receptor-A message, suggesting that the observed effect was specific.
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Uptake of modified lipoproteins by matrix-activated monocytes
The presence of functional scavenger receptors was confirmed using flow cytometry in which incubation of matrix-activated monocytes with Dil-labelled Ac-LDL led to an increase in MFI (Figure 6). This effect was largely reversed by the addition of an excess of unlabelled ligand. The MFI of THP-1 cells incubated with Dil-labelled Ac-LDL after exposure to matrix increased to 373 ± 34.8% (P < 0.005) as compared to cells exposed to BSA (100%). PMA pre-stimulation of monocytes increased MFI to 423 ± 55.5% (P < 0.005). These increases in MFI induced by matrix and PMA activation were inhibited by the addition of excess unlabelled Ac-LDL to 134 ± 12.1% (P < 0.001 versus no excess of unlabelled lipoprotein) and to 170 ± 16.1% (P < 0.001) respectively, suggesting specific binding of Dil-labelled Ac-LDL to scavenger receptors.
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Incubation of monocytes with unlabelled Ac-LDL following stimulation by exposure to matrix for 120 h led to intracellular accumulation of Oil Red O-stained lipid droplets (Figure 7A). Lipid uptake did not occur following BSA stimulation (Figure 7B). Prior exposure to PMA was also associated with intracellular lipid deposition but no intracellular lipid staining was observed when Poly I was added with Ac-LDL following activation of monocytes by matrix or PMA (not shown).
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Oxidation of LDL by mesangial cell matrix
Incubation of LDL with mesangial cell matrix in the absence of cells led to enhanced electrophoretic mobility of recovered lipoprotein on agarose gel (Figure 8). A similar shift in mobility was seen when LDL was exposed to copper sulphate, a powerful oxidizing agent, but was blocked when the anti-oxidants EDTA (100 µM) and BHT (20 µM) were added, suggesting that matrix induces LDL oxidation. In contrast, incubation with BSA did not change the electrophoretic mobility of the lipoprotein.
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Identification of macrophage activation markers in human kidney biopsy material
CD-68 positive cells were extremely difficult to identify in the non-inflamed kidney sections and there was no staining for PPAR-
or scavenger receptor. In contrast, all three markers were readily detected in the inflamed kidneys, predominantly within the glomeruli (Figure 9). CD68 and scavenger receptor were located in a cytoplasmic distribution and PPAR- within nuclei in keeping with the cellular location of these markers.
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Discussion |
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These results demonstrate that exposure of monocytes to mesangial cell matrix in vitro induces expression of PPAR-
, CD36 and scavenger receptor-A and promotes phagocytic activity, characteristics usually associated with a macrophage phenotype. This would suggest that conversion of infiltrating monocytes to mature tissue macrophages within the glomerular mesangium in vivo may not necessarily depend on the presence of mesangial cells, but may result from direct interactions with matrix components. While circulating monocytes are relatively inert, the activated tissue macrophage may take on a destructive role, inducing cell death by apoptosis and degrading extracellular matrix. Alternatively, these cells may facilitate repair by inducing cell proliferation and secretion of replacement matrix components [2]. Macrophages also play a key role in the phagocytosis of cellular debris, lipids and denatured proteins in inflamed tissue, a process that may result in the formation of foam cells. Foam cells are characteristically seen at sites of tissue injury, for example in the arterial intima in atherosclerosis, and are recognized in the kidney in damaged glomeruli [13]. Given the pivotal role of the monocyte/macrophage in modulating tissue injury and the diverse biological activities of these cells, our findings may have important implications in the context of glomerular disease as supported by our demonstration of both PPAR- and scavenger receptor expression in inflamed human kidney sections. Thus, matrix-mediated activation may influence the behaviour of monocytes that infiltrate the glomerular mesangium and thereby potentially modify the outcome of an inflammatory process.
It is now recognized that the phenotypic state adopted by a tissue macrophage is influenced by the activation signals that naive monocytes receive and that broadly two distinct activated phenotypes can be identified. The classical activation pathway results from exposure to Th-1 type cytokines such as TNF-, IL-1β and IL-6 and results in a macrophage with pro-inflammatory properties, capable of further generation of pro-inflammatory cytokines and the degradation of normal and abnormal matrix components [14]. Classically activated cells also possess the ability to take up modified lipoproteins, potentially resulting in the formation of lipid-laden foam cells. In contrast, the alternative activation pathway induced by Th-2-type cytokines such as IL-4 and IL-13 produces a macrophage that generates anti-inflammatory cytokines, suppresses the synthesis of pro-inflammatory cytokines and is resistant to re-activation, thus being responsible for coordinating the resolution of an inflammatory process [2]. Taken together with our previous experiments, which demonstrated that incubation of monocytes with mesangial cell matrix stimulates secretion of the pro-inflammatory cytokines IL-6, IL-1β and TNF- as well as matrix-degrading metalloproteinases [5], it seems reasonable to conclude that mesangial cell matrix activates macrophages via the classical pathway. We believe that these findings are highly relevant to the fate of monocytes that undergo transmigration to become tissue macrophages, but not to cells that undergo reverse transmigration since these adopt the phenotype of an immature or mature dendritic cell (depending on the absence or presence of inflammatory stimuli, respectively) [15].
Since most of our experiments were performed over a maximum period of 7 days, we cannot exclude the possibility that at later time points, matrix-activated macrophages lose their pro-inflammatory capacity and take on the alternatively activated anti-inflammatory phenotype. In vivo, both cell phenotypes are likely to be present in inflamed tissue and the balance between them may be critical in determining the extent of subsequent fibrosis. It is also possible that a classically activated macrophage programmed by exposure to matrix does not respond to the signals usually associated with the alternative activation pathway, potentially resulting in an uncontrolled inflammatory response with limited subsequent tissue repair [2].
We accept that there are limitations with our experimental system that was based on the THP-1 cell line rather than peripheral blood monocytes. However, the use of THP-1 cells as a monocyte model is well established [16]. We should stress that we have previously demonstrated that matrix activation of THP-1 cells leads to the production of monocyte chemoattractant protein-1, IL1-β, IL-6, IL-8 and MMP-9 in similar concentrations when compared to peripheral blood monocytes. Furthermore, our previous work has demonstrated similarities between monocyte cell lines and cells isolated from peripheral blood in terms of surface markers and binding characteristics [8]. We also used matrix elaborated by healthy mesangial cells and did not take account of the fact that disease-specific matrix modification may influence monocyte activation. For example, matrix glycation that occurs in diabetes mellitus influences the balance between matrix synthesis and degradation by mesangial cells, promoting accumulation [17]. Matrix proteins may be particularly prone to glycation or oxidation as a result of their prolonged lifespan. Finally, we cannot rule out the possibility that our results are at least in part explained by retention of low concentrations of cytokines within the matrix material.
When designing our experiments, one challenge was the identification of a suitably robust marker of monocyte to macrophage conversion. Other macrophage-specific markers which we had previously explored included CD69 and the HLA-DR antigens [5]. However we found both to be expressed at low levels on THP-1 monocytes with no significant up-regulation occurring following stimulation with PMA, an accepted and potent activator of monocytes. Other investigators have observed that HLA-DR expression varies with the source of the macrophage, such that 15% of peritoneal macrophages express the antigen compared to 50% of spleen and thymus-derived cells [18]. We also studied Mac-1, a member of the β2 integrin family also known as CD11b/CD18; however, this cell surface adhesion receptor did not prove to be specific to macrophages and was also expressed at low levels by freshly isolated monocytes as demonstrated by other workers [19]. PPAR- expression proved to be a more useful indicator since this intracellular receptor showed very low levels of expression in monocytes, but was strongly induced during their differentiation into mature macrophages, suggesting that it may be involved in the differentiation process. In addition, PPAR- has been implicated in the modulation of several macrophage functions including the regulation of pro-inflammatory activities and stimulation of ox-LDL uptake further strengthening the use of this factor as a macrophage marker [6]. PPAR- is also abundantly expressed in lesions such as atherosclerotic plaques where the formation of foam cells is observed [20]. It should be emphasized that PPAR- was used simply as a macrophage marker in these studies and that ligand-induced activation of this receptor was not examined. It seems likely that other signalling pathways are activated by matrix–monocyte interactions, particularly since PPAR- activation does not explain the increase in cytokine production that we have previously reported [5].
Scavenger receptor-A is a macrophage-specific cell surface protein that specifically binds and internalizes oxidized and chemically modified LDL particles, similar to the class B scavenger receptor (CD36), which also binds modified forms of LDL [7]. Scavenger receptor expression is restricted to macrophages thereby providing a reliable marker for the purpose of these studies. Scavenger receptor-A has been implicated in mediating a variety of macrophage functions, including intracellular signalling, endocytosis, adhesion and phagocytosis. Unlike uptake of native LDL via the LDL receptor, which is tightly controlled, scavenger receptor-A mediated uptake of modified lipoprotein is not regulated by intracellular cholesterol levels and can therefore potentially lead to intracellular cholesterol accumulation and the formation of foam cells [21]. The class B scavenger receptor CD36 has also been implicated in the process of lipid accumulation in macrophages and serves as an adhesion receptor on macrophages for matrix components such as collagen and thrombospondin [22].
In keeping with the changes in lipoprotein receptor expression observed, matrix-activated monocytes accumulated intracellular lipid when incubated with Ac-LDL, a synthetic scavenger receptor ligand, as demonstrated by intracellular Oil Red O staining. This phagocytic capacity was further confirmed by flow cytometry of matrix-activated monocytes exposed to Dil-labelled Ac-LDL. Uptake of Ac-LDL was shown to be specific, since it could be inhibited by the addition of an excess of unlabelled acetylated lipoprotein, thus confirming receptor involvement. Induction of phagocytic activity was also observed following PMA-mediated activation, but not when an irrelevant protein (BSA) was added.
Lipoproteins, including LDL, infiltrate the normal mesangium and are found deposited in diseased glomeruli [23]. Having previously shown that mesangial cells oxidize LDL in vitro [8], we here demonstrate that exposure of LDL to mesangial matrix has a similar effect. Thus, not only does matrix exposure induce a phagocytic macrophage phenotype in monocytes, but also converts LDL to an appropriate scavenger receptor ligand thereby potentially contributing to the development of foam cells. The mechanisms by which matrix promotes LDL oxidation were not explored but may involve entrapment of lipoprotein by glycosaminoglycans, thereby rendering particles more susceptible to the effects of reactive oxidative species [24].
In conclusion, mesangial matrix has the capacity to convert monocytes to macrophages displaying characteristics associated with a classically activated phenotype. By inducing macrophage scavenger receptor expression and converting LDL to an oxidized product, matrix may also play a key role in the formation of foam cells within the glomerular mesangium. The impact of changes in matrix composition on these interactions and the potential for such changes to modify the outcome of an inflammatory process within the glomerulus warrant further investigation.
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Abbreviations |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAbbreviationsReferences |
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PPAR-
, peroxisome proliferator-activated receptor-; SCr-A, scavenger receptor-A; Ac-LDL, acetylated-low density lipoprotein; Ox-LDL, oxidized-low density lipoprotein; LPS, lipopolysaccharide; TNF, tumour necrosis factor ; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-4, interleukin-4; IL-10, interleukin-10; IL-13, interleukin-13; Dil, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; RT-PCR, reverse transcriptase-polymerase chain reaction; SDS– PAGE, sodium dodecyl sulphate– polyacrylamide gel electrophoresis; BSA, bovine serum albumin; MFI, mean fluorescence intensity; CD-69, cluster of differentiation-69; Th, T-helper; HLA-DR, human histocompatability leukocyte antigen-differentiation region; EDTA, ethylenediaminetetraacetic acid; BHT, butylated hydroxytoluene.
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Acknowledgments |
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This work was presented in part at the 2002 and 2003 Annual meetings of the American Society of Nephrology and published in abstract form (J Am Soc Nephrol 2002; 13: 556A and J Am Soc Nephrol 2003; 14: 593A). We would like to thank Dr Jill Norman for her support and for help in preparing the manuscript and to Professor Alec Howie for his help with staining the human kidney sections.
Conflict of interest statement. None declared.
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References |
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Accepted in revised form: 27.11.07
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