Enhanced chemokine-receptor expression, function, and signaling in healthy African American and scleroderma-patient monocytes are regulated by caveolin-1
© Lee et al. 2015
Received: 19 March 2015
Accepted: 8 June 2015
Published: 20 June 2015
A major health disparity suffered by African Americans (AA) is a predisposition toward fibrotic diseases of the skin, lung, and other organs. We previously showed that healthy AA and scleroderma (systemic sclerosis (SSc)) patient monocytes share biochemical and functional differences from control Caucasian (C) monocytes that may predispose AA to SSc. The central difference is a decrease in caveolin-1. Low caveolin-1 levels promote monocyte migration, their differentiation into fibrocytes, and fibrocyte recruitment into fibrotic tissues. Here we have greatly expanded our studies on the mechanism of action in fibrosis of caveolin-1 in AA and SSc monocytes.
Expression of chemokine receptors (CCR1, CCR2, CCR3) is enhanced in healthy AA monocytes compared to healthy C monocytes and further increased in SSc monocytes. A parallel increase in function occurs assessed by migration toward chemokines MCP-1 and MCP-3. Chemokine-receptor expression and function are inhibited by the caveolin-1 scaffolding domain peptide (CSD) via its action as a surrogate for caveolin-1. Cells bearing chemokine receptors accumulate to high levels in fibrotic lung and skin tissue from SSc patients and from mice treated with bleomycin. This accumulation is almost completely blocked in mice treated with CSD. In signaling studies, Src activation is enhanced in AA monocytes compared to C monocytes and further increased in SSc monocytes. Lyn is also highly activated in SSc monocytes. Src and Lyn activation are inhibited by CSD. Src and Lyn’s roles in monocyte migration were demonstrated using specific inhibitors.
To the best of our knowledge, this is the first report that the expression and function of CCR1, CCR2, and CCR3 are upregulated in monocytes from healthy AA and from SSc patients via molecular mechanisms involving caveolin-1, Src/Lyn, and MEK/ERK. The results suggest that the migration/recruitment of monocytes and fibrocytes into fibrotic tissues, mediated at least in part by CCR1, CCR2, and CCR3, plays a major role in the progression of lung and skin fibrosis and in the predisposition of AA to fibrotic diseases. Our findings further suggest that chemokine receptors and signaling molecules, particularly caveolin-1, that control their expression/function are promising targets for treating fibrotic diseases.
A major health disparity affecting African Americans (AA) is a predisposition toward fibrotic diseases of the skin, lung, and other organs. AA scleroderma (systemic sclerosis, SSc) patients have a younger age of disease onset, higher probability of the more severe diffuse cutaneous form of the disease, and higher mortality. AA SSc patients are significantly more likely than Caucasian (C) SSc patients to exhibit impaired lung function [1–8]. While there has been a considerable focus on AA SSc patients, there have been few studies on underlying differences between healthy AA and C that might explain the predisposition of AA to SSc and interstitial lung disease (ILD). In one study, levels of the profibrotic cytokine transforming growth factor β (TGFβ) were twice as high in serum from healthy AA compared to healthy C .
We recently identified several parameters in which healthy AA are similar to SSc patients that may predispose AA to fibrosing diseases, e.g., SSc . The central observation was a diminution in the master regulatory protein caveolin-1 in monocytes from healthy AA compared to healthy C. A greater loss of monocyte caveolin-1 is linked to lung and skin fibrosis in bleomycin-treated mice and in SSc-ILD and IPF patients [11–14]. The low level of caveolin-1 in AA and SSc monocytes strongly promotes their migration toward several chemokines and their differentiation into α-smooth muscle actin (ASMA)-positive fibrocytes. Both of these functions are blocked by the caveolin-1 scaffolding domain peptide (CSD), which enters cells and compensates for the lack of caveolin-1.
Monocyte migration in vitro models their recruitment in vivo into tissues undergoing inflammation and fibrosis. In both cases, chemokines provide a chemotactic signal to cells by binding to their specific cell-surface receptors. The molecular mechanism through which low caveolin-1 enhances monocyte migration involves the accumulation of chemokine receptors such as CXCR4 and CCR5 [12, 15]. This accumulation may result from either enhanced expression or decreased turnover. Signaling downstream from the chemokine receptor-ligand interaction is mediated by several pathways including G protein-coupled receptor signaling, Src-family signaling, and MAPK family signaling [16, 17]. Src-family kinases are also important in fibrosis due to their ability to regulate ECM protein expression by dermal fibroblasts.
Here we expand our study of the regulation of AA and SSc monocyte migration to additional chemokines, chemokine receptors, and signaling pathways. In particular, we have studied chemokine receptors CCR1, CCR2, and CCR3 and the chemokines MCP-1 (also known as CCL2, binds to CCR2) and MCP-3 (also known as CCL7; binds to CCR1, CCR2, and CCR3). Both MCP-1 and MCP-3 are upregulated in SSc . To the best of our knowledge, this is the first report that the expression and function of CCR1, CCR2, and CCR3 are upregulated in monocytes from healthy AA and from SSc patients via molecular mechanisms involving caveolin-1, Src/Lyn, and MEK/ERK signaling.
CCR1, CCR2, and CCR3 expression and function are enhanced in monocytes from healthy AA and SSc patients
CCR1, CCR2, and CCR3 are upregulated in SSc patient lung and skin tissue
Upregulation of CCR1, CCR2, and CCR3 in fibrotic mouse tissues is reversed by CSD
Enhanced Src and Lyn activation in AA and SSc monocytes
MEK/ERK and Src-family inhibitors inhibit monocyte migration
In previous studies, we showed that caveolin-1 levels are low in monocytes from SSc patients and from healthy AA compared to healthy C [10, 12]. Profibrotic features associated with monocytes from both SSc-ILD and healthy AA subjects include the enhanced expression of chemokine receptors CXCR4 and CCR5, enhanced migration toward their respective chemokine ligands (SDF-1 for CXCR4, MIP-1α and MIP-1β for CCR5), and enhanced differentiation into fibrocytes [10, 12, 19]. All of these features were linked to low caveolin-1 expression by the fact that they were reversed by restoring caveolin-1 function with the CSD. Here we greatly expand on these studies, demonstrating: (1) the enhanced expression of chemokine receptors CCR1, CCR2, and CCR3 by AA and SSc-ILD monocytes and the reversal of the enhanced expression by restoring caveolin-1 function with CSD; (2) the enhanced migration of AA and SSc-ILD monocytes toward chemokines MCP-1 and MCP-3 and the reversal of the enhanced migration by CSD; (3) enhanced Lyn/Src signaling in AA and SSc-ILD monocytes, its reversal by CSD, and the use of specific inhibitors to demonstrate the regulation of monocyte migration by Lyn/Src and MEK/ERK; (4) the overexpression of CCR1, CCR2, and CCR3 in SSc skin and lung tissue; and (5) the overexpression of CCR1, CCR2, and CCR3 in fibrotic murine skin and lung tissue generated by systemic bleomycin delivery using implanted osmotic minipumps, and the reversal of this overexpression by treatment with CSD.
CCR1, CCR2, and CCR3 are expressed on a variety of classes of leukocytes. Here we have focused on their expression by monocytes. To the best of our knowledge, there have been few previous reports related to their enhanced expression by monocytes from SSc patients and healthy AA subjects. At the mRNA level, CCR1 expression was enhanced in monocytes from SSc patients with PAH [21, 22]. No data was presented at the protein level. CCR2 expression detected by immunohistochemistry was observed to be upregulated in early-stage diffuse cutaneous SSc skin by a variety of cell types including macrophages, myofibroblasts, pericytes, lymphocytes, and endothelial cells . We also find a major upregulation of CCR2 in SSc skin in the fibrocyte-fibroblast lineage (HSP47+ cells). Interestingly, the number of double-positive cells in this lineage in the lungs is greater for CCR1 and CCR3 than for CCR2. Overall, comparing double staining with the monocyte/macrophage marker CD68 to double staining with HSP47 suggests that the various cell types that express chemokine receptors accumulate differentially during fibrosis.
Although key receptors can be differentially expressed on human and murine cells [24, 25], we find that CCR1, CCR2, and CCR3 are all expressed at high levels in both human and mouse fibrotic skin and lung tissue. Moreover, we find that the overexpression of these receptors is inhibited when mice are treated with CSD. Previous studies in mouse model systems are also consistent with the importance of these chemokine receptors in fibrosing disease. For example, antibodies against CCR1 delivered i.v. enhanced the survival of mice treated with bleomycin while inhibiting the accumulation in their lungs of collagen and inflammatory cells . Experiments using CCR2 knockout mice demonstrated that CCR2 plays a major role in the recruitment of fibrocytes into the airspace of mice in which fibrosis was induced using FITC . In yet another model, the overproduction of collagen induced by injection of TGFβ into the skin was significantly reduced in CCR2 knockout mice .
Chemokines MCP-1 and MCP-3 are present at high levels in the serum and bronchoalveolar lavage fluid of SSc patients and are expressed at high levels by SSc fibroblasts [23, 29–32]. Among SSc patients, high levels of MCP-1 and MCP-3 are associated with worse clinical outcomes. In addition to their role as chemoattractants of inflammatory cells into target tissues, MCP-1 and MCP-3 may be important in fibrosis as initiators of signaling cascades resulting in collagen overexpression [18, 30, 33, 34]. While some studies show direct effects of MCP-1 and MCP-3 on collagen expression by fibroblasts [30, 34, 35], another study proposes that MCP-1 indirectly promotes the expression of collagen by fibroblasts by activating the expression of IL-4 by T cells . In turn, this IL-4 is responsible for increasing collagen production by fibroblasts.
Relatively little is known about the signaling pathways that link relative caveolin-1 deficiency in SSc and AA monocytes to the enhanced ability of these cells to migrate toward various chemokines and to differentiate into fibrocytes. We reported the importance of MEK/ERK signaling [10, 12] in these cell functions. Here we have studied the Src-family kinases Src and Lyn. We find that Src and Lyn are hyperactivated in SSc monocytes and that Src is activated in AA monocytes. In both cases, Src and Lyn activation are reversed by treating cells with CSD. In addition, we find that the Src/Lyn inhibitors PP2 and SU6656 (as well as a MEK/ERK inhibitor U0126) block the enhanced migration of SSc and AA monocytes. Not surprisingly, CSD (which blocks multiple signaling pathways) was slightly more effective than these inhibitors that block only one pathway. Src-family kinases have been implicated in a variety of activities relevant to monocyte biology including innate immune signaling, responses to cytokines and growth factors, apoptosis, and G protein-coupled signaling . To the best of our knowledge, the current study is the first to link caveolin-1, Src-family signaling, and monocyte migration.
Caveolin-1 and Src have been studied more extensively in other cell types in the context of SSc and fibrosis. Most of these studies involve signaling initiated by TGFβ. In one study focusing on the role of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI) in regulating the epithelial-mesenchymal transformation (EMT) of alveolar type II epithelial cells into myofibroblasts, it was proposed that CSD blocked EMT by inhibiting Src leading to the enhanced expression of uPA and the inhibition of PAI expression . These effects were observed whether EMT was induced by bleomycin, TGFβ, or cigarette smoke. Other studies focus on fibroblasts. TGFβ receptor internalized through caveolin-1 lipid rafts undergoes rapid degradation, thereby decreasing TGFβ signaling . This mechanism links low caveolin-1 to enhanced TGFβ signaling. It is noteworthy that TGFβ is present at high levels in the circulation of healthy AA  and SSc patients , and TGFβ treatment decreases caveolin-1 levels in a variety of cell types [12, 38]. Thus, the combination of low caveolin-1 and high TGFβ may be particularly likely to cause fibrosis because their effects appear to be mutually reinforcing. Src has also been directly linked to TGFβ signaling. Stimulation of human dermal fibroblasts with TGFβ activated Src signaling . Treatment of these cells with SU6656 inhibited collagen expression both at the mRNA and protein levels. Similarly, dermal fibrosis induced in mice by bleomycin injection was inhibited by SU6656. Finally, it was observed that TGFβ can signal through the Src family member c-Abl and that this signaling is independent of canonical TGFβ signaling through Smad2/3 .
In summary, the current study strongly supports and extends our observations on the role of monocytes and cells derived from monocytes (e.g., fibrocytes) in lung and skin fibrosis and on the predisposition of AA to fibrotic diseases. Our findings highlight the idea that chemokine receptors (e.g., CCR1, CCR2, CCR3) and signaling molecules that control their expression/function (e.g., caveolin-1, MEK/ERK, Src/Lyn) are promising targets for novel treatments for fibrotic diseases such as SSc.
Under a protocol approved by the Medical University of South Carolina (MUSC) Institutional Review Board for Human Research, SSc-ILD patients were recruited from the MUSC Scleroderma Clinic. All patients provided written informed consent before enrollment in the study, fulfilled the American College of Rheumatology criteria for SSc , and had evidence of ILD . Demographic data for SSc patients and healthy control donors are summarized in Additional file 1: Tables S1 and S2. Note that Additional file 1: Table S1 describes the combined data for all the patients that participated in the entire study, not the patients that participated in a particular experiment.
PBMC and monocyte isolation
Peripheral blood mononuclear cells (PBMC) were isolated by standard methods  by centrifugation on density 1.083 Histopaque cushions. Monocytes were isolated from the PBMC by immunodepletion using a Dynal Monocyte Negative Isolation Kit (Invitrogen, Carlsbad, CA) resulting in a cell population about 95 % Mac-1+ monocytes .
The CSD peptide (amino acids 82–101 of caveolin-1; DGIWKASFTTFTVTKYWFYR) was synthesized as a fusion peptide to the C terminus of the Antennapedia Internalization Sequence (RQIKIWFQNRRMKWKK). The Antennapedia Internalization Sequence (AP) alone was used as control peptide and showed no effect on cell behavior when compared to no added peptide. When treating cells with peptides, stock solutions of peptides (10 mM in 100 % DMSO) were diluted to the indicated final concentrations.
Monocyte migration assays
Were performed as described . Briefly, SDF-1 (100 ng/ml in RPMI 1640/1 % BSA), MCP-1 or MCP-3 (50 ng/ml in RPMI 1640/1 % BSA), or unsupplemented RPMI 1640/1 % BSA were placed into the lower wells of Neuro Probe Multiwell Chemotaxis Chambers (Neuro Probe, Gaithersburg, MD) fitted with 5-μm pore size polycarbonate filters. With or without TGFβ pretreatment (45 min, 10 ng/ml in RPMI 1640/1 % BSA), 25 μl of cell suspension (5 × 105 cells/ml) was placed in the upper wells. Peptides (0.1 μM) or inhibitors (U0126, 0.1 μM; PP2, 10 μM; SU6656, 10 μM) were added to the cell suspension prior to placement in the upper chamber. After incubation (2.5 h, 37 ° C, 5 % CO2), filters were removed, fixed, and stained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA). Cells on the underside of the membrane were photographed and counted in six high power fields per condition.
Monocyte signaling/Western blots
Chemokine-receptor levels and levels of total and activated Src and Lyn were determined by Western blot of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer extracts of freshly isolated monocytes. For CSD treatment, monocytes were cultured overnight in 6-well tissue culture plates (2 × 106 cells per well) in RPMI 1640/20 % fetal calf serum (FCS). Attached cells were then treated for 3 h with fresh medium (RPMI/1 % BSA) supplemented with 0.1 μM CSD or control peptide. Cells were next washed twice with PBS then extracted with SDS-PAGE sample buffer. Western blots were performed using the indicated antibodies.
Images were collected using a Leica DMI 4000B fluorescence microscope. To detect caveolin-1, CCR1, CCR2, and CCR3, cells isolated as described above were cultured overnight in 6-well tissue culture plates (1 × 106 cells per well) on coverslips in RPMI 1640/20 % FCS. Cells were then fixed and permeabilized, labeled with appropriate primary and secondary antibodies, and counterstained with the nuclear stain DAPI.
Immunohistochemistry of human lung tissue sections was performed as described . Briefly, paraffin sections were stained with primary antibodies, appropriate AlexaFluor647- or AlexaFluor555-conjugated secondary antibodies and the nuclear stain DAPI (Invitrogen, Carlsbad, CA). Images were collected using a Leica DMI 4000B fluorescence microscope. Primary antibodies were: rabbit anti-CCR1 (Thermo Fisher Scientific, Rockford, IL, USA; PA1-21629), rabbit anti-CCR2 (Abcam, Cambridge, MA, USA; ab32144), rabbit anti-CCR3 (Abcam, Cambridge, MA, USA; ab36827), rabbit anti-MCP-1 (Abcam, Cambridge, MA, USA; ab 9669), rabbit anti-MCP-3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; SC-374002), rabbit anti-pSrc-Tyr416 (Cell Signaling Technology, Inc., Danvers, MA, USA; #2101S), and rabbit anti-pLyn-Tyr507 (Cell Signaling Technology, Inc., Danvers, MA, USA; 04–375).
Mice were treated systemically with bleomycin or vehicle and received CSD or vehicle as recently described [15, 19]. These studies were performed under protocols approved by the MUSC Institutional Animal Care and Use Committee (AR#3134, AR#3029, AR#3323).
Immunoreactive bands were quantified by densitometry using Image J 1.32 NIH software. Raw densitometric data were processed and analyzed using Prism 3.0 (GraphPad Software Inc.). ANOVA with post hoc Tukey’s test was used to evaluate Western blots and monocyte migration. In all figures, ***indicates p < 0.002, **indicates p < 0.01, and *indicates p < 0.05.
analysis of variance
Antennapedia internalization sequence
α-smooth muscle actin
bovine serum albumin
7, Chemokine (C-C motif) ligand 2, 7
2, 3, 5, C-C chemokine receptor types 1, 2, 3, 5
caveolin-1 scaffolding domain peptide
C-X-C receptor type 4
extracellular signal-regulated kinase
fetal calf serum
forced vital capacity
interstitial lung disease
idiopathic pulmonary fibrosis
mitogen-activated protein kinase
MCP-3, monocyte chemotactic protein-1, monocyte chemotactic protein-3
β, macrophage inflammatory protein-1α, β
Medical University of South Carolina
pulmonary arterial hypertension
plasminogen activator inhibitor
peripheral blood mononuclear cells
phosphate buffered saline
stromal derived factor-1, also known as CXCL12
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
standard error of the mean
scleroderma (systemic sclerosis)
transforming growth factor β
urokinase-type plasminogen activator
The authors thank coordinators Dana Rosson and Kelley Gibson for recruiting donors and collecting blood samples. This work was supported by grants: NIH NIAMS R01 AR062078, R03 AR056767, and K01 AR054143 (to ET); USARMY/USAMRAA W81XWH-11-1-0508 (to SH), NIH NIAMS P60 AR049459 (Multidisciplinary Clinical Research Center, to RMS), and an NIH NCRR Construction Grant C06 RR015455. ET also received a grant and the Marta Marx Award from the Scleroderma Foundation.
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