Background
Rheumatoid arthritis (RA) is a chronic autoimmune disease that affects the peripheral joints, leading to joint deformity and destruction [
1]. Synovial macrophages, through their ability to release inflammatory cytokines, matrix metalloproteinases (MMPs), chemokines, reactive oxygen and nitrogen intermediates, and prostanoids, play a central role in the pathological process of RA [
2]. Importantly, macrophage numbers and cytokine products are associated with disease activity and disease progression in RA, and decreases in synovial sublining CD68+ macrophages represent a sensitive biomarker of effective treatment [
2]. Therefore, strategies specifically designed to target the activation, survival, or pro-inflammatory differentiation of macrophages in synovial tissue may be of therapeutic benefit in RA [
2,
3].
Colony-stimulating factors (CSF) and functionally related cytokines regulate myeloid lineage cell development, proliferation, survival, mobilization, differentiation, and activation in both health and disease [
4]. Granulocyte-macrophage CSF (GM-CSF) plays an important role in promoting the differentiation of granulocytes and macrophages from hematopoietic precursors [
4]. During inflammation this property of GM-CSF may be important for the sustained recruitment of immature monocytes to affected tissues [
5‐
7]. GM-CSF may also directly contribute to inflammation by polarizing macrophages into a pro-inflammatory phenotype, and participating in cytokine networks with other pro-inflammatory cytokines such as TNF and interleukin (IL)-1β [
8,
9]. Administration of exogenous GM-CSF has been demonstrated to exacerbate collagen-induced arthritis (CIA) in mice, while mice deficient in GM-CSF expression are protected against disease in this model of arthritis [
6,
10‐
13]. Additionally, neutralizing antibodies (Abs) against GM-CSF display both prophylactic and therapeutic efficacy in experimental arthritis [
6,
14]. The direct relevance of these findings to RA is exemplified in recent clinical trials in which a blocking Ab directed at the alpha chain of the GM-CSF receptor has demonstrated safety and efficacy [
15,
16].
Colony-stimulating factor-1 (CSF-1), through binding to the tyrosine kinase receptor CSF1R (also known as c-fms), also promotes the survival, proliferation and differentiation of myeloid cells, including monocytes, macrophages and osteoclasts [
4,
17]. CSF-1 plays a distinctly different role from GM-CSF in myelopoiesis, acting as a survival and differentiation factor for mature circulating monocytes (Ly6C
lo in mice, CD16
+ in humans) and resident tissue macrophages [
6,
17,
18]. In RA, CSF-1 is produced mainly by synovial endothelial cells, but in vitro data indicate that synovial fibroblasts and chondrocytes stimulated with TNF or IL-1β also produce CSF-1 [
19‐
21]. Animal models have shown that exogenous administration of CSF-1, like GM-CSF, exacerbates the incidence and severity of CIA, while anti-CSF-1 Ab, genetic deletion of CSF-1, and pharmacological inhibitors of CSF1R reduce the severity of experimental arthritis [
11,
12,
22,
23]. CSF-1 also has an essential function in joint destruction, as CSF-1 is required for osteoclastogenesis and TNF-induced osteolysis [
24,
25].
IL-34 was recently discovered as a novel ligand of CSF1R through its ability to support monocyte viability [
26]. CSF-1 and IL-34 share structural, but not sequence homology, and have largely overlapping effects on CSF1R downstream signaling, regulation of monocyte survival, macrophage polarization, and osteoclastogenesis [
27‐
32]. However, during murine development, IL-34 plays a non-redundant role in the generation of Langerhans cells and microglia [
33,
34]. Recent studies have reported that IL-34 levels are elevated in the serum, synovial fluid, and synovial tissue of RA patients, and like CSF-1, IL-34 is induced by TNF and IL-1β in RA fibroblast-like synoviocytes (FLS) [
35‐
38]. The goal of this study was to determine the relative contributions of CSF-1 and IL-34 to CSF1R-dependent inflammation in RA.
Methods
Patients and tissue donors
Synovial biopsies were obtained by needle arthroscopy as previously described from clinically active joints in patients with RA (n = 15), psoriatic arthritis (PsA) (n = 15), or osteoarthritis (OA) (n=7), as previously described [
39]. RA and PsA patients fulfilled the 1987 American College of Rheumatology criteria for RA and the classification of psoriatic arthritis study (CASPAR) criteria, respectively [
40,
41]. Patient clinical characteristics are detailed in Additional file
1: Tables S1 and S2. All patients gave their written informed consent prior to study inclusion, and this study was approved by the Medical Ethics Committee of the Academic Medical Center, University of Amsterdam, and in accordance with the Declaration of Helsinki.
Immunohistochemical and digital imaging analysis
Serial sections from six different TissueTek-embedded biopsy samples per patient were cut with a cryostat (5 μm), fixed with acetone, and endogenous peroxidase activity blocked with 0.3 % hydrogen peroxide in 0.1 % sodium azide/phosphate-buffered saline. Sections were stained overnight at 4°C with Abs against CSF-1 (R&D), IL-34 (M4, provided by Five Prime Therapeutics Inc.) and CD68 (Dako). Equivalent concentrations of irrelevant mouse monoclonal Abs were used as negative controls. Sections were then washed and incubated with goat anti-mouse-horseradish peroxidase (HRP) (Dako), except in the case of anti-IL-34 Ab, which was followed by incubation with biotinylated tyramide and streptavidin-HRP. Sections were developed with amino-ethylcarbazole (AEC, Vector Laboratories), counterstained with Gill’s hematoxylin (Klinipath), and mounted in Kaiser’s glycerol gelatine (Merck). Stained sections were analyzed by computer-assisted image analysis using the Qwin analysis system (Leica) as previously described in detail [
42]. Values of integrated optical densities (IOD)/mm
2 were obtained and corrected for the total number of nuclei/mm
2. Data were presented as the number of positive cells/mm
2 for quantitative analysis of cell-type-specific markers.
Monocyte purification and macrophage differentiation
Human mononuclear cells were isolated from volunteer donor blood buffy coats (Sanquin), peripheral blood from healthy controls and RA patients, and from synovial fluid of RA patients by gradient centrifugation with Lymphoprep (Axis-Shield PoPAS) and monocytes were further isolated by Percoll gradient separation (GE Healthcare). Monocytes were differentiated into macrophages in IMDM/10 % fetal calf serum (FCS) supplemented with 100 μg/ml gentamycin (Invitrogen), in the presence of human GM-CSF (5 ng/ml, R&D Systems), CSF-1 (25 ng/ml, R&D Systems), or IL-34 (25 ng/ml, provided by Five Prime Therapeutics Inc.) for 7 days.
Flow cytometry
Macrophage purity and differentiation were assessed by flow cytometry (FACS Canto Flow Cytometer, BD Biosciences). Fluorochrome-labeled monoclonal Abs against CD14 (eBiosciences), CD206 and CD163 (both from BD Pharmingen), CD64 (Biolegend), and equivalent concentrations of isotype control Abs were used. Before staining, Fc receptors were blocked with 10 % human serum (Lonza). Data were analyzed with FlowJo Flow Cytometry Analysis software (Tree Star). Values were expressed as the ratio of the geometric mean fluorescence intensity (geomean) of the marker of interest over that of the isotype control.
Macrophage cell death and viability
Macrophage cell death was assessed by Annexin V-propidium iodide (PI) staining. GM-CSF-, IL-34-, and CSF-1-differentiated macrophages in peripheral blood from healthy donors and in peripheral blood and synovial fluid from RA patients were stained with Annexin V (1:100 dilution, iQ products) and PI (1:100 dilution, iQ products) and measured using a FACS Canto Flow Cytometer. Data were analyzed with FlowJo Flow Cytometry Analysis software. Values were expressed as the percentage of Annexin V-PI double-positive cells. Macrophage viability was assessed by the calcein assay. Monocytes from the buffy coat were differentiated into macrophages with medium alone, CSF-1 or IL-34 and cell proliferation was analyzed at days 1, 3 and 7 by staining with Calcein-AM (1 μM, BD Bioscience) and analysis using a multi-label reader Victor3™ (PerkinElmer Inc.). Data were expressed as signal in arbitrary units.
Real-time (RT) PCR and quantitative (q)PCR arrays
RNA from frozen synovial biopsies or from GM-CSF-, IL-34- and CSF-1-differentiated macrophages was isolated using the RNeasy Kit and RNase-Free DNase Set (Qiagen). Patient clinical characteristics are detailed in Additional file
1: Table S3. Total RNA was reverse-transcribed using SuperScript™ II RT (Invitrogen). Duplicate PCR reactions were performed using SYBR green (Applied Biosystems) with an ABI Prism® 7000 sequence detection system (Applied Biosystems). Complementary DNA (cDNA) was amplified using specific primers (Invitrogen) (see Additional file
1: Table S4). Relative levels of gene expression were normalized to
glyceraldehyde-3-phosphate dehydrogenase (
GAPDH) housekeeping gene. Messenger RNA (mRNA) expression was presented as 2
-ΔCt or relative quantity (RQ, 2
-ΔΔCt). Alternatively, total RNA was subjected to cDNA synthesis using an RT
2 First Strand Kit (Qiagen) and mRNA expression of 336 inflammation-related genes was analyzed by RT-qPCR using low density qPCR arrays (Human Angiogenic Growth Factors & Angiogenesis Inhibitors, Human Extracellular Matrix & Adhesion Molecules and Human TGFß/BMP Signaling Pathway PCR Arrays, Qiagen). Relative levels of gene expression were normalized to five housekeeping genes and RQ values determined as above.
Antibodies
Anti-human CSF-1 Ab, clone 26730, a neutralizing mouse IgG2a monoclonal Ab, was purchased from R&D Systems, Minneapolis, MN, USA. Anti-human IL-34 Abs M2 and M4 were provided by Five Prime Therapeutics. M2 and M4 monoclonal Abs were prepared by immunization of Balb/c mice with purified human IL-34 produced at Five Prime Therapeutics, followed by standard hybridoma procedures and protein A purification. All animal experiments in this study were performed in compliance with appropriate protocols approved by the Institutional Animal Care and Use Committee at Five Prime Therapeutics and Bolder Biopath (Boulder, CO, USA). The M2 Ab was determined to be a blocking Ab, and the M4 Ab was determined to be a non-blocking Ab by testing hybridoma supernatant for the ability to block biotinylated IL-34-binding to human CSF1R-Fc in an ELISA format, and by testing the ability of purified Ab to neutralize IL-34-mediated survival of primary human monocytes. Anti-human CSF1R Ab huAB1, a humanized IgG4, monoclonal Ab, was provided by Five Prime Therapeutics. huAB1 blocks the binding of both CSF-1 and IL-34 to the human CSF1R, thereby inhibiting CSF1R-mediated signaling pathways (see Additional file
2: Figure S1a). Anti-mouse CSF1R Ab muAB5, was provided by Five Prime Therapeutics and is a chimeric IgG1, monoclonal Ab consisting of rat V regions and murine C regions. muAB5 blocks the binding of both mouse CSF-1 and mouse IL-34 to the mouse CSF1R, thereby inhibiting CSFR-mediated responses (see Additional file
2: Figure S1b). Anti-CSF1R treatment in mice has been reported to selectively reduce peripheral Ly6C
lo monocytes while having no effect on Ly6C
hi monocyte numbers [
6,
18]. To assess the impact of anti-mouse CSF1R muAB5 on peripheral monocytes, we treated mice with 20 mg/kg of muAB5 or saline and 7 days later quantified monocyte populations in the blood. Anti-CSF1R muAB5 selectively reduced the number of Ly6Clo monocytes while having little to no effect on Ly6Chi monocytes (see Additional file
2: Figure S2).
Synovial biopsy explant assays
Intact synovial biopsies (5 mm
3) were obtained from the knees of patients with RA and were cultured, three biopsies per condition, for 24 h in complete DMEM supplemented with 10 % FCS and stimulated for 24 h with increasing concentrations of human IL-34 or CSF-1. Alternatively, explants were cultured for 96 h with anti-human CSF-1 (5 μg/ml), anti-IL-34 (5 μg/ml, M2), increasing concentrations of anti-human CSF1R Ab (huAB1), or their respective human isotype controls (Eureka Therapeutics). Cell-free tissue culture supernatants were harvested for cytokine analysis. Patient clinical characteristics are detailed in Additional file
1: Table S4.
Measurement of cytokine production
Cell-free supernatants from synovial biopsies were analyzed by ELISA for IL-6 (PelKine Compact™ ELISA kits, Sanquin Reagents). CCL2, CCL-7, ENA-78, SDF-1, MIG, GCP-2, TAC, NAP-2, IP-10, IL-1β, TNF-α, CXCL-8, MMP-2, MMP-7, and MMP-9 were measured using human single-plex assays (Bio-Rad) and read on a Bio-Plex 200 system (Bio-Rad).
Effects of anti-CSF1R Ab on murine myeloid homeostasis
CB17 SCID mice (3–5/group) were injected intravenously with 20 mg/kg of muAB5 or saline. Seven days later 50 μl of whole blood was harvested into FACS buffer containing ethylenediaminetetraacetic acid (EDTA) (MACs Rinsing Buffer with BSA; Miltenyi Biotech) to prevent clotting. Red blood cells were lysed by hypotonic lysis (0.16M NH4Cl, 0.01M KCO3 and 0.1mM EDTA). Samples were washed and Fc receptors blocked by staining with 5 μg/ml anti-CD16/32 Ab (Clone: 93, eBioscience). Cells were washed and stained with the following Abs: anti-mF4/80-FITC (Clone: BM/8; Biolegend), anti-mCD11b-PE (Clone: M1/70; BD Biosciences), anti-mLy6C-PerCP (Clone: HK1.4; Biolegend) and anti-mLy6G-Alexa700 (Clone: 1A8; Biolegend). To facilitate identification of dead cells the samples were stained with Aqua Live/Dead© (Invitrogen) according to the manufacturer’s recommendations. Cells were washed, pellets were resuspended in 50 μl of FACs buffer, and 50 μl of CountBright™ Absolute Counting Beads (Invitrogen) were added to each sample. Samples were thoroughly mixed and run on a BD LSRII flow cytometer (BD Biosciences). Data files were analyzed using Flow Jo software v.7.6.4 (Treestar Inc.)
Induction and assessment of CIA and histopathological analysis
The mouse CIA models were established at Bolder Biopath. Male DBA/1 mice (12/group) were injected intradermally at the base of the tail with 150 μl of Freund’s Complete Adjuvant containing bovine type II collagen (2 mg/ml) on day 0 and day 21. For prophylactic treatment mice were dosed starting on day 0 with vehicle, Enbrel at 10 mg/kg, or muAB5 at 30 mg/kg. Treatment continued three times weekly through day 32. Clinical sores on a scale of 0–5 were determined for each of the paws on study days 18–35. The study was terminated on day 35 and blood was collected. Hind paws and knees were collected at termination and processed for histological analysis, as detailed in Additional file
3: Supplementary methods. For therapeutic treatment, mice were immunized as outlined above and randomized into treatment groups once swelling was obviously established in at least one paw. Group mean scores were 0.5–1.0 (out of a possible maximal score of 5.0) at the time of enrollment. Treatment with vehicle, Enbrel at 10 mg/kg, or muAB5 at 30 mg/kg was initiated after enrollment and continued three times weekly through day 20 of arthritis with mice terminated on day 23 of arthritis. Drug exposure as measured by the terminal plasma concentration was below the limit of detection in 3 out of 12 animals in the muAB5-treated group. These samples tested positive for anti-drug antibodies (data not shown), which likely contributed to the poor exposure. Based on these findings, these 3 animals with undetectable drug levels were excluded from any of the study analysis and interpretation: 7 the 12 animals in the Enbrel-treated group also had no detectable drug at termination and were excluded from the analysis.
TRAP5b analysis
Mouse serum band 5 tartrate-resistant acid phosphatase isoform b (TRAP5b) was measured in mouse EDTA plasma using a commercial ELISA kit (MouseTRAP™ Assay, Immunodiagnostic Systems, Inc.) as per the manufacturer’s instructions, including a 1:4 dilution of all samples, using plasma instead of serum. Each sample was tested in singlet. The standard curve absorbance vs. concentration data were fit to a four-parameter logistic for calculation of the test results. Results are reported in units/liter (U/L).
Statistical analyses
Statistical analysis was performed using Windows GraphPad Prism 5 (GraphPad Software, Inc.). Potential differences between experimental groups were analyzed by the non-parametric Mann-Whitney U test, Kruskal-Wallis test, or Friedman paired test, as appropriate. P values <0.05 were considered statistically significant.
Discussion
In the present study we demonstrated that specific antibodies against CSF1R, which prevent binding of both CSF-1 and IL-34 to their receptor, reduce the severity of CIA and the production of inflammatory mediators in RA synovial tissue explants ex vivo. Conversely, neutralization of CSF-1 or IL-34 individually had no detectable effect on inflammatory gene expression in RA synovial tissue. This latter observation was surprising, as CSF-1 deficiency or neutralization in animal models of RA has clear therapeutic effects [
11,
12]. Our data might suggest that in CIA, CSF-1 plays an important role in promoting the differentiation and survival of mature monocytes in the periphery before or as they are entering the inflamed tissue, as observed in certain murine models of lung and peritoneal inflammation [
6], while in RA synovial explants, synovial macrophages are no longer dependent upon CSF-1 alone. Instead, synovial macrophages may also utilize IL-34 as a survival factor. In line with this, CSF-1 and IL-34 are detected at equivalent concentrations in synovial fluid from patients with RA, and here we confirm and extend previous observations that both CSF-1 and IL-34 are produced locally in RA synovial tissue [
35,
36]. However, stimulation of RA synovial biopsies with either CSF-1 or IL-34 failed to influence acute expression of inflammatory mediators, possibly indicating that endogenous synovial production of CSF-1 and/or IL-34 in RA is saturating in regard to CSF1R availability.
Our inability to modulate RA synovial explant gene expression by neutralizing CSF-1 and IL-34 individually further supports the notion that these two cytokines are largely redundant in supporting the differentiation and survival of most tissue macrophage populations. Some subtle differences have been noted between CSF-1 and IL-34 in the strength and kinetics with which they activate CSFR1 [
30‐
32]. Indeed, global gene expression analysis of human monocytes differentiated with IL-34 and M-CSF revealed that of the genes regulated by these cytokines, quantitative differences in the induction were noted for approximately 30 % of the genes [
45]. Consistent with this, of 336 genes involved in processes contributing to pathological changes in RA, such as angiogenesis, inflammation, and tissue remodeling, we observed only 18 genes that were differentially expressed by CSF-1 and IL-34 Mφ. However, as we observe that CSF-1 (detected in cells surrounding the blood vessels) and IL-34 (throughout the synovial sublining and intimal lining layers) are expressed in different regions of the synovium, these cytokines could potentially contribute to the phenotypic heterogeneity of macrophages observed in the sublining and intimal lining layers of synovium in inflammatory arthritis [
46].
Our results provide the first direct evidence that targeting of CSF1R has anti-inflammatory effects in not only animal, but also human models of established RA. Previous studies have observed protective effects against pathological changes in animal models of RA using pharmacological inhibitors of CSF1R kinase activity [
22,
23]. However, concerns have been raised that these compounds can also target other tyrosine kinases relevant to pathological change in RA [
17]. While this manuscript was in preparation, Toh and colleagues reported that another CSF1R-specific Ab could prophylactically prevent inflammation and joint damage in CIA [
47]. Intriguingly, the same antibody did not suppress inflammation in the murine serum transfer model of arthritis, while retaining its capacity to block cartilage and bone destruction. The authors attributed this discrepancy to potentially differential roles for macrophage recruitment and activation in these animal models [
47].
Similarly, conflicting results using different CSF1R-blocking antibodies in other animal disease models have led to questions about the feasibility of this strategy [
17]. For instance, the anti-CSFR1 Ab AFS98, which rapidly reduces monocytes and tissue macrophages in vivo, confers protection in murine models of lung and peritoneal inflammation [
6]. However, another Ab M279, which reduces the F4/80
hiLy6
lo monocyte population and tissue macrophages more slowly, does not display anti-inflammatory effects in similar models, but exacerbates graft-versus-host disease [
18]. The difference in activity of AFS98 and M279 could be due to differences in Fc effector function [
17]. AFS98 is a rat IgG2a with greater potential for Fc effector function compared to M279, which is a rat IgG1. muAb5 is a mouse IgG1 and expected to have low Fc effector function, similar to M279. In our study, muAb5 reduced the same circulating monocyte population observed with M279, but provided almost complete protection against all parameters of disease in CIA.
These studies have led to the suggestion that in some inflammatory diseases, anti-CSF1R therapy might aggravate pathological changes by depleting the tissue of the tolerizing resident tissue macrophages and allowing their replacement by pro-inflammatory monocyte populations, or by promoting pro-inflammatory re-polarization of tissue macrophages [
17]. These concerns can only be addressed empirically in the clinic, but it is noteworthy that huAB1 treatment reduced the production of pro-inflammatory cytokines (IL-6, TNF, and IL-1β), chemokines (CXCL-8, GCP-2, MIG, IP-10, CCL-2, and CCL7), and MMPs (MMP-2 and MMP-9) in RA synovial tissue. This suggests that CSF1R blockade in the inflamed tissue does not result in conversion of synovial macrophages to a more pro-inflammatory functional phenotype, consistent with recent observations that environmental stimuli present in synovial tissue, such as IgG complexes and angiopoietins, can override polarization conditions to regulate macrophage gene expression [
48,
49]. Experimentation in synovial biopsies cannot address the possibility that circulating pro-inflammatory monocytes might replace synovial macrophages following anti-CSF1R treatment, but studies of monocyte migration using scintigraphy in RA patients has indicated that macrophage turnover in synovial tissue is slow, and even during successful therapy, the rate of monocyte immigration into synovial tissue is unchanged [
50,
51], raising the possibility that CSF-1 maintains distinct monocyte populations in mouse and man.
Competing interests
This study was funded by an open research grant from Five Prime Therapeutics Inc. to KAR. HL, JW, LL, JAZ, ALR, ELM, and BRW are or were employees of Five Prime Therapeutics Inc. HL, JW, JAZ, ALR, ELM, and BRW own stock or stock options in Five Prime Therapeutics Inc. None of the other authors have any competing interests to declare.
Authors’ contributions
SG contributed to the design of the studies, performed and interpreted the ELISA, flow cytometry, and gene expression experiments, and drafted and revised the manuscript. LMH and IEvE designed, performed and analyzed immunohistochemistry experiments. BMF contributed to designing, performing, and interpreting immunohistochemistry and gene expression experiments. HL participated in the development of the anti-IL34 antibodies and participated in the design and interpretation of the CIA studies. JW carried out the development of the anti-human and anti-mouse CSF1R antibodies and participated in the writing and revision of the manuscript. LL participated in the design and interpretation of the CIA studies and helped design and perform experiments determining the effects of anti-mouse CSF1R on murine myeloid homeostasis. JAZ participated in the development of the anti-IL34 antibodies and helped design and perform experiments determining the effects of anti-mouse CSF1R on murine myeloid homeostasis. ALR helped design and perform experiments determining the effects of anti-mouse CSF1R on murine myeloid homeostasis. ELM, BRW, PPT, and KAR helped conceive the study and participated in its design, coordination, and interpretation and helped to draft and revise the manuscript. TRDJ participated in the coordination and interpretation of the study, and revised the manuscript. All authors read, corrected and approved the final manuscript.