Induction of tumour necrosis factor receptor-expressing macrophages by interleukin-10 and macrophage colony-stimulating factor in rheumatoid arthritis

The total patient population consisted of 30 patients with RA (24 women and 6 men; mean ± standard deviation age, 57.2 ± 13.6 years) diagnosed according to the revised 1987 criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) [24]. Patients were receiving prednisolone (≤ 7.5 mg/day; n = 22) and disease-modifying antirheumatic drugs such as methotrexate (n = 12) and salazosulfapyridine (n = 8). They were divided into groups with low, moderate, and high disease activity using the 28-joint disease activity score (DAS28) system [25]; the demographic and laboratory data in these three groups are shown in Table 1. Twenty-six healthy volunteers (20 women and 6 men) matched for age (58.8 ± 15.5 years) served as controls. ST samples were obtained from five patients with RA (4 women and 1 man; age 61.6 ± 3.5 years; disease duration 14.0 ± 2.0 years) at the time of total knee joint replacement. Their clinical parameters were as follows: tender joint count (0–28), 4.2 ± 1.1; swollen joint count (0–28), 2.8 ± 1.5; erythrocyte sedimentation rate, 49 ± 6 mm/hour; serum C-reactive protein, 22 ± 5 mg/liter; immunoglobulin (Ig) M class rheumatoid factor (RF) titer, 61 ± 17 units per ml; visual analogue scale, 40 ± 3 mm; and DAS28, 4.7 ± 0.1. All patients gave informed consent.

Peripheral blood mononuclear cells (PBMCs) were prepared from heparinised blood samples by centrifugation over Ficoll-Hypaque density gradients (Pharmacia, Uppsala, Sweden). Cells were washed well with RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) and were resuspended in phosphate-buffered saline (PBS) with 1% heat-inactivated fetal calf serum (FCS) (Invitrogen). Cell surface expression of IL-10R1, IL-10R2, M-CSF receptor (M-CSFR), TNFR1, and TNFR2 was analysed by cell surface staining and flow cytometry as described previously [22, 23]. PBMC suspensions were incubated with mouse IgG₁ anti-IL-10R1 monoclonal antibody (mAb) (R&D Systems, Minneapolis, MN, USA), mouse IgG₁ anti-IL-10R2 mAb (R&D Systems), rabbit IgG anti-M-CSFR (c-fms) polyclonal Ab (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse IgG_2a anti-TNFR1 mAb (BD Biosciences, San Jose, CA, USA), fluorescein isothiocyanate (FITC)-conjugated mouse IgG_2a anti-TNFR2 mAb (Genzyme/Techne, Minneapolis, MN, USA), or isotype-matched control Abs. Cells were washed and then incubated with rhodamine-conjugated goat anti-mouse IgG₁ mAb (Santa Cruz Biotechnology, Inc.) for anti-IL-10R1 mAb and anti-IL-10R2 mAb, phycoerythrin-conjugated goat anti-rabbit IgG mAb (Santa Cruz Biotechnology, Inc.) for anti-M-CSF Ab, or FITC-conjugated anti-mouse IgG_2a mAb (Santa Cruz Biotechnology, Inc.) for anti-TNFR1 mAb. After washing, cells were resuspended in 1% FCS/PBS. Analysis was performed on a FACScan flow cytometer (BD Biosciences). The monocytes were specifically analysed by selective gating based on parameters of forward and side light scatter. By flow cytometric analysis of PBMCs with anti-CD14 mAb (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), the scattered light-based monocyte population from three patients with RA and three healthy controls was found to contain more than 93% of CD14⁺ cells, and there was no significant difference in their CD14⁺ cell frequencies.

PBMCs were resuspended at a density of 5 × 10⁶ cells in culture medium (RPMI 1640 medium supplemented with 25 mM HEPES, 2 mM L-glutamine, 2% nonessential amino acids, 100 U/ml penicillin, and 100 μg/ml streptomycin) with 10% FCS. Monocytes were purified from PBMCs by negative selection using a cocktail of Abs against CD3, CD7, CD16, CD19, CD56, CD123, and glycophorin A (monocyte isolation kit II; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's instructions. The cell suspensions were adjusted at a density of 5 × 10⁵ cells per ml in culture medium with 10% FCS and were incubated with or without 20% RA ST cell culture supernatants, 25 ng/ml human rIL-10 (R&D Systems), 50 ng/ml human rM-CSF (R&D Systems), or rIL-10 plus rM-CSF in the wells of 48-well plates (Ultra Low Attachment Clusters; Corning Incorporated, Corning, NY, USA) in a humidified atmosphere containing 5% CO₂. Three days later, the cells were harvested, and cell surface expression of cytokine receptors was determined by flow cytometric analysis.

In addition, monocytes were preincubated with or without 25 ng/ml rIL-10, 50 ng/ml rM-CSF, or rIL-10 plus rM-CSF, and the cells were washed with culture medium and were incubated at a density of 5 × 10⁴ cells per 200 μl in 96-well plates (Corning Incorporated) with or without 10 ng/ml human rTNF-α (Sigma-Aldrich, St. Louis, MO, USA), 30 μM N6,2'-O-dibutyryladenosine-3'-5'-cyclic monophosphate sodium (dbcAMP) (BIOMOL International, L.P., Exeter, UK), or rTNF-α plus dbcAMP. Twenty-four hours later, cell-free culture supernatants samples were stored at -30°C until the cytokine assay.

To determine IL-10-induced mRNA expression of cytokine receptors, PBMC suspensions were incubated in six-well plates (Corning Incorporated) at 37°C for 2 hours, and adherent monocytes, after removal of nonadherent cells, were gently harvested using a rubber spatula; the purity of monocytes was found to be more than 95%, as determined by flow cytometric analysis of CD14 and CD3 expression. The cells suspensions, adjusted at a density of 5 × 10⁵ cells per ml in culture medium with 10% FCS, were incubated with or without 25 ng/ml rIL-10. Twelve hours later, the cells were immediately lysed using a reagent for RNA isolation (TRIzol reagent; Invitrogen) and were stored at -80°C until RNA isolation.

RA ST cell culture supernatants were prepared as previously described [26]. Briefly, ST samples from patients with RA were fragmented and digested with collagenase and DNase for 1 hour at 37°C. After tissue debris was removed, cells were resuspended in culture medium with 10% FCS. RA ST cells were incubated at a density of 1 × 10⁶ cells per ml in 10% FCS/culture medium in six-well plates; 72 hours later, culture supernatants were harvested and stored at -30°C.

Total cellular RNA was extracted from adhered monocytes lysed in TRIzol reagent according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesised from total RNA with Molony murine leukemia virus reverse transcriptase (USB Corporation, Cleveland, OH, USA) and oligo-(dT)₁₅ primers (Promega Corporation, Madison, WI, USA). To semiquantify the amounts of TNFR1 and TNFR2 mRNAs, real-time polymerase chain reaction (PCR) was performed with the LightCycler Instrument (Roche Diagnostics, Penzberg, Germany) in glass capillaries. The reaction mix containing DNA double-strand-specific SYBR green I dye (Roche Diagnostics) and specific primers for human TNFR1 and TNFR2 (Nihon Gene Research Laboratories, Sendai, Japan) and β-actin (Search-LC GmbH, Heidelberg, Germany) were added to cDNA dilutions. The cDNA samples were amplified and analysed according to the manufacture's instructions. TNFR1 and TNFR2 expression was determined by normalisation against β-actin expression. The sequences of oligonucleotide primers were as follows: for TNFR1, 5' primer GGT GAC TGT CCC AAC TTT GC and 3' primer GGG TCA TCA GTG TCT AGG CT 3'; for TNFR2, 5' primer CTT CGC TCT TCC AGT TGG and 3' primer AGG CAA GTG AGG CAC CTT.

Concentrations of IL-1β and IL-6 in monocyte culture supernatants were measured in triplicate by the quantitative sandwich enzyme-linked immunosorbent assay (ELISA) using cytokine-specific capture and biotinylated detection mAbs and recombinant cytokine proteins (all from BD Biosciences) according to the manufacturer's protocol. The detection limits for these cytokines were 7.8 pg/ml.

Cryostat sections (4 μm) from RA ST samples were fixed in acetone and blocked with 10% rabbit or goat serum for 30 minutes. Double immunofluorescence was performed by serially incubating sections with 10 μg/ml of mouse IgG₁ anti-IL-10R1 mAb, rabbit IgG anti-TNFR1 Ab, rabbit IgG anti-TNFR2 Ab (Santa Cruz Biotechnology, Inc.), rabbit IgG anti-M-CSFR Ab, mouse IgG₁ anti-CD68 mAb (BD Biosciences), and isotype-matched control Abs at 4°C overnight, followed by incubation with rhodamine-conjugated goat anti-mouse IgG₁ mAb (Santa Cruz Biotechnology, Inc.) and FITC-conjugated anti-rabbit IgG mAb (Santa Cruz Biotechnology, Inc.) for 30 minutes at room temperature. The double immunofluorescence of sections was examined with an LSM510 inverted laser-scanning confocal microscope (Carl Zeiss, Jena, Germany) and illuminated with 488 nm and 568 nm of light. Images decorated with FITC and rhodamine were recorded simultaneously through separate optical detectors with a 530 nm band-pass filter and a 590 nm long-pass filter, respectively. Pairs of images were superimposed for colocalisation analysis.

IL-10-induced gene expression profiles in monocytes were determined by microarray analysis using the gene expression array kit (SuperArray Bioscience Corporation, Frederick, MD, USA), which detects 367 known genes that encode for inflammatory cytokines, chemokines and their receptors, and cell surface markers, as well as representative signaling proteins and downstream targets of the signal transduction pathways that mediate the immune response. RNA preparation, probe synthesis, hybridisation, chemiluminescent detection, and image and data acquisition and analysis were performed according to the manufacture's protocol. All signal intensities of the genes were normalised to those of β-actin.

Samples with values below the detection limit for the assay were regarded as negative and assigned a value of zero. Data were expressed as the mean ± standard error of the mean. The statistical significance of differences between two groups was determined by the Mann-Whitney U test. P < 0.05 was considered significant. The correlation coefficient was obtained by Spearman's rank correlation test.

The IL-10 receptor is a heterodimer consisting of two subunits, and expression of the inducible IL-10R1 seems critical in cellular responses to IL-10 [11]. The cell surface expression of IL-10R1 and IL-10R2 on peripheral blood monocytes from patients with RA and healthy controls was determined by flow cytometric analysis. Figure 1a shows representative histographic patterns of IL-10R1 expression on monocytes. The mean fluorescence intensity (MFI) ratio of IL-10R1 was significantly higher in patients with RA than in controls (RA 2.92 ± 0.42, n = 30; controls 1.93 ± 0.16, n = 25; P < 0.05), but the MFI ratio of IL-10R2 was lower in patients with RA (RA 8.26 ± 0.59; controls 11.81 ± 0.94; P < 0.01). To determine the correlation of IL-10R1 levels and disease activity, we divided patients with RA into three groups according to disease activity using the DAS28 system – low activity (DAS28 < 3.2), moderate activity (3.2–5.1), and high activity (>5.1) – and compared the IL-10 R1 intensity of monocytes from these RA groups and controls. As shown in Figure 1b, the levels of IL-10R1 expression incrementally increased in patients with RA with higher disease activity. However, IL-10R1 levels did not correlate with patients' age, disease duration, or the drug therapy employed. These results indicate that RA blood monocytes may become responsive to IL-10 during active disease in terms of receptor expression.

To corroborate the correlation between the high expression of IL-10R1 on monocytes and disease activity, purified monocytes were incubated for 3 days with or without 20% RA ST cell culture supernatants, and the induction of cell surface IL-10R1 and IL-10R2 was determined by flow cytometry. Figure 2 shows the ratio of the MFI of IL-10R1 and IL-10R2 expression with culture supernatants to the MFI with culture medium alone. The levels of IL-10R1, but not IL-10R2, were significantly augmented in the presence of culture supernatants. These results indicate that the increased IL-10R1 on monocytes found in active RA may be induced by a spillover effect of the particular stimuli produced in the inflamed joint, such as cytokines.

To examine whether the expression of two distinct receptors for TNF-α, TNFR1 and TNFR2, and of M-CSFR was also increased in RA monocytes, the cell surface expression of these receptors on blood monocytes from patients with RA and healthy controls was determined by flow cytometry. As shown in Figure 3, there was no significant difference in TNFR1 expression between patients with RA and controls; however, TNFR2 expression was lower and M-CSFR expression was higher in the patients. Therefore, RA monocytes appear to partially mature into the cells with high levels of IL-10R1 and M-CSFR expression while in the blood circulation.

Previous studies have demonstrated that IL-10 stimulates cell surface expression of both TNF receptors, in particular TNFR2 on monocytes [20]. To confirm this finding, monocytes from patients with RA and controls were incubated for 12 hours, and TNFR1 and TNFR2 mRNA expression was measured by real-time PCR. As shown in Figure 4, expression of both TNFR receptors was augmented at the transcriptional level by IL-10. RA monocytes were more responsive to IL-10 in terms of TNFR2 induction than were normal monocytes, although TNFR1 induction was weaker in RA monocytes.

IL-10 has been shown to enhance M-CSF-induced macrophage differentiation in part by M-CSFR upregulation [21]. Normal monocytes were incubated for 3 days with or without M-CSF, IL-10, or IL-10 plus M-CSF, and their expression of M-CSF and TNF receptors was analysed by flow cytometry. M-CSFR expression was increased by IL-10 and, more significantly, by IL-10 and M-CSF (Figure 5a). Accordingly, both TNFR1 and TNFR2 expression was increased by IL-10 alone, which was enhanced in the presence of M-CSF (Figure 5b,c). These results indicate that cell surface expression of TNF receptors on monocytes/macrophages may be effectively increased by a combination of IL-10 and M-CSF.

Preliminary culture experiments showed that TNF-α alone failed to stimulate cytokine production by adhered monocytes. In agreement with this finding, previous studies have demonstrated that TNF-α could induce IL-1β production by monocytes when intracellular cAMP levels were elevated by prostaglandin or in the presence of the cAMP analogue dbcAMP and have proved a crucial role of cAMP signaling in the activation of IL-1β gene transcription by site-directed mutagenesis analysis of a CRE (cAMP response element) site in the IL-1β promoter [27].

To assess the functional significance of increased TNF receptor expression induced by IL-10 and M-CSF, normal monocytes were preincubated for 3 days with or without M-CSF, IL-10, or IL-10 plus M-CSF, then incubated for 24 hours with or without TNF-α stimulation in the presence or absence of dbcAMP, and their IL-1β and IL-6 production was measured by ELISA. In the absence of dbcAMP, adhered monocytes were unable to produce detectable amounts of cytokines in response to TNF-α. Although the stimulatory effect of dbcAMP alone was limited at a concentration of 30 μM, the combination of TNF-α and dbcAMP induced IL-1β and IL-6 production by monocytes preincubated with M-CSF and, more prominently, by monocytes with IL-10 and M-CSF (Figure 6). However, such cytokine production was rather diminished when monocytes were preincubated with IL-10. These results suggest that IL-10, despite its inhibitory potential, may facilitate monocyte differentiation into TNF-α-responsive macrophages in the presence of M-CSF by increasing surface TNF receptor expression.

High levels of IL-10, M-CSF, and TNF-α expression have all been detected in the inflamed joint of RA [14, 23, 28, 29]. To confirm the interaction of these cytokines in macrophage differentiation at the site of inflammation, ST samples from five patients with RA were analysed by two-color immunofluorescence labeling using Abs against IL-10R1, M-CSFR, and TNF receptors, as well as anti-CD68 Ab. Figure 7 shows representative staining patterns in RA ST. The majority of M-CSFR⁺ and IL-10R1⁺ cells were localised to the lining layer and the endothelial cells, whereas TNFR1⁺ and TNFR2⁺ cells were more widely distributed throughout the tissue, including the lining layer and lymphocyte infiltrates. Colocalisation analysis revealed that the CD68⁺ synovial lining layer contained a large number of cells stained intensely for IL-10R1, M-CSFR, and both TNF receptors, but such cells were sparsely distributed in the sublining layer. Coexpression of these cytokine receptors in lining macrophages suggests that costimulation with IL-10 and M-CSF signals may be involved in the high expression of TNF receptor in lining macrophages in the inflamed ST of RA.

To further elucidate the roles of IL-10 in monocyte maturation, blood monocytes from two patients with RA were incubated for 12 hours with or without IL-10 and the gene expression profile was analysed by an microarray that detects 367 known genes that encode for cytokines, chemokines and their receptors, cell surface markers, representative signaling proteins, and downstream targets of the signal transduction pathway. Seventeen genes were identified as IL-10-inducible (> twofold increase in gene expression) in both patients with RA, although a total of 87 gene signals were increased by IL-10 in either patient. As shown in Table 2, these genes include the TNF receptor superfamily, chemokine receptors, growth factor receptors, TLRs, and TNF receptor-associated factors (TRAFs), although it also induced the endogenous JAK (Janus kinase) kinase inhibitor SOCS (suppressor of cytokine signaling) 3, as has previously been described [30]. Thus, IL-10 can activate various genes essential for macrophage functions.