A novel mechanism for the regulation of IFN-γ inducible protein-10 expression in rheumatoid arthritis

Completed medium consisted of Dulbecco's modified Eagle's medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 2 mmol/l L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (Gibco Laboratories, Grand Island, NY, USA). Monoclonal and biotinylated polyclonal antibodies against human IP-10 and recombinant human IP-10 were purchased from Genzyme/Techne (Cambridge, MA, USA). Monoclonal antibodies against human CD11b and CD18 were purchased from Ancell Corporation (Bayport, MN, USA), and those against intercellular adhesion molecule (ICAM)-1 were purchased from R&D Systems (Minneapolis, MN, USA).

RA or osteoarthritis (OA) synovial fluid (SF) was obtained from knee punctures in 32 RA patients and 10 OA patients. No patient received more than 5 mg oral prednisolone/day or intra-articular injections of glucocorticoids within 1 month of SF sample aspiration.

RA SF monocytes and polymorphonuclear neutrophils (PMNs) were obtained from knee punctures in 23 RA patients. Normal peripheral blood monocytes and PMNs were obtained from 10 age-matched and sex-matched healthy individuals. PMNs were isolated by centrifugation on a Ficoll-Hypaque (Pharmacia LKB Biotechnology Inc, Piscataway, NJ, USA) density gradient, after which they were separated from erythrocytes by lysing the erythrocytes in a solution of 0.15 mol/l NH₄Cl, 0.01 mol/l NaHCO₃, and 0.01 mol/l tetra EDTA. The recovered PMNs (purity 96–98%, viability 98%) were washed three times and resuspended at a density of 5 × 10⁶ cells/ml in completed medium. The mononuclear cells, isolated by centrifugation on a Ficoll-Hypaque, were then separated by centrifugation on a density gradient (1.068 g/ml; Nycodenz, Nycomed AS Oslo, Norway), as described previously [18, 19]. The isolated monocytes were washed, cytospun onto a glass slide, stained with Diff-Quik (Baxter, McGaw, IL, USA), and differentially counted using non-specific esterase staining. The final cell preparations contained more than 75–80% monocytes, based on their morphology and nonspecific esterase staining; their viability was greater than 98%, as assessed by trypan blue exclusion. The recovered monocytes were washed three times and resuspended at a density of 1 × 10⁶ cells/ml in completed medium.

All human experiments were performed in accordance with protocols approved by the Human Subjects Research Committee at our institution, and informed consent was obtained from all patients and volunteers.

Synovial tissues were obtained from seven RA patients (five women and two men; mean age 63.5 years, range 48–72 years) with active synovitis, as determined by serum C-reactive protein levels (mean 3.3 mg/dl), who fulfilled the 1987 American College of Rheumatology criteria for RA [20], all of whom underwent joint replacement surgery. Synovial membrane cell suspension cultures were prepared by collagenase and DNase digestion of minced membranes, as described previously [21]. Isolated fibroblast-like synoviocytes (FLSs) were cultured in completed medium in 75-mm tissue culture flasks. The cells were used from passages 3 through to 10, when they morphologically resembled FLSs and were negative for Mo-1 and major histocompatibility complex class II, indicating the absence of type A or 'macrophage-like' synoviocytes.

SF monocytes or PMNs were layered onto unstimulated semiconfluent FLS monolayers in 48-well plates (Nalge-Nunc International, Tokyo, Japan), and culture supernatants were collected at selected times thereafter. In some experiments, a transwell membrane (pore size 0.45 μm; Becton Dickinson, Bedford, MA, USA) was used to separate the two cell groups, whereas in others anti-integrin antibodies or adhesion molecules were added to the cocultures.

IP-10 was specifically quantified using the double-ligand enzyme-linked immunosorbent assay method, in a modification to a previously reported assay [22]. Monoclonal murine antihuman IP-10 (1 μg/ml) served as the primary antibody, and biotinylated polyclonal goat anti-IP-10 (0.1 μg/ml) served as the secondary antibody. The sensitivity limit for the IP-10 enzyme-linked immunosorbent assay was approximately 50 pg/ml.

Cell-associated IP-10 was visualized immunohistochemically in a modification to a previously reported assay [22]. Briefly, FLSs were grown to near confluence in an 8-well LabTeK chamber slide (Nalge Nunc International), and then incubated for 24 hours with or without either monocytes and PMNs. The slides were then incubated with polyclonal rabbit anti-IP-10 antibody (1:500 dilution; purchased from PeproTech EC, London, UK) or in preimmune rabbit IgG. Biotinylated goat antirabbit IgG (1:20; Biogenex Laboratories Inc, Burlingame, CA, USA) and peroxidase-conjugated streptavidin served as second and third reagents, respectively.

Total cellular RNA was isolated as previously described [22]. Briefly, samples were dispersed in a solution of 25 mmol/l Tris (pH 8.0) that also contained 4.2 mol/l guanidine isothiocyanate, 0.5% sarkosyl, and 0.1 mol/l 2-mercaptoethanol. The RNA was further extracted with chloroform-phenol and then alcohol precipitated.

Semiquantitative reverse transcription (RT)-PCR was performed as previously described [23]. Briefly, 2-μg samples of total RNA were reverse transcribed using M-MLV reverse transcriptase (GIBCO BRL). The primers used in the PCR reaction were 5'-TGA-CTC-TAA-GTG-GCA-TTC-AAG-G (sense) and 5'-GAT-TCA-GAC-ATC-TCT-TCT-CAC-CC (antisense) for IP-10 [24], and 5 '-GTG-GGG-CGC-CCC-AGG-CAC-CA (sense) and 5'-CTC-CTT-AAT-GTC-ACG-CAC-GAT-TTC (antisense) for β-actin, which served as an internal control. The amplification buffer contained 50 mmol/l KCl, 10 mmol/l Tris-HCL (pH 8.3), and 1.5 mmol/l MgCl₂. Specific oligonucleotide primer was added (200 ng/sample) to the buffer, along with 1 μl of the reverse transcribed cDNA samples. The cDNA was amplified after determining the optimal number of cycles. The mixture was first incubated for 5 min at 94°C; it was then cycled 35 times at 95°C for 30 s and at 58°C for 60 s, and elongated at 72°C for 75 s. This format allowed optimal amplification with little or no nonspecific amplification of contaminating DNA. The amplified products were separated on 2% agarose gels containing 0.3 μg/ml ethidium bromide, and were visualized and photographed using ultraviolet transillumination.

Data were analyzed on a Power Macintosh computer using a statistical software package (Statview 4.5; Abacus Concept Inc, Berkeley, CA, USA) and expressed as mean ± SEM. Groups of data were compared by analysis of variance; the means of groups with variances that were determined to be significantly different were then compared using Student's t-test for comparison of the means of multiple groups. P < 0.05 was considered statistically significant.

We first investigated the concentrations of IP-10 in SF from patients with RA (n = 32) or OA (n = 10) using enzyme-linked immunosorbent assay. As shown in Fig. 1, the IP-10 concentrations in the SF from patients with RA were significantly greater than those in the SF of patients with OA (RA 6.05 ± 0.86 ng/ml versus OA 2.32 ± 1.28 ng/ml), which is in agreement with previous findings [25]. We next examined the in situ expression of IP-10 in RA synovial tissue. Immunolocalization indicated that IP-10 was associated mainly with infiltrating macrophage-like and fibroblast-like cells of chronically inflamed synovial tissues (Fig. 2); there was little or no nonspecific staining in tissue sections incubated with control IgG (Fig. 2).

We next assessed the induction of IP-10 expression mediated by the interaction of FLSs and leukocytes (monocytes or PMNs). When plated alone, unstimulated FLSs and leukocytes derived from either RA SF or peripheral blood secreted very small amounts of IP-10 (Fig. 3). On the other hand, when unstimulated RA SF monocytes, and to a lesser extent RA SF PMNs, were cocultured with FLSs, significantly greater amounts of IP-10 (FLS monocytes 5698.0 ± 865.0 pg/ml, FLS PMNs 417.0 ± 48.5 pg/ml) were secreted into the supernatant (Fig. 3). In addition, in order to determine whether the augmented production of IP-10 was specific to leukocytes in the RA SF, we tested the capacity of FLSs and either peripheral blood monocytes or PMNs obtained from healthy individuals to produce IP-10. Although enhanced production of IP-10 was observed in RA FLS peripheral blood leukocyte cocultures, the enhancement was less pronounced than in RA FLS SF leukocyte cocultures (Fig. 3). In addition, IFN-γ and, to a lesser extent, tumor necrosis factor (TNF)-α are potent inducers of IP-10 [8, 24, 26, 27]. Therefore, IFN-γ and TNF-α were neutralized with monoclonal antibodies (obtained from Chemicon International, Temecula, CA, USA, and from R & D Systems, respectively) in order to eliminate the effects of newly synthesized IFN-γ and TNF-α by in situ cell–cell interactions. IP-10 concentrations in the medium with FLS and leukocyte coculture in the presence or absence of either neutralizing antibody were measured, and no significant stimulatory or inhibitory effects were observed (Fig. 3).

Because FLS–lymphocyte interactions induce inflammatory mediators [28], it was important to rule out contaminating lymphocytes as a major source of IP-10 in the FLS–monocyte interactions. We examined the effect of mononuclear lymphocytes on IP-10 secretion in FLS lymphocytes. SF monocytes were depleted from mononuclear cell suspension by adhesion to a plastic dish for 2 hours. Although monocyte-depleted nonadherent lymphocytes (5 × 10⁵/ml; monocyte contamination 5%) were added to unstimulated FLS cultures, stimulatory effects of lymphocytes on IP-10 secretion were not observed (FLS <50 pg/ml, monocyte-depleted lymphocyte <50 pg/ml, FLS plus monocyte-depleted lymphocyte 146.0 ± 66.2 pg/ml), suggesting that contaminated lymphocytes were not involved in the increased IP-10 secretion.

Steady-state expression of IP-10 mRNA in the cocultures was assessed using RT-PCR. Consistent with the expression of IP-10 protein, RT-PCR revealed that substantial steady-state expression of IP-10 mRNA was significantly upregulated in either monocytes or PMNs cocultured with FLSs (Fig. 4). Immunohistochemical analysis confirmed that IP-10 was upregulated in leukocytes when cocultured with FLSs (Fig. 5). Although small amounts of IP-10 antigen were present in both unstimulated leukocytes and FLSs, markedly greater amounts were observed in leukocytes cocultured with FLSs, indicating that the major cellular sources of IP-10 are probably either monocytes or PMNs during coculture.

In order to gain a better understanding of the mechanism whereby the interaction between leukocytes and FLSs induces IP-10 expression in the leukocytes, the two cell groups were cultured together in a chamber in which they were separated by a transwell membrane (pore size 0.45 μm) that allowed passage of soluble factors but prevented physical contact between the cell groups. As shown in Table 1, augmentation of IP-10 secretion was completely blocked by the transwell membrane, suggesting that direct cell–cell contact is important for this process. Fibroblasts interact with monocytes or PMNs via pathways that are mediated by adhesion molecules, including the ICAM-1–integrin pathway [19, 29]. The potential role of these molecules in FLS–leukocyte interactions was investigated by assessing the capacity of specific antibodies to inhibit IP-10 production by cocultured leukocytes. Although control mouse IgG had no significant effects on IP-10 secretion, the addition of anti-CD11b, anti-CD18, or anti-ICAM-1 monoclonal antibody (5 μg/ml) to either FLS–monocyte or FLS–PMN coculture reduced production of IP-10 by 59.1%, 56.5%, and 53.0%, and by 79.0%, 87.4%, and 54.0%, respectively (Fig. 6). Addition of the antibodies to the cells when cultured individually had little effect on IP-10 production (Fig. 6).