SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

Hyperactivation of innate immunity by Toll-like receptors (TLRs) can contribute to the development of autoinflammatory or autoimmune diseases. This study evaluated the activation of Tyro3, Axl, Mer (TAM) receptors, physiologic negative regulators of TLRs, by their agonists, growth arrest–specific protein 6 (GAS-6) and protein S, in the prevention of collagen-induced arthritis (CIA).

Methods

Adenoviruses overexpressing GAS-6 and protein S were injected intravenously or intraarticularly into mice during CIA. Splenic T helper cell subsets from intravenously injected mice were studied by flow cytometry, and the knee joints of mice injected intravenously and intraarticularly were assessed histologically. Synovium from mice injected intraarticularly was evaluated for cytokine and suppressor of cytokine signaling (SOCS) expression.

Results

Protein S significantly reduced ankle joint swelling when overexpressed systemically. Further analysis of knee joints revealed a moderate reduction in pathologic changes in the joint and a significant reduction in the number of splenic Th1 cells when protein S was overexpressed systemically. Local overexpression of GAS-6 decreased joint inflammation and joint pathology. Protein S treatment showed a similar trend of protection. Consistently, GAS-6 and protein S reduced cytokine production in the synovium. Moreover, levels of messenger RNA for interleukin-12 (IL-12) and IL-23 were reduced by GAS-6 and protein S treatment, with a corresponding decrease in the production of interferon-γ and IL-17. TAM ligand overexpression was associated with an increase in SOCS-3 levels, which likely contributed to the amelioration of arthritis.

Conclusion

This study provides the first evidence that TAM receptor stimulation by GAS-6 and protein S can be used to ameliorate arthritis when applied systemically or locally. TAM receptor stimulation limits proinflammatory signaling and adaptive immunity. This pathway provides a novel strategy by which to combat rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease that is manifested in articulating joints, causing destruction of cartilage and bone. The cause of this disease is still unknown, and treatment has focused on down-regulating inflammation by blocking downstream signaling or by neutralizing harmful cytokines. Although successful in the clinic, these therapies have substantial side effects and a high rate of nonresponders among treated patients. Natural negative feedback mechanisms can potentially be used therapeutically in order to halt the progression of the inflammatory process and initiate recovery. This approach could possibly limit side effects, since the body's own self-regulating responses are enhanced, rather than uncontrolled systemic blocking of cytokines, which is important in host defense.

One such system for controlling inflammation is that of the Tyro3, Axl, Mer (TAM) receptors. Tyro3, Axl, and Mer comprise a family of tyrosine kinase receptors, and they have been implicated in the negative regulation of inflammation. The regulatory role of TAM receptors in inflammation was found in triple-knockout mice for the TAM receptors; these animals showed excessive lymphocyte proliferation and autoimmunity (1). Moreover, proinflammatory cytokine expression by macrophages is inhibited upon treatment with growth arrest–specific protein 6 (GAS-6) (2). Two ligands have been described for the TAM receptor family, GAS-6 and protein S (3). Both of these ligands bind to phosphatidylserine on cell membranes and subsequently stimulate TAM receptors (4).

GAS-6 has been shown to regulate Toll-like receptor (TLR) signaling in dendritic cells via activation of the Axl receptor (5). Stimulation of cells via the Axl receptor in conjunction with interferon-α/β receptor (IFNAR) leads to up-regulation of suppressor of cytokine signaling (SOCS) proteins 1 and 3 (5, 6), which are inhibitors of inflammation. SOCS-1 blocks intracellular signaling, since SOCS-1 can directly inhibit myeloid differentiation factor 88 adaptor–like protein [Mal], an adapter molecule for TLR-2 and TLR-4 (7). TLRs have also been implicated in maintenance of the chronic inflammatory loop in RA synovium (8, 9), and TLR-2 and TLR-4 play an important role in arthritis (10, 11). SOCS-3 prevents the binding of tumor necrosis factor receptor–associated factor 6 (TRAF6) to transforming growth factor β (TGFβ)–activated kinase 1, a key signaling molecule in TLR, interleukin-1 (IL-1) receptor, and tumor necrosis factor (TNF) receptor signaling (12, 13). The protective role of SOCS proteins in experimental models of inflammation in mice has been shown by ectopic overexpression of SOCS-3 in collagen-induced arthritis (CIA) (14). This resulted in altered splenic T helper cell responses to antigens and ameliorated the arthritis.

Taking into account that inflammation in mice with CIA can be resolved by SOCS-3 treatment, we set out to determine if overexpression of GAS-6 or protein S can ameliorate experimental arthritis. To our knowledge, this study is the first to show that TAM stimulation can ameliorate arthritis. We found that systemic overexpression of protein S decreased arthritis severity and was able to reduce splenic Th1 cell numbers. Both GAS-6 and protein S were also capable of decreasing arthritis when overexpressed intraarticularly, since joint pathology and synovial proinflammatory cytokine production were significantly reduced in the inflamed joints.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Mice.

Male DBA/1 mice ages 10–12 weeks (Janvier) were housed in filter-top cages and fed a standard diet with freely available food and water. All in vivo studies complied with national legislation and were approved by local authorities for the care and use of animals, with related codes of practice.

Cloning strategy.

The constructs pcDNA6AmGas6 and pcDNA6AmPros1 were cloned with Kpn I and Xba I in the pShuttle vector behind the cytomegalovirus (CMV) promoter. Then, pShuttleCMVmGas6 and pShuttleCMVmPros1 were cloned into the E1-deleted region of the Ad5 virus backbone pAdEasyI.

Construction of adenoviral vectors.

Viral vectors were E1A,B- and E3-deleted and were produced according to the method described elsewhere (15). The purified recombinant adenoviral vector DNA was transfected into N52E6 viral packaging cells using Lipofectamine 2000 (Invitrogen). Virus was purified using 2 CsCl gradient centrifugations and then stored in small aliquots at −80°C. The viral titer of the purified viral vectors was determined in human embryonic retinoblastoma 911 indicator cells by immunohistochemical detection of viral capsid protein at 20 hours after transfection.

Induction of CIA.

CIA induction has been described in detail previously (14). Briefly, bovine type II collagen was dissolved in 0.05M acetic acid to a concentration of 2 mg/ml and was then emulsified in equal volumes of Freund's complete adjuvant (2 mg/ml of Mycobacterium tuberculosis strain H37Ra; Difco). Mice were immunized intradermally at the base of the tail with 100 μl of emulsion (50 μg of bovine type II collagen). On day 21 after immunization, mice were given an intraperitoneal booster injection of 100 μg of type II collagen dissolved in phosphate buffered saline. One day after the booster injection, immunized mice were injected intravenously with 3 × 108 focus-forming units (FFU) of adenovirus; for intraarticular injection into both knees, 1 × 107 FFU of either Ad5.Gas6, Ad5.Pros1, or Ad5.Luc was used. Two independent observers (BTvdB and MBB) monitored macroscopic clinical signs of arthritis in the paws and ankle joints. Redness, swelling, and in later stages, ankylosis were scored as follows (maximum score of 2 per limb): 0 = no changes, 0.25 = redness or swelling of 1–2 toes, 0.5 = redness or swelling of 3–5 toes, 0.5 = swelling of the ankle, 0.5 = swelling of the footpad, and 0.5 = severe swelling and ankylosis (redness, excessive edema, and deformation).

Histologic analysis.

Whole knee joints were dissected and fixed in phosphate buffered 4% paraformaldehyde followed by decalcification with 5% formic acid, and embedded in paraffin. Serial tissue sections (7 μm) were stained with Safranin O (BDH Chemicals) and counterstained with fast green (BDH Chemicals) or with hematoxylin and eosin (Merck). Histopathologic changes seen in serial sections were scored on a scale of 0–3. Joint inflammation was determined by the presence of synovial cell infiltrates and inflammatory cell exudates. Connective tissue destruction was determined by the depletion of cartilage proteoglycan (loss of Safranin O staining of the noncalcified upper cartilage layer) and by cartilage and bone erosion.

RNA isolation and quantitative polymerase chain reaction (PCR) analysis.

Synovium and liver samples were disrupted using a MagNaLyser (Roche). Total RNA was extracted from the tissue homogenates and from cells using TRI Reagent (Sigma) according to the manufacturer's protocol. Isolated RNA was treated with DNase, followed by reverse transcription of 1 μg of RNA into complementary DNA (cDNA) using 0.5 μg of Moloney murine leukemia virus reverse transcriptase per microliter of oligo(dT) primers, and 12.5 mM dNTPs (Invitrogen). Real-time quantitative PCR was performed using a StepOnePlus sequence detection system (Applied Biosystems). PCR was performed in a total reaction volume of 12.5 μl consisting of appropriate cDNA and a 5 μM concentration of forward and reverse primers and SYBR Green PCR Master Mix (Applied Biosystems). The PCR protocol consisted of 2 minutes at 50°C and 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. PCR signals were quantified by calculating the difference between the Ct value for the gene of interest and the Ct value for the reference gene GAPDH for each sample (ΔCt).

Immunohistochemistry.

Local expression of SOCS-3 was evaluated in paraffin sections of the knee joints. Sections were deparaffinized and rehydrated. Endogenous peroxidase was blocked for 15 minutes with 1% hydrogen peroxide. Tissue sections were incubated overnight with anti–SOCS-3 (Abbiotec), followed by incubation with biotinylated goat anti-rabbit IgG. Peroxidase-labeled streptavidin (Vectastain; Vector) and diaminobenzidine were used for staining. Hematoxylin was used as a counterstain. At least 2 sections per joint were arbitrarily scored on a scale of 0–3, where 0 = normal and 3 = most intense staining.

Assessment of anticollagen antibody titers.

Concentrations of anti–bovine type II collagen antibodies types IgG1 and IgG2a were determined by enzyme-linked immunosorbent assay. Briefly, 96-well plates were coated with 0.1 μg of bovine type II collagen. Nonspecific binding sites were blocked with a 5% solution of powdered milk. Serial dilutions of mouse sera were added, followed by incubation with isotype-specific goat anti-mouse antibodies (peroxidase labeled) and 5-aminosalicylic acid as substrate. Absorbance was measured at 450 nm.

Flow cytometric analysis of splenic T cells.

Spleens from mice were mashed and filtered, and erythrocytes were removed by osmotic shock. After washing, CD3+ cells were isolated using a pan–T cell isolation kit (Miltenyi Biotec) according to the manufacturer's instructions. Purified CD3+ cells were stimulated in RPMI 1640 (Invitrogen) supplemented with 10% fetal calf serum, penicillin/streptomycin, and pyruvate, with 50 ng/ml of phorbol myristate acetate, Brefeldin A (1 μl/ml/106 cells; BD PharMingen), and 1 μg/ml of ionomycin. After 4 hours of stimulation, cells were stained with allophycocyanin-conjugated anti-mouse CD4 (1:200 dilution) or allophycocyanin-conjugated IgG2a (control) (both from BD PharMingen) for 30 minutes at 4°C. Cells were subsequently stained intracellularly with fluorescein isothiocyanate (FITC)–conjugated anti–IL-17 and PE-conjugated anti–interferon-γ (anti-IFNγ) or with their respective controls, PE-conjugated IgG1 and FITC-conjugated IgG1, according to the manufacturer's instructions (BD PharMingen). Stained cells were analyzed using a FACSCalibur with FlowJo software (Becton Dickinson).

Fluorescence imaging.

On day 28 of CIA, mice received an intravenous injection with activatable ProSense 680 near-infrared fluorescent probe (150 μl, 2 nmoles; PerkinElmer). The probe becomes activated upon enzymatic cleavage by cathepsins (B, K, L, and S) that are present in arthritic joints. Cathepsin activity is a measure of inflammation, especially macrophage infiltration. At the time of imaging (24 hours after intravenous injection), mice were anesthetized with 2.5% isoflurane in oxygen, the knee joints were shaved, and mice were placed on their back inside the light-tight chamber and imaged with the Lumina In Vivo Imaging System (Caliper Life Sciences), using a Cy5.5 filter. The collected data were analyzed using Living Image 3.0 software (Caliper Life Sciences). Two-dimensional regions of interest were drawn around the knee and ankle joints, and the fluorescent signal intensity was measured and then corrected for background and autofluorescence signals.

Measurement of serum IL-6 and keratinocyte-derived chemokine (KC) levels.

IL-6 and KC levels in serum were measured with a Luminex 100 system, using a magnetic bead–based multiplex immunoassay (Milliplex; Merck Millipore). Data analysis was performed with Bio-Plex Manager software (Bio-Rad).

Statistical analysis.

All data are represented as the mean ± SEM and were analyzed with the use of GraphPad 5.0 software. Statistical significance was determined by either one-way analysis of variance (ANOVA) or two-way ANOVA with the Bonferroni post hoc test, comparing AdGas6 and AdPros1 groups with the AdLuc group.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Moderately reduced arthritis severity following systemic overexpression of GAS-6 and protein S.

Adenoviruses expressing luciferase, GAS-6, or protein S were administered intravenously to mice that had been immunized with bovine type II collagen. As shown in Figure 1, overexpression of either GAS-6 or protein S did not affect the incidence of arthritis. However, arthritis severity was slightly reduced when GAS-6 was overexpressed. Moreover, protein S treatment resulted in a significant decrease in arthritis severity. In addition to scoring the macroscopic swelling and redness of the joints, the knee joints were isolated to enable detailed examination of the effects of TAM activation on cell influx as well as on bone and cartilage. This revealed a trend toward decreased inflammation, cartilage erosion, and bone erosion when GAS-6 or protein S was overexpressed systemically (Figures 1B and C). These data indicate a protective role of TAM activation in experimental arthritis.

thumbnail image

Figure 1. Effect of systemically overexpressed growth arrest–specific protein 6 (GAS-6) or protein S on arthritis. Mice with collagen-induced arthritis (CIA) were intravenously injected with adenovirus encoding for luciferase (Luc), GAS-6 (Gas6), or protein S (Pros1) after immunization, and arthritis development was monitored over time. A, Arthritis incidence and severity in the hind paws of mice treated with the indicated adenovirus. Severity was determined macroscopically (see Materials and Methods for details). Shown are the mean cumulative scores for the ankle joints per animal. Values are the mean ± SEM of 9 mice per group. ∗ = P < 0.05 versus the Ad-Luc group, by two-way analysis of variance with the Bonferroni post hoc test. B, Histologic analysis of knee joints for inflammation (hematoxylin and eosin–stained sections) and bone and cartilage erosion (Safranin O–stained sections), scored as described in Materials and Methods. Values are the mean ± SEM of 9 mice per group. C, Representative images of Safranin O–stained sections of knee joints from mice treated with the indicated adenovirus, showing cartilage erosions (solid arrows) and bone erosions (open arrows). P = patella; F = femur. Bars = 100 μm. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

Download figure to PowerPoint

Suppression of the proinflammatory immune response by systemic overexpression of GAS-6 and protein S.

To study the effects of GAS-6 and protein S overexpression on macrophage activity, serum was evaluated for circulating levels of cytokines. TLR-inducible cytokines IL-6 and KC were detected in serum, and overexpression of GAS-6 and protein S significantly reduced the circulating levels of IL-6 (by 59% and 78%, respectively). In addition, protein S caused a 68% decline in circulating KC levels (Figure 2A), which potentially explains a decrease in inflammatory cell influx into the inflamed joints. Moreover, serum IL-6 and KC levels were significantly correlated with macroscopic arthritis scores (r2 = 0.41, P = 0.001 for IL-6 and r2 = 0.33, P = 0.004 for KC). This indicates that GAS-6 and protein S decrease the levels of systemically produced cytokines under inflammatory conditions and possibly control antigen-presenting cell (APC) activation and function.

thumbnail image

Figure 2. Analysis of serum cytokine levels, antibody titers, and splenic Th1 and Th17 cell numbers in mice with CIA injected intravenously with adenovirus encoding for luciferase, GAS-6, or protein S. A, Serum levels of interleukin-6 (IL-6) and keratinocyte-derived chemokine (KC) on day 36 of CIA, as measured using a multiplex bead array system. B, Serum levels of IgG1 and IgG2a anti–bovine type II collagen antibodies (abCII), as determined by enzyme-linked immunosorbent assay. C, Percentages of Th1 (CD4+/interferon-γ–positive) and Th17 (CD4+/IL-17+) cells in spleens obtained on day 36 of CIA, as determined by flow cytometry, with gating on CD4+ T cells. D, Relative expression (2math image) of mRNA for the transcription factors T-bet (Th1) and retinoic acid receptor–related orphan nuclear receptor γt (RORγt) (Th17) in spleens obtained on day 36 of CIA, as determined by polymerase chain reaction analysis. Values are the mean ± SEM of 9 mice per group. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus the Ad-Luc group, by one-way analysis of variance with the Bonferroni post hoc test. See Figure 1 for other definitions.

Download figure to PowerPoint

To investigate the systemic effects of TAM ligand overexpression on B cells, titers of antibody against bovine type II collagen were determined (Figure 2B). Neither GAS-6 nor protein S had an effect on the titers of type II collagen–specific IgG1 or IgG2a antibodies. This suggests that TAM ligands did not influence B cell function. To further analyze the effects of GAS-6 and protein S overexpression on adaptive immunity, splenic CD3+ cells were isolated, and T cell differentiation was determined. TAM receptor stimulation significantly reduced the levels of Th1 cells, whereas the levels of Th17 cells were unaffected by either treatment (Figure 2C). Similarly, the expression of messenger RNA (mRNA) for T-bet was decreased significantly, but the expression of retinoic acid receptor–related orphan nuclear receptor γt (RORγt) was unchanged (Figure 2D). This indicates that TAM activation has a clear effect on T cell immunity by diminishing the development of Th1 cells, resulting in a reduction of arthritis severity.

Decreased inflammation and joint pathology following local overexpression of TAM ligands.

GAS-6 and protein S showed clear effects on Th1 cell development, but failed to significantly ameliorate inflammation and joint pathology. To study the effects of GAS-6 and protein S directly at the site of inflammation, adenoviruses were injected intraarticularly into both knee joints prior to the onset of CIA. During arthritis development, inflammation was measured with a ProSense probe on day 29 (Figure 3A). TAM activation significantly reduced inflammation in treated knee joints. Further analysis of inflammation, cartilage destruction, and bone destruction revealed that TAM activation was beneficial in halting joint destruction (Figures 3B and C). Inflammation of the untreated ankle joints was unaltered by either treatment (data not shown), indicating that TAM activation occurred only locally in the knee joint. This indicates that TAM activation directly at the site of inflammation can be applied for the treatment of inflammatory diseases.

thumbnail image

Figure 3. Effects of locally overexpressed GAS-6 and protein S on joint inflammation and histologic features. Mice were given an intraarticular injection of adenovirus encoding for luciferase, GAS-6, or protein S into both knee joints prior to the onset of CIA, and arthritis development was monitored over time. A, Knee inflammation on day 29 of CIA, as measured with a cathepsin-inducible fluorescent probe (scored with a Lumina In Vivo Imaging System [IVIS]), and knee swelling on day 31 of CIA, determined macroscopically (scored on a 0–2 scale). B, Histologic analysis of knee joint sections on day 31 for inflammation (hematoxylin and eosin staining), cartilage erosion (Safranin O staining), and bone erosion (Safranin O staining). Values are the mean ± SEM of 20 mice per group for knee inflammation and 10 mice per group for knee swelling in A, and 14 mice per group for histologic analyses in B. ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus the Ad-Luc group, by one-way analysis of variance with the Bonferroni post hoc test. C, Representative images of Safranin O–stained sections of knee joints from mice treated with the indicated adenovirus, showing cartilage erosions (solid arrows) and bone erosions (open arrows). P = patella; F = femur. Bars = 100 μm. See Figure 1 for other definitions. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

Download figure to PowerPoint

Analysis of mRNA expression in synovium showed that both GAS-6 and protein S were up-regulated 2 days after virus injection (data not shown). Further analysis revealed that both GAS-6 and protein S reduced the expression of matrix metalloproteinases (MMPs) in synovium (Figures 4A–C). GAS-6 and protein S significantly reduced MMP-13 mRNA expression, whereas MMP-9 expression was significantly diminished by overexpressing protein S and MMP-14 by overexpressing GAS-6. Taken together, these data show that direct, local activation of TAM in inflamed joints decreases joint destruction by reducing the expression of MMPs.

thumbnail image

Figure 4. Matrix metalloproteinase (MMP) expression in the synovium of mice treated with local injections of adenovirus encoding for luciferase, GAS-6, or protein S into both knee joints prior to the onset of CIA. The relative expression (2math image) of mRNA for A, MMP-9, B, MMP-13, and C, MMP-14 was determined in synovium harvested on day 24 and day 31 of CIA. Values are the mean ± SEM of 4 mice per group for day 24 analyses and 6 mice per group for day 31. ∗ = P < 0.05; ∗∗ = P < 0.01 versus the Ad-Luc group, by two-way analysis of variance with the Bonferroni post hoc test. See Figure 1 for other definitions.

Download figure to PowerPoint

Decreased cytokine production in synovium due to GAS-6 and protein S overexpression.

To study the effects of TAM activation on local cytokine production prior to clinical manifestations of arthritis, synovium was isolated on day 24 of CIA. Interestingly, TNFα production was detected before any clinical manifestation and was significantly inhibited by protein S (87%) and GAS-6 (62%). IL-1β and IL-6 were only marginally produced on day 24, but were markedly induced by day 31, when synovitis had occurred (Figure 5A). GAS-6 and protein S overexpression decreased the production of IL-1β by inflamed synovium on day 31 of CIA (by 65% and 78% respectively). In addition, IL-6 production returned to near basal expression levels by overexpression of GAS-6 and protein S, as indicated by the significant reduction in IL-6 mRNA expression (74% and 92%, respectively).

thumbnail image

Figure 5. Cytokine expression in the synovium of mice treated with local injections of adenovirus encoding for luciferase, GAS-6, or protein S into both knee joints prior to the onset of CIA. Synovium was isolated 2 days after virus injection (day 24 of CIA) and at the end of the study (day 31 of CIA), and mRNA was determined by quantitative polymerase chain reaction analysis. A, Expression of mRNA for tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and IL-6 on days 14 and 31 in the 3 treatment groups. Values are the mean ± SEM of 4 mice per group for day 24 analyses and 6 mice per group for day 31. ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus the Ad-Luc group, by two-way analysis of variance (ANOVA) with the Bonferroni post hoc test. B–D, Expression of mRNA for cytokines IL-12p35, IL-12p40, and IL-23p19 (B), cytokines interferon-γ (IFNγ) and IL-17 (C), and transcription factors T-bet and retinoic acid receptor–related orphan nuclear receptor γt (RORγt) (D) on day 31 of CIA. Values are the mean ± SEM of 6 mice per group. ∗ = P < 0.05; ∗∗ = P < 0.01 versus the Ad-Luc group, by one-way ANOVA with the Bonferroni post hoc test. See Figure 1 for other definitions.

Download figure to PowerPoint

We also observed antiinflammatory effects of GAS-6 and protein S on the production of the T cell–activating cytokines IL-12 and IL-23. Overexpression of GAS-6 and protein S caused a decline in IL-12 and IL-23 production in the synovium (Figure 5B) and resulted in reduced levels of IFNγ and IL-17 in the synovium (Figure 5C). In addition, expression of mRNA for the T cell transcription factors T-bet and RORγt, which are responsible for the development of Th1 cells and Th17 cells, respectively, was significantly reduced by GAS-6 and protein S (Figure 5D). These cytokine expression profiles support the findings of reduced pathologic changes in the joint, since IL-1 and IL-17 are key factors in cartilage and bone destruction. These data show that local TAM activation by GAS-6 and protein S reduces proinflammatory cytokine production in inflamed synovium. This probably led to subsequently hampered T cell activation and proliferation at sites of inflammation.

SOCS-mediated antiinflammatory effects of GAS-6 and protein S.

To unravel the inhibitory mechanism of TAM receptor stimulation, we evaluated the expression of mRNA for SOCS-1 and SOCS-3 (Figure 6A). SOCS-1 mRNA expression was up-regulated 2.3-fold in the synovium of mice injected with GAS-6 or protein S, whereas control animals showed a slight down-regulation. In contrast, SOCS-3 mRNA expression was marginally affected by GAS-6 overexpression and was even slightly down-regulated by protein S overexpression. Since this is in contrast to previously reported findings (5), we sought to determine SOCS-3 protein levels by immunohistochemistry. Representative images of SOCS-3 staining shown in Figure 6B reveal a clear trend in the up-regulation of SOCS-3 protein by GAS-6 and protein S (Figure 6C). This suggests that SOCS-1 and SOCS-3 mediate the antiinflammatory effects of TAM activation by GAS-6 and protein S.

thumbnail image

Figure 6. GAS-6 and protein S up-regulation of suppressor of cytokine signaling (SOCS) proteins in the synovium of mice treated with local injections of adenovirus encoding for luciferase, GAS-6, or protein S into both knee joints prior to the onset of CIA. Synovium was isolated 2 days after virus injection (day 24 of CIA) and at the end of the study (day 31 of CIA). A, Fold increase in expression of mRNA for SOCS-1 and SOCS-3 on day 31 of CIA as compared to day 24 of CIA. Values are the mean ± SEM of 4 mice per group on day 24 and 6 mice per group on day 31. ∗ = P < 0.05; ∗∗∗ = P < 0.001 versus the Ad-Luc group, by one-way analysis of variance with the Bonferroni post hoc test. B, Representative images showing immunohistochemistry staining for SOCS-3 (solid arrows) in sections of knee joints from mice treated with the indicated adenovirus. P = patella; F = femur. Bars = 100 μm. C, SOCS-3 staining scores (0–3 scale) in mice injected with the indicated adenovirus. Values are the mean ± SEM of 14 mice per group. See Figure 1 for other definitions. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

A novel inhibitory pathway for TLR and cytokine signaling by TAM receptor activation was used in this study to inhibit experimental arthritis. We found that enhancing the negative feedback for inflammation by activating TAM receptors could be used to treat arthritis in a prophylactic setting. Systemic overexpression of protein S affected the T cell immune response by decreasing Th1 cell numbers, which had a moderately ameliorating effect on experimental arthritis. Intraarticular overexpression of GAS-6 and protein S reduced proinflammatory cytokine production in synovium, which was likely mediated by SOCS-1 and SOCS-3. GAS-6 also significantly decreased joint destruction when overexpressed in the inflamed joint. This study is the first to show that TAM receptor activation by GAS-6 and protein S in vivo ameliorates arthritis. This puts the TAM pathway forward as a new therapeutic pathway to be exploited for the treatment of arthritis.

In our study, protein S overexpression decreased splenic Th1 cells by 40% while leaving Th17 levels unaffected. This is consistent with previous studies in Axl and Mer double-knockout animals (16). Naive splenic CD4+ T cells from double-knockout mice show a remarkable increase in IFNγ production when stimulated with anti-CD3 and anti-CD28, with no change in IL-17 production. In addition, immunized double- knockout mice show increased development of Th1 cells and normal levels of Th17 cells in spleen and draining lymph nodes (16). In animals that lack the Mer receptor on the diabetes-prone NOD background, a strong Th1 response was observed when beta cells underwent apoptosis (17). Combined with our data, it appears that TAM activation on APCs primarily affects the Th1 cell response in vivo while not influencing the Th17 cell response. Since circulating IL-6 levels were significantly decreased by GAS-6 or protein S overexpression in our study, an effect on Th17 cells could be expected. However, previous studies have shown that GAS-6 can regulate TGFβ expression. Clauser et al (18) showed that increased GAS-6 secretion from carotid plaques correlated with increased TGFβ secretion. In addition, GAS-6–knockout animals produce less TGFβ upon induction of liver damage (19). If GAS-6, and perhaps protein S, increase TGFβ levels, this could compensate for the reduced IL-6 levels and leaving Th17 cell levels unaffected.

GAS-6 and protein S appeared to have differential effects, depending on local or systemic overexpression. When overexpressed systemically, protein S seemed slightly more efficacious than GAS-6, and when overexpressed locally, the reverse effect was observed. But, no significant differences between the effects of GAS-6 and protein S on CIA were found. The trends observed between GAS-6 and protein S could be attributable to different target cells. Systemically overexpressed TAM ligands will affect systemic adaptive immunity by APC activity modulation in the spleen, which was also observed in our study. At the site of inflammation, however, TAM ligands are expressed and secreted into the joint cavity, affecting all the cells present, such as infiltrated macrophages, T cells, and the synovial lining. Fibroblasts in the synovial lining are active contributors to the inflammation (20), and the effects of TAM ligands and TAM receptor expression on synovial fibroblasts is unknown and warrants further investigation.

The antiinflammatory effects of TAM receptors have been reported to be mediated by SOCS-1 and SOCS-3 (5, 6). Rothlin et al (5) found that stimulation of the Axl receptor in conjunction with IFNAR-1 led to up-regulation of SOCS-1 and SOCS-3 in dendritic cells, which interfere with intracellular signaling and NF-κB activation. The effects of local GAS-6 or protein S overexpression appear to be mediated via SOCS-1 and SOCS-3. Overexpression resulted in up-regulation of SOCS-1 expression during arthritis, whereas control animals without CIA showed a slight down-regulation of SOCS-1.

The pivotal role of SOCS-1 in controlling inflammation has been shown in macrophages from SOCS-1 conditional-knockout animals, in which TNFα and IL-6 expression was down-regulated upon lipopolysaccharide (LPS) challenge (21). In our study, we also observed a decrease in proinflammatory cytokine production in synovium by overexpressing GAS-6 or protein S in the joint cavity. In contrast to SOCS-1 up-regulation, little regulation of SOCS-3 mRNA by TAM receptor activation was found. However, immunohistologic staining revealed a trend toward increased SOCS-3 protein levels after GAS-6 or protein S overexpression. SOCS-3 mRNA levels are partly controlled by TNFα (22) and IL-6 (23), for which we found significant differences on day 24 and day 31 of CIA, respectively. Therefore, mRNA expression at the time of euthanasia could deviate from the protein levels. In addition, cytokine signaling has been suggested to prevent SOCS-3 turnover (24). The increase in SOCS-1 and SOCS-3 are also consistent with previous studies (5) showing the involvement of SOCS-1 and SOCS-3 in TAM-mediated down-regulation of inflammation. Taken together, a significant increase in SOCS-1 mRNA in synovium and a clear trend in increased SOCS-3 protein could partly account for the antiinflammatory effects of GAS-6 and protein S we observed.

Another possible mechanism by which GAS-6 and protein S exert their antiinflammatory effects is by inducing phagocytosis. GAS-6 and protein S can opsonize apoptotic cells by binding to phosphatidylserine displayed on apoptotic cells. It has previously been shown that joint inflammation can be reduced by prophylactic injection of apoptotic cells directly into the joint (25). Clearance of apoptotic leukocytes by lining layer macrophages decreases their chemotactic activity and thereby limits inflammation. Mer is predominantly involved in phagocytosis (26) and plays a role in inflammation as well. It has been shown that Mer down-regulates TNFα production upon LPS stimulation (27) and is also involved in LPS-induced lung injury (6). Both GAS-6 and protein S are ligands for the Mer receptor and could therefore increase TAM signaling via apoptotic cells or via direct stimulation of the Mer receptor on macrophages. However, the exact role of exogenous GAS-6 and protein S in mediating phagocytosis of apoptotic cells to facilitate resolution of joint inflammation needs further investigation.

Axl and GAS-6 have been implicated in the maintenance of abnormal vasculature in RA (28) and thereby contribute to inflammation. Here, we show that the net effect of increasing TAM signaling is beneficial in experimental arthritis. TAM ligands could potentially induce SOCS-1 and SOCS-3 expression in the RA synovium in humans and thereby decrease inflammation. With decreasing inflammation, the process of angiogenesis will also be halted, and TAM stimulation by GAS-6 or protein S could potentially be used to treat RA by controlling inflammation, irrespective of its putative effect on angiogenesis.

In summary, we provide herein the first evidence that enhancing natural negative feedback for inflammation by TAM stimulation is efficacious in the treatment of inflammatory arthritis. TAM receptors and their ligands GAS-6 and protein S offer many possibilities and options for fine-tuning the negative feedback for inflammation in order to resolve autoinflammatory and autoimmune diseases.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. van de Loo had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Van den Brand, Abdollahi-Roodsaz, van de Loo.

Acquisition of data. Van den Brand, Vermeij, Bennink, Arntz.

Analysis and interpretation of data. Van den Brand, Abdollahi-Roodsaz, Vermeij, Bennink, Rothlin, van den Berg, van de Loo.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We would like to thank Richard Huijbens for performing the Luminex assays.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
  • 1
    Lu Q, Lemke G. Homeostatic regulation of the immune system by receptor tyrosine kinases of the Tyro 3 family. Science 2001; 293: 30611.
  • 2
    Alciato F, Sainaghi PP, Sola D, Castello L, Avanzi GC. TNF-α, IL-6, and IL-1 expression is inhibited by GAS6 in monocytes/macrophages. J Leukoc Biol 2010; 87: 86975.
  • 3
    Stitt TN, Conn G, Gore M, Lai C, Bruno J, Radziejewski C, et al. The anticoagulation factor protein S and its relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine kinases. Cell 1995; 80: 66170.
  • 4
    Uehara H, Shacter E. Auto-oxidation and oligomerization of protein S on the apoptotic cell surface is required for Mer tyrosine kinase-mediated phagocytosis of apoptotic cells. J Immunol 2008; 180: 252230.
  • 5
    Rothlin CV, Ghosh S, Zuniga EI, Oldstone MB, Lemke G. TAM receptors are pleiotropic inhibitors of the innate immune response. Cell 2007; 131: 112436.
  • 6
    Lee YJ, Han JY, Byun J, Park HJ, Park EM, Chong YH, et al. Inhibiting Mer receptor tyrosine kinase suppresses STAT1, SOCS1/3, and NF-κB activation and enhances inflammatory responses in lipopolysaccharide-induced acute lung injury. J Leukoc Biol 2012; 91: 92132.
  • 7
    Yoshimura A, Naka T, Kubo M. SOCS proteins, cytokine signalling and immune regulation. Nat Rev Immunol 2007; 7: 45465.
  • 8
    Radstake TR, Roelofs MF, Jenniskens YM, Oppers-Walgreen B, van Riel PL, Barrera P, et al. Expression of Toll-like receptors 2 and 4 in rheumatoid synovial tissue and regulation by proinflammatory cytokines interleukin-12 and interleukin-18 via interferon-γ. Arthritis Rheum 2004; 50: 385665.
  • 9
    Abdollahi-Roodsaz S, van de Loo FA, van den Berg WB. Trapped in a vicious loop: Toll-like receptors sustain the spontaneous cytokine production by rheumatoid synovium. Arthritis Res Ther 2011; 13: 105.
  • 10
    Abdollahi-Roodsaz S, Joosten LA, Roelofs MF, Radstake TR, Matera G, Popa C, et al. Inhibition of Toll-like receptor 4 breaks the inflammatory loop in autoimmune destructive arthritis. Arthritis Rheum 2007; 56: 295767.
  • 11
    Abdollahi-Roodsaz S, Joosten LA, Koenders MI, Devesa I, Roelofs MF, Radstake TR, et al. Stimulation of TLR2 and TLR4 differentially skews the balance of T cells in a mouse model of arthritis. J Clin Invest 2008; 118: 20516.
  • 12
    Baetz A, Frey M, Heeg K, Dalpke AH. Suppressor of cytokine signaling (SOCS) proteins indirectly regulate toll-like receptor signaling in innate immune cells. J Biol Chem 2004; 279: 5470815.
  • 13
    Jacobsen ML, Ronn SG, Bruun C, Larsen CM, Eizirik DL, Mandrup-Poulsen T, et al. IL-1β-induced chemokine and Fas expression are inhibited by suppressor of cytokine signalling-3 in insulin-producing cells. Diabetologia 2009; 52: 2818.
  • 14
    Veenbergen S, Bennink MB, de Hooge AS, Arntz OJ, Smeets RL, van den Berg WB, et al. Splenic suppressor of cytokine signaling 3 transgene expression affects T cell responses and prevents development of collagen-induced arthritis. Arthritis Rheum 2008; 58: 374252.
  • 15
    Chartier C, Degryse E, Gantzer M, Dieterle A, Pavirani A, Mehtali M. Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli. J Virol 1996; 70: 480510.
  • 16
    Ye F, Han L, Lu Q, Dong W, Chen Z, Shao H, et al. Retinal self-antigen induces a predominantly Th1 effector response in Axl and Mertk double-knockout mice. J Immunol 2011; 187: 417886.
  • 17
    Wallet MA, Sen P, Flores RR, Wang Y, Yi Z, Huang Y, et al. MerTK is required for apoptotic cell-induced T cell tolerance. J Exp Med 2008; 205: 21932.
  • 18
    Clauser S, Meilhac O, Bieche I, Raynal P, Bruneval P, Michel JB, et al. Increased secretion of Gas6 by smooth muscle cells in human atherosclerotic carotid plaques. Thromb Haemost 2012; 107: 1409.
  • 19
    Fourcot A, Couchie D, Chobert MN, Zafrani ES, Mavier P, Laperche Y, et al. Gas6 deficiency prevents liver inflammation, steatohepatitis, and fibrosis in mice. Am J Physiol Gastrointest Liver Physiol 2011; 300: G104353.
  • 20
    Ospelt C, Brentano F, Rengel Y, Stanczyk J, Kolling C, Tak PP, et al. Overexpression of Toll-like receptors 3 and 4 in synovial tissue from patients with early rheumatoid arthritis: Toll-like receptor expression in early and longstanding arthritis. Arthritis Rheum 2008; 58: 368492.
  • 21
    Sachithanandan N, Graham KL, Galic S, Honeyman JE, Fynch SL, Hewitt KA, et al. Macrophage deletion of SOCS1 increases sensitivity to LPS and palmitic acid and results in systemic inflammation and hepatic insulin resistance. Diabetes 2011; 60: 202331.
  • 22
    Ehlting C, Lai WS, Schaper F, Brenndorfer ED, Matthes RJ, Heinrich PC, et al. Regulation of suppressor of cytokine signaling 3 (SOCS3) mRNA stability by TNF-α involves activation of the MKK6/p38MAPK/MK2 cascade. J Immunol 2007; 178: 281326.
  • 23
    Yang XP, Schaper F, Teubner A, Lammert F, Heinrich PC, Matern S, et al. Interleukin-6 plays a crucial role in the hepatic expression of SOCS3 during acute inflammatory processes in vivo. J Hepatol 2005; 43: 70410.
  • 24
    Babon JJ, McManus EJ, Yao S, DeSouza DP, Mielke LA, Sprigg NS, et al. The structure of SOCS3 reveals the basis of the extended SH2 domain function and identifies an unstructured insertion that regulates stability. Mol Cell 2006; 22: 20516.
  • 25
    Van Lent PL, Licht R, Dijkman H, Holthuysen AE, Berden JH, van den Berg WB. Uptake of apoptotic leukocytes by synovial lining macrophages inhibits immune complex-mediated arthritis. J Leukoc Biol 2001; 70: 70814.
  • 26
    Scott RS, McMahon EJ, Pop SM, Reap EA, Caricchio R, Cohen PL, et al. Phagocytosis and clearance of apoptotic cells is mediated by MER. Nature 2001; 411: 20711.
  • 27
    Camenisch TD, Koller BH, Earp HS, Matsushima GK. A novel receptor tyrosine kinase, Mer, inhibits TNF-α production and lipopolysaccharide-induced endotoxic shock. J Immunol 1999; 162: 3498503.
  • 28
    O'Donnell K, Harkes IC, Dougherty L, Wicks IP. Expression of receptor tyrosine kinase Axl and its ligand Gas6 in rheumatoid arthritis: evidence for a novel endothelial cell survival pathway. Am J Pathol 1999; 154: 117180.