ARTHRITIS & RHEUMATOLOGY Vol. 67, No. 4, April 2015, pp 903–913 DOI 10.1002/art.38996 © 2015, American College of Rheumatology

Mast Cell Promotion of T Cell–Driven Antigen-Induced Arthritis Despite Being Dispensable for Antibody-Induced Arthritis in Which T Cells Are Bypassed Nadja Schubert,1 Jan Dudeck,1 Peng Liu,2 Anna Karutz,1 Stephan Speier,3 Marcus Maurer,4 Jan Tuckermann,2 and Anne Dudeck1 and effector T cells. In addition to diminished joint inflammation in the absence of MCs, we found a dramatic loss of T cell expansion upon immunization, accompanied by reduced T cell cytokine responses. Conclusion. In this analysis of an arthritis model in which the cellular arm of adaptive immunity was not bypassed, we identified MCs as important promoters of T cell–conditioned autoimmune disorders and, consequently, as potential therapeutic targets in rheumatoid arthritis.

Objective. The function of mast cells (MCs) in autoimmune disorders has been a subject of controversy recently. MC-deficient KitW/W-v mice were found to be resistant to K/BxN serum–transfer arthritis, whereas KitW-sh/W-sh mice and a genetic model of MC deficiency independent of the Kit mutation were found to be fully susceptible. This debate might lead to the assumption that MCs are dispensable in autoimmunity in general. Thus, the purpose of this study was to examine the relevance of MCs to arthritis using a genetic model of inducible MC deficiency without compromised Kit signaling. Methods. We compared MC functions in K/BxN serum–induced arthritis and in collagen-induced arthritis (CIA) in a mouse model of inducible MC deficiency by analyzing joint inflammation, parameters of cartilage degradation and bone erosion, and the autoreactive adaptive immune response. Results. We observed a redundant role of MCs in K/BxN serum–induced arthritis, where joint inflammation is triggered by cartilage-bound immune complexes independently of T cells. In contrast, we found MCs to be critically relevant in CIA, which is provoked by two arms of autoimmune attack: autoreactive antibodies

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease with progressive cartilage and bone destruction in the proximal joints. Massive infiltration of immune cells into the joint is accompanied by extensive hyperplasia of synovial macrophages. Consequently, an invasive structure, called synovial pannus, is formed that destroys cartilage and bone. Numerous studies have demonstrated an impressively expanded population of mast cells (MCs) in the inflamed synovium of RA patients, constituting as much as 5% of synovial cells (1–5). MC accumulation has been found especially around blood vessels in the synovial sublining, at the cartilage–pannus junction at sites of cartilage erosions, and in joint fluid (1,5,6). Increased numbers of MCs are closely correlated to the clinical activity of the disease. Moreover, profound MC activation has been observed in RA tissues in association with increased levels of various MC mediators, including histamine, proteases, and cytokines (7–10). Among the variety of animal models used in the study of RA, the two most common ones are serumtransfer–induced arthritis (STIA) using sera from K/BxN mice and collagen-induced arthritis (CIA) in mice (11). K/BxN mice (i.e., F1 offspring of T cell receptor–transgenic KRN mice crossed with NOD mice) spontaneously develop progressive arthritis that is me-

Supported by the German Research Foundation (DFG Priority Program 1468 grants Tu220/6 to Dr. Tuckermann and DU1172/2 to Dr. A. Dudeck, and Priority Program 1394 grant DU1172/3 to Dr. A. Dudeck). 1 Nadja Schubert, Dipl Biol, Jan Dudeck, Dipl Mineral, Anna Karutz, MSc, Anne Dudeck, PhD: Technische Universität Dresden, Dresden, Germany; 2Peng Liu, MSc, Jan Tuckermann, PhD: University of Ulm, Ulm, Germany; 3Stephan Speier, PhD: Technische Universität Dresden and German Center for Diabetes Research, Dresden, Germany; 4Marcus Maurer, MD: Charite´Universitätsmedizin Berlin, Berlin, Germany. Address correspondence to Anne Dudeck, PhD, Institute for Immunology, Technische Universität Dresden, Medical Faculty Carl Gustav Carus, Fetscherstrasse 74, 01307 Dresden, Germany. E-mail: [email protected]. Submitted for publication September 22, 2014; accepted in revised form December 9, 2014. 903

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diated by autoantibodies directed against glucose-6phosphate isomerase. Serum from arthritic K/BxN mice can passively transfer disease to recipient mice of almost any background, where joint inflammation is induced by cartilage-bound antigen–antibody immune complexes, subsequently activating innate immune cells (12–14). In the CIA model of antigen-induced arthritis, joint inflammation is elicited by immunization with type II collagen (CII) emulsified in Freund’s complete adjuvant (CFA). This immunization generates autoreactive CII-specific effector T cells, which attack the joints and cause inflammation via antibody cross-reactivity with cartilage-restricted CII (15), as well as CII-specific autoantibodies. Joint inflammation in CIA is therefore provoked by two arms of adaptive immunity—T cells and autoantibodies—that subsequently activate synovial macrophages, leading to cartilage degradation and bone erosion in a T cell–dependent way (15,16). The relevance of MCs to experimental arthritis has been a topic of controversy. Several studies found MCs to be essential for the induction of STIA in MCdeficient KitW/W-v mice (17,18). Contradictory results were obtained in a study of another antibody-mediated arthritis model, in which MC-deficient KitW-sh/W-sh mice, but not KitW/W-v mice, developed disease (19). Recently, Feyerabend et al (20) disproved MC functions in STIA by the use of a novel genetic approach to MC deficiency that is independent of Kit mutations. Since Kit is expressed on hematopoietic stem cells and almost all myeloid progenitor cells, mice with Kit mutations exhibit severe alterations of the immune system beyond MC deficiency. Therefore, the in vivo function of MCs, which were determined in Kit-mutant mouse lines, should be reinvestigated in mouse models that are independent of compromised Kit signaling. In this study, we used Mcpt5-Cre;iDTR mice as a Kit-independent model of inducible MC deficiency (21–23) to analyze MC functions in antibody-induced arthritis and in T cell–driven antigen-induced arthritis. MATERIALS AND METHODS Mice. Mcpt5-Cre mice crossed with the iDTR or R26tdRFP line, C57BL/6 mice, C57BL/6 KitW-sh/W-sh mice, KRN mice, NOD mice, and B6.CD4-GFP mice were housed under specific pathogen–free conditions at the Experimental Centre, Technische Universität Dresden. Mcpt5-Cre mice were kindly provided by Axel Roers (Institute for Immunology, Technische Universität Dresden, Dresden, Germany). KRN mice were kindly provided by Diane Mathis and Christophe Benoist (Harvard Medical School, Boston, MA). NOD mice, KitW-sh/W-sh mice (B6.Cg-KitW-sh/HNihrJaeBsmGlliJ), and B6.CD4-GFP mice (B6.NOD-Tg[Cd4-EGFP]1Lt/J) for breeding were purchased from The Jackson Laboratory.

Cre–;iDTR⫹ littermates were used as the control for MCdepleted Mcpt5-Cre⫹;iDTR⫹ mice. Age- and sex-matched C57BL/6 wild-type mice were used as controls for the KitW-sh/W-sh mice. In all experiments, mice were ages 8–16 weeks when used in the experiments. All procedures were performed in accordance with institutional guidelines on animal welfare and were approved by the Landesdirektion Dresden (no. 11-1/2009-34). Chemicals. CII, CFA, Freund’s incomplete adjuvant (IFA), and diphtheria toxin (DT) were purchased from Sigma. T cell proliferation–grade CII was obtained from Chondrex. The following monoclonal antibodies directed against mouse antigens were obtained from eBioscience: CD11c (clone N418), CD4 (clone RM4-5), CD8a (clone 53-6.7), CD19 (clone eBio1D3), CD3 (clone 145-2C11), MHCII I-A/I-E (clone M5/114.15.2), CD117 (clone 2B8), and Fc␧ receptor type I (Fc␧RI; clone MAR-1). Purified antibodies directed against CD3 (clone 17A2) and CD28 (clone 37.51) (both from eBioscience) were used for restimulation of lymph node (LN) cells. Mast cell depletion. For complete ablation of peripheral connective tissue–type MCs, Mcpt5-Cre mice were crossed with mice of the iDTR line (24), and litters were given an intraperitoneal injection of DT (25 ng/gm of body weight) at weekly intervals for 4 weeks before all of the experimental protocols (22). Cre–;iDTR⫹ littermates were treated in the same manner and served as controls for the MC-depleted Mcpt5-Cre⫹;iDTR⫹ mice. DT treatment was continued over the whole period of the experiments (depending on the duration of each experiment) to minimize recruitment of MCs to inflamed tissues. Initiation of STIA. K/BxN mice of F1 generation were produced by crossing the KRN line (25) with the NOD line, and arthritogenic serum was obtained when the mice were 10 weeks old. Antibody-induced arthritis was evoked in Mcpt5Cre;iDTR mice by an intraperitoneal injection of 150 ␮l of pooled sera from K/BxN mice or from C57BL/6 control mice on day 0 and on day 2. Joint inflammation was assessed every second day by measuring ankle thickness of the hind paws and by clinical scoring of the front and hind paws. Clinical scoring was performed as described elsewhere (26). Briefly, each paw was scored on a scale of 0–3, where 0 ⫽ no evidence of erythema and swelling, 1 ⫽ erythema and mild swelling, 2 ⫽ erythema and pronounced edematous swelling, and 3 ⫽ ankylosis of the limb. Scores for all 4 paws were then summed (maximum score 12 for each mouse). Joint swelling was defined as an increase in ankle thickness over that noted on day 0 (baseline). Initiation of CIA. For immunization, 4 mg/ml (weight/ volume) of chicken CII was dissolved in 0.1M acetic acid and emulsified in an equal volume of CFA. A total volume of 100 ␮l of emulsified CII/CFA per mouse was injected intradermally at 2 sites at the base of the tail (27). On day 14 following the primary immunization, mice were given a booster immunization with CII emulsified in IFA. Control mice received an intradermal injection of 100 ␮l of NaCl. Joint inflammation was assessed every second day after the booster immunization by measuring the hind paw footpad thickness and by clinical scoring of the front and hind paws. Clinical scoring of each paw was performed as described by Inglis et al (27), using a 0–3 scale (maximum score 12 for each mouse).

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Joint swelling was defined as increase in footpad thickness as compared to that obtained on day 0 (baseline). We studied CIA in male Mcpt5-Cre;iDTR mice that were at least 12 weeks old because the disease incidence was ⬃80%, in contrast to females, which had a disease incidence of 50%. Analysis of lymph node hypertrophy and T cell restimulation. The left and right inguinal LNs were isolated on day 40 after immunization. LN cells were resuspended in phosphate buffered saline/2% bovine serum albumin and stained with monoclonal antibodies for 30 minutes at 4°C. Cell suspensions were washed twice and quantified using a Miltenyi MACSQuant flow cytometer with either MACSQuantify or FlowJo software. For cytokine response analysis, LN cells were resuspended in complete RPMI medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin at a density of 2 ⫻ 106 cells/ml, and 200 ␮l of cell suspension per well was plated in 96-well plates. LN cells were restimulated for 4 days at 37°C with monoclonal antibodies directed against CD3 and CD28 at a final concentration of 2 ␮g/ml each or with 10 ␮g/well of T cell–grade CII. LN cells incubated in medium alone served as an unstimulated control. Following incubation, cell culture supernatants were collected, and cytokine levels were analyzed by enzyme-linked immunosorbent assay (ELISA; Chondrex) according to the manufacturer’s protocol. ELISA for CII-specific antibodies. Sera from CIIimmunized mice were obtained on day 40 postimmunization, and anti-CII antibodies (IgG1 and IgG2a) were measured by ELISA (Chondrex) according to the manufacturer’s guidelines. Histologic assessment. Ankle joints were harvested from arthritic mice on day 28 after serum transfer (for STIA) or day 40 after immunization (for CIA), then fixed in 4% formalin, and decalcified for 14 days in OsteoSoft (Merck). Samples were embedded in paraffin, and 5-␮m sections were cut and stained with Giemsa, hematoxylin and eosin (H&E), or Safranin O. Histologic scoring of joint inflammation was evaluated in H&E-stained sections from both ankles. A score of 0–3 was assigned according to the grade of leukocyte infiltration (0 ⫽ no evidence of inflammation, 1 ⫽ mild leukocyte infiltration, 2 ⫽ pronounced leukocyte infiltration and mild destruction of cartilage and bone, and 3 ⫽ intense leukocyte infiltration and pronounced destruction of cartilage and bone). Cartilage degradation was evaluated in Safranin O–stained ankle joint sections. Sections were stained with Weigert’s iron hematoxylin solution for 4 minutes, rinsed in acidulated ethanol, and washed in tap water. Slides were stained for 3 minutes with 0.02% fast green solution, rinsed with 1% acetic acid, stained with 1% Safranin O solution for 5 minutes, and dehydrated. Cartilage degradation is represented by loss of Safranin O staining and was quantified by use of Zeiss AxioVision software as the cartilage area of the calcaneus. Micro–computed tomography (micro-CT). Ankle joints and femurs were scanned using a SkyScan 1174 compact micro-CT apparatus (Bruker) with a resolution of 6.2 ␮m. For determination of calcaneus erosions, a region of interest was defined that spanned 2.4 mm from the distal end. The percentage of eroded bone was quantified by determining grayscale values indicative of eroded bone (70–100) as compared to complete bone (70–255), using digital image analysis software (SkyScan CTAn software). For measurement of bone density, a region of interest spanning 1.8 mm was defined as extending 0.3 mm from the distal growth plate into the diaphysis. The

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trabecular bone volume/tissue volume, trabecular thickness, trabecular separation, and trabecular number were determined according to the methods described by the ASBMR Histomorphometry Nomenclature Committee (28,29). Intravital 2-photon microscopy of inguinal LNs. Mcpt5-Cre mice were crossed with the tandem-dimer red fluorescent protein (tdRFP) excision reporter line (30) and with B6.CD4-GFP mice. Mice were prepared for intravital microscopy as previously described (22). Briefly, animals were intubated and subjected to inhalation narcosis with a mixture of isoflurane (3.0%) and oxygen (97%). Surgical exposure of an inguinal LN was performed using a mouse heating pad. The inguinal LN located at the inside of the abdominal skin was exposed from the fatty tissue without injuring the blood and lymph vessels and imaged for a maximum of 2 hours. Two-photon intravital microscopy was performed with a Zeiss LSM 780 microscope with simultaneous detection via 4 external nondescanned detectors. Illumination was performed at 920 nm with a Chameleon Vision II laser (at 15–20 mW) via a 20⫻ water immersion lens with a 1.0 numerical aperture. Vidisic eye gel (Bausch & Lomb) was used as long-term stable immersion medium to avoid desiccation. Green fluorescent protein (GFP)–expressing CD4⫹ T cells were detected between 500 and 550 nm, and tdRFP-expressing MCs between 575 and 610 nm. Blood vessels were stained by intravenous injection of Qtracker 705 nanocrystals (detection at 690–730 nm; Invitrogen) and phycoerythrin-conjugated anti– intercellular adhesion molecule 1 monoclonal antibodies (detection at 575–610 nm; eBioscience). Macrophages were stained with Alexa Fluor 680–labeled 10,000 MW Dextran (detection at 690–730 nm; Invitrogen). Collagen was visualized by its second harmonic generation signal (⬍480 nm). Fixed images of inguinal LNs were collected as z-stacks up to a depth of 150 ␮m with an x/y resolution of 1,024 ⫻ 1,024 pixels, size of 425 ⫻ 425 ␮m, and z-spacing of 4 ␮m. Close-ups were made with a size of 40 ⫻ 40 ␮m to 160 ⫻ 160 ␮m, and a z-spacing of 1–2 ␮m. Data were visualized as maximum intensity projection or surface-rendered z-stacks using Imaris (Bitplane) and Fiji (31) software. Statistical analysis. Data are reported as the mean ⫾ SD except where indicated otherwise. Statistical analysis was performed using Student’s t-test or two-way analysis of variance.

RESULTS Novel genetic mouse model of inducible mast cell deficiency. In this study, we compared the role of mast cells in passive antibody-induced arthritis and in active antigen-induced arthritis by studying a model of inducible MC deficiency that was independent of Kit mutations. Mcpt5-Cre mice (21) were bred with mice of the iDTR line (24). In the iDTR line, Cre recombinase– mediated deletion of a loxP-flanked stop element results in the selective expression of a simian DT receptor in the Cre-expressing cell subset. Since wild-type mouse cells are resistant to DT, only MCs were sensitive to DTinduced cell death in the Mcpt5-Cre⫹;iDTR⫹ offspring. We previously demonstrated the efficient and specific depletion of connective tissue–type MCs in peritoneal

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lavage fluid and in skin, without alterations in other immune cell subsets (22). Redundant role of MCs in antibody-induced experimental arthritis. We first analyzed MC functions in STIA as a model of antibody-induced arthritis by intraperitoneal injection of serum obtained from arthritic F1 generation K/BxN mice into MC-depleted DT-treated

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Mcpt5-Cre⫹;iDTR⫹ animals as well as DT-treated MCcompetent Cre–;iDTR⫹ littermate controls. Joint inflammation was evaluated by measurement of ankle thickness in the hind limbs and clinical scoring of front and hind paws. In both MC-competent Cre–;iDTR⫹ mice and MC-depleted Mcpt5-Cre⫹;iDTR⫹ mice, arthritis developed rapidly, with disease onset on days 2–3

Figure 1. Dispensability of mast cells (MCs) for antibody-induced joint inflammation. A, Progression of serum-transfer–induced arthritis in MC-depleted Cre–;iDTR⫹ mice and Mcpt5-Cre⫹;iDTR⫹ mice (n ⫽ 14 per group) treated with sera from K/BxN mice, as indicated by ankle joint swelling and clinical scores. Control mice (n ⫽ 12) received sera from healthy wild-type donors (n ⫽ 6). Values are the mean ⫾ SD. B, Histologic scores in Cre–;iDTR⫹ mice, MCpt5-Cre⫹;iDTR⫹ mice, and control mice. Leukocyte infiltration in hematoxylin and eosin (H&E)–stained sections from both ankle joints obtained on day 28 after K/BxN or control serum transfer was scored on a scale of 0–3. C, Histologic analysis of leukocyte infiltration in H&E-stained sections of ankle joints obtained on day 28 after K/BxN or control serum transfer. Bars ⫽ 1,000 ␮m. D, Numbers of MCs in Cre–;iDTR⫹ mice and Mcpt5-Cre⫹;iDTR⫹ mice before and 28 days after K/BxN serum transfer and in control mice 28 days after control serum transfer. MCs were quantified in Giemsa-stained sections of ankle joints isolated after treatment with diphtheria toxin (DT) but before serum transfer (n ⫽ 6 mice per group) or after DT treatment and after K/BxN serum transfer (n ⫽ 10 mice per group) or control serum transfer in healthy controls (n ⫽ 4 mice). Data in B and D are shown as box plots. Each box represents the range of values. Lines inside the boxes represent the mean. ⴱ ⫽ P ⬍ 0.05; ⴱⴱⴱ ⫽ P ⬍ 0.001. NS ⫽ not significant.

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(Figures 1A and B) and with a similar incidence. Ankle thickness values and clinical scores revealed that joint inflammation peaked between day 11 and day 13, declining thereafter until day 28. Comparison of MC-competent Cre–;iDTR⫹ mice and MC-depleted Mcpt5-Cre⫹;iDTR⫹ mice revealed no impact of MCs on the incidence, onset time, or severity of inflammation (Figure 1). Joint inflammation was further evaluated by histologic assessment and scoring of ankle joints obtained on day 28 after serum transfer (Figure 1C). Both the Cre–;iDTR⫹ mice and Mcpt5-Cre⫹;iDTR⫹ mice that received K/BxN sera showed massive infiltration of leukocytes into the ankle joints and severe destruction of cartilage and bones. No signs of pathologic alteration were detected in joints

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isolated from wild-type mice that received control sera. MC depletion in the ankle joints proved to be highly efficient, as indicated by histologic quantification of MCs after systemic DT treatment (Figure 1D). Importantly, STIA resulted in the accumulation of MCs in the inflamed joints of MC-competent Cre–;iDTR⫹ mice on day 28 posttransfer, as well as the reappearance and survival of MCs in the MC-depleted Mcpt5-Cre⫹; iDTR⫹ mice despite continuing DT treatment. MC numbers in the ankle joints of Mcpt5-Cre⫹;iDTR⫹ mice were nevertheless significantly reduced to even lower numbers than in saline-treated controls (Figure 1D). To address previously suspected effects of MCs on cartilage degradation and bone resorption (10), we further assessed cartilage degradation in Safranin O–

Figure 2. No effect of mast cells (MCs) on cartilage degradation and bone destruction in antibody-induced arthritis. A, Histologic analysis of cartilage degradation in Safranin O–stained sections of ankle joints from Cre–;iDTR⫹ mice, MCpt5-Cre⫹;iDTR⫹ mice, and control mice obtained on day 28 after K/BxN or control serum transfer. Cartilage degradation is indicated by loss of Safranin O staining. Bars ⫽ 200 ␮m. Cartilage destruction was quantified as the cartilage area of the calcaneus (n ⫽ 6 mice per group) (shown at the right). ⴱ ⫽ P ⫽ 0.034; # ⫽ P ⫽ 0.054. B, Analysis of the degree of bone erosion by micro–computed tomography (micro-CT). The areas of bone destruction are labeled in red in this 3-dimensional model. Cartilage erosion was quantified as the percentage of eroded bone volume per the total bone volume (n ⫽ 6 mice per group) (shown at the right). # ⫽ P ⫽ 0.053; ⴱ ⫽ P ⫽ 0.037. C, Analysis of bone density by micro-CT. Bone volume/tissue volume (BV/TV), trabecular (Tb) thickness, trabecular separation, and trabecular number were determined in femurs obtained on day 28 after transfer of K/BxN serum or control serum (n ⫽ 6 mice per group). ⴱ ⫽ P ⬍ 0.05. Data are shown as box plots. Each box represents the range of values. Lines inside the boxes represent the mean.

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Figure 3. Mast cell (MC) enhancement of joint inflammation in collagen-induced arthritis (CIA). A, Progression of CIA-induced joint inflammation, as indicated by measurement of joint swelling and scoring of clinical features, in type II collagen (CII)/Freund’s complete adjuvant (CFA)–immunized, MC-depleted Cre–;iDTR⫹ mice and MCpt5-Cre⫹;iDTR⫹ mice (n ⫽ 10 mice per group) and in saline-treated controls (n ⫽ 4). Values are the mean ⫾ SD. B, Quantification of IgG1 and IgG2a isotype antibodies directed against CII (Coll II) in serum obtained on day 40 after immunization from immunized Cre–;iDTR⫹ mice and Mcpt5-Cre⫹;iDTR⫹ mice (n ⫽ 10 per mice group) and from saline-treated controls (n ⫽ 4). Values are the mean ⫾ SD. C, Numbers of MCs in Giemsa-stained sections of ankle joints obtained on day 40 after CII/CFA immunization from Cre–;iDTR⫹ mice and MCpt5-Cre⫹;iDTR⫹ mice (n ⫽ 6 mice per group) and from saline-treated controls (n ⫽ 4 mice). Data are shown as box plots. Each box represents the range of values. Lines inside the boxes represent the mean. ⴱⴱⴱ ⫽ P ⬍ 0.001.

stained sections of the calcaneus (Figure 2A). We found a significantly decreased cartilage area in the calcaneus, indicating profound cartilage degradation, in the K/BxN serum–treated animals as compared to the controls, but we did not detect significant differences between MCdepleted and MC-competent mice. Moreover, we evaluated the effects of MCs on arthritis-induced bone erosion by means of micro-CT analysis of calcaneus samples isolated from K/BxN serum–treated mice and wild-type serum–treated control mice on day 28 after transfer. Both the Cre–;iDTR⫹ mice and Mcpt5-Cre⫹; iDTR⫹ mice that received K/BxN sera had clear evidence of bone erosions (⬃5% of bone eroded) as compared to the control mice (Figure 2B). General loss of trabecular bone induced by inflammation was determined by micro-CT analysis of mouse femurs obtained on day 28 after serum transfer (Figure 2C). K/BxN serum–treated mice irrespective of their genotype displayed a reduction of the bone volume/tissue volume, trabecular thickness, and trabecular number as compared with controls. However, we

found no significant difference in bone destruction or bone density parameters in K/BxN serum–treated MCdepleted mice as compared to the MC-competent controls. Taken together, our results demonstrate that MCs are dispensable for joint inflammation in STIA as a model of antibody-induced experimental arthritis. MCs as critical promoters of effector T cell activation and T cell–driven joint inflammation in CIA. In STIA, joint inflammation is induced by cartilagebound antigen–antibody immune complexes that activate innate immune cells in a T cell–independent manner (11). Since in humans, RA is driven by both autoantibodies and autoreactive effector T cells (16), we further studied the role of MCs in CIA as a model of antigen-induced arthritis. In CIA, the immune response is induced by immunization with CII, and the joint inflammation is provoked by CII-specific effector T cells and by antibodies cross-reacting with cartilage– restricted CII (16). Immunized C57BL/6 mice showed disease onset around day 14 following primary immunization, pro-

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Figure 4. Critical need for mast cells (MCs) in the promotion of collagen-induced arthritis–generated lymph node (LN) hypertrophy, T cell expansion, and T cell cytokine response. A, Assessment of LN hypertrophy in type II collagen (CII)/Freund’s complete adjuvant–immunized Cre–;iDTR⫹ mice and MCpt5-Cre⫹;iDTR⫹ mice (n ⫽ 10 mice per group) and in saline-treated control mice (n ⫽ 4). Total leukocyte counts and numbers of the indicated cell subsets were determined by flow cytometry of inguinal LNs on day 40 postimmunization. Data are shown as box plots. Each box represents the range of values. Lines inside the boxes represent the mean. B, Measurement of cytokine release from inguinal LN cells obtained from arthritic Cre–;iDTR⫹ mice and MCpt5-Cre⫹;iDTR⫹ mice (n ⫽ 10 mice per group) isolated on day 40 postimmunization and restimulated ex vivo with anti-CD3/anti-CD28 (aCD3/aCD28) antibodies or with specific CII immunogen (Coll II). Unstimulated LN cells from arthritic Cre– mice served as the control (⭋). Values are the mean ⫾ SD. ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001. IFN␥ ⫽ interferon-␥; IL-17 ⫽ interleukin-17; TNF ⫽ tumor necrosis factor.

gressing to peak ankle swelling around day 30 and declining thereafter until day 40. Surprisingly, the joint inflammation, which was assessed as ankle swelling of hind paws and clinical scoring of front and hind paws, was markedly reduced in the absence of MCs in Mcpt5Cre⫹;iDTR⫹ mice as compared to Cre–;iDTR⫹ mice (Figure 3A). Quantification of serum levels of IgG1- and IgG2a-specific isotype antibodies directed against CII did not reveal any significant differences between MC-

depleted Mcpt5-Cre⫹;iDTR⫹ mice and Cre–;iDTR⫹ control mice (Figure 3B). The efficiency of MC depletion was determined by histologic quantification of MCs in ankle joint sections obtained on day 40 postimmunization. We found that DT treatment resulted in the highly efficient depletion of MCs from the ankle joints of Mcpt5-Cre⫹;iDTR⫹ mice even after joint inflammation had occurred (Figure 3C). To characterize MC functions in CIA in more

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Figure 5. Complete susceptibility of KitW-sh/W-sh mice to collagen-induced arthritis (CIA)–associated joint inflammation but with delayed onset of disease. A, Progression of CIA-induced joint inflammation, as indicated by measurement of joint swelling and scoring of clinical features, in type II collagen (CII)/Freund’s complete adjuvant (CFA)–immunized KitW-sh/W-sh mice and in age- and sex-matched C57BL/6 wild-type controls (n ⫽ 5 mice per group). Values are the mean ⫾ SD. B, Assessment of lymph node (LN) hypertrophy in CII/CFA-immunized C57BL/6 wild-type controls and KitW-sh/W-sh mice (n ⫽ 5 mice per group). Total cell counts in inguinal LNs were determined by flow cytometry. Data are shown as box plots. Each box represents the range of values. Lines inside the boxes represent the mean. ⴱ ⫽ P ⬍ 0.05; ⴱⴱⴱ ⫽ P ⬍ 0.001.

detail, we used flow cytometry to analyze hypertrophic changes in draining inguinal LNs at the site of immunization in Mcpt5-Cre⫹;iDTR⫹ and Cre–;iDTR⫹ mice on day 40 postimmunization. Arthritic Cre–;iDTR⫹ animals exhibited a 10-fold increase in total LN cell numbers as compared to controls. In contrast, the total number of LN cells was increased only 4-fold in Mcpt5Cre⫹;iDTR⫹ mice and was therefore significantly reduced as compared to controls (Figure 4A). Specifically, Mcpt5-Cre⫹;iDTR⫹ mice showed considerably diminished expansion of CD4⫹ and CD8⫹ T cells and B cells as compared to Cre–;iDTR⫹ mice. The absence of MCs further resulted in lower numbers of CD11c⫹MHCIIhigh dendritic cells, suggesting that MCs function in the migration of dendritic cells from immunized skin to draining LNs. MCs were found in very low numbers in immunized Cre–;iDTR⫹ mice but were significantly increased as compared to those in saline-treated controls. Importantly, MCs were found to have been efficiently depleted from the LNs of Mcpt5-Cre⫹;iDTR⫹ mice (Figure 4A). We further analyzed the cytokine response of CD4⫹ T cells isolated from the inguinal LNs of MCdepleted mice or MC-competent control mice upon nonspecific restimulation with anti-CD3/CD28 antibodies or specific restimulation with the immunogenic CII. We found that the nonspecific as well as collagenspecific production of interferon-␥ and interleukin-17 (IL-17) by CD4⫹ LN T cells isolated from Mcpt5-Cre⫹; iDTR⫹ mice was significantly reduced as compared to that in cells from control mice, indicating a reduced number or impaired activation of Th1 and Th17 effector T cell subsets (Figure 4B). Moreover, CD4⫹ LN T cells isolated from Mcpt5-Cre⫹;iDTR⫹ mice secreted lower levels of tumor necrosis factor and IL-2 upon nonspe-

cific restimulation than did CD4⫹ T cells from the control animals, which suggests a further contribution to LN edema, circulating lymphocyte recruitment, and T cell expansion (Figure 4B). The functions of MCs in CIA are at present ill defined, with one study reporting that MC-deficient KitW-sh/W-sh mice were fully susceptible to CIA (32) but another reporting that mice deficient in the MC-specific chymase Mcpt4 exhibited reduced joint inflammation (33). We therefore analyzed CIA in the same MCdeficient KitW-sh/W-sh mice as used in the study by Pitman et al (32) and compared them with age- and sex-matched C57BL/6 wild-type controls. We detected no difference in disease incidence or severity. However, MC-deficient KitW-sh/W-sh mice exhibited slightly decelerated joint inflammation at early onset of disease (days 16–22), accompanied by delayed abatement of inflammation (Figure 5A). The shift in time course of joint inflammation in KitW-sh/W-sh mice was accompanied by a slight reduction in LN hyperplasia as compared to the wildtype controls (Figure 5B). Taken together, our data identify MCs as important promoters of effector T cell expansion and polarization to Th1 and Th17. The reduced numbers of dendritic cells in inguinal LNs from immunized MCdeficient mice suggest that the impact of MCs on T cell expansion is due to effects on dendritic cell migration. Surprisingly, intravital 2-photon–based imaging analysis of Mcpt5-Cre⫹tdRFP⫹ MC reporter mice crossed with CD4-GFP T cell reporter mice showed that as early as day 6 postimmunization, there was a massive increase in MC numbers (by 121%) in draining inguinal LNs at the site of intradermal immunization as compared to the numbers in saline-treated control mice (Figure 6). Importantly, upon immunization, MCs were found in close

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Figure 6. Accumulation of mast cells (MCs) in T cell zones of draining lymph nodes (LNs) at the site of type II collagen (CII)/Freund’s complete adjuvant (CFA) immunization. Intravital imaging was performed on inguinal LNs in CII/CFA-immunized Mcpt5-Cre⫹tdRFP⫹/CD4-GFP double-reporter mice. A, Inguinal LN under steady-state conditions (cortex/ventral side, 10–70-␮m depth). B, Inguinal LN on day 6 after CII/CFA immunization at the base of the tail (cortex/ventral side, 20–80-␮m depth). C, Quantification of MC numbers in the inguinal LNs of mice with collagen-induced arthritis on day 6 after CII/CFA immunization and in the inguinal LNs of healthy reporter mice (n ⫽ 3 per group). Data are shown as box plots. Each box represents the range of values. Lines inside the boxes represent the mean. ⴱⴱⴱ ⫽ P ⬍ 0.001. D, Multicolor staining of an inguinal LN obtained on day 6 after CII/CFA immunization. High endothelial venules were stained with an anti–intercellular adhesion molecule 1 monoclonal antibody (red) and a vessel tracer (Qtracker; blue). Arrows indicate MCs (red) lying in close proximity to CD4⫹ T cells (green). Macrophages were stained with Alexa Fluor 680–labeled Dextran. Collagen fibers are shown in gray. Yellow indicates autofluorescence. Imaging parameters were as follows: for A and B, field of view 425.1 ⫻ 425.1 ␮m, resolution 512 ⫻ 512 pixels, and pixel size 0.83 ⫻ 0.83 ␮m; for D, field of view 425.1 ⫻ 425.1 ␮m, resolution 1,024 ⫻ 1,024 pixels, and pixel size 0.42 ⫻ 0.42 ␮m.

proximity to CD4⫹ T cells, whereas in the steady state, they were restricted to the LN capsule (Figure 6D). DISCUSSION The role of MCs in arthritis has been a controversial topic of discussion. MCs have been shown to accumulate in the synovial tissue of RA patients, where their numbers correlate with disease severity (11,34,35). However, the mechanisms by which MCs act on disease are poorly understood. Analyses of the in vivo functions of MCs in mouse models of antibody-induced arthritis have yielded conflicting results. MC-deficient KitW/W-v mice have been shown to be resistant to STIA, and their disease susceptibility could be restored by engraftment of wild-type, but not IL-1–deficient, bone marrow– derived MCs (17,18). In contrast, MC-deficient KitW-sh/ W-sh mice developed robust arthritis upon transfer of K/BxN serum (36,37). A similar discrepancy between the two lines was also shown in studies of anti-CII antibody– induced arthritis, in which MC-deficient KitW-sh/W-sh mice, but not KitW/W-v mice, developed disease (19). The reason for the discrepancies may be attributed to pleiotropic effects of Kit beyond MC deficiency. Specifically, KitW/W-v mice display a profound reduction in neutro-

phil numbers, whereas KitW-sh/W-sh mice exhibit neutrophilia and splenomegaly (38). To distinguish MC functions from pleiotropic effects of Kit in arthritis and other models of adaptive immune responses, we and other investigators (20–23,39) have generated genetic models of MC deficiency without a compromised c-Kit–stem cell factor axis. Mice exhibiting Cre-mediated MC eradication have been shown to be fully susceptible to STIA (20). To address the conflicting evidence of the relevance of MCs to autoimmune arthritis, we studied MC functions in both antibody-induced and antigen-induced experimental arthritis in Mcpt5-Cre;iDTR mice as a model of inducible MC-deficiency that is independent of Kit mutations and reflects a normal immune system despite the absence of MCs (22). Our findings are consistent with those reported by Feyerabend et al (20), in that we detected a redundant role of MCs in K/BxN serum–induced joint inflammation. We have now extended previous studies by demonstrating that MCs also have no effects on cartilage degradation and bone destruction following the induction of arthritic joint inflammation. Taken together, our findings indicate that MCs do not significantly contribute to the susceptibility, severity, or progression of STIA.

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The data derived from our inducible model of MC deficiency that is independent of the Kit mutation confirm the findings in the KitW-sh/W-sh mice and contrast with the conclusions drawn from studies of KitW/W-v mice. Since we clearly demonstrated the irrelevance of MCs to cartilage degradation, our findings also contrast with the report by Shin et al (40), who showed a contribution of MC-restricted tryptase–heparin complexes in K/BxN serum–transfer arthritis. Joint inflammation in the STIA model of RA is induced by the activation of innate immune cells by cartilage-bound immune complexes in a T cell–independent mechanism (11). There is clear evidence, however, that in humans, RA is driven by two arms of the immune system: self-directed autoantibodies and autoreactive effector T cells (16). We therefore studied the role of MCs in CIA as a model of antigen-induced arthritis in which joint inflammation is provoked by CII-specific effector T cells and antibodies cross-reacting with cartilage (16). We unexpectedly found that MCs critically promote disease severity, since CIA-induced joint inflammation was markedly reduced in Mcpt5-Cre⫹;iDTR mice. The functions of MCs in CIA are as yet ill defined. Pitman et al (32) reported that MC-deficient KitW-sh/W-sh mice were fully susceptible to CIA. In contrast, mice deficient in the MC-specific chymase Mcpt4 were shown to exhibit reduced joint inflammation (33). We therefore analyzed our MC-depleted mouse model in addition to the CIA-associated joint inflammation in the same MC-deficient KitW-sh/W-sh mice as used in the study by Pitman et al. Confirming the findings by Pitman et al, but in contrast to our findings in Kit mutation–independent MC-deficient mice, we detected no reduction of disease incidence or severity in KitW-sh/W-sh mice and only a mild delay in disease progression. Consequently, Kit mutation–driven alterations of the immune system beyond MC deficiency (most likely, increased numbers of blood and bone marrow neutrophils) may substitute for MCs in promoting the progression of joint inflammation in KitW-sh/W-sh mice. Detailed analysis of inguinal LNs revealed that the absence of MCs resulted in diminished expansion of CD4⫹ T cells and release of Th1 and Th17 cytokines, T cell subsets that have previously been shown to be key players in joint inflammation. Although the frequency of B cells was also dramatically reduced in the absence of MCs, B cell numbers were still sufficient to attain similar serum levels of CII-directed antibodies in the MCdeficient mice as in the MC-competent controls. Consequently, the reduced joint inflammation in the absence of MCs is most likely connected to the effects of MCs on T cell expansion and activation. This may explain the

SCHUBERT ET AL

redundancy of MCs in STIA, where the effects of T cells are completely bypassed by immune complex targeting of innate immune cells. The reduced dendritic cell numbers in inguinal LNs from immunized MC-deficient mice suggest the prominent functions of MCs on LN hypertrophy as well as the linking of T cell expansion to the effects of MCs on dendritic cell migration. However, intravital imaging of LNs showed a profound accumulation of MCs following immunization, particularly their presence in close proximity to T cell zones. MCs may therefore also affect LN hypertrophy and effector T cell expansion by direct effects on the LN microenvironment via the release of proinflammatory mediators in proximity to T cell zones or even via their antigen-presenting capacity. The controversial conflicting data about MC functions in autoimmune disorders led to a debate questioning the relevance of MCs in autoimmune responses in general (20,41–44). By analyzing a Cre-mediated model of MC deficiency without compromised Kit signaling, we have confirmed that MCs are dispensable in antibodyinduced arthritis. In contrast, our findings define MCs as critical promoters of T cell–driven autoreactive responses in CIA. MC redundancy in STIA most likely results from the circumvention of MC-conditioned autoreactive T cell responses. Since in humans, RA includes the humoral and cellular arms of adaptive immunity, the contribution of MCs to the autoattack cannot therefore be judged by studying animal models that rely exclusively on the humoral arm. The critical MC functions in the induction of autoreactive T cell responses indicate MCs as potential therapeutic targets for the prevention and treatment of RA in its early stages. ACKNOWLEDGMENTS We cordially thank Axel Roers for providing the Mcpt5-Cre mice and Christophe Benoist and Diane Mathis for providing the KRN mice used in this study. Expert technical assistance by Christa Haase, Tobias Haering, and Christina Hiller is gratefully acknowledged. AUTHOR CONTRIBUTIONS 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. A. Dudeck 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. Tuckermann, A. Dudeck. Acquisition of data. Schubert, J. Dudeck, Liu, Karutz, Speier, Tuckermann, A. Dudeck. Analysis and interpretation of data. Schubert, J. Dudeck, Liu, Maurer, A. Dudeck.

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