Published online before print June 15, 2007
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* Atopy (Allergy) Research Center, Departments of
Dermatology and
Immunology,
Division of Molecular and Biochemical Research, Biomedical Center, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan
1 Correspondence: Atopy (Allergy) Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan. E-mail: hushio{at}med.juntendo.ac.jp
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, IL-6, and IL-1ß), similar to the pattern produced by LPS. FN-EDA-induced cytokine production was mediated by TLR4, as cytokine production by FN-EDA was absent in TLR4-deficient (TLR4–/–) BMMCs. We examined the roles of TLR4-mediated mast cell activation by this form of fibronectin fragment in the pathogenesis of RA in vivo. The injection of FN-EDA, but not FN-III11and FN-III12, to joints resulted in joint swelling of mice in vivo. Genetically mast cell-deficient WBB6F1-W/Wv mice exhibited significantly less swelling and cytokine production compared with mast cell-sufficient +/+ mice, suggesting that swelling and inflammatory cytokine production were partially dependent on tissue mast cells. Reduced swelling and cytokine production were recovered by the reconstitution of tissue mast cells by the injection of BMMCs from wild-type mice but not from TLR4–/– mice. Altogether, these results suggest that the TLR4-mediated activation of mast cells by endogenous ligand FN-EDA might contribute to the pathogenesis of RA through proinflammatory cytokine production.
Key Words: basophil • rheumatoid arthritis
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In addition to a wide variety of bacterial and virus products, endogenous components including heat shock protein (Hsp)22, -60, and -70, gp96, high-mobility group box 1 protein, cellular fragment of fibronectin, degraded product of hyaluronic acid, and heparan sulfate have been reported to be capable of stimulating the immune system through specific TLR [6 7 8 9 10 11 12 13 14 15 16 ]. Cellular fibronectin, which contains an alternatively spliced exon coding Type III repeat extra domain A (EDA), is produced in response to tissue injury. Hyaluronic acid, one of the major glycosaminoglycans of the extracellular matrix (ECM), has been shown to undergo rapid degradation at inflammation sites. Heparan sulfate is shed rapidly from cell surfaces and basement membranes as a result of tissue injury. Thus, TLR is thought to contribute to the initiation of primary immune responses by recognizing abnormal tissue components.
Rheumatoid arthritis (RA) is characterized by chronic inflammatory infiltration of the synovium, leading to eventual cartilage and bone destruction [17 ]. Although many mechanisms leading to the pathogenesis of RA have been suggested, the innate immune system-mediated pathway is one of the most probable mechanisms in establishing and propagating synovitis [18 19 20 ]. Disruption of IL-1R signaling, such as MyD88–/– mice or IL-1R–/– mice in the transgenic RA model, KBxN mice, diminished the extent of spontaneous disease [21 ]. Furthermore, TLR4 mutant mice had a shortened course of paw swelling, suggesting that TLR signaling might additionally activate MyD88 and perpetuate joint inflammation [21 ]. Also, the ability of TLR signaling to lead to destructive arthritis has been demonstrated by direct intra-articular injection of TLR ligands [22 23 24 25 ] or by findings that several TLR ligands were highly expressed in synovial tissue from RA patients compared with that from healthy donors [6 , 26 , 27 ]. Although it has been reported that TLR-mediated dendritic cells (DCs) or synovial fibroblast activation may contribute to the inflammatory loop in RA [26 , 28 , 29 ], it is still largely unknown whether these endogenous ligands of TLRs act on mast cells in vivo as an amplifier of an inflammatory loop in certain diseases including RA. In this study, we focus on the mast cells, which play an important role in allergic and autoimmune diseases, and investigate whether mast cells can be activated by an endogenous TLR4 ligand, such as fibronectin (FN)-EDA via TLR4, and whether this activation contributes to the pathogenesis of joint inflammation in RA.
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Generation of BMMCs
BMMCs were generated from the femoral BM cells of mice and maintained in RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% heat-inactivated FCS, 100 µM 2-ME, 10 µM MEM-nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% pokeweed mitogen-stimulated, spleen-conditioned medium (PWM-SCM) as a source of mast cell growth factors as described previously [4
, 5
]. After 4 weeks of culture, more than 98% of cells were identifiable as mast cells, as determined by toluidine blue staining and FACS analysis of cell surface expressions of c-kit and Fc
RI.
Preparation of recombinant fibronectin proteins
The bacterial expression vector pET-22b (Novagen, Madison, WI, USA), which contains individual human fibronectin Type III repeats and EDA was described originally and kindly provided by Yamanouchi Pharmaceutical Co. (Ibaraki, Japan) [13
], which enables the expression of fusion proteins carrying six additional histidine residues (6x His tags) at the carboxyl terminus. Individual fibronectin Type III protein genes with 6x His tags were expressed in Escherichia coli BL21 (DE3) and purified using a TALON metal column (Clontech, Takara Bio Inc., Japan). The levels of endotoxin in the purified protein were evaluated by Limulus amebocyte lysate assay, and contaminated endotoxin was removed by endotoxin affinity resin, END-X (Associates of Cape Cod, Inc., Seikagaku Corp., East Falmouth, MA, USA). The highest concentration of endotoxin in the purified EDA used in the experiment was under 0.6 pg/µg protein, which is insufficient to affect cytokine production from BMMC [5
].
Measurement of cytokine concentrations
BMMCs (2x106 cells/ml) in complete culture medium were stimulated with the indicated concentration of EDA, III11, or III12 for 3 h for TNF- or 6 h for IL-1ß and IL-6. LPS (100 ng/ml), from E. coli (Serotype 0111:B4, Sigma-Aldrich) in the presence of 10% FCS and PWM-SCM, was used as a positive control. To exclude the possibility that contaminated LPS in the fibronectin preparations was responsible for BMMC stimulatory activity, some experiment was done in the presence of polymixin B (Sigma-Aldrich). For the IgE-mediated stimulation, BMMCs (5x106/ml) were incubated with 1 µg antitrinitrophenyl (anti-TNP) mouse IgE (BD Biosciences, San Jose, CA, USA) on ice for 2 h. After washing the cells twice, BMMCs (2x106 cells/ml) in complete culture medium were stimulated with 250 ng/ml anti-mouse IgE (BD Biosciences). In some experiments, cells were stimulated in the medium without FCS, together with a soluble form of recombinant (s)CD14 (R&D Systems, Minneapolis, MN, USA). ELISA was used to measure the level of each cytokine in the supernatant (R&D Systems).
ß-Hexosaminidase release assay
In vitro degranulation of mast cells was determined by ß-hexosaminidase release assay, as described previously [4
]. Briefly, BMMCs were sensitized with TNP-specific mouse IgE (BD PharMingen, San Diego, CA, USA), as described in the cytokine measurement section. BMMCs (1x106 cells/ml) in Tyrode’s buffer (10 mM HEPES, 130 mM NaCl, 5 mM KCl, and 5.6 mM glucose containing 0.1% BSA, 1 mM CaCl2, and 0.6 mM MgCl2) were stimulated with 0.5 µg/ml anti-mouse IgE (BD PharMingen) for 40 min at 37°C. Cell supernatants and total cell lysate solubilized with 1% Tween 20 were collected, and ß-hexosaminidase in the supernatants and cell lysate was quantified by spectrophotometrical measurement of the hydrolysis of p-nitrophenyl-N-acetyl ß-D-glucopyranoside (Sigma-Aldrich Japan KK, Tokyo, Japan) in 0.1 M sodium citrate buffer (pH 4.5). The reaction was terminated by the addition of 0.2 M glycine (pH 10.7). The percentage of ß-hexosaminidase release was calculated using the following formula: percent release = (OD of the stimulated supernatant–OD of the unstimulated supernatant) x 100/(OD of the total cell lysate–OD of the unstimulated supernatant).
Western blot analysis
A total of 5 x 106 BMMCs/ml was stimulated with FN-EDA for the indicated time period. The reaction was stopped with cold PBS. The cells were lysed with 20 µl lysis buffer [1% Triton X-100, 150 mM NaCl, 25 mM Tris-HCl (pH 7.5), 1 mM EDTA] containing 1 µM PMSF, 10 µg/ml leupeptin, 10 µg/ml pepstatin-A, 50 µg/ml aprotinin, and 2 mM sodium orthovanadate, and the lysates were subjected to 12% SDS-PAGE (Bis Tris, Novex, San Diego, CA, USA). Immunoblotting using a polyclonal antibody to Phospho-I
B- (Ser32) and IB- (Cell Signaling Technology, Danvers, MA, USA) was performed according to the manufacturer’s instructions [5
]. The membrane was developed with an ECL detection kit (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA).
Reconstitution of W/Wv mice with BMMCs
Mast cell deficiency of W/Wv mice was reconstituted selectively by the injection of 1 x 106 BMMCs from +/+, TLR4–/– mice into the ankle joint. Five weeks after the injection of BMMCs, the mice were used for experiments. The reconstitution of mast cells was confirmed by toluidine blue or alcian blue/safranin staining of the formalin-fixed tissue section of the joint.
Induction of joint inflammation
FN-EDA-induced joint inflammation was elicited by an injection of 10 µg EDA into the murine ankle joint as described previously [25
]. In time-course experiments, joint swelling was measured using a dial thickness gauge (Peacock, Ozaki Mfg. Co., Japan). At various times after EDA injections, joints were dissected from the mice and weighed, snap-frozen in liquid nitrogen, and stored at –80°C until further analysis. The dissected ankle joints were homogenized in 500 µl buffered solution (137 mM NaCl, 20 mM Tris-Hcl, 1% Nonidet P-40, and 10% glycerol) containing protease inhibitors and then centrifuged at 800 g for 20 min. The resultant supernatant was used for the determination of cytokine concentration using ELISA.
Histological examination
Various times after EDA injection, the mouse legs were dissected and fixed in 20% formalin, and decalcified tissues were embedded in paraffin. Sections of 4 µM were stained with H&E or toluidine blue.
Statistical analysis
Statistical analysis was performed using Student’s t-test or two-way ANOVA, as indicated in the figure legends. P values less than 0.05 were considered to be significant.
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, IL-1ß, and IL-6. The amounts of cytokines produced by 50 µg/ml FN-EDA were almost the same as those from BMMCs stimulated with 100 ng/ml LPS (Fig. 1a
1b
1c
). Also, similar to the effect of LPS on BMMCs, none of fibronectin fragments induced the degranulation of mast cells, although IgE receptor cross-linking induced significant degranulation when evaluated by ß-hexosaminidase release (Fig. 1d)
. Longer incubation of BMMCs with FN-EDA did not lead to degranulation (data not shown). To exclude the possibility that contaminated LPS in our fibronectin preparations was responsible for this mast cell activation, we stimulated BMMCs with FN-EDA in the presence of polymixin B, a natural, LPS-binding protein (Fig. 2
). Indicated concentrations of polymixin B (1 µg/ml), which completely inhibited the cytokine productions from BMMCs stimulated by LPS, did not affect those stimulated by FN-EDA, suggesting the activation of mast cells by FN-EDA was not a result of contaminated LPS.
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Figure 1. FN-EDA induces cytokine production but not degranulation of BMMCs from C57BL/6 mice, were stimulated with indicated concentrations of FN-EDA, FN-III11 and FN-III12, or LPS, 3 h for TNF- (a) and 6 h for IL-1ß (b) and IL-6 (c). The concentrations of cytokine in the supernatant were determined as described in Materials and Methods. Degranulation of mast cells was evaluated by ß-hexosaminidase release after stimulation with 10 µg/ml FN-EDA, FN-III11, FN-III12, or IgE plus anti-IgE (0.5 µg/ml) for 40 min as described in Materials and Methods (d). Data are the mean ± SD of three to four experiments conducted with different BMMC preparations. Statistical analysis was performed using Student’s t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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Figure 2. FN-EDA stimulates BMMCs to produce cytokines in the presence of polymixin B. BMMCs from C57BL/6 mice were stimulated with indicated concentrations of FN-EDA or LPS in the absence or the presence of polymixin B (1 µg/ml) for 6 h. The concentration of IL-6 in the supernatant was determined by ELISA. Data are the mean ± SD of three to four experiments conducted with different BMMC preparations. Statistical analysis was performed using two-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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, IL-1ß, IL-6, and IL-13) from BMMCs in a TLR4-dependent manner [4
, 5
], and it has been reported that FN-EDA was an endogenous ligand for TLR4 [14
], we examined whether the observed FN-EDA responsiveness of BMMCs was dependent on the TLR4 of mast cells. FN-EDA induced the secretion of these cytokines from the BMMCs of wild-type and TLR2–/– mice but not from those of TLR4–/– mice (Fig. 3a
3b
3c
). In contrast, there were no significant differences in TNF-, IL-1ß, and IL-6 secretions by BMMCs of these mice when stimulated by IgE receptor cross-linking (Fig. 3d
, and data not shown). As expected, the result that LPS or PGN stimulation showed similar defects in cytokine production from TLR4–/– or TLR2–/– BMMCs, but not from wild-type BMMCs, was consistent with previous results (Fig. 3d)
[4
].
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Figure 3. FN-EDA stimulates BMMCs to produce cytokines via TLR4. BMMCs from wild-type (open bars), TLR4–/– (solid bars), or TLR2–/– (hatched bars) mice were stimulated with indicated concentrations of FN-EDA, 3 h for TNF- (a) and 6 h for IL-1ß (b) and IL-6 (c). IL-6 production of BMMCs from each mouse stimulated with LPS (100 ng/ml), peptidoglycan (PGN; 100 µg/ml), or IgE plus anti-IgE (250 ng/ml) was shown as positive controls (d). Data are the mean ± SD of three to five experiments conducted with different BMMC preparations. Statistical analysis was performed using two-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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RI-mediated pathway was not affected by withdrawal of FCS or by addition of sCD14 (Fig. 4d)
. These results again indicate that mast cell-stimulating activity by FN-EDA preparation is not a result of contaminated LPS, as LPS cannot stimulate BMMCs in the absence of FCS or sCD14.
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Figure 4. sCD14 is not required for the activation of mast cells by FN-EDA. BMMCs from C57BL/6 mice were stimulated with indicated concentrations of FN-EDA, LPS, PGN, or anti-IgE in the medium without FCS or in the medium containing sCD14 (1 µg/ml) for 6 h. The concentration of IL-6 in the supernatant was determined as described in Materials and Methods. Data are the mean ± SD of three to five experiments conducted with different BMMC preparations. Statistical analysis was performed using two-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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B signaling in a TLR4-dependent mannerB activation was also triggered in mast cells via TLR4 upon FN-EDA stimulation. The phosphorylation of IB at Ser32, which is essential for the release of active NF-B and is a marker of NF-B activation, was induced in mast cells of the wild-type but not those of TLR4–/– mice at a maximum of 10 min upon FN-EDA stimulation and then gradually decreased within 30 min (Fig. 5
). These results again support the idea that FN-EDA signals via TLR4 and is followed by the activation of NF-B.
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Figure 5. Activation of mast cells by FN-EDA leads to NF-B signaling in a TLR4-dependent manner. A total of 5 x 106 BMMCs/ml was stimulated with FN-EDA (10 µg/ml) for the indicated time period. Cell lysates were prepared as described in Materials and Methods and were subjected to 12% SDS-PAGE. Immunoblotting using a polyclonal antibody to Phospho-IB (Ser32) and IB was performed according to the manufacturer’s instructions [5
]. The membrane was developed with an ECL detection kit. Data are representative of five independent experiments, which had almost similar results. WT, Wild-type.
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and IL-1ß was well-correlated with the ankle swelling of these mice (Fig. 7c
and 7d)
. Although cytokine production and ankle swelling were partially dependent on tissue mast cells, mast cell deficiency did not lead to the total loss of inflammation, as observed in mast cell-deficient mice, suggesting that FN-EDA also stimulated cells other than mast cells, such as tissue macrophages or fibroblasts, to secrete these cytokines and to induce inflammation. We did not observe significant differences in the number of tissue mast cells between W/Wv mice reconstituted with wild-type and TLR4–/– BMMCs after 5 weeks (data not shown). More than 95% of these mast cells showed positive staining with alcian blue/safranin, which was the property of connective, tissue-typed mast cells (data not shown). These results indicate that mast cell activation by an endogenous ligand, FN-EDA, via TLR4 might contribute to the pathogenesis of RA.
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Figure 6. Injection of FN-EDA, but not FN-III11and FN-III12, induces joint swelling. (a) FN-III11, FN-III12, FN-EDA (10 µg), or LPS (6 pg) was injected into the ankle joint of C57BL/6 mice, and dissected ankle weight was measured 24 h after injection. Ankle swelling was expressed as percent increase of the weight of the PBS-injected ankle. (b) FN-EDA (10 µg)-induced joint swelling was measured using a dial-thickness gauge following 72 h. The subtracted values (EDA-injected joint–PBS-injected joint) are shown. (a and b) Data are the mean ± SD of three to five mice. Statistical analysis was performed using Student’s t-test. **, P < 0.01; ***, P < 0.001. Histological examination of PBS (c, d)- or EDA-injected joints (e, f) was performed after staining tissue sections with H&E (H. E; c, e) or toluidine blue (T. B; d, f). Arrowheads indicate mast cells in the synovium.
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Figure 7. FN-EDA induces joint swelling in a mast cell TLR4-dependent manner. PBS or 10 µg FN-EDA was injected into the ankle joint of WBB6F1-+/+, mast cell-deficient WBB6F1-W/Wv, and W/Wv mice, whose mast cell deficiency was reconstituted by the injection of 1 x 106 BMMCs from +/+ or TLR4–/– mice into the ankle joint 5 weeks before experiments. (a) The ankle weight was measured at 24 h after injection and expressed as percent increase of the weight of a PBS-injected ankle. (b) Histological examinations of FN-EDA-injected joints of WBB6F1-+/+ mice or -W/Wv mice or whose mast cell deficiency was reconstituted with BMMCs of +/+ or TLR4–/– mice were performed after staining tissue sections with H&E. Tissue extracts were prepared from ankle joints dissected from the respective mice 24 h after injection of FN-EDA or PBS, weighed, and used for the measurement of TNF- (c) and IL-1ß (d) as described in Materials and Methods. Data are the mean ± SD of four to six mice. Student’s t-test was performed to analyze the differences of each group. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, Not significant.
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Our observations of significantly reduced joint swelling and tissue cytokine production in mast cell-deficient mice by FN-EDA injection clearly suggest that this way of mast cell activation in the absence of infection might play roles in the pathogenesis of RA. However, swelling was not completely absent in mast cell-deficient mice, indicating that FN-EDA also works for cells other than mast cells, such as DCs, macrophages, and fibroblasts. As TLRs are expressed on immune and nonimmune cells in a synovial compartment, which can also be activated by several molecular entities ranging from degraded ECM, antibodies, necrotic cell products, and Hsps, all are released in large quantities in damaged tissues. Roelofs et al. [6 , 26 , 27 ] reported up-regulated expression of various TLRs in synovium tissues of RA patients and suggested a possible involvement of DC activation by these TLR ligands, including Hsp22, in the inflammatory process of RA. As they have reported that stimulation of DCs from RA patients by these TLR ligands resulted in markedly higher production of inflammatory mediators compared with DCs from healthy controls [27 ], it is tempting to speculate that mast cells of RA patients may have different responsiveness against these TLR ligands by changing levels of their TLR expressions.
The activation of mast cells by FN-EDA via TLR4 leads to cytokine production but not to degranulation. This result was similar to that of LPS. Among the cytokines produced, TNF- has been reported to increase the expression of matrix metalloproteinase-9 (gelatinase B), which is known to be involved in the pathogenesis of RA by promoting the migration of mast cell progenitors to sites of inflammation or by contributing to local tissue damage [37
]. Although the amount of cytokines produced by BMMCs stimulated with FN-EDA was not as much as that stimulated with LPS or IgE plus anti-IgE, mast cell deficiency clearly showed reduced swellings of ankle in vivo, indicating that the initial activation of mast cells via TLR4 by FN-EDA plays a crucial step in the complicated, inflammatory process in RA. Because of the short period of inflammation in the FN-EDA-injected joint, we did not observe destruction of cartilage or bone erosions, as has been observed in the chronic RA model. Also, consistent with the in vitro result, we have not observed degranulated mast cells at FN-EDA-injected sites by histological examination.
Although various structurally unrelated ligands of TLR4 have been reported, the precise mechanisms for recognition of these ligands by TLR4 are not known. Myeloid differentitation protein-2 (MD-2), an extracellular-associated molecule for TLR4, which is essential for the recognition of LPS and the cell surface expression of TLR4, is reported to be required for the recognition of FN-EDA [14 ]. As we have shown that MD-2 was essential for the full responsiveness of mast cells to LPS and gram-negative bacteria in vitro and in vivo, a similar recognition mechanism may exist in the activation of mast cells by FN-EDA via TLR4 [38 ]. CD14 is another interesting cofactor required for the recognition of LPS by TLR4 in many cells. As mast cells do not express CD14, the presence of FCS as a source of sCD14 is essential for the responsiveness of mast cell to LPS as shown in Figure 4b . It is interesting that responsiveness to FN-EDA was retained in the absence of FCS (Fig. 4a) , suggesting that FN-EDA may be recognized by TLR4 in a different manner from LPS.
In this experiment, we used recombinant products, which are produced by genetically engineered E. coli; thus, final preparations may be contaminated with bacterial products. Studies using highly purified Hsp, essentially free of LPS, suggest that the previous reported, cytokine-producing effect of Hsp may be a result of contaminants [39 ]. We have examined the concentration of LPS in our FN-EDA preparations, and the contaminants were usually less than 0.6 pg/µg protein (30 pg/50 µg, highest concentration of protein used for the experiment), which did not induce cytokine production and activation of mast cells previously [5 ]. A similar preparation of other fragments, III11 or III12, did not lead to mast cell activation, and the activation of mast cells by FN-EDA occurred in the presence of polymixin B, suggesting that the observed activation by FN-EDA was not the result of LPS contamination but the putative effect of FN-EDA via TLR4. Although we cannot exclude the possibility that some protein fragments may bind LPS tightly and render LPS removal ineffective, additional data—that the requirement of sCD14 for the responsiveness of BMMCs to LPS, but not to FN-EDA—clearly support that the stimulating effect of FN-EDA was not a result of contaminated LPS. Although we only demonstrate that TLR4, not TLR2, is required for BMMC activation by FN-EDA, it is unlikely that activation of mast cells may occur via other TLRs by other contaminants from bacteria, such as CpG DNA or ribosomal components, as previous reports and our unpublished results show that BMMCs do not lead to activation by TLR3, TLR7, and TLR9 ligands [40 ].
Taken together, this study first shows that mast cells are activated by an endogenous tissue ligand, FN-EDA, a danger signal from damaged and inflamed tissue through TLR4 and possible involvement of this way of mast cell activation in the pathogenesis of joint inflammation in RA via inflammatory cytokine production. As the production of an endogenous ligand for TLRs is not limited to synovial fluid, as FN-EDA has been observed in the skin tissue from psoriasis patients, where proliferating keratinocytes are reported to be a putative source [41 , 42 ], this way of mast cell activation may occur in many diseases. Furthermore, a recent report documented in vivo crucial roles of TLR2 and -4 in recognition of hyaluronan degradation products for the regulation of epithelial cell integrity [43 , 44 ]. Thus, clarification of the detailed mechanisms of mast cell activation by endogenous ligands in the absence of IgE would be important for understanding the roles played by mast cells, not only in pathological but also in physiological conditions, where the triggering mechanisms that lead to mast cell activation have not been defined.
Received December 15, 2006; revised January 25, 2007; accepted May 21, 2007.
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