Published online before print May 30, 2008
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, and CXCL-1
* Departamento de Farmacologia Aplicada, Farmanguinhos, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil;
Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, Brazil;
Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil; and
Departamento de Farmacologia, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil
2 Correspondence: Farmacologia Aplicada, Farmanguinhos–Fiocruz, Rua Sizenando Nabuco, 100, Manguinhos, Rio de Janeiro, RJ, Brazil. CEP: 21041-250. E-mail: gracahenriques{at}fiocruz.br
ABSTRACT
Endothelins (ETs) are involved in inflammatory events, including pain, fever, edema, and cell migration. ET-1 levels are increased in plasma and synovial membrane of rheumatoid arthritis (RA) patients, but the evidence that ETs participate in RA physiopathology is limited. The present study investigated the involvement of ETs in neutrophil accumulation and edema formation in the murine model of zymosan-induced arthritis. Intra-articular (i.a.) administration of selective ETA or ETB receptor antagonists (BQ-123 and BQ-788, respectively; 15 pmol/cavity) prior to i.a. zymosan injection (500 µg/cavity) markedly reduced knee-joint edema formation and neutrophil influx to the synovial cavity 6 h and 24 h after stimulation. Histological analysis showed that ETA or ETB receptor blockade suppressed zymosan-induced neutrophil accumulation in articular tissue at 6 h. Likewise, dual blockade of ETA/ETB with bosentan (10 mg/kg, i.v.) also reduced edema formation and neutrophil counts 6 h after zymosan stimulation. Pretreatment with BQ-123 or BQ-788 (i.a.; 15 pmol/cavity) also decreased zymosan-induced TNF-
production within 6 h, keratinocyte-derived chemokine/CXCL1 production within 24 h, and leukotriene B4 at both time-points. Consistent with the demonstration that ET receptor antagonists inhibit zymosan-induced inflammation, i.a. injection of ET-1 (1–30 pmol/cavity) or sarafotoxin S6c (0.1–30 pmol/cavity) also triggered edema formation and neutrophil accumulation within 6 h. Moreover, knee-joint synovial tissue expressed ETA and ETB receptors. These findings suggest that endogenous ETs contribute to knee-joint inflammation, acting through ETA and ETB receptors and modulating edema formation, neutrophil recruitment, and production of inflammatory mediators.
Key Words: neutrophil • cytokine • chemokine • lipid mediator • bosentan • sarafotoxin s6c
INTRODUCTION
The endothelins (ETs) constitute a family of three 21 aa isopeptides (ET-1, ET-2, and ET-3), synthesized from distinct pre-pro-ET precursors, which are each cleaved to a specific Big-ET, which is in turn converted to ETs by action of an ET-converting enzyme. These peptides typically present are characterized by two disulfide bridges and six conserved amino acid residues at the COOH terminus [1 ] and are closely related in structure to sarafotoxins found in the venom of the burrowing asp Atractaspis engaddensis [2 ].
ETs exert their effects through two distinct cell surface-specific G protein-coupled receptors, type A (ETA) [3 ] and type B (ETB) [4 ]. These receptors display distinct molecular and pharmacological characteristics and signal different functions, depending on their location. ET-1 and ET-2 show higher affinity for ETA receptors than ET-3, whereas all three ETs have similar affinity for ETB receptors [5 ].
In addition to its well-recognized, vasoconstrictive properties, ET-1 has been demonstrated to participate in the pathogenesis of sepsis, bronchial asthma, and pulmonary hypertension among others [6 ]. In addition to its well-recognized, vasoconstrictive properties, ETs exert an important role in inflammatory reactions. ET-1 modulates the expression of adhesion molecules on endothelial cells and on fibroblast-like synovial cells [7 ], induces plasma exudation and edema formation [8 , 9 ], stimulates cytokine production [10 , 11 ], and regulates neutrophil adhesion and migration [9 , 12 ].
Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation as a result of a chronic inflammatory process of the synovial membrane, caused mainly by the proliferation of resident fibroblast-like cells and synoviocytes, by angiogenesis and by the infiltration of macrophages, lymphocytes, and polymorphonuclear cells into the synovial tissue [13 ]. Few studies have reported increased ET-1 levels in serum [14 , 15 ] and synovial fluid [16 ] of RA patients when compared with healthy subjects. In addition, sera samples from RA patients with systemic involvement showed higher ET-1 concentrations than those from RA patients without systemic inflammation [14 ]. Increased ET-1 levels were also found in periarticular tissue from rats submitted to antigen-induced arthritis [17 ]. Although several of the proinflammatory effects of ET-1 are well-described, its correlation with RA joint inflammation remains poorly understood. In this study, we investigated the participation of ETs in a murine model of zymosan-induced arthritis, evaluating edema formation, leukocyte migration, and production of inflammatory mediators.
MATERIALS AND METHODS
Animals
Male C57BL/6 mice (20–25 g) from Oswaldo Cruz Foundation Breeding Unit (Fiocruz, Rio de Janeiro, Brazil) were kept in plastic cages with free access to food and fresh water in a room with controlled temperature (22°C–24°C) and light (12 h light/dark cycle) at Farmanguinhos’ experimental animal facility until use. All experimental procedures performed were approved by the institution’s ethical Committee for Animal Care and Use.
Treatments
The selective ETA receptor antagonist (BQ-123) or the selective ETB receptor antagonist (BQ-788) was injected in situ in doses ranging from 0.15 to 150 pmol/cavity in a final volume of 25 µl in sterile saline 5 min before intra-articular (i.a.) zymosan stimulation. Bosentan, a nonpeptidic, dual ETA/ETB receptor antagonist, was administered i.v. at 10 mg/kg, 15 min before i.a. stimulation in a final volume of 100 µl. Cromolyn sodium salt (0.2 mg/kg in 25 µl sterile saline), a mast cell stabilizer, was i.a.-injected 30 min before zymosan i.a. stimulation. Respective control groups were injected with the same volume of sterile saline.
Induction of joint inflammation
Joint inflammation was induced by i.a. injection of zymosan (500 µg/cavity in 25 µl sterile saline), ET-1 (1, 10, and 30 pmol/cavity), or the selective ETB receptor agonist, sarafotoxin S6c (SRTX; 0.1, 1, 10, and 30 pmol/cavity) by the insertion of a 27.5-G needle through the suprapatellar ligament into the left knee joint cavity as described previously [18
]. Control animals received an i.a. injection of an equal volume of sterile saline.
Measurement of knee-joint swelling
Knee-joint swelling was evaluated by measurement of the transverse diameters of left knee joints using a digital caliper (Digmatic Caliper, Mitutoyo Corp., Kanagawa, Japan). Values of knee-joint thickness are expressed as the difference (
) between the diameter measured before (basal) and after induction of articular inflammation in millimeters.
Collection of synovial fluid and leukocyte counts
Animals were killed by an excess of CO2 at 6 h and/or 24 h after i.a. injection of zymosan, ET-1, or SRTX. Knee synovial cavities were washed with 300 µl PBS containing EDTA (10 mM) by the insertion of a 21-G needle into mice knee joints, and the synovial washes were recovered by aspiration. Total leukocyte counts were made in a Neubauer chamber under optical microscope after dilution in Türk fluid (2% acetic acid). Differential counts of neutrophils were made using May-Grünwald-Giemsa-stained cytospins (Cytospin 3, Shandon Inc., Pittsburgh, PA, USA), and values are reported as numbers of cells per cavity (x105).
Real-time PCR
Quantitative PCR (QPCR) was performed as described previously [19
]. Briefly, mice were killed by excessive CO2 inhalation 2 h after i.a. injection of zymosan (500 µg), and knee-joint complexes were harvested. Samples were homogenized in Trizol reagent, and total RNA was extracted using the SV Total RNA isolation system (Promega Biosciences, San Luis Obispo, CA, USA). QPCR was performed in an ABI Prism 7500 sequence detection system using the SYBR Green fluorescence (Applied Biosystems, Wilmington, NC, USA). The following primers were used: pre-pro-ET-1, sense: 5'-TGT GTC TAC TTC TGC CAC CT-3', antisense: 5'-CAC CAG CTG CTG ATA GAT AC-3'; β-actin, sense: 5'-AGC TGC GTT TTA CAC CCT TT-3', antisense: 5'-AAG CCA TGC CAA TGT TGT CT-3' [20
]. The expression of β-actin mRNA was used as a control for tissue integrity in all samples.
Enzymatic immune assay for leukotriene B4 (LTB4)
LTB4 levels were evaluated in cell-free synovial washes recovered from zymosan-stimulated C57BL/6 mice at 6 h and 24 h after zymosan stimulation (500 µg/cavity). LTB4 was assayed by enzyme immunosorbent assay (EIA) according to the manufacturer’s protocol (Cayman Chemical, Ann Arbor, MI, USA).
Histology
Whole knee joints obtained from C57BL/6 mice at 6 h after i.a. administration of zymosan or saline were removed, dissected, and fixed in 10% formalin for 12 h. After decalcification in 10% EDTA in PBS solution for 1–2 weeks, the specimens were processed for paraffin embedding. Tissue sections (5 µm) were stained with H&E and mounted on permanent glass slides and analyzed under optic microscope (Olympus BX41, Olympus, Japan; original magnification, 100x). Alternatively, for mast cell visualization, 18 µm-thick tissue slides were stained in acetate buffer (pH 1.42) containing 0.36% Alcian blue/0.02% safranin/0.01% Trypan blue (AB/S/TB), as described previously [21
], prior to mounting on slides.
Preparation of knee-joint extracts
Knee-joint extracts were prepared as described previously by Rosengren et al. [22
] for rats and modified by us for application in mice, which were killed 6 h and 24 h after zymosan i.a. injection, and muscular and bone tissues extraneous to the joint were carefully removed to provide a well-defined, triangular knee-joint specimen. Knee joints were then frozen immediately in liquid nitrogen, pulverized with a hammer, and homogenized using a glass potter homogenizer (Kontes Glass Co., Vineland, NJ, USA) in 1 ml HBSS containing 0.4% of Triton X-100 and 0.2% of protease inhibitor cocktail (protease inhibitor cocktail, Sigma Chemical Co., St. Louis, MO, USA) in a proportion of 50 µl per 10 mg tissue. The homogenate was then centrifuged at 5000 g for 10 min, at 4°C, and the supernatant was filtered (0.2 µm) and stored at –70°C.
ELISA
Levels of TNF-, IL-1β, and keratinocyte-derived chemokine (KC)/CXCL1 in the knee-joint extracts were evaluated by sandwich ELISA, using matched antibody pairs from R&D Systems (Minneapolis, MN, USA; Quantikine), according to the manufacturer’s instructions.
Isolation of murine knee-joint capsule and protein determination
Joint capsule (including adjacent synovium, patellae, and patellar ligament) was isolated in a standardized manner as described by Van Meurs et al. [23
]. Joint capsule was recovered at 6 h or 24 h after i.a. injection of zymosan or vehicle and frozen immediately in liquid nitrogen. Tissue samples were homogenized by hand using a glass potter homogenizer (Kontes Glass Co.) and lysed for 15 min in ice-cold buffer [20 mM HEPES, 350 mM NaCl, 20% (v/v) glycerol, 1% (v/v) Nonidet P-40 (NP-40), 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT], supplemented with 0.5 PMSF, 1 nM benzamidine, 1 µM leupeptin, and 1 mM protease inhibitor cocktail (Sigma Chemical Co.), and stored at –70°C. Insoluble material was removed by centrifugation (10,000 g for 5 min at 4°C), and the total protein content in the supernatant was determined by the Bradford method.
Immunoblotting analysis
Knee-joint capsule extracts were denatured in Laemmli sample buffer (50 mM Tris-HCl, pH 6.8,1% SDS, 5% 2-ME, 10% glycerol, 0.001% bromophenol blue) and heated in a boiling water bath for 3 min. Samples (40 µg total protein) were run on SDS-12% polyacrylamide gel, and proteins were electrophoretically transferred onto nitrocellulose membranes (Hybond-C Pure, Amersham Pharmacia Biotech, San Francisco, CA, USA). Membranes were blocked with Tween-PBS (0.5% Tween-20) containing 2% BSA and probed with specific, primary polyclonal anti-ETA or anti-ETB receptor antibodies (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After extensive washing in Tween-PBS, nitrocellulose sheets were incubated with anti-goat IgG biotin-conjugated antibody (1:10,000, Sigma Chemical Co.) for 1 h and developed by an ECL system (SuperSignal West Pico chemiluminescent substrate kit, Pierce Biotechnology, Rockford, IL, USA). To confirm the amounts of protein loaded, the membrane was reprobed with anti-murine β-actin (1:1000, Sigma Chemical Co.). The bands were quantified by densitometry, using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA).
Statistical analysis
When pertinent, results are reported as the mean ± SEM and were statistically analyzed by means of ANOVA followed by Newman-Keuls-Student test or Student’s t-test. Values of P < 0.05 were regarded as significant.
Drugs and reagents
PBS, Tween-20, o-phenylenediamine dihydrochloride, EDTA, HBSS, BSA, diaminobenzidine, NP-40, DTT, PMSF, saponin from Quillaja bark, a protease inhibitor cocktail, Bradford reagent, aprotinin, and cromolyn sodium salt were purchased from Sigma Chemical Co. Polyacrylamide and dextran 500 were obtained from Amersham Biosciences (Buckinghamshire, UK). TNF-, IL-1β, and KC/CXCL1-matched antibody pairs were obtained from R&D Systems. Goat anti-mouse ETA (C-19) or ETB (C-20) receptor polyclonal antibodies and biotinylated rabbit anti-goat IgG were obtained from Santa Cruz Biotechnology, and streptavidin-conjugated HRP was purchased from Caltag Laboratories (Burlingame, CA, USA). BQ-123 [cyclo(DTrp-DAsp-Pro-DVal-Leu)] and BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-
-methylleucyl-D-1-methoxyxarboyl-D-norleucine) were purchased from American Peptide Co. (Sunnyvale, CA, USA). Bosentan was kindly donated by Dr. Martine Clozel (Actelion Ltd., Allschwil, Switzerland).
RESULTS
ET receptor antagonists reduced zymosan-induced articular inflammatory response
Previous results from our laboratory demonstrated that the i.a. zymosan administration into the knee enhances joint thickness and induces neutrophil migration into the synovial cavity within 6 h and 24 h [18
]. To address the role of ETs in this phenomenon, C57BL/6 mice were treated in situ with selective antagonists of an ETA (BQ-123; 0.15–150 pmol/cavity) or ETB (BQ-788; 0.15–150 pmol/cavity) receptor 5 min before i.a. zymosan stimulation. Blockade of ETA receptors markedly reduced knee-joint thickness at 6 h, and 150 pmol BQ-123 provided 65% inhibition (Fig. 1A
). Interestingly, selective blockade of ETB receptors with BQ-788 was also effective in reducing knee-joint edema formation, such that at 150 pmol/cavity, the percentage of inhibition induced by this antagonist was identical to that seen with BQ-123 (Fig. 1D)
. Treatments with either antagonist also inhibited total leukocyte and neutrophil infiltration within 6 h of i.a. zymosan injection. However, the doses of BQ-123 needed to inhibit total leukocyte and neutrophil migration (15–150 pmol/cavity; Fig. 1B
and 1C
) were clearly greater than those of BQ-788 (
0.15 pmol/cavity; Fig. 1E
and 1F
). Consistent with these findings, we detected an early and significant increase in pre-pro-ET-1 mRNA expression in knee joints at 2 h after zymosan injection, as shown in Figure 1G
. Histological analysis confirmed an intense, inflammatory cell infiltration into synovial tissue at 6 h after stimulation with i.a. zymosan (Fig. 2B
), which was inhibited by pretreatment with BQ-123 or BQ-788 (15 pmol/cavity, i.a.; Fig. 2C
and 2D
). By means of a mast cell staining [21
], we observed the presence of mast cells in sections obtained from saline- and zymosan-injected knees, with a predominance of degranulated mast cells in those from the later group (Fig. 3
). Interestingly, we also observed that prior to i.a. injection of the mast cell stabilizer, cromolyn sodium salt significantly inhibited zymosan-induced knee-joint edema, demonstrating that mast cells contribute importantly to edema formation in this experimental model of articular inflammation (Fig. 3C)
.
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Figure 1. ETs mediate 6 h zymosan-induced articular inflammation. Selective ETA or ETB receptor antagonists (BQ123 or BQ788, respectively) reduced zymosan-induced joint edema formation (A and D), total leukocyte (B and E), and neutrophil migration (C and F) into C57BL/6 mice. BQ-123 or BQ-788 (0.15–150 pmol/knee, i.a., 25 µl) or vehicle (saline) was administered 5 min before zymosan injection (500 µg/knee, i.a., 25 µl). Knee-joint diameter was evaluated with a digital caliper, and knee synovial cells were recovered 6 h after zymosan stimulation. Results are expressed as the mean ± SEM from at least 10 animals per group. *, Statistically significant differences (P 0.05) between stimulated and nonstimulated groups; #, differences between treated and untreated groups. pre-pro-ET-1 mRNA expression in knee joints 2 h after zymosan (500 µg/knee, i.a., 25 µl) or saline injection (G) was evaluated by real-time PCR. Results are expressed as the mean ± SEM from four animals per group. *, P < 0.05, test.
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Figure 2. H&E-stained representative of histological joint sections of knee joints injected with saline (A) and zymosan (B–D) collected 6 h after stimulation. Pharmacological blockade of ETA (C) or ETB (D) receptors (BQ-123 or BQ-788, respectively) reduced zymosan-induced joint leukocyte infiltration. Original magnification, 100x. Black arrows show inflammatory cell infiltration. C, Cartilage; MT, muscle tissue; ST, synovial tissue; BM, bone marrow.
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Figure 3. Mast cell staining. Decalcified, 18 µm-thick knee-joint slices from saline (A)- or zymosan (B)-injected mice within 6 h were stained with AB/S/TB for identification of mast cells (arrows). Original magnification, 400x (A and B). Graph shows the effect of in vivo pretreatment with cromolyn sodium salt (0.2 mg/kg, i.a., 30 min before stimulation) on zymosan-induced knee-joint edema formation (C). Statistically significant differences (P 0.05) between stimulated and non-stimulated groups are indicated by an asterisk; plus sign represents differences between treated and untreated groups.
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Figure 4. ET receptor blockade impairs 24 h zymosan-induced articular inflammation. Selective ETA or ETB receptor antagonists (BQ-123 or BQ-788, 15 pmol/knee, i.a., 25 µl) reduced zymosan-induced joint edema formation (A) and neutrophil migration (B) induced by zymosan (500 µg/knee, i.a., 25 µl) in C57BL/6 mice. Knee-joint diameter was evaluated with a digital caliper, and knee synovial cells were recovered 24 h after zymosan stimulation. Results are expressed as the mean ± SEM from at least 10 animals per group. *, Statistically significant differences (P 0.05) between stimulated and nonstimulated groups; #, differences between treated and untreated groups.
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, IL-1β, and KC/CXCL1 in knee-joint extracts and of LTB4 in synovial washes obtained at 6 h and 24 h after i.a. zymosan stimulation, respectively. Levels of TNF-, IL-1β, KC/CXCL1, and LTB4 were elevated in samples from stimulated joints at both time-points (Fig. 5
). In situ pretreatment with BQ-123 or BQ-788 significantly inhibited the zymosan-induced increase in the levels of LTB4 (at 6 h and 24 h), TNF- (at 6 h only), and CXCL1 (at 24 h only), but neither antagonist affected IL-1β levels.
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Figure 5. Blockade of ET receptors reduces zymosan-induced inflammatory mediators. Protein levels of TNF-, IL-1β, and KC/CXCL1 were determined in the supernatant of knee-joint tissue extracts by ELISA. Levels of LTB4 were measured in cell-free supernatant of knee-joint synovial washes by EIA. Analysis was performed 6 h and 24 h after zymosan (500 µg/cavity; i.a., 25 µl) or vehicle (saline) injection. Mice were pretreated with BQ-123 or BQ-788 (15 pmol/knee, i.a., 25 µl) or with vehicle 5 min before i.a. stimulation. Results are expressed as the mean ± SEM from at least 10 animals per group. *, Statistically significant differences (P0.05) between stimulated and nonstimulated groups; #, differences between treated and untreated groups.
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Figure 6. ET-1 and SRTX induce knee-joint edema formation and neutrophil influx. i.a. injection of ET-1 (1–30 pmol/cavity) or SRTX (0.1–30 pmol/cavity) induces inflammatory reaction in C57BL/6 mice knee joints characterized by edema formation (A and D) and leukocyte accumulation (B and E), mainly as a result of neutrophils (C and F). Knee-joint diameter was evaluated with a digital caliper, and knee synovial cells were recovered 6 h after i.a. stimulation. Results are expressed as the mean ± SEM from at least 10 animals per group. Expression levels of ETA (G) and ETB (H) receptors in knee-joint capsule of zymosan- or vehicle-injected animals. Knee-joint capsules were recovered 6 h after stimulation and analyzed by Western blotting, as described in Materials and Methods. Results are expressed as the mean ± SD from one out of four independent experiments, each in triplicate. *, Statistically significant differences (P 0.05) between stimulated and nonstimulated groups.
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Figure 7. Bosentan pretreatment (10 mg/kg, i.v., 100 µl) inhibited knee-joint edema formation (A), total leukocyte (B), and neutrophil (C) influx in C57BL/6 mice stimulated with zymosan (500 µg/knee, i.a.). Analysis was performed 24 h after stimulation. Knee-joint diameter was evaluated with a digital caliper, and knee synovial cells were recovered 24 h after i.a. injection of zymosan. Results are expressed as the mean ± SEM from at least 10 animals per group. *, Statistically significant differences (P0.05) between stimulated and nonstimulated groups; #, differences between treated and untreated groups.
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The results of the current study demonstrate that endogenous ETs participate in zymosan-induced articular inflammation in mice. The various effects of endogenous ETs in this model, namely, synovial edema formation, neutrophil accumulation, as well as modulation of TNF-, KC/CXCL1, and LTB4 production, are mediated via ETA and ETB receptor-operated mechanisms.
A few clinical studies have encountered elevated levels of ET-1 in synovial fluid samples [16 , 24 ] and plasma of RA patients [14 , 15 ], thus indicating a correlation between ETs and this pathophysiological condition. It is well established that zymosan induces acute arthritis that mimics features of rheumatoid synovitis, such as synovial edema and neutrophil influx, among others [18 , 25 ]. In this regard, the present study clearly shows that i.a. zymosan induced an early up-regulation of pre-pro-ET-1 mRNA expression in synovial tissue, as well as a pronounced inflammatory response, which was markedly attenuated by prior i.a. administration of selective ETA or ETB receptor antagonists (BQ-123 or BQ-788) or i.v. injection of bosentan, a dual ETA/ETB receptor antagonist. Further evidence that mechanisms operated by ETA and ETB receptors contribute to zymosan-induced articular inflammation includes the expression of both receptor subtypes in synovial tissue of naïve and zymosan-stimulated mice and the fact that i.a. injections of exogenous ET-1 or of the selective ETB receptor agonist SRTX closely mimic zymosan in promoting synovial edema and leukocyte accumulation. By activating ETA (but not ETB) receptor-mediated mechanisms, ETs also contribute to hind-paw edema induced by antigen or formalin and to leukocyte infiltration into the pleural cavity in response to antigen or LPS in mice [9 , 26 27 28 29 ]. The fact that neither ETA nor ETB receptor blockade influenced zymosan-induced hind-paw edema or pleural leukocyte accumulation in these studies further highlights the importance of ETs to the particular context of articular inflammation.
It is interesting to note that BQ-123 and BQ-788 were equieffective in inhibiting zymosan-induced joint swelling, but the ETB receptor antagonist was clearly more potent in blocking early (6 h), total leukocyte and neutrophil accumulation in the synovial cavity. This dichotomy could suggest that leukocyte infiltration triggered by zymosan was dependent only on activation of ETB receptor-mediated mechanisms in such a way that at higher doses, the selectivity of action of BQ-123 toward ETA receptors was lost. However, this clearly was not the case, as BQ-123 (at 150 pmol/cavity) did not affect leukocyte infiltration (or edema) induced by i.a. sarafotoxin, a selective ETB receptor agonist [2 ].
It is important to mention that similar levels of expression of ETA and ETB receptors were detected by Western blot assay of synovial tissue homogenates of control, saline-treated and zymosan-treated joints. However, it should be borne in mind that discrete alterations in the levels of expression of such receptors by particular cell types in the synovial tissue may have gone undetected because of the restraints of this type of assay. On the other hand, the present study detected a significant, 3.5-fold up-regulation in synovial tissue pre-pro-ET-1 mRNA levels at 2 h after zymosan, but it remains to be seen how quickly and effectively increased messenger levels translate into increased, mature ET-1 production in the tissue and which cells are implicated in this activity. ET-1 is produced by several articular cell types, including chondrocytes [30
], macrophage-like type A synoviocytes [31
], and synovial blood vessel endothelium [32
]. Another potential candidate cell is the mast cell, which can produce ET-1 and expresses ETA receptors coupled to degranulation [33
]. In fact, mast cells can act as major contributors to ET-1-induced inflammation [34
, 35
]. Although ET-1 production by mast cells of the sublining synovial tissue or perivascular areas of the synovium has yet to be demonstrated, these cells play a pivotal role in the development of inflammatory arthritis [36
], during which they increase in number, produce inflammatory mediators such as LTB4 and TNF-, and release their granular content [37
]. Our observations that a large proportion of mast cells in synovial tissue following i.a. zymosan injection was clearly degranulated and that knee-joint edema was inhibited by the mast stabilizing drug cromolyn lend support for an active role of mast cells in zymosan-induced, articular inflammation.
Another relevant finding of the present study was that blockade of ETA or ETB receptors inhibited not only zymosan-induced leukocyte infiltration but also the augmented production of TNF- at 6 h, KC/CXCL1 at 24 h, and LTB4 at both time-points after i.a. stimulation. In agreement, rheumatic inflammatory diseases such as RA and gout are associated with a dramatic influx of neutrophils into joints [13
, 38
]. Therapeutic blockade of TNF- or IL-1β suppresses RA in animal models and humans [39
, 40
]. Indeed, we have previously shown that ETs modulate the production of TNF- and CXCL1 in other inflammatory models [26
]. In murine arthritis models, LTB4, acting on LTB4 receptor 1, is important for disease development, and neutrophil migration into the joints [41
42
43
] and administered i.a. into the knee joint is, per se, capable of inducing neutrophil influx [41
]. In addition, LTB4 modulates the production of IL-1β and chemokines active on neutrophils, such as epithelial-derived neutrophil-activating factor-78/CXCL5 and MIP-1β/CCL4 [41
]. Somewhat surprisingly, blockade of ET receptors caused no changes in IL-1β content in knee extracts, although IL-1β exerts a pivotal role in promoting the chronic arthritis and cartilage erosion associated with different experimental models [44
, 45
]. In zymosan-induced arthritis, increased levels of IL-1β have been demonstrated previously, which were associated with nociception [46
, 47
]. However, based on our results, it seems that edema formation and cell recruitment induced by zymosan seem to occur independently of IL-1β modulation, although ETs contribute to the development of articular inflammation in this model. It remains to be assessed if the situation would be similar at later stages after i.a. zymosan injection. Hence, our data strongly suggest that during zymosan-induced, articular inflammation, neutrophil migration into joints requires a concerted interaction of ETs with receptors coupled to distinct cytokine/chemokine signaling mechanisms at different time-points of the response. More specifically, ETA and ETB receptors are involved in the early enhancement of TNF- production, in the augmentation of LTB4 levels throughout the early and later stages of the inflammatory response, and in increased KC/CXCL1 levels only at the later stage. However, as ETs are able to directly induce neutrophil activation and chemotaxis in vitro [12
, 48
], it is possible that part of the neutrophil accumulation induced by zymosan may have been a result of direct activation of neutrophils by ETs.
In conclusion, the current study demonstrates an important role of ETs in zymosan-induced arthritis, acting through receptors ETA and ETB. The mechanisms triggered by ETs to induce edema formation and neutrophil migration seem to rely on the modulation of TNF-, LTB4, and KC/CXCL1 production.
ACKNOWLEDGEMENTS
This work was supported by grants from Conselho Nacional de Pesquisa (CNPq; Brazil). F. d. P. C. is a fellow of CNPq. The authors are grateful to Daniela Pacheco and Marcelo Meuser-Batista (MSc) for histological analysis, Simone Vargas da Silva for Western blots, Actelion Pharmaceutical Ltd. (Allschwil, Switzerland) for the generous gift of bosentan, and Fernanda Schnoor for language revision.
FOOTNOTES
1 These authors contributed equally to this work. 
Received December 12, 2007; revised April 8, 2008; accepted April 22, 2008.
REFERENCES
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