Transplant Immunology 16 (2006) 105 111 www.elsevier.

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Antiinf lammatory effects of Tacrolimus in a mouse model of pleurisy

Robson Pereira a,c , Yara Santos Medeiros b,d , Tnia Silvia Frde c,
b

Department of Medical Science, Universidade Federal de Santa Catarina, Campus Universitrio Trindade, 88040-970, Florianpolis, SC, Brazil Pharmacology and Clinical Analysis, Universidade Federal de Santa Catarina, Campus Universitrio Trindade, 88040-970, Florianpolis, SC, Brazil c Department of Clinical Analysis, Center for Health Sciences and Center, Universidade Federal de Santa Catarina, Campus Universitrio Trindade, 88040-970, Florianpolis, SC, Brazil d Center for Biological Science, Universidade Federal de Santa Catarina, Campus Universitrio Trindade, 88040-970, Florianpolis, SC, Brazil
a

Received 17 January 2006; received in revised form 18 April 2006; accepted 25 April 2006

Abstract Introduction: Tacrolimus is an antibiotic macrolide with immunosuppressant properties isolated from Streptomyces tsukubaensis. Objectives: This study evaluated whether the acute and systemic administration of Tacrolimus significantly interfered in leukocyte migration, exudation, myeloperoxidase and adenosine-deaminase and nitric oxide levels, as well as Interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF) levels in a mouse model of pleurisy in comparison to those obtained with dexamethasone. Materials and methods: Pleurisy was induced by carrageenan (Cg, 1%), bradykinin (BK, 10 nmol), histamine (HIS, 1 mol) or substance P (PS, 20 nmol) administered by intrapleural route (ipl.) and the inflammatory parameters (cell migration and exudation) were analyzed 4 h after. In the model of pleurisy induced by carrageenan, other markers in the pleural fluid, such as cytokines (TNF and Il-1), nitrite/nitrate (NOx), myeloperoxidase (MPO) and adenosine-deaminase (ADA) levels, were also studied. Dexamethaseone (0.5 mg/kg, i.p., 0.5 h before) was also analyzed in all protocols. Results: In the pleurisy induced by carrageenan, Tacrolimus (1 mg/kg, i.p.) and dexamethasone (0.5 mg/kg, i.p.) administered 0.5 h before caused a significant decrease in leukocytes, neutrophils and exudation (P b 0.01). Under the same conditions, Tacrolimus and dexamethasone did not modify the blood's white or red cells (P N 0.05). Tacrolimus showed a long lasting antiinflammatory effect, inhibiting leukocytes and neutrophils for up to 24 h (P b 0.01), whereas the inhibition of exudation was less marked (up to 2 h) (P b 0.01). These drugs caused a marked reduction in MPO activity, as well as IL-1 and TNF levels (P b 0.01), but only Tacrolimus inhibited ADA activity (P b 0.01). On the other hand, dexamethasone, but not Tacrolimus, inhibited NOx levels (P b 0.01). In the same conditions, Tacrolimus significantly inhibited cell migration induced by either bradykinin, histamine or substance P (P b 0.05). In a similar manner, dexamethasone inhibited leukocyte influx induced by bradykinin and histamine (P b 0.05). Regarding exudation effects, dexamethasone markedly inhibited this parameter induced by BK, HIS or SP, whereas Tacrolimus only inhibited exudation caused by HIS (P b 0.05). Conclusions: The results of the present work indicate that Tacrolimus showed important antiinflammatory properties against pleurisy in mice that are different from those caused by dexamethasone. The inhibition of proinflammatory cytokine (TNF, IL-1), enzyme (myeloperoxidase, adenosine-deaminase) and mediator (bradykinin, histamine, substance P) release and/or action appears to account for Tacrolimus's actions. 2006 Elsevier B.V. All rights reserved.
Keywords: Tacrolimus; Mouse pleurisy; Leukocytes; Cytokines; Mediators of inflammation

1. Introduction Tacrolimus (FK 506) is an antibiotic macrolide isolated from Streptomyces tsukubaensis [1]. It is an immunosuppressive agent with an increasing number of clinical applications including pre Corresponding author. Tel.: +55 48 9961 48 46; fax: +55 48 32 44 09 36. E-mail addresses: saleh@ccs.ufsc.br, taniafrode@zipmail.com.br (T.S. Frde). 0966-3274/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2006.04.001

vention of acute rejection of allograft transplants [26] and atopic dermatitis [7]. Other preclinical studies have demonstrated that Tacrolimus exerts antirheumatic effects in an arthritis model [8]. The immunosuppressive properties of Tacrolimus result primarily from its action on T-helper lymphocytes. Tacrolimus binds to intracellular proteins termed immunophilins. The drugimmunophilin complex inhibits calcineurin phosphatase, an enzyme involved in the activation of the transcription nuclear factor of activated T cells (NF-AT) that is linked to the expression of

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cytokine genes [9]. This results in the promotion of a reduction in proinflammatory cytokines such as interleukin-2 (IL-2) and interferon-gamma (IFN-) [10,11]. 2. Objectives This study was designed to examine the effects of Tacrolimus on acute inflammation in a mouse model of pleurisy compared to those obtained with a classical steroidal antiinflammatory drug (dexamethasone). Specifically, we evaluated the effects of this drug on the pleurisy induced by different phlogogens (carrageenan, bradykinin, histamine and substance P) where several proinflammatory markers are released. 3. Materials and methods 3.1. Animals Swiss mice, weighing 1822 g, were housed under standardized conditions (room at constant temperature (25 C) with alternating 12-h periods of light and darkness) and fed a standard mouse diet with water ad libitum before use. This study was approved by the Committee for Ethics in Animal Research (CEUA) of our university and performed in accordance with norms of the Brazilian College of Animal Experimentation (COBEA) [12].

3.2. Experimental protocol Pleurisy experiments were carried out as previously described [1316] and different phlogistic agents were employed (Cg; BK; HIS and SP). Cell migration and exudation were evaluated 4 h after phlogogen administration. Myeloxperoxidase (MPO), adenosine deaminase (ADA), nitrate/nitrate concentrations (NOx), tumor necrosis factor alpha (TNF) and interleukin-1 beta (IL-1) levels were evaluated only in the protocols of carrageenan-induced pleurisy. Samples of the fluid collected from the pleural cavity were stored in a freezer ( 20 C) and determinations of the exudation (amount of Evans Blue dye) were made as scheduled on different days. Enzymes and cytokine assays were processed on the same day as the pleurisy protocols. Initially, to establish a standard Tacrolimus dose and timing to be used in the experiments, a group of animals was treated (0.5 h before) with either different doses of Tacrolimus (0.51.5 mg/kg) administered by intraperitoneal route (i.p.) or treated with sterile saline administered by intrapleural route (ipl.), and the inflammatory parameters (cell migration and exudation) were analyzed 4 h after carrageenan injection (Cg 1%, ipl.). In another set of experiments, animals were pretreated with Tacrolimus (1.0 mg/kg, i.p.) at different time points (0.572 h) and the same inflammatory parameters were evaluated 4 h after pleurisy induction. According to this protocol, Tacrolimus (1.0 mg/kg,

Fig. 1. Effects of different doses of Tacrolimus (TAC: 0.51.5 mg/kg, i.p.) on the mouse model of pleurisy induced by carrageenan (Cg, 1%). Effects of this drug upon leukocytes (A), neutrophils (B), mononuclears (C) and exudation (D). C = control = responses in animals treated only with sterile saline (NaCl, 0.9%); Cg = responses in animals treated only with carrageenan, DEX = responses in animals treated with dexamethasone (0.5 mg/kg, i.p.) plus carrageenan. Each group represents the mean of 3 to 6 animals and the vertical bars the S.E.M. Significant differences determined by ANOVA complemented with Dunnett's or Student's t tests. P b 0.01.

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0.5 h before) was elected as the dose to be used in the experiments below. Animals were randomly allocated in twelve groups: (1) Control-treated with sterile saline (NaCl, 0.9%, ipl.), (2) Cg (1%, ipl.), (3) Cg (1%, ipl.) plus Tacrolimus (0.51.5 mg/kg, i.p.), (4) Cg (1%, ipl.) plus dexametahasone (0.5 mg/kg, i.p.), (5) BK (10 nmol, ipl.), (6) BK (10 nmol, ipl.) plus Tacrolimus (1.0 mg/kg, i.p.), (7) BK (10 nmol, ipl.) plus dexamethasone (0.5 mg/kg, i.p.), (8) HIS (1 g, ipl.), HIS (1 g, ipl.) plus Tacrolimus (1.0 mg/kg, i. p.), (9) HIS (1 g, ipl.) plus dexamethasone (0.5 mg/kg, i.p.), (10) SP (20 nmol, ipl., (11), SP (20 nmol, ipl.) plus Tacrolimus (1.0 mg/ kg, i.p.), and (12) SP (20 nmol, ipl.) plus dexamethasone (0.5 mg/ kg, i.p.). Tacrolimus and dexamethasone were administered 0.5 h prior to pleurisy induction. In the pleurisy induced by bradykinin, the animals were also treated with captopril (5 mg/kg, i.p., 0.5 h before) to prevent the action of kininases [17]. In some groups of animals, blood samples by cardiac puncture were also collected for determination of white and red cells. 3.3. Quantification of cell migration and exudation After killing the animals (4 h after pleurisy), samples of the pleural cavity fluid were collected for determinations of total and differential leukocyte contents and of exudation. Total leukocyte counts were performed on an automatic counting machine (Beckman Coulter Inc., Brea, CA, USA), while cytospin preparations of

pleural wash were stained with MayGrnwaldGiemsa for the differential count, which was performed under an oil immersion objective. The degree of exudation was determined by the measurement of the amount of Evans Blue dye extravasation in the pleural liquid as previously described [1316]. Thus, in each experimental group, animals were previously challenged (1 h) with a solution of Evans blue dye (25 mg/kg) administered by intravenous route (i.v.) in order to evaluate the degree of exudation in the pleural cavity. On the day of the experiments, a batch of stored samples was thawed at room temperature and the amount of dye was estimated by colorimetry using an Elisa plate reader (Organon Teknika, Roseland, New Jersey, USA) at 600 nm, by interpolation from a standard curve of Evans blue dye in the range of 0.01 to 50 g/ml. 3.4. Quantification of nitrate/nitrite concentrations in carrageenan-induced pleurisy Nitric oxide was measured as its breakdown products nitrite (NO2 ) and nitrate (NO3 ) using the Griess method [18]. Samples of the pleural fluid obtained from control (treated with sterile saline), carrageenan-treated animals and animals pretreated with either Tacrolimus or dexamethasone were collected, separated and stored at 20 C. The levels of nitrate/nitrite were determined as previously described by Saleh et al. [19]. Results were expressed as M.

Fig. 2. Effects of Tacrolimus (TAC: 1 mg/kg, i.p.) administered 0.5 h to 72 h before carrageenan administration on the mouse model of pleurisy induced by carrageenan (Cg, 1%). Effects of this drug upon leukocytes (A), neutrophils (B), mononuclears (C) and exudation (D). C = control = responses in animals treated only with sterile saline (NaCl, 0.9%); Cg = responses in animals treated only with carrageenan, DEX = responses in animals treated with dexamethasone (0.5 mg/kg, i.p.) plus carrageenan. Each group represents the mean of 3 to 6 animals and the vertical bars the S.E.M. Significant differences determined by ANOVA complemented with Dunnett's or Student's t tests. P b 0.01.

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3.5. Quantification of myeloperoxidase and adenosine-deaminase activities in carrageenan-induced pleurisy In-house assays of both MPO and ADA were employed according to the methods developed by Rao et al. [20] and Giusti and Galanti [21]. Using conventional reagents, the concentration of each enzyme was estimated by means of colorimetric measurements (absorbances of 450 and 630 nm, respectively) in an ELISA plate reader (Organon Tecknica). One unit of MPO is defined as the activity of the enzyme that oxidizes 1 mol of H2O2/ min, whereas one unit of ADA is equivalent to the amount of enzyme required to release 1 mmol of ammonia/min. Results were expressed as mU/ml (MPO) and U/l (ADA). Detailed descriptions of these assays have been previously published [19,22]. 3.6. Quantification of TNF and IL-1 in carrageenan-induced pleurisy Samples of the pleural fluid obtained from control (treated with sterile saline) and carrageenan-treated animals, and animals pretreated with either Tacrolimus or dexamethasone, were collected and immediately prepared for the analysis of cytokine levels. In this protocol, commercially available kits were used with monoclonal specific antibodies for each cytokine. The cytokine levels

were measured by enzyme-linked immunosorbent assay (ELISA), using Bioscience Pharmigen, USA (for TNF) and Immuno Biological Laboratories Co. Ltd., Japan (for IL-1) kits according to the manufacturers' instructions. The ranges of the values detected by these assays were: TNF (52000 pg/ml) and IL-1 (1006400 pg/ml). The intra and interassay coefficients of variation (CV) for TNF and IL-1 were: intra CV: TNF = 7.8 0.9%; and IL-1: = 6.2 0.4%; inter CV: TNF = 9.6 2.1% and IL-1 = 5.1 0.6%, sensitivities of TNF = 5 pg/ml and IL1 = 1.67 pg/ml. All cytokine concentrations were estimated by means of colorimetric measurement at 450 nm on an ELISA plate reader (Organon Teknika, Roseland, NJ, USA) by interpolation from a standard curve. 3.7. Drugs The following drugs and reagents were used: Tacrolimus (Prograf, Fujisawa Ireland Ltd., Killorgin, Ireland), carrageenan (degree IV), bradykinin, histamine, substance P, human neutrophil myeloperoxidase (Sigma Chemical Co., St. Louis, MO, USA), dexamethasone (Prodome Qumica e Farmacutica Ltda, Campinas, SP, Brazil), captopril (Ranbaxy Laboratories Limited Industrial, Dewas Madhya Pradesh, India), enzyme-linked immunosorbent assay (ELISA) for quantitative determination of mouse

Fig. 3. Effects of Tacrolimus (TAC: 1 mg/kg, i.p.) administered 0.5 h before carrageenan administration on the mouse model of pleurisy induced by carrageenan (Cg, 1%). Effects of this drug upon myeloperoxidase (A) and adenosine-deaminase (B) activities and nitrite/nitrate levels. (C). C = control = responses in animals treated only with sterile saline (NaCl, 0.9%); Cg = responses in animals treated only with carrageenan, DEX = responses in animals treated with dexamethasone (0.5 mg/kg, i. p.) plus carrageenan. Each group represents the mean of 3 to 6 animals and the vertical bars the S.E.M. Significant differences determined by ANOVA complemented with Dunnett's or Student's t tests. P b 0.01.

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Fig. 4. Effects of Tacrolimus (TAC: 1 mg/kg, i.p.) administered 0.5 h before carrageenan administration on the mouse model of pleurisy induced by carrageenan (Cg, 1%). Effects of this drug upon IL-1 (A) and TNF (B) levels. C = control = responses in animals treated only with sterile saline (NaCl, 0.9%); Cg = responses in animals treated only with carrageenan, DEX = responses in animals treated with dexamethasone (0.5 mg/kg, i.p.) plus carrageenan. Each group represents the mean of 3 to 6 animals and the vertical bars the S.E.M. Significant differences determined by ANOVA complemented with Dunnett's or Student's t tests. P b 0.01.

TNF (BD Biosciences Pharmingen, San Diego, CA, USA) and rat IL-1 (IBL Immuno Biological Laboratories Co. Ltd., Fujioka city, Gunma, Japan). MayGrnwald dye (Newprov, Pinhais, PR, Brazil) and Giemsa dye (Laborclin, Pinhais, PR, Brazil). Other reagents used were of analytical grade and were obtained from different commercial sources. 3.8. Statistical analysis Data are reported as mean SEM. Significant differences between groups were determined by analysis of variance (ANOVA) complemented with Dunnett's and/or Student's t tests. p b 0.05 was considered as indicative of significance. 4. Results 4.1. Pleurisy induced by carrageenan 4.1.1. Effects of Tacrolimus and dexamethasone on cellular infiltration and exudation Tacrolimus (1.0 mg/kg, i.p., 0.5 h) caused a significant decrease in leukocyte migration when it was administered 0.5 h before carrageenan (% of inhibition: 31.1 4.6) (P b 0.01) (Fig. 1A). This reduction was attributed to inhibition of neutrophil influx (% of inhibition: 33.9 4.9) (P b 0.01) (Fig. 1B). Tacrolimus did not alter mononuclear migration (P N 0.05) (Fig. 1C). Under the same conditions, exudation was also inhibited by Tacrolimus (1 mg/kg) (% of inhibition: 27.3 8.1) (P b 0.01) (Fig. 1D). Dexamethasone caused a similar effect on all studied parameters. This drug inhibited leukocytes (% of inhibition: 64.5 5.8), neutrophils (% of inhibition: 68.1 4.2), and exudation (% of inhibition: 32.6 7.8) (P b 0.01) (Figs. 1A, B, D, 2A, B and D). The time course analysis also showed that the pretreatment of animals with Tacrolimus (1.0 mg/kg) had a more marked effect on cell migration (maximal inhibition up to 24 h) (P b 0.01) than exudation (maximal inhibition up to 2 h) (P b 0.01) (Figs. 2A and D). Tacrolimus significantly decreased the leukocyte influx (% of inhibition: 2 h: 44.8 8.0; 4 h: 34.4 15.4 and 24 h: 25.6 5.6)

(P b 0.01) (Fig. 2A). This inhibitory effect was due to inhibition of neutrophil migration (% of inhibition 2 h: 55.1 4.1; 4 h: 38.5 16.0 and 24 h: 31.8 6.9) (P b 0.01) (Fig. 2B). The exudation was also inhibited by Tacrolimus (% of inhibition: 2 h: 21.0 8.2) (P b 0.01) (Fig. 2D). Again, no effect was detected on mononuclear cell influx (P N 0.05) (Fig. 2C).
Table 1 Effects of Tacrolimus and dexamethasone upon cell migration and exudation levels in the pleurisy induced by bradykinin, histamine or substance P Group/dose Absolute values Bradykinin (10 nmol/cav.) Leukocytes (10 ) Ca TAC (1.0 mg/kg)b DEX (0.5 mg/kg)b Neutrophils (106) Ca TAC (1.0 mg/kg)b DEX (0.5 mg/kg)b Mononuclears (106) Ca TAC (1.0 mg/kg)b DEX (0.5 mg/kg)b Exudation (g/ml) Ca TAC (1.0 mg/kg)b DEX (0.5 mg/kg)b
6

Histamine (1 g/cav.) 1.95 0.15 1.64 0.05 1.26 0.15

Substance P (20 nmol/cav.) 2.38 0.25 1.65 0.18 2.00 0.17

2.12 0.07 1.54 0.09 1.00 0.04

0.17 0.04 0.17 0.03 0.05 0.08

0.40 0.01 0.35 0.09 0.19 0.03

0.81 0.06 0.52 0.06 0.80 0.40

1.94 0.11 1.37 0.16 0.95 0.70

1.55 0.15 1.31 0.06 1.07 0.13

1.58 0.19 1.21 0.11 1.20 0.50

1.96 0.05 2.23 0.18 1.40 0.3

2.20 0.24 1.54 0.14 1.45 0.11

1.66 0.07 1.78 0.15 1.3 0.50

Tacrolimus (1 mg/kg i.p.) or dexamethasone (0.5 mg/kg) administered to different groups of animals 0.5 h before pleurisy induction by bradykinin, histamine or substance P. C = control = responses in animals treated only with bradykinin (10 nmol/cav.), histamine (100 g/cav.) or substance P (20 nmol/ cav.). TAC = responses in animals pretreated with Tacrolimus (1 mg/kg, i.p., 0.5 h). DEX = responses in animals pretreated with dexamethasone (0.5 mg/kg, i.p., 0.5 h). Each value represents the mean S.E.M. of 5 to 6 animals. P b 0.05 and P b 0.01. , indicate statistical significance. a = administered by intrapleural route; b = administered by intraperitoneal route.

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4.1.2. Effect of Tacrolimus and dexamethasone on blood cells Tacrolimus (0.51.5 mg/kg, i.p.) or dexamethasone (0.5 mg/ kg, i.p.) did not change the blood's white or red cells in this model of pleurisy (results not shown). 4.1.3. Effects of Tacrolimus and dexamethasone on nitrite/nitrate concentrations, myeloperoxidase and adenosine-deaminase activities Tacrolimus (1 mg/kg) caused a marked reduction in MPO and ADA activities (% of inhibition: MPO: 50.7 6.3; ADA: 39.4 3.5) (P b 0.01) (Figs. 3A and B), but not in nitrite/nitrate concentrations (P N 0.05) (Fig. 3C) when compared with carrageenan-treated animals. On the other hand, dexamethasone (0.5 mg/kg) significantly inhibited both MPO (% of inhibition: 44.3 2.6) (P b 0.01) and nitrite/nitrate concentrations (% of inhibition: 51.6 4.0) (P b 0.01) (Figs. 3A and C), but not ADA levels (P N 0.05) (Fig. 3B). 4.1.4. Effects of Tacrolimus and dexamethasone on cytokine levels In this protocol, Tacrolimus (1 mg/kg), as well as dexamethasone (0.5 mg/kg), significantly decreased both IL-1 (% of inhibition: Tacrolimus: 47.8 1.9; dexamethasone: 41.4 2.0) (P b 0.01) (Fig. 4A) and TNF levels (% of inhibition: Tacrolimus: 41.6 3.9; dexamethasone: 30.9 4.2) (P b 0.01) (Fig. 4B). 4.2. Pleurisy induced by bradykinin, histamine or substance P 4.2.1. Effect of Tacrolimus and dexamethasone on cellular infiltration and exudation Tacrolimus (1 mg/kg, i.p., 0.5 h) significantly reduced leukocyte influx in the pleurisy induced by either BK, HIS or SP (P b 0.05) (Table 1). This inhibitory effect was due to inhibition of mononuclear cells in the pleurisy induced by either bradykinin or histamine (P b 0.05) and of neutrophils in the pleurisy induced by substance P (P b 0.01) (Table 1). However, Tacrolimus only inhibited the exudation induced by histamine (P b 0.05) (Table 1). In relation to dexamethasone (0.5 mg/kg, i.p., 0.5 h), this drug significantly inhibited the cell migration (neutrophils, mononuclears) and exudation stimulated by either bradykinin or histamine (P b 0.05) (Table 1). In contrast to Tacrolimus effects, dexamethasone was able to inhibit exudation induced by all phlogogens (P b 0.05) (Table 1). 4.2.2. Effect of Tacrolimus and dexamethasone on blood cells Neither Tacrolimus nor dexamethasone at the studied doses changed the blood's white or red cells when the pleurisy was triggered by the studied phlogogistic agents (results not shown). 5. Discussion This study shows that Tacrolimus exerts an acute anti-inflammatory effect in a model of inflammation caused by different phlogogens. The results allowed us to characterize a marked inhibitory profile upon cell migration, regarding both neutrophils and mononuclears, besides a slight but significant inhibition of exudation. Since dexamethasone presented the same antiinflam-

matory profile, it is possible that both drugs may be acting via common and distinct pathways. The antiinflammatory and immunosuppressant effects of Tacrolimus and dexamethasone rely on several molecular mechanisms. In common, both drugs reduced transcriptional activation of Activating protein-1 (AP-1) and NF-kB factors that are linked to the activation of early cytokine genes [9,10,2326]. Furthermore, nongenomic mechanisms may also be involved in these antiinflammatory effects [27]. In this study, both drugs significantly decreased TNF and IL-1 levels which are potent triggers of many of the actions involved in leukocyte migration [2831]. Both drugs inhibited cell migration by decreasing the influx of neutrophils, the most representative cells of carrageenan pleurisy, and of mononuclears, in the inflammation caused by both bradykinin and histamine [1315]. Analysis of their effects on this model also showed that the decrease of neutrophil influx in the carrageenan model was also associated with an inhibition of myeloperoxidase, corroborating their inhibitory effect upon neutrophils [19]. On the other hand, distinct effects of these drugs were observed in relation to other inflammatory parameters such as ADA levels and nitrate/nitrite concentrations in the pleural fluid, indicating that different pathways were activated. To support this hypothesis, we noted that dexamethasone inhibitory effects on exudation were associated with a decrease in nitrate/ nitrite levels, an effect that involves, among others, the activation of endothelial nitric oxide synthetase [27], whereas Tacrolimus did not modify this parameter. It is suggested that in this model, Tacrolimus may be exerting its antiinflammatory effects via other mediator pathways besides nitric oxide. This lack of effect of Tacrolimus upon nitrate/nitrite levels is in great contrast to the inhibition caused by other immunosuppressant drugs such as cyclosporine and methotrexate in the same model [32,33]. On the other hand, whereas Tacrolimus was able to inhibit ADA levels in the pleural fluid, no effect was detected when dexamethasone was employed. The effects of Tacrolimus upon cell migration was longlasting in comparison to its effects on exudation. Again, both Tacrolimus and dexamethasone inhibited this parameter in the pleurisy induced by carrageenan, bradykinin and histamine. A possible explanation for these findings may be the fact that Tacrolimus inhibits the cyclooxygenase-2 mRNA expression and prostaglandins synthesis that is involved in the exudate formation in the inflammatory process [34]. In relation to the pleurisy model induced by substance P, opposite inhibitory effects were observed. Dexamethasone only inhibited exudation, whereas Tacrolimus only inhibited cell migration. These data reinforce the hypothesis that these drugs may be acting via different pathways. In addition, Tacrolimus's inhibitory effects upon cell migration and exudation induced by histamine are in agreement with the reports that this drug inhibits the exocytosis of mast cells in rat basophilic leukemia cells, promoting a decrease in the release of many mediators including histamine [35]. In conclusion, Tacrolimus showed important antiinflammatory properties against pleurisy in mice. The inhibition of proinflammatory cytokine (TNF, IL-1), enzyme (myeloperoxidase, adenosine-deaminase) and mediator (bradykinin,

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histamine, substance P) release and/or action appears to account for Tacrolimus's actions. In this model of inflammation, the majority of its action was similar to that induced by dexamethasone. However, the limitations observed in this model warrant the continued study of the antiinflammatory effects of Tacrolimus. Acknowledgement

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The authors thank Jansen-Cilag, the Universidade Federal de Santa Catarina and the Universidade do Panalto Catarinense for technical support. R. Pereira is a Master's degree student. References
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