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Acute pulmonary inflammation induced by exposure of the airways to staphylococcal enterotoxin type B in rats
Toxicology and Applied Pharmacology 217 (2006) 107 – 113 www.elsevier.com/locate/ytaap
Acute pulmonary inflammation induced by exposure of the airways to staphylococcal enterotoxin type B in rats Ivani A. Desouza a,⁎,1 , Carla F. Franco-Penteado a , Enilton A. Camargo a , Carmen S.P. Lima b , Simone A. Teixeira c , Marcelo N. Muscará c , Gilberto De Nucci a , Edson Antunes a b
a Department of Pharmacology, State University of Campinas (UNICAMP), P.O. Box 6111, 13084-971, Campinas (SP), Brazil Laboratory of Haemathology of Faculty of Medical Sciences, State University of Campinas (UNICAMP), P.O. Box 6111, 13084-971, Campinas (SP), Brazil c Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo (USP), São Paulo (SP), Brazil
Received 11 May 2006; revised 30 June 2006; accepted 6 July 2006 Available online 14 July 2006
Abstract Staphylocococcus aureus is a gram-positive bacterium that produces several enterotoxins, which are responsible for most part of pathological conditions associated to staphylococcal infections, including lung inflammation. This study aimed to investigate the underlying inflammatory mechanisms involved in leukocyte recruitment in rats exposed to staphylococcal enterotoxin B (SEB). Rats were anesthetized with pentobarbital sodium and intratracheally injected with either SEB or sterile phosphate-buffered saline (PBS, 0.4 ml). Airways exposition to SEB (7.5–250 ng/ trachea) caused a dose- and time-dependent neutrophil accumulation in BAL fluid, the maximal effects of which were observed at 4 h post-SEB exposure (250 ng/trachea). Eosinophils were virtually absent in BAL fluid, whereas mononuclear cell counts increased only at 24 h post-SEB. Significant elevations of granulocytes in bone marrow (mature and immature forms) and peripheral blood have also been detected. In BAL fluid, marked elevations in the levels of lipid mediators (LTB4 and PGE2) and cytokines (TNF-α, IL-6 and IL-10) were observed after SEB instillation. The SEB-induced neutrophil accumulation in BAL fluid was reduced by pretreatment with dexamethasone (0.5 mg/kg), the COX-2 inhibitor celecoxib (3 mg/kg), the selective iNOS inhibitor compound 1400 W (5 mg/kg) and the lipoxygenase inhibitor AA-861 (200 μg/kg). In separate experiments carried out with rat isolated peripheral neutrophils, SEB failed to induce neutrophil adhesion to serum-coated plates and chemotaxis. In conclusion, rat airways exposition to SEB causes a neutrophil-dependent lung inflammation at 4 h as result of the release of proinflammatory (NO, PGE2, LTB4, TNF-α, IL-6) and anti-inflammatory mediators (IL-10). © 2006 Elsevier Inc. All rights reserved. Keywords: Cyclooxygenase-2; Cytokines; Enterotoxins; Neutrophil influx; Nitric oxide
Introduction Staphylocococcus aureus is a gram-positive bacterium often found in the normal microflora of skin, upper respiratory and Abbreviations: BAL, brochoalveolar lavage; COX-2, cyclooxygenase-2; fMLP, N-formyl-methionyl-leucyl-phenilalanine; IL, interleukin; iNOS, inducible nitric oxide synthase; L-NAME, N ω-nitro-L-arginine methyl ester; LTB4, leukotriene B4; MEM, Eagle's minimum essential medium; MPO, myeloperoxidase; NO, nitric oxide; PBS, phosphate-buffered saline; PGE2, prostaglandin E2; SEB, Staphylococcal enterotoxin type B; TNF-α, tumoral necrosis factor-α ⁎ Corresponding author. Fax: +55 19 32892968. E-mail address: [email protected] (I.A. Desouza). 1 Current address: Department of Pharmacology; Faculty of Medical Sciences, State University of Campinas (UNICAMP), PO Box 6111, CEP 13084-971, Campinas, SP, Brazil. 0041-008X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2006.07.001
gastrointestinal tracts in humans (Bachert et al., 2002, 2003). This bacterium produces and secretes the so-called staphylococcal enterotoxins, which are a family of structurally related heat-stable 23 to 29-kDa proteins. They comprise several serological types, namely the classical types A to E, and the newly characterized types G to K (Balaban and Raooly, 2000; Abe et al., 2000; Orwin et al., 2001). Staphylococcal enterotoxins often lead to multiorgan dysfunction in humans and animals (Kotzin et al., 1993; Lowy, 1998; Müller-Alouf et al., 2001) which may be related to their superantigenic properties (Balaban and Raooly, 2000; Yarwood et al., 2001). Clinical evidences suggest a link between bacterial organisms and pathogenesis and/or exacerbation of human upper airway disease (Kraft, 2000; Bachert et al., 2003; Rossi and Monasterolo, 2004). In addition, several studies report that
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IgE-independent bronchial asthma can be provoked by bacterial infections, including Gram-positive bacteria (Tuner et al., 1995; Wenzel et al., 1999; Douwes et al., 2002). Recently, airways exposition to staphylococcal enterotoxin type A (SEA) in rats has been shown to evoke a large influx of neutrophils in bronchoalveolar lavage fluid by mechanisms involving complex interactions of different signaling pathways including overexpression of cytokine-induced neutrophil chemoattractant (CINC-2), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), as well as enhanced production of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 (Desouza et al., 2005). Staphylococcal enterotoxin type B (SEB) can be easily obtained in relatively large amounts and purity, but few studies have attempted to investigate the pulmonary inflammation induced by airways exposure to this enterotoxin. Intranasal administration of staphylococcal enterotoxin B (SEB) in mice has been shown to induce mucosal and airway recruitment of polymorphonuclear and mononuclear cells (Neumann et al., 1997; Herz et al., 1999) that was associated with increased levels of IL-4 and TNF-α production in bronchoalveolar lavage (BAL) fluid (Herz et al., 1999), but no further studies have explored the mechanisms underlying the SEB-induced pulmonary inflammation, particularly the airways neutrophil infiltration. Therefore, in this study, rat airways have been exposed to SEB in a dose- and time-dependent manner in order to understand the underlying inflammatory mechanisms determining the pulmonary neutrophil infiltration.
Materials and methods Materials. Staphylococcal enterotoxin B (SEB), minimum essential medium (MEM), Dextran, Nω-nitro-L-arginine methyl ester (L-NAME), AA-861, Nformyl-L-methionyl-L-leucyl-phenylalanine (fMLP), hexadecytrimethyl-ammonium bromide (HTAB) and dexamethasone were purchased from Sigma Chemical Co. (St. Louis, MO, USA). N-(3-(aminomethyl)benzyl)acetamidine (1400 W) was purchased from Alexis (Nottingham, UK). Celecoxib was obtained from Laboratórios Pfizer Ltd. (São Paulo, Brazil). Enzyme-linked immunosorbent assays (ELISA) for rat TNF-α were obtained from BD Biosciences (CA, USA), whereas ELISA for rat LTB4, PGE2, IL-6 and IL-10 were obtained from R&D Systems (MN, USA). Animal experimentation guidelines. The experimental protocols were approved by the Ethical Principles in Animal Research adopted by the Brazilian College for Animal Experimentation (COBEA). Male Wistar rats (250–300 g; 7–8 weeks age) were housed in temperature-controlled rooms and received water and food ad libitum until used. Intratracheal injection of SEB. Rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and intratracheally injected with SEB (7.5–250 ng/ trachea, 0.4 ml). Control animals received 0.4 ml of sterile phosphate-buffered saline (PBS) alone. At selected time thereafter (4, 16, 24 and 48 h), animals were sacrificed by overdose of inhaled halothane, and then peripheral blood, bronchoalveolar lavage (BAL) fluid and bone marrow were collected to assess the total and differential leukocyte counts. Leukocyte counts in BAL fluid. Bronchoalveolar lavage was performed at 4 to 48 h after SEB (or PBS) intratracheal injection. Briefly, the trachea was exposed and cannulated with a polyethylene tube (1 mm in diameter) connected to a syringe. The lungs were washed by flushing with PBS solution containing heparin (20 UI/ml). The PBS buffer was instilled through the tracheal cannula as
one 10-ml aliquot followed by three 5-ml aliquots (making a total of approximately 25 ml). The first 10-ml aliquot was centrifuged (1000×g for 10 min at 20 °C), and the supernatant recovered and stored at − 80 °C for measurement of inflammatory mediators. The three 5-ml aliquots were pooled and centrifuged (1000×g for 10 min at 20 °C). The cell pellet obtained from all aliquots (the 10-ml aliquot together with those of the 5-ml aliquots) were combined and resuspended in 2 ml of PBS solution. Total cell counts were done with an automated cell counter (CELL-DYN 1700) while differential counts were carried out on a minimum of 200 cells using cytospin preparation stained with May-Grünwald. The cells were classified as neutrophils, eosinophils, mast cells, lymphocytes and macrophages based on normal morphological criteria. Leukocyte counts in peripheral blood and bone marrow. Blood samples were obtained from the abdominal artery after the SEB (or PBS) injection into the airways. Total cell counts were done with an automated cell counter (CELL-DYN 1700, USA) while differential counts were carried out on blood smears stained by the May-Grünwald method. The cells were classified as neutrophils, eosinophils, mast cells, lymphocytes, and mononuclear based on normal morphological criteria. For measurement of cells in bone marrow, femurs were removed from rats immediately after killing. The epiphyses were cut transversely and bone marrow cells were flushed out with PBS containing heparin (20 IU/ml). Differential count was carried out on a minimum of 200 cells using cytospin preparation stained with Leishman. Results are expressed as the number of leukocytes per femur. Cells were classified as immature neutrophils (myeloblast, promyelocyte and myelocyte), mature neutrophils (metamyelocyte, band and mature), eosinophils, mast cells, lymphocytes and monocytes based on normal morphological criteria. Measurement of leukotriene B4 (LTB4), TNF-α, IL-10, IL-6 and prostaglandin E2 (PGE2) in BAL fluid. Rat LTB4, TNF-α, IL-10, IL-6 and PGE2 were measured in BAL fluid supernatant using commercially available enzymelinked immunosorbent assays (ELISA) according to the manufacturer instructions. Pharmacological investigation with different drugs. The efficacy of doses and schedules of administration for each drug in particular were chosen according to previous studies, as follows: (1) dexamethasone (0.5 mg/kg) was administered i.v. 1 h before SEB exposition (Cunha and Ferreira, 1986); (2) the lypoxygenase inhibitor AA-861 (200 μg/kg) was administered i.v. 15 min before SEB exposition (Filliatre et al., 2001); (3) the selective cyclooxygenase-2 inhibitor celecoxib (3 mg/kg) was administered i.p. 1 h before SEB exposition (Filliatre et al., 2001); (4) the non-selective NOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg) was administered i.v. immediately before SEB exposition (Parmentier et al., 1999); (5) the iNOS selective inhibitor compound 1400 W (5 mg/kg) was administered i.v. immediately before SEB exposition (Franco-Penteado et al., 2001). Isolation of peripheral blood neutrophils. Neutrophils were separated from naïve rat peripheral blood in 3.13% (w/v sodium citrate (10:1) and obtained by Dextran sedimentation by Ficcol (1.077 g/l) gradient. For each experiment, a pool of blood of 5 rats was used. After separation of monocytes and granulocytes by centrifuging at 400×g for 30 min, the granulocyte layer was washed once in Eagle's minimum essential medium (MEM; pH 7.2) before performing a hypotonic lysis to disrupt the red cells. Cells were washed once again in MEM and resuspended in MEM/0.1% ovalbumin. Samples of cell suspension were used to determine total cell number using an improved Neubauer hemocytometer, and then cytospinned onto slides for a differentiation count. The final cell suspension contained 89% of neutrophils. Cell viability (>95%) was assessed by Trypan blue dye exclusion test. Neutrophil adhesion assays in vitro. 96-well plates were prepared by coating individual wells with 60 μl of serum (1:10 dilution in PBS) overnight at 4 °C. Wells were then washed twice with PBS before blocking non-coated sites with 0.1% (w/v) bovine serum albumin for 60 min at 37 °C. Wells were washed twice again with PBS before allowing plates to dry. Neutrophils (50 μl of 1 × 106 cells/ ml in MEM/ovalbumin) were seeded onto the coated wells alone or with SEB (0.3 to 10 ng/well) and cells were allowed to adhere for 30 min at 37 °C, 5% CO2. A positive control was run using the N-formyl-methionyl-leucyl-phenilalanine (fMLP). Following incubation non-adhered cell were washed twice with PBS.
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Fifty microliters of MEM was added to each well and varying numbers of the parent stock neutrophil cell suspension were added to empty wells to form a standard curve. Plates were then stored frozen overnight before measuring the myeloperoxidase (MPO) content of adherent cells (Bradley et al., 1982). Plates were defrosted on ice before extracting with hexadecytrimethyl-ammonium bromide in 50 mM potassium phosphate buffer, pH 6.0. Twenty microliters of each well sample to be measured were mixed with 200 μl of o-dianisidine solution (0.167 mg/ml o-dianiidine dihydrochloride, 0.0005% hydrogen peroxide in 50 mM phosphate buffer, pH 6.0) immediately prior to reading change of absorbance at 460 nm over 5 minutes in a microplate reader (Multiscan MS, Labsystems, CA, USA). Adherence was calculated by comparing absorbance changes of unknowns to those of the standard curve. In separate experiments, the MPO assay was carried out in presence of SEB (10 ng/well) to exclude the possibility that SEB interferes at the enzyme-substrate level. Neutrophil chemotaxis assays in vitro. Neutrophil migration assays were performed using a 96-well chemotaxis chamber (NeuroProbe, MD, USA). Twenty-five microliters of 8 × 106 cell/ml neutrophils (prepared in MEM containing 0.1% ovalbumin) were added to the upper compartment of the chamber and separated by a polycarbonate filter (5-μm pore) from the lower chamber containing 29 μl of MEM, SEB (0.01 to 1.0 ng/well) or fMLP (1 × 10− 7 M). The chambers were incubated at 37 °C in a 5% CO2 atmosphere for 120 min. The wells of the upper compartment were emptied by aspiration, and then disassembled. To detach adherent neutrophil from the filter, the microtiter plate with attached filter was centrifuged at 1200 rpm for 5 min at room temperature. The MPO activity was determined as described above. Migrated neutrophils were calculated by comparing absorbance changes of unknowns to those of the standard curve. Statistical analysis. Data are presented as the mean values ± SEM and were analyzed by analysis of variance (ANOVA) for multiple comparisons followed Bonferroni test, or Student's unpaired t-test where appropriate. In both cases, the level of significance was set at P < 0.05.
Results Neutrophil counts in BAL fluid Rat airway exposure to SEB (7.5–250 ng/trachea) caused a marked and dose-dependent neutrophil accumulation in BAL fluid with maximal responses achieved with 250 ng/trachea of SEB (Fig. 1A). Maximal neutrophil accumulation in BAL fluid (22-fold increase) was observed at 4 h after SEB injection (250 ng/trachea), decaying thereafter (at 16 and 24 h). At 48 h, the number of neutrophils in BAL fluid returned to basal counts (Fig. 1B). Eosinophils were virtually absent in BAL fluid in any time and dose studied. The number of mononuclear cells increased only at 24 h-post-SEB (250 ng/trachea) injection (n = 5; data not shown). Neutrophil counts in bone marrow and peripheral blood Airways exposition to SEB (250 ng/trachea) induced significant increases in the immature forms of granulocytes in bone marrow at 4 h thereafter, whereas the mature forms of granulocytes increased significantly at later times (24 h). A significant elevation in counts of mononuclear cell lineage in bone marrow was observed at 4 h after SEB exposition (Table 1). In peripheral blood, significant elevations of neutrophils were observed at 4 h and 16 h (data not shown). The mononuclear cell counts (lymphocytes and monocytes) in peripheral blood of SEB-exposed rats did not change significantly in comparison
Fig. 1. Neutrophil influx in BAL fluid from rats exposed to SEB. (Panel A) Rats were intratracheally injected with PBS or SEB at the indicated doses, and BAL fluid was collected at 4 h. (Panel B) Rats were intratracheally injected with PBS or SEB (250 ng/trachea) and BAL fluid was collected at the indicated times. The black bars represent the counts obtained with PBS alone. The data are the mean values ± SEM of 6 rats. *P < 0.05 compared with respective PBS group.
with PBS-injected rats in any time evaluated (data not shown, n = 5–6). Measurements of PGE2, LTB4, TNF-α, IL-6 and IL-10 levels in BAL fluid Rats were exposed to SEB (250 ng/trachea), and at 1 h (for PGE2) or 4 h (for LTB4, TNF-α, IL-6 and IL-10) later, BAL fluid was recovered in order to measure the inflammatory mediators. Fig. 2 shows that concentrations of lipid mediators (LTB4 and PGE2) and cytokines (TNF-α, IL-6 and IL-10) were significantly higher after SEB instillation when compared with control groups. Effect of different in vivo drug treatments on pulmonary neutrophil influx The neutrophil counts in BAL fluid of in non-treated and treated rats were performed at 4 h post-intratracheal injection of 250 ng/trachea of SEB. Pretreatment of rats with dexamethasone (0.5 mg/kg, i.p) nearly abolished the SEB-induced neutrophil accumulation in BAL fluid (Fig. 3). Similar inhibitory responses in the neutrophil influx were observed with the COX-2 inhibitor
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Table 1 Leukocyte counts in bone marrow of rats after the airways exposition to SEB Cells (×106/femurs) Immature granulocytes Mature granulocytes Mononuclear cells PBS SEB 4 h SEB 16 h SEB 24 h SEB 48 h
4.2 ± 0.4 7.4 ± 1.8 ⁎ 6.0 ± 0.5 5.3 ± 1.7 3.0 ± 0.2
36 ± 2.8 32 ± 6.2 46 ± 5.4 60 ± 6.5 ⁎ 48.7 ± 4.9
1.7 ± 0.3 3.8 ± 1.6 ⁎ 1.0 ± 0.3 1.7 ± 0.5 2.0 ± 0.2
Rat bone marrows were obtained after intratracheal injection of SEB (250 ng/ trachea) at the indicated time periods. The data are the mean values ± SEM of six rats. ⁎ P < 0.05 compared with respective PBS group. PBS values presented were pooled (4, 16, 24 and 48 h) since they did not significantly differ among each other.
celecoxib (3 mg/kg, i.p), the non-specific nitric oxide synthase inhibitor L-NAME (20 mg/kg, i.v.), the selective iNOS inhibitor compound 1400W (5 mg/kg, i.v.) and the lipoxygenase inhibitor AA-861 (200 μg/kg, i.v.) (Fig. 3).
Fig. 3. The effect of different pharmacological agents in the neutrophil influx in BAL fluid from rats exposed to SEB. Dexamethasone (DEXA, 0.5 mg/kg), AA-861 (AA, 200 μg/kg), celecoxib (3 mg/kg), L-NAME (20 mg/kg) and 1400 W (5 mg/kg) were administered as described in Materials and methods. Neutrophil counts were performed at 4 h after SEB injection (250 ng/trachea). Control (untreated) animals received PBS instead of SEB. The data are the mean values ± SEM of 4–6 rats. *P < 0.05, compared with the untreated SEBexposed group.
Fig. 2. Measurements of levels of LTB4 (panel A), PGE2 (panel B), TNF-α (panel C), IL-6 (panel D) and IL-10 (panel E) in BAL fluid after rat airways exposition with SEB. Rats were intratracheally injected with PBS or SEB (250 ng/trachea), and BAL fluid was collected at 1 h (PGE2) or 4 h (LTB4, TNF-α, IL-6 and IL-10) thereafter. Results are mean values ± SEM from 4 animals. *P < 0.05 compared with respective PBS group.
I.A. Desouza et al. / Toxicology and Applied Pharmacology 217 (2006) 107–113 Table 2 Rat neutrophil chemotaxis and adhesion in vitro induced by staphylococcal enterotoxin B (SEB) Chemotaxis (×105/ml) MEM fMLP (10− 7 M) SEB (0.01 ng/well) SEB (0.10 ng/well) SEB (1.00 ng/well)
Adhesion (%) 5.6 ± 0.60 12.1 ± 0.80 ⁎ 4.6 ± 0.17 5.4 ± 0.38 4.6 ± 0.36
MEM fMLP (10− 5 M) SEB (0.30 ng/well) SEB (1.00 ng/well) SEB (10.0 ng/well)
49.7 ± 0.6 65.8 ± 2.6 ⁎ 53.4 ± 0.5 54.8 ± 0.8 55.5 ± 0.5
Neutrophils were isolated from rat peripheral blood in 3.13 % (w/v) sodium (10:1) and obtained by Dextran using Ficcol (1.077 g/l) gradient (see Materials and methods). The myeloperoxidase (MPO) activity was measured, and results were expressed as absolute number of neutrophils that migrated through the filter (chemotaxis) or % neutrophil adhesion. The data represent the mean values ± SEM of 3 experiments (each in duplicate). ⁎ P < 0.05 compared with respective MEM.
In vitro neutrophil chemotaxis and adhesion Incubation of rat peripheral neutrophils with SEB (0.01 to 1.0 ng/well) did not significantly induce cell chemotaxis when compared with untreated neutrophils (not shown; n = 3, each in duplicate). Similarly, SEB (0.3 to 10 ng/well) failed to affect the neutrophil adhesion to serum-coated plates when compared with untreated neutrophils (not shown; n = 3, each in duplicate). Additionally, SEB itself (10 ng/well) did not interfere with the MPO assay (not shown). In the same experimental conditions, neutrophil activation with fMLP significantly increased (P < 0.05) both neutrophil chemotaxis (12.1 ± 0.8 × 105/ml) and adhesion (65.8 ± 2.6%) in comparison with spontaneous chemotaxis (5.6 ± 0.6 × 105/ml) and adhesion (49.7 ± 0.6), respectively (Table 2). Discussion The bacterium Staphylocococcus aureus can be found in normal microflora of upper respiratory tract where it secretes the so-called staphylococcal enterotoxins, which may contributes to pathogenesis of pulmonary inflammatory conditions such as bronchial asthma and rhinitis (Bachert et al., 2002; Rossi and Monasterolo, 2004). Recently, exposition of rat airways to staphylococcal enterotoxin type A (SEA) has been shown to induce a marked pulmonary neutrophil infiltration by mechanisms involving the release of multiple inflammatory mediators including cytokines, PGE2, LTB4 and NO (Desouza et al., 2005). Although the staphylococcal enterotoxin type B (SEB) is the largest product of Staphylocococcus aureus and possibly the main toxin responsible for the pathological conditions during Staphylocococcus aureus infection (Chesney, 1991; Micusan and Thibodeau, 1993), the mechanisms involved in the SEB-induced pulmonary inflammation have been poorly investigated. Our present study shows that rat airways exposition to SEB evoked a large neutrophil infiltration in BAL fluid at early stages (4 h), maintaining markedly elevated at 16 and 24 h after SEB exposition. At 4 h, elevated levels of PGE2, LTB4, TNF-α, IL-6 and IL-10 in BAL fluid were detected. At later stages post-SEB exposition (24 h), BAL fluid
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showed the presence of mononuclear cells (lymphocytes and macrophages), but not of eosinophils, thus suggesting that mechanisms triggering SEB-induced pulmonary inflammation differ from the classical IgE-dependent allergic responses. It is known that bone marrow regulates the homeostatic release of mature neutrophils and the accelerated production of granulocytes in response to inflammatory signals and infections (Terashima et al., 1996). In our study, significant elevations of immature forms of granulocytes in bone marrow were detected at 4 h, whereas its mature forms were detected at 24 h. With regard to the mononuclear cell lineage in bone marrow, significant elevations were detected at 4 h post-SEB exposition. Since cytokines such as TNF-α and granulocyte–macrophage colony stimulation factor regulates the granulocyte production in bone marrow (Ueda et al., 2004; Jacobsen et al., 1992), we speculate that modifications in bone marrow cell pattern reflect the pulmonary inflammation and hence the release of inflammatory mediators after SEB exposition. We next attempted to investigate the mechanisms involved in the neutrophil influx induced by SEB at 4 h. Our findings that SEB failed to induce in vitro chemotaxis and adhesion of neutrophils suggest that in vivo neutrophil recruitment in airways takes place by indirect mechanisms. A number of inflammatory mediators have been implicated in the movement of neutrophils across the endothelium to sites of injury, including cytokines, chemokines, lipid-derived mediators and nitric oxide (NO), amongst others (Wagner and Roth, 2000). It is known that enterotoxins possess superantigenic properties (Balaban and Raooly, 2000) and high affinity to class II major histocompatibility complex (MHC). Interaction of enterotoxins with the MHC-class II molecules expressed in different pulmonary cells can result in a large production of inflammatory mediators and leukocyte accumulation (Müller-Alouf et al., 2001; Bachert et al., 2002). TNF-α is an important cytokine involved in leukocyte recruitment through both upregulation of adhesion molecules on vascular endothelial cells and induction of other cytokine and chemokine synthesis (Kips, 2001). In our study, the levels of TNF-α in BAL fluid increased by approximately 170% at 4 h, which is in accordance with previous studies in the mice air pouch exudates (Tessier et al., 1998). It has been shown that neutrophil migration in response to TNF-α in mice takes place by LTB4-dependent mechanisms (Canetti et al., 2001). Thus, our findings that LTB4 levels are elevated in BAL fluid at 4 h after SEB exposition and that the animal pretreatment with the lipoxygenase inhibitor AA-861 largely reduces the neutrophil infiltration suggest that SEB-induced pulmonary responses may be secondary to enhanced TNF-α synthesis. Interleukin6 is a pleiotropic cytokine that plays an important role during lung inflammatory conditions, including bacterial infections, and can be stimulated by TNF-α and IL-1 (Song and Kellum, 2005). On the other hand, IL-10 is a wellknown anti-inflammatory cytokine described to inhibit the release and/or action of proinflammatory cytokines such as IL-1β, IL-6 and TNF-α from monocytes/macrophages
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(Bhatia and Moochhala, 2004). Thus, IL-10 has been associated with clinical protection and reduction of lung pathology (Morrison et al., 2000). In our present study, besides increasing TNF-α levels, exposure of rat airways to SEB significantly increased the levels of both IL-6 and IL10 in BAL fluid at 4 h. It is plausible therefore that elevation of IL-10 levels in SEB-exposed rats act to downregulate the production and action of TNF-α and IL6. Interestingly, rats exposed to staphylococcal enterotoxin type A failed to release IL-10 but presented higher levels of TNF-α in BAL fluid in comparison with SEB (Desouza et al., 2005). Besides cytokines, NO and PGE2 derived from iNOS and COX-2, respectively, have been recognized as important mediators of inflammatory conditions. The anti-inflammatory properties of corticosteroids have been attributed in part to its ability to inhibit the induction and/or activity of such enzymes (Goodwin et al., 1999; Uno et al., 2004). In our study, animals pretreated with the selective iNOS inhibitor 1400 W and the selective COX-2 inhibitor celecoxib, as well as dexamethasone markedly reduced SEB-induced neutrophil influx into the airways. Additionally, high levels of PGE2 were detected in BAL fluid of SEB-treated rats. Together, this indicates that NO and PGE2 are also important mediators to modulate the pulmonary neutrophil influx induced by enterotoxins, which is consistent with previous studies reporting high detectable expression of mRNA for iNOS and COX-2 in mouse microvascular endothelial cells in vitro and rat lungs treated with staphylococcal enterotoxins (Leclaire et al., 1995; Desouza et al., 2005). In conclusion, this study shows that the neutrophilic lung inflammation induced by SEB is a consequence of the release of proinflammatory (TNF-α and IL-6) and antiinflammatory cytokines (IL-10). Besides cytokines, other mediators seem to play an important role in a SEB-induced lung inflammation such as NO, PGE2 and LTB4. Our findings highlight the importance in developing specific and effective anti-inflammatory therapy against to Staphylococcus aureus-induced lung pathogenesis. Acknowledgment Ivani A. De Souza thanks Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the financial support. References Abe, J., Ito, Y., Onimaru, M., Kohsaka, T., Takeda, T., 2000. Characterization and distribution of new enterotoxin-related superantigen produced by Staphylococcus aureus. Microbiol. Immunol. 44, 79–88. Bachert, C., Gevaert, P., Van Cauwenberge, P., 2002. Staphylococcus aureus enterotoxins: a key in airway disease? Allergy 57, 480–487. Bachert, C., Gevaert, P., Van Cauwenberge, P., 2003. Staphylococcus aureus superantigens and airway disease. Curr. Allergy Asthma Rep. 2, 252–258. Balaban, N., Raooly, A., 2000. Staphylococcal enterotoxins. Int. J. Food Microbiol. 61, 1–10. Bhatia, M., Moochhala, S., 2004. Role of inflammatory mediators in the
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I.A. Desouza et al. / Toxicology and Applied Pharmacology 217 (2006) 107–113 Rossi, R.E., Monasterolo, G., 2004. Prevalence of serum IgE antibodies to the Staphylococcal aureus enterotoxins (SAE, SEB, SEC, SED, TSST-1) in patients with persistent allergic rhinitis. Int. Arch. Allergy Immunol. 133, 261–266. Song, M., Kellum, J.A., 2005. Interleukin-6. Crit. Care Med. 33, S463–S465. Terashima, T.B., English, D., Hogg, J.C., Van Eeden, S.F., 1996. Polymorphonuclear leukocyte transit times in bone narrow during streptococcal pneumonia. Am. J. Physiol. 271, L587–L592. Tessier, P.A., Naccache, P.H., Diener, K.R., Gladue, R.P., Neote, K.A., ClarkLewis, I., Mccoll, S.R., 1998. Induction of acute inflammation in vivo by staphylococcal superantigens: II. Critical role for chemokines, ICAM-1, and TNF-α. J. Immunol. 161, 1204–1211. Tuner, M.O., Hussack, P., Sears, M.R., Dolovich, J., Hargreave, F.E., 1995. Exacerbation of asthma without sputum eosinophilia. Thorax 10, 1057–1061. Ueda, Y., Yang, K., Foster, S.J., Kondo, M., Kelsoe, G., 2004. Inflammation
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controls B lymphopoiesis by regulating chemokine CXCL12 expression. J. Exp. Med. 199, 47–58. Uno, K., Iuchi, Y., Fujii, J., Sugata, H., Iijima, K., Kato, K., Shimosegawa, T., Yoshimura, T., 2004. In vivo study on cross talk between inducible nitricoxide synthase and cyclooxygenase in rat gastric mucosa: effect of cyclooxygenase activity on nitric oxide production. Pharmacol. Exp. Ther. 309, 995–1002. Wagner, J.G., Roth, R.A., 2000. Neutrophil migration mechanisms with an emphasis on pulmonary vasculature. Pharmacol. Rev. 52, 349–374. Wenzel, S.E., Schwartz, L.B., Langmack, L.E., Halliday, J.L., Trudeau, J.B., Gibbs, R.L., Chu, H.W., 1999. Evidence that severe asthma can be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics. Am. J. Respir. Crit. Care Med. 160, 1001–1008. Yarwood, J.M., McCormick, J.K., Schlievert, P.M., 2001. Identification of a novel two-component regulatory system that acts in global regulation of virulence factors of Staphylococcus aureus. J. Bacteriol. 183, 1113–1123.
Acute pulmonary inflammation induced by exposure of the airways to staphylococcal enterotoxin type B in rats Ivani A. Desouza a,⁎,1 , Carla F. Franco-Penteado a , Enilton A. Camargo a , Carmen S.P. Lima b , Simone A. Teixeira c , Marcelo N. Muscará c , Gilberto De Nucci a , Edson Antunes a b
a Department of Pharmacology, State University of Campinas (UNICAMP), P.O. Box 6111, 13084-971, Campinas (SP), Brazil Laboratory of Haemathology of Faculty of Medical Sciences, State University of Campinas (UNICAMP), P.O. Box 6111, 13084-971, Campinas (SP), Brazil c Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo (USP), São Paulo (SP), Brazil
Received 11 May 2006; revised 30 June 2006; accepted 6 July 2006 Available online 14 July 2006
Abstract Staphylocococcus aureus is a gram-positive bacterium that produces several enterotoxins, which are responsible for most part of pathological conditions associated to staphylococcal infections, including lung inflammation. This study aimed to investigate the underlying inflammatory mechanisms involved in leukocyte recruitment in rats exposed to staphylococcal enterotoxin B (SEB). Rats were anesthetized with pentobarbital sodium and intratracheally injected with either SEB or sterile phosphate-buffered saline (PBS, 0.4 ml). Airways exposition to SEB (7.5–250 ng/ trachea) caused a dose- and time-dependent neutrophil accumulation in BAL fluid, the maximal effects of which were observed at 4 h post-SEB exposure (250 ng/trachea). Eosinophils were virtually absent in BAL fluid, whereas mononuclear cell counts increased only at 24 h post-SEB. Significant elevations of granulocytes in bone marrow (mature and immature forms) and peripheral blood have also been detected. In BAL fluid, marked elevations in the levels of lipid mediators (LTB4 and PGE2) and cytokines (TNF-α, IL-6 and IL-10) were observed after SEB instillation. The SEB-induced neutrophil accumulation in BAL fluid was reduced by pretreatment with dexamethasone (0.5 mg/kg), the COX-2 inhibitor celecoxib (3 mg/kg), the selective iNOS inhibitor compound 1400 W (5 mg/kg) and the lipoxygenase inhibitor AA-861 (200 μg/kg). In separate experiments carried out with rat isolated peripheral neutrophils, SEB failed to induce neutrophil adhesion to serum-coated plates and chemotaxis. In conclusion, rat airways exposition to SEB causes a neutrophil-dependent lung inflammation at 4 h as result of the release of proinflammatory (NO, PGE2, LTB4, TNF-α, IL-6) and anti-inflammatory mediators (IL-10). © 2006 Elsevier Inc. All rights reserved. Keywords: Cyclooxygenase-2; Cytokines; Enterotoxins; Neutrophil influx; Nitric oxide
Introduction Staphylocococcus aureus is a gram-positive bacterium often found in the normal microflora of skin, upper respiratory and Abbreviations: BAL, brochoalveolar lavage; COX-2, cyclooxygenase-2; fMLP, N-formyl-methionyl-leucyl-phenilalanine; IL, interleukin; iNOS, inducible nitric oxide synthase; L-NAME, N ω-nitro-L-arginine methyl ester; LTB4, leukotriene B4; MEM, Eagle's minimum essential medium; MPO, myeloperoxidase; NO, nitric oxide; PBS, phosphate-buffered saline; PGE2, prostaglandin E2; SEB, Staphylococcal enterotoxin type B; TNF-α, tumoral necrosis factor-α ⁎ Corresponding author. Fax: +55 19 32892968. E-mail address: [email protected] (I.A. Desouza). 1 Current address: Department of Pharmacology; Faculty of Medical Sciences, State University of Campinas (UNICAMP), PO Box 6111, CEP 13084-971, Campinas, SP, Brazil. 0041-008X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2006.07.001
gastrointestinal tracts in humans (Bachert et al., 2002, 2003). This bacterium produces and secretes the so-called staphylococcal enterotoxins, which are a family of structurally related heat-stable 23 to 29-kDa proteins. They comprise several serological types, namely the classical types A to E, and the newly characterized types G to K (Balaban and Raooly, 2000; Abe et al., 2000; Orwin et al., 2001). Staphylococcal enterotoxins often lead to multiorgan dysfunction in humans and animals (Kotzin et al., 1993; Lowy, 1998; Müller-Alouf et al., 2001) which may be related to their superantigenic properties (Balaban and Raooly, 2000; Yarwood et al., 2001). Clinical evidences suggest a link between bacterial organisms and pathogenesis and/or exacerbation of human upper airway disease (Kraft, 2000; Bachert et al., 2003; Rossi and Monasterolo, 2004). In addition, several studies report that
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IgE-independent bronchial asthma can be provoked by bacterial infections, including Gram-positive bacteria (Tuner et al., 1995; Wenzel et al., 1999; Douwes et al., 2002). Recently, airways exposition to staphylococcal enterotoxin type A (SEA) in rats has been shown to evoke a large influx of neutrophils in bronchoalveolar lavage fluid by mechanisms involving complex interactions of different signaling pathways including overexpression of cytokine-induced neutrophil chemoattractant (CINC-2), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), as well as enhanced production of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 (Desouza et al., 2005). Staphylococcal enterotoxin type B (SEB) can be easily obtained in relatively large amounts and purity, but few studies have attempted to investigate the pulmonary inflammation induced by airways exposure to this enterotoxin. Intranasal administration of staphylococcal enterotoxin B (SEB) in mice has been shown to induce mucosal and airway recruitment of polymorphonuclear and mononuclear cells (Neumann et al., 1997; Herz et al., 1999) that was associated with increased levels of IL-4 and TNF-α production in bronchoalveolar lavage (BAL) fluid (Herz et al., 1999), but no further studies have explored the mechanisms underlying the SEB-induced pulmonary inflammation, particularly the airways neutrophil infiltration. Therefore, in this study, rat airways have been exposed to SEB in a dose- and time-dependent manner in order to understand the underlying inflammatory mechanisms determining the pulmonary neutrophil infiltration.
Materials and methods Materials. Staphylococcal enterotoxin B (SEB), minimum essential medium (MEM), Dextran, Nω-nitro-L-arginine methyl ester (L-NAME), AA-861, Nformyl-L-methionyl-L-leucyl-phenylalanine (fMLP), hexadecytrimethyl-ammonium bromide (HTAB) and dexamethasone were purchased from Sigma Chemical Co. (St. Louis, MO, USA). N-(3-(aminomethyl)benzyl)acetamidine (1400 W) was purchased from Alexis (Nottingham, UK). Celecoxib was obtained from Laboratórios Pfizer Ltd. (São Paulo, Brazil). Enzyme-linked immunosorbent assays (ELISA) for rat TNF-α were obtained from BD Biosciences (CA, USA), whereas ELISA for rat LTB4, PGE2, IL-6 and IL-10 were obtained from R&D Systems (MN, USA). Animal experimentation guidelines. The experimental protocols were approved by the Ethical Principles in Animal Research adopted by the Brazilian College for Animal Experimentation (COBEA). Male Wistar rats (250–300 g; 7–8 weeks age) were housed in temperature-controlled rooms and received water and food ad libitum until used. Intratracheal injection of SEB. Rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and intratracheally injected with SEB (7.5–250 ng/ trachea, 0.4 ml). Control animals received 0.4 ml of sterile phosphate-buffered saline (PBS) alone. At selected time thereafter (4, 16, 24 and 48 h), animals were sacrificed by overdose of inhaled halothane, and then peripheral blood, bronchoalveolar lavage (BAL) fluid and bone marrow were collected to assess the total and differential leukocyte counts. Leukocyte counts in BAL fluid. Bronchoalveolar lavage was performed at 4 to 48 h after SEB (or PBS) intratracheal injection. Briefly, the trachea was exposed and cannulated with a polyethylene tube (1 mm in diameter) connected to a syringe. The lungs were washed by flushing with PBS solution containing heparin (20 UI/ml). The PBS buffer was instilled through the tracheal cannula as
one 10-ml aliquot followed by three 5-ml aliquots (making a total of approximately 25 ml). The first 10-ml aliquot was centrifuged (1000×g for 10 min at 20 °C), and the supernatant recovered and stored at − 80 °C for measurement of inflammatory mediators. The three 5-ml aliquots were pooled and centrifuged (1000×g for 10 min at 20 °C). The cell pellet obtained from all aliquots (the 10-ml aliquot together with those of the 5-ml aliquots) were combined and resuspended in 2 ml of PBS solution. Total cell counts were done with an automated cell counter (CELL-DYN 1700) while differential counts were carried out on a minimum of 200 cells using cytospin preparation stained with May-Grünwald. The cells were classified as neutrophils, eosinophils, mast cells, lymphocytes and macrophages based on normal morphological criteria. Leukocyte counts in peripheral blood and bone marrow. Blood samples were obtained from the abdominal artery after the SEB (or PBS) injection into the airways. Total cell counts were done with an automated cell counter (CELL-DYN 1700, USA) while differential counts were carried out on blood smears stained by the May-Grünwald method. The cells were classified as neutrophils, eosinophils, mast cells, lymphocytes, and mononuclear based on normal morphological criteria. For measurement of cells in bone marrow, femurs were removed from rats immediately after killing. The epiphyses were cut transversely and bone marrow cells were flushed out with PBS containing heparin (20 IU/ml). Differential count was carried out on a minimum of 200 cells using cytospin preparation stained with Leishman. Results are expressed as the number of leukocytes per femur. Cells were classified as immature neutrophils (myeloblast, promyelocyte and myelocyte), mature neutrophils (metamyelocyte, band and mature), eosinophils, mast cells, lymphocytes and monocytes based on normal morphological criteria. Measurement of leukotriene B4 (LTB4), TNF-α, IL-10, IL-6 and prostaglandin E2 (PGE2) in BAL fluid. Rat LTB4, TNF-α, IL-10, IL-6 and PGE2 were measured in BAL fluid supernatant using commercially available enzymelinked immunosorbent assays (ELISA) according to the manufacturer instructions. Pharmacological investigation with different drugs. The efficacy of doses and schedules of administration for each drug in particular were chosen according to previous studies, as follows: (1) dexamethasone (0.5 mg/kg) was administered i.v. 1 h before SEB exposition (Cunha and Ferreira, 1986); (2) the lypoxygenase inhibitor AA-861 (200 μg/kg) was administered i.v. 15 min before SEB exposition (Filliatre et al., 2001); (3) the selective cyclooxygenase-2 inhibitor celecoxib (3 mg/kg) was administered i.p. 1 h before SEB exposition (Filliatre et al., 2001); (4) the non-selective NOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg) was administered i.v. immediately before SEB exposition (Parmentier et al., 1999); (5) the iNOS selective inhibitor compound 1400 W (5 mg/kg) was administered i.v. immediately before SEB exposition (Franco-Penteado et al., 2001). Isolation of peripheral blood neutrophils. Neutrophils were separated from naïve rat peripheral blood in 3.13% (w/v sodium citrate (10:1) and obtained by Dextran sedimentation by Ficcol (1.077 g/l) gradient. For each experiment, a pool of blood of 5 rats was used. After separation of monocytes and granulocytes by centrifuging at 400×g for 30 min, the granulocyte layer was washed once in Eagle's minimum essential medium (MEM; pH 7.2) before performing a hypotonic lysis to disrupt the red cells. Cells were washed once again in MEM and resuspended in MEM/0.1% ovalbumin. Samples of cell suspension were used to determine total cell number using an improved Neubauer hemocytometer, and then cytospinned onto slides for a differentiation count. The final cell suspension contained 89% of neutrophils. Cell viability (>95%) was assessed by Trypan blue dye exclusion test. Neutrophil adhesion assays in vitro. 96-well plates were prepared by coating individual wells with 60 μl of serum (1:10 dilution in PBS) overnight at 4 °C. Wells were then washed twice with PBS before blocking non-coated sites with 0.1% (w/v) bovine serum albumin for 60 min at 37 °C. Wells were washed twice again with PBS before allowing plates to dry. Neutrophils (50 μl of 1 × 106 cells/ ml in MEM/ovalbumin) were seeded onto the coated wells alone or with SEB (0.3 to 10 ng/well) and cells were allowed to adhere for 30 min at 37 °C, 5% CO2. A positive control was run using the N-formyl-methionyl-leucyl-phenilalanine (fMLP). Following incubation non-adhered cell were washed twice with PBS.
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Fifty microliters of MEM was added to each well and varying numbers of the parent stock neutrophil cell suspension were added to empty wells to form a standard curve. Plates were then stored frozen overnight before measuring the myeloperoxidase (MPO) content of adherent cells (Bradley et al., 1982). Plates were defrosted on ice before extracting with hexadecytrimethyl-ammonium bromide in 50 mM potassium phosphate buffer, pH 6.0. Twenty microliters of each well sample to be measured were mixed with 200 μl of o-dianisidine solution (0.167 mg/ml o-dianiidine dihydrochloride, 0.0005% hydrogen peroxide in 50 mM phosphate buffer, pH 6.0) immediately prior to reading change of absorbance at 460 nm over 5 minutes in a microplate reader (Multiscan MS, Labsystems, CA, USA). Adherence was calculated by comparing absorbance changes of unknowns to those of the standard curve. In separate experiments, the MPO assay was carried out in presence of SEB (10 ng/well) to exclude the possibility that SEB interferes at the enzyme-substrate level. Neutrophil chemotaxis assays in vitro. Neutrophil migration assays were performed using a 96-well chemotaxis chamber (NeuroProbe, MD, USA). Twenty-five microliters of 8 × 106 cell/ml neutrophils (prepared in MEM containing 0.1% ovalbumin) were added to the upper compartment of the chamber and separated by a polycarbonate filter (5-μm pore) from the lower chamber containing 29 μl of MEM, SEB (0.01 to 1.0 ng/well) or fMLP (1 × 10− 7 M). The chambers were incubated at 37 °C in a 5% CO2 atmosphere for 120 min. The wells of the upper compartment were emptied by aspiration, and then disassembled. To detach adherent neutrophil from the filter, the microtiter plate with attached filter was centrifuged at 1200 rpm for 5 min at room temperature. The MPO activity was determined as described above. Migrated neutrophils were calculated by comparing absorbance changes of unknowns to those of the standard curve. Statistical analysis. Data are presented as the mean values ± SEM and were analyzed by analysis of variance (ANOVA) for multiple comparisons followed Bonferroni test, or Student's unpaired t-test where appropriate. In both cases, the level of significance was set at P < 0.05.
Results Neutrophil counts in BAL fluid Rat airway exposure to SEB (7.5–250 ng/trachea) caused a marked and dose-dependent neutrophil accumulation in BAL fluid with maximal responses achieved with 250 ng/trachea of SEB (Fig. 1A). Maximal neutrophil accumulation in BAL fluid (22-fold increase) was observed at 4 h after SEB injection (250 ng/trachea), decaying thereafter (at 16 and 24 h). At 48 h, the number of neutrophils in BAL fluid returned to basal counts (Fig. 1B). Eosinophils were virtually absent in BAL fluid in any time and dose studied. The number of mononuclear cells increased only at 24 h-post-SEB (250 ng/trachea) injection (n = 5; data not shown). Neutrophil counts in bone marrow and peripheral blood Airways exposition to SEB (250 ng/trachea) induced significant increases in the immature forms of granulocytes in bone marrow at 4 h thereafter, whereas the mature forms of granulocytes increased significantly at later times (24 h). A significant elevation in counts of mononuclear cell lineage in bone marrow was observed at 4 h after SEB exposition (Table 1). In peripheral blood, significant elevations of neutrophils were observed at 4 h and 16 h (data not shown). The mononuclear cell counts (lymphocytes and monocytes) in peripheral blood of SEB-exposed rats did not change significantly in comparison
Fig. 1. Neutrophil influx in BAL fluid from rats exposed to SEB. (Panel A) Rats were intratracheally injected with PBS or SEB at the indicated doses, and BAL fluid was collected at 4 h. (Panel B) Rats were intratracheally injected with PBS or SEB (250 ng/trachea) and BAL fluid was collected at the indicated times. The black bars represent the counts obtained with PBS alone. The data are the mean values ± SEM of 6 rats. *P < 0.05 compared with respective PBS group.
with PBS-injected rats in any time evaluated (data not shown, n = 5–6). Measurements of PGE2, LTB4, TNF-α, IL-6 and IL-10 levels in BAL fluid Rats were exposed to SEB (250 ng/trachea), and at 1 h (for PGE2) or 4 h (for LTB4, TNF-α, IL-6 and IL-10) later, BAL fluid was recovered in order to measure the inflammatory mediators. Fig. 2 shows that concentrations of lipid mediators (LTB4 and PGE2) and cytokines (TNF-α, IL-6 and IL-10) were significantly higher after SEB instillation when compared with control groups. Effect of different in vivo drug treatments on pulmonary neutrophil influx The neutrophil counts in BAL fluid of in non-treated and treated rats were performed at 4 h post-intratracheal injection of 250 ng/trachea of SEB. Pretreatment of rats with dexamethasone (0.5 mg/kg, i.p) nearly abolished the SEB-induced neutrophil accumulation in BAL fluid (Fig. 3). Similar inhibitory responses in the neutrophil influx were observed with the COX-2 inhibitor
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Table 1 Leukocyte counts in bone marrow of rats after the airways exposition to SEB Cells (×106/femurs) Immature granulocytes Mature granulocytes Mononuclear cells PBS SEB 4 h SEB 16 h SEB 24 h SEB 48 h
4.2 ± 0.4 7.4 ± 1.8 ⁎ 6.0 ± 0.5 5.3 ± 1.7 3.0 ± 0.2
36 ± 2.8 32 ± 6.2 46 ± 5.4 60 ± 6.5 ⁎ 48.7 ± 4.9
1.7 ± 0.3 3.8 ± 1.6 ⁎ 1.0 ± 0.3 1.7 ± 0.5 2.0 ± 0.2
Rat bone marrows were obtained after intratracheal injection of SEB (250 ng/ trachea) at the indicated time periods. The data are the mean values ± SEM of six rats. ⁎ P < 0.05 compared with respective PBS group. PBS values presented were pooled (4, 16, 24 and 48 h) since they did not significantly differ among each other.
celecoxib (3 mg/kg, i.p), the non-specific nitric oxide synthase inhibitor L-NAME (20 mg/kg, i.v.), the selective iNOS inhibitor compound 1400W (5 mg/kg, i.v.) and the lipoxygenase inhibitor AA-861 (200 μg/kg, i.v.) (Fig. 3).
Fig. 3. The effect of different pharmacological agents in the neutrophil influx in BAL fluid from rats exposed to SEB. Dexamethasone (DEXA, 0.5 mg/kg), AA-861 (AA, 200 μg/kg), celecoxib (3 mg/kg), L-NAME (20 mg/kg) and 1400 W (5 mg/kg) were administered as described in Materials and methods. Neutrophil counts were performed at 4 h after SEB injection (250 ng/trachea). Control (untreated) animals received PBS instead of SEB. The data are the mean values ± SEM of 4–6 rats. *P < 0.05, compared with the untreated SEBexposed group.
Fig. 2. Measurements of levels of LTB4 (panel A), PGE2 (panel B), TNF-α (panel C), IL-6 (panel D) and IL-10 (panel E) in BAL fluid after rat airways exposition with SEB. Rats were intratracheally injected with PBS or SEB (250 ng/trachea), and BAL fluid was collected at 1 h (PGE2) or 4 h (LTB4, TNF-α, IL-6 and IL-10) thereafter. Results are mean values ± SEM from 4 animals. *P < 0.05 compared with respective PBS group.
I.A. Desouza et al. / Toxicology and Applied Pharmacology 217 (2006) 107–113 Table 2 Rat neutrophil chemotaxis and adhesion in vitro induced by staphylococcal enterotoxin B (SEB) Chemotaxis (×105/ml) MEM fMLP (10− 7 M) SEB (0.01 ng/well) SEB (0.10 ng/well) SEB (1.00 ng/well)
Adhesion (%) 5.6 ± 0.60 12.1 ± 0.80 ⁎ 4.6 ± 0.17 5.4 ± 0.38 4.6 ± 0.36
MEM fMLP (10− 5 M) SEB (0.30 ng/well) SEB (1.00 ng/well) SEB (10.0 ng/well)
49.7 ± 0.6 65.8 ± 2.6 ⁎ 53.4 ± 0.5 54.8 ± 0.8 55.5 ± 0.5
Neutrophils were isolated from rat peripheral blood in 3.13 % (w/v) sodium (10:1) and obtained by Dextran using Ficcol (1.077 g/l) gradient (see Materials and methods). The myeloperoxidase (MPO) activity was measured, and results were expressed as absolute number of neutrophils that migrated through the filter (chemotaxis) or % neutrophil adhesion. The data represent the mean values ± SEM of 3 experiments (each in duplicate). ⁎ P < 0.05 compared with respective MEM.
In vitro neutrophil chemotaxis and adhesion Incubation of rat peripheral neutrophils with SEB (0.01 to 1.0 ng/well) did not significantly induce cell chemotaxis when compared with untreated neutrophils (not shown; n = 3, each in duplicate). Similarly, SEB (0.3 to 10 ng/well) failed to affect the neutrophil adhesion to serum-coated plates when compared with untreated neutrophils (not shown; n = 3, each in duplicate). Additionally, SEB itself (10 ng/well) did not interfere with the MPO assay (not shown). In the same experimental conditions, neutrophil activation with fMLP significantly increased (P < 0.05) both neutrophil chemotaxis (12.1 ± 0.8 × 105/ml) and adhesion (65.8 ± 2.6%) in comparison with spontaneous chemotaxis (5.6 ± 0.6 × 105/ml) and adhesion (49.7 ± 0.6), respectively (Table 2). Discussion The bacterium Staphylocococcus aureus can be found in normal microflora of upper respiratory tract where it secretes the so-called staphylococcal enterotoxins, which may contributes to pathogenesis of pulmonary inflammatory conditions such as bronchial asthma and rhinitis (Bachert et al., 2002; Rossi and Monasterolo, 2004). Recently, exposition of rat airways to staphylococcal enterotoxin type A (SEA) has been shown to induce a marked pulmonary neutrophil infiltration by mechanisms involving the release of multiple inflammatory mediators including cytokines, PGE2, LTB4 and NO (Desouza et al., 2005). Although the staphylococcal enterotoxin type B (SEB) is the largest product of Staphylocococcus aureus and possibly the main toxin responsible for the pathological conditions during Staphylocococcus aureus infection (Chesney, 1991; Micusan and Thibodeau, 1993), the mechanisms involved in the SEB-induced pulmonary inflammation have been poorly investigated. Our present study shows that rat airways exposition to SEB evoked a large neutrophil infiltration in BAL fluid at early stages (4 h), maintaining markedly elevated at 16 and 24 h after SEB exposition. At 4 h, elevated levels of PGE2, LTB4, TNF-α, IL-6 and IL-10 in BAL fluid were detected. At later stages post-SEB exposition (24 h), BAL fluid
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showed the presence of mononuclear cells (lymphocytes and macrophages), but not of eosinophils, thus suggesting that mechanisms triggering SEB-induced pulmonary inflammation differ from the classical IgE-dependent allergic responses. It is known that bone marrow regulates the homeostatic release of mature neutrophils and the accelerated production of granulocytes in response to inflammatory signals and infections (Terashima et al., 1996). In our study, significant elevations of immature forms of granulocytes in bone marrow were detected at 4 h, whereas its mature forms were detected at 24 h. With regard to the mononuclear cell lineage in bone marrow, significant elevations were detected at 4 h post-SEB exposition. Since cytokines such as TNF-α and granulocyte–macrophage colony stimulation factor regulates the granulocyte production in bone marrow (Ueda et al., 2004; Jacobsen et al., 1992), we speculate that modifications in bone marrow cell pattern reflect the pulmonary inflammation and hence the release of inflammatory mediators after SEB exposition. We next attempted to investigate the mechanisms involved in the neutrophil influx induced by SEB at 4 h. Our findings that SEB failed to induce in vitro chemotaxis and adhesion of neutrophils suggest that in vivo neutrophil recruitment in airways takes place by indirect mechanisms. A number of inflammatory mediators have been implicated in the movement of neutrophils across the endothelium to sites of injury, including cytokines, chemokines, lipid-derived mediators and nitric oxide (NO), amongst others (Wagner and Roth, 2000). It is known that enterotoxins possess superantigenic properties (Balaban and Raooly, 2000) and high affinity to class II major histocompatibility complex (MHC). Interaction of enterotoxins with the MHC-class II molecules expressed in different pulmonary cells can result in a large production of inflammatory mediators and leukocyte accumulation (Müller-Alouf et al., 2001; Bachert et al., 2002). TNF-α is an important cytokine involved in leukocyte recruitment through both upregulation of adhesion molecules on vascular endothelial cells and induction of other cytokine and chemokine synthesis (Kips, 2001). In our study, the levels of TNF-α in BAL fluid increased by approximately 170% at 4 h, which is in accordance with previous studies in the mice air pouch exudates (Tessier et al., 1998). It has been shown that neutrophil migration in response to TNF-α in mice takes place by LTB4-dependent mechanisms (Canetti et al., 2001). Thus, our findings that LTB4 levels are elevated in BAL fluid at 4 h after SEB exposition and that the animal pretreatment with the lipoxygenase inhibitor AA-861 largely reduces the neutrophil infiltration suggest that SEB-induced pulmonary responses may be secondary to enhanced TNF-α synthesis. Interleukin6 is a pleiotropic cytokine that plays an important role during lung inflammatory conditions, including bacterial infections, and can be stimulated by TNF-α and IL-1 (Song and Kellum, 2005). On the other hand, IL-10 is a wellknown anti-inflammatory cytokine described to inhibit the release and/or action of proinflammatory cytokines such as IL-1β, IL-6 and TNF-α from monocytes/macrophages
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(Bhatia and Moochhala, 2004). Thus, IL-10 has been associated with clinical protection and reduction of lung pathology (Morrison et al., 2000). In our present study, besides increasing TNF-α levels, exposure of rat airways to SEB significantly increased the levels of both IL-6 and IL10 in BAL fluid at 4 h. It is plausible therefore that elevation of IL-10 levels in SEB-exposed rats act to downregulate the production and action of TNF-α and IL6. Interestingly, rats exposed to staphylococcal enterotoxin type A failed to release IL-10 but presented higher levels of TNF-α in BAL fluid in comparison with SEB (Desouza et al., 2005). Besides cytokines, NO and PGE2 derived from iNOS and COX-2, respectively, have been recognized as important mediators of inflammatory conditions. The anti-inflammatory properties of corticosteroids have been attributed in part to its ability to inhibit the induction and/or activity of such enzymes (Goodwin et al., 1999; Uno et al., 2004). In our study, animals pretreated with the selective iNOS inhibitor 1400 W and the selective COX-2 inhibitor celecoxib, as well as dexamethasone markedly reduced SEB-induced neutrophil influx into the airways. Additionally, high levels of PGE2 were detected in BAL fluid of SEB-treated rats. Together, this indicates that NO and PGE2 are also important mediators to modulate the pulmonary neutrophil influx induced by enterotoxins, which is consistent with previous studies reporting high detectable expression of mRNA for iNOS and COX-2 in mouse microvascular endothelial cells in vitro and rat lungs treated with staphylococcal enterotoxins (Leclaire et al., 1995; Desouza et al., 2005). In conclusion, this study shows that the neutrophilic lung inflammation induced by SEB is a consequence of the release of proinflammatory (TNF-α and IL-6) and antiinflammatory cytokines (IL-10). Besides cytokines, other mediators seem to play an important role in a SEB-induced lung inflammation such as NO, PGE2 and LTB4. Our findings highlight the importance in developing specific and effective anti-inflammatory therapy against to Staphylococcus aureus-induced lung pathogenesis. Acknowledgment Ivani A. De Souza thanks Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the financial support. References Abe, J., Ito, Y., Onimaru, M., Kohsaka, T., Takeda, T., 2000. Characterization and distribution of new enterotoxin-related superantigen produced by Staphylococcus aureus. Microbiol. Immunol. 44, 79–88. Bachert, C., Gevaert, P., Van Cauwenberge, P., 2002. Staphylococcus aureus enterotoxins: a key in airway disease? Allergy 57, 480–487. Bachert, C., Gevaert, P., Van Cauwenberge, P., 2003. Staphylococcus aureus superantigens and airway disease. Curr. Allergy Asthma Rep. 2, 252–258. Balaban, N., Raooly, A., 2000. Staphylococcal enterotoxins. Int. J. Food Microbiol. 61, 1–10. Bhatia, M., Moochhala, S., 2004. Role of inflammatory mediators in the
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