Ivani A. DeSouza

Inflammatory mechanisms underlying the rat pulmonary neutrophil influx induced by airway exposure to staphylococcal enterotoxin type A

Association between staphylococcal infection and pathogenesis of upper airways disease has been reported. This study aimed to investigate the mechanisms underlying the rat pulmonary inflammation induced by airway exposure to staphylococcal enterotoxin A (SEA). SEA (0.3–10 ng trachea−1) caused dose‐dependent neutrophil accumulation in BAL fluid, reaching maximal responses at 4 h (25‐fold increase for 3 ng trachea−1). Significant accumulation of both lymphocytes and macrophages in BAL fluid was also observed at 4 h (2.1‐ and 1.9‐fold increase, respectively, for 3 ng trachea−1). At later times (16 h), neutrophil counts in bone marrow (immature forms) and peripheral blood increased by 63 and 81%, respectively. SEA failed to directly induce chemotaxis and adhesion of isolated neutrophils. Analysis of mRNA expression for iNOS, COX‐2 and CINC‐2 in lung tissue showed an upregulation of these enzymes, which paralleled elevated levels of LTB4, PGE2, TNF‐α, IL‐6 and NO2− in BAL fluid. Expression of CINC‐1 was unchanged, whereas CINC‐3 was reduced in SEA‐treated rats. Incubation of isolated alveolar macrophages with SEA (3 μg ml−1) resulted in significant elevations of TNF‐α and NO2− levels in the cell supernatants. Dexamethasone (0.5 mg kg−1), celecoxib (3 mg kg−1) and compound 1400 W (5 mg kg−1) markedly reduced SEA‐induced lung neutrophil influx and NO2− levels in BAL fluid. The lipoxygenase inhibitor AA‐861 (100 μg kg−1) partly inhibited the neutrophil influx in SEA‐treated rats without modifying the NO2− levels. None of these treatments reduced the number of mononuclear cells in BAL fluid (except of dexamethasone, which abolished the increased lymphocyte counts). Our study shows that airways exposure to SEA results in marked neutrophil influx through mechanisms involving increased expressions of CINC‐2, iNOS and COX‐2, as well as enhanced production of NO, PGE2, LTB4, TNF‐α and IL‐6.

Role of nitric oxide on the increased vascular permeability and neutrophil accumulation induced by staphylococcal enterotoxin B into the mouse paw.

The role of nitric oxide (NO) on the increase in vascular permeability and neutrophil migration induced by staphylococcal enterotoxin B (SEB; 25 microgram/paw) in the mouse was investigated in this study. The NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) [but not its inactive enantiomer N(omega)-nitro-D-arginine methyl ester (D-NAME)], given intravenously (25-100 micromol/kg) or subplantarly (0.25-1.0 micromol/paw), reduced SEB-induced paw oedema significantly. A similar response was observed with aminoguanidine, given either intravenously (200-600 micromol/kg) or subplantarly (2 micromol/paw). In contrast to paw oedema, the plasma exudation in response to SEB was not affected by the subplantar injection of L-NAME or aminoguanidine. The inhibition of oedema and plasma exudation by systemic treatment with L-NAME or aminoguanidine was reversed by co-injection of the vasodilator iloprost (0.3 nmol/paw). Subplantar injection of SEB (25 microgram/paw) increased by 69% the myeloperoxidase (MPO) activity of SEB-treated paws, indicating the presence of neutrophils. Intravenous (12.5-50 micromol/kg) or subplantar (0.125-0.5 micromol/paw) administration of L-NAME (but not of its inactive enantiomer, D-NAME) largely reduced the MPO activity in SEB-treated paws. Similarly, intravenous (200-600 micromol/kg) or subplantar (2 micromol/paw) administration of aminoguanidine significantly reduced the MPO values of the SEB-injected paws. The vasodilator iloprost (0.3 nmol/paw) completely reversed the inhibition by L-NAME or aminoguanidine of the MPO activity in SEB-injected paws. Our results show that the increased vascular permeability and neutrophil accumulation in response to subplantar injection of SEB in the mouse are inhibited by L-NAME and aminoguanidine by mechanisms probably involving reduction of local microvascular blood flow.

Inflammatory responses induced in mice by lectin from Talisia esculenta seeds

A novel lectin from Talisia esculenta seeds (TEL) has recently been purified and characterized. In this study we investigated the proinflammatory activity of TEL in mice using both the air-pouch and peritoneal cavity as well as paw oedema models. TEL (10-40 microg) induced significant neutrophil and mononuclear cell recruitment when injected into either mouse air-pouch or peritoneal cavity. The neutrophil accumulation into the air-pouch was dose- and time-dependent with a maximal response at 16 h, returning to control levels at 72 h whereas maximal mononuclear cell accumulation was observed at 24 h after TEL injection. The same profile of neutrophil accumulation was observed when this lectin was injected into mouse peritoneal cavity, although the maximal mononuclear cell recruitment was observed 48 h after TEL injection. Additionally, TEL (12.5-200 microg/paw) caused a dose-dependent mice paw, as evaluated at 4 h after the lectin injection. D-mannose, better than D-glucose, significantly inhibited TEL-induced neutrophil migration into the peritoneal cavity or air-pouch. D-galactose had no effect on TEL-induced neutrophil migration in either cavity studied. On the other hand, D-mannose slightly inhibited the TEL-induced paw oedema, whereas neither D-glucose nor D-galactose affected this phenomenon. In conclusion, our data show that TEL induces neutrophil and mononuclear cell accumulation by a mechanism related to their specific sugar-binding properties.

Involvement of nitric oxide on the peritoneal neutrophil influx induced by staphylococcal enterotoxin B in mouse.

In this study, the role of nitric oxide (NO) on neutrophil migration induced by staphylococcal enterotoxin B (SEB) in the mouse peritoneal cavity was investigated. The NO synthase inhibitors L-NAME and aminoguanidine, as well as dexamethasone, markedly reduced SEB-induced neutrophil influx. In mice with an increased population of peritoneal macrophages, the inhibition of SEB-induced neutrophil influx by these agents was significantly lower. The in vivo treatment with aminoguanidine inhibited only the iNOS activity, whereas L-NAME inhibited both the cNOS and iNOS activities. In conclusion, NO modulates the neutrophil migration in response to SEB through the activity of an iNOS isoform.

Evidence for the involvement of a macrophage-derived chemotactic mediator in the neutrophil recruitment induced by staphylococcal enterotoxin B in mice.

Staphylococcus aureus secretes enterotoxins which are superantigens and the major cause of food poisoning in man. Staphylococcal enterotoxins types A and B can induce neutrophil migration into the peritoneal cavity of mice through sensory C-fiber neuropeptides, lipoxygenase or cyclooxygenase metabolites, nitric oxide, histamine, platelet-activating factor and resident macrophages. In this work, we examined the influence of macrophage-derived products on neutrophil migration during peritonitis induced by staphylococcal enterotoxin type B (SEB) in mice. Macrophages stimulated with SEB released a thermolabile neutrophil chemotactic protein with a molecular weight of 1,000-3,000 (by ultrafiltration). This release was inhibited 30% by dexamethasone (an inhibitor of cytokine synthesis and phospholipase A(2) activity), but not by indomethacin (a cyclooxygenase inhibitor) or BW755C (a dual cyclo- and lipoxygenase inhibitor). Dexamethasone also inhibited (100%) the neutrophil migration induced by the chemotactic protein. Similar inhibition occurred in mice pretreated with BWA4C (lipoxygenase inhibitor; 90%), BW755C (99%), BN52021 (platelet-activating factor-acether receptor antagonist; 93%), cimetidine (histamine H(2) receptor antagonist; 76%), capsaicin (a depletor of sensory C-fiber neuropeptides; 82%) and the neurokinin-1 receptor antagonist SR140333 (71%), but not by indomethacin or the neurokinin(2) receptor antagonist SR48968. These results confirm that macrophages are involved in the neutrophil recruitment induced by SEB, and that the chemotactic protein apparently induces neutrophil migration by a mechanism mediated by platelet-activating factor, histamine H(2) receptors, lipoxygenase products and substance P.

Pre-exposure to Staphylococcal enterotoxin A exacerbates the pulmonary allergic eosinophil recruitment in rats.

Gram-positive Staphylococcus aureus releases classical enterotoxins which aggravates allergic airway diseases. However, little is known about the mechanisms underlying the cell influx exacerbation in asthmatic individuals under exposure to Staphylococcal enterotoxins. We therefore aimed to investigate the effects of airways exposure to Staphylococcal enterotoxin A (SEA) to pulmonary leukocyte recruitment in rats sensitized and challenged with ovalbumin (OVA). Rats were exposed to SEA at 4h prior to OVA challenge or at 4h post-OVA challenge. Bronchoalveolar lavage (BAL) fluid, bone marrow and lung tissue were obtained at 24h after OVA challenge. Pre-exposure to SEA markedly enhanced the eosinophil counts in both BAL fluid and pulmonary tissue in OVA-challenged rats, whereas neutrophil and mononuclear cell counts remained unchanged. In bone marrow, pre-exposure to SEA alone significantly increased the number of eosinophils, and that was further increased in OVA-challenged rats. Exposure to SEA post-OVA challenge did not affect the number of eosinophils, neutrophils and mononuclear cells in BAL fluid. Pre-exposure to the endotoxin lipopolyssacharide (LPS) in OVA-challenged animals rather enhanced the neutrophil number in BAL fluid. In rats pre-exposed to SEA and OVA-challenged, a marked elevation in the levels of TNF-alpha and eotaxin (but not of IL-10) in BAL fluid was observed. The eotaxin levels increased by about of 3-fold in alveolar macrophages treated with SEA in vitro. In conclusion, airways pre-exposure to SEA causes a selective increase in eosinophil number in BAL fluid and bone marrow of OVA-challenged rats by mechanisms involving enhancement of TNF-alpha and eotaxin synthesis.

Mouse macrophages release a neutrophil chemotactic mediator following stimulation by staphylococcal enterotoxin type A

Abstract:Objective and Design: To examine the role of macrophages in the neutrophil migration induced by staphylococcal enterotoxin type A (SEA) in mice.¶Materials and Methods: Peritoneal macrophages were harvested from male Swiss mice pre-treated with thioglycollate. After adhering to plastic tissue culture dishes, the cells were washed and incubated with RPMI or SEA (0.62-2.5 μg/ml) and washed again prior to further incubation with RPMI alone. The medium was then collected, sterilized and assayed for promigratory activity in the mouse peritoneal cavity.¶Results: Mouse macrophage monolayers stimulated with SEA secreted a thermolabile neutrophil chemotactic component (MNCC-SEA) with a molecular mass >100 kDa (by ultrafiltration). This release was dose- and time-dependent and was inhibited by dexamethasone but not by indomethacin or BW755C. Dexamethasone, indomethacin, BWA4C, BW755C, BN52021, cimetidine and SR48968 had no effect on the neutrophil migration induced by MNCC-SEA while capsaicin and SR140333 reduced this phenomenon.¶Conclusions: Macrophages play a key role in the neutrophil recruitment induced by SEA probably by releasing an MNCC-SEA that presumably induces neutrophil migration via a mechanism mediated by substance P.¶

Mechanisms involved in the rat peritoneal leukocyte migration induced by a Kunitz-type inhibitor isolated from Dimorphandra mollis seeds

DMTI-II is a Kunitz-type inhibitor isolated from Dimorphandra mollis seeds that causes rat inflammatory edema by mechanisms involving activation of mast cells and sensory C-fibers. The present study aimed to further explore the inflammatory mechanisms involved in DMTI-II-induced inflammation, focusing to the leukocyte migration in vivo. Male Wistar rats (250-280 g) were injected with DMTI-II (1-100microg/cavity), and at 4-24h thereafter the leukocyte counts in peritoneal lavage were evaluated. DMTI-II caused dose- and time-dependent accumulation of neutrophils and eosinophils. The peritoneal neutrophil influx initiated at 4h, achieving maximal responses at 16 h after DMTI-II injection (16- and 22-fold increase, respectively). The DMTI-II-induced eosinophil recruitment was observed as early as 4h achieving the maximal responses at 16 h (12- and 17-fold increase, respectively). The mononuclear cell number increased at 4h and 16 h (1.5-fold and 1.6-increase, respectively). Prior treatments with dexamethasone, the cyclooxygenase (COX) inhibitors indomethacin and celecoxib, as well as the PAF receptor antagonist PCA4248 largely reduced the neutrophil and eosinophil accumulation. The selective lypoxygenase inhibitor AA861, the tachykinin NK(1) antagonist SR-140333 and the nitric oxide inhibitor L-NAME reduced only the eosinophil number. The eotaxin levels were significantly higher in DMTI-II-injected rats compared with control animals. In conclusion, DMTI-II causes an early migration of eosinophils and neutrophils by mechanisms involving COX-2- and lipoxygenase-derived metabolites, PAF, substance P and NO. The capacity of DMTI-II to recruit eosinophils at early times is likely to reflect the allergen properties of proteinase inhibitors belonging to Kunitz family.

Role of sensory innervation in the rat pulmonary neutrophil recruitment induced by staphylococcal enterotoxins type A and B.

Rat airways exposure to Staphylococcal enterotoxin A (SEA) and B (SEB) induces marked neutrophil influx. Since sensory neuropeptides play important roles in cell infiltration, in this study we have investigated its contribution in triggering SEA- and SEB-induced pulmonary neutrophil infiltration. Male Wistar rats were exposed intratracheally with SEA (3 ng/trachea) or SEB (250 ng/trachea). Animals received different in vivo pretreatments, after which the neutrophil counts and levels of substance P and IL-1 in bronchoalveolar lavage fluid were evaluated. Alveolar macrophages and peritoneal mast cells were incubated with SEA and SEB to determine the IL-1 and TNF-alpha levels. Capsaicin pretreatment significantly reduced SEA- and SEB-induced neutrophil influx in bronchoalveolar lavage fluid, but this treatment was more effective to reduce SEA responses. Treatments with SR140333 (tachykinin NK(1) receptor antagonist) and SR48968 (tachykinin NK(2) receptor antagonist) decreased SEA-induced neutrophil influx, whereas SEB-induced responses were inhibited by SR140333 only. Cyproheptadine (histamine/5-hydroxytriptamine receptor antagonist) and MD 7222 (5-HT(3) receptor antagonist) reduced SEA- and SEB-induced neutrophil influx. The substance P and IL-1 levels in bronchoalveolar lavage fluid of SEA-exposed rats were significantly higher than SEB. In addition, SEA (but not SEB) significantly released mast cell TNF-alpha. Increased production of TNF-alpha and IL-1 in alveolar macrophages was observed in response to SEA and SEB. In conclusion, sensory neuropeptides contribute significantly to SEA- and SEB-induced pulmonary neutrophil recruitment, but SEA requires in a higher extent the airways sensory innervation, and participation of mast cells and alveolar macrophage products.

Airway exposure to staphylococcal enterotoxin A potentiates allergen-induced bone marrow eosinophilia and trafficking to peripheral blood and airways

Bone marrow (BM) eosinopoiesis is a common feature during allergen exposure in atopic individuals. Airway exposure to staphylococcal superantigens aggravates allergic airway disease and increases the output of BM eosinophils. However, the exact mechanisms regulating eosinophil mobilization and trafficking to the peripheral circulation and airways remain to be elucidated. Therefore, this study aimed to investigate the mechanisms determining the BM eosinopoiesis in allergic mice under exposure to staphylococcal enterotoxin A (SEA). Ovalbumin (OVA)-sensitized male BALB/C mice were intranasally exposed to SEA (1 μg), and at 4, 12, 24, and 48 h later animals were challenged with OVA (10 μg, twice a day). Measurement of IL-5, eotaxin, and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels, flow cytometry for CCR3(+), VLA4(+), and CCR3(+)VLA4(+), as well as adhesion assays to VCAM-1 were performed in BM. Prior airway exposure to SEA time dependently increased the BM eosinophil number in OVA-challenged mice. Eosinophils gradually disappear from peripheral blood, being recruited over time to the airways, where they achieve a maximal infiltration at 24 h. SEA exposure increased the levels of IL-5 and eotaxin (but not GM-CSF) in BM of OVA-challenged mice. Marked increases in CCR3(+) and CCR3(+)VLA4(+) expressions in BM eosinophils of OVA-challenged mice were observed, an effect largely reduced by prior exposure to SEA. Adhesion of BM eosinophils to VCAM-1 was increased in OVA-challenged mice, but prior SEA exposure abrogated this enhanced cell adhesion. Accumulation of BM eosinophils by airway SEA exposure takes place through IL-5- and CCR3-dependent mechanisms, along with downregulation of CCR3/VL4 and impaired cell adhesion to VCAM-1.