W. Tavares de Lima

Subchronic effects of nasally instilled diesel exhaust particulates on the nasal and airway epithelia in mice

Diesel exhaust is the major source of ultrafine particles released during traffic-related pollution. Subjects with chronic respiratory diseases are at greater risk for exacerbations during exposure to air pollution. This study evaluated the effects of subchronic exposure to a low-dose of diesel exhaust particles (DEP). Sixty male BALB/c mice were divided into two groups: (a) Saline: nasal instillation of saline (n = 30); and (b) DEP: nasal instillation of 30 µg of DEP/10 µl of saline (n = 30). Nasal instillations were performed 5 days a week, over 30 and 60 days. Animals were anesthetized with pentobarbital sodium (50 mg/kg intraperitoneal [i.p.]) and sacrificed by exsanguination. Bronchoalveolar lavage (BAL) fluid was performed to evaluate the inflammatory cell count and the concentrations of the interleukin (IL)-4, IL-10, and IL-13 by enzyme-linked immunosorbent assay (ELISA). The gene expression of oligomeric mucus/gel-forming (Muc5ac) was evaluated by real-time polymerase chain reaction (PCR). Histological analysis in the nasal septum and bronchioles was used to evaluate the bronchial and nasal epithelium thickness as well as the acidic and neutral nasal mucus content. The saline group (30 and 60 days) did not show any changes in any of the parameters. However, the instillation of DEP over 60 days increased the expression of Muc5ac in the lungs and the acid mucus content in the nose compared with the 30-day treatment, and it increased the total leukocytes in the BAL and the nasal epithelium thickness compared with saline for 60 days. Cytokines concentrations in the BAL were detectable, with no differences among the groups. Our data suggest that a low-dose of DEP over 60 days induces respiratory tract inflammation.

Contractile Responses of Proximal and Distal Trachea Segments Isolated from Rats Subjected to Immunological Stimulation: Role of Connective Tissue Mast Cells

1. Anaphylaxis-induced contractions of proximal and distal tracheal segments isolated from 14-day ovalbumin (OA)-sensitized rats were studied. 2. OA-induced contractions in distal segments were significantly greater than those observed in proximal segments. 3. Pretreatment of the rats with compound 48/80 or with sodium cromoglycate (SCG) aerosolization significantly reduced OA-induced contractions of trachea distal segments, whereas the contractions of proximal segments were reduced only by compound 48/80. 4. Mepacrine reduced and indomethacin increased the OA-induced contractions in all tracheal segments. Nor-dihydroguaiaretic acid increased the OA-induced contractions in distal tracheal segments, whereas dazoxiben inhibited the contractions in these same segments; neither of these drugs had any effect on the contractions in proximal tracheal segments. 5. The depletion of connective tissue mast cells and subsequent in vitro treatment with indomethacin increased the OA-induced contractions in both segments. 6. We conclude that the contractions of tracheal muscle from OA-sensitized rats depends on the topographic and anatomical origin of the airway tissue. 7. Mediators released by connective tissue mast cells in proximal and distal segments play a pivotal role in this response and may account for variations in the intensity of contraction seen after the addition of OA.

Effects of Amphetamine on Immune-Mediated Lung Inflammatory Response in Rats

Objective: The present study analyzed the effects of acute amphetamine (AMPH) treatment on immune-mediated lung inflammatory response in rats. Methods: There were four experiments. In the first and second experiments, rats were treated with AMPH (1 mg/kg) or 0.9% NaCl, and locomotor activity (experiment 1) and serum AMPH concentrations (experiment 2) were measured 1 or 12 h after treatment. In the third experiment, rats which were immunized with ovalbumin (OVA) were treated 14 days later with 0.9% NaCl or AMPH (1 mg/kg). Twelve hours after these treatments, all animals were submitted to challenge by 1% OVA inhalation being analyzed afterwards for bronchoalveolar lavage fluid (BAL), peripheral blood and bone marrow cellularity. In the fourth and final experiment, rats were treated and studied as for experiment 3, except that half of the animals within each group were previously treated with metyrapone prior to the OVA challenge. Results: In the non- immunized rats, AMPH treatment induced an increase in locomotor activity synchronized to high serum AMPH concentrations 1 h after, but not 12 h after treatment. In OVA-challenged rats, AMPH treatment decreased the total number of inflammatory cells, recovered in both BAL and peripheral blood and increased the total number of bone marrow cells. These effects, observed 1 day after OVA challenge, were abrogated by previous metyrapone treatment. Conclusion: AMPH treatment changed HPA-axis responsiveness to the stress condition imposed by the OVA challenge decreasing lung and blood leukocytes cellularity most probably via corticosterone actions on bone marrow activity.

Effects of single or repeated amphetamine treatment and withdrawal on lung allergic inflammation in rats

The effects of single or repeated amphetamine (AMPH) treatment and those of AMPH withdrawals on immune-mediated lung inflammatory response were studied in rats. Two experiments were done. In the first, rats egg-albumin (OVA) sensitized were singularly or repeatedly (21 days, once daily) treated with AMPH (1.0 mg/kg) or with a similar number and volume of 0.9% NaCl. The OVA aerosol challenge was performed 12 h after the single or last repeated AMPH treatment and also 72 and 120 h after AMPH withdrawal. In the second experiment, the effects of reserpine (1.0 mg/kg/day for 5 consecutive days) on single AMPH actions on lung allergic response of rats were analyzed. Single and repeated AMPH treatment induced opposite actions on Bronchoalveolar lavage fluid (BAL) cellularity of allergic rats: single treatment decreased and repeated treatment increased the total number of cells as well as those of macrophages, neutrophils and eosinophils. Our data also showed that single but not repeated AMPH treatment decreased the number of neutrophils, monocytes and lymphocytes in the peripheral blood, and increased the total number of bone marrow cells in rats sensitized and challenged with OVA. Furthermore, it was shown that reserpine treatment precluded the effects of single AMPH treatment on cellular migration to the lung of OVA-sensitized and challenged rats. It was concluded that AMPH effects on lung inflammatory response and cell recruitment to the lung in allergic rats rely at least partially on corticosterone serum levels. The possible involvement of vesicular monoamine transporter type 2 (VMAT2) with these observed effects was discussed.

Studies on the mechanism of PAF-induced vasopermeability in rat lungs.

The present study evaluated the effect of platelet activating factor (PAF) instilled into rat airways on vascular permeability assessed in isolated lung tissues by Evans blue (EB)-labelled plasma protein extravasation. It was found that intratracheal instillation of PAF induces a dose-dependent increase of EB extravasation in the bronchi (upper and inner) but not in the lung parenchyma. The contribution of eicosanoids to PAF-induced increase of vascular permeability was investigated by treating the animals with selected inhibitors prior to PAF administration. Mepacrine (5 mg/kg), L-663,536 (10 mg/kg), indomethacin (4 mg/kg) and dazoxiben (10 mg/kg) significantly reduced EB extravasation in the bronchi. The PAF antagonists BN-52021 (5 mg/kg), WEB-2086 (1 mg/kg), WEB-2170 (5 mg/kg) and PCA-4248 (3 mg/kg) were all effective in reducing the extravasation. These results suggest that PAF-induced increase of vascular permeability in rat bronchi is mediated by cyclooxygenase and lipoxygenase products of arachidonic acid metabolism.

Impairment in connective tissue mast cells degranulation in spontaneously hypertensive rats: stimulus dependent resistance

Microvascular permeability in the mesentery and consequent leakage of protein into the peritoneum of spontaneously hypertensive rats (SHR) and normotensive rats (NTR) was measured in vivo by the extravasation of Evans blue dye. In sensitized NTR, challenge with antigen produced extensive increases in dye extravasation in the mesentery and in peritoneal lavage fluid within 10 min. In sensitized SHR there was no increase in the permeability of the mesentery and a very weak increase in dye extravasation in the peritoneal cavity following challenge. The glucocorticoid antagonist RU38486 did not change the permeability response induced by antigen in sensitized NTR and SHR. However, compound 48/80 was equally effective in either NTR or SHR in causing increased vasopermeability. Mesenteric mast cells in the NTR were degranulated after immunological challenge, whereas those in the SHR were resistant, as measured histologically. Similarly, challenge ex vivo of mesentery from sensitized NTR induced contraction of guinea‐pig ileum in co‐incubation experiments, whereas SHR mesentery was unresponsive. Plasma levels of antigen‐specific IgE and IgG2a in sensitized NTR and SHR were identical. Immune serum from SHR was unable to induce a passive cutaneous anaphylaxis (PCA) reaction in the skin of NTR and SHR did not develop a PCA reaction upon passive sensitization with NTR immune serum. We conclude that the mast cells of SHR are resistant to degranulation following immunological challenge, although the relevant antibodies are present.

MODULATION OF HYPERSENSITIVITY REACTION BY LIPIDS GIVEN ORALLY

The effect of lipids administration by gavage (0.4% body weight) given daily during four weeks on the hypersensitivity reaction in trachea, upper and lower bronchi, liver, kidney, mesentery, and pancreas was investigated in male rats. The plasma exudation was assessed by using Evans blue (EB) dye extravasation method. There was a significant difference in the permeability of the organs in nonimmunized rats. The immunization increased the vascular permeability and the response with the organs varied greatly. The effect of lipids on anaphylactic reaction was compared to those of untreated rats (control group). The EB extravasation was significantly increased in the trachea obtained from rats treated with cocoa butter and soybean oil. In the upper bronchi of rats treated with soybean oil, the EB extravasation was increased. However, in the lower bronchi, none of the treatments with lipids changed the extravasation of EB. The same was observed in the liver and kidney. The animals treated with lipids by gavage did not present differences in EB extravasation in the mesentery. However, in the pancreas and duodenum, the treatment with fish and soybean oils and cocoa butter markedly lowered EB extravasation.

Inhibitory effect of econazole on the release of thromboxanes

The effect of econazole on the release of thromboxanes was investigated. It was found that econazole inhibited concentration-dependently the aggregation of guinea pig platelets stimulated with arachidonic acid. The compound also reduced significantly the LTB4-induced contraction of guinea pig lung parenchyma strips and the contraction of rabbit aorta to the effluent of LTD4-stimulated guinea pig lungs, both effects mediated mostly by thromboxane generation. The concentration of TXB2 in the effluents from LTD4 stimulated lungs, assayed by EIA, was significantly reduced following pretreatment of the lungs with 10−4M and 10−5M of econazole, whereas the levels of PGE2 were increased. These results demonstrate that econazole is a selective inhibitor of thromboxane synthesis.

44. Cohabitation with a sick-cage mate exacerbates allergic lung inflammatory response in mice

The objective of this study was analyze the effects of cohabitation with sick partner on allergic lung inflammatory response in ovalbumin (OVA) sensitized and challenged mice. Pairs of mice were divided into control and experimental groups. Within each group, one mouse from each pairs was immunized with a solution containing (OVA). The remaining mouse from the experimental group was injected with Ehrlich tumor cells being designated as “companion of sick partner” (CSP); the remaining mouse of the control group was injected with PBS being designated as “companion of healthy partner” (CHP). CSP mice presented in relation to CHP mice: increased number of eosinophils and neutrophils in the BAL; increased IL-4 and IL-5 levels and decreased IL-10 and INF-γ in the BAL supernatant; increased IgG1-OVA and decrease IgG2a-OVA levels on peripheral blood; increased expression of L-selectin in the BAL granulocytes; decreased in vitro tracheal reactivity to metacholine; no changes on plasma corticosterone levels and increased levels of plasmatic noradrenaline. The results suggest that the exacerbation of the allergic lung inflammatory response in CSP mice is a consequence of the psychological stress induced by forced long-term cohabitation with sick the partner, a fact that might ultimately depend on the changes induced by catecholamines on Th1/Th2 balance, toward a Th2 cytokine profile and finally the recruitment and activation of eosinophils in the airways. FAPESP: 2012/03372-3; 2009/51886-3.