Ana Cristina Breithaupt-Faloppa

Formaldehyde induces lung inflammation by an oxidant and antioxidant enzymes mediated mechanism in the lung tissue

Formaldehyde (FA) is an indoor and outdoor pollutant widely used by many industries, and its exposure is associated with inflammation and oxidative stress in the airways. Our previous studies have demonstrated the role of reactive oxygen species (ROS) in lung inflammation induced by FA inhalation but did not identify source of the ROS. In the present study, we investigate the effects of FA on the activities and gene expression of glutathione peroxidase (GPX), glutathione reductase (GR), glutathione S-transferase (GST), superoxide dismutase (SOD) 1 and 2, catalase (CAT), nitric oxide synthase (iNOS and cNOS) and cyclooxygenase (COX) 1 and 2. The hypothesized link between NADPH-oxidase, nitric oxide synthase and cyclooxygenase, the lung inflammation after FA inhalation was also investigated. For experiments, male Wistar rats were submitted to FA inhalation (1%, 90 min daily) for 3 consecutive days. The treatments with apocynin and indomethacin before the FA exposure reduced the number of neutrophils recruited into the lung. Moreover, the treatments with apocynin and indomethacin blunted the effect of FA on the generation of IL-1β, while the treatments with L-NAME and apocynin reduced the generation of IL-6 by lung explants when compared to the untreated group. FA inhalation increased the levels of NO and hydrogen peroxide by BAL cells cultured and the treatments with apocynin and l-NAME reduced these generations. FA inhalation did not modify the activities of GPX, GR, GST and CAT but reduced the activity of SOD when compared to the naïve group. Significant increases in SOD-1 and -2, CAT, iNOS, cNOS and COX-1 expression were observed in the FA group compared to the naïve group. The treatments with apocynin, indomethacin and L-NAME reduced the gene expression of antioxidant and oxidant enzymes. In conclusion, our results indicate that FA causes a disruption of the physiological balance between oxidant and antioxidant enzymes in lung tissue, most likely favoring the oxidant pathways and thus positively modulating lung inflammation.

Reduced allergic lung inflammation in rats following formaldehyde exposure: Long-term effects on multiple effector systems

Clinical and experimental evidences show that formaldehyde (FA) exposure has an irritant effect on the upper airways. As being an indoor and outdoor pollutant, FA is known to be a causal factor of occupational asthma. This study aimed to investigate the repercussion of FA exposure on the course of a lung allergic process triggered by an antigen unrelated to FA. For this purpose, male Wistar rats were subjected to FA inhalation for 3 consecutive days (1%, 90-min daily), subsequently sensitized with ovalbumin (OVA)-alum via the intraperitoneal route, and 2 weeks later challenged with aerosolized OVA. The OVA challenge in rats after FA inhalation (FA/OVA group) evoked a low-intensity lung inflammation as indicated by the reduced enumerated number of inflammatory cells in bronchoalveolar lavage as compared to FA-untreated allergic rats (OVA/OVA group). Treatment with FA also reduced the number of bone marrow cells and blood leukocytes in sensitized animals challenged with OVA, which suggests that the effects of FA had not been only localized to the airways. As indicated by passive cutaneous anaphylactic reaction, FA treatment did not impair the anti-OVA IgE synthesis, but reduced the magnitude of OVA challenge-induced mast cell degranulation. Moreover, FA treatment was associated to a diminished lung expression of PECAM-1 (platelet-endothelial cell adhesion molecule 1) in lung endothelial cells after OVA challenge and an exacerbated release of nitrites by BAL-cultured cells. Keeping in mind that rats subjected solely to either FA or OVA challenge were able to significantly increase the cell influx into lung, our study shows that FA inhalation triggers long-lasting effects that affect multiple mediator systems associated to OVA-induced allergic lung such as the reduction of mast cells activation, PECAM-1 expression and exacerbation of NO generation, thereby contributing to the decrease of cell recruitment after the OVA challenge. In conclusion, repeated expositions to air-borne FA may impair the lung cell recruitment after an allergic stimulus, thereby leading to a non-responsive condition against inflammatory stimuli likely those where mast cells are involved.

Protective effect of estradiol on acute lung inflammation induced by an intestinal ischemic insult is dependent on nitric oxide.

ABSTRACT Introduction: It has been shown that the innate immune system mediates acute lung inflammation triggered by intestinal trauma. Sexual dimorphism modulates the profile of TH1 and TH2 lymphocytes, and accordingly sex hormones may modulate acute lung inflammation by intestinal ischemia/reperfusion (I/R). Studies indicate that female rats are relatively resistant to organ injury caused by hemorrhagic shock and that the gut of female is more resistant than that of the male to deleterious effects of ischemic injury. At the present study, we investigated the effect of estradiol (E2) on the lung inflammation after intestinal I/R and its interaction with the nitric oxide (NO) pathway. Methods: Anesthetized female rats submitted or not to 7 days ovariectomy (OVx) were subjected to occlusion of the superior mesenteric artery during 45 min, followed by 2 h of reperfusion. Groups of rats were treated with E2 (17&bgr;-estradiol, 280 &mgr;g/kg, s.c.) 24 h before ischemia and/or with the nonselective NO synthase inhibitor L-NAME (N&ohgr;-nitro-L-arginine methyl ester hydrochloride) (5 mg/kg, i.v.). In a parallel set of experiments, the selective NO synthase inhibitor, aminoguanidine (50 mg/kg i.v.), was given 1 h before ischemia. In all groups, lung vascular permeability (LVP) was assessed using the Evans blue dye extravasation method, neutrophil recruitment to the tissues by the standard myeloperoxidase (MPO) method, and endothelial NO synthase (eNOS) protein expression by Western blot. Results: In OVx rats, LVP and MPO were increased after intestinal I/R as compared with intact controls. Estradiol reverted the LVP, but not MPO. Aminoguanidine reduced LVP in OVx rats. The E2 protective effect on LVP was abolished by L-NAME; moreover, an increase in LVP even when compared with OVx rats treated only with L-NAME was observed. In addition, lung eNOS protein expression was reduced in OVx-I/R rats in comparison to intact controls and the E2 inhibited this effect. Conclusions: Estradiol treatment is able to reduce lung inflammation due to intestinal I/R, but with the concomitant blockade of NOS activity, this effect is abolished. Nitric oxide probably reduces the vascular deleterious effects of intestinal I/R, and E2 pretreatment reduces lung inflammation after intestinal I/R and exerts these effects by modulating eNOS protein expression in the lungs.

Nitric oxide mediates lung vascular permeability and lymph-borne IL-6 after an intestinal ischemic insult.

Acute lung injury following intestinal I/R depends on neutrophil-endothelial cell interactions and on cytokines drained from the gut through the lymph. Among the mediators generated during I/R, increased serum levels of IL-6 and NO are also found and might be involved in acute lung injury. Once intestinal ischemia itself may be a factor of tissue injury, in this study, we investigated the presence of IL-6 in lymph after intestinal ischemia and its effects on human umbilical vein endothelial cells (HUVECs) detachment. The involvement of NO on the increase of lung and intestinal microvascular permeability and the lymph effects on HUVEC detachment were also studied. Upon anesthesia, male Wistar rats were subjected to occlusion of the superior mesenteric artery during 45 min, followed by 2-h intestinal reperfusion. Rats were treated with the nonselective NO synthase (NOS) inhibitor l-NAME (N&ohgr;-nitro-l-arginine methyl ester) or with the selective inhibitor of iNOS aminoguanidine 1 h before superior mesenteric artery occlusion. Whereas treatment with l-NAME during ischemia increased both IL-6 levels in lymph and lung microvascular permeability, aminoguanidine restored the augmented intestinal plasma extravasation due to ischemia and did not induce IL-6 in lymph. On the other hand, IL-6 and lymph of intestinal I/R detached the HUVECs, whereas lymph of ischemic rats upon l-NAME treatment when incubated with anti-IL-6 prevented HUVEC detachment. It is shown that the intestinal ischemia itself is sufficient to increase intestinal microvascular permeability with involvement of iNOS activation. Intestinal ischemia and absence of constitutive NOS activity leading to additional intestinal stress both cause release of IL-6 and increase of lung microvascular permeability. Because anti-IL-6 prevented the endothelial cell injury caused by lymph at the ischemia period, the lymph-borne IL-6 might be involved with endothelial cell activation. At the reperfusion period, this cytokine does not seem to be modulated by NO.

Intestinal Lymph-Borne Factors Induce Lung Release of Inflammatory Mediators and Expression of Adhesion Molecules After an Intestinal Ischemic Insult

BACKGROUND Intestinal ischemia and reperfusion (I/R) is a documented cause of acute lung injury (ALI) and systemic inflammation. We previously reported that obstruction of thoracic lymphatic flow during intestinal I/R blunts pulmonary neutrophil recruitment and microvascular injury and decreases the systemic levels of tumor necrosis factor. Here, we consider the existence of a gut-lung axis promoting the induction of systemic inflammation, whereby drained intestinal lymph stimulates lung expression of adhesion molecules and matrix components and generation of inflammatory mediators. MATERIAL AND METHODS Upon administration of anesthesia, male Wistar rats were subjected to occlusion of the superior mesenteric artery for 45 min, followed by 2 h of intestinal reperfusion (I/R); groups of rats were subjected to I/R with or without thoracic lymphatic duct ligation immediately before the procedure. The non-manipulated rats were used to investigate basal parameters. RESULTS Obstruction of thoracic lymphatic flow before intestinal I/R decreased the ability of cultured lung tissue explants to release IL-1β, IL-10, and VEGF. In contrast, lymphatic obstruction normalized the elevated lung expression of PECAM-1 caused by intestinal I/R. On the other hand, lung E-selectin expression was significantly reduced, whereas fibronectin expression and collagen synthesis were not affected. Lymph levels of LTB(4) and TXB(2) were found to be significantly increased. CONCLUSIONS These data suggest that lymph factors drained from the intestine during ischemic trauma stimulate the lung to generate inflammatory mediators and alter the expression of adhesion molecules. Disturbances in lung homeostasis mediated by lymph might contribute to the spread of inflammatory processes, thereby accounting for the systemic inflammation induced by intestinal I/R.

Acute effects of estradiol on lung inflammation due to intestinal ischemic insult in male rats.

ABSTRACT Intestinal ischemia and reperfusion (intestinal I/R) causes acute lung inflammation that is characterized by leukocyte migration, increased lung microvascular permeability, and, in severe forms, noncardiogenic pulmonary edema and acute respiratory distress syndrome. Female sex hormones interfere with immune response, and experimental and clinical evidence shows that females are more resistant than males to organ injury caused by gut trauma. To reduce the lung inflammation caused by intestinal I/R, we have acutely treated male rats with estradiol. Intestinal I/R was performed by the clamping (45 min) of the superior mesenteric artery (SMA), followed by 2 h of intestinal reperfusion (unclamping SMA). Groups of rats received 17&bgr; estradiol (E2, 280 µg/kg, i.v., single dose) 30 min after the SMA occlusion (ischemia period) or 1 h after the unclamping of SMA (reperfusion period). Leukocytes influx into the lung and microvascular leakage were assessed by lung myeloperoxidase activity and Evans blue dye extravasation, respectively. The lung expression of adhesion molecules (intercellular adhesion molecule 1, platelet endothelial cell adhesion molecule 1, and vascular cell adhesion molecule [VCAM]) was evaluated by immunohistochemistry. Interleukin 1&bgr; (IL-1&bgr;), IL-10, and NOx− concentrations were quantified in supernatants of cultured lung tissue. We have found that intestinal I/R increased the lung myeloperoxidase activity and Evans blue dye extravasation, which were reduced by treatment of rats with E2. Intestinal I/R increased ICAM-1 expression only, and it was decreased by E2 treatment. However, E2 treatment reduced the basal expression of platelet endothelial cell adhesion molecule 1. E2 treatment during intestinal ischemia was effective to reduce the levels of IL-10 and IL-1&bgr; in explant supernatant, but only IL-10 levels were reduced by E2 at reperfusion phase. The treatment with E2 did not affect NOx− concentration. Taken together, our data suggest that estradiol modulates the lung inflammatory response induced by lung injury, likely by acute effects. Thus, acute estradiol treatment could be considered as a potential therapeutic agent in ischemic events.

Connective tissue mast cells are the target of formaldehyde to induce tracheal hyperresponsiveness in rats: Putative role of leukotriene B4 and nitric oxide

Formaldehyde (FA) exposure induces upper airways irritation and respiratory abnormalities, but its mechanisms are not understood. Since mast cells are widely distributed in the airways, we hypothesized that FA might modify the airways reactivity by mechanism involving their activation. Tracheal rings of rats were incubated with Dulbeccos modified medium culture containing FA (0.1 ppm) in 96-well plastic microplates in a humid atmosphere. After 30 min, 6h, and 24-72 h, the rings were suspended in an organ bath and dose-response curve to methacholine (MCh) were determined. Incubation with FA caused a transient tracheal hyperresponsiveness to MCh that was independent from tracheal epithelium integrity. Connective tissue mast cell depletion caused by compound 48/80 or mast cell activation by the allergic reaction, before exposure of tracheal rings to FA prevented the increased responsiveness to MCh. LTB(4) concentrations were increased in the culture medium of tracheas incubated with FA for 48 h, whereas the LTB(4)-receptor antagonist MK886 (1 microM) added before FA exposure rendered the tracheal rings normoreactive to MCh. In addition, FA exposure did not cause hyperresponsiveness in tracheal segments incubated with l-arginine (1 microM). We suggest that airway connective tissue mast cells constitute the target and may provide the increased LTB(4) generation as well as an elevated consumption of NO leading to tracheal hyperresponsiveness to MCh.

In vitro behaviour of endothelial cells on a titanium surface

BackgroundEndothelial cells play an important role in the delivery of cells to the inflammation site, chemotaxis, cell adhesion and extravasation. Implantation of a foreign material into the human body determines inflammatory and repair reactions, involving different cell types with a plethora of released chemical mediators. The evaluation of the interaction of endothelial cells and implanted materials must take into account other parameters in addition to the analysis of maintenance of cell viability.MethodsIn the present investigation, we examined the behavior of human umbilical vein endothelial cells (HUVECs) harvested on titanium (Ti), using histological and immunohistochemical methods. The cells, after two passages, were seeded in a standard density on commercially plate-shaped titanium pieces, and maintained for 1, 7 or 14 days.ResultsAfter 14 days, we could observe a confluent monolayer of endothelial cells (ECs) on the titanium surface. Upon one-day Ti/cell contact the expression of fibronectin was predominantly cytoplasmatic and stronger than on the control surface. It was observed strong and uniform cell expression along the time of α5β1 integrin on the cells in contact with titanium.ConclusionThe attachment of ECs on titanium was found to be related to cellular-derived fibronectin and the binding to its specific receptor, the α5β1 integrin. It was observed that titanium effectively serves as a suitable substrate for endothelial cell attachment, growth and proliferation. However, upon a 7-day contact with Ti, the Weibel-Palade bodies appeared to be not fully processed and exhibited an anomalous morphology, with corresponding alterations of PECAM-1 localization.

Formaldehyde inhalation reduces respiratory mechanics in a rat model with allergic lung inflammation by altering the nitric oxide/cyclooxygenase-derived products relationship.

Bronchial hyperresponsiveness is a hallmark of asthma and many factors modulate bronchoconstriction episodes. A potential correlation of formaldehyde (FA) inhalation and asthma has been observed; however, the exact role of FA remains controversial. We investigated the effects of FA inhalation on Ovalbumin (OVA) sensitisation using a parameter of respiratory mechanics. The involvement of nitric oxide (NO) and cyclooxygenase-derived products were also evaluated. The rats were submitted, or not, to FA inhalation (1%, 90 min/day, 3 days) and were OVA-sensitised and challenged 14 days later. Our data showed that previous FA exposure in allergic rats reduced bronchial responsiveness, respiratory resistance (Rrs) and elastance (Ers) to methacholine. FA exposure in allergic rats also increased the iNOS gene expression and reduced COX-1. L-NAME treatment exacerbated the bronchial hyporesponsiveness and did not modify the Ers and Rrs, while Indomethacin partially reversed all of the parameters studied. The L-NAME and Indomethacin treatments reduced leukotriene B₄ levels while they increased thromboxane B₂ and prostaglandin E₂. In conclusion, FA exposure prior to OVA sensitisation reduces the respiratory mechanics and the interaction of NO and PGE₂ may be representing a compensatory mechanism in order to protect the lung from bronchoconstriction effects.

Estradiol Modulates Local Gut Injury Induced by Intestinal Ischemia-Reperfusion in Male Rats.

Intestinal ischemia and reperfusion (I/R) triggers a systemic inflammatory response characterized by leukocyte mobilization from the bone marrow, release of cytokines to the circulation, and increased microvascular permeability, leading to high mortality. Females have shown attenuated inflammatory response to trauma when compared with males, indicating a role for female sex hormones in this process. Here, we have evaluated the effect of estradiol on the local gut injury induced by I/R in male rats. I/R was induced by the clamping of the superior mesenteric artery for 45 min, followed by 2 h of reperfusion. A group received 17β-estradiol (280 μg/kg, i.v., single dose) at 30 min of ischemia. Morphometric analysis of the gut showed I/R induced a reduction of villous height that was prevented by estradiol. White blood cells, notably granulocytes, were mobilized from the circulation to the intestine by I/R, which was also prevented by estradiol treatment. Groups had the intestine wrapped in a plastic bag to collect intestinal fluid, where leukocytes count, TNF-α, and IL-10 levels were increased by I/R. Serum chemokines (CINC-1, MIP-1α, MIP-2), ICAM-1 expression in the mesenteric tissue, and neutrophils spontaneous migration measured in vitro were also increased after I/R. Estradiol treatment reduced leukocytes numbers and TNF-α on intestinal fluid, serum chemokine release and also downregulated MIP-1α, MIP-2 gene expression, and spontaneous in vitro neutrophil migration. In conclusion, estradiol blunts intestinal injury induced by I/R by modulating chemokines release and leukocyte trafficking.ABSTRACT Intestinal ischemia and reperfusion (I/R) triggers a systemic inflammatory response characterized by leukocyte mobilization from the bone marrow, release of cytokines to the circulation, and increased microvascular permeability, leading to high mortality. Females have shown attenuated inflammatory response to trauma when compared with males, indicating a role for female sex hormones in this process. Here, we have evaluated the effect of estradiol on the local gut injury induced by I/R in male rats. I/R was induced by the clamping of the superior mesenteric artery for 45 min, followed by 2 h of reperfusion. A group received 17&bgr;-estradiol (280 &mgr;g/kg, i.v., single dose) at 30 min of ischemia. Morphometric analysis of the gut showed I/R induced a reduction of villous height that was prevented by estradiol. White blood cells, notably granulocytes, were mobilized from the circulation to the intestine by I/R, which was also prevented by estradiol treatment. Groups had the intestine wrapped in a plastic bag to collect intestinal fluid, where leukocytes count, TNF-&agr;, and IL-10 levels were increased by I/R. Serum chemokines (CINC-1, MIP-1&agr;, MIP-2), ICAM-1 expression in the mesenteric tissue, and neutrophils spontaneous migration measured in vitro were also increased after I/R. Estradiol treatment reduced leukocytes numbers and TNF-&agr; on intestinal fluid, serum chemokine release and also downregulated MIP-1&agr;, MIP-2 gene expression, and spontaneous in vitro neutrophil migration. In conclusion, estradiol blunts intestinal injury induced by I/R by modulating chemokines release and leukocyte trafficking.