Oral feeding of Lactobacillus bulgaricus N45.10 inhibits the lung inflammation and airway remodeling in murine allergic asthma: Relevance to the Th1/Th2 cytokines and STAT6/T-bet.

Asthma is a chronic disease with impacts on public health. It affects the airways causing pulmonary inflammation mediated by CD4 T cells type Th2, eosinophilia, mucus hypersecretion, and elevated IgE. The unbalance between cytokines and transcription factors is an important feature in asthma. Probiotics has gaining highlight as a therapy for chronic diseases. Thus, we investigate the Lactobacillus bulgaricus (Lb) effect in murine allergic asthma. BALB/c-mice were sensitized to ovalbumin (OA) on days 0 and 7 and were challenged from day 14-28 with OA. Mice received Lb seven days prior to sensitization and it was kept until day 28. The Lb attenuated the eosinophils infiltration, mucus and collagen secretion, IgE production, pro-inflammatory cytokines, TLR4 expression, GATA3, STAT6 and RORγt in lung. Otherwise, Lb increased the anti-inflammatory cytokines, the T-bet and foxp3. Finally, Lb attenuated the allergic asthma-induced inflammation and airway remodeling by interfering on Th1/Th2 cytokines and STAT6/T-bet transcription factors.

Beneficial effect of low-level laser therapy in acute lung injury after i-I/R is dependent on the secretion of IL-10 and independent of the TLR/MyD88 signaling.

The use of low-level laser for lung inflammation treatment has been evidenced in animal studies as well as clinical trials. The laser action mechanism seems to involve downregulation of neutrophil chemoattractants and transcription factors. Innate immune responses against microorganisms may be mediated by toll-like receptors (TLR). Intestinal ischemia and reperfusion (i-I/R) lead to bacterial product translocation, such as endotoxin, which consequently activates TLRs leading to intestinal and lung inflammation after gut trauma. Thus, the target of this study was to investigate the role of TLR activation in the laser (660 nm, 30 mW, 67.5 J/cm2, 0.375 mW/cm2, 5.4 J, 180 s, and spot size with 0.08 cm2) effect applied in contact with the skin on axillary lymph node in lung inflammation induced by i-I/R through a signaling adaptor protein known as myeloid differentiation factor 88 (MyD88). It is a quantitative, experimental, and laboratory research using the C57Bl/6 and MyD88-/- mice (n = 6 mice for experimental group). Statistical differences were evaluated by ANOVA and the Tukey-Kramer multiple comparisons test to determine differences among groups. In order to understand how the absence of MyD88 can interfere in the laser effect on lung inflammation, MyD88-/- mice were treated or not with laser and subjected to occlusion of the superior mesenteric artery (45 min) followed by intestinal reperfusion (4 h). In summary, the laser decreased the MPO activity and the lung vascular permeability, thickened the alveolar septa, reduced both the edema and the alveolar hemorrhage, as well as significantly decreased neutrophils infiltration in MyD88-deficient mice as well in wild-type mice. It noted a downregulation in chemokine IL-8 production as well as a cytokine IL-10 upregulation in these animals. The results also evidenced that in absence of IL-10, the laser effect is reversed. Based on these results, we suggest that the beneficial effect of laser in acute lung injury after i-I/R is dependent on the secretion of IL-10 and independent of the TLR/MyD88 signaling.

PPAR gamma activation protects the brain against microvascular dysfunction in sepsis.

Sepsis is a severe disorder characterized by systemic inflammatory responses in the presence of an infection and may progress to multiple organ dysfunction and death. Alterations in cerebral microcirculation fulfill a crucial role in the pathogenesis of severe sepsis, and include a decrease in capillary density and disturbances in leukocyte movement along capillaries. Nevertheless, the mechanisms involved in sepsis-associated cerebral microcirculatory alterations have so far not been defined. We investigated the effect of the peroxisome proliferator-activated receptor gamma (PPARγ) selective agonist rosiglitazone on leukocyte/endothelial cell interaction and functional capillary density in the brain in the cecal ligation and puncture (CLP) model of sepsis. Anti-inflammatory effects of rosiglitazone on the cerebral microcirculation were marked. Functional capillary density increased and leukocyte rolling and adhesion were decreased in animals submitted to CLP and treated with rosiglitazone. Our data provide evidence for involvement of PPARγ activation in leukocyte-endothelium interactions and alterations in capillary density. Improved cerebral perfusion in animals treated with rosiglitazone, suggests that PPARγ activation is protective against cerebral microvascular dysfunction in sepsis.

Effects of chronic ethanol consumption in experimental sepsis.

AIMS: To evaluate the effects of chronic ethanol consumption on the development and the pathophysiology of sepsis, using an experimental model of polymicrobial peritonitis by feces i.p. injection. METHODS: Forty-day-old male Wistar rats were divided into groups for two experiments: A and B. Experiment A was performed for determination of mortality rates, while experiment B was designed for biochemical analysis and measurement of cytokines before and after sepsis. In both the experiments, treated animals were exposed to a 10% ethanol solution as the single drinking source for 4 weeks, while untreated animals were exposed to tap water over the same period. Food was provided ad libitum. After this period, the animals underwent i.p. fecal injection for induction of sepsis. RESULTS: Experiment A showed that higher doses of ethanol resulted in early mortality from sepsis that was correlated with the alcohol consumption (high dose = 85.7%, low dose = 14.3%, P = 0.027). In experiment B, cytokine analysis demonstrated important changes resulting from sepsis, which were further affected by ethanol exposure. In addition, glucose and creatinine levels decreased and increased, respectively, after sepsis, but a significant change occurred only in the ethanol group (P < 0.003 glucose, P < 0.01 creatinine). The levels of pro-inflammatory cytokines, interleukin-6 and tumor necrosis factor-α, increased after sepsis, but were less evident after ethanol exposure. CONCLUSION: These differences may be the result of either early mortality or an increase in the severity of the septic process. Taking into account the high mortality rate and the extreme severity of sepsis after alcohol consumption, often encouraged by advertising, a caution should be given to patients with severe infections and a history of alcohol abuse.

Leptospira and inflammation.

Leptospirosis is an important zoonosis and has a worldwide impact on public health. This paper will discuss both the role of immunogenic and pathogenic molecules during leptospirosis infection and possible new targets for immunotherapy against leptospira components. Leptospira, possess a wide variety of mechanisms that allow them to evade the host immune system and cause infection. Many molecules contribute to the ability of Leptospira to adhere, invade, and colonize. The recent sequencing of the Leptospira genome has increased our knowledge about this pathogen. Although the virulence factors, molecular targets, mechanisms of inflammation, and signaling pathways triggered by leptospiral antigens have been studied, some questions are still unanswered. Toll-like receptors (TLRs) are the primary sensors of invading pathogens. TLRs recognize conserved microbial pattern molecules and activate signaling pathways that are pivotal to innate and adaptive immune responses. Recently, a new molecular target has emerged--the Na/K-ATPase--which may contribute to inflammatory and metabolic alteration in this syndrome. Na/K-ATPase is a target for specific fatty acids of host origin and for bacterial components such as the glycolipoprotein fraction (GLP) that may lead to inflammasome activation. We propose that in addition to TLRs, Na/K-ATPase may play a role in the innate response to leptospirosis infection.

The MAPKinase Signaling and the Stimulatory Protein-1 (Sp1) Transcription Factor Are Involved in the Phototherapy Effect on Cytokines Secretion from Human Bronchial Epithelial Cells Stimulated with Cigarette Smoke Extract.

The present study was aimed to investigate the phototherapy effect with low-level laser on human bronchial epithelial cells activated by cigarette smoke extract (CSE). Phototherapy has been reported to actuate positively for controlling the generation/release of anti-inflammatory and pro-inflammatory mediators from different cellular type activated by distinct stimuli. It is not known whether the IL-8 and IL-10 release from CSE-stimulated human bronchial epithelium (BEAS) cells can be influenced by phototherapy. Human bronchial epithelial cell (BEAS) line was cultured in a medium with CSE and irradiated (660 nm) at 9 J. Apoptosis index was standardized with Annexin V and the cellular viability was evaluated by MTT. IL-8, IL-10, cAMP, and NF-κB were measured by ELISA as well as the Sp1, JNK, ERK1/2, and p38MAPK. Phototherapy effect was studied in the presence of mithramycin or the inhibitors of JNK or ERK. The IL-8, cAMP, NF-κB, JNK, p38, and ERK1/2 were downregulated by phototherapy. Both the JNK and the ERK inhibitors potentiated the phototherapy effect on IL-8 as well as on cAMP secretion from BEAS. On the contrary, IL-10 and Sp1 were upregulated by phototherapy. The mithramycin blocked the phototherapy effect on IL-10. The results suggest that phototherapy has a dual effect on BEAS cells because it downregulates the IL-8 secretion by interfering with CSE-mediated signaling pathways, and oppositely upregulates the IL-10 secretion through of Sp1 transcription factor. The manuscript provides evidence that the phototherapy can interfere with MAPK signaling via cAMP in order to attenuate the IL-8 secretion from CSE-stimulated BEAS. In addition, the present study showed that phototherapy effect is driven to downregulation of the both the IL-8 and the ROS secretion and at the same time the upregulation of IL-10 secretion. Besides it, the increase of Sp-1 transcription factor was crucial for laser effect in upregulating the IL-10 secretion. The dexamethasone corticoid produces a significant inhibitory effect on IL-8 as well as ROS secretion, but on the other hand, the corticoid blocked the IL-10 secretion. Taking it into consideration, it is reasonable to suggest that the beneficial effect of laser therapy on lung diseases involves its action on unbalance between pro-inflammatory and anti-inflammatory mediators secreted by human bronchial epithelial cells through different signaling pathway.

Oral feeding with probiotic Lactobacillus rhamnosus attenuates cigarette smoke-induced COPD in C57Bl/6 mice: Relevance to inflammatory markers in human bronchial epithelial cells.

COPD is a prevalent lung disease with significant impacts on public health. Affected airways exhibit pulmonary neutrophilia and consequent secretion of pro-inflammatory cytokines and proteases, which result in lung emphysema. Probiotics act as nonspecific modulators of the innate immune system that improve several inflammatory responses. To investigate the effect of Lactobacillus rhamnosus (Lr) on cigarette smoke (CS)-induced COPD C57Bl/6 mice were treated with Lr during the week before COPD induction and three times/week until euthanasia. For in vitro assays, murine bronchial epithelial cells as well as human bronchial epithelial cells exposed to cigarette smoke extract during 24 hours were treated with Lr 1 hour before CSE addition. Lr treatment attenuated the inflammatory response both in the airways and lung parenchyma, reducing inflammatory cells infiltration and the production of pro-inflammatory cytokines and chemokines. Also, Lr-treated mice presented with lower metalloproteases in lung tissue and lung remodeling. In parallel to the reduction in the expression of TLR2, TLR4, TLR9, STAT3, and NF-κB in lung tissue, Lr increased the levels of IL-10 as well as SOCS3 and TIMP1/2, indicating the induction of an anti-inflammatory environment. Similarly, murine bronchial epithelial cells as well as human bronchial epithelial cells (BEAS) exposed to CSE produced pro-inflammatory cytokines and chemokines, which were inhibited by Lr treatment in association with the production of anti-inflammatory molecules. Moreover, the presence of Lr also modulated the expression of COPD-associated transcription found into BALF of COPD mice group, i.e., Lr downregulated expression of NF-κB and STAT3, and inversely upregulated increased expression of SOCS3. Thus, our findings indicate that Lr modulates the balance between pro- and anti-inflammatory cytokines in human bronchial epithelial cells upon CS exposure and it can be a useful tool to improve the lung inflammatory response associated with COPD.

Low-level laser therapy decreases levels of lung neutrophils anti-apoptotic factors by a NF-kappaB dependent mechanism.

BACKGROUND AND OBJECTIVE: Low-level laser therapy (LLLT) is a known modulator of inflammatory process. Herein we studied the effect of 660 nm diode laser on mRNA levels of neutrophils anti-apoptotic factors in lipopolysaccharide (LPS)-induced lung inflammation. STUDY DESIGN/METHODOLOGY: Mice were divided into 8 groups (n=7 for each group) and irradiated with energy dosage of 7.5 J/cm(2). The Bcl-xL and A1 mRNA levels in neutrophils were evaluated by Real Time-PCR (RT-PCR). The animals were irradiated after exposure time of LPS. RESULTS: LLLT and an inhibitor of NF-kappaB nuclear translocation (BMS 205820) attenuated the mRNA levels of Bcl-xL and A1 mRNA in lung neutrophils obtained from mice subjected to LPS-induced inflammation. CONCLUSION: LLLT reduced the levels of anti-apoptotic factors in LPS inflamed mice lung neutrophils by an action mechanism in which the NF-kappaB seems to be involved.

Effect of low-level laser therapy on hemorrhagic lesions induced by immune complex in rat lungs.

OBJECTIVE: The aim of this study was to investigate if low-level laser therapy (LLLT) can modulate formation of hemorrhagic lesions induced by immune complex. BACKGROUND DATA: There is a lack of information on LLLT effects in hemorrhagic injuries of high perfusion organs, and the relative efficacy of LLLT compared to anti-inflammatory drugs. METHODS: A controlled animal study was undertaken with 49 male Wistar rats randomly divided into seven groups. Bovine serum albumin (BSA) i.v. was injected through the trachea to induce an immune complex lung injury. The study compared the effect of irradiation by a 650-nm Ga-Al-As laser with LLLT doses of 2.6 Joules/cm(2) to celecoxib, dexamethasone, and control groups for hemorrhagic index (HI) and myeloperoxide activity (MPO) at 24 h after injury. RESULTS: The HI for the control group was 4.0 (95% CI, 3.7-4.3). Celecoxib, LLLT, and dexamethasone all induced significantly (p < 0.01) lower HI than control animals at 2.5 (95% CI, 1.9-3.1), 1.8 (95% CI, 1.2-2.4), and 1.5 (95% CI, 0.9-2.1), respectively, for all comparisons to control. Dexamethasone, but not celecoxib, induced a slightly, but significantly lower HI than LLLT (p = 0.04). MPO activity was significantly decreased in groups receiving celecoxib at 0.87 (95% CI, 0.63-1.11), dexamethasone at 0.50 (95% CI, 0.24-0.76), and LLLT at 0.7 (95% CI, 0.44-0.96) when compared to the control group, at 1.6 (95% CI, 1.34-1.96; p < 0.01), but there were no significant differences between any of the active treatments. CONCLUSION: LLLT at a dose of 2.6 Joules/cm(2) induces a reduction of HI levels and MPO activity in hemorrhagic injury that is not significantly different from celecoxib. Dexamethasone is slightly more effective than LLLT in reducing HI, but not MPO activity.

Inflammatory response and bacterial dissemination after laparotomy and abdominal CO2 insufflation in a murine model of peritonitis.

BACKGROUND: The immunologic repercussions due to cavity insufflation are the focus of great discussion. The aim of this study was to compare the inflammatory response and bacterial dissemination after laparotomy and abdominal CO2 insufflation in a murine model of peritonitis. METHODS: Swiss mice were inoculated intraperitoneally with 0.5 ml of a solution containing 1 x 10(8) colony-forming units (CFU)/ml of Escherichia coli and were divided into three groups as follow: control (anesthesia for 30 min), laparotomy (2.5-cm midline incision for 30 min), and CO2 pneumoperitoneum (CO2 cavity insufflation for 30 min). The number of leukocytes, CFU/ml counting, and the levels of interleukin (IL)-6, tumor necrosis factor-alpha (TNF-alpha), and IL-10 were evaluated in blood, peritoneal, and pleural fluid samples obtained at 90 min and 18 h after the procedures. RESULTS: The laparotomy group showed a greater bacterial dissemination to the blood, peritoneum, and pleural cavity and also greater neutrophil migration to the peritoneal cavity compared to the CO2 insufflated and control groups. The 24-h mortality was also significantly higher in the laparotomy group. The IL-6 levels showed a precocious rise in all groups submitted to bacterial inoculation at the 90-min time point. At the 18-h time point, IL-6 levels in the peritoneum were significantly higher in the laparotomy group than in the control or CO2 insufflated groups. At the same time, TNF-alpha levels were higher in the laparotomy and CO2 insufflated groups than in controls; IL-10 levels showed no differences among the groups. CONCLUSIONS: Our results suggest that cavity insufflation with CO2 is a more effective method of access, inducing less bacterial dissemination and also a less intense inflammatory response. Cavity insufflation with CO2 may present a good option for the surgical treatment of patients with bacterial peritonitis.

Low-level laser therapy induces dose-dependent reduction of TNFalpha levels in acute inflammation.

OBJECTIVE: The aim of this study was to investigate if low-level laser therapy (LLLT) can modulate acute inflammation and tumor necrosis factor (TNFalpha) levels. BACKGROUND DATA: Drug therapy with TNFalpha-inhibitors has become standard treatment for rheumatoid arthritis, but it is unknown if LLLT can reduce or modulate TNFalpha levels in inflammatory disorders. METHODS: Two controlled animal studies were undertaken, with 35 male Wistar rats randomly divided into five groups each. Rabbit antiserum to ovalbumin was instilled intrabronchially in one of the lobes, followed by the intravenous injection of 10 mg of ovalbumin in 0.5 mL to induce acute lung injury. The first study served to define the time profile of TNFalpha activity for the first 4 h, while the second study compared three different LLLT doses to a control group and a chlorpromazine group at a timepoint where TNFalpha activity was increased. The rats in LLLT groups were irradiated within 5 min at the site of injury by a 650-nm Ga-Al-As laser. RESULTS: There was a time-lag before TNFalpha activity increased after BSA injection. TNFalpha levels increased from < or =6.9 (95% confidence interval [CI], 5.6-8.2) units/mL in the first 3 h to 62.1 (95% CI, 60.8-63.4) units/mL (p < 0.001) at 4 h. An LLLT dose of 0.11 Joules administered with a power density of 31.3 mW/cm(2) in 42 sec significantly reduced TNFalpha level to 50.2 (95% CI, 49.4-51.0), p < 0.01 units/mL versus control. Chlorpromazine reduced TNFalpha level to 45.3 (95% CI, 44.0-46.6) units/mL, p < 0.001 versus control. CONCLUSION: LLLT can reduce TNFalpha expression after acute immunocomplex lung injury in rats, but LLLT dose appears to be critical for reducing TNFalpha release.

Increase in the rat blood leukocyte counts induced by PAF-acether is suppressed by general anesthesia.

Blood leukocyte count alterations induced by PAF-acether in anesthetized and nonanesthetized rats were investigated. Intravenous injection of increasing amounts of PAF-acether (1.5-8 micrograms/kg) in nonanesthetized animals induced dose-dependent hemoconcentration and leukocytosis. The former was apparent within 10 min, peaked from 30 min to 1 h, and diminished thereafter. The leukocytosis was noted within 30 min, was maximal at 1 h, and was over 4 h after injection of PAF-acether (4 micrograms/kg). It was characterized by a marked increase in the blood neutrophil counts under conditions in which the number of lymphocytes, monocytes, and eosinophils remained unchanged. PAF-acether-induced leukocytosis occurred in parallel with a marked decrease in the number of bone marrow nucleated cells, suggesting that the latter phenomenon may determine the former one. Leukocytosis by PAF-acether was inhibited dose-dependently by specific PAF-acether antagonists including BN 52021 (median effective dose ED50 = 4.99 mg/kg), WEB 2086 (ED50 = 4.59 mg/kg), and 48740 RP (ED50 = 9.02 mg/kg). General anesthesia by either pentobarbital, urethane, or ether inhalation, but not by ketamine, also impaired the PAF-acether-induced blood leukocytosis under conditions in which the hemoconcentration was not modified. In addition, pentobarbital-anesthetized rats did not have reduced bone marrow nucleated cell counts after PAF-acether stimulation. These findings are consistent with the assessment that PAF-acether-induced rat leukocytosis is accounted for by a bone marrow neutrophil mobilization process that is clearly suppressed in animals anesthetized by pentobarbital.

LPS-induced blood neutrophilia is inhibited by alpha 1-adrenoceptor antagonists: a role for catecholamines.

A role for catecholamines in the regulation of the blood neutrophilia induced by intravenous (i.v.) injection of lipopolysaccharide (LPS; 250 micrograms/kg) was examined in Wistar rats by means of surgical adrenalectomy or pretreatment with adrenergic and dopaminergic antagonists into naive animals. Treatment of animals with a single dose (250 micrograms/kg) of LPS caused a dramatic increase in the number of circulating neutrophils concomitant with a decrease in the number of these cells in the bone marrow. These effects were partially reversed when catecholamine stores were depleted with reserpine. It was found that neither adrenalectomy nor pretreatment with the dopaminergic antagonists, chlorpromazine and pimozide, affected the changes in neutrophil counts induced by LPS. The injection of the alpha 1/alpha 2-adrenoceptor antagonist, phentolamine, and the selective alpha 1-adrenoceptor antagonist, prazosin, significantly decreased blood neutrophilia induced by LPS. However, neither the selective alpha 2-adrenoceptor antagonist, yohimbine, nor the beta-adrenoceptor antagonist, propranolol, had any effect on LPS response. Taken together, these findings support the hypothesis that the catecholamine norepinephrine plays a role in the regulation of the LPS-induced neutrophilia through activation of alpha 1-adrenoceptors.

Requirement for lymphocytes and resident macrophages in LPS-induced pleural eosinophil accumulation.

In this study we investigated the involvement of inflammatory cells in the pleural accumulation of eosinophils induced by lipopolysaccharide (LPS). Intrathoracic (i.t.) injection of LPS (250 ng/cavity) into rats induced a significant eosinophil accumulation that developed within 24 h, was maximal at 48 h, and returned to control values within 120 h. This eosinophil influx was preceded by a huge neutrophil influx within 4 h and accompanied by a mononuclear cell accumulation between 24 and 48 h. Pretreatment with an antineutrophil monoclonal antibody (RP-3, 2 ml per animal) selectively reduced the number of circulating neutrophils within 8 h but failed to alter the LPS-induced eosinophilia. Similarly, platelet depletion with an anti-rat platelet antiserum did not alter the LPS-induced eosinophil accumulation. Cyclosporine (50 mg/kg, 12 and 2 h before) partially inhibited (51%) the LPS-induced pleural eosinophilia, whereas the eosinophilia was not changed by prior degranulation of pleural mast cells with polymyxin B (10 micrograms/cavity, 24 h before). Moreover, selective depletion of T lymphocytes using an anti-Thy 1.0 monoclonal antibody significantly inhibited the eosinophilia triggered by LPS. The i.t. injection of liposomes containing dichloromethylene diphosphonate significantly reduced (65%) the number of resident macrophages after 5 days. Under this condition, the eosinophil infiltration induced by LPS was completely inhibited. Accordingly, the i.t. injection of supernatant from macrophage monolayers, obtained from the pleural cavities of LPS-injected rats, into naive recipient animals led to a twofold increase in the number of pleural eosinophils. In conclusion, our data suggest an important role for resident macrophages and T lymphocytes in the eosinophil accumulation induced by LPS.

Role of PAF in the allergic pleurisy caused by ovalbumin in actively sensitized rats.

Selective platelet-activating factor (PAF) antagonists and autodesensitization to this lipid were used to investigate the role of PAF in antigen-induced pleurisy in the rat. Pleural inflammation was triggered by the intrathoracic (i.t.) injection of ovalbumin (12 micrograms/cavity) into animals actively sensitized 14 days before. Successive daily i.t. injections of PAF (1 microgram/cavity) led to selective autodesensitization, which was apparent after the third injection and maximal after the fifth. The PAF antagonists BN 52021 and WEB 2086 inhibited the late pleural eosinophil accumulation caused by antigen but, as also noted with WEB 2170, failed to modify the early antigen-induced plasma exudation and leukocyte infiltration. In contrast to the antagonists, desensitization to PAF was clearly effective against these early alterations. To further investigate this discrepancy, the antigenic challenge was performed 24 h after a single prestimulation with PAF, when sensitivity to the lipid was still intact. Under this condition, plasma exudation and cellular influx triggered by the antigen were also abrogated, indicating that this protective effect was accounted for by a mechanism other than refractoriness to PAF. Because 24 h after PAF injection only eosinophil counts remained elevated, an alternative eosinophilotactic substance was used to further study the mechanism of PAF versus antigen-induced pleural inflammation. Prior treatment with the peptide Ala-Gly-Ser-Glu (ECF-A, 20 micrograms/cavity) also inhibited the allergic pleurisy, whereas the noneosinophilotactic substances histamine (200 micrograms/cavity) and serotonin (100 micrograms/cavity) were inactive. Furthermore, drugs that share the ability to impair PAF-induced eosinophilia, including azelastine and cetirizine, prevented the inhibitory effect of PAF on the antigen-induced pleurisy. These findings suggest that PAF may account for the late eosinophilia, but not for the acute phase of the rat allergic pleurisy, which is clearly attenuated by PAF or ECF-A pretreatment.

A role for adrenoceptors in the regulation of pleural neutrophilia induced by LPS.

The role of catecholamines in regulating pleural neutrophilia evoked by intrathoracic (i.t.) injection of lipopolysaccharide (LPS) was investigated in Wistar rats by means of surgical adrenalectomy, depletion of catecholamine stores or adrenoceptor blockade. Treatment of animals with a single dose of LPS evoked a dramatic increase in the number of pleural neutrophils concomitant with an increase in the number of these cells in blood at 4 h. Although blood neutrophilia was drastically reduced when catecholamine stores were depleted with intraperitoneal (i.p.) injection of reserpine, pleural neutrophilia was not modified. However, the i.t. injection of reserpine reduced the increase in pleural neutrophils after LPS stimulation. Adrenalectomy failed to inhibit the increase in neutrophil counts in the blood or pleural cavity after LPS challenge. Pretreatment with intravenous (i.v.) injection of prazosin, an alpha(1)/alpha(2B) antagonist, reduced LPS-induced blood but not pleural neutrophilia. On the other hand, although pleural neutrophilia was not affected by systemic pretreatment with the alpha(2)-adrenoceptor antagonist, yohimbine, the local treatment (i. t. injection) with this antagonist markedly reduced the increase in pleural neutrophil counts observed after stimulation by LPS. In contrast, pleural neutrophilia induced by i.t injection of formyl-methionyl-leucyl-phenylalanine (fMLP) was not modified by local treatment with yohimbine. Taken together, our results suggest that catecholamines, through activation of alpha(1) and alpha(2)-adrenoceptors, play a role in the regulation of blood and pleural neutrophilia observed during the inflammatory response evoked by LPS in the pleural cavity.

Platelet mobilization induced by PAF and its role in the thrombocytosis triggered by adrenaline in rats.

The injection of PAF (6 micrograms/kg, i.v.) induced, in rats, haemoconcentration accompanied by an increase in the platelet number, as attested by the counts of platelets in blood samples diluted in formalin-free EDTA solution. This increase was significant at 15 min, peaked from 1 to 4 h and returned to basal levels 24 h after the lipid administration. The release of platelets induced by PAF was inhibited dose-dependently by specific PAF receptor antagonist such as WEB 2086 (0.5-2 mg/kg), BN 52021 and 48740 RP (5-25 mg/kg). Furthermore, platelet mobilization was clearly impaired in splenectomized animals stimulated by PAF, whereas thrombocytopenia and haemoconcentration by the same stimulus were intact. It was also noted that a second injection of PAF, 24 h after the initial stimulation with the lipid, failed to induce an increase in platelet counts, indicating autodesensitization. Desensitization to PAF or pretreatment with PAF antagonists clearly prevented the increase in the platelet counts after stimulation by adrenaline (15 micrograms/kg). These findings suggest that, in rats, PAF can induce release of platelets by a spleen-dependent mechanism and that this lipid may be relevant to the thrombocytosis triggered by adrenaline.

Nedocromil sodium prevents in vivo generation of the eosinophilotactic substance induced by PAF but fails to antagonize its effects.

1 The intrathoracic injection of platelet activating factor (PAF) into rats induced a decrease in the pleural leucocyte numbers within 15 min, accompanied by a marked exudation, maximal 1 h later. After 6 h, concomitantly with the reduction of exudation, a marked increase in the number of mononuclear cells, neutrophils and eosinophils was observed. Within 24 h, the pleural eosinophil accumulation peaked and persisted up to 96 h. 2 Topical treatment with nedocromil sodium affected pleural exudation by PAF under conditions where systemic meclizine was ineffective. Nedocromil sodium blocked, dose-dependently, the increase in the pleural content of mononuclear cells, neutrophils and eosinophils, observed 6 h after PAF administration, as well as the eosinophilia 24 h later. Moreover, the co-incubation of peritoneal eosinophils with nedocromil sodium did not interfere with the migration triggered by PAF. 3 The transfer of the 6 h-PAF pleural washings from donor to recipient rats caused a selective pleural eosinophilia, which was clearly inhibited when nedocromil sodium was administered to donor, but not to recipient animals, showing that this drug interferes with the generation rather than with the expression of the eosinophilotactic activity(ies). 4 These findings indicate that the nedocromil sodium interferes with PAF-induced exudation and leucocyte accumulation, by a mechanism other than its ability to reduce the local effects of histamine and which may relate to suppression of the eosinophilotactic principle generation.

Bradykinin down-regulates LPS-induced eosinophil accumulation in the pleural cavity of mice through type 2-kinin receptor activation: a role for prostaglandins.

1. The role of both exogenously administered and endogenously generated bradykinin (BK) on LPS-induced eosinophil accumulation in the mice pleural cavity was investigated by means of treatment with BK selective receptor agonists/antagonists and captopril. 2. Intrathoracic (i.t.) injection of LPS (250 ng cavity(-1)) induced eosinophil influx at 24 h as previously described (Bozza et al., 1993). Pretreatment with the B1 receptor antagonist des-Arg9-[leu-8]BK (0.025 and 0.25 nmol cavity(-1)) showed no effect on this phenomenon, whereas pretreatment with the B2 receptor antagonists, NPC 17731 (0.025 and 0.25 nmol cavity(-1)) or HOE 140 (2.5 nmol cavity(-1)), increased LPS-induced eosinophil influx. Accordingly, pretreatment with captopril at 10 mg kg(-1) i.p., inhibited eosinophil infiltration induced by LPS in the pleural cavity, suggesting that endogenous BK is down-regulating LPS-induced eosinophil accumulation. 3. BK administered at 15 and 25 nmol cavity(-1), i.t. or i.p. also inhibited LPS-induced eosinophil accumulation. BK alone had no effect on the basal number of leucocytes in the pleural or peritoneal cavity in doses up to 25 nmol cavity(-1). Nevertheless, when injected at doses of 50 and 100 nmol cavity(-1) BK induced leucocyte influx characterized by neutrophil and eosinophil accumulation at 24 h. 4. Similarly to what was observed with BK, a specific B2 receptor agonist, Tyr8BK, administered at 0.25 nmol cavity(-1) i.p., significantly inhibited the eosinophil influx induced by LPS. 5. The mechanism by which B2 receptor agonists inhibit LPS-induced eosinophil accumulation was investigated by pretreating the animals with indomethacin or a selective cyclooxygenase-2 inhibitor, NS-398. Pretreatment with either indomethacin or NS-398 had no effect on eosinophil influx induced by LPS alone, but those drugs were able to restore the LPS-induced eosinophil influx in Tyr8BK (0.25 nmol cavity(-1)) injected mice. 6. In conclusion, endogenously generated bradykinin seems to modulate, through activation of B2 receptors, eosinphil accumulation induced by LPS via a mechanism dependent on prostanoid synthesis.

Long-lasting inhibitory activity of the hetrazepinic BN 50730 on exudation and cellular alterations evoked by PAF and LPS.

1. Inhibitory effects of the hetrazepinic derivative BN 50730 on the rat pleural inflammatory response, triggered by PAF or lipopolysaccharides (LPS), were examined. The type of pharmacological blockade exerted by this antagonist in in vitro assays of eosinophil chemotaxis and platelet aggregation were also investigated. 2. Intrathoracic injection of PAF (1 microgram per cavity) caused a 4 fold increase in the extravasated protein within 15 min and led to a marked eosinophil accumulation 24 h post-challenge. BN 50730 (0.5-10 micrograms per cavity) inhibited exudation by PAF dose-dependently without modifying the response induced by histamine, bradykinin or 5-hydroxytryptamine (5-HT). 3. The kinetics of the inhibitory effect on exudation revealed that the actions of WEB 2086 and BN 52021 (10 micrograms per cavity) were over within 2 and 4 h respectively, whereas BN 50730 (10 micrograms per cavity) retained 80% of its inhibitory activity for 4 days. 4. Oral treatment with BN 50730 (10-20 mg kg-1, 1 h beforehand) suppressed the leucocyte accumulation and late eosinophilia observed 6 and 24 h after PAF respectively, but did not modify the eosinophilia induced by leukotriene B4 (LTB4) or bradykinin. BN 50730 also failed to reduce the eosinophil accumulation induced by LPS but drastically inhibited the neutrophil influx. 5. The pre-incubation of rat peritoneal eosinophils for 10 min with BN 50730 (30 nM-1 microM) dose-dependently inhibited the chemotaxis induced by PAF (0.1 microM) in vitro. The IC50 values for BN 52021, WEB 2086 and BN 50730 in this system were 5, 5 and 0.05 microM respectively. 6. In separate assays, rat peritoneal eosinophils and rabbit washed platelets were preincubated with BN 50730 or WEB 2086 (1 pM) then subjected to a series of at least two consecutive washings in order to remove the antagonist from the receptor environment. Under such conditions, only the cells pretreated with WEB 2086 recovered the sensitivity to the lipid.7. We conclude that BN 50730 is a potent, specific and long-acting PAF antagonist and its effect seems to result from a high affinity and non-competitive interaction of the drug with the PAF receptor.