Inflammatory Cytokines Contribute to Asbestos-Induced Injury of Mesothelial Cells.

BACKGROUND: Several diseases have been related to asbestos exposure, including the pleural tumor mesothelioma. The mechanism of pleural injury by asbestos fibers is not yet fully understood. The inflammatory response with release of mediators leading to a dysregulation of apoptosis may play a pivotal role in the pathophysiology of asbestos-induced pleural disease. OBJECTIVE: To determine whether pro-inflammatory cytokines produced by asbestos-exposed pleural mesothelial cells modify the injury induced by the asbestos. METHODS: Mouse pleural mesothelial cells (PMC) were exposed to crocidolite or chrysotile asbestos fibers (3.0 µg/cm(2)) for 4, 24, or 48 h and assessed for viability, necrosis and apoptosis, and the production of cytokines IL-1ß, IL-6 and macrophage inflammatory protein-2 (MIP-2). Cells exposed to fibers were also treated with antibodies anti-IL-1ß, anti-IL-6, anti- IL-1ß+anti-IL-6 or anti-MIP-2 or their irrelevant isotypes, and assessed for apoptosis and necrosis. Non-exposed cells and cells treated with wollastonite, an inert particle, were used as controls. RESULTS: Mesothelial cells exposed to either crocidolite or chrysotile underwent both apoptosis and necrosis and released cytokines IL-1ß, IL-6 and MIP-2. In the crocidolite group, apoptosis and the levels of all cytokines were higher than in the chrysotile group, at comparable concentrations. Neutralization of IL-1ß andIL-6, but not MIP-2, inhibited apoptosis and necrosis, especially in the cells exposed to crocidolite fibers. CONCLUSIONS: Both crocidolite and chrysotile asbestos fibers induced apoptosis and produced an acute inflammatory response characterized by elevated levels of IL-1ß, IL-6 and MIP-2 in cultured mouse PMC. IL-1ß and IL-6, but not MIP-2, were shown to contribute to asbestos-induced injury, especially in the crocidolite group.

Avaliação ultrassonográfica torácica em cães com efusão pleural e/ou periocárdica / Thoracic ultrasonographyc evaluation of dogs with pleural and/or pericardial effusion

Com o objetivo de avaliar a ultrassonografia torácica (incluindo a ecocardiografia) como método de exame complementar em pacientes portadores de efusão pleural e/ou pericárdica, realizou-se a ultrassonografia torácica em 30 cães, machos e fêmeas de raças e idades variadas. Animais atendidos nos serviços de Pronto-Atendimento, Clínica Médica ou Clínica Cirúrgica do HOVET/USP com efusão pleural e/ou pericárdica, detectadas por meio de exame radiográfico, ultrassonografia emergencial ou toracocentese exploratória foram incluídos no estudo. Observou-se efusão pleural em 12 cães (40%), efusão pericárdica em oito (26,66%) e efusão pleural e pericárdica em outros dez cães (33,33%). A causa da efusão pleural e/ou pericárdica foram avaliadas ultrassonograficamente como sendo: nódulo/tumor cardíaco (5 - 16,66%), nódulo/tumor intratorácico (5 – 16,66%), insuficiência cardíaca congestiva por cardiomiopatia dilatada (4 – 13,33%) ou endocardiose de mitral e tricúspide (3 – 10%), efusão pericárdica idiopática (3 - 10%), li fossarcoma (2 - 6,66%), piotórax (2 – 6,66%), ruptura diafragmática (1 – 3,33%), hérnia peritônio-pericárdica (1 –3,33%), pneumonia e pleurite (1 - 3,33%), tumor de ovário com metástases torácicas (1 – 3,33%), pericardite infecciosa (cinomose) (1 – 3,33%) e hipoalbuminemia (1 – 3,33%).

To evaluate diagnostic accuracy of thoracic ultrasonography (including echocardiography) of patients with pleural and/or pericardial effusion, thoracic ultrasonography was performed in 30 dogs, males and females, of different breeds and ages. The animals were admitted to the Emergency, Internal medicine or Surgery department of the Faculty of Veterinary Medicine, University of São Paulo, presenting with pleural and/or pericardial effusion, diagnosed by thoracic radiography, emergency thoracic ultrasonography or exploratory thoracocentesis. Twelve (40%) dogs had pleural effusion, 9 (30%) had pericardial effusion and 9 (30%) had both pleural and pericardial effusions. The definitive cause of effusion were obtained by thoracic ultrasonography as follows: heart mass (5 –16,66%), intrathoracic mass (5 – 16,66%), congestive heart failure by dilated cardiomyopathy (4 – 13,33%) or mitral and tricuspid insufficiency (3 – 10%), idiopathic pericardial effusion (3 – 10%), lymphosarcoma (2 – 6,66%), pyothorax (2 - 6,66%), traumatic diaphragmatic hernia (1 – 3,33%), congenital peritoneopericardial hernia (1 – 3,33%), pneumonia and pleuritis (1 – 3,33%), ovarian neoplasia and thoracic metastasis (1 – 3,33%), infectious pericarditis (distemper) (1 – 3,33%) and hypoalbuminemia (1 – 3,33%). The conclusion was that thoracic ultrasonography was an excellent auxiliary exam in animals with pleural and/or pericardial effusion, and it’s not invasive and safe for the patient, allowing to guide biopsies and perform the exam in different decubits, avoiding patient stress.

Avaliação ultrassonográfica torácica em cães com efusão pleural e/ou periocárdica / Thoracic ultrasonographyc evaluation of dogs with pleural and/or pericardial effusion

Com o objetivo de avaliar a ultrassonografia torácica (incluindo a ecocardiografia) como método de exame complementar em pacientes portadores de efusão pleural e/ou pericárdica, realizou-se a ultrassonografia torácica em 30 cães, machos e fêmeas de raças e idades variadas. Animais atendidos nos serviços de Pronto-Atendimento, Clínica Médica ou Clínica Cirúrgica do HOVET/USP com efusão pleural e/ou pericárdica, detectadas por meio de exame radiográfico, ultrassonografia emergencial ou toracocentese exploratória foram incluídos no estudo. Observou-se efusão pleural em 12 cães (40%), efusão pericárdica em oito (26,66%) e efusão pleural e pericárdica em outros dez cães (33,33%). A causa da efusão pleural e/ou pericárdica foram avaliadas ultrassonograficamente como sendo: nódulo/tumor cardíaco (5 - 16,66%), nódulo/tumor intratorácico (5 16,66%), insuficiência cardíaca congestiva por cardiomiopatia dilatada (4 13,33%) ou endocardiose de mitral e tricúspide (3 10%), efusão pericárdica idiopática (3 - 10%), li fossarcoma (2 - 6,66%), piotórax (2 6,66%), ruptura diafragmática (1 3,33%), hérnia peritônio-pericárdica (1 –3,33%), pneumonia e pleurite (1 - 3,33%), tumor de ovário com metástases torácicas (1 3,33%), pericardite infecciosa (cinomose) (1 3,33%) e hipoalbuminemia (1 3,33%).(AU)

To evaluate diagnostic accuracy of thoracic ultrasonography (including echocardiography) of patients with pleural and/or pericardial effusion, thoracic ultrasonography was performed in 30 dogs, males and females, of different breeds and ages. The animals were admitted to the Emergency, Internal medicine or Surgery department of the Faculty of Veterinary Medicine, University of São Paulo, presenting with pleural and/or pericardial effusion, diagnosed by thoracic radiography, emergency thoracic ultrasonography or exploratory thoracocentesis. Twelve (40%) dogs had pleural effusion, 9 (30%) had pericardial effusion and 9 (30%) had both pleural and pericardial effusions. The definitive cause of effusion were obtained by thoracic ultrasonography as follows: heart mass (5 –16,66%), intrathoracic mass (5 16,66%), congestive heart failure by dilated cardiomyopathy (4 13,33%) or mitral and tricuspid insufficiency (3 10%), idiopathic pericardial effusion (3 10%), lymphosarcoma (2 6,66%), pyothorax (2 - 6,66%), traumatic diaphragmatic hernia (1 3,33%), congenital peritoneopericardial hernia (1 3,33%), pneumonia and pleuritis (1 3,33%), ovarian neoplasia and thoracic metastasis (1 3,33%), infectious pericarditis (distemper) (1 3,33%) and hypoalbuminemia (1 3,33%). The conclusion was that thoracic ultrasonography was an excellent auxiliary exam in animals with pleural and/or pericardial effusion, and its not invasive and safe for the patient, allowing to guide biopsies and perform the exam in different decubits, avoiding patient stress.(AU)

Involvement of CC chemokines in gammadelta T lymphocyte trafficking during allergic inflammation: the role of CCL2/CCR2 pathway.

In the present study, we show that the intra-thoracic injection of ovalbumin (OVA, 12.5 microg per cavity) into C57BL/10 mice induced a significant increase in gammadelta T lymphocyte numbers in the pleural cavity, blood and thoracic lymph node of challenged mice. Such increase was significant within 12 h, peaked within 48 h and returned to basal counts within 120 h. Levels of CC chemokine ligand (CCL)-2/monocyte chemotactic protein-1, CCL5/regulated upon activation, normal T cell expressed and secreted, CCL3/macrophage inflammatory protein-1 alpha and CCL25/thymus-expressed chemokine were above control values in pleural washes recovered 24 h after OVA challenge (OPW) and were likely produced by pleural macrophages and mesothelial cells. Antigenic challenge also induced an up-regulation in CC chemokine receptor (CCR)-2, CCR5 and CCR9 on gammadelta T cells from pleural cavities, blood and lymph nodes, suggesting that cells found in mice pleural cavity migrate from secondary lymphoid organs into the inflammatory site via blood stream. The in vitro neutralization of CCL2 (but not of CCL3, CCL5 or CCL25) abrogated OPW-induced gammadelta T lymphocyte transmigration. Confirming such results, the in vivo administration of alpha-CCL2 mAb inhibited gammadelta T lymphocyte accumulation in the pleural cavity of challenged mice, whereas the blockade of CCL3, CCL5 or CCL25 showed no effect on gammadelta T cell mobilization. In addition, OVA challenge failed to induce gammadelta T lymphocyte accumulation in the pleural cavity of C57BL/6 CCR2 knockout mice, which also showed decreased numbers of these cells in blood and lymph nodes when compared with wild-type mice. Overall, such results demonstrate that CCR2/CCL2 pathway is crucial for gammadelta T lymphocyte mobilization during the allergic response.

Increased levels of cyclic adenosine monophosphate contribute to the hyporesponsiveness of mast cells in alloxan diabetes.

In this study, we investigated the influence of intracellular cyclic adenosine monophosphate (cAMP) changes on the rat mast cell hyporesponsiveness following immunological and non-immunological stimuli. Compared with mast cells from normal rats, those recovered from 21-day diabetic animals showed a significant augmentation in the intracellular levels of cAMP, in directly correlated with secretion of lower amounts of histamine after stimulation with antigen, bradykinin and compound 48/80 in vitro. Incubation of normal mast cells with selective inhibitors of phosphodiesterase type 4 (PDE 4) rolipram, NCS 613 and RP 73401, or the cell permeable analogue N6-2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (db cAMP), led to a decrease of histamine secretion in vitro. However, the effectiveness of either NCS 613 or db cAMP in inhibiting antigen-induced degranulation is comparable in both normal and diabetic mast cells. We suggest that (a) there is a close correlation between higher levels of intracellular cAMP and hyporesponsiveness of diabetic mast cells, phenomena probably associated with a reduction in the expression and/or activity of PDE 4 and that (b) the mechanism of cAMP-mediated down-regulation of mast cell function is saturated in diabetic rats.

Importance of the vagus nerve for fever and neutrophil migration induced by intraperitoneal LPS injection.

OBJECTIVE: We investigated the importance of the vagus nerve in fever, neutrophil migration and neutrophilia simultaneously induced by intraperitoneal injection of endotoxin (lipopolysaccharide, LPS) and in terms of the production of pre-formed pyrogenic factor (PFPF) and of the fever induced by this factor. METHODS: Naïve, sham-operated or subdiaphragmatically vagotomized male Wistar rats received either LPS (i.p. or i.pl.) or PFPF (i.v., i.c.v., i.p.). The number of neutrophils was evaluated in peritoneal or pleural fluid and in blood. Fever was monitored using a rectal probe. RESULTS: In naïve animals, LPS (0.02-200 microg kg(-1), i.p.) induced dose-related neutrophilia and fever while on neutrophil migration it resulted in a bell-shaped curve. Vagotomy reduced the peritoneal resident cell population (56%), fever (71%) and neutrophil migration (43%) but not the neutrophilia or neutrophil migration to the pleural cavity. Vagotomy did not affect the PFPF production or PFPF-induced fever. CONCLUSIONS: Vagus nerve integrity is important not only for fever but also for the neutrophil influx to the peritoneal cavity by controlling the number of resident cells in this cavity.

LPS induces eosinophil migration via CCR3 signaling through a mechanism independent of RANTES and Eotaxin.

Mounting evidence suggests that lipopolysaccharide (LPS) modulates bronchoconstriction and eosinophil function in asthma. We have investigated the role of different chemokines in the eosinophil influx to the pleural cavity after LPS stimulation. Expression of mRNA for eotaxin, regulated on activation, normal T cells expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, and monocyte chemotactic protein (MCP)-1 was increased in cells recovered from the mouse pleural cavity 6 h after LPS administration. Eotaxin and RANTES, but not MIP-1alpha, protein levels were also increased in cell-free pleural washes recovered 6 h after LPS stimulation (LPW). Antimurine eotaxin and antimurine RANTES antibodies (Abs) failed to inhibit LPS-induced eosinophil influx into mouse pleural cavity in vivo. Pertussis toxin inhibited LPW-induced eosinophil shape change in vitro, suggesting the involvement of G protein-coupled receptors in LPW signaling. Blockade of CCR3 receptors diminished eosinophil shape change induced by LPW fractions in vitro and LPS-induced eosinophil accumulation in vivo. To investigate further contribution of CC chemokines, we administered a 35-kD CC chemokine neutralizing protein (vCKBP) in vivo. vCKBP inhibited the eosinophil accumulation induced by eotaxin and ovalbumin, but did not block that induced by LPS or LPW. Our data suggest that LPS-induced eosinophil accumulation depends on G protein-coupled CCR3 receptor activation, through a mechanism independent of eotaxin, RANTES, or other vCKBP-inhibitable CC chemokines.

Mechanisms of allergen- and LPS-induced bone marrow eosinophil mobilization and eosinophil accumulation into the pleural cavity: a role for CD11b/CD18 complex.

OBJECTIVE: The mechanisms involved in bone marrow eosinophil emigration and recruitment to inflammatory sites are not fully understood. The involvement of CD11b/CD18 in marrow eosinophil release induced by lipopolysaccharide (LPS) or allergen was investigated in mice. METHODS: Eosinophil and neutrophil counts in the pleural cavity, blood and bone marrow were performed at different time intervals after the intrathoracic injection of LPS (250 ng/cavity) or ovalbumin (OVA, 12 microg/cavity; into actively sensitized mice) and compared to anti-CD11b/CD 18 (5C6, 1 mg/mouse) or anti-IL-5 (TRFK-5, 500 microg/kg) treated mice. RESULTS: LPS induced local eosinophil influx, that peaked within 24 h and that was preceded by a decrease in marrow eosinophils at 4 h. Antigenic challenge induced a decrease in marrow eosinophils within 4 h, followed by a long lasting pleural eosinophil accumulation and a persistent increase in marrow eosinophil numbers. Pretreatment with anti-CD11b/CD18 abolished LPS-induced neutrophil and eosinophil accumulation in the pleural cavity at 4 and 24 h, respectively. This pretreatment failed to modify neutrophil emigration from bone marrow, but significantly inhibited marrow eosinophil release at 4 h post-LPS or OVA challenge. Anti-IL-5 pretreatment failed to inhibit LPS-induced pleural eosinophil accumulation and mobilization from bone marrow, but it abolished allergen-induced effects, indicating a role for IL-5 in marrow eosinophil mobilization induced by antigen, but not by LPS challenge. CONCLUSIONS: Our results suggest that eosinophil migration induced by antigen or LPS into the pleural cavity is preceded by bone marrow eosinophil release through a mechanism that depends on CD11b/CD18.

Contribution of C-fibers to leucocyte recruitment in bronchoalveolar lavage fluid and pleural cavity in the rat.

The effect of neonatal capsaicin (8 methyl-N-vanillyl-6-nonenamide) treatment on the leucocyte infiltration into the airways and pleural cavity was investigated in rats actively sensitized with ovalbumin. The animals were neonatally injected with either capsaicin (50 mg/kg, s.c., 2nd day of life) or vehicle (10% ethanol and 10% Tween 80). At adult ages, the animals were actively sensitized with ovalbumin (200 microg, s.c.) and 14 days later they were intratracheally (or intrapleurally) challenged with ovalbumin. The substance P level in bronchoalveolar lavage fluid of the capsaicin group was reduced by >90% compared to control group (vehicle), confirming the efficacy of capsaicin treatment. In the capsaicin group, the number of neutrophils (but not of eosinophils and mononuclear cells) in bronchoalveolar lavage fluid of sensitized animals was significantly higher than the control group. Intrapleural injection of ovalbumin in sensitized rats caused a significant neutrophil influx at 6 h that was markedly increased in the capsaicin-pretreated animals compared to control group. The counts of eosinophils and mononuclear cells in the pleural exudates did not differ significantly between capsaicin and control groups. The increased levels of immunoglobulin (Ig)E, IgG1 and IgG2a anti-ovalbumin antibodies in serum of sensitized rats did not differ between capsaicin and control groups. In conclusion, the exacerbated pulmonary neutrophil recruitment caused by the capsaicin neonatal treatment is unrelated to increase in serum immunoglobulin antibodies, and suggests a protective role for C-fibers in attenuating the allergic neutrophil infiltration.

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.

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.

Opposite cellular accumulation and nitric oxide production in vivo after pleural immunization with M. leprae or M. bovis BCG.

Mycobacteria as intracellular pathogens have evolved mechanisms to survive within macrophages. Our previous data showed that M. leprae (ML), unlike M. bovis BCG, did not induce an inflammatory response in the mice subcutaneous tissue. Further, ML inhibited BCG-induced foot pad oedema and seemed to transform macrophages in epithelioid cells. Since these mycobacteria share common antigens, here we seeked to compare the acute and chronic cellular response evoked by ML and BCG in pleurisy of a mycobacteria-susceptible mice (BALB/c). The total leukocytes, the cell type that migrated to the pleural cavity and macrophage activation assayed by nitric oxide release were determined. Live or dead BCG Moreau recruited the same extent of cells, essentially monocytes and neutrophils, dose-dependently, in both acute and chronic pleurisy. BCG-induced eosinophilia was observed only in the acute response (after 24 h of injection). A significant nitric oxide release by pleural macrophages was triggered by BCG Moreau without previous activation. Nevertheless, ML failed to recruit leukocytes to the pleural space or to lead to nitric oxide production despite the number of bacilli used and the time studied (1, 7 or 14 days after injection). Although these mycobacteria have common antigens that cross-react, these data show a distinct ability of ML or BCG to recruit cells to the pleural space and to activate pleural macrophage for nitric oxide production in vivo.

Characterization of the receptor and the mechanisms underlying the inflammatory response induced by des-Arg9-BK in mouse pleurisy.

1 The characterization of the B1 kinin receptor, and some mediators involved in the inflammatory response elicited by intrathoracic (i.t.) administration of des-Arg9-bradykinin (BK) in the mouse model of pleurisy, was investigated. 2 An i.t. injection of des-Arg9-BK (10-100 nmol per site), a selective B1 agonist, caused a significant and dose-related increase in the vascular permeability observed after 5 min, which peaked at 1 h, associated with an increase in cell influx, mainly neutrophils, and, to a lesser extent, mononuclear cell influx, peaking at 4 h and lasting for up to 48 h. The increase in fluid leakage caused by des-Arg9-BK was completely resolved 4 h after peptide injection. I.t. injection of Lys-des-Arg9-BK (30 nmol per site) caused a similar inflammatory response. 3 Both the exudation and the neutrophil influx elicited by i.t. injection of des-Arg9-BK were significantly antagonized (P<0.01) by an i.t. injection of the selective B1 antagonists des-Arg9-[Leu8]-BK (60 and 100 nmol per site) or des-Arg9-NPC 17731 (5 nmol per site), administered in association with des-Arg9-BK (P<0.01), or 30 and 60 min before the cellular peak, respectively. In contrast, an i.t. injection of the B2 bradykinin selective receptor antagonist Hoe 140 (30 nmol per site), at a dose which consistently antagonized bradykinin (10 nmol per site)-induced pleurisy, had no significant effect on des-Arg9-BK-induced pleurisy. 4 An i.t. injection of the selective tachykinin receptor antagonists (NK1) FK 888 (1 nmol per site), (NK2) SR 48968 (20 nmol per site) or (NK3) SR 142801 (10 nmol per site), administered 5 min before pleurisy induction, significantly antagonized neutrophil migration caused by i.t. injection of des-Arg9-BK. In addition, FK 888 and SR 142801, but not SR 48968, also prevented the influx of mononuclear cells in response to i.t. injection of des-Arg9-BK (P<0.01). However, the NK3 receptor antagonist SR 142801 (10 nmol per site) also significantly inhibited des-Arg9-BK-induced plasma extravasation. An i.t. injection of the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP8-37 (1 nmol per site), administered 5 min before pleurisy induction, inhibited des-Arg9-BK-induced plasma extravasation (P<0.01), without significantly affecting the total and differential cell migration. 5 The nitric oxide synthase inhibitors L-NOARG and L-NAME (1 pmol per site), administered 30 min beforehand, almost completely prevented des-Arg9-BK (i.t.)-induced neutrophil cell migration (P<0.01), and, to a lesser extent, mononuclear cell migration (P<0.01). The D-enantiomer D-NAME had no effect on des-Arg9-BK-induced pleurisy. At the same dose range, L-NOARG and L-NAME inhibited the total cell migration (P<0.01). L-NAME, but not L-NOARG caused significant inhibition of des-Arg9-BK-induced fluid leakage. Indomethacin (1 mg kg(-1), i.p.), administered 1 h before des-Arg9-BK (30 nmol per site), inhibited the mononuclear cell migration (P<0.05), but, surprisingly, increased the neutrophil migration at 4 h without interfering with plasma extravasation. The administration of terfenadine (50 mg kg(-1), i.p.), 30 min before des-Arg9-BK (30 nmol per site), did not interfere significantly with the total cell migration or with the plasma extravasation in the mouse pleurisy caused by i.t. injection of des-Arg9-BK. 6 Pretreatment of animals with the lipopolysaccharide of E. coli (LPS; 10 microg per animal, i.v.) for 24 h did not result in any significant change of the inflammatory response induced by i.t. injection of des-Arg9-BK compared with the saline treated group. However, the identical treatment of mice with LPS resulted in a marked enhancement of des-Arg9-BK induced paw oedema (P<0.01). 7 In conclusion, we have demonstrated that the inflammatory response induced by i.t. injection of desArg9-BK, in a murine model of pleurisy, is mediated by stimulation of constitutive B1 receptors. (These responses are largely mediated by release of neuropeptides such as substanceP or CGRP and also by NO, but products derived from cyclo-oxygenase pathway and histamine seem not to be involved. Therefore, these results further support the notion that the B1 kinin receptor has an important role in modulating inflammatory responses, and it is suggested that selective B1 antagonists may provide therapeutic benefit in the treatment of inflammatory and allergic conditions.

Organization of collagen and elastic fibers studied in stretch preparations of whole mounts of human visceral pleura.

Fibers of the collagenous and elastic systems are most relevant in the double mechanical action of visceral pleura (VP), i.e. volume limitation and the generation of elastic recoil pressure. In this work we studied the organization of these fibrous components of VP in two situations: normal lungs and bullous disease. We employed histochemical methods on conventional histological slides and on thin spreads of whole mounts of visceral pleura. In addition, the scanning electron microscope was also used. According to our results, pleural function is made possible by the combination of both the elastic and collagenous fiber systems, each one having as intrinsic organizational pattern. Marked alterations of pleural bullous structure are observed with changes in lung volume. Fibers of the elastic and collagenous systems are clearly interdependent elements. Collagenous fibers are interwoven in a plaited structure that closely resembles the osiers of a wicker basket, indicating that collagen fibers allow for lung volume increase up to a point of maximal stretching of the system. The pleural contribution to lung elastic recoil pressure originates from the elastic network which turns back to its resting position when inspiratory pressures are negligible. The pleural immobility in bullous disease is associated with an almost complete absence of elastic fibers and the presence of very thick collagen fibers, suggestive of a cicatricial process, devoid of any characteristic pattern of distribution.

Pro-inflammatory effects induced by bradykinin in a murine model of pleurisy.

Bradykinin caused a dose-related increase in cell influx 4 h after its administration into the mouse pleural cavity (ED50 = 3.2 nmol/cav., 95% confidence limits = 0.6-15.5). Cell influx peaked at 4 h and remained elevated for up to 72 h, whereas exudation was detected between 2 and 6 h after bradykinin administration. Both HOE 140 (D-Arg-[Hyp3,Thi5,D-Tic7, Oic8]bradykinin) and NPC 17731 (D-Arg0-[Hyp3 D-HypE(transpropyl7)Oic8]bradykinin) inhibited bradykinin-induced cell influx (ID50 0.028 (0.05-0.16) and 0.4 (0.3-0.7) pmol/cav., respectively). Des-Arg9-[Leu8]bradykinin (0.1 and 3.0 nmol/cav., 30 min before) did not inhibit the effects of bradykinin. Pre-treatment of animals with either indomethacin, terfenadine, dexamethasone, N(omega)-nitro-L-arginine benzyl ester, cromolyn, theophylline, salbutamol, FK 888 (N2-[(4R)-4-hydroxy-1-(1-methyl-1H-indol-3-yl)carbonyl-L-propyl]N-met hyl-N-phenyl-methyl-3-(2-naphthyl)-L-alaninamide) or SR 142801 ((N)-(1-[3-[1-benzoyl-3-(3,4-dichloro-phenyl)-piperidin-3-yl]pr opy l]-4-phenyl-piperidin-4-yl)-N-methyl-acetamide) significantly inhibited cell migration (P < 0.01). These results indicate that bradykinin had a significant pro-inflammatory effect on the pleural cavity of the mice. This effect seems to be primarily mediated via activation bradykinin B2 receptors which trigger the release of other mediators.

Citología del lavado pleural intraoperatorio en patología sin derrame, con especial referencia al cáncer del pulmón / Citology of the intraoperatory pleural lavage in pathology without effusion, specially in lung cancer

Objetivo: determinar la existencia de células neoplásicas en la cavidad pleural de pacientes sin derrame. Diseño: estudio prospectivo y ciego en 50 toracotomías sucesivas. Población: tres grupos de pacientes: Grupo I, control, con enfermedades no neoplásicas (n = 19); Grupo II con cáncer del pulmón (n = 22) y Grupo III, otras enfermedades neoplásicas (n = 9). Método: una vez abierta la cavidad pleural, antes de cualquier maniobra, se virtió 300 cc de solución fisiológica. Luego se repitió el procedimiento antes del cierre de la toracotomía. Se estudió la citología de ambos lavados. Se clasificó a los resultados de la citología en positivos, sospechosos y negativos. Resultados: en el Grupo I, todos los controles fueron negativos. En el Grupo II, hubo 7 positivos, 31,8 por ciento (p = 0,007). En el Grupo III, hubo un lavado +, 11 por ciento (p = 0,03) y 3 sospechosos (33 por ciento). Luego de una observación media de 11 meses (rango 7-15 meses), excluyendo la mortalidad por causas ajenas al cáncer, cursan la enfermedad todos los que presentaron citología + y el 14 por ciento de los pacientes con citología negativa (p = 0,0007). Conclusión: el estudio citológico intraoperatorio permite con un bajo costo, agregar una evaluación de extensión de la enfermedad de aparente valor pronóstico. El método es confiable y disponible. Su aplicación sistemática constituiría un excelente complemento de la estadificación habitual

Citología del lavado pleural intraoperatorio en patología sin derrame, con especial referencia al cáncer del pulmón / Citology of the intraoperatory pleural lavage in pathology without effusion, specially in lung cancer

Objetivo: determinar la existencia de células neoplásicas en la cavidad pleural de pacientes sin derrame. Diseño: estudio prospectivo y ciego en 50 toracotomías sucesivas. Población: tres grupos de pacientes: Grupo I, control, con enfermedades no neoplásicas (n = 19); Grupo II con cáncer del pulmón (n = 22) y Grupo III, otras enfermedades neoplásicas (n = 9). Método: una vez abierta la cavidad pleural, antes de cualquier maniobra, se virtió 300 cc de solución fisiológica. Luego se repitió el procedimiento antes del cierre de la toracotomía. Se estudió la citología de ambos lavados. Se clasificó a los resultados de la citología en positivos, sospechosos y negativos. Resultados: en el Grupo I, todos los controles fueron negativos. En el Grupo II, hubo 7 positivos, 31,8 por ciento (p = 0,007). En el Grupo III, hubo un lavado +, 11 por ciento (p = 0,03) y 3 sospechosos (33 por ciento). Luego de una observación media de 11 meses (rango 7-15 meses), excluyendo la mortalidad por causas ajenas al cáncer, cursan la enfermedad todos los que presentaron citología + y el 14 por ciento de los pacientes con citología negativa (p = 0,0007). Conclusión: el estudio citológico intraoperatorio permite con un bajo costo, agregar una evaluación de extensión de la enfermedad de aparente valor pronóstico. El método es confiable y disponible. Su aplicación sistemática constituiría un excelente complemento de la estadificación habitual (AU)

Alloxan diabetes reduces pleural mast cell numbers and the subsequent eosinophil influx induced by allergen in sensitized rats.

Alloxan damages insulin-producing cells and has been used as an inducer of experimental diabetes in several animal species. In this study, administration of alloxan (40 mg/kg, i.v.) to rats was followed by a selective and time-dependent reduction in the number of pleural mast cells (50 +/- 2.2%, p < 0.01; mean +/- SEM), while mononuclear cell and eosinophil counts were not altered. As compared to naive rats, the reduction in mast cell numbers was first noted 48 h following alloxan administration and remained unaltered for at least 60 days. It is noteworthy, that the depletion in the mast cell population was not accompanied by alterations in the total amount of histamine stored per cell. Sensitized rats turned diabetic by alloxan treatment performed 72 h before challenge showed a less pronounced antigen-induced mast cell degranulation compared to nondiabetic rats. Moreover, rats injected with alloxan 72 and 48 but not 24 h before challenge, reacted to allergenic challenge with 50% reduction in the number of eosinophils recruited to the pleural cavity within 24 h. We found that the less pronounced eosinophil accumulation did not relate to an intrinsic cell locomotor abnormality since eosinophils from diabetic rats presented similar chemotactic responses to LTB4 and PAF in vitro as compared to matching controls. Insulin (3 IU/rat) restored basal levels of mast cells and reversed the subsequent inhibition of allergen-induced pleural eosinophilia, suggesting a causative relationship between these phenomena. Treatment with insulin also significantly increased the number of mast cells in the pleural cavity of naive rats (from 637 +/- 57 to 978 +/- 79 x 10(3) cells/cavity, p < 0.001). Consistently, previous depletion of mast cells by means of local treatment with compound 48/80 significantly reduced the antigen-induced eosinophil recruitment in sensitized animals. We conclude that the reduction in the pleural mast cell population noted in alloxan-treated rats could be directly implicated in the diminished pleural eosinophil influx following allergen challenge. This hyporesponsiveness is independent of an intrinsic abnormality of cell chemotaxis, but can be imitated by local mast cell depletion.

Inhibition of eosinophil chemotaxis by chronic blockade of nitric oxide biosynthesis.

The effect of chronic N omega-nitro-L-arginine methyl ester (L-NAME) treatment on the in vivo eosinophil migration induced by bradykinin, platelet-activating factor (PAF), lipopolysaccharide and carrageenin has been investigated in the rat using the pleurisy model. The in vitro (microchemotaxis chamber) eosinophil migration induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP), PAF and zymosan-activated serum was also evaluated in the rat. The eosinophils were obtained from the peritoneal cavity of male Wistar rats and isolated on a discontinuous metrizamide gradient. Chronic inhibition of nitric oxide biosynthesis was achieved by adding L-NAME to the drinking water to give an intake of approximately 75 mumol/rat/day for 4 weeks. Rats treated chronically with L-NAME developed a significant level of hypertension (163 +/- 4.8 mmHg; P < 0.01) compared with animals which received either the same dose of the inactive enantiomer D-NAME (124 +/- 3.2 mmHg) or tap water alone (119 +/- 1.6 mmHg). The intrapleural injection of bradykinin (50 micrograms), PAF (1 microgram), lipopolysaccharide (0.25 microgram) and carrageenin (125 micrograms) into untreated rats in vivo induced a significant level of eosinophil migration by 24 h post-injection. This migration was markedly reduced in L-NAME-treated rats. Eosinophils obtained from untreated rats showed a significant level of migration in vitro in response to fMLP (5 X 10(-8) M), PAF (10(-8) M) and zymosan-activated serum (27 microliters). In contrast, the migration induced by these chemotactic agents was markedly reduced in cells isolated from animals treated chronically with L-NAME. L-Arginine (5.5 mM), but not D-arginine (5.5 mM), restored the ability of eosinophils from L-NAME-treated animals to migrate in response to fMLP. Our results indicate that nitric oxide plays a major role in the in vivo and ex vivo migration of eosinophils.