Airway hyperresponsiveness and remodeling in antigen-challenged guinea pigs

Airway hyperresponsiveness (AHR) is the main feature of allergic subjects/animals, and its underlying mechanism is not clear. We explored whether antigen-induced AHR is associated with cytokine generation, inflammatory cell infiltration, and/or remodeling of airway smooth muscle. Guinea pigs were divided into three groups: control-1, control-2, and ovalbumin (OA). Animals in the control-1 group were not sensitized, while those in the control-2 and the OA group were sensitized with OA. Forty to forty-two days after the initial sensitization or equivalent time, animals in the control-2 group inhaled saline aerosol and those in the OA group inhaled OA aerosol for 30 min. Twenty-four h after OA challenge or equivalent time, animals in each group were further divided into two subgroups: methacholine and hyperventilation. Functional tests were carried out before and after the methacholine or hyperventilation treatment. Immediately after the functional study, bronchoalveolar lavage fluid was collected for determination of inflammatory cells and tumor necrosis factor-alpha (TNF-alpha. The trachea was then removed to determine smooth muscle mass. In both the methacholine and hyperventilation subgroups, significantly more severe airway constriction was found in the OA group, indicating OA-induced AHR. Eosinophil accumulation increased in the control-2 group and this increase was further augmented in the OA group. In addition, TNF-alpha level and smooth muscle mass significantly increased in the OA group. These results suggest that OA challenge-induced AHR is associated with increases in TNF-alpha level, cellular infiltration, and airway smooth muscle mass.     

Effect of pericardial fluid pro-inflammatory cytokines on hemodynamic parameters

We investigated the effects of pro-inflammatory cytokines of pericardial fluid on hemodynamic parameters in patients undergoing coronary artery surgery. Seventy-eight patients were included in the study and they were allocated to three groups: group 1, stable angina pectoris (SAP, n = 15); group 2, unstable angina pectoris (USAP, n = 34); group 3, post-myocardial infarction (PMI, n = 29). Pericardial fluid and arterial blood samples were obtained from all patients and interleukin (IL)-1beta, IL-2 receptor, IL-6, IL-8 and tumor necrosis factor-alpha (TNF-alpha) levels were measured. Pericardial IL-1beta concentration (pg/mL) was significantly higher in the USAP group (26.6 +/- 10.9) compared to the SAP (5.0 +/- 0.1) and PMI (5.8 +/- 1.0) groups. IL-2R, IL-6, IL-8 and TNF-alpha concentrations of pericardial fluid were significantly higher than serum in all groups; difference was more prominent in the PMI group compared to the SAP and the USAP groups. Serum IL-1beta concentrations (pg/mL) were significantly higher in the USAP group (21.8 +/- 3.4) compared to the SAP group (5.0 +/- 0.1) and the PMI group (5.4 +/- 1.6). Cardiac index (CI) before opening the pericardial sac was found to be lower in the USAP group (1.6 +/- 0.3 L/min/m2) compared to the SAP (2.2 +/- 0.5 L/min/m2) and the PMI (2.1 +/- 0.5 L/min/m2) groups (p = 0.028 and p = 0.011, respectively). In the USAP group, there was a relationship between reduction of CI and increase of IL-1beta levels in serum and pericardial fluid.     

Attenuation of IL-2-induced multisystem organ edema by phalloidin and antamanide

Interleukin 2 (IL-2) is a potent cytokine with diverse effects, including the ability to stimulate lymphocyte differentiation into cells capable of lysing tumor. Its therapeutic efficacy is limited because of side effects such as breakdown of the microvascular barrier and edema. Control of the microvascular barrier is in part regulated by endothelial cell cytoskeletal contractile proteins. This study tests whether the cyclopeptides that maintain actin filament organization and distribution and reduce macromolecular flux across the endothelial cell junction in vitro would similarly maintain barrier tightness and prevent early edema produced by IL-2 in vivo. Anesthetized rats were treated at 30-min periods with intravenous saline (0.5 ml, n = 41), phalloidin (20 micrograms in 0.5 ml, n = 21), or antamanide, (20 micrograms in 0.5 ml, n = 21), starting 30 min before the 1-h infusion of 10(6) U of recombinant human IL-2 or saline. Six hours after the start of IL-2, there was edema in the saline/IL-2 group, as measured by increased wet-to-dry ratios (W/D) in the lungs, heart, and kidney. With saline/IL-2, bronchoalveolar lavage (BAL) fluid contained an elevated protein concentration and higher plasma thromboxane levels compared with controls. The number of neutrophils sequestered in the lungs was more than twice that of saline controls. Phalloidin significantly attenuated edema in lung and reduced BAL protein leak. Antamanide treatment was as effective in limiting lung and heart edema, but, in contrast to phalloidin, antamanide prevented kidney edema and did not lead to an alteration in the liver W/D. Antamanide also prevented BAL fluid protein leak.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interference of the PAF-acether antagonist BN 52021 with passive anaphylaxis in the guinea-pig

PAF-acether may be involved in anaphylaxis and asthma. We tested the new PAF-acether antagonist BN 52021 against the effects of antigen in passively sensitized guinea-pigs. Bronchoconstriction by ovalbumin administered i.v. (1 mg/kg) or by aerosol (1 or 10 mg/ml for a period of 1 min) was significantly reduced by BN 52021 (1–10 mg/kg), which did not inhibit drop of leukocyte counts after the i.v. challenge. In both cases, when the guinea-pigs were pretreated by propranolol, high amounts of BN 52021 became ineffective against shock. The reduction of the anaphylactic bronchoconstriction, induced by the combination of mepyramine, aspirin and FPL 55712 was not improved by BN 52021. Tested on isolated lung strips from passively sensitized guinea-pig, BN 52021, at a concentration which inhibits PAF-induced contraction (0.1 mM), did not inhibit the anaphylactic contraction triggered by the administration of ovalbumin (10 μg/ml) nor the accompanying release of histamine and thromboxane. In contrast, BN 52021 (30 μM) significantly reduced the anaphylactic release of histamine and of thromboxane from perfused lungs of passively sensitized guinea-pigs. The results with the isolated lung strips and the propranolol-treated guinea-pigs in vivo suggest a dissociation between the anti-anaphylactic and the anti-PAF-acether properties of BN 52021.     

Differential effects of ebselen on neutrophil recruitment,chemokine, and inflammatory mediator expression in a rat model of lipopolysaccharide-induced pulmonary inflammation

We postulated that the seleno-organic compound ebselen would attenuate neutrophil recruitment and activation after aerosolized challenge with endotoxin (LPS) through its effect as an antioxidant and inhibitor of gene activation. Rats were given ebselen (1-100 mg/kg i.p.) followed by aerosolized LPS exposure (0.3 mg/ml for 30 min). Airway inflammatory indices were measured 4 h postchallenge. Bronchoalveolar lavage (BAL) fluid cellularity and myeloperoxidase activity were used as a measure of neutrophil recruitment and activation. RT-PCR analysis was performed in lung tissue to assess gene expression of TNF-alpha, cytokine-induced neutrophil chemoattractant-1 (CINC-1), macrophage-inflammatory protein-2 (MIP-2), ICAM-1, IL-10, and inducible NO synthase. Protein levels in lung and BAL were also determined by ELISA. Ebselen pretreatment inhibited neutrophil influx and activation as assessed by BAL fluid cellularity and myeloperoxidase activity in cell-free BAL and BAL cell homogenates. This protective effect was accompanied by a significant reduction in lung and BAL fluid TNF-alpha and IL-1 beta protein and/or mRNA levels. Ebselen pretreatment also prevented lung ICAM-1 mRNA up-regulation in response to airway challenge with LPS. This was not a global effect of ebselen on LPS-induced gene expression, because the rise in lung and BAL CINC-1 and MIP-2 protein levels were unaffected as were lung mRNA expressions for CINC-1, MIP-2, IL-10, and inducible NO synthase. These data suggest that the anti-inflammatory properties of ebselen are achieved through an inhibition of lung ICAM-1 expression possibly through an inhibition of TNF-alpha and IL-1 beta, which are potent neutrophil recruiting mediators and effective inducers of ICAM-1 expression.     

Interference of the PAF-acether antagonist BN 52021 with passive anaphylaxis in the guinea-pig

PAF-acether may be involved in anaphylaxis and asthma. We tested the new PAF-acether antagonist BN 52021 against the effects of antigen in passively sensitized guinea-pigs. Bronchoconstriction by ovalbumin administered i.v. (1 mg/kg) or by aerosol (1 or 10 mg/ml for a period of 1 min) was significantly reduced by BN 52021 (1-10 mg/kg), which did not inhibit drop of leukocyte counts after the i.v. challenge. In both cases, when the guinea-pigs were pretreated by propranolol, high amounts of BN 52021 became ineffective against shock. The reduction of the anaphylactic bronchoconstriction, induced by the combination of mepyramine, aspirin and FPL 55712 was not improved by BN 52021. Tested on isolated lung strips from passively sensitized guinea-pig, BN 52021, at a concentration which inhibits PAF-induced contraction (0.1 mM), did not inhibit the anaphylactic contraction triggered by the administration of ovalbumin (10 micrograms/ml) nor the accompanying release of histamine and thromboxane. In contrast, BN 52021 (30 microM) significantly reduced the anaphylactic release of histamine and of thromboxane from perfused lungs of passively sensitized guinea-pigs. The results with the isolated lung strips and the propranolol-treated guinea-pigs in vivo suggest a dissociation between the anti-anaphylactic and the anti-PAF-acether properties of BN 52021.     

Cellular recruitment and cytokine generation in a rat model of allergic lung inflammation are differentially modulated by progesterone and estradiol

We evaluated the role of estradiol and progesterone in allergic lung inflammation. Rats were ovariectomized (Ovx) and, 7 days later, were sensitized with ovalbumin (OA) and challenged after 2 wk with inhaled OA; experiments were performed 1 day thereafter. Ovx-allergic rats showed reduced cell recruitment into the bronchoalveolar lavage (BAL) fluid relative to sham-Ovx allergic rats, as was observed in intact allergic rats treated with ICI-182,780. Estradiol increased the number of cells in the BAL of Ovx-allergic rats, whereas progesterone induced an additional reduction. Cells of BAL and bone marrow (BM) of Ovx-allergic rats released elevated amounts of IL-10 and reduced IL-1 and TNF-. BM cells of Ovx-allergic rats released increased amounts of IL-10 and lower amounts of IL-4. Estradiol treatment of Ovx-allergic rats decreased the release of IL-10 but increased that of IL-4 by BM cells. Estradiol also caused an increased release of IL-1 and TNF- by BAL cells. Progesterone significantly increased the release of IL-10, IL-1, and TNF- by BAL cells and augmented that of IL-4 by BM cells. Degranulation of bronchial mast cells from Ovx rats was reduced after in vitro challenge, an effect reverted by estradiol but not by progesterone. We suggest that the serum estradiol-to-progesterone ratio might drive cellular recruitment, modulating the pulmonary allergy and profile of release of anti-inflammatory or inflammatory cytokines. The existence of such dual hormonal effects suggests that the hormone therapy of asthmatic postmenopausal women and of those suffering of premenstrual asthma should take into account the possibility of worsening the pulmonary conditions. asthma; eosinophils     

Antigen challenge of sensitized rats increases airway responsiveness to methacholine

We measured airway responsiveness to methacholine (MCh) of highly inbred rats before and after six inhalational challenges with antigen. Ten Brown-Norway rats (130-216 g) that were actively sensitized to ovalbumin (OA) received six challenges with OA at 5-day intervals beginning 19 days after sensitization. An aerosol of OA (5% wt/vol) was inhaled for 1, 2, 5, and 10 min or until pulmonary resistance (RL) increased by at least 50%. Challenges with aerosolized MCh were performed immediately before and 14 days after sensitization, 2 days after the 3rd OA exposure, and 2, 7, 12, and 17 days after the 6th OA challenge. Four unsensitized rats underwent inhalational challenges with MCh over an equivalent time period. Responsiveness to MCh was calculated as the concentration of MCh required to increase RL to 200% of the control value (EC200RL). Seven out of 10 rats in the experimental group reacted to the first OA challenge with an immediate increase in RL of greater than 50% of control (range 70-550%). Three animals were unreactive to OA. Base-line EC200RL for all rats undergoing sensitization was 2.13 mg/ml (geometric mean), and it did not change significantly after sensitization (2.05 mg/ml). However, EC200RL of the rats that reacted to OA (n = 7) decreased significantly after 3 (1.11 mg/ml; P less than 0.005) and 6 OA exposures (0.96 mg/ml; P less than 0.005). The increase in responsiveness to inhaled MCh was present 17 days after the last OA exposure (EC200RL = 1.40 mg/ml; P less than 0.05). EC200RL of neither the unreactive sensitized rats (n = 3) nor the control rats (n = 4) changed after OA challenges.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased synovial fluid levels of interleukin-12, sCD25 and sTNF-RII/sTNF-RI ratio delineate a cytokine pattern characteristic of immune arthropathies

The assessment of cytokines and their soluble receptors in the synovial fluid (SF) of inflammatory arthropathies may be useful in studying pathogenetic and immunoregulatory mechanisms underlying different diseases. The aim of this work was to study the cytokine network occurring in inflammatory arthropathies and to identify a cytokine profile which is characteristic of an immune-mediated synovitis. Levels of IL-12, as well as IL-4, IL-8, IL-10, IFN-gamma, sCD25, TNF-alpha and its soluble receptors were measured in the SF of various arthropathies, i.e. non-inflammatory arthropathies: "control" meniscus pathology (n = 21), osteoarthritis (n = 22) and chronic crystal arthritis (n = 9); a non-immune inflammatory arthropathy: acute crystal arthritis (n = 11); 2 immune inflammatory arthropathies: reactive arthritis (ReA) (n = 23) and rheumatoid arthritis (RA) (n = 44). SF levels of IL-10, TNF-alpha and sTNF-RII were found to be increased in the three inflammatory arthropathies compared to the "control" meniscus group. Within the inflammatory group, acute crystal arthritis was characterized by a significantly higher sTNF-RI/TNF-alpha ratio and ReA by a significantly lower sTNF-RII/TNF-alpha ratio compared to the two other diseases. The two immune arthropathies, RA and ReA, were characterized by increased SF levels of IL-12, sCD25 and of the sTNF-RII/sTNF-RI ratio. ReA differed however from RA by showing lower IL-8 and IL-4 levels, higher IFN-gamma levels and a higher IL-12/IL-10 ratio, suggesting a more prevalent Th1 profile in ReA SF. Our data indicate that the measurement of SF cytokines and soluble receptors may discriminate between each inflammatory arthropathy and might be useful in clinical practice.     

Interleukin 5 release into asthmatic airways 4 and 24 hours after endobronchial allergen challenge: its relationship with eosinophil recruitment.

Interleukin 5 (IL-5), a cytokine with a range of activities on eosinophils, has been implicated in the allergic asthmatic reaction. We have investigated the kinetics of release of this cytokine into asthmatic airways as well as its relationship to eosinophil recruitment following allergen challenge. Twelve asthmatic patients underwent endobronchial allergen challenge and bronchoalveolar lavage (BAL) fluid was obtained either 4 h (n=6) or 24 h (n=6) after challenge. Four hours after challenge, levels of IL-5 were significantly increased in BAL fluid (10-fold concentration obtained from the allergen-challenge site compared with the saline control (median 2.67 pg/ml, range 1.0-7.4 pg/ml vs 1.0 pg/ml <1.0-2.4 pg/ml, P<0.05). At 24 h levels of IL-5 increased further at the allergen site but not at the saline control lavage (31.1 pg/ml, range 3.6-59. 0 pg/ml vs 1.5 pg/ml, range <1.5-4.9 pg/ml, respectively P<0.02). At 4h there was almost a three fold increase in IL-5 level, whereas at 24 h IL-5 levels were 20-fold greater. Differential cell counts showed that eosinophil numbers obtained 4 and 24 h after allergen challenge were 7 and 32 times higher than numbers after saline challenge. The parallel increase of eosinophil numbers and IL-5 concentrations in BAL fluid suggests that this cytokine may contribute to the eosinophil recruitment observed into asthmatic airways after allergen challenge.     

Acute and long-term TNF-alpha administration increases pulmonary vascular reactivity in isolated rat lungs.

Tumor necrosis factor-alpha (TNF-alpha) causes pulmonary hypertension and arterial hypoxemia, but the mechanisms are unknown. We conducted two experiments to test the hypothesis that TNF-alpha alters pulmonary vascular reactivity, which in turn could cause either pulmonary hypertension or arterial hypoxemia. In experiment 1, rats were given acute or long-term injections of TNF-alpha (recombinant human) in vivo. Rats treated acutely received either saline or TNF-alpha (40 micrograms/kg iv in saline) 3 min (TNF-3 min; n = 8), 20 min (TNF-20 min; n = 8), or 24 h (TNF-24 h; n = 5) before the lungs were isolated. Rats treated chronically received injections of either saline or TNF-alpha (250 micrograms/kg ip in saline) two times per day for 7 days (TNF-7 days; n = 9). Lungs were isolated and perfused with Earle's salt solution (+2 g/l NaHCO3 + 4 g/100 ml Ficoll), and vascular reactivity was tested with acute hypoxia (3 min; 3% O2) and angiotensin II (ANG II; 0.025-0.40 micrograms). Pulmonary pressor responses to hypoxia were greater (P less than 0.05) in TNF-20 min and TNF-7 day groups. ANG II responses were increased (P less than 0.05) in TNF-7 day rats. In experiment 2, lungs were isolated and perfused and received direct pulmonary arterial injections of TNF-alpha (0.2, 2.0, and 20 micrograms) or saline, after stable responses to hypoxia and ANG II (0.10 microgram) were attained. Reactivity was not different between control and TNF-alpha rats before the injections, but TNF-alpha increased (P less than 0.05) responses to hypoxia and ANG II.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of upper and lower airway responses of two sensitized rat strains to inhaled antigen.

The purpose of the study was to investigate the relationships between upper airways responses and pulmonary responses of two strains of highly inbred rats to inhaled antigen. To do this we measured the upper and lower airways resistance for 60 min after challenge of Brown-Norway rats (BN; n = 13) and an inbred rat strain (MF; n = 11), derived from Sprague-Dawley, with aerosolized ovalbumin (OA). Rats were actively sensitized with OA (1 mg sc) using Bordetella pertussis as an adjuvant. Two weeks later the animals were anesthetized and challenged. Tracheal pressure, esophageal pressure, and airflow were measured, from which total pulmonary resistance was partitioned into upper airway and lower pulmonary resistance (RL). The peak upper airway response to inhaled OA was similar in BN (1.89 +/- 0.66 cmH2O.ml-1.s; n = 7) and MF (2.85 +/- 0.68 cmH2O.ml-1.s; n = 6). The lower airway response to OA challenge was substantially greater in BN, and RL changed from 0.07 +/- 0.01 to 0.34 +/- 0.13 (n = 6; P < 0.05). The MF did not have any significant increase in RL after challenge; the baseline RL was 0.12 +/- 0.02 and only reached a peak value of 0.15 +/- 0.05 (n = 5; P = NS). Lower airway responsiveness of BN (n = 10) to serotonin, an important mediator early allergic airway responses, was similar to MF (n = 7).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhaled nitric oxide attenuates acute lung injury via inhibition of nuclear factor-kappa B and inflammation.

The effect of inhaled nitric oxide (NO) on inflammatory process in acute lung injury (ALI) is unclear. The aims of this study were to 1) examine whether inhaled NO affects the biochemical lung injury parameters and cellular inflammatory responses and 2) determine the effect of inhaled NO on the activation of nuclear factor-kappa B (NF-kappa B) in lipopolysaccharide (LPS)-induced ALI. Compared with saline controls, rabbits treated intravenously with LPS showed increases in total protein and lactate dehydrogenase in the bronchoalveolar lavage (BAL) fluid, indicating ALI. LPS-treated animals with NO inhalation (LPS-NO) showed significant decreases in these parameters. Neutrophil numbers in the BAL fluid, the activity of reactive oxygen species in BAL cells, and the levels of interleukin (IL)-1 beta and IL-8 in alveolar macrophages were increased in LPS-treated animals. In contrast, neutrophil numbers and these cellular activities were substantially decreased in LPS-NO animals, compared with LPS-treated animals. NF-kappa B activation in alveolar macrophages from LPS-treated animals was also markedly increased, whereas this activity was effectively blocked in LPS-NO animals. These results suggest that inhaled NO attenuates LPS-induced ALI and pulmonary inflammation. This attenuation may be associated with the inhibition of NF-kappa B activation.     

Beneficial effects of ApoA-I on LPS-induced acute lung injury and endotoxemia in mice

High density lipoprotein (HDL) binds lipopolysaccharide (LPS) and neutralizes its toxicity. The aim of our study was to investigate the effects of Apolipoprotein (ApoA-I), the major apolipoprotein of HDL, on LPS-induced acute lung injury (ALI) and endotoxemia. BALB/c mice were challenged with LPS, followed by ApoA-I or saline administration for 24h. The mice were then sacrificed and histopathological analysis of the lung was performed. We found that ApoA-I could attenuate LPS-induced acute lung injury and inflammation. To investigate the mechanisms, we measured tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) levels in the serum and bronchoalveolar lavage (BAL) fluid and found that ApoA-I could significantly inhibit LPS-induced increases in the IL-1beta and TNF-alpha levels in serum (P<0.05, respectively), as well as in the IL-1beta, TNF-alpha, and IL-6 levels in BAL fluid (P<0.01 and P<0.05, P<0.05, respectively). Moreover, we evaluated the effect of ApoA-I on the mortality of L-929 cells which were attacked by LPS-activated peritoneal macrophages. We found that ApoA-I could significantly inhibit the LPS-induced cell death in a dose-dependent fashion. Furthermore, we investigated in vivo the effects of ApoA-I on the mortality rate and survival time after LPS administration and found that ApoA-I significantly decreased the mortality (P<0.05) and increased the survival time (P<0.05). In summary, the results suggest that ApoA-I could effectively protect against LPS-induced endotoxemia and acute lung damage. The mechanism might be related to inhibition of inflammatory cytokine release from macrophages.     

Contribution of antigen-primed CD8+ T cells to the development of airway hyperresponsiveness and inflammation is associated with IL-13

The role of Th2/CD4 T cells, which secrete IL-4, IL-5, and IL-13, in allergic disease is well established; however, the role of CD8(+) T cells (allergen-induced airway hyperresponsiveness (AHR) and inflammation) is less clear. This study was conducted to define the role of Ag-primed CD8(+) T cells in the development of these allergen-induced responses. CD8-deficient (CD8(-/-)) mice and wild-type mice were sensitized to OVA by i.p. injection and then challenged with OVA via the airways. Compared with wild-type mice, CD8(-/-) mice developed significantly lower airway responsiveness to inhaled methacholine and lung eosinophilia, and exhibited decreased IL-13 production both in vivo, in the bronchoalveolar lavage (BAL) fluid, and in vitro, following Ag stimulation of peribronchial lymph node (PBLN) cells in culture. Reconstitution of sensitized and challenged CD8(-/-) mice with allergen-sensitized CD8(+) T cells fully restored the development of AHR, BAL eosinophilia, and IL-13 levels in BAL and in culture supernatants from PBLN cells. In contrast, transfer of naive CD8(+) T cells or allergen-sensitized CD8(+) T cells from IL-13-deficient donor mice failed to do so. Intracellular cytokine staining of lung as well as PBLN T cells revealed that CD8(+) T cells were a source of IL-13. These data suggest that Ag-primed CD8(+) T cells are required for the full development of AHR and airway inflammation, which appears to be associated with IL-13 production from these primed T cells.     

Caffeic acid phenethyl ester attenuates allergic airway inflammation and hyperresponsiveness in murine model of ovalbumin-induced asthma

Caffeic acid phenethyl ester (CAPE) is a biologically active ingredient of propolis, which has several interesting biological properties, including antioxidant and anti-inflammatory; however, its anti-allergic effects are poorly understood. The objective of this study was to determine whether treatment with CAPE results in significant inhibition of asthmatic reactions in a mouse model. Mice sensitized and challenged with ovalbumin (OVA) had the following typical asthmatic reactions: an increase in the number of eosinophils in bronchoalveolar lavage (BAL) fluid; a marked influx of inflammatory cells into the lung around blood vessels and airways, and airway luminal narrowing; the development of airway hyperresponsiveness (AHR); the presence of tumor necrosis factor-alpha (TNF-alpha) and Th2 cytokines, including IL-4 and IL-5, in the BAL fluid; and the presence of allergen-specific IgE in the serum. Five successive intraperitoneal administrations of CAPE before the last airway OVA challenge resulted in significant inhibition of characteristic asthmatic reactions. We determined that increased generation of reactive oxygen species (ROS) by inhalation of OVA was diminished via the administration of CAPE in BAL fluid, as well as nuclear factor-kappaB (NF-kappaB) DNA binding activity. These findings indicate that oxidative stress may have a crucial function in the pathogenesis of bronchial asthma, and that CAPE may be useful as an adjuvant therapy for the treatment of bronchial asthma.     

Transient neutrophil infiltration after allergen challenge is dependent on specific antibodies and Fc gamma III receptors

Following allergen challenge of sensitized mice, neutrophils are the first inflammatory cells found in bronchoalveolar lavage (BAL) fluid. To determine the underlying mechanism for their accumulation, mice were sensitized to OVA on days 0 and 14, and received, on day 28, a single intranasal challenge (s.i.n.) with either OVA or ragweed. Eight hours after the s.i.n., BAL fluid was obtained. BALB/c mice sensitized and challenged with OVA showed significantly higher total cell counts and numbers of neutrophils in BAL fluid compared to the OVA-sensitized and ragweed-challenged or nonsensitized mice. Levels of neutrophil chemokines in BAL fluid supernatants were markedly elevated in the sensitized and OVA-challenged mice; Fc epsilon RI-deficient mice showed comparable numbers of neutrophils and neutrophil chemokines in BAL fluid after s.i.n. But in sensitized mice lacking the Fc common gamma-chain and B cell-deficient mice, the number of neutrophils and levels of neutrophil chemokines in BAL fluid were significantly lower. Further, mice lacking the FcgammaRIII did not develop this early neutrophil influx. Neutrophil infiltration could be induced in naive mice following intranasal instillation of allergen combined with allergen-specific IgG1. In addition, macrophages from sensitized mice were stimulated with allergen and activated to produce neutrophil chemokines. These results demonstrate that neutrophil influx after allergen challenge requires prior sensitization, is allergen-specific, is mediated through FcgammaRIII, and is dependent on the presence of Ab.     

Neovastat (AE-941) inhibits the airway inflammation and hyperresponsiveness in a murine model of asthma

Matrix metalloproteinase (MMP)-9 plays an important role in the pathogenesis of bronchial asthma. Neovastat, having significant antitumor and antimetastatic properties, is classified as a naturally occurring multifunctional antiangiogenic agent. We evaluated the therapeutic effect of Neovastat on airway inflammation in a mouse model of asthma. BALB/c mice were immunized subcutaneously with ovalbumin (OVA) on days 0, 7, 14, and 21 and challenged with inhaled OVA on days 26, 29, and 31. Neovastat was administrated by gavage (5 mg/kg body weight) three times with 12 h intervals, beginning 30 min before OVA inhalation. On day 32, mice were challenged with inhaled methacholine, and enhanced pause (Penh) was measured as an index of airway hyperresponsiveness. The severity of airway inflammation was determined by differential cell count of bronchoalveolar lavage (BAL) fluid. The MMP-9 concentration in BAL fluid samples was measured by ELISA, and MMP-9 activity was measured by zymography. The untreated asthma group showed an increased inflammatory cell count in BAL fluid and Penh value compared with the normal control group. Mice treated with Neovastat had significantly reduced Penh values and inflammatory cell counts in BAL fluid compared with untreated asthmatic mice. Furthermore, mice treated with Neovastat showed significantly reduced MMP-9 concentrations and activity in BAL fluid. These results demonstrate that Neovastat might have new therapeutic potential for airway asthmatic inflammation.     

Splenic denervation worsens lipopolysaccharide-induced hypotension, hemoconcentration, and hypovolemia

During lipopolysaccharide (LPS)-induced endotoxemia, increased intrasplenic fluid efflux contributes to a reduction in plasma volume. We hypothesized that splenic sympathetic nerve activity (SSNA), which increases during endotoxemia, limits intrasplenic fluid efflux. We reasoned that splenic denervation would exaggerate LPS-induced intrasplenic fluid efflux and worsen the hypotension, hemoconcentration, and hypovolemia. A nonlethal dose of LPS (150 microg x kg(-1) x h(-1) for 18 h) was infused into conscious male rats bearing transit time flow probes on the splenic artery and vein. Fluid efflux was estimated from the difference in splenic arterial inflow and venous outflow (A-V). LPS significantly increased the (A-V) flow differential (fluid efflux) in intact rats (saline -0.01 +/- 0.02 ml/min, n = 8 vs. LPS +0.21 +/- 0.06 ml/min, n = 8); this was exaggerated in splenic denervated rats (saline -0.03 +/- 0.01 ml/min, n = 7 vs. LPS +0.41 +/- 0.08 ml/min, n = 8). Splenic denervation also exacerbated the LPS-induced hypotension, hemoconcentration, and hypovolemia (peak fall in mean arterial pressure: denervated 19 +/- 3 mmHg, n = 10 vs. intact 12 +/- 1 mmHg, n = 8; peak rise in hematocrit: denervated 6.7 +/- 0.3%, n = 8 vs. intact 5.0 +/- 0.3%, n = 8; decrease in plasma volume at 90-min post-LPS infusion: denervated 1.08 +/- 0.15 ml/100 g body wt, n = 7 vs. intact 0.54 +/- 0.08 ml/100 g body wt, n = 8). The exaggerated LPS-induced hypovolemia associated with splenic denervation was mirrored in the rise in plasma renin activity (90 min post-LPS: denervated 11.5 +/- 0.8 ng x ml(-1) x h(-1), n = 9 vs. intact 6.6 +/- 0.7 ng x ml(-1) x h(-1), n = 8). These results are consistent with our proposal that SSNA normally limits LPS-induced intrasplenic fluid efflux.     

Increased level of cytokines and matrix metalloproteinases in osteoarthritic subchondral bone

OBJECTIVE: The aim of this study was to investigate the expression of several cytokines, matrix metalloproteinases (MMPs), and tissue inhibitor of matrix metalloproteinases (TIMP)-1 in osteoarthritis (OA) and control sera and different joint tissues. METHODS: Serum, synovial fluid, cartilage, synovial and subchondral bone tissues were examined in OA and control subjects. The protein level of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, IL-8, IL-10 and MMP-2, MMP-3, MMP-9, and TIMP-1 were measured by immunoanalysis. RESULTS: Serum levels of TNF-alpha, MMP-3 and -9 were significantly higher in OA patients than in controls. Conversely, serum IL-10 was decreased in OA patients. CRP was elevated when compared to healthy controls and decreased significantly 6 months after the surgery. In contrast to control samples, OA cartilage and synovium revealed significantly higher MMP-2, -3, -9 and IL-10. IL-1alpha was significantly higher in OA cartilage and IL-8 in OA synovium. Interestingly, MMP-3, -9, TIMP-1 and all tested cytokines were up-regulated in OA subchondral bone. DISCUSSION: This study demonstrates pro-inflammatory condition of OA pathology and supports the idea that vascularized subchondral region may increase the synthesis of cytokines and MMPs leading to degradation of adjacent cartilage.