Systemic FasL neutralization increases eosinophilic inflammation in a mouse model of asthma.

BACKGROUND: Eosinophils and lymphocytes are pathogenically important in allergic inflammation and sensitive to Fas-mediated apoptosis. Fas ligand (FasL) activity therefore should play a role in regulating the allergic immune response. We aimed to characterize the role of FasL expression in airway eosinophilia in Aspergillus fumigatus (Af)-induced sensitization and to determine whether FasL neutralization alters the inflammatory response. METHODS: Sensitized Balb/c mice were killed before (day 0) and 1, 7 and 10 days after a single intranasal challenge with Af. Animals received either neutralizing antibody to FasL (clone MFL4) or irrelevant hamster IgG via intraperitoneal injection on days -1 and 5. FasL expression, BAL and tissue inflammatory cell and cytokine profile, and apoptosis were assessed. RESULTS: Postchallenge FasL gene expression in BAL cells and TUNEL positivity in the airways coincided with the height of inflammatory cell influx on day 1, while soluble FasL protein was released on day 7, preceding resolution of the inflammatory changes. Although eosinophil numbers showed a negative correlation with soluble FasL levels in the airways, MBP(+) eosinophils remained TUNEL negative in the submucosal tissue, throughout the 10-day period after Af challenge. Systemic FasL neutralization significantly enhanced BAL and tissue eosinophil counts. This effect was associated with increased activation of T cells and release of IL-5, IL-9, and GM-CSF in the BAL fluid of mice, indicating an involvement of pro-eosinophilic survival pathways. CONCLUSIONS: FasL activity may play an active role in resolving eosinophilic inflammation through regulating T cells and pro-eosinophilic cytokine release during the allergic airway response.

Ozone inhalation induces exacerbation of eosinophilic airway inflammation and hyperresponsiveness in allergen-sensitized mice.

BACKGROUND: Ozone (O(3)) exposure evokes asthma exacerbations by mechanisms that are poorly understood. We used a murine model to characterize the effects of O(3) on allergic airway inflammation and hyperresponsiveness and to identify factors that might contribute to the O(3)-induced exacerbation of asthma. METHODS: BALB/c mice were sensitized and challenged with Aspergillus fumigatus (Af). A group of sensitized and challenged mice was exposed to 3.0 ppm of O(3) for 2 h and studied 12 h later (96 h after Af challenge). Naive mice and mice exposed to O(3) alone were used as controls. Bronchoalveolar lavage (BAL) cellular and cytokine content, lung function [enhanced pause (P(enh))], isometric force generation by tracheal rings and gene and protein expression of Fas and FasL were assessed. Apoptosis of eosinophils was quantified by FACS. RESULTS: In sensitized mice allergen challenge induced a significant increase of P(enh) and contractile force in tracheal rings that peaked 24 h after challenge and resolved by 96 h. O(3) inhalation induced an exacerbation of airway hyperresponsiveness accompanied by recurrence of neutrophils and enhancement of eosinophils 96 h after allergen challenge. The combination of allergen and O(3) exposure inhibited Fas and FasL gene and protein expression and eosinophil apoptosis and increased interleukin-5 (IL-5), granulocyte-macrophage-colony stimulating factor (GM-CSF) and G-CSF protein levels. CONCLUSIONS: O(3) affects airway responsiveness of allergen-primed airways indirectly by increasing viability of eosinophils and eosinophil-mediated pathological changes.

Bronchoalveolar lavage fluid concentrations of transforming growth factor (TGF)-beta1, TGF-beta2, interleukin (IL)-4 and IL-13 after segmental allergen challenge and their effects on alpha-smooth muscle actin and collagen III synthesis by primary human lung fibroblasts.

RATIONALE: Asthmatic airway remodelling is characterized by myofibroblast hyperplasia and subbasement membrane collagen deposition. We hypothesized that cytokines and growth factors implicated in asthmatic airway remodelling are increased in bronchoalveolar lavage (BAL) fluid of asthmatics after segmental allergen challenge (SAC), and that these growth factors and cytokines increase alpha-smooth muscle actin (alpha-SMA) and collagen III synthesis by human lung fibroblasts (HLFs). METHODS: Transforming growth factor (TGF)-beta1, TGF-beta2, IL-4 and IL-13 levels were measured in BAL fluid from 10 asthmatics and 9 non-asthmatic controls at baseline and then 1 day, 1 week and 2 weeks after SAC. Confluent cultures of HLFs were stimulated by exogenous addition of TGF-beta1, TGF-beta2, IL-4 or IL-13 (concentration range 0.01-10 ng/mL) over 48 h. Collagen III was measured in culture supernates and alpha-SMA in cell lysates by Western blot. RESULTS: At baseline, there was no difference in BAL fluid concentrations of TGF-beta1, IL-4 and IL-13 between asthmatics and controls; however, non-asthmatics had higher concentrations of total TGF-beta2. In asthmatics, BAL fluid concentrations of all four factors increased significantly 1 day after SAC. TGF-beta1, TGF-beta2 and IL-13 concentrations returned to baseline by 1 week after SAC, but BAL fluid IL-4 concentration remained elevated for at least 2 weeks. TGF-beta1, TGF-beta2 and IL-4 significantly increased alpha-SMA in fibroblasts, but only IL-4 caused corresponding increases in collagen III synthesis. IL-13 had no direct effects on collagen III synthesis and alpha-SMA expression. CONCLUSIONS: Because IL-4 caused a dose-dependent increase in alpha-SMA and collagen III synthesis, it may be an important cytokine mediating asthmatic airway remodelling. TGF-beta1 and TGF-beta2 may also play a role in airway remodelling by stimulating phenotypic change of fibroblasts to myofibroblasts. Additionally, collagen III synthesis appears to be independent of myofibroblast phenotype and is apparently regulated by different growth factors and cytokines.

Kinetics of IL-10 production after segmental antigen challenge of atopic asthmatic subjects.

BACKGROUND: IL-10 is an anti-inflammatory cytokine made by lymphocytes, monocytes-macrophages, and eosinophils, and it may have an important role in regulating the asthmatic inflammatory response. IL-10 levels have been reported to be reduced in asthmatic airways, potentially contributing to more intense inflammation. OBJECTIVE: We sought to determine whether IL-10 levels were deficient in patients with mild asthma compared with controls and to determine whether IL-10 levels were associated with the resolution of eosinophilic inflammation. METHODS: We quantified IL-10 levels in the bronchoalveolar lavage (BAL) fluid (ELISA), BAL cells (quantitative immunocytochemistry), purified alveolar macrophages-monocytes studied ex vivo (ELISA), before (day 1) and after (24 hours [day 2], 1 week [day 9], and 2 weeks [day 16]) segmental antigen challenge (SAC), and investigated the effect of glucocorticoid treatment on ex vivo macrophage-monocyte IL-10 production. RESULTS: IL-10 levels were significantly higher in the BAL fluid of mild asthmatic subjects who demonstrated a dual reaction (both early and late) after whole lung ragweed inhalation challenge compared with nonallergic, nonasthmatic control subjects before and 24 hours and 1 week after SAC. Macro-phages-monocytes obtained before and after SAC from asthmatic patients also secreted increased amounts of IL-10 ex vivo than those from controls. Dexamethasone did not significantly change spontaneous IL-10 secretion from macrophages-monocytes in vitro. Quantitative immunocytochemical analysis of BAL cells demonstrated increased IL-10 in macrophages 24 hours after SAC and a similar trend in eosinophils. CONCLUSION: IL-10 is not deficient in mild asthma. Furthermore, BAL IL-10 levels are significantly higher in asthmatic subjects with a dual response than in control subjects before and after SAC. The increase in IL-10 was coincident with the initial increase in BAL eosinophils, although BAL eosinophilia persisted after IL-10 levels had returned to baseline, suggesting that the increased IL-10 levels could not promptly terminate this localized eosinophilic response.

Effect of IL-5, glucocorticoid, and Fas ligation on Bcl-2 homologue expression and caspase activation in circulating human eosinophils.

IL-5 is a potent eosinophil viability-enhancing factor that has been strongly implicated in the pathogenesis of IgE-mediated inflammation in vivo. Recently published data have suggested that IL-5 (and related cytokines) may act by altering the expression of the anti-apoptotic regulator Bcl-2 or its homologues, but this is controversial. The behaviour of the recently described pro-apoptotic cysteine proteases (caspases) in eosinophils after IL-5 treatment has not been explored. We examined the effect of IL-5 on the expression of four major Bcl-2 homologues, as well as on the expression/activation of key members of the caspase cell death cascade in cultured circulating human eosinophils. The effect of relevant inducers of eosinophil apoptosis (glucocorticoid and Fas ligation) on these regulatory proteins was also examined. We observed baseline expression of the anti-apoptotic Mcl-1 and pro-apoptotic Bax proteins in immunoblots of eosinophil lysates, but not Bcl-x, Bcl-2. IL-5 treatment had the effect of maintaining this basal level of expression over time without altering the balance of Bcl-2 homologues. The (upstream) caspase 8 and (downstream) caspase 3 proenzymes were detected in eosinophils at baseline, and were processed during spontaneous and stimulated eosinophil death. IL-5 completely blocked caspase processing in spontaneous and dexamethasone-induced cell death, and significantly slowed processing during Fas ligation. Our data do not support the theory that IL-5 acts by altering the balance of anti-apoptotic and pro-apoptotic Bcl-2 homologues, but suggest that it may act by regulating activation of the caspase cell death cascade.

HSP27 elevated in mild allergic inflammation protects airway epithelium from H2SO4 effects.

Inflammation in allergic individuals is hypothesized to elevate stress proteins [heat shock proteins (HSP)] in airway epithelium, which may protect cells from further adverse conditions. Allergic, either asthmatic or not, and normal volunteers participated in a 2-day segmental allergen challenge bronchoscopic procedure. Bronchial epithelium was obtained before and after challenge. Epithelium was exposed to medium with H2SO4 (pH5), returned to medium at pH 7.4, and finally harvested for Western blotting with anti-27-kDa HSP (HSP27) antibody. Prechallenge epithelium of all subjects had significantly inhibited ciliary function by H2SO4 (pH 5) conditions (P < 0.001); only epithelium of normals recovered (P = 0.02). Allergic subjects with mild inflammation (< 50 micrograms/ml increase in albumin in bronchoalveolar lavage) had significantly increased HSP27 postchallenge (P = 0.01) and little ciliary dysfunction at pH 5, whereas subjects with severe inflammation (> 50 micrograms/ml increase in albumin) had little change in HSP27 and significant ciliary inhibition (P = 0.02). Normal epithelium had similar trends in HSP27 and equivalent inhibition of ciliary activity at pH 5 before and after allergen challenge. These data indicate that mild inflammation to allergen elevates HSP27 stress protein levels, thereby potentially protecting epithelial function from additional adverse conditions.

Kinetics of the development and recovery of the lung from IgE-mediated inflammation: dissociation of pulmonary eosinophilia, lung injury, and eosinophil-active cytokines.

Events occurring up to 16 d after antigen challenge were characterized using a novel protocol employing four bronchoscopies, two segmental antigen challenge (SAC) procedures (on Days 1 and 2), and six bronchoalveolar lavages (BALs) (on Days 1, 2, 9, and 16) in three groups: ragweed allergic asthmatics with dual phase airway reactions (AA-D), allergic asthmatics with a single early airway reaction (AA-S), and nonallergic nonasthmatic control subjects. In AA-D subjects, SAC produced a marked eosinophilic inflammatory response at 24 h associated with eosinophil degranulation (eosinophil cationic protein [ECP] in BAL fluid) and lung injury, which largely resolved by Day 16. When the second antigen-challenged segment (SAC performed on Day 2) was lavaged 7 d after challenge (Day 9), a persistent pulmonary eosinophilia was noted accompanied by minimal elevations in ECP and albumin. Eosinophil-active cytokines showed unique patterns: interleukin-5 (IL-5) increased in the antigen segment on Day 2 then returned to baseline after 7 d; granulocyte-macrophage colony-stimulating factor (GM-CSF) peaked at Day 2 but was persistently elevated throughout Day 16 in antigen segments, and increased in control segments at late time points; IL-3 levels were constant and similar in antigen and control segments. Changes were specific to AA-D subjects in comparison with control subjects. Elements of the IgE-mediated pulmonary inflammatory response differ markedly in their development and resolution.

Baculovirus P35 inhibits the glucocorticoid-mediated pathway of cell death.

Recent evidence suggests that members of the interleukin-1-beta-converting enzyme (ICE)/Ced-3 family are key mediators of mammalian apoptosis. The known members of the ICE/Ced-3 cysteine protease family are synthesized as proenzymes and require proteolytic processing to produce active, heterodimeric enzymes. The baculovirus protein P35 has recently been shown to inhibit several members of the ICE/Ced-3 cysteine protease family. The importance of ICE/Ced-3 cysteine proteases in programmed cell death prompted us to investigate the role of the apoptotic mediator, CPP32, in the glucocorticoid-mediated cell death pathway. Glucocorticoids induce growth inhibition and apoptosis in sensitive leukemic cell lines, immature thymocytes, and eosinophils. In this report, we demonstrate the enzymatic cleavage of proCPP32 to its active subunits in cells undergoing glucocorticoid-induced apoptotic cell death. Concurrently, in apoptotic cells, PARP, a 116-kilodalton (kDa) human poly(ADP-ribose) polymerase, is proteolytically cleaved to its signature 85-kDa fragment. The proteolytic processing of PARP (the nuclear DNA repair enzyme known to be cleaved in association with apoptosis) is catalyzed by members of the ICE/Ced-3 family. Importantly, stable transfection of the antiapoptotic baculovirus P35 inhibits glucocorticoid-induced apoptotic cell death, proteolytic processing of proCPP32, and cleavage of the 116kDa PARP. We conclude that activation of CPP32 is a critical event in glucocorticoid-induced apoptosis and that this pathway is inhibited at or upstream of CPP32 by baculovirus P35. These data demonstrate that PARP cleavage occurs during glucocorticoid-induced apoptotic cell death and show that this proteolytic process is blocked by the expression of baculovirus P35, supporting a role for activation of the ICE/Ced-3-like cysteine protease during glucocorticoid-induced apoptosis.

Beta 2-agonist-elevated stress response in human bronchial epithelial cells in vivo and in vitro.

Recent studies report high baseline levels of stress (heat shock) proteins in bronchial epithelial cells from asthmatic individuals. The promoter of the gene encoding the 72-kDa heat shock protein has an element responsive to cAMP, which may be affected by beta-agonists. This study examined stress protein levels in subjects enrolled in a segmental lung allergen challenge study to determine whether beta-agonist medication could contribute to a stress response. Subjects were divided on the basis of no premedication (n = 17), metered dose inhalations of albuterol (n = 24), or placebo inhalation (n = 3) prior to bronchoscopy. Levels of the inducible stress protein Hsp72 and constitutive Hsp73 were quantitated in bronchial epithelial cells from brush biopsy of allergic nonasthmatic, allergic asthmatic, and normal individuals. Mean levels were increased significantly (p < 0.003 and p < 0.004, respectively) in those subjects who received albuterol premedication. No significant differences were found between clinical groups of individuals or for placebo inhalation vs nonpremedication. Albuterol in vitro increased the levels of Hsp72 and Hsp73 in epithelial cells from either nonpremedicated or placebo-treated donors; the Hsp72 levels correlated linearly with increased albuterol concentration (r = 0.81, p < 0.01). Therefore, beta-agonists elevate or prolong an elevated stress response in epithelial cells, possibly through cAMP-mediated effects.

The Ced-3/interleukin 1beta converting enzyme-like homolog Mch6 and the lamin-cleaving enzyme Mch2alpha are substrates for the apoptotic mediator CPP32.

Recent evidence suggests that CPP32 is an essential component of an aspartate-specific cysteine protease (ASCP) cascade responsible for apoptosis execution in mammalian cells. Activation of CPP32 could lead to activation of other downstream ASCPs, resulting in late morphological changes such as lamin cleavage and DNA fragmentation, observed in cells undergoing apoptosis. Here we describe the identification and cloning of a novel human ASCP named Mch6 from Jurkat T lymphocytes. We demonstrate that the pro-enzymes of Mch6 and the lamin-cleaving enzyme Mch2alpha are substrates for mature CPP32. Site-directed mutagenesis revealed that CPP32 processes pro-Mch6 preferentially at Asp330 to generate two subunits of molecular masses 37 kDa (p37) and 10 kDa (p10). However, CPP32 processes pro-Mch2alpha at three aspartate processing sites (Asp23, Asp179, and Asp193) to produce the large (p18) and small (p11) subunits of the mature Mch2alpha enzyme. The CPP32-processed Mch2alpha is capable of cleaving the VEIDN lamin cleavage site, indicating that CPP32 can, in fact, activate pro-Mch2alpha. Granzyme B at a concentration that allows processing and activation of CPP32 failed to process pro-Mch2alpha. However, incubation of pro-Mch2alpha with granzyme B in the presence of a cellular extract containing pro-CPP32 resulted in activation of pro-CPP32 and subsequent processing of pro-Mch2alpha. Interestingly, granzyme B can also process pro-Mch6 but at a site N-terminal to that cleaved by CPP32. These data suggest that Mch2alpha and Mch6 are downstream proteases activated in CPP32- and granzyme B-mediated apoptosis. This is the first demonstration of a protease cascade involving granzyme B, CPP32, Mch2alpha, and Mch6 and evidence that the lamin-cleaving enzyme Mch2 is a target of mature CPP32.

Effects of inflammation and acute beta-agonist inhalation on beta 2-AR signaling in human airways.

Although alterations in beta 2-adrenergic receptor (AR) responsiveness may in part explain reports linking deterioration of asthma control with beta-agonist treatment of asthmatics, few data exist on beta 2-AR regulation in human airway cells. We have employed a bronchoscopy model to examine inflammation- and beta-agonist-induced alterations in human bronchial epithelial cell beta 2-AR density and responsiveness. Allergic asthmatic subjects participated in 2-day protocols examining airways before and 24 h after segmental antigen challenge (SAC) with ragweed. To assess the effect of acute beta-agonist exposure, bronchoscopies were performed both with (+ beta-Ag) and without (-beta-Ag) inhalation of beta-agonist 30 min before the procedure. Measurements of inflammatory cell infiltration were obtained by analysis of bronchoalveolar lavage fluid, and beta 2-AR density and responsiveness were examined in bronchial epithelial cells obtained by bronchoscopic brushing. Neither SAC nor acute beta-agonist administration alone significantly affected epithelial cell beta 2-AR density. beta-Agonist-stimulated adenosine 3', 5'-cyclic monophosphate (cAMP) generation was significantly lower in the + beta-Ag groups compared with the-beta-Ag group, demonstrating acute agonist-specific beta 2-AR desensitization in vivo. SAC caused a small, statistically insignificant reduction in beta-Agonist-stimulated cAMP production in both -beta-Ag or + beta-Ag groups. These lata suggest that acute beta-agonist inhalation, but not airway inflammation, significantly reduces maximal beta 2-AR responsiveness in airway cells.

IL-1 beta release from cultured bronchial epithelial cells and bronchoalveolar lavage cells from allergic and normal humans following segmental challenge with ragweed.

This work investigated whether interleukin 1 beta (IL-1 beta) release from epithelial cells is modulated by antigen challenge in vivo, and inflammatory cells in vitro. Bronchial epithelial cells were obtained before and after segmental allergen challenge in allergic and normal individuals, and were cultured with and without autologous bronchoalveolar lavage (BAL) cells. IL-1 beta in culture medium was quantitated by enzyme-linked immunoabsorbent assay (ELISA). Pre-challenge IL-1 beta levels from epithelial cells were similar in allergic (4.4 +/- 0.8 pg/ml, n = 32) and normal (6.8 +/- 1.7 pg/ml, n = 17) subjects. IL-1 beta levels were significantly elevated from epithelium with BAL cell co-culture vs without co-culture in both subject groups (allergic, 13.2 +/- 2.3 pg/ml, P = 0.006; normal: 16.4 +/- 4.0 pg/ml, P = 0.007). Post-challenge IL-1 beta from epithelial cells without BAL cells was increased in both groups, but significant only for allergic subjects (P = 0.003). Post-challenge IL-1 beta from epithelial with BAL cells changed little for allergic subjects (13.8 +/- 2.4 pg/ml), and increased for normal subjects (20.0 +/- 4.8 pg/ml). Decreased production of tumour necrosis factor alpha (TNF-alpha) was observed in allergic subjects (day 1: 447 pg/ml vs day 2: 258 pg/ml). Moreover, pre-challenge release of TNF-alpha and IL-1 beta levels from epithelial + BAL cells correlated well for allergic (r = 0.84) and normal subjects (r = 0.6), but post-challenge release correlated only in normal subjects (r = 0.90). These results indicate that bronchial epithelial cells and BAL cells interact, modulating release of these inflammatory cytokines.

1,1'-Ethylidenebis[L-tryptophan], an impurity in L-tryptophan associated with eosinophilia-myalgia syndrome, stimulates type I collagen gene expression in human fibroblasts in vitro.

Eosinophilia-myalgia syndrome (EMS), a recently described inflammatory disorder characterized by myalgia, peripheral eosinophilia, and multisystem inflammation is associated with L-tryptophan consumption. Fibrosis of various tissues due to excessive accumulation of type I collagen is a prominent late manifestation of the syndrome. 1,1'-Ethylidenebis[L-tryptophan] (EBT), an impurity distinct from L-tryptophan found in case-associated lots, has been implicated in function in vitro. Incubation of confluent fibroblasts with EBT, but not its hydrolysis product 1-methyl-tetrahydro-beta-carboline-3-carboxylic acid, caused a dose-dependent increase in collagen synthesis and in type I collagen mRNA levels independent of its effect on proliferation. In contrast, expression mRNA for fibronectin was not affected. These findings indicate that EBT stimulates type I collagen production by human fibroblast, and suggest that EBT may be involved in the development of fibrosis in EMS.

sVCAM-1 levels after segmental antigen challenge correlate with eosinophil influx, IL-4 and IL-5 production, and the late phase response.

Evidence from in vitro studies suggests a potential role for vascular cell adhesion molecule-1 (VCAM-1) in eosinophil trafficking. We hypothesized that induction of VCAM-1 occurs in the lung during IgE-mediated airway inflammation in humans. The technique of segmental antigen provocation followed by bronchoalveolar lavage (BAL) at 24 h was used to study 27 ragweed-allergic asthmatics (AA) and 18 atopic nonasthmatics (ANA). Total and differential cell counts were performed, and IL-4, IL-5, and soluble (VCAM) (sVCAM) levels in concentrated BAL fluid were measured by ELISA. A large increase in sVCAM levels after segmental challenge in both AA and ANA (1.79 +/- 0.31 to 139.39 +/- 68.58 ng/ml, p < 0.0005 and 2.85 +/- 0.80 to 98.25 +/- 77.35 ng/ml, p < 0.05, respectively) was observed. BAL IL-4 and IL-5 also increased after challenge (IL-4: 51.7 +/- 17.72 to 150.1 +/- 58.82 pg/ml, 0.05 < p < 0.10, n = 20 for AA, and 36.6 +/- 9.05 to 116.8 +/- 51.5 pg/ml, 0.05 < p < 0.10, n = 15 for ANA; IL-5: 0 to 2.67 +/- 1.62 ng/ml, p < 0.01, n = 16 for AA, and 0 to 2.87 +/- 2.16 ng/ml, 0.05 < p < 0.10, n = 10 for ANA). In both groups, the majority of the increase in sVCAM, IL-4, and IL-5 was accounted for by subjects who displayed a dual phase response after whole-lung antigen inhalation. This fact, plus the strong correlation observed between postchallenge sVCAM, IL-4, and IL-5 levels and eosinophil influx, suggests that VCAM, IL-4, and IL-5 play important roles in the recruitment of eosinophils to the lung of humans after antigen challenge.

Eosinophils recruited to the lung by segmental antigen challenge show a reduced chemotactic response to leukotriene B4.

Inflammatory cells, particularly eosinophils, are thought to be important in allergic airway disease. However, the mechanisms responsible for their recruitment to the lung have not been well established in humans, in vivo. In this report we demonstrate that eosinophils recruited to the lung of ragweed allergic volunteers by segmental antigen challenge have a decreased chemotactic response to leukotriene (LT) B4 ex vivo, in comparison with eosinophils isolated from peripheral blood (80% inhibition at 10(-8) M LTB4, the optimal concentration, p < 0.01). Their chemotactic response to formyl-methionyl-leucyl-phenylalanine (FMLP) was normal. Although the response to platelet activating factor also appeared to be reduced, this reduction was not statistically significant. These data suggest that lung eosinophils were exposed to LTB4 in vivo and are consistent with the hypothesis that LTB4 is important in the recruitment of eosinophils to the lung during IgE-mediated reactions in the lung in humans.