IL-15-deficient mice develop enhanced allergic responses to airway allergen exposure.

BACKGROUND: Interleukin-15 is a pleiotropic cytokine that is critical for the development and survival of multiple haematopoietic lineages. Mice lacking IL-15 have selective defects in populations of several pro-allergic immune cells including natural killer (NK) cells, NKT cells, and memory CD8+ T cells. We therefore hypothesized that IL-15-/- mice will have reduced inflammatory responses during the development of allergic airway disease (AAD). OBJECTIVE: To determine whether IL-15-/- mice have attenuated allergic responses in a mouse model of AAD. METHODS: C57BL/6 wild-type (WT) and IL-15-/- mice were sensitized and challenged with ovalbumin (OVA), and the development of AAD was ascertained by examining changes in airway inflammatory responses, Th2 responses, and lung histopathology. RESULTS: Here, we report that IL-15-/- mice developed enhanced allergic responses in an OVA-induced model of AAD. In the absence of IL-15, OVA-challenged mice exhibited enhanced bronchial eosinophilic inflammation, elevated IL-13 production, and severe lung histopathology in comparison with WT mice. In addition, increased numbers of CD4+ T and B cells in the spleens and bronchoalveolar lavage (BAL) were also observed. Examination of OVA-challenged IL-15Rα-/- animals revealed a similar phenotype resulting in enhanced airway eosinophilia compared to WT mice. Adoptive transfer of splenic CD8+ T cells from OVA-sensitized WT mice suppressed the enhancement of eosinophilia in IL-15-/- animals to levels observed in WT mice, but had no further effects. CONCLUSION AND CLINICAL RELEVANCE: These data demonstrate that mice with an endogenous IL-15 deficiency are susceptible to the development of severe, enhanced Th2-mediated AAD, which can be regulated by CD8+ T cells. Furthermore, the development of disease as well as allergen-specific Th2 responses occurs despite deficiencies in several IL-15-dependent cell types including NK, NKT, and γδ T cells, suggesting that these cells or their subsets are dispensable for the induction of AAD in IL-15-deficient mice.

Tolerance induced by chronic inhaled antigen in a murine asthma model is not mediated by endotoxin.

Ovalbumin (OVA)-sensitized wildtype (WT) and endotoxin-resistant (ER) mice developed similar degrees of airways eosinophilia and serum OVA-specific IgE levels after acute aerosolized OVA challenge. WT mice demonstrated methacholine hyperreactivity, whereas ER mice showed no change in responsiveness. With chronic aerosolized OVA challenge, both WT and ER mice developed local tolerance, with resolution of airway eosinophilia but persistence of anti-OVA IgE in serum. Thus, the development of local tolerance with chronic aerosol exposure to OVA is independent of any potential effects of endotoxin in the OVA aerosol solution.

Restrictive lung disease following treatment for malignant brain tumors: a potential late effect of craniospinal irradiation.

PURPOSE: To examine the effects of lomustine (CCNU), a commonly used nitrosourea, and craniospinal radiation therapy on the subsequent development of restrictive lung disease (RLD) following treatment for malignant brain tumors. PATIENTS AND METHODS: Pulmonary function testing with measurement of lung volume, spirometry, and diffusion capacity was performed in 28 patients who had received CCNU and/or radiation therapy as treatment for a malignant brain tumor. The median age at the time of treatment was 11.4 years (range, 3.9 to 36.7) and radiation therapy was completed 6 months to 11.6 years (median, 2.6 years) before testing. Patients were divided into four groups based on prior therapy. Group 1 received involved-field irradiation and a CCNU-containing chemotherapy regimen (n = 7); group 2, craniospinal irradiation with a boost to the primary tumor site and a CCNU-containing chemotherapy regimen (n = 6); group 3, craniospinal irradiation with a boost to the primary tumor site and a non-CCNU-containing chemotherapy regimen (n = 7); and group 4, craniospinal irradiation with a boost to the primary tumor site without chemotherapy (n = 8). RESULTS: Fourteen patients (50%) had findings consistent with RLD. One of seven patients (14.3%) who received CCNU without spinal irradiation had RLD, whereas 13 of 21 (61.9%) who received spinal irradiation with or without CCNU had RLD (P = .038), including four of eight patients treated with craniospinal irradiation alone. Logistic regression analysis showed that only spinal irradiation was a significant predictor for RLD. Patients who received spinal irradiation were 4.3 times more likely to have RLD than those who did not receive spinal irradiation. CONCLUSION: Spinal irradiation may be a risk factor for the development of RLD.

Corticosteroid modulation of Na(+)-K+ pump-mediated relaxation in maturing airway smooth muscle.

1. The ontogeny of the relaxant influence of the airway electrogenic Na(+)-K+ pump and its potential modulation by corticosteroids were examined in airway smooth muscle (ASM) segments isolated from newborn and adult rabbits. 2. Control and methylprednisolone-treated (MP) ASM segments were half-maximally contracted with methacholine in K(+)-free buffer and the ASM relaxant responses to Na(+)-K+ pump activation were subsequently evaluated. Relative to adult ASM, control newborn ASM showed significantly enhanced maximal relaxation (Rmax) to KCl (62.5 +/- 5.2% vs. 47.8 +/- 5.2%), but no difference in sensitivity (pC2 = -log concentration producing 50% Rmax: 2.18 +/- 0.12 vs. 2.29 +/- 0.09-log M). 3. Exposure of ASM segments to 500 microM methylprednisolone for 1 h potentiated the airway Na(+)-K+ pump activity. A more pronounced effect was obtained in newborn ASM, where both the Rmax and pC2 values were significantly enhanced. In mature ASM, only the Rmax response to KCl was increased in the presence of MP. 4. Collectively, these data demonstrate that: (i) the functional activity of the airway electrogenic Na(+)-K+ pump decreases with post-natal maturation in the rabbit: (ii) corticosteroid treatment potentiates Na(+)-K+ pump activity in rabbit ASM; and (iii) the latter effect of corticosteroids is enhanced in immature airways. 5. The above findings provide new evidence that the airway relaxant response to activation of the electrogenic Na(+)-K+ pump varies ontogenetically and that corticosteroids potentiate the Na(+)-K+ pump activity in an age-dependent manner.

Use of TAO without methylprednisolone in the treatment of severe asthma.

The antimicrobial agent troleandomycin (TAO) has been shown to be effective in reducing corticosteroid requirements in patients with corticosteroid-dependent asthma. To our knowledge, the efficacy of TAO without concomitant use of corticosteroids has never been documented. We report the case of a 12-year-old patient with corticosteroid-dependent asthma who has remained asymptomatic and without any evidence of pulmonary deterioration during treatment with TAO without concomitant use of corticosteroids.

Respiratory influence of peripheral chemoreceptor stimulation in maturing rabbits.

To evaluate whether the influence of peripheral chemoreceptor (PCR) stimulation is centrally modulated by hypoxia, the respiratory effects of sodium cyanide (NaCN) infusion were compared during room air and 10% O2 inhalation in 18 lightly anesthetized, tracheotomized rabbits of varying postnatal age (1-33 days). During normoxia, noncumulative infusions of NaCN (5-400 micrograms/kg body wt) produced dose-dependent ventilatory (VE) stimulation. Maximal VE stimulation (VEmax) and ventilatory sensitivity to NaCN [i.e., log dose producing 50% of VEmax (log ED50)] did not significantly vary with age, with the average VEmax and log ED50 values amounting to 238% above base line and 1.564 micrograms/kg, respectively. During hypoxia, after initial stimulation (average: 152%), VE progressively decreased and stabilized to 67% above the normoxic base-line level. In contrast to normoxia, subsequent NaCN administration during steady-state hypoxia produced dose-dependent VE depression, occasionally manifested by abrupt apnea. The NaCN effect during hypoxia was significantly related to age (P less than 0.05), as well as to the estimated change in CO2 production during hypoxia (P less than 0.01). Both the respiratory depressant effects of hypoxia alone and in combination with NaCN were abolished after denervation of the peripheral chemoreceptors. These findings demonstrate that while PCR stimulation during normoxia produces ventilatory stimulation, the influence of enhanced PCR input during hypoxia is centrally modulated to produce ventilatory depression.

Mechanisms of protein kinase C regulation of airway contractility.

To elucidate the role of protein kinase C (PK-C) in regulating airway contractility, the effects of PK-C activation with phorbol esters, 12-deoxyphorbol 13-isobutyrate (DPB), and phorbol 12-myristate 13-acetate (PMA), and with the diacylglycerol analogue 1-oleoyl-2-acetate-rac-glycerol (OAG) were separately evaluated in isolated rabbit tracheal smooth muscle (TSM) segments. The latter agents produced dual and opposing contractile effects, with DPB being the most potent. Lower doses of DPB (less than or equal to 10(-6) M) elicited significant increases in isometric tension in both untreated TSM, as well as in TSM half-maximally precontracted with methacholine. These potentiated TSM contractions were inhibited by the Ca2+ channel blockers, nifedipine (10(-4) M) and diltiazem (10(-5) M). In contrast, higher doses of DPB (greater than or equal to 10(-6) M) induced airway relaxation, which was ablated by preinhibition of the electrogenic Na+-K+ pump with ouabain (5 x 10(-6) M) or K+-free buffer. Indeed, in separate experiments DPB (10(-7) M) was found to significantly potentiate the functional activity of the Na+-K+ pump, an effect occurring independent of inhibition of Na+-H+ exchange with amiloride (10(-4) M) or extracellular Ca2+ influx with nifedipine (10(-4) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Expiratory volume clamping: a new method to assess respiratory mechanics in sedated infants.

During breathing under sedation via a two-way valve, airflow (V), volume (delta V), and airway pressure (P) were recorded in eight normal (N) infants, seven with reversible obstructive airway disease (ROAD), and seven with chronic lung disease (CLD). Intermittently, expiratory volume clamping (EVC) was applied, involving selective occlusion of the expiratory valve for three to five breaths. The latter produced cumulative increases in delta V that, due to progressive recruitment of the Hering-Breuer reflex, were accompanied by increasing expiratory plateaus in P (i.e., apneas). The resultant passive inflation delta V-P relationships were closely approximated by the expression: delta V = aP2 + bP + c, wherein a represented the pressure-related changes in chord compliance (Crs), b the Crs at P = 0, and c the difference between the dynamic end-expiratory and relaxation volumes of the respiratory system. Relative to N, the ROAD and CLD infants had significantly reduced weight-specific values of a/kg, their b/kg values were increased, whereas the c/kg measurements did not significantly vary. Moreover, for each subject we determined the net Crs/kg obtaining at P = 20 cmH2O (i.e., Crs20/kg), an estimate of the net deflation compliance; the passive respiratory time constant (tau rs) based on the slope of the expired delta V/V relationship; and the respiratory system conductance (Grs/kg). Relative to N, the mean Crs20/kg was significantly reduced only in the infants with CLD and, due to increases in tau rs, both patient groups depicted significantly diminished values of Grs/kg, suggesting the presence of airways obstruction.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of protein kinase C potentiation of airway beta-adrenergic relaxation.

To evaluate the regulatory action of protein kinase C (PKC) on airway beta-adrenergic function, the relaxant effects of isoproterenol (ISO) and 8 bromo-cyclic AMP (BrcAMP) were examined in tracheal smooth muscle (TSM) segments half-maximally contracted with acetylcholine in the absence (control) and presence of PKC activation with the phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB). Relative to control tissues, TSM treated with 0.1 microM DPB depicted significantly enhanced maximal relaxation and sensitivity to ISO but not to BrcAMP. The enhancing effect of DPB on ISO responsiveness was completely inhibited in the presence of the PKC antagonist H-7. Inhibition of the Na(+)-K+ pump with either ouabain or K(+)-free buffer diminished the TSM relaxant response to ISO but not to BrcAMP. Inhibition of the Na(+)-K+ pump also ablated the DPB-induced potentiation of beta-adrenoceptor responsiveness. Collectively, these data demonstrate that: 1) PKC activation enhances TSM relaxant responsiveness to beta-adrenoceptor stimulation; 2) inhibition of the airway Na(+)-K+ pump markedly blunts the relaxant response to beta-adrenoceptor stimulation; and 3) inhibition of the Na(+)-K+ pump abolishes the above potentiating effect of DPB on beta-adrenoceptor-mediated relaxation of rabbit TSM. Thus, the above findings provide new evidence that PKC activation enhances the airway relaxant response to beta-adrenoceptor stimulation, and that the latter effect is dependent on potentiated stimulation of the airway electrogenic Na(+)-K+ pump.

Methylprednisolone and isoproterenol inhibit airway smooth muscle proliferation by separate and additive mechanisms.

To determine whether glucocorticoids and beta-adrenoceptor agonists act independently to inhibit airway smooth muscle (ASM) proliferation, the present study investigated the effects of methylprednisolone (MP) and isoproterenol (ISO) alone and in combination on leukotriene D4-induced ASM proliferation. MP and ISO had no effect on unstimulated ASM cell growth. In contrast, MP and ISO demonstrated dose-dependent inhibition of LTD4-induced proliferation, and the inhibitory effect was additive for combinations of MP and ISO. The competitive cAMP receptor antagonist, Rp-cAMPS, ablated the ISO-induced inhibition but had no affect on the inhibitory response to MP. In cells exposed to both ISO and MP, Rp-cAMPS attenuated the growth inhibition to levels achieved by MP alone. Accordingly, these findings demonstrate that glucocorticoids and beta-adrenergic agonists inhibit LTD4-induced ASM proliferation, and that their inhibitory effects are mediated by different signaling pathways.

Predictive value of pulmonary function testing during pulmonary exacerbations in cystic fibrosis.

The optimal duration of therapy for acute exacerbations of cystic fibrosis (CF) has not been defined, and the utility of serial pulmonary function testing in predicting the duration of therapy has yet to be established. In a review of 90 pulmonary exacerbations of 39 patients with CF requiring hospitalization, we found that 72% of the patients recovered following 2 weeks of intravenous antibiotics and aggressive chest physiotherapy, and that 28% required an extended third week of therapy. Recovery was delayed in patients with more severe chronic pulmonary disease, but the rate of improvement was independent of the degree of pulmonary deterioration with the acute exacerbation. A 40% recovery of FEV1 at 1 week was found to correlate significantly with the duration of hospitalization in the 90 patients. When prospectively applied to a second series of consecutively hospitalized patients with CF, 25/28 patients admitted for 2 weeks demonstrated > 40% improvement in FEV1 at 1 week, as compared to 5/10 patients subsequently treated for > or = 3 weeks (P = 0.030). The predictive values for 2- or 3-week hospitalizations with 1-week interval recovery of > 40% or < or = 40% in FEV1 were 79% and 62%, respectively. These findings suggest that the response to intensive therapy in CF exacerbations is variable and that improvements in pulmonary function after 1 week of therapy may be used to predict the subsequent duration of therapy in the majority of CF patients with pulmonary exacerbations.

Ontogeny of beta-adrenergic desensitization in rabbit tracheal smooth muscle.

Prolonged or repeated exposure to beta-agonist medications may result in a desensitization of the agonist-mediated response. Under certain conditions, such agonist-induced desensitization may limit the efficacy of administered beta-adrenergic agonists to elicit bronchodilation. Accordingly, the present study was designed to study the mechanism of acute beta-adrenergic desensitization in maturing rabbit tracheal smooth muscle (TSM). Isometric tension was measured in tracheal ring segments isolated from newborn and mature rabbits and half-maximally contracted with Methacholine (Meth) or KCl. TSM segments were serially relaxed with repetitive single doses of isoproterenol (ISO: 0.1, 1.0, 10, or 100 microM) or prostaglandin E2 (PGE2: 0.1 or 10 microM). Serial administration of ISO-elicited dose-dependent desensitization of relaxation in mature and newborn TSM, contracted with either Meth or KCl. In contrast, the relaxant response to PGE2 was retained in the ISO-desensitized tissue. Repeated administration of PGE2 elicited no desensitization of PGE2 responsiveness, but did induce some dose-dependent desensitization of the ISO response in mature TSM. Compared to mature tissues, newborn TSM developed subtotal desensitization to 100 microM ISO and no ISO desensitization in response to PGE2. Thus, these findings demonstrate that (1) beta-adrenoceptor responsiveness undergoes dose-dependent homologous and, to a lesser extent, heterologous desensitization in rabbit TSM; and (2) both beta-adrenergic desensitization mechanisms increase with postnatal maturation.

Regulatory B cells from hilar lymph nodes of tolerant mice in a murine model of allergic airway disease are CD5+, express TGF-ß, and co-localize with CD4+Foxp3+ T cells.

In a biphasic, ovalbumin (OVA)-induced murine asthma model where allergic airway disease is followed by resolution and the development of local inhalational tolerance (LIT), transforming growth factor (TGF)-ß-expressing CD5(+) B cells were selectively expanded locally in hilar lymph nodes (HLN) of LIT mice. LIT HLN CD5(+) B cells, but not LIT HLN CD5(-) B cells, induced expression of Foxp3 in CD4(+)CD25(-) T cells in vitro. These CD5(+) regulatory B cells (Breg) and CD4(+)Foxp3(+) T cells demonstrated similar increases in expression of chemokine receptors (CXCR4 and CXCR5) and co-localized in HLN B cell zones of LIT mice. The adoptive transfer of LIT HLN CD5(+) B cells, but not LIT HLN CD5(-) B cells, increased the number of CD4(+)Foxp3(+) T cells in the lung and inhibited airway eosinophilia in this OVA model. Thus, Breg in HLNs of LIT mice reside in a CD5(+) TGF-ß-producing subpopulation and co-localize with CD4(+)Foxp3(+) T cells.

Current concepts of respiratory complications of neuromuscular disease in children.

Chronic neuromuscular diseases affect the respiratory muscles in varying patterns and degrees. As a result, patients with these disorders develop restrictive pulmonary disease, ineffective cough, atelectasis and pneumonia, and chronic respiratory insufficiency leading to respiratory failure. Therapeutic strategies are evolving to augment cough and airway clearance, improve lung volumes, and support the patient with progressive ventilatory failure. These techniques have improved longevity and quality of life for many patients with neuromuscular disease.

beta-adrenergic relaxation of rabbit tracheal smooth muscle: a receptor deficit that improves with corticosteroid administration.

beta-Adrenergic agonists are potent relaxing agents of airway smooth muscle; however, they are often incapable of fully reversing agonist-mediated contractions. The present study was designed to quantitate the relationship between beta-adrenoceptor binding, signal transduction, and relaxation in rabbit tracheal smooth muscle (TSM). TSM segments contracted with acetylcholine to 25 to 75% maximal contraction were relaxed with cumulative administration of isoproterenol (ISO). A beta-adrenergic receptor "deficit" was found, such that incomplete relaxation was achieved with full receptor occupancy. Binding studies with [(3)H]dihydroalprenolol demonstrated a beta-adrenoceptor density of 33.1 +/- 8.6 fmol/mg protein in control TSM. Paired studies were performed in TSM from rabbits treated with dexamethasone. Relative to control tissues, dexamethasone-treated TSM displayed twice as much relaxation and cAMP production in response to ISO and twice the beta-adrenoceptor density (82.2 +/- 12.3 fmol/mg protein). Dexamethasone did not affect G(i) function, as assessed by the degree of functional antagonism exerted by acetylcholine on ISO-induced relaxations, or beta-adrenoceptor-G(s) coupling, as reflected in high-affinity beta-agonist binding. Collectively, these results demonstrate that corticosteroid administration exerts parallel potentiating effects on beta-adrenoceptor expression and function in rabbit airway smooth muscle.

Maturation of catecholamine response and extraneuronal uptake in rabbit trachea.

The present study investigated whether maturational changes in extraneuronal catecholamine clearance accounted for the ontogenetic attenuation of isoproterenol responsiveness in rabbit tracheal segments. Following half-maximal contraction with acetylcholine (ACh) or KCl, tracheal ring segments (TS) isolated from newborn, 1-mo, and adult rabbits were relaxed with cumulative administration of isoproterenol. With postnatal maturation, there occurred a significant decrease in both maximal relaxation and sensitivity to isoproterenol in both ACh- and KCl-contracted TS. Inhibition of extraneuronal catecholamine uptake with methylprednisolone resulted in enhanced sensitivity to isoproterenol in 1-mo and adult, but not in newborn tracheal segments. In separate studies, extraneuronal catecholamine uptake was directly assayed in tracheal segments isolated from rabbits of similar age and pretreated with neuronal uptake and monoamine oxidase inhibitors. Maximal extraneuronal uptake of [3H]norepinephrine significantly decreased with age in tracheal tissues. In contrast, the sensitivity of the uptake process to norepinephrine increased with age. The relative catecholamine reserve markedly decreased with age in rabbit tracheal segments and was directly related to the maximal degree of relaxation elicited with the beta-adrenergic catecholamine, isoproterenol, in age-matched TS precontracted with either ACh or KCl. These findings suggest that, with maturation, extraneuronal uptake more effectively competes with beta-adrenoreceptor stimulation and, accordingly, may contribute to the ontogenetic attenuation of beta-adrenergic responsiveness in rabbit tracheal tissues.

Dexamethasone potentiates high-affinity beta-agonist binding and g(s)alpha protein expression in airway smooth muscle.

Corticosteroids enhance beta-adrenergic responses by actions at both beta-adrenoceptor (beta-AR) and post-beta-AR sites. The present study investigated the effects of dexamethasone on beta-AR density, high-affinity beta-agonist binding, G(s)alpha and G(i)alpha protein expression, and cAMP responses in bovine tracheal smooth muscle (bTSM). Dexamethasone treatment of cultured bTSM cells increased total beta-AR density 1.6- to 1.9-fold as assessed by the saturation binding of [(3)H]CGP-12177 and by displacement of radioligand binding with isoproterenol. Isoproterenol bound to the beta-AR at two sites, a high-affinity site with a density of 5.9 +/- 1.2 fmol/mg protein and a low-affinity site with a density of 16.9 +/- 1. 0 fmol/mg protein. Dexamethasone increased both high- and low-affinity isoproterenol binding sites to 11.1 +/- 2.2 and 25.9 +/- 2.1 fmol/mg protein, respectively, without influencing agonist binding affinities. Dexamethasone also selectively increased G(s)alpha protein levels from 0.99 +/- 0.14 to 1.46 +/- 0.17 microg/mg protein without affecting G(i)alpha levels. The net effect of these changes was a 1.8-fold increase in maximal isoproterenol-induced cAMP generation in dexamethasone-treated bTSM cells. These findings provide new insights into the corticosteroid regulation of beta-adrenergic signaling pathways in airway smooth muscle.

Assessment of signal transduction mechanisms regulating airway smooth muscle contractility.

Agonist-receptor interactions regulate airway smooth muscle tone through activation of guanine nucleotide binding proteins (G proteins) which are coupled to second-messenger pathways that mediate changes in the tissue's contractile state. Various methods have been applied to identify the structure/function characteristics of G proteins and their role in signal transduction in airway smooth muscle, including the use of exotoxins, nonhydrolyzable analogs of guanosine-triphosphate (GTP), antibodies to purified G proteins, and membrane reconstitution studies. In elucidating mechanisms of airway smooth muscle relaxation, considerable progress has been made in identifying the molecular basis for receptor/G protein coupling and other regulatory processes leading to both the activation and down-regulation of the adenylate cyclase/adenosine 3',5'-cyclic monophosphate system. Further, with respect to airway smooth muscle contraction, various approaches have been used to evaluate the role of membrane phosphoinositide turnover and the mechanisms of action of the bifurcating signal transduction pathways associated with the production and metabolism of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, and activation of protein kinase C. This review identifies much of the information gained to date on the above signal transduction pathways, with an emphasis placed on various methodological approaches used to determine membrane and transmembrane signaling processes in airway smooth muscle.