Studying human airway pharmacology in microsections: application of videomicrometry.

The influence of endogenously-released mediators and activated eosinophils on the airway lumen and the effect of passive sensitization on anti-immunoglobulin (Ig)-E-induced contractile responses was investigated by videomicrometry. Human bronchial sections of 2-3 mm internal diameter, placed in 250 microL Hank's balanced salt solution on microtitre plates, were monitored and recorded by digitized image analysis. Airway preparations exhibited a spontaneous narrowing (mean+/-SEM -33+/-5% of the luminal area). Removal of the bronchial epithelium almost completely prevented the development of spontaneuous narrowing (-6+/-3%; p<0.001). The addition of platelet-activating factor stimulated human eosinophils to the bronchial sections led to significant narrowing of the airway lumen (-39+/-9%; p<0.05). Passive sensitization induced hyperresponsiveness to polyclonal anti-IgE (-35+/-8%; p<0.01). It is concluded that videomicrometry is suitable for studying interactions between human airways and inflammatory cells, as well as the effect of passive sensitization on smooth muscle reactivity in vitro, without the imposition of preload. Under these conditions, human airways exhibited a spontaneous decrease of the airway lumen over time suggesting a role for epithelium-derived mediators because the development of spontaneous tone was epithelium dependent.

The effect of the vasoactive intestinal polypeptide agonist Ro 25-1553 on induced tone in isolated human airways and pulmonary artery.

Ro 25-1553 is a metabolically stable analogue of endogenous vasoactive intestinal polypeptide (VIP). This compound is a potent bronchodilator in vitro as well as in vivo. Moreover, Ro 25-1553 has been shown to be highly selective of the VPAC2 receptor. We assessed the effect of Ro 25-1553 on isolated human bronchi and pulmonary arteries in vitro. Macroscopically normal human airways and pulmonary arteries were obtained from patients undergoing surgery for lung cancer. The relaxing capability of Ro 25-1553 on bronchial and pulmonary artery tone was measured using standard techniques. Bronchial rings were pre-contracted with 0.1 mM histamine, and tone in pulmonary artery rings was induced with 10 microM PGF2alpha. Increasing concentrations of Ro 25-1553 within a range of 1 pM to 10 microM were added and isometric tension changes were recorded. Ro 25-1553 caused a concentration-dependent relaxation of airway and pulmonary artery preparations, with an EC50 of approximately 10 nM and a maximal relaxation of 70%-75% of the induced tone. The presence of VPAC2 receptors in the two tissues, though low in density, was confirmed by in situ hybridization, immunocytochemistry and ligand binding. These findings indicate that the VIP analogue Ro 25-1553 may be useful in the treatment of asthma and/or chronic obstructive pulmonary diseases.

Isolated airways from current smokers are hyper-responsive to histamine.

Epidemiological studies suggest that bronchial hyper-responsiveness (BHR) and elevated levels of serum IgE are more frequently found in current smokers than in ex-smokers. Since elevated serum IgE is associated with BHR under both in vivo and in vitro conditions, we aimed to assess whether smoking affects BHR independently from IgE. Lung resection material was obtained from 27 current smokers and 11 non-smokers with low serum IgE (< 100 U/mL). Peripheral airways were cut into rings and incubated overnight in the presence (passively sensitized) or absence (non-sensitized) of serum containing IgE levels above 250 U/mL. Isometric contractile responses to histamine were assessed in the organ bath. Compared with non-smokers, isolated airways from smokers showed significantly increased responses to histamine (P < 0.05, ANOVA). Passive sensitization enhanced responses in both groups by about the same amount (P < 0.05, both). In patients with low serum IgE current smoking is associated with increased bronchial responsiveness to histamine in vitro, which can be further enhanced by passive sensitization. These findings suggest that both smoking and serum IgE contribute to non-specific airway hyper-responsiveness.

Involvement of protein tyrosine kinases in activation of human eosinophils by platelet-activating factor.

Activation of human eosinophils by platelet-activating factor (PAF) involves multiple signal transduction pathways. Among these, protein kinase C has been demonstrated both to mediate respiratory burst and to suppress an alternative pathway of activation of respiratory burst and arachidonic acid metabolism in eosinophils. We utilized inhibitors of protein tyrosine kinases (PTK) to elucidate the role of PTK in PAF-induced activation of eosinophils. Eosinophils were isolated from peripheral blood of atopic donors and stimulated with PAF in the absence or presence of broad-spectrum PTK inhibitors-genistein or lavendustin A; an inhibitor of mitogen-activated protein (MAP) kinase activation-tyrphostin AG126; or an inhibitor of Janus kinase 2 (Jak2)-tyrphostin B42 (AG490). PAF induced superoxide anion (O2-*) generation, leukotriene C4 (LTC4) release, intracellular calcium ion mobilization and tyrosine phosphorylation of multiple eosinophil proteins in a concentration-dependent manner. All of these responses were concentration-dependently inhibited by genistein; lavendustin A also exhibited potent inhibition of PAF-induced LTC4 release. AG126 had no effect on either O2-* generation or LTC4 release, while AG490 inhibited both responses, albeit less effectively than genistein. We conclude that PAF activates PTK in human eosinophils and that this signalling pathway is involved in eliciting respiratory burst and leukotriene production. The specific PTK(s) involved are unknown but may include Jak2.

Up-regulation of human eosinophil leukotriene C4 generation through contact with bronchial epithelial cells.

OBJECTIVE AND DESIGN: We studied the effect of contact with bronchial epithelial cells on the functional activity of human eosinophils, measured as the production of a bronchoconstrictor lipid mediator, leukotriene C4 (LTC4) to determine the role of cell-cell interaction in activation of airway eosinophils. MATERIALS AND METHODS: Eosinophils were isolated from peripheral blood of atopic donors. Epithelial cells were obtained from the bronchi of surgically resected lung lobes and cultured to confluence on collagen-coated plates. Eosinophils were stimulated with platelet activating factor (PAF) or serum opsonized zymosan (SOZ) after incubation with or without epithelial cells. Leukotriene C4 (LTC4) was assayed in supernatants by enzyme immunoassay. RESULTS: Bronchial epithelial cells did not produce LTC4 in response to PAF or SOZ. Eosinophils pre-incubated in collagen-coated plates for 1 h produced LTC4 in response to both PAF (130 +/- 53 fmol/10(6) eosinophils at 10 micromol/l PAF, 5 min) and SOZ (1,900 +/- 550 fmol/10(6) eosinophils at 2 mg/ml SOZ, 15 min). Eosinophils co-incubated with bronchial epithelial cells for 1 h produced significantly higher quantities of LTC4 in response to both PAF (310 +/- 94 fmol/10(6) eosinophils; P<0.01) and SOZ (5,500 +/- 1,500 fmol/10(6) eosinophils; P<0.001). Ligation of the common beta2 integrin subunit (CD18) with a monoclonal antibody inhibited PAF-stimulated and augmented SOZ-stimulated LTC4 generation by eosinophils alone but had marginal effects on the epithelium-dependent up-regulation. CONCLUSIONS: Contact with bronchial epithelial cells up-regulates the responsiveness of human eosinophils, a finding that has significant implications in the pathology of asthma.

The effect of selective and non-selective phosphodiesterase inhibitors on allergen- and leukotriene C(4)-induced contractions in passively sensitized human airways.

Non-selective inhibitors of cyclic nucleotide phosphodiesterase (PDE) block allergen-induced contraction of passively sensitized human airways in vitro by a dual mechanism involving a direct relaxant effect on smooth muscle and inhibition of histamine and cysteinyl leukotriene (LT) release from airways. We investigated the effects of non-selective PDE inhibitors and selective inhibitors of PDE3 and PDE4 in order to determine the involvement of PDE isoenzymes in the suppression of allergic bronchoconstriction. Macroscopically normal airways from 76 patients were sensitized with IgE-rich sera (>250 u ml(-1)) containing specific antibodies against allergen (Dermatophagoides farinae). Contractile responses of bronchial rings were assessed using standard organ bath techniques. Passive sensitization caused increased contractile responses to allergen, histamine and LTC(4). Non-selective PDE inhibitors (theophylline, 3-isobutyl-1-methylxanthine [IBMX]), a PDE3-selective inhibitor (motapizone), PDE4-selective inhibitors (RP73401, rolipram, AWD 12-281) and a mixed PDE3/4 inhibitor (zardaverine) all significantly relaxed inherent bronchial tone at resting tension and to a similar degree. Theophylline, IBMX, zardaverine and the combination of motapizone and RP73401 inhibited the contractile responses to allergen and LTC(4). Pre-treatment with motapizone, RP73401, rolipram or the methylxanthine adenosine receptor antagonist, 8-phenyltheophylline, did not significantly decrease responses to either allergen or LTC(4). We conclude that combined inhibition of PDE3 and PDE4, but not selective inhibition of either isoenzyme or antagonism of adenosine receptors, is effective in suppressing allergen-induced contractions of passively sensitized human airways. The relationship between allergen- and LTC(4)-induced responses suggests that PDE inhibitors with PDE3 and PDE4 selectivity are likely to act in part through inhibition of mediator release and not simply through direct relaxant actions on airway smooth muscle.

Histamine hypersensitivity induced by passive sensitization of human bronchus: effect of serum IgE depletion.

BACKGROUND: Sensitivity to specific allergens and increased sensitivity to common spasmogens are characteristic features of allergic asthma and are also features demonstrated by tissues passively sensitized with serum from atopic donors, displaying high levels of IgE. It is evident that the specific response to allergen is related to circulating levels of allergen-specific IgE, but it is unclear whether the histamine hypersensitivity is also related to this immunoglobulin. OBJECTIVE: The objective was to deplete IgE in the serum of a donor with high levels of total and allergen-specific IgE and compare specific-allergen sensitivity and sensitivity to histamine in tissues passively sensitized with either the whole serum or the IgE-depleted serum. METHODS: Serum from a Dermatophagoides farinae-sensitive asthmatic (total IgE = 1047 U/mL, D. farinae-specific IgE > 17.5 U/mL) was subjected to an immunomagnetic separation technique to reduce the levels of IgE (total and specific) to below 10 U/mL. Bronchial tissue from six non-atopic donors was then passively sensitized overnight with either the whole serum or IgE-depleted serum and D. farinae and histamine sensitivity were evaluated the next day using standard organ bath techniques. RESULTS: Passive sensitization with the whole serum resulted in the development of sensitivity to D. farinae and increased sensitivity to histamine (750+/-169 mg contraction to 10 U/mL D. farinae, histamine pEC50 5.64+/-0.16 and max. 813+/-109 mg in sensitized vs 37+/-34 mg contraction to 10 U/mL D. farinae histamine pEC50 5.05+/-0.23 and max. 490+/-84 in non-sensitized tissues, P>0.05). Incubation with IgE-depleted serum still produced histamine hypersensitivity (histamine pEC50 5.57+/-0.16 and max. 737+/-70 mg P>0.05), but no significant response to allergen was detected (20+/-13 mg contraction to 10 U/mL D. farinae). CONCLUSION: These results demonstrate, that increased reactivity to histamine and airway contraction to allergen induced by passive sensitization, occur through independent mechanisms and that, unlike allergen-sensitivity, histamine hypersensitivity is caused by a serum factor other than IgE.

Protein kinase C inhibition enhances platelet-activating factor-induced eicosanoid production in human eosinophils.

Previous investigations have suggested that protein kinase C (PKC) may regulate guinea pig eosinophil responses through a suppressive "negative feedback" mechanism. Using the selective PKC inhibitors bisindolylmaleimide I (Bis I, GF 109203X) and calphostin C, we examined the role of PKC in platelet-activating factor (PAF)-induced respiratory burst and generation of arachidonic acid metabolites in human peripheral blood eosinophils. Bis I inhibited PAF-induced generation of superoxide anion with substantially lower potency (geometric mean IC50 = 1.41 microM, 95% CI 0.94-2.11 microM) than it exhibited against responses to the phorbol esters 4-beta-phorbol 12-myristate 13-acetate (PMA; IC50 = 0.25 microM, 0.09-0.72 microM; P < 0.01) and 4-beta-phorbol 12,13-dibutyrate (IC50 = 0.48 microM, 0.20-1.14 microM; P < 0.05). The production of thromboxane (measured as TxB2) induced by 1 microM PAF was increased significantly by Bis I at concentrations of 1 microM (162 +/- 7.5% of control PAF response; P < 0.01) and 10 microM (194 +/- 17%; P < 0.001); TxB2 release induced by PMA was unaffected by concentrations of Bis I up to 1 microM and inhibited by 10 microM Bis I (48 +/- 11%; P < 0.05). Bis I (1 microM) significantly increased both thromboxane and leukotriene C4 (LTC4) production induced by 2 microM (P < 0.01 and P < 0.05, respectively) or 20 microM PAF (both P < 0.001). The actions of Bis I on PAF-stimulated thromboxane and leukotriene production were mimicked by a second PKC inhibitor, calphostin C, whereas the non-PKC-inhibitory analog, bisindolylmaleimide V, caused no enhancement of TxB2 or LTC4 production. The increase in intracellular free calcium induced by 1 microM PAF was heightened and prolonged in cells pre-treated with 1 microM Bis I or 1 microM calphostin C (peak increase, P < 0.05 for both drugs; level 60 s after addition of PAF, P < 0.001 and P < 0.05 for Bis I and calphostin C, respectively; time to return to 50% of peak, P < 0.05 for Bis I). We conclude that PKC inhibition causes augmentation of thromboxane and LTC4 production in PAF-stimulated human eosinophils despite suppressing respiratory burst activity, indicating that different signaling pathways predominate in these two responses and that PKC mediates a suppression of an early stage in an alternative pathway of activation.

Role of IgE in hyperresponsiveness induced by passive sensitization of human airways.

Incubation of airways from nonatopic patients with serum from patients with high IgE levels confers responsiveness to "specific" (allergen) and hyperresponsiveness to "nonspecific" (histamine) stimuli. We have tested the hypothesis that the level of IgE determines the degree of specific and nonspecific responsiveness. Bronchial rings from nonatopic patients were sensitized overnight with serum containing high levels of allergen-specific IgE, or with an allergen-specific chimeric IgE (JW8) in physiologic buffer. In vitro responsiveness to allergen and histamine was evaluated and compared with non-sensitized tissues from the same patients. Responses to specific allergen were demonstrated in all tissues sensitized with atopic serum or chimeric IgE, but not in nonsensitized tissues. Allergen responses were specific, since tissues sensitized using serum containing high Dermatohagoides farinae-specific IgE only, did not respond to either horse or dog allergens. The potency and magnitude of the maximal contraction to histamine was significantly (p < 0.05) increased in tissues sensitized using atopic serum with high total IgE concentrations compared with nonsensitized preparations, but was unchanged in tissues sensitized using chimeric IgE or serum with low total IgE levels. Therefore, specific IgE determines allergen responsiveness in passively sensitized human airways, but histamine hyperresponsiveness is independent of specific IgE and appears to be related to some other factor associated with serum containing high concentrations of total IgE.

Conservation of bronchiolar wall area during constriction and dilation of human airways.

We assessed the effect of smooth muscle contraction and relaxation on airway lumen subtended by the internal perimeter (Ai) and total cross-sectional area (Ao) of human bronchial explants in the absence of the potential lung tethering forces of alveolar tissue to test the hypothesis that bronchoconstriction results in a comparable change of Ai and Ao. Luminal area (i.e., Ai) and Ao were measured by using computerized videomicrometry, and bronchial wall area was calculated accordingly. Images on videotape were captured; areas were outlined, and data were expressed as internal pixel number by using imaging software. Bronchial rings were dissected in 1.0- to 1.5-mm sections from macroscopically unaffected areas of lungs from patients undergoing resection for carcinoma, placed in microplate wells containing buffered saline, and allowed to equilibrate for 1 h. Baseline, Ao [5.21 +/- 0.354 (SE) mm2], and Ai (0.604 +/- 0.057 mm2) were measured before contraction of the airway smooth muscle (ASM) with carbachol. Mean Ai narrowed by 0.257 +/- 0.052 mm2 in response to 10 microM carbachol (P = 0.001 vs. baseline). Similarly, Ao narrowed by 0.272 +/- 0.110 mm2 in response to carbachol (P = 0.038 vs. baseline; P = 0.849 vs. change in Ai). Similar parallel changes in cross-sectional area for Ai and Ao were observed for relaxation of ASM from inherent tone of other bronchial rings in response to 10 microM isoproterenol. We demonstrate a unique characteristic of human ASM; i.e., both luminal and total cross-sectional area of human airways change similarly on contraction and relaxation in vitro, resulting in a conservation of bronchiolar wall area with bronchoconstriction and dilation.

Passive sensitization of human bronchi augments smooth muscle shortening velocity and capacity.

We assessed whether incubation with human serum from atopic individuals containing high concentrations of immunoglobulin E (IgE) causes augmentation of maximal contraction of human bronchial smooth muscle from non-atopic subjects in vitro. Bronchi were obtained from eight patients undergoing lung resection, and force-velocity relationships were determined for eight pairs of epithelium-intact bronchial rings of generations 6-7 using an electromagnetic lever system, which allowed isotonic shortening when load-clamps [from 0 to maximal isometric force (P0)] were applied at specific times after onset of contraction. Contractions were elicited by supramaximal electrical field stimulation (50 Hz, 10 s train duration, 25 V). Optimal length (Lo) for each tissue was determined during equilibration. After resection, tissues were sensitized passively with human sera containing high titers (> 1,000 U/ml) of IgE by incubation for 16 h at 20 degrees C. Maximal shortening velocity (Vmax) was increased for passively sensitized bronchi [0.1150 +/- 0.0240 1/2 circumferences/s (1/2Cir/s)] compared with sham-sensitized bronchi [0.0731 +/- 0.0152 1/2Cir/s, P = 0.038]. Similarly, maximal shortening (delta Lmax) was augmented in sensitized bronchial rings (11.27 +/- 1.80 %Lo) compared with sham-sensitized tissues (8.19 +/- 1.39 %Lo, P = 0.012). However, P0 did not differ between sensitized (122.5 +/- 24.4 mN/cm2) compared with sham-sensitized tissues (138.4 +/- 32.1 mN/cm2, P = 0.642). Our data are the first demonstration that Vmax and delta Lmax are augmented in sensitized but not challenged human bronchial rings after passive sensitization using human serum containing high concentrations of IgE.

A rapid and sensitive immunoassay for antibodies against alloantigens on human platelet glycoproteins (BIPA).

A rapid and sensitive enzyme immunoassay has been developed for the detection and specification of alloantibodies against human platelet antigens (HPA) on platelet membrane glycoproteins. Human platelet glycoprotein complexes were coupled by monoclonal antibodies to immunomagnetic beads with sheep anti-mouse IgG directed antibodies. These beads served as solid-phase target antigens for human alloantibodies in a modified MAIPA assay. This test provided high sensitivity, permitting the identification of antibodies against membrane receptors and required only 1 h of assay time. The specificity and sensitivity of this assay were evaluated in serum samples from polytransfused patients and compared with standard immunoassays employing antigen coated immunowells.