Role of leukotrienes in bronchial hyperresponsiveness and cellular responses in airways.
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Animals↗
Biomedical subjects
A R Leff
Publications and source records attributed to A R Leff.
Cytosolic phospholipase A2 activation is essential for beta 1 and beta 2 integrin-dependent adhesion of human eosinophils.
We examined the role of cytosolic phospholipase A2 (cPLA2) during human eosinophil adherence to ICAM-1- or VCAM-1-coated plates. IL-5-stimulated eosinophils adhered to ICAM-1 through the beta 2 integrin CD11b/CD18, while nonstimulated eosinophils did not. By contrast, nonstimulated eosinophils adhered to VCAM-1 through the beta 1-integrin VLA-4/CD29. Both IL-5-induced adhesion to ICAM-1 and spontaneous adhesion to VCAM-1 corresponded temporally to cPLA2 phosphorylation, which accompanied enhanced catalytic activity of cPLA2. The structurally unrelated cPLA2 inhibitors, arachidonyl trifluoromethylketone and surfactin, significantly inhibited eosinophil adhesion to ICAM-1 and VCAM-1 in a concentration-dependent manner. Inhibition of secretory PLA2, 5-lipoxygenase, or cyclooxygenase did not affect eosinophil adhesion. Addition of arachidonic acid to eosinophils after cPLA2 inhibition with arachidonyl trifluoromethylketone or surfactin did not reverse the blockade of adhesion to ICAM-1 or VCAM-1. However, CV-6209, a receptor-specific antagonist of platelet-activating factor, inhibited all integrin-mediated adhesion. The activated conformation of CD11b as identified by the mAb, CBRM1/5, as well as quantitative surface CD11b expression were up-regulated after IL-5 stimulation. However, cPLA2 inhibition neither prevented CBRM1/5 expression nor blocked surface Mac-1 up-regulation caused by IL-5. Our data suggest that cPLA2 activation and its catalytic product platelet-activating factor play an essential role in regulating beta 1 and beta 2 integrin-dependent adhesion of eosinophils. This blockade occurs even in the presence of up-regulated eosinophil surface integrin.
Arachidonic Acids↗
Roles of Trp31 in high membrane binding and proinflammatory activity of human group V phospholipase A2.
Group V phospholipase A2 is a recently discovered secretory phospholipase A2 (PLA2) that has been shown to be involved in eicosanoid formation in inflammatory cells, such as macrophages and mast cells. We have demonstrated that human group V PLA2 (hsPLA2-V) can bind phosphatidylcholine (PC) membranes and hydrolyze PC substrates much more efficiently than human group IIa PLA2, which makes it better suited for acting on the outer plasma membrane (Han, S.-K., Yoon, E. T., and Cho, W. (1998) Biochem. J. 331, 353-357). In this study, we demonstrate that exogenous hsPLA2-V has much greater activity than does group IIa PLA2 to release fatty acids from various mammalian cells and to elicit leukotriene B4 formation from human neutrophils. To understand the molecular basis of these activities, we mutated two surface tryptophans of hsPLA2-V to alanine (W31A and W79A) and measured the effects of these mutations on the kinetic activity toward various substrates, on the binding affinity for vesicles and phospholipid-coated beads, on the penetration into phospholipid monolayers, and on the activity to release fatty acids and elicit eicosanoid formation from various mammalian cells. These studies show that the relatively high ability of hsPLA2-V to induce cellular eicosanoid formation derives from its high affinity for PC membranes and that Trp31 on its putative interfacial binding surface plays an important role in its binding to PC vesicles and to the outer plasma membrane.
Cell Membrane↗
Augmentation of eosinophil degranulation and LTC(4) secretion by integrin-mediated endothelial cell adhesion.
We examined the effect of eosinophil ligation to cultured human umbilical vein endothelial cells (HUVECs) in augmenting the stimulated secretion of leukotriene (LT) C(4) and eosinophil peroxidase (EPO). The effects of adhesion were compared before and after specific blockade with monoclonal antibodies directed against eosinophil surface integrins or endothelial counterligands. Adhesion to HUVECs augmented EPO release caused by formyl-methionyl-leucyl-phenylalanine plus cytochalasin B from 403 +/- 15.3 (BSA control) to 778 +/- 225 ng/10(6) cells for eosinophils exposed to interleukin-1alpha-treated HUVECs (P < 0.05) and also caused a twofold increase in stimulated LTC(4) secretion (P < 0.05). To determine whether augmented secretion resulted directly from adhesive ligation, studies were also performed with paraformaldehyde-treated HUVECs; stimulated secretion of LTC(4) from eosinophils was comparable to that for living HUVECs. Our study is the first demonstration that adhesion to HUVECs through ligation to alpha(4)- or beta(2)-integrin on the eosinophil surface causes augmentation of stimulated secretion of both EPO and LTC(4) and that blockade of adhesion molecules on either eosinophils or HUVECs prevents the priming effect on eosinophil secretion.
Antibodies, Monoclonal↗
Inhibition of cPLA2 translocation and leukotriene C4 secretion by fluticasone propionate in exogenously activated human eosinophils.
We examined the effect of the highly lipophilic corticosteroid, fluticasone propionate (FP), in causing (1) inhibition of nuclear translocation of cytosolic phospholipase A2 (cPLA2), and (2) blockade of leukotriene C4 (LTC4) synthesis in isolated human eosinophils in vitro. Eosinophils were isolated from peripheral blood, treated with either buffer or 10(-)10 M to 10(-)6 M FP in the presence of 10 pg/ml human recombinant interleukin-5 (rhIL-5) and activated with formyl-met-leu-phe (FMLP) + cytochalasin B (CB). At 24 h, stimulated LTC4 secretion from eosinophils was unchanged; however, when corrected for cell viability, LTC4 secretion decreased from 1,429 +/- 327 pg/10(6) cells to 762 +/- 113 pg/10(6) cells for eosinophils treated for 48 h with >/= 10(-)8 M FP (p < 0.003). FMLP/CB-stimulated translocation of cPLA2 to the nuclear envelope assessed by specific immunohistochemical staining also was blocked by FP. By contrast, membrane expression of annexin-1, which was not minimal at 30 min, was substantial at 48 h for eosinophils treated with > 10(-)10 M FP, and inhibition of LTC4 synthesis was reversed by exogenous arachidonic acid (AA). We find that FP causes a decrease in stimulated eosinophil secretion of LTC4 that is regulated by phospholipase A2 (PLA2). Inhibition of LTC4 synthesis precedes the global cytotoxic effects of FP as indicated by the simultaneous upregulation of annexin-1 expression. Inhibited stimulated secretion corresponds to inhibited translocation of cPLA2 to the nuclear envelope during cellular activation.
Administration, Topical↗
Relationship of cellular transmigration and airway response after allergen challenge.
We examined the relationship between eosinophil migration into the bronchoalveolar space and change in FEV(1) after endobronchial allergen challenge (EBAC) in atopic asthmatic (AA) and atopic nonasthmatic (ANA) subjects. The purpose of this study was to obtain continuous, intrasubject controlled assessment of the relationship between cell migration in control and allergen-challenged segments in the same individuals over 96 h. In AA subjects, the eosinophil (Eos) count in the bronchoalveolar lavage fluid (BALF) increased from a baseline of 7,896 +/- 3,865 to 416,476 +/- 231,012 Eos/ml by 72 h (p = 0.001) in the challenged segment post-EBAC. For ANA subjects, the postsegmental challenge count was 29,874 +/- 474 Eos/ml (p = 0.03 versus baseline and p < 0.05 AA peak versus ANA peak). In both groups, there was a comparable decrease in peripheral blood eosinophil count beginning 5 h after challenge, which resolved at 24 h. In AA subjects, 416,476 +/- 231,012 Eos/ml was obtained from the allergen-challenged segment and 23,522 +/- 8,298 Eos/ml was obtained from the sham-challenged segment (p < 0.001) at 72 h. In contrast, there was no difference in the Eos count obtained from the BALF between the antigen- and sham-challenged segments of ANA subjects. We also found that increased airway neutrophils were present in equal numbers in allergen-challenged and sham-challenged segments in both AA and ANA subjects. We conclude that augmented eosinophil migration after EBAC is a characteristic of atopic asthma and is not present in atopic subjects who do not have asthma. We find that BAL eosinophilia in ANA patients as well as neutrophilia in both ANA and AA subjects are nonspecific consequences of bronchoscopy. Finally, we find no relationship between specific airway eosinophil migration into the BALF and FEV(1) < 72 h after challenge; however, at 96 h, there is a substantial decrease in FEV(1) that accompanies BALF eosinophilia.
Allergens↗
Intracellular expression of Fc gamma RIII (CD16) and its mobilization by chemoattractants in human eosinophils.
We characterized the existence, translocation, and reabsorption during cellular activation of a constitutively expressed intracellular CD16 in the human eosinophil. By two-color flow cytometry, we showed that 6.5+/-0.3% of nonpurified eosinophils expressed surface CD16. After digestion with phosphatidylinositol-specific phospholipase C, surface CD16 on both neutrophils and eosinophils decreased substantially, suggesting that eosinophil CD16 is a glycosyl-phosphatidylinositol-linked isoform. However, CD16 was substantially expressed intracellularly in human eosinophils. Epitope-specific binding to CLB-gran11 mAb from non-NA2/NA2 donors demonstrated that intracellular eosinophil CD16 also differed from the transmembrane isoform of CD16 expressed on NK cells or macrophages. Western blot analysis performed with 3G8 or DJ130c mAb showed a broad band at approximately 65 to 80 kDa, which was the same as neutrophil CD16 from the same NA2/NA2 donors. Upon stimulation by chemoattractants C5a, FMLP, or platelet-activating-factor, eosinophilic intracellular CD16 was rapidly translocated to the eosinophil surface, expressed maximally at 30 s, and then gradually disappeared from the cell surface during the next 10 min. Intracellular flow cytometry of stimulated eosinophils and sandwich ELISA of stimulated eosinophil supernatants demonstrated that the disappearance was due to its rapid release into medium and reabsorption by the cells. Our data identify a CD16B that is consistently expressed intracellularly but only rarely on the surface of nonactivated human eosinophils. This CD16 is transiently expressed during stimulation by chemoattractants.
Blotting, Western↗
Cyclic nucleotide phosphodiesterase in human bronchial epithelial cells: characterization of isoenzymes and functional effects of PDE inhibitors.
Cyclic AMP (adenosine 3':5'-cyclic monophosphate, cAMP) is an intracellular second messenger that mediates the actions of endogenous hormones and neurotransmitters and also of drugs such as beta-adrenoceptor agonists. The presence of functional beta-adrenoceptors on human airway epithelial cells has been demonstrated but the expression of the cAMP-metabolizing enzyme, cyclic nucleotide phosphodiesterase (PDE) in these cells has not been studied. We investigated the profile of activity of the different PDE isoenzymes in lysates of a pulmonary epithelial cell line, A549, and of human bronchial epithelial (HBE) cells grown in primary culture. The effects of non-selective and isoenzyme-selective PDE inhibitors on beta-agonist-induced elevations in intracellular cAMP concentrations and the production of interleukin (IL) 8 and prostaglandin (PG) E2 was also investigated. A549 cells expressed a high level of PDE4, lower levels of PDE1 and PDE3, and minor PDE5 activity. Primary HBE cultures expressed PDE4 and PDE1 activity at approximately equal levels with small additional PDE3 and PDE5 activities. The total PDE activity of the HBE cells was approximately nine-fold lower than that of A549 cells. The beta-adrenoceptor agonist salbutamol, caused a slow, concentration-dependent increase in intracellular cAMP levels in HBE cells which was not affected by a non-selective PDE inhibitor, IBMX (100 microM), or by a selective PDE4 inhibitor, rolipram (100 microM). Zardaverine, a dual-selective PDE3/PDE4 inhibitor, had no effect on cAMP levels at 10 microM but did cause a significant enhancement of salbutamol-induced elevations at 100 microM (150+/-36 pmol/10(5) cells at 10 microM salbutamol vs. 64+/-25 pmol/10(5) cells in the absence of zardaverine; n=3,P<0.01). Neither basal nor tumour necrosis factor alpha (10 ng/ml)-induced IL8 secretion was affected by salbutamol (10 microM) in the absence or presence of IBMX (100 microM). Salbutamol (10 microM), alone or in the presence of IBMX (100 microM) or rolipram (100 microM), also failed to affect basal or bradykinin (1 microM)-induced PGE2 release. Zardaverine (100 microM) caused a significant increase in basal PGE2 release but this was not enhanced in the presence of salbutamol (10 microM) and was not related to changes in cAMP levels. We conclude that HBE cells express a low total PDE activity, made up predominantly of PDE1 and PDE4 isoenzymes, and that intracellular cAMP levels in HBE cells are not related to the production of IL8 or PGE2.
Arachidonic Acid↗
Pharmacologic management of asthma.
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Adrenergic beta-Agonists↗
Differential effects of cyclosporine A after acute antigen challenge in sensitized cats in vivo and ex vivo.
1. We determined the effect of cyclosporine A (CsA) treatment on mast cell degranulation and lung resistance (R(L)) in vivo, and tracheal smooth muscle (TSM) contraction ex vivo after antigen challenge in sensitized cats. We also determined the direct effects of addition of CsA to the tissue bath on antigen-induced responses of TSM in vitro. 2. Cats (n=10) were sensitized by i.m. injection of Ascaris suum antigen (AA); 5 cats (CsA+) received CsA twice daily for 2 weeks before acute antigen challenge in doses sufficient to suppress interleukin-2 secretion from feline peripheral blood mononuclear cells ex vivo. 3. Lung resistance increased comparably within 10 min of exposure to AA (P<0.03). Histamine content in bronchoalveolar lavage fluid from both groups increased comparably within 30 min of antigen challenge, from undetectable levels to 542+/-74 pg ml(-1) post AA for CsA+ and from 74+/-19 pg ml(-1) at baseline, to 970+/-180 pg ml(-1) post AA CsA- (P<0.05; P=NS vs CsA+). 4. In excised TSM, active tension elicited by exposure to AA in vitro was 107+/-38% KCl in the CsA+ group vs 144+/-56% KCl in the CsA- group (P=NS). However, contraction of TSM (n=4) harvested from both groups was abolished or greatly diminished after AA challenge when tissues were pre-incubated with 1 microM CsA in vitro (8+/-8% KCl, P<0.05 vs CsA+ and CsA-). This was associated with inhibited release of 5-hydroxytryptamine into the organ bath fluid of tissues treated with CsA in vitro only. 5. We demonstrated that CsA treatment in vivo does not inhibit the early phase asthmatic response or mast cell degranulation following antigen challenge in sensitized cats. Additionally, the effects of CsA on mast cell function ex vivo do not reflect lack of effects of CsA on mast cell function in vivo in this animal model of atopic asthma.
Animals↗
Biology of airway smooth muscle. Conference summary.
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Animals↗
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.
Arachidonic Acid↗
Effect of enantiomeric forms of albuterol on stimulated secretion of granular protein from human eosinophils.
We studied the effect of R-, S- and R,S-albuterol in inhibiting the eosinophil peroxidase (EPO) secretion caused by 10(-10) to 10(-6) M formyl-met-leu-phe + 5 micrograms/ml cytochalasin B (FMLP/CB) in non-allergic and allergic subjects. Total RAST score obtained for allergic subjects was 4.12 +/- 0.21 vs 0.36 +/- 0.17 for non-allergic subjects (P < 0.0001). Stimulated EPO secretion was comparable in allergic [2,051 +/- 567 ng/10(6) eosinophils (eos)] and non-allergic subjects [2,337 +/- 488 ng/10(6) eos (P = NS)]. At all concentrations used, both R- and R,S-enantiomers caused comparable (27-32%) inhibition of FMLP/CB stimulated secretion of EPO in allergic and non-allergic subjects. Pretreatment with S-albuterol caused no augmentation of EPO secretion in either allergic (115 +/- 34.6%) or non-allergic subjects (114 +/- 23.7%) subjects, and there was no significant difference in secretion caused by FMLP/CB alone in either experimental group. Similar results were obtained for subjects stratified according to serum IgE concentration. Our data demonstrate that both R- and R,S-albuterol are equivalently effective in inhibiting stimulated secretion of EPO in both normal and allergic subjects and that there is no paradoxical augmenting effect of S-albuterol in stimulated eosinophil secretion.
Adrenergic beta-Agonists↗
A platelet activating factor antagonist attenuates the effects of testicular ischemia.
PURPOSE: Platelet activating factor, a biochemical marker and lipid mediator of ischemic injury, has been demonstrated in several organ systems. The objective of this study was to investigate the possible role of platelet activating factor in testicular ischemic injury. MATERIALS AND METHODS: Five groups of 6 male Sprague-Dawley rats were studied, including group 1-nonoperated controls, group 2-sham operated controls, group 3-those that underwent administration of 10 micrograms./kg. exogenous platelet activating factor into the left testicular artery, group 4-those that underwent 4 hours of testicular ischemia and group 5-those that received pretreatment with 0.4 mg./kg. of the platelet activating factor antagonist CV-6209 intravenously before 4 hours of testicular ischemia. Ipsilateral and contralateral testes were examined histologically and seminiferous tubular diameters were measured. RESULTS: Exogenous platelet activating factor administration in group 3 and 4 hours of ischemia in group 4 resulted in a similar extent of histological degeneration of the experimental testicle. Pretreatment with CV-6209 in group 5 resulted in a marked decrease in hemorrhagic discoloration, vascular congestion and histological changes noted with ischemia in group 4. CONCLUSIONS: The results of this study suggest that platelet activating factor has a biochemical role in tissue injury associated with testicular ischemia. Also, administration of a platelet activating factor antagonist before the ischemic event decreases seminiferous tubule degeneration.
Animals↗
Association of granular exocytosis with Ca(2+)-activated K+ channels in human eosinophils.
We studied the mechanism of degranulation caused by Ca(2+)-activated K+ channels (KCa channels) in eosinophils isolated from mildly atopic donors using negative immunoselection. Stimulation of eosinophils with 0.1 microM platelet-activating factor (PAF) caused activation of single channels as recorded by the cell-attached patch-clamp technique. These channels were selectively permeable to K+ because the reversal potential was close to the equilibrium potential for K+. However, the channels were not permeable to Na+ or Cl- as demonstrated by ion substitution experiments. The calcium ionophore A-23187, at 1 microM, increased the K+ channel activity in the presence of Ca2+ in the external perfusate but did not induce channel activity in the absence of Ca2+. Similar results were obtained with another calcium ionophore, ionomycin (1 microM), and the Ca(2+)-releasing agent thapsigargin (10 microM). K+ channels activated by PAF and A-23187 had similar characteristics: two levels of single-channel conductances were observed, 10 +/- 1.5 and 22 +/- 1.7 pS as induced by PAF and 11 +/- 1.3 and 24 +/- 1.9 pS by A-23187; the mean open times of the large-conductance channels were 1.45 +/- 0.3 ms as induced by PAF and 1.26 +/- 0.5 ms by A-23187. These results indicate that PAF activates KCa channels. Both KCa currents and major basic protein release caused by A-23187 were blocked by quinidine. It is suggested that KCa channels are associated with granule secretion in human eosinophils.
Calcimycin↗
EP1 receptor blockade attenuates both spontaneous tone and PGE2-elicited contraction in guinea pig trachealis.
We assessed the effect of prostaglandin (PG) E2 on tone of guinea pig tracheal smooth muscle (TSM) strips in vitro. In the presence of spontaneous tone [ST; i.e., no indomethacin (-Indo)], exogenous PGE2 caused a significant relaxation of ST at concentrations > 10(-6) M [to -127 +/- 40.8% electric field stimulation (EFS); P = 0.001 vs. baseline ST] and at concentrations < 10(-6) M caused a variable change in contractile force (51.6 +/- 29.6% EFS; P = NS vs. baseline ST). In the absence of ST (i.e., +Indo) 10(-10) to 10(-7) M PGE2 elicited contraction of TSM to 126.3 +/- 10.5% EFS (P = 0.001 vs. baseline) and no relaxation. Addition of prostanoid EP1 receptor antagonist (either AH-6809 or SC-19220) to Indo-treated TSM caused a substantial rightward shift and attenuation of contraction in response to PGE2 (55.9 +/- 16.8% EFS for 10(-5) MAH-6809; P = 0.007 vs. +Indo alone, and 80.5 +/- 12.7% EFS for 10(-5) M SC-19220, P = 0.03 vs. +Indo alone). We further assessed the effect of EP1 and EP4 receptor antagonism on the ST of guinea pig TSM strips. Concentration-response curves to the EP1 receptor-specific antagonists AH-6809 or SC-19220 and the EP4 receptor-specific antagonist AH-23848B were generated (10(-7) to 10(-5) M); AH-6809 caused relaxation of ST to 11.4 +/- 2.9% ST (P = 0.001 vs. initial ST) and SC-19220 caused relaxation to 31.0 +/- 12.7% ST (P = 0.02 vs. initial ST). However, AH-23848B did not significantly affect ST (to 60.9 +/- 7.7% ST; P = 0.07 vs. initial ST). Furthermore, AH-6809 specifically inhibited contraction elicited by the EP1 receptor agonist Iloprost but had no effect on contraction elicited by the EP3 receptor agonist Enprostil. We demonstrate that in the presence of ST (-Indo), exogenous PGE2 elicits a biphasic response in guinea pig TSM in which relaxation predominates. In the absence of ST (+Indo), exogenous PGE2 elicits contraction of guinea pig TSM strips that is inhibited by EP1 receptor-specific antagonism. Spontaneous tone of guinea pig TSM strips also is inhibited by EP1 receptor antagonism. Our data suggest that both PGE2-elicited contraction and ST of guinea pig TSM are mediated through activation of EP1 prostanoid receptors.
Acetylcholine↗
Activated eosinophils elicit substance P release from cultured dorsal root ganglion neurons.
This study was performed to test the hypothesis that activated eosinophils or their secretory products can directly stimulate sensory neurons to release their neuropeptides. Neurons derived from neonatal rat dorsal root ganglia (DRG), which synthesize and store sensory neuropeptides, were placed in primary cell culture and were exposed to eosinophils or their bioactive mediators. The resultant release of substance P (SP) was measured by enzyme-linked immunosorbent assay and was expressed as a percent (mean +/- SE) of total neuronal SP content. Eosinophils were isolated from human volunteers with a history of allergic rhinitis and/or mild asthma and were activated by incubation with cytochalasin B (5 micrograms/ml) and N-formyl-methionyl-leucyl-phenylalanine (FMLP, 1 microM). Activated eosinophils [6 x 10(6)/ml, suspended in Hanks' buffered salt solution (HBSS)] applied to cultured DRG neurons for 30 min increased basal SP release 2.4-fold compared with HBSS-exposed neurons (activated eosinophils 11.10 +/- 2.48% vs. HBSS 4.59 +/- 0.99%; P = 0.002), whereas neither nonactivated eosinophils nor cytochalasin B and FMLP in HBSS influenced SP release. Additional cultured DRG neurons were exposed to soluble products made by eosinophils. Compared with SP release under control conditions (2.37 +/- 0.34%), major basic protein (MBP) increased release in a concentration-related fashion (e.g., 3 microM MBP: 6.23 +/- 0.67%, P = 0.006 vs. control), whereas neither eosinophil cationic protein (3 microM), eosinophil-derived neurotoxin (3 microM), leukotriene D4 (500 nM), platelet-activating factor (100 nM), nor H2O2 (100 microM) affected SP release. These studies demonstrate that activated eosinophils can stimulate cultured DRG neurons directly and suggest that MBP may be the responsible mediator.
Animals↗
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.
Bronchi↗