Upregulated surface expression of intracellularly sequestered Igepsilon receptors (FcepsilonRII/CD23) following activation in human peripheral blood eosinophils.

We investigated the regulation, secretion, and surface expression of the low-affinity FcepsilonRII receptor (CD23) in eosinophils isolated from human blood using multiple monoclonal antibodies (mAbs) directed at different epitopes of human CD23. Substantial surface expression of CD23 was not demonstrated in the resting state. Mean fluorescence intensity (MFI) measured by flow cytometry was 7. 1 +/- 0.8 for 9P25 mAb (p = NS) and 15.7 +/- 3.8 for BU38 mAb (p <. 04) versus 5.3 +/- 1.0 for IgG1 isotype control Ab. By contrast, MFI using BU38 mAb was 154 +/- 18 for JY-B lymphocytes (p <.0001 versus eosinophils). Despite weak surface expression, eosinophil permeabilization demonstrated substantial intracellular expression of CD23; MFI was 33.6 +/- 5.2 for 9P25 mAb versus 4.4 +/- 0.43 for IgG control (p <.001). Western blot analysis using both positive and negative controls demonstrated immunological identity with CD23 on JY-B lymphocytes. Activation of eosinophils caused rapid translocation of CD23 to the surface membrane (160 +/- 33 MFI; p <. 005), which was maximal within 30 sec. Secretory CD23 was detected within the perfusate also at 30 sec and was fully reinternalized at 10 min. This is the first demonstration of the presence of intracellular CD23 in human eosinophils. Our data indicate that eosinophils rarely express CD23 on their surface but are capable of transient high-level expression and secretion with rapid reuptake of intracellular stores of CD23.

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.

Eosinophil chemotaxis inhibited by 5-lipoxygenase blockade and leukotriene receptor antagonism.

We studied the effects of the 5-lipoxygenase inhibition and sulfidopeptidyl leukotriene receptor antagonism on lumenal chemotaxis of eosinophils in 124 guinea pig tracheal explant preparations from 62 animals. Cell migration was assessed histologically and by differential cell count, and airway narrowing was measured by calibrated micrometry. Intralumenal instillation of the chemotaxin, formyl-met-leu-phe (FMLP) caused migration of 163,509 +/- 18,103 eosinophils/cm segment (eos/cm) versus 15,443 +/- 3,557 eos/cm for segments receiving vehicle only (p < 0.001). Coincubation of FMLP with zileuton, a selective inhibitor of 5-lipoxygenase, caused a concentration-related inhibition of eosinophil migration. At 10(-10) M zileuton, cell migration caused by FMLP was decreased by 57% and nearly complete reduction to 17,200 +/- 3,620 eos/cm resulted after 10(-6) M zileuton (p < 0.001 versus FMLP). Lumenal narrowing caused by FMLP (15.3 +/- 3.4%) was attenuated maximally to 1.15 +/- 2.51% after 10(-8) M zileuton (p < 0.02). In 36 preparations, concentration of leukotriene B4 (LTB4) was measured in treated tracheal perfusate. LTB4 secretion caused by FMLP was 6.4 +/- 0.48 pg/ml versus 3.32 +/- 0.89 pg/ml for buffer control at 5 min (p < 0.02) and was undetectable 120 min after activation with FMLP. Blockade of LTB4-receptor with the selective antagonist, LTB4 dimethyl amide, caused > 90% inhibition of eosinophil migration (p < 0.001). Comparable results were obtained with zafirlukast, an LTD4-receptor antagonist. Our data demonstrate that both LTB4 and LTD4 facilitate eosinophil migration from lamina propria to lumen caused by the chemotaxin, FMLP, and that LTB4-induced eosinophil migration is accompanied by initial lumenal secretion of LTB4.

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.

Quantitation of the cytosolic phospholipase A2 (type IV) in isolated human peripheral blood eosinophils by sandwich-ELISA.

Sandwich enzyme-linked immunosorbent assay (sELISA) was developed for precise quantitation of cytosolic phospholipase A2 (cPLA2 type IV) concentration in isolated human peripheral blood eosinophils as an alternative to semiquantitative chemiluminescent assay employing immunoprecipitation/Western blot analysis. In this assay, monoclonal mouse anti-human cPLA2 antiserum was used as the capture antibody, polyclonal rabbit anti-human cPLA2 antiserum as the secondary antibody, and alkaline phosphatase-conjugated goat anti-rabbit IgG as the tertiary, reporter antibody. Purified human cPLA2 (0-1000 ng/ml) dissolved in Tris-HCl buffered saline was used as the standard protein. The detection limit for cPLA2 in 10(6) eosinophils was 0.109 ng/ml, and coefficients of inter- and intra-assay variation were 4.23% and 7.07%, respectively. There was no cross-reactivity with other (secretory) isoforms of PLA2 (sPLA2 types I-III) either from porcine pancreas, human synovial fluid, or bee venom. In separate studies, the recovery of cPLA2 was > 83% when eosinophil lysate was supplemented exogenously with two different concentrations of cPLA2. From a total protein content of 22.3 +/- 1.7 micrograms/10(6) cells, the baseline concentration of cPLA2 was 0.38 +/- 0.18 ng/10(6) cells in eosinophils obtained from mildly atopic donors. Immunoblotting studies confirmed the complete specificity for the type IV isoform as detected by sELISA. This sELISA method permits the precise quantitative assessment of cPLA2 in nanogram quantities per million cells, which has not previously been possible by immunoblotting analysis.

Eosinophil VLA-4 binding to fibronectin augments bronchial narrowing through 5-lipoxygenase activation.

We examined the effect of ligation of human eosinophils activated by platelet-activating factor (PAF) to soluble human fibronectin (FN) on the augmented contractile response of human bronchial explants. Styrene microplate wells were FN-coated and eosinophils were allowed to adhere in the presence of 1) buffer control, 2) 20 micrograms/ml monoclonal antibody (HP2/1) to the alpha 4 beta 1 ligand (VLA-4) on the eosinophils, 3) 20 micrograms/ml anti-CD18 R15.7, 4) 20 micrograms/ml anti-CD16 3G8, or 5) 10(-6) M A63162, a 5-lipoxygenase inhibitor. Sixty minutes later, treated cells were activated with either buffer or 10(-6) M PAF. Airway luminal diameter was assessed by computerized videomicrometry as a function of pixel number, and activation of eosinophils was confirmed by measurement of leukotriene C4 (LTC4) secretion. Ligation with FN caused an increase in PAF-stimulated LTC4 secretion from 276 +/- 75.6 pg/10(6) cell at baseline to 606 +/- 90.2 pg/10(6) cell (P < 0.01). This corresponded to augmented luminal narrowing of human bronchial explants from 25.3 +/- 9.39% (PAF activation alone) to 42.9 +/- 8.0% (PAF-activated eosinophils + FN) (P < 0.01). Both augmented airway luminal narrowing and increased LTC4 secretion caused by PAF-activated cells after FN ligation were blocked completely by anti-VLA-4 MAb (P < 0.05 vs. control). Pretreatment with 10(-6) MA63162 inhibited completely the PAF-stimulated LTC4 secretion to baseline level ( P < 0.001). Inhibition of 5-lipoxygenase similarly blocked luminal narrowing caused by eosinophils stimulated by PAF by > 95% (P < 0.001). We demonstrate that the binding of human eosinophils to the matrix protein FN causes augmented secretion of LTC4 which, in turn, causes augmented luminal narrowing of explanted human bronchi in vitro. We also demonstrate that the augmented activity is blocked selectively by pretreatment with specific monoclonal antibody against VLA-4 and blockade of eosinophil 5-lipoxygenase inhibits both LTC4 secretion and airway narrowing after PAF-stimulation.

Differential tachykinin receptor subtype activation in capsaicin and KCl contractions of guinea pig trachealis.

We assessed the role of endogenously secreted tachykinins in mediating contraction caused by potassium chloride (KCl) in guinea pig tracheal smooth muscle (TSM) strips in vitro. Maximal isometric contraction was elicited with approximately 45 mM KCl and was 196 +/- 8% of the response to electrical field stimulation (% EFS) in the same tissues. Muscarinic receptor blockade with atropine modestly attenuated this contraction caused by KCl to 175 +/- 9 %EFS (P < 0.05), and treatment with a selective neurokinin subtype 1 (NK1) receptor antagonist, LY-297911, caused even greater inhibition of KCl-elicited contraction to 124 +/- 8 %EFS (P < 0.001). By contrast, SR-48968, a selective NK2 antagonist, had no effect on contraction caused by KCl (183 +/- 9 %EFS; P = NS vs. KCl alone). However, given together at the same concentration, SR-48968 augmented the inhibition of contraction caused by LY-297911 to 93 +/- 15 %EFS (P < 0.05 vs. LY-297911 alone). In contrast to the effect on KCl-induced contraction, LY-297911 caused only moderate inhibition of the contraction caused by capsaicin to 81 +/- 13 %EFS (P < 0.05 vs. control, 114 +/- 15 %EFS), whereas SR-48968 caused substantial attenuation of contraction caused by capsaicin to 23 +/- 5 %EFS (P < 0.005 vs. LY-297911). We demonstrate that a significant portion of the contraction caused by KCl, in addition to capsaicin, is elicited in guinea pig TSM through neurokinin secretion. However, NK1 receptors predominantly mediate contraction caused by KCl, and NK2 receptors predominantly mediate contraction elicited by capsaicin in guinea pig airway smooth muscle.

Assessment of agonist- and cell-mediated responses in airway microsections by computerized videomicrometry.

The objective of this investigation was to develop a method for real-time measurement of changes in luminal area in microexplants of airways during pharmacological and physiological interventions. After guinea pigs were killed, tracheal rings (1- to 2-mm thick) were excised and placed in 300-microliters chambers. The area of the airway lumen was calculated as pixel number with the use of computerized videomicrometry. In 29 epithelium-intact airways, 10(-3) M acetylcholine (ACh) caused decrease in luminal area of 38.1 +/- 2.80% (P < 0.001 vs. 10(-9) M). Spontaneous tone also was demonstrated in 34 preparations from 4 guinea pigs; decrease in area of 17.0 +/- 1.45% after 60-min incubation in buffer alone was blocked completely by 10(-5) M indomethacin (P = 0.01). Luminal narrowing caused by < or = 10(-6) M ACh was reversed completely by 10(-6) M albuterol (P = 0.002). Addition of 100,000 activated human eosinophils caused 24.7 +/- 4.41% decrease in luminal area vs. 7.24 +/- 5.51% for nonactivated cells (P = 0.048). We demonstrate a real-time method for the assessment of auxotonic changes in airway caliber that utilizes microsections of explanted airways and permits the use of extremely small numbers of isolated cells to achieve physiological activation. Concentration-response characteristics and spontaneous tone are similar to those of large chamber preparations, and narrowing is reversed by beta 2-adrenoceptor activation.