Eosinophils, major basic protein, and polycationic peptides augment bovine airway myocyte Ca2+ mobilization

Mark E. Wylam1,2, Nesli Gungor1, Richard W. Mitchell2, and Jason G. Umans2,3

Departments of 1 Pediatrics and 2 Medicine, and 3 Committee on Clinical Pharmacology, Division of Biological Sciences, University of Chicago, Chicago, Illinois 60637

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Previous studies in vivo or in isolated airway preparations have suggested that eosinophil-derived polycationic proteins enhance airway smooth muscle tone in an epithelium-dependent manner. We assessed the direct effects of activated human eosinophil supernatant, major basic protein (MBP), and polycationic polypeptides on basal and agonist-stimulated intracellular Ca2+ concentrations ([Ca2+]i) in cultured bovine tracheal smooth muscle (TSM) cells. A 1-h incubation of myocytes with activated eosinophil buffer resulted in a doubling of basal [Ca2+]i and increased responsivity to histamine compared with myocytes that were exposed to sham-activated eosinophil buffer. In addition, concentration-dependent acute transient increases and subsequent 1-h sustained elevations of basal [Ca2+]i were observed immediately after addition of MBP and model polycationic proteins. Finally, both peak and plateau [Ca2+]i responses to bradykinin addition were augmented significantly in cultured myocytes that had been exposed to low concentrations of MBP or model polycationic proteins but were inhibited at greater concentrations. This elevated [Ca2+]i to polycationic proteins was manifest in epithelium-denuded bovine TSM strips as concentration-dependent increased basal tone. We conclude that activated eosinophil supernatant, MBP, and other polycationic proteins have a direct effect on both basal and subsequent agonist-elicited Ca2+ mobilization in cultured TSM cells; TSM strips in vitro demonstrated, respectively, augmented and diminished responses to the contractile agonist acetylcholine. It is possible that alteration in myocyte Ca2+ mobilization induced by these substances may influence clinical states of altered airway tone, such as asthma.

polycationic proteins; intracellular calcium; airway smooth muscle

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

IT HAS BEEN SUGGESTED THAT eosinophil-derived cationic proteins might be involved in the pathogenesis of airway hyperresponsiveness. For example, in airway epithelial cells, major basic protein (MBP) increases prostaglandin synthesis (35), including E2 and F2alpha , reduces ciliary motility (15), and induces cytological damage. Prior investigations in vivo (7) and in vitro (5, 8, 11, 24) have suggested that the effect of MBP on smooth muscle cell contraction is indirect and likely mediated via barrier interruption of the airway epithelium or by altering epithelial mediator release (34). However, in some cells, cationic proteins may directly alter cellular Ca2+ homeostasis (4, 14); this suggested the possibility that they may directly augment smooth muscle contraction. We sought to test explicitly the hypothesis that products of activated eosinophils, including MBP, and synthetic cationic polypeptides directly mobilize Ca2+ in cultured airway smooth muscle. Furthermore, using the fura 2 fluorescent dye technique, we tested the hypothesis that cationic proteins would augment receptor-coupled Ca2+ mobilization. We found that MBP and other polycationic polypeptides directly mobilize intracellular Ca2+ in a concentration-dependent manner and that, after a 1-h incubation at lesser concentrations, these compounds augment bovine tracheal smooth muscle (TSM) responsiveness to receptor-coupled agonists. These findings were paralleled in studies conducted using epithelium-denuded bovine tracheal muscle strips in vitro in which polycationic proteins increased basal tone and augmented muscarinic responsiveness. These data suggest that products of eosinophil activation may directly augment airway smooth muscle responsiveness in inflammatory disease states such as asthma.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Isolation of human eosinophils and eosinophil activation. Activated eosinophil supernatant was a gift from Dr. Steven R. White (University of Chicago, Chicago, IL). Briefly (28), human eosinophils were isolated from volunteers according to a protocol approved by the University of Chicago Institutional Review Board. Whole blood was collected, white blood cells were isolated, and eosinophils were separated over discontinuous colloidal Percoll gradients. The interface containing eosinophils (1.095-1.000 g/ml Percoll) was collected and diluted in Hanks' balanced salt solution containing 1 mM Ca2+ and 0.1% gelatin. Eosinophil purity was >90%. Eosinophils were kept on ice and used on the same day of activation; 3.8 million eosinophils were incubated with 10-6 M formyl-methionyl-leucyl-phenylalanine (fMLP) plus 5 mM cytochalasin B (cytB) in polypropylene tubes for 30 min at 37°C. After activation, the tube was placed on ice and then centrifuged at 4°C and 400 g for 10 min. The activated supernatant was then separated and stored at -70°C for subsequent use.

Cell culture. Bovine tracheae were obtained from a local abattoir and transported to the laboratory in ice-cold N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline [HBS (in mM): 130 mM NaCl, 5.0 mM KCl, 1.0 mM CaCl2, 1.0 mM MgSO4, 10 mM dextrose, and 10.0 mM HEPES; pH 7.4] supplemented with antibiotics (see below). TSM was dissected from the mucosa, cut into ~1-mm3 pieces, and washed two times in HBS. Tissue was then incubated in 2 ml of HBS containing 0.2% collagenase (type IV; Sigma, St. Louis, MO) and 0.05% elastase (type IV; Sigma) for 30 min at 37°C. Approximately 1 g of dissociated tissue was filtered through a nylon mesh, washed two times with HBS, and then resuspended in medium 199 (M199) with 1% fetal bovine serum, 100 µl/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B. After 24-h incubation, the medium was changed to M199 with 20% FBS, and cells were grown to confluence. Isolated cells were identified by morphological appearance on light microscopy and by immunofluorescent staining for alpha -smooth muscle actin (Sigma). Cells were maintained at 37°C in a 5% CO2-95% O2 environment, passaged every 5-7 days, and grown on eight-well Lab-Tek glass coverslides (Nunc, Naperville, IL) for use in experiments at 1-3 days postconfluence. Cells from passages 2-6 were used in these studies; all responses were stable over these passages.

Measurement of intracellular Ca2+ concentration. In each well of the coverslide, incubation medium was replaced for 1 h with activated eosinophil supernatant (100 µl in 300 µl HBS) or 400 µl of HBS alone. For experiments using polycationic proteins, individual wells were incubated for 1 h in the presence of 10-9 to 10-5 M MBP, 10-8 to 10-3 M poly-L-arginine (poly-A), 10-8 to 10-5 M poly-L-lysine (poly-L), or 10-8 to 10-5 M melittin. After incubation, the cells were rinsed and incubated in HBS with 0.1% bovine serum albumin containing 5 µM fura 2-AM for 30 min at room temperature. Cells were washed two times with fresh buffer and incubated for an additional 30 min to allow for complete hydrolysis of the ester. The slide was then transferred to the stage of an inverted Nikon Diaphot microscope for microspectrofluorimetery using a ×10 objective, with the results being taken from confluent fields containing 50-80 cells. The microscope was coupled to an air turbine spectrofluorimeter (Biomedical Instrumentation Group, University of Pennsylvannia, Philadelphia, PA). The fluorimeter filter wheel contained 340- and 380-nm excitation filters and was adjusted to spin at 100 ± 10 Hz. The wheel was coupled to the microscope via a fiber-optic light guide. Emitted fluorescence, gated to the position of the filter wheel, passed through a dichroic mirror and a 510-nm filter and was measured by a photomultiplier tube (PMT); the current output of the PMT was converted to voltage input into an encoding amplifier. Amplifier output was then digitized and sampled with software (Lakeshore Technologies, Chicago, IL) at 1 Hz by a microcomputer. Ca2+ concentration (nM) was calculated using an in vitro calibration using known free Ca2+ (0-1.35 µM) and pentapotassium fura 2 (5 µM). The linear plot of the log of the Ca2+ concentration versus the log of the 340- to 380-nm fluorescence ratio [(R - Rmin)/(Rmax - R) × (380min/380max), where R is ratio, Rmin is the minimum ratio, Rmax is maximum ratio, 380min is minimum fluorescence at 0 Ca2+, and 380max is the maximum fluorescence at 1.35 µM Ca2+] was plotted to determine the dissociation constant (Kd) after subtraction of unloaded cell and system background at each wavelength. Total field intracellular Ca2+ was calculated from experimental ratios by the equation of Grynkiewicz et al. (12) using Rmax, Rmin, and Kd from the calibration plot; selected standards were run daily. In all cells, Ca2+ responses were acquired for 20 s to establish average baseline intracellular Ca2+ and for 300 s to characterize peak and plateau responses after the acute addition of activated eosinophil supernatant, MBP, and model cationic proteins and the subsequent addition of bradykinin (BK; 10-5 M) or histamine (Hist; 10-4 M) after the 1-h incubation period (see above).

Preparation of TSM strips. Bovine tracheae were obtained from a local abattoir. The tracheae were placed in 4°C Krebs-Henseleit (KH) solution of the following composition (in mM): 115 NaCl, 25 NaHCO3, 1.38 NaH2PO4, 2.51 KCl, 2.46 MgSO4 · 7H2O, 1.92 CaCl2, and 11.2 dextrose. The KH solution was gassed with a mixture of 95% O2-5% CO2 gas to maintain a pH of 7.35-7.45 (19, 20). A 10-cm midtracheal segment was cleaned of overlying connective tissue. The posterior aspect of the tracheal cartilage was clipped away, and the connective tissue lying between the cartilage rings and the smooth muscle was cut. The opened posterior aspect of the TSM was cleaned of overlying connective tissue by careful dissection, and the ends of the rings were cut away. The major portion of the ventral cartilage rings was cut away, and the remaining segment of trachea was pinned epithelium side up to a dissecting tray. Under cold KH, the epithelium was dissected away from the smooth muscle; care was taken not to overstretch the underlying smooth muscle layer. Eight tracheal preparations (~2 mm wide and 10 mm in length) were dissected. Each tracheal ring preparation was tethered at the junctions of the posterior membrane and adjacent cartilage with 3-0 silk thread (Deknatel). Each TSM preparation was randomly assigned to one of the treatment groups described below.

Tissue perfusion. The TSM strips were mounted in 15-ml glass tissue baths (Easterbrooke, Winnipeg, MB) containing 10 ml of continuously gassed KH at 37°C. One end of the TSM strip was attached with a loop of 3-0 braided silk suture to a rigidly held glass rod within the bath. The upper end was fastened to a Grass model FT.03 force displacement transducer. This transducer was mounted on a rack-and-pinion system to enable the muscle preparation to be stretched to optimal length (20). Isometric tension was recorded digitally with a microcomputer. An initial resting load (isoproterenol insensitive) of 1.0-2.0 g was applied to each TSM strip; this resting load stretched bovine TSM preparations to approximately optimal length to allow for optimal contractile responses to electrical field stimulation (EFS) and acetylcholine (ACh).

Active equilibration with EFS. To establish maximal and reproducible isometric contraction, tissues were equilibrated initially for 60 min as follows (20, 29). Each TSM strip received supramaximal EFS (60-Hz alternating current, 10 V, 10-s duration) applied through platinum wire electrodes placed on either side and parallel to the TSM preparation. Strips were stimulated every 15 min and rinsed with fresh KH every 15 min (20, 29). During this time, a limited length-tension study for each strip was conducted to determine optimal length and response to EFS. All subsequent data were normalized to this maximal contractile response to EFS (20).

Response to model polycationic proteins. Bovine TSM strips were randomly assigned to receive no polycationic protein (control), a concentration of poly-A (10-6 to 10-4 M), or a concentration of poly-L (10-7 to 10-5 M). These concentrations were determined based on the Ca2+ studies. Tissues were incubated for 1 h in the presence or absence of protein. Resting tone was noted before and after polycationic protein exposure. All TSM preparations were then washed with fresh KH buffer.

Concentration-response curves to ACh. After incubation with polycationic protein, each bovine TSM strip (n = 80 preparations from 10 animals) was subjected to an ACh concentration-response study. Cumulative concentration-response curves were generated with 10-9 to 10-3 M ACh. The next greater concentration of ACh was not added until either 5 min had elapsed and/or a plateau response to the previous concentration had been achieved.

Reagents. MBP was a gift from Dr. Gerald J. Gliech (Mayo Clinic and Mayo Foundation, Rochester, MN) and was isolated as described previously (1). BK, Hist, poly-A HCl (mean mol wt = 10,800), poly-L hydrobromide (poly-L; mean mol wt = 123,900), and melittin (purity 85%, phospholipase A2 impurity <5 U/mg; mol wt = 2,848) were dissolved in HBS and were obtained from Sigma. Fura 2-AM and Ca2+ standards were obtained from Molecular Probes (Eugene, OR).

Statistical analysis. Data are expressed as group means ± SE where n is the number of fields of 50-80 cells analyzed. Group comparisons were by ANOVA with Bonferroni correction for multiple comparisons; P values <=  0.05 were considered significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Acute and 1-h exposure to eosinophil-activated buffer on basal and Hist-elicited Ca2+ mobilization. The acute addition of fMLP, cytB, or eosinophil-activated buffer did not immediately alter basal intracellular Ca2+ concentration ([Ca2+]i). There were no effects on basal or agonist-stimulated [Ca2+]i after a 1-h incubation of myocytes with fMLP or cytB. However, a 1-h incubation of myocytes with eosinophil-activated buffer resulted in an increased basal level (Fig. 1) compared with myocytes exposed to sham-activated buffer. In preliminary studies, BK alone (10-5 M) elicited an increase in peak [Ca2+]i above basal level of 477 ± 79 nM, which was not significantly different from the effect of 10-5 M BK in cells incubated in eosinophil-activated buffer (475 ± 88 nM, n = 4). However, eosinophil-activated buffer incubation significantly increased peak [Ca2+]i elicited by 10-4 M Hist (Fig. 1).


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Fig. 1.   Influence of 1-h incubation with either sham (open bars) or eosinophil-activated (filled bars) buffer on basal and histamine (10-4 M)-elicited incremental increase in peak intracellular Ca2+ concentration ([Ca2+]i). Values ± SE indicate the increase in [Ca2+]i (n = 4 in each group). * P < 0.05 and dagger  P < 0.001 compared with respective control.

Effect of acute exposure to MBP and cationic polypeptides on Ca2+ transients. After the acute addition of HBS (100-µl control addition), [Ca2+]i increased maximally 5 ± 1 nM above resting values of 45 ± 2 nM (n = 35). Low concentrations of (10-7 M) poly-A, MBP, and melittin led to significant rapid, although transient, increases in [Ca2+]i compared with buffer. Peak increases were 20 ± 13 nM (n = 5) for poly-A, 18 ± 8 nM (n = 6) for MBP, and 26 ± 9 nM (n = 4) for melitin (P < 0.007, 0.002, and 0.0001 vs. control, respectively; Fig. 2; sample traces 10-7 and 10-5 M MBP). Poly-L (10-7 M) did not elicit an acute increase in basal [Ca2+]i. However, greater concentrations (10-5 M) of poly-L, poly-A, and MBP elicited significant increases in basal [Ca2+]i compared with control myocytes [poly-A, 18 ± 5 nM (n = 7); poly-L, 128 ± 27 nM (n = 7); MBP, 78 ± 28 nM (n = 5); P < 0.0002, 0.0001, and 0.0001 vs. control, respectively]. Moreover, melittin (10-5 M) elicited increases in basal [Ca2+]i by >1 µM, an amount that exceeds precise quantification using fura 2 (P < 0.00001 vs. control, n = 13), and was associated with morphological cellular injury.


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Fig. 2.   Sample traces of [Ca2+]i vs. time, showing the transient effect of 10-7 M (A) and 10-5 M (B) major basic protein (MBP; arrow) in confluent bovine tracheal smooth muscle cells on [Ca2+]i.

Effect of 1-h incubation with MBP and cationic polypeptides on basal [Ca2+]i. In control cells incubated for 1 h with HBS, basal [Ca2+]i remained unchanged (Figs. 3 and 4; control). However, after incubation for 1 h, poly-A, poly-L, and melittin elicited, at sufficient concentration, an increase in basal [Ca2+]i. This increase was concentration dependent and significant at concentrations of >= 10-5 M for poly-A and MBP, and 10-6 M for poly-L and melittin (Fig. 4).


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Fig. 3.   Sample traces of effect of addition (first arrow) of either control buffer or MBP (10-9 M to 10-5 M) on [Ca2+]i. Subsequent to 1-h incubation (axis break), the influence of increasing concentrations of MBP are noted upon addition (second arrow) of bradykinin (BK; 10-5 M). A: control; B: 10-9 M MBP; C: 10-8 M MBP; D: 10-7 M MBP; E: 10-6 M MBP; F: 10-5 M MBP.


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Fig. 4.   Influence of 1-h incubation with poly-L-arginine (poly-A; A), poly-L-lysine (poly-L; B), MBP (C), or melittin (D) on basal [Ca2+]i. Values indicate the increase above basal [Ca2+]i (n = 5-11 for poly-A, 4-15 for poly-L, 6-22 for MBP, and 6-15 for melittin). * P < 0.05, ** P < 0.001, and dagger  P < 0.0001 compared with respective control.

Effect of 1-h incubation with MBP and cationic polypeptides on BK-elicited peak and plateau Ca2+ mobilization. A 1-h incubation of bovine airway myocytes with polycationic proteins produced a biphasic response to subsequent BK-elicited Ca2+ mobilization (Fig. 5). In control cells, BK (10-5 M) elicited a peak [Ca2+]i of 336 ± 19 nM (n = 20). At relatively lower concentrations of poly-A (10-8 to 10-7 M), poly-L (10-7 to 10-6 M), and MBP (10-9 to 10-8 M), BK-elicited peak [Ca2+]i was significantly augmented compared with control cells (Fig. 4). However, at relatively high concentrations of each agent (poly-A >=  10-4 M, poly-L 10-5 M, and MBP 10-5 M), the magnitude of BK-elicited peak [Ca2+]i was substantially attenuated compared with control cells (Fig. 5). At relatively lower concentrations of melittin (10-8 to 10-7 M), there was a tendency for BK-elicited peak [Ca2+]i to be enhanced in some cells compared with controls; however, the response was widely variable. In addition, at concentrations greater than 10-7 M, melittin caused a direct and irreversible release of Ca2+ such that there was no discernible response to the subsequent addition of BK. In control cells 300 s after the addition of BK, the sustained plateau elevation of [Ca2+]i [68 ± 3 nM (n = 43)] was significantly greater than basal level [45 ± 2 (n = 62); P < 0.0001]. This value was concentration dependently augmented by poly-L, increased significantly only at the highest concentrations of poly-A and MBP tested, and was concentration dependently increased at low concentrations (10-8 and 10-7 M) of melittin, with no response obtained at higher concentrations (Fig. 6).


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Fig. 5.   Effect of 1-h incubation with poly-A (A), poly-L (B), MBP (C), or melittin (D) on the incremental increase in peak [Ca2+]i elicited by 10-5 M BK. Values indicate the increase above basal [Ca2+]i (n = 4-11 for poly-A, 4-11 for poly-L, 8-22 for MBP, and 6-13 for melittin). * P < 0.05, ** P < 0.001, and dagger  P < 0.0001 compared with respective control.


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Fig. 6.   Effect of 1-h incubation with poly-A (A), poly-L (B), MBP (C), or melittin (D) on the incremental increase in the plateau value of [Ca2+]i elicited by 10-5 M BK (n = 5-20 for control, 4-16 for poly-A, 6-13 for poly-L, 8-24, and 6-10 for melittin). * P < 0.05, ** P < 0.001, and dagger  P < 0.0001 compared with respective control.

Response of TSM strips to model polycationic proteins. Over the incubation period of 1 h, basal tone of bovine TSM strips increased in response to polycationic proteins (Fig. 7); control tissues maintained baseline resting tone (Fig. 8). The increase in basal tone was dependent on the exposure concentration of polycationic protein. For 10-7 M poly-L, resting tone did not increase significantly (4.4 ± 4.3% EFS). However, for 10-5 M poly-L, resting tone increased to 28.5 ± 9.9% EFS (P < 0.001 vs. initial; P < 0.05 vs. 10-7 M poly-L). For 10-6 M poly-A, resting tone increased to 9.9 ± 4.1% EFS (P < 0.01); for 10-4 M poly-A, resting tone increased to 36.3 ± 8.9% EFS (P < 0.0005 vs. initial; P = 0.01 vs. 10-6 M poly-A).


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Fig. 7.   Representative myogram of the effect of 10-5 M poly-L on bovine tracheal smooth muscle (TSM) responsiveness. After equilibration and excitation by electrical field stimulation (EFS), 10-5 M poly-L was added, and basal tone was monitored for 1 h. After 5 min, basal tone (1.5 g) began to increase; after 60 min, tone was elevated to 5.9 g. The bovine TSM strip was rinsed with buffer 3 times, and responses to increasing concentrations of ACh were elicited.


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Fig. 8.   Effect of polycationic protein exposure on basal tone of bovine TSM. Basal tone (expressed a percent of each tissue's maximal response to EFS; %EFS) increased in response to exposure to both poly-L and poly-A in concentration-dependent manners for bovine TSM strips. Control tissues did not demonstrate significant changes in resting tone over the 1-h incubation period (n = 6-10 for each group). * P < 0.05 and ** P < 0.001 compared with control.

Subsequent response to ACh. Lesser concentrations of polycationic proteins augmented and greater concentrations reduced maximal contractile responses of bovine TSM preparations to ACh (Fig. 9). Tissues incubated with 10-7 M poly-L demonstrated a maximal contractile response to 10-3 M ACh of 194.0 ± 6.2% EFS compared with 165.3 ± 9.3% EFS (P = 0.04) for control TSM strips, respectively. However, greater concentrations (10-5 M) of poly-L reversed this augmentation (Fig. 9).


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Fig. 9.   Effect of polycationic protein exposure on bovine TSM response to ACh. Lesser concentrations of poly-L and poly-A augmented and greater concentrations reduced contractile responses of bovine TSM preparations to ACh compared with control tissues (n = 6-10 for each group). * P < 0.05 and ** P < 0.01 vs. preincubation values for basal tone.

Tissues incubated with 10-6 M poly-A demonstrated a similar trend as 10-7 M poly-L in that a maximal response to 10-3 M ACh (181.7 ± 10.4% EFS) was augmented; however, this increase in contraction was not significantly different from the control responses (Fig. 8). However, preparations incubated with 10-5 M poly-A (126.1 ± 12.6% EFS, P < 0.05 vs. control; P = 0.01 vs. 10-6 M poly-A; Fig. 9) demonstrated a reduction in the maximal contractile response.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Evidence of increased epithelial permeability (36), cytotoxicity (18), and/or bronchoconstrictor mediator release (27, 28, 30, 34) clearly supports an indirect effect of polycations, favoring smooth muscle contraction in intact airway preparations. By contrast, we sought to determine the direct effects of products of activated eosinophils, graded concentrations of MBP, and other model polycations on cultured bovine tracheal myocyte Ca2+ mobilization. The synthetic polycations poly-A and poly-L are of similar size and cationic charge as eosinophil MBP, and in past studies (7), the indirect changes in airway hyperresponsiveness have been similar for proteins of similar charge, although the precise mechanism(s) of action of MBP and the role of its cationic charge remain to be defined. In addition, we compared the above results with the direct effect of the amphiphilic peptide melittin, which is known to increase the activity of phosphoinositide phospholipase through a cationic interaction (22). A further objective was to assess the effects of these substances on agonist-elicited Ca2+ mobilization and airway smooth muscle contraction. We used cultured airway smooth muscle cells so that responses could be studied independent of other airway cell types, particularly epithelium and neuronal tissue. We also used epithelium-denuded bovine TSM strips to assess the direct effects of model polycationic proteins on these tissue preparations. Organ bath volumes and availability of MBP precluded the use of this eosinophil-derived polycationic protein in studies on bovine TSM strips.

Activated eosinophils applied to guinea pig tracheal epithelium increase basal tone and muscarinic responsiveness (13, 28). Because this effect is blocked by pretreatment with a 5-lipoxygenase inhibitor, epithelial synthesis of leukotriene C4 may mediate the enhanced airway responsiveness in this species. In contrast, when A-23187-stimulated eosinophils are added either luminally or adventially to intact bovine bronchial segments, there is no effect on either basal tone or ACh responsiveness (24). Because the mode of eosinophil activation may alter its effects on smooth muscle (28), we used human peripheral blood eosinophils stimulated by both fMLP and cytB. This method of eosinophil activation stimulates production of eosinophil peroxidase and leads to increases in both basal tone and muscarinic responsiveness for epithelium-intact tissues in guinea pigs (28). A new finding of this investigation is a direct effect of activated eosinophil supernatant on cultured airway myocytes. One hour of incubation of cultured bovine tracheal myocytes with this activated eosinophil supernatant not only doubled resting myocyte [Ca2+]i but also augmented Hist-elicited Ca2+ mobilization. However, subsequent myocyte responses to BK were not affected by incubation with eosinophil supernatant; this differs from results obtained with MBP (see below). Dissimilar effects on the agonist Ca2+ response elicited by BK vs. Hist may be due to differing signal transduction mechanisms or competitive signalling due to other smooth muscle modifying mediators found in activated eosinophil supernatant.

Second, we found that both MBP and model polycations led to concentration-dependent acute transient increases and a subsequent 1-h sustained elevation in basal [Ca2+]i in these same airway myocytes (Figs. 1-3). We also found that peak and plateau values for [Ca2+]i in response to BK were significantly greater in cultured myocytes exposed to relatively low concentrations of MBP and other polycationic proteins (Figs. 4 and 5). Incubation with greater concentrations of MBP and polycationic proteins increased basal [Ca2+]i to the extent that subsequent responses to BK were attenuated (Fig. 4). As with melittin, greater concentrations of MBP and analogs may adversely affect cellular and Ca2+ homeostasis. In the absence of other agonists, MBP and other model polycations led to concentration-dependent acute and sustained Ca2+ mobilization in these cultured cells.

A third finding of our study was that the elevated [Ca2+]i could be manifest functionally by increased basal tone in epithelium-denuded bovine TSM preparations (Fig. 6). Parallel to our findings for [Ca2+]i, basal tone increased with exposure to model polycationic proteins in a concentration-dependent manner (Fig. 7). Also, low concentrations of poly-L augmented and high concentrations of poly-A reduced subsequent responses to ACh (Fig. 8).

These findings contrast with conclusions of prior investigations carried out in intact tissue models that suggest an intact airway epithelium is required for the effect of MBP on enhanced airway responsiveness (5, 11, 34). Several studies have examined the effect of MBP on airway responsiveness only in epithelium-intact preparations (7, 30). Others have clearly demonstrated an epithelium dependence to MBP enhancement of airway responsiveness in some species. Flavahan et al. (11) in vitro and White et al. (34) in situ noted that, in epithelium-denuded preparations, MBP did not directly affect the tone of guinea pig trachea and did not increase sensitivity to contractile agonist. Similarly, Brofman et al. (5) noted that, although the intraepithelial administration of MBP augmented contraction of underlying canine TSM, this effect was absent after epithelial removal or direct injection of MBP into trachealis muscle. However, none of these previous studies was able to assess the direct effects of MBP or polycationic proteins on airway smooth muscle myocytes, and conclusions drawn were limited by the potential effects of these substances on neuronal tissue and other cells, such as mast cells, in the whole airway preparation.

The synthetic cationic proteins poly-A, poly-L, and melittin are of similar size and cationic charge as eosinophil-derived MBP and have been shown to augment muscarinic airway hyperresponsiveness (7, 32), an effect that may be related to cationic charge density. However, as with MBP, previous studies have either examined only epithelium-intact tissues (27, 30, 32) or determined that model cationic proteins influence airway hyperresponsivenss in an epithelium-dependent manner (8, 24). In addition to an epithelium-mediated effect, Spina and Goldie (27) found that poly-A caused tracheal contraction via the release of ACh from parasympathetic nerves and/or by the release of mast cell-derived serotonin. Similarly, Strek et al. (30) determined that model cationic proteins may mediate airway contraction via the cyclooxygenase pathway (30). Although the present results do not diminish the contributing effects of epithelium-dependent and neuron-dependent influences, they point to a significant possibility for an additional direct effect of eosinophil-derived cationic proteins on airway myocyte stability and activity in vivo.

Eosinophil-derived cationic proteins appear to be released continually into the blood of patients with stable persistent asthma (6), and eosinophil infiltration may be found in the lamina propria and smooth muscle layer of subjects with asthma (10, 16, 23). Thus MBP may be continually released in high concentrations by eosinophils interspersed and migrating among airway myocytes or in adjacent subepithelium. Therefore, the acute experimental addition of MBP or model cationic proteins to intact tissue preparations may fail to permeate myocytes sufficiently to produce a direct effect on myocyte contractility.

In addition to direct effects on airway myocyte Ca2+ and contraction, we also found a biphasic effect of MBP and cationic proteins on receptor-stimulated Ca2+ mobilization; each agent tested augmented peak BK-elicited Ca2+ responses. By contrast, progressively increasing concentrations of these substances inhibited peak Ca2+ mobilization in response to BK while continuing to elicit sustained elevation in myocyte Ca2+ concentration. In the case of melittin, this was associated with morphologically evident cell injury.

In a variety of cells, MBP and polycations may interact with cell-surface anions and integral membrane proteins and ultimately may lead to cytotoxicity. Like our results, Ayars et al. (3) found that MBP effects, in their case epithelial cytotoxicity after high doses, occurred only after a significant time delay. Similarly, we noted that persisting MBP effects on myocyte Ca2+ metabolism required at least a 1-h exposure to 10-7 M. Perhaps continuous exposure to even lesser concentrations of MBP at the level of the individual myocyte in vivo may alter airway contractility. Cationic proteins may induce myoepithelial cellular contraction by histochemical alteration of actin filaments as has been shown in canine kidney epithelial cells (25). These effects are often associated with receptor inhibition or synthesis and release of other mediators. Others have shown that MBP or cationic proteins cause prostaglandin synthesis in cultured guinea pig tracheal epithelium (35), rat glomerular mesangial cells (2), fibroblasts (26), and endothelial cells (21). Thus it is possible that these agents stimulated eicosanoid synthesis/release in our cultured airway myocytes, perhaps through a Ca2+- and phospholipase C-dependent mechanism (17). As well, polycations may specifically inhibit Ca2+-ATPase (4), inhibit sarcolemmal Na+-Ca2+ exchange (22), stimulate the activity of phophatidylinositol 4-kinase (33), or activate phospholipase A2- and glibenclamide-sensitive potassium channels (9). In addition, MBP may activate a pertussis toxin-sensitive G protein (31). All of these potential mechanisms would lead to augmented contractile responses in airway smooth muscle cells.

In summary, we found that eosinophil supernatant, MBP, and model polycations cause elevation in basal airway myocyte Ca2+. Moreover, in a biphasic manner, low concentrations of these agents augmented receptor-coupled Ca2+ mobilization, whereas high concentrations inhibited these events, and, in the case of melittin, induced cytotoxic damage. These changes in Ca2+ mobilization to MBP were manifest functionally in bovine TSM strips by 1) concentration-dependent, increased basal tone with exposure to model polycationic proteins, 2) augmentation of contractile response to low concentrations of polycationic protein, and 3) reduced contractile response to ACh after exposure to high concentrations of poly-L or poly-A. In airways, these events could lead to direct contraction of airway smooth muscle. Although these events may, in other preparations, be influenced by epithelial or neuronal factors, they do not depend exclusively on contributions by these other cells. Moreover, the in vivo time-dependent effects of continuous or repeated exposure of myocytes to cationic proteins remain unknown. However, our data demonstrate the potential for products of eosinophil inflammation to directly alter airway smooth muscle tone in disease states such as asthma.

    ACKNOWLEDGEMENTS

We thank Claire Buchanan who performed preliminary studies on the effect of activated eosinophil supernatant on cultured bovine tracheal myocyte Ca2+ mobilization. We thank Gerald J. Gleich, Department of Immunology, Mayo Clinic, Rochester, MN, for the purified major basic protein used in this study.

    FOOTNOTES

This research was supported in part by the Ralph S. Zitnik Clinical Investigatorship Award from the Chicago Heart Association (to M. E. Wylam), by a career development award in clinical pharmacology from the Pharmaceutical Research and Manufacturers of America Foundation (to J. G. Umans), and by National Heart, Lung, and Blood Institute Grant HL-48302 (to J. G. Umans).

Address for reprint requests and present address of M. E. Wylam: Mayo Clinic and Mayo Foundation, 200 First St., SW, Rochester, MN 55905-0001.

Received 15 April 1997; accepted in final form 18 February 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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