Acetylcholine stimulates bronchial epithelial cells to release neutrophil and monocyte chemotactic activity SEKIYA KOYAMA, STEPHEN I. RENNARD, AND RICHARD A. ROBBINS Research Service, Omaha Veterans Affairs Medical Center, and Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska 68198-2465 Koyama, Sekiya, Stephen I. Rennard, and Richard A. Robbins. Acetylcholine stimulates bronchial epithelial cells to release neutrophil and monocyte chemotactic activity. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L466-L471, 1992.-Bronchial asthma is accompanied by inflammatory cell infiltration in the airway. Increased bronchial reactivity to cholinergic stimulation is well recognized in patients with bronchial asthma. Thus, we postulated that acetylcholine (ACh) stimulates bronchial epithelial cells (BEC) to release neutrophil and monocyte chemotactic activity (NCA and MCA). To test this hypothesis, bovine BEC monolayers were tested for NCA and MCA by a blind-well chemotactic chamber technique. BEC released NCA and MCA in response to ACh in a dose-dependent and time-dependent manner. Molecular sieve column chromatography revealed that ACh induced a single low-molecularweight peak (near 400) for NCA and two low-molecular-weight peaks (near 12,000 and 400) for MCA. The release of NCA and MCA was inhibited by the lipoxygenase inhibitors, nordihydroguaiaretic acid and diethylcarbamazine. Cigarette smoke is a well-recognized stimulus for airway inflammation. To determine whether smoke might activate BEC to release NCA by stimulating nicotinic ACh receptors, we further characterized the ACh receptors, using nicotine and nicotinic and muscarinic receptor antagonists. Nicotine, the nicotinic receptor antagonist d-tubocurarine, and the M2 receptor antagonist gallamine did not modulate the release of NCA in response to ACh. In contrast, atropine and the M, receptor antagonist, pirenzepine, inhibited the release of NCA. These data demonstrate that ACh stimulates BEC to release lipoxygenase-derived NCA and MCA through the muscarinic receptor. neutrophil

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of the underlying pathology of asthma are unclear. However, nonspecific airway reactivity is thought to be a central feature of the disease (51). The association between the late asthmatic response (LAR) and increases in airway reactivity has been demonstrated (6, 9, 12). There is accumulating evidence that inflammation may lead to an increase in airway reactivity and may play an integral role in the pathophysiology of asthma (14,19,24,38). The presence of neutrophils in bronchoalveolar lavage fluid during a period of increased airway reactivity support a relationship between the role of the neutrophil in the production of the LAR and the subsequent increase in airway reactivity (4,34). The role of the neutrophil in the LAR has also been suggested by the demonstration that the LAR can be ablated by neutrophil depletion but restored with homologous neutrophi1 transfusion (34). The potential capacity for neutrophils and macrophagesto participate in asthmatic responses has been suggested by their capability of producing and releasing several distinct mediators. Neutrophils and macrophages are capable of generating several substances, including prostaglandins, platelet activating factor, and MANY

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leukotrienes, many of which can potentiate bronchospasm (28, l&31,46,47). The role of these neutrophilderived mediators is supported by the observation that aerosolized supernatant fractions from activated neutrophils result in increased airway reactivity (35). Anticholinergic drugs can dilate airways (1,44). However, atropine sulfate, an anticholinergic, has a number of other effects on airway functions including decreases in mucus secretion, transmucosal electrolyte, and water flux, ciliary motility, and mucus transport (5, 18). Bronchial epithelial cells have the potential to participate in the inflammatory response in the lower respiratory tract by releasing neutrophil and monocyte chemotactic activity (NCA and MCA) in response to a variety of stimuli (26,28). Therefore, we postulated that bronchial epithelial cells (BEC) might release NCA and MCA in response to acetylcholine (ACh) stimulation and that anticholinergics might modulate this release. To test the hypothesis, bovine BEC were evaluated for their capacity to release NCA and MCA in response to ACh. The results demonstrated that ACh stimulated BEC to release lipoxygenase-derived NCA and MCA. Furthermore, addition of the M1 muscarinic receptor antagonist, pirenzepine, was able to attenuate the release of NCA and MCA in response to ACh. These data may suggest the potential for ACh in inflammatory cell recruitment into the airway.

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Culture and identification of bronchial epithelial cells. BECs were prepared by modification of the methods of Wu et al. (26, 28, 52). The BEC were cultured in medium 199 (GIBCO, NY) supplemented with hydrocortisone (0.1 pg/ml, Sigma Chemical, St. Louis, MO), epidermal growth factor (25 rig/ml, Sigma), transferrin (5 pg/ml, Sigma), insulin (5 pg/ml, Sigma), penicillin (50 U/ml, GIBCO), streptomycin (50 pg/ml, GIBCO), fungizone (2 bGc/mL GIBCO), and 10% fetal calf serum (FCS, GIBCO) at 37°C in a 5% CO, atmosphere. After 3-5 days in culture, the cells had reached confluence. The medium was replaced with 1.5 ml of medium 199 supplemented as above and incubated one additional day. On the following day the culture was washed twice and used for experiments. With the use of this technique, >98% of the cells cultured were viable, and >98% were identified as epithelial cells by staining with antikeratin antibody (ICN Immunological, Lisle, IL). There were no cells that reacted with antivimentin monoclonal antibody (DAKO, Santa Barbara, CA), The antivimentin antibody stained both HFL-1 fibroblasts (American Type Culture Collection, Rockville, MD) and bovine lung fibroblasts (H. Takizawa, personal communication). Cultured bronchial epithelial cells were cobblestone in appearance by phase-contrast microscopy. Transmission and scanning electron microscopy revealed that only a small amount of cells maintained cilia (90% of the migrated cells appeared to be monocytes morphologically by light microscopy; 2) >9O% of the migrated cells were esterase positive; and 3) lymphocytes purified by allowing the monocytes to attach to plastic and tested in the chemotaxis assay yielded O-20% of the chemotactic activity of the monocyte preparation. Partial characterization of the released neutrophil and monocyte chemotactic actiuity. The released neutrophil

and monocyte chemotactic activity obtained from bronchial epithelial cell supernatant fluids incubated with 100 PM of ACh for 72 h was evaluated by molecular sieve chromatography using Sephadex G-75. These experiments revealed that the released NCA was homogeneous in size, with the estimated molecular weight after quinacrine being 450 (Fig. 3). However, the released MCA was heterogeneous in size in unstimulated supernatant fluids (Fig. 4, dotted line). At least three separate peaks of activity were separated by column chromatography, with the estimated molecular weight at or near bovine serum albumin (mol wt 66,200), after cytochrome c (mol wt 12,300)

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Fig. 2. Time-related release of neutrophil chemotactic activity (A) and monocyte chemotactic activity (B) in response to 100 uM of acetylcholine from bronchial epithelial cell monolayers (n = 8). Closed circles and solid line, neutrophil chemotattic activity in response to 100 uM of acetylcholine. Open circles and dotted line, neutrophil chemotactic activity from supernatant fluids without acetylcholine. * P < 0.05 compared with baseline supernatant fluids. * P < 0.05 compared with supernatant fluids without acetylcholine .

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Fig. 4. Molecular sieve column chromatographic finding of released monocyte chemotactic activity in response to acetylcholine from bronchial epithelial cell monolayers. Closed circles and solid line, released monocyte chemotactic activity in response to incubation with 100 uM of acetylcholine for 72 h. Open circles and dotted line, release of monocyte chemotactic activity in steady state after a 72-h incubation.

and additional peak which eluted near quinacrine (mol wt 450). When incubated with 100 PM ACh, the low-molecular-weight peak became more prominent. The bronchial epithelial cell supernatant fluids incubated with 100 PM of ACh in the presence of the lipoxygenase inhibitors, NDGA and DEC, showed a significant decrease in the chemotactic activity for both neutrophils and monocytes (Figs. 5 and 6). Thus the NCA and MCA from bronchial epithelial cell in response to ACh was

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Fig. 5. Effects of nordihydroguaiaretic acid and diethylcarbamazine on release of neutrophil chemotactic activity in response to acetylcholine from bronchial epithelial cell monolayers (n = 6). * P < 0.05 compared with acetylcholine-exposed supernatant fluids.

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Fig. 6. Effects of nordihydroguaiaretic acid and diethylcarbamazine on release of monocyte chemotactic activity in response to acetylcholine from bronchial epithelial cell monolayers (n = 6). * P < 0.05 compared with acetylcholine-exposed supernatant fluid.

consistent with a low-molecular-weight, rived chemotactic activity.

Fig. 7. Effects of acetylcholine muscarinic receptor antagonists on release of neutrophil chemotactic activity in response to acetylcholine from bronchial epithelial cell monolayers (n = 6). * P < 0.05 compared with acetylcholine-exposed supernatant fluid.

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Effects of Ach receptor agonists and antagonists on the release of chemotactic activity from bronchial epithelial ceUs in response to Ach. The ACh nicotinic receptor was

examined by using nicotine and the nicotinic receptor antagonist d-tubocurarine. Nicotine did not stimulate the release of NCA from bronchial epithelial cells (data not shown, P > 0.05 compared with BEC cultured in media alone). Consistent with these results, the nicotinic receptor antagonist did not inhibit the release of NCA from bronchial epithelial cells in response to ACh (data not shown, P > 0.05 compared with BEC cultured in 100 PM ACh). However, the ACh muscarinic receptor antagonist, atropine, and the muscarinic M1 receptor antagonist, pirenzepine, inhibited the release of NCA from bronchial epithelial cells in response to ACh (Fig. 7). In contrast, the muscarinic M2 receptor antagonist, gallamine, did not inhibit the release of NCA (Fig. 7). These data are consistent with the concept that the receptor responsible for the release of NCA in response to ACh may be muscarinic M1 receptor.

The potential for BEC to participate in inflammation and repair is suggested by the observation that BEC can release chemotactic activity for monocytes, lymphocytes, and fibroblasts (28, 39, 41). In the present study, BEC released neutrophil and monocyte chemotactic activity in response to ACh in a dose- and time-dependent manner. The released activity was chemotactic by checkerboard analysis and consistent with a low-molecular-weight lipoxygenase-derived activity. Studies using receptor antagonists suggest that the ACh receptor responsible for the release of the chemotactic activity was the muscarinic M1 receptor rather than the M2 or nicotinic receptors. These data suggest that ACh may play a role in bronchial inflammation by recruiting inflammatory cells into the bronchial lumen. Increased bronchial reactivity is thought to play an integral role in bronchial asthma. The accumulating evidence that inflammation may lead to an increase in airway reactivity was supported by several lines of evidence. First, a mixed cellular infiltrate is found in the airway tissue during LAR and an increased numbers of neutrophils are recovered in bronchoalveolar lavage fluid which has been associated with increased airway reactivity (4, 34). Second, the depletion of neutrophils can ablate the LAR airway reactivity and homologous neutrophil transfusion can restore the bronchial reactivity (34). Third, aerosolized supernatant fractions from activated neutrophils can result in increased airway reactivity (35). The mechanism(s) accounting for the recruitment of inflammatory cells to the airway is controversial. Mast cells have been implicated and can release a high-molecular-weight, heat-stable protein neutrophil chemotactic activity (29, 36). Leukotriene B4 (LTBJ, a potent neutrophil and monocyte chemotactic factor, can be released from a variety of cells including neutrophils, eosinophils, alveolar macrophages, (43) and mononuclear cells and is present in the bronchoalveolar fluid recovered from asthmatic patients (34). Platelet-ac tivating factor or activated complement has also been suggested to pl .aY a role

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in the inflammatory cell recruitment observed in asthma (11,42,48). The results of the present study indicate that in addition to these potential mechanisms, BEC can release lipoxygenase-derived NCA in response to ACh stimulation in airway inflammation. Characterization of the exact identity of the chemotactic activity released from BEC in response to ACh is not complete. DEC and NDGA, two known inhibitors of the lipoxygenase pathway (33), inhibited the release of the chemotactic activity. The released activity was also low in molecular weight, suggesting the activity may be derived from the lipoxygenase pathway. In support of the concept, LTB4 and 12-lipoxygenase products can be released from bovine airway cells (21, 43), and previous investigations have demonstrated that LTB4 and 15dihydroxyeicosatetraenoic acid (SHETE) are released from dog and human tracheal epithelial cells (23, 25), respectively. These lipoxygenase metabolites have been reported as neutrophil and monocyte chemoattractants (3, 15, 17). Consistent with the concept that ACh can induce the release of lipoxygenase products, Salari et al. (41) have reported that ACh stimulates the release of 15-HETE from human airway epithelial cells. Although the released lipoxygenase metabolites appear to be species specific, most epithelial cells release lipoxygenase-derived products. Thus ACh may play an important role in the release of lipoxygenase-derived chemotactic activity from bronchial epithelial cells. The present studies are consistent with the concept of a lipoxygenase product being responsible, at least in part, for the neutrophil and monocyte chemotactic activity released from BEC. However, there are at least two limitations to the present studies. First, bovine epithelial cells were used with human neutrophils and monocytes as the responding inflammatory cells. It is possible that human neutrophils and monocytes might not recognize the bovine equivalent of a chemoattractant. Second, NDGA and DEC may not selectively inhibit cellular lipoxygenase activity. Rankin et al. (39) have recently reported that NDGA also inhibits release of interleukin8-like activity from alveolar macrophages. In this context, it should be noted that NDGA was more effective than DEC in inhibiting the release of neutrophil and monocyte chemotactic activity. Although the ACh receptor of the airway epithelial cell is not identified and the amount of ACh that BEC are exposed to in vivo is unclear, the present study suggests a potential role for ACh in the release of chemotactic activity via the muscarinic M1 receptor. BEC appear to release NCA by way of the activation of calcium-dependent protein kinase C in response to endotoxin, opsonized zymosan, calcium ionophore, and phorbol myristate acetate (27). It is reported that the ACh M1 receptor (especially high affinity for pirenzepine) acts on the cell by the activation of phosphatidyl inositol turnover (38). Since inositol turnover results in protein kinase C activation and increased intracellular calcium concentration, these data are consistent with increased inositol turnover facilitation leading to the release of chemotactic activity. Anticholinergic drugs often dilate airways in persons who have asthma and decrease mucus secretions, trans-

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mucosal electrolyte and water flux, ciliary motility, and mucus transport (1, 5, 18, 44). Since ACh stimulated the release of inflammatory cell chemotactic activity and since inflammatory cells may play an important role in bronchial hyperreactivity, the present results suggest that anticholinergic drugs may additionally effect airway inflammation. In conclusion, BEC released NCA and MCA in response to ACh stimulation. The released activity was chemotactic by checkerboard analysis and low-molecularweight lipoxygenase-derived activity. The receptor responsible for the release of chemotactic activity in response to ACh was muscarinic M1 receptor. These results suggest that BEC may play a role in inflammatory cell recruitment into airways. Present address of S. Koyama: The First Dept. of Internal Medicine, Shinshu University School of Medicine, 3-l-l Asahi, Matsumoto 390, Japan. Address for reprint requests: R. A. Robbins, Pulmonary and Critical Care Section, Univ. of Nebraska Medical Center, 600 South 42nd St., Omaha, NE 68198-2465. Received 7 June 1991; accepted in final form 8 October 1991. REFERENCES 1. Altounyan, R. E. C. Variation of drug action on airway obstruction in man. Thorax 19: 405-415,1964. 2. Arnoux, B., M. H. S Caerio, A. Landes, M. Mathieu, P. Duroux, and J. Benveniste. Alveolar macrophage from asthmatic patients release platelet-activating factor (PAF-acether) and lyso-paf-acether when stimulated with the specific allergen (Abstract). Am. Rev. Respir. Dis. 125: A70,1982. 3. Atkins, P. C., M. Valenzano, E. J. Goetzl, W. D Ratnoff, F. M. Graziano, and B. Zweiman. Identification of leukotriene B, as the neutrophil chemotactic factor released by antigen challenge from passively sensitized guinea pig. J. AZZergy CZin. Immunol. 83: 136-143, 1989. 4. Behrens, B. L., R. A. F. Clark, D. C. Feldsien, D. M. Presley, L. S. Glezen, J. P. Graves, and G. L. Larsen. Comparison of the histopathology of immediate and late asthmatic and cutaneous responses in a rabbit model. Chest 87: 153%155S, 1985. 5. Bethel, R. A., and C. G. Irvine. Anticholinergic drugs and asthma. Semin. Respir. Med. 8: 336-371, 1987. 6. Boulet, L. P., A. Cartier, N. C. Thomson, R. S. Roberts, and F. E. Hargreave. Asthma and increases in nonallergic bronchial responsiveness from seasonal pollen exposure. J. Allergy CZin. ImmunoZ. 71: 399-406, 1983. 7. Boyum, A. Isolation of mononuclear cells and granulocytes from human blood. Stand. J. Clin. Invest. 21: 77-98, 1968.. 8. Bunning, S., S. Moncade, and J. R. Vane. The prostacyclinthromboxane AZ balance: pathophysiological and therapeutic implications. Br. Med. BUZZ. 39: 271-276,1983. 9. Cartier, A., N. C. Thompson, P. A Frith, R. Roberts, and F. E. Hargreave. Allergen-induced increase in bronchial responsiveness to histamine: Relationship to the late asthmatic response and change in airway caliber. J. Allergy CZin. Immunol. 70: 170177, 1982. 10. Clark, A. L, and F. Mitchelson. The inhibitory effect of gallamine on muscarinic receptors. Br. J. PharmacoZ. 58: 323-331, 1976. 11. Clark, P. O., D. J. Hanahan, and R. N. Pinckard. Physical and chemical properties of platelet-activating factor obtained from human neutrophils and monocytes and rabbit neutrophils and basophils. Biochem. Biophys. Acta 628: 69-75, 1980. 12. Cockcroft, D. W., R. E. Ruffin, J. Dolovich, and F. E. Allergen-induced increase in non-allergic bronchial Hargreave. reactivity. CZin. Allergy 7: 503-513, 1977. 13. Coleman, D. L., J. K. Tuet, and J. H. Widdicombe. Electrical properties of dog tracheal epithelial cells grown in monolayer culture. Am. J. Physiol. 246 (Cell Physiol. 15): C355-C359, 1984.

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Acetylcholine stimulates bronchial epithelial cells to release neutrophil and monocyte chemotactic activity.

Bronchial asthma is accompanied by inflammatory cell infiltration in the airway. Increased bronchial reactivity to cholinergic stimulation is well rec...
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