Cationic Proteins and Bronchial Hyperresponsiveness

J. Mark Madison and Craig M. Schramm

Division of Pulmonary, Allergy, and Critical Care Medicine, University of Massachusetts Medical School, Worcester, Massachusetts; and Pediatric Pulmonary Division, University of Connecticut School of Medicine, Farmington, Connecticut

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Chronic airway inflammation in asthma is characterized by the preponderance of eosinophils in the airway walls and lumens. Eosinophils are unique among granulocytes in that they contain intracytoplasmic granules, which stain with eosin. These granules contain a number of preformed enzymes and other proteins, including cationic proteins such as major basic protein (MBP), eosinophil cationic protein, eosinophil-derived neurotoxin, and eosinophil peroxidase. Other granule enzymes include lysophospholipase, collagenase, -glucuronidase, and arylsulfatase B (1).

The principal granule protein in eosinophils is MBP, a highly positively charged, 117-amino acid polypeptide with a molecular weight of 14 kD. It is named for its highly basic isoelectric point (calculated to be 10.9) that is due to its large number of arginine residues (2). It is encoded from a 3.3-kb, five-intron gene located on chromosome 11 (3). MBP is synthesized in the cell as a pre-proprotein and is then processed and transported as a relatively neutral pH, 33-kD proprotein, presumably to protect the eosinophil from the toxic effects of MBP (4). Pro-MBP is converted into mature MBP after its sequestration in the granule crystalloid cores. Upon release, physiologic concentrations of MBP upregulate expression of intercellular adhesion molecule-1 on cultured human nasal epithelial cells (5). Moreover, MBP can induce eosinophil degranulation and the production of interleukin 8 by these cells (6). Thus, MBP can act as an autocrine factor to potentiate further eosinophil recruitment and activation in the airways. In addition, the highly cationic MBP reacts readily with acidic lipids, disordering lipid membranes and resulting in fusion and lysis (4). Accordingly, MBP is a potent bacteriocidal and helminthotoxic agent that is also toxic to tumor cells and other mammalian cells, including the airway epithelium.

Naturally occurring cationic polypeptides such as MBP have diverse cytotoxic and noncytotoxic effects on many different cell types due to interactions with cell-surface anion charges. The importance of the cationic charge is supported by the observation that synthetic polycations of similar size and charge, such as poly-L-arginine and poly-L-lysine, mimic many of the effects of MBP. Specific mechanisms are not known, but interactions of polycations with cell-surface anionic charges could interfere with normal cell function in a variety of ways, including the blockade of ion channels, inhibition of transporters, compromise of membrane integrity, and binding to or aggregation of integral membrane proteins (7). However, cationic charge alone may not be the only mechanism of action for naturally occurring cationic polypeptides. For example, contractions of guinea pig tracheal smooth muscle by MBP are inhibited by atropine, whereas the contractions due to synthetic polycations of similar charge are not (12). Thus, all of the effects of MBP may not be attributed to cationic charge.

Interest in eosinophil-derived cationic proteins, especially MBP, was stimulated by evidence implicating them in the pathogenesis of bronchial hyperresponsiveness (13, 14) (Table 1). In ovalbumin-sensitized guinea pigs and in patients with asthma, a significant correlation exists between the concentrations of MBP and methacholine hyperresponsiveness (15, 16). Additional evidence suggests that neutralization of endogenously secreted MBP, either with a polyanionic peptide or with antibodies to MBP, can prevent antigen-induced bronchial hyperreactivity in sensitized animals. Specifically, the polyanion poly-L-glutamic acid inhibits the bronchial hyperresponsiveness induced by MBP in primates (17) as well as the increase in vagal responsiveness induced by antigen provocation in guinea pigs (18). Similarly, anti-MBP antibodies suppress the development of bronchial hyperreactivity after antigen challenge in sensitized guinea pigs without affecting airway eosinophilia (19). Such studies suggest that MBP plays a key role in the alterations of airway function that accompany eosinophilic airway inflammation.

Initial in vivo (20) and in vitro studies (21, 22, 23) suggested that the effects of MBP on airway smooth-muscle contractility occurred indirectly, via effects on the airway epithelium. Exposure to MBP or synthetic polycations damages the airway epithelial barrier (24, 25) and increases epithelial permeability to hydrophilic tracers (26). Such actions would expose the underlying airway smooth muscle to higher concentrations of bronchoactive substances. Nevertheless, MBP also exerts important nontoxic effects on the airway epithelium. It has been shown that MBP affects epithelial eicosanoid metabolism, altering the balance between constricting and dilating prostaglandins (27, 28). Furthermore, recent evidence shows that polycations inhibit L-arginine uptake by airway epithelial cells (8) and that a deficiency in nitric oxide production contributes to polycation-induced airway hyperreactivity (9).

In addition to actions on the airway epithelium, cationic polypeptides such as MBP exert neuromodulatory influences on airway reactivity. An important observation has been that eosinophils cluster around airway nerves in patients with asthma. In one study of three patients with fatal asthma, 196 of 637 eosinophils (30%) were associated with nerves, and release of eosinophil MBP was evident. Conversely, in three control patients, 1 of 11 (9%) eosinophils were in contact with nerves (29). This increase in nerve- associated eosinophils was similar to the threefold increase seen in antigen-challenged guinea pigs (29). This histologic evidence makes it plausible that concentrations of MBP around nerves could be sufficient to affect neural function. Several neural pathways may be affected, but the most well characterized is the augmentation of cholinergic neurotransmission by MBP. A feature of cholinergic neurotransmission in the airways is that prejunctional neuronal M2 muscarinic receptors normally function to inhibit the release of acetylcholine from parasympathetic nerves in the airways. This negative feedback mechanism is attenuated in asthmatic individuals (30) and in animal models of asthma (31). In such animal models, the degree of M2 receptor dysfunction correlates with the number of eosinophils clustered around airway neurons (29), and good evidence suggests that MBP contributes to this M2 receptor dysfunction. Specifically, MBP is an allosteric antagonist at M2 muscarinic receptors in vitro (10), and pretreatment with anti-MBP antibodies prevents loss of neuronal M2 receptor function and the development of hyperreactivity to vagal nerve stimulation (32, 19). MBP may also modulate other neural pathways in the airways. MBP activates sensory nerves in the airways (33) and can induce release of substance P from cultured dorsal root ganglion cells (34). Substance P is a potent spasmogen for airway smooth muscle and a neuromodulator, potentiating the release of acetylcholine from cholinergic nerves in the airway. Bradykinin generation may be involved in this neuronal response in that the development of airway hyperreactivity in response to MBP or synthetic polycations can be blocked by pretreatment with a selective bradykinin-2 receptor antagonist (35).

However, not all the effects of MBP on airway smooth muscle are indirect. Recent evidence has demonstrated that cationic polypeptides may also directly influence smooth-muscle contractility. The first evidence for a direct effect on airway smooth muscle was a study showing that MBP and other cationic polypeptides increase cytosolic calcium in cultured bovine tracheal myocytes (36). Relatively low concentrations of MBP and poly-L-lysine cause transient increases in cytosolic calcium. Higher concentrations cause larger transient increases in calcium, which are followed by sustained increases in calcium at one hour. These findings suggest that cationic polypeptides from eosinophils could have direct effects on calcium homeostasis in airway smooth-muscle cells. Moreover, an important observation was made that cationic polypeptides also alter subsequent calcium mobilization by agonists. One hour of exposure to MBP augments calcium mobilization by bradykinin, such that both the peak of the calcium transient and the subsequent sustained plateau in calcium are increased. These responses to agonists vary among different cationic polypeptides. Also, higher concentrations of these peptides inhibit, rather than augment, responses to bradykinin. All these findings are interesting given the evidence that cationic polypeptides induce bronchial hyperresponsiveness. Although epithelium-dependent and neural-dependent mechanisms clearly play major, and probably dominant, roles in the development of bronchial hyperresponsiveness after cationic polypeptide exposure, findings for cultured tracheal myocytes raise the possibility that direct effects on smooth-muscle cells may be an additional contributing factor.

In this issue of the Red Journal, Oshiro and colleagues provide additional evidence that cationic polypeptides act directly on airway smooth-muscle cells, and they describe two potentially important mechanisms by which cationic polypeptides could regulate calcium in smooth muscle (37). Applying patch clamp techniques to freshly isolated tracheal myocytes, Oshiro and coworkers report that the synthetic polycations poly-L-arginine and poly-L-lysine rapidly and reversibly inhibit maxi-K⁺ channel (large conductance, calcium- and voltage-activated potassium channel) activity and depolarize cells by suppressing voltage-dependent, delayed rectifier potassium channel activity. These findings are notable for several reasons. First, the delayed rectifier potassium channel is a major determinant of resting membrane potential in airway smooth muscle (38). Thus, inhibition of this channel could conceivably contribute to the increases in cytosolic calcium reported by Wylam and coworkers (36). Second, maxi-K⁺ channels are coupled to -adrenergic signaling pathways in airway smooth muscle (39). And third, these findings also raise the possibility that inhibition of potassium channels might underlie some of the diverse effects that cationic polypeptides have on other cell types as well.

In the study by Oshiro and associates, the most characterized observation is the suppression of maxi-K⁺ channel activity by cationic peptides. Spontaneous transient outward currents (STOCs) represent the transient opening of maxi-K⁺ channels in response to the spontaneous and sporadic release of calcium from intracellular calcium stores (40). Poly-L-arginine and poly-L-lysine rapidly and reversibly suppress STOCs in the whole cell configuration and decrease the conductance and open-state probabilities of maxi-K⁺ channels in excised patch experiments. The findings are interesting because there has been much learned about maxi-K⁺ channels in airway smooth muscle (39). Convincing evidence shows that these abundant, plasma-membrane, potassium channels are coupled to -adrenergic receptors both directly (via guanine nucleotide binding proteins) and indirectly (via the synthesis of cyclic adenosine monophosphate) in airway smooth muscle (41). Although the role of hyperpolarization in -adrenergic relaxation remains controversial, evidence does indicate that -adrenergic agonists promote opening of maxi-K⁺ channels, with the consequent change in membrane potential favoring closure of voltage-gated calcium channels and lowering of intracellular calcium (39). Because of this link between maxi-K⁺ channels and -adrenergic relaxant mechanisms, Oshiro and associates suggest that the reversible inhibition of maxi-K⁺ channels by cationic proteins could attenuate relaxation of airway smooth muscle by -adrenergic agonists.

The new findings raise many questions. For example, is the inhibition of maxi-K⁺ and delayed rectifier potassium channels important for the enhanced mobilization of calcium by agonists (36)? Do native, rather than synthetic, polycations (such as MBP) also inhibit these potassium channels? What are the mechanisms underlying the direct effect of these peptides on potassium channels? In this regard, it is notable that maxi-K⁺ channel function in artificial membranes can be inhibited by removing anionic charges near the surface of the channel pore (7). Possibly, cationic polypeptide binding to these surface charges underlies the inhibition of maxi-K⁺ channel activity in airway smooth muscle.

Finally, to assess the implications of these new findings for -adrenergic relaxation, it will be important to establish that cationic polypeptides inhibit maxi-K⁺ channel opening in response to -adrenergic agonists. Even if this can be shown directly, however, it is notable that the role of maxi-K⁺ channels in -adrenergic relaxation of airway smooth muscle remains controversial and may be species- and preparation-dependent (39). Charybdotoxin, a potent inhibitor of maxi-K⁺ channels, strongly antagonizes relaxant responses to -adrenergic agonists in guinea pig trachealis (42). In contrast, the role of maxi-K⁺ channels in human airway smooth muscle is less clear, with charybdotoxin inhibiting relaxant responses in one study (43) but not others (44, 45). Therefore, if cationic proteins such as MBP do inhibit maxi-K⁺ channels in human airway smooth-muscle cells, the effect that this would have on -adrenergic receptor-mediated relaxation may or may not be large.

In summary, during airway inflammation, eosinophils release a variety of highly cationic proteins, particularly MBP, which have been implicated in the pathogenesis of bronchial hyperresponsiveness. Many mechanisms may underlie this augmentation of smooth-muscle contractility. Emerging evidence raises the possibility that one of these mechanisms involves direct effects of cationic proteins on calcium homeostasis in airway smooth-muscle cells.

	Footnotes

Address correspondence to: J. Mark Madison, M.D., Pulmonary, Allergy, and Critical Care Medicine, UMass Memorial Health Care-University Campus, 55 Lake Ave., North, Worcester, MA 01655.

(Received in original form February 14, 2000).

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