Human Nasal Respiratory Secretions and Host Defense1 •2

MICHAEL A. KALINER

The Human Nasal Mucous Membrane Mucous membranes are named for their capacity to generate mucus. Respiratory mucous membranes begin at the nasal vestibule and continue through the nose, pharynx, larynx, trachea, bronchi, and bronchioles. Although many studies have focused on airway secretions (1, 2), many recent investigations have employed human nasal secretions. For the purpose of this review, information derived from studies of human nasal secretions will be employed with the expectation that the information reflects, at least in part, lower airway secretions as well. Nasal airway secretions and their constituent proteins derive from epithelial cells (including goblet cells), submucosal glands (including both serous and mucous cells), blood vessels, and secretory cells resident in the mucosa (including plasma cells, mast cells, lymphocytes, and fibroblasts). Respiratory secretions consist of a mixture of mucous glycoproteins (3), glandular products, and plasma proteins (table 1). Baseline, resting secretions include the following major proteins: albumin (representing about 15070 of total protein), immunoglobulin (lg)G (2 to 4070), secretory IgA (15070), lactoferrin (2 to 4070), lysozyme (15 to 30070), non secretory IgA (about 1070), IgM « 1070), and mucous glycoproteins (about 10 to 15070). Careful immunohistochemical analysis has shown that plasma proteins such as albumin and IgG are found throughout the lamina propria of the mucosa, with an apparent increased concentration at the basement membrane (4). Some albumin can also be found staining submucosal gland lumens and tracking between epithelial cells, presumably being exported toward the airway lumen. Bycontrast, staining for lysozyme, lactoferrin, and S-IgA is seen exclusively in the serous cells, serous crescents, and ducts of the submucosal cells (5-8). More recently, several enzymes important in metabolizing neuropeptides also have been localized to the serous cells(9). Neutral endopeptidase, which metabolizes substance P and endothelin as well as other neuropeptides is also found in glands, as well as the nasal epithelium and in the endothelium of small blood vessels. Not only is neutral endopeptidase localized to serous cells, but NEP is also secreted in response to a variety of stimuli. Thus, by analysis of proteins found in nasal secretions one is able to determine the source of the proteins and to deduce the un552

SUMMARY The largest human body surface is the lining of the respiratory tract, gastrointestinal tract, and reproductive system each of which is covered by mucous membranes, named for their capacity to secrete mucus. Recent studies of mucus have defined some of the physiologic and pharmacologic controls of secretions. However,the constituents that are found in mucus and their roles in human health and disease are still in the initial phases of exploration. Human nasal respiratory secretions provide one convenient source of mucous membrane secretions. Nasal secretions include a variety of proteins, which appear to serve important functions in host-defense. Most, if not all, of the antiphlogistic products are synthesized and secreted by serous cells in the submucous glands, and it appears that the serous cell is the resident antimicrobial cell in mucous AM REV RESPIR DIS 1991; 144:S52-S56 membranes.

derlying mechanisms responsible for their secretion.

The Constituents of Human Nasal Secretions and Their Functions Mucous Glycoproteins The major proteins and other molecules found in nasal secretions are listed in table 1. It is clear that this complex mixture of molecules serves a variety of functions. Our current understanding of the roles and functions of respiratory secretions is summarized in table 2. Because the mucous membrane has no keratin layer to protect it from various exogenous stresses, mucus provides many of these functions. Thus, mucous glycoproteins (MOP) provide many of the protective functions of the outer layer of skin. Because' of the large size of the mucous glycoprotein molecule (200 to 400,000 D) and its capacity to polymerize extensively into a size greater than 2,000,000 D (3), MOP provides a replaceable, flexible, continuous extracellular surface coating and protecting the mucous membranes. This gelatinous layer insulates the epithelium, waterproofs it by trapping an aqueous layer beneath it, lubricates the surface, and humidifies the inspired air. Each MOP molecule absorbs waters of hydration onto itself, providing a generous source of humidification for inspired air. Temperature transfer through the airway secretions to inspired air is facilitated by the gel-like structure of mucus, which allows for a gradual transfer of heat to inspired air while protecting the underlying mucosa from excessive cooling. In a subject breathing nasally at rest, the transit time through the nose is estimated to take about 0.01 s. In this period of time, the inspired air is warmed from room temperature to 30 0 C (23). Humidification is completed by the time inspired air reaches the pharynx. The reverse process takes place dur-

ing exhalation. Air is cooled about 50 C by the time it reaches the nasal vestibule, and water content is reduced by about 25070. The mucosa and its surface secretions absorb the temperature and redistribute it to the next inspiration, while water exchanges with the mucus blanket.

Uric Acid The respiratory mucous membrane is constantly exposed to oxygen on its luminal surface. In order to restrict opportunities for oxygen-induced injury, nasal secretions include antioxidants. A number of antioxidants have been found in airway secretions, including lactoferrin, glutathione, transferrin, ceruloplasmin, and vitamin C, but the major antioxidant found in human nasal secretions was recently found to be uric acid (10, 11). Nasal secretions are approximately 5 ~M in respect to uric acid content, and this concentration can achieve 16~M when submucosal gland secretion is elicited. The source of uric acid appears to be glandular secretions, although the precise biochemical mechanism for uric acid production is unknown at this time. However, conditions that lead to glandular secretions enrich the surface fluid with uric acid, which greatly increases the antioxidant activity of secretions. Epithelial Lining Fluid The secretory blanket is thought to consist of two separable layers: the surface mucus (or 1 From the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland. 2 Correspondence and requests for reprints should be addressed to Michael A. Kaliner, M.D., Head, Allergic Diseases Section, Building 10, Room llC205, National institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.

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HUMAN NASAL RESPIRATORY SECRETIONS AND HOST DEFENSE

TABLE 1 CONSTITUENTS OF NASAL SECRETIONS Mucous cell products Mucous glycoproteins (3) Serous cell products Lactoferrin (4, 5) Lysozyme (5, 6) Secretory IgA and secretory component (7, 8) Neutral endopeptidase (9) Aminopeptidase Uric acid (10, 11) Glandulin (12) Peroxidase (13) Plasma proteins Albumin (8, 14) Immunoglobulins: IgG, IgA (monomeric, 7S), IgM, IgE (15) Carboxypeptidase N (16, 17) Angiotensin-converting enzyme (16) Kallikrein (17) Indeterminate sources CGRP (18) Urea (19) Inflammatory mediators Histamine (20) TAME esterase (20) PGD2 (20) Bradykinin (20) LTC.. (20) Tryptase (21) Major basic protein (22) Eosinophil-derived neurotoxin (22) Definition of abbreviations: Ig = immunoglobulin; CGRP = calcitonin gene-related peptide; PG0 4 = prostaglandin 0 4 ; LTC4 = leukotriene C4 -

gel) layer and a deeper aqueous (or serous) layer in which the base of the cilia is located. The concept of a two-layered epithelial lining fluid is quite old, dating back more than 50 yr. In the nose, particles trapped in the surface mucus layer are transported by mucociliary action to the posterior pharynx at the rate of 1 ern/min, The surface mucus blanket is then swallowed, and it is constantly being replaced, about every 10to 20 min under resting conditions. Analysis of lavage proteins indicates that only about 15070 of the total pro-

tein is attributable to MGP, although this figure fails to take into account that MGP is at least 80070 carbohydrate (3). Thus, the mucus blanket of MGP is secreted constantly and is constantly removed and replaced. This rapid turnover contributes to the barrier functions of the mucus blanket. Microorganisms and particulate materials are trapped in the mucus and passively removed by these processes. The blanket is selective, as large particles never reach the mucous membrane, whereas smaller molecules do and are readily ab-

TABLE 2 ROLES AND FUNCTIONS OF HUMAN NASAL SECRETIONS Protective functions Antioxidant (uric acid) Humidification Lubrication Waterproofing Insulation Provide proper medium for ciliary actions Barrier functions Macromolecular sieve Entrapment of microorganisms and particulates Transport media for elimination of entrapped materials Host defense functions Extracellular source of IgAllgG Extracellular site for multiple enzyme actions Antimicrobial functions Lysozyme Lactoferrin IgAllgG Glandulin Rapid deployment of multiple plasma proteins

sorbed. Thus, the mucus layer is a selective sieve. The layer on which the mucus floats and in which the cilia beat is the epithelial lining layer.On the basis of preliminary experiments, this layerof fluid appears to follow verydifferent kinetics than does the mucus blanket. The epithelial lining fluid (ELF) contains most of the aqueous proteins listed in table 1, many of which derive from the serous cells of the submucous glands. The proteins within the ELF appear to reconstitute themselves rapidly after repeated lavages, and may become concentrated over a 4- to 24-h period (19).The anterior portion of the nose is lined by about 100 JlI of ELF. The ELF layer does not turn over quickly, and the stability of this layer of fluid may provide many of the protective functions of secretions in host defense.

Host Defense Functions of Human Respiratory Secretions Inspiration of toxic or infectious materials deposits these potential pathogens onto the mucus blanket of the nose. The capacity of nasal secretions to neutralize or eliminate these potentially harmful pathogens is evident by the relative health that most of us enjoy as an ordinary part of life. The specific molecules in respiratory secretions that mediate the defensive capabilities of secretions are currently being defined, but it is quite clear that much remains to be identified.

Immunoglobulins The major specific mediators of host defense in secretions are the immunoglobulins. IgA and IgG are the major immunoglobulins in secretions, and they appear to act quite differently. IgG is a plasma protein that is distributed in the nasal mucosa by microvascular permeability. IgG is found diffusely throughout the mucosa, but in highest concentration near the basement membrane (15). Analysis of IgGproducing plasma cellsrevealsthat about 25070 of the plasma cells in the human nasal mucosa produce IgG, whereas the remainder are IgA-producing (15). IgA is largely produced locally by plasma cells located within 50 urn of submucous glands. The locally produced IgA is dimeric, being joined by a J chain before secretion. Dimeric IgA binds to secretory component produced by serous cells and forms secretory IgA. The S-IgA is transported transcellularly through the serous cells into glandular secretions and becomes part of the glandular secretions. S-IgA acts primarily by binding microorganisms in the airway lumen and preventing attachment of these potential pathogens to the mucosa. By contrast, IgG acts primarily in the mucosa itself to limit invasion by microorganisms that reach the epithelium. Although IgG is present in secretions (about 2 to 4070 of total protein in baseline secretions), it is found in a much higher concentration in the tissue fluid itself and in secretions after the process of vascular permeability has been increased. The

554

nonspecific antimicrobial properties of mucus are able to compensate for the functions of S-IgA as IgA-deficient patients are generally asymptomatic, suffering from a normal incidence of infections. By contrast, patients deficient in IgG present for medical help because of recurring respiratory infections. It therefore appears that the protective mechanism served in the mucosa by IgG are of greater importance in preventing the development of respiratory infections than is S-IgA.

Lysozyme There are several nonspecific antimicrobial proteins in nasal secretions that have broad spectrum antimicrobial actions. Lysozyme is a relatively small protein (14,000 D) found in all body secretions. Fleming (6) discovered lysozyme in human nasal secretions while searching for molecules capable of killingbacteria. He recognized that lysozyme killed most bacteria found in the air, but only some bacteria that ordinarily reside on the mucosa. Thus, lysozyme, which represents 15 to 30070 of the protein normally found in nasal secretions, effectively prevents mucosal infections from most airborne bacteria. Lysozymeis synthesized and secreted by the serous cell of the submucosal gland. Lactoferrin Lactoferrin is another antimicrobial protein made by serous cells that is both bacteriostatic and bacteriocidal to susceptible bacteria. Lactoferrin binds iron and it is presumably this action that kills bacteria. Lactoferrin constitutes about 2 to 4070 of nasal proteins. Glandulin In searching for additional antimicrobial factors in nasal secretions, a novel new activity has recently been described (12). Glandulin is a small « 1,000D), potent molecule secreted in glandular secretions that is bactericidal to multiple bacteria, particularly gram-negative bacteria such as Pseudomonas. The structure of glandulin is not known as yet, but its'presence suggests that searching secretions for antimicrobial factors may lead to additional molecules of potential importance. There are multiple specific and nonspecific antimicrobial factors in human plasma. Processes that lead to increased microvascular permeability (see below) cause the outpouring of plasma proteins into the nasal mucosa and then into nasal secretions. The increased volume of secretions may lead to clearance of particulate materials, as well as the availability of increased amounts of IgG, albumin (which may nonspecifically bind particulate materials), and other plasma proteins. It has been suggested that this outpouring of plasma proteins might represent the first Iine of host-defense at mucosal surfaces (24). Regulation of Human Nasal Secretions Adrenergic and Cholinergic Control After washing the nose to remove existing secretions, the relative amounts of proteins

MICHAEL A. KALINER

in nasal secretions is remarkably reproducible from patient to patient. However, initial washes reveal remarkable variability, probably reflecting the concentration of ELF over time. The innervation of the nose includes parasympathetic nerves that innervate the glands and vascular bed, sympathetic nerves that innervate the vascular bed, and sensory nerves that originate in the epithelium and arborize to include the vascular beds and glands (25). Parasympathetic nerves are the major motor nerves in the nose. Stimulation of parasympathetic nerves leads to increased secretions, whereas addition of cholinomimetics onto the nasal mucosa or initiation of oral gustatory reflexes leads to secretions (8, 26). Parasympathetic stimulation causes secretions enriched for the glandular proteins (table 3), although plasma proteins also are included in these secretions. When the relative amounts of proteins in secretions are determined by dividing the individual protein by the total protein concentration, a figure is generated that has been termed the "protein" percentage. The baseline protein percentages in nasal secretions were provided earlier, and changes in these percentages can provide information about the source of secretions. Thus, cholinergic stimulation of glandular secretion may change the relative percentage of lysozyme in secretions from 15 to 20070 to as much as 30070; lactoferrin from 2 to 4070 to 8070, and S-IgA from 15 to 25070. The plasma proteins found in glandular secretions do not change. Thus, albumin remains at 15070 of total protein and IgG at 2 to 4070. Adrenergic stimulation of the mucosa either has no effect on secretions (l3-adrenergic stimulation) or mildly stimulates glandular secretions (a-adrenergic stimulation) (27). The effects of adrenergic stimulation have been studied both in vivo and in vitro with similar findings.

Neuropeptides Sensory nerves contain several associated neuropeptides, including calcitonin gene-related

peptide (CGRP), gastrin-releasing peptide (GRP), substance P (SP), and neurokinin A (NKA) (28). The distribution of these neuropeptides has been carefully studied in the nasal mucosa by immunohistochemistry. Of the sensory nerves, all have some fibers apparently innervating submucosal glands. However, CGRP and GRP (as well as the parasympathetic neuropeptide vasoactive intestinal peptide [VIP]) are most evident with regard to glandular innervation (29-31). The presence of neuropeptide receptors was examined in the nasal mucosa by binding 125I-Iabeled neuropeptides to their binding sites. It was found that GRP had the most intense binding to glands, but both VIP and SP also had gland receptors (32). The capacity of these neuropeptides to cause mucous and serous cell secretion of specific products has been examined in vitro employing human nasal turbinates in short-term culture. GRP and VIP were the most potent stimuli for glandular secretion, although both SP and NKA had some activity as well. These data are summarized in table 4. In vivo challenge with neuropeptides with measurement of secretions has been reported for several potentially important peptides. For example, SP evokes nasal secretion in rats and dogs, but not in humans (33). SP and several other neuropeptides, including endothelin, VIP, and NKA, are metabolized by peptidases found in nasal secretions (9). Thus, the levelsof these neuropeptide-degrading enzymes may have profound influences on airway reactivity to released or secreted (18) neuropeptides. Animal models clearly reveal that modulation of neutral endopeptidase activity influences the reactivity of airways to the release of neuropeptides (34). Bradykinin is a mediator generated by action of the plasma or tissue enzyme kallikrein on the substrate kininogen. Bradykinin has been found in nasal secretions (16), has its receptors exclusivelyon blood vessels (35), and topical application onto the mucosa results in secretions rich in vascular proteins and is also associated with increased nasal blood flow (36).

TABLE 3 NASAL SECRETORY RESPONSES Protein

Stimulation

Response

Parasympathetic Plasma proteins Glandular proteins

+ +++ Histamine or allergen challenge

Ipsilateral Plasma proteins Glandular proteins Contralateral Plasma proteins Glandular proteins

++++ + + +++ Upper respiratory infection

Early phase Plasma proteins Glandular proteins Later stage Plasma proteins Glandular proteins

++++ + + +++

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HUMAN NASAL RESPIRATORY SECRETIONS AND HOST DEFENSE

TABLE 4 NEUROPEPTIDES AND HUMAN NASAL SUBMUCOSAL GLAND SECRETION

Neuropeptide GRP CGRP SP NKA VIP

Secretion

Glandular Innervation

Glandular Receptors

Mucous

Serous

++ ++ + + ++

+++

++

++

+

+ + ++

++ ++

++

Definition of abbreviations: GRP = gastrin-related peptide; CGRP = calcitonin gene-related peptide; SP = substance P; NKA = neurokinin A; VIP = vasoactive intestinal polypeptide.

Thus, it is likely that several neuropeptides play an important role in regulating the volume and nature 0 f nasal secretions. Mechanical stimulation of the mucosa leads to activation of irritant, sensory receptors with the release of SP, NKA, CORP, and ORP. SP, NKA, and ORP are each capable of stimulating secretions, and the degrees of secretions that result might be influenced by the capacity of several neuropeptide-degrading enzymes to metabolize these molecules. Parasympathetic discharge leads to the concomitant release of acetylcholine and VIP, both of which are potent secretagogues. VIP also is susceptible to degradation, and therefore, the relativeactivity of degrading enzymes might influence parasympathetic discharges as well. A new peptide, endothelin, is also found in the nasal epithelium and is secreted in response to a number of stimuli (37). Endothelin is also a secretagogue and is susceptible to neutral endopeptidase degradation. Information currently being generated from several laboratories indicates that neutral endopeptidase is modulated by conditions including viral infections and certain pharmaceutical agents (34). The response to neuropeptide release will therefore be determined by the number and affinity of receptors available for stimulation, the influence of inhibitory counter forces, and the presence of neuropeptide-degrading enzymes.

Summary and Conclusions Human respiratory secretions subserve many critical functions, both protecting the mucosa and providing essential host defense roles. It appears that the serous cell in the submucosal glands is the source of most of the protective proteins that prevent infections of the mucosa. Moreover, the serous cell also appears to secrete a number of enzymes, among which are those that metabolize neuropeptides, thereby preventing excessand perhaps inappropriate glandular secretions. The mucus in secretions is secreted by both goblet cells and mucous cells in the glands. The major component, the mucous glycoprotein, is a huge molecule consisting of a protein core to which multiple long-chain polysaccharides are attached. The individual glycoprotein molecules polymerize to form a family of proteins varying in size up to 2 million or more. The mucus floats on the ELF, which exists

in the space between the surface of the epithelium and the tips of the cilia. Although the mucus itself is rapidly transported to the pharynx and swallowed, the ELF .layer appears to be quite stable, providing a pool of molecules on the surface of the epithelium that can interact with any foreign proteins that manage to reach the mucosa. Although glandular secretions are an essential product of the mucosa, secretions are also derived from the vascular bed as a result of increased vascular permeability. These secretions can be recognized by a relative enrichment with vascular proteins such as albumin and IgO. Clinically, it appears that secretions derived from vascular permeability are somewhat thinner than glandular secretions, which might account for the variability of the rheologic assessment of secretions. Analysis of secretory responses to topical histamine (14), allergen challenge (38), and after an experimentally induced upper respiratory tract infection (39) reveals that vascular permeability is the under lying mechanism responsible. Thus, in those circumstances in which patients complain most commonly of increased secretions (as during a cold or in response to allergen exposure), it is really increased vascular permeability, and not glandular secretion, that is responsible. The proteins increased in secretions as a consequence of increased vascular permeability include IgO, IgM, IgA, albumin, and many other plasma proteins. These proteins mediate both specific (IgO) and nonspecific (albumin) antimicrobial functions. The sum results of stimulating nasal secretion is the outpouring of fluids capable of preventing or limiting infections, neutralizing toxic materials that may impact on the secretions, and elimination of the particulate materials that are trapped. Most humans have a limited number of viral infections and fleetingly few bacterial infections of their respiratory mucosa, speaking eloquently of the effectiveness of the host defense mechanisms involved. Identification of new and novel molecules in secretions (12)may provide useful information and novel direction to future pharmaceutical approaches to treat respiratory infections. Because secretions are designed to introduce molecules onto the mucosal surface that provide host-defense functions, it seems somewhat paradoxical to pharmaco-

logically restrict this secretion. Thus, it may be more useful in the course of a self-limited upper respiratory infection to increase glandular secretions (perhaps by eating hot soup, as my mother always suggests) and stimulating a gustatory reflex (26) rather than reducing secretions. In allergic rhinitis, where the secretions serve no apparent function, it seems appropriate to make patients comfortable by inhibiting vascular permeability.

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