- Email: [email protected]
Studies on the allergic and nonallergic nasal inflammation
Studies on the allergic and nonallergic nasal inflammation* A. Togias, MD, R. M. Naclerio, MD, ** D. Proud, PhD, U. Pipkorn, MD,*** R. Bascom, MD, 0. Iliopoulos, MD, A. Kagey-Sobotka, PhD, P. S. Norman, MD, and L. M. Lichtenstein, MD, PhD**** Baltimore, Md. Nasal lavage after antigenic and nonantigenic nasal stimulation has become an important tool for the study of injlammatory phenomena in the upper airway. Biochemical and cytologic information is relatively easily obtainable, and pharmacologic manipulations can be readily monitored. This article is of several studies aiming toward a more profound understanding of the mechanisms of allergic and nonallergic rhinitis by the use of laboratory-challenge procedures and nasal-lavage techniques. An early and a late reaction are detected clinically in the nose after antigen challenge of allergic individuals. In addition, the sensitivity to antigen significantly increases after the initial challenge, and this phenomenon is not obligatorily linked to the presence of a late-phase reaction (LPR). Inflammatory mediators, mostly mast cell- andlor basophil-derived, are detected in the nasal washes and correlate with the symptomatology in both the early and the late reactions. The allergen-induced LPR is marked by an early influx of eosinophils and, later, basophils and neutrophils. Elevation of major basic protein and histamine, but not prostaglandin D, , is detected during the LPR, giving evidence of active eosinophil and basophil participation. Systemic steroids can effectively suppress the clinical, biochemic, and cellular manifestations of antigen-induced LPR. Topical steroids have a similar effect but are also capable of suppressing the early reaction to antigen. A nonallergic form of rhinitis can be induced in the laboratory by nasal inhalation of dry air at freezing temperatures in individuals who report sensitivity to cold and windy environments. Early and late reactions are also detectable with this stimulus, and the panel of mediators appears to be identical with that of antigen-induced rhinitis, indicating activation of the same cell type(s). This stimulus, however, appears to act through dtflerent mechanisms, since pharmacologic agents that control the allergen response are inactive against cold, dry air (CDA) reactions. Studies on the mechanism of CDA-induced rhinitis have detected elevations in the osmolality of nasal secretions after CDA challenge. This occurs only in CDA-sensitive subjects and not in CDA nonresponders or antigen-challenged allergic individuals. Elevated osmolality correlates with mediator release, suggesting a role of nasal fluid tonicity in mast cell activation. The above pieces of information offer signijicant insight in the mechanisms of allergic and nonallergic intammation of the nose and the upper airways. Reconstitution of that information into a persuasive model of the naturally occurring disease and development of new therapeutic modalities will be our tasks in the future. (J ALLERGYCLIN IMMUNOL 1988;81:782-90.)
From the Department of Medicine, Division of Clinical Immunology, and the Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine, and the Center for Occupational and Environmental Health of the Johns Hopkins Medical Institutions, Baltimore, Md. Supported by National Institutes of Health Grants AI 07290, AI 08270, HL 32272, NS 22488, and HL 37119. Presented at the Fifth Annual Aspen Allergy Conference, Aspen, Colo., July 1987. Reprint requests: Alkis Togias, MD, Nasal Laboratory, The Good Samaritan Hospital, 5601 Loch Raven Blvd., Baltimore, MD 21239.
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Publication number from the O’Neill Laboratories at the Good Samaritan Hospital, Baltimore, Md. *Sponsored by the Colorado Allergy Society. **Recipient of Teacher Investigator Development Award 5 K07 NS 00811 from the National Institute of Neurological and Communicative Disorders and Stroke. *** Recipient of the International Research Fellowship Award (NIH F05 TWO 3516-01) from the Fogarty International Center. ****Recipient of a Pfizer Biomedical Research Award.
VOLUME NllMRER
8I 5 PART 1
Allergic and nonallergic rhinitis is a wellrecognized entity affecting a significant percentage of the population.’ Allergic rhinitis has, of course, been attributed to the inhalation of specific antigens to which susceptible individuals are sensitized. Basic experimental knowledge of type I immunologic reactions has ied to the assumption that tissue anaphylaxis involves activation of stationary mast cells by IgEantigen interaction.’ Furthermore, since it has been well recognized that histamine is a major inflammatory mediator released by triggered mast cells, antihistamine medications (H, antagonists) have, for years, constituted our major pharmacologic means for the control of allergic rhinitis. In the past two decades, several other inflammatory compounds, such as arachidonic acid metabolites, were found in the supernatants of antigenically activated mast cells from rodents,’ but only recently have human mast cells from different sources been purified and their inflammatory mediator armamentarium explored.4-6The emergence of this knowledge has shifted our therapeutic strategy toward mast cell stabilizing agents in order to avoid the task of simultaneous use of multiple antagonists for all the mediators involved in allergic reactions. However, not only does the above strategy appear difficult to expedite but also our understanding of the natural history of allergic rhinitis appears to be far from clear. Deliberate efforts to reproduce the disease in the laboratory, in humans, extend back to the late nineteenth century.’ These efforts have become much more sophisticated in our time,X-‘”and the method of nasal lavage has been recently applied in our laboratory to quantitate several mediators of inflammation after nasal antigen challenge of allergic individuals. “-z However, (1) the immediate clinical response of the nose to antigen lasts about 30 minutes, whereas naturally occurring allergic rhinitis is a chronic disease, and (2) the dose of pollen needed to induce a nasal allergic reaction in the laboratory frequently far exceedsthat inhaled by an atopic individual in a single day during the pollen season. The immediate reaction that occurs after antigen challenge in the lung, skin, or nose of allergic patients is followed by a quiescent period lacking symptoms or mediator peaks. In many cases, a recrudescenceof the symptoms and signs, as well as of the physiologic changes of the immediate reaction, will be observed several hours later. “-I7 This is known as the LPR. The clinical significance of those reactions is intriguing, and the pathogenesisis poorly understood. The belief that LPRs might represent the naturally occurring allergic disease much more closely than does the acute response” has led to extensive investigations of this
Allergic
and nonallergic
nasal ?nftar~mat~on
Abbre~htions used LPR: MBP:
Late-phase reaction
LTB.,. LTC,, LTD,: CDA: WMA:
Leukotricne B,, Q ’ :txtd D. Cold, dry a:r Warm. moix: .ilr
783
Major basic protern PGD,: ProstaglandinD.
phenomenon during the past few years. This concept is mainly substantiated by the fact that LPRs are suppressed by pretreatment with steroids. whereas early reactions are not. Steroids are, indeed. our most efficacious therapeutic modality in the treatment of allergic diseases. W e have applied the nasal lavage techniques to study LPRs in the nose. This has enabled us to quantitate the amount of inflammatory mediators produced and to simultaneously quantify the cellular components of the lavage fluids. Furthermore, we have been able to study the effects of pharmacologic agents, such as steroids, on the clinical, htochemical, and cellular aspects of nasal LPRs. The clinical picture of several other forms of chronic nasal disease is very similar to that of aliergic rhinitis.‘9-2’This has tempted us to investigate the role of mast cells and their mediators in these pathologic entities. Since there appears to be a large spectrum of precipitating factors, some of which are not well documented, we have focused on the group of patients who report symptoms of rhinitis on exposure to cold and windy environments and not to any other climatic condition. A nasal challenge model with ClDA as the stimulus and nasal lavage for the detection of inflammatory mediators has been developed.-” METHODS Subjects These studies have involved three groups of volunteers: (1) Individuals had documented seasonalallergic rhinitis to ragweed and/or to grass pollens. Documentation was based on both their history and the results of intradexmal skin testing with a panel of aeroallergens that are prominent in the Baltimore/Washington, D.C. area. Those who participated in the trials of steroids in the LPR previously had been demonstrated to develop an LPR in response to nasal antigen challenge. All studies were performed during pollen-free months. (2) Subjects who participated in the CDA nasal challenges had a history of rhinitis on exposure to cold and windy environments, regardless ot their atopic status and the possible concomitant seasonal or perennial, allergic or nonallergic rhinitis. (3) Appropriate control volunteers without allergic rhinitih or without Gensitivity to
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Togias et al.
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CLIN. IMMUNOL. MAY 1988
FIG. 1. An example of an immediate and an LPR to antigen challenge. (From Togias A, Naclerio RM, Proud D, et al. Mediator release during nasal provocation: a model to investigate the pathophysiology of rhinitis. Am J Med 1985;79(suppl 6A):26-33.)
CDA were used in several studies. All our volunteers gave informed consent, and the studies were approved by the Joint Committee on Clinical Investigation of the Johns Hopkins Medical Institutions. Antigen challenges were performed with the use of either ragweed or mixed grass-pollen extract or ragweed pollen at doses ranging from IO to 1000 protein nitrogen units and 10 to 10,000 grains of ragweed pollen. The antigen was insufflated in the nostrils of participating volunteers. CDA ( - 3” to - 10’ C temperature and 0% to 10% relative humidity) was inhaled through the nose at a flow of 12.5 L/min and was exhaled through the mouth. Control challenges either with the pollen-extract vehicle (normal saline with phenol) or with lactose powder for antigen provocations and with WMA (29” to 35” C temperature and 95% to 100% relative humidity) for CDA challenges were performed. Nasal secretions were obtained by instilling 2.5 to 5 ml of normal saline or lactate Ringer’s solution into each nostril. These nasal lavages were always performed at baseline and after any manipulations, such as control or antigen challenge, WMA or CDA challenge, or drug administration. During the LPR studies, a nasal lavage was performed every hour for 11 to 12 hours after the initial nasal challenge. All mediator assays on aliquots of nasal lavage fluids used in these studies have been previously described in detail. Histamine was measured with a spectrofluorometric assay sensitive to 1 ng/mI,*3 TAME-esterase measurements required radiochemical assay,24and PGD2, leukotrienes C,, D,, and E,, and MBP were quantitated with the use of radioimmunoassays. ‘*. “-” In addition, bradykinin and lysylbradykinin were measured with radioimmunoassay,* as was albumin (a marker of plasma transudation).gO
FIG. 2. Initial challenge and rechallenge mediator response in a group of 13 allergic volunteers. The vertical dashed/he indicates that 11 hours have elapsed between the last step (1000 protein nitrogen unit [PNU]) of the initial challenge and rechallenge. Note that the dose of antigen used in the rechallenge is 10 PNUs, that is the lowest dose of the initial, three-step, provocation. NS = not significant; *p < 0.05; **p < 0.01.
The processing of the cells found in the nasal washes also has been previously described.3’ Briefly, the returned nasal lavage fluids (lactated Ringer’s) were initially centrifuged, and the pellets were resuspended in RPM1 with n-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid. Thereafter, they were vigorously shaken and exposed to N-acetyl cysteine for 45 minutes. After resuspension with the previous medium, cell counts were performed by hemocytometer, and differential counts for neutrophils, monocytes, eosinophils and epithelials required cytospin slides stained with Diff-Quick (Scientific Products, McGraw Park, Ill.), a modified Wright stain, whereas alcian blue-staining positive cells were identified from separate aliquots with alcian blue staining. Morphologic criteria under light microscopy were used to differentiate between basophils and mast cells among the alcian blue-positive cells.32 Osmolality measurements in the nasal lavage fluids were performed with vapor pressure osmometry. In situ osmolality of the nasal secretions was measured by direct application of filter paper disks over the inferior turbinate. After being saturated with nasal secretions, these disks were immediately transferred to the vapor pressure osmometer a (SlOO B microosmometer, Wescor, Inc., Logan, Utah).
VOLUME NUMBER
81 5, PART 1
Allergic and nonallergic
nasal rnfl
785
TABLE1. Mediators detected in nasal lavage fluids Antigen challenge Early phase
Histamine TAME-esterasets) Kinins pcm Leukotrienes(C,, D,, E,) MBP Other prostaglandins LeukotrieneB,
+” +” + 29 +” + II + 34 + ‘I + 3.5
Late phase
+ ia -t i1 + ‘I - 42 & ii + 34 NT + ih
CDA challenge -- ._.. “-.. _ -- ..-_- ._ Early phase Late phase --“-
.+ ‘2 + 11 t 12 + 12 ,. 1;; NT NT
.)“i k’i :i;y N’i 2‘1 4’1
NT = not tested
Nasal antigen provocation
mediatorsfound in the LPR lavagefluids and that they may play a significant role in this reaction.” a characteristicexampleof the eventsthat follow nasalchalLPRs and nasal priming. If we are to define the LPR as a twofold or greater increaseabove baselinein symptom lenge of an allergic individual with antigen. Serial nasal scoresand in two of the three mediatorsthat we commonly lavagesare performedbefore.the challengeto reducesmall, measure(histamine,TAME-esterase(s).and kinins), a 37% resting-phasemediatorlevels to a stablebaseline.A control prevalencecan be demonstratedin a pKviously unscreened challengewith the vehicle usedfor the subsequentantigenic group of patients with ragweedhay fever.4’So far, no alprovocationis performedto rule out nonspecificreactivity. lergic individual without an early nasalreactionto antigen An immediate response,a quiescentperiod, and an LPR areeasily recognizable.In this case,only histamine,TAMEhas demonstratedevidenceof an LPR. Thus, althoughit is not a ubiquitousphenomenonunder our laboratorycondiesterase(s),PCD,, and kinins were measured.However, tions, nasal LPR can be observedonly when a previous. severalother inflammatorymediatorshavebeendetectedin the nasal lavage fluids, as presentedin the left panel of biochemically demonstrableearly reaction occurs. NeverTable I. Among thesemediators,histamineis the only one theless,there is no significant correlation betweenthe inthat can be confidently attributed to m&t cells and/or batensity of the early and the late reaction, suggestingthat sophils. PGD, is the major cycloxygenaseproduct of mast factors other than simple mediator releasemay affect.the cells and is not produced by basophils.25, 33 TAMEmagnitudeof this inflammatoryprocess,” esterase(s)activity in the nasal fluids representsa mixture Another, potentially very important, phenomenonis obof the activity of plasma kallikrein, glandular kallikrein, served. After the initial antigen challenge,rhe sensitivity and, to a lesserdegree,mast cell tryptase, which can be a of the nasal mucosato antigen increases.This is demonmarkerof mast cell degranulation.34.j5 Thesekinin-forming strablein Fig. 2 where, after an initial three-stepchallenge enzymesappearin the nasal sectionstogetherwith highprotocol, an antigenchallengewith the lowest doseinitially and low-molecular-weightkininogens30 and are responsible used is repeatedat 11 hours. This rechallengereaction is for the formation of kinins, the pharmacologicpropertiesof significantly intensifiedwith regardta symptamsand to the which include vasodilationand increasedcapillary flow, as levelsof mediatorsin the lavagefluids, as comparedto those well as increasedvascularpermeability.36.37Recentstudies obtainedwith the sameantigendosein the initial challenge. indicate that nasal provocation with bradykinin induces This phenomenonbecomeseven more obvious when the three-or four-stepdose-response curveto antigenis repeated symptoms of rhinitis and a sore throat.” The source of every 24 hours for 3 consecutivedays. In that case, a sigleukotrienesin the nasalsecretionsis unknown, sincemany nificant shift in the dose-response curve is obtained,and a cell types presentin the nasalmucosa,such as mast cells, basophils,eosinophils,and neutrophils, and macrophages largepercentageof the participatingvolunteersbecomeable to respondin the laboratoryto antigendosesthat could easily are capableof generatingthose substances.‘39-4’ 3. MBP is be inhaledduring the ragweedhay fever seasonwithin minreleasedby eosinophilson activation. It is a potent toxin The intensificationof the clinical for epithelial cells42and has recently been demonstrated utesof naturalexposure.46 by the numberof sneezes,appearsto capableof causingmediatorreleasefrom rodent basophils response,as assessed and mast cells.43W ith regard to the panel of mediators correlatewell with the increasein the numberof white blood cells in the nasalfluids of the lavagesperformedeveryday obtainedin the immediatereaction and the LPR of experbefore the challenge.4s This observation. termed ‘%asal imental allergic rhinitis, a striking differenceis notedin that PGDZ,while it is presentin the early phase,is absentin the priming,” has beenpreviouslyreportedin both a laboratory and a clinical setting.“’It appearsthat it can provide the LPR. This has temptedus to postulatethat basophils,and means to close the gap between experimental antigen not mast cells, may be the source of at least part of the Mediators of the early and late reaction. Fig. 1 represents
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J. ALLERGY
Togias et al.
II EP
EP
LP
CLIN. IMMUNOL. MAY 1988
LP
FIG. 3. Mean net increases in mediator levels (-+ SEM) in nasal lavage fluids and the total number of sneezes in 13 patients that participated in the study of the topical steroid (flunisolide) effect on the early phase (EP) and late phase (LP) reactions. Mediator levels in the EP are calculated by pooling of lavage samples obtained 10 minutes after each of three consecutive antigen challenges with increasing doses, as illustrated in Fig. 2, plus the sample from a lavage performed 20 minutes after the third antigen challenge. LP values represent pooled hourly samples during postchallenge hours 2 to 11. All variables were significantly reduced by pretreatment with topical steroids; *p < 0.05; **p < 0.01.
TABLE II. Effect of systemic
giucocorticoid
treatment
on nasal
secretion
Early reaction Placebo
Histamine (rig/ml) Kinins (ng / ml) TAME (cpm x 10e3) LTC, (D,, Ed) WO.1 ml)
6.8 7.4 48.4 606
k k
1.8 1.7
+ 10 ? 110
mediator
levels
Late-phase Steroids
6.6 3.3 38.8 373
Placebo
reaction Steroids
k 3.1 f 0.6* t 4.0
6.7 k 3.0 3.8 * 1.3 18.0 k 6.0
2.3 5 0.8* 0.9 k 0.3* 5.4 + 1.5*
k 100
271 +- 50
143 * 25t
*p < 0.05. tp < 0.06.
challenge and the naturally occurring allergic disease with regard to the doses of antigen used. Interestingly enough, an obligatory link between priming and the occurrence of an LPR under the conditions in which our studies where carried out does not appear to exist.45 However, as illustrated in Fig. 2, the fact that the only mediator not following the intensification trend is PGD, suggests that the increased sensitivity to antigen may partially result from the recruitment and activation of basophils in the nasal mucosa and that the pathogenesis of LPR and priming may be closely related. Pharmacologic manipulations with steroids. We have recently concluded two studies evaluating the effects of systemic and local steroids on the early reaction and the LPR in the nose.48.49 Oral prednisone was administered to 13 allergic individuals in a double-blind, placebo-controlled, crossover study (2 days pretreatment with 20 mg, three times
a day). In the second study, topical flunisolide was insufflated in both nostrils twice a day (total dose was 200 pg/day) for 1 week before nasal antigen challenge. This was also a double-blind, placebo-controlled, crossover study involving 13 allergic subjects. Table II and Fig. 3 present the results from the oral and the local steroid-treatment studies, respectively. As expected, oral steroids significantly suppressed the LPR with regard to both mediators and symptoms but did not affect the immediate reaction to antigen challenge. Since in vitro data with purified human lung mast cells and purified human peripheral blood basophils demonstrate steroid inhibition of mediator release in response to antigen only for the latter,50,51this in vivo study offers additional evidence of the active participation of basophils in the human nasal LPR. In contrast to oral steroids, topical flunisolide effectively inhibited not only the late phase but
VOLLiME 81 hllJMI
also the immediate allergic response to antigen. It is, in fact. difficult to assesswhether the ablation of the LPR was an independent effect or a result of the early phase suppression Either way, this study offers additional reasons for preferring topical administration of corticosteroids in allergic rhinitis and substantiates the common practice of initiation of’topical steroid treatment several days before the pollen season begins. It is notable that with both oral and local steroids, the increased sensitivity to antigen, as detected at 1 I hours after the initial challenge, was found to be completely suppressed. thus offering more evidence for the importance of this phenomenon in the natural history of allergic rhinitis. Cellular studies qf the LPR. One of the important advantages of the nasal lavage technique is that it provides the ability for simultaneous measurement of mediators and characterization of the cellular components in the lavage tluids. Thus. not only can we comment on the influx of specific cell types but we can also follow their degree of activatron. if specific biochemical markers are detectable. We have completed a series of studies of the cellular pattern in the nasal washes during the LPR.3’.5’ A number of inflammatory cells appears to populate the surface of the nasal mucosa during that reaction. The pattern of this influx is highly variable among individuals, as is the case for mediators. Eosinophils demonstrate a significant increase in the nasal secretions within 1 to 2 hours after antigen challenge and reach their peak at 7 to 10 hours. The detection of increased levels of MBP in the LPR correlates with the eosinophil influx, suggesting eosinophil activation in the LPR .” The role of the eosinophil becomes even more pronounced by the fact that treatment of allergic subjects with systemic or topical steroids not only ablates the LPR but also significantly inhibits eosinophil recruitment and MBP release.” Neutrophils enter the nasal secretions somewhat later than eosinophils, but they represent the greatest number of infiltrating cells in the LPR. Their significance is, however, uncertain. Steroid treatment does not appear to affect either the number of neutrophils in the lavage fluids of the LPR” or the levels of leukotriene B,, which is believed to be partially neutrophil derived.53.54Nevertheless, assessment of neutrophil activation status is difficult, since we did not identify a specific marker in nasal washes. Mononuclear cells also increase, albeit not significantly, 9 to 10 hours after antigen challenge. Epithelial cells remain unaffected by the LPR. Since our early speculations regarding the participation of basophils in the LPR,‘” we have been particularly interested in the detection of these cells in nasal fluids during the course of this reaction. We have observed that alcian blue-positive staining cells significantly increase in percentage and total number in the LPR, as compared to baseline or immediate postchallenge periods. Nevertheless, they do not exceed I % of the total cells.3’ These cells were studred under light microscopy, with previously established morphologic criteria? Approximately 70% of the cells were believed to be compatible with basophils, < 10% were identified as mast cells, and the remainder were indeterminate.” The number of alcian blue-staining cells correlated signif-
Allergic
and nonallergic nasa\
Intim*matior:
787
* 4 H 3 2 I! R
:
FIG. 4. An example of mediator release in nasal lavage fluids and symptom scores from one subject chahenged sequentially with W M A and CDA. Downward arrows represent nasal washes; dashed lines represent the degrees of sensitivity of the assays used. (From Togias AG, Naclerio RM, Peters SP, et al. Local generation of sulfidopeptide leukotrienes upon nasal provocation with Cold, dry air. Am Rev Respir Dis 1986;133:1133-7 1
icantly with the levels of histamine in the LPR. Furthermore. topical steroid pretreatment prevented the influx of alcian blue-positive staining cells, since it prevented mediator elevation and ablated the symptoms of this inflammatory process.” These findings have also strengthened our hypothesis of active basophil participation in the LPR.
Nasal provocation
with CDA
An example of the response to nasal challenge with CDA of an individual who gave a history of nasal symptoms after
788 Togias et al.
exposure to cold and windy environments is presented in Fig. 4. Initial challenge with WMA had no effect on these subjects. Their specific sensitivity to CDA is obvious by the fact that all mediators measured in the returned lavage fluids are elevated after this challenge and correlate significantly with symptom scores and with the clinical evaluation by the observer.**. *‘. 55 No symptoms or mediator elevations are detected in individuals who deny any sensitivity to CDA when they are challenged in the laboratory. As noted in the right panel of Table I, all the mediators that have been detected so far in the nasal secretions during the immediate reaction to CDA are also produced during the early reaction to antigen in atopic subjects. This certainly suggests common cellular sources for the two different reactions. Furthermore, recent studies have demonstrated the presence of an LPR in certain individuals after CDA challenge.* The cellular components of nasal secretions in the LPR of CDAinduced reactions have not been studied yet. Nevertheless, it is tempting to speculate that mast cells lining the mucosal surface may be largely responsible for the immediate CDA reaction. The question that has risen, therefore, involves the mechanism of mast cell and/or other cell activation during the response to CDA. This appears to be different from the antigen-IgE interaction. Strong evidence is offered by the fact that pretreatment of CDA-sensitive volunteers with azatadine, a tricyclic antihistamine previously demonstrated to inhibit the nasal reaction to antigen challenge,56 has no effect on CDA-induced rhinitis and mediator release.55 Since one of the expected effects of CDA would be drying of the nasal mucosa through evaporation of water needed for maximum humidification of inspired air, we attempted to measure the osmolality of the returned lavage fluids before and after CDA challenge. Several other observations led us to those studies: (1) Human lung mast cells are triggered to release mediators when they are exposed to hyperosmolar media.57 (2) Instillation of hyperosmolar solutions in the nasal cavities of certain individuals will result in mast cell mediator release.” We have now demonstrated that the osmolality of the nasal secretions after CDA challenge of CDAsensitive subjects significantly increases from baseline and correlates with mediator release. In contrast, osmolality remains stable after WMA challenge of CDA-sensitive volunteers, as well as after CDA or WMA challenge of individuals who deny sensitivity to CDA.59 Furthermore, no changes in the osmolality are detected in atopic subjects when they receive antigen challenge and demonstrate significant mediator elevations. The changes in the osmolality are more pronounced when in situ measurements are obtained by saturation of small filter paper disks with nasal secretions after CDA challenge.59These findings are leading us to the assumption that elevated osmolality may be the cause of mast cell activation in CDA-induced rhinitis. Furthermore, they tempt us to generate several other speculations: (1) The lack of elevation in the osmolality of the nasal
*Iliopoulos 0. Personalcommunication.
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secretions in the control group of volunteers points to a specific defect in the ability of the nasal mucosa of CDAsensitive subjects to humidify inhaled air under extreme conditions. Mast cell mediator release may act to overcome this “malfunction” by increasing vessel permeability and glandular secretions. (2) If a spectrum of response of CDA exists among the population, the role of nasal secretion tonicity and subsequent mast cell mediator release may be important in the natural homeostasis of air humidification by the upper airways. (3) Since the stimulus of CDA in the nose appears to be the same as that causing exercise-induced bronchospasm in subjects with asthma, a similar mechanism of mediator release may account for that pathologic entity.
CONCLUSIONS With different types of nasalprovocationfollowed by nasal lavage, we have engagedin studies of the pathophysiologyof upper airway disease.The combination of information on the biochemical,cellular, and physiologic characteristicsof severalaspectsof allergic and nonallergicrhinitis has alreadyled to improved knowledgeabout thoseconditions. The question remains whether this knowledge will generate new and moreefficaciousconceptsregardingour therapeutictools. This will, of course,be a challengefor years to come. REFERENCES I. National Health Survey. Series 10; No. 100, September1975. 2. Plaut M, Lichtenstein LM. Cellular and chemical basisof the allergic inflammatory response.In: Middleton E Jr, ReedCE, Ellis EF, eds. Allergy: principles and practice, vol 1. 2nd ed. St. Louis: The CV Mosby Co, 1983:119-46. 3. WassermanSI. The mast cell and the inflammatory response. In: PepysJ, EdwardsAM, eds. The mast cell: its role in health and disease.London: Pitmaa Medical, 1979:9-20. 4. Lichtenstein LM, Kagey-Sobotka A, MacGlashan D W Jr, PetersSP, KazimierczakW, SchleimerRP, SchulmanES. Mediator release:studieswith purified human basophilsand mast cells. In: Kern JW, GandertonMA, eds. Proceedingsof the Eleventh International Congressof Allergology and Clinical Immunology. London: The MacMillan PressLtd, 1983:11-15. 5. Pox CC, Dvorak AM, PetersSP, Kagey-SobotkaA, Lichtenstein LM. Isolation and characterization of human intestinal mucosal mast cells. J Immunol 1985;135:483-91. 6. Lawrence ID, Warner JA, Cohan VL, Hubbard WC, KageySobotka A, Lichtenstein LM. Purification and characterization of human skin mast cells: evidence for human mast cell heterogeneity. J Immunol 1987;139:3062-9. 7. Blackley CH. Experimental researcheson the cause and nature of catarrhusaeatinus(hay fever or hay asthma).London: Bailliere, Tindal and Cox Ltd, 1873.
8. Taylor G, Shivalkar PR. Changesin nasal airways resistance on antigenic challenge in allergic rhinitis. Clin Allergy 1971;1:63. 9. McLean J, Clarkowski A, Solomon W, Mathews K. An improved technique for nasal inhalation challenge tests. J ALLERGY CLIN hiMUNOL 1976;57:153-63. 10. Connell J. Quantitative intranasal pollen challenge. J ALLERGY 1968:41:123-39.
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1 I Naclrrio RM, Meier HL, Kagey-Sobotka A, Adkinson NF Jr, Meyers DA, Norman PS, Lichtenstein LM. Mediator release after nasal airway challenge with allergen. A m Rev Respir Dis
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36. 37.
1981;67:253-62.
22. Togias AC. Naclerio RM, Proud D, Fish JE, Adkinson NF Jr, Kagey-Sobotka A, Norman PS, Lichtenstein LM. Nasal chalienge with cold, dry air results in release of inflammatory mediators: possible mast cell involvement. J Clin Invest 1985;76: 1375-x I. 13. Firaganian RP. An automated continuous flow system for the extraction and fluorometric analysis of histamine. Anal Bioihem 1974:57:283-94. 24. lmanari T, Kaizu T, Yoshida H, Yates J, Pierce V, Pisano JJ. Radiochemical assaysfor human urinary, salivary, and plasma kallikreins. In: Pisano JJ. Austen KF, eds. Chemistry and biology of kallikrein-kinin system in health and disease. Washtngton, D.C.: Department of Health, Education, and Welfare, publication No. (NIH) 76-791, 1976:205-13. 25. Schulman ES, Newball HH, Demers LM, Fitzpatrick FD. Adkinson NF Jr. Anaphylactic release of thromboxane A,, pros:agIandin D,, and prostacyclin from human lung parenchyma. 4 m Rev Respir Dis 1981;124:402-6. 26. Have< EC, Lombard0 DL, Girard Y, et al. Measuring leuCotrienes of slow-reacting substancesof anaphylaxis: develi)pment of specific radioimmunoassay. J lmmunol 1983:13 I:
38.
79:x4. 39. Conroy MC, Orange RP, Lichtenstem 1.M Release of slow-
40.
41.
42.
43.
-329-33.
27. Togias AG, Naclerio RM, Peters SP, Nimmagadda 1, Proud D, Kagey-Sobotka A, Adkinson NF Jr, Norman PS, Lichten>tein LM. Local generation of sulfidopeptide leukotrienes upon nasal provocation with cold, dry air. A m Rev Respir Dis 1986;133:1133-7. 28. WassomDL, Loegering DA, Solley GO, Moore SB, Schooley KT. Fauci AS. Gleich GJ. Elevated serum levels of the eosin-
ophil granule major basic protein in ‘wticnt\ ,,:,ttir :lks;nbmhilla. J Clin Invest 1981:67:651-61. Proud D. Togias A. Naclerio RM. crush :?:\. torman I’S, Lichtenstein LM. Kinins are generated in v:\t’ !‘oll~>wrngnaqal airway challenge of allergic individualr u 1111 ali:~en I Cbn Invest 1983;72:1678.85. Baumgarten CR, To~las AG, Naclerlo K M l.&ccnstem L.M. Norman PS. Proud D. Influx ofkinino~cns ultr; ~sai secretion5 after antigen challenge of allergic ~ndivrd,~:::.. j i..‘lt:l !nvert 1985:76:191-7. Bascom R, Wachs M. Naclerio KM. Pipk\ml I,.,. (;alli SJ, Lichtenstein LM. Basophil influx occur, artrr nasal ,mrigen challenge. effects of topical cortictrsrcroiti prtirrearmcni J AI LEKGY CLIN IMMUN~L.1988;81:580. Galli SJ. Dvorak AM, Dvorak HF. Basoplril~ and mast cells: morphologic insights into their biology. secreiorb patternr. and functions. Prog Allergy 1984;34: I -!-li MacClashan D Jr. Schleimer R, Peters S. L’~xI~ D. Nacleno R, Toglas A, Lichtenstein L. The mediators ot ailergic diseases. In: SeraIini U. Errigo E, eds. Proceedingsof the XII Congress of the European Academy 01’Allergo& and Clinical In!munology. Firenze: O.I.C. Medical Prcsa. ;Qi(.?:l67-7 3 Baumgarten CR, Nichols RC. Naclerio KM, I;.!chtensternLM. Norman PS. Proud D. Plasma kallikrem dilrmg experimentally induced allergic rhinitis: role in himn forntai~orl and contribution to TAME-esterase activity in nasal ~.t’crctllmx 1 lmmunol 1986;137:977-82. Schwarz LB, Metcalfe DD, Miller JS. Ear! Ii, Sulhvan T. Tryptase levels as an indicator of mast cell acrivation m systemic anaphylaxis and mastocytos:i ri rkl ! bkii 19x7; 316:1622-h. Keele CA. Climcal and pathological aspect%Ilj kmtn\ in man. Proc R Sot [Biol] 1969;173:361-70 Garcia-Leme J. Bradykinin system in: Vane .iR. krrzlra SH, eds. Handbook of experimental pharmacy@!\ l;g>l50 Berlin: Springer-Verlag, 1978:464-522. Proud D. Reynolds CJ, La Carpa S. Kagey-b$,hotka A. Lichtenstein LM, Nacleno RM. The responseto nasal provocation with bradykinin [Abstract). I Al [ ~.t~;i (-,.I\ !M\?I.?.~II l987:
44.
45.
reacting substance of anaphylaxis from human lrukocytes by the calcium ionophore A23187. J lmmunol 1976;i 16: !677-81. Weller P, Lee C. Foster D. Corey EJ. Austere KF. Lewis R. Generation and metabolism of 5 lipoxygenase pathway leukotrtenes by human eosinophils: predominant production of leukotrlene C,. Proc Nat1 Acad Sci USA 19X~.XO:7636-30. Rankin JA, Hitchcock M, Merrill W . Bach MK. Brashler JR, Askenase PW. IgE-dependent release of lcukotrrene (‘. from alveolar macrophagea. Nature 1982.29?:.129: 1. Gleich GJ, Frigas E, Loegering DA. nlassotn rX,. Stemmuller D. Cytotoxic propertles of the eosinophd ma!:it basic protein. J lmmunol 1979;123.2925-7. O ’Donnell MC, Ackerman SJ. Glersh GJ. ‘Thomas LL. Activation of basophil and mast cell histamine xleasc by cosinophil granule major basic protein. J Ewp X&d iY~?:I 47: 14i(l91. Naclerio RM. Proud D, Togias A. Adkmson NI. Jr. Meyers D. Kagey-Sobotka A. Plaut M, Norman PS. Lrchtenalem LM. Inflammatory mediator in late antigen-mducekrrhinitis. N Engl J Med 1985;313:65-70. lliopoulos 0, Proud D, Lichtenstein L.M. Kagev-Sohotka A, Creticos PS. Adkinson NF Jr, Mac Donald SM, Normal PS. Naclerio RM, Relationships hetweeu early l:ite. JnJ ri&chal-
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lenge responses to nasal challenge [Abstract]. J ALLERGYCLIN
IMMUNOL1987;79:253. 46. Wachs M, Proud D, Lichtenstein LM, Kagey-Sototka A, Norman PS, Naclerio RM. Nasal priming [Abstract]. J ALLERGY CLIN IMMUNOL1987;79:253. 47. Connell JT. The priming effect and its relationship to histopathologic changes in allergic rhinitis [Abstract]. J ALLERGY 1968;41:101-2. 48. Pipkom U, Proud D, Lichtenstein LM, Schleimer RP, Peters SP, Adkinson NF Jr, Kagey-Sobotka A, Norman PS, Naclerio RM. Effect of short-term systemic glucocorticoid treatment on human nasal mediator release after antigen challenge. J Clin Invest 1988;80:957-61. 49. Pipkom U, Proud D, Lichtenstein LM, Kagey-Sobotka A, Norman PS, Naclerio RM. Inhibition of mediator release in allergic rhinitis by pretreatment with topical glucocorticosteroids. N Engl J Med 1987;3166:1506-10. 50. Schleimer RP, Schulman ES, MacGlashan D W Jr, Peters SP, Adams GK II, Lichtenstein LM, Adkinson NF Jr. Effects of dexamethasone on mediator release from human lung fragments and purified human lung mast cells. J Clin Invest 1983;71:1830-5. 5 1. Schleimer RP, Lichtenstein LM, Gillespie E. Inhibition of basophil histamine release by anti-inflammatory steroids. Nature 1981;292:454-5. 52. Bascom R, F’ipkom U, Gleich J, Lichtenstein LM, Naclerio RM. Effect of systemic steroids on eosinophils and major basic protein during nasal antigen challenge [Abstract]. J ALLERGY CLIN IMMUNOL1986;77:246. 53. Freeland HS, Pipkom U, Naclerio RM, Adkinson NF Jr, Lich-
54.
55.
56.
57.
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59.
CLIN. IMMUNOL. M A Y 1988
tenstein LM, Peters SP. The role of leukotriene B, (LTB,) in human allergic late-phase reactions [Abstract]. J ALLERGYCLIN IMMUNOL1986;77:244. Freeland HS, Schleimer RP, Alger M, Peters SP. Leukotriene B, (LTB,) generation by human PMNs is not inhibited by glucocorticosteroids in vivo or in vitro [Abstract]. J ALLERGY CLIN IMMLINOL1987;79: 157, Togias A, Proud D, Kagey-Sobotka A, Norman PS, Lichtenstein LM, Naclerio RM. The effect of a topical tricyclic antihistamine on the response of the nasal mucosa to challenge with cold, dry air and histamine. J ALLERGYCLIN IMMUNOL 1987;79:599-604. Togias A, Naclerio RM, Warner J, Proud D, Kagey-Sobotka A, Nimmagadda I, Norman PS, Lichtenstein LM. Demonstration of inhibition of mediator release from human mast cells by azatadine base. In vivo and in vitro evaluation. J A M A 1986;255:225-9. Eggleston PA, Kagey-Sobotka A, Schleimer RP, Lichtenstein LM. Interaction between hyperosmolar and IgE-mediated histamine release from basophils and mast cells. A m Rev Respir Dis 1983;130:86-91. Silbcr G, Naclerio RM, Eggleston P, Proud D, Togias A, Lichtenstein LM, Norman PS. In vivo release of histamine by hyperosmolar stimuli [Abstract]. J ALLERGYCLIN IMMUNOL 1985;75(suppl):176. Togias AG, Proud D, Kagey-Sobotka A, Adams GK III, Norman PS, Lichtenstein LM, Naclerio RM. The osmolality of nasal secretions increases in the clinical response to inhalation of cold, dry air. A m Rev Respir Dis 1988;137:625-9.
From the Department of Medicine, Division of Clinical Immunology, and the Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine, and the Center for Occupational and Environmental Health of the Johns Hopkins Medical Institutions, Baltimore, Md. Supported by National Institutes of Health Grants AI 07290, AI 08270, HL 32272, NS 22488, and HL 37119. Presented at the Fifth Annual Aspen Allergy Conference, Aspen, Colo., July 1987. Reprint requests: Alkis Togias, MD, Nasal Laboratory, The Good Samaritan Hospital, 5601 Loch Raven Blvd., Baltimore, MD 21239.
782
Publication number from the O’Neill Laboratories at the Good Samaritan Hospital, Baltimore, Md. *Sponsored by the Colorado Allergy Society. **Recipient of Teacher Investigator Development Award 5 K07 NS 00811 from the National Institute of Neurological and Communicative Disorders and Stroke. *** Recipient of the International Research Fellowship Award (NIH F05 TWO 3516-01) from the Fogarty International Center. ****Recipient of a Pfizer Biomedical Research Award.
VOLUME NllMRER
8I 5 PART 1
Allergic and nonallergic rhinitis is a wellrecognized entity affecting a significant percentage of the population.’ Allergic rhinitis has, of course, been attributed to the inhalation of specific antigens to which susceptible individuals are sensitized. Basic experimental knowledge of type I immunologic reactions has ied to the assumption that tissue anaphylaxis involves activation of stationary mast cells by IgEantigen interaction.’ Furthermore, since it has been well recognized that histamine is a major inflammatory mediator released by triggered mast cells, antihistamine medications (H, antagonists) have, for years, constituted our major pharmacologic means for the control of allergic rhinitis. In the past two decades, several other inflammatory compounds, such as arachidonic acid metabolites, were found in the supernatants of antigenically activated mast cells from rodents,’ but only recently have human mast cells from different sources been purified and their inflammatory mediator armamentarium explored.4-6The emergence of this knowledge has shifted our therapeutic strategy toward mast cell stabilizing agents in order to avoid the task of simultaneous use of multiple antagonists for all the mediators involved in allergic reactions. However, not only does the above strategy appear difficult to expedite but also our understanding of the natural history of allergic rhinitis appears to be far from clear. Deliberate efforts to reproduce the disease in the laboratory, in humans, extend back to the late nineteenth century.’ These efforts have become much more sophisticated in our time,X-‘”and the method of nasal lavage has been recently applied in our laboratory to quantitate several mediators of inflammation after nasal antigen challenge of allergic individuals. “-z However, (1) the immediate clinical response of the nose to antigen lasts about 30 minutes, whereas naturally occurring allergic rhinitis is a chronic disease, and (2) the dose of pollen needed to induce a nasal allergic reaction in the laboratory frequently far exceedsthat inhaled by an atopic individual in a single day during the pollen season. The immediate reaction that occurs after antigen challenge in the lung, skin, or nose of allergic patients is followed by a quiescent period lacking symptoms or mediator peaks. In many cases, a recrudescenceof the symptoms and signs, as well as of the physiologic changes of the immediate reaction, will be observed several hours later. “-I7 This is known as the LPR. The clinical significance of those reactions is intriguing, and the pathogenesisis poorly understood. The belief that LPRs might represent the naturally occurring allergic disease much more closely than does the acute response” has led to extensive investigations of this
Allergic
and nonallergic
nasal ?nftar~mat~on
Abbre~htions used LPR: MBP:
Late-phase reaction
LTB.,. LTC,, LTD,: CDA: WMA:
Leukotricne B,, Q ’ :txtd D. Cold, dry a:r Warm. moix: .ilr
783
Major basic protern PGD,: ProstaglandinD.
phenomenon during the past few years. This concept is mainly substantiated by the fact that LPRs are suppressed by pretreatment with steroids. whereas early reactions are not. Steroids are, indeed. our most efficacious therapeutic modality in the treatment of allergic diseases. W e have applied the nasal lavage techniques to study LPRs in the nose. This has enabled us to quantitate the amount of inflammatory mediators produced and to simultaneously quantify the cellular components of the lavage fluids. Furthermore, we have been able to study the effects of pharmacologic agents, such as steroids, on the clinical, htochemical, and cellular aspects of nasal LPRs. The clinical picture of several other forms of chronic nasal disease is very similar to that of aliergic rhinitis.‘9-2’This has tempted us to investigate the role of mast cells and their mediators in these pathologic entities. Since there appears to be a large spectrum of precipitating factors, some of which are not well documented, we have focused on the group of patients who report symptoms of rhinitis on exposure to cold and windy environments and not to any other climatic condition. A nasal challenge model with ClDA as the stimulus and nasal lavage for the detection of inflammatory mediators has been developed.-” METHODS Subjects These studies have involved three groups of volunteers: (1) Individuals had documented seasonalallergic rhinitis to ragweed and/or to grass pollens. Documentation was based on both their history and the results of intradexmal skin testing with a panel of aeroallergens that are prominent in the Baltimore/Washington, D.C. area. Those who participated in the trials of steroids in the LPR previously had been demonstrated to develop an LPR in response to nasal antigen challenge. All studies were performed during pollen-free months. (2) Subjects who participated in the CDA nasal challenges had a history of rhinitis on exposure to cold and windy environments, regardless ot their atopic status and the possible concomitant seasonal or perennial, allergic or nonallergic rhinitis. (3) Appropriate control volunteers without allergic rhinitih or without Gensitivity to
784
Togias et al.
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CLIN. IMMUNOL. MAY 1988
FIG. 1. An example of an immediate and an LPR to antigen challenge. (From Togias A, Naclerio RM, Proud D, et al. Mediator release during nasal provocation: a model to investigate the pathophysiology of rhinitis. Am J Med 1985;79(suppl 6A):26-33.)
CDA were used in several studies. All our volunteers gave informed consent, and the studies were approved by the Joint Committee on Clinical Investigation of the Johns Hopkins Medical Institutions. Antigen challenges were performed with the use of either ragweed or mixed grass-pollen extract or ragweed pollen at doses ranging from IO to 1000 protein nitrogen units and 10 to 10,000 grains of ragweed pollen. The antigen was insufflated in the nostrils of participating volunteers. CDA ( - 3” to - 10’ C temperature and 0% to 10% relative humidity) was inhaled through the nose at a flow of 12.5 L/min and was exhaled through the mouth. Control challenges either with the pollen-extract vehicle (normal saline with phenol) or with lactose powder for antigen provocations and with WMA (29” to 35” C temperature and 95% to 100% relative humidity) for CDA challenges were performed. Nasal secretions were obtained by instilling 2.5 to 5 ml of normal saline or lactate Ringer’s solution into each nostril. These nasal lavages were always performed at baseline and after any manipulations, such as control or antigen challenge, WMA or CDA challenge, or drug administration. During the LPR studies, a nasal lavage was performed every hour for 11 to 12 hours after the initial nasal challenge. All mediator assays on aliquots of nasal lavage fluids used in these studies have been previously described in detail. Histamine was measured with a spectrofluorometric assay sensitive to 1 ng/mI,*3 TAME-esterase measurements required radiochemical assay,24and PGD2, leukotrienes C,, D,, and E,, and MBP were quantitated with the use of radioimmunoassays. ‘*. “-” In addition, bradykinin and lysylbradykinin were measured with radioimmunoassay,* as was albumin (a marker of plasma transudation).gO
FIG. 2. Initial challenge and rechallenge mediator response in a group of 13 allergic volunteers. The vertical dashed/he indicates that 11 hours have elapsed between the last step (1000 protein nitrogen unit [PNU]) of the initial challenge and rechallenge. Note that the dose of antigen used in the rechallenge is 10 PNUs, that is the lowest dose of the initial, three-step, provocation. NS = not significant; *p < 0.05; **p < 0.01.
The processing of the cells found in the nasal washes also has been previously described.3’ Briefly, the returned nasal lavage fluids (lactated Ringer’s) were initially centrifuged, and the pellets were resuspended in RPM1 with n-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid. Thereafter, they were vigorously shaken and exposed to N-acetyl cysteine for 45 minutes. After resuspension with the previous medium, cell counts were performed by hemocytometer, and differential counts for neutrophils, monocytes, eosinophils and epithelials required cytospin slides stained with Diff-Quick (Scientific Products, McGraw Park, Ill.), a modified Wright stain, whereas alcian blue-staining positive cells were identified from separate aliquots with alcian blue staining. Morphologic criteria under light microscopy were used to differentiate between basophils and mast cells among the alcian blue-positive cells.32 Osmolality measurements in the nasal lavage fluids were performed with vapor pressure osmometry. In situ osmolality of the nasal secretions was measured by direct application of filter paper disks over the inferior turbinate. After being saturated with nasal secretions, these disks were immediately transferred to the vapor pressure osmometer a (SlOO B microosmometer, Wescor, Inc., Logan, Utah).
VOLUME NUMBER
81 5, PART 1
Allergic and nonallergic
nasal rnfl
785
TABLE1. Mediators detected in nasal lavage fluids Antigen challenge Early phase
Histamine TAME-esterasets) Kinins pcm Leukotrienes(C,, D,, E,) MBP Other prostaglandins LeukotrieneB,
+” +” + 29 +” + II + 34 + ‘I + 3.5
Late phase
+ ia -t i1 + ‘I - 42 & ii + 34 NT + ih
CDA challenge -- ._.. “-.. _ -- ..-_- ._ Early phase Late phase --“-
.+ ‘2 + 11 t 12 + 12 ,. 1;; NT NT
.)“i k’i :i;y N’i 2‘1 4’1
NT = not tested
Nasal antigen provocation
mediatorsfound in the LPR lavagefluids and that they may play a significant role in this reaction.” a characteristicexampleof the eventsthat follow nasalchalLPRs and nasal priming. If we are to define the LPR as a twofold or greater increaseabove baselinein symptom lenge of an allergic individual with antigen. Serial nasal scoresand in two of the three mediatorsthat we commonly lavagesare performedbefore.the challengeto reducesmall, measure(histamine,TAME-esterase(s).and kinins), a 37% resting-phasemediatorlevels to a stablebaseline.A control prevalencecan be demonstratedin a pKviously unscreened challengewith the vehicle usedfor the subsequentantigenic group of patients with ragweedhay fever.4’So far, no alprovocationis performedto rule out nonspecificreactivity. lergic individual without an early nasalreactionto antigen An immediate response,a quiescentperiod, and an LPR areeasily recognizable.In this case,only histamine,TAMEhas demonstratedevidenceof an LPR. Thus, althoughit is not a ubiquitousphenomenonunder our laboratorycondiesterase(s),PCD,, and kinins were measured.However, tions, nasal LPR can be observedonly when a previous. severalother inflammatorymediatorshavebeendetectedin the nasal lavage fluids, as presentedin the left panel of biochemically demonstrableearly reaction occurs. NeverTable I. Among thesemediators,histamineis the only one theless,there is no significant correlation betweenthe inthat can be confidently attributed to m&t cells and/or batensity of the early and the late reaction, suggestingthat sophils. PGD, is the major cycloxygenaseproduct of mast factors other than simple mediator releasemay affect.the cells and is not produced by basophils.25, 33 TAMEmagnitudeof this inflammatoryprocess,” esterase(s)activity in the nasal fluids representsa mixture Another, potentially very important, phenomenonis obof the activity of plasma kallikrein, glandular kallikrein, served. After the initial antigen challenge,rhe sensitivity and, to a lesserdegree,mast cell tryptase, which can be a of the nasal mucosato antigen increases.This is demonmarkerof mast cell degranulation.34.j5 Thesekinin-forming strablein Fig. 2 where, after an initial three-stepchallenge enzymesappearin the nasal sectionstogetherwith highprotocol, an antigenchallengewith the lowest doseinitially and low-molecular-weightkininogens30 and are responsible used is repeatedat 11 hours. This rechallengereaction is for the formation of kinins, the pharmacologicpropertiesof significantly intensifiedwith regardta symptamsand to the which include vasodilationand increasedcapillary flow, as levelsof mediatorsin the lavagefluids, as comparedto those well as increasedvascularpermeability.36.37Recentstudies obtainedwith the sameantigendosein the initial challenge. indicate that nasal provocation with bradykinin induces This phenomenonbecomeseven more obvious when the three-or four-stepdose-response curveto antigenis repeated symptoms of rhinitis and a sore throat.” The source of every 24 hours for 3 consecutivedays. In that case, a sigleukotrienesin the nasalsecretionsis unknown, sincemany nificant shift in the dose-response curve is obtained,and a cell types presentin the nasalmucosa,such as mast cells, basophils,eosinophils,and neutrophils, and macrophages largepercentageof the participatingvolunteersbecomeable to respondin the laboratoryto antigendosesthat could easily are capableof generatingthose substances.‘39-4’ 3. MBP is be inhaledduring the ragweedhay fever seasonwithin minreleasedby eosinophilson activation. It is a potent toxin The intensificationof the clinical for epithelial cells42and has recently been demonstrated utesof naturalexposure.46 by the numberof sneezes,appearsto capableof causingmediatorreleasefrom rodent basophils response,as assessed and mast cells.43W ith regard to the panel of mediators correlatewell with the increasein the numberof white blood cells in the nasalfluids of the lavagesperformedeveryday obtainedin the immediatereaction and the LPR of experbefore the challenge.4s This observation. termed ‘%asal imental allergic rhinitis, a striking differenceis notedin that PGDZ,while it is presentin the early phase,is absentin the priming,” has beenpreviouslyreportedin both a laboratory and a clinical setting.“’It appearsthat it can provide the LPR. This has temptedus to postulatethat basophils,and means to close the gap between experimental antigen not mast cells, may be the source of at least part of the Mediators of the early and late reaction. Fig. 1 represents
786
J. ALLERGY
Togias et al.
II EP
EP
LP
CLIN. IMMUNOL. MAY 1988
LP
FIG. 3. Mean net increases in mediator levels (-+ SEM) in nasal lavage fluids and the total number of sneezes in 13 patients that participated in the study of the topical steroid (flunisolide) effect on the early phase (EP) and late phase (LP) reactions. Mediator levels in the EP are calculated by pooling of lavage samples obtained 10 minutes after each of three consecutive antigen challenges with increasing doses, as illustrated in Fig. 2, plus the sample from a lavage performed 20 minutes after the third antigen challenge. LP values represent pooled hourly samples during postchallenge hours 2 to 11. All variables were significantly reduced by pretreatment with topical steroids; *p < 0.05; **p < 0.01.
TABLE II. Effect of systemic
giucocorticoid
treatment
on nasal
secretion
Early reaction Placebo
Histamine (rig/ml) Kinins (ng / ml) TAME (cpm x 10e3) LTC, (D,, Ed) WO.1 ml)
6.8 7.4 48.4 606
k k
1.8 1.7
+ 10 ? 110
mediator
levels
Late-phase Steroids
6.6 3.3 38.8 373
Placebo
reaction Steroids
k 3.1 f 0.6* t 4.0
6.7 k 3.0 3.8 * 1.3 18.0 k 6.0
2.3 5 0.8* 0.9 k 0.3* 5.4 + 1.5*
k 100
271 +- 50
143 * 25t
*p < 0.05. tp < 0.06.
challenge and the naturally occurring allergic disease with regard to the doses of antigen used. Interestingly enough, an obligatory link between priming and the occurrence of an LPR under the conditions in which our studies where carried out does not appear to exist.45 However, as illustrated in Fig. 2, the fact that the only mediator not following the intensification trend is PGD, suggests that the increased sensitivity to antigen may partially result from the recruitment and activation of basophils in the nasal mucosa and that the pathogenesis of LPR and priming may be closely related. Pharmacologic manipulations with steroids. We have recently concluded two studies evaluating the effects of systemic and local steroids on the early reaction and the LPR in the nose.48.49 Oral prednisone was administered to 13 allergic individuals in a double-blind, placebo-controlled, crossover study (2 days pretreatment with 20 mg, three times
a day). In the second study, topical flunisolide was insufflated in both nostrils twice a day (total dose was 200 pg/day) for 1 week before nasal antigen challenge. This was also a double-blind, placebo-controlled, crossover study involving 13 allergic subjects. Table II and Fig. 3 present the results from the oral and the local steroid-treatment studies, respectively. As expected, oral steroids significantly suppressed the LPR with regard to both mediators and symptoms but did not affect the immediate reaction to antigen challenge. Since in vitro data with purified human lung mast cells and purified human peripheral blood basophils demonstrate steroid inhibition of mediator release in response to antigen only for the latter,50,51this in vivo study offers additional evidence of the active participation of basophils in the human nasal LPR. In contrast to oral steroids, topical flunisolide effectively inhibited not only the late phase but
VOLLiME 81 hllJMI
also the immediate allergic response to antigen. It is, in fact. difficult to assesswhether the ablation of the LPR was an independent effect or a result of the early phase suppression Either way, this study offers additional reasons for preferring topical administration of corticosteroids in allergic rhinitis and substantiates the common practice of initiation of’topical steroid treatment several days before the pollen season begins. It is notable that with both oral and local steroids, the increased sensitivity to antigen, as detected at 1 I hours after the initial challenge, was found to be completely suppressed. thus offering more evidence for the importance of this phenomenon in the natural history of allergic rhinitis. Cellular studies qf the LPR. One of the important advantages of the nasal lavage technique is that it provides the ability for simultaneous measurement of mediators and characterization of the cellular components in the lavage tluids. Thus. not only can we comment on the influx of specific cell types but we can also follow their degree of activatron. if specific biochemical markers are detectable. We have completed a series of studies of the cellular pattern in the nasal washes during the LPR.3’.5’ A number of inflammatory cells appears to populate the surface of the nasal mucosa during that reaction. The pattern of this influx is highly variable among individuals, as is the case for mediators. Eosinophils demonstrate a significant increase in the nasal secretions within 1 to 2 hours after antigen challenge and reach their peak at 7 to 10 hours. The detection of increased levels of MBP in the LPR correlates with the eosinophil influx, suggesting eosinophil activation in the LPR .” The role of the eosinophil becomes even more pronounced by the fact that treatment of allergic subjects with systemic or topical steroids not only ablates the LPR but also significantly inhibits eosinophil recruitment and MBP release.” Neutrophils enter the nasal secretions somewhat later than eosinophils, but they represent the greatest number of infiltrating cells in the LPR. Their significance is, however, uncertain. Steroid treatment does not appear to affect either the number of neutrophils in the lavage fluids of the LPR” or the levels of leukotriene B,, which is believed to be partially neutrophil derived.53.54Nevertheless, assessment of neutrophil activation status is difficult, since we did not identify a specific marker in nasal washes. Mononuclear cells also increase, albeit not significantly, 9 to 10 hours after antigen challenge. Epithelial cells remain unaffected by the LPR. Since our early speculations regarding the participation of basophils in the LPR,‘” we have been particularly interested in the detection of these cells in nasal fluids during the course of this reaction. We have observed that alcian blue-positive staining cells significantly increase in percentage and total number in the LPR, as compared to baseline or immediate postchallenge periods. Nevertheless, they do not exceed I % of the total cells.3’ These cells were studred under light microscopy, with previously established morphologic criteria? Approximately 70% of the cells were believed to be compatible with basophils, < 10% were identified as mast cells, and the remainder were indeterminate.” The number of alcian blue-staining cells correlated signif-
Allergic
and nonallergic nasa\
Intim*matior:
787
* 4 H 3 2 I! R
:
FIG. 4. An example of mediator release in nasal lavage fluids and symptom scores from one subject chahenged sequentially with W M A and CDA. Downward arrows represent nasal washes; dashed lines represent the degrees of sensitivity of the assays used. (From Togias AG, Naclerio RM, Peters SP, et al. Local generation of sulfidopeptide leukotrienes upon nasal provocation with Cold, dry air. Am Rev Respir Dis 1986;133:1133-7 1
icantly with the levels of histamine in the LPR. Furthermore. topical steroid pretreatment prevented the influx of alcian blue-positive staining cells, since it prevented mediator elevation and ablated the symptoms of this inflammatory process.” These findings have also strengthened our hypothesis of active basophil participation in the LPR.
Nasal provocation
with CDA
An example of the response to nasal challenge with CDA of an individual who gave a history of nasal symptoms after
788 Togias et al.
exposure to cold and windy environments is presented in Fig. 4. Initial challenge with WMA had no effect on these subjects. Their specific sensitivity to CDA is obvious by the fact that all mediators measured in the returned lavage fluids are elevated after this challenge and correlate significantly with symptom scores and with the clinical evaluation by the observer.**. *‘. 55 No symptoms or mediator elevations are detected in individuals who deny any sensitivity to CDA when they are challenged in the laboratory. As noted in the right panel of Table I, all the mediators that have been detected so far in the nasal secretions during the immediate reaction to CDA are also produced during the early reaction to antigen in atopic subjects. This certainly suggests common cellular sources for the two different reactions. Furthermore, recent studies have demonstrated the presence of an LPR in certain individuals after CDA challenge.* The cellular components of nasal secretions in the LPR of CDAinduced reactions have not been studied yet. Nevertheless, it is tempting to speculate that mast cells lining the mucosal surface may be largely responsible for the immediate CDA reaction. The question that has risen, therefore, involves the mechanism of mast cell and/or other cell activation during the response to CDA. This appears to be different from the antigen-IgE interaction. Strong evidence is offered by the fact that pretreatment of CDA-sensitive volunteers with azatadine, a tricyclic antihistamine previously demonstrated to inhibit the nasal reaction to antigen challenge,56 has no effect on CDA-induced rhinitis and mediator release.55 Since one of the expected effects of CDA would be drying of the nasal mucosa through evaporation of water needed for maximum humidification of inspired air, we attempted to measure the osmolality of the returned lavage fluids before and after CDA challenge. Several other observations led us to those studies: (1) Human lung mast cells are triggered to release mediators when they are exposed to hyperosmolar media.57 (2) Instillation of hyperosmolar solutions in the nasal cavities of certain individuals will result in mast cell mediator release.” We have now demonstrated that the osmolality of the nasal secretions after CDA challenge of CDAsensitive subjects significantly increases from baseline and correlates with mediator release. In contrast, osmolality remains stable after WMA challenge of CDA-sensitive volunteers, as well as after CDA or WMA challenge of individuals who deny sensitivity to CDA.59 Furthermore, no changes in the osmolality are detected in atopic subjects when they receive antigen challenge and demonstrate significant mediator elevations. The changes in the osmolality are more pronounced when in situ measurements are obtained by saturation of small filter paper disks with nasal secretions after CDA challenge.59These findings are leading us to the assumption that elevated osmolality may be the cause of mast cell activation in CDA-induced rhinitis. Furthermore, they tempt us to generate several other speculations: (1) The lack of elevation in the osmolality of the nasal
*Iliopoulos 0. Personalcommunication.
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secretions in the control group of volunteers points to a specific defect in the ability of the nasal mucosa of CDAsensitive subjects to humidify inhaled air under extreme conditions. Mast cell mediator release may act to overcome this “malfunction” by increasing vessel permeability and glandular secretions. (2) If a spectrum of response of CDA exists among the population, the role of nasal secretion tonicity and subsequent mast cell mediator release may be important in the natural homeostasis of air humidification by the upper airways. (3) Since the stimulus of CDA in the nose appears to be the same as that causing exercise-induced bronchospasm in subjects with asthma, a similar mechanism of mediator release may account for that pathologic entity.
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