Inhibitory effect of oral cetirizine on in vivo antigen-induced histamine and PAF-acether release and eosinophil recruitment in human skin

Inhibitory effect of oral cetiririne on in vivo antigen-induced histamine and PAF-acether re#ease and eosinophil recruitment in human skin Laurence Michel, PhD,* Christine De Vos, PhD,** Jean-Pierre Rihoux, MD,** Claude Burtin, MD,*** Jacques Benveniste, MD,*** and Louis Dubertret, MD* Bra&

L’Alleud, Belgium, and Creteil and Clamart, France

The use of a noninvasive skin chamber technique in vivo in pollen-sensitive patients allowed us to quanttfi the time-course release of histamine and the recruitment of inflammatory cells (i.e., neutrophils, monocytes,and eosinophils) in skin sites challenged with pollen, histamine, and compound48180. The new H,-receptor antagonist, cetirizine 2 HCl, orally administered with IO mg once a day to pollen-sensitive patients in a double-blind, crossover study versus placebo, induced a significant decreasein the wheal-and-flare cutaneousreaction induced by intradermal injection of pollen, histamine, and compound48180. it also signt&ntly inhibited the immediate histamine release occurring in skin chambersafter pollen introduction, whereas it did not significantly inhibit the late release. In patients receiving placebo, we detected platelet-activating factor-acether in media collected at the sixth hour from chambersfilled with pollen. With cetirizine 2 NC1 treatment, platelet-activating factor-acether was not detected in chamber media. Interestingly, cetirizine 2 HCEsignificantly reduced the eosinophil recruitment observedon the superficial dermis 24 hours after pollen challenge. (J ALLERGYCLIN IMMUNOL 1988;82:101-9.j

Human cutaneous immediate hypersensitivity results from the antigen-induced releaseof biologically active materialsfrom dermal mastcells sensitizedwith specificIgE antibody.’ The prediction of IgE-mediated reactions for antigens in a sensitive subject is very commonly studied by skin testing, such as intradermal injection or prick test, with various protein antigens.‘33The cutaneousreaction occurring rapidly after the exposure to the appropriate antigen is characterized by a transient pruritus and the so-called whealand-flarereaction, sometimesfollowed by a late-phase developmentof an indurated poorly demarcatedlesion that fades in 18 to 24 houmh6 The immediate events are induced by the primary release of the mast cell From the *Unit6 INSERM U 312, Service de Dermatologie, Hbpital

Hem-iMmdor, Crete&France,**UCB, PhmmceuticalSector, Braine L’Alleud, Belgium, and ***Unite INSERM U 200, Clamart, France. Supported by a grant from CNRS-INSERM: Lipides Pharmacolcgiquement Actifs, and by UCB, Secteur Phannaceutique, Belgique. Received for publication July 6, 1987. Accepted for publication Jan. 20, 1988. Reprint requests: Louis Dubertret, MD, INSERM U. 312. See Dermatologie, Hapita Hem? Mondor, 94010 Creteil, France.

Abbreviations used

HBSS: Hank’sbalancedsalt solution HSA: Humanserumal&unin HBSS-HSA: HBSScontairping0.25%NSA PAF: Platelet-activating factor IR: Index of reactivity

mediators,7such as histamine, that induces a typical wheal-and-flareskin reaction.’ The mediatorsreleased by this primary effector system recruit and activate other effector cells, such as eosinophils, neutrophils, basophils, and monocytes.g-” These activated ceils can amplify the inflammatory process by generating and releasing their mediators, including RAF-acether among many other mediators, with the consequent developmentof a late-phaseallergic reaction. Moreover, as human eosinophils, macrophages,and platelets possessspecific receptor for IgE (Fc RZ),” they can be also directly activated by allergen and might be primarily involved in the initiation of inflammatory responses. H,-receptor antagonists have been developedto in101

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TABLE I. Protocol

1*

2*

3*

4*

5

Blister formation

Laying of skin chamber Introduction of the to agonists Renewal of the t 30 min agonists Renewal by t 6 hr HBSS-HSA

t 24 hr Collection of HBSS-HSA from chambers Application of Rebuck skin windows every hour for 6 hr Intradermal injections of the agonists

t = Time. *Oral administrationof cetirizine2 HCI or placebo.

hibit the end organ effects of the main mast cell mediator, histamine, thus inhibiting allergen-induced wheal-and-flare skin reaction.13. I4 However, the in vivo effect of antihistamines on antigen-induced mediator release and consequent inflammatory cell attraction has not been intensively studied. A new potent H,-receptor antagonist, cetirizine 2 HCl, or {2-(4-[(4-chlorophenyl) phenylmethyllI-piperazinyl)ethoxy}acetic acid dihydrochloride strongly inhibits cutaneous reactions induced by histamine and its releaser, compound 48 / 80, in animalsI and in humans.‘6’ ” With a noninvasive skin chamber technique, we quantitatively studied, in six pollen-sensitive subjects, the in vivo effect of cetirizine 2 HCl, administered orally, on the histamine and PAF-acether time-course release and on the inflammatory cell recruitment induced by challenge with either specific antigen, histamine, or compound 48 I 80.

MATERIAL AND METHODS Subjects Six patients (two female and four male subjects,aged 18 to 25 years, weighing 56 to 75 kg) were investigated. Before investigation, informed consent of the patients was obtained. All patients were pollen-sensitive on the basis of a typical history of allergic rhinitis/conjunctivis, with or without asthma, during the pollen season, and demonstrated positive whealing response20 minutes after intradermal injection of pollen extract (10 ~1) with IR of 0.01 per injection (Laboratories Stallergenes, Fresnes,France). (The IR of a given pollen extract is defined by Stallergenes as the ratio X 100 of the cutaneous response induced after 20 minutes by a Stallerpoint (Laboratories Stallergenes)prick test with the pollen extract to that of a reference pollen extract that induces a 6 mm diameter wheal in a population of 30 patients sensitive to the primary whole aIIergen.) The concentrated pollen extract 100 IR (1 ml) that we used contained 80 pg of protein nitrogen.

All experiments were performed outside the pollen season.

Reagents Cetirizine dihydrochloride (2 HCI), 10 mg tablets, placebo, and histamine dihycrochloride solution were provided by UCB, Brussels, Belgium. Compound 48/80 and HSA were obtained from Sigma Chemical Co., St. Louis, MO. HBSS-HSA (Diagnostics Pasteur,Paris, France) was used asa vehicle for the pollen extract, histamine, and compound 48/80. HSA, known as a protein carrier of PAF-acether,” was added to the dilution medium in order to measurethe releaseof this mediator.

Product administration The study was conductedas a randomized, double-blind trial. Each patient was twice investigated with the same experimental procedure, receiving, at random, cetirizine 2 HCI, by mouth, in one set of experiment, and plac+o in the other set of experiment, in a crossoverfashion. A period of 4 weeksseparatedthe two setsof experiments. Cetirizine 2 HCl (10 mg) and placebo, both in tablets of absolutely identical appearance,were numbered according to a predetermined code. In the two sets of experiments, tablets were administeredin the,evening,once a day, during 4 days, Blood sampleswere obtained before each treatment and then in the morning of the fifth day, that is, after 4 days of oral treatment. The plasma level of cetirizine 2 HCl was measuredby gas chromatography analysis.* Blood histamine levels were determined by spectrofluorometry, according to the method describedby LebeI.‘9 A control did not permit cetirizine 2 HCl to influence the spectrofluorometric measurementof histamine.

Skin chamber technique On the third day of the experiment (Table I), that is, after two days of oral treatment, five suction blisters, 0.8 cm *BakesE. Unpublishedobservation.

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diameter base, three on one forearm and two on the other arm, were induced simultaneously by vacuum suction with a negative pressureof 300 mm Hg. Blisters were left untouched until the next day. In the morning of the fourth day, the blister fluid was collected and cooled at - 20” C for measurementof the level of cetirizine 2 HCl by gaschromatographyanalysis. Each blister was unroofed aseptically. Sterile siliconed Macrolon skin chambers(Trametal, Saint Maur, France) were placed djrectly over the obtained cutaneouswindows (0.5 cm’ area) open on the superficial dermis and were kept in place by appropriatetaping.20 Time 0. Each chamber was filled with 1 ml of the following solutions: (I) control chamber:HBSS-HSA, the dilution medium of the other products, (2) compound 48/80 (1 pgi ml), (3) histamine dihydrochloride (1 pg/ml), (4) pollen solution at 0.001 IR per milliliter, and (5) pollen solution at 0.01 IR per milliliter. Time 30 minutes. Thirty minutes after the first introduction, chamberfluids were renewed by 1 ml of freshly preparedidentical solutions. Collected fluids were sampledfor further measurements. Time 6 hours. Six hours after the first introduction, chamber fluids were collected and sampled. At this time, all chamberswere filled with 1 ml of pure HBSS-HSA. Time 24 hours. Chamber fluids were collected in the morning of the fifth day, namely, 24 hours after the first introduction of the various solutions into the skin chambers. The skin chamberswere removed.

Application of Rebuck skin windows superficial dermis to analyze the recruitment of inflammatory cells

on the

Each fibrin layer was unglued by use of two tweezers. According to the method describedby Rebuck and Crowley,” a sterile glasscoverslip was applied over eachdenuded areaand was replacedhourly by a new one during 6 hours. Removed coverslips were air-dried, stained with MayGtinwald-Giemsa solution, and mounted on microscope slides. The percentageof each type of inflammatory cells was determinedfrom a total of 500 counted cells.

Measurement of skin test responses On the fifth day, five 10 ~1 intradermal injections were performed on the outer face of each patient’s upper arm with the samesolutions introduced into the skin chambers, namely, per site: (1) HBSS-HSA, (2) compound 48/80 (1 p.g), (3) histamine (1 Fg), (4) pollen, 0.001 IR, and (5) pollen, 001 IR. Ten minutes after injection of (l), (2), and (3), and 20 minutes after pollen injection (4 and 5), the areasof the skin whealing and flare at the cutaneoustest sites were outlined on the skin, reproducedon an overlying sheet of transparentpaper, and measuredin square millimeterswith a computerized planimetry analyzer.

Measurement of mediators in skin chamber-collected media Skin chamber media collected at 30 minutes and 6 and 24 hours were immediately centrifuged for 10 minutes at

HESS -WA

3 E E il 2 2

CompMIndHislamine

4&80

Pollen ii(TliR

‘Pollen

;)(IlIR

2200 zoo0 1800 1600 1400 1200 loo0 800 a0 400 200 0 WSS

-FlSA

CampoudHistamine

48l80

Fallen

0.001 IR

‘POki

0.01 IR

FIG. 1. Mean area of wheals and flares (square miliimeters -+ standard deviation) in response to 10 pi intradermal injection of HESS-HSA, compound 4W60 (I&, histamine (lpg), polien, 0.001 IR, and pollen, 0.01 1R, in pollen-sensitive patients (N = 6) with oral administration of placebo (lightsquare) and cetirizine 2 HCI (dwksquare). (NS = non significant.)

400 g. Cell-free supcmatants were sampled and treated as in the following manner:(1) a 500 ~1 afiquot of supernataut was deproteinizedby precipitation with 500 ~1 of perchloric acid (0.4 N final) and kept for spectrofluorometric measurementof histamine.” Results were expressedas nanogramsof histamine baseper milliliter per squairecentimeter of skin-denudedarea,and (2) a 200 ~1 aliquot of supernatant was frozen at -20” C until it was tested for PAF-acether content by rabbit-platelet aggregationassay.‘”

Statistics Results were expressedas mean values .Z standarddeviations. The statistical significance of the results was estimated with Wilcoxon’s test.*’

RESULTS Measurement of cetirizine 2 HCI in the plasma and in the Mister fluid: Comp@ance with treatment In the six allergic patients receiving cetitizine 2 HCI cetirizine 2 HCI mean mounts were not significantly different according to the Wilcoxon’s test in plasma (0.156 -+ 0.080 ygiml) as compared to treatment,

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0

30 min

6h

BBS!&ISA

24 h

30 min

6h

24 h

Compound 48180

30 min Polten

6 h

24 h

0.001IR

30 mln

6 h

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IMMUNOL. JULY 1999

24 h

Pollen O.OlIR

FIG. 2. Measurement of histamine release (nanograms per milliliter per square centimeter) in skin chambers challenged with HBSS-HSA, compound 48/80 (1 Fg), pollen, 0.001 IR, and pollen, 0.01 IR, in pollen-sensitive patients (N = 6; mean values r standard deviation) with oral administration of placebo (light square) and cetirizine 2 HCI (dark square). (NS = non significant.)

blister fluid (0.101 +- 0.035 pg/ml). No cetirizine 2 HCl was detectedin the plasma and the blister fluid from the samepatients during oral treatmentwith placebo (results not presented). Measurement of wheel-ancbflare responses In two of six patients, HBSS-HSA intradermal injection induced a small skin wheal sometimescircled by a tiny etythema. Theseskin wheals and erythemas were not significantly modified by treatment with cetirizine 2 HCI (Fig. 1). In all patients, pollen at the high dose elicited skin whealing responsesandperilesional erythemasof similar surface area as the ones induced by compound 48/ 80 and histamine, whereasthe low dose of pollen induced smaller ones. After 4 days of oral treatmentwith cetirizine 2 HCl, the skin-whealing responsesand the erythemas induced by compound 48 / 80, histamine, and pollen at 0.001 IR and 0.01 IR per milliliter were inhibited 84%, 78%, 88% and 90% (p 6 0.05) and 75%, 76%, 55% and 76% (p 6 0.05), respectively, as compared with the placebo sequence. Measurement of mediators: Histamine concentration In blood. Blood histamine levels were measured before any treatment and after 5 days of placebo or cetirizine 2 HCl oral administration (Table II). Levels were not influenced by placebo or cetirizine 2 HC1 treatment. In skin chamber$uids after stimulation by HBSSHSA, compound 48180, or pollen (Fig. 2). When

patients were receiving placebo, a nonimmunologic histamine release was measured at 30 minutes in chambersfilled with HBSS-HSA. Histamine was still

detected at the sixth and twenty-fourth hour. Cetirizine 2 HCl did not significantly inhibit the inflammatory histamine release occurring in HBSS-HSAcontaining chambersat 30 minutes and 24 hours, but it significantly decreased( -47%; p G 0.05) the histamine releaseoccurring at 6 hours, ascomparedwith histamine amountsmeasuredat the sametime during the placebo sequence. When chamberswere filled with compound 48 / 80, the amountsof histamine detectedin media collected at 30 minutes and6 and 24 hours were not significantly different from amounts measured in HBSS-HSAcontaining chambers. The effect of cetirizine 2 HCl administration on compound 48/ 80-induced histamine releasewas analogousto that obtained with control HBSS-HSA. When chamberswere filled with pollen at 0.001 IR or 0.01 IR per milliliter, histamine releaseat 30 minutes was significantly increased 53.8% (p < 0.02) and 195%(p G 0.05), respectively, ascomparedwith that of control chambers containing HBSS-HSA. Treatment with cetirizine 2 HCl significantly decreased18% (p < 0.05) and 21% (p G 0.05) the 30minute histamine release induced by the pollen extracts at 0.001 and 0.01 IR per milliliter, respectively, as comparedwith that of the placebo sequence. Six hours after the first introduction of both doses of pollen, histamine levels in the chamberswere not significantly different from the levels measured in HBSS-MA-containing chamber at the same time. Cetirizine 2 HCl did not significantly decreasethe 6-hour histamine release induced by pollen at either 0.001 IR per milliliter ( - 7%; p 6 0.10) or 0.01 IR per milliliter ( -26%; p G O.lO), comparedwith the 6-hour pollen-induced histamine release measured during the placebo sequence.

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In media collected 24 hours after the initial pollen introduction, histamine amounts were significantly higher than amounts obtained in control HBBS-HSA collected at the sametime (p 4 0.05 for both pollen concentrations), despite the replacementof pollen by HBSS-HSA alone in all the chamberssince the sixth hour. Histamine release induced by the low dose of pollen was not significantly different from that induced by the high dose. Cetirizine 2 HCl did not significantly decreasethe 24-hour histamine levels obtained after pollen at 0.001 IR per milliliter (- 28?~; p G 0.1) or after pollen at 0.01 IR per milliliter ( - 16%;p G 0. I ), comparedwith the corresponding placebo sequence PAF-acether When patients were receiving placebo treatment, low amounts of PAF-acetherwere detected in fluids collected at 6 hours from the chamberscontaining the high dose of pollen (0.01 IR per milliliter). The meanvalue of PAF-acetheramount was 95.0 & 34.5 pgi ml/cm’ (n = 6). With cetirizine 2 HCl administration, PAF-acetherwas not detectedin the 6-hour media. No PAF-acether was detected in other collected media with either placebo or cetirizine 2 HCl treatment. Analysis of the type of inflammatory cells recruited from the superficial dermis The percentageof different types of inflammatory cells collected from the superficial dermis by the Rebuck skin window technique is schematizedin Fig. 3. After HBSS-HSA, recruited inflammatory cells were mainly neutrophils (93.5% to 99.1%). A few monocytes(0.9% to 6.0%) and occasionaleosinophils (0% to 0.8%) were counted. After compound 48/80 or histamine, percentagesof recruited cells were similar to those obtained after HBSS-BSA. In thesethree studies (HBSS-BSA, compound 48180, and histamine), treatment with cetirizine 2 HCl did not significantly modify cellular recruitments observed with placebo. After challenge with the low dose of pollen, the percentage of recruited eosinophils (1.1% to 1.6%) was twice as high as the one obtained after HBSS-BSA. After the high doseof pollen, eosinophils represented3.3% to 4.9% of the various recruited cell types, percentage about four times higher than the value found after HBSS-BSA. Monocyte accumulation increased from the first hour to the sixth hour, but not significantly, whereas neutrophil recruitment decreased,also not significantly. When results obtained with placebo and cetirizine 2 HC1are compared, treatment with cetirizine 2 HCl

TABLE Ii. Measurement

of blood histarnine levels before each treatment and after 4 days of oral administration of pkwebo or cetirizine 2 HCI in six pollen-sensitive patients Blood histamine level (rig/ml t SD) Sequence

Before treatment

After treatment

69.5 + IY.5 NS 5d.J 7: LO.6 NS Placebo Cetirizine2 HCI 63.0 -t 17.8NS h!.ir !- 18.3NS _____-_ -..-. . ..---..-__ NS = not significant.

decreasedthe eosinophii recruitment induced by the low concentration of pollen (down to 0.2% to 0.5% eosinophils), significantly at the first hour (p G 0.05) and not significantly at the sixth hour. Cetirizine 2 HCl also significantly inhibited the eosinophil recruitment induced by the high dose of pollen at both the first and the sixth hour (p < 0.05) (down to 0.4% to 1.5% eosinophils). DISCUSSJON In the present study, we evaluatedthe time-course releaseof histamine for 24 hours. Histamine was releasedin the chambersas early as the first 30 minutes after pollen challenge in pollen-sensitive patients. This result is in accord with the concept that IgE and a specific antigen combine together on mast cell surfacesto lead to an immediate exocytotic secretion of preformed mediators.‘4.2sThereafter, the rate of histamine delivery from the dermis in the chamber decreasedby time unit. However, the long-lasting diffusion of histamine in the chambers,even after pollen removal, suggests that besides the immediate degranulation of mast cells that release the preformed granule-associatedmediators, other cellular origin of histamine might be involved. Histamine could be newly generatedby activated mast cells after the primary degranulation. Circulating cells, such as basophils, attractedto the inflammatory cutaneousreaction site, could be also activated to release the histamine content.” Oral treatment with 10 mglday of cetirizine 2 HCI significantly reducedin allergic patientsthe 30-minute allergen-induced histamine release, but, not significantly, the later histamine releases.By useof a similar in vivo technique, anotheranti-H, molecule, hydroxyzine, has been previously reported as an inhibitor of immediate histamine releasein skin anaphylactic reaction.” The moderateinhibition by cetirizine 2 l-ICI

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T=lh

T=lh

T=6h

T=6h

T=lh

ll8.S -1ISA

4W.O HisCanine Pdlm Pdlen a.cw IP 0.01 IR

HBSS -WA

4WO Hirtaminc Pollen Pdlcn O.M)l IR 0.01 IR

FIG. 3. Percentage of recruited inflammatory cells (neutrophils, monocytes, and eosinophils) on the superficial dermis 24 hours after challenge with HBSS-HSA, compound 48180 (1 kg), histamine (1 kg), pollen, 0.001 IR, and pollen, 0.01 IR, in pollen-sensitive patients (N = 6; mean percent k standard deviation) with oral administration of placebo (light square) and cetirizine 2 HCI (dark square], Unless p values are indicated, there is no significant inhibition of recruited cell percentage with cetirizine 2 HCI treatment as compared with the placebo sequence, according to Wilcoxon’s statistical test.

of the late histamine release could be explained by the fact that the primary effector system, involving essentially dermal mast cells, immediately releases many mediators that can induce histamine production from other cells by mechanismsthat do not involve H, receptors. Previousstudieshave also demonstrated that H, blockers significantly inhibited the immediate wheal-and-flare reaction and the histamine-induced cutaneousreaction, but failed to inhibit the late cutaneousreaction.I4 The absenceof effect of compound 48180 on histamine releasein the skin chambersis in contrastwith the wheal-and-flare reaction that we observed after

intradermal injection of the same compound and with in vitro results on purified human skin mast cells .*‘. ** This could be explained by the instability of compound48 / 80 in solution and by its nondiffusion through the superficial dermis. In the skin chamber, HBSS-HSA also induced a low histamine appearance.This nonimmunologic releasecould be due to the traumatisminduced by blister formation and removal of the epidermal roof just before the chamber laying. This underlined the high reactivity of dermal mast cells to degranulate in responseto a nonspecificinjury. Thus, histamine release can occur in the superficial dermis after any nonspe-

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citic traumatism, inducing increase in vascular permeability and formation of an inflammatory edema. Interestingly, cetirizine 2 HCl was able to inhibit this nonspecific cutaneous reaction and, as recently demonstrated by Juhlin et al. ,I6 cetirizine 2 HCl might be of interest for the treatment of nonimmunologic reaction like dermographism.‘9 Interestingly, 6 hours after pollen introduction in skin chambers, we were able to detect PAF-acether in the chamber-collected media despite the poor diffusibility and high hydrophobic property of the phospholipid. This suggests that PAF-acether was produced in sufficiently high amounts in the dermis to diffuse through the superficial dermis toward the chambers. In fact, various cell types may contribute to the presence of PAF-acether at the cutaneous reaction site. After in vitro anti-IgE stimulation, human lung mast cells are able to synthetize PAF-acether,‘O but they release little or none of the generated PAFacether into the extracellular medium. However, technical conditions may influence the ability of various cells to release the PAF-acether that they synthetize. It could be that, in vivo, stimulated cutaneous mast cells are able to generate and release the phospholipid within tissues. However, many inflammatory cells, including eosinophils, neutrophils, basophils, and monocytes”’ can release PAF-acether and, since they are recruited at the challenge site, they may be the main sources of PAF-acether within the dermis. The lack of the phospholipid in 24-hour media could be due either to the termination of antigen stimulation or to an acetylhydrolase activity responsible for PAFacether degradation, as described in eosinophils from patients with eosinophilia.3’ According to our results, PAF-acether release in skin chamber fluids overlayed on atopic subjects has been observed 5 hours after pollen challenge in vivo.33 The formation of PAF-acether in addition to histamine release from human lung has been reported in other studies, and this lipidic mediator actively participates in allergic bronchial asthma.34Moreover, intradermal injection of PAF-acether elicited in normal subjects a wheai-and-flare reaction similar to the one induced by moderate doses of allergen in sensitized patients3’ PAF-acether induced an accumulation of eosinophils on the dermis in allergic patients.36 Thus, PAF-acether might account for many cellular and vascular features of hypersensitivity and might play an important local role in the development of cutaneous allergic reactions. PAF-acether was not detected in cetirizine 2 HCItreated patients. This suggests that the drug was able to decrease the activation of mast cells or inhibit the production of PAF-acether-releasing agents or the re-

cruitment and activation of inflammatory cells able to generate PAF-acether, such as eosinophiis. ah discussed later. The use of Rebuck skin window technique demonstrated a significant recruitment of eosinophils 24 to 30 hours after the pollen-induced reaction. This eosinophil recruitment was not observed after challenge with histamine or compound 48 ! X0. other studies on late-phase hypersensitivity reactions have also noted the appearance of eosinophils 4 or 6 hours after intradermal injection of allergens” or PAl--accther in atopic subjects.“’ Recent data demonstrated that PAFacether in vitro was a potent chemotactic and themokinetic factor for human eosinophils, ” ‘i Moreover, in animal models, PAF-acether antagonists bave been demonstrated to inhibit antigen-induced eosinophil intiltration.“” Compared with PAF-acether, histamine, and mast cell-derived eosinophil ihemotactic factor of anaphylaxis induced negligibie eosinophil migration. “-“’ Nevertheless, histamine ha\ been also reported to be directly chemotactic for eobinophils at low doses by its action on H, receptors, whereas at higher concentrations, it was an inhibitor of this recruitment by an HZ action.” ” We did not observe significant accumulation of eosinophils after challenge with 1 p-g of histamine. Therefore. the release of PAF-acether after pollen challenge, and perhaps the release of low amounts of histamine, might be responsible for the eosinophil rccntitmcni that we observed. Eosinophilia is associated with many c\linicai disorders in humans, especially with allergic inflammation and parasitic infections.43 However, the precise role of the eosinophil is still the subject of somespeculation. In IgE-mediated allergic disease. it had been suggested that eosinophils, once they arrive at the reaction site, may have a regulatory role in dampening mast cell-mediator activity by releasing their content of histaminase, peroxidase, and other cationic proteins.# However, more and more data cast additional evidence on the toxic potential of eosinophils in atopic patients.‘: 46 In fact, these cells are able to release several inflammatory mediators 1 inciudinp PAFacether”. 4:.48and leukotriene C,,“” and chcir granulederived cationic proteins can induce mast cell degranulation and stimulate histamine release from human basophils.‘“- 5’ Therefore, eosinophil accumulation in the dermis appears to play a pathogenic role in allergic IgEmediated reactions, and the properties ibl cetirizine 2 HCI to antagonize this accumulation is of particular interest. Interestingly, cetirizine 2 HCl inhibits both PAF-acether appearance and eosinophil recruitment in allergic patients, suggesting that these IW) events are

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108 Michel et al.

closely associated during in vivo allergic cutaneous reaction. In conclusion, with an original experimental approach associating a new skin chamber technique and the Rebuck skin window technique, we have demonstrated in vivo in man that cetirizine 2 HCl inhibits not only the early allergic response but also the late inflammatory cell recruitment after IgE-mediated reactions in the skin. We thank Regine Merval for expert technical assistance in histamine analysis and Eugene Bakes for gas chromatography analysis of cetirizine 2 HCI. REFERENCES 1. Ishizaka T, Ishizaka K. Activation of mast cells for mediator releasethrough IgE receptors. In: Progressin allergy, vol 34. Basel: S Karger AG, 1984:188. 2. Walker IC. Studies on the sensitization of patients with bronchial asthmato bacterial proteins as demonstratedby the skin reaction and the methodsemployed in the preparation of these proteins. J Med Res 1917;35:487. 3. Bryant DH, Bums MW, Lazarus L. The correlation between skin tests, bronchial provocation tests, and the serum level of IgE specific for common allergens in patients with asthma. Clin Allergy 1975;5:145. 4. Levine BB, RedmondAP. The nature of the antigen-antibody complexes initiating the specific wheal-and-flare reaction in sensitized man. J Clin Invest 1968;47:556. 5. Solley GO, Gleich J, JordonE, SchroetterAL. The late phase of the immediate wheal-and-Aareskin reaction. J Clin Invest 1976;58:408. 6. Dolovich J, Hargreave FE, ChalmersR, Skier KJ, Gauldie J, Bienenstock J. Late cutaneousallergic responsesin isolated IgE-dependent reactions. J ALLERGYCLIN IMMUNOL1973; 52:38. 7. WassermanSI. The mast cell and its mediators. In: Goldsmith LA, ed. Biochemistry and physiology of the skin, vol 2. Oxford: University Press, 1983:878. 8. Lewis T. The blood vessels of the human skin and their responses.London: Shaw and Sons, 1927. 9. Fowler JW III, Lowe1FC. The accumulationof eosinophils as an allergic responseto allergen applied to the denuded skin surface. J ALLERGYCLIN IMMUNOL1966,37:19. 10. Durham SR, Carroll M, Walsh GM, Kay AB. Leukocyte activation in allergen-induced late-phase asthmatic reactions. N Engl J Med 1984;311:1398. 11. Wei Y, Heghinian K, Bell RL, Jakschik BA. Contribution of macrophages to immediate hypersensitivity reaction. J Immunol 1986;137:1993. 12. Capron A. Dessaint JP, Capron M, Joseph M, Ameisen JC, Tonne1AB. From parasites to allergy: a secondreceptor for IgE. Immunol Today 1986;7:15. 13. Douglas WW. Histamine and antihistamines.In: GoodmanLS, Gilman A, eds. The pharmacologicalbasis of therapeutics.4th ed. New York: Macmillan, 1970. 14. Smith JA, Mansfield LE, deShazoRD. An evaluation of the pharmacologicinhibition of the immediate and late cutaneous reaction to allergen. J AUERGY C~nu IMMUNOL 198@,65:118. 15. De Vos C, Maleux MR, Baltes E, Gobert J. Inhibition of histamine and allergen skin wheal by cetirizine in four animal species. Ann Allergy 1987;59:278.

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