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 compound 48180. 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 decrease in the wheal-and-flare cutaneous reaction induced by intradermal injection of pollen, histamine, and compound 48180. it also signt&ntly inhibited the immediate histamine release occurring in skin chambers after 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 chambers filled with pollen. With cetirizine 2 NC1 treatment, platelet-activating factor-acether was not detected in chamber media. Interestingly, cetirizine 2 HCE significantly reduced the eosinophil recruitment observed on the superficial dermis 24 hours after pollen challenge. (J ALLERGY CLIN IMMUNOL 1988;82:101-9.j

Human cutaneous immediate hypersensitivity re- sults from the antigen-induced release of biologically active materials from dermal mast cells sensitized with specific IgE antibody.’ The prediction of IgE-mediated reactions for antigens in a sensitive subject is very commonly studied by skin testing, such as intrader- mal injection or prick test, with various protein anti- gens . ‘3 3 The cutaneous reaction occurring rapidly after the exposure to the appropriate antigen is character- ized by a transient pruritus and the so-called wheal- and-flare reaction, sometimes followed by a late-phase development of an indurated poorly demarcated lesion 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-i Mmdor, Crete& France, **UCB, Phmmceutical Sector, Braine L’Alleud, Belgium, and ***Unite INSERM U 200, Cla- mart, France.

Supported by a grant from CNRS-INSERM: Lipides Pharmaco- lcgiquement 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’s balanced salt solution HSA: Human serum al&unin

HBSS-HSA: HBSS contairping 0.25% NSA PAF: Platelet-activating factor

IR: Index of reactivity

mediators,7 such as histamine, that induces a typical wheal-and-flare skin reaction.’ The mediators released 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 development of a late-phase allergic reaction. More- over, as human eosinophils, macrophages, and plate- lets possess specific 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 developed to in-

101

102 Michel et al. J. ALLERGY CLIN. IMMUNOL. JULY 1989

TABLE I. Protocol

1* 2* 3*

Blister formation

4*

Laying of skin chamber to Introduction of the

agonists t 30 min Renewal of the

agonists t 6 hr Renewal by

HBSS-HSA

5

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 administration of cetirizine 2 HCI or placebo.

hibit the end organ effects of the main mast cell me- diator, 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) phenylmethyll- I-piperazinyl)ethoxy}acetic acid dihydrochloride strongly inhibits cutaneous reactions induced by his- tamine 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 in- duced by challenge with either specific antigen, his- tamine, 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 ob- tained. All patients were pollen-sensitive on the basis of a typical history of allergic rhinitis/conjunctivis, with or with- out asthma, during the pollen season, and demonstrated positive whealing response 20 minutes after intradermal in- jection 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, pla-

cebo, 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 as a 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 measure the release of this mediator.

Product administration The study was conducted as 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 crossover fashion. A period of 4 weeks separated the two sets of experiments. Cetirizine 2 HCl (10 mg) and placebo, both in tablets of absolutely identical appearance, were numbered according to a pre- determined code. In the two sets of experiments, tablets were administered in the,evening, once a day, during 4 days,

Blood samples were 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 measured by gas chromatography analysis.* Blood hista- mine levels were determined by spectrofluorometry, ac- cording to the method described by LebeI.‘9 A control did not permit cetirizine 2 HCl to influence the spectrofluoro- metric measurement of 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

*Bakes E. Unpublished observation.

VOLUME 82 NUMBER 1

diameter base, three on one forearm and two on the other arm, were induced simultaneously by vacuum suction with a negative pressure of 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 measurement of the level of cetirizine 2 HCl by gas chromatography analysis. Each blis- ter was unroofed aseptically. Sterile siliconed Macrolon skin chambers (Trametal, Saint Maur, France) were placed dj- rectly over the obtained cutaneous windows (0.5 cm’ area) open on the superficial dermis and were kept in place by appropriate taping. 20

Time 0. Each chamber was filled with 1 ml of the fol- lowing solutions: (I) control chamber: HBSS-HSA, the di- lution 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 introduc- tion, chamber fluids were renewed by 1 ml of freshly pre- pared identical solutions. Collected fluids were sampled for further measurements.

Time 6 hours. Six hours after the first introduction, cham- ber fluids were collected and sampled. At this time, all chambers were 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 chambers were removed.

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

Each fibrin layer was unglued by use of two tweezers. According to the method described by Rebuck and Crow- ley,” a sterile glass coverslip was applied over each denuded area and was replaced hourly by a new one during 6 hours. Removed coverslips were air-dried, stained with May- Gtinwald-Giemsa solution, and mounted on microscope slides. The percentage of each type of inflammatory cells was determined from 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 same solutions 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 areas of the skin whealing and flare at the cutaneous test sites were outlined on the skin, reproduced on an overlying sheet of transparent paper, and measured in square milli- meters with 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 CompMIndHislamine Pollen ‘Pollen

-WA 4&80 ii(TliR ;)(IlIR

2200 zoo0

3 1800 E E

1600 1400

il 1200 loo0 2 800 2 a0

400 200

0 WSS CampoudHistamine Fallen ‘POki

-FlSA 48l80 0.001 IR 0.01 IR

FIG. 1. Mean area of wheals and flares (square miliime- ters -+ standard deviation) in response to 10 pi intrader- mal injection of HESS-HSA, compound 4W60 (I&, his- tamine (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 deproteinized by precipitation with 500 ~1 of perchloric acid (0.4 N final) and kept for spectrofluorometric mea- surement of histamine.” Results were expressed as nano- grams of histamine base per milliliter per squaire centimeter of skin-denuded area, and (2) a 200 ~1 aliquot of supernatant was frozen at -20” C until it was tested for PAF-acether content by rabbit-platelet aggregation assay.‘”

Statistics Results were expressed as mean values .Z standard de-

viations. The statistical significance of the results was es- timated 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 treatment, 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

104 Michel et al. J. ALLERGY CLIN. IMMUNOL.

JULY 1999

0 30 min 6 h 24 h 30 min 6 h 24 h 30 min 6 h 24 h 30 mln 6 h 24 h

BBS!&ISA Compound 48180 Polten 0.001 IR 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 ad- ministration 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 detected in the plasma and the blister fluid from the same patients during oral treatment with pla- cebo (results not presented).

Measurement of wheel-ancbflare responses

In two of six patients, HBSS-HSA intradermal in- jection induced a small skin wheal sometimes circled by a tiny etythema. These skin wheals and erythemas were not significantly modified by treatment with ce- tirizine 2 HCI (Fig. 1).

In all patients, pollen at the high dose elicited skin whealing responses and perilesional erythemas of sim- ilar surface area as the ones induced by compound 48/ 80 and histamine, whereas the low dose of pollen induced smaller ones.

After 4 days of oral treatment with cetirizine 2 HCl, the skin-whealing responses and the erythemas in- duced 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 HBSS- HSA, compound 48180, or pollen (Fig. 2). When patients were receiving placebo, a nonimmunologic histamine release was measured at 30 minutes in chambers filled with HBSS-HSA. Histamine was still

detected at the sixth and twenty-fourth hour. Cetiri- zine 2 HCl did not significantly inhibit the inflamma- tory histamine release occurring in HBSS-HSA- containing chambers at 30 minutes and 24 hours, but it significantly decreased ( -47%; p G 0.05) the his- tamine release occurring at 6 hours, as compared with histamine amounts measured at the same time during the placebo sequence.

When chambers were filled with compound 48 / 80, the amounts of histamine detected in media collected at 30 minutes and 6 and 24 hours were not significantly different from amounts measured in HBSS-HSA- containing chambers. The effect of cetirizine 2 HCl administration on compound 48 / 80-induced hista- mine release was analogous to that obtained with con- trol HBSS-HSA.

When chambers were filled with pollen at 0.001 IR or 0.01 IR per milliliter, histamine release at 30 min- utes was significantly increased 53.8% (p < 0.02) and 195% (p G 0.05), respectively, as compared with that of control chambers containing HBSS-HSA. Treatment with cetirizine 2 HCl significantly de- creased 18% (p < 0.05) and 21% (p G 0.05) the 30- minute histamine release induced by the pollen ex- tracts at 0.001 and 0.01 IR per milliliter, respectively, as compared with that of the placebo sequence.

Six hours after the first introduction of both doses of pollen, histamine levels in the chambers were not significantly different from the levels measured in HBSS-MA-containing chamber at the same time. Cetirizine 2 HCl did not significantly decrease the 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), compared with the 6-hour pollen-induced histamine release measured during the placebo sequence.

VOLUME 82 NUMBER 1

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 same time (p 4 0.05 for both pollen concentrations), despite the replacement of pollen by HBSS-HSA alone in all the chambers since the sixth hour. Histamine release induced by the low dose of pollen was not significantly different from that in- duced by the high dose. Cetirizine 2 HCl did not significantly decrease the 24-hour histamine levels ob- tained 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 ), compared with the corresponding placebo sequence

PAF-acether

When patients were receiving placebo treatment, low amounts of PAF-acether were detected in fluids collected at 6 hours from the chambers containing the high dose of pollen (0.01 IR per milliliter). The mean value of PAF-acether amount was 95.0 & 34.5 pgi ml/cm’ (n = 6). With cetirizine 2 HCl admin- istration, PAF-acether was not detected in the 6-hour media. No PAF-acether was detected in other col- lected media with either placebo or cetirizine 2 HCl treatment.

Analysis of the type of inflammatory cells recruited from the superficial dermis

The percentage of different types of inflammatory cells collected from the superficial dermis by the Re- buck skin window technique is schematized in 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 occasional eosinophils (0% to 0.8%) were counted. After compound 48/80 or histamine, percentages of recruited cells were sim- ilar to those obtained after HBSS-BSA. In these three studies (HBSS-BSA, compound 48180, and hista- mine), treatment with cetirizine 2 HCl did not sig- nificantly 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 dose of pollen, eosinophils represented 3.3% to 4.9% of the various recruited cell types, percentage about four times higher than the value found after HBSS-BSA. Monocyte accumula- tion 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 HC1 are 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

Placebo 69.5 + IY.5 NS 5d.J 7: LO.6 NS Cetirizine 2 HCI 63.0 -t 17.8 NS h!.ir !- 18.3 NS

_____-_ -. .-. . ..---.. -__ NS = not significant.

decreased the 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 re- cruitment 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 evaluated the time-course release of histamine for 24 hours. Histamine was re- leased in the chambers as 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 sur- faces to lead to an immediate exocytotic secretion of preformed mediators.‘4. 2s Thereafter, the rate of his- tamine delivery from the dermis in the chamber de- creased by time unit. However, the long-lasting dif- fusion of histamine in the chambers, even after pollen removal, suggests that besides the immediate de- granulation of mast cells that release the preformed granule-associated mediators, other cellular origin of histamine might be involved. Histamine could be newly generated by activated mast cells after the pri- mary degranulation. Circulating cells, such as baso- phils, attracted to the inflammatory cutaneous reaction site, could be also activated to release the histamine content. ”

Oral treatment with 10 mglday of cetirizine 2 HCI significantly reduced in allergic patients the 30-minute allergen-induced histamine release, but, not signifi- cantly, the later histamine releases. By use of a similar in vivo technique, another anti-H, molecule, hydroxy- zine, has been previously reported as an inhibitor of immediate histamine release in skin anaphylactic re- action.” The moderate inhibition by cetirizine 2 l-ICI

106 Michel et al.

T=lh

J. ALLERGY CLIN. IMMUNOL. JULY 1988

T=lh

T=lh

T=6h

T=6h

ll8.S 4W.O HisCanine Pdlm Pdlen

-1ISA a.cw IP 0.01 IR

HBSS 4WO Hirtaminc Pollen Pdlcn

-WA 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), his- tamine (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 mechanisms that do not involve H, receptors. Previous studies have also demonstrated that H, blockers significantly inhibited the immediate wheal-and-flare reaction and the histamine-induced cutaneous reaction, but failed to inhibit the late cu- taneous reaction. I4

The absence of effect of compound 48180 on his- tamine release in the skin chambers is in contrast with 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 compound 48 / 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 re- lease could be due to the traumatism induced by blister formation and removal of the epidermal roof just be- fore the chamber laying. This underlined the high reactivity of dermal mast cells to degranulate in re- sponse to a nonspecific injury. Thus, histamine release can occur in the superficial dermis after any nonspe-

VOLUME 82 NUMBER i

citic traumatism, inducing increase in vascular per- meability and formation of an inflammatory edema. Interestingly, cetirizine 2 HCl was able to inhibit this nonspecific cutaneous reaction and, as recently dem- onstrated by Juhlin et al. ,I6 cetirizine 2 HCl might be of interest for the treatment of nonimmunologic re- action 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 diffu- sibility and high hydrophobic property of the phos- pholipid. This suggests that PAF-acether was pro- duced 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 re- action 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 PAF- acether into the extracellular medium. However, tech- nical 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 PAF- acether 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.34 Moreover, 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 re- actions.

PAF-acether was not detected in cetirizine 2 HCI- treated 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 dis- cussed later.

The use of Rebuck skin window technique dem- onstrated a significant recruitment of eosinophils 24 to 30 hours after the pollen-induced reaction. This eosinophil recruitment was not observed after chal- lenge with histamine or compound 48 ! X0. other stud- ies 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 PAF- acether in vitro was a potent chemotactic and the- mokinetic factor for human eosinophils, ” ‘i More- over, in animal models, PAF-acether antagonists bave been demonstrated to inhibit antigen-induced eosin- ophil intiltration.“” Compared with PAF-acether, his- tamine, 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 re- cruitment 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 dis- orders in humans, especially with allergic inflam- mation and parasitic infections.43 However, the precise role of the eosinophil is still the subject of some spec- ulation. 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 pro- teins.# 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 PAF- acether”. 4:. 48 and leukotriene C,,“” and chcir granule- derived cationic proteins can induce mast cell de- granulation and stimulate histamine release from hu- man basophils.‘“- 5’

Therefore, eosinophil accumulation in the dermis appears to play a pathogenic role in allergic IgE- mediated 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

108 Michel et al. J. ALLERGY CLIN. IMMUNOL. JULY 1988

closely associated during in vivo allergic cutaneous reaction.

In conclusion, with an original experimental ap- proach associating a new skin chamber technique and the Rebuck skin window technique, we have dem- onstrated 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 re- actions in the skin.

We thank Regine Merval for expert technical assistance in histamine analysis and Eugene Bakes for gas chroma- tography analysis of cetirizine 2 HCI.

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