B-
`a‘“
`
`VOLUME 104 No.3 PART 1
`
`SEPTEMBER 199
`
`CURRENT REVIEWS
`OF ALLERGY AND CLINICAL
`
`IMMUNOLOGY
`Airway remodeling and
`persistent obstruction in
`"asthma
`
`MOLECULAR MECHANISMS
`IN ALLERGY AND CLINICAL
`IMMUNOLOGY
`gMust cell-T cell interactions
`
`EDITORIALS
`
`New understanding of
`disease mechanisms:
`‘1; PF, citement and caution
`
`Beyond breast-feeding
`
`BalanceIn asthma between
`matrix metalloproteinases
`and the" '“h'b'l°"5
`
`Mast cell-T cell interactions
`(a detailed explanation of the figure appears on page 13A)
`
`THE JOURNAL OF
`Allerg WClinical
`Immunology
`
`
`
`OFFICIAL JOURNAL OF
`
`0
`AMERICAN Ammam or ALLERLIY
`ASTHMA ;\ lllellNOLOUY
`
`'
`
`Published Monthly by
`V
`‘
`l‘ A Mosby
`ISSN 00916749
`
`0 CT O 4 1999
`"I
`J5/120 .Cl
`"
`500 HlliHLn«L )W-Joni/131m. m -...
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`;'
`
`GSK Exhibit 1017 - Page 1 of 14
`
`

`

`THE JOURNAL OF
`
`AllergyAND Clinical
`Immunology
`
`VOLUME 104
`
`NUMBER 3, PART 1
`
`OFFICIAL JOURNAL OF THE AMERICAN ACADEMY OF AWRGY, AsTHMA AND IMMUNOLOGY
`
`The editors' choice
`Donald Y. M. Leung, MD, PhD, Harold S. Nelson, MD, and Stanley J S:ufler, MD
`Current reviews· of allergy and clinical immunology
`Airway remodeling_ and persistent airway obstruction in asthma
`James E. Fish, MD, and Stephen P. Peters, MD, PhD, Philadelphia, Pa
`Moiecula.r mechanisms in allergy and clinical immunology
`Mast cell-T cell interactions
`Yoseph A. Mekori, MD, and Dean D. Metcalfe, MD, Kjar-Saba, Israel and Bethesda, Md
`
`Editorials
`New understanding of disease mechanisms: Excitement and caution
`David A. Stempel MD, Seattle, Wash
`
`Beyond breast-feeding
`Jmniftr K Peat, PhD, Jane Allen, PhD, and Wmdy Oddy, MPH. Westmead and West Perth, Australia
`Balance in asthma between matrix metalloproteinases and their inhibitors
`Gisele Mautino, PhD, Franfoise Capony, PhD, jean Bousquet, MD, and Antonio M. Vignola, MD, Montpellier, France, and
`Palermo, Italy
`
`Rostrum
`Allergic rhinobronchitis: The asthma-allergic rhinitis link
`F Estelle R. Simons, MD, FRCPC, Winnipeg, Manitoba, Canada
`
`507
`
`509
`
`517
`
`524
`
`526
`
`530
`
`534
`
`~•,1 Mosby Copyright© 1999 by Mosby, Inc.
`Vol. 10-l. No. 3. PL I, ScpLcmbcr 1999. The Jnumal of Allergy nnd Clinicnl lmmunology (ISSN 0091-6749) Is publbhcd mcm1hly, cxccptscn1imon1hly in
`Junuiuy (thirteen i.sues per ycnr). by Mosby. Inc., 11830 Westline lndusuial Dr., SL Louis, MO 63 146-33 18; phone I (800) 453-4351 or {3 14) 453•4351.
`Peciodicul~ posrng.c paid at St. Louis, Mo .• and nddi1ionul mailing office..~. Pos1mnstcr: Send change tir udch'e~-- 10 The J11umnl of 1\llcrg)' aml Clinical
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`for individual, ;ind $280.00 for ins1i1u1ions. Printed in the 'U.S.A. Copyright © 1999 by Mo,by. Inc.
`
`Continued on page 7 A
`
`J ALLERGY CUN IMMUNOL
`
`September 1999 5A
`
`

`

`CONTENTS
`
`CONTINUED
`
`Original articles
`Asthma, rhinitis, other respiratory diseases
`
`541
`
`547
`
`554
`
`559
`
`565
`
`~ Treatment of late-onset asthma with fluconazole
`Ge_,orge W Ward Jr, MD, Judith A. Woodfolk, MB, ChB, Mary L. Hayden, RN. MS, Sandra Jackson, RN, and
`Thomas A. E. Platts-Mills, MD, PhD, Charlottesville, Vtz
`Montelukast versus salmeterol in patients with asthma and exercise-induced
`bronchoconstriction
`Cesar Villaran, MD, Shane j. O'Neill, MD, FRCPL Arthur Helbling, MD, Jan A. van Noord, MD, PhD, Tak H. Lee,
`MD, Alexander G. Chuchalin, MD, PhD, Stephen J. Langley, MB, K11/asiri A. Gunawnrdena, MD, Stanis/av Suskovic,
`MD, PhD, Martino La11renzi, MD, Jay Jnsan, VMD, Joris Menten, MSc, Jonathan A. Leff, MD, and the
`Mo11teluknst/Snlmeterol Exercise Study Group, Lima, Peru, Dublin, Ireland, Bern, Switzerland, Heer/en, The Netherlands,
`London, Manchester, and Stoke Poges, United Kingdom, Moscow, Russia, Ljubljana, Slovenia, and Whitehouse Station, NJ
`The magnitude of the effect of smaller family sizes on the increase in
`the prevalence of asthma and hay fever in the United Kingdom and
`New Zealand
`lfristin Wickens, DPH, Julian Crane, FRAC!? Neil Pearce, PhD, and Richard Bensley, MD, Wellington, New Zealand
`Association of urinary leukotriene E4 excretion during aspirin challenges
`with severity of respiratory responses
`Pamela J. Daffern, MD, Diego Muilenburg, BA, Tony E. Hugli, PhD, and Donald D. Stevenson, MD, La Jolla, Calif
`In vivo resistance to corticosteroids in bronchial asthma is associated with
`enhanced phosyphorylacion of JUN N-terminal kinase and failure of
`prednisolone to inhibit JUN N-terminal kinase phosphorylation
`Ann R. Sousa, PhD, Stephen j. Lane, MRCPL PhD, Cecilia Soh, PhD, and Tak H. Lee, ScD, FRCP, London,
`United Kingdom
`Altered expression and action of the low-affinity IgE receptor FcERII (CD23) 575
`in asthmatic airway smooth muscle
`Hakon Hakonarson, MD, Carrie Carter, BA, Cecilia Kim, BA, and Michael M Grunstein, MD, PhD, Philadelphia, Pa
`Prevention of severe premenstrual asthma attacks by leukotriene
`receprnr antagonisr
`Hiroko Nakasato, MD, Taknshi Ohrui, MD, Kiyohisa Sekiznwa, MD, Toshifi,mi MatJu1; MD, Mutsuo Yamnya, MD,
`Gen Tamura, MD, and Hidetada Sasaki, MD, Sendai, Japan
`
`585
`
`Mechanisms of allergy
`
`Breast-feeding, maternal IgE, and total serum IgE in childhood
`Anne L. Wright, PhD, Duane Sherrill, PhD, Catharine J. Holberg, PhD, Marilyn Halonen, PhD, and
`Fernando D. Martinez, MD, Tucson, Ariz
`
`589
`
`J ALLERGY CUN IMMUNOL
`
`Continued on page 8A
`September 1999 7A
`
`

`

`CONTENTS
`
`CONTINUED
`
`Allergic airway inflammation in hypothyroid rats
`Simone Manr,olli, MSc, Maria Fernanda Macedo-Soares, PhD, Ekio 0. Vianna, MD, PhD, and
`Pauli1111 Sannomiya, PhD, Siio Paulo, Brazil
`Skin rest reactivity to 2 recombinant Aspergillus fitrnigatus allergens in A
`famigatus-sensicized asthmatic subjects allows diagnostic separation of
`allergic bronchopulmonary aspergillosis from fungal sensitization
`Stefanie Hemmann, PhD, Gunter Menz, MD, Chaim Ismail, MD, Kurt Blaser, PhD, and Reto Crameri, PhD,
`Davos, Switzerland
`
`Isolation and characterization of the ·mpuntain cedar (Juniperus ashei)
`pollen major allergen, Jun a 1
`Terumi Midoro-Horiuti, MD, PhD, Randall M. Go/db/um, MD, Alexander Kurosky. PhD, David W. Goetz, MD, PhD,
`and Edward G. Brooks, MD, Galveston and Lackland Air Force Base, Tex
`
`Molecular cloning of the mountain c-~dar (Juniperus ashei) pollen major
`allergen, Jun a 1
`Terumi Midoro-Horiuti, MD, PhD, Randall M. Go/db/um, MD, Alexamkr Kurosky. PhD, Thomas G. Wood PhD,
`Catherine H Schein, PhD, and Edward G. Brooks, MD, Galveston, Tex
`
`595
`
`601
`
`608
`
`613
`
`Linkage between lgE receptor-mediated histamine releasability from
`basophils and gene marker of chromosome llq13
`.
`Yoon-Keun Kim, MD, Sang-Heon Cho, MD, Young-Yul/ Koh, MD, Jee-Woong Son, MD, Byung-Jae Lee, MD,
`Kyung-Up Min, MD, and You-Young Kim, MD, Seoul Korea
`~ An in vitro comparison of commonly used topical glucocorticoid preparations 623
`Cmtiana Stellato, MD, PhD, Jun Atsuta, MD, PhD, Carol A. Bickel, MS, and Rob~rt P. Schleimer, PhD,
`Baltimore, Md and Tm Mie, Japan
`
`618
`
`IgE-binding proliferative responses and skin test reactivity to Cop c 1, the
`first recombinant allergen from the basidjomycete Coprinus comatus
`Karl A. Brander, PhD, Peter Borbely, MD, Reto Crameri, PhD, ~mer J Pichler, MD, and Arthur Helbling, MD,
`Bern and Davos, Switzerland
`
`630
`
`Basic and clinical immunology
`
`Immunopathogenesis of human gastrointestinal infection by Anisakis simplex 637
`Victoria de/ Pozo, PhD, Ignacio Arrieta, MD, Teresa Tunon, MD, PhD, Isabel Cortegano, BS, Belen Gomez, MD,
`Blanca Cdrdaba, PhD, Soledad Gallardo, PhD, Marta Rojo, BS, Guadalupe Rmedo, MD, PhD, Pilar Palomino, PhD,
`Ana l Tabar, MD, PhD, and Carlos Lahoz, MD, PhD, Madrid and Pamplona, Spain
`Differential regulatioJ1 of cyrokine expression after allergen exposure of
`~
`sensitized rats by cyclosporin A and corticosteroids: Relationship to .
`bronchial hyperresponsiveness
`Tung-Jung Huang, MD, Robert Newton, PhD, EI-Bda~ui Haddad. PhD, and K Fan Chung, MD, London, United King(cid:173)
`dom, and Keelung, Republic of China
`
`644
`
`SA September 1999
`
`Continued on page 1 OA
`J ALLERGY CUN IMMUNOL
`
`

`

`CONTENTS
`
`CONTINUED
`
`Gonadotropin-releasing hormone increases CD4+ T-lymphocyte numbers in
`an animal model of immunodeficiency
`Jill D. Jacobson, MD, Mansoor A. Amari, PhD, Matthew E. Mansfield, MS, Carole P McArthur, MD, and
`Loran T. Clement, MD, Kamas City. Mo, and Los Angeles, Calif
`
`653
`
`Dermatologi,c and ocular diseases
`
`Increased production of macrophage migration inhibitory factor by PBMCs
`of atopic dermatitis
`Tndamichi Shimizu, MD, Riichiro Abe, MD, Akim Ohkawam, MD, and ]1111 Nishi him, MD, Sapporo, Japan
`
`659
`
`Environmental and occupational disorders
`
`Molecular cloning of a major Alternaria alternata allergen, rAlt a 2
`Robert K B11Sh, MD, Hiram Sa11chez, BS, and David Geisler, AAS, Madison, Wis
`
`Measurement and characterization of cockroach allergens detected during
`normal domestic activity
`Sandra D. De Lucca, Bsc (Hons), David]. M. Taylor, Bsc (Hom), Timothy]. O'Menm, PhD, A/11111 S. Jones, PhD, 1111d
`Euan R. Tovey. PhD, Sydney, A11Strali11
`
`Food and drug reactions and anaphylaxis
`
`Cross-reactions in the latex-fruit syndrome: A relevant role of chitinases but
`not of complex asparagine-linked glycans
`Araceli Diaz-Perales, BS, Carmen Collado, PhD, Carlos Bl1111co, PhD, Rosa Sanchez-Monge, PhD, Teresa Carrillo, PhD,
`Cipriano Amgo11cillo, PhD, and Gabriel Salcedo, PhD, Madrid and Las Pa/mas de Gran Canaria, Spain
`
`Pollen allergy in peach-allergic patients: Sensitization and cross-reactivity to
`taxonomically unrelated pollens
`Javier C11esta-Herra11z, MD, PhD, Milagros Ldzaro, MD, PhD, Alberto Martinez, PhD, Elena Figueredo, MD, Ricardo
`Palncios, PhD, Manuel de-Las-Heras, MD, a11d]orge Martinez, PhD, Madrid, Salnma11ca, and Bilbao, Spain
`
`665
`
`672
`
`681
`
`688
`
`IgE reactivity to al and a2 chains of bovine type 1 collagen in children with 695
`bovine gelatin allergy
`Masahiro Sakaguchi, PhD, Hisae Hori, PhD, Shunji Hattori, PhD, Shinkichi Irie, PhD, Atsushi Imai, DVM, Makoto
`Ytt1111gida, PhD, Hiroshi Miyazawa, PhD, Masako Toda, PhD, and Sakae Inouye, MD, Tokyo a11d Gunma, japan
`
`Brief communications
`
`Allergy to proteases in medical laboratory technicians: A new occupational
`disease?
`Wemer Kempfi MD, Ham Oman, PhD, and Brune/lo Wiithrich, MD, Ziirich 1111d Uppsaln, Switzerland
`
`700
`
`10A September 1999
`
`J ALLERGY CLIN IMMUNOL
`
`

`

`CONTENTS
`
`CONTINUED
`
`Cockroach extermination does not rapidly reduce allergen in settled dust
`Larry W. Williams, MD, Patrick Reinfried, BA, and Richard J Brenner, PhD, Durham, NC. and Gainesville, Fla
`Allergic rhinoconjunctivitis caused by Harmonia axyridis (Asian lady
`beetle, Japanese lady beetle, or lady bug)
`john A. Yarbrough, MD, jack L. Armstrong, MD, Michael Z. Blumberg, MD, Amy E Phillips, BS,
`Edna McGahee, RN, and William K Dolen, MD, Gainewi/le and Auguita, Ga, and Richmond, ¼z
`
`Skeeter syndrome
`F. Emile R. Simom, MD, FRCPC. and Zhikang Peng, MD, Winnipeg, Manitoba, Canada
`
`Quantitation of dust mites and allergen in small dust samples
`Larry G. Arlian, PhD, Marjorie S. Morgan, PhD, and John F. Goelz, PhD, Dayton, Ohio, and Racine, Wu
`
`Pollen asthma in the deep
`Gennaro D:Amato, MD, Paolo Noschese, MD, Maria Russo, PhD, Jean Gilder, and Gennaro Liccardi, MD,
`Naples, Italy
`Inhibition of the seasonal IgE increase to Dactylis glomerata by daily
`sodium chloride nasal-sinus irrigation during the grass pollen season
`Jose L. Subiza: MD, PhD, Javier Subiza, MD, Marla C. Ba,jau, MD, Rosa Rodriguez, PhD, and
`Marla J GaviUn, MD, Madrid, Spain
`
`Correspondence
`Effects of oral and inhaled corticosteroids on the hypothalamic(cid:173)
`pituitary-adrenal axis
`B. J Lipworth, MD, FRCPE, and C. M. Jackson, MBCHB, MRCGP, Dundee, United Kingdom
`
`Reply
`James T. Li, MD, PhD, Rochester, Minn
`
`Corrections
`
`The editors' choice
`(1999;104:1-2)
`IgE antithyroid microsomal antibodies in a patient with chronic urticaria
`(Bar-Sela S, ReshefT. Mekori YA. 1999;103:1216-7)
`
`702
`
`704
`
`705
`
`707
`
`710
`
`711
`
`713
`
`714
`
`652
`
`699
`
`J ALLERGY CUN IMMUNOL
`
`Continued on page 13A
`
`September 1999 11A
`
`

`

`CONTENTS
`
`CONTINUED
`
`Reader service
`
`Instruction to authors
`
`Information for readers
`
`Newsview-American Academy of Allergy, Asthma and Immunology
`
`CME calendar-American Academy of Allergy, Asthma and Immunology
`
`Acknowledgements
`
`Professional opportunities
`
`23A
`
`28A
`
`31A
`
`37A
`
`40A
`
`59A
`
`About the cover
`Mast-T cell interactions are bidirectional, fulfilling regulatory or modulatory roles affecting various
`aspects of the immune response. These include mast cell and T cell proliferation and activation and T cell
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`J ALLERGY CUN IMMUNOL
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`Septembe r 1999 13A
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`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`An in vitro comparison of commonly
`used topical glucocort~coid preparations
`
`Cristian a Stellato, MD, PhD,a Jun Atsuta, MD, PhD,b Carol A. Bickel, MS,a and
`Robert P. Schleimer, PhD8 Baltimore, Md, and Tsu Mie, Japan
`
`Background: Glucocorticoids (GC) are potent inhibitors of
`peripheral blood eosinophil, basophil, and airway epithelial
`cell function.
`Objectives: We compared in vitro the inhibitory activity of
`synthetic GC used for topical treatment in asthma and rhinitis
`on basophil histamine release (HR), eosinophil viability, and
`expression of vascular cell adhesion molecule-I (VCAM-1) in
`the human bronchial epithelial cell line BEAS-2B.
`Methods: Cells were treated for 24 hours with increasing con(cid:173)
`centrations (range I0-13 to I0-6 mol/L) offluticasone propi(cid:173)
`onate (FP), mometasone furoate (MF), budesonide (BUD),
`beclomethasone dipropionate (BDP), triamcinolone acetonide
`(TAA), hydrocortisone (HC), or dimethyl sulfoxide diluent
`before challenge. HR was measured by a fluorometric assay,
`viability of purified eosinophils was assessed by erythrosin B
`dye exclusion, and expression ofVCAM-1 was measured by
`flow cytometry.
`Results: GC induced a concentration-dependent inhibition of
`anti-IgE-induced HR. Maximum inhibition ranged from
`59.7% to 81 %, with a rank order of GC potency of FP >MF>
`BUD > BDP = TAA >> HC. Three-day treatment of eosinophils
`with GC concentration-dependently inhibited IL-5-induced
`eosinophil viability, with a rank of potency almost identical to
`that observed with basophil HR. The rank order of potency of
`GC for inhibition of the expression of VCAM-1 in BEAS-2B
`cells was MF = FP » BUD > TAA > HC = BDP. Inhibitory
`concentration of 50% values revealed that epithelial cells were
`the most sensitive and eosinophils were the least sensitive.
`Conclusions: These data, combined with information on phar(cid:173)
`macodynamics of these drugs in vivo, may be useful in estimat(cid:173)
`ing GC local anti-inflammatory effects. (J Allergy Clin
`lmmunol 1999;104:623-9.)
`
`Key words: Gl11cocor1icoids, basophi/, eosinophi/, epithelial cells,
`allergy
`
`Glucocorticoids (GC) have been the most potent and
`effective drugs for the treatment of severe asthma since
`their discovery and synthesis in the late 1940s. Over the
`last 2 decades the use of potent inhaled GC preparations,
`which are metabolized rapidly once absorbed, has
`expanded widely. For many physicians treating asthma,
`
`From the Johns Hopkins Asthma and Allergy Center, Baltimore, Md,• and the
`Mic National Hospital, Tsu Mie, Japan.b
`Supported by grants from the National Institutes of Health and by gifts from
`Astra, Glaxo, and Schering-Plough.
`Received for publication Dec 21, 1998; revised June 7, 1999; accepted for
`pllblication June 7, 1999.
`Reprint requests: Cristiana Stellato, MD, PhD, Johns Hopkins Asthma and
`Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224-
`6801.
`Copyright © 1999 by Mosby, Inc.
`0091-6749/99 $8.00 + 0 1/1/100691
`
`Abbreviations used
`BDP: Beclomethasone dipropionate
`17-BMP: Beclomethasone-17-monopropionate
`BUD: Budesonide
`DMEM: Dulbecco's modified Eagle's medium
`DMSO: Dimethyl sulfoxide
`~-E: ~-Estradiol
`FP: Fluticasone propionate
`GC: Glucocorticoid
`HC: Hydrocortisone
`HR: Histamine release
`IC50: Concentration at which each GC produced 50%
`inhibition
`MF: Mometasone furoate
`PIPES: Piperazine- N,N'-bis-(2-ethanesulfonic acid)
`TAA: Triamcinolone acetonide
`VCAM-1: Vascular cell adhesion molecule-I
`
`inhaled GCs have now become a drug of first choice for
`even mild to moderate asthma because of the relative
`safety and clear efficacy of these compounds. Although a
`considerable amount is known about the mechanism of
`GC actions as anti-inflammatory drugs in general and for
`the treatment of asthma, relatively few studies have been
`performed to compare the available inhaled GC prepara(cid:173)
`tions that are being widely used.1,2 It is not a certainty
`that all GC will behave similarly, either in vivo or in dif(cid:173)
`ferent in vitro cellular systems. Variation in lipid solubil(cid:173)
`ity, receptor affinity, binding to lung tissue at sites other
`than the glucocorticoid receptor, and other factors can
`dramatically influence the potency and duration of action
`of GC in the lungs. We have recently shown that human
`lung tissue is active in metabolizing the endogenous GC
`hydrocortisone (HC) because of the presence of the
`enzyme 11 ~-hydroxysteroid dehydrogenase in epithelial
`cells and perhaps other cells in the lung. 3.4 Thus local
`metabolism may also influence the effectiveness of
`inhaled or endogenous GC in the airways. The purpose
`of this study was to compare commonly used inhaled
`GCs for efficacy in cellular assays with several important
`targets of GC action in the airway. The goal was to estab(cid:173)
`lish a rank order of potency of the following GCs in
`assays of steroid inhibition of basophil histamine release,
`eosinophil survival, and vascular cell adhesion molecule(cid:173)
`! (VCAM-1) expression in airway epithelial cells:
`mometasone furoate (MF), fluticasone propionate (FP),
`(BDP), budesonide
`beclomethasone dipropionate
`(BUD), triamcinolone acetonide (TAA), and HC. We set
`out to determine whether there are differences in their
`rank order of potency and concentration at which each
`623
`
`

`

`624 Stellato et al
`
`J A LLERGY CUN IM MUNOL
`SEPTEMBER 1999
`
`GC produced 50% inhibition (IC50) in these systems. We
`have observed that basophils are slightly more sensitive
`to GCs than eosinophils in the assays used and that air(cid:173)
`way epithelial cells are particularly sensitive to these
`inhaled GCs. The rank order of potency was relatively
`similar with all 3 assays.
`
`MATERIAL AND METHODS
`The following materials were purchased: piperazine- N,N'-bis(cid:173)
`(2-ethanesulfonic acid) (PIPES), o-glucose, BSA, heparin, dimethyl
`sulfoxide (DMSO), erythrosin B (Sigma, St Louis, Mo), Percoll
`(Pharmacia, Uppsala, Sweden), RPMI containing 25 mmol/L
`HEPES, penicillin-streptomycin solution, FBS, L-glutamine
`(Gibco, Grand Island, NY), rhu-IL-5 (R&D Systems, Minneapolis,
`Minn), goat antihuman IgE (Kirkegaard and Perry Laboratories,
`Gaithersburg, Md), alcian blue dye (Fisher Scientific, Pittsburgh,
`Pa), Dynabeads (Dynal, Great Neck, NY), CD16 mAB
`(Immunotech, Westbrook, Me), Dulbecco's modified Eagle's medi(cid:173)
`um (DMEM), HAM's F-12 medium, Ca+t- and Mg++-free HBSS,
`Versene (Ca++- and Mg++-free HBSS with 0.02% EDTA), trace ele(cid:173)
`ments, phosphoethanolamine-ethanolamine, retinoic acid (Bioflu(cid:173)
`ids, Rockville, Md), insulin, HC, epidermal growth factor, and
`endothelial cell growth supplement (Collaborative Research, Bed(cid:173)
`ford, Mass), cholera toxin, tri-iodothyronine (Sigma); heat-inacti(cid:173)
`vated FCS, penicillin-streptomycin solution (Life Technologies,
`Gaithersburg, Md), recombinant human TNF-a (R&D Systems).
`The following GCs were provided or purchased from the indi(cid:173)
`cated sources: FP, BDP, and beclomethasone-17-monopropionate
`(17-BMP) (Glaxo); MF and TAA (Schering-Plough); BUD (Astra
`Draco); and HC and P-estradiol (P-E) (Sigma). Stock solutions of
`the GCs were prepared in DMSO at 0.1 mol/L concentration and
`stored at -20°C. Buffers used for basophil histamine release were
`PAG (PIPES buffer [25 mmol/L] containing 110 mmol/L sodium
`chloride, 5 mmol/L potassium chloride, 0.003% BSA, 0.1 % D-glu(cid:173)
`cose), PAGCM (PAG containing calcium chloride 1 mmol/L and
`magnesium chloride l mmol/L).
`
`Preparation, culture, and histamine release
`from basophil-enriched leukocyte
`suspensions
`Venous blood was obtained from adult donors who had given
`informed consent and basophils were isolated with double-step Per(cid:173)
`coll gradients as previously described.5 Briefly, whole blood was
`anticoagulated with 0.1 mol/L EDTA and diluted with an equal vol(cid:173)
`ume of PIPES buffer, and the density was adjusted to 1.065 g/mL
`with 100% isotonic Percoll. The blood-Percoll mixture was layered
`over a cushion of Percoll of density 1.079 g/cm3 and then cen(cid:173)
`trifuged at 400g for 15 minutes at 22°C. The cells were collected
`from the supernatant and from the plasma-Percoll interface, washed
`once in EDTA- sodium chloride solution to remove the Percoll and
`washed twice in PAG-EDTA, and the number and percentage of
`basophils in each fraction was determined. The mean percentage of
`basophils found in these fractions ranged from 5% to 12%, as deter(cid:173)
`mined by cell counts in Spiers-Levy chambers with use of Alcian
`blue.6 Cells were then_ washed and cultured in replicates of l mL in
`24-well plates (Costar, Cambridge, Mass) at cell densities of l to I 0
`. x 106 cells, depending on basophil purity, as described.7 Briefly,
`cells resuspended in RPMI 1640 media supplemented with l % glu(cid:173)
`tamine, I% penicillin-streptomycin, and 10% FBS were cultured ih
`various concentrations of GC or DMSO diluent for 24 hours at
`37°C. Previous studies have shown that GCs have no effect on
`basophil viability during a 24-hour culture (not shown). Cells were
`then harvested, washed, and challenged with 0.1 µg/mL of anti-IgE
`
`(45 minutes, 37°C). At the end of the incubation, cell-free super(cid:173)
`natants were assayed for histamine release with the automated flu(cid:173)
`orometric technique of Siraganian. 8 Basophil histamine release was
`calcu)ated as the percent of total cellular histamine after subtraction
`of spontaneous release (5%-8% ).
`
`Preparation, culture, and viability
`assessment of peripheral blood eosinophils
`Human granulocytes were isolated from EDTA-anticoagulated
`venous blood of normal donors or patients with asymptomatic aller(cid:173)
`gic rhinitis or asthma by Percoll (1.090 g/mL) gradient centrifuga(cid:173)
`tion at room temperature. After centrifugation, all procedures were
`carried out at 4 °C to minimize cell activation. Red blood cells were
`removed by hypotonic lysis followed by removal of CD16-positive
`cells (neutrophils) with an immunomagnetic bead technique.9
`Eosinophil purity (on the basis of examination of Diff-Quik-stained
`cytocentrifugation preparations) was 99% ± l % and viabili_ty (on
`the basis of erythrosin B dye exclusion) was 99% ± l %. Cells were
`resuspended in RPMI 1640 medium supplemented with 1 % gluta(cid:173)
`mine, 1 % penicillin-streptomycin, 10% FBS, and IL-5 (0.01
`ng/mL) and placed (in replicates of 100 µL) in 96-we]l flat-bottom
`plates (Cqstar) at a cell density of 2.5 x 105 eosinophils per milli(cid:173)
`liter in the presence of various concentrations of GC, DMSO dilu(cid:173)
`ent, or medium alone for 3 days at 37°C. After the incubation peri(cid:173)
`od 70 µL of supernatant was_ carefully removed, without disturbing
`the eosinophils settled at the bottom of the we11s. Erythrosin B (2-3
`µL) was added to wells with gentle pipette mixing, and staining was
`allowed for precisely 5 minutes. A cell sample was then counted at
`x40 magnification. Viability of cells was expressed as percent of
`total ce11s counted.
`
`Culture and challenge of BEAS-2B epithelial
`cells
`BEAS-2B cells were a generous gift from Dr Curtis Harris
`(National Cancer Institute, Bethesda, Md). This c~ll line was
`derived from human bronchial epithelium transformed by an aden(cid:173)
`ovirus 12-SV40 hybrid virus.IO These cells retain electron micro(cid:173)
`scopic features of epithelial cells and show positive staining with
`antibodies to cytokeratin but do not form tightjunctions 11 (data not
`shown). BEAS-2B cells were cultured in 25-cm2 tissue culture
`flasks and maintained in Fl2/10x medium consisting of Ham's Fl2
`nutrient medium containing penicillin (100 U/mL) and strepto(cid:173)
`mycin (100 U/mL) and supplemented with insulin (5 µg/mL), HC
`(I0-7 mol/L), tri-iodothyronine (3.1 x I0-9 mol/L), cholera toxin
`(10 ng/mL), endothelial cell growth supplement (3.75 µg/mL), epi(cid:173)
`dermal growth factor (12.5 ng/mL), phosphoethanolamine(cid:173)
`ethanolamine (5 x IQ-6 mol/L), trace element~ ( l x), and retinoic
`acid (0.1 µg/mL) . Cells were used between passages 35 and 4 7 and
`were plated on 6-well cluster plates (Costar) and cultured for at least
`48 hours in F12/DMEM medium (which lacks added HC) contain(cid:173)
`ing 5% heat-inactivated FCS, penicillin ( 100 U/mL), and strepto(cid:173)
`mycin (100 U/mL) before harvesting.
`Flow cytometric analysis
`In the experiments assessing the inhibitory effects of glucocorti(cid:173)
`coids on VCAM-1 expression on BEAS-2B cells, cells were prein(cid:173)
`cubated with increasing concentrations of GCs or an equivalent
`concentration of DMSO diluent for 24 hours and then incubated
`with TNF-a (10 ng/mL) for 24 hours. These methods are described
`in detail elsewhere.12.13 Cells were then washed 3 times with Ca++(cid:173)
`and Mg++-free HBSS and treated for 10 minutes with Versene (Ca++
`and Mg++-free HBSS containing 0.02% EDTA) without trypsin and
`then removed from plates by repeated pipetting. For each analysis I
`x 1 Q6 cells were incubated in 30 µL of PBS/0.2% BSA containing
`
`

`

`J ALLERGY CUN IMMUNOL
`VOLUME 104, NUMBER 3, PART 1
`
`Stellat o et al 625
`
`TABLE I. Concentration of glucocorticoids required for
`inhibition of basophil HR, eosinophil survival, and
`epithelial VCAM-1 expression
`Eosinophil
`survival
`(mol/L)
`
`BEAS-2B VCAM-1
`expression
`(mol/L)
`
`GC
`
`Basophil HR
`(mol/L)
`
`FP
`MF
`BUD
`BDP
`TAA
`HC
`
`7xJO-11 *
`2 X JQ--10
`5.9 X JQ--I0
`1 X JO-9
`3 X 10-9
`1.5 X 10-7
`
`2.5 X JQ--10t
`7 X 10--10
`5.9 X JQ--9
`3 X 10--8
`IX 10-8
`2.5 X 10--6
`
`4.6 x JO-I2t
`1.8 X 10-12
`3 X 10--10
`3 X JO-R
`6.3 X 10-IO
`1.1 X 10-8
`
`*IC50 of HR.
`tConcenlration for half maximal reduction in eosinophil survival.
`UC50 ofTNF-a - induced VCAM-1 expression.
`
`saturating concentrations of each mAb and 4 mg/mL of human IgG
`(to reduce nonspecific binding) on ice for 30 minutes, as previous(cid:173)
`ly described.1 4 ,15 mAb for detection of intercellular adhesion mole(cid:173)
`cule-I was RR! (AMAC, Westbrook, Me) and for VCAM-1 was
`BBIG-Vl (R&D Systems). The cells were washed, resuspended in
`saturating amounts of fluorescein-conjugated goat F(ab ' )z anti(cid:173)
`mouse IgG antibody (Bio Source, Camarillo, Calif) for another 30
`minutes, and then washed again. Negative staining with propidium
`iodide (2 µg/mL) and a combination of scatter characteristics were
`used to identify a uniform population of viable cells. Fluorescence
`was measured with an EPICS Profile II flow cytometer (Coulter
`Electronics, Hialeah, Fla) and was expressed as percent of control
`IgG mean fluorescence intensity by comparison to control staining
`with an irrelevant isotype-matched mouse mAb. For each sample, at
`least 5000 events were collected.
`
`Statistical analysis
`Analysis of data was performed with use of Statview II software
`(Abacus Concepts, Berkeley, Calif) on a Macintosh Ilsi computer.
`Data are expressed as mean ± SEM. Statistical analysis between
`groups was performed with the ANOVA test with a post-hoc analy(cid:173)
`sis (Fisher PLSD test). A P value <.05 was considered significant.
`
`RESULTS
`Glucocorticoids are first-line drugs in the therapy of
`airway allergic diseases such as asthma and rhinitis.2 The
`inflammatory cells infiltrating the site of allergic reac(cid:173)
`tions from peripheral blood, such as eosinophils and
`basophils, are all targets of the anti-inflammatory activi(cid:173)
`ty of GCs. 16 These compounds are effective inhibitors of
`lgE-mediated mediator release by basophils.7,16 It has
`been shown that several GCs used by oral administration
`exert their inhibitory effect on basophils with a rank of
`potency that parallels that found in vivo. 14 GCs also exert
`profound inhibitory effects on the recruitment, activa(cid:173)
`tion, and survival of eosinophils.17 In the current in vitro
`study we compared the effect of several synthetic GCs
`currently used for topical treatment in asthma and rhini(cid:173)
`tis on basophil mediator release, eosinophil viability, and
`epithelial cell activation. Among the resident cells in the
`airways, epithelial cells are a particularly important tar(cid:173)
`get of GC action.18
`Fig 1 shows the results of a series of experiments (n =
`2-6) in which basophil-enriched cell suspensions were
`
`a: 100
`:c
`"C
`Cl)
`u ::, 80
`"C
`.5
`I.LI
`Cl 60
`"T
`;
`r:::
`
`CII -0 40
`
`r:::
`0
`!E
`.Cl 20
`:E
`.5
`#-
`
`0
`
`e FP
`~MF
`■ BUD
`■BDP
`1:!,,.TAA
`eHC
`
`10 -11
`
`10 -10
`
`10-9
`
`10~
`
`10•7
`
`10~
`
`Glucocorticold (M)
`FIG 1. Inhibition of basophil histamine release with GC. Basophils
`were cultured for 24 hours in presence of indicated GC or DMSO
`diluent and subsequently challenged with 0.1 µg/ ml anti-lgE.
`Data shown are mean ± SEM inhibition of histamine release (HR)
`from 2 to 6 experiments; percent HR induced by anti-lgE in
`OM SO-treated cells was 36.1 % ± 12.2%.
`
`incubated for 24 hours in the presence of increasing con(cid:173)
`centrations (10--11 to Io---6 mol/L) of the indicated GC or
`DMSO diluent before challenge with 0.1 µg/mL anti-IgE.
`Treatment with GC induced a concentration-dependent
`inhibition of anti-IgE-induced histamine release, with
`maximum inhibition ranging from 59.7%, achieved by HC
`at 10--0 mol/L, to 81 %, induced by BDP at 10--7 mol/L.
`Because a maximum effect was not reached in every
`experiment, for each GC we arbitrarily chose a defined net
`effect-50% inhibition of histamine release-<:alculated
`on the mean ± SEM of all experiments to express the con(cid:173)
`centration at which each GC produced IC50. These values
`are indicated in Table I, showing a rank order of potency
`of FP > MF> BUD > BDP = TAA >> HC.
`We next compared the ability of the various GCs to
`affect IL-5-sustained eosinophil survival by assessing
`eosinophil viability by erythrosin B dye exclusion after
`culture in the presence of GC. In a series of preliminary
`experiments, to identify a concentration of IL-5 able to
`prolong eosinophil survival without overriding the
`inhibitory activity of GC, we cultured eosinophils for 3
`days with a broad spectrum of IL-5 concentrations
`(0.002-5 ng/mL) in medium only, DMSO diluent, or 10--7
`mol/L BUD. A suboptimal concentration of IL-5 (0.01
`ng/mL) was chosen, at which both eosinophil viability
`and the inhibitory effect of the steroid were optimal (Fig
`2). The presence of DMSO did not affect eosinophil via(cid:173)
`bility (Fig 2). Subsequent