identification of messenger rna for il-4 in human ... · identification of messenger rna for 11-4...

of April 17, 2017.This information is current as

and release of the translated product.human eosinophils with granule localization Identification of messenger RNA for IL-4 in

Durham and A B KayWakelin, L Taborda-Barata, Q Meng, C J Corrigan, S R R Moqbel, S Ying, J Barkans, T M Newman, P Kimmitt, M

http://www.jimmunol.org/content/155/10/49391995; 155:4939-4947; ;J Immunol

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. All rights reserved.Copyright © 1995 by American Association of Immunologists1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on April 17, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from


http://ww

w.jim

munol.org/

Dow

nloaded from

http://www.jimmunol.org/cgi/adclick/?ad=50971&adclick=true&url=http%3A%2F%2Fmyriadrbm.com%2Fsimoa-technology%2Fjimmunol%2F

http://www.jimmunol.org/content/155/10/4939

http://jimmunol.org/subscription

http://www.aai.org/About/Publications/JI/copyright.html

http://jimmunol.org/alerts

http://www.jimmunol.org/


Identification of Messenger RNA for 11-4 in Human Eosinophils with Granule localization and Release of the Translated Product'

Redwan Moqbel,* Sun Ying,* Julia Barkans,* Terence M. Newman,+ Patrick Kimmitt,* Matthew Wakelin,* Luis Taborda-Barata,* Qiu Meng,* Christopher J. Corrigan,* Stephen R. Durham,* and A. Barry Kay2*

Human eosinophils are cytokine-producing cells that are prominent in IgE-dependent allergic tissue reactions. 11-4 promotes the development of the Th2-type phenotype in T cells and is an essential cofactor for IgE production by B cells. We detected mRNA for 11-4 by reverse transcription-PCR in blood eosinophils from atopic asthmatics. By specific ELISA, 108 -C 20 pg of 11-4 protein/lO' cells could be extracted from whole cells, and approximately 30% of the 11-4 was released after incubation with serum-coated particles. Using immunocytochemistry, eosinophils from atopic asthmatics and nonatopic controls showed 11-4 immunoreactivity using an anti-11-4 mAb. 11-4 was located predominantly in the eosinophil granules, as shown by both immunogold electron microscopy and a cell fractionation technique that dissociated cell granules from membrane and cytosolic components. 11-4 mRNA colocalized with eosinophils (using sequential immunocytochemistry with an eosinophil-specific (EG2) mAb and in situ hybridization using an 11-4-specific antisense riboprobe) in both cell cytospins from bronchoalveolar lavage fluid from asthmatics as well as skin biopsies obtained from allergen-induced late phase (6-h) reactions in atopic subjects. Using double immunocytochemistry on skin biopsies with eosinophil- and 11-4-specific mAb, 83.5 -C 3.5% of eosinophils were I1-4+. Conversely, eosinophils accounted for 46.5 f 3.9% of the total cells expressing 11-4 immunoreactivity. Thus, human eosinophils express mRNA for 11-4, and the translated product is contained within the crystalloid granule from which it is released after stimulation with serum-coated particles. These observations are consistent with the hypothesis that eosinophils contribute to the development of the Th2 phenotype by T cells infiltrating atopic allergic reactions as well as to IgE synthesis. The Journal of Immunology, 1995,155: 4939-4947.

E osinophils are prominent cells in IgE-mediated allergic tissue reactions, including asthma, and in immune and inflammatory reactions against parasitic helminths (1, 2).

Recent studies have shown that eosinophils synthesize and trans- late a number of cytokines, including IL-la, transforming growth factor-a, transforming growth factor-pl, GM-CSF, IL-3, IL-5, IL-6, IL-8, and TNF-a, and the chemokine macrophage inflammatory protein-la (reviewed in Ref. 3) and have recently been shown to synthesize and store IL-2 (4,5). Thus, eosinophil-derived cytokines have the propensity to perform important auto- and para- crine regulatory roles (3, 6, 7).

Since eosinophils are intimately associated with IgE-dependent inflammation, it is likely that they may exert some influence on the development and/or the maintenance of the IgE-dependent allergic response. Synthesis of IgE requires IL-4 as an essential cofactor (8). IL-4 is a product of activated CD4+ T lymphocytes (8) of the Th2 subset (9, 10). IL-4 also up-regulates the expression of FceRII (CDZ3) on B cells and monocytes (11) and costimulates the via-

tional Heart and Lung Institute, Royal Brompton Hospital, London, United Kingdom Departments of *Allergy and Clinical Immunology, and 'Thoracic Medicine, Na-

Received for publication March 27, 1995. Accepted for publication August 21, 1995.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by a program grant from the Medical Research Coun- CII of the United Kingdom and a Medical Research Council Clinician Scientist Fellowship awarded to C.J.C.

' Address correspondence and reprint requests to Dr. A. 8. Kay, Allergy and Clinical Immunology, National Heart and Lung Institute, Dovehouse Street, Lon- don, United Kingdom SW3 6LY.

Copyright 0 1995 by The American Association of Immunologists

bility and growth of normal resting T cells and some T cell lines (12). The ability of eosinophils to contribute to IL-4 synthesis has not yet been evaluated. Thus, we hypothesized that eosinophils have the capacity to synthesize, store, and release IL-4 both in vitro and in the context of allergic inflammation in vivo. As a precedent for this hypothesis, it has recently been shown that eosinophils contain IL-5 mRNA and protein product (13, 14). Since the genes encoding IL-4 and IL-5 are situated close together on chromosome 5 (15), it is possible that eosinophils co-ordinately express these cytokines, as is the case for Th2-type T lymphocytes.

In this study, we present evidence consistent with transcription of IL-4 mRNA by human peripheral blood eosinophils. We also show localizationistorage of IL-4 protein with the crystalloid granule as well as IL-4 release following an appropriate stimulus. We also identified IL-4 mRNA and translated product in BAL3 fluid eosinophils from atopic asthmatics as well as tissue eosinophils from biopsies of cutaneous allergen-induced LPR in atopic subjects.

Materials and Methods Isolation of peripheral blood eosinophils

Peripheral blood (100 ml) was obtained from mild atopic asthmatics not receiving oral corticosteroids (eosinophils >lo% of the total leukocyte count) as well as from normal nonatopic healthy subjects. After dextran sedimentation of the erythrocytes, the granulocyte pellet was obtained by

Abbreviations used in this paper: BAL, bronchoalveolar lavage; LPR, late phase reaction; ECP, eosinophil cationic proteln; LDH, lactate dehydrogenase; TBS, Tris-buffered saline; RT, room temperature; APAAP, alkaline phosphatase-anti- alkaline phosphatase, DAB, diaminobenzidine; K C , immunocytochemistry;

protein. ISH, in situ hybridization; RT-PCR, reverse transcription-PCR; MBP, major basic

0022-1 767/95/$02.00


http://ww

w.jim

munol.org/

Dow

nloaded from


4940 DETECTION OF IL-4 IN HUMAN EOSINOPHILS

density centrifugation on Percoll. Eosinophils were purified by immune- magnetic selection using the magnetic activated cell separator system (Bec- ton Dickinson, Cowley, UK) as previously described (16). To eliminate contamination of the eosinophils with mononuclear cells, anti-CD14- and anti-CD3-coated micromagnetic beads (Lab Impex, Teddington, UK) were also added to the anti-CD16/granulocyte mixture. By negative selection, highly purified CD16- eosinophils (>99%) depleted of magnetically positive neutrophils (CD16+) and any contaminating mononuclear cells (CD14+/CD3+) were routinely obtained. The few contaminating cells were neutrophils.

The IL-4 content of blood eosinophils was estimated as follows. Freshly isolated and purified cells from atopic asthmatics were washed once by sedimentation (5 min, 120 X g ) in PBS and resuspended in the Same buffer at a concentration of 5 X 106/ml. Purified eosinophils from each donor were divided. One aliquot was disrupted with three cycles of freeze-thawing, and IL-4 was measured in the lysate using a specific ELISA (CLB, Amsterdam, The Netherlands) according to the manufacturer’s instructions. The other aliquot of cells was incubated with serum-treated Seph- adex G-15 beads for 45 min at 37°C as previously described (17), and IL-4 was measured in the supernatant. The sensitivity of the assay was 2.3 pgiml.

Fractionation of human eosinophils

The method for separation of granule-, plasma membrane-, and cytosol- rich fractions of highly purified human eosinophils (99% pure) from atopic asthmatics has been described previously (18). Briefly, 5 X lo7 cells were sedimented by centrifugation at 240 X g for 5 min, and the pellet was resuspended in ice-cold 0.25 M sucrose buffer containing 10 mM HEPES, 1 mM EDTA, and 5 pg/ml each of leupeptin, aprotinin, and tosyl-L-argi- nine methyl ester (TAME), pH 7.4, (all from Sigma Chemical Co., Poole, UK) and repelleted at 4°C. For homogenization, the cells were subjected to 8 to 12 passes through a high-precision ball bearing device (EMBL, Hei- delberg, Germany) with a clearance of 11 p m (19). The postnuclear supernatant (3-4 ml) obtained by centrifugation at 400 X g for 10 min was layered gently onto an 8-ml linear Nycodenz (Nycomed AS Pharma, Oslo, Norway) gradient (0-45% Nycodenz dissolved in HEPES-buffered sucrose) and centrifuged to equilibrium density at 100,000 X g (1 h, 4°C). Fractions (n = 24; 0.4 ml) were collected from each preparation. These were stored at 4°C for no longer than overnight and later at -80°C until used.

Protein detection in eosinophil fractions

IL-4 was detected in duplicate samples of Nycodenz-separated cell fractions and freeze-thaw-disrupted eosinophils, using a Quantikine ELISA kit (R&D Systems, Abingdon, UK). Dot blotting was used as previously described (17) to detect ECP recognized by the mAb EG2 (Supernatant IgG1, Pharmacia, Uppsala, Sweden) and CD9 (purified IgGl monoclonal anti- CD9, Becton Dickinson, Abingdon, UK), which are associated with eosinophil granules and plasma membrane, respectively. Volumes (2 pl) of eosinophil fractions were placed on a nitrocellulose membrane, allowed to dry, and blocked in 5% milk powder (Sigma Chemical Co.). The blotted membrane strips were incubated with the appropriate mAb (EG2 at 11250, anti-CD9 at 11500, or irrelevant isotype identical control Ab, including anti-CD3 IgGl supernatant and anti-CDS purified IgGl (Becton Dickinson, Mountain View, CA), both at 1/500). After extensive washings in PBS- Tween 20, strips were incubated at room temperature with a biotinylated rabbit anti-mouse IgG (at 11200) followed by streptavidin-alkaline phosphatase (1175). The strips were developed with Fast Red. Recombinant IL-4 was used as a positive control for anti-IL-4. The concentrations of IL-4, ECP, and CD9 in the fractions were assessed by assigning arbitrary units to the staining density of each fraction and converting the data to a percentage of the total activity.

The cytosolic enzyme LDH was measured in eosinophil fractions using an established technique (19) adapted for use with a microtiter plate reader. Absorbance (340 nm) was recorded at 1-min intervals over 20 min, and the rate of enzyme reaction was calculated by regression analysis over the 20-min period for each sample.

Immunocytochemistry

Cytospins. Cytospins of magnetic activated cell separator-purified (>99% pure) eosinophils from documented atopic asthmatics and normal healthy nonatopic subjects were fixed for 90 s in acetone. IL-4 was detected by overlaying cells with 100 pl of a biotinylated rat anti-human IL-4 mAb (PharMingen, San Diego, CA; a blocking anti-IL-4 Ab, MP4-25D2, previously validated (20-22)) in TBS at a final concentration of 10 pgiml and incubating overnight at RT. Slides were washed thoroughly in TBS, and streptavidin alkaline phosphatase (Amersham International, Aylesbury,

UK) was added at a 1/75 dilution for 30 min at RT. After an additional wash in TBS, the slides were developed with Fast Red tablets (Sigma Chemical Co.) and then counterstained with hematoxylin. Positive cells stained red. Appropriate negative controls (TBS only, an irrelevant biotinylated rat Ab of the same isotype, anti-IL-4 in the absence of the second layer, and second layer alone) were routinely included. The coefficient of repeated measurements (23) was 11.5 (95% confidence limits). Skin biopsies of LPR. These biopsies were performed as previously described (24). Six atopic subjects (two females and four males; mean age, 38.5 * 2.8 yr) were recruited from the Allergy Clinic of the Royal Bromp- ton Hospital (London, UK) and from the staff of the National Heart and Lung Institute (London, UK). Inclusion criteria were as follows: age between 18 and 55 yr, history of seasonal and/or perennial allergic rhinitis and/or asthma, absence of any other illness, and positive skin prick tests (weal diameter, >5 mm) to timothy grass pollen (Phleurn prutense; Solu- prick, ALK, Horsholm, Denmark) or house dust mite extract in the presence of positive histamine and negative vehicle controls. Patients taking oral medication were not included in the study. The total IgE levels ranged from 101 to 1780 IUlml, and all patients had positive RAST results to P. pratense (6.3-100 IU/ml).

The study was approved by the Royal Brompton Hospital ethics com- mittee and was performed with the patients’ written informed consent. Thirty biologic units of timothy grass pollen extract (0.02 ml) or diluent was injected intradermally on the extensor aspect of the forearms of each subject. Macroscopic responses were measured by evaluating skin indura- tion by resistance to the movement of a sharpened pencil point with which the reaction was outlined. The site injected with allergen was biopsied at 6 h, immediately after measurement of the size of the reaction. The control site injected with diluent was biopsied at 24 h. A 4-mm disposable biopsy punch was used to take biopsies after using 1% plain lidocaine for local anesthesia.

The biopsies were snap-frozen in isopentane (BDH Chemicals, Poole, UK) and cooled in liquid nitrogen. Cryostat sections (6 pm thick) were cut from biopsies, air-dried overnight, and then fixed in acetone for 10 min. Endogenous peroxidase was blocked using 1% H202 (plus 0.02% sodium azide in TBS) for 30 min. After a brief wash in TBS, sections were incubated in 1% BSA (Sigma Chemical Co.) in TBS for 30 min. A mixture of the primary mAb was added. This consisted of the biotinylated rat anti- human IL-4 mAb (150) used to detect IL-4 product and a 1/30 dilution of BMK-13 (a mouse anti-MBP) used to determine the eosinophil phenotype (25). The slides were left to incubate overnight at RT. After washing in TBS, a secondary layer, rabbit anti-mouse IgG (1140; Dako, High Wy- combe, UK), was applied to the slides (30 min, RT), which were then washed in TBS and incubated (30 min, RT) with a tertiary layer of a mixture of streptavidin peroxidase (Amersham; 11150) and murine APAAP conjugate (Dako; 1/40). Sections were developed sequentially in Fast Red (an APAAP substrate) followed by DAB (a peroxidase substrate; Sigma Chemical Co.). IL-4-producing cells stained brown, eosinophils stained red, and eosinophils producing IL-4 stained reddish brown. Appropriate negative controls were included. These were TBS alone, an irrelevant biotinylated rat Ab of the same isotype, an irrelevant mouse IgG, omission of anti-IL-4 Ab from the entire protocol, and omission of anti-MBP from the entire protocol. Cells were enumerated as described below under double ICC1ISH. Immunokzbefing for electron microscopy. This was performed as previously described (18). Briefly, pelleted isolated eosinophils were fixed in freshly prepared formaldehyde (2% in PBS, 0.1 M, pH 7.2) for 2 h and embedded in Lowicryl K4M resin using the Balzers FSU 010 preparation plant, equipped with an UV lamp, and a progressive lowering of temperature infiltration procedure. Silver sections were cut and picked up onto thin bar (460 TB Hex) nickel grids. Before labeling, sections were blocked and then labeled by immersion in a solution containing biotinylated rat anti-human IL-4 Ab at (PharMingen; 1:200 in PBS) for 16 h at RT. After a 1-min wash in PBS, label was visualized by immersion in streptavidin- gold (10 nm; Biocell, Cardiff, UK) at 1:20 in PBS for 1 h. Sections were then immersed in 1% glutaraldehyde (1 min) before being jet washed with distilled water. For negative controls, we substituted biotinylated rat IgG (1:lOO) for anti-IL-4 or performed labeling with 1% BSA PBS without a primary Ab. Grids were examined without counterstaining, as the location of gold particles was obscured by the dense crystalloid granule in stained sections.

RNA extraction and reverse transcription

Total RNA was extracted from batches of 2 X lo6 eosinophils (>99% pure) using TRI Reagent (Molecular Research Center, Cincinnati, OH)

22 pl of diethyl pyrocarbonate (DEPCf-treated water, heated to 65°C for according to the manufacturer’s instructions. The RNA was suspended in


http://ww

w.jim

munol.org/

Dow

nloaded from


The Journal of Immunology 4941

5 min, and reverse transcribed in a final volume of 40 yI. The reverse transcription mix contained 40 U of avian myeloblastosis virus reverse transcriptase (Pharmacia, Milton Keynes, UK); 1X PCR buffer (50 mM Tris-HCI (pH 8.3), 50 mM KCI, and 50 mM MgCI,); 0.6 mM each of d m , dATP, dCTP, dGTP, and d’ITP (Pharmacia); 200 ng of BSA (Sigma Chemical Co., Poole, UK); 5 y g poly(dT),,,, (Pharmacia); and 32.4 U of RNA-guard (Pharmacia). The mix was incubated at RT for 10 min followed by 42°C for 1 h. Following reverse transcription, the enzyme was inactivated by heating the mix to 80°C for 10 min. The resulting cDNA was stored at -80°C until used.

PCR amplification

PCR was performed on 1/20 dilutions of cDNA from the reverse transcription reaction (above). A 5-y1 aliquot of this dilution was used in a 50-yl PCR reaction containing 2.5 U of Taq DNA polymerase (Applied Biosys- tems, Warrington, UK), 1X PCR buffer, 0.2 mM dNTP, 0.1% Triton X-100 (Sigma Chemical Co.), and 0.4 p M each of appropriate forward (5’) and reverse ( 3 ’ ) primers. The IL-4 primer sequences were 5”CGGCAACTIT GACCACGGACACAAGTGCGATA-3‘ (5’ primer) and 5”ACGTACTC TGGTTGGC’ITCCTTCACAGGACAG-3’ (3’ primer), which span a 344-bp region of the IL-4 cDNA. This primer crosses a genomic intron, thus precluding amplification of the genomic sequence. Positive and negative controls were included in each set of reactions, and amplification of the housekeeping gene p-actin was employed as an endogenous control. The p-actin primers were 5‘-AAGGCCAACCGCGAGAAGAAGATG-3‘ (5’ primer) and 5’-ACAGGACTCCATGCCCAGGAA-3’ (3’ primer), which span a 479-bp region of the p-actin cDNA. Samples were overlaid with 50 yl of light mineral oil (Sigma Chemical Co.) and transferred to an thermal cycler (Omnigene, Hybaid, UK). After an initial denaturation step at 95°C for 5 min, the samples were subjected first to four cycles of heating to 94°C for 1 min, 60°C for 5 min, and 72°C for 1 min, followed by an additional 36 cycles of heating to 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min. A final extension step was then performed by heating to 72°C for 7 min. The amplified products were visualized by electrophoresis of a 10-yl aliquot on a 2% ethidium bromide-stained agarose gel.

Analysis of PCR-amplified products

Southern dot blotting was employed to confirm the identity of PCR products. Aliquots (5 pl) of PCR samples were denatured by the addition of 100 pl of 0.4 M NaOH and 10 mM EDTA and boiling for 10 min. Samples were immediately cooled on ice and neutralized by the addition of an equal volume of 2 M ammonium acetate, pH 7.0, and applied to a nitrocellulose membrane (Hybond-N, Amersham) using a dot blotting apparatus (Bio- Rad, Watford, UK). The cDNA was cross-linked to the membrane using a Spectrolinker XL-1000 (Spectronics Corp., New York, NY). The membrane was prehybridized for 1 h at 55°C in 6X standard saline citrate (SSC), 10 mM EDTA (pH 8.0), 2X Denhardt’s solution, 0.5% SDS, and 100 yglml denatured salmon sperm DNA (Sigma Chemical Co.). An internal IL-4 oligonucleotide probe end labeled with [y-”P]ATP (Amer- sham) using polynucleotide kinase (Promega, Southampton, UK) was added and allowed to hybridize for 20 h at 55°C. The sequence of the IL-4 probe was 5’-GTCCTTCTCATGGTGGCTGTAGAACTGCCG-3’. The membrane was then rinsed several times in 2X SSC-0.05% SDS at room temperature and washed twice in 2X SSC-0.05% SDS for 20 min at 55°C. Autoradiography was performed using Kodak X-Omat AR film (Eastman Kodak, Hemel Hempstead, Herts., UK).

F iberoptic bronchoscopy, BAL cells

Bronchoscopy and BAL were performed in subjects with documented atopic asthma, and BAL cells were processed as previously described (26, 27). Briefly, cytospins were prepared from BAL cells (at a concentration of 0.5X 10‘ cellsiml) using RNase-free slides coated with 0.1% poly-L-lysine (Sigma Chemical Co.) using a Shandon 2 cytospin device (Shandon South- ern Instruments, Runcorn, UK). Samples were air-dried for 10 min, fixed in 4% paraformaldehyde in PBS (pH 7.4) for 30 min, and washed in 15% sucrose (Sigma Chemical Co.) in PBS. Slides were incubated at 37°C overnight, then stored at -80°C until used.

Sequential immunocytochemical staining and in situ hybridization

ICC. BAL cytospins or sections from skin biopsies were warmed to RT. ICC using EG2 Ab and the APAAP technique was performed as previously described (28-30). For negative control preparations, the primary Ab was replaced by either an irrelevant mouse Ig or TBS. BAL cytospins or skin biopsies were stained with EG2 or MBP, respectively, and then probed with an IL-4 riboprobe as described below,

ISH. After immunochemical staining, BAL cells or skin biopsy specimens were immediately refixed by immersion in 4% paraformaldehyde in PBS for 5 min and then permeabilized by immersion in 0.3% Triton X-100 in PBS for 10 min. After a brief wash in PBS, specimens were further permeabilized by exposure to proteinase K (Promega) solution (1 mg/ml in 20 mM Tris-HCI and 1 mM EDTA, pH 7.2) for 10 min at 37”C, the activity of which was terminated by immersion in 4% paraformaldehyde in PBS for 5 min. After a brief rinse in PBS, slides were air-dried. Radiolabeled (Sulphur-35, Amersham) riboprobes (sense and antisense) were prepared from IL-4 cDNA in pGEM-type vectors as previously described (26, 28, 29, 31). As a positive control for IL-4 mRNA+ cells, cytospins from a peripheral blood T lymphocyte clone obtained from a patient with the hyper-IgE syndrome known to express IL-4 mRNA were prepared (27). For negative controls, slides were hybridized to sense probes and/or pretreated with RNase A, then hybridized to antisense probes (27, 30, 31).

Quantitation of numbers of cells expressing cytokine mRNA and phenotypic markers In BAL cytospins, at least two slides from each of the BAL preparations were stained with EG2 mAb and subsequently probed for expression of IL-4 mRNA. EG2+ cells were stained red, while hybrids between cytokine mRNA and cRNA probes were localized as dense collections of silver grains in the photographic emulsion overlying individual cells. Two observers quantified the numbers of eosinophils and those showing positive IL-4 signals in 1000 BAL cells/slide independently using a Zeiss microscope (Germany) with bright and dark fields. In the skin biopsies, numbers were expressed per square millimeter of biopsy area using a computerized program (Apple H e , Apple Computer, Cupertino, CA). The interobserver coefficient of variability for BAL and skin biopsy counts was <5%.

Data analysis

Data were analyzed using a statistical package (Minitab Release 7, Minitab, State College, PA). All between-subject and within-subject com- parisons were analyzed using the Mann-Whitney U test. Correlation coef- ficients were obtained by Kendall’s rank method with correction for tied values. P < 0.05 was considered significant. Despite the nonparametric analysis, data are quoted as the mean t SEM for clarity.

The bioactivities of eosinophil granule, membrane, and cytosol constit- uents following fractionation, including IL-4 quantitation by ELISA (in pglml) and dot blot, are expressed as frequency distributions according to the method of Beaufay et al. (32), by which the fractional activities of the markers are presented as a function of the density for each fraction.

Results Peripheral blood eosinophils

Using RT-PCR amplification under the stated conditions, mRNA encoding IL-4 was detected in highly purified (>99%) peripheral blood eosinophils from five of seven atopic asthmatics (Fig. 1). The identity of the amplified IL-4 cDNA was verified by Southern dot blotting using internal oligonucleotide probes (Fig. 2).

Using the APAAP technique, freshly prepared cytospins of eosinophils (>99% pure) were examined for IL-4 immunoreactivity using a monoclonal anti-human IL-4 Ab (Fig. 3a). Cytoplasm- or granule-associated IL-4 immunoreactivity was observed in 21.8 ? 5% of eosinophils from atopic asthmatics and 38.0 ? 13% of eosinophils from nonatopic healthy controls (n = 6 in each case; Fig. 4). Relevant negative controls (irrelevant biotinylated rat Ab of the same isotype and omission of the primary Ab) showed no immunoreactivity. Purified eosinophils from six subjects contained 108 2 20 pgi106 cells of total extractable IL-4. Of these, approximately 30% was released after incubation with serum-coated Sephadex beads using a single 45 min point (Table I).

The subcellular localization of intracellular IL-4 in peripheral blood eosinophils (5 X 10’; >99% pure) obtained ex vivo from an atopic asthmatic subject was determined by homogenizing the cells and fractionating them on a linear Nycodenz gradient. Figure 5 shows the elution profile of fractionation in which the major peak of IL-4 immunoreactivity was detected in fractions of equivalent density (1.2 dml) to those containing the granule marker ECP.


http://ww

w.jim

munol.org/

Dow

nloaded from



FIGURE 1. RT-PCR of eosinophil mRNA using primers specific for p-actin (first lane) and IL-4 (second lane). Third lane, m.w. markers to confirm the predicted sizes of amplified segments of f3-actin (479 bp) and IL-4 (344 bp). Fourth lane, IL-4 control cDNA amplified using the same primers. Fifth lane, PCR negative control (RNA only).

j ) A=subject 1 B = subject 2 C = control (H2O) D = L-4 cDNA

@ E = irrelevant cDNA

3 4 4 bp")

/I' /I' D E

FIGURE 2. Southern blotting analysis of reverse transcribed eosinophil mRNA amplified by PCR using IL-4-specific primers.

There was a smaller peak of activity detectable in fractions with a density range of 1.04 to 1.17 glml and corresponding to fractions containing the CD9 plasma membrane marker. No IL-4 was detected in the cytosolic fractions of the lowest (1.03-1.07 g/ml) density containing the LDH marker.

Immunogold labeling of eosinophils with an anti-IL-4 mAb showed gold particles in association with eosinophil secretory granules, particularly the crystalline granule cores, with minimal labeling of the cytoplasm or nucleus. The amount of granule labeling was greater than that observed in control experiments using a biotinylated normal rat IgG or secondary streptavidin-gold only, where few gold particles of uniform distribution were seen (Fig. 6).

Bronchoalveolar lavage

Using sequential ICC and ISH, the expression of mRNA encoding IL-4 in EG2-immunoreactive eosinophils was determined using BAL cells from baseline (unchallenged) atopic asthmatics (Fig. 3). BAL contained a mean of 7.7 t 1.4% EG2+ eosinophils ( n = 7). Of the EG2' cells, 1.6 2 0.5% coexpressed IL-4 mRNA.

Cutaneous allergen-induced L PR

Sequential ICC and ISH were performed as described for BAL, except that eosinophils were identified by immunoreactivity with anti-MBP mAb (BMK-13). Skin biopsies obtained 6 h after allergen challenge of sensitized atopic subjects ( n = 6) contained a mean of 26.7 eosinophils/mm2, of which 22.4 2 3.3% coexpressed IL-4 mRNA, while 52.7 t 4.4% of cells expressing IL-4 mRNA

were MBP+ (Table I1 and Fig. 3). IL-4 protein was also detected by a double immunostaining method in additional sections from the same biopsies (Table 111 and Fig. 3). A mean of 83.5% t 3.5% of all eosinophils counted (MBP+) were IL-4+, while 46.5 t 3.9 of IL-4+ cells were identified as MBP+ eosinophils. There was an approximate association between IL-4 mRNA and protein expression especially when the percentages of eosinophils expressing IL-4 mRNA and protein (rhird columns of Tables I1 and 111) were compared (r = 0.54).

Discussion We have demonstrated, using several approaches, that human eosinophils express mRNA encoding IL-4, store translated IL-4 protein in association with secretory granules, and release IL-4 in vitro following an appropriate stimulus (serum-coated particles). These findings are of clear relevance to the pathogenesis of allergic inflammation.

The detection of mRNA encoding IL-4 by RT-PCR in highly purified peripheral blood eosinophils from a majority of atopic asthmatics suggests that this cytokine is constitutively expressed in at least some of these patients in eosinophils. Contamination of eosinophils in these experiments was minimized by removal of CD16+ neutrophils, CD3+ T lymphocytes, and CD14' monocytes. This procedure resulted in >99% purity of eosinophils obtained by the immunomagnetic microbead technique. Furthermore, we were unable to demonstrate IL-4 mRNA by RT-PCR analysis of similar small numbers of contaminating cells under the same experimental conditions.

To demonstrate unequivocally the presence of IL-4 mRNA in eosinophils, we used ISH combined with ICC to identify the cell phenotype. We have been able to present clear evidence that eosinophils express mRNA encoding IL-4 (BAL cells from unchallenged atopic asthmatics) and both IL-4 mRNA protein (allergen- induced cutaneous LPR; Fig. 3 and Tables I1 and 111). In the latter case, allergen challenge of skin was associated with substantial infiltration with eosinophils, high percentages of which expressed IL-4 mRNA and immunoreactivity (in the case of IL-4 protein, substantially higher than the percentages of positive cells in the peripheral blood). Since, as we have shown previously (1, 24), diluent challenge control skin sites contain very few eosinophils, this observation is compatible with the hypothesis that recruitment of eosinophils to sites of allergic inflammation is associated with increased IL-4 immunoreactivity of the infiltrating cells. We are currently evaluating the possible effects of agents previously shown to modulate eosinophil surface marker expression (33) or eosinophil expression of mRNA encoding cytokines other than IL-4 (34, 35) on IL-4 mRNA expression by eosinophils in vitro.


http://ww

w.jim

munol.org/

Dow

nloaded from



F'

1

Immunocytochemical staining of peripheral blood eosinophils This was conlirmed by detection of E-4 in eosinophils disrupted indicated that IL-4 inkmoreactivity was detectable in a propor- by repetitive freeze-thawing (Table I). The amount of IL-4 in these tion of these cells, principally associated with the granules (Fig. 3). cells was substantial (mean, 108 pg/106 cells) and within the


http://ww

w.jim

munol.org/

Dow

nloaded from



80

+ * I -1

)P

- 20

0

e

e

_L

e

e e e -

8

Non Ato ic Atopic

Control Normar Acthmatlc

FIGURE 4. Percentages of peripheral blood eosinophils obtained from atopic asthmatics and nonatopic healthy controls showing imrnunoreac- tivity to IL-4 using the anti-IL-4 mAb and the APAAP technique.

Table I . IL-4 immunoreactivity in whole blood eosinophils and in the supernatants of eosinophils stimulated with serum-coated particles

Experiment Whole Cell Lysates Serum-Coated Beadsb Supernatants from

Subject 1 124 pg/l O6 cells 32 pg/l Oh cells Subject 2 145 pg/l O6 cells 38 pg/l Oh cells Subject 3 59 pg/l o6 cells 25 pg/l O6 cells Subject 4 68 pg/l Oh cells 23 pg/l O6 cells Subject 5 75 pg/l o6 cells 25 pg/l O6 cells Subject 6 1 78 pg/l O6 cells 61 pg/106 cells Mean + SEM 108 2 20 34 + 6

with three cycles of freeze-thawing (whole cell lysates) or incubated with serum- " Purified cells from each individual donor were divided and either disrupted

treated Sephadex (2-15 beads (or medium alone) for 45 min at 3 7 T , centrifuged, and the supernatants assayed for IL-4.

tions of <5 pg1Oh cells. Supernatants from cells incubated with medium alone had IL-4 concentra-

physiologically relevant range. Similar percentages of eosinophils from both atopic asthmatics and normal controls showed IL-4 immunoreactivity, while in some subjects no immunoreactivity was observed (Fig. 4). These observations further validate the ICC technique in the sense that they clearly show that the Ab used in this technique did not adhere nonspecifically to the eosinophil granules. The significance of the variability in the percentages of immunoreactive cells in the asthmatics and controls is difficult to assess based on single observations from limited numbers of subjects and does not support the view that there is overproduction of IL-4 by eosinophils in atopic asthma. On the other hand, the skin studies suggest that infiltration of eosinophils into sites of allergic inflammation might be associated with up-regulation of IL-4 synthesis. We were unable to observe a correlation between the percentages of peripheral blood eosinophils showing IL-4 immunoreactivity and their total IL-4 content measured after freeze- thawing, bearing in mind the fact that ICC gives no indication, beyond a certain threshold, of the amount of cytokine stored in individual cells. It does not detract from our basic observation that eosinophils contained preformed IL-4 protein. The release (-30% of the total) of IL-4 by eosinophils following stimulation with a physiologic stimulus (serum-coated beads resemble opsonized phagocytosable particles) is of particular interest, since it implies that eosinophils may represent an important repository for rapidly mobilizable IL-4, which may maintain a local environment con-

3 5 1 A t

- 25 0

3 5 1 R

granule membrane cytosol

FIGURE 5. Fractionation profile of human eosinophils showing the elution of ECP, CD9, and LDH together with IL-4 immunoreactivity, as determined by ELSA and dot blot analysis. Eosinophil subcellular fractions were separated on the basis of their density (g/ml) on Nycodenz gradient.

ducive to the development of Th2-type T lymphocytes and IgE synthesis.

The granule-associated nature of IL-4 staining in peripheral blood eosinophils was confirmed by the cell fractionation technique employed here (Fig. 5). We previously used this method to identify GM-CSF localization to the eosinophil granule compartment (25). The technique permits the collection of subcellular fractions and separation into distinct subcellular compartments, i.e., secondary crystalloid granules, plasma membrane, and cytosol. The clear coelution of IL-4 with eosinophil granule markers, with negligible amounts found in association with plasma membrane (CD9+), and its absence in the cytosol confirmed the association of this cytokine with the eosinophil crystalloid granule. How far this reflects storage of IL-4 following de novo synthesis, as opposed to uptake by endocytosis via IL-4 receptors on eosinophils (36), re- mains to be determined. The demonstration of mRNA in eosinophils, both ex vivo and in vitro, is, however, compatible with the hypothesis that the intracellular protein detected is derived at least partly from de novo synthesis of IL-4.

Immunogold labeling of intact eosinophils examined under electron microscopy (Fig. 6) showed gold particles in association principally with the crystalline core of the eosinophil granule, thus providing further confirmation of the presence of IL-4 in human eosinophils and its close association with the crystalloid granules (Fig. 6). The immunogold labeling method was also used to confirm the association between eosinophil-derived GM-CSF and the crystalloid granules of these cells (18). Others have described the presence of TNF-a and IL-5 in association with eosinophil granules in patients with blood eosinophilia (15, 37).


http://ww

w.jim

munol.org/

Dow

nloaded from



a

.1 .

b

. .. : . . . . . . . .

. . . .

. .

FIGURE 6. lmmunogold labeling of Lowicryl-embedded intact purified human eosinophils fixed with 2% formaldehyde. The sections were not counterstained, allowing the gold-labeled granules to be clearly observed. a, Rat anti-human IL-4 labeling (magnification, X15,500); 6, high magnification of a (magnification, X34,OOO) to show distribution of gold particles over the granules; c, rat IgC control (magnification, X 15,500); d, secondary Ab gold only control (magnification, X1 5,500).


http://ww

w.jim

munol.org/

Dow

nloaded from



Table II. M B P eosinophils expressing IL-4 mRNA in skin biopsies from atopic subjects 6 h after allergen challenge

MBP+/IL-4- MBP+/IL-4+ MBP-/IL-4+ Subject (cells/mm2) (cells/mm*) % Eos/lL-4+ (cells/mm2) % IL-4/Eosf

1 2 3 4 5 6

Mean 5 SEM

15.4 4.8 23.7 3.3 34.0 6.0 22.7 9.0 24.8 12.3 18.3 5.0

23.2 6.7 t 2.6 5 1.4

23.8% 12.2% 15.0% 28.4% 33.2% 21.5%

22.4% +- 3.3

4.4 4.7 3.8

10.2 14.8

2.2

6.7 +- 1.9

52.2% 41.3% 61.2% 46.9% 45.4% 69.4%

52.7% 5 4.4

Table 111. M B P eosinophils expressing IL-4 protein in skin biopsies from atopic subjects 6 hr after allergen challenge

MBP+/IL-4- MBP+/lL-4+ MBP-/IL-4+ Subject (+ve/mm2) (+ve/mrn2) % Eos/lL-4+ (+ve/mm') % IL-4+/Eost

1 2 3 4 5 6

Mean +- SEM

2 8 3 3 4 7

4.5 t- 0.9

29 18 18 23 21 24

22.2 ? 1.7

93.5% 69.2% 85.7% 88.5% 84.0% 77.4%

83.1% +- 3.5

24 23 44 20 21 24

26.3 t 3.6

54.7% 43.9% 29.0% 53.5% 50.0% 48.0%

46.5% _t 3.9

The present finding adds to previously established links between eosinophils and IL-4. For example, Dubois et al. (38) reported that IL-4 was chemotactic for eosinophils (but not neutrophils) obtained from patients with atopic dermatitis. In a preliminary report, eosinophils were found to express IL-4 receptors (36). IL-4 may also induce selective eosinophil infiltration in allergic inflammatory reactions indirectly by up-regulating the expression of VCA"1 on endothelial cells (39), since VLA-4, the ligand for VCA"1, is expressed on human eosinophils, but not neutrophils (40).

In addition to T cells, IL-4 is also a product of human leukemic and activated normal basophils (41) as well as mast cell lines (42). IL-4 has also been immunolocalized in human mast cells in nasal mucosa (43). Human basophils, with or without stimulation (44, 45), would appear to synthesize and secrete more IL-4 than eosinophils (46). It has been suggested that basophil-derived IL-4 is synthesized de novo and secreted, but not stored, after IgE-dependent stimulation (45, 47), although others have demonstrated intracellular immunolocalization of IL-4 in normal peripheral blood basophils (48). The precise bioavailability and bioactivity of inflammatory cell-derived IL-4 cornpared with those of T cell production are unknown and difficult to document. However, the presence of this cytokine in a stored granule-associated form, at least in the eosinophil, suggests that this cytokine may exert its effect on the inflammatory reaction possibly via mechanisms involving juxtacrine intracellular signaling (49).

In conclusion, we have provided several lines of evidence sug- gesting that human eosinophils can synthesize IL-4 and store it in association with their crystalloid granules. Furthermore, we have shown that IL-4 is released from highly purified eosinophils following an appropriate stimulus. Thus, eosinophils have the poten- tial to participate in allergic inflammatory reactions both by pro- moting IgE synthesis and by maintaining the Th2-type cytokine environment.

Acknowledgments The authors are grateful to Prof. B. D. Gomperts and Dr. P. Lacy for their help and guidance with cell fractionation, and Dr. Nicholas M. Severs for his helpful advice with electron microscopy.

References 1. Frew, A. J., and A. B. Kay. 1988. The relationship between infiltrating CD4+

lymphocytes, activated eosinophils and the magnitude of the allergen induced late-phase response in man. J. lmmunol. 141:4158.

2. Wardlaw, A. J., and R. Moqbel. 1992. The eosinophil in allergic and helminth related inflammatory responses. In Allergy and Immunity IO Helminfhs.

3. Moqbel, R., F. Levi-Schaffer, and A. B. Kay. 1994. Cytokine generation by R. Moqbel, ed. Taylor and Francis, London, p. 154.

4. Levi-Schaffer, F., J. Barkans, T. M. Newman, M. Wakelin, R. Hohenstein, eosinophils. J Allergy Clin. lmmunol. 94(SuppI.):lJ83.

V. Barak, A. B. Kay, and R. Moqbel. 1995. Identification of interleukin-2 (IL-2)

806). in human peripheral blood eosinophils. J. Allergy Clin. lmmunol. 951342 (Abstr.

5 . BossC, M., M. Audette, C. Ferland, G. Pelletier, A. Dakhama, S. Lavigne, and M. Laviolette. 1994. Detection of interleukin-2 (IL-2) cDNA in peripheral blood

A718 (Abstr.). eosinophils by RT-PCR and in cell RT-PCR. Am. J. Resp. Crit. Care Med. 149:

6. Kita, H., T. Ohnishi, Y. Okubo, D. Weiler, J. S. Abrams, and G. J. Gleich. 1991. GM-CSF and interleukin 3 release from human peripheral blood eosinophils and neutrophils. J. Exp. Med. 174:743.

7. Anwar, A. R. E., R. Moqbel, G. M. Walsh, A. B. Kay, and A. I. Wardlaw. 1993. Adhesion to fibronectin prolongs eosinophil survival. J. Exp. Med. 177:83Y.

8. Del Prete, G., E. Maggi, P. Parronchi, I. Chretien, A. Tiri, D. Macchia, M. Ricci, J. Banchereau, J. E. de Vries, and S. Romagnani. 1988. IL-4 is an essential factor in the IgE synthesis induced in vitro by human T cell clones and their supernatants. J. Immunol. 140:4193.

9. Mosmann, T. R., and R. L. Coffman. 1989. Thl and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Im-

10. Romagnani, S. 1991. Human Thl and Tb2: doubt no more. Immunol. Toduy munol. 7:145.

11. Vercelli, D., H. H. Jabara, B. W. Lee, N. Woodland, R. S. Geha, and D. Y. h u n g . 12:256.

1988. Human recombinant interleukin-4 induces FcaRIIICD23 on normal human monocytes. J. Exp. Med. 167:1406.

12. Hu-Li, J., E. M. Shevach, J. Mizuguchi, J . Ohara, T. R. Mosmann, and W. E.

normal resting T lymphocytes. J. Exp. Med. 165:157. Paul. 1987. B-cell stimulatory factor 1 (interleukin 4) is a potent costimulant for

13. Desreumaux, P., A. Janin, J. F. Colotnbel, L. Prin, J. Plumas, G. Torpier, A. Capron, and M. Capron. 1992. Interleukin-5 mRNA expression by eosinophils in the intestinal mucosa of patients with coeliac disease. J. Exp. Med. 175:293.

14. van Leuwen, B. H., M. E. Martinsen, C. G. Webb, and I. G. Young. 1989. Molecular organization of the cytokine gene cluster, involving the human IL-3,

15. Dubucquoi, S., P. Desreumaux, A. Janin, 0. Klein, M. Goldman, J. Tavernier, IL-4, IL-5 and GM-CSF genes on human chromosome 5 . Blood 73:1142.

A. Capron, and M. Capron. 1994. Interleukin-5 synthesis by eosinophils: association with granules and immunoglobulin-dependent secretion. J. Exp. Med.

16. Hansel, T. T., I. J. M. De Vries, T. Iff, S. Rihs, M. Wandzilak, S. Betz, K. BbdSer, 179:703.

and C. Walker. 1991. An improved immunomagnetic procedure for the isolation of highly purified human blood eosinophils. J. Immunol. Methods 145:105.

17. Winqvist, I., T. Olofsson, and I. Olsson. 1984. Mechanisms for eosinophil de- granulation: release of the eosinophil cationic protein. 1mmunoiog.v 5 l : l .

18. Levi-Schaffer, F., P. Lacy, N. J. Severs, T. M. Newman, J. North, B. Gomperts, A. B. Kay, and R. Moqbel. 1995. Association of granulocyte-macrophage colony- stimulating factor (GM-CSF) with the crystalloid granules of human eosinophils.

19. Tatham, P. E. R., and B. D. Gomperts. 1990. Cell permeabilization. In Peptide Blood 85.2579.

Hormone Secretion: A Practical Approach. J. C. Hutton and K. Siddle, eds. IRL

20. Chretien, I., A. V. Kimmenade, M. K. Pearce, J. Brancheredu, and J. S. Abrams. Press, Oxford University, p. 257.

1989. Development of polyclonal and monoclonal antibodies for immunoassay

21. Abrams, J. S., M.-G. Roncarolo, H. Yssel, U. Andersson, G . J. Gleich, and J. E. and neutralization of human interleukin-4. J. lmmunol. Methods 11757.

Silver, 1992. Strategies of anti-cytokine monoclonal antibody development: im-

22. Andersson, J., J. Abrams, L. Bjork, K. Funa, M. Litton, K. Agren, and munoassay of IL-10 and IL-5 in clinical samples. lmmunol. Rev. 127:5.

U. Andersson. 1994. Concomitant in vivo production of 19 different cytokines in human tonsils. Immunology 83:16.

23. Bland, J. M., and D. G. Altman. 1986. Statistical methods for assessing agree- ment between two methods of clinical measurements. Lancet 1.307.

24. Ying, S., L. Tahorda-Barata, Q. Meng, M. Humbert, and A. B. Kay. 1995. The kinetics of allergen-induced transcription of messenger RNA for monocyte chemotactic protein-3 (MCP-3) and RANTES in the skin of human atopic subjects: relationship to eosinophil, T-cell and macrophage recruitment. J. Exp. Med. 181:

25. Moqbel, R., J. Barkans, B. L. Bradley, S. R. Durham, and A. B. Kay. 1992. 2153.

Application of monoclonal antibodies against major basic protein (BMK-13) and

chid biopsies from atopic asthma. Ciin. Exp. Allergy 221265. eosinophil cationic protein (EGI and EG2) for quantifying eosinophils in bron-


http://ww

w.jim

munol.org/

Dow

nloaded from



26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

Hamid, Q., M. Azzawi, S. Ying, R. Moqbel, A. J. Wardlaw, C. J. Corrigan, 8. Bradley, S. R. Durham, J . V. Collins, P. K. Jeffery, D. J. Quint, and A. B. Kay.

asthma. J. Clin. Invest. 87:1541. 1991. Expression of mRNA for interleukin-5 in mucosal bronchial biopsies from

Robinson, D. S., Q. Hamid, S. Ying, A. Tsicopoulos, J. Barkans, A. M. Bentley, C. Corrigan, S. R. Durham, and A. B. Kay. 1992. Predominant Th2-like bronchoalveolar T-lymphocyte population in atopic asthma. N. Engl. J . Med. 326:298.

0. Lowhagen, R. Moqbel, A. B. Kay, and Q. Hamid. 1993. T cells are the principal Ying, S. , S. R. Durham, J. Barkans, K. Masuyama, M. Jacobson, S. Rak,

source of interleukin-5 mRNA in allergen-induced rhinitis. Am. J. Respir. Cell Mol. Biol. Y:356.

A. B. Kay, and Q. Hamid. 1994. T lymphocytes and mast cells express messenger Ying, S. . S. R. Durham, M. R. Jacobson, S. Rak, K. Masuyama, 0. Lowbagen,

nology 82:200. RNA for interleukin-4 in the nasal mucosa in allergen-induced rhinitis. lmmu-

Robinson, D. S., S . Ying, 1. K. Taylor, A. Wangoo, D. M. Mitchell, A. B. Kay, Q. Hamid, and R. J. Shaw. 1994. Evidence for a Thl-like bronchoalveolar T-cell subset and predominance of interferon-gamma gene activation in pulmonary tu- berculosis. Am. J. Respir. Crit. Care Med. 14Y:Y89.

Q. Hamid. 1991. Messenger RNA expression of the cytokine gene cluster, IL-3, Kay, A. B., S. Ying, V. Varney, S . R. Durham, R. Moqbel, A. J. Wardlaw, and

IL-4, IL-5, and GM-CSF, in allergen-induced late-phase reactions in atopic subjects. J. Exp. Med. 173:775. Beaufay, H., P. Jacques, P. Baudhuin, 0. 2. Sellinger, I. Berthet, and C. de Duve.

ulations of cytoplasmic particles by means of density equilibration in various 1964. Rcsolution of mitochondrial fractions from rat liver into three distinct pop-

gradients. Biochem. J. 92:184. Hartnell, A., A. B. Kay, and A. J. Wardlaw, A. J. 1992. Interferon-gamma expression of FcyRIII (CD16) on human eosinophils. J. lmmunol. 148:1471.

Wardlaw, and A. B. Kay. 1991. Expression of mRNA and immunoreactivity for Moqbel, R., Q. Hamid, S . Ying, J. Barkans, A. Hartnell, A. Tsicopoulos, A. J.

the granulocyteimacrophage-colony stimulating factor in activated human eosinophils. J. Exp. Med. 174:74Y.

T. Takaishi, Y . Morita, K. Ohta, T. Kasahara, and K. Ita. 1995. Chemotactic Miyamasu, M., K. Hirai, Y. Takashi, M. lida, M. Yamaguchi, T. Koshino,

agents induce cytokine generation in eosinophils. J . lmmunol. 154:1339. Dubois, G. R., C. A. F. M. Broijnzeel-Koomen, and P. L. B. Bruijnzeel. 1995. Human eosinophils express interleukin-4 receptors. J . Allergy Clin. lmmunol. 95:340 (Abstr. 800). Bcil, W. J., P. F. Weller, D. M. Tzizik, S . J. Galli, and A. M. Dvorak. 1993. Ultrastructural immunogold localization of tumor necrosis factor-alpha to the matrix compartment of eosinophil secondary granules in patients with idiopathic hypereosinophilic syndrome. J. Hi.rlochen1. Cyrochem. 41:1611.

38. Dubois, G. R., C. A. F. M. Bruijnzeel-Koomen, and P. L. B. Bruijnzeel. 1994. IL-4 induces chemotaxis of blood eosinophils from atopic dermatitis patients, but

39. Schleimer, R. P., S. A. Sterbinsky, J. Kaiser, C. A. Bickel, D. A. Klunk, not from normal individuals. J. Invest. Dermatol. 102:843.

K. Tamioka, W. Newman, F. W. Luscinskas, M. A. Gimbrone, Jr., B. W. Mcln- tyre, and B. S. Bochner. 1992.1L-4 induces adherence of human eosinophils and basophils but not neutrophils to endothelium. Association with expression of VCAM-1. J. lmmunol. 148:1086.

40. Walsh, G. M., A. Hartnell, J.J. Mermod, A. B. Kay, and A. J. Wardlaw. 1991. Human eosinophil, but not neutrophil, adherence to IL-1-stimulated HUVEC is a 4 p l (VLA-4) dependent. J. lmmunol. 1463419.

41. Arock, M., H. Merle-Nrd, B. Dugas, F. Ouaaz, L. Le Go& 1. Vouldoukis, J:M. Mencia-Heurta, C. Schmitt, V. Leblond-Missenard, P. Debre, and D. Mossalayi. 1993. IL-4 release by human leukemic and activated normal basophils. J. lmmunol. 151:1441.

42. Plaut, M., J. H. Pierie, C. J. Watson, J. Hanky-Hyde, R. P. Nordan, and W. E. Paul. 1989. Mast cell lines produce lymphokines in response to cross-linkage of FceRl or to calcium ionophores. Nature 339.44.

43. Bradding, P., 1. H. Feather, S. Wilson, P. G. Bardin, C. H. Heusser, S. T. Holgate,

of normal and perennial rhinitic subjects. J. lmmunol. 151:3853. and P. H. Howarth. 1993. lmmunolocalization of cytokines in the nasal mucosa

44. Brunner, T., C. H. Heusser, and C. A. Dahinden. 1993. Human peripheral blood basophils primed by interleukin 3 (IL-3) produce IL-4 in response to immunoglobulin E receptor stimulation. J. Exp. Med. 177:605.

45. MacGlashan, D. W., Jr., J. M. White, S.-K. Huang, S. J . Ono, J. T. Schroeder, and L. M. Lichtenstein. 1994. Secretion of 1L-4 from human basophils. The relationship between IL-4 mRNA and protein in resting and stimulated basophils. J . Immunol. 152:3006.

46. Nonaka, M., R. Nonaka, K. Woolley, E. Adelroth, K. Miura, P. O'Byme, J. Dolovich, and M. Jordana. 1995. Localization of interleukin-4 in eosinophils in nasal polyps and asthmatic bronchial mucosa. J . Allergy Clin. lmmunol. Y5:220 (Ahstr. 320).

47. Schroeder, I. T., D. W. MacGlashan, Jr., A. Kagey-Sobotka, J. M. White, and L. M. Lichtenstein. 1994. IgE-dependent secretion by human basophils: the relationship between cytokine production and histamine release in mixed leukocyte cultures. J . lmmunol. 153:1808.

48. Mueller, R., C. H. Heusser, S. Rihs, T. Brunner, G. R. Bullock, and C. A. Da- binden. 1994. Immunolocalization of intracellular interleukin-4 in normal human peripheral blood basophils. Eur. J . lmmunol. 24:2Y35.

49. Zimmerman, G . M., D. E. Lorant, M. T. McIntyre, and S. M. Prescott. 1993. Juxtacrine intracellular signaling: another way to do it. Am. J. Re,spir. Cell Mol. Biol. Y:573.


http://ww

w.jim

munol.org/

Dow

nloaded from


identification of messenger rna for il-4 in human ... · identification of messenger rna for 11-4...

Documents