Functional expression of the eotaxin receptor CCR3 in T lymphocytes co-localizing with eosinophils

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Research Paper

Functional expression of the eotaxin receptor CCR3 in T lymphocytes co-localizing with eosinophils B.O. Gerber*, M.P. Zanni†, M. Uguccioni*, M. Loetscher*, C.R. Mackay‡, W.J. Pichler†, N. Yawalkar†, M. Baggiolini* and B. Moser* Background: The chemokine eotaxin is produced at sites of allergic inflammation, binds selectively to the chemokine receptor CCR3 and attracts eosinophil and basophil leukocytes, which express high numbers of this receptor. Responses of T lymphocytes to eotaxin have not been reported so far. We have investigated the expression of CCR3 in T lymphocytes and analysed the properties and in vivo distribution of T lymphocytes expressing this receptor. Results: In search of chemokine receptors with selective expression in T lymphocytes, we have isolated multiple complementary DNAs (cDNAs) encoding CCR3 from a human CD4+ T-cell cDNA library. T-lymphocyte clones with selectivities for protein and non-protein antigens were analysed for expression of CCR3 and production of Th1- and Th2-type cytokines. Of 13 clones with surface CCR3, nine secreted enhanced levels of interleukin-4 and/or interleukin-5, indicating that CCR3 predominates in Th2-type lymphocytes. CCR3+ T lymphocytes readily migrated in response to eotaxin, and showed the characteristic changes in cytosolic free calcium. Immunostaining of contact dermatitis, nasal polyp and ulcerative colitis tissue showed that CCR3+ T lymphocytes are recruited together with eosinophils and, as assessed by flow cytometry, a large proportion of CD3+ cells extracted from the inflamed skin tissue were CCR3+. By contrast, CCR3+ T lymphocytes were absent from tissues that lack eosinophils, as demonstrated for normal skin and rheumatoid arthritis synovium.

Addresses: *Theodor Kocher Institute, University of Bern, P.O. Box, CH-3000 Bern 9, Switzerland; †Institute for Immunology and Allergology, Inselspital, CH-3010 Bern, Switzerland; ‡LeukoSite, Inc., 215 First Street, Cambridge, Massachusetts 02142, USA. Correspondence: Bernhard Moser E-mail: [email protected] Received: 5 August 1997 Revised: 11 September 1997 Accepted: 22 September 1997 Published: 14 October 1997 Current Biology 1997, 7:836–843 http://biomednet.com/elecref/0960982200700836 © Current Biology Ltd ISSN 0960-9822

Conclusions: We show that T lymphocytes co-localizing with eosinophils at sites of allergic inflammation express CCR3, suggesting that eotaxin/CCR3 represents a novel mechanism of T-lymphocyte recruitment. These cells are essential in allergic inflammation, as mice lacking mature T lymphocytes were insensitive to allergen challenge. Surface CCR3 may mark a subset of T lymphocytes that induce eosinophil mobilization and activation through local production of Th2-type cytokines.

Background The typical cellular infiltrate of allergic inflammation consists mainly of eosinophils, T lymphocytes and occasional basophils. These leukocytes are recruited by chemokines that are produced in the affected tissue by resident or immigrant cells [1–3]. Eosinophils migrate in response to the CC chemokines eotaxin [4,5] and RANTES [6,7] and the monocyte chemotactic proteins MCP-2 [8], MCP-3 [9] and MCP-4 [10]. Eotaxin was originally identified in the bronchoalveolar lavage fluid of ovalbumin-sensitized guinea-pigs [11–13] and was later found in the mouse [14] and in man [5]. Human eotaxin is structurally related to the MCPs sequence identity), and is a potent chemoattractant for eosinophils which express large numbers of the eotaxin receptor CCR3 [15–17]. Like all other known chemokine

receptors, CCR3 belongs to the family of seven-transmembrane domain receptors, which couple to G proteins for signal transduction [1,2,18,19]. In addition to eotaxin, CCR3 binds RANTES, MCP-2, MCP-3 and MCP-4 [10,15–17,20]. However, eotaxin is highly selective for CCR3, whereas RANTES and the MCPs recognize additional chemokine receptors that are expressed on monocytes, granulocytes, natural killer cells and T lymphocytes [1–3]. Eotaxin was recently shown to mediate histamine release in basophils, which also express CCR3 [20–22], but responses of T lymphocytes were not reported. Studies of eosinophil mobilization in guinea-pig and murine models of allergic inflammation emphasize the critical role of eotaxin in the regulation of allergic responses [4,12,23–25]. Notably, in eotaxin-deficient mice and in animals treated with neutralizing antibodies to eotaxin, allergen-induced eosinophil invasion of the airways was drastically reduced [24,26].

Research Paper Functional expression of CCR3 in T lymphocytes Gerber et al.

837

Figure 1 (a) OFB2

B22

B24

(b) D21

D21

18S CCR3 B24 28S

50 cells

CCR3 expression in human T-lymphocyte clones. (a) Northern blot of CCR3 (top panel) and CCR2 (middle panel) mRNA. Ethidium bromide staining of total RNA in the agarose gel prior to blotting is shown in the bottom panel. One of two independent experiments is shown. (b) Flow cytometric analysis with anti-CCR3 monoclonal antibody (bold trace) or isotype control immunoglobulins (fine trace). One of three independent experiments is shown.

CCR2 B22

OFB2

10 Current Biology

Unlike monocytes and granulocytes, the majority of freshly isolated peripheral T lymphocytes do not respond to chemokines [1,2,27]. Culturing in the presence of interleukin-2 (IL-2), however, results in polyclonal expansion of activated CD45RO+ T cells that express the relevant chemokine receptors and readily respond to chemokines [28,29]. The IL-2-treated T lymphocytes resemble the lymphocytes that are recruited through locally produced chemokines during immune reactions. In search of chemokine receptors with selective expression in T lymphocytes, we have recovered full-length cDNAs for known and novel chemokine receptors from a human CD4+ T-cell line cDNA library. One cDNA encoded CXCR3, the receptor for the CXC chemokines IP10 and Mig and which is exclusively found on activated T cells [29]. In addition, we have isolated several cDNA clones encoding CCR3. In view of the current understanding that eotaxin is selective for eosinophils and basophils, this finding was surprising. Here we demonstrate that CCR3 expression defines a subset of T lymphocytes that respond to eotaxin and co-localize with eosinophils at sites of allergic inflammation.

Results and discussion Cloning of CCR3 and functional expression in T lymphocytes

In the course of screening a human CD4+ T-cell cDNA library with a PCR-amplified DNA probe we identified no

100

Fluorescence intensity

fewer than eight cDNA clones encoding the eotaxin receptor CCR3. A nucleotide fragment corresponding to part of the CCR3 coding sequence was used as a hybridization probe in northern blot analysis. In agreement with previous reports [15–17], abundant CCR3 transcripts could be demonstrated in eosinophils, whereas neutrophils, monocytes or freshly isolated blood lymphocytes were negative (not shown). T lymphocyte responses to chemokines depend on cell activation, and IL-2 was shown to induce the expression of CCR1 and CCR2 [28] as well as CXCR3 [29]. IL-2 treatment did not enhance the expression of CCR3. Occasionally, however, low levels of CCR3 transcripts were observed in these cultures, suggesting that CCR3 is present in a subset of circulating T lymphocytes. Skin hypersensitivity responses to various drugs, such as antibiotics, anaesthetics or anti-epileptics, are characterized by a cellular infiltrate consisting mainly of eosinophils and T lymphocytes [30]. Cloned T lymphocytes generated from peripheral blood lymphocytes of patients who develop contact dermatitis after topical application of lidocaine [31] were analysed for CCR3 expression. On the basis of their clonality, cytokine pattern and in vitro cell growth, four CD4+ T-lymphocyte clones — B22, OFB2, B24 and D21 — were selected for further study. Clonality was determined with a panel of 17 different monoclonal antibodies recognizing the Vβ chain of the T-cell receptor (TCR) and

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PCR-based TCR Vβ expression analysis. The TCR specificity of the clones was Vβ17 for OFB2, Vβ12 for B22, Vβ5.1 for B24 and Vβ13.1 and 13.2 for D21. Northern blot analysis showed the presence of transcripts for CCR3 in all four clones (Figure 1a). Transcripts for CCR2, which is upregulated in T lymphocytes by stimulation with IL-2 [28], were also detected. In addition, the expression of CCR3 was studied by flow cytometry using the selective antibody 7B11 [32]. The clones B24, B22 and OFB2 were strongly positive, whereas D21 expressed only low levels of CCR3 protein (Figure 1b). To show that CCR3 was functional, [Ca2+]i changes and chemotaxis were measured after stimulation with the selective ligand eotaxin. As shown in Figure 2, Ca2+ mobilization was observed in all four clones, whereas chemotaxis was observed in B22, B24 and OFB2, the clones expressing high levels of CCR3. The migration response was typically bimodal with respect to chemokine concentration and maximum efficacy was reached at 1 nM. The responses were prevented by pretreatment of the cells with the CCR3-blocking antibody 7B11 [32]. All four clones also responded to MCP-1, which acts through CCR2 [33], and this response was not affected by the anti-CCR3 monoclonal antibody (Figure 2a). Eotaxin and MCP-1 were equally effective in inducing [Ca2+]i changes. As expected, in view of the receptor selectivity of the two chemokines, no cross-desensitization was found (Figure 2b).

CCR3 expression in cultured T-lymphocyte clones

A total of 32 T-lymphocyte clones with selectivity for lidocaine (11 clones), sulfamethoxazole (7 clones), purified protein derivative of Mycobacterium tuberculosis (4 clones) or tetanus toxoid protein (10 clones) were analysed for expression of CCR3 (Table 1). The production of interferon-γ (IFN-γ) as marker for Th1 cells, or IL-4 and IL-5 as markers for Th2 cells was determined in a total of 24 clones. Of these, 13 expressed high levels of CCR3, and nine of them also secreted enhanced levels of IL-4 and/or IL-5. This indicates that CCR3 expression predominates in T lymphocytes that produce Th2-type cytokines. The frequent expression of CCR3 in T lymphocytes specific for topically applied lidocaine may imply that these cells are important in allergic inflammation of the skin or mucosal/epithelial tissue. CCR3+ T lymphocytes in inflammatory diseases

Skin sections from patients with contact dermatitis induced by topical application of lidocaine or nickel were analysed by immunohistochemistry for the presence of infiltrating cells that were positive for CD3, CCR3 and eotaxin. In the perivascular areas, eosinophils (identified by haematoxylin/eosin staining) and T lymphocytes (CD3+) were frequent. Most cells in these areas were also positive for CCR3, and eotaxin was readily detected throughout the lesions. T lymphocytes, eosinophils and CCR3+ cells

Figure 2

Responses of human T-lymphocyte clones to eotaxin and MCP-1. (a) In vitro chemotaxis. The assay was performed in the presence (open symbols) or absence (closed symbols) of anti-CCR3 monoclonal antibody and increasing concentrations of eotaxin (triangles) or MCP-1 (circles). The number of cells that migrated in the absence of chemokines

is also shown (diamonds). Mean number (± s.d.) of migrating cells per five high-power fields are presented from one out of three independent experiments. (b) Eotaxin- and MCP-1-induced [Ca2+]i changes. Agonists were added at 100 nM at 0 sec and 80 sec as indicated by the arrowheads. One of two independent experiments is shown.

Research Paper Functional expression of CCR3 in T lymphocytes Gerber et al.

were counted in serial sections and their frequency was calculated (Table 2). In contrast, only few CD3+ cells Table 1 CCR3 expression by human T-lymphocyte clones. Clone

Ref.*

B24

[31]

Antigen† Cluster‡ IFN-γ§ LC

CD4+

211

IL-4§

IL-5§

CCR3¶

12

17

+

B22

[31]

CD4+

11

39

285

+

D21

[31]

CD4+

225

35

15

+/–

OFB2

[31]

CD4+

15

67

210

+

G6

[31]

CD4+

4

5.4

20.6

+

[31]

CD4+

13

8.7

52.2

+

I39

[31]

CD4+

6.5

19.5

83.7

+

LI35

[31]

CD4+

3.6

11.9

113.3

+

LK19

[31]

CD4+

3.6

33

177

+/–

K58

CD4+

42.3

3.5

60.5

+/–

OFZ45

CD4+

13.5

3.7

127

+

P102B

CD4+

14

12

<1

+

P103C

CD8+

87.2

0.3

7.1

–

P21E

CD4+

22

28

16

–

P26B

[39]

CD8+

183

0.5

26

–

S1011E [39]

CD4+

40

68

25

–

S211A

CD8+

83

0.7

36.6

+

CD4+

17

13

15

–

G7

S23E

SM

[39]

CD4+

–

PD22

CD4+

–

PD24

CD4+

–

PD20

CD4+

–

CD4+

–

PD4

PPD**

C6

[40]

C24

[40]

CD8+

50

48

+

[40]

CD4+

150

26

–

[41]

CD4+

C38 KT30

TT

839

and no CCR3+ cells were found in normal skin. Clustering of the infiltrating cells prevented a clear-cut identification of cells positive for both CD3 and CCR3 in double-stained sections. However, CCR3+ T lymphocytes were readily recovered by trypsin treatment of a contact dermatitis biopsy as shown by flow cytometry (Figure 3). About 20% of CD3+ T lymphocytes derived from inflamed skin expressed CCR3, which is in keeping with the frequency of CCR3+ infiltrating cells other than eosinophils (Table 2). The phenotype of T lymphocytes co-localizing with eosinophils was further examined in biopsies from nasal polyps and ulcerative colitis, which are inflammatory conditions with marked eosinophil infiltration [5,34]. Analysis of tissue sections from nasal polyps revealed large numbers of T lymphocytes and eosinophils. Serial sections stained for CD3 or CCR3 are shown in Figure 4 a,b. Double staining for CD3 and CCR3 was also performed. Double-positive T lymphocytes (T+) along with eosinophils (E) and CD3+/CCR3- cells (T–) were present (Figure 4c). Eosinophils are recognized by their typical nucleus and blue staining for surface CCR3 and CCR3-negative T lymphocytes by their bright red staining for surface CD3. A double-positive T lymphocyte (T+) is shown at higher magnification (Figure 4c inset). T lymphocytes expressing CCR3 were always found in association with eosinophils, whereas T lymphocyte clusters without eosinophils were always CCR3-negative. T lymphocytes in areas rich in

Figure 3

+

Q16

CD4+

95

0.9

2.3

–

O35

CD4+

5

10

41

–

O45

CD4+

172

13.8

38.2

–

TT10

CD4+

TT21

CD4+

TT24

CD4+

+/– – 118.6

24

68.3

+

*No reference means that the clone is unpublished. †T-cell clones are specific for lidocaine (LC); sulfamethoxazole (SM); purified protein derivative of M. tuberculosis (PPD); tetanus toxoid (TT). ‡Cluster of differentiation antigen. §Cytokine production in culture (ng ml–1). ¶CCR3 expression < D21 (–); CCR3 expression = D21 (+/–); CCR3 expression < D21 (+) (see Figure 1). **The majority of T-cell clones specific for PPD have a Th1 profile as previously shown [46].

Identification of CCR3+ T lymphocytes extracted from a contact dermatitis lesion. Flow cytometric analysis with (a) anti-CD3, and (b) anti-CCR3 monoclonal antibodies (bold trace) or isotype control immunoglobulins (fine traces).

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Table 2 Analysis of CCR3+ cells in serial tissue sections obtained from patients with allergic and non-allergic inflammation. Contact dermatitis

Ulcerative colitis

Rheumatoid arthritis

CCR3+

Eosinophils

CD3+

Patient

CCR3+

Eosinophils

CD3+

Patient

CCR3+

Eosinophils

CD3+

1

166

59

337

1

625

263

651

1

0

0

416

2

88

20

644

2

307

121

536

2

0

0

569

3

83

22

283

3

275

222

340

Patient

T cells, eosinophils and CCR3+ cells were counted in serial sections of contact dermatitis, ulcerative colitis, and rheumatoid arthritis samples. The same fields were evaluated at 40x magnification. The numbers of cells per mm2 are shown.

eosinophils as well as the CD3+/CCR3+ cells were counted and found to amount to 12–15% of the total count (Table 3). In confirmation of previous results, nasal polyp specimens were also regularly positive for eotaxin [5]. Colon mucosa biopsies from patients with ulcerative colitis showed massive infiltration of CD3+ and CCR3+ cells (Table 2) and high levels of eotaxin (Figure 4d–f). In this case, double staining could not be performed because endogenous alkaline phosphatase interferes with the detection system used. As in contact dermatitis and nasal polyps, CCR3+ T lymphocytes and eosinophils were found again in close proximity. In contrast, no CCR3+ cells were found in synovial tissue from patients with rheumatoid arthritis, which is strongly infiltrated by T lymphocytes but lacks eosinophils (Table 2). This result further indicates that infiltration by CCR3+ T lymphocytes is characteristic of allergic inflammation. Which types of T lymphocyte express CCR3 and are attracted by eotaxin? Chemokine receptors, including CXCR3, CCR1, CCR2 and CCR5, are expressed by the majority of peripheral blood CD45RO+ T lymphocytes after IL-2 stimulation [28,29], whereas CCR3+ T lymphocytes constituted a minor fraction in these cultures. Screening of 32 T-lymphocyte clones with specificity for various peptide and non-peptide antigens revealed that all

lidocaine-specific T-cell clones were CCR3+ (Table 1). CCR3 expression was much less frequent in T lymphocyte clones specific for sulfamethoxazole or protein antigens. Possibly, the route of antigen application (topical as opposed to systemic) rather than the chemical nature of the antigen determines the expansion and mobilization of CCR3+ T lymphocytes.

Conclusions Our findings demonstrate that in allergic inflammation, T lymphocytes are recruited together with eosinophils by way of CCR3. T lymphocytes have a decisive role in allergic inflammation, as documented in CD3- and CD4-deficient mice, which show severely reduced eosinophil mobilization after allergen challenge [24,35]. Through local production of Th2-type cytokines, CCR3+ T lymphocytes are likely to fulfil several functions, including the induction of adhesion molecules in the microvasculature that are required for eosinophil diapedesis and for the priming and prolonged survival of eosinophils [24]. We propose that these essential functions are triggered and maintained by CCR3+ T lymphocytes, whose population is expanded and recruited as a consequence of allergen challenge. CCR3 expression in T lymphocytes may serve as a marker for evaluating an individual’s predisposition to allergic inflammation and as a target for therapeutic intervention.

Materials and methods Cell isolation and T-lymphocyte clones

Table 3 Analysis of CCR3+ cells in double-stained tissue sections of nasal polyps. CCR3+/CD3+

Eosinophils

CD3+

1

40 (14)

148 (52)

135 (48)

2

37 (12)

135 (45)

167 (55)

3

38 (15)

133 (46)

145 (54)

Patient

Co-localizing T cells and eosinophils and cells co-expressing CD3 and CCR3 were counted in nasal polyp sections. Nine fields of each double-stained section were analysed at 40x magnification. The numbers of cells per mm2 and the percentage of the total number of counted cells (in brackets) are shown.

Human neutrophils, monocytes and lymphocytes were isolated from buffy coats [36–38]. T-lymphocyte clones were established and characterized as described [31,39–41]. IL-4, IL-5 and INF-γ were determined in the supernatant of the T-cell clones (106 cells) stimulated with 10 ng ml–1 PMA and 0.5 µg ml–1 ionomycin for 24 h [31,39,40]. For the characterization of CCR3 expression, clones were cultured for 6–7 days in the presence of 20 U ml–1 recombinant and 20 U ml–1 natural IL-2. For isolation of resident leukocytes from inflamed skin, a 5 mm punch biopsy was taken from a patient with contact dermatitis and kept in 0.25% trypsin for 8 h at 4°C [42]. A cell suspension was obtained by mechanical disaggregation. T cells were resuspended in RPMI 1640, 10% human AB serum (Swiss Red Cross Laboratory) and stimulated with 1 µg ml–1 phytohaemagglutinin (PHA) in the presence of irradiated feeder cells for 24 h. After 3 days of culture in the presence of 20 U ml–1 rIL-2, cells were analysed for CCR3 and CD3 expression by flow cytometry.

Research Paper Functional expression of CCR3 in T lymphocytes Gerber et al.

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Figure 4 CCR3+ T lymphocytes in nasal polyps and ulcerative colitis. (a–c) Immunohistochemical analysis of serial sections of nasal polyp tissue (a) stained for CD3, (b) for CCR3 and (c) double-stained for CD3 (red) and CCR3 (blue). Arrowheads designate a CCR3+ T lymphocyte (T+), a CCR3– T lymphocyte (T–) and a CCR3+ eosinophil (E). The inset at bottom right shows a magnification of the designated CCR3+ T lymphocyte. (d–f) Immunohistochemical analysis of sequential colon mucosa tissue sections from a patient with ulcerative colitis stained for (d) CD3, (e) CCR3 and (f) eotaxin. Representative fields are shown.

Nasal polyp

Ulcerative colitis

(a)

(d)

(b)

(e)

(c)

(f) T–

T+

E

50 m

100 m Current Biology

CCR3 cDNA isolation and northern blot analysis

T lymphocyte responses

For generation of CCR3 DNA fragments, two degenerate primers: 5′-GGGCTGCAG(C/A)GITAC(C/G)TGGCCATIGT(C/G)C-3′; 5′-GGGTCTAGAIGGGTTIAI(G/A)CA(G/A)C(T/A)(G/A)(T/C)G-3′; (I = inosine) corresponding to conserved sequence motifs in known chemokine receptors were used in PCR amplifications with human genomic DNA as template, and products of expected size were subcloned and sequenced [29]. An amplified CCR3 DNA was used as probe to isolate full-length cDNAs from a human tetanus toxoid-specific CD4 + T-cell cDNA library [29]. The eight individual cDNA clones contained three different 5′-untranslated regions (UTRs) with two putative mRNA splicing sites, indicating that the variant 5′-UTRs were derived from alternative mRNA processing. For northern blot analysis, samples of 15 µg total RNA were fractionated by agarose gel electrophoresis and vacuum blotted [29]. As hybridization probes, a 1.3 kb BamHI–XbaI fragment of the CCR3 cDNA and a 0.5 kb BstXI–BglII fragment of the CCR2 cDNA were used. Blots were analysed on a PhosphorImager (Molecular Dynamics).

Chemotaxis was performed in 48-well microchambers (NeuroProbe) with 105 cells per well in RPMI 1640, 20 mM Hepes pH 7.4 and 1% pasteurized human plasma protein solution (Swiss Red Cross Laboratory) using collagen-precoated polycarbonate membranes (Poretics) with 3 µm pores [28]. Where indicated, cells were preincubated at room temperature for 20 min with anti-CCR3 monoclonal antibody 7B11 at 10 µg ml–1.

Flow cytometry T lymphocytes were incubated with anti-CCR3 monoclonal antibody 7B11 or anti-CD3 (Dako) or IgG2 a mouse immunoglobulins (Sigma) on ice and washed before addition of fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse antibody (DAKO). Paraformaldehyde-fixed cells were analysed by flow cytometry (FACScan, Becton Dickinson).

Changes in cytosolic free calcium concentrations ([Ca2+]i) were determined in Fura-2-loaded cells as described [29]. Briefly, T-lymphocyte clones (3 × 106 ml–1) were incubated with 0.1 nmol Fura-2/AM for 30 min at 37°C, and after washing stimulated with a chemokine as indicated. [Ca2+]i-related fluorescence changes were recorded [43].

Immunohistochemistry Tissue samples obtained from patients with contact dermatitis, nasal polyps, ulcerative colitis and rheumatoid arthritis were analysed. Cryostatic sections of 4 µm (8 µm for nasal polyps) were fixed and stained as described [30,44]. For contact dermatitis, nasal polyp and rheumatoid arthritis samples, an alkaline phosphatase detection system was used; for ulcerative colitis a peroxidase detection system was used. Endogenous peroxide activity in colon mucosa was blocked with 1% hydrogen peroxide in PBS/saponin 0.1% at room temperature for 1 h. The tissue sections were incubated overnight with one of the following monoclonal antibodies: anti-CCR3 7B11 [32] at 5 µg ml–1, antieotaxin 6H9 at 1:200 [5] or anti-CD3 (Dako) at 1:50. As controls,

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species- and isotype-matched immunoglobulins (Sigma) were used. After washing, the slides were incubated with biotin-labelled sheep anti-mouse antibody (Amersham) at 1:800 for 30 min. After additional washes, the slides were incubated with either streptavidin–biotin–horseradish peroxidase (Dako) or streptavidin–biotin–alkaline phosphatase (Dako) for 30 min. All incubations were performed at room temperature. The colour reaction was developed with 3-amino-9-ethyl-carbazole (AEC, Sigma) for peroxidase or with New Fuchsin (Dako) containing levamisole (Dako) for alkaline phosphatase detection. Slides were counterstained with Mayer’s haematoxylin. Nasal polyps were double stained [45]. Sections were first incubated with anti-CD3 monoclonal antibody as described above, and were then treated with 0.1 M glycine for 5 min at room temperature before incubation with anti-CCR3 monoclonal antibody overnight. CCR3+ cells were detected with Fast Blue (Sigma), an alternative substrate for alkaline phosphatase. Nuclei were counterstained with methyl green. Slides were analysed with a Leitz Dialux 20EB microscope using a 40x magnification and a 0.09 mm2 grid.

Acknowledgements The authors thank R. Stuber Roos and W. Liu for help with PCR, P. Gionchetti, M. Caversaccio and S. Negri for providing tissue samples for immunohistochemical analysis, A. Lanzavecchia for the T-cell clone KT30, and G. Bilbe for help in the generation of the KT30 cDNA library. Human recombinant and natural IL-2 were kindly provided by A. Cerny and U. Schwulèra, respectively, and the chemokines eotaxin and MCP-1 were obtained from I. Clark-Lewis. The Master Cycler PCR amplification machine was financed through the ‘Hochschulstiftung’ of the University of Bern. Donor blood buffy coats were provided by the Swiss Central Laboratory Blood Transfusion Service, SRK. This work was supported by grant 31-039744.93 to M.B. and B.M. from the Swiss National Foundation. B.M. is a recipient of a career development award from the Prof. Max Cloetta Foundation.

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