INTERLEUKINS 4 AND 13 UPREGULATE EXPRESSION OF CD44 IN HUMAN COLONIC EPITHELIAL CELL LINES

Article No. ck980361

INTERLEUKINS 4 AND 13 UPREGULATE EXPRESSION OF CD44 IN HUMAN COLONIC EPITHELIAL CELL LINES Ludwik K. Trejdosiewicz, Ruth Morton, Yaoquin Yang,1 Roz E. Banks, Peter J. Selby, Jennifer Southgate Interleukin 4 (IL-4) inhibits carcinoma cell growth and promotes expression of differentiation-associated products by normal and malignant epithelial cells. The effects of IL-4 and IL-13 on expression of the CD44 transmembrane adhesion receptor were examined in human epithelial cell lines of colonic (HT-29, CaCo-2, DLD-1, T84), breast (MCF-7, ZR75-1) and liver (Hep-G2, PLC/PRF/5) origins as well as mitogen-activated and resting peripheral blood lymphocytes (PBL) and T cell lines (Jurkat, HUT78). Liver and Jurkat cells were negative for CD44. Colonic, breast and HUT78 cells expressed CD44 constitutively and all except DLD-1 and HUT78 also expressed CD44 splice variant (CD44v) epitopes. All cell lines expressed IL-4 receptors, but IL-4 and IL-13 induced upregulation of CD44 only in the colonic cell lines. CD44v was also upregulated, but there was no de novo induction of CD44v in variant-negative cells and no de novo expression of CD44 in the CD44− lines. CD44 upregulation in mitogen-activated PBL was not increased by IL-4 and IL-13 and was not inhibited by neutralizing antibodies. Other cytokines tested [interferon g (IFN-g, tumour necrosis factor a (TNF-a), transforming growth factor b1 (TGF-b1) and IL-6] did not affect CD44 core epitope expression in the cell lines tested. 7 1998 Academic Press

IL-4 plays a central role in immunoregulation by polarizing the immune system towards Th2-type responses, promoting B cell differentiation and isotype switching and downregulating Th1-type responses (recently reviewed in Ref. 1). IL-13 is a closelyrelated cytokine of high sequence homology and near-identical biological action, with the important exception of having no effect on T cell differentiation.2,3 In addition to direct actions on immunocytes and lymphomyeloid cells, IL-4 and IL-13 are known to have effects on other cell types involved in the immune response. For example, IL-4 and IL-13 promote adhesiveness of leukocytes to endothelial cells by upregulation of vascular cell adhesion molecule 1 (VCAM-1).4–6 IL-4 has been shown to directly upregulate the expression of intercellular adhesion molecule 1 (ICAM-1) in human dermal fibroblasts7 and From the ICRF Cancer Medicine Research Unit, St James’s University Hospital, Leeds LS9 7TF, UK Correspondence and reprint requests to: LK Trejdosiewicz, ICRF Cancer Medicine Research Unit, St James’s University Hospital, Leeds LS9 7TF, UK; E-mail: l.k.trejdosiewicz.leeds.ac.uk 1 On sabbatical from: Tumor Cell Research Institute, Department of Histology and Embryology, Shanghai Railway Medical College, People’s Republic of China Received 3 November 1997; accepted for publication 28 March 1998 7 1998 Academic Press 1043–4666/98/100756 + 10 $30.00/0 KEY WORDS: CD44/ colon/ IL-4/ IL-13 756

modulate the effects of pro-inflammatory cytokines on adhesion molecule expression in endothelial cells.8,9 There is also considerable literature on the effects of IL-4 on epithelial cells in vitro. Overall, IL-4 appears to inhibit the growth of many carcinoma cell types, including those of colorectal origins (e.g. Refs 10–12). IL-4 also generally appears to promote expression of functional or differentiation-associated epithelial proteins. Thus, IL-4 induces differentiation at the expense of proliferation in colorectal carcinoma cells13 and can upregulate secretory component (poly-Ig receptor) in intestinal HT-29 cells14,15 and in normal and transformed endometrial cells.16 IL-4 has also been reported to upregulate peptidases in renal cell carcinoma cell lines and renal tubular epithelial cell lines,17 C3 complement production by human pulmonary18 and alveolar epithelial cells,19 CD23 in nasopharyngeal carcinoma cells20 and in keratinocytes21 and lipoxygenase in airway epithelial cells.22 Considerably less is known of the effects of IL-13 on epithelial cells. The emerging evidence suggests that endothelial and other cell types express common receptor components for IL-4 and IL-13,6,23,24 signalling through the JAK Janus kinase and STAT transcription factor families.25,26 However, the situation is complex, as cells can respond to IL-4 and IL-13 in the absence of the common g-chain6,25 and different cell types express different binding affinities for IL-4 and CYTOKINE, Vol. 10, No. 10 (October), 1998: pp 756–765

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IL-13.27,28 A distinct, high-affinity soluble IL-13 binding protein has also been described.29 In this report, we demonstrate that the CD44 molecule implicated in cell–matrix interactions, and in metastatic tumour progression as well as in lymphocyte homing and lymphocyte activation, is upregulated by IL-4 and IL-13 in epithelial cells of colonic origins, but not in other cell types, including T lymphocytes.

showed weak labelling with monoclonal antibodies directed against CD44 splice variant epitopes v3, v4, v5 and intense labelling with v6-specific antibodies.

Effects of cytokines on CD44 expression in cell lines Following 72-h culture of HT-29 and DLD-1 cell lines with various doses of IFN-g, TNF-a or TGF-b1, there was no increase in expression of core CD44 epitopes as shown by flow cytometry (Fig. 1). As positive controls, the effects of IFN-g and TNF-a on HLA-DR and ICAM-1 expression were also assessed. HLA-DR and ICAM-1 expression were upregulated by IFN-g treatment, whereas TNF-a only upregulated ICAM-1, but not HLA-DR expression (Fig. 1). IL-4-induced upregulation of CD44 expression in all four colorectal cell lines (Table 1). The effect was dose dependent: measurable upregulation was seen with as little as 0.1 U/ml and r90% of maximal upregulation occurred with 10 U/ml. All three CD44 core epitopes tested were upregulated equally. By contrast, IL-4 at concentrations of up to and including 1000 U/ml had no effect on CD44 expression in the other CD44+ cell lines (MCF-7, ZR75-1 and HUT78) and did not induce CD44 expression in the CD44− cell lines (PLC/PRF/5 and Hep-G2). Several of the cell lines were further examined by immunofluorescence microscopy to assess the effects of IL-4 on CD44 splice variant expression. In HT-29 cells, a marked increase in labelling intensity was noted with antibodies against v3, v4/5, v5 and v6 (similar results were obtained for all three v6 antibodies VFF7, 2F10, 2G9) (Fig. 2). ZR75-1 and T84 also showed an increase in intensity of labelling with the CD44v antibodies

RESULTS CD44 core and splice variant expression by cell lines With the exception of the PLC/PRF/5, Hep-G2 and Jurkat cells, all cell lines were positive by flow cytometry for cell surface expression of core CD44 epitopes (Table 1). Using the KZ-1 anti-CD44 antibody, median fluorescence values varied from low (Q100) in CaCo-2, DLD-1, MCF-7 and ZR75-1 cells, to moderate (100–300) in T84 cells, high (q300) in HT-29, HUT78 and resting PBL cells and very high (q1000) in activated PBL. Immunofluorescence performed on the three negative cell lines confirmed that Jurkat, PLC/PRF/5 and Hep-G2 cell lines were r99% CD44−. Of the CD44+ cell lines, only DLD-1 and HUT78 did not show CD44 splice variant (CD44v) expression when tested by immunofluorescence using splice variant-specific antibodies (Table 1). Cell lines which did express CD44v were always positive for multiple splice variant epitopes, although there was some variation in the labelling intensity between different epitopes. In particular, the HT-29 cell line TABLE 1.

CD44 expression by cell lines CD44 expression

Cell line

Core*

Colon adenocarcinoma derived HT-29 High CaCo-2 Low DLD-1 Low T84 Moderate Hapatocellular carcinoma derived PLC/PRF/5 Negative Hep-G2 Negative Breast carcinoma derived MCF-7 Low ZR75-1 Low T cell derived Jurkat J6 Negative HUT78 High

Variant†

Mean (range) of change in CD44 core expression induced by IL-4‡

+ + − +

2.0-fold 1.3-fold 4.8-fold 2.3-fold

increase increase increase increase

− −

no change no change

+ ++

no change no change

− −

no change no change

(1.38–3.11) (1.09–2.00) (3.25–6.09) (2.13–2.52)

*Based on flow cytometry median fluorescence values obtained with antibody KZ-1 (CD44 core epitope 3), scored as Low (median fluorescence R100), Moderate (100–300) and High (r300). Mean derived from a minimum of 3 independent determinations. Results with all other CD44 core epitope antibodies were comparable. †Splice variant expression qualitatively assessed by immunofluorescence as negative (−), moderate (+) or strong (++) using antibodies to v3, v4/5, v5 and v6. In all cases, either no CD44v epitopes were detected or multiple splice variants were present. ‡Comparable results were obtained with IL-13.

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A

of the CD44 increase was either unaltered or even reduced when PBL were activated in the presence of IL-4 (Fig. 3). IL-4 did not cause upregulation of CD44 expression in resting (unstimulated) PBL in culture. Addition of anti-IL-4 or anti-IL-13 neutralizing Ab at up to 100 mg/ml (see below) had no effect on CD44 expression in resting PBL and did not alter upregulation of CD44 as a consequence of PHAinduced activation.

*

1500 1000 500

Median fluorescence channel

0 150

Kinetics of CD44 upregulation

B

*

100

*

50

0 200

C

*

150 100 50 0

Control IFN-γ

TNF-α

IL-4

TGF-β

Figure 1. Effects of cytokines on surface antigen expression by HT-29 intestinal cells. Cells were cultured with cytokines for 72 h before analysis by flow cytometry. Cytokines were used at 200 U/ml (IFN-g); 250 U/ml (TNF-a); 10 U/ml (IL-4) and 200 U/ml (TGF-b1). CD44 expression (A) was constitutive, but upregulated only by IL-4; whereas in the positive controls, upregulation was seen as expected for ICAM-1 (B) and HLA-DR (C) with IFN-g and ICAM-1 with TNF-a. Bars show replicate means (295% confidence intervals) of the median fluorescence channel of nine independent experiments. Asterisks indicate values significantly different from controls (P Q 0.001, two-tailed t-test).

following IL-4 administration. However, no change in labelling intensity for CD44v antibodies was apparent in MCF-7 and Caco-2 cells and no de novo expression of splice variant epitopes was induced in either the DLD-1 or the HUT78 cell lines.

Effects of IL-4 on CD44 expression by resting and activated lymphocytes Forty-eight to 72 hours after phytohaemagglutinin (PHA) stimulation, PBL showed a 2–3-fold increase in cell surface binding of core CD44 antibodies, compared to non-activated control cells (Fig. 3). The magnitude

IL-4 induced a stable upregulation of CD44 in the colon cells, which was detectable at 24 h and reached a plateau at 72 h. CD44 expression remained elevated for as long as the cells were cultured in presence of the IL-4 (or IL-13) and returned to baseline some 24–48 h after removal of the cytokine (not shown). Pulsing with IL-4 for 24 h was sufficient to induce approximately 50% of maximal upregulation, peaking at 72 h and returning to baseline some 24–48 h later. In order to determine whether IL-4 induced de novo transcription and translation of CD44, as opposed to conformational changes resulting in increased exposure of antigenic epitopes, the effects of cycloheximide and actinomycin D, respectively, were studied on HT-29 cells. Both inhibitors reduced the baseline expression of CD44 of control cells relative to untreated cells and completely abolished the IL-4 induced upregulation (Fig. 4). Hybridization of Northern blots with a CD44 probe showed there to be three main transcripts of approximately 4.4, 2.8 and 2.1 kb (Fig. 5). When cultured in the presence of IL-4, CD44 transcripts were increased at 24 h relative to no IL-4 controls. The increased CD44 transcription was maintained in cells grown for 72 h with IL-4.

IL-4 receptor expression IL-4R protein was detected in approximately equal amounts in all of the cell lines used in the study, irrespective of their epithelial, T cell or B cell origins. By immunoprecipitation followed by immunoblotting, three bands were detected, comprising two intense bands of Mr approximately 140 000 and 120 000 with a weaker band of 65 000 (Fig. 6). By contrast, direct immunoblotting of whole cell lysates revealed only 120 000 and 65 000 bands with no 140 000 product. This supports previous work suggesting that the IL-4R is exquisitely sensitive to degradation (discussed in Ref. 30). The same three bands were detected with the anti-IL-4R monoclonal antibody on immunoblots of PBL (not shown). The amount of IL-4R expressed by PBL was low and increased markedly at 72 or 96 h post-activation, although it was always less than that detected on an equivalent number of cells from cell lines.

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Figure 2. Indirect immunofluorescence of CD44s and CD44v antibodies on HT-29 cells cultured for 72 h without (left column: A, C, E) and with (right column: B, D, F) IL-4 at 10 U/ml. Cells were labelled with antibodies F10.44.2 to CD44s epitope 1 (top row: A and B), 7B8 to CD44v4/5 (middle row: C and D) and 2G9 to CD44v6 (bottom row: E and F). In all cases, IL-4 upregulated expression of CD44, even where baseline expression was weak. All micrographs taken with timed exposures set for the IL-4-induced cells. Scale bar = 10 mm.

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1400

Effect of IL-13

1200

IL-13 showed the same effect as IL-4 and upregulated CD44 expression on the same cell lines which were upregulated by IL-4. No cell line showed a differential effect with either of the two cytokines and IL-13 acted over the same concentration range as IL-4. There was no additive or synergistic effect of IL-4 and IL-13, suggesting that both cytokines competed for the same receptors.

Neutralization of IL-4 and IL-13 activities The cytokine-induced upregulation of CD44 expression was abrogated completely by preincubation of the cytokine with cognate neutralizing Ab (Fig. 7). Anti-IL-4 Ab (10 mg/ml) completed blocked the effects of 10 U IL-4 with a specific activity of 1.3 × 107 U/mg. Similarly, the upregulation of CD44 by IL-13 could be neutralized by preincubation of 10 U IL-13 of specific activity of 2.5 × 105 U/mg with 50 mg anti-IL-13 Ab (Fig. 7).

Median fluorescence channel

detected on an equivalent number of cells from cell lines.

1000 800 600 400 200 0

Control

Cycloheximide Actinomycin D

Figure 4. The effects of transcriptional and translational inhibitors on CD44s upregulation by IL-4 on HT-29 cells. Flow cytometry data is illustrated as the median fluorescence channel using KZ-1 antibody. q: no IL-4 control; Q: with 10 U/ml IL-4. Cycloheximide and actinomycin D abrogated the IL-4 induced upregulation of CD44s expression; as expected, both inhibitors also reduced the baseline expression of CD44.

DISCUSSION

Median fluorescence ratio

3

2

1

0 Resting

Activated

Figure 3. CD44 expression on PHA-activated PBL measured by flow cytometry with antibody KZ-1. CD44 was upregulated on 72-h PHA blasts (closed symbols and solid lines); addition of exogenous IL-4 (open symbols and dashed lines) or neutralizing anti-IL-4 Abs (not shown) had no significant effect on CD44 expression. Data shown from representative experiments; results were calculated as the ratio of median fluorescence channels for resting:activated cells.

We have presented evidence that interleukins 4 and 13 are key modulators of CD44 expression in cultured epithelial cells of large bowel origins. By contrast, other pleiotropic cytokines tested did not affect expression of CD44 in other epithelial or T lymphocyte-derived cells tested. Other investigators have also found that cytokines such as IFN-g did not upregulate CD44 core epitope expression in colorectal HT29 cells, although some upregulation of CD44v6 has been reported.31 This may be the mechanism of upregulation of CD44v6 observed in ulcerative colitis.32 Specific upregulation of CD44 by IL-4 and IL-13 implies that colonic epithelial cells are responsive to immunomodulatory mechanisms. The central role of IL-4 in the intestinal microenvironment is underlined by the demonstration that IL-4 ‘‘knockout’’ mice develop Peyer’s patches without germinal centres and have profoundly defective mucosal immune responses.33 IL-4R are expressed by both normal intestinal mucosae and the majority of colorectal tumours.34 The IL-4R is a member of the haemopoietin receptor superfamily and is expressed at low copy numbers on the cell surface.30 Our data suggest that colorectal cells do not express separate receptors for IL-4 and IL-13, as the effects of both cytokines were identical and no additive effects were noted. This would confirm an

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0

12

12

24

24

48

48

72

72

4.4

2.8

2.1

A

1.9

B Figure 5.

Northern blot of HT-29 cells extracted 12, 24, 48 and 72 h post-addition of 10 U/ml IL-4 (arrowed tracks).

Parallel cultures of HT-29 cells without IL-4 were extracted at the same time points as control (no arrows). The blot was hybridized with P-labelled DNA probes to: A: A 664 bp PCR fragment generated from plasmid clone pCD44 and containing a 1341 bp insert of unspliced CD44s. B: A 615 bp b-actin PCR fragment as a control for RNA quality and gel loading. The CD44s probe hybridized to three mRNA species of approximately 4.4, 2.8 and 2.1 kb. IL-4 had a biphasic effect on CD44 transcription. Expression of all three transcripts peaked at 12 h, falling to just above control levels by 24 h. The two smaller transcripts showed a secondary increase at 48 h which was still sustained at 72 h. This may be due to mRNA stabilization.

32

earlier report, which demonstrated that both IL-4 and IL-13 acted through the IL-4 receptor on colon carcinoma cell lines.26 All carcinoma lines tested expressed the high affinity 140 000 a-chain of the IL-4R at approximately equal densities and it is known that IL-4R a-chain alone is functional for signal transduction.35 Thus, the lack of apparent response to IL-4 by non-colorectal cell lines is not accounted for by differences in IL-4R expression. Rather, it would appear that the phenomenon of CD44 regulation by IL-4 and IL-13 reflects a tissue-specific difference in the response elements downstream to receptor binding. Increased CD44 expression by T cells is known to be associated with a memory phenotype and arises as a consequence of T cell activation. However, our data clearly show that IL-4 and IL-13 do not participate in CD44 upregulation during activation of T-cells. The available data suggest that CD44 expression by different cell types may be regulated by separate mechanisms. We could demonstrate no effects of IL-4 or IL-13 on the other cell types tested and no cytokine-mediated mechanism has been discovered for fibroblasts36 to date. However, CD44 upregulation in

1

2

3

4

5

6

Figure 6. Immunoprecipitations of MCF-7 (tracks 1 & 2), HT-29 (tracks 3 & 4) and DLD-1 (tracks 5 & 6) cell lines with antibody M57 against the IL-4R (tracks 1, 3, 5) and a matched antibody control (tracks 2, 4, 6). After separation on a 7.5% polyacrylamide gel, proteins were transferred to nitrocellulose membrane and probed with M57 antibody. In all cell lines, three specific bands were detected (arrowed), representing the 140 000 IL-4R a-chain and 120 000 and 75 000 degradation products. Immunoblots of whole cell lysates labelled with the M57 antibody revealed only 75 000 and 120 000 bands due to degradation of the 140 000 protein (not shown).

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Figure 7. CD44 upregulation in HT-29 cells and the effects of neutralizing antibodies by flow cytometry. A: Constitutive expression of core CD44 on HT-29 cells (shaded histogram) labelled with antibody KZ1 to CD44 core epitope 3 compared to no-antibody control (open histogram). B: The IL-4 upregulated CD44 expression on HT-29 cells (shaded histogram) was wholly inhibited with neutralizing Ab (open histogram). C: IL-13 also upregulated CD44 expression on HT-29 cells (shaded histogram) which was wholly inhibited by anti-IL-13 neutralizing Ab (open histogram).

endothelial cells can be mediated by hepatocyte growth factor37 and by TNF-a in Langerhans cells,38 whereas TNF-a had no effect in our system. There is also evidence that CD44 can be functionally distinct in different cell types39,40 perhaps through mechanisms that involve post-translational modification.40,41 Glyco-

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sylation has been shown to reduce the binding affinity of CD44 for hyaluronan40,41 and expression of splice variant exons exposes further glycosylation sites.42 Reduced hyaluronate binding has been proposed as the basis for the association between CD44v expression and colorectal cancer progression.43 Thus, tissuespecific expression of splice variants and differential glycosylation may be mechanisms for ‘‘fine-tuning’’ cell adhesiveness to the stromal ground substance. Although it is often assumed that altered expression of CD44 is an inherent property of tumour cells, our data suggest that the cytokine milieu of the host tissue microenvironment may be another determining or modulatory factor. Taken together, the data suggest that CD44 gene expression may be differentially modulated by local immune reactions and the balance of Th1 and Th2-type cytokines. This finding has implications for the cytokine therapy of colorectal carcinomas. Increased CD44v expression has been associated with a more aggressive phenotype,44 although more recent work has cast doubts on this premise.45 Increased CD44 expression also has been associated with colonocyte proliferation.46 However, a recent study has provided direct evidence that CD44 expression is not correlated with cell division in colorectal carcinomas47 and it has been shown that restoration of CD44 core sequences into colorectal carcinoma cells increased adhesion to hyaluronan, thereby inhibiting cell division.43 Furthermore, restoration of CD44 core protein reduced the tumorigenicity of colorectal carcinoma cells in vivo.48 Our data could strengthen the rationale for the therapeutic use of IL-4, particularly in colorectal tumours, as upregulation of CD44 should have a negative effect on tumour progression by increasing cell–matrix adhesion. Indeed, recent experimental evidence has demonstrated that adhesiveness of HT-29 cells was increased and the metastatic potential of the cells was reduced by IL-449 and IL-4 expression by tumour-infiltrating lymphocytes in human colon carcinoma is associated with significantly improved patient survival.50 Because IL-13 was equally effective at upregulating CD44 core expression and shares essentially all the biological activities of IL-4, but without promoting the development of Th2 cells,1–3 IL-13 may prove to be a superior agent to IL-4, particularly in colorectal cancer. These implications, if sustained, could suggest new approaches for immunotherapy in this condition.

MATERIALS and METHODS Cell culture The established human cell lines used in the study are detailed in Table 1. All cell lines were obtained from the American Type Culture Collection (ATCC) or the European

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Collection of Animal Cell Cultures (Porton Down, UK). Cells were maintained in growth medium comprising a 1:1 mixture of RPMI 1640 and DMEM (Sigma, Poole, UK) without antibiotics, supplemented with 5% FCS (Sera-Lab, Crawley Down, UK). Adherent cells were harvested by incubation for 5 min in PBS containing 0.1% (W/V) EDTA followed by incubation in 0.25% (W/V) trypsin in versene at 37°C, for the minimum period until cells detached. Cells were resuspended in growth medium and replated or analysed as described below. Cytokines The cytokines used included rIFN-g (gift from Biogen Research Corporation, Cambridge, MA), rIL-4 (gift from Schering-Plough Ltd., Bury St Edmunds, UK), rIL-6, rIL-13, TGF-b1 and rTNF-a (all obtained from R&D Systems, Abingdon, UK). Unless otherwise stated, cells were plated and precultured for 24 h prior to aspiration of media and replacement with fresh medium containing cytokine at doses of 0.1–1000 U/ml. Growth in the presence of cytokine was continued for the times indicated. In some experiments, 10 mg/ml cycloheximide (Sigma) or 1 mg/ml actinomycin D (Sigma) were added to cell cultures 10 min before addition of cytokines. PBL were isolated by density barrier centrifugation (Lymphoprep; Nycomed (UK) Ltd., Sheldon) and cultured at 5 × 105 cells/ml in RPMI 1640 containing 10% FCS, with or without 2.5 mg/ml PHA (Murex Diagnostics Ltd., Dartford, UK) as mitogen. This concentration was previously found optimal for inducing T cell activation, assessed by dual fluorescence flow cytometry for upregulation of CD69 at 24 h and CD25 at 72 h in the CD2+ subset.51 Antibodies and immunolabelling procedures Monoclonal antibodies to the extracellular domain of CD44 comprised F10.44.2 (Serotec, Oxford, UK), Bric 222, Bric 235, KZ-1 (IBGRL Research Products, Bristol, UK) and Hermes 3 (gift from Dr S. Jalkanen). The CD44 antibodies were classified into epitope groups 1 (F10.44.2 and Bric 222), 2 (Bric 235) and 3 (KZ-1 and Hermes 3), according to Anstee et al.52 Monoclonal antibodies specific to the splice variant exons of CD44 included 3G5 (v3), 7B8 (v4/5), 2F10 and 2G9 (v6), purchased from R&D Systems Europe Ltd (Oxon, UK) and VFF8 (v5) and VFF7 (v6) purchased from BioWhittaker UK Ltd (Wokingham, UK). Also used were monoclonal antibodies to HLA-DR (clone RFDR1; gift of Professor G. Janossy) and ICAM-1 (clone 84H10 from Immunotech, supplied by The Binding Site, Birmingham) in positive control experiments. Antibodies were titrated separately for flow cytometry and indirect immunofluorescence techniques. For specificity controls, primary antibodies were either omitted or were substituted with an irrelevant, isotypematched control antibody.51,53,54 For flow cytometry, cultured cells were harvested and immunolabelled in suspension at 4°C using an indirect technique.53 Cells (5 × 104) were resuspended in pretitred primary monoclonal antibody and incubated for 30 min, washed by centrifugation in PBS containing 1% FCS and 0.1% NaN3, resuspended in 2 mg goat anti-mouse Ig conjugated to FITC (Southern Biotechnology Associates, supplied by Eurogenetics, Teddington, UK) and incubated

for 30 min at 4°C. After washing as before, cells were resuspended in 500 ml PBS and analysed on a Becton Dickinson FACScan flow cytometer using Lysis II acquisition software and analysed using the Cell Quest program. Baseline fluorescence medians were established for each cell line using negative controls (no primary antibody) and were found not to alter following culture with cytokines. Median fluorescence values of cells labelled with specific antibodies grown with and without cytokine were compared using an unpaired Student’s t-test. Indirect immunofluorescence of cultured cells plated on ‘‘Multitest’’ slides (Hendley, Essex, UK) was performed as previously described.54 Briefly, slides were fixed by permeabilisation in a 1:1 mixture of methanol:acetone followed by air-drying. The slides were incubated in primary antibodies for 60 min, rinsed in 10 mM Tris-buffered saline pH 7.4 (TBS), refixed in methanol:acetone, air-dried and incubated for 30 min in goat FITC-conjugated anti-mouse Ig (Southern Biotechnology Associates). After washing in TBS containing 0.25% Tween 20, slides were rinsed in distilled water, air-dried and mounted in glycerol containing 2.5% (W/V) 1,4-diazabicyclo[2.2.2]octane to prevent photobleaching. Slides were viewed on a Zeiss Axioplan microscope fitted with immersion objectives and epifluorescent illumination. Neutralization of IL-4 and IL-13 activity Neutralizing rabbit heteroantisera against IL-4 (Genzyme Diagnostics, West Malling, UK) and IL-13 (R&D Systems, Abingdon, UK) were used as specificity controls for IL-4 and IL-13 activity, respectively. Cytokine (10 U) was preincubated with a 0–100 mg/ml dilution range of neutralizing antibody for 60 min at 37°C before addition to cells. Cultures were maintained for a further 72 h prior to flow cytometric analysis of CD44 expression relative to untreated controls (above). In order to determine whether upregulation of CD44 on activated PBL occurred due to autocrine IL-4 and/or IL-13 secretion, neutralizing Abs were added as above to PBL cultures simultaneously stimulated with PHA. RNA extraction and Northern blotting Total RNA was isolated with ‘‘Ultraspec’’ (Biogenesis, Bournemouth, UK) according to the manufacturer’s instructions. The RNA was dissolved in diethylpyrocarbonate (DEPC)-treated water and the concentration and purity was estimated from the ratio of absorbance ratio of 260 nm and 280 nm. RNA was aliquoted and stored frozen at −20°C in 70% ethanol. ‘‘RNAguard’’ at 0.25 U/ml (Pharmacia, St Albans, UK) was included in all subsequent steps involving RNA manipulation. 15 mg of RNA was electrophoretically separated per track on formaldehyde agarose gels alongside 0.28–6.58 kb RNA size markers (Promega, Southampton, UK) and transferred and UV cross-linked onto Hybond-N membrane (Amersham, Little Chalfont, UK). A 664-bp DNA fragment was generated from plasmid clone pCD44 containing a 1341-bp insert of unspliced CD4455 by PCR, using forward primer 5'-CTC CGG ACA CCA TGG ACA AGT-3' and reverse primer 5'-TCT GTC TGT GCT GTC GGT GAT-3'. The PCR product was excised and eluted from the gel and labelled with 32P using the ‘‘Rediprime’’ random-primed

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DNA labelling kit (Amersham). Membranes were prehybridized for 1 h at 65°C and incubated overnight with the 32 P-labelled denatured DNA probe in 0.25 M phosphate buffer pH 7.2 containing 7% (W/V) SDS and 10 mg/ml denatured salmon sperm DNA. The integrity and loading of the RNA samples was tested by reprobing the Northern blot with a 32P-labelled 615-bp b-actin cDNA probe generated by PCR from a human melanoma cell line cDNA using forward primer 5'-GGC ATC GTG ATG GAC TCC G-3' and reverse primer 5'-GCT GGA AGG TGG ACA GCG A-3'. The RNA size markers were visualized on the blot by hybridization with 32P-labelled Lambda DNA (Promega). IL-4 receptor analysis IL-4R were isolated by immunoprecipitation. Raji B-lymphoblastoid cells were included as IL-4R positive control cells.30 Briefly, cells were lysed by 15 min incubation on ice at 5 × 106 cells/ml of lysis buffer comprising 50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% Nonidet P40, 0.1 mM NaF, 0.1 mM Na3VO4, 20 kIU/ml aprotinin and 2 mM PMSF. Lysates were clarified by microcentrifugation and 900 ml aliquots were incubated with 3 mg of purified M57 monoclonal antibody against the human CDw124 IL-4R (Serotec, Oxford, UK) and 20 ml of protein A beads [50% slurry, ‘‘Immunopure’’, Pierce and Warriner (UK) Ltd., Chester, UK] overnight at 4°C on a mixer. The beads were collected by centrifugation, washed 4 times in lysis buffer and once in 50 mM Tris–HCl pH 7.5 and resuspended in 20 ml of twice-concentrated standard SDS PAGE sample buffer containing 0.2 M dithiothreitol (DTT). Negative control precipitations were included with each experiment, substituting an equal amount of an isotype-matched irrelevant antibody. IL-4R from immunoprecipitations (and from whole cell lysates) were detected by immunoblotting. Following separation by SDS PAGE on 7.5% mini-gels (Protean II; BioRad Laboratories Ltd., Hemel Hempstead, UK), proteins were transferred by semi-dry electroblotting [Milliblot; Millipore (UK) Ltd., Watford] to nitrocellulose (Hybond ECL; Amersham). The membranes were blocked for 30 min in 2% (W/V) non-fat skimmed milk in PBS and incubated overnight at 4°C in either blocking buffer alone (non-specific control) or in blocking buffer containing 10 mg/ml M57 antibody. Membranes were washed in PBS with 0.2% (W/V) Tween 20 and bound antibody was detected using an ABC-immunoperoxidase kit (Dako) and visualized by an enhanced chemiluminescence detection system (Amersham), used according to the manufacturer’s instructions.

Acknowledgements We thank Dr G.J. Dougherty of the British Columbia Cancer Research Center, Canada, Dr S. Jalkanen, National Public Health Institute, Turku, Finland, Professor G. Janossy of the Royal Free Hospital, London; Biogen Research Corporation, Cambridge, MA, and Schering-Plough Ltd., Bury St. Edmunds, UK for generously providing reagents. We are grateful to Mrs B. Smith and Dr A. Wood for assistance with the Northern blotting experiments.

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