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A novel, rapid and sensitive heterotypic cell adhesion assay for CD2–CD58 interaction, and its application for testing inhibitory peptides
Journal of Immunological Methods 291 (2004) 39 – 49 www.elsevier.com/locate/jim
Research paper
A novel, rapid and sensitive heterotypic cell adhesion assay for CD2–CD58 interaction, and its application for testing inhibitory peptides Jining Liu a, Vincent T.K. Chow b, Seetharama D.S. Jois a,* b
a Department of Pharmacy, 18 Science Drive 4, National University of Singapore, Singapore 117543, Singapore Human Genome Laboratory, Department of Microbiology, 18 Science Drive 4, National University of Singapore, Singapore 117543, Singapore
Received 8 January 2004; accepted 24 April 2004 Available online 15 June 2004
Abstract The immunoglobulin CD2 is a cell adhesion molecule that mediates T-cell activation by binding to its receptor CD58 on antigen-presenting cells (APCs). Modulation or inhibition of this interaction has been shown to be therapeutically useful. Erosetting assay is usually applied in the study of the modulation of CD2 – CD58 interaction. In this study, we demonstrated a novel, rapid and sensitive heterotypic cell adhesion assay for CD2 – CD58 interaction. The CD2 expression on the surface of Jurkat cells and the CD58 expression on the Caco-2 cells were confirmed by flow cytometry and ELISA studies, respectively. Then Jurkat cells were fluorescent-labeled with 2 AM of BCECF-AM for 45 min at 37 jC before adding to confluent Caco-2 monolayers cultured in 96-well culture dishes. After 30 min, non-adherent Jurkat cells were removed by washing with PBS, while the monolayer-associated Jurkat cells were lysed with 0.5 ml of 2% Triton X-100 in 0.1 M NaOH. Fluorescence (FL) was quantitated using a microplate fluorescence analyzer with BCECF’s excitation maximum of 485 nm and emission maximum of 535 nm. This method was successfully applied for testing inhibitory peptides to CD2 – CD58 interaction. D 2004 Elsevier B.V. All rights reserved. Keywords: CD2; CD58; Jurkat cells; Caco-2 cells; Lymphocyte – epithelial adhesion assay; E-rosetting assay
1. Introduction Cell-surface proteins mediate intercellular recognition and adhesion. In the immune response, a host of proteins is involved in the recognition of antigen-
* Corresponding author. Tel.: +65-6-874-2653; fax: +65-6-7791554. E-mail address: [email protected] (S.D.S. Jois). 0022-1759/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2004.04.026
presenting cells (APCs) by T-cells. These include the T-lymphocyte adhesion receptor CD2 and its ligands, CD48 in rodents and CD58 in humans (Davis et al., 1998). CD2, a 50- to 55-kDa glycoprotein, is expressed on T-cells and natural killer (NK) cells and distinguishes them from other leukocytes (Aiuti et al., 1975; Dustin et al., 1987). CD58, a 55- to 70kDa glycoprotein, is expressed on both hematopoietic and non-hematopoietic cells (Arulanandam et al., 1993). The interaction between CD2 on T-cells and
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CD58 on APCs is believed to augment the adhesion between T-cells and APCs (Davis et al., 1998; Moigneon et al., 1989). In particular, this heterotypic cell adhesion facilitates initial cell – cell contact before specific antigen recognition and also enhances T-cell receptor (TcR) triggering by fostering interaction with peptide-class II MHC complex (pMHC) (Kim et al., 2001). Although the affinity of CD2 – CD58 interaction is relatively low (Kd f 1 AM), the very rapid koff and kon supports dynamic binding with rapid counterreceptor exchange, and creates an optimal intercellular ˚ ) on opposing cell membrane distance ( f 135 A surfaces suitable for TcR – pMHC or NK receptor – MHC interactions to foster immune recognition. Hence, in the presence of hCD2 – hCD58 interaction, T-cells recognize the correct pMHC with a 50- to 100fold greater efficiency than in the absence of hCD2 – hCD58 interaction. In addition, endothelial cells (EC) in rheumatoid arthritis (RA) have been shown to express elevated levels of CD58, and RA lymphocytes in synovial fluid (SF) express increased levels of CD2 and CD58 relative to RA or normal peripheral blood lymphocytes (Bachmann et al., 1999; Mojcik and Shevach, 1997). These findings led to postulation that the modulation of T-cell responses in vivo after blockade of the interaction between CD58 and CD2 can potentially be applied in the treatment of autoimmune diseases and transplantation. It has been shown that blockade of the CD2 – CD58 interaction (Kaplon et al., 1995; Sultan et al., 1997) and/or modulation of the CD2 costimulatory pathway (Qin et al., 1994; Hirahara et al., 1995; Sido et al., 1996, 1997) can result in prolonged tolerance towards allografts. The soluble CD58 – Ig fusion protein Amevive (LFA3TIP) has been employed in the treatment of psoriasis (Aruffo and Hollenbaugh, 2001). The humanized versions of antibodies BTI-322 (Przepiorka et al., 1998) and MEDI-507 (Branco et al., 1999) have been tested for the treatment of acute organ rejection and graftversus-host disease. In previous CD2/CD58 studies, the modulation of CD2 – CD58 interaction was mainly investigated by E-rosetting, either by counting bound cells visually under a microscope, or radiolabeling of cells with 125I and measuring the residual radioactivity after separating non-E-rosette forming cells. The first method is less expensive and simple, but very labor-intensive.
Although the second method is sensitive, rapid and easier than counting, it requires the use of hazardous radiochemicals. In this study, using a heterologous system formed by human T-lymphocytes (Jurkat cells) and human epithelial cells (Caco-2 cells) as an adhesion model, we provided evidence that adhesion between Jurkat and Caco-2 cells is modulated by the interaction between CD2 in lymphocytes and CD58 expression on the cell surface of epithelial cells. This model was used in the evaluation of peptides derived from CD2 protein for the modulation of CD2 – CD58 interaction.
2. Materials and methods 2.1. Immunological reagents Human recombinant IFN-g was purchased from Sigma, mouse anti-human CD58 monoclonal antibody (mAb), clone 1C3 (AICD58.6) (IgG2a), and fluorescent-conjugated mAb to human CD58 (FITCanti-CD58) were purchased from Becton Dickinson; murine anti-human CD58mAb, clone TS2/9 (IgG1) was purchased from Ancell; and rabbit anti-human CD2mAb (IgG) was obtained from Immunotech. Stock solution of phorbol ester, 12-myristate-13acetate (PMA) (Sigma) was prepared by dissolving 1 mg in 1.6 ml of dimethyl sulfoxide (DMSO), and frozen at 20 jC. Stock solution of phytohemagglutinin (PHA) (Murex) was prepared by dissolving 2 mg powder in 2 ml of RPMI, and frozen at 20 jC. Bis-carboxyethyl-carboxyfluorescein, acetoxymethyl (BCECF-AM) (Molecular Probes) solution was freshly prepared by dissolving 50 Ag in 50 Al of dimethyl sulfoxide (DMSO), and stored frozen at 20 jC. 2-Aminoethylisothiouonium hydrobromide (AET) solution was prepared by dissolving 0.402 g of AET in 10 ml distilled water and adjusting pH to 9.0. 2.2. Cell lines and culture conditions The human colon adenocarcinoma (Caco-2) cell line and the T-leukemia Jurkat cell line were obtained from the American Type Culture Collection (Rockville, MD). Jurkat cells were maintained in suspension in RPMI 1640 supplemented with 10% heat-inacti-
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vated fetal bovine serum (FBS), 2 mM L-glutamine and 100 mg/l of penicillin/streptomycin. Caco-2 cells were maintained in minimum essential medium-a (MEM-a) containing 10% FBS, 1% non-essential amino acids, 1 mM sodium pyruvate, 1% L-glutamine and 100 mg/l of penicillin/streptomycin. Caco-2 cells were used between passages 50 and 60. 2.3. Peptides Control peptide KGKTDAISVKAI (lKI) was generated by random arrangement of the sequence from the ‘‘hotspot’’ region of human CD2 protein (Kim et al., 2001) and prepared by solid-phase synthesis using Fmoc strategy with PAL resin as solid support. After cleavage by reacting with TFA/thioanisole/ EDT (90:5:5) and precipitating in cold ether, the peptide was purified by preparative HPLC (Waters 600 HPLC system), on reverse-phase C18 column ˚ ) with a linear (Inertsil, 10 250 mm, 5 Am, 300 A gradient of solvent A (0.1% TFA/H2O) and solvent B (0.1% TFA/acetonitrile). The purity was verified by an analytical HPLC (Shimadzu LC-10AT VP) using a reverse-phase C18 column (Lichrosorb RP18, 4.6 200 mm, 10 Am) with the same solvent system as in the preparative HPLC. The HPLC chromatogram showed that the purity was more than 95% and electro-spray ionization mass spectrometry (ESI-MS, Finnigon MAT) showed the correct molecular ion for the peptide. The peptides cyclo(1,12) PenERGSTLVAEFC (cER); cyclo(1,6) ERGSTL (cEL) were designed based on the structure of rat CD2 and purchased from Multiple Peptide Systems (San Diego, CA). 2.4. CD58 detection and ELISA assay Caco-2 cells were seeded at a density of 104 cells/well in a 96-well tissue culture plate and incubated at 37 jC with 5% CO2 for about 48 h or until almost confluent. The monolayers were then washed with fresh medium, before adding IFNg (250, 1000 and 1500 U/ml) to appropriate wells, and incubating at 37 jC with 5% CO2 for indicated periods. For detection of CD58 expression, cells were rinsed with 0.3% BSA/PBS and fixed with 3% paraformaldehyde/PBS for 15 min at room temperature.
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Cells were then washed and blocked for 20 min with 0.05 M Tris – HCl (pH 7.4), and incubated with primary antibody CD58 (1:500) for 2 h. The cells were washed three times with BSA/PBS before incubating with anti-mouse Ig – horseradish peroxidase (HRP) conjugate (1:1000). Following incubation for 2 h, the cells were washed five times, and incubated with substrate solution (ABTS) for 30 min at room temperature. Optical density readings were taken at 430 nm. 2.5. CD2 induction and flow cytometry assay Jurkat cells were plated at a density of 2 106 cells/well in a 96-well tissue culture plate. For activation, various concentrations of PMA, PHA, or combination of PMA and PHA were added to appropriate well and incubated at 37 jC with 5% CO2 for indicated periods. To detect CD2 expression, 106 cells were washed with 0.5% BSA/PBS, and incubated with FITC-conjugated CD2 mAb for 1 h at 37 jC. After washing three times with 0.5% BSA/10 mM HEPES/PBS, the cells were fixed using 1% paraformaldehyde/PBS and analyzed with a flow cytometer (FACScan apparatus, Becton Dickinson) equipped with the Cell Quest software program. During acquisition, 10,000 cells were counted for every sample. 2.6. Fluorochrome-labeling procedure The Jurkat cells were propagated in medium containing 10 ng/ml of PMA for 48 h. The cells were then washed with PBS containing 0.5% BSA twice and adjusted to 3 106 cells/ml. Viability was greater than 95% as determined by trypan blue exclusion. BCECF – AM solution was then added to the Jurkat cell suspension to achieve the desired final concentration of BCECF – AM. The mixture was then incubated at 37 jC for various times. Labeled cells were washed twice and resuspended with RPMI containing 1% FBS at 4 jC for the adhesion assay. 2.7. Cell – cell adhesion assay Caco-2 cells were seeded onto 96-well culture dishes at approximately 104 cells/well and incubated
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at 37 jC with 5% CO2 for about 72 h (or until complete confluence was observed). After a gentle wash with MEM-a/FBS, serial dilutions of stained Jurkat cells from 4 106 to 0.5 106 cells/ml were added into each well in 100 Al aliquots. The plate was incubated at 37 jC with 5% CO2 for appropriate periods. Non-adherent Jurkat cells were removed by washing thrice with PBS, while the monolayer-associated Jurkat cells were lysed with 0.1 ml of 2% Triton X-100 in 0.1 M NaOH. Fluorescence (FL) was quantified using a Spectra Fluo fluorescent plate reader (Tecan). The excitation filter was a 20-nm bandwidth filter centered at 485 nm, while the emission filter was a 25-nm bandwidth filter centered at 535 nm, which corresponds to well with BCECF’s excitation maximum of 500 nm and emission maximum of 535 nm. 2.8. Inhibition of lymphocyte – epithelial adhesion by antibody or peptides based on CD2 protein Monolayers of Caco-2 were prepared and serial dilutions of antibody or peptides in RPMI/FBS were added. After incubation at 37 jC with 5% CO2 for 30 min, 2.5 105 fluorescent-labeled Jurkat cells were Inhibition ð%Þ ¼
added to each well. The plate was incubated at 37 jC for an additional 30 min. The remainder of the assay was completed as described above. Data are presented as relative fluorescence or percent inhibition. Relative fluorescence (FL) was found by reading the values of fluorescence intensity corrected for the reading of background (cell monolayers only): Inhibition ð%Þ 1 FLAdherent with peptide or mAb treatment 100 ¼ FLAdherent without peptide or mAb treatment 2.9. E-rosetting assay of inhibitory peptides Pretreated sheep red blood cells (SRBCs) with AET were incubated with peptides at various concentrations at 37 jC for 30 min, and Jurkat cells (1 105 cells) were then added to preincubated SRBCs for another 15 min. The cells were pelleted by centrifugation (200 g, 5 min, 4 jC) and incubated at 4 jC for 1 h. The cell pellet was gently resuspended and the E-rosettes were counted in a hemocytometer. Rosettes containing less than five SRBCs were considered as non-E-rosette forming cells (non-E-RFC).
non E RFC %treated with peptide non E RFC %blank 100 E RFC %blank
3. Results 3.1. Constitutive expression of CD58 on Caco-2 cells First, CD58 expression on the surface of Caco-2 cells was examined. Flow cytometry assay is a very sensitive and simple technique to study surface antigens on single cells. To avoid damage to surface proteins by trypsin, Caco-2 monolayers were detached by treating with 10 mM EDTA/2% HEPES/ PBS at 37 jC for 30 min. However, it was found that the efficacy was low since the cells were severely damaged when attempting to resuspend into single cells. The application of ELISA to study membrane CD58 expression indicated that CD58 was constitutively expressed on the surface of Caco-2 monolayers, a finding confirmed by confocal microscopy.
The effect of IFN-g treatment on the regulation of CD58 expression was investigated. On day 1, there was no difference in CD58 expression among cells incubated in medium alone compared with those treated with different doses of IFN-g. However, CD58 expression decreased by 20% on day 2; and diminished further on day 3. Cells treated with higher doses of IFN-g exhibited greater decrease in CD58 expression compared with untreated controls (Fig. 1). Since CD58 expression on Caco-2 cells was not upregulated by IFN-g, confluent Caco-2 monolayers cultivated in medium were selected for further investigations. 3.2. Cytokine regulation of CD2 expression on Jurkat cells Flow cytometric analysis revealed that more than 90% of Jurkat cells (both resting and activated)
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Fig. 1. CD58 surface-expression of Caco-2 cells and regulation by IFN-g treatment assayed by ELISA. On day 1, there was no difference in CD58 expression among cells incubated in medium alone compared with those treated with different doses of IFN-g. However, CD58 expression decreased by 20% on day 2 and diminished further on day 3. Cells treated with higher doses of IFN-g exhibited greater decrease in CD58 expression compared with untreated controls.
expressed the cell-surface marker CD2 (Fig. 2), an observation confirmed by confocal microscopy. To investigate whether the surface-expression of CD2 could be enhanced, Jurkat cells were treated with three different inducers. We found no evidence of enhancement following: (1) exposure to PHA (at 1, 2 Ag/ml) for 1 –3 days; (2) exposure to PMA (at 1 – 5, 20– 100 ng/ ml) for 1– 3 days; or (3) exposure to a combination of PHA (at 1, 2 Ag/ml) and PMA (at 1, 2 ng/ml) for 1 – 3 days (data not shown). High doses of PMA (50 – 100 ng/ml) even reduced CD2 expression. Only activation
by a low dose of PMA (of 10 ng/ml) for 48 h enhanced the expression of CD2 on Jurkat cells marginally (Fig. 2). Furthermore, CD2 expression levels decreased greatly following all the treatments on day 3. Finally, PMA (at 10 ng/ml for 48 h) was selected to activate Jurkat cells prior to the adhesion assay. 3.3. Determination of lymphocyte labeling The optimal conditions for labeling of Jurkat cells were determined using single-cell suspensions. The
Fig. 2. Regulation of CD2 expression on Jurkat cells by PHA, PMA and a combination of PHA and PMA. Jurkat cells (2 106/ml) were cultivated for 48 h in the presence PMA (10 ng/ml). The cells were stained with FITC-labeled anti-CD2. Histograms representing CD2-stained cells treated with PMA (3) were compared to those representing CD2-labeled cells incubated in culture medium alone (2) and unspecific labeling (1). Results are shown as cell number ( y-axis) versus log of fluorescence intensity (x-axis).
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Fig. 3. Fluorescent labeling of Jurkat cells is dependent on BCECF – AM concentrations. Standard curve showing the fluorescent intensity of Jurkat cells labeled with increasing concentrations of BCECF – AM (0.5 – 8 AM for 3 106/ml). The data points are the means of 10 determinations. All measurements were performed at excitation/emission of 485/530 nm.
fluorescent signals of Jurkat cells (3 106/ml) labeled with a wide range of fluorochrome concentrations were plotted (Fig. 3). The standard curve indicated that a concentration of 4 AM BCECF – AM was close to the saturation level (r2 = 0.9993 in the range of 0.5– 4 AM). However, confocal microscopic observations revealed that Jurkat cells labeled with 2 AM BCECF – AM already exhibited a bright and uniform cytoplasmic staining pattern. No fluorescence was detected in the medium after plating, and less than 6% of BCECF was released under adhesion assay conditions (data not shown). Thus, a concentration of 2 AM BCECF – AM was applied in subsequent experiments. The kinetics of BCECF loading was analyzed by flow cytometry. The uptake of the probe was rapid enough to be completed by 45 min. Moreover, incubation with 2 AM BCECF at 37 jC for 45 min did not affect the viability of Jurkat cells as determined by
trypan blue staining (data not shown). Consequently, to control for cell damage due to high concentrations of BCECF, incubation with 2 AM BCECF for 45 min was selected as the standard labeling condition for Jurkat cells. 3.4. Determination of optimal conditions for the cell – cell adhesion assay The optimal conditions for the Jurkat adhesion assay were determined by incubating Jurkat cells in wells coated with confluent Caco-2 monolayers for 60 min. Initially, the linearity of the adhesion assay was determined. Fig. 4 shows the correlation between Jurkat cell number (0.5 105 – 4 105 cells/well) and fluorescence signal in a 45-min adhesion assay. The fluorescence arising from adherent Jurkat cells revealed that 2 105 Jurkat cells/well was close to the
Fig. 4. Linearity of Jurkat cell adhesion assay. Varying numbers of BCECF-labeled Jurkat cells in a standard volume were pipetted into wells. Means and S.D. of eight separate measurements are plotted.
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saturation level, and exhibited a good linear relationship (r2 = 0.9972, in the range of 0.5 105 –2 105 cells/well) with the original cell concentration in the wells. Thus, a standard cell concentration of 2.5 105 cells/well was chosen for further experiments. To ascertain the time required for Jurkat cells to attach to Caco-2 cells, BCECF-loaded Jurkat cells were added to confluent Caco-2 monolayers, and were allowed to adhere for various time periods at 37 jC. No significant differences in the fluorescence arising from adherent Jurkat cells were observed among cells incubated for 15, 30, 45 and 60 min (data not shown). Thus, the kinetics of Jurkat cell adhesion to Caco-2 monolayers was rapid enough to be completed by 15 min. Since BCECF has higher emission efficiency in alkaline solution, the attached Jurkat cells were lysed in NaOH solution. To test the stability of the fluorescence signal after lysis, plates loaded with BCECFlabeled Jurkat cells and lysed in 2% Triton X-100/0.1 M NaOH were first assayed, and then kept overnight at room temperature before reanalysis. There was no
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detectable loss of fluorescence after the incubation (data not shown). 3.5. Application of lymphocyte – epithelial adhesion assay Since Jurkat and Caco-2 cells express CD2 and CD58 protein, respectively, the lymphocyte – epithelial adhesion assay was applied to assess the inhibitory effect of peptides designed based on the CD2 protein against CD2 – CD58 interaction. For example, cER is a cyclic 12-mer peptide derived from the CD2 h-turn region. To validate the function of the residues in the h-turn region, a cyclic hexapeptide cEL with h-turn was constructed and compared with the parent peptide cER. The lymphocyte – epithelial adhesion assay revealed that there was no significant difference between cER and cEL at concentrations as low as 10 AM (20 F 11% and 24 F 4%, respectively) (Fig. 5A). However, when the concentration was increased to 25 AM, cEL showed 39 F 3% inhibition of lym-
Fig. 5. (A) Dose – response curves for the inhibitory activity of peptides cER, cEL and negative control peptide by the lymphocyte – epithelial adhesion. (B) Dose – response curves for the inhibitory activity of peptides cER cEL and negative control peptide by the E-rosetting assay.
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phocyte – epithelial adhesion, while cER showed 24 F 9% inhibition. The fact that cEL had greater inhibitory activity than cER was clearly demonstrated at a concentration of 90 AM (45 F 3% and 31 F 5%, respectively). Compared with the above results, the Erosetting assay exhibited less sensitivity (Fig. 5B). There was no significant difference in inhibition at concentrations of less than 200 AM. CD and NMR studies indicated that cEL exhibited a stable h-turn structure, while cER formed a mixed conformation (h-turn and random structure) in solution. This may suggest that the residues in h-turn regions of CD2 are important for its interaction with CD58. Moreover, the h-turn conformation helps the peptide to mimic the CD2 protein. In addition, negative control peptide exhibited very low inhibitory activity; while the adhesion was markedly reduced (51% at 10 Ag/ml and 90% at 25 Ag/ml) when Caco-2 monolayers were preincubated with a monoclonal antibody, TS2/9, directed against the CD2-binding site in CD58. These data indicated the specificity of this assay.
4. Discussion To maximally induce T-cell proliferation and cytokine secretion, an APC must provide two independent intercellular signals. For CD4+ T cells, signal 1 is provided via the interaction between the HLA class II peptide complex (on the APC) and the T-cell receptor. Signal 2 is delivered via a so-called ‘‘costimulatory’’ molecule. The best-characterized costimulatory molecules are CD80 (B7-1) and CD86 (B7-2) (Lenschow et al., 1996). One attractive alternative to CD80 or CD86 as a costimulatory molecule is CD58. Like other costimulatory molecules, CD58 complements HLA-peptide-mediated signals to fully induce T-cell proliferation and cytokine production via interaction with the T-cell surface molecule CD2. In contrast to the restricted expression of CD80 and CD86, CD58 expression has been detected not only on various lymphoid populations, but also on several non-lymphoid cell types in the eye (Iwata et al., 1997; Liversidge et al., 1996), lung (Cunningham and Kirby, 1995; Bloemen et al., 1993), skin (Singer et al., 1990), and blood vessels (Wesphal et al., 1993; Hughes et al., 1990).
As indicated earlier, modulation of CD2 – CD58 interaction has very important therapeutic applications. Previous studies on the modulation of CD2 – CD58 interactions by antibodies report the inhibition of CD2 – CD58 by E-rosetting assay. Recently, however, a new method for directly measuring heterotypic adhesion has been developed. Lymphocyte –epithelial cell adhesion has been applied to study the interaction between adhesion molecules, such as LFA-1/ICAM-1 (Yusuf-Makagiansar et al., 2001) and CD26 – adenosine deaminase (ADA) (Gines et al., 2002). In this study, we investigated the existence of the CD2– CD58 module at the cell surface via human lymphocyte –epithelial cell adhesion and developed a model for screening of inhibitory compounds. Jurkat cells express CD2 on their surface. For epithelial cells, Caco-2 cells were selected. The polarized intestinal epithelial cells (IECs) that line the gastrointestinal tract are exposed to a high concentration of foreign antigens (e.g., food and bacterial antigens) at their apical (luminal) surface. In addition, they are in direct contact with several distinct subsets of T-lymphocytes at their basolateral surface, both within the epithelium and the underlying lamina propria. In this context, several groups have suggested that IECs function as APCs and modulate their proliferation, cytolytic activity and/or cytokine production (Bland and Warren, 1986; Mayer and Shlien, 1987; Kaiserlian et al., 1989; Hershberg et al., 1997). Typically, APCs are bone marrow-derived cells such as dendritic cells, monocytes, or B-lymphocytes (the so-called ‘‘professional’’ APCs) whose role in immuno-modulation is well established. Certain other cell types including IECs (Bland and Warren, 1986; Mayer and Shlien, 1987; Kaiserlian et al., 1989; Hershberg et al., 1997), renal tubular epithelial cells (Kelly and Singer, 1993), keratinocytes (Nickoloff and Turka, 1994), and endothelial cells (Savag et al., 1995) have been suggested to function in a limited context as the so-called ‘‘nonprofessional’’ APCs. The lack of ‘‘professional’’ stature reflects both the relative inefficiency of antigen processing and presentation compared with their professional counterparts, and the fact that these cells serve additional physiological (‘‘professional’’) functions independent of their role in T-lymphocyte regulation. Thus, Caco-2 cells with CD58 expression may serve as good model for T-cell – epithelial cell adhesion assay.
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To establish the characteristics of adhesion between Jurkat and Caco-2 cells, CD2 and CD58 expression on the two cell lines were determined, respectively. Using two independent techniques, i.e., confocal microscopy and ELISA, we demonstrated constitutive expression of a functional T-cell costimulatory molecule, CD58, on the surface of the human Caco-2 cell line in the absence of cytokines or other stimulation. Adhesion molecules such as ICAM-1 and CD58 may be upregulated by treatment of cytokines. Most studies on CD58 used IFN-g, while some studies used TNF-a to enhance CD58 expression on endothelial cells (Omari and Dorovini-Zis, 1999). However, it was generally found that no significant enhancement of CD58 expression was observed by incubation of cells with IFN-g or TNF-a. The effects of varying IFN-g concentrations (250, 1000 and 1500 U/ml) on CD58 expression on Caco-2 cells cultivated for 3 days was investigated. In similar studies with endothelial cells, IFN-g could not enhance and even down-regulated CD58 expression. CD2 is a surface marker of T-cells, and is reported to be present on more than 80% of T-cells. In our experiments, FACS revealed that almost 100% of Jurkat cells expressed CD2. To enhance CD2 expression, Jurkat cells were activated with PHA and PMA. Mitogenic lectin PHA can deliver a signal via the TcR/CD3 complex (Wiskocil et al., 1984), thus generating the first signal necessary for T-cell activation. The phorbol ester PMA can directly activate the second messenger pathway by mimicking diacylglycerol, a natural ligand and activator of PKCs (Robb et al., 1981). However, neither PHA nor a combination of low doses of PHA and PMA could augment CD2 expression on Jurkat cells. There was only marginal enhancement after treatment with 10 ng/ml of PMA for 48 h. Since surface proteins cluster at the surface after activation and PMA rapidly up-regulates CD2 avidity (William et al., 1993), PMA (at 10 ng/ml for 48 h) was selected to activate Jurkat cells prior to the adhesion assay. After confirming CD2 and CD58 expression on the surface of Jurkat and Caco-2 cells, respectively, a rapid and sensitive method for the measurement of lymphocyte – epithelial adhesion, i.e., quantitation of CD2 –CD58 interaction was developed. The advent of the fluorescent plate reader combines the sensitivity inherent with fluorochromes and the speed of spec-
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trophotometric plate readers that allows the screening of large numbers of samples. Lymphocytes can be conveniently labeled with non-polar BCECF – AM before they are used in the attachment assay. The AM derivatives of the non-fluorescent indicator BCECF rapidly permeate the cell membrane and are readily cleaved by high activity and large capacity cytoplasmic esterase into fluorescent free-acid form. Thus, BCECF is widely used in cell adhesion and cell viability assays. A major advantage of using acetoxymethyl esters (AM) of BCECF is that only living cells are stained with this dye. In addition, most fluorometers and fluorescence microscopes are equipped with a filter set for FITC, which can be used for detecting this dye. Although calcein –AM is currently more popular for assaying cell adhesion, BCECF –AM is relatively less expensive and can be loaded into cells at low concentration. Moreover, by taking advantage of the pH sensitivity of BCECF, lower concentration of the dye could be used and still obtain a higher fluorescence intensity compared to cells loaded with calcein– AM. A potential problem with BCECF-loaded cells is dye leakage from cells (William et al., 1993). However, the epithelial cells investigated in our assay are human colonic adenocarcinoma cells, which stably express the human multidrug transporter, P-glycoprotein, in the plasma membrane. P-glycoprotein can actively extrude the hydrophobic AM form of the fluorescent indicator BCECF (Rink et al., 1982; Homoloya et al., 1993). Moreover, since the hydrophilic free-acid form of BCECF is not membranepermanent, Caco-2 cells will not be fluorescentlabeled. In addition, BCECF release was found to be less than 3% after plating monolayers for 60 min. Therefore, the likelihood of BCECF leakage may be excluded under our adhesion assay conditions. In initial studies, certain experimental parameters that influence labeling were determined. A standard loading concentration of 2 AM BCECF –AM was adopted as this provided sufficient labeling, but was low enough to avoid unnecessary washing steps and accumulation of large amounts of the potentially toxic hydrolysis product, formaldehyde, in lymphocytes (Hollo et al., 1994). The accumulation of BCECF was rapid, and the spontaneous release of BCECF from labeled cells was low over the test period.
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Jurkat cell adhesion was linear in the range of 0.5 105 –2.5 105 cells/well. The saturated density of 2.5 105 Jurkat cells/well was selected for the competitive adhesion assay. Although it is noteworthy that maximum adhesion of Jurkat cells to Caco-2 monolayers occurred within 15 min, an incubation time of 30 min was adopted for the peptide/Jurkat cell competitive adhesion assay to ensure sufficient exchange time and adhesion. Finally, this assay was applied to screen peptides with inhibitory activities against CD2 – CD58 interaction. Although another major known mechanism of T-cell adhesion is mediated by LFA-1 –ICAM-1 interaction and ICAM-1 has been reported to be expressed on Caco-2 cells, the results that the anti-human CD58, TS2/9, directed against the CD2-binding site in CD58, markedly reduced the Jurkat – Caco-2 adhesion and negative control peptide from CD2 protein exhibited very low inhibitory activity suggest that cell-surface CD58 from Caco-2 cells, via binding to CD2 on Jurkat cells, regulated lymphocyte– epithelial cell adhesion. Moreover, this assay is more rapid and sensitive, and the results are consistent with those obtained by the E-rosetting assay.
Acknowledgements We would like to thank Miss Jessie Lim for technical assistance. Funding for this research was provided by Academic Research grant (R-148-000-026-112), National University of Singapore, Singapore.
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J. Liu et al. / Journal of Immunological Methods 291 (2004) 39–49 Kim, M., Sun, Z.Y., Byron, O., Campbell, G., Wagner, G., Wang, J., Reinherz, E.L., 2001. Molecular dissection of the CD2 – CD58 counter-receptor interface identifies CD2 Tyr86 and CD58 Lys34 residues as the functional ‘‘hot spot’’. J. Mol. Biol. 314, 331. Lenschow, D.J., Walunas, T.L., Bluestone, J.A., 1996. CD28/B7 system of T cell costimulation. Annu. Rev. Immunol. 14, 233. Liversidge, J., Dawson, R., Hoey, S., Mckay, D., Grabowski, P., Forrester, J.V., 1996. CD59 and CD48 expressed by rat retinal pigment epithelial cells are major ligands for CD2-mediated alternative pathway of T cell activation. J. Immunol. 156, 3696. Mayer, L., Shlien, R., 1987. Evidence for function of Ia molecules on gut epithelial cells in man. J. Exp. Med. 166, 1471. Moigneon, P.H., Chang, B.P., Wallner, C., Stebbins, A.Z., Reinherz, E.L., 1989. CD2-mediated adhesion facilitates T lymphocyte antigen recognition function. Nature (Lond.) 339, 312. Mojcik, C.F., Shevach, E., 1997. Adhesion molecules: a rheumatologic perspective. Arthritis Rheum. 6, 991. Nickoloff, B.J., Turka, L.A., 1994. Immunological functions of non-professional antigen-presenting cells: new insight from studies of T-cell interactions with keratinocytes. Immunol. Today 15, 464. Omari, K.I., Dorovini-Zis, K., 1999. Expression and function of lymphocyte function associated antigen-3 (LFA-3) at the blood – brain barrier. Cell. Mol. Biol. 45, 25. Przepiorka, D., Phillips, G.L., Ratanatharathorn, V., Michele, C.F., Sehn, L.H., Antin, J.H., Lebherz, D., Awwad, M., Hope, J., McClain, J.B., 1998. A phase II study of BTI-322, a monoclonal anti-CD2 antibody, for treatment of steroid-resistant acute graftversus-host disease. Blood 92, 4066. Qin, L.H., Chavin, K.D., Lin, J.X., Yagita, H., Bromberg, J.S., 1994. Anti-CD2 receptor and anti-CD2 ligand (CD48) antibodies synergize to prolong allograft survival. J. Exp. Med. 179, 341. Rink, T.J., Tsien, R.Y., Pozzan, T., 1982. Cytoplasmic pH and free Mg2 + in lymphocytes. J. Cell. Biol. 95, 189. Robb, R.J., Munck, A., Smith, K.A., 1981. T cell growth factor receptors. Quantitation, specificity, and biological relevance. J. Exp. Med. 154, 1455.
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Savag, C.O., Brooks, C.J., Harcourt, G.C., Picard, J.K., King, W., Sansom, D.M., Willcox, N., 1995. Human vascular endothelial cells process and present autoantigen to human T cell lines. Int. Immunol. 7, 471. Sido, B., Otto, G., Zimmermann, R., Mu¨ller, P., Meuer, S., Dengler, T.J., 1996. Prolonged allograft survival by the inhibition of costimulatory CD2 signals but not by modulation CD48 (CD2 ligand) in the rat. Transplant. Int. 9, S323. Sido, B., Otto, G., Zimmermann, R., Mu¨ller, P., Meuer, S., Dengler, T.J., 1997. Modulation of the CD2 receptor and not disruption of the CD2/CD48 interaction is the principal action of CD2-mediated immunosuppression in the rat. Cell. Immunol. 182, 57. Singer, K.H., Le, P.T., Denning, S.M., Whichard, L.P., Haynes, B.F., 1990. The role of adhesion molecules in epithelial – T-cell interactions in thymus and skin. J. Invest. Dermatol. 94, 85S. Sultan, P., Schechner, J.S., McNiff, J.M., Hochman, P.S., Hughes, C.C.W., Lorber, M.I., Askenase, P.W., Pober, J.S., 1997. Blockade of CD2 – LFA-3 interactions protects human skin allografts in immunodeficient mouse/human chimeras. Nat. Biotechnol. 15, 759. Wesphal, J.R., Willems, H.W., Tax, W.J., Koene, R.A., Ruiteer, D.J., de Waal, R.M., 1993. The proliferative response of human T cells to allogeneic IFN-gamma-treated endothelial cells is mediated via both CD2/LFA-3 and LFA-1/ICAM-1 and -2 adhesion pathways. Transplant. Immunol. 1, 183. William, C.H., Steven, J.B., Barbara, E.B., 1993. Signal transduction pathways involved in T cell receptor-induced regulation of CD2 avidity for CD58. J. Immunol. 150, 2607. Wiskocil, R., Weiss, A., Imboden, J., Kamin-Lewis, R., Stobo, J., 1984. Activation of a human T cell line: a two-stimulus requirement in the pretranslational events involved in the coordinate expression of interleukin 2 and gamma-interferon genes. J. Immunol. 134, 1599. Yusuf-Makagiansar, H., Makagiansar, I.T., Siahaan T.J., 2001. Inhibition of the adherence of T-lymphocytes to epithelial cells by a cyclic peptide derived from inserted domain of lymphocyte function-associated antigen-1. Inflammation 25, 203.
Research paper
A novel, rapid and sensitive heterotypic cell adhesion assay for CD2–CD58 interaction, and its application for testing inhibitory peptides Jining Liu a, Vincent T.K. Chow b, Seetharama D.S. Jois a,* b
a Department of Pharmacy, 18 Science Drive 4, National University of Singapore, Singapore 117543, Singapore Human Genome Laboratory, Department of Microbiology, 18 Science Drive 4, National University of Singapore, Singapore 117543, Singapore
Received 8 January 2004; accepted 24 April 2004 Available online 15 June 2004
Abstract The immunoglobulin CD2 is a cell adhesion molecule that mediates T-cell activation by binding to its receptor CD58 on antigen-presenting cells (APCs). Modulation or inhibition of this interaction has been shown to be therapeutically useful. Erosetting assay is usually applied in the study of the modulation of CD2 – CD58 interaction. In this study, we demonstrated a novel, rapid and sensitive heterotypic cell adhesion assay for CD2 – CD58 interaction. The CD2 expression on the surface of Jurkat cells and the CD58 expression on the Caco-2 cells were confirmed by flow cytometry and ELISA studies, respectively. Then Jurkat cells were fluorescent-labeled with 2 AM of BCECF-AM for 45 min at 37 jC before adding to confluent Caco-2 monolayers cultured in 96-well culture dishes. After 30 min, non-adherent Jurkat cells were removed by washing with PBS, while the monolayer-associated Jurkat cells were lysed with 0.5 ml of 2% Triton X-100 in 0.1 M NaOH. Fluorescence (FL) was quantitated using a microplate fluorescence analyzer with BCECF’s excitation maximum of 485 nm and emission maximum of 535 nm. This method was successfully applied for testing inhibitory peptides to CD2 – CD58 interaction. D 2004 Elsevier B.V. All rights reserved. Keywords: CD2; CD58; Jurkat cells; Caco-2 cells; Lymphocyte – epithelial adhesion assay; E-rosetting assay
1. Introduction Cell-surface proteins mediate intercellular recognition and adhesion. In the immune response, a host of proteins is involved in the recognition of antigen-
* Corresponding author. Tel.: +65-6-874-2653; fax: +65-6-7791554. E-mail address: [email protected] (S.D.S. Jois). 0022-1759/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2004.04.026
presenting cells (APCs) by T-cells. These include the T-lymphocyte adhesion receptor CD2 and its ligands, CD48 in rodents and CD58 in humans (Davis et al., 1998). CD2, a 50- to 55-kDa glycoprotein, is expressed on T-cells and natural killer (NK) cells and distinguishes them from other leukocytes (Aiuti et al., 1975; Dustin et al., 1987). CD58, a 55- to 70kDa glycoprotein, is expressed on both hematopoietic and non-hematopoietic cells (Arulanandam et al., 1993). The interaction between CD2 on T-cells and
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CD58 on APCs is believed to augment the adhesion between T-cells and APCs (Davis et al., 1998; Moigneon et al., 1989). In particular, this heterotypic cell adhesion facilitates initial cell – cell contact before specific antigen recognition and also enhances T-cell receptor (TcR) triggering by fostering interaction with peptide-class II MHC complex (pMHC) (Kim et al., 2001). Although the affinity of CD2 – CD58 interaction is relatively low (Kd f 1 AM), the very rapid koff and kon supports dynamic binding with rapid counterreceptor exchange, and creates an optimal intercellular ˚ ) on opposing cell membrane distance ( f 135 A surfaces suitable for TcR – pMHC or NK receptor – MHC interactions to foster immune recognition. Hence, in the presence of hCD2 – hCD58 interaction, T-cells recognize the correct pMHC with a 50- to 100fold greater efficiency than in the absence of hCD2 – hCD58 interaction. In addition, endothelial cells (EC) in rheumatoid arthritis (RA) have been shown to express elevated levels of CD58, and RA lymphocytes in synovial fluid (SF) express increased levels of CD2 and CD58 relative to RA or normal peripheral blood lymphocytes (Bachmann et al., 1999; Mojcik and Shevach, 1997). These findings led to postulation that the modulation of T-cell responses in vivo after blockade of the interaction between CD58 and CD2 can potentially be applied in the treatment of autoimmune diseases and transplantation. It has been shown that blockade of the CD2 – CD58 interaction (Kaplon et al., 1995; Sultan et al., 1997) and/or modulation of the CD2 costimulatory pathway (Qin et al., 1994; Hirahara et al., 1995; Sido et al., 1996, 1997) can result in prolonged tolerance towards allografts. The soluble CD58 – Ig fusion protein Amevive (LFA3TIP) has been employed in the treatment of psoriasis (Aruffo and Hollenbaugh, 2001). The humanized versions of antibodies BTI-322 (Przepiorka et al., 1998) and MEDI-507 (Branco et al., 1999) have been tested for the treatment of acute organ rejection and graftversus-host disease. In previous CD2/CD58 studies, the modulation of CD2 – CD58 interaction was mainly investigated by E-rosetting, either by counting bound cells visually under a microscope, or radiolabeling of cells with 125I and measuring the residual radioactivity after separating non-E-rosette forming cells. The first method is less expensive and simple, but very labor-intensive.
Although the second method is sensitive, rapid and easier than counting, it requires the use of hazardous radiochemicals. In this study, using a heterologous system formed by human T-lymphocytes (Jurkat cells) and human epithelial cells (Caco-2 cells) as an adhesion model, we provided evidence that adhesion between Jurkat and Caco-2 cells is modulated by the interaction between CD2 in lymphocytes and CD58 expression on the cell surface of epithelial cells. This model was used in the evaluation of peptides derived from CD2 protein for the modulation of CD2 – CD58 interaction.
2. Materials and methods 2.1. Immunological reagents Human recombinant IFN-g was purchased from Sigma, mouse anti-human CD58 monoclonal antibody (mAb), clone 1C3 (AICD58.6) (IgG2a), and fluorescent-conjugated mAb to human CD58 (FITCanti-CD58) were purchased from Becton Dickinson; murine anti-human CD58mAb, clone TS2/9 (IgG1) was purchased from Ancell; and rabbit anti-human CD2mAb (IgG) was obtained from Immunotech. Stock solution of phorbol ester, 12-myristate-13acetate (PMA) (Sigma) was prepared by dissolving 1 mg in 1.6 ml of dimethyl sulfoxide (DMSO), and frozen at 20 jC. Stock solution of phytohemagglutinin (PHA) (Murex) was prepared by dissolving 2 mg powder in 2 ml of RPMI, and frozen at 20 jC. Bis-carboxyethyl-carboxyfluorescein, acetoxymethyl (BCECF-AM) (Molecular Probes) solution was freshly prepared by dissolving 50 Ag in 50 Al of dimethyl sulfoxide (DMSO), and stored frozen at 20 jC. 2-Aminoethylisothiouonium hydrobromide (AET) solution was prepared by dissolving 0.402 g of AET in 10 ml distilled water and adjusting pH to 9.0. 2.2. Cell lines and culture conditions The human colon adenocarcinoma (Caco-2) cell line and the T-leukemia Jurkat cell line were obtained from the American Type Culture Collection (Rockville, MD). Jurkat cells were maintained in suspension in RPMI 1640 supplemented with 10% heat-inacti-
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vated fetal bovine serum (FBS), 2 mM L-glutamine and 100 mg/l of penicillin/streptomycin. Caco-2 cells were maintained in minimum essential medium-a (MEM-a) containing 10% FBS, 1% non-essential amino acids, 1 mM sodium pyruvate, 1% L-glutamine and 100 mg/l of penicillin/streptomycin. Caco-2 cells were used between passages 50 and 60. 2.3. Peptides Control peptide KGKTDAISVKAI (lKI) was generated by random arrangement of the sequence from the ‘‘hotspot’’ region of human CD2 protein (Kim et al., 2001) and prepared by solid-phase synthesis using Fmoc strategy with PAL resin as solid support. After cleavage by reacting with TFA/thioanisole/ EDT (90:5:5) and precipitating in cold ether, the peptide was purified by preparative HPLC (Waters 600 HPLC system), on reverse-phase C18 column ˚ ) with a linear (Inertsil, 10 250 mm, 5 Am, 300 A gradient of solvent A (0.1% TFA/H2O) and solvent B (0.1% TFA/acetonitrile). The purity was verified by an analytical HPLC (Shimadzu LC-10AT VP) using a reverse-phase C18 column (Lichrosorb RP18, 4.6 200 mm, 10 Am) with the same solvent system as in the preparative HPLC. The HPLC chromatogram showed that the purity was more than 95% and electro-spray ionization mass spectrometry (ESI-MS, Finnigon MAT) showed the correct molecular ion for the peptide. The peptides cyclo(1,12) PenERGSTLVAEFC (cER); cyclo(1,6) ERGSTL (cEL) were designed based on the structure of rat CD2 and purchased from Multiple Peptide Systems (San Diego, CA). 2.4. CD58 detection and ELISA assay Caco-2 cells were seeded at a density of 104 cells/well in a 96-well tissue culture plate and incubated at 37 jC with 5% CO2 for about 48 h or until almost confluent. The monolayers were then washed with fresh medium, before adding IFNg (250, 1000 and 1500 U/ml) to appropriate wells, and incubating at 37 jC with 5% CO2 for indicated periods. For detection of CD58 expression, cells were rinsed with 0.3% BSA/PBS and fixed with 3% paraformaldehyde/PBS for 15 min at room temperature.
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Cells were then washed and blocked for 20 min with 0.05 M Tris – HCl (pH 7.4), and incubated with primary antibody CD58 (1:500) for 2 h. The cells were washed three times with BSA/PBS before incubating with anti-mouse Ig – horseradish peroxidase (HRP) conjugate (1:1000). Following incubation for 2 h, the cells were washed five times, and incubated with substrate solution (ABTS) for 30 min at room temperature. Optical density readings were taken at 430 nm. 2.5. CD2 induction and flow cytometry assay Jurkat cells were plated at a density of 2 106 cells/well in a 96-well tissue culture plate. For activation, various concentrations of PMA, PHA, or combination of PMA and PHA were added to appropriate well and incubated at 37 jC with 5% CO2 for indicated periods. To detect CD2 expression, 106 cells were washed with 0.5% BSA/PBS, and incubated with FITC-conjugated CD2 mAb for 1 h at 37 jC. After washing three times with 0.5% BSA/10 mM HEPES/PBS, the cells were fixed using 1% paraformaldehyde/PBS and analyzed with a flow cytometer (FACScan apparatus, Becton Dickinson) equipped with the Cell Quest software program. During acquisition, 10,000 cells were counted for every sample. 2.6. Fluorochrome-labeling procedure The Jurkat cells were propagated in medium containing 10 ng/ml of PMA for 48 h. The cells were then washed with PBS containing 0.5% BSA twice and adjusted to 3 106 cells/ml. Viability was greater than 95% as determined by trypan blue exclusion. BCECF – AM solution was then added to the Jurkat cell suspension to achieve the desired final concentration of BCECF – AM. The mixture was then incubated at 37 jC for various times. Labeled cells were washed twice and resuspended with RPMI containing 1% FBS at 4 jC for the adhesion assay. 2.7. Cell – cell adhesion assay Caco-2 cells were seeded onto 96-well culture dishes at approximately 104 cells/well and incubated
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at 37 jC with 5% CO2 for about 72 h (or until complete confluence was observed). After a gentle wash with MEM-a/FBS, serial dilutions of stained Jurkat cells from 4 106 to 0.5 106 cells/ml were added into each well in 100 Al aliquots. The plate was incubated at 37 jC with 5% CO2 for appropriate periods. Non-adherent Jurkat cells were removed by washing thrice with PBS, while the monolayer-associated Jurkat cells were lysed with 0.1 ml of 2% Triton X-100 in 0.1 M NaOH. Fluorescence (FL) was quantified using a Spectra Fluo fluorescent plate reader (Tecan). The excitation filter was a 20-nm bandwidth filter centered at 485 nm, while the emission filter was a 25-nm bandwidth filter centered at 535 nm, which corresponds to well with BCECF’s excitation maximum of 500 nm and emission maximum of 535 nm. 2.8. Inhibition of lymphocyte – epithelial adhesion by antibody or peptides based on CD2 protein Monolayers of Caco-2 were prepared and serial dilutions of antibody or peptides in RPMI/FBS were added. After incubation at 37 jC with 5% CO2 for 30 min, 2.5 105 fluorescent-labeled Jurkat cells were Inhibition ð%Þ ¼
added to each well. The plate was incubated at 37 jC for an additional 30 min. The remainder of the assay was completed as described above. Data are presented as relative fluorescence or percent inhibition. Relative fluorescence (FL) was found by reading the values of fluorescence intensity corrected for the reading of background (cell monolayers only): Inhibition ð%Þ 1 FLAdherent with peptide or mAb treatment 100 ¼ FLAdherent without peptide or mAb treatment 2.9. E-rosetting assay of inhibitory peptides Pretreated sheep red blood cells (SRBCs) with AET were incubated with peptides at various concentrations at 37 jC for 30 min, and Jurkat cells (1 105 cells) were then added to preincubated SRBCs for another 15 min. The cells were pelleted by centrifugation (200 g, 5 min, 4 jC) and incubated at 4 jC for 1 h. The cell pellet was gently resuspended and the E-rosettes were counted in a hemocytometer. Rosettes containing less than five SRBCs were considered as non-E-rosette forming cells (non-E-RFC).
non E RFC %treated with peptide non E RFC %blank 100 E RFC %blank
3. Results 3.1. Constitutive expression of CD58 on Caco-2 cells First, CD58 expression on the surface of Caco-2 cells was examined. Flow cytometry assay is a very sensitive and simple technique to study surface antigens on single cells. To avoid damage to surface proteins by trypsin, Caco-2 monolayers were detached by treating with 10 mM EDTA/2% HEPES/ PBS at 37 jC for 30 min. However, it was found that the efficacy was low since the cells were severely damaged when attempting to resuspend into single cells. The application of ELISA to study membrane CD58 expression indicated that CD58 was constitutively expressed on the surface of Caco-2 monolayers, a finding confirmed by confocal microscopy.
The effect of IFN-g treatment on the regulation of CD58 expression was investigated. On day 1, there was no difference in CD58 expression among cells incubated in medium alone compared with those treated with different doses of IFN-g. However, CD58 expression decreased by 20% on day 2; and diminished further on day 3. Cells treated with higher doses of IFN-g exhibited greater decrease in CD58 expression compared with untreated controls (Fig. 1). Since CD58 expression on Caco-2 cells was not upregulated by IFN-g, confluent Caco-2 monolayers cultivated in medium were selected for further investigations. 3.2. Cytokine regulation of CD2 expression on Jurkat cells Flow cytometric analysis revealed that more than 90% of Jurkat cells (both resting and activated)
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Fig. 1. CD58 surface-expression of Caco-2 cells and regulation by IFN-g treatment assayed by ELISA. On day 1, there was no difference in CD58 expression among cells incubated in medium alone compared with those treated with different doses of IFN-g. However, CD58 expression decreased by 20% on day 2 and diminished further on day 3. Cells treated with higher doses of IFN-g exhibited greater decrease in CD58 expression compared with untreated controls.
expressed the cell-surface marker CD2 (Fig. 2), an observation confirmed by confocal microscopy. To investigate whether the surface-expression of CD2 could be enhanced, Jurkat cells were treated with three different inducers. We found no evidence of enhancement following: (1) exposure to PHA (at 1, 2 Ag/ml) for 1 –3 days; (2) exposure to PMA (at 1 – 5, 20– 100 ng/ ml) for 1– 3 days; or (3) exposure to a combination of PHA (at 1, 2 Ag/ml) and PMA (at 1, 2 ng/ml) for 1 – 3 days (data not shown). High doses of PMA (50 – 100 ng/ml) even reduced CD2 expression. Only activation
by a low dose of PMA (of 10 ng/ml) for 48 h enhanced the expression of CD2 on Jurkat cells marginally (Fig. 2). Furthermore, CD2 expression levels decreased greatly following all the treatments on day 3. Finally, PMA (at 10 ng/ml for 48 h) was selected to activate Jurkat cells prior to the adhesion assay. 3.3. Determination of lymphocyte labeling The optimal conditions for labeling of Jurkat cells were determined using single-cell suspensions. The
Fig. 2. Regulation of CD2 expression on Jurkat cells by PHA, PMA and a combination of PHA and PMA. Jurkat cells (2 106/ml) were cultivated for 48 h in the presence PMA (10 ng/ml). The cells were stained with FITC-labeled anti-CD2. Histograms representing CD2-stained cells treated with PMA (3) were compared to those representing CD2-labeled cells incubated in culture medium alone (2) and unspecific labeling (1). Results are shown as cell number ( y-axis) versus log of fluorescence intensity (x-axis).
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Fig. 3. Fluorescent labeling of Jurkat cells is dependent on BCECF – AM concentrations. Standard curve showing the fluorescent intensity of Jurkat cells labeled with increasing concentrations of BCECF – AM (0.5 – 8 AM for 3 106/ml). The data points are the means of 10 determinations. All measurements were performed at excitation/emission of 485/530 nm.
fluorescent signals of Jurkat cells (3 106/ml) labeled with a wide range of fluorochrome concentrations were plotted (Fig. 3). The standard curve indicated that a concentration of 4 AM BCECF – AM was close to the saturation level (r2 = 0.9993 in the range of 0.5– 4 AM). However, confocal microscopic observations revealed that Jurkat cells labeled with 2 AM BCECF – AM already exhibited a bright and uniform cytoplasmic staining pattern. No fluorescence was detected in the medium after plating, and less than 6% of BCECF was released under adhesion assay conditions (data not shown). Thus, a concentration of 2 AM BCECF – AM was applied in subsequent experiments. The kinetics of BCECF loading was analyzed by flow cytometry. The uptake of the probe was rapid enough to be completed by 45 min. Moreover, incubation with 2 AM BCECF at 37 jC for 45 min did not affect the viability of Jurkat cells as determined by
trypan blue staining (data not shown). Consequently, to control for cell damage due to high concentrations of BCECF, incubation with 2 AM BCECF for 45 min was selected as the standard labeling condition for Jurkat cells. 3.4. Determination of optimal conditions for the cell – cell adhesion assay The optimal conditions for the Jurkat adhesion assay were determined by incubating Jurkat cells in wells coated with confluent Caco-2 monolayers for 60 min. Initially, the linearity of the adhesion assay was determined. Fig. 4 shows the correlation between Jurkat cell number (0.5 105 – 4 105 cells/well) and fluorescence signal in a 45-min adhesion assay. The fluorescence arising from adherent Jurkat cells revealed that 2 105 Jurkat cells/well was close to the
Fig. 4. Linearity of Jurkat cell adhesion assay. Varying numbers of BCECF-labeled Jurkat cells in a standard volume were pipetted into wells. Means and S.D. of eight separate measurements are plotted.
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saturation level, and exhibited a good linear relationship (r2 = 0.9972, in the range of 0.5 105 –2 105 cells/well) with the original cell concentration in the wells. Thus, a standard cell concentration of 2.5 105 cells/well was chosen for further experiments. To ascertain the time required for Jurkat cells to attach to Caco-2 cells, BCECF-loaded Jurkat cells were added to confluent Caco-2 monolayers, and were allowed to adhere for various time periods at 37 jC. No significant differences in the fluorescence arising from adherent Jurkat cells were observed among cells incubated for 15, 30, 45 and 60 min (data not shown). Thus, the kinetics of Jurkat cell adhesion to Caco-2 monolayers was rapid enough to be completed by 15 min. Since BCECF has higher emission efficiency in alkaline solution, the attached Jurkat cells were lysed in NaOH solution. To test the stability of the fluorescence signal after lysis, plates loaded with BCECFlabeled Jurkat cells and lysed in 2% Triton X-100/0.1 M NaOH were first assayed, and then kept overnight at room temperature before reanalysis. There was no
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detectable loss of fluorescence after the incubation (data not shown). 3.5. Application of lymphocyte – epithelial adhesion assay Since Jurkat and Caco-2 cells express CD2 and CD58 protein, respectively, the lymphocyte – epithelial adhesion assay was applied to assess the inhibitory effect of peptides designed based on the CD2 protein against CD2 – CD58 interaction. For example, cER is a cyclic 12-mer peptide derived from the CD2 h-turn region. To validate the function of the residues in the h-turn region, a cyclic hexapeptide cEL with h-turn was constructed and compared with the parent peptide cER. The lymphocyte – epithelial adhesion assay revealed that there was no significant difference between cER and cEL at concentrations as low as 10 AM (20 F 11% and 24 F 4%, respectively) (Fig. 5A). However, when the concentration was increased to 25 AM, cEL showed 39 F 3% inhibition of lym-
Fig. 5. (A) Dose – response curves for the inhibitory activity of peptides cER, cEL and negative control peptide by the lymphocyte – epithelial adhesion. (B) Dose – response curves for the inhibitory activity of peptides cER cEL and negative control peptide by the E-rosetting assay.
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phocyte – epithelial adhesion, while cER showed 24 F 9% inhibition. The fact that cEL had greater inhibitory activity than cER was clearly demonstrated at a concentration of 90 AM (45 F 3% and 31 F 5%, respectively). Compared with the above results, the Erosetting assay exhibited less sensitivity (Fig. 5B). There was no significant difference in inhibition at concentrations of less than 200 AM. CD and NMR studies indicated that cEL exhibited a stable h-turn structure, while cER formed a mixed conformation (h-turn and random structure) in solution. This may suggest that the residues in h-turn regions of CD2 are important for its interaction with CD58. Moreover, the h-turn conformation helps the peptide to mimic the CD2 protein. In addition, negative control peptide exhibited very low inhibitory activity; while the adhesion was markedly reduced (51% at 10 Ag/ml and 90% at 25 Ag/ml) when Caco-2 monolayers were preincubated with a monoclonal antibody, TS2/9, directed against the CD2-binding site in CD58. These data indicated the specificity of this assay.
4. Discussion To maximally induce T-cell proliferation and cytokine secretion, an APC must provide two independent intercellular signals. For CD4+ T cells, signal 1 is provided via the interaction between the HLA class II peptide complex (on the APC) and the T-cell receptor. Signal 2 is delivered via a so-called ‘‘costimulatory’’ molecule. The best-characterized costimulatory molecules are CD80 (B7-1) and CD86 (B7-2) (Lenschow et al., 1996). One attractive alternative to CD80 or CD86 as a costimulatory molecule is CD58. Like other costimulatory molecules, CD58 complements HLA-peptide-mediated signals to fully induce T-cell proliferation and cytokine production via interaction with the T-cell surface molecule CD2. In contrast to the restricted expression of CD80 and CD86, CD58 expression has been detected not only on various lymphoid populations, but also on several non-lymphoid cell types in the eye (Iwata et al., 1997; Liversidge et al., 1996), lung (Cunningham and Kirby, 1995; Bloemen et al., 1993), skin (Singer et al., 1990), and blood vessels (Wesphal et al., 1993; Hughes et al., 1990).
As indicated earlier, modulation of CD2 – CD58 interaction has very important therapeutic applications. Previous studies on the modulation of CD2 – CD58 interactions by antibodies report the inhibition of CD2 – CD58 by E-rosetting assay. Recently, however, a new method for directly measuring heterotypic adhesion has been developed. Lymphocyte –epithelial cell adhesion has been applied to study the interaction between adhesion molecules, such as LFA-1/ICAM-1 (Yusuf-Makagiansar et al., 2001) and CD26 – adenosine deaminase (ADA) (Gines et al., 2002). In this study, we investigated the existence of the CD2– CD58 module at the cell surface via human lymphocyte –epithelial cell adhesion and developed a model for screening of inhibitory compounds. Jurkat cells express CD2 on their surface. For epithelial cells, Caco-2 cells were selected. The polarized intestinal epithelial cells (IECs) that line the gastrointestinal tract are exposed to a high concentration of foreign antigens (e.g., food and bacterial antigens) at their apical (luminal) surface. In addition, they are in direct contact with several distinct subsets of T-lymphocytes at their basolateral surface, both within the epithelium and the underlying lamina propria. In this context, several groups have suggested that IECs function as APCs and modulate their proliferation, cytolytic activity and/or cytokine production (Bland and Warren, 1986; Mayer and Shlien, 1987; Kaiserlian et al., 1989; Hershberg et al., 1997). Typically, APCs are bone marrow-derived cells such as dendritic cells, monocytes, or B-lymphocytes (the so-called ‘‘professional’’ APCs) whose role in immuno-modulation is well established. Certain other cell types including IECs (Bland and Warren, 1986; Mayer and Shlien, 1987; Kaiserlian et al., 1989; Hershberg et al., 1997), renal tubular epithelial cells (Kelly and Singer, 1993), keratinocytes (Nickoloff and Turka, 1994), and endothelial cells (Savag et al., 1995) have been suggested to function in a limited context as the so-called ‘‘nonprofessional’’ APCs. The lack of ‘‘professional’’ stature reflects both the relative inefficiency of antigen processing and presentation compared with their professional counterparts, and the fact that these cells serve additional physiological (‘‘professional’’) functions independent of their role in T-lymphocyte regulation. Thus, Caco-2 cells with CD58 expression may serve as good model for T-cell – epithelial cell adhesion assay.
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To establish the characteristics of adhesion between Jurkat and Caco-2 cells, CD2 and CD58 expression on the two cell lines were determined, respectively. Using two independent techniques, i.e., confocal microscopy and ELISA, we demonstrated constitutive expression of a functional T-cell costimulatory molecule, CD58, on the surface of the human Caco-2 cell line in the absence of cytokines or other stimulation. Adhesion molecules such as ICAM-1 and CD58 may be upregulated by treatment of cytokines. Most studies on CD58 used IFN-g, while some studies used TNF-a to enhance CD58 expression on endothelial cells (Omari and Dorovini-Zis, 1999). However, it was generally found that no significant enhancement of CD58 expression was observed by incubation of cells with IFN-g or TNF-a. The effects of varying IFN-g concentrations (250, 1000 and 1500 U/ml) on CD58 expression on Caco-2 cells cultivated for 3 days was investigated. In similar studies with endothelial cells, IFN-g could not enhance and even down-regulated CD58 expression. CD2 is a surface marker of T-cells, and is reported to be present on more than 80% of T-cells. In our experiments, FACS revealed that almost 100% of Jurkat cells expressed CD2. To enhance CD2 expression, Jurkat cells were activated with PHA and PMA. Mitogenic lectin PHA can deliver a signal via the TcR/CD3 complex (Wiskocil et al., 1984), thus generating the first signal necessary for T-cell activation. The phorbol ester PMA can directly activate the second messenger pathway by mimicking diacylglycerol, a natural ligand and activator of PKCs (Robb et al., 1981). However, neither PHA nor a combination of low doses of PHA and PMA could augment CD2 expression on Jurkat cells. There was only marginal enhancement after treatment with 10 ng/ml of PMA for 48 h. Since surface proteins cluster at the surface after activation and PMA rapidly up-regulates CD2 avidity (William et al., 1993), PMA (at 10 ng/ml for 48 h) was selected to activate Jurkat cells prior to the adhesion assay. After confirming CD2 and CD58 expression on the surface of Jurkat and Caco-2 cells, respectively, a rapid and sensitive method for the measurement of lymphocyte – epithelial adhesion, i.e., quantitation of CD2 –CD58 interaction was developed. The advent of the fluorescent plate reader combines the sensitivity inherent with fluorochromes and the speed of spec-
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trophotometric plate readers that allows the screening of large numbers of samples. Lymphocytes can be conveniently labeled with non-polar BCECF – AM before they are used in the attachment assay. The AM derivatives of the non-fluorescent indicator BCECF rapidly permeate the cell membrane and are readily cleaved by high activity and large capacity cytoplasmic esterase into fluorescent free-acid form. Thus, BCECF is widely used in cell adhesion and cell viability assays. A major advantage of using acetoxymethyl esters (AM) of BCECF is that only living cells are stained with this dye. In addition, most fluorometers and fluorescence microscopes are equipped with a filter set for FITC, which can be used for detecting this dye. Although calcein –AM is currently more popular for assaying cell adhesion, BCECF –AM is relatively less expensive and can be loaded into cells at low concentration. Moreover, by taking advantage of the pH sensitivity of BCECF, lower concentration of the dye could be used and still obtain a higher fluorescence intensity compared to cells loaded with calcein– AM. A potential problem with BCECF-loaded cells is dye leakage from cells (William et al., 1993). However, the epithelial cells investigated in our assay are human colonic adenocarcinoma cells, which stably express the human multidrug transporter, P-glycoprotein, in the plasma membrane. P-glycoprotein can actively extrude the hydrophobic AM form of the fluorescent indicator BCECF (Rink et al., 1982; Homoloya et al., 1993). Moreover, since the hydrophilic free-acid form of BCECF is not membranepermanent, Caco-2 cells will not be fluorescentlabeled. In addition, BCECF release was found to be less than 3% after plating monolayers for 60 min. Therefore, the likelihood of BCECF leakage may be excluded under our adhesion assay conditions. In initial studies, certain experimental parameters that influence labeling were determined. A standard loading concentration of 2 AM BCECF –AM was adopted as this provided sufficient labeling, but was low enough to avoid unnecessary washing steps and accumulation of large amounts of the potentially toxic hydrolysis product, formaldehyde, in lymphocytes (Hollo et al., 1994). The accumulation of BCECF was rapid, and the spontaneous release of BCECF from labeled cells was low over the test period.
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Jurkat cell adhesion was linear in the range of 0.5 105 –2.5 105 cells/well. The saturated density of 2.5 105 Jurkat cells/well was selected for the competitive adhesion assay. Although it is noteworthy that maximum adhesion of Jurkat cells to Caco-2 monolayers occurred within 15 min, an incubation time of 30 min was adopted for the peptide/Jurkat cell competitive adhesion assay to ensure sufficient exchange time and adhesion. Finally, this assay was applied to screen peptides with inhibitory activities against CD2 – CD58 interaction. Although another major known mechanism of T-cell adhesion is mediated by LFA-1 –ICAM-1 interaction and ICAM-1 has been reported to be expressed on Caco-2 cells, the results that the anti-human CD58, TS2/9, directed against the CD2-binding site in CD58, markedly reduced the Jurkat – Caco-2 adhesion and negative control peptide from CD2 protein exhibited very low inhibitory activity suggest that cell-surface CD58 from Caco-2 cells, via binding to CD2 on Jurkat cells, regulated lymphocyte– epithelial cell adhesion. Moreover, this assay is more rapid and sensitive, and the results are consistent with those obtained by the E-rosetting assay.
Acknowledgements We would like to thank Miss Jessie Lim for technical assistance. Funding for this research was provided by Academic Research grant (R-148-000-026-112), National University of Singapore, Singapore.
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