The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ariel, A.
Right arrow Articles by Lider, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ariel, A.
Right arrow Articles by Lider, O.
The Journal of Immunology, 2001, 166: 3052-3060.
Copyright © 2001 by The American Association of Immunologists

Cell Surface-Expressed Moesin-Like Receptor Regulates T Cell Interactions with Tissue Components and Binds an Adhesion-Modulating IL-2 Peptide Generated by Elastase1

Amiram Ariel*, Rami Hershkoviz{dagger}, Idit Altbaum-Weiss*, Sharon Ganor* and Ofer Lider2,*

* Department of Immunology, Weizmann Institute of Science, Rehovot, Israel; and {dagger} Department of Internal Medicine B, Meir Hospital, Kfar-Saba, Israel


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The adhesion of leukocytes to the extracellular matrix (ECM) depends on their responses to variations in the chemotactic signals in their milieu, as well as on the functioning of cytoskeletal and context-specific receptors. Ezrin, radixin, and moesin constitute a family of proteins that link the plasma membrane to the actin cytoskeleton. The surface expression of moesin on T cells and its role in cell adhesion has not been fully elucidated. Recently, we found that IL-2 peptides generated by elastase modified the adhesion of activated T cells to ECM ligands. Here, we further examined the adhesion regulatory effects of EFLNRWIT, one of the IL-2 peptides, as well as the existence and putative function of its receptor on T cells. We found that when presented to T cells in the absence of another activator, the EFLNRWIT peptide induced cell adhesion to vessel wall and ECM components. Binding of a radiolabeled peptide to T cells, precipitation with the immobilized peptide, and amino acid sequencing of the precipitated protein revealed that EFLNRWIT exerts its function via a cell surface-expressed moesin-like moiety, whose constitutive expression on T cells was increased after activation. This notion was further supported by our findings that: 1) anti-moesin mAb inhibited the binding of T cells to the immobilized EFLNRWIT peptide, 2) immobilized recombinant moesin bound the IL-2 peptide, and 3) soluble moesin inhibited the EFLNRWIT-induced T cell adhesion to fibronectin. Interestingly, moesin appears to be generally involved in T cell responses to adhesion-regulating signals. Thus, the IL-2 peptide EFLNRWIT appears to exert its modulating capacities via an adhesion-regulating moesin-like receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The migration of activated leukocytes to inflamed foci involves both extravasation through vascular barriers and migration through the extracellular matrix (ECM)3 (1). This migration is regulated by a variety of cytokines and chemokines that induce adhesion and chemotaxis and by adhesion molecules, including integrins, on the cell surface. These receptors mediate the interaction of leukocytes with endothelial cell ligands and ECM components (2, 3). Because the ECM is a substrate for enzymes secreted by migrating cells, the immunologically relevant context (in this case, inflammatory) also includes degradation fragments of ECM components and cytokines. Previously, we showed that elastase, an ECM-degrading enzyme secreted by neutrophils, not only degrades the pleiotropic cytokine IL-2, thereby modifying its actions, but also generates small IL-2-derived peptides that are biologically active. At pM concentrations, these IL-2 peptides (EFLNRWIT, RMLT, and IVL) can down-regulate T cell adhesion to ECM glycoproteins induced by intact IL-2, as well as PMA (4). We suggest that the degradation products of cytokines generated by inflammatory enzymes are part of an intrinsic functional program, not just molecular waste. Here, we investigated whether, in the presence or absence of any additional proadhesive moieties, different amounts of the elastase-generated IL-2 peptides can differentially affect the interactions of T cells with components of blood vessel walls, including ECM glycoproteins, hyaluronic acid (HA), ICAM-1, and VCAM-1.

The adhesion of T cells to components of blood vessel walls is an activation-dependent process (1, 5). Activation of T cells by various mediators leads to a rapid change in the adhesive potential of two subfamilies of adhesion molecules, namely the {beta}1 and {beta}2 integrins (1), and thereby increases the ability of T lymphocytes to bind fibronectin (FN), laminin (LN), collagen (CO)-IV, and the endothelial cell ligands VCAM-1 and ICAM-1 (5). HA, a blood vessel wall-associated glycosaminoglycan involved in leukocyte migration, is expressed both on the surface of endothelial cells and in the ECM (5, 6).

Recently, it has been shown that the membrane spike protein moesin, as well as its cytoskeleton-based related proteins, radixin and ezrin, can associate with cell surface protrusions (at the tip of microvilli) and actin microextensions (7). Moesin has been found in epithelial cells, lymphocytes, endothelial cells, and certain types of tumor cells and has been described as a receptor for measles and rabies viruses (8, 9). However, to date, the direct involvement of moesin in CD4+ T cell-tissue interactions during chemokine- and cytokine-induced migration has not been fully clarified.

We examined whether the elastase-generated IL-2 peptides can induce receptor-specific T cell adhesion to vessel wall and ECM ligands in the absence of other proadhesive moieties. We found that the IL-2 peptides, like the intact IL-2 molecule, induced adhesion of T cells to FN, LN, CO-IV, VCAM-1, ICAM-1, and HA in a time- and dose-dependent manner. Moreover, the adhesive effects of the peptides depended on their interactions with a moesin-like receptor expressed on the surface of T cells; blockage of moesin inhibited T cell adhesion to tissue ligands. We discuss the possibility that the behavior of T cells in inflamed areas is affected by receptor-specific recognition of the molecular breakdown products of the very signals that elicit the inflammatory reaction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Human rIL-2 (Chiron B.V., Amsterdam, The Netherlands); macrophage-inflammatory protein-1{beta} (MIP-1{beta}; PeproTech, Rocky Hill, NJ); PMA, PHA, keyhole limpet hemocyanin (KLH), BSA, and HA (Sigma, St. Louis, MO); FN (Chemicon, Temecula, CA); LN, CO type IV (ICN Pharmaceuticals, Costa Mesa, CA); calyculin A (Alexis Biochemicals, San Diego, CA), and antibiotics and RPMI 1640 (Beit-Haemek, Israel) were obtained as indicated. The mouse anti-human moesin mAb clone 38/87 was obtained from NeoMarkers (Fremont, CA); mAb anti-CD3 (clone UCHT-1; PharMingen, San Diego, CA), anti-CD69 (clone CH/4), anti-CD49e (VLA-5, clone JBS5), CD29 (clone 3S3), CD44 (clone 5035-41.1D), and -LFA-1 (CD11a, clone 121/7) were obtained from Serotec (Oxford, U.K.), and FITC-conjugated rabbit anti-human Ab from Dako (Glostrup, Denmark). All protected amino acids, coupling reagents, and polymers were obtained from Nova Biochemicals (Läufelfingen, Switzerland); synthesis-grade solvents were obtained from Labscan (Dublin, Ireland); and HPLC solvents and columns were obtained from Merck (Darmstadt, Germany). Ig-ICAM-1 and the recombinant soluble (extracellular portion) VCAM-1 were kindly provided by Dr. R. Alon (The Weizmann Institute, Rehovot, Israel). Unless mentioned otherwise, standard biological chemicals were purchased from Sigma.

IL-2 peptides

The IL-2 peptides IVL, RMLT, and EFLNRWIT, as well as EFLNRWITAY, TIWRNLFE, and EFLNRWITC were synthesized using an ABIMED AMS-422-automated solid phase, multiple peptide synthesizer (Langenfeld, Germany). The peptides were purified and analyzed as previously described (4). In some experiments, the peptides were modified by adding protecting groups at their C-terminal sites. The protecting groups used were tert-butyloxycarbonyl for Lys and Trp; trityl for Asn, Cys, Gln, and His; tert-butyl-ester for Asp and Glu; and tert-butyl ether for Ser, Thr, and Tyr. Usually, coupling was achieved using two successive reactions for 20–45 min at 22°C, with the time varying due to the length of the peptide and the amino acid derivative type. Coupling was conducted in 50 µmol (4 eq) of N-Fmoc-protected amino acid, 50 µmol of PyBop (4 eq) reagent, and 100 µmol (8 eq) of N-methylmorpholine, all dissolved in dimethylformamide (Labscan). The peptide was cleaved from the resin polymer by incubating (2 h, 22°C) with trifluoroacetic acid diluted in H2O (5:5, v/v). The crude, unprotected peptides thus released were cooled to 4°C, precipitated with ether, and centrifuged (15 min, 5000 rpm, 4°C). The resulting pellets were washed with ether and recentrifuged (three times), dissolved in 30% acetonitrile in H2O, and lyophilized.

Binding of EFLNRWIT to T cells

The EFLNRWIT-AY peptide was radioactively labeled with 125I as previously described (9). The 125I-labeled EFLNRWIT-AY (40 nM) was added to human Jurkat T cells (0.5 x 106 cells/well) that had been seeded in flat-bottom microtiter plates in the presence of increasing concentrations of unlabeled EFLNRWIT or TIWRNLFE. After 90 min at 4°C, the cells were washed with cold PBS to remove the unbound ligand; the cells were then solubilized, and the amount of radioactivity associated with the cells was determined. Mathematical analysis, description, and figure presentation of peptide binding to T cells were performed using the GraphPad Prism Software (http://www.graphpad.com/prism/Prism.htm) (San Diego, CA).

For binding assays to recombinant human moesin, flat-bottom, 96-microtiter well plates were precoated (18 h, 4°C) with the recombinant molecule (18.5 ng/well) and then blocked with 1% BSA. Next, radiolabeled EFLNRWIT (40 nM) was added to the well in the presence or absence of mAb anti-moesin, -CD3, -CD69, or -CD49e ({alpha}5{beta}1 integrin), soluble recombinant moesin, or KLH. After 90 min (at 4°C), the cells were washed with cold PBS and the radioactivity associated with moesin was determined.

Peptide conjugation with KLH

KLH (5 mg in a 3-ml solution containing 50 mM NaHCO3) was mixed (4 h, 22°C, gentle agitation) with 1 ml bromoacetic acid N-hydroxysuccinimide ester (Sigma) in 1 ml of dimethylformamide, followed by an extensive dialysis against PBS (18 h, 4°C). The dialyzed solution was then divided into three equal aliquots, each of which was reacted (4 h, 22°C, gentle agitation) with 2.5 mg (in 250 µl of dimethylformamide) of the HPLC-purified cysteine-containing peptide (EFLNRWITC). After dialysis (18 h, 4°C), the peptide solutions were passed through a 0.22-µm filter. The final amounts of the peptide-KLH conjugates were 0.5–1 mg/ml.

T cells and T cell adhesion assays

Human T cells were purified from the peripheral blood of healthy donors as previously described (4, 10, 11). Briefly, human leukocytes were isolated on a Ficoll gradient, washed, and incubated (2 h, 37°C, 7.5% CO2, humidified atmosphere) on petri dishes. The nonadherent cells were then collected and incubated (1 h, 37°C, 7.5% CO2, humidified atmosphere) on nylon wool columns (Novamed, Jerusalem, Israel). Unbound cells were eluted from the columns by extensive washings. The resulting cell population was always >90% T cells, as determined by FACS analysis. The Jurkat T cell clone, which consists of CD4+ leukemic T cells, was used in some experiments.

T cell adhesion to immobilized protein substrates was also examined as previously described (11). Briefly, flat-bottom microtiter plates were precoated with FN, LN, CO-IV (all at 0.5 µg/well), HA (5 µg/well), and Ig-conjugated ICAM-1, and VCAM-1 (5 µg/well). The remaining binding sites were blocked with 0.1% BSA. 51Cr-labeled T cells (105 cells in 100 µl of adhesion medium (RPMI 1640 containing antibiotics and 0.1% BSA) (21)) were then added to the coated wells followed by the addition of IL-2, PMA, MIP-1{beta}, or IL-2 peptides. In some experiments, soluble recombinant moesin had been preincubated (240 min, 37°C) with IL-2 or the IL-2 peptide and added to the wells with the T cells. Where indicated, T cells were pretreated with mAb anti-moesin (clone 38/87), anti-LFA-1, anti-CD29, and anti-CD44 (all at 1 µg/ml), and then activated by various activators. The microtiter plates were then incubated (37°C in a 7.5% CO2 humidified atmosphere) for 60 min for assaying T cell adhesion to FN, ICAM-1, and VCAM-1, and for 3 h for assaying T cell adhesion to HA. At the conclusion of the incubation periods, the microtiter plates were gently washed with warm PBS, the adherent cells were lysed with 1% Tween 20 in 1 N NaOH, and the radioactivity associated with the resulting supernatants was determined using a gamma counter. For each experimental group, the results were expressed as the mean percentage (± SD) of bound T cells from quadruplicate wells. The percentage of cells that adhered was calculated as follows: [cpm of residual bound cells in the well/(total cpm of cells added to the well - spontaneous release of 51Cr)] x 100.

Peptide binding assays

The EFLNRWIT-AY peptide was iodinated with 125I using the chloramine T labeling method (9). The labeled peptide was isolated from the solution using a Sephadex G-10 column (Pharmacia Biotech, Uppsala, Sweden). The specific activity of the 125I-labeled peptide thus obtained was 0.6 µCi/µg peptide. The labeled peptide was incubated (40 nM; 4°C, 90 min) with Jurkat cells (106) in the presence of increasing amounts of unlabeled EFLNRWIT, RMLT, and IVL or TIWRNLFE peptides. The cells were then washed three times with cold PBS, and the radioactivity associated with the cells was determined using a gamma counter. In other experiments, KLH or KLH-conjugated IL-2 peptides (0.25 µg/well) that were immobilized (18 h, 4°C) on microtiter plates (Maxisorp, Nunc, Denmark). The unbound areas of the wells were blocked with KLH (1 µg/well; 1 h, 25°C). 51Cr-labeled Jurkat cells (105 cells/well) were then added to the plates and incubated (4 h, 4°C) in the presence of anti-moesin or anti-CD3 mAb. The plates were then washed with PBS, the adherent cells lysed, and the amount of radioactivity in the resulting lysates was measured.

Protein precipitation and Western blotting

Affi-Gel 10 beads were washed with MOPS (0.1 M, pH 7.5) and incubated (18 h, 4°C) with KLH (100 µg) or KLH-conjugated IL-2 peptides (100 µg) in 500 µl of MOPS containing 40 mM CaCl2. These KLH-coupled beads were washed with PBS and loaded with precleared lysates of Jurkat cells (5 x 106 cells per lane). T cells were lysed by incubating them with H2O containing sucrose (0.15 M), {beta}-glycerophosphate (80 mM), EDTA (2 mM), EGTA (2 mM), NaVO3 (10 mM), Triton X-100 (1%), pepstatin (10 µg/ml), leupeptin (10 µg/ml), and PMSF (2 mM). Next, the lysates of the T cells were centrifuged (15 min, 15000 rpm); the resulting supernatant was collected and precleared with KLH-coupled beads (1 h, 4°C). Finally, the beads were precipitated by centrifugation and the supernatants were collected and incubated (4 h, 4°C) with the KLH-conjugated EFLNRWIT beads or KLH beads as controls. In some experiments, T cell lysates were incubated (30 min, 4°C) with soluble EFLNRWIT (400 µg/ml), TIWRNLFE (400 µg/ml), or KLH (2 mg/ml) before the precipitation. The peptide- and KLH-conjugated beads that were incubated with the precleared lysates were washed (three times) with PBS containing 0.5% Triton X-100. The material thus bound (i.e., the material precipitated by the conjugated beads) was added to the reducing sample buffer and was boiled for 5 min. These eluted samples were applied onto SDS-PAGE; material from the equivalent of 5 x 106 Jurkat cells was applied to each lane. After analysis with the SDS-PAGE, the gels were stained with Coomasie blue, scanned, and analyzed densitometrically. The resulting major protein band was cut out, processed, and subjected to peptide sequencing (see next section). In addition, proteins precipitated by the IL-2 peptide-conjugated beads were immunoblotted with anti-moesin mAb (diluted 1:1000). The immunoblots were subjected to autoradiography, and the autoradiograms were scanned.

Amino acid sequencing of the EFLNRWIT-binding molecule

The IL-2 peptide-binding molecule was sequenced at the Protein Center of the Technion Institute of Technology (Haifa, Israel) as follows. After routine destaining procedures to remove the Coomassie blue stained molecules, the relevant protein band (i.e., the most pronounced band of the precipitated proteins) was gently removed from the gel using a razor blade. The gel-embedded protein was reduced with DTT (5 mM) and carboxymethylated using 10 mM iodoacetamide. The gel was then further destained in 50% acetonitrile containing 100 mM ammonium bicarbonate, cut into little pieces that were lyophilized, and then rehydrated (18 h, 37°C) in 10 mM ammonium bicarbonate (pH 7.4) containing trypsin. The resulting peptide was eluted from the gel pieces using 60% acetonitrile containing 0.1% trifluoroacetic acid and analyzed. For liquid chromatography-mass spectrometer (MS) analysis, the peptide was resolved by reverse phase HPLC with a 1 x 150-mm Vydac C18 column using a linear gradient of 4–65% acetonitrile in 0.025% trifluoroacetic acid, with 1%/min increments and at a flow rate of 40 µl/min. Approximately 20% of the sample eluted from the HPLC column was microsprayed directly into an electrospray ion trap MS (LCQ, Finnigan-MAT, San Jose, CA), and the remaining 80% was collected manually into microfuge tubes for automated Edman sequencing, which was performed with an automated Perkin-Elmer sequencer. The MS analysis was performed in the positive ion mode, using a repetitive full MS scan followed by an MS/MS experiment (collision-induced fragmentation) on the most abundant ion selected from the MS scan. The MS and MS/MS data from the run were compared with the simulated proteolysis and fragmentation of the protein in the owl database using Sequest software (J. Eng and J. Yates, University of Washington, unpublished observations). The sequencing of the IL-2 peptide-binding molecule yielded the following four peptides. GSELDLGVDALGLNIYEQNDR, FYPEDVSEELIQDITQR, and ESPLLFK were found to be unique to human moesin. The fourth peptide, LFFLQVK, is present in moesin homologues as well, such as ezrin and radixin.

Expression in bacteria and purification of recombinant human moesin

Recombinant human (full-length) moesin was expressed in Escherichia coli as a fusion protein with GST. E. coli transfected with the pGhuMo plasmid (pGEX-KG-human moesin residues 1–577, or GST-moesin) (kindly provided by Dr. Furthmayr, Stanford, CA) was grown in L-broth containing ampicillin. The expression of fusion protein was induced with 100 µM isopropyl {beta}-D-thiogalactopyranoside. Recombinant GST-fusion protein was bound to a glutathione-agarose (Sigma) column and cleaved with thrombin (Pharmacia, Piscataway, NJ) as previously described (12, 13). The purified moesin protein thus obtained was dialyzed against PBS (at 4°C) and stored at -70°C. The purity and integrity of the recombinant moesin was determined and confirmed by SDS-PAGE and Western blotting with the anti-moesin mAb clone 38/87. The amount of recombinant moesin was measured by the densitometry of Coomassie stained SDS-PAGE of the moesin and known amounts of BSA, which were subsequently used to construct a standard curve.

FACS analysis of the expression of moesin

We determined the cell surface expression of moesin on freshly purified human PBL (isolated and purified as previously described) (4) after their stimulation with different stimulators. The cells were maintained in RPMI 1640 medium containing 1% HEPES, 1% L-glutamine, sodium pyruvate (200 mM), and 10% FCS and antibiotics (Kibbutz Beit-Haemek, Israel). Where indicated, the T cells were activated (48 h in tissue culture condition) with PMA (10 ng/ml), PHA (8 µg/ml), or calyculin A (100 nM). All staining protocols were performed with staining buffer (PBS/0.1% BSA and 0.01% sodium azide). For direct staining, the freshly purified human T cells were stained (45 min, 4°C) with PE-conjugated anti-CD3 mAb (Serotec) and anti-moesin Abs and then, with a FITC-conjugated secondary Ab (diluted 1:200; 30 min, 4°C). In other experiments, the T cells were stained (45 min, 4°C) with mAb anti-moesin (250 ng/ml per 0.5 x 106 cells). The cells were then washed with PBS, incubated (30 min, 4°C) with FITC-conjugated rabbit anti-mouse Ab (diluted 1:200 in PBS; Dako), washed, and subjected to FACScan (BD Becton Dickinson, San Jose, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of T cell adhesion to ECM components and endothelial cell ligands by the EFLNRWIT peptide

Previously, we showed that the peptides IVL, RMLT, and EFLNRWIT, generated from rIL-2 by elastase, inhibit the adhesion of IL-2 and PMA-activated T cells to FN, LN, and CO type IV (4). Thus, the IL-2 peptides manifest an anti-adhesive effect if the targeted T cells are coincidentally exposed to proinflammatory signals. However, while migrating, T cells may encounter cleaved IL-2 peptides in the absence of an adhesion- or migration-strengthening stimulus in the form of chemokines or cytokines. Therefore, we first elucidated the effects of the IL-2 peptide, EFLNRWIT, in the absence of other stimuli, on T cell adhesion to ECM glycoproteins and molecular components of blood vessel walls. When T cells were exposed to the IL-2 peptide alone, a substantial cell adhesion was observed not only to immobilized FN, LN, CO-IV, and HA (Fig. 1GoA), but also to the bound vessel wall molecules, ICAM-1 and VCAM-1 (Fig. 1GoB). The EFLNRWIT-induced T cell adhesion to VCAM-1 was greater than that to ICAM-1. Note that T cell adhesion to FN, LN, CO, as well as to ICAM-1 and VCAM-1 was induced by relatively low amounts (0.001–0.1 pg/ml) of EFLNRWIT, but this slowly decreased when higher concentrations were used. However, with HA, exposure of the T cells to increasing amounts of the peptide resulted in a marked and consistent increase of T cell adhesion to the ligand, with maximal adhesion occurring at 1 to 10 pg/ml. A similar pattern of results was obtained with Jurkat cells, a CD4+ T cell clone. The control peptide TIWRNLFE, used in equal concentrations, had no apparent effect on T cell adhesion to the various ligands. Adhesion of T cells to BSA-coated microtiter wells, which were used as a control, was not affected by the IL-2 peptide (data not shown).



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 1. Induction, by IL-2 peptide, of T cell adhesion to ECM and endothelial cell ligands. A, Labeled human T cells were activated with the IL-2 peptides and seeded onto FN-, LN-, CO type-IV, and HA-coated flat-bottom microtiter wells. T cell adhesion was measured after 1 (for FN, LN, and CO) or 3 (for HA) h. B, Identical studies were conducted with T cells seeded into microtiter wells onto which recombinant human, Ig-conjugated ICAM-1 or VCAM-1 molecules had been preimmobilized. Mean percentages (and SD) of T cell adhesion for quadruplicate wells are depicted. One experiment representative of four.

 
The modulation of the observed adhesion was peptide and sequence specific; substituting the two N-terminal amino acids of EFLNRWIT with EA or AF abrogated the effectiveness of the molecule, and the reversely synthesized peptide (TIWRNLFE) did not induce the adhesion of T cells to FN (data not shown). It is noteworthy that the IL-2 peptide-induced T cell adhesion to FN and HA was specifically blocked by mAb anti-{alpha}4{beta}1 and anti-{alpha}5{beta}1 integrins, or anti-CD44, respectively, but not by control mAb. Furthermore, the peptide-induced T cell adhesion to ICAM-1 was inhibited by anti-LFA-1, but not by anti-CD44 or {beta}1 integrin mAb (data not shown). Thus, depending on the amounts of the IL-2 peptides and the absence or presence of other adhesion-inducing mediators, these peptides can induce (Fig. 1Go) or inhibit (4) T cell adhesion not only to ECM ligands, but also to ICAM-1 and VCAM-1.

Specificity of the binding of the IL-2 peptide EFLNRWIT to Jurkat T cells

The results of our previous study (4) and of the foregoing experiments suggest that the elastase-generated IL-2 peptide EFLNRWIT exerts its immuno-modulatory functions by interacting with a T cell surface-expressed receptor (other than the IL-2R) (4). This possibility was examined by adding two amino acids, A and Y, to the C-terminal end of the EFLNRWIT peptide, radioactively labeling the modified peptide, and adding it to the Jurkat T cells together with increasing amounts of unlabeled EFLNRWIT or its reversely synthesized counterpart. The two other unlabeled elastase-generated IL-2 peptides, i.e., IVL and RMLT, were also used to compete on the binding of EFLNRWIT to the T cells. Human Jurkat T cells were chosen based on their clonotype characteristics (e.g., durability, cell-to-cell similarity, and accessibility), upon verifying their positive response to the adhesion-promoting amounts of the EFLNRWIT peptide. The amount of peptide that bound to the T cells was plotted and the binding characteristics and specificity of the receptor-ligand interactions were analyzed. The binding curve indicates that the EFLNRWIT peptide specifically bound to the T cells; the unlabeled EFLNRWIT, but not the TIWRNLFE peptide competed, in a dose-dependent manner, with the binding of the labeled peptide to the cells (Fig. 2Go). Interestingly, competition was not observed with soluble IVL and RMLT peptides, suggesting that although the three peptides exert comparable effects on T cells (4), they may interact with distinct cell surface binding sites or receptors. Moreover, the pattern of the displacement curve revealed that the EFLNRWIT peptide interacts with two distinctive binding sites on the human Jurkat cells, with an estimated EC50 of 9.7 x 10-9 M for one binding site and 2.5 x 10-6 M for the other. Thus, the IL-2 peptides exert their functions on T cells by binding to cell surface-expressed moieties with medium-to-high binding affinities.



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 2. Competition on the binding of the soluble EFLNRWIT peptide to Jurkat T cells. Analysis of the binding (90 min, 4°C) of 125I-labeled EFLNRWIT peptide (40 nM) to Jurkat cells (0.5 x 106 cells/sample) in the presence of increasing amounts of unlabeled EFLNRWIT, its control peptide (TIWRNLFE), or two other elastase-generated IL-2 peptides, RMLT and IVL. Analysis of the binding of the peptide to T cells and plotting of the binding curves was done using GraphPad Prism software. One experiment representative of three.

 
Moesin was identified as the IL-2 peptide-binding moiety on T cells

The chemical nature of the putative IL-2-peptide binding moiety was examined by lysing Jurkat T cells, preclearing the cell lysate with KLH-coupled beads, and incubating the unbound material with beads coupled with either KLH or EFLNRWIT conjugated to KLH. The unbound material (soluble proteins) was washed, and the precipitated proteins were subjected to SDS-PAGE, and stained for proteins. A major protein band, of an apparent molecular mass of 78 kDa, was specifically precipitated from Jurkat lysates with KLH-EFLNRWIT-conjugated beads (Fig. 3GoA). The binding of KLH-EFLNRWIT to the 78-kDa protein was substantially reduced in the presence of soluble EFLNRWIT, but not by the reversed soluble peptide or KLH (see the densitometric analysis of Fig. 3GoA).



View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 3. Binding of the IL-2 peptides to the T cell moiety moesin. A, SDS-PAGE. Jurkat cells that were precleared with KLH (0.4 mg/ml)-coupled beads were incubated with KLH (50 µg)- or KLH-conjugated EFLNRWIT (50 µg)-coupled beads in the presence of soluble EFLNRWIT, TIWRNLFE (both at 400 µg/ml), or KLH (2 mg/ml). The protein molecules that bound were eluted and subjected to SDS-PAGE, followed by staining and scanning. Densitometric analysis of lanes 1–4 is shown in the right hand corner of the gel. B, Immunoblot (I.B.). Precleared lysates of Jurkat cells (see A) were incubated with KLH-conjugated EFLNRWIT, or KLH-conjugated with beads. The proteins that bound were eluted and subjected to immunoblotting with anti-moesin mAb (clone 38/87). One experiment representative of five for both A and B.

 
Next, the 78-kDa protein band was removed from the gel and a peptide-sequencing analysis of different protein fragments of the molecule was performed. The resulting amino acid sequence was found to be identical with that of the ezrin, radixin, moesin (ERM) protein moesin implicated in cell adhesion (15, 16). Whether moesin is indeed precipitated by the IL-2 peptide was verified by using the anti-moesin mAb clone 38/87 in Western blot analysis. The major moiety in the cell lysates that were bound (precipitated) by the EFLNRWIT molecule was clearly recognized by the anti-moesin mAb (Fig. 3GoB). Although mAb 38/87 also recognizes radixin, it is highly unlikely that this is the precipitated molecule, because T cells do not express radixin (15).

Analysis of the surface expression of moesin on resting and activated T cells

Can an ERM moesin-like molecule be expressed on the surface of human T cells? Moesin has been characterized mainly in the interior parts of different types of cells. Recently, however, the expression of a molecule identical with moesin on the membrane of leukocytes and other cell types was demonstrated (7, 8, 17, 18, 19). Therefore, we examined the expression of moesin on the membrane of resting or activated T cells by binding anti-moesin mAb and anti-actin mAb (isotype-matched control mAb) to human T cells (PBL). First, we verified, using a double fluorescence staining technique of resting human PBL, that most of the cells expressed both the CD3 and moesin markers on their surfaces (Fig. 4GoA).



View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 4. Analysis of the expression of moesin on resting or activated T cells. The cell surface expression of moesin on freshly purified resting human T cells (PBL) or after their stimulation (48 h) with the following stimulators: PMA (10 ng/ml), PHA (8 µg/ml), or calyculin A (100 nM). A, Direct double staining. T cells were stained with PE-conjugated anti-CD3 mAb and washed. Next, the treated cells were stained with mAb anti-moesin, and then, with a FITC-conjugated rabbit anti-mouse IgG. B–D, Staining of resting or activated T cells. Gray areas and dotted lines represent the staining of resting or activated T cells, respectively, only with the FITC-conjugated Ab. Moesin staining of T cells is represented by the thin and thick lines corresponding to resting and activated T cells, respectively. E, Mean fluorescence intensity staining of moesin on resting or activated T cells. One experiment representative of 5.

 
Next, we examined the expression of moesin on resting and PHA- or PMA-activated T cells. The results, shown in Fig. 4Go, B and C, indicate that resting T cells expressed a significant number of moesin-like molecules on their membranes. However, as seen in Fig. 4Go, B and C, the activation of the T cells for 48 h, by PMA and PHA, induced a pronounced elevation (i.e., 2-fold and >4-fold, respectively; Fig. 4GoE) in the expression of this molecule above the control. As expected, if the membranes of the T cells were permeabilized before adding the mAb anti-moesin, a substantial amount of positive staining occurred (data not shown), indicating that the mAb anti-moesin used here indeed interacts with the internal ERM molecule.

It has recently been shown that calyculin A, a selective inhibitor of Ser/Thr phosphatase 1 and 2A, can phosphorylate and subsequently induce the association of moesin with F-actin and its redistribution in the cytoplasms of platelets and macrophages (20). Therefore, the effect of PBL treatment with calyculin A on the cell surface expression of moesin was examined. The results, shown in Fig. 4GoC, indicate that the expression of moesin on T cells treated with calyculin A (100 nM, 48 h) was increased by almost twofold over the control (Fig. 4GoE). Thus, it appears that resting, and to a much greater degree, activated human T cells express moesin on their cell surfaces.

Interaction of the IL-2 peptide EFLNRWIT with recombinant human moesin

The results of the foregoing studies indicate that the EFLNRWIT peptide binds to a moesin-like molecule, or alternatively, to a moesin-associated molecule on T cells. This assumption was further examined, on the molecular level, in the following set of experiments.

First, we assumed that if indeed the IL-2 peptide binds a T cell surface-expressed moesin, then anti-moesin mAb would be expected to competitively inhibit the binding of the peptide to T cells. Such competition was studied by analyzing the binding of labeled human T cells to immobilized KLH-conjugated EFLNRWIT in the presence of increasing amounts of either anti-moesin or anti-CD3 mAb. We found that the binding of 51Cr-labeled T cells to the immobilized EFLNRWIT was abrogated by the anti-moesin mAb clone 38/87 in a dose-dependent manner, but not by anti-CD3 mAb (Fig. 5GoA), indicating that the major IL-2 peptide-binding moiety on T cells is indeed a moesin-like molecule.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 5. Analysis of the binding of EFLNRWIT to Jurkat cells and recombinant (rec.) moesin and the inhibition of this binding by anti-moesin mAb. A, Labeled Jurkat cells were incubated (4 h, 4°C) in microtiter wells precoated with the KLH-conjugated EFLNRWIT (0.25 µg/well) in the presence of increasing amounts of anti-moesin mAb, clone 38/87, and a control, anti-CD3 mAb. The plates were washed, the adherent cells lysed, and the radioactivity in the lysates determined. Radioactivity due to T cell binding to KLH-coated wells was subtracted. One experiment representative of three. B and C, Recombinant human moesin (18.5 ng/well) was applied (18 h, 4°C) onto microtiter well plates. The unbound protein was removed, and the wells were blocked by BSA (0.1% in PBS) before the binding (90 min, 4°C) of 125I-labeled EFLNRWIT-AY peptide (40 nM). The binding was inhibited by mAb against moesin, CD3, CD69, or CD49e (B), or soluble recombinant moesin or KLH (C). One experiment representative of three.

 
Next, the binding of 125I-labeled EFLNRWIT-AY peptide to immobilized recombinant human moesin was tested in the absence or presence of increasing amounts of different T cell-specific mAbs, including anti-moesin mAb. As shown in Fig. 5GoB, in the absence of mAb, apparently a considerable amount of the labeled peptide was found to be bound to the recombinant immobilized moesin. The binding of 125I-labeled EFLNRWIT-AY was not affected by increasing amounts of anti-CD3, -CD69, or -VLA5 mAb. However, the moesin-specific mAb, clone 38/87, inhibited, in a dose-dependent manner, the binding of the labeled EFLNRWIT peptide to its immobilized ligand; a complete inhibition occurred at 1 µg/ml.

Finally, we examined the binding of radiolabeled EFLNRWIT-AY to immobilized recombinant moesin in the presence of increasing amounts of soluble recombinant moesin as a competitor. The results, shown in Fig. 5GoC, indicate that the recombinant soluble moesin, but not the control molecule KLH, inhibited, in a dose-dependent manner, the binding of the labeled EFLNRWIT to the immobilized ERM molecule. Taken together, these studies strongly suggest that the EFLNRWIT peptide interacts with moesin (or a closely related molecule) that appears to be expressed on the membranes of human T cells.

Inhibition of EFLNRWIT-induced T cell adhesion to FN by soluble recombinant moesin

Because we demonstrated that the IL-2 peptide EFLNRWIT regulates T cell adhesion to vessel wall and tissue components by interacting with a T cell surface-expressed moesin-like moiety, the involvement of moesin in the EFLNRWIT-induced T cell adhesion to FN was investigated. We assumed that the presence of soluble moesin in the T cell adhesion assay should interfere with IL-2 peptide-induced cell adhesion to FN by interacting directly with the peptide, and thus block its interactions with membranal moesin.

Hence, adhesion to FN of T cells pretreated with adhesion-inducing amounts of intact IL-2 or EFLNRWIT was examined by using increasing concentrations of recombinant moesin. The results indicate that T cell adhesion to FN induced by EFLNRWIT, but not by intact IL-2, was blocked, in a dose-dependent manner, by recombinant human moesin (Fig. 6Go). Recombinant soluble human moesin did not affect T cell adhesion to the identical, immobilized moesin (data not shown). This suggests that moesin binds the IL-2 peptide (but not the intact IL-2 molecule from which it is derived), and thereby, interferes with its adhesion-promoting abilities. Alternatively, the IL-2 peptide may bind to an undefined moiety other than moesin itself, and this binding may be interrupted by the binding of the anti-moesin mAb to T cells.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 6. Recombinant (rec.) moesin inhibits the EFLNRWIT-induced T cell adhesion to FN. Human T cells were radiolabeled and then exposed to the EFLNRWIT peptide (1 fg/ml) or intact IL-2 (10 U/ml) that were preincubated (37°C, 4 h) with the indicated increasing amounts of soluble recombinant moesin, before being added to the wells. T cell adhesion (1 h) to immobilized FN was analyzed as described in Fig. 1Go. One experiment representative of four.

 
Involvement of moesin in modulating the adhesion of activated T cells to FN and HA

Because the foregoing experimental findings indicate that a moesin-like molecule is involved in IL-2 peptide-mediated T cell adhesion, we further investigated whether the same anti-moesin mAb also interferes with the induction, by various proinflammatory mediators (that interact with nonmoesin moieties on T cells), of T cell adhesion to FN and HA. For this purpose, T cells were activated by various physiological and nonphysiological stimuli, then treated with anti-moesin mAb, and seeded onto the ligand-coated plates.

Surprisingly, we found that the adhesion of T cells activated with the EFLNRWIT peptide, IL-2, as well as CD3 cross-linking by the anti-CD3 mAb, PMA, and the proadhesive chemokine MIP-1{beta}, to FN and HA, was significantly abrogated by the anti-moesin mAb, clone 38/87, but not by the anti-LFA-1 mAb (Table IGo). Note that mAb anti-CD29 and -CD44 specifically inhibited T cell adhesion to FN or HA, respectively. Interestingly, the same mAb anti-moesin did not inhibit the binding of PMA-activated human monocytes (of the MonoMax-6 cell line) to FN. However, these cells did not express moesin on their surface and they did not respond to the EFLNRWIT peptide (data not shown).


View this table:
[in this window]
[in a new window]
  		Table I. Percentage of inhibition of T cell adhesion to FN and HA1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The accuracy, effectiveness, and time-limited characteristics of the inflammatory reaction are facilitated by the rapid recognition of the inflamed context by tissue-specific receptors on immunocytes. Molecular interactions with such receptors, which influence the activation, adhesion, and migration of immune cells, are affected by inflammatory-regulating signals (21). These signals can be generated by (and their biological nature determined by) products of the migrating leukocytes themselves (e.g., cytokines, chemokines, growth factors, and enzymes) from the ECM (e.g., intact, degraded immobilized, or degraded soluble components), or, as shown here, products of the enzymatic degradation of inflammatory cytokines. During migration, leukocytes, especially T cells, can encounter, either sequentially or simultaneously, several of these soluble or immobilized modulators (3, 21, 22, 23), whereupon the chemical complexity of these signals is increased. Recently, we isolated, sequenced, and synthesized peptides resulting from the degradation of IL-2 by human neutrophil elastase (4). In this study, a role for one of these degradation products of inflammation and for the receptor that recognizes it in mediating the behavior of T cells in inflamed areas was demonstrated. We showed that 1) the effects (pro- or anti-adhesive) of the IL-2 peptide EFLNRWIT depend on its concentration and on the presence or absence of other signals and 2) the peptide exerts its functions by interacting with moesin, which is involved in cell adhesion.

First, we showed that even at concentrations as low as few fg/ml, the IL-2 peptide induced the adhesion of human T cells to FN, LN, CO, HA, VCAM-1, and ICAM-1. Interestingly, the dose-response curve of peptide-induced adhesion to FN was different from that of the intact IL-2-induced adhesion. In addition, the adhesion pattern of T cells induced by the elastase-generated IL-2 peptide is similar to that induced by certain cytokines and chemokines (22), which induce the adhesion of T cells by regulating their shapes and the avidities of adhesion receptors on their surfaces (3, 11). The movement of leukocytes depends on local concentrations of different chemotactic mediators; when the concentration of migratory mediators, such as IL-1{beta}, IL-2, or IL-8, surpasses that of their maximal effective dose, the migration of lymphocytes and neutrophils is abrogated (23, 24). This phenomenon may be due to the loss of a gradient of a single chemokine or a complex and opposing effect of two proadhesive or promigratory signals operating together. Future studies should aim to better understand the mechanism of the context- and concentration-dependent inhibition of lymphocyte adhesion by the EFLNRWIT peptide and to investigate whether this inhibition is due to interventions in the adhesive process by receptor desensitization (3). Recently, fMLP was observed to inhibit leukocyte migration induced by the subsequent exposure of the cells to either IL-8 or leukotriene B4 (22). Opiates, which induce chemotaxis of monocytes, inhibit cell migration induced by chemokines (25). Substance P, which by itself does not induce T cell adhesion to ECM, inhibits RANTES- and MIP-1{beta}-induced T cell adhesion to FN (26).

HA, a glycosaminoglycan expressed on cell surfaces and complexed within the ECM, has been implicated in leukocyte migration; CD44-HA interactions are instrumental in recruiting T cells to inflammatory locations (6, 27). Therefore, we studied whether the IL-2 peptide also induces T cell adhesion to HA and the dose dependence and kinetics of this interaction. Although the IL-2 peptides induced T cell adhesion to immobilized HA, the kinetics of T cell adhesion to HA were different from those of T cell interactions with ECM glycoproteins, which are mediated by {beta}1 integrins. More specifically, the T cell adhesion to FN occurred earlier than the adhesion to HA, and then decreased to background levels while appreciable adhesion to HA was detected. Moreover, the dose dependence of T cell adhesion to HA, induced by EFLNRWIT, showed an increasing-with-dose type of kinetic curve; its effect on T cell adhesion to FN showed a bell-shaped curve. These different patterns suggest that during migration to inflamed areas, T cells probably initially encounter low amounts of the IL-2 peptide, which induce adhesion and migration. However, as the T cells get closer to the inflammatory loci, they probably encounter increasingly higher concentrations of the mediator, and while their integrins are first turned off, the mediation of CD44-HA movement of the T cells is affected. These separate adhesive responses can be combined to ensure continuous and prolonged adhesive interaction of T cells with the ECM.

Regulation (induction or inhibition) of T cell adhesion (and probably also of migration) induced by EFLNRWIT appears to depend on the context in which the migrating T cells encounter this peptide. The peptide manifests its effect rapidly and functions at relatively low concentrations, which suggests that an IL-2 peptide-specific receptor is present on the T cells. Indeed, we found that two IL-2 peptide-specific receptors appear to be present on the T cells (Figs. 2Go and 3Go), one of which is moesin, a protein that is intracellularly and extracellularly associated with the membrane of various cells. Here, moesin is implicated in the regulation of T cell adhesion induced by various physiological and nonphysiological activators (Fig. 6Go). Thus, moesin on T cells appears to be involved in dynamic changes in the function of T cells that occur during cell-substratum attachment and movement.

Moesin, a 78-kDa protein, has been characterized as being one of the ERM family of proteins (along with ezrin and radixin) involved in cell adhesion and membrane dynamics, probably because of their ability to link plasma membrane components with the actin cytoskeleton (7, 8, 15, 28). Although the functions of ERM proteins have not yet been fully delineated, these proteins are known to be key effector molecules of the downstream signal transduction pathways that modulate the plasticity of the membranes of cells (15, 29). In addition to its structural role as a cellular stabilizer, moesin was recently shown to interact, via its N-terminal domains, with cell surface-expressed adhesion receptors (CD44 and ICAM-3) on mobile (T cells) and nonmobile (fibroblast) cells (16, 30, 31, 32). Hence, moesin is involved not only in the formation of uropods, microvilli, and ruffling membranes, but also in establishing firm adhesive contacts between cells. By interacting with the actin filaments and certain signaling molecules associated with plasma membranes, such as Rho-associated kinases, moesin and related proteins have indeed been implicated in the structural and functional responses of different cells (19).

Accordingly, we suggest the existence of cell surface-expressed moesin on the freshly isolated, human PBL. Previously, the cellular localization of moesin was examined in lymphoblasts in which moesin was detected in the intracellular but not in the extracellular compartment (16). We have found that the expression of moesin on resting PBL (but not on human T cell lines; data not shown) can be up-regulated by activation (for 48 h) of the cells, not only by PHA and PMA, but also by calyculin A, a specific inhibitor of phosphatase 1 and 2A, which was shown to induce moesin phosphorylation (20). However, the expression level of moesin on activated lymphocytes was reduced after 5 days of PHA activation (data not shown). Our results imply that moesin, or a moesin-like moiety, is capable of acting as a cell surface receptor.

Recently, other research groups also reported the existence of moesin on macrophages, lymphocytes, fibroblasts, endothelial, and epithelial cells, and examined both the ligand-binding specificity and putative signal transduction capacities of moesin (17, 33, 34, 35). A similar phenomenon was observed with the cytoplasmic protein annexin II, which is expressed on the surface of various cell types (36). Interestingly, cell surface-expressed moesin interacts with heparan sulfate, LPS, and components of rabies and measles viruses (8, 18, 19, 28, 37). If moesin is indeed also a cell surface receptor, this implies that moesin, whether an intracellular or extracellular moiety, is a permissive receptor with a wide ligand-recognition specificity. Interestingly, a recent report demonstrates that moesin possesses a chemically complexed N-terminal FERM domain, which is often present in cell signaling and cytoskeletal proteins. Upon binding other peptides and/or phospholipid ligands, this moiety could produce varying levels of activation (38).

This study suggests that during inflammatory reactions, the products of enzymatic degradation of proinflammatory mediators can regulate the behavior of immune cells. Additional studies are needed to elucidate the mechanisms underlying the regulation of immune cell adhesion by moesin (and IL-2 peptides). One such research direction may be to clarify the functional association of moesin and T cell adhesion receptors. Our preliminary findings indicate that moesin, upon responding to the IL-2 peptides, associates physically with surface {beta}1 integrins (data not shown), thereby up-regulating their ligand-binding functions.


   Acknowledgments
 
We thank Drs. M. Fridkin and E. Yavin (Weizmann Institute of Science) for their scientific assistance for kindly providing the anti-moesin mAb, and Dr. H. Furthmayr for his helpful discussions.


   Footnotes
 
1 This study was supported by research grants from the Israeli Cancer Research Funds, the Robert Koch-Minerva Center for Research in Autoimmune Diseases (Weizmann Institute), and the Center for Study of Emerging Diseases. The fellowship of A.A. is funded by the Samara Jan Turkel Scholarship Fund for Autoimmune Diseases. O.L. is the incumbent of the Weizmann League Career Development Chair in Children’s Diseases. Back

2 Address correspondence and reprint requests to Dr. Ofer Lider, Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. Back

3 Abbreviations used in this paper: ECM, extracellular matrix; HA, hyaluronic acid; FN, fibronectin; LN, laminin; CO, collagen; MIP-1{beta}, macrophage-inflammatory protein-1{beta}; KLH, keyhole limpet hemocyanin; MS, mass spectrometer; ERM, ezrin, radixin, moesin. Back

Received for publication October 27, 2000. Accepted for publication December 20, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Butcher, E. C., L. J. Picker. 1996. Lymphocyte homing and homeostasis. Science 272:60.[Abstract]
  2. Schall, T. J., K. Bacon, K. J. Toy, D. V. Goeddel. 1990. Selective attraction of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES. Nature 347:669.[Medline]
  3. Ben Baruch, A., D. F. Michiel, J. J. Oppenheim. 1995. Signals and receptors involved in recruitment of inflammatory cells. J. Biol. Chem. 270:11703.[Free Full Text]
  4. Ariel, A., E. J. Yavin, R. Hershkoviz, A. Avron, S. Franitza, I. Hardan, L. Cahalon, M. Fridkin, O. Lider. 1998. IL-2 induces T cell adherence to extracellular matrix: inhibition of adherence and migration by IL-2 peptides generated by leukocyte elastase. J. Immunol. 161:2465.[Abstract/Free Full Text]
  5. Springer, T. A.. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76:301.[Medline]
  6. DeGrendele, H. C., P. Estess, M. H. Siegelman. 1997. Requirement for CD44 in activated T cell extravasation into an inflammatory site. Science 278:672.[Abstract/Free Full Text]
  7. Sato, N., N. Funayama, A. Nagafuchi, S. Yonemura, S. Tsukita, S. Tsukita. 1992. A gene family consisting of ezrin, radixin, and moesin: its specific localization at actin filament/plasma membrane association sites. J. Cell Sci. 103:131.[Abstract/Free Full Text]
  8. Sagara, J., S. Tsukita, S. Yonemura, S. Tsukita, A. Kawai. 1995. Cellular actin-binding ezrin-radixin-moesin (ERM) family proteins are incorporated into the rabies virion and closely associated with viral envelope proteins in the cell. Virology 206:485.[Medline]
  9. Ariel, A., R. Hershkoviz, L. Cahalon, C. Chen, D. E. Williams, S. K. Akiyama, K. M. Yamada, R. Alon, T. Lapidot, O. Lider. 1997. Induction of T cell adhesion to extracellular matrix and endothelial cell ligands by soluble or matrix-bound IL-7. Eur. J. Immunol. 27:2562.[Medline]
  10. Franitza, S., R. Alon, O. Lider. 1999. Dynamic mechanisms of integrin-mediated migration of T lymphocytes along chemotaxis gradients of IL-2 and RANTES within 3-dimensional extracellular matrix-like gels. J. Immunol. Methods 225:9.[Medline]
  11. Vaday, G. G., O. Lider. 2000. Extracellular matrix moieties, cytokines, and enzymes: dynamic effects on lymphocyte behavior and inflammation. J. Leukocyte Biol. 67:149.[Abstract]
  12. Pestonjamasp, K., M. R. Amieva, C. P. Strassel, W. M. Nauseef, H. Furthmayr, E. J. Luna. 1995. Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes. Mol. Biol. Cell 6:247.[Abstract]
  13. Guan, K., J. E. Dixon. 1991. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal. Biochem. 192:262.[Medline]
  14. Serrador, J. M., M. Nieto, F. Sanchez-Madrid. 1999. Cytoskeletal rearrangement during migration and activation of T lymphocytes. Trends Cell Biol. 9:228.[Medline]
  15. Mangeat, P., C. Roy, M. Martin. 1999. ERM proteins in cell adhesion and membrane dynamics. Trends Cell Biol. 9:187.[Medline]
  16. Alonso-Lebrero, J. L., M. A. del Pozo, H. Furthmayer, R. Schwartz-Albiez, J. Calvo, F. Lozano, F. Sanchez-Madrid. 1997. Moesin interacts with the cytoplasmatic region of intracellular adhesion molecule-3 and is redistributed to the uropod of T lymphocytes during cell polarization. J. Cell Biol. 138:1409.[Abstract/Free Full Text]
  17. Lankes, W., A. Griesmacher, J. Grunwald, R. Schwartz-Albiez, R. Keller. 1988. A heparin-binding protein involved in inhibition of smooth-muscle cell proliferation. Biochem. J. 251:831.[Medline]
  18. Dunster, L. M., J. Schneider-Schaulies, S. Loffler, W. Lankes, R. Schwartz-Albiez, F. Lottspeich, V. T. Meulen. 1994. Moesin: a cell membrane protein linked with susceptibility to measles virus infection. Virology 198:265.[Medline]
  19. Thome, Z. N., S. Amar, T. E. van Dyke. 1999. Moesin functions as a LPS receptor on human monocytes. Infect. Immun. 67:3215.[Abstract/Free Full Text]
  20. Nakamura, F., M. R. Amieva, C. Hirota, Y. Mizuno, H. Furthmayr. 1996. Phosphorylation of 558T of moesin detected by site-specific antibodies in RAW264.7 macrophages. Biochem. Biophys. Res. Commun. 226:650.[Medline]
  21. Nathan, C., M. Sporn. 1991. Cytokines in context. J. Cell Biol. 113:981.[Free Full Text]
  22. Campbell, J. J., E. F. Foxman, E. C. Butcher. 1997. Chemoattractant receptor cross talk as a regulatory mechanism in leukocyte adhesion and migration. Eur. J. Immunol. 27:2571.[Medline]
  23. Foxman, E. F., J. J. Campbell, E. C. Butcher. 1997. Multistep navigation and the combinatorial control of leukocyte chemotaxis. J. Cell Biol. 139:1349.[Abstract/Free Full Text]
  24. Pleass, R., R. Camp. 1994. Cytokines induce lymphocyte migration in vitro by direct, receptor-specific mechanisms. Eur. J. Immunol. 24:273.[Medline]
  25. Grimm, M. C., A. Ben-Baruch, D. D. Taub, O. M. Howard, J. M. Wang, J. J. Oppenheim. 1998. Opiate inhibition of chemokine-induced chemotaxis. Ann. NY Acad. Sci. 840:9.[Abstract/Free Full Text]
  26. Levite, M., L. Cahalon, R. Hershkoviz, L. Steinman, O. Lider. 1998. Neuropeptides, via specific receptors, regulate T cell adhesion to fibronectin. J. Immunol. 160:993.[Abstract/Free Full Text]
  27. Lesley, J., N. Howes, A. Perschl, R. Hyman. 1994. Hyaluronan binding function of CD44 is transiently activated on T cells during an in vivo immune response. J. Exp. Med. 180:383.[Abstract/Free Full Text]
  28. Nakamura, F., L. Huang, K. Pestonjamasp, E. J. Luna, H. Furthmayr. 1999. Regulation of F-actin binding to platelet moesin in vitro by both phosphorylation of threonine 558 and phosphosphatidylinositides. Mol. Biol. Cell 10:2669.[Abstract/Free Full Text]
  29. Hirao, M., N. Sato, T. Kondo, S. Yonemura, M. Monden, T. Sasaki, Y. Takai, S. Tsukita, S. Tsukita. 1996. Regulation mechanism of ERM (ezrin/radixin/oesin) protein/plasma membrane association: possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway. J. Cell Biol. 135:37.[Abstract/Free Full Text]
  30. Tsukita, S., K. Oishi, N. Sato, J. Sagara, A. Kawai, S. Tsukita. 1994. ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeleton. J. Cell Biol. 126:391.[Abstract/Free Full Text]
  31. Oshiro, N., Y. Fukata, K. Kaibuchi. 1998. Phosphorylation of moesin by Rho-associated kinase (Rho-kinase) plays a crucial role in the formation of mcrovilli-like structures. J. Biol. Chem. 273:34663.[Abstract/Free Full Text]
  32. Del Pozo, M. A., M. Vicente-Manzanares, R. Tejedor, J. M. Serrador, F. Sanchez-Madrid. 1999. Rho GTPases control migration and polarization of adhesion molecules and cytoskeletal ERM components in T lymphocytes. Eur. J. Immunol. 29:3609.[Medline]
  33. Nakamura, F., M. Amieva, C. Hirota, Y. Mizuo, H. Futhmayr. 1996. Phosphorylation of 558T of moesin detected by site-specific antibodies in RAW264.7 macrophages. Biochem. Biophys. Res. Commun. 226:650.
  34. Takahashi, K., T. Sasaki, A. Mammoto, K. Takaishi, T. Kameyama, S. Tsukita, Y. Takai. 1997. Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein. J. Biol. Chem. 272:23371.[Abstract/Free Full Text]
  35. Matsui, T., M. Maeda, Y. Doi, S. Yonemura, M. Amano, K. Kaibuchi, S. Tsukita, S. Tsukita. 1998. Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. J. Cell Biol. 140:647.[Abstract/Free Full Text]
  36. Harder, T., R. Kellner, R. G. Parton, J. Gruenberg. 1997. Specific release of membrane-bound annexin II and cortical cytoskeletal elements by sequestration of membrane cytoskeleton. Mol. Biol. Cell 8:533.[Abstract]
  37. Dunster, L., J. Schneider-Schaulies, M. Dehof, V. Holers, R. Schwartz-Albiez, V. Ter Mulen. 1995. Moesin, and not the murine functional homologue (Crry/p65) of human membrane cofactor protein (CD46), is involved in the entry of measles virus (strain Edmonston) into susceptible murine cell lines. J. Gen. Virol. 76:2085.[Abstract/Free Full Text]
  38. Pearson, M. A., D. Reczek, A. Brescher, P. A. Karplus. 2000. Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain. Cell 101:259.[Medline]



This article has been cited by other articles:


Home page
 J. Lipid Res.Home page
A. Matsuyama, N. Sakai, H. Hiraoka, K.-i. Hirano, and S. Yamashita
Cell surface-expressed moesin-like HDL/apoA-I binding protein promotes cholesterol efflux from human macrophages
J. Lipid Res., January 1, 2006; 47(1): 78 - 86.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
I. Iontcheva, S. Amar, K. H. Zawawi, A. Kantarci, and T. E. Van Dyke
Role for Moesin in Lipopolysaccharide-Stimulated Signal Transduction
Infect. Immun., April 1, 2004; 72(4): 2312 - 2320.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ariel, A.
Right arrow Articles by Lider, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ariel, A.
Right arrow Articles by Lider, O.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS