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(Journal of Leukocyte Biology. 2002;72:199-206.)
© 2002 by Society for Leukocyte Biology

Differential localization of IL-2- and -15 receptor chains in membrane rafts of human T cells

Jens Goebel*, Kathy Forrest*, Lorri Morford{dagger} and Thomas L. Roszman{dagger}

* Departments of Pediatrics and
{dagger} Immunology, University of Kentucky Medical Center, Lexington

Correspondence: Jens Goebel, M.D., Section of Pediatric Nephrology, Room J455, Kentucky Clinic Building, Department of Pediatrics, University of Kentucky, Lexington, KY 40536-0284. E-mail: jwgoeb0{at}uky.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
We studied whether cytokine receptors (Rs) on T cells associate with lipid microdomains ("rafts"). Low-dose phytohemagglutinin (PHA)-stimulated human T cells were separated into cytoplasmic, membrane, and raft fractions by buoyant density centrifugation. Examination of these fractions for the presence of interleukin (IL)-2- and -15R chains and associated signaling molecules by Western blotting revealed marked, selective enrichment of the IL-2/15R ß-chain in rafts before IL-2 stimulation. After IL-2 stimulation, a substantial amount of the ß-chain was found in the membrane fraction. This partial translocation was also observed for the ß-chain-associated molecules JAK-1, p56lck, and grb-2. Finally, raft disruption with methyl-ß-cyclodextrin (MBCD) attenuated IL-2-induced tyrosine phosphorylation events and selectively decreased the surface expression of the IL-2/15R ß-chain detected by flow cytometry. These results show that the IL-2/15R ß-chain is enriched in rafts obtained from low-dose, PHA-stimulated T cells, that IL-2 binding alters this enrichment, and that this enrichment may be functionally relevant as a possible mechanism to ensure cytokine selectivity and specificity.

Key Words: T lymphocytes • signal transduction • cytokine receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The formation of lipid microdomains ("rafts") is a prerequisite for the initiation of signaling through the T cell antigen receptor (TCR) and for associated cytoskeletal reorganization [1 ]. Other T cell plasma membrane receptors (Rs), such as the interleukin (IL)-2R, have been described to form "caps" after cell activation [2 3 4 ], and the presence of IL-2R chains in detergent-resistant membrane complexes has been proposed [5 6 7 ]. The IL-2R and the TCR are similar in several aspects, including their shared association with signaling components, such as non-R tyrosine kinases (NRTKs), e.g., p56lck, which is concentrated in rafts [8 ], or adapter molecules, such as grb-2, which is recruited to these domains during T cell activation [9 ]. Additionally, interferon (IFN) R chains have recently been detected in caveolar membrane domains of HeLa cells [10 ]. Caveolae are lipid-enriched plasma membrane compartments and are considered to be a type of raft [11 ]. T cells, however, do not express caveolin [12 ], a key protein component of caveolae. Furthermore, T cells may require special, raft-mediated "clustering" for optimal spatial interactions between presented antigen/major histocompatibility complexes and the TCR/CD3 complex to form immunological synapses [13 ]. Such distinct localization may be less necessary for cytokine Rs, as T cells are surrounded by a milieu containing cytokine molecules. Available data on the possible localization of IL-2R chains in rafts are scarce and not entirely consistent [5 6 7 , 14 15 16 17 ; also see Discussion).

We therefore examined whether IL-2R chains are found in rafts of human T cells before or after IL-2 stimulation. Because the IL-2R differs from the IL-15R on these cells only in its {alpha}-chain [18 ], we also determined whether the IL-15R {alpha}-chain is found in rafts. We found that the IL-2/15R ß-chain, but not the {alpha}- or {gamma}-chain, is enriched in rafts of T cells prior to IL-2 binding and that cytokine ligation of the IL-2R is accompanied by a partial translocation of the IL-2/15R ß-chain and associated signaling molecules to the membrane fraction. Furthermore, this selective enrichment of the IL-2R ß-chain in rafts appears to have functional relevance, as raft disruption attenuated IL-2 signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Cells and cell preparation
Human T cells were isolated by gradient-density centrifugation and sheep erythrocyte rosetting essentially as described [19 ] and were cultured as described previously [20 ]. Unless otherwise specified, expression of the IL-2R {alpha}-chain was induced by addition of 1–2 µg/ml phytohemagglutinin (PHA) for 72 h (cell concentration 1–2x106/ml medium). Cells were briefly exposed to a pH of 6.5, resuspended in fresh medium, and rested for 24 h, resulting in T cells that express the high-affinity IL-2R [21 ]. As an alternative method of stimulation, we also applied a combination of anti-CD3 [5 µg/ml; antibody-containing supernatant of OKT3 cell line, American Type Culture Collection (ATCC), Manassas, VA] and anti-CD28 (1 µg/ml; PharMingen, San Diego, CA) antibodies for 48 h, essentially as described by Sundvold et al. [22 ]. The cells were then resuspended at 5 x 106/ml in medium containing 2% fetal bovine serum (FBS). Where indicated, they were stimulated with 200 U/ml IL-2 for 20 min in the presence or absence of methyl-ß-cyclodextrin (MBCD; Sigma Chemical Co., St. Louis, MO; 0.1 or 1 mM) or nystatin (Sigma Chemical Co.; 300 or 1000 µg/ml) and were added 30 min before IL-2 stimulation. Cell viability was assessed by trypan blue exclusion. Jurkat cells (ATCC) were cultured in RPMI supplemented with penicillin/streptomycin (Gibco, Grand Island, NY), 10 mM HEPES (Gibco), and 10% FBS (Summitt Biotechnology, Fort Collins, CO). The cells were passaged twice weekly. Unless noted otherwise, all experiments were repeated with cells isolated from several different donors, and the results were consistently reproduced.

Preparation of cytoplasmic, plasma membrane, and raft fractions
Cell fractionation was performed at 4°C as described by Xavier et al. [23 ]. Approximately 2 x 108 T cells each were prepared from healthy volunteer donors and treated as indicated (see above). After acid-stripping and rest, they were resuspended in 1 ml ice-cold MES [2-(N-morpholino)ethanesulfonic acid]-buffered saline [MBS; 25 mM MES and 150 mM NaCl mixed together at a pH of 6.5 followed by the addition of 0.5% Triton X-100, 1 mM sodium vanadate, 2 mM ethylenediaminetetraacetate (EDTA), 1 mM phenylmethylsulfonly fluoride (PMSF), and 1 µg/ml aprotinin]. After a 30-min incubation on ice, the cells were homogenized, and the material from the different donors was pooled and mixed with an equal volume of 85% sucrose (w/v) in MBS before placement in the bottom of a SW40 centrifuge tube. The sample was overlaid with 6 ml 35% sucrose and 4 ml 5% sucrose in MBS and spun for 14–16 h at 200,000 g. Rafts were harvested by collecting the band visible at the 35/5% sucrose interface, pelleted by microcentrifugation at 14,000 g for 30–40 min, and resuspended in 100–300 µl MBS buffer.

Postnuclear supernatant (PNS) was prepared by resuspending at least 1 x 108 cells in 1 ml homogenization buffer (0.25 M sucrose, 1 mM EDTA, 20 mM tricine, pH 7.8, 1 mM sodium vanadate, 1 mM PMSF, and 1 µg/ml aprotinin), followed by homogenization and centrifugation at 1000 g for 10 min. The supernatant was removed and stored on ice. The pellet was resuspended in 1 ml homogenization buffer, homogenized, and centrifuged as described above. The resulting supernatant was combined with the initial supernatant to form the PNS. The cytosol was collected by dilution of 0.5 ml PNS with 0.5 ml homogenization buffer and centrifugation for 1 h at 108,000 g. Plasma membrane was prepared by overlaying 30% Percoll in homogenization buffer with 1.5 ml PNS and centrifuging at 84,700 g for 35 min in a Beckman Ti 80 rotor. The opaque membrane band was then collected from the PNS/Percoll interface by aspiration with a long-tip Pasteur pipette. The protein concentrations of the obtained cytoplasmic, plasma membrane, and raft samples were determined by appropriate protein assays, using the BCATM assay (Pierce, Rockford, IL) for the lipid-rich raft samples and the DC assay (BioRad, Hercules, CA) for the other fractions because of their detergent content.

Slot-blot analysis of subcellular fractions for ganglioside GM1
Each subcellular fraction (5 µg; cytosol, plasma membrane, and rafts) was slot-blotted onto nitrocellulose in Tris-buffered saline (TBS; 20 mM Tris, pH 7.6, 137 mM NaCl), and the membranes were blocked in TBS containing 5% bovine serum albumin (BSA) for 1 h. Ganglioside GM1 was detected on the membrane by incubation with cholera-toxin B subunit (CTB)-conjugated horseradish peroxidase (HRP; Sigma Chemical Co.) in TBS-BSA for 1 h. Membranes were washed with TBS, and detection of the HRP-conjugated CTB was performed using enhanced chemiluminescence (ECL; Amersham, Piscataway, NJ) on Cronex film.

Immunoblots
Where needed, cells were lysed in lysis buffer (1% Triton X-100, 20 mM Tris, pH 7.4, 150 mM NaCl) containing protease and phosphatase inhibitors (10 µg/ml leupeptin and aprotinin, 1 mM PMSF, and 200 µM sodium vanadate). Cell lysates or cell fractions (10 µg) prepared as described above were submitted to polyacrylamide gel electrophoresis (PAGE) and were transferred to a nitrocellulose membrane. After transfer, membranes were blocked and washed extensively in TBS/0.1% Tween before hybridization with antibodies against the IL-2- or -15R chains, CD45, p56lck, JAK-1, JAK-3, Pan-STAT5, and grb-2 (all from Santa Cruz Biotechnology, Santa Cruz, CA), ZAP-70 (Transduction Laboratories, Lexington, KY), phospho-STAT5 A/B (Upstate Biotechnology, Lake Placid, NY), or actin (Sigma Chemical Co.), followed by repeat washes and incubation with appropriate HRP-conjugated secondary antibody. An HRP-conjugated antibody against phosphotyrosine (PY20, Transduction Laboratories) was used for antiphosphotyrosine blotting. The targeted proteins were visualized by ECL, all as described previously [24 ]. The detected bands were further quantitated by densitometry using the ChemiImager 4000 according to the manufacturer’s instructions (Alpha Innotech, San Leandro, CA).

Flow cytometry
Flow cytometric analysis was performed on fixed cells in the University of Kentucky’s flow cytometry core facility using fluorescein isothiocyanate (FITC)- and R-phycoerythrin-conjugated isotype controls or antibodies against the human IL-2R chains essentially according to the manufacturer’s instructions (PharMingen). The raft "marker" GM1 was stained with FITC-labeled CTB as described by Tuosto et al. [25 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Association of IL-2- and -15R chains with rafts
Experiments were conducted to determine the cellular localization of IL-2- and -15R chains in T cells stimulated with low-dose PHA to express the high-affinity IL-2R. Extracts of these cells were subjected to sucrose buoyant density separation to yield cytoplasmic, plasma membrane, and raft fractions. Subsequently, these cellular fractions were separated by PAGE, and Western blotting was performed to detect proteins characteristically associated with these fractions. As expected [8 , 11 , 23 ], the results show that ZAP-70 and CD45 are found predominantly in the cytoplasmic and membrane fractions, respectively, whereas p56lck is highly enriched in the raft fraction (Fig. 1 A ). Furthermore, we confirmed the enrichment of GM1 in the raft fraction obtained from Jurkat cells in slot blots (Fig. 1B) .



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  		Figure 1. Isolation and characterization of rafts. (A) Representative Western blots of homogenates of human T cells treated with low-dose PHA and separated into cytoplasmic (C), membrane (M), and raft (R) fractions as outlined in Materials and Methods, documenting the localization of ZAP-70 (cytoplasmic "marker"), CD45 (membrane "marker"), and p56lck (lck; raft "marker") in the respective fractions. (B) Representative slot blot demonstrating the relative enrichment of GM1 in raft fractions of unstimulated Jurkat cells, further confirming the fidelity of the fractionation protocol.

 
Having established the fidelity of cell fractionation procedures, we next performed experiments to identify the subcellular location of the IL-2- and -15R chains in low-dose, PHA-stimulated T cells. Thus, T cell extracts were processed as described above, and Western blotting was done to detect the presence of the IL-2- and -15R chains. The results show that before IL-2 stimulation, the {alpha}- and {gamma}-chains are found predominantly in the cytoplasmic and membrane fractions. However, the IL-2/15R ß-chain is enriched in the raft fraction (Fig. 2 ). For the other chains, similar results were obtained examining Jurkat cells, whereas no IL-2/15R ß-chain was detected in any of the Jurkat cell fractions (not shown), consistent with the lack of IL-2/15R ß-chain expression in these cells [26 ].



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  		Figure 2. Selective enrichment of the IL-2/15R ß-chain in rafts of T cells treated with low-dose PHA, documented in representative Western blots localizing the IL-2/15R chains in the cytoplasmic (C), membrane (M), and raft (R) fractions (also see Figs. 1 and 3 and Materials and Methods).

 
To exclude the possibility that the enrichment of the IL-2R ß-chain in rafts is a nonspecific effect of the PHA used for cell stimulation, we also examined fractions from T cells treated with a combination of anti-CD3 and anti-CD28 antibodies instead of PHA. Again, the IL-2R ß-chain—as well as p56lck—was found in rafts (not shown), but in a distribution pattern similar to that seen in PHA-treated cells after IL-2 stimulation (see below, Fig. 3A and Fig. 5 ). We are therefore investigating whether stimulation with anti-CD3 and anti-CD28 antibodies as compared with PHA results in the generation and binding of significantly increased amounts of IL-2 by the examined T cells.



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  		Figure 3. IL-2-induced, partial translocation of the IL-2/15R ß-chain from the raft to the plasma membrane fraction. (A) Representative Western blot of plasma membrane (M), cytosol (C), and raft (R) fractions obtained from human T cells treated with low-dose PHA and IL-2 (200 U/ml for 20 min). The results show that IL-2 stimulation induces the appearance of a substantial amount of IL-2R ß-chain in the plasma membrane fraction (compare with Fig. 2 ). (B) Representative densitometric analysis of the IL-2/15R ß-chain bands in the respective subcellular fractions before and after IL-2 treatment.

 


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  		Figure 5. Subcellular distribution of IL-2/15R ß-chain-associated signaling molecules before and after IL-2 stimulation of low-dose, PHA-treated T cells. Representative Western blots showing the relative distribution of p56lck, JAK-1, and grb-2 before and after IL-2 treatment of such cells. Cell lysates were separated by buoyant density centrifugation to obtain cytosol, plasma membrane, and raft fractions as described in Materials and Methods. Numbers below each panel indicate their relative optical density as percent of total signal in all three fractions.

 
Effect of IL-2 stimulation on the enrichment of the IL-2R ß-chain in rafts
Because IL-2 binding to its R induces the heterodimerization of the IL-2R ß- and {gamma}-chains [18 ], we asked whether this process involved the redistribution of the ß- or {gamma}-chain, out of or into rafts. Thus, low-dose, PHA-stimulated T cells were also treated with IL-2 and were fractionated as described above. The results show partial translocation of the ß-chain into the plasma membrane fraction after IL-2 stimulation (Fig. 3 ; compare Fig. 3A with Fig. 2 ). This observation suggests that IL-2 "recruits" IL-2/15R ß-chain molecules from rafts to the plasma membrane to facilitate its ligand-induced association with the {gamma}-chain. This possibility is further supported by the only modest colocalization of the IL-2R ß-chain with the raft marker GM1, which we found by confocal microscopy after rafts were "patched" (and the T cells thus further stimulated and altered in their membrane structure) with anti-CTB antibody to allow their microscopic detection (data not shown).

Quantitation of GM1 expressed by human T cells before and after PHA and IL-2 stimulation
To examine whether the IL-2-induced translocation of the IL-2R ß-chain from rafts occurs as a consequence of alterations in the overall integrity of rafts, experiments were done to quantitate and compare plasma membrane GM1 in resting or PHA-treated T cells with or without IL-2 stimulation using flow cytometry and slot blotting. As shown in Figure 4 , we found that membrane expression of GM1 is minimal in resting T cells, highest in T cells stimulated with 20 µg/ml PHA, and intermediate in T cells stimulated with 2 µg/ml PHA with or without the addition of IL-2. Furthermore, membrane expression of GM1 in unstimulated Jurkat cells is approximately threefold higher than in primary human T cells stimulated with high-dose PHA.



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  		Figure 4. Quantitation of the raft "marker" GM1 in the plasma membrane of T cells. Flow cytometric examination of cells with FITC-labeled CTB after the following treatments: immediately after sheep red blood cell rosetting (Resting), after a subsequent 3-day stimulation with 2 or 20 µg/ml PHA only (PHA 2 and PHA 20, respectively), or after additional IL-2 treatment (200 U/ml for 20 min; PHA 2 + IL-2). Untreated Jurkat cells were studied for comparison (Jurkat), and background fluorescence (omission of FITC-labeled CTB; Background) was also studied. The geometric mean of the fluorescence intensity (MFI) in each sample is indicated in the top right corner of each panel. The results represent one of at least three independent experiments with identical results.

 
As these flow cytometric studies only document the lack of effect of IL-2 on the overall membrane expression of GM1, we also performed a slot-blotting experiment comparing the relative GM1 content of rafts with nonraft membrane fractions before and after IL-2 stimulation and demonstrated that IL-2 did not alter this distribution in a manner comparable with its effect on the localization of the IL-2R ß-chain. Specifically, the relative amount of GM1 in the raft fraction as determined by densitometry of the blots was essentially identical before and after IL-2 exposure (50 and 51%, respectively, blots not shown).

These results indicate that addition of IL-2 to low-dose, PHA-stimulated, primary human T cells neither alters the overall amount of GM1 expressed on the cell surface nor shifts its relative distribution from raft to nonraft membrane fractions, suggesting that the IL-2-induced translocation of the IL-2R ß-chain from rafts does not occur as a consequence of alterations in overall raft integrity.

Subcellular distribution of IL-2/15R ß-chain-associated signaling molecules before and after addition of IL-2 to low-dose, PHA-stimulated T cells
If the IL-2R ß-chain partially translocates from rafts to the plasma membrane during IL-2 binding, signaling molecules associated with this chain and rafts, such as the kinase p56lck or the adapter molecule grb-2, should follow the chain to facilitate IL-2R signal transduction. This question was examined by separating lysates of low-dose, PHA-stimulated T cells with or without subsequent IL-2 treatment into cytosol, plasma membrane, and raft fractions, which were analyzed by Western blot for the presence of p56lck, JAK-1, and grb-2. The results show that in T cells stimulated only with low-dose PHA, the majority of p56lck as well as approximately 30% of JAK-1 and grb-2 is found in the raft fraction (Fig. 5 ). However, IL-2 stimulation triggers a substantial shift of all three of these proteins out of the raft fraction similar to that observed for the IL-2R ß-chain with which they associate (Figs. 2 3A and 5) . In contrast, we failed to detect the {gamma}-chain-associated kinase JAK-3 in rafts before or after IL-2 ligation (data not shown).

Effects of raft disruption of IL-2 signaling
To determine whether the localization of the IL-2/15R ß-chain to rafts in T cells is important for IL-2R signaling, we treated T cells with MBCD to induce cholesterol depletion. As shown in Figure 6A and 6B , IL-2-induced tyrosine phosphorylation of several proteins including STAT5 is reduced in MBCD-pretreated T cells. Specifically, 1 mM MBCD decreased the amount of tyrosine-phosphorylated STAT5 following IL-2 stimulation to 65 ± 19% (mean±SD) of that seen in the absence of MBCD when tyrosine phosphorylation was measured as the densitometric ratio of phospho-STAT5 to Pan-STAT5 in three separate experiments. The MBCD concentrations used in these experiments did not affect cell viability as determined by Trypan blue exclusion. MBCD itself did not induce obvious Tyrosine phosphorylation events either (first, third, and fifth lanes in Fig. 6A and 6B ), an effect of this agent previously observed in Jurkat cells and at higher MBCD concentrations than the ones used by us [27 ].



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  		Figure 6. Attenuation of IL-2-induced tyrosine phosphorylation events by disruption of rafts with MBCD in low-dose, PHA-treated T cells. The cells were induced to express the high-affinity IL-2R and were stimulated with IL-2 in the presence or absence of the raft-disrupting agent MBCD as described in Materials and Methods. (A) Representative antiphosphotyrosine immunoblot showing that MBCD treatment of the cells inhibits IL-2-induced tyrosine phosphorylation of proteins (top panel; arrows indicating several proteins phosphorylated by IL-2). The same membrane was then reprobed for actin to confirm consistent protein loading (bottom panel). (B) Inhibition of IL-2-induced STAT5 tyrosine phosphorylation by MBCD. Representative immunoblot demonstrating that the amount of tyrosine-phosphorylated STAT5 (Phospho-STAT5; top panel) decreases with increasing concentrations of MBCD, whereas the amount of total STAT5 detected by reprobing the membrane for Pan-STAT5 (bottom panel) does not. This observation is confirmed by densitometric analysis of the Phospho-STAT5/Pan-STAT5 ratio (Ratio; calculated as the quotient of densitometric values for Phospho-STAT5 and Pan-STAT5), indicating that in the experiment shown, the presence of 1 mM MBCD results in the reduction of the relative amount of tyrosine-phosphorylated STAT5 to almost half of its baseline amount. (C) Flow cytometric analysis of the effect of MBCD treatment on the membrane expression of IL-2R chains by human T cells, demonstrating that 1 mM of this raft-disrupting agent selectively reduces IL-2R ß-chain expression to almost half of its baseline value. The MFI in each sample is indicated in the top right corner of each panel.

 
To further confirm that MBCD attenuated IL-2 signaling through a raft (and thus IL-2/15R ß-chain)-specific effect, we assessed the effect of MBCD on the surface expression of the IL-2R chains by flow cytometry. As shown in Figure 6C , MBCD selectively attenuated expression of the IL-2R ß-chain, indicating that raft disruption with MBCD attenuates IL-2 signaling by altering the surface expression of the raft-enriched IL-2R ß-chain. Identical results were obtained when rafts were altered with nystatin (not shown), again without concomitant effects on cell viability.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
To our knowledge, this is the first demonstration that in human T cells, the IL-2/15R ß-chain, but not the {alpha}- and {gamma}-chains, is enriched in rafts. Using biochemical and morphological approaches, we show that the IL-2R ß-chain is present in rafts of T cells treated with low-dose PHA and that it can leave these rafts during IL-2 signaling. These data may explain previous observations, showing what appears to be the IL-2/15R ß-chain, but not the IL-2R {alpha}-chain, enriched in detergent-resistant membrane microdomains of IL-2-stimulated, murine lymphocytes [5 ]. This work was done not only before the current nomenclature for IL-2R chains had been established and at a time when the existence of the {gamma}-chain was just beginning to be appreciated, but also using experimental approaches rather different from those applied by us. Moreover, the absence of the IL-2R {alpha}-chain from rafts was recently shown by Saint-Ruf et al. [14 ] in murine splenocytes. Conversely, another study suggests a functional association of the IL-2R {alpha}-chain with rafts in murine lymphoma cell lines [7 ]. During the revision of this paper, Marmor and Julius [16 ] and Lamaze et al. [17 ] published papers that complicated the picture still further. The former group found only the IL-2R {alpha}-chain to be enriched in lipid rafts prepared from an IL-2-dependent, murine T cell line using a fractionation strategy different from ours. Marmor and Julius [16 ] did not find JAK-1 in rafts or observe any effects of IL-2 stimulation on the distribution of any of the molecules studied. In contrast, Lamaze et al. [17 ] demonstrated the presence of the IL-2R ß-chain in rafts isolated from YT cells before IL-2 stimulation. Furthermore, this group found additional recruitment of the ß-chain into rafts after IL-2 treatment, but this recruitment was most apparent when the cells were exposed to IL-2 at 12°C. More in agreement with our data, these investigators also observed the absence of any effects of IL-2 on the relative subcellular distribution of GM1. As discussed by Marmor and Julius [16 ], these fairly discrepant observations may be related to the type of cells examined, their degree of activation or IL-2 stimulation, or the experimental methods used. For example, our flow cytometric data indicate that there are significant differences between primary human T cells and Jurkat cells in terms of the amount of GM1 and thus presumably rafts present in the plasma membrane. Furthermore, several transformed T cell lines are known to feature constitutive IL-2R signaling [25 ], thus limiting the comparability of IL-2R signal transduction in such cell lines with that in primary T cells.

Other cytokine R chains, the IFN-{alpha}/ß R 1 and the IFN-{gamma} R 2, have been detected in caveolar membrane domains of HeLa cells [10 ]. Caveolae, which are not found in T cells [12 ], are considered a type of lipid rafts and play important roles in signal transduction and possibly protein trafficking in many different cells [11 , 28 ]. It is interesting that Takaoka et al. [10 ] also found several IFN R-associated NRTKs, JAK-1 and -2, in caveolae, paralleling the observation that NRTKs are found in lymphocyte rafts [8 ]. In support of this, we detected JAK-1, but not JAK-3, in rafts, further suggesting a relationship between rafts and caveolae. Additionally, our observation that IL-2 binding results in only partial translocation of IL-2R ß-chain-associated and raft-enriched signaling molecules to the plasma membrane may be explained by the association of such molecules with other Rs, e.g., the predominant association of p56lck with CD4 [29 ] or that of JAK-1 with certain IFN R chains [10 ]. These associations obviously would not be affected by IL-2 treatment, and they would thus serve to maintain a substantial amount of protein kinase molecules within the raft fraction. Nonetheless, our data indicate that the localization of the IL-2/15R ß-chain, but not the {alpha}- or {gamma}-chains and the {gamma}-chain-associated kinase JAK-3, in rafts is related to the chain’s known association with the NRTKs p56lck and JAK-1 and the adapter molecule grb-2 [30 ], all of which are also enriched in rafts or caveolae [8 , 9 ]. Our data are also consistent with the recent demonstration that H-ras is enriched in rafts and translocates to the nonraft plasma membrane after cell stimulation [31 ]. This is noteworthy because ras is involved in signal transduction through the IL-2R, specifically via the IL-2R ß-chain and the ß-chain-downstream adapter grb-2 [30 ].

CD4 has not only been demonstrated to be highly enriched in rafts [8 ], but CD4 and signaling through this co-R are also linked to the cytoskeleton [20 , 32 ], suggesting a connection between the cytoskeleton and rafts [33 34 35 ]. A similar interaction of the raft-enriched IL-2/15R ß-chain with the cytoskeletal system may also exist, because we have recently found an association between the IL-2/15R ß-chain and the tubulin cytoskeleton (see ref [36 ], and unpublished results). A direct relationship between the tubulin cytoskeleton and rafts has also been suggested recently [37 ], further supporting the possibility that the IL-2R ß-chain could be involved in an interaction between rafts and cytoskeletal tubulin. The significance of this interaction remains to be further explored.

The biological relevance of the association of the IL-2/15R ß-chain with rafts and cytoskeletal tubulin remains unclear, although our finding of modestly attenuated IL-2 signaling after raft disruption suggests that the enrichment of the IL-2R ß-chain in rafts may have functional significance. This latter observation supports the findings of Zhao et al. [6 ], who described attenuated IL-2 signal transduction in spleen cells from mice who lack complex ganglioside and thus intact rafts. It is possible that in the absence of IL-2, the IL-2/15R ß-chain is protected from internalization by its sequestration in rafts and that cytoskeletal tubulin participates in this process. This would resemble the involvement of microtubules in the kinetics of other R chains such as the transferrin or the ß2-adrenergic R described by others [38 39 40 ]. Thus, IL-2 binding may initiate a process that alters the sequestration of the IL-2R ß-chain in rafts, allowing it to move out of rafts in preparation for subsequent internalization and degradation steps described by Hémar et al. [42 ]. In support of this, we have observed increased IL-2R ß-, but not {alpha}- or {gamma}-, chain surface expression on human T cells treated with taxol to enhance the polymerization of their cytoskeletal tubulin (see ref [36 ], and manuscript in preparation).

An area requiring further clarification is the effect of MBCD on IL-2R signal transduction. MBCD is well established as an agent capable of disrupting rafts by preferential cholesterol extraction [28 , 43 ]. Thus, the MBCD-induced, selective reduction in IL-2/15R ß-chain surface expression, which we detected by flow cytometry, strongly suggests that the attenuation of IL-2 signaling by MBCD is indeed a consequence of MBCD-induced raft disruption. The decreased amount of ß-chain detectable by flow cytometry after MBCD treatment further indicates that the chain does not merely "leak" into nonraft areas of the cell membrane. Rather, the chain may be internalized or degraded, raising the possibility that localization in rafts may be important in the regulation of its turnover as discussed above and suggested by Lamaze et al. [17 ]. Alternatively, MBCD could extract the ß-chain from the membrane, resulting in its release into the medium. To further clarify whether raft alterations indeed modulate IL-2 signaling by affecting the enrichment of the IL-2/15R ß-chain in these domains, we are initiating additional studies using alternative methods of raft modification, e.g., the addition of exogenous gangliosides [28 ], internalization studies of the IL-2R ß-chain similar to those done by others [17 , 42 ] but in the presence or absence of MBCD or other raft-altering agents, and work aimed at detecting the chain in the culture medium before and after raft disruption.

Finally, it becomes important to assess whether the selective enrichment of the IL-2R ß-chain in rafts may be an example of a novel mechanism to preserve cytokine selectivity in signaling through a limited and redundant number of R chains. As the IL-2R ß-chain pairs up with the {gamma}-chain for IL-2 signal transduction, and the {gamma}-chain is associated with several other IL R chains as the common cytokine R {gamma}-chain [18 ], it is possible that raft enrichment of less "promiscuous" cytokine R chains serves to control specific cytokine responsiveness in T cells. Accordingly, preliminary work by us (not shown) indicates raft enrichment of the IL-4R {alpha}-chain, which is similar in structure and function to the IL-2R ß-chain and heterodimerizes with the common cytokine R {gamma}-chain to form the IL-4R [18 ]. Thus, sequestration of certain cytokine R chains in rafts before ligand binding could allow their subsequent, selective recruitment out of these domains by specific cytokines. Alternatively, this binding and the associated heterodimerization of the IL-2R ß-chain (and presumably other chains initially enriched in rafts) with chains not found in rafts, such as the common cytokine R {gamma}-chain, could merely result in a loss of buoyancy and thus of raft localization.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
Our data support the existence of a biologically relevant relationship among the IL-2R ß-chain, associated signaling molecules, lipid-enriched membrane domains, and the cytoskeleton in human T cells. We propose that these relationships may serve to preserve ligand selectivity and specificity, especially in the redundant area of cytokine signaling, and to down-regulate signaling by modulating R chain internalization and degradation. Further clarification and characterization of these relationships and their functional implications in future studies as suggested above should elucidate their exact role in the regulation of lymphocyte activation and homeostasis.


   ACKNOWLEDGEMENTS
 
This work was supported by the Department of Pediatrics, University of Kentucky, Lexington. We also gratefully acknowledge J. G.’s support by the University of Kentucky Hospital under the Physician-Scientist Program and by the Bristol Myers Squibb Young Investigator Grant of the National Kidney Foundation.

Received August 25, 2001; revised January 23, 2002; accepted February 20, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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