Published online before print December 4, 2003
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,
,
,1
* Research Center on Aging, Geriatric Institute,
Graduate Program in Immunology, Clinical Research Center, and Departments of
Biochemistry, Faculty of Medicine, and
Medicine, Geriatric Division, University of Sherbrooke, Quebec, Canada
1 Correspondence: Research Center on Aging, University of Sherbrooke, 1036 Belvedere St. South, Sherbrooke, QC, Canada J1H 4C4. E-mail: tamas.fulop{at}usherbrooke.ca
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Key Words: T cell receptor • LAT • p56Lck • aging • cholesterol
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-dimer (reviewed in ref. [7
]). -Associated protein-70 (ZAP-70) is recruited to the -dimer and phosphorylated by Lck [8
]. Activated ZAP-70 phosphorylates the linker of activated T cells (LAT), which becomes a scaffold protein for the recruitment of multiple partners [9
] including the adaptor proteins Gads and Grb2 and the enzymes of phospholipid metabolism, phosphatidylinositol 3-kinase and phospholipase-C
1 (reviewed in ref. [10
]). LAT-associated Gads bring SLP-76 to the plasma membrane, where it becomes phosphorylated, allowing its interactions with the exchange factor Vav and the adaptor proteins SLAP-130/Fyb, which provide a link among T cell activation, up-regulation of integrin affinity/avidity, and reorganization of the cell cytoskeleton (reviewed in ref. [11
]). Although the engagement of the TCR provides an essential signal to T cells, commitment to proliferation and differentiation will not occur unless a secondary signal is provided by ligation of CD28 [12
, 13
].
A number of components of the T cell activation complex are recruited to plasma membrane microdomains that are termed lipid rafts or detergent-resistant microdomains (DRM; reviewed in ref. [14
]). These domains are composed primarily of high-melting sphingolipids packed with cholesterol that generate a liquid-ordered phase arrangement [15
, 16
]. DRM can act as molecular filters, in which case, some plasma membrane proteins having an affinity for DRM are brought into molecular contacts, whereas others are excluded [16
17
18
]. Current evidence suggests that DRM provide a platform for the recruitment of proteins involved in the formation of the initial steps of T cell signaling [19
]. For instance, proteins critical to T cell activation, such as src, PTK, LAT, protein kinase C (PKC)-
, Gads, Wiskott-Aldrich syndrome protein [18
, 20
], and a host of other proteins [21
], are recruited to DRM following TCR ligation. Current data support the notion that DRM are essential for T cell activation and interleukin-2 (IL-2) production [22
] and that ligation of CD28 is required to enhance the coalescence of DRM [23
].
A decrease in the immune response is generally associated with aging (reviewed in ref. [24 ]). A host of data supports the notion that T cell functions such as proliferation and cytokine production are perturbed in aged individuals [25 ]. The cause of these age-related alterations in T cell functions is not fully understood. However, atrophy of the thymus, a shift in T cell subpopulation toward memory cell types, cytokine shift toward T helper cell type 2, and alterations in T cell-associated signal transduction have been suggested as possible causes (reviewed in ref. [26 ]). Furthermore, there is a decline in CD28 but not TCR expression [27 , 28 ]. A number of studies have reported defects in the early events of the T cell signaling cascade with aging in humans [25 , 27 , 29 ] and mice [30 , 31 ]. These defects observed include altered tyrosine phosphorylation of signaling proteins, calcium mobilization, activation of the mitogen-activated protein kinase and c-jun NH2-terminal kinase pathways, translocation of nuclear factor of activated cells to the nucleus, IL-2 production, and less efficient immune synapse formation as a result of defects in signaling protein recruitment to DRM (reviewed in ref. [31 ]). In this connection, the cholesterol content of T cells is increased with aging in humans [32 , 33 ], and that may be related to the decrease in plasma membrane fluidity observed in aged human T cells (reviewed in ref. [34 ]).
We have previously reported alterations in signal transduction in T lymphocytes with aging in human T cells [35 ]. In the present work, we extend our studies to the cholesterol content of DRM as well as signaling molecule recruitment in DRM. We observed that the cholesterol content of DRM was significantly higher in T cells from elderly subjects as compared with young individuals, whereas there were the opposite findings in the case of plasma membrane and DRM fluidity. Confocal microscopy analysis revealed a defect in the coalescence of DRM in T cells of aged subjects exposed to monoclonal antibody (mAb) directed against CD3 or a combination of CD3 and CD28. In addition, the association of phosphorylated Lck (pLck) and pLAT in DRM was significantly decreased in the case of T cells from elderly subjects. Our data suggest that the recruitment of key signaling proteins associated with the early events of T cell activation is impaired with age, and this finding may be of importance in the decline of T cell functions associated with aging.
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Subjects
Twenty elderly volunteers, aged 65–78 years (mean, 73 years), participated in the study, which included 17 women. The cohort of 20 young, healthy subjects, aged 19–25 years (mean, 22 years), included 14 women. A local institutional (Research Center on Aging, Sherbrooke, Quebec, Canada) ethics committee approved the research protocol. All subjects gave written, informed consent. The lipid profile was determined by routine biochemical analysis at the Clinical Biochemistry Laboratory of the Centre Hospitalier Universitaire de Sherbrooke (Quebec, Canada). All the subjects were in good health, normolipidemic, and satisfied the inclusion criteria of the SENIEUR protocol for immune investigations of human, elderly subjects [36
].
T lymphocyte preparation
Heparinized blood was obtained by veinpuncture and diluted twofold with RPMI-1640 medium containing 2% fetal bovine serum. Ficoll-Hypaque density sedimentation isolated lymphocytes [32
]. The resulting mononuclear cell fraction was depleted of monocytes by adhesion to plastic tissue-culture flasks coated with autologous serum (1 h, 37°C). B lymphocytes and residual phagocytic cells were removed by absorption to a prewarmed, nylon wool column (1 h, 37°C). The resulting, highly enriched T cell population was phenotyped by flow cytometry and shown to contain less than 3% contaminating B or natural killer (NK) cells. T cell preparations used in the assays consisted of greater than 96% CD3-positive cells with less than 1.0% surface immunoglobulin M (B cells)-, CD16 (NK cells)-, and CD14 (monocytes)-positive, contaminating cells. Cell viability was greater than 95% (Trypan blue staining). Identical numbers of T cells from young and aged donors were used in each comparative experiment.
T cell stimulation and isolation of DRM on sucrose gradients
Freshly prepared T cells were kept for 1 h in RPMI medium at 37°C. The cells (20x106 lymphocytes) were then exposed to a combination of anti-CD3 (5 µg/ml) and anti-CD28 (5 µg/ml) mAb for various periods of time at 37°C as described [33
]. Control cells (20x106 lymphocytes) were left untreated. The cells were lysed (4°C, for 30 min) by treatment with 300 µl Hepes-buffered saline (25 mM Hepes, 100 mM NaCl, pH 6.9) containing 0.5% (v/v) Triton X-100, 2 mM EDTA, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 10 mM phenylmethane sulfonate, and 200 mM orthovanadate. An 85% (w/v) solution of sucrose in Hepes-buffered saline was added to a final concentration of 42.5%, and the solution transferred to 2 ml ultracentrifuge tubes. The lysate was gently overlayed with 1 ml sucrose, 35% (w/v), and 300 µl sucrose of 5% dissolved in Hepes-buffered saline [25
]. Centrifugations were performed at 4°C for 16 h at 200,000 g in a Beckman TLA-100.4 rotor (Beckman Instruments, Montreal, QC). Nine fractions of 200 µl each were collected from the top of the gradient. DRM fractions were selected on the basis of their content in GM1, which was revealed as follows: CTxB (10 µg/ml) was added to 20 x 106 lymphocytes, which were lysed as above. The lysate was sized on SDS-PAGE, transferred to nitrocellulose membranes, and Western blotted as described in the next section using indirect immunodetection with an anti-CTxB polyclonal Ab.
Western blotting
Proteins (20 µg) from total cell lysates and 25 µl from sucrose gradient fractions were diluted in Laemmli’s buffer under reducing (DL-dithiothreitol) conditions. The samples were separated by SDS-PAGE (10% acrylamide gels) as described [33
]. Proteins were transferred to nitrocellulose membranes, which were treated (1 h, room temperature) with Tris-buffered saline (20 mM Tris, 137 mM NaCl, pH 7.6) containing 0.1% (v/v) Tween 20 and a solution of 5% (w/v) skimmed, powdered milk. They were then incubated with the relevant antibodies, antiphosphotyrosine (1:1000), anti-CD45 (1:1000), anti-LAT (1:1000), anti-Lck (1:1000), or anti-pLAT (1:200). The membranes were placed on a rotary end-over-end mixer and incubated overnight at 4°C under constant rotary movement. The membranes were washed, and the corresponding secondary antibody conjugated to horseradish peroxidase was added (1:2000) for 1 h. The membranes were then washed, and ECL revealed proteins. Low-density fractions 1–3 corresponded to DRM. Densitometric analyses were performed using an image analyzer, Chemigenius2 Bio Imaging System (Syngene, Frederick, MD).
Quantification of cholesterol
Cholesterol concentrations were measured in sucrose gradient fractions of T cell lysates using a modification of the procedure of Katsanidis and Addis [37
]. Briefly, 100 µl each fraction, from 20 x 106 cells, was added to a mixture of chloroform/methanol (2:1) and vigorously mixed for 2 min. The tubes were centrifuged, and the organic layer was collected and analyzed by HPLC. The chromatographic system consisted of a Shimadzu pump and a photo-diode array detector (Shimadzu Scientific Instruments, Guelph, ON). Samples (50 µL) were applied to a silica-packed column (Zorbax RX-SIL, 5 µm particle size, 4.6x250 mm). The mobile phase was a mixture of hexane-isopropanol (99:1), and the flow rate was 1.3 ml/min. Effluents were monitored at a wavelength of 202 nm. The retention time of cholesterol was 4 min.
Fluorescence anisotropy measurements in T cells and DRM derived from these cells
Cell membrane anisotropy was determined using the fluorescent probe diphenylhexatriene (DPH) dissolved in tetrahydofuran (4 mM) [38
]. The cell suspension (5x105 lymphocytes/ml) was incubated in the presence of 2 µM DPH for 30 min at 37°C, washed, and resuspended in buffered saline solution (BSS; 130 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1.2 mM MgCl2, 10 mM Hepes, and 10 mM glucose, pH 7.2). In the case of measurement of anisotropy of DRM, 100 µl of the pooled fractions (1–3) from 20x106 T cells was diluted in 1 ml BSS, and the lipid probe was added, as described in the case of whole T cells. The DRM samples were centrifuged (1 h, 100,000 g), the supernatants discarded, and the cell fractions resuspended in BSS as described [39
]. Fluorescence was recorded on a HITACHI spectrofluorimeter F-4500 coupled to a Vextra polarizer (Oriental Motor Co. Ltd., Japan). The lipid probe was excited by a vertically polarized light at 360 nm, and the emitted light was recorded at 430 nm through a polarizer orientated parallel and perpendicular to the direction of polarization of the excitation beam. Steady-state fluorescence anisotropy (r) was calculated as follows: r = (Iv-GIp)/(Iv+2GIp), where Iv and Ip are, respectively, the parallel and perpendicular polarized fluorescence intensities, whereas G is the monochromator grating correction factor. Fluidity (f) was derived from the inverse value of anisotropy (f=1/r).
Laser scanning confocal microscopy (LSCM)
Circular glass coverslips (22 mm) were coated with poly-L-lysine (0.1 mg/ml) for 15 min at 37°C. The coverslips were then coated with an anti-CD3 mAb (5 µg/ml) or a combination of anti-CD3 (5 µg/ml) and anti-CD28 (5 µg/ml) for 1 h at 37°C and were washed. T cells (1x106 lymphocytes) in phosphate-buffered saline (PBS; 1 ml) were treated with Alexa 594-conjugated CTxB (10 µg) for 15 min at 4°C, washed, and resuspended in the same medium. The cells were allowed to adhere to the coverslips (10 min), and fluorescence was recorded along the z-axis after 5 min of contact. The LSCM system was from Thermo Noran (Middleton, WI) and has already been described in detail [40
]. Optical sections (0.2-µm thick) corresponding to the region of contact of the cell with the coverslip were recorded. The CTxB-labeled cells were excited at 568 nm through a 10-µm pinhole aperture, and the emitted fluorescence was measured through a long-pass filter (>590 nm). Image processing and surface quantification of pixel intensities were done using the NIH Image freeware (http://rsb.info.nih.gov/nih-image/). Representations of fluorescence intensities according to a palette of pseudocolors were generated by means of the NIH Image freeware, which was also used to quantify surface pixel intensities.
Statistical analysis
Results were analyzed using Student’s t-test for paired data, and significance was set for P < 0.05 or as indicated.
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Table 1. Cholesterol Concentration in T Cells and in DRM and Non-DRM Fractions Derived from T Cells of Young and Elderly Subjects
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Figure 1. Membrane fluidity of T cells and DRM derived from T cells of young and elderly donors. (A) T cells (5x105 lymphocytes) were labeled with the fluorescent probe DPH. Fluorescence was measured by excitation at 360 nm and emission at 430 nm through a polarizer. *, Significant differences (P<0.002, n=5) between the fluidity (f) of T cells from young (solid bar) and elderly (open bar). (B) Similar analysis performed using DRM isolated from 20 x 106 T cells. *, Significant differences (P<0.05, n=4) between DRM from T cells of young (solid bar) and elderly (open bar) donors.
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Figure 2. LSCM analysis of the distribution of GM1-associated fluorescence in T cells from young and elderly subjects. T lymphocytes were labeled with an Alexa 594-CTxB conjugate and exposed to coverslips coated with poly-L-lysine (Poly K) or additionally, with an anti-CD3 mAb or a mixture of anti-CD3 and anti-CD28 mAb. The cells were maintained at 37°C in PBS. The solid rectangles shown under each figure correspond to a scale of 2 µm. The palette of pseudocolor intensities is indicated on the figure, on a scale of 0–32 arbitrary units. Pixel intensities were recorded in 0.2-µm-thick sections along the z-axis corresponding to contact of the cell with the coverslip. Representations in pseudocolors of pixel intensities recorded in the case of a T cell from a young (A) and an elderly (D) individual in contact with a poly K-coated coverslip. The next series of images are representations in pseudocolors of pixel intensities recorded in the case of a T cell from a young (B) and an elderly (E) individual in contact for 5 min with a poly K- and anti-CD3-coated coverslip. The intensities of pixel intensities in pseudocolors in the case of a T cell from a young (C) and an elderly (F) individual in contact with a coverslip that had been coated with a mixture of poly K, anti-CD3, and anti-CD28 mAb. (G) The graph corresponds to pixel intensities integrated over the surface for young (solid bars) and elderly donors (open bars). Data are shown as mean ± SD (indicated by the error bars). *, Significant differences between the two groups (P<0.05). Data are representative of five donors.
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Figure 3. Distribution of Lck and pLck in DRM of T cells of young and elderly subjects. A T cell (20x106 lymphocytes) lysate was separated on a sucrose density gradient. Fractions corresponding to DRM (fractions 1–3) were collected from the top of the gradient, pooled, and analyzed by Western blotting. (A) Results showing the distribution of Lck in pooled DRM fractions of resting [unstimulated (none)] and stimulated [anti ( )CD3/CD28, 5-min treatment] T cells obtained from young and elderly donors. (B) Corresponding densitometric results of Western blots of Lck in DRM of resting and activated T cells from young (Y, solid bars) and elderly (E, open bars) donors. Data are expressed as the mean ± SD (n=4). (C) Distribution of pLck in pooled DRM fractions in the case of resting [unstimulated (none)] and stiumlated (CD3/CD28, 5-min treatment) T cells from young and elderly donors. (D) Corresponding densitometric results of Western blots of pLck in DRM of resting (unstimulated) and activated (CD3/CD28) T cells from (Y, solid bars) and elderly (E, open bars) donors. Data are shown as the mean ± SD (n=6). Statistical significance between the two groups is indicated. *, P < 0.01; **, P < 0.001.
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Figure 4. Distribution of LAT and pLAT in T cells of young and elderly subjects. T cell lysates (20x106 T lymphocytes) were separated on a sucrose density gradient. Fractions corresponding to DRM (fractions 1–3) were collected from the top of the gradient and analyzed by Western blotting. (A) Distribution of LAT in DRM fractions of resting [unstimulated (none)] and stimulated (CD3/CD28) T cells. (B) Corresponding densitometric analysis of the Western blots of LAT in DRM from resting and activated T cells from young (Y, solid bars) and elderly donors (E, open bars). (C) Distribution of pLAT in DRM fractions in the case of resting [unstimulated (none)] and activated (CD3/CD28) T cells for both groups of donors. (D) Corresponding densitometric analysis of the Western blots of pLAT in DRM from resting and activated T cells from young (Y, solid bars) and elderly donors (E, open bars). The graphs are representative of four independent experiments shown as the mean ± SD. Statistical significance is indicated: *, P < 0.01.
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Figure 5. Distribution of CD45 in DRM of T cells of young and elderly subjects. The cells were left untreated (none; A and B) or exposed (5 min) to a mixture of CD3 and CD28 mAb (C and D) and were lysed, and the lysates were separated on a sucrose density gradient. Fractions 1–9 were recovered, and CD45 (A-D) was revealed by Western blotting using the appropriate mAb. The blots shown are representative of five independent experiments.
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Aging has been reported to be associated with a decrease in the fluidity of the T cell plasma membrane [34 ]. We measured the fluidity of T cells from young and elderly donors and DRM derived from these cells. The differences in the protocols of labeling did allow a direct comparison between T cells and T cell fractions. Data showed that the decrease in fluidity of T cells observed with aging [34 ] was also found in DRM, which are key elements of T lymphocyte activation [14 , 42 ]. These alterations in the physicochemical properties of T cell plasma membrane could affect the formation of DRM and their lateral mobility as a result of the high levels of plasma membrane cholesterol [42 , 53 ]. We used LSCM to obtain evidence of decreased mobility of DRM in T cells from aged individuals. T cells from both groups of donors were challenged with insolubilized mAb directed against the TCR and CD28 or control, and the location of DRM was visualized with an Alexa 594-labeled CTxB, a marker of the GM1 ganglioside [40 ]. Viola et al. [23 ] have shown that labeled CTxB did not induce lipid raft coalescence in resting T cells. We confirmed these results in resting T cells (Fig. 2A and 2D) . The levels of pixel intensities were higher in the case of T cells from young as opposed to elderly individuals, whether an anti-CD3 or a combination of anti-CD3 and anti-CD28 was used to activate the cells (Fig. 2G) . The combination of anti-CD3 and anti-CD28 mAb was more than twice as effective as the anti-CD3 mAb in triggering the coalescence of fluorescence (Fig. 2G) , in agreement with the findings of Viola et al. [23 ], who showed that ligation of the TCR complex and CD28 was needed to induce maximal coalescence of lipid rafts in human T cells. The decrease in DRM movement in the membrane bilayer observed here may contribute to the defects in T cell proliferation seen with aging [33 ], as plasma membrane and especially DRM integrity are needed for T cell activation through the immunological synapse [54 ].
Recent data suggest that the differential localization of signaling proteins to DRM plays a key role in T cell activation [18 , 21 ]. Miller and his group [30 ] have reported an alteration in several components of the signaling complex in memory and naïve T cells from aged mice. They observed a reduced activation of several DRM-associated proteins (LAT, PKC, and Vav) and a decline in the proportion of cells that induces a redistribution of LAT and Vav to the T cell-APC synapse upon ligation of the TCR. Studies performed here revealed that exposure of T cells to a combination of anti-CD3 and anti-CD28 mAb triggered an increase of Lck redistribution to DRM fractions, which was higher in the case of young as compared with elderly donors (Fig. 3) . These differences were not related to the levels of expression of Lck, as we have reported that its expression did not change with aging [29 ]. There is a decline in the levels of tyrosine-phosphorylated proteins following the activation of T cells from aged humans [27 ] and mice [55 ], and these differences could be explained by a differential distribution in DRM. This possibility was investigated, and data shown here clearly indicated that the activation of T cells resulted in increased levels of association of Lck and pLck in DRM of young, but this level was lower in the case of elderly subjects (Fig. 3) . Our group and others [29 , 43 ] have already observed the low levels of activation of Lck in T cells at the whole cell level. Here, we show for the first time that this decline also occurs in specific membrane microdomains. The decline in Lck association to lipid rafts may also be explained by an altered interaction with LAT, as it has been shown that LAT regulates Lck in T cells [56 ].
LAT is an essential component of the assembly of the machinery of signal transduction in T lymphocytes [9 , 57 ]. We found that LAT was distributed approximately to the same extent in DRM derived from T cell lysates of young and elderly subjects (Fig. 4) . However, the activation of the T lymphocytes resulted in lower association of LAT with DRM in T cells from elderly donors as compared with young donors. There were no changes in the total expression of the protein. Data were even more striking when the distribution of pLAT in DRM was analyzed. Results showed more than a tenfold increase in DRM-associated pLAT in the case of activated T cells from young subjects (Fig. 4) . In contrast, this increase in DRM-associated pLAT was significantly less in T cells from elderly subjects. These findings are in agreement with those observed in the case of T cells from aged mice [30 , 58 ] and may be part of the defects in downstream pathways of T cell activation and the chronic inflammatory process associated with aging [24 , 44 45 46 , 59 ].
CD45 relieves the self-inhibition of Lck by dephosphorylating a single tyrosine residue located in the C-terminal of the kinase [60 ]. The regulation of Lck activity may be provided by transient localization of CD45 in DRM or by sequestering these interacting proteins into distinct compartments [61 ]. However, most reports have concluded that CD45 is excluded from DRM in various experimental models [6 ]. Here, we found that CD45 was exclusively located in the heavy density sucrose fractions in resting and activated T cells from young and elderly individuals, independently of the state of activation of the T cells (Fig. 5) . This lack of differences between the two groups of donors suggests that CD45 may not play a major role in the defects in T cell signaling that is observed with aging.
Defects in the transduction of mitogenic signals following TCR stimulation are attractive candidates to explain the decrease in IL-2 production that has been observed with aging. DRM are involved in several processes leading to T cell activation and IL-2 production via communication with the immunological synapse [62 ]. The bulk of our data reveals alterations in the properties of DRM with aging, which include an increase in cholesterol content, impaired DRM coalescence, and selective differences in the recruitment and activation of key proteins involved in T cell signaling. This could affect the formation of the immune synapse, which seems to be linked to lipid raft coalescence [63 ]. These alterations could explain, in part, the downstream defects in T cell signaling in aged humans. Further studies on the regulation of cholesterol metabolism in aged T cells may help to understand these alterations and to better define the role of DRM in aging and age-related diseases (reviewed in ref. [63 ]). Hence, studies in T cell subsets taking into account the specific senescence markers (including CD57 and CD28) will help to determine whether their responsiveness is selectively affected in aged humans. Current investigations are addressing these questions in our laboratories.
Received July 9, 2003; revised September 19, 2003; accepted October 6, 2003.
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to membrane rafts is required for T cell activation Nat. Immunol. 2,556-563[CrossRef][Medline]2ß1 and 4ß1 integrins to lipid rafts in Jurkat T lymphocytes exposed to collagen type IV and fibronectin J. Leukoc. Biol. 73,243-252This article has been cited by other articles:
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