(Received for publication, April 22, 1997, and in revised form, May 28, 1997)
From the Department of Internal Medicine III,
University of Vienna, Währinger Gürtel 18-20, A-1090
Vienna, Austria, ¶ Institute of Immunology, Vienna International
Research Cooperation Center, University of Vienna, Brunnerstrasse 59, A-1235 Vienna, Austria and
Institute of Molecular Genetics,
Academy of Sciences of the Czech Republic, Víde
ská
1083, 14220 Prague 4, Czech Republic
Glycosylphosphatidylinositol (GPI)-anchored proteins can deliver costimulatory signals to lymphocytes, but the exact pathway of signal transduction involved is not yet characterized. GPI-anchored proteins are fixed to the cell surface solely by a phospholipid moiety and are clustered in distinct membrane domains that are formed by an unique lipid composition requiring cholesterol. To elucidate the role of membrane lipids for signal transduction via GPI-anchored proteins, we studied the influence of reduced cellular cholesterol content on calcium signaling via GPI-anchored CD59 and CD48 in Jurkat T cells. Lowering cholesterol by different inhibitors of cellular cholesterol synthesis suppressed calcium response via GPI-anchored proteins by about 50%, whereas stimulation via CD3 was only minimally affected (<10%). The decrease in overall calcium response via GPI-anchored proteins was reflected by inhibition of calcium release from intracellular stores. Cell surface expression of GPI-anchored proteins was not changed quantitatively by lowering cellular cholesterol, and neither was the pattern of immunofluorescence in microscopic examination. In addition, the distribution of GPI-anchored proteins in detergent-insoluble complexes remained unaltered. These results suggest that cellular cholesterol is an important prerequisite for signal transduction via GPI-anchored proteins beyond formation of membrane domains.
Activation of lymphocytes is the first step in any immune reaction. Apart from the specific interaction of major histocompatibility complex-coupled antigens with the T cell receptor complex, costimulatory factors are effective modulators of the immune response. Several glycosylphosphatidylinositol (GPI)1-anchored proteins can provide costimulatory signals to T cells (1-5). On human lymphocytes, CD59 (5, 6), CD55 (decay accelerating factor (7)), CD52 (8), and CD73 (9, 10) were shown to be involved in activation. On murine T cells, evidence for signal-transducing GPI-anchored proteins include Ly-6 (11-14), the major histocompatibility complex Ib molecule Qa-2 (15, 16), Thy-1 (17, 18), and sgp60, the murine analogue to CD48 (19-22). A variety of responses could be elicited by cross-linking of different GPI-anchored proteins on T lymphocytes, which included early events such as rise in intracellular calcium (6, 10, 11, 16), and protein tyrosine phosphorylation (1, 4, 23, 24). In addition, several late events have been observed as interleukin-2 production and proliferation (6, 7, 9, 11-13, 15, 16, 19, 21). Thus, GPI-anchored proteins may play a role in modulating the immune response.
GPI-anchored proteins are linked to the plasma membrane by a phospholipid moiety residing in the outer leaflet of the lipid bilayer (25). Since GPI-anchored proteins have no direct connection to the cytoplasm, the mechanism by which signal transduction can occur is not yet elucidated. However, GPI-linked proteins are clustered in membrane domains (26-28) that may be involved in T lymphocyte activation by mediating the association with Src family tyrosine kinases p56lck and p59fyn (1, 5, 26, 28-31). GPI-anchored protein complexes exhibit a particular lipid composition enriched in cholesterol and sphingolipids (32) that appears to be important for their clustering in living cells and the insolubility of GPI-anchored protein complexes in mild non-ionic detergents in vitro (32-35). Cellular transport and function of GPI-anchored folate receptor and alkaline phosphatase was abolished by sequestrating plasma membrane cholesterol by specific detergents (35-38). Accordingly, exogenously added CD59 was prevented from entering GPI complexes by filipin (5), and internalization of endogenous CD59 was partially inhibited by nystatin in lymphocytes (39), which suggests that membrane cholesterol plays an important role for the distribution and function of GPI-anchored proteins.
Here we studied the influence of membrane cholesterol on signal transduction via GPI-anchored CD59 and CD48 in T cells. To this end, cellular cholesterol content was reduced by culture in the presence of cholesterol synthesis inhibitors. Subsequently the rise in cytoplasmic calcium concentration was analyzed following cross-linking of GPI-anchored proteins or stimulation via the T cell receptor-CD3 complex, which served as a control. The calcium response was chosen for quantitative analysis of signal transduction since this parameter can be measured with high reliability. We show that cholesterol lowering particularly inhibits signal transduction via GPI-anchored proteins, whereas no changes were found in the distribution of these proteins in detergent-insoluble membrane domains.
All reagents were obtained from
Sigma unless stated otherwise. Antibodies were used as follows: mouse
monoclonal antibodies MEM-43 (IgG2a), MEM-43/5 (IgG2b), MEM-125 (IgM),
MEM-129 (IgM, all four CD59), MEM-102 (IgG1, CD48), MEM-57 (IgG2a),
MEM-92 (IgM, both CD3; all generated in the lab of Václav
Hoej
í and partly obtained from Monosan, Uden, The
Netherlands), BRIC 216 (IgG1, CD55) (Haran Sera-Lab, Crawley Down,
Sussex, UK), OKT3 (IgG2a, CD3) (Ortho Pharmaceuticals, Raritan, NJ),
monoclonal antibody AAA6 (IgG1, CD147), and monoclonal antibody 0662 (IgG3, CD99; kindly provided by O. Majdic and A. Bernard,
respectively); F(ab
)2-fragments of goat-anti-mouse (GAM)
IgG (Sigma or Jackson ImmunoResearch Laboratories, Inc., West Grove,
PA), fluorescein isothiocyanate-labeled F(ab
)2-fragments
of GAM Ig (Dako, Glostrup, Denmark), tetramethylrhodamine isothiocyanate-labeled F(ab
)2-fragments of GAM IgG
(Accurate Chemical & Science Corp., Westbury, NY), GAM IgG labeled with horseradish peroxidase (Bio-Rad).
The human T cell line Jurkat E6-1 (American Type Culture Collection, Rockville, MD) was grown under standard conditions in RPMI 1640 medium supplemented with 10% heat-inactivated bovine calf serum (HyClone, Logan, UT), penicillin/streptomycin (50 units/ml and 50 µg/ml, respectively, Life Technologies, Inc.), and 2 mM glutamine (Life Technologies, Inc.) at 37 °C in humidified atmosphere in the presence of 5% CO2. For modifications of cellular lipids, cells were incubated for 3 days in serum-free Iscove's modified Dulbecco's medium (Life Technologies, Inc.) supplemented with 0.4% (w/v) bovine serum albumin (fraction V), 1 mg/liter transferrin, 8.1 mg/liter monothioglycerol, 2 mM glutamine, and antibiotics as above (40). Cellular cholesterol synthesis was blocked by addition of 2 µM lovastatin (Merck), an inhibitor of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase, or squalestatin 1 (50 µg/ml) (Glaxo Wellcome), an inhibitor of squalene synthase, both generous gifts from the respective companies. The drugs were added to the culture medium from stock solutions in double distilled water (for squalestatin), or dimethyl sulfoxide (maximal solvent concentration 0.2%), respectively. Dimethyl sulfoxide was used as solvent control instead of lovastatin and had no effect on signal transduction (not shown). Sodium mevalonate was generated from mevalonolactone (Fluka Chemie AG, Buchs, Switzerland) by hydrolysis in NaOH. Cell viability was >90% as determined by trypan blue exclusion, and since INDO-1 also served as a vital dye only living cells were assessed with respect to their calcium response.
Quantitation of Calcium ResponseJurkat cells were labeled
with the fluorescent Ca2+ indicator indo-1-AM (2 µM) (Molecular Probes, Inc., Eugene, OR) by incubation at
37 °C for 30 min in serum-free culture medium. Cells were primed with about 10 µg of antibodies to GPI-anchored proteins or
Hanks'-buffered salt solution including 1% bovine serum albumin for
20 min followed by a 7 min preequilibration period at 37 °C.
Subsequently, measurement of [Ca2+]i by flow
cytometry was started at 37 °C constant temperature, and after
60 s 20 µg cross-linking F(ab)2-fragments of GAM
IgG were added and measurement continued for another 5 min unless stated otherwise. For CD3 stimulation 2 µg of OKT3 was added directly without cross-linking. Cross-linking did not result in a strong calcium
response in any OKT3 concentration with absence of calcium response by
adding the primary antibody solely. Flow cytometric analysis was
performed on a FACStarplus (Becton Dickinson, San Jose,
CA) using excitation by argon laser with 50-milliwatt multiline UV,
emission 530 nm (Fl1, calcium-free indo-1) and 395 nm (Fl2, calcium
bound form). The fluorescence ratio Fl2/Fl1 (FR), which is a direct
estimate of the cytoplasmic calcium concentration (41), was computed in
real time by a pulse processing unit and expressed as arbitrary units.
The FR of the unstimulated control (FRus) was set to about 200 arbitrary units. For quantitation of stimulation, maximal FR was
measured at 1-3 min after adding the stimulating antibody. Since CD3
stimulation of cells often induced a calcium response somewhat higher
than stimulation via GPI-anchored proteins, results from modified cells were usually expressed as percentage of that achieved with the solvent
control to allow comparability: relative stimulation in percent of
control was calculated as (FRs - FRus)/(FRco - FRus) × 100%, with
FRco representing FR of the solvent control, and FRs representing FR of
the sample. Data are presented as means ± S.D. unless stated
otherwise.
Cells were prepared for stimulation of calcium response as described above. After preequilibration, SKF 96365 hydrochloride (Calbiochem) was added at optimal concentration (100 µM) to completely inhibit calcium entry from the medium (42). Two min afterward, cells were stimulated by antibodies as described, and changes in cytoplasmic calcium concentration were determined by flow cytometry over a 3-min period.
PI-PLC Treatment106 cells were treated with 0.5 units of phosphatidylinositol-specific phospholipase C (PI-PLC) (Boehringer Mannheim) for 60 min at 37 °C in Hanks'-buffered salt solution including 0.5% bovine serum albumin. Cells were simultaneously loaded with INDO-1 for subsequent analysis of calcium response.
Analysis of Detergent-insoluble ComplexesCells were washed
three times in Hanks'-buffered salt solution and lysed for 30 min on
ice with lysis buffer containing 20 mM Tris-HCl, pH 8.2, 140 mM NaCl, 1% Nonidet P-40 (Pierce), 10 µg/ml
aprotinin (Bayer, Leverkusen, Germany), 5 mM
iodoacetamide, 10 µg/ml leupeptin, and 0.4 mM Pefabloc
(both Boehringer Mannheim), 1 µM pepstatin, and 0.1 mM
N-p-tosyl-L-lysine
chloromethyl ketone,
N-p-tosyl-L-phenylalanine chloromethyl ketone, N-CBZ-L-phenylalanine chloromethyl
ketone, and quercetin. Lysates were then applied to Sepharose 4B
minigel filtration columns as described earlier (26) with fractions 4 and 5 referring to the void volume. Aliquots of the fractions 3 to 11 were separated by 12% non-reducing SDS-polyacrylamide gel
electrophoresis (43) and blotted on nitrocellulose membrane (Hybond
ECL, Amersham International, UK) using a semidry blotting system
(C.B.S. Scientific Co., Inc., Del Mar, CA). Membranes were developed
according to standard Western blotting procedures, and detection was
performed by the chemiluminescence system from Boehringer Mannheim.
Indirect immunofluorescence for cell surface expression of GPI-anchored proteins on unfixed cells was performed by standard procedures with tetramethylrhodamine isothiocyanate-labeled second antibody for flow cytometric analysis. For microscopic examination, cells were first fixed for 20 min in 4% formaldehyde followed by 0.1 M glycine before immunofluorescence staining. Photographs were taken from immunofluorescence with tetramethylrhodamine isothiocyanate-labeled GAM Ig, since fluorescein isothiocyanate fluorescence faded before adequate exposure of the film (Kodak EPH1600) could be achieved. For determination of cholesterol content, 3 × 107 cells were twice extracted with chloroform/methanol (2:1). Dried lipid extracts were dissolved in ethanol, and cholesterol was quantified by a commercial enzymatic colorimetric test according to the manufacturers instructions (Boehringer Mannheim).
Cross-linking of
GPI-anchored proteins CD59 or CD48 resulted in a marked rise in
intracellular calcium as measured by flow cytometry (Fig.
1). The signal via CD59 quantified by
INDO-1 fluorescence ratio was consistently at least half of that
elicited by CD3, which was used as a positive control. No calcium
signal was obtained by cross-linking of antibodies directed to CD147 or
CD99 molecules, which were well expressed on the Jurkat cell line used,
or with isotype controls (not shown). Addition of CD59 or CD48
antibodies without cross-linking had no direct effect on the
cytoplasmic calcium concentration (not shown).
To test for the specificity of the calcium response for GPI-anchored
proteins, cells were treated with PI-PLC to cleave the glycoproteins
from their phospholipid anchor. Following treatment, CD59 or CD48 could
no longer be detected on the cell surface and the calcium signal was
essentially abolished, whereas surface expression and calcium response
elicited via CD3 remained unchanged (Fig.
2, A and B).
Cholesterol Depletion Inhibits Calcium Signaling via GPI-anchored CD59
Cellular cholesterol content was lowered by blocking
endogenous cholesterol biosynthesis by lovastatin, an inhibitor of
3-hydroxy-3-methylglutaryl-coenzyme A reductase catalyzing the reaction
to mevalonate (44). Treatment of cells with 2 µM
lovastatin for a 3-day period decreased cellular cholesterol content by
28% compared with the solvent control (1.71 ± 0.09 nmol
versus 1.24 ± 0.15 nmol/106 cells). Under
the same conditions, lovastatin inhibited CD59-driven calcium response
by 52 ± 9%, whereas the response via CD3 was only minimally
affected (7 ± 8% inhibition; Fig.
3, A and B). The
inhibition by lovastatin could be reversed by added mevalonate (7.5 mM) showing that no unspecific toxicity was responsible for the drug effect. Accordingly, lovastatin did not alter calcium response
of Jurkat cells when added to serum-supplemented medium (91 and 97% of
solvent control for CD59 and CD3, respectively), presumably because of
the presence of serum lipoproteins providing exogenous cholesterol
(data not shown).
Lovastatin inhibits production of mevalonate that can be further
processed not only to cholesterol but also to other lipophilic products
(44) including farnesyl and geranylgeranyl moieties needed for
prenylation of proteins, e.g. of the Ras family or G-protein
subunits. To further prove lovastatin's inhibitory effect to be
due to cholesterol lowering, squalestatin 1, an inhibitor of the
squalene synthase, was also tested for its effect on signal transduction via GPI-anchored proteins. Squalestatin 1 blocks cholesterol biosynthesis at a step distal to mevalonate synthesis and,
therefore, does not affect side products of the mevalonate pathway
(45). Squalestatin 1 inhibited signal transduction via CD59 similarly
to lovastatin (47 ± 14% inhibition; Fig.
4) with minimal effect on CD3 signaling
(2 ± 3% inhibition). The associated block in cholesterol
synthesis downstream from mevalonate could not be reverted by
mevalonate but only by cholesterol.
Inhibition of Calcium Response by Cholesterol Lowering is Common to Different GPI-anchored Proteins and Is Not Due to Their Altered Surface Expression
The inhibitory effect of cholesterol depletion on
calcium response is not restricted to CD59 but shared by CD48, another
GPI-anchored protein (Fig. 5). Thus,
inhibition by cholesterol depletion seems to be characteristic for
signal transduction via GPI-anchored proteins.
Immunofluorescence analysis of lovastatin-treated cells revealed no
alterations in cell surface expression of GPI-anchored proteins CD59
and CD48, and CD3 (Fig. 6). The
expression of at least two different epitopes on CD59 (epitope 1:
MEM-43; epitope 2: MEM-43/5, MEM-125 (46)) was unaltered upon blockade
of cellular cholesterol synthesis, suggesting absence of gross
conformational changes of the protein part of CD59 (Fig. 6). The
markedly diminished surface fluorescence detected by monoclonal
antibodies MEM-125, MEM-129 (CD59), and MEM-92 (CD3) compared with
other antibodies of the same antigen is due to their different isotype
(IgM).
Influence of Cholesterol Depletion on Calcium Release from Intracellular Stores
Transient release of calcium from
intracellular stores into the cytoplasm precedes capacitative calcium
entry from the environment leading to further activation of T
lymphocytes (47, 48). To test whether inhibition of the release of
intracellularly sequestered calcium underlies the total decrease in
calcium response, cells were treated with SKF 96365 prior to
stimulation. The maximal concentration of cytoplasmic calcium achieved
following stimulation via GPI-anchored CD59 was decreased by about 40%
after blockade of cholesterol synthesis (Fig.
7A). In contrast, stimulation
via CD3 yielded no difference between lovastatin-treated and untreated cells (Fig. 7B).
Cholesterol Lowering Does Not Alter Clustering of GPI-anchored Proteins and Distribution in Detergent-insoluble Complexes
Cholesterol lowering may interfere with clustering of
GPI-anchored proteins and distribution in detergent-insoluble
complexes. Cells fixed in formaldehyde prior to indirect
immunofluorescence to avoid artificial clustering by antibodies, showed
a punctate pattern of CD59 and CD48 expression (Fig.
8, A and C) that
was maintained after pretreatment of cells with lovastatin (Fig. 8, B and D). Investigating the impact of cholesterol
depletion on the distribution of CD59 in large detergent-insoluble
complexes, cells cultured with or without lovastatin were lysed in
buffer containing non-ionic detergents Brij-58 or Nonidet P-40 and
analyzed by size fractionation and Western blotting. Thereby most of
GPI-anchored proteins CD59 and CD48 were found in large membrane
complexes although after lysis with Nonidet P-40 a significant amount
of CD59 was recovered in lower molecular weight fractions (Fig.
9). However, the distribution of
GPI-anchored proteins was identical irrespective of whether cells have
been cholesterol depleted or not. Thus, the influence of cholesterol
depletion on signal transduction via GPI-anchored proteins seems not to
be due to destruction of GPI-anchored protein complexes.
The obtained data show that cholesterol lowering in living cells by metabolic intervention effectively inhibits signal transduction via GPI-anchored proteins without changing their clustering or distribution in large detergent-insoluble complexes. In contrast, signaling via the T cell receptor (TCR)-CD3 complex was hardly affected by cholesterol deprivation.
Since GPI-anchored proteins lack a transmembrane domain, the clustering of these proteins to specific membrane regions seems to be prerequisite for signal transduction. Cholesterol is an essential component for the formation of these detergent-insoluble complexes (5, 35-37, 39) that mediate the association with possible signal-transducing molecules, e.g. Src family kinases p56lck and p59fyn (1, 29-31) or G-proteins (49). It has been shown that exogenously added fluorescent CD59 needs to aggregate into clusters before signal transduction is enabled (5). Lowering cholesterol by 60% in living epithelial cells by blocking its endogenous synthesis resulted in strong inhibition of folate uptake via the GPI-linked folate receptor along with variable differences in clustering of these receptors (50). In our study, reduction in lymphocyte cholesterol content by about 30% did not disclose any alterations in the distribution of GPI-anchored proteins when examined by immunofluorescence or gel filtration of membrane complexes. In addition, lowering cell cholesterol had no influence on overall cell surface expression of GPI-anchored proteins as quantitated by flow cytometry in contrast to previous publications (51-53). Though delipidation may cause some conformational changes in GPI-anchored proteins (54) this seems to be unlikely in our experiments, since antibodies against at least two different epitopes on CD59 (46) bound to cholesterol-depleted and control cells to a similar extent (Fig. 5). Thus, in contrast to rather harsh treatments with sterol-sequestrating detergents, which are able to destroy detergent-insoluble complexes in situ (5, 36, 37, 39), the signal-transducing function of these proteins seems to be particularly sensitive to a disturbed cholesterol availability.
Although GPI-anchored proteins partly share signaling events with the
TCR, there may be unique steps in the signaling pathways for either
type of activation. Signal transduction via the TCR-CD3 complex but
also distinct signaling events induced via GPI-anchored proteins
including interleukin-2 secretion, CD69 expression, and activation of
nuclear factor-B, require expression of the TCR-
chain (14,
55-58). On the other hand, induction of calcium response (55),
activation of p56lck, protein kinase C, and ERK-2,
expression of CD25 (58), and inhibition of CD3-driven interleukin-2
production (57, 59) were shown to be independent of the expression of
TCR-
. Furthermore, activation of murine T cells via GPI-anchored
Thy-1 but not via CD3 requires expression of p59fyn (60).
Thus, signaling via GPI-anchored proteins seems to differ from TCR/CD3
signaling with regard to the involvement of the TCR-
chain and
particular Src protein kinases. Despite these differences, both
signaling pathways were shown to interact with each other since
adequate expression of GPI-anchored proteins is needed for signaling
via the TCR-CD3 complex (61, 62). It appears from this that T cell
activation via GPI-anchored proteins differs from TCR/CD3-driven
activation, and each pathway may require components of the other for
optimal stimulation (2, 61).
Our findings, that activation of calcium response in T cells via GPI-anchored proteins and the TCR is differentially affected by cholesterol depletion, supports the concept of separate signaling pathways to be involved. Moreover, since activation via CD59 and CD48 were suppressed to a similar extent, cholesterol lowering seems to impair signaling pathways common to GPI-anchored proteins. Absence of structural homologies between both GPI-linked molecules beyond the phospholipid anchor suggests that the GPI-anchor itself could play an important role in signal transduction via these proteins, as proposed previously by others (30, 61, 63, 64).
Rescue by appropriate downstream products of cholesterol synthesis proves that the drugs applied are actually effective via inhibition of cholesterol biosynthesis. Such inhibition of cholesterol synthesis in the endoplasmic reticulum may directly interfere with more subtle associations of lipids and proteins during aggregation to detergent-insoluble complexes. How a disturbed lipid composition can alter the signal transduction process remains to be elucidated. Conceivably, spatial relationships between GPI-anchored proteins and candidate signal-transducing molecules like the Src family protein tyrosine kinases could play a role in this process as well as changes in lateral mobility of components within the complexes.
In conclusion, our data suggest that cholesterol lowering affects signal transduction via GPI-anchored proteins in a very early stage up to the release of calcium without apparent structural alterations. Thus, lipids may play an intriguing role in signal transduction via these surface molecules whose exact molecular mechanisms remain to be elucidated.
We are grateful to Samuel Godár and Dr. Winfried Pickl for helpful discussions, and to Dr. Otto Majdic for the provision of antibodies.