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*
Department of Medicine, The University of Alabama at Birmingham, Birmingham, AL 35294;
Veterans Administration Medical Center, Birmingham, AL 35233
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussion References |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
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Fas/Apo-1 (CD95) Ag, a cell-surface protein expressed on a variety of human tissues and cell lines, belongs to the TNF/nerve growth factor (NGF)3 receptor family (10, 11). Cross-linking of Fas with anti-Fas Ab or binding of Fas with Fas ligand (FasL), a member of the TNF family, mediates apoptosis (12, 13, 14). Loss-of-function mutations of murine fas and fasL are associated with lymphoproliferation and a generalized lymphoproliferative disease in lpr and gld mice, respectively (15, 16, 17). Mutations of fas and fasL genes also have been found in patients with autoimmune lymphoproliferative disease (18, 19, 20, 21). Thus, Fas and FasL are important proapoptotic molecules involved in the regulation of T cell apoptosis.
Stimulation of T cells via their Ag receptors leads to cell proliferation and differentiation (22) as well as apoptosis (23, 24, 25, 26). Activation-induced cell death (AICD) has been proposed to be an important mechanism in the maintenance of T cell tolerance in the periphery (27). Activation of T cells causes coexpression of Fas and FasL on the cell surface, and the interaction of these two death-inducing proteins triggers autocrine apoptosis (28, 29).
We and others have shown that not all T cells are susceptible to Fas-mediated apoptosis and that the T cells that are resistant to Fas-mediated apoptosis express lower levels of Fas or have defective Fas signaling (12, 13, 30). Although the Fas-FasL system plays an important role in maintaining peripheral T cell tolerance, it is not clear if FasL activity is differentially regulated in the heterogeneous population of mature T cells. The present experiments utilized a 2.3-kb fragment of human FasL promoter capable of driving the expression of GFP to allow identification and analysis of FasL activity in Jurkat cells. Our results indicated that resting Jurkat T cells can be divided into two distinct populations according to FasL production and that these two populations display differences in susceptibility to Fas-mediated apoptosis. The interactions between Fas and FasL in these two populations determine whether apoptosis occurs by an autocrine or paracrine pathway in the susceptible population.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
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The human fasL gene was isolated from two human placental genomic DNA libraries constructed in the Lambda FIXII vector (Stratagene, La Jolla, CA) and in the EMBL-SP6/T7 vector (Clontech Laboratories, Palo Alto, CA) using 32P-labeled segments of human FasL cDNA (Su et al., manuscript in preparation). After the positive clones were identified, the inserts of these clones were analyzed by restriction mapping. The DNA fragment containing the exon I 5'-end cDNA sequence and upstream 5'-flanking region was prepared by BglII digestion and subcloned into the pBluescript vector (Stratagene). The human FasL reporter construct was generated by cloning the HindIII-flanked 2.3-kb human FasL 5'-flanking region upstream of the FasL translational start site into the pEGFP-1 reporter vector (Clontech Laboratories), which contains a neomycin resistance (neor) gene as a selection marker for stable transfectants.
Cell culture and stable transfection
The Jurkat human leukemic T cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), and glutamine (2 mM) in a humidified CO2 incubator at 37°C. For stable transfection, 5 x 106 Jurkat cells were incubated with 10 µg of DNA in 1 ml of RPMI 1640 medium and subjected to electroporation with a Gene Pulser (Bio-Rad Laboratories, Hercules, CA) at 250 V/960 µF. Forty-eight hours after transfection, cells were plated in a 24-well microtiter plate (Costar, Cambridge, MA) in medium containing 2 mg/mg of G418 (Clontech). Two weeks later, cells from different wells were transferred to corresponding 6-cm dishes for expansion. The level of GFP expression in individual cell culture was analyzed by fluorescent microscopy and flow cytometry with excitation and emission wavelengths of 485 nm and 510 nm, respectively. G418-resistant cells expressing higher levels of GFP (GFP+) and lower levels of GFP (GFP-) were separated by FACS and maintained in complete RPMI 1640 medium containing 2 mg/ml of G418. The levels of GFP expression in the two cell populations were stable as determined by flow cytometric analysis.
Abs and reagents
Anti-CD3 (clone: HIT3a), phycoerythrin (PE)-conjugated anti-CD4 (clone: RPA-T4), PE-conjugated anti-CD45RO-PE (clone: UCHL1), and biotinylated anti-Fas (clone: DX2) mAbs were obtained from PharMingen (San Diego, CA). Phorbol myristyl acetate (PMA), ionomycin, and Con A were purchased from Sigma Chemical (St. Louis, MO). Fas fusion (Fas-Fc) protein was prepared as described previously (31).
RT-PCR
Total RNA was isolated from GFP+ and GFP- Jurkat cells by RNA STAT-60 (Tel-Test "B", Friendswood, TX) according to the manufacturer’s instructions. Two micrograms of RNA was used as a template for cDNA synthesis in a reaction mixture with 50 ng of oligo(dT) primers and 50 U of reverse transcriptase (Promega, Madison, WI) in a 20-µl reaction volume. A 2-µl aliquot was used in 50 µl of PCR reaction mixture containing 20 pmol of the FasL primers (forward, 5'-GGAATTCCAGCTCTTCCACCTACAG-3'; reverse, 5'-ATTTGCGGCCGCTTAGAGCTTATATAAGCC-3'), GFP primers (forward, 5'-TGAAGTTCATCTGCACCACC-3'; reverse, 5'-ACGAACTCCAGCAGGACCA-3'), neor primers (forward, 5'-GAGGCTATTCGGCTATGACT-3'; reverse, 5'-GATATTCGGCAAGCAGGCAT-3'), or ß-actin primers (forward, 5'-GCGGGAAATCGTGCGTGACATT-3'; reverse, 5'-GTGGACTTGGGAGAGGACTGGG-3') in the presence of 2.5 U of Taq polymerase (Promega). The conditions for PCR were 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C for 30 cycles. The samples were then subjected to electrophoresis on a 1% agarose gel and visualized with ethidium bromide staining and UV illumination.
FasL activity
The cell death of Fas-positive SKW6.4 cells was used as an assay of the FasL activity of GFP+ and GFP- cells. SKW6.4 cells were labeled with 700 µCi of [51Cr]sodium chromate (Amersham, Arlington Heights, IL) for 1 h at 37°C. After washing three times with RPMI 1640 medium supplemented with 10% dialyzed FCS, 1 x 105 of 51Cr-labeled cells were incubated with GFP+ and GFP- cells in a 96-well plate (E:T ratio of 10:1). The supernatants were collected after 6 h and the amount of released 51Cr was measured using a gamma counter (Beckman Instruments, Fullerton, CA). For the GFP+ and GFP- cell mixing experiment, 1 x 105 of 51Cr-labeled GFP- and GFP+ cells were added to GFP+ cells at different E:T ratios in a 96-well plate. The percentage of specific 51Cr release was determined as described previously (32).
Fas-mediated cytotoxicity assay
The GFP+ and GFP- Jurkat cells were labeled with [51Cr]sodium chromate as described above. Then 1 x 105 of 51Cr-labeled cells were incubated with different concentrations of soluble FasL (Alexis, San Diego, CA) in a 96-well plate. The supernatant was collected after 12 h, and the percentage of specific 51Cr release was determined as described above.
Stimulation of GFP+ and GFP- Jurkat cells
GFP+ and GFP- Jurkat cells (1 x 106/ml) were incubated with anti-CD3 (5 µg/ml), PMA (100 ng/ml) plus ionomycin (500 ng/ml), or Con A (5 µg/ml) in a 24-well microtiter plate (Costar) with or without Fas-Fc protein (100 µg/ml) for different time periods. GFP expression was analyzed 12 h later by flow cytometric analysis, and cell viability was analyzed by flow cytometric analysis of 7-amino-actinomycin D (7-AAD) staining as described below.
Flow cytometric selection of viable cells
Flow cytometric selection of viable cells was conducted after staining with 7-AAD. Briefly, cells were stained with 10 µg/ml of 7-AAD (Calbiochem, San Diego, CA) in PBS on ice for 30 min. After washing twice with PBS, cells were fixed in 1% paraformaldehyde supplemented with 50 µg/ml actinomycin D. Nongated cells (10,000) were analyzed using FACScan (Becton Dickinson, Mountain View, CA). Nonapoptotic cells are 7-AAD negative (32).
Flow cytometric analysis
The GFP+ and GFP- Jurkat cells were stained with optimal concentrations of Abs in FACS buffer (PBS with 5% FCS and 0.1% sodium azide) as described previously (33). Biotinylated Abs were revealed by PE-streptavidin (Southern Biotechnology Associates, Birmingham, AL). Viable cells (10,000 per sample) were analyzed by flow cytometry on a FACScan (Becton Dickinson).
Hoechst 33342 (HO342) staining
GFP+ and GFP- cells (1 x 106/ml) were cocultured at the ratio of 1:1 in a 6-well plate (Costar) for 12 h. Cells were stained with HO342 (1 µg/ml in FACS buffer) on ice for 7 min. Cells were washed twice with FACS buffer and then fixed with 0.5% paraformaldehyde. Stained cells were analyzed by microscopy with UV illumination. Nonapoptotic cells are HO342 negative (34).
Measurement of IL-2 concentration by ELISA
GFP+ and GFP- Jurkat cells (1 x 106/ml) were incubated with PMA (100 ng/ml) and ionomycin (500 ng/ml) in a 24-well microtiter plate (Costar). Culture supernatants were harvested at different time points, and the IL-2 concentration was measured in duplicate supernatant samples using an ELISA assay kit according to the procedure recommended by the supplier (Endogen, Woburn, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
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A human FasL promoter (hFasLpro)/GFP reporter construct was
generated by cloning a 2.3-kb human FasL 5'-flanking region upstream of
the fasL translational start site into the pEGFP-1 reporter vector.
Human Jurkat T cells were transfected with this hFasLpro/GFP construct,
and the stable transfectants expressing neor
gene were selected after short-time culture in a 24-well microtiter
plate with G418. Interestingly, all the wells of the microtiter plate
contained two populations of G418-resistant Jurkat cells with different
levels of GFP expression (Fig. 1
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A and B). Approximately 20% of Jurkat
cells expressed higher levels of GFP. Cells were then separated by FACS
based on their levels of GFP expression and maintained in culture
medium containing G418. The levels of GFP expression were stable for at
least 10 days in the two cell populations as determined by fluorescent
microscopy and flow cytometric analysis. As observed by flow cytometric
analysis, one population of Jurkat cells expressed high levels of GFP
(GFP+), whereas the other expressed nearly undetectable
levels of GFP (GFP-) (Fig. 1C). This was
further demonstrated by fluorescent microscopy, which indicated that
both GFP+ and GFP--cells existed in the
G418-resistant Jurkat cells obtained after transfection (Fig. 1D).
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A). Furthermore, both
GFP+ and GFP- Jurkat cells expressed
equivalent level of neor mRNA, indicating that the failure
of GFP- cells to express GFP mRNA was not due to the
unsuccessful transfection of hFasLpro/GFP into these cells. The
functional capacity of the FasL on the GFP+ and
GFP- cells was further determined by evaluation of the
ability of GFP+ and GFP- cells to lyse
51Cr-labeled SKW6.4 target cells. GFP+ cells
exhibited greater cytotoxicity against SKW6.4 cells compared with
GFP- cells, indicating that the GFP+ cells
produce higher levels of functional FasL (Fig. 2B).
These results indicated that Jurkat T cells can be divided into
FasL-positive and FasL-negative subpopulations that can be separated
based on their GFP expression.
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We then determined whether the FasL promoter activity in
GFP- Jurkat cells could be induced by different
stimuli. Stimulation of GFP- cells with anti-CD3, PMA
plus ionomycin, or Con A for 1 and 2 days resulted in an increase in
the percentage of GFP+ cells (Fig. 3A). The induction of
GFP expression in GFP- cells was also demonstrated by
increased GFP fluorescence density after PMA and ionomycin stimulation
(Fig. 3B). Similar stimulation of GFP+
cells had no effect on GFP expression, which was consistent with
constitutive expression of GFP by these cells. These results indicated
that one population of Jurkat cells constitutively expressed high
levels of FasL while the other population exhibited lower but inducible
levels of FasL.
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The existence of a stable population of GFP+,
FasL-positive Jurkat cells in the absence of exogenous stimulation
suggests that these cells may exhibit decreased sensitivity to
FasL-induced apoptosis. To explore this possibility, GFP+
and GFP- cells were labeled with 51Cr and
incubated with different concentrations of soluble FasL. The
sensitivity of cells to FasL-mediated apoptosis was determined by the
specific lysis of GFP+ and GFP- cells after
12 h. GFP- cells exhibited a dose-dependent
sensitivity to FasL-mediated apoptosis (Fig. 4A). In contrast, FasL
did not induce significant apoptosis in GFP+ cells even at
a higher concentration (200 ng/ml).
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B). PMA and
ionomycin stimulation induced greater levels of apoptosis than Con A or
anti-CD3 stimulation, which correlated with the relative ability of
the agents to induce GFP expression in GFP- cells (Fig. 3A). The activation-induced apoptosis in
GFP- cells was inhibited by coincubation with soluble
Fas-Fc protein, indicating that AICD in GFP- cells was
mediated by Fas-FasL interaction. In contrast, AICD was not observed in
GFP+ cells. These results indicated that GFP+
cells exhibit a lower sensitivity to FasL and activation-induced
apoptosis than GFP- cells. Apoptosis mediated by a paracrine pathway in GFP- Jurkat cells
Since GFP+ cells expressed high levels of FasL,
we determined whether unstimulated GFP+ cells can induce
apoptosis in GFP- cells through interaction of FasL with
Fas. GFP- cells were labeled with 51Cr and
cocultured with GFP+ cells at different E:T ratios, and the
cytotoxic activity of GFP+ cells against GFP-
cells was determined by the specific lysis of GFP- cells
12 h after incubation. GFP+ cells induced paracrine
apoptosis of nearly 30% of the GFP- target cells as the
ratio of E:T was increased, but there was no detectable autocrine
apoptosis of the GFP+ cells (Fig. 5A). The lysis of
GFP- cells by GFP+ cells also was demonstrated
by HO342 staining, since HO342-positive apoptotic cells in the
coculture were GFP-negative (Fig. 5B). The killing of
GFP- cells by GFP+ cells was inhibited by
Fas-Fc protein (data not shown), further confirming that FasL
interaction with Fas was required for the induction of apoptosis in
GFP- cells. These results indicated that apoptosis of
GFP- cells can be induced by GFP+ cells
through a paracrine pathway.
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GFP+ cells expressed lower levels of Fas Ag on
the cell surface than GFP- cells (Fig. 6). GFP+ cells also expressed
lower levels of CD4 than GFP- cells but expressed higher
levels of CD45RO. There was no difference in the expression of CD3 in
GFP+ and GFP- cells, and CD8 was undetectable
in both populations (data not shown). These results suggested that the
lower levels of Fas expression on GFP+ cells might account
for the lower sensitivity of these cells to FasL-induced cytotoxicity.
They also indicate that Fas-apoptosis-resistant GFP+ cells
exhibit a phenotype that is similar to that of the
CD3+CD4-CD8-B220+ T
cells that accumulate in the periphery of lpr and
gld mice, and exhibit an increased number in FasL transgenic
mice (35).
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We then evaluated the response of GFP+ and
GFP- cells to PMA and ionomycin stimulation.
GFP+ and GFP- cells were stimulated with PMA
and ionomycin for 12 h and 24 h, and the concentrations of
IL-2 in the culture supernatants were measured as an indicator of T
cell activation. After stimulation, lower quantities of IL-2 were
secreted by GFP+ cells than GFP- cells,
indicating that GFP+ cells exhibited a lower response to
PMA and ionomycin activation than GFP- cells (Fig. 7). These results suggest that
GFP+ cells are less responsive to PMA and ionomycin
stimulation, which may result in decreased activation-induced
apoptosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
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Our functional analyses of GFP+ and GFP- cells have clarified the previous observations that regulation of T cell growth by apoptosis can occur by both autocrine and paracrine pathways. It is known that stimulation of T cells with excessive levels of Ags leads to up-regulation of FasL. FasL may be cleaved from the cell surface and act as a soluble factor on neighboring cells in a paracrine fashion. Alternatively, in either the cleaved or uncleaved form, the FasL can act in an autocrine fashion on the same cell that generates it. (23, 36, 37, 38, 39). Our finding, that GFP- cells were sensitive to FasL cytotoxicity produced by GFP+ cells, suggests that T cells can regulate apoptosis of other T cells by a paracrine mechanism and that T cell apoptosis can be regulated by an autocrine pathway in AICD and by paracrine pathways involving other T cells as well other cell types.
The FasL-negative and FasL-positive Jurkat cells are phenotypically distinct, in that the FasL-positive cells express lower levels of Fas and CD4 but increased levels of CD45RO. Jurkat cells constitutively expressing FasL may be derived from Jurkat cells that have been activated to express a higher level of FasL but fail to undergo apoptosis due to down-regulation of Fas expression or defective Fas apoptosis signaling. This is consistent with the lower level of IL-2 produced by GFP+ cells after PMA and ionomycin stimulation. A second possibility is that, during the development of Jurkat cells, certain cells undergo a differentiation step that results in constitutive expression of higher levels of FasL. Down-regulation of expression of Fas and CD4 and up-regulation of CD45RO expression is also associated with this differentiation step. Other Jurkat cells differentiate to express a lower level of FasL but a higher level of Fas on the cell surface. Upon activation, these FasL-negative cells are capable of expressing increased levels of FasL and undergo apoptosis. We favor this second mechanism, since it is consistent with a previous study showing that FasL expression in T cells is regulated by TCR-mediated signaling events and that the transcription factor NF-AT, a key element in the regulation of IL-2 expression (40), is involved in this TCR-mediated FasL expression (41). On the other hand, Jurkat cells that constitutively express higher levels of FasL may have defective TCR signaling, which is suggested by decreased activation-induced apoptosis. Thus, it is possible that FasL expression is regulated by different regulatory factors during different stages of cell differentiation in Jurkat cells. Future characterization of the factors that regulate FasL expression in Jurkat cells will certainly aid in elucidating the mechanisms by which FasL is differentially expressed in T cells.
A stable proportion of FasL-positive and FasL-negative Jurkat cells was
maintained in the mixed cell cultures, despite the fact that
FasL-positive cells have the ability to induce apoptosis in
FasL-negative cells. The FasL-negative cells may regenerate in the
mixed cultures. The balance between the populations also may be
regulated and maintained by factors in addition to FasL. This concept
is supported by our preliminary data, which show that FasL-positive
cells are more sensitive to TNF-
-induced apoptosis than
FasL-negative cells (Su et al., unpublished observation). The balance
between the two cell populations may be dictated by a complex
interaction of factors acting through autocrine and paracrine
regulation to regulate the numbers of cells that are sensitive to
Fas/FasL-mediated and TNF--mediated apoptosis.
The coexistence of two functionally different populations of T cells supports the notion that there is a social control of cell survival and cell death in the immune system (2). FasL-negative cells express an inducible level of FasL after stimulation and are highly susceptible to FasL-mediated apoptosis induced by both autocrine and paracrine pathways. In contrast, FasL-positive T cells, which are resistant to FasL-mediated apoptosis, induce apoptosis of FasL-negative cells in a paracrine fashion. We propose that FasL-negative T cells are the primary effector cells during an immune response, whereas FasL-positive T cells play an important regulatory role in maintaining the steady state or equilibrium of cell number within a mixed population by inducing apoptosis of FasL-negative cells. Therefore, FasL-mediated apoptosis induced by autocrine and paracrine fashion is a critical mechanism for down-regulation of the effector T cell subpopulation and the maintenance of homeostasis of the immune system.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John D. Mountz, The University of Alabama at Birmingham, 701 South 19th Street, LHRB 473, Birmingham, AL 35294. E-mail address:
3 Abbreviations used in this paper: NGF, nerve growth factor; GFP+, green fluorescence protein positive; GFP-, green fluorescence protein negative; PMA, phorbyl myristyl acetate; AICD, activation-induced cell death; FasL, Fas ligand; 7-AAD, 7-amino-actinomycin D; neor, neomycin resistant; HO342, Hoechst 33342; PE, phycoerythrin; Fas-Fc, Fas-Fc fusion protein.
Received for publication October 31, 1997. Accepted for publication January 30, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
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