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Cell Growth & Differentiation Vol. 10, 575-582, August 1999
© 1999 American Association for Cancer Research

Interferon-{alpha} Inhibits Proliferation in Human T Lymphocytes by Abrogation of Interleukin 2-induced Changes in Cell Cycle-regulatory Proteins1

Sven Erickson, Olle Sangfelt, Juan Castro, Mats Heyman, Stefan Einhorn and Dan Grandér2

Department of Oncology and Pathology, Radiumhemmet, Karolinska Hospital and Institute, S-171 76 Stockholm, Sweden


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
IFN-{alpha} exerts prominent regulatory functions on the immune system. One such effect is the inhibition of proliferation of in vitro stimulated T lymphocytes. The exact physiological function of this activity is not known, but it has been implicated in the antiviral effects of IFN, its antitumor action in T-cell malignancies, and the regulation of the in vivo T-cell response. Here, we have investigated the mechanism underlying the IFN-{alpha}-mediated growth inhibition of normal human PHA- and IL-2-stimulated T lymphocytes by an analysis of how IFN-{alpha} treatment influences known molecular events that normally accompany the transition from quiescence to proliferation in these cells.

IFN-{alpha} treatment was found to profoundly block S-phase entry of stimulated T lymphocytes. This correlated with a strong inhibition of IL-2-induced changes in G1-regulatory proteins, including the prevented up-regulation of G1 cyclins and cyclin-dependent kinases as well as an abrogation of mitogen-induced reduction of p27Kip1 levels. This latter effect was due to a maintained stability of the p27Kip1 protein in the IFN-{alpha}-treated cells. In line with these findings, phosphorylation of the pocket proteins was abrogated in IFN-{alpha}-treated cells. Furthermore, our data indicate that IFN-{alpha} has selective effects on the pathways that emerge from the IL-2 receptor because IFN-{alpha} treatment does not block IL-2-induced up-regulation of c-myc or Cdc25A.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
An essential feature of the cellular immune response is the activation of T lymphocytes as a consequence of TCR3 binding to HLA-associated antigens. Ligation of TCR/CD3 provides an activation signal involving induction of a number of genes, and it is also a prerequisite for further cell cycle progression (1 , 2) . IL-2 binding to its receptor is an essential event for TCR-stimulated cells to further progress from G1 into the S phase of the cell cycle (2 , 3) . IFN-{alpha} is a potent modulator of these events: it has been shown to abrogate mitogen-induced proliferation of T lymphocytes (4 , 5) .

A paradigm for the molecular regulation of cell proliferation and the molecular control of G1-S transition has emerged during recent years. The unifying cell cycle regulating molecules in all eukaryotes are the Cdks and their regulatory partners, the cyclins (6) . Different kinase complexes, such as cyclin D-Cdk4, cyclin D-Cdk6, and cyclin E-Cdk2, regulate the G1-S transition in mammalian cells by the phosphorylation and inactivation of substrates such as the pocket proteins (pRb, p130, and p107). Additionally, two families of low molecular weight CKIs have been identified, the Cip/Kip and Ink4 families (7) . The Cip/Kip members, including p21, p27, and p57, associate with both cyclin E and cyclin D complexes, whereas the Ink4 members, including p15, p16, p18, and p19, inhibit cyclin D-Cdk4/6 complex formation (7) . Together, these inhibitors are critical regulators at the G1-S border in response to both exogenous and endogenous signals.

p27 has been implicated as a central regulator of quiescence in different systems. Overexpression of p27 results in G1 arrest (8) , whereas inhibition of p27 expression by antisense oligonucleotides may result in the failure of the cell to exit from the cell cycle and enter into quiescence (9) . Any one of a number of antimitogenic conditions or agents, including contact inhibition, cyclic AMP, transforming growth factor-ß, and IFN-{alpha}, results in formation of inactive cyclin-Cdk complexes, attributed to the binding of p27 (8 , 10, 11, 12) .

Recent elucidation of the essential mechanisms involved in the regulation of the eukaryotic cell cycle has also contributed to an increased understanding of the molecular events leading to T-cell proliferation. A hallmark of the unstimulated, quiescent T lymphocyte is a high expression of the CKI p27 (13 , 14) . Mitogenic stimulation with PHA and IL-2 results in reduction of p27 protein levels, allowing cell proliferation to occur. Furthermore, a number of cell cycle-regulatory proteins, including cyclin D3, cyclin E, Cdk2, Cdk6, and pRb, are up-regulated upon mitogenic stimulation (13, 14, 15) .

Recently, some of the upstream events evolving from the IL-2R have been coupled to the above-mentioned cell cycle effects. The high-affinity IL-2R consists of at least three subunits ({alpha}, ß, and {gamma}c; Refs. 2 and 3 ). Whereas the {alpha} chain (CD25) is important for formation of the high-affinity receptor, the other two subunits are sufficient to mediate IL-2-dependent signaling (16 , 17) . Like many other hematopoietic receptors, the IL-2R has no intrinsic kinase activity but rather relies on the activity of associated kinases. The Jak kinases, Jak1 and Jak3, activated by ligand binding and IL-2R oligomerization, form one such group of enzymes (3) . Jak3 associates with the {gamma}c-chain of the IL-2R, whereas Jak1 associates with the ß chain. Although Jak3 is required for proper lymphoid development and T-cell proliferation, the precise role of this kinase has yet to be defined. Furthermore, the PI3K pathway is activated by IL-2 stimulation. Interestingly, IL-2-induced activation of the PI3K pathway has recently been linked to modulation of the basal cell cycle machinery in T cells because PI3K activation seems to be necessary for the down-regulation of p27 and the up-regulation of cyclin D3 (18) .

IFN-{alpha} belongs to a family of secreted proteins produced by eukaryotic cells when challenged by viruses and other infectious agents, with the common denominator of inhibiting viral replication. In addition to the antiviral effects, IFN-{alpha} exerts pleiotropic cellular effects, such as inhibition of cellular proliferation, induction of apoptosis, and differentiation as well as modulation of the immune system. The latter effect includes inhibition of T-lymphocyte activation, enhancement of the cytotoxic activity of NK cells and T lymphocyte, and inhibition of antibody production by B cells (4 , 5 , 19 , 20) . IFN-{alpha} also exerts an antitumor activity in several malignancies, with the antiproliferative effect of IFN-{alpha} being one possible explanation for this antineoplastic effect (20 , 21) .

The CKIs p15, p21, and p27 have all been shown to be subject to regulation by IFN-{alpha} in malignant cell lines, and the accumulation of these proteins in G1 Cdk-complexes leads to decreased kinase activity and subsequent G1 arrest (10 , 22) . Regulation of other central cell cycle proteins, such as Rb, c-myc, Cdc25A, and cyclins D and E, have also been suggested to be important for IFN's growth-suppressive effects (22 , 23) . To obtain a better understanding of how IFN-{alpha} blocks the induction of proliferation in primary human T lymphocytes, we have focused on investigating the molecular effects of this cytokine on the cell cycle machinery in human primary T lymphocytes following mitogen- and IL-2-induced proliferation. Here, we have used a stimulation procedure previously described by Firpo et al. (13) , in which the general effects on the G1-regulatory proteins are well characterized. In this system, T cells are activated by PHA treatment for 72 h, and subsequent IL-2 addition provides the signal necessary for S-phase entry as well as allowing evaluation of possible interference with IL-2 action. Our results show that IFN-{alpha} selectively blocks downstream events from the IL-2R, preventing p27 down-regulation and inhibiting the activation of the basic cell cycle machinery, whereas other events, such as c-myc and Cdc25A up-regulation, are not affected.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
IFN-{alpha} Arrests T Lymphocytes in G0/G1 and Inhibits Entry into S Phase.
Following PHA stimulation for 72 h, 1–2% of the T lymphocytes entered S phase (Fig. 1)Citation . Addition of IL-2 at day 3 allowed the majority of the cells to enter into a exponential-growth phase with {approx}90% actively proliferating cells, as judged by bromodeoxyuridine labeling (Ref. 24 ; data not shown), and an S-phase fraction of 20–30% (Fig. 1)Citation . To characterize the transition from quiescence (G0) to S in greater detail, the cellular protein content was measured because an increased cellular protein content is considered to be a marker for exit from quiescence (G0). Cellular protein content increased only slightly after PHA stimulation compared with quiescent cells, whereas subsequent IL-2 stimulation for 72 h resulted in a significant increase in cellular protein content (Fig. 1)Citation . The effect of IFN-{alpha} on cell cycle progression was investigated by cotreatment with IFN-{alpha} (5000 units/ml) in parallel with IL-2 at day 3. IFN-{alpha} treatment resulted in an almost complete suppression of S-phase entry, with only {approx}2–5% S-phase cells at day 6 (Fig. 1)Citation . Analysis of protein content also indicated that the majority of the IFN-{alpha}-treated cells remained in G0/G1 because there were only minor changes in this parameter in the IFN-{alpha}-treated cells compared with unstimulated cells (Fig. 1)Citation . Thus, IFN-{alpha} abrogates IL-2-dependent S-phase entry, arresting the T cells in a G0/G1-like state.



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Fig. 1. Flow cytometric analysis illustrating DNA and protein content at indicated time points in primary human T lymphocytes from a representative experiment. Top, cell cycle distributions of quiescent (Day 0 Untreated) and PHA-treated (Day 3 PHA), IL-2-treated (Day 6 IL-2), and IL-2-/IFN-{alpha}-treated (Day 6 IL-2+IFN-{alpha}) cells, determined by plotting the intensity of 4',6-diamidino-2-phenylindole propidium iodide fluorescence as a function of the cell number. Bottom, DNA and protein content in quiescent and PHA-, IL-2-, and IL-2-/IFN-{alpha}-treated cells, determined by plotting the intensity of 4',6-diamidino-2-phenylindole and SR101 fluorescence, respectively. Cells appearing in G2 in the quiescent (Day 0 Untreated), PHA-treated (Day 3 PHA), and IL-2-/IFN-{alpha}-treated (Day 6 IL-2+IFN-{alpha}) groups probably represent aggregated cells from G1 because no G2 cells were detected at these time points (top).

 
IFN-{alpha} Suppresses the IL-2-mediated Decline of p27 by Posttranscriptional Regulation.
The down-regulation of p27 upon mitogenic stimulation of T lymphocytes is a primary event that is obligate for the activation of G1 kinase activity and subsequent S-phase entry (14) . Because p27 acts as a target for IFN-{alpha}-mediated cell growth inhibition in other cellular systems (10) , we sought to examine whether p27 was also subjected to regulation in this system. Quiescent and PHA-stimulated cells contained high amounts of p27, correlating with the G0/G1 status of these cells. IL-2 addition resulted in a rapid decline of the level of p27 protein over a period of 48 h (Fig. 2A)Citation . After 72 h of IL-2 stimulation (day 6), p27 was very weakly expressed, and this weak band probably reflects a minor fraction of the purified T cells still remaining in G0/G1. IFN-{alpha}-cotreated cells, on the other hand, displayed high, unchanged levels of p27 after 72 h (day 6; Fig. 2ACitation ). Expression of the closely related Cip/Kip family member, p21, was also examined. p21 was barely detectable at any time point, and the level did not seem to be affected by IFN-{alpha} treatment in this system (data not shown). The Ink4 family member p16 is not expressed, and p15 is barely detectable in quiescent or early proliferating T cells, whereas both of these proteins accumulate as cells approach senescence (24) . However, faint bands of p15 and p16 proteins appear at day 6 in IL-2-stimulated cells, but this increase does not occur in IFN-{alpha}-cotreated cells (data not shown).



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Fig. 2. Effect of IFN-{alpha} and IL-2 on p27 expression levels and stability. Analysis of p27 regulation in quiescent (Day 0), PHA-treated (Day 3), IL-2-treated (Days 4–6), and IL-2/IFN-{alpha}-treated (Days 4–6) cells by immunoblotting (A), Northern blotting (B), and pulse-chase analysis (Day 3; C) of p27 synthesis (4-h pulse) and turnover rate (20-h chase). 18S and 28S rRNA served as controls for equal loading because the GAPDH expression varies between cells cultured in the absence or presence of IFN-{alpha}.

 
We further tried to determine the mechanism by which IFN-{alpha} modulates the amount of p27 in IL-2-stimulated T cells. The effect of IFN-{alpha} cotreatment on p27 mRNA levels in T cells was investigated by Northern blot analysis. As seen in Fig. 2BCitation , p27 mRNA was unaffected by PHA, IL-2, and IFN-{alpha} treatment. Because the housekeeping genes GAPDH and ß-actin (Fig. 2BCitation and data not shown) were also subjected to regulation during T-cell activation, we used ribosomal 18S and 28S RNA species as a control for RNA loading. Next, a pulse-chase experiment was performed to determine the rate of [35S]methionine and [35S]cysteine incorporation into p27 as well as the turnover rate of the labeled protein. PHA-treated cells were labeled and stimulated with IL-2 (at day 3) in the presence or absence of IFN-{alpha}. IFN-{alpha} did not affect the rate of p27 synthesis during the 4-h labeling period (Fig. 2CCitation , Lanes Pulse). During the 20-h chase period, the amount of labeled p27 became significantly reduced in the IL-2-treated cells, whereas it was unchanged in the IL-2-/IFN-{alpha}-treated cells (Fig. 2CCitation , Lanes Chase). Together, these data indicate that IFN-{alpha} affects p27 expression through the rate of its degradation rather than affecting its rate of synthesis.

IFN-{alpha} Abrogates IL-2-dependent Up-Regulation and Activation of G1 Cyclins, Cdks, and Pocket Proteins.
In addition to the down-regulation of p27, IL-2 stimulation of T cells results in the induction of a number of cell cycle-regulatory proteins. To investigate the effect of IFN-{alpha} on these events, we analyzed the expression levels of the cyclins and their catalytic partners, the G1 Cdks, by Western blotting. Quiescent and PHA-stimulated cells did not express detectable protein levels of cyclin D3, and a readily visible band was first detected 24–48 h after IL-2 addition (day 6; Fig. 3ACitation ). Cdk4 was weakly expressed both in unstimulated, IL-2/IFN-{alpha}-treated as well as in proliferating T cells (data not shown). In contrast, Cdk6 protein levels were rapidly up-regulated within 24 h after IL-2 stimulation and were expressed constantly thereafter (Fig. 3B)Citation . IFN-{alpha} strongly inhibited the IL-2-dependent up-regulation of cyclin D3 and Cdk6 (Fig. 3, A and B)Citation .



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Fig. 3. Effects of IFN-{alpha} on IL-2-mediated up-regulation of cell cycle proteins. Immunoblot analysis of cyclin D3 and cyclin E (A) and Cdk2 and Cdk6 (B) expression in quiescent (Day 0), PHA-treated (Day 3), IL-2-treated (Days 4–6), and IL-2/IFN-{alpha}-treated (Days 4–6) cells.

 
It has recently been shown that cyclin E/Cdk2 activity alone is sufficient to force cells into S phase (25) . These findings prompted us to investigate the regulation of these proteins in our system. The cyclin E protein was weakly expressed in quiescent cells as a single Mr 55,000 band. PHA stimulation resulted in up-regulation of this protein (Fig. 3A)Citation . IL-2 addition further increased the intensity of the cyclin E band, and a steady level was accomplished at 48 h after IL-2 addition (day 5; Fig. 3ACitation ). Cdk2 protein levels were low in unstimulated and PHA-stimulated cells and expressed exclusively as the inactive, slowly migrating form (Fig. 3B)Citation . After only 24 h of IL-2 stimulation, the faster-migrating Cdk2 species, corresponding to the active CAK-phosphorylated form (26) , appeared and was gradually up-regulated until 72 h (day 6). IFN-{alpha}-treated cells expressed the same cyclin E levels as PHA-treated cells and completely lacked the IL-2-dependent increase in cyclin E expression. IFN-{alpha} also prevented up-regulation of Cdk2 expression as well as the shift toward the active CAK-phosphorylated form (Fig. 3B)Citation .

Because the pocket proteins pRb, p130, and p107 are substrates for the G1 kinases and critical regulators of the G1-S transition (7 , 27) , the total protein levels and phosphorylation status of these proteins were also analyzed by Western blotting. Quiescent T cells express p130 entirely in its hypophosphorylated form (form 1), whereas PHA-treated cells also express a phosphorylated form of p130 (form 2; Ref. 10 ). p130 protein levels increase somewhat following IL-2 stimulation, and a slower-migrating hyperphosphorylated form (form 3) of p130 was clearly detected on day 5 and thereafter (Fig. 4)Citation . IFN-{alpha} nearly completely abolished the up-regulation and phosphorylation of p130 (Fig. 4)Citation . In contrast to p130, pRb and p107 are not expressed at detectable levels until day 4, 24 h post-IL-2 stimulation, after which the expression of both pRb and p107 are gradually up-regulated and shifted to their hyperphosphorylated forms. In line with the findings on cyclin/Cdk expression, IFN-{alpha} interfered with both the up-regulation and the phosphorylation of all three pocket proteins (Fig. 4)Citation . Together, these results show that inhibition of S-phase entry by IFN-{alpha} correlates with the prohibited activation and/or expression of the pocket proteins.



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Fig. 4. Effects of IFN-{alpha} on IL-2-mediated up-regulation and phosphorylation of the pocket proteins. Immunoblot analysis of pRb, p130, and p107 expression in quiescent (Day 0), PHA-treated (Day 3), IL-2-treated (Days 4–6), and IL-2/IFN-{alpha}-treated (Days 4–6) cells. Tubulin served as control for equal loading in each lane. The monoclonal p130 antibody used also detected an nonspecific band (*); this band has been described previously (27) .

 
Because IFN-{alpha}-treated cells expressed some cyclin E as well as Cdk2, we decided to analyze the kinase activity of these complexes by performing an in vitro Cdk2 kinase assay using histone H1 as a substrate to determine whether or not the cells contained active Cdk2 complexes. Both unstimulated and IFN-{alpha}-cotreated cells possessed a low kinase activity, corresponding in both cases to {approx}20% of that in actively proliferating cells treated with IL-2 alone (data not shown).

IFN-{alpha} Effects on c-myc and Cdc25A Expression.
We have found that IFN-{alpha} has profound effects on the IL-2-dependent up-regulation of important G1-regulatory cell cycle proteins. We, therefore, wanted to investigate whether other downstream events emerging from the IL-2R were inhibited by IFN-{alpha}. Two of the downstream targets, c-myc and Cdc25A, were analyzed by Northern and Western blotting. c-myc and Cdc25A are two central regulators of proliferation in human cells, and it has recently been stipulated that c-myc exerts one of its functions by regulating Cdc25A expression. In accordance with previous studies, mitogenic stimulation of quiescent T cells results in c-myc induction at both the mRNA and protein levels (Fig. 5, A and B)Citation . The basal levels of c-myc expression in unstimulated cells varied slightly between different donors; however, the mitogen induced up-regulation was reproducible in each experiment. Interestingly, IFN-{alpha} did not prevent this up-regulation of c-myc following IL-2 stimulation at either the mRNA or protein level; rather, it resulted in slightly enhanced expression (Fig. 5, A and B)Citation . In addition, IFN-{alpha} did not inhibit the up-regulation of Cdc25A following IL-2 stimulation (Fig. 5C)Citation . We also analyzed the expression of the high-affinity subunit of the IL-2R (IL-2R{alpha}) by flow cytometry. This receptor subunit was up-regulated following IL-2 stimulation, and no major inhibitory effect was detected following IFN cotreatment (data not shown).



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Fig. 5. IFN-{alpha} effects on c-myc and Cdc25A expression. Analysis of c-myc and Cdc25A regulation in quiescent (Day 0), PHA-treated (Day 3), IL-2-treated (Days 4–6), and IL-2/IFN-{alpha}-treated (Days 4–6) cells by immunoblotting (A), Northern blot analysis (B), and immunoblotting (C). Tubulin (A and C) and 18S and 28S rRNA (B) served as controls for equal loading.

 
Effects of IFN-{gamma} on T-Cell Activation.
IFN-{alpha} and -{gamma} have similar cellular effects in many systems, including their antiviral action, induction of differentiation, and cell growth-inhibitory effects. IFN-{alpha} and -{gamma} share certain signaling components such as Jak1 and Stat1, and they have both been shown to regulate CKI expression and exert antiproliferative effects in various cell systems; however, they have profoundly different effects on S-phase entry in T cells because IFN-{gamma} may, instead, function as a T-cell stimulator (4 , 28) . We analyzed the basis for this difference by investigating the effects of IFN-{gamma} on IL-2-mediated cell cycle progression in PHA-stimulated cells. IFN-{gamma} was not found to abrogate IL-2-dependent S-phase entry and, in contrast to IFN-{alpha}, did not abrogate any of the IL-2-induced effects on the cell cycle-regulatory proteins analyzed in this study (data not shown).


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Proliferation and differentiation within the immune system are tightly regulated by a combination of stimulatory signals derived from antigens and costimulatory molecules in concert with different cytokines. Stimulation of resting T lymphocytes is mainly dependent on the binding of HLA-associated antigens to the TCR and further stimulation by the autocrine growth factor IL-2, although a number of other molecules can modulate the T-cell response as well (29) . The early events of T-cell activation and induction of proliferation have been largely defined by in vitro models, which allow T cells to be stimulated in a controlled manner. A number of studies have pointed out crucial molecular events taking place during T-cell activation, resulting in exit from G0 and activation of the cell cycle machinery (13, 14, 15 , 27) . It has also been demonstrated that activation of T-cell proliferation via the IL-2R is a multistep event, involving several different pathways (3) . For a proper proliferative response, these pathways must be activated in concert, and accordingly, inactivation of one pathway can completely abrogate the proliferative response to IL-2 (3) . It is also important to stress that, although our understanding of the molecular mechanisms underlying IL-2-induced proliferation has increased greatly during recent years, a complete picture has not yet emerged.

IFN-{alpha} has previously been demonstrated to possess T lymphocyte-regulatory functions, including the inhibition of T-cell proliferation (4) . However, little is known about the molecular basis of these effects. In this study, we examined the growth-inhibitory effects of IFN-{alpha} during stimulation of primary T lymphocytes and, in parallel, studied the effects on cell cycle-regulatory proteins.

PHA stimulation of T cells provides an activation signal, but alone, it is not sufficient to induce proliferation, which was clearly demonstrated by Firpo et al. (13) ; instead, it results in exit from G0 and entry into G1. IL-2 addition results in S-phase entry and activation of cyclin E-Cdk2 complexes (13) . Here, we used the same experimental model. We found this experimental model useful because it is possible to study the sequential activation of the cell cycle machinery during the transition from G0 to proliferation. Furthermore, this model provides a comparative basis for studying the effects of IFN-{alpha} on T-cell proliferation because the molecular cell cycle events are well characterized in this system (13) . Upon IL-2 addition, the signals necessary for proliferation are provided, and within 24 h, cells begin to enter S-phase, and the majority of cells stimulated with IL-2 are proliferating within 72 h. The general effects of PHA/IL-2 stimulation on the cell cycle machinery in our system are in accordance with previous studies using a similar stimulation protocol (13) . In other studies, in which a faster activation of the cell cycle machinery has been observed, an alternative stimulation procedure is the most probable explanation for this discrepancy (15) .

Activation of the cell cycle machinery is a crucial event for progression into S phase. To search for a molecular explanation for the potent antiproliferative effect of IFN-{alpha} in T cells, we performed a detailed analysis of its effects on different cell cycle-regulatory proteins. In several lymphoid cell lines, IFN-{alpha} seems to exert its antiproliferative action via the up-regulation of CKIs, resulting in pocket protein dephosphorylation and G1 arrest (10 , 22 , 23) . Quiescent T cells express p130 but not pRb and p107 (27) . Stimulation with PHA and IL-2 up-regulates all three pocket proteins, an effect that is also accompanied by the increased phosphorylation of these proteins (Fig. 4)Citation , coinciding with the onset of proliferation. Pocket protein up-regulation and phosphorylation is prevented by cotreatment with IFN-{alpha}. Furthermore, IL-2-induced expression of both early and late G1 Cdk complexes (cyclin D3-Cdk6 and cyclin E-Cdk2) was also inhibited by IFN-{alpha} (Fig. 3, A and B)Citation , and Cdk2 activity was kept at the same level as that in unstimulated cells. As has been shown previously (15) , cyclin D2 is expressed prior to cyclin D3 during T-cell stimulation, indicating that D-type cyclins are also present in our system before 48 h, although they were not analyzed. Furthermore, IFN-{alpha} treatment blocked a shift toward the more rapidly migrating active form of Cdk2, which corresponds to the CAK-phosphorylated (Thr-160) Cdk2 species in IL-2-stimulated cells (26) . This effect was probably due to the maintenance of high p27 levels during IFN-{alpha} treatment, which may interfere with phosphorylation of Cdk2 by CAK (30) . It is of interest to note that IFN-{gamma}, which does not block T-cell proliferation, had no effect on the studied cell cycle-regulatory proteins.

The CKI p27 has been shown to be essential for maintenance of quiescence in several cellular systems (9 , 10) . In line with this, unstimulated quiescent T lymphocytes contain high p27 levels. Upon mitogen stimulation of cells, p27 is phosphorylated, possibly by activated Cdk2, thus targeting this CKI for proteosomal degradation (31) . The IL-2-dependent down-regulation of p27, in the present in vitro system, is almost completely abrogated by IFN-{alpha}. The mechanism behind the effect of IFN-{alpha} on p27 expression was mainly due to a maintained stability of the p27 protein following IFN-{alpha} treatment (Fig. 2C)Citation . One possible explanation for this finding could be that the inhibition of G1 cyclin expression and, therefore, Cdk activation prevent phosphorylation and subsequent elimination of p27. However, we cannot exclude the possibility that IFN-{alpha} inhibits the activation of another kinase(s) necessary for p27 degradation.

Immunosuppressive drugs such as rapamycin (14) and undeculprodigiosin (32) also inhibit antigen-stimulated T-cell proliferation. However, the molecular effects seen in these systems are only partial in comparison with IFN-{alpha}. The T-cell suppressive action of rapamycin is not due to an inhibited expression of either Cdk2/4/6 or any of cyclins D2/D3 or E. However, the IL-2-mediated down-regulation of p27 is completely blocked, demonstrating that this event is a prerequisite for S-phase entry in this system. In undeculprodigiosin-treated cells, the picture is different because the down-regulation of p27 is not affected, whereas induction of cyclin E/A and Cdk2/4 are strongly blocked. Our data indicate that IFN-{alpha} acts further upstream than these other immunosuppressive drugs because it shows a broader inactivating effect on the cell cycle machinery.

The mechanism underlying IL-2-mediated activation of the cell cycle machinery and subsequent induction of E2F-driven transcription has recently been investigated by Cantrell and colleagues (18) . They suggest that IL-2-dependent induction of E2F activity occurs through stimulation of the PI3K pathway, involving activation of protein kinase B because expression of active protein kinase B alone is sufficient to induce E2F activity. Furthermore, inhibition of PI3K by a specific inhibitor prevents IL-2-dependent cyclin D3 up-regulation, p27 elimination, and pRb/p130 phosphorylation (18) . At what point IFN-{alpha} interacts with the IL-2 signaling pathways, leading to a blocked activation of the cell cycle machinery, was not determined here. However, several possibilities exist because both these signaling pathways use some of the same Jak and Stat molecules and also interact with the PI3K pathway (3 , 19 , 28 , 33 , 34) .

Activation of the PI3K pathway by IL-2 is not sufficient to trigger proliferation in T cells (21) ; additional pathways must be activated for S-phase entry. One such important pathway is mediated by the IL-2R-associated protein kinase Syk, which has been suggested to induce the expression of c-myc (3 , 35) . In contrast to the other cell cycle-regulatory genes examined in this study, the PHA-/IL-2-mediated up-regulation of c-myc was not inhibited but rather slightly enhanced by IFN-{alpha} treatment (Fig. 5, A and B)Citation . It has also been shown that supranormal expression of c-myc can lead to S-phase entry by activation of Cdk2 complexes (36) . However, the maintenance of high p27 levels in IFN-{alpha}-treated T cells remains a sound explanation for the inability of c-myc to mediate S-phase entry in these cells.

The exact physiological role of the potent influence of IFN-{alpha} on T-lymphocyte activation is not known. Because IFN-{alpha} is commonly produced during viral infections, this may, for example, be a way by which the immune system avoids a general nonspecific activation of resting T lymphocytes during infection. Although IFN-{alpha} has been shown to be immunosuppressive under some circumstances, patients treated with IFN-{alpha} do not generally show symptoms associated with potent immunosuppression, such as opportunistic infections. One reason for this paradox may be that IFN-{alpha} in itself provides protection to many infectious agents via its antimicrobial action. Furthermore, IFN-{alpha} activates other immunologically active cells, such as granulocytes, monocytes, and NK cells. It is also possible that the potent effect of type I IFNs on T-lymphocyte activation may add to the clinical effects observed in T-cell malignancies as well as in multiple sclerosis and other diseases in which autoreactive T cells have been implicated (37 , 38) . Somewhat paradoxically, IFN-{alpha} treatment has also been shown to trigger autoimmune disorders, such as thyroiditis and autoimmune hepatitis, in rare cases (39) . Thus, further studies are required to elucidate the physiological role of IFN-{alpha} in the regulation of the immune system. This study may also provide a tool for defining how IL-2 signaling leads to T-cell proliferation.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
T-Lymphocyte Isolation and Cell Culture.
Buffy coats from healthy blood donors were heparinized, mononuclear cells were isolated by Lymphoprep gradient centrifugation (Nycomed, Oslo, Norway), and T lymphocytes were isolated using nylon wool columns, as described previously (24) . In each experiment performed, >95% of the cells were viable, as determined by trypan blue exclusion. The nylon wool-enriched human T lymphocytes were tested for purity before stimulation, as described below.

The lymphocytes were seeded at a density of 5 x 105 cells per ml in complete MEM (MEM supplemented with 10% heat inactivated AB+ serum, 2 mM glutamine, 50 µg/ml streptomycin, and 50 µg/ml penicillin) and kept in a humid incubator with 5% CO2. The cells were counted and reseeded every second day (5 x 105 cells per ml), when fresh medium was added. The resting T lymphocytes were stimulated to proliferation in a two-stage process by the sequential addition of 0.8 µg/ml PHA (Sigma) for 72 h and, subsequently, IL-2 at a concentration of 100 units/ml (13 , 24 , 40) . Restimulation was performed every second day by inclusion of IL-2 (100units/ml) in the fresh medium added to the cells. IFN-{alpha} (5000 units/ml, similar inhibitory effects on T-cell proliferation was observed with 500 units) and IFN-{gamma} (5000 units/ml) were added to the cultures together with IL-2.

Each experiment was repeated with cells from two to five different donors. All figures show the results from representative experiments.

Antibodies and Cytokines.
Recombinant IFN-{alpha}2b (Schering-Plough, Kenilworth, NJ) and IFN-{gamma} (Imukin; Boehringer Ingelheim AB, Stockholm, Sweden) were used in the different experiments. Recombinant IL-2 was a generous gift from Dr. A. Österborg (Karolinska Institute, Stockholm, Sweden). The following antibodies were used in this study. pRb, Cdk4, Cdk6, and cyclin E from PharMingen (San Diego, CA); p27, p21, p130, and Cdk2 were from Transduction Laboratories (Lexington, KY); p130, p107, cyclin D3, p27, and c-Myc were from Santa Cruz Biotechnology (Santa Cruz, CA); and tubulin was from Sigma Chemical Co. (St. Louis, MO). Cdk2 and Cdc25A were generous gifts from D. Beach (CSH Laboratories, Cold Spring Harbor, NY) and I. Hoffman (GCRC, Heidelberg, Germany), respectively.

Flow Cytometric Analysis.
Fixation and analysis of DNA histograms were performed as described previously (22) . The presence of non-T cells in the culture and CD25 expression were determined by flow cytometry. The following monoclonal antibodies were purchased from Becton Dickinson (Mountain View, CA): CD3, CD16, CD56, CD14, CD19, and CD25. The cells were stained as follows and analyzed in a FACScan flow cytometer using the CellQuest software. Statistics were calculated based on an ungated cell population. The cells (0.5 x 106) were mixed with 10 µl of fluorochrome-conjugated antibody, incubated in the dark at 4°C for 30 min, and washed twice with PBS. Cells were resuspended in 1 ml of PBS and analyzed.

Quiescent T-cell cultures contained <5% NK cells (CD56 and CD16 positive and CD3 negative) and <1% B cells (CD19 positive). The remaining >94% cells were CD3 positive (data not shown).

Bromodeoxyuridine labeling and subsequent flow cytometric analysis were performed as described previously (41) . For protein content measurement, cells from two different donors were fixed and treated as described previously (42) . The ACAS (Ahrens Flow System, Bargteheide/Hamburg, Germany) program was used for the contour plot analysis.

Western Blot Analysis.
Western blot analysis was performed essentially as described previously (22) . Briefly, whole cell extracts were prepared by lysis through sonication in LSLD buffer containing protease inhibitors. The protein concentration was determined spectrophotometrically with the Bradford method, according to instructions of the manufacturer (Bio-Rad, Hercules, CA). Seventy µg of protein were loaded in each well. Proteins were resolved by SDS-PAGE on 6% (for pocket proteins, c-myc, and Cdc25A) or 12% (for all other proteins) gels and electroblotted to polyvinylidene difluoride membranes (Boehringer Mannheim, Mannheim, Germany) by semidry transfer. The filters were subsequently stained with Ponceau S (Sigma; 0.1% in 5% acetic acid) to determine transfer efficiency and even loading. For protein detection, we hybridized the filters for 1 h with monoclonal antibodies anti-pRb (1:1000), anti-p130 (1:2000), anti-c-myc (1:200), anti-p27 (1:2000), anti-Cdk2 (1:2000), anti-p21 (1:500), anti-cyclin E (1:1000), and antitubulin (1:4000) and polyclonal antibodies anti-Cdk4 (1:1000), anti-Cdk6 (1:2000), anti-cyclin D3 (1:200), anti-Cdc25A (1:2000), anti-p107 (1:200), and anti-p130 (1:1000). Antibody-antigen interaction was detected by incubation with horseradish peroxidase-conjugated antimouse or antirabbit IgG antibodies, and subsequent detection was performed by enhanced chemiluminescence (ECL; Amersham, Aylesbury, United Kingdom).

Immunoprecipitation and in Vitro Kinase Assay.
Cdk2 immunoprecipitations as well as subsequent Cdk2 kinase assays using histone H1 as substrate were performed as described previously (10 , 22) .

Northern Blot Analysis.
Total cellular RNA isolation and Northern blot analysis was performed as described (25) . Northern filters (Hybond C-extra; Amersham) were hybridized with a 600-bp NheI/XhoI restriction fragment of the p27 cDNA (a generous gift from Dr. J. Massagué, Memorial Sloan-Kettering Cancer Center, New York, NY) and a 800-bp c-myc cDNA probe (kindly provided by Dr. M. Henriksson, Karolinska Institute, Stockholm, Sweden) as well as with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß-actin probes. All probes were labeled as described previously (22) .

Measurement of p27 Biosynthesis and Turnover Rate.
Twenty million purified and PHA-stimulated cells (day 3) were washed twice in methionine- and cysteine-free RPMI 1640 at room temperature and incubated 30 min at a concentration of 10 x 106 cells/ml. The medium was removed by centrifugation, and cells were labeled (pulse) for 4 h at 37°C in labeling medium containing 0.2 mCi of 35S labeling mix (Redivue Pro-mix; Amersham) and IL-2 (100 units/ml) in the absence or presence IFN-{alpha} (5000 units/ml). After removal of labeling medium, cells were incubated in the presence of excess nonradioactive methionine and cysteine (in complete medium), also containing IL-2 with or without IFN-{alpha}, for 20 h (chase). Cells were harvested and washed in ice-cold PBS. Cell pellets were frozen in liquid nitrogen and subsequently lysed in LSLD buffer by sonication. Five hundred µg of labeled protein extract were subjected to immunoprecipitation with polyclonal p27 antibodies, as described previously (10 , 22) . The immunoprecipitated proteins were separated by 12% SDS-PAGE and blotted to a polyvinylidene difluoride membrane by semidry transfer. The filter was exposed to X-ray film (Amersham) at room temperature with an intensifying screen (Hyperscreen; Amersham) for examination of p27 synthesis and stability. After exposure, the filter was subjected to Western blot analysis with monoclonal p27 antibodies as a control for the total amount of p27 loaded onto each well (data not shown).

Quantitation of Band Intensity.
Hybridization signals were quantitated by scanning densitometry using an Ultroscan XL (Pharmacia LKB Biotechnology, Uppsala, Sweden).


    Acknowledgments
 
The excellent technical assistance of Elisabet Anderbring, Ann-Charlotte Björklund, and Ingrid Eriksson is gratefully acknowledged. We thank Drs. Niel Portwood and Sampsa Matikainen for helpful discussions and critical reading of this manuscript.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the Swedish Cancer Society, the Cancer Society of Stockholm, and the Felix Mindus Fund for Leukemia Research. Back

2 To whom requests for reprints should be addressed, at Research Laboratory of Radiumhemmet, Cancer Center Karolinska, Karolinska Hospital and Institute, S-171 76 Stockholm, Sweden. Phone: 46-8-5177 6262; Fax: 46-8-33 90 31; E-mail: Dan.Grander{at}cck.ki.se Back

3 The abbreviations used are: TCR, T-cell receptor; Cdk, cyclin-dependent kinase; CKI, Cdk inhibitor; PHA, phytohemagglutinin; IL-2, interleukin 2; IL-2R, IL-2 receptor; PI3K, phosphatidylinositol 3-kinase; NK, natural killer; CAK, cdk-activating kinase; Thr, threonine. Back

Received for publication 1/ 4/99. Revision received 6/ 1/99. Accepted for publication 6/23/99.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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