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The Journal of Immunology, 2000, 164: 2296-2302.
Copyright © 2000 by The American Association of Immunologists

IFN-{alpha}2b Reduces IL-2 Production and IL-2 Receptor Function in Primary CD4+ T Cells

Davide Zella1,*, Fabio Romerio*, Sabrina Curreli*, Paola Secchiero*,{dagger}, Claudia Cicala{ddagger}, Daniel Zagury§ and Robert C. Gallo*

* Institute of Human Virology, University of Maryland Biotechnology Institute and University of Maryland Medical Center, Baltimore, MD 21201; {dagger} Department of Morphology and Embryology, Human Anatomy Section, University of Ferrara, Ferrara, Italy; {ddagger} Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and § Institute Pierre and Marie Curie, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Initially described as an antiviral cytokine, IFN-{alpha} has been subsequently shown to affect several cellular functions, including cellular differentiation and proliferation. For these reasons, IFN-{alpha} is currently used in clinical practice for the treatment of viral infections and malignancies. In this manuscript, we show two novel mechanisms concomitantly responsible for the antiproliferative effect of IFN-{alpha}. First, long-term treatment with IFN-{alpha} of primary CD4+ T cells reduced surface expression of CD3 and CD28. These events resulted in decreased phosphorylation of the mitogen-activated extracellular signal-regulated activating kinase and its substrate extracellular signal-regulated kinase, leading to diminished production of IL-2. Second, IFN-{alpha} treatment of primary CD4+ T cells reduced proliferative response to stimulation in the presence of exogenous IL-2 by markedly decreasing mRNA synthesis and surface expression of CD25 ({alpha}-chain), a critical component of the IL-2R complex. These results may be relevant for the antitumor effects of IFN-{alpha} and may help us to better understand its detrimental role in the inhibition of proliferation of the bulk of CD4+ T cells (uninfected cells) in HIV-infected persons, who are known to overproduce IFN-{alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The network of protein organization, which transduces signals from the external environment to activate specific cellular pathways, has several levels of complexity. The first level can be seen as a simple execution of a cellular program upon stimulation of a single receptor. However, this ideal situation is virtually absent in the life span of a cell. Instead, a continuous array of stimuli and multiple protein-protein interactions, which often allow intra- and interpathway communications, renders the outcome of a signal (or a series of signals) difficult to predict. Nonetheless, disclosing these levels of complexity is of fundamental importance to understand the processes governing cellular activity.

IFN-{alpha}, like many cytokines, transduces regulatory signals through the Janus tyrosine kinase(Jak)2/STAT pathway (1). Receptor-associated, ligand-activated Jak kinases phosphorylate STAT proteins on tyrosine residues. Subsequently, activated STATs translocate into the nucleus to initiate transcriptional activation. IFN-{alpha} has been initially described as an antiviral cytokine (2), conferring a state of resistance at one or more phases of the viral cycle, thus gaining potential relevance for the treatment of chronic infectious diseases such as hepatitis (3). Subsequently, it has been shown that IFN-{alpha} can also affect the growth, differentiation, and function of various cell types (4, 5, 6, 7), thus acquiring relevance for the treatment of malignancies, including hairy cell leukemia, Kaposi’s sarcoma, chronic myeloid leukemia, B and T cell lymphomas, myelomas, melanomas, and renal carcinomas (for reviews, see Refs. 8, 9, 10). Understanding the mechanisms of action of this multifunctional cytokine is then of fundamental importance both for our comprehension of immune function and to better take advantage of the therapeutic potential of IFN-{alpha}.

The signaling pathways employing MEK and ERK are critical in growth factor signaling. Engagement of the receptors initiates a phosphorylative cascade leading to activation of several proteins among which MEK and ERK play a central role in routing signals critical in controlling cell development, activation, and proliferation (11, 12, 13, 14). In addition, modifications in this well-conserved regulatory cascade often lead to cellular transformation or to uncontrolled cellular proliferation (15, 16, 17, 18, 19, 20). In CD4+ T cells, upon triggering of the TCR, activation of the MEK/ERK pathway routes signals which are critical for cellular activation, proliferation, and production of cytokines important for the regulation of immune responses (21, 22, 23, 24, 25, 26, 27). In contrast, inhibition of the MEK/ERK pathway activity has been shown to result in 1) impaired proliferative response of primary human CD4+ T cells upon costimulation with anti-CD3 and anti-CD28 and diminished IL-2 production (28), and 2) reduced maturation of thymocytes (29, 30).

In CD4+ T cells, simultaneous stimulation of the TCR and CD28 is necessary for production of IL-2 (24, 25, 26, 31). Subsequently, proliferation of T lymphocytes is triggered by the interaction of IL-2 with its specific receptor complex (IL-2R), composed of three transmembrane proteins (namely, {alpha}-, ß-, and {gamma}-chain), which make up two forms of receptor: intermediate affinity (ß + {gamma}) and high affinity ({alpha}, ß, and {gamma}) (32). Once properly assembled, the IL-2R complex allows intracellular transduction of the signals generated upon IL-2 stimulation (33).

Previous studies demonstrated that IFN-{alpha} reduces proliferative response of CD4+ T cells stimulated through the TCR in the presence of IL-2 (34). Consequently, in an attempt to provide a molecular basis for this antiproliferative effect, we tested the hypotheses that IFN-{alpha} could affect 1) the function of the MEK/ERK pathway, triggered by TCR stimulation; and 2) the expression of components of the IL-2R complex, namely, the {alpha}-, ß-, and {gamma}-chain.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Purification of CD4+ T cells

CD4+ T cells were purified by negative selection from Ficoll-separated PBMCs obtained from healthy donors. Briefly, PBMCs were incubated with anti-CD8, -CD14, -CD16, -CD19, -CD20, -CD56, and -CD57 for 60 min at 4°C in RPMI 1640 and then washed with 1x PBS (both reagents from Life Technologies, Rockville, MD). Subsequently, cells were incubated with magnetic beads conjugated to anti-mouse IgG for 60 min at 4°C (Polysciences, Warrington, PA). Bead-bound cells were removed from PBMCs with a magnetic device. Purity of CD4+ T cells was then assessed by flow cytometry and resulted to be >92% in all of the experiments described.

Cross-stimulation of CD4+ T cells and measurement of IFN-mediated antiproliferative activity

Recombinant human IL-2 was obtained from Boheringer Mannheim (Indianapolis, IN). Recombinant human IFN-{alpha}2b was purchased from Biosidus (Buenos Aires, Argentina). The protein was >98% pure as assessed by gel electrophoresis. IFN-{alpha} antiviral activity was assessed in culture with the standard biological test by using MDBK cells and vesicular stomatitis virus as described previously (35). The activity was in the range of 2–3 x 108 U/mg. Anti-IFN-{alpha} polyclonal Abs were obtained from BioSource International (Camarillo, CA).

Aliquots (1 x 106 cells/ml) of purified CD4+ T cells were treated with IFN-{alpha}2b (50–100 ng/ml) for 18 h before cross-stimulation. Cross-stimulation was conducted in 6-well plates coated with anti-CD3 (1 µg/ml) plus anti-CD28 (1 µg/ml) in the presence of IL-2 (40 U/ml) and IFN-{alpha}2b (100 ng/ml) plates from Costar (Cambridge, MA), mAb anti-CD3 (clone UCHT1) was from Coulter Pharmaceutical (Palo Alto, CA), and anti-CD28 was from Becton Dickinson (San Jose, CA). Viable cells were counted by trypan blue exclusion at different time points. Experiments were performed in duplicate on CD4+ T cells purified from four to six different donors.

Preparation of the protein lysates

Aliquots of cells were resuspended in cell lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerolphosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM PMSF), and the lysate was incubated at 4°C for 15 min with moderate shaking. Cell debris were pelleted by centrifugation and the supernatant was collected and frozen at -80°C.

Western blot analysis

For the Western blot analysis, 25 µg of cellular lysate was run on a 12% SDS-polyacrylamide gel in Tris-glycine buffer. The gels were transferred to polyvinylidene fluoride using a SemiDry Blotting apparatus (Pharmacia, Piscataway, NJ) in 25 mM Tris, 192 mM glycine, and 20% methanol. After blocking in Blotto-Tween 20 (10 mM Tris, 0.9% NaCl, 0.1% Tween 20, and 5% nonfat dry milk), the membranes were first probed with the specific mAb detecting the protein (according to the manufacturer’s instructions). The blots were then incubated with a specific HRP-linked secondary Ab and developed using the enhanced chemiluminescence plus kit from Amersham (Arlington Heights, IL). Subsequently, the blots were stripped in 0.1 M glycine (pH 2.9), blocked in Blotto-Tween 20, and reprobed with Abs detecting total protein levels. Anti-MEK1/2 and anti-ERK 1/2 Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-MEK1/2 and anti-phospho-ERK 1/2 Abs were purchased from New England Biolabs (Beverly, MA).

Flow cytometry assay

Approximately 1 x 106 cells/sample were pelleted in a round-bottom centrifuge tube at 200 x g for 5 min. Cells were resuspended in 100 µl of PBS containing 1% FBS (both reagents from Life Technologies) and incubated with the appropriate Abs on ice for 30 min in the dark. Aliquots of cultures were washed in supplemented PBS before analysis of cellular markers by three-color flow cytometry (FACSan; Becton Dickinson). mAbs used were anti-CD3, -CD4, -CD25, -CD122, and -CD132 (all from Immunotech, Westbrook, ME). Anti-CD28 was obtained from PharMingen (San Diego, CA). The appropriate isotype mAb conjugate controls were used to determine the levels of background fluorescence. Viable cell gates were used to collect 10,000 events within the T cell populations.

Semiquantitative RT-PCR analysis

Semiquantitative mRNA analysis was performed by using end-point dilution PCR. Direct comparison of the samples allowed the quantification of the specific mRNA. This method is utilized when no internal competitor or external standard is available. Aliquots of cells (5 x 106) were collected at the indicated time points. Total cellular RNA was isolated by TRIzol LS reagent (Life Technologies) following the manufacturer’s protocol. Samples of RNA were treated by DNase/RNase-free (Life Technologies). Concentration of RNA was determined by spectrophotometric analysis. Synthesis of first-strand DNA was performed in 20 µl of reaction mix (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 500 µM of each dNTPs, 2.5 µM random hexamer primers (PE Applied Biosystems, Branchburg, NJ), 1.5 µg of RNA, and 200 U Superscript II RT (Life Technologies) for 60 min at 37°C, followed by a heating/inactivation step at 99°C for 5 min. Amplification was performed with 10 µl of RT mixture in a total volume of 100 µl using 10 µM dNTPs, 40 pM primers, and 5 U of AmpliTaq polymerase (PE Applied Biosystems). After a common initial denaturing step of 5 min at 92°C, amplification reaction for the components of the IL-2R was conducted as follows with specific conditions for each amplification products which allowed us to detect as low as 50 copies: for the {alpha}-chain, 94°C for 60 s, 60°C for 45 s, 72°C for 60 s; for the ß-chain, 94°C for 60 s, 64°C for 60 s, 72°C for 60 s; for the {gamma}-chain: 94°C for 60 s, 60°C for 60 s, 72°C for 60 s; for the ß- actin, 94°C for 60 s, 60°C for 60 s, 72°C for 60 s. After a common step at 72°C for 5 min, the resulting PCR products were separated on a 2% SeaKem GTG agarose (FMC, Rockland, ME). As a control for genomic DNA contamination, equal amounts of RNA extraction products were used for each sample assessed and PCR amplification was performed without the addition of RT to the first-strand synthesis step. The following sets of primers were used for the amplification of the IL-2R components: {alpha}-chain (36): sense 5'-CAAAGTCCAATGCAGCCAGT-3' and antisense 5'-TCACCTGTGCATATGAGCTG-3' yielding a PCR product of 232 bp; ß-chain (37): sense 5'-GCGTGGCTCGGCCACCTC-3' and antisense 5'-GACGATGAGGGGAAGGGCGAAGA-3' yielding a PCR product of 211 bp; and {gamma}-chain (38): sense: 5'-GTCCCAGAGAACCTAACACT-3' and antisense 5'-GATCCTCTAGGTTCTTCAGG-3' yielding a PCR product of 409 bp. As control, the ß-actin mRNA was amplified: sense 5'- CGAGCGGGAAATCGTGCGTGACATTAAGGA-3' and antisense 5'- CGTCATACTCCTGCTTGCTGATCCACATCT-3' yielding a PCR product of 478 bp.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Recombinant human IFN-{alpha}2b reduces proliferative ability of primary CD4+ T cells in response to anti-CD3 plus anti-CD28 in the presence of IL-2

It has been previously demonstrated that primary CD4+ T cells have reduced proliferative ability when stimulated with PHA (or anti-CD3) plus IL-2 in the presence of IFN-{alpha} (34). We reasoned that, since triggering of costimulatory molecules (e.g., CD28) could provide additional signals to promote CD4+ T cell proliferation, such costimulation could possibly overcome the antiproliferative effect of IFN-{alpha}. Cross-linked anti-CD3 plus anti-CD28 were thus used to stimulate primary, peripheral blood-purified CD4+ T cells in the presence of IL-2. A significant reduction (about 50% after 72 h) of cell proliferative ability was observed when recombinant human IFN-{alpha}2b was added to the cultures, as opposed to the untreated samples (Fig. 1Go). Addition of polyclonal Abs against IFN-{alpha} reversed the antiproliferative effect (data not shown). Based on these data, we hypothesized that IFN-{alpha} could act on the components of one of the pathways involved in our conditions of stimulation, namely, the TCR, CD28, and/or IL-2R complex. For this reason, we proceeded to test both the surface expression and the ability of such cellular components to deliver the proper intracellular signals.



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  		FIGURE 1. Proliferative response of purified CD4+ T cells to anti-CD3 plus anti-CD28 costimulation in the presence of IFN-{alpha}2b. CD4+ T cells were purified by negative selection from total PBMC of healthy seronegative donors (see Materials and Methods). A total of 106 cells/ml was treated for 18 h with IFN-{alpha} (50–100 ng/ml). Subsequently, cells were plated in anti-CD3 plus anti-CD28-coated plates in the presence of IFN-{alpha}. No IFN-{alpha} was added to the untreated cultures. At the indicated times, samples of the cultures were taken and the cells were counted. Viable cell number (>95%) was determined by trypan blue staining. Results are representative of experiments conducted in duplicate with CD4+ cells purified from six donors. Mean and SD are shown.

 
IFN-{alpha}2b reduces IL-2 production in primary CD4+ T cells cross-stimulated with anti-CD3 plus anti-CD28

One of the most important consequences following CD3 plus CD28 cross-stimulation of CD4+ T cells is the production of IL-2 (24, 25, 26, 31). For this reason, as a functional assay for the TCR and CD28 pathway, we sought to assess whether cross-stimulation in the presence of IFN-{alpha} resulted in reduced IL-2 production. Primary CD4+ T cells were thus stimulated with cross-linked anti-CD3 plus anti-CD28. Subsequently, we determined intracellular IL-2 production by using a standardized commercial ELISA kit. Treatment with IFN-{alpha} markedly reduced production of intracellular IL-2, as opposed to untreated aliquots (Fig. 2Go). Addition of polyclonal Abs against IFN-{alpha} prevented the reduction of IL-2 production (data not shown). This would indeed indicate that IFN-{alpha} induces defect(s) at a certain point of one (or both) pathway triggered by the anti-CD3 and/or the anti-CD28.



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  		FIGURE 2. Analysis of intracellular IL-2 production of purified CD4+ T cells cross-stimulated with anti-CD3 plus anti-CD28 in the presence of IFN-{alpha}2b. CD4+ T cells were purified by negative selection from total PBMC of healthy seronegative donors (see Materials and Methods). A total of 106 cells/ml was treated for 18 h with IFN-{alpha} (50–100 ng/ml). Subsequently, cells were plated in anti-CD3 plus anti-CD28-coated plates in the presence of IFN-{alpha}. Control aliquots were treated in the same way, except that no IFN-{alpha} was added to the cultures. At the indicated times, samples of the cultures (106 cells) were collected, washed, and resuspended in lysis buffer for the intracellular IL-2 protein analysis with a standardized ELISA kit (R&D Systems, Minneapolis, MN). Results are representative of experiments conducted in duplicate with CD4+ cells purified from four donors. Mean and SD are shown.

 
IFN-{alpha}2b treatment of CD4+ T cells reduces surface expression of CD3 and CD28

A possible explanation of these results could be that IFN-{alpha}-mediated down-regulation of surface TCR and/or CD28, the two receptors stimulated in our system. For this reason, we used flow cytometry analysis to determine their expression on cell surface. Aliquots of primary, purified CD4+ T cells were stimulated with IFN-{alpha} for 18–20 h and subsequently stimulated with cross-linked CD3 plus CD28. Upon treatment with IFN-{alpha} and subsequent costimulation with anti-CD3 and anti-CD28, we observed a slight reduction in the surface expression of these molecules (Fig. 3Go). This difference was quantified by comparison with standard labeled microbeads as described previously (39). CD3 expression in the untreated cells ranged between 9,000 and 12,000 molecules of equivalent soluble fluorochrome (MESF) vs 3,000 and 5,000 MESF expressed by the IFN-treated cells (data not shown). CD28 expression ranged between 13, 000 and 15,000 MESF for the untreated cells vs 7,000 and 9,000 MESF expressed by the IFN-treated ones (data not shown).



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  		FIGURE 3. Surface analysis of CD3 and CD28 in purified CD4+ T cells cross-stimulated with anti-CD3 plus anti-CD28 in the presence of IFN-{alpha}2b. Purified CD4+ T cells (106/ml) were treated for 18–20 h with IFN-{alpha} (50–100 ng/ml). Subsequently, cells were stimulated with immobilized anti-CD3 plus anti-CD28 as described in Materials and Methods. For the flow cytometry analysis, cells were collected at the indicated time and treated as described in Materials and Methods. Histograms are representative of experiments conducted in three different donors. Thin line, untreated cells; thick line, IFN-{alpha}-treated cells; and dotted lines, isotype controls. anti-CD3 x anti-CD28, cross-stimulation with immobilized anti-CD3 plus anti-CD28.

 
IFN-{alpha}2b reduces phosphorylation of MEK1 and ERK1/2 in primary CD4+ T cells

Because of the critical importance of the MEK/ERK pathway in the transduction of the signals necessary for IL-2 production, we reasoned that IFN-{alpha} could alter the activity of important components in this cascade. Phosphorylation of MEK by autophosphorylation or upstream kinases (40, 41, 42, 43, 44, 45) is required for its enzymatic activity, and it is the main upstream mechanism leading to phosphorylation of both tyrosine and serine/threonine residues and subsequent activation of ERK (46). This, in turn, results in the regulation of a large number of proteins both in the cytoplasm and, following ERK translocation, in the nucleus (47, 48, 49). We thus determined the effect of IFN-{alpha} on MEK and ERK phosphorylation. Using Abs that can specifically recognize the phosphorylated form of these proteins, we found a decreased level of phosphorylation of MEK1 and ERK1/2 upon long-term treatment (12–48 h) with IFN-{alpha} (Fig. 4Go, upper and middle panels). Western blot analysis demonstrated that this reduction was not due to a decreased level of protein (Fig. 4Go, upper and middle panels). In contrast, short-term treatment (1–30 min) did not have any effect on ERK1/2 phosphorylation (Fig. 4Go, lower panel). Also, in this case Western blot analysis showed the presence of equal amounts of ERK1/2 in all lanes (Fig. 4Go, lower panel). In addition, during this time (1–30 min) the phosphorylated form of MEK1 was barely detectable and remained unchanged upon IFN-{alpha} treatment (data not shown). The effect of IFN-{alpha} on MEK phosphorylation and activity, in addition with reduced phosphorylation and activity of its substrate, ERK, has been reproduced in lymphocytoid and monocytoid cell lines (data not shown).



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  		FIGURE 4. Phosphorylation of MEK and ERK upon IFN-{alpha}2b treatment of purified CD4+ T cells. CD4+ T cells were purified by negative selection from total PBMC of healthy seronegative donors (see Materials and Methods) and stimulated for the indicated time (long term, upper and middle panels; short term, lower panel) with anti-CD3 in the presence (+) or absence (-) of IFN-{alpha} (50–100 ng/ml). Subsequently, cellular extracts were prepared and equal amounts of total proteins were analyzed by Western blot for the phosphorylation of ERK1/2 and MEK1. The same blot was then stripped and reprobed to assay the protein levels of MEK1 and ERK1/2. Results are representative of experiments conducted with three different donors. P-ERK1/2 and P-MEK1, phosphorylated ERK1/2 and phosphorylated MEK1, respectively.

 
IFN-{alpha} 2b reduces surface density and mRNA expression of IL-2R {alpha}-chain on primary CD4+ T cells

Although the above described results could explain the lack of proliferative response of CD4+ T cells to CD3/CD28 cross-stimulation, they did not explain the lack of cell proliferation when IL-2 also was added to the culture. In fact, even with the inactive MEK/ERK pathway, proliferation of CD4+ T cells has been achieved if cells were provided with signals generated by stimulation of the IL-2R complex (29).

With this in mind, we assessed whether IFN-{alpha} reduces the surface density of molecules constituting the IL-2R complex in CD4+ T cells cross-stimulated with anti-CD3 and anti-CD28. Flow cytometry analysis revealed a marked delay in surface expression of the {alpha}-chain in the IFN-{alpha}-treated aliquots, as opposed to the untreated control (Fig. 5Go). This delay was observed as early as 10 h after cross-stimulation and persisted during the 24 h of observation (Fig. 5Go). This reduced surface density was paralleled by a 5-fold decrease in the levels of mRNA expression, as determined by end-point dilution RT-PCR (Fig. 6Go). Analysis of the ß-chain revealed no differences in surface expression (Fig. 5Go). Surface expression analysis of the {gamma}-chain showed a slight reduction after 24 h of cross-stimulation in IFN-{alpha}-treated aliquots (Fig. 5Go), whereas no difference in mRNA expression was observed using RT-PCR analysis (data not shown). This could indicate either that 1) the difference in mRNA expression was too little (<3-fold) to be detected by our RT-PCR assay or 2) IFN-{alpha} acts at a posttranscriptional level to reduce surface expression of the {gamma}-chain.



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  		FIGURE 5. Surface analysis of IL-2R {alpha}-, ß-, and {gamma}-chains in CD4+ T cells treated with IFN-{alpha}2b and subsequently cross-stimulated with anti-CD3 plus anti-CD28. Purified CD4+ T cells (106/ml) were treated for 18–20 h with IFN-{alpha} (50–100 ng/ml). Subsequently, cells were stimulated with immobilized anti-CD3 plus anti-CD28 as described in Materials and Methods. For the flow cytometry analysis, cells were collected at the indicated time and treated as described in Materials and Methods. For the flow cytometry analysis, cells were collected at the indicated time and treated as described in Materials and Methods. Histograms are representative of experiments conducted in three different donors. Thin line, untreated cells; thick line, IFN-{alpha}-treated cells; and dotted lines, isotype controls. Anti-CD3 x anti-CD28, cross-stimulation with immobilized anti-CD3 plus anti-CD28.

 


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  		FIGURE 6. End-point dilution RT-PCR analysis of IL-2 {alpha}-chain mRNA in CD4+ T cells pretreated with IFN-{alpha}2b and subsequently cross-stimulated with anti-CD3 plus anti-CD28. For the preparation of the template for RT-PCR analysis, cells were pretreated with IFN-{alpha} (100 ng/ml), cross-stimulated, and collected as described in Materials and Methods. Amplification was performed on samples collected before IFN-{alpha} treatment and subsequently at 1, 3, and 5 h after anti-CD3 plus anti-CD28 cross-stimulation. The major difference between treated and untreated samples was observed at 5 h. Consequently, we decided to determine the difference in the relative copy number of mRNA molecules at this time point by using end-point dilution RT-PCR. Samples were amplified using serial dilution (1:5) of the cDNA template. ß-actin amplification was used to compare samples. To highlight the differences among samples in relative copy number of the template, amplification reaction was conducted for 35 cycles as described in Materials and Methods. To exclude contamination by amplified PCR products, appropriate negative controls were amplified and particular care was taken in separating the different areas of the sample treatment and handling. As the amplification products spanned between exon 3 and exon 4 of the {alpha}-chain gene, this allowed us to discriminate between the expected product and amplification of possible contaminating genomic DNA. To further confirm the lack of contaminating genomic DNA, we also amplified samples of mRNA in the absence of the RT process (lane negative control (NC)). Results are representative of experiments conducted with three different donors. Lane 1, undiluted sample; lane 2, dilution 1:5; lane 3, dilution 1:25; and lane 4, dilution 1:125.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A number of studies have been performed in an attempt to dissect the molecular mechanisms involved in the pleiotropic activity of IFN-{alpha}, and the combined efforts of several laboratories have allowed a partial understanding of the mechanisms by which IFN-{alpha} exerts its functions. To this regard, several experimental evidences demonstrated that the antiviral and the antiproliferative effects of IFN-{alpha} are mediated by different, although partially overlapping, pathways (50, 51, 52). In addition, it is becoming clear that IFN-{alpha} is able to recruit additional components of the cellular machinery to exert its physiological functions. For example, it was recently demonstrated that an association existed between components of the TCR complex (Zap-70, p56-LCK, and CD45) and IFN-{alpha} receptor. Lack of these components resulted in a reduced antiproliferative effect of IFN-{alpha}, whereas its antiviral effect was not modified (53). However, another group reported that p56-LCK is not necessary for the antiproliferative activity of IFN-{alpha} (54).

Although the Jak/STAT pathway is clearly important in the establishment of the IFN-mediated effects, a number of recent studies suggest that additional signaling pathways may also be important for IFN-dependent biological response. In fact, both type I and II IFNs have been shown to stimulate Raf and ERK activation in a Jak-dependent, but Ras-independent manner (55, 56). The cross-talk between the Jak/STAT and MEK/ERK pathway is further demonstrated by the observation that IFN-ß stimulation results in ERK activation and its direct association with Stat1, as revealed by coimmunoprecipitation studies (57). In other reports, IFN-{alpha} has been shown to directly activate both MEK/ERK (52, 54) and phosphatidylinositol 3-kinase (53). In contrast, treatment of cells with the phosphatidylinositol 3 kinase inhibitor wortmannin appears to inhibit type I IFN-regulated ERK activation (58).

It is important to note that all of these experiments analyzed events occurring early after the stimulation of cells with IFN-{alpha}. Certainly, this is a notable contribution to understand all of the molecular players involved in the establishment of the IFN-mediated effects. Nonetheless, biochemical and functional characterizations of the late events occurring upon long-term treatment with IFN-{alpha} are also needed to better comprehend the mechanisms of action of this multifunctional cytokine. To this regard, a number of experiments investigated the changes occurring in a cell following long-term exposure to IFN-{alpha}. Examples of such changes include modulation of protein kinase C isotypes, down-regulation of c-myc and cyclin A, and hypophosphorylation of the retinoblastoma gene product (59). In addition, it was demonstrated that transcriptionally active Stat1 is required for the antiproliferative effect of IFN-{alpha}, and this antiproliferative activity was established after long-term treatment of the cells with IFN-{alpha} (60). Consequently, proliferation of Stat1-deficient cells was not inhibited by IFN-{alpha} (60, 61). Finally, failure of IFN-{alpha} to reduce proliferative ability of a cutaneous T cell lymphoma cell line was correlated to lack of Stat1 expression (62). These observations strongly suggest that 1) activation of the Jak/STAT pathway is crucial to establish the antiproliferative effect of IFN-{alpha}, and 2) it is likely that events occurring downstream of this pathway, and triggered by long-term treatment with IFN-{alpha}, are relevant for the establishment of its antiproliferative effect.

Our results indicate that long-term exposure of CD4+ T lymphocytes to IFN-{alpha} affects two of the main functions of these cells, namely, production of endogenous IL-2 and cellular proliferative response to exogenous IL-2. Further investigations into the molecular mechanisms responsible for these effects revealed 1) a slight reduction of surface expression of CD3 and CD28, important for the production of exogenous IL-2; 2) a change in the status of phosphorylation of MEK1 and ERK1/2, molecules fundamental to signal transduction upon TCR triggering; and 3) a reduced expression of the {alpha}-chain of the IL-2R complex, possibly altering the IL-2R function. These latter data could help to explain the recent observation that IFN-{alpha} treatment caused a strong inhibition of IL-2-induced changes in G1 regulatory proteins, thus resulting in a block of the S-phase entry (7).

A direct effect of IFN-{alpha} at the transcriptional level seemed to account for the reduction of the IL-2R {alpha}-chain. However, we cannot exclude additional possibilities such as an effect of IFN-{alpha} on the stability of the protein. Since the surface level of the CD3 molecule was slightly decreased, this could partially explain the decreased MEK1 and ERK1/2 phopshorylation following TCR engagement. The effect of IFN-{alpha} on the MEK/ERK-signaling cascade could also be a consequence of an alteration in the level and/or the function of kinases upstream of MEK (such as Raf-1) (63, 64). Alternatively, a specific phosphatase, such as CD45 or PP2A (47), may be induced by IFN-{alpha}, thus resulting in the inactivation of MEK (or kinases upstream of MEK). Another possibility is that levels of docking proteins (65, 66, 67) could be regulated by IFN-{alpha}, eventually affecting the formation of the complex between MEK and ERK or between MEK and upstream kinases.

Activation of the MEK/ERK pathway and assembly of IL-2Rs are functionally linked, as they both are required for T cell activation and subsequent proliferation. It thus is possible that IFN-{alpha} transient stimulation of CD4+ T cells confers a status of resistance to viral infection, and the temporary arrest of cellular proliferation and immediate response against foreigner pathogens would likely result in a favorable situation for the host. On the contrary, repeated exposure of CD4+ T cells to IFN-{alpha} may seriously hamper cellular proliferation, eventually resulting in a detrimental effect to the immune system. Overproduction of IFN-{alpha} correlates with disease progression in HIV-seropositive subjects (68) and could be detrimental for the immune system by contributing to the two major defects, reduced IL-2 production and regulatory dysfunction of the IL-2R complex, described in this report on in vitro studies and already known in HIV-infected subjects (69, 70).

Further studies into the cellular mechanisms responsible for the antiproliferative activity of IFN-{alpha} may be relevant for its antitumor effects as well as its putative role in inhibition of growth of uninfected CD4+ T cells in HIV-infected persons.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Davide Zella, Institute of Human Virology, 725 West Lombard Street, Baltimore, MD 21201. E-mail address: Back

2 Abbreviations used in this paper: Jak, Janus tyrosine kinase; ERK, extracellular-regulated kinase; MEK, mitogen-activated ERK-activating kinase; MESF, molecules of equivalent soluble fluorochrome. Back

Received for publication August 18, 1999. Accepted for publication December 13, 1999.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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