Vimentin expression is differentially regulated by IL-2 and IL-4 in murine T cells

0022-1 767/93/1518-4013$02.00/0

Copyright 0 1993 by The American Association of Immunologists The Journal of Immunology Vol 151, 4013-4021, No. 8, October 15, 1993

Printed in U.S.A.

Vimentin Expression I s Differentially Regulated by IL-2 and IL=4 in Murine T Cells'

Peter V. Hornbeck,** James 1. Carrels,+ Yassemi Capetanaki,* and Susan Heimer*

*Division of Rheumatology and Clinical Immunology, Department of Medicine, University of Maryland School of Medicine, Baltimore, M D 21 201 ; T o l d Spring Harbor Laboratory, Cold Spring Harbor, NY 11 724; and *Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030

ABSTRACT. IL-2 and IL-4 are T cell growth factors that are produced by different T cell subsets and have distinct roles in lymphocyte biology. Despite their importance in the immune system, little is known about the genes that these lymphokines may specifically control and the interaction of these lymphokines in regulating the expression of their target genes. In this paper, we use the factor-dependent murine T cell line (CT.4R) to investigate the interaction of IL-2 and IL-4 in regulatinggeneexpression. We report that the intermediate filament protein vimentin is differentially regulated by these lymphokines. Cells grown in IL-2 typically express 10- to 20-fold more vimentin and vimentin RNA than those grown in IL-4, but express similar levels of other cytoskeletal proteins including actin and tubulin. Vimentin was specifically induced by IL-2 and apparently suppressed by IL-4 in normal lymph node T cells, suggesting that its differential regulation by these lymphokines is physiologically relevant. We investigated the synergy between IL-2 and IL-4 in regulating the expression of vimentin RNA and compared it to that of two other lymphokine-responsive genes, pancreatic lipase and the IL-2Ra subunit. Complex regulatory interactions were revealed: IL-4 suppressed the ability of IL-2 to induce vimentin RNA but not IL-2Ra RNA, whereas IL-2 inhibited the ability of IL-4 to induce lipase RNA. These results indicate that IL-2 and IL-4 can cross-regulate lymphokine- responsive genes and can simultaneously exert both positive and negative regulation of different genes within the same cell. lournal of Immunology, 1993, 151 : 401 3.

T he lymphokines IL-2 and IL-4 are T cell growth factors that induce distinct patterns of T cell growth and development. IL-2 is a potent growth

factor for all activated T cells (l), whereas IL-4 appears to function as a growth factor for only a subset of normal T cells (2, 3). IL-2 and IL-4 are secreted by distinct sub- populations of T cells (4) and differentially regulate the growth and differentiation of thymocytes (5 ,6) , Th cells (7, 8), and CTL (9, 10). IL-2 and IL-4 play crucial roles in shaping the nature of immune responses (1, ll), yet little is known about the specific influences that these lymphokines may have upon gene expression.

Received for publication April 7, 1993. Accepted for publication July 17, 1993.

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.

' This work was supported in part by ACS JFRA-353, NIH R29A130822-01 A l , and the Maryland Chapter of the Arthritis Foundation.

Address correspondence and reprint requests to Dr. Peter V. Hornbeck, Uni- versity of Maryland School of Medicine, Department of Medicine, 8-34 MSTF, 10 South Pine Street, Baltimore, M D 21201.

The physiologic milieu in which lymphocytes mature undoubtedly includes a complex mixture of lymphokines that varies depending upon the location of the cell, the types of immune responses being mounted by the organism, other types of cells that are locally present, and genetic factors. Understanding how lymphokines interact in regulating cellular processes is important in understanding the outcomes of immune responses and other lymphokine-dependent processes. IL-4 has been previously shown to have either in- hibitory or stimulatory effects upon IL-2-induced signaling in a number of cell types. IL-4 inhibits the ability of IL-2 to induce IL-2Ra in thymocytes, B cells, T cells, and macrophages (12-15), to induce J-chain transcription and proliferation in a B cell line (16), and to induce proliferation of Tcells (14). On the other hand, IL-4 augments the ability of IL-2 to induce IL-2RP in B and T cells (17) and to stimulate pro-T cell proliferation (12).

The CT.4R cell line offers an excellent model system for identifying and characterizing genes that are specifically regulated by IL-2 or IL-4. CT.4R cells, a T cell line apparently derived from the IL-2-dependent mouse line

401 3

401 4 CROSS-TALK BETWEEN IL-2 AND IL-4

CTLL, grow at similar rates in either IL-2 or IL-4 (18). Thus, genes that are differentially regulated by these lymphokines are likely to reflect lymphokine-specific regulation rather than simply reflecting growth-responsive regulation. Previously, differential hybridization screening of a CT.4R cDNA library has been used to identify T lymphocyte genes that are specifically induced by IL-4 (19).

The cytoskeleton of lymphocytes, like that of all higher eukaryotic cells, is composed of three different filamentous networks: microtubules, actin microfilaments, and IE3 IF are composed of distinct IFP that are encoded by a mul- tigene family, the members of which are regulated developmentally in a tissue-specific fashion (20, 21). The IFP vimentin is predominantly expressed in cells of mesenchy- mal origin, in undifferentiated cells, and in most cancer cells. It is growth-regulated in many cell types (22) and belongs to the early response competence gene family (23). Vimentin, thought to be the sole IFP of lymphocytes and other hematopoietic cells, is retained throughout all stages of T cell development, yet is lost during terminal differ-

sion of its RNA by IL-2 and IL-4 with that of IL-4- responsive lipase and IL-2Ra, genes previously shown to be induced by IL-4 or IL-2, respectively (18, 19, 38, 39). We show that each of these genes exhibits a distinct pattern of synergistic regulation by IL-2 and IL-4. The first pattern, in which IL-4 suppresses the inductive ability of IL-2, is represented by vimentin. The second pattern, in which IL-2 suppresses the inductive ability of IL-4, is represented by lipase. The third pattern, in which IL-4 transiently augments the inductive ability of IL-2, is represented by 1L- 2Ra. Time course studies indicate that each of these genes is regulated with different kinetics, implying that the molecular mechanisms that regulate the expression of each gene are distinct. Taken together, these results demonstrate complex patterns of cross-talk between IL-2 and IL-4 in regulating the expression of lymphokine-responsive genes.

Materials and Methods Reagents and materials

entiation of B cells into plasma cells (24). Vimentin is induced by IL-6 during terminal differentiation of M1 myeloid leukemia cells (25), is induced during EBV infection (26), and is transactivated by the T cell leukemia virus type I Tax protein (27). Previous studies, showing that vimentin is induced in normal resting murine T lymphocytes during the Go to S phase transition by mitogens and growth factors including Con A (28), polyamines (28), and IL-2 (29), demonstrated that vimentin is a growth-responsive gene in T cells.

The function of vimentin during growth and differen-

PMA (P-8139), PMSF (P-7626), leupeptin (L-2884), and aprotinin (A-6012) were from Sigma (St. Louis, MO). Human rIL-2 was provided by Dr. Craig Reynolds (Bio- logical Resources Branch, National Cancer Institute- Frederick Cancer Research and Development Center, Frederick, MD). Mouse rIL-4 was a gift from Ms. Cynthia Watson and Dr. William Paul (National Institute of Allergy and Infectious Diseases, Bethesda, MD). Methionine-free RPMI 1640 was prepared from the Select-Amine Kit (GIBCO 300-7402AV).

tiation is presently not understood (30). Vimentin appears to link the nucleus to the plasma membrane (31) and closely associates with microtubules (32), apparently through interactions with microtubule-associated protein 2 (33) and kinesin (34). Vimentin is phosphorylated by ~ 3 4 " ~ " ' during mitotic reorganization (35), transiently co-localizes with and is phosphorylated by cGMP-dependent protein kinase during neutrophil activation (36), and shares significant ho- mology with DNA binding domains of several transcriptional regulators includingfos, jun, and CREB (37). Based upon the above properties, it has been postulated that vimentin plays a role in cell cycle progression, in signal transduction and transport processes between the cell surface and the nucleus, and in gene expression.

In this report, we describe our investigations into the abilities of IL-2 and IL-4 to differentially regulate gene expression in CT.4R cells. We used high resolution 2-D analysis to identify vimentin as a protein that is specifically suppressed by IL-4 and compare the regulation of expres-

Abbreviations used in this paper: IF, intermediate filament; 2-D, two-dimensional; IFP, intermediate filament protein; 3'S-met, [35S]methionine; CRPMI, complete RPMI.

Cells and cell culture

Cell lines include: CT.4R, a factor-dependent murine T cell line that requires either IL-2 or IL-4 for growth (18); M5/ 114.15.2 (40) (AmericanType Culture CollectionTIB 120), a rat hybridoma that secretes anti-Ia antibody; and MAR 18.5 (41) (American Type Culture Collection TIB 216), a mouse hybridoma that secretes anti-rat K-light chain antibody.

Normal T cells were prepared as described previously (42). Briefly, mesenteric lymph node cells from female BALB/c mice, 8 to 12 wk old, were passed through nylon wool columns followed by two cycles of cytolysis using M5/114 and MAR 18.5 supernatants followed by rabbit C (Cedarlane Low-Tox-M CL3051). Resting cells were isolated on Percoll gradients by pooling cells with densities greater than 60% Percoll.

Cells were cultured in CRPMI (RPMI 1640 supplemented with 6% FCS, 2 nM L-glutamine, penicillin (100 U/ml), streptomycin (100 pg/ml), and 2-ME (5 X M). CT.4R cells were grown in CRPMI supplemented with either IL-2 (50 U/ml) or IL-4 (1000 U/ml). These concen- trations of lymphokines, determined in preliminary dose-

Journal of Immunology 401 5

response analyses, produce similar levels of near-maximal proliferation of CT.4R cells.

Biosynthetic labeling and whole cell lysis

Cells were biosynthetically labeled as described previously (43). Briefly, cells were incubated at 37°C in 12-well tissue culture plates for 4 h in methionine-deficient CRPMI containing 0.5 mCi 35S-met/ml (Amersham SJ.1015) and 5% FCS dialyzed against normal saline. Cells were washed twice in PBS and lysed in ice-cold lysis buffer (0.4% SDS, 50 mM Tris (pH €LO), 2% 2-ME, 1 mM PMSF) at 10’ cells/0.25 ml, heated to 95°C for 10 min, cooled on ice, and treated with DNase and RNase as described previously (44).

IF preparation

tween treatment groups. Where indicated, ”S-met-labeled samples were run and analyzed using the Quest system for quantitative analysis of 2-D gels as described previously (44). Spot intensities are expressed as parts per million, which are determined by dividing the dpm in each spot by the total TCA-precipitable dpm applied to the gel and multiplying by lo6.

RNA preparation and analysis

Cellular RNA was extracted using RNAzol B (CS-105; Biotecx Laboratories, Houston, TX) following the manufacturer’s instructions. Northern analysis was performed as described (47). Briefly, 10 p g of total RNA were electro- phoresed through 1.5% agarose/formaldehyde gels, trans- ferred to MAGNAnylon membranes (N04HY00010; MSI, Westboro, MA) UV-cross-linked at 0.3 joules/cm2 using a

Cellular fractions enriched in IF were prepared by lysis in high salt buffer using a protocol similar to one described previously (45). Cells were biosynthetically labeled by culturing for 16 h in methionine-deficient CRPMI containing 0.2 mCi 35S-met/ml and supplemented with 15 &ml cold methionine. Cells were pelleted by centrifugation in 15-ml tubes and lysed for 10 min at room temperature at 106/ml in 0.6 M KCl, 1% Triton-X 100, 10 mM MgC12, 1 mM PMSF, 10 mdml leupeptin, and 100 U/ml aprotinin. DNase was added to a final concentration of 0.25 mdml and incubated 5 min at 21°C with frequent pipetting during the incubation. Lysates were centrifuged at top speed for 5 min in a tabletop microcentrifuge (Eppendorf 5415C). The pellets, enriched in IFP, were washed twice by resuspending

Bioslinker (Bios Corp., New Haven, CT), and probed with 32P-labeled murine vimentin (37), IL-4-responsive lipase (19), actin (48), and IL-2Ra (49) probes. Probes were prepared by electrophoretic purification of specific inserts from restriction digests and labeling with [a3”P]dCTP by random priming using an Oligolabelling Kit (27-9250-01; Pharmacia LKB, Piscataway, NJ) following the manufacturer’s instructions. After hybridization and washing, blots were analyzed directly on a Molecular Dynamics PhosphorImager or autoradiograms were analyzed on a Molecular Dynamics Scanning Densitometer. For densitometric analysis, only autoradiographic exposures lying within the linear dynamic range of the film were used in quantitative studies.

in wash buffer (PBS, 5 mM EDTA, 0.1 mM PMSF) and centrifuged. If the pellets were viscous after centrifugation, 50 p g of DNase were added directly to the pellet, which was mixed by repeated pipetting and incubated for an additional 5 min at 21°C before washing.

2-D analysis

2-D analysis of biosynthetically labeled cellular proteins was routinely performed using the Investigator 2-D Elec- trophoresis System (Millipore, Bedford, MA) according to protocols provided by the manufacturer. This system was used because its geometry is equivalent to that used in the Quest Facility at Cold Spring Harbor Laboratory (44), ren- dering the observed electrophoretic mobilities of cellular proteins nearly identical to those in a previously published protein database (46). Dried gels were analyzed directly on a Molecular Dynamics PhosphorImager. Autoradiograms were analyzed on a Molecular Dynamics Scanning Den- sitometer. For densitometric analysis, only exposures lying within the linear dynamic range of the film were analyzed. To quantitatively compare spots between gels, the gels were first normalized to 10 reference proteins that are known from previous experiments to be relatively invariant be-

Results Vimentin, but not other major cytoskeletal proteins, is differentially regulated by IL-2 and IL-4 in CT.4R cells

2-D analysis was used to identify proteins that are differentially regulated by IL-2 and IL-4 in CT.4R cells. Analyses of whole cell lysates of cells grown in either IL-2 or IL-4 and labeled with ”S-met indicated that a protein with an electrophoretic mobility slightly slower than that of a-tubulin was consistently expressed at higher levels in cells grown in IL-2 than those grown in IL-4 (Fig. 1). Com- parison with the protein database previously published (46) suggested that this protein was the IFPvimentin. To directly test this possibility, IFP were extracted from CT.4R cells grown in IL-2 or IL-4 for 72 h and analyzed by 2-D analysis. As shown in Figure 2, the protein tentatively identified as vimentin was greatly enriched in the IF fraction of cells grown in IL-2, providing strong evidence that this protein is indeed vimentin.

To determine whether other major cytoskeletal proteins are differentially regulated by IL-2 and IL-4, the gels shown in Figure 1 were analyzed by PhosphorImager (Figure 3).

401 6 CROSS-TALK BETWEEN IL-2 A N D IL-4

FIGURE 1. 2-D analysis of whole cell lysates of cells grown in IL-2 or IL-4. CT.4R cells were cultured for 3 days in 50 U/ml IL-2 ( A ) or 1000 U/ml IL-4 (B ) , biosynthetically labeled with j5S-rnet (0.5 mCi/ml) for 4 h, lysed in hot SDS buffer, and analyzed by 2-D electrophoresis as described in Materials and Methods. Similar results were obtained in four separate experiments.

vimenlin A /

B 4 - p n

tubulin

\

-c, I' Y actin

FIGURE 2. 2-D analysis of IFP from CT.4R cells grown in IL-2 or IL-4. CT.4R cells were cultured for 3 days in 50 U/ml IL-2 ( A ) or 1000 U/ml IL-4 (6 ) and biosynthetically labeled with -?5-met (0.2 mCi/ml) for 1 2 h. Fractions enriched in IFP were isolated and analyzed by 2-D electrophoresis as described in Materials and Methods.

P- and y-actin and a- and P-tubulin were synthesized at similar levels in cells grown in IL-2 and IL-4, whereas the synthesis of vimentin was 38-fold higher in cells grown in IL-2 than those grown in IL-4. On average, however, vimentin is expressed 10- to 20-fold higher in cells grown in IL-2. Thus, vimentin appears to be the only major cytoskeletal protein that is differentially regulated by IL-2 and IL-4.

Vimentin is differentially regulated by IL-2 and IL-4 during activation of resting T cells

To determine whether vimentin is differentially regulated in normal T cells, resting lymph node T cells were treated with IL-2 or IL-4 alone or in combination with PMA. Be- cause treatment with IL-2 or IL-4 activates the cells but does not effectively induce proliferation, PMA was added to provide a second signal that drives the cells through proliferation (50). After culturing for 24 h, cells were biosynthetically labeled with "S-met, lysed, and analyzed by computer-assisted high resolution 2-D analysis as described previously (44). Autoradiography of the gels revealed that the synthesis of vimentin is induced by IL-2 and

Cytoskeletal Proteins in CT.4R Cells

vlmentin EiLA Ea IL-4 IL-2

p-tubulin

13 1 0 6 10'

Integrated Intensity

FIGURE 3. Analysis of cytoskeletal proteins in whole cell lysates of CT.4R cells grown in IL-2 or IL-4. CT.4R cells were cultured for 3 days in IL-2 (solid bars) or IL-4 (hatched bars) and biosynthetically labeled with j5S-met (0.5 mCi/ml) for 4 h. Whole cell lysates were prepared and analyzed for expression of vimentin, p-actin, y-actin, a-tubulin, and p-tubulin by 2-D electrophoresis. Level of expression was determined by Phosphorlmager analysis and is expressed as integrated intensity in arbitrary units. Similar results were obtained in a separate experiment in which autoradiograms were analyzed using a scanning densitometer.

suppressed by IL-4 (Fig. 4). Cells cultured with IL-2 alone or IL-2 plus PMAexpressed 3.7- and 10-fold more vimentin than their counterparts cultured with IL-4 alone (Fig. 4B) or IL-4 plus PMA (Fig. 3C). Control proteins a- and P-tubulin are expressed at similar levels in these groups. Cells cultured with IL-4 alone or IL-4 plus PMAexpressed half as much vimentin as control groups that received no lymphokine (Fig. 4, B and C ) . Collectively, these data suggest that vimentin is specifically induced by IL-2 and suppressed by IL-4 during lymphokine-induced activation of normal resting T cells.

The pattern of regulation of vimentin RNA by IL-2 and IL-4 is distinct from that of IL-4-responsive lipase and IL-2Ra

The identification of vimentin as a lymphokine-responsive protein (Figs. 1 to 4) suggested that IL-2 and IL-4 might be regulating vimentin expression at the RNA level. Further- more, the low level of vimentin in cells grown in IL-4 alone could indicate either that IL-4 fails to induce or that it actively suppresses vimentin RNA expression. To discrimi- nate between these possibilities, RNA was extracted from CT.4R cells grown in IL-2, IL-4, or a combination of both for 72 h and analyzed for vimentin RNA content (Fig. 5, top). Vimentin RNA expression was highest in cells grown in IL-2,1&fold less in cells grown in IL-4 alone, and three- fold less in cells grown in the combination of IL-2 plus IL-4. These results indicate that vimentin expression is regulated by IL-2 and IL-4 at the RNA level and that IL-4

journal of Immunology

A IL-2 11-4

401 7

I vimentin

1 I

i I 0 100 200 300 400

PPM

C. (+) PMA

P-tubulin

0 200 4 0 0 600 800

PPM

FIGURE 4. Vimentin is differentially regulated by IL-2 and IL-4 in normal T cells. Resting mesenteric lymph node T cells (1 X 1 06/ml) were cultured with IL-4 or IL-2 ? PMA. After 20 h, cells were biosynthetically labeled with 35S-met for 4 h. Whole cell lysates were prepared and analyzed by computer- assisted high resolution 2-D analysis using the Quest system as described (45). A, Regions of autoradiograms of 2-D gels containing vimentin (arrows). T cells were cultured with no added lymphokine (left), 30 U/ml IL-2 (center), or 300 U/ml IL-4 (right). Cells on the bottom row were treated with PMA (20 ng/ml) in addition to IL-2 or IL-4. B, Analysis of vimentin, a-tubulin, and P-tubulin expression in T cells shown in the top row o f A above. Cells were cultured with no added

IL-2 IL-4 + IL-2 IL-4

LIPASE I

I

0 20 40 60 80 100 120

RNA (% maximal expression)

FIGURE 5. Synergy between IL-2 and IL-4 in regulating vimentin, IL-Cresponsive lipase, IL-2Ra, and actin RNA expression. CT.4R cells were cultured with 50 U/ml IL-2 (filled bars), 100 U/ml IL-4 (light hatched bars), or a combination of IL-2 and IL-4 (dark hatched bars). After 72 h of culture, RNA was isolated and analyzed by Northern analysis. Relative RNA expression was determined directly by Phosphorlmager analysis. Autoradiographic images of Northern blots for each group are inset on the right side. Similar results were obtained in two separate experiments.

actively suppresses the ability of IL-2 to induce vimentin RNA.

The regulation of vimentin RNA by IL-2 and IL-4 was compared to that of two other known lymphokine- responsive genes, IL-2Ra (38) and IL-4 responsive lipase (19). It has been previously reported that IL-2Ra RNA, like vimentin RNA, is low in CT.4R cells grown in IL-4 and high in those grown in IL-2 (18), suggesting that IL-2Ra RNA might be regulated in a manner similar to that of vimentin RNA. To investigate this possibility, RNA from CT.4R cells grown in IL-2, IL-4 or both was probed for IL-2Ra RNA expression. As expected based upon previous observations (18), IL-2Ra RNA was low in cells grown in IL-4 and high in cells grown in IL-2 (Fig. 5). However, unlike the dominant suppression of vimentin RNA by IL-4, the addition of IL-4 to cells growing in IL-2 did not suppress the expression of IL-2Ra RNA.

While previous reports have indicated that IL-4- responsive lipase RNA is expressed at high levels in CT.4R cells grown in IL-4 but not in IL-2 (19), the interaction between IL-2 and IL-4 in regulating this lipase gene has not been reported previously. Northern analysis of RNA from

lymphokine (open bars), with IL-2 (solid bars), or with IL-4 (hatched bars). Protein expression is expressed as parts per million (PPM) based upon normalization algorithms as described (45). C, Analysis of vimentin, a-tubulin, and P-tubulin in T cells shown in the bottom row o f A above. Cells were cultured as in B but with the addition of PMA.

401 8

CT.4R cells grown in IL-2, IL-4, or both indicated that the regulation of lipase RNA is nearly a mirror-image of the regulation of vimentin RNA: lipase RNA was highest in cells grown in IL-4, 40-fold less in cells grown in IL-2 alone, and 3.4-fold less in cells grown in the combination of IL-4 and IL-2 (Fig. 5). Thus, IL-2 partially suppresses the ability of IL-4 to induce lipase RNA. As a control, actin RNA expression was independent of whether cells were grown in IL-2 or IL-4.

The kinetics of synergy between IL-2 and IL-4 in regulating lymphokine-responsive genes

To investigate the kinetics of synergy between IL-2 and IL-4 in regulating lymphokine-responsive gene expression, IL-4 was added at various time points to CT.4R cells growing in IL-2. After 72 h, cells in all groups were harvested and analyzed for vimentin, IL-2Ra, and IL-4-responsive lipase RNA content. Each of the three RNA species examined displayed a unique kinetic response to co- stimulation with IL-2 and IL-4 (Fig. 6). Vimentin RNA expression is rapidly suppressed after the addition of IL4, achieving half-maximal inhibition by 7 h and maximal inhibition by 48 h. In contrast to vimentin, both IL-2Ra and lipase RNA are induced after the addition of IL-4, but the kinetics of induction vary markedly between these the two RNA species. The increase in IL-2Ra-RNA proceeded in two stages: a rapid and transient 2.5-fold increase that peaked at or before 7 h, and a latter phase that stabilized at a 1.5-fold increase by 48 to 72 h. It should be noted that, in other experiments, no long term increase in IL-2Ra- RNA was observed during co-culture with IL-2 and IL-4 (Fig. 5). The induction of lipase RNA by IL-4 was much slower than either the suppression of vimentin RNA (Fig. 6) or the transient elevation of IL-2Ra-RNA and had not achieved maximal induction even after 72 h. The distinct kinetics associated with the suppression of vimentin RNA and the induction of IL-2Ra and lipase RNA implies that each of these processes proceeds by separate molecular mechanisms.

Vimentin expression directly correlates with vimentin RNA content

To determine whether vimentin expression reflects the amount of vimentin RNA, CT.4R cells were cultured with IL-2, IL-4, or for various periods of time with both IL-2 and IL-4. As seen in Figure 6, this protocol produced a series of cells that expresses widely varying amounts of vimentin RNA. Aliquots of cells were removed at various times, biosynthetically labeled with [35S]met, lysed, and analyzed for vimentin synthesis using 2-D electrophoresis. Comparison of protein synthesis with RNA content indicated that the amount of vimentin synthesized directly correlated with the amount of vimentin RNA (Fig. 7). For example, 50% in-

CROSS-TALK BETWEEN IL-2 AND IL-4

A.

11-2: + + + + + + - 11-4: - + + + + + +

VlMENTlN

LIPASE

lL-2R

ACTIN

0 7 13 24 48 72 72 TIME IN 11-4 (HRS)

B. KINETICS OF RNA EXPRESSION

120 IL-2R

100 ' * LIPASE "0- VIMENTIN

- u

80 - 4 1

60 - L 1

20 I L 6

" 8

0 24 48 72

TIME IN IL-4 (H) FIGURE 6. Kinetics of expression of vimentin, IL-4-responsive lipase, IL-2Ra, and actin RNA in CT.4R cells cultured in IL-2, IL-4, or a combination of IL-2 and IL-4 for various periods of time. A, Northern analysis of CT.4R cells grown in IL-2 (50 U/ml) for 72 h (lane I), IL-4 (1000 U/ml) for 72 h (lane 7), or both IL-2 and IL-4 for various periods of time as indicated (lanes 2 to 6) . Vimentin, lipase, IL-2Ra, and actin RNA were visualized by autoradiography. Two additional experiments at selected time points confirmed these results (data not shown). B, Comparison of the relative expression of vimentin, lipase, and IL-2Ra RNA determined by scanning densitometry from the autoradiograms shown in A above. Points at time 0 represent cells grown only in IL-2 for 72 h, and points at the extreme right represent cells grown only in IL-4 for 72 h. The integrated intensities for each time point were normalized to the percentage of the maximal signal. Assays for IL-2Ra and vimentin RNA were performed in duplicate. Symbols, mean; error bars, range.


z v) v) W U

X w

0

n

VlMENTlN EXPRESSION CORRELATES WITH VlMENTlN RNA CONTENT

4 I

looL -0- PROTEIN -t- RNA -0- PROTEIN -t- RNA

20 - 40/ 20 b i 0 0

0 12 24

TIME IN IL-2 + IL-4 (H) FIGURE 7. Amount of vimentin synthesized directly correlates with vimentin RNA content. CT.4R cells were cultured in IL-2 (50 U/ml), IL-4 (1000 U/ml), or a combination of IL-2 and IL-4. At various times, aliquots of cells were harvested and analyzed for vimentin RNA content as in Figure 6 or labeled with 35S-met for 4 h and analyzed for vimentin content as described in Figures 1 and 3 above. Points at time 0 represent cells grown only in IL-2 for 72 h and points on the extreme right represent cells grown only in IL-4 for 72 h. Vimentin expression was measured using the Phosphorlm- ager and RNA expression was measured using the scanning densitometer. The integrated intensities of each signal was normalized to the percentage of the maximal signal. North- ern assays were performed in duplicate, and the mean and range are shown.

hibition for both vimentin RNA content and synthesis oc- curred after 8 h of culture with IL-4. These results, coupled with those from pulse-chase studies (data not shown), indicate that the regulation of vimentin expression by IL-2 and IL-4 is determined at the level of RNA expression and is not due to translational or post-translational regulation.

Discussion

We have demonstrated that IL-4 dominantly suppresses the ability of IL-2 to induce vimentin expression in CT.4R cells. This suppression could reflect either that IL-4 is suppressive of all signaling pathways through the IL-2R or that IL-4 specifically suppresses vimentin while leaving intact other pathways that are coupled to the IL-2R. To distinguish between these alternatives, we examined the influence of IL-4 upon the ability of IL-2 to induce IL-2Ra RNA. This strategy was chosen because it is known that efficient expression of IL-2Ra RNA in CT.4R cells and other T cells requires positive inductive signaling through the IL-2R (18, 38). The results presented in Figures 5 and 6 demonstrated that IL-4 does not interfere with the ability of IL-2 to induce

IL-2R RNA. Rather, IL-4 appears to transiently augment the ability of IL-2 to induce its receptor. Thus, the ability of IL-4 to dominantly suppress the expression of vimentin by IL-2 does not reflect a general suppression of positive inductive signaling through the IL-2R and indicates that IL-4 acts at a point distal to the IL-2R to suppress the induction of vimentin.

To further characterize the signals that are involved in regulating vimentin RNA expression, the interaction between IL-2 and IL-4 in regulating IL-4-responsive lipase RNA expression was compared to the regulation of vimentin RNA expression. The regulation of lipase RNA by IL-2 and IL-4 is nearly a mirror image of the regulation of vimentin RNA: IL-4 induces lipase RNAexpression, whereas IL-2 partially suppresses its induction by IL-4 (Figs. 5 and 6). Kinetic analysis indicated that IL-4 rapidly suppressed vimentin RNA expression but slowly induced lipase RNA, suggesting that distinct regulatory pathways are involved in each of these processes. Collectively, these data demonstrate that IL-2 and IL-4 exhibit complex patterns of cross- regulation and can simultaneously suppress or stimulate the expression of different genes within the same cell type. These results suggest that it is the ratio between IL-2 and IL-4 and not simply the absolute amount of lymphokine that may be critical in determining the levels of expression of certain lymphokine-responsive genes.

The differential regulation of vimentin RNA by IL-2 and IL-4 may reflect transcriptional regulation. In this case, IL-4 either could interfere with positive regulation by IL-2 or could evoke dominant negative regulation by inducing activities that directly suppress the transcription of vimentin. Since the vimentin promoter is known to contain regions that are targets of both positive (27,51,52) and negative control (27, 51-53), either of these mechanisms might be account for the suppressive effects of IL-4. The possibility that IL-4 regulates the transcription of the vimentin gene is the subject of further investigation in our laboratory.

The demonstration in this report that CT.4R cells growing in IL-4 express vanishingly small amounts of vimentin demonstrates that vimentin is not an obligate growth- responsive gene in T cells. Furthermore, these results have revealed that physiologically relevant ligands induce an un- expected plasticity of IFP expression in T cells. If these observations are relevant to the intact organism, then vimentin may underlie distinct properties of cells that are driven to mature in environments rich in IL-4 vs environments poor in IL-4.

Although the function of vimentin in lymphocytes is not understood, vimentin cocaps with 1ymphocyteAg receptors (54) and undergoes extensive cytoplasmic reorganization in B cells after cross-linking Ag receptors (55). Collectively, these data suggest that vimentin might influence some as- pect of cell-cell or cell-substrate interactions, Ag receptor signal transduction, or lymphocyte effector mechanisms.

402 0

This latter possibility is especially appealing in light of recent reports showing that T cells orient their cellular ar- chitecture and secretory apparatus in a polar fashion during Ag-specific cellular contacts (56) and that vimentin reor- ganizes into the contact region between target cells and NK cells during NK cell-mediated cytolysis (57). Thus, the differential expression of cytoskeletal elements in cells re- sponding to IL-2 or IL-4 may affect the quality of the interactions of T cells with cellular targets or with the extracellular matrix.

Acknowledgments

We thank Dr. William Paul and Ms. Cynthia Watson for kind gifts of the CT.4R cell line and rIL-4; Dr. Steve Desiderio for providing the IL-2Ra DNA probe; Dr. Michael Grusby for the IL-4-inducible lipase cDNA clone; Mr. Steve P. Donald for preparation of probes; Dr. Ziyu Zhang for technical assistance; Ms. Cecile Chang and Ms. Phyllis Myers for assistance with the QUEST analysis; Ms. Fay Chaires for excellent secretarial help; and Dr. William E. Paul and Dr. Barry S. Handwerger for reading various versions of the manuscript.

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Vimentin expression is differentially regulated by IL-2 and IL-4 in murine T cells