FcϵRII expression in human B cells: Post-transcriptional modulation by interferon-γ

MolecularImmunology, Vol. 30, No. 3, pp. 301-307, 1993

0161~5890/93 $6.00 + 0.00 Pergamon Press Ltd

Printed in Great Britain.

MECHANISM OF INTERFERON-y DOWN-REGULATION OF THE INTERLEUKIN 4-INDUCED CD23/Fc,RII EXPRESSION IN HUMAN B CELLS: POST-TRANSCRIPTIONAL MODULATION BY INTERFERON-y CHOONG-EUNLEE,* SUK-RANYOONand KWANG-HOPYUN Immunology Research Laboratory, Genetic Engineering Research Institute, KIST, P.O. Box 17, Taedok Science Town, Taejon, Korea (First received 15 May 1992; accepted in revised form 7 August 1992)

Abstract-It has been reported that the interleukin 4 (IL-4) specific induction of cell surface CD23 (Fc,RII) is down-regulated by interferon-y (IFN-7) in monocytes and B cells. However, the molecular level at which the inhibition occurs seems to vary depending on the cell types. In normal human B cells, IFN-7 inhibits the IL-4 induced de nova synthesis of CD23 at the level of gene expression. Analysis of inhibition kinetics suggested a rapid signal transmission by IFN-)I. Yet the inhibitory action of IFN-y on CD23 mRNA accumulation appeared as a secondary response requiring a new protein synthesis. Through nuclear run-on transcription and mRNA stability studies, we further demonstrate that the IL-4 induced CD23 gene expression is down-regulated by IFN-y mainly at post-transcriptional levels by decreasing mRNA stability.

INTRODUCTION One of the pleiotropic responses of B cells invoked by interleukin 4 (IL-4) is the up-regulation of CD23, also known as Fc,RII, the low affinity receptor for IgE (Defiance et al., 1987; Yukawa et al., 1987). CD23 is a B cell differentiation specific antigen with a molecular weight of 45 KD (Kikutani et al., 1986). It has been found to function in B cell activation, auto-stimulation, IgE-dependent antigen focusing, and possibly in cell to cell adhesions (Delespesse et al., 1988; Kehry and Yamashita, 1989; Bonnefoy et al., 1988a; Cairns and Gordon, 1990). Its proteolytic cleavage products, soluble CD23, have been identified (Bonnefoy et al., 19883), which also exhibit multi-functional activities, such as IL-4 induced IgE production and B cell proliferation and differentiation (Sarfati and Delespesse, 1988; Bonnefoy et al., 1990; Liu et al., 1991; Callard et al., 1988). While CD23 is expressed on a variety of hematopoietic cell, regulation mechanisms appear different depending on the biochemical stimuli and cell types (Willheim et al., 1991; Te Velde et al., 1990; Kawabe et al., 1988). Interferon-y (IFN-y) and interferon-a (IFN-a) are shown to inhibit the CD23 expression and IgE production induced by IL-4 in B cell enriched cultures (Pene et al., 1988). In B lymphoblast cell line, Jijoye, IFN-), enhances the IL-4 induced CD23 mRNA expression but down-regulates the cell surface CD23 with concomitant increase in soluble CD23 release (Kawabe et al., 1988). In normal B cells, however, IFN-7 down-regulation of the IL-4 induced CD23 occurs both at cell surface and soluble molecules (Galizzi et al., 1988; Delespesse et al., *Author to whom correspondence should be addressed: Dr Choong-Eun Lee, Genetic Engineering Research Institute, KIST, P.O. Box 17, Taedok Science Town, Taejon, Korea 305-606.

1989). A recent report by Denoroy et al. (1990) has demonstrated that IFN-1, and IFN-a inhibit the IL-4 induced CD23 expression at the level of mRNA in normal B cells. In addition, studies on the mechanism of IFN-7 regulation of the IL-4 induced CD23 have indicated that IFN-y does not act through increasing intracellular CAMP levels, nor does it alter binding characteristics of the IL-4 receptor (Galizzi et al., 1988). In the present study we have investigated the kinetics and molecular mechanism of IFN-y inhibition for the counter-regulation of the IL-4 activated CD23 expression. The results demonstrate that the signal transmission of IFN-7 is completed within minutes, which leads to the inhibition of the IL-4 induced de nova synthesis of CD23 via post-transcriptional down-regulation of CD23 mRNA by decreasing the mRNA stability, an effect likely exerted via newly synthesized protein factors. MATERIALS AND METHODS Preparation

of B cells and B cell culture

Quiescent human B cells were prepared from tonsils by Ficoll-Hypaque (Sigma, d = 1.077) density gradient centrifugation and negative selection upon rosetting twice with AET-treated sheep RBC. After removing adherent cells, 97% purity of B cells was obtained as confirmed by staining with anti-Leu16, anti-Leu4, or anti-Leu MS (Be&on Dickinson). Purified resting B cells were cultured in RPM1 media (GIBCO) containing 10% FBS (Hyclone), 1OmM Hepes, 2 mM L-glutamine, 5 x 10v5 M 2-mercaptoethanol and antibiotics. Recombinant human IL-4 (Genzyme, 100-4OOu/ml), IFN-7 (Shering Plough Research, lO-10,000 u/ml), actinomycin D (Sigma, 5 pg/ml) and cycloheximide (Sigma, 30 pg/ml), were added to cells at indicated times and the cells were cultured in humidified 5% CO, at 37°C.

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C.-E. LEE et al.

302

Membrane CD23 (Fc,RII) measurement Membrane CD23 expression in cultured B cells (1 x lo6 cells) was analyzed by staining with anti-Leu 16 (pan B marker)-FITC and anti Leu20 (CD23)-PE (Becton Dickinson) in HBSS containing 3% FBS and 0.1% NaN, for 30 min at 4°C using fluorescence activated cell scanner (FACscan, Becton Dickinson). CD23 positivity among B cells was determined as CD23 positive B cells x 1oo 9 Total B cells and values represent a mean of three independent determinations in duplicate cultures. Soluble CD 23 measurement Levels of soluble CD23 in the B cell culture supernatant were analyzed by ELISA using monoclonal 3-514 anti CD23 and MHM6 anti CD23 conjugated with alkaline phosphatase (Dr Kishimoto, Osaka, Japan). The ELISA standard for soluble CD23 was also provided by Dr Kishimoto. CD23 mRNA analysis Total cellular RNA from B cells (1 x lo8 cells) were isolated after treatment with IL-4, or IL-4 plus IFN-y, with and without actinomycin D or cycloheximide for various durations as indicated, using guanidinium isothiocyanate and cesium chloride through ultracentrifugation (Chirgwin et al., 1979). For Northern blot, 10 pg of total RNA from each preparation were separated on a 1% agarose-formaldehyde gel, transferred to nylon membranes (Genescreen Plus, NEN). A full-length CD23 cDNA probe (from Dr Kishimoto, Osaka, Japan) was labelled with [a-32P]dCTP (Amersham, 3,000 Ci/ mmol) at specific activity of 5 x 10’ cpm/pg and used for hybridization. RNA concn was determined by O.D. measurement and the amount of loaded RNA on the gel was confirmed by ethidium bromide (EtBr) staining. Densitometric analyses of autoradiograms were performed using Soft Laser scanning densitometer (Biomed Instrument). Nuclear run -on transcription Nuclei were prepared by incubating 1 x lo* B cells for 5 min on ice in lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl,, 0.5 mM DTT, 0.3M sucrose, and 0.2% NP-40. In vitro transcription reactions were then performed with 10’ nuclei in 200 ~1 reaction buffer {10 mM Tris-Hcl pH 7.5, 35% glycerol, 5 mM MgCl,, 80 mM KCl, 0.1 mM EDTA, 0.5 mM DTT, 4mM each of ATP, CTP, GTP and 200 PCi[a -32P]UTP (Amersham, 3,000 Ci/mmol)} . The nuclei were digested with RNase-free DNase I and proteinase K. Nuclear RNA was then purified exactly as described (Chomczynski and Sacchi, 1987) and used to hybridize linearized plasmids containing appropriate cDNA (2 hg), which were previously blotted on nylon membranes. Conditions for hybridization and washing were as described by Celano et al. (1989).

RESULTS

Inhibition kinetics of the IL-4 induced CD23 expression by IFN-y Studies on the cell surface CD23 expression by IL-4 in B cells and cell lines have shown that CD23 induction becomes noticeable after 8-24 h exposure of cells to IL-4 (Hudak et al., 1987; Galizzi et al., 1989). Cotreatment with IFN-y has been reported to significantly decrease the IL-4 induced expression of CD23. To elucidate the mechanism of IFN-y action in this process, we have analyzed inhibition kinetics by exposing cells to IFN-y for various durations before or after IL-4 treatment. As shown in Fig. 1, the IL-4 induced CD23 expression was effectively blocked by cotreatment of B cells with IFN-y. However, a continuous presence of IFN-y in the culture was not necessary to induce its inhibitory effect. In fact, pretreatment of cells with IFN-y as short as 10 min and subsequent removal by washing, was sufficient to lead to the maximum inhibitory effect by IFN-y on the IL-4 induced CD23 expression (Fig. 1, panel D). This suggests that unlike the case of IL-4, the signal transmitted by IFN-y for a brief period of time is capable of inducing the effect of IFN-y to the full extent. In contrast, delayed addition of IFN-y after the IL-4 treatment resulted in a differential regulation. A prolonged delay in IFN-y addition impaired its action to downregulate the IL-4 induced CD23 expression (Fig. 1, panels G and H), i.e. after a significant level of CD23 induction was allowed by IL-4 for 12 to 20 h, IFN-y exerted progressively less inhibitory effects. The results suggest that IFN-y down-regulation of cell surface CD23 is not through accelerating modulation or degradation of CD23 protein, but through interfering with the de novo synthesis process. In fact, when we analyzed the levels of soluble CD23 in the culture supernatant of our purified tonsillar B cells, it was observed that, upon IFN-y treatment, the IL-4 induced soluble CD23 production was reduced concomitantly with the decrease in membrane CD23 expression, further suggesting that IFN-y modulation of surface CD23 in human B cells occurs via inhibition of CD23 biosynthesis rather than via membrane CD23 shedding into soluble forms (Table 1). B cell response to IFN-y for the down-regulation of CD23 mRNA is delayed and requires new protein synthesis The inhibition of the IL-4 induced de novo CD23 synthesis by IFN-y was further confirmed by RNA Northern blot analysis where effects of IL-4 and varying doses of IFN-y on the steady state CD23 mRNA levels were examined. It is shown that IL-4 caused a significant up-regulation of CD23 mRNA, and pretreatment with IFN-y for 1 h substantially reduced the IL-4 induced CD23 mRNA accumulation for the following 16 h period in a dose-dependent manner (Fig. 2). The action of IL-4 on the increased mRNA induction was impaired by actinomycin D but not by cycloheximide (data not shown). On the other hand, the inhibitory effect of IFN-y on the mRNA accumulation was completely

Post-tran~~ptional

modulation

303

of CD23 by interferon-y

(a) Control (6)

(c) IFN-r

pretreatment 122)

I h

(d) IFN-r

(f)

igl IFN-r

past tmatment (38)

pretreatment 125)

IFN-r

pasttreatment (28)

4h

12 h I

d 100

IO'

IO2

IO3

10416

IO'

102

IO4

Fluorescence intensity for CD 23

Fig. 1. Ef%ct of differential time treatment of IFN-y on the IL-4 induced CD23 expression in tonsillar B cells. Purified B cells were treated with media alone (a), IL-4 100 u/ml (B), pretreated with IFN-y 1,000 u/ml and washed prior to IL-4 addition (c, d), cotreated with IL-4 and IFN-y (e), or treated with IFN-y after IL-4 addition (f, 8, h) for indicated durations, and cultured for a total of 48 h. Cells were collected and stained with anti-Leu 20 (CD23) for analysis by FACScan. The limit of background fluorescence intensity is indicated with a solid perpendicular line on the histogram. The numbers in ~renthe~s represent percent CD23 positive B cells among total B cells.

abolished by cycloheximide (Fig. 2, lane 6). The results imply that while CD23 induction by IL-4 is a primary response, the inhibitory action of IFN-y is a secondary response requiring synthesis of new proteins, i.e. IFN-y induced factors which in turn, regulate transcription or stability of CD23 mRNA. We then examined the kinetics of B cell response to IFN-y for the down-regulation of CD23 mRNA. B cells were initially treated with IL-4 for 4 h, which resulted in a significant level of CD23 mRNA induction. IFN-y or media alone was then added to the cells and harvested after various time periods to assess the kinetics of IFN-y effect on the IL-4 induced CD23 mRNA levels. Don-relation of CD23 mRNA by

IFN-y was not observed by 4 h and became evident by 8 h incubation of cells after IFN-7 treatment, indicating that a lag period of 4-8 h was necessary for the manifestation of the inhibitory effect (Fig. 3). This observation is in line with the result of cycloheximide treatment, and points to the fact that new protein synthesis is required for the IFN-y down-regulation of CD23 mRNA expression. IFN-y post-transcriptionally regulates CD23 by afecting mRNA stability We have further examined whether the observed down-regulation of the IL-4 induced CD23 mRNA by

304

C.-E. LEE et al. Table

1. IFN-y

does not stimulate

soluble cells

Membrane CD23 (percent positivity)

Treatment” Control IL-4 20 u/ml 100 u/ml IL-4 (100 u/ml) + IFN-y (1,000 u/ml) + IFN-y (10,000 u/ml)

CD23 release in tonsillar

Shedded

soluble

B

CD23

(u/ml)

3.5 31.2 61.3

U.D. 20.5 116.4

30.9 22.4

68.9 45.8

“Purified tonsillar B cells were cultured for 24 h in the presence of IL-4 or IL-4 plus IFN-y as indicated. Soluble CD23 in the culture supernatant was analyzed by ELISA and compared with mCD23 expressed on the cells from the same culture as analyzed by FACScan, as in Materials and Methods. bU.D.; under detection limit.

IFN-y at steady state levels is due to the inhibition of the IL-4 induced transcriptional activation, or due to mRNA degradation. Nuclear run-on transcriptions were thus performed to assess the effect of IFN-y on the IL-4 induced transcriptional activation of CD23 gene. As shown in Fig. 4, we observed an enhancement of CD23 gene transcription occurring within 3 h of IL-4 treatment. Densitometric analysis revealed that IL-4 induced a significant increase (2.0 fold) in nuclear CD23 transcription over the untreated sample, while the levels of control APRT transcripts were not affected by IL-4 treatment (0.9 fold over the untreated sample). The pretreatment of cells with IFN-1; for up to 16 h, a duration sufficient for the manifestation of the IFN-y induced down-regulation of CD23 mRNA and surface protein expression, did not affect the IL-4 activated 1

2

3

4

5

CD23 gene transcription. Hence, mRNA stability studies were subsequently conducted to examine a posttranscriptional control by IFN-y. Cells were first treated with IL-4 or IL-4 plus IFN-y for 6 h to induce the IL-4 activation of CD23 gene expression. Actinomycin D was then added to block further synthesis of CD23 mRNA, and time-dependent changes in mRNA levels by degradation were analyzed by Northern blots. An accelerated decrease of CD23 mRNA levels in cells treated with IL-4 plus IFN-y was noted compared to the cells treated with IL-4 alone (Fig. 5a). The half life of CD23 mRNA was found to be 5.9 h and 2.7 h in IL-4 and IL-4 plus IFN-y treated cells, respectively (Fig. 5b). These data suggest that IFN-y post-transcriptionally rather than transcriptionally modulates CD23 through destabilization of CD23 mRNA in human B cells.

6 DISCUSSION

CD 23

RNA

Fig. 2. Down-regulation of the IL-4 induced CD23 mRNA by IFN-y and effect of cycloheximide. Purified B cells were pretreated with varying doses of IFN-y with or without cycloheximide, or media alone for 1 h and washed prior to IL-4 (100 u/ml) treatment. Cells were cultured for additional 16 h in the presence of IL-4 after which total cellular RNAs were isolated and subjected to Northern analysis, lane 1. IL-4, lane 2. IFN-y 100 u/ml + IL-4, lane 3. IFN-y 1,000 u/ml + IL-4, lane 4. IFN-y lO,OOOu/ml+ IL-4, lane 5. Media, lane 6. Cycloheximide 30 pg/ml + IFN-y 10,000 u/ml + IL-4.

As a part of our investigation on the cytokine regulation of B cell differentiation, the present study aimed to elucidate the molecular mechanism of IFN-y modulation of CD23 expression in human B cells. Our results demonstrate that unlike the IL-4 signaling process leading to CD23 induction, which requires a prolonged exposure (8-24 h) of cells to IL-4, only a transient cell exposure (10 min) to IFN-y was sufficient to deliver IFN-y signal to regulate CD23 expression. Delayed addition of IFN-y to IL-4 stimulated B cell cultures resulted in progressively less inhibition of CD23 expression, indicating that TFN-y acts in an early stage of CD23 biosynthesis rather than affecting CD23 protein degradation or shedding. This notion was further supported by the observation that IFN-y did not reduce surface CD23 levels with an accelerated release and enhanced production of soluble CD23 (Table 1). A dose-dependent downregulation of CD23 mRNA by IFN-y agrees well with the study by Denoroy et al. (1990) and clearly indicates that the inhibition of CD23 biosynthesis by IFN-y is manifested at the level of gene expression. The initial 48 h lag period appears to be necessary for production of proteins that mediate mRNA down-

Post-transcriptional IL-4

(4h)

IFN-r Time (h)

modulation

of CD23 by interferon-y

-

+

+

+

+

+

+

+

+

-

-

-

+

-

+

-

+

-

4

0

6

12

305 + t 20

CD 23

RNA

Fig. 3. Kinetics of IFN-y inhibition of the IL-4 induced CD23 mRNA levels. B cells were treated with IL-4 (100 u/ml) for 4 h after which IFN-y (1,000 u/ml) or media was added. Cells were then cultured for the indicated periods and harvested for mRNA analysis. Indicated times represent the duration of cell culture after the IFN-), treatment.

regulation because cycloheximide blocked the inhibition of CD23 mRNA expression induced by IL-4. We have CD 23

APRT

PGEM

Control

IL-4

IL-4 + IFN-r 1,000 u/ml

IL-4 + IFN-r IO ,000 u/ml

Fig. 4. Effect of IFN-y on the IL-4 induced CD23 gene transcription. Nuclear run-on transcription assays were performed with nuclei prepared from B cells stimulated with 400 u/ml IL-4 for 3 h. Where indicated, IFN-7 treatment was done for 16 h prior to IL-4 stimulation. Isolated [3zP]-RNA was hybridized with 2 ,ug of linearized plasmid containing CD23 cDNA. Adenosine phosphoribosyl transferase (APRT) cDNA and pGEM plasmid DNA were used as internal controls. Autoradiograms were subjected to densitometric analysis using Soft Laser scanning densitometer.

further delineated the molecular mechanism by which IFN-y down-regulates CD23 gene expression, showing that regulation by IFN-7 occurs through a post-transcriptional down-modulation rather than through a direct or indirect transcriptional repression. Possible mediators responsible for the IFN-y induced instability of CD23 mRNA would include selective ribonucleases or RNA destabilizers, as proposed for the degradation of certain proinflammatory cytokine mRNAs with the consensus AU-rich sequences in the 3’-untranslated region (Shaw and Kamen, 1986; Caput et al., 1986). A liable cytosolic destabilizer accelerating c-myc mRNA degradation, which is found depleted in cycloheximidetreated cells (Brewer and Ross, 1988) shares resemblance with the IFN-y induced CD23 mRNA destabilizing activities described here, in that a continuous protein synthesis is required for both cases. Differential regulation of CD23 expression by IFN-)J has been noted in various cell types. In particular, reports on the effect of IFN-y on the IL-4 induced CD23 expression in monocytes or monocytic cell lines are seemingly contradictory, demonstrating either upregulation (Kawabe et al., 1988; Mayumi et al., 1989) or down-regulation (Tevelde et al., 1990) of the IL-4 induced CD23 at mRNA and/or protein levels. Although B cell response to IL-4 and IFN-y for CD23 expression has been found to be more general in that IFN-1/ results in the inhibition of the IL-4 induced surface CD23 protein levels, the molecular mechanisms by which such modulation occurs appear to be diverse and under cell-type specific regulations. For example, in B lymphoblast cell lines, Jijoye, IFN-7 enhances CD23 mRNA expression, yet stimulates shedding of CD23 protein into soluble forms resulting in a net decrease in membrane CD23 levels. In contrast, in normal human resting B cells we have found that IFN-), mediated down-regulation of cell surface CD23 is due to the perturbation of CD23

C.-E.

306

(a)

IL-~ ACD

0

2

6 h

ACD 0

2

4

6

et a/

(PKC) pathway. Nonetheless, IL-4 or IFN-7 mediated regulation of CD23 appears to operate through a mechanism distinct from PKC activation (Lee et al., 1993). Although no intrinsic tyrosine kinase activities of the cloned IFN-1/ receptor (Aguet et al., 1988) have been demonstrated, much effort is being directed to investigate the role of tyrosine phosphorylation together with other novel signaling components participating in IFN-y signal transduction for its diverse immune regulation.

IL-4tIFN-r 4

LEE

h

Acknowledgements-We thank Dr T. Kishimoto for providing us CD23 cDNA probe and monoclonal antibodies. Also Mr Y. H. Choi, Dr B. S. Cho and MS H. R. Yoon for technical assistance in preparing this manuscript.

REFERENCES

(b)

/

I

0

2

4

6

Time after ACD addition (h)

Fig. 5. Effect of IFN-y on the stability of the IL-4 induced CD23 mRNA. B cells were treated with IL-4 (100 u/ml) or with IL-4 plus IFN-y (1,000 u/ml) for 6 h. Actinomycin D (5 pgg/ml) was then added and cultured for indicated durations after which RNAs were isolated and subjected to Northern analysis. Densitometric analysis of CD23 mRNA was performed as in Fig. 4 to determine relative intensity of radioactive bands. Panel a: Northern blot autoradiogram; Panel b: Decay curve for CD23 mRNA.

biosynthesis by mRNA destabilization through a process likely involving IFN-7 induced protein factor(s). Whether or not such IFN-), mediated induction of CD23 mRNA destabilizing factor is a distinct feature of normal resting B cells, which is absent or inactive in transformed B cell line (Jijoye) is not clear and needs to be further explored. In addition, the triggering mechanism for the activation of such a destabilizer by IFN-7 and second messengers mediating this process need to be investigated in detail to further decipher IFN-y signal transduction in normal human B cells. In fact, very little is known regarding the transmembrane signaling involved in IFN-y regulation of CD23 expression. The CAMP-protein kinase A pathway may operate to independently down-regulate CD23 expression, but this pathway is not likely utilized for IFN-1/ or IL-4 signaling (Galizzi et al., 1988). A recent study in our laboratory has revealed that CD23 gene expression in human

B cells can be modulated

via protein

kinase

C

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