Eur. J. Immunol. 1990.20: 2191-2199

Gang Qiu, Jean-Francois Gauchat", Monique Vogel, Michele Mandallaz, Main L. De Weck and Beda M. Stadler Institute of Clinical Immunology, Inselspital, University of Bern, Bern, Switzerland

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Human IgE mRNA expression by peripheral blood lymphocytes stimulated with interleukin 4 and pokeweed mitogen* Expression of human IgE mRNA by peripheral blood lymphocytes (PBL) and an IgE-producing myeloma cell line, U-266, was examined by Northern blot hybridization and compared with IgE levels in culture supernatants. A 2.35-kb IgE mFWA was detected in unstimulated atopic PBL and U-266 cells but not in normal PBL, and its levels correlated with IgE protein levels in the supernatant. Upon stimulation with interleukin 4, a new 1.75-kb transcript was revealed in both atopic and normal PBL but not in U-266 cells. Its expression did not correlate with IgE levels in the supernatant. Pokeweed mitogen also induced the expression of the 1.75-kb transcript without concomitant induction of IgE synthesis by normal PBL and even suppressed the spontaneous expression of the 2.35-kb transcript and IgE protein synthesis by atopic PBL. Interferon-y, which suppressed both the 2.35-kb transcript and IgE protein production, had no inhibitory effect on the 1.75-kb transcript. Expression of the 1.75-kb transcript was already high after 2 days of stimulation and peaked around day 4. The length of the transcript is smaller than that of mRNA coding for secreted human IgG and IgA and contains all four C, exon sequences, suggesting it might be a truncated transcript without v region and might be a human counterpart of the murine germ-line C, transcript.

1 Introduction Due to its important clinical significance [ l , 21, much effort has been made to understand the regulatory mechanisms governing the production of IgE antibody. A real breakthrough was made in this field when recent evidence showed that the two lymphokines, i.e. IL 4 and IFN-y, are the major modulators for IgE synthesis. Studies in the mouse have demonstrated that IL 4 is required for induction of IgE synthesis both in vitro and in vivo [3-51; IL 4 instructs uncommitted B lymphocytes to switch to IgE (and IgG1) production [6]; IL 4 induces generation of a germline E chain transcript before isotype switching to IgE [7,8]; IFN-y specifically antagonizes the activity of IL 4 for the induction of IgE antibody production [9, 101. In vitro studies with human lymphocytes also demonstrated the important role played by these two lymphokines in the regulation of IgE antibody production: IL4 induces de novo IgE synthesis by PBL from normal donors and enhances spontaneous IgE production by atopic PBL. These effects of I L 4 can be inhibited by the addition of I F N y to the culture system [ll-l3].The abilityof clonedTh cells to induce IgE production by purified human B cells is directly related to the quantity of IL 4 and inversely related to the quantity of IFN-y produced by the Tcell clones [14]. Furthermore, the steady state level of IL 4 transcripts in stimulated Tcells from atopic patients is reduced compared to that seen in normal Tcells and that of IFN-y is increased in atopic T cells [15]. [I 85241

* This work was supported by Swiss National Science Foundation, Grant No. 3.998-084 and No. 3.512.086. Correspondence: Beda M. Stadler, Institute of Clinical Immunology, Inselspital, University of Bern, CH-3010 Bern, Switzerland 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

In spite of great progress made in elucidating human in vitro IgE synthesis, many basic questions concerning the regulatory mechanism of human IgE synthesis still remain. First, unlike in the murine system where LPS stimulation is necessary for IL 4 to induce IgE production by purified B cells and where no Tcells or Tcell products other than IL 4 are needed [3, 41, IL 4 alone is sufficient for induction of IgE production by human PBL and failed to support IgE synthesis by purified human B cells in the presence or absence of mitogens [ll, 161. Second, the amount of IgE induced by I L 4 in in virro cultured normal PBL varied considerably among different laboratories as well as among different donors [ll-131. PBL from some normal donors even failed to synthesize IgE in the presence of IL 4 [12]. Finally, the molecular mechanism by which IL 4 induces human PBL to synthesize IgE is still to be determined. One of the difficulties in the search for the regulatory mechanism of human IgE synthesis is the accurate measurement of the very low levels of IgE synthesized in vitro. All information published so far was obtained by measuring IgE protein levels in culture SN with various immunoassay systems using polyclonal or monoclonal anti-human IgE antibodies. The specificity and sensitivity of these immunoassay systems was sometimes questionable and intrinsically prone to interference by for example in vivo preformed IgE [17] or naturally occurring anti-IgE autoantibodies present in the same culture SN (our unpublished results). Furthermore, a multicenter study showed great variation in the performances of the immunoassay systems used by different laboratories, which constituted a major source of controversy in this field [18]. To remove some uncertainty related to the immunoassay system and t o investigate the regulatory mechanism of IgE synthesis by human lymphocytes at the gene level, we decided to measure IgE mRNA levels by Northern blot 0014-2980/90/1010-2191$3.50+.25/0

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IgE mRNA expression by peripheral blood lymphocytes

hybridization in addition to the IgE protein levels in culture SN. In this report, we present evidence that several species of IgE mRNA can be detected in PBL and in an IgEproducing human B cell line. In normal PBL, IL 4 mainly induces a 1.75-kb IgE transcript which is shorter than the IgE mRNA spontaneously produced by atopic PBL or by an IgE-producing human B cell line and did not correlate with IgE protein levels in the same culture SN. The shorter IgE transcript was also induced by PWM, without concomitant induction of IgE protein synthesis while IFN-y suppressed IgE protein production but had no inhibitory effect on the expression of the 1.75-kb IgE mRNA.

2 Materials and methods

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incubated at room temperature overnight. After blocking plates with 10% FCS in PBS for 2 h at 37°C and washing with PBS containing 0.1% Tween 20,100 pl of appropriately diluted serum or undiluted culture SN as well as a series of twofold dilutions of a standard IgE preparation (Pharmacia) was added to each well followed by 25 pl(5 X 105cpm) of BSW17 labeled with 12510dine (100 mCi/ml = 3.7 GBq/ml, Amersham Int., Amersham, GB) by the chloramine-T method. The plate was then incubated at room temperature overnight. After six washes, individual wells were cut off and radioactivity was determined in a y counter. A linear curve in the range of standard IgE from 50-25600 pg/ml was obtained. The lowest standard IgE (50 pg/ml) gave rise to a cpm value higher than the sum of background (usually below 300 cpm) plus 2 SD < 10%).

2.1 Lymphokines and reagents 2.4 Probes

Human rIL 4 with a specific activity of lo6U/mg was kindly provided by Dr. M. Schreier (Sandoz AG, Basel, Switzerland) and used at a final concentration of 200 U/ml. Human rIFN-y was kindly provided by Drs. G . Garotta and E . Herzog (Hoffmann-La Roche Ltd., Basel, Switzerland) and used at a final concentration of 200 U/ml. PWM obtained from Gibco (Grand Island, NY) was used at a final concentration of 1% (v/v). PMA purchased from Sigma Chemie GmbH (Deisenhofen, FRG) was used at a final concentration of 10 ng/ml. 2.2 PBL preparation and culture Heparinized blood was obtained from 23 normal healthy individuals with serum IgE levels between 3.3 to 24.6 IU/ml (14.8 f 8.3 IU/ml, 1IU = 2.42 ng), 7 atopic patients with serum IgE levels from 235 to 1575 IU/ml (1030 f 658 IU/ml) and an 18-year-old male patient with hyper IgE syndrome (serum IgE level: 40766 IU/ml). The blood from the patient with hyper IgE syndrome was kindly provided by Dr. R. Seger, Children's Hospital, Zurich, Switzerland. PBL were isolated from the blood by centrifugation on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradients at 400 x g for 30 min, washed twice in HBSS without Ca2+ and Mg2+ and resuspended in RPMI 1640 supplemented with 100 U/ml penicillin G , 100 pg/ml streptomycin, 2 m~ L-glutamine and 10% heat-inactivated FCS. PBL were cultured at a concentration of 1.5 x lo6 cells/ml in 75-cm2 tissue culture flask (Falcon No. 3024, Oxnard, CA) in the presence or absence of various reagents for 5 days. At the end of culture, cells were harvested by centrifugation, washed twice in HBSS without Ca2+/Mg2+and subjected to RNA purification (see below). The culture SN was also collected and stored at - 20°C before quantitation of IgE protein levels by RIA (see below).

2.3 Microtiter solid-phase radioimmunoassay The two mouse anti-human IgE mAb used in microtiter solid-phase RIA, Le27 and BSW17, have been described [19], and the procedure of the RIA was modified from that of Eisenbrey et al. [20]. Briefly, flexible plastic U-bottom plates (Dynatech Laboratories, Alexandria, VA) were coated by adding 100 pl of Le27 solution (20 pg/ml diluted in 50 mM sodium carbonate buffer, pH 9.6) to each well and

Plasmid pH Ige-11 and pCe-spl. were kindly provided by Dr. T. Honjo. The pH . Ige-11 plasmid contains a 3-kb Bam HI fragment of human C, genomic DNA [21,22] cloned in pBR322.The pCE-spl. plasmid contains a type of cDNA of the human C, sequence which was not made by reverse transcription of IgE mRNA but by first cloning the same 3-kb Bam HI fragment of human C, genomic DNA into the Bgl I1 site of chicken retrovirus vector pTK.2 ter (R) [23], and then removing the intervening sequences by in vivo splicing [24, 251. The Bam HI fragment from the resultant viral vector was then recloned into pBR322 (T. Honjo, personal communication). The human C, probes used in this study are shown in Fig. 1.The E 1probe is a 389-bp Bam H I - Sal I fragment isolated from the pH Ige-11 plasmid and cloned into the polylinker region of pBS (-) plasmid (Stratagene, La Jolla, CA).The E 2-3 probe is a 240-bp Sac I - Sma I fragment isolated from the pCe-spl. plasmid and cloned into Sac I - Hinc I1 sites in pBS (+). This probe contains the sequence encoding the FceR I-binding region of the IgE protein [26] and thus was the one most often used in this study.The E 4 probe is a 785-bp Bgl I -Sac I fragment isolated from pCe-spl.

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Figurel. Map of the IgE genomic DNA fragment cloned in pH-Iga-11 (top) and IgE cDNA fragment cloned in pC&-spl. (bottom). Only restriction sites relevant to this report are indicated. The probes specific for human IgE are indicated as black bars below the cloned DNA fragment from which they were isolated.

Eur. J. Immunol. 1990. 20: 2191-2199

The probe for human C, chain is a 2.0-kb Eco RI -Hind I11 fragment isolated from plasmid pSH3Cy3 [27] obtained from the American Type Culture Collection (ATCC No. 59624 Rockville, MD) and has been shown to be able to hybridize with the gene of all four human IgG subclasses [27].The probe for human C, chain is a 4.84-kb Sac I - Hind 111 fragment isolated from Ch.H.Iga-25 phage DNA [28] obtained from the Japanese Cancer Research Resources Bank Tokyo (JCRB No. HG068) and is able to hybridize with both C,1 and C,2 genes [28]. The probe for human C, chain is a 1.2-kb Eco RI fragment isolated from phage H24 DNA [29] (ATCC No. 57430).

2.5 Isolation of RNA and Northern blot hybridization

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2.6 S1 nuclease protection assay Published procedures [38] were followed with slight modification. Briefly, pBS( -)/~2-3 plasmid was transformed into the XL1- Blue script [39] Stratagene) and singlestranded DNA (ssDNA) was purified by PEG 6000 precipitation [40] from SN of the XL-1 Blue culture infected with R408 helper phage ([40], Stratagene). The ssDNA was annealed with T3 primer, extended with the Klenow fragment of E. coZi DNA polymerase in the presence of [32P]dCTP (800 Ci/mmol, NEN Research Products), cut with Eco RI and separated on 1% agarose gel. The gel was then exposed to X-ray film wrapped with aluminium foil and the labeled ssDNA of 288 nucleotides (nt) containing 240 nt of E 2-3 insert and 48 nt of sequence derived from the polylinker region of pBS (-) was isolated from the gel. For the S1 nuclease protection assay, 1.5 x lo5 cpm of labeled ssDNA was mixed with 120 pg of RNA, precipitated with ethanol, redissolved in hybridization buffer (0.4 M NaCl, 40 mM Pipes, pH 6.5, 1 mM EDTA and 50% formamide), boiled for 5 min, incubated at 70°C overnight, digested with 150 U S1 nuclease at room temperature for 1 h and after ethanol precipitation was separated on an 8% polyacrylamide gel containing 8 M urea. Bsp RI-digested pBR322 DNA (Biofinex, Praroman, Switzerland) was used as molecular weight standard. The gel was then dried on a gel dryer (Pharmacia LKB Biotechnology) and exposed to X-ray film at - 70°C for 19 to 24 h.

Total RNA was isolated from cultured PBL or cell lines by the guanidinium isothiocyanate-CsC1 procedure [30]. Samples of RNA (2 yg for each lane) or an RNA molecular weight standard (RNA ladder, Gibco/BRL AG, Basel, Switzerland) were subjected to horizontal electrophoresis in 1.2% agarose gels containing 6% formaldehyde [31] and electrophoretically transferred to Genescreen nylon membrane (NEN Research Products, Boston, MA) as described [32]. RNA was fixed to the membrane by UV irradiation [32]. Specific mRNA were revealed by hybridization either with antisense RNA probes ( ~ and l ~ 2 - 3probes) labeled with 32P by in vitro transcription [33, 341 of pBS (+/-) plasmids containing appropriate insert, or with DNA probes ( ~ 4y,, a and p probes) labeled with 32Pby random priming [35] of isolated DNA fragments. Prehybridizations 3 Results and hybridizations were carried out overnight either at 65 "C (for RNA probes) or at 60 "C (for DNA probes) in a 3.1 Spontaneous and induced IgE mRNA expression by solution containing 0.75 M NaCl, 50 mM sodium citrate (pH in vitro cultured human PBL 7.0), 25 mM sodium phosphate (pH 6.8), 1mM EDTA, 50% formamide, 10% PEG 6OOO [36], 0.2% Ficoll 400, PBL were isolated from atopic patients showing elevated 0.2% polyvinylpyrrolydone, 0.2% BSA, 1% SDS, 0.1% serum IgE levels (patients with mild atopic disease and a sodium pyrophosphate, 200 pg/ml salmon sperm DNA, 50 patient with hyper IgE syndrome) as well as from normal pglml yeast RNA, 10 pglml poly (A) and 10 pglml poly (C) donors.The cells were cultured for 5 days in the presence or [33] in the absence or presence of 2 x 106 c p d m l of probe, absence of recombinant lymphokines and PWM. Total respectively. The membranes hybridized with RNA probes RNA was then isolated and IgE mRNA expression was were rinsed twice with 0.1 x SSC/l% SDS, washed 3 times visualized by Northern blot hybridization. As a control, we with 0.1 x SSC/l% SDS for 30 min each at 70 "C and rinsed also included total RNA from U-266 cells, a human twice with 0.1 X SSC.The membranes were then incubated myeloma cell line which spontaneously secretes high levels for 30 min at 37°C with 2 yg/ml RNase A , rinsed and of IgE protein (see [41]). Results shown in Fig. 2 are washed with 0.1 x SSC/1% SDS for 30 min at 70°C. representative of more than 30 experiments and can be Because RNase treatment prevents the rehybridization summarized as follows: with actin or ribosome RNA probe, the amount of total RNA on the membrane was monitored by staining with In unstimulated atopic PBL a 2.35-kb IgE mRNA was methylene blue before hybridization [31] and was demon- detected. The level of this "spontaneously" expressed IgE strated to be the same in different lanes in all of the mRNA was relatively low in PBL from patients with experiments presented in this report (data not shown).The common atopic diseases (Fig. 2A) but was higher in PBL membranes hybridized with DNA probes were treated in from a patient with hyper IgE syndrome (Fig. 2B). the same way except that washings were carried out in 1 x Stimulation with IL 4 enhanced the level of the 2.35-kb IgE SSC/l% SDS at 60°C and RNase A treatment was omitted. mRNA and, more importantly, induced a strong expression Radioactivity was revealed by exposure of membranes to of a transcript with an apparent molecular weight of Kodak (Rochester, NY) X-Omat SX-ray films at - 70 "C in 1.75 kb. the presence of an intensifying screen [37].When necessary, the signals on the films were assessed by a CAMAG No detectable IgE mRNA expression was observed in (Muttenz, Switzerland) reflection densitometer. For rehy- unstimulated PBL from most normal donors tested while bridization the previous DNA probes were removed by stimulation with IL 4 induced the expression of a 1.75-kb boiling membranes twice in 0.1% SDS for 10 min each and species (Fig. 2). Occasionally the 2.35-kb transcript could prehybridization and hybridization were carried out as also be detected in a few normal donors (e.g. donor 2 in Fig. mentioned above. 2A) and reason for this disparity is not clear. I L 4 also

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IgE mRNA expression by peripheral blood lymphocytes

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induced the expression of the 2.35-kb transcript but the level of this transcript was very low in these experiments and could not always be detected in all donors. (but see Sect. 4).

I F N y by itself did not induce the expression of either IgE mRNA species in normal PBL and inhibited expression of the 2.35-kb transcript in atopic PBL (Fig. 2 A) but not of the 1.75-kb transcript induced by IL 4 in normal PBL (Fig. 2B). Interestingly, PWM like IL 4 also induced expression of the 1.75-kb IgE mRNA in both atopic and normal PBL but, like IFN-y, inhibited the spontaneous expression of the 2.35 kb IgE mRNA in atopic PBL (Fig. 2A). Additional experiments with PBL from different donors including both normal and atopic patients (31 donors in total, data not shown) yielded the same results. In U-266 cells, several species of IgE mRNA were detected (Fig. 2 B).The 3.7- and 3.3-kb transcripts observed in U-266 were also detected in some atopic and normal PBL as a large band of 3.5-kb (Fig. 2 A). The 2.35-kb transcript corresponding to the spontaneously expressed IgE mRNA by atopic PBL was the major band in U-266 cells, while the 1.75-kb transcript (which is the one induced by IL 4) was not observed. Some additional IgE transcripts (2.85, 1.85, 1.65 and 1.40-kb) were expressed by U-266 cells but were not detectable in PBL samples.

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cultured human PBL. Total RNA isolated either from PBL cultured in vitro for 5 days with the indicated agents (IL 4, 200 Ulml; IFN-y, 200 U/ml; PWM, 10 pUml), or from human B cell lines were fractionated in a 1.2% agarose gel, transferred to Genescreen membrane and hybridized with the ~2-3RNA probe. (A) Each lane contains 2 pg of PBL RNA. Autoradiography was camed out at - 70°C for 14 days. (B) Each lane contains either 2 pg of PBL or F1 cell RNA, or 20 ng of U-266 cell RNA plus 2 p g of yeast RNA. Autoradiography was camed out at - 70°C for 18 days. The equality of the total RNA between each lanes was confirmed by methylene blue staining.

donors, were capable of spontaneously synthesizing IgE in vitro (for review, see [42]). Assuming that this was also the case in the present investigation, we therefore considered that the IgE detected in SN of normal PBL cultures was preformed in vivo and that of atopic PBL was partly synthesized in vitro. Results shown in Fig. 3 demonstrate that as expected from our previous observation [13] as well as from the results reported by others (for review see [16], I L 4 enhanced spontaneous IgE secretion by atopic PBL and induced IgE production by normal PBL. IFN-y and PWM did not induce IgE production by normal PBL but inhibited spontaneous

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3.2 Correlation between IgE mRNA levels in PBL and IgE protein levels in culture SN IgE protein levels in culture SN were determined by an isotype-specific RIA and were compared with the levels of the 1.75-kb as well as of the 2.35-kb IgE mRNA measured by scanning the autoradiography of the Northern blot. Comparison with the 3.5-kb IgE mRNA was not made because this RNA species was not detectable in PBL from all donors. Furthermore, no control for in vivo preformed IgE [17] was carried out in order to obtain from the same cultures the cells for isolation of RNA and the SN for IgE protein determination. However, well-controlled experiments performed by several laboratories, including our own [19], have established that PBL from atopic patients with elevated serum IgE levels, but not PBL from normal

Figure 3. IgE protein secretion and mRNA expression by in vitro cultured human PBL. PBL from three atopic patients and three normal donors were cultured in vitro for 5 days with or without stimulation. IgE protein levels in culture SN were measured by a microtiter solid-phase RIA using two different mouse anti-human IgE mAb. IgE mRNA levels were determined by scanning autoradiographies of Northern blot hybridizations using the ~ 2 - 3 RNA probe.The scanning units shown were estimated according to the signals from a fixed amount of U-266 RNA used as a standard.

G . Qiu, J.-F. Gauchat, M.Vogel et al.

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IgE secretion by atopic cells. IFN-y also inhibited IgE production by normal PBL stimulated with IL 4. When IgE levels in culture SN were compared with levels of IgE mRNA derived from the same cultures, it became evident that the IgE levels in the SN correlate better with the level of 2.35-kb transcript than with that of the 1.75-kb species. In IL 4-stimulated cultures, the increase of IgE levels in SN was small and seemed incompatible with the high levels of expression of the 1.75-kb transcript, but more consistent with levels of expression of the 2.35-kb transcript, which was only slightly increased compared to the control. IFN-y, which inhibits both spontaneous and IL 4induced IgE production, inhibited only the expression of the 2.35-kb transcript but not that of the 1.75-kb transcript induced by IL 4 (Fig. 2B). Similarly, the inhibitory effects of PWM on spontaneous IgE protein production by atopic PBL was consistent with the inhibition of the 2.35-kb transcript by this reagent but opposite to its stimulatory effect on the expression of the 1.75-kb species. These observations were verified by regression analysis of the data from 14 donors. The results shown in Fig. 4 demonstrated that in both unstimulated and stimulated cultures, the levels of the 2.35-kb transcript correlated with IgE protein levels in the culture SN, while levels of the 1.75-kb species showed no correlation, suggesting that only the 2.35-kb RNA species might have been translated into IgE protein detectable by our immunoassay. 3.3 Differences between the 1.75-kb species and the 2.35-kb IgE mRNA

The finding that the levels of the 1.75-kb mRNA species did not correlate with IgE protein levels in the SN would argue against the identification of this species of mRNA as IgE mRNA and thus question the specificity of our Northern blot system. However, several observations support the conclusion that our ~2-3probe specifically detected human

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IgE mRNA. First, hybridization of our C, probes with a Southern blot containing genomic DNA isolated from U-266 and U-937 cells and digested with Bam HI, Eco RI and Hind I11 revealed the same pattern (data not shown) as those published previously [21, 411, suggesting that they hybridized only with genomic C, sequences. Furthermore, in both Southern and Northern blots the hybridizations were observed only when an antisense probe but not the sense probe was used (data not shown), suggesting the probe is specific for IgE mRNA. Second, a search in a computer gene bank (EMBLC) for DNA sequences homologous to our ~ 2 - 3probe found significant homology only with chimpanzee and orangutan IgE [43]. Third, the same mRNA bands were also detected by probes specific for IgE sequence other than the C,2-3 region. Thus, identical Northern blot membranes were hybridized with probes specific for C,2-3 region. The autoradiography of Northern blot probed with a CE4exon probe is shown in Fig. 5 which demonstrates identical patterns of hybridization as those seen with the C,2-3 probe. Identical patterns of hybridization were also observed when a CE1probe was used (data not shown), which was derived from another clone than ~ 2 - 3 and ~4 probes (see Sect. 2.4), indicating that the shorter IgE mRNA also contains C,1 and C,4 exon transcripts in addition to the C,2-3 exon transcript. Finally a S1 nuclease protection assay shown in Fig. 5 demonstrates that the RNA samples containing almost exclusively the 1.75-kb mRNA species (RNA from IL 4and PWM-stimulated normal PBL) protected an IgE DNA fragment which has the same length as that protected by the RNA samples containing apparently only the 2.35-kb species (RNA from unstimulated PBL of a patient with hyper IgE syndrome, see Fig. 2). Moreover, an RNA

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Figure 4. Correlation of IgE protein levels in culture SN with levels of expression of the 1.75-kb (dotted lines) and 2.35-kb (solid lines) transcripts in PBL. The correlation coefficient (r) is shown close to each line in the figures. Data from 14 donors are shown, which had been stimulated with either IL 4 (200 Ulml), IFN-y (200 U/ml) or PWM (10 pl/ml) for 5 days.

Figure 5. Identification of IgE mRNa by S1 nuclease protection assay. The 32P-labeled single-stranded ~2-3probe was hybridized with total RNA isolated from the indicated cells, digested with S1 nuclease and separated on an 8% polyacrylamide gel containing 8M urea.Tota1 amount of RNA in each lane was 120 lGmg consisting of either 120 pg of yeast RNA, 120 pg of total RNA from normal PBL (no. 4) and F1 cells, 30 pg of total RNA from PBL of a patient with hyper IgE syndrome and from normal PBL (no. 3 and S ) , or 3 pg of total RNA from U-266 cells, plus 0, 90 or 117 pg of yeast RNA, respectively. The dried gel was exposed to X-ray film at - 70°C for 19 h.

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IgE mRNA expression by peripheral blood lymphocytes Normal donor

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Figure 6. Hybridization of IgE mRNA with €2-3 and €4 probes. PBL total RNA (2 pg) or U-266 total RNA (20 ng) were applied to each lane and the identical membranes were hybridized with random labeled probes specific for either ~ 2 - 3 or €4 exons. Autoradiography was carried out at - 70°C for 24 h.

sample containing approximately equal amounts of the 1.75- and 2.35-kb mRNA (RNA from IL 4-stimulated PBL of the patient with hyper IgE syndrome) also protected only a single IgE DNA fragment of the same length as that protected by U-266 RNA as well as by other RNA samples (Fig. 6). These observations unambiguously prove the “inducible” 1.75-kb mRNA is identical for the constant E domains with the 2.35-kb mRNA spontaneously expressed in atopic PBL and in the U-266 myeloma cell line.

level in unstimulated PBL. These changes were not due to differences in the amount of RNA added in each lane because a probe specific for 28 S ribosomal RNA gave rise to identical signals in all the lanes (data not shown). In U-266-derived RNA, the IgA probe revealed a 3.2-kb transcript which may code for membrane-bound IgA [44, 451 and a 1.1-kb band of unknown nature.When the same membrane was re-hybridized with the IgE probe, a hybridization pattern identical to that seen with the membrane hybridized with the E probe as that shown in Fig. 7 was revealed (data not shown), excluding the possibility that RNA degradation was the reason for the appearance of a 1.1-kb IgA mRNA in U-266.

In PBL-derived RNA the IgG probe detected a 1.85-kb band,whose intensity increased after PWM stimulation and which might code for secreted IgG [45, 471. In F1 cellderived RNA a 3.5-kb band coding for membrane-bound IgG [46,47] was detected in addition to the 1.85-kb band. In PBL- and F1-derived RNA the IgM probe detected a strong band of 2.35-kb for secreted IgM [47,48] and a weak band of 1.85-kb whose nature is not known.The IgE probe detected again the 1.75-kb band in IL 4-stimulated PBL, and the major 3.5- and 1.85-kb bands in U-266 cells. Re-hybridization of the E probe-hybridized membrane with the IgG probe and all other membranes with the IgE probe detected the expected band for IgG and IgE, respectively (data not shown). In summary, the 1.75-kb IgE mRNA is shorter than any other human Ig mRNA tested and may represent a truncated mRNA. 3.5 Time-course study of the IL 4induced IgE mRNA expression

3.4 The 1.75-kb IgE mRNA is shorter than human IgG, IgA and IgM mRNA Identical Northern blot membranes were also hybridized with specific probes for human IgG, IgA and IgM CHgenes (Fig. 7). In normal PBL and F1 cells (an EBV transformed human B lymphoblastoid cell line) the a probe detected a 1.8-kb band probably coding for the secreted form of IgA [44]. I L 4 down-regulated while PWM enhanced IgA mRNA expression in PBL as compared to the IgA mRNA

Normal PBL were cultured in the presence or absence of IL 4 for different lengths of time and IgE mRNA expression was visualized by Northern blot hybridization. Fig. 8 shows the autoradiography of one such experiment using RNA isolated from the cells cultured for a period of 2-8 days and demonstrates that expression of the 1.75-kb IgE Donor A

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Figure 7. Autoradiography of Northern blots of normal PBL RNA hybridized with human Ig CHgene probes. Total RNA (2 pgnane) isolted from either normal PBL stimulated in vitro for 5 days with the indicated reagents or from cell lines were fractionated on a 0.8% agarose gel and transferred t o Genescreen membrane. The four identical membranes were hybridized with the DNA probe indicated on the top of each membrane. Exposure time was 17 h at - 70 “C.

Figure 8. Time - course study of IL 4 - induced expression of the 1.75-kb human transcript by normal PBL. PBL from normal donors were cultured for different times in the absence or presence of 200 U/ml of human rIL 4.Total RNA isolated from the resultant cells was subject to Northern blot hybridization using the ~ 2 - 3 RNA probe. Exposure time was 15 days at - 70°C.

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mRNA was already high on day 2 of I L 4 stimulation, peaked around day 4 and decreased afterward.

3.6 IL 4 failed to induce the expression of the 1.75-kb IgE mRNA in U-266cells In order to test if the expression of the 1.75-kb mRNA could also be induced in U-266 cells, we stimulated U-266 cells with I L 4 , PWM and PMA for 1 or 2 days and the resultant RNA was subjected to Northern blot hybridization. Fig. 9 shows that neither IL 4 nor PWM (or PMA) induced detectable amounts of the 1.75-kb IgE mRNA, suggesting that under these conditions this cell line may not be capable of accumulating the shorter IgE mRNA species detected in untransformed lymphocytes.

4 Discussion

2.35 1.75

Figure 9. IgE mRNA expression b y U-266 cells stimulated with IL 4, PWM or PMA. U-266 cells were cultured in the presence or absence of the stimuli for the indicated times. Total RNA (50 ng) was separated in each lane for subsequent Northern blot hybridization. Total RNA from normal, long-term cultured U-266 cells (unstimulated) and from IL 4-stimulated normal PBL are also included as controls.

4.1 The 2.35 kb transcript codes for secreted IgE In this study we demonstrated that several species of IgE mRNA could be detected by Northern blot hybridization of total RNA from human PBL and from an IgE-producing B cell line. A 2.35-kb mRNA was detected mainly in unstimulated atopic PBL, and U-266 cells but not in unstimulated PBL from most normal donors tested. The 2.35-kb mRNA levels correlated with IgE protein levels in the culture SN. Its length is similar to that of the mRNA coding for secreted mouse [49] and rat [50]IgE and secreted human IgM [51], Fig. 7). Taken together these observations suggested that the 2.35-kb transcript most likely codes for secreted IgE. More interesting is the finding that upon stimulation with IL 4, a 1.75-kb transcript was detected in both atopic and normal PBL and that its levels did not correlate with IgE protein levels in the SN. PWM also induced the expression of the 1.75-kb transcript without concomitant induction of IgE synthesis by normal PBL and even suppressed the spontaneous expression of the 2.35-kb transcript and IgE protein synthesis by atopic PBL. m y , which suppressed both expression of the 2.35-kb IgE mRNA and IgE protein synsthesis, had no inhibitory effect on the expression of the 1.75-kb IgE mRNA. Several high-molecular weight IgE mRNA were also detected (3.5-kb IgE mRNA in PBL and 3.7-, 3.3- and 2.85-kb IgE mRNA in U-266 cells) and might code for the membranebound form of IgE protein because their length is similar t o the mRNA coding for human membrane-bound form of IgG, IgA and IgM as well as mRNA coding for rat and mouse membrane-bound IgE [41, 44-51]. The possibility that some of them might be the precursor for mature mRNA could also be considered [49]. The nature of the 1.85, 1.65- and 1.40-kb transcript detected in U-266 cells only is not known. Although the half-life of IgE mRNA in vivo has not been determined, it seems unlikely that the IgE mRNA detected in unstimulated atopic PBL was “pre-formed”; because the half-life of the mRNA coding for secreted IgM in murine plasmacytoma has been reported to be 20 [52] to 23 h [53] whereas we routinely cultured PBL in vitro for 5 days.

4.2 The 1.75-kb mRNA is a germ-line C, transcript The nature of the 1.75-kb transcript is not clear at the present time. Several observations suggest that it might be a counte art of the germ-line C, transcript found in the mouseT7, 81. The mouse germ-line C , transcript was reported to be induced by IL 4 plus LPS in normal spleen cells as well as in transformed cell lines. Its length (1.9-kb) is shorter than that of authentic E mRNA (2.2-kb) and its expression was detectable after 2 days of stimulation, peaked at day 4 and decreased afterward [7]. All of these observations parallel our findings for the 1.75-kb transcript. In mice, the expression of the germ-line C, transcript was followed by the expression of the mature transcript [7, 81. This is also the case for human IgE mRNA in that prolonged kinetic studies showed that the expression of the 2.35-kb transcript was detectable after 8 days of I L 4 stimulation and peaked around day 12, much later than the 1.75-kb transcript whose expression was already high after 2 days of stimulation and peaked around day 4. The mouse germ-line C, transcript lacks the v region and thus is also a truncated mRNA [7, 81. Our finding that the 1.75-kb transcripts can hybridize with probes specific for all four C, exons and has a length slightly shorter than that of mRNA coding for human y and a chain, which are known to consist of only three c domains and a short hinge region, suggests that the fragment which is missing in this transcript is most likely that coding for the v region. Cloning and sequencing of the 1.75-kb IgE mRNA is now in progress in our laboratory and preliminary results demonstrated that it indeed contains no v region but a stretch of novel sequence not found in coding IgE mRNA (Qiu et al., manuscript in preparation). The expression of a germ-line transcript whose length is shorter than or similar to authentic mRNA is not unique to IgE and has also been reported for IgA, IgG1, IgG2a and IgGzb [8, 54, 551. In most cases transcription of the germ-line gene occurs prior to the expression of the

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IgE mRNA expression by peripheral blood lymphocytes

authentic mRNA, starts of the switching region and thus is thought to be the result of the activation of the corresponding Ig CHgene prior to the actual switch recombination [7, 8, 551. In this respect, several findings of this study are of interest. First, it has been reported [13, 19,421 and also observed in our study that PWM did not induce normal PBL to synthesize IgE in vitro. However, we found that PWM induced a 1.75-kb transcript, suggesting that PWM may be able to activate the C , gene but fails to lead to actual IgE synthesis. Similarly, IFN-y suppressed IL 4-induced IgE synthesis but had no effect on IL 4-induced expression of the 1.75-kb transcript, suggesting that it probably acts at a stage later than activation of the germ-line C, gene. These results also implied that activation of the germ-line gene and actual switching to IgE synthesis might be separately regulated. 4.3 The 1.75-kb transcript is not necessarily limited to IgE synthesis

In our initial experiments, we also included the total RNA from Fl cells, an EBV-transformed human lymphoblastoid cell line, and intended to use it as a negative control. However, we could always detect a weakly expressed 1.75-kb band in unstimulated F1 cells (Fig. 2). S1 protection assay failed to give an unambiguous answer regarding the nature of this band apparently due to insufficient levels of the 1.75-kb species in this cell line (Fig. 6). In subsequent experiments, however,we found that stimulation of F1 cells with IL 4 dramatically enhanced expression of the 1.75-kb but failed to induce that of the 2.35-kb transcript even after prolonged stimulation (Qiu et al., manuscript in preparation). Since EBV-transformed human B cells have been shown to produce IgE only when stimulated b y IL 4 [56],we speculate that the C, gene in F1 cells had already been activated to some extent by EBV infection so that a weak production of the 1.75-kb IgE mRNA is maintained in unstimulated cells. The enhancement of the 1.75-kb transcript but the failure to induce the 2.35-kb IgE mRNA expression in F1 cells upon IL 4 stimulation again implies a separate regulation of germ-line gene activation and switching to IgE synthesis. It will be interesting to know if the 2.35-kb transcript can be induced in these cells by other cytokines and the appropriate experiments are in progress in our laboratory. The level of expression of the 1.75-kb transcript is much higher than that of the 2.35-kb transcript. This could stem from either a prolonged half-life of the 1.75-kb species, a more active transcription, a higher frequency of cells capable of producing the 1.75-kb transcript compared to those producing the 2.35-kb or a combination of these reasons. The fact that we could detect the 1.75-kb but not the 2.35-kb transcript in appropriately stimulated F1 cells as mentioned above suggests the possibility that the relative higher level of expression of the 1.75-kb transcript might be, at least in part, due to the higher frequency of cells producing this species. If this is also the case for normal cells in vivo, then there might be some mechanism(s) which prevents the majority of cells, whose germ-line C, are activated, to become IgE-producing cclls. The failure to induce the 1.75-kb transcript in U-266 cells by IL 4 is also

consistent with the notion mentioned above, because the class switching process in this cell line is already completed and the S, region and the intervening sequence 5' of it on the expressed chromosome has been deleted [21, 221. However, the possibility that human I L 4 does not act directly on B cells must also be considered. Detection of IgA mRNA expression in U-266 was not surprising, because the same phenomenon has been reported by Hellman et al. who detected a 3.5-kb transcript for membrane-bound and a transcript of about 2-kb for secreted IgA in their U-266 cells [41]. In our U-266 cells, a 3.2-kb transcript was detected which might code for membrane-bound IgA. However, no band corresponding to the 1.8-kb transcript coding for secreted IgA was detected but a 1.1-kb band was detected instead (Fig. 7). This small band did not result from degradation of RNA because both ribosomal FWA and IgE mRNA on the same membrane were intact (data not shown). In the light of the model mentioned above one explanation for this difference might be that our U-266 culture contain cells which are at the stage of undergoing germ-line C , gene activation (the 1.1-kb RNA hybridizing with the a probe would be a germ-line C, transcript), while U-266 cells used by Hellman et al. contain mainly cells in which the activating process has already been completed and hence only authentic a mRNA was produced. In this respect it will be interesting to investigate whether it is possible to induce expression of mature a transcripts in our U-266 cells. We thank Dr. i? Honjo for providing the plasmids PH.IgE-11 and pCE-spl., Drs. M . Schreier, G . Garotta and E. Herzog for supplying recombinant human IL 4 and IFN y, Dr. R . Seger for help in obtaining blood from a hyper IgE patient, Dr. A . Walz for help to search the computer gene bank and Dr. S. Miescher for helpful discussion and critical reading of the manuscript.

Received April 24, 1990.

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