Immunology 1991 74, 703-708
Comparison of IgE expression at the mRNA and protein levels in vitro K. J. TURNER, J. CREANY, R. J. COELEN, K. J. CAMERON, B. J. HOLT & M. W. BEILHARZ Department of Microbiology, University of Western Australia, QEII Medical Centre, Australia Acceptedfor publication 2 August 1991
SUMMARY The regulating effects of IL-4 and pokeweed mitogen on IgE synthesis in vitro by human peripheral blood leucocytes has been compared with the corresponding effect of these regulators on the expression of IgE mRNA. The latter was measured by dot blot hybridization with an oligonucleotide coding for a unique six amino acid region of the CHe2 domain. Specificity of the oligonucleotide probe was established by its inability to hybridize with RNA extracted from HMY-2 (IgG) and XQ15 (IgM) secreting cell lines whilst producing intense signals with RNA extracted from the IgE secreting cell line U266. Whilst IgE mRNA was detected in RNA extracted from PBL of both atopic and control subjects, spontaneous IgE synthesis was restricted to atopic PBL. IL-4 increased both IgE mRNA and IgE synthesis in all PBL samples but PWM, while significantly increasing IgE mRNA expression either failed to modify IgE synthesis or actively suppressed it. The assay system employed to quantitate IgE synthesis in vitro was shown to be inhibited by both IgE binding factors and IgG anti-IgE autoantibodies which are produced in PBL cultures. IgE mRNA levels might therefore more accurately monitor the regulatory effects of IL-4 and PWM on IgE synthesis than quantitation of the IgE by radioimmunoassay. and of the relative effect of IL-4 on cells from atopic and nonatopic donors.'2 15 This study was motivated by the need to provide an alternative procedure for investigating IgE regulation based on measurement of IgE mRNA levels by dot blot hybridization with an oligonucleotide coding for a unique six amino acid region of the CHa2 domain of the £ chain in parallel with quantitation of synthesized IgE by radioimmunoassay. IgE mRNA was identified in RNA extracted from PBL of all donors both atopic and control. IL-4 increased both IgE mRNA and IgE protein synthesis but PWM, while significantly increasing IgE mRNA expression, failed to induce IgE protein synthesis as measured by radioimmunoassay. It is likely that this inability to assay induced IgE synthesis is due to the formation of immune
INTRODUCTION
The in vitro models employed to investigate mechanisms regulating the human IgE antibody response require the accurate measurement of the very low levels of this immunoglobulin synthesized by culture of peripheral blood leucocytes (PBL), tonsils or other lymphoid tissue and myeloma cells. This measurement and consequently interpretation of the regulatory mechanisms based thereon is subject to at least four major problems (i) the requirement for stringent specificity and sensitivity in the immunoassays employed, (ii) the presence and subsequent fate in culture of preformed cell associated IgE,' 2 (iii) the possible instability of IgE in some cultures attributed to active degradation or sequestration and (iv) the presence in culture of substances such as the various IgE binding factors3 and naturally occurring auto IgG anti-IgE antibodies4.5 which by complexing with IgE may mask accurate quantitation. These problems may account at least in part for the controversy surrounding reports of the effect of regulatory factors on both spontaneous and induced IgE synthesis. For example, pokeweed mitogen (PWM) has been reported to stimulate,6 8 inhibit2.9 or to have no effect'" I on IgE synthesis by PBL in vitro. More recently, IL-4 has been reported to stimulate IgE synthesis although the data have been inconsistent in terms of isotypic specificity and quantity of the antibody produced
complexes. MATERIALS AND METHODS Mononuclear cell lines The human mononuclear cell lines U266, HMY-2 and XQ- 15 spontaneously secrete the immunoglobulins IgE, IgG and 1gM respectively. U266 was established in 1970 by Nilsson et al.'6 from patient ND, the IgGl secreting cell line HMY-2 from a patient with plasma cell leukaemia'7 and XQ15 was kindly provided by Dr U. Kees of Western Australian Research Institute of Child Health. All cell lines, which were established free from mycoplasma by Dr G. Harnett, State Health Laboratories, were maintained in RPMI containing 10% FCS with 2 mM L-glutamine. The medium used for XQ15 cells was also supplemented with 50 pM 2-mercaptoethanol.
Correspondence: Professor K. J. Turner, Department of Microbiology, University of Western Australia, QEII Medical Centre, Nedlands, WA 6009, Australia.
703
704
K. J. Turner et al.
Peripheral blood leucocyte (PBL) cultures PBL were isolated from 40 ml blood, donated by atopic and control patients, by centrifugation on Ficoll-Paque. 1 x 106 cells were established at 370 for 6 days in a humidified atmosphere of 5% CO2 in 1 ml Iscove's Modified Dulbecco's medium (IMDM) supplemented with 10% FCS, 0-5% BSA, 50 pg/ml human transferrin, 5 pg/ml each of oleic, linoleic and palmitic acids.'5 Stimulated cultures contained 100 U/ml recombinant IL-4 (Sandoz Ltd, Basel, Switzerland) or 10 ,ul/ml pokeweed mitogen (PWM-Gibco, Grand Island, NY). These levels of IL-4 and PWM were chosen as optimal on an empirical basis. At the conclusion of culture the cells were removed by centrifugation at 400 g for 5 min and the supernatant, together with acid extracts of the cells prior to and at the conclusion of culture2 stored at -20C until assayed for IgE content. Immunoglobulin assays IgE content of supernatants and cell extracts was determined by a double radioimmune assay8 to provide the derivation net IgE = (IgE supernatant + IgE in cells at conclusion of culture) - (IgE in cells at commencement of culture). A positive figure for net IgE indicates actual synthesis, while a negative value indicates loss during the culture period. IgG in culture supernatants was assayed according to the method of Platts-Mills and Ishizakal8 and was sensitive to 20 ng IgG/ml. IgG-IgE complexes in culture supernatants were assayed by absorption onto protein A Sepharose followed by detection with 1251 anti-IgE as described by Inganais et al.5 Two hundred microlitres of protein A Sepharose (Pharmacia) was added to 200 pl of serum or tissue culture supernatants and incubated for 30 min whilst shaking at room temperature. After washing three times by centrifugation with cold saline containing 0-3% tween 20, 200 yl of normal rabbit serum was added to block unoccupied protein A sites. Following a further 30-min incubation at room temperature the gel was washed as before and incubated for 1 hr whilst shaking at room temperature with 50 pl. 1251 mouse mAb to human IgE (2-1-5) diluted in 20%/, normal mouse serum. After extensive washing the radioactivity bound to the gels was determined in a Beckman Gamma Counter. IgE binding factors (IgE-BF) IgE binding factors (IgE-BF) were prepared by culturing T cells, prepared by rosetting PBLs from atopic patients, at a density of 6 x 105 cells per ml. RPMI+10% FCS supplemented with HEPES for 18 hr at 37°. The cell-free supernatant was then fractionated by centrifugation through an Amicon centriflo membrane followed by pressure dialysis through Sartorius collodian membranes to retain a fraction with a molecular weight between 12 and 50,000 daltons. This was incubated by rotation for 18 hr at 4° with an IgE-Sepharose complex prepared from WT IgE myeloma. The gel was then washed and the IgE-BF eluted by rapid exposure to 0-1 M glycine/HCl buffer pH 2 2 followed by dialysis and concentration in RPMI 10% FCS to a final volume of 5 ml. This IgE-BF, obtained by overnight culture of 1-3 x 107 T cells, was assigned an arbitrary titre of 10,000 U/ml. The capacity of IgE-BF to inhibit quantitation of IgE by the double antibody radioimmunoassay was measured by prior incubation of 100 yl IgE-BF for 1 hr at room temperature, with 100 pl 2-0 ng/ml IgE. A CNBr activated paper disc coupled to monoclonal anti-IgE was then added and
shaken for 3 hr at room temperature, then 18 hr at 40. After washing, the discs were incubated overnight at room temperature with polyvalent 1251 anti-IgE, washed and counted. Production of oligonucleotides Synthetic oligonucleotide sequences directed against IgE and IgG mRNAs were designed by analysing the published sequence data in the Gen Bank Version 63 computer database. The probe sequence PJE-2 was chosen from the CH92 domain of the E chain of IgE and PJGl-1 from the CHy, domain of IgG. The sequences for these oligonucleotides are: 5'-AGG GTG AGC TCG CTT TGT GT-3' PJE-2 PJG1-1 5'-GCT CTG CCC CCA GAG GTG CR-3'
Comparison with entries in the Gen Bank nucleic acid database showed that y and p chain genetic sequences had greater than fine mismatches over the lengths of the E specific probe. The oligonucleotides were synthesized on an Applied Biosystems Model 380-B DNA Synthesizer (Applied Biosystems Inc., U.S.A.) The two oligonucleotides migrated at similar rates on polyacrylamide electrophoresis in a Protean II (Bio Rad) System confirming similarity in size with no underlength fragments representing breakdown products. The probes were labelled with [y32P]-adenosine triphosphate using T4 polynucleotide kinase (PNK) to a specific activity of 8 x 108 c.p.m./Pg. RNA extraction and hybridization Total RNA was extracted from the three mononuclear cell lines and from PBL by the acid guadinium iso thiocyanate/phenol/ chloroform (AGPC) extraction procedure. The RNA preparations were heat denatured at 95' for 5 min followed by quenching on ice and immobilized on Zeta-Probe Membranes (Bio-Rad, Richmond, CA). Hybridization with the mRNA was carried out as recommended by the manufacturers of ZetaProbe, but modified with respect to the hybridization and washing temperatures which were 37 and 42° respectively. Following hybridization the membranes were washed, dried, mounted on cardboard, placed in an X-ray film cassette with Fuji RX-100 X-ray film plus intensifying screen, exposed at - 70c for periods of 2-10 days and scanned on a Bio-Rad Model 620 densitometer to quantitate intensity of the signals.
RESULTS Hybridization under the conditions described here using the PJE-2 probe resulted in intense signals directed against RNA preparations from U266 (IgE) cells while no signals were seen against RNA extracted from HMY-2 (IgG) and XQ- 15 (IgM) cells (Fig. 1). The intensity of the signals correlated (r=0 92) with the U266 RNA content, the limit of detection being less than 0 1 pg RNA. The oligonucleotide probe PJG 1-I hybridized only with RNA extracted from the IgG producing cell line HMY-2 (data not shown). RNA was extracted from the PBLs at the conclusion of 6 days in culture from five unselected samples and hybridized against the oligonucleotide probe PJE-2. Five micrograms U266 RNA and 5 pg Escherichia coli mRNA were blotted as controls resulting in an intense signal directed against the IgE secreting cell U266 RNA but no signal against E.coli RNA. IgE specific mRNA was detected in all RNA samples extracted from PBL of
IgE expression at mRNA and protein levels in vitro
705
Table 1. Interteukin 4 (IL-4) and pokeweed mitogen (PWM) regulation of IgE synthesis in vitro by human peripheral blood leucocytes (PBL) Nett IgE in vitro (pg/ml)
Serum IgE
Patient
Figure 1. Autoradiograph of membranes blotted with 2-0,1 -0 and 0 1 Pg RNA extracted from the cell lines U266 (IgE), HMY-2 (IgG) and XQ 15 (IgM) hybridized at 37° against the oligonucleotide probe PJE-2.
(U/ml)
Unstimulated
IL-4 stimulated
PWM stimulated
640 490 320 230 120 54 15 5
435+95 295+35 135 + 70 60+20 75 + 25 < 40
Comparison of IgE expression at the mRNA and protein levels in vitro K. J. TURNER, J. CREANY, R. J. COELEN, K. J. CAMERON, B. J. HOLT & M. W. BEILHARZ Department of Microbiology, University of Western Australia, QEII Medical Centre, Australia Acceptedfor publication 2 August 1991
SUMMARY The regulating effects of IL-4 and pokeweed mitogen on IgE synthesis in vitro by human peripheral blood leucocytes has been compared with the corresponding effect of these regulators on the expression of IgE mRNA. The latter was measured by dot blot hybridization with an oligonucleotide coding for a unique six amino acid region of the CHe2 domain. Specificity of the oligonucleotide probe was established by its inability to hybridize with RNA extracted from HMY-2 (IgG) and XQ15 (IgM) secreting cell lines whilst producing intense signals with RNA extracted from the IgE secreting cell line U266. Whilst IgE mRNA was detected in RNA extracted from PBL of both atopic and control subjects, spontaneous IgE synthesis was restricted to atopic PBL. IL-4 increased both IgE mRNA and IgE synthesis in all PBL samples but PWM, while significantly increasing IgE mRNA expression either failed to modify IgE synthesis or actively suppressed it. The assay system employed to quantitate IgE synthesis in vitro was shown to be inhibited by both IgE binding factors and IgG anti-IgE autoantibodies which are produced in PBL cultures. IgE mRNA levels might therefore more accurately monitor the regulatory effects of IL-4 and PWM on IgE synthesis than quantitation of the IgE by radioimmunoassay. and of the relative effect of IL-4 on cells from atopic and nonatopic donors.'2 15 This study was motivated by the need to provide an alternative procedure for investigating IgE regulation based on measurement of IgE mRNA levels by dot blot hybridization with an oligonucleotide coding for a unique six amino acid region of the CHa2 domain of the £ chain in parallel with quantitation of synthesized IgE by radioimmunoassay. IgE mRNA was identified in RNA extracted from PBL of all donors both atopic and control. IL-4 increased both IgE mRNA and IgE protein synthesis but PWM, while significantly increasing IgE mRNA expression, failed to induce IgE protein synthesis as measured by radioimmunoassay. It is likely that this inability to assay induced IgE synthesis is due to the formation of immune
INTRODUCTION
The in vitro models employed to investigate mechanisms regulating the human IgE antibody response require the accurate measurement of the very low levels of this immunoglobulin synthesized by culture of peripheral blood leucocytes (PBL), tonsils or other lymphoid tissue and myeloma cells. This measurement and consequently interpretation of the regulatory mechanisms based thereon is subject to at least four major problems (i) the requirement for stringent specificity and sensitivity in the immunoassays employed, (ii) the presence and subsequent fate in culture of preformed cell associated IgE,' 2 (iii) the possible instability of IgE in some cultures attributed to active degradation or sequestration and (iv) the presence in culture of substances such as the various IgE binding factors3 and naturally occurring auto IgG anti-IgE antibodies4.5 which by complexing with IgE may mask accurate quantitation. These problems may account at least in part for the controversy surrounding reports of the effect of regulatory factors on both spontaneous and induced IgE synthesis. For example, pokeweed mitogen (PWM) has been reported to stimulate,6 8 inhibit2.9 or to have no effect'" I on IgE synthesis by PBL in vitro. More recently, IL-4 has been reported to stimulate IgE synthesis although the data have been inconsistent in terms of isotypic specificity and quantity of the antibody produced
complexes. MATERIALS AND METHODS Mononuclear cell lines The human mononuclear cell lines U266, HMY-2 and XQ- 15 spontaneously secrete the immunoglobulins IgE, IgG and 1gM respectively. U266 was established in 1970 by Nilsson et al.'6 from patient ND, the IgGl secreting cell line HMY-2 from a patient with plasma cell leukaemia'7 and XQ15 was kindly provided by Dr U. Kees of Western Australian Research Institute of Child Health. All cell lines, which were established free from mycoplasma by Dr G. Harnett, State Health Laboratories, were maintained in RPMI containing 10% FCS with 2 mM L-glutamine. The medium used for XQ15 cells was also supplemented with 50 pM 2-mercaptoethanol.
Correspondence: Professor K. J. Turner, Department of Microbiology, University of Western Australia, QEII Medical Centre, Nedlands, WA 6009, Australia.
703
704
K. J. Turner et al.
Peripheral blood leucocyte (PBL) cultures PBL were isolated from 40 ml blood, donated by atopic and control patients, by centrifugation on Ficoll-Paque. 1 x 106 cells were established at 370 for 6 days in a humidified atmosphere of 5% CO2 in 1 ml Iscove's Modified Dulbecco's medium (IMDM) supplemented with 10% FCS, 0-5% BSA, 50 pg/ml human transferrin, 5 pg/ml each of oleic, linoleic and palmitic acids.'5 Stimulated cultures contained 100 U/ml recombinant IL-4 (Sandoz Ltd, Basel, Switzerland) or 10 ,ul/ml pokeweed mitogen (PWM-Gibco, Grand Island, NY). These levels of IL-4 and PWM were chosen as optimal on an empirical basis. At the conclusion of culture the cells were removed by centrifugation at 400 g for 5 min and the supernatant, together with acid extracts of the cells prior to and at the conclusion of culture2 stored at -20C until assayed for IgE content. Immunoglobulin assays IgE content of supernatants and cell extracts was determined by a double radioimmune assay8 to provide the derivation net IgE = (IgE supernatant + IgE in cells at conclusion of culture) - (IgE in cells at commencement of culture). A positive figure for net IgE indicates actual synthesis, while a negative value indicates loss during the culture period. IgG in culture supernatants was assayed according to the method of Platts-Mills and Ishizakal8 and was sensitive to 20 ng IgG/ml. IgG-IgE complexes in culture supernatants were assayed by absorption onto protein A Sepharose followed by detection with 1251 anti-IgE as described by Inganais et al.5 Two hundred microlitres of protein A Sepharose (Pharmacia) was added to 200 pl of serum or tissue culture supernatants and incubated for 30 min whilst shaking at room temperature. After washing three times by centrifugation with cold saline containing 0-3% tween 20, 200 yl of normal rabbit serum was added to block unoccupied protein A sites. Following a further 30-min incubation at room temperature the gel was washed as before and incubated for 1 hr whilst shaking at room temperature with 50 pl. 1251 mouse mAb to human IgE (2-1-5) diluted in 20%/, normal mouse serum. After extensive washing the radioactivity bound to the gels was determined in a Beckman Gamma Counter. IgE binding factors (IgE-BF) IgE binding factors (IgE-BF) were prepared by culturing T cells, prepared by rosetting PBLs from atopic patients, at a density of 6 x 105 cells per ml. RPMI+10% FCS supplemented with HEPES for 18 hr at 37°. The cell-free supernatant was then fractionated by centrifugation through an Amicon centriflo membrane followed by pressure dialysis through Sartorius collodian membranes to retain a fraction with a molecular weight between 12 and 50,000 daltons. This was incubated by rotation for 18 hr at 4° with an IgE-Sepharose complex prepared from WT IgE myeloma. The gel was then washed and the IgE-BF eluted by rapid exposure to 0-1 M glycine/HCl buffer pH 2 2 followed by dialysis and concentration in RPMI 10% FCS to a final volume of 5 ml. This IgE-BF, obtained by overnight culture of 1-3 x 107 T cells, was assigned an arbitrary titre of 10,000 U/ml. The capacity of IgE-BF to inhibit quantitation of IgE by the double antibody radioimmunoassay was measured by prior incubation of 100 yl IgE-BF for 1 hr at room temperature, with 100 pl 2-0 ng/ml IgE. A CNBr activated paper disc coupled to monoclonal anti-IgE was then added and
shaken for 3 hr at room temperature, then 18 hr at 40. After washing, the discs were incubated overnight at room temperature with polyvalent 1251 anti-IgE, washed and counted. Production of oligonucleotides Synthetic oligonucleotide sequences directed against IgE and IgG mRNAs were designed by analysing the published sequence data in the Gen Bank Version 63 computer database. The probe sequence PJE-2 was chosen from the CH92 domain of the E chain of IgE and PJGl-1 from the CHy, domain of IgG. The sequences for these oligonucleotides are: 5'-AGG GTG AGC TCG CTT TGT GT-3' PJE-2 PJG1-1 5'-GCT CTG CCC CCA GAG GTG CR-3'
Comparison with entries in the Gen Bank nucleic acid database showed that y and p chain genetic sequences had greater than fine mismatches over the lengths of the E specific probe. The oligonucleotides were synthesized on an Applied Biosystems Model 380-B DNA Synthesizer (Applied Biosystems Inc., U.S.A.) The two oligonucleotides migrated at similar rates on polyacrylamide electrophoresis in a Protean II (Bio Rad) System confirming similarity in size with no underlength fragments representing breakdown products. The probes were labelled with [y32P]-adenosine triphosphate using T4 polynucleotide kinase (PNK) to a specific activity of 8 x 108 c.p.m./Pg. RNA extraction and hybridization Total RNA was extracted from the three mononuclear cell lines and from PBL by the acid guadinium iso thiocyanate/phenol/ chloroform (AGPC) extraction procedure. The RNA preparations were heat denatured at 95' for 5 min followed by quenching on ice and immobilized on Zeta-Probe Membranes (Bio-Rad, Richmond, CA). Hybridization with the mRNA was carried out as recommended by the manufacturers of ZetaProbe, but modified with respect to the hybridization and washing temperatures which were 37 and 42° respectively. Following hybridization the membranes were washed, dried, mounted on cardboard, placed in an X-ray film cassette with Fuji RX-100 X-ray film plus intensifying screen, exposed at - 70c for periods of 2-10 days and scanned on a Bio-Rad Model 620 densitometer to quantitate intensity of the signals.
RESULTS Hybridization under the conditions described here using the PJE-2 probe resulted in intense signals directed against RNA preparations from U266 (IgE) cells while no signals were seen against RNA extracted from HMY-2 (IgG) and XQ- 15 (IgM) cells (Fig. 1). The intensity of the signals correlated (r=0 92) with the U266 RNA content, the limit of detection being less than 0 1 pg RNA. The oligonucleotide probe PJG 1-I hybridized only with RNA extracted from the IgG producing cell line HMY-2 (data not shown). RNA was extracted from the PBLs at the conclusion of 6 days in culture from five unselected samples and hybridized against the oligonucleotide probe PJE-2. Five micrograms U266 RNA and 5 pg Escherichia coli mRNA were blotted as controls resulting in an intense signal directed against the IgE secreting cell U266 RNA but no signal against E.coli RNA. IgE specific mRNA was detected in all RNA samples extracted from PBL of
IgE expression at mRNA and protein levels in vitro
705
Table 1. Interteukin 4 (IL-4) and pokeweed mitogen (PWM) regulation of IgE synthesis in vitro by human peripheral blood leucocytes (PBL) Nett IgE in vitro (pg/ml)
Serum IgE
Patient
Figure 1. Autoradiograph of membranes blotted with 2-0,1 -0 and 0 1 Pg RNA extracted from the cell lines U266 (IgE), HMY-2 (IgG) and XQ 15 (IgM) hybridized at 37° against the oligonucleotide probe PJE-2.
(U/ml)
Unstimulated
IL-4 stimulated
PWM stimulated
640 490 320 230 120 54 15 5
435+95 295+35 135 + 70 60+20 75 + 25 < 40
