Journal of Immunological Methods, 130 (1990) 9-13
9
Elsevier JIM05574
The determination of IgE synthesis in vitro Methodological aspects Amiran Gamkrelidze
*
and Bengt Bjt~rkst6n
Department of Pediatrics, Faculty of Health Sciences, University Hospital, S-581 85 Linki~ping, Sweden
(Received 25 July 1989, revised received 23 January 1990, accepted 29 January 1990)
Various methods to determine net synthesis of IgE in cell cultures and to differentiate this from preformed IgE have been evaluated. The most accurate values for net synthesis were obtained by adding the IgE levels in supernatants to the level of cell-bound acid extractable IgE at the end of the culture period and subtracting the IgE levels present in cultures subject to acid treatment on day 0. Key words: lgE synthesis; lgE, preformed; Acid extraction
Introduction Over the past few years several research groups have evaluated IgE formation in vitro by blood mononuclear cells (MNC) from atopic and nonatopic subjects (Sampson and Buckley, 1981; H e m a d y et al., 1983). These in vitro models are potentially important tools for the study of IgE regulatory mechanisms in humans. The results of these studies are, however, equivocal, at least in
Correspondence to: B. Bj~rkst6n, Department of Pediatrics, Faculty of Health Sciences, University Hospital, S-581 85 LinkiSping, Sweden. * Present address: Department of Allergology, State Medical Institute, Vaja Pshavela av. 33, 380077 Tbilisi. Georgia, U.S.S.R. Abbreviations: AC7, cell-peUet treated with acid on day 7 and then harvested; ACO, cell cultures treated with acid on day 0 and then harvested; ACO7, cell cultures treated with acid on day 0 and harvested on day 7; C7, cell cultures harvested on day 7 by centrifugation; CY, cell cultures treated with cycloheximideon day 0 and harvested on day 7; FTO, cell cultures frozen and thawed five times on day 0 and then harvested; FTO7, cell cultures frozen and thawed five times on day 0 and harvested on day 7; MNC, mononuclear cells; PRIST, paper radioimmunosorbent test.
part because of methodological problems. There are several possible explanations for the technical problems, including the specificity of the anti-IgE antibody used in the assay and poor discrimination between preformed and de novo synthesized IgE (Massicot and Ishizaka, 1986). Methods for the discrimination between preformed and de novo synthesized IgE include repeated freezing and thawing of cell pellets to extract all preformed IgE (Sampson and Buckley, 1982), use of the protein synthesis inhibitor cycloheximide (Knutti-Miiller, 1986) and acid treatment (Turner et al., 1983). We have compared various methods for the evaluation of de novo (net) synthesis of IgE in cell cultures and the discrimination of this from preformed IgE. Materials and methods Cell cultures
Heparinized venous blood was obtained from 33 allergic adolescents with atopic asthma, hay fever a n d / o r eczema, and from nine non-allergic healthy adolescents with no history of allergy. The M N C were isolated by Ficoll-Hypaque (Pharma-
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
10 cia, Uppsala, Sweden) gradient centrifugation of sterile heparinized blood, washed three times in Hanks' balanced salt solution and resuspended in complete medium - RPMI 1640 (Dutch modification, Flow laboratories, U.K.), with 10% heat inactiv~,ed fetal calf serum, penicillin 100 ~ g / m l , streptomycin 5 0 / t m o l / m l and L-glutamine 2 raM. Aliquots of 2 ml cell suspension at a concentration of 1 x 106 were added to 16 mm diameter flat bottom culture wells (Co 3524, Cambridge, MA, U.S.A.) and cultured for 7 days at 3 7 ° C in an incubator containing 5% CO 2. The supernatants were collected from cultures by centrifugation at 700 z g for 10 min and stored at - 2 0 ° C until assayed for IgE content. The viability of the cells was tested by vital staining with trypan blue.
Measurement of IgE protein Immunoglobulin E levels in the culture supernatants were determined by a modification of the Phadebas IgE PRIST assay (Hemady et al., 1983). Briefly, the assay was performed according to the routine procedure, except that all incubations were done overnight with continuous shaking. The antiIgE of the commercial Phadebas RAST kit was used as tracer. A standard curve was obtained by diluting the reference serum with medium to 2 k U / l , corresponding to approximately 4.8 ng I g E / m l and then diluting six times to 2.4, 1.2, 0.6, 0.24, 0.12 and 0.06 ng/ml. Complete culture medium was used as diluent for the standards. The detection limit of this modified procedure was always less than 100 pg I g E / m l and usually around 60 pg/ml. Several methods for the differentiation between preformed IgE and de novo synthesized IgE in the cultures were compared. (I) Net IgE synthesis calculated as the IgE level in the 7 day cultures (C7), minus IgE levels in cultures subject to freezing and thawing ( - 70 o C, + 37 o C) on day 0 (FTO). (II) Determination of preformed IgE as described by Sampson and Buckley (1981). In this procedure control cultures were freeze-thawed five times on day 0 and then incubated for 7 days under normal culture conditions (FTO7). The net IgE synthesis was calculated as IgE in the C7 cultures, minus the IgE levels in the FTO7 cultures.
(III) Cultures with and without cycloheximide at a concentration of 50-100 /xmol/ml (Sigma Chemical Company, St. Louis, MO, U.S.A.). The net IgE synthesis was calculated as the IgE levels in the untreated C7 cultures, minus the IgE in cycloheximide treated cultures on day 7 (CY). (IV) Acid treatment of cultures on day 0 (ACO) as described by Turner et al. (1983). Cell pellets were adjusted to pH 2.0-2.5 with 1 M HCI, thoroughly mixed by vortexing and then the pH was rapidly restored to 7.4 with NaOH. IgE synthesis was calculated as the IgE levels in the C7 cultures, minus the IgE levels in acid treated ACO cultures. (V) Calculation of de novo synthesis according to the formula described by Turner et al. (1983), i.e., net IgE synthesis = C7 + AC7 - ACO, with AC7 corresponding to acid treatment of cell pellets after 7 days cultivation.
Statistical methods Most cell cultures and all IgE determinations were performed in duplicate. The mean values were then used for the calculations. Student's t test and the Wilcoxon two-sample test, as well as the sign test were used for statistical analysis. Results obtained with t tests were confirmed by Wilcoxon non-parametric tests for all groups in which the numbers in the compared groups were small, i.e., less than 10.
Results
A comparison of the levels of preformed IgE in cultures that were freeze-~awed five times and then incubated for 7 days (method II), and in cycloheximide-treated cultures (method Ill), revealed that the results were influenced by the number of cells in the control cultures surviving the freeze-thawing. Thus, in cultures in which the number of MNC was less than 0.2 x 106/ml, the amount of preformed IgE was 559-1238 p g / m l (mean and mean + SD), while the corresponding figures for method IIl were 545-1065 pg/ml. The corresponding figures for synthesized IgE were 836-1908 and 850-1973 pg/ml. The correlation coefficient for preformed IgE was r = 0.914 and for de novo synthesis r = 0.965. However, in freeze-thawed cultures in which there were higher
11
numbers of surviving MNC, the correlation with CY-treated cultures was less; 1297-2626 pg/ml of preformed IgE in the former and 561-1007 (r = 0.481) in the latter. For de novo synthesis the levels were 836-1908 and 850-1973 pg/ml (r = 0.646). Consequently, freeze-thawing could result in overestimation of preformed IgE and underestimation of net IgE in vitro in cultures with surviving MNC. The fact that cells sometimes survived after repeated freezing and thawing was confirmed by trypan blue dying. The IgE levels in CY cultures was similar for cycloheximide concentrations of 50 and 100 # g / m l with an r value of 0.940. The inter-assay reproducibility for 4-8 parallel cycloheximide-treated cultures was 0.956-0.996 in 15 experiments using various donors. Although the drug blocks protein synthesis, another 50% of preformed IgE could be released from the cell-pellets of CY-treated cultures on day 7 with acid treatment. As shown in Table I the IgE levels in acid treated controls were roughly equal to the IgE levels in CY supernatants plus the amounts that were acid extracted from the cell pellets of CY cultures. The correlation coefficient was 0.932. Determination of preformed IgE with acid treatment consistently yielded higher levels than
the cycloheximide-treated MNC cultures (P < 0.01 and < 0.05 as compared to methods IV and V respectively). Furthermore, 20 out of 21 MNC cultures from allergic donors yielded higher total IgE levels after acid treatment as compared to cycloheximide treated cultures (P < 0.01, sign test). Calculation of de novo synthesis of IgE by subtracting lgE present in acid treated cultures on day 0 from the levels in acid treated controls on day 7 can result in underestimation of de novo synthesis, since there was always some (104-522 pg/ml; GM + SD) measurable cell-associated IgE in the cell pellet after 7 days' incubation and removal of the supernatant (data not shown). To evaluate the possibility that cells in some cultures could release preformed IgE even after acid extraction, similar to the observation in freeze-thawed cultures, cells from 12 allergic donors were acid treated on day 0 and then cultured for 7 days in another set of experiments. The amount of IgE in the supernatants from these cultures was not significantly higher than in ACO cultures, as judged by a sign test. Preformed and de novo synthesized IgE in cell cultures from 15 allergic individuals was assessed by the five methods in parallel. As shown in Fig. 1, merely freezethawing the controls on day 7 resulted in a great
TABLE 1 THE RELATIONSHIP BETWEEN CYCLOHEXIMIDE CONTROLS (CY), CY-TREATED CULTURES SUBJECT TO ACID TREATMENT OF CELL PELLET ON DAY 7 (CY/AC7) AND ACID TREATED CONTROLS (ACO) IN TEN EXPERIMENTS WITH CELLS FROM ALLERGIC INDIVIDUALS The values are expressed in p g / m l
Experiment 1 2 3 4 5 6 7 8 9 10 Geom. mean SD
Culture
A CY
B CY/AC7
A+B
ACO
450 120 950 3,100 2,050 1,500 4,000 1,200 255 875
420 60 340 1,050 340 750 2,000 825 305 200
450 110 135 625 260 440 675 60 270 240
860 170 475 1,675 600 1,190 2,675 885 575 440
850 200 675 2,350 975 1,200 2,600 500 762 550
937 312-2,818
432 163-1,146
257 117-584
741 344-1,574
839 337-1,774
12 of Ig[ I00
°°1 oo] 40
20
II III IV v Method Fig. 1. Determination of preformed IgE and de novo synthesis in vitro comparing several methods in 15 experiments with cells from allergic patients. The amount of preformed IgE is given as mean percentage of the total amount ol IgE in the cultures after 7 days in the respective experiments. Methods I-V are described in detail in the materials and methods section. Briefly, preformed IgE was determinated in control cultures subject to freeze thawing five times (I, 11), cycloheximide treatment (111)or acid treatment (IV, V) I
overestimate of IgE synthesis. Falsely high levels of IgE synthesis were also seen in cycloheximide treated cultures.
Discussion
Many technical problems have to be considered when IgE antibody synthesis is assessed, including individual variations among cell donors and the specificity of the anti-IgE antibody used in the test. The source of IgE present in the cell cultures may also be difficult to assess accurately. In particular, net IgE synthesis in vitro can be influenced by preformed IgE present in the cells already at the time of initiation of cell culture. Freeze-thawing of cell cultures on day 0 has been recommended as a measure of preformed IgE (Sampson and Buckley, 1981). However, even after properly preformed freeze-thawing five times some M N C may survive. If these M N C have the capacity to produce IgE in vitro, then the amount of IgE present in the cultures after 7 days of incuba-
tion will not exclusively represent preformed IgE, but also a certain net synthesis. This in turn means that the true IgE synthesis in the test cultures will be underestimated. Thus, freezethawing on day 0 and then incubating for 7 days as a means of determining the amount of preformed IgE will be valid only when the number of cells surviving the procedure is low. In our hands only freeze-thawed cultures with less than 0.2 × 106 suriving cells/ml truly revealed the amount of preformed IgE. Treatment of the cultures with cycloheximide blocks protein synthesis. However, the demonstration of additional lgE in cell pellets of cycloheximide-treated cultures that could be acid extracted after 7 days indicates incomplete release of preformed IgE from the cells in the cultures. As a consequence, the use of cycloheximide-treated cultures as a means to determine preformed IgE will result in falsely low levels of preformed IgE and the amount of newly formed IgE will consequently be overestimated. Calculation of de novo synthesis merely by subtracting the levels in acid treated cells on day 0 from the total lgE levels in 7 days cultures would underestimate the true net synthesis, since some IgE remains tightly cell-bound even after culture (Turner et al., 1983; Knutti-Miiller et ai., 1986). As a consequence, the formula suggested by Turner et al. would give the most accurate values for net synthesis of IgE in vitro. In conclusion, the methodology for the determination of IgE production in vitro must be carefully defined. The most reliable method to calculate net synthesis seems to be to determine the levels in supernatants plus cell-bound acid extractable IgE in cultures after completion of the culture period and to subtract the IgE levels in cultures subject to acid treatment on day 0.
Acknowledgements The technical assistance of Ms. Kerstin Hagersten and Mrs. Lisbeth Hj~ille is gratefully acknowledged. The work was supported by a grant from the Swedish Medical Research Council (no. 7510). Dr.
13
Gamkrelidze had a scholarship from the Swedish Institute.
References Hemady, Z., Blomberg, F., Gellis, S. and Rocklin, R.E. (1983) IgE production in vitro by human blood mononuclear cells: a comparison between atopic and non-atopic subjects. J. Allergy Clin. lmmunol. 71, 324. Knutti-Mfiller, J.M., Stadler, B.M., Magnusson, G.M. and De
Weck, L. (1986) Human lgE-synthesis in vitro. Allergy 41, 457. Massicot, J.G. and Ishizaka, K. (1986) Workshop on measurement of in vitro IgE-synthesis and regulation of lgE°synthesis. J. Allergy Clin. Immunol. 77, 544. Sampson, H. and Buckley, R.H. (1981) Human lgE-synthesis in vitro: a reassessment. J. lmmunol. 127, 829. Turner, K.J., Holt, P.G., Holt, B.J. and Cameron, K.J. (1983) In vitro synthesis of IgE by human peripheral blood leukocytes. Ill. Release of a pre-formed antibody. Clin. Exp. Immunol. 51,387.
9
Elsevier JIM05574
The determination of IgE synthesis in vitro Methodological aspects Amiran Gamkrelidze
*
and Bengt Bjt~rkst6n
Department of Pediatrics, Faculty of Health Sciences, University Hospital, S-581 85 Linki~ping, Sweden
(Received 25 July 1989, revised received 23 January 1990, accepted 29 January 1990)
Various methods to determine net synthesis of IgE in cell cultures and to differentiate this from preformed IgE have been evaluated. The most accurate values for net synthesis were obtained by adding the IgE levels in supernatants to the level of cell-bound acid extractable IgE at the end of the culture period and subtracting the IgE levels present in cultures subject to acid treatment on day 0. Key words: lgE synthesis; lgE, preformed; Acid extraction
Introduction Over the past few years several research groups have evaluated IgE formation in vitro by blood mononuclear cells (MNC) from atopic and nonatopic subjects (Sampson and Buckley, 1981; H e m a d y et al., 1983). These in vitro models are potentially important tools for the study of IgE regulatory mechanisms in humans. The results of these studies are, however, equivocal, at least in
Correspondence to: B. Bj~rkst6n, Department of Pediatrics, Faculty of Health Sciences, University Hospital, S-581 85 LinkiSping, Sweden. * Present address: Department of Allergology, State Medical Institute, Vaja Pshavela av. 33, 380077 Tbilisi. Georgia, U.S.S.R. Abbreviations: AC7, cell-peUet treated with acid on day 7 and then harvested; ACO, cell cultures treated with acid on day 0 and then harvested; ACO7, cell cultures treated with acid on day 0 and harvested on day 7; C7, cell cultures harvested on day 7 by centrifugation; CY, cell cultures treated with cycloheximideon day 0 and harvested on day 7; FTO, cell cultures frozen and thawed five times on day 0 and then harvested; FTO7, cell cultures frozen and thawed five times on day 0 and harvested on day 7; MNC, mononuclear cells; PRIST, paper radioimmunosorbent test.
part because of methodological problems. There are several possible explanations for the technical problems, including the specificity of the anti-IgE antibody used in the assay and poor discrimination between preformed and de novo synthesized IgE (Massicot and Ishizaka, 1986). Methods for the discrimination between preformed and de novo synthesized IgE include repeated freezing and thawing of cell pellets to extract all preformed IgE (Sampson and Buckley, 1982), use of the protein synthesis inhibitor cycloheximide (Knutti-Miiller, 1986) and acid treatment (Turner et al., 1983). We have compared various methods for the evaluation of de novo (net) synthesis of IgE in cell cultures and the discrimination of this from preformed IgE. Materials and methods Cell cultures
Heparinized venous blood was obtained from 33 allergic adolescents with atopic asthma, hay fever a n d / o r eczema, and from nine non-allergic healthy adolescents with no history of allergy. The M N C were isolated by Ficoll-Hypaque (Pharma-
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
10 cia, Uppsala, Sweden) gradient centrifugation of sterile heparinized blood, washed three times in Hanks' balanced salt solution and resuspended in complete medium - RPMI 1640 (Dutch modification, Flow laboratories, U.K.), with 10% heat inactiv~,ed fetal calf serum, penicillin 100 ~ g / m l , streptomycin 5 0 / t m o l / m l and L-glutamine 2 raM. Aliquots of 2 ml cell suspension at a concentration of 1 x 106 were added to 16 mm diameter flat bottom culture wells (Co 3524, Cambridge, MA, U.S.A.) and cultured for 7 days at 3 7 ° C in an incubator containing 5% CO 2. The supernatants were collected from cultures by centrifugation at 700 z g for 10 min and stored at - 2 0 ° C until assayed for IgE content. The viability of the cells was tested by vital staining with trypan blue.
Measurement of IgE protein Immunoglobulin E levels in the culture supernatants were determined by a modification of the Phadebas IgE PRIST assay (Hemady et al., 1983). Briefly, the assay was performed according to the routine procedure, except that all incubations were done overnight with continuous shaking. The antiIgE of the commercial Phadebas RAST kit was used as tracer. A standard curve was obtained by diluting the reference serum with medium to 2 k U / l , corresponding to approximately 4.8 ng I g E / m l and then diluting six times to 2.4, 1.2, 0.6, 0.24, 0.12 and 0.06 ng/ml. Complete culture medium was used as diluent for the standards. The detection limit of this modified procedure was always less than 100 pg I g E / m l and usually around 60 pg/ml. Several methods for the differentiation between preformed IgE and de novo synthesized IgE in the cultures were compared. (I) Net IgE synthesis calculated as the IgE level in the 7 day cultures (C7), minus IgE levels in cultures subject to freezing and thawing ( - 70 o C, + 37 o C) on day 0 (FTO). (II) Determination of preformed IgE as described by Sampson and Buckley (1981). In this procedure control cultures were freeze-thawed five times on day 0 and then incubated for 7 days under normal culture conditions (FTO7). The net IgE synthesis was calculated as IgE in the C7 cultures, minus the IgE levels in the FTO7 cultures.
(III) Cultures with and without cycloheximide at a concentration of 50-100 /xmol/ml (Sigma Chemical Company, St. Louis, MO, U.S.A.). The net IgE synthesis was calculated as the IgE levels in the untreated C7 cultures, minus the IgE in cycloheximide treated cultures on day 7 (CY). (IV) Acid treatment of cultures on day 0 (ACO) as described by Turner et al. (1983). Cell pellets were adjusted to pH 2.0-2.5 with 1 M HCI, thoroughly mixed by vortexing and then the pH was rapidly restored to 7.4 with NaOH. IgE synthesis was calculated as the IgE levels in the C7 cultures, minus the IgE levels in acid treated ACO cultures. (V) Calculation of de novo synthesis according to the formula described by Turner et al. (1983), i.e., net IgE synthesis = C7 + AC7 - ACO, with AC7 corresponding to acid treatment of cell pellets after 7 days cultivation.
Statistical methods Most cell cultures and all IgE determinations were performed in duplicate. The mean values were then used for the calculations. Student's t test and the Wilcoxon two-sample test, as well as the sign test were used for statistical analysis. Results obtained with t tests were confirmed by Wilcoxon non-parametric tests for all groups in which the numbers in the compared groups were small, i.e., less than 10.
Results
A comparison of the levels of preformed IgE in cultures that were freeze-~awed five times and then incubated for 7 days (method II), and in cycloheximide-treated cultures (method Ill), revealed that the results were influenced by the number of cells in the control cultures surviving the freeze-thawing. Thus, in cultures in which the number of MNC was less than 0.2 x 106/ml, the amount of preformed IgE was 559-1238 p g / m l (mean and mean + SD), while the corresponding figures for method IIl were 545-1065 pg/ml. The corresponding figures for synthesized IgE were 836-1908 and 850-1973 pg/ml. The correlation coefficient for preformed IgE was r = 0.914 and for de novo synthesis r = 0.965. However, in freeze-thawed cultures in which there were higher
11
numbers of surviving MNC, the correlation with CY-treated cultures was less; 1297-2626 pg/ml of preformed IgE in the former and 561-1007 (r = 0.481) in the latter. For de novo synthesis the levels were 836-1908 and 850-1973 pg/ml (r = 0.646). Consequently, freeze-thawing could result in overestimation of preformed IgE and underestimation of net IgE in vitro in cultures with surviving MNC. The fact that cells sometimes survived after repeated freezing and thawing was confirmed by trypan blue dying. The IgE levels in CY cultures was similar for cycloheximide concentrations of 50 and 100 # g / m l with an r value of 0.940. The inter-assay reproducibility for 4-8 parallel cycloheximide-treated cultures was 0.956-0.996 in 15 experiments using various donors. Although the drug blocks protein synthesis, another 50% of preformed IgE could be released from the cell-pellets of CY-treated cultures on day 7 with acid treatment. As shown in Table I the IgE levels in acid treated controls were roughly equal to the IgE levels in CY supernatants plus the amounts that were acid extracted from the cell pellets of CY cultures. The correlation coefficient was 0.932. Determination of preformed IgE with acid treatment consistently yielded higher levels than
the cycloheximide-treated MNC cultures (P < 0.01 and < 0.05 as compared to methods IV and V respectively). Furthermore, 20 out of 21 MNC cultures from allergic donors yielded higher total IgE levels after acid treatment as compared to cycloheximide treated cultures (P < 0.01, sign test). Calculation of de novo synthesis of IgE by subtracting lgE present in acid treated cultures on day 0 from the levels in acid treated controls on day 7 can result in underestimation of de novo synthesis, since there was always some (104-522 pg/ml; GM + SD) measurable cell-associated IgE in the cell pellet after 7 days' incubation and removal of the supernatant (data not shown). To evaluate the possibility that cells in some cultures could release preformed IgE even after acid extraction, similar to the observation in freeze-thawed cultures, cells from 12 allergic donors were acid treated on day 0 and then cultured for 7 days in another set of experiments. The amount of IgE in the supernatants from these cultures was not significantly higher than in ACO cultures, as judged by a sign test. Preformed and de novo synthesized IgE in cell cultures from 15 allergic individuals was assessed by the five methods in parallel. As shown in Fig. 1, merely freezethawing the controls on day 7 resulted in a great
TABLE 1 THE RELATIONSHIP BETWEEN CYCLOHEXIMIDE CONTROLS (CY), CY-TREATED CULTURES SUBJECT TO ACID TREATMENT OF CELL PELLET ON DAY 7 (CY/AC7) AND ACID TREATED CONTROLS (ACO) IN TEN EXPERIMENTS WITH CELLS FROM ALLERGIC INDIVIDUALS The values are expressed in p g / m l
Experiment 1 2 3 4 5 6 7 8 9 10 Geom. mean SD
Culture
A CY
B CY/AC7
A+B
ACO
450 120 950 3,100 2,050 1,500 4,000 1,200 255 875
420 60 340 1,050 340 750 2,000 825 305 200
450 110 135 625 260 440 675 60 270 240
860 170 475 1,675 600 1,190 2,675 885 575 440
850 200 675 2,350 975 1,200 2,600 500 762 550
937 312-2,818
432 163-1,146
257 117-584
741 344-1,574
839 337-1,774
12 of Ig[ I00
°°1 oo] 40
20
II III IV v Method Fig. 1. Determination of preformed IgE and de novo synthesis in vitro comparing several methods in 15 experiments with cells from allergic patients. The amount of preformed IgE is given as mean percentage of the total amount ol IgE in the cultures after 7 days in the respective experiments. Methods I-V are described in detail in the materials and methods section. Briefly, preformed IgE was determinated in control cultures subject to freeze thawing five times (I, 11), cycloheximide treatment (111)or acid treatment (IV, V) I
overestimate of IgE synthesis. Falsely high levels of IgE synthesis were also seen in cycloheximide treated cultures.
Discussion
Many technical problems have to be considered when IgE antibody synthesis is assessed, including individual variations among cell donors and the specificity of the anti-IgE antibody used in the test. The source of IgE present in the cell cultures may also be difficult to assess accurately. In particular, net IgE synthesis in vitro can be influenced by preformed IgE present in the cells already at the time of initiation of cell culture. Freeze-thawing of cell cultures on day 0 has been recommended as a measure of preformed IgE (Sampson and Buckley, 1981). However, even after properly preformed freeze-thawing five times some M N C may survive. If these M N C have the capacity to produce IgE in vitro, then the amount of IgE present in the cultures after 7 days of incuba-
tion will not exclusively represent preformed IgE, but also a certain net synthesis. This in turn means that the true IgE synthesis in the test cultures will be underestimated. Thus, freezethawing on day 0 and then incubating for 7 days as a means of determining the amount of preformed IgE will be valid only when the number of cells surviving the procedure is low. In our hands only freeze-thawed cultures with less than 0.2 × 106 suriving cells/ml truly revealed the amount of preformed IgE. Treatment of the cultures with cycloheximide blocks protein synthesis. However, the demonstration of additional lgE in cell pellets of cycloheximide-treated cultures that could be acid extracted after 7 days indicates incomplete release of preformed IgE from the cells in the cultures. As a consequence, the use of cycloheximide-treated cultures as a means to determine preformed IgE will result in falsely low levels of preformed IgE and the amount of newly formed IgE will consequently be overestimated. Calculation of de novo synthesis merely by subtracting the levels in acid treated cells on day 0 from the total lgE levels in 7 days cultures would underestimate the true net synthesis, since some IgE remains tightly cell-bound even after culture (Turner et al., 1983; Knutti-Miiller et ai., 1986). As a consequence, the formula suggested by Turner et al. would give the most accurate values for net synthesis of IgE in vitro. In conclusion, the methodology for the determination of IgE production in vitro must be carefully defined. The most reliable method to calculate net synthesis seems to be to determine the levels in supernatants plus cell-bound acid extractable IgE in cultures after completion of the culture period and to subtract the IgE levels in cultures subject to acid treatment on day 0.
Acknowledgements The technical assistance of Ms. Kerstin Hagersten and Mrs. Lisbeth Hj~ille is gratefully acknowledged. The work was supported by a grant from the Swedish Medical Research Council (no. 7510). Dr.
13
Gamkrelidze had a scholarship from the Swedish Institute.
References Hemady, Z., Blomberg, F., Gellis, S. and Rocklin, R.E. (1983) IgE production in vitro by human blood mononuclear cells: a comparison between atopic and non-atopic subjects. J. Allergy Clin. lmmunol. 71, 324. Knutti-Mfiller, J.M., Stadler, B.M., Magnusson, G.M. and De
Weck, L. (1986) Human lgE-synthesis in vitro. Allergy 41, 457. Massicot, J.G. and Ishizaka, K. (1986) Workshop on measurement of in vitro IgE-synthesis and regulation of lgE°synthesis. J. Allergy Clin. Immunol. 77, 544. Sampson, H. and Buckley, R.H. (1981) Human lgE-synthesis in vitro: a reassessment. J. lmmunol. 127, 829. Turner, K.J., Holt, P.G., Holt, B.J. and Cameron, K.J. (1983) In vitro synthesis of IgE by human peripheral blood leukocytes. Ill. Release of a pre-formed antibody. Clin. Exp. Immunol. 51,387.
