Stimulation and regulation of human IgE production in vitro using peripheral blood lymphocytes

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

14, 474-488 (4979)

Stimulation and Regulation of Human IgE Production in Vitro Using Peripheral Blood Lymphocytes’ ANDREW

SAXONS

AND RONALD

H. STEVENS

Division of Clinicul Immuno/ogylAl~ergy, Department oj’ Medicinr and DepcrrtmcInt of Microbiology and Immunology, UCLA School of Medicine. Los Angeles. Calijbrnia 90024 Received May II, 1979 The ability of human peripheral blood mononuclear cells (PBL) to produce IgE in vitro was examined. The amount of IgE produced was quantitated using a solid-phase double-antibody radioimmunoassay. While cultures of PBL synthesized IgE without pokeweed mitogen (PWM) stimulation (mean 871 pg per culture), the addition of PWM enhanced IgE synthesis in all cultures from 125 to greater than 617% (mean 2502 pg per culture). IgE was first detectable in culture between Days 3 and 4 and steadily increased thereafter up to Day 7. That this was in vitro synthesized IgE and not cytophilic IgE carried over into the cultures was demonstrated by the fact that IgE production was completely inhibited by addition of cycloheximide to the cultures or irradiation of the B cells. This B-lymphocyte IgE production was dependent on the presence of T lymphocytes. However, in contrast to IgG and IgM, high T/B-cell ratios in culture only modestly inhibited IgE production. Irradiated T lymphocytes provided helper function without suppression of IgE at high T/B-cell ratios. This effect of high numbers of irradiated T lymphocytes on IgE synthesis was distinguishable from their effect on IgG and IgM. There was no correlation between the amount of IgE produced in vitro and the individual’s serum IgE level, nor was there a direct relationship between the amounts of IgE. IgG, or IgM produced within the cultures.

INTRODUCTION

The development of in vitro models analogous to in V~VU aspects of immune system has been responsible for much of the recent progress in immunology. Such in vitro systems permit the manipulation and dissection of the complex interacting events which comprise even simple immune responses (1, 2). This is especially true in the study of human immune reactions where in V~VOstudies are difficult to perform (3). The discovery that human peripheral blood lymphocytes (PBL)” can be stimulated to synthesize IgG, IgM, and IgA in vitro by pokeweed mitogen (PWM) has lead to numerous investigations which have provided insight into the normal (i) T-cell regulation of B cells, (ii) events involved in maturation and differentiation of B cells into immunoglobulin (Ig)-producing cells and, (iii) role of adherent cells in Ig production (4-9). Furthermore, the PWM-driven synthesis of Ig has provided valuable information about the possible pathophysiology of a number of conditions in which Ig production is abnormal (10-13). ’ Supported by USPHS Grants AI 12521. AI 15332, CA 12800. *Dr. A. Saxon is the recipient of an Allergic Diseases Academic Award AI 00326-01 from the National Institute of Allergy and Infectious Diseases. 3 Abbreviations used: PBL. peripheral blood lymphocytes; Ig, immunoglobulin; PWM, pokeweed mitogen; BSA, bovine serum albumin: PBS, phosphate-buffered saline. 474 0090-12291791120474-15$01.00/O Copyright @ 1979 by Academic Press. Inc All rights of reproduction in any form reserved.

HUMAN

IgE

PRODUCTION

IN VITRO

475

Although IgE production and regulation has been extensively studied in rodent systems (reviewed in Refs. 14 and 15), a method for the stimulation and manipulation of human IgE production by normal human lymphocytes has been lacking. In this paper we report the conditions for stimulating and regulating the in vitro synthesis of IgE by human PBL. Lymphocyte

Preparation

MATERIALS AND METHODS and Separation Procedures

Human PBL suspensions were prepared by Ficoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, N.J.) differential sedimentation (16) of heparinized blood obtained from normal volunteers between the ages of 25 and 35 years. Donors with known allergic rhinitis or asthma were not excluded. All donors had a serum IgE level of less than 500 IU/ml. T- and B-lymphocyte fractions were separated by density sedimentation of spontaneous rosettes formed by T lymphocytes and sheep erythrocytes. The procedure was modified in that the sheep erythrocytes had been pretreated with 2-aminoethyl-isothiouronium bromide (17). PBL suspensions contained greater than 95% mononuclear cells with 0.7 ? 0.2% basophils. Separated T-lymphocyte fractions had greater than 90% sheep erythrocyte rosette-forming cells and less than 2% membrane immunoglobulinbearing cells. The B-cell fraction contained 5% or less T cells, 51-66% membrane immunoglobulin-bearing cells, and 23 -34% monocytes as determined by nonspecific esterase staining. These procedures are reported in detail elsewhere (18). Lymphocyte

Cultures

Either unfractionated, fractionated, or fractionated and recombined PBL were cultured in RPM1 1640 medium buffered with NaHCO, and supplemented with L-glutamine (10 mM), gentamicin (0.05 mgml), and 15% heat-inactivated fetal calf serum. All cultures were done in a final volume of 1.5 ml in 13 x 100 mm plastic tubes (Falcon 2027, Falcon Plastics, Division BioQuest, Oxnard, Calif.). The tubes were incubated in a humidified atmosphere at 37°C with 5% CO, for l-9 days. Preliminary experiments with dilutions of PWM (Grand Island Biological Company, Grand Island, N.Y.) from l/25 to l/500 v/v showed a maximum response between l/50 and 11200. Experiments with PWM, therefore, employed a dose of l/100 as it was in the middle of the optimum range. Furthermore, this level had been used in our previous studies of IgG, IgM and IgA in vitro responses and allowed for comparison with those experiments (5). A number of other mitogens (Concanavalin A, lipopolysaccharide, calcium ionophore A 23187, nocardia extract, phytohemagglutinin, oubina, and staphylococcal protein A) were also tested over various concentrations. None stimulated IgE synthesis in vitro. Radioimmunoassays

The quantitative radioimmunoassays for IgE, IgG, and IgM were performed in microtiter plates (19). The exact techniques for the measurement of IgG and IgM have been reported in detail previously and are similar to the assay for IgE except that the specificity (anti-human IgG vs IgM vs IgE) of the primary coating and secondary labeling antibodies were different (13, 20).

476

SAXON

AND

STEVENS

For measurement of IgE produced in culture, the individual wells in microtiter plates were filled with monospecific goat anti-human IgE (Research Products International, Elk Grove Village, Ill., Lot 105) at a concentration of 0.5 mg antibody/ml. After incubation in a humidified chamber at 23°C overnight, the coating antibody solution was removed and saved for reuse. The wells were washed individually three times with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and then 10% BSA in PBS was added to the wells for 1 hr to coat any remaining protein-binding surface. After another wash with 1% BSA in PBS, 300 ~1 of the samples to be assayed were added to individual wells and allowed to incubate overnight in a humidified chamber at 23°C. The following day the samples were removed and discarded. After three more washes with 1% BSA in PBS an IgG fraction of monospecific 1251-labeled (21) goat anti-human IgE (Meloy Laboratories, Springfield, Va.) was added to each well in a final volume of 300 ~1 and allowed to incubate at 23°C for 5 hr in a humidified chamber. Thereafter the plates were washed three times in 1% BSA in PBS and eight times in running tap water. The individual wells were cut apart and the bound radioactivity was determined. For each radioimmunoassay a 7-point standard curve at IgE was performed in parallel with the culture samples on each plate. Generally, each culture sample was run as a single sample but triplicate cultures were performed at each experimental point as we found that the variability between culture tubes (coefficient of variation 14.3% range 5.2-23%) was greater than the variability in the radioimmunoassay, (coefficient of variation 3.9%, range 1.3-7.5%). The IgE standard was a myeloma protein, PS (22), which was purified from serum by DEAE-52 column chromatography. After elution of IgG in 0.005 M borate buffer, pH 8.0, the IgE was eluted in 0.025 M borate buffer. This was then rechromatographed on Sephadex G200 with the protein eluting as one sharp peak. Only the leading half of this peak was used as purified IgE protein. Analysis by radioimmunoassay showed no IgG, IgM, or IgA present and sodium dodecycl sulfate-polyacrylamide gel electrophoresis demonstrated a single protein with a molecular weight of 180,000-200,000 daltons. This IgE protein was standardized in our radioimmunoassay against WHO IgE standard NC1 68/341 (kindly supplied by Dr. Gerald Gleich, Mayo Medical School and Mayo Foundation). This avoided the possibility of anti-idiotypic binding by the Meloy antiserum (raised against protein PS) causing a falsely elevated standard curve. Serum IgE levels were determined using the Prist technique (Pharmacia Fine Chemicals, Piscataway, N.J.). RESULTS Radioimmunoassay

for Human

IgE

The IgE binding capacity of the anti-IgE coated plates was determined by incubating triplicate wells with increasing amounts of purified IgE. Figure 1 demonstrates that increasing quantities of IgE added in the first incubation resulted in a subsequent increased binding of the 1251-anti-IgE second antibody with saturation being reached between 35 and 40 ng of IgE. IgM- and IgG-purified proteins were not detected when up to 5000 ng of purified polyclonal IgG or 1000 ng of IgM were added. As it was necessary to detect small quantities of IgE in the culture supernatants, the linear part of the curve between 0 and 2.0 ng was utilized with the second antibody labeled at 3000-6000 cpm/ng (Fig. 1, insert).

HUMAN

A

A I IO

b

A 20

IgE PRODUCTION

A , 30

477

IA’ VITRO

A

A

40

50

60

4 LA!-+* 701%Y 5000

IMIllUNOGLOBULlN

ng

1. Radioimmunoassay for IgE. Saturation of anti-IgE-coated plates with increasing amounts of human IgE ( 0 ). Assay did not detect human IgG (0) or human IgM (A ). Linearity of assay for IgE at levels up to 2.0 ng is shown in insert. FIG.

The binding of lz51-anti-human IgE to the wells during the second incubation could be inhibited by the addition of purified human IgE but not with IgG or IgM (Fig. 2). Fifty nanograms of IgE were added to each anti-IgE coated well and incubated overnight in order to saturate the wells. Immediately prior to the addition of the radiolabeled anti-IgE, increasing amounts of either IgE or IgG were added to triplicate wells as competitors. Increasing quantities of the IgE competitor gave progressive inhibition of binding of the second antibody to the wells with 50% inhibition being achieved with 124 ng of IgE added. No inhibition of

‘i

, I

LOG

ng

,

,

2

3

COMPETITOR

FIG. 2. Competition by IgE added with the second antibody. Competition of the detection of IgE by either IgE (O), IgG (0). or IgM (A) when added with the second antibody (*251-anti-human IgE) reaction. The 100% binding value was 26,592 cpm for 40 ng of IgE.

478

SAXON

AND

STEVENS

binding was observed when up to 5000 ng of the IgG competitor or 1000 ng of IgM competitor were added. Similar lack of inhibition occurred when IgG or IgM was added to 2 ng of IgE (12,540 cpm). In Vitro IgE Production by Unfractionated PEL and Stimulation by PWh4 Pairs of triplicate cultures of 2 x lo6 unfractionated PBL were initiated in the presence or absence of PWM and harvested on Day 7. In the 42 individuals studied in this manner, all individuals produced greater quantities of IgE when stimulated by PWM (Table 1). The level of this stimulation by PWM was from 125% to greater than 617%. Several individuals (Table 1, Nos. 39-42) were studied on multiple occasions over a 4-month interval (Table 1). The level of TABLE IgE PRODUCED OF POKEWEED

IA VITR(J MITOGEN

IN THE

I PRESENCE

(PICOGRAMS

Individual

- PWM”

+ PWM”

individual

I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

520 1135 830 1630 3100 500 1290 1180 870 540 865 670 940 766 520 640 590 765 625 c 500 585 630 955 c500 710 1070 610 <500 650
1530 4085 4030 2110 3310

IX I9 20 21 22 23 24 25 26 27 28 29 30 31 3’i 33 34 4?2. 42 42

17

35 36 37 38 39 39 39 39 40 40 40 41 41 41 42 42

1590

3650 I460 1760 2050

2770 3620 1870 1125 930 1120 875 2580 2905 1590 311.5 1520

OR ABSENTS PER CULTURE)

Mean

+ SE

by three

identical

- PWM” 1500

925 1415 990 1520 ‘C .(og 1100 835 c-500 540 570 -500 935 1625 -500 500 510 1330 143s 1100 871 5 73

2800 1600 1890 2650 1670 1340 2685 1530 2240 3605 3995

” Each point represents the mean IgE produced * Values less than 500 ng taken as 500 ng.

cultures

+ PWM” 2415 2285 2155 4215 4020 1120 2195 2225 28.55 1370 2160 1945 3265 2550 3085 2090 52ts 2965 3150 2930 2502 f

1%

HUMAN

IgE PRODUCTION TABLE

IgE PRODUCTION B AND Experiment No. 1

2 3 4 5 6 I 8 9 10 11 12 13 Mean

k SE

BY STANDARDIZED T LYMPHOCYTES

479

IN VITRO

2 CULTURES

(0.40.6

IgE produced - PWM <500

625 930 400 560
OF RECOMBINED

x IO6 CELLS) (pgkulture) + PWM 2785 4770 2520 3205 3370 2640 2400 2785 1375 2295 3465 4040 1130 2502 k 158

increased IgE with PWM showed a maximum range of 220-437% for an individual (No. 42) with the absolute IgE level being 2930 to 3995 pg for that individual. Thus, while on each occasion there was greater IgE production with PWM, there was a considerable range observed for any one individual. We further confirmed this stimulation of IgE production in vitro utilizing paired sets of cultures of separated B cells (0.4 x log) to which a standard number (1.6 x 106) of T lymphocytes had been added (Table 2). Again standardized B- and T-lymphocyte cultures with PWM consistently produced greater amounts of IgE. Next we sought to assess whether the amount of IgE produced in vitro by an individual’s PBL correlated with their serum level of IgE. Simultaneous serum IgE levels were determined on individuals whose cultured PBL (2 x 10s) were assayed for IgE production on Day 7 (Fig. 3). There was no observed correlation (r = +0.45). Only two individuals did not produce greater than 500 pg of IgE per culture when stimulated with PWM. Both of these subjects suffer from adult onset hypo-y-globulinemia and have been shown to have defective B-cell IgG, IgM, and IgA production in vitro as well (Ashman, Saxon, and Stevens, unpublished observation). Kinetics of ZgE Production in Vitro The kinetics of the in vitro production of IgE was determined by initiating triplicate cultures of 2 x lo6 unfractionated PBL or B and T (0.411.4 x 106) lymphocytes plus PWM and harvesting them over a Pday time course. Four representative exneriments are shown in Fig. 4. IgE production was first detectable between Days 3 and 4 and reached a maximum on Days 7-9. As a number of individuals showed a plateau or decline on Day 8 or 9, all cultures in other experiments were harvested and assayed for IgE on Day 7. The evidence that PWM

480

SAXON AND STEVENS --------------1

I

l __

. .

.

e

0

2 Ill/ml

I

SERUM

IgE

- ..-,

/------

---

3

c j

4

(xd)

FIG. 3. Relationship between IgE produced in vitro by 2 x 106 unfmctionated PBL and serum IgE.

stimulated IgE production in vitro and the delayed appearance of this IgE in culture both suggested that the IgE detected was synthesized in vitro and was not cytophilic IgE carried over into the cultures. To exclude the possibility that the IgE detected was due to cytophilic Ig, PWM-stimulated cultures of I3 cells and irradiated T lymphocytes (3000 rad) were initiated in the presence and absence of cycloheximide (75 ,ug per culture). This dose of cycloheximide has been previously shown by us to inhibit SO% of the protein synthesis by such cultures (23). The inclusion of cycloheximide decreased the IgE detected to background levels

5 DAYS

IN

6

7-8

9

CULTURE

FIG. 4. Kinetics of IgE production in vitro. Cultures of 2 x lo6 unfractionated PBL (0, 0) or recombined B and T lymphocytes (0.4 x 10611.6 x lo”) (A, X) were harvested on sequential days and assayed for IgE.

HUMAN

IgE PRODUCTION TABLE

IgE PRODUCED IN THE

Experiment

No. I 2 3 4 s 6 7 8 9 10 11 11 11 11 11 11 11

IN CULTURE ABSENCE

AND

Day harvested

7 7 7 7 7 7 7 7 7 7 1

2 3 4 5 6 7

IN

481

VITRO

3

BY B AND

IRRADIATED

PRESENCE

OF CYCLOHEXIMIDE

T LYMPHOCYTES (0.4/1.6 (75 &CULTURE)

(PP) Control

4,370 13,525 3,090 7,975 5,380 3,570 3.870 2,990 8.460 4.770 (500 1500 <500 880 1,250 1,680 2,940

x 106)

Percentage

+ Cyclohexamide

inhibition

<500 840 <500 585 <500 620 <500 <500 <500 <500 <500 <500 <500 <500 <500 <500 c500

>88 93 >54 93 91 83 >87 >83 >94 >90

of

>83

(Table 3). Irradiation (3000 rad) of the B lymphocytes also prevented the appearance of detectable amounts of IgE on Days 1 through 7 (results not shown). Finally 2 x lo6 PBC from each of 10 donors were freeze thawed three times in 0.5 ml of culture medium and the supernatant was assayed for IgE. The mean released IgE was 441 pg with a range of 132-621 pg. T Lymphocyte

Regulation

for IgE Production

in Vitro

The requirement for T lymphocytes in in vitro IgE production was explored by initiating cultures of B-fraction cells (0.4 x 106) plus PWM with or without autochonous T lymphocytes (1.6 x 106). In a series of 15 such experiments, 12 of the B-cell fractions alone produced less than 500 pg per culture while the remaining three experiments produced 740,880, and 560 pg per culture. The matching series of B/T (0.4/1.6 x 106) lymphocytes produced a mean of 2651 pg (+ 1 SD = 263). The low levels of IgE detected in the B-cell fractions alone probably represented contamination with a low but functionally detectable level of T lymphocytes but could be due to some cytophilic IgE. The background levels of IgE for 1.6 x lo6 unirradiated T lymphocytes alone for 13 experiments was less than 500 pg while in 2 experiments 520 and 640 pg were detected, here representing higher B-cell contamination in these 2 experiments. Next we undertook a series of experiments to determine the effect of increasing numbers of untreated or irradiated (3000 rad) T lymphocytes on IgE production by a constant number of B-fraction cells (0.4 x 106). T lymphocytes were irradiated at 3000 rad in order to remove possible suppressor influences (6, 8). Four such experiments are shown in Fig. 5. Increasing numbers of unirradiated T lympho-

SAXON

AND

STEVENS

__--__

1

B I

6

c / i-

6-

0

0.6

1.6 2.4 32 T LYMPHOCYTES

FIG. 5. Effect of T lymphocytes irradiated (0) T lymphocytes were for IgE 7 days later. IgE production shown by the horizontal bars ( 1 shown.

40 ADDED

0 PER

t

--L--..----j

0.6 CULTURE

1.6 (x10-‘)

24

!I

32

40

on 1gE production in rJitn>. Increasing number of untreated (0) or added to B-fraction cells (0.4 x 10’3 and supematants were assayed by B cells alone is represented by the asterisk (‘) while I 1 SD is ). Four experiments (A, B, C, D) with different individuals are

cytes enhanced IgE production. This helper effect was maximal between 0.4 and 1.6 x lo6 T lymphocytes. At higher numbers of T cells (up to 4 x lo”), IgE production declined slightly from the maximum (average = 20%, range l l -36%). Irradiated T lymphocytes provided a consistent enhancing effect on B-cel1 IgE production. However, IgE synthesis rose as irradiated T lymphocytes were increased up to 3-4 x 10Vml. The maximum IgE production achieved by B cells with irradiated T lymphocytes was always greater than that seen with unirradiated T lymphocytes. To further define the relationship between these T lymphocyte effects (untreated vs irradiated), in 16 experiments we compared the amount of IgE produced by identical cultures with 1.6 x 106 of either untreated or irradiated T lymphocytes (Table 4). Fourteen experiments showed IgE production greater with the irradiated T lymphocytes (T/irradiated T mean ratio = .59) whiie two experiments did not (T/irradiated T = 1.05 and 1.09). As all these experiments were performed with autochthonous T- and B-cell combinations, similar untreated and irradiated T-cell titrations were established with allogeneic cell combinations. These allogeneic cultures showed IgE production indistinguishable from that seen with the autochthonous mixes (Table 5). Comparison of IgE, IgG, and IgM Produced in Vitro In viva human IgE levels vary to a much greater degree than any other Ig

isotype (24) and regulation of IgE in murine systems appears to be under differential control from IgG or IgM (25,27). To examine whether the in vitro synthesis of IgE was coordinated with IgM or IgG synthesis, we assayed cl-day culture super-

HUMAN

DIFFERENTIAL

EFFECTS

IgE PRODUCTION

483

IN VITRO

TABLE 4 OF UNTREATED VERSUS IRRADIATED

T LYMPHOCYTES

(1.6 x 106) ON IgE PRODUCTION BY B LYMPHOCYTES Experiment No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

IgE produced per culture (pg) T irradiated

T

4500 2500 4500 2700 8460 4770 12,525 4305 6255 5050 701 7350 5380 3570 4560 2410

2750 1400 3000 1750 4685 4990 2520 3205 4040 2230 585 7975 2640 2400 2785 1375

TABLE

Ratio of T/irradiated

T

.61 -56 .67 .65 .55

1.05 .20 .74 .65 .44 .83 1.09 .49 .67 .61 .57

5

EFFECT OF ALLOGENEIC OR AUTOCHTHONOUS PRODUCTION

Experiment No.

T CELLS (1.6 x 106) ON IgE BY B CELLS (0.4 x 10”)

T cells

Irradiation

W (pg)

1 1 1 1

Autochthonous Autochthonous Allogeneic Allogeneic

+ +

1670

2 2 2 2

Autochthonous Autochthonous Allogeneic Allogeneic

+ +

2430 4375

3 3 3 3

Autochthonous Autochthonous Allogeneic Allogeneic

+ +

3010 4185 2805 4055

4 4 4 4

Autochthonous Autochthonous Allogeneic Allogeneic

+ +

3565 2300 3320

2785 1890 2835

2190 3835

1980

484

SAXON

AND

STEVENS

natants for the quantitatives of IgE, IgG, and IgM produced. There was no interrelationship between the amount of IgE produced in PWM-stimulated unfractionated PBL cultures and the amounts of IgG or IgM synthesized over 7 days (Table 6). Titrations of untreated and irradiated T lymphocytes plus a constant number of B-fraction cells (0.4 x 106) were also examined for the different classes of Ig produced (Fig. 6). As previously shown (5), IgG and IgM production peaked at l/l untreated T/B cell ratio (0.4/0.4 x 106) with inhibition at higher T/B cell ratios. IgG was inhibited 80 and 89% while IgM was inhibited 59% from maximum production in the experiments shown. IgE production was only modestly inhibited (8 and 9%, respectively) at the same points (B/T = 0.4/4.0 x 106). Irradiation removed suppressor T-cell influences for IgG, IgM, and IgE. However, while the resulting immunoglobulin production curves were similar, they were not identical (Fig. 6). While in all experiments there tended to be some decline from maximum IgG and IgM production when 4.0 x 10” irradiated T cells were added, IgE production rarely declined at all and often continued to rise. DISCUSSION

The amounts of IgE we have detected in culture (up to a maximum of 12,500 pg or 1.25 x 10wg)are minute enough that it was crucial to establish that this IgE was synthesized in vitro and not cytophilic Ig carried over into the cultures. This was suggested by the delayed appearance of detectable IgE in culture. That the IgE was in fact biosynthesized in vitro was shown by the experiments where (i) inclusion of the protein synthesis inhibitor cycloheximide reduced IgE in vitro over a 7-day time course to background levels, (ii) irradiation of the B cells prevented the appearance of IgE in culture as a round of DNA synthesis is known to be required for PWM driven Ig synthesis (7), and (iii) freeze-thaw lysis of PBL on Day 0 failed to yield amounts of IgE comparable to that achieved in culture. Furthermore, in vitro IgE levels might be expected to correlate with serum levels if it were cytophilic Ig, a relationship we did not observe. Another important point was the specificity of our radioimmunoassay. We were not detecting synthesized IgG or IgM or IgA assay as shown by (i) failure to detect up to 5000 ng of these isotypes in direct binding (Fig. 1), (ii) failure of IgG or IgM to compete with the second

RELATIONSHIP

Experiment No. 1 2 3 4 5 6 I 8 9 10

BETWEEN

IgE,

IgG,

AND

TABLE 6 IgM PRODUCED

IN CULTURES

OF PBL

(2 x IW)

W (pgkulture)

I& @kulture)

Wf (ngkulturei

3620 2650 1870 1125 1120 930 875 4070 2995 2585

6920 3115 4370 4290 5575 3090 3420 2915 7005 3910

2043 2090 3835 6250 2250 4490 3165 5215 3875 4510

HUMAN

IgE PRODUCTION

IN VZTRO

485

antibody (Fig. 2), and (iii) lack of correlation between IgG, IgM, and IgE synthesized in individual cultures (Table 5). Furthermore, the IgG we used was polyclonal and represents all four subclasses. The in vitro system we employed utilizing PBL from normal or atopic donors with serum IgE levels ~500 III/ml consistently showed stimulation of IgE biosynthesis in the presence of PWM. This was true whether unfractionated PBL or separated and recombined B- and T-lymphocyte cultures were established. This observation is in accord with results from many laboratories where PWM has been shown to stimulate in vitro IgG, IgM, and IgA production (4- 11). Furthermore, we have shown that the ability of the B cells to synthesize IgE in vitro in our system was dependent on T-lymphocyte helper effects. B-cell cultures alone produced only minimal amounts of IgE (Figs. 5 and 6). This T-lymphocyte helper effect on IgE production did not show major histocompatibility restriction nor do other PWM-driven Ig systems. The IgE measured in the combined T- and B-cell cultures was not due to the detection of some T lymphocyte product as T fractions alone failed to produce IgE and irradiated T lymphocytes (3000 rad) provided for even greater amounts of IgE in combined B and T cultures. At this point it is important to define how our studies relate to those of Buckley and Becker (27). We, like they, were able to detect increased IgE synthesis in vitro by PBL from normals and atopics with low serum levels of IgE in the presence of PWM. The amount of IgE detected per PWM-stimulated culture of 2 X lo6 normal

T

LYMPHOCYTES

ADDED

(X IO-1

FIG. 6. Relationship of IgE, IgG, and IgM produced in vitro. Titrations of untreated (open symbols) or irradiated (closed symbols) T lymphocytes were added to 0.4 x 10s B-fraction cells and the amount of IgE (A, A), IgG (0, l ), and IgM (0, n ) produced per culture was determined.

486

SAXON

AND

STEVENS

PBL was twofold or more than that observed by those authors. When we established cultures of standard numbers of B and irradiated T lymphocytes, far greater amounts of IgE were produced by the same number of “normal” blood lymphocytes (Table 4, mean 4970 pg/culture). Furthermore, using recombined T- and B-cell cultures we were able to define regulatory events relating to the amount of IgE produced per culture as will be discussed later. While Buckley and Becker showed increased amounts of IgE produced by cultures from individuals with disease processes associated with elevated levels of serum IgE neither we, nor they, found such a relationship for low (~500 IU/ml) serum IgE subjects. Fiser and Buckley (28) later reported that the IgE they detected was not dependent on T-cell help. The other difference that is important is that those investigators observed an inhibition of IgE production in cultures of cells from subjects with serum levels > 1000 &ml in the presence of PWM and allogeneic T lymphocytes. This lack of dependence on T-cell help, inhibition by PWM, and the ability to be inhibited by T lymphocytes suggests to us that at least part of the peripheral blood cells responsible for the IgE produced in vitro by PBL from individuals with such elevated serum IgE levels are not the same functional subpopulation of B cells which go on to make IgE in the system we have reported in this paper. Recently we have examined a number of antigen-specific B-cell subpopulations which can be found in the circulation after in vivo booster immunization (20, 30). In vivo activated B lymphocytes, which we have chosen to call lymphoblastoid B cells, exhibit just the characteristics mentioned above. Furthermore, they are distinguishable on the basis of their increased size and density and the rapid kinetics of their Ig production in vitro (29). Indeed IgE-producing cells of this nature were probably identified by Patterson et al. (30) in the circulation of two individuals with very high serum IgE levels. For the time being, it is important to be cognizant of the potential differences observed between “normal” and “high” IgE individuals’ IgE production in vitro, and the possibility that it is due to distinct B-lymphocyte populations being examined. Experiments are underway in our laboratory to explore this possibility. The characteristics of in vitro PWM-driven IgE biosynthesis by human PBL parallel many of the features of the PWM-induced production of IgG and IgM (4-9). However, certain differences should be noted. While a high 10 to 1 ratio of T to B cells gave greater than 60% inhibition of IgG and IgM synthesis, IgE production fell only modestly from maximum levels (l l -36%). This could be due to IgE production being less sensitive to T-suppressor effects. This is unlikely in that T suppression is probably the dominant theme in IgE production in almost all systems examined (reviewed in Ref. 31). Alternatively, T-lymphocyte help and suppression for IgE may be more balanced than for IgM or IgG thereby preventing culture of untreated T/B cells at low ratio from producing levels of IgE comparable to IgG or IgM. That this latter interpretation is correct is suggested by our experiments comparing the amount of IgE produced by B-cell cultures containing 1.6 x 10” of either untreated or irradiated T lymphocytes (Table 4). The average ratio between these cultures was .fS-very similar to that for IgG as we have previously reported (3). Two subjects did have a T to irradiation-resistant T suppres-

HUMAN

IgE PRODUCTION

IN VZTRO

487

sion which is known to occur in activated suppressor systems (32). In titrations using T lymphocytes irradiated to remove suppressor influences, IgE production proportionally increased at the higher T-cell numbers in comparison with IgM or IgG. This effect and the other differences between IgE, IgG, and IgM could be due to separate isotype regulatory populations or differential sensitivity of isotypespecific precursor cells in our system. The development of this in vitro system for the stimulation and analysis of IgE production will allow for (i) further analysis of the regulatory events controlling IgE production, (ii) comparison with the regulatory events and cells modulating other Ig isotypes, (iii) analysis of the nature of B-cell precursors of IgE production, and (iv) evaluation of the role of these factors in states of altered in vivo and in vitro IgE production. ACKNOWLEDGMENTS The authors wish to thank Mark Kaplan, Alice Carter, and Howard Sofen for their excellent techmcal assistance in the performance of this work, and Dr. John L. Fahey for his enthusiastic support.

REFERENCES 1. Feldman, M., Baverley, P. C. L., Woody, J., and McKenzie, I. F. C., J. Exp. Med. 145, 793, 1977. 2. Herzenberg, L. A., Okamora, K., Cantor, H., Sato, V. L.. Shen, F., Boyse, E. A., and Herzenberg, L. A., J. Exp. Med. 144, 330, 1976 3. Stevens, R. H., and Saxon, A., .I. Clin. Invest. 62, 1154, 1978. 4. Keightley, R., Cooper, M. D., and Lawton, A. R., J. Zmmunol. 117, 1538, 1976. 5. Saxon, A., Stevens, R. H., and Ashman, R. F., J. Immunol. 118, 1872, 1977. 6. Siegal, F., and Siegal, M., J. Zmmunol. 118, 642, 1977. 7. Janossy, G., and Greaves, M., Transplant. Rev. 24, 177, 1975. 8. Saxon, A.. and Stevens, R. H., Clin. Immunol. Immunopathol. 10, 427, 1978. 9. Knapp, W., and Baumgartner, G., J. Immunol. 121, 1177, 1978. 10. Waldman, T. A., Durm, M., Broder, S., Blaese, R. M., Blackman, M., and Strober, W., Lancet 2, 609, 1974. 11. De la Concha, E. G., Oldham, G., Webster, A. B. D., Asherton, G. L., and Platts-Mills, T. A. E., Clin. Exp. Immunol. 27, 208, 1977. 12. Buckley, R. H., Gilbertsen, R. B., Schiff, R. I., Ferreira, E., Sanal, S. O., and Waldman, T. A., J. Clin. Invest. 58, 130, 1976. 13. Broder, S., Humphrey, A., Durm, M., Blackman, M., Meade, B., Goldman, C., Strober, W., and Waldman, T. A., N. Engl. J. Med. 293, 887, 1975. 14. Ovary, Z., Itaya, T., Watanabe, N., and Kojima, S., Immunol. Rev. 41, 26, 1978. 15. Jarrett, E. E. E., Immunol. Rev. 41, 52, 1978. 16. Boyum, A., Stand. J. Clin. Lab. Invest. Suppl. 97, 77, 1968. 17. Saxon, A., Feldhaus, J. L., and Robbins, R. A., J. Zmmunol. Methods 12, 285, 1976. 18. Saxon, A., and Portis, J., Cancer Res. 37, 1154, 1977. 19. Zollinger, F. P., Palyrymple, J. M., and Artenstein, M. S., J. Zmmunol. 117, 1788, 1976. 20. Stevens, R. H., and Saxon, A., Cell Zmmunol. 45, 142, 1979. 21. Marchalonis, J., Cone, R., and Santer, V., Biochem. J. 113, 229, 1971. 22. Ishizaka, K., Ishizaka, F., and Lee, E. H., Immunochemistry 7, 687, 1970. 23. Saxon, A., and Stevens, R. H., C/in. Zmmunol. Zmmunopathol. 12, 82, 1979. 24. Ishizaka, T., and Ishizaka, K., Progr. Allergy 19, 60, 1973. 25. Kimoto, M., Kishimoto, T., Noguchi, S., Watanabe, T., and Yamamura, Y., J. Zmmunol. 118, 840, 1977. 26. Chiorazzi, N., Fox, D. A., and Katz, D. H., J. Zmmunol. 118, 840, 1977. 27. Buckley, R. H., and Becker, G. W., Zmmunol. Rev. 41, 228, 1978.

488 28. 29. 30. 31. 32.

SAXON Fiser, P. Stevens, Patterson, Ishimka, Rich, R.

AND

STEVENS

M., and Buckley, R. H., J. Allergy Clin. R. H., Macy, E., Morrow, C., and Saxon, R., Suszko, I. M., Metzger, J. W., and K., and Ishizaka, T., Zmmunol. Rev. 41, R., and Pierce, C. W., J. Exp. Med. 137,

Immunol. (Abstract) 63, 145, 1979. A., J. Immunol. 122,2498, 1979. Roberts, M., J. Immunol. 117, 97, 1976 109, 1978. 649, 1973.