IgE production in vitro by human blood mononuclear cells: a comparison between atopic and nonatopic subjects

Zuhayr Hemady, M.D., Fred Blomberg, and Ross E. Rocklin, M.D. Boston, Muss.,

Ph.D., Stephen and

Uppsula,

Gellis, M.D.,

Sweden

In r?tro IgE synthesis by blood mononucleur cells fi-om atopic patients ctnd nonatoprc su&ec II IVLU examined. A totul of 1 x IO” mononuclear cells cultured in RPMI-1640 anti IO‘% ,titcrl c,tr!/ serum with or \isithout cycloheximide was f&nd to be optimul to detect de no\~~ Jynthesis. A modified Phudebas IgE paper radioi~rrmunosorbent test MU employeci,f& the quclrrtitution cr/ .supernutunt IgE concentration. Kinetic studies indicated thut about half the peak trmount oj I$ is secreted within the first 2 daxs und the maximum concentrcrtion is reached at dr+ 7 Mononucleur cells obtained from six of six atopic putients with eczema und eleiwted .serum I,# levels and 22133 atopic patients without eczema spontaneousls synthesized .sign$cunt umount.\ (I/ IgE in vitro. We ,failed to detect de now JgE synthesis by the c~ei1.s obtained.from 40 ntmu/opic controls. Polyclonal crctivutors Such us pokeweed mitogen, Staphylococcusaureus Con~rl I, concanavulin A, and phytohemagglutinin ,failed to induce or enhance in \Ytro JgE swthesis it; normul and atopic subjects. These findings indicate that the ,study of immunorr~~ulntion of I,pE synthesis in man will be d@cult to uccomplish until new’ methods ure developed that ullo~ induction qf’ the IgE response in LYtro in nonatopic subjects. (.I ALLERGY CLIN IMMIINOI. 71~~24.

1983.)

In the past decade much has been learned about the immunologic mechanisms controlling IgE biosynthesis in experimental animals.” 2 Studies of rodent models in vivo have revealed that the IgE-producing B cell is highly dependent on T-cell regulation.” The effect of T cells seems to be mediated by soluble factors with either helper or suppressor activities.2 Similar in vivo investigations have not been conducted in humans. Therefore our understanding of the regulation of IgE production in man derives mainly from recently developed in vitro systems. Such in vitro studies were first attempted by Geha et al.” and Patterson et al .3 Subsequently, various laboratories have evaluated the biosynthesis of IgE by blood mononuclear cells in both atopic and nonatopic subjects. +16 However, discrepant results have been reported and controversy remains with regard to the -~.From the Allergy and Dermatology Divisions, Department of Medicine, Tufts New England Medical Center, Boston, Mass., and BioCell Laboratories HB, Uppsala, Sweden. Received for publication April 12, 1982. Accepted for publication Sept. 27, 1982. Reprint requests to: Ross E. Rocklin, M.D., Allergy Division, Box 30, Tufts New England Medical Center, Boston, MA 021 I I. Vol. 71, No. 3, pp. 324-330

production of IgE by MNC from normal donors, the effect of PWMR-‘” andStuphylocc-,ccus uztreu.s Cowan 1,‘;’ and the role of T cells. L Using a sensitive radioimmunoassay. we have examined in vitro IgE biosynthesis by blood mononuclear cells from atopic patients with eczema and elevated serum IgE, atopic patients without eczema, and nonatopic individuals. Our results indicate that de novo IgE synthesis can be detected in the supernatants of MNC cultures from atopic patients with eczema and from the majority of atopic patients withoui eczema during their allergy season, but not from normal nonatopic controls. The effect of varying culture conditions and polyclonal stimulants on IgE synthesk is also examined. MATERJALS ANQ METHODS Blood donors The patients included six subjects with atopic dermatitis (mean age 14.18 yr, median age 19 yr) and elevated serum IgE level (>2000 kU/L) and 33 adult subjects with atopic asthma and/or hay fever (without eczema) and positive skin tests. Control subjects consisted of 40 adults who had no history of atopy Informed consent was obtained from all individuals stud.

VOLUME 71 NUMBER 3

IgE production

Ahhre~iations

MNC: PWM: PRIST: RAST: FCS: HSA: Con-A: PHA:

used

Mononuclear cells Pokeweed mitogen Paper radioimmunosorbent Radioallergosorbent test Fetal calf serum Human serum albumin Concanavalin A Phytohemagglutinin

test

ied. None of the patients were taking oral steroids and none had been on immunotherapy for at least 2 yr prior to the study.

MNC isolation MNC suspensions were isolated by Ficoll-Hypaque (LSM; Litton Bionetics, Kensington, Md.) gradient centrifugation of sterile heparinized blood after a modification of the method of Boyum. I7 Cells were washed three times in RPM1 1640 (Grand Island Biological Co., Grand Island, N.Y .) before being resuspended in RPM1 1640 supplemented with L-glutamine (10 mM), gentamicin (0.05 mgiml), penicillin (100 U/ml), streptomycin (50 pgiml), and 10% heat-inactivated FCS (complete medium). In some experiments the culture medium consisted of Iscove’s medium supplemented with L-glutamine (10 mM), antibiotics, and 0.5% HSA (Sigma Chemical Co., St. Louis, MO.).

MNC cultures Unless otherwise indicated in the text, unfractionated MNC were cultured in 12 by 75 mm polystyrene tubes (Falcon, Oxnard, Calif.) for 7 days in a 5% CO2 humidified environment at 37” C. In experiments with PWM (Grand Island Biological Co.) and S. aureus Cowan I rich in protein A (IgG sorb; the Enzyme Center, Boston, Mass.), a wide range of concentrations was employed, including a final concentration of l/l00 and 111000, respectively, which was found in previous experiments to be optimal for stimulation of in vitro IgG biosynthesis by human MNC.‘” Two other mitogens, PHA (Difco, Detroit, Mich.) and Con-A (Miles Laboratories, Elkhart, Ind.) were also tested over various concentrations. In all experiments, MNC were cultured in the absence or presence of cycloheximide (50 pgiml).

Measurement

of IgE protein

Serum IgE levels of blood donors were determined with Phadebas IgE PRIST (Pharmacia Diagnostics AB, Uppsala, Sweden). The same technique with some modifications was used for quantitation of IgE produced from MNC in vitro. The reagents used in the modified assay were as follows: Phadebas IgE PRIST discs, the 100 kU IgEiL standard point, the Phadebas RAST 12”1-anti-IgE tracer, and complete medium as diluent for the standards. Reconstituted standard (100 kU/L) was diluted to 2 kU IgEiL (1 kU IgEiL corresponds approximately to 2.4 ng

in vitro

325

100 1 :R1.0A 8 1 0 L-4

Diluent

1 06

IgE

I I2

I .24

PROTEIN

I 60

I 1.2

I 2.4

1) 4.8

(rig/ml)

FIG. 1. Dose-response curve for modified Phadebas PRIST IgE assay. Phadebas IgE standard (100 kU/L) was diluted in complete medium to 2 kU/L (4.8 ngiml) and seven standard points were made as dilutions from 4.8 ngiml. Triangle, Mean + 2 SD of percent total counts bound in diluent samples (complete culture medium).

lgE/ml’Y~ 2”). Seven standard points were made as dilutions from4.8ng/ml(2.4, l.2,0.6,0.24,0.12,and0.06ng/ml). Polystyrene tubes with round bottoms (12 by 55 mm; Pharmacia, Inc., Piscataway, N. J.) were used for the test. Discs were added and thereafter 300 ~1 of standards or culture supematants were added to the tubes. Incubation was carried out at room temperature for 20 to 24 hr on a horizontal shaker (Lab-line, Melrose Park, Ill.) at 300 rpm with an amplitude of 3 cm. Thereafter, three washings (10 min each) with 0.9% NaCl and 0.5% Tween 20 (Sigma Chemical Co., St. Louis, MO.) were done according to the Phadebas IgE PRIST instructions. Five minutes afterwards the discs were aspirated again to carefully remove any excess moisture in the tubes. For the second incubation, reconstituted Phadebas RAST tracer was diluted four times with phosphate-buffered saline containing 0. I % Tween 20 and 4 ng (200 ~1) of the diluted tracer were added to each tube. The tubes were again shaken for 20 to 24 hr at room temperature then washed with 0.9% NaCl and 0.5% Tween 20 exactly as described for the first incubation. The tubes were counted in a Beckman gamma counter for 10 min or until a 2% error level was reached. Fig. 1 shows a typical standard curve, plotting percent total counts bound, which represent counts per minute in each tube X 100 total counts added/tube against IgE protein concentration

(ngiml)

on log-log axes.

326

Hemady

J. ALLERGY WN.

et al.

KINETICS

OF IgE PRODUCT/ON iN VITRO

TABLE

I. Comparison

by atopic 1640

MNC

of

in vitro

cultured

or serum-free

"0

I2

3

4 Days

of

5 6 Culture

7

8

No.

Iscove’s

13 i

The

RPMl1640 + 10% FCS

k3

.

. *

t 2

t

NON-ATOPIC SUBJECTS

(O/22,

* -

:

ATOPIC WITHOUT ECZEMA (221331

ATOPIC WITH ECZEMA

(6/6)

FIG. 3. Spontaneous de novo IgE synthesis in normal subjects and in atopic patients with or without eczema. Unfractionated MNC were cultured in RPM1 1640 + 10% FCS, and supernatant IgE was assayed at day 7. Net IgE synthesis was observed in 616 atopic patients with eczema and in 22/33 atopic patients without eczema. No de novo IgE synthesis was noted in normal subjects.

The detection limit of the modified Phadebas IgE PRIST assay was less than 100 pg IgEiml as calculated from diluent background values +2 SD. The coefficient of variation ranged from 2.5% to 10% between the levels of 2400 and 240 pg IgEiml and increased at 120 pg IgEiml to 10% to 20%.

(pglmll

Iscove’s medium t O.!i% HSA -p,-..I-.

of results

All experiments were performed at least x11dupircates. student’s t test was used for statistical analyses.

RESULTS Specificity and technical radioimmunoassay

tf

tgE synthesis

To compare our results to those of other mvcutrgators. we tested several dilutions of World Health Organization reference standard IgE (75!502), ranging from 0.12 KI 3 X rig/ml, in our assay. Using our standard curve. we found that the amount of TgE in each dilution of the WHO standard was very close to the quantity of IgE known to be present rn each sample. To determine whether or not de novo IgE synthesis occurred in MNC cultures, we employed the Ihltowing for. mula: net IgE synthesis equals total supematant IgE concentration in untreated cultures minus total supematant IgE, concentration in cycloheximide-treated cultures.

Analysis

-4 c

RPM1

medium

9

FW. 2. Kinetics of spontaneous de novo IgE synthesis in an atopic patient with eczema. Parallel unstimulated (a--- .--0) and cycloheximide-treated MNC cultures (o---o) (1 x 106cells/ml) were initiated on day 0 and harvested on sequential days, and supernatant IgE was assayed. Net IgE synthesis (A-A) peaked at day 7.

z5

IgE synthesis

net

in serum-containing

Net

Experiment

IMWJhlOL. MARF,W t983

aspects

of the IgE

To confirm the specificity of the IgE radioimmunoassay, we added several dilutions of highly purified immunoglobulins (G, M, A, and D) to the assay in order to test their reactivity with the affinity-purified radiolabeled anti-IgE (RAST tracer). None of’ the immunoglobulins in concentration up to 20 @g/ml for polyclonal IgG and 10 pg/ml for monoclonal IgM, IgA, and IgD had any significant effect in our assay (data not shown). To exclude the possibility that IgE was being consumed either during the 7 day culture period or during the assay procedure, we added two dilutions of polyclonal IgE PRIST standard (0.5 and 0.8 kU/L) tcr unstimulated and PWM-stimulated cultures, which were established in parallel with identical cultures to which no exogenous IgE was added. The difference in supernatant IgE content at day 7 between these two sets of cultures was equal to the amount of exogenous IgE added at day 0, implying that the IgE in culture supernatants was not being bound to the tubes 01 otherwise affected by the culture conditions used (data not shown).

VOLUME 71 NUMBER 3

TABLE

positive

IgE production

II. Supernatant IgE concentration net IgE synthesis

in unstimulated

Without

cycloheximide

With

Net

Range

2857,200

652 k 90

60-I ,400

1,833-12,600

701 k 133

Range

cultures

cycloheximide

X-~SEM

x 2 SEM

Atopic patients without eczema 1,985 t 404

and cycloheximide-treated

in vitro

327

with

IgE synthesis

X~SEM

Range

1,380 ? 374

IO@6,100

5,360 2 997

1,686-l 1,600

(n = 22)

Atopic patients with eczema

6,289 t

1,058

l47-2,050

(n = 6)

Effect of changes in culture conditions the detection of de novo IgE synthesis culture supernatants

on in

In an attempt to optimize culture conditions, we compared two different culture media: RPM1 + 10% FCS and Iscove’s medium supplemented with 0.5% HSA. The data from these experiments are shown in Table I. The results suggest that there is no advantage to the use of enriched serum-free Iscove’s medium, since a higher concentration of IgE was detected when the more conventional RPM1 1640 + 10% FCS medium was employed. To examine the influence of cell concentration on the net quantity of IgE measured at 7 days, duplicate cultures containing various concentrations of MNC ranging from 0.5 X lo6 to 3 X lo6 cells/ml were simultaneously initiated in the presence or absence of cycloheximide (50 pug/ml). In four experiments (data not shown) there was a proportionate increase in net IgE synthesis when the cell concentration was increased from 0.5 million to 1 million cells/ml. However, there was no further rise in net IgE synthesis at a cell concentration of 2 or 3 million cells/ml possibly as a result of cellular overcrowding. In other preliminary experiments (data not shown) MNC were freeze-thawed three times on day 0 and held in culture for 7 days in parallel with intact unstimulated and cycloheximide-treated cultures. The concentration of IgE detected at 7 days in the supernatant of these freeze-thawed cultures was not significantly different from the level of IgE in cycloheximide-treated cultures. We then determined the kinetics of in vitro IgE synthesis in atopic patients with eczema and elevated serum IgE by measuring net IgE synthesis in MNC culture supematants harvested on sequential days. As depicted in a representative experiment in Fig. 2, net IgE synthesis increased from time 0 until day 7 of culture, at which time the highest concentration of spontaneously produced IgE was reached. It is of interest that about half of the peak amount of IgE detected was made within the first 2 days. In all subsequent experiments, MNC were cultured at 1 million

TABLE III. Comparison of serum IgE concentration and quantity of IgE synthesized in vitro by atopic eczema patients Experiment

No. 1

2 3 4 5 6

Serum IgE (klJ/L)

Net

20,800 2,800 22,800 5,800 17,000 2,500

IgE synthesis (pglml)

4,200 2,650 10,290 1 1,600 1,686

1,750

cells/ml and supematants were collected by centrifugation at day 7. A set of cycloheximide-treated cultures was included in each experiment to determine in vivo preformed IgE carried over into the cultures. In vitro IgE biosynthesis MNC

by unfractionated

In 40 normal nonatopic subjects (the results of 22 are shown in Fig. 3) there was virtually no difference between the level of IgE in supematants of unstimulated 7 day MNC cultures and their cycloheximidetreated counterparts. In contrast, supernatants of 7 day MNC cultures from 6/6 patients with atopic dermatitis and elevated serum IgE contained significantly higher levels of IgE than analogous cycloheximidetreated cultures (Table II). Spontaneous de novo IgE synthesis also occurred in supernatants of 7 day MNC cultures from 22/33 atopic subjects without eczema (Table II). Most of the latter patients were studied during their allergy season. A comparison of serum IgE concentrations and net IgE synthesis in vitro by MNC from atopic patients with eczema is shown in Table III. It can be seen that there is poor individual correlation between the IgE level in the serum and the quantity of IgE synthesized in vitro. Effect of PWM and other stimulants

polyclonal

Fig. 4 illustrates the effect of PWM, S. uureus Cowan I, Con-A, and PHA on de novo IgE synthesis

J. ALLERGY CLIM. IWVJWL. MARCH ‘1983

338 Xemady et al. -PWMI

I

, I

c

1 I

4

+ PWM 2 4? $ ;;;

- Staph.

aweus

t Staph.

aureqr

-r--$---l

B

- CON. A 1

4

t CON. A

-----S

*.g 8

- PHA +PHA I 500

1 1000

Net IgE FIG. 4. Effect Unfractionated Cowan I, Con-A, mean (tSEM).

of

I 1500

Antibody

I 2000

1 2500

Synthesized

.-L--. 3000

--__

(pg/ml)

polyclonal stimulants on net IgE synthesis in atopic patients with eczema. MNC were cultured in RPM1 1640 + 10% FCS with or without PWM, S. aureus or PHA, and supernatant IgE was assayed at day 7. The data are depicted as the

I

. IO % FCS

1300 - x-x,

1,

x

1100 -*--.--Xmw--*--.--X-~-~

l ----__

900700

--_

-

1 0.5% a IOYFCS*O.l .0.5XHSA*O.IM

-----__

HSA M

aMM #MM

-V

SOO- _

\

I

300 loot, 0

,

*

IO

20

100 CON

A

(~Phl)

FIG. 5. Effect of Con-A on the measurement of IgE by radioimmunoassay. A known quantity of IgE was assayed with or without increasing concentrations of Con-A (IO to 100 wgiml) either in RPM1 1640 + 10% FCS or in Iscove’s medium + 0.5% HSA. Con-A interfered with the detection of IgE at concentrations >20 pglml. Simultaneous addition of a-methylmannoside (cuMM) cornpletely reversed the effect of the highest concentration of Con-A used (100 pgiml) whether the assay was conducted in RPM1 1640 + 10% FCS or in Iscove’s medium i- 0.5% HSA.

by 7 day MNC cultures from atopic patients. In all cases, polyclonal stimulants appeared to have a variable inhibitory effect on spontaneous in vitro IgE production. When the above polyclonal activators were added to cultures from nonatopic controls, or atopic patients whose MNC failed to form IgE in vitro, no induction of de novo IgE synthesis was observed. In subsequent experiments (data not shown), blood MNC from normal donors were cultured with and without an optimal dose of PWM for 3 days and washed thoroughly before being transferred into V-shaped microwell culture plates for another 6 day culture period. In none of the subjects studied did we observe any significant stimulation of IgE synthesis in the presence of PWM. Ascaris larvae extract (a generous gift of Dr. Bert Stomberg) and ragweed Ag E

(Sigma) were tested over various concentrations in a number of atopic patients and normal controls, but both reagents neither induced nor enhanced total IgE production in vitro. It should be pointed out that supernatants from PWM-stimulated MNC cultures did contain markedly elevated amounts of IgC; even though there was no increase in IgE synthesis above baseline (Lima and Rocklin, unpublished data). Evaluation of possiWe intarference polycionat stimulants in the IgE radioimmunoassay

of

When the polyclonal stimulants employed in the study were added at various concentrations to known quantities of polyclonal IgE, only Con-A in relatively high concentrations significantly interfered with the

VOLUME 71 NUMBER 3

measurement of IgE in our radioimmunoassay. As can be seen from a representative experiment shown in Fig. 5, the effect of Con-A is dose dependent and is negligible at concentrations equal to or less than 10 pgiml. Furthermore, the Con-A effect was completely reversed when a-methylmannoside was simultaneously added, implying that its effect is attributable to its sugar binding moiety. DISCUSSION In vitro production of IgE antibody by cultured human mononuclear cells has been the subject of several recent reports.*-‘” Conflicting data have been reported with regard to de novo IgE production by atopic and nonatopic individuals, the effect of PWM and T cells, and the kinetics of in vitro IgE synthesis. Initial attempts by Geha et al.” and Patterson et a1.j revealed that IgE production by MNC from atopic patients and hyper-IgE patients can be measured in vitro. Buckley and associates,‘j, ’ using a sensitive radioimmunoassay, originally found that MNC from some normal subjects and from atopic donors with eczema manufacture IgE antibody in vitro. More recently, Sampson and Buckley8 have reassessed their previous findings in light of the observation that measurement of IgE protein in freeze-thawed supernatants on day 0 underestimates “true baseline” IgE because incubation of these freeze-thawed cultures for 7 days leads to the release of higher quantities of IgE into the supematant. They also noted that the concentration of supematant IgE in cycloheximide-treated cultures at 7 to 10 days was virtually the same as the concentration of IgE in freeze-thawed cultures held for the same period of time. Using this revised method for assessing “baseline” preformed IgE, Sampson and BuckleyH observed that MNC from normal donors do not synthesize IgE in vitro, whereas MNC from atopic patients with eczema and elevated serum IgE spontaneously produce IgE in vitro. Saxon et al. ,15 studying patients with markedly elevated serum IgE, showed that B cells from these patients spontaneously produce large quantities of IgE in vitro, whereas B cells from normal controls did not spontaneously produce IgE. Tjio et al.” studied in vitro production of IgE in nonatopic and atopic subjects using the IgE concentration in freeze-thawed culture supernatants at day 0 as the baseline level. They concluded that nonatopic subjects do not form IgE in vitro, while ragweedallergic patients spontaneously produce both IgE protein and ragweed-specific IgE in vitro during the ragweed season. Romagnani et al.“’ l2 evaluated in vitro IgE synthesis in nonatopic controls and in atopic subjects

IgE production

in vitro

329

with normal or slightly elevated serum IgE levels and found that MNC from most nonatopic donors and from some atopic patients fail to synthesize IgE in vitro. However, most grass-sensitive individuals in their study synthesized significant amounts of IgE protein and grass-specific IgE antibodies. The results described in the present article confirm in general the findings of the above investigators. By including a set of cycloheximide-treated cultures in each experiment, we have clearly demonstrated that blood MNC from a large group of non-atopic donors do not spontaneously produce de novo IgE in vitro. In contrast, we found that blood MNC from atopic eczema patients and from the majority of atopic patients without eczema during their allergy season are able to spontaneously secrete IgE in vitro. Our findings in non-atopic donors are at variance with the data presented by Saxon and Stevens,‘O Ghory et al., I3 Pryjma et al., I4 and Zuraw et al. I6 The reason for these differences among various laboratories is unclear and requires further investigation. The results of our experiments in which PWM and S. aureus Cowan I were used suggest that these polyclonal mitogens do not stimulate IgE synthesis in vitro in both atopic and nonatopic subjects. A controversy exists in the literature regarding this issue. Whereas most investigators have found that PWM depresses or does not significantly affect in vitro IgE synthesis by atopic MNC,Sx 9, 11, 12, 15 conflicting reports have appeared on the effect of PWM and S. aureus in normal donors. The data presented in this article regarding the lack of a stimulating effect of PWM on IgE synthesis in nonatopic donors concur with the findings of several previous studies8T$3 11, l* but differ from those of Saxon and Stevens,‘O Pryjma et a1.,14 and Ghory et a1.13In addition, we were unable to confirm the report of Zuraw et al. l6 that consistent stimulation of IgE biosynthesis in vitro can be seen with a two-stage in vitro culture system consisting of a 3 day incubation period in the presence of PWM followed by thorough washing, and another 6 day incubation in the absence of PWM. The failure of many workers, including ourselves, to find significant augmentation of IgE synthesis by PWM stimulation has not been fully investigated. Saxon et al.‘” have suggested that the variability in reported studies is due to the use of different batches of FCS. Another possibility is the difference in the specificity of the anti-IgE antisera used in various studies or in the lots of PWM used, all of which could also account for the conflicting results reported to date. The results of this study also indicate that such factors as the culture medium employed and the cell

330

Hemedy

J. ALLERGY CLIN. IMIMUNOL. MARCH f&U

et al.

concentration significantly affect supernatant IgE levels at the termination of culture. Furthermore, we were able to demonstrate that by means of the commercially available Phadebas PRIST and RAST reagents, a radioimmunoassay can be developed that is highly specific and at least as sensitive as all the previously reported IgE radioimmunoassays, with the exception of the assay described by Zuraw et al .I6 This should allow any investigator with interest in the field to conduct studies of IgE biosynthesis in vitro without having to contend with the tedious task of developing anti-IgE antisera with high affinity and specificity. Finally, the usefulness of our in vitro system for studying IgE immunoregulation in man is limited by the fact that only spontaneous IgE synthesis by lymphocytes from atopic patients could be detected. To further our understanding of the mechanisms controlling the IgE response, we need to develop a system whereby in vitro IgE production can be induced in nonatopic subjects and augmented in atopic subjects. The regulatory role of lymphocyte subsets and possibly of cell-derived soluble factors can then be studied through appropriate manipulations.

7

8 9 10

II

12

13

14

15

REFERENCES 1. Ishizaka K, Ishizaka T: Mechanisms of reaginic hypersensitivity and IgE antibody response. Immunol Rev 41: 109, 1978. 2. Katz DH: Recent studies on the regulation of IgE antibody synthesis in experimental animals and man. Immunology 41: 1, 1980. 3. Tada T: Regulation of reaginic antibody formation in animals. Prog Allergy 19:122, 1975. 4. Geha R, Colten H, Schneeberger E, Merler E: Cooperation between human thymus-derived and bone marrow-derived lymphocytes in the antibody response to ragweed antigen E in vitro. J Clin Invest 56:386, 1975. 5. Patterson R, Suszko I, Metzger WJ, Roberts M: In vitro production of IgE by lymphocytes from a patient with hyperimmunoglobulinemia E, eosinophilia and increased lymphocytes carrying surface IgE. CIin Exp Immunol 20:265, 1975. 6. Buckley RH, Becker WG: Abnormalities in the regulation of human IgE synthesis. Immunol Rev 41:288. 1978.

16

17.

18.

19.

20.

Fiser PM, Buckley RH: Human IgE biosynthesis in vitro: studies with atopic and normal blood mononuclear cefls and suhpopulations. J Immunol 123: 1788, 1979. Sampson HA, Buckley RH: Human IgE synthesrs iti I’IWC~: :I reassessment. J Immunol 127:829, 1981, Tjio AH, Hull WM, Gleich GJ: Production of human immunoglobulin E antibody in t?tro. J lmmunol 122:213 I 1979. Saxon A, Stevens RH: Stimulation and regulation of human IgE production in virro using peripheral blood lymphocytes. Clin immunol Immunopathol 14:974, 1979. Romagnani S, Maggi E, DeIPrete GF, Troncone R. Krcci M: In t3ro production of IgE by human peripheral blnud mononuclear cells. I. Rate of 1gE biosynthesis. Clin Exp immunoi 42:167, 1980. Romagnani S, DelPrete GF, Maggi E, Troncone K; Giudtr (i, Almerigogua F, Ricci M: In ~ifro production of IgE by human peripheral blood mononuclear cells. II, Cells imolvcd in spontaneous IgE production in atopic patients. Clin Exp lmmunol 42:579, 1980. Ghory NC, Patterson R, Roberts M, Suszko 1: Ifr i’~r‘ti 1gF formation by peripheral blood lymphocytes from normal incli. viduals and patients with allergic bronchopulmonary nspergiilosis. Clin Exp Immunol 40~581, 1980. Pryjma J, Munoz J, Virella 0, Fudenberg HH: Evaluation ot lgM, IgG, IgA, IgD, and IgE secretion by human pcrrpheral blood lymphocytes in cultures stimulated with pokeweed mitogen and Staphylococcus aurcu.~ Cowan I. Celi lmmunol 50~115, 1980. Saxon A, Morrow C, Stevena RH: Subpopulations ctt c~culaaing B cells and regulatory T ceils involved in in yirr~ immunoglobulin E production in atopic patients with elevated serum immunoglobulin E. J Clin Invest 651457, 19811. Zuraw BL, Nonaka M, O’Hair C, Katz DH: Human IgE antibody synthesis in l,irru: stimulation of IgE responses by pokeweed mitogen and selective inhibition of such responses by human suppressive factor of allergy (SFA). J Immunol 127:1169, 1981. Boyum A: Isolation of mononuclear cella and granuiocytes from human blood. Stand J Clin Lab Invest [Suppl] 97:?1. 1968. Lima M, Rocklin RE: Histamine modulates in ~/trt, IgE production by pokeweed mitogen-stimulated human motionuctear cells. Cell Immunol 64:324, 1981. Yunginger JW, Gleich GJ: Seasonal changea in serum and nasal IgE concentrations. J ALLERGY CLIN IMMMWOL 51: 174. 1973. Bazaral M, Hamburger RN: Standardization and stability ot immunoglobulin E (IgE). J ALLERGY CLIN ~MMUNCIL 49: 1%~ 1972.