Synthesis of IgE by the human conceptus

Synthesis of IgE by the human conceptus D. lee

Miller,

M.D.,

Turku,

Pinland

Toivo

Hirvonen,

M.D.,

and

David

Gitlin,

M.D.

The capability of the hwman conceptus in synthesising IgE was investigated in this study. Embryonic and fetal tissues obtained from spontaneously or therapeutically aborted conoeptwes were cd&red in the presence of MXabeled amino acids, and the czllture flzlias were then analyaed by radioimmnzlnoelectrophore&v for the presence of radioactive IgE and IgG. Synthesis of IgE was noted aa early as 11 weelcs in fetal lung and liver, and by $1 weeks in fetal spleen. Interestingly, synthesis of IgG was also fownd as early as 11 weelcs of gestation. The data indicate: (1) that the conceptus has the potential, in terms of IgE synthesis, for developing reaginmediated sensitivity in utero; and (8) that each of the immunoglobulin classes of man may be synthesized by separate lymphoid cell lines rather than from a single cell line that develops immunoglobulin synthesis sequentially.

Previous studies have revealed that the human fetus can synthesize IgM as early as 10.5 weeks of gestation and IgG by 12 weeks of gestati0n.l Neither synthesis of IgAll 2 nor synthesis of IgD2 has been demonstrated in the human conceptus, although small amounts of IgA may appear in fetal serumlj 2; IgD has not been detected in cord sera2 or in the sera of young infants.3 IgE, which has been shown to be responsible for reagin-mediated hypersensitivity in man,’ is present in cord serum, and some investigator+ 6 have suggested that the human fetus may synthesize this immunoglobulin, since there appears to be a lack of correlation between maternal and cord IgE levels. The present study was undertaken to determine if the human conceptus can indeed synthesize IgE. MATERIALS Fractionation

AND METHODS of IgE myeloma

plasma

IgE was partially purified from the plasma of a patient with IgE myeloma,r employing DEAE (Brown Co., Berlin, N. H.) and Sephadex G-200 (Pharmacia, Uppsala, Sweden) column chromatography as outlined by Ishizaka and co-w0rkers.s The partially purified material contained small amounts of IgG and several proteins that were not identified, and therefore this preparation was further fractionated by zone electrophoresis on Geon (B. F. Goodrich Chemical Co., Avon Lake, Ohio) in O.lM borate buffer, pH 8.6, to obtain immunoelectrophoretically pure IgE.

Antisera 0.25

Antiserum mg. of

against partially

IgE was prepared by purified IgE emulsified

injecting the hind footpads of in complete Freund’s adjuvant.

rabbits Two

with weeks

From the Department of Pediatrics, University of Pittsburgh School of Medicine, and the Department of Obstetrics and Gynecology, University of Turku School of Medicine. Supported by grants (HD-00652 and HD-01031) from the National Institutes of Health, United States Public Health Service, and the Anna Randolph Darlington Gillespie and David Lindsay Gillespie Fund of the Children’s Hospital of Pittsburgh. Received for publication April 4, 1973. Reprint requests to: David Gitlin, M.D., Children’s Hospital of Pittsburgh, 125 DeSoto St., Pittsburgh, Pa. 15213.

Vol. 5.9, No. 3, pp. 186-188

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TABLE

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52 3

I. Summary

Concephs

T2 T4

Synthesis

Et Tl T6

IgE by

conceptus

183

of IgE synthesis* AS* (wk.1

Ddhwyt

Uver

Lung

H H H

0 3+ 3+ 3+ 0 2+ -

0 0

8.5 11 11 14 14.5 15.516 21

Ti

of

; + to 3+ =

synthesis of IgE and relative zz not tested. tH = hysterotokotomy; V = per vagina. ZProtein synthesis minimal.

if H V density

of the radioactive

Sple0n

0 0 0

ii 0 0 2+ bands;

i 0 + 0 =

no

synthesis;

-

later, 1 mg. of the protein emulsion was injected subcutaneously, and the animals were bled 3 weeks after this injection. The resulting antiserum was found to contain not only strong precipitating antibodies against IgE, but also against several other serum proteins including IgG. Aliquots of the antiserum were mixed with small amounts of pooled human serum and purified human IgG to adsorb precipitating antibodies against proteins other than IgE. Antiserum against the proteins of human serum was prepared by immunizing rabbits with pooled human serum emulsified in complete Freund’s adjuvant; this antiserum contained antibodies against more than 17 different serum proteins including IgG but not IgE.

Conceptuses The conceptuses studied, which were the products of spontaneous or therapeutic abortions, are listed in Table I. They were obtained within 15 minutes to one hour following delivery and were clinically dead at the time of study as evidenced by no respiratory effort, heartbeat, or motor activity. T6, which was a product of spontaneous abortion, was noted to have a marked difference in the lenghts of the two feet, and T5 was delivered dead following intra-amniotic prostaglandin F,, induction. The remaining conceptuses were delivered by hysterotokotomy, and all appeared to be grossly normal. The gestational ages of the eonceptuses were estimated (Table I) from the crown-rump length,9 crown-heel length,ls foot length,il or a combination of these parameters.

Tissue

cultures

Selected embryonic and fetal tissues were minced in 2 ml. of Eagle’s basal medium containing Hanks’ salts and L-glutamine (Grand Island Biological Co., Grand Island, New York), and the tissues were then transferred together with the culture medium to roller tubes. Approximately 4 pCi of uniformly labeled amino i*C:-acids (ICN, Irvine, California) were then added to each tube. As controls, aliquots of fetal spleen, liver, lung, gastrointestinal tract, and bone marrow, which had been stored following slow freezing at -20” C., were then minced in culture medium and placed in roller tubes with added l4CXabeled amino acids. All cultures were incubated at 37” C. for 3 to 5 days, after which the tube contents were dialyzed against B or more changes of distilled water over a 3 day period. After dialysis, the cultures were concentrated by lyophilization and resolution in 0.2 C.C. of O.lM borate buffer, pH 8.6; insoluble components were removed by centrifugation.

kadioimmunoelectrophoresis Aliquots of the concentrated culture fluids were eleetrophoresed in 1.5 per cent agar in OJM borate buffer, pH 8.6, using either partially purified IgE, the purified IgE obtained from zone electrophoresis or pooled human serum as carriers. The immunoelectrophoresis patterns were developed with either the unadsorbed rabbit antiserum against IgE, the same rabbit antiserum rendered specific for IgE by absorption, or the rabbit antiserum against

184

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J. ALLERGY

CLIN. IMMUNOL. SEPTEMBER 1973

FIG.

1. lmmunoelectrophoresis of lung culture fluids using either partially purified IgE (A and C) or pooled human serum (E) as carriers and unadsorbed rabbit anti-IgE (A and C) and rabbit antihuman serum for development (E). B, D, and F are the corresponding autoradiographs. The gestational age of A and E was 11 weeks and that of C, 21 weeks. Arrows: e = IgE and g = IgG.

pooled human serum. The slides were placed in a humid chamber for 48 to 72 hours at room temperature, and were washed in 6 or more changes of O.lM NaCl over a period of 3 days. The slides were dried overnight at 42” C., and autoradiography was performed by inverting the dried slides on sheets of Tri-X Ortho photographic film (Eastman Kodak, Rochester, New York) for 8 to 10 weeks.

RESULTS Radioactive IgE appeared in cultures of lung and liver from embryos as early as 11 weeks gestation (Table I and Figs. 1 and 2), and it was found in cultures of lung (Fig. 1) and cultures of spleen (Fig. 3) from a fetus of 21 weeks gestation. Interestingly, radioactive IgG was also found as early as 11 weeks gestation in cultures of the yolk sac, lung, and gastrointestinal tract, but not in cultures of the spleen until 15 to 16 weeks gestation. On the other hand, incubation fluids containing labeled amino acids and the tissues that had been frozen and stored, including lung, liver, and spleen, failed to reveal radioactive IgE or IgG. T5, a 14.5 week fetus that had been aborted with prosta-

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Synthesis

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FIG. 2. lmmunoelectrophoresis of liver culture fluids from two different ceptuses using either IgE (A and C) or pooled human serum (E) as carriers (A and C) and antihuman serum (E) for development. B, D, and F ore the autoradiographs. Arrows: e = IgE, a = albumin, and t = transferrin.

185

11 week

conand anti-IgE corresponding

glandin F,, and was delivered dead, also failed to synthesize radioactive IgG or IgE; the only evidence of protein synthesis in cultures of tissues from this fetus was the production of a few faint radioactive bands in the beta region on the radioimmuneolectrophoresis of liver culture fluid. Cultures of heart from Tl, T4, and T6, pancreas from Tl and T6, kidney, adrenal, and thymus from Tl, T2, T4, and T6, mesentery from Tl, T4, and T6, bone marrow from T2, T3, T4, and T6, blood from T6, choroid plexus, testes, yolk sac, and placenta from T2 and T6, umbilical cord from T4, and gastrointestinal tract including the stomach, small intestine, large intestine, cecum, and appendix from Tl, T2, T4, T5, T6, and T7 were all negative for radioactive IgE. DISCUSSION

The radioactivity found to be associated with IgE on radioimmunoelectrophoresis is indicative of tissue synthesis of IgE and is not due to simple Ending of radiolabeled amino acids with carrier proteins : (1) many tissue culture fluids

186

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CLIN. IMMUNOL. SEPTEMBER 1973

FIG. 3. lmmunoelectrophoresis of culture fluid supernates of spleen from a 21 week fetus using either partially purified IgE (A) or pooled human serum (C) as carriers and unadsorbed anti-lgf (A) and antihuman serum (C) for development. 6 and D are the corresponding autoradiographs. Arrows: e = IgE and g = IgG.

did not yield radioactive IgE although the same carrier proteins were used throughout; (2) stored, frozen tissues incubated with labeled amino acids did not produce radioactive IgE; (3) all culture fluids developed with antisera that did not contain precipitating antibodies to IgE did not yield radioactive IgE bands; (4) carrier IgE incubated with the culture medium containing radiolabeled amino acids did not become radioactive. Using the fluorescent antibody technique, Tada and IshizakalZ investigated the distribution of IgE-forming cells in lymphoid tissues that had been removed surgically from humans and monkeys. They noted IgE-staining plasma cells primarily in the respiratory and gastrointestinal mucosa and in the regional lymph nodes including the peribronchial area, and relatively few IgE plasma cells in the spleen and subcutaneous lymph nodes. In the study reported here, it was found that cultures of human fetal lung, which necessarily included some of the hilar lymph nodes, produced IgE. Although synthesis of IgE occurred as early as 11 weeks gestation in lung and liver, splenic synthesis of IgE was not observed until 21 weeks gestation. Synthesis of IgE in the fetal liver was somewhat surprising, although it should be noted that the liver does show prominent hematopoietic activity from the second to the seventh months of gestation.13 Our failure to find IgE synthesis in the fetal gastrointestinal tract would seem to indicate either that this immunoglobulin is not produced in gut until later in development, perhaps postnatally, or that the technique used was too insensitive to detect it. Detection of IgG and IgE synthesis at 11 weeks and IgM synthesis at 10.5 weeks1 suggests that synthesis of these three immunoglobulins commences at

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about the same time, and would seem to offer support for the theory that each of the immunoglobulin classes of man develops from separate and distinct kymphoid cell lines.14 This would seem to be in distinct contradiction to the concept that the different human immunoglobulin classes arise from differentiation of a single cell line in which the stem cell first develops into IgM-producing cells that in turn later develop into IgG-producing cells and then into IgA-producing cells.15 Disease due to an immediate hypersensitivity reaction can appear in the neonatal period. I* That sensitization could occur in utero by maternofetal transfer of maternal IgE is indicated by the observation that human IgE can cross the simian placenta in small amounts from mother to fetus by simple diffusi0n.l’ However, the data in the present report reveal that synthesis of IgE in utero also occurs, and that the fetus is thus endowed with the potential, in terms of IgE synthesis, for developing active sensitization to antigens that may cross the maternofetal barrier. The authors wish to thank Professor M. L. Rauramo, Chief of Obstetrics and Gynecology, the University of Turku School of Medicine, Turku, Finland, for permitting study of the embryonic and fetal tissues used in this research and for his generosity in allowing us to use the facilities of his department. Also, we wish to thank Professor R. Kouvalainen, formerly of the University of Turku School of Medicine, and presently Chief of Pediatrics at the University of Oulu School of Medicine, Oulu, Finland, for his assistance in the performance of this study.

REFERENCES 1

2 3 4 5 6 7 8

9 10

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Gitlin, D., and Biasueci, A.: Development of 70, yA, yM, &/&, C’l c&erase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, plasminogen, cu,-antitrypsin, orosomucoid, P-lipoprotein, cY,-macroglobulin and prealbumin in the human conceptus, J. Clin. Invest. 48: 1433, 1969. van Furth, R., Schuit, H. R. E., and Hijmans, W.: The immunological development of the human fetus, J. Exp. Med. 122: 1173, 1965. Rowe, D. S., and Fahey, J. L.: A new class of human immunoglobulins. II. Normal serum IgD, J. Exp. Med. 121: 185, 1965. Ishizaka, K., and Ishizaka, T.: Identification of YE-antibodies as a carrier of reaginic activity, J. Immunol. 99: 1187, 1967. Johnasson, S. G. 0.: Serum IgND levels in healthy children and adults, Int. Arch. Allergy 34: 1, 1968. Bazaral, M., Orgel, H. A., and Hamburger, R. N.: IgE levels in normal infants and mothers and an inheritance hypothesis, J. Immunol. 107: 794, 1971. Ogawa, M., Kochwa, S., Smith, C., Ishizaka, K., and McIntyre, 0. R.: Clinical aspects of IgE myeloma, N. Engl. J. Med. 281: 121’7, 1969. Ishizaka, T., Ishizaka, K., Orange, R. P., and Austen, K. R.: The capacity of human immunoglobulin E to mediate the release of histamine and slow reacting substance of anaphylaxis (SRS-A) from monkey lung, J. Immunol. 104: 335, 1979. Patten, B. M.: Human embryology, New York, 1968, McGraw-Hill Book Co., p. 143. Shepard, T. H., Anderson, H. J., and Anderson, H.: The human fetal thyroid. Its weight in relation. to body weight, crown-rump length, foot length and estimated gestation age, Anat. Rec. 148: 123, 1964. Wagner G., and Fuchs, F.: Volume of amniotic fluid in the first half of human pregnancy, J. Obstet. Gynaec. Br. Commonw. 69: 131, 1962. Tada, T., and Ishizaka, K.: Distribution of YE-forming cells in lymphoid tissues of the human and monkey, 5. Immunol. 104: 377, 1970. Arey, L. B. : Developmental anatomy, Philadelphia, 1954, W. B. Saunders Co., p. 253. Gitlin, D.: The differentiation and maturation of specific immune mechanisms, Acta Paediat. Stand. (Suppl.) 172: 60, 1967.

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15 Cooper, M. D., Kincade, P. W., and Lawton, A. R.: Thymus and bursal function in immunologic development, in Kazan, B. M., and Steihm, E. R., editors: Immunologic incompetence, Chicago, 1971, Year Book Medical Publishers, Inc., pp. 81-101. 16 Ingall, M., Glaser, J., Meltzer, R. S., and Dreyfuss, E. M.: Allergic rhinitis in early infancy. Review of the literature and report of a case in a newborn, Pediatrics 35: 108, 1965. 17 Miller, D. L., Zapata, R., Hutchinson, D. L., and Gitlin, D.: Maternofetal passage of human IgE in the pregnant monkey, mouse, rat and guinea pig, Fed. Proc. 32: 1013, 1973.