Endocrine gland function during intrauterine life is very important for fetal survival and development. As a result, inadequate immune protection of the endocrine system during this period could result in serious disruption of fetal development (Filiushkin et al., 1998; Harrell and Murray, 1998). The largest immune-protective mechanism in adults is the secretory (mucosal) immune system (SIS) which has been found in the gastrointestinal, respiratory and urogenital tracts (Brandtzaeg, 1992; Goldblum et al., 1996; Kondi-Paphitis et al., 1999; McGhee and Kiyono, 1999). It consists of protein components, such as the secretory component (SC) of the poly-immunoglobulin receptor (Brandtzaeg et al., 1992), joining (J) chain, different immunoglobulins (Igs), and immune-competent cells, particularly Ig-secreting ones (Brandtzaeg, 1995; Goldblum et al., 1996; McGhee and Kiyono, 1999). Suchcomponents have been described recently in the thyroid of adults as Igs (Davila et al., 1988) and SC (Kondi-Paphitis et al., 2000).

Reports on the formation of the SIS during prenatal life are limited to several publications that describe the SIS of the gastrointestinal and respiratory tracts and their glands during the second half of the gestation period (Thrane et al., 1991; Ben-Hur et al., 1997; Hayashi et al., 1999). In principle, this SIS is similar to those of adults. However, many lymphoid structures, such as the tonsils, Peyer’s patches, solitary follicles of the intestine and plasma cells, are absent or poorly developed before birth (Hayward and Lawton, 1977; Gurevich et al., 1997, 2001). We are not aware of any reports on SIS components in the fetal endocrine system. Because of its ontogenic importance, we investigated, by immunohistochemical means, the components of the SIS in fetal endocrine glands and their embryonic precursors throughout intrauterine life.

Avidin-biotin complexation and peroxidase technique with commercial markers were used to evaluate the various components of the SIS, namely the SC and J chain, IgA, IgM, IgG, macrophages, and different subsets of lymphocytes. The monoclonal markers were obtained from Novocastra Labs, Newcastle, UK (NCL05 for SC, PS1 for CD3, IF6 for CD4, NCL295 for CD8, MJ1 for CD20, NCL for CD68), BioGenex, San Ramon, CA (374M for J chain), Zymed Labs, San Francisco, CA (Z003 for IgA, HP6083 for IgM), and Dako, Denmark (polyclonal marker AO423 for IgG). High-temperature (10 min at 92°C) antigen unmasking was used in most reactions. For evaluation of SC, sections were treated with trypsin, whereas J chains were immunostained according to the standard protocol without additional treatment. Antibodies were diluted according to manufacturer recommendations: anti-SC and J chain 1:20, ant-IgA and IgM 1:50, ant-IgG 1:500, anti-CD 1:20 to 1:200.Sections were incubated for 60 min at 37°C and were also stained with hematoxylin.

The anterior portion of the pituitary body of 8- to 9-week-old embryos is a tubular structure that makes two or three turns in the Turkish saddle, and is lined with multiple layers of epithelium. Of these cells, which are derived from the Rathke’s pouch, 53% to 68% were strongly immunopositive for SC (Figure 1A). In the second trimester, SC was seen in 15% to 23% of these cells, and in the third trimester in 5% to 8% of them (Figure 1B). In glandular cells of the pituitary pars intermedia, immunostaining for SC was positive in 68% to 88% of them during the second and third trimesters, but no SC was present in the neurohypophysis. J chain (Figure 1C), IgG and IgA were observed in all epithelial cells of the pars anterior and intermedia of the pituitary body, but only weakly in the neurohypophysis. In a few instances, weak positive immunostaining for IgM was observed.

The thyroid gland at 7 to 9 weeks of development consists of trabecular and alveolar groups of epithelial cells, and 85% to 97% of them contained SC and J chain and were weakly positive to IgG, IgA and IgM. When its follicular structure became defined at 10 to 11 weeks, the follicular epithelium waspositive for SC, J chain (Figure 2A,B) and Igs. The follicular colloid was, however, more immunopositive for IgG, IgA and IgM than the follicular epithelium (Figure 2C), and negative for SC and J chain (Figure 2A,B).

Parathyroid glands were found in seven cases; in three of them, weak positive immunostaining for SC, J chain and Igs was observed in the light chief cells.

Epithelial cells of pancreatic acini and ducts were strongly positive for SC, J chain (Figure 3A,B) and Igs. SC immunostaining of pancreatic islets cells was negative in most specimens, but a few of them exhibited weak staining (Figure 3A). Islet cells were more positive to J chain and Igs than acinar and ductal cells (Figure 3B).

Adrenal fetal cortex cells in the second and third trimesters were weakly positive for J chain, IgA, IgG and IgM, and negative for SC. In definitive cortex cells and in developing medullary cells, the immunostaining for SC, J chain and Igs was negative.

Table 1 demonstrates the increasing numbers of immunocompetent cells in the various endocrine glands as development progressed. Lymphocytes secreting IgA and IgM appeared after 10 to 11 weeks of pregnacy.

Massive antigenic exposure due to chorioamnionitis at any time during ontogeny (Group II) caused little change in the distribution and immunoreactivity of SC and J chain in the endocrine cells (Figure 3A,B). However, with chorioamnionitis, immunoreactivity of Igs declined relative to Group I (Figure 3C), especially if infection occurred during the second or third trimesters. A parallel decrease was observed in the number of Ig-positive endocrine cells. In the thyroid, Ig-positive follicular cells amounted to less than 12%, compared to 85% to 97% in Group I, and weak Ig-immunoreactivity was present in the follicular colloid. In the pancreas, Igs were found in 45% to 59% of islet cells and in 2% to 5% of acinar cells. These values are much lower than in pancreatic cells of Group I (69%-88% and 53-62%, respectively).

Chorioamnionitis also resulted in the reduced presence of different subsets lymphocytes in the stroma of the endocrine glands, relative to Group I (Table 1). In contrast, more lymphocytes that secrete IgA and especially IgM were present in the regional (cervical and retroperitoneal) lymph nodes, from 0.1 and 0.4/50,000 µm² in Group I, to 2.8 and 3.9/50,000 µm² in Group II, respectively.

Not all SIS components appear to be same extent in the different endocrine organs. J chain is constantly present in all endocrine cells of developing fetuses and in the precursor cells of embryos. The content and reactivity of Igs change with maturation of the embryos or fetuses and with the functional status of the endocrine glands. Embryos 4 to 7 weeks into pregnancy do not have their own Ig-producing lymphocytes, and all endocrine gland precursors contain maternal IgG passing through the placental barrier (Madani and Heiner, 1989). Maternal IgA has been found to be potentially available to embryos in the first trimester of gestation (Jauniaux et al., 1995). Together, these data show that the embryonic SIS functions via maternal antibodies. In the adrenals, Ig immunoreactivity decreases during the second and third trimesters. Massive foreign antigenic effects, as a consequence of chorioamnionitis, also decrease Ig immunoreactivity in other endocrine cells, and frequently in the colloid of the thyroid.

The third protein component, the SC, is present in some endocrine organs (thyroid, pars intermedia of the pituitary body, sometimes in the anterior lobe of the hypophysis and the pancreatic islets) and consistently in cells of the endocrine gland precursors. The differential presence of SC in different endocrine glands is closely related to changes in organs’ cellular activity during intrauterine life. Cells of pancreatic acini and ducts that perform excretory functions, including excretion of various Igs, consistently contain SC. Pancreatic islet cells, which are derived from pancreatic acini, perform only endocrine functions and contain no, or just trace amounts of SC. These pancreatic islet cells have lost the ability to secrete Igs, storing them instead in the cytoplasm. This is very pronounced in specimens from Group II where massive antigenic stimulation caused large secretion of Igs from acinar and ductal cells containing SC, as witnessed by their negative staining for Igs; however this event had no influence on the Ig content of islet cells (Figure 3C).

A similar pattern of the SIS was found during the transformation of the Rathke’s pouch epithelium into cells of the adenohypophysis. During this process, loss of cellular excretory function is accompanied by loss of SC. Thus, we might conclude that the absence of SC and storage of Igs in pancreatic islet and adenohypophysis cells are related events. However, epithelial cells of the pars intermedia of the pituitary body and thyroid follicular cells contain SC during their entire intrauterine life and still preserve the ability to secrete Igs. After secretion, these Igs are stored in the follicular colloid, which contains more of them than the follicular epithelium (Figure 2C). These data reflect the close relationship between the presence of SC in endocrine cells and their ability to exocrine secrete Igs. This allows us to link the presence of SC in the endocrine cells with their ability to excrete, and loss of SC parallels a decrease in this ability. In adrenals, the absence of SC is, perhaps, connected with the mesenchymal origin of cortical cells.

We suggest that accumulation of Igs in cells of the main endocrine glands may act as a local protective mechanism against foreign antigens. Decreased immunoreactivity of Igs in endocrine cells following chorioamnionitis supports this assumption. The protein components of the SIS are detected as early as one month into development, and are present within the endocrine organs during the rest of the gestational period. Thus, the SIS in endocrine organs appears much earlier than the common immune system and its organs (thymus, spleen, lymph nodes), and acquires functional activity (Vetro and Bellanti, 1989; Burgio et al., 1990; Ben-Hur et al., 1998).

Ben-Hur, H., Gurevich, P., Huszar, M., Ziv-Sokolovsky, N., Hagay, Z., Isaegson, I., Berman, V. and Zusman, I. (1997). Immunoglobulin A in the respiratory tract and intrahepatic bile ducts of fetuses and newborns with pneumonia and sepsis. Human Antibodies, 8, 119-23

Brandtzaeg, P. (1992). Humoral immune response patterns of human mucosae: Induction and relation to bacterial respiratory tract infections. J . Infect. Dis., 165, S167-76

Brandtzaeg, P., Halstensen, T.S., Huitfeldt, H.S., Krajci, P., Kvale, D., Scott, H. and Thrane, P.S. (1992). Epithelial expression of HCA, secreting component (poly-Ig receptor) and adhesion molecules in the human alimentary tract. Ann. N.Y. Acad. Sci., 664, 157-79

Burgio, G.R., Ugazio, A.G. and Notarangelo, L.D. (1990). Immunology of the neonate. Curr. Opin. Immunol., 2, 770-777

Davila, R.M., Bedrossian, C.W. and Silverberg, A.B. (1988) Immunocytochemistry of the thyroid in surgical and cytologic specimens. Arch. Pathol. Lab. Med., 112, 51-6

Filiushkin, I.V., Ivanov, A.N., Leshchenko, M.V., Makashina, O.M., Kashirin, V.S., Stetsenko, A.V., Gruden, M.A., Shumova, E.A. and Belchenko, A.N. (1998). Several parameters of the state of the nervous, immune and endocrine system in newborn rats exposed to irradiation during the preimplantation period of embryogenesis. Radiat. Biol. Radioecol., 38, 15-26

Gurevich, P., Ben-Hur, H., Szvalb, S., Moldavsky, M. and Zusman, I. (2001). The lymphoid- epithelial secretory immune system in human fetuses in the second trimester of gestation. Ped. Dev. Pathol., in press

Gurevich, P., Erina, S., Gershon, S. and Zusman, I. (1997) The role of the fetal immune system in the pathogenesis of RhD-hemolytic disease of newborns. Human Antibodies, 8, 76-89

Harrell, G. and Murray, P. (1998) Diagnosis and management of congenital hypothyroidism. J. Prenat. Neonat. Nursing, 11, 75-83

Hayashi, Y., Kurashima, C., Takemura, T. and Hirokawa, K. (1989). Ontogenic development of the secretory immune system in human fetal salivary glands. Pathol. Immunopathol. Res., 8, 314-20

Hayward, A.R. and Lawton, A.R. (1977). Induction of plasma cell differentiation of human fetal lymphocytes: Evidence for functional immaturity of T and B cells. J. Immunol., 119, 1213-17

Jauniaux, E., Jurkovic, D., Gulbis, B., Liesnard, C., Lees, C. and Campbell, S. (1995). Materno-fetal immunoglobulin transfer and passive immunity in the first trimester of human pregnancy. Hum. Reprod., 10, 3297-300

Kondi-Paphitis, A., Carvounis, H., Kairi, E., Frangou, M., Papayanopoulou, A. and Deligeorgi, H. (1999). Expression of a local immune defence system in the female genital tract. An immunohistochemical study. Eur. J. Gynaecol. Oncol., 20, 141-43

Kondi-Paphitis, A., Smyrniotis, V., Frangou, M., Papayanopoulou, A., Englezou, M. and Deligeorgi, H. (2000). Immunohistochemical study of ceruloplasmin, lactoferrin and secretory component expression in neoplastic and non-neoplastic thyroid gland diseases. Acta Oncol., 39, 753-6

Madani, G. and Heiner, D.C. (1989). Antibody transmission from mother to fetus. Curr. Opin. Immunol., 1, 1157-64

McGhee, J.R. and Kiyono, H. (1999). The mucosal immune system. In Paul, W.E. (ed) Fundamental Immunology, edn 4, pp. 909-46. (Philadelphia-N.Y.: Lippincott-Raven Publ.)

Thrane, P.S., Rognum, T.O. and Brandtzaeg, P. (1991). Ontogenesis of the secretory immune system and innate defense factor in human parotid glands. Clin. Exp. Immunol., 86, 342-8

Vetro, S.W. and Bellanti, J.A. (1989) Fetal and neonatal immunoincompetence. Fetal Ther., 4, 82-91

Figure 1
Anterior lobe of the pituitary body. A 9-week-old fetus from a medically indicated abortion. Note SC (arrows) in about 57% of cells (A, ´ 200) and J chain (arrows) in about 82% of epithelial cells (B, ´ 200). A 38-week-old fetus that died of aspiration syndrome. SC is seen in single cells (arrows) (C, ´ 400).

Figure 2
Same 9-weeks-old fetus as in Fig. 1. Note SC in the epithelium of the thyroid (large arrows) and of the trachea (thin arrows). 1, the cartilaginous rings of the trachea. (A, x 200). A 13-week-old fetus after a rupture of uterine horn. Note SC (B, x 400) and J chain (C, x 1000) in the epithelium of thyroid follicles but not in the colloid (small arrows). IgM was seen both in the epithelium of thyroid follicles and at a higher concentration in the colloid (large arrows) (D, ´ 400).

Figure 3
A 16-week-old fetus from a septic abortion. Note the high immunostaining of SC in the epithelium of pancreatic ducts and acini (thin arrows) and very low - in the cells of pancreatic islets (large arrows) (A, ´ 200). A 13-week-old fetus that died of chorioamnionitis. B, J chain in the cells of pancreatic islets (1), acini (2) and ducts (3). C, IgA in the cells of pancreatic islets (large arrow) and in single cells of acini (thin arrows). ´ 400