Eur. J. Immunol. 1991. 21: 3049-3052
Polarized expression of MHC class I
3049
Short paper Marion A.Vm Eijkeren*+, Peter J. Peterso and Hans J. GeuzeO Department of Obstetrics and Gynaecologyn and Laboratory of Utrecht, Utrecht
Polarized expression of major histocompatibility complex class I molecules in human endometrial and endocervical epithelial cells The localization of major histocompatibility complex (MHC) class I molecules in human endometrial and endocervical epithelial cells was studied with an immunogoldtechnique on ultrathin cryosections. At the cell surface, MHC class I molecules are delivered solely to the basolateral plasma membrane in human epithelia. Therefore, only the basolateral domain of epithelial cells is capable of presenting antigen to MHC class I-restricted cytotoxic T cells.
1 Introduction
2 Materials and methods
MHC class I molecules play a major role in the surveillance against virus-infected and malignant-transformed cells.CytotoxicT cells recognize antigens in the framework of MHC class I molecules [l]. In the course of biosynthesis, MHC class I molecules follow the constitutive secretory pathway. They are synthesized in the endoplasmic reticulum (ER) and travel through the Golgi complex to the plasma membrane [2,3]. The association with antigen-derived peptide most likely takes place in the ER [4].
2.1 Patients
For a better understanding of immunological processes involved in oncogenesis, detailed studies on the site of expression of MHC class I molecules are mandatory. MHC class I molecules, as studied with indirect immunoperoxidase and immunofluorescencetechniques, are expressed in normal human tissue and in some malignant transformed tissues, though often to a lesser extent [5,6]. However,with these light-microscopical techniques, it is not possible to determine the precise intra- and extracellular localization of MHC class I molecules.The latter is of major importance since surface disposition of MHC class1 molecules is a prerequisite for T cell surveillance. Therefore, we decided to study MHC class I expression in normal human endometrium and endocervix with the immunogold technique on ultrathin cryosections, which allows a precise localization of proteins [7]. In this study polarized expression of MHC class I molecules in human endometrial and endocervical epithelium is demonstrated.
We studied two hysterectomy specimens. Indications for hysterectomy were dysmenorrhea and menorrhagia, respectively.Thepatients were 35 and 37 years of age. Both were healthy and had a regular menstrual cycle. They did not use hormonal medication, nor did they have an intra-uterine device. Recent cervix smears did not reveal signs of viral infection or transformation of cervical epithelial cells. Both uteri were removed at the 10th day of the menstrual cycle. At hysterectomy, one uterus had a normal appearance, the other was mildly leiomyomatously engrossed. 2.2 Tissue processing
Immediately after removal, the cavum uteri was opened and fractions of endometrium and endocervix were cut off and emerged in Karnovsky's fixative solution for 2 h. The tissue blocks were then further processed for cryo-immuno electron microscopy, as described previously [7]. Ultrathin cryosections were incubated for 60 min at room temperature with an antibody derived in rabbit against the H chain of the MHC class I molecule [8] and with protein A gold complexes for 20min. The sections were studied with a JEOL 1200 electron microscope (Tokyo, Japan).
3 Results
Since malignant tumors of endometrium and endocervix derive predominantly from glandular tissue, our interest was focused on the MHC class1 expression in these epithelial cells. Background labeling on nuclei and mito[I 97761 chondria was negligible with an antibody dilution of 1:300. The most striking finding in both patients was the exclusive Supported by a research grant of Parke Davis, Mitchell House, localization of MHC class I molecules on the basolateral Southampton Road, Eastleigh, Hampshire, GB. Dlasma membrane of these eDithelial cells UD to the Cofiespondence: Marion A.Vm Eijkeren, Department of Obstet- junctional complexes. NO MHi3 class I moleckes were rics and Gynaecology, University Hospital Utrecht, Heidelberg- detected on the apical plasma membrane (Figs. 1-4 and laan 100, 3584 CX Utrecht, The Netherlands Table 1). Labeling for MHC class I molecules was more +
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
+
0014-2980/91/1212-3049$3.50 .25/0
3050
M. A.Van Eijkeren, I! J. Peters and H. J. Geuze
Eur. J. Immunol. 1991.21: 3049-3052
Figure 1. This micrograph of endometrial glandular cells shows labeling for MHC class I molecules at the basolateral (thick arrows), but not at the apical plasma membrane. Arrowhead = tight junction; thin arrows = desmosomes; L = luminal side of the cell with microvilli; m = mitochondria. mvb = multivesicular body; bars = 200 nm. Figure 2. See legend to Fig. 1.
Eur. J. Immunol. 1991.21: 3049-3052
Polarized expression of MHC class I
3051
Figure 3. This micrograph shows endometrial glandular cells. Polarized expression of MHC class I molecules at the basolateral plasma membrane (thick arrows) is depicted. Thin arrows = desmosomes; L = luminal side of the cells: m = mitochondria. Figure 4. A micrograph showing endometrial glandular cells. Labeling for MHC class I molecules along the constitutive secretory pathway from the golgi complex (g) towards the plasma membrane (pm) in two adjacent cells. Thin arrow = desmosome. Bars = 200 nm.
3052
M. A.Van Eijkeren, F! J. Peters and H. J. Geuze
Table 1. Expression of MHC class I molecules at the basolateral and apical Plasma membrane (PM) in human t~dometrialand endocervical epithelial cells
Basolateral
ma)
Apical PMa)
Backgroundb)
Endometrium
490
27
16
Endocervix
202
8
7
MHc class I labeling is expressed as number Of gold particles per 100 pm PM. From various endometrial and endocervical cells of both patients, 15 randomly selected micrographs of magruficationsbetween loooo and 15ooo were used for the quantificationof gold particles. Approximately200 ym PM was counted. Background label is expressed as number of gold particles per 100 ym, as counted along a random line through the same 15 micrographs of endometrium and endocervix. The total length of the counted line was approximately 200 ym.
dense in endometrial epithelial cells than in endocervical epithelial cells (Table 1). For this reason only micrographs derived from endometrial tissue are shown in the figures. Intracellularly, labeling for MHC class I molecules was found along the constitutive secretory pathway, i.e. in the ER, the Golgi complex (Fig. 4) and in electron-dense multivesicular bodies (Fig. 1). The tissues described in this study are derived from two patients at the same, i.e. the tenth day of their menstrual cycle, but an influence of hormonal status on MHC class I expression seems unlikely, since no change in immunological status during the menstrual cycle is known.
4 Discussion The exclusively basolateral localization of MHC class I molecules in human endometrial and endocervical epithelia found in this study support and extent previous immunofluorescenceobservations on epithelial cells from human jejunum [9], and indicate that MHC class I molecules are
Eur. J. Immunol. 1991.21: 3049-3052
expressed in a polarized way in human epithelia. Polarized expression of glycoproteins in epithelial cells is a wellknown phenomenon, although the target mechanisms towards either the basolateral or the apical plasma mernbrane are still obscure. Sorting to the apical plasma membrane apparently requires a specific signal, while for sorting to the basolateral plasma membrane no specific signalhas been reported [101. How and where the sorting of MHC class I molecules takes place in epithelial cells are questions that remain to be addressed. The polarized expression of MHC class I molecules in epithelial cells makes Sense from a teleological point of view, since T cells are transported by the bloodstream and hence can only approach epithelial cells at their basolateral and not at their side. The authors wish to thank H . L. Ploegh of the Netherlands Cancer Institute, Amsterdam, The Netherlands, for kindly providing the antibody against human class Z heavy chain, and A . l? M . Heintz, H. L. Ploegh and J. W Slot for critically reading the manuscript.
Received July 15,1991.
5 References 1 Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 1989. 7: 601. 2 Neefjes, J. J., Stollorz,V., Peters, F! J., Geuze, H. J. and Ploegh, H. L., Cell 1990. 62: 171. 3 Peters, I! J., Neefjes, J. J., Oorschot,V., Ploegh, H. L. and Geuze, H. J., Nature 1991. 349: 669. 4 Yewdell, J. W. and Bennink, J. R., Cell 1990. 62: 203. 5 Doyle, A., Martin, W. J., Funa, K., Gazdar, A., Carney, D., Martin, S. E., Linnoila, I., Cuttitta, E, Mulshine, J., Bunn, €! and Minna, J., J. Exp. Med. 1985. 161: 1135. 6 Natali, F! G., Nicotra, M. R., Bigotti, A., Venturo, I., Marcenaro, L., Giacomini, F! and Russo, C., Proc. Natl. Acad. Sci. USA 1989. 86: 6719. 7 Slot, J.W., Geuze, H. J., Gigengack, S., Leinhard, G. E. and James, D., J. Cell Biol. 1991. 113: 123. 8 Neefjes, J. J. and Ploegh, H. L., Eur. J. Immunol. 1988. 18: 801. 9 Gorvel, J. €?,Sarles, J., Maroux, S., Olive, D. and Mawas, C., Biol. Cell 1984. 52: 249. 10 Simons, K. and Wandiger-Ness, A., Cell 1990. 62: 207.
Polarized expression of MHC class I
3049
Short paper Marion A.Vm Eijkeren*+, Peter J. Peterso and Hans J. GeuzeO Department of Obstetrics and Gynaecologyn and Laboratory of Utrecht, Utrecht
Polarized expression of major histocompatibility complex class I molecules in human endometrial and endocervical epithelial cells The localization of major histocompatibility complex (MHC) class I molecules in human endometrial and endocervical epithelial cells was studied with an immunogoldtechnique on ultrathin cryosections. At the cell surface, MHC class I molecules are delivered solely to the basolateral plasma membrane in human epithelia. Therefore, only the basolateral domain of epithelial cells is capable of presenting antigen to MHC class I-restricted cytotoxic T cells.
1 Introduction
2 Materials and methods
MHC class I molecules play a major role in the surveillance against virus-infected and malignant-transformed cells.CytotoxicT cells recognize antigens in the framework of MHC class I molecules [l]. In the course of biosynthesis, MHC class I molecules follow the constitutive secretory pathway. They are synthesized in the endoplasmic reticulum (ER) and travel through the Golgi complex to the plasma membrane [2,3]. The association with antigen-derived peptide most likely takes place in the ER [4].
2.1 Patients
For a better understanding of immunological processes involved in oncogenesis, detailed studies on the site of expression of MHC class I molecules are mandatory. MHC class I molecules, as studied with indirect immunoperoxidase and immunofluorescencetechniques, are expressed in normal human tissue and in some malignant transformed tissues, though often to a lesser extent [5,6]. However,with these light-microscopical techniques, it is not possible to determine the precise intra- and extracellular localization of MHC class I molecules.The latter is of major importance since surface disposition of MHC class1 molecules is a prerequisite for T cell surveillance. Therefore, we decided to study MHC class I expression in normal human endometrium and endocervix with the immunogold technique on ultrathin cryosections, which allows a precise localization of proteins [7]. In this study polarized expression of MHC class I molecules in human endometrial and endocervical epithelium is demonstrated.
We studied two hysterectomy specimens. Indications for hysterectomy were dysmenorrhea and menorrhagia, respectively.Thepatients were 35 and 37 years of age. Both were healthy and had a regular menstrual cycle. They did not use hormonal medication, nor did they have an intra-uterine device. Recent cervix smears did not reveal signs of viral infection or transformation of cervical epithelial cells. Both uteri were removed at the 10th day of the menstrual cycle. At hysterectomy, one uterus had a normal appearance, the other was mildly leiomyomatously engrossed. 2.2 Tissue processing
Immediately after removal, the cavum uteri was opened and fractions of endometrium and endocervix were cut off and emerged in Karnovsky's fixative solution for 2 h. The tissue blocks were then further processed for cryo-immuno electron microscopy, as described previously [7]. Ultrathin cryosections were incubated for 60 min at room temperature with an antibody derived in rabbit against the H chain of the MHC class I molecule [8] and with protein A gold complexes for 20min. The sections were studied with a JEOL 1200 electron microscope (Tokyo, Japan).
3 Results
Since malignant tumors of endometrium and endocervix derive predominantly from glandular tissue, our interest was focused on the MHC class1 expression in these epithelial cells. Background labeling on nuclei and mito[I 97761 chondria was negligible with an antibody dilution of 1:300. The most striking finding in both patients was the exclusive Supported by a research grant of Parke Davis, Mitchell House, localization of MHC class I molecules on the basolateral Southampton Road, Eastleigh, Hampshire, GB. Dlasma membrane of these eDithelial cells UD to the Cofiespondence: Marion A.Vm Eijkeren, Department of Obstet- junctional complexes. NO MHi3 class I moleckes were rics and Gynaecology, University Hospital Utrecht, Heidelberg- detected on the apical plasma membrane (Figs. 1-4 and laan 100, 3584 CX Utrecht, The Netherlands Table 1). Labeling for MHC class I molecules was more +
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
+
0014-2980/91/1212-3049$3.50 .25/0
3050
M. A.Van Eijkeren, I! J. Peters and H. J. Geuze
Eur. J. Immunol. 1991.21: 3049-3052
Figure 1. This micrograph of endometrial glandular cells shows labeling for MHC class I molecules at the basolateral (thick arrows), but not at the apical plasma membrane. Arrowhead = tight junction; thin arrows = desmosomes; L = luminal side of the cell with microvilli; m = mitochondria. mvb = multivesicular body; bars = 200 nm. Figure 2. See legend to Fig. 1.
Eur. J. Immunol. 1991.21: 3049-3052
Polarized expression of MHC class I
3051
Figure 3. This micrograph shows endometrial glandular cells. Polarized expression of MHC class I molecules at the basolateral plasma membrane (thick arrows) is depicted. Thin arrows = desmosomes; L = luminal side of the cells: m = mitochondria. Figure 4. A micrograph showing endometrial glandular cells. Labeling for MHC class I molecules along the constitutive secretory pathway from the golgi complex (g) towards the plasma membrane (pm) in two adjacent cells. Thin arrow = desmosome. Bars = 200 nm.
3052
M. A.Van Eijkeren, F! J. Peters and H. J. Geuze
Table 1. Expression of MHC class I molecules at the basolateral and apical Plasma membrane (PM) in human t~dometrialand endocervical epithelial cells
Basolateral
ma)
Apical PMa)
Backgroundb)
Endometrium
490
27
16
Endocervix
202
8
7
MHc class I labeling is expressed as number Of gold particles per 100 pm PM. From various endometrial and endocervical cells of both patients, 15 randomly selected micrographs of magruficationsbetween loooo and 15ooo were used for the quantificationof gold particles. Approximately200 ym PM was counted. Background label is expressed as number of gold particles per 100 ym, as counted along a random line through the same 15 micrographs of endometrium and endocervix. The total length of the counted line was approximately 200 ym.
dense in endometrial epithelial cells than in endocervical epithelial cells (Table 1). For this reason only micrographs derived from endometrial tissue are shown in the figures. Intracellularly, labeling for MHC class I molecules was found along the constitutive secretory pathway, i.e. in the ER, the Golgi complex (Fig. 4) and in electron-dense multivesicular bodies (Fig. 1). The tissues described in this study are derived from two patients at the same, i.e. the tenth day of their menstrual cycle, but an influence of hormonal status on MHC class I expression seems unlikely, since no change in immunological status during the menstrual cycle is known.
4 Discussion The exclusively basolateral localization of MHC class I molecules in human endometrial and endocervical epithelia found in this study support and extent previous immunofluorescenceobservations on epithelial cells from human jejunum [9], and indicate that MHC class I molecules are
Eur. J. Immunol. 1991.21: 3049-3052
expressed in a polarized way in human epithelia. Polarized expression of glycoproteins in epithelial cells is a wellknown phenomenon, although the target mechanisms towards either the basolateral or the apical plasma mernbrane are still obscure. Sorting to the apical plasma membrane apparently requires a specific signal, while for sorting to the basolateral plasma membrane no specific signalhas been reported [101. How and where the sorting of MHC class I molecules takes place in epithelial cells are questions that remain to be addressed. The polarized expression of MHC class I molecules in epithelial cells makes Sense from a teleological point of view, since T cells are transported by the bloodstream and hence can only approach epithelial cells at their basolateral and not at their side. The authors wish to thank H . L. Ploegh of the Netherlands Cancer Institute, Amsterdam, The Netherlands, for kindly providing the antibody against human class Z heavy chain, and A . l? M . Heintz, H. L. Ploegh and J. W Slot for critically reading the manuscript.
Received July 15,1991.
5 References 1 Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 1989. 7: 601. 2 Neefjes, J. J., Stollorz,V., Peters, F! J., Geuze, H. J. and Ploegh, H. L., Cell 1990. 62: 171. 3 Peters, I! J., Neefjes, J. J., Oorschot,V., Ploegh, H. L. and Geuze, H. J., Nature 1991. 349: 669. 4 Yewdell, J. W. and Bennink, J. R., Cell 1990. 62: 203. 5 Doyle, A., Martin, W. J., Funa, K., Gazdar, A., Carney, D., Martin, S. E., Linnoila, I., Cuttitta, E, Mulshine, J., Bunn, €! and Minna, J., J. Exp. Med. 1985. 161: 1135. 6 Natali, F! G., Nicotra, M. R., Bigotti, A., Venturo, I., Marcenaro, L., Giacomini, F! and Russo, C., Proc. Natl. Acad. Sci. USA 1989. 86: 6719. 7 Slot, J.W., Geuze, H. J., Gigengack, S., Leinhard, G. E. and James, D., J. Cell Biol. 1991. 113: 123. 8 Neefjes, J. J. and Ploegh, H. L., Eur. J. Immunol. 1988. 18: 801. 9 Gorvel, J. €?,Sarles, J., Maroux, S., Olive, D. and Mawas, C., Biol. Cell 1984. 52: 249. 10 Simons, K. and Wandiger-Ness, A., Cell 1990. 62: 207.
