Immunology 1992 76 636-641
Regulation of leucocyte subpopulations in the sheep endometrium by progesterone S. L. GOTTSHALL & P. J. HANSEN Dairy Science Department, University of Florida, Gainesville, Florida, U.S.A.
Acceptedfor publication 22 March 1992
SUMMARY To determine whether progesterone causes a change in lymphocyte subpopulations in the endometrium, frozen sections of intercaruncular and caruncular endometrium from ewes receiving daily i.m. injections of 100 mg/day progesterone were evaluated by immunohistochemistry for the presence of lymphoid cells bearing CD45, major histocompatibility complex (MHC) class II, CD45R, CD4 and CD8 antigens. The pattern of lymphocyte distribution in the uterine endometrium of untreated ewes was similar to previous reports. Progesterone treatment, particularly after 60 days, caused reductions in numbers of CD45 + cells in the glandular epithelium and associated subepithelial stroma, MHC class II+ cells in all regions of the intercaruncular endometrium and CD45R+ cells in all epithelial regions of intercaruncular and caruncular endometrium. These data demonstrate a role for progesterone in regulating migration or proliferation of endometrial lymphocyte populations; this action of progesterone may represent an important mechanism by which progesterone modifies uterine immune function.
INTRODUCTION One of the proposed functions of progesterone during pregnancy is to inhibit immune responses directed against the allogeneic conceptus.' Indeed, administration of progesterone to ovariectomized animals inhibits uterine responses to tissue grafts2' and bacterial contamination.5'6 There are several possible mechanisms by which progesterone could regulate uterine immune function. It is well documented that progesterone can induce the appearance of uterine secretory products that inhibit mitogen and mixed lymphocyte culture (MLC)induced lymphocyte proliferation in vitro4'7 '9 and antibody responses in vivo.8 Progesterone can also directly inhibit lymphocyte blastogenesis'0 but the supraphysiological concentrations required are higher than concentrations of progesterone causing alterations of uterine immune function in ovariectomized animals.4 Another possibility that has not been examined is that progesterone, or uterine products induced by progesterone, may alter lymphocyte subpopulations within the uterus. In the ewe, the major type of lymphocyte present in the uterine endometrial epithelium is a granulated, CD45R+/major histocompatibility complex (MHC) class II-/CD5- lymphocyte-like cell that may represent a natural killer (NK) cell population.""2 At Day 19 of pregnancy, the number of CD45R+ cells in the endometrium is slightly greater on the side ipsilateral to the conceptus;'3 subsequently, however, there is a depletion of lymphocytes in the regions of the endometrium that combine
with foetal cotyledonary placenta to form the placentomes.'2 The objective of the current experiment was to evaluate whether progesterone treatment causes a change in lymphocyte subpopulations in the endometrium. If so, such an effect could represent one mechanism by which progesterone limits uterine immune responses. MATERIALS AND METHODS
Materials Progesterone and glycerol-gelatin were obtained from Sigma (Chemical Co., St Louis, MO), OCT embedding compound was purchased from American Scientific (Illinois) and Histoscan Universal Monoclonal Detector Kit was purchased from Biomeda (Foster City, CA). Normal goat serum was obtained from Pel-Freeze. Hybridoma supernatants recognizing ovine leucocyte common antigen CD45 [SBU-LCA, monoclonal antibody (mAb) 1-28], CD4 (SBU-T4, mAb 44-38+44-97), CD8 (SBUT8, mAb 38-65), CD45R (SBU-p220, mAb 20-96) and MHC class II (SBU-II, mAb 49-1) were generously provided by Dr K. J. Gogolin-Ewens (Center for Animal Biotechnology, University of Melbourne, Parkville, Australia). Details on preparation of antibodies can be found elsewhere.'2 A supernatant from cultures of non-producer BALB/c myeloma fusion partner (HL-4 cells) was provided by the Hybridoma Facility of the University of Florida Interdisciplinary Core for Biotechnology Research (Gainesville, FL). Collection of uterine tissues One month prior to the experimental period, Rambouillet-type ewes were ovariectomized and randomly assigned to receive
Correspondence: Dr P. J. Hansen, PO Box 110920, University of Florida, Gainesville, FL 32611-0920, U.S.A.
636
Effects of progesterone on endometrial leucocytes daily i.m. injections of 100 mg progesterone in 2 ml 90:10 corn oil:ethanol (v/v) for 0 (n=3), 10 (n=3), 30 (n=4) or 60 days (n = 3). Each ewe was killed 1 day after the conclusion of the treatment period and the uterus removed and immediately processed. The uterine lumen was flushed with 20-30 ml of sterile saline before collection of tissues. Four samples, two from each horn, of intercaruncular and caruncular endometrium were collected, embedded in OCT compound, and snap frozen in liquid nitrogen-isopentane. Caruncular endometrium represents the portion of the endometrium that, together with the foetal cotyledons, form the placentomes, while intercaruncular endometrium is the region of the endometrium rich in uterine glands.
Immunohistochemistry Tissue blocks were stored at -70° until sectioned. Sections of 6 gm thickness were cut with a cryostat microtome, allowed to air dry for at least 1 hr, and then fixed with 95% ethanol for a minimum of 10 min and stored at -20° until immunostaining was performed. Immunohistochemistry for localization of surface antigens was performed via an indirect peroxidase technique using a streptavidin-peroxidase conjugate detection kit (Histoscan, Biomeda) using instructions provided by the manufacturer. Briefly, sections were rehydrated in phosphatebuffered saline (PBS) containing 1% (v/v) normal goat serum and treated for 15 min with a 3% (v/v) H202 solution to quench endogenous peroxidase activity. Sections were incubated with undiluted primary antibody for 30 min, washed with PBS containing 1% (v/v) normal goat serum and incubated with biotin-labelled second antibody for 30 min. Sections were then incubated with streptavidin-peroxidase, exposed to chromogen and counterstained with haematoxylin. Coverslips were mounted with glycerol-gelatin. All incubations were performed at room temperature. A negative control for non-specific binding was carried out by substituting culture supernatants of myeloma fusion partner for specific antibody.
Analysis of immunostaining The relative numbers of cells staining positive for CD45, MHC class II and CD45R antigens were estimated subjectively. For each ewe, two tissue samples of caruncular endometrium and two tissue samples of intercaruncular endometrium were examined. One of the samples was from the left uterine horn and the other was from the right uterine horn. The number of cells was estimated by assigning an intensity score of from 0 (no staining) to 4 (most intense staining). Cell numbers were estimated in three separate areas of intercaruncular endometrium. These areas were: (1) the lumenal epithelium and immediately adjacent stroma; (2) the glandular epithelium and adjacent stroma (intercaruncular endometrium only); and (3) the remaining stroma (termed the stroma proper). For CD45R+ cells, cells were overwhelmingly in epithelia and only these regions were scored. Each tissue section was evaluated independently by two observers. For each area, values for staining intensity were averaged across tissue samples and observers. To determine if there was a significant effect of length of progesterone treatment on intensity score, simple regression analysis was performed to determine the linear relationship between days of progesterone treatment and staining intensity.
637
RESULTS CD45
Cells staining with antibody to CD45 were located in the lumenal and glandular epithelium and immediately adjacent stroma (Fig. IC). For lumenal regions, the number of CD45+ cells in the underlying stroma was greater than the number of cells staining within the epithelium itself, whereas the opposite was true for glandular areas. There were also some CD45 + cells scattered in the stroma proper. In all regions, the majority of CD45+ cells had a lymphocyte-like appearance. In the intercaruncular endometrium, increasing length of exposure to progesterone was associated with a decrease (P < 0-05) in the number of CD45 + cells in the glandular epithelium and associated subepithelial stroma (Fig. 2). A reduction in intensity was only seen after 60 days of progesterone treatment. There was no significant effect of treatment on numbers of CD45+ cells in other regions of the endometrium, however (Fig. 2).
MHC class II Cells staining for MHC class II antigen were widely distributed throughout the intercaruncular and caruncular endometrium (Fig. 1 B). Staining was most intense in the lumenal epithelium
and associated subepithelial layers of intercaruncular and caruncular endometrium. In many sections the entire lumenal epithelium stained intensely red, making it impossible to discern individual cells. Most positive cells of the stroma proper were like macrophages (large in size and irregularly shaped). Positive cells were also occasionally associated with vascular elements, in particular within the endothelium itself and in the musculature of arterioles. There was also some staining of granular, extracellular material, especially in the glandular epithelium and lumens. In the intercaruncular endometrium, ewes treated with progesterone for 60 days had fewer MHC class II+ cells in all regions, resulting in a significant effect of duration of progesterone treatment on staining intensity in lumenal epithelium and associated subepithelial stroma (P < 0-06), glandular epithelium and subepithelial stroma (P < 0-05) and stroma proper (P < 0-06) (Fig. 3). There was, however, no effect of progesterone treatment on staining intensity for any regions of the caruncular endometrium (Fig. 3).
CD45R For ewes treated with progesterone for 0 or 10 days, CD45R+ cells were numerous in both intercaruncular and caruncular endometrium. Positive cells were located almost exclusively in lumenal and glandular epithelium. Only a few cells were seen in the stroma and these were usually adjacent to epithelium. Most cells had a lymphocyte-like appearance (Fig. I D). Similar staining patterns were present in ewes treated with progesterone for 30 or 60 days except that the number of cells was greatly reduced, especially after 60 days of treatment (effect of days of progesterone treatment: P
Regulation of leucocyte subpopulations in the sheep endometrium by progesterone S. L. GOTTSHALL & P. J. HANSEN Dairy Science Department, University of Florida, Gainesville, Florida, U.S.A.
Acceptedfor publication 22 March 1992
SUMMARY To determine whether progesterone causes a change in lymphocyte subpopulations in the endometrium, frozen sections of intercaruncular and caruncular endometrium from ewes receiving daily i.m. injections of 100 mg/day progesterone were evaluated by immunohistochemistry for the presence of lymphoid cells bearing CD45, major histocompatibility complex (MHC) class II, CD45R, CD4 and CD8 antigens. The pattern of lymphocyte distribution in the uterine endometrium of untreated ewes was similar to previous reports. Progesterone treatment, particularly after 60 days, caused reductions in numbers of CD45 + cells in the glandular epithelium and associated subepithelial stroma, MHC class II+ cells in all regions of the intercaruncular endometrium and CD45R+ cells in all epithelial regions of intercaruncular and caruncular endometrium. These data demonstrate a role for progesterone in regulating migration or proliferation of endometrial lymphocyte populations; this action of progesterone may represent an important mechanism by which progesterone modifies uterine immune function.
INTRODUCTION One of the proposed functions of progesterone during pregnancy is to inhibit immune responses directed against the allogeneic conceptus.' Indeed, administration of progesterone to ovariectomized animals inhibits uterine responses to tissue grafts2' and bacterial contamination.5'6 There are several possible mechanisms by which progesterone could regulate uterine immune function. It is well documented that progesterone can induce the appearance of uterine secretory products that inhibit mitogen and mixed lymphocyte culture (MLC)induced lymphocyte proliferation in vitro4'7 '9 and antibody responses in vivo.8 Progesterone can also directly inhibit lymphocyte blastogenesis'0 but the supraphysiological concentrations required are higher than concentrations of progesterone causing alterations of uterine immune function in ovariectomized animals.4 Another possibility that has not been examined is that progesterone, or uterine products induced by progesterone, may alter lymphocyte subpopulations within the uterus. In the ewe, the major type of lymphocyte present in the uterine endometrial epithelium is a granulated, CD45R+/major histocompatibility complex (MHC) class II-/CD5- lymphocyte-like cell that may represent a natural killer (NK) cell population.""2 At Day 19 of pregnancy, the number of CD45R+ cells in the endometrium is slightly greater on the side ipsilateral to the conceptus;'3 subsequently, however, there is a depletion of lymphocytes in the regions of the endometrium that combine
with foetal cotyledonary placenta to form the placentomes.'2 The objective of the current experiment was to evaluate whether progesterone treatment causes a change in lymphocyte subpopulations in the endometrium. If so, such an effect could represent one mechanism by which progesterone limits uterine immune responses. MATERIALS AND METHODS
Materials Progesterone and glycerol-gelatin were obtained from Sigma (Chemical Co., St Louis, MO), OCT embedding compound was purchased from American Scientific (Illinois) and Histoscan Universal Monoclonal Detector Kit was purchased from Biomeda (Foster City, CA). Normal goat serum was obtained from Pel-Freeze. Hybridoma supernatants recognizing ovine leucocyte common antigen CD45 [SBU-LCA, monoclonal antibody (mAb) 1-28], CD4 (SBU-T4, mAb 44-38+44-97), CD8 (SBUT8, mAb 38-65), CD45R (SBU-p220, mAb 20-96) and MHC class II (SBU-II, mAb 49-1) were generously provided by Dr K. J. Gogolin-Ewens (Center for Animal Biotechnology, University of Melbourne, Parkville, Australia). Details on preparation of antibodies can be found elsewhere.'2 A supernatant from cultures of non-producer BALB/c myeloma fusion partner (HL-4 cells) was provided by the Hybridoma Facility of the University of Florida Interdisciplinary Core for Biotechnology Research (Gainesville, FL). Collection of uterine tissues One month prior to the experimental period, Rambouillet-type ewes were ovariectomized and randomly assigned to receive
Correspondence: Dr P. J. Hansen, PO Box 110920, University of Florida, Gainesville, FL 32611-0920, U.S.A.
636
Effects of progesterone on endometrial leucocytes daily i.m. injections of 100 mg progesterone in 2 ml 90:10 corn oil:ethanol (v/v) for 0 (n=3), 10 (n=3), 30 (n=4) or 60 days (n = 3). Each ewe was killed 1 day after the conclusion of the treatment period and the uterus removed and immediately processed. The uterine lumen was flushed with 20-30 ml of sterile saline before collection of tissues. Four samples, two from each horn, of intercaruncular and caruncular endometrium were collected, embedded in OCT compound, and snap frozen in liquid nitrogen-isopentane. Caruncular endometrium represents the portion of the endometrium that, together with the foetal cotyledons, form the placentomes, while intercaruncular endometrium is the region of the endometrium rich in uterine glands.
Immunohistochemistry Tissue blocks were stored at -70° until sectioned. Sections of 6 gm thickness were cut with a cryostat microtome, allowed to air dry for at least 1 hr, and then fixed with 95% ethanol for a minimum of 10 min and stored at -20° until immunostaining was performed. Immunohistochemistry for localization of surface antigens was performed via an indirect peroxidase technique using a streptavidin-peroxidase conjugate detection kit (Histoscan, Biomeda) using instructions provided by the manufacturer. Briefly, sections were rehydrated in phosphatebuffered saline (PBS) containing 1% (v/v) normal goat serum and treated for 15 min with a 3% (v/v) H202 solution to quench endogenous peroxidase activity. Sections were incubated with undiluted primary antibody for 30 min, washed with PBS containing 1% (v/v) normal goat serum and incubated with biotin-labelled second antibody for 30 min. Sections were then incubated with streptavidin-peroxidase, exposed to chromogen and counterstained with haematoxylin. Coverslips were mounted with glycerol-gelatin. All incubations were performed at room temperature. A negative control for non-specific binding was carried out by substituting culture supernatants of myeloma fusion partner for specific antibody.
Analysis of immunostaining The relative numbers of cells staining positive for CD45, MHC class II and CD45R antigens were estimated subjectively. For each ewe, two tissue samples of caruncular endometrium and two tissue samples of intercaruncular endometrium were examined. One of the samples was from the left uterine horn and the other was from the right uterine horn. The number of cells was estimated by assigning an intensity score of from 0 (no staining) to 4 (most intense staining). Cell numbers were estimated in three separate areas of intercaruncular endometrium. These areas were: (1) the lumenal epithelium and immediately adjacent stroma; (2) the glandular epithelium and adjacent stroma (intercaruncular endometrium only); and (3) the remaining stroma (termed the stroma proper). For CD45R+ cells, cells were overwhelmingly in epithelia and only these regions were scored. Each tissue section was evaluated independently by two observers. For each area, values for staining intensity were averaged across tissue samples and observers. To determine if there was a significant effect of length of progesterone treatment on intensity score, simple regression analysis was performed to determine the linear relationship between days of progesterone treatment and staining intensity.
637
RESULTS CD45
Cells staining with antibody to CD45 were located in the lumenal and glandular epithelium and immediately adjacent stroma (Fig. IC). For lumenal regions, the number of CD45+ cells in the underlying stroma was greater than the number of cells staining within the epithelium itself, whereas the opposite was true for glandular areas. There were also some CD45 + cells scattered in the stroma proper. In all regions, the majority of CD45+ cells had a lymphocyte-like appearance. In the intercaruncular endometrium, increasing length of exposure to progesterone was associated with a decrease (P < 0-05) in the number of CD45 + cells in the glandular epithelium and associated subepithelial stroma (Fig. 2). A reduction in intensity was only seen after 60 days of progesterone treatment. There was no significant effect of treatment on numbers of CD45+ cells in other regions of the endometrium, however (Fig. 2).
MHC class II Cells staining for MHC class II antigen were widely distributed throughout the intercaruncular and caruncular endometrium (Fig. 1 B). Staining was most intense in the lumenal epithelium
and associated subepithelial layers of intercaruncular and caruncular endometrium. In many sections the entire lumenal epithelium stained intensely red, making it impossible to discern individual cells. Most positive cells of the stroma proper were like macrophages (large in size and irregularly shaped). Positive cells were also occasionally associated with vascular elements, in particular within the endothelium itself and in the musculature of arterioles. There was also some staining of granular, extracellular material, especially in the glandular epithelium and lumens. In the intercaruncular endometrium, ewes treated with progesterone for 60 days had fewer MHC class II+ cells in all regions, resulting in a significant effect of duration of progesterone treatment on staining intensity in lumenal epithelium and associated subepithelial stroma (P < 0-06), glandular epithelium and subepithelial stroma (P < 0-05) and stroma proper (P < 0-06) (Fig. 3). There was, however, no effect of progesterone treatment on staining intensity for any regions of the caruncular endometrium (Fig. 3).
CD45R For ewes treated with progesterone for 0 or 10 days, CD45R+ cells were numerous in both intercaruncular and caruncular endometrium. Positive cells were located almost exclusively in lumenal and glandular epithelium. Only a few cells were seen in the stroma and these were usually adjacent to epithelium. Most cells had a lymphocyte-like appearance (Fig. I D). Similar staining patterns were present in ewes treated with progesterone for 30 or 60 days except that the number of cells was greatly reduced, especially after 60 days of treatment (effect of days of progesterone treatment: P
