human uterus. The greatest number of MCs was found in chymase. Those cells that contain only tryptase are designated the inner (i.e. luminal) half of the ...
hrep$$0206
Human Reproduction vol.12, no.2 pp.368–372, 1997
Distribution and heterogeneity of mast cells in the human uterus
Atsushi Mori, Ya li Zhai, Toshihiko Toki, Toshio Nikaido and Shingo Fujii1 Department of Obstetrics and Gynecology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan 1To
whom correspondence should be addressed
Mast cells (MCs) are widely distributed in most human tissues. Those cells that contain only tryptase are designated as T-MCs, while those that also contain chymase are referred to as TC-MCs. This study uses immunohistochemical staining for tryptase and chymase to assess the distribution and heterogeneity of these two types of MCs in the human uterus. The greatest number of MCs was found in the inner (i.e. luminal) half of the myometrium, with this area containing approximately equal proportions of T-MCs and TC-MCs. There were fewer MCs in the outer half of the myometrium and the cervix, but the proportion of TCMCs in both of these areas was substantially higher. In contrast, the endometrium contained significantly fewer MCs, but proportionally more T-MCs. There was no change in the number of MCs between the proliferative and secretory phases of the menstrual cycle; however, there was a significantly lower number in all areas after menopause. Most of the MCs were observed in close association with uterine smooth muscle cells, as well as in the vicinity of fibroblasts and collagen, and it appears they may play an important role in the reconstruction of uterine tissues during the menstrual cycle. Key words: chymase/mast cell/tryptase/uterus
Introduction Mast cells (MCs) are widely distributed in most human tissues. The human myometrium has a considerable number of MCs, but the function of these cells in the uterus has not been clearly established (Crow et al., 1991). A variety of observations has been made in regard to the nature of MCs in the uterus. For example, it has been suggested that degranulation of myometrial MC mediators such as histamine and serotonin might facilitate strong contractions at parturition (Rudolph et al., 1993). Also, the number of MCs in the uterus of rodents is known to decrease around the time of implantation (Hore and Mehrotra, 1988). On the other hand, the insertion of an intrauterine contraceptive device into the hamster leads to an increase in the number of myometrial MCs (Moore et al., 1979). Also, there is a high incidence of uterine inversions in mutant strains of mice that lack MCs, and this suggests that MCs may be associated with the occurrence of placental 368
accretion, or with relaxation of the myometrium and cervix (Yokoi et al., 1994). Although it is possible that MCs have a wide variety of roles in the female reproductive tract, there is only limited information about the distribution and heterogeneity of these cells in human uterine tissue. Previously, in rodents, MCs have been classified into either mucosal mast cells (MMC), or connective tissue mast cells (CTMC), depending on their histological location and on their size and mediator composition (Enerback, 1966). However, in humans, MCs cannot be clearly classified by location, but instead are categorized according to their content of the specific neutral proteases tryptase and chymase. Those cells that contain only tryptase are designated T-MCs, while those that contain chymase in addition to tryptase are designated as TC-MCs (Irani et al., 1986, 1989; Irani and Schwartz, 1989). These cells appear to be analogous to the rodent MMC and CTMC, respectively. Because these two types of MCs have different biochemical compositions (Bradding et al., 1995) and respond differently to stimulatory agents (Galli, 1990), a more thorough analysis of their distribution is essential for a better understanding of their functions in the human uterus. For this purpose, the present study uses immunohistochemical staining for tryptase and chymase to further assess the distribution and heterogeneity of MCs in the human uterus.
Materials and methods Experimental tissue Tissue was collected from 24 women who had been diagnosed with any of several different gynaecological disorders (i.e. seven with leiomyomas, eight with carcinoma in situ, six with utero-vaginal prolapse, and three with benign ovarian tumours). Uterine (n 5 24) and cervical (n 5 17) samples were extirpated in a manner that included the mucosa. Tissue from the small intestine (n 5 2) was obtained from the jejunum at the time of surgery for a gastric bypass procedure in order to have at least one extra-uterine source to examine for MC distribution. The tissues were used with the approval of the Ethical Committee of Shinshu University, Japan, and only after obtaining written consent from the patients. All experimental tissues were fixed immediately in Carnoy’s fixative overnight and embedded in paraffin by standard procedures. The stage of the menstrual cycle was determined by histological analysis of the endometrium (Noyes et al., 1950). The uterine tissues (i.e. both endometrial and myometrial samples) were obtained from 11 women in the proliferative phase and five in the secretory phase, while eight women were postmenopausal. The cervical tissue was obtained from six women in the proliferative phase, five in the secretory phase, and six were postmenopausal. © European Society for Human Reproduction and Embryology
Mast cells in the human uterus
Double-staining for chymase and tryptase To determine the phenotypes of the MCs, sections of tissue were double-stained for two proteases that are known to be present in MCs. For this purpose, tissue sections were deparaffinized in xylene and rehydrated in graded concentrations of ethanol. Endogenous peroxidase activity was blocked by treating the tissue with 0.03% hydrogen peroxide in methanol for 20 min. The tissue was exposed to 10% normal rabbit serum to minimize non-specific reactivity. Then, the sections were incubated with anti-chymase monoclonal antibody (0.2 µg/ml; Chemicon International Inc., Temecula, CA, USA), or with non-immunized mouse serum (as a control) at 4°C overnight. After rinsing with 0.05 M Tris–HCl (pH 7.4), the tissue was exposed to anti-mouse immunoglobulin (Ig)G at room temperature for 30 min. After rinsing again, the tissue was exposed to an alkaline phosphatase–antialkaline phosphatase (APAAP) solution (Dako, Glostrup, Denmark) for 30 min. Subsequently, the tissue was treated with nitroblue tetrazolium (NTB) in order to visualize the histological distribution of the chymase antibody. To dissociate the anti-chymase antibody from the enzyme, the sections were rinsed twice with 0.1 M glycine–HCl buffer (pH 2.2) for 60 min. Then, they were exposed again to 10% normal rabbit serum. Subsequently, the sections were incubated with anti-tryptase monoclonal antibody (0.2 µg/ml; Chemicon), or with non-immunized mouse serum (as a control) at room temperature for 60 min. After rinsing with phosphate-buffered saline (PBS), the tissue was treated with peroxidase-conjugated anti-mouse IgG (Amersham, Buckinghamshire, UK) for 60 min and then exposed to 0.05% 3,39-diaminobenzidine (DAB) to visualize the tryptase. This technique allowed the detection of tryptase as a brown colour that was apparent after treatment of the tissue with the DAB chromagen. In contrast, chymase was visualized by treatment of the tissue with the NBT chromagen, which produced a strong blue stain that usually masked the brown colour of DAB. Double-staining for α-smooth muscle actin and tryptase, von Willebrand factor and tryptase, and collagen type III and tryptase Since the bioactive agents in MCs are thought to have a paracrine effect on surrounding cells, the specific location of MCs is usually associated with the function of these cells. To analyse the distribution of MCs in the myometrium, sections of uterine tissue were stained for tryptase to identify the MCs, and then double-stained with either α-smooth muscle actin to distinguish smooth muscle cells, von Willebrand factor to reveal endothelial cells, or for collagen type III to assess the distribution of MCs among collagenous tissue in the uterus. Tissue sections were processed as described above. The sections that were prepared for immunohistochemical staining for α-smooth muscle actin and von Willebrand factor were incubated in 0.1% trypsin (Gibco BRL, Gaithersburg, PA, USA) in PBS at 37°C for 30 min. The sections for staining collagen type III were incubated in 0.25% trypsin in PBS at 37°C for 60 min. After blocking endogenous peroxidase activity, the sections were exposed to 10% normal rabbit serum, and then incubated with either anti-α-smooth muscle actin monoclonal antibody (1:100 dilution, Dako), anti-von Willebrand factor monoclonal antibody (1:30 dilution, Dako), anti-collagen type III antibody (1:200 dilution; Fujiyakuhin Co, Takaoka, Japan), or non-immunized mouse serum (as a control) at 4°C overnight. After rinsing with PBS, the sections were treated with peroxidase-conjugated anti-mouse IgG for 30 min and then exposed to DAB for visualization of the distribution of the specific monoclonal antibodies. After the sections were rinsed twice in 0.1 M glycine–HCl buffer (pH 2.2) for 60 min, they were treated once again with normal rabbit serum. Subsequently, the sections were incubated with anti-tryptase
monoclonal antibody, or with non-immunized mouse serum (as a control) at room temperature for 60 min. After rinsing the sections in 0.05 M Tris–HCl (pH 7.4), they were exposed to anti-mouse IgG at room temperature for 30 min. Then, they were treated with APAAP solution at room temperature for 30 min and visualized by exposure to naphthol AS-MX phosphate and Fast Red TR reagents. Finally, the sections were counter-stained with Mayer haematoxylin. Mast cell counts and statistical analysis The number of MCs were counted in five high-power fields of the endometrium, the inner and outer half of the myometrium, and the cervix. The results are expressed as the number of MCs (mean 6 SD) per one high-power field. Statistical comparisons of the means were carried out by the unpaired t-test and one-way analysis of variance followed by the Scheffe´ test. Computations were performed using StatView 4.0A (Abacus Concept Inc., Berkeley, CA, USA). Differences were considered to be significant when P ,0.05.
Results Double-staining for tryptase and chymase Following staining, the T-MCs were brown (Figure 1, arrow), whereas the TC-MCs were dark blue, or dark bluish-brown (Figure 1, arrow head). T-MCs occasionally contained a few scattered blue granules (usually inside the cell membrane), but such cells were easily distinguishable from the chymasepositive cells. Also, brown cells were not found in negative controls. In each of the sites from which experimental tissue was taken, there were no significant differences between the values for the proliferative phase versus the secretory phase of the menstrual cycle. However, there was a significantly (P ,0.03) lower number in the endometrium, the inner and outer half of the myometrium after the menopause (Table I). In the pre-menopausal endometrium, almost all of the MCs were located in the basal layer, with significantly (P ,0.005) fewer of these cells compared with the cervix, the inner and outer half of the myometrium. The greatest number of MCs was found in the inner half of the pre-menopausal myometrium, and this number was significantly (P ,0.005) larger than in the outer half of the myometrium. Also, in post-menopausal tissue, the number of MCs in the inner half of the myometrium was significantly (P ,0.01) larger than in the outer half of the myometrium (Table I). The numbers of MCs were also tabulated as a ratio of TCMCs to total MCs (i.e. total MCs 5 TC-MCs 1 T-MCs) (Table II). This was a convenient way to assess the heterogeneity of the distribution of the two different phenotypes of MCs in the various areas of the uterus. The data show that TC-MCs are the more dominant phenotype in the outer half of the myometrium (Figure 1C) and in the cervix. In contrast, TCMCs were proportionately lower in the endometrium (Figure 1A). The ratios of TC-MCs to total MCs and of T-MCs to total MCs were essentially equivalent in the inner half of the myometrium (Figure 1B). A statistical analysis of the data showed that the ratio of TC-MCs to total MCs was significantly (P ,0.01) greater in the outer half of the myometrium compared with the ratio in the inner half of the myometrium. There were no significant differences in the values of the 369
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Figure 1. Double-staining for tryptase and chymase in mast cells (MCs). The endometrium (A), inner one-half of the myometrium (B), and outer half of the myometrium (C) are visible. T-MCs are stained brown (arrow). TC-MCs are dark blue, or dark bluishbrown (arrow head). Original magnification3400.
ratios in any of the uterine areas when the proliferative phase was compared to the secretory phase. However, the ratio of TC-MCs to total MCs was significantly (P ,0.00001) greater in the post-menopausal endometrium compared with the ratio in the pre-menopausal endometrium. In the limited analysis of the smooth muscle layers of the small intestines, the mean number of MCs was 3.6/high-power field, with TC-MCs comprising 72% of this number. Double-staining for α-smooth muscle actin and tryptase, von Willebrand factor and tryptase, and collagen type III and tryptase Sections of the myometrium that were double-stained for α-smooth muscle actin and tryptase revealed that some of the 370
Figure 2. Double-staining for tryptase and α-smooth muscle actin (A) in the myometrium. Mast cells (MCs), which are tryptasepositive, are red. Smooth muscle cells are stained brown. Doublestaining for tryptase and collagen type III (B), or for tryptase and von Willebrand factor (C) in the myometrium. MCs are red. Collagen type III and the von Willebrand factor are stained brown. Original magnification 3400.
MCs were located among smooth muscle cells, while others were among the spindle-shaped cells negative for α-smooth muscle actin (Figure 2A). The sections that were doublestained for collagen type III and tryptase showed that some MCs appeared to be embedded among the collagen fibres of the myometrium (Figure 2B). Also, on the basis of staining for the von Willebrand factor, some MCs were located around blood vessels in the myometrium (Figure 2C). Discussion The present study shows that the greatest number of MCs is localized in the inner half of the myometrium, with this area
Mast cells in the human uterus
Table I. Distribution of mast cells in the human uterus. Values are mean 6 SD Endometrium Pre-menopause (n 5 16) Proliferative (n 5 11) Secretory (n 5 5) Post-menopause (n 5 8)
6.7 6.0 8.3 1.5
6 6 6 6
3.8 2.3 6.2 0.9
Inner myometrium 36.9 40.0 30.1 23.9
6 6 6 6
11.5a,b 10.2 12.4 15.0c,d
Outer myometrium 20.5 21.0 20.0 7.6
6 6 6 6
7.1a 5.6 10.3 2.5
Cervix 22.8 20.7 25.4 15.0
6 6 6 6
13.0a (n 5 11) 14.5 (n 5 6) 12.0 (n 5 5) 6.5 (n 5 6)
,0.005 versus endometrium. ,0.005 versus outer myometrium. ,0.0005 versus post-menopausal endometrium. dP ,0.01 versus post-menopausal outer myometrium. aP
bP cP
Table II. Tryptase and chymase-containing mast cell:total mast cell ratio in the human uterus Endometrium Pre-menopause (n 5 16) Proliferative (n 5 11) Secretory (n 5 5) Post-menopause (n 5 8) aP bP cP
0.16 0.16 0.17 0.74
6 6 6 6
0.22 0.21 0.28 0.28c
Inner myometrium 0.52 0.50 0.56 0.73
6 6 6 6
0.34ab 0.34 0.38 0.36
Outer myometrium 0.90 0.96 0.78 0.95
6 6 6 6
0.18a 0.10 0.27 0.10
Cervix 0.60 0.72 0.45 0.82
6 6 6 6
0.46a (n 5 11) 0.43 (n 5 6) 0.51 (n 5 5) 0.40 (n 5 6)
,0.01 versus endometrium. ,0.01 versus outer myometrium. ,0.00001 compared with pre-menopause endometrium.
containing approximately equal proportions of T-MCs and TCMCs. There were fewer MCs in the outer half of the myometrium and the cervix, but the proportion of TC-MCs in both of these areas was substantially higher. In contrast, the endometrium contained a significantly lower total number of MCs, but proportionally more T-MCs. Moreover, MCs rarely exist in the functional layer of the endometrium. In comparison with the granulated lymphocytes which exist in the functional layer of the endometrium and interact with the endometrial stromal cells during the menstrual cycle (Bulmer et al., 1991, Bhartiya et al., 1996), the interaction of MCs with the endometrial stromal cells in the functional layer is negligible. T-MCs are known to be especially abundant in the gastrointestinal mucosa, in the subepithelial and epithelial linings of the bronchi and bronchioles, and in the alveolar walls of the lungs, whereas TC-MCs are more numerous in the gastrointestinal submucosa and in the skin (Irani et al., 1986, 1989; Irani and Schwartz, 1989). Thus, the heterogeneity of MC distribution in the uterus is comparable with that in the gastrointestinal tract and the respiratory tract. However, the present study reveals that the concentration of MCs in the myometrium of the uterus is substantially higher than that in the smooth muscle layer of the intestines, because the adjusted number of MCs/high power field in that layer is ~3.0 (Befus et al., 1985). This study also shows that there is no significant change in the number of MCs between the proliferative and secretory phases of the menstrual cycle. This observation is not congruent with an earlier report which found that the concentration of MCs in the hamster uterus varied during the sexual cycle, with the lowest number of MCs being present on the fourth day of the cycle, shortly before ovulation (Brandon and Evans, 1983). Thus, it appears that there may be variations in the distribution of uterine MCs in different species. It was also noted that there is a significantly lower number
of MCs in the post-menopausal uterus compared with the pre-menopausal uterus. This suggests that, at least in the human species, there is a decline in the number of MCs concomitant with the general atrophy of the uterus that occurs with ageing and particularly following menopause. The myometrial MCs were distributed in close association with smooth muscle cells, as well as with connective tissue. It has been reported that the MCs in the myometrial connective tissue are different in size and in staining intensity for toluidine blue when compared to those associated with smooth muscle tissue (Rudolph et al., 1993). However, the present study did not confirm any such phenotypic variation. Recently, it has been reported that MCs contribute to the reconstruction of traumatized connective tissues. For example, MC tryptase (Ruoss et al., 1991), tumour necrosis factor-α (TNFα) (Sugarman et al., 1985), and histamine (Jordana et al., 1988) all act as mitogens on fibroblasts. In addition, tryptase (Krejci et al., 1992) and TNFα (Dayer et al., 1985) degrade components of the extracellular matrix, whereas histamine enhances the production of collagen by fibroblasts (Hatamochi et al., 1985). Also, it has been reported that serotonin induces collagenase production in human myometrial smooth muscle cells (Jeffrey et al., 1991). Therefore, in view of the substantial number of MCs in the uterus, it is possible that these cells may have an important functional role in the remodelling and reconstruction of uterine smooth muscle tissue and connective tissue during the menstrual cycle and following parturition. It is well known that histamine and eicosanoids from MCs are important in the regulation of vascular tone and permeability. Moreover, it has been reported recently that chymase promotes the conversion of angiotensin I to angiotensin II (Urata et al., 1990), as well as the processing of bigendothelin-1 to endothelin-1 (Wypij et al., 1992). Therefore, since the present study shows that MCs are common along the vascular compartment of the uterus, it is likely that MCs 371
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also regulate the vascular tone and permeability of blood vessels in the human uterus. In summary, the results show that MC distribution and heterogeneity in the human uterus resembles that of the gastrointestinal and respiratory tracts. However, the uterus is characterized by relatively large numbers of MCs, and these cells are especially numerous in the inner half of the myometrium. In addition, the study shows that uterine MCs are closely associated not only with fibrous connective tissue elements, but also with bundles of smooth muscle cells. Therefore, the results suggest that uterine MCs may have a special function in the female reproductive tract, and that myometrial smooth muscle cells and the vascular network supplying these cells may be a principal target of the bioactive agents in MCs. Acknowledgements We sincerely appreciate Professor Lawrence L.Espey of Trinity University for critical review of the manuscript. This work was supported in part by Grants (No. 07671772) from the Ministry of Education, Science, Sports and Culture of Japan.
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