Immunosuppressor activity of rat endometrial granulated cells and their differentiation

Natural killer and natural suppressor activities of the rat endometrial granulated cells were assayed on day 13 of pregnancy or pseudopregnancy. Metrial gland granulated cells were used as endometrial granulated cells. The natural killer activities of metrial gland granulated cells and other cells were determined by means of Hashimoto-Sudo test with K562 cells as targets. The estimation of natural killer activity included removal of the cells sticking to glass from a suspension of material gland granulated cells. Cytochemically, metrial gland granulated cells were identified by the presence of PAS-positive granules in the cytoplasm after treatment of the cells with diastase and identification of a specific antigen with the help of specific antisera. The natural killer activity of metrial gland granulated cells was twice weaker than that of splenocytes from the same pregnant or pseudopregnant females. The level of natural killer activity was proportional to the content of metrial gland granulated cells in a cell system. These data suggest that the natural killer activity of metrial gland granulated cells is realized via their contact with cell targets. Natural killer and suppressor activities were determined simultaneously for metrial gland granulated cells and splenocytes of the same rat with common cell targets. When estimating the nuclear suppressor activity of metrial gland granulated cells, the splenocytes of the same rat were used as an effector in a natural killer test. Various amounts of metrial gland granulated cells were added to the effector : target system at a ratio of 50:1. The natural suppressor activity of metrial gland granulated cells did not depend on the amount of metrial gland granulated cells present in a natural killer system. After fractionation in a Percoll gradient, the highest natural killer activity was recorded in a 30% Percoll fraction. The highest and lowest natural suppressor activities were recorded in 30% and 60% Percoll fractions, respectively. The culture medium was characterized by natural suppressor activity as well. The differences in mean areas of metrial gland granulated cells in 30 and 60% Percoll fractions between the pregnant (144.7 +/- 13.4 and 75.0 +/- 12.5 microm2, respectively) and pseudopregnant (97.5 +/- 4.9 and 69.2 +/- 3.5 microm2) females were reliable. The natural killer activity was estimated in all studied 23 samples of metrial gland granulated cells, among which 18 (79.6 +/- 7.8%) displayed the natural suppressor activity as well. The absence of natural suppressor activity in five samples was combined with the absence of this activity in their culture medium and with a reduction in the mean area of metrial gland granulated cells in 30% Percoll fraction to 109.1 +/- 5.2 microm2. The data obtained confirm the known data on a low activity of metrial gland granulated cells and demonstrated for the first time the natural suppressor activity of these cells. It was concluded that the natural suppressor activity of metrial gland granulated cells is due to their differentiation from metrial gland granulated cells with natural killer activity.     

Measurement of natural killer activity and target cell binding by mouse metrial gland cells isolated by enzymic or mechanical methods

Cells of the metrial glands of mice were isolated by enzymic or mechanical dissociation procedures. Morphological observations indicated that up to half of the enzymically dissociated cells and nearly all of the mechanically dissociated cells were granulated metrial gland cells, but the presence of some fibroblast-like stromal cells among the latter population was not ruled out. Moreover, the granulated metrial gland cells had lost a substantial part of their granule content during isolation. Both cell preparations had little or no natural killer (NK) activity, indicating either that granulated metrial gland cells are not NK-like or that their NK activity was impaired by loss of granule-associated lytic substances or by other factors. Enzymically dissociated metrial gland cells did not bind significantly to the NK target cell YAC-1, nor did they develop granules, NK activity, or the ability to bind YAC-1 cells during culture in vitro, either in normal medium or with the addition of indomethacin or lymphokines. Mechanically dissociated metrial gland cells bound avidly to YAC-1 cells but not to P815 cells or adult thymus cells, which are not NK target cells. Since many if not most of the mechanically dissociated metrial gland cells appeared morphologically to be granulated metrial gland cells, their selective binding to an NK target cell suggests that granulated metrial gland cells may be related in some way to NK cells.     

Lectin histochemical studies of mouse granulated metrial gland cells

A lectin histochemical study has been carried out on mouse granulated metrial gland cells, the major leucocyte population that differentiates in the uterine wall in pregnancy. The binding characteristics of 26 lectins were examined using light microscopical methods. Fourteen of the lectins, with affinities ranging through N-acetylgalactosamine, galactose, N-acetylglucosamine, mannose and sialic acid residues, bound to the cytoplasmic granules of granulated metrial gland cells, and each appeared to bind to the limiting membrane of the granules. The binding characteristics of three of these lectins (Wheat germ agglutinin, Concanavalin A and Helix pomatia agglutinin) were examined using electron microscopical methods. These showed a different binding pattern to the cytoplasmic granules of granulated metrial gland cells compared with that found using light microscopical methods, as they appeared to bind evenly across the granule's matrix. This binding pattern corresponds to the reactivity of the granule matrix in the periodic acid--Schiff technique. Six lectins bound to the cell membranes of granulated metrial gland cells. These included the E and L isoforms of Phaseolus vulgaris agglutinin, with affinities for complex carbohydrates, whose binding differences were related to the stage of differentiation of the granulated metrial gland cells. The lectin binding described presents additional markers of granulated metrial gland cells and tools for investigating carbohydrate moieties in the functional activities of granulated metrial gland cells     

Lectin histochemical studies of mouse granulated metrial gland cells

A lectin histochemical study has been carried out on mouse granulated metrial gland cells, the major leucocyte population that differentiates in the uterine wall in pregnancy. The binding characteristics of 26 lectins were examined using light microscopical methods. Fourteen of the lectins, with affinities ranging through N-acetylgalactosamine, galactose, N-acetylglucosamine, mannose and sialic acid residues, bound to the cytoplasmic granules of granulated metrial gland cells, and each appeared to bind to the limiting membrane of the granules. The binding characteristics of three of these lectins (Wheat germ agglutinin, Concanavalin A and Helix pomatia agglutinin) were examined using electron microscopical methods. These showed a different binding pattern to the cytoplasmic granules of granulated metrial gland cells compared with that found using light microscopical methods, as they appeared to bind evenly across the granule's matrix. This binding pattern corresponds to the reactivity of the granule matrix in the periodic acid--Schiff technique. Six lectins bound to the cell membranes of granulated metrial gland cells. These included the E and L isoforms of Phaseolus vulgaris agglutinin, with affinities for complex carbohydrates, whose binding differences were related to the stage of differentiation of the granulated metrial gland cells. The lectin binding described presents additional markers of granulated metrial gland cells and tools for investigating carbohydrate moieties in the functional activities of granulated metrial gland cells This revised version was published online in November 2006 with corrections to the Cover Date.     

Functional and phenotypic analyses of interleukin 2-activated tumor-infiltrating lymphocytes

The tumor-infiltrating lymphocytes (TILs) were cultured with interleukin 2 (IL-2) to induce the activated killer cells possessing autologous tumor-killing activity, and analysed their cell surface phenotypes and assessed anti-tumor killing activity. Furthermore, the activated TILs were transferred into 7 patients adoptively resulting in complete remission in a patient with pancreatic cancer and partial remission in another patient with gastric cancer.The cytotoxic activities of activated TILs at 3 weeks-incubation was 72 ± 15, 42 ± 26, 27 ± 21 and 25 ± 15% against K562, Daudi, KATO-III and autologous tumor, respectively. The negative selection method, indicated that the killer cells recognizing autologous tumor cells consisted of CD4- or CD8-positive T lymphocytes and CD16- or CD56-positive natural killer cells. The activated TILs could not only lyse cultured tumor cell lines, but also autologous tumor cells.     

Localization of a pore-forming protein (perforin) in granulated metrial gland cells

Several studies have suggested that the granulated metrial gland (GMG) cells of the metrial gland (MG) may be natural killer (NK)-like cells. The cytotoxicity of NK cells involves secretion of a pore-forming protein termed perforin, which can polymerize on the target cell membrane to form transmembrane pores that are thought to be involved in target cell death. In the present study, we used an antiserum against perforin to determine whether this protein can be detected immunohistochemically in GMG cells. Mouse uteri were fixed by vascular perfusion with several fixatives on Day 14 of pregnancy, and tissue sections were labeled by an indirect immunofluorescence method. Specific perforin labeling was detected in GMG cells throughout the MG, in the decidua basalis, and in the labyrinthine placenta. The presence of perforin in GMG cells supports the suggestion that they may be NK-like cells.     

Synergistic antitumor activity of interleukin-2 and cimetidine against syngeneic murine tumor

Summary Cimetidine, an H2 histamine receptor antagonist, is a potent immunomodulating agent, which acts by inhibiting suppressor T lymphocyte function. The present work investigated the effect, if any, of cimetidine on interleukin-2 (IL-2)-induced natural killer (NK) and lymphokine-activated killer (LAK) cell activities, and on in vivo antitumor activity using syngeneic colon 26 adenocarcinoma as the model. Mimicking the clinical conditions, all in vitro experiments were evaluated with the splenocytes prepared from tumor-bearing BALB/c mice. Ten days after subcutaneous inoculation of tumor cells (5 × 10⁵), animals were treated intraperitoneally daily with phosphate-buffered saline (PBS), cimetidine (2 mg kg^–1 day^–1), IL-2 (300 000 IU/day), or cimetidine plus IL-2 for 7 consecutive days. The treatment of IL-2 plus cimetidine increased NK and LAK cell activities significantly and synergistically at the end of the treatment (i.e. on day 18) as well as 1 week after the treatment (i.e. on day 25), in comparison with those of the control groups (PBS, cimetidine alone, IL-2 alone). Also, in vivo antitumor activity, as analyzed by a Kaplan-Meier life table with the log-rank test, revealed a significantly prolonged survival in the group treated with IL-2 plus cimetidine compared to the control groups. Phenotyping performed on the murine splenocytes on day 18 indicated a significant reduction in Lyt2-positive cells in the cimetidine-treated group in comparison with the PBS group. A significant increase in asialo GM1-positive cells and IL-2-receptor-positive cells was detected in the group treated with IL-2 plus cimetidine in comparison with the PBS and IL-2 control groups. Therefore, this study indicates a synergistic enhancement of IL-2-induced NK and LAK cell activities in tumor-bearing hosts by cimetidine, a noncytotoxic inhibitor of suppressor T function, and a significantly prolonged survival of tumor-bearing animals treated by IL-2 plus cimetidine. It also suggests the clinical potential of combination therapy of IL-2 with cimetidine.     

The structure and differentiation of granulated metrial gland cells of the pregnant mouse uterus

Summary A study was made with the light and electron microscopes of the granulated metrial gland cells of the decidua basalis of the pregnant mouse uterus, up to day 11 of pregnancy. The granulated metrial gland cells are large, up to 50 in diameter, mono- or binucleate and the glycogen rich cytoplasm typically contains many large glycoprotein granules which may be up to 5 in diameter. Morphological evidence is described in support of a lymphocyte-like cell being the precursor to the granulated metrial gland cell. This differentiation sequence is similar to that already proposed in the rat but differences between the ultrastructure of the mature metrial gland cells of rats and mice were noted.     

Granulated metrial gland cells in the pregnant uterus of mice expressing the collagen mutation tight-skin (Tsk/+)

Summary Influences of the extracellular matrix (ECM) on the differentiation and distribution of granulated metrial gland (GMG) cells, a uterine natural killer (NK)-like cell subset, were studied by histological examination of implantation sites in the mouse mutant Tsk/+. Tsk/+ mice overproduce collagens I and III. GMG cell differentiation appeared to progress normally in Tsk/+ mice between days 6.5 and 12.5 of gestation. The distribution of GMG cells, however, was abnormal. Significant numbers of GMG cells were found in the antimesometrial and lateral decidual regions at day 8.5 of gestation and in the regions between implantation sites until day 10.5 of gestation. Loss of GMG cells from these regions normally occurs by day 6.5 of gestation. These data suggest that alterations to the ECM change the migration properties or life span of GMG cells.     

Effects of phenytoin on cell-mediated immunity

Summary The effects of phenytoin on cellular immunity were examined in murine models. Fresh splenocytes were obtained from mice which had received 1 mg/day of phenytoin i.p. for 28 days. The serum concentration of phenytoin in these animals was 10–10 g/ml. The proliferative response of splenocytes to mitogens was assessed by ³H-thymidine incorporation. The cytotoxic activities of cells such as natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and lymphokine-activated killer (LAK) cells were estimated by a 4-h ⁵¹Cr release assay. The ³H-thymidine incorporation of splenocytes was reduced significantly (P<0.01) in phenytoin-treated mice. The NK and CTL activities of splenocytes from phenytoin-treated mice were significantly suppressed. However, the LAK activity of phenytoin-treated mice was equal to that of control mice.     

The metrial gland

The information available about the metrial gland of the pregnant rodent uterus with its content of granulated metrial gland (GMG) cells is reviewed. Recent research shows that GMG cells differentiate from bone marrow cells and supports the suggestion that GMG cells are involved in the immunological relationship between mother and foetus. There is probably a complex association between GMG cells and stromal cells of the metrial gland, and it is suggested that the association between GMG cells and the placental labyrinthine cells represents a functional interaction.     

Isolation of serine protease from granulated metrial gland cells of mice and rats with lectin from Dolichos biflorus.

Granulated metrial gland cells were the only cells in the endometria of pregnant mice and rats that reacted histochemically with fluoresceinated lectin (DBA) from Dolichos biflorus. Cell extracts of uteri of pregnant animals, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and analysed by lectin overlay blotting, contained DBA-reactive, 40-50 kDa, doublet glycoprotein bands. This glycoprotein was purified on a DBA agarose affinity column. It was identified by amino acid sequencing as a serine protease closely related to granzymes of T lymphocytes. We conclude that this granzyme accounts for the selective reactivity of granulated metrial gland cells with fluoresceinated DBA in histological sections of uteri of pregnant rodents and show that DBA affinity columns can be used for purification of granzyme derived from granulated metrial gland cells.     

Decidualisation: origin and role of associated cells

Proliferation of peri- and subluminal stroma following a stimulus from the blastocyst leads to the appearance of decidual cells in the mammalian endometrium. Decidualisation can also be elicited by artificial stimuli in pseudopregnant animals. A variety of histophysiological reactions accompany decidualisation and distinct morphological features characterize decidual cells localized in the antimesometrial and mesometrial area. Granulated metrial gland cells, arising in the endometrium of decidualising uterus, form a separate class of cells and become prominent in the mesometrial triangle as pregnancy advances. This review deals with factors related to induction of decidualisation; structural characteristics of decidual and metrial gland cells; the origin and postulated roles of decidualisation-associated cells. The functional role of decidual and metrial gland cells is discussed in relation to their structural complexity and recent observations on the haemopoietic origin of these cells.     

Human perforin (PRF1) maps to 10q22, a region that is syntenic with mouse chromosome 10.

Perforin (PRF1) is a cytolytic, channel-forming protein of cytolytic T cells, natural killer cells, and granulated metrial gland cells and plays a crucial role in the killer cell-mediated elimination of virally infected host cells, tumor cells, and allotransplants. Two-thirds of the perforin sequence is homologous to the lytic, channel-forming complement proteins C6, C7, C8 alpha, C8 beta, and C9. Using cosmid DNA containing the PRF1 gene as a probe for fluorescence in situ hybridization, we have reevaluated its chromosomal location. Previously assigned to chromosome 17q11-q21, it has now been mapped to 10q22. The human PRF1 locus lies within a conserved synteny segment present on mouse chromosome 10, consistent with the previous chromosomal assignment of mouse perforin. The perforin locus is not linked to any of the genes of the terminal complement system.     

Acetylesterase isoenzymes in rat uterus and placenta

The occurrence of acetylesterase activity in the uterus and placenta of the rat has been investigated using a general histochemical simultaneous coupling technique after separation on polyacrylamide gradient gels. apart from a complex band associated with serum esterases which was demonstrated in all the tissues studied, several other isoenzyme bands were demonstrable in differing degrees in the yolk sac and the virgin uterus. Two of these bands were evident in metrial gland up to day 16 of pregnancy, and a third became present by day 17. Unlike the other two bands, this new band did not seem to be associated with the large granules of the granulated metrial gland cells. None of these bands were detected in trophoblast. The metrial gland isoenzymes reacted as well at acid pH as at neutral pH. The yolk sac isoenzymes reacted either as well or slightly better at acid pH, and one extra band was demonstrable under acid conditions.     

妊娠小鼠子宫腺细胞区的碱性磷酸酶与酸性磷酸酶的研究

小鼠子宫系膜三角区在妊娠后出现上皮样细胞群,群内的细胞称颗粒子宫腺细胞(granu-lated metriial gland cells,GMG细胞),该区改称子宫腺细胞区(metriial gland cell area,MGCA).取孕12～19天MGCA,液氮速冻,恒冷箱切片,偶氮偶联法显示碱性磷酸酶(ALP)与酸性磷酸酶(ACP).结果为,ALP主要分布于GMG细胞群间的疏松结缔组织中;GMG细胞为阴性反应.ACP主要位于GMG细胞内,群间结缔组织含量较少.两种酶的活性随胎龄增加而减弱.ALP与ACP的定位与活性变化特性显示它们与GMG细胞功能关系密切.     

A comparison of the suppressor and cytotoxic activities of the blood mononuclear cells during the adoptive immunotherapy of cancer patients using lymphokine-activated killers with a low dose of recombinant interleukin-2]

The suppressor and cytotoxic activities of mononuclear blood cells (MNC) were studied in 70 cancer patients (melanoma, renal carcinoma) undergoing adoptive immunotherapy (AIT). In the course of AIT the patients' MNC were treated in vitro with the recombinant interleukin-2 (RIL-2) in order to generate the lymphokine-activated killer (LAK) cells. Then patients received i/v 2.5-13.6 10(9) autologous LAK cells and RIL-2 (75000 u). Each course included 2-3 repeated infusions; the patients received 1-5 courses according to their clinical conditions. The cytotoxic activity of MNC was assessed by a routine method; but for evaluation of the suppressor activity we used a new technique based on separation of MNS populations in the Percoll gradient. Twenty-four hours after the completion of each AIT course the suppressor activity of MNC decreased drastically up to the zero level in some patients. The decrease in the suppressor activity inversely correlated with the rise in the cytotoxic activity on Mel-I (LAK-sensitive) and K-562 (natural killer-sensitive) target cells. The level of cytotoxicity in some patients reached 51.2%.     

Uptake of immunoglobulin and albumin by granulated metrial gland cells in vitro

Single cell suspensions of metrial gland tissue from rats at Day 14 of pregnancy were prepared for maintenance in vitro. During the first 2 days of culture IgG was detected in glycoprotein granule-containing granulated metrial gland (GMG) cells. Albumin was also detected in GMG cells at the same stages. The IgG and albumin were not detected during the next 4 days in culture. When metrial gland cells, maintained in vitro for 5 days, were incubated with rat serum for a further 24 h, IgG and albumin were detected in GMG cells. When similar cultures were incubated for 24 h with purified rat IgG or purified rat albumin, GMG cells were positive for IgG and albumin respectively. Albumin was not detected in GMG cells in wax sections of metrial gland tissue, although IgG has previously been demonstrated. The uptake of serum proteins by GMG cells in vitro has been clearly shown but the difference in IgG and albumin content of these cells in paraffin-wax sections indicates that the means by which IgG accumulates intracellularly may be different in vitro and in vivo.     

Natural cytotoxic and antibody-dependent cellular cytotoxic activity of cells in the decidua basales and metrial glands of pseudopregnant rats with deciduomata

Cytotoxic cells are present in the uterine wall of pregnant rats. To determine if the cytotoxic activity arises in response to semen or the products of conception, the profile of cytotoxic activity in deciduomata of pseudopregnant rats was examined. To examine NK activity, Yac-1 cells were used as targets in chromium release cytotoxicity assays and an antibody to Yac-1 cells was included in some assays to determine antibody-dependent cellular cytotoxic (ADCC) activity. Cells from the metrial glands and deciduae of deciduomata of rats at days 10 and 13 of pseudopregnancy did not show NK activity but ADCC activity was present. To examine natural cytotoxic (NC) activity, Wehi 164 cells were used as targets in chromium release cytotoxicity assays. Cells isolated from the metrial glands and deciduae of rats at day 10 of pseudopregnancy were able to kill Wehi 164 cells after 21 h assays, thus demonstrating NC activity. The profile of cytotoxic activity in the uterine wall of pseudopregnant rats with deciduomata is similar to that found in pregnancy and is thus independent of semen or the products of conception.     

The selective growth of murine newborn-derived suppressor cells and their probable mode of action

Naturally occurring suppressor cells residing in the spleens of newborn mice of less than 5 days old are known to suppress various lymphocyte activities. A population of these suppressor cells can be maintained and expanded in the supernatants derived from Wehi-3 cells. These suppressor cells, designated as Wehi-3-expanded neonatal splenocytes (WENS), can suppress mixed lymphocyte reactions (MLR) and T and B cell mitogen responses without any genetic restrictions. The WENS bear the Ly-5, J11d, and class I molecules. WENS suppression is not mediated through an interleukin 1 or interleukin 2 absorptive mechanism. To achieve maximum suppression of MLR, WENS must be present for at least 24 hr. WENS inhibited the proliferation of Wehi-164 cells but not other tumor cells. The inhibition of Wehi-164 growth was due to the action of natural cytotoxic cells, because WENS lysed Wehi-164 cells but not the natural killer target cell YAC-1. Maximum lysis of Wehi-164 by WENS required 18 to 24 hr. Five WENS cell lines were cultured for more than 6 mo; three of the cell lines lost their capacity to lyse Wehi-164 targets (natural cytotoxicity) and simultaneously lost their natural suppressor activity. The two WENS lines that retained natural cytotoxicity also retained natural suppressor activity. Thus, natural suppressor cells may manifest their suppression through a natural cytotoxicity mechanism.