Activation of maternal killer cells in the pregnant uterus with chronic indomethacin therapy, IL-2 therapy, or a combination therapy is associated with embryonic demise

We have previously shown that NK lineage cells migrate to the murine decidua of pregnancy; but with advancing gestation, they are progressively inactivated in situ by prostaglandins of the E series (PGE2) secreted by decidual cells and decidual macrophages. We have also shown that the same mechanism inactivates all killer lineage cells in the human decidua, and that this inactivation is at least in part due to a down-regulation of IL-2 receptors and an inhibition of IL-2 production in situ. We examined whether chronic indomethacin therapy (to block prostaglandin synthesis), or a systemic administration of a high dose of IL-2, or a combination of both agents administered to pregnant mice could activate killer cells in situ and interfere with the progress of pregnancy; and if so, whether there was a causal relationship between the two events. Pregnant CD1 mice (Day 5 of gestation) were subjected to chronic indomethacin therapy (14 micrograms/ml in drinking water up to Day 15, or 50 micrograms twice daily sc or ip up to Day 10), high dose IL-2 therapy (25,000 Cetus U of human recombinant IL-2, ip every 8 or 12 hr for 3-5 days), or a combination of the two. These treatments led to pregnancy loss in 89-100% of mice, in contrast to 1% loss in control, vehicle-treated mice. Uterine mononuclear cells isolated from the embryo resorption sites exhibited high killer activity against YAC-1 lymphoma as well as murine trophoblast targets, with NK-like phenotype (Asialo GM-1+, Thy-1-) after indomethacin therapy and LAK-like phenotype (AGM-1+, Thy-1+) after IL-2 or indomethacin + IL-2 therapy. That AGM-1+ killer cells resulted in the pregnancy loss was suggested by the findings that in two of three separate experiments, iv injections of AGM-1 ab into pregnant indomethacin + IL-2-treated mice nearly completely prevented the fetoplacental demise (reducing it to 7.7% from 100%). These results reveal that PGE2-mediated inactivation of killer lineage cells in the decidua in situ is conducive to the survival of the conceptus.     

Changes in the host natural killer cell population in mice during tumor development. 1. Kinetics and in vivo significance

Our earlier studies revealed an increase in the level of null (surface IgM-negative, Thy 1-negative) lymphocytes in mice shortly after tumor transplantation and before the clinical appearance of spontaneous mammary tumors. The present study examined the splenic natural killer (NK) cell activity as well as the incidence of NK lineage cells in these hosts, since NK cells are considered to be a subset of null lymphocytes. Splenic NK activity against YAC-1 lymphoma targets was measured with a 4-hr 51Cr-release assay in CBA mice transplanted ip with 10(6) Ehrlich ascites tumor (EAT) cells, in elderly C3H mice prior to and during the growth of spontaneous mammary tumors (SMT) and in young C3H mice transplanted sc with 5 X 10(6) SMT cells or 10(6) cells from two syngeneic mammary tumor lines (T-58 and MT-2) of recent origin. In EAT-transplanted mice total NK activity in the spleen increased rapidly to a peak (11-fold) at 3 days, coincident with the null cell rise, but then declined to subnormal levels by Day 7 when the null cell level was still high. A similar pattern of activity was exhibited by intratumor lymphocytes isolated from the EAT. In SMT-transplanted mice splenic NK activity showed a small rise at Day 3, followed by a drop to below normal at Day 7, subnormal levels lasting for the tumor life span. Similar results were noted in T-58- or MT-2-transplanted mice. Null lymphocytes recovered during the peak NK activity from the spleen of 3-day EAT-bearing mice, when mixed with 10(6) EAT cells at 25:1 E:T ratio and adoptively transferred into fresh mice in a Winn type assay either ip or sc, completely prevented tumor development indicating a high enrichment of NK cells functionally effective in vivo. Elderly clinically tumor-free C3H mice showed measurable NK activity, which dropped after the appearance of spontaneous mammary tumors to very low levels, the magnitude of decline rising with increasing tumor age (1-11 weeks) or size. The incidence of NK lineage cells was measured from the tumor target (YAC-1 lymphoma)-binding ability of the splenic null cells, identified with a radioautographic technique. Null target-binding cells (TBC) were NK-1+ and included both active as well as inactive NK lineage cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of immune effector cells present in early murine decidua

It has been suggested that murine decidual cells act as an important immunoregulatory population localized to the pregnant uterus. We have examined early murine decidua to determine if immune effector cells occur in the decidual environment in proximity to the conceptus. High levels of natural killer (NK) cell activity were found consistently in decidual cell suspensions with peak activity occurring on Day 6.5 of gestation. NK activity declined as pregnancy proceeded and was not significant by Day 12.5 of gestation. Decidual cell suspensions did not appear to contain significant numbers of functional B or T effector cells. No antipaternal T-cell response could be demonstrated even in the decidua of immune mice. Lack of T-cell responses was attributed to the absence of T cells from decidua rather than to their inactivation because precursors of cytotoxic T lymphocytes (pCTL) could not be detected in decidual cell suspensions. Furthermore, the levels of pCTL detectable in spleen cell suspensions could not be reduced by mixing spleen cells with 7.5-day decidual cells. These results suggest that B cells and T cells may not occur in early decidua while NK cells are present and regulated independently.     

Murine granulated metrial gland cells at uterine implantation sites are natural killer lineage cells

Granulated metrial gland (GMG) cells, a population of morphologically distinct, bone marrow-derived cells in murine decidua that react with mAb 4H12, are shown in this report to express NK-specific Ag and to become cytolytic to the NK cell target YAC-1 when cultured in the lymphokine IL-2. When 1-mm3 explants of 8-day decidual tissue were cultured with IL-2, large numbers of 4H12+ GMG cells migrated out of the tissue. Migration was dependent on the amount of IL-2 used. This explant technique was used to isolate a pure population of GMG cells. The migratory activated GMG cells were phenotypically 4H12+, NK1.1+, LGL-1+/-, CD3-, and MAC-1-. Furthermore, the IL-2-activated GMG cells killed YAC-1 but not P815 cells in a 4-h 51Cr-release cytotoxicity assay. 4H12+ GMG cells from collagenase-digested decidual tissue also were analyzed for the presence of NK lineage Ag by flow cytometry and shown to coexpress the NK-associated Ag NK1.1 and ASGM1 but not the T cell Ag CD3 or macrophage Ag MAC-1 or F4/80. GMG cells isolated by collagenase digestion did not express LGL-1, an Ag associated with lytic NK cells. Our results demonstrate that GMG cells express Ag and functions characteristic of NK cells, and thus GMG cells can be assigned to the NK lineage. The possible relevance of NK cells at implantation sites is discussed.     

An evaluation of the maternal natural killer cell population during the course of murine pregnancy

Earlier work from this laboratory revealed an increase in the level of null (Thy-1-, IgM-) lymphocytes in the maternal lymphoid organs during the first pregnancy in the mouse that was more pronounced during allogeneic pregnancy than during syngeneic pregnancy. In view of the suggestive evidence for the bone marrow origin of these null cells, the functional significance of the null cell rise was explored in this study by an examination of (1) splenic NK activity as measured by the 51Cr-release assay using YAC-1 lymphoma targets, (2) the incidence of NK lineage cells in the spleen as measured by the ability of splenic null lymphocytes to bind YAC-1 lymphoma targets, and (3) the possible presence of a NK target structure on placental trophoblast cells, studied with a cold target competition assay. Results revealed that the absolute levels of null lymphocytes, NK lineage cells, and NK activity in the spleen increased moderately and nearly at the same time during syngeneic pregnancy. During allogeneic pregnancy all three parameters increased more significantly, the rise in the levels of NK activity and NK lineage cells somewhat preceding the null cell rise, suggesting preferential recruitment of active NK cells within the null lymphocyte population of the spleen. Trophoblast cells appeared to share NK target structures with YAC-1 lymphoma cells, suggesting that a rise in the NK cell level in both pregnancy types may represent a response of the mother to such target structures. Since the density of such moieties was similar for homozygous and heterozygous trophoblasts, a higher NK cell response during allogeneic pregnancy is considered to result indirectly from an alloreactive response of the mother to the paternally encoded antigens on the fetoplacental unit, possibly from a stimulation of interferon producing cells such as macrophages. Nevertheless, such a response appears to be harmless for the allogeneic conceptus.     

Suppression of lymphocyte alloreactivity by early gestational human decidua. I. Characterization of suppressor cells and suppressor molecules

We examined the immunosuppressor role of the first trimester human decidua on lymphocyte alloreactivity in vitro in order to identify (1) the major cell classes in the decidua mediating the suppressor effect; (2) the stages in the lymphocyte alloreactive responses susceptible to the suppressor effects of the decidua; and (3) the precise nature of the suppressor molecules. Irradiated (2800 R), Ficoll-Paque-separated nucleated cells of the collagenase-dispersed early gestational (6.5-9.5 weeks menstrual age) decidua containing 70-94% typical decidual cells (identified on the basis of distinctive morphology and numerous cytoplasmic or surface markers) or their plastic-nonadherent fractions further enriched for decidual cells (approximately 96% pure) caused a strong dose-dependent suppression of the one way mixed lymphocyte reaction (MLR, i.e., proliferative response measured on Days 3, 4, or 5), when added at the onset of the mixed lymphocyte cultures (MLC). As few as 10(3) decidual cells caused a detectable inhibition of the MLR exhibited by 10(5)-1.5 X 10(5) responder lymphocytes. A smaller degree of suppression was noted with the plastic-adherent fractions of the early decidua (which retained all macrophages and granulocytes, but still included many decidual cells) or unfractionated cells of later gestational (10-13 weeks) decidua containing a higher incidence of leukocytes, granulocytes, and macrophages in particular, or the plastic-adherent fraction thereof, enriched for macrophages. Thus, decidual cells seem to represent an important suppressor cell class in the early gestational human decidua; however, suppression by decidual leukocytes, macrophages in particular, was also evident. The suppressor effect was unrelated to the major histocompatibility phenotype of the responder or the stimulator cells. It was not caused by cell crowding, since an equivalent number of irradiated K562 erythroleukemia cells had little effect on the MLR. The effect was exerted during both the initiation and the progression of the MLR. A delay in the addition of regulator cells progressively minimized the effect on the Day 4 MLR, but did not abolish it completely even when added as late as on Day 3. The major class of mediator molecules was identified as prostaglandins, primarily PGE2, on the basis of the following results: (1) the presence of indomethacin (10(-5) M) or varying dilutions of an anti-PGE2 antibody abrogated this suppression substantially or completely. (2) Addition of pure PGE2 (3 X 10(-7) to 1.1 X 10(-5) M), but not PGF2 alpha, reproduced a dose-dependent suppressor effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of murine decidual natural killer (NK) cells and their relevance to the success of pregnancy

The identification of lytic cells in 6.5-day to 9.5-day murine decidua as NK cells has been extended. The cells with natural killer (NK) activity in early decidua were nonphagocytic and heterogeneous in size as assessed by velocity sedimentation at unit gravity. The numbers of lytic cells were reduced by treatment with anti-asialo GM1 in vivo and they were absent from the decidua of bg/bg mice. Thus, decidual NK cells were not distinct from NK cells in other tissues. The decline in the levels of decidual NK activity as pregnancy progressed was attributed to their regulation by other cells present in decidua by midgestation. The development of NK activity in decidua was dependent upon the presence of an embryo, however, decidual NK cells were not essential for successful pregnancy because viable offspring were obtained from mice lacking decidual NK activity. It was shown that NK cells from either spleen or decidua were unlikely to cause damage to embryos during the first half of pregnancy as freshly dissociated 9.5- and 11.5-day embryonic cells resisted NK lysis. Furthermore, blastocysts were not damaged by coincubation with splenic or decidual NK cells and were viable upon subsequent embryo transfer. These studies indicate that decidual NK cells are not essential for successful pregnancy and are not necessarily detrimental to early embryos. It is suggested that decidual NK cells may play other nonimmunological roles during embryonic development.     

Indomethacin therapy abrogates the prostaglandin-mediated suppression of natural killer activity in tumor-bearing mice and prevents tumor metastasis

We have shown earlier that a decline in splenic natural killer (NK) activity during the development of transplanted or spontaneous tumors in mice results from an inactivation of NK lineage cells, mediated by prostaglandins (primarily PGE2) secreted by NK suppressor cells of the monocyte-macrophage lineage. In the present study we have used a C3H mouse mammary carcinoma model to examine whether this mechanism of NK suppression is conducive to tumor metastasis in vivo and whether a reversal of this suppression by a chronic indomethacin therapy can prevent metastatic spread from the primary tumor site. Three mammary tumor lines, all derived in our laboratory from a spontaneous C3H mammary tumor were employed: T-58 (uncloned parental line, having weak lung metastasizing ability from the subcutaneous site), C3 (a clone of T-58, showing high metastatic ability), and C10 (a nonmetastatic clone of T-58). Although the degree of NK susceptibility of these lines varied inversely with their metastatic potential, none was NK resistant. A chronic administration of indomethacin in the drinking water (14 micrograms/ml) to mice beginning on Day 4 after subcutaneous transplantation of 10(6) tumor cells resulted in a significant reduction in the growth rate of primary tumors in all hosts and led to a complete or nearly complete abrogation of lung metastasis in T-58- or C3-transplanted hosts examined at 1 month after tumor transplantation; C10-transplanted mice showed no metastasis in the control or the treated group. Concomitantly, there was a substantial restoration of splenic NK activity in all indomethacin-treated hosts. Plastic-adherent cells (greater than 95% macrophages) isolated from tumors growing in control mice, when coincubated for 20 hr with normal splenic effector cells caused a suppression of NK activity, reversible in the presence of indomethacin (10(-5) M) in vitro. Similar cells recovered from the residual primary tumors in indomethacin-treated mice had no suppressor ability. Chemically pure PGE2 (at concentrations of 0.5 to 1 X 10(-6) M, but not 0.25 X 10(-6) to 10(-8) M) also caused a suppression of NK activity of normal splenic effector cells, when added during the 4-hr 51Cr-release assay or allowed to interact with effector cells alone for a 20-hr incubation period; a removal of the cell-free PGE2 in the latter case prior to the NK assay did not relieve the suppression.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human trophoblast cell resistance to decidual NK lysis is due to lack of NK target structure.

We have previously shown that human cultured trophoblast cells are resistant to lysis by natural killer (NK) cells from both peripheral blood and decidua although cells are present in decidua which do exhibit NK activity against K562(1). Using a cold-target inhibition assay and a single-cell conjugate assay we have now examined whether these trophoblast cells have NK target structures on their surfaces. Our findings indicate that first-trimester human trophoblast cells do not express surface structures recognized by decidual Leu19+ (CD56+) large granular lymphocytes (LGLs) isolated from human decidua. Immunostaining of the conjugates formed between decidual NK effectors and K562 cells confirmed that these effector cells are CD56+ LGLs.     

Natural killer (NK) cell activity of first trimester human decidua

The NK cell functional capacity of first trimester human decidua against K562 targets was assessed in a 3-hr CRA. Collagenase dispersal combined with plastic adherence, nylon wool passage, and density gradient centrifugation yielded NKH-1 (Leu 19) positive enriched decidual large granular lymphocyte fraction (mean 75% positive). Decidual effectors displayed reduced lytic activity compared with autologous PB effectors at every effector:target ratio but this difference in cytotoxicity was abolished by short-term culture before the CRA. Decidual effectors treated with 50 units rIL-2 showed increased lytic activity compared to untreated decidual cells. By FACS analysis the majority of NKH-1 positive decidual effectors were CD3 and CD16 negative which corresponds with a minority PB NK population. The implications of a population of functional NK cells in early pregnancy decidua for the materno-fetal relationship is discussed.     

Population dynamics of "null" and Thy1lo lymphocytes in mouse bone marrow: genesis of cells with natural killer cell lineage characteristics.

The population dynamics of "null" small lymphocytes lacking B and T lineage markers in mouse bone marrow have been examined using a combination of immunolabeling and hydroxyurea (HU) deletion techniques. The binding of the B lineage-associated mAb, 14.8, and anti-Thy1.2 to bone marrow cells has been detected radioautographically. Null cells lacking 14.8 and Thy1.2 determinants (14.8- Thy1-) formed a substantial subset (12-14%) of bone marrow small lymphocytes, representing 0.5 x 10(6) cells per femur (2-3% of nucleated cells). HU treatment revealed an exceptionally rapid turnover of the null small lymphocyte population (T1/2, 7.5 hr) compared with 14.8+ cells (T1/2, 20.5 hr) and Thy1+ cells (T1/2, 53 hr). Small lymphocytes bearing low intensities of Thy1 (Thy1lo) were also rapidly renewed (T1/2, 28 hr) whereas those with high intensities of Thy1 (Thy1hi) were renewed only slowly (T1/2, 123 hr). During ontogeny, null small lymphocytes first appeared in the fetal liver by Day 11 and the fetal spleen by Day 16, but increased rapidly in the bone marrow in early postnatal life. Double immunolabeling techniques demonstrated that 10% of null small lymphocytes in the bone marrow expressed NK1.1 antigen, while larger proportions bound to tumor (YAC.1) cells in vitro and displayed Fc receptors. The NK1.1-bearing fraction of null small lymphocytes in bone marrow was depleted by HU treatment only after an initial delay. NK1.1 was also expressed on subsets of Thy1lo cells and Thy1hi cells. The results have revealed the continuous production in mouse bone marrow of null and Thy1lo small lymphocytes, totaling 1-3 x 10(7) cells/day and 1.2 x 10(6) cells/day, respectively. The findings suggest that the large-scale production of null lymphocytes in mouse bone marrow includes the genesis of NK lineage cells which express NK1.1 and Thy1lo during a period of terminal maturation.     

Human decidual natural killer cells express the receptor for and respond to the cytokine interleukin 15

The natural killer (NK) cells that are present in the uterine mucosa (decidua) during early pregnancy have a distinctive phenotype, CD56(bright) CD16(-). These cells have previously been shown to proliferate and be activated by interleukin (IL)-2. However, IL-2 is absent from the decidua and placenta, and we have therefore investigated whether IL-15 is present in the uterus and can act on decidual NK cells. Both IL-15 mRNA and protein were found in a variety of cells but particularly in decidual macrophages. IL-15 induced a proliferative response in decidual NK cells that was blocked by anti-IL-15 and was augmented by stem cell factor. The cytolytic activity of decidual NK cells against K562 was augmented. Interestingly, in contrast to IL-2, although activation with IL-15 resulted in some killing of JEG-3 choriocarcinoma cells, normal trophoblast cells remained resistant to lysis. These findings suggest that IL-15 is a candidate cytokine responsible for NK cell proliferation in vivo in the progesterone-dominated secretory endometrium and early decidua.     

Inhibition of human NK cell and LAK cell cytotoxicity and differentiation by PGE2

Activation of natural killer (NK) activity K562 target cells from nonadherent (NA) lymphocytes by interleukin 2 (IL-2) was inhibited marginally PGE2 (30-3000 nM). PGE2 did not effectively suppress the NK activity of IL-2-activated cells. The NK activation and acquisition of resistance to PGE2-mediated suppression of NK activity were dependent on protein synthesis. When NA cells were incubated with IL-2 for 3 or more days to generate lymphokine-activated killer (LAK) activity against Raji target cells, PGE2 only partially inhibited the activation of NK/LAK activity by an optimal dose of IL-2 (10 U/ml). The activation of NK/LAK activity by a suboptimal dose of IL-2 (0.1 U/ml) was inhibited by PGE2. When the NK/LAK activity of IL-2-activated cells was assessed in the presence or absence of PGE2, the LAK activity was more sensitive than the NK activity to PGE2-mediated suppression.     

PGE2-mediated immunosuppression by first trimester human decidual cells blocks activation of maternal leukocytes in the decidua with potential anti-trophoblast activity

We have earlier shown that first trimester human decidual cells and decidual macrophages suppress T lymphocyte alloreactivity in an MHC-unrestricted manner by secreting PGE2, which blocks the generation of IL-2 receptors (IL-2R) and production of IL-2 by lymphocytes but does not interfere with the interaction between IL-2 and IL-2R or the lytic function of CTL, once generated. The present study examined whether these events constituted a physiological, immunoprotective mechanism in situ against the activation of maternal decidua-infiltrating leukocytes with potential anti-trophoblast cytocidal function. We examined (1) whether there was IL-2R expression, IL-2 production, or anti-trophoblast killer activity in short-term (0-3 day) cultures of collagenase-dispersed first trimester human decidua inclusive of leukocytes; (2) if not, whether any of these parameters could be stimulated in these cultures by blocking PGE2 synthesis with indomethacin, or neutralizing PGE2 with anti-PGE2 antibody; (3) whether exogenously added recombinant IL-2 in the presence or absence of indomethacin stimulated IL-2R expression or anti-trophoblast killer function in these cultures. IL-2R (as defined by Tac antigen) was measured in the whole cell population by a radioimmunoassay and further examined at the cellular level with radioautography. IL-2 production in culture supernatants was measured from the proliferative response (3HTdR uptake) of an IL-2-dependent (CTLL) cell line. Killer activity in fresh or cultured decidua-associated cells as well as PBL of normal or pregnant subjects was measured against 51Cr-labeled targets inclusive of autologous cytotrophoblast cells or long-term human trophoblast cell lines, K562 and Daudi cells. Results revealed a complete absence of IL-2R expression, IL-2 production, or anti-trophoblast killer activity in the untreated cultures of the decidua, but all these parameters were significantly stimulated in the presence of indomethacin or anti-PGE2 antibody. The indomethacin-stimulated killer cells had NK-like activity. Presence of high dose exogenous IL-2 alone in these cultures strongly stimulated IL-2R expression and anti-trophoblast killer function, which were augmented further in the additional presence of indomethacin. The resultant killer cells had LAK cell-like activity. These findings suggest that PGE2 secretion by first trimester human decidual cells blocks activation of maternal leukocytes in the decidua with potential anti-trophoblast killer function, by inhibiting IL-2 receptor generation and IL-2 production in situ.     

Changes in the host natural killer cell population in mice during tumor development. 2. The mechanism of suppression of NK activity

Our earlier studies revealed that a rapid and progressive loss of splenic NK activity in mice during the development of a number of transplanted tumors as well as of spontaneous tumors was due to an inactivation of natural killer (NK) lineage cells rather than to their disappearance. The mechanism of this inactivation have now been explored in CBA/J mice receiving transplanted Ehrlich ascites tumors and in C3H/HeJ mice bearing spontaneous mammary tumors or receiving transplants of syngeneic mammary tumor lines of recent origin. A poor activation state or maturation arrest of NK lineage cells due to a low interferon level in vivo was ruled out, since the host NK activity could not be restored after administration of either an interferon inducer poly(I:C) or interferon-alpha, although such treatments enhanced the activity in tumor-free mice by four- to eightfold. Possible presence of host suppressor cells acting on the effector or preeffector stage of NK cells was explored by mixing spleen cells from tumor bearers with normal spleen cells either during the NK assay, or for a 20-hr period of in vitro short-term culture prior to the NK assay. Mixing during the NK assay led to a reduction of NK activity explicable by a simple dilution of active NK cell concentration rather a suppression of active NK cells. On the other hand, a 20-hr coculture of the mixed population at various ratios led to a complete abrogation of the NK activity, indicating that the suppressor cells acted on the preeffector stage of the NK Lineage. A further characterization of suppressor cells revealed that they were (1) contained in the adherent fraction of the spleen of tumor bearers, (2) of monocyte/macrophage morphology, (3) capable of phagocytosing latex particles, and (4) positive for surface Mac-1 antigen, as noted from a radioautographic binding of 125I-labeled monoclonal anti-Mac-1 antibody. The mechanism of the suppression was identified, at least in part, as being mediated by prostaglandin-like molecules, since the presence of indomethacin, a prostaglandin-inhibitor, during the 20-hr coculture period completely abrogated the suppression. Indomethacin exerted no direct effect on the recruitment or killer activity of NK lineage cells in vitro. NK cell suppression may be another normal immunoregulatory mechanism which alters the host-tumor balance in favor of the tumor rather than the host.     

Cytotoxic activity against trophoblast and choriocarcinoma cells of large granular lymphocytes from human early pregnancy decidua

Large granular lymphocytes (LGL) are the most abundant cell type in first trimester human pregnancy decidua. We have shown previously that CD56-positive decidual LGL have cytotoxic activity against the natural killer (NK) target K562, and that this cytotoxicity is augmented by pretreatment with interleukin-2 (IL-2). We now report that flow cytometrically purified populations of CD56-positive decidual LGL have no cytotoxic activity against either the BeWo choriocarcinoma cell line or freshly isolated term trophoblast. Incubation of unfractionated decidual cells with IL-2 induced cytotoxicity against BeWo, but term trophoblast remained resistant to lysis. Both BeWo and trophoblast showed much lower binding frequencies to decidual or peripheral blood cells than K56 targets, and excess trophoblast did not inhibit cytotoxic activity against K562. This suggests that the resistance of trophoblast to lysis by either decidual or peripheral blood LGL is due to the lack of accessible NK target structures on the surface of trophoblast.     

Regulation of human natural killing. II. Protective effect of interferon on NK cells from suppression by PGE2

Activation of human natural killer (NK) cells in vitro with interferon (IFN) and poly I:C results in a partial loss of sensitivity of these cells to suppression by PGE2. The acquired resistance to suppression can be induced with the large granular lymphocytes (LGL) in the absence of monocytes. With K562, HSB, and CEM used as NK target cells, the IFN-induced resistance to suppression by PGE2 is observed with all three target cells. Furthermore, ADCC activity of IFN-activated cells against tumor (SB-TNP) and erythroid (CRC-TNP) target cells is also less susceptible to suppression by PGE2. The dual effect of IFN on NK cells is prompt; the augmentation of NK activity and the acquired resistance to suppression by PGE2 can be seen after 3 hr of treatment with IFN. Both of these characteristics seem to be quite stable for at least 24 hr. Spleen cells from mice (CBA, C3H, and BALB/c nude) treated in vivo with poly I:C also acquire partial resistance to suppression by PGE2. Our data therefore suggest that IFN-stimulated NK cells are protected from suppression by PGE2. Biologically, the IFN-induced protective effect may be beneficial to host resistance to neoplasia.     

Separation of a population of human T lymphocytes that bind prostaglandin E2 and exert a suppressor activity

Prostaglandin E2 (PGE2) is a potent inhibitor of immune functions. Two possible mechanisms of PGE2-mediated suppression have been proposed: one is a direct inhibitory effect exerted on interleukin 2-producing T cells; the second is mediated by the activation of nonspecific suppressor T lymphocytes. We previously showed that PGE2 can directly activate human T lymphocytes to suppress lymphocyte proliferation and B lymphocyte maturation. Herein is described the binding of 10 to 30% of human peripheral blood T lymphocytes to insolubilized PGE2 coated to albumin-Sepharose. The T lymphocytes that bound PGE2 (PGE2(+] could be eluted by the addition of serum and gentle shaking of the beads. The following data indicated the specificity of the binding: i) T lymphocytes after an overnight incubation, a condition known to abolish sensitivity to PGE2, lost their affinity for PGE2; ii) preincubation of T lymphocytes with PGE2 blocked the binding; iii) PGE2(+) T cells bound PGE after a 24-hr incubation, whereas PGE2(-) T cells did not. Few T cells bound albumin, and only a small percentage (7 to 9%) bound 6-keto-prostaglandin F1 alpha-coated beads. Among PGE2(+) T cells, there was a slight increase in the percentage of OKT8+ cells. Although T cells that had no affinity for PGE2 (PGE2(-] proliferated as well as unseparated T lymphocytes when stimulated with mitogens or antigens, the proliferative response of the PGE2(+) subset was poor. Moreover, PGE2(+) T lymphocytes did exert a strong suppressor activity on mitogen- or allogeneic cell-induced lymphocyte proliferation as well as on pokeweed mitogen-driven B cell maturation into Ig-containing cells. PGE2(-) T lymphocytes were shown not to exert a significant suppressor activity in these assays. The PGE2(+) subset-mediated suppression was not secondary to a carry-over of PGE2 released from the beads, because its suppressor activity was not altered by the addition of an anti-PGE2 serum. Moreover, PGE2(-) T lymphocytes were not sensitive to the inhibitory activity on cell proliferation of PGE2. These results indicate that a given functional subset of peripheral blood T lymphocytes binds PGE2, and that at least some of them are activated into suppressor T cells. The relationship between the PGE2-activatable T suppressor subset and other functionally defined suppressor T cells remains to be clarified; it is suggested, however, that PGE2 can act as an immunoregulator through the activation of identifiable suppressor T cells.     

The inhibitory effect of cytosolic fraction of human decidua on prostaglandin synthesis

To elucidate the mechanism of suppression of prostaglandin (PG) production in decidua in early pregnancy, the PG synthetase activity of decidua and mid-secretory endometrium was studied. The microsomal fractions and their supernatants were prepared from the tissue by ultracentrifugation at 105,000 g. The standard incubation mixture consisted of the microsomal fraction and 14C arachidonic acid with cofactors, with incubation being carried out for 10 minutes at 37 degrees C. After extraction, the radioactivity of PGE2 was measured and PG synthetase activity was assayed. The apparent Km value for PG synthetase in decidua was 4.6 +/- 0.14 x 10(-6) M (n = 4), whereas that in endometrium was 4.6 +/- 1.18 x 10(-6) M (n = 3). Subsequently, kinetic studies on PG synthetase inhibitor in decidua were carried out. When sheep seminal vesicle was used as an enzyme source, the decidual supernatant showed competitive inhibition. The inhibitory substance in decidua was inactivated after incubation for 15 minutes at 65 degrees C. It seems likely that the suppression of PG biosynthesis in human decidua in early pregnancy is not due to the difference in PG synthetase found in decidua and in endometrium, but due to the existence of PG synthetase inhibitor in decidua.