Molecular mechanisms in IL-1ß-mediated decorin production by decidual cells.

Decorin, a small leucine-rich proteoglycan produced by decidual cells restrains trophoblast differentiation, migration and invasiveness of extra-villous trophoblast cells. Decidual overproduction of decorin is associated with preeclampsia, and elevated decorin levels in maternal plasma are a predictive biomarker of preeclampsia. Furthermore, decorin plays an autocrine role in maturation of human endometrial stromal cells into decidual cells. Thus, a balanced decorin production by the decidua is critical for healthy pregnancy. However, the molecular mechanisms regulating decorin production by the decidua are unclear. Interleukin-1 beta is an inflammation-associated multi-functional cytokine, and is reported to induce decidualization in primates. Hence, the present study was designed: (i) to test if exogenous Interleukin-1 beta stimulated decorin production by human endometrial stromal cells; and if so, (ii) to identify the cellular source of Interleukin-1 beta in first trimester decidual tissue; (iii) to identify the downstream molecular partners in Interleukin-1 beta mediated decorin production by human endometrial stromal cells. Results revealed that (i) amongst multiple pro-inflammatory cytokines tested, Interleukin-1 beta alone stimulated decorin production by these cells; (ii) both macrophages and decidual cells in first trimester decidua produced Interleukin-1 beta; (iii) Interleukin-1 beta mediated decorin production was dependent on Interleukin-1 receptor activation, followed by activation and nuclear translocation of nuclear factor kappa B and its binding to the decorin promoter. These results reveal that Interleukin-1 beta plays a novel role in inducing decorin production by human endometrial stromal cells by activating nuclear factor kappa B.

Decorin production by the human decidua: role in decidual cell maturation.

Decidualization involves the proliferation and differentiation of fibroblast-like endometrial stromal cells into epithelioid-shaped and secretory 'decidual' cells in response to steroid hormones. Human decidual cells produce insulin-like growth factor-binding protein-1 and prolactin (PRL), two well-recognized markers of decidual cell maturation and a proteoglycan decorin (DCN). We reported that DCN restrains the human trophoblast renewal, migration, invasion and endovascular differentiation needed for uterine arterial remodeling during normal pregnancy. DCN overproduction by the decidua is associated with a hypo-invasive placenta and a serious pregnancy disorder, pre-eclampsia (PE). Furthermore, elevated maternal plasma DCN levels during the second trimester is a predictive biomarker of PE. While these paracrine roles of decidua-derived DCN on trophoblast physiology and pathology have been well-defined, it remains unknown whether DCN plays any autocrine role in decidual cell development. The objectives of this study were to examine: the kinetics of DCN production during decidualization of human endometrial stromal cells; gestational age-related changes in DCN production by the first trimester decidua; and a possible autocrine role of DCN on decidual cell maturation. We found that DCN production is enhanced during decidualization of both primary and immortalized human endometrial stromal cells in vitro and during early gestation in decidual samples tested ex vivo, and that it is important for endometrial stromal cell maturation into a decidual phenotype. Decorin-depleted human endometrial stromal cells exposed to decidualizing stimuli failed to mature fully, as evidenced by fibroblastoid morphology, reduced insulin-like growth factor-binding protein-1 and PRL expression, and reduction in cellular ploidy. We identified heart and neural crest derivatives-expressed protein 2, and progesterone receptor as potential downstream mediators of DCN effects.

Bone marrow origin of decidual cell precursors in the pseudopregnant mouse uterus.

Decidual cells are considered to be the endproduct of a hormonally induced transformation of endometrial stromal cells of the uterus. However, the source of these precursors remains unknown. This study of evaluated the possibility of their bone marrow origin by an examination of the H-2 phenotype of decidual cells in pseudopregnant bone marrow chimeras. These chimeras were produced by repopulating lethally irradiated CBA/J female (H-2k) mice with bone marrow from (CBA/J x C57BL/6J) F1 female (H-2kb) mice. Pseudopregnancy was produced with a hormonal regimen followed by an oil-induced decidual stimulus. Chimerism was evaluated radioautographically by an identification of the donor-specific Kb phenotype on cells with an immunolabeling technique with monospecific anti-H-2 serum followed by radioiodinated protein A. The extent of chimerism as indicated by the degree of Kb labeling on decidual cells as well as macrophages contained within the decidual nodules was quantitatively compared with that seen on splenic lymphocytes. Fair to good chimerism, as reflected by labeling for the donor-specific marker (Kb), was seen on splenic lymphocytes and macrophages within the decidual nodules in 6 out of 11 animals. A similar level of chimerism was detected on decidual cells in all but one of these six, in which case this was low. One animal showed low chimerism in the spleen but good chimerism on the decidual cells. The remaining four mice were nonchimeric for all three cell types. These results indicate that decidual cells and macrophages appearing within the decidual nodules of pseudopregnant mice are ultimate descendants of bone marrow cells.

Monoclonal origin of B lymphocyte colony-forming cells in spleen colonies formed by multipotential hemopoietic stem cells.

Spleen colonies produced by transplanting lethally irradiated mice with either 12 day fetal liver or adult bone marrow cells were found to contain B- lymphocyte colony-forming cells (BL-CFC) . The proportion of BL-CFC positive spleen colonies did not increase substantially between 8 and 14 days after transplantation, the range being 18-45 percent. However, the absolute number of BL-CFC per spleen colony varied considerably (between 1 and 10,318), although the majority of colonies contained less than 200 BL-CFC. Irrespective of the time after transplantation, smaller spleen colonies were found to have a higher frequency of BL-CFC than larger spleen colonies. To determine the possible clonal origin of BL-CFC from spleen colony- forming unit (CFU-S), CBA mice were injected with equal numbers of CBA and CBA T(6)/T(6) fetal liver or adult bone marrow cells. Analysis of 7-15-day spleen colonies demonstrated that 90 percent were either exclusively T(6) positive or T(6) negative and approximately equal numbers ofboth colony types were observed. B-lymphocyte colonies were grown and successfully karyotyped from 19 spleen colonies. When compared with the original spleen colony karyotype the B-lymphocyte colony cells karyotype was identical in all 19 cases. In 3 of the 19 colonies analyzed a mixture of T(6) positive and T(6) negative karyotypes was present and identical proportions of the karyotypes were present in the pooled B-lymphocyte colony cells and spleen colony cells. The data indicate that the B-lymphocyte colony-forming cells detected in spleen colonies are genuine members of the hemopoietic clone derived from the initiating hemopoietic stem cell (CFU-S).

Localization of H-2 antigens on mouse trophoblast cells.

The presence of H-2 antigens of the paternal and maternal haplotypes on mouse trophoblast cells was examined at several stages of pregnancy by using a sensitive immunolabeling technique followed by quantitative radioautography. Results revealed the presence of H-2 antigens (determined by the K or D loci) of both parental haplotypes on the F1 trophoblast cells. At 14-16 d of gestation, the antigen density was equivalent to that on adult thymocytes and there was a further 50% increase on day 18. H-2 antigens of both parental haplotypes are also found to be expressed on 11-13 d trophoblast cells.

Localization of paternal H-2K antigens on murine trophoblast cells in vivo.

We have previously shown the presence of H-2K and D antigens of both parental haplotypes on dispersed murine trophoblast cells. The question still remained whether such antigens are sequestered away from the sinusoidal face of these cells making them inert as allografts. The in vivo expression of H-2 antigens on these cells was therefore examined radioautographically after perfusion of 125I-labeled monoclonal and anti-H-2Kk (anti-paternal) antibody directly into individual placental branches of the uterine artery suppling 15-d-old (C57BL/6J female) X CBA/J male) placentae. Syngeneic C57BL/6J placentae served as negative controls. A radioautographic examination of 0.5-micrometer-thick sections revealed specific labeling of labyrinthine trophoblasts lining the sinusoids of allogeneic placentae. Most of this labeling was localized to the sinusoidal face of the cells as opposed to a weak labeling of the intracellular aspect. Spongiotrophoblasts and trophoblast giant cells did not label, but specific labeling of fetal capillary endothelium and some macrophages was also noted.

Amelioration of B16F10 melanoma lung metastasis in mice by a combination therapy with indomethacin and interleukin 2.

Our earlier work revealed that PGE-mediated inactivation of NK cells in tumor-bearing mice by host macrophages promoted spontaneous lung metastasis that could be prevented or ameliorated by chronic indomethacin therapy. Since PGE was found to suppress the in vitro development and/or activation of a family of tumoricidal lymphocytes such as CTL, NK, and LAK cells by one or both of two mechanisms, that is to say, a down regulation of IL-2-R and an inhibition of IL-2 production, the present study tested whether a combined therapy with indomethacin and IL-2 was more effective than one with indomethacin or IL-2 alone in ameliorating established experimental lung metastasis. B6 mice injected intravenously with 10(6) highly metastatic B16F10 melanoma cells showed profuse micrometastases in the lungs by day 5, and macrometastases by day 10 which were confluent on day 21. Chronic indomethacin therapy by the oral route (14 micrograms/ml in drinking water) starting on day 0 or day 5, or a single round of IL-2 therapy (25,000 U rIL-2, every 8 h for 5 d on days 10-14) reduced the number of metastatic nodules by two-thirds (from a median of 473 in control mice receiving vehicles alone) by day 21. A single round of IL-2 as above, combined with either protocol of indomethacin therapy, completely or nearly completely irradicated the lung metastases, corroborated by a histological examination. An evaluation of splenic killer cell activity measured with a 4-h 51Cr-release assay against NK-sensitive YAC-1 lymphoma and B16F10 melanoma or NK-resistant thymic lymphoma 9705 targets revealed negligible activity in control tumor-bearing mice, and a good restoration of activity against NK-sensitive targets with either protocols of indomethacin therapy. IL-2 alone or a combination of IL-2 and indomethacin given by either protocol generated strong killer activity against all these targets, most marked with the combination therapy. Splenic killer cell phenotype in normal as well as all treated animals was ASGM1+, Thy-1-, and Lyt-2-. The combination therapy resulted in the strongest mononuclear cell infiltration in the lungs, with areas of young granulation tissue suggestive of repair sites of original metastases.

Antisense RNA-induced reduction in murine TIMP levels confers oncogenicity on Swiss 3T3 cells.

Mouse 3T3 cell lines capable of constitutively synthesizing an RNA complementary to the messenger RNA encoding TIMP, tissue inhibitor of metalloproteinases, were constructed by transfection with appropriate plasmid constructs. Many of the lines were down-modulated for TIMP messenger RNA levels and secreted less TIMP into the culture medium. In comparison to noninvasive, nontumorigenic controls, these cells not only were invasive in a human amnion invasion assay, but also were tumorigenic and metastatic in athymic mice. These results indicate that TIMP suppresses oncogenicity, at least in immortal murine 3T3 cells.

Decorin-mediated inhibition of proliferation and migration of the human trophoblast via different tyrosine kinase receptors.

Decorin (DCN), a decidua-derived TGFbeta-binding proteoglycan, negatively regulates proliferation, migration, and invasiveness of human extravillous trophoblast (EVT) cells in a TGFbeta-independent manner. The present study examined underlying mechanisms, in particular possible roles of epidermal growth factor receptor (EGFR), IGF receptor (IGFR)-I, and vascular endothelial growth factor receptor (VEGFR)-2. EVT cell sprouting from first-trimester chorionic villus explants in the presence or absence of TGFbeta-neutralizing antibody was inhibited with DCN, suggesting its negative regulatory role in situ. Inhibition of migration of the human EVT cell line HTR-8/SVneo in transwells undercoated with fibronectin was stronger when cells were briefly preincubated with DCN at 4 C (known to retard dissociation of receptor-ligand complex) than at 37 C, suggesting possible DCN action by cell membrane binding. Pretreatment of cells with an IGFR-I blocking agent, but not two EGFR blocking agents or a VEGFR blocking agent, significantly abrogated migration inhibitory effects of DCN, suggesting the involvement of IGFR-I but not EGFR or VEGFR in migration inhibition by DCN. On the other hand, pretreatment with either of the EGFR blocking agents, or the VEGFR blocking agent but not the IGFR-I blocking agent, blocked proliferation inhibitory effects of DCN, indicating the roles of EGFR and VEGFR, but not IGFR-I in antiproliferative action of DCN. EVT cells expressed EGFR, IGFR-I, and VEGFR-2. IGFR-I and VEGF-R2 were phosphorylated in the presence of their natural ligands as well as DCN, and these events were blocked by pretreatment with respective receptor blocking agents indicating DCN-mediated activation of these receptors. In conclusion, DCN effects on EVT cells are mediated selectively by multiple tyrosine kinase receptors.

Suppression of invasion by inducible expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in B16-F10 melanoma cells.

BACKGROUND: We have previously shown that down-modulation (i.e., by antisense expression vector) of tissue inhibitor of metalloproteinase-1 (TIMP-1) in a noninvasive, nontumorigenic cell line led to an acquisition of an invasive, tumorigenic, and metastatic ability in these cells. PURPOSE: Our purpose was to examine whether increased levels of murine TIMP-1 can directly suppress the invasive ability of malignant cells. METHODS: Murine B16-F10 melanoma cells were transfected with an expression vector to overproduce TIMP-1. Among these transfectants, we isolated five clonal cell lines (2-5, 2-8, 2-10, 6-5, and 6-9) that showed upregulation (i.e., overexpression) of TIMP-1. RESULTS: These cell lines had an increased basal level of TIMP-1 messenger RNA. TIMP-1 expression was under the control of the mouse metallothionein-I promoter, and four of these five clones (2-5, 2-8, 6-5, and 6-9) showed a threefold to 10-fold induction of TIMP-1 message when they were treated with 20 microM cadmium for 4 hours. An increase in TIMP-1 message led to an increase in TIMP-1 protein activity measured in the conditioned medium of clones 2-10 and 6-5. The invasive ability of the TIMP-1-upregulated cells was tested in a matrigel transwell invasion assay. All of the upregulated clones showed a significant reduction in their invasive ability, relative to the invasive ability of parental B16-F10 and the control 1-2 cell line; this reduction correlated with the level of TIMP-1 overexpression. Cadmium induction of TIMP-1 messenger RNA resulted in a further suppression of the invasive ability of the two inducible cell lines tested (i.e., 2-8 and 6-5). CONCLUSIONS: Our data demonstrate that a specific upregulation of murine TIMP-1 expression in B16-F10 melanoma cells directly suppresses their invasive ability.

Mechanisms of cellular invasiveness: a comparison of amnion invasion in vitro and metastatic behavior in vivo.

We employed a sensitive in vitro amnion invasion assay to examine the relationship of the invasive ability of numerous mouse and human tumor cell lines and their variants to their ability to spontaneously or artificially metastasize; we also studied possible enzymatic activities involved in the in vitro invasion process. In vitro invasive ability of tumor cells was strongly correlated with spontaneous metastatic ability from the subcutaneous site, regardless of the ability of tumor cells to form artificial metastases when introduced intravenously. However, normal nontumorigenic human trophoblast cells were also highly invasive. Various collagenase inhibitors totally abrogated amnion penetration by all invasive cells; various inhibitors of plasmin, plasminogen, and plasminogen activators prevented invasion in most, but not all, cases. Thus, amnion penetration provides a rigorous test for tumor cell invasiveness required for spontaneous metastasis in vivo, and invasiveness is strongly dependent on metalloproteinase activity, which usually follows plasmin activation.

Changes in the host lymphocyte subsets during chemical carcinogenesis.

Changes in small lymphocyte subsets in the lymphoid organs of young C3H mice were studied following i.m. injection of a carcinogenic dose of 3-methylcholanthrene in trioctanoin oil. Using monoclonal anti-Lyt antibodies and a sandwich radiolabeling method with 125I-labeled rabbit anti-mouse Immunoglobulin, the lymphocyte subpopulations in the thymus, spleen, and draining lymph node were examined by radioautography. During the fifth week following the administration of the carcinogen and prior to the appearance of histologically evident tumor cells, a sharp decrease in the level of Ly-1,2+ small lymphocyte population in the thymus was noted which coincided with a considerable increase (10-fold) in the Ly-2+ and a small increase (1.7-fold) in the Ly-1+ population. During the same period, a similar increase in the Ly-2+ population was also observed in the draining but not in the contralateral lymph node. The high levels of Ly-2+ cells lasted for more than 4 weeks in the thymus while, in the draining node, they lasted for 2 weeks and dropped to normal levels (0 to 2%) simultaneously with the appearance of tumor cells identified in histological preparations. Smaller increases (3-fold) in the Ly-2+ subset were also noted in the spleen and contralateral node, but they were seen later (7 to 8 weeks) and were shorter in duration. These systemic increases coincided with the appearance of macroscopic tumor nodules. The relative incidence of immunoglobulin+ cells did not change significantly in any of the organs tested during the entire tumor induction phase, while the level of null cells underwent a slight increase (approximately 2-fold) in all organs tested immediately prior to and following the appearance of macroscopic tumors. None of these changes was observed in trioctanoin oil-injected control animals. The mixed lymphocyte reaction response of the draining node cells, but not of the spleen, was suppressed during the period of increased level of Ly-2+ cells. Furthermore, during this period, s.c. transplantation of a syngeneic mammary tumor in the same leg resulted in enhanced local growth as well as metastatic spread of the tumor to the lungs in 3-methylcholanthrene-treated mice. These findings suggest that a localized immunosuppression associated with the rise in the Ly-2+ cells may be of functional significance during carcinogen-induced tumor development.

Cure of B16F10 melanoma lung metastasis in mice by chronic indomethacin therapy combined with repeated rounds of interleukin 2: characteristics of killer cells generated in situ.

We had earlier shown that a single round of interleukin 2 (IL-2) in chronically indomethacin treated mice can totally or nearly totally ameliorate established, experimental lung metastases and reactivate natural killer (NK) and lymphokine activated killer-like cells in the spleen. The present study examined whether a lasting cure of metastasis is obtainable by chronic indomethacin therapy (CIT) combined with single or multiple rounds of IL-2, and if so, what are the morphological, phenotypic, and functional properties of tumoricidal cells generated in situ. Experimental lung metastasis was produced in B6 mice by an i.v. injection of 10(6) B16F10 melanoma cells to compare therapeutic effects of six protocols: (a) CIT (14 micrograms/ml via drinking water) starting on day 5 when pulmonary micrometastases are well established; (b) CIT combined with a single round of IL-2 (25,000 units, i.p., every 8 h for 5 days on days 10 through 14); (c) CIT combined with two rounds of IL-2 (days 10-14 and 20-24); (d) two rounds of IL-2 alone; (e) two rounds of IL-2 plus indomethacin given only during the IL-2 therapy; and (f) which was similar to (c), but in addition, followed by repeated injections of IL-2 (25,000 units twice a day on Mondays and Fridays for 8 consecutive weeks). Results revealed that chronic indomethacin therapy alone or two rounds of IL-2 alone, or two rounds of IL-2 plus discontinuous indomethacin therapy reduced the lung metastases (examined at 21-25 days) by about two-thirds. In contrast, both single and multiple rounds of IL-2 in chronically indomethacin treated mice totally or nearly totally eradicated the lung metastases. However, long-term disease-free survival (greater than 13-16 months) resulted only with multiple rounds of IL-2. With chronic indomethacin therapy alone, NK-like (AGM-1+, Thy-1-,Lyt-2-) killer lymphocytes (capable of killing NK sensitive YAC-1 lymphoma and B16F10 melanoma targets) appeared in the spleen, but not lungs; no killer activity was generated in macrophages at either site. Addition of a single round of IL-2 generated lymphokine activated killer-like killer lymphocytes (also capable of killing an NK resistant target) of the same phenotype, but of higher activity in the spleen; some lymphokine activated killer-like killer function was generated among pulmonary lymphocytes which were AGM-1+, Thy-1+,Lyt-2-, as well as among splenic but not pulmonary macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of cancer immunotherapy with indomethacin and interleukin-2 on murine hemopoietic stem cells.

We examined: (a) whether in vitro-generated lymphocyte-activated killer (LAK) cells from normal mice and splenic killer cells from tumor-bearing mice subjected to interleukin-2 (IL-2) therapy alone or in combination with chronic indomethacin therapy have any detrimental effects on the spleen colony-forming units (CFU-S) of the normal bone marrow (BM); and (b) the effects of these immunotherapy protocols on CFU-S numbers in host hemopoietic organs. Effects of in vitro-generated LAK cells (normal C3H/HeN mouse splenocytes cultured with 1000 units IL-2/10(6) cells for 72 h) on BM CFU-S were examined by incubating macrophage-depleted BM cells with LAK cells at 1:2.5 and 1:5 BM:LAK cell ratios or with LAK cell supernatant for 4 h. The cells were washed and subsequently injected into irradiated mice. Irradiated mice were also reconstituted with BM cells or LAK cells incubated alone. Spleen colonies were scored macroscopically and microscopically on day 7 after reconstitution of lethally irradiated mice with the various cell combinations. A comparison of colony numbers produced by LAK and BM cell mixture revealed that LAK cells at either dose had no suppressive effect on the colony-forming ability of BM at the macroscopic and microscopic levels of analysis. The supernatant of cultured LAK cells had a minor suppressive effect on colony formation at the macroscopic but not the microscopic level of analysis, indicating the presence of one or more suppressive factors capable of mediating a short-term inhibitory effect. In the immunotherapy experiment, C3H/HeN mice transplanted s.c. with 5 x 10(5) C3L5 mammary adenocarcinoma cells received either vehicle alone (controls), IL-2 (1.5 x 10(4) Cetus units i.p. every 8 h on days 10-14 and days 20-25), or chronic indomethacin therapy (10 micrograms/ml in drinking water from day 5 onwards) plus IL-2 as above. Animals were killed 24-25 days after tumor transplantation to examine: (a) the number of metastatic lung nodules; (b) the effects of co-incubating therapy-generated splenic effector cells with normal BM cells for 4 h on BM CFU-S, and (c) the CFU-S content of host BM and spleen. Results revealed a drop in spontaneous lung metastases from a mean of 50 in control mice to 18 with IL-2 therapy alone, and to 5 with chronic indomethacin therapy plus IL-2 therapy. Splenocytes from normal and tumor-bearing control or treated mice, when incubated with normal BM, had no effect on spleen colony formation at the macroscopic level.(ABSTRACT TRUNCATED AT 400 WORDS)

Suppression by cathepsin L inhibitors of the invasion of amnion membranes by murine cancer cells.

Cysteine proteinases, particularly cathepsins B and L, have been strongly implicated in fostering metastasis in mice. In this work four different inhibitors of cysteine proteinases have been shown to inhibit the invasion of the human amnion by murine melanoma and mammary carcinoma cells in vitro. Two of the inhibitors are synthetic peptides [ZPhePheCHN2 (benzyloxycarbonyl-L-phenylalanyl-L-phenylalanyldiazomethane) and ZPheAlaCH2F [3-(N-benzyloxycarbonylphenylalanylamido)-DL-1-fluoro-2-butanone]] and two are thiol protease inhibitors (TPIn, TPId) isolated from the skeletal muscle of the hind limbs of normal and dystrophic mice, respectively. The inhibitors (ZPhePheCHN2, TPId), with apparent selectivity for cathepsin L, blocked invasion as effectively as inhibitors (ZPheAlaCH2F, TPIn) effective on both cathepsins. The data reveal that in these cell lines the cysteine proteinases contribute significantly to the invasive capacity of the cells, but to a lesser extent than do the metalloproteinases. We suggest that the cysteine proteinases facilitate the action of metalloproteinases (collagenase, gelatinase, and stromelysin), possibly by activating them, by inactivating the tissue inhibitor of metalloproteinases, and/or by making basement membrane matrix more accessible.

Prostaglandin E2-mediated inactivation of various killer lineage cells by tumor-bearing host macrophages.

We have previously reported that natural killer (NK) lineage cells are progressively inactivated during tumor development by prostaglandin E2 (PGE2) secreted by host macrophages; that this facilitates spontaneous tumor metastases, which can be prevented by chronic indomethacin therapy (CIT); and that CIT combined with multiple rounds of interleukin 2 (IL-2) can cure experimental metastases and activate all killer lineage cells in situ including NK cells, lymphokine-activated killer (LAK) cells, and tumoricidal macrophages. The present study tested whether PGE2 secreted by tumor-bearing host macrophages exerts pansuppressor effects against the activation of T cells, NK cells, LAK cells, and tumoricidal macrophages from normal splenic cell populations. Macrophages isolated from CBA mice bearing 21-day intraperitoneal Ehrlich ascites tumors (EAT) or C3H/HeJ mice bearing 21-day subcutaneous T58 mammary adenocarcinomas were added (+/- 10(-5) M indomethacin, or a monoclonal anti-PGE2 ab) to syngeneic splenic lymphocytes to examine the effects on 1) polyclonal activation (3-d 3H-thymidine [3H-TdR] uptake) with concanavalin A (Con A); 2) one-way (CBA alpha BALB/C or C3H alpha BALBC) MLR (5-d 3H-TdR uptake) and subsequent CTL generation (tested against 51Cr-labeled Con A blasts of the stimulator phenotype); 3) NK activity (after 24-h co-culture) against YAC-1 targets; 4) generation of LAK cell activity (in the presence of 200 or 2,000 units recombinant IL-2 for 3 or 5 days), tested against NK-sensitive and NK-resistant targets. Similar effects were also noted on the generation of tumoricidal activity in normal splenic macrophages cultured for 3 days in the presence of LPS. Normal splenic macrophages added under the same conditions served as controls. Effects of pure PGE2 or PGF2 alpha (10(-6) M) were also examined on these activation events. Results revealed that tumor-host-derived macrophages (but not normal macrophages) markedly suppressed all these activation events and this suppression was abrogated nearly totally by indomethacin and totally by anti-PGE2 ab, indicating its mediation by PGE2. This finding ran parallel with high levels of PGE2 production by tumor-host-derived but not normal splenic macrophages. Pure PGE2 but not PGF2 alpha mimicked these suppressor effects. While tumoricidal activity was generated in normal macrophages in the presence of LPS, IL-2, or IFN-gamma or their various combinations (which led to further augmentation), these agents required the presence of indomethacin to generate significant killer activity in tumor-host-derived macrophages. macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)

Down-regulation of macrophage I-A expression in tumor-bearing mice.

During the subcutaneous growth of a highly metastatic mammary adenocarcinoma line, C3-L4 in C3H/HeJ mice, there was a rapid decline in macrophage I-A expression in vivo. The incidence of I-A+ macrophage subset in the spleen declined from 90% to 10% or less within 5 days of tumor transplantation, with a parallel decline in their absolute number. I-A expression in these cells remained suppressed for a long time until tumors became necrotic and ulcerated. In spite of a low incidence (15-20%) of I-A+ macrophages in the normal peritoneal space, tumor transplantation caused a long-lasting decline in their incidence to one-fourth of the original level. Tumor-associated macrophages were predominantly I-A- throughout the tumor life span. Thus I-A- macrophages dominated in all anatomical compartments in tumor-bearing mice. Macrophage I-A expression was substantially restored (spleen) or stimulated (peritoneal space and tumor) in tumor-transplanted mice subjected to chronic indomethacin therapy in the drinking water, indicating a reversal of prostaglandin-mediated down-regulation of macrophage I-A expression in situ. Concomitantly, this therapy caused regression of the primary tumors and prevented lung metastasis. These results are relevant to the understanding of tumor-host interactions and its exploitation for immunotherapy.

Characterization of macrophage subsets regulating murine natural killer cell activity.

We examined the relationship of I-A expression by normal murine macrophages to their immunoregulatory role on natural killer cell activity. Macrophages were isolated on the basis of plastic adherence; characterized on the basis of conventional markers such as phagocytic ability, cytoplasmic non-specific esterase activity, surface MAC-1 and F4/80 antigen expression; and then used for functional studies relative to their expression of surface I-A. Two functional macrophage subsets were identified: NK-stimulatory and NK-suppressive subsets. The former function was associated with splenic macrophages, which were predominantly I-A+ as identified with a radioautographic immunolabeling technique; the latter function with peritoneal macrophages which were predominantly I-A-. Loss of macrophage I-A expression in vitro was delayed in the presence of indomethacin and enhanced in the presence of PGE2, indicating that PGE2 down-regulates I-A expression on macrophages. The NK stimulatory function of I-A+ macrophages was attributable to a soluble mediator, identified as IFN-gamma, since the stimulatory ability was abrogated with an anti-IFN-gamma antibody. I-A expression appears to be important for the stimulatory function, since some interference with this function was noted in the presence of anti-I-A antibody. The NK-suppressor function of I-A- macrophages was attributable to the soluble mediator PGE2, since this function was abrogated with indomethacin or anti-PGE2 antibody. These results are relevant to the understanding of normal in vivo immunoregulation by macrophages.

Insulin-like growth factor-binding protein 1 stimulates human trophoblast migration by signaling through alpha 5 beta 1 integrin via mitogen-activated protein Kinase pathway.

A highly migratory subpopulation of the human placental trophoblast, known as the extravillous trophoblast (EVT), invades the uterus and its vasculature, to establish adequate exchange of key molecules between the maternal and fetal circulations. During their formation, EVT cells selectively acquire alpha 5 beta 1 integrin. We had shown that alpha 5 beta 1 is required for their migratory function, and that EVT cell migration is stimulated by insulin-like growth factor-binding protein (IGFBP)-1 produced by the uterine decidua. The present study examined whether this stimulation is dependent on binding of the Arg-Gly-Asp (RGD) domain of IGFBP-1 to an RGD binding site on the alpha 5 beta 1 integrin, followed by activation of focal adhesion kinase (FAK) and stimulation of the mitogen-activated protein kinase (MAPK) pathway. IGFBP-1 treatment increased migration of EVT cells, whereas an anti-alpha 5 beta 1 integrin antibody blocked migration regardless of IGFBP-1 treatment. Migration stimulation by IGFBP-1 was abrogated by pretreatment with a Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), but not a Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) hexapeptide, and by mutation of the RGD domain of IGFBP-1 to Trp-Gly-Asp (WGD). IGFBP-1 treatment caused a rapid localization of immunoreactive FAK to cellular lamellipodia, a rapid increase in phosphorylation of FAK and extracellular-signal regulated kinases 1 and 2. Preincubation of EVT cells with Herbimycin A, a tyrosine kinase inhibitor, abrogated IGFBP-1 effects; whereas an MAPK kinase inhibitor, PD 98059, reduced migration regardless of IGFBP-1 treatment. These results indicate that IGFBP-1 stimulation of EVT cell migration occurs by binding of its RGD domain to the alpha 5 beta 1 integrin, leading to activation of FAK and stimulation of MAPK pathway.

Interleukin-1 stimulates human chorionic gonadotropin secretion by first trimester human trophoblast.

Interleukin-1 (IL-1) has been reported to stimulate LH, GH, ACTH, and TSH release from cultured pituitary cells. IL-1 also has been found to be secreted in significant amounts by placental macrophages. To determine the possible role of IL-1 within the placenta, we studied the effects of human recombinant IL-1 on hCG release by long term cultures of human first trimester trophoblast and the JAR (human choriocarcinoma) cell line. IL-1 in concentrations ranging from 10(-11)-10(-9) mol/L stimulated hCG release from trophoblast cultures. This stimulatory effect was not mediated by prostaglandin E2 (PGE2), as indicated by the inability of indomethacin to block this stimulation as well as a lack of IL-1 effect on PGE2 release by trophoblast cells. At these doses, IL-1 exerted no effect on hCG release by JAR cells. PGE2, when used in high concentrations (10(-6)-10(-5) mol/L), stimulated the release of hCG by the trophoblasts as well as by the JAR cells. Neither IL-1 nor PGE2 stimulated the proliferation, [3H]thymidine incorporation, or differentiation (syncytium formation) of trophoblast or JAR cells. These results suggest that IL-1 may be an important local regulator of hCG secretion by first trimester trophoblasts.