A Hypothetical Model Suggesting Some Possible Ways That the Progesterone Receptor May Be Involved in Cancer Proliferation.

Cancer and the fetal-placental semi-allograft share certain characteristics, e.g., rapid proliferation, the capacity to invade normal tissue, and, related to the presence of antigens foreign to the host, the need to evade immune surveillance. Many present-day methods to treat cancer use drugs that can block a key molecule that is important for one or more of these characteristics and thus reduce side effects. The ideal molecule would be one that is essential for both the survival of the fetus and malignant tumor, but not needed for normal cells. There is a potential suitable candidate, the progesterone induced blocking factor (PIBF). The parent 90 kilodalton (kDa) form seems to be required for cell-cycle regulation, required by both the fetal-placental unit and malignant tumors. The parent form may be converted to splice variants that help both the fetus and tumors escape immune surveillance, especially in the fetal and tumor microenvironment. Evidence suggests that membrane progesterone receptors are involved in PIBF production, and indeed there has been anecdotal evidence that progesterone receptor antagonists, e.g., mifepristone, can significantly improve longevity and quality of life, with few side effects.

Therapy Aimed to Suppress the Production of the Immunosuppressive Protein Progesterone Induced Blocking Factor (PIBF) May Provide Palliation and/or Increased Longevity for Patients With a Variety of Different Advanced Cancers - A Review.

Progesterone induced blocking factor (PIBF) is a unique protein that is not present in normal cells, but is found predominantly in rapidly growing cells of the fetal placental unit or cancer cells. There is a larger "parent" form that is a nuclear protein involved in cell to cell regulation, allowing tumor cells to proliferate and invade tissues. The parent compound is cleaved into smaller intracytoplasmic isoforms that can suppress cellular immune response, especially, but not limited to natural killer cells. The progesterone receptor antagonist mifepristone can suppress messenger RNA for PIBF, but can also suppress the intracytoplasmic protein. Treating cancer cell lines, intact animals with a variety of spontaneous cancers, and people with various cancers with mifepristone, has been found to inhibit cancer growth, and provide both palliation of symptoms and longevity possibly by suppressing this unique immunomodulatory protein.

Soluble Immune Response Suppressor (SIRS): Reassessing the immunosuppressant potential of an elusive peptide.

A previously studied immunosuppressive cytokine, Soluble Immune Response Suppressor (SIRS), may have relevance to current studies of immune suppression in a variety of human disease states. Despite extensive efforts using experimental models, mainly in mice, much remains to be discovered as to how autoimmune cells in mice and humans escape normal regulation and, conversely, how tumor cells evade evoking an immune response. It is the contention of this commentary that the literature pre-2000 contain results that might inform current studies. The broadly immunosuppressive protein, SIRS, was studied extensively from the 1970s to 1990s and culminated in the determination of the n-terminal 21mer sequence of this 15kDa protein which had high homology to the short neurotoxins from sea snakes, that are canonical members of the three finger neurotoxin superfamily (3FTx). It was not until 2007 that the prophylactic administration of the synthetic N-terminal peptide of the SIRS 21mer, identical to the published sequence, was reported to inhibit or delay the development of two autoimmune diseases in mice: experimental allergic encephalomyelitis (EAE) and type I diabetes (T1D). These findings were consistent with other studies of the 3FTx superfamily as important probes in the study of mammalian pharmacology. It is the perspective of this commentary that SIRS, SIRS peptide and the anti-peptide mAb, represent useful, pharmacologically-active probes for the study of the immune response as well as in the potential treatment of autoimmune, inflammatory diseases and cancer.

Ubiquitin-like protein MNSFß covalently binds to Bcl-G and enhances lipopolysaccharide/interferon γ-induced apoptosis in macrophages.

Monoclonal non-specific suppressor factor ß (MNSFß) is a ubiquitously expressed member of the ubiquitin-like family that is involved in various biological functions. Previous studies have demonstrated that MNSFß covalently binds to intracellular pro-apoptotic protein Bcl-G and regulates the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade in the mouse macrophage cell line Raw264.7. In this study, we demonstrate that MNSFß promotes lipopolysaccharide (LPS)/interferon γ (IFNγ)-induced apoptosis of Raw264.7 macrophages. In Raw264.7 cells treated with MNSFß small interfering RNA (siRNA), LPS/IFNγ- or NO donor S-nitrosoglutathione-induced apoptosis was inhibited. siRNA-mediated knockdown of MNSFß did not affect inducible nitric-oxide synthase (iNOS) expression in LPS/IFNγ-stimulated Raw264.7 cells. Conversely, co-transfection with MNSFß and Bcl-G greatly enhanced LPS/IFNγ- induced apoptosis in Raw264.7 cells, accompanied by increased expression of p53 and decreased Cox-2 activity. Unlike co-transfection with wild-type MNSFß, co-transfection of a mutant MNSFß (G74A) and Bcl-G did not result in enhancement of LPS/IFNγ-induced apoptosis. Co-over-expression of MNSFß and Bcl-G reduced S-nitrosoglutathione-induced ERK1/2 phosphorylation. Furthermore, electrophoretic mobility shift assay experiments revealed that MNSFß down-regulates the ERK/activator protein 1 (AP-1) signaling cascade which leads to Cox-2 activation. We also observed that MNSFß-Bcl-G promotes LPS/IFNγ-induced apoptosis of mouse peritoneal macrophages, together with a decrease in Cox-2 expression. Taken together, our data indicate an apoptosis-enhancing effect of MNSFß-Bcl-G is due in part to down-regulation of Cox-2 activation in macrophages.

Immunoneutralization of endometrial monoclonal nonspecific suppressor factor beta (MNSFbeta) inhibits mouse embryo implantation in vivo.

Successful embryo implantation and pregnancy in mammals depends on the establishment of immune tolerance between the maternal immune system and fetal cells. Monoclonal nonspecific suppressor factor beta (MNSFbeta), a cytokine produced by suppressor T cells in various tissues, possesses an antigen-nonspecific immune-suppressive function, and may be involved in the regulation of the uterine immune response during embryo implantation. In this study, anti-MNSFbeta IgG administered directly into the uterine lumen, significantly inhibited mouse embryo implantation in a dose-dependent manner in vivo, and this effect was reversed by co-administration of recombinant MNSFbeta. The effects of anti-MNSFbeta IgG on the gene pattern profiles in mouse uterine tissues were examined by cDNA microarray and several changes were confirmed by real-time PCR. Anti-MNSFbeta IgG caused up-regulation (> or = 2-fold) of 71 known genes and 17 unknown genes, and decreased expression (> or = 2-fold) of 74 known genes and 43 unknown genes, including several genes previously associated with embryo implantation or fetal development. Most of the known genes are involved in immune regulation, cell cycle/proliferation, cell differentiation/apoptosis, and lipid/glucose metabolism. These results demonstrate that MNSFbeta plays critical roles during the early pregnancy via multiple pathways.

Tumor-infiltrating lymphocyte secretion of IL-6 antagonizes tumor-derived TGF-beta 1 and restores the lymphokine-activated killing activity.

IL-6 is a multifunctional cytokine that regulates cell growth, differentiation, and cell survival. Many tumor cells produce TGF-beta1, which allows them to evade CTL-mediated immune responses. IL-6 antagonizes TGF-beta1 inhibition of CD3 cell activation. However, whether IL-6 restores NK activity, which also is suppressed by TGF-beta1, is not known. We used canine transmissible venereal tumor (CTVT), which produces TGF-beta1, as a model to determine whether IL-6 restores lymphokine-activated killer (LAK) activity. During the progression phase, CTVT cells stop expressing MHC molecules. During the regression phase, the number of surface MHC molecules increases dramatically on about one-third of tumor cells. Tumor cells that stop expressing MHC should be targeted by NK cells. In this study, we found that TGF-beta1 secreted by CTVT cells suppressed LAK cytotoxicity. Interestingly, tumor-infiltrating lymphocytes (TIL) isolated from regressing CTVT secrete high concentrations of IL-6 and antagonize the anti-LAK activity of tumor cell TGF-beta1. TIL also produce IL-6 during progression phase, but the concentration is too low to block the anti-LAK activity of TGF-beta1. There is probably a threshold concentration of IL-6 needed to reverse TGF-beta1-inhibited LAK activity. In addition, in the absence of TGF-beta1, IL-6 derived from TIL does not promote the activity of LAK. This new mechanism, in which TIL manufacture high concentrations of IL-6 to block tumor TGF-beta1 anti-LAK activity, has potential applications in cancer immunotherapy and tumor prognosis.

Cloning and characterization of GITR ligand.

The gene encoding the natural ligand of murine glucocorticoid-induced tumor necrosis factor receptor (GITR) was cloned and characterized. The putative GITR ligand (GITRL) is composed of 173 amino acids with features resembling those of type II membrane proteins and is 51% identical to the human activation-inducible TNF receptor (AITR) ligand, TL6. Expression of the GITRL is restricted to immature and mature splenic dendritic cells. GITRL binds GITR expressed on HEK 293 cells and triggers NF-kappaB activation. Functional studies reveal that soluble CD8-GITRL prevents CD4+CD25+ regulatory T-cell-mediated suppressive activities.

Glucocorticoid involvement in suppression of NK activity following surgery in rats.

We studied plasma factors mediating suppression of NK activity (NKA) following surgery. Plasma from operated rats suppressed NKA of splenocytes, leukocytes, and purified natural killer (NK) cells, and charcoal stripping nullified suppression. The glucocorticoid antagonist mifepristone prevented suppression, whereas blockers of reactive oxygen metabolites, opioids, catecholamines, prostaglandin-E2, and histamine did not. NKA dropped as corticosterone levels peaked postoperatively, and administration of relevant doses of corticosterone suppressed NKA. Inhibition of glucocorticoid synthesis prevented plasma from suppressing NKA but merely attenuated NKA suppression in operated rats. Thus, postoperative concentrations of corticosterone can directly suppress NKA but additional factors probably act in vivo.

[Effect of inhibitors of cyclooxygenase and lypoxygenase pathway of metabolism of arachidonic acid on the development and suppression of the immune response in mice]. / Vplyv blokatoriv tsyklo- ta lipoksyhenaznoho shliakhiv metabolizmu arakhidonovoï kysloty na rozvytok i supresiiu imunnoï vidpovidi u myshei.

The influence of indomethacin (IM) and nordihydroguaiaretic acid (NDGA) as inhibitors of cyclooxygenase and lypoxygenase pathways of arachidonic acid metabolism, accordingly, on the development of the immune response (IR) to sheep red blood cells (SRBC), as well as on the formation and functional activity of the antigen-induced (by the tolerogenous dose of SRBC) T-suppressors (AITs) was studied. Investigation was carried out on mice of line CBA. The IR was estimated by quantity of antibody-forming cells in the mouse spleen. The functional activity of AITs was determined in the transfer adopting system by the intensity of IR suppression in mice--recipients. The data obtained have demonstrated, that both inhibitors mainly stimulated the IR which was more expressed at NDGA application and depended on a phase of the IR. The stimulation of the IR was related with a suppression of AITs and a decrease in their functional activity.

B7-H1 is expressed by human endothelial cells and suppresses T cell cytokine synthesis.

Human endothelial cells (ECs) provide costimulatory signals sufficient to activate resting memory T cells to produce IL-2 and IFN-gamma, at least in part through CD58-CD2 interactions. Recently, the B7-like molecule, B7-H1 (PD-L1), was described and shown to regulate T cell activation; however, there are conflicting reports on whether it stimulates or inhibits T cell cytokine synthesis. B7-H1 is not expressed constitutively by ECs; however, it is rapidly induced by IFN-gamma, and synergistically by IFN-gamma and TNF. In inflamed skin, B7-H1 is expressed by a subset of microvessels, and by keratinocytes, but is barely detectable in normal skin. Blocking the interaction of EC-expressed B7-H1 with its T cell ligand, programmed death-1 (PD-1), using a PD-1-Fc fusion protein, or by blocking B7-H1 expression with morpholino antisense oligonucleotides, augments expression of IL-2 and IFN-gamma, implicating B7-H1 as a negative regulator of cytokine synthesis. However, signaling through PD-1 does not affect induction of the activation markers CD25 or CD69 on T cells, suggesting that its effects are specific to cytokine synthesis. The suppressive effects of B7-H1 on cytokine expression are proportional to the strength of the primary stimulus, allowing for B7-H1 to determine the level of T cell activation in response to ECs. Our results demonstrate that B7-H1 negatively regulates cytokine synthesis in T cells activated by ECs.

Progesterone and non-specific immunologic mechanisms in pregnancy.

PROBLEM: Progesterone-dependent immunomodulation is one of the mechanisms that enables pregnancy to proceed to term. Immunologic effects of progesterone are mediated by a protein named the progesterone-induced blocking factor (PIBF). Among other effects this protein inhibits natural killer (NK) activity and displays an anti-abortive effect in mice. Recently, we have shown that PIBF induces a Th2 shift in vitro. The present study was aimed at investigating the in vivo effect of PIBF on cytokine production, as well as the relationship between cytokine production, NK activity, and pregnancy loss. METHOD OF STUDY: Balb-c mice on day 8.5 of pregnancy were injected intraperitoneally with 0.5 mg of rabbit anti-PIBF immunoglobulin G (IgG). Another group of mice was simultaneously treated with anti-NK monoclonal antibodies. Mice treated with the same amount of normal rabbit serum or untreated mice of similar gestational age were used as controls. The animals were sacrificed and their uteri were inspected. The ratio of living and resorbed embryos was determined. NK activity as well as cytokine expression on the spleen cells were determined by immunocytochemistry and enzyme-linked immunoadsorbent assay (ELISA). RESULTS: Mitogen-activated spleen cells from anti-PIBF-treated mice produced significantly (P < 0.001) less IL-10 than those of pregnant control mice. A significantly higher percentage (P < 0.001) of spleen cells from anti-PIBF-treated mice expressed interferon-gamma (IFN gamma) as determined by immunocytochemistry, than those of untreated pregnant mice. There was a positive relationship between the percentage of IFN gamma-positive spleen cells and resorption rates, and an inverse relationship between the latter and interleukin-10 (IL-10) production. All these effects were corrected by treatment with anti-NK antibodies. CONCLUSION: Our data suggest that PIBF contributes to the success of gestation via cytokine-mediated inhibition of NK activity.

The antiabortive effect of progesterone-induced blocking factor in mice is manifested by modulating NK activity.

Immunologic effects of progesterone are mediated by a protein named the progesterone-induced blocking factor (PIBF), which inhibits NK activity and displays an antiabortive effect in mice. Our previous data provide indirect evidence for the importance of PIBF in the maintenance of normal gestation. This study was aimed at investigating whether neutralization of endogenous PIBF production influences pregnancy outcome and if so, what are the mechanisms that participate in this process. Syngeneically pregnant Balb/c mice on Day 8.5 of pregnancy were injected ip with 0.3 mg/kg of RU 486 or with 0.5 mg of rabbit anti-PIBF IgG alone, or together with anti-NK monoclonal antibodies. Mice treated with the same amount of normal rabbit serum or untreated mice of similar gestational age were used as controls. On Day 10.5 the ratio of living and resorbed embryos and NK activity of the spleen cells were determined. In mice treated with anti-PIBF the ratio of resorbed fetuses was significantly higher than that in untreated controls. In RU 486-treated mice we also observed significantly increased resorption rate, which was associated with the inability of spleen cells to produce PIBF. Both anti-PIBF treatment and that with progesterone receptor blocker resulted in increased splenic NK activity. There was a positive relationship between NK activity and the rate of resorptions. All the above effects were corrected by simultaneous treatment with anti-NK or anti-NC (natural cytotoxic) antibodies. These data allow the conclusion that PIBF contributes to normal gestation in mice and that the effect of PIBF is manifested via blocking NK and/or NC activity.

Cell lines derived from human autorosette-forming cells with suppressive activity.

Concanavalin-A-stimulated human T lymphocytes from healthy donors and from patients suffering from diverse immune disorders were fractionated into rosette-forming (R) and nonrosette-forming (NR) cells. The separation method is based upon the ability of the lymphocytes to bind autologous erythrocytes and form autorosettes. Long-term cultures of the R and NR subpopulations were established. The activity of the culture supernatants on the T cell proliferation of normal human phytohemagglutinin (PHA)-induced lymphocytes and of a murine, interleukin-2 (IL-2)-dependent cytotoxic T cell line (CTLL) was investigated. Only the R cell line-derived supernatants from almost all patients tested evinced potent suppressor activity, those from healthy donors less so. The suppressive function was demonstrated not to be due to a cytotoxic effect since preincubation of the PHA-induced lymphocytes and CTLL cells with the factor did not diminish their proliferative capacity. Our study indicates the existence of a competitive relationship between the suppressor factor and IL-2. We found that inhibition of the proliferation decreased with the addition of increasing quantities of exogenous IL-2. We also observed that preincubating the CTLL cells with IL-2 prior to exposing them to the suppressive factor precludes inhibition of their proliferation. Phenotypic analysis of the suppressor cell line revealed that they were comprised of a T cell population which included OKT4+ and OKT8+ cells and that 99% of the cells formed autorosettes. Preliminary purification of the suppressive factor was performed by ultrafiltration and maximal suppression was exhibited by the fraction of less than 10,000 daltons. The development of suppressor cell lines from the unique population of autologous rosette-forming cells may be very helpful in studying the immunoregulatory properties of these cells and their suppressor activity.

The molecular basis of granuloma formation in schistosomiasis. III. In vivo effects of a T cell-derived suppressor effector factor and IL-2 on granuloma formation.

Granuloma formation in schistosomiasis is characterized by the formation of a large lesion in acutely infected animals which subsequently decreases in size as disease progresses into the chronic phase. These in vivo studies confirm and extend previous in vitro observations on the regulation of granulomatous hypersensitivity by a T cell-derived suppressor effector factor (TseF). TseF regulation of granuloma formation in vivo and DTH are shown to be both antigenically and genetically restricted. This suppression is accompanied by a suppression of the ability of cells derived from TseF recipients to function in an in vitro assay of granuloma formation. Antigenic recognition, defined by cellular proliferation in response to antigenic stimulation, is uneffected by TseF administration. Administration of IL-2 reduces TseF function in acutely infected mice and results in increased liver granuloma size. However, the ability of cells derived from these animals to form granulomas in vitro is uneffected. Cells obtained from chronically infected IL-2 recipients do not produce TseF in vitro and granuloma size is increased in these animals. Animals receiving both IL-2 and TseF continue to demonstrate decreased granuloma formation, indicating that IL-2 does not effect the ability of preformed TseF to function. These observations suggest that TseF modulates granuloma formation in vivo and may interact with IL-2 in a dynamic process which determines the intensity of the granulomatous response.

Characterization of a suppressor factor that regulates phagocytosis by macrophages in murine cryptococcosis.

A T-suppressor factor which inhibits the phagocytic activity of a macrophage subset has been further characterized. This suppressor factor was first described for a murine model of cryptococcosis but was later found to be common to models of immunologic unresponsiveness. The suppressor factor was produced when suppressor cells were cultured in the presence of specific cryptococcal antigen. It could not be extracted from spleen cells and was not induced by antigen in cultures of lymph node cells. The suppressor factor was filtered through Amicon filters of 100-kilodalton (kDa) exclusion limit but was retained by filters excluding molecules of less than 50 kDa. By Sephadex G-100 chromatography, the factor eluted just ahead of bovine serum albumin (68 kDa). The activity of the suppressor factor could not be inhibited by anticryptococcal antibody, but it was inhibited by anti-I-J alloantiserum of the same genotype as the lymphocyte which produced the factor. Absorption with an encapsulated strain of Cryptococcus neoformans removed the suppressor factor from culture supernatants, while absorption with a nonencapsulated mutant or an unrelated yeast cell had not effect. On the basis of these observations, it was apparent that the suppressor factor was idiotypic in nature and that I-J and/or the I-J-interactive molecule played a role in the function of the suppressor factor. The requirement for antigenic stimulation for the production of suppressor factor in vitro distinguished it from the T-suppressor factor 3 described by others which regulates delayed-type hypersensitivity in cryptococcosis.

T-cell suppressor factors play an integral role in preventing fetal rejection.

In previous studies we showed that the blocking of T-cell suppressor factors (TsF) with monoclonal antibody could completely ablate pregnancy, and demonstrated the presence of TsF in fetal and maternal tissues. In our current study we used a monoclonal antibody specific for TsF to determine the time during gestation when TsF is most integral in the maintenance of pregnancy. Significant decreases in the number of viable pregnancies when monoclonal antibody was administered on days 3, 4 and 5 were demonstrated. In addition, ELISAs were used to measure the levels of TsF in tissues from pregnant and non-pregnant mice. Increased levels of TsF in the uterus and the lymph nodes draining the uterus were observed when compared to the same tissues of non-pregnant animals. These data strengthen the hypothesis that TsF is in part responsible for the fetal specific immune supression during pregnancy in allogeneic mice and more clearly define the importance of TsF in the implantation of the embryo.

Mode of action of monoclonal-nonspecific suppressor factor (MNSF) produced by murine hybridoma.

Monoclonal-nonspecific suppressor factor (MNSF), a product of murine T cell hybridoma, suppresses antibody response to lipopolysaccharide. In an attempt to clarify the functional mechanisms in vitro, we investigated the mode of action of MNSF. This factor inhibited the antibody response by B cells (depleting T cells and Mø), thereby indicating that the lymphokine acts directly on B cells, without interaction between B and T cells or Mø. MNSF activity was absorbed by mitogen-stimulated T or B cells, but not by resting lymphocytes. Proliferative responses to T cell and B cell mitogens were inhibited dose dependently by the addition of MNSF. Kinetic studies showed that MNSF suppressed the antibody response, in all culture periods, thereby indicating that immunoglobulin secretion and proliferation were inhibited. The effect of growth factor on MNSF-mediated suppression was investigated to search for a possible suppression of MNSF action. Interleukin 2 (IL-2) remarkably inhibited MNSF activity, and the effect of IL-1 or IL-4 was less. IL-2 was most effective when added on the fourth day of culture. MNSF also inhibits division in the plasmacytoma line MOPC-31C or in thymoma EL4, but not in L929 fibroblasts. Tumor necrosis factor (TNF) inhibits cell division of various tumor cells and suppresses the pokeweed mitogen-induced antibody response, without cytotoxic action, as does MNSF. While MNSF and TNF have similar biochemical and physiochemical properties, the cross-reaction tests showed that both are antigenically discrete lymphokines. Although MNSF lacks TNF activity, the concomitant addition of both factors to L929 increases the cytotoxic action, a finding indicative of a synergistic effect.

Monoclonal anti-idiotypic antibodies to an I-J interacting molecule inhibit suppression in an H-2 restricted way.

We have obtained 10 mAb from two independent fusions that are anti-idiotypic to the combining site of an anti-I-Jk antibody. These mAb block Ts cell function isn a genetically restricted manner in vitro and in vivo and recognize a determinant on macrophage membranes. In addition, they do not affect the I-Ek-restricted activation of a Th cell line specific for pigeon heart cytochrome c. We conclude that these mAb may recognize a molecule other than conventional I-Ek on cells interacting with Ts cells that is involved in mediating Ts activity. The molecule recognized may be a modified I-Ek molecule or a molecule not encoded by the genes encoding I-Ek.

Induction of suppression by a murine nonspecific suppressor-inducer cell line (M1-A5). II. The role of prostaglandins.

A murine nonspecific suppressor-inducer cell line (M1-A5) was generated from the spleen cells of a mouse bearing an advanced methylcholanthrene-induced fibrosarcoma. We previously demonstrated the capability of M1-A5 cells to activate suppressor cells from the spleen cells of unprimed mice. We demonstrate here that induction of suppression by M1-A5 cells was blocked by acetylsalicylic acid (ASA) and ibuprofen at concentrations which block prostaglandin (PG) synthesis. Maximal blockade of the induction of suppression by M1-A5 cells was seen when ASA was added at the initiation of culture, and it required inhibition of PG synthesis at the level of the inducer (M1-A5 cells) population. However, ASA blockade of suppressor cell activation by M1-A5 cells was not due to ASA acetylating suppressor-inducing factor. Exogenously added PGE1, PGE2, and PGI2, but not PGF2 alpha or PGD2, were able to restore the inducing capability of M1-A5 cells, which had been blocked by ASA. However, PGE1, PGE2, or PGI2 did not reconstitute an inactive suppressor-inducing factor. These results suggest that PG act to modulate the release of suppressor-inducing factor from M1-A5 cells.