A proof of concept study: human C48-placenta immunoregulatory factor is an effective, single therapeutic agent enabling allogeneic, nonmanipulated murine bone marrow transplantation.

OBJECTIVE: Cloned placenta immunoregulatory ferritin (PLIF) contains a novel, nonferritin bioactive domain (C-48) with immunodulatory activity. We documented that treatment of whole human bone marrow cells with PLIF and its subcloned C48 proteins resulted in myeloid progenitor cell growth and differentiation and T-cell suppression via an effect on the cytokine network. We tested whether this differential effect supports allogeneic bone marrow transplantation with long-lasting tolerance without any further treatments. MATERIALS AND METHODS: Splenocyte-enriched C3H (H2(k)) whole bone marrow was transplanted into C57Bl (H2(b)) recipients after total body irradiation. Recipients were injected with recombinant C48 (3 mg/kg, intraperitoneal) for 21 days or with glutathione S-transferase. Animals were monitored for survival, chimerism, and clinical signs of graft-vs-host disease (GVHD). Next, chimera whole bone marrow was transplanted to secondary myeloablated C57Bl (H2(b)) hosts without treatment. RESULTS: Mice that received C48 treatment following allogeneic splenocyte-enriched bone marrow transplantation demonstrated full-donor chimerism without GVHD mortality, and normal blood cell counts in 75% of recipients. Secondary transplants from the full chimera to myeloblated C57Bl hosts showed 100% engraftment, no GVHD mortality, and no impairment in the long-term hematopoietic reconstitution potential. Allogeneic response of spleen cells from secondary chimeras against donor C3H (H2(k)) and recipient C57Bl (H2(b)) were similar to syngeneic response, whereas reactivity to third party (DBA H2(d)) was significantly enhanced. CONCLUSIONS: Findings of this study provide the proof of concept that C48-a novel, single, bifunctional therapeutic modality enabled successful allogeneic, unmanipulated bone marrow transplantation without GVHD, and with lasting specific tolerance.

Antibodies to placental immunoregulatory ferritin with transfer of polyclonal lymphocytes arrest MCF-7 human breast cancer growth in a nude mouse model.

The recently cloned human gene named "placental immunoregulatory ferritin" (PLIF) is a pregnancy-related immunomodulator. Recombinant PLIF and its bioactive domain C48 are immune-suppressive and induce pronounced IL-10 production by immune cells. PLIF is expressed in the placenta and breast cancer cells. Blocking PLIF in pregnant mice by anti-C48 antibodies inhibited placental and fetal growth and modulated the cytokine network. It has been revealed that anti-C48 treatment inhibited MCF-7 tumor growth in nude mice. However, this significant effect was observed only in those transfused with human peripheral blood mononuclear cells. Blocking PLIF in tumor-engrafted human immune cell transfused mice resulted in massive infiltration of human CD45+ cells (mainly CD8+ T cells), both intratumorally and in the tumor periphery, and a significant number of caspase-3+ cells. In vitro, anti-C48 treatment of MCF-7 tumor cells cocultured with human lymphocytes induced a significant increase in interferon-gamma secretion. We conclude that blocking PLIF inhibits breast cancer growth, possibly by an effect on the cytokine network in immune cells and on breakdown of immunosuppression.

Treatment of human bone marrow with recombinant placenta immunoregulator ferritin results in myelopoiesis and T-cell suppression through modulation of the cytokine-chemokine networks.

OBJECTIVE: Placenta immunomodulator ferritin (PLIF) is a cloned human chimeric ferritin H chain with a novel non-ferritin C-terminal 48 amino acid sequence (C48). Recombinant PLIF-C48 exhibited cell-mediated immunosuppression. The aim of the current study was to investigate the regulatory effects of native placental ferritin (PLF), recombinant PLIF, and C48 on hematopoiesis of human bone marrow (BM). METHODS: BM mononuclear cells (BM-MNCs) and CD34(+) selected cells were treated in vitro with either PLF, PLIF, or C48 without and in combination with granulocyte (G)-monocyte (M) colony-stimulating factor (GM-CSF) and subjected to hematopoietic progenitor cell assay. Cytokines and chemokines secreted by the treated cells were evaluated in culture supernatant using antibody array assays to determine mechanism of action. RESULTS: In vitro treatment of BM-MNCs with PLF, PLIF, or C48 induced significant growth of myeloid colonies and when mixed with GM-CSF or Granulocyte-Colony Stimulating Factor (G-CSF) exhibited additive enhanced colony forming units-granulocyte monocyte growth. Yet, C48 treatment of selected CD34(+) cells did not yield colony formation and did not affect their response to GM-CSF. Treatment of BM-MNCs with C48 for 48 hours induced secretion of marked levels of GM-CSF, interleukin (IL)-6, IL-1, and IL-10. These cytokines were secreted primarily by C48-treated BM adherent cells and partly by nonadherent cells, whereas the CD34(+) selected cells secreted IL-6 only. CONCLUSION: C48-PLIF enhancement of myelopoiesis resulted from cross talk between BM accessory cells and progenitor cells. The differential PLIF-C48 effects (i.e., myeloid progenitor cell growth and T-cell suppression) are due to their effect on the cytokine-chemokine networks.

PLIF induces IL-10 production in monocytes: a calmodulin-p38 mitogen-activated protein kinase-dependent pathway.

Recently, we reported the cloning and preliminary characterization of a novel human immunomodulator named PLIF (placenta immunomodulatory ferritin). PLIF has a unique molecular structure, which is composed of a ferritin heavy chain-like domain and a novel cytokine-like domain called C48. Both intact molecule and C48 inhibit T cell proliferation following allogeneic or anti-CD3 stimuli. PLIF is localized at the fetal-maternal interface of human placenta and might play a role in down-modulating the maternal immune reaction toward the embryo. The inhibitory effect of PLIF on T cell activation can be direct, indirect through cytokine mediators, or both. In the present study we investigated the possible indirect effects of PLIF by using its bioactive domain C48. Measurement of various cytokines revealed that C48, predominantly, induce pronounced and rapid IL-10 production in monocytes, which is immune activation-independent. Further, we discovered that C48-induced IL-10 production is mediated through a calcium/calmodulin-p38 mitogen-activated protein (MAP) kinase signaling pathway. However, extracellular signal-related kinases1,2 (ERK1,2), also activated by C48 stimulation, exhibited a limiting effect on IL-10 production.

Placental immunomodulator ferritin, a novel immunoregulator, suppresses experimental arthritis.

OBJECTIVE: To determine the effect of treatment with C48, the recombinant cytokine-like domain of the novel human placental immunomodulator ferritin (PLIF) immunoregulator, on zymosan-induced arthritis (ZIA) in mice and on adjuvant-induced arthritis (AIA) in rats. METHODS: The in vitro effect of PLIF/C48 was tested in mixed lymphocyte cultures (MLCs) of allogeneic mouse splenocytes. Arthritis was induced by intraarticular injection of zymosan into naive mice and by subcutaneous injection of Mycobacterium tuberculosis into rats. C48 was injected intraperitoneally daily from day 3 to day 9 or from day 7 to day 13 after induction of synovitis by zymosan, and every other day from day 2 to day 14 after induction of AIA. Swelling of the joints and histologic features of the synovium were assessed. Th1 and Th2 cytokines were quantified by enzyme-linked immunosorbent assay. RESULTS: Both PLIF and C48 significantly inhibited the in vitro immunoreactivity of mouse splenocytes in MLCs. Treatment of ZIA mice and AIA rats with C48 effectively reduced joint swelling. C48 treatment reduced synovial lining thickening, numbers of mononuclear cells and histiocytes, as well as cartilage destruction and bone erosions. In vitro, activated splenocytes from C48-treated ZIA and AIA animals produced significantly higher levels of interleukin-10 (IL-10). In animals with ZIA, this was accompanied by lower levels of tumor necrosis factor and IL-2. CONCLUSION: Human PLIF and C48 were shown to exert cross-species immunosuppressive activity in vitro. The in vivo suppression of articular inflammation in the experimental models of ZIA and AIA was the result of treatment with the antiinflammatory human C48. These results suggest that treatment with C48 may offer an effective immunotherapeutic means of controlling inflammatory polyarthritis.

PLIF, a novel human ferritin subunit from placenta with immunosuppressive activity.

Ferritin is a ubiquitous iron storage protein existing in multiple isoforms composed of 24 heavy and light chain subunits. We describe here a third ferritin-related subunit cloned from human placenta cDNA library and named PLIF (placental immunomodulatory ferritin). The PLIF coding region is composed of ferritin heavy chain (FTH) sequence lacking the 65 C-terminal amino acids, which are substituted with a novel 48 amino acid domain (C48). In contrast to FTH, PLIF mRNA does not include the iron response element in the 5'-untranslated region, suggesting that PLIF synthesis is not regulated by iron. The linkage between the FTH and C48 domains created a restriction site for EcoRI. PLIF protein was found to localize in syncytiotrophoblasts of placentas (8 weeks of gestation) at the fetal-maternal interface. Increased levels of PLIF transcript and protein were also detected in the breast carcinoma cell lines T47D and MCF-7 but not in the benign corresponding cell line HBL-100. In vitro, PLIF was shown to down-modulate mixed lymphocyte reactions and to inhibit the proliferation of peripheral blood mononuclear cells stimulated with OKT3. The accumulated data indicate that PLIF is an embryonic immune factor involved in down-modulating the maternal immune recognition of the embryo toward anergy. This mechanism may have been adapted by breast cancer cells over expressing PLIF.