Regulation of IL-27 p28 gene expression in macrophages through MyD88- and interferon-gamma-mediated pathways.

Interleukin (IL)-27 is the newest member of the IL-12 family of heterodimeric cytokines composed of the Epstein-Barr virus-induced gene 3 and p28 chains. IL-27 not only plays an important role in the regulation of differentiation of naive T helper cells but also possesses antiinflammatory properties. IL-27 is an early product of activated monocytes/macrophages and dendritic cells. However, the mechanisms whereby inflammatory signals stimulate IL-27 production have not been explored. In this study, we investigated the transcriptional regulation of the mouse IL-27 p28 gene in macrophages in response to lipopolysaccharide (LPS) and interferon (IFN)-gamma. We found that LPS-stimulated p28 production was completely dependent on the Toll-like receptor 4/myeloid differentiation factor 88 (MyD88)-mediated pathway but only partially dependent on nuclear factor kappaB c-Rel. IFN-gamma-induced p28 production/secretion was also partially dependent on MyD88 but independent of c-Rel. We then cloned the mouse p28 gene promoter and mapped its multiple transcription initiation sites. Furthermore, we identified critical promoter elements that mediate the inductive effects of LPS and IFN-gamma, separately and synergistically, on p28 gene transcription in a c-Rel- and interferon regulatory factor 1-dependent manner, respectively.

Autonomous growth potential of leukemia blast cells is associated with poor prognosis in human acute leukemias.

We have described a severe combined immunodeficiency (SCID) mouse model that permits the subcutaneous growth of primary human acute leukemia blast cells into a measurable subcutaneous nodule which may be followed by the development of disseminated disease. Utilizing the SCID mouse model, we examined the growth potential of leukemic blasts from 133 patients with acute leukemia, (67 acute lymphoblastic leukemia (ALL) and 66 acute myeloid leukemia (AML)) in the animals after subcutaneous inoculation without conditioning treatment. The blasts displayed three distinct growth patterns: "aggressive", "indolent", or "no tumor growth". Out of 133 leukemias, 45 (33.8%) displayed an aggressive growth pattern, 14 (10.5%) displayed an indolent growth pattern and 74 (55.6%) did not grow in SCID mice. The growth probability of leukemias from relapsed and/or refractory disease was nearly 3 fold higher than that from patients with newly diagnosed disease. Serial observations found that leukemic blasts from the same individual, which did not initiate tumor growth at initial presentation and/or at early relapse, may engraft and grow in the later stages of disease, suggesting that the ability of leukemia cells for engraftment and proliferation was gradually acquired following the process of leukemia progression. Nine autonomous growing leukemia cell lines were established in vitro. These displayed an aggressive proliferation pattern, suggesting a possible correlation between the capacity of human leukemia cells for autonomous proliferation in vitro and an aggressive growth potential in SCID mice. In addition, we demonstrated that patients whose leukemic blasts displayed an aggressive growth and dissemination pattern in SClD mice had a poor clinical outcome in patients with ALL as well as AML. Patients whose leukemic blasts grew indolently or whose leukemia cells failed to induce growth had a significantly longer DFS and more favorable clinical course.

Serum proteomic profiling and haptoglobin polymorphisms in patients with GVHD after allogeneic hematopoietic cell transplantation.

We studied serum proteomic profiling in patients with graft versus host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) by two-dimensional gel electrophoresis (2-DE) and mass spectrometry analysis. The expression of a group of proteins, haptoglobin (Hp), alpha-1-antitrypsin, apolipoprotein A-IV, serum paraoxonase and Zn-alpha-glycoprotein were increased and the proteins, clusterin precursor, alpha-2-macroglobulin, serum amyloid protein precursor, sex hormone-binding globulin, serotransferrin and complement C4 were decreased in patients with extensive chronic GVHD (cGVHD). Serum haptoglobin (Hp) levels in patients with cGVHD were demonstrated to be statistically higher than in patients without cGVHD and normal controls (p < 0.01). We used immunoblotting and PCR in combination with 2-DE gel image analysis to determine Hp polymorphisms in 25 allo-HCT patients and 16 normal donors. The results demonstrate that patients with cGVHD had a higher incidence of HP 2-2 phenotype (43.8%), in comparison to the patients without cGVHD (0%) and normal donors (18.7%), suggesting the possibility that specific Hp polymorphism may play a role in the development of cGVHD after allo-HCT. In this study, quantitative serum Hp levels were shown to be related to cGVHD development. Further, the data suggest the possibility that specific Hp polymorphisms may be associated with cGVHD development and warrant further investigation.

Interferon regulatory factor 1 is an essential and direct transcriptional activator for interferon {gamma}-induced RANTES/CCl5 expression in macrophages.

Interferon regulatory factor 1 (IRF-1) is an important transcription factor in interferon gamma (IFNgamma)-mediated signaling in the development and function of NK cells and cytotoxic T lymphocytes. RANTES (regulated on activation normal T cell expressed and secreted; CCL5) is a member of the CC chemokine family of proteins, which is strongly chemoattractant for several important immune cell types in host defense against infectious agents and cancer. However, the role of IFNgamma and IRF-1 in the regulation of RANTES gene expression and their operative mechanisms in macrophages have not been established. We report here that RANTES expression in IRF-1-null mice, primarily in macrophages, in response to carcinogenic stimulation in vivo and in vitro and to IFNgamma but not to lipopolysaccharide in vitro, was markedly decreased. As a result, RANTES-mediated chemoattraction of CCR5(+) target cells was also severely impaired. Adenovirus-mediated gene transduction of IRF-1 in primary macrophages resulted in enhanced RANTES expression. The IFNgamma and IRF1 response element was localized to a TTTTC motif at -147 to -143 of the mouse RANTES promoter, to which endogenous or recombinant IRF-1 can physically bind in vitro and in vivo. This study uncovers a novel IFNgamma-induced pathway in RANTES expression mediated by IRF-1 in macrophages and elucidates an important host defense mechanism against neoplastic transformation.

Interleukin-12: an update on its immunological activities, signaling and regulation of gene expression.

Interleukin-12 (IL-12) is a heterodimeric cytokine composed of the p35 and p40 subunits. It is produced by antigen-presenting cells and plays a critical role in host defense against intracellular microbial infection and control of malignancy via its ability to stimulate both innate and adaptive immune effector cells. The potency of IL-12 renders itself to stringent regulation of the timing, locality and magnitude of its production during an immune response. Subversion of the delicate control and balance frequently leads to immunologic disorders. In this article, we provide an update, since our last review of the subject four years ago, on recent advances in: (1) uncovering of novel activities of IL-12 and related molecules in various immunological settings and models; and (2) dissection of the physiological pathways involved in the modulation of IL-12 production by pathogens and immune regulators. The increased understanding of IL-12 immunobiology and expression will likely benefit the development of therapeutic modalities to correct immune dysfunctions.

Synergistic activation of interleukin-12 p35 gene transcription by interferon regulatory factor-1 and interferon consensus sequence-binding protein.

Interferon regulatory factor-1 (IRF-1) and interferon consensus sequence-binding protein (ICSBP or IRF-8) are two members of the IRF family of transcription factors that play critical roles in interferon signaling in a wide range of host responses to infection and malignancy. Interleukin-12 (IL-12) is a key factor in the induction of innate resistance and generation of T helper type 1 cells and cytotoxic T lymphocytes. In this work, we find that ICSBP-deficient macrophages are highly defective in the production of IL-12. The defect is also observed at the level of IL-12 p40 and p35 mRNA expression. Transcriptional analyses revealed that ICSBP is a potent activator of the IL-12 p35 gene. It acts through a site localized to -226 to -219, named ICSBP-response element (ICSBP-RE), in the human IL-12 p35 promoter through physical association with IRF-1 both in vitro and in vivo. Co-expression of ICSBP and IRF-1 synergistically stimulates the IL-12 p35 promoter activity. Mutations at the ICSBP-RE results in the loss of protein binding as well as transcriptional activation by ICSBP alone or together with IRF-1. This study provides novel mechanistic information on how signals initiated during innate and adaptive immune responses synergize to yield greater IL-12 production and sustained cellular immunity.