Ke Fu Wu

IL-18 increases invasiveness of HL-60 myeloid leukemia cells: up-regulation of matrix metalloproteinases-9 (MMP-9) expression.

Similar to matrix metalloproteinases (MMP-9/-2), IL-18 was overexpressed in some hematologic malignancies such as acute myeloid leukemia (AML), which is associated with a poor clinical outcome. To establish a possible functional relationship between IL-18 and MMPs in myeloid leukemia, we used semi-quantitative PCR and zymographic analysis to examine whether IL-18 stimulates human myeloid leukemia cell line HL-60 to produce MMPs and/or specific tissue inhibitors (TIMPs), and to degrade extracellular matrix (ECM) gel in vitro. In the ECM invasion assay IL-18 significantly up-regulated transmigration of HL-60 cells, which in turn was inhibited by a synthetic MMP inhibitor: O-phenanthroline (o-PE), anti-MMP-9, anti-MMP-2 as well as anti-IL-18 monoclonal antibody (McAb), respectively, suggesting that induction of gelatinases by IL-18 leads to ECM degradation by these cells. Moreover, IL-18 could significantly increase MMP-9 but not MMP-2 production at both mRNA and/or protein level, slightly up-regulate TIMP-1 mRNA, and clearly induce TIMP-2 mRNA secretion. We postulate that IL-18 may in part play a role in the clinical aggressiveness of human myeloid leukemia by stimulating MMP-9 production.

Expression and production of bioactive human interleukin-18 in transgenic tobacco plants

The cDNA of human interleukin-18 (hIL-18) was successfully inserted into the genome of tobacco plant, Nicotiana tabacum cv. NC-89, using Agrobacterium tumefaciens-mediated transformation. Insertion and translation of hIL-18 in transformants were confirmed by PCR, ELISA, and Western blot, respectively. The transformed extracts contained the recombinant hIL-18 protein up to 0.05% of total soluble protein. Activity of the recombinant hIL-18 in plant cells was confirmed by the induction of IFN-γ on IL-18-responsive J6-1 cells by the extracts obtained from the transformants. The expression level of hIL-18 (351 ngxa0g−1 tobacco tissue) obtained in the present study may be sufficient to induce responses/effects inxa0vivo.

Expression of IL-18 and its receptor in human leukemia cells

The importance of IL-18, although clearly established in solid tumors, has not been fully elucidated in human hematopoietic neoplasms. Here we examined the mRNA and protein for IL-18 in eight human hematopoietic cell lines representing different lineages and neoplasms including leukemia, lymphoma and others. Our results revealed that IL-18 mRNA was expressed in these cells and that the corresponding protein was found in the cytoplasm. Seven of eight cell lines were also found to express two subunits of the IL-18 receptor (IL-18R) at varied levels. Furthermore, 29 out of 51 leukemia patients tested were observed to express IL-18R with 18/29 (62%) co-expression of both receptor and ligand. By blocking the IL-18 loop using specific antisense oligodeoxynucleotide (ASON) for IL-18 mRNA or anti-human IL-18R monoclonal antibody (McAbR), we were not able to demonstrate a marked inhibition on the most leukemic cell lines growth. Moreover, the potential proliferation in vitro of primary AML cells co-expressing IL-18 and its receptor was not significantly enhanced by recombinant human IL-18, suggesting that IL-18 is not apparently implicated in the proliferation of the leukemia cells via an autocrine loop. Additionally, we also found the effective modulating effect of M-CSF, IFN-alpha and TNF-alpha on IL-18R expression, implying an important in vivo effect of cytokines on IL-18-induced reaction. Moreover, the modulation of IL-18R expression was possibly irrelevant to IFN-gamma secretion induced by these cytokines.

Clinical significance of IL-18 gene over-expression in AML

Little is known about the clinical significance of interleukin (IL)-18, a novel immunoregulatory cytokine, in acute myeloid leukemia (AML). Using reverse transcriptase polymerase chain reaction (RT-PCR) analysis, levels of IL-18 mRNA were assessed in bone marrow mononuclear cells (BMMC) from 47 adult patients with de novo or CR AML in order to explore the clinical significance of IL-18. The relationship between expression levels and the established prognostic factors such as age, cytogenetic aberrations, CD34 expression and FAB subtypes was investigated. Either disease status, age or CD34 expression were found to significantly correlate with the expression of IL-18. With respect to FAB cytotypes, expression of IL-18 gene in M4/M5 (n=15) was statistically higher than that in other subtypes (n=32, P<0.001). Moreover, a significant difference in IL-18 gene expression was obtained between the high risk group and the intermediate risk group (0.5627 versus 0.3111, P=0.038). In addition, a relationship between IL-18 expression of BMMC and initial white blood cell (WBC) was clearly demonstrated by a statistical analysis (r=0.806, P<0.001). These observations suggest that IL-18 gene over-expression might reflect the convergence of several important unfavorable prognostic factors in AML.

Expression of LL-37/hCAP-18 gene in human leukemia cells

LL-37/hCAP-18 is an important part of host defense. Several diseases in human are characterized by impairment in the function of LL-37/hCAP-18 peptide. We examined the expression of LL-37/hCAP-18 in a panel of hematopoietic cell lines representing multiple cell lineages. LL-37/hCAP-18 expression at mRNA level was detected and varied among six of nine cell lines. The level of Raji cells was about eight folds higher than that of Ramos cells. However, only two cell lines, J6-1 and U937, expressed protein products. We also investigated LL-37/hCAP-18 protein in nine leukemia and three idiopathic thrombocytopenic purpura (ITP) patients via immunocytochemical staining. The rate of LL-37/hCAP-18 positive cells ranged from 60 (ITP) to 0.5% (M5). These data suggested that the low translation efficiency of LL-37/hCAP-18 expresses in some leukemia cells might be one of the reasons that leukemia patients were susceptible to infection.

Expression and regulation of interleukin-23 subunits in human peripheral blood mononuclear cells and hematopoietic cell lines in response to various inducers

IL‐23 is a novel cytokine composed of an IL‐12 p40 and a p19 subunit. We analyzed human peripheral blood mononuclear cells (PBMC) and hematopoietic cell lines for constitutive expression of IL‐23 and IL‐12 subunits and expression following exposure to various stimuli, and investigated the mechanisms of their induction by LPS and SAC. IL‐23 p19 and IL‐12 p35 mRNAs were expressed in fresh PBMC, and expression of all IL‐23 and IL‐12 subunits was up‐regulated by LPS and SAC. LPS‐induced increase of IL‐23 and IL‐12 subunits expression was CD14‐dependent, while CD14 was not involved in SAC‐induced p19 transcription. Both LPS‐ and SAC‐induced subunits expression required p38 MAPK pathway. PHA effected an increase of p19 mRNA in both CD4+ and CD8+ T cells, whereas p35 was minimally regulated by PHA. IFN‐γ primed monocytes for LPS stimulation of p19, p35 and p40 expression. p19 mRNA was detectable in most hematopoietic cell lines tested but p35 distribution was more restricted. A phorbol diester enhanced p19, p35 and p40 expression in EBV‐positive cell lines. Our results suggest that both the subunits of IL‐23 are tightly regulated; the expression pattern and regulation mode of IL‐23 p19 is similar to as well as distinct from that of IL‐12 p35.