Structure, function and biology of tissue factor pathway inhibitor-2.

Tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa matrix-associated Kunitz-type serine proteinase inhibitor consisting of a short amino-terminal region,three tandem Kunitz-type domains and a positively charged carboxy-terminal tail. Human TFPI-2, previously designated as placental protein 5, inhibits a broad spectrum of serine proteinases almost exclusively through its first Kunitz-type domain, and is thought to play an important role in the regulation of extracellular matrix digestion and remodeling. In this context, reduced synthesis of TFPI-2 has been related to numerous pathophysiological processes such as inflammation, angiogenesis, atherosclerosis, retinal degeneration and tumor growth/metastasis. In this review, we document current information regarding the expression of TFPI-2 by various tissues, its inhibitory activity and proteinase specificity in-vitro, and discuss possible physiological roles for this inhibitor based on in-vivo studies.

Interleukin-21 is a T-helper cytokine that regulates humoral immunity and cell-mediated anti-tumour responses.

Cytokines and their receptors represent key targets for therapeutic intervention. Ligands are being used to supplement cell numbers that become depleted as a result of disease (organ failure, infection) or subsequent disease treatments (i.e. chemotherapy). Conversely, the inhibition of target cell binding by cytokines is an established strategy for abrogating pathologic cellular activities common to many immunological diseases. Considerable effort in biomedical research is being focused on the cytokine families that play a dominant role in regulating immunity and then prioritizing each member for its therapeutic potential. Currently, the interleukin-2 (IL-2) family of cytokines is widely recognized for its central involvement in controlling lymphocyte function and is the most explored for medical utility. Collectively, these proteins (or their antagonists) are either marketed drugs or have received advanced testing for an impressive array of indications including cancer, infectious disease, transplantation, inflammation and allergic asthma. Here we review the current understanding of IL-21, the most recent member of this cytokine family to be discovered. As will be discussed, IL-21 shares many of the same attributes as its relatives in that it has broad immunoregulatory activity and can modulate both humoral and cell-mediated responses. Its ability to stimulate durable anti-tumour responses in mice defines one therapeutic indication that merits clinical development.

Interleukin-21 inhibits dendritic cell-mediated T cell activation and induction of contact hypersensitivity in vivo.

Interleukin (IL)-21 is a newly described cytokine that is produced by activated T cells and displays structural homology to IL-4 and IL-15. We here analyzed the role of IL-21 in dendritic cell (DC)-induced, T cell-mediated contact hypersensitivity (CHS) in vivo and on T cell activation and unspecific mixed lymphocyte reaction in vitro. By PCR, we demonstrate here constitutive expression of the specific IL-21 receptor and the common gamma-chain in DC, which together are able to mediate IL-21 signaling. Short-time incubation of in vitro generated DC with IL-21 significantly reduced their potential to induce an antigen-specific CD8+ T cell proliferation. Interestingly, 2h incubation of these DC with IL-21 before injection completely inhibited the potential of these DC to induce a CHS reaction to the hapten fluorescein 5-isothiocyanate in vivo. Mice injected with IL-21-treated DC even failed to mount a CHS response after repetitive injection of non-IL-21-treated DC 2 weeks later, suggesting that an antigen-specific unresponsiveness can be induced by IL-21-treated DC. Our data demonstrate that IL-21 is a new modulator of DC-T cell interaction with the potential to induce DC-mediated antigen-specific tolerance.

Interleukin-21 inhibits dendritic cell activation and maturation.

Interleukin 21 (IL-21) is a newly described cytokine with homology to IL-4 and IL-15. They belong to a cytokine family that uses the common gamma chain for signaling but also have their private high-affinity receptors. Since it is well known that IL-4 modulates differentiation and activation of dendritic cells (DCs), we analyzed effects of IL-21 compared with IL-15 on DC differentiation, maturation, and function. Here we show that DCs generated with granulocyte-macrophage colony-stimulating factor (GMCSF) in the presence of IL-21 (IL-21DCs) differentiated into phenotypically and functionally altered DCs characterized by reduced major histocompatibility complex class II (MHCII) expression, high antigen uptake, and low stimulatory capacity for T-cell activation in vitro. Additionally, IL-21DCs completely failed to induce antigen (Ag)-specific T-cell mediated contact hypersensitivity. Furthermore, IL-21 blocked lipopolysaccharide (LPS)-induced activation and maturation of DCs, which was not mediated by release of the anti-inflammatory cytokine IL-10. In contrast, when supplementing GMCSF with IL-15, DCs differentiated into mature antigen-presenting cells (APCs) with low antigen uptake and highly significant increased capacities to stimulate T cells in vitro and in vivo. Taken together, these results identify a dichotomous action of these structurally related cytokines on DCs, establishing IL-21 as inhibitory cytokine on DC activation and IL-15 as potent stimulator of DC function, making both cytokines interesting targets for therapeutic manipulation of DC-induced immune reactions.

Promoter methylation and silencing of the tissue factor pathway inhibitor-2 (TFPI-2), a gene encoding an inhibitor of matrix metalloproteinases in human glioma cells.

We have shown previously that the tissue factor pathway inhibitor-2 (TFPI-2), a broad range proteinase inhibitor, is highly expressed in low-grade gliomas, but, minimally expressed or undetectable in glioblastomas, and that enforced expression of this gene reduces the invasive properties of brain tumor cells. Here, we examined the role of promoter methylation as a mechanism of TFPI-2 gene silencing. In SNB19 glioblastoma cells, which have no detectable TFPI-2 expression, 5-aza-2'-deoxycytidine (5aC), an inhibitor of DNA methyltransferase, induced TFPI-2 mRNA in a dose-dependent manner. Trichostatin A (TSA), the histone deacetylase (HDAC) inhibitor, by itself, was more efficient than 5aC in inducing TFPI-2 transcripts, and the 5aC+TSA combination resulted in highly synergistic reactivation of the gene, both at the transcript and protein levels. In Hs683 glioma cells, which express the TFPI-2 gene at high levels, transfection of the in vitro methylated TFPI-2 promoter constructs resulted in a drastic decrease of promoter activity compared to the unmethylated promoter. Further, the methylation-specific PCR in SNB19 and Hs683 cells showed that TFPI-2 gene repression was closely linked with methylation of the CpG islands in the promoter. Finally, the chromatin immunoprecipitation assays in SNB19 cells showed that the methylated and repressed TFPI-2 promoter was associated with the methyl-CpG binding protein 2 (MeCP2), and that gene reactivation resulted in the loss of MeCP2 from this site. These studies establish that TFPI-2 is transcriptionally silenced through promoter methylation in SNB19 cells.

Physiological and chemical inducers of tissue factor pathway inhibitor-2 in human glioma cells.

Tissue factor pathway inhibitor-2 (TFPI-2), a serine protease inhibitor abundant in the extracellular matrix, is expressed in high amounts in low-grade, non-invasive glioma cells but in low amounts in high-grade, highly invasive glioma cells. Overexpression of TFPI-2 by highly invasive glioma cells reduces their invasiveness and thus may be useful in cancer therapy. The mechanisms underlying the transcriptional regulation of TFPI-2 are not well elucidated. We previously reported that the -312 to +1 region of TFPI-2 was critical for the minimal, inducible regulation of TFPI-2 in gliomas. This region harbors sites for several transcription factors, including SP1 (-192 to -183 and -135 to -128), AP-1 (-310 to -300, -213 to -204, and -163 to -154), NF-kappaB (-229 to -221), an NF-kappaB-like site (-291 to -281), and Lyf-1 (-260 to -252). Here we transiently transfected low-grade Hs683 glioma cells with mutant constructs to clarify the role of these transcription factors in TFPI-2 regulation. Addition of phorbol 12-myristate 13-acetate, 1,2-diacyl-sn-glycerol, IFN-gamma, or IFN-alpha induced the expression of TFPI-2 wild-type promoter construct as well as TFPI-2 protein and mRNA in Hs683 cells. Mutations at either of two AP-1 sites (-310 to -300 and -163 to -154) or either of two SP1 sites (-192 to -183 and -135 to -128) resulted in reduced TFPI-2 activity, regardless of the presence of stimulator compounds, and reduction in DNA-protein binding (by electrophoretic mobility shift assay).

Interleukin-21 and the IL-21 receptor: novel effectors of NK and T cell responses.

Interleukin (IL)-21 was recently discovered using a functional cloning approach based on expression of its receptor. It is similar in domain organization and primary sequence to IL-2 and IL-15. Like these cytokines, IL-21 uses the common gamma chain of the IL-2/15 receptor, which forms a heterodimeric receptor complex with IL-21R. IL-21 is produced by activated T cells, and it influences proliferation of T and B cells and cytolytic activity of natural killer cells. The elucidation of the unique biological effects of IL-21 represents an intense area of interest in current cytokine biology.

Interleukin 21 prevents antigen-induced IgE production by inhibiting germ line C(epsilon) transcription of IL-4-stimulated B cells.

Interleukin 21 (IL-21) has recently been identified as a multifunctional cytokine that induces the proliferation of T cells and B cells and differentiation of natural killer cells. To determine whether IL-21 regulates IL-4-mediated immune responses, we examined the effect of IL-21 on antigen-specific IgE production in mice. We also examined the effect of IL-21 on IL-4-induced IgE production from B cells and antigen-induced T-helper 2 (T(h)2) cell differentiation. The in vivo injection of IL-21 prevented antigen-specific IgE but not IgG2a production on immunization. IL-21 did not affect T(h)2 cell differentiation or IL-4 production from CD4(+) T cells but directly inhibited IL-4-induced IgE production from B cells at single-cell levels. Moreover, IL-21 inhibited IL-4-induced germ line C(epsilon) transcription in B cells without the inhibition of signal transducer and activator of transcription 6 (Stat6) activation. Taken together, these results indicate that IL-21 down-regulates IgE production from IL-4-stimulated B cells through the inhibition of germ line C(epsilon) transcription and thus suggest that IL-21 may be useful for the treatment of IgE-dependent allergic diseases.

Interleukins 19, 20, and 24 signal through two distinct receptor complexes. Differences in receptor-ligand interactions mediate unique biological functions.

Cytokines that signal through Class II receptors form a distinct family that includes the interferons and interleukin 10 (IL-10). Recent identification of several IL-10 homologs has defined a cytokine subfamily that includes AK155, IL-19, IL-20, IL-22, and IL-24. Within this subfamily, IL-19, IL-20, and IL-24 exhibit substantial sharing of receptor complexes; all three are capable of signaling through IL-20RA/IL-20RB, and IL-20 and IL-24 both can also use IL-22R/IL-20RB. However, the biological effects of these three cytokines appear quite distinct: immune activity with IL-19, skin biology with IL-20, and tumor apoptosis with IL-24. To more fully elucidate their interactions with the receptor complexes, we have performed a series of in vitro assays. Reporter, proliferation, and direct STAT activation assays using cell lines expressing transfected receptors revealed differences between the receptor complexes. IL-19 and IL-24 also exhibited growth inhibition on a cell line endogenously expressing all three receptor subunits, an effect that was seen at cytokine levels two orders of magnitude above those required for STAT activation or proliferation. These results demonstrate that, although this subclass exhibits receptor complex redundancy, there are differences in ligand/receptor interactions and in signal transduction that may lead to specificity and a distinct biology for each cytokine.

Minimal and inducible regulation of tissue factor pathway inhibitor-2 in human gliomas.

Tissue factor pathway inhibitor-2 (TFPI-2), a serine protease inhibitor abundant in the extra cellular matrix, is highly expressed in non-invasive cells but undetectable levels in highly invasive human glioma cells. The mechanisms responsible for its transcriptional regulation are not well elucidated. In this study, we made several deletion constructs from a 3.6 kb genomic fragment from Hs683 cells containing the 5'-flanking region of the TFPI-2 gene, transiently transfected with these constructs into non-invasive (Hs683) and highly invasive (SNB19) human glioma cells, and assessed their expression by using a luciferase reporter gene. Three constructs showed high promoter activity (pTF5, -670 to +1; pTF6, -312 to +1; pTF2, -1511 to +1). Another construct, pTF8 (-81 to +1), showed no activity. PTF9, a variant of pTF5 in which a further 231 bp fragment (-312 to -81) was deleted, from the [-670 to +1] pTF5 region, also showed no promoter activity. Hence, (-312 to -81) this region is essential for the transcription of TFPI-2 in glioma cells. Sequencing of this promoter region revealed that it has a high G+C content, contains potential SP1 and AP1 binding motifs, and lacks canonical TATA and CAAT boxes immediately upstream of the major transcriptional initiation site, although CAAT boxes were found about -3000 bp upstream of the transcription start site. We also found a strong repressor in the region between -927 to -1181, upstream of the major transcriptional initiation site, followed by positive elements or enhancers between -1511 to -1181. These positive elements masked the silencer effect. Finally TFPI-2 was induced in Hs683 cells transfected with the pTF6 construct (-312 to +1) and stimulated with phorbol-12-myristate-13-acetate (PMA). We conclude that the -312 to +1 region is critical for the minimal and inducible regulation of TFPI-2 in non-invasive (Hs683) and highly invasive (SNB19) human glioma cell lines.