Muneo Numasaki

IL-17 Enhances the Net Angiogenic Activity and In Vivo Growth of Human Non-Small Cell Lung Cancer in SCID Mice through Promoting CXCR-2-Dependent Angiogenesis

In this study, we examined the biological action of IL-17 on human non-small cell lung cancer (NSCLC). Although IL-17 had no direct effect on the in vitro growth rate of NSCLC, IL-17 selectively augmented the secretion of an array of angiogenic CXC chemokines, including CXCL1, CXCL5, CXCL6, and CXCL8 but not angiostatic chemokines, by three different NSCLC lines. Endothelial cell chemotactic activity (as a measure of net angiogenic potential) was increased in response to conditioned medium from NSCLC stimulated with IL-17 compared with those from unstimulated NSCLC. Enhanced chemotactic activity was suppressed by neutralizing mAb(s) to CXCL1, CXCL5, and CXCL8 or to CXCR-2 but not to vascular endothelial growth factor-A. Transfection with IL-17 into NSCLC had no effect on the in vitro growth, whereas IL-17 transfectants grew more rapidly compared with controls when transplanted in SCID mice. This IL-17-elicited enhancement of NSCLC growth was associated with increased tumor vascularity. Moreover, treatment with anti-mouse CXCR-2-neutralizing Ab significantly attenuated the growth of both neomycin phosphotransferase gene-transfected and IL-17-transfected NSCLC tumors in SCID mice. A potential role for IL-17 in modulation of the human NSCLC phenotype was supported by the findings that, in primary NSCLC tissues, IL-17 expression was frequently detected in accumulating and infiltrating inflammatory cells and that high levels of IL-17 expression were associated with increased tumor vascularity. These results demonstrate that IL-17 increases the net angiogenic activity and in vivo growth of NSCLC via promoting CXCR-2-dependent angiogenesis and suggest that targeting CXCR-2 signaling may be a novel promising strategy to treat patients with NSCLC.

IL-28 Elicits Antitumor Responses against Murine Fibrosarcoma

IL-28 is a recently described antiviral cytokine. In this study, we investigated the biological effects of IL-28 on tumor growth to evaluate its antitumor activity. IL-28 or retroviral transduction of the IL-28 gene into MCA205 cells did not affect in vitro growth, whereas in vivo growth of MCA205IL-28 was markedly suppressed along with survival advantages when compared with that of controls. When the metastatic ability of IL-28-secreting MCA205 cells was compared with that of controls, the expression of IL-28 resulted in a potent inhibition of metastases formation in the lungs. IL-28-mediated suppression of tumor growth was mostly abolished in irradiated mice, indicating that irradiation-sensitive cells, presumably immune cells, are primarily involved in the IL-28-induced suppression of tumor growth. In vivo cell depletion experiments displayed that polymorphonuclear neutrophils, NK cells, and CD8 T cells, but not CD4 T cells, play an equal role in the IL-28-mediated inhibition of in vivo tumor growth. Consistent with these findings, inoculation of MCA205IL-28 into mice evoked enhanced IFN-γ production and cytotoxic T cell activity in spleen cells. Antitumor action of IL-28 is partially dependent on IFN-γ and is independent of IL-12, IL-17, and IL-23. IL-28 increased the total number of splenic NK cells in SCID mice and enhanced IL-12-induced IFN-γ production in vivo and expanded spleen cells in C57BL/6 mice. Moreover, IL-12 augmented IL-28-mediated antitumor activity in the presence or absence of IFN-γ. These findings indicate that IL-28 has bioactivities that induce innate and adaptive immune responses against tumors.

A distinct regulatory role of Th17 cytokines IL-17A and IL-17F in chemokine secretion from lung microvascular endothelial cells.

Th17 cytokines IL-17A and IL-17F play a critical role in the activation and recruitment of neutrophils at airway inflammation mainly through the induction of CXC chemokines in the lungs. Vascular endothelial cells belong to the category of major CXC chemokine-producing cells. However, until now, the precise role of Th17 cytokines in CXC chemokine secretion in lung microvascular endothelial cells (LMVECs) has not been fully elucidated. In this study, we examined the biological effects of Th17 cytokines IL-17A and IL-17F on CXCL1, CXCL5, and CXCL8 release in LMVECs. Both IL-17 receptor A (IL-17RA) and IL-17RC are expressed on the surface of LMVECs. In contrast to IL-17F, IL-17A significantly upregulated CXCL1 mRNA expression and protein release, whereas both IL-17A and IL-17F did not have the ability to induce CXCL5 and CXCL8 secretion in LMVECs. IL-17A and IL-17F displayed positive regulatory effects on IL-1β-induced CXCL1, CXCL5, and CXCL8 secretion. On the other hand, IL-17A enhanced the upregulating effect of TNF-α on CXCL1, CXCL5, and CXCL8 release, whereas IL-17F had a negative regulatory effect on TNF-α-mediated secretion. Th2 cytokines IL-4 and IL-13 showed an inhibitory effect on IL-1β plus IL-17A-induced CXCL1, CXCL5, and CXCL8 secretion, but displayed a positive regulatory effect on TNF-α plus IL-17A-induced secretion. These results provide evidence that Th17 cytokines IL-17A and IL-17F have a distinct regulatory role in CXCL1, CXCL5, and CXCL8 expression in LMVECs stimulated either with IL-1β or with TNF-α. Our findings also suggest that CXC chemokine secretion in LMVECs may be complicatedly regulated by Th17 cytokines, Th2 cytokines, and macrophage-associated cytokines in pathological conditions such as bronchial asthma.

Toll-like receptor-2-activating bifidobacteria strains differentially regulate inflammatory cytokines in the porcine intestinal epithelial cell culture system: finding new anti-inflammatory immunobiotics

A total of 23 strains of bifidobacteria taxonomically belonging to five species were tested for their potent immunomodulatory effect using a combination of two methods: the NF-κB-reporter assay using a toll-like receptor 2-expressing transfectant (HEK(pTLR2) system) and the mitogenic assay using porcine Peyers patches immunocompetent cells. Among the four preselected strains from different immunomodulatory groups, Bifidobacterium breve MCC-117 was able to efficiently modulate the inflammatory response triggered by enterotoxigenic Escherichia coli (ETEC) in a porcine intestinal epithelial (PIE) cell line. Moreover, using PIE cells and swine Peyers patches immunocompetent cell co-culture system, we demonstrated that the immunoregulatory effect of B. breve MCC-117 was related to the capacity of the strain to influence PIE and immune cell interactions, leading to the stimulation of regulatory T cells. The results suggested that bifidobacteria that express high activity in both the HEK(pTLR2) and the mitogenic assays may behave like potential anti-inflammatory strains. The combination of the HEK(pTLR2) system, the evaluation of mitogenic activity and PIE cells will be of value for the development of new immunologically functional foods and feeds that could prevent inflammatory intestinal disorders. Although our findings should be proven in appropriate experiments in vivo, the results of the present work provide a scientific rationale for the use of B. breve MCC-117 to prevent ETEC-induced intestinal inflammation.

Antitumor activity of type III interferon alone or in combination with type I interferon against human non‐small cell lung cancer

The antitumor activities of type III interferon (IFN) (interleukin [IL]‐28 and IL‐29) and the combination of type III IFN and type I IFN (IFN‐α) were evaluated using human non‐small cell lung cancer (NSCLC). The expression of type III and type I receptor complexes was detected in NSCLC lines. IL‐29 significantly inhibited the in vitro growth of a wide range of NSCLC lines in a dose‐dependent fashion. To a lesser degree, IL‐28A also displayed growth inhibitory activity. Antitumor activity of type III IFN is associated with cell cycle arrest at the G1 phase and apoptosis. IL‐29 upregulated cyclin‐dependent kinase inhibitor p21Waf1/Cip1 in cells sensitive, but not insensitive, to antiproliferative activity, and knockdown of p21 with small interfering RNA largely attenuated the antiproliferative effect. Intratumoral and systemic administration of IL‐29 inhibited OBA‐LK1 and LK‐1, but not A549, tumor growth in severe combined immunodeficiency mice. Immunohistochemical analyses demonstrated marked upregulated p21 and downregulated Ki‐67 expression in tumors treated with IL‐29. The interferon combination of IL‐29 and IFN‐α displayed a more effective antiproliferative effect and a more intense p21 expression than each reagent alone in vitro. Furthermore, interferon combination therapy suppressed in vivo NSCLC growth more effectively than interferon monotherapy. These findings demonstrate that type III IFN can mediate direct antitumor activities via increased p21 expression and induction of apoptosis and cooperate with type I IFN to elicit more efficient direct antitumor activities, and suggest the possibility that type III IFN might improve the efficacy and reduce the side‐effects of type I IFN cancer therapy. (Cancer Sci 2011; 102: 1977–1990)

Submucosal gland cells in human lower airways produce MUC5AC protein

Background and objective:  It is now considered that the major component of mucus, MUC5AC, is mainly produced by goblet cells but not submucosal glands, and the role of the submucosal glands in the production of MUC5AC is unclear. The aim of this study was to clarify whether human submucosal glands produce MUC5AC.

Molecular cloning, tissue expression, and subcellular localization of porcine peptidoglycan recognition proteins 3 and 4.

Peptidoglycan recognition proteins (PGRPs) are innate immune molecules that are present in most invertebrates and vertebrates. Mammals have four PGRPs, PGLYRP1-4. In the present study, we cloned the cDNAs encoding porcine PGLYRP3 and 4 from the esophagus of adult swine. The length of the complete open reading frames of porcine PGLYRP3 and 4 are identical and contain 1125bp encoding 374 amino acid residues. The amino acid sequences of these two proteins were more similar to their human orthologs (78.9% [PGLYRP3] and 73.9% [PGLYRP4]) than to their mouse orthologs (71.3% [PGLYRP3] and 67.9% [PGLYRP4]). Expression analysis revealed that both PGLYRP3 and 4 were more strongly expressed in digestive tract, especially the esophagus, than in immune organs such as spleen or mesenteric lymph nodes in both newborn and adult swine. To analyze the subcellular distribution of porcine PGLYRP1-4, we constructed transfectant cell lines. Western blot and flow cytometric analyses revealed that porcine PGLYRP3 and 4 are not only secreted, but also expressed on the cell surface, unlike PGLYRP1 and 2. These results should help contribute to the understanding of PGLYRP3- and 4-mediated immune responses via their recognition of intestinal microorganisms in newborn and adult swine.

A Heterodimeric Cytokine, Consisting of IL-17A and IL-17F, Promotes Migration and Capillary-Like Tube Formation of Human Vascular Endothelial Cells

The interleukin (IL)-17 family, consisting of six homodimeric cytokines IL-17A, IL-17B, IL-17C, IL-17D, IL-17E/IL-25, and IL-17F, mediates a variety of biological activities including regulation of chemokine secretion and angiogenesis. Among the IL-17 family members, IL-17A and IL-17E/IL-25 are angiogenesis stimulators, while IL-17B and IL-17F are angiogenesis inhibitors. Recently, IL-17A/F heterodimer, comprised of the IL-17A and IL-17F subunits, was found as another member of the IL-17 cytokine family. However, to date, it has been unknown whether IL-17A/F has biological actions to affect the angiogenesis-related vascular endothelial functions. Therefore, in this study, we investigated the biological effects of IL-17A/F on the growth, migration and capillary-like tube formation of vascular endothelial cells. Recombinant IL-17A/F protein had no direct effects on the growth of human dermal microvascular endothelial cells (HMVECs), whereas, after 4-hour incubation in a modified Boyden Chemotaxicell chamber, IL-17A/F significantly induced migration of HMVECs over a wide range of doses via the phosphatidylinositol-3 kinase (PI3K) signaling pathway. We further investigated the biological effect of IL-17A/F on capillary-like tube formation using a co-culture system of human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs), which mimicked the in vivo microenvironment. In this co-culture system, IL-17A/F significantly promoted capillary-like endothelial tube formation in a dose-dependent fashion via the PI3K and extracellular signal-regulated kinase (ERK) signaling pathways. Additionally, IL-17A/F up-regulated secretion of angiogenic growth factors such as IL-8 and growth-related oncogene (GRO)-α by HDFs. These findings identify a novel biological function for IL-17A/F as an indirect angiogenic agent.

Anti-tumor immunity elicited by direct intratumoral administration of a recombinant adenovirus expressing either IL-28A/IFN-λ2 or IL-29/IFN-λ1

Interleukin (IL)-28A/interferon (IFN)-λ2 and IL-29/IFN-λ1 have been demonstrated to elicit direct and indirect anti-tumor actions. In this study, we constructed an adenovirus vector expressing either IL-28A/IFN-λ2 (AdIL-28A) or IL-29/IFN-λ1 (AdIL-29) to evaluate the therapeutic properties of intratumoral injection of recombinant adenovirus to apply for the clinical implementation of cancer gene therapy. Despite the lack of an anti-proliferative effect on MCA205 and B16-F10 cells, a retarded growth of established subcutaneous tumors was observed following multiple injections of either AdIL-28A or AdIL-29 when compared with AdNull. In vivo cell depletion experiments displayed that both NK cells and CD8+ T cells have a major role in AdIL-28A-mediated tumor growth suppression. A significant increase in the number of infiltrating CD8+ T cells into the tumors treated with either AdIL-28A or AdIL-29 was observed. Moreover, specific anti-tumor cytotoxic T lymphocyte reactivity was detected in spleen cells from animals treated with either AdIL-28A or AdIL-29. In IFN-γ-deficient mice, anti-tumor activities of AdIL-28A were completely impaired, indicating that IFN-γ is critically involved in the tumor growth inhibition triggered by AdIL-28A. IL-12 provided a synergistic anti-tumor effect when combined with AdIL-28A. These results indicate that AdIL-28A and AdIL-29 could be successfully utilized as an alternative cancer immunogene therapy.

IL-28 and IL-29 in Regulation of Antitumor Immune Response and Induction of Tumor Regression

Type III interferons (IFNs), also known as IFN-λs, are recently described as a novel group of the cytokine family that shares with type I IFNs the same Jak/STAT intracellular signaling pathway driving the expression of a common set of IFN-stimulated genes despite the distinct receptor system usage from type I IFNs. Accordingly, type III IFNs exhibit multiple common biological features with type I IFNs including antiviral activity and antitumor activity in more restricted cell types. Early in vitro studies using human neuroendocrine and colorectal tumor cell lines displayed that, like type I IFNs, type III IFNs can exert direct biological effects on tumor cell growth and functions, such as an induction of apoptosis, by multiple mechanisms. Subsequently, an ensemble of studies based on the use of genetically modified murine tumor cells producing IFN-λ2 (IL-28A) or the in vivo delivery of naked plasmid DNA provided important information on the host-mediated antitumor mechanisms induced by locally produced IL-28A. Of note, these studies have revealed the immunomodulatory functions of type III IFNs, including biological effects on polymorphonuclear neutrophils, NK cells, and T cells, which mainly contribute to type III IFN-induced in vivo antitumor immunity. IFN-γ is partially involved in type III IFN-induced antitumor activity. Additionally, IL-12 enhances type III IFN-mediated antitumor action in the presence or absence of IFN-γ. On the whole, these findings provide clear evidence that type III IFNs have bioactivities to elicit antitumor immune response and indicate the possibility for the application of type III IFNs to cancer immunotherapy.