Diverse and divergent functions of IL-32ß and IL-32γ isoforms in the regulation of malignant pleural mesothelioma cell growth and the production of VEGF-A and CXCL8.

In this study, we sought to elucidate the roles of the interleukin (IL)-32ß and IL-32γ in mesothelioma cell growth, and vascular endothelial growth factor (VEGF)-A and C-X-C motif chemokine ligand 8 (CXCL8) expression. IL-32 elicited a growth-promoting effect against one of the six mesotheliomas lines and exerted diverse regulatory functions in VEGF-A and CXCL8 secretion from mesotheliomas stimulated with or without IL-17A. Retroviral-mediated transduction of mesothelioma lines with IL-32γ resulted in enhanced IL-32ß expression, which facilitated or suppressed the in vitro growth, and VEGF-A and CXCL8 expression. Overexpressed IL-32ß-augmented growth and VEGF-A and CXCL8 production were mainly mediated through the phosphatidylinositol-3 kinase (PI3K) signaling pathway. On the other hand, overexpressed IL-32ß-deceased growth was mediated through mitogen-activated protein kinase (MAPK) pathway. NCI-H2373IL-32γ tumors grew faster than NCI-H2373Neo tumors in a xenograft model, which was associated with increased vascularity. These findings indicate that IL-32 are involved in the regulation of growth and angiogenic factor production in mesotheliomas.

Impaired antigen-specific lymphocyte priming in mice after Toll-like receptor 4 activation via induction of monocytic myeloid-derived suppressor cells.

In sepsis, the pathology involves a shift from a proinflammatory state toward an immunosuppressive phase. We previously showed that an agonistic anti-TLR4 antibody induced long-term endotoxin tolerance and suppressed antigen-specific secondary IgG production when primed prior to immunization with antigen. These findings led us to speculate that TLR4-induced innate tolerance due to primary infection causes an immunosuppressive pathology in sepsis. Therefore, the mechanism underlying impaired antigen-specific humoral immunity by the TLR4 antibody was investigated. We showed, in a mouse model, that primary antigen-specific IgG responses were impaired in TLR4 antibody-induced tolerized mice, which was the result of reduced numbers of antigen-specific GC B cells and plasma cells. Ovalbumin-specific CD4 and CD8 T-cell responses were impaired in TLR4 antibody-injected OT-I and -II transgenic mice ex vivo. Adoptive transfer studies demonstrated suppression of OVA-specific CD4 and CD8 T-cell responses by the TLR4 antibody in vivo. The TLR4 antibody induced Gr1+ CD11b+ myeloid-derived suppressor cell (MDSC) expansion with suppression of T-cell activation. Monocytic MDSCs were more suppressive and exhibited higher expression of PD-L1 and inducible nitric oxidase compared with granulocytic MDSCs. In conclusion, immune tolerance conferred by TLR4 activation induces the expansion of monocytic MDSCs, which impairs antigen-specific T-cell priming and IgG production.

Lipopolysaccharide (LPS)-binding protein stimulates CD14-dependent Toll-like receptor 4 internalization and LPS-induced TBK1-IKKϵ-IRF3 axis activation.

Toll-like receptor 4 (TLR4) is an indispensable immune receptor for lipopolysaccharide (LPS), a major component of the Gram-negative bacterial cell wall. Following LPS stimulation, TLR4 transmits the signal from the cell surface and becomes internalized in an endosome. However, the spatial regulation of TLR4 signaling is not fully understood. Here, we investigated the mechanisms of LPS-induced TLR4 internalization and clarified the roles of the extracellular LPS-binding molecules, LPS-binding protein (LBP), and glycerophosphatidylinositol-anchored protein (CD14). LPS stimulation of CD14-expressing cells induced TLR4 internalization in the presence of serum, and an inhibitory anti-LBP mAb blocked its internalization. Addition of LBP to serum-free cultures restored LPS-induced TLR4 internalization to comparable levels of serum. The secretory form of the CD14 (sCD14) induced internalization but required a much higher concentration than LBP. An inhibitory anti-sCD14 mAb was ineffective for serum-mediated internalization. LBP lacking the domain for LPS transfer to CD14 and a CD14 mutant with reduced LPS binding both attenuated TLR4 internalization. Accordingly, LBP is an essential serum molecule for TLR4 internalization, and its LPS transfer to membrane-anchored CD14 (mCD14) is a prerequisite. LBP induced the LPS-stimulated phosphorylation of TBK1, IKKϵ, and IRF3, leading to IFN-ß expression. However, LPS-stimulated late activation of NF-κB or necroptosis were not affected. Collectively, our results indicate that LBP controls LPS-induced TLR4 internalization, which induces TLR adaptor molecule 1 (TRIF)-dependent activation of the TBK1-IKKϵ-IRF3-IFN-ß pathway. In summary, we showed that LBP-mediated LPS transfer to mCD14 is required for serum-dependent TLR4 internalization and activation of the TRIF pathway.

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.

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.

VEGF-A, HGF and bFGF are Involved in IL-17A-mediated Migration and Capillary-like Vessel Formation of Vascular Endothelial Cells.

BACKGROUND: Interleukin (IL)-17A possesses biological activities to promote vascular endothelial cell migration and microvessel development. OBJECTIVE: To clarify which angiogenic factors are involved in IL-17A-modified angiogenesis-related functions of vascular endothelial cell migration and microtube development or not. METHODS: The potential contribution of various angiogenic stimulators to in vitro angiogenic activities of IL-17A was assessed with both modified Boyden Chemotaxicell chamber assay and in vitro angiogenesis assay. RESULTS: The addition of a neutralizing antibody (Ab) for hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF)-A to the upper and lower compartments in a modified Boyden Chemotaxicell chamber significantly attenuated human dermal microvascular endothelial cell (HMVEC) migration elicited by IL-17A. Moreover, IL-17A-induced capillary-like microvessel development in human umbilical vein endothelial cell (HUVEC) and human dermal fibroblast (HDF) co-culture system was significantly impaired by a neutralizing Ab against HGF, bFGF, VEGF-A, cysteine-x-cysteine ligand 8 (CXCL8)/IL-8 or cysteine-x-cysteine (CXC) chemokine receptor (CXCR)-2. CONCLUSION: Our findings demonstrate the involvement of HGF, bFGF, VEGF-A and/or CXCL8/IL-8, to various degrees, in migration and microvessel development of vascular endothelial cells mediated by IL-17A.

Could problem lists summarize comprehensive geriatric assessments? A nationwide cross-sectional survey on geriatricians' attitudes towards problem lists.

AIM: The use of problem lists is encouraged to overcome the inconsistency in reporting comprehensive geriatric assessment results. The present study aimed to identify the latent variables influencing the use of geriatrician problem lists. METHODS: Surveys were sent to all geriatricians registered with the Japan Geriatrics Society (n = 1439) as of November 2015, and responses (n = 204) were analyzed with univariate and exploratory factor analyses. To account for active, inactive and tentative items, the survey addressed "disease," "symptom" and "condition" separately. RESULTS: Most geriatricians (34.8%) composed problem lists for interdisciplinary information sharing. Nearly half of the respondents (46.6%) created problem lists for every patient. Information omissions were mainly due to the exclusion of information from other specialties (26% for omitted diseases and 12.3% for omitted symptoms), lack of time (25.5% for omitted diseases, 22.1% for omitted symptoms and 26.5% for omitted conditions), and lack of standardization of terminologies regarding observed diseases, symptoms and conditions (12.3% for omitted diseases, 19.6% for omitted symptoms and 16.7% for omitted conditions). An exploratory factor analysis, based on 20 predefined symptoms and conditions that are frequently omitted from problem lists, showed that considering the symptom "geriatric syndromes" and the condition "assistance needs in medication management" are crucial for improving problem list comprehensiveness. CONCLUSIONS: Geriatricians commonly use problem lists; however, there is considerable variation regarding the problems listed and their relationships. The listings of "geriatric syndrome" and "assistance needs in medication management" are crucial for improving problem list comprehensiveness. Geriatr Gerontol Int 2019; 19: 159-164.

Erythromycin attenuates MUC5AC synthesis and secretion in cultured human tracheal cells infected with RV14.

BACKGROUND AND OBJECTIVE: The common cold is a major cause of asthma exacerbation and chronic obstructive lung disease. Rhinovirus is reported to be responsible for more than 50% of cases of the common cold. In a previous study, we reported that rhinovirus infection of cultured airway cells induced MUC5AC mucin overproduction and hypersecretion by activating the p44/42 mitogen-activated protein kinase (p44/42 MAPK) pathway. The aim of this study was to examine the effect of erythromycin on RV14-induced airway mucin overproduction and hypersecretion. METHODS: RV14-infected human tracheal epithelial cells were treated with erythromycin. RESULTS: Erythromycin blocked RV14-induced MUC5AC protein overproduction and hypersecretion, and also blocked RV14-induced p44/42 MAPK activation in the cells. CONCLUSIONS: Erythromycin may attenuate RV14-induced MUC5AC overproduction and hypersecretion by blocking the p44/42 MAPK pathway or its upstream regulators.

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. METHODS: Immunohistochemical analysis with MUC5AC antibody of lower airways resected from six lung cancer patients and three patients with acute myocardial infarction was performed. RESULTS: The submucosal glands contained both MUC5AC-positive cells and non-positive cells. CONCLUSIONS: These data suggest that MUC5AC protein is produced in both goblet cells and in airway submucosal glands.

An inhibitory epitope of human Toll-like receptor 4 resides on leucine-rich repeat 13 and is recognized by a monoclonal antibody.

Lipopolysaccharide (LPS)-induced activation of Toll-like receptor 4 (TLR4) elicits the innate immune response and can trigger septic shock if excessive. Two antibodies (HT4 and HT52) inhibit LPS-induced human TLR4 activation via novel LPS binding-independent mechanisms. The HT52 epitope resides on leucine-rich repeat 2 (LRR2) and is a feature of many inhibitory antibodies; antigen specificity of HT4 does not reside in LRR2. Here, we identified an HT4 epitope on LRR13 located close to the TLR4 dimerization interface that plays a role in NFκB activation. HT4 and HT52 mutually enhanced TLR4 inhibition. LRR13 is a novel inhibitory epitope and may be useful for developing anti-TLR4 antibodies. Combination therapy with LRR2 and LRR13 may effectively inhibit TLR4 activation.

Mechanisms of mucin production by rhinovirus infection in cultured human airway epithelial cells.

Mucus hypersecretion relates to exacerbations of bronchial asthma and chronic obstructive pulmonary disease (COPD) caused by rhinovirus (RV) infection. We examined the mechanisms of RV infection-induced mucin production in human tracheal surface epithelial cells and submucosal gland cells. RV14 up-regulated the mRNA expression of MUC2, MUC3, MUC5AC, MUC5B and MUC6, and increased MUC5AC and total mucin concentration in supernatants and lysates of the surface cells. An inhibitor of the nuclear factor kappaB caffeic acid phenylethyl ester, inhibitors of selective p44/42 mitogen-activated protein kinase-kinase PD98059 and U0126, and a selective Src inhibitor PP1 attenuated MUC5AC mRNA expression, and secretion and production of MUC5AC and total mucin glycoprotein in the surface cells. In the gland cells, RV14 also increased mRNA expression of MUC2, MUC5AC, MUC5B and MUC7, and the inhibitors attenuated the secretion of total mucin glycoprotein. Src-related p44/42 mitogen-activated protein kinase pathway may be associated with RV-induced mucin hypersecretion in human airways.

Interleukin-17 enhances bFGF-, HGF- and VEGF-induced growth of vascular endothelial cells.

Interleukin-17 (IL-17) is a CD4 T cell-derived proinflammatry and proangiogenic cytokine. In this study, we investigated the effects of this cytokine on vascular endothelial cell growth induced by a well-known direct angiogenic factor bFGF, HGF, VEGF, CXCL5/ENA-78 or CXCL8/IL-8. While a wide range of doses of IL-17 alone did not show the ability to stimulate the growth of human dermal microvascular endothelial cells (HMVECs), bFGF, HGF, VEGF, CXCL5 or CXCL8 significantly induced the growth of HMVECs in vitro. When bFGF and IL-17 were used in combination, 10 or 100 ng/ml IL-17 enhanced 10 ng/ml bFGF-induced growth of HMVECs. Similarly, when HGF and IL-17 were combined together, 10 or 100 ng/ml IL-17 potentiated 10 ng/ml HGF-induced growth of HMVECs. When VEGF and IL-17 were used together, 10 ng/ml IL-17 did not significantly enhance 10 ng/ml VEGF-induced growth, whereas 100 ng/ml IL-17 clearly promoted 10ng/ml VEGF-mediated proliferation of HMVECs. On the contrary, IL-17 did not augment CXCL5- and CXCL8-mediated growth. These results indicate that IL-17 itself does not have the capability to stimulate the growth of vascular endothelial cells, whereas IL-17 is able to selectively enhance the mitogenic activity of bFGF, HGF, and VEGF for vascular endothelial cells. Our findings also suggest that IL-17 may promote bFGF-, HGF- and VEGF-mediated angiogenesis through enhancing bFGF-, HGF- and VEGF-induced growth of vascular endothelial cells.

Bone marrow-derived cells contribute to lung regeneration after elastase-induced pulmonary emphysema.

All-trans retinoic acid (ATRA) is known to reverse the anatomic and physiologic signs of pulmonary emphysema. However, the origin of the progenitor cells involved in this lung regeneration remains unclear. Recently, it was shown that bone marrow could be the source of progenitor cells for several cell types. Mice with elastase-induced emphysema were treated with ATRA, granulocyte colony-stimulating factor (G-CSF), or a combination of both. ATRA or G-CSF promoted lung regeneration and increased bone marrow-derived cell (BMC) numbers in alveoli. Combined treatment of both had an additive effect, which indicated that BMC mobilization might be important in lung regeneration.

IL-17 and IL-17F modulate GM-CSF production by lung microvascular endothelial cells stimulated with IL-1beta and/or TNF-alpha.

In this study, we investigated the roles of CD4 T cell cytokines IL-17 and IL-17F in GM-CSF production from lung microvascular endothelial cells (LMVECs). While a wide range of doses of IL-17 or IL-17F alone did not induce GM-CSF release from LMVECs, IL-17 had an enhancing effect on macrophage-derived IL-1beta- and TNF-alpha-induced GM-CSF mRNA expression and production, whereas IL-17F had an enhancing effect on IL-1beta-induced GM-CSF production, but a marked inhibitory effect on TNF-alpha-induced secretion. GM-CSF production was further enhanced with the combination of three cytokines IL-1beta, TNF-alpha and IL-17 or IL-17F. Additionally, when Th1 or Th2 cytokine was combined with IL-1beta or TNF-alpha, both Th1 and Th2 cytokines had a modest stimulatory effect on TNF-alpha-induced GM-CSF production, whereas IL-4 and IFN-gamma profoundly attenuated IL-1beta-induced secretion. Moreover, the regulation by IL-17 plus Th1 or Th2 cytokine of GM-CSF production from LMVECs treated with IL-1beta or TNF-alpha was dependent on the concentration of IL-17. Our findings indicate that IL-17 and IL-17F play a differential regulatory role in GM-CSF production by LMVECs stimulated with IL-1beta and/or TNF-alpha, which is sensitive to Th1 and Th2 cytokine modulation.

Interleukin-17 augments tumor necrosis factor-alpha-induced elaboration of proangiogenic factors from fibroblasts.

Interleukin-17 (IL-17) is a CD4 T cell cytokine. In this report, we investigated the effects of this cytokine on the elaboration of proangiogenic factors by lung fibroblasts. After stimulation with a wide range of doses of IL-17, fibroblasts produced more amount of various kinds of angiogenic factors including NO, HGF, MCP-1, KC, MIP-2, PGE1, PGE2 and VEGF in a dose-dependent manner. Treatment with a COX-1 and COX-2 inhibitor indomethacin did not impair IL-17-induced HGF and VEGF secretion in fibroblasts. In addition, TNF-alpha alone stimulated the elaboration of KC, MIP-2, PGE2 and VEGF in fibroblasts. IL-17 and TNF-alpha in combination up-regulated elaboration of these proangiogenic factors additively or synergistically. Moreover, conditioned media (CM) from IL-17-stimulated fibroblasts showed significantly higher activity on endothelial cell growth than those from non-treated control cells. These results indicate that IL-17 up-regulates elaboration of various proangiogenic factors, and modulates macrophage-derived TNF-alpha-induced production of KC, MIP-2, PGE2 and VEGF by fibroblasts. Our findings also demonstrate that IL-17 might be a potential contributor to the inflammatory angiogenesis via induction of proangiogenic factors by stromal fibroblasts.

Regulatory roles of IL-17 and IL-17F in G-CSF production by lung microvascular endothelial cells stimulated with IL-1beta and/or TNF-alpha.

The role of the interleukin (IL)-17 family members in the regulation of G-CSF production by lung microvasculature has not been elucidated yet. We therefore investigated the effects of IL-17 and IL-17F on the regulation of G-CSF production by lung microvascular endothelial cells (LMVECs). While a wide range of doses of IL-17 or IL-17F alone did not up-regulate G-CSF production from primary human LMVECs, IL-17 had an enhancing effect on macrophage-derived IL-1beta- and TNF-alpha-induced G-CSF production, whereas IL-17F had an enhancing effect on IL-1beta-induced production, but an inhibitory effect on TNF-alpha-induced secretion. G-CSF production was further enhanced with the combination of three cytokines IL-1beta, TNF-alpha and IL-17. In contrast, three cytokines IL-1beta, TNF-alpha and IL-17F were combined together, G-CSF production was less than that induced by IL-1beta or IL-1beta plus TNF-alpha or IL-17F. Moreover, IL-17 plus Th1 or Th2 cytokine had a modest stimulatory effect on TNF-alpha-induced G-CSF production, whereas IL-17 plus IFN-gamma had an inhibitory effect on IL-1beta-induced release. Similarly, IL-17F plus IL-10, IL-13 or IFN-gamma had an inhibitory effect on IL-1beta-induced production. Our findings indicate that CD4 T cell cytokines IL-17 and IL-17F play a differential regulatory role in G-CSF production by LMVECs stimulated with IL-1beta and/or TNF-alpha, which is also sensitive to Th1 and Th2 cytokine modulation.

[Criteria of impairment of pulmonary function using Japanese standard values].

According to the law of functional impairment, cases of impairment of pulmonary function are designated as 1st, 3rd and 4th grade impairment. The criteria of pulmonary function a based on Baldwin's predicted values. Recently, the Japanese Respiratory Society reported the Japanese standard values for pulmonary function. In the present study, we compared the numbers of patients classified into the three grades using the Japanese values and Baldwin's values. We found that from about 10 to 20 percent of patients appeared in a higher grade when Japanese standard values were used instead of Baldwin's values. We suggest that Japanese standard values should be used in order to judge the impairment of these patients.

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-1ß-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-1ß 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-1ß 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.