Fumihiko Makino

Wound-induced TGF-β1 and TGF-β2 enhance airway epithelial repair via HB-EGF and TGF-α

The abundance of transforming growth factor-beta (TGF-β) in normal airway epithelium suggests its participation in physiological processes to maintain airway homeostasis. The current study was designed to address the hypothesis that TGF-β1 and TGF-β2 might contribute to normal reparative response of airway epithelial cells (AECs). Treatments with exogenous TGF-β1 or TGF-β2 significantly enhanced wound repair of confluent AEC monolayers. Mechanical injury of AEC monolayers induced production of both TGF-β1 and TGF-β2. Wound repair of AECs was significantly reduced by a specific inhibitor of TGF-β type I receptor kinase activity. We investigated whether the TGF-β-enhanced repair required epidermal growth factor receptor (EGFR) transactivation and secretion of EGFR ligands. Both TGF-β1 and TGF-β2 enhanced EGFR phosphorylation and induced production of heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor-alpha (TGF-α) in AECs. Moreover, treatment with a broad-spectrum metalloproteinase inhibitor or anti-HB-EGF and anti-TGF-α antibodies inhibited the wound repair and the EGFR phosphorylation by TGF-β1 and TGF-β2, indicating that the TGF-β1 and TGF-β2 effects on wound repair required the release of HB-EGF and TGF-α. Our data, for the first time, have shown that both TGF-β1 and TGF-β2 play a stimulatory role in airway epithelial repair through EGFR phosphorylation following autocrine production of HB-EGF and TGF-α. These findings highlight an important collaborative mechanism between TGF-β and EGFR in maintaining airway epithelial homeostasis.

TIM-4 Has Dual Function in the Induction and Effector Phases of Murine Arthritis

T cell Ig and mucin domain (TIM)-4 is involved in immune regulation. However, the pathological function of TIM-4 has not been understood and remains to be clarified in various disease models. In this study, DBA/1 mice were treated with anti–TIM-4 mAb during the induction or effector phase of collagen-induced arthritis (CIA). Anti–TIM-4 treatment in the induction phase exacerbated the development of CIA. In vitro experiments suggest that CD4 T cells bind to TIM-4 on APCs, which induces inhibitory effect to CD4 T cells. In contrast, therapeutic treatment with anti–TIM-4 mAb just before or after the onset or even at later stage of CIA significantly suppressed the development and progression by reducing proinflammatory cytokines in the ankle joints without affecting T or B cell responses. Consistently, clinical arthritis scores of collagen Ab-induced arthritis, which is not mediated by T or B cells, were significantly reduced in anti–TIM-4–treated mice with a concomitant decrease of proinflammatory cytokines in the joints. In vitro, macrophages secreted proinflammatory cytokines in response to TIM-4-Ig protein and LPS, which were reduced by the anti–TIM-4 mAb. The anti–TIM-4 mAb also inhibited the differentiation and bone-resorbing activity of osteoclasts. These results indicate that TIM-4 has two distinct functions depending on the stage of arthritis. The therapeutic effect of anti–TIM-4 mAb on arthritis is mediated by the inhibition of proinflammatory cytokine production by inflammatory cells, osteoclast differentiation, and bone resorption, suggesting that TIM-4 might be an appropriate target for the therapeutic treatment of arthritis.

Anti-T cell immunoglobulin and mucin domain-2 monoclonal antibody exacerbates collagen-induced arthritis by stimulating B cells

IntroductionT cell immunoglobulin and mucin domain-2 (TIM-2) has been shown to regulate CD4 T cell activation. However, the role of TIM-2 in the autoimmune disease models has not been clarified yet. In this study, we investigated the effects of anti-TIM-2 monoclonal antibodies (mAbs) in collagen-induced arthritis (CIA) to determine whether TIM-2 contributes to the development of T helper (Th) 1 or Th17 cells and joint inflammation.MethodsDBA/1 mice were treated with anti-TIM-2 mAbs during the early or late phase of CIA. Type II collagen (CII)-specific CD4 T-cell proliferative response and cytokine production were assessed from lymph node cell culture. The serum levels of CII-specific antibody were measured by ELISA. The expression of TIM-2 on CD4 T cells or B cells was determined by flow cytometric analysis.ResultsAdministration of anti-TIM-2 mAbs in early phase, but not late phase, significantly exacerbated the development of CIA. Although anti-TIM-2 mAbs treatment did not affect the development of Th1 or Th17 cells in the draining lymph node, the serum levels of anti-CII antibodies were significantly increased in the anti-TIM-2-treated mice. TIM-2 expression was found on splenic B cells and further up-regulated by anti-immunoglobulin (Ig)M, anti-CD40, and interleukin(IL)-4 stimulation. In contrast, CD4 T cells did not express TIM-2 even when stimulated with both anti-CD3 and anti-CD28 mAbs. Interestingly, anti-TIM-2 mAbs enhanced proliferation and antibody production of activated B cells in vitro.ConclusionsTIM-2 signaling influences both proliferation and antibody production of B cells during the early phase of CIA, but not induction of Th1 or Th17 cells.

Characteristics of alveolar macrophages from murine models of OVA-induced allergic airway inflammation and LPS-induced acute airway inflammation

ABSTRACT Background: Macrophages include the classically activated pro-inflammatory M1 macrophages (M1s) and alternatively activated anti-inflammatory M2 macrophages (M2s). The M1s are activated by both interferon-γ and Toll-like receptor ligands, including lipopolysaccharide (LPS), and have potent pro-inflammatory activity. In contrast, Th2 cytokines activate the M2s, which are involved in the immune response to parasites, promotion of tissue remodeling, and immune regulatory functions. Although alveolar macrophages (AMs) play an essential role in the pulmonary immune system, little is known about their phenotypes. Methods: Quantitative reverse transcription polymerase chain reaction and flow cytometry were used to define the characteristics of alveolar macrophages derived from untreated naïve mice and from murine models of both ovalbumin (OVA)-induced allergic airway inflammation and LPS-induced acute airway inflammation. AMs were co-cultured with CD4+ T cells and were pulsed with tritiated thymidine to assess proliferative responses. Results: We characterized in detail murine AMs and found that these cells were not completely consistent with the current M1 versus M2-polarization model. OVA-induced allergic and LPS-induced acute airway inflammation promoted the polarization of AMs towards the current M2-skewed and M1-skewed phenotypes, respectively. Moreover, our data also show that CD11c+ CD11b+ AMs from the LPS-treated mice play a regulatory role in antigen-specific T-cell proliferation in vitro. Conclusions: These characteristics of AMs depend on the incoming pathogens they encounter and on the phase of inflammation and do not correspond to the current M1 versus M2-polarization model. These findings may facilitate an understanding of their contributions to the pulmonary immune system in airway inflammation.

Blockade of CD70-CD27 interaction inhibits induction of allergic lung inflammation in mice.

The interaction between the TNF receptor family member CD27 and its ligand CD70 provides a costimulatory signal for T-cell activation. In this study, we investigated the effects of neutralizing anti-CD70 monoclonal antibody (mAb) in a murine model of allergic lung inflammation to determine whether CD27 contributes to the development of pathogenic Th2 cells and pulmonary inflammation. BALB/c mice were immunized by an injection of ovalbumin (OVA) with alum adjuvant and challenged with aerosolized OVA in PBS. Some groups of mice were treated with anti-CD70 mAb or control rat IgG during the induction or effector phase. The administration of anti-CD70 mAb during the induction phase, but not the effector phase, reduced eosinophil infiltration in lung tissue compared with control IgG-treated mice. Treatment with anti-CD70 mAb also resulted in the decreased production of Th2 cytokines (IL-4, IL-5, and IL-13) in the bronchoalveolar lavage fluid and draining lymph node cell cultures. We further revealed that antigen-specific CD4 T cells were separated into CD27(+) and CD27(-) populations in the lymph nodes of OVA-immunized DO11.10/Rag-2(-/-) mice. The CD27(+) CD4 T cells produced a high concentration of IFN-γ, representing Th1 cells. In contrast, CD27(-) CD4 T cells produced high concentrations of IL-4, IL-5, and IL-13, representing Th2 cells. Moreover, the population of CD27(-) Th2 cells was significantly reduced by the anti-CD70 mAb treatment. These results indicate an important role for CD27 in the development of pathogenic Th2 cells in a murine model of allergic lung inflammation.

Evaluation of switching low-dose inhaled corticosteroid to pranlukast for step-down therapy in well-controlled patients with mild persistent asthma

Abstract Objective: Treatment guidelines for asthma recommend step-down therapy for well-controlled asthma patients. However, the precise strategy for step-down therapy has not been well defined. We investigated whether well-controlled patients with mild persistent asthma can tolerate a step-down therapy of either a reduced dose of inhaled corticosteroid (ICS) or a switch to a leukotriene receptor antagonist (LTRA), pranlukast hydrate. Methods: We recruited 40 adult patients with mild persistent asthma who were well-controlled for at least 3 months with a low-dose ICS therapy. The patients were randomly assigned to either an ICS dose reduction or a switch to pranlukast for 6 months. Results: FeNO levels in the pranlukast group were significantly increased over that in the ICS group. There were no significant differences between the two groups for lung function, FOT, at the endpoint. The percentage of patients with controlled asthma was 72.2% in the pranlukast group and 90% in the ICS group. No statistically significant difference between the two groups in the percentages of patients with treatment failure was observed. Conclusions: Patients with mild persistent asthma that is well-controlled by a low dose of ICS can be switched to pranlukast safely for at least 6 months. However, 27.8% of the pranlukast group failed to maintain well-control, and FeNO levels increased with the switch to pranlukast at 6 months. This study was been limited by the small sample size and should therefore be considered preliminary. Further studies are needed to investigate the therapeutic efficacy of LTRA monotherapy as a step-down therapy.

Difference between two exhaled nitric oxide analyzers, NIOX VERO® electrochemical hand-held analyzer and NOA280i® chemiluminescence stationary analyzer

Abstract Background: Fractional exhaled nitric oxide (FENO) is useful for the evaluation of eosinophilic airway inflammation, including that seen in asthma. Although a new electrochemical hand-held FENO analyzer, the NIOX VERO® (Aerocrine AB, Solna, Sweden), is clinically convenient to use, it has not been fully compared with the chemiluminescence stationary electrochemical analyzer NOA280i® (Sievers Instruments, Boulder, CO, USA) in terms of the level of measured FENO. The aim of this study was to determine whether there is a difference between the two analyzers. Methods: The FENO levels measured with both NIOX VERO® and NOA280i® were evaluated in 1,369 adults at Juntendo University Hospital from May 2016 to October 2016. Results: The median FENO level measured with the NIOX VERO® was significantly lower than that measured with the NOA280i® (41 ppb, range 5–368 ppb vs. 29 ppb, range 5–251 ppb; p < 0.001). There was a strong positive correlation in the measurement of FENO level between the NOA280i® and the NIOX VERO® (r = 0.942, p < 0.001). The following conversion equation was calculated: FENO (NOA280i®) = 1.362 (SE, 0.661) + 1.384 (SE, 0.021) × FENO (NIOX VERO®). Conclusions: To our best knowledge, we have provided the first report showing that the measured FENO level with the NIOX VERO® was approximately 30% lower than that with the NOA280i® and that there was a significant correlation between the measurements of these two devices. The correction equation that we provided may help assess the data obtained by these two analyzers. AbbreviationsATS American Thoracic SocietyBMI Body mass indexERS European Respiratory SocietyFENO Fractional exhaled nitric oxideGINA Global Initiative for AsthmaNO Nitric oxideppb Parts per billionROC Receiver operating characteristicSD Standard deviation