Hirofumi Kamata

Elevated CC Chemokine Level in Bronchoalveolar Lavage Fluid Is Predictive of a Poor Outcome of Idiopathic Pulmonary Fibrosis

Background: CC chemokines play important roles in the pathogenesis of interstitial lung diseases. Elevated CC chemokine levels have been observed in bronchoalveolar lavage (BAL) fluid of patients with idiopathic pulmonary fibrosis (IPF). Objectives:We aimed to examine whether the levels of four CC chemokines, i.e. monocyte chemoattractant protein-1 (MCP-1/CCL2), macrophage inflammatory protein-1α (MIP-1α/CCL3), thymus- and activation-regulated chemokine (TARC/CCL17), and macrophage-derived chemokine (MDC/CCL22), in BAL fluid are predictive of the prognosis of IPF patients. Methods: We compared the chemokine levels of patients alive 5 years after diagnosis and those who had died. Lung function data, CT scores, and serum markers were also compared. Results: Among 39 patients (29 males, median age, 60 years), 19 patients (48%) died within 5 years after the diagnosis. Whereas percent vital capacity was not different, percent lung diffusion capacity for carbon monoxide was significantly higher in the surviving patients than in the nonsurviving patients (p < 0.01). Median CCL2 levels of surviving and nonsurviving patients were 154.3 (interquartile range, IQR: 67.3–381.8) and 427.2 (IQR: 329.2–1184.1) pg/ml, respectively (p < 0.02). CCL3 levels in BAL fluid did not differ between the surviving and nonsurviving patients. CCL17 was detected in BAL fluid of 7 patients, 6 of whom died within 5 years. CCL22 was detectable in BAL fluid of 10 patients, only 1 of whom survived. Serum levels of KL-6 and lactate dehydrogenase did not differ between the surviving and nonsurviving patients. Conclusion: Elevated levels of CCL2, CCL17 and CCL22 in BAL fluid might be predictive of a poor outcome in patients with IPF.

CRTH2 Is A Critical Regulator of Neutrophil Migration and Resistance to Polymicrobial Sepsis

Although arachidonic acid cascade has been shown to be involved in sepsis, little is known about the role of PGD2 and its newly found receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), on the septic response. Severe sepsis is associated with the failure of neutrophil migration. To investigate whether CRTH2 influences neutrophil recruitment and the lethality during sepsis, sepsis was induced by cecal ligation and puncture (CLP) surgery in mice. CRTH2 knockout (CRTH2−/−) mice were highly resistant to CLP-induced sepsis, which was associated with lower bacterial load and lower production of TNF-α, IL-6, and CCL3. IL-10, an anti-inflammatory cytokine, was higher in CRTH2−/− mice, blunting CLP-induced lethality in CRTH2−/− mice. Neutrophil accumulation in the peritoneum was more pronounced after CLP in CRTH2−/− mice, which was associated with higher CXCR2 levels in circulating neutrophils. Furthermore, sepsis caused a decrease in the level of acetylation of histone H3, an activation mark, at the CXCR2 promoter in wild-type neutrophils, suggesting that CXCR2 expression levels are epigenetically regulated. Finally, both pharmacological depletion of neutrophils and inhibition of CXCR2 abrogated the survival benefit in CRTH2−/− mice. These results demonstrate that genetic ablation of CRTH2 improved impaired neutrophil migration and survival during severe sepsis, which was mechanistically associated with epigenetic-mediated CXCR2 expression. Thus, CRTH2 is a potential therapeutic target for polymicrobial sepsis.

Carbon black nanoparticles enhance bleomycin-induced lung inflammatory and fibrotic changes in mice

With the recent increasing use of nanoparticles, there is concern that they may become an environmental risk factor as airborne particles. However, the impact of these particles on susceptible subjects with predisposing lung disease have not been sufficiently elucidated. In the present study, we investigated the effects of nanoparticles on pulmonary inflammatory and fibrotic changes induced by intratracheal bleomycin (BLM) challenge in mice. Mice were intratracheally administered either vehicle, 14-nm carbon black nanoparticles (CBNPs), BLM or BLM plus CBNP. First, we assessed lung collagen content, lung compliance and fibrotic changes in histopathology on day 21 after instillation. Then, to elucidate how CBNP contributes to the development of BLM-induced fibrosis, we collected bronchoalveolar lavage (BAL) fluid on days 2, 7, 14 and 21 and determined the total and differential cell counts and concentrations of two proinflammatory cytokines (keratinocyte chemoattractant [KC] and interleukin [IL]-6) and two fibrogenic mediators (CC chemokine ligand 2 [CCL2] and transforming growth factor-β 1 [TGF-β 1]). Expression of nitrotyrosine, an indicator of oxidant injury, was also evaluated on days 7 and 21. CBNP, when combined with BLM, significantly enhanced BLM-induced increase in lung collagen content, decrease in lung compliance, and fibrotic changes in histopathology. CBNP significantly augmented BLM-induced increase in the numbers of inflammatory cells in BAL fluid on days 2 and 7 and levels of KC and IL-6 on day 2. In addition, CBNP administered in combination with BLM significantly elevated the levels of CCL2 on days 2, 7 and 14, and TGF-β 1 on day 14 in BAL fluid as compared with BLM alone. Nitrotyrosine expression was also increased by BLM plus CBNP compared with BLM alone. In contrast, CBNP did not exert any significant effect on these parameters by itself. These results indicate that CBNP can exaggerate BLM-induced inflammatory and fibrotic changes in the lung, suggesting the potential impact of nanoparticles on lung inflammation and fibrosis.

Cytokine profiles of bronchoalveolar lavage fluid in patients with pneumocystis pneumonia

The clinical features of PCP differ according to the factors responsible for the predisposing immunosuppression. Although the diagnosis of PCP often requires BAL, the profiles of the inflammatory mediators in the BAL fluid are not thoroughly documented. The aim of the current study was to characterize the profiles of inflammatory mediators in BAL fluid during PCP in patients with underlying autoimmune diseases, malignancies, or AIDS. The medical records of 14 patients with autoimmune diseases, 10 with malignancies, and 8 with AIDS, all of whom had been diagnosed with PCP by microscopic examination of BAL fluid, were reviewed. The concentrations of TNF‐α, MCP‐1, HMGB1, IL‐8, IL‐6, IL‐10, and IFN‐γ in the BAL fluid that had been obtained for the diagnosis of PCP were measured. The concentrations of MCP‐1, IL‐8, and IL‐6 differed according to the underlying disease, tending to be higher in patients with autoimmune diseases and lower in those with AIDS. The concentrations of HMGB1, IL‐8, and IL‐6 were positively correlated with the proportion of neutrophils in BAL fluid and inversely with the oxygenation index. Although the serum concentrations of CRP and LDH were positively correlated with those of IL‐8 and MCP‐1, none of the mediators in BAL fluid was correlated with the serum β‐D‐glucan concentration. The production of inflammatory mediators in the lung differed between the patient groups with different underlying disorders. The modest upregulation of IL‐8 and IL‐6 might be associated with the milder clinical manifestations of PCP in AIDS patients.

Intratracheal synthetic CpG oligodeoxynucleotide causes acute lung injury with systemic inflammatory response

Bacterial genome is characterized by frequent unmethylated cytosine-phosphate-guanine (CpG) motifs. Deleterious effects can occur when synthetic oligodeoxynucleotides (ODN) with unmethylated CpG dinucleotides (CpG-ODN) are administered in a systemic fashion. We aimed to evaluate the effect of intratracheal CpG-ODN on lung inflammation and systemic inflammatory response. C57BL/6J mice received intratracheal administration of CpG-ODN (0.01, 0.1, 1.0, 10, or 100 μM) or control ODN without CpG motif. Bronchoalveolar lavage (BAL) fluid was obtained 3 or 6 h or 1, 2, 7, or 14 days after the instillation and subjected to a differential cell count and cytokine measurement. Lung permeability was evaluated as the BAL fluid-to-plasma ratio of the concentration of human serum albumin that was injected 1 h before euthanasia. Nuclear factor (NF)-κB DNA binding activity was also evaluated in lung homogenates. Intratracheal administration of 10 μM or higher concentration of CpG-ODN induced significant inflammatory cell accumulation into the airspace. The peak accumulation of neutrophils and lymphocytes occurred 1 and 2 days after the CpG-ODN administration, respectively. Lung permeability was increased 1 day after the 10 μM CpG-ODN challenge. CpG-ODN also induced nuclear translocation of NF-κB and upregulation of various inflammatory cytokines in BAL fluid and plasma. Histopathology of the lungs and liver revealed acute lung injury and liver damage with necrosis, respectively. Control ODN without CpG motif did not induce any inflammatory change. Since intratracheal CpG-ODN induced acute lung injury as well as systemic inflammatory response, therapeutic strategies to neutralize bacterial DNA that is released after administration of bactericidal agents should be considered.

Elevated serum adiponectin level in patients with mycobacterium avium-intracellulare complex pulmonary disease

Background: Patients with Mycobacterium avium-intracellulare complex (MAC) pulmonary disease often suffer from weight loss. Adipokines are factors secreted by adipocytes, including leptin and adiponectin, as well as some inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6). Body mass index (BMI) is known to be inversely correlated with adiponectin and positively with leptin, TNF-α, and IL-6. Objective: We aimed to evaluate the levels of serum adipokines, including adiponectin, leptin, TNF-α, and IL-6 in patients with MAC pulmonary disease. Methods: Forty consecutive patients with MAC pulmonary disease (8 males; median age 62 years; median BMI 18.1) were examined. Serum levels of adiponectin, leptin, TNF-α, and IL-6 were measured with ELISA. Age-, sex- and BMI-matched healthy subjects served as controls. Results: Serum adiponectin was significantly elevated in patients with MAC pulmonary disease compared with the controls (p < 0.01). In both the patients and controls, serum adiponectin levels were inversely correlated with BMI (p < 0.05). No significant correlation was observed between serum adiponectin levels and C-reactive protein or lung function. Serum leptin levels, which were positively correlated with BMI, did not differ between patients and controls. Serum levels of TNF-α and IL-6 were significantly greater in patients with MAC pulmonary disease than in controls. The levels of TNF-α and IL-6 were not correlated with BMI and other adipokines examined. Conclusion: The results of the present study indicate that, in patients with MAC pulmonary disease, adiponectin is inappropriately secreted and may play a role in the pathophysiology of the disease.

Role of interleukin-6 in elastase-induced lung inflammatory changes in mice

ABSTRACT Interleukin-6 (IL-6) is known to be involved in the pathogenesis of various inflammatory diseases, but its role in the development of pulmonary emphysema remains unclear. Wild-type (WT) and IL-6–deficient mice received either phosphate-buffered saline (PBS) or porcine pancreatic elastase (PPE) intratracheally. The development of emphysema was determined by measuring the mean linear intercept (Lm). The lung specimens were also subjected to immunohistochemistry for single-stranded DNA to detect apoptotic cells. Lung mechanics and airway responsiveness to inhaled methacholine were analyzed. Bronchoalveolar lavage (BAL) fluid was subjected to evaluation of inflammatory cell accumulation and cytokine measurement. PPE treatment caused significant increases in Lm and lung compliance, which was attenuated by IL-6 deficiency. The increases in apoptotic cells in the lung were attenuated in IL-6 null mice. Airway responsiveness was not affected by PPE challenge or IL-6 deficiency. Intratracheal PPE increased the cell counts in BAL fluid throughout the observation, which was suppressed in IL-6 null mice. In BAL fluid, PPE-induced increases in the levels of macrophage inflammatory protein (MIP)-1α and eotaxin were mitigated by IL-6 deficiency. PPE-induced up-regulation of matrix metalloproteinase (MMP)-12 in the lung was attenuated by IL-6 deficiency. These results indicate that IL-6 may play an important role in the development of elastase-induced lung inflammatory changes.

Clarithromycin expands CD11b+Gr-1+cells via the STAT3/Bv8 axis to ameliorate lethal endotoxic shock and post-influenza bacterial pneumonia

Macrolides are used to treat various inflammatory diseases owing to their immunomodulatory properties; however, little is known about their precise mechanism of action. In this study, we investigated the functional significance of the expansion of myeloid-derived suppressor cell (MDSC)-like CD11b+Gr-1+ cells in response to the macrolide antibiotic clarithromycin (CAM) in mouse models of shock and post-influenza pneumococcal pneumonia as well as in humans. Intraperitoneal administration of CAM markedly expanded splenic and lung CD11b+Gr-1+ cell populations in naïve mice. Notably, CAM pretreatment enhanced survival in a mouse model of lipopolysaccharide (LPS)-induced shock. In addition, adoptive transfer of CAM-treated CD11b+Gr-1+ cells protected mice against LPS-induced lethality via increased IL-10 expression. CAM also improved survival in post-influenza, CAM-resistant pneumococcal pneumonia, with improved lung pathology as well as decreased interferon (IFN)-γ and increased IL-10 levels. Adoptive transfer of CAM-treated CD11b+Gr-1+ cells protected mice from post-influenza pneumococcal pneumonia. Further analysis revealed that the CAM-induced CD11b+Gr-1+ cell expansion was dependent on STAT3-mediated Bv8 production and may be facilitated by the presence of gut commensal microbiota. Lastly, an analysis of peripheral blood obtained from healthy volunteers following oral CAM administration showed a trend toward the expansion of human MDSC-like cells (Lineage−HLA-DR−CD11b+CD33+) with increased arginase 1 mRNA expression. Thus, CAM promoted the expansion of a unique population of immunosuppressive CD11b+Gr-1+ cells essential for the immunomodulatory properties of macrolides.

Anti-inflammatory roles of mesenchymal stromal cells during acute Streptococcus pneumoniae pulmonary infection in mice

BACKGROUND Pneumonia is the fourth leading cause of death worldwide, and Streptococcus pneumoniae is the most commonly associated pathogen. Increasing evidence suggests that mesenchymal stromal cells (MSCs) have anti-inflammatory roles during innate immune responses such as sepsis. However, little is known about the effect of MSCs on pneumococcal pneumonia. METHODS Bone marrow-derived macrophages (BMDMs) were stimulated with various ligands in the presence or absence of MSC-conditioned medium. For in vivo studies, mice intranasally-inoculated with S. pneumoniae were intravenously treated with MSCs or vehicle, and various parameters were assessed. RESULTS After stimulation with toll-like receptor (TLR) 2, TLR9 or TLR4 ligands, or live S. pneumoniae, TNF-α and interleukin (IL)-6 levels were significantly decreased, whereas IL-10 was significantly increased in BMDMs cultured in MSC-conditioned medium. In mice, MSC treatment decreased the number of neutrophils in bronchoalveolar lavage fluid (BALF) after pneumococcal infection, and this was associated with a decrease in myeloperoxidase activity in the lungs. Levels of proinflammatory cytokines, including TNF-α, IL-6, GM-CSF and IFN-γ, were significantly lower in MSC-treated mice, and the bacterial load in the lung after pneumococcal infection was significantly reduced. In addition, histopathologic analysis confirmed a decrease in the number of cells recruited to the lungs; however, lung edema, protein leakage into the BALF and levels of the antibacterial protein lipocalin 2 in the BALF were comparable between the groups. CONCLUSIONS These results indicate that MSCs could represent a potential therapeutic application for the treatment of pneumonia caused by S. pneumoniae.

A Phase II study of S-1 and irinotecan combination therapy in previously treated patients with advanced non-small cell lung cancer

OBJECTIVE This Phase II study was conducted to evaluate the efficacy and safety of S-1 and irinotecan combination therapy as a second-line treatment in patients with advanced non-small cell lung cancer. METHODS Irinotecan was administered at 60 mg/m(2) on Days 1 and 8. Oral S-1 was administered on Days 1-14 every 3 weeks at 80 mg/day for patients with a body surface area of <1.25 m(2), 100 mg/day for patients with a body surface area of 1.25-1.5 m(2) and 120 mg/day for patients with a body surface area of >1.5 m(2). The primary endpoint was response rate, while the secondary endpoints were progression-free survival, overall survival and safety. RESULTS Thirty-one patients were enrolled in this study. The response and disease control rates were 6.5 and 58.1%, respectively. Progression-free survival and median survival time were 2.8 and 12.6 months, respectively. Grade 3-4 adverse events were reported for 29.0% of the patients. Hematological toxicities of Grade 3 or 4 included leukopenia (9.7%), neutropenia (9.7%), febrile neutropenia (3.2%), thrombopenia (3.2%) and anemia (6.5%). Non-hematological toxicities of Grade 3 or 4 included pneumonitis (6.5%), diarrhea, colitis, dyspnea, rash, oral mucositis, anorexia and pulmonary thromboembolism/deep vein thrombosis (3.2% each). CONCLUSIONS S-1 and irinotecan combination therapy at the present dose and schedule exhibited only modest efficacy with mild toxicities in previously treated patients with non-small cell lung cancer. No further clinical investigation with current dose and schedules is warranted for patients with non-small cell lung cancer who failed first-line platinum-based doublet chemotherapy.