Contribution of antigen-primed CD8+ T cells to the development of airway hyperresponsiveness and inflammation is associated with IL-13

The role of Th2/CD4 T cells, which secrete IL-4, IL-5, and IL-13, in allergic disease is well established; however, the role of CD8(+) T cells (allergen-induced airway hyperresponsiveness (AHR) and inflammation) is less clear. This study was conducted to define the role of Ag-primed CD8(+) T cells in the development of these allergen-induced responses. CD8-deficient (CD8(-/-)) mice and wild-type mice were sensitized to OVA by i.p. injection and then challenged with OVA via the airways. Compared with wild-type mice, CD8(-/-) mice developed significantly lower airway responsiveness to inhaled methacholine and lung eosinophilia, and exhibited decreased IL-13 production both in vivo, in the bronchoalveolar lavage (BAL) fluid, and in vitro, following Ag stimulation of peribronchial lymph node (PBLN) cells in culture. Reconstitution of sensitized and challenged CD8(-/-) mice with allergen-sensitized CD8(+) T cells fully restored the development of AHR, BAL eosinophilia, and IL-13 levels in BAL and in culture supernatants from PBLN cells. In contrast, transfer of naive CD8(+) T cells or allergen-sensitized CD8(+) T cells from IL-13-deficient donor mice failed to do so. Intracellular cytokine staining of lung as well as PBLN T cells revealed that CD8(+) T cells were a source of IL-13. These data suggest that Ag-primed CD8(+) T cells are required for the full development of AHR and airway inflammation, which appears to be associated with IL-13 production from these primed T cells.     

Necrosis is associated with IL-6 production but apoptosis is not

Due to loss of cell membrane integrity, necrotic cells passively release several cytosolic factors that can activate antigen presenting cells and other immune cells. In contrast, cells dying by apoptosis do not induce an inflammatory response. Here we show that necrotic cell death induced by several stimuli, such as TNF, anti-Fas or dsRNA, coincides with NF-kappaB-and p38MAPK-mediated upregulation and secretion of the pro-inflammatory cytokine IL-6. This event is greatly reduced or absent in conditions of apoptotic cell death induced by the same stimuli. This demonstrates that besides the capacity of necrotic cells to induce an inflammatory response due to leakage of cellular contents, necrotic dying cells themselves are involved in the expression and secretion of inflammatory cytokines. Moreover, inhibition of NF-kappaB and p38MAPK activation does not affect necrotic cell death in all conditions tested. This suggests that the activation of inflammatory pathways is distinct from the activation of necrotic cell death sensu strictu.     

Decreased alveolar macrophage apoptosis is associated with increased pulmonary inflammation in a murine model of pneumococcal pneumonia

Regulation of the inflammatory infiltrate is critical to the successful outcome of pneumonia. Alveolar macrophage apoptosis is a feature of pneumococcal infection and aids disease resolution. The host benefits of macrophage apoptosis during the innate response to bacterial infection are incompletely defined. Because NO is required for optimal macrophage apoptosis during pneumococcal infection, we have explored the role of macrophage apoptosis in regulating inflammatory responses during pneumococcal pneumonia, using inducible NO synthase (iNOS)-deficient mice. iNOS(-/-) mice demonstrated decreased numbers of apoptotic macrophages as compared with wild-type C57BL/6 mice following pneumococcal challenge, greater recruitment of neutrophils to the lung and enhanced expression of TNF-alpha. Pharmacologic inhibition of iNOS produced similar results. Greater pulmonary inflammation was associated with greater levels of early bacteremia, IL-6 production, lung inflammation, and mortality within the first 48 h in iNOS(-/-) mice. Labeled apoptotic alveolar macrophages were phagocytosed by resident macrophages in the lung and intratracheal instillation of exogenous apoptotic macrophages decreased neutrophil recruitment in iNOS(-/-) mice and decreased TNF-alpha mRNA in lungs and protein in bronchial alveolar lavage, as well as chemokines and cytokines including IL-6. These changes were associated with a lower probability of mice becoming bacteremic. This demonstrates the potential of apoptotic macrophages to down-regulate the inflammatory response and for the first time in vivo demonstrates that clearance of apoptotic macrophages decreases neutrophil recruitment and invasive bacterial disease during pneumonia.     

Antigen-induced airway hyperresponsiveness and pulmonary inflammation in allergic dogs

We studied whether antigen-induced airway hyperresponsiveness was associated with pulmonary inflammation in 11 anesthetized ragweed-sensitized dogs. Airway responsiveness to acetylcholine aerosol was determined before and at 2, 6, and 24 h after ragweed or sham aerosol challenge. Pulmonary inflammation was assessed by bronchoalveolar lavage (BAL) performed at either 2 or 6 h. Total pulmonary resistance increased 11-fold at 5 min after ragweed. Airway responsiveness was unchanged at 2 h but was increased 6.6-fold at 6 h in 8 of 11 dogs (P less than 0.001); hyperresponsiveness persisted from 4 days to 4 mo. Airway responsiveness was unchanged by aerosols of diluent. Neutrophils in BAL fluid increased approximately sixfold at 2 h (P less than 0.02) and at 6 h (P less than 0.02) after antigen challenge. There were fewer eosinophils in fluid recovered at 6 h after antigen compared with 2 h lavages (P less than 0.05). In three nonresponders, BAL showed no significant changes in neutrophils and eosinophils after antigen. Thus antigen-induced hyperresponsiveness is associated with the presence of pulmonary inflammation, presumably arising from the airways and involving both neutrophils and eosinophils.     

Resolution of LPS-induced airway inflammation and goblet cell hyperplasia is independent of IL-18

Background

The resolution of inflammatory responses in the lung has not been described in detail and the role of specific cytokines influencing the resolution process is largely unknown.

Methods

The present study was designed to describe the resolution of inflammation from 3 h through 90 d following an acute injury by a single intratracheal instillation of F344/N rats with LPS. We documented the inflammatory cell types and cytokines found in the bronchoalveolar lavage fluid (BALF), and epithelial changes in the axial airway and investigated whether IL-18 may play a role in the resolution process by reducing its levels with anti-IL-18 antibodies.

Results

Three major stages of inflammation and resolution were observed in the BALF during the resolution. The first stage was characterized by PMNs that increased over 3 h to 1 d and decreased to background levels by d 6–8. The second stage of inflammation was characterized by macrophage influx reaching maximum numbers at d 6 and decreasing to background levels by d 40. A third stage of inflammation was observed for lymphocytes which were elevated over d 3–6. Interestingly, IL-18 and IL-9 levels in the BALF showed a cyclic pattern with peak levels at d 4, 8, and 16 while decreasing to background levels at d 1–2, 6, and 12. Depletion of IL-18 caused decreased PMN numbers at d 2, but no changes in inflammatory cell number or type at later time points.

Conclusion

These data suggest that IL-18 plays a role in enhancing the LPS-induced neutrophilic inflammation of the lung, but does not affect the resolution of inflammation.     

Mitochondrial ROS and NLRP3 inflammasome in acute ozone-induced murine model of airway inflammation and bronchial hyperresponsiveness

Oxidative stress is a key mechanism underlying ozone-induced lung injury. Mitochondria can release mitochondrial reactive oxidative species (mtROS), which may lead to the activation of NLRP3 inflammasome. The goal of this study was to examine the roles of mtROS and NLRP3 inflammasome in acute ozone-induced airway inflammation and bronchial hyperresponsiveness (BHR). C57/BL6 mice (n?=?8/group) were intraperitoneally treated with vehicle (phosphate buffered saline, PBS) or mitoTEMPO (mtROS inhibitor, 20?mg/kg), or orally treated with VX-765 (caspse-1 inhibitor, 100?mg/kg) 1?h before the ozone exposure (2.5?ppm, 3?h). Compared to the PBS-treated ozone-exposed mice, mitoTEMPO reduced the level of total malondialdehyde in bronchoalveolar lavage (BAL) fluid and increased the expression of mitochondrial complexes II and IV in the lung 24?h after single ozone exposure. VX-765 inhibited ozone-induced BHR, BAL total cells including neutrophils and eosinophils, and BAL inflammatory cytokines including IL-1α, IL-1β, KC, and IL-6. Both mitoTEMPO and VX-765 reduced ozone-induced mtROS and inhibited capase-1 activity in lung tissue whilst VX-765 further inhibited DRP1 and MFF expression, increased MFN2 expression, and down-regulated caspase-1 expression in the lung tissue. These results indicate that acute ozone exposure induces mitochondrial dysfunction and NLRP3 inflammasome activation, while the latter has a critical role in the pathogenesis of ozone-induced airway inflammation and BHR.     

Theophylline induces apoptosis of the IL-3 activated eosinophils of patients with bronchial asthma

In bronchial asthma, eosinophils are upregulated and their survival is suggested to be prolonged by the action of some cytokines such as Interleukin (IL)-3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF). We find here that the survival of eosinophils in the peripheral blood of patients with asthma is correlated with the serum levels of IL-3 but not of IL-5 and GM-CSF. Interestingly, theophylline is revealed to induce apoptosis of the prolonged survival eosinophils by IL-3, as judged by morphological changes and nucleosomal DNA fragmentation. During the apoptosis, caspase-3 in eosinophils stimulated by IL-3 is activated by theophylline. The substrate of caspase-3, poly (ADP-ribose) polymerase (PARP), is cleaved in the eosinophils after theophylline treatment. These results suggest that theophylline is able to induce apoptosis of the IL-3 activated eosinophils in patients with bronchial asthma, and that its clinical effectiveness may be due to the reduction of inflammatory cells in the airway.     

Airway inflammation in cadmium-exposed rats is associated with pulmonary oxidative stress and emphysema

The aim of this study was to test the hypothesis that pulmonary inflammation and emphysema induced by cadmium (Cd) inhalation are associated with pulmonary oxidative stress. Two groups of Sprague Dawley rats were used: one vehicle-exposed group undergoing inhalation of NaCl (0.9%, n = 24) and one Cd-exposed group undergoing inhalation of CdCl(2) (0.1%, n = 24). The animals in the vehicle-and Cd-exposed groups were divided into 4 subgroups (n = 6 per group), which underwent either a single exposure (D2) of 1H or repeated exposures 3 times/week for 1H for a period of 3 weeks (3W), 5 weeks (5W) or 5 weeks followed by 2 weeks without exposure (5W + 2). At sacrifice, the left lung was fixed for histomorphometric analysis (median inter-wall distance, MIWD), whilst bronchoalveolar lavage fluid (BALF) was collected from the right lung. Cytological analysis of BALF was performed and BALF was analysed for oxidant markers 8-iso-PGF(2a), uric acid (UA), reduced (AA) and oxidised ascorbic acid (DHA) and reduced (GSH) and oxidised glutathione (GSSG). Cd-exposure induced a significant increase of BALF macrophages and neutrophils. 8-iso-PGF(2a), UA, GSH and GSSG were significantly increased at D2. At 5W and 5W + 2, AA and GSH were significantly lower in Cd-exposed rats, indicating antioxidant depletion. MIWD significantly increased in all repeatedly Cd-exposed groups, suggesting development of pulmonary emphysema. 8-iso-PGF(2a) and UA were positively correlated with macrophage and neutrophil counts. GSH, GSSG and 8-iso-PGF(2a) were negatively correlated with MIWD, indicating that Cd-induced emphysema could be associated with pulmonary oxidative stress.     

Plasma leukocyte cell-derived chemotaxin 2 is associated with the severity of systemic inflammation in patients with sepsis

The aim of the present study was to determine the correlations between leukocyte cell-derived chemotaxin 2 (LECT2) and inflammation-related variables in human inflammatory disease. Plasma samples from 23 septic patients who had been admitted to the intensive care unit (ICU) of our institution and 31 volunteers were used. Plasma LECT2 concentrations were examined retrospectively and compared with those of various inflammatory cytokines and routine laboratory data. The LECT2 concentrations of the septic patients at the time of ICU entry (5.3 ± 4.1 ng/mL) were significantly lower than those of the volunteers (19.7 ± 3.4 ng/mL) and these concentrations had significantly increased by the time of ICU discharge. Individual analyses showed that the LECT2 concentrations of all 19 patients had increased by the time of ICU discharge. A combination of LECT2 and C-reactive protein (CRP) concentrations was capable of discriminating the acute and recovery phases of sepsis to a degree similar to those of the combinations of CRP concentration and percentage of neutrophils, CRP concentration and percentage of immature white blood cells, or CRP and interleukin-6 concentrations. Thus, the LECT2 concentration correlates with the severity of systemic inflammation in patients with sepsis. LECT2 may be a reliable diagnostic indicator of human inflammatory diseases.     

Airway hyperresponsiveness is associated with airway remodeling but not inflammation in aging Cav1-/- mice

Background

Airway inflammation and airway remodeling are the key contributors to airway hyperresponsiveness (AHR), a characteristic feature of asthma. Both processes are regulated by Transforming Growth Factor (TGF)-β. Caveolin 1 (Cav1) is a membrane bound protein that binds to a variety of receptor and signaling proteins, including the TGF-β receptors. We hypothesized that caveolin-1 deficiency promotes structural alterations of the airways that develop with age will predispose to an increased response to allergen challenge.

Methods

AHR was measured in Cav1-deficient and wild-type (WT) mice 1 to 12 months of age to examine the role of Cav1 in AHR and the relative contribution of inflammation and airway remodeling. AHR was then measured in Cav1^-/- and WT mice after an ovalbumin-allergen challenge performed at either 2 months of age, when remodeling in Cav1^-/- and WT mice was equivalent, and at 6 months of age, when the Cav1^-/- mice had established airway remodeling.

Results

Cav1^-/- mice developed increased thickness of the subepithelial layer and a correspondingly increased AHR as they aged. In addition, allergen-challenged Cav1^-/- mice had an increase in AHR greater than WT mice that was largely independent of inflammation. Cav1^-/- mice challenged at 6 months of age have decreased AHR compared to those challenged at 2 months with correspondingly decreased BAL IL-4 and IL-5 levels, inflammatory cell counts and percentage of eosinophils. In addition, in response to OVA challenge, the number of goblet cells and α-SMA positive cells in the airways were reduced with age in response to OVA challenge in contrast to an increased collagen deposition further enhanced in absence of Cav1.

Conclusion

A lack of Cav1 contributed to the thickness of the subepithelial layer in mice as they aged resulting in an increase in AHR independent of inflammation, demonstrating the important contribution of airway structural changes to AHR. In addition, age in the Cav1^-/- mice is a contributing factor to airway remodeling in the response to allergen challenge.     

CD8+ T cell-mediated airway hyperresponsiveness and inflammation is dependent on CD4+IL-4+ T cells

CD4+ T cells, particularly Th2 cells, play a pivotal role in allergic airway inflammation. However, the requirements for interactions between CD4+ and CD8+ T cells in airway allergic inflammation have not been delineated. Sensitized and challenged OT-1 mice in which CD8+ T cells expressing the transgene for the OVA(257-264) peptide (SIINFEKL) failed to develop airway hyperresponsiveness (AHR), airway eosinophilia, Th2 cytokine elevation, or goblet cell metaplasia. OT-1 mice that received naive CD4+IL-4+ T cells but not CD4+IL-4- T cells before sensitization developed all of these responses to the same degree as wild-type mice. Moreover, recipients of CD4+IL-4+ T cells developed significant increases in the number of CD8+IL-13+ T cells in the lung, whereas sensitized OT-1 mice that received primed CD4+ T cells just before challenge failed to develop these responses. Sensitized CD8-deficient mice that received CD8+ T cells from OT-1 mice that received naive CD4+ T cells before sensitization increased AHR and eosinophil numbers in bronchoalveolar lavage fluid when challenged with allergen. In contrast, sensitized CD8-deficient mice receiving CD8+ T cells from OT-1 mice without CD4+ T cells developed reduced AHR and eosinophil numbers in bronchoalveolar lavage fluid when challenged. These data suggest that interactions between CD4+ and CD8+ T cells, in part through IL-4 during the sensitization phase, are essential to the development of CD8+IL-13+ T cell-dependent AHR and airway allergic inflammation.     

Esophagitis-related esophageal shortening in opossum is associated with longitudinal muscle hyperresponsiveness

Acute intraluminal acid perfusion induces esophageal shortening in humans and opossums. Lower esophageal sphincter (LES) hypotension and peristaltic dysfunction occur in patients and animal models of reflux esophagitis. This study examined whether similar shortening and motor dysfunction occur in anesthetized opossums after repeated esophageal acid exposure and whether this is associated with longitudinal muscle (LM) hyperresponsiveness. Manometry used before and after 3 consecutive days of 45-min perfusion with 100 mmol/l HCl or normal saline measured esophageal length and motor responses to induced swallows. LM electrical and mechanical responses were assessed using standard isometric tension and intracellular recording techniques. Compared with controls, repeated acid perfusion induced erosive esophagitis and significant esophageal shortening, associated with enhanced LM responses to carbachol, a significantly depolarized resting membrane potential, and abnormal spike patterns. LES resting pressure and swallow-induced peristalsis were unaffected. In this model of reflux esophagitis, marked persistent esophageal shortening and associated LM hyperresponsiveness occur before significant LES or peristaltic dysfunction, suggesting that esophageal shortening is the earliest motor disorder induced by acid injury.     

Administration of pentoxifylline during allergen sensitization dissociates pulmonary allergic inflammation from airway hyperresponsiveness

Asthma, a chronic inflammatory disease characterized by intermittent, reversible airflow obstruction and airway hyperresponsiveness (AHR), is classically characterized by an excess of Th2 cytokines (IL-13, IL-4) and depletion of Th1 cytokines (IFN-gamma, IL-12). Recent studies indicating an important role for Th1 immunity in the development of AHR with allergic inflammation suggest that Th1/Th2 balance may be important in determining the association of AHR with allergic inflammation. We hypothesized that administration of pentoxifylline (PTX), a phosphodiesterase inhibitor known to inhibit Th1 cytokine production, during allergen (OVA) sensitization and challenge would lead to attenuation of AHR in a murine model of allergic pulmonary inflammation. We found that PTX treatment led to attenuation of AHR when administered at the time of allergen sensitization without affecting other hallmarks of pulmonary allergic inflammation. Attenuation of AHR with PTX treatment was found in the presence of elevated bronchoalveolar lavage fluid levels of the Th2 cytokine IL-13 and decreased levels of the Th1 cytokine IFN-gamma. PTX treatment during allergen sensitization leads to a divergence of AHR and pulmonary inflammation following allergen challenge.     

Protectin D1 is generated in asthma and dampens airway inflammation and hyperresponsiveness

Protectins are newly identified natural chemical mediators that counter leukocyte activation to promote resolution of inflammation. In this study, we provide the first evidence for protectin D1 (PD1, 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid) formation from docosahexaenoic acid in human asthma in vivo and PD1 counterregulatory actions in allergic airway inflammation. PD1 and 17S-hydroxy-docosahexaenoic acid were present in exhaled breath condensates from healthy subjects. Of interest, levels of PD1 were significantly lower in exhaled breath condensates from subjects with asthma exacerbations. PD1 was also present in extracts of murine lungs from both control animals and those sensitized and aerosol challenged with allergen. When PD1 was administered before aeroallergen challenge, airway eosinophil and T lymphocyte recruitment were decreased, as were airway mucus, levels of specific proinflammatory mediators, including IL-13, cysteinyl leukotrienes, and PGD(2), and airway hyperresponsiveness to inhaled methacholine. Of interest, PD1 treatment after aeroallergen challenge markedly accelerated the resolution of airway inflammation. Together, these findings provide evidence for endogenous PD1 as a pivotal counterregulatory signal in allergic airway inflammation and point to new therapeutic strategies for modulating inflammation in asthmatic lung.     

Endothelial lipase is associated with inflammation in humans

The aim of this study was to investigate the extent to which inflammation is linked with plasma endothelial lipase (EL) concentrations among healthy sedentary men. Plasma C-reactive protein (CRP) concentrations were measured with a highly sensitive commercial immunoassay, plasma interleukin-6 (IL-6) concentrations were measured using a commercial ELISA, and plasma secretory phospholipase A(2) type IIA (sPLA(2)-IIA) concentrations were measured using a commercial assay in a sample of 74 moderately obese men (mean body mass index, 29.8 +/- 5.2 kg/m(2)). Plasma EL concentrations were positively correlated with various indices of obesity, fasting plasma insulin, and plasma CRP, IL-6, and sPLA(2)-IIA concentrations. Multiple regression analyses revealed that plasma CRP concentrations explained 14.5% (P = 0.0008) of the variance in EL concentrations. When entered into the model, LPL activity accounted for 16.1% (P < 0.0001) and plasma CRP concentrations accounted for 20.9% (P < 0.0001) of the variance in EL concentrations. The combined impact of visceral adipose tissue (VAT) and of an inflammation score on EL concentrations was investigated. Among subjects with high or low VAT, those having a high inflammation score based on plasma CRP, IL-6, and sPLA(2)-IIA concentrations had increased plasma EL concentrations (P = 0.0005). In conclusion, our data reveal a strong association between proinflammatory cytokines and plasma EL concentrations among healthy people with low or high VAT levels.     

Attenuation of antigen-induced airway hyperresponsiveness and inflammation in CXCR3 knockout mice

Background

CD8+ T cells participate in airway hyperresponsiveness (AHR) and allergic pulmonary inflammation that are characteristics of asthma. CXCL10 by binding to CXCR3 expressed preferentially on activated CD8+ T cells, attracts T cells homing to the lung. We studied the contribution and limitation of CXCR3 to AHR and airway inflammation induced by ovalbumin (OVA) using CXCR3 knockout (KO) mice.

Methods

Mice were sensitized and challenged with OVA. Lung histopathological changes, AHR, cellular composition and levels of inflammatory mediators in bronchoalveolar lavage (BAL) fluid, and lungs at mRNA and protein levels, were compared between CXCR3 KO mice and wild type (WT) mice.

Results

Compared with the WT controls, CXCR3 KO mice showed less OVA-induced infiltration of inflammatory cells around airways and vessels, and less mucus production. CXCR3 KO mice failed to develop significant AHR. They also demonstrated significantly fewer CD8+ T and CD4+ T cells in BAL fluid, lower levels of TNFα and IL-4 in lung tissue measured by real-time RT-PCR and in BAL fluid by ELISA, with significant elevation of IFNγ mRNA and protein expression levels.

Conclusions

We conclude that CXCR3 is crucial for AHR and airway inflammation by promoting recruitment of more CD8+ T cells, as well as CD4+ T cells, and initiating release of proinflammatory mediators following OVA sensitization and challenge. CXCR3 may represent a novel therapeutic target for asthma.