Jiyoun Kim

Anti‐tumor necrosis factor‐α antibody treatment reduces pulmonary inflammation and methacholine hyper‐responsiveness in a murine asthma model induced by house dust

Background/Aims Recent studies documented that sensitization and exposure to cockroach allergens significantly increase childrens asthma morbidity as well as severity, especially among inner city children. TNF‐α has been postulated to be a critical mediator directly contributing to the bronchopulmonary inflammation and airway hyper‐responsiveness in asthma. This study investigated whether an anti‐TNF‐α antibody would inhibit pulmonary inflammation and methacholine (Mch) hyper‐responsiveness in a mouse model of asthma induced by a house dust extract containing both endotoxin and cockroach allergens.

Keratinocyte growth factor pretreatment is associated with decreased macrophage inflammatory protein-2α concentrations and reduced neutrophil recruitment in acid aspiration lung injury

A two-hit model of acid aspiration was used to examine the effect of keratinocyte growth factor (KGF) on chemokine levels and neutrophil recruitment into the lung. Mice were subjected to cecal ligation and puncture and then either KGF or saline, intratracheally (IT). Forty-eight hours later, the mice were given IT acid. After 8 h, neutrophil counts in bronchoalveolar lavage (BAL) fluid were significantly decreased in animals pretreated with KGF (23 ± 4 × 103/mouse) compared with saline (74 ± 2 × 103/mouse). In addition, the BAL fluid IL-6 levels were decreased in the KGF-treated group (88 ± 44 pg/mL) compared with the saline group (166 ± 34 pg/mL). To examine the mechanism behind the KGF-induced reduction in neutrophil influx, the murine chemokines KC and macrophage inflammatory protein (MIP)-2&agr; were measured. KC levels in plasma and BAL fluid were not significantly different between the treatment groups. Likewise, levels of MIP-2&agr; in plasma were not affected by KGF treatment. However, 8 h after acid aspiration, MIP-2&agr; concentrations were significantly lower in the KGF-treated group. The ratio of MIP-2&agr; in BAL fluid versus plasma was lower in the KGF group (0.72 ± 0.28) than in the saline group at 3 h (2.23 ± 0.93) and also significantly lower in the KGF group (3.02 ± 0.78) compared with the saline group (6.23 ± 1.19) at 8 h. In this study, KGF pretreatment after acid aspiration was associated with reduced neutrophil recruitment into the lung and a decrease in MIP-2&agr; gradients between BAL fluid and plasma.

Eotaxin Represents the Principal Eosinophil Chemoattractant in a Novel Murine Asthma Model Induced by House Dust Containing Cockroach Allergens

Asthma represents a serious health problem particularly for inner city children, and recent studies have identified that cockroach allergens trigger many of these asthmatic attacks. This study tested the concept that asthma-like pulmonary inflammation may be induced by house dust containing cockroach allergens. An aqueous extract was prepared from a house dust sample containing endotoxin and high levels of cockroach allergens. BALB/c mice were immunized with the house dust extract (HDE) and received two additional pulmonary challenges. Bronchoalveolar lavage (BAL) eosinophil counts and eotaxin levels were significantly increased in immunized mice exposed to the HDE, whereas neutrophils were the predominant BAL inflammatory cell in the unimmunized mice. Kinetics studies in immunized mice demonstrated a peak pulmonary inflammatory response 48 h after the last challenge. The allergic response in this model was further confirmed by histological and physiological studies demonstrating a significant influx of eosinophils and lymphocytes in the peribronchial area, and severe airway hyperreactivity through whole-body plethysmography. The specificity of the response was established by immunizing with HDE and challenging with purified cockroach allergen, which induced pulmonary eosinophilia and airway hyperreactivity. Ab inhibition of eotaxin significantly inhibited the number of BAL eosinophils. These data describe a novel murine model of asthma-like pulmonary inflammation induced by house dust containing endotoxin and cockroach allergens and further demonstrate that eotaxin represents the principal chemoattractant for the recruitment of the pulmonary eosinophils.

Critical role of CD14 for production of proinflammatory cytokines and cytokine inhibitors during sepsis with failure to alter morbidity or mortality.

ABSTRACT We investigated the immunopathophysiologic responses during sepsis induced by cecal ligation and puncture (CLP) in CD4-deficient (CD14 knockout [CD14KO]) mice. Our studies were designed to specifically test the role of CD14 in the inflammatory response to sepsis and to ascertain if alterations would improve morbidity or mortality. Sepsis was induced using the CLP model with appropriate antibiotic treatment. The severity of sepsis increased in the CD14KO mice with increasing puncture size (18 gauge [18G], 21G, and 25G). Following CLP, body temperature (at 12 h) and gross motor activity levels of the sham and 25G CLP groups recovered to normal, while the 21G and 18G CLP groups exhibited severe hypothermia coupled with decreased gross motor activity and body weight. There were no significant differences in survival, temperature, body weight, or activity levels between CD14KO and control mice after 21G CLP. However, CD14KO mice expressed two- to fourfold less pro-inflammatory (interleukin-1β [IL-1β], tumor necrosis factor [TNF], and IL-6) and anti-inflammatory (IL-10, IL-1 receptor antagonist, and TNF receptors I and II) cytokines in the blood after 21G CLP. Plasma levels of the chemokines macrophage inflammatory protein 2α and KC were similarly reduced in CD14KO mice. A similar trend of decreased cytokine and cytokine inhibitor levels was observed in the peritoneal cavity of CD14KO mice. Our results indicate that the CD14 pathway of activation plays a critical role in the production of both pro-inflammatory cytokines and cytokine inhibitors but has minimal impact on the morbidity or mortality induced by the CLP model of sepsis.

CECAL LIGATION AND PUNCTURE INDUCED MURINE SEPSIS DOES NOT CAUSE LUNG INJURY

Objective:The cause of death in murine models of sepsis remains unclear. The primary purpose of this study was to determine if significant lung injury develops in mice predicted to die after cecal ligation and puncture-induced sepsis compared with those predicted to live. Design:Prospective, laboratory controlled experiments. Setting:University research laboratory. Subjects:Adult, female, outbred Institute of Cancer Research mice. Interventions:Mice underwent cecal ligation and puncture to induce sepsis. Two groups of mice were euthanized at 24 and 48 hrs postcecal ligation and puncture and samples were collected. These mice were further stratified into groups predicted to die (Die-P) and predicted to live (Live-P) based on plasma interleukin-6 levels obtained 24 hrs postcecal ligation and puncture. Multiple measures of lung inflammation and lung injury were quantified in these two groups. Results from a group of mice receiving intratracheal normal saline without surgical intervention were also included as a negative control. As a positive control, bacterial pneumonia was induced with Pseudomonas aeruginosa to cause definitive lung injury. Separate mice were followed for survival until Day 28 postcecal ligation and puncture. These mice were used to verify the interleukin-6 cutoffs for survival prediction. Measurements and Main Results:After sepsis, both the Die-P and Live-P mice had significantly suppressed measures of respiratory physiology but maintained normal levels of arterial oxygen saturation. Bronchoalveolar lavage levels of pro- and anti-inflammatory cytokines were not elevated in the Die-P mice compared with the Live-P. In addition, there was no increase in the recruitment of neutrophils to the lung, pulmonary vascular permeability, or histological evidence of damage. In contrast, all of these pulmonary injury and inflammatory parameters were increased in mice with Pseudomonas pneumonia. Conclusions:These data demonstrate that mice predicted to die during sepsis have no significant lung injury. In murine intra-abdominal sepsis, pulmonary injury cannot be considered the etiology of death in the acute phase.

Acute Pulmonary Lipopolysaccharide Tolerance Decreases TNF-α without Reducing Neutrophil Recruitment

Pulmonary LPS exposure plays a key role in exacerbation of lung diseases such as chronic obstructive pulmonary disease and asthma. However, little is known about the effects of repeated LPS exposure in the lung microenvironment. We have developed a novel murine model of pulmonary LPS tolerance induced by intratracheal (i.t.) administration of LPS. First, we show that pulmonary LPS exposure does not induce whole-body refractoriness to systemic LPS, because i.t. administration followed by i.p. administration did not decrease plasma TNF-α. However, a local refractory state can be induced with two i.t. LPS exposures. Pulmonary LPS tolerance was induced by i.t. administration of 100 ng LPS at time 0 and 48 h. Nontolerant mice received PBS at time 0 and LPS at 48 h. Bronchoalveolar lavage levels of TNF-α were significantly attenuated in tolerant mice vs nontolerant mice (1597 pg/ml vs 7261 pg/ml). TNF-α mRNA was significantly reduced in bronchoalveolar lavage cells (5-fold) and lung tissue (10-fold). No reduction was seen in neutrophil numbers in the bronchoalveolar lavage fluid, myeloperoxidase activity, or expression of neutrophil chemoattractants CXCL1 and CXCL2, reflecting the specificity of the response. The reduction in TNF-α was accompanied by a significant increase in soluble receptors, TNF-SRI (159 pg/ml vs 206 pg/ml) and TNF-SRII (1366 pg/m vs 2695 pg/ml). In conclusion, pulmonary LPS tolerance results in a specific reduction in TNF-α expression, while the neutrophilic response is unaffected. This response may be a mechanism to limit tissue damage by reducing TNF-α levels, while still maintaining the antimicrobial capacity of the lung.

Enhanced fibrinolysis protects against lung ischemia-reperfusion injury

OBJECTIVE Ischemia-reperfusion injury continues to plague the field of lung transplantation, resulting in suboptimal outcomes. In acute lung injury, processes such as ventilator-induced injury, sepsis, or acute respiratory distress syndrome, extravascular fibrin has been shown to promote lung dysfunction and the acute inflammatory response. This study investigates the role of the fibrinolytic cascade in lung ischemia-reperfusion injury and investigates the interplay between the fibrinolytic system and the inflammatory response. METHODS Mice lacking the plasminogen activator inhibitor-1 gene (PAI-1 knock out, PAI-1 KO; and thus increased lysis of endogenous fibrin) and wild-type mice underwent in situ left lung ischemia and reperfusion. Fibrin content in the lung was evaluated by immunoblotting. Reperfusion injury was assessed by histologic and physiologic parameters. Proinflammatory mediators were measured in bronchoalveolar lavage fluid and plasma using enzyme-linked immunosorbent assays. RESULTS Ischemia-reperfusion causes fibrin deposition in murine lungs. Less fibrin was seen in PAI-1 KO mice than in wild-type mice subjected to the same ischemia-reperfusion conditions. By histologic criteria, more evidence of ischemia-reperfusion injury was noted (thickening of the interstium, cellular infiltration in the alveoli) in the wild-type than in PAI-1 KO mice. Physiologic parameters also revealed more ischemia-reperfusion injury in the wild-type than in PAI-1 KO mice. Cytokine and chemokines were elevated more in the wild-type group than the PAI-1 KO group. CONCLUSIONS Lung ischemia-reperfusion injury triggers fibrin deposition in the murine lungs and fibrin creates a proinflammatory environment. Preventing fibrin deposition may reduce ischemia-reperfusion injury and inflammation. This finding may lead to novel treatment strategies for ischemia-reperfusion.

Diesel Exhaust Particulates Exacerbate Asthma-Like Inflammation by Increasing CXC Chemokines

Particulate matter heavily pollutes the urban atmosphere, and several studies show a link between increased ambient particulate air pollution and exacerbation of pre-existing pulmonary diseases, including asthma. We investigated how diesel exhaust particulates (DEPs) aggravate asthma-like pulmonary inflammation in a mouse model of asthma induced by a house dust extract (HDE) containing cockroach allergens and endotoxin. BALB/c mice were exposed to three pulmonary challenges via hypopharyngeal administration of an HDE collected from the home of an asthmatic child. One hour before each pulmonary challenge, mice were exposed to DEP or PBS. Pulmonary inflammation was assessed by histological features, oxidative stress, respiratory physiological features, inflammatory cell recruitment, and local CXC chemokine production. To prove the role of CXC chemokines in the augmented inflammation, CXC chemokine-specific antibodies were delivered to the lungs before DEP exposure. DEP exacerbated HDE-induced airway inflammation, with increased airway mucus production, oxidative stress, inflammatory cell infiltration, bronchoalveolar lavage concentrations of CXC chemokines, and airway hyperreactivity. Neutralization of airway keratinocyte-derived chemokine and macrophage inflammatory protein-2 significantly improves the respiratory function in addition to decreasing the infiltration of neutrophils and eosinophils. Blocking the chemokines also decreased airway mucus production. These results demonstrate that DEP exacerbates airway inflammation induced by allergen through increased pulmonary expression of the CXC chemokines (keratinocyte-derived chemokine and macrophage inflammatory protein-2).

Allergens induce enhanced bronchoconstriction and leukotriene production in C5 deficient mice

BackgroundPrevious genetic analysis has shown that a deletion in the complement component 5 gene-coding region renders mice more susceptible to allergen-induced airway hyperresponsiveness (AHR) due to reduced IL-12 production. We investigated the role of complement in a murine model of asthma-like pulmonary inflammation.MethodsIn order to evaluate the role of complement B10 mice either sufficient or deficient in C5 were studied. Both groups of mice immunized and challenged with a house dust extract (HDE) containing high levels of cockroach allergens. Airways hyper-reactivity was determined with whole-body plesthysmography. Bronchoalveolar lavage (BAL) was performed to determine pulmonary cellular recruitment and measure inflammatory mediators. Lung homogenates were assayed for mediators and plasma levels of IgE determined. Pulmonary histology was also evaluated.ResultsC5-deficient mice showed enhanced AHR to methylcholine challenge, 474% and 91% increase above baseline Penh in C5-deficient and C5-sufficient mice respectively, p < 0.001. IL-12 levels in the lung homogenate (LH) were only slightly reduced and BAL IL-12 was comparable in C5-sufficient and C5-deficient mice. However, C5-deficient mice had significantly higher cysteinyl-leukotriene levels in the BAL fluid, 1913 +/- 246 pg/ml in C5d and 756 +/- 232 pg/ml in C5-sufficient, p = 0.003.ConclusionThese data demonstrate that C5-deficient mice show enhanced AHR due to increased production of cysteinyl-leukotrienes.

Attenuating burn wound inflammation improves pulmonary function and survival in a burn-pneumonia model.

Objective:We previously showed that topical inhibition of inflammatory signaling in burn wounds reduced systemic inflammatory response and burn-induced pulmonary inflammation. We hypothesized that this topical intervention would attenuate burn-induced lung injury, improve pulmonary function, protect lungs from bacterial invasion, and reduce mortality. Design:Controlled, in vivo, laboratory study. Setting:University laboratory. Subjects:Female mice, 8–10 wks old. Interventions:Animals received 30% total body surface area burn followed by topical application of a specific inhibitor of p38 mitogen-activated protein kinase, a key inflammatory signaling pathway, or vehicle to the wound. Twenty-four hours after injury, pulmonary collagen deposition and pulmonary function were assessed. One day postburn, some of the animals received intratracheal instillation of Klebsiella pneumoniae and were subsequently monitored for 7 days. Measurements and Main Results:Topical inhibition of p38 mitogen-activated protein kinase significantly decreased pulmonary collagen deposition and prevented a decline in pulmonary function at 1 day after burn injury. Compared with sham controls, animals with burn injury had a significantly higher mortality in response to intratracheal bacterial challenge. Application of p38 mitogen-activated protein kinase inhibitor to the burn wound attenuated pulmonary neutrophil infiltration and reduced the mortality rate to a level experienced by sham controls. Conclusions:Inflammatory source control in burn wounds with topical p38 mitogen-activated protein kinase inhibition attenuates acute lung injury, avoids pulmonary dysfunction, protects lungs from bacterial challenge, and improves survival.