Events such as a nuclear meltdown accident or nuclear attack have potential for severe radiation injuries. Radiation injury frequently occurs in combination with other forms of trauma, most often burns. Thus far, combined injury studies have focused mainly on skin wound healing and damage to the gut. Since both radiation exposure and remote burn have pulmonary consequences, we examined the early effects of combined injury on the lung. C57BL/6 male mice were subjected to 5 Gy of total body irradiation followed by a 15% total body surface area scald burn. Lungs from surviving animals were examined for evidence of inflammation and pneumonitis. At 48 hours post-injury, pathology of the lungs from combined injury mice showed greater inflammation compared to all other treatment groups, with marked red blood cell and leukocyte congestion of the pulmonary vasculature. There was excessive leukocyte accumulation, primarily neutrophils, in the vasculature and interstitium, with occasional cells in the alveolar space. At 24 and 48 hours post-injury, myeloperoxidase levels in lungs of mice given combined injury were elevated compared to all other treatment groups (p<0.01), confirming histological evidence of neutrophil accumulation. Pulmonary levels of the neutrophil chemoattractant KC (CXCL1) were 3 times above that of either injury alone (p<0.05). Further, monocyte chemotactic protein-1 (MCP-1, CCL2) was increased 2-fold and 3-fold compared to burn injury or radiation injury, respectively (p<0.05). Together, these data suggest that combined radiation and burn injury augments early pulmonary congestion and inflammation.. Currently, countermeasures for this unique type of injury are extremely limited. Further research is needed to elucidate the mechanisms behind the synergistic effects of combined injury in order to develop appropriate treatments.
radiation injury; burn injury; severe combined injury; cytokines; pneumonitis
Pulmonary infections are a major cause of mortality in the critically ill burn patient. Alcohol consumption before burn increases the risk of pulmonary infection. Previously, we have shown an elevated mortality and lung pathology in mice given ethanol before burn and intratracheal infection relative to controls. Here we examine the cellular composition at 24 and 48 h in the circulation and the alveoli of infected mice given alcohol and burn. At 24 h after injury, blood neutrophils obtained from mice exposed to ethanol before burn and infection were 2-fold above those of the experimental controls (P < 0.05). By 48 h, the number of circulating neutrophils decreased and was comparable to levels found in untreated animals. Moreover, at 24 h, bronchoalveolar lavage cells obtained from all treatment groups had similar frequencies and contained 80% neutrophils regardless of treatment. In contrast, the following day, neutrophils were elevated 2-fold only in the alveoli of infected burn animals and 5-fold when ethanol preceded the injury (P < 0.05). These data were confirmed by immunofluorescence microscopy using a neutrophil-specific marker (P < 0.05). Levels of neutrophil chemoattractants, KC and macrophage inflammatory protein 2, and the cytokine, IL-1β, were 2-fold greater in the lungs of infected mice given burn, regardless of ethanol exposure, relative to infected sham injured animals (P < 0.05). Like the number of neutrophils, by the second day after injury, KC and macrophage inflammatory protein 2 remained 5-fold higher in the animals given ethanol, burn, and infection, when compared with other groups (P < 0.05). A similar pattern was seen for pulmonary levels of IL-1β (P < 0.05). Additionally, a reduction in neutrophil apoptosis was observed at the 24-h time point in infected mice exposed to ethanol and burn (P < 0.05). Targeting proinflammatory mediators in mice exposed to ethanol before burn and infection may help alleviate prolonged neutrophil accumulation in the lungs.
Alcohol; inflammation; KC; macrophage inflammatory protein 2; cytokines; apoptosis; Pseudomonas aeruginosa; acute lung injury
Clinical and laboratory evidence suggests that alcohol consumption dysregulates immune function. Burn patients who consume alcohol before their injuries demonstrate higher rates of morbidity and mortality, including acute respiratory distress syndrome, than patients without alcohol at the time of injury. Our laboratory observed higher levels of proinflammatory cytokines and leukocyte infiltration in the lungs of mice after ethanol exposure and burn injury than with either insult alone. To understand the mechanism of the increased pulmonary inflammatory response in mice treated with ethanol and burn injury, we investigated the role of intercellular adhesion molecule (ICAM)-1. Wild-type and ICAM-1 knockout (KO) mice were treated with vehicle or ethanol and subsequently given a sham or burn injury. Twenty-four hours postinjury, lungs were harvested and analyzed for indices of inflammation. Higher numbers of neutrophils were observed in the lungs of wild-type mice after burn and burn with ethanol treatment. This increase in pulmonary inflammatory cell accumulation was significantly lower in the KO mice. In addition, levels of KC, interleukin-1β, and interleukin-6 in the lung were decreased in the ICAM-1 KO mice after ethanol exposure and burn injury. Interestingly, no differences were observed in serum or lung tissue content of soluble ICAM-1 24 hours postinjury. These data suggest that upregulation of adhesion molecules such as ICAM-1 on the vascular endothelium may play a critical role in the excessive inflammation seen after ethanol exposure and burn injury.
Clinical and laboratory evidence suggests that alcohol consumption prior to burn injury leads to dysregulated immune function and subsequent higher rates of morbidity and mortality. Our laboratory previously observed higher levels of pro-inflammatory cytokines and leukocyte infiltration in the lungs of mice following ethanol and burn injury. To understand the mechanism of the increased inflammatory response, we looked at different signaling initiators of inflammation including toll-like receptors 2 and 4 (TLR2 and 4) pathways.
Wild-type, TLR2, and TLR4 knockout mice were treated with vehicle or a single binge dose of ethanol (1.11 g/kg) and subsequently given a sham or burn injury. Twenty-four hours postinjury, systemic and pulmonary levels of pro-inflammatory cytokines were quantified, and differences in neutrophil infiltration were determined by histological examination.
Higher numbers of neutrophils were observed in the lungs of wild-type mice following the combined insult of ethanol and burn injury relative to either injury alone. This increase in leukocyte accumulation was absent in the TLR4 knockout mice. Circulating levels of IL-6 and tumor necrosis factor-α were also elevated in wild-type mice but not in TLR4 knockout mice. Consistent with these findings, pulmonary levels of KC and IL-6 were increased in wild-type mice following burn and ethanol compared to burn injury alone as well as to their TLR4 knockout counterparts. In contrast, TLR2 knockout mice displayed similar levels, to wild-type mice, of neutrophil infiltration as well as IL-6 and KC in the lung.
These data suggest that TLR4 signaling is a crucial contributory component in the exuberant inflammation after ethanol and burn injury. However, TLR2 does not appear to play a vital role in the aberrant pulmonary inflammation.
Lungs; Toll-Like Receptor; Inflammation; Ethanol; Burn
Neutrophil infiltration is one of the critical cellular components of an inflammatory response during peritonitis. The adhesion molecules, P-selectin and intercellular adhesion molecule (ICAM)-1, mediate neutrophil-endothelial cell interactions and the subsequent neutrophil transendothelial migration during the inflammatory response. Despite very strong preclinical data, recent clinical trials failed to show a protective effect of anti-adhesion therapy, suggesting that the length of injury might be a critical factor in neutrophil infiltration. Therefore, the objective of this study was to determine the role of P-selectin and ICAM-1 in neutrophil infiltration into the peritoneal cavity during early and late phases of peritonitis.
Peritonitis was induced in both male wild-type and P-selectin/ICAM-1 double deficient (P/I null) mice by cecal ligation-puncture (CLP). Peripheral blood and peritoneal lavage were collected at 6 and 24 hours after CLP. The total leukocyte and neutrophil contents were determined, and neutrophils were identified with the aid of in situ immunohistochemical staining. Comparisons between groups were made by applying ANOVA and student t-test analysis.
CLP induced a severe inflammatory response associated with a significant leukopenia in both wild-type and P/I null mice. Additionally, CLP caused a significant neutrophil infiltration into the peritoneal cavity that was detected in both groups of mice. However, neutrophil infiltration in the P/I null mice at 6 hours of CLP was significantly lower than the corresponding wild-type mice, which reached a similar magnitude at 24 hours of CLP. In contrast, in peritonitis induced by intraperitoneal inoculation of 2% glycogen, no significant difference in neutrophil infiltration was observed between the P/I null and wild-type mice at 6 hours of peritonitis.
The data suggest that alternative adhesion pathway(s) independent of P-selectin and ICAM-1 can participate in neutrophil migration during peritonitis and that the mode of stimuli and duration of the injury modulate the neutrophil infiltration.
peritonitis; sepsis; transgenic mice; adhesion molecules
Acute neutrophil (PMN) recruitment to postischemic cardiac or pulmonary tissue has deleterious effects in the early reperfusion period, but the mechanisms and effects of neutrophil influx in the pathogenesis of evolving stroke remain controversial. To investigate whether PMNs contribute to adverse neurologic sequelae and mortality after stroke, and to study the potential role of the leukocyte adhesion molecule intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of stroke, we used a murine model of transient focal cerebral ischemia consisting of intraluminal middle cerebral artery occlusion for 45 min followed by 22 h of reperfusion. PMN accumulation, monitored by deposition of 111In-labeled PMNs in postischemic cerebral tissue, was increased 2.5-fold in the ipsilateral (infarcted) hemisphere compared with the contralateral (noninfarcted) hemisphere (P < 0.01). Mice immunodepleted of neutrophils before surgery demonstrated a 3.0-fold reduction in infarct volumes (P < 0.001), based on triphenyltetrazolium chloride staining of serial cerebral sections, improved ipsilateral cortical cerebral blood flow (measured by laser Doppler), and reduced neurological deficit compared with controls. In wild-type mice subjected to 45 min of ischemia followed by 22 h of reperfusion, ICAM-1 mRNA was increased in the ipsilateral hemisphere, with immunohistochemistry localizing increased ICAM-1 expression on cerebral microvascular endothelium. The role of ICAM-1 expression in stroke was investigated in homozygous null ICAM-1 mice (ICAM-1 -/-) in comparison with wild-type controls (ICAM-1 +/+). ICAM-1 -/- mice demonstrated a 3.7-fold reduction in infarct volume (P < 0.005), a 35% increase in survival (P < 0.05), and reduced neurologic deficit compared with ICAM-1 +/+ controls. Cerebral blood flow to the infarcted hemisphere was 3.1-fold greater in ICAM-1 -/- mice compared with ICAM-1 +/+ controls (P < 0.01), suggesting an important role for ICAM-1 in the genesis of postischemic cerebral no-reflow. Because PMN-depleted and ICAM-1-deficient mice are relatively resistant to cerebral ischemia-reperfusion injury, these studies suggest an important role for ICAM-1-mediated PMN adhesion in the pathophysiology of evolving stroke.
We previously reported that massive infiltration of neutrophils in C3H/HeN (C3H) mice could not efficiently control Chlamydia muridarum (Cm) infection and might contribute to the high susceptibility of these mice to lung infection. To further define the nature of neutrophil responses in C3H mice during chlamydial infection, we examine the expression of adhesion molecules and CD11b related to neutrophils infiltration and activation, respectively, following intranasal Cm infection. The results showed that the expression of selectins (E-selectin, P-selectin and L-selectin), and intercellular cell adhesion molecule-1 (ICAM-1) in the lung of C3H mice increased more significantly than in C57BL/6 (B6) mice, the more resistant strain. These results correlated well with the massive neutrophils infiltration in C3H mice. In contrast, CD11b expression on peripheral blood and lung neutrophils in C3H mice exhibited a significant reduction compared with B6 mice during the late phage of infection (day 14). These findings suggest that the high-level expression of adhesion molecules in C3H mice may enhance neutrophils recruitment to the lung, but the decline of CD11b expression on neutrophils may attenuate neutrophil function. Therefore, CD11b down-regulation on neutrophils may contribute to the failure of C3H mice to control chlamydial lung infection.
Chlamydia trachomatis; neutrophils; selectin; ICAM-1; CD11b
Blunt chest trauma resulting in pulmonary contusion is a common but poorly understood injury. We previously demonstrated that lung contusion activates localized and systemic innate immune mechanisms and recruits neutrophils to the injured lung. We hypothesized that the innate immune and inflammatory activation of neutrophils may figure prominently in the response to lung injury. To investigate this, we used a model of pulmonary contusion in the mouse that is similar to that observed clinically in humans and evaluated postinjury lung function and pulmonary neutrophil recruitment. Comparisons were made between injured mice with and without neutrophil depletion. We further examined the role of chemokines and adhesion receptors in neutrophil recruitment to the injured lung. We found that lung injury and resultant physiological dysfunction after contusion was dependent upon the presence of neutrophils in the alveolar space. We show that CXCL1, CXCL2/3, and CXCR2 are involved in neutrophil recruitment to the lung after injury, and that ICAM-1 is locally expressed and actively participates in this process. Injured gp91phox deficient mice showed improved lung function, indicating that oxidant production by neutrophil NADPH oxidase mediates lung dysfunction after contusion. These data suggest that both neutrophil presence and function are required for lung injury after lung contusion.
Neutrophil; pulmonary contusion; inflammation; chemokine; cytokine; mouse model
Intercellular adhesion molecule-1 (ICAM-1) is expressed on a variety of cells including endothelial cells, alveolar epithelial cells, and alveolar macrophages. Endothelial/epithelial cell ICAM-1 participates in the migration of leukocytes out of the blood in response to pulmonary inflammation, whereas alveolar macrophage ICAM-1 may represent cell activation. Our previous studies have shown that there is increased expression of ICAM-1 in lung tissue during acute inflammation following intratracheal injection with silica particles (2 mg/mouse). This increased expression was shown to play a role, in part, in the migration of neutrophils from the circulation into the tissue parenchyma. The aim of the current work is to localize expression of ICAM-1 during acute inflammation in lungs of mice exposed to either silica or the nuisance dust, titanium dioxide. In silica-exposed mice, a significant increase in ICAM-1 was detected on day-1 and localized by immunohistochemistry to aggregates of pulmonary macrophages and to type II epithelial cells. Areas of the lung with increased ICAM-1 expression also showed increased tumor necrosis factor alpha expression. Immunocytochemical staining of bronchoalveolar lavage (BAL) cells demonstrated increased ICAM-1 expression associated with alveolar macrophages 3, 5, and 7 days following silica exposure. Finally, soluble ICAM-1 levels in the BAL fluid were significantly increased in mice exposed to silica on the same days. Titanium dioxide exposure elicited a minimal increase in expression of ICAM-1 in the lungs. These data demonstrate that exposure to the toxic particle silica specifically increases ICAM-1 expression localized to pulmonary macrophages and type II epithelial cells.
Acute lung injury (ALI) is associated with high mortality due to the lack of effective therapeutic strategies. Mechanical ventilation itself can cause ventilator-induced lung injury. Pulmonary vascular barrier function, regulated in part by Src kinase-dependent phosphorylation of caveolin-1 and intercellular adhesion molecule-1 (ICAM-1), plays a crucial role in the development of protein-/neutrophil-rich pulmonary edema, the hallmark of ALI. Amide-linked local anesthetics, such as ropivacaine, have anti-inflammatory properties in experimental ALI. We hypothesized ropivacaine may attenuate inflammation in a “double-hit” model of ALI triggered by bacterial endotoxin plus hyperinflation via inhibition of Src-dependent signaling.
C57BL/6 (WT) and ICAM-1
mice were exposed to either nebulized normal saline (NS) or lipopolysaccharide (LPS, 10 mg) for 1 hour. An intravenous bolus of 0.33 mg/kg ropivacaine or vehicle was followed by mechanical ventilation with normal (7 ml/kg, NTV) or high tidal volume (28 ml/kg, HTV) for 2 hours. Measures of ALI (excess lung water (ELW), extravascular plasma equivalents, permeability index, myeloperoxidase activity) were assessed and lungs were homogenized for Western blot analysis of phosphorylated and total Src, ICAM-1 and caveolin-1. Additional experiments evaluated effects of ropivacaine on LPS-induced phosphorylation/expression of Src, ICAM-1 and caveolin-1 in human lung microvascular endothelial cells (HLMVEC).
WT mice treated with LPS alone showed a 49% increase in ELW compared to control animals (p = 0.001), which was attenuated by ropivacaine (p = 0.001). HTV ventilation alone increased measures of ALI even more than LPS, an effect which was not altered by ropivacaine. LPS plus hyperinflation (“double-hit”) increased all ALI parameters (ELW, EVPE, permeability index, MPO activity) by 3–4 fold compared to control, which were again decreased by ropivacaine. Western blot analyses of lung homogenates as well as HLMVEC treated in culture with LPS alone showed a reduction in Src activation/expression, as well as ICAM-1 expression and caveolin-1 phosphorylation. In ICAM-1
mice, neither addition of LPS to HTV ventilation alone nor ropivacaine had an effect on the development of ALI.
Ropivacaine may be a promising therapeutic agent for treating the cause of pulmonary edema by blocking inflammatory Src signaling, ICAM-1 expression, leukocyte infiltration, and vascular hyperpermeability.
Acute lung injury; ARDS; Ventilator-induced lung injury; Local anesthetics; Endothelium; Caveolin-1; Src protein tyrosine kinase
Mechanical ventilation, often required to maintain normal gas exchange in critically ill patients, may itself cause lung injury. Lung-protective ventilatory strategies with low tidal volume have been a major success in the management of acute respiratory distress syndrome (ARDS). Volutrauma causes mechanical injury and induces an acute inflammatory response. Our objective was to determine whether neutrophil elastase (NE), a potent proteolytic enzyme in neutrophils, would contribute to ventilator-induced lung injury. NE-deficient (NE−/−) and wild-type mice were mechanically ventilated at set tidal volumes (10, 20, and 30 ml/kg) with 0 cm H2O of positive end-expiratory pressure for 3 hours. Lung physiology and markers of lung injury were measured. Neutrophils from wild-type and NE−/− mice were also used for in vitro studies of neutrophil migration, intercellular adhesion molecule (ICAM)-1 cleavage, and endothelial cell injury. Surprisingly, in the absence of NE, mice were not protected, but developed worse ventilator-induced lung injury despite having lower numbers of neutrophils in alveolar spaces. The possible explanation for this finding is that NE cleaves ICAM-1, allowing neutrophils to egress from the endothelium. In the absence of NE, impaired neutrophil egression and prolonged contact between neutrophils and endothelial cells leads to tissue injury and increased permeability. NE is required for neutrophil egression from the vasculature into the alveolar space, and interfering with this process leads to neutrophil-related endothelial cell injury.
neutrophil elastase; ventilator-induced lung injury; endothelial injury; emigration
Pasteurella haemolytica is an important respiratory pathogen of cattle that incites extensive infiltrates of neutrophils into the lung. In addition to the parenchymal damage caused by factors released by P. haemolytica, neutrophils contribute to the pathologic changes in the lungs. Molecules which mediate neutrophil infiltration into the lungs during P. haemolytica pneumonia are poorly characterized. To determine whether the CD18 family (β2-integrin) of leukocyte adhesion molecules mediates initial passage of neutrophils into the pulmonary bronchi and bronchioles of lungs infected with P. haemolytica, three Holstein calves homozygous for bovine leukocyte adhesion deficiency (BLAD) (CD18-deficient neutrophils), and three age- and breed-matched control calves (normal CD18 expression) were inoculated with P. haemolytica A1 via a fiberoptic bronchoscope and euthanized at 2 h postinoculation. Sections of lung were stained for neutrophils, and the intensity of neutrophilic infiltration was determined by computerized image analysis. Significantly fewer (P < 0.05) neutrophils infiltrated the lumen, epithelium, and adventitia of bronchioles and bronchi in lungs of calves with BLAD compared to normal calves, which had dense infiltrates within these sites at 2 h postinoculation. The reduced infiltration in calves with BLAD occurred despite the presence of an extremely large number of neutrophils in peripheral blood that is typical for these calves. The large number of neutrophils in the blood of calves with BLAD is probably a physiologic response that can occur without microbial colonization, since one calf with BLAD that was raised under germ-free conditions had large numbers of neutrophils in the blood that were similar to those in a calf with BLAD that was raised conventionally. Neutrophil counts in the germ-free and conventionally reared calves with BLAD were much higher than those in the three normal calves raised under germ-free conditions. The work in this study demonstrates that during the initial inflammatory response, neutrophils with normal CD18 expression pass more readily than CD18-deficient neutrophils into the walls and lumen of bronchi and bronchioles. It suggests that CD18 is needed for initial passage through the extensive extracellular matrix of the bronchi and bronchioles. This has potential importance for the development of therapies to direct or inhibit neutrophil infiltration into conducting airways rather than alveolar spaces.
Neutrophil adhesion and migration are critical in hepatic ischemia and reperfusion injury (I/R). P-selectin and the intercellular adhesion molecule (ICAM)-1 can mediate neutrophil-endothelial cell interactions, neutrophil migration, and the interactions of neutrophils with hepatocytes in the liver. Despite very strong preclinical data, recent clinical trials failed to show a protective effect of anti-adhesion therapy in reperfusion injury, indicating that the length of injury might be a critical factor in neutrophil infiltration. Therefore, the aim of this study was to assess the role of P-selectin and ICAM-1 in neutrophil infiltration and liver injury during early and late phases of liver I/R.
Adult male wild-type and P-selectin/ICAM-1-deficient (P/I null) mice underwent 90 minutes of partial liver ischemia followed by various periods of reperfusion (6, 15 h, and a survival study). Liver injury was assessed by plasma level of alanine aminotransferase (ALT) and histopathology. The plasma cytokines, TNF-α, IL-6, MIP-2 and KC, were measured by ELISA.
Reperfusion caused significant hepatocellular injury in both wild-type and P/I null mice as was determined by plasma ALT levels and liver histopathology. The injury was associated with a marked neutrophil infiltration into the ischemic livers of both wild-type and P/I null mice. Although the levels of ALT and neutrophil infiltration were slightly lower in the P/I null mice compared with the wild-type mice the differences were not statistically significant. The plasma cytokine data of TNF-α and IL-6 followed a similar pattern to ALT data, and no significant difference was found between the wild-type and P/I null groups. In contrast, a significant difference in KC and MIP-2 chemokine levels was observed between the wild-type and P/I null mice. Additionally, the survival study showed a trend towards increased survival in the P/I null group.
While ICAM-1 and P-selectin does not appear to be critical for neutrophil infiltration and I/R injury in the liver, they may regulate CXC-chemokine production. Blockage of these adhesion molecules may improve survival and remote organ injury that often accompanies liver I/R injury, through chemokine regulation.
Recognition of microbial patterns by host receptors is the first step in a multistep sequence leading to neutrophil-dependent host resistance. Although the role of membrane-bound sensors in bacterial recognition has been examined in detail, the importance of cytosolic sensors in the lungs is largely unexplored. In this context, there is a major lack of understanding related to the downstream signaling mediators, such as cells and/or molecules, during acute extracellular Gram-negative bacterial pneumonia. In order to determine the role of NOD-like receptors (NLRs), we used an experimental Escherichia coli infection model using mice deficient in the gene coding for the NLR adaptor, receptor-interacting protein 2 (RIP2). RIP2−/− mice with E. coli infection displayed higher bacterial burden and reduced neutrophil recruitment and tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), macrophage inflammatory protein 2 (MIP-2), and CXCL5/LIX expression, along with attenuated histopathological changes in the lungs. Decreased IL-17A levels were observed, along with lower numbers of IL-17A-producing T cells, in RIP2−/− mice after infection. RIP2−/− mice also show reduced IL-6 and IL-23 levels in the lungs, along with decreased activation of STAT3 after infection. Furthermore, activation of NF-κB and mitogen-activated protein kinases (MAPKs) and expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in the lungs of infected RIP2−/− mice were attenuated following infection. Although neutrophil mobilization to the blood was impaired in RIP2−/− mice following infection, the expression of CD62P, CD11a/18, CD11b, and CXCR2 on blood and lung neutrophils was not altered between infected wild-type (WT) and RIP2−/− mice. Thus, RIP2 contributes to neutrophil-dependent host defense against an extracellular Gram-negative pathogen via (i) IL-17A regulation and (ii) neutrophil mobilization to the blood.
BACKGROUND--The transit of neutrophils through the pulmonary microvasculature is prolonged compared with red blood cells and is increased further during cigarette smoking and in exacerbations of chronic obstructive pulmonary disease. The increased residence time (sequestration) of neutrophils in the pulmonary capillaries in these conditions may be the first step leading to the accumulation of cells within the lung interstitium and in the bronchoalveolar space, so potentiating lung damage. A rat model has been developed to investigate the factors which may influence neutrophil transit through the lung microvasculature. METHODS--Intratracheal instillation of the heat killed organism Corynebacterium parvum was used to induce an acute neutrophil alveolitis. Neutrophils and red blood cells were isolated from donor rats, labelled with two distinct radioisotopes, and then reinjected into recipient rats to assess their transit through the pulmonary circulation. To ascertain whether peripheral blood neutrophils were minimally altered by the isolation procedure their functional status in vitro was compared with that of inflammatory neutrophils in a number of assays commonly used as descriptors of neutrophil activation. The influence of neutrophil activation on the accumulation of cells in the lungs was assessed by comparing the lung sequestration of control neutrophils, isolated from peripheral blood, with that of inflammatory neutrophils obtained from bronchoalveolar lavage of inflamed rat lungs. Lung sequestration of neutrophils was defined as the fold increase in the ratio of neutrophils labelled with chromium-51 to red blood cells labelled with technetium-99m in lung tissue compared with the same ratio in peripheral blood. RESULTS--Sequestration of peripheral blood neutrophils occurred in control rat lungs as shown by a 17.5 (2.1) fold increase in the ratio of neutrophils to red blood cells in the pulmonary circulation compared with the ratio of these cells in the peripheral circulation. When inflammatory neutrophils, obtained by bronchoalveolar lavage from C parvum-treated animals, were injected into control rats, the increase was 90.6 (11.0) fold. Induction of an inflammatory response in the lung tissue of the recipient rat also caused an increase in the sequestration of control neutrophils compared with the same cells in control rat lungs which was, however, less marked than when inflammatory neutrophils were used (34.7 (4.7) fold). The mean (SE) pressure developed on filtration of inflammatory neutrophils in vitro through a millipore filter (7.53 (0.2) cm H2O) was greater than that of peripheral blood neutrophils (1.18 (0.2) cm H2O). Increased filtration pressure indicates a decrease in cell deformability and suggests that this may be a contributory factor to the increased sequestration of inflammatory neutrophils in the pulmonary vasculature. CONCLUSIONS--This study shows that there is sequestration of neutrophils in the pulmonary vasculature in normal rat lungs which increases in acute lung inflammation and when inflammatory neutrophils are injected into control animals. In this model changes in the neutrophil, such as cell deformability, may have a more important role in inducing increased neutrophil sequestration than the inflammatory response in the lungs.
COPD exacerbations are associated with neutrophilic airway inflammation. Adhesion molecules on the surface of neutrophils may play a key role in their movement from blood to the airways. We analysed adhesion molecule expression on blood and sputum neutrophils from COPD subjects and non-obstructed smokers during experimental rhinovirus infections.
Blood and sputum were collected from 9 COPD subjects and 10 smoking and age-matched control subjects at baseline, and neutrophil expression of the adhesion molecules and activation markers measured using flow cytometry. The markers examined were CD62L and CD162 (mediating initial steps of neutrophil rolling and capture), CD11a and CD11b (required for firm neutrophil adhesion), CD31 and CD54 (involved in neutrophil transmigration through the endothelial monolayer) and CD63 and CD66b (neutrophil activation markers). Subjects were then experimentally infected with rhinovirus-16 and repeat samples collected for neutrophil analysis at post-infection time points.
At baseline there were no differences in adhesion molecule expression between the COPD and non-COPD subjects. Expression of CD11a, CD31, CD62L and CD162 was reduced on sputum neutrophils compared to blood neutrophils. Following rhinovirus infection expression of CD11a expression on blood neutrophils was significantly reduced in both subject groups. CD11b, CD62L and CD162 expression was significantly reduced only in the COPD subjects. Blood neutrophil CD11b expression correlated inversely with inflammatory markers and symptom scores in COPD subjects.
Following rhinovirus infection neutrophils with higher surface expression of adhesion molecules are likely preferentially recruited to the lungs. CD11b may be a key molecule involved in neutrophil trafficking in COPD exacerbations.
Chronic obstructive pulmonary disease; Exacerbations; Respiratory viruses; Neutrophils
The role of the Toll-like receptor 4 (TLR4), a component of the innate immune system, in the development of burn-induced acute lung injury (ALI) has not been completely defined. Recent data suggested that an intact TLR4 plays a major role in the development of organ injury in sterile inflammation. We hypothesized that burn-induced ALI is a TLR4-dependent process. Male C57BL/6J (TLR4 wild-type [WT]) and C57BL/10ScN (TLR4 knockout [KO]) mice were subjected to a 30% total body surface area steam burn. Animals were killed at 6 and 24 h after the insult. Lung specimens were harvested for histological examination after hematoxylin-eosin staining. In addition, lung myeloperoxidase (MPO) and intercellular adhesion molecule 1 immunostaining was performed. Lung MPO was measured by an enzymatic assay. Total lung keratinocyte-derived chemoattractant (IL-8) content was measured by enzyme-linked immunosorbent assay. Western blot was performed to quantify phosphorylated IκBα, phosphorylated nuclear factor κB p65 (NF-κBp65), and high mobility group box 1 expression. Acute lung injury, characterized by thickening of the alveolar-capillary membrane, hyaline membrane formation, intraalveolar hemorrhage, and neutrophil infiltration, was seen in WT but not KO animals at 24 h. Myeloperoxidase and intercellular adhesion molecule 1 immunostaining of KO animals was also similar to sham but elevated in WT animals. In addition, a reduction in MPO enzymatic activity was observed in KO mice as well as a reduction in IL-8 levels compared with their WT counterparts. Burn-induced ALI develops within 24 h after the initial thermal insult in our model. Toll-like receptor 4 KO animals were clearly protected and had a much less severe lung injury. Our data suggest that burn-induced ALI is a TLR4-dependent process.
TLR4; HMGB-1; burn; lung injury; neutrophil
AIM: To investigate the role of intercellular adhesion molecule-1 (ICAM-1) and its counter receptors LFA-1 and Mac-1 in acute pancreatitis (AP).
METHODS: SD rats were allocated to AP group and control group randomly (25 rats each). AP was induced by infusion of 5% chenodeoxycholic acid into the pancreatic duct, followed by ligation of pancreatic duct. The rats were sacrificed at 1, 3, 6, 12 and 24 h after induction of pancreatitis. Five rats were sacrificed at one time point in the two groups before the blood and specimens from pancreas and lung were obtained. Serum amylase and ascitic fluid were measured at each time point. Expression of ICAM-1 at different time points was assessed by immunohistochemistry in pancreas and lung, and the expression of LAF-1 and Mac-1 on neutrophils at different time points was detected by flow cytometer.
RESULTS: Induction of AP was confirmed by the serum levels of amylase and histological studies. The expression of ICAM-1 in pancreas increased significantly than that in the control group at all time points (P < 0.05 or P < 0.01), as well as the expression in lung except at 1 h. The expression of LFA-1 and Mac-1 on neutrophil in blood increased significantly in AP group than that in control group at several time points (P < 0.05 or P < 0.01). The amount of ascitic fluid and serum amylase level of AP group increased significantly than that of control group at all time points (P < 0.05 or P < 0.01). Parallel to these results, a significant neutrophil infiltration was found in pancreas and lung tissues of AP group rats.
CONCLUSION: Our findings suggest the important role for ICAM-1, LFA-1 and Mac-1 in mediating the development of AP from a local disease to a systemic illness. Upregulation of ICAM-1, LFA-1, Mac-1 and subsequent leukocyte infiltration appear to be significant events of pancreatic and pulmonary injuries in AP.
Acute pancreatitis; ICAM-1; LAF-1; Mac-1
Smoke inhalation injury frequently increases the risk of pneumonia and mortality in burn patients. The pathophysiology of acute lung injury secondary to burn and smoke inhalation is well studied, but long-term pulmonary function, especially the process of lung tissue healing following burn and smoke inhalation, has not been fully investigated. By contrast, early burn excision has become the standard of care in the management of major burn injury. While many clinical studies and small-animal experiments support the concept of early burn wound excision, and show improved survival and infectious outcomes, we have developed a new chronic ovine model of burn and smoke inhalation injury with early excision and skin grafting that can be used to investigate lung pathophysiology over a period of 3 weeks.
Materials and methods
Eighteen female sheep were surgically prepared for this study under isoflurane anesthesia. The animals were divided into three groups: an Early Excision group (20% TBSA, third-degree cutaneous burn and 36 breaths of cotton smoke followed by early excision and skin autografting at 24 h after injury, n = 6), a Control group (20% TBSA, third-degree cutaneous burn and 36 breaths of cotton smoke without early excision, n = 6) and a Sham group (no injury, no early excision, n = 6). After induced injury, all sheep were placed on a ventilator and fluid-resuscitated with Lactated Ringers solution (4 mL/% TBS/kg). At 24 h post-injury, early excision was carried out to fascia, and skin grafting with meshed autografts (20/1000 in., 1:4 ratio) was performed under isoflurane anesthesia. At 48 h post-injury, weaning from ventilator was begun if PaO2/FiO2 was above 250 and sheep were monitored for 3 weeks.
At 96 h post-injury, all animals were weaned from ventilator. There are no significant differences in PaO2/FiO2 between Early Excision and Control groups at any points. All animals were survived for 3 weeks without infectious complication in Early Excision and Sham groups, whereas two out of six animals in the Control group had abscess in lung. The percentage of the wound healed surviving area (mean ± SD) was 74.7 ± 7.8% on 17 days post-surgery in the Early Excision group. Lung wet-to-dry weight ratio (mean ± SD) was significantly increased in the Early Excision group vs. Sham group (p < 0.05). The calculated net fluid balance significantly increased in the early excision compared to those seen in the Sham and Control groups. Plasma protein, oncotic pressure, hematocrit of % baseline, hemoglobin of % baseline, white blood cell and neutrophil were significantly decreased in the Early Excision group vs. Control group.
The early excision model closely resembles practice in a clinical setting and allows long-term observations of pulmonary function following burn and smoke inhalation injury. Further studies are warranted to assess lung tissue scarring and measuring collagen deposition, lung compliance and diffusion capacity.
Wound healing; Wet-to-dry weight ratio; Net fluid balance; Plasma protein; Oncotic pressure; Hematocrit; Neutrophils
Exaggerated inflammatory responses and the resultant increases in alveolar-capillary permeability underlie the pathogenesis of acute lung injury during sepsis. This study examined the functions of aldose reductase (AR) in mediating acute lung inflammation. Transgenic mice expressing human AR (ARTg) were used to study the functions of AR since mice have low intrinsic AR activity. In a mild cecal ligation and puncture model, ARTg mice demonstrated an enhanced AR activity and a greater inflammatory response as evaluated by circulating cytokine levels, neutrophil accumulation in the lungs, and activation of Rho kinase in lung endothelial cells (ECs). Compared with WT lung cells, ARTg lung cells produced more IL-6 and showed augmented JNK activation in response to LPS stimulation ex vivo. In human neutrophils, AR activity was required for fMLP-included CD11b activation and up-regulation, respiratory burst, and shape changes. In human pulmonary microvascular ECs, AR activity was required for TNF-α-induced activation of the Rho kinase/MKK4/JNK pathway and IL-6 production, but not p38 activation or ICAM-1 expression. Importantly, AR activity in both human neutrophils and ECs was required for neutrophil adhesion to TNF-α-stimulated ECs. These data demonstrate a novel role for AR in regulating the signaling pathways leading to neutrophil-EC adhesion during acute lung inflammation.
Neutrophil accumulation is a critical event to clear bacteria. Since uncontrolled neutrophil recruitment can cause severe lung damage, understanding neutrophil trafficking mechanisms is important to attenuate neutrophil-mediated damage. While monocyte chemoattractant protein 1 (MCP-1) is known to be a monocyte chemoattractant, its role in pulmonary neutrophil-mediated host defense against Gram-negative bacterial infection is not understood. We hypothesized that MCP-1/chemokine (C-C motif) ligand 2 is important for neutrophil-mediated host defense. Reduced bacterial clearance in the lungs was observed in MCP-1−/− mice following Escherichia coli infection. Neutrophil influx, along with cytokines/chemokines, leukotriene B4 (LTB4), and vascular cell adhesion molecule 1 levels in the lungs, was reduced in MCP-1−/− mice after infection. E. coli-induced activation of NF-κB and mitogen-activated protein kinases in the lung was also reduced in MCP-1−/− mice. Administration of intratracheal recombinant MCP-1 (rMCP-1) to MCP-1−/− mice induced pulmonary neutrophil influx and cytokine/chemokine responses in the presence or absence of E. coli infection. Our in vitro migration experiment demonstrates MCP-1-mediated neutrophil chemotaxis. Notably, chemokine receptor 2 is expressed on lung and blood neutrophils, which are increased upon E. coli infection. Furthermore, our findings show that neutrophil depletion impairs E. coli clearance and that exogenous rMCP-1 after infection improves bacterial clearance in the lungs. Overall, these new findings demonstrate that E. coli-induced MCP-1 causes neutrophil recruitment directly via chemotaxis as well as indirectly via modulation of keratinocyte cell-derived chemokine, macrophage inflammatory protein 2, and LTB4.
Intravenous salbutamol (albuterol) reduces lung water in patients with the acute respiratory distress syndrome (ARDS). Experimental data show that it also reduces pulmonary neutrophil accumulation or activation and inflammation in ARDS.
To investigate the effects of salbutamol on neutrophil function.
The in vitro effects of salbutamol on neutrophil function were determined. Blood and bronchoalveolar lavage (BAL) fluid were collected from 35 patients with acute lung injury (ALI)/ARDS, 14 patients at risk from ARDS and 7 ventilated controls at baseline and after 4 days' treatment with placebo or salbutamol (ALI/ARDS group). Alveolar–capillary permeability was measured in vivo by thermodilution (PiCCO). Neutrophil activation, adhesion molecule expression and inflammatory cytokines were measured.
In vitro, physiological concentrations of salbutamol had no effect on neutrophil chemotaxis, viability or apoptosis. Patients with ALI/ARDS showed increased neutrophil activation and adhesion molecule expression compared with at risk‐patients and ventilated controls. There were associations between alveolar–capillary permeability and BAL myeloperoxidase (r = 0.4, p = 0.038) and BAL interleukin 8 (r = 0.38, p = 0.033). In patients with ALI/ARDS, salbutamol increased numbers of circulating neutrophils but had no effect on alveolar neutrophils.
At the onset of ALI/ARDS, there is increased neutrophil recruitment and activation. Physiological concentrations of salbutamol did not alter neutrophil chemotaxis, viability or apoptosis in vitro. In vivo, salbutamol increased circulating neutrophils, but had no effect on alveolar neutrophils or on neutrophil activation. These data suggest that the beneficial effects of salbutamol in reducing lung water are unrelated to modulation of neutrophil‐dependent inflammatory pathways.
It has been hypothesized that the destruction of lung tissue observed in smokers with chronic obstructive pulmonary disease and emphysema is mediated by neutrophils recruited to the lungs by smoke exposure. This study investigated the role of the chemokine receptor CXCR2 in mediating neutrophilic inflammation in the lungs of mice acutely exposed to cigarette smoke. Exposure to dilute mainstream cigarette smoke for 1 hour, twice per day for 3 days induced acute inflammation in the lungs of C57BL/6 mice, with increased neutrophils and neutrophil chemotactic CXC chemokines MIP-2 and KC. Treatment with SCH-N, an orally active small molecule inhibitor of CXCR2, reduced the influx of neutrophils into the bronchoalveolar lavage (BAL) fluid. Histologic changes were seen, with drug treatment reducing perivascular inflammation and the number of tissue neutrophils. β-glucuronidase activity was reduced in the BAL fluid of mice treated with SCH-N, indicating that the reduction in neutrophils was associated with a reduction in tissue damaging enzymes. Interestingly, while MIP-2 and KC were significantly elevated in the BAL fluid of smoke exposed mice, they were further elevated in mice exposed to smoke and treated with drug. The increase in MIP-2 and KC with drug treatment may be due to the decrease in lung neutrophils which either are not present to bind these chemokines or which fail to provide a feedback signal to other cells that produce these chemokines. Overall, these results demonstrate that inhibiting CXCR2 reduces neutrophilic inflammation and associated lung tissue damage due to acute cigarette smoke exposure.
neutrophil chemokines; emphysema; COPD; MIP-2; KC
BACKGROUND--Pulmonary neutrophilia characterises both the relatively transient inflammation associated with infant respiratory distress syndrome (IRDS) and the persistent inflammation of chronic lung disease. The possibility that persistently raised markers of inflammation indicate the development of chronic lung disease in low birth weight (< 1730 g) preterm (< 31 weeks) infants was therefore investigated. METHODS--Soluble ICAM-1 (sICAM-1) levels in plasma, and interleukin (IL)-8 and myeloperoxidase (MPO) levels in bronchial lavage fluid (BLF) obtained from 17 infants on days 1, 5, and 14 following birth were measured and correlations with the number of neutrophils in BLF sought. Peripheral neutrophils were isolated on Polymorphoprep and chemotactic responsiveness to IL-8 was assessed using micro Boyden chambers. RESULTS--Sixteen infants developed IRDS and, of these, 10 infants subsequently developed chronic lung disease. Levels of IL-8 in BLF at 14 days of age correlated with the long term requirement for intermittent positive pressure ventilation (IPPV). Interleukin 8 levels in BLF correlated with neutrophil numbers and MPO concentration, suggesting both recruitment and activation in response to this cytokine. Antibody depletion studies showed that approximately 50% of total neutrophil chemotactic activity in BLF was due to IL-8. No difference in peripheral neutrophil chemotactic responsiveness at any age was observed for infants with IRDS or chronic lung disease. Plasma soluble intercellular adhesion molecule (sICAM-1) was higher at 14 days of age in infants who developed chronic lung disease than in those with resolving IRDS, and correlated with severity of disease, as indicated by duration of IPPV. CONCLUSIONS--The results indicate that high levels of plasma sICAM-1 and IL-8 in BLF at day 14 correlate with the development of chronic lung disease and indicate the severity of disease.
Monocytes and neutrophils are examples of phagocytic leukocytes, with neutrophils being considered as the 'chief' phagocytic leukocyte. Both monocytes and neutrophils have been implicated to play a key role in the development of ischaemia-reperfusion injury, where they are intrinsically involved in leukocyte-endothelial cell interactions. In this pilot study we hypothesised that mild episodes of tourniquet induced forearm ischaemia-reperfusion injury results in leukocyte activation and changes in inflammatory and coagulation markers.
Ten healthy human volunteers were recruited after informed consent. None had any history of cardiovascular disease with each subject volunteer participating in the study for a 24 hour period. Six venous blood samples were collected from each subject volunteer at baseline, 10 minutes ischaemia, 5, 15, 30, 60 minutes and 24 hours reperfusion, by means of a cannula from the ante-cubital fossa. Monocyte and neutrophil leukocyte sub-populations were isolated by density gradient centrifugation techniques. Leukocyte trapping was investigated by measuring the concentration of leukocytes in venous blood leaving the arm. The cell surface expression of CD62L (L-selectin), CD11b and the intracellular production of hydrogen peroxide (H2O2) were measured via flow cytometry. C-reactive protein (CRP) was measured using a clinical chemistry analyser. Plasma concentrations of D-dimer and von Willebrand factor (vWF) were measured using enzyme-linked fluorescent assays (ELFA).
During ischaemia-reperfusion injury, there was a decrease in CD62L and an increase in CD11b cell surface expression for both monocytes and neutrophils, with changes in the measured parameters reaching statistical significance (p =< 0.05). A significant decrease in peripheral blood leukocyte concentration was observed during this process, which was measured to assess the degree of leukocyte trapping in the micro-circulation (p =< 0.001). There was an increase in the intracellular production of H2O2 production by leukocyte sub-populations, which was measured as a marker of leukocyte activation. Intracellular production of H2O2 in monocytes during ischaemia-reperfusion injury reached statistical significance (p = 0.014), although similar trends were observed with neutrophils these did not reach statistical significance. CRP was measured to assess the inflammatory response following mild episodes of ischaemia-reperfusion injury and resulted in a significant increase in the CRP concentration (p =< 0.001). There were also increased plasma concentrations of D-dimer and a trend towards elevated vWF levels, which were measured as markers of coagulation activation and endothelial damage respectively. Although significant changes in D-dimer concentrations were observed during ischaemia-reperfusion injury (p = 0.007), measurement of the vWF did not reach statistical significance.
Tourniquet induced forearm ischaemia-reperfusion injury results in increased adhesiveness, trapping and activation of leukocytes. We report that, even following a mild ischaemic insult, this leukocyte response is immediately followed by evidence of increased inflammatory response, coagulation activity and endothelial damage. These results may have important implications and this pilot study may lead to a series of trials that shed light on the mechanisms of ischaemia-reperfusion injury, including potential points of therapeutic intervention for pathophysiological conditions.