Search

Rationale: Recruited leukocytes play an important role in ventilator-induced lung injury, although studies have focused predominantly on neutrophils. Inflammatory subset Gr-1(high) monocytes are recruited to sites of inflammation and have been implicated in acute lung injury induced by systemic endotoxin.Objectives: To investigate the recruitment and role of Gr-1(high) monocytes in an in vivo mouse model of ventilator-induced lung injury.Methods: Anesthetized mice were ventilated with low or high stretch. Flow cytometry was used to quantify monocyte subset margination to the lungs, and to assess their in situ cellular activation in response to mechanical stretch. To investigate monocyte involvement in lung injury progression, a two-hit model was used, with a subclinical dose of lipopolysaccharide (intraperitoneal) given 2 hours prior to high-stretch ventilation. In some animals, monocytes were depleted using intravenous clodronate liposomes. Development of lung injury was assessed in ventilated animals by peak inspiratory pressure and respiratory system mechanics.Measurements and Main Results: High-stretch ventilation induced significant pulmonary margination of Gr-1(high) but not Gr-1(low) monocytes compared with nonventilated mice. These monocytes displayed increased activation status, with higher CD11b (vs. nonventilated mice) and lower L-selectin expression (vs. low-stretch ventilation). Lipopolysaccharide challenge led to enhanced lung margination of Gr-1(high) monocytes and neutrophils, and sensitized the lungs to high stretch-induced pulmonary edema. Clodronate-liposome pretreatment depleted lung monocytes (but not neutrophils) and significantly attenuated lung injury.Conclusions: High-stretch mechanical ventilation promotes pulmonary margination of activated Gr-1(high) monocytes, which play a role in the progression of ventilator-induced lung injury. [ABSTRACT FROM AUTHOR]

Mechanical ventilation contributes to the morbidity and mortality of patients in intensive care, likely through the exacerbation and dissemination of inflammation. Despite the proximity of the pleural cavity to the lungs and exposure to physical forces, little attention has been paid to its potential as an inflammatory source during ventilation. Here, we investigate the pleural cavity as a novel site of inflammation during ventilator-induced lung injury. Mice were subjected to low or high tidal volume ventilation strategies for up to 3 hours. Ventilation with a high tidal volume significantly increased cytokine and total protein levels in BAL and pleural lavage fluid. In contrast, acid aspiration, explored as an alternative model of injury, only promoted intraalveolar inflammation, with no effect on the pleural space. Resident pleural macrophages demonstrated enhanced activation after injurious ventilation, including upregulated ICAM-1 and IL-1β expression, and the release of extracellular vesicles. In vivo ventilation and in vitro stretch of pleural mesothelial cells promoted ATP secretion, whereas purinergic receptor inhibition substantially attenuated extracellular vesicles and cytokine levels in the pleural space. Finally, labeled protein rapidly translocated from the pleural cavity into the circulation during high tidal volume ventilation, to a significantly greater extent than that of protein translocation from the alveolar space. Overall, we conclude that injurious ventilation induces pleural cavity inflammation mediated through purinergic pathway signaling and likely enhances the dissemination of mediators into the vasculature. This previously unidentified consequence of mechanical ventilation potentially implicates the pleural space as a focus of research and novel avenue for intervention in critical care.

Colorectal cancer (CRC) is the third most predominant malignancy in the world. Although the importance of immune system in cancer development has been well established, the underlying mechanisms remain to be investigated further. Here we studied a novel protein prokineticin 2 (Prok2, also known as Bv8) as a key pro-tumoral factor in CRC progression in in vitro and ex vivo settings. Human colorectal tumor tissues, myeloid cell lines (U937 cells and HL60 cells) and colorectal cancer cell line (Caco-2 cells) were used for various studies. Myeloid cell infiltration (especially neutrophils) and Bv8 accumulation were detected in human colorectal tumor tissue with immunostaining. The chemotactic effects of Bv8 on myeloid cells were presented in the transwell assay and chemotaxis assy. Cultured CRC cells treated with myeloid cells or Bv8 produced reactive oxygen species (ROS) and vascular endothelial growth factor (VEGF). Furthermore, ROS and VEGF acted as pro-angiogenesis buffer in myeloid cell-infiltrated CRC microenvironment. Moreover, myeloid cells or Bv8 enhanced energy consumption of glycolysis ATP and mitochondria ATP of CRC cells. Interestingly, myeloid cells increased CRC cell viability, but CRC cells decreased the viability of myeloid cells. ERK signalling pathway in CRC cells was activated in the presence of Bv8 or co-cultured myeloid cells. In conclusion, our data indicated the vital roles of Bv8 in myeloid cell infiltration and CRC development, suggesting that Bv8 may be a potential therapeutic target for colorectal cancer-related immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Chang, Cui, Zhao, Hu, O’Dea, Tirlapur, Balboni, Zhang, Ying and Ma.)

Blood-borne myeloid cells, neutrophils and monocytes, play a central role in the development of indirect acute lung injury (ALI) during sepsis and noninfectious systemic inflammatory response syndrome. By contrast, the contribution of circulating myeloid cell-derived extracellular vesicles (EVs) to ALI is unknown, despite acute increases in their numbers during sepsis and systemic inflammatory response syndrome. Here, we investigated the direct role of circulating myeloid-EVs in ALI using a mouse isolated perfused lung system and a human cell coculture model of pulmonary vascular inflammation consisting of lung microvascular endothelial cells and peripheral blood mononuclear cells. Total and immunoaffinity-isolated myeloid (CD11b + ) and platelet (CD41 + ) EVs were prepared from the plasma of intravenous LPS-injected endotoxemic donor mice and transferred directly into recipient lungs. Two-hour perfusion of lungs with unfractionated EVs from a single donor induced pulmonary edema formation and increased perfusate concentrations of RAGE (receptor for advanced glycation end products), consistent with lung injury. These responses were abolished in the lungs of monocyte-depleted mice. The isolated myeloid- but not platelet-EVs produced a similar injury response and the acute intravascular release of proinflammatory cytokines and endothelial injury markers. In the in vitro human coculture model, human myeloid- (CD11b + ) but not platelet- (CD61 + ) EVs isolated from LPS-stimulated whole blood induced acute proinflammatory cytokine production and endothelial activation. These findings implicate circulating myeloid-EVs as acute mediators of pulmonary vascular inflammation and edema, suggesting an alternative therapeutic target for attenuation of indirect ALI.

Intra-alveolar microvesicles (MVs) are important mediators of inter-cellular communication within the alveolar space, and are key components in the pathophysiology of lung inflammation such as acute respiratory distress syndrome (ARDS). Despite the abundance of data detailing the pro-inflammatory effects of MVs, it remains unclear how MVs interact or signal with target cells in the alveolus. Using both in vivo and in vitro alveolar models, we analyzed the dynamics of MV uptake by resident alveolar cells: alveolar macrophages and epithelial cells. Under resting conditions, the overwhelming majority of MVs were taken up by alveolar macrophages. However, following lipopolysaccharide (LPS)-mediated inflammation, epithelial cells internalized significantly more MVs (p<0.01) whilst alveolar macrophage internalization was significantly reduced (p<0.01). We found that alveolar macrophages adopted a pro-inflammatory phenotype after internalizing MVs under resting conditions, but reduction of MV uptake following LPS pre-treatment was associated with loss of inflammatory phenotype. Instead, MVs induced significant epithelial cell inflammation following LPS pre-treatment, when MV internalization was most significant. Using pharmacological inhibitors, we interrogated the mechanisms of MV internalization to identify which endocytic pathways and cell surface receptors are involved. We demonstrated that epithelial cells are exclusively dependent on the clathrin and caveolin dependent endocytotic pathway, whereas alveolar macrophage uptake may involve a significant phagocytic component. Furthermore, alveolar macrophages predominantly engulf MVs via scavenger receptors whilst, epithelial cells internalize MVs via a phosphatidylserine/integrin receptor mediated pathway (specifically alpha V beta III), which can be inhibited with phosphatidylserine-binding protein (i.e. annexin V). In summary, we have undertaken a comprehensive evaluation of MV internalization within the alveolar space. Our results demonstrate that different environmental conditions can modulate MV internalization, with inflammatory stimuli strongly enhancing epithelial cell uptake of MVs and inducing epithelial cell activation. Our data reveal the unique mechanisms by which alveolar macrophages and epithelial cells internalize MVs thereby elucidating how MVs exert their pathophysiological effect during lung inflammation and injury. As MVs are potential novel therapeutic targets in conditions such as ARDS, these data provide crucial insights into the dynamics of MV-target cell interactions and highlight potential avenues for researchers to modulate and inhibit their pro-inflammatory actions within the alveolar space., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Soni, O’Dea, Abe, Khamdan, Shah, Sarathchandra, Wilson and Takata.)

Extracellular microvesicles (MVs) are released into the circulation in large numbers during acute systemic inflammation, yet little is known of their intravascular cell/tissue-specific interactions under these conditions. We recently described a dramatic increase in the uptake of intravenously injected MVs by monocytes marginated within the pulmonary vasculature, in a mouse model of low-dose lipopolysaccharide-induced systemic inflammation. To investigate the mechanisms of enhanced MV uptake by monocytes, we developed an in vitro model using in vivo derived monocytes. Although mouse blood is a convenient source, monocyte numbers are too low for in vitro experimentation. In contrast, differentiated bone marrow monocytes are abundant, but they are rapidly mobilized during systemic inflammation, and thus no longer available. Instead, we developed a protocol using marginated monocytes from the pulmonary vasculature as an anatomically relevant and abundant source. Mice are sacrificed by terminal anesthesia, the lungs inflated and perfused via the pulmonary artery. Perfusate cell populations are evaluated by flow cytometry, combined with in vitro generated fluorescently labelled MVs, and incubated in suspension for up to one hour. Washed cells are analyzed by flow cytometry to quantify MV uptake and confocal microscopy to localize MVs within cells (O'Dea et al., 2020). Using this perfusion-based method, substantial numbers of marginated pulmonary vascular monocytes are recovered, allowing multiple in vitro tests to be performed from a single mouse donor. As MV uptake profiles were comparable to those observed in vivo, this method is suitable for physiologically relevant high throughput mechanistic studies on mouse monocytes under in vitro conditions. Graphic abstract: Figure 1. Schematic of lung perfusate cell harvest and co-incubation with in vitro generated MVs. Created with BioRender.com., Competing Interests: Competing interestsThe authors declare no competing interests., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

Rationale: Mechanical ventilation is a mainstay of intensive care but contributes to the mortality of patients through ventilator-induced lung injury. eCypA (extracellular CypA [cyclophilin A]) is an emerging inflammatory mediator and metalloproteinase inducer, and the gene responsible for its expression has recently been linked to coronavirus disease (COVID-19). Objectives: To explore the involvement of eCypA in the pathophysiology of ventilator-induced lung injury. Methods: Mice were ventilated with a low or high Vt for up to 3 hours, with or without blockade of eCypA signaling, and lung injury and inflammation were evaluated. Human primary alveolar epithelial cells were exposed to in vitro stretching to explore the cellular source of eCypA, and CypA concentrations were measured in BAL fluid from patients with acute respiratory distress syndrome to evaluate the clinical relevance. Measurements and Main Results: High-Vt ventilation in mice provoked a rapid increase in soluble CypA concentration in the alveolar space but not in plasma. In vivo ventilation and in vitro stretching experiments indicated the alveolar epithelium as the likely major source. In vivo blockade of eCypA signaling substantially attenuated physiological dysfunction, macrophage activation, and MMPs (matrix metalloproteinases). Finally, we found that patients with acute respiratory distress syndrome showed markedly elevated concentrations of eCypA within BAL fluid. Conclusions: CypA is upregulated within the lungs of injuriously ventilated mice (and critically ill patients), where it plays a significant role in lung injury. eCypA represents an exciting novel target for pharmacological intervention.

Despite advances in the pathophysiology of chronic obstructive pulmonary disease (COPD), there is a distinct lack of biochemical markers to aid clinical management. Microvesicles (MVs) have been implicated in the pathophysiology of inflammatory diseases including COPD, but their association to COPD disease severity remains unknown. We analyzed different MV populations in plasma and bronchoalveolar lavage fluid (BALF) taken from 62 patients with mild to very severe COPD (51% male; mean age: 65.9 yr). These patients underwent comprehensive clinical evaluation (symptom scores, lung function, and exercise testing), and the capacity of MVs to be clinical markers of disease severity was assessed. We successfully identified various MV subtype populations within BALF [leukocyte, polymorphonuclear leukocyte (PMN; i.e., neutrophil), monocyte, epithelial, and platelet MVs] and plasma (leukocyte, PMN, monocyte, and endothelial MVs) and compared each MV population to disease severity. BALF neutrophil MVs were the only population to significantly correlate with the clinical evaluation scores including forced expiratory volume in 1 s, modified Medical Research Council dyspnea score, 6-min walk test, hyperinflation, and gas transfer. BALF neutrophil MVs, but not neutrophil cell numbers, also strongly correlated with BODE index. We have undertaken, for the first time, a comprehensive evaluation of MV profiles within BALF/plasma of COPD patients. We demonstrate that BALF levels of neutrophil-derived MVs are unique in correlating with a number of key functional and clinically relevant disease severity indexes. Our results show the potential of BALF neutrophil MVs for a COPD biomarker that tightly links a key pathophysiological mechanism of COPD (intra-alveolar neutrophil activation) with clinical severity/outcome.

In our earlier work, we found that intrauterine (i.u.) and intraperitoneal (i.p.) injection of LPS (10-μg serotype 0111:B4) induced preterm labor (PTL) with high pup mortality, marked systemic inflammatory response and hypotension. Here, we used both i.u. and i.p. LPS models in pregnant wild-type (wt) and CCR2 knockout (CCR2-/-) mice on E16 to investigate the role played by the CCL2/CCR2 system in the response to LPS. Basally, lower numbers of monocytes and macrophages and higher numbers of neutrophils were found in the myometrium, placenta, and blood of CCR2-/- vs. wt mice. After i.u. LPS, parturition occurred at 14 h in both groups of mice. At 7 h post-injection, 70% of wt pups were dead vs. 10% of CCR2-/- pups, but at delivery 100% of wt and 90% of CCR2-/- pups were dead. Myometrial and placental monocytes and macrophages were generally lower in CCR2-/- mice, but this was less consistent in the circulation, lung, and liver. At 7 h post-LPS, myometrial ERK activation was greater and JNK and p65 lower and the mRNA levels of chemokines were higher and of inflammatory cytokines lower in CCR2-/- vs. wt mice. Pup brain and placental inflammation were similar. Using the IP LPS model, we found that all measures of arterial pressure increased in CCR2-/- but declined in wt mice. These data suggest that the CCL2/CCR2 system plays a critical role in the cardiovascular response to LPS and contributes to pup death but does not influence the onset of inflammation-induced PTL., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Our previous work has shown that pregnancy exacerbates the hypotensive response to both infection and lipopolysaccharide (LPS). The high levels of progesterone (P4) associated with pregnancy have been suggested to be responsible for the pregnancy-induced changes in the cardiovascular response to infection. Here, we test the hypothesis that P4 supplementation exacerbates the hypotensive response of the maternal cardiovascular to LPS.Female CD1 mice had radiotelemetry probes implanted to measure hemodynamic function noninvasively and were time-mated. From day 14 of pregnancy, mice received either 10 mg of P4 or vehicle alone per day and on day 16, intraperitoneal LPS (10 μg of serotype 0111:B4) was injected. In two identically treated cohorts of mice, tissue and serum (for RNA, protein studies) were collected at 6 and 12 h.Administration of LPS resulted in a fall in blood pressure in vehicle treated, but not P4 supplemented mice. This occurred with similar changes in the circulating levels of cytokines, vasoactive factors and in both circulating and tissue inflammatory cell numbers, but with reduced left ventricular expression of cytokines in P4-supplemented mice. However, left ventricular expression of markers of cardiac dysfunction and apoptosis were similar.This study demonstrates that P4 supplementation prevented LPS-induced hypotension in pregnant mice in association with reduced myocardial inflammatory cytokine gene expression. These observations suggest that rather than being detrimental, P4 supplementation has a protective effect on the maternal cardiovascular response to sepsis.

Microvesicles (MVs), a plasma membrane-derived subclass of extracellular vesicles, are produced and released into the circulation during systemic inflammation, yet little is known of cell/tissue-specific uptake of MVs under these conditions. We hypothesized that monocytes contribute to uptake of circulating MVs and that their increased margination to the pulmonary circulation and functional priming during systemic inflammation produces substantive changes to the systemic MV homing profile. Cellular uptake of i.v.-injected, fluorescently labelled MVs (J774.1 macrophage-derived) in vivo was quantified by flow cytometry in vascular cell populations of the lungs, liver and spleen of C57BL6 mice. Under normal conditions, both Ly6C high and Ly6C low monocytes contributed to MV uptake but liver Kupffer cells were the dominant target cell population. Following induction of sub-clinical endotoxemia with low-dose i.v. LPS, MV uptake by lung-marginated Ly6C high monocytes increased markedly, both at the individual cell level (~2.5-fold) and through substantive expansion of their numbers (~8-fold), whereas uptake by splenic macrophages was unchanged and uptake by Kupffer cells actually decreased (~50%). Further analysis of MV uptake within the pulmonary vasculature using a combined model approach of in vivo macrophage depletion, ex vivo isolated perfused lungs and in vitro lung perfusate cell-based assays, indicated that Ly6C high monocytes possess a high MV uptake capacity (equivalent to Kupffer cells), that is enhanced directly by endotoxemia and ablated in the presence of phosphatidylserine (PS)-enriched liposomes and β3 integrin receptor blocking peptide. Accordingly, i.v.-injected PS-enriched liposomes underwent a redistribution of cellular uptake during endotoxemia similar to MVs, with enhanced uptake by Ly6C high monocytes and reduced uptake by Kupffer cells. These findings indicate that monocytes, particularly lung-marginated Ly6C high subset monocytes, become a dominant target cell population for MVs during systemic inflammation, with significant implications for the function and targeting of endogenous and therapeutically administered MVs, lending novel insights into the pathophysiology of pulmonary vascular inflammation., (© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.)

Introduction : Acute respiratory distress syndrome (ARDS) is a heterogeneous and multifactorial disease; it is a common and devastating condition that has a high mortality. Treatment is limited to supportive measures hence novel pharmacological approaches are necessary. We propose a new direction in ARDS research; this means moving away from thinking about individual inflammatory mediators and instead investigating how packaged information is transmitted between cells. Microvesicles (MVs) represent a novel vehicle for inter-cellular communication with an emerging role in ARDS pathophysiology. Areas covered : This review examines current approaches to ARDS and emerging MV research. We describe advances in our understanding of microvesicles and focus on their pro-inflammatory roles in airway and endothelial signaling. We also offer reasons for why MVs are attractive therapeutic targets. Expert opinion : MVs have a key role in ARDS pathophysiology. Preclinical studies must move away from simple models toward more realistic scenarios while clinical studies must embrace patient heterogeneity. Microvesicles have the potential to aid identification of patients who may benefit from particular treatments and act as biomarkers of cellular status and disease progression. Understanding microvesicle cargoes and their cellular interactions will undoubtedly uncover new targets for ARDS.

Cellular stress or injury induces release of endogenous danger signals such as ATP, which plays a central role in activating immune cells. ATP is essential for the release of nonclassically secreted cytokines such as IL-1β but, paradoxically, has been reported to inhibit the release of classically secreted cytokines such as TNF. Here, we reveal that ATP does switch off soluble TNF (17 kDa) release from LPS-treated macrophages, but rather than inhibiting the entire TNF secretion, ATP packages membrane TNF (26 kDa) within microvesicles (MVs). Secretion of membrane TNF within MVs bypasses the conventional endoplasmic reticulum- and Golgi transport-dependent pathway and is mediated by acid sphingomyelinase. These membrane TNF-carrying MVs are biologically more potent than soluble TNF in vivo , producing significant lung inflammation in mice. Thus, ATP critically alters TNF trafficking and secretion from macrophages, inducing novel unconventional membrane TNF signaling via MVs without direct cell-to-cell contact. These data have crucial implications for this key cytokine, particularly when therapeutically targeting TNF in acute inflammatory diseases.-Soni, S., O'Dea, K. P., Tan, Y. Y., Cho, K., Abe, E., Romano, R., Cui, J., Ma, D., Sarathchandra, P., Wilson, M. R., Takata, M. ATP redirects cytokine trafficking and promotes novel membrane TNF signaling via microvesicles.

Clinical investigations lack predictive value when diagnosing pneumonia, especially when patients are ventilated and develop ventilator associated pneumonia (VAP). New tools to aid diagnosis are important to improve outcomes. This pilot study examines the potential for a panel of inflammatory mediators to aid in the diagnosis. Forty-four ventilated patients, 17 with pneumonia and 27 with brain injuries, eight of whom developed VAP, were recruited. 51 inflammatory mediators, including cytokines and oxylipins, were measured in patients' serum using flow cytometry and mass spectrometry. The mediators could separate patients admitted to ICU with pneumonia compared to brain injury with an area under the receiver operating characteristic curve (AUROC) 0.75 (0.61-0.90). Changes in inflammatory mediators were similar in both groups over the course of ICU stay with 5,6-dihydroxyeicosatrienoic and 8,9-dihydroxyeicosatrienoic acids increasing over time and interleukin-6 decreasing. However, brain injured patients who developed VAP maintained inflammatory profiles similar to those at admission. A multivariate model containing 5,6-dihydroxyeicosatrienoic acid, 8,9-dihydroxyeicosatrienoic acid, intercellular adhesion molecule-1, interleukin-6, and interleukin-8, could differentiate patients with VAP from brain injured patients without infection (AUROC 0.94 (0.80-1.00)). The use of a selected group of markers showed promise to aid the diagnosis of VAP especially when combined with clinical data.

Rationale: Primary graft dysfunction in lung transplant recipients derives from the initial, largely leukocyte-dependent, ischaemia-reperfusion injury. Intravascular lung-marginated monocytes have been shown to play key roles in experimental acute lung injury, but their contribution to lung ischaemia-reperfusion injury post transplantation is unknown., Objective: To define the role of donor intravascular monocytes in lung transplant-related acute lung injury and primary graft dysfunction., Methods: Isolated perfused C57BL/6 murine lungs were subjected to warm ischaemia (2 hours) and reperfusion (2 hours) under normoxic conditions. Monocyte retention, activation phenotype and the effects of their depletion by intravenous clodronate-liposome treatment on lung inflammation and injury were determined. In human donor lung transplant samples, the presence and activation phenotype of monocytic cells (low side scatter, 27E10+, CD14+, HLA-DR+, CCR2+) were evaluated by flow cytometry and compared with post-implantation lung function., Results: In mouse lungs following ischaemia-reperfusion, substantial numbers of lung-marginated monocytes remained within the pulmonary microvasculature, with reduced L-selectin and increased CD86 expression indicating their activation. Monocyte depletion resulted in reductions in lung wet:dry ratios, bronchoalveolar lavage fluid protein, and perfusate levels of RAGE, MIP-2 and KC, while monocyte repletion resulted in a partial restoration of the injury. In human lungs, correlations were observed between pre-implantation donor monocyte numbers/their CD86 and TREM-1 expression and post-implantation lung dysfunction at 48 and 72 hours., Conclusions: These results indicate that lung-marginated intravascular monocytes are retained as a 'passenger' leukocyte population during lung transplantation, and play a key role in the development of transplant-associated ischaemia-reperfusion injury., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

The role of progesterone (P4) in the regulation of the local (uterine) and systemic innate immune system, myometrial expression of connexin 43 (Cx-43) and cyclooxygenase 2 (COX-2), and the onset of parturition was examined in (i) naïve mice delivering at term; (ii) E16 mice treated with RU486 (P4-antagonist) to induce preterm parturition; and (iii) in mice treated with P4 to prevent term parturition. In naïve mice, myometrial neutrophil and monocyte numbers peaked at E18 and declined with the onset of parturition. In contrast, circulating monocytes did not change and although neutrophils were increased with pregnancy, they did not change across gestation. The myometrial mRNA and protein levels of most chemokines/cytokines, Cx-43, and COX-2 increased with, but not before, parturition. With RU486-induced parturition, myometrial and systemic neutrophil numbers increased before and myometrial monocyte numbers increased with parturition only. Myometrial chemokine/cytokine mRNA abundance increased with parturition, but protein levels peaked earlier at between 4.5 and 9 h post-RU486. Cx-43, but not COX-2, mRNA expression and protein levels increased prior to the onset of parturition. In mice treated with P4, the gestation-linked increase in myometrial monocyte, but not neutrophil, numbers was prevented, and expression of Cx-43 and COX-2 was reduced. On E20 of P4 supplementation, myometrial chemokine/cytokine and leukocyte numbers, but not Cx-43 and COX-2 expression, increased. These data show that during pregnancy P4 controls myometrial monocyte infiltration, cytokine and prolabor factor synthesis via mRNA-dependent and independent mechanisms and, with prolonged P4 supplementation, P4 action is repressed resulting in increased myometrial inflammation.

Objective: Obesity has a complex impact on acute respiratory distress syndrome patients, being associated with increased likelihood of developing the syndrome but reduced likelihood of dying. We propose that such observations are potentially explained by a model in which obesity influences the iatrogenic injury that occurs subsequent to intensive care admission. This study therefore investigated whether fat feeding protected mice from ventilator-induced lung injury., Design: In vivo study., Setting: University research laboratory., Subjects: Wild-type C57Bl/6 mice or tumor necrosis factor receptor 2 knockout mice, either fed a high-fat diet for 12-14 weeks, or age-matched lean controls., Interventions: Anesthetized mice were ventilated with injurious high tidal volume ventilation for periods up to 180 minutes., Measurements and Main Results: Fat-fed mice showed clear attenuation of ventilator-induced lung injury in terms of respiratory mechanics, blood gases, and pulmonary edema. Leukocyte recruitment and activation within the lungs were not significantly attenuated nor were a host of circulating or intra-alveolar inflammatory cytokines. However, intra-alveolar matrix metalloproteinase activity and levels of the matrix metalloproteinase cleavage product soluble receptor for advanced glycation end products were significantly attenuated in fat-fed mice. This was associated with reduced stretch-induced CD147 expression on lung epithelial cells., Conclusions: Consumption of a high-fat diet protects mice from ventilator-induced lung injury in a manner independent of neutrophil recruitment, which we postulate instead arises through blunted up-regulation of CD147 expression and subsequent activation of intra-alveolar matrix metalloproteinases. These findings may open avenues for therapeutic manipulation in acute respiratory distress syndrome and could have implications for understanding the pathogenesis of lung disease in obese patients.

Sepsis is the leading cause of direct maternal mortality, but there are no data directly comparing the response to sepsis in pregnant and nonpregnant (NP) individuals. This study uses a mouse model of sepsis to test the hypothesis that the cardiovascular response to sepsis is more marked during pregnancy. Female CD1 mice had radiotelemetry probes implanted and were time mated. NP and day 16 pregnant CD-1 mice received intraperitoneal lipopolysaccharide (LPS; 10 μg, serotype 0111: B4). In a separate study, tissue and serum (for RNA, protein and flow cytometry studies), aorta and uterine vessels (for wire myography) were collected after LPS or vehicle control administration. Administration of LPS resulted in a greater fall in blood pressure in pregnant mice compared to NP mice. This occurred with similar changes in the circulating levels of cytokines, vasoactive factors, and circulating leukocytes, but with a greater monocyte and lesser neutrophil margination in the lungs of pregnant mice. Baseline markers of cardiac dysfunction and apoptosis as well as cytokine expression were higher in pregnant mice, but the response to LPS was similar in both groups as was the ex vivo assessment of vascular function. In pregnant mice, nonfatal sepsis is associated with a more marked hypotensive response but not a greater immune response. We conclude that endotoxemia induces a more marked hypotensive response in pregnant compared to NP mice. These changes were not associated with a more marked systemic inflammatory response in pregnant mice, although monocyte lung margination was greater. The more marked hypotensive response to LPS may explain the greater vulnerability to some infections exhibited by pregnant women., (© The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Microvesicles are cell-derived signaling particles emerging as important mediators and biomarkers of systemic inflammation, but their production in severe burn injury patients has not been described. In this pilot investigation, we measured circulating microvesicle levels following severe burns, with severe sepsis patients as a comparator group. We hypothesized that levels of circulating vascular cell-derived microvesicles are elevated acutely following burns injury, mirroring clinical severity due to the early onset and prevalence of systemic inflammatory response syndrome (SIRS) in these patients. Blood samples were obtained from patients with moderate to severe thermal injury burns, with severe sepsis, and from healthy volunteers. Circulating microvesicles derived from total leukocytes, granulocytes, monocytes, and endothelial cells were quantified in plasma by flow cytometry. All circulating microvesicle subpopulations were elevated in burns patients on day of admission (day 0) compared to healthy volunteers (leukocyte-microvesicles: 3.5-fold, p = 0.005; granulocyte-microvesicles: 12.8-fold, p<0.0001; monocyte-microvesicles: 20.4-fold, p<0.0001; endothelial- microvesicles: 9.6-fold, p = 0.01), but decreased significantly by day 2. Microvesicle levels were increased with severe sepsis, but less consistently between patients. Leukocyte- and granulocyte-derived microvesicles on day 0 correlated with clinical assessment scores and were higher in burns ICU non-survivors compared to survivors (leukocyte MVs 4.6 fold, p = 0.002; granulocyte MVs 4.8 fold, p = 0.003). Mortality prediction analysis of area under receiver operating characteristic curve was 0.92 (p = 0.01) for total leukocyte microvesicles and 0.85 (p = 0.04) for granulocyte microvesicles. These findings demonstrate, for the first time, acute increases in circulating microvesicles following burns injury in patients and point to their potential role in propagation of sterile SIRS-related pathophysiology., Competing Interests: The authors have declared that no competing interests exist.

Inflammation plays a key role in human term and preterm labor (PTL). Intrauterine LPS has been widely used to model inflammation-induced complications of pregnancy, including PTL. It has been shown to induce an intense myometrial inflammatory cell infiltration, but the role of LPS-induced inflammatory cell activation in labor onset and fetal demise is unclear. We investigated this using a mouse model of PTL, where an intrauterine injection of 10 μg of LPS (serotype 0111:B4) was given at E16 of CD1 mouse pregnancy. This dose induced PTL at an average of 12.7 h postinjection in association with 85% fetal demise. Flow cytometry showed that LPS induced a dramatic systemic inflammatory response provoking a rapid and marked leucocyte infiltration into the maternal lung and liver in association with increased cytokine levels. Although there was acute placental inflammatory gene expression, there was no corresponding increase in fetal brain inflammatory gene expression until after fetal demise. There was marked myometrial activation of NFκB and MAPK/AP-1 systems in association with increased chemokine and cytokine levels, both of which peaked with the onset of parturition. Myometrial macrophage and neutrophil numbers were greater in the LPS-injected mice with labor onset only; prior to labor, myometrial neutrophils and monocytes numbers were greater in PBS-injected mice, but this was not associated with an earlier onset of labor. These data suggest that intrauterine LPS induces parturition directly, independent of myometrial inflammatory cell infiltration, and that fetal demise occurs without fetal inflammation. Intrauterine LPS provokes a marked local and systemic inflammatory response but with limited inflammatory cell infiltration into the myometrium or placenta., (© 2016 by the Society for the Study of Reproduction, Inc.)

Background: Microvesicles (MVs) are important mediators of intercellular communication, packaging a variety of molecular cargo. They have been implicated in the pathophysiology of various inflammatory diseases; yet, their role in acute lung injury (ALI) remains unknown., Objectives: We aimed to identify the biological activity and functional role of intra-alveolar MVs in ALI., Methods: Lipopolysaccharide (LPS) was instilled intratracheally into C57BL/6 mice, and MV populations in bronchoalveolar lavage fluid (BALF) were evaluated. BALF MVs were isolated 1 hour post LPS, assessed for cytokine content and incubated with murine lung epithelial (MLE-12) cells. In separate experiments, primary alveolar macrophage-derived MVs were incubated with MLE-12 cells or instilled intratracheally into mice., Results: Alveolar macrophages and epithelial cells rapidly released MVs into the alveoli following LPS. At 1 hour, the dominant population was alveolar macrophage-derived, and these MVs carried substantive amounts of tumour necrosis factor (TNF) but minimal amounts of IL-1β/IL-6. Incubation of these mixed MVs with MLE-12 cells induced epithelial intercellular adhesion molecule-1 (ICAM-1) expression and keratinocyte-derived cytokine release compared with MVs from untreated mice (p<0.001). MVs released in vitro from LPS-primed alveolar macrophages caused similar increases in MLE-12 ICAM-1 expression, which was mediated by TNF. When instilled intratracheally into mice, these MVs induced increases in BALF neutrophils, protein and epithelial cell ICAM-1 expression (p<0.05)., Conclusions: We demonstrate, for the first time, the sequential production of MVs from different intra-alveolar precursor cells during the early phase of ALI. Our findings suggest that alveolar macrophage-derived MVs, which carry biologically active TNF, may play an important role in initiating ALI., Competing Interests: Conflicts of Interest: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

The lung has a unique structure consisting of three functionally different compartments (alveolar, interstitial, and vascular) situated in an extreme proximity. Current methods to localize lung leukocytes using bronchoalveolar lavage and/or lung perfusion have significant limitations for determination of location and phenotype of leukocytes. Here we present a novel method using in vivo antibody labeling to enable accurate compartmental localization/quantification and phenotyping of mouse lung leukocytes. Anesthetized C57BL/6 mice received combined in vivo intravenous and intratracheal labeling with fluorophore-conjugated anti-CD45 antibodies, and lung single-cell suspensions were analyzed by flow cytometry. The combined in vivo intravenous and intratracheal CD45 labeling enabled robust separation of the alveolar, interstitial, and vascular compartments of the lung. In naive mice, the alveolar compartment consisted predominantly of resident alveolar macrophages. The interstitial compartment, gated by events negative for both intratracheal and intravenous CD45 staining, showed two conventional dendritic cell populations, as well as a Ly6C(lo) monocyte population. Expression levels of MHCII on these interstitial monocytes were much higher than on the vascular Ly6C(lo) monocyte populations. In mice exposed to acid aspiration-induced lung injury, this protocol also clearly distinguished the three lung compartments showing the dynamic trafficking of neutrophils and exudative monocytes across the lung compartments during inflammation and resolution. This simple in vivo dual-labeling technique substantially increases the accuracy and depth of lung flow cytometric analysis, facilitates a more comprehensive examination of lung leukocyte pools, and enables the investigation of previously poorly defined "interstitial" leukocyte populations during models of inflammatory lung diseases., (Copyright © 2015 the American Physiological Society.)

Objectives: To determine the effect of severe sepsis on monocyte tumor necrosis factor-α-converting enzyme baseline and inducible activity profiles., Design: Observational clinical study., Setting: Mixed surgical/medical teaching hospital ICU., Patients: Sixteen patients with severe sepsis, 15 healthy volunteers, and eight critically ill patients with noninfectious systemic inflammatory response syndrome., Interventions: None., Measurements and Main Results: Monocyte expression of human leukocyte antigen-D-related peptide, sol-tumor necrosis factor production, tumor necrosis factor-α-converting enzyme expression and catalytic activity, tumor necrosis factor receptor 1 and 2 expression, and shedding at 48-hour intervals from day 0 to day 4, as well as p38-mitogen activated protein kinase expression. Compared with healthy volunteers, both sepsis and systemic inflammatory response syndrome patients' monocytes expressed reduced levels of human leukocyte antigen-D-related peptide and released less sol-tumor necrosis factor on in vitro lipopolysaccharide stimulation, consistent with the term monocyte deactivation. However, patients with sepsis had substantially elevated levels of basal tumor necrosis factor-α-converting enzyme activity that were refractory to lipopolysaccharide stimulation and this was accompanied by similar changes in p38-mitogen activated protein kinase signaling. In patients with systemic inflammatory response syndrome, monocyte basal tumor necrosis factor-α-converting enzyme, and its induction by lipopolysaccharide, appeared similar to healthy controls. Changes in basal tumor necrosis factor-α-converting enzyme activity at day 0 for sepsis patients correlated with Acute Physiology and Chronic Health Evaluation II score and the attenuated tumor necrosis factor-α-converting enzyme response to lipopolysaccharide was associated with increased mortality. Similar changes in monocyte tumor necrosis factor-α-converting enzyme activity could be induced in healthy volunteer monocytes using an in vitro two-hit inflammation model. Patients with sepsis also displayed reduced shedding of monocyte tumor necrosis factor receptors upon stimulation with lipopolysaccharide., Conclusions: Monocyte tumor necrosis factor-α-converting enzyme catalytic activity appeared altered by sepsis and may result in reduced shedding of tumor necrosis factor receptors. Changes seemed specific to sepsis and correlated with illness severity. A better understanding of how tumor necrosis factor-α-converting enzyme function is altered during sepsis will enhance our understanding of sepsis pathophysiology, which will help in the assessment of patient inflammatory status and ultimately may provide new strategies to treat sepsis.

Mechanical ventilation, through overdistension of the lung, induces substantial inflammation that is thought to increase mortality among critically ill patients. The mechanotransduction processes involved in converting lung distension into inflammation during this ventilator-induced lung injury (VILI) remain unclear, although many cell types have been shown to be involved in its pathogenesis. This study aimed to identify the profile of in vivo lung cellular activation that occurs during the initiation of VILI. This was achieved using a flow cytometry-based method to quantify the phosphorylation of several markers (p38, ERK1/2, MAPK-activated protein kinase 2, and NF-κB) of inflammatory pathway activation within individual cell types. Anesthetized C57BL/6 mice were ventilated with low (7 ml/kg), intermediate (30 ml/kg), or high (40 ml/kg) tidal volumes for 1, 5, or 15 min followed by immediate fixing and processing of the lungs. Surprisingly, the pulmonary endothelium was the cell type most responsive to in vivo high-tidal-volume ventilation, demonstrating activation within just 1 min, followed by the alveolar epithelium. Alveolar macrophages were the slowest to respond, although they still demonstrated activation within 5 min. This order of activation was specific to VILI, since intratracheal lipopolysaccharide induced a very different pattern. These results suggest that alveolar macrophages may become activated via a secondary mechanism that occurs subsequent to activation of the parenchyma and that the lung cellular activation mechanism may be different between VILI and lipopolysaccharide. Our data also demonstrate that even very short periods of high stretch can promote inflammatory activation, and, importantly, this injury may be immediately manifested within the pulmonary vasculature., (Copyright © 2015 the American Physiological Society.)

Whilst lung transplantation is a viable solution for end-stage lung disease, donor shortages, donor lung inflammation and perioperative lung injury remain major limitations. Ex vivo lung perfusion has emerged as the next frontier in lung transplantation to address and overcome these limitations, with multicentre clinical trials ongoing in the UK, rest of Europe and North America. Our research seeks to identify the poorly understood cellular and molecular mechanisms of primary graft dysfunction through the development of an isolated perfused lung model of transplantation and investigation of the role of pulmonary inflammation in this paradigm.

Objective: Postoperative cognitive decline is emerging as a significant complication of surgery among older adults. Animal models indicate a central role of hippocampal inflammatory responses in the pathophysiology of postoperative cognitive decline. We hypothesized that atorvastatin, shown to exert neuroprotective potential in central nervous system (CNS) disorders, would attenuate neuroinflammation and improve cognitive function in mice after surgery and anesthesia., Methods: C57BL6 adult mice were pretreated with atorvastatin (250 μg) or vehicle, orally, for 5 days before undergoing unilateral nephrectomy under isoflurane anesthesia. We evaluated behavioral parameters related to cognitive function (fear conditioning and Morris Water Maze) and determined systemic and hippocampal interleukin-1β levels, postoperatively. Endothelial COX-2 expression, gross NF-κB and microglial (IBA1, CD68) activation, synaptic function (synapsin-1, PSD95, COX-2), heme oxygenase-1, and GSK3β were also examined., Results: Surgery induced a significant reduction in hippocampal-dependent fear response that was attenuated by treatment with atorvastatin, which also preserved spatial memory on day 7 after surgery. Atorvastatin evoked significant protection from hippocampal interleukin-1β production, but not systemic interleukin-1β production, accompanied by a marked reduction in hippocampal endothelial COX-2, NF-κB activation and decreased microglial reactivity. Surgery triggered an acute decline in synapsin-1, paralleled by an increase in postsynaptic COX-2 that was partially attenuated by atorvastatin. Furthermore, phosphorylation and inactivation of neuronal GSK3β was significantly enhanced after atorvastatin treatment., Conclusions: These findings indicate that cognitive decline is very likely associated with synaptic pathology after systemic and central inflammation induced by peripheral surgery/isoflurane anesthesia and suggest that the anti-inflammatory and neuroprotective properties of atorvastatin provide a rationale for its use as a therapeutic strategy for postoperative cognitive decline.

Objectives: Ventilator-induced lung injury has substantive impact on mortality of patients with acute respiratory distress syndrome. Although low tidal volume ventilation has been shown to reduce mortality, clinical benefits of open-lung strategy are controversial. In this study, we investigated the impact of two distinct forms of ventilator-induced lung injury, i.e., volutrauma and atelectrauma, on the progression of lung injury and inflammation, in particular alveolar and systemic cytokine production., Design: Ex vivo study., Setting: University research laboratory., Subjects: C57BL/6 mice., Interventions: Isolated, buffer-perfused lungs were allocated to one of three ventilatory protocols for 3 hours: control group received low tidal volume (7 mL/kg) with positive end-expiratory pressure (5 cm H2O) and regular sustained inflation; high-stretch group received high tidal volume (30-32 mL/kg) with positive end-expiratory pressure (3 cm H2O) and sustained inflation; and atelectasis group received the same tidal volume as control but neither positive end-expiratory pressure nor sustained inflation., Measurements and Main Results: Both injurious ventilatory protocols developed comparable levels of physiological injury and pulmonary edema, measured by respiratory system mechanics and lavage fluid protein. High-stretch induced marked increases in proinflammatory cytokines in perfusate and lung lavage fluid, compared to control. In contrast, atelectasis had no effect on perfusate cytokines compared to control but did induce some up-regulation of lavage cytokines. Depletion of monocytes marginated within the lung microvasculature, achieved by pretreating mice with i.v. liposome-encapsulated clodronate, significantly attenuated perfusate cytokine levels, especially tumor necrosis factor, in the high-stretch, but not atelectasis group., Conclusions: Volutrauma (high-stretch), but not atelectrauma (atelectasis), directly activates monocytes within the pulmonary vasculature, leading to cytokine release into systemic circulation. We postulate this as a potential explanation why open-lung strategy has limited mortality benefits in ventilated critically ill patients.

Prolonged hypothermic storage elicits severe ischemia-reperfusion injury (IRI) to renal grafts, contributing to delayed graft function (DGF) and episodes of acute immune rejection and shortened graft survival. Organoprotective strategies are therefore needed for improving long-term transplant outcome. The aim of this study is to investigate the renoprotective effect of xenon on early allograft injury associated with prolonged hypothermic storage. Xenon exposure enhanced the expression of heat-shock protein 70 (HSP-70) and heme oxygenase 1 (HO-1) and promoted cell survival after hypothermia-hypoxia insult in human proximal tubular (HK-2) cells, which was abolished by HSP-70 or HO-1 siRNA. In the brown Norway to Lewis rat renal transplantation, xenon administered to donor or recipient decreased the renal tubular cell death, inflammation, and MHC II expression, while delayed graft function (DGF) was therefore reduced. Pathological changes associated with acute rejection, including T-cell, macrophage, and fibroblast infiltration, were also decreased with xenon treatment. Donors or recipients treated with xenon in combination with cyclosporin A had prolonged renal allograft survival. Xenon protects allografts against delayed graft function, attenuates acute immune rejection, and enhances graft survival after prolonged hypothermic storage. Furthermore, xenon works additively with cyclosporin A to preserve post-transplant renal function.

The ability of the alveolar epithelium to prevent and resolve pulmonary edema is a crucial determinant of morbidity and mortality in acute lung injury (ALI). TNF has been implicated in ALI pathogenesis, but the precise mechanisms remain undetermined. We evaluated the role of TNF signaling in pulmonary edema formation in a clinically relevant mouse model of ALI induced by acid aspiration and investigated the effects of TNF p55 receptor deletion, caspase-8 inhibition, and alveolar macrophage depletion on alveolar epithelial function. We found that TNF plays a central role in the development of pulmonary edema in ALI through activation of p55-mediated death signaling, rather than through previously well-characterized p55-mediated proinflammatory signaling. Acid aspiration produced pulmonary edema with significant alveolar epithelial dysfunction, as determined by alveolar fluid clearance (AFC) and intra-alveolar levels of the receptor for advanced glycation end-products. The impairment of AFC was strongly correlated with lung caspase-8 activation, which was localized to type 1 alveolar epithelial cells by flow cytometric analysis. p55-deficient mice displayed markedly attenuated injury, with improved AFC and reduced caspase-8 activity but no differences in downstream cytokine/chemokine production and neutrophil recruitment. Caspase-8 inhibition significantly improved AFC and oxygenation, whereas depletion of alveolar macrophages attenuated epithelial dysfunction with reduced TNF production and caspase-8 activity. These results provide in vivo evidence for a novel role for TNF p55 receptor-mediated caspase-8 signaling, without substantial apoptotic cell death, in triggering alveolar epithelial dysfunction and determining the early pathophysiology of ALI. Blockade of TNF-induced death signaling may provide an effective early-phase strategy for ALI.

Background: Peripheral nerve injury is associated with a spinal microglial response that has been correlated with the development of behaviours reflective of neuropathic pain., Methods: To examine whether this phenomenon is generalizable to neuropathic pain of non-traumatic aetiology, this study investigated the association between spinal microgliosis and behavioural measures of neuropathic hypersensitivity and pain-related anxiety behaviour in four distinct rat models of peripheral neuropathic pain. These were traumatic neuropathy [L5 spinal nerve transection (SNT)], HIV-related neuropathies (either treatment with the antiretroviral drug Zalcitabine (ddC) or combination of perineural exposure to the HIV-gp120 protein and ddC treatment) and varicella zoster virus (VZV) infection., Results and Conclusion: Persistent mechanical hypersensitivity developed in all 'neuropathic' rats. However, spinal microgliosis, as measured by increased CD11b/c immunohistochemical staining and increased numbers of cells expressing CD11b measured by flow cytometry, was evident in the SNT and to a lesser extent in the HIV neuropathy models but not the VZV model. These results suggest that behavioural hypersensitivity and thigmotaxis can only be linked to a microglial response in certain models of neuropathy., (© 2012 European Federation of International Association for the Study of Pain Chapters.)

Margination and activation of monocytes within the pulmonary microcirculation contribute substantially to the development of acute lung injury in mice. The enhanced LPS-induced TNF expression exhibited by Gr-1(high) compared with Gr-1(low) monocytes within the lung microvasculature suggests differential roles for these subsets. We investigated the mechanisms responsible for such heterogeneity of lung-marginated monocyte proinflammatory response using a combined in vitro and in vivo approach. The monocyte subset inflammatory response was studied in vitro in mouse peripheral blood mononuclear cell-lung endothelial cell coculture and in vivo in a two-hit model of intravenous LPS-induced monocyte margination and lung inflammation in mice, by flow cytometry-based quantification of proinflammatory genes and intracellular phospho-kinases. With LPS stimulation in vitro, TNF expression was consistently higher in Gr-1(high) than Gr-1(low) monocytes, markedly enhanced by coculture with endothelial cells, and abrogated by p38 MAPK inhibitors. Expression of IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) was only detectable under coculture conditions, was substantially higher in Gr-1(high) monocytes, and was attenuated by p38 inhibition. Consistent with these differential responses, phosphorylation of p38 and its substrate MAPK-activated protein kinase 2 (MK2) was significantly higher in the Gr-1(high) subset. In vivo, p38 inhibitor treatment significantly attenuated LPS-induced TNF expression in "lung-marginated" Gr-1(high) monocytes. LPS-induced p38/MK2 phosphorylation was higher in lung-marginated Gr-1(high) than Gr-1(low) monocytes and neutrophils, mirroring TNF expression. These results indicate that the p38/MK2 pathway is a critical determinant of elevated Gr-1(high) subset responsiveness within the lung microvasculature, producing a coordinated proinflammatory response that places Gr-1(high) monocytes as key orchestrators of pulmonary microvascular inflammation and injury.

Tumor necrosis factor α-converting enzyme (TACE) is responsible for the shedding of cell surface TNF. Studies suggest that reactive oxygen species (ROS) mediate up-regulation of TACE activity by direct oxidization or modification of the protein. However, these investigations have been largely based upon nonphysiological stimulation of promonocytic cell lines which may respond and process TACE differently from primary cells. Furthermore, investigators have relied upon TACE substrate shedding as a surrogate for activity quantification. We addressed these concerns, employing a direct, cell-based fluorometric assay to investigate the regulation of TACE catalytic activity on freshly isolated primary human monocytes during LPS stimulation. We hypothesized that ROS mediate up-regulation of TACE activity indirectly, by activation of intracellular signaling pathways. LPS up-regulated TACE activity rapidly (within 30 min) without changing cell surface TACE expression. Scavenging of ROS or inhibiting their production by flavoprotein oxidoreductases significantly attenuated LPS-induced TACE activity up-regulation. Exogenous ROS (H(2)O(2)) also up-regulated TACE activity with similar kinetics and magnitude as LPS. H(2)O(2)- and LPS-induced TACE activity up-regulation were effectively abolished by a variety of selective p38 MAPK inhibitors. Activation of p38 was redox-sensitive as H(2)O(2) caused p38 phosphorylation, and ROS scavenging significantly reduced LPS-induced phospho-p38 expression. Inhibition of the p38 substrate, MAPK-activated protein kinase 2, completely attenuated TACE activity up-regulation, whereas inhibition of ERK had little effect. Lastly, inhibition of cell surface oxidoreductases prevented TACE activity up-regulation distal to p38 activation. In conclusion, our data indicate that in primary human monocytes, ROS mediate LPS-induced up-regulation of TACE activity indirectly through activation of the p38 signaling pathway.

Elevated soluble tumor necrosis factor-α receptor (sTNFR) levels in bronchoalveolar lavage fluid (BALF) are associated with poor patient outcome in acute lung injury (ALI). The mechanisms underlying these increases are unknown, but it is possible that pulmonary inflammation and increased alveolar epithelial permeability may individually contribute. We investigated mechanisms of elevated BALF sTNFRs in two in vivo mouse models of ALI. Anesthetized mice were challenged with intratracheal lipopolysaccharide or subjected to injurious mechanical ventilation. Lipopolysaccharide instillation produced acute intra-alveolar inflammation, but minimal alveolar epithelial permeability changes, with increased BALF sTNFR p75, but not p55. Increased p75 levels were markedly attenuated by alveolar macrophage depletion. In contrast, injurious ventilation induced substantial alveolar epithelial permeability, with increased BALF p75 and p55, which strongly correlated with total protein. BALF sTNFRs were not increased in isolated buffer-perfused lungs (devoid of circulating sTNFRs) subjected to injurious ventilation. These results suggest that lipopolysaccharide-induced intra-alveolar inflammation upregulates alveolar macrophage-mediated production of sTNFR p75, whereas enhanced alveolar epithelial permeability following mechanical ventilation leads to increased BALF p75 and p55 via plasma leakage. These data provide new insights into differential regulation of intra-alveolar sTNFR levels during ALI and may suggest sTNFRs as potential markers for evaluating the pathophysiology of ALI.

TNF plays a crucial role in the pathogenesis of acute lung injury. However, the expression profile of its two receptors, p55 and p75, on pulmonary endothelium and their influence on TNF signaling during lung microvascular inflammation remain uncertain. Using flow cytometry, we characterized the expression profile of TNF receptors on the surface of freshly harvested pulmonary endothelial cells (PECs) from mice and found expression of both receptors with dominance of p55. To investigate the impact of stimulating individual TNF receptors, we treated wild-type and TNF receptor knockout mice with intravenous TNF and determined surface expression of adhesion molecules (E-selectin, VCAM-1, ICAM-1) on PECs by flow cytometry. TNF-induced upregulation of all adhesion molecules was substantially attenuated by absence of p55, whereas lack of p75 had a similar but smaller effect that varied between adhesion molecules. Selective blockade of individual TNF receptors by specific antibodies in wild-type primary PEC culture confirmed that the in vivo findings were due to direct effects of TNF receptor inhibition on endothelium and not other cells (e.g., circulating leukocytes). Finally, we found that PEC surface expression of p55 dramatically decreased in the early stages of endotoxemia following intravenous LPS, while no change in p75 expression was detected. These data demonstrate a crucial in vivo role of p55 and an auxiliary role of p75 in TNF-mediated adhesion molecule upregulation on PECs. It is possible that the importance of the individual receptors varies at different stages of pulmonary microvascular inflammation following changes in their relative expression.

Background: Pulmonary inflammation is a major contributor to morbidity in a variety of respiratory disorders, but treatment options are limited. Here we investigate the efficacy, safety and mechanism of action of low dose inhaled carbon monoxide (CO) using a mouse model of lipopolysaccharide (LPS)-induced pulmonary inflammation., Methodology: Mice were exposed to 0-500 ppm inhaled CO for periods of up to 24 hours prior to and following intratracheal instillation of 10 ng LPS. Animals were sacrificed and assessed for intraalveolar neutrophil influx and cytokine levels, flow cytometric determination of neutrophil number and activation in blood, lung and lavage fluid samples, or neutrophil mobilisation from bone marrow., Principal Findings: When administered for 24 hours both before and after LPS, inhaled CO of 100 ppm or more reduced intraalveolar neutrophil infiltration by 40-50%, although doses above 100 ppm were associated with either high carboxyhemoglobin, weight loss or reduced physical activity. This anti-inflammatory effect of CO did not require pre-exposure before induction of injury. 100 ppm CO exposure attenuated neutrophil sequestration within the pulmonary vasculature as well as LPS-induced neutrophilia at 6 hours after LPS, likely due to abrogation of neutrophil mobilisation from bone marrow. In contrast to such apparently beneficial effects, 100 ppm inhaled CO induced an increase in pulmonary barrier permeability as determined by lavage fluid protein content and translocation of labelled albumin from blood to the alveolar space., Conclusions: Overall, these data confirm some protective role for inhaled CO during pulmonary inflammation, although this required a dose that produced carboxyhemoglobin values close to potentially toxic levels for humans, and increased lung permeability.

Reduced HLA-DR expression on monocytes has been suggested as a predictive marker of immunosuppression following very high risk surgery, but there are few reports in lower risk surgery. In 32 patients undergoing low to intermediate risk surgery, blood samples were analysed by flow cytometry for HLA-DR expression and numbers in both CD14(high) and CD14(low)CD16+ monocyte subsets. The numbers of CD14(high) monocytes increased at 24 h (mean (SD), 5.0 (2.2) vs 7.6 (3.9) x 10(5) cells.ml(-1); p < 0.01) while CD14(low)CD16+ monocytes decreased (0.68 (0.36) vs 0.44 (0.36) x 10(5) cells.ml(-1); p < 0.01). HLA-DR expression was significantly reduced in both subsets by 24 h (mean (SD) fluorescent intensity 440 (310) vs 160 (130) for CD14(high) and 1000 (410) vs 560 (380) for CD14(low)CD16+ subsets; p < 0.01). This reduction of monocyte HLA-DR expression 24 h following lower risk surgery raises questions about the purported clinical utility of this biomarker as an early predictor of postoperative complications. Our results also suggest that surgery induces significant trafficking (i.e. mobilisation, margination and extravasation) of monocyte subsets, and that monocyte HLA-DR depression is the result of a down-regulatory phenomenon (decreased protein expression on each cell) rather than the differential trafficking of monocyte subsets.

The specialized role of mouse Gr-1(high) monocytes in local inflammatory reactions has been well documented, but the trafficking and responsiveness of this subset during systemic inflammation and their contribution to sepsis-related organ injury has not been investigated. Using flow cytometry, we studied monocyte subset margination to the pulmonary microcirculation during subclinical endotoxemia in mice and investigated whether marginated monocytes contribute to lung injury in response to further septic stimuli. Subclinical low-dose i.v. LPS induced a rapid (within 2 h), large-scale mobilization of bone marrow Gr-1high monocytes and their prolonged margination to the lungs. With secondary LPS challenge, membrane TNF expression on these premarginated monocytes substantially increased, indicating their functional priming in vivo. Zymosan challenge produced small increases in pulmonary vascular permeability, which were markedly enhanced by the preadministration of low-dose LPS. The LPS-zymosan-induced permeability increases were effectively abrogated by pretreatment (30 min before zymosan challenge) with the platelet-activating factor antagonist WEB 2086 in combination with the phosphatidylcholine-phospholipase C inhibitor D609, suggesting the involvement of platelet-activating factor/ceramide-mediated pathways in this model. Depletion of monocytes (at 18 h after clodronate-liposome treatment) significantly attenuated the LPS-zymosan-induced permeability increase. However, restoration of normal LPS-induced Gr-1high monocyte margination to the lungs (at 48 h after clodronate-liposome treatment) resulted in the loss of this protective effect. These results demonstrate that mobilization and margination of Gr-1high monocytes during subclinical endotoxemia primes the lungs toward further septic stimuli and suggest a central role for this monocyte subset in the development of sepsis-related acute lung injury.

The immunological basis of the aberrant immune response in hyperreactive malarial splenomegaly (HMS) is poorly understood, but believed to be associated with polyclonal B cell activation by an unidentified malaria mitogen, leading to unregulated immunoglobulin and autoantibody production. HMS has been previously reported in Yanomami communities in the Upper Orinoco region of the Venezuelan Amazon. To investigate a possible association between antibody responses against Plasmodium falciparum and uninfected red blood cell (URBC) glycolipids and splenomegaly, a direct comparison of the parasite versus host anti-glycolipid antibody responses was made in an isolated community of this area. The anti-P. falciparum glycolipid (Pfglp) response was IgG3 dominated, whereas the uninfected red blood cell glycolipid (URBCglp) response showed a predominance of IgG1. The levels of IgG1 against Pfglp, and of IgG4 and IgM against URBCglp were significantly higher in women, while the anti-Pfglp or URBCglp IgM levels were inversely correlated with the degree of splenomegaly. Overall, these results suggest differential regulation of anti-parasite and autoreactive responses and that these responses may be linked to the development and evolution of HMS in this population exposed to endemic malaria. The high mortality rates associated with HMS point out that its early diagnosis together with the implementation of malaria control measures in these isolated Amerindian communities are a priority.