Imbalance of Pulmonary Microvascular Endothelial Cell-Expression of Metalloproteinases and Their Endogenous Inhibitors Promotes Septic Barrier Dysfunction.

Sepsis is a life-threatening disease characterized by excessive inflammation leading to organ dysfunction. During sepsis, pulmonary microvascular endothelial cells (PMVEC) lose barrier function associated with inter-PMVEC junction disruption. Matrix metalloproteinases (MMP) and a disintegrin and metalloproteinases (ADAM), which are regulated by tissue inhibitors of metalloproteinases (TIMPs), can cleave cell-cell junctional proteins, suggesting a role in PMVEC barrier dysfunction. We hypothesize that septic PMVEC barrier dysfunction is due to a disruption in the balance between PMVEC-specific metalloproteinases and TIMPs leading to increased metalloproteinase activity. The effects of sepsis on TIMPs and metalloproteinases were assessed ex vivo in PMVEC from healthy (sham) and septic (cecal ligation and perforation) mice, as well as in vitro in isolated PMVEC stimulated with cytomix, lipopolysaccharide (LPS), and cytomix + LPS vs. PBS. PMVEC had high basal Timp expression and lower metalloproteinase expression, and septic stimulation shifted expression in favour of metalloproteinases. Septic stimulation increased MMP13 and ADAM17 activity associated with a loss of inter-PMVEC junctional proteins and barrier dysfunction, which was rescued by treatment with metalloproteinase inhibitors. Collectively, our studies support a role for metalloproteinase-TIMP imbalance in septic PMVEC barrier dysfunction, and suggest that inhibition of specific metalloproteinases may be a therapeutic avenue for septic patients.

Quantification of adherens junction disruption and contiguous paracellular protein leak in human lung endothelial cells under septic conditions.

OBJECTIVE: Sepsis is associated with dysfunction of MVEC resulting in organ edema and inflammation. VE-cadherin, a component of MVEC adherens junctions, may be disrupted in sepsis. However, the direct connection between individual MVEC VE-cadherin disruption and increased paracellular permeability is uncertain. METHODS: Human pulmonary MVEC were cultured on a biotin matrix and treated with cytomix, as a model of sepsis, vs PBS. MVEC permeability was assessed by trans-MVEC monolayer leak of Oregon green 488-conjugated avidin, which bound subcellular biotin to localize sites of paracellular leak. Leak was correlated with individual cell-specific MVEC surface VE-cadherin continuity by fluorescence microscopy. RESULTS: Cytomix treatment reduced total MVEC VE-cadherin density, disrupted surface VE-cadherin continuity, was associated with intercellular gap formation, and enhanced paracellular avidin leak. Cytomix-induced MVEC paracellular avidin leak was strongly correlated temporally and was highly contiguous with focal MVEC surface VE-cadherin disruption. Total cellular VE-cadherin density was less strongly correlated with MVEC paracellular avidin leak and individual cell-specific focal surface VE-cadherin discontinuity. CONCLUSIONS: These data support a mechanistic link between septic human lung MVEC VE-cadherin disruption and contiguous paracellular protein leak, and will permit more detailed assessment of individual cell-specific mechanisms of septic MVEC barrier dysfunction.

The effects of aging and exercise on lung mechanics, surfactant and alveolar macrophages.

Purpose: Advancing age leads to changes to the respiratory system associated with increased susceptibility to lung diseases, and exercise may counteract this effect. To explore the underlying processes, we investigated the effects of aging and exercise on lung mechanics, alveolar macrophage function, and surfactant pools and activity, in mice. It was hypothesized that aging would impact lung mechanics, macrophage polarization, and the status of the surfactant system, and that these changes would be mitigated by exercise. Methods: Male C57BL/6 mice were housed from 2-3 to 22 months, for the aged group, or until 4 months of age for young mice. Mice in both groups were randomized to voluntarily running exercise or to non-exercise, for a 2-month period. Mice were euthanized and lung mechanics were analyzed using a flexiVent ventilator. Subsequently, the lungs were lavaged to obtain pulmonary surfactant and alveolar macrophages. Pulmonary surfactant was analyzed for pool sizes and activity whereas alveolar macrophages were examined for response to pro and anti-inflammatory stimuli. Results: Changes in lung mechanics, such as increased compliance and decreased airway resistance, were associated with aging but were not affected by exercise. The quantity as well as the biophysical activity of the pulmonary surfactant system was unaffected by either aging or exercise. More alveolar macrophages were recovered from exercising aged mice compared to both the young and non-exercising groups. Macrophages in this aged exercise group were more responsive to an anti-inflammatory stimulus. Conclusions: Our data supports previous literature that suggest the development of emphysema-like alterations to lung mechanics with aging. This effect was independent of exercise. Our data also indicates that surfactant is unaffected by aging and exercise. Alveolar macrophage properties and numbers were affected by exercise in the aging lung and may represent the main, potentially beneficial, effect of exercise on the pulmonary system.

Autocrine GABA signaling distinctively regulates phenotypic activation of mouse pulmonary macrophages.

In response to micro-environmental cues such as microbial infections or T-helper 1 and 2 (TH1 and TH2) cytokines, macrophages (Mϕs) develop into M1- or M2-like phenotypes. Phenotypic polarization/activation of Mϕs are also essentially regulated by autocrine signals. Type-A γ-aminobutyric acid receptor (GABAAR)-mediated autocrine signaling is critical for phenotypic differentiation and transformation of various cell types. The present study explored whether GABAAR signaling regulates lung Mϕ (LMϕ) phenotypic activation under M1/TH1 and M2/TH2 environments. Results showed that GABAAR subunits were expressed by primary LMϕ of mice and the mouse Mϕ cell line RAW264.7. The expression levels of GABAAR subunits in mouse LMϕs and RAW264.7 cells decreased or increased concurrently with classical (M1) or alternative (M2) activation, respectively. Moreover, activation or blockade of GABAARs distinctively influenced the phenotypic characteristics of Mϕ. These results suggested that microenvironments leading to LMϕ phenotypic polarization concurrently modulates autocrine GABA signaling and its role in Mϕ activation.

Lung remodeling associated with recovery from acute lung injury.

Acute respiratory distress syndrome (ARDS) is a disease with a variety of causes and is defined by severe hypoxemia. Whereas ARDS carries a mortality of approximately 30 %, patients that survive may ultimately regain near normal pulmonary physiology. The critical pathophysiological processes in ARDS are alveolar barrier dysfunction and overwhelming inflammation. This encompasses damage to the epithelial and endothelial layers, thickening of the interstitial matrix, edema with inactivation of pulmonary surfactant at the alveolar surface and marked inflammation mediated by infiltrating neutrophils and pro-inflammatory macrophages. For patients that survive the disease, these are the critical processes that require repair and remodeling to allow for the recovery of ARDS. As such, the current review focuses on the experimental studies that have begun to elucidate the mechanisms involved in restoring the alveolar barrier following injury.

Voluntary running exercise protects against sepsis-induced early inflammatory and pro-coagulant responses in aged mice.

BACKGROUND: Despite many animal studies and clinical trials, mortality in sepsis remains high. This may be due to the fact that most experimental studies of sepsis employ young animals, whereas the majority of septic patients are elderly (60 - 70 years). The objective of the present study was to examine the sepsis-induced inflammatory and pro-coagulant responses in aged mice. Since running exercise protects against a variety of diseases, we also examined the effect of voluntary running on septic responses in aged mice. METHODS: Male C57BL/6 mice were housed in our institute from 2-3 to 22 months (an age mimicking that of the elderly). Mice were prevented from becoming obese by food restriction (given 70-90% of ad libitum consumption amount). Between 20 and 22 months, a subgroup of mice ran voluntarily on wheels, alternating 1-3 days of running with 1-2 days of rest. At 22 months, mice were intraperitoneally injected with sterile saline (control) or 3.75 g/kg fecal slurry (septic). At 7 h post injection, we examined (1) neutrophil influx in the lung and liver by measuring myeloperoxidase and/or neutrophil elastase in the tissue homogenates by spectrophotometry, (2) interleukin 6 (IL6) and KC in the lung lavage by ELISA, (3) pulmonary surfactant function by measuring percentage of large aggregates, (4) capillary plugging (pro-coagulant response) in skeletal muscle by intravital microscopy, (5) endothelial nitric oxide synthase (eNOS) protein in skeletal muscle (eNOS-derived NO is putative inhibitor of capillary plugging) by immunoblotting, and (6) systemic blood platelet counts by hemocytometry. RESULTS: Sepsis caused high levels of pulmonary myeloperoxidase, elastase, IL6, KC, liver myeloperoxidase, and capillary plugging. Sepsis also caused low levels of surfactant function and platelet counts. Running exercise increased eNOS protein and attenuated the septic responses. CONCLUSIONS: Voluntary running protects against exacerbated sepsis-induced inflammatory and pro-coagulant responses in aged mice. Protection against pro-coagulant responses may involve eNOS upregulation. The present discovery in aged mice calls for clinical investigation into potential beneficial effects of exercise on septic outcomes in the elderly.

Inhibition of Murine Pulmonary Microvascular Endothelial Cell Apoptosis Promotes Recovery of Barrier Function under Septic Conditions.

Sepsis is characterized by injury of the pulmonary microvasculature and the pulmonary microvascular endothelial cells (PMVEC), leading to barrier dysfunction and acute respiratory distress syndrome (ARDS). Our recent work identified a strong correlation between PMVEC apoptosis and microvascular leak in septic mice in vivo, but the specific role of apoptosis in septic PMVEC barrier dysfunction remains unclear. Thus, we hypothesize that PMVEC apoptosis is likely required for PMVEC barrier dysfunction under septic conditions in vitro. Septic stimulation (mixture of tumour necrosis factor α, interleukin 1ß, and interferon γ [cytomix]) of isolated murine PMVEC resulted in a significant loss of barrier function as early as 4 h after stimulation, which persisted until 24 h. PMVEC apoptosis, as reflected by caspase activation, DNA fragmentation, and loss of membrane polarity, was first apparent at 8 h after cytomix. Pretreatment of PMVEC with the pan-caspase inhibitor Q-VD significantly decreased septic PMVEC apoptosis and was associated with reestablishment of PMVEC barrier function at 16 and 24 h after stimulation but had no effect on septic PMVEC barrier dysfunction over the first 8 h. Collectively, our data suggest that early septic murine PMVEC barrier dysfunction driven by proinflammatory cytokines is not mediated through apoptosis, but PMVEC apoptosis contributes to late septic PMVEC barrier dysfunction.

Shedding of Syndecan-1/CXCL1 Complexes by Matrix Metalloproteinase 7 Functions as an Epithelial Checkpoint of Neutrophil Activation.

Although neutrophils play critical roles in innate immunity, in excess these cells cause severe tissue damage. Thus, neutrophil activation must be tightly regulated to prevent indiscriminant damage. Previously, we reported that mice lacking matrix metalloproteinase (MMP) 7 are protected from lung injury owing to markedly impaired neutrophil movement from the interstitium into mucosal lumenal spaces. This phenotype resulted from a lack of MMP7 shedding of syndecan-1, a heparan sulfate proteoglycan that carries the neutrophil chemokine CXCL1 as cargo. Here, we assessed if shedding syndecan-1/CXCL1 complexes affects neutrophil activation. Whereas injured monolayers of wild-type alveolar type II cells potently stimulated neutrophil activation, as gauged by release of myeloperoxidase, cells from Mmp7(-/-) or syndecan-1-null (Sdc1(-/-)) mice or human cells with MMP7 knockdown did not. In vivo, we observed reduced myeloperoxidase release relative to neutrophil numbers in bleomycin-injured Mmp7(-/-) and Sdc1(-/-) mice. Furthermore, we determined that soluble syndecan-1 directly stimulated neutrophil activation in the absence of cellular damage. These data indicate that MMP7 shedding of syndecan-1/CXCL1 complexes functions as a checkpoint that restricts neutrophil activation at sites of epithelial injury.

Tissue inhibitor of metalloproteinases 3-dependent microvascular endothelial cell barrier function is disrupted under septic conditions.

Sepsis is associated with dysfunction of microvascular endothelial cells (MVEC) leading to tissue edema and multiple organ dysfunction. Metalloproteinases can regulate MVEC function through processing of cell surface proteins, and tissue inhibitor of metalloproteinases 3 (TIMP3) regulates metalloproteinase activity in the lung following injury. We hypothesize that TIMP3 promotes normal pulmonary MVEC barrier function through inhibition of metalloproteinase activity. Naive Timp3(-/-) mice had significantly higher basal pulmonary microvascular Evans blue (EB) dye-labeled albumin leak vs. wild-type (WT) mice. Additionally, cecal-ligation/perforation (CLP)-induced sepsis significantly increased pulmonary microvascular EB-labeled albumin leak in WT but not Timp3(-/-) mice. Similarly, PBS-treated isolated MVEC monolayers from Timp3(-/-) mice displayed permeability barrier dysfunction vs. WT MVEC, evidenced by lower transendothelial electrical resistance and greater trans-MVEC flux of fluorescein-dextran and EB-albumin. Cytomix (equimolar interferon γ, tumor necrosis factor α, and interleukin 1ß) treatment of WT MVEC induced significant barrier dysfunction (by all three methods), and was associated with a time-dependent decrease in TIMP3 mRNA and protein levels. Additionally, basal Timp3(-/-) MVEC barrier dysfunction was associated with disrupted MVEC surface VE-cadherin localization, and both barrier dysfunction and VE-cadherin localization were rescued by treatment with GM6001, a synthetic metalloproteinase inhibitor. TIMP3 promotes normal MVEC barrier function, at least partially, through inhibition of metalloproteinase-dependent disruption of adherens junctions, and septic downregulation of TIMP3 may contribute to septic MVEC barrier dysfunction.

Role of pulmonary microvascular endothelial cell apoptosis in murine sepsis-induced lung injury in vivo.

BACKGROUND: Sepsis remains a common and serious condition with significant morbidity and mortality due to multiple organ dysfunction, especially acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Sepsis-induced ALI is characterized by injury and dysfunction of the pulmonary microvasculature and pulmonary microvascular endothelial cells (PMVEC), resulting in enhanced pulmonary microvascular sequestration and pulmonary infiltration of polymorphonuclear leukocytes (PMN) as well as disruption of the normal alveolo-capillary permeability barrier with leak of albumin-rich edema fluid into pulmonary interstitium and alveoli. The role of PMVEC death and specifically apoptosis in septic pulmonary microvascular dysfunction in vivo has not been established. METHODS: In a murine cecal ligation/perforation (CLP) model of sepsis, we quantified and correlated time-dependent changes in pulmonary microvascular Evans blue (EB)-labeled albumin permeability with (1) PMVEC death (propidium iodide [PI]-staining) by both fluorescent intravital videomicroscopy (IVVM) and histology, and (2) PMVEC apoptosis using histologic fluorescent microscopic assessment of a panel of 3 markers: cell surface phosphatidylserine (detected by Annexin V binding), caspase activation (detected by FLIVO labeling), and DNA fragmentation (TUNEL labeling). RESULTS: Compared to sham mice, CLP-sepsis resulted in pulmonary microvascular barrier dysfunction, quantified by increased EB-albumin leak, and PMVEC death (PI+ staining) as early as 2 h and more marked by 4 h after CLP. Septic PMVEC also exhibited increased presence of all 3 markers of apoptosis (Annexin V+, FLIVO+, TUNEL+) as early as 30 mins--1 h after CLP-sepsis, which all similarly increased markedly until 4 h. The time-dependent changes in septic pulmonary microvascular albumin-permeability barrier dysfunction were highly correlated with PMVEC death (PI+; r = 0.976, p < 0.01) and PMVEC apoptosis (FLIVO+; r = 0.991, p < 0.01). Treatment with the pan-caspase inhibitor Q-VD prior to CLP reduced PMVEC death/apoptosis and attenuated septic pulmonary microvascular dysfunction, including both albumin-permeability barrier dysfunction and pulmonary microvascular PMN sequestration (p < 0.05). Septic PMVEC apoptosis and pulmonary microvascular dysfunction were also abrogated following CLP-sepsis in mice deficient in iNOS (Nos2 (-/-)) or NADPH oxidase (p47 (phox-/-) or gp91 (phox-/-)) and in wild-type mice treated with the NADPH oxidase inhibitor, apocynin. CONCLUSIONS: Septic murine pulmonary microvascular dysfunction in vivo is due to PMVEC death, which is mediated through caspase-dependent apoptosis and iNOS/NADPH-oxidase dependent signaling.

Tissue inhibitor of metalloproteinases-3 moderates the proinflammatory status of macrophages.

Tissue inhibitor of metalloproteinases-3 (TIMP-3) has emerged as a key mediator of inflammation. Recently, we reported that the resolution of inflammation is impaired in Timp3(-/-) mice after bleomycin-induced lung injury. Here, we demonstrate that after LPS instillation (another model of acute lung injury), Timp3(-/-) mice demonstrate enhanced and persistent neutrophilia, increased numbers of infiltrated macrophages, and delayed weight gain, compared with wild-type (WT) mice. Because macrophages possess broad immune functions and can differentiate into cells that either stimulate inflammation (M1 macrophages) or are immunosuppressive (M2 macrophages), we examined whether TIMP-3 influences macrophage polarization. Comparisons of the global gene expression of unstimulated or LPS-stimulated bone marrow-derived macrophages (BMDMs) from WT and Timp3(-/-) mice revealed that Timp3(-/-) BMDMs exhibited an increased expression of genes associated with proinflammatory (M1) macrophages, including Il6, Il12, Nos2, and Ccl2. Microarray analyses also revealed a baseline difference in gene expression between WT and Timp3(-/-) BMDMs, suggesting altered macrophage differentiation. Furthermore, the treatment of Timp3(-/-) BMDMs with recombinant TIMP-3 rescued this altered gene expression. We also examined macrophage function, and found that Timp3(-/-) M1 cells exhibit significantly more neutrophil chemotactic activity and significantly less soluble Fas ligand-induced caspase-3/7 activity, a marker of apoptosis, compared with WT M1 cells. Macrophage differentiation into immunosuppressive M2 cells is mediated by exposure to IL-4/IL-13, and we found that Timp3(-/-) M2 macrophages demonstrated a lower expression of genes associated with an anti-inflammatory phenotype, compared with WT M2 cells. Collectively, these findings indicate that TIMP-3 functions to moderate the differentiation of macrophages into proinflammatory (M1) cells.

Pericyte TIMP3 and ADAMTS1 modulate vascular stability after kidney injury.

Kidney pericytes are progenitors of scar-forming interstitial myofibroblasts that appear after injury. The function of kidney pericytes as microvascular cells and how these cells detach from peritubular capillaries and migrate to the interstitial space, however, are poorly understood. Here, we used an unbiased approach to identify genes in kidney pericytes relevant to detachment and differentiation in response to injury in vivo, with a particular focus on genes regulating proteolytic activity and angiogenesis. Kidney pericytes rapidly activated expression of a disintegrin and metalloprotease with thrombospondin motifs-1 (ADAMTS1) and downregulated its inhibitor, tissue inhibitor of metalloproteinase 3 (TIMP3) in response to injury. Similarly to brain pericytes, kidney pericytes bound to and stabilized capillary tube networks in three-dimensional gels and inhibited metalloproteolytic activity and angiogenic signaling in endothelial cells. In contrast, myofibroblasts did not have these vascular stabilizing functions despite their derivation from kidney pericytes. Pericyte-derived TIMP3 stabilized and ADAMTS1 destabilized the capillary tubular networks. Furthermore, mice deficient in Timp3 had a spontaneous microvascular phenotype in the kidney resulting from overactivated pericytes and were more susceptible to injury-stimulated microvascular rarefaction with an exuberant fibrotic response. Taken together, these data support functions for kidney pericytes in microvascular stability, highlight central roles for regulators of extracellular proteolytic activity in capillary homoeostasis, and identify ADAMTS1 as a marker of activation of kidney pericytes.

Sex-based analysis of treatment responses in animal models of sepsis: a preclinical systematic review protocol.

BACKGROUND: The importance of investigating sex- and gender-dependent differences has been recently emphasized by major funding agencies. Notably, the influence of biological sex on clinical outcomes in sepsis is unclear, and observational studies suffer from the effect of confounding factors. The controlled experimental environment afforded by preclinical studies allows for clarification and mechanistic evaluation of sex-dependent differences. We propose a systematic review to assess the impact of biological sex on baseline responses to disease induction as well as treatment responses in animal models of sepsis. Given the lack of guidance surrounding sex-based analyses in preclinical systematic reviews, careful consideration of various factors is needed to understand how best to conduct analyses and communicate findings. METHODS: MEDLINE and Embase will be searched (2011-present) to identify preclinical studies of sepsis in which any intervention was administered and sex-stratified data reported. The primary outcome will be mortality. Secondary outcomes will include organ dysfunction, bacterial load, and IL-6 levels. Study selection will be conducted independently and in duplicate by two reviewers. Data extraction will be conducted by one reviewer and audited by a second independent reviewer. Data extracted from included studies will be pooled, and meta-analysis will be conducted using random effects modeling. Primary analyses will be stratified by animal age and will assess the impact of sex at the following time points: pre-intervention, in response to treatment, and post-intervention. Risk of bias will be assessed using the SYRCLE's risk-of-bias tool. Illustrative examples of potential methods to analyze sex-based differences are provided in this protocol. DISCUSSION: Our systematic review will summarize the current state of knowledge on sex-dependent differences in sepsis. This will identify current knowledge gaps that future studies can address. Finally, this review will provide a framework for sex-based analysis in future preclinical systematic reviews. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42022367726.

Development and characterization of a fecal-induced peritonitis model of murine sepsis: results from a multi-laboratory study and iterative modification of experimental conditions.

BACKGROUND: Preclinical sepsis models have been criticized for their inability to recapitulate human sepsis and suffer from methodological shortcomings that limit external validity and reproducibility. The National Preclinical Sepsis Platform (NPSP) is a consortium of basic science researchers, veterinarians, and stakeholders in Canada undertaking standardized multi-laboratory sepsis research to increase the efficacy and efficiency of bench-to-bedside translation. In this study, we aimed to develop and characterize a 72-h fecal-induced peritonitis (FIP) model of murine sepsis conducted in two independent laboratories. The experimental protocol was optimized by sequentially modifying dose of fecal slurry and timing of antibiotics in an iterative fashion, and then repeating the experimental series at site 1 and site 2. RESULTS: Escalating doses of fecal slurry (0.5-2.5 mg/g) resulted in increased disease severity, as assessed by the modified Murine Sepsis Score (MSS). However, the MSS was poorly associated with progression to death during the experiments, and mice were found dead without elevated MSS scores. Administration of early antibiotics within 4 h of inoculation rescued the animals from sepsis compared with late administration of antibiotics after 12 h, as evidenced by 100% survival and reduced bacterial load in peritoneum and blood in the early antibiotic group. Site 1 and site 2 had statistically significant differences in mortality (60% vs 88%; p < 0.05) for the same dose of fecal slurry (0.75 mg/g) and marked differences in body temperature between groups. CONCLUSIONS: We demonstrate a systematic approach to optimizing a 72-h FIP model of murine sepsis for use in multi-laboratory studies. Alterations to experimental conditions, such as dose of fecal slurry and timing of antibiotics, have clear impact on outcomes. Differences in mortality between sites despite rigorous standardization warrants further investigations to better understand inter-laboratory variation and methodological design in preclinical studies.

Effect of insulin-like growth factor blockade on hyperoxia-induced lung injury.

Insulin-like growth factor (IGF)-1 is increased in different models of acute lung injury, and is an important determinant of survival and proliferation in many cells. We previously demonstrated that treatment of mice with IGF-1 receptor-blocking antibody (A12) improved early survival in bleomycin-induced lung injury. We have now examined whether administration of A12 improved markers of lung injury in hyperoxia model of lung injury. C57BL/6 mice underwent intraperitoneal administration of A12 or control antibody (keyhole limpet hemocyanin [KLH]), then were exposed to 95% hyperoxia for 88-90 hours. Mice were killed and bronchoalveolar lavage (BAL) and lung tissue were obtained for analysis. Hyperoxia caused a significant increase in IGF levels in BAL and lung lysates. Peripheral blood neutrophils expressed IGF-1R at baseline and after hyperoxia. BAL neutrophils from hyperoxia-treated mice and patients with acute lung injury also expressed cell surface IGF-1R. A12-treated mice had significantly decreased polymorphonuclear cell (PMN) count in BAL compared with KLH control mice (P = 0.02). BAL from A12-treated mice demonstrated decreased PMN chemotactic activity compared with BAL from KLH-treated mice. Pretreatment of PMNs with A12 decreased their chemotactic response to BAL from hyperoxia-exposed mice. Furthermore, IGF-1 induced a dose-dependent chemotaxis of PMNs. There were no differences in other chemotactic cytokines in BAL, including CXCL1 and CXCL2. In summary, IGF blockade decreased PMN recruitment to the alveolar space in a mouse model of hyperoxia. Furthermore, the decrease in BAL PMNs was at least partially due to a direct effect of A12 on PMN chemotaxis.

Pulmonary macrophage subpopulations in the induction and resolution of acute lung injury.

Macrophages are key orchestrators of the inflammatory and repair responses in the lung, and the diversity of their function is indicated by their polarized states and distinct subpopulations and localization in the lung. Here, we characterized the pulmonary macrophage populations in the interstitial and alveolar compartments during the induction and resolution of acute lung injury induced by Pseudomonas aeruginosa infection. We identified macrophage subpopulations and polarity according to FACS analysis of cell surface protein markers, combined with cell sorting for gene expression using real-time PCR. With these techniques, we validated a novel, alternatively activated (M2) marker (transferrin receptor), and we described three interstitial and alveolar macrophage subpopulations in the lung whose distribution and functional state evolved from the induction to resolution phases of lung injury. Together, these findings indicate the presence and evolution of distinct macrophage subsets in the lung that serve specific niches in regulating the inflammatory response and its resolution. Alterations in the balance and function of these subpopulations could lead to nonresolving acute lung injury.

Syndecan-4 regulates early neutrophil migration and pulmonary inflammation in response to lipopolysaccharide.

Proteoglycans (PGs) and their associated glycosaminoglycan side chains are effectors of inflammation, but little is known about changes to the composition of PGs in response to lung infection or injury. The goals of this study were to identify changes to heparan sulfate PGs in a mouse model of gram-negative pneumonia, to identify the Toll-like receptor adaptor molecules responsible for these changes, and to determine the role of the heparan sulfate PG in the innate immune response in the lungs. We treated mice with intratracheal LPS, a component of the cell wall of gram-negative bacteria, to model gram-negative pneumonia. Mice treated with intratracheal LPS had a rapid and selective increase in syndecan-4 mRNA that was regulated through MyD88-dependent mechanisms, whereas expression of several other PGs was not affected. To determine the role of syndecan-4 in the inflammatory response, we exposed mice deficient in syndecan-4 to LPS and found a significant increase in neutrophil numbers and amounts of CXC-chemokines and total protein in bronchoalveolar lavage fluid. In studies performed in vitro, macrophages and epithelial cells treated with LPS had increased expression of syndecan-4. Studies performed using BEAS-2B cells showed that pretreatment with heparin and syndecan-4 decreased the expression of CXCL8 mRNA in response to LPS and TNF-α. These findings indicate that the early inflammatory response to LPS involves marked up-regulation of syndecan-4, which functions to limit the extent of pulmonary inflammation and lung injury.

Fas-deficient mice have impaired alveolar neutrophil recruitment and decreased expression of anti-KC autoantibody:KC complexes in a model of acute lung injury.

BACKGROUND: Exposure to mechanical ventilation enhances lung injury in response to various stimuli, such as bacterial endotoxin (LPS). The Fas/FasL system is a receptor ligand system that has dual pro-apoptotic and pro-inflammatory functions and has been implicated in the pathogenesis of lung injury. In this study we test the hypothesis that a functioning Fas/FasL system is required for the development of lung injury in mechanically ventilated mice. METHODS: C57BL/6 (B6) and Fas-deficient lpr mice were exposed to either intra-tracheal PBS followed by spontaneous breathing or intra-tracheal LPS followed by four hours mechanical ventilation with tidal volumes of 10 mL/kg, respiratory rate of 150 breaths per minute, inspired oxygen 0.21 and positive end expiratory pressure (PEEP) of 3 cm of water. RESULTS: Compared with the B6 mice, the lpr mice showed attenuation of the neutrophilic response as measured by decreased numbers of BAL neutrophils and lung myeloperoxidase activity. Interestingly, the B6 and lpr mice had similar concentrations of pro-inflammatory cytokines, including CXCL1 (KC), and similar measurements of permeability and apoptosis. However, the B6 mice showed greater deposition of anti-KC:KC immune complexes in the lungs, as compared with the lpr mice. CONCLUSIONS: We conclude that a functioning Fas/FasL system is required for full neutrophilic response to LPS in mechanically ventilated mice.

Kinetics of chemokine-glycosaminoglycan interactions control neutrophil migration into the airspaces of the lungs.

Chemokine-glycosaminoglycan (GAG) interactions are thought to result in the formation of tissue-bound chemokine gradients. We hypothesized that the binding of chemokines to GAGs would increase neutrophil migration toward CXC chemokines instilled into lungs of mice. To test this hypothesis we compared neutrophil migration toward recombinant human CXCL8 (rhCXCL8) and two mutant forms of CXCL8, which do not bind to heparin immobilized on a sensor chip. Unexpectedly, when instilled into the lungs of mice the CXCL8 mutants recruited more neutrophils than rhCXCL8. The CXCL8 mutants appeared in plasma at significantly higher concentrations and diffused more rapidly across an extracellular matrix in vitro. A comparison of the murine CXC chemokines, KC and MIP-2, revealed that KC was more effective in recruiting neutrophils into the lungs than MIP-2. KC appeared in plasma at significantly higher concentrations and diffused more rapidly across an extracellular matrix in vitro than MIP-2. In kinetic binding studies, KC, MIP-2, and rhCXCL8 bound heparin differently, with KC associating and dissociating more rapidly from immobilized heparin than the other chemokines. These data suggest that the kinetics of chemokine-GAG interactions contributes to chemokine function in tissues. In the lungs, it appears that chemokines, such as CXCL8 or MIP-2, which associate and disassociate slowly from GAGs, form gradients relatively slowly compared with chemokines that either bind GAGs poorly or interact with rapid kinetics. Thus, different types of chemokine gradients may form during an inflammatory response. This suggests a new model, whereby GAGs control the spatiotemporal formation of chemokine gradients and neutrophil migration in tissue.

Overexpression of urokinase by plaque macrophages causes histological features of plaque rupture and increases vascular matrix metalloproteinase activity in aged apolipoprotein e-null mice.

BACKGROUND: The mechanisms of atherosclerotic plaque rupture are poorly understood. Urokinase-type plasminogen activator (uPA) is expressed at elevated levels by macrophages in advanced human plaques. Patients with evidence of increased plasminogen activation have an elevated risk of major cardiovascular events. We used atherosclerotic mice to test the hypothesis that increased macrophage uPA expression in advanced plaques would cause histological features similar to those in ruptured human plaques. METHODS AND RESULTS: Bone marrow from transgenic mice with increased macrophage uPA expression or nontransgenic controls (all apolipoprotein E-null [Apoe(-/-)]) was transplanted into 35-week-old Apoe(-/-) recipients, and innominate lesions and aortas were examined 8 to 13 weeks later. Donor macrophages accumulated in innominate lesions adjacent to plaque caps and in aortas, increasing uPA expression at both sites. Recipients of uPA-overexpressing macrophages had an increased prevalence of intraplaque hemorrhage (61% versus 13%; P=0.002) as well as increased lesion fibrin staining and fibrous cap disruption (P=0.06 for both). Transplantation of uPA-overexpressing macrophages increased aortic matrix metalloproteinase activity (40%; P=0.02). This increase was independent of matrix metalloproteinase-9. CONCLUSIONS: In advanced plaques of Apoe(-/-) mice, macrophage uPA overexpression causes intraplaque hemorrhage and fibrous cap disruption, features associated with human plaque rupture. uPA overexpression also increases vascular matrix metalloproteinase activity. These data provide a mechanism that connects macrophage uPA expression, matrix metalloproteinase activity, and plaque rupture features in mice. The data also suggest that elevated plaque plasminogen activator expression and plasminogen activation in humans may be causally linked to plaque rupture and cardiovascular events.