Pulmonary contusion induces alveolar type 2 epithelial cell apoptosis: role of alveolar macrophages and neutrophils

Alveolar type 2 (AT-2) cell apoptosis is an important mechanism during lung inflammation, lung injury, and regeneration. Blunt chest trauma has been shown to activate inflammatory cells such as alveolar macrophages (AMs) or neutrophils (polymorphonuclear granulocytes [PMNs]), resulting in an inflammatory response. The present study was performed to determine the capacity of different components/cells of the alveolar compartment (AMs, PMNs, or bronchoalveolar lavage [BAL] fluids) to induce apoptosis in AT-2 cells following blunt chest trauma. To study this, male Sprague-Dawley rats were subjected to either sham procedure or blunt chest trauma induced by a single blast wave. Various time points after injury (6 h to 7 d), the lungs were analyzed by immunohistochemistry, for AT-2 cells, or with antibodies directed against caspase 3, caspase 8, Fas, Fas ligand (FasL), BAX, and BCL-2. Bronchoalveolar lavage concentrations of TNF-alpha, IL-1beta, and soluble FasL were determined by enzyme-linked immunosorbent assay. Furthermore, cultures of AT-2 cells isolated from healthy rats were incubated with supernatants of AMs, PMNs, or BAL fluids obtained from either trauma or sham-operated animals in the presence or absence of oxidative stress. Annexin V staining or TUNEL (terminal deoxynucleotidyl transferase) assay was used to detect apoptotic AT-2 cells. Histological evaluation revealed that the total number of AT-2 cells was significantly reduced at 48 h following trauma. Fas, FasL, active caspase 8, and active caspase 3 were markedly up-regulated in AT-2 cells after chest trauma. BAX and BCL-2 did not show any significant changes between sham and trauma. IL-1beta, but not TNF-alpha, levels were markedly increased at 24 h after the injury, and soluble FasL concentrations were significantly enhanced at 6, 12, 24, and 48 h after the insult. Apoptosis of AT-2 cells incubated with supernatants from cultured AMs, isolated at 48 h following chest trauma was markedly increased when compared with shams. In contrast, no apoptosis was induced in AT-2 cells incubated with supernatants of activated PMNs or BAL fluids of traumatized animals. In summary, blunt chest trauma induced apoptosis in AT-2 cells, possibly involving the extrinsic death receptor pathway. Furthermore, mediators released by AMs appeared to be involved in the induction of AT-2 cell apoptosis.     

Keratinocyte-derived chemokine plays a critical role in the induction of systemic inflammation and tissue damage after trauma-hemorrhage

Neutrophil infiltration is a crucial step in the development of organ dysfunction after trauma. We have previously shown that keratinocyte-derived chemokine (KC), a chemoattractant for neutrophils, is up-regulated after trauma-hemorrhage. To determine the role of KC after trauma-hemorrhage, the effect of a KC-neutralizing antibody on the posttraumatic inflammatory response was examined. One hour before surgery, male C3H/HeN mice were treated with an anti-KC antibody or isotype control. Animals were subjected to sham operation or trauma-hemorrhage and resuscitated with Ringer lactate thereafter. They were killed 2 h later, and Kupffer cells were isolated. Plasma levels, Kupffer cell production, and lung and liver content of TNF-alpha, IL-6, IL-10, monocyte chemoattractant protein 1, macrophage inflammatory protein 1alpha, and KC were determined by BD cytometric bead arrays. Myeloperoxidase content in lung and liver were measured as a parameter for neutrophil infiltration, and wet-to-dry weight ratios of these organs were also determined. Hepatocyte damage was assessed by measuring alpha-gluthathione S-transferase concentration. Administration of the anti-KC antibody before trauma-hemorrhage prevented increases in KC plasma levels, which was accompanied by amelioration of neutrophil infiltration and edema formation in lung and liver after trauma-hemorrhage. No effect on other cytokines in plasma or Kupffer cell release was observed. These results suggest that KC plays a pivotal role in neutrophil infiltration and organ damage after trauma-hemorrhage and resuscitation.     

Role of activated neutrophils in chest trauma-induced septic acute lung injury

More than 50% of severely injured patients have chest trauma. Second insults frequently result in acute lung injury (ALI), with sepsis being the main underlying condition. We aimed to develop a standardized, reproducible, and clinically relevant double-hit mouse model of ALI induced by chest trauma and polymicrobial sepsis and to investigate the pathophysiologic role of activated neutrophils. Lung contusion was applied to C57Bl/6 mice via a focused blast wave. Twenty-four hours later, sepsis was induced by cecal ligation and puncture. For polymorphonuclear leukocyte (PMN) depletion, animals received intravenous injections of PMN-depleting antibody. In response to blunt chest trauma followed by sepsis as well as after sepsis alone, a significant local and systemic inflammatory response with increased cytokine/chemokine levels in lung and plasma was observed. In contrast, lung apoptosis was markedly elevated only after a double hit. Intra-alveolar neutrophils and total bronchoalveolar lavage protein concentrations were markedly increased following isolated chest trauma or the combined insult, but not after sepsis alone. Lung myeloperoxidase activity was enhanced only in response to the double hit accompanied by histological disruption of the alveolar architecture, lung congestion, and marked cellular infiltrates. Neutrophil depletion significantly diminished lung interleukin 1β and interleukin 6 concentrations and reduced the degree of septic ALI. Here we have established a novel and highly reproducible mouse model of chest trauma-induced septic ALI characterizing a clinical relevant double-hit scenario. In particular, the depletion of neutrophils substantially mitigated the extent of lung injury, indicating a pathomechanistic role for neutrophils in chest trauma-induced septic ALI.     

Compartmentalization of macrophage inflammatory protein-2, but not cytokine-induced neutrophil chemoattractant, in rats challenged with intratracheal endotoxin

An important feature of the pulmonary inflammatory response is that the production of certain cytokines and chemokines is largely confined to the lung. This study investigated the local and systemic responses of macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) in rats administered with either intratracheal or intravenous lipopolysaccharide (LPS). Intratracheal LPS induced a significant increase in MIP-2 in bronchoalveolar lavage (BAL) fluid with no detectable MIP-2 in the plasma. In contrast, CINC was significantly increased in both BAL fluid and the plasma after intratracheal LPS challenge. Cell-associated MIP-2 was increased in the pulmonary-recruited neutrophils (PMNs) but not in the circulating PMNs in rats given intratracheal LPS. Cell-associated CINC was increased in both the recruited and circulating PMNs in these animals. Intravenous LPS caused a marked increase in plasma MIP-2 and CINC, whereas only a small elevation of both MIP-2 and CINC concentrations in BAL fluid was observed. The lack of CINC compartmentalization compared to MIP-2 implies that these C-X-C chemokines are regulated differentially and may have different effects upon polymorphonuclear leukocyte (PMN) recruitment into the alveolar space in response to intrapulmonary LPS or bacterial challenge.     

Neutrophil modulation of the pulmonary chemokine response to lipopolysaccharide

When cells within the intrapulmonary compartment are exposed to pathogens or their products such as lipopolysaccharide, they produce CXC chemokines in order to attract circulating neutrophils into the lower respiratory tract. Previous studies have shown that as neutrophils (PMNs) enter the lung, bronchoalveolar lavage (BAL) chemokine levels are decreased. In this study, we determined the intrapulmonary and systemic responses to two important rat chemokines, cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 (MIP-2), to intratracheal (i.t.) LPS (100 microg in 0.5 mL of phosphate-buffered saline) under neutropenic (cyclophosphamide [CPA]) and neutrophilic (G-CSF) conditions. By 4 h after i.t. LPS, CPA pretreatment decreased PMN recruitment 83% and G-CSF increased PMN recruitment 91% compared with recruitment into the lung in vehicle-pretreated rats (42.7 +/- 19.3 million PMNs). Neutropenic rats had increased CINC and MIP-2 concentrations in BAL fluid 4 h after i.t. LPS when compared with levels seen in vehicle controls (P < 0.05). In vitro LPS-stimulated chemokine production by alveolar macrophages obtained from CPA- and vehicle-pretreated animals did not differ. The increase in BAL fluid chemokine levels in neutropenic rats corresponded to increased chemotaxis of neutrophils to BAL fluid from CPA-pretreated rats as compared with the chemotaxis response of PMN to BAL fluid from vehicle-pretreated rats. In contrast, G-CSF enhancement of neutrophil recruitment decreased chemotactic activity of BAL fluid collected 4 h after i.t. LPS. These data show that as neutrophils are recruited into the lung, they alter chemokine levels, which most likely serves to down-regulate the inflammatory response.     

Alveolar macrophage phagocytosis is enhanced after blunt chest trauma and alters the posttraumatic mediator release

Blunt chest trauma is known to induce a pulmonary invasion of short-lived polymorphonuclear neutrophils and apoptosis of alveolar epithelial type 2 (AT2) cells. Apoptotic cells are removed by alveolar macrophages (AMΦ). We hypothesized that chest trauma alters the phagocytic response of AMΦ as well as the mediator release of AMΦ during phagocytosis. To study this, male Sprague-Dawley rats were subjected to blunt chest trauma. Phagocytosis assays were performed in AMΦ isolated 2 or 24 h after trauma with apoptotic cells or opsonized beads. Phagocytosis of apoptotic AT2 cells by unstimulated AMΦ was significantly increased 2 h after trauma. At 24 h, AMΦ from traumatized animals, stimulated with phorbol-12-myristate-13-acetate, ingested significantly more apoptotic polymorphonuclear neutrophils than AMΦ from sham animals. Alveolar macrophages after trauma released significantly higher levels of tumor necrosis factor α, macrophage inflammatory protein 1α, and cytokine-induced neutrophil chemoattractant 1 when they incorporated latex beads, but significantly lower levels of interleukin 1β and macrophage inflammatory protein 1α when they ingested apoptotic cells. In vivo, phagocytosis of intratracheally instilled latex beads was decreased in traumatized rats. The bronchoalveolar lavage concentrations of the phagocytosis-supporting surfactant proteins A and D after blunt chest trauma were slightly decreased, whereas surfactant protein D mRNA expression in AT2 cells was significantly increased after 2 h. These findings indicate that chest trauma augments the phagocytosis of apoptotic cells by AMΦ. Phagocytosis of opsonized beads enhances and ingestion of apoptotic cells downregulates the immunologic response following lung contusion. Our data emphasize the important role of phagocytosis during posttraumatic inflammation after lung contusion.     

Interleukin-1-dependent sequential chemokine expression and inflammatory cell infiltration in ischemia-reperfusion injury

OBJECTIVE: Ischemia-reperfusion injury is known to cause organ failure, but the mechanisms of pathogenesis remain unclear. Inflammation is a factor in tissue destruction in ischemia reperfusion injury, and interleukin (IL)-1 is a key promoter of inflammation. DESIGN: Prospective, randomized, and controlled study. SETTING: University laboratory. SUBJECTS: Male mice 6-8 wks of age, in which genes for IL-1alpha and IL-1beta (IL-1alpha/beta deficient) and IL-1 receptor antagonist (IL-1RA deficient) are deleted by homologous recombination, and wild-type controls on a Balb/c background. INTERVENTIONS: In this study, the role of IL-1 on inflammatory cascades, including chemokine expression, inflammatory cell infiltration, and tissue destruction, was investigated in 45 mins of unilateral renal ischemic injury using IL-1alpha/beta-deficient mice and IL-1RA-deficient mice. In addition, the effects of IL-1 on chemokine expression in cultured tubular epithelial cells were investigated. MEASUREMENTS AND MAIN RESULTS: In vivo study revealed that the number of interstitial infiltrated neutrophils and macrophages, which accompanied the increase of the serum levels of keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-1alpha, respectively, significantly increased in IL-1RA-deficient mice. The number of interstitial infiltrated neutrophils correlated well with serum levels of KC at 24 hrs after reperfusion, whereas the number of interstitial infiltrated macrophages correlated well with the serum levels of MIP-1alpha and monocyte chemoattractant protein (MCP)-1 at 24 and 48 hrs after reperfusion, respectively. Likewise, in vitro study revealed that stimulation of tubular epithelial cells by IL-1beta and/or H2O2 sequentially induced KC, MIP-1alpha, and MCP-1 in both protein and messenger RNA levels, which is consistent with in vivo results. CONCLUSION: IL-1-dependent inflammatory cascades, followed by inflammatory cell infiltration and subsequent tissue destruction, may affect pathogenesis of renal ischemia-reperfusion injury.     

The evolution of isolated bilateral lung contusion from blunt chest trauma in rats: cellular and cytokine responses

Lung contusion is the leading cause of death from blunt thoracic trauma in adults, but its mechanistic pathophysiology remains unclear. This study uses a recently developed rat model to investigate the evolution of inflammation and injury in isolated lung contusion. Bilateral lung contusion with minimal cardiac trauma was induced in 54 anesthetized rats by dropping a 0.3-kg hollow cylindrical weight onto a precordial shield (impact energy, 2.45 Joules). Arterial oxygenation, pressure-volume (P-V) mechanics, histology, and levels of erythrocytes, leukocytes, albumin, and inflammatory mediators in bronchoalveolar lavage (BAL) were assessed at 8 min, at 4, 12, 24, and 48 h, and at 7 days after injury. The role of neutrophils in the evolution of inflammatory injury was also specifically studied by depleting these cells with intravenous vinblastine before lung contusion. Arterial oxygenation was severely reduced at 8 min to 24 h postcontusion, but became almost normal by 48 h. Levels of erythrocytes, leukocytes, and albumin in BAL were increased at 相似文献   

Molecular hydrogen ameliorates lipopolysaccharide-induced acute lung injury in mice through reducing inflammation and apoptosis

Acute lung injury (ALI) is still a leading cause of morbidity and mortality in critically ill patients. Recently, our and other studies have found that hydrogen gas (H?) treatment can ameliorate the lung injury induced by sepsis, ventilator, hyperoxia, and ischemia-reperfusion. However, the molecular mechanisms by which H? ameliorates lung injury remain unclear. In the current study, we investigated whether H? or hydrogen-rich saline (HS) could exert protective effects in a mouse model of ALI induced by intratracheal administration of lipopolysaccharide (LPS) via inhibiting the nuclear factor κB (NF-κB) signaling pathway-mediated inflammation and apoptosis. Two percent of H? was inhaled for 1 h beginning at 1 and 6 h after LPS administration, respectively. We found that LPS-challenged mice exhibited significant lung injury characterized by the deterioration of histopathology and histologic scores, wet-to-dry weight ratio, and oxygenation index (PaO?/FIO?), as well as total protein in the bronchoalveolar lavage fluid (BALF), which was attenuated by H? treatment. Hydrogen gas treatment inhibited LPS-induced pulmonary early and late NF-κB activation. Moreover, H? treatment dramatically prevented the LPS-induced pulmonary cell apoptosis in LPS-challenged mice, as reflected by the decrease in TUNEL (deoxynucleotidyl transferase dUTP nick end labeling) staining-positive cells and caspase 3 activity. Furthermore, H? treatment markedly attenuated LPS-induced lung neutrophil recruitment and inflammation, as evidenced by downregulation of lung myeloperoxidase activity, total cells, and polymorphonuclear neutrophils in BALF, as well as proinflammatory cytokines (tumor necrosis factor α, interleukin 1β, interleukin 6, and high-mobility group box 1) and chemokines (keratinocyte-derived chemokine, macrophage inflammatory protein [MIP] 1α, MIP-2, and monocyte chemoattractant protein 1) in BALF. In addition, i.p. injection of 10 mL/kg hydrogen-rich saline also significantly attenuated the LPS-induced ALI. Collectively, these results demonstrate that molecular hydrogen treatment ameliorates LPS-induced ALI through reducing lung inflammation and apoptosis, which may be associated with the decreased NF-κB activity. Hydrogen gas may be useful as a novel therapy to treat ALI. munosorbent assay; H?-hydrogen gas; HMGB1-high-mobility group box 1; HS-hydrogen-rich saline; i.t.-intratracheal; KC-keratinocyte-derived chemokine; LPS-lipopolysaccharide; MCP-1-monocyte chemoattractant protein 1; MIP-1α-macrophage inflammatory protein 1α; MIP-2-macrophage inflammatory protein 2; MPO-myeloperoxidase; PBS-phosphate-buffered saline; PMNs-polymorphonuclear neutrophils; TUNEL-deoxynucleotidyl transferase dUTP nick end labeling; W/D-wet-to-dry.     

Differential effects of macrophage inflammatory chemokine-2 and keratinocyte-derived chemokine on hemorrhage-induced neutrophil priming for lung inflammation: assessment by adoptive cells transfer in mice

Prior studies have shown that hemorrhage (Hem) can serve as a priming stimulus for acute lung injury (ALI) triggered by subsequent septic challenge (cecal ligation and puncture, CLP). Furthermore, we have reported that in vivo antibody neutralization of the chemokines, macrophage inflammatory chemokine-2 (MIP-2) and keratinocyte-derived chemokine (KC), immediately after Hem appears to differentially effect the onset of ALI. However, although we hypothesize that this is due to divergent effects of MIP-2 and KC on Hem-induced neutrophil (PMN) priming, this has not been tested. To examine this hypothesis, PMN donor mice were Sham-Hem or Hem for 90 min at 35 +/- 5 mmHg and were then administered anti-MIP- 2 (Hem/anti-MIP2), anti-KC (Hem/anti-KC), or nonspecific immunoglobulin (Ig) G (Hem/IgG) during resuscitation (Ringer's lactate = four times the amount of drawn blood volume). Twenty-four hours post-Hem, the peripheral blood PMN were purified from these donor animals and were introduced into PMN-depleted recipient mice [depleted by prior anti-Gr1 (mouse PMN-specific marker) antibody treatment]. One hour after PMN transfer, recipient mice were subjected to CLP, euthanized 24 h later, and plasma as well as lung tissue samples were collected. PMN influx was assessed by myeloperoxidase assay (MPO; microU/mg protein) and histologically (IL-6, MIP-2, KC, and IL-10 levels) by enzyme-linked immunoabsorbant assay (ELISA; ng/mg). The results show that donor PMN from Hem/IgG but not Sham-Hem mice produce increased PMN influx (increased MPO, increased % esterase+ cells in tissue) into the lung and local tissue inflammation (increased IL-6/MIP-2, decreased IL-10) in PMN-depleted CLP recipient mice, which was attenuated in mice receiving cells from Hem/anti-MIP-2 but not Hem/anti-KC treated donors. Interestingly, although Hem/anti-MIP-2 donor PMN produced comparable effects on blood IL-6/MIP-2 levels, they were ineffective in altering the change in plasma IL-10/KC levels induce by Hem. Taken together, these data demonstrate that Hem-induced priming of PMN not only mediates ALI in the mouse, but also that this process is differentially effected by MIP2 and KC, despite the fact that both signal through CXCR2.     

The pulmonary and hepatic immune microenvironment and its contribution to the early systemic inflammation following blunt chest trauma

OBJECTIVE: Blunt chest trauma is accompanied by an early increase in plasma cytokine concentrations. However, the local sources of these mediators are poorly defined. We investigated the impact of blunt chest trauma on the inflammatory mediator milieu in different compartments (lung tissue, bronchoalveolar lavage, liver tissue, Kupffer cells, plasma) along with the time course of trauma-induced pulmonary endothelial barrier dysfunction to elucidate potential relationships. In addition, the correlation between intratracheally instilled interleukin-6 and its systemic release were studied. DESIGN: Prospective, randomized, controlled animal study. SETTING: Basic science laboratory of a university affiliated level 1 trauma center. SUBJECTS: Male C3H/HeN mice, 8-9 wks old, n = 141. INTERVENTIONS: Blunt chest trauma induced by a focused blast wave, intravenous injection of Evans blue, and intratracheal instillation of recombinant human interleukin-6. MEASUREMENTS AND MAIN RESULTS: Two hours after blunt chest trauma, plasma interleukin-6 was markedly increased. Simultaneously, interleukin-6, tumor necrosis factor-alpha, macrophage inflammatory protein-2, monocyte chemotactic polypeptide-1 and neutrophil/monocyte accumulation in bronchoalveolar lavage and interleukin-6, monocyte chemotactic polypeptide-1, and myeloperoxidase activity in lung tissue were significantly increased. This was accompanied by a coinciding elevation in the Evans blue lung-plasma ratio. Recombinant human interleukin-6, instilled intratracheally before blunt chest trauma, was detected in a dose-dependent manner in the plasma of the mice. Additionally, Kupffer cell interleukin-6, tumor necrosis factor-alpha, and interleukin-10 production was significantly augmented as early as 30 mins after the insult. CONCLUSIONS: These results indicate that early increased cytokine concentrations in the lung, particularly interleukin-6, are important mediator sources as their local peak coincides with the systemic inflammatory response and is accompanied by a simultaneous impaired function of the pulmonary endothelial barrier. A direct relationship between their local and systemic concentrations can be established. Furthermore, this is the first study to show that Kupffer cells are activated early after blunt chest trauma.     

Direct effects of protein S in ameliorating acute lung injury

Summary. Objective: Protein S may exert an anticoagulant activity by enhancing the anticoagulant activity of activated protein C and/or by directly inhibiting the prothrombinase complex. Protein S itself may also directly regulate inflammatory responses and apoptosis. The role of protein S in acute lung injury (ALI) was unknown. This study evaluated the effect of protein S on ALI in the mouse. Methods: Animal ALI was induced in C57/BL6 mice by intratracheal instillation of lipopolysaccharide (LPS). Mice were treated with protein S or saline by intraperitoneal injection 1 h before LPS instillation. Results: Activated protein or protein S alone and combined activated protein C + protein S therapy decreased inflammatory markers and cytokines in mice with acute lung injury. In LPS‐treated mice compared with controls ALI was induced as shown by significantly increased levels of total protein, tumor necrosis factor‐α, interleukin‐6 and monocyte chemoattractant protein‐1 in the bronchoalveolar lavage fluid. Mice with ALI treated with protein S had significantly decreased concentrations of tumor necrosis factor‐α and interleukin‐6 in the lung compared with untreated animals. Thrombin‐antithrombin III, a marker of the activity of the coagulation cascade, was unchanged. Protein S inhibited the expression of cytokines in vitro and increased activation of the Axl tyrosine kinase pathway in A549 epithelial cells. Conclusion: Protein S protects against LPS‐induced ALI, possibly by directly inhibiting the local expression of inflammatory cytokines without affecting coagulation.     

Inhaled hydrogen sulfide induces suspended animation, but does not alter the inflammatory response after blunt chest trauma

The treatment of acute lung injury and septic complications after blunt chest trauma remains a challenge. Inhaled hydrogen sulfide (H?S) may cause a hibernation-like metabolic state, which refers to an attenuated systemic inflammatory response. Therefore, we tested the hypothesis that inhaled H?S-induced suspended animation may attenuate the inflammation after pulmonary contusion. Male Sprague-Dawley rats were subjected to blunt chest trauma (blast wave) or sham procedure and subsequently exposed to a continuous flow of H?S (100 ppm) or control gas for 6 h. Body temperature and activity were measured by an implanted transmitter. At 6, 24, or 48 h after trauma, animals were killed, and the cellular contents of bronchoalveolar lavage (BAL) as well as cytokine concentrations in BAL, plasma, and culture supernatants of blood mononuclear cells, Kupffer cells, splenic macrophages, and splenocytes were determined. Hydrogen sulfide inhalation caused a significant reduction in body temperature and activity. The trauma-induced increase in alveolar macrophage counts was abrogated 48 h after trauma when animals received H?S, whereas the trauma-induced increase in neutrophil counts was unaltered. Furthermore, H?S inhalation partially attenuated the mediator release in BAL and culture supernatants of Kupffer cells as well as splenic cells; it altered plasma cytokine concentrations but did not affect the trauma-induced changes in mononuclear cell culture supernatants. These findings indicate that inhaled H?S induced a reduced metabolic expenditure and partially attenuated inflammation after trauma. Nevertheless, in contrast to hypoxic- or pathogen-induced lung injury, H?S treatment appears to have no protective effect after blunt chest trauma.     

Protective effects of aerobic exercise on acute lung injury induced by LPS in mice

Introduction

The regular practice of physical exercise has been associated with beneficial effects on various pulmonary conditions. We investigated the mechanisms involved in the protective effect of exercise in a model of lipopolysaccharide (LPS)-induced acute lung injury (ALI).

Methods

Mice were divided into four groups: Control (CTR), Exercise (Exe), LPS, and Exercise + LPS (Exe + LPS). Exercised mice were trained using low intensity daily exercise for five weeks. LPS and Exe + LPS mice received 200 µg of LPS intratracheally 48 hours after the last physical test. We measured exhaled nitric oxide (eNO); respiratory mechanics; neutrophil density in lung tissue; protein leakage; bronchoalveolar lavage fluid (BALF) cell counts; cytokine levels in BALF, plasma and lung tissue; antioxidant activity in lung tissue; and tissue expression of glucocorticoid receptors (Gre).

Results

LPS instillation resulted in increased eNO, neutrophils in BALF and tissue, pulmonary resistance and elastance, protein leakage, TNF-alpha in lung tissue, plasma levels of IL-6 and IL-10, and IL-1beta, IL-6 and KC levels in BALF compared to CTR (P ≤0.02). Aerobic exercise resulted in decreases in eNO levels, neutrophil density and TNF-alpha expression in lung tissue, pulmonary resistance and elastance, and increased the levels of IL-6, IL-10, superoxide dismutase (SOD-2) and Gre in lung tissue and IL-1beta in BALF compared to the LPS group (P ≤0.04).

Conclusions

Aerobic exercise plays important roles in protecting the lungs from the inflammatory effects of LPS-induced ALI. The effects of exercise are mainly mediated by the expression of anti-inflammatory cytokines and antioxidants, suggesting that exercise can modulate the inflammatory-anti-inflammatory and the oxidative-antioxidative balance in the early phase of ALI.     

Infiltration of COX-2-expressing macrophages is a prerequisite for IL-1 beta-induced neovascularization and tumor growth

Inflammatory angiogenesis is a critical process in tumor progression and other diseases. The inflammatory cytokine IL-1beta promotes angiogenesis, tumor growth, and metastasis, but its mechanisms remain unclear. We examined the association between IL-1beta-induced angiogenesis and cell inflammation. IL-1beta induced neovascularization in the mouse cornea at rates comparable to those of VEGF. Neutrophil infiltration occurred on day 2. Macrophage infiltration occurred on days 4 and 6. The anti-Gr-1 Ab-induced depletion of infiltrating neutrophils did not affect IL-1beta- or VEGF-induced angiogenesis. The former was reduced in monocyte chemoattractant protein-1-deficient (MCP-1(-/-)) mice compared with wild-type mice. After day 4, clodronate liposomes, which kill macrophages, reduced IL-1beta-induced angiogenesis and partially inhibited VEGF-induced angiogenesis. Infiltrating macrophages near the IL-1beta-induced neovasculature were COX-2 positive. Lewis lung carcinoma cells expressing IL-1beta (LLC/IL-1beta) developed neovasculature with macrophage infiltration and enhanced tumor growth in wild-type but not MCP-1(-/-) mice. A COX-2 inhibitor reduced tumor growth, angiogenesis, and macrophage infiltration in LLC/IL-1beta. Thus, macrophage involvement might be a prerequisite for IL-1beta-induced neovascularization and tumor progression.     

Neutrophil chemotactic activity produced by normal and activated human bronchoalveolar lavage cells

Activated macrophages can secrete a number of mediators that can attract inflammatory cells and enhance secretion of phlogistic substances from these cells. The ultimate effect of activated bronchoalveolar lavage (BAL) cells may be fibrotic lung injury. Inasmuch as pulmonary sarcoidosis is a disease associated with spontaneous activation of macrophages and lymphocytes among BAL cells, cells obtained from patients with sarcoidosis were compared with normal cells. We report that adherent BAL cells in culture from patients with sarcoidosis (n = 21) release during a resting period in vitro more chemotactic activity for neutrophils (PMNs) than do BAL cells from normal individuals (n = 14). After density fractionation of the respiratory cells by albumin gradient, cells from high-density fractions in the group with sarcoidosis secrete more chemotactic activity for neutrophils than cells from less dense fractions. The PMN chemotactic activity spontaneously released in vitro by BAL cells from patients with sarcoidosis correlates with the percentage of PMNs recovered by BAL. Immunochemical bioassay and high-performance liquid chromatographic (HPLC) analysis of BAL cell supernatants revealed a complex pattern of chemotactic factors to be present. Generally, three peaks of chemotactic activity were noted on HPLC 1-60 separations at greater than 20 kd, 8 to 10 kd, and less than 1 kd apparent molecular weights. Significantly, interleukin-1 was present in these supernatants, whereas complement components and leukotriene B4 were absent. Sarcoid BAL cells, principally alveolar macrophages, are activated in vivo as manifested by spontaneous secretion of chemotactic factors for PMNs in vitro. Interleukin-1 and other less well characterized molecules were detected. The presence of PMNs among the lavage cells of some patients with sarcoidosis appears to be an in vivo biologic correlate of this activation. These data provide additional criteria of BAL cell activation in patients with pulmonary sarcoidosis and provide further evidence concerning factors that attract inflammatory cells into the lung.     

神经降压素对内毒素诱导的急性肺损伤的保护作用及机制

目的探讨神经降压素在内毒素（LPS）所诱导的小鼠急性肺损伤（ALI）中的保护作用,并明确其在炎症反应中的作用。 方法100只健康无特异病原级性C57BL/6小鼠（雄性50只,雌性50只）,体质量（20~25 g）,由上海南方模式实验动物中心提供。所有小鼠随机分为以下5组：①正常对照组：60 μl无菌磷酸缓冲盐溶液（PBS）滴鼻处理小鼠;②急性肺损伤模型组：60 μg/只LPS滴鼻处理;③20 mg/kg神经降压素组：先使用LPS诱导肺损伤,诱导后1 h通过尾静脉注射的方式给予20 mg/kg神经降压素处理;④40 mg/kg神经降压素给药组：先使用LPS诱导肺损伤,诱导后1 h通过尾静脉注射的方式给予40 mg/kg神经降压素处理;⑤80 mg/kg神经降压素给药组：先使用LPS诱导肺损伤,诱导后1 h通过尾静脉注射的方式给予80 mg/kg神经降压素处理。每组20只。肺损伤诱导后24 h,检测不同处理组小鼠肺损伤程度、髓过氧化物酶（MPO）活性、炎症细胞的浸润、肺水肿程度以及肺泡灌洗液中促炎症细胞的分泌水平。 结果与正常对照组相比,LPS的滴鼻处理显著提高了小鼠肺组织的损伤程度,包括MPO活性、炎症细胞的浸润、肺泡灌洗液内促炎症细胞因子［肿瘤坏死因子（TNF-α）、白介素（IL）-6、IL-1b以及单核细胞趋化因子（MCP）-1］的分泌等（P<0.05）;与内毒素诱导的急性肺损伤模型组相比,神经降压素的给药处理明显减轻了小鼠肺组织损伤的程度,包括MPO活性、炎症细胞的浸润、肺泡灌洗液内促炎症细胞因子（TNF-α、IL-6、IL-1b以及MCP-1）的分泌等（P<0.05）,且这种保护性作用呈现剂量依赖性。 结论神经降压素能够有效减轻由内毒素诱导的ALI,其机制可能是通过封闭由速激肽所介导的炎症反应信号通路,进而减轻内毒素引起的炎症反应对肺组织所造成的炎症损伤来实现。     

Polyclonal antibody directed against human RANTES ameliorates disease in the Lewis rat adjuvant-induced arthritis model.

Adjuvant-induced arthritis (AIA) is one of many animal models of rheumatoid arthritis, a disease characterized by a T-lymphocyte and macrophage cellular infiltrate. We have characterized the development of this disease model with respect to chemokine expression. Increased levels of two chemokines, RANTES, a T-lymphocyte and monocyte chemo-attractant, and KC a chemoattractant for neutrophils, were found in whole blood and in the joint. Surprisingly, levels of MIP-1alpha, another T-lymphocyte and monocyte chemoattractant, were unchanged throughout the course of the disease in whole blood and only slightly elevated in the joint. RANTES expression plays an important role in the disease since a polyclonal antibody to RANTES greatly ameliorated symptoms in animals induced for AIA and was found to be as efficacious as treatment with indomethacin, a non-steroidal anti inflammatory. Polyclonal antibodies to either MIP-1alpha or KC were ineffective. This is the first report to show the importance of RANTES in the development of AIA.     

CCR2b和CCR1拮抗剂RS504393对LPS诱导小鼠急性肺损伤的保护作用

目的 探讨CC趋化因子受体(CCR)2b和CCR1的特异性拈抗剂RS504393在细菌脂多糖(LPS)诱导急性肺损伤中的作用及其机制.方法 体外实验选用A549细胞系,应用LPS(10 μg/mL)刺激合并RS504393(10 μg/mL)治疗6 h后,流式细胞仪技术检测CCR1和CCR2的表达,ELISA法检测细胞培养上清液内白介素(IL)-8的浓度;体内实验选用C57BL/6J小鼠,腹腔注射RS504393(5 mg/kg)预处理30 min后经鼻滴入LPS(5 mg/kg),LPS刺激4 h后收集BALF和肺脏标本,计数BALF内细胞总数,应用BCA法检测BALF内蛋白浓度,Realtime-RT PCR和ELISA法检测BALF和肺脏IL-1β、凝血酶原激活物抑制物(PAI)-1和单核细胞趋化蛋白(MCP)-2的表达.多组间差别比较采用单因素方差分析(One-way ANOVA procedures),两组间差别比较采用成组t检验,以P<0.05为差异具有统计学意义.结果 A549细胞经LPS刺激后与对照组相比,CCR1、CCR2和IL-8的表达明显升高,RS504393治疗可明显降低它们的表达.在LPS诱导肺损伤模型中,与对照组相比,BALF内细胞总数和蛋白浓度明显升高,BALF和肺脏内IL-1β、PAI-1的mRNA和蛋白表达显著增加,经RS504393治疗后均显著下降;LPS刺激使BALF内MCP-2升高,RS504393治疗使其进一步增加.结论 CCR2和CCR1在急性肺损伤的发病中充当着重要角色,应用CCR2b和CCR1的拈抗剂RS504393治疗急性肺损伤有一定的应用前景.