Pulmonary and extrapulmonary acute lung injury: inflammatory and ultrastructural analyses.

To test whether pulmonary and extrapulmonary acute lung injury (ALI) of identical mechanical compromise would express diverse morphological patterns and immunological pathways. For this purpose, a model of pulmonary (p) and extrapulmonary (exp) ALI with similar functional changes was developed and pulmonary morphology (light and electron microscopy), cytokines levels, and neutrophilic infiltration in the bronchoalveolar lavage fluid (BALF), elastic and collagen fiber content in the alveolar septa, and neutrophil apoptosis in the lung parenchyma were analyzed. BALB/c mice were divided into four groups. In control groups, saline was intratracheally (it, 0.05 ml) instilled and intraperitoneally (ip, 0.5 ml) injected, respectively. In the ALIp and ALIexp groups, mice received E. coli lipopolysaccharide (10 microg it and 125 microg ip, respectively). The changes in lung resistive and viscoelastic pressures and in static elastance, alveolar collapse, and cell content in lung tissue were similar in the ALIp and ALIexp groups. The ALIp group presented a threefold increase in KC (murine function homolog to IL-8) and IL-10 levels in the BALF in relation to ALIexp, whereas IL-6 level showed a twofold increase in ALIp. Neutrophils in the BALF were more frequent in ALIp than in ALIexp. ALIp showed more extensive injury of alveolar epithelium, intact capillary endothelium, and apoptotic neutrophils, whereas the ALIexp group presented interstitial edema and intact type I and II cells and endothelial layer. In conclusion, given the same pulmonary mechanical dysfunction independently of the etiology of ALI, insult in pulmonary epithelium yielded more pronounced inflammatory responses, which induce ultrastructural morphological changes.     

Pulmonary angiotensin-converting enzyme kinetics after acute lung injury in the rabbit

Depression of lung endothelial cell metabolic function may be an early and sensitive indicator of lung damage. When such functions are measured in vivo, substrates injected usually must be limited to "trace" doses due to the significant hemodynamic effects of high doses of substrate. Under first-order conditions (i.e., trace doses) the enzyme or transport system rate constant Vmax/Km may be calculated, but independent estimates of each variable (Vmax and Km) are not available. We therefore used multiple indicator-dilution methods and higher substrate concentrations to apply a mathematical model, based on saturable kinetics that yield independent estimates of the apparent kinetic parameters Vmax and Km for pulmonary angiotensin-converting enzyme (ACE). We used the ACE substrate, [3H]benzoyl-phenylalanyl-alanyl-proline ([3H]BPAP) and made these measurements and also estimates of serotonin [5-hydroxytryptamine (5-HT)] removal, before and after acute lung injury induced by intratracheal administration of phorbol myristate acetate (PMA). PMA significantly depressed the percent 5-HT removal (62 +/- 3 to 44 +/- 4%) and BPAP percent metabolism (74 +/- 2 to 66 +/- 2), when trace amounts of either compound were injected as a bolus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immature lung and acute lung injury

Acute lung injury occurs mostly in the very low birth weight and extremely low birth weight infants. The pathological process leading to acute lung injury includes immature and/or diseased lung that experienced oxidative stress, inflammation and mechanical insult with the bronchial, alveolar and capillary injuries and cell death. It may be the first step to the subsequent development of chronic lung disease of prematurity or bronchopulmonary dysplasia. The mechanisms of lung injury are extensively investigated in the experimental models and clinical studies, mostly performed on the adult patients. At present, the explanations of the mechanism(s) leading to lung tissue injury in tiny premature babies are just derived from these studies. Acute lung injury seems to be rather a syndrome than a well-defined nosological unit and is of multifactorial etiology. The purpose of this review is to discuss the main factors contributing to the development of acute lung injury in the very low or extremely low birth weight infants--lung immaturity, mechanical injury, oxidative stress and inflammation. Nevertheless, numerous other factors may influence the status of immature lung after delivery.     

Pulmonary clearance of radiotracers after positive end-expiratory pressure or acute lung injury

In anesthetized rabbits we measured clearance from lung to blood of eight aerosolized technetium-99m-labeled compounds: diethylenetriaminepentaacetate (99mTc-DTPA); cytochrome c; myoglobin; a myoglobin polymer; albumin; and anionic, cationic, and neutral dextrans of equivalent molecular size. We investigated the effect of applying positive end-expiratory pressure (PEEP) and, on a subsequent occasion, of injecting oleic acid intravenously to produce acute lung injury on the pulmonary clearance rate. Base-line clearance rates were monoexponential and varied with the molecular weights of the radiotracers. For each tracer the rate of clearance was increased a similar degree by either PEEP or oleic acid. However, with PEEP, clearance remained monoexponential, whereas after oleic acid, smaller molecular-weight radiotracers had multiexponential clearance curves. This suggests that after oleic acid the alveolar epithelium breaks down in a nonuniform fashion. We conclude that differentiation of the effect of PEEP from that of severe lung injury caused by oleic acid is not readily accomplished by either increasing the size of the tracer molecule or by varying the molecular charge.     

Normal tissue complication probability models for severe acute radiological lung injury after radiotherapy for lung cancer

PurposeTo derive Normal Tissue Complication Probability (NTCP) models for severe patterns of early radiological radiation-induced lung injury (RRLI) in patients treated with radiotherapy (RT) for lung tumors. Second, derive threshold doses and optimal doses for prediction of RRLI to be used in differential diagnosis of tumor recurrence from RRLI during follow-up.Methods and materialsLyman-EUD (LEUD), Logit-EUD (LogEUD), relative seriality (RS) and critical volume (CV) NTCP models, with DVH corrected for fraction size, were used to model the presence of severe early RRLI in follow-up CTs. The models parameters, including α/β, were determined by fitting data from forty-five patients treated with IMRT for lung cancer. Models were assessed using Akaike information criterion (AIC) and area under receiver operating characteristic curve (AUC). Threshold doses for risk of RRLI and doses corresponding to the optimal point of the receiver operating characteristic (ROC) curve were determined.ResultsThe α/βs obtained with different models were 2.7–3.2 Gy. The thresholds and optimal doses curves were EUDs of 3.2–7.8 Gy and 15.2–18.1 Gy with LEUD, LogEUD and RS models, and μ_d of 0.013 and 0.071 with the CV model. NTCP models had AUCs significantly higher than 0.5. Occurrence and severity of RRLI were correlated with patients’ values of EUD and μ_d.ConclusionsThe models and dose levels derived can be used in differential diagnosis of tumor recurrence from RRLI in patients treated with RT. Cross validation is needed to prove prediction performance of the model outside the dataset from which it was derived.     

Pulmonary stromal-derived factor-1 expression and effect on neutrophil recruitment during acute lung injury

The severe and protracted inflammation that characterizes acute lung injury (ALI) is driven by the ongoing recruitment of neutrophils to the lung. Although much of the cytokine signaling responsible for the initial phase of ALI has been elaborated, relatively little is known about the mechanisms governing the recruitment of neutrophils from the bone marrow to the lung in the later period of this disease. Given its previously described chemoattractant effects on marrow neutrophils, we investigated whether stromal-derived factor-1 (SDF-1) (CXCL12) might participate in this later phase of recruitment. Using immunohistochemistry to examine both banked human lung specimens from patients with ALI and lungs from mice with LPS-induced pneumonitis, we found that pulmonary SDF-1 expression increases during ALI. We further determined that both lung SDF-1 protein expression and mRNA expression rise in a delayed but sustained pattern in this mouse model and that the major source of the increase in expression appears to be the lung epithelium. Lastly, we found that expression of the SDF-1 receptor CXCR4 rises in a similar temporal pattern on neutrophils in both the blood and airspace of LPS-injured mice and that Ab-mediated SDF-1 blockade significantly attenuates late but not early pulmonary neutrophilia in this model. These results implicate SDF-1 in neutrophil recruitment to the lung in the later period of acute lung injury and suggest a novel role for this cytokine in coordinating the transition from the inflammatory response to the initiation of tissue repair.     

Microparticles and acute lung injury

The pathophysiology of acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), is characterized by increased vascular and epithelial permeability, hypercoagulation and hypofibrinolysis, inflammation, and immune modulation. These detrimental changes are orchestrated by cross talk between a complex network of cells, mediators, and signaling pathways. A rapidly growing number of studies have reported the appearance of distinct populations of microparticles (MPs) in both the vascular and alveolar compartments in animal models of ALI/ARDS or respective patient populations, where they may serve as diagnostic and prognostic biomarkers. MPs are small cytosolic vesicles with an intact lipid bilayer that can be released by a variety of vascular, parenchymal, or blood cells and that contain membrane and cytosolic proteins, organelles, lipids, and RNA supplied from and characteristic for their respective parental cells. Owing to this endowment, MPs can effectively interact with other cell types via fusion, receptor-mediated interaction, uptake, or mediator release, thereby acting as intrinsic stimulators, modulators, or even attenuators in a variety of disease processes. This review summarizes current knowledge on the formation and potential functional role of different MPs in inflammatory diseases with a specific focus on ALI/ARDS. ALI has been associated with the formation of MPs from such diverse cellular origins as platelets, neutrophils, monocytes, lymphocytes, red blood cells, and endothelial and epithelial cells. Because of their considerable heterogeneity in terms of origin and functional properties, MPs may contribute via both harmful and beneficial effects to the characteristic pathological features of ALI/ARDS. A better understanding of the formation, function, and relevance of MPs may give rise to new promising therapeutic strategies to modulate coagulation, inflammation, endothelial function, and permeability either through removal or inhibition of "detrimental" MPs or through administration or stimulation of "favorable" MPs.     

Volume loading reduces pulmonary vascular resistance in ventilated animals with acute lung injury: evaluation of RV afterload

During mechanical ventilation, increased pulmonary vascular resistance (PVR) may decrease right ventricular (RV) performance. We hypothesized that volume loading, by reducing PVR, and, therefore, RV afterload, can limit this effect. Deep anesthesia was induced in 16 mongrel dogs (8 oleic acid-induced acute lung injury and 8 controls). We measured ventricular pressures, dimensions, and stroke volumes during positive end-expiratory pressures of 0, 6, 12, and 18 cmH(2)O at three left ventricular (LV) end-diastolic pressures (5, 12, and 18 mmHg). Oleic acid infusion (0.07 ml/kg) increased PVR and reduced respiratory system compliance (P < 0.05). With positive end-expiratory pressure, PVR was greater at a lower LV end-diastolic pressure. Increased PVR was associated with a decreased transseptal pressure gradient, suggesting that leftward septal shift contributed to decreased LV preload, in addition to that caused by external constraint. Volume loading reduced PVR; this was associated with improved RV output and an increased transseptal pressure gradient, which suggests that rightward septal shift contributed to the increased LV preload. If PVR is used to reflect RV afterload, volume loading appeared to reduce PVR, thereby improving RV and LV performance. The improvement in cardiac output was also associated with reduced external constraint to LV filling; since calculated PVR is inversely related to cardiac output, increased LV output would reduce PVR. In conclusion, our results, which suggest that PVR is an independent determinant of cardiac performance, but is also dependent on cardiac output, improve our understanding of the hemodynamic effects of volume loading in acute lung injury.     

Intrapleural delivery of MSCs attenuates acute lung injury by paracrine/endocrine mechanism

Two different repair mechanisms of mesenchymal stem cells (MSCs) are suggested to participate in the repair of acute lung injury (ALI): (i) Cell engraftment mechanism, (ii) Paracrine/endocrine mechanism. However, the exact roles they play in the repair remain unclear. The aim of the study was to evaluate the role of paracrine/endocrine mechanism using a novel intrapleural delivery method of MSCs. Either 1 × 10⁶ MSCs in 300 μl of PBS or 300 μl PBS alone were intrapleurally injected into rats with endotoxin‐induced ALI. On days 1, 3 or 7 after injections, samples of lung tissues and bronchoalveolar lavage fluid (BALF) were collected from each rat for assessment of lung injury, biochemical analysis and histology. The distribution of MSCs was also traced by labelling the cells with 4′,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI). MSCs intrapleural injection significantly improved LPS‐induced lung histopathology compared with PBS‐treated group at day 3. There was also a significant decrease in total cell counts and protein concentration in BALF at day 7 in the MSCs ‐treated rats compared to PBS control group. Tracking the DAPI‐marked MSCs showed that there were no exotic MSCs in the lung parenchyma. MSCs administration resulted in a down‐regulation of pro‐inflammatory response to endotoxin by reducing TNF‐α both in the BALF and in the lung, while up‐regulating the anti‐inflammatory cytokine IL‐10 in the lung. In conclusion, treatment with intrapleural MSCs administration markedly attenuates the severity of endotoxin‐induced ALI. This role is mediated by paracrine/endocrine repair mechanism of MSCs rather than by the cell engraftment mechanism.     

Polyketal copolymers: a new acid-sensitive delivery vehicle for treating acute inflammatory diseases

Acute inflammatory diseases are a major cause of death in the world, and effective treatments are greatly needed. Macrophages play a central role in causing acute inflammatory diseases, and there is currently great interest in developing drug delivery vehicles that can target therapeutics to macrophages. Microparticles formulated from aliphatic polyketals have great potential to enhance the treatment of acute inflammatory diseases, due to their ability to passively target therapeutics to macrophages, their acid sensitivity, and their biocompatible degradation products. However, existing aliphatic polyketals are unsuitable for treating acute inflammatory diseases because they require weeks to hydrolyze, and strategies for accelerating their hydrolysis kinetics are greatly needed. In this report, we demonstrate that the hydrolysis kinetics of aliphatic polyketals can be accelerated by increasing their hydrophilic/hydrophobic balance. Aliphatic polyketals of varying hydrophobicity were synthesized, via the acetal exchange reaction, and their hydrolysis kinetics were investigated at the pH values of 4.5 and 7.4. A polyketal termed PK3 was developed, which had the hydrolysis kinetics suitable for treating acute inflammatory diseases. PK3 has a hydrolysis half-life of 2 days at pH 4.5, but requires several weeks to hydrolyze at pH 7.4. Microparticles were formulated with PK3, which encapsulated the anti-inflammatory drug, imatinib. In vivo experiments demonstrated that PK3 microparticles were able to significantly improve the efficacy of imatinib in treating acute liver failure. We anticipate that aliphatic polyketals will have numerous applications for the treatment of acute inflammatory diseases, given their pH sensitivity, tunable hydrolysis kinetics, and biocompatible degradation products.     

Gamma-enolase predicts lung damage in severe acute pancreatitis-induced acute lung injury

Severe acute pancreatitis (SAP) associated acute lung injury (ALI) accounts for about 70% mortality of SAP patients. However, there are no precise biomarkers for the disease currently. Herein, we evaluated the potential of gamma-enolase (ENO2), against its universal isoform alpha-enolase (ENO1), as a marker of SAP–ALI in a rat model. Firstly, 16 male Sprague–Dawley rats were randomly divided into two groups, Sham (n?=?8) and SAP–ALI (n?=?8), for pancreatitis induction. Ultra-structure examination by electron microscopy and HE staining were used for lung injury assessment. Lung tissue expressions of alpha-enolase and gamma-enolase were evaluated by qRT-PCR and immunohistochemistry. In a prospective validation experiment, 28 rats were used: sham (n?=?8), SAP–ALI at 3 h (3 h, n?=?10), and SAP–ALI at 24 h (24 h, n?=?10). Lung tissue damage, tissue expression and circulating alpha-enolase and gamma-enolase levels were evaluated. Elevated serum levels of α-amylase and TNF-α were observed in SAP rats but not in sham-operated rats. Histological examination of pancreatic and lung tissues indicated marked damage in SAP rats. While alpha-enolase was universally expressed, gamma-enolase was expressed only in damaged lung tissues. Gamma-enolase was detected in lung tissues, BALF, and serum as early as 3 h post-surgery when physical pathological damage was not apparent. Unlike alpha-enolase, secreted and/or circulating gamma-enolase level progressively increased, especially in serum, as lung damage progressed. Thus, gamma-enolase may signal and correlate lung tissue damage well before obvious physical pathological tissue damage and might be a candidate diagnostic and/or prognostic marker.     

Monokine-induced acute lung injury in rabbits

Interleukin-1 (IL-1) mediates components of the acute phase response, stimulates granulocyte metabolism, and induces endothelial cell surface changes. We studied in unanesthetized rabbits the effects of intravenous divided dose infusions of a murine monokine preparation containing IL-1 activity, on circulating granulocytes, their sequestration within the pulmonary microvasculature, pulmonary edema formation, and changes in pulmonary vascular permeability. Monokine administration induced significant (P less than 0.01) granulocytopenia as well as a significant (P less than 0.001) increase in mean alveolar septal wall granulocytes per high power field (HPF) compared with saline-injected controls. Infusions of the monokine preparation significantly (P less than 0.005) increased lung wet-to-dry weight ratios as well as significantly (P less than 0.025) increased pulmonary extravasation of radiolabeled albumin. Electron microscopic analysis of lung sections obtained from monokine-infused animals demonstrated endothelial injury, perivascular edema, and extravasation of an ultrastructural tracer. We conclude that a monokine preparation containing IL-1 activity can induce profound granulocytopenia, pulmonary leukostasis, and acute pulmonary vascular endothelial injury.     

Cytokine-mediated inflammation in acute lung injury

Clinical acute lung injury (ALI) is a major cause of acute respiratory failure in critically ill patients. There is considerable experimental and clinical evidence that pro- and anti-inflammatory cytokines play a major role in the pathogenesis of inflammatory-induced lung injury from sepsis, pneumonia, aspiration, and shock. A recent multi-center clinical trial found that a lung-protective ventilatory strategy reduces mortality by 22% in patients with ALI. Interestingly, this protective ventilatory strategy was associated with a marked reduction in the number of neutrophils and the concentration of pro-inflammatory cytokines released into the airspaces of the injured lung. Further research is needed to establish the contribution of cytokines to both the pathogenesis and resolution of ALI.     

Pulmonary lymphatics and edema accumulation after brief lung injury

In a past study of hyperoxia-induced lung injury, the extensive lymphatic filling could have resulted from lymphatic proliferation or simple lymphatic recruitment. This study sought to determine whether brief lung injury could produce similar changes, to show which lymphatic compartments fill with edema, and to compare their three-dimensional structure. Tracheostomized rats were ventilated at high tidal volume (12-16 ml) or low tidal volume (3-5 ml) or allowed to breathe spontaneously for 25 min. Light microscopy showed more perivascular, interlobular septal, and alveolar edema in the animals ventilated at high tidal volume (P < 0.0001). Scanning electron microscopy of lymphatic casts showed extensive filling of the perivascular lymphatics in the group ventilated at high tidal volume (P < 0.01), but lymphatic filling was greater in the nonventilated group than in the group that was ventilated at low tidal volume (P < 0.01). The three-dimensional structures of the cast interlobular and perivascular lymphatics were similar. There was little filling and no difference in pleural lymphatic casts among the three groups. More edema accumulated in the surrounding lymphatics of larger blood vessels than smaller blood vessels. Brief high-tidal-volume lung injury caused pulmonary edema similar to that caused by chronic hyperoxic lung injury, except it was largely restricted to perivascular and septal lymphatics and prelymphatic spaces.     

Knockdown of POLA2 increases gemcitabine resistance in lung cancer cells

Background

Gemcitabine is used as a standard drug treatment for non-small cell lung cancer (NSCLC), but treatment responses vary among patients. Our previous studies demonstrated that POLA2 + 1747 GG/GA single nucleotide polymorphism (SNP) improves differential survivability and mortality in NSCLC patients. Here, we determined the association between POLA2 and gemcitabine treatment in human lung cancer cells.

Results

Human PC9, H1299 and H1650 lung cancer cell lines were treated with 0.01-100 μM gemcitabine for 72 h. Although all 3 cell lines showed decreased cell viability upon gemcitabine treatment, H1299 was found to be the most sensitive to gemcitabine treatment. Next, sequencing was performed to determine if POLA2 + 1747 SNP might be involved in gemcitabine sensitivity. Data revealed that all 3 cell lines harbored the wild-type POLA2 + 1747 GG SNP, indicating that the POLA2 + 1747 SNP might not be responsible for gemcitabine sensitivity in the cell lines studied. Silencing of POLA2 gene in H1299 was then carried out by siRNA transfection, followed by gemcitabine treatment to determine the effect of POLA2 knockdown on chemosensitivity to gemcitabine. Results showed that H1299 exhibited increased resistance to gemcitabine after POLA2 knockdown, suggesting that POLA2 does not act alone and may cooperate with other interacting partners to cause gemcitabine resistance.

Conclusions

Collectively, our findings showed that knockdown of POLA2 increases gemcitabine resistance in human lung cancer cells. We propose that POLA2 may play a role in gemcitabine sensitivity and can be used as a prognostic biomarker of patient outcome in NSCLC pathogenesis.

     

Intrapulmonary delivery of bone marrow-derived mesenchymal stem cells improves survival and attenuates endotoxin-induced acute lung injury in mice

Recent in vivo and in vitro work suggests that mesenchymal stem cells (MSC) have anti-inflammatory properties. In this study, we tested the effect of administering MSC directly into the airspaces of the lung 4 h after the intrapulmonary administration of Escherichia coli endotoxin (5 mg/kg). MSC increased survival compared with PBS-treated control mice at 48 h (80 vs 42%; p < 0.01). There was also a significant decrease in excess lung water, a measure of pulmonary edema (145 +/- 50 vs 87 +/- 20 microl; p < 0.01), and bronchoalveolar lavage protein, a measure of endothelial and alveolar epithelial permeability (3.1 +/- 0.4 vs 2.2 +/- 0.8 mg/ml; p < 0.01), in the MSC-treated mice. These protective effects were not replicated by the use of further controls including fibroblasts and apoptotic MSC. The beneficial effect of MSC was independent of the ability of the cells to engraft in the lung and was not related to clearance of the endotoxin by the MSC. MSC administration mediated a down-regulation of proinflammatory responses to endotoxin (reducing TNF-alpha and MIP-2 in the bronchoalveolar lavage and plasma) while increasing the anti-inflammatory cytokine IL-10. In vitro coculture studies of MSC with alveolar macrophages provided evidence that the anti-inflammatory effect was paracrine and was not cell contact dependent. In conclusion, treatment with intrapulmonary MSC markedly decreases the severity of endotoxin-induced acute lung injury and improves survival in mice.     

Systemic complement activation and acute lung injury

Experimental studies of rats have provided significant evidence that intravascular complement activation after i.v. injection of cobra venom factor (CVF) or thermal injury of skin can result in acute lung injury. This has been determined by morphological changes in lung and increases in lung vascular permeability. Systemic complement activation is associated with an early appearance of C5-derived chemotactic activity in the circulation coincident with the development of transient neutropenia, followed by extensive granulocytosis and sequestration of neutrophils in lung interstitial capillaries. The acute pulmonary injury depends on availability of complement and neutrophils. Depletion of either complement or blood neutrophils before CVF injection or thermal injury will prevent development of lung injury. Interventional studies with catalase, scavengers of hydroxyl radical OH., and iron chelators have revealed that the acute pulmonary injury is related to production of oxygen-derived free radicals by activated neutrophils. OH. appears to be the key mediator involved in the acute lung microvascular injury.     

Heat shock response and acute lung injury

All cells respond to stress through the activation of primitive, evolutionarily conserved genetic programs that maintain homeostasis and assure cell survival. Stress adaptation, which is known in the literature by a myriad of terms, including tolerance, desensitization, conditioning, and reprogramming, is a common paradigm found throughout nature, in which a primary exposure of a cell or organism to a stressful stimulus (e.g., heat) results in an adaptive response by which a second exposure to the same stimulus produces a minimal response. More interesting is the phenomenon of cross-tolerance, by which a primary exposure to a stressful stimulus results in an adaptive response whereby the cell or organism is resistant to a subsequent stress that is different from the initial stress (i.e., exposure to heat stress leading to resistance to oxidant stress). The heat shock response is one of the more commonly described examples of stress adaptation and is characterized by the rapid expression of a unique group of proteins collectively known as heat shock proteins (also commonly referred to as stress proteins). The expression of heat shock proteins is well described in both whole lungs and in specific lung cells from a variety of species and in response to a variety of stressors. More importantly, in vitro data, as well as data from various animal models of acute lung injury, demonstrate that heat shock proteins, especially Hsp27, Hsp32, Hsp60, and Hsp70 have an important cytoprotective role during lung inflammation and injury.     

Stat3 activation in acute lung injury

Stat3 plays diverse roles in biological processes including cell proliferation, survival, apoptosis, and inflammation. Very little is known regarding its activation and function in the lung during acute inflammation. We now show that Stat3 activation was triggered in lungs and in alveolar macrophages after intrapulmonary deposition of IgG immune complexes in rats. Low levels of constitutive Stat3 were observed in normal rat lungs as determined by the EMSA. Stat3 activity in whole lung extracts increased 2 h after initiation of IgG immune complex deposition, reaching maximal levels by 4 h, whereas Stat3 activation was found in alveolar macrophages as early as 30 min after onset of injury. Expression and activation of Stat3 mRNA, protein, and protein phosphorylation was accompanied by increased gene expression of IL-6, IL-10, and suppressor of cytokine signaling-3 in whole lung tissues. Both Tyr(705) and Ser(727) phosphorylation were involved in Stat3 activation as assessed in whole lung extracts. C5a (complement 5, fragment a) per se can induce phosphorylation of Ser(727) of Stat3. In vivo, Stat3 activation was dramatically suppressed by depletion of neutrophils or lung macrophages, resulting in reduced gene expression of IL-6 and IL-10 in whole lung tissues. Using blocking Abs to IL-6, IL-10, and C5a, Stat3 activation induced by IgG immune complexes was markedly diminished. These data suggest in the lung injury model used that activation of Stat3 in lungs is macrophage dependent and neutrophil dependent. IL-6, IL-10, and C5a contribute to Stat3 activation in inflamed rat lung.