Endotoxin-induced Pulmonary Inflammation Research Articles

In Vivo Efficacy and Toxicity of an Antimicrobial Peptide in a Model of Endotoxin-Induced Pulmonary Inflammation.

SET-M33 is a synthetic peptide that is being developed as a new antibiotic against major Gram-negative bacteria. Here we report two in vivo studies to assess the toxicity and efficacy of the peptide in a murine model of pulmonary inflammation. First, we present the toxicity study in which SET-M33 was administered to CD-1 mice by snout inhalation exposure for 1 h/day for 7 days at doses of 5 and 20 mg/kg/day. The results showed adverse clinical signs and effects on body weight at the higher dose, as well as some treatment-related histopathology findings (lungs and bronchi, nose/turbinates, larynx and tracheal bifurcation). On this basis, the no observable adverse effect level (NOAEL) was considered to be 5 mg/kg/day. We then report an efficacy study of the peptide in an endotoxin (LPS)-induced pulmonary inflammation model. Intratracheal administration of SET-M33 at 0.5, 2 and 5 mg/kg significantly inhibited BAL neutrophil cell counts after an LPS challenge. A significant reduction in pro-inflammatory cytokines, KC, MIP-1α, IP-10, MCP-1 and TNF-α was also recorded after SET-M33 administration.

Design, Synthesis, and Biological Characterization of Inhaled p38α/β MAPK Inhibitors for the Treatment of Lung Inflammatory Diseases.

The identification of novel inhaled p38α/β mitogen-activated protein kinases (MAPK) (MAPK14/11) inhibitors suitable for the treatment of pulmonary inflammatory conditions has been described. A rational drug design approach started from the identification of a novel tetrahydronaphthalene series, characterized by nanomolar inhibition of p38α with selectivity over p38γ and p38δ isoforms. SAR optimization of 1c is outlined, where improvements in potency against p38α and ligand-enzyme dissociation kinetics led to several compounds showing pronounced anti-inflammatory effects in vitro (inhibition of TNFα release). Targeting of the defined physicochemical properties allowed the identification of compounds 3h, 4e, and 4f, which showed, upon intratracheal instillation, low plasma levels, prolonged lung retention, and anti-inflammatory effects in a rat acute model of a bacterial endotoxin-induced pulmonary inflammation. Compound 4e, in particular, displayed remarkable efficacy and duration of action and was selected for progression in disease models of asthma and chronic obstructive pulmonary disease (COPD).

Rho-kinase inhibitor treatment prevents pulmonary inflammation and coagulation in lipopolysaccharide-induced lung injury

IntroductionIn the pathogenesis of sepsis-induced acute lung injury (ALI), the crosstalk between inflammation and coagulation plays a pivotal role. The aim of this study was to investigate the role of Rho kinase (ROCK) inhibitor in alleviating pulmonary inflammation and coagulation in lipopolysaccharide (LPS)-induced acute lung injury (ALI) models. MethodsIn the in vivo study, mice were randomized to four different groups: Control, Y-27632 (Y), LPS, and LPS+Y-27632 (LPS+Y). ALI was induced by intranasally administering LPS (10μg in 50μL PBS). Y-27632 (10mg/kg body weight,) was injected intraperitoneally at 18h and 1h before LPS challenge. Mice were euthanized at 3h or 8h post LPS challenge (N=8 per group). In the in vitro study, human pulmonary microvascular endothelial cells (HPMECs) were incubated with LPS alone (1μg/mL) or in combination with 10μM Y-27632 or 50μM BAY11-7082. Cells were pretreated with the inhibitors 30min before exposure to LPS. Three hours later, cells were isolated for subsequent analysis. ResultsThe myeloperoxidase (MPO) activity and fibrinogen deposits in the lung tissue significantly decreased and the lung damage in ALI mouse was attenuated. Pretreatment with Y-27632 markedly reduced the LPS-induced expression of interleukins 1β and 6, and the activation of nuclear factor (NF)-κB. Furthermore, ROCK inhibitor treatment antagonized the expression of tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 in lung tissue and HPMECs. ConclusionsROCK inhibition protects against the endotoxin-induced pulmonary inflammation and coagulation via NF-kappaB pathway modulation.

Deletion of Src family kinase Lyn aggravates endotoxin-induced lung inflammation.

Overwhelming acute inflammation often leads to tissue damage during endotoxemia. In the present study, we investigated the role of Lyn, a member of the Src family tyrosine kinases, in modulating inflammatory responses in a murine model of endotoxemia. We examined lung inflammatory signaling in Lyn knockout (Lyn(-/-)) mice and wild-type littermates (Lyn(+/+)) during endotoxemia. Our data indicate that Lyn deletion aggravates endotoxin-induced pulmonary inflammation and proinflammatory signaling. We found increased activation of proinflammatory transcription factor NF-κB in the lung tissues of Lyn(-/-) mice after endotoxin challenge. Furthermore, during endotoxemia, the lung tissues of Lyn(-/-) mice showed increased inflammasome activation indicated by augmented caspase-1 and IL-1β cleavage and activation. The aggravated lung inflammatory signaling in Lyn(-/-) mice was associated with increased production of proinflammatory mediators and elevated matrix metallopeptidase 9 and reduced VE-cadherin levels. Our results suggest that Lyn kinase modulates inhibitory signaling to suppress endotoxin-induced lung inflammation.

Hemokinin-1 is an important mediator of endotoxin-induced acute airway inflammation in the mouse

ObjectiveHemokinin-1, the newest tachykinin encoded by the preprotachykinin C (Tac4) gene, is predominatly produced by immune cells. Similarly to substance P, it has the greatest affinity to the tachykinin NK1 receptor, but has different binding site and signaling mechanisms. Furthermore, several recent data indicate the existence of a not yet identified own receptor and divergent non-NK1-mediated actions. Since there is no information on its functions in the airways, we investigated its role in endotoxin-induced pulmonary inflammation. MethodsAcute pneumonitis was induced in Tac4 gene-deleted (Tac4−/−) mice compared to C57Bl/6 wildtypes by intranasal E. coli lipopolysaccharide (LPS). Airway responsiveness to inhaled carbachol was measured with unrestrained whole body plethysmography 24h later. Semiquantitative histopathological scoring was performed; reactive oxygen species (ROS) production was measured with luminol bioluminescence, myeloperoxidase activity with spectrophotometry, and inflammatory cytokines with Luminex. ResultsAll inflammatory parameters, such as histopathological alterations (perivascular edema, neutrophil/macrophage accumulation, goblet cell hyperplasia), myeloperoxidase activity, ROS production, as well as interleukin-1beta, interleukin-6, tumor necrosis factor alpha, monocyte chemoattractant protein-1 and keratinocyte chemoattractant concentrations were significantly diminished in the lung of Tac4−/− mice. However, bronchial hyperreactivity similarly developed in both groups. Interestingly, in LPS-treated Tac4−/− mouse lungs, bronchus-associated, large, follicle-like lymphoid structures developed. ConclusionsWe provide the first evidence that hemokinin-1 plays a crucial pro-inflammatory role in the lung by increasing inflammatory cell activities, and might also be a specific regulator of lymphocyte functions.

The TRPM2 ion channel, an oxidative stress and metabolic sensor regulating innate immunity and inflammation

TRPM2 (transient receptor potential melastatin 2) is the unique fusion of a Ca(2+)-permeable pore with an enzymatic domain that binds the NAD(+)-metabolite ADP-ribose (ADPR), resulting in channel opening. ADPR formation is a metabolic corollary of cellular stress, but can also be elicited enzymatically through NAD glycohydrolases like CD38. TRPM2 thus functions as a metabolic and oxidative stress sensor and translates this information into ion fluxes that can affect Ca(2+) signaling and the membrane potential. TRPM2 is strongly represented in immune cells of the phagocytic lineage, themselves professional generators of oxidants. The recent characterization of TRPM2-deficient mouse models has revealed the involvement of this channel in various aspects of immunity. Monocytes lacking TRPM2 show reduced production of the CXCL2 chemokine, resulting in diminished neutrophilic influx to the colon in chemically induced colitis, and thus protection against tissue ulceration in TRPM2(-/-) mice. However, the insufficient production of proinflammatory cytokines leads to high morbidity and lethality of the TRPM2(-/-) mice following infection with the bacterial pathogen Listeria monocytogenes. In the context of endotoxin-induced pulmonary inflammation, TRPM2's absence was found to promote inflammation and ROS production. TRPM2 acts thereby as a negative feedback loop by interfering through membrane depolarization with ROS generation by NADPH oxidases. In dendritic cells, TRPM2 is a lysosomal Ca(2+)-release channel that promotes chemokine responsiveness and cell migration, which is reminiscent of CD38-mediated functions. The discovery of TRPM2 has unveiled an unsuspected signaling pathway and established ADPR as a novel second messenger. Understanding TRPM2's complex involvement in inflammation is crucial to evaluating the potential of manipulating TRPM2 activity and ADPR metabolism for therapeutic intervention.

Adora2b Adenosine Receptor Engagement Enhances Regulatory T Cell Abundance during Endotoxin-Induced Pulmonary Inflammation

Anti-inflammatory signals play an essential role in constraining the magnitude of an inflammatory response. Extracellular adenosine is a critical tissue-protective factor, limiting the extent of inflammation. Given the potent anti-inflammatory effects of extracellular adenosine, we sought to investigate how extracellular adenosine regulates T cell activation and differentiation. Adenosine receptor activation by a pan adenosine-receptor agonist enhanced the abundance of murine regulatory T cells (Tregs), a cell type critical in constraining inflammation. Gene expression studies in both naïve CD4 T cells and Tregs revealed that these cells expressed multiple adenosine receptors. Based on recent studies implicating the Adora2b in endogenous anti-inflammatory responses during acute inflammation, we used a pharmacologic approach to specifically activate Adora2b. Indeed, these studies revealed robust enhancement of Treg differentiation in wild-type mice, but not in Adora2b −/− T cells. Finally, when we subjected Adora2b-deficient mice to endotoxin-induced pulmonary inflammation, we found that these mice experienced more severe inflammation, characterized by increased cell recruitment and increased fluid leakage into the airways. Notably, Adora2b-deficient mice failed to induce Tregs after endotoxin-induced inflammation and instead had an enhanced recruitment of pro-inflammatory effector T cells. In total, these data indicate that the Adora2b adenosine receptor serves a potent anti-inflammatory role, functioning at least in part through the enhancement of Tregs, to limit inflammation.

1H and hyperpolarized 3He magnetic resonance imaging clearly detect the preventative effect of a glucocorticoid on endotoxin-induced pulmonary inflammation in vivo

Proton (¹H ) magnetic resonance imaging (MRI) can be utilized to quantify pulmonary edema in endotoxin-induced pulmonary inflammation and hyperpolarized (HP) ³He MRI can assess pulmonary ventilation. Neither of the methods has been applied to assess the impact of a drug on endotoxin-induced pulmonary inflammation in vivo. The aim of the current study was to evaluate the capability of ¹H and HP ³He MRI to assess the effects of a glucocorticoid on endotoxin-induced pulmonary inflammation in vivo. Mice were exposed to an aerosol of either saline or endotoxin (5 mg/ml) for 10 min. Half of the endotoxin-exposed mice were pretreated with a glucocorticoid (budesonide 3 mg/kg; 2 times/day) and the other half with vehicle p.o. The first budesonide treatment was administered 1 h prior to the aerosol inhalation. Forty-eight hours after the aerosol exposure, the mice were anaesthetized for subsequent imaging. Hyperpolarized ³He was administered and axial MR images of the lungs obtained. Matching ¹H MR images were then acquired. The mice were sacrificed and broncho-alveolar lavage (BAL) samples were harvested to determine total and cell differential counts. The lesion volume on both ¹H and ³He MRI, were markedly increased by endotoxin exposure (P<0.001). Budesonide strongly reduced lesion volume (P<0.001). The BAL cell count correlated strongly with both (3)He (P<0.001; r = 0.96) and ¹H lesion volumes (P<0.001; r = 0.97). Hyperpolarized ³He MRI and ¹H MRI clearly visualized the preventative effect of budesonide on the impact of endotoxin on pulmonary ventilation and edema, respectively. The fact that ventilation defects on ³He MRI corresponded to findings from conventional ¹H MRI, as well as to counts of BAL inflammatory cells suggests that these imaging techniques constitute promising tools for non-invasive monitoring of pulmonary inflammation in vivo.

The complex role of fibrin in acute lung injury

in the last decade there have been many advances in our understanding of the pathogenesis of acute lung injury (ALI), but there have been frustratingly few successful therapeutic interventions. In fact, the only treatment proven to reduce mortality is low tidal volume lung-protective mechanical

Surfactant Protein-D and Surfactant Inhibit Endotoxin-Induced Pulmonary Inflammation

Acute lung injury is a common cause of morbidity and mortality following pulmonary or systemic infections. Surfactant protein-D is a member of the collectin family of proteins, which play important roles in innate host defense of the lung. In this study, the effect of exogenous recombinant human SP-D (rhSP-D) on protection of the adult mouse lung from lipopolysaccharide (LPS)-induced and lipoteichoic acid (LTA)-induced injury was assessed. The effect of rhSP-D on LPS-induced and LTA-induced lung inflammation and injury was assessed with and without exogenous pulmonary surfactant in Sftpd+/+ and Sftpd-/- mice. A total of 204 mice (6 mice per group) were used for the present study. Sftpd-/- mice were more susceptible to intratracheal LPS than were Sftpd+/+ mice. rhSP-D decreased neutrophilic infiltrates induced by LPS and LTA in the lungs of both Sftpd+/+ and Sftpd-/- mice. The addition of exogenous pulmonary surfactant to rhSP-D further decreased LPS-induced and LTA-induced pulmonary inflammation in Sftpd-/- and Sftpd+/+ mice. Intratracheal rhSP-D inhibited inflammation induced by intratracheal LPS and LTA instillation in the lung. The antiinflammatory effects of rhSP-D were enhanced by the addition of pulmonary surfactant, providing a potential therapy for the treatment of lung inflammation.

Recombinant Soluble Forms of Extracellular TLR4 Domain and MD-2 Inhibit Lipopolysaccharide Binding on Cell Surface and Dampen Lipopolysaccharide-Induced Pulmonary Inflammation in Mice

In this study, we sought the possibility of a new therapeutic strategy for dampening endotoxin-induced inflammation using soluble form of extracellular rTLR4 domain (sTLR4) and soluble form of rMD-2 (sMD-2). Addition of sTLR4 plus sMD-2 was significantly effective in inhibiting LPS-elicited IL-8 release from U937 cells and NF-kappaB activation in the cells transfected with TLR4 and MD-2 when compared with a single treatment with sTLR4 or sMD-2. Thus, we investigated the role of the extracellular TLR4 domain in interaction of lipid A with MD-2. Biotinylated sTLR4 failed to coprecipitate [(3)H]lipid A when it was sedimented with streptavidin-agarose, demonstrating that the extracellular TLR4 domain does not directly bind lipid A by itself. The amounts of lipid A coprecipitated with sMD-2 significantly increased when coincubated with sTLR4, and sTLR4 increased the affinity of lipid A for the binding to sMD-2. Soluble CD14 is required for the sTLR4-stimulated increase of lipid A binding to sMD-2. We also found that addition of sTLR4 plus sMD-2 inhibited the binding of Alexa-conjugated LPS to the cells expressing TLR4 and MD-2. Murine lungs that had received sTLR4 plus sMD-2 with LPS did not show any findings indicative of interstitial edema, neutrophil flux, and hemorrhage. Co-instillation of sTLR4 plus sMD-2, but not sTLR4 or sMD-2 alone, significantly decreased neutrophil infiltration and TNF-alpha levels in bronchoalveolar lavage fluids from LPS-treated mice. This study provides novel usage of sTLR4 and sMD-2 as an antagonist against endotoxin-induced pulmonary inflammation.

Monitoring of Endotoxin-Induced Pulmonary Inflammation In Vivo in NF- kB Luciferase Transgenic Mice

Rationale: NF-kappaB is necessary for coordinating the activation of many genes involved in inflammatory responses in the lungs. We explored the use of NF-kB-luciferase BALB/c transgenic mice to evaluate the relationship between inhaled endotoxin and the activation of NF-kB signals in lungs in vivo. Methods: Xenogen NF-kB BALB/c mice with 3 luciferase reporter elements were exposed to N. meningitidis endotoxin (0-300, 000 EU/mouse) and saline (controls) by nasal aspiration. Before exposure and after certain time points (4, 8hr) luciferin was injected intraperitoneally and mice were imaged with an ultrasensitive CCD camera (IVIS-100, Xenogen). After imaging the thoracic region, mice were euthanized and lungs were rapidly excised and imaged. The oxyluciferin signals from the thoracic region and lungs were quantified using Living Image Software. Results: The imaging of exposed transgenic NF-kB-luc BALB/c mice demonstrated a quantifiable endotoxin induced signal in the thoracic region in vivo over controls at doses of endotoxin above 3, 000 EU/mouse. The signal in the lungs of exposed mice imaged ex vivo showed high values (5 to 12-fold increases) over control and provided documentation of NF-kB activation in the lung. Conclusions: Markers of lung inflammation from bronchoalveolar lavage such as neutrophil recruitment and cytokine concentrations were more sensitive biomarkers than imaging oxyluciferin photonic yield in vivo. A lower dose of inhaled endotoxin was capable of inducing measurable lung luminescence compared with controls when lungs were imaged ex vivo. This study provides new data regarding the sensitivity of NF-kB-luc transgenic mice and their utility for in vivo monitoring of lung inflammation. Funded By: NIH P30 ES05605-15

Shen-Fu Attenuates Endotoxin-Induced Acute Lung Injury in Rats

Sepsis is associated with the highest risk of progression to acute lung injury or acute respiratory distress syndrome. Shen-Fu has been advocated to treat many severely ill patients. Our study was designed to investigate the effect of Shen-Fu on endotoxin-induced acute lung injury in vivo. Adult male Wistar rats were randomly divided into 6 groups: controls; those challenged with endotoxin (5 mg/kg) and treated with saline; those challenged with endotoxin (5 mg/kg) and treated with Shen-Fu (1 mg/kg); those challenged with endotoxin (5 mg/kg) and treated with Shen-Fu (10 mg/kg); increase challenged with endotoxin (5 mg/kg) and treated with Shen-Fu (100 mg/kg); saline injected and treated with Shen-Fu (100 mg/kg). TNF-alpha, IL-6, and NF-kappa B were investigated in the lung two hours later. Myeloperoxidase (MPO) activity and wet/dry weight ratio were investigated six hours later. Intravenous administration of endotoxin provoked significant lung injury, which was characterized by increment increase of MPO activity and wet/dry lung weight ratio, and TNF-alpha and IL-6 expression and NF-kappa B activation. Shen-Fu (10,100 mg/kg) decreased MPO activity and wet/dry weight ratio and inhibited TNF-alpha and IL-6 production, endotoxin-induced NF-kappa B activation. Our results indicated that Shen-Fu at a dose of higher than 10 mg/kg inhibited endotoxin-induced pulmonary inflammation in vivo.

Large dose ketamine inhibits lipopolysaccharide-induced acute lung injury in rats

Sepsis is associated with the highest risk of progression to acute lung injury or the acute respiratory distress syndrome. Ketamine has been advocated for anesthesia in endotoxemic and other severely ill patients because it is a cardiovascular stimulant. Our study was designed to investigate the effect of ketamine on the endotoxin-induced acute lung injury in vivo. Adult male Wistar rats were randomly divided into 6 groups: saline controls; rats challenged with endotoxin (5 mg/kg) and treated with saline; challenged with endotoxin (5 mg/kg) and treated with ketamine (0.5 mg/kg); challenged with endotoxin (5 mg/kg) and treated with ketamine (5 mg/kg); challenged with endotoxin (5 mg/kg) and treated with ketamine (50 mg/kg); saline injected and treated with ketamine (50 mg/kg). TNF-alpha, IL-6 and NF-kappa B were investigated in the tissues of the lung after 2 h. Myeloperoxidase (MPO) activity and wet/dry weight ratio were investigated 6 h later. We demonstrated that intravenous administration of endotoxin could provoke significant lung injury, which was characterized by increase of MPO activity and wet/dry weight ratio, TNF-alpha and IL-6 expression and NF-kappa B activation. Ketamine (5, 50 mg/kg) inhibited endotoxin-induced NF-kappa B activation. Ketamine only at a dose of 50 mg/kg inhibited TNF-alpha and IL-6 production, and decreased MPO activity and wet/dry weight ratio after endotoxin challenge. Ketamine, only at a supra-anesthetic dosage, could inhibit endotoxin-induced pulmonary inflammation in vivo.

Recombinant human activated protein C reduces human endotoxin-induced pulmonary inflammation via inhibition of neutrophil chemotaxis

AbstractRecombinant human activated protein C (rhAPC) is a natural anticoagulant with potentially important anti-inflammatory properties. In humans with severe sepsis, rhAPC treatment reduces mortality, but mechanisms responsible have not been well characterized. Accumulation of activated neutrophils in the lungs and other organs during severe infection contributes to sepsis-induced organ dysfunction, including acute inflammatory lung injury. Because neutrophils express an APC receptor, we hypothesized that immunomodulatory effects of rhAPC occur, in part, via modulation of neutrophil responses. To examine this issue, we performed a double-blinded, placebo-controlled study of rhAPC in a human model of endotoxin-induced pulmonary inflammation. Administration of rhAPC significantly reduced leukocyte accumulation to the airspaces, independent of pulmonary cytokine or chemokine release. Neutrophils recovered from bronchoalveolar lavage fluid of volunteers receiving rhAPC demonstrated decreased chemotaxis ex vivo. Decreased neutrophil chemotaxis following exposure to rhAPC was confirmed in vitro. No differences were detected in gene expression, kinase activation, cytokine release, cell survival, or apoptosis of neutrophils recovered in the presence or absence of rhAPC. These studies demonstrate that rhAPC reduces both endotoxin-induced accumulation of leukocytes in the airspaces and neutrophil chemotaxis. These rhAPC-induced effects on neutrophil function may represent a mechanism by which rhAPC improves survival in patients with sepsis. (Blood. 2004;104:3878-3885)

Induction of neutrophil apoptosis and secondary necrosis during endotoxin-induced pulmonary inflammation in mice.

The present study investigated the relationship between apoptotic and necrotic cell death and their role in pulmonary inflammatory response to endotoxin. Pulmonary administration of lipopolysaccharide (LPS) caused a rapid increase in the levels of pro-inflammatory cytokine TNF-alpha and inflammatory cell influx in the bronchoalveolar lavage (BAL) fluids. Control mice showed only resident alveolar macrophages with no apoptosis, whereas LPS-treated mice showed clear apoptosis of BAL cells. Microscopic studies confirmed the presence of apoptotic neutrophils and macrophages ingesting apoptotic bodies. The number of apoptotic neutrophils increased concomitantly with the increase in neutrophil influx which peaked 1 day after the treatment. However, necrosis was not detected at this early time, but increased subsequently and peaked at day 3. The levels of necrosis and apoptosis were both elevated and prolonged at high LPS doses. Treatment of mice with phosphatidylserine (PS)-containing liposome, known to inhibit macrophage phagocytosis of apoptotic cells, increased the level of apoptosis and necrosis caused by LPS, whereas control non-PS liposome or saline treatment had no effects. We conclude that necrosis occurs secondary to apoptosis in LPS-treated lung model and that this development is not the result of direct insult by LPS. Instead, our results and previous studies suggest that inefficient clearance of apoptotic cells by macrophages contributes, at least in part, to the levels of apoptosis and necrosis induced by LPS. Because necrosis is associated with cell damage and release of histotoxic contents, this development is likely to play a role in determining the severity and duration of lung toxicity induced by endotoxin.

Pulmonary phenotype of CCSP/UG deficient mice: a consequence of CCSP deficiency or altered Clara cell function?

Clara cell secretory protein (CCSP) is the most abundant secreted protein within airways of the lung. Moreover, CCSP levels are modulated in human lung disease, supporting a potentially important role for CCSP and/or Clara cells in lung homeostasis. However, in vivo roles for CCSP remain elusive. A popular hypothesis is that CCSP is a regulator of the inflammatory response. The purpose of this review is to provide an overview of the phenotype of CCSP null mice and relate this phenotype to proposed functions for the protein. Phenotypic analysis of mice homozygous for the CCSP-1 null allele of the CCSP gene (CCSP-/-1) revealed susceptibility to inhaled oxidant gases. Sensitivity of CCSP-/-1 mice to inhaled ozone is unrelated to alterations in antioxidant defenses, but is associated with increased cellular injury. Additional studies investigating inflammatory control in CCSP deficient mice found no differences between wild-type and CCSP-/-1 mice in their inflammatory response to low-dose inhaled endotoxin exposure, arguing against a role for CCSP in regulation of pulmonary inflammation. The findings among CCSP-/-1 mice of ultrastructural alterations to Clara cell secretory apparatus, with associated changes in airway lining fluid protein composition, demonstrate that the CCSP-/-1 genotype results in more complex changes to airways than CCSP deficiency per se. It can be concluded that CCSP does not regulate endotoxin-induced pulmonary inflammation. Moreover, CCSP-/-1 mice represent a valuable tool for probing functional roles for Clara cells in regulation of airway lining fluid composition and lung pollutant susceptibility.