Pulmonary lipopolysaccharide (LPS)-binding protein inhibits the LPS-induced lung inflammation in vivo

LPS-binding protein (LBP) facilitates the interaction of the Gram-negative cell wall component LPS with CD14, thereby enhancing the immune response to LPS. Although lung epithelial cells have been reported to produce LBP in vitro, knowledge of the in vivo role of pulmonary LBP is limited. Therefore, in the present study we sought to determine the function of pulmonary LBP in lung inflammation induced by intranasal administration of LPS in vivo. Using LBP-deficient (LBP-/-) and normal wild-type mice, we show that the contribution of LBP to pulmonary LPS responsiveness depended entirely on the LPS dose. Although the inflammatory response to low dose (1 ng) LPS was attenuated in LBP-/- mice, neutrophil influx and cytokine/chemokine concentrations in the bronchoalveolar compartment were enhanced in LBP-/- mice treated with higher (>10 ng) LPS doses. This finding was specific for LBP, because the exogenous administration of LBP to LBP-/- mice reversed this phenotype and reduced the local inflammatory response to higher LPS doses. Our results indicate that pulmonary LBP acts as an important modulator of the LPS response in the respiratory tract in vivo. This newly identified function of pulmonary LBP might prove beneficial by enabling a protective immune response to low LPS doses while preventing an overwhelming, potentially harmful immune response to higher doses of LPS.     

Comparison of the effect of recombinant bovine wild and mutant lipopolysaccharide-binding protein in lipopolysaccharide-challenged bovine mammary epithelial cells

Lipopolysaccharide (LPS)-binding protein (LBP) plays a crucial role in the recognition of bacterial components, such as LPS that causes an immune response. The aim of this study was to compare the different effects of recombinant bovine wild LBP and mutant LBP (67 Ala?→?Thr) on the LPS-induced inflammatory response of bovine mammary epithelial cells (BMECs). When BMECs were treated with various concentrations of recombinant bovine lipopolysaccharide-binding protein (RBLBP) (1, 5, 10, and 15 μg/mL) for 12 h, RBLBP of 5 μg/mL increased the apoptosis of BMECs induced by LPS without cytotoxicity, and mutant LBP resulted in a higher cell apoptosis than wild LBP did. By gene-chip microarray and bioinformatics, the data identified 2306 differentially expressed genes that were changed significantly between the LPS-induced inflamed BMECs treated with 5 μg/mL of mutant LBP and the BMECs only treated with 10 μg/mL of LPS (fold change ≥2). Meanwhile, 1585 genes were differently expressed between the inflamed BMECs treated with 5 μg/mL of wild LBP and 10 μg/mL of LPS-treated BMECs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that these differentially expressed genes were involved in different pathways that regulate the inflammation response. It predicted that carriers of this mutation increase the risk for a more severe inflammatory response. Our study provides an overview of the gene expression profile between wild LBP and mutant LBP on the LPS-induced inflammatory response of BMECs, which will lead to further understanding of the potential effects of LBP mutations on bovine mammary glands.     

Diet-induced bacterial immunogens in the gastrointestinal tract of dairy cows: Impacts on immunity and metabolism

Dairy cows are often fed high grain diets to meet the energy demand for high milk production or simply due to a lack of forages at times. As a result, ruminal acidosis, especially subacute ruminal acidosis (SARA), occurs frequently in practical dairy production. When SARA occurs, bacterial endotoxin (or lipopolysaccharide, LPS) is released in the rumen and the large intestine in a large amount. Many other bacterial immunogens may also be released in the digestive tract following feeding dairy cows diets containing high proportions of grain. LPS can be translocated into the bloodstream across the epithelium of the digestive tract, especially the lower tract, due to possible alterations of permeability and injuries of the epithelial tissue. As a result, the concentration of blood LPS increases. Immune responses are subsequently caused by circulating LPS, and the systemic effects include increases in concentrations of neutrophils and the acute phase proteins such as serum amyloid-A (SAA), haptoglobin (Hp), LPS binding protein (LBP), and C-reactive protein (CRP) in blood. Entry of LPS into blood can also result in metabolic alterations. Blood glucose and nonesterified fatty acid concentrations are enhanced accompanying an increase of blood LPS after increasing the amount of grain in the diet, which adversely affects feed intake of dairy cows. As the proportions of grain in the diet increase, patterns of plasma β-hydoxybutyric acid, cholesterol, and minerals (Ca, Fe, and Zn) are also perturbed. The bacterial immunogens can also lead to reduced supply of nutrients for synthesis of milk components and depressed functions of the epithelial cells in the mammary gland. The immune responses and metabolic alterations caused by circulating bacterial immunogens will exert an effect on milk production. It has been demonstrated that increases in concentrations of ruminal LPS and plasma acute phase proteins (CRP, SAA, and LBP) are associated with declines in milk fat content, milk fat yield, 3.5% fat-corrected milk yield, as well as milk energy efficiency.     

LPS-binding protein mediates LPS-induced liver injury and mortality in the setting of biliary obstruction

It is generally accepted that low levels of lipopolysaccharide (LPS)-binding protein (LBP) augment the cell's response to LPS, whereas high levels of LBP have been shown to inhibit cell responses to LPS. Clinical studies and in vitro work by our group have demonstrated that, in the setting of liver disease, increased or acute-phase levels of LBP may actually potentiate rather than inhibit an overwhelming proinflammatory response. Therefore, in the present studies we sought to determine the role of acute-phase LBP in mediating morbidity and mortality in animals challenged with LPS in the setting of biliary obstruction. Using LBP-deficient mice and LBP blockade in wild-type mice, we demonstrate that high levels of LBP are deleterious in the setting of cholestasis. Following biliary obstruction and intraperitoneal LPS challenge, hepatic injury, hepatic neutrophil infiltration, and mortality were significantly increased in animals with an intact LBP acute-phase response. Kupffer cell responses from these animals demonstrated a significant increase in several inflammatory mediators, and Kupffer cell-associated LBP appears to be responsible for these differences, at least in part. Our results indicate that the role of LBP signaling in inflammatory conditions is complex and heterogeneous, and elevated levels of LBP are not always protective. Increased LBP production in the setting of cholestatic liver disease appears to be deleterious and may represent a potential therapeutic target for preventing overwhelming inflammatory responses to LPS in this setting.     

Lipopolysaccharide stimulates HepG2 human hepatoma cells in the presence of lipopolysaccharide-binding protein via CD14.

Lipopolysaccharide (LPS)-binding protein (LBP), an opsonin for activation of macrophages by bacterial LPS, is synthesized in hepatocytes and is known to be an acute phase protein. Recently, cytokine-induced production of LBP was reported to increase 10-fold in hepatocytes isolated from LPS-treated rats, compared with those from normal rats. However, the mechanism by which the LPS treatment enhances the effect of cytokines remains to be clarified. In the present study, we examined whether LPS alone or an LPS/LBP complex directly stimulates the hepatocytes, leading to acceleration of the cytokine-induced LBP production. HepG2 cells (a human hepatoma cell line) were shown to express CD14, a glycosylphosphatidylinositol-anchored LPS receptor, by both RT/PCR and flow cytometric analyses. An LPS/LBP complex was an effective stimulator for LBP and CD14 production in HepG2 cells, but stimulation of the cells with either LPS or LBP alone did not significantly accelerate the production of these proteins. The findings were confirmed by semiquantitative RT/PCR analysis of mRNA levels of LBP and CD14 in HepG2 cells after stimulation with LPS alone and an LPS/LBP complex. In addition, two monoclonal antibodies (mAbs) to CD14 (3C10 and MEM-18) inhibited LPS/LBP-induced cellular responses of HepG2 cells. Furthermore, prestimulation of HepG2 cells with LPS/LBP augmented cytokine-induced production and gene expression of LBP and CD14. All these findings suggest that an LPS/LBP complex, but not free LPS, stimulates HepG2 cells via CD14 leading to increased basal and cytokine-induced LBP and CD14 production.     

Lipopolysaccharide (LPS)-binding protein inhibits responses to cell-bound LPS

Lipopolysaccharide (LPS)-binding protein (LBP) is an acute phase reactant that may play a dual role in vivo, both potentiating and decreasing cell responses to bacterial LPS. Whereas low concentrations of LBP potentiate cell stimulation by transferring LPS to CD14, high LBP concentrations inhibit cell responses to LPS. One inhibitory mechanism involves the ability of LBP to neutralize LPS by transferring it to plasma lipoproteins, whereas other inhibitory mechanisms, such as the one described here, do not require exogenous lipoproteins. Here we show that LBP can inhibit monocyte responses to LPS that has already bound to membrane-bound CD14 (mCD14) on the cell surface. LBP caused rapid dissociation of LPS from mCD14 as measured by the ability of LBP to inhibit cross-linking of a radioiodinated, photoactivatable LPS derivative to mCD14. Whereas LBP removed up to 75% of the mCD14-bound LPS in 10 min, this was not accompanied by extensive release of the LPS from the cells. The cross-linking data suggest that much of the LPS that remained bound to the cells was associated with LBP. The ability of LBP to inhibit cell responses could not be explained by its effect on LPS internalization, because LBP did not significantly increase the internalization of the cell-bound LPS. In cell-free LPS cross-linking experiments, LBP inhibited the transfer of LPS from soluble CD14 to soluble MD-2. Our data support the hypothesis that LBP can inhibit cell responses to LPS by inhibiting LPS transfer from mCD14 to the Toll-like receptor 4-MD-2 signaling receptor.     

褪黑素对内毒素致大鼠急性肺损伤的保护作用

目的:研究内毒素(LPS)致大鼠急性肺损伤(ALI)时,p-p38蛋白激酶(p-p38MAPK)在肺组织的表达及褪黑素(MT)对肺组织的保护作用及其机制。方法:将72只健康雄性SD大鼠随机分为3组,每组24只,对照组(Control)、模型组(LPS)和褪黑素干预组(LPS+MT),采用气管内滴注LPS的方法建立大鼠ALI的模型,通过免疫组织化学染色和Western blot技术检测大鼠肺组织中p-p38蛋白激酶的表达变化,并在光镜下观察大鼠肺组织形态学变化。结果:Control组气道和肺组织可见反应极弱的p-p38蛋白激酶阳性细胞,散在分布于气道上皮细胞和肺泡上皮细胞;LPS组p-p38蛋白激酶阳性细胞较对照组明显增多(P0.05或P0.01),主要分布于浸润的炎症细胞、气道上皮细胞、肺泡上皮细胞和血管内皮细胞;LPS+MT组气道和肺组织中阳性细胞数较LPS组明显减少(P0.05或P0.01),Western blot结果与免疫组织化学一致。结论:LPS致大鼠急性肺损伤模型中,肺内炎性、非炎性细胞均有p38MAPK信号通路的激活;MT对急性肺损伤的保护机制可能与其抑制p38 MAPK信号通路的过度激活有关。     

Surfactant lipids regulate LPS-induced interleukin-8 production in A549 lung epithelial cells by inhibiting translocation of TLR4 into lipid raft domains

In addition to providing mechanical stability, growing evidence suggests that surfactant lipid components can modulate inflammatory responses in the lung. However, little is known of the molecular mechanisms involved in the immunomodulatory action of surfactant lipids. This study investigates the effect of the lipid-rich surfactant preparations Survanta®, Curosurf®, and the major surfactant phospholipid dipalmitoylphosphatidylcholine (DPPC) on interleukin-8 (IL-8) gene and protein expression in human A549 lung epithelial cells using immunoassay and PCR techniques. To examine potential mechanisms of the surfactant lipid effects, Toll-like receptor 4 (TLR4) expression was analyzed by flow cytometry, and membrane lipid raft domains were separated by density gradient ultracentrifugation and analyzed by immunoblotting with anti-TLR4 antibody. The lipid-rich surfactant preparations Survanta®, Curosurf®, and DPPC, at physiological concentrations, significantly downregulated lipopolysaccharide (LPS)-induced IL-8 expression in A549 cells both at the mRNA and protein levels. The surfactant preparations did not affect the cell surface expression of TLR4 or the binding of LPS to the cells. However, LPS treatment induced translocation of TLR4 into membrane lipid raft microdomains, and this translocation was inhibited by incubation of the cells with the surfactant lipid. This study provides important mechanistic details of the immune-modulating action of pulmonary surfactant lipids.     

Native high-density lipoprotein augments monocyte responses to lipopolysaccharide (LPS) by suppressing the inhibitory activity of LPS-binding protein

High-density lipoprotein (HDL) is an abundant plasma lipoprotein that is generally thought to be anti-inflammatory in both health and infectious disease. It binds and neutralizes the bioactivity of the potent bacterial lipids, LPS and lipoteichoic acid, that stimulate host innate immune responses. LPS-binding protein (LBP) plays an important role in augmenting leukocyte responses to LPS, whereas high concentrations of LBP, in the range of those found in plasma, can be inhibitory. We found that native HDL (nHDL) augmented human monocyte responses to LPS in the presence of inhibitory concentrations of LBP as measured by production of TNF and other cytokines. HDL did not stimulate cells in the absence of LPS, and it did not augment responses that were stimulated by IL-1beta or lipoteichoic acid. This activity of HDL was inhibited by trypsin treatment, suggesting that one or more protein constituents of HDL are required. In contrast to nHDL, low-density lipoprotein, and reconstituted HDL did not possess this activity. The total lipoprotein fraction of normal plasma had activity that was similar to that of nHDL, whereas lipoproteins from septic patients with reduced HDL levels had a reduced ability to augment responses to LPS; this activity was restored by adding normal HDL to the patient lipoproteins. Our results demonstrate a novel proinflammatory activity of HDL that may help maintain sensitive host responses to LPS by suppressing the inhibitory activity of LBP. Our findings also raise the possibility that the decline of HDL during sepsis may help control the response to LPS.     

Lycium barbarum polysaccharide protects against LPS-induced ARDS by inhibiting apoptosis,oxidative stress,and inflammation in pulmonary endothelial cells

Acute respiratory distress syndrome (ARDS) is a heterogenous syndrome characterised by diffuse alveolar damage, with an increase in lung endothelial and epithelial permeability. Lycium barbarum polysaccharide (LBP), the most biologically active fraction of wolfberry, possesses antiapoptotic and antioxidative effects in distinct situations. In the present study, the protective effects and potential molecular mechanisms of LBP against lipopolysaccharide (LPS)-induced ARDS were investigated in the mice and in the human pulmonary microvascular endothelial cells (HPMECs). The data indicated that pretreatment with LBP significantly attenuated LPS-induced lung inflammation and pulmonary oedema in vivo. LBP significantly reversed LPS-induced decrease in cell viability, increase in apoptosis and oxidative stress via inhibiting caspase-3 activation and intracellular reactive oxygen species (ROS) production in vitro. Moreover, the scratch assay verified that LBP restored the dysfunction of endothelial cells (ECs) migration induced by LPS stimulation. Furthermore, LBP also significantly suppressed LPS-induced NF-κB activation, and subsequently reversed the release of cytochrome c. These results showed the antiapoptosis and antioxidant LBP could partially protect against LPS-induced ARDS through promoting the ECs survival and scavenging ROS via inhibition of NF-κB signalling pathway. Thus, LBP could be potentially used for ARDS against pulmonary inflammation and pulmonary oedema.     

Regulation of angiopoietin expression by bacterial lipopolysaccharide

Angiopoietins are ligands for Tie-2 receptors and play important roles in angiogenesis and inflammation. While angiopoietin-1 (Ang-1) inhibits inflammatory responses, angiopoietin-2 (Ang-2) promotes cytokine production and vascular leakage. In this study, we evaluated in vivo and in vitro effects of Escherichia coli lipopolysaccharides (LPS) on angiopoietin expression. Wild-type C57/BL6 mice were injected with saline (control) or E. coli LPS (20 mg/ml ip) and killed 6, 12, and 24 h later. The diaphragm, lung, and liver were excised and assayed for mRNA and protein expression of Ang-1, Ang-2, and Tie-2 protein and tyrosine phosphorylation. LPS injection elicited a severalfold rise in Ang-2 mRNA and protein levels in the three organs. By comparison, both Ang-1 and Tie-2 levels in the diaphragm, liver, and lung were significantly attenuated by LPS administration. In addition, Tie-2 tyrosine phosphorylation in the lung was significantly reduced in response to LPS injection. In vitro exposure to E. coli LPS elicited cell-specific changes in Ang-1 expression, with significant induction in Ang-1 expression being observed in cultured human epithelial cells, whereas significant attenuation of Ang-1 expression was observed in response to E. coli LPS exposure in primary human skeletal myoblasts. In both cell types, E. coli LPS elicited substantial induction of Ang-2 mRNA, a response that was mediated in part through NF-kappaB. We conclude that in vivo endotoxemia triggers functional inhibition of the Ang-1/Tie-2 receptor pathway by reducing Ang-1 and Tie-2 expression and inducing Ang-2 levels and that this response may contribute to enhanced vascular leakage in sepsis.     

Soluble MD2 increases TLR4 levels on the epithelial cell surface

The accessory protein MD2 has been implicated in LPS-mediated activation of the innate immune system by functioning as a co-receptor with TLR4 for LPS binding at the cell surface. Epithelial cells that play a role in primary immune response, such as in the lung or gut, often express TLR4, but are dependent on circulating soluble MD2 (sMD2) to bind TLR4 to assemble the functional receptor. In this study, we show that sMD2 incubation with HEK293 epithelial cells transfected with TLR4 increases the cell surface levels of TLR4 in the absence of LPS. Dose response studies reveal that a threshold sMD2 concentration (approximately 450 nM) stimulates maximal TLR4 levels on the cell surface, whereas higher concentrations of sMD2 (approximately 1800 nM) reduce these enhanced TLR4 levels. We show evidence that MD2 multimer formation is increased at these higher concentrations of sMD2 and that addition of LPS to sMD2-stimulated cells masks the enhanced TLR4 cell surface levels, most likely due to the LPS-induced downregulation of TLR4 by endocytosis following receptor stimulation. All together, these results support a model in which sMD2 binds to TLR4 and increases TLR4 levels at the cell surface by preventing TLR4 turnover through the endocytic pathway. Thus, sMD2 may prime epithelial cells for enhanced immunoresponsive function prior to LPS exposure.     

Inflammatory response of tracheobronchial epithelial cells to endotoxin

Respiratory epithelial cells play a crucial role in the inflammatory response in endotoxin-induced lung injury, an experimental model for acute lung injury. To determine the role of epithelial cells in the upper respiratory compartment in the inflammatory response to endotoxin, we exposed tracheobronchial epithelial cells (TBEC) to lipopolysaccharide (LPS). Expression of inflammatory mediators was analyzed, and the biological implications were assessed using chemotaxis and adherence assays. Epithelial cell necrosis and apoptosis were determined to identify LPS-induced cell damage. Treatment of TBEC with LPS induced enhanced protein expression of cytokines and chemokines (increases of 235-654%, P < 0.05), with increased chemotactic activity regarding neutrophil recruitment. Expression of the intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was enhanced by 52-101% (P < 0.0001). This upregulation led to increased adhesion of neutrophils, with >95% adherence to TBEC after LPS stimulation, which could be blocked by either ICAM-1 (69%) or VCAM-1 antibodies (55%) (P < 0.05). Enhanced neutrophil-induced necrosis of TBEC was observed when TBEC were exposed to LPS. Reduced neutrophil adherence by ICAM-1 or VCAM-1 antibodies resulted in significantly lower TBEC death (52 and 34%, respectively, P < 0.05). Therefore, tight adherence of neutrophils to TBEC appears to promote epithelial cell killing. In addition to indirect effector cell-induced TBEC death, direct LPS-induced cell damage was seen with increased apoptosis rate in LPS-stimulated TBEC (36% increase of caspase-3, P < 0.01). These data provide evidence that LPS induces TBEC killing in a necrosis- and apoptosis-dependent manner.     

Lipopolysaccharide binding protein potentiates airway reactivity in a murine model of allergic asthma

The development of allergic asthma is influenced by both genetic and environmental factors. Epidemiologic data often show no clear relationship between the levels of allergen and clinical symptoms. Recent data suggest that bacterial LPS may be a risk factor related to asthma severity. Airborne LPS is typically present at levels that are insufficient to activate alveolar macrophages in the absence of the accessory molecule LPS binding protein (LBP). LBP levels are markedly elevated in bronchoalveolar lavage fluids obtained from asthmatic subjects compared with those in normal controls. We hypothesized that LBP present in the lung could augment the pulmonary inflammation and airway reactivity associated with allergic asthma by sensitizing alveolar macrophages to LPS or other bacterial products and triggering them to release proinflammatory mediators. We compared wild-type (WT) and LBP-deficient mice using a defined Ag immunization and aerosol challenge model of allergic asthma. Immunized LBP-deficient mice did not develop substantial Ag-induced airway reactivity, whereas WT mice developed marked bronchoconstriction following aerosol Ag sensitization and challenge with methacholine. Similarly, production of NO synthase 2 protein and the NO catabolite peroxynitrite was dramatically higher in the lungs of WT mice following challenge compared with that in LBP-deficient mice. Thus, NO production appears to correlate with airway reactivity. In contrast, both mice developed similar pulmonary inflammatory cell infiltrates and elevated mucin production. Thus, LBP appears to participate in the development of Ag-induced airway reactivity and peroxynitrite production, but does not seem to be required for the development of pulmonary inflammation.     

Plasma lipopolysaccharide (LPS)-binding protein. A key component in macrophage recognition of gram-negative LPS.

LPS-binding protein (LBP) binds with high affinity (Kd approximately equal to 10(-9) M) to lipid A of LPS isolated from rough (R)- or smooth (S)-form Gram-negative bacteria as well as to lipid A partial structures such as precursor IVA. To define the role of LBP in regulating responses to LPS we have examined TNF release in rabbit peritoneal exudate macrophages (M phi) stimulated with LPS or with complete or partial lipid A preparations in the presence or absence of LBP. In the presence of LBP, M phi showed increased sensitivity to S- and R-form LPS as well as synthetic lipid A. Compared with LPS or lipid A, up to 1000-fold greater concentrations of partial lipid A structures were required to induce TNF production. However, consistent with our previous observations that these structures bind to LBP, TNF production was increased in the presence of LBP. In contrast, LBP did not enhance or inhibit TNF production produced by heat-killed Staphylococcus aureus, peptidoglycan isolated from S. aureus cell walls, or PMA. Potentiated M phi responsiveness to LPS was observed with as little as 1 ng LBP/ml. Heat-denatured LBP (which no longer binds LPS), BPI (an homologous LPS-binding protein isolated from neutrophils), or other serum proteins were without effect. LBP-treated M phi also showed a more rapid induction of cytokine mRNA (TNF and IL-1 beta), higher steady-state mRNA levels and increased TNF mRNA stability. These data provide additional evidence that LBP is part of a highly specific recognition system controlling M phi responses to LPS. The effects of LBP are lipid A dependent and importantly, extend to LPS preparations isolated from bacteria of R- and S-form phenotype.     

Lipopolysaccharide-coated erythrocytes activate human neutrophils via CD14 while subsequent binding is through CD11b/CD18

Interaction of LPS with monocytes and neutrophils is known to occur via CD14 and is strongly enhanced by LPS-binding protein (LBP). Integrins as well as CD14 play a role in the interaction of erythrocytes (E) coated with LPS or whole Gram-negative bacteria with phagocytes. We reasoned that the density of LPS on a particle is an important determinant in these interactions. Therefore, E were coated with different concentrations of LPS (ELPS). The binding of these ELPS to neutrophils was evaluated by flow cytometry. Simultaneously, we measured fMLP receptor expression to evaluate neutrophil activation. ELPS only bound to neutrophils in the presence of LBP. Blocking CD14 inhibited both activation and binding, whereas blocking complement (C) receptor 3 (CR3) inhibited binding but not activation. TNF activation restored ELPS binding in CD14-blocked cells but not in cells in which CR3 was blocked. Salmonella minnesota did bind to neutrophils independent of CR3 or CD14. The addition of LBP enhanced binding twofold, and this surplus was dependent upon CD14 but not on CR3. We conclude that ELPS interact with neutrophils via CD14, initially giving rise to cell activation; subsequently, binding is solely mediated by activated CR3.     

Surfactant protein D is expressed and modulates inflammatory responses in human coronary artery smooth muscle cells

Surfactant protein D (SP-D) is a constituent of the innate immune system that plays a role in the host defense against lung pathogens and in modulating inflammatory responses. While SP-D has been detected in extrapulmonary tissues, little is known about its expression and function in the vasculature. Immunostaining of human coronary artery tissue sections demonstrated immunoreactive SP-D protein in smooth muscle cells (SMCs) and endothelial cells. SP-D was also detected in isolated human coronary artery SMCs (HCASMCs) by PCR and immunoblot analysis. Treatment of HCASMCs with endotoxin (LPS) stimulated the release of IL-8, a proinflammatory cytokine. This release was inhibited >70% by recombinant SP-D. Overexpression of SP-D by adenoviral-mediated gene transfer in HCASMCs inhibited both LPS- and TNF-alpha-induced IL-8 release. Overexpression of SP-D also enhanced uptake of Chlamydia pneumoniae elementary bodies into HCASMCs while attenuating IL-8 production induced by bacterial exposure. Both LPS and TNF-alpha increased SP-D mRNA levels by five- to eightfold in HCASMCs, suggesting that inflammatory mediators upregulate the expression of SP-D. In conclusion, SP-D is expressed in human coronary arteries and functions as an anti-inflammatory protein in HCASMCs. SP-D may also participate in the host defense against pathogens that invade the vascular wall.