Surfactant protein A modulates neuroinflammation in adult mice upon pulmonary infection.

BACKGROUND: One of the most common entry gates for systemic infection is the lung. In humans, pulmonary infections can lead to significant neurological impairment, ranging from acute sickness behavior to long-term disorders. Surfactant proteins (SP), essential parts of the pulmonary innate immune defense, have been detected in the brain of rats and humans. Recent evidence suggests that SP-A, the major protein component of surfactant, also plays a functional role in modulating neuroinflammation. This study aimed to determine whether SP-A deficiency affects the inflammatory response in the brain of adult mice during pulmonary infection. EXPERIMENTAL PROCEDURE: Adult male wild-type (WT, n = 72) and SP-A-deficient (SP-A-/-, n = 72) mice were oropharyngeally challenged with lipopolysaccharide (LPS), Pseudomonas aeruginosa (P. aeruginosa), or PBS (control). Both, behavioral assessment and subsequent brain tissue analysis, were performed 24, 48, and 72 h after challenge. The brain concentrations of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß were determined by ELISA. Quantitative rtPCR was used to detect SP-A mRNA expression in brain homogenates and immunohistochemistry was applied for the detection of SP-A protein expression in brain coronal slices. RESULTS: SP-A mRNA and histological evidence of protein expression were detected in both the lungs and brains of WT mice, with significantly higher amounts in lung samples. SP-A-/- mice exhibited significantly higher baseline concentrations of brain TNF-α, IL-6, and IL-1ß compared to WT mice. Oropharyngeal application of either LPS or P. aeruginosa elicited significantly higher brain levels of TNF-α and IL-1ß in SP-A-/- mice compared to WT mice at all time points. In comparison, behavioral impairment as a measure of sickness behavior, was significantly stronger in WT than in SP-A-/- mice, particularly after LPS application. CONCLUSION: SP-A is known for its anti-inflammatory role in the pulmonary immune response to bacterial infection. Recent evidence suggests that in an abdominal sepsis model SP-A deficiency can lead to increased cytokine levels in the brain. Our results extend this perception and provide evidence for an anti-inflammatory role of SP-A in the brain of adult WT mice after pulmonary infection.

Signaling Pathways That Mediate Alveolar Macrophage Activation by Surfactant Protein A and IL-4.

Activation of tissue repair program in macrophages requires the integration of IL-4/IL-13 cytokines and tissue-specific signals. In the lung, surfactant protein A (SP-A) is a tissue factor that amplifies IL-4Rα-dependent alternative activation and proliferation of alveolar macrophages (AMs) through the myosin18A receptor. However, the mechanism by which SP-A and IL-4 synergistically increase activation and proliferation of AMs is unknown. Here we show that SP-A amplifies IL-4-mediated phosphorylation of STAT6 and Akt by binding to myosin18A. Blocking PI3K activity or the myosin18A receptor abrogates SP-A´s amplifying effects on IL-4 signaling. SP-A alone activates Akt, mTORC1, and PKCζ and inactivates GSK3α/ß by phosphorylation, but it cannot activate arginase-1 activity or AM proliferation on its own. The combined effects of IL-4 and SP-A on the mTORC1 and GSK3 branches of PI3K-Akt signaling contribute to increased AM proliferation and alternative activation, as revealed by pharmacological inhibition of Akt (inhibitor VIII) and mTORC1 (rapamycin and torin). On the other hand, the IL-4+SP-A-driven PKCζ signaling axis appears to intersect PI3K activation with STAT6 phosphorylation to achieve more efficient alternative activation of AMs. Consistent with IL-4+SP-A-driven activation of mTORC1 and mTORC2, both agonists synergistically increased mitochondrial respiration and glycolysis in AMs, which are necessary for production of energy and metabolic intermediates for proliferation and alternative activation. We conclude that SP-A signaling in AMs activates PI3K-dependent branched pathways that amplify IL-4 actions on cell proliferation and the acquisition of AM effector functions.

Surfactant Protein A Enhances the Degradation of LPS-Induced TLR4 in Primary Alveolar Macrophages Involving Rab7, ß-arrestin2, and mTORC1.

Respiratory infections by Gram-negative bacteria are a major cause of global morbidity and mortality. Alveolar macrophages (AMs) play a central role in maintaining lung immune homeostasis and host defense by sensing pathogens via pattern recognition receptors (PRR). The PRR Toll-like receptor (TLR) 4 is a key sensor of lipopolysaccharide (LPS) from Gram-negative bacteria. Pulmonary surfactant is the natural microenvironment of AMs. Surfactant protein A (SP-A), a multifunctional host defense collectin, controls LPS-induced pro-inflammatory immune responses at the organismal and cellular level via distinct mechanisms. We found that SP-A post-transcriptionally restricts LPS-induced TLR4 protein expression in primary AMs from healthy humans, rats, wild-type and SP-A-/- mice by further decreasing cycloheximide-reduced TLR4 protein translation and enhances the co-localization of TLR4 with the late endosome/lysosome. Both effects as well as the SP-A-mediated inhibition of LPS-induced TNF-α release are counteracted by pharmacological inhibition of the small GTPase Rab7. SP-A-enhanced Rab7 expression requires ß-arrestin2 and, in ß-arrestin2-/- AMs and after intratracheal LPS challenge of ß-arrestin2-/- mice, SP-A fails to enhance TLR4/lysosome co-localization and degradation of LPS-induced TLR4. In SP-A-/- mice, TLR4 levels are increased after pulmonary LPS challenge. SP-A-induced activation of mechanistic target of rapamycin complex 1 (mTORC1) kinase requires ß-arrestin2 and is critically involved in degradation of LPS-induced TLR4. The data suggest that SP-A post-translationally limits LPS-induced TLR4 expression in primary AMs by lysosomal degradation comprising Rab7, ß-arrestin2, and mTORC1. This study may indicate a potential role of SP-A-based therapeutic interventions in unrestricted TLR4-driven immune responses to lower respiratory tract infections caused by Gram-negative bacteria.

LAMP3 deficiency affects surfactant homeostasis in mice.

Lysosome-associated membrane glycoprotein 3 (LAMP3) is a type I transmembrane protein of the LAMP protein family with a cell-type-specific expression in alveolar type II cells in mice and hitherto unknown function. In type II pneumocytes, LAMP3 is localized in lamellar bodies, secretory organelles releasing pulmonary surfactant into the extracellular space to lower surface tension at the air/liquid interface. The physiological function of LAMP3, however, remains enigmatic. We generated Lamp3 knockout mice by CRISPR/Cas9. LAMP3 deficient mice are viable with an average life span and display regular lung function under basal conditions. The levels of a major hydrophobic protein component of pulmonary surfactant, SP-C, are strongly increased in the lung of Lamp3 knockout mice, and the lipid composition of the bronchoalveolar lavage shows mild but significant changes, resulting in alterations in surfactant functionality. In ovalbumin-induced experimental allergic asthma, the changes in lipid composition are aggravated, and LAMP3-deficient mice exert an increased airway resistance. Our data suggest a critical role of LAMP3 in the regulation of pulmonary surfactant homeostasis and normal lung function.

Local amplifiers of IL-4Rα-mediated macrophage activation promote repair in lung and liver.

The type 2 immune response controls helminth infection and maintains tissue homeostasis but can lead to allergy and fibrosis if not adequately regulated. We have discovered local tissue-specific amplifiers of type 2-mediated macrophage activation. In the lung, surfactant protein A (SP-A) enhanced interleukin-4 (IL-4)-dependent macrophage proliferation and activation, accelerating parasite clearance and reducing pulmonary injury after infection with a lung-migrating helminth. In the peritoneal cavity and liver, C1q enhancement of type 2 macrophage activation was required for liver repair after bacterial infection, but resulted in fibrosis after peritoneal dialysis. IL-4 drives production of these structurally related defense collagens, SP-A and C1q, and the expression of their receptor, myosin 18A. These findings reveal the existence within different tissues of an amplification system needed for local type 2 responses.

Surfactant Protein A Enhances Constitutive Immune Functions of Clathrin Heavy Chain and Clathrin Adaptor Protein 2.

NF-κB transcription factors are key regulators of pulmonary inflammatory disorders and repair. Constitutive lung cell type- and microenvironment-specific NF-κB/inhibitor κBα (IκB-α) regulation, however, is poorly understood. Surfactant protein (SP)-A provides both a critical homeostatic and lung defense control, in part by immune instruction of alveolar macrophages (AMs) via clathrin-mediated endocytosis. The central endocytic proteins, clathrin heavy chain (CHC) and the clathrin adaptor protein (AP) complex AP2, have pivotal alternative roles in cellular homeostasis that are endocytosis independent. Here, we dissect endocytic from alternative functions of CHC, the α-subunit of AP2, and dynamin in basal and SP-A-modified LPS signaling of macrophages. As revealed by pharmacological inhibition and RNA interference in primary AMs and RAW264.7 macrophages, respectively, CHC and α-adaptin, but not dynamin, prevent IκB-α degradation and TNF-α release, independent of their canonical role in membrane trafficking. Kinetics studies employing confocal microscopy, Western analysis, and immunomagnetic sorting revealed that SP-A transiently enhances the basal protein expression of CHC and α-adaptin, depending on early activation of protein kinase CK2 (former casein kinase II) and Akt1 in primary AMs from rats, SP-A(+/+), and SP-A(-/-) mice, as well as in vivo when intratracheally administered to SP-A(+/+) mice. Constitutive immunomodulation by SP-A, but not SP-A-mediated inhibition of LPS-induced NF-κB activity and TNF-α release, requires CHC, α-adaptin, and dynamin. Our data demonstrate that endocytic proteins constitutively restrict NF-κB activity in macrophages and provide evidence that SP-A enhances the immune regulatory capacity of these proteins, revealing a previously unknown pathway of microenvironment-specific NF-κB regulation in the lung.

Postnatal development of the bronchiolar club cells of distal airways in the mouse lung: stereological and molecular biological studies.

Club (Clara) cells are nonciliated secretory epithelial cells present in bronchioles of distal pulmonary airways. So far, no information is available on the postnatal differentiation of club cells by a combination of molecular biological, biochemical, and stereological approaches in the murine lung. Therefore, the present study was designed to investigate the changes in the club cell secretory proteins (CC10, surfactant proteins A, B and D) and club cell abundance within the epithelium of bronchioles of distal airways during the postnatal development of the mouse lung. Perfusion-fixed murine lungs of three developmental stages (newborn, 15-day-old and adult) were used. Frozen, unfixed lungs were used for cryosectioning and subsequent laser-assisted microdissection of bronchiolar epithelial cells and RT-PCR analyses. High resolution analyses of the three-dimensional structures and composition of lung airways were obtained by scanning electron microscopy. Finally, using design-based stereology, the total and average club cell volume and the volume of secretory granules were quantified by light and transmission electron microscopy. Our results reveal that murine club cells are immature at birth and differentiate postnatally. Further, increase of the club cell volume and number of intracellular granules are closely correlated to the total lung volume enlargement. However, secretory granule density was only increased within the first 15 days of postnatal development. The differentiation is accompanied by a decrease in glycogen content, and a close positive relationship between CC10 expression and secretory granule abundance. Taken together, our data are consistent with the concept that the morphological and functional differentiation of club cells is a postnatal phenomenon.

C22-bronchial and T7-alveolar epithelial cell lines of the immortomouse are excellent murine cell culture model systems to study pulmonary peroxisome biology and metabolism.

In pulmonary research, temperature-sensitive immortalized cell lines derived from the lung of the "immortomouse" (H-2k(b)-tsA58 transgenic mouse), such as C22 club cells and T7 alveolar epithelial cells type II (AECII), are frequently used cell culture models to study CC10 metabolism and surfactant synthesis. Even though peroxisomes are highly abundant in club cells and AECII and might fulfill important metabolic functions therein, these organelles have never been investigated in C22 and T7 cells. Therefore, we have characterized the peroxisomal compartment and its associated gene transcription in these cell lines. Our results show that peroxisomes are highly abundant in C22 and T7 cells, harboring a common set of enzymes, however, exhibiting specific differences in protein composition and gene expression patterns, similar to the ones observed in club cells and AECII in situ in the lung. C22 cells contain a lower number of larger peroxisomes, whereas T7 cells possess more numerous tubular peroxisomes, reflected also by higher levels of PEX11 proteins. Moreover, C22 cells harbor relatively higher amounts of catalase and antioxidative enzymes in distinct subcellular compartments, whereas T7 cells exhibit higher levels of ABCD3 and plasmalogen synthesizing enzymes as well as nuclear receptors of the PPAR family. This study suggest that the C22 and T7 cell lines of the immortomouse lung are useful models to study the regulation and metabolic function of the peroxisomal compartment and its alterations by paracrine factors in club cells and AECII.

Lung cell-specific modulation of LPS-induced TLR4 receptor and adaptor localization.

Lung infection by Gram-negative bacteria is a major cause of morbidity and mortality in humans. Lipopolysaccharide (LPS), located in the outer membrane of the Gram-negative bacterial cell wall, is a highly potent stimulus of immune and structural cells via the TLR4/MD2 complex whose function is sequentially regulated by defined subsets of adaptor proteins. Regulatory mechanisms of lung-specific defense pathways point at the crucial role of resident alveolar macrophages, alveolar epithelial cells, the TLR4 receptor pathway, and lung surfactant in shaping the innate immune response to Gram-negative bacteria and LPS. During the past decade intracellular spatiotemporal localization of TLR4 emerged as a key feature of TLR4 function. Here, we briefly review lung cell type- and compartment-specific mechanisms of LPS-induced TLR4 regulation with a focus on primary resident hematopoietic and structural cells as well as modifying microenvironmental factors involved.

Surfactant protein-A modulates LPS-induced TLR4 localization and signaling via ß-arrestin 2.

The soluble C-type lectin surfactant protein (SP)-A mediates lung immune responses partially via its direct effects on alveolar macrophages (AM), the main resident leukocytes exposed to antigens. SP-A modulates the AM threshold of lipopolysaccharide (LPS) activity towards an anti-inflammatory phenotype both in vitro and in vivo through various mechanisms. LPS responses are tightly regulated via distinct pathways including subcellular TLR4 localization and thus ligand sensing. The cytosolic scaffold and signaling protein ß-arrestin 2 acts as negative regulator of LPS-induced TLR4 activation. Here we show that SP-A neither increases TLR4 abundancy nor co-localizes with TLR4 in primary AM. SP-A significantly reduces the LPS-induced co-localization of TLR4 with the early endosome antigen (EEA) 1 by promoting the co-localization of TLR4 with the post-Golgi compartment marker Vti1b in freshly isolated AM from rats and wild-type (WT) mice, but not in ß-arrestin 2(-/-) AM. Compared to WT mice pulmonary LPS-induced TNF-α release in ß-arrestin 2(-/-) mice is accelerated and enhanced and exogenous SP-A fails to inhibit both lung LPS-induced TNF-α release and TLR4/EEA1 positioning. SP-A, but not LPS, enhances ß-arrestin 2 protein expression in a time-dependent manner in primary rat AM. The constitutive expression of ß-arrestin 2 in AM from SP-A(-/-) mice is significantly reduced compared to SP-A(+/+) mice and is rescued by SP-A. Prolonged endosome retention of LPS-induced TLR4 in AM from SP-A(-/-) mice is restored by exogenous SP-A, and is antagonized by ß-arrestin 2 blocking peptides. LPS induces ß-arrestin 2/TLR4 association in primary AM which is further enhanced by SP-A. The data demonstrate that SP-A modulates LPS-induced TLR4 trafficking and signaling in vitro and in vivo engaging ß-arrestin 2.

Pulmonary surfactant protein A enhances endolysosomal trafficking in alveolar macrophages through regulation of Rab7.

Surfactant protein A (SP-A), the most abundant pulmonary soluble collectin, modulates innate and adaptive immunity of the lung, partially via its direct effects on alveolar macrophages (AM), the most predominant intra-alveolar cells under physiological conditions. Enhanced phagocytosis and endocytosis are key functional consequences of AM/SP-A interaction, suggesting a SP-A-mediated modulation of small Rab (Ras related in brain) GTPases that are pivotal membrane organizers in both processes. In this article, we show that SP-A specifically and transiently enhances the protein expression of endogenous Rab7 and Rab7b, but not Rab5 and Rab11, in primary AM from rats and mice. SP-A-enhanced GTPases are functionally active as determined by increased interaction of Rab7 with its downstream effector Rab7 interacting lysosomal protein (RILP) and enhanced maturation of cathepsin-D, a function of Rab7b. In AM and RAW264.7 macrophages, the SP-A-enhanced lysosomal delivery of GFP-Escherichia coli is abolished by the inhibition of Rab7 and Rab7 small interfering RNA transfection, respectively. The constitutive expression of Rab7 in AM from SP-A(-/-) mice is significantly reduced compared with SP-A(+/+) mice and is restored by SP-A. Rab7 blocking peptides antagonize SP-A-rescued lysosomal delivery of GFP-E. coli in AM from SP-A(-/-) mice. Activation of Rab7, but not Rab7b, by SP-A depends on the PI3K/Akt/protein kinase Cζ (PKCζ) signal transduction pathway in AM and RAW264.7 macrophages. SP-A induces a Rab7/PKCζ interaction in these cells, and the disruption of PKCζ by small interfering RNA knockdown abolishes the effect of SP-A on Rab7. The data demonstrate a novel role for SP-A in modulating endolysosomal trafficking via Rab7 in primary AM and define biochemical pathways involved.

Role of clathrin-mediated endocytosis of surfactant protein A by alveolar macrophages in intracellular signaling.

We recently provided evidence that anti-inflammatory macrophage activation, i.e., the inhibition of constitutive and signal-induced NF-kappaB activity by the pulmonary collectin surfactant protein (SP)-A, critically involves a promoted stabilization of IkappaB-alpha, the predominant inhibitor of NF-kappaB, via posttranscriptional mechanisms comprising the activation of atypical (a)PKCzeta. SP-A uptake and degradation by alveolar macrophages (AMphi) occur in a receptor-mediated, clathrin-dependent manner. However, a mutual link between endocytosis of and signaling by SP-A remains elusive. The aim of this study was to investigate whether clathrin-mediated endocytosis (CME) of SP-A by AMphi is a prerequisite for its modulation of the IkappaB-alpha/NF-kappaB pathway. The inhibition of clathrin-coated pit (CCP) formation and clathrin-coated vesicle (CCV) formation/budding abrogates SP-A-mediated IkappaB-alpha stabilization and SP-A-mediated inhibition of LPS-induced NF-kappaB activation in freshly isolated rat AMphi, as determined by Western analysis, fluorescence-activated cell sorting, confocal microscopy, and EMSA. Actin depolymerization and inhibition of CCP formation further abolished SP-A-mediated inhibition of LPS-induced TNF-alpha release, as determined by ELISA. In addition, SP-A-induced atypical PKCzeta activation was abolished by pretreatment of AMphi with CCV inhibitors as determined by in vitro immunocomplex kinase assay. Although CME is classically considered as a means to terminate signaling, our results demonstrate that SP-A uptake via CME by AMphi has to precede the initiation of SP-A signaling.

Pulmonary cytokine responses during mechanical ventilation of noninjured lungs with and without end-expiratory pressure.

BACKGROUND: Positive end-expiratory pressure (PEEP) during mechanical ventilation may impose different degrees of stress on healthy lungs. On the assumption that stress is reflected by cytokine production, we performed a translational study investigating the effect of PEEP on bronchoalveolar and systemic mediator levels in isolated perfused mouse lungs (IPL) and in patients with healthy lungs. METHODS: (Part I) IPL were ventilated with end-expiratory pressures of 0, 3, 6, or 10 cm H2O and end-inspiratory pressure (EIP) levels of 10 or 25 cm H2O. Interleukin (IL)-6 and macrophage inflammatory protein-2 concentrations in the venous effluate were monitored. (Part II) Patients (nonsmokers) scheduled for elective otorhinolaryngology surgery (duration>90 min) were randomized to receive either ventilation with zero end-expiratory pressure or PEEP (10 cm H2O). Mediators in bronchoalveolar lavage, nuclear factor kappaB, (NF-kappaB)-activation in alveolar macrophages and circulating systemic mediators were monitored. Control patients underwent bronchoalveolar lavage after intubation. RESULTS: In the IPL, mediator concentrations increased with increasing end-expiratory pressure at an EIP of 10 cm H2O, but decreased at 25 cm H2O EIP. In patients, bronchoalveolar IL-6, monocyte chemoattractant protein-1, and granulocyte monocyte-colony stimulating factor were increased by ventilation regardless of the PEEP level. IL-6 and IL-8 levels were moderately increased by PEEP but not zero end-expiratory pressure. Nuclear factor kappaB DNA binding activity in alveolar macrophages and systemic mediator levels did not change. CONCLUSIONS: On the basis of the premise that cytokine levels may indicate mechanical stress, our findings indicate that even low tidal volume ventilation causes some stress. PEEP is beneficial at high inspiratory pressure, but imposes moderate stress at low inspiratory pressure.

Surfactant protein A activation of atypical protein kinase C zeta in IkappaB-alpha-dependent anti-inflammatory immune regulation.

The pulmonary collectin surfactant protein (SP)-A has a pivotal role in anti-inflammatory modulation of lung immunity. The mechanisms underlying SP-A-mediated inhibition of LPS-induced NF-kappaB activation in vivo and in vitro are only partially understood. We previously demonstrated that SP-A stabilizes IkappaB-alpha, the primary regulator of NF-kappaB, in alveolar macrophages (AM) both constitutively and in the presence of LPS. In this study, we show that in AM and PBMC from IkappaB-alpha knockout/IkappaB-beta knockin mice, SP-A fails to inhibit LPS-induced TNF-alpha production and p65 nuclear translocation, confirming a critical role for IkappaB-alpha in SP-A-mediated LPS inhibition. We identify atypical (a) protein kinase C (PKC) zeta as a pivotal upstream regulator of SP-A-mediated IkappaB-alpha/NF-kappaB pathway modulation deduced from blocking experiments and confirmed by using AM from PKCzeta-/- mice. SP-A transiently triggers aPKCThr(410/403) phosphorylation, aPKC kinase activity, and translocation in primary rat AM. Coimmunoprecipitation experiments reveal that SP-A induces aPKC/p65 binding under constitutive conditions. Together the data indicate that anti-inflammatory macrophage activation via IkappaB-alpha by SP-A critically depends on PKCzeta activity, and thus attribute a novel, stimulus-specific signaling function to PKCzeta in SP-A-modulated pulmonary immune response.

MaxiK blockade selectively inhibits the lipopolysaccharide-induced I kappa B-alpha /NF-kappa B signaling pathway in macrophages.

Macrophages have a pivotal function in innate immunity against bacterial infections. They are present in all body compartments and able to detect invading microorganisms with high sensitivity. LPS (endotoxin) of Gram-negative bacteria is among the most potent stimuli for macrophages and initiates a wide panel of cellular activation responses. The release of mediators such as TNF-alpha and ILs is essential for the initiation of a proinflammatory antibacterial response. Here, we show that blockade of the large-conductance Ca2+ -activated potassium channel MaxiK (BK) inhibited cytokine production from LPS-stimulated macrophages at the transcriptional level. This inhibitory effect of channel blockade was specific to stimulation with LPS and affected neither stimulation of macrophages with the cytokine TNF-alpha nor LPS-induced activation of cells that do not express MaxiK. Investigation of the upstream intracellular signaling pathways induced by LPS revealed that the blockade of MaxiK selectively inhibited signaling pathways leading to the activation of the transcription factor NF-kappaB and the MAPK p38, whereas activation of ERK was unaffected. We present data supporting that proximal regulation of the inhibitory factor IkappaB-alpha is critically involved in the observed inhibition of NF-kappaB translocation. Using alveolar macrophages from rats, we could show that the necessity of MaxiK function in activation of NF-kappaB and subsequent cytokine production is not restricted to in vitro-generated monocyte-derived macrophages but also can be observed in primary cells. Thus, MaxiK appears to be a central molecule in the NF-kappaB-dependent inflammatory response of macrophages to bacterial LPS.

Cell activation of human macrophages by lipoteichoic acid is strongly attenuated by lipopolysaccharide-binding protein.

Lipoteichoic acid (LTA) represents immunostimulatory molecules expressed by Gram-positive bacteria. They activate the innate immune system via Toll-like receptors. We have investigated the role of serum proteins in activation of human macrophages by LTA from Staphylococcus aureus and found it to be strongly attenuated by serum. In contrast, the same cells showed a sensitive response to LTA and a significantly enhanced production of tumor necrosis factor alpha under serum-free conditions. We show that LTA interacts with the serum protein lipopolysaccharide-binding protein (LBP) and inhibits the integration of LBP into phospholipid membranes, indicating the formation of complexes of LTA and soluble LBP. The addition of recombinant human LBP to serum-free medium inhibited the production of tumor necrosis factor alpha and interleukins 6 and 8 after stimulation of human macrophages with LTA in a dose-dependent manner. Using anti-LBP antibodies, this inhibitory effect could be attributed to soluble LBP, whereas LBP in its recently described transmembrane configuration did not modulate cell activation. Also, using primary alveolar macrophages from rats, we show a sensitive cytokine response to LTA under serum-free culture conditions that was strongly attenuated in the presence of serum. In summary, our data suggest that innate immune recognition of LTA is organ-specific with negative regulation by LBP in serum-containing compartments and sensitive recognition in serum-free compartments like the lung.

A frequent toll-like receptor (TLR)-2 polymorphism is a risk factor for coronary restenosis.

Restenosis is a major problem for patients undergoing percutaneous transluminal coronary angioplasty (PTCA). Inflammatory processes and genetic factors have been suggested to be involved in the pathogenesis of both atherosclerosis and restenosis. The recently discovered family of Toll-like receptors (TLRs) consists of molecules that initiate signaling after host-pathogen interactions. Recently it has been shown that the TLRs are involved in the development and progression of atherosclerosis by interfering with lipid metabolisms and by mediating inflammation. TLR-2 is a key innate immunity receptor for sensing both endogenous inflammatory mediators and ligands of several microbial pathogens postulated to be involved in atherosclerosis. A frequent single nucleotide polymorphism (SNP) for the TLR-2 gene, resulting in a non-functional receptor, has been described. By genotyping two independent groups of patients receiving PTCA, followed by stent implantation in one group, we found a significantly enhanced frequency of the TLR-2 Arg753Gln SNP in patients with restenosis as compared to those without restenosis (PTCA: 7.21 versus 2.45%, P = 0.014; PTCA/stent: 6.86 versus 1.53%, P = 0.013). In contrast, a common TLR-4 SNP was similarly distributed among the patient groups investigated. We furthermore compared the frequency of both SNPs in the patients with an age-matched group of individuals without atherosclerosis and found a trend towards a lower frequency of the TLR-4 SNP in the atherosclerotic group (PTCA: 5.58; PTCA/stent: 3.85 versus 7.14%). We conclude that in restenosis a functional TLR-2 is protective and potentially involved in a reaction pattern preventing restenosis. Screening for the TLR-2 Arg753Gln SNP may be of importance for stratifying a patient's risk and for preventive and therapeutic measures.

Acute-phase concentrations of lipopolysaccharide (LPS)-binding protein inhibit innate immune cell activation by different LPS chemotypes via different mechanisms.

The chain length of bacterial lipopolysaccharide (LPS) is a crucial factor for host-pathogen interaction during bacterial infection. While rough (R)-type and smooth (S)-type LPSs have been shown to differ in their ability to interact with the bactericidal/permeability-increasing protein, little is known about the differential mode of interaction with the acute-phase reactant LPS-binding protein (LBP). At lower concentrations, LBP catalyzes the binding of LPS to CD14 and enhances LPS-induced cellular activation via Toll-like receptor 4. In humans, however, concentrations of LBP in serum increase during an acute-phase response, and these LBP concentrations exhibit inhibitory effects in terms of cellular activation. The mechanisms of inhibition of LPS effects by LBP are not completely understood. Here, we report that human high-dose LBP (hd-LBP) suppresses binding of both R-type and S-type LPS to CD14 and inhibits LPS-induced nuclear translocation of NF-kappaB, although cellular uptake of R-type LPS was found to be increased by hd-LBP. In contrast, we found that hd-LBP enhanced the binding and uptake of S-type LPS only under serum-free conditions, whereas in the presence of serum, hd-LBP inhibited cellular binding and uptake. This inhibitory effect of serum could be mimicked by the addition of purified high-density lipoprotein (HDL) to serum-free medium, indicating an LBP-mediated transfer of preferentially S-type LPS to plasma lipoproteins such as HDL. A complete understanding of the host's mechanisms to modulate the proinflammatory effects of LPS will most likely help in the understanding of inflammation and infection and may lead to novel therapeutic intervention strategies.

Accumulation of inhibitory kappaB-alpha as a mechanism contributing to the anti-inflammatory effects of surfactant protein-A.

The collectin surfactant protein (SP)-A has been implicated in multiple immunoregulatory functions of innate pulmonary host defense via modulating immune responses both in vitro and in vivo. The aim of the present study was to investigate mechanisms responsible for the anti-inflammatory effects of human (hu) SP-A on the inhibitory kappaB (IkappaB)/nuclear factor (NF)-kappaB signaling pathway in alveolar macrophages (AMs). Initial CD25 expression analysis by flow cytometry of CD14/hu Toll-like receptor 4-transfected Chinese hamster ovary reporter cells demonstrated that SP-A alone does not induce any NF-kappaB-dependent CD25 expression in these cells. In AMs, SP-A pretreatment caused a marked inhibition of lipopolysaccharide (LPS)-induced NF-kappaB activation independent of the LPS chemotype used as determined by electrophoretic mobility shift assay. Western blot analysis revealed that SP-A by itself increased the protein expression of IkappaB-alpha, the predominant regulator for rapidly induced NF-kappaB, in a dose- and time-dependent manner without enhancing IkappaB-alpha messenger RNA as determined by reverse transcription-polymerase chain reaction. SP-A did not interfere with LPS-induced serine(32) phosphorylation of IkappaB-alpha but significantly enhanced IkappaB-alpha abundance under LPS-coupled conditions. The data suggest that anti-inflammatory effects of SP-A on LPS-challenged AMs are associated with a SP-A-mediated direct modulation of the IkappaB-alpha turnover in these cells.

Effect of surfactant on ventilation-induced mediator release in isolated perfused mouse lungs.

The human acute respiratory distress syndrome (ARDS) is a severe pulmonary complication with high mortality rates. To support their vital functions, patients suffering from ARDS are mechanically ventilated. Recently it was shown that low tidal volume ventilation reduces mortality and pro-inflammatory mediator release in these patients, suggesting biotrauma as a side effect of mechanical ventilation. Because the application of exogenous surfactant has been proposed as a treatment for ARDS, we investigated the effect of surfactant on ventilation-induced release of tumor necrosis factor (TNF), interleukin-6 (IL-6) and 6-keto-PGF(1 alpha) (the stable metabolite of prostacyclin) in isolated perfused mouse lungs ventilated with high end-inspiratory pressures. Instillation of 100mg/kg surfactant into the lungs was well tolerated and improved tidal volume, pulmonary compliance and alveolar expansion. Exogenous surfactant increased the ventilation-induced liberation of TNF and IL-6 into the perfusate, but had no effect on the release of 6-keto-PGF(1 alpha). The surfactant preparation used reduced baseline TNF production by murine alveolar macrophages, indicating that the exaggeration of ventilation-induced TNF release cannot be explained by a direct effect of surfactant on these cells. We hypothesize that ventilation-induced mediator release is explained by stretching of lung cells, which is reinforced by surfactant. The findings that in this model of ventilation-induced lung injury exogenous surfactant at the same time improved lung functions and enhanced mediator release suggest that surfactant treatment may prevent barotrauma and augment biotrauma.