Surfactant Proteins A/D-CD14 on Alveolar Macrophages Is a Common Pathway Associated With Phagocytosis of Nanomaterials and Cytokine Production.

Alveolar macrophages are responsible for clearance of airborne dust and pathogens. How they recognize and phagocytose a variety of engineered nanomaterials (ENMs) with different properties is an important issue for safety assessment of ENMs. Surfactant-associated proteins, specifically existing in the pulmonary surfactant, are important opsonins for phagocytosis of airborne microorganisms. The purposes of the current study are to understand whether opsonization of ENMs by surfactant-associated proteins promotes phagocytosis of ENMs and cytokine production, and to determine whether a common pathway for phagocytosis of ENMs with different properties exists. For these purposes, four ENMs, MWCNT-7, TiO2, SiO2, and fullerene C60, with different shapes, sizes, chemical compositions, and surface reactivities, were chosen for this study. Short-term pulmonary exposure to MWCNT-7, TiO2, SiO2, and C60 induced inflammation in the rat lung, and most of the administered ENMs were phagocytosed by alveolar macrophages. The ENMs were phagocytosed by isolated primary alveolar macrophages (PAMs) in vitro, and phagocytosis was enhanced by rat bronchioalveolar lavage fluid (BALF), suggesting that proteins in the BALF were associated with phagocytosis. Analysis of proteins bound to the 4 ENMs by LC/MS indicated that surfactant-associated proteins A and D (SP-A, SP-D) were common binding proteins for all the 4 ENMs. Both BALF and SP-A, but not SP-D, enhanced TNF-α production by MWCNT-7 treated PAMs; BALF, SP-A, and SP-D increased IL-1ß production in TiO2 and SiO2 treated PAMs; and BALF, SP-A, and SP-D enhanced IL-6 production in C60 treated PAMs. Knockdown of CD14, a receptor for SP-A/D, significantly reduced phagocytosis of ENMs and SP-A-enhanced cytokine production by PAMs. These results indicate that SP-A/D can opsonize all the test ENMs and enhance phagocytosis of the ENMs by alveolar macrophages through CD14, suggesting that SP-A/D-CD14 is a common pathway mediating phagocytosis of ENMs. Cytokine production induced by ENMs, however, is dependent on the type of ENM that is phagocytosed. Our results demonstrate a dual role for surfactant proteins as opsonins for both microbes and for inhaled dusts and fibers, including ENMs, allowing macrophages to recognize and remove the vast majority of these particles, thereby, greatly lessening their toxicity in the lung.

Localization, Occurrence, and CSF Changes of SP-G, a New Surface Active Protein with Assumable Immunoregulatory Functions in the CNS.

Conventional surfactant proteins (A, B, C, and D) are important players of the innate immunity in the central nervous system and serve as effective regulators of cerebrospinal fluid rheology, probably being involved in clearance of detrimental metabolites like beta-amyloid and phospho-tau. Recently, a novel surfactant protein, SP-G, was described in kidneys and peripheral endocrine and exocrine glands. So far, its presence and possible functions in the central nervous system are unknown. Therefore, our study aimed to elucidate the presence of SP-G in the brain and its concentration in normal and pathologic samples of cerebrospinal fluid in order to gain first insight into its regulation and possible functions. A total of 121 samples of human cerebrospinal fluid (30 controls, 60 hydrocephalus patients, 7 central nervous system infections, and 24 brain hemorrhage patients) and 21 rat brains were included in our study. CSF samples were quantified using a commercially available ELISA system. Results were analyzed statistically using SPSS 22, performing Spearman Rho correlation and ANOVA with Dunnett's post hoc analysis. Rat brains were investigated via immunofluorescence to determine SP-G presence and colocalization with common markers like aquaporin-4, glial fibrillary acidic protein, platelet endothelial adhesion molecule 1, and neuronal nuclear antigen. SP-G occurs associated with brain vessels, comparable to other conventional SPs, and is present in a set of cortical neurons. SP-G is furthermore actively produced by ependymal and choroid plexus epithelium and secreted into the cerebrospinal fluid. Its concentrations are low in control subjects and patients suffering from aqueductal stenosis, higher in normal pressure hydrocephalus (p < 0.01), and highest in infections of the central nervous system and brain hemorrhage (p < 0.001). Interestingly, SP-G did correlate with total CSF protein in patients with CNS infections and hemorrhage, but not with cell count. Based on the changes in CSF levels of SP-G in hydrocephalus, brain hemorrhage, and CNS infections as well as its abundance at CSF flow-related anatomical structures closely associated with immunological barrier systems, importance for CSF rheology, brain waste clearance, and host defense is assumable. Thus, SP-G is a potential new CSF biomarker, possibly not only reflecting aspects of CNS innate immune responses, but also rheo-dynamically relevant changes of CSF composition, associated with CSF malabsorbtion. However, further studies are warranted to validate our findings and increase insight into the physiological importance of SP-G in the CNS.

The importance of surfactant proteins-New aspects on macrophage phagocytosis.

Surfactant and its components have multiple functions. The so called collectins are surfactant proteins which opsonize bacteria and improve pulmonary host defense via the phagocytosis and clearance of microorganisms and particles. In this special issue of the Annals of Anatomy a new surfactant protein, Surfactant Associated 3, is highlighted. As outlined in this mini review Surfactant Associated 3 is regarded as an enhancer of phagocytosis. In addition, the role played by SP-A is updated and open research questions raised.

Effect of natural porcine surfactant in Staphylococcus aureus induced pro-inflammatory cytokines and reactive oxygen species generation in monocytes and neutrophils from human blood.

Surfacen® is a clinical surfactant preparation of porcine origin. In the present study, we have evaluated the effect of Surfacen® in the modulation of oxidative burst in monocytes and neutrophils in human blood and pro-inflammatory cytokine production in peripheral blood mononuclear cells (PBMC). Reactive oxygen species (ROS) level was measured in monocytes and neutrophils by flow cytometry using 2,7-dichlorofluorescein diacetate (DCFH-DA) as substrate, while, tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels were estimated in PBMC supernatant by enzyme-linked immunosorbent assays (ELISA). Our results show that Staphylococcus aureus-induced ROS level was slightly affected by Surfacen® added to whole blood monocytes and neutrophils. The time course experiments of pre-incubation with Surfacen® showed no significant increase of ROS level at 2h; however, the ROS level decreased when pre incubated for 4h and 6h with Surfacen®. Pre-incubation of PBMC cells with Surfacen® at 0.125 and 0.5mg/mL showed a dose-dependent suppression of TNF-α levels measured after 4h of S. aureus stimulation, an effect less impressive when cells were stimulated for 24h. A similar behavior was observed in IL-6 release. In summary, the present study provides experimental evidence supporting an anti-inflammatory role of Surfacen® in human monocytes and neutrophils in vitro.

Ontogeny of the immune response in the ovine lung.

Perinatal lambs are increasingly appreciated as a model to study respiratory infections of premature and newborn human infants. To explore the relationship between developmental age and immunological competence in the respiratory tract, the basal levels of expression of genes involved in innate and adaptive immune functions in the lung were examined in pre-term lambs (115 days and 130 days), at birth (145 days) and post-partum (15 days and 3 years old). Our results show that innate immune genes (TLRs-3, -4, -7, -8; SP-A, SP-D, and SBD1) were differentially expressed through development; cytokines (IFN-γ, IL-6, TNF-α) and chemokines (IL-8, MCP-1) were low during gestation and post-partum but maximal at birth; genes involved in adaptive immunity (PD-1, PD-L1, TGF-ß) were present in pre-term and newborn lung, but were lower in adult lung. The results suggest that pre-term and neonatal lambs may be able to mount an immune response following infection, but that the response may not be optimal. Our studies provide an important set of comparative data on the ontogeny of lung immunity in sheep and set a framework for studies on age-dependent susceptibility to respiratory pathogens.

Pulmonary surfactant: an immunological perspective.

Pulmonary surfactant has two crucial roles in respiratory function; first, as a biophysical entity it reduces surface tension at the air water interface, facilitating gas exchange and alveolar stability during breathing, and, second, as an innate component of the lung's immune system it helps maintain sterility and balance immune reactions in the distal airways. Pulmonary surfactant consists of 90% lipids and 10% protein. There are four surfactant proteins named SP-A, SP-B, SP-C, and SP-D; their distinct interactions with surfactant phospholipids are necessary for the ultra-structural organization, stability, metabolism, and lowering of surface tension. In addition, SP-A and SP-D bind pathogens, inflict damage to microbial membranes, and regulate microbial phagocytosis and activation or deactivation of inflammatory responses by alveolar macrophages. SP-A and SP-D, also known as pulmonary collectins, mediate microbial phagocytosis via SP-A and SP-D receptors and the coordinated induction of other innate receptors. Several receptors (SP-R210, CD91/calreticulin, SIRPalpha, and toll-like receptors) mediate the immunological functions of SP-A and SP-D. However, accumulating evidence indicate that SP-B and SP-C and one or more lipid constituents of surfactant share similar immuno-regulatory properties as SP-A and SP-D. The present review discusses current knowledge on the interaction of surfactant with lung innate host defense.

Pulmonary surfactant and tuberculosis.

Mycobacterium tuberculosis comes in contact with pulmonary surfactant, alveolar macrophages and type II epithelial cells. Alveolar type II epithelial cells secrete pulmonary surfactant, a complex mixture of phospholipids and proteins lining the alveolar surface, while alveolar macrophages are involved in surfactant catabolism. Surfactant proteins SP-A and SP-D modulate phagocytosis of M. tuberculosis by alveolar macrophages. We have reported that mice with decreased surfactant catabolism resulting from GM-CSF deficiency are highly susceptible to acute aerosol infection with 100 cfu of M. tuberculosis. Here, we evaluated the lungs of WT, GM-CSF-deficient, and GM-CSF-corrected mice surviving six months after sub-acute aerosol infection of 5-10 cfu M. tuberculosis. We show that GM-CSF-deficient mice develop intra-bronchial and intra-alveolar tuberculosis lesions with numerous mycobacteria, inflammatory cells, and extracellular proteinaceous material containing surfactant protein B (SP-B). In contrast, WT and GM-CSF-corrected mice develop typical epithelioid granulomas containing lymphocytes, SP-B positive cells, and M. tuberculosis bacilli inside macrophages. Our findings support the concept that whole pulmonary surfactant is an important component of host mycobacterial infection in the distal lung.

Therapeutic use of surfactant components in allergic asthma.

Pulmonary surfactant is a complex mixture of lipids and proteins that reduces the surface tension at the air-liquid interface. In addition to its biophysical function, some surfactant components play an important role for the innate and adaptive immunity of the lung. A negative modulation of the surfactant function was observed in allergic asthma leading to the assumption that the therapeutic application of surfactant components might be beneficial in this disease. So far, there are a number of preclinical and already some clinical studies demonstrating various effects of different surfactant components that were administered with preventive or therapeutic aim in allergic asthma. This review summarizes the current knowledge on the possibilities to treat allergic asthma with surfactant components.

Host antimicrobial proteins as endogenous immunomodulators.

Host defense mechanisms are multilayered and involve physical as well as chemical barriers, antimicrobial factors as well as a broad set of immunocompetent cells. The mode of action of antimicrobial factors is variable, ranging from opsonisation and agglutination to direct killing of pathogens. In the last years it has become increasingly clear that some of these factors act as endogenous ligands that bind to distinct host receptors, as for example pathogen recognition receptors (PRRs), thereby influencing distinct immunological processes like chemotaxis, modulation of phagocytosis, dendritic cell maturation or the production of cytokines. By that way, these factors are implicated to protect the host by preventing and clearing of microbial infections.

Surfactant proteins in inflammatory skin diseases: controlled study.

Surfactant proteins (SP) have recently been reported to be expressed in human skin tissue. SP is thought to play an essential role in the firstline defense of skin. In this study, we aimed to investigate if the SP may play a role in inflammatory skin diseases. Seven volunteers with psoriasis (n = 3), atopic dermatitis (n = 2), lichen planus (n = 1) and Behcet's disease (n = 1) participated in the study. Biopsies from each lesion and adjacent (approximately 2 cm distant) normal-appearing skin in patients were performed. Expression and localization of the SP-A, -B, -C, and -D in fresh tissues were studied by an immunohistochemical technique. In all patients, there was a weak cytoplasmic staining with SP-A and SP-D and nuclear staining with SP-B and SP-C in the epidermis of normal-appearing skin samples. However, epidermal staining with SP was observed to be stronger in all lesional samples. In addition, there was a prominent staining in inflammatory cells infiltrating dermis. This expression represents a previously unknown immunologic response in the inflammatory skin diseases and may represent an important step in the pathogenesis of these disorders.

Analysis of the immunogenicity and stability of a porcine pulmonary surfactant preparation administered in rabbits.

PURPOSE: To study the immunogenicity and the stability of the porcine pulmonary surfactant preparation produced by the Instituto Butantan. METHOD: Immunogenicity assay: Sixteen New-Zealand-White rabbits (1000 g body weight) were divided into 4 study groups. Each group was assigned to receive either a) Butantan surfactant, b) Survanta (Abbott Laboratories), c) Curosurf (Farmalab Chiesi), or d) no surfactant. The surfactants were administered intratracheally, and the animals were collected immediately before and 60 and 180 days after surfactant administration. Sera were assayed for the presence of antisurfactant antibodies by enzyme-linked immunosorbent assay (ELISA). Stability assay: The Butantan surfactant used in this assay had been stored for one year in the refrigerator (4 to 8 degrees C) and its stability was evaluated in distinct assay conditions using a premature rabbit model. RESULTS: Immunogenicity assay: None of the surfactants analyzed triggered antibody immune responses against their components in any of the animals. Stability assay: The results of this study demonstrate that Butantan surfactant was as effective as Curosurf when both were submitted to the adverse circumstance of short- and long-term storage at room temperature. A similar level of efficacy for the Butantan surfactant, as compared to Curosurf was demonstrated by the pulmonary dynamic compliance, ventilatory pressure, and pressure-volume curve results. CONCLUSION: The results of our study demonstrate that Butantan surfactant may be a suitable alternative for surfactant replacement therapy.

Surfactant protein levels in bronchoalveolar lavage after segmental allergen challenge in patients with asthma.

BACKGROUND: Allergic asthma is associated with airway inflammation and dysfunction of pulmonary surfactant. Because surfactant proteins (SP) account for immunomodulatory functions as well as biophysical functions, we hypothesized that the allergic response in asthma might be accompanied by a dysregulation of SPs. METHODS: We measured levels of SP-A, SP-B, SP-C and SP-D by enzyme-linked immunosorbent assay in bronchoalveolar lavage (BAL) fluid of 23 asthma patients and 10 healthy control subjects under well-controlled conditions before and 24 h after segmental allergen provocation. These data were related to surfactant function, Th(2) cytokine levels in BAL fluid and to the degree of eosinophilic inflammation. RESULTS: In patients with asthma, allergen challenge increased BAL levels of SP-B, SP-C and SP-D while SP-A was decreased. For SP-B and SP-D, a moderate increase was also observed after saline challenge. In contrast, no alterations were observed in healthy control subjects. Levels of SP-B and SP-C in asthmatics correlated with the ratio of small to large surfactant aggregates (SA/LA ratio) and correlated negatively with BAL surface activity. Furthermore, increased SP-C but not SP-B levels after allergen challenge correlated with eosinophil numbers, interleukin (IL)-5, and IL-13 in BAL while increased SP-D levels only correlated with eosinophil numbers. CONCLUSIONS: This study demonstrates significant alterations of all SPs in BAL fluid after allergen challenge of which SP-C was most closely related to surfactant dysfunction and the degree of the allergic inflammation.

Analysis of the immunogenicity and stability of a porcine pulmonary surfactant preparation administered in rabbits

OBJETIVO: Estudar a imunogenicidade e a estabilidade do surfactante de origem porcina produzido pelo Instituto Butantan. MÉTODO: Experimento imunogenicidade: 16 coelhos da raça New-Zealand-White (Peso de 1000g) foram divididos em grupos de 4 animais. Cada grupo foi designado para receber: a) Surfactante do Butantan, b) Survanta® (Abbott Laboratories), c) Curosurf (Farmalab Chiesi) e d) nenhum tratamento com surfactante. Os surfactantes foram administrados via intratraqueal e o sangue dos animais foi coletado antes, 60 e 180 dias após a administração do surfactante. O soro obtido foi analisado quanto a presença de anticorpos anti-surfactante pelo método ELISA (enzyme-linked immunosorbent assay). Experimento estabilidade: O surfactante do Butantan usado neste experimento tinha sido armazenado por um ano em refrigerador (4 a 8°C) e sua estabilidade foi analisada em condições distintas de experimentação, usando o modelo de coelho prematuro. RESULTADOS: Experimento imunogenicidade: Nenhum dos surfactantes analisados determinou a produção de anticorpos contra seus constituintes. Experimento estabilidade: Os resultados deste estudo demonstraram que o surfactante do Instituto Butantan mostrou eficácia semelhante a do Curosurf após ter sido submetido à condições adversas ao longo do tempo. A eficácia foi demonstrada através da complacência pulmonar dinâmica, pressão ventilatória e da curva pressão-volume. CONCLUSÃO: Os resultados deste estudo demonstraram que o surfactante do Instituto Butantan pode representar um tratamento alternativo de reposição de surfactante.

Immunodetection of surfactant proteins in human organ of Corti, Eustachian tube and kidney.

The presence of surfactant proteins was investigated in the human organ of Corti, Eustachian tube and kidney tissues. It has previously been shown that lamellar bodies are present in hairy cells of organ of Corti, in the cytoplasm of secretory and lumen of tubal glands of Eustachian tube and kidney renal basement membrane. No evidence for the presence of surfactant proteins in the organ of Corti and kidney has been presented until now. The aim of this study was to find out if surfactant proteins were expressed in other epithelia such as organ of Corti, Eustachian tube and kidney. Surfactant proteins were identified using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. On one-dimensional Western blots, bands for surfactant protein A in human Eustachian tube (SP-A, 34 kDa) and in kidney extracts, and for surfactant protein D (SP-D, 43 kDa) in Eustachian tube and in kidney extracts (SP-D, 86 kDa), and for surfactant protein B (SP-B, 8 kDa) in human Eustachian tube and organ of Corti extracts were detected. Bands corresponded to monomeric forms of lung surfactant proteins. These results indicate the presence of SP-A and SP-D in kidney epithelium, SP-A, SP-B and SP-D in Eustachian tube and SP-B in the organ of Corti.

Lung surfactant protein D (SP-D) and the molecular diverted descendants: conglutinin, CL-43 and CL-46.

Lung surfactant protein D (SP-D), conglutinin, CL-43 and CL-46 belong to a group of proteins designated collectins that, besides a common structure made of a collagen-like region and a C-type lectin domain, are important components of the innate immune defence. They all bind complex glycoconjugates on microorganisms thereby inhibiting infection, enhancing the clearance by phagocytes and modulating the immune response. In addition, SP-D inhibits the generation of radical oxygen species or the propagation of lipid peroxidation. Knock-out mice deficient in SP-D have a disturbed homeostasis of pulmonary surfactant and suffer from oxidative stress leading to pulmonary inflammation upon microbial challenge. Conglutinin, CL-43 and CL-46 have in contrast to the rest of the collectin family only been found in cattle. During the characterization of the genes encoding conglutinin, CL-43 and CL-46 we observed several features showing that the additional bovine collectins are diverted molecular descendants of an ancestral SP-D gene. Since structural similarity often associates with common functionality, some of SP-D's effector mechanisms may apply to conglutinin, CL-43 and CL-46--and vice versa. This review focus on the structural and functional relationship of this group of collectins.