Established ARDS is often refractory to treatment. Clinical trials have demonstrated modest treatment effects, and mortality remains high. Ventilator strategies must be developed to prevent ARDS.
Early ventilatory intervention will block progression to ARDS if the ventilator mode: 1) maintains alveolar stability and 2) reduces pulmonary edema formation.
Yorkshire Pigs (38–45kg) were anaesthetized and subjected to "2-hit" Ischemia-Reperfusion and Peritoneal Sepsis. Following injury, animals were randomized into two groups: Early Preventative Ventilation (Airway Pressure Release Ventilation- APRV) vs. Non-Preventative Ventilation (NPV) and followed for 48hr. All animals received anesthesia, antibiotics, and fluid/vasopressor therapy per Surviving Sepsis Campaign. Ventilation parameters: 1) NPV Group - Tidal volume (Vt): 10cc/kg + PEEP- 5 cm/H2O volume-cycled mode, 2) APRV Group - Vt: 10–15 cc/kg; Phigh, Plow, Thigh, Tlow were titrated for optimal alveolar stability. Physiologic data and plasma were collected throughout the 48hr study period, followed by BAL and necropsy.
APRV prevented development of ARDS (p<0.001 vs NPV) by PaO2/FiO2 ratio. Quantitative histological scoring showed APRV prevented lung tissue injury (p<0.001 vs. NPV). BALF showed APRV lowered total protein and IL-6, while preserving surfactant proteins A & B (p<0.05 vs. NPV). APRV significantly lowered lung water (p<0.001 vs. NPV). Plasma IL-6 concentrations were similar between groups.
Early preventative mechanical ventilation with APRV blocked ARDS development, preserved surfactant proteins, and reduced pulmonary inflammation and edema, despite systemic inflammation similar to NPV. These data suggest early preventative ventilation strategies stabilizing alveoli and reducing pulmonary edema can attenuate ARDS after ischemia-reperfusion-sepsis.
Sepsis; Shock; ARDS; ALI; Ventilator Induced Lung Injury; Airway Pressure Release Ventilation
The first half of the surfactant protein B (SP-B) gene intron 4 is a CA-repeat-rich region that contains 11 motifs. To study the role of this region on SP-B mRNA splicing, minigenes were generated by systematic removal of motifs from either the 5′ or 3′ end. These were transfected in CHO cells to study their splicing efficiency. The latter was determined as the ratio of completely to incompletely spliced SP-B RNA. Our results indicate that SP-B intron 4 motifs differentially affect splicing. Motifs 8 and 9 significantly enhanced and reduced splicing of intron 4, respectively. RNA mobility shift assays performed with a Motif 8 sequence that contains a CAUC cis-element and cell extracts resulted in a RNA:protein shift that was lost upon mutation of the element. Furthermore, in silico analysis of mRNA secondary structure stability for minigenes with and without motif 8 indicated a correlation between mRNA stability and splicing ratio. We conclude that differential loss of specific intron 4 motifs results in one or more of the following: a) altered splicing, b) differences in RNA stability and c) changes in secondary structure. These, in turn, may affect SP-B content in lung health or disease.
Moths depend on olfactory cues such as sex pheromones to find and recognize mating partners. Pheromone receptors (PRs) and Pheromone binding proteins (PBPs) are thought to be associated with olfactory signal transduction of pheromonal compounds in peripheral olfactory reception. Here six candidate pheromone receptor genes in the diamondback moth, Plutella xyllostella were identified and cloned. All of the six candidate PR genes display male-biased expression, which is a typical characteristic of pheromone receptors. In the Xenopus-based functional study and in situ hybridization, PxylOR4 is defined as another pheromone receptor in addition to the previously characterized PxylOR1. In the study of interaction between PRs and PBPs, PxylPBPs could increase the sensitivity of the complex expressing oocyte cells to the ligand pheromone component while decreasing the sensitivity to pheromone analogs. We deduce that activating pheromone receptors in olfactory receptor neurons requires some role of PBPs to pheromone/PBP complex. If the chemical signal is not the pheromone component, but instead, a pheromone analog with a similar structure, the complex would have a decreased ability to activate downstream pheromone receptors.
Male moths can accurately perceive the sex pheromone emitted from conspecific females by their highly accurate and specific olfactory sensory system. Pheromone receptors are of special importance in moth pheromone reception because of their central role in chemosensory signal transduction processes that occur in olfactory receptor neurons in the male antennae. There are a number of pheromone receptor genes have been cloned, however, only a few have been functionally characterized. Here we cloned six full-length pheromone receptor genes from Helicoverpa armigera male antennae. Real-time PCR showing all genes exhibited male-biased expression in adult antennae. Functional analyses of the six pheromone receptor genes were then conducted in the heterologous expression system of Xenopus oocytes. HarmOR13 was found to be a specific receptor for the major sex pheromone component Z11-16:Ald. HarmOR6 was equally tuned to both of Z9-16: Ald and Z9-14: Ald. HarmOR16 was sensitively tuned to Z11-16: OH. HarmOR11, HarmOR14 and HarmOR15 failed to respond to the tested candidate pheromone compounds. Our experiments elucidated the functions of some pheromone receptor genes of H. armigera. These advances may provide remarkable evidence for intraspecific mating choice and speciation extension in moths at molecular level.
Female mice exhibited higher survival rate than males after pneumonia, with a reversal of this pattern following ozone exposure. Surfactant protein A (SP-A) plays an important role in innate immunity and SP-A (−/−) mice were more susceptible to pneumonia than wild type mice. Here, we investigated underlying mechanisms of the differential susceptibility of mice to pneumonia. Wild type and SP-A (−/−) C57BL/6J male and female mice were exposed to ozone or filtered air (FA) and then infected intratracheally with Klebsiella pneumoniae. Blood, spleen, and lung were analyzed for bacterial counts, lung and spleen weights, and sex hormone and cortisol levels were measured in plasma within two days post-infection. We found: 1) in the absence of ozone-induced oxidative stress, males had higher level of bacterial dissemination compared to females; ozone exposure decreased pulmonary clearance in both sexes and ozone-exposed females were more affected than males; 2) ozone exposure increased lung weight, but decreased spleen weight in both sexes, and in both cases ozone-exposed females were affected the most; 3) plasma cortisol levels in infected mice changed: ozone-exposed > FA-exposed, females > males, and infected > non-infected; 4) no major sex hormone differences were observed in the studied conditions; 5) differences between wild type and SP-A (−/−) mice were observed in some of the studied conditions. We concluded that reduced pulmonary clearance, compromised spleen response to infection, and increased cortisol levels in ozone-exposed females, and the higher level of lung bacterial dissemination in FA-exposed males, contribute to the previously observed survival outcomes.
Pneumonia infection; Lung inflammation; Spleen; Hormones; CFU; Cortisol
The genetic contribution to the development of bronchopulmonary dysplasia (BPD) in prematurely born infants is substantial, but information related to the specific genes involved is lacking. We conducted a case-control single nucleotide polymorphism (SNP) association study of candidate genes (n=601) or 6,324 SNPs in 1,091 prematurely born infants with gestational age <35 weeks, with or without neonatal lung disease including BPD. BPD was defined as need for oxygen at 28 days. Genotype analysis revealed, after multiple comparisons correction, two significant SNPs, rs3771150 (IL-18RAP) and rs3771171 (IL-18R1), in African Americans (AA) with BPD (vs. AA without BPD; q<0.05). No associations with Caucasian (CA) BPD, AA or CA RDS, or prematurity in either AA or CA, were identified with these SNPs. Respective frequencies were 0.098 and 0.093 without BPD and 0.38 for each SNP in infants with BPD. In the replication set (82 cases; 102 controls), the p-values were 0.012 for rs3771150 and 0.07 for rs3771171. Combining p-values using Fisher's method, overall p-values were 8.31E-07 for rs3771150, and 6.33E-06 for rs3771171. We conclude, IL-18RAP and IL-18R1 SNPs identify AA infants at risk for BPD. These genes may contribute to AA BPD pathogenesis via inflammatory-mediated processes and require further study.
Surfactant protein A (SP-A), encoded by two functional genes, SP-A1 and SP-A2, is essential for the inflammatory process and host defence in the lungs. Recent studies have demonstrated the extrapulmonary expression of SP-A. Similar to the lungs, the kidneys are organs exposed to external pathogens. The present study evaluated the expression and location of SP-A in the kidneys. The effect of lipopolysaccharide (LPS) on the expression of SP-A subtypes was also studied in renal tubular epithelial (HK-2) cells.
Immunohistochemical staining was performed using polyclonal antibody against SP-A. RT-PCR was also performed using mRNA from normal human renal tissues and HK-2 cells. The expressions of the SP-A1 and SP-A2 genes were determined by PCR-based RFLP analysis, gene-specific amplification, and direct sequencing of RT-PCR products. Western blot was conducted to analyse the SP-A protein. HK-2 cells were treated with LPS at various concentrations (0, 0.1, 1, 2, 5, and 10 μg/mL) for 8 h and at 5 μg/mL at various time points (0, 2, 4, 8, 16, and 24 h). The LPS-induced expressions of SP-A1 and SP-A2 mRNA and protein were analysed by RT-PCR and Western blot.
SP-A was localised in the renal tubular epithelial cells in the proximal and distal convoluted tubules. SP-A1 and SP-A2 mRNA and protein were expressed in HK-2 cells and human renal tissues, which were significantly increased in time- and dose-dependent manners after LPS treatment (P < 0.05).
Human renal tubular epithelial cells can express both SP-A1 and SP-A2 genes, which may play important roles in the inflammatory modulation of the kidney.
Surfactant protein A1; Surfactant protein A2; Human renal tubular epithelial cells; Lipopolysaccharide; Inflammatory modulation
Antennal olfaction is extremely important for insect survival, mediating key behaviors such as host preference, mate choice, and oviposition site selection. Multiple antennal proteins are involved in olfactory signal transduction pathways. Of these, odorant receptors (ORs) and ionotropic receptors (IRs) confer specificity on olfactory sensory neuron responses. In this study, we identified the olfactory gene repertoire of the economically important agricultural pest moth, Helicoverpa armigera, by assembling the adult male and female antennal transcriptomes. Within the male and female antennal transcriptomes we identified a total of 47 OR candidate genes containing 6 pheromone receptor candidates. Additionally, 12 IR genes as well as 26 odorant-binding proteins and 12 chemosensory proteins were annotated. Our results allow a systematic functional analysis across much of conventional ORs repertoire and newly reported IRs mediating the key olfaction-mediated behaviors of H. armigera.
Aedes aegypti and Anopheles gambiae are among the best-characterized mosquito species within the Culicinae and Anophelinae mosquito clades which diverged ∼150 million years ago. Despite this evolutionary distance, the olfactory systems of these mosquitoes exhibit similar morphological and physiological adaptations. Paradoxically, mosquito odorant receptors, which lie at the heart of chemosensory signal transduction pathways, belong to a large and highly divergent gene family. We have used 2 heterologous expression systems to investigate the functional characteristics of a highly conserved subset of Ors between Ae. aegypti and An. gambiae to investigate whether protein homology correlates with odorant-induced activation. We find that these receptors share similar odorant response profiles and that indole, a common and ecologically relevant olfactory cue, elicits strong responses from these homologous receptors. The identification of other highly conserved members of this Or clade from mosquito species of varying phylogenetic relatedness supports a model in which high sensitivity to indole represents an ancient ecological adaptation that has been preserved as a result of its life cycle importance. These results provide an understanding of how similarities and disparities among homologous OR proteins relate to olfactory function, which can lead to greater insights into the design of successful strategies for the control of mosquito-borne diseases.
Aedes aegypti; Anopheles gambiae; indole; mosquito; odorant-receptor; olfaction; oviposition
It has been shown that female mice with pneumonia have a survival advantage over males, but this is reversed if ozone exposure precedes infection. The purpose of this study was to investigate factors that underlie these observations, by studying histopathologic changes in lung and extrapulmonary (spleen and liver) tissues after ozone or filtered air (FA) exposure followed by pulmonary bacterial infection. Male and female wild type C57BL/6J mice were exposed to ozone or FA, then anesthetized and infected intratracheally with Klebsiella pneumoniae bacteria. Tissues (lung, spleen, and liver) were subjected to histopathologic analysis at 48 h post-infection. We found that after infection, 1) the severity of inflammation was higher, the affected area of the lung was larger, and spleen red pulp myelopoiesis was lower in ozone-exposed mice compared to FA-exposed animals in both sexes; 2) more pronounced extrapulmonary lesions (in liver and spleen) were observed in FA-exposed males compared to FA-exposed females; and 3) excessive lung inflammatory response was detected in ozone-exposed females compared to ozone-exposed males. We concluded that different risk factors contribute to the differential outcome of pneumonia between sexes in the presence or absence of ozone-induced oxidative stress. In specific, the excessive lung inflammation and higher risk for extrapulmonary lesions in ozone-exposed infected females and in FA-exposed infected males appear to play, respectively, a dominant role in the previously observed respective survival outcomes.
Sex; infection; lung inflammation; spleen function; pathology
Many species of mosquitoes, including the major malaria vector Anopheles gambiae, utilize carbon dioxide (CO2) and 1-octen-3-ol as olfactory cues in host-seeking behaviors that underlie their vectorial capacity. However, the molecular and cellular basis of such olfactory responses remains largely unknown.
Here, we use molecular and physiological approaches coupled with systematic functional analyses to define the complete olfactory sensory map of the An. gambiae maxillary palp, an olfactory appendage that mediates the detection of these compounds. In doing so, we identify three olfactory receptor neurons (ORNs) that are organized in stereotyped triads within the maxillary-palp capitate-peg-sensillum population. One ORN is CO2-responsive and characterized by the coexpression of three receptors that confer CO2 responses, whereas the other ORNs express characteristic odorant receptors (AgORs) that are responsible for their in vivo olfactory responses.
Our results describe a complete and highly concordant map of both the molecular and cellular olfactory components on the maxillary palp of the adult female An. gambiae mosquito. These results also facilitate the understanding of how An. gambiae mosquitoes sense olfactory cues that might be exploited to compromise their ability to transmit malaria.
Heat sensitivity is a sensory modality that plays a critical role in close-range host-seeking behaviors of adult female Anopheles gambiae, the principal Afrotropical vector for human malaria. An essential step in this activity is the ability to discriminate and respond to increases in environmental temperature gradients through the process of peripheral thermoreception. Here, we report on the characterization of the anopheline homolog of the transient receptor potential (TRP) A1/ANKTM1 channel that is consistent with its role as a heat-sensor in host-seeking adult female mosquitoes. We identify a set of distal antennal sensory structures that specifically respond to temperature gradients and express AgTRPA1. Functional characterization of AgTRPA1 in Xenopus oocytes supports its role in the molecular transduction of temperature gradients in An. gambiae, providing a basis for targeting mosquito heat responses as a means toward reducing malaria transmission.
coeloconic sensilla; temperature receptor; TRP channel
Innate immunity mechanisms play a critical role in the primary response to invading pathogenic microorganisms and other insulting agents. The innate lung immune system includes lung surfactant, a lipoprotein complex that carries out a function essential for life, that is, reduction of the surface tension at the air–liquid interphase of the alveolar space. By means of this function, pulmonary surfactant prevents lung collapse, therefore ensuring normal lung function and lung health. Pulmonary surfactant contains a number of host-defense molecules that are involved in the elimination of pathogens, viruses, particles, allergens, and other insults, as well as in the control of inflammation. This review is concerned with one of the surfactant proteins, the human (h) surfactant protein A (hSP-A), which, in addition to its role in surfactant-related functions, plays an important role in the modulation of lung host defense. The hSP-A locus has been identified with extensive complexity that may have an impact on its function, structure, and regulation. In humans, two genes—SP-A1 (SFTPA1) and SP-A2 (SFTPA2)—encode SP-A, with SP-A2 gene products being more biologically active than SP-A1 in most of the in vitro assays investigated. Although the two hSP-A genes share a high level of sequence similarity, differences in the structure and function between SP-A1 and SP-A2 have been observed in recent studies. In this review, we discuss the human SP-A complexity and how this may affect SP-A function.
innate immunity; phagocytosis; genetic variants; post-translational modifications; structure–function correlations
Surfactant protein A (SP-A), the most abundant protein in the lung alveolar surface, has multiple activities, including surfactant-related functions. SP-A is required for the formation of tubular myelin and the lung surface film. The human SP-A locus consists of two functional SP-A genes, SP-A1 and SP-A2, with a number of alleles characterized for each gene. We have found that the human in vitro expressed variants, SP-A1 (6A2) and SP-A2 (1A0), and the coexpressed SP-A1/SP-A2 (6A2/1A0) protein have a differential influence on the organization of phospholipid monolayers containing surfactant protein B (SP-B). Lipid films containing SP-B and SP-A2 (1A0) showed surface features similar to those observed in lipid films with SP-B and native human SP-A. Fluorescence images revealed the presence of characteristic fluorescent probe-excluding clusters coexisting with the traditional lipid liquid-expanded and liquid-condensed phase. Images of the films containing SP-B and SP-A1 (6A2) showed different distribution of the proteins. The morphology of lipid films containing SP-B and the coexpressed SP-A1/SP-A2 (6A2/1A0) combined features of the individual films containing the SP-A1 or SP-A2 variant. The results indicate that human SP-A1 and SP-A2 variants exhibit differential effects on characteristics of phospholipid monolayers containing SP-B. This may differentially impact surface film activity.
Surfactant protein A (SP-A) enhances phagocytosis of Pseudomonas aeruginosa. SP-A1 and SP-A2 encode human (h) SP-A; SP-A2 products enhance phagocytosis more than SP-A1. Oxidation can affect SP-A function. We hypothesized that in vivo and in vitro ozone-induced oxidation of SP-A (as assessed by its carbonylation level) negatively affects its function in phagocytosis (as assessed by bacteria cell association). To test this, we used P. aeruginosa, rat alveolar macrophages (AMs), hSP-As with varying levels of in vivo (natural) oxidation, and ozone-exposed SP-A2 (1A, 1A0) and SP-A1 (6A2, 6A4) variants. SP-A oxidation levels (carbonylation) were measured; AMs were incubated with bacteria in the presence of SP-A, and the phagocytic index was calculated. We found: 1) the phagocytic activity of hSP-A is reduced with increasing levels of in vivo SP-A carbonylation; 2) in vitro ozone exposure of hSP-A decreases its function in a dose-dependent manner as well as its ability to enhance phagocytosis of either gram-negative or gram-positive bacteria; 3) the activity of both SP-A1 and SP-A2 decreases in response to in vitro ozone exposure of proteins with SP-A2 being affected more than SP-A1. We conclude that both in vivo and in vitro oxidative modifications of SP-A by carbonylation reduce its ability to enhance phagocytosis of bacteria and that the activity of SP-A2 is affected more by in vitro ozone-induced oxidation. We speculate that functional differences between SP-A1 and SP-A2 exist in vivo and that the redox status of the lung microenvironment differentially affects function of SP-A1 and SP-A2.
air pollution; host defense; carbonylation; macrophage
The mosquito Anopheles gambiae is the major vector of malaria in sub-Saharan Africa. It locates its human hosts primarily through olfaction, but little is known about the molecular basis of this process. Here we functionally characterize the Anopheles gambiae Odourant Receptor (AgOr) repertoire. We identify receptors that respond strongly to components of human odour and that may act in the process of human recognition. Some of these receptors are narrowly tuned, and some salient odourants elicit strong responses from only one or a few receptors, suggesting a central role for specific transmission channels in human host-seeking behavior. This analysis of the Anopheles gambiae receptors permits a comparison with the corresponding Drosophila melanogaster odourant receptor repertoire. We find that odourants are differentially encoded by the two species in ways consistent with their ecological needs. Our analysis of the Anopheles gambiae repertoire identifies receptors that may be useful targets for controlling the transmission of malaria.
A combination of gene silencing and behavioral studies in the malaria vector mosquito Anopheles gambiae sheds light on the olfactory basis of DEET repulsion as well as reveals the role of another family of chemosensory receptors that facilitate olfaction in An. gambiae.
Anopheles gambiae is the principal Afrotropical vector for human malaria, in which olfaction mediates a wide range of both adult and larval behaviors. Indeed, mosquitoes depend on the ability to respond to chemical cues for feeding, host preference, and mate location/selection. Building upon previous work that has characterized a large family of An. gambiae odorant receptors (AgORs), we now use behavioral analyses and gene silencing to examine directly the role of AgORs, as well as a newly identified family of candidate chemosensory genes, the An. gambiae variant ionotropic receptors (AgIRs), in the larval olfactory system. Our results validate previous studies that directly implicate specific AgORs in behavioral responses to DEET as well as other odorants and reveal the existence of at least two distinct olfactory signaling pathways that are active in An. gambiae. One system depends directly on AgORs; the other is AgOR-independent and requires the expression and activity of AgIRs. In addition to clarifying the mechanistic basis for olfaction in this system, these advances may ultimately enhance the development of vector control strategies, targeting olfactory pathways in mosquitoes to reduce the catastrophic effects of malaria and other mosquito-borne diseases.
Anopheles gambiae, the principal Afrotropical mosquito vector for human malaria, uses olfaction to respond to chemical cues that are required for feeding, host preference, and mate selection. At the heart of this process is a large family of An. gambiae odorant receptors (AgORs) that respond to these olfactory cues in peripheral sensory neurons. We have now taken advantage of the relative simplicity of the larval olfactory system to develop a novel behavioral paradigm for individual larva that has been used together with gene silencing approaches in vivo to examine directly the role of AgORs in the olfactory system of An. gambiae. In addition to supporting and extending previous studies on the general olfactory response properties of AgORs, our data directly implicate the activity of specific AgORs in mediating the behavioral responses to the commercial insect repellent DEET and reveal the existence of at least two distinct olfactory signaling pathways in An. gambiae. One system depends directly on AgORs, while the other is AgOR-independent and requires the expression and activity of a newly identified family of candidate chemosensory genes, the An. gambiae variant ionotropic receptors (AgIRs). In addition to clarifying the mechanistic basis for olfaction in this system and the basis of DEET repulsion, these advances may ultimately enhance the development of vector control strategies, targeting olfactory pathways in mosquitoes to reduce the catastrophic effects of malaria and other mosquito-borne diseases.
Surfactant protein A (SP-A) inhibits hemagglutination (HA) activity and infectivity of Influenza A viruses (IAV). As we have showed before in different assays, SP-A2 gene products are more active than SP-A1. Here, we hypothesized that SP-A1 and SP-A2 mammalian CHO-cell-expressed proteins also differentially modulate HA inhibition of IAV. We found that both SP-A1 and SP-A2 equally displayed α(2,3)-linked sialic acids, and had similar activity against a strain (PR-8) that preferentially binds to α(2,3)-linked sialic acids. Based on these Findings, we speculate that in human lung SP-A1 and SP-A2 will not be different in their activity against IAV that preferably bind to α(2,3)-linked sialic acids (like avian strains).
Surfactant protein A variants; Collectins; IAV
Four “core” amino acid differences within the collagen-like domain distinguish the human surfactant proteins A1 (SP-A1) variants from the SP-A2 variants. One of these, cysteine 85 that could form intermolecular disulfide bonds, is present in SP-A1 (Cys85) and absent in SP-A2 (Arg85). We hypothesized that residue85 affects both structure and function of SP-A1 and SP-A2 variants. To test this, wild type (WT) variants, 6A2 of SP-A1 and 1A0 of SP-A2, and their mutants (6A2(C85R) and 1A0(R85C)), were generated and studied. We found: 1) Residue85 affected the binding ability to mannose and the oligomerization pattern of SP-As. The 1A0(R85C) and 6A2(C85R) patterns were similar and/or resembled those of WT 6A2 and 1A0, respectively. 2) Both SP-A WT and mutants differentially induced rough LPS and Pseudomonas aeruginosa aggregation in the following order: 1A0 > 6A2 > 6A2(C85R) > 1A0(R85C) for Re-LPS aggregation, and 1A0 > 6A2 = 6A2(C85R) = 1A0(R85C) for bacterial aggregation. 3) SP-A WT and mutants enhanced phagocytosis of P. aeruginosa by rat alveolar macrophages. Their phagocytic index order was: 6A2(C85R) > 1A0 > 6A2 = 1A0(R85C). The activity of mutant 1A0(C85R) was significantly lower from WT 1A0 but similar to 6A2. Compared to WT 6A2, the 6A2(C85R) mutant exhibited a significantly higher activity. These results indicate that SP-A variant/mutant with Arg85 exhibits higher ability to enhance bacterial phagocytosis than that with Cys85. Residue85 plays a important role in the structure and function of SP-A, and is a major factor for the differences between SP-A1 and SPA2 variants.
Aggregation; Macrophage; Oligomerization; Phagocytosis; Pseudomonas aeruginosa; Surfactant protein A (SP-A); Variant
Surfactant protein A (SP-A) enhances phagocytosis of Pseudomonas aeruginosa. Two functional genes, SP-A1 and SP-A2, encode human SP-A. As we showed before, baculovirus-mediated insect cell-expressed SP-A2 enhances the association of P. aeruginosa with rat alveolar macrophages (rAMs) more than does SP-A1. However, true phagocytosis (internalization) was not shown, and insect cell derived proteins lack or are defective in certain mammalian posttranslational modifications that may be important for SP-A1 and SP-A2 activity and specificity. Here we used SP-A1 (6A2, 6A4) and SP-A2 (1A0, 1A1) allele variants expressed by CHO (Chinese hamster ovary) mammalian cells to study their effect on association and/or internalization of P. aeruginosa by rAMs and/or human AMs (hAMs) and to study if phagocytosis can be modulated differentially and/or more effectively by CHO cell-expressed SP-A variants than by insect-cell expressed SP-A variants. For cell association and internalization assessments, light microscopy and fluorescence-activated cell sorter analyses were used, respectively. We found the following for the first time. (i) SP-A2 variants enhanced phagocytosis (cell association and/or internalization) of P. aeruginosa more than SP-A1 variants did, and the cell association correlated with internalization. (ii) Differences in the activities of SP-A variants were observed in the following order: 1A1>1A0>6A2>6A4. (iii) rAMs, although more active than hAMs, are an appropriate model, as SP-A2 variants exhibited activity higher than that seen for SP-A1 variants with either rAMs or hAMs. (iv) CHO cell-expressed SP-A was considerably more active than insect cell-expressed variants. We conclude that SP-A2 variants stimulate phagocytosis of P. aeruginosa more effectively than SP-A1 variants and that posttranslational modifications positively influence the phagocytic activity of SP-A.
Ozone exposure can cause inflammation and impaired lung function. Human surfactant protein A (SP-A) may play a role in inflammation by modulating cytokine production by macrophages. SP-A is encoded by two genes, SP-A1 and SP-A2, and several allelic variants have been characterized for each gene. These allelic variants differ among themselves in amino acids that may exhibit differential sensitivity to ozone-induced oxidation and this may produce functional differences. We studied the effects of SP-A variants before and after ozone exposure on the production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-8. These are important proinflammatory cytokines and are expressed by the macrophage-like THP-1 cells. Eight variants were expressed in vitro, characterized by gel electrophoresis, and studied. These included six single-gene SP-A alleles and two SP-A variants derived from both genes. Variants were exposed to ozone at 1 ppm for 4 hr at 37 degrees C, and we compared their ability to stimulate cytokine (TNF-alpha and IL-8) production by THP-1 cells to air-exposed and unexposed SP-A variants. We found that a) SP-A2 variants (1A, 1A(0), 1A(1) stimulate significantly more TNF-alpha and IL-8 production than SP-A1 variants (6A, 6A(2), 6A(4); b) coexpressed SP-A variants (1A(0)/6A(2), 1A(1)/6A(4) have significantly higher activity than single gene products; c) after ozone exposure, all SP-A variants showed a decreased ability to stimulate TNF-alpha and IL-8 production, and the level of the decrease varied among SP-A variants (26-48%); and d) human SP-A from patients with alveolar proteinosis exhibited a minimal decrease (18% and 12%, respectively) in its ability to stimulate TNF-alpha and IL-8 after in vitro ozone exposure. We conclude that biochemical and functional differences exist among SP-A variants, that ozone exposure modulates the ability of SP-A variants to stimulate cytokines by THP-1 cells, and that SP-As from bronchoalveolar lavage (BAL) fluid of certain alveolar proteinosis patients may be oxidized in vivo.