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Surfactant protein D binding to alveolar macrophages.

ABSTRACT: Surfactant protein D (SP-D) is a lung-specific protein, synthesized and secreted by lung epithelial cells. It belongs to group III of the family of C-type lectins; each member of this group has an unusual overall structure consisting of multiple globular 'head' regions (which contain the C-type lectin domains) linked by triple-helical, collagen-like, strands. This group includes the surfactant protein A (SP-A) and the serum proteins mannan-binding protein, conglutinin and collectin-43, all of which have been shown to bind to the C1q receptor found on a wide variety of cells, including macrophages. Both SP-D and SP-A have been shown to enhance oxygen radical production by alveolar macrophages. Although this strongly suggests a direct interaction between SP-D and a specific receptor on alveolar macrophages, it is still unclear whether SP-D binds to the same receptor used by SP-A and/or C1q. Human SP-D was isolated from amniotic fluid and was radiolabelled using 125I. Alveolar macrophages were isolated from human bronchioalveolar lavage fluid, and also from bovine lung washings, by differential adhesion to 24-well tissue-culture plates. The study was carried out using EDTA-containing buffers, to eliminate Ca(2+)-dependent C-type lectin binding, and was also carried out at 4 degrees C to eliminate possible internalization by the cells. 125I-SP-D showed specific binding to alveolar macrophages in both a time- and concentration-saturable manner. The binding was inhibited, by approx. 90%, on addition of a 200-fold excess of unlabelled SP-D. The apparent dissociation constant (Kd) was (3.6 +/- 1.3) x 10(-11) M, based on the assumption that native SP-D is assembled as a dodecamer of 12 identical polypeptides of 43 kDa to yield a protein of 516 kDa. C1q was also shown to bind alveolar macrophages (Kd 3 x 10(-6) M), but addition of C1q did not show inhibition of the binding of 125I-SP-D to the macrophages. We conclude that SP-D binds specifically to alveolar macrophages and the receptor involved is different from that utilized by C1q.

SUBMITTER: Miyamura K

PROVIDER: S-EPMC1138147 | biostudies-other | 1994 May

REPOSITORIES: biostudies-other

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Project description:BackgroundSurfactant protein-A (SP-A) plays a critical role in lung innate immunity by regulating alveolar macrophages (AM), expression of inflammatory mediators, and other host defense proteins. The toponome imaging system (TIS), a serial immunostainer, was used to study the AM toponome because it characterizes the localization of multiple markers and identifies marker combinations in each pixel as combinatorial molecular phenotypes (CMPs). We used TIS to study the AM toponome from wild type (WT) and SP-A knockout (KO) mice and changes following Klebsiella pneumoniae exposure.MethodsWT or KO mice received intratracheal K. pneumoniae or vehicle and AM were obtained by bronchoalveolar lavage after one hour. AM were attached to slides and underwent TIS analysis. Images were analyzed to characterize all pixels. AM CMPs from WT vehicle (n=3) and infected (n=3) mice were compared to each other and to AM from KO (n=3 vehicle; n=3 infected). Histograms provided us with a tool to summarize the representation of each marker in a set of CMPs.ResultsUsing the histograms and other tools we identified markers of interest and observed that: 1) Both comparisons had conserved (present in all group members) CMPs, only in vehicle AM and only in infected AM, or common to both vehicle and infected AM, (i.e., unaffected by the condition). 2) the CMP number decreased with infection in WT and KO versus vehicle controls. 3) More infection-specific CMPs in WT vs KO AM. 4) When AM from WT and KO vehicle or infected were compared, there were more unique CMPs exclusive to the KO AM. 5) All comparisons showed CMPs shared by both groups.ConclusionsThe decrease of CMPs exclusive to infected AM in KO mice may underlie the observed susceptibility of KO mice to infection. However, both KO groups had more exclusive CMPs than the corresponding WT groups, perhaps indicating a vigorous effort by KO to overcome deficits in certain proteins and CMPs that are dysregulated by the absence of SP-A. Moreover, the presence of shared CMPs in the compared groups indicates that regulation of these CMPs is not dependent on either infection or the presence or absence of SP-A.

Project description:Surfactant protein A (SP-A) in addition to its surfactant-related functions interacts with alveolar macrophages (AM), the guardian cells of innate immunity in the lungs, and regulates many of its functions under basal condition and in response to various pressures, such as infection and oxidative stress. The human SP-A locus consists of two functional genes, SFTPA1 and SFTPA2, and one pseudogene. The functional genes encode human SP-A1 and SP-A2 proteins, respectively, and each has been identified with several genetic variants. SP-A variants differ in their ability to regulate lung function mechanics and survival in response to bacterial infection. Here, we investigated the effect of hSP-A variants on the AM gene expression profile in response to Klebsiella pneumoniae infection. We used four humanized transgenic (hTG) mice that each carried SP-A1 (6A2, 6A4) or SP-A2 (1A0, 1A3), and KO. AM gene expression profiling was performed after 6 h post-infection. We found: (a) significant sex differences in the expression of AM genes; (b) in response to infection, 858 (KO), 196 (6A2), 494 (6A4), 276 (1A0), and 397 (1A3) genes were identified (P < 0.05) and some of these were differentially expressed with ≥2 fold, specific to either males or females; (c) significant SP-A1 and SP-A2 variant-specific differences in AM gene expression; (d) via Ingenuity Pathway Analysis (IPA), key pathways and molecules were identified that had direct interaction with TP53, TNF, and cell cycle signaling nodes; (e) of the three pathways (TNF, TP-53, and cell cycle signaling nodes) studied here, all variants except SP-A2 (1A3) female, showed significance for at least 2 of these pathways, and KO male showed significance for all three pathways; (f) validation of key molecules exhibited variant-specific significant differences in the expression between sexes and a similarity in gene expression profile was observed between KO and SP-A1. These results reveal for the first time a large number of biologically relevant functional pathways influenced in a sex-specific manner by SP-A variants in response to infection. These data may assist in studying molecular mechanisms of SP-A-mediated AM gene regulation and potentially identify novel therapeutic targets for K. pneumoniae infection.

Project description:ImportanceSurfactant protein C (SP-C) dysfunction is a rare disease associated with interstitial lung disease. Early therapies may improve outcomes but the diagnosis is often delayed owing to variability of manifestations.ObjectiveTo investigate the manifestations and outcomes of SP-C dysfunction.MethodsWe retrospectively analyzed the records of five pediatric patients who were diagnosed with SP-C dysfunction between February 2014 and April 2017 at Beijing Children's Hospital.ResultsThe five patients included two boys and three girls with a median age at diagnosis of 1.3 years. All patients presented with interstitial lung disease and had a heterozygous SFTPC mutation, including an I73T mutation in three patients, a V39L mutation in one patient, and a Y104H mutation in one patient. In addition to common respiratory manifestations, hemoptysis and anemia were observed in one patient with the I73T mutation. Elevated levels of autoantibodies and a large number of hemosiderin-laden macrophages in bronchoalveolar lavage fluid were found in two patients with the I73T mutation, suggesting the presence of diffuse alveolar hemorrage and autoimmunity. Chest high-resolution computed tomography features included ground-glass opacities, reticular opacities, cysts, and pleural thickening. Transbronchial lung biopsy was performed in one patient with the I73T mutation, which revealed the presence of some hemosiderin-laden macrophages in alveolar spaces. All patients received treatment with corticosteroids; two received combined treatment with hydroxychloroquine. During follow-up, the two patients who received hydroxychloroquine showed improved symptoms; of the remaining three patients, two died after their families refused further treatment, while the final patient was lost to follow-up.InterpretationThis is the first report to describe a new phenotype of diffuse alveolar hemorrhage with autoimmunity in patients with I73T SFTPC mutation. Treatment with hydroxychloroquine should be considered for patients with SP-C dysfunction.

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Surfactant protein D binding to alveolar macrophages.

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