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1.
Am J Respir Crit Care Med ; 196(12): 1526-1534, 2017 12 15.
Article in English | MEDLINE | ID: mdl-28915062

ABSTRACT

RATIONALE: Recombinant fragment of human surfactant protein D (rfhSP-D) has been shown to suppress house dust mite- and Aspergillus fumigatus-induced allergic inflammation in murine models. OBJECTIVES: We sought to elucidate the effect of rfhSP-D on high-affinity IgE receptor- and CD23-mediated, grass pollen-induced allergic inflammatory responses. METHODS: rfhSP-D, containing homotrimeric neck and lectin domains, was expressed in Escherichia coli BL21(λDE3)pLysS cells. Peripheral blood mononuclear cells and sera were obtained from individuals with grass pollen allergy (n = 27). The effect of rfhSP-D on basophil activation and histamine release was measured by flow cytometry. IgE-facilitated allergen binding and presentation were assessed by flow cytometry. T-helper cell type 2 (Th2) cytokines were measured in cell culture supernatants. The effect of rfhSP-D on IgE production by B cells when stimulated with CD40L, IL-4, and IL-21 was also determined. MEASUREMENTS AND MAIN RESULTS: rfhSP-D bound to Phleum pratense in a dose- and calcium-dependent manner. Allergen-induced basophil responsiveness and histamine release were inhibited in the presence of rfhSP-D, as measured by CD63, CD203c (P = 0.0086, P = 0.04205), and intracellularly labeled diamine oxidase (P = 0.0003, P = 0.0148). The binding of allergen-IgE complexes to B cells was reduced by 51% (P = 0.002) in the presence of rfhSP-D. This decrease was concomitant with reduction in CD23 expression on B cells (P < 0.001). rfhSP-D suppressed allergen-driven CD27-CD4+CRTh2+ T-cell proliferation (P < 0.01), IL-4, and IL-5 levels (all P < 0.01). Moreover, rfhSP-D inhibited CD40L/IL-4- and IL-21-mediated IgE production (77.12%; P = 0.02) by B cells. CONCLUSIONS: For the first time, to our knowledge, we show that rfhSP-D inhibited allergen-induced basophil responses at a single-cell level and suppressed CD23-mediated facilitated allergen presentation and Th2 cytokine production. In addition, rfhSP-D inhibited IgE synthesis by B cells, which is also a novel observation.


Subject(s)
B-Lymphocytes/immunology , Basophils/immunology , Hypersensitivity/immunology , Inflammation/immunology , Poaceae/immunology , Pollen/immunology , Pulmonary Surfactant-Associated Protein D/immunology , Adult , Allergens/blood , Allergens/immunology , Female , Flow Cytometry , Humans , Hypersensitivity/blood , Hypersensitivity/prevention & control , Immunoglobulin E/blood , Immunoglobulin E/immunology , Inflammation/blood , Inflammation/prevention & control , Male , Middle Aged , Pulmonary Surfactant-Associated Protein D/blood , Receptors, IgE/blood , Receptors, IgE/immunology , Th2 Cells , Young Adult
2.
PLoS One ; 9(7): e102395, 2014.
Article in English | MEDLINE | ID: mdl-25036364

ABSTRACT

Surfactant Protein SP-D, a member of the collectin family, is a pattern recognition protein, secreted by mucosal epithelial cells and has an important role in innate immunity against various pathogens. In this study, we confirm that native human SP-D and a recombinant fragment of human SP-D (rhSP-D) bind to gp120 of HIV-1 and significantly inhibit viral replication in vitro in a calcium and dose-dependent manner. We show, for the first time, that SP-D and rhSP-D act as potent inhibitors of HIV-1 entry in to target cells and block the interaction between CD4 and gp120 in a dose-dependent manner. The rhSP-D-mediated inhibition of viral replication was examined using three clinical isolates of HIV-1 and three target cells: Jurkat T cells, U937 monocytic cells and PBMCs. HIV-1 induced cytokine storm in the three target cells was significantly suppressed by rhSP-D. Phosphorylation of key kinases p38, Erk1/2 and AKT, which contribute to HIV-1 induced immune activation, was significantly reduced in vitro in the presence of rhSP-D. Notably, anti-HIV-1 activity of rhSP-D was retained in the presence of biological fluids such as cervico-vaginal lavage and seminal plasma. Our study illustrates the multi-faceted role of human SP-D against HIV-1 and potential of rhSP-D for immunotherapy to inhibit viral entry and immune activation in acute HIV infection.


Subject(s)
CD4 Antigens/metabolism , Cytokines/biosynthesis , HIV Envelope Protein gp120/metabolism , HIV-1/drug effects , Pulmonary Surfactant-Associated Protein D/pharmacology , Adult , CD4 Antigens/chemistry , Cervix Uteri/virology , Cytokines/metabolism , Female , HIV Envelope Protein gp120/chemistry , HIV-1/metabolism , HIV-1/physiology , Humans , Inflammation/metabolism , Jurkat Cells , Male , Mitogen-Activated Protein Kinases/metabolism , Molecular Docking Simulation , Monocytes/drug effects , Monocytes/virology , Phosphorylation/drug effects , Protein Binding/drug effects , Protein Conformation , Proto-Oncogene Proteins c-akt/metabolism , Pulmonary Surfactant-Associated Protein D/chemistry , Pulmonary Surfactant-Associated Protein D/metabolism , Semen/virology , T-Lymphocytes/drug effects , T-Lymphocytes/virology , Vagina/virology , Virus Internalization/drug effects
3.
Methods Mol Biol ; 1100: 257-72, 2014.
Article in English | MEDLINE | ID: mdl-24218266

ABSTRACT

Surfactant protein SP-A is a hydrophilic glycoprotein, similar to SP-D, which plays an important role in pulmonary surfactant homeostasis and innate immunity. SP-A is actively expressed in the alveolar type II cells and Clara cells. Their basic structure consists of triple-helical collagen region and a C-terminal carbohydrate recognition domain (CRD). By binding to the infectious microbes, SP-A (like SP-D) are involved in pathogen opsonization and agglutination and subsequent clearance of the microorganism, via recruitment of phagocytic cells via receptors for the collagen region. SP-A has also been localized at extra-pulmonary sites such as salivary epithelium, amniotic fluid, prostate glands, and semen. The presence of SP-A in fetal and maternal tissue and amniotic fluid suggests it is involved in pregnancy and labor. Native SP-A can be purified from amniotic fluid and bronchiolar lavage fluid (BALF) via affinity chromatography. In addition, we also report here a procedure to express and purify a recombinant form of trimeric CRD in Escherichia coli. The availability of highly pure native SP-A and CRD region can be central to studies that examine the diverse roles that SP-A play in surfactant homeostasis, pulmonary infection and inflammation and pregnancy.


Subject(s)
Amniotic Fluid/chemistry , Bronchoalveolar Lavage Fluid/chemistry , Pulmonary Surfactant-Associated Protein A/isolation & purification , Batch Cell Culture Techniques , Chromatography, Affinity , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Protein Denaturation , Protein Refolding , Pulmonary Surfactant-Associated Protein A/biosynthesis , Pulmonary Surfactant-Associated Protein A/chemistry , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification
4.
Methods Mol Biol ; 1100: 273-90, 2014.
Article in English | MEDLINE | ID: mdl-24218267

ABSTRACT

Surfactant protein SP-D is a multimeric collagenous lectin, called collectin. SP-D is a multifunctional, pattern recognition innate immune molecule, which binds in a calcium dependent manner to an array of carbohydrates and lipids, thus offering resistance to invading pathogens, allergen challenge, and pulmonary inflammation. SP-D is predominantly found in the endoplasmic reticulum of type 2 pneumocytes and in the secretory granules of Clara or non-ciliated bronchiolar cells. The highest expression of SP-D is observed in the distal airways and alveoli. There is also an extra pulmonary existence of SP-D. The common sources of native full-length human SP-D are bronchoalveolar lavage (BAL) washings from normal or preferably patients suffering from alveolar proteinosis who overproduce SP-D in the lungs. Amniotic fluid collected at the term during parturition is another reasonable source. Here, we describe a simple and rapid method of purifying native SP-D away from SP-A which is also present in the same source. We also describe procedures of expressing and purifying a recombinant fragment of human SP-D (rhSP-D) comprising trimeric neck and carbohydrate recognition domains that has been shown to have therapeutic effects in murine models of allergy and infection.


Subject(s)
Amniotic Fluid/chemistry , Bronchoalveolar Lavage Fluid/chemistry , Pulmonary Surfactant-Associated Protein D/isolation & purification , Chromatography, Affinity/methods , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Humans , Pulmonary Surfactant-Associated Protein D/biosynthesis , Pulmonary Surfactant-Associated Protein D/chemistry , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification
5.
Front Biosci (Landmark Ed) ; 18(3): 1129-40, 2013 06 01.
Article in English | MEDLINE | ID: mdl-23747872

ABSTRACT

Surfactant Protein A (SP-A) and D (SP-D) are calcium-dependent collagen-containing lectins, also called collectins, which play a significant role in surfactant homeostasis and pulmonary immunity. The role of SP-A and SP-D in immune defence is well- established. They are known to bind to a range of microbial pathogens that invade the lungs and target them for phagocytic clearance by resident alveolar macrophages. They are also involved in the clearance of apoptotic and necrotic cells and subsequent resolution of pulmonary inflammation. To date, the molecular mechanisms by which SP-A and SP-D interact with various immune cells are poorly understood. In spite of overall structural similarity, SP-A and SP-D show a number of functional differences in their interaction with surface molecules of microorganisms and host cells. The aim of this review is to provide an overview of the current knowledge of ligands and receptors that are known to interact with SP-A and SP-D.


Subject(s)
Pulmonary Surfactant-Associated Protein A/metabolism , Pulmonary Surfactant-Associated Protein D/metabolism , Receptors, Cell Surface/metabolism , Humans , Ligands
6.
Mol Immunol ; 54(1): 98-107, 2013 May.
Article in English | MEDLINE | ID: mdl-23220073

ABSTRACT

Surfactant protein D (SP-D) is an innate immune molecule that plays a protective role against lung infection, allergy, asthma and inflammation. In vivo experiments with murine models have shown that SP-D can protect against allergic challenge via a range of mechanisms including inhibition of allergen-IgE interaction, histamine release by sensitised mast cells, downregulation of specific IgE production, suppression of pulmonary and peripheral eosinophilia, inhibition of mechanisms that cause airway remodelling, and induction of apoptosis in sensitised eosinophils. SP-D can also shift helper T cell polarisation following in vivo allergenic challenge, from pathogenic Th2 to a protective Th1 cytokine response. Interestingly, SP-D gene deficient (-/-) mice show an IL-13 over-expressing phenotype. IL-13 has been shown to be involved in the development of asthma. Transgenic mice over-expressing IL-13 in the lung develop several characteristics of asthma such as pulmonary eosinophilia, airway epithelial hyperplasia, mucus cell metaplasia, sub-epithelial fibrosis, charcot-Leyden-Like crystals, airways obstruction, and non-specific airways hyper-responsiveness to cholinergic stimulation. Although both IL-4 and IL-13 are capable of inducing asthma like phenotype, the effector activity of IL-13 appears to be greater than that of IL-4. SP-D -/- mice seem to express considerably higher levels of IL-13, which is consistent with increased sensitivity and exaggerated immune response of the mice to allergenic challenge. Allergenic exposure also induces elevation in SP-D protein levels in an IL-4/IL-13-dependent manner, which prevents further activation of sensitised T cells. This negative feedback loop seems essential in protecting the airways from inflammatory damage after allergen inhalation. Here, we examine this link between IL-13 and SP-D, and its implications in the progression/regulation of asthma and allergy.


Subject(s)
Asthma/genetics , Hypersensitivity/genetics , Interleukin-13/physiology , Pulmonary Surfactant-Associated Protein D/physiology , Animals , Asthma/immunology , Eosinophils/immunology , Eosinophils/metabolism , Eosinophils/physiology , Gene Expression Regulation/genetics , Gene Expression Regulation/immunology , Humans , Hypersensitivity/immunology , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Interleukin-13/genetics , Interleukin-13/metabolism , Mice , Models, Biological , Pulmonary Surfactant-Associated Protein D/genetics , Pulmonary Surfactant-Associated Protein D/metabolism
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