Structural characterisation of ligand-binding determinants in human lung surfactant protein D: influence of Asp325.

The crystal structures of a biologically and therapeutically active recombinant homotrimeric fragment of human lung surfactant protein D with a series of bound ligands have been determined. While the structures reveal various different binding modes, all utilise a similarly positioned pair of mannose-type O3' and O4' hydroxyls with no direct interaction between any non-terminal sugar and protein. The orientation, position, and interactions of the bound terminal sugar depend on the sugar itself, the presence and form of glycosidic linkage, and the environment in the crystal, which, via Asp325, places stereochemical and electronic constraints, different for the three different subunits in the homotrimer, on the ligand-binding site. As a direct consequence of this influence, the other binding-pocket flanking residue, Arg343, exhibits variable conformation and variable interactions with bound ligand and leaves open to question which orientation of terminal mannobiose, and of other terminal disaccharides, may be present in extended physiological ligands. The combined structural evidence shows that there is significant flexibility in recognition; that Asp325, in addition to Arg343, is an important determinant of ligand selectivity, recognition, and binding; and that differences in crystal contact interfaces exert, through Asp325, significant influence on preferred binding modes.

Elevated plasma surfactant protein D (SP-D) levels and a direct correlation with anti-severe acute respiratory syndrome coronavirus-specific IgG antibody in SARS patients.

Pulmonary SP-D is a defence lectin promoting clearance of viral infections. SP-D is recognized to bind the S protein of SARS-CoV and enhance phagocytosis. Moreover, systemic SP-D is widely used as a biomarker of alveolar integrity. We investigated the relation between plasma SP-D, SARS-type pneumonia and the SARS-specific IgG response. Sixteen patients with SARS, 19 patients with community-acquired pneumonia (CAP) (Streptococcus pneumonia) and 16 healthy control subjects were enrolled in the study. Plasma SP-D and anti-SARS-CoV N protein IgG were measured using ELISA. SP-D was significantly elevated in SARS-type pneumonia [median (95% CI), 453 (379-963) ng/ml versus controls 218 (160-362) ng/ml, P < 0.05] like in patients with CAP. SP-D significantly correlated with anti-SARS-CoV N protein IgG (r(2) = 0.5995, P = 0.02). The possible re-emergence of SARS or SARS-like infections suggests a need for minimal traumatic techniques for following the alveolar compartment, e.g. during testing of antivirals. We suggest that monitoring systemic SP-D may be useful in monitoring the alveolar integrity in SARS-type pneumonia. The significant correlation between plasma SP-D and anti-SARS-CoV-specific antibodies support the role for SP-D in interlinking innate and adaptive immune pathways.

Role of collectins in innate immunity against aspergillosis.

The protective role of lung surfactant proteins SP-A, SP-D and MBL in the host defense against both allergic and invasive aspergillosis was identified and established by a series of in vitro and in vivo studies. Therapeutic administration of SP-D and MBL proteins in a murine model of pulmonary invasive aspergillosis rescued mice from death. In mice mimicking human allergic bronchopulmonary aspergillosis, SP-A and SP-D suppressed IgE levels, eosinophilia, pulmonary cellular infiltration and cause a marked shift from a pathogenic Th2 to a protective Th1 cytokine profile. SP-A and SP-D knock-out mice studies made significant contributions in understanding the mechanisms by which SP-A and SP-D modulate the host defense response in patients suffering from pulmonary allergies and infections. The results suggested that individuals with any structural or functional defects in these innate immune molecules due to genetic variations might be susceptible to aspergillosis. SNPs in SP-A2 and MBL genes showed significant associations with patients of allergic bronchopulmonary aspergillosis in an Indian population. The patients carrying either one or both of GCT and AGG alleles of SP-A2 and patients with A allele at position 1011 of MBL had markedly higher eosinophilia, total IgE antibodies and lower FEV1 (the clinical markers of ABPA). Our results show that collectins play an important role in Aspergillus mediated allergies and infections.

Therapeutic effect of surfactant protein D in allergic inflammation of mite-sensitized mice.

BACKGROUND: Surfactant protein D (SP-D) is involved in the innate immunity within the lung and may have important roles in modulating the inflammatory process of asthma. OBJECTIVE: To examine the potential immunomodulating role of SP-D on the allergic response in mice, and its interaction with the alveolar macrophages (AMs) during allergic inflammation. METHODS: A recombinant 60 kDa fragment of human SP-D (rfh SP-D), Survanta, and budesonide were administrated, respectively, to Der p-sensitive BALB/c mice before or after allergen challenge (AC). Total and differential cell counts, levels of cytokines in bronchoalveolar lavage fluids(BALFs), and levels of Der p-specific IgE and IgG1 antibodies in sera, were assayed. The production of nitric oxide (NO), and inducible NO synthase (iNOS) expression, in AMs, were determined by ELISA and RT-PCR, respectively. RESULTS: Instillation of rfh SP-D to sensitized mice 6 h after AC (therapeutic), but not 24 h before AC (preventive), markedly reduced infiltration of eosinophils, and also reduced levels of IL-4, IL-5, eotaxin, and TNF-alpha but elevated levels of IFN-gamma in the BALF. These effects were comparable with those obtained with budesonide treatment, whereas Survanta did not have a suppressive effect, either before or after AC. There was significant inhibition of NO production in the rfh SP-D pre-treated AMs of allergen-sensitized mice, but not in naive mice. CONCLUSIONS: These results indicate that rfh SP-D has a therapeutic effect on allergen-induced bronchial inflammation, and that this might be because of its inhibitory effect on NO and TNF-alpha production by AMs, and it thus prevents the development of T-helper type 2 cytokine response.

Mite allergen induces nitric oxide production in alveolar macrophage cell lines via CD14/toll-like receptor 4, and is inhibited by surfactant protein D.

BACKGROUND: Previously, we have found that dust mite allergens can directly activate alveolar macrophages (AMs), induce inflammatory cytokines, and enhance T-helper type 2 cytokine production. A molecule of innate immunity in the lung, surfactant protein D (SP-D), is able to bind mite allergens and alleviates allergen-induced airway inflammation. OBJECTIVES: This study was aimed at investigating the activation pathway of mite allergen (Dermatophagoides pteronyassinus, Der p)-induced nitric oxide (NO) production by AMs, and the role of SP-D in the modulation of activated AMs by mite allergens. METHODS: Porcine SP-D was purified from bronchoalveolar lavage fluids of Lan-Yu mini-pigs, by affinity chromatography on maltose-sepharose. NO production, inducible expression of lipopolysaccharides (LPS)-related binding and responding surface receptors complex, CD14 and toll-like receptor 4 (TLR4), as well as inducible NO synthase (iNOs) and nuclear factor-kappaB activation were studied in two AMs cell lines, MH-S (BALB/c strain),and AMJ2-C11 (C57BL/6 strain), and one peritoneal macrophage cell line (RAW264.7), after stimulation with LPS, or Der p. RESULTS: LPS and Der p elicited different responses of NO production in the different cell lines, and the response might depend upon the expression of the cell surface CD14/TLR4 complex in different genetic backgrounds of macrophage cell lines. Pretreatment of macrophages with SP-D could inhibit NO production from Der p or LPS-stimulated alveolar macrophages. CONCLUSION: Mite allergen-induced alveolar macrophage activation is mediated by CD14/TLR4 receptors and can be inhibited by SP-D; it further supports the concept that SP-D may be an important modulator of allergen-induced pulmonary inflammation.

A recombinant fragment of human SP-D reduces allergic responses in mice sensitized to house dust mite allergens.

C57Bl6 mice sensitized to Dermatophagoides pteronyssinus and challenged with D. pteronyssinus allergen extract given intranasally followed by treatment with intranasal applications of a 60-kDa truncated, trimeric recombinant form of human SP-D (rfhSP-D) showed a significant reduction in serum IgE, IgG1, peripheral blood eosinophilia and airway hyperresponsiveness compared to saline or bovine serum albumin-treated controls. Intracellular cytokine staining of lung and spleen homogenates showed increases in interleukin (IL)-12 production in lung tissue and normalization of IL-12 and interferon (IFN)-gamma in spleen tissue. In previous studies we demonstrated the effectiveness of native SP-D and rfhSP-D in down-regulating allergic responses to allergens of Aspergillus fumigatus. The results reported here indicate that rfhSP-D can suppress the development of allergic symptoms in sensitized mice challenged with allergens of the common house dust mite.

The scavenger receptor, cysteine-rich domain-containing molecule gp-340 is differentially regulated in epithelial cell lines by phorbol ester.

Gp-340 is a glycoprotein belonging to the scavenger receptor cysteine rich (SRCR) group B family. It binds to host immune components such as lung surfactant protein D (SP-D). Recent studies found that gp-340 interacts directly with pathogenic microorganisms and induces their aggregation, suggesting its involvement in innate immunity. In order to investigate further its potential immune functions in the appropriate cell lines, the expression of gp-340 in four conventional immune cell lines (U937, HL60, Jurkat, Raji), and two innate immune-related epithelial cell lines (A549 derived from lung and AGS from stomach), was examined by RT-PCR and immunohistochemistry. The resting immune cell lines showed weak or no gp-340 mRNA expression; while the two epithelial cell lines expressed gp-340 at much higher level, which was differentially regulated by phorbol myristate acetate (PMA) treatment. In the A549 cells, gp-340 was up-regulated along with the PMA-induced proinflammatory expression of both IL-6 and IL-8. In AGS cells, PMA down-regulation of gp-340 was seen in parallel with an up-regulation of the two mature gastric epithelial specific proteins TFF1 (trefoil factor 1) and TFF2, which are implicated as markers of terminal differentiation. Analysis of the distribution of gp-340, together with the TFFs and SP-D in normal lung and gastric mucosa, supported further our in vitro data. We conclude that the differential regulation of gp-340 in the two epithelial cell lines by PMA indicates that gp-340 s involvement in mucosal defence and growth of epithelial cells may vary at different body locations and during different stages of epithelial differentiation.

Intranasal delivery of a truncated recombinant human SP-D is effective at down-regulating allergic hypersensitivity in mice sensitized to allergens of Aspergillus fumigatus.

C57BL/6 mice were sensitized to Aspergillus fumigatus 1-week culture filtrate, which is rich in the non-glycosylated allergen Asp f1, a major allergen in allergic bronchopulmonary aspergillosis (ABPA). A comparison of the effect of treatment of allergen challenged mice by intranasal administration of a 60-kDa truncated recombinant form of human SP-D (rfhSP-D) or recombinant full length SP-A (rhSP-A) was undertaken. Treatment with rfhSP-D produced significant reduction in IgE, IgG1 and peripheral blood eosinophilia and treatment with rfhSP-D, but not rhSP-A resulted in a significant reduction in airway hyperresponsiveness as measured by whole body plethysmography. Lung histology revealed less peribronchial lymphocytic infiltration in mice treated with rfhSP-D. Intracellular cytokine staining of spleen homogenates showed increases in IL-12 and IFN-gamma and decrease in IL-4. The level of endogenous mouse SP-D was elevated sixfold in the lungs of sensitized mice and was not affected by treatment with rfhSP-D. Taken with our previous studies, with a BALB/c mouse model of ABPA using a 3-week A. fumigatus culture filtrate, the present results show that rfhSP-D can suppress the development of allergic symptoms in sensitized mice independent of genetic background and using a different preparation of A. fumigatus allergens.

Expression and characterisation of the thrombospondin type I repeats of human properdin.

Properdin, an upregulator of the alternative complement pathway, is central to deposition of the activated complement fragment C3b on the surfaces of the pathogens, which it achieves by preventing the dissociation of the Bb catalytic subunit from the inherently labile C3bBb complexes. It is also known to bind sulphated glycoconjugates, such as sulphatides. Properdin has an unusual structure formed by oligomerisation of a rod-like monomer into cyclic dimers, trimers and tetramers. The monomer (approximately 53 kDa) contains an N-terminal region of no known homology, followed by six non-identical repeats of 60 amino acids (based on exon/intron boundaries), called 'thrombospondin type I repeats' or TSR modules. We have expressed and purified the N-terminal region and each of the individual TSR repeats in Escherichia coli. Although the individual recombinant TSRs, after a denaturation-renaturation cycle, appeared to be correctly folded modules, as judged by the one-dimensional (1D)- and 2D-nuclear magnetic resonance spectra of TSR3, they did not show binding to either C3b or sulphatide. Polyclonal antibodies were raised against each TSR and were found to be module-specific. The anti-TSR5 polyclonal antibody was found to inhibit binding of native human properdin to solid-phase C3b, or sulphatides. It could also block properdin-dependent haemolysis of rabbit erythrocytes. These results are consistent with the view that the TSR5 contains the major site in properdin which is involved in both C3b and sulphatide binding. It also suggests that a co-operative intramolecular interaction between TSRs, as found in the native molecule, is required for TSR5 to bind either C3b or sulphatides.

Protective role of lung surfactant protein D in a murine model of invasive pulmonary aspergillosis.

The protective effects of intranasal administration of amphotericin B (AmB), human SP-A, SP-D and a 60-kDa fragment of SP-D (rSP-D) were examined in a murine model of invasive pulmonary aspergillosis (IPA). The untreated group of IPA mice showed no survival at 7 days postinfection. Treatment with AmB, SP-D, and rSP-D increased the survival rate to 80, 60, and 80%, respectively, suggesting that SP-D (and rSP-D) can protect immunosuppressed mice from an otherwise fatal challenge with Aspergillus fumigatus conidia.

Surfactant proteins A and D protect mice against pulmonary hypersensitivity induced by Aspergillus fumigatus antigens and allergens.

Allergic bronchopulmonary aspergillosis (ABPA) is an allergic disorder caused by an opportunistic fungal pathogen, Aspergillus fumigatus (AFU:). Lung surfactant proteins SP-A and SP-D can interact with the glycosylated antigens and allergens of AFU:, inhibit specific IgE binding to these allergens, and block histamine release from sensitized basophils. We have now examined the therapeutic effect of exogenous administration of human SP-A, SP-D, and a recombinant fragment of SP-D (rSP-D), in a murine model of pulmonary hypersensitivity induced by AFU: antigens and allergens, which resembles human ABPA immunologically. The ABPA mice exhibited high levels of AFU:-specific IgG and IgE, blood eosinophilia, extensive infiltration of lymphocytes and eosinophils in the lung sections, and a Th2 cytokine response. Treatment with SP-A, SP-D, and rSP-D lowered blood eosinophilia, pulmonary infiltration, and specific Ab levels considerably, which persisted up to 4 days in the SP-A-treated ABPA mice, and up to 16 days in the SP-D- or rSP-D-treated ABPA mice. The levels of IL-2, IL-4, and IL-5 were decreased, while the level of IFN-gamma was raised in the splenic supernatants of the treated mice, indicating a marked shift from Th2 to Th1 response. These results clearly implicate pulmonary SP-A and SP-D in the modulation of allergic reactions.

A recombinant homotrimer, composed of the alpha helical neck region of human surfactant protein D and C1q B chain globular domain, is an inhibitor of the classical complement pathway.

The first step in the activation of the classical complement pathway by immune complexes involves the binding of the six globular heads of C1q to the Fc regions of IgG or IgM. The globular heads of C1q (gC1q domain) are located C-terminal to the six triple-helical stalks present in the molecule, each head being composed of the C-terminal halves of one A, one B, and one C chain. The gC1q modules are also found in a variety of noncomplement proteins, such as type VIII and X collagens, precerebellin, hibernation protein, multimerin, Acrp-30, and saccular collagen. In several of these proteins, the chains containing these gC1q modules appear to form a homotrimeric structure. Here, we report expression of an in-frame fusion of a trimerizing neck region of surfactant protein D with the globular head region of C1q B chain as a fusion to Escherichia coli maltose binding protein. Following cleavage by factor Xa and removal of the maltose binding protein, the neck and globular region, designated ghB(3), formed a soluble, homotrimeric structure and could inhibit C1q-dependent hemolysis of IgG- and IgM-sensitized sheep erythrocytes. The functional properties of ghB(3) indicate that the globular regions of C1q may adopt a modular organization in which each globular head of C1q may be composed of three structurally and functionally independent domains, thus retaining multivalency in the form of a heterotrimer. The finding that ghB(3) is an inhibitor of C1q-mediated complement activation opens up the possibility of blocking activation at the first step of the classical complement pathway.

Collectin structure: a review.

Collectins are animal calcium dependent lectins that target the carbohydrate structures on invading pathogens, resulting in the agglutination and enhanced clearance of the microorganism. These proteins form trimers that may assemble into larger oligomers. Each polypeptide chain consists of four regions: a relatively short N-terminal region, a collagen like region, an alpha-helical coiled-coil, and the lectin domain. Only primary structure data are available for the N-terminal region, while the most important features of the collagen-like region can be derived from its homology with collagen. The structures of the alpha-helical coiled-coil and the lectin domain are known from crystallographic studies of mannan binding protein (MBP) and lung surfactant protein D (SP-D). Carbohydrate binding has been structurally characterized in several complexes between MBP and carbohydrate; all indicate that the major interaction between carbohydrate and collectin is the binding of two adjacent carbohydrate hydroxyl group to a collectin calcium ion. In addition, these hydroxyl groups hydrogen bond to some of the calcium amino acid ligands. While each collectin trimer contains three such carbohydrate binding sites, deviation from the overall threefold symmetry has been demonstrated for SP-D, which may influence its binding properties. The protein surface between the three binding sites is positively charged in both MBP and SP-D.

Altered airway surfactant phospholipid composition and reduced lung function in asthma.

Pulmonary surfactant in bronchoalveolar lavage fluid (BALF) and induced sputum from adults with stable asthma (n = 36) and healthy controls (n = 12) was analyzed for phospholipid and protein compositions and function. Asthmatic subjects were graded as mild, moderate, or severe. Phospholipid compositions of BALF and sputum from control subjects were similar and characteristic of surfactant. For asthmatic subjects, the proportion of dipalmitoyl phosphatidylcholine (16:0/16:0PC), the major phospholipid in surfactant, decreased in sputum (P < 0.05) but not in BALF. In BALF, mole percent 16:0/16:0PC correlated with surfactant function measured in a capillary surfactometer, and sputum mole percent 16:0/16:0PC correlated with lung function (forced expiratory volume in 1 s). Neither surfactant protein A nor total protein concentration in either BALF or sputum was altered in asthma. These results suggest altered phospholipid composition and function of airway (sputum) but not alveolar (BALF) surfactant in stable asthma. Such underlying surfactant dysfunction may predispose asthmatic subjects to further surfactant inhibition by proteins or aeroallergens in acute asthma episodes and contribute to airway closure in asthma. Consequently, administration of an appropriate therapeutic surfactant could provide clinical benefit in asthma.

Distinct pathways of mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine protease-2.

Mannan-binding lectin (MBL) plays a pivotal role in innate immunity by activating complement after binding carbohydrate moieties on pathogenic bacteria and viruses. Structural similarities shared by MBL and C1 complexes and by the MBL- and C1q-associated serine proteases, MBL-associated serine protease (MASP)-1 and MASP-2, and C1r and C1s, respectively, have led to the expectation that the pathways of complement activation by MBL and C1 complexes are likely to be very similar. We have expressed rMASP-2 and show that, whereas C1 complex autoactivation proceeds via a two-step mechanism requiring proteolytic activation of both C1r and C1s, reconstitution with MASP-2 alone is sufficient for complement activation by MBL. The results suggest that the catalytic activities of MASP-2 split between the two proteases of the C1 complex during the course of vertebrate complement evolution.

C1q: structure, function, and receptors.

C1q is the first subcomponent of the C1 complex of the classical pathway of complement activation. Several functions have been assigned to C1q, which include antibody-dependent and independent immune functions, and are considered to be mediated by C1q receptors present on the effector cell surface. There remains some uncertainty about the identities of the receptors that mediate C1q functions. Some of the previously described C1q receptor molecules, such as gC1qR and cC1qR, now appear to have less of a role in C1q functions than in functions unrelated to C1q. The problem of identifying receptor proteins with complementary binding sites for C1q has been compounded by the highly charged nature of the different domains in C1q. Although newer candidate receptors like C1qR(p) and CR1 have emerged, full analysis of the C1q-C1q receptor interactions is still at an early stage. In view of the diverse functions that C1q is considered to perform, it has been speculated that several C1q-binding proteins may act in concert, as a C1q receptor complex, to bring about C1q mediated functions. Some major advances have been made in last few years. Experiments with gene targeted homozygous C1q-deficient mice have suggested a role for C1q in modulation of the humoral immune response, and also in protection against development of autoimmunity. The recently described crystal structure of Acrp-30, which is a serum protein secreted from adipocytes, has revealed a new C1q/TNF superfamily of proteins. Although the members of this superfamily may have diverse functions, there may be a common theme in their phylogeny and modular organisation of their distinctive globular domains.