Ida Tornøe

Localization of Lung Surfactant Protein D on Mucosal Surfaces in Human Tissues

Lung surfactant protein-D (SP-D), a collectin mainly produced by alveolar type II cells, initiates the effector mechanisms of innate immunity on binding to microbial carbohydrates. A panel of mRNAs from human tissues was screened for SP-D mRNA by RT-PCR. The lung was the main site of synthesis, but transcripts were readily amplified from trachea, brain, testis, salivary gland, heart, prostate gland, kidney, and pancreas. Minor sites of synthesis were uterus, small intestine, placenta, mammary gland, and stomach. The sequence of SP-D derived from parotid gland mRNA was identical with that of pulmonary SP-D. mAbs were raised against SP-D, and one was used to locate SP-D in cells and tissues by immunohistochemistry. SP-D immunoreactivity was found in alveolar type II cells, Clara cells, on and within alveolar macrophages, in epithelial cells of large and small ducts of the parotid gland, sweat glands, and lachrymal glands, in epithelial cells of the gall bladder and intrahepatic bile ducts, and in exocrine pancreatic ducts. SP-D was also present in epithelial cells of the skin, esophagus, small intestine, and urinary tract, as well as in the collecting ducts of the kidney. SP-D is generally present on mucosal surfaces and not restricted to a subset of cells in the lung. The localization and functions of SP-D indicate that this collectin is the counterpart in the innate immune system of IgA in the adaptive immune system.

A Common Polymorphism in the SFTPD Gene Influences Assembly, Function, and Concentration of Surfactant Protein D

Surfactant protein D (SP-D) plays important roles in the host defense against infectious microorganisms and in regulating the innate immune response to a variety of pathogen-associated molecular pattern. SP-D is mainly expressed by type II cells of the lung, but SP-D is generally found on epithelial surfaces and in serum. Genotyping for three single-nucleotide variations altering amino acids in the mature protein in codon 11 (Met11Thr), 160 (Ala160Thr), and 270 (Ser270Thr) of the SP-D gene was performed and related to the SP-D levels in serum. Individuals with the Thr/Thr11-encoding genotype had significantly lower SP-D serum levels than individuals with the Met/Met11 genotype. Gel filtration chromatography revealed two distinct m.w. peaks with SP-D immunoreactivity in serum from Met/Met11-encoding genotypes. In contrast, Thr/Thr11 genotypes lacked the highest m.w. form. A similar SP-D size distribution was found for recombinant Met11 and Thr11 expressed in human embryonic kidney cells. Atomic force microscopy of purified SP-D showed that components eluting in the position of the high m.w. peak consist of multimers, dodecamers, and monomers of subunits, whereas the second peak exclusively contains monomers. SP-D from both peaks bound to mannan-coated ELISA plates. SP-D from the high m.w. peak bound preferentially to intact influenza A virus and Gram-positive and Gram-negative bacteria, whereas the monomeric species preferentially bound to isolated LPS. Our data strongly suggest that polymorphic variation in the N-terminal domain of the SP-D molecule influences oligomerization, function, and the concentration of the molecule in serum.

Tissue Localization of Human Trefoil Factors 1, 2, and 3

Trefoil factors (TTFs) are small, compact proteins coexpressed with mucins in the gastrointestinal tract. Three trefoil factors are known in mammals: TFF1, TFF2, and TFF3. They are implicated to play diverse roles in maintenance and repair of the gastrointestinal channel. We compared the expression pattern of the three trefoil factors analyzing mRNA from a panel of 20 human tissues by conventional reverse transcriptase (RT) PCR and, in addition, by real-time PCR. These findings were supported by immunohistochemical analysis of paraffin-embedded human tissues using rabbit polyclonal antibodies raised against these factors. TFF1 showed highest expression in the stomach and colon, whereas TFF2 and TFF3 showed highest expression in stomach and colon, respectively. All three TFFs were found in the ducts of pancreas. Whereas TFF2 was found to be restricted to these two tissues, the structurally more closely related TFF1 and TFF3 showed a more general tissue distribution and were found to colocalize on an array of mucosal surfaces. This is the first thorough parallel description of the tissue distribution of TFFs in normal tissues, and it provides a baseline for similar analysis in diseased tissues.

Detection of novel biomarkers of liver cirrhosis by proteomic analysis

Hepatic cirrhosis is a life‐threatening disease arising from different chronic liver disorders. One major cause for hepatic cirrhosis is chronic hepatitis C. Chronic hepatitis C is characterized by a highly variable clinical course, with at least 20% developing liver cirrhosis within 40 years. Only liver biopsy allows a reliable evaluation of the course of hepatitis C by grading inflammation and staging fibrosis, and thus serum biomarkers for hepatic fibrosis with high sensitivity and specificity are needed. To identify new candidate biomarkers for hepatic fibrosis, we performed a proteomic approach of microdissected cirrhotic septa and liver parenchyma cells. In cirrhotic septa, we detected an increasing expression of cell structure associated proteins, including actin, prolyl 4‐hydroxylase, tropomyosin, calponin, transgelin, and human microfibril–associated protein 4 (MFAP‐4). Tropomyosin, calponin, and transgelin reflect a contribution of activated stellate cells/myofibroblasts to chronic liver injury. The expression of tropomyosin, transgelin, and MFAP‐4, an extracellular matrix associated protein, were further evaluated by immunohistochemistry. Tropomyosin and MFAP‐4 demonstrated high serum levels in patients with hepatic cirrhosis of different causes. Conclusion: A quantitative analysis of MFAP‐4 serum levels in a large number of patients showed MFAP‐4 as novel candidate biomarker with high diagnostic accuracy for prediction of nondiseased liver versus cirrhosis [area under receiver operating characteristic curve (AUC) = 0.97, P < 0.0001] as well as stage 0 versus stage 4 fibrosis (AUC = 0.84, P < 0.0001), and stages 0 to 3 versus stage 4 fibrosis (AUC = 0.76, P < 0.0001). (HEPATOLOGY 2009.)

Surfactant protein D (SP-D) serum levels in patients with community-acquired pneumonia.

SP-D is a lectin involved in the first line of defense against microorganisms. It is primarily found in the lung but also at extrapulmonary sites and in the circulation. An immunoassay for the quantification of SP-D in serum was established and the SP-D concentration was measured in consecutive blood samples from 61 patients hospitalized for community-acquired pneumonia of suspected bacterial origin. On the day of admission to the hospital the serum SP-D concentration was significantly lower than that in healthy subjects. On day 5, the SP-D concentration had increased on average three times the concentration on admission and then slowly declined toward normal levels. CRP was measured simultaneously but no correlation was observed between concentrations of SP-D and CRP. The results show a wide range of serum SP-D concentration in healthy volunteers and indicate that significant changes occur during pulmonary infection.

CRP-ductin, the mouse homologue of gp-340/deleted in malignant brain tumors 1 (DMBT1), binds gram-positive and gram-negative bacteria and interacts with lung surfactant protein D.

CRP‐ductin is a protein expressed mainly by mucosal epithelial cells in the mouse. Sequence homologies indicate that CRP‐ductin is the mouse homologue of human gp‐340, a glycoprotein that agglutinates microorganisms and binds the lung mucosal collectin surfactant protein‐D (SP‐D). Here we report that purified CRP‐ductin binds human SP‐D in a calcium‐dependent manner and that the binding is not inhibited by maltose. The same properties have previously been observed for gp‐340 binding of SP‐D. CRP‐ductin also showed calcium‐dependent binding to both gram‐positive and ‐negative bacteria. A polyclonal antibody raised against gp‐340 reacted specifically with CRP‐ductin in Western blots. Immunoreactivity to CRP‐ductin was found in the exocrine pancreas, in epithelial cells throughout the gastrointestinal tract and in the parotid ducts. A panel of RNA preparations from mouse tissues was screened for CRP‐ductin and SP‐D expression by reverse transcription‐PCR. The pancreas was the main site of synthesis of CRP‐ductin, but transcripts were also readily amplified from salivary gland, the gastrointestinal tract, liver, testis, uterus and lung. Lung was the main site of synthesis of SP‐D, but transcripts were also amplified from uterus, salivary gland, thymus, thyroid gland, pancreas and testis. We conclude that CRP‐ductin is the mouse homologue of human gp‐340 and that its capacity to bind SP‐D as well as gram‐negative and gram‐positive bacteria suggests a role in mucosal immune defense.

Characterization of FIBCD1 as an Acetyl Group-Binding Receptor That Binds Chitin

Chitin is a highly acetylated compound and the second most abundant biopolymer in the world next to cellulose. Vertebrates are exposed to chitin both through food ingestion and when infected with parasites, and fungi and chitin modulate the immune response in different directions. We have identified a novel homotetrameric 55-kDa type II transmembrane protein encoded by the FIBCD1 gene and highly expressed in the gastrointestinal tract. The ectodomain of FIBCD1 is characterized by a coiled-coil region, a polycationic region and C-terminal fibrinogen-related domain that by disulfide linkage assembles the protein into tetramers. Functional analysis showed a high-affinity and calcium-dependent binding of acetylated components to the fibrinogen domain, and a function in endocytosis was demonstrated. Screening for ligands revealed that the FIBCD1 is a high-affinity receptor for chitin and chitin fragments. FIBCD1 may play an important role in controlling the exposure of intestine to chitin and chitin fragments, which is of great relevance for the immune defense against parasites and fungi and for immune response modulation.

Reduced influenza viral neutralizing activity of natural human trimers of surfactant protein D

BackgroundSurfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection. Common human polymorphisms of SP-D have been found in many human populations and associated with increased risk of certain infections. We recently reported that the Thr/Thr 11 form of SP-D is associated with low serum levels and assembles predominantly as trimers as opposed to the more common multimeric forms of SP-D.MethodsPreliminary experiments were done to establish the effects of different monoclonal antibodies against SP-D on ability of SP-D to bind to or neutralize the virus. We then purified natural human trimeric and multimeric forms of SP-D from amniotic fluid and tested ability of these preparations to bind to IAV, to inhibit infectivity and hemagglutination activity of IAV in vitro.ResultsIn initial experiments mAbs directed against different areas on the CRD of SP-D were found to have differing effects on antiviral activity. Using an mAb that did not interfere with antiviral activity of SP-D, we confirm that natural SP-D trimers had reduced ability to bind to IAV. In addition, the trimers had reduced ability to neutralize IAV as compared to natural human SP-D multimers as well as reduced hemagglutination inhibiting activity against several strains of IAV. Natural SP-D trimers also had different interactions with human neutrophil peptide defensins (HNPs) in viral neutralization assays as compared to multimeric SP-D.ConclusionThese studies indicate that a common human polymorphic form of SP-D may modulate host defense against IAV and give impetus to clinical studies correlating this genotype with risk for IAV infection in susceptible groups. We also show that mAbs directed against different areas on the carbohydrate recognition domain of SP-D can be useful for dissecting out different functional properties of the protein.

Microfibril-associated Protein 4 Binds to Surfactant Protein A (SP-A) and Colocalizes with SP-A in the Extracellular Matrix of the Lung

Pulmonary surfactant protein A (SP‐A) is an oligomeric collectin that recognizes lipid and carbohydrate moieties present on broad range of micro‐organisms, and mediates microbial lysis and clearance. SP‐A also modulates multiple immune‐related functions including cytokine production and chemotaxis for phagocytes. Here we describe the molecular interaction between the extracellular matrix protein microfibril‐associated protein 4 (MFAP4) and SP‐A. MFAP4 is a collagen‐binding molecule containing a C‐terminal fibrinogen‐like domain and a N‐terminal located integrin‐binding motif. We produced recombinant MFAP4 with a molecular mass of 36 and 66 kDa in the reduced and unreduced states respectively. Gel filtration chromatography and chemical crosslinking showed that MFAP4 forms oligomers of four dimers. We demonstrated calcium‐dependent binding between MFAP4 and human SP‐A1 and SP‐A2. No binding was seen to recombinant SP‐A composed of the neck region and carbohydrate recognition domain of SP‐A indicating that the interaction between MFAP4 and SP‐A is mediated via the collagen domain of SP‐A. Monoclonal antibodies directed against MFAP4 and SP‐A were used for immunohistochemical analysis, which demonstrates that the two molecules colocalize both on the elastic fibres in the interalveolar septum and in elastic lamina of pulmonary arteries of chronically inflamed lung tissue. We conclude, that MFAP4 interacts with SP‐A via the collagen region in vitro, and that MFAP4 and SP‐A colocates in different lung compartments indicating that the interaction may be operative in vivo.

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.