Jens Madsen

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.

N-Linked Glycosylation Attenuates H3N2 Influenza Viruses

ABSTRACT Over the last four decades, H3N2 subtype influenza A viruses have gradually acquired additional potential sites for glycosylation within the globular head of the hemagglutinin (HA) protein. Here, we have examined the biological effect of additional glycosylation on the virulence of H3N2 influenza viruses. We created otherwise isogenic reassortant viruses by site-directed mutagenesis that contain additional potential sites for glycosylation and examined the effect on virulence in naïve BALB/c, C57BL/6, and surfactant protein D (SP-D)-deficient mice. The introduction of additional sites was consistent with the sequence of acquisition in the globular head over the past 40 years, beginning with two sites in 1968 to the seven sites found in contemporary influenza viruses circulating in 2000. Decreased morbidity and mortality, as well as lower viral lung titers, were seen in mice as the level of potential glycosylation of the viruses increased. This correlated with decreased evidence of virus-mediated lung damage and increased in vitro inhibition of hemagglutination by SP-D. SP-D-deficient animals displayed an inverse pattern of disease, such that more highly glycosylated viruses elicited disease equivalent to or exceeding that of the wild type. We conclude from these data that increased glycosylation of influenza viruses results in decreased virulence, which is at least partly mediated by SP-D-induced clearance from the lung. The continued exploration of interactions between highly glycosylated viruses and surfactant proteins may lead to an improved understanding of the biology within the lung and strategies for viral control.

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.

Review: Gp-340/DMBT1 in mucosal innate immunity.

Deleted in Malignant Brain Tumour 1 (DMBT1) is a gene that encodes alternatively spliced proteins involved in mucosal innate immunity. It also encodes a glycoprotein with a molecular mass of 340 kDa, and is referred to as gp-340 (DMBT1gp340) and salivary agglutinin (DMBT1SAG). DMBT1gp340 is secreted into broncho-alveolar surface lining fluid whereas DMBTSAG is present in the saliva. The two molecules were shown to be identical and both interact with and agglutinate several Gram-negative and Gram-positive bacteria including Streptococcus mutans, a bacterium responsible for caries in the oral cavity. DMBT1gp340 interacts with surfactant proteins A and D (SP-D). DMBT1gp340 and SP-D can individually and together interact and agglutinate influenza A virus. DMBT1gp340 also binds to HIV-1 and facilitates transcytosis of the virus into epithelial cells. DMBT1 binds to a variety of other host proteins, including serum and secretory IgA, C1q, lactoferrin, MUC5B and trefoil factor 2 (TFF2), all molecules with involvement in innate immunity and/or wound-healing processes. Recent generation of Dmbt1-deficient mice has provided the research field of DMBT1 with a model that allows research to progress from in vitro studies to in vivo functional studies of the multifunctional proteins encoded by the DMBT1 gene.

The genomic structure of the DMBT1 gene: evidence for a region with susceptibility to genomic instability

Increasing evidence has accumulated for an involvement of the inactivation of tumour suppressor genes at chromosome 10q in the carcinogenesis of brain tumours, melanomas, and carcinomas of the lung, the prostate, the pancreas, and the endometrium. The gene DMBT1 (Deleted in Malignant Brain Tumours 1) is located at chromosome 10q25.3 – q26.1, within one of the putative intervals for tumour suppressor genes. DMBT1 is a member of the scavenger-receptor cysteine-rich (SRCR) superfamily and displays homozygous deletions or lack of expression in glioblastoma multiforme, medulloblastoma, and in gastrointestinal and lung cancers. Based on these properties, DMBT1 has been proposed to be a candidate tumour suppressor gene. We have determined the genomic sequence of DMBT1 to allow analyses of mutations. The gene has at least 54 exons that span a genomic region of about 80 kb. We have identified a putative exon with coding potential for a transmembrane domain. Our data further suggest that alternative splicing gives rise to isoforms of DMBT1 with a differential utilization of SRCR domains and SRCR interspersed domains. The major part of the gene harbours locus specific repeats. These repeats may point to the DMBT1 locus as a region susceptible to chromosomal instability.

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.

Frequent downregulation of DMBT1 and galectin-3 in epithelial skin cancer

DMBT1 and galectin‐3 are potential interacting proteins with presumably complex roles in tumorigenesis. While at present a variety of mechanisms are discussed for DMBT1 and its participation in cancer, galectin‐3 is commonly known to exert tumor‐promoting effects. However, in vitro studies in a rodent system have suggested that DMBT1/galectin‐3 interaction in the ECM triggers epithelial differentiation, which would point to tumor‐suppressive properties. To improve the understanding of DMBT1/galectin‐3 action in cancer, we carried out studies in skin cancer of different origins. Mutational analyses of DMBT1 identified a missense mutation in 1 of 13 melanoma cell lines. It led to an exchange of an evolutionary conserved proline residue for serine and located within the second CUB domain of DMBT1. Immunohistochemical analyses demonstrated absence of DMBT1/galectin‐3 expression from melanocytes but induction of DMBT1 expression in 1 of 8 nevi and 1 of 11 melanomas and of galectin‐3 expression in 3 of 8 nevi and 4 of 8 melanomas. These data suggest that DMBT1 and galectin‐3 are unlikely to act as classical tumor suppressors in melanomas. DMBT1 and galectin‐3 appear to be secreted to the ECM by epithelial cells within the epidermis and the hair follicle. Compared to the flanking normal epidermis, skin tumors of epithelial origin frequently displayed downregulation of DMBT1 (18 of 19 cases) and galectin‐3 (12 of 12 cases). Thus, loss of DMBT1/galectin‐3 expression may play a role in the genesis of epithelial skin cancer. This would support the view that galectin‐3 can exert tumor‐suppressive effects in certain scenarios, and DMBT1/galectin‐3‐mediated differentiation represents a candidate mechanism for this effect.

The SRCR/SID region of DMBT1 defines a complex multi-allele system representing the major basis for its variability in cancer.

Deleted in malignant brain tumors 1 (DMBT1) at 10q25.3–q26.1 has been proposed as a candidate tumor‐suppressor gene for brain and epithelial cancer. DMBT1 encodes a multifunctional mucin‐like protein presumably involved in epithelial differentiation and protection. The gene consists of highly homologous and repeating exon and intron sequences. This specifically applies to the region coding for the repetitive scavenger receptor cysteine‐rich (SRCR) domains and SRCR‐interspersed domains (SIDs) that constitutes the major part of the gene. This particular structure may previously have interfered with the delineation of DMBT1 alterations in cancer. Uncovering these, however, is of mechanistic importance. By a combined approach, we conducted a detailed mutational analysis, starting from a panel of 51 tumors, including 46 tumor cell lines and five primary tumors. Alterations in the repetitive region were present in 22/31 (71%) tumors that were investigated in detail. Six tumors showed presumably de novo mutations, among these three with point mutations in combination with a loss of heterozygosity. However, none of the alterations unambiguously would be predicted to lead to an inactivation of DMBT1. We define seven distinct DMBT1 alleles based on variable numbers of tandem repeats (VNTRs). At least 11 tumors exclusively harbored these VNTRs. The data suggest that the SRCR/SID region defines a complex multi‐allele system that has escaped previous analyses and that represents the major basis for the variability of DMBT1 in cancer. DMBT1 thus compares to mucins rather than to conventional tumor suppressors.

Carcinogen inducibility in vivo and down-regulation of DMBT1 during breast carcinogenesis

Deleted in malignant brain tumors 1 (DMBT1) has been proposed as a candidate tumor suppressor for brain and epithelial cancer. Initial studies suggested loss of expression rather than mutation as the predominant mode of DMBT1 inactivation. However, in situ studies in lung cancer demonstrated highly sophisticated changes of DMBT1 expression and localization, pointing to a chronological order of events. Here we report on the investigation of DMBT1 in breast cancer in order to test whether these principles might also be attributable to other tumor types. Comprehensive mutational analyses did not uncover unambiguous inactivating DMBT1 mutations in breast cancer. Expression analyses in the human and mouse mammary glands pointed to the necessity of DMBT1 induction. While age‐dependent and hormonal effects could be ruled out, 9 of 10 mice showed induction of Dmbt1 expression after administration of the carcinogen 7,12‐dimethybenz(α)anthracene prior to the onset of tumorigenesis or other histopathological changes. DMBT1 displayed significant up‐regulation in human tumor–flanking tissues compared to in normal breast tissues (P < 0.05). However, the breast tumor cells displayed a switch from lumenal secretion to secretion to the extracellular matrix and a significant down‐regulation compared to that in matched normal flanking tissues (P < 0.01). We concluded that loss of expression also is the predominant mode of DMBT1 inactivation in breast cancer. The dynamic behavior of DMBT1 in lung carcinoma is fully reflected in breast cancer, which suggests that this behavior might be common to tumor types arising from monolayered epithelia.