Surfactant protein D (SP-D) belongs to the collectin family and has pro-and anti-inflammatory capacities depending on its oligomerization. Previously, circulating SP-D was shown to be decreased in early rheumatoid arthritis (RA) and negatively correlated to disease activity. This study aimed at assessing the diurnal rhythmicity and the influence of physical activity on circulating SP-D in patients with RA at different stages compared with healthy individuals. Patients with early RA (ERA) with disease duration <6 months and with long-standing RA (LRA) with disease duration 5-15 years were included in two sub-studies. Healthy individuals served as controls. Diurnal variation: blood samples were collected every 3 h from 7 a.m to 10 p.m and the following morning. Physical activity: blood sampling was done before and after standardized physical challenge. SP-D was measured by ELISA. SP-D exhibited diurnal variation in healthy controls (n = 15) and in patients with ERA (n = 9) and LRA (n = 9) with peak values at 10 a.m. and nadir in the evening (controls: P < 0.001, ERA: P = 0.004 and LRA: P = 0.009). Three hours after cessation of physical activity, SP-D decreased below pre-exercise levels in both ERA (n = 10), LRA (n = 10) and controls (n = 13) (ERA: P < 0.001, LRA: P < 0.001 and controls: P = 0.005). In patients with RA, the decline was already observed 1 h post-exercise. Circulating SP-D exhibits diurnal variation both in patients with RA at different stages and in healthy controls. SP-D in serum decreases following physical activity in health and RA disease. This study underscores the need of standardized blood sampling conditions in future studies on SP-D.

Surfactant protein D (SP-D) is a member of the collectin family and is an important component of the pulmonary innate host defence. The protein has a widespread distribution in the human body and is present in multiple epithelia, in endothelium and in blood. Various studies have looked at the relationship between serum SP-D levels and pulmonary inflammatory diseases. The SP-D distribution has been most thoroughly described in European populations and appears with a broad range of serum values highly influenced by genetic factors. In the present study, we investigated the plasma SP-D distribution in a Chinese population from the Tai An region comprising 268 individuals. We found that (i) plasma SP-D in the Chinese population was distributed with a median value of 380.2 ng/ml (324.9; 418.7) and a range from 79.4 to 3965.3 ng/ml, (ii) significantly higher plasma SP-D in men than in women, and no significant effect of age, and (iii) a significant inverse association between serum SP-D and body mass index (BMI) (P = 0.012). The data indicate that racial differences in SP-D expression exist as the median plasma SP-D in the Chinese population was approximately two times lower than the median serum SP-D previously measured in a Danish population using the same immuno-assay. The inverse association between serum SP-D and BMI found in the Chinese population indicates that serum SP-D is related to obesity in similar ways in Chinese and Danes.

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 envelope protein (gp120) of human immunodeficiency virus (HIV) contains highly conserved mannosylated oligosaccharides. These glycoconjugates contribute to resistance to antibody neutralization, and binding to cell surface lectins on macrophages and dendritic cells. Mannose-binding lectin (MBL) binds to gp120 and plays a role in defence against the virus. In this study it is demonstrated that surfactant protein D (SP-D) binds to gp120 and inhibits HIV infectivity at significantly lower concentrations than MBL. The binding of SP-D was mediated by its calcium-dependent carbohydrate-binding activity and was dependent on glycosylation of gp120. Native dodecameric SP-D bound to HIV gp120 more strongly than native trimeric SP-D. Since one common polymorphic form of SP-D is predominantly expressed as trimers and associated with lower blood levels, these individuals may have less effective innate defence against HIV. A chimeric protein containing the N-terminal and collagen domains of SP-D linked to the neck and carbohydrate-recognition domains of MBL (called SP-D/MBLneck+CRD) had greater ability to bind to gp120 and inhibit virus replication than either SP-D or MBL. The enhanced binding of SP-D/MBLneck+CRD was dependent on assembly into higher molecular mass multimers (i.e. a trimeric form of the chimera did not bind to a greater extent than MBL). Hence, the enhanced binding of SP-D compared with MBL results from distinctive properties of its N-terminal and/or collagen domains. SP-D is present in lung and airway fluids, as well as in blood and various mucosal locations, and could, like MBL, play a role in restricting HIV transmission or replication in vivo.

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 (Met(11)Thr), 160 (Ala(160)Thr), and 270 (Ser(270)Thr) of the SP-D gene was performed and related to the SP-D levels in serum. Individuals with the Thr/Thr(11)-encoding genotype had significantly lower SP-D serum levels than individuals with the Met/Met(11) genotype. Gel filtration chromatography revealed two distinct m.w. peaks with SP-D immunoreactivity in serum from Met/Met(11)-encoding genotypes. In contrast, Thr/Thr(11) genotypes lacked the highest m.w. form. A similar SP-D size distribution was found for recombinant Met(11) and Thr(11) 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.

The surfactant collectins, surfactant proteins A and D (SP-A and D), and scavenger receptor-rich glycoprotein 340 (gp340) inhibit influenza A virus (IAV) in the following order of potency: SP-D>gp340>SP-A. SP-D binds in a calcium-dependent manner to carbohydrate attachments on the viral hemagglutinin (HA) and neuraminidase (NA). By contrast, gp340 and SP-A act like mucins in that they provide sialic acid ligands that bind to the viral HA. In this study, SP-D, SP-A, and gp340 showed cooperative antiviral interactions. These cooperative effects were most evident in viral aggregation but were also observed in at least some hemagglutination inhibition and viral neutralization assays. The mechanism of binding between gp340 and SP-D was further characterized using monoclonal antibodies. Although gp340 can bind to SP-D at a site distinct from the mannan-binding site, binding of gp340 to SP-D did not contribute to cooperative antiviral interactions. SP-D and mucin showed cooperative interactions, apparently dependent on NA inhibition by SP-D. The commercial NA inhibitor oseltamivir had a similar effect and also enhanced the neutralizing activity of SP-A and bronchoalveolar lavage fluid. Hence, oseltamivir collaborates with innate immune proteins in inhibiting the initial infection of epithelial cells.

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 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.

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.

Salivary agglutinin is a 300–400kDa salivary glycoprotein that binds to antigen B polypeptides of oral streptococci, thereby playing a role in their colonization and the development of caries. A mass spectrum was recorded of a trypsin digest of agglutinin. A dominant peak of 1460Da was sequenced by quadrupole time-of-flight (Q-TOF) tandem MS. The sequence showed 100% identity with part of the scavenger receptor cysteine-rich (‘SRCR’) domain found in gp-340/DMBT1 (deleted in malignant brain tumours-1). The mass spectrum revealed 11 peaks with an identical mass as a computer-simulated trypsin digest of gp-340. gp-340 is a 340kDa glycoprotein isolated from bronchoalveolar lavage fluid that binds specifically to lung surfactant protein-D. DMBT1 is a candidate tumour suppressor gene. A search in the human genome revealed only one copy of this gene. The molecular mass, as judged from SDS/PAGE and the amino acid composition of agglutinin, was found to be nearly identical with that of gp-340. It was shown by Western blotting that monoclonal antibodies against gp-340 reacted with salivary agglutinin, and monoclonals against agglutinin reacted with gp-340. It was demonstrated that gp-340 and agglutinin bound in a similar way to Streptococcus mutans and surfactant protein-D. Histochemically, the distribution of gp-340 in the submandibular salivary glands was identical with the agglutinin distribution, as shown in a previous paper [Takano, Bogert, Malamud, Lally and Hand (1991) Anat. Rec. 230, 307–318]. We conclude that agglutinin is identical with gp-340, and that this molecule interacts with S. mutans and surfactant protein-D.

Surfactant protein D (SP-D) is an oligomeric C type lectin that promotes phagocytosis by binding to microbial surface carbohydrates. A 340-kDa glycoprotein (gp-340) has been shown to bind SP-D in the presence of calcium but does so independently of carbohydrate recognition. This protein exists both in a soluble form and in association with the membranes of alveolar macrophages. The primary structure of gp-340 has been established by molecular cloning, which yielded a 7,686-bp cDNA sequence encoding a polypeptide chain of 2, 413 amino acids. The domain organization features 13 scavenger receptor cysteine-rich (SRCR) domains, each separated by an SRCR-interspersed domain, except for SRCRs 4 and 5, which are contiguous. The 13 SRCR domains are followed by two C1r/C1s Uegf Bmp1 domains separated by a 14th SRCR domain and a zona pellucida domain. gp-340 seems to be an alternative spliced form of DMBT1. Reverse transcription-PCR analysis showed that the main sites of synthesis of gp-340 are lung, trachea, salivary gland, small intestine, and stomach. Immunohistochemistry revealed strong staining for gp-340 in alveolar and other tissue macrophages. Immunostaining of the macrophage membrane was either uniform or focal in a way that suggested capping, whereas other macrophages showed strong intracellular staining within the phagosome/phagolysosome compartments. In some macrophages, SP-D and gp-340 were located in the same cellular compartment. Immunoreactive gp-340 was also found in epithelial cells of the small intestine and in the ducts of salivary glands. The distribution of gp-340 in macrophages is compatible with a role as an opsonin receptor for SP-D.

We have purified a glycoprotein from bovine lung washings using affinity chromatography on a maltose-affinity column. On SDS-polyacrylamide gel electrophoresis the protein showed a molecular mass of 36 kDa in the reduced state and 66 kDa in the unreduced state. On gel permeation chromatography the apparent molecular mass was 250 kDa. N-terminal sequencing showed homology to the human matrix protein microfibril-associated protein (hMFAP4), and the glycoprotein was designated bovine MFAP4 (bMFAP4). Lung surfactant protein D (SP-D) was also purified from lung washings, and calcium-dependent binding was demonstrated between bMFAP4 and SP-D. hMFAP4 was cloned, and recombinant hMFAP4 showed the same binding pattern to SP-D as bMFAP4. No binding was seen to recombinant SP-D composed of the neck region and carbohydrate recognition domain of SP-D, indicating that the interaction between MFAP4 and SP-D is mediated via the collagen region of SP-D. MFAP4 also showed calcium-dependent binding to mannan, which was partially inhibited by maltose. Our findings indicate that MFAP4 has two binding specificities, one for collagen and one for carbohydrate, and we suggest that MFAP4 may fix the collectins in the extracellular compartment during inflammation.

No standard procedure is available for the purification of human monoclonal antibodies for human i.v. administration. Here we describe the procedure developed for pilot scale purification of the human IgM monoclonal antibody COU-1 directed against a cancer-associated antigen. The hybridoma cells were grown in protein-free medium and purification from the clarified culture supernatant was carried out in 4 simple chromatographic steps: (1) hydroxylapatite chromatography; (2) hydrophobic interaction chromatography on phenyl-Sepharose: (3) cation-exchange chromatography on sulphonyl-Sepharose; and (4) anion-exchange chromatography on tetraethylamino-Sepharose. The product was substantially pure with regard to protein after step 3, but contained DNA which was removed in step 4. The average recovery of the IgM was 54% with a range of 40-65%. Importantly, the ability of the antibody to bind to its antigen in ELISA was fully maintained during the purification. Subsequently, the purified antibody was isotope labelled and successfully used for in vivo detection of colon, rectal and pancreas carcinomas in patients. The purification procedure described appears to compare favourably with previously published methods, but a critical comparison is not possible due to the lack of necessary information in the available literature.

We have purified a previously unknown glycoprotein (designated gp-340) from human bronchioalveolar lung washings from a patient with alveolar proteinosis. gp-340 was identified by its calcium-dependent binding to lung surfactant protein D (SP-D) and by its molecular mass of 340 kDa in the reduced state on SDS-polyacrylamide gel electrophoresis (PAGE). gp-340 was purified from the 10,000 x g pellet of the lavage fluid by ion-exchange and gel permeation chromatography. On SDS-PAGE, gp-340 showed an apparent molecular mass of 290 kDa in the unreduced state. On gel chromatography under non-dissociating conditions, the apparent molecular mass of gp-340 was >1000 kDa. The presence of N-linked glycosylation was shown by digestion with N-glycosidase F, which reduced the apparent molecular mass of gp-340 under reducing condition to about 300 kDa. Partial amino acid sequence data showed the presence of scavenger-receptor type domains. Monoclonal and polyclonal antibodies were raised against gp-340, and their specificities were confirmed by Western blotting. The antibodies were used for immunohistochemical localization of gp-340 in the lung, where it was found on the surface of and within alveolar macrophages. Direct binding between gp-340 and SP-D took place at physiological ionic strength, required the presence of calcium, and was not inhibited by maltose. The binding between SP-D and mannan also required the presence of calcium, but this interaction was completely inhibited by maltose. The same binding pattern was seen between gp-340 and recombinant human SP-D composed of the trimeric neck region and three carbohydrate recognition domains. These findings indicate that the binding between gp-340 and SP-D is a protein-protein interaction rather than a lectin-carbohydrate interaction and that the binding to gp-340 takes place via the carbohydrate recognition domain of SP-D. We conclude that gp-340 is a new member of the scavenger-receptor superfamily and likely to be a truncated form of a receptor for SP-D.

Aims: Lung surfactant-associated protein D (SP-D) is an oligomeric molecule with lectin (carbohydrate-binding) domains and collagen regions which has been demonstrated in lung washings. SP-D is believed to be of importance in innate immunity primarily through lectin-like activity and to participate in the formation of lung surfactant and mucus. SP-D is synthesized by pulmonary alveolar type II cells and non-ciliated bronchial cells. Recently, SP-D was demonstrated in the gastric mucosa (Fisher and Mason, 1995). The aim of this study was to investigate the distribution of SP-D throughout the gastrointestinal tract. Methods: Monoclonal antibodies were raised against SP-D and their specificity was confirmed by Western blotting. One antibody was subsequently used for immunohistochemistry. Reverse transcriptase PCR with an antisense SP-D primer and appropriate controls was used to look for SP-D mRNA. The presence of SP-D in saliva was investigated using affinity purification on maltose-agarose columns followed by separation on polyacryl amide gel (SDS-PAGE), Western blotting and detection with antibody. Results: Immunohistochemical localization of SP-D was observed within pulmonary alveolar type II cells, alveolar macrophages and in parotid and submandibular glands. Furthermore, SP-D was found in normal gastric mucosal lining cells and in epithelial cells of small intestinal and colonic tissue (8 subjects). By reverse transcriptase PCR products of approx. 300 base pairs identical to the finding in pulmonary tissue were detected in tissue from parotid gland, stomach, small intestine, colon and pancreas. SP-D was observed in saliva with a molecular mass of 43 kDa under reducing conditions, identical to the findings of SP-D from lung washings. Conclusions: These data indicate that the occurrence of SP-D is not restricted to pulmonary cells, but is found widely distributed at mucosal surfaces. SP-D may play a significant role in mucosal immunity of the gastrointestinal tract.

Conglutinin, like mannan-binding lectin (MBL) and CL-43, is a serum collection involved in the innate immune defence system. In man, low serum MBL concentrations, resulting from mutations in the collagen region, are associated with a common opsonic defect. Plasma levels of conglutinin in cattle were assayed by rocket immunoelectrophoresis to examine whether they were genetically determined. Samples were collected from calves (309 bull-calves and 260 heifers with complex pedigree relationships). The number of respiratory infections from the 42nd to 336th day of life was recorded. The number of infections was found to be genetically determined (heritability: h2 = 0.31 +/- 0.07). A wide concentration range of conglutinin was found in plasma (< 1.25-35 micrograms/ml for females, geometric mean 8.1 micrograms/ml, and < 1.25-47 micrograms/ml for males, geometric mean 15.5 micrograms/ml), and the concentrations was found to be genetically determined (heritability, h2 = 0.52 +/- 0.07). The analysis revealed a negative association between disease frequency and the conglutinin levels (-0.56 +/- 0.18 for female; -0.50 +/- 0.18 for male). Levels of conglutinin below the detection limit of the assay (1.25 micrograms/ml) were found in 2% of the animals. If these animals are assumed to be homozygous for a single recessive allele causing low concentrations a gene frequency of 0.15 could be calculated. These findings suggests that selection for resistance against infectious disease is possible in cattle and that the level of plasma conglutinin may be a helpful trait in such a breeding scheme.

Collectin-43 (CL-43) is a recently described bovine plasma protein containing both collagenous regions and C-type-lectin domains [Holmskov, Teisner, Willis, Reid and Jensenius (1993) J. Biol. Chem. 268, 10120-10125; Lim, Willis, Reid, Lu, Laursen, Jensenius and Holmskov (1994) J. Biol. Chem. 269, 11820-11824]. CL-43 was purified by affinity chromatography on mannan-Sepharose. On SDS/PAGE under reducing conditions the purified lectin showed a double band at about 43 kDa, with the upper band representing the intact molecule and the lower band a truncated form that lacked the N-terminal nine amino acid residues. Under non-reducing conditions, only one band was seen at 120 kDa. Analytical gel chromatography and sucrose-density-gradient centrifugation of the purified molecule, showed a Stokes radius of 9.1 +/- 0.3 nm (91 +/- 3 A) and a sedimentation coefficient (s20,w) of 3.6 +/- 0.1 S. These values correspond to a molecular mass of 119-138 kDa under non-denaturing condition in solution. The frictional coefficient (f/f0) was 2.7, indicating extreme elongation due to the collagenous segment. Only monomer subunits, with 37.4 +/- 1.7-nm-long rods, were seen by electron microscopy. These findings indicate that CL-43, in contrast with the other circulating collectins, is found only as a single subunit composed of three polypeptide chains. Two-dimensional gel electrophoresis showed that CL-43 has two isoforms, with pI values of 4.9 and 5.3, corresponding to the native form and the truncated form of the molecule respectively. CL-43, like conglutinin, lung surfactant protein A and mannan-binding protein (MBP), was shown to bind to the collectin receptor. Bovine MBP caused the activation of the complement system as revealed by the deposition of complement component C4 upon incubation of diluted serum in wells containing MBP bound to solid-phase mannan. CL-43, lung surfactant protein D (SP-D) and conglutinin showed no complement-activating properties under the same conditions. Conglutinin binds fluid- and solid-phase iC3b, while CL-43 and MBP do not show such reactivity.