ARTICLE |
Correspondence to: Mildred T. Stahlman, Vanderbilt U. Medical Center, Dept. of Pediatrics/Div. of Neonatology, A-0126 Medical Center North, Nashville, TN 37232-2370. E-mail: mildred.stahlman@mcmail.vanderbilt.edu
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Summary |
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Immunoreactive surfactant protein-D (SP-D) was assessed in human fetal, newborn, and adult tissues. In the fetal lung, SP-D was detected on airway surfaces by 10 weeks' gestation, staining increasing in the distal airways, decreasing in the proximal conducting airways with advancing gestation. In lungs from near-term infants and adults, SP-D was detected in Type II cells, serous cells of tracheobronchial glands, and subsets of cells lining peripheral airways. Immunostaining was decreased or absent in areas of lungs of neonates after injury to Type II cells, infection, or hemorrhage and was decreased in collapsed or unseptated airways from older infants with bronchopulmonary dysplasia. SP-D was also detected in many organs at all ages. SP-D was readily detected in epithelial cells and luminal material in lacrimal glands, salivary glands, pancreas, bile ducts, renal tubules, esophageal muscle and glands, parietal cells of the stomach, crypts of Lieberkuhn, sebaceous and eccrine sweat glands, Von Ebner's glands, endocervical glands, seminal vesicles, adrenal cortex, myocardium, and anterior pituitary gland. SP-D is a widely distributed member of the "collectin" family of polypeptides secreted onto luminal surfaces by epithelial cells lining ducts of many organs, where it likely plays a role in innate host defense.
(J Histochem Cytochem 50:651660, 2002)
Key Words: surfactant protein D, collectin, bronchopulmonary dysplasia, (BP-D)
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Introduction |
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SP-D, also termed collectin-7 (col-7), is a 43-kD member of the C-type lectin family of polypeptides, sharing considerable homology with other mammalian lectins, including SP-A, conglutinin, and mannose-binding protein (
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Materials and Methods |
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Tissue Preparation
This was a retrospective study that was approved by the Committee for the Protection of Human Subjects, Vanderbilt University Medical Center. Fetal tissues were obtained immediately after hysterotomy, hysterectomy, or spontaneous abortion. Infant samples were obtained at surgical biopsy, or by postmortem sampling within 2 hr of death. Child and adult samples were obtained postmortem. Tissues were fixed in 10% phosphate-buffered formalin, dehydrated through graded ethanols, and embedded in paraffin. Four-µm-thick serial sections were cut and mounted separately on Superfrost Plus (Fisher; Atlanta, GA) glass sides.
Purification of Mouse SP-D and Production of Antiserum
Lung lavage fluid from granulocyte-macrophage colony-stimulating factor (GM-CSF) and SP-A double-null mutant mice was used as a source of SP-D (
Purification of Antiserum
Lungs were removed from SP-D-null mutant mice (
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Specificity of SP-D ELISA
Microtiter plates were coated with 0.2 µg material from alveolar proteinosis of mouse SP-D overnight. Human SP-A or mouse SP-D (0.0011 µg/ml) was incubated overnight with rabbit anti-mouse SP-D (1:20,000). Antibodyantigen mixtures were incubated in the SP-D-coated microtiter plate for 2 hr. After washing, goat anti-rabbit IgGperoxidase conjugate (1:1000) was added to the microtiter plate for 1 hr. After washing, the color was visualized using ortho-phenylenediamine in the presence of hydrogen peroxide and the absorbance was determined at 490 nm. The absorbance was graphed vs the concentration of SP-A or SP-D (Fig 1B).
Immunolocalization
Slides were deparaffinized by placing in three xylene baths, 10 min each, and then rehydrated through two baths of ethanol (5 min each in 100%). Endogenous peroxide was quenched for 20 minutes in a bath of 0.3% H2O2 in methanol followed by 95% and then 80% ethanol 5 min each and finally placed in PBS, pH 7.2. Nonspecific staining was blocked by exposing slides for 30 minutes to prediluted normal goat serum (BioGenex catalog HK112-9K; San Ramon, CA). The appropriately diluted primary antibody (1:5000 for SPD) was then applied and allowed to incubate overnight at 4C. Slides were washed three times in PBS, pH 7.2, 5 minutes each and then a prediluted biotinylated goat anti-rabbit immunoglobulin reagent was applied to the slides for 30 min (BioGenex SS Kit; catalog AP500-5R).
After washing again, a prediluted peroxidase-conjugated streptavidin (BioGenex kit) was applied for 30 min. After more washes, the peroxidase activity was then localized by reaction with 0.5% 3,3'-diaminobenzidine0.01% H2O2 and counterstained with hematoxylin.
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Results |
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Characterization of the SP-D Antiserum
To minimize crossreactivity with SP-A, the initial SP-D antiserum was prepared against mouse SP-D purified from a double transgenic mouse lacking both SP-A (
Distribution of SP-D in Adult and Term Newborn Lung
SP-D immunoreactivity was initially assessed in adult human lung, demonstrating diffuse intracellular staining in Type II epithelial cells. Characteristic extracellular luminal staining was observed along the surfaces of the airspaces (Fig 2). It was also demonstrated around lamellar bodies in Type II cells of near-term infants, as well as rimming their terminal air spaces (Fig 3).
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SP-D in Non-pulmonary Tissues
SP-D was readily detected in a variety of non-pulmonary tissues from fetuses, children, and adults. The presence of SP-D staining in non-pulmonary tissues was distinct from our previous findings demonstrating the lack of staining for SP-A in those tissues. Although SP-D reacted strongly in eccrine sweat glands and gastric fundus, no staining was seen in the same samples with anti SP-A antiserum (data not shown). In initial studies, intense SP-D staining was noted in the intercalated ducts of the exocrine pancreas and in the lumen of larger ducts (Fig 4). To further assess the specificity of reactivity in non-pulmonary tissue, reactivity was assessed in the presence of excess purified mouse SP-D. Co-incubation with exogenous SP-D ablated immunoreactivity against pancreatic tissue (Fig 5).
SP-D was detected in epithelial cells and in luminal material of the ducts of many adult tissues, including lacrimal glands, salivary glands, intercalated ducts of the pancreas, hepatocytes and intra- and extrahepatic bile ducts, esophageal glands, breast, sebaceous and eccrine sweat glands of the skin, and Von Ebner's glands of the tongue. Parietal cells of the stomach, proximal and distal renal tubules, and podocytes of the glomeruli were immunostained. In the reproductive tract, seminal vesicles and endocervical glands stained. SP-D was also observed in endocrine tissues, including adrenal cortex, and in follicular stellate cells of the anterior pituitary gland. Myocardial cells from the right atrium also stained intensely for SP-D (Fig 6 Fig 7 Fig 8 Fig 9 Fig 10 Fig 11 Fig 12 Fig 13 Fig 14). Consistent with widespread distribution of staining, SP-D was detected in human amniotic fluid and saliva but was not detected by ELISA in seminal fluid, urine, breast milk, or tears.
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SP-D in Fetal Tissue and Neonatal Lung
SP-D was detected in lungs of all fetuses and newborns. In 1020-week fetuses, all open airways were rimmed with SP-D (Fig 15 and Fig 16). Thereafter, bronchioles were lightly rimmed or unstained. With advancing gestation there was a distalproximal gradient of SP-D staining, terminal airways being most heavily stained. Staining was less intense in larger bronchioles. Tracheas were rimmed with SP-D up to 15 weeks, and some term fetal tracheas showed staining of basal and intermediate cells (Fig 19). Serous cells of tracheal glands were stained from 18 weeks onward (Fig 17). In the near-term infants, SP-D was detected in the cytoplasm of Type II cells (Fig 18), and airways were rimmed with stain (Fig 3). In lungs from infants with hyaline membrane disease (HMD) and bronchopulmonary dysplasia (BPD), only open terminal airways were rimmed with SP-D. Injured areas lined with hyaline membranes, or alveoli filled with hemorrhage, infection, or edema fluid, were lightly stained or unstained (Fig 20 and Fig 21) SP-D was not detected in bronchioles and bronchi from these infants. However, serous cells of bronchial and tracheal glands were consistently well stained, particularly in infants with lung inflammation. In infants, both layers of striated muscle surrounding the upper esophagus were immunostained but staining was not uniform in distribution (Fig 14). Lungs from older infants with BPD were lightly stained around dilated, unseptated open airways, as were lungs of older infants without a history of HMD whose lungs remained unseptated (Fig 22 and Fig 23). As seen in adult tissues, pancreas, stomach, and duodenum were stained in fetuses and newborns, as were distal convoluted kidney tubules, seminal vesicles and anterior pituitary gland (Table 1).
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A number of tissues tested remained unstained (Table 2), and all tissues showed some variability in the degree of staining, not related to age but most probably to the patients' illness and its inflammatory response in various tissues.
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Discussion |
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SP-D was readily detected in fetal and postnatal human lung as intracellular staining in Type II epithelial cells, and in serous cells of tracheobronchial glands. Staining was prominent along luminal surfaces of terminal airspaces. Intense and specific SP-D staining was also observed in duct cells and in open ducts in various organs and glands in fetuses, infants, and adults. In infants, SP-D was decreased in pulmonary tissues in areas of acute and chronic injury or collapse of alveoli. The abundance of SP-D in luminal material in various organs, including lung, liver, pancreas, lacrimal and salivary glands, tongue, and cervix, supports its proposed role in innate defense at potential sites of invasion by pathogens. The expression of SP-D at unexpected sites, including myocardium, and in the striated muscle surrounding the esophagus supports a potential anti-inflammatory or host defense role for SP-D in non-epithelial organs.
SP-D in the Developing Lung
Early in gestation, SP-D was detected in open airways throughout the lung. As gestation progressed, SP-D staining increased in the lung periphery, with terminal airways being stained most heavily near term and thereafter. Tracheobronchial glands were stained from 18 weeks' gestation and thereafter. In late gestation, fetal lung, and adult lung, SP-D was detected primarily in Type II epithelial cells, consistent with previous studies in rodents in which SP-D mRNA and protein were detected in alveolar cells in both rat and mouse (
In the human lung, serous cells and luminal contents of tracheobronchial glands were immunostained for SP-D, suggesting that the primary source of SP-D in the conducting regions of the human lung may be derived from secretions of tracheobronchial glands. This finding may provide a basis for the recently demonstrated lack of SP-D in lung washings from patients with cystic fibrosis (
Pulmonary findings in SP-D -/- null mice support a critical and unexpected role for SP-D in the regulation of oxidant generation, metalloproteinase activation, and cytokine responses in alveolar macrophages, effects that are independent of exposure to pathogens (B in alveolar macrophages from SP-D-deficient mice (
The distribution of SP-D immunostaining seen in various tissues is consistent with that of SP-D mRNA seen in other species (
The abundance of SP-D on luminal surfaces is consistent with its role in innate defense against pathogens at sites of entry into various organs. The finding that SP-D was expressed at high concentrations in secretory cells and in luminal contents of secretory ducts of liver, pancreas, salivary glands, glands of the skin, and other tissues would provide concentrations of this collectin at sites of potential invasion by microorganisms. The paucity of SP-D staining of lungs of infants with acute HMD and in advancing BPD in areas with alveolar hemorrhage, infection, edema, or alveolar collapse, where Type II cell function may be compromised, may render the lung susceptible to secondary infection and inflammation. The abundant expression of SP-D in tissues such as myocardium, anterior pituitary, and esophageal muscle however, is more difficult to explain on the basis of innate host defense function for SP-D. The finding that SP-D regulates NF-B translocation, oxidant production, metalloproteinase activation, and cytokine responses in the lung may indicate that similar roles are played by SP-D in other tissues, in which SP-D may regulate inflammation and remodeling involved in the pathogenesis of various acute and chronic disorders.
In the present study, considerable variability in SP-D staining was seen in pathological samples from the lungs and other tissues obtained postmortem. It is unlikely that this variability relates to sample collection or fixation because tissues were obtained and fixed rapidly after death, especially in fetuses and newborns. The variability in SP-D staining is likely related to the biological differences in this heterogeneous population of infants. The heterogeneity of SP-D staining is probably influenced by developmental, spatial, and inflammatory stimuli that influence SP-D gene expression or stability. In vivo studies support the concept that SP-D gene expression is strongly increased by endotracheal endotoxin (
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Footnotes |
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Acknowledgments |
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Supported by HL56387 and HL61646.
We wish to thank Ms Ann Maher, Ms Robin Roller, and Mr Jeffrey Phillips for secretarial help, Mr Brent Weedman for photography, Mr Terry Johnson for preparation of illustrations, and Ms Sandra Olson for immunohistochemistry.
Received for publication February 15, 2001; accepted November 28, 2001.
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