Purification, characterization and immunolocalization of porcine surfactant protein D

Abstract

Summary Surfactant protein D (SP‐D) is a collectin believed to play an important role in innate immunity. SP‐D is characterized by having a collagen‐like domain and a carbohydrate recognition domain (CRD), which has a specific Ca2+‐dependent specificity for saccharides and thus the ability to bind complex glycoconjugates on micro‐organisms. This paper describes the tissue immunolocalization of porcine SP‐D (pSP‐D) in normal slaughter pigs using a monoclonal antibody raised against purified pSP‐D. Porcine SP‐D was purified from porcine bronchoalveolar lavage (BAL) by maltose‐agarose and immunoglobulin M affinity chromatography. The purified protein appeared on sodium dodecyl sulphate–polyacrylamide gel electrophoresis as a band of ∼53 000 MW in the reduced state and ∼138 000 MW in the unreduced state. Porcine SP‐D was sensitive to collagenase digestion and N‐deglycosylation, which reduced the molecular mass to ∼24 000 MW and ∼48 000 MW respectively, in the reduced state. N‐deglycosylation of the collagen‐resistant fragment, reduced the molecular mass to ∼21 000 MW showing the presence of an N‐glycosylation site located in the CRD. Porcine SP‐D bound to solid‐phase mannan in a dose and Ca2+‐dependent manner with a saccharide specificity similar to rat and human SP‐D. The purified protein was used for the production of a monoclonal anti‐pSP‐D antibody. The antibody reacted specifically with pSP‐D in the reduced and unreduced state when analysed by Western blotting. Immunohistochemical evaluation of normal porcine tissues showed pSP‐D immunoreactivity predominantly in Clara cells and serous cells of the bronchial submucosal glands, and to a lesser extent in alveolar type II cells, epithelial cells of the intestinal glands (crypts of Lieberkühn) in the duodenum, jejunum and ileum and serous cells of the dorsolateral lacrimal gland.

Pulmonary infections in swine induce altered porcine surfactant protein D expression and localization to dendritic cells in bronchial‐associated lymphoid tissue

“…15 Comparing normal and diseased lung tissue from the same animals, pSP-D was found to be expressed excessively in the surfactant in both acute and chronic bronchopneumonic lesions, in particular those caused by A. pleuropneumoniae. Embolic pneumonias caused by Staph.…”

“…Sections of normal porcine lung tissue, with previously identified pSP-D immunoreactivity, 15 were included as positive staining controls in each test round of pSP-D staining (i.e. only rounds with immunoreactivity in control slides were regarded as valid).…”

“…The specificity of the monoclonal antibody (mAb) 1Á7 for purified pSP-D and pSP-D in bronchoalveolar lavage fluid has previously been shown by Western blot and N-terminal sequencing. 15 Negative-staining controls of all tissues were generated by substituting the primary mAbs, all of which were of the IgG1 isotype, with an irrelevant monoclonal IgG1 (DAKOCytomation, no. X0931; Glostrup, Denmark) of identical concentration, and by substituting the primary polyclonal antibodies with normal rabbit immunoglobulins (DAKOCytomation, no.…”

“…The pSP-D cDNA sequence has been determined, 14 the pSP-D protein has been purified and characterized, 15,16 and the interaction between pSP-D and influenza A virus described. 17,18 Furthermore, pSP-D was found to be immunolocalized in normal tissues (predominantly in Clara cells and in serous cells of the bronchial submucosal glands) and, to a lesser extent, in alveolar type II cells, in epithelial cells of the intestinal glands (crypts of Lieberkühn) in the duodenum, jejunum and ileum, and in serous cells of the dorsolateral lacrimal gland.…”

“…17,18 Furthermore, pSP-D was found to be immunolocalized in normal tissues (predominantly in Clara cells and in serous cells of the bronchial submucosal glands) and, to a lesser extent, in alveolar type II cells, in epithelial cells of the intestinal glands (crypts of Lieberkühn) in the duodenum, jejunum and ileum, and in serous cells of the dorsolateral lacrimal gland. 15 DCs are the most potent antigen-presenting cells known. 19 DCs exist in an immature state at mucosal surfaces, including the lungs, where they constantly survey the environment for the presence of foreign antigen.…”