CL-L1 and CL-K1 Exhibit Widespread Tissue Distribution With High and Co-Localized Expression in Secretory Epithelia and Mucosa

Abstract

Collectin liver 1 (CL-L1, alias collectin 10) and collectin kidney 1 (CL-K1, alias collectin 11) are oligomeric pattern recognition molecules associated with the complement system, and mutations in either of their genes may lead to deficiency and developmental defects. The two collectins are reportedly localized and synthesized in the liver, kidneys, and adrenals, and can be found in the circulation as heteromeric complexes (CL-LK), which upon binding to microbial high mannose-like glycoconjugates activates the complement system via the lectin activation pathway. The tissue distribution of homo- vs. heteromeric CL-L1 and -K1 complexes, the mechanism of heteromeric complex formation and in which tissues this occurs, is hitherto incompletely described. We have by immunohistochemistry using monoclonal antibodies addressed the precise cellular localization of the two collectins in the main human tissues. We find that the two collectins have widespread and almost identical tissue distribution with a high expression in epithelial cells in endo-/exocrine secretory tissues and mucosa. There is also accordance between localization of mRNA transcripts and detection of proteins, showing that local synthesis likely is responsible for peripheral localization and eventual formation of the CL-LK complexes. The functional implications of the high expression in endo-/exocrine secretory tissue and mucosa is unknown but might be associated with the activity of MASP-3, which has a similar pattern of expression and is known to potentiate the activity of the alternative complement activation pathway.

Keywords: 3MC syndrome; collectin; complement system; innate immunity; mucosal immunology.

Figure 1

Structure of CL-LK and antibody specificity. (A) Schematic illustration of the domain organization of CL-L1 and CL-K1 polypeptide chains. The symbol “*”on the CL-L1 polypeptide chain represent two N-linked glycosylation sites in the carbohydrate recognition domain. CL-K1 is found in the circulation in the form of two isoforms: CL-K1a represents full-length and CL-K1d represents an alternative spliced form devoid of a part of the collagen-like region. (B) Subunit of CL-LK and oligomeric structures. A total of three polypeptide chains of CL-K1 and CL-L1 join to form a heteromeric subunit, which may further oligomerize into structures ranging from dimers to hexamers of subunits, here illustrated by a tetramer. (C) Analysis of purified CL-LK by SDS-PAGE and visualization by silver staining. The three bands of CL-L1 represent non-, partially, and fully glycosylated forms of CL-L1. (D,E) Specificity of monoclonal antibodies by Western blotting of serum under. reducing conditions and visualization by ECL. (F–H) SPR analyses of monoclonal antibodies with immobilized CL-K1, CL-L1, and CL-LK as antigen, respectively. MAbs 16-13 (--) and 11-1 (---) were analyzed for binding to immobilized purified CL-L1 (F), CL-K1 (G), or CL-KL (H) MAb using concentrations ranging from 0.1 to 10 µg/ml.

Figure 2

Characterization of mAbs’ impact on the ligand-binding and complement activity mediated by CL-LK. (A) Impact on ligand-binding. CL-LK was incubated on ligand-coated plates (mannan or DNA) in the presence or absence of the mAbs used for IHC (mAb 11-1 and mAb 16-13), mannose, or serum (known to interfere with the binding of CL-LK to ligands). mAb 131-1 (anti-MBL) was included as control. Bound CL-LK was detected with biotinylated mAb-anti-CL-K1 (compatible with the applied mAbs) and HRP-streptavidin. (B) Impact on CL-LK-mediated complement activation. Prior to incubation with MASP-2 and C4, plates were prepared with CL-LK and coated ligands (mannan or DNA) as above. Deposition of C4b was detected with biotin-labeled anti-C4b mAb and HRP-streptavidin. The results shown are representative of three independent experiments. Error bars refer to max and min of triplicate measurements. None of the tested mAbs interfered with ligand binding or complement activation. CL-LK binding to mannan and DNA occurs via two separate binding site, and the latter is not inhibited by mannose, whereas uncharacterized blood components inhibit both types of interaction (10, 11).

Figure 3

Immunohistochemical localization of CL-K1 and CL-L1 in formalin fixed and paraffin embedded sections of the liver (A,B), kidney (D,E), and lung (H,I). Italic letters within images refer to: for the liver h: hepatocytes, ch: centrilobular hepatocytes, and kc: Kupffer cells; kidney g: glomerulus, dt: distal tubules, pt: proximal tubules, and bc: Bowman’s capsule; lung I: type I pneumocytes, II: type II pneumocytes, and m: alveolar macrophages. Scale bars in large sections and in isotype control sections (C,F,G) correspond to 500 µm and in small sections to 50 µm.

Figure 4

Immunohistochemical localization of CL-K1 and CL-L1 in formalin fixed and paraffin embedded sections of the thyroid gland (A,B), pancreas (D,E), and adrenal gland from a patient diagnosed with pheochromocytoma, (H,I). Italic letters within images refer to: for the thyroid gland tf, thyroid follicles and c, parafollicular C cells; pancreas il: islets of Langerhans and a: acinar epithelial cells. Scale bars in large sections and in isotype control sections (C,F,G) correspond to 500 µm and in small sections to 50 µm.

Figure 5

Immunohistochemical localization of CL-K1 and CL-L1 in formalin-fixed and paraffin-embedded sections of the gallbladder (A,B), duodenum (D,E), and colon (H,I). Italic letters within images refers to: for the gallbladder s: serosa layer, ms: muscularis layer, m: mucosa layer, lp: lamina propria, ce: columnar epithelial cells, and f: fibroblasts; duodenum and colon me: muscularis externica layers, sm: submucosa, mu: mucosa, cl: crypts of Lieberkuhn (tubular glands), mm: muscularis mucosa, and v/a: vein/artery. Scale bars in large sections and in isotype control sections (C,F,G) correspond to 500 µm and in small sections to 50 µm.

Figure 6

Immunohistochemical localization of CL-K1 and CL-L1 in formalin-fixed and paraffin-embedded sections of the testis (A,B), prostate (D,E), and uterus corpus (H,I). Italic letters within images refers to: for the testis st: seminiferous tubules, e: endothelial cells, l: Leydig cell, tp: tunicapropria, ge: germinal epithelial cells, s: spermatogonia, sy: spermatocytes, and s: spermatids; prostate g: gland, d: duct, c: “concretized” material: a: acininar cells/acinus, s: stroma, and e: basal epithelial cells; uterus corpus sb: stratum basale, sf: stratum functionalis, ls: luminal surface, g: gland, d: duct, se: stratified columnar epithelial cells, and ce: ciliated epithelial cells. Scale bars in large sections and in isotype control sections (C,F,G) correspond to 500 µm and in small sections to 50 µm.

Figure 7

Immunohistochemical localization of CL-K1 and CL-L1 in formalin fixed and paraffin embedded sections of the skin (A,B), parotid gland (D,E), and placenta (H,I). Italic letters within images refers to: for the skin ed: epidermis, de: dermis, hd: hypodermis, sg: sweat glands, me: myoepithelial cells, and se: stratified cuboidal epithelia cells; partoid salivary gland d: duct, ed: epithelial ductal cells, sa: serous acini, ma: mucous acini, mj: major duct, il: intralobulated duct, and ic: intercalated duct; placenta v: villus, st: syncytiotrophoblast, ct: cytotrophoblast, and md: mesoderm. Scale bars in large sections and in isotype control sections (C,F,G) correspond to 500 µm and in small sections to 50 µm.