Immunolocalization of a novel collectin CL-K1 in murine tissues

Abstract

We have recently identified a novel collectin, CL-K1, that may play a role in innate immunity as a member of the collectin family. In this study using mice, we investigated the tissue distribution of CL-K1 for better understanding of its pathophysiological relevance. Real-time PCR analyses demonstrated that CL-K1 mRNA was expressed in all tissues tested. Immunohistochemical analyses demonstrated that CL-K1 was expressed in proximal tubules of kidney, in mucosa of the gastrointestinal tract, and in bronchial glands of bronchioles similar to the localization of SP-A and SP-D in these pulmonary structures. Immunohistochemistry also showed that CL-K1 was highly expressed in hepatocytes around the central veins in liver, which suggests that murine CL-K1 may be mainly produced in the liver and secreted into the blood stream as is human CL-K1. CL-K1 was especially detected in vascular smooth muscle in several types of tissues. In addition, it was also expressed in intestinal Paneth cells, in mesangial cells of kidney, in pancreatic islet D cells, and in neurons of the brain. It is of interest that this profile of CL-K1 expression is unique among the collectins. Together these histological findings may be useful for understanding the biological function of this novel collectin.

Figure 1

Estimation of the amount of CL-K1 mRNA in different tissues. Relative mRNA levels were measured by TaqMan RT-PCR. Data were normalized based on the value of 18S rRNA.

Figure 2

Specificity of our CL-K1 polyclonal antibody was analyzed by ELISA, immunocytochemistry, and immunohistochemistry (IHC). The anti-CL-K1 IgG fraction (IgG) was purified from rabbit serum. After IgG purification, the affinity antibody (postaffinity) was purified on an antigen column, and the pass-through IgG was used as the control IgG (pass-through IgG). (A) Results of ELISA analysis using anti-CL-K1 IgG, postaffinity antibody, or pass-through IgG. ELISA analyses of anti-CL-K1 antibodies against human CL-K1. (B) Results of ELISA analyses of anti-CL-K1 affinity antibody reactivity with other collectins, namely, CL-L1, CL-P1, and MBL. (C) Cross-reactivity between human and murine CL-K1 recombinant protein. (D) Immunofluorescence in CHO cells overexpressing CL-K1 (left and middle panels) as well as in empty vector expressed CHO cells (mock cells) (right panel). (E) IHC staining and immunofluorescence staining with affinity antibody or control IgG in murine testis.

Figure 3

IHC of murine renal cortex (A) and vascular smooth muscle cells in kidney (B). CL-K1 protein was expressed in mesangial cells in glomerulus (red arrow in A) and in brush border of proximal tubules (yellow arrow in A). Double immunofluorescence staining (C) demonstrates that CL-K1 was not colocalized in microvascular endothelial cell.

Figure 4

IHC of vascular cells in heart (A) and small intestine (B). CL-K1 expression was detected in vascular portion in heart (A) and small intestine (B). Double immunofluorescence staining (C,D) demonstrates that CL-K1 was colocalized in vascular smooth muscle cells but not in endothelial cells.

Figure 5

IHC localization of CL-K1 in murine lung, heart, testis, and brain. CL-K1 expression was especially strong in bronchial glands of bronchium (red arrow in A,B). In peripheral lung (C), CL-K1 was also expressed in respiratory bronchioles (black arrow). In heart and testis, CL-K1 was expressed in lamina elastica of coronary artery in myocardium (D) and in cytoplasm of spermatocytes (E). (F) Representative neurons stained with CL-K1 antibody in the reticular formation of the medulla oblongata.

Figure 6

IHC localization of CL-K1 in liver and pancreas. In liver (A), CL-K1 was expressed in hepatocytes. A relatively high expression of CL-K1 was seen in hepatocytes around the central vein (black arrow). In pancreas (B), CL-K1 was expressed not only in acinar cells but also in islet cells (C). Double immunofluorescence staining (D) demonstrates that CL-K1 was colocalized in somatostatin-containing D cells but not in glucagon-containing α-cells or insulin-containing β-cells.

Figure 7

IHC localization of CL-K1 in gastrointestinal tract. In esophagus (A), stomach (B), small intestine (C), and large intestine (D), CL-K1 was expressed in epithelium. In stomach, CL-K1 was colocalized with somatostatin in somatostatin-containing cells (E). In small intestine, CL-K1 was expressed in Paneth cells (yellow arrow in C). In large intestine, CL-K1 was expressed in epithelial mucosa (D).