Expression of Toll-like receptors 2 and 4 on human intestinal lymphatic vessels

Abstract

The Toll-like receptor (TLR) is a part of the innate immune system sensing pathogen-associated molecular patterns (PAMPs). Recently, TLRs 2 and 4 have been demonstrated for the ligand engagements, which result in the induction of cytokines. Here we investigated the expression of TLRs 2 and 4 on lymphatic vessels producing cys–cys chemokine ligand 21 (CCL21) in the human small intestine. The specificity of antibodies to TLRs was tested on a human monocyte leukemia cell line, umbilical vein endothelial cells (HUVEC), and periodontal ligament fibroblasts (PDLF) with the examination for the TLR gene expression by the reverse transcription-polymerase chain reaction (RT-PCR), and lymphatic vessels were identified by antibodies for platelet-endothelial cell adhesion molecule-1 (PECAM-1) and desmoplakin. The expression of CCL21 was not clearly detected on collecting lymphatic vessels in the submucosa while it was generally observed on the central lacteals of villi and lymphatic capillaries in the lamina propria mucosae. The reaction of antibodies to TLRs 2 and 4 was also not clearly detected on collecting lymphatic vessels in the submucosa and central lacteals of villi, but generally observed on lymphatic capillaries expressing CCL21 in the lamina propria mucosae of tissue where the expression of CCL21 and TLRs was not clearly observed in blood vessels. These may suggest that the expression of CCL21, and TLRs 2 and 4 is predominantly induced in the peripheral lymphatic endothelium of the small intestinal microcirculation. The lymphatic endothelium may contribute to allow dendritic cells to home into secondary lymphoid tissue through the expression of TLRs, the ligand engagements of which result in the induction of chemokines.

Introduction

Human Toll-like receptors (TLRs) 1–10, designated to 10 human homologues of Drosophila Toll, are part of the innate immune system sensing the presence of conserved pathogen-associated molecular patterns (PAMPs). The TLRs are type I transmembrane proteins with leucine-rich repeat extracellular domains and conserved cytoplasmic domains with homology to interleukin 1 receptor Aderem and Ulevitch, 2000, Janssens and Beyaert, 2002, Martin and Wesche, 2002, Ozinsky et al., 2000. Recently, TLRs 2 and 4 have been demonstrated for the ligand engagements which result in the induction of cytokines and co-stimulatory molecules required for the adaptive immune response through the activation of at least NF-kappa B following the MyD-88-dependent and -independent signaling pathway Horng et al., 2002, Kawai et al., 2001, Schnare et al., 2001. The TLR2 recognizes lipoteichoic acid of Gram-positive bacteria, macrophage-activating lipopeptide from Mycoplasma fermentans, phenol-soluble modulin secreted by Staphylococcus epidermidis, yeast cell-wall particles (zymosan), glycosylphosphatidylinositol anchors from parasitic protozoa, and a collectin family member of preimmune opsonins (surfactant protein A). The TLR4 recognizes lipopolysaccharide (LPS) of Gram-negative bacteria, a plant diterpene sharing a number of macrophage stimulatory functions with LPS (Taxol), and autologous heat shock protein 60 that serves as a danger signal to the innate immune system Kadowaki et al., 2001, Kaisho and Akira, 2002, Lien and Ingalls, 2002, Muzio et al., 2000, Sabroe et al., 2002. In the small intestine, pathogens are transported through microfold cells in the simple epithelium into lamina propria mucosae by endocytosis and exocytosis. The lymphatic endothelium may have recognition mechanisms for PAMPs by TLRs, the ligand engagements of which result in induction of lymphoid tissue chemokines like cys–cys chemokine ligand 21 (CCL21; Gunn et al., 1999, Kriehuber et al., 2001).

The identification of lymphatic vessels were performed by monoclonal antibodies for desmoplakin and platelet-endothelial cell adhesion molecule-1 (PECAM-1). It has been revealed that lymphatic but not blood endothelium expresses desmoplakin in human small intestine and tongue by immuno-electron microscopic studies, and it is known that lymphatic vessels also express PECAM-1 Ebata et al., 2001a, Ebata et al., 2001b. Desmoplakin is a desmosome-associated protein found in epithelia, and lymphatic vessels were identified by anti-desmoplakin after the discrimination from the intestinal epithelia by anti-PECAM-1. This study aimed to examine the possibility that lymphatic endothelium expresses TLRs 2 and 4, and to investigate the distribution of the intestinal lymphatic vessels with the production of CCL21 and the TLRs.