Introduction

Localization of lymphatic vessels in hyperplastic polyps to villous adenoma of the colon has been hindered so far because of a lack of suitable markers for the vessels. By immunocytochemical staining using lymphatic vessels endothelial hyaluronan receptor-1 (LYVE-1) for lymphatic vessels and factor-8 (F-8) for venous vessels, localization of lymphatic and venous vessels in normal colon and colonic adenomas was the object of this study. Immunocytochemical staining for lymphatic vessels has been reported using D2-40 as a marker, which reacts with a O-linked sialoglycoprotein found on lymphatic endothelium: lymphatic vessels are located beneath the muscularis mucosa with rare branching through the muscularis mucosa to the most basal aspects of the colonic crypts [1, 2], whereas lymphatic vessels are identified only in the superficial stroma of the colonic adenomatous stalk [1]. Immunocytochemical staining using antipodoplanin also revealed lymphatic vessels in the narrow bands of muscularis mucosa but not in the mucosa or lamina propria [3]. The third marker for lymphatic vessels is LYVE-1, and our study was aimed at detecting lymphatic vessels in normal colon, hyperplastic polyp, tubular adenoma to villous adenoma using polyclonal LYVE-1 antibody. The previous studies for quantitative analysis using LYVE-1 RNA reported below-detectable levels of LYVE-1 RNA in normal colon [4]. Thus, we aimed to immunocytochemically identify lymphatic vessels in normal colon, polyps, and adenomas using polyclonal antihuman LYVE-1 [5–7].

Materials and methods

All cases of colonic polyps and adenomas with adjacent normal colon were from the University of Kansas Medical Center, collected between 1985 and 2001 during tenure at the medical center and consisted of four cases each of hyperplastic adenoma, tubular adenoma, tubovillous adenoma, and villous adenoma, as previously reported by us [8, Table 1]. All tissues were routinely fixed in buffered formalin and embedded in paraffin. The majority of the tissue samples were obtained by endoscopy. Deparaffinized sections were treated with antigen retrieval procedure using citrate buffer pH 6.2. Immunocytochemical staining was performed for LYVE-1 and F-8 using goat antihuman LYVE-1 (R & D System, Minneapolis, MN, USA) and rabbit antihuman F-8 (Dako System, Carpenteria, CA, USA) at 1:100 dilution, respectively [7]. Only tissue sections, which were sufficiently stained for both LYVE-1 and F-8, were included in this study. We had previously tested frozen liver sections for LYVE-1 immunostaining [7] and also tested frozen sections of normal colon and jejunum for LYVE-1 immunostaining.

Table 1 Colonic polyps and adenomas
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Results

Normal colon: Lymphatic vessels identified by LYVE-1 immunostaining were slender, irregularly shaped spaces lying parallel to the transverse muscularis mucosa, which consistently extended into the base and deeper ends of colonic crypts while extending numerously into submucosa in the majority of sections (Fig. 1). In a few sections, small, round, lymphatic-like vessels were also identified in the mid portion and even superficial part of lamina propria surrounded by numerous LYVE-1-positive macrophages (Fig. 1). In muscularis mucosa, venous vessels were relatively fewer, with round and plump lumens rather than slender and irregular-shaped lymphatic vessels, and extended abundantly and longitudinally coiled into lamina propria and sparingly into submucosa (Fig. 1).

Fig. 1
figure 1

Normal colon (a–e) and jejunum (f): Slender, irregularly shaped lymphatic spaces (*) are seen in the deeper ends of colonic crypts (a). Many slender lymphatic vessels (l) are present in muscularis mucosa, which extend into submucosa and lymphoid follicle (L) (c). In a few sections, there are lymphatic vessels endothelial hyaluronan receptor-1 (LYVE-1)-positive macrophages (*) in the lamina propria, some of which tend to form slender spaces, similar to small, slender lymphatic vessels (*) (c). Many round, dilated venous vessels (V) are present in muscularis mucosa, which extend into submucosa and longitudinally coiled into the full thickness of lamina propria (v) L-lymphoid follicle (d). Frozen colon section reveals large lymphatic vessels beneath muscularis mucosa and many small slender lymphatic vessels extending into the entire lamina propria (e). Frozen jejunum section reveals an elongated, dilated lymphatic vessel at each villus, which lies along the longitudinal length of the villus (f). a, c, e, f: Immunostained for LYVE-1; b, d: Immunostained for factor-8

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Hyperplastic polyp: Lymphatic vessels were usually limited in the deep ends of colonic crypts, being parallel to the transverse muscularis mucosa (Fig. 2). Dilated venous vessels were present in muscularis mucosa and in submucosa, and smaller and less darkly immunostained venous vessels were present in the entire lamina propria, which contained minimal fibrous stroma from the base of the crypts to just beneath the surface epithelium (Fig. 2).

Fig. 2
figure 2

Hyperplastic polyp, 0.3 cm in diameter, case 2: Slender lymphatic vessels (l) are noted in the base of colonic crypts, and venous vessels (v) are mostly in muscularis mucosa, the latter extending into submucosa and the entire thickness of lamina propria (*). There are crushing artifacts in this section, as the tissue was obtained by endoscopic biopsy. a: Immunostained for lymphatic vessels endothelial hyaluronan receptor-1; b: immunostained for factor-8

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Tubular adenoma: Both lymphatic and venous vessels were weakly immunocytochemically stained in the fine fibrous, intermucosal stroma of small tubular adenoma (Fig. 3), where lymphatic vessels were present in the base to upper one half of the fibrous stalk, whereas small, less darkly stained, more abundant venous vessels were noted in the mid to top of the stalk (Fig. 3). The stalk of larger tubular adenoma contained scarce lymphatic vessels compared with more abundant slender venous vessels (Fig. 4). The fine fibrous stroma between tubular glands contained practically no lymphatic vessels but a few venous vessels (Fig. 4).

Fig. 3
figure 3

Small tubular adenoma, 0.4 cm in diameter, case 3. Both small, slender lymphatic (*) and venous vessels (*) are present in the deeper ends of colonic crypts, whereas plump venous vessels (v) are present up the mid portion of the fibrous stalk. L-lymphoid follicle. a: Immunostained for lymphatic vessels endothelial hyaluronan receptor-1; b immunostained for factor-8

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Fig. 4
figure 4

Large tubular adenoma, 1.2 × 0.8 cm in diameter, case 1. Stalk of large adenoma contains a few slender lymphatic vessels (l) and abundant, round, venous vessels (V) with much fewer, small lymphatic (*) and venous vessels (*) in lamina propria. a: Immunostained for lymphatic vessels endothelial hyaluronan receptor-1; b: immunostained for factor-8

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Tubovillous adenoma: Tubular part was the same as the tubular adenoma containing lymphatic and venous vessels in superficial fibrous stalk stroma, whereas thin fibrous stroma between the villi had practically no lymphatic vessels but had less darkly immunostained numerous dilated venous vessels (Fig. 5).

Fig. 5
figure 5

Tubovillous adenoma, 2.5 × 2.0 cm, case 1. The villous part reveals thin fibrous stroma containing no lymphatic vessels but abundant round, dilated, venous vessels (v) of less factor-8 (F-8) immunostaining. The deep broader stalk contains many small lymphatic vessels (l) and dilated venous vessel (V). a: Immunostained for lymphatic vessels endothelial hyaluronan receptor-1; b: immunostained for F-8

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Villous adenoma: There were very small lymphatic and venous vessels in the majority of slender, superficial fibrous intervillous stroma, some of which were stained for both LYVE-1 and F-8 (Fig. 6), whereas broader, deep, stalk stroma contained slender lymphatic vessels and round, dilated, venous vessels (Fig. 6).

Fig. 6
figure 6

Villous adenoma, 6.5 × 5.2 cm, case 4. This thin, superficial fibrous stroma between the villi contains slender lymphatic vessels (l) and round, dilated, venous vessels (V) along the villus, some of which (*) are stained weakly for both lymphatic vessels endothelial hyaluronan receptor-1 (LYVE-1) and a little stronger for factor-8 (F-8) in the superficial part of villi. The round, venous vessel is positively stained for F-8 but is negative for LYVE-1. a: Immunostained for LYVE-1; b: immunostained for F-8

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Thus, there were no increased lymphatic or venous vessels in intermucosal stroma and stalks of adenomas as compared with normal colon. Frozen sections of normal colon and jejunum were also stained for LYVE-1 after being fixed in paraformaldehyde, as previously reported [7]. The frozen sections were stained with more diluted antibody for immunostaining for LYVE-1, up to 1:6,000 dilution. Large lymphatic vessels were located in muscularis mucosa, which extended into the base of colonic crypts, very similar to the formalin-fixed and paraffin-embedded sections, and smaller and slender lymphatic vessels extended into the entire lamina propria just beneath the surface endothelium (Fig. 1). Jejunal mucosa contained elongated, dilated, lymphatic vessels in each villus, which extended along the longitudinal length of the villus (Fig. 1).

Discussion

In the majority of colon sections, LYVE-1-positive lymphatic vessels were located in the muscularis mucosa, which consistently spread into the deep end of each colonic crypt and abundantly spread into the submucosa. Thus, this immunostaining for LYVE-1 revealed more lymphatic vessels than that by D2-40 immunostaining [1, 2]. The study reported here also revealed in a few colonic sections that lymphatic vessels were present in the mid portion and even in the upper part of lamina propria surrounded by numerous LYVE-1-positive macrophages (Fig. 1). In normal colon, there were a few lymphatic vessels compared with abundant, longitudinally coiled venous vessels in lamina propria (Fig. 1). Furthermore, frozen sections of normal colon revealed smaller and slender lymphatic vessels in the entire thickness of lamina propria (Fig. 1).

Localization of lymphatic and venous vessels was similar in normal colon and hyperplastic polyp, except more lymphatic vessels were noted up to the mid portion of the lamina propria in hyperplastic polyps (Fig. 2). In tubular adenomas, lymphatic and venous vessels were scarce in fine fibrous, intermucosal stroma, but both lymphatic and venous vessels were abundant in the deeper parts of broader stalks, and further small lymphatic and venous vessels were also noted in the superficial stalk.

In the villous part of tubovillous adenomas and villous adenomas, there were practically no lymphatic vessels but some venous vessels in thin, fibrous stalks. In deeper, broader fibrous stalks, there were large lymphatic and venous vessels, which were strongly stained for LYVE-1 and F-8 (Fig. 4). In occasional superficial fibrous stroma between the villi, small lymphatic and venous vessels were noted, which weakly stained for both LYVE-1 and F-8, probably representing newly forming vessels with little LYVE-1 and F-8 antigens.

In colonic mucosa where both lymphatic vessels and venous vessels were present, lymphatic vessels mostly absorbed and venous vessels mostly secreted interstitial fluid [8]. In colonic adenomas where a few lymphatic vessels and abundant venous vessels were present, venous vessels may well have a dual function of some absorbing and more secreting interstitial fluid [8]. Large adenomas may cause watery diarrhea and electrolyte loss in some patients, especially those with large villous adenomas [9]. These adenomatous glands of superficial villi correspond to the atypical growing cells with high percentage of proliferating cell nuclear antigen (PCNA)-positive immunostaining, as reported by us [10].

More lymphatic vessels have been reported in cases of ulcerative colitis than in normal colon by D2-40 immunostaining, which consistently extended into lamina propria as a result of mucosal inflammation [2, 3]. Few but definite lymphatic vessels have also been reported in the margin of colonic adenocarcinoma but not within the carcinoma using D2-40 immunostaining [1, 11]. By immunofluorescence study, Fiedler et al. detected that CD-34-positive vessels were LYVE-1+/Prox-1+/lymphatic vessels in peritumoral and intratumoral colonic tissue from patients with colonic carcinoma [12]. Lymphatic vessels are readily identifiable by immunocytochemical staining as follows: (1) D2-40 [1, 2]; (2) podoplanin, a 43-kDa glycoprotein cloned as a cell-surface protein expressed on glomerular podocytes [3, 11]; (3) LYVE-1 [5–7, 13]; (4) Prox-1, a transmembrane factor, that is expressed in lens, heart, liver, and nervous system [14]; (5) vascular endothelial growth factor-receptor 3 (VEGF-R3), a receptor tyrosine kinase, that is mainly expressed on lymphatic vessels [15]; and (6) desmoplakin, present in endothelial adhering junction, that is immunostained in small lymphatic vessel endothelium [16]. Thus, lymphatic vessels comprise a complex vascular system as characterized by many markers, as listed above.

Further studies are warranted to convincingly identify colonic lymphatic vessels, especially in lamina propria and in fine fibrous stroma between tubular and villous glands in addition to abundant lymphatic vessels in muscularis mucosa, submucosa, muscle layers, and subserosa, preferably using frozen sections [7].