CD11c immunopositive cells in the human fetal vermiform appendix

Introduction/Aim. A vermiform appendixis an abdominal organ which contains the elements of gut-associated lymphoid tissue and carries out important immunological functions as a reservoir of intestinal microbial flora. It also has a role in the normal development of gut-associated lymphatic tissue. The aim of this study was to examine the distribution of dendritic cell marker C D11c in the human fetal vermiform appendix from the 13th to the 23rd week of development. Methods. The material in this study consisted of 28 human fetal vermiform appendixes from the 13th to the 23rd week of gestagion. The tissue samples were routinely processed to obtain paraffin blocks, and 5 ?m thick tissue sections were stained with hematoxylin and eosin, and with rabbit monoclonal antibody against CD11c antigen and mouse monoclonal antibody against desmin. Results. The first CD11c immunopositive cells appear in the 14th week of development. They are present in the mucosa/submucosa and are interconnected via their cytoplasmic processes. Around these cells, a small number of lymphocytes can be seen. The first lymphoid aggregations appear in the 16th week of development, and lymphocytes are organized around the network made of CD11c immunopositive cells. From the 18th week of development, the lymphoid aggregations are organized in the form of primary lymphoid follicles, containing an extensive network made of CD11c immunopositive cells. Conclusion. CD11c immunopositive cells appear first in the process of primary lymphoid follicle generation and have a role in forming a lattice which will serve as the basis for lymphocyte migration.


Introduction
The vermiform appendix is an abdominal organ, classified as a part of the large intestine, that was considered for a long time to be a vestigial remnant of the caecum, without any specific functions in humans, especially having in mind that it is not involved in the processes of digestion and intestinal peristaltics. 1 However, some more recent studies have shed a different light on the function of this organ, suggesting the importance of its immunological roles, especially regarding its being a haven for microbial flora of the intestines, releasing it in cases when its repopulation is required. 1 This is especially important in cases when the normal intestinal flora has been destroyed after some viral or bacterial infections. Further, it is believed that the presence of bacterial flora in the vermiform appendix is important for tolerance induction against the indigenous flora, as well as for the stimulation and normal development of gut-associated lymphoid tissue. 2 The vermiform appendix is the intestinal derivative of the midgut. The development of the vermiform appendix is closely related to the midgut development, which can be summarized to take place in three phases. 3 In phase one, that occurs during the sixth week of development, the midgut elongates considerably, which results in the formation of the hairpin-shaped loop. Due to insufficient space in the embryo, this loop extends into the extraembryonic coelom of the umbilical cord forming the physiological umbilical hernia. 3 During this phase, the intestinal loop rotates 90 degrees around the superior mesenteric artery. The phase two occurs during the tenth week of development and is characterized by back-positioning of the instestinal loop inside the embryonal cavity, closure of the physiological umbilical hernia and by additional rotation of the intestinal convolute for 180 degrees. 3,4 During the third phase that occurs in the twelfth week of gestation, the midgut is fixated in the peritonal cavity. The bud-like complex of the early cecum and appendix appears during the phase two of midgut development on the right-hand side of the upper abdominal cavity. 3,4,5 The process of colon elongation consequently leads to the descent of cecum and appendix, as they become finally positioned in the right iliac fossa. 3,4 The appendix vermiformis can be observed at the eight week of gestation, while the first accumulations of the lymphatic tissue develop during the weeks 14 and 15, at first as a few lymphatic cells located below the epithelium. 3,5 Lymph nodules appear approximately during the 4th and 5th months of development and they continue to increase, with a peak in the 28th week of development. The components of GALT continue to grow up to puberty. 3,5,6 The colonization of vermiform appendix with bacterial flora begins approximately two weeks after birth. 2 The lymphoid tissue of vermiform appendix consists of multiple solitary lymphoid follicles, whose cellular content comprises B lymphocytes and a small number of T lymphocytes, dendritic cells and macrophages. 7 Some authors suggest that the nerves of the enteric nervous system (ENS) might play a role in the regulation of immunological functions of the gut-associated lymphoid tissue, including the vermiform appendix. 8 The immune system and ENS interact with each other and some studies have revealed that nerves can act as inflammation modulators in the intestinal tissue. 9 Moreover, it has been reported that higher levels of neuropeptides in the vermiform appendix might provoke acute abdominal pain without any signs of acute appendicitis. 10 Bearing in mind the importance of lymphoid tissue in the vermiform appendix, the aim of this paper was to examine the expression of CD11c antigen in its tissue during the second trimester of fetal development. CD11c is one of the classical markers of dendritic cells, but is also expressed on the neutrophils, macrophages and some B lymphocytes. 11,12 6

Methods
The material consisted of 28 vermiform appendixes of the fetuses from 13 th to 23 rd week of gestational age (Table 1)  All vermiform appendix samples were fixed in 10% buffered formalin and routinely processed to paraffin blocks. From each paraffin block, 5 µm-thick sections were obtained using a Leica microtome. The obtained paraffin sections were deparaffinized (in the thermostat at 64°C and xylene) and rehydrated in a series of descending concentrations of alcohol (100%, 96%, and 75%) and distilled water. The sections were stained with hematoxylin and eosin (HE), and immunohistochemically by using the rabbit monoclonal antibody against CD11c antigen (Abcam, ab52632, 1:100) and the mouse monoclonal antibody against desmin (Dako, M0760). The incubation with the antibody was performed overnight at 4°C. As a visualization system, EnVisionFLEX, HighpH (Agilent, K8000/8002) was used. The photo documentation, used for microscopic analysis, was obtained using an Olympus BX50 light microscope equipped with a Leica DFC295 digital camera (Leica Microsystems, Germany).

Results
The analysis of HE sections of vermiform appendix samples showed that their histological structure corresponded well to the week of gestational age. In the 13 th week of development, all the layers of the wall (mucosa, submucosa, muscularis and serosa) were present on all the examined samples. All vermiform appendixes had the intestinal glands with crypts present in their mucosa; however, the lamina muscularis mucosae was absent. The lamina muscularis mucosae appeared first in the 18 th week of development in the form of scattered smooth muscle cells between the mucosa and submucosa. The muscular layer was composed of well-developed circular and thin longitudinal sublayers (Fig 1A). Until the 23 rd week of development, the lamina muscularis mucosae was well developed and the longitudinal muscle sublayer became thicker (Fig 1F).
Lymphatic tissue or CD11c immunopositive cells were absent in the samples in the 13 th week of development. Rare CD11c immunopositive cells were first observed in the vermiform appendixes in the 14 th week of development (Fig. 1B). These cells were seen in small groups with focal distribution in the mucosa/submucosa layer. Their morphology revealed the presence of cytoplasmic processes with whom they were interconnected.
CD11c immunopositive cells were surrounded by rare, scarce lymphocytes that were not seen in the areas between the groups of CD11c immunopositive cells. The first lymphocyte aggregations, resembling the primary lymphoid follicles, were observed in the samples in the 16 th week of development ( Fig 1C). The lymphocyte aggregations were organized around the network made by CD11c immunopositive cells. The number of CD11c immunopositive cells was markedly higher and the network they form was more extensive.
As the development continued, the number of CD11c immunopositive cells and lymphocytes gradually increased (Fig 1D). Until the 20 th week, the primary lymphoid follicles were completely formed (Fig. 1E, F). CD11c immunopositive cells were present inside the lymphoid follicle, where they formed a network around which the lymphocytes were situated.

Discussion
The morphology of the vermiform appendix from 13 th to 23 rd week of development showed that all the layers of its wall were present, as well as the intestinal glands and two sublayers 9 in its muscular layer. The lamina muscularis mucosae was observed only in the samples from 18 th week of development. The longitudinal muscle sublayer was very thin in the 13 th week of development, but it became thicker and both muscle sublayers were clearly visible until the 23 rd week. These findings were in accordance with the results of other authors who studied the development of this organ. 13,14 However, in the literature there have been different information concerning the appearance of lymphatic tissue in its mucosal/submucosal layer. 15,16,17 Our findings suggest that the first lymphocytes can be CD11c. 19 Experimental models suggest that the formation of primary lymphoid follicles is a multistep process that involves both B lymphocytes and dendritic cells. 20,21,22 The first step in this process appears to depend on the secretion of LTα1β2 by B lymphocytes, which is responsible for the formation of a network of dendritic cells. 22,23 When the network of dendritic cells is formed, the migration of B lymphocytes is facilitated and the quantity of lymphatic tissue increases, thus forming a morphologically distinctive primary lymphoid follicle. 22 In conclusion, our results give the morphological evidence that a network of CD11c immunopositive cells of the future primary lymphoid follicle arises before the appearance of lymphoid aggregates in the vermiform appendix, suggesting thus that these cells are crucial in the process of lymphoid follicle generation.