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Journal of the Anatomical Society of India

Morphology and Distribution of Human Colonic Dendritic Cells: A Light Microscopic Zinc Iodide-Osmium Study

Author(s): Indrasingh, I., Koshy, S. and Vettivel, S.

Vol. 52, No. 1 (2003-01 - 2003-12)

Department of Anatomy, Christian Medical College, Vellore INDIA

Abstract

Dendritic cells have been demonstrated in human and animal epithelium and subepithelial tissue. The present studyused human colonic specimens, resected for carcinoma and revealed zinc iodide osmium positive dendritic cells beneath the basementmembrane in the crypt of Lieberkuhn with the dendritic process toward the crypt lumen and throughout in the lamina propria. The cellsappeared to be more in the lamina propria than in the crypt. Dendritic cells were polygonal without obvious processes and triangular withone or two (often a single) slender, short processes. The cells do not seem to be of typical, mature phenotype.

Key words: Colon, crypt, dendritic cell, human, lamina propria, zinc iodide-osmium.

Introduction:

The intestinal immune system is to eliminate pathogenic microorganisms and to avoid unnecessary immune responses to orally ingested antigens and commensal bacteria. Luminal antigens, which have penetrated into the intestinal lamina propria, interact with components of the immune system including dendritic cells (DCs). DCs are potent immunostimulatory cells (Steinman 1991); they are bone marrow-derived, thymus independent cells (Steinman and Nussenzweig, 1980); they can take up and present both orally and intestinally administered antigens to naive T cells (Liu and MacPherson, 1991) DCs are the most important and the only antigen presenting cell type capable of presenting peptides to virgin T cells, thereby initiating cell mediated immunity to newly encountered antigens. Efficient capture and presentation of antigens by DCs is central to the induction of an immune response (Colaco, 1999). DCs are probably the key factors in controlling T cell responses in the gut wall. These are present in all organized lymphoid tissues and may be also present in gut epithelium (Maric et al, 1996).

Various markers have been used to identify DCs in the digestive tract: vimentin (Olah and Glick 1995); S100 (Pavli et al, 1996); ATPase (Bykov, 1997); and fractalkine (Papadopoulos et al, 1999). Zinc iodide-osmium (ZIO) has been used to identify DCs (Crocker and Hopkins, 1984; Chandi et al, 1988, 1989; Dagdeviren et al, 1994; Breathnach and Goodwin, 1965; Niebauer et al, 1969; Rodriguez and Caorsi, 1978; Hart and Fabre, 1981; Sertle et al, 1986; Prickett et al, 1988; Steinman, 1991; Abraham et al, 1996, 2000; Indrasingh et al, 2001).

DCs have been found in human tissues: epidermis (Mishima and Millar-Miniska, 1961; Zelickson and Mottaz, 1968; Rodriguez and Caorsi 1978), pigmented and unpigmented skin (Riley, 1967); oesophagus (Al Yassin and Toner, 1976), exocervix (Figueroa and Caorsi, 1980), tonsil (Crocker and Hopkins 1984; Chandi et al, 1988, 1989; Nobel et al, 1996, Papadopoulos et al, 1999), liver Prickett et al, 1988); dermis (Lenz et al 1993), decidua (Abraham et al, 1996; 2000), colon (Pavli et al, 1996), skin and tonsil (Papadopoulos et al, 1999), respiratory tract (Holt and Stumbles, 2000), and artery intima (Millonig, et al, 2001),

The present study was to demonstrate, using zinc iodide-osmium, the morphology and distribution of the DCs in the human colon.

Materials and Methods:

Full thickness specimens of colon were obtained from patients undergoing surgery for colorectal cancer (n = 2) in Christian Medical College Hospital, Vellore.

The tissue pieces were immersed in a solution of veronal buffered zinc iodide osmium tetroxide at pH 7.4 (Figueroa and Caorsi, 1980) for 48 hours at 4°C in the dark, washed in distilled water, dehydrated in graded ethanol, cleared in xylene and embedded in paraffin wax. Serial sections of seven- micron thickness were cut and the sections were transferred to glass slides, deparaffinised, mounted in Canada balsam, without counter staining (Chandi et al, 1988; Abraham et al, 1996; Indrasingh et al, 2001) and viewed under light microscope.

Results:

ZIO positive DCs were located in the crypts of Lieberkuhn as well as in the lamina propria (Fig. 1). The cells were polygonal and triangular. Many cells did not show the dendritic processes. The triangular cells showed one or two processes. The processes were thin and short. More DCs were present in the lamina propria than in the crypt (Fig. 1).

Where infiltration of lymphocytes from lymphoid follicle in the submucosa to the lamina propria occurred, a few ZIO-positive DCs, possibly follicular dendritic cells (FDC), were present (Fig. 2)

In the crypts, DCs had the process directed towards the lumen of the crypt (Figs. 3, 4) and in the lamina propria dendritic cells were present (Fig. 4).

Discussion:

ZIO has been extensively used to identify the DCs. Cellular reactivity to ZIO is attributed to certain reducing substances such as catecholamines and ascorbic acid (Stockinger and Graf, 1965) and to lipid moieties unmasked from lipoprotein (Niebauer et al. 1969). The ZIO technique, with marked deposition of reaction product in the mitochondrial granules, indicates the presence of lipids and/or precursor proteins (Taffarel et al, 1984).

DCs. which belong to mononuclear phagocyte family, initiate immune reactions in lymphocytes and have a critical role in antigen handling (Nossal et al, 1968; Veerman and van Rooijen, 1975). Penetration of the gut mucosa by pathogens is believed to occur mainly through specialized epithelial cells, called M cells that are located in Peyer's patches. An alternative route for bacterial uptake in the mucosal tissues of the gut is mediated by dendritic cells (Rescigno et al, 2001). A new mechanism for the bacterial uptake in mucosal tissues is mediated by DCs, endowed with the ability to make intercellular adhesive links directly with epithelial cells, thereby preserving the integrity of the gut barrier; DCs can penetrate the epithelium to directly take up bacteria from the gut lumen without compromising the barrier function of the gut (Collins, 2002). DCs are probably the key factors in controlling T cell responses in the gut wall.

In colon, the colonic DCs have a distinctive distribution pattern; they form a reticular framework throughout the lamina propria and beneath the basement membrane of the colonic crypts (Pavli et al, 1996). This pattern of dendritic cell distribution in human colon is similar to that seen in other sites, e.g. skin (Hume et al, 1983) and large airways of the lung (Holt, et al,1989). The observation in this differs from Pavli et al. (1996) and does not seem to show the reticular framework pattern. The stimulated release of DCs from intestine may be important In regulating antigen presentation in lymph nodes draining inflammatory sites (MacPherson et al, 1995). DCs in the colon may regulate intestinal immunity but remain poorly characterized (Bell et al, 2001). DCs that infiltrate colon cancer are of mature dendritic cell phenotype, the density of the cells in colorectal cancer primaries is three times lower than that seen in normal colonic mucosa (Schwaab et al, 2001). However, in the present study, the ZIO positive polygonal DCs do not show processes and the triangular cells show one or two processes; the processes are thin and short; the DCs do not seem to have a typical, mature phenotype. The phenotype of the DC may probably depend on the state and development of the DCs as well as on the state and stage of the cancer. Production of immunosuppressive factors is one of the mechanisms by which tumors evade immunosurveilance; primary tumors negatively impact DC development (Sombroek et al, 2002).

DCs can be tested as immunotherapeutic agents for cancer and in relation to the pathogenesis of human immunodeficiency virus (HIV) infection (Barratt-Boyes et al, 1996). DCs can stimulate protective antitumor responses (Morse and Lyerly, 1998). immunotherapy, using autologous DCs loaded with unfractionated tumor derived antigens in the form of RNA, is a powerful, useful vaccination strategy for cancer (Gilboa et al, 1998). DCs have an emerging role in novel cancer therapies (Hermans et al, 1998). DCs have the capacity to induce responses and are used as potent adjuvant for the treatment of human cancer (Nestle and Burge, 1999). DC tumor vaccines for cancer immunotherapy reverse T cell energy and result in tumor rejection (Avigan, 1999). Human tumors express a number of protein antigens recognized by T cells. DCs are potent to present antigens to T cells. DCs are applied to cancer vaccines (Timmerman and Levy, 1999). Intratumoral injection of bone marrow DCs, engineered to produce interleukin-12, induces complete regression of established murine transplantable colon adenocarcinomas (Melaro et al, 1999). Immunotherapy of cancer can be improved by protecting DC from tumor-induced apoptosis. (Esche et al, 1999). The ability to immunize against endogenous retroviral tumor antigens may have relevance in the induction of antitumor immunity for some human cancers (Kershaw et al, 2001). Activated and mature DCs may have a role in the induction of an exacerbated immune response in ulcerative colitis (Ikeda et al, 2001). DCs transfected with total tumor RNA, may represent a method for inducing immune responses against the entire repertoire of tumor antigens of surgically resected malignancies (Nair et al, 2002).

To respond to orally ingested antigens and commensal bacteria, presence of DCs in the colon is possible. DCs are the major antigen-presenting cells of the human colonic lamina propria (Pavli et al, 1993). Colonic lamina propria DCs have an important role in the immunological pathogenesis of ulcerative colitis (Kimura and Morisa, 1990).

Distribution of DCs in the colon mucosa, as seen in this study, is important because of the antigens that enter through mouth and of the intestinal bacteria. DCs are potent stimulators of primary T cell responses (Steinman, 1991). They reside in the interstitium of many tissues and epithelium of mucosa, where they take up and process both soluble and particulate antigens. Following exposure to antigens, DCs mature and develop potent immunostimulatory activity whilst migrating to draining lymph nodes; there they interact with T cells to initiate T cell responses (Pavli et al. 1996).

Acknowledgements:

The authors thank Dr. Aravindan Nair for providing and permitting us to use the colon specimens resected during surgery for colorectal cancer from his patients and Fluid Research Committee of Christian Medical College for funding this study.

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Fig. 1. Longitudinal section of the colon. Dendritic cell in the crypt (thin arrow); Dendritic cell in lamina propria (thick arrow). ZIO 290 X.

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Fig. 2. Infiltration of lymphocytes from lymphatic follicle in submucosa into lamina propria. S - submucosa; F - follicle; L - lamina propria; Dendritic cell (arrow). ZIO 150 X.

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Fig. 3. Cross section of colon. Dendritic cell in the crypt with the process toward the lumen of the crypt (thin arrow). ZIO 565 X.

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Fig. 4. Longitudinal section of the colon. Dendritic cell in the crypt with the process toward the lumen of the crypt (thin arrow); Dendritic cell in the lamina propria (thick arrow).

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