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Journal of the Anatomical Society of India

Dendritic Cells in the Airways of Laboratory Guinea Pig and Rabbit: A Light Microscopic Zinc lodide-Osmium Study

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

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

Department of Anatomy, Christian Medical College, Vellore, INDIA.

Abstract

Dendritic cells are the principal antigen presenting cells. Dendritic cells have been demonstrated in human and animalepithelium and subepithelial tissue. The present study used trachea and lung specimens, collected from laboratory guinea pig and rabbit thatwere sacrificed after routine practical classes, which revealed zinc iodide-osmium (ZIO) positive cells. In guinea pig trachea, dendritic cellswere absent in the epithelium or subepithelial tissue. In rabbit trachea, dendritic cells were absent in the epithelium; while in the subepithelialtissue, polygonal cells without dendritic processes were present. Chondrocytes in the tracheal hyaline cartilage showed ZIO positivegranules. In guinea-pig lung, dendritic cells were present in the parenchyma and formed network. In rabbit lung, dendritic cells were absentin the epithelium but were present in the subepithelial tissue of the terminal bronchiole; dendritic cells were present also in the alveolarepithelium. Absence of dendritic cells or of dendritic processes is unique in these animals and suggests that, on exposure to antigens, thecells should form and mature.

Key words: Dendritic cell, guinea pig, lung, rabbit, trachea, zinc iodide osmium

Introduction:

Inhaled antigens must be identified and an immunologic response mounted without disrupting pulmonary gas exchange. T cells recognize the antigen bound to major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells (APC) known as dendritic cells (DCs), which take up and process the antigen. Because the lung is an immunologically active organ in which a primary immune response can be created, dendritic cells should occur in the lung (Toews; 1991). DCs are the principal APC in the human and animal respiratory tract tissues. These are potent immunostimulatory cells; being bone marrow-derived & thymus-independent cells (Steinman 1991; Steinman and Nussenzweig, 1980); they can take up and present antigens to naive T cells (Liu and MacPherson, 1991).

DCs have been, found in mouth of rhesus monkey (Hutchens et al, 1971) infant rat respiratory tract (Nelson et al, 1994), stratified squamous epithelium of rat (Muller, 1996), mucous membranes of laboratory mice and rat (Bykov, 1997), trachea and bronchi of the guinea pig (Lawrence et al, 1997), epithelium of respiratory tract of the rat (McWilliam and Holt, 1998), respiratory tract of the human and animal (Holt and Stumbles, 2000) middle ear and auditory mucosa of the rat (Jecker et al, 2001)

Zinc iodide-osmium (ZIO) has been used to identify the DCs: (Crocker and Hopkins, 1984; Dagdeviren et al, 1994; Breathnach and Goodin, 1965; Niebauer et al, 1969; Rodriguez and Caorsi 1978; Hart and Fabre, 1981; Sertl et al, 1986; Prickett et al, 1988; Steinman, 1991).

Previous studies in this department, demonstrated DCs in human palatine tonsil (Chandi et al. 1988, 1989), human decidua (Abraham et al. 1996, 2000), and lip, tongue and oesophagus in monkey (Indrasingh et al, 2001)

Since not much work has been done in airways of guinea pig and rabbit, the present study was made to verify, using zinc iodide osmium, the presence, location, and morphology of DCs in the airways of those animals.

Materials and Methods:

Fresh specimens of trachea and lung were obtained from laboratory guinea pig and rabbit, which were sacrificed after rountine practical classes for the postgraduate students in the Department of Pharmacology, Christian Medical College, Vellore.The guinea pigs and rabbits were kept in ideal laboratory conditions in the animal house.

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. Seven micron thick serial sections 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:

Trachea of guinea pig: ZIO positive DCs were absent in the epithelium and subepithelial tissue. Chondrocytes, in the hyaline cartilage, showed ZIO positive granules (Fig. 1)

Trachea of rabbit: ZIO positive DCs were absent in the epithelium. ZIO positive polygonal cells, without dendritic processes, were present in the subepithelial tissue (Fig. 2). Chondrocytes, in the hyaline cartilage, were ZIO positive (Fig. 3)

Lung of guinea pig: ZIO positive DCs were present in reticular network pattern in the parenchyma (Fig. 4).

Lung of rabbit: ZIO positive DCs were absent in the epithelium but were present in the subepithelial tissue of the terminal bronchioles (Fig. 5). ZIO positive DCs were present also in the alveolar epithelium (Fig. 6)

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, probably, indicated the presence of lipids and/ or precursor proteins (Taffarel et al, 1984).

Chondrocytes are marked by ZIO due to the presence in them of some reactive substance like alkaline phosphatase, chondroitin sulphate, protein, or lipid. This needs to be ascertained.

In the human and mouse lung epithelium of large airways, DCs were interspersed perpendicularly between the columnar epithelial cells with extending processes, not extending to bronchial lumen; in smaller airways, DCs appeared to extend towards the lumen; DCs were present in the vascular walls and peripherally as far as the alveolar septa and the visceral pleura with the processes arranged parallel to the pleura; DCs are the only antigen presenting cells in the bronchopulmonary epithelium (Seertl et al, 1986). Pulmonary DCs are very effective APC. Langerhans cells are detected in tracheal tissue in vitamin A deficiency (Wong and Buck, 1971) and in alveolar tissue in certain disease states (Kewanami et al, 1971).

Reticular framework of dendritic cell distribution was in the large airways of the lung (Holt et al. 1989). A network of DCs in the epithelium of the respiratory tract detect foreign antigens, process these antigens to be transported to local lymph nodes and presented to naive T cells (McWilliam and Holt, 1998) DCs, in the parenchymal tissues of the peripheral lung and in the epithelium of the conducting airways, are distributed as a network; under conditions of local stress (inflammation), their turnover accelerates, reflecting their role in local surveillance (Holt and Stumbles, 2000). The observation in this study shows the reticular framework pattern in the parenchyma of guinea-pig lung.

In rat, DCs are the only resident cells present in the alveolar septa (Holt et al, 1985) or airway epithelium (Holt et al, 1988). Dcs are essential for the recongnition and presentation of allergens to the cells of the immune system; airway DCs capture allergen in the mucosa and present it to naive T cells after migration into lymph nodes; DCs have an essential role in the induction and maintenance of eosinophilic airway inflammation (Lambrecht et al, 2001).

Chronic (eosinophilic) inflammation and acute (neutrophilic) inflammation are accompanied by increased intra epithelial DCs in the large airways (Schon-Hegrad et al, 1991). Failure of T cell system to discriminate between pathogenic antigens (bacteria and viruses) and non-pathogenic antigens (pollens) appears to underlie immunoinflammatory diseases in the respiratory tract (Holt and McMenamin, 1989).

The location of the palatine tonsil at the gateway to the respiratory and digestive tracts suggests a functional role in generating an immune response to inhaled or swallowed antigens. To respond to inhaled or swallowed antigens, DCs are present in the tongue and oesophagus (Indrasingh et al. 2001); Similarly, to respond to inhaled antigens, presence of DCs in the airways is required.

The distribution of DCs in airways found in this study is important because of the antigens that enter through nose and mouth. 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 (Pavli et al, 1996). Absence of DCs in the epithelium and subepithelial tissue of guinea pig trachea, of DCs in the epithelium and dendritic processes of ZIO positive cells in the subepithelial tissue of rabbit trachea, and of DCs in epithelium of terminal bronchiole of rabbit lung in this study is unique. Absence of DCs from airway epithelia in these two species would represent a significant finding at odds with established knowledge from the past two decades. Absence of DCs and dendritic processes in airway epithelium or subepithelial tissue in this study needs explanation. The guinea pig and rabbit were kept from birth ih ideal hygienic laboratory conditions in the animal house. Possibly, they were not sufficiently exposed to inhaled antigens and DCs were not yet formed or mature. Following exposure to antigens, DCs should 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). In animals, housed in dust-controlled conditions, airway epithelial and alveolar DCs are not detectable in infancy (Nelson et al, 1994):

Acknowledgements:

The authors thank Dr. Kalpana Earnest for permitting us to collect the trachea and lung specimens of guinea pig and rabbit, which were used for routine practical classes in the Department of Pharmacology and Fluid Research Committee of Christian Medical College Vellore for funding this study.

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Fig.1. Trachea of guinea pig. (E - epithelium; H - hyaline cartilage) absence of dendritic cells in epithelium and subepithelial tissue; chondrocytes (arrow) with ZIO positive granules. ZIO 220 X.

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Fig. 2. Trachea of rabbit. (E - epithelium; S - subepithelial tissue) absence of dendritic cells in epithelium; polygonal cells without dendritic processes in subepithelial tissue. ZIO 330 X.

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Fig. 3. Hyaline cartilage (H) of trachea. ZIO positive granules in chondrocytes. ZIO 440 X.

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Fig. 4. Lung of guinea pig. Dendritic cells in reticular network pattern in parenchyma. ZIO 410 X.

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Fig. 5. Lung of rabbit. Absence of ZIO positive dendritic cells in epithelium; dendritic cells (arrow) in subepithelial tissue of terminal bronchiole. ZIO 185 X.

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Fig. 6. Lung of rabbit. Dendritic cells in alveolar epithelium. ZIO 520 X

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