Bezuidenhout_175-180.qxd INTRODUCTION The gross, light and electron microscopic structure of the respiratory air sacs of the fowl are well docu- mented (Carlson & Beggs 1973). A simple epitheli- um resting on a basal lamina supported by a layer of fibro-elastic connective tissue lines the respirato- ry surface of the air sacs. The epithelium varies from simple squamous to a ciliated columnar epithelium with goblet cells. Air sacs of birds are prone to infec- tion by various microorganisms, including fungi, bac- teria and viruses. Some microorganisms or their toxins need to attach to glycoproteins or glycolipids on cell surfaces in order to cause disease, e.g. the S glycoproteins of the infectious bronchitis virus bind to neuraminic acid-containing glycans (Schultze, Cavanagh & Herrler 1992). The relative expression of glycoconjugates is important because of the sig- nificant role membrane glycoconjugates appear to play in respiratory host defenses (Lasky 1992; Abdi, Kobzik, Li & Mentzer 1995). The diversity of glycoconjugates and the applica- tion of lectin histochemistry is extensively reviewed by Spicer & Schulte (1992). Most lectins are multi- meric proteins that share the common property of binding to defined sugar structures (Spicer & Schulte 1992). The assumed monosaccharide specificity of a lectin can be very different from the actual com- plex oligosaccharide(s) it recognizes in histochem- ical preparations. Because of the specificity that 175 Onderstepoort Journal of Veterinary Research, 72:175–180 (2005) A lectin histochemical study of the thoracic respiratory air sacs of the fowl A.J. BEZUIDENHOUT Department of Biomedical Sciences, College of Veterinary Medicine Cornell University, Ithaca, New York 14850 ABSTRACT BEZUIDENHOUT, A.J. 2005. A lectin histochemical study of the thoracic respiratory air sacs of the fowl. Onderstepoort Journal of Veterinary Research, 72:175–180 The lectin-binding characteristics of the epithelial lining of the thoracic air sacs of the chicken were determined. Con A, LCA and PSA bound to the apical membrane as well as to the cytoplasm distal to the nucleus of the surface epithelium, indicated the presence of α-linked mannose as well as N- acetylchitobiose-linked α-fucose residues in the glycoproteins. GSL I bound to the apical membrane and cytoplasm distal to the nucleus, but not to the cilia of the epithelium, where-as MPL, DBA and RCA120 bound to the apical membrane, cilia and cytoplasm, indicated the presence of α-linked N- acetylgalactosamine residues. However, neither SJA or SBA showed any binding, indicating the absence of β anomers of galactosyl (β1.3)N-acetylgalactosamine and β-linked N-acetylgalactosamine residues. UEA I bound to the apical membrane and cilia, as well as to the cytoplasm of a few cells, indicated the presence of α-linked fucose residues. PNA bound to the apical membrane of some, but not all, surface epithelium cells, indicated the presence of galactosyl (β1.3)N-acetylgalactosamine residues. WGA bound to the apical membrane and cilia, as well as to the cytoplasm of a few cells, indicated the presence of neuraminic acid residues. Keywords: Fowl, lectin binding, thoracic air sacs Accepted for publication 15 April 2005—Editor each lectin has toward a particular carbohydrate structure, even oligosaccharides with identical sugar compositions can be distinguished. Some lectins will bind only to structures with mannose or glucose residues, while others may recognize only galac- tose residues. Some lectins require that the partic- ular sugar be in a terminal non-reducing position in the oligosaccharide, while others can bind to sugars within the oligosaccharide chain. Some lectins do not distinguish between α or β anomers while others require not only the correct anomeric structure, but also a specific sequence of sugars for binding. Some glycoproteins have terminal sialic acid residues that can block lectin binding and require desialylation before binding. No data could be found in the existing literature on the lectin-binding glycoproteins or glycolipids of the epithelium lining the respiratory surface of air sacs in chickens. The present study was undertaken to determine the lectin-binding characteristics of the thoracic air sacs of the chicken using lectin histo- chemical techniques. MATERIALS AND METHODS Source and type of birds For this study a total of 25 specific pathogen free (SPF) White Leghorn chickens were obtained from the Poultry Research Farm, Cornell University, Ithaca, New York. The birds were clinically healthy, of both sexes, 42–84 days old and weighed 140– 900 g. Collection of the air sac membranes The birds were euthanased with CO2 in a chamber designed for this purpose. After euthanasia they were placed in dorsal recumbency, the skin over the ventral abdomen and thorax was removed and the hips dislocated to provide stability in dorsal re- cumbency. The ventral abdominal wall was incised just caudal to the sternum to open the ventral hepat- ic peritoneal cavities. From here the incision was extended cranially along the lateral borders of the pectoral muscles, transecting the sternal ribs close to their junctions with the sternum. Both clavicles and coracoids were cut and the sternum reflected cra- nially. The ventral abdominal wall was removed and the gizzard freed from its attachments to facilitate access to the thoracic air sacs. The medial wall of the thoracic air sacs was visualized by cutting the left and right hepatic ligaments and reflecting the liver and gut medially. The septum separating the cranial and caudal thoracic air sacs was located and a small transverse incision was made over the septum along their ventral borders. The septum was then incised along its entire length to create one cavity consisting of both the cranial and caudal thoracic air sacs. A thin (2 mm thick) stainless steel ring was passed through the incision and placed so as to include the medial wall of both cranial and caudal air sacs. A thick (3 mm) stainless steel ring was placed on the peritoneal surface of the air sac and aligned with the thin ring. The two rings were clamped together with a modified hemostat and the membrane was removed by cutting the tissue along the outer circumference of the rings. The rings were then clamped in a small binder clamp and the hemo- stat removed. The entire specimen (clamp, rings and membrane) was placed in either 10 % phosphate- buffered formalin or Bouin’s fixative. This ensured that the membranes did not shrink during fixing and processing. Processing of specimens for light microscopy For paraffin wax embedding the tissues were dehy- drated through a graded series of ethanol, cleared in Propar (Anatech Ltd., Lake Rd., Battle Creek, MI 49015) and infiltrated with paraffin wax. After infiltra- tion the membrane was cut from the rings, embed- ded in paraffin wax and sectioned at 4 µm on a rotary microtome. Paraffin wax-embedded sections were mounted on slides coated with Poly-L-lysine (Vector Laboratories, Burlingame, California). Lectins used Thirteen biotinylated lectins (Kit I and Kit II, Vector Laboratories) were screened. These lectins were obtained from the plants Maclura pomifera (MPL), Dolichos biflorus (DBA), Glycine maxi (SBA), Ara- chis hypogaea (PNA), Ricinus communis (RCA), Triticum vulgaris (WGA), Ulex europaeas (UEA), Concanavalin A (CON A), Griffonia simplicifolia (GSL), Pisum sativum (PSA), Lens culinaris (LCA) and Sophora japonica agglutinin (SJA), and wheat germ agglutinin (WGA). Optimal dilution concentra- tion for each lectin was determined and varied from 5–50 µg/ml. Lectin staining Tissue sections were de-paraffinated in xylene and re-hydrated through a graded series of ethanol. En- dogenous peroxidase activity was blocked by incu- bation of sections for 10 min at room temperature in 0.5 % (v/v) hydrogen peroxide in methanol. After rinsing the sections in 0.01 mol phosphate buffer 176 Thoracic respiratory air sacs of the fowl solution (PBS), the specific biotinylated lectin at the appropriate dilution in PBS was added to cover the tissue sections, and then incubated at 37 °C in a humid chamber for 1 h. Negative controls for each tissue were incubated with buffer only, and for bind- ing specificity the lectins were blocked with their inhibitory sugars. The sections were washed with PBS and covered with pre-diluted streptavidin-per- oxidase conjugate (Vector Laboratories) for 15 min at room temperature in a humid chamber. The sec- tions were then washed six times with PBS and incubated with aminoethyl carbazole (AEC) sub- strate (Zymed Laboratories Inc., San Francisco, California) for 2–20 min at room temperature. Colour development was monitored under the microscope. Sections were washed in distilled water to stop fur- ther colour development, and then counterstained with haematoxylin. All sections were examined under an Olympus BH-2 light microscope and relevant areas photographed. Lectin staining was recorded as positive or negative. RESULTS AND DISCUSSION The results of staining with various lectins are sum- marized in Table I. Processing paraffin wax embedded tissues for light microscopy requires removing the wax from the tis- sues prior to staining. This process also removes most of the glycolipids (Brooks, Leathem & Schu- macher 1997) that are therefore excluded in the results of this study. It is generally agreed that a squamous epithelium lines the air sacs, and that many of the cells contain surfactant in the form of myeloid inclusions. Well- defined areas of ciliated cuboidal to columnar cells, as well as goblet cells are randomly distributed over the surface of the membrane (Lucas 1970; Smith, Meier, Lamke, Neill & Box 1986). The results of the present study showed that areas of squamous epithelium reacted evenly for all the lectins, except for SJA and SBA that did not show any positive bind- ing, irrespective of the concentration of the lectin that was used. Due to the very attenuated nature of the squamous epithelium it was not possible to dis- tinguish between staining of the apical membrane and staining of the cytoplasm distal to the nucleus. Contrary to the squamous epithelium, areas of columnar and ciliated epithelium did not react uni- formly with all the lectins that showed positive bind- ing. Some lectins bound to the apical membrane of ciliated and non-ciliated cells, while others only bound to the apical membrane of non-ciliated cells. Binding to the cytoplasm also varied between the different lectins. The lectins Con A, LCA and PSA bind to a wide variety of membrane glycoproteins that have a core structure that includes a-linked mannose. LCA rec- 177 A.J. BEZUIDENHOUT TABLE 1 Results of lectin binding Lectin Binding Sugar specificity Blocking sugar MPL 6.25 µg/ml Positive N-acetyl galactosamine D+ galactose UEA 50 µg/ml Positive Fucose Fucose PNA 50 µg/ml Positive Galactose D+ galactose RCA 50 µg/ml Positive Galactose and N- D+ galactose acetylgalactosamine Con A 50 µg/l Positive Glucose and mannose Methyl mannoside and methyl glucoside DBA 25 µg/ml Positive N-acetyl galactosamine N-acetyl galactosamine GSL I 25 µg/ml Positive Galactose and N- Galactose and/or N-acetyl acetylgalactosamine galactosamine did not block WGA 12.5 µg/ml Positive N-acetylglucosamine Chitobiose PSA 12.5 µg/ml Positive Glucose and mannose N-methyl mannoside and methyl glucoside LCA 12.5 µg/ml Positive Glucose and mannose N-methyl mannoside and methyl glucoside SBA up to 100 µg/ml Negative N-acetyl galactosamine SJA up to 100 µg/ml Negative N-acetyl galactosamine ognizes additional sugars as part of the receptor structure and is therefore more specific than Con A. PSA is almost identical to LCA and binds to α-linked mannose with an N-acetylchitobiose-linked α-fucose residue included in the receptor sequence (Spicer & Schulte 1992; Brooks et al. 1997). All three lectins 178 Thoracic respiratory air sacs of the fowl A B C D E F G H I J K L require Ca and Mg cations for binding. Con A (Fig. 1L), LCA (Fig. 1A) and PSA (Fig. 1B) showed strong binding to the apical membrane, and to a lesser degree to the cytoplasm distal to the nucleus of the surface epithelium of the thoracic air sacs. This would indicate the presence of a-linked mannose, as well as α-linked mannose with an N-acetylchito- biose-linked α-fucose residue in the glycoproteins of the apical membrane and in the cytoplasm of the epithelium. The cytoplasm of the surface epithelium contains large amounts of glycogen (Carlson & Beggs 1973). Therefore the affinity of the cytoplasm for Con A is probably due to the presence of glucose residues in the glycogen (Pedini, Ceccarelli & Gar- giulo 1994; Nadel 2003). N-methyl mannoside and methyl glucoside effectively blocked binding of the three lectins. The lectins GSL I, MPL, RCA120 SJA, SBA and DBA preferentially bind to α-linked N-acetylgalactosamine residues of glycoproteins (Spicer & Schulte 1992). GSL I (Fig. 1H) showed binding to the apical mem- brane and to the cytoplasm distal to the nucleus of a few cells, but did not bind to the cilia of the epithe- lium. MPL (Fig. 1K), DBA (Fig. 1G) and RCA120 (Fig. 1E) showed binding to the apical membrane and cilia, as well as to the cytoplasm proximal to the nu- cleus of some cells. Neither SJA (Fig. 1C) nor SBA (Fig. 1D) showed any binding to the epithelium. This may be due to the fact that SJA preferentially binds to β anomers of galactosyl (β1.3)N-acetylgalacto- samine and that SBA preferentially binds to oligo- saccharide structures with terminal α or β-linked N- acetylgalactosamine residues (Spicer & Schulte 1992). This would indicate that a-linked N-acetyl- galactosamine residues are present on the glyco- proteins of the epithelium, but that it is neither pres- ent as β anomers, nor as terminal α or β residues. Binding of GSL I, MPL, DBA and RCA120 could effectively be blocked with galactose. UEA I binds to glycoproteins and glycolipids con- taining α-linked fucose residues at a branch or ter- minal position on the glycoprotein (Spicer & Schulte 1992). The lectin showed positive binding to the apical membrane and cilia of the epithelium (Fig. 1I). A few cells also showed positive binding to the cytoplasm distal to the nucleus. This would indicate that the glycoproteins of the apical membrane and the cytoplasm of some cells contain α-linked fucose residues. Binding could effectively be blocked with fucose. PNA binds specifically to galactosyl (β1.3)N-acetyl- galactosamine (T-antigen) present in many glyco- conjugates in soluble and membrane associated glycoproteins (Lotan, Skutelsky, Danon & Sharon 1975). The lectin showed positive binding to the apical membrane and cytoplasm of some cells of the epithelium, but not to all of them (Fig. 1F). This would indicate that the specific receptor is not pres- ent on all surface-lining cells. Binding could effec- tively be blocked with galactose. WGA preferentially binds to oligosaccharides con- taining terminal dimers and trimers of N-acetylglu- cosamine or chitobiose, and can also interact with some glycoproteins via sialic (neuraminic) acid res- idues (Spicer & Schulte 1992). The lectin showed binding to the apical membrane and cilia, as well as to the cytoplasm of a few cells (Fig. 1J). This would 179 A.J. BEZUIDENHOUT FIG. 1 Results of lectin binding A LCA bound to the apical membrane and cytoplasm distal to the nucleus of the surface epithelium B PSA bound to the apical membrane and cytoplasm distal to the nucleus of the surface epithelium C SJA showed no affinity to any part of the surface epithelium D SBA showed no affinity to any part of the surface epithelium E RCA 120 bound to the apical membrane, cilia and cytoplasm distal to the nucleus of the surface epithelium F PNA bound to the apical membrane and cytoplasm of some, but not all, cells G DBA bound to the apical membrane and cilia, and to the cytoplasm of some of the surface epithelium H GSL bound to the apical membrane, but not to the cilia of the surface epithelium I UEA I bound to the apical membrane and cilia, as well as to the cytoplasm distal to the nucleus of some cells of the surface epithelium J WGA bound to the apical membrane and cilia of the surface epithelium K MPL bound to the apical membrane and cilia, as well as to the cytoplasm distal to the nucleus of some surface epithe- lial cells L Con A bound to the apical membrane of the surface epithelium indicate that the glycoproteins of the apical mem- brane and cytoplasm of some cells contain dimers and trimers of N-acetylglucosamine or chitobiose, or sialic acid residues as part of their structure. Binding was effectively blocked with chitin hydrolysate. In conclusion, most cells of the epithelium lining the respiratory surface of the thoracic air sacs of the fowl showed an affinity to a wide variety of lectins. This indicated that the apical cell membrane and the cytoplasm distal to the nucleus contained α-linked mannose, α-linked mannose with an N-acetylchitobi- ose-linked α-fucose, α-linked N-acetylgalactosamine, α-linked fucose, galactosyl (β1.3)N-acetylgalactos- amine and dimers and trimers of N-acetylglucos- amine or chitobiose, or sialic acid residues as part of their glycoconjugate structure. This information will be useful in further studies of air sacculitis in poul- try. REFERENCES ABDI, K., KOBZIK, L., LI, X. & MENTZER, S.J. 1995. Expression of membrane glycoconjugates on the sheep lung endotheli- um. Laboratory Investigation, 72:445–452. BROOKS, S.A., LEATHEM, A.J.C. & SCHUMACHER, U. (Eds) 1997. Lectin histochemistry, a concise practical handbook. Oxford: Bios Scientific Publishers Ltd. CARLSON, H.C. & BEGGS, E.C. 1973. Ultrastructure of the ab- dominal air sac of the fowl. Research in Veterinary Science, 14:148–150. LASKY, L.A. 1992. Selectins: interpreters of cell-specific carbo- hydrate information during inflammation. Science, 258:964– 969. LOTAN, H., SKUTELSKY, E., DANON, D. & SHARON, N. 1975. The purification, composition, and specificity of the anti-T lectin from peanut (Arachis hypogaea). Journal of Biological Chemistry, 250:8518–8523. LUCAS, A.M. 1970. Avian functional anatomic problems. Fed- eration Proceedings, 29(5):1641–1648. NADEl, E. 2003. Metabolism and nutrition, in Medical Physiol- ogy, edited by W.F. Boron & E.L. Boulpaep. Philadephia: Saunders. PEDINI, V., CECCARELLI, P. & GARGIULO, A.M. 1994. Glyco- conjugates in the mandibular salivary gland of adult dogs revealed by lectin histochemistry. Research in Veterinary Science, 57:353–357. SCHULTZE, B., CAVANAGH, D. & HERRLER, G. 1992. Neur- amindase treatment of avian infectious bronchitis corona- virus reveals a hemagglutinating activity that is dependent on sialic acid-containing receptors on erythrocytes. Virology, 189:792–794. SMITH, J.H., MEIER, J.L., LAMKE, C., NEILL, P.J. & BOX, E.D. 1986. Microscopic and submicroscopic anatomy of the para- bronchi, air sacs and respiratory space of the budgerigar (Melopsittacus undulatus). American Journal of Anatomy, 177:221–242. SPICER, S.S. & SCHULTE, B.A. 1992. Diversity of cell glyco- conjugates shown histochemically: a perspective. Journal of Histochemistry and Cytochemistry, 40:1–38. 180 Thoracic respiratory air sacs of the fowl