Developmentaland ComparativeImmunology,Vol. 16, p p 209- 219, 1992 Printed in the USA. All rights reserved.
0145-305X/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
ULTRASTRUCTURAL LOCALIZATION OF A SOLUBLE ANTIGEN IN THE CHICKEN HARDERIAN GLAND Emilio del Cacho, Margarita Gallego, and Jose Antonio Bascuas Department of Animal Pathology, Universityof Zaragoza, Zaragoza,Spain (Submitted January 1991; Accepted July 1991) [~Abstract--The relationship between plasma cells, macrophages, B and T cells, dendritic cells, and epithelium in the chicken Harderian gland have been studied by means of ultrastructural localization of the horseradish peroxidase following local immunization. After 5 d, peroxidase activity was found in vesicles located in macrophages and immature plasma cells. On day 7, peroxidase-antiperoxidase complexes were found in vesicles of the epithelial cells lining the secondary ducts and the acini, in the lumina of the ducts, and on the surface of lymphocytes located among these epithelial cells. Dendritic cells showing peroxidase activity on their surface were seen in the subepitheliai lymphoid tissue and in the lymphoid follicles. On day 9, peroxidase activity was found as iccosomes on the surface of dendritic cells and lymphoblasts. These results indicate that immature plasma cells in the Harderian gland can take up antigen and may have a role in presenting it to T cells. Further, our results suggest that intraepithelial lyrnphocytes might be involved in antigen transportation from the epithelium to the subepithelial lymphoid tissue. 7qKeywords--Follicular dendritic cell; Plasma cell; Intraepithelial lymphocyte; Horseradish peroxidase; Ultrastructure; Gland of Harder; Chicken.
Introduction The Harderian gland (HG) contains the largest concentration of plasmacytic cells within avian lymphoid tissue. MuelSupported by Grant No. PB86-0532 from DGICYT. Address correspondence to Margarita Gallego, Dpto. Patologia Animal, Facultad de Veterinaria, Miguel Servet 177, 50013-Zaragoza, Spain.
ler et al. (1) failed to show cells capable of antibody production in the HG after systemic stimulation via intravenous injection, but succeeded when stimulation was applied locally by dropping onto the eyeball. Survashe et al. (2) demonstrated an increased lymphoid activity and a rise in plasma cell numbers following eyedrop antigen application and neither effect was impaired b y s p l e n e c t o m y . Therefore, it can be assumed that the plasma cell population of the HG is activated by antigen located on the eyeball, and that the spleen is not a major source of immunocompetent cells of the gland in response to an immunogenic stimulus (3). Bell (4) found evidence that many of the plasmablasts that appeared in the gland as a response to antigenic stimulation initiated its differentiation by contact with antigen at sites outside the gland, and further migrate via the vascular system to enter the gland. Moreover, Gallego et al. (5) suggested that macrophages in the lymphoid agreggates within the fornix migrate to the gland after antigen uptake. The excellent response of the HG to a T-dependent antigen (1), in spite of the small population of T cells identified in the gland (6), suggests that a special environment for the B and plasmacytic cells activation and differentiation might exist (7). This unique environment might be maintained by the lymphoid cell interactions with the epithelium, the interstitium, or both. Microscopic and electron microscopic features of the HG, as well as the immunoglobulin classes in this gland, have been widely studied by several groups (7-13).
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However, limited information relevant to the relationship between plasma, lymphoid, dendritic, and epithelial ceils is very limited. The high proliferative capacity of the HG plasma cells (6) and the efficiency of the gland in the production of antibodies during a local immune response (7,14) indicate that a specific relationship might exist between the aforementioned cells. This relationship may help explain the effective antigen presentation and high antibody titers in tears associated with the local immune response to T-dependent antigens in spite of the reduced T-cell population. In the present study, the relationship between immunocompetent cells of the gland of Harder has been investigated by means of the ultrastructural localization of peroxidase after local immunization with horseradish peroxidase (HRP).
Materials and Methods In these experiments, 7-week-old White Leghorn chickens were used. Twelve chickens were injected in the nictitating membrane (as close as possible to the fornix) with 0.02 mL 2% HRP (Sigma) in sterile saline. Three saline control birds were injected in the nictitating membrane with 0.02 mL sterile saline. Another three animals were used as a noninjected control group. Before injecting HRP and sterile saline, the chickens were anesthetized with sodium thiopental (30 mg/kg body weight). Four of the immunized animals, one bird from the control group, and another from the non-injected group were euthanized by cervical dislocation at 5, 7, and 9 d after the injection. Both Harderian glands from each chicken were removed immediately after death, fixed in 1% buffered glutaraldehyde for 90 min, and washed in cacodylate buffer. To develop peroxidase activity, samples were embedded in 7% agar at 40°C and cut at 100 ~m. These sec-
E. del Cacho et al.
tions were incubated in diaminobenzidine hydrochloride (DAB: 0.5 mg/mL) in Tris-HCl buffer containing 0.01% HzOz plus 10 mM 3 - a m i n o - l , 2 , 4 - t r i a z o l e (Sigma) (15) to inhibit endogenous peroxidase activity for 10 min at room temperature. Further, the glands were postfixed for 30 min in 1% osmium tetroxide. After final washing in buffer and dehydration in ethanol, fixed glands were cleared in propylene oxide and embedded in Epon-Araldite (1:1). Selected blocks were thin sectioned (400-600/~), stained with uranyl acetate exclusively, and examinated in a Jeol T8 electron microscope.
Results Five days after antigen injection, peroxidase activity was found in the phagosomes located in the cytoplasm of the macrophages, which were surrounded by immature plasma cells (Fig. 1). These immature plasma cells showed peroxidase activity in vesicles (Fig. 1) and in Golgi saccules at the maturing face (Fig. 2). The clusters, made up of macrophages and immature plasma cells, were seen in the diffuse lymphoid tissue beneath the epithelium lining the glandular ducts. Lymphocytes, iymphoblasts, and follicular dendritic cells gave a negative reaction, as well as the epithelial ceils lining the intraglandular ducts. Lumina of the ducts were negative for peroxidase activity. Seven days after HRP injection, peroxidase activity was detected (i) on the basolateral surface of the epithelial cells lining the secondary ducts and the acini, (ii) in vesicles distributed throughout the cytoplasm of the aforementioned cells, and (iii) within the lumina of the abovementioned ducts [Fig. 3(A)]. Peroxidasepositive particles observed in the vesicles and in the duct lumina were pentagonal in shape with diameters of 205 /~ (average of 45 particles) [Fig. 3(B)]. Per-
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Figure 1. Electron micrograph of a cellular cluster located in the subepithelial lymphoid tissue of a Harderian gland taken 5 d after injection of HRP. The cluster is made up of a macrophage (M), with peroxidase activity in its phagosomes, and of immature plasma cells showing peroxidase activity in vesicles (>-). Mag. ×6,000. Bar = 2 p,m.
oxidase activity was also identified on the surface of the cells seen between the epithelial cells lining the secondary ducts and the distal part of the acini, within the subepithelial diffuse lymphoid tissue, and in the lymphoid follicles. The cells with peroxidase-positive activity on their surface were classified into three types depending on their ultrastructural features. • T y p e / - - C e l l s ultrastructurally similar
to small lymphocytes [Fig. 3(A)]. • Type H m R o u n d cells with a patchy
chromatin pattern of the nucleus similar to that of lymphocytes. Their cytoplasm showed several electron-dense granules located around the nucleus [Fig. 3(A)]. This cell type shows ultrastructural characteristics intermediary
between lymphocyte and chicken dendritic cell. Type II might correspond to an immature dendritic cell. • Type llI---Cells showing the ultrastructural features of a dendritic cell such as an eccentric nucleus with a chromatin pattern similar to that of a small lymphocyte, an elongated cytoplasm with one or more cell processes, and electron-dense cytoplasmic granules in one of the processes (Fig. 4). Cell type I was observed exclusively between the epithelial cells lining the secondary ducts and the acini. In contrast, types II and III were seen either between epithelial cells (Figs. 3, 4) or within the subepithelial diffuse lymphoid tissue (Fig. 4) or within the lymphoid follicles [Fig. 5(A,B)]. Macrophages and
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Figure 2. Golgi complex of an immature plasma cell in a cellular cluster of a Harderian gland taken 5 d after injection of HRP. Peroxidase activity was found in the saccules and within vesicles associated to the maturing face. Mag. ×44,000. Bar = 0.5 i~m.
immature plasma cells were negative for peroxidase activity. Nine days after antigen injection, peroxidase activity was found in germinal centers on the surface of both lymphoblasts and follicular dendritic cells (Fig. 6) mainly as spherical particles containing material of varying electron density (Figs. 6, 7). Size estimates of 20 particles gave a mean diameter of 0.4 +- 0.08 (SD) p.m. These particles with peroxidase activity were also observed intracellularly in vesicles located in the peripheral area of the cytoplasm (Fig. 7). Structures with peroxidase activity were seen in small invaginations of the cell membrane and having a dash-like appearance either straight or U-shaped (Fig. 6).
Discussion The DAB technique for the ultrastructural localization of peroxidase activity, initially introduced by Graham and Karnowsky (16), is not specific for exogenous peroxidase, as endogenous peroxidase can also react positively. Herzog and Miller (15) demonstrated that 10 -2 M aminotriazole completely inhibits the endogenous peroxidase, while the exogenous peroxidase remains unaffected. Endogenous peroxidase was completely inhibited in our samples with 10 -2 M aminotriazole since it was not observed either in epithelial cells or in macrophages, which are the localizations described for this enzyme (15,17).
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Figure 3. (A) Electron micrograph of a secondary duct of a Harderian gland taken 7 d after injection of HRP. Peroxidase activity ( ~ ) is located in the duct lumen (~), in vesicles in the cytoplasm of epithelial cells (E), and on the basolateral surface of these cells. Lymphocyte-like (L) and immature dendritic cell (D) showing peroxidase activity on their surface. Note the electron-dense cytoplasmic granules that chicken dendritic cells exhibit. Mag. x5,000. Bar = 2 p.m. (B) Detail of region in 3A. Peroxidase-positive particles pentagonal in shape ( ~ ) located in theMuct lumen and within cytoplasmic vesicles in the epithelial cells. Mag. x8,000. Bar = 1 p.m.
Plasma cells and macrophages in the clusters observed 5 d after HRP administration showed peroxidase activity in their cytoplasms. As peroxidase activity was observed exclusively in the cytoplasm of macrophages within the 72 h following HRP administration (5), it can be assumed that macrophages may have a role in presenting antigen to immature plasma cells. In mammals, macrophages have been demonstrated to have a role as antigen-presenting cells to B cells (18,19). B cells have been established as being able to internalize low-molecular-weight soluble peptides (34,000-44,000 Da) released by macrophages, i.e., exocytosed, as the protein antigen is being processes (20). Therefore, since the HRP MW is 40,000 Da (21) it can be assumed
that the Harderian gland plasma cells endocytose nonprocessed or slightly processed HRP, which may have been released by the macrophages observed in the above-mentioned cellular clusters. Our results clearly show that immature plasma cells in these clusters internalize peroxidase. There are two ways in which B-cells can take up antigen, both nonspecific and specific. In the nonspecific uptake, B-cells behave as antigen-presenting cells by internalizing the antigen, processing it, and reexpressing its fragments on the cell surface bound to class II MCH molecules (22). In the specific uptake, B-cells bind the antigen via their immunoglobulin receptor, which concentrates the antigen as the cell becomes activated to endocytose it (23,24). Previ-
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Figure 4. Electron micrograph of a secondary duct of a Harderian gland taken 7 d after injection of HRP. Note the peroxidase activity on the surface of mature dendritic cells (MD) located between the epithelial cells (E) lining the duct and on the surface of an immature dendritic cell (Ib) in the subepithelial lymphoid tissue. -~, duct lumen. Mag. x4,000. Bar = 2 p.m.
ous studies have demonstrated a high frequency of plasma cells in the Harderian gland possessing a high density of immunoglobulin receptor (25,26). Both frequency of cells and density of immunoglobulin receptors were reported to be higher than those found in any other lymphoid organ in the chicken (26). These reports and the existence of Ia ÷ plasma
cells in the Harderian gland of the chicken (6) might indicate that plasma cells in this localization are more efficient in endocytosing and presenting antigen than B cells and plasma cells in other lymphoid tissues. Therefore, the excellent response of the Harderian gland to a T-dependent antigen may be explained by the fact that Harderian
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Figure 5. Electron micrographs of two lymphoid follicles in a Harderian gland taken 7 d after injection of HRP. (A) Immature dendritic cell (ID) showing peroxidase activity on its surface. Mag. x4,000. Bar = 2 i~m. (B) Mature dendritic cell (MD) with peroxidase activity on its surface. Mag. ×3,000, Bar = 2 p,m.
gland immature plasma cells behave as antigen-presenting cells to T lymphocytes. The association of the peroxidase with the Golgi complex, which may provide a mechanism for reexpression of antigen fragments, suggests that the interaction of processed protein antigen with MHC may occur in this cellular compartment as proposed by Chain et al. (27). The pentagonal particles that were peroxidase positive are similar to those described by Sternberger et al. (28) for peroxidase- antiperoxidase complexes (PAP). The ultrastructural sites for PAP complexes in the Harderian gland were found to be analogous to those described for secretory IgA in the bronchial and bronchiolar mucosa (29,30) and in the acini of the lacrimal gland (31), where the IgA locally produced is transported through the epithelial cells and then discharged into the lumen by exocytosis. In light of these reports and through our findings, we suggest that transport mech-
anisms of PAP complexes in the Harderian gland may be similar to IgA transport, through the epithelial cells into the lumen. Peroxidase-positive lymphocytes located between the epithelial cells lining the ducts correspond to the intraepithelial lymphocytes (IELs). Our findings suggest that IELs are able to attach antigen to their surface. Antigen attachment to the IEL surface seems to involve the modification of the lymphocyte ultrastructural features into those described for the chicken dendritic cell (32-34). The localization of peroxidase on the surface of the apparent transitional cell from IEL to dendritic cell, which was observed both among the epithelial cells and in the subepithelial lymphoid tissue, suggests that this cell may transport antigen. Our results further support the findings of Lawn and Rose (35), who demonstrated that IELs located in the chicken gut and provided with cytoplasmic processes and electron-dense gran-
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Figure 6. Electron micrograph of a germinal center of a Harderian gland taken 9 d after injection of HRP. Lymphoblast (L) and follicular dendritic cell (D) with peroxidase activity, mainly as spherical particles, on their surface. Peroxidase-positive structures with a dash-like appearance either straight or U-shaped can be observed in invaginations of the dendritic cells membrane ( ~.- ). Mag. x4,000. Bar = 2 p.m.
ules are capable of transporting antigen. According to our results, to perform this function IELs might leave the intraepithelial location and further migrate into the lymphoid follicles from the diffuse lymphoid tissue.
The spherical peroxidase-positive particles we found on the surface of lymphoblasts and follicular dendritic cells were ultrastructuraUy similar to the iccosomes described by Szakal et al. (19). Therefore, we suggest that the immune
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Figure 7. Electron micrograph of a germinal center of a Harderian gland taken 9 d after injection of HRP. Spherical particles showing peroxidase activity located both on the surface of a dendritic cell (~) and in the peripheral area of its cytoplasm (~-). mt, lymphoid cell undergoing mitosis; L, lymphoblasts. Mag. x12,000. Bar = 1 p.m.
complexes in the chicken are retained on the dendritic cell surface as iccosomes, as proposed for mammalian species. Iccosomes have been demonstrated to mediate the delivery of antigen in the form of immune complexes from follicular dendritic cells to germinal center B-lymphocytes and macrophages. It has also been demonstrated that these germinal center events lead to the production of B memory cells and a possible increase of the antibody levels in the anamnestic response (19). According to our findings, these events seem to be
similar in chickens and mammals. The amount of iccosomes observed was lower than that reported by Szakal et al. (19). This may be due to the fact that chicken follicular dendritic cells have less filiform processes than mouse follicular dendritic cells. Further, the I g A m the more abundant immunoglobulin in the Harder gland after antigen stimulat i o n m h a s scarce biological properties in the complement activation and memory B-cells formation, and it is hardly retained on the follicular dendritic cell surface (36).
References 1. Mueller, A. E; Sato, K.; Glick, B. The chicken lacrimal gland, gland of Harder, caecal tonsil, and accessory spleens as sources of antibody producting cells. Cell Immunol. 2:140-153; 197 l.
2. Survashe, B. D.; Aitken, I. D.; Powell, J. R. The response of the Harderian gland of the fowl to antigen given by the ocular route. I. Histological changes, Avian Pathol. 8:77-93; 1979.
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3. Powell, J. R.; Aitken, 1. D.; Survashe, B. D. The response of the Harderian gland of the fowl to antigen given by the ocular route. II. Antibody production. Avian Pathol. 8:363376; 1979. 4. Bell, R. G. Histology of the local cellular response of ducks to infections of antigenic material. J. Rec. Soc. 15:213-224; 1974. 5. Gallego, M.; del Cacho, E.; Arnal, C.; Bascuas, J. A. Local immune response in the chicken Harderian gland to antigen given by different ocular routes. Res. Vet. Sci. (in press). 6. Gallego, M.; Glick, B. The proliferative capacity of the cells of the avian Harderian gland. Dev. Comp. Immunol. 12:157-166; 1988. 7. Mansikka, A.; Sandberg, M.; Verona, T.; Vainio, O.; Ganfors, K.; Toivanen, P. B-cell maturation in the chicken Harderian gland. J. Immunol. 142:1826-1833; 1989. 8. Wight, P. A. L.; Burns, R. B.; Rothwell, B.; Mackenzie, G. M. The Harderian gland of the domestic fowl. I. Histology. J. Anat. 110:307315; 1971. 9. Burns, R. B.; Maxwell, M. H. The structure of the Harderian and lacrimal gland ducts of the turkey, fowl and duck. A light microscope study. J. Anat. 128:285-292; 1979. 10. RothweU, B.; Wight, P. A. L.; Burns, R. B.; Mackenzie, G. M. The Harderian gland of the domestic fowl. III. Ultrastructure. J. Anat. 112:233-250; 1972. 11. Schramm, V. Lymphoid cells in the Harderian gland of birds. Cell Tiss. Res. 205:85-94; 1980. 12. Davelaar, E G.; Noordzij, A.; Van der Donk, J. A. A study on the synthesis and secretion of immunoglobulins by the Harderian gland of the fowl after eyedrop vaccination against infectious bronchitis at l-day-old. Avian Pathol. 11:63-79; 1982. 13. Baba, T.; Kawata, T.; Masumoto, K.; Kajikawa, T. Role of the Harderian gland in immunoglobulin A production in chicken lacrimal fluid. Res. Vet. Sci. 49:20-24; 1990. 14. Parry, S. H.; Aitken, i. D. lmmunoglobulin A in the respiratory tract of the chicken following exposure to Newcastle disease virus. Vet. Rec. 1:258-260; 1973. 15. Herzog, V.; Miller, F. Endogenous peroxidase in the lacrimal gland of the rat and its differentiation against infected catalase and horseradi s h - p e r o x i d a s e . H i s t o c h e m i e 30:235-246; 1972. 16. Graham, R. C.; Karnowsky, M. J. The early stages of absorption of infected horseradishperoxidase in the proximal tubules of mouse kidney. J. Histochem. Cytochem. 14:291-302; 1966. 17. Contran, R. S.; Citt, M. Ultrastructural localization of horseradish peroxidase and endogenous peroxidase activity in the guinea pig peritoneal macrophages. J. lmmunol. 105:15361546; 1970.
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18. Humphrey, J. H.; Grennan, D. Different macrophages populations distinguished by means of fluorescent polysacharides. Recognition and properties of marginal-zone macrophages. Eur. J. lmmunol. 11:221-228; 1981. 19. Szakal, A. K.; Kosko, M. H.; Tew, J. G. A novel in vivo follicular dendritic cell-dependent iccosome-mediated mechanism for delivery of antigen-processing cells..I. Immunol. 140:341-353; 1988. 20. Alien, P. M.; Belier, D. I.; Braun, J.; Unanne, E. R. The handling of Listeria rnonocytogenes by macrophages: the search for an immunogenic molecule in antigen presentation. J. lmmunol. 132:323-330; 1984. 21. Bergmeyer, H. U., ed. Methods of enzymatic analysis. Berlin: Verlag Chemie Weinheim; 1974. 22. Klein, J. Immunology. Oxford, England: Blackwell Scientific Publications; 1990. 23. Chesnut, R. W.; Grey, H. M. Studies on the capacity of B-cells to serve as antigen-presenting cells. J. Immunol. 126:1075-1079; 1981. 24. Abbas, A. K.; Liano, D.; Kurt Jones, E. A.; Lichtman, A. H. Antigen presentation by B lymphocytes. In: Schook, L. B.; Tew, J. G., eds. Antigen presenting cells: diversity, differentiation and regulation. New York: Alan R. Liss, Inc.; 1988:2269-2279. 25. Albini, B.; Wich, G. Immunoglobulin determinants on the surface of chicken lymphoid cells. Int. Arch. Allergy. 44:804-822; 1973. 26. Glick, B.; Subba Rao, D. S.; Stinson, R.; McDuffie, F. C. Immunoglobulin-positive cells from the gland of Harder and bone marrow of the chicken. Cell Immunol. 31:177-181; 1977. 27. Chain, B. M.; Kaye, P. M.; Shaw, M. A. The biochemistry and cell biology of antigen processing. Immunol. Rev. 106:33-58; 1988. 28. Sternberger, L. A.; Hardy, P. H.; Cuculis, J. J.; Meyer, H. G. The unlabelled antibody enzyme method of immunohistochemistry. J. Histochem. Cytochem. 118:315-333; 1970. 29. Goodman, M. R.; Link, D. W.; Brower, W. R.; Nakane, P. K. Ultrastructural evidence of transport of secretory lgA across bronchial epithelium. Am. Rev. Respir. Dis. 123:115-119; 1981. 30. Haimoto, H.; Nagura, H.; lmaizumi, M,; Watanabe, K.; Ijimma, S. Immunoelectronmicroscopic study on the transport of secretory IgA in the lower respiratory tract and alveoli. Wirch Arch. (Pathol. Anat.) 404:369-380; 1984. 31. Franklin, R. M. ; Kenyan, K. R.; Tomasi, T. B. Immunohistologic studies of human lacrimal gland: localization of immunoglobulins, secretory component and lactoferrin. J. Immunol. 110:984-992; 1973. 32. Olah, I.; Glick, B. Structure of the germinal centers in the chicken caecal tonsil: light and
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electron microscopic and autorradiographic studies. Poultry Sci. 58:195-210; 1979. 33. Olah, I.; Glick, B. Splenic white pulp and associated vascular channels in chicken spleen. Am. J. Anat. 165:445-480; 1982. 34. Olah, I.; Glick, B. Bursal secretory cells: an electron microscope study. Anat. Rec. 219: 268-274; 1987.
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35. Lawn, A. M.; Rose, M. E. Mucosal transport of Eimeria tenella in the cecum of chicken. J. Parasitol. 68: 1117-1123; 1982. 36. Klaus, G. B.; Humffrey, J. H.; Kunkl, A.; Dongworth, D. W. The follicular dendritic cell: its role in antigen presentation in the generation of immunological memory. Immunol. Rev. 53:3-28; 1980.
0145-305X/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
ULTRASTRUCTURAL LOCALIZATION OF A SOLUBLE ANTIGEN IN THE CHICKEN HARDERIAN GLAND Emilio del Cacho, Margarita Gallego, and Jose Antonio Bascuas Department of Animal Pathology, Universityof Zaragoza, Zaragoza,Spain (Submitted January 1991; Accepted July 1991) [~Abstract--The relationship between plasma cells, macrophages, B and T cells, dendritic cells, and epithelium in the chicken Harderian gland have been studied by means of ultrastructural localization of the horseradish peroxidase following local immunization. After 5 d, peroxidase activity was found in vesicles located in macrophages and immature plasma cells. On day 7, peroxidase-antiperoxidase complexes were found in vesicles of the epithelial cells lining the secondary ducts and the acini, in the lumina of the ducts, and on the surface of lymphocytes located among these epithelial cells. Dendritic cells showing peroxidase activity on their surface were seen in the subepitheliai lymphoid tissue and in the lymphoid follicles. On day 9, peroxidase activity was found as iccosomes on the surface of dendritic cells and lymphoblasts. These results indicate that immature plasma cells in the Harderian gland can take up antigen and may have a role in presenting it to T cells. Further, our results suggest that intraepithelial lyrnphocytes might be involved in antigen transportation from the epithelium to the subepithelial lymphoid tissue. 7qKeywords--Follicular dendritic cell; Plasma cell; Intraepithelial lymphocyte; Horseradish peroxidase; Ultrastructure; Gland of Harder; Chicken.
Introduction The Harderian gland (HG) contains the largest concentration of plasmacytic cells within avian lymphoid tissue. MuelSupported by Grant No. PB86-0532 from DGICYT. Address correspondence to Margarita Gallego, Dpto. Patologia Animal, Facultad de Veterinaria, Miguel Servet 177, 50013-Zaragoza, Spain.
ler et al. (1) failed to show cells capable of antibody production in the HG after systemic stimulation via intravenous injection, but succeeded when stimulation was applied locally by dropping onto the eyeball. Survashe et al. (2) demonstrated an increased lymphoid activity and a rise in plasma cell numbers following eyedrop antigen application and neither effect was impaired b y s p l e n e c t o m y . Therefore, it can be assumed that the plasma cell population of the HG is activated by antigen located on the eyeball, and that the spleen is not a major source of immunocompetent cells of the gland in response to an immunogenic stimulus (3). Bell (4) found evidence that many of the plasmablasts that appeared in the gland as a response to antigenic stimulation initiated its differentiation by contact with antigen at sites outside the gland, and further migrate via the vascular system to enter the gland. Moreover, Gallego et al. (5) suggested that macrophages in the lymphoid agreggates within the fornix migrate to the gland after antigen uptake. The excellent response of the HG to a T-dependent antigen (1), in spite of the small population of T cells identified in the gland (6), suggests that a special environment for the B and plasmacytic cells activation and differentiation might exist (7). This unique environment might be maintained by the lymphoid cell interactions with the epithelium, the interstitium, or both. Microscopic and electron microscopic features of the HG, as well as the immunoglobulin classes in this gland, have been widely studied by several groups (7-13).
209
210
However, limited information relevant to the relationship between plasma, lymphoid, dendritic, and epithelial ceils is very limited. The high proliferative capacity of the HG plasma cells (6) and the efficiency of the gland in the production of antibodies during a local immune response (7,14) indicate that a specific relationship might exist between the aforementioned cells. This relationship may help explain the effective antigen presentation and high antibody titers in tears associated with the local immune response to T-dependent antigens in spite of the reduced T-cell population. In the present study, the relationship between immunocompetent cells of the gland of Harder has been investigated by means of the ultrastructural localization of peroxidase after local immunization with horseradish peroxidase (HRP).
Materials and Methods In these experiments, 7-week-old White Leghorn chickens were used. Twelve chickens were injected in the nictitating membrane (as close as possible to the fornix) with 0.02 mL 2% HRP (Sigma) in sterile saline. Three saline control birds were injected in the nictitating membrane with 0.02 mL sterile saline. Another three animals were used as a noninjected control group. Before injecting HRP and sterile saline, the chickens were anesthetized with sodium thiopental (30 mg/kg body weight). Four of the immunized animals, one bird from the control group, and another from the non-injected group were euthanized by cervical dislocation at 5, 7, and 9 d after the injection. Both Harderian glands from each chicken were removed immediately after death, fixed in 1% buffered glutaraldehyde for 90 min, and washed in cacodylate buffer. To develop peroxidase activity, samples were embedded in 7% agar at 40°C and cut at 100 ~m. These sec-
E. del Cacho et al.
tions were incubated in diaminobenzidine hydrochloride (DAB: 0.5 mg/mL) in Tris-HCl buffer containing 0.01% HzOz plus 10 mM 3 - a m i n o - l , 2 , 4 - t r i a z o l e (Sigma) (15) to inhibit endogenous peroxidase activity for 10 min at room temperature. Further, the glands were postfixed for 30 min in 1% osmium tetroxide. After final washing in buffer and dehydration in ethanol, fixed glands were cleared in propylene oxide and embedded in Epon-Araldite (1:1). Selected blocks were thin sectioned (400-600/~), stained with uranyl acetate exclusively, and examinated in a Jeol T8 electron microscope.
Results Five days after antigen injection, peroxidase activity was found in the phagosomes located in the cytoplasm of the macrophages, which were surrounded by immature plasma cells (Fig. 1). These immature plasma cells showed peroxidase activity in vesicles (Fig. 1) and in Golgi saccules at the maturing face (Fig. 2). The clusters, made up of macrophages and immature plasma cells, were seen in the diffuse lymphoid tissue beneath the epithelium lining the glandular ducts. Lymphocytes, iymphoblasts, and follicular dendritic cells gave a negative reaction, as well as the epithelial ceils lining the intraglandular ducts. Lumina of the ducts were negative for peroxidase activity. Seven days after HRP injection, peroxidase activity was detected (i) on the basolateral surface of the epithelial cells lining the secondary ducts and the acini, (ii) in vesicles distributed throughout the cytoplasm of the aforementioned cells, and (iii) within the lumina of the abovementioned ducts [Fig. 3(A)]. Peroxidasepositive particles observed in the vesicles and in the duct lumina were pentagonal in shape with diameters of 205 /~ (average of 45 particles) [Fig. 3(B)]. Per-
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Figure 1. Electron micrograph of a cellular cluster located in the subepithelial lymphoid tissue of a Harderian gland taken 5 d after injection of HRP. The cluster is made up of a macrophage (M), with peroxidase activity in its phagosomes, and of immature plasma cells showing peroxidase activity in vesicles (>-). Mag. ×6,000. Bar = 2 p,m.
oxidase activity was also identified on the surface of the cells seen between the epithelial cells lining the secondary ducts and the distal part of the acini, within the subepithelial diffuse lymphoid tissue, and in the lymphoid follicles. The cells with peroxidase-positive activity on their surface were classified into three types depending on their ultrastructural features. • T y p e / - - C e l l s ultrastructurally similar
to small lymphocytes [Fig. 3(A)]. • Type H m R o u n d cells with a patchy
chromatin pattern of the nucleus similar to that of lymphocytes. Their cytoplasm showed several electron-dense granules located around the nucleus [Fig. 3(A)]. This cell type shows ultrastructural characteristics intermediary
between lymphocyte and chicken dendritic cell. Type II might correspond to an immature dendritic cell. • Type llI---Cells showing the ultrastructural features of a dendritic cell such as an eccentric nucleus with a chromatin pattern similar to that of a small lymphocyte, an elongated cytoplasm with one or more cell processes, and electron-dense cytoplasmic granules in one of the processes (Fig. 4). Cell type I was observed exclusively between the epithelial cells lining the secondary ducts and the acini. In contrast, types II and III were seen either between epithelial cells (Figs. 3, 4) or within the subepithelial diffuse lymphoid tissue (Fig. 4) or within the lymphoid follicles [Fig. 5(A,B)]. Macrophages and
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Figure 2. Golgi complex of an immature plasma cell in a cellular cluster of a Harderian gland taken 5 d after injection of HRP. Peroxidase activity was found in the saccules and within vesicles associated to the maturing face. Mag. ×44,000. Bar = 0.5 i~m.
immature plasma cells were negative for peroxidase activity. Nine days after antigen injection, peroxidase activity was found in germinal centers on the surface of both lymphoblasts and follicular dendritic cells (Fig. 6) mainly as spherical particles containing material of varying electron density (Figs. 6, 7). Size estimates of 20 particles gave a mean diameter of 0.4 +- 0.08 (SD) p.m. These particles with peroxidase activity were also observed intracellularly in vesicles located in the peripheral area of the cytoplasm (Fig. 7). Structures with peroxidase activity were seen in small invaginations of the cell membrane and having a dash-like appearance either straight or U-shaped (Fig. 6).
Discussion The DAB technique for the ultrastructural localization of peroxidase activity, initially introduced by Graham and Karnowsky (16), is not specific for exogenous peroxidase, as endogenous peroxidase can also react positively. Herzog and Miller (15) demonstrated that 10 -2 M aminotriazole completely inhibits the endogenous peroxidase, while the exogenous peroxidase remains unaffected. Endogenous peroxidase was completely inhibited in our samples with 10 -2 M aminotriazole since it was not observed either in epithelial cells or in macrophages, which are the localizations described for this enzyme (15,17).
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Figure 3. (A) Electron micrograph of a secondary duct of a Harderian gland taken 7 d after injection of HRP. Peroxidase activity ( ~ ) is located in the duct lumen (~), in vesicles in the cytoplasm of epithelial cells (E), and on the basolateral surface of these cells. Lymphocyte-like (L) and immature dendritic cell (D) showing peroxidase activity on their surface. Note the electron-dense cytoplasmic granules that chicken dendritic cells exhibit. Mag. x5,000. Bar = 2 p.m. (B) Detail of region in 3A. Peroxidase-positive particles pentagonal in shape ( ~ ) located in theMuct lumen and within cytoplasmic vesicles in the epithelial cells. Mag. x8,000. Bar = 1 p.m.
Plasma cells and macrophages in the clusters observed 5 d after HRP administration showed peroxidase activity in their cytoplasms. As peroxidase activity was observed exclusively in the cytoplasm of macrophages within the 72 h following HRP administration (5), it can be assumed that macrophages may have a role in presenting antigen to immature plasma cells. In mammals, macrophages have been demonstrated to have a role as antigen-presenting cells to B cells (18,19). B cells have been established as being able to internalize low-molecular-weight soluble peptides (34,000-44,000 Da) released by macrophages, i.e., exocytosed, as the protein antigen is being processes (20). Therefore, since the HRP MW is 40,000 Da (21) it can be assumed
that the Harderian gland plasma cells endocytose nonprocessed or slightly processed HRP, which may have been released by the macrophages observed in the above-mentioned cellular clusters. Our results clearly show that immature plasma cells in these clusters internalize peroxidase. There are two ways in which B-cells can take up antigen, both nonspecific and specific. In the nonspecific uptake, B-cells behave as antigen-presenting cells by internalizing the antigen, processing it, and reexpressing its fragments on the cell surface bound to class II MCH molecules (22). In the specific uptake, B-cells bind the antigen via their immunoglobulin receptor, which concentrates the antigen as the cell becomes activated to endocytose it (23,24). Previ-
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Figure 4. Electron micrograph of a secondary duct of a Harderian gland taken 7 d after injection of HRP. Note the peroxidase activity on the surface of mature dendritic cells (MD) located between the epithelial cells (E) lining the duct and on the surface of an immature dendritic cell (Ib) in the subepithelial lymphoid tissue. -~, duct lumen. Mag. x4,000. Bar = 2 p.m.
ous studies have demonstrated a high frequency of plasma cells in the Harderian gland possessing a high density of immunoglobulin receptor (25,26). Both frequency of cells and density of immunoglobulin receptors were reported to be higher than those found in any other lymphoid organ in the chicken (26). These reports and the existence of Ia ÷ plasma
cells in the Harderian gland of the chicken (6) might indicate that plasma cells in this localization are more efficient in endocytosing and presenting antigen than B cells and plasma cells in other lymphoid tissues. Therefore, the excellent response of the Harderian gland to a T-dependent antigen may be explained by the fact that Harderian
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Figure 5. Electron micrographs of two lymphoid follicles in a Harderian gland taken 7 d after injection of HRP. (A) Immature dendritic cell (ID) showing peroxidase activity on its surface. Mag. x4,000. Bar = 2 i~m. (B) Mature dendritic cell (MD) with peroxidase activity on its surface. Mag. ×3,000, Bar = 2 p,m.
gland immature plasma cells behave as antigen-presenting cells to T lymphocytes. The association of the peroxidase with the Golgi complex, which may provide a mechanism for reexpression of antigen fragments, suggests that the interaction of processed protein antigen with MHC may occur in this cellular compartment as proposed by Chain et al. (27). The pentagonal particles that were peroxidase positive are similar to those described by Sternberger et al. (28) for peroxidase- antiperoxidase complexes (PAP). The ultrastructural sites for PAP complexes in the Harderian gland were found to be analogous to those described for secretory IgA in the bronchial and bronchiolar mucosa (29,30) and in the acini of the lacrimal gland (31), where the IgA locally produced is transported through the epithelial cells and then discharged into the lumen by exocytosis. In light of these reports and through our findings, we suggest that transport mech-
anisms of PAP complexes in the Harderian gland may be similar to IgA transport, through the epithelial cells into the lumen. Peroxidase-positive lymphocytes located between the epithelial cells lining the ducts correspond to the intraepithelial lymphocytes (IELs). Our findings suggest that IELs are able to attach antigen to their surface. Antigen attachment to the IEL surface seems to involve the modification of the lymphocyte ultrastructural features into those described for the chicken dendritic cell (32-34). The localization of peroxidase on the surface of the apparent transitional cell from IEL to dendritic cell, which was observed both among the epithelial cells and in the subepithelial lymphoid tissue, suggests that this cell may transport antigen. Our results further support the findings of Lawn and Rose (35), who demonstrated that IELs located in the chicken gut and provided with cytoplasmic processes and electron-dense gran-
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Figure 6. Electron micrograph of a germinal center of a Harderian gland taken 9 d after injection of HRP. Lymphoblast (L) and follicular dendritic cell (D) with peroxidase activity, mainly as spherical particles, on their surface. Peroxidase-positive structures with a dash-like appearance either straight or U-shaped can be observed in invaginations of the dendritic cells membrane ( ~.- ). Mag. x4,000. Bar = 2 p.m.
ules are capable of transporting antigen. According to our results, to perform this function IELs might leave the intraepithelial location and further migrate into the lymphoid follicles from the diffuse lymphoid tissue.
The spherical peroxidase-positive particles we found on the surface of lymphoblasts and follicular dendritic cells were ultrastructuraUy similar to the iccosomes described by Szakal et al. (19). Therefore, we suggest that the immune
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Figure 7. Electron micrograph of a germinal center of a Harderian gland taken 9 d after injection of HRP. Spherical particles showing peroxidase activity located both on the surface of a dendritic cell (~) and in the peripheral area of its cytoplasm (~-). mt, lymphoid cell undergoing mitosis; L, lymphoblasts. Mag. x12,000. Bar = 1 p.m.
complexes in the chicken are retained on the dendritic cell surface as iccosomes, as proposed for mammalian species. Iccosomes have been demonstrated to mediate the delivery of antigen in the form of immune complexes from follicular dendritic cells to germinal center B-lymphocytes and macrophages. It has also been demonstrated that these germinal center events lead to the production of B memory cells and a possible increase of the antibody levels in the anamnestic response (19). According to our findings, these events seem to be
similar in chickens and mammals. The amount of iccosomes observed was lower than that reported by Szakal et al. (19). This may be due to the fact that chicken follicular dendritic cells have less filiform processes than mouse follicular dendritic cells. Further, the I g A m the more abundant immunoglobulin in the Harder gland after antigen stimulat i o n m h a s scarce biological properties in the complement activation and memory B-cells formation, and it is hardly retained on the follicular dendritic cell surface (36).
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35. Lawn, A. M.; Rose, M. E. Mucosal transport of Eimeria tenella in the cecum of chicken. J. Parasitol. 68: 1117-1123; 1982. 36. Klaus, G. B.; Humffrey, J. H.; Kunkl, A.; Dongworth, D. W. The follicular dendritic cell: its role in antigen presentation in the generation of immunological memory. Immunol. Rev. 53:3-28; 1980.
