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A Giant Cell with Dendritic Cell Properties in Spleens of the Anuran Amphibian Xenopus Laevis
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 5, pp. 461-473, 19B1. 0145-305X!B1!030461-13$02.00!0 Printed in the USA. Copy~ight (c) Pergamon Press Ltd. All rights reserved.
A GIANT CELL WITH DENDRITIC CELL PROPERTIES IN SPLEENS OF THE ANURAN AMPHIBIAN XENOPUS LAEVISI
William M. Baldwin 1112 and Nicholas Cohen Department of Pathology, Peter Bent Brigham Hospital Boston, Mass. 02115, and Department of Microbiology, University of Rochester School of ~1edicine and Dentistry, Rochester, New York 14642
ABSTRACT
Numerous large, mitotically active cells with abundant electron lucent cytoplasm, large hyperlobated nuclei and prominent nucleoli are found in the periphery of the splenic white pulp follicles of healthy Xenopus Zaevis. Like mammalian dendritic cells, these cells are located in B-Iymphocyte-rich splenic follicles and have long cytoplasmic processes that are in intimate contact with adjacent lymphocytes. Some of the cytoplasmic processes extend through the Tlymphocyte-rich marginal zone into red pulp and appear to trap and transport foreign material (colloidal carbon and human IgG) from its initial site of entry in the splenic red pulp into the white pulp. In contrast to macrophages, these cells do not phagocytose large quantities of carbon. They also fail to stain for the non-specific esterase, either in a diffuse pattern characteristic for macrophages or in the punctate pattern found in most lymphoid cells in this species. In contrast to adjacent B-lymphocytes in the white pulp follicle, they do not stain for cytoplasmic Ig. Unlike T-lymphocytes, early thymectomy does not interfere with their development (1). Thus, this cell may well be a primitive follicular dendritic cell. If so, it offers insights into the function of the Xenopus spleen, and the phylogenetic origin of dendritic cells. INTRODUCTION
About 3% of the cells in the splenic white pulp of healthy juvenile
Xenopus Zaevis frogs are giant cells with abundant electron lucent cytoplasm,
hyperlobated nuclei and prominent nucleoli. In formalin-fixed sections, their cytoplasm often retracts, giving them a lacunar appearance. Based on this Supported, in part, by USPHS grants HD 07901 (NC) and NRSA S-T32-HL07066
(WMB)
2 Address reprint requests to: W. M. Baldwin, Dept. of Nephrology, University
Hospital, 2333 AA Leiden, The Netherlands. 461
462
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Vol. 5, NO. 3
appearance, these cells have been described previously as "degenerating macrolymphocytes" and have been considered to be effete by-products of lymphocyte proliferation (1,2). Our observations on glutaraldehyde-fixed sections indicate that these cells are neither degenerating nor lymphocytic. Instead, they are mitotically active cells that trap foreign material on the surface of their cytoplasmic extensions. Since this is the primary functional characteristic of follicular dendritic cells in mammals (3,4), these amphibian cells may be one of the phylogenetically early forms of dendritic cells. MATERIALS AND METHODS Tissue fixation Slices of spleen, thymus, liver, kidney, and bone marrow from healthy, laboratory-raised, .6-8 month old juvenile Xenopu8 laevi8 were fixed in cold (4°C) acidified formalin (10% formalin with 1.5% acetic acid) for light microscopy or in 2% paraformaldehyde and 2.5% glutaraldehyde in cacodylate buffer for electron microscopy (5). Sections of acidified formalin fixed tissues were either stained with hematoxylin and eosin or by the immunoperoxidase technique for Ig (6). Immunoperoxidase staining For immunoperoxidase staining, the primary reagent was a rabbit anti-
Xenopu8 Ig antiserum (kindly provided by Paul Bleicher) raised against Seph-
arose 6B fractionated, ammonium sulphate precipitated euglobulin that was twice absorbed against Xenopu8 erythrocytes. This antiserum, which forms a strong line of precipitation with Xenopu8 Ig on immunoelectrophoresis, was used in dilutions of 1:10, 1:20, and 1:40, and was then "sandwiched" with swine anti-rabbit Ig 1:60 and rabbit anti-peroxidase complexed to peroxidase 1:80 (Dakkopatts, Denmark). The peroxidase was reacted with a solution of 3-3' diaminobenzidene tetrahydrochloride (Sigma, St. Louis, Missouri) plus 0.3% H202 (6 mg DAB plus 3 drops H202 per 10 ml of 0.5 M Tris buffer). Sectioning of tissue
Paraformaldehyde and glutaraldehyde fixed tissues were post fixed for 1 hour in osmium collidine at 4°C and embedded in epon. One micron sections were stained with toluidine blue. Selected blocks were thin sectioned, stained with uranyl acetate and lead citrate, and examined in a Joel JEM-lOOS electron microscope. Cytocentrifuge preparations Additional splenic tissue was either frozen and sectioned or teased into single cell suspensions for cytocentrifuge preparations. Some of these preparations were fixed in formal-acetone solution and used for localization of the nonspecific esterase (alpha-naphthyl esterase) by standard techniques (7). Other single cell suspensions were reacted with neuraminidase-treated sheep erythrocytes (SRBC) or with SRBC coated with rabbit IgG or IgM (7), in an effort to determine the presence of erythrocyte and Fc receptors. Phagocytosis To determine the distribution of colloidal carbon particles (20-30 ~), Pelikan India ink (Gunther Wagner, Hannover, Germany) was administered by
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intracardiac injection at the time of sacrifice or by dorsal lymph sac and s.c. injection 1 or 48 hours prior to sacrifice (8). Carbon localization was evaluated by light microscopy. To determine the distribution of an antigenic protein, 0.5 m1 of isolated human IgG (2 mg(ml) in phosphate buffered saline was injected into the dorsal lymph sac 1-4 hours prior to sacrifice (9, 10). Frozen sections of spleens from these Xenopus were stained with fluorescence conjugated swine anti-human Ig (Nordic, Tilburg, The Netherlands).
RESULTS Morphology of Xenopus spleen The Xenopus spleen is divided into two distinct compartments by a double layer of endothelial-like "boundary cells" that surround an anastomosing network of spherical and ovoid white pulp follicles (Fig. 1). White pulp follicles encircle centrally located arteries and are traversed by penetrating arteriolar branches (Fig. 2). Within the white pulp follicles are scattered pigment-laden cells and numerous lymphocytes (4-5.5 ~ in diameter) which are interspersed between an interconnecting framework of peripherally located, large (12.5-20 ~ in diameter) cells 'with multilobed nuclei (provisionally called XL cells) (Figs. 3-4). Lymphocytes also rim the exterior of the boundary cells forming a marginal zone which merges with the red pulp. The red pulp is composed of thin-walled vascular sinuses with intervening macrophages and erythrocytes. Morphology of XL cells The XL cells have abundant e~ectron lucent cytoplasm, large multilobed nuclei, and prominent nucleoli. In formalin fixed sections, the cytoplasm retracts resulting in a lacunar appearance of these cells (Fig. 2). The nuclear chromatin is finely granular and generally evenly distributed with some condensation around the nuclear membrane (Fig. 5). Within the white pulp follicles, the XL cells form frequent interconnections with each other either along broad adjoining cell surfaces or through long (5-15 ~) narrow cytoplasmic bridges. Lymphocytes line the free surfaces of the XL cells and penetrate Figures 1 and 2
Xenopus spleen stained for Ig by immunoperoxidase (dark cells) and counter-
stained with hematoxylin (40X and 400X). Lymphocytes that stain positively for Ig in the white pulp follicles are distinctly separated from the surrounding marginal zone and red pulp by boundary cell layers. Large peripherally located XL cells do not stain for Ig. Figure 3 White pu!'p follicle with XL cell in mitosis (Toluidine blue, 400X). Figures 4a and b
Serial sections through one follicle. XL cells interconnect with each other and extend pseudopods (arrows) through the boundary cell layer into the red pulp. In each section, prominent nucleoli, lobated nuclei and centrioles are evident in some XL cells (Toluidine blue, I ~ thick section; 500X).
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FIGURES 1-4:
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See bottom of preceding page for legends.
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between adjoining XL cells (Figs 3-5). Many of these lymphocytes have microvilli that interdigitate with the surface of the XL cells (Fig. 5). These interdigitations are most evident in the cytoplasmic bridges between XL cells. XL cells also have long (10-40 ~) pseudopod-like cytoplasmic extensions which protrude through "ports" in the shell of boundary ceJ.ls into the red pulp (Figs. 4 and 6). Lymphocytes squeeze through these "ports" being compressed against the pseudopods (Figs. 4 and 6). Prominent large (0.7-1.2 ~ diameter) centrioles are in the cytoplasm of most XL cells between the nucleus and the boundary of the white pulp. Frequently they are located at the origin of the pseudopods (Figs. 4-6). Microtubules radiate from the centrioles extending to the nucleus in one direction and streaming down the pseudopod in the opposite direction. Many of the organelles appear to be oriented along these microtubules. Although mitotic figures were frequently present (Fig. 3), the majority of centrioles were in cells that were not in mitosis. Organ distribution of XL cells XL cells were not found in the other hematopoietic or lymphopoietic organs (liver, kidney, thymus and bone marrow) of this anuran species. XL cells did not migrate to sites of immune or non-immune inflammation such as to skin allo- or autografts or to subcutaneouns deposits of carbon. Instead, these sites contained accumulations of lymphocytes, blasts, and macrophages, many of which contained easily detectable cytoplasmic Ig. Immunohistochemical comparison of XL cells and B-lymphocytes Immunoperoxidase staining also demonstrated that a majority of the small lymphocytes and large blasts ~ithin the white pulp follicle contained cytoplasmic Ig, while only a few of the lymphocytes in the marginal zone or red pulp did so. None of the XL cells were stained by this method (Figs. 1 and 2). Histochemical comparison of XL cells, macrophages, and lymphocytes Three patterns of nonspecific esterase staining were detected on frozen sections and cytocentrifuge preparations. Macrophages, large clusters of which encircle the periphery of the marginal zone, had a very dense stain in frozen sections, and a diffuse, finely granular stain over most of their cytoplasm on cytocentrifuge preparations (Fig. 7). Most lymphocytes (70-90%) contained 1 or 2 brightly staining, punctate granules in their cytoplasm (Fig. 7), while the remainder had no stain. Macrophage, but not lymphocyte, staining was resistant to fluoride treatment. XL cells had either no or very faint staining Figure 5 XL cell with a large biolobed nucleus. A centriole with radiating micro tubules is located in the lucent cytoplasm between the nucleus and the boundary cells (B). Microvilli (arrows) of adjacent lymphocytes invaginate into the XL cell surface giving the cytoplasmic bridge, which extends to the right, a segmented appearance (uranyl acetate and lead citrate; 8000X). Figures 6a and b Pseudopod-like extensions of XL cells that protrude from the white pulp through the boundary cell layer (B) into the red pulp. Microtubules extend the length of both pseudopods and a centriole is at the origin of (a). A lymphocyte is traversing the boundary cell layer next to the pseudopod in (b) (uranyl acetate and lead citrate; 6000X).
466
XENOPUS DENDRITIC CELLS
FIGURES 5 and 6:
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See bottom of preceding page for legends.
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in frozen sections and no staining in cytocentrifuge preparations (Fig. 7). Surface receptor studies Preliminary attempts to demonstrate surface receptors for erythrocytes or for the Fc portion of IgG or M were unsuccessful. However, this may reflect a less than optimal selection of reagents (rabbit IgG and M, and ~heep erythrocytes) or conditions, rather than a total absence of such receptors from the XL cell surface. Carbon localization in relation to XL cells and macrophages Both i.v. and S.C. injected colloidal carbon was initially deposited in large quantities in the spleen just peripheral to the marginal zone in the macrophage-rich region of the red pulp and none was directly extravasated from the blood vessels into the white pulp (Fig. 2). Some carbon was phagocytosed and retained as aggregates in the red pulp macrophages for at least 2 days. However, some carbon particles were transported within I hour into the white pulp. Initially, most of the carbon particles that had penetrated into the marginal zone and white pulp, were localized on the surfaces of the XL cell bodies and their pseudopods (Fig. 8a). After 2 days, aggregates of carbon were within the few macrophages located centrally in the white pulp (Fig. 8b). Antigen localization in relation to XL cells Similarly, human IgG was initially deposited in a finely granular pattern between the cells in the peripheral marginal zone. Granules were also evident on large stellate cells located in the periphery of the white pulp follicle. On some of these cells, which had the same distribution, size, and shape of XL cells, the granules adhered to cellular extensions that reached through the boundary cell layer into the marginal zone (Fig. 9). Figure 7 Composite picture of cytocentrifuge preparations stained for nonspecific esterase. Macrophages (M) have diffuse cytoplasmic staining, lymphocytes (L) have punctate staining and XL cells no staining. Sheep erythrocytes in the background were markers for an IgG Fc binding assay; XL cells did not convincingly bind these markers (750X). figure 8a XL cells 1 hour after dorsal lymph sac injection of colloidal carbon. Carbon particles are on the surfaces of the pseudopod and cell bodies of the XL cells (IOOOX). Figure 8b XL cells 2 days after dorsal lymph sac injection of colloidal carbon. Only occasional small carbon particles are sill associated with the surface of the XL cell while adjacent macrophages are filled with large carbon particles (1,200X). figures 9a and b Indirect immunofluorescent stain for human IgG 4 hours after injection. Granular staining is scattered in the peripheral marginal zone and concentrated on large cells in the periphery of the follicle (a, 20X; b, 40X).
468
XENOPUS DENDRITIC CELLS
FIGURES 7-9:
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See bottom of preceding page for legends.
priminent
B-lymphocytes with extensions to T-Iymphocytes
contacts
d
c
b
See ref (21)
See ref (20)
See ref (19)
See ref (3)
independent
Thymus dependency
a
no
Predominantly phagocytic
weak-none
present
long extensions
non-specific esterase
present
present
micro tubules
?
no
weak diffuse
B-Iymphocytes
present
?
prominent
centrioles
few
few
lysosomes
Cytoplasm
clear
not prominent
hyperlobulated
Nucleus
clear
lobulated
splenic follicles
Location
Nucleoli
splenic follicles
XL cell
Follicular a b dendritic cells ,
independent
no d
focal positive
T-Iymphocytes
present
present
present
few
clear
not prominent
lobulated
T-cell areas
Interdigitating cells c
independent
no
positive
epidermal cells + lymphocytes (T?)
present
present
?
variable
clear
not prominent
lobulated
skin
Langerhans cells
Characteristics of XL cells, follicular dendritic cells, interdigitating cells, and Langerhans cells
Characteristic
TABLE I.
~
1.0
'"
til
~
n n
H
~
~
t:I
~
til
~
o '"d c:::
w
~
VI
......
(f
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XENOPUS DENDRITIC CELLS
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DISCUSSION Several observations suggest that XL cells are distinct from both lymphoid cells and macrophages. Manning (1) has reported that the histogenesis of the XL cells is not affected by thymectomy at early stages of lymphoid organ maturation. Yet, thymectomy at this stage significantly depletes the lymphoid population in the marginal zone and inhibits manifestations of T~lymphocyte function (I,ll). Unlike the B-lymphocytes in the white pulp surrounding them. the XL cells contain no demonstrable cytoplasmic Ig. In addition, XL cells fail to stain for the non-specific esterase either in the punctate pattern found in most lymphoid cells or in a diffuse pattern characteristic of macrophages in this species. In further distinction to macrophages, they do not phagocytose and retain large quantities of colloidal carbon (8). Thus, XL cells appear to be distinct from cells of both the lymphoid and macrophage lineage. XL cells have some morphological similarities to splenic follicular dendritic cells in mammals (Table 1). Both are large cells with clear cytoplasm containing few lysosomes; both have long cytoplasmic extensions in intimate contact with adjacent B-lymphocytes of the splenic follicles. Although mammalian dendritic cells have large lobulated nuclei, the hyperlobulated nuclei and prominent nucleoli of the XL cells more closely resemble those of ReedSternberg type giant cells found in malignant and reactive lymphoid tissues of humans (12-14), some of which may be derived from dendritic cells (15). XL cells also share the characteristic function of dendritic cells, namely trapping foreign material on the surface of their cytoplasmic extensions for potential presentation to adjacent lymphocytes (3,4). Additionally, these XL cells may be involved in transportating carbon and human IgG from their initial site of deposition in the red pulp through the marginal zone and into the white pulp. Although lymphoid cells are thought to be responsible for antigen redistribution in mammalian spleens (16), dendritic cells may serve this function in lower animals. In chickens, for example, dendritic-type cells trap antigen on their surface in the peripheral white pulp and then migrate along arteriolar sheaths to initiate germinal center formation in the central white pulp (17). Our working hypothesis to explain the present observations on the XL cells is diagrammed in Fig. 10. Antigens and other materials are extravasated in the macrophage-rich region of the red pulp encircling the marginal zone. Large particles are phagocytosed by macrophages. Small particles and macrophage-processed material may be trapped on the surface of the XL cell, pseudopods and then transported through the marginal zone into the white pulp. It is possible that the elaborate microtubular system that is anchored by the large centriole and extends the length of the pseudopod, may be involved in transporting the trapped material. T-lymphocytes may assist XL cells in trapping and transporting antigen from the marginal zone to the white pulp. This would correlate both with the movement of lymphocytes along the XL cell pseudopods (Fig. 6b) and the inhibition of antigen retention caused by early thymectomy (10). Once within the white pulp, the material may be temporarily retained in the invaginated surfaces of the XL cell that interdigitate with the microvilli of adjacent lymphocytes (10). Possibly when an appropriate antigenic stimulus is presented to the adjacent lymphocytes, they divide and transform. This was not observed with the colloidal carbon tracer, but does occur following skin grafting (Baldwin and Cohen, unpublished observations) or immunization with sheep erythrocytes. In these situations, mitotic activity was largely confined to lymphocytes adjacent to the XL cells in the peripheral white pulp but was absent from the lymphocytes in the central white pulp (18). Xhus, by serving to trap and transport antigen, the XL cell may function as a bridge between T-cells in
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the marginal zone and B-ce11s in the white pulp follicle. To determine whether XL cells can perform the functions hypothesized in this model, we have isolated XL cells and have begun to determine their ability to actively trap and transport antigenic material in vitro.
Figure 10 Diagram of Xenopus splenic white pulp follicle following an i.v. injection of carbon particles. Carbon is extravasated in the red pulp (1) where it is phagocytosed by macrophages (2) or trapped on XL cell pseudopods (3). Trapped material is drawn towards the white pulp follicle being presented to T-1ymphocytes in the 'margina1 zone (4) and B-1ymphocytes in the follicle (5). Finally, it is phagocytosed by macrophages in the central white pulp (6).
ACKNOWLEDGEMENTS The authors are grateful to Dr. Jonathan Said and Mrs. Christine Rudo1fi for their aid in interpreting and preparing the electronmicrographs; to Dr. Geraldine Pinkus and Ms. Janet McLeod for their help in interpreting and preparing the naphthyl acetate esterase stains; to Paul Bleicher for his generous gift of rabbit anti-Xenopus Ig; and to the Hubrecht Laboratory, Utrecht, The Netherlands, for supplying some of the animals used in these studies.
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REFERENCES 1.
Manning, M.J. The effect of early thymectomy on histogenesis of the lymphoid organs in Xenopus Zaevis. J. EmbryoZ. expo Morph. 26, 219, 1971.
2.
Sterba, G. Untersuchungen an der Milts des Krallenfrosches (Xenopus Zaevis Daudin). Gegenbaurs. Morph. Jahrbuah 90, 221, 1950.
3.
Chen, L.L., Adams, J.C., Steinman, R.M. Anatomy of germinal centers in mouse spleen, with special reference to "follicular dendritic cells". J. CeZZ BioZ. 77, 148, 1978.
4.
Nossal, G.J.V., Abbot, A., Mitchell, J., Lumnes, Z. Antigens in immunity. XV. Ultrastructural features of antigen capture in primary and secondary lymphoid follicles. J. expo Med. 127, 277, 1968.
5.
Karnovsky, M.J. A formaldehyde glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. CeZZ BioZ. 27, 137a, 1965.
6.
Pinkus, G.S., Said, J.W. Specific identification of intracellular immunoglobulin in paraffin sections of multiple myeloma and macroglobulinemia using an immunoperoxidase technique. Am. J. PathoZ. 87, 47, 1977.
7.
Pinkus, G.S., Hargreaves, H.K., McLeod, J.A., Nadler, L.M., Rosenthal, D.S., Said, S.W. Naphthyl acetate esterase activity - a cytochemical marker for T lymphocytes; correlation with immunologic studies of normal tissues, lymphocytic leukemias, non-Hodgkin's lymphomas, Hodgkin's disease, and other lymphoproliferative disorders. Am. J. PathoZ. 97, 17, 1979.
8.
Turner, R.J. The functional development of the reticuloendothelial system in the toad, Xenopus Zaevis (Daudin). J. Exp. ZooZ. 170, 467, 1969.
9.
Collie, M.H.
Zaevis.
The location of soluble antigen in the spleen of Xenopus
Experientia 30, 1205, 1974.
10.
Horton, J.D., Manning, M.J. Effect of early thymectomy on the cellular changes occurring in the spleen of the clawed toad following administration of soluble antigen. ImmunoZogy 26, 797, 1974.
11.
Cohen, N., Turpen, J.B. Experimental analysis of lymphocyte ontogeny and differentiation in an amphibian model system. In: BioZogicaZ Basis.of Immunodeficiency. (E. W. Gelfand and H.-M. Dosch (Eds.) Raven Press, New York, 1980, p. 25.
12.
Mann, R.B., Jaff, E.S., Beard, C.W. Malignant lymphomas. A conceptual understanding of morphologic diversity. Am. J. PathoZ. 94, 103, 1979.
13.
Carr, I. disease.
14.
Glick, A.D., Leech, J.H., Flexner, J.M., Collins, R.D. Ultrastructural study of Reed-Sternberg cells: comparison with transformed lymphocytes and histocytes. Am. J. PathoZ. 85, 195, 1976.
15.
Curran, R.C., Jones, E.L. Hodgkin's disease: an immuno-histochemical and histological study. J. PathoZ. 125, 39, 1978.
The ultractructure of the abnormal reticulum cells in Hodgkin's
J. PathoZ. lIS, 45, 1975.
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16.
Veerman, A.J.P., Rooijen, N.V. Lymphocyte capping and lymphocyte migration as associated events in the in vitro antigen trapping process. An electromicroscopic autoradiographic study in the spleen of mice. CeZZ Tiss. Res. 161, 211, 1975.
17.
White, R.G., Henderson, D.C., Eslami, M.B., Nielsen, K.H. Localization of a protein antigen in the chicken spleen: Effects of various manipulative procedures on the morphogenesis of the germinal center. ImmunoZogy 28, 1, 1975.
18.
Turner, R.J., Manning, M.J. Responses of the toad, Xenopus Zaevis, to circulating antigens. I. Cellular changes in the spleen. J. Exp. ZooZ. 183, 21, 1972.
19.
Eike1enboom, P. Characterization of non-lymphoid cells in the white pulp of the mouse spleen: An in vivo and in vitro study. CeZZ Tiss. Res. 195, 445, 1978.
20.
Drexhage, H.A., Mullink, H., Groot, J. de, Clarke, J., Balfour, B.M. A study of cells present in peripheral lymph of pigs with special reference to a type of cell resembling the Langerhans cell. CeZZ Tiss. Res. 202, 407, 1979.
21.
Fossum, S., Smith, M.E., Bell, E.B., Ford, W.L. The architecture of rat lymph nodes. III. The lymph nodes and lymph-borne cells of the congenitally athymic nude rat (rnu). Scand. J. ImmunoZ. 12, 421, 1980.
-------------------------
Received for Publication September 1980 Accepted for Publication January 1981
A GIANT CELL WITH DENDRITIC CELL PROPERTIES IN SPLEENS OF THE ANURAN AMPHIBIAN XENOPUS LAEVISI
William M. Baldwin 1112 and Nicholas Cohen Department of Pathology, Peter Bent Brigham Hospital Boston, Mass. 02115, and Department of Microbiology, University of Rochester School of ~1edicine and Dentistry, Rochester, New York 14642
ABSTRACT
Numerous large, mitotically active cells with abundant electron lucent cytoplasm, large hyperlobated nuclei and prominent nucleoli are found in the periphery of the splenic white pulp follicles of healthy Xenopus Zaevis. Like mammalian dendritic cells, these cells are located in B-Iymphocyte-rich splenic follicles and have long cytoplasmic processes that are in intimate contact with adjacent lymphocytes. Some of the cytoplasmic processes extend through the Tlymphocyte-rich marginal zone into red pulp and appear to trap and transport foreign material (colloidal carbon and human IgG) from its initial site of entry in the splenic red pulp into the white pulp. In contrast to macrophages, these cells do not phagocytose large quantities of carbon. They also fail to stain for the non-specific esterase, either in a diffuse pattern characteristic for macrophages or in the punctate pattern found in most lymphoid cells in this species. In contrast to adjacent B-lymphocytes in the white pulp follicle, they do not stain for cytoplasmic Ig. Unlike T-lymphocytes, early thymectomy does not interfere with their development (1). Thus, this cell may well be a primitive follicular dendritic cell. If so, it offers insights into the function of the Xenopus spleen, and the phylogenetic origin of dendritic cells. INTRODUCTION
About 3% of the cells in the splenic white pulp of healthy juvenile
Xenopus Zaevis frogs are giant cells with abundant electron lucent cytoplasm,
hyperlobated nuclei and prominent nucleoli. In formalin-fixed sections, their cytoplasm often retracts, giving them a lacunar appearance. Based on this Supported, in part, by USPHS grants HD 07901 (NC) and NRSA S-T32-HL07066
(WMB)
2 Address reprint requests to: W. M. Baldwin, Dept. of Nephrology, University
Hospital, 2333 AA Leiden, The Netherlands. 461
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Vol. 5, NO. 3
appearance, these cells have been described previously as "degenerating macrolymphocytes" and have been considered to be effete by-products of lymphocyte proliferation (1,2). Our observations on glutaraldehyde-fixed sections indicate that these cells are neither degenerating nor lymphocytic. Instead, they are mitotically active cells that trap foreign material on the surface of their cytoplasmic extensions. Since this is the primary functional characteristic of follicular dendritic cells in mammals (3,4), these amphibian cells may be one of the phylogenetically early forms of dendritic cells. MATERIALS AND METHODS Tissue fixation Slices of spleen, thymus, liver, kidney, and bone marrow from healthy, laboratory-raised, .6-8 month old juvenile Xenopu8 laevi8 were fixed in cold (4°C) acidified formalin (10% formalin with 1.5% acetic acid) for light microscopy or in 2% paraformaldehyde and 2.5% glutaraldehyde in cacodylate buffer for electron microscopy (5). Sections of acidified formalin fixed tissues were either stained with hematoxylin and eosin or by the immunoperoxidase technique for Ig (6). Immunoperoxidase staining For immunoperoxidase staining, the primary reagent was a rabbit anti-
Xenopu8 Ig antiserum (kindly provided by Paul Bleicher) raised against Seph-
arose 6B fractionated, ammonium sulphate precipitated euglobulin that was twice absorbed against Xenopu8 erythrocytes. This antiserum, which forms a strong line of precipitation with Xenopu8 Ig on immunoelectrophoresis, was used in dilutions of 1:10, 1:20, and 1:40, and was then "sandwiched" with swine anti-rabbit Ig 1:60 and rabbit anti-peroxidase complexed to peroxidase 1:80 (Dakkopatts, Denmark). The peroxidase was reacted with a solution of 3-3' diaminobenzidene tetrahydrochloride (Sigma, St. Louis, Missouri) plus 0.3% H202 (6 mg DAB plus 3 drops H202 per 10 ml of 0.5 M Tris buffer). Sectioning of tissue
Paraformaldehyde and glutaraldehyde fixed tissues were post fixed for 1 hour in osmium collidine at 4°C and embedded in epon. One micron sections were stained with toluidine blue. Selected blocks were thin sectioned, stained with uranyl acetate and lead citrate, and examined in a Joel JEM-lOOS electron microscope. Cytocentrifuge preparations Additional splenic tissue was either frozen and sectioned or teased into single cell suspensions for cytocentrifuge preparations. Some of these preparations were fixed in formal-acetone solution and used for localization of the nonspecific esterase (alpha-naphthyl esterase) by standard techniques (7). Other single cell suspensions were reacted with neuraminidase-treated sheep erythrocytes (SRBC) or with SRBC coated with rabbit IgG or IgM (7), in an effort to determine the presence of erythrocyte and Fc receptors. Phagocytosis To determine the distribution of colloidal carbon particles (20-30 ~), Pelikan India ink (Gunther Wagner, Hannover, Germany) was administered by
Vol. 5, No. 3
XENOPUS DENDRITIC CELLS
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intracardiac injection at the time of sacrifice or by dorsal lymph sac and s.c. injection 1 or 48 hours prior to sacrifice (8). Carbon localization was evaluated by light microscopy. To determine the distribution of an antigenic protein, 0.5 m1 of isolated human IgG (2 mg(ml) in phosphate buffered saline was injected into the dorsal lymph sac 1-4 hours prior to sacrifice (9, 10). Frozen sections of spleens from these Xenopus were stained with fluorescence conjugated swine anti-human Ig (Nordic, Tilburg, The Netherlands).
RESULTS Morphology of Xenopus spleen The Xenopus spleen is divided into two distinct compartments by a double layer of endothelial-like "boundary cells" that surround an anastomosing network of spherical and ovoid white pulp follicles (Fig. 1). White pulp follicles encircle centrally located arteries and are traversed by penetrating arteriolar branches (Fig. 2). Within the white pulp follicles are scattered pigment-laden cells and numerous lymphocytes (4-5.5 ~ in diameter) which are interspersed between an interconnecting framework of peripherally located, large (12.5-20 ~ in diameter) cells 'with multilobed nuclei (provisionally called XL cells) (Figs. 3-4). Lymphocytes also rim the exterior of the boundary cells forming a marginal zone which merges with the red pulp. The red pulp is composed of thin-walled vascular sinuses with intervening macrophages and erythrocytes. Morphology of XL cells The XL cells have abundant e~ectron lucent cytoplasm, large multilobed nuclei, and prominent nucleoli. In formalin fixed sections, the cytoplasm retracts resulting in a lacunar appearance of these cells (Fig. 2). The nuclear chromatin is finely granular and generally evenly distributed with some condensation around the nuclear membrane (Fig. 5). Within the white pulp follicles, the XL cells form frequent interconnections with each other either along broad adjoining cell surfaces or through long (5-15 ~) narrow cytoplasmic bridges. Lymphocytes line the free surfaces of the XL cells and penetrate Figures 1 and 2
Xenopus spleen stained for Ig by immunoperoxidase (dark cells) and counter-
stained with hematoxylin (40X and 400X). Lymphocytes that stain positively for Ig in the white pulp follicles are distinctly separated from the surrounding marginal zone and red pulp by boundary cell layers. Large peripherally located XL cells do not stain for Ig. Figure 3 White pu!'p follicle with XL cell in mitosis (Toluidine blue, 400X). Figures 4a and b
Serial sections through one follicle. XL cells interconnect with each other and extend pseudopods (arrows) through the boundary cell layer into the red pulp. In each section, prominent nucleoli, lobated nuclei and centrioles are evident in some XL cells (Toluidine blue, I ~ thick section; 500X).
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FIGURES 1-4:
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See bottom of preceding page for legends.
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XENOPUS DENDRITIC CELLS
465
between adjoining XL cells (Figs 3-5). Many of these lymphocytes have microvilli that interdigitate with the surface of the XL cells (Fig. 5). These interdigitations are most evident in the cytoplasmic bridges between XL cells. XL cells also have long (10-40 ~) pseudopod-like cytoplasmic extensions which protrude through "ports" in the shell of boundary ceJ.ls into the red pulp (Figs. 4 and 6). Lymphocytes squeeze through these "ports" being compressed against the pseudopods (Figs. 4 and 6). Prominent large (0.7-1.2 ~ diameter) centrioles are in the cytoplasm of most XL cells between the nucleus and the boundary of the white pulp. Frequently they are located at the origin of the pseudopods (Figs. 4-6). Microtubules radiate from the centrioles extending to the nucleus in one direction and streaming down the pseudopod in the opposite direction. Many of the organelles appear to be oriented along these microtubules. Although mitotic figures were frequently present (Fig. 3), the majority of centrioles were in cells that were not in mitosis. Organ distribution of XL cells XL cells were not found in the other hematopoietic or lymphopoietic organs (liver, kidney, thymus and bone marrow) of this anuran species. XL cells did not migrate to sites of immune or non-immune inflammation such as to skin allo- or autografts or to subcutaneouns deposits of carbon. Instead, these sites contained accumulations of lymphocytes, blasts, and macrophages, many of which contained easily detectable cytoplasmic Ig. Immunohistochemical comparison of XL cells and B-lymphocytes Immunoperoxidase staining also demonstrated that a majority of the small lymphocytes and large blasts ~ithin the white pulp follicle contained cytoplasmic Ig, while only a few of the lymphocytes in the marginal zone or red pulp did so. None of the XL cells were stained by this method (Figs. 1 and 2). Histochemical comparison of XL cells, macrophages, and lymphocytes Three patterns of nonspecific esterase staining were detected on frozen sections and cytocentrifuge preparations. Macrophages, large clusters of which encircle the periphery of the marginal zone, had a very dense stain in frozen sections, and a diffuse, finely granular stain over most of their cytoplasm on cytocentrifuge preparations (Fig. 7). Most lymphocytes (70-90%) contained 1 or 2 brightly staining, punctate granules in their cytoplasm (Fig. 7), while the remainder had no stain. Macrophage, but not lymphocyte, staining was resistant to fluoride treatment. XL cells had either no or very faint staining Figure 5 XL cell with a large biolobed nucleus. A centriole with radiating micro tubules is located in the lucent cytoplasm between the nucleus and the boundary cells (B). Microvilli (arrows) of adjacent lymphocytes invaginate into the XL cell surface giving the cytoplasmic bridge, which extends to the right, a segmented appearance (uranyl acetate and lead citrate; 8000X). Figures 6a and b Pseudopod-like extensions of XL cells that protrude from the white pulp through the boundary cell layer (B) into the red pulp. Microtubules extend the length of both pseudopods and a centriole is at the origin of (a). A lymphocyte is traversing the boundary cell layer next to the pseudopod in (b) (uranyl acetate and lead citrate; 6000X).
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XENOPUS DENDRITIC CELLS
FIGURES 5 and 6:
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See bottom of preceding page for legends.
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XENOPUS DENDRITIC CELLS
467
in frozen sections and no staining in cytocentrifuge preparations (Fig. 7). Surface receptor studies Preliminary attempts to demonstrate surface receptors for erythrocytes or for the Fc portion of IgG or M were unsuccessful. However, this may reflect a less than optimal selection of reagents (rabbit IgG and M, and ~heep erythrocytes) or conditions, rather than a total absence of such receptors from the XL cell surface. Carbon localization in relation to XL cells and macrophages Both i.v. and S.C. injected colloidal carbon was initially deposited in large quantities in the spleen just peripheral to the marginal zone in the macrophage-rich region of the red pulp and none was directly extravasated from the blood vessels into the white pulp (Fig. 2). Some carbon was phagocytosed and retained as aggregates in the red pulp macrophages for at least 2 days. However, some carbon particles were transported within I hour into the white pulp. Initially, most of the carbon particles that had penetrated into the marginal zone and white pulp, were localized on the surfaces of the XL cell bodies and their pseudopods (Fig. 8a). After 2 days, aggregates of carbon were within the few macrophages located centrally in the white pulp (Fig. 8b). Antigen localization in relation to XL cells Similarly, human IgG was initially deposited in a finely granular pattern between the cells in the peripheral marginal zone. Granules were also evident on large stellate cells located in the periphery of the white pulp follicle. On some of these cells, which had the same distribution, size, and shape of XL cells, the granules adhered to cellular extensions that reached through the boundary cell layer into the marginal zone (Fig. 9). Figure 7 Composite picture of cytocentrifuge preparations stained for nonspecific esterase. Macrophages (M) have diffuse cytoplasmic staining, lymphocytes (L) have punctate staining and XL cells no staining. Sheep erythrocytes in the background were markers for an IgG Fc binding assay; XL cells did not convincingly bind these markers (750X). figure 8a XL cells 1 hour after dorsal lymph sac injection of colloidal carbon. Carbon particles are on the surfaces of the pseudopod and cell bodies of the XL cells (IOOOX). Figure 8b XL cells 2 days after dorsal lymph sac injection of colloidal carbon. Only occasional small carbon particles are sill associated with the surface of the XL cell while adjacent macrophages are filled with large carbon particles (1,200X). figures 9a and b Indirect immunofluorescent stain for human IgG 4 hours after injection. Granular staining is scattered in the peripheral marginal zone and concentrated on large cells in the periphery of the follicle (a, 20X; b, 40X).
468
XENOPUS DENDRITIC CELLS
FIGURES 7-9:
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See bottom of preceding page for legends.
priminent
B-lymphocytes with extensions to T-Iymphocytes
contacts
d
c
b
See ref (21)
See ref (20)
See ref (19)
See ref (3)
independent
Thymus dependency
a
no
Predominantly phagocytic
weak-none
present
long extensions
non-specific esterase
present
present
micro tubules
?
no
weak diffuse
B-Iymphocytes
present
?
prominent
centrioles
few
few
lysosomes
Cytoplasm
clear
not prominent
hyperlobulated
Nucleus
clear
lobulated
splenic follicles
Location
Nucleoli
splenic follicles
XL cell
Follicular a b dendritic cells ,
independent
no d
focal positive
T-Iymphocytes
present
present
present
few
clear
not prominent
lobulated
T-cell areas
Interdigitating cells c
independent
no
positive
epidermal cells + lymphocytes (T?)
present
present
?
variable
clear
not prominent
lobulated
skin
Langerhans cells
Characteristics of XL cells, follicular dendritic cells, interdigitating cells, and Langerhans cells
Characteristic
TABLE I.
~
1.0
'"
til
~
n n
H
~
~
t:I
~
til
~
o '"d c:::
w
~
VI
......
(f
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XENOPUS DENDRITIC CELLS
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DISCUSSION Several observations suggest that XL cells are distinct from both lymphoid cells and macrophages. Manning (1) has reported that the histogenesis of the XL cells is not affected by thymectomy at early stages of lymphoid organ maturation. Yet, thymectomy at this stage significantly depletes the lymphoid population in the marginal zone and inhibits manifestations of T~lymphocyte function (I,ll). Unlike the B-lymphocytes in the white pulp surrounding them. the XL cells contain no demonstrable cytoplasmic Ig. In addition, XL cells fail to stain for the non-specific esterase either in the punctate pattern found in most lymphoid cells or in a diffuse pattern characteristic of macrophages in this species. In further distinction to macrophages, they do not phagocytose and retain large quantities of colloidal carbon (8). Thus, XL cells appear to be distinct from cells of both the lymphoid and macrophage lineage. XL cells have some morphological similarities to splenic follicular dendritic cells in mammals (Table 1). Both are large cells with clear cytoplasm containing few lysosomes; both have long cytoplasmic extensions in intimate contact with adjacent B-lymphocytes of the splenic follicles. Although mammalian dendritic cells have large lobulated nuclei, the hyperlobulated nuclei and prominent nucleoli of the XL cells more closely resemble those of ReedSternberg type giant cells found in malignant and reactive lymphoid tissues of humans (12-14), some of which may be derived from dendritic cells (15). XL cells also share the characteristic function of dendritic cells, namely trapping foreign material on the surface of their cytoplasmic extensions for potential presentation to adjacent lymphocytes (3,4). Additionally, these XL cells may be involved in transportating carbon and human IgG from their initial site of deposition in the red pulp through the marginal zone and into the white pulp. Although lymphoid cells are thought to be responsible for antigen redistribution in mammalian spleens (16), dendritic cells may serve this function in lower animals. In chickens, for example, dendritic-type cells trap antigen on their surface in the peripheral white pulp and then migrate along arteriolar sheaths to initiate germinal center formation in the central white pulp (17). Our working hypothesis to explain the present observations on the XL cells is diagrammed in Fig. 10. Antigens and other materials are extravasated in the macrophage-rich region of the red pulp encircling the marginal zone. Large particles are phagocytosed by macrophages. Small particles and macrophage-processed material may be trapped on the surface of the XL cell, pseudopods and then transported through the marginal zone into the white pulp. It is possible that the elaborate microtubular system that is anchored by the large centriole and extends the length of the pseudopod, may be involved in transporting the trapped material. T-lymphocytes may assist XL cells in trapping and transporting antigen from the marginal zone to the white pulp. This would correlate both with the movement of lymphocytes along the XL cell pseudopods (Fig. 6b) and the inhibition of antigen retention caused by early thymectomy (10). Once within the white pulp, the material may be temporarily retained in the invaginated surfaces of the XL cell that interdigitate with the microvilli of adjacent lymphocytes (10). Possibly when an appropriate antigenic stimulus is presented to the adjacent lymphocytes, they divide and transform. This was not observed with the colloidal carbon tracer, but does occur following skin grafting (Baldwin and Cohen, unpublished observations) or immunization with sheep erythrocytes. In these situations, mitotic activity was largely confined to lymphocytes adjacent to the XL cells in the peripheral white pulp but was absent from the lymphocytes in the central white pulp (18). Xhus, by serving to trap and transport antigen, the XL cell may function as a bridge between T-cells in
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XENOPUS DENDRITIC CELLS
471
the marginal zone and B-ce11s in the white pulp follicle. To determine whether XL cells can perform the functions hypothesized in this model, we have isolated XL cells and have begun to determine their ability to actively trap and transport antigenic material in vitro.
Figure 10 Diagram of Xenopus splenic white pulp follicle following an i.v. injection of carbon particles. Carbon is extravasated in the red pulp (1) where it is phagocytosed by macrophages (2) or trapped on XL cell pseudopods (3). Trapped material is drawn towards the white pulp follicle being presented to T-1ymphocytes in the 'margina1 zone (4) and B-1ymphocytes in the follicle (5). Finally, it is phagocytosed by macrophages in the central white pulp (6).
ACKNOWLEDGEMENTS The authors are grateful to Dr. Jonathan Said and Mrs. Christine Rudo1fi for their aid in interpreting and preparing the electronmicrographs; to Dr. Geraldine Pinkus and Ms. Janet McLeod for their help in interpreting and preparing the naphthyl acetate esterase stains; to Paul Bleicher for his generous gift of rabbit anti-Xenopus Ig; and to the Hubrecht Laboratory, Utrecht, The Netherlands, for supplying some of the animals used in these studies.
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XENOPUS DENDRITIC CELLS
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REFERENCES 1.
Manning, M.J. The effect of early thymectomy on histogenesis of the lymphoid organs in Xenopus Zaevis. J. EmbryoZ. expo Morph. 26, 219, 1971.
2.
Sterba, G. Untersuchungen an der Milts des Krallenfrosches (Xenopus Zaevis Daudin). Gegenbaurs. Morph. Jahrbuah 90, 221, 1950.
3.
Chen, L.L., Adams, J.C., Steinman, R.M. Anatomy of germinal centers in mouse spleen, with special reference to "follicular dendritic cells". J. CeZZ BioZ. 77, 148, 1978.
4.
Nossal, G.J.V., Abbot, A., Mitchell, J., Lumnes, Z. Antigens in immunity. XV. Ultrastructural features of antigen capture in primary and secondary lymphoid follicles. J. expo Med. 127, 277, 1968.
5.
Karnovsky, M.J. A formaldehyde glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. CeZZ BioZ. 27, 137a, 1965.
6.
Pinkus, G.S., Said, J.W. Specific identification of intracellular immunoglobulin in paraffin sections of multiple myeloma and macroglobulinemia using an immunoperoxidase technique. Am. J. PathoZ. 87, 47, 1977.
7.
Pinkus, G.S., Hargreaves, H.K., McLeod, J.A., Nadler, L.M., Rosenthal, D.S., Said, S.W. Naphthyl acetate esterase activity - a cytochemical marker for T lymphocytes; correlation with immunologic studies of normal tissues, lymphocytic leukemias, non-Hodgkin's lymphomas, Hodgkin's disease, and other lymphoproliferative disorders. Am. J. PathoZ. 97, 17, 1979.
8.
Turner, R.J. The functional development of the reticuloendothelial system in the toad, Xenopus Zaevis (Daudin). J. Exp. ZooZ. 170, 467, 1969.
9.
Collie, M.H.
Zaevis.
The location of soluble antigen in the spleen of Xenopus
Experientia 30, 1205, 1974.
10.
Horton, J.D., Manning, M.J. Effect of early thymectomy on the cellular changes occurring in the spleen of the clawed toad following administration of soluble antigen. ImmunoZogy 26, 797, 1974.
11.
Cohen, N., Turpen, J.B. Experimental analysis of lymphocyte ontogeny and differentiation in an amphibian model system. In: BioZogicaZ Basis.of Immunodeficiency. (E. W. Gelfand and H.-M. Dosch (Eds.) Raven Press, New York, 1980, p. 25.
12.
Mann, R.B., Jaff, E.S., Beard, C.W. Malignant lymphomas. A conceptual understanding of morphologic diversity. Am. J. PathoZ. 94, 103, 1979.
13.
Carr, I. disease.
14.
Glick, A.D., Leech, J.H., Flexner, J.M., Collins, R.D. Ultrastructural study of Reed-Sternberg cells: comparison with transformed lymphocytes and histocytes. Am. J. PathoZ. 85, 195, 1976.
15.
Curran, R.C., Jones, E.L. Hodgkin's disease: an immuno-histochemical and histological study. J. PathoZ. 125, 39, 1978.
The ultractructure of the abnormal reticulum cells in Hodgkin's
J. PathoZ. lIS, 45, 1975.
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16.
Veerman, A.J.P., Rooijen, N.V. Lymphocyte capping and lymphocyte migration as associated events in the in vitro antigen trapping process. An electromicroscopic autoradiographic study in the spleen of mice. CeZZ Tiss. Res. 161, 211, 1975.
17.
White, R.G., Henderson, D.C., Eslami, M.B., Nielsen, K.H. Localization of a protein antigen in the chicken spleen: Effects of various manipulative procedures on the morphogenesis of the germinal center. ImmunoZogy 28, 1, 1975.
18.
Turner, R.J., Manning, M.J. Responses of the toad, Xenopus Zaevis, to circulating antigens. I. Cellular changes in the spleen. J. Exp. ZooZ. 183, 21, 1972.
19.
Eike1enboom, P. Characterization of non-lymphoid cells in the white pulp of the mouse spleen: An in vivo and in vitro study. CeZZ Tiss. Res. 195, 445, 1978.
20.
Drexhage, H.A., Mullink, H., Groot, J. de, Clarke, J., Balfour, B.M. A study of cells present in peripheral lymph of pigs with special reference to a type of cell resembling the Langerhans cell. CeZZ Tiss. Res. 202, 407, 1979.
21.
Fossum, S., Smith, M.E., Bell, E.B., Ford, W.L. The architecture of rat lymph nodes. III. The lymph nodes and lymph-borne cells of the congenitally athymic nude rat (rnu). Scand. J. ImmunoZ. 12, 421, 1980.
-------------------------
Received for Publication September 1980 Accepted for Publication January 1981