Ontogeny of Human Fetal Lymph Nodes'n2 R A Y M O N D P. BAILEY AND LEON WEISS Department of A n a t o m y a n d Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsyluania 15261 and Department of A n a t o m y , School of Medicine, T h e J o h n s Hopkins University, Baltimore, Maryland 21205
ABSTRACT Developing lymph nodes from 30 human embryos and fetuses with crown-rump lengths (CRL) of 18 mm (5.6 wk) to 245 mm (26 wk) were examined by light microscopy. The nodes were embedded in araldite, and the sections examined were approximately 1 p in thickness. The development of nodes was divided into three stages: 1. the lymphatic plexus and connective tissue invagination (30 mm to 67 mm CRL]; 2. the early fetal lymph node (43 mm to 95 mm CRL); and 3. the late fetal lymph node (CRL greater than 75 mm). The lymphatic plexus was formed by connective tissue invaginations and bridges which divided a lymph sac into a meshwork of channels and spaces. Connective tissue invaginations were endothelially-lined and were surrounded by lymphatic space. Reticular cells, macrophages, and blood vessels were found in these invaginations. Early fetal lymph nodes were formed from invaginations when the cellular density and lymphocyte content increased. The lymphatic space surrounding the early node was the developing subcapsular sinus. With further development the early node became packed with lymphocytes, increasing the cellular density and size of the node. The connective tissue surrounding the subcapsular sinus condensed to form the capsule. Afferent lymphatic vessels pierced the capsule. Capillaries, veins, postcapillary venules, and occasional arteries were found in early and late nodes.
This paper constitutes a light microscopic description of the histogenesis of human fetal lymph nodes, describing stages heretofore not reported. It is a supplement to light microscopic studies of developing mammalian lymph nodes (Gulland, 1894; Lewis, '09; Sabin, '09, '12; Downey, '22; Wischnewezkaja, '32; Ackerman, '67; Soderstrom, '67; Hostetler and Ackerman, '69; Bryant and Shifrine, '72) and adult lymph nodes (Sainte-Marie and Sin, '70). The cell population of the nodes is described, and inferences are made on the development of the capsule, sinuses, trabeculae, and parenchyma. The use, in this investigation, of sections one micron in thickness for light microscopic observations has helped to clarify much of the work previously done on the development of mammalian lymph nodes. M A T E R I A L S AND METHODS
Human embryos and fetuses were obtained from abdominal hysterectomies, hysterotomies, or therapeutic abortions. AM. J. ANAT., 142: 15-28.
Twenty-seven embryos and fetuses were used in this study, and their crown-rump lengths (CRL) ranged from 18 m m to 245 mm. Their ages, determined by correlating CRL with the "age vs. CRL" charts of Streeter ('20) ranged from 5.6 weeks to 26 weeks. An attempt was made to recover most of the tissues which are in either the lymphoid or the hemopoietic compartment, including lymph nodes, thymus, spleen, bone marrow, liver, ileum (Peyer's patches), and appendix. Not all of these tissues were excised from each embryo or fetus. The excised tissues were fixed by immersion in Karnovsky's glutaraldehydeparaformaldehyde mixture for two to five hours at 4°C. Tissues to be embedded in araldite were cut into small pieces (1-2 1 Supported by U. S. Public Health Service Grants AM-11381, GM00286, and General Research Grant
RR05416-12. 2Parts of this work are included in a dissertation submitted in 1973 in partial fulfillment of the re-
quirements for the Doctor of Philosophy degree at the Johns Hopkins University School of Medicine, Baltimore, Maryland.
15
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RAYMOND P. BAILEY AND LEON WEISS
(fig. 2), indicate the direction of lymph flow, and thereby differentiate afferent and efferent lymphatic vessels. The channels and spaces of the plexus also give rise to afferent and efferent lymphatic vessels, and to the subcapsular sinus that forms around the early node (figs. 3, 4, 5, 9, 10). A few macrophages and lymphocytes are found in the lumina of the afferent and efferent lymphatic vessels and in the channels and spaces of the lymphatic plexus (figs. 2, 3). Connective tissue invaginations, the anlagen of lymph nodal parenchyma, bulge into the lumina of the spaces and channels of the plexus (figs. 1, 3, 9). Invaginations are covered by an endothelium (fig. 3 ) and an underlying basement membrane (fig. 9 ) . Connective tissue invaginations contain macrophages, fibroblasts, reticular cells, capillaries, vascular loops, and abundant intercellular ground substance. Occasional small lymphocytes occur in the connective tissue invaginations, but they are uncommon and are observed in the blood vascular system before they are observed in the OBSERVATIONS extravascular areas. There are more cells Lymphatic plexus and connective in the invaginations than in the surroundtissue inwaginations ing connective tissue (fig. 3). In the forMammalian lymph nodes develop in mation of a connective tissue invagination, regions occupied by lymph sacs (Sabin, the locus of invagination of a lymph chan'09), broad thin-walled spaces that lie nel wall becomes the hilus, is a connective close to large veins. It was found that dur- tissue stalk that contains the blood vessels ing the initial development of human and nerves supplying the invagination, and lymph nodes the lymph sac was trans- joins the invagination to the wall of the formed into a system of anastomosing lymph channel (figs. 1, 9 ) . channels called a lymphatic plexus (fig 1), Lymphatic plexuses and connective tissue The connective tissue of the wall of a sac invaginations are found in human eminvaginates and bridges the sac, and the bryos and fetuses 30 mm to 67 mm CRL. resulting network of interlacing channels Early fetal l y m p h nodes formed by these invaginations and bridges is the lymphatic plexus. The channels and Several distinctive characteristics mark spaces of the plexus vary in size, and serial the early node. It projects into the lumen sections show that they anastomose freely. of a lymph channel or space (figs. 4, 5, 10) Each plexus gives rise to several lymph and lacks a capsule. The early node has an nodes. The lumina and walls of the chan- oval shape, with a diameter of 50 to 300 p . nels and spaces of the plexus, and the con- The long axis of the node is oriented along nective tissue invaginations and bridges the long axis of a lymph channel (figs. that partition the plexus contribute to the 4, 5). The hilus projects out from the nodal formation of the node. The lymphatic parenchyma and connects it to the survessels that lead into the plexus become rounding connective tissue (figs. 4, 5, 10). the afferent lymphatic vessels to the node, Like an invagination, an early node is and those that lead away from the plexus primarily a cellular reticulum interspersed become the efferent lymphatic vessels. with free cells and blood vessels. However, Occasional valves, present in these vessels early nodes differ from invaginations in
mm2) while immersed in fixative. After fixation, the tissues were rinsed overnight in 0.05 M cacodylate buffer (pH 7.1) and then post-fixed in 1% osmium tetroxide for two hours at room temperature. The tissues were then rinsed again with 0.5 M cacodylate buffer, dehydrated in ethanol, rinsed in propylene oxide, and embedded in araldite (durcupan ACM). Thin sections ( 1 p ) were cut on a Porter-Blum ultramicrotome, mounted on glass slides, stained with toluidine blue, and examined by light microscopy. Tissues to be embedded in paraffin were dehydrated in ethanol after fixation, then cleared overnight in methyl benzoate and parloiden, washed twice in xylene and infiltrated with three changes in paraffin. The embedded tissues were sectioned at six sL, fixed to glass slides, and stained either with hematoxylin and eosin or by silver impregnation of the reticulum. The stained sections were examined by light microscopy.
ONTOGENY OF HUMAN FETAL LYMPH NODES
that they possess more extracellular reticulum (fig. l o ) , more lymphocytes and more blood vessels (figs. 4, 5, 6). The space between the early node and the wall of the lymph channel is the forming subcapsular sinus, 20 to 100 wide (figs. 4, 5). The subcapsular sinus has a parietal and a visceral cellular lining (figs. 4, 5, 6). The visceral lining covers the early node; the parietal lining forms the inner surface of the future capsule. A basement membrane, visualized by silver impregnation, underlies the endothelium of both these linings (fig. 10). The visceral endothelium, originally the lining of a lymph channel, has processes or branches that traverse the sinus (figs. 4, 5, 10). Sinus reticular cells appear in the subcapsular sinus and interconnect with one another and with the endothelium. Small lymphocytes and macrophages are found in the subcapsular sinus of the early node (figs. 4, 5). The first lymphocytes to appear in the early node are small, 5 to 8 in diameter (fig. 6). In addition to the capillaries and vascular loops found in the early node, blood vessels whose lumina contain lymphocytes are common (fig. 6 ) ; these blood vessels may be post-capillary venules (PCV). During the course of development of early nodes, the cellular density increases due to an increase in the number of leucocytes, most of which are small lymphocytes. However, some medium-sized lymphocytes and granulocytes appear late in the development of an early node (fig. 6). It is presumed that lymph flows through the channels and spaces of the plexus. When an early node is present in a channel, one end of the channel can be considered afferent; the other end, efferent. Consequently, from the first appearance of the node, the afferent vessel, the subcapsular sinus, and the efferent vessel are segments of a continuous channel. Early fetal lymph nodes are found in human fetuses 43 mm to 95 mm CRL.
Late fetal l y m p h nodes The transition from early fetal lymph node to late fetal lymph node is gradual. Because of this gradual transition, an intermediate stage of nodal development cannot be described. Early nodes become filled with small lymphocytes, increasing
17
the cellular density and volume of the node. As the node increases in size, the parietal endothelium of the subcapsular sinus and the underlying connective tissue are pushed out and away from the original locus of invagination. In the late node the parenchyma is no longer located in the lumen of a lymph channel but rather is enclosed in an outpocketing of the lymph channel (figs. 7, 11). The connective tissue that surrounds the outpocketing is the capsule of the node. With further development the cellular and extracellular density of the capsule increases, and additional condensation of the surrounding connective tissue causes the capsule to thicken. Afferent lymphatic vessels pierce the capsule at several points (figs. 7, 12). An efferent lymphatic vessel(s) is located at one end of the late node, near the region of the hilus (figs. 7, 11). The subcapsular sinus of the late node is 20 to 100 wide (figs. 7, 11, 12), much less than the diameter of the nodal parenchyma (approximately 300 /.). The inner lining of the capsule, the parietal endothelium of the sinus, becomes irregular (figs. 11, 12). Well developed trabeculae are sparse. When present, they are endothelial-lined, connective tissue bands that span the subcapsular sinus, joining the capsule to the nodal parenchyma. Some trabeculae contain veins (fig. 12). Radial sinuses, representing branches of the subcapsular sinus, are occasionally evident (fig. 12). In the late node, the hilus appears as a stalk of connective tissue (figs. 7, S ) , traversed by a plexus of lymphatic vessels or spaces (fig. 8). A lymphatic space surrounds the hilus. The efferent lymphatic drainage of the late node is through this lymphatic space. Blood vessels and nerves are prominent features of the hilus of late nodes (fig. 8). Macrophages, lymphocytes, and reticular cells are present but are less concentrated than in the parenchyma. There is little cortico-medullary differentiation in the late node. The parenchyma contains primarily lymphocytes, interspersed with reticular cells, macrophages, and blood vessels. PCV are present, being the more prevalent of the non-capillary blood vessels (figs. 7, 12, 13). Neither plasma cells nor primary and secondary
18
RAYMOND P. BAILEY AND LEON WEISS
nodules are found. The extracellular reticulum of the subcapsular sinus and nodal parenchyma is well developed (fig. 11) . Late fetal lymph nodes are found in fetuses measuring more than 75mm CRL. DISCUSSION
Mammalian lymph nodes develop in regions occupied by lymph sacs (Sabin, '09), and lymph sacs are transformed into lymphatic plexuses (Lewis, '09). We have divided the ontogeny of human fetal lymph nodes into three stages: ( 1 ) the development of a lymphatic plexus (30 to 67 m m CRL); ( 2 ) the formation of early lymph nodes ( 4 3 to 95 m m CRL); and ( 3 ) the formation of late fetal lymph nodes (CRL greater than 75 m m ) . Lymph nodes develop in different regions of the fetus at different ages, and in any specific region, lymph nodes are found i n various stages of development at any given fetal age. Thus in a single fetus, lymph nodes i n various stages of development are observed. There is little information available regarding the development of the nodal medulla. Few arteries or arterioles are found in the fetal lymph nodes before the formation of the medulla. When the medullary cords do appear they contain arterioles and are surrounded by medullary sinuses (Sainte-Marie and Sin, '70). In earlier reports, it has been concluded that the medullary and radial sinuses form a s the result of sinus growth away from the hilus and toward the parenchyma, with the sinuses etching their way through the parenchyma (Gulland, 1894; Rothermel, '29; Bloom, '38). We think it more likely, however, that late in nodal development there is a branching and growth of the arterioles to the node. The arteriolar branches extend into the connective tissue that surrounds the plexus of lymphatic vessels within the hilus. With further growth, each arteriole grows out from the hilus toward the parenchyma. These arterioles are enclosed in a sheath of lymphoid tissue, which in turn is covered by the lymphatic vessels and spaces that are eventually identified as medullary sinuses. The late development of many arterioles, medullary cords, and medullary sinuses can be explained in this way. None of the theories proposed thus far
adequately explains the formation of trabeculae (Gulland, 1894; Rothermel, '29). Trabeculae are among the earliest structures of the fetal node to form. They are not late acquisitions but they do become more evident with development. In early nodal development the trabeculae join the forming capsule to the nodal parenchyma. As nodal development proceeds, particularly during the late fetal and postnatal periods, the cortex differentiates and rapidly increases in bulk. As the cortex proliferates, it increases in volume and acquires nodules (postnatally) thereby forcing the subcapsular sinus and capsule to expand. Trabeculae seem to be anchored to the subcortical parenchyma, and the cortex anpears to grow around the trabeculae leaving a space between the trabeculae and the enlarging cortex. This para-trabecular space becomes a radial or trabecular sinus. In this fashion, the trabecular sinuses develop from the subcapsular sinus. 1,ITERATURE CITED Ackerman, G . A. 1967 Developmental relationship between the appearance of lymphocytes and lymphoietic activity i n the thymus and lymph nodes of the fetai cat. Anat. Rec., 158: 387-400. Bloom, W. 1938 Lymphatic tissue: Lymphatic organs. In: Handbook o f Hematology. Vol. 2. 13. Downey Hoeber, ed. New York, pp. 14271462. Bryant, B. J., and M. Shifrine 1972 Histogenesis of lymph nodes during development of h e dog. J. Reticulo-endothelial SOC., 12: 96107. Downey, H. 1922 The structure and origin o f the lymph sinuses of mammalian lymph nodes and their relations to endothelium and reticulum. Hematologica, 33: 431-468. Gulland, G. L. 1894 The development of lymphatic glands, J. Path. Bact., 2: 447-485. Hostetler, J. R., and G. A. Ackerman 1969 Lyniphopoiesis and lymph node histogenesis i n the embrycnic and neonatal rabbit. Am. J. Anat., 124: 57-75. Lewis, F. T. 1909 The first lymph glands in rabbits and human embryos. Anat. Rec., 3: 341-353. Rothermel, J. E. 1929 A developmental study o f the medial retropharyngeal lymphatic node of the calf (Bos taurus). Am. J. Anat., 43: 461496. Sabin, F. R. 1909 On the development of the lymphatic system in human embryos with a consideration of the morphology of the system as a whole. Am. J. Anat., 9: 43-91. 1912 The development of the lymphatic system. In: Manual of Human Embry-
ONTOGENY OF HUMAN FETAL LYMPH NODES ology. Vol. 11. F. Keibel and Franklin P. Mall, eds. J. B. Lippincott Co., pp. 709-744. Sainte-Marie, G., and Y. M. Sin 1970 The Lymph Node: Structure and possible function during the immune response. In: Regulation of Hematopoiesis. Vol. 11. A. S . Gordon, ed. Appleton-Century-Crofts, New York, pp. 1339-1382. Soderstrom, N. 1967 Postcapillary venules as basic structural units in the development of
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lymphoglandular tissue. Scand. J. Hemat., 4: 411429. Streeter, G. L. 1920 Weight, sitting height, head size, foot length, and menstrual age of the human embryo. Contribution to Embryology, Vol. xi. pp. 143-170. Wischnewezkaja, L. J. 1932 Beitrag zur Entwicklungsgeschichte der Lymphdrusen. Z. Mikr. Anat. Forsch, 3 1 : 175-192.
PLATE 1 EXPLANATION O F FIGURES
1 The lymphatic plexus is a system of anastomizing channels and spaces. Bridges and invaginations of connective tissue (arrows) partition the plexus. Some of the invaginations are anlagen of lymph nodes. Toluidine blue. 67 mm CRL. x 210.
20
2
The afferent and efferent lymphatic vessels of lymphatic plexus have valves. The valves and the vessels are lined by endothelium. Occasional nucleated cells (arrow) circulate in the lymphatic system of the young fetus. Toluidine blue. 43 mm CRL. x 550.
3
The connective tissue invagination projects into the lumen of a lymph channel. The developing subcapsular sinus (SS) is the portion of the channel that surrounds the invagination. The cellular density of the invagination is greater than that of the surrounding connective tissue, but is not as great as in early nodes (figs. 4, 5 ) . Reticular cells, fibroblasts, macrophages, occasional lymphocytes, and vascular endothelium are found i n connective tissue invaginations. The developing subcapsular sinus has an outer, or parietal lining (arrow), and an inner, or visceral lining. Toluidine blue. 47 m m CRL. X 550.
4
The early fetal lymph node is a connective tissue invagination that, in addition to reticular cells, macrophages, and vascular endothelial cells, possesses several lymphocytes. The blood vessels are randomly distributed throughout the nodal parenchyma. Note the vascular loops ( V ) and capillaries (C). Many of the red cells are nucleated. The node is not encapsulated. The developing subcapsular sinus (SS) surrounds the early node. Sinus reticular cells (arrows) span the sinus. The hilus ( H ) projects out from the nodal parenchyma. Toluidine blue. 57 mm CRL. x 350.
ONTOGENY OF HUMAN FETAL LYMPH NODES Raymond P. Bailey and Leon Weiss
PLATE 1
21
PLATE 2 EXPLANATION O F FIGURES
22
5
The cellular density of the early fetal lymph node increases with development. There are fewer cells in the hilus ( H ) than in the nodal parenchyma. Lymphatic vessels (LV), blood vessels, and nerves ( N ) are found in the region of the hilus. The visceral endothelium of the subcapsular sinus is more irregular than the parietal endothelium. Toluidine blue. 75 mm CRL. x 350.
6
Higher magnification of a portion of figure 5. The parenchymal of the early fetal lymph node are reticular cells ( R ) , lymphocytes macrophages ( M ) , and granulocytes ( G ) . Nerves ( N ) , arteries veins ( V ) , and capillaries ( C ) are present. Some of the veins be postcapillary venules ( P C V ) . Toluidine blue. 75 mm CRL. X
cells
(L), (A), may 875.
7
The late fetal lymph node consists of capsule ( C ) , subcapsular sinus (SS), hilus ( H ) , and parenchyma. The cellular density of the late node is greater than in the early node. The nodal blood vessels (arrows) are more common near the edge of the parenchyma. The subcapsular sinus surrounds the nodal parenchyma, and is continuous with the efferent lymphatic vessels ( E L ) . The nodal capsule is pierced by afferent lymphatics (AL). The lack of radial sinuses indicates that this late node is not advanced i n its development (fig. 12). Toluidine blue. 145 mm CRL. x 130.
8
The hilus ( H ) connects the nodal parenchyma with the surrounding connective tissue. The hilus is traversed by a plexus of lymphatic vessels (arrows). The cellular density of the hilus is less than that of the parenchyma. Arteries (A) and veins ( V ) are found in the hilus. Toluidine blue. 145 mm CRL. X 170.
ONTOGENY OF HUMAN FETAL LYMPH NODES Raymond P. Bailey and Leon Weiss
PLATE 2
23
PLATE 3 EXPLANATION O F FIGURES
9
The connective tissue invagination (CTI) is surrounded by spaces ( S ) of a lymphatic plexus. The hilus ( H ) appears as a stalk of tissue connecting the invagination with the surrounding connective tissue. The thin and coarse black lines appear to be silver-impregnated reticulum. Note the silver-impregnated basement membranes (arrows) that underlie the parietal and visceral endothelia of the developing subcapsular sinus ( S ) . Reticulum stain. 47 mm CRL. x 220.
10
The early fetal lymph node is often no larger than a connective tissue invagination (compare with fig. 9). Extracellular reticulum in the form of reticular fibers and basement membranes of blood vessels and lymphatic sinuses appear silver-impregnated. The extracellular reticulum of the surrounding connective tissue is not as extensive as in the early node. Reticulum stain. 75 mm CRL. x 220.
11 Late nodes are larger than early nodes. The reticular framework is well developed. Reticular fibers and trabeculae span the subcapsular sinus (SS). A n efferent lymphatic vessel (ELV) is shown near the hilar region. The capsule consists of densely packed reticular elements. Reticulum stain. 105 mm CRL. X 220.
24
ONTOGENY OF HUMAN FETAL LYMPH NODES Raymond P. Bailey and Leon Weiss
PLATE 3
25
PLATE 4 EXPLANATION O F FIGURES
12 The capsule ( C ) , afferent lymphatics ( A L ) , subcapsular sinus ( S S ) , trabeculae ( T ) , radial sinuses ( R S ) , and parenchyma of a late fetal lymph node are shown. The subcapsular sinus contains free cells and is traversed by reticular cells. The parenchyma contains several blood vessels. PCV are distinguished from veins (arrows) by their luminal contents. Toluidine blue. 245 mm CRL. x 340.
13 Postcapillary venules in late fetal lymph nodes are characterized by a high endothelium ( E ) , luminal lymphocytes ( L ) , and lymphocytes in diapedesis (arrows). Toluidine blue. 165 m m CRL. X 1;750.
26
ONTOGENY OF HUMAN FETAL LYMPH NODES Raymond P. Bailey and Leon Weiss
PLATE 4
27
ABSTRACT Developing lymph nodes from 30 human embryos and fetuses with crown-rump lengths (CRL) of 18 mm (5.6 wk) to 245 mm (26 wk) were examined by light microscopy. The nodes were embedded in araldite, and the sections examined were approximately 1 p in thickness. The development of nodes was divided into three stages: 1. the lymphatic plexus and connective tissue invagination (30 mm to 67 mm CRL]; 2. the early fetal lymph node (43 mm to 95 mm CRL); and 3. the late fetal lymph node (CRL greater than 75 mm). The lymphatic plexus was formed by connective tissue invaginations and bridges which divided a lymph sac into a meshwork of channels and spaces. Connective tissue invaginations were endothelially-lined and were surrounded by lymphatic space. Reticular cells, macrophages, and blood vessels were found in these invaginations. Early fetal lymph nodes were formed from invaginations when the cellular density and lymphocyte content increased. The lymphatic space surrounding the early node was the developing subcapsular sinus. With further development the early node became packed with lymphocytes, increasing the cellular density and size of the node. The connective tissue surrounding the subcapsular sinus condensed to form the capsule. Afferent lymphatic vessels pierced the capsule. Capillaries, veins, postcapillary venules, and occasional arteries were found in early and late nodes.
This paper constitutes a light microscopic description of the histogenesis of human fetal lymph nodes, describing stages heretofore not reported. It is a supplement to light microscopic studies of developing mammalian lymph nodes (Gulland, 1894; Lewis, '09; Sabin, '09, '12; Downey, '22; Wischnewezkaja, '32; Ackerman, '67; Soderstrom, '67; Hostetler and Ackerman, '69; Bryant and Shifrine, '72) and adult lymph nodes (Sainte-Marie and Sin, '70). The cell population of the nodes is described, and inferences are made on the development of the capsule, sinuses, trabeculae, and parenchyma. The use, in this investigation, of sections one micron in thickness for light microscopic observations has helped to clarify much of the work previously done on the development of mammalian lymph nodes. M A T E R I A L S AND METHODS
Human embryos and fetuses were obtained from abdominal hysterectomies, hysterotomies, or therapeutic abortions. AM. J. ANAT., 142: 15-28.
Twenty-seven embryos and fetuses were used in this study, and their crown-rump lengths (CRL) ranged from 18 m m to 245 mm. Their ages, determined by correlating CRL with the "age vs. CRL" charts of Streeter ('20) ranged from 5.6 weeks to 26 weeks. An attempt was made to recover most of the tissues which are in either the lymphoid or the hemopoietic compartment, including lymph nodes, thymus, spleen, bone marrow, liver, ileum (Peyer's patches), and appendix. Not all of these tissues were excised from each embryo or fetus. The excised tissues were fixed by immersion in Karnovsky's glutaraldehydeparaformaldehyde mixture for two to five hours at 4°C. Tissues to be embedded in araldite were cut into small pieces (1-2 1 Supported by U. S. Public Health Service Grants AM-11381, GM00286, and General Research Grant
RR05416-12. 2Parts of this work are included in a dissertation submitted in 1973 in partial fulfillment of the re-
quirements for the Doctor of Philosophy degree at the Johns Hopkins University School of Medicine, Baltimore, Maryland.
15
16
RAYMOND P. BAILEY AND LEON WEISS
(fig. 2), indicate the direction of lymph flow, and thereby differentiate afferent and efferent lymphatic vessels. The channels and spaces of the plexus also give rise to afferent and efferent lymphatic vessels, and to the subcapsular sinus that forms around the early node (figs. 3, 4, 5, 9, 10). A few macrophages and lymphocytes are found in the lumina of the afferent and efferent lymphatic vessels and in the channels and spaces of the lymphatic plexus (figs. 2, 3). Connective tissue invaginations, the anlagen of lymph nodal parenchyma, bulge into the lumina of the spaces and channels of the plexus (figs. 1, 3, 9). Invaginations are covered by an endothelium (fig. 3 ) and an underlying basement membrane (fig. 9 ) . Connective tissue invaginations contain macrophages, fibroblasts, reticular cells, capillaries, vascular loops, and abundant intercellular ground substance. Occasional small lymphocytes occur in the connective tissue invaginations, but they are uncommon and are observed in the blood vascular system before they are observed in the OBSERVATIONS extravascular areas. There are more cells Lymphatic plexus and connective in the invaginations than in the surroundtissue inwaginations ing connective tissue (fig. 3). In the forMammalian lymph nodes develop in mation of a connective tissue invagination, regions occupied by lymph sacs (Sabin, the locus of invagination of a lymph chan'09), broad thin-walled spaces that lie nel wall becomes the hilus, is a connective close to large veins. It was found that dur- tissue stalk that contains the blood vessels ing the initial development of human and nerves supplying the invagination, and lymph nodes the lymph sac was trans- joins the invagination to the wall of the formed into a system of anastomosing lymph channel (figs. 1, 9 ) . channels called a lymphatic plexus (fig 1), Lymphatic plexuses and connective tissue The connective tissue of the wall of a sac invaginations are found in human eminvaginates and bridges the sac, and the bryos and fetuses 30 mm to 67 mm CRL. resulting network of interlacing channels Early fetal l y m p h nodes formed by these invaginations and bridges is the lymphatic plexus. The channels and Several distinctive characteristics mark spaces of the plexus vary in size, and serial the early node. It projects into the lumen sections show that they anastomose freely. of a lymph channel or space (figs. 4, 5, 10) Each plexus gives rise to several lymph and lacks a capsule. The early node has an nodes. The lumina and walls of the chan- oval shape, with a diameter of 50 to 300 p . nels and spaces of the plexus, and the con- The long axis of the node is oriented along nective tissue invaginations and bridges the long axis of a lymph channel (figs. that partition the plexus contribute to the 4, 5). The hilus projects out from the nodal formation of the node. The lymphatic parenchyma and connects it to the survessels that lead into the plexus become rounding connective tissue (figs. 4, 5, 10). the afferent lymphatic vessels to the node, Like an invagination, an early node is and those that lead away from the plexus primarily a cellular reticulum interspersed become the efferent lymphatic vessels. with free cells and blood vessels. However, Occasional valves, present in these vessels early nodes differ from invaginations in
mm2) while immersed in fixative. After fixation, the tissues were rinsed overnight in 0.05 M cacodylate buffer (pH 7.1) and then post-fixed in 1% osmium tetroxide for two hours at room temperature. The tissues were then rinsed again with 0.5 M cacodylate buffer, dehydrated in ethanol, rinsed in propylene oxide, and embedded in araldite (durcupan ACM). Thin sections ( 1 p ) were cut on a Porter-Blum ultramicrotome, mounted on glass slides, stained with toluidine blue, and examined by light microscopy. Tissues to be embedded in paraffin were dehydrated in ethanol after fixation, then cleared overnight in methyl benzoate and parloiden, washed twice in xylene and infiltrated with three changes in paraffin. The embedded tissues were sectioned at six sL, fixed to glass slides, and stained either with hematoxylin and eosin or by silver impregnation of the reticulum. The stained sections were examined by light microscopy.
ONTOGENY OF HUMAN FETAL LYMPH NODES
that they possess more extracellular reticulum (fig. l o ) , more lymphocytes and more blood vessels (figs. 4, 5, 6). The space between the early node and the wall of the lymph channel is the forming subcapsular sinus, 20 to 100 wide (figs. 4, 5). The subcapsular sinus has a parietal and a visceral cellular lining (figs. 4, 5, 6). The visceral lining covers the early node; the parietal lining forms the inner surface of the future capsule. A basement membrane, visualized by silver impregnation, underlies the endothelium of both these linings (fig. 10). The visceral endothelium, originally the lining of a lymph channel, has processes or branches that traverse the sinus (figs. 4, 5, 10). Sinus reticular cells appear in the subcapsular sinus and interconnect with one another and with the endothelium. Small lymphocytes and macrophages are found in the subcapsular sinus of the early node (figs. 4, 5). The first lymphocytes to appear in the early node are small, 5 to 8 in diameter (fig. 6). In addition to the capillaries and vascular loops found in the early node, blood vessels whose lumina contain lymphocytes are common (fig. 6 ) ; these blood vessels may be post-capillary venules (PCV). During the course of development of early nodes, the cellular density increases due to an increase in the number of leucocytes, most of which are small lymphocytes. However, some medium-sized lymphocytes and granulocytes appear late in the development of an early node (fig. 6). It is presumed that lymph flows through the channels and spaces of the plexus. When an early node is present in a channel, one end of the channel can be considered afferent; the other end, efferent. Consequently, from the first appearance of the node, the afferent vessel, the subcapsular sinus, and the efferent vessel are segments of a continuous channel. Early fetal lymph nodes are found in human fetuses 43 mm to 95 mm CRL.
Late fetal l y m p h nodes The transition from early fetal lymph node to late fetal lymph node is gradual. Because of this gradual transition, an intermediate stage of nodal development cannot be described. Early nodes become filled with small lymphocytes, increasing
17
the cellular density and volume of the node. As the node increases in size, the parietal endothelium of the subcapsular sinus and the underlying connective tissue are pushed out and away from the original locus of invagination. In the late node the parenchyma is no longer located in the lumen of a lymph channel but rather is enclosed in an outpocketing of the lymph channel (figs. 7, 11). The connective tissue that surrounds the outpocketing is the capsule of the node. With further development the cellular and extracellular density of the capsule increases, and additional condensation of the surrounding connective tissue causes the capsule to thicken. Afferent lymphatic vessels pierce the capsule at several points (figs. 7, 12). An efferent lymphatic vessel(s) is located at one end of the late node, near the region of the hilus (figs. 7, 11). The subcapsular sinus of the late node is 20 to 100 wide (figs. 7, 11, 12), much less than the diameter of the nodal parenchyma (approximately 300 /.). The inner lining of the capsule, the parietal endothelium of the sinus, becomes irregular (figs. 11, 12). Well developed trabeculae are sparse. When present, they are endothelial-lined, connective tissue bands that span the subcapsular sinus, joining the capsule to the nodal parenchyma. Some trabeculae contain veins (fig. 12). Radial sinuses, representing branches of the subcapsular sinus, are occasionally evident (fig. 12). In the late node, the hilus appears as a stalk of connective tissue (figs. 7, S ) , traversed by a plexus of lymphatic vessels or spaces (fig. 8). A lymphatic space surrounds the hilus. The efferent lymphatic drainage of the late node is through this lymphatic space. Blood vessels and nerves are prominent features of the hilus of late nodes (fig. 8). Macrophages, lymphocytes, and reticular cells are present but are less concentrated than in the parenchyma. There is little cortico-medullary differentiation in the late node. The parenchyma contains primarily lymphocytes, interspersed with reticular cells, macrophages, and blood vessels. PCV are present, being the more prevalent of the non-capillary blood vessels (figs. 7, 12, 13). Neither plasma cells nor primary and secondary
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RAYMOND P. BAILEY AND LEON WEISS
nodules are found. The extracellular reticulum of the subcapsular sinus and nodal parenchyma is well developed (fig. 11) . Late fetal lymph nodes are found in fetuses measuring more than 75mm CRL. DISCUSSION
Mammalian lymph nodes develop in regions occupied by lymph sacs (Sabin, '09), and lymph sacs are transformed into lymphatic plexuses (Lewis, '09). We have divided the ontogeny of human fetal lymph nodes into three stages: ( 1 ) the development of a lymphatic plexus (30 to 67 m m CRL); ( 2 ) the formation of early lymph nodes ( 4 3 to 95 m m CRL); and ( 3 ) the formation of late fetal lymph nodes (CRL greater than 75 m m ) . Lymph nodes develop in different regions of the fetus at different ages, and in any specific region, lymph nodes are found i n various stages of development at any given fetal age. Thus in a single fetus, lymph nodes i n various stages of development are observed. There is little information available regarding the development of the nodal medulla. Few arteries or arterioles are found in the fetal lymph nodes before the formation of the medulla. When the medullary cords do appear they contain arterioles and are surrounded by medullary sinuses (Sainte-Marie and Sin, '70). In earlier reports, it has been concluded that the medullary and radial sinuses form a s the result of sinus growth away from the hilus and toward the parenchyma, with the sinuses etching their way through the parenchyma (Gulland, 1894; Rothermel, '29; Bloom, '38). We think it more likely, however, that late in nodal development there is a branching and growth of the arterioles to the node. The arteriolar branches extend into the connective tissue that surrounds the plexus of lymphatic vessels within the hilus. With further growth, each arteriole grows out from the hilus toward the parenchyma. These arterioles are enclosed in a sheath of lymphoid tissue, which in turn is covered by the lymphatic vessels and spaces that are eventually identified as medullary sinuses. The late development of many arterioles, medullary cords, and medullary sinuses can be explained in this way. None of the theories proposed thus far
adequately explains the formation of trabeculae (Gulland, 1894; Rothermel, '29). Trabeculae are among the earliest structures of the fetal node to form. They are not late acquisitions but they do become more evident with development. In early nodal development the trabeculae join the forming capsule to the nodal parenchyma. As nodal development proceeds, particularly during the late fetal and postnatal periods, the cortex differentiates and rapidly increases in bulk. As the cortex proliferates, it increases in volume and acquires nodules (postnatally) thereby forcing the subcapsular sinus and capsule to expand. Trabeculae seem to be anchored to the subcortical parenchyma, and the cortex anpears to grow around the trabeculae leaving a space between the trabeculae and the enlarging cortex. This para-trabecular space becomes a radial or trabecular sinus. In this fashion, the trabecular sinuses develop from the subcapsular sinus. 1,ITERATURE CITED Ackerman, G . A. 1967 Developmental relationship between the appearance of lymphocytes and lymphoietic activity i n the thymus and lymph nodes of the fetai cat. Anat. Rec., 158: 387-400. Bloom, W. 1938 Lymphatic tissue: Lymphatic organs. In: Handbook o f Hematology. Vol. 2. 13. Downey Hoeber, ed. New York, pp. 14271462. Bryant, B. J., and M. Shifrine 1972 Histogenesis of lymph nodes during development of h e dog. J. Reticulo-endothelial SOC., 12: 96107. Downey, H. 1922 The structure and origin o f the lymph sinuses of mammalian lymph nodes and their relations to endothelium and reticulum. Hematologica, 33: 431-468. Gulland, G. L. 1894 The development of lymphatic glands, J. Path. Bact., 2: 447-485. Hostetler, J. R., and G. A. Ackerman 1969 Lyniphopoiesis and lymph node histogenesis i n the embrycnic and neonatal rabbit. Am. J. Anat., 124: 57-75. Lewis, F. T. 1909 The first lymph glands in rabbits and human embryos. Anat. Rec., 3: 341-353. Rothermel, J. E. 1929 A developmental study o f the medial retropharyngeal lymphatic node of the calf (Bos taurus). Am. J. Anat., 43: 461496. Sabin, F. R. 1909 On the development of the lymphatic system in human embryos with a consideration of the morphology of the system as a whole. Am. J. Anat., 9: 43-91. 1912 The development of the lymphatic system. In: Manual of Human Embry-
ONTOGENY OF HUMAN FETAL LYMPH NODES ology. Vol. 11. F. Keibel and Franklin P. Mall, eds. J. B. Lippincott Co., pp. 709-744. Sainte-Marie, G., and Y. M. Sin 1970 The Lymph Node: Structure and possible function during the immune response. In: Regulation of Hematopoiesis. Vol. 11. A. S . Gordon, ed. Appleton-Century-Crofts, New York, pp. 1339-1382. Soderstrom, N. 1967 Postcapillary venules as basic structural units in the development of
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lymphoglandular tissue. Scand. J. Hemat., 4: 411429. Streeter, G. L. 1920 Weight, sitting height, head size, foot length, and menstrual age of the human embryo. Contribution to Embryology, Vol. xi. pp. 143-170. Wischnewezkaja, L. J. 1932 Beitrag zur Entwicklungsgeschichte der Lymphdrusen. Z. Mikr. Anat. Forsch, 3 1 : 175-192.
PLATE 1 EXPLANATION O F FIGURES
1 The lymphatic plexus is a system of anastomizing channels and spaces. Bridges and invaginations of connective tissue (arrows) partition the plexus. Some of the invaginations are anlagen of lymph nodes. Toluidine blue. 67 mm CRL. x 210.
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2
The afferent and efferent lymphatic vessels of lymphatic plexus have valves. The valves and the vessels are lined by endothelium. Occasional nucleated cells (arrow) circulate in the lymphatic system of the young fetus. Toluidine blue. 43 mm CRL. x 550.
3
The connective tissue invagination projects into the lumen of a lymph channel. The developing subcapsular sinus (SS) is the portion of the channel that surrounds the invagination. The cellular density of the invagination is greater than that of the surrounding connective tissue, but is not as great as in early nodes (figs. 4, 5 ) . Reticular cells, fibroblasts, macrophages, occasional lymphocytes, and vascular endothelium are found i n connective tissue invaginations. The developing subcapsular sinus has an outer, or parietal lining (arrow), and an inner, or visceral lining. Toluidine blue. 47 m m CRL. X 550.
4
The early fetal lymph node is a connective tissue invagination that, in addition to reticular cells, macrophages, and vascular endothelial cells, possesses several lymphocytes. The blood vessels are randomly distributed throughout the nodal parenchyma. Note the vascular loops ( V ) and capillaries (C). Many of the red cells are nucleated. The node is not encapsulated. The developing subcapsular sinus (SS) surrounds the early node. Sinus reticular cells (arrows) span the sinus. The hilus ( H ) projects out from the nodal parenchyma. Toluidine blue. 57 mm CRL. x 350.
ONTOGENY OF HUMAN FETAL LYMPH NODES Raymond P. Bailey and Leon Weiss
PLATE 1
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PLATE 2 EXPLANATION O F FIGURES
22
5
The cellular density of the early fetal lymph node increases with development. There are fewer cells in the hilus ( H ) than in the nodal parenchyma. Lymphatic vessels (LV), blood vessels, and nerves ( N ) are found in the region of the hilus. The visceral endothelium of the subcapsular sinus is more irregular than the parietal endothelium. Toluidine blue. 75 mm CRL. x 350.
6
Higher magnification of a portion of figure 5. The parenchymal of the early fetal lymph node are reticular cells ( R ) , lymphocytes macrophages ( M ) , and granulocytes ( G ) . Nerves ( N ) , arteries veins ( V ) , and capillaries ( C ) are present. Some of the veins be postcapillary venules ( P C V ) . Toluidine blue. 75 mm CRL. X
cells
(L), (A), may 875.
7
The late fetal lymph node consists of capsule ( C ) , subcapsular sinus (SS), hilus ( H ) , and parenchyma. The cellular density of the late node is greater than in the early node. The nodal blood vessels (arrows) are more common near the edge of the parenchyma. The subcapsular sinus surrounds the nodal parenchyma, and is continuous with the efferent lymphatic vessels ( E L ) . The nodal capsule is pierced by afferent lymphatics (AL). The lack of radial sinuses indicates that this late node is not advanced i n its development (fig. 12). Toluidine blue. 145 mm CRL. x 130.
8
The hilus ( H ) connects the nodal parenchyma with the surrounding connective tissue. The hilus is traversed by a plexus of lymphatic vessels (arrows). The cellular density of the hilus is less than that of the parenchyma. Arteries (A) and veins ( V ) are found in the hilus. Toluidine blue. 145 mm CRL. X 170.
ONTOGENY OF HUMAN FETAL LYMPH NODES Raymond P. Bailey and Leon Weiss
PLATE 2
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PLATE 3 EXPLANATION O F FIGURES
9
The connective tissue invagination (CTI) is surrounded by spaces ( S ) of a lymphatic plexus. The hilus ( H ) appears as a stalk of tissue connecting the invagination with the surrounding connective tissue. The thin and coarse black lines appear to be silver-impregnated reticulum. Note the silver-impregnated basement membranes (arrows) that underlie the parietal and visceral endothelia of the developing subcapsular sinus ( S ) . Reticulum stain. 47 mm CRL. x 220.
10
The early fetal lymph node is often no larger than a connective tissue invagination (compare with fig. 9). Extracellular reticulum in the form of reticular fibers and basement membranes of blood vessels and lymphatic sinuses appear silver-impregnated. The extracellular reticulum of the surrounding connective tissue is not as extensive as in the early node. Reticulum stain. 75 mm CRL. x 220.
11 Late nodes are larger than early nodes. The reticular framework is well developed. Reticular fibers and trabeculae span the subcapsular sinus (SS). A n efferent lymphatic vessel (ELV) is shown near the hilar region. The capsule consists of densely packed reticular elements. Reticulum stain. 105 mm CRL. X 220.
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ONTOGENY OF HUMAN FETAL LYMPH NODES Raymond P. Bailey and Leon Weiss
PLATE 3
25
PLATE 4 EXPLANATION O F FIGURES
12 The capsule ( C ) , afferent lymphatics ( A L ) , subcapsular sinus ( S S ) , trabeculae ( T ) , radial sinuses ( R S ) , and parenchyma of a late fetal lymph node are shown. The subcapsular sinus contains free cells and is traversed by reticular cells. The parenchyma contains several blood vessels. PCV are distinguished from veins (arrows) by their luminal contents. Toluidine blue. 245 mm CRL. x 340.
13 Postcapillary venules in late fetal lymph nodes are characterized by a high endothelium ( E ) , luminal lymphocytes ( L ) , and lymphocytes in diapedesis (arrows). Toluidine blue. 165 m m CRL. X 1;750.
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ONTOGENY OF HUMAN FETAL LYMPH NODES Raymond P. Bailey and Leon Weiss
PLATE 4
27
