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Thymic cell migration in the subnodular spaces of draining lymph nodes of rats
CELLULAR
52, 211-217 (1980)
IMMUNOLOGY
Thymic Cell Migration in the Subnodular Spaces of Draining Lymph Nodes of Rats1 G. SAINTE-MARIE Dkpartement
d’dnatomie,
Universite
AND
F. S. PENG
de Montreal,
MontrCal,
Qukbec, Canada
Received October 15, 1979
Little is known about the pathway of possible lymphocyte traftic in the nodules (germinal centers) of the node. Observations reported here indicate the involvement of the subnodular spaces. These spaces, which constitute the inner limit of the nodules, are contiguous to the perivascular channels of the postcapillary venules which partially encircle each nodule. Twelve hours after a local transfer of labeled thymic cells, they were observed in the subnodular spaces and in the perivascular channels of the draining nodes. It is proposed that the spaces and the channels provide a pathway for the rapid migration of lymphocytes entering a node via the afferent lymph and, probably, carrying an immunogenic information. The pathway would permit these cells to transmit the information rapidly to the appropriate cell population(s) of a node draining a tissue undergoing an immunological process.
INTRODUCTION Previous study (1) has demonstrated the presence of “subnodular spaces” along the inner margin of folliculonodules (Fig. 1). In other investigations, a few transfused labeled lymphocytes were seen in nodules (2-5), however, the observation provided no information about the possible pathway of cell migration via the nodules. Moreover, no findings have thus far suggested the involvement of the subnodular spaces in this migration. The present work, based on observations made following local transfer of labeled thymic cells, provides evidence of such involvement. MATERIALS
AND METHODS
Treatment of Donors
Three lo-week-old inbred male Wistar Lewis rats, weighing 250 g, received injections of 5-[3H]deoxycytidine (sp act 38 Ci/mmol), dissolved in saline at a concentration of 1 mCi/ml. The first injection was given intravenously at a dose of 1 $X/g body wt. Thereafter, a subcutaneous dose of 0.5 &i/g body wt was repeated every 8 hr for 5 days. One hour after the last injection, the animals were sacrificed. The thymuses of the donors were kept at 4°C in Earle’s medium 199, and the parathymic nodes were discarded. The cells were liberated by scraping a piece of 1 Supported by funds from the Medical Research Council of Canada and CAFIR de 1’UniversitC de Montrkal. 211 0008-8749/80/070211-07$02.00/O Copyright 0 1980by Academic Press, Inc. All rights of reproduction in any form reserved.
212
SHORT COMMUNICATIONS
FIG. 1. Schematic illustration of a segment of the peripheral cortex of the rat lymph node. A folliculonodule is seen with the extrafollicular zone on both sides. The nodule (germinal center) of latter structure is made up of a dark (D) and light (L) zone, the light zone being capped by a follicle (F). The extrafollicular zone has abundant parallel reticular fibers (R) which cross the subcapsular sinus ($5) and fuse with the capsule (C). Fibers encircle the folliculonodule, and suspend it from the capsule; they also delimit a subnodular space (S) under the dark zone. Fibers also form perivascular channels (P) cuffing the postcapillary venules (V) partially encircling the folliculonodule. The space and the channels connect together.
stainless-steel screen over the thymuses, as described previously (6). The cell suspension was filtered through progressively finer screens. A cell count was performed as well as a viability test using erythrosin B. The concentration of the suspension was adjusted to 8 x lo7 viable cells per milliliter. Prior to the preparation of the suspension, a sample of a donor’s thymus was set aside and processed for radioautography. Smears of the suspension were also processed for radioautography. The preparations were exposed for 30 days and then stained with Giemsa. Treatment of Recipients Under ether anesthesia, each of nine syngeneic male recipients received 0.1 ml of cell suspension in the lingual frenulum, and 0.5 ml in the mediastinum using a specially designed needle (7). Three recipients were sacrificed 6, 12, and 24 hr later, respectively. Each one was perfused with a 10% neutral formaldehyde solution; the draining cervical and parathymic nodes as well as other nodes were removed and fixed in Carnoy’s fluid for 8 hr. A series of consecutive 5-pm-thick paraffin sections were cut at various levels of each draining node. The central section of each series was processed for radioautography, using Kodak NTB3 nuclear emulsion. The radioautographs were exposed for 150 days and were then developed and stained with Harris hematoxylin. The remaining sections of each series were kept as extras for a possible tridimensional analysis of the distribution of labeled cells in the subnodular spaces of some nodules. Tridimensional Reconstruction A reconstruction of the tridimensional distribution of labeled cells in subnodular spaces was performed occasionally. To do so, the radioautographs of serial sections
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213
were projected on paper at a 70x magnification; the margin of the investigated structures as well as the location of the associated labeled cells was outlined. The margins and cells were then reproduced on glass plates; when superposed, they provided the desired image. RESULTS Labeling
Index and Viability
Test
The radioautographs of the donor thymuses showed that nearly all cortical and medullary lymphocytes were labeled. On the radioautographs of the cell suspension, at least 97% of the thymic cells exhibited a moderate to strong reaction, about 88% of them being small lymphocytes. According to the erythrosin B test, 85 to 95% of these cells were viable. Draining
Nodes
Practically all labeled cells involved in the following observations exhibited a moderate to strong radioautographical reaction comparable to that of small lymphocytes. Six hours after the transfer, labeled cells were present in the subcapsular sinus (Fig. 2) and scattered throughout the extrafollicular zone. A few labeled cells were detected in the follicles and nodules (Figs. 2, 3) while some adhered to the outline of cross-cut to obliquely cut dark zones of nodules (Figs. 2, 3). Labeled cells were also seen in subnodular spaces of nodules which were sometimes cut longitudinally. Likewise, labeled cells were present in the perivascular channels of postcapillary venules. After 12 hr, there were fewer labeled cells in the subcapsular sinus and the extrafollicular zone than after 6 hr, but the subnodular spaces of the few longitudinally cut nodules exhibited a greater accumulation of them (Figs. 4, 5). Labeled cells were seen also along the outline of the cross-cut to obliquely cut nodules. In order to determine whether these cells were part of an accumulation of labeled cells in the subnodular space of these nodules, we carried out a tridimensional reconstruction. For this, we processed for radioautography the extra paraffin sections, specifically those which neighbored a section exhibiting cross-sectioned adjacent nodules showing labeled cells along their outlines. The tridimensional analysis revealed that the labeled cells were also part of a row of cells which had accumulated in the subnodular space of the nodules. A concentration of labeled cells was also observed in the perivascular channels of the postcapillary venules, primarily in those venules which were adjacent to the nodules containing labelled cells in their subnodular space. Labeled cells were also found in the perivascular channels of the postcapillary venules running over deep cortex units whenever such a structure was seen below the nodules (Fig. 6, U). In a recent work clarifying the morphology of the deep cortex (paracortex), the deep cortex units were found to be its basic elements. They are semirounded structures contiguous to the peripheral cortex and bulging into the medulla (Belisle and Sainte-Marie, 1979, unpublished observations). The deep cortex units were referred to previously as “pseudofollicles” (1). After 24 hr, only a few labeled cells remained in the subcapsular sinus or in the extrafollicular zone. The concentration of labeled cells in the subnodular spaces
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FIG. 2. Radioautograph of a draining parathymic node, removed 6 hr after a local transfer of labeled thymic cells. Labeled cells are present in the subcapsular sinus (arrowheads) and the extrafollicular zone. A partial row of labeled cells (vertical arrows) outlines the inner margin of an obliquely cut dark zone (d). A few labeled cells are present in the follicle (upper horizontal arrow) and the nodule (lower horizontal arrow). Exposure time: 150 days. 140x.
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and perivascular channels had disappeared. Most of the labeled cells remaining in the draining nodes were in the medullary cords. Any node, not involved in draining a site of cell transfer, contained only negligible amounts of labeled cells at all times. DISCUSSION The present results are in line with previous observations (6) which revealed that locally transferred cells migrate from the subcapsular sinus into, and through, the extrafollicular zone. Thereafter, some cells enter deep cortex units while others enter medullary cords or leave the organ via medullary sinuses. Transferred cells have been observed in perivascular channels (Figs. 8 and 9, (6)) and along the outline of the nodules, however, they attracted little attention at that time since the nodules had not been cut in a plane across their subnodular spaces. Again in agreement with previous observations (2,4,5, S- 1l), we found that only a few cells migrated in the follicles and nodules. In the case of the follicles, this is probably because we did not transfer the proper cell population, earlier findings indicated that their cells are B lymphocytes (2,5, 12). By contrast, it is probably a reflect of the reality that the nodules contained only a few labeled cells, since it is unlikely that a substantial amount of traffic can occur through active nodules. Indeed, the dark zone of the nodules contains a particular type of cells, the nodulocytes, which proliferate intensely in active nodules. It is difficult to conceive that a heavy migration of other types of cells could occur through the resulting compact population. As to the few thymic cells seen in the nodules, they remind the results of Kotani et al. (13) and of Jacobson et al. (14) indicating that thymocytes accelerate or restore the formation of nodules in nodes of thymectomized rats and mice. But, it was not determined then whether this occurred by way of a direct cell participation to the nodulocytic populations. In accordance with known histological features and the present observations, we propose that lymphocytes migrate according to the pathways represented in Fig. 6. The figure illustrates the role of the perivascular channels and subnodular spaces in the cell migration from the subcapsular sinus across the cortex. First of all, the cells poured in the subcapsular sinus are slowed down by the abundant fibers crossing the sinus above the extrafollicular zone. This favors entry of the cells into the zone rather than into the folliculonodules. FIG. 3. Similar to node in Fig. 2. The lower limit of an obliquely cells (arrows). Exposure time: 150 days. 220x.
cut dark zone (d) is lined with labeled
FIG. 4. Radioautograph of a draining cervical node, removed 12 hr after local transfer of labeled thymic cells. An accumulation of labeled cells is seen in the subnodular space (arrows) of two nodules (d), the left one being rather longitudinally cut. The right nodule is cut in an oblique plane passing only through the bottom part of its subnodular space so that fewer labeled cells can be seen. A concentration of labeled cells is present in an area of the extrafollicular zone in between the two nodules. High magnification revealed that many of these cells were concentrated around postcapillary venules. Exposure time: 150 days. 90x. FIG. 5. Similar to node in Fig. 4. The nearly longitudinally cut right nodule (d) exhibits a row of labeled cells in its subnodular space. Exposure time: 150 days. 90x.
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FIG. 6. Schematic illustration of the probable pathways of migration, through the cortex, of lymphocytes poured in the subcapsular sinus by the afferent lymph. The darkened circles illustrate the passage of cells from the sinus directly into the medullary cord (C) or sinuses (S) by migration between the parallel fibers of the extrafollicular zone. The shaded circles illustrate a similar migration pathway of cells from the subcapsular sinus along the perivascular channels of the postcapillary venules running beneath the sinus directly into medulla. The blank circles illustrate the migration of cells entering the perivascular channels of the postcapillary venules encircling a folliculonodule. From the channels some cells may enter the subnodular space and penetrate into the dark zone of the nodule. Other cells continue their migration in the channels of the venules which vascularize the periphery of a deep cortex unit(U). From these channels, the cells can enter the unit center.
Once in the zone, the cells are directed toward the medulla, or toward the deep cortex unit, by the walls of the fibers crossing the peripheral cortex. Some of these fibers support the venules in the zone (l), whereas a portion of them continue on to form perivascular channels and, therefore, can direct some of the cells into the channels. Venules run from the vicinity of the subcapsular sinus toward the medulla; their channels provide a second pathway for the rapid migration of cells from one site to the other. Since the channels of the venules surrounding a nodule connect with its subnodular space, some cells could enter the space from the channels. Labelled cells are hence funnelled from the channels into the space and accumulate there. Some cells may migrate from a subnodular space into a nodule. This is supported by the fact that a few labelled cells do appear in the nodules and the fact that small lymphocytic cells are known to undergo diapedesis across the boundary between a nodule and its subnodular space (1). From beneath nodules, the venules branch off to curve around and vascularize the periphery of a deep cortex unit. The labeled cells could continue to migrate along the channels of the venules and a proportion of these would then pass into the
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unit center, where lymphocytes are known to accumulate temporarily during migration through a node (6, U-17). Lymphocytes, coming from a tissue undergoing an immunological process and pouring into the subcapsular sinus of a node, are likely to carry on an immunogenic information. The cells, migrating along the proposed pathways of perivascular channels and subnodular spaces, could transmit the information very rapidly to the concerned nodal cell populations. They could also influence the endothelial cells of the postcapillary venules and thus stimulate their hypertrophy and the entry of blood lymphocytes into the node (1, 18). If this is the case, then the immunogenic information could be simultaneously transmitted to the blood lymphocytes, which explains how they would receive an information when entering a node via the venules at a distance from the subcapsular sinus. From the subnodular space, some cells may transmit an information to the nodulocytes, which would explain why the nodulocytes proliferate in the inner part of the nodule away from the subcapsular sinus. The.cells migrating in channels of the venules along the periphery of a deep cortex unit may transmit information to the unit lymphocytes which are involved in cellular immune responses (19, 20, Belisle and Sainte-Marie, unpublished data, 1979). However, other cells which migrate in the channels of the venules running from the vicinity of a subcapsular sinus directly into medullary cords may transmit information to the plasmocyte precursors of the cords which participate in humoral immune responses. The narrowness of the channels and subnodular spaces would enhance contact between migrating cells. Therefore, they would provide favorable sites in which T and B cells could meet and interact. ACKNOWLEDGMENTS The authors wish to thank Mrs. S. Loiselle for her skillful technical assistance and Miss E. Lambertfor her artistic assistance in the preparation of the drawings. They also thank Dr. M. Pelletier for reviewing the paper.
REFERENCES 1. Sainte-Marie, G., and Sin, Y. M., In “Regulation of Haematopoiesis” (A. S. Gordon, Ed.), pp. 1339-1383. Appleton-Century-Crofts, New York, 1970. 2. Gutman, Cl. A., and Weissman, I. L., Trunsplnntation 16, 621, 1973. 3. Sainte-Marie, G., Peng, F. -S., and Denis, G., Ann. Zmmunol. Inst. Pasteur 126C, 481, 1976. 4. Brahim, F., and Osmond, D. G., Clin. Exp. Immunol. 24, 515, 1976. 5. Nieuwenhuis, P., and Ford, W. L., Cell. Immunol. 23, 254, 1976. 6. Sainte-Marie, G., and Peng, F. -S., Ann. Immunol. Inst. Pasteur 126C, 501, 1975. 7. Sainte-Marie, G., and Messier, B., Stand. J. Haematol. 7, 163, 1970. 8. Parrott, D. M. V., and De Sousa, M., Clin. Exp. Immunol. 8,663, 1971. 9. Gowans, J. L., and Knight, E. J., Proc. Roy. Sot. London Ser. B 159, 257, 1964. 10. Parrott, D. M. V., De Sousa, M., and East, J., J. Exp. Med. 123, 191, 1966. 11. Sprent, J., and Miller, J. F. A. P., Cell. Zmmunol. 3, 385, 1972. 12. Howard, J. C., Hunt, S. V., and Gowans, J. L., J. Exp. Med. 135, 200, 1972. 13. Kotani, M., Nawa, Y., Fujii, H., Fukumoto, T., Miyamoto, M., and Yamashita, A. ,Acfa Anat. 90, 585, 1974. 14. Jacobson, E. B., and Caporale, L. H., and Thorbecke, G. J., Cell. Immunol. 13, 416, 1974. 15. Sainte-Marie, G., and Peng, F.-S., Rev. Canad. Biol. 34, 205, 1975. 16. Sainte-Marie, G., and Peng, F.-S., J. Reticuloendothel. Sot. 20, 51, 1976. 17. Sainte-Marie, G., and Peng, F.-S., In “Function and Structure of the Immune System” (W. MtlllerRuchholtz and H. K. Milller-Hermelink, Eds.), pp. 59-64. Plenum, New York, 1979. 18. Sainte-Marie, G., Rev. Canad. Biol. 25, 263, 1966. 19. Oott, J., and Turk, J. L., Brit. J. Exp. Pathol. 46, 147, 1965. 20. Parrott, D. M. V., and De Sousa, M. A. B., Nature (London) 212, 1316, 1966.
52, 211-217 (1980)
IMMUNOLOGY
Thymic Cell Migration in the Subnodular Spaces of Draining Lymph Nodes of Rats1 G. SAINTE-MARIE Dkpartement
d’dnatomie,
Universite
AND
F. S. PENG
de Montreal,
MontrCal,
Qukbec, Canada
Received October 15, 1979
Little is known about the pathway of possible lymphocyte traftic in the nodules (germinal centers) of the node. Observations reported here indicate the involvement of the subnodular spaces. These spaces, which constitute the inner limit of the nodules, are contiguous to the perivascular channels of the postcapillary venules which partially encircle each nodule. Twelve hours after a local transfer of labeled thymic cells, they were observed in the subnodular spaces and in the perivascular channels of the draining nodes. It is proposed that the spaces and the channels provide a pathway for the rapid migration of lymphocytes entering a node via the afferent lymph and, probably, carrying an immunogenic information. The pathway would permit these cells to transmit the information rapidly to the appropriate cell population(s) of a node draining a tissue undergoing an immunological process.
INTRODUCTION Previous study (1) has demonstrated the presence of “subnodular spaces” along the inner margin of folliculonodules (Fig. 1). In other investigations, a few transfused labeled lymphocytes were seen in nodules (2-5), however, the observation provided no information about the possible pathway of cell migration via the nodules. Moreover, no findings have thus far suggested the involvement of the subnodular spaces in this migration. The present work, based on observations made following local transfer of labeled thymic cells, provides evidence of such involvement. MATERIALS
AND METHODS
Treatment of Donors
Three lo-week-old inbred male Wistar Lewis rats, weighing 250 g, received injections of 5-[3H]deoxycytidine (sp act 38 Ci/mmol), dissolved in saline at a concentration of 1 mCi/ml. The first injection was given intravenously at a dose of 1 $X/g body wt. Thereafter, a subcutaneous dose of 0.5 &i/g body wt was repeated every 8 hr for 5 days. One hour after the last injection, the animals were sacrificed. The thymuses of the donors were kept at 4°C in Earle’s medium 199, and the parathymic nodes were discarded. The cells were liberated by scraping a piece of 1 Supported by funds from the Medical Research Council of Canada and CAFIR de 1’UniversitC de Montrkal. 211 0008-8749/80/070211-07$02.00/O Copyright 0 1980by Academic Press, Inc. All rights of reproduction in any form reserved.
212
SHORT COMMUNICATIONS
FIG. 1. Schematic illustration of a segment of the peripheral cortex of the rat lymph node. A folliculonodule is seen with the extrafollicular zone on both sides. The nodule (germinal center) of latter structure is made up of a dark (D) and light (L) zone, the light zone being capped by a follicle (F). The extrafollicular zone has abundant parallel reticular fibers (R) which cross the subcapsular sinus ($5) and fuse with the capsule (C). Fibers encircle the folliculonodule, and suspend it from the capsule; they also delimit a subnodular space (S) under the dark zone. Fibers also form perivascular channels (P) cuffing the postcapillary venules (V) partially encircling the folliculonodule. The space and the channels connect together.
stainless-steel screen over the thymuses, as described previously (6). The cell suspension was filtered through progressively finer screens. A cell count was performed as well as a viability test using erythrosin B. The concentration of the suspension was adjusted to 8 x lo7 viable cells per milliliter. Prior to the preparation of the suspension, a sample of a donor’s thymus was set aside and processed for radioautography. Smears of the suspension were also processed for radioautography. The preparations were exposed for 30 days and then stained with Giemsa. Treatment of Recipients Under ether anesthesia, each of nine syngeneic male recipients received 0.1 ml of cell suspension in the lingual frenulum, and 0.5 ml in the mediastinum using a specially designed needle (7). Three recipients were sacrificed 6, 12, and 24 hr later, respectively. Each one was perfused with a 10% neutral formaldehyde solution; the draining cervical and parathymic nodes as well as other nodes were removed and fixed in Carnoy’s fluid for 8 hr. A series of consecutive 5-pm-thick paraffin sections were cut at various levels of each draining node. The central section of each series was processed for radioautography, using Kodak NTB3 nuclear emulsion. The radioautographs were exposed for 150 days and were then developed and stained with Harris hematoxylin. The remaining sections of each series were kept as extras for a possible tridimensional analysis of the distribution of labeled cells in the subnodular spaces of some nodules. Tridimensional Reconstruction A reconstruction of the tridimensional distribution of labeled cells in subnodular spaces was performed occasionally. To do so, the radioautographs of serial sections
SHORT COMMUNICATIONS
213
were projected on paper at a 70x magnification; the margin of the investigated structures as well as the location of the associated labeled cells was outlined. The margins and cells were then reproduced on glass plates; when superposed, they provided the desired image. RESULTS Labeling
Index and Viability
Test
The radioautographs of the donor thymuses showed that nearly all cortical and medullary lymphocytes were labeled. On the radioautographs of the cell suspension, at least 97% of the thymic cells exhibited a moderate to strong reaction, about 88% of them being small lymphocytes. According to the erythrosin B test, 85 to 95% of these cells were viable. Draining
Nodes
Practically all labeled cells involved in the following observations exhibited a moderate to strong radioautographical reaction comparable to that of small lymphocytes. Six hours after the transfer, labeled cells were present in the subcapsular sinus (Fig. 2) and scattered throughout the extrafollicular zone. A few labeled cells were detected in the follicles and nodules (Figs. 2, 3) while some adhered to the outline of cross-cut to obliquely cut dark zones of nodules (Figs. 2, 3). Labeled cells were also seen in subnodular spaces of nodules which were sometimes cut longitudinally. Likewise, labeled cells were present in the perivascular channels of postcapillary venules. After 12 hr, there were fewer labeled cells in the subcapsular sinus and the extrafollicular zone than after 6 hr, but the subnodular spaces of the few longitudinally cut nodules exhibited a greater accumulation of them (Figs. 4, 5). Labeled cells were seen also along the outline of the cross-cut to obliquely cut nodules. In order to determine whether these cells were part of an accumulation of labeled cells in the subnodular space of these nodules, we carried out a tridimensional reconstruction. For this, we processed for radioautography the extra paraffin sections, specifically those which neighbored a section exhibiting cross-sectioned adjacent nodules showing labeled cells along their outlines. The tridimensional analysis revealed that the labeled cells were also part of a row of cells which had accumulated in the subnodular space of the nodules. A concentration of labeled cells was also observed in the perivascular channels of the postcapillary venules, primarily in those venules which were adjacent to the nodules containing labelled cells in their subnodular space. Labeled cells were also found in the perivascular channels of the postcapillary venules running over deep cortex units whenever such a structure was seen below the nodules (Fig. 6, U). In a recent work clarifying the morphology of the deep cortex (paracortex), the deep cortex units were found to be its basic elements. They are semirounded structures contiguous to the peripheral cortex and bulging into the medulla (Belisle and Sainte-Marie, 1979, unpublished observations). The deep cortex units were referred to previously as “pseudofollicles” (1). After 24 hr, only a few labeled cells remained in the subcapsular sinus or in the extrafollicular zone. The concentration of labeled cells in the subnodular spaces
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FIG. 2. Radioautograph of a draining parathymic node, removed 6 hr after a local transfer of labeled thymic cells. Labeled cells are present in the subcapsular sinus (arrowheads) and the extrafollicular zone. A partial row of labeled cells (vertical arrows) outlines the inner margin of an obliquely cut dark zone (d). A few labeled cells are present in the follicle (upper horizontal arrow) and the nodule (lower horizontal arrow). Exposure time: 150 days. 140x.
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and perivascular channels had disappeared. Most of the labeled cells remaining in the draining nodes were in the medullary cords. Any node, not involved in draining a site of cell transfer, contained only negligible amounts of labeled cells at all times. DISCUSSION The present results are in line with previous observations (6) which revealed that locally transferred cells migrate from the subcapsular sinus into, and through, the extrafollicular zone. Thereafter, some cells enter deep cortex units while others enter medullary cords or leave the organ via medullary sinuses. Transferred cells have been observed in perivascular channels (Figs. 8 and 9, (6)) and along the outline of the nodules, however, they attracted little attention at that time since the nodules had not been cut in a plane across their subnodular spaces. Again in agreement with previous observations (2,4,5, S- 1l), we found that only a few cells migrated in the follicles and nodules. In the case of the follicles, this is probably because we did not transfer the proper cell population, earlier findings indicated that their cells are B lymphocytes (2,5, 12). By contrast, it is probably a reflect of the reality that the nodules contained only a few labeled cells, since it is unlikely that a substantial amount of traffic can occur through active nodules. Indeed, the dark zone of the nodules contains a particular type of cells, the nodulocytes, which proliferate intensely in active nodules. It is difficult to conceive that a heavy migration of other types of cells could occur through the resulting compact population. As to the few thymic cells seen in the nodules, they remind the results of Kotani et al. (13) and of Jacobson et al. (14) indicating that thymocytes accelerate or restore the formation of nodules in nodes of thymectomized rats and mice. But, it was not determined then whether this occurred by way of a direct cell participation to the nodulocytic populations. In accordance with known histological features and the present observations, we propose that lymphocytes migrate according to the pathways represented in Fig. 6. The figure illustrates the role of the perivascular channels and subnodular spaces in the cell migration from the subcapsular sinus across the cortex. First of all, the cells poured in the subcapsular sinus are slowed down by the abundant fibers crossing the sinus above the extrafollicular zone. This favors entry of the cells into the zone rather than into the folliculonodules. FIG. 3. Similar to node in Fig. 2. The lower limit of an obliquely cells (arrows). Exposure time: 150 days. 220x.
cut dark zone (d) is lined with labeled
FIG. 4. Radioautograph of a draining cervical node, removed 12 hr after local transfer of labeled thymic cells. An accumulation of labeled cells is seen in the subnodular space (arrows) of two nodules (d), the left one being rather longitudinally cut. The right nodule is cut in an oblique plane passing only through the bottom part of its subnodular space so that fewer labeled cells can be seen. A concentration of labeled cells is present in an area of the extrafollicular zone in between the two nodules. High magnification revealed that many of these cells were concentrated around postcapillary venules. Exposure time: 150 days. 90x. FIG. 5. Similar to node in Fig. 4. The nearly longitudinally cut right nodule (d) exhibits a row of labeled cells in its subnodular space. Exposure time: 150 days. 90x.
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SHORT COMMUNICATIONS
FIG. 6. Schematic illustration of the probable pathways of migration, through the cortex, of lymphocytes poured in the subcapsular sinus by the afferent lymph. The darkened circles illustrate the passage of cells from the sinus directly into the medullary cord (C) or sinuses (S) by migration between the parallel fibers of the extrafollicular zone. The shaded circles illustrate a similar migration pathway of cells from the subcapsular sinus along the perivascular channels of the postcapillary venules running beneath the sinus directly into medulla. The blank circles illustrate the migration of cells entering the perivascular channels of the postcapillary venules encircling a folliculonodule. From the channels some cells may enter the subnodular space and penetrate into the dark zone of the nodule. Other cells continue their migration in the channels of the venules which vascularize the periphery of a deep cortex unit(U). From these channels, the cells can enter the unit center.
Once in the zone, the cells are directed toward the medulla, or toward the deep cortex unit, by the walls of the fibers crossing the peripheral cortex. Some of these fibers support the venules in the zone (l), whereas a portion of them continue on to form perivascular channels and, therefore, can direct some of the cells into the channels. Venules run from the vicinity of the subcapsular sinus toward the medulla; their channels provide a second pathway for the rapid migration of cells from one site to the other. Since the channels of the venules surrounding a nodule connect with its subnodular space, some cells could enter the space from the channels. Labelled cells are hence funnelled from the channels into the space and accumulate there. Some cells may migrate from a subnodular space into a nodule. This is supported by the fact that a few labelled cells do appear in the nodules and the fact that small lymphocytic cells are known to undergo diapedesis across the boundary between a nodule and its subnodular space (1). From beneath nodules, the venules branch off to curve around and vascularize the periphery of a deep cortex unit. The labeled cells could continue to migrate along the channels of the venules and a proportion of these would then pass into the
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unit center, where lymphocytes are known to accumulate temporarily during migration through a node (6, U-17). Lymphocytes, coming from a tissue undergoing an immunological process and pouring into the subcapsular sinus of a node, are likely to carry on an immunogenic information. The cells, migrating along the proposed pathways of perivascular channels and subnodular spaces, could transmit the information very rapidly to the concerned nodal cell populations. They could also influence the endothelial cells of the postcapillary venules and thus stimulate their hypertrophy and the entry of blood lymphocytes into the node (1, 18). If this is the case, then the immunogenic information could be simultaneously transmitted to the blood lymphocytes, which explains how they would receive an information when entering a node via the venules at a distance from the subcapsular sinus. From the subnodular space, some cells may transmit an information to the nodulocytes, which would explain why the nodulocytes proliferate in the inner part of the nodule away from the subcapsular sinus. The.cells migrating in channels of the venules along the periphery of a deep cortex unit may transmit information to the unit lymphocytes which are involved in cellular immune responses (19, 20, Belisle and Sainte-Marie, unpublished data, 1979). However, other cells which migrate in the channels of the venules running from the vicinity of a subcapsular sinus directly into medullary cords may transmit information to the plasmocyte precursors of the cords which participate in humoral immune responses. The narrowness of the channels and subnodular spaces would enhance contact between migrating cells. Therefore, they would provide favorable sites in which T and B cells could meet and interact. ACKNOWLEDGMENTS The authors wish to thank Mrs. S. Loiselle for her skillful technical assistance and Miss E. Lambertfor her artistic assistance in the preparation of the drawings. They also thank Dr. M. Pelletier for reviewing the paper.
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