Effect of blockage of the afferent lymphatic vessels on the development of popliteal lymph nodes in the rat

The effect of blockage of the afferent lymphatic vessels on the development of popliteal lymph nodes in the rat was studied. The afferent lymphatic vessels to each popliteal node were surgically interrupted at the lowest edge of the popliteal fossa at 3, 7 or 28 days after birth and the popliteal nodes were obtained from treated animals at 4, 8 or 16 weeks after the operation. At 7 days after birth, each popliteal node was small and weighed 0.2 mg. Its parenchyma consisted of reticular cells and a small number of dispersed lymphoid elements. Four weeks after birth, each node weighed 4-5 mg, and its parenchyma comprised two layers, an outer continuous layer of peripheral cortex containing 40-50 lymph follicles and an inner discontinuous layer of deep cortex made up of 4-5 deep cortical units. At 10-12 weeks after birth, each node weighed about 10 mg and showed full structural development; the peripheral cortex contained 100-130 lymph follicles and the deep cortex consisted of 4-6 well developed units. The popliteal node was drained by 4-6 afferent lymphatic vessels, which opened into the subcapsular sinus of the node. Each lymphatic opening was topographically associated with a respective deep cortical unit, as previously described by Bélisle and Sainte-Marie (1982). In animals treated at 3 or 7 days after birth, the development of the popliteal nodes was considerably inhibited. Four weeks after surgery, each node showed 10-30 lymph follicles in the peripheral cortex and 1-3 small units in the deep cortex. Sixteen weeks after surgery, the node weighed about 4 mg and its cortex exhibited about 50 lymph follicles in the peripheral cortex and only 2 units in the deep cortex. The popliteal nodes of the treated animals generally received 2 afferent lymphatic vessels. In animals treated at 4 weeks after birth, the popliteal nodes showed no gain in weight for following 16 weeks. Four weeks after surgery, each node usually had 4-5 deep cortical units and 50-60 lymph follicles. Thereafter, some units and their overlaying peripheral cortex underwent atrophy, while others persisted. Sixteen weeks after surgery, the popliteal node showed only 2 deep cortical units and 50-60 lymph follicles, and was drained by 2 afferent lymphatic vessels. Surgical interruption of the afferent lymphatic vessels to the popliteal node at the lowest edge of the popliteal fossa did not obliterate all the draining lymphatic vessels , but reduced the number of vessels opening into the node.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of T and B cell areas in peripheral lymphoid organs of the rat

In the present study the early development of peripheral lymphoid organs (spleen, popliteal lymph node, mesenteric lymph node and Peyer's patches) is described in terms of homing patterns of T and B cells, demonstrated with immunohistoperoxidatic detection of characteristic membrane antigen in normal rats and with routine histology in neonatally thymectomized rats. In the first days after birth the peripheral lymphoid organs are almost exclusively populated by T cells. After neonatal thymectomy lymphocytes appear in the dome areas of Peyer's patches from four to six days after birth, in mesenteric and popliteal lymph nodes lymphocytes are found in the outer cortex from day 6 and day 8 respectively and in the marginal zone of the spleen from eight days onwards. These lymphocytes showed no membrane staining when reacted for T antigen with immunohistoperoxidatic techniques. The morphological evidence for considering Peyer's patches of rats as central inductive sites for the generation of B cells is poor. The discrepancy in the order of appearance of T and B cell (sub)populations in spleen compartments in normal ontogenetic development and lethally irradiated, stem cell reconstituted animals is discussed.     

Development of T and B cell areas in peripheral lymphoid organs of the rat.

In the present study the early development of peripheral lymphoid organs (spleen, popliteal lymph node, mesenteric lymph node and Peyer's patches) is described in terms of homing patterns of T and B cells, demonstrated with immunohistoperoxidatic detection of characteristic membrane antigen in normal rats and with routine histology in neonatally thymectomized rats. In the first days after birth the peripheral lymphoid organs are almost exclusively populated by T cells. After neonatal thymectomy lymphocytes appear in the dome areas of Peyer's patches from four to six days after birth, in mesenteric and popliteal lymph nodes lymphocytes are found in the outer cortex from day 6 and day 8 respectively and in the marginal zone of the spleen from eight days onwards. These lymphocytes showed no membrane staining when reacted for T antigen with immunohistoperoxidatic techniques. The morphological evidence for considering Peyer's patches of rats as central inductive sites for the generation of B cells is poor. The discrepancy in the order of appearance of T and B cell (sub)populations in spleen compartments in normal ontogenetic development and lethally irradiated, stem cell reconstituted animals is discussed.     

The migration of lymphocytes from bone marrow to popliteal lymph nodes demonstrated by selective bone marrow labeling with 3H-thymidine in vivo

Guinea pig popliteal lymph nodes were examined by DNA radioassay and radioautography following the selective labeling of tibial and femoral marrow cells by intramyeloid injections of ³H-thymidine. The DNA radioactivity of the node increased for the first four days and at four to seven days exceeded that seen after an intraperitoneal injection of the same total dose of ³H-thymidine, indicating an export of radioactivity from the labeled marrow to the node. Simultaneously, radioautographic sections of the node revealed labeled cells indicative of an origin from marrow precursors. Small lymphocytes constituted 60–90% of the labeled cells and reached maximal numbers at four to six days. Most of them were observed in the cortex, including the subcapsular sinus, primary follicles, mantle zones around germinal centers, and the lumen and walls of post-capillary venules. However, the highest labeling indices of small lymphocytes occurred in the medulla, including the medullary cords, medullary sinuses and efferent lymphatic vessels. Labeled large mononuclear cells, including large lymphoid cells, monocytes and large blast cells, were confined almost exclusively to the cortex. A small number of labeled plasma cells was observed in the medullary cords. It is concluded that newly-formed bone marrow lymphocytes migrate continuously into immunologically quiescent lymph nodes and become widely distributed throughout the cortex and medulla, while some enter the recirculating small lymphocyte pool.     

大鼠腘窝淋巴结内淋巴滤泡的生后发育

目的:研究大鼠淋巴结淋巴滤泡的生后发育.方法:采用常规组织学、免疫细胞化学及三维重建技术研究了大鼠腘窝淋巴结内淋巴滤泡的发生.结果:生后18天始,淋巴结浅层皮质内sIgM阳性B淋巴细胞聚集形成初级淋巴滤泡.生后3周ED-5阳性滤泡树突状细胞出现.随着鼠龄及体重的增长,初级淋巴滤泡不断扩大,每个淋巴结内淋巴滤泡数亦增多.生后13周滤泡数达高峰,平均每个淋巴结86个,13周以后体重缓慢增长,淋巴滤泡数逐渐下降.生后8周时,次级淋巴滤泡出现,ED-5阳性滤泡树突状细胞主要分布于亮区.结论:初级淋巴滤泡形成过程中B淋巴细胞聚集可能诱导局部网状细胞分化成滤泡树突状细胞;发育期间淋巴滤泡数随体重增长而增加可能与某些决定机体生长的因素作用有关.     

Antigen localization in lymphopenic states: II. Further studies on whole body X-irradiation

The gross and microscopic distribution of ¹²⁵I polymerized flagellin from Salmonella adelaide was studied in adult rats at various times following 800 r whole body X-irradiation. Injections of radioactive antigen were made in both hind footpads, and the popliteal lymph nodes were excised for autoradiographic study 1 day later. This dose of irradiation caused a progressive decline in the ability of lymphoid follicles of popliteal nodes to capture and retain antigen. Irradiation had no detectable effect upon antigen uptake by whole lymph nodes or upon the number of grains overlying the phagocytic cells of the medullary sinuses of popliteal nodes.

Various substances capable of restoring follicular antigen uptake in the irradiated rat were studied by means of injecting the test substance into one hind footpad 1 hour prior to the injection of antigen into both feet. The distribution of antigen in each popliteal node was compared, each animal thus acting as its own control. It was found that 0.01 ml of specific anti-flagellar immune serum, or 0.25 ml of normal adult rat serum significantly improved follicular antigen uptake when tested ten days after irradiation. Foetal calf serum, homologous lymphocytes, and the media from pooled concentrated lymphocyte cultures were without demonstrable effects when given by regional injection. Shielding of the popliteal nodes at the time of irradiation improved follicular antigen uptake, whereas shielding of the femoral bone marrow and appendix was ineffective. No agent found capable of improving follicular antigen capture in the irradiated rat significantly altered footpad retention of antigen, whole organ counts of the popliteal nodes, or antigen localization in the phagocytic cells of the lymph node medulla.

The results favour the interpretation that the follicular antigen trapping mechanism is extremely sensitive to changes in levels of opsonins; that substances present in normal adult rat serum act as `follicular opsonins'; that these substances decline exponentially following irradiation; and that these substances are secreted by small lymphocytes or their progeny.

     

Lymph node localization and whole body distribution of radioiodinated encephalitogenic polypeptide in guinea-pigs

A bovine encephalitogenic polypeptide (BEP) labelled with radioiodide retained its capacity to induce experimental encephalomyelitis (EAE). Guinea-pigs were injected with ¹²⁵I BEP in Freund's complete adjuvant (FCA), to study changes in the architecture and the distribution of radioactivity in draining lymph nodes, and the amount of radioactivity in various organs.

After injection of BEP in FCA the lymph node rapidly enlarged. Within 48 hr there was depletion of lymphocytes, the enlarging lymphoid follicles had become confluent and there was proliferation of large `epithelioid' cells throughout the node. At 5 days the lymph node architecture was disorganized and lymph follicles with germinal centres could not be recognized; similar but less pronounced changes were present in regional nodes. By contrast, after injection of flagellin in FCA, there were numerous lymphocytes, plasmablasts and pyroninophilic cells, germinal centres were prominent, and the architecture was preserved.

From 0·5 to 0·8% of the total injected radioactivity was concentrated in the popliteal lymph node 2–5 days after injection of ¹²⁵I BEP in FCA. No radioactivity was concentrated in the node after injection of ¹²⁵I BEP without FCA, and animals thus immunized did not develop encephalomyelitis.

The popliteal lymph node was examined by autoradiography after injection of ¹²⁵I BEP in FCA. At 24 hr radioactive encephalitogen associated with droplets of adjuvant was present mainly in the peripheral sinus and at 48 hr encephalitogen–adjuvant droplets were deposited randomly throughout cortex and medulla. These droplets appeared to represent sites where lymphoid cells acquired their capacity for pathogenic reactivity with their target antigen in the central nervous system.

     

Oil-induced arthritis in da rats: Tissue distribution of arthritogenic 14C-labelled hexadecane

As part of the study of the pathogenesis of oil-induced arthritis in DA rats, the tissue dissemination of arthritogenic oil labelled with ¹⁴C has been determined. Rats received labelled hexadecane in arthritogenic doses by the intradermal route and were killed at I and 6 h, 2, 10, 14, 18 and 27 days after injection. Whole-body autoradiography was performed and localization of radioactivity in different organs was investigated. With the exception of the injection site, the lymph nodes showed the highest content of radioactivity throughout the study. Radioactivity could be seen in the popliteal, inguinal and axillary lymph nodes. Detailed examination of lymph nodes revealed at I h radioactivity in the subcapsular sinus of the lymph node. By 10 days the activity had spread to the cortex and paracortex, apart from the subcapsular sinus. The activity in other lymphoid organs, such as the bone marrow and spleen, was more transient, with a peak at 2 days. Oil disseminating to joints was not prominent: very little radioactivity was observed in the knee joints of both non-arthritic and arthritic animals. From these data it is suggested that the adjuvant oil exerts its major proarthritogenic activity in the lymph nodes rather than directly in the joints.     

Immunohistochemical study of lymphoid tissues in adjuvant arthritis (AA) by image analysis; relationship with synovial lesions

The aim of this study was to examine leucocyte populations in lymphoid organs during AA and to ascertain the relationship with lesions in synovial joints. Popliteal lymph nodes, spleen and knee synovial membranes were removed from both healthy and AA rats at intervals of 3-4 days over a 3-week period. Cryostat sections were stained with MoAbs directed against lymphocyte and macrophage subpopulations, and studied by image analysis. Throughout the arthritic period, high numbers of ED1+ and ED3+ macrophages were seen in both lymphoid compartments and intercellular adhesion molecule-1 (ICAM-1) expression also increased in some zones of lymph nodes and spleen. The percentages of CD4+ and CD8+ cells rose in the splenic zones studied but fell in the lymph node cortex. Very few natural killer (NK) cells were found in lymphoid tissues, but the number rose after AA induction. In synovia from AA rats, ED2+ macrophages proliferated but alpha/beta T cell infiltration was only occasionally observed, accompanied by ED1+ cells and ICAM-1 expression. In conclusion, synovitis developing after AA induction seems to be caused directly by macrophages and indirectly by lymphocytes placed both in popliteal lymph nodes and spleen.     

Tridimensional study of the deep cortex of the rat lymph node. VI. The deep cortex units of the germ-free rat

The deep cortex of the normal rat lymph node consists of semirounded lymphocytic structures, termed deep cortex "units," each being centered on the opening of an afferent lymphatic. The aim of the present work was to investigate the morphologic features of the units in germ-free animals, in an attempt to evaluate the influence on natural exogenous antigenic stimulation on the development of the units. For this, the lymph nodes from various anatomic locations from 8-week-old Sprague-Dawley germ-free rats were analysed tridimensionally. The observations revealed that, in comparison with the lymph nodes of normal rats, the units of the cervical and mesenteric lymph nodes of the germ-free animals were underdeveloped, while those of the brachial, inguinal, and popliteal lymph nodes were unchanged. Moreover, the germ-free state modified the units of the mesenteric lymph nodes in a manner not encountered in the remaining lymph nodes. Other morphologic features of the peripheral cortex of the lymph nodes of germ-free rats also differed from those of normal ones. The significance of these differences is discussed with respect to immune responses and the process of lymphocyte recirculation. They are of interest because they support previous proposals regarding some aspects of the functioning of the normal lymph node, accounting for the features of the structures and overall architecture of the organ.     

The ovine immune response to Chlamydia psittaci; histopathology of the lymph node

The histopathological response of the ovine popliteal lymph node to infection by an ovine abortion strain of Chlamydia psittaci was studied. After infection of 10 seronegative sheep by the subcutaneous route, the draining popliteal lymph nodes enlarged considerably. By day 6, expansion was more marked in the medulla than in the cortex but, by day 18, cortical follicles were prominent. Immunoglobulin-containing cells increased in number both in the medulla and cortex between days 6 and 18. C. psittaci was re-isolated from three nodes on day 6 and one on day 12, but at no stage was it demonstrated in tissue sections by an immunoperoxidase method. Thus it was shown that while C. psittaci could apparently become "latent" in lymphoid tissue, it could also stimulate a profound response at the same site.     

Effect of interferon-alpha-2a on the output of recirculating lymphocytes from single lymph nodes.

The output of recirculating lymphocytes from cannulated popliteal lymph nodes in sheep was measured after administration of human recombinant interferon (rIFN)-alpha-2a. Interferon (IFN) injection caused a dramatic decrease in lymphocyte output from lymph nodes. Following a single s.c. or i.d. injection of 2 x 10(7) U IFN into the drainage area of the popliteal lymph node, lymphocyte output fell to below 1% of the pre-treatment level and remained depressed for up to 35 hr. A substantial decrease in lymphocyte output from cannulated nodes also occurred after IFN was injected either i.v., into the skin of the opposite non-cannulated hind leg or into an afferent lymphatic vessel leading to the popliteal lymph node. After the period of depressed lymphocyte output, a seemingly compensatory surge of cell traffic occurred that lasted 2-3 days. During this phase there was a relative increase in the proportion of CD4+ T cells in lymph. Similar changes occurred after each treatment in animals given multiple doses of IFN. These effects are unlikely to be antigen-induced since there was no blast cell response in any treated animal. The analysis of blood and lymph plasma samples showed that the most severe depression of lymphocyte output was associated with high levels of IFN, while there was no apparent correlation between the reduction in lymphocyte traffic and concentrations of cortisol in plasma. These results suggest that IFN-alpha-2a is involved directly in the regulation of lymphocyte output from lymph nodes.     

Hydrocortisone and the antibody response in mice. I. Correlations between serum cortisol levels and cell numbers in thymus, spleen, marrow and lymph nodes.

Mice injected with a single well tolerated dose of hydrocortisone acetate were observed over 2--3 weeks for serum cortisol levels and for cell depletion in thymus, spleen, femoral marrow, mesenteric, inguinal and popliteal lymph nodes. Serum cortisol peaked within 24 h and declined to normal after 4 days. Total marrow cell numbers were relatively unaffected, but in all other tissues studied, cell depletion was severe and prolonged. B lymphocytes were affected more severely than T lymphocytes. There was a transient increase in the percentage of marrow T lymphocytes but otherwise little change. The percentage of node T lymphocytes increased while that of B lymphocytes decreased. The percentage of spleen B lymphocytes was reduced severely but transiently during the period of serum cortisol elevation. Spleen T lymphocyte percentages rose steadily between the fourth and seventh days after treatment, then returned to normal. Representatives of most types of lymphoid tissue were studied. As cell losses in any one were not compensated by gains in any other, most were probably due to destruction rather than redistribution. The slow rates of recovery were also more consistent with regeneration than with reappearance after redistribution.     

Migration of blood-borne lymphocytes into antigen-stimulated lymph nodes. A study of the kinetics of lymphocyte recruitment throughout an immune response

The kinetics of lymphocyte recruitment into rat popliteal lymph nodes stimulated with varying doses of allogeneic lymphocytes or purified protein derivative of tuberculin differed markedly. The number of blood-borne lymphocytes entering lymph nodes stimulated with allogeneic lymphocytes was greater and the duration of their entry more prolonged than in lymph nodes stimulated with the soluble antigen purified protein derivative of tuberculin. When individual antigenic doses were compared, lymphocyte recruitment into popliteal lymph nodes was shown to be dependent upon antigenic dose; higher doses of antigen resulting in increased levels of lymphocyte recruitment. In addition it was found that there is no linear relationship between lymph node weight changes and the extent of lymphocyte recruitment.     

Tridimensional study of the deep cortex of the rat lymph node. V: Postnatal development of the deep cortex units

ABSTRACT Recently, the deep cortex of the adult rat lymph node was shown to be made up of semirounded lymphocytic “units.” Each unit is contiguous to the peripheral cortex, bulges into the medulla, and is centered on the opening(s) of an afferent lymphatic vessel of a node. Furthermore, each unit comprises a “center” and a “periphery,” bearing distinct morphological features. The present study investigated the postnatal development of the units in rats of various ages. One minute after birth, no lymphocytic structures were detected in the nodes. One day after birth, tiny rounded lymphocytic areas were detected in the developing cortex. These areas were topographically related to the openings of afferent lymphatic vessels. One week after birth, small semirounded lymphocytic areas with some morphological features of the adult deep cortex units were observed. Two weeks after birth, typical units were present in the nodes. The observations indicated that the rounded lymphocytic areas observed in nodes of rats aged 1 week or less were actually developing deep cortex units. The overall findings further provided information on the morphological processes involved during the postnatal development of the deep cortex units. Key words: lymph node, deep cortex, development of deep cortex, rat.     

Tumor-induced sentinel lymph node lymphangiogenesis and increased lymph flow precede melanoma metastasis

Lymphangiogenesis is associated with human and murine cancer metastasis, suggesting that lymphatic vessels are important for tumor dissemination. Lymphatic vessel alterations were examined using B16-F10 melanoma cells implanted in syngeneic C57Bl/6 mice, which form tumors metastasizing to draining lymph nodes and subsequently to the lungs. Footpad tumors showed no lymphatic or blood vessel growth; however, the tumor-draining popliteal lymph node featured greatly increased lymphatic sinuses. Lymph node lymphangiogenesis began before melanoma cells reached draining lymph nodes, indicating that primary tumors induce these alterations at a distance. Lymph flow imaging revealed that nanoparticle transit was greatly increased through tumor-draining relative to nondraining lymph nodes. Lymph node lymphatic sinuses and lymph flow were increased in mice implanted with unmarked or with foreign antigen-expressing melanomas, indicating that these effects are not due to foreign antigen expression. However, tumor-derived immune signaling could promote lymph node alterations, as macrophages infiltrated footpad tumors, whereas lymphocytes accumulated in tumor-draining lymph nodes. B lymphocytes are required for lymphangiogenesis and increased lymph flow through tumor-draining lymph nodes, as these alterations were not observed in mice deficient for B cells. Lymph node lymphangiogenesis and increased lymph flow through tumor-draining lymph nodes may actively promote metastasis via the lymphatics.     

Response in distant lymph nodes of mice to infection in the hind footpad with Mycobacterium marinum.

In an attempt to demonstrate the importance of the popliteal lymph node in limiting the progress of infection with Mycobacterium marinum in the hind footpads of C57BL mice, such infections were studied in mice subjected to popliteal or popliteal and inguinal adenectomies. In the absence of the popliteal node, the footpad infection was only slightly enhanced compared with infections of sham-operated control mice; the inguinal node was found to be greatly enlarged and appeared to have substituted for the absent popliteal node. In the absence of both popliteal and inguinal nodes, the disease process in the footpads was again only slightly enhanced, and the axillary node appeared to have enlarged greatly and to have functionally replaced the missing, more proximate nodes. In additional experiments, mice subjected to adenectomy only on one side and injected in that hind footpad with phytohemagglutinin or India ink demonstrated hypertrophy or deposition of carbon particles in the more distant node only on the side of the injection. Thus, there appear to be rather direct functional connections among popliteal, inguinal, and axillary nodes that do not depend on blood circulation.     

Patterns of age-dependent changes in the numbers of lymph follicles and germinal centres in somatic and mesenteric lymph nodes in growing C57Bl/6 mice

The timing of the first appearance of lymph follicles and germinal centres in various lymph nodes, and the ways in which numbers of these and IgM-synthesising cells increase within the nodes, were investigated in male and female C57Bl/6N mice aged from 4 d to 16 wk. The lymphoid organs examined were the Peyer's patches, spleen, somatic (submandibular, deep cervical, brachial, axillary, inguinal and popliteal) and visceral (mesenteric and lumbar) lymph nodes. Primary follicles appeared in most somatic lymph nodes 6 d after birth. The number of follicles per node then increased rather sharply in larger lymph nodes and slowly in smaller nodes, up to 28 d of age, reaching a level which varied according to the location of the node. Thereafter, the number of follicles in the somatic lymph nodes increased only slightly to moderately, reaching a peak or plateau at 8–12 wk. In the mesenteric (ileocaecal) nodes, primary follicles first appeared at 12 d, then increased linearly during the suckling period and after weaning to reach a plateau at 8 wk of age. Germinal centres appeared in the submandibular and mesenteric nodes at 28 d and their numbers increased consistently in the latter, while remaining low in the former. The impact of possible 'natural' exogenous antigen stimulation of the various lymph nodes was estimated from the presence of IgM-synthesising cells and germinal centres. Differences between the patterns of age-dependent changes in the numbers of lymph follicles observed in the somatic and mesenteric lymph nodes during their ontogeny are discussed in relation to differences in the magnitude of the exogenous antigen stimulatory effect. We also found that the variations in the numbers of lymph follicles produced in somatic lymph nodes at different locations during the first 28 d after birth reflected differences in the dimensions of the body regions drained by a particular somatic lymph node at this stage of development.     

Fate of Autogenous Canine Lymphocytes after Injection into an Afferent Lymphatic of the Popliteal Lymph Node

Dog thoracic duct lymphocytes were labeled in vitro with ³H-uridine and infused into an afferent lymphatic of the popliteal lymph node of the same dog. Ten minutes after infusion nearly all the injected radioactivity was recovered from the lymph node. An effect of infusion flow rate on the percentage of cells retained by the lymph node was observed ½ to 3½ hours after infusion, and was probably mediated by the tendency of the node to become edematous after infusions at a rate exceeding 0.045 ml/min. Edematous nodes retained 83.7% of the cells, as compared to 47.5% for nonedematous nodes. As early as 30 minutes after infusion a small amount of ³H-radioactivity was found in the spleen and thoracic duct lymph. The deep iliac and paraaortic nodes on the side of the infusion contained significant amounts of ³H-radioactivity, while negligible amounts were detected in the contralateral popliteal node at any time. The intranodal localization of the ³H-labeled cells was studied by radioautography. All labeled cells remained intrasinusoidal during the first 4 hours after infusion. At 9 and 21 hours some labeled cells were located in the extrasinusoidal parenchymal lymphoid tissue of the cortex and the medulla, but the majority still remained intrasinusoidal.     

Tridimensional study of the deep cortex of the rat lymph node. II: Relation of deep cortex units to afferent lymphatic vessels

Recently, we reported that the deep cortex of the rat lymph node is formed of semi-rounded structures, the “deep cortex units,” contiguous to the peripheral cortex and bulging into the medulla. It was suggested that a unit represents an accumulation of lymphocytes centered on the opening of an afferent lymphatic vessel. To verify the proposal, we carried out a tridimensional analysis of serially sectioned rat nodes, fixed by perfusion and trimmed in such a way as to preserve their lymphatics. The tridimensional analysis revealed that a constant topographical relationship exists between the units and the openings of the afferent lymphatics. The results demonstrated that the topographical organization of the deep cortex of a rat node correlates with the distribution pattern of the opening(s) of its afferent lymphatic(s). The overall observations suggested the following explanation for the shape and topography of the units: factor(s) present in the lymph would spread in a radial manner from the opening(s) of an afferent lymphatic through the underlying cortex. The factor(s) would induce morphological modifications in the stimulated semi-rounded area which, in turn, would provoke a local accumulation of circulating lymphocytes.