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The influence of afferent lymphatic vessel interruption on vascular addressin expression.

Tissue-selective lymphocyte homing is directed in part by specialized vessels that define sites of lymphocyte exit from the blood. These vessels, the post capillary high endothelial venules (HEV), are found in organized lymphoid tissues, and at sites of chronic inflammation. Lymphocytes bearing specific receptors, called homing receptors, recognize and adhere to their putative ligands on high endothelial cells, the vascular addressins. After adhesion, lymphocytes enter organized lymphoid tissues by migrating through the endothelial cell wall. Cells and/or soluble factors arriving in lymph nodes by way of the afferent lymph supply have been implicated in the maintenance of HEV morphology and efficient lymphocyte homing. In the study reported here, we assessed the influence of afferent lymphatic vessel interruption on lymph node composition, organization of cellular elements; and on expression of vascular addressins. At 1 wk after occlusion of afferent lymphatic vessels, HEV became flat walled and expression of the peripheral lymph node addressin disappeared from the luminal aspect of most vessels, while being retained on the abluminal side. In addition, an HEV-specific differentiation marker, defined by mAb MECA-325, was undetectable at 7-d postocclusion. In vivo homing studies revealed that these modified vessels support minimal lymphocyte traffic from the blood. After occlusion, we observed dramatic changes in lymphocyte populations and at 7-d postsurgery, lymph nodes were populated predominantly by cells lacking the peripheral lymph node homing receptor LECAM-1. In addition, effects on nonlymphoid cells were observed: subcapsular sinus macrophages, defined by mAb MOMA-1, disappeared; and interdigitating dendritic cells, defined by mAb NLDC-145, were dramatically reduced. These data reveal that functioning afferent lymphatics are centrally involved in maintaining normal lymph node homeostasis.

L PHOCYTES migrate continuously between various lymphoid and extra-lymphoid tissues of the body by way of the blood and lymph vascular systems. Lymphocyte entry into peripheral lymph nodes, mucosal lymphoid tissues, and sites of chronic inflammation is directed in part by tissue-selective interactions between blood-borne lymphocytes and specialized cells lining postcapillary high endothelial venules (HEV) . I Functional studies have revealed that lymphocyte adhesion to HEV is governed at the lymphocyte level by homing receptors (6,12,13,18), and at the endothelial cell level by vascular addressins (21,22). In the mouse system, two structurally and functionally distinct vascular addressins have been described. The peripheral lymph node addressin mediates the interaction of lymphocytes with HEV in peripheral lymphoid organs (22), and the mucosal addressin directs equivalent cellular interactions in mucosal lymphoid tissues (21). The peripheral lymph node and mucosal addressins are defined, and the adhesive interactions directed by these molecules are functionally 1 flat walled and expression of the peripheral lymph node addressin disappeared from the luminal aspect of most vessels, while being retained on the abluminal side . In addition, an HEVspecific differentiation marker, defined by mAb MECA 325, was undetectable at 7-d postocclusion. In vivo homing studies revealed that these modified vessels support minimal lymphocyte traffic from the blood. After occlusion, we observed dramatic changes in lymphocyte populations and at 7-d postsurgery, lymph nodes were populated predominantly by cells lacking the peripheral lymph node homing receptor LECAM-1. In addition, effects on nonlymphoid cells were observed : subcapsular sinus macrophages, defined by mAb MOMA-1, disappeared ; and interdigitating dendritic cells, defined by mAb NLDC-145, were dramatically reduced. These data reveal that functioning afferent lymphatics are centrally involved in maintaining normal lymph node homeostasis.
Several reports indicate the importance of afferent lymph in lymphocyte migration into lymph nodes (3,11). Hendriks et al. described a model in which interruption of afferent lymphatic vessels of rat popliteal lymph nodes results in a flattening of high endothelial venule cells, and a decline in lymphocyte influx into modified lymph nodes (11). Within 1 d after afferent lymphatic occlusion lymphocyte influx is observed to decrease and this decrease is followed by the flattening of high endothelial cells (3). Here we have adapted this model to the mouse system, to study the influence of afferent lymphatic vessel interruption on vascular addressin expression, and the presence and distribution oflymph node leukocyte populations.

Materials and Methods
Animals BALB/c mice were either purchased from Bomholtgard (Ry, Denmark), or were obtained from the animal colony maintained at SySternix . All animals were kept under routine laboratory conditions .

85
Occlusion ofAf ferent Lymphatics Mice were anaesthetized by intramuscular injection of 1 pl/g body weight of a 4 :3 mixture of Aescoket (Aesculaap N. V., Gent, Belgium) and Rompun (Bayer, Leverkussen, Germany) . The operative procedure was adapted from the procedure developed for the rat (9) . Briefly, popliteal lymph nodes were exteriorized, and afferent lymphatic vessels were severed. Efferent lymphatic vessels and blood vessels, emerging from the hilus, were left intact . Mice were killed at different timepoints after afferent lymphatic vessel interruption . Before excision of the operated lymph node, 0.05 ml of a 10 % India ink solution was injected into the ipsilateral footpad to determine the completeness of the operation. Lymph nodes with intact afferent lymphatics, as demonstrated by the uptake of ink, were discarded . The contralateral popliteal lymph node served as unoperated control tissue. Excised lymph nodes were snap-frozen in liquid nitrogen and stored at -20°C until use.
In Vitro HEV-binding Assay Lymphocyte adherence to HEV in vitro was assayed by the method of Butcher et al. (1), a modification of the assay originally described by Stamper and Woodruff (20) . Briefly, lymphocyte suspensions, containing 1 x 106 cells/100 ul were incubated at 4°C on unfixed frozen sections (8 Pm) of operated and control lymph nodes . During the assay, lymph node sections were rotated for 30 min . Upon completion of the assay, unbound The Journal of Cell Biology, Volume 115, 1991 cells were decanted, and sections were fixed in PBS containing 1% glutaraldehyde . Cell binding to HEV was assessed microscopically under darkfield or normal illumination after staining by methyl green-pyronin, and the mean number of bound cells per HEV was determined .

Flow Cytometric Analysis
Cell fluorescence was analyzed with a FACStar°(a registered trademark of Becton-Dickinson, Mountain View, CA). Single-cell suspensions were made from control and operated lymph nodes. For triple-color immunofluorescence staining, the following procedure was used . Briefly, cells were treated with the mAb MEL-14, washed in HBSS containing 2 k FCS, and treated with Texas red-conjugated goat anti-rat IgG (Caltag Laboratories, San Francisco, CA) in HBSS/FCS containing 5% normal mouse serum. After washing in HBSS/FCS the cells were incubated for 5 min with an excess of rat Ig. The cells were then incubated with FITC-conjugated 53-2 .1 and biotin-conjugated 6B2. After an additional wash, cells were incubated with phycoerytherin conjugated streptavidin (Biomeda, Foster City, CA). Each incubation step took 20 min and was performed at 4°C. In addition to the above three colors, forward angle scatter was measured and used to gate out debris and dead cells.

Lymphocyte Homing
In Intro Labeling of Lymphocytes with s'Cr. Lymph node cells (obtained from axillary, brachial, inguinal, and mesenteric lymph nodes) and spleen cells from BALB/c mice were pooled and labeled with S'Cr as described earlier (21) . Briefly, lymphocytes were labeled by incubation of 2.5 x 107 cells/ml for 1.5 hat 37°C, with mixing every 15 min, in DME supplemented with 20 mM Hepes, 5% FCS, and 100 pCi/nil sodium chromate (Na2 5'Cr04; New England Nuclear, Boston, MA). After labeling, the cells were centrifuged through a layer of FCS, and washed twice with HBSS . 2.5 x 107 chromium-labeled cells were injected intravenously into each mouse, which were operated on 5 h, 1, 5, or 8 d previously. Lymphocyte localization was determined by quantitating the cpm in the operated and the contralateral control node . Results are presented as percent of contralateral control (±SD).

Results
The Influence ofAfferent Lymphatic Occlusion on the Lymph Node When afferent lymphatic vessels of popliteal lymph nodes were interrupted, and the nodes replaced in their original position (i .e ., within the popliteal fossa), afferent lymphatic Mebius et al . Afferent Lymph Supply and Vascular Adressin Expression vessels were found to reconnect to the operated lymph nodes within 7 d. To prevent this reassociation, we attached the operated lymph nodes, with intact efferent lymphatics and  blood supply, onto the fascia directly under the skin . In this subcutaneous position afferent lymphatic vessels draining the footpad were not able to reassociate with the node. The original blood supply remained intact during these manipulations. Reconnection of afferent lymphatic vessels was assessed by injection of India ink into footpads 20 min before lymph nodes excision .
The Journal of Cell Biology, Volume 115, 1991 Removal of afferent lymphatics had an effect on lymph node size, and within 1 wk of interruption, the number of lymphocytes present within manipulated lymph nodes had diminished considerably. At 7 d after lymphatic vessel interruption, T cell zones were markedly depleted of T cells, and the follicles within the lymph nodes had decreased in size ( Fig . 1) . In contrast to normal lymph node compartmentalization, increased numbers of B cells were observed in T cell zones in manipulated lymph nodes. The number of interdigitating cells in the T-cell dependent areas, stained by mAb NLDC-145, had diminished by day 4 after occlusion ( Fig . 2) . At 7 d after surgery, weakly NLDC-145-positive cells were present in the paracortical area, while strong positive cells, characteristically found in the cortical area, were not detectable. MOMA1-positive subcapsular sinus macrophages were observed in the paracortical area at 4 d after surgery (Fig.  3), and were not detectable by day 7 after lymphatic vessel occlusion (Fig. 4) .
The Influence ofAfferent Lymphatic Occlusion on Lymph Node High Endothelial Venules Hendriks et al . have reported that interruption of lymph node afferent lymphatic vessels has anegative effect on HEV function and morphology (11). Their study was done in rats, however, where mAb defining vascular addressins or specific markers of HEV differentiation are not available . In the present investigation, involving mice rather than rats, the effects of afferent lymphatic vessel occlusion on HEV was assessed . Expression of both an HEV specific differentiation marker, defined by mAb MECA325, and the peripheral lymph node addressin as recognized by mAb MECA-79 was evaluated at 88 Figure 5. Expression ofthe peripheral lymph node addressin in control and operated lymph nodes at day 4 after afferent lymphatic interruption. A shows the pronounced addressin expression on high walled endothelial cells in the control nodes. At 4 d after occlusion of afferent lymphatics more flat walled vessels could be observed (B) . Some vessels showed a loss ofthe peripheral lymph node addressin (C) . In most cases the addressin was still expressed on the luminal side of the venules . Bar, 40 um . 4 d after afferent lymphatic vessel interruption, expression of the peripheral lymph node addressin was observed on both high-and flatwalled vessels (Fig . 5) . These vessels were also weakly positive for the HEV differentiation antigen MECA-325, with expression sometimes restricted to the luminal side of the endothelium (Fig . 6) . It was striking that some high endothelial cells of several individual HEV expressed the mucosal addressin (Fig . 7) . While normal peripheral lymph node HEV can express low levels of the mucosal addressin (21), increased numbers of mucosal addressin expressing high endothelial cells were observed in lymph nodes 4 d after afferent lymphatic occlusion . 7 d after occlusion of afferent lymphatic vessels, low levels of the MECA-325 antigen were observed on rare HEV; peripheral lymph node addressin expression appeared to be restricted, in most instances, to the abluminal aspect of relatively flat-walled endothelium (Fig. 8) . The abluminal expression was confirmed by the intravenous injection of MECA-79, which failed to localize at high levels in operated lymph nodes, but did localize in the contralateral control nodes of the same animal (Fig. 9) . Restriction of the peripheral lymph node addressin to the abluminal side of the vessels, with no expression of the mucosal addressin, suggested Mebius et al . Afferent Lymph Supply and Vascular Adressin Expression that these vessels would not support high levels of lymphocyte adhesion or lymphocyte trafficking from the blood into operated lymph nodes . Indeed, in vivo homing studies revealed that homing into operated lymph nodes at 8 d after operation was decreased by >80% (Fig. 10) . Decreased entrance of lymphocytes started at around day 5. At this timepoint, trafficking of labeled cells into operated lymph nodes was comparable to the contralateral control nodes, but varied greatly between animals . The binding of lymphocytes to these vessels at day 4, measured in an in vitro binding assay, was decreased by >30 % (Fig . 11) . At 7 d after lymphatic occlusion HEV failed to support lymphocyte adhesion .

The Effect ofAfferent Lymphatic Occlusion on Peripheral Lymph Node Homing Receptor Expression
The rapid disappearance of lymphocytes from afferent lymphatic vessel occluded lymph nodes prompted us to study the phenotype of residual lymph node cell populations by  fascia of four animals was opened, and the nodes were replaced in the popliteal fossa, the original popliteal lymph node environment . During this manipulation the efferent lymphatics and the blood supply were unaffected . 2 wk after replacing the nodes into the popliteal fossa, we examined the nodes of the two groups by immunchistochemistry. 20 min before excision of the nodes, India ink was injected into the ipsilateral footpad to detect reconnected afferent lymphatic vessels . In the group that had their lymph nodes replaced in the popliteal fossa, three out of four lymph nodes contained a small amount of India ink, demonstrating partial restoration of afferent lymphatic vessels . Although these nodes were small in size, staining with mAb specific for T and B cells showed a well-compartmentalized T cell zone, and a B Figure 8 . Expression of the peripheral lymph node addressin and MECA-325 antigen by vessels in control and operated lymph nodes (day 7) . The morphologically distinct HEV of control lymph nodes, which express high levels of the peripheral lymph node addressin (A), are quite distinct from their counterparts in operated lymph nodes . B illustrates the modified expression of the peripheral lymph node addressin in lymph nodes 7 d after interruption of afferent lymphatics . The vessels became flat walled, and expression of the peripheral lymph node addressin appeared restricted to the abluminal aspect of the vessels. At 7 d after occlusion of afferent lymphatics the vessels did not show any expression of the MECA-325 antigen (C) . Bar, 40 pm .
cell area comparable to normal control lymph nodes . Interdigitating cells in the T cell dependent areas within these nodes were strongly positive for NLDC-145, and these cells were abundant (Fig. 14) . Within this relatively short timeperiod, we also observed a restoration of the MOMA-1-positive subpopulation of macrophages in the subcapsular sinus (Fig . 14) . Staining with the HEVspecific marker MECA-325 showed high walled HEV, which were strongly positive for the peripheral lymph node addressin . The node replaced in the popliteal fossa that had not reconnected its afferent lymphatics was too small for analysis . The lymph nodes which were left on top of the fascia for 3 wk, rather than being replaced in the popliteal fossa, showed small B cell compart-Mebius et al . Afferent Lymph Supply and Vascular Adressin Expression ments, with B cells present in the T cell zones . No MOMA-1positive subcapsular sinus macrophages were seen in these nodes and NLDC-145-positive cells were not as well represented as in nodes which had been replaced in the popliteal fossa . Interestingly, while India ink did not drain from the footpad into these nodes, we observed an intermediate phenotype of HEV, with both high and flat walled vessels expressing the peripheral lymph node addressin and the MECA-325 antigen (Fig . 15) .

Discussion
Interruption of afferent lymphatic vessels to mouse popliteal lymph nodes affected the function and phenotype of HEV. 7 d after occlusion of the lymph supply to lymph nodes, HEV became flat walled and expression of the HEV specific differentiation marker MECA-325 had disappeared . At this time, the peripheral lymph node addressin was restricted in most cases to the abluminal side of the vessels, a site where it would not be available to circulating lymphocytes, and functional in vitro and in vivo assays confirmed that these vessels did not support normal levels of lymphocyte adhesions and homing, respectively.
Removing afferent lymphatic vessels had a traumatic effect on lymphocyte entry into lymph nodes : 5 h after lymphatic vessel interruption the entrance of labeled cells had diminished by 70% compared to the contralateral control node in the same animal . This early effect of surgery, likely related to trauma to operated nodes, was short lived, with recovery occurring 4-5 d after surgery. After 5 d, however, the lack of incoming lymph appeared to begin to have an effect on lymph node function . At this timepoint the entrance of lymphocytes was still comparable to the contralateral control nodes. However, lymphocyte homing in different animals was highly variable, suggesting that changes occur around this time . Immunohistologic assessment of lymph nodes at 4 d after surgery also showed significant variation : both high and flatwalled vessels were observed, and some venules exhibited a partial loss of the peripheral lymph node addressin . In most cases, however, peripheral addressin was still expressed on the luminal side of the venules .
The observed abluminal expression of the peripheral lymph node addressin, the flat appearance of the HEV, and the low trafficking of lymphocytes into operated lymph nodes at 8 d after afferent lymphatic occlusion are all in agreement with each other. Absence of the peripheral lymph node addressin on the luminal aspect of the vessels prevents the adhesion of lymphocytes to HEV. As a consequence lymphocytes cannot enter the nodes, which is shown in in vivo homing studies . Previous studies have shown that decreased migration of lymphocytes across HEV can cause flattening of HEV, which become high walled again after reconstitution with cells that migrate across HEV (7,23) . Our observation of flat walled venules in lymph nodes not receiving afferent lymph could therefore be a consequence of impaired lymphocyte migration across the venular wall . Our experiments however showed that the effect of afferent lymphatic vessel interruption on HEV morphology could be reversed when the nodes were left on top of the fascia for an additional 2 wk . These data suggest that, if lymph-borne factors or  Figure 10. In vivo lymphocyte homing to operated lymph nodes. Afferent lymphatics to popliteal lymph nodes were interrupted and "Cr-labeled lymphocytes were injected intravenously at 5 h, 1, 5, or 8 d after operation . 1 h after injection of labeled lymphocytes recipients were killed and cell localization in operated and contralateral control nodes was determined by quantitating the cpm in these lymph nodes. Relative lymphocyte localization in operated lymph nodes is expressed as a percent of lymphocytes localizing in the contralateral control nodes . Three to six animals were included in each group . Occlusion of afferent lymphatic vessels resulted in an early reduction in lymphocyte homing into operated lymph nodes. This reduction was reversed during the days following occlusion. A pronounced effect of lymphatic occlusion on lymphocyte homing into operated lymph nodes was observed at day 8 after afferent lymphatic vessel occlusion . Figure 11. In vitro binding of lymphocytes to HEV in operated and control lymph nodes . Interruption of afferent lymphatics induced a 30% decrease (±SD) in binding of lymphocytes to HEV at day 4 compared to control nodes (day 0), measured in an in vitro binding assay. At 7 d after afferent lymphatic occlusion no specific lymphocyte binding to vessels in the nodes was observed .
cells are responsible for HEV function, they may be arriving by way of newly connected lymphatics . Alternatively, HEV morphology and function could be regulated by resident cells within lymph nodes, or factors Mebius  produced by such cells. The effects of afferent lymph on lymph node macrophage populations have been described previously in rats (10) and it was suggested that macrophages are directly or indirectly responsible for HEV function . Previously we reported in the rat system that subcapsular ED3 macrophages disappeared upon interruption of afferent lymphatic vessels (17) . In the present study, this finding was confirmed for MOMA-1 positive subcapsular macrophages in the mouse. However, when HEV regained their characteristic morphology and phenotype in lymph nodes in the subcutaneous site at 3 wk after surgery, this population of macrophages was not replaced, revealing that the presence of this lymph node macrophage population is not required for HEV differentiation . The MOMA-1-positive subcapsular macrophage population was restored rapidly however, when lymph nodes were replaced in the popliteal fossa, and afferent lymphatics allowed to connect . These data suggest that MOMA-1-positive cells in the lymph node turn over rapidly, and that they can be replaced by cells entering via afferent lymphatics draining the footpad .  While the physiological processes controlling HEV induction and vascular addressin expression are unclear, a role for interdigitating cells in the HEV induction process has been suggested (3) . Our experiments, in which lymph nodes were left on top of the fascia for longer periods support these findings . 7 d after disconnection of afferent lymphatics, the NLDC-145 antigen was weakly expressed on interdigitating cells and HEV had become flat walled . Interdigitating cells might either have left the lymph node or lost their ability to express the NLDC-145 antigen as a consequence of afferent lymphatic vessel occlusion . At 3 wk after surgery increased levels, as compared to 1 wk after occlusion, of the NLDC-145 antigen was observed in lymph nodes with both high and flat walled vessels expressing the MECA-325 antigen andthe peripheral lymph node addressin . The correlation between NLDC-145 antigen expression and HEV induction, including peripheral lymph node addressin expression, suggests a role for interdigitating cells in this inductive process .
The importance of soluble factors in HEV differentiation and function should also be considered . Experiments in which we diminished the entrance of soluble factors and/or cells to a greatest extent by surrounding the nodes with parafilm, revealed lymph nodes with HEV which became flat walled more rapidly, and had fewer LECAM-1 expressing cells than lymph nodes without parafilm (results not shown) . 94 4 d after interruption of the afferent lymph supply to lymph nodes, a decrease in the percentage of LECAM-1 expressing B cells was observed. By contrast, a decrease in the percentage ofLECAM-1 expressing T cells was not observed until day 7 after occlusion . It should be mentioned that operated lymph nodes as a whole had decreased in size by day 4 postsurgery. The observed early decrease in the percentage of LECAM-1 expressing B cells in operated lymph nodes could be explained by a direct effect of lymph flow on lymphocyte migration to B cell follicles . B cells home to follicles in a highly selective manner, migrating against lymph flow (8), and having a variable transit time (5) . Interruption ofthe lymph flow may disrupt lymph node compartmentalization by preventing the directed migration of B cells to follicular regions . Those B cells not localizing in follicles may leave the node more rapidly. Our observation of increased numbers of B cells within the T cell regions is consistent with an inability of B cells to localize in follicular regions . Alternatively, interruption of lymph flow may directly induce a loss of follicular integrity, a scenario which would also explain both the presence of B cells in the T cell zones, and the rapid depletion of B cells from the lymph node. In contrast to our studies, investigations ofprolonged thoracic duct cannulation indicated that T cells leave lymph nodes more rapidly than B cells (19) . These studies differ from our own, since the afferent lymph supply was intact .
In conclusion, our experiments demonstrate the crucial role of lymph-borne cells and/or factors in the presence, and in some cases, the phenotype of cell populations within lymph nodes . Upon afferent lymphatic occlusion, venules that normally support lymphocyte migration lose their high walled phenotype, they show decreased expression ofthe peripheral lymph node addressin on the luminal side, and they do not support high levels of lymphocyte homing. Lymph node leukocyte populations are also influenced, with dramatic losses of B cells, T cells, subcapsular sinus macrophages, and interdigitating cells. While these leukocyte population shifts are due, at least in part, to reduced cellular trafficking into lymph nodes via the blood, a direct effect of lymph components on cellular compartmentalization and cellular retention within lymph nodes is also possible . These observations reveal that lymph node homeostasis is maintained by the complex cellular and molecular interactions that occur in normal lymph nodes ; interactions that are dramatically altered by afferent lymphatic occlusion .