248
Agents and Actions vol. 6 . 1 / 2 / 3 (1976) Birkh~iuser Verlag, Basel
The Migration of Lymphocytes across Vascular Endothelium W. L. Ford and M. Sedgely Department of Pathology, Stopford Building, University of Manchester, U K
Abstract According to Morris and his colleagues the migration of lymphocytes from the blood into inflammatory sites is a morphologically similar process to the large scale migration of lymphocytes into the paracortex of lymph-nodes which occurs under normal conditions. Little is known of the nature of the selective interaction between lymphocytes and specialized endothelial cells although several investigators have tested the migratory properties of lymphocytes which have been treated in vitro with enzymes or lectins and injected i.v, Such results are difficult to inerpret because decreased lymph-node localization may be secondary to increased localization elsewhere, To overcome these difficulties the mesenteric lymphnode chain of the rat was perfused at 37~ for 30-60 minutes with rat serum at 0.33 ml/min, W h e n labelled lymphocytes were added to the perfusate the uptake of cells into the lymph-nodes was 2.134-0.63% of the number of which had passed through the preparation and this fraction was independent of the concentration in the perfusate over a wide range. No release of lymphocytes from the isolated node into the perfusate could be detected. Neuraminidase treated lympbocytes localized in the perfused lymph-node at least as well as did untreated lymphocytes in contrast to their deficient lymph-node localization following i.v. injection into intact recipients as originally found by Gesner. Other evidence has been found that the primary effect of neuraminidase treatment is to increase hepatic localization. Large lymphocytes migrate into the isolated node more readily than do small lymphocytes which is also in contrast to results in intact recipients. The migration of lymphocytes treated with trypsin or concanavalin A has also been studied.
Results Lymphocytes migrate from the blood into lymphoid organs such as the spleen and lymphnodes in large numbers [1]. However under normal conditions lymphocyte migration from the blood into non-lymphoid tissue is on a much smaller scale as has been shown by the ex-
periments of Morris and his colleagues who measured the flux of tymphocytes in peripheral lymph [2]. By contrast in chronically inflamed tissues the influx of lymphocytes from the blood is enormously increased and this has been associated with a morphological change in the endothelium of the venules so that it comes to resemble the cuboidal endothelium which is seen in the paracortical region of lymph-nodes [3]. There is no proof that identical mechanisms produce a high flux of lymphocytes into normal lymph-nodes and into non-lymphoid tissues which are inflamed. In both cases the endothelial factors permitting the adherence and the transport of increased numbers of lymphocytes are completely unknown. This preliminary report deals with the mechanism of lymphocyte migration into lymph-nodes. We intend to apply these findings to the problem of lymphocyte migration into inflamed, non-lymphoid tissues at a later stage. The selective interaction between lymphocytes and specialized endothelial ceils in lymphnodes was studied by Gesner and his colleagues [4-6] who found that the treatment of lymphocytes with several enzymes altered their migration pattern when injected i.v. into recipients. However these interesting results are difficult to interpret with certainty since decreased localization of the treated cells in one organ may be a consequence of increased localization in another organ such as the liver. To overcome these difficulties we have studied the migration of enzyme treated rat lymphocytes into an isolated lymph-node preparation and also in vivo after i.v. injection into syngeneic recipients. In the latter case control
The Migration of Lymphocytesacross Vascular Endothelium lymphocytes alternatively labelled to the treated lymphocytes (3H or 14C) were always injected simultaneously and the ratio of the two labels was measured in the blood as well as in several lymphoid tissues. This gives a precise indication of any alteration of the migratory pattern of the treated lymphocytes. The mesenteric lymph-node chain of the rat was perfused at 37 ~ for 30-60 minutes with rat serum; some surrounding connective tissue was inevitably perfused as well. In most experiments the labelled lymphocytes were added to the perfusate. At the end of the 30-60minute perfusion period and after washing out the node with cell free serum the retention of labelled cells in the lymph-node was measured by scintillation counting of the isolated node dissected free of the surrounding connective tissue. The uptake of cells into the lymph-node was 2.13+0.63% of the number which had passed through the preparation and this fraction was independent of the number of lymphocytes in the perfusate over the range of 3 • 106/ml to 40 • 106/ml. Autoradiography of the perfused node showed that the localization of labelled lymphocytes was in and around the post-capillary venules as is seen at early intervals after i.v. injection into intact recipients. By light and electron microscopy the perfused nodes were normal except for some oedema of the medulla. The isolated lymph-node did not release any lymphocytes into a cell-free perfusate. This is telling evidence against a 'direct-entry' of lymphocytes from the lymph-nodes into the blood especially when compared to earlier experiments on the isolated perfused rat spleen [7] which released large numbers of lymphocytes into the venous effluent. Neuraminidase-treated lymphocytes were added to serum along with alternatively labelled (3H/14C) control lymphocytes and perfused through an isolated node. The treated lymphocytes entered the node at least as well as did control lymphocytes in all five experiments. In contrast after i.v. injection into intact recipients neuraminidase-treated lymphocytes localized poorly in the lymph-nodes and spleen [5]. However the ratio of treated/control cells was just as low in the blood as it was in the lymphnodes and spleen. Neuraminidase treated cells are sequestered in the liver and the deficient localization in other tissues appears to be a consequence of this. Thus there is no evidence
249 that the surface structure responsible for lymphnode localization is neuraminidase sensitive. Large lymphocytes were selectively labelled in vitro using 3H-thymidine or 14Cthymidine. When injected i.v. they localized mainly in the lamina propria of the small gut and very poorly in lymph-nodes. However they migrated into the isolated lymph-node slightly better than did small lymphocytes suggesting that during lymphoblastic transformation lymphocytes acquire an affinity for vessels in the gut without losing their affinity for lymphnodes. After i.v. injection large lymphocytes may be removed by the gut before most of them have the opportunity to reach lymph-nodes. Surface membrane vesicles were prepared from lymphocytes by mechanical disruption and differential centifugation. When large numbers of such vesicles were perfused through an isolated node there was no sign of any adhesion of the vesicles to specialized endothelium. Technical factors may have militated against the adhesion of vesicles but a more likely explanation is that selective adhesion and migration require metabolically intact lymphocytes. We have confirmed that lightly trypsinized lymphocytes localize poorly in lymph-nodes after i.v. injection [6]. There is little change in hepatic and splenic localization. In the blood the ratio of trypsinized/control cells is greater than unity which suports the view that the action of trypsin is to selectively remove a recognition structure for lymph-node endothelium on the surface of lymphocytes. We have also confirmed Gillette's finding that concanavalin A treated lymphocytes show a decreased localization in lymph-nodes and an increased localization in the spleen 24 hours after injection [8]. However at earlier intervals treated cells localize poorly in both the spleen and lymph-nodes and are in excess in the liver. Concanavalin A may have at least two distinct effects on lymphocyte migration. In conclusion trypsin appears to be the only agent we have studied so far which directly interferes with lymphocyte/endothelial cell interaction. We intend to screen a number of other agents before building hypotheses of the mechanism of this interaction. This work was supported by Programme Grant G972/455/B from the Medical Research Council.
250
References [1] Gowans, J.L., and Knight, E.J., The route ofrecirculation of lymphocytes in the rat, Proc. Roy. Soc. [B] 159, 257 (1964). [2] Smith, J.B., McIntosh, G.H., and Morris, B, The traffic of cells through tissues: a study of peripheral lymph in sheep, J. Anat. 107, 87 (1970). [3] Smith, J.B., McIntosh, G.H., and Morris, B., The migration of cells through chronically inflamed tissues, J. Pathol. 100, 21 (1970). [4] Gesner, B.M., and Ginsburg, V., Effect of glycosidases on the fate of transfused lymphocytes, Proc. nat. Acad. Sci. 52, 750 (1964). [5] Woodruff, J.J., and Gesner, B.M., The effect ofneuraminidase on the fate of transfused lymphocytes, J. exp. Med. 129, 551 (1969). [6] Woodruff, J.J., and Gesner, B,M., Lymphocytes: Circulation altered by trypsin, Science 161, 176 (1968). [7] Ford, W.L., The immunological and migratory properties of the lymphocytes recirculating through the rat spleen, Br. J. exp. Path. 50, 257 (1969). [8] Gillette, R.W., McKenzie, G.O., and Swanson, M.H., Effect of eonvanavalin A on the homing of labelled T lymphocytes, J. Immunol. 111, 1902 (1973).
Discussion
Parrot (UK) This is not really a question but an additional comment to your last slide, with regard to the trypsin-treated lymphocytes. A young worker in our department has shown with, in vivo experiments, with trypsinized lymphocytes, and as you may, anticipate, if you splenectomize such mice, then not having the obstacle of the spleen to get over, they go to the lymph nodes.
W.L. Ford and M. Sedgley
Ford (UK) In fact we have injected some trypsinized lymphocytes into splenectomized recipients but have not got the results, yet.
Ziff(USA) I am just curious about the figure of 20:1 small: large 1 hour later. I don't understand that. Is that because they became small in the hour, or what? Ford (UK) These are the experiments on the isolated lymph-nodes, where we labelled large lymphocytes. We inject them into intact recipients, we found that the ratio of large to small in gut was about six or seven, and in lymph-nodes was about 0.06. So there is an enormous difference. Whether they become large or small we don't really know. If one looks autoradiographically at the earlier stages, and I think Delphine will agree with me, they still look large. But what happens to them eventually, we don't really know that. In the perfused lymph-nodes the large lymphocytes go in much better than small lymphocytes, in contrast to what tends to happen when you inject them into intact recipients, when if you look at the lymph-nodes the small go in twenty times better than the large. But what I am saying, is that it is simply because the large cells are picked out as they perfuse through the gut, and they never get a chance to go the lymph-nodes.
Agents and Actions vol. 6 . 1 / 2 / 3 (1976) Birkh~iuser Verlag, Basel
The Migration of Lymphocytes across Vascular Endothelium W. L. Ford and M. Sedgely Department of Pathology, Stopford Building, University of Manchester, U K
Abstract According to Morris and his colleagues the migration of lymphocytes from the blood into inflammatory sites is a morphologically similar process to the large scale migration of lymphocytes into the paracortex of lymph-nodes which occurs under normal conditions. Little is known of the nature of the selective interaction between lymphocytes and specialized endothelial cells although several investigators have tested the migratory properties of lymphocytes which have been treated in vitro with enzymes or lectins and injected i.v, Such results are difficult to inerpret because decreased lymph-node localization may be secondary to increased localization elsewhere, To overcome these difficulties the mesenteric lymphnode chain of the rat was perfused at 37~ for 30-60 minutes with rat serum at 0.33 ml/min, W h e n labelled lymphocytes were added to the perfusate the uptake of cells into the lymph-nodes was 2.134-0.63% of the number of which had passed through the preparation and this fraction was independent of the concentration in the perfusate over a wide range. No release of lymphocytes from the isolated node into the perfusate could be detected. Neuraminidase treated lympbocytes localized in the perfused lymph-node at least as well as did untreated lymphocytes in contrast to their deficient lymph-node localization following i.v. injection into intact recipients as originally found by Gesner. Other evidence has been found that the primary effect of neuraminidase treatment is to increase hepatic localization. Large lymphocytes migrate into the isolated node more readily than do small lymphocytes which is also in contrast to results in intact recipients. The migration of lymphocytes treated with trypsin or concanavalin A has also been studied.
Results Lymphocytes migrate from the blood into lymphoid organs such as the spleen and lymphnodes in large numbers [1]. However under normal conditions lymphocyte migration from the blood into non-lymphoid tissue is on a much smaller scale as has been shown by the ex-
periments of Morris and his colleagues who measured the flux of tymphocytes in peripheral lymph [2]. By contrast in chronically inflamed tissues the influx of lymphocytes from the blood is enormously increased and this has been associated with a morphological change in the endothelium of the venules so that it comes to resemble the cuboidal endothelium which is seen in the paracortical region of lymph-nodes [3]. There is no proof that identical mechanisms produce a high flux of lymphocytes into normal lymph-nodes and into non-lymphoid tissues which are inflamed. In both cases the endothelial factors permitting the adherence and the transport of increased numbers of lymphocytes are completely unknown. This preliminary report deals with the mechanism of lymphocyte migration into lymph-nodes. We intend to apply these findings to the problem of lymphocyte migration into inflamed, non-lymphoid tissues at a later stage. The selective interaction between lymphocytes and specialized endothelial ceils in lymphnodes was studied by Gesner and his colleagues [4-6] who found that the treatment of lymphocytes with several enzymes altered their migration pattern when injected i.v. into recipients. However these interesting results are difficult to interpret with certainty since decreased localization of the treated cells in one organ may be a consequence of increased localization in another organ such as the liver. To overcome these difficulties we have studied the migration of enzyme treated rat lymphocytes into an isolated lymph-node preparation and also in vivo after i.v. injection into syngeneic recipients. In the latter case control
The Migration of Lymphocytesacross Vascular Endothelium lymphocytes alternatively labelled to the treated lymphocytes (3H or 14C) were always injected simultaneously and the ratio of the two labels was measured in the blood as well as in several lymphoid tissues. This gives a precise indication of any alteration of the migratory pattern of the treated lymphocytes. The mesenteric lymph-node chain of the rat was perfused at 37 ~ for 30-60 minutes with rat serum; some surrounding connective tissue was inevitably perfused as well. In most experiments the labelled lymphocytes were added to the perfusate. At the end of the 30-60minute perfusion period and after washing out the node with cell free serum the retention of labelled cells in the lymph-node was measured by scintillation counting of the isolated node dissected free of the surrounding connective tissue. The uptake of cells into the lymph-node was 2.13+0.63% of the number which had passed through the preparation and this fraction was independent of the number of lymphocytes in the perfusate over the range of 3 • 106/ml to 40 • 106/ml. Autoradiography of the perfused node showed that the localization of labelled lymphocytes was in and around the post-capillary venules as is seen at early intervals after i.v. injection into intact recipients. By light and electron microscopy the perfused nodes were normal except for some oedema of the medulla. The isolated lymph-node did not release any lymphocytes into a cell-free perfusate. This is telling evidence against a 'direct-entry' of lymphocytes from the lymph-nodes into the blood especially when compared to earlier experiments on the isolated perfused rat spleen [7] which released large numbers of lymphocytes into the venous effluent. Neuraminidase-treated lymphocytes were added to serum along with alternatively labelled (3H/14C) control lymphocytes and perfused through an isolated node. The treated lymphocytes entered the node at least as well as did control lymphocytes in all five experiments. In contrast after i.v. injection into intact recipients neuraminidase-treated lymphocytes localized poorly in the lymph-nodes and spleen [5]. However the ratio of treated/control cells was just as low in the blood as it was in the lymphnodes and spleen. Neuraminidase treated cells are sequestered in the liver and the deficient localization in other tissues appears to be a consequence of this. Thus there is no evidence
249 that the surface structure responsible for lymphnode localization is neuraminidase sensitive. Large lymphocytes were selectively labelled in vitro using 3H-thymidine or 14Cthymidine. When injected i.v. they localized mainly in the lamina propria of the small gut and very poorly in lymph-nodes. However they migrated into the isolated lymph-node slightly better than did small lymphocytes suggesting that during lymphoblastic transformation lymphocytes acquire an affinity for vessels in the gut without losing their affinity for lymphnodes. After i.v. injection large lymphocytes may be removed by the gut before most of them have the opportunity to reach lymph-nodes. Surface membrane vesicles were prepared from lymphocytes by mechanical disruption and differential centifugation. When large numbers of such vesicles were perfused through an isolated node there was no sign of any adhesion of the vesicles to specialized endothelium. Technical factors may have militated against the adhesion of vesicles but a more likely explanation is that selective adhesion and migration require metabolically intact lymphocytes. We have confirmed that lightly trypsinized lymphocytes localize poorly in lymph-nodes after i.v. injection [6]. There is little change in hepatic and splenic localization. In the blood the ratio of trypsinized/control cells is greater than unity which suports the view that the action of trypsin is to selectively remove a recognition structure for lymph-node endothelium on the surface of lymphocytes. We have also confirmed Gillette's finding that concanavalin A treated lymphocytes show a decreased localization in lymph-nodes and an increased localization in the spleen 24 hours after injection [8]. However at earlier intervals treated cells localize poorly in both the spleen and lymph-nodes and are in excess in the liver. Concanavalin A may have at least two distinct effects on lymphocyte migration. In conclusion trypsin appears to be the only agent we have studied so far which directly interferes with lymphocyte/endothelial cell interaction. We intend to screen a number of other agents before building hypotheses of the mechanism of this interaction. This work was supported by Programme Grant G972/455/B from the Medical Research Council.
250
References [1] Gowans, J.L., and Knight, E.J., The route ofrecirculation of lymphocytes in the rat, Proc. Roy. Soc. [B] 159, 257 (1964). [2] Smith, J.B., McIntosh, G.H., and Morris, B, The traffic of cells through tissues: a study of peripheral lymph in sheep, J. Anat. 107, 87 (1970). [3] Smith, J.B., McIntosh, G.H., and Morris, B., The migration of cells through chronically inflamed tissues, J. Pathol. 100, 21 (1970). [4] Gesner, B.M., and Ginsburg, V., Effect of glycosidases on the fate of transfused lymphocytes, Proc. nat. Acad. Sci. 52, 750 (1964). [5] Woodruff, J.J., and Gesner, B.M., The effect ofneuraminidase on the fate of transfused lymphocytes, J. exp. Med. 129, 551 (1969). [6] Woodruff, J.J., and Gesner, B,M., Lymphocytes: Circulation altered by trypsin, Science 161, 176 (1968). [7] Ford, W.L., The immunological and migratory properties of the lymphocytes recirculating through the rat spleen, Br. J. exp. Path. 50, 257 (1969). [8] Gillette, R.W., McKenzie, G.O., and Swanson, M.H., Effect of eonvanavalin A on the homing of labelled T lymphocytes, J. Immunol. 111, 1902 (1973).
Discussion
Parrot (UK) This is not really a question but an additional comment to your last slide, with regard to the trypsin-treated lymphocytes. A young worker in our department has shown with, in vivo experiments, with trypsinized lymphocytes, and as you may, anticipate, if you splenectomize such mice, then not having the obstacle of the spleen to get over, they go to the lymph nodes.
W.L. Ford and M. Sedgley
Ford (UK) In fact we have injected some trypsinized lymphocytes into splenectomized recipients but have not got the results, yet.
Ziff(USA) I am just curious about the figure of 20:1 small: large 1 hour later. I don't understand that. Is that because they became small in the hour, or what? Ford (UK) These are the experiments on the isolated lymph-nodes, where we labelled large lymphocytes. We inject them into intact recipients, we found that the ratio of large to small in gut was about six or seven, and in lymph-nodes was about 0.06. So there is an enormous difference. Whether they become large or small we don't really know. If one looks autoradiographically at the earlier stages, and I think Delphine will agree with me, they still look large. But what happens to them eventually, we don't really know that. In the perfused lymph-nodes the large lymphocytes go in much better than small lymphocytes, in contrast to what tends to happen when you inject them into intact recipients, when if you look at the lymph-nodes the small go in twenty times better than the large. But what I am saying, is that it is simply because the large cells are picked out as they perfuse through the gut, and they never get a chance to go the lymph-nodes.
