Topographical studies of lymphocyte localization using an intracellular fluorochrome.код для вставкиСкачать
THE ANATOMICAL RECORD 213~421-428 (1985) Topographical Studies of Lymphocyte Localization Using an lntracellular Fluorochrome M. BRENAN, C.R. PARISH, AND G.I. SCHOEFL Departments of Microbiology (M.B., C. R.P.) and Experimental Pathology (G.IS.), John Curtin School of Medical Research, Australian National University, Canberra, A . C. I: 2601, Australia ABSTRACT A procedure for analysing the topographical localization in tissue sections or whole-organ mounts of lymphocytes labelled with a n intracellular DNAbinding fluorochrome, Hoechst dye No. 33342, is described. The localization of intravenously injected lymphocytes in spleen, popliteal lymph nodes, and Peyer’s patches was followed up to 7 days. In the case of spleen, both B and T lymphocytes initially localised in the marginal zone. Subsequently, B cells appeared to exit via the red pulp, while T cells aggregated around vessels in the white pulp. In Peyer’s patches, B and T lymphocytes localized to different lymphoid areas. The advantages and potential applications of this technique are discussed. Lymphocytes are mobile cells which migrate from blood to lymphoid tissues (Gowans and Knight, 1964). Determination of their localization in specialized lymphoid tissue microenvironments is essential for establishing the mechanisms involved in selective migration of subpopulations of lymphoid cells. Most previous studies have utilized radioactive markers to establish the percentage of cells migrating to a particular organ and the position of these cells within the organ (reviewed in de Sousa, 1981). To overcome the problems associated with radioisotopes, fluorochromes such a s fluorescein isothiocyanate, which bind covalently to cellular proteins, have been utilized (Butcher and Weissman, 1980; Butcher et al., 1980a,b). The major disadvantage of fluorescein isothiocyanate is that the fluorescence intensity is weak and deteriorates on illumination (Yamaguchi and Schoefl, 198313). In contrast, H33342, a n intracellular DNA-binding fluorochrome which has been extensively used for flow cytometry (Arndt-Jovin and Jovin, 1977; Loken, 1980) is strongly fluorescent. This paper describes its use to determine the positioning of intravenously injected lymphocytes within the spleen, Peyer’s patches, and popliteal lymph nodes. MATERIALS AND METHODS Animals CBA/H (H-Zk)mice of either sex and aged between 6 and 12 weeks were used. All mice were bred at the John Curtin School of Medical Research. Preparation of Lymphocyre Suspensions for Labelling Standard cell suspension medium was Eagle’s minimal essential medium (F15) (Grand Island Biological Co., Grand Island, NY). Spleen cells were pressed gently through a fine wire mesh and depleted of red and dead cells by isopaque-ficoll separation to yield a lymphocyte population (Davidson and Parish, 1975). In some experiments, T-cells, i.e., immunoglobulin-negative (Ig-) spleen cells, were prepared by rosetting surface Ig-bear0 1985 ALAN R. LISS. INC. ing cells with sheep antimouse Ig-coupled erythrocytes and separating rosetting and nonrosetting cells by centrifugation on isopaque-ficoll (Parish et al., 1974). To obtain B-cells, spleen cells were depleted of Thy 1’ cells by being treated with monoclonal rat anti-Thy-1.2 antibody (clone 30H12) and guinea pig complement (McKenzie and Parish, 19761, the resulting dead cells were removed by centrifugation on isopaque-ficoll (Davidson and Parish, 1975). All lymphocytes were washed three times with medium prior to labelling. Labelling Lymphocytes With H33342 Lymphocytes were resuspended a t 5 x lo7 lymphocyteslml in F15 to which was then added 6 pglml of H33342 (Calbiochem-Behring, Kingsgrove, N.S.W.) and the lymphocytes were incubated in a 37°C water bath for 15 minutes. Labelling was stopped by adding cold F15. Lymphocytes were washed twice and resuspended ready for injection. In Vivo Localization and Tissue Preparation In all experiments 2-4 x lo7 labelled lymphocytes were injected intravenously in 0.2 ml F15 into syngeneic sex- and age-matched recipients. At specified time intervals the spleen, lymph nodes, and Peyer’s patches were removed and prepared for fluorescence microscopy. With epi-illumination, the thickness of the tissue is not critical so that it is possible to examine the surface of rather bulky organs and also internal levels after the tissue has been planed to the desired depth. This latter method was used for the spleen and lymph nodes. These tissues were frozen onto specimen chucks (Sorvall, Newtown, CT) in 30% aqueous sucrose and planed by hand on a mounted skin graft knife blade. In the case of spleen, Received J a n u a r y 28, 1985; accepted J u n e 4, 1985. M. Brenan i s the recipient of a Royal Society of London Florey fellowship. 422 M. BRENAN, C.R. PARISH, AND G.I. SCHOEFL the exposed surface included areas of red and white pulp and for lymph nodes approximately half was planed off. For Peyer’s patches it was only necessary to cut open the gut segments and to pin them out on nonfluorescent silicone rubber (Silastic, Dow Corning, Midland, MI). For cell counts spleen cell suspensions were prepared by gently pressing the minced spleen through a fine wire mesh. All suspensions were centrifuged and resuspended in recorded volumes ready for counting (Brenan and Parish, 1984). Fluorescent Microscopy An Olympus microscope (BH series) with a n HBO 100W mercury vapour lamp for epi-illumination and appropriate exciter and barrier filter combinations for H33342 (365-nm excitation and > 435-nm emission) was used. The tissues were examined and photographed unfixed and immersed in saline. Fluorescent cells in the splenic cell suspensions were counted in haemocytometer chambers and the percentage of injected cells which had localized in the spleen was calculated. RESULTS Analysis of Localization of 6 and T Lymphocytes in Spleen Figures 2 and 3 show the localization patterns of H33342-labelled B and T lymphocytes in spleen sections at selected time points taken from a n analogous time course. In the case of B lymphocytes at 15 minutes (Fig. 2A) and 30 minutes (Fig. 2B) localization was predominantly in the marginal zones. By 2 hours, a large proportion of these cells were still present in the marginal zones, but some were now also scattered in the adjacent areas, presumably the red pulp (Fig. 2C). This pattern at 2 hours was similar to that a t 12 hours (Fig. 2D). However, by 72 hours the number of B lymphocytes in the red pulp had declined although cells were still present in the marginal zones a t 7 days, when the experiment was terminated. In the case of T lymphocytes, a t 15 minutes cells were present in the marginal zones but some were also present in the white pulp (Fig. 3A) and their numbers in the white pulp had increased by 30 minutes (Fig. 3B). At 2 hours, few fluorescent cells remained in the marginal zones but they now formed distinctive dense clusters in the white pulp (Fig. 3C). The dramatic localization pattern a t 12 hours (Fig. 3D) showed T lymphocytes aggregating around vessels within the white pulp; by 16 hours the number of cells surrounding these vessels had declined. At later time points fluorescent cells were less distinct, possibly reflecting uptake by other cells of released H33342. The localization patterns after the intravenous injection of unseparated lymphocytes were characteristic of both B and T lymphocytes (data not shown). Figure 1 shows the percentage of the total injected unseparated lymphocytes recovered from spleen at time intervals ranging from 15 minutes t o 7 days. By 1hour, the number of lymphocytes in the spleen was maximal at 23.2% and remained constant for 8 hours, after which Analysis of Localization of B and T Lymphocytes in Peyer’s the numbers declined from 20.7 to 5.1% by 72 hours. Patches and Lymph Nodes Fluorescent cells (1.1%) were still visible at 7 days. These In the case of Peyer’s patches, whole-organ mounts data show that large numbers of labelled lymphocytes are retained in the spleen but they give no information were used to examine the localization of lymphocytes. where the cells localize. The topographical distribution Figure 4 shows the localization of unseparated lymphoof these cells was therefore examined in spleens planed cytes at 15, 30, 45, and 60 minutes after intravenous injection. At 15 minutes, lymphocytes had localized prefto various depths. 24r f i 4 t I I I I L 1 1 I b & 1 2 3 4 5 6 7 8 9 L 10 I I I I I , , , I I 11 12 13 14 15 16”24 48 ,+ 72’168 HOURS Fig. 1. Kinetics of mean percentage localization of H33342-labelled CBA/H lymphocytes to spleen; 2 x lo7 CBA/H lymphocytes were injected intravenously into syngeneic recipients. Spleens were removed at specified times after injection and cell suspensions prepared for counting. Results represent mean percentage lymphocytes injected of three recipients. LYMPHOCYTE LOCALIZATION STUDIES Fig. 2. Areas of spleen A) 15 minutes, B) 30 minutes, C) 2 hours, and D) 12 hours after intravenous injection of 2 x lo7 labelled B lymphocytes. Note localization in marginal zone (arrow) and red pulp (double arrow). x55. 423 424 M. BRENAN, C.R. PARISH, AND G.I. SCHOEFL Fig. 3. Areas of spleen A) 15 minutes, B) 30 minutes, C) 2 hours, and D) 12 hours after intravenous injection of 2 x lo7 labelled T lymphocytes. Note localization in marginal zone (arrow) and dense clustering in white pulp (double arrow). X55. LYMPHOCYTE LOCALIZATION STUDIES Fig. 4. Peyer’s patches at A) 15 minutes, B) 30 minutes, C) 45 minutes, and D) 60 minutes after intravenous injection of 4 X lo7 labelled unseparated lymphocytes. ~ 3 5 . 425 426 M. BRENAN, C.R. PARISH, AND G.I. SCHOEFL erentially in blood vessels (Fig. 4A) which have previously been shown to be high endothelial venules (Yamaguchi and Schoefl, 1983a). The distribution of these cells at 30 minutes (Fig. 4B) and 45 minutes (Fig. 4C) suggested emigration into the adjacent lymphoid tissues. No further change in distribution was noted a t 60 minutes (Fig. 4D), but the fluorescent intensity had declined. It is noteworthy that labelled cells were not seen in the centre of the follicles even after 24 hours (data not shown). Since previous work has suggested that B cells localize preferentially in Peyer’s patches (Stevens et al., 1982), it was of interest to examine the localization of B and T lymphocytes using this technique (Fig. 5). At 15 minutes, both B and T lymphocytes were present in blood vessels surrounding the follicles (Fig. 5A,C), but the distribution of T lymphocytes appeared more extensive. By 45 minutes, both B (Fig. 5B) and T (Fig. 5D) lymphocytes were also seen in the adjacent lymphoid tissue but the distribution of T lymphocytes was more diffuse compared to that of the B lymphocytes. These differences in localization were still apparent at 2 hours. A similar study of the localization of lymphocytes was undertaken in popliteal lymph nodes. At 15 minutes lymphocytes had localized in blood vessels of the node (Fig. 6A) in areas in which high endothelial venules have previously been described (Anderson and Anderson, 1975). By 45 minutes they were also present in the adjacent lymphoid tissue (Fig. 6B). DISCUSSION In this paper the topographical localization in tissue slices or whole-organ mounts of lymphocytes labelled with the intracellular DNA-binding fluorochrome H33342 in vitro prior to injection into syngeneic mice is described. It is a n extension of previous work (Brenan and Parish, 1984), where migration of H33342-labelled lymphocytes into different organs was quantified by preparing cell suspensions and counting the fluorescent cells. The advantages of H33342 for localization studies over conventional autoradiography, which is often used to establish the unique positioning of lymphocytes within organs, are: I) the problems associated with radioisotopes, which have been described previously (Butcher et al., 1980a,b; Brenan and Parish, 19841, are eliminated 2) compared with other fluorochromes, such as fluorescein isothiocyanate (Butcher and Weissman, 1980; Butcher et al., 1980a,b), the fluorescent intensity is much greater, does not visibly deteriorate on illumination, and appears unchanged in aldehyde-fixed tissues (Brenan and Parish, 1984); 3) the method is not time consuming; 4) it is possible to visualize the cells associated with the H33342; 5) both quantitative (Brenan and Parish, 1984) and topographical data can be obtained using H33342. It should be emphasized that a comparison of quantitative and topographical data is especially important where differences in positioning within one organ may not result in a change in the total number of lymphocytes within that organ; 6) false positives due to leakage of 51Cr into the blood is avoided (unpublished data). Disadvantages of the method are that some leakage of H33342 is possible because H33342 is not covalently linked to cells and that the dye may be taken up by bystander cells. Also, the dye is diluted by cell divi- sion (Durand and Olive, 19821, which complicates interpretation of localization patterns a t later time points. We have used this method to study the localization of labelled lymphocytes at various times after intravenous injection in a n attempt to elucidate the pathways of migration through spleen, Peyer’s patches, and popliteal lymph nodes. In the case of spleen, B and T lymphocytes showed distinct localization patterns in tissue slices. B lymphocytes localized preferentially in and adjacent to the marginal zones (Fig. 2C) and T lymphocytes preferentially in the white pulp (Fig. 3C). These findings are in agreement with previously reported B and T-dependent areas (reviewed in de Sousa, 1981). In contrast to entry, the routes by which lymphocytes leave the spleen are not clearly defined (reviewed in de Sousa, 1981).Our results indicate that B lymphocytes may preferentially leave through the red pulp, since large numbers of lymphocytes were present in that area (Fig. 2D). The striking localization a t 12 hours showing lymphocytes surrounding a vessel in the while pulp suggests T lymphocytes may leave through or along these vessels (Fig. 3D). The number of these lymphocytes declined with time. Figure 6, which shows the localization of lymphocytes in sections of popliteal lymph nodes a t different times, represents another example of the application of this method to a different organ. Lymphocytes were initially present at sites which are characteristic for high endothelial venules (Fig. 6A) (Anderson and Anderson, 1975) and later appeared in the surrounding lymphoid tissue (Fig. 6B), in agreement with previous reports. Peyer’s patches are particularly advantageous for topographic studies of lymphocyte localization, since it is possible to visualize their lymphoid tissue and most of the blood vessels in one plane in the whole organ (Yamaguchi and Schoefl, 1983a-c). This permits analysis at sequential time points of stages interpreted as adhesion, migration across the vascular wall, and localization of the lymphocytes within this lymphoid tissue (Fig. 4).At 15 minutes (Fig. 4A), the pattern of localization of these cells was identical to that shown by Yamaguchi and Schoefl (198313) with fluorescein-isothiocyanate-labelled cells and corresponded to the distribution of high endothelial venules. By 45 minutes, the distribution suggested emigration into the adjacent lymphoid tissue (Fig. 4C). Analysis of the localization and migration of B and T lymphocytes through Peyer’s patches clearly showed that both B and T lymphocytes localize to different lymphoid areas (Fig. 5 ) .These findings are in agreement with thymus-dependent and thymus-independent areas demonstrated by neonatal thymectomy and hydrocortisone treatment (Abe and Ito, 1978), but they are at variance with other reports indicating that B lymphocytes preferentially localize to Peyer’s patches (Stevens et al., 1982). Comparison of the distribution of B and T lymphocytes even at 15 minutes suggests that either each lymphocyte subpopulation recognizes different sites in the high endothelial venules or they migrate at different speeds (Fig. 6A,B). The latter seems to be supported by the distribution of both types of lymphocytes a t later time points, B lymphocytes appearing to migrate more slowly than T lymphocytes. It is noteworthy that neither B nor T lymphocytes entered the central follicle region (Fig. 5B,D). LYMPHOCYTE LOCALIZATION STUDIES Fig. 5. Peyer’s patches after intravenous injection of 2 x lo7 labelled lymphocytes. A,B) Blymphocytes and C,D) T-lymphocytesat A,C) 15 minutes and B,D) 45 minutes. x35. 427 428 M. BRENAN, C.R. PARISH, AND G.I. SCHOEFL Fig. 6 . Areas of opliteal lymph nodes A) 15 minutes and B) 45 minutes after intravenous injection of 2 x 10.plabelled unseparated lymphocytes. ~ 5 5 . This paper demonstrates the potential use of H33342 as a fluorescent label of lymphocytes. This fluorochrome can be used to analyse the topographical localization of lymphocytes within organs and help to define the pathways of migration of these cells. It should also be applicable to cells other than lymphocytes. Finally, Reinhold and Visser (1983) have shown that it is possible to label cells in vivo by injecting the dye directly into animals. LITERATURE CITED Abe, K., and T. 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