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Topographical studies of lymphocyte localization using an intracellular fluorochrome.

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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.
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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. Ito (1978) Qualitative and quantitative morphologic
study of Peyer’s patches of t h e mouse after neonatal thymectomy
and hydrocortisone injection. Am. J. Anat., 151:227-238.
Anderson, A.O., and N.D. Anderson (1975) Studies on the structure
and permeability of the microvasculature in normal r a t lymph
nodes. Am. J. Pathol., 80:387-418.
Arndt-Jovin, D.J., and T.M. Jovin (1977) Analysis and sorting of living
cells according to deoxyribonucleic acid content. J. Histochem. Cytochem., 25585-589.
Brenan, M.. and C.R. Parish (1984) Intracellular fluorescent labelling
of cells for analysis of lymphocyte migration. J. Immunol. Method;
74:31-38.
Butcher, E.C., R.G. Scollay, and I.L. Weissman (1980a) Organ specificity of lymphocyte migration: Mediation by highly selective lymphocyte interaction with organ-specific determinants on high
endothelial venules. Eur. J. Immunol., 10:556-561.
Butcher, E.C., R.G. Scollay, and I.L. Weissman (1980b) Direct fluorescent labeling of cells with fluorescein or rhodamine isothiocyanate.
11. Potential application to studies of lymphocyte migration and
maturation. J. Immunol. Methods, 37:109-121.
Butcher, E.C., and I.L. Weissman (1980) Direct fluorescent labeling of
cells with fluorescein or rhodamine isothiocyanate. I. Technical
aspects. J. Immunol. Methods, 37:97-108.
Davidson, W.F., and C.R. Parish (1975) A procedure for removing red
cells and dead cells from lymphoid cells suspensions. J. Immunol.
Methods, 7:291-300.
de Sousa, M. (1981) Lymphocyte Circulation: Experimental and Clinical Aspects. John Wiley and Sons.
Durand, R.E., and P.L. Olive (1982) Cytotoxicity, mutagenicity and
DNA damage by Hoechst 33342. J. Histochem. Cytochem., 30:111116.
Gowans, J.L., and E.J. Knight (1964) The route of re-circulation of
Lond. [Biol.] 109:257-282.
lymphocytes in the rat. Proc. R. SOC.
Loken, M.R. (1980) Simultaneous quantitation of Hoechst H33342 and
immunofluorescence on viable cells using a fluorescence activated
cell sorter. Cytometry, I :136-142.
McKenzie, I.F.C., and C.R. Parish (1976) Secretion of I a antigens by a
subpopulation of T cells which are Ly-l+, Ly-2- and Ia-. J. Exp.
Med., 144:847-851.
Parish, C.R., S.M. Kirov, N. Bowern, and R.V. Blanden (1974) A one
step procedure for separating mouse T and B lymphocytes. Eur. J.
Immunol., 4:808-815
Reinhold, H.S., and J.W.M. Visser (1983) In vivo fluorescence of endothelial cell nuclei stained with the dye Bis-benzamide H33342. Int.
J. Microcirc. Clin. Exp., 2143-146.
Stevens, S.K., I.L. Weissman, and E.C. Butcher (1982) Differences in
the migration of B and T lymphocytes: Organ selective localization
in vivo and the role of lymphocyte-endothelial cell recognition. J.
Immunol., 128:884-851.
Yamaguchi, K., and G.I. Schoefl (1983a) Blood vessels of the Peyer’s
patch in the mouse: I. Topographic studies. Anat. Rec., 206:391401.
Yamaguchi, K., and G.I. Schoefl (1983b) Blood vessels of the Peyer’s
patch i n the mouse: 11. In vivo observations. Anat. Rec., 206:403417.
)
vessels of the Peyer’s
Yamaguchi, K., and G.I. Schoefl ( 1 9 8 3 ~Blood
patch in the mouse: 111. High endothelial venules. Anat. Rec.,
206419-438.
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