Download AUTORADIOGRAPHIC OBSERVATIONS ON THE LOCATION OF

AUTORADIOGRAPHIC OBSERVATIONS ON THE LOCATION
OF DNA— AND RNA— SYNTHESIZING CELLS IN RAT
POPLITEAL LYMPH NODES^
by J. J. MILLER III^
(From tlie Walter and Eliza Hall Institute, tlie Department of Exixrimentd
Medicine, the University of Melbi)urne, Melbourne, Victoria).
(Accepted for publication 1st November, 1964.)
Summary. ''H-thymidine and •''H-cytidine have bct'ii usfd to study tlic histohiHical localization of DNA- and RNA-synthesizin^ cells in ptipHteal lymph
nodes from untreated, imiuuiiized and ut'onatally tliyineftoinizt'tl rats. Aetive
cell proliffration, as measured by •ill-thyinidine incorijoration, was diuiionstrated
in tlu' medulla of imstimnlaled nodes from normal and from nconatally tbymeetoniizcd rats. After an immnnolojfical stinuikis, an early increase in medullary
cell proliferation was associated with a similar but less intense reaction in the
cortex. A partial dissociation between the sites of cell proliferation and the
sites of most aetive HNA synthesis was noted. Cells with the highest apparent
RNA-syrithetie rates were found predominantly in the cortex. Reticnlar cells,
histiocytes, und pliisma eells were found to retain labelled RNA longer than the
otlier cells of the lymph nodes.
The ability to achieve coasiderably more intense labelling in popliteal
lymph node eells than in the rest of the lymphoid system by iiiiid foot-pad injeetions of ''H-nueleosides was confinned. However, attempts to use liiis finding
for a study of lympli node eell migration were eomplieateil i>y a failure to olitain
adequate numbers of labelled eells after injection of •'H-t!iymidiiic, am! by
the diffieiilty t)f fintliiig a dose of •^Il-cytidine wliieli Ialielled small lymphocytes in the popliteal nodes but not blast eells in other organs. It was possible,
however, to demonstrate lympliocyte migration within 30 minutes of selective
labelling of one popliteal node. Small numbers of migrant cells were found
in other lymph nodes, spleen, thymiis, and ileal subniiieosa.
INTRODUCTION.
The use of ^H-thymidinc to study lymphopoiesis and plasmacytojioiesis in
rats has been reported frtjm this laboratory (Nossal UTid Miikela, 1962; Mitchell,
MeDonald and Nossal, 1963; Nossal, Mitchell and McDonald, 1963). These
1 This work was supported in part lu' Grant AI-()3y58 from the National Institutes uf
Hi'alth, U.S.A., to Dr. C. J. V. Nossal, and in part by a grant from the National Health and
Rc'Seareh Conncil. Canberra, to Sir Maefarlunc Bnrnet.
-The author was snpported by rost-d<ietora! Grant .5 F2 GM 18, 2fi2-02 from the
National Institute.s of Health, U.S.A.
Aust. J. exp. Biol. med. Sci. (1965) 4.3, pp. 107-122.
108
j . J. MILLER III
studies used autoradiographs of smears aud isolated tells to follow the kinetics
of cell proliferation. Aiitoradiographs of smears have also been used to study
RNA metabolism iu lymphoid cells (Mitchell, 1964a, 1964b). Since tiiese studies
were reported, we have become increasingly interested in the microenvironnieutal aspects of lyuiph node function (Miller and Nossal, 1964). The obscrvations reported in this paper deal with tbe detailed histological location within
rat popliteal lymph ntnles of those eells studied on smejirs in earlier reports.
MATERIALS AXD METHODS.
Animals. Male Wistar albino rats, randomly bred at ttie Hall Institnte, and fed on
"Barastoe Dog Gulws", eabbage and tap water ad tihitum were used, l-'ifty rats of varied
ages were studied. The details of the different e\periniental eonditions are shown in Table 1.
Immunizations. Stoek suspensions of Salmonella adelakh flagellar antigen, prepared as
(leserihcd hy Ada, Nossal. Pye, and Abbot (1964), and of proven good antigenieity, were
nsed. All injeetions were given subentaneously into the hind foot-pads. Doses of either
10 ug. or 50 fig. per foot-pad were used as shown in Table I. These doses are well ahove
the minimum required for an immune response (Nossal, Ada and Austin, 1964),
Tritiatcd micleosidcs. ''H-thymidine and ''H-eytidine were used as DNA and RNA
precursors respeetivcly. Tliey were obtained from the Radioehemical Gentre, Amersham.
England, and consisted of : •'H-thymidine, batch 21, 9-5 euries/millimole, and -'H-cytidine,
hateh 9, 1-9 enries/mfllimole, and batch 10, 2'() ciiries/millimole. The nneleosides were
injeeted snbeutaneonsly into both or only the left hind foot-pads in O'O.'i to 0-2.5 ml. of saline.
The doses, sites and eomses oC injections are shown in Table 1.
Neonatallij tlujmvctomizvd rats. Tliree rats from one litter were tliymectomized when
less than 24 hours okl. The>' were iinaestlietizetl by heinti placed on iec; cnbes until all
movement eeased. The sternnm was split and retracted, the th>inus identified anti remo\e(l
by snetion throngh a glass pipette with a 1 mm. orifice. The sternum was elosed with a
single silk sntnre, and the overlying skin with fonr interrupted silk sntnres and eoilodion.
Similarly treated rats had a redncetl or aKseiit primary re.sponse to Salmonella adelaide
ilagellur antigen dnrhig at least the first six weeks of life, hnt gave normal secondary
responses (Miller, unpublished). Three litter mates were subjeeted to the same procedure
except that the thyinns was not. in fact, removed. At four weeks of age these animals
were injeeted with ''H-thymidine intravenously via the dorsal hind foot vein witli the dose
indicated in Table 1. Examination of serial seetions of the mediastinal contents re\eaie(l
that one of the three "thymectomized" rats had microscopic residual thymif (issue, and
tliis animal was excluded from the stiuK'.
Pi-cparatkm af uuturudiograph.s. Hats were killed by a hlow on the head nnder ether
anaesthesia at varied times after nueleosidc and antigen injection. The popliteal lymph
nodes were removed, fixed in 10 per cent neutral formalin for 24 hours, and then dehydrated,
cleared, and paraffin emhedtled by standard tcchni<ines. Sections were eiit at 5-7 microns
and prepared for autr)racliograpliy witli Kodak AR 10 stripping film nsing the method of
Pelc (iy.5G). In must instanecs aiitoiadiograplis of seetions of aortic nodes, mescnteric
nodes, thymus. spleen, and ilenm were also prepared. After e.xposnre at 4° for appropriate
periods, (ieterniined empirically for each experiment, the autoradiojiraphic film was de\elope(l, fixed and washed, anti the sections were stained with methyl green-pyronin.
Extiminathm of autorddiogruiiha. In most instances aiitoradiographs of sections of
l)opliteal nodes were simply observed for location iA labelled eells, type of cell labelled, and
AUTORADIOGRAPHIC OBSERVATIONS OF LYMPH NODES
109
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relative degree of labelling. In some cases grain counts over labelled cells were determined,
and mean values were calculated after counting 40 to 50 cells nf a given type. Similarly,
in some cases, the percentage of labelled cells of a given t>'pe was determined hy counting
100 to 200 cells of that type.
Organs other than the poplileal ncidcs were examined to determine the degree of
labelling of resident cells, and then were scanned for po.ssible migrating cells from the
popliteal nodes as previously de.seril)ed (Miller, 1964a). \VIien ''H-eytidine was injected
only in the left foot-pad, the right popliteal node was examined for migrant cells.
RESULTS.
1. The Location of •''H-thymidinc lucorporafing Cells in Popliteal Nodes.
A. Nodes from untreated rats.
All tlie normal unstimulated popliteal lymph node.s had labelled large pyroninophilic cells (blast cells) in berth medulla and cortex .sliortly after injection
of •4I-thymidine (Eig. 1). Labelled retieular cells and histiocvte.s were less
Fij;. 1, An antoradiograph of a section of a normal imstimulatcd 6-7-week-old rat's
popliteal lymph node 2)2 hours after a foot-pad injection of •'H-thymidine. Labelled cells,
seen as black spots, are present scattered throngh both the medulla and the cortex. (X 150.)
common and were mo.st easily distinguished in the medulla and along tbe peripheral sinus. Endothehal cells of the cortical venules and of the efferent
lymphatics were oceasionalh" labelled. Labelled mast cells were rare. Small
lympboeytes and the rare mature plasma cells were not labelled.
AUTORADIOGRAPHIC OHSERVATIONS OF LYMPH NODES
IU
Those IKKICS fnjm rats which hud received multiple injections of •'*Hthymidine, and ihu.se from rats whieli were killed after periods of mor(^ than
one day after nucleoside injection, had small numbers of labelled small lymphocytes tiirouiiliout the cortex. They also had a relatively greater proportion of
labelled retitular cells and medullary histiocxtes. Small numbers of labelled
mature plasma cells were present in tlie medulla.
B. Nodes from antigeniatlhj-stimnhtcd rats.
In the primariU- slimuiated rats, the popliteal nodes showed several
sequential clianges in the distribution of cells capable of taking up ^H-thymidine
following antigen injectiou. liy the seeoud da\ there was an increased density
FiR. 2. Till' distrihntinn of labelled cells oO mimites after the injection of -Ul-thymidine
on the second da\' ot a niimar>- respon.se is Mcn in this autoracliograpti of a popliteal node.
Increased numbers of labelled'ceUs can be seen iu both cortex and medulla, especially in
the medullary cords. (X 150.)
of labelled cells in both cortex and medulla, but the density was greater in the
medulla than in the cortex (Fig. 2). On the tliird day after stimulation, small
germinal centres appeared in the eortex. These grew in size until the si.xth day,
J. j . MILLEH HI
112
the last time-point includt-d. Most of the cells of these jierniinal centres were
lul)elled, hilt usually more lightly than cells in the surnninding cortex. After
the third dii\'. iiceiinnilalions of mature plasma cells were found in the medullary
cords. These failed to take up 'H-thxiiiidiiie. and tlie density of DNA-synthesizini; cells appeared lower (Fig. 3). Mitotic figures could he found in the medulla
throughout the course of the experiment.
Fig. !3. An iiiitoradioj-'riipli (it ;i scdioii (if ;i poplitciil node 30 miiuitt'S nftcr ;in injection
(if •Ul-tln'[iiidiiK' (Ml tlif fiflli (iiiy of ;i primary rcspmisf. A siiuill H'Tininul triitrf lias appeared in the tortcx. Tin- iiidi\idu;il ci'lls of the utTiiiiiud tcntrc arc not ii.s liiKlilv labelled
a.s Cfils rlsewliiTi-. Laryf miinlicrs of Kil)fllcd cells arc still present in the nu'diilla, but the
concentration, cell for cell, is less than that seen un the second day of a response (see
Kin. 3).
(X 150.)
The nodes from those rats given •'H-thymidine on tlie third day after
antigen, and killed at duiK inter\als until the sixth da\-. showed continued label.
gcneralK- witli lower grain counts, in most of the hiast cells of the node. Lahelled
small hmphocytes and mature plasma cells appeared in the cortex and medulla
respectively. Some of these cells ujipeared iu clumps, but more commonly were
scattered siugK. The small Kuiphocytes surrounding gerininal centres never
showed sigm'ficant degrees of lahelling. The cells of the genninal centres them-
AUTORADIOGRAPHIG OBSERVATIONS OE LYMPH NODES
113
selves showed widely divergent grain counts as time passed, suggesting heterogeneous rates of division.
G. Nodes from neonatally thymectoviized rats.
The popliteal nodes of the neonatally thymectomized rats showed those
changes previously described by Waksman, Arnason and Jankovic (1962). The
nodes had thinner cortices than the sham-operated controls, and the internally
oriented, large cortical "nodules" were markedly depleted of small lymphocytes. Primary follicles appeared normal in the popliteal nodes. In other
lymphoid organs germinal centres and plasma cells were abundant, but small
lymphocytes always appeared decreased in numbers. The location of labelled
cells after administration of '^H-thymidine was essentially the same as in unstimulated, normal popliteal nodes. There were scattered labelled blast cells
in both cortex and medulla (Eig. 4). These cells were particularly frequent
Fig. 4. An autoradiograph of a section of a popliteal node from a four-week-old neonatally thymectomized rat one hour after injection of '^H-thymidine. A thinner than normal
cortex is present. Labelled cells are again seen scattered through both cortex and medulla.
(XlOO.)
in the large "nodules" (Eig. 5). Labelled reticular cells and histiocytes were
more apparent in the cortex than in the normal nodes.
2. The Location of 'H-cytidine Incorporating Cells in Popliteal Nodes.
A. Nodes from untreated rats.
Essentially all cells in popliteal lymph nodes were labelled during the first
four hours after an injection of ''H-cytidine (Eig. 6). Label appeared largely
114
J. J. MILLER III
Fig. 5. A high power view of an autoradioniapli of an iiiUriuil tiutical "nodule" in a
popliteal node from a four-week-old neonatally thymectomized rat one hour after injection
of ''H-thymidine. The camera was focused on the plane of the grains in the autoradiographic film, thus the cells are not clearly seen. However, tbe relative lack of small lymphocytes can be noted by the size and loose arrangement of the visible nuclei. There are many
labelled cells present, inchuliii" on" '" T tvpiral lymph node venulc. (X800.)
Fig. 6. An autoradiograph of the cortex of a normal, unstimulated, five-week-old rat's
popliteal lymph node one hour after an injection of ^H-cytidine. Label is present in all cell
types. The camera was focused on the plane of grains in the autoradiographic film, so cell
details are not obvious. The dense, round groups of grains are over blast cells (a). Moderate
degrees of labelling can be seen over the venule endothelial cells (b) and reticular cells (c).
The small lymphocytes have the least label of those cells seen. (X500.)
AUTORADIOGRAPHIC OBSERVATIONS OF LYMPH NODES
115
limited to nuclei. A progression in degree of labelling was apparent: cortical
blast cells > reticular cells, histiocytes, and mast cells > small lymphocytes.
The blast cells had highly variable counts. The cortical blast cells tended to
have higher individual counts than medullary blast cells. Mature plasma cells
had the lowest grain counts of any cell present in the nodes and frequently
were unlabelled.
B. Nodes from antigenically stimulated rats.
When ^H-cytidine was given at intervals after primary or secondary antigenic stimulation, and rats were killed shortly after isotope injection, there
was an increase in the number of heavily labelled blast cells in both cortex
Fig. 7.
An autoradiogiai)li ol u .>ti.ii,,,i ,.i .i piipliUal uoJc 31) luinutcs after injection
of ''H-cytidine on the second day of a secondary response. The cortex has a concentrated
population of heavily labelled cells in contrast to the relative lack of highly labelled cells
in the medulla. (X 150.)
and medulla (Fig. 7). The number of cells heavily labelled with ''H-cytidine
was greater than the number of cells labelled with -'H-thymidine, and these
cells tended to be found in greater density in the cortex. Most medullary cells
were labelled, but to lesser degrees. Germinal centres which appeared during
116
J. J. MILLER III
the course of immune responses had high concentrations of heavily labelled cells
(Fig. 8). Germinal centres found in unstimulated nodes did not have such
high concentrations of heavily labelled cells, although some were always present.
When ^H-cytidine was given on the first day of a primary response, and
rats were killed at daily intervals thereafter, a progressively greater proportion
of label was found in the cytoplasm of the popliteal lymph node cells. The most
Fig. 8. An autoradiograph of a section of a popliteal node 30 minutes after injection
of 3H-cytidine on the seventh day of a primary response. A high density of labelled cells
is present in the germinal centre, but individual germinal centre cells vary widely in degree
of labelling. The difFerence in the concentration of heavily labelled cells in the diffuse
cortex as eompared to the medulla is not as marked as is seen in Fig. 7. (X 150.)
obvious labelled cells were the dendritic reticular cells and histiocytes of the
open sinuses, Cortical cells, both blast cells and lymphocytes, had lost most
of their label, but a patterned network of label persisted in the cortex (probably
overlying) undefined reticular cell processes. Mast cells and some of the mature
plasma cells also retained label, the latter presumably having "matured" in the
period after ^H-cytidine injection. This is consistent with a prior report (Miller,
1964b).
AUTORADIOGRAPHIC OBSERVATIONS OF LYMPH NODES
117
Fig. 9. An aiitimuliogniph of ii popliteal node from tlie rat which received three injections of •*H-cytidiiie duriiiji a Sfcondary inuniine respiin.sp. Dense labellinil covrrs a germinal
centre. Bla.st cells outside tlir gcTininal crntn- tire seen us rnimd, dense groups of grains.
The groups of Krains formiiiji monr irregular iiiitterns suggest t]i;it label is persisting jn
dendritic reticular cell processes. (\25().)
The j)()pl!teal nodes from the rat which w;is killed eight hours after the
last of a series of injoctinns of ''Il-cytidine during a second a r\' response had
dense labelling over all uerminal centres and over the mc'chilla. The diffuse
cortex showed light labelling of most cells with scattered den.sely labelled cells.
Some of these were round blast cells, but others appeared to have irregular,
dendritic shapes, suggesting that tliey represented reticular cells (ir histiocytes
(Fig. 9). Very lightly labelled (ir uiilahelled mature iilasma eells were scattered
sinj4y through the medulla (Fig. 10).
3. ObservaHons of Lymphoid Cell Mis,ration.
The selective labelling of p{)j-)Iite;il h'mpli imde colls after foot-pad injections of -'H-thymidine, and the use of this teclinicjuc to follow migration of these
cells has been reported previously (Miller, 1964a). In the studies reported
here, selective labelling of popliteal node cells was also olitained, and migrant
cells were found in all lymphoid organs studied, bnt too few for quantitative
study.
Studies of popliteal lymph node cell migration nsing ''H-cytidine were
expected to be more snccessful because more cells, specifically small lymphocytes, were labelled by this nncleoside. However, these studies were compli-
118
j . J. MILU^R 111
Fig. 10. An iiiitnr;uli()gr;i]ih of tlic iiK'diilhi of the .same niiclc jiichircd in Fiy, 9. Note
the iiiilabclk'c! liiatiirc plasma cell siirroundfc! hy larj^c iiuin!)ers of heavily labcUed cells.
(X200().)
eated by the faet that small lymphocytes exhibited much lower grain counts
following -'H-cytidine than did blast cells. Thus it was not found possible to
label sinall lymphoeytes in one popliteal Kinpli n(Kle without also labelling
ijlust cells in the opposite popliteal lymph node to some degree. This is shown
ill Table 2, which presents data from four representative experiments. The
percentage of blast cells labelled in the left popliteal node (injected side), and
TABLK 2.
Di.iirihutiaii of hibel nfter injection of ^ff-ri^li'luip Inlo thr. left liiml fool-pnil of uttttimulatcd rats.
Doac of nut'lcosido given.
[nterval bptween injection
iind death
Loft jioplitfal node
Blast colls
Small Iymphooytes
Right popliteal node
Blast cells
% laliplled
Menu ^min
eomit.
".;, Inbpllpd
Mean grain
count
% labelled
Mt'iiii grain
count
O-.VC/g
30 min
0-5 ^C/g
60 min
98%
100%
73 3
30 mill
60 mill
18'S
1^7 • ( !
81-5
1'7
:t4
78
8-6
6L'
r.4
54
8-6
5 3
U
r. • 8
18
:! 7
9-8
AUTORADIOCRAPHIC OBSERVATIONS OF LYMPH NODES
119
their mean grain eounts, are eompared with the same data for the small lymphocytes in the left popliteal node and for the blast cells in the right popliteal node.
The grain counts of the small lymphocytes in the right nodes were not significantly above background counts. The ratio of mean grain counts of blast
cells ; small lymphocytes, was quite high on the injected side. The mean grain
counts of the small lymphocytes in the left popliteal node were slightly higher
than the mean grain counts of the blast cells in the right popliteal node. It was
therefore thought that study of the migration of small lymphocytes might be
possible. A few highly labelled small lymphoeytes could be fcmnd in all
lymphoid organs within 30 minntes of injection of -'H-cytidine into one hind
foot-pad, and grain count data indicated that these cells were very probably
migrants. However, in no instance could this be demonstrated with complete
objectivity. The problem is illnstrated in Fig. 11. Moreover, the number of
cells found which appeared to be migrants was never large, making significant
observations impossible.
Fig. 11. An luitoradiiijjriiph of a .section of a right popliteal node from a normal, nnstiniiilatecl rat which had rfceivt'd an injection of -^H-fytidinc in the left hind foot-pad 30
minutes curlier. A cortical veniile is shown. There is a very low degree of labelling of
endothelial eells in the veniile ( a ) . Two hl.ist eells adjacent to the vemde hav(^ denser
labelling ( h ) . A small cell just oiit.side the \eniile lia.s denser lahe] yet ( c ) . Is the Kinall
cell a migrant from the left popliteal node, or was it lahellrd in .situ a.s the endothelial cells
and hiast eell prohahly wore? (X 1000.)
As far AS they went, the studies of migration using b(jth •'H-thymidine and
•'H-cytidine were consistent with eacii other. The cells believed to be migrants
in lymph nodes distant from the injection site, were found in the walls of venules
or in the adjacent cortex. In the spleen they were found at the border of the
lymphocyte cuffs and marginal zones, and less commonly in the red pnlp. In
the thymus they were most commonly found in the cortex, occasionally at the
120
J. J. MILLER III
cortico-medullary border. In all instances the greatest number of possible
migrant cells was found in the subimicosa of the ileum and in the diffuse
lymphocyte mass of Feyer's patches.
DISCUSSION.
These observations support the prior reports on the persistence of small
lymphocytes and plasma cells in rat popliteal nodes (Miller, 1964a), the lack
of incorporation of RNA precursors into mature plasma cells {Mitchell, 1964a,
1964b), and the stability of plasma cell RNA once it is labelled (Miller, 1964b).
They also confirm the obsei-vations of Craddoek, Nakai, Fukuta and Vanslager
(1964) on the presence of active ''H-thymidinc incorporating cells in the medulhiry cords of rats, and the relatively low uptake of -^H-thymidine by germinal
centre cells in comparison with the blast cells in the difiFuse cortex. Tliese
workers believe that germinal centre eells may have a different pattern of DNA
metabolism than other lymphoid blast Cflls. An alternative explanation would
be that -^H-thymidine has a slower rate of diffusion into germinal centres than
into other parts of the lymph nodes.
Horowitz, Bauer, Paranetto, Abrams, Watkins and Popper (1964) have
reported the location of ''H-thymidine incorporating cells in immunologically
reacting lymph nodes in germ-free and "normal" mice. They found that the
first increase in numbers of sueh eells occurred in the diffuse cortex, and later
an increase appeared in the medulla. In the rats studied in these experiments,
this increase occurred simultaneously in cortex and medulla, and the eoneentration of -'H-thymidine ineorjiorating cells was greater in the medulla in the
early stages of the primary- response. In both rats and mice active cell proliferation was established in the medulla well before the appearance of germinal
centres, indicating that this strueture is not necessary for plasma cell differentiation in a given organ. It remains possible that germinal centime cells from some
other lymphoid organ migrated to the popliteal nodes and served as plasma cell
precursors. The rat experiments indicated also that germinal centres do not
give rise directly to the surrounding lymphocyte cuffs, confirming the findings
of Fliedner, Kesse, Cronkite and Robertson (1964).
Nossal and Miikela (1962) and Nossal et al (1963) have presented evidence
that plasma cells may arise from cells which are actively dividing prior to antigenic stimulation. In the unstimnlated rats studied in tlie.se e.xperiments, proliferating cells, as indieated by ''H-thymidine incorporation, were always present
in the medulla. Such cells eould be the stem eells postulated by Nossal's group.
On the other hand, none of the popliteal nodes studied here, even from the
youngest rats used, was comjiletely free of medullary plasma cells, and it
remains possible that the '"'H-thymidine incorporating cells found in the medulla
of these rats were present as a part of a naturally-oceuning immunologica]
reaction and did not represent a true "stem cell" population.
AUTORADIOGRAPHIC OBSERVATIONS OF LYMPH NODES
121
The l(Kation of the most heavily labelled cells after injection of -'H-cytidine
appeared to indicate a partial dissociation between the sites of proliferating
cells and the sites of cells most active in RNA synthesis. In contrast to the
greater concentration of -'H-thymidine incorporating cells in the medulla, those
cells most actively incorjiorating 'II-Lytidine were found predominantly in the
cortex. Mitchell (1964a, 1964b) demonstrated the relative absence of new
RNA s\'ntlit'sis in the maturer elements of tho plasma cell line. The findings
reported here would be consistent witli the argument tliat the basic stem cells
of the plasma cell line arise in the cortex, where they are active in RNA synthcsi.s, and then move to the medulla, where this activity decreases as the cells
"mature". However, there is no proof in any of these oliservations that such a
migration occurs.
The finding of the labelling of dendritic cytoplasmic processes of reticular
cells and liistiocytes at periods of one or more days after the injection of -'Hcytidine indicates that studies of the RNA of tliese cells might be possible in vico.
This would be of interest in light of Fishman's (1961) evidence that RNA
transfer between histiocytes and lymphocytes is an important step in the initiation of immunological reactions.
Achnowlcdgnicnts. The author wishes to thank Dr. G. J. V. Nossal and Sir Macfarlane
Burnet for tlieir help and critici.sm of tliis work and puper, and Miss Judy Thompson for
painstaking technical assistance.
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