Spector 1966
Spector 1966
Spector 1966
GRANULOMATA
80 -
2 70-
.-l -
ga 60- --*-.-._,_. U.
%,
J
-I 5.
-,
m
50- -,
-. *,-,
2
%. -,
e 40- *.*‘
0. . -. - 0
Y 30-
fa’*
2
z 20-
2 ),”l’.*.”-*”r
,()’
,--”,.*..-”-~-
.” ”...~......
.~..
---------
-*--C.I
y.----~-----------’”-------------------*-----------------~
,,,,.,,”
-,-------
-
”..
l,,,,..,.,,,,~, *,...a
“-‘-*.***111
-. -.
’HT-labelled blood monocytes
0
3HT-labelled exudate mononuclears
0-0
. Carbon-labelled blood monocytes
0-
0 ---
-
Carbon-labelled exudate mononuclears
0 3HT-labelled blood small lymphocytes
FIG. 1 .-The percentage of labelled mononuclear cells in blood and granuloma at various
times after induction of the lesion, labelling being completed before initiation of
the granuloma.
(fig. 2). By comparison, the percentage of small lymphocytes in the
blood so labelled is very small. Large lymphocytes give an inter-
mediate value (Spector et a[.). Fig. 1 also shows the close corre-
spondence between the proportion of blood monocytes that took up
colloidal carbon and the proportion of exudate mononuclears containing
the marker. These findings are very similar to those obtained with
other types of inflammatory stimuli (Spector and Coote; Spector et 01.).
declined to 5 grains per nucleus (fig. 8). Most of the cells that still
contain demonstrable tritium are histiocytes or macrophages. The
proportion of labelling amongst the cells with small round nuclei is
lower than that in the mononuclear population as a whole, the average
value being 20 per cent. The average grain count is also lower at
4 grains per nucleus, but highly labelled nuclei are occasionally seen.
Blood smears taken at the time of death show that 3HT labelling of
blood monocytes and polymorphs has fallen to less than 2 per cent.
and of small lymphocytes to less than 1 per cent.
1
I I 1 I
7 I4 21 28
DAYS
0 -. -. Percentage 3HT-labelled cells
0-0 Average nuclear grain aunts ( x 10)
0 ..... 0 Percentage of carbon-labelled cells
FIG. 8.-The decline in the percentage of labelled mononuclear cells and in their average
nuclear grain counts, in granulomata of various ages. All labelling was completed
before initiation of the granuloma.
FIG.4. - 3H-thymidine-labelled
mononuclear cells in a 2-day-
old granuloma. Labelling was
completed before initiation of
the granuloma and the cells are
therefore haematogenous.
Autoradiograph, HHE. X 1OOO.
SPECTOR
AND LYKKE PLATEXLVIII
EVOLUTION
OF INFLAMMATORY
GRANULOMATA
FIG.5.-’H-thymidine-labelled peri-
cyte in the wall of a venule in a 2-
day-old granuloma, A single dose
of isotope was given 1 h r before
death. Autoradiograph, HHE.
x 1000.
T ’
hm, +I- I%
FIG.6. - 3H-thymidine-labelled
histiocytes and macrophages in
a 2-day-old granuloma. A
single dose of isotope was given
1 hr before death. Autoradio-
graph, HHE. x 1OOO.
...
r) a
EVOLUTION OF INFLAMMATORY GRANULOMATA 169
injection of 3HT and death, the proportion of labelled cells in the small-
round-cell collections was similar to or slightly lower than that found
at 3 days. The average grain count of the labelled cells, however, had
fallen to 4 per nucleus. In addition, within the small-round-cell areas
the proportion of tritiated cells that resemble histiocytes or reticulo-
endothelial cells is now 60 per cent. and the corresponding proportion
of labelled cells of the small round cell type has fallen to 40 per cent.
DISCUSSION
Comparison of tritium and carbon labelling indicates that most
mononuclear cells that infiltrated the 24-hr-old lesion were blood
monocytes. This finding accords with the similar results obtained with
double-labelling techniques in inflammation induced by injections of
fibrinogen and paraffin oil (Spector and Coote, 1965; Spector et al.,
1965). It conforms also with the findings of Volkman and Gowans
(1965~and b) who used tritium labelling in conjunction with parabiosis.
All these papers agree in opposing the view that lymphocytes play a
major role in this phase of inflammation.
A major feature of the reaction was the onset at 48 hr of cell prolifera-
tion, which 24 hr earlier had been almost completely absent. At 48 hr
this proliferation was present particularly in vessel walls. Of the
vascular cells synthesising DNA, some could have been endothelium,
but most resembled pericytes or closely applied histiocytes (fig. 5).
Reactions labelled with tritium at 48 hr and then allowed to proceed
for a further 24 hr before the animal was killed showed that on the
whole vascular cells that took up 3HT remained part of the vascular
structure after mitosis. It was apparent that perithelial cell multiplica-
tion inevitably contributed to perivascular cell collections, and that had
the inflammatory stimulus ceased to operate at this time before general
cellular proliferation commenced (as perhaps happens in the tuberculin
reaction), such collections would have been more prominent than they
were.
It is possible that some of the cellular division in vessel walls was
associated with the proliferation of new blood vessels, but no unequi-
vocal evidence of this was found. Of more importance than the blood
vessel changes, however, was the general proliferation of histiocytes
and macrophages that commenced at 48 hr. This proliferation persisted
at a rate that declined only a little throughout the 12 weeks’ duration
of the experiment. Initially, division amongst vascular cells was a
major component of this cell multiplication. By the end of the week,
however, such vascular change had become of negligible proportions.
EVOLUTION OF INFLAMMATORY CRANULOMATA 173
CELL
DEATH
PHAGOCYTOSIS
...-
: b '
FREE
H IST IOCYTE CARBON
I
CEYL
CELL
DEATH
..a.
. *e
1,
EL000
MONOCYTE
FIG. 19.--Schematic presentation of two possible pathways for the derivation of local
small-round-cell or lymphoid collections in granulomata.
may have occurred in the granulomata. This however did not appear
to be extensive. Thus the massive death of tritiated polymorphs was
not followed by the appearance of significant numbers of labelled
epithelium or mesenchymal cells, even though both these cell types
were dividing. There is therefore no evidence that re-utilisation of
isotopic thymidine played a significant part in the results described.
SUMMARY
Granulomata of up to 12 weeks’ duration have been induced in rats
by injecting dead tubercle bacilli in mineral oil (Complete Freund’s
adjuvant). Their development has been studied by labelling of blood
and tissue cells with tritiated thymidine given before or after initiation
of the granuloma and similar labelling with colloidal carbon. The
results show that the mononuclear infiltration of the reaction sits is
due mainly to blood monocytes. Thisis followedby mitotic proliferation,
first of cells in blood-vessel walls, then of histiocytes and macrophages.
The proliferation of histiocytes and macrophages persists for the
duration of the lesion, i.e. 12 weeks or more, and the dividing cells are
largely derived from blood monocytes, although the latter first
acquire the characteristics of histiocytes or macrophages before
dividing. This sustained proliferation, aided probably to some extent
by further emigration, is responsible for the persistence of the lesion.
Cells laden with particulate matter, e.g. carbon, do not divide, but
when they die their cytoplasmic inclusions are taken up by young
histiocytes, etc. In this way insoluble matter is retained in the granuloma
for its duration.
As well as histiocytes, macrophages, epithelioid cells and giant cells,
the granulomata contain small round cells, many of which would
normally be identified as lymphocytes. Evidence was found that in
many cases these round cells are the progeny of histiocytes, etc., and
they subsequently change to a histiocytic or macrophage form, thus
acting as intermediary varieties. Large collections of small round cells,
probably lymphocytes, were seen in later granulomata and these
were at least partly derived by division in siru of larger precursor
cells.
The authors express their gratitude to The Life Insurance Medical Research
Fund, The Medical Research Council and the Nuffield Foundation for their
contributions to the expenses of this investigation. Thanks are also due to Mr P.
Cull for the diagrams and to Mr R. Dunbar for technical assistance. Dr A. W. J.
Lykke held a Royal Society and Nuffield Foundation Bursary.
REFERENCES
BRYANT,
B. J. . . . . . . 1962. E x p . Cell Res., 27, 70.
CRADDOCK, C . G., NAKAI,
G. S., 1964. J. Exp. Med., 120, 389.
FUKUTA,H., AND VANSLAGER,
LOUISE
M.
SPECTOR
AND LYKKE PLATEXLIX
EVOLUTION
OF INFLAMMATORY GRANULOMATA
FIG.9.-jH-thymidine: labelling of a
macrophage in a 1-wk-old granu-
loma. A single dose of isotope
was given 1 hr before death. Auto-
radiograph, HHE. X 900.
FIG.12.-3H-thymidine labelling
of small mononuclear cells
(“ lymphocytes ”) in an 8-wk-
old granuloma. The isotope
was given as a single dose 24
hr before death (cf. fig. 11).
Autoradiograph, HHE. x 1OOO.
FIG.13.-3H-thymidine uptake by
a macrophage in a 12-wk-old
granuloma. A single dose of
isotope was given 1 hr before
death. Autoradiograph, HHE.
x 1OOo.
EVOLUTION
OF INFLAMMATORY GRANULOMATA
FIG. 16.-Carbon-ladem giant cell in a 12-wk-old granuloma. The carbon was given
prior to initiation of this Itsion. HHE. X 1250.
EVOLUTION OF ZNFLAMMATOR Y GRANULOMATA 177