THE CELLULAR EVOLUTION OF INFLAMMATORY

GRANULOMATA

W. G . SPECTOR AND A. W. J. LYKKE*

Pathology Department, St Bartholomew's Hospital Medical College, London

INspite of the importance of chronic inflammation in human pathology,

little is known about the natural history of inflammatory granulomata,
i.e. the origin of the cells of which they are composed and the way in
which the infiltration survives, develops and changes over a period of
months or years. It is for example an open question whether the cells
of granulomata are long-lived survivors of an initial reaction, their
lineal descendants by mitotic division or the representatives of continual
emigration from the blood.
The study of short-lived inflammatory reactions with the aid of tritium-labelled
leucocytes has shown that mononuclear cells in various types of exudate are
haematogenous (Cronkite et al., 1960; Kosunen et al., 1963; Spector and Coote,
1965; Spector, Walters and Willoughby, 1965; Volkman and Gowans, 1965a and
b). Ebert and Florey (1939) had shown that carbon-labelled monocytes emigrated
into inflamed tissues and changed into histiocytes. Volkman and Gowans ( 1 9 6 5 ~
and b) using tritium-labelled cells showed that most macrophages in the exudate
they studied were derived from monocytes. Spector et al. and Spector and Coote
using a double-labelling technique were able to demonstrate that virtually all
mononuclear cells in inRammatory exudates up to 3 days old had originally left
the blood as monocytes.
These experiments left unsolved the question of the survival of the
granulomatous reactions for months or years. The present paper
describes an investigation of this problem in which we made use of the
subcutaneous reaction to dead tubercle bacilli in mineral oil (Freund's
adjuvant) and of cells doubly labelled with 3H-thymidine and colloidal
carbon.
METHODS
The rats used were adult albinos (150-250 g.) of the Wistar strain, and the mice
were adult albino females (2C25 g.) of the Strong A strain. Tritiated thymidine
(3HT), specific activity 5c per mM, was obtained from the Radiochemical Centre,
Amersham. 3H labelling of white cells was achieved by means of a course of 3
injections, each of 0.5 pc per g., as previously described (Spector et al.). To secure
adequate labelling of small lymphocytes, a course of 28 daily intraperitoneal
injections, each of 0.5 pc per g., was given to the mice. Colloidal carbon was
injected intravenously as before (Spector et al.).
Granulomatous inflammation was produced by injecting Complete Freund's
adjuvant subcutaneously and intradermally into the dorsum of the feet. The
adjuvant was emulsified before use with an equal volume of sterile isotonic saline
in an ultra-turrax homogeniser; the quantity of emulsion injected was 0.05 ml. per
Present address: Pathology Dept, University of Adelaide, Adelaide, S. Australia.
1. mm. BACT.-VOL. 92 (1966) 163
164 W. C. SPECTOR A N D A . W. J . LYKKE

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-. -.
’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.).

24 hr after injection of adjuvant

3HT labelling before initiation of granuloma. When labelling of
blood cells was completed 5 hr before induction of the granuloma, 50
per cent. of the large numbers of polymorphs in the 24-hr exudate
were labelled with tritium. Similarly a mean of 60 per cent. of the
exudate mononuclears had incorporated the isotope (fig. 1). These
values correspond closely to those observed in blood polymorphs and
monocytes respectively in the initial 24 hr after injection of adjuvant.
 EVOLUTION OF INFLAMMATORY GRANULOMATA 165

Morphologically almost all the tritiated mononuclear cells resemble

blood monocytes. Most of the tissue histiocytes are unlabelled, but a
small number (about 4 per cent.) contain the isotope. The average
grain count of the radioactive-labelledmononuclears is 8 grains per cell
nucleus.
3HT labelling I hr before death. Animals given a single dose
of tritiated thymidine 1 hr before death revealed no significant
uptake of isotope by any cells in the reaction site. A proportion of
epithelial cells, however, contain the radioactive marker.
Carbon labelling. When carbon labelling of blood cells and endo-
thelial cells was completed 5 hr before initiation of the granuloma, a
mean of 15 per cent. of the exudate mononuclears contain carbon
(fig. 1). Almost all the labelled cells are of the free rounded variety
that make up the bulk of the mononuclear exudate. In addition many
polymorphs are labelled. There is no free carbon in the vessel walls,
as the substance had been cleared from the circulation by the time the
inflammatory reaction was instituted. Carbon given after initiation of
the granuloma 5 hr before death leads to extensive blackening of venules,
indicating increased permeability (Majno, Palade and Schoefl, 1961)
(fig. 3). Much of the carbon has escaped to be taken up by monocytes
and histiocytes in the perivascular tissues.

Two days after injection of adjuvant

Histological appearances. Emigration of polymorphs and mono-
nuclear cells is still apparent as judged by their presence in the vessel
walls. Large dense collections of neutrophils are present, some of
them being degenerate. The rest of the dermis shows a diffuse heavy
infiltration of leucocytes, mononuclear cells now predominating over
polymorphs. The morphology of the mononuclear cells has changed
since 24 hr. More of the cells are large, elongated, fusiform or irregular
in shape with large oval or round nuclei and variable amounts of
eosinophilic cytoplasm. These cells are described as histiocytes or
macrophages. Conversely a smaller proportion of the cells are of the
free rounded variety dominant 24 hr earlier. A number of these have
small dark rounded nuclei, some with relatively scanty cytoplasm.
3HT labelling before initiation of granuloma. The percentage of
neutrophils containing tritium is somewhat higher (60 per cent.) at
2 days than at 24 hr. The proportion of all mononuclear cells iso-
topically labelled (fig. 4)is little changed. The average grain count per
nucleus is not significantly different from that found 24 hr earlier.
Some of the tritiated mononuclears have small round dark nuclei and
sometimes relatively scanty cytoplasm, so that they resemble small
lymphocytes (fig. 4). The endothelium and perithelium of vessel walls
do not contain the marker, but 15 per cent. of the histiocytes of the
upper dermis are labelled, possibly indicating that some of these cells
originate from monocytes, whereas vascular cells do not.
1. PATH. BACT.-VOL. 92 (1966) L2
166 W. G. SPECTOR AND A . W. J. LYkXE

3HT labelling I hr before death. In contrast to the findings at 24 hr,

2-day-old reactions show extensive uptake of tritiated thymidine by
mononuclear cells, indicating the onset of cell multiplication in the
exudate. Of all mononuclear cells a mean of 2 per cent. have incor-
porated the isotope. Some of the mononuclears are degenerate, and
none of these contain tritium. Of all the cells labelled about 50 per
cent. form part of vessel walls (fig. 5). Most of these appear to be
pericytes, but some uptake by endothelium cannot be excluded. Free
rounded cells with round or reniform nuclei (unaltered monocytes)
show labelling only rarely. Apart from the vascular structures the
great majority of 3HT-labelled cells are elongated, fusiform or pyriform
in shape with oval or rounded nuclei (histiocytes) or less commonly,
large round cells with abundant cytoplasm and round or oval nuclei
(macrophages: fig. 6). Morphologically all the cell varieties in 2-day
reactions that have taken up 3HT given 1 hr before death are also
labelled when isotope administration was completed before initiating
the granuloma. Another feature is the presence of labelled cells with
round nuclei apparently in the lumen of blood vessels (fig. 7). Blood
smears contain no labelled cells so that the intravascular tritiated cells
were presumably anchored " monocytes ".
3HT labelling 28 hr before death. There is a fall in the average
grain count of labelled cells to 8 grains per nucleus compared with 16
per nucleus 1 hr after administration of isotope. The percentage of
labelled mononuclear cells has, however, not risen to the extent that the
count might have led one to expect (Koburg, 1963), suggesting that
labelled cells had been diluted by entry of unlabelled cells. Polymorphs
containing 3HT are visible, indicating emigration of granulocytes
within the previous 24 hr. Morphologically, the tagged mononuclear
cells include many more small cells with round nuclei (including some
that resembled small lymphocytes) than they did 1 hr after labelling.
Of the labelled mononuclears 50 per cent. continue to form part of the
vascular wall and the number of labelled cells in vessel walls has risen
since one hour.
Carbon labelling. Carbon given 5 hr before the intradermal
adjuvant is widely distributed in mononuclear cells of all morphological
types. In particular the cytoplasmic marker is detectable in many
histiocytes and macrophages as well as in the smaller mononuclear
phagocytes and in small round cells with small dark round nuclei and
relatively scanty cytoplasm. Carbon given 5 hr before death blackens
venules extensively, indicating the persistence of increased vascular
permeability.
3 days after injection of adjuvant
3 HT labelling before initiation of granuloma. The proportion of
mononuclear cells containing tritium remains at 60 per cent., the
average grain count falling fractionally to 7 grains per nucleus. A high
proportion of the mononuclears are large histiocytes or macrophages,
 EVOLUTION OF INFLAMMATORY GRANULOMATA 167

of the types that in other experiments actively incorporated 3HT.

Even in the upper dermis 30 per cent. of these cells are labelled as
compared with 15 per cent. at 2 days. Considerable numbers of small
round cells with round dark nuclei and often with scanty cytoplasm
contain 3HT and sometimes carbon also. Many of these cells would
have been identified as small lymphocytes in the absence of cell markers.
Endothelium and perithelial cells never contain isotope, an indication
that re-utilisation of 3HT is minimal, since these cells had been actively
synthesising DNA in the previous 24 hr.
3HT labelling I hr before death. Uptake of tritium by mononuclear
cells is of a similar order in 3-day as in 2-day exudates, indicating a
sustained high rate of cell multiplication. Unaltered monocytes are
almost never labelled. Most of the cells that have incorporated the
marker are histiocytes or macrophages. There is a striking diminution
in the number of vascular pericytes and possible endothelial cells
incorporating 3HT. No more than 5-10 per cent. of labelled cells
form part of the vascular structures.
Carbon labelling. When carbon was given 5 hr before inoculation
of adjuvant a high proportion (10 per cent.) of mononuclears in the
exudate contained the marker. Cells heavily laden with carbon do not
take up 3HT given 1 hr before death, but those with finer cytoplasmic
deposits of small quantities do so. By now a good deal of the carbon
is present in histiocytes and macrophages, and a smaller proportion
than at 2 days in unaltered mononuclear phagocytes.

5 days after initiation of granuloma

Almost all the mononuclear phagocytes of the original exudate are
now replaced by histiocytes and macrophages. Many of these can now
be aptly described as epithelioid cells, being elongated, with foamy
eosinophilic cytoplasm which merges with that of its neighbour to
form a syncytium.

7 days after initiation of granuloma

Histological appearances. Most of the reaction is occupied by
histiocytes of the epithelioid type. Scattered amongst the histiocytes
are macrophages with an irregular rounded outline, large round nuclei
and abundant foamy cytoplasm. Also present are numbers of cells
with small round nuclei with a dense chromatin pattern, which at first
sight resemble small lymphocytes. Some of these cells, however,
unlike lymphocytes have relatively abundant cloudy cytoplasm and the
nucleus in many cases lacks the characteristic lymphocyte pattern of
angular chromatin pieces. The formation of new blood vessels or
collagen or increased numbers of fibroblasts is not in evidence.
3HT labelling before initiation of granuloma. The proportion of
labelled mononuclear cells has fallen to 35 per cent. from its peak of
65 per cent. (fig. 8). The average grain count of the labelled cells has
I68 W . G. SPECTOR A N D A . W. J. LYKKE

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.

3HT labelling I hr before death. The mean proportion of mono-

nuclear cells labelled is 2 per cent. Almost all the cells that incorporate
the isotope have eosinophilic lipid-laden cytoplasm and fall into the
morphological category of histiocyte or macrophage (fig. 9). A small
proportion of the labelled cells have large pale round nuclei with one
or two prominent nucleoli, and scanty cytoplasm. The cells with small
dark rounded nuclei practicallynever takeup the 3HT. Many of the cells
that have incorporated the nuclear marker are lining fatty vacuoles.
Only 5 per cent. (or less) of the labelled cells form part of the vessel
walls.
Carbon labelling. When carbon labelling was completed before
initiation of the granuloma, the 1-wk reaction still showed extensive
labelling (5-10 per cent. of mononuclear cells). Most of the labelled
cells are either histiocytes or macrophages, but an occasional smaller
mononuclear phagocyte containing carbon is present, although
only if it has fairly abundant cytoplasm. Large elongated cells
with elongated nuclei resembling endothelial cells or fibroblasts
 AND LYKKE
SPEC~OR XLW
PLATE
EVOLUTION
OF INFLAMMATORY GRANULOMATA

FIG. 2.-A 1 typical blood monocyte

doubly labelled with 3 1i-thymidine
and colloidal carbon. Autoradio-
graph, Giemsa. x 1OOO.

FIG.3.4olloidal carbon confined

within the wall of an abnormally
permeable small venule in a 24-
hr-old granuloma. Carbon was
injected 5 hr before death. Harris’
haematoxylin and eosin. X 1OOO.

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.

...

FIG. 7.~H-thymidine-1abelled mono-

nuclear cells within the lumen and
apparently moored to the wall of a
, venule in a 2-day-old granuloma. A
single dose of isotope was given 1 hr
before death. Autoradiograph, HHE.
x 1m.

r) a
 EVOLUTION OF INFLAMMATORY GRANULOMATA 169

do not show carbon labelling. On the whole the morphological

appearances of cells containing the marker are identical with those of
the cells that incorporate tritiated thymidine given just before death.
However, cells heavily laden with carbon do not take up the isotope,
whereas similar cells lightly labelled with carbon do.
When carbon was given 5 hr before death there was evidence of
extensive leakage of the colloidal particles from the venules. In
contrast to the findings at 24 hr, however, there is little or no blackening
of the vessel wall due to accumulation of carbon between endothelium
and basement-membrane. There is, however, extensive uptake of
carbon by mononuclear cells in and outside the vessel wall, and also
by mononuclears, apparently free in the vessel lumen. Carbon is
sometimes seen in such cells within the lumen, but is apparently
anchored to the vessel wall.

2 weeks after initiation of granuloma

3HT labelling before initiation of granuloma. In the two-week-old
reactions the proportion of all mononuclear cells labelled with 3HT has
fallen to a mean value of 25 per cent.; the average grain count is
4 grains per nucleus (fig. 8). Epithelioid cells, histiocytes and macro-
phages contain a similar proportion of tritiated cells. Examination of
the non-degenerate polymorphs lining the central vacuole reveals that
few of these cells contain isotopic thymidine, indicating that many of
them had emigrated in the previous week.
3HT labelling I hr before death. About 2 per cent. of all mono-
nuclears incorporate 3HT. The isotope is distributed evenly among
epithelioid cells, histiocytes and macrophages ; small round cells are
almost never labelled. Large round cells with scanty cytoplasm
and prominent nucleoli appear, as at 1 wk, to have taken up the
marker.
Carbon labelling. Carbon given 5 hr before initiation of the granu-
loma is clearly visible in the two-week-reactions, being present in
about 10 per cent. of histiocytes, epithelioid cells and macrophages
and in an occasional pseudo-lymphocyte. Carbon given 5 hr before
death fails to accumulate in the vessel walls, indicating the absence of
increased vascular permeability of the usual type. On the other hand
carbon has clearly passed through the vessel wall and is present in a
number of mononuclear cells, often some distance from the nearest
vessel.
4 weeks after initiation of granuloma
3HT labelling before initiation of granuloma. At 4 wk the over-all
proportion of labelled mononuclear cells has fallen to a mean of 7 per
cent. with an average grain count of 3 grains per nucleus (fig. 8). An
occasional highly labelled histiocyte or macrophage is present, possibly
indicating re-utilisation of isotopic thymidine.
170 W. G. SPECTOR AND A . W.J. LYKKE
3HTgiven I hr before death. The percentages and types of labelled
cell are in general similar to those in the 2-wk reaction. The 4-wk granu-
lomas are, however, characterised by the presence of focal collections
of cells resembling small lymphocytes. The foci include also a number
of larger cells with paler nuclei and some typical macrophages. In
these small round cell collections the small round-cells themselves
virtually never incorporate 3H-thymidine. On the other hand the
larger cells with paler nuclei in these areas contain a higher proportion
of labelled cells than the population of the histiocytic areas.
Carbon labelling. Carbon given a few hours before initiating the
reaction is still detectable as fine, dust-like particles in the cytoplasm
of histiocytes, epithelioid cells and macrophages scattered through the
granuloma. Carbon given a few hours before death fails to blacken
vessel walls. Nevertheless some carbon particles pass through the
vessel wall and as at earlier times are taken up by mononuclear phago-
cytes of various types lying beyond (fig. 10).

8 weeks after initiation of granuloma

3HT labelling before initiation of granuloma. The over-all proportion
of mononuclear cells containing the isotopic marker is 3 per cent. and
the average grain count 2.5 grains per nucleus-the lowest value
feasible to measure. The morphological varieties of cells that incor-
porate the isotope are similar to those at 2 and 4 weeks.
3HTgiven I hr before death. The findings were similar to those at
4 wk. The over-all proportion of mononuclear cells labelled is 2 per
cent., rather higher (3 per cent.) in the small-round-cell areas. Within
these areas tritium incorporation is not seen in the small round-cells
themselves, but is present in a variety of larger cells with oval, round
or reniform nuclei (fig. 11). These cells could have been described
variously as histiocytes, reticulo-endothelial cells, haemocytoblasts or
lymphoblasts. The average grain count is 16 per nucleus.
3HT given 24 hr-7 days before death. To follow the fate of the
labelled cells described above, 24 hr were allowed to lapse between
administration of isotope and killing the animal. Within the small-
round-cell areas, the distribution of tritium is now quite different and
the number of labelled cells has risen to 8 per cent.; of the cells that
have incorporated tritium, 65 per cent. are small round cells (fig. 12)
as compared with less than 1 per cent at 1 hr. The average grain count
of the labelled cells shows a corresponding drop to 8 per nucleus.
When 3 days passed between labelling and death, the picture was
found to have changed further. The percentage of labelled cells in the
small-round-cell areas remains around 8 per cent. and the average grain
count has fallen to 6 per nucleus. However, in the same area the propor-
tion of labelled cells categorised as small round cells has fallen to about
50 per cent. and a corresponding higher proportion resemble histiocytes
or reticulo-endothelial cells. When 7 days were allowed to pass between
 EVOLUTION OF INFLAMMATORY GRANULOMATA 171

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.

12 weeks after initiation of granuloma

3HT labelling before initiation of granuloma. The proportion of
cells containing tritium and their average grain count are now too low
for study.
3HTgiven 1 hr before death. The proportion of cells that incorporate
the isotope is 1.5 per cent. The cell types observed to do so are similar
to those at 8 wk (fig. 13)--in particular labelling is seldom found
amongst the many small round cells in the sections. Isotopic incor-
poration is however seen in larger cells amongst the small-round-cell
collection (fig. 14). These larger cells often have large rounded nuclei
and prominent nucleoli and sometimes scanty cytoplasm. Langhans’
giant cells never possess labelled nuclei.
Carbon. Carbon given shortly before initiation of the lesion is still
visible in macrophages and epithelioid cells 12 wk later (fig. 15). As
at 8 wk the marker is present chiefly in the form of fine dust-like
particles in the cytoplasm. Many carbon-laden Langhans’ giant cells
are present (fig. 16). If given shortly before the death of the animal,
carbon was not visible either in or beyond the vessel walls. An
occasional carbon-laden monocyte is seen within the vascular lumen.

3HT labelling of small lymphocytes prior to induction of granuloma

Mice were given a course of single daily intraperitoneal injections
of 3H thymidine (0.5 pc per g.) for 28 days. Blood smears were
examined from 9 to 23 days after the end of the course. The proportion
of small lymphocytes labelled was 12 per cent. at 9 days, 8 per cent. at
16 days and 6 per cent. at 23 days. The proportions of monocytes
containing tritium at the same times were 14, 9 and 4 per cent.
Nine days after the last injection of isotope, complete adjuvant was
injected intradermally into the dorsum of the hind feet. Two weeks
later the animals were killed and the granulomata examined. Basically
the histology is similar to that of comparable lesions in rats. The
sections are distinguished, however, by the presence of large numbers
of small cells with dark round nuclei, many of which resemble small
lymphocytes. These cells tend to be diffuse rather than localised into
foci. In the granulomata of animals whose blood-smear autoradiograph
analyses are quoted above, the proportion of small round cells labelled
with tritium is 3 per cent. The proportion of all other mononuclear
172 W. G. SPECTOR A N D A . W. J. LYKKE

cell types including epithelioid cells, histiocytes and macrophages

similarly labelled is 1 per cent.
For comparison a further group of mice were given the usual 3-dose
course of 3HT, and adjuvant was then injected 8 hr after the last dose
of isotope. Blood smears at this time show the expected high rate of
incorporation into monocytes and a low rate of 2 per cent. in small
lymphocytes. Two weeks later when the animals were killed, autoradio-
graphs of the blood revealed that tritium labelling of both monocytes
and small lymphocytes was less than 1 per cent. Similar examination
of the 2-week-old granulomata indicated that 0.5 per cent. of the small
round cells were labelled and that 2 per cent. of the other mononuclear
cells (e.g. histiocytes) had incorporated the nuclear marker.

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

As judged by 3HT incorporation after 1 hr, proliferation was

confined to cells with the morphology of histiocytes and macrophages.
24 hr after the single dose of isotope, however, the morphology of the
labelled cells had changed, and their number had increased so that as
well as histiocytes and macrophages there were many smaller cells
containing 3HT. In particular a number of small round cells resembling
lymphocytes were labelled, and thus were apparently derived from
dividing histiocytes or macrophages. On the other hand most of these
small round cells differed from lymphocytes in having a more abundant
cytoplasm. From these findings therefore it seems that at least some
of the progeny of dividing histiocytes and macrophages pass through a
period when their morphology is that of a small round cell and they
might be said to resemble small lymphocytes (fig. 17). The key to the
origin of the proliferating histiocytes and macrophages is provided by
HISTIOCYTE
,. ..;.*.
,
......
' . .:\.
. . .
MONOCYTE
FROM
BLOOD

FIG. 17.-Schematic presentation of the suggested sequence of events whereby initial

colonisation by monocytes is followed by sustained but not necessarily by
expanding granuloma formation.
the experiments in which tritium labelling was completed before
inflammation was induced. In these animals there was in the first
week of granuloma development a striking fall in the proportion of
labelled mononuclear cells and in the average grain count. Taken
together these two measurements indicate that a large part of the
decline in labelled cells was due to their division with resulting dilution
of the isotope to the point of extinction (Koburg,1963). The morphology
of the labelled cells in these experiments was similar to that of the
cells that took up 3HT within 1 hr of its injection. These observa-
tions provide clear evidence that blood monocytes undergo proliferation
after migrating into chronic inflammatory areas and that such pro-
liferation does not occur until the monocytes have become transformed
to the morphological types known as histiocytes or macrophages,
a process completed in the first week. The actual values indicate that
monocytes are the major source of dividing cells in the granuloma, with
pre-existing tissue histiocytes as a secondary source.
The behaviour of carbon injected into rats with granulomata of
1-4 weeks' duration is of interest. In these animals the particles did
not cause massive blackening of vessels with accumulation beneath the
basement-membrane as at earlier times. Instead a much smaller
174 W. G. SPECTOR AND A . W. J. LYKKE

quantity of carbon passed through the basement-membrane and was

taken up by phagocytes in the perivascular tissues. This difference in the
behaviour of carbon in the earliest and in the later phase of the reaction
seems likely to be due to persistent alteration in the retentive function
of the basement-membrane.
Carbon injected before the granuloma was induced did not enter
the reaction site except in the circulating phagocytes. Large numbers
of carbon-laden monocytes entered the area in the first 24 hr, and 12
weeks later a significant proportion of histiocytes or macrophages
contained the intracytoplasmic carbon. The pattern appears to be
that carbon-bearing monocytes died at the end of their natural life-span,

CELL
DEATH

PHAGOCYTOSIS
...-
: b '
FREE
H IST IOCYTE CARBON
I
CEYL
CELL
DEATH

..a.
. *e

FIG. 18.-Schematic presentation of the suggested sequence of events whereby insoluble

particulate matter is retained within a granuloma.

whereupon the particles were ingested by histiocytes or macrophages

in the immediate vicinity, which, when they eventually died, in turn
yielded the cytoplasmic carbon to a new generation of similar cells
(fig. 18). Studies with 3HT indicate that cells laden with carbon are
incapable of mitosis. Uptake of colloidal particles by newly formed
phagocytes seems to explain phenomena such as cutaneous tattooing,
where pigmented particles may persist and retain their original pattern
for a lifetime.
A feature of granulomata of 4-12 weeks' duration was the presence
of focal collections of small round cells. Some of these cells had
relatively abundant cytoplasm, but many were indistinguishable from
small lymphocytes. In earlier lesions, some small round cells were seen
and many of these at least were derived from monocytes or histiocytes
with or without an intervening mitosis (fig. 17). A single " pulse " of
3HT in rats bearing 4-12-week lesions revealed that the lymphocytes
or pseudo-lymphocytes almost never incorporated the isotope, but that
larger paler-staining cells in the foci frequently did so (figs. 11 and 14).
 EVOLUTION OF INFLAMMATORY GRANULOMATA 175

24 hr later, however, the tritium was found widely distributed among

the small round cells, indicating that they originated from the larger
paler cells by mitotic division (figs. 17 and 19). The origin of these
larger cells is uncertain, but the experiments with labelling of small
lymphocytes in mice suggest that some of them may have been lympho-
blasts derived from small lymphocytes that emigrate relatively late in
granuloma development (fig. 19).
Nevertheless it is not yet clear what proportion, if any, of the small-
round-cell collections that form such a well known feature of granulo-
mata are in fact lymphocytes. It is hoped that future experiments may
SMALL "LYMPHOBIAST"
_.,. .
SMALL
LYMPHOCYTE
FROM BLOOD
....... 111,

1,

EL000
MONOCYTE

FIG. 19.--Schematic presentation of two possible pathways for the derivation of local
small-round-cell or lymphoid collections in granulomata.

clarify this point. It is plain, however, that many of thesecellsareformed

in situ from precursor cells, even though the origin of the latter is in
doubt. However, whether derived from lymphoblasts or histiocytes of
monocytic origin or histiocytes of tissue origin, many of the small
round cells of the granulomata seem to be an intermediate stage in the
proliferation of the larger cells. Thus after a single dose of 3HT in
8-wk granulomata, the label appears first in the larger precursor cells
at 1 hr, then at 24 hr in the small round cells, and then 2 days later it
is shared between small round cells and larger precursor-type cells.
Since the small round cells do not appear to divide, the reappearance
of isotope in the larger cells at 3 days presumably represents " modula-
tion " of the smaller to the larger cell type (figs. 17 and 19). This
cyclical sequence of cell development and division is reminiscent of
events in lymphoid tissues (Gowans et al., 1962; Craddock et al., 1964;
Turk and Heather, 1965).
It seems likely from other work (Bryant, 1962; Diderholm,
Fichtelius and Linder, 1962) that some re-utilisation of labelled DNA
176 W . G. SPECTOR A N D A. W. J. LYKKE

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.

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 SPECTOR
AND LYKKE PLATEXLIX
EVOLUTION
OF INFLAMMATORY GRANULOMATA

FIG.9.-jH-thymidine: labelling of a
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FIG. lO.--Colloidal carbon within

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FIG. 11 .-3H-thymidine labelling of large

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asingle dose 1 hr beforedeath. Auto-
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 SPECTOR A N D LYKKE PLATEL
EVOLUTION
OF INFLAMMATORY GRANULOMATA

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FIG. 14. -3H-thymidine uptake

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SPECTOR A N D LYKKE PLATELI

EVOLUTION
OF INFLAMMATORY GRANULOMATA

FIG. 15.-Carbon-laden macrophages in a 12-wk-old granuloma. The carbon was

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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

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