Cell Tissue Kinet. (1975) 8,491-502.
STUDIES O N THE REGULATION O F LYMPHOCYTE PRODUCTION I N THE M U R I N E T H Y M U S A N D SOME EFFECTS O F A C R U D E T H Y M U S EXTRACT LENNART OLSSONA N D MOGENSH. CLAESSON Institute of Medical Anatomy A , Copenhagen, Denmark (Received 20 December 1974; revision received 24 March 1975)
ABSTRACT
Cell proliferation in the murine thymus was studied in viuo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the fist 24 hr the mitotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0-8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering G,/G, cells into DNA synthesis, the inhibition of G2 efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed. INTRODUCTION It is generally accepted that the production of erythrocytes and leucocytes is under homeostatic regulation. The small lymphocytes have their origin in the bone marrow, the thymus, and peripheral lymphoid tissues, where blast cells are transformed to small lymphocytes by proliferation and differentiation (Fliedner et al., 1964; Cronkite & Chanana, 1970; Bryant, 1972). The major part of the thymus consists of proliferating lymphoid blast cells and small non-DNA synthesizing lymphocytes (Metcalf, 1966). Since the organ contains both the generative and differentiatedcell populations it is suitable to studies on the mechanisms controlling 1ymphopoiesis. Weiss & Kavanau (1957) proposed a template-antitemplate model for the regulation of cell production, suggesting an autoregulation based on cybernistic principles with the negative feedback mechanism acting in the link between the generative and differentiated Correspondence: Dr L. Olson, The Institute of Medical Anatomy A, University of Copenhagen, 71Raadmandsgade, DK-2200 Copenhagen N, Denmark. 33 491
492
Lennart Olsson and Mogens H. Claesson
cells. The model implies that the differentiated cells secrete substances that regulate by inhibition the rate of cell renewal. Experiments with crude water-soluble extracts of proliferating tissues have shown that these extracts may inhibit the mitotic activity of the tissues from which the extracts have been recovered (Bullough, 1971; Elgjo, 1972; Iversen, 1973). Bullough (1962) proposed the name ‘chalone’ to such an extract. In lymphoid systems mitotic inhibitors have been recovered from lymph nodes and spleen. In uitro these agents induce inhibition of DNA synthesis in human blood lymphocytes (Moorhead et al., 1969; Houck, Irausquin & Leikin, 1971; Math6, 1972). Recently it has been shown that a crude extract from calf thymus inhibits some immune functions of the T-cells in the mouse (Kiger et al., 1973). In the present work we have studied the effects in vivo of a crude water-soluble extract of mouse thymus, spleen, and skin on the proliferation of thymic blast cells. The parameters are mitotic activity, 3H-TdR initial labelling index (LI), initial average grain counts, distribution (in per cent) of cells within the cell cycle, and cell death. MATERIALS A N D METHODS Animals. Inbred conventional Balb/c mice of both sexes were used. The mice were 6-7 weeks old and were kept in plastic cages with four animals in each cage. The light was automatically switched on and off at 6 a.m. and 6 p.m., respectively. Preparation of extracts. Extracts from thymus, spleen and skin were prepared as follows : Immediately after the mice were killed by ether narcosis the organs were removed, frozen in liquid nitrogen and ground to powder in a mill cooled by liquid nitrogen. The powder was suspended in ice cold distilled water and centrifuged at 15,000 g for 30 min at 4°C. The supernatant was Iyophilized and stored at -70°C until used for injection. The injected dose of lyophilized powder per mouse was : thymus extract (TE) 1SOmg, spleen extract (SE) 1.5 mg, and skin extract (SkE) 5.0 mg. The doses correspond to extracts obtained from one thymus, one spleen, and the skin of one mouse. All doses were dissolved in 0.25 ml physiological saline and injected intraperitoneally. Microscopical slides. The thymus was minced with fine needles in ten drops of Hanks’ solution at 4”C, washed, and the cell number adjusted to 20 x 106/ml. Supravitally stained preparations were used for the mitotic counts. A few drops of the thymus suspension were stained with a mixture of Giemsa and methyl green dissolved in acetic acid (Nakamura & Metcalf, 1961). Normal smears for autoradiography and microspectrophotometry were fixed in 4°C cold Carnoy fixative for 10 min and stained (see below). Smears to microspectrophotometry were made on < I mm thick glass slides. Skin biopsies were taken from the lumbar region, fixed in Bouin’s fixative for 24 hr and prepared routinely taking 4 pm sections. Mitotic activity. The stathmokinetic method (Dustin, 1959) was used to calculate the mitotic rate. Pilot experiments showed that 0.15 mg Cokemid@(Ciba) per animal is an optimal dose to arrest thymic blast cell mitoses in the metaphase. The increase in the number of metaphases was approximately linear to a 4 hr period after injection of colcemid. Mitotic rate was estimated as the increment in the number of metaphases per hour in the 4 hr period. We have demonstrated earlier that these conditions also are suitable for the basal cell layer of epidermis (Olsson, 1972). In thymus the mitotic index was estimated by counting
Lymphocyte production in murine thymus
493
1000 consecutive cells in a supravitally stained smear. In epidermis the number of mitoses per 2000 interfollicular basal cells was estimated on each biopsy. Microspectrophotometry. The fixed smears were stained by Eeulgen method as follows : The slides were hydrolysed for 25 min in 1 N HC1 at 20°C, stained in a Schiff reagent (1 g pararosaniline (Chroma) in 30 ml 1 N HCI mixed with 1 g potassium bisulphate dissolved in 170 ml distilled water) for 30 min and rinsed three times (3 x 10 min) in a freshly prepared bisulphite solution. A Zeiss Universal Microspectrophotometer (UMSP I) incorporating an occular grid for cell diameter measurements was as described by Sordat et al. (1972). Our classification of thymic blast cells in GI cells, S cells and G2 cells was based on the fact that all small lymphocytes (nuclear diameter 4 3 pm) contained about 2N (arbitrary unit) DNA and all mitoses about 4N DNA. According to Metcalf (1966), thymic cells with a nuclear diameter 28 pm were recorded as blast cells. 200 blast cells from each thymic smear were measured. Autoradiography. The mice were injected i.p. with pCi/g 3H-TdR (spact. 6.7 Ci/mmol, NEN) 30 min before sacrifice. The fixed smears were dipped in Kodak K2 emulsion, exposed for 30 days at 4"C, developed, fixed, and stained with haematoxylin and eosin. The initial labelling index was estimated in the large blast cell population (nuclear diameter > 12 pm) and in the medium sized blast cell population (8 pm < nuclear diameter < 12 pm). 400 cells of each category were counted per thymus smear. Cells with four grains or more were considered as labelled. The average grain count was estimated from 150 consecutive labelled blast cells. Supravitally dye exclusion. 25 p1 of the thymus suspension was mixed with 100 pl of a 0-1% nigrosin solution in phosphate buffered saline and stained for 5 min at 4°C. The number of viable and dead (stained) cells were counted within 10 min. At least 100 stained cells were registered and the percentage of dead cells calculated. RESULTS Mitotic activity. The number of mitoses in the thymus lymphoid cells was measured over 4 hourly iniervals between 0-4 hr, 4-8 hr, 8-12 hr and 20-24 hr afcer injection of TE, SE, SkE, or physiological saline starting at 5 a.m. In the text and in the Tables only the time of killing is given. Thus, the mitotic rate at 4 hr means the average mitotic rate from t = 0 to t = 4 hr. The last group served as controls. All animals were injected with colcemid 4 hr prior to sacrifice. The results are shown in Table 1; from these results the mitotic rate was calculated. TE induced a significant reduction in the mitotic activity 4,8,12 and 24 hr afcer the injection as compared to controls. The depression was at average 30 %. SE induced a significantly lower mitotic activity 8, 12 and 24 hr after administration as compared to controls. In SkE treated animals no effect was detected on the mitotic activity. On the other hand, SkE induced a decrease in the mitotic activity in the basal cell layer of epidermis, while TE, SE, and physiological saline had no effect on the mitotic activity in this tissue (Table 2). SkE induced a 45 % depression in the mitotic activity of the basal cells 4 hr afcer the injection and a 20 % depression 4 hr later. The mitotic activity was normal 12 and 24 hr afLer treatment with SkE. Mirrospectrophotometric and airtoradiographic data. The effects of TE, SE, and physiological saline on the pattern of proliferation of thymic blast cells were analysed. The results from the groups treated with saline (controls) are shown in Fig. 1.
4.0 & 0.3 2.6 -f 0.1 35 0.001 < P < 0.01 0.49 %/hr 3.0 & 0.3 25 0.001 < P < 0.01 0.53 %/hr
3.1 & 0.3 31 0.001 < P < 0.01 0.53 %/hr 4.3 40.2
MI rf: SEM (%) Depressionf ('4 Probability oft8 MRll MI&SEM("%) Depressionf Probability oft§ MRlf MIkSEM(%) Depression$. (%) Probability oft$ MRll
Thymus
Spleen
Skin
0.78 %/hr
0.73 %/hr
0.15 %/hr
3.9
3.8 & 0.2
2.1 & 0.2 34 P < 0.001 0.45 %/hr
2.7 L- 0.1 34 P < 0.001 0 4 5 %/hr
4.1 & 0.2 0.80 %/hr
24
*0 1 -
2.4 ? 0.2 38 P < 0.001 0.38 %/hr
2.9 & 0.1 26 0.001 < P < 0.01 0.50%/hr
3.9 If:0.3 0.75 %/hr
12
7 Mitotic rate (MR) was estimated as the increase in the number of metaphases per hour in the 4 hr period.
$ Compared to the corresponding group treated with physiological saline. Q Tested with the corresponding group treated with physiological saline.
t Each group consisted of eight animals.
-
0.83 %/hr
-
4.0 5 0.1
4.2 & 0.2
0-83%/hr
-
* All animals received a colcemid injection 4 hr before sacrifice.
(x)
0.85 %/hr
*
4.5 0.2 0.90%/hr
MI k SEM ( MRll
Physiological saline O h
8
4
Source of extract
Hours after injection of extracts
TABLE 1. Effects of the extracts on the mitotic activity in thymus*.?
-m: F
9 n F
3
tJ.
5
%
5SL
3
g
0
Y
P
MI f SEM (%) Depression1 (%) Probability of tQ MI+SEM(%) Depression$ (%) Probability oft§
Spleen
Skin
11.4 f 0.8
-
27 0.05 > P > 0.02
-
$ Compared to the corresponding group treated with physiological saline.
Q Tested with the corresponding group treated with physiological saline.
-
11.5 f 1.2
-
11.9 L- 0.9
-
11.8 f 1.5
11.5 k 1.1
-
11.6 f 1-1
24
11.3 If: 0.8
-
11.8 f 1.3
12
8.3 k 1-1
-
11.6 f 1.3
-
12.0 f 1.1
11.4 f 0-6
8
* All animals received a colcemid injection 4 hr before sacrifice. t Each group consisted of eight animals.
6.6 f 1.4 45 0.05 > P > 0.02
-
12.4 f 1.8
0.7
MI f SEM (%) Depression1 ('A Probability of t Q
Thymus
-
12.1 f 1.1
MIfSEM(%)
Physiological saline 11.8
4
Source of extract
Hours after injection of extracts
TABLE 2. Effects of the extracts on the mitotic activity in the basal cell layer of epidermis*, t
E
3
-c8.
5
3
s.
3;31
i2$
2
% 0
3
L
6'
496
Lennart Olsson and Mogens H. Claesson NaCl
-.
----. ........... ia
C : large blast cells
.
4 hr . 8 hr .. 12 hr : 24 hr
m :medium blast cells
17.9+_04 15.7f0.616.820.4 15,4205
90 -
I-\
I
I
\ '
I€
--
14
f
12
70
-
x U W
S 50 m ._ 2 40-
b 10
-
0 W
E
-
3 60-
W
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80
Average grain counts
8
:6
U
a
5 30H
4
'.
2
..-
20
-
10
L
16
2 0
2 4
20
3 2
36
DNA-content
4 0
4 4 48"
lm 4hr
!1 Lm
Lm
Lm
8hr
12hr
24hr
FIG.1. Left: The curves show the distribution of thymic lymphoid blast cells as a function of their DNA-content at various time intervals after injection of physiological saline. The amount of DNA is given in arbitrary units. GI cells have a DNA-content of 2N or a little less, G, cells 4N or a little more, and the DNA-content of S cells is 2N-4N. Each curve is the result of an equalization by least squares method of the mean values for four animals. The average SEM value was +_1-0%.Right: The initial labelling index in the population of large blast cells (I) and the medium sized blast cells (m) is shown at the columns. Each value represents the mean of the values from four animals. Vertical bars represent SEM values. Finally the average grain counts values +SEM are shown.
The number of blast cells in each of the three interphase compartments varied as a function of time, but it is evident that the majority of blast cells are almost always in the S phase. Furthermore, it is strongly suggested from the figure that the blast cells are in partial synchrony. The percentage of labelled large and medium sized blast cells is also shown in Fig. 1. Only minor variations were observed during the 24 hr. The medium sized blast cells constituted about 90 % of the total blast cell population. At average 70 % of the medium sized blast cells were labelled, while LI of the large blast cell population was about 75 %. Fig. 2 shows the results in the groups treated with TE. 4 hr after administration blast cells accumulated in the late G , and early S phase. This was still evident 4 hr later, when most of the blast cells were found between the first and second third of S . 12 hr after the injection of TE the wave of synchrony had reached the end of the S phase, and 12 hr later the bulk of cells had apparently passed through mitosis. Treatment with TE induced marked alterations in initial LI. 4 hr after the injection only 45 % of both the medium and large blast cells were labelled. These low values were also found at 8 and 12hr. 24 hr after administration of TE the initial LI was at control levels. TE also inhibited DNA synthesis in the blast cell
497
Lymphocyte production in murine thymus Thymic- extracr
-.
----........... -.-.-
18 -
L : large blast cells m : medium blast cells
.
4 hr : 8 hr : 12 hr : 24hr
12.8fb2 11.5t0.4 11.1 + I 2 15;1+07
------.90
Average grain counts
16 14
-
II)
8 12-
c
In
54
10-
-E
8-
L
0
'
0 C
2
6 4-
2L
I
I
I
I
I
-
I
DNA content
I
I
I
4hr
8 hr
I
I2hr
24hr
FIG.2. Left: The curves show the distribution of thymic lymphoid blast cells as a function of their DNA-content at various time intervals after injection of the thymic extract. Right: The initial labelling index in the population of large blast cells (1) and the medium sized blast cells (m) is shown at the columns. Each value represents the mean of the values from four animals. Vertical bars represent SEM values. Finally the average grain counts values fSEM are shown.
population as judged by grain counts analyses. The average grain counts were reduced about 30 % (P < 0.01) 4,8 and 12 hr after the injection of TE. Grain counts in the 24 hr group were similar to controls. The ratio between large and medium sized blast cells was not changed by TE. The results from animals treated with spleen extract are shown in Fig. 3. 4 hr after the injection of SE the blast cells accumulated in early S phase, but by 8 hr the wave of synchrony had passed through most of the S phase. 4 hr later the majority of the blast cells was in early S, and thus they must have passed through mitosis and GI. 24 hr after treatment with SE most of the cells was in early S . The spleen extract also induced changes in initial LI of the medium sized blast cells 4 and 8 hr after the injection, while LI was normal in the 12 and 24 hr groups. 4 hr after the injection of SE 48 % of the medium sized blast cells was labelled, and 4 hr later only 40% was labelled. The average grain counts was reduced only in the 4 hr group (about 20%, P < 0-01). The ratio between large and medium sized lymphocytes did not differ from the ratio in controls. Planimetric measurements on the microspectrophotometric curves was used to estimate the distribution (in per cent) of interphase blast cells in the three interphase compartments (Table 3). Under normal conditions about 10 of the cells are in GI, 85 % in S, and 5 % in G,. TE induced an initial increase of G, cells, a decrease in S cells, and an increase in G, cells. 8,12 and 24 hr after TE treatment the percentage of cells in G1and S was reduced, and
498
Lennart Olsson and Mogens H . Claesson Spleen- extract
-. ----
1:large blast cells
.
4 hr : 8 hr ........... : 12 hr -.-.- : 2 4 h r
rn:mediurn blast cells 13.6k0.7 142f0.9 15920.2 16.8k1.1
gOl Average grain counts
16
i
.,’
80 \.
-s? 70 -
14 -
- 60-
Ln
--d U Ln
12-
x
’p
._
10 -
p 50-
._ n 40-
L
o
-F
0
0
8-
-
C
0 ._
4-
20 -
2-
10 -
”,
6-
n
H
I
I
I
I
I
I
I
I
1.6
2.0
2.4
28
3.2
3.6
4.0
4.4
30
4.8
-
DNA content
m
Lrn
Lrn
Lm
4hr
8hr
12 hr
24hr
FIG.3. Left: The curves show the distribution of thymic blast cells as a function of their DNAcontent at various time intervals after injection of the spleen extract. Right: The initial labelling index in the population of large blast cells (1) and the medium sized blast cells (m) is shown at the columns. Each value represents the mean of the values from four animals. Vertical bars represent SEM values. Finally the average grain counts values +SEM are shown.
those in G2increased. However, the pile up of cells in G 2 at 12 hr (35%) decreased at 24 hr (19%). SE induced similar changes, though less marked, in the pattern of proliferation as those induced by TE. From the microspectrophotometric measurements for the controls it is possible to evaluate the average cell cycle time (t,) and the average duration of S (Ts).Fig. 1 shows that the wave of synchrony passed through all phases of the cell cycle within 10-12 hr, which thus is the average value oft,. The top of the wave of synchrony passed from 2N to 4N in about 6 hr (=Ts).From the experimental curves it is seen that the wave moves very slowly at the beginning of the experiment-especially in TE treated animals-later it accelerates to normal values. TABLE 3. Effects of the extracts on the distribution (in per cent) of blast cells in G , , S and GZphases Physiological saline
Thymus extract
Spleen extract
Phase
4hr
8hr
12hr
24hr
4hr
8hr
12hr
24hr
4hr
8hr
12h
24h
GI S
8 85 7
11 87 2
8 81
10 87
6 83
11
3
17 70 13
4 60 35
5 76 19
13 76 11
11 60 29
16 75 7
6 79 15
~~
Gz
11
Lymphocyte production in murine thymus
499
The effect of TE on the viability of thymic lymphoid cells is shown in Table 4.4,8 and 12 hr after treatment no differences were found as compared to controls. Only in the 24 hr group a significant (P< 0.001) higher percentage of non-viable cells were present in the group treated with TE compared to animals treated with physiological saline or SkE. TABLE 4. Effects of the extracts on the percentage of non-viable cells*
Hours after injection of extract Source of extract Physiological saline Skin Thymus
4
8
12
24
4-8 f 0.5 4.2 f 0.6 4-2 k 0.5
4.8 f 0-6 4.4 f 1.0 4.3 5 0.8
4.5 -+ 0-5 4.2 f 0.3 4-1 f 1.0
4.6 If: 0-4 4.5 k 0.2 7.7 k 0.5t
* Each value is the mean of eight animals k SEM. t P i0.001 by testing the value vs. corresponding control value. DISCUSSION
In the present study mitotic activity of thymic blast cells as well as initial LIs in control groups were comparable to values obtained by other authors (Nakamura & Metcalf, 1961; Metcalf & Wiadrowsky, 1966; Bryant, 1972). DNA synthesis, estimated by average grain counts, was about the same rate throughout the S phase with a tendency to a higher activity in the last third part of the phase. This is in agreement with studies on calf thymocytes (Sordat er al., 1972). By microspectrophotometry it was possible to study directly the percentage distribution of blast cells within the mitotic cell cycle. The blast cells showed different DNA distribution profiles at various time intervals after saline treatment, suggesting a partial synchrony in the pattern of proliferation. The t, value of 10-12 hr indicates a biphasic diurnal rhythm of the mitotic activity in thymic blast cells. Other studies have shown a monophasic diurnal rhythm in mitotic index in the thymus (Kirk, 1972). The discrepancy might be due to measuring the mitotic index as the only parameter of mitotic activity. The different values of the percentage of S cells obtained by autoradiography and microspectrophotometry might reflect the fact that the microspectrophotometric method does not sharply delimit S cells, and that some S cells synthesize DNA very slowly, resulting in lack of labelling in the autoradiographs. The water-soluble thymus extract obviously inhibited mitotic activity in thymic blast cells. As TGzof these blast cells is 1-2 hr (Metcalf & Wiadrowsky, 1966; Bryant, 1972), the mitotic inhibition observed 4 hr after the injection of the extract is caused by a decrease in the rate of the progression of G2cells and/or S cells toward mitosis. The inhibition observed in the 8,12 and 24 hr groups could be explained by an inhibited progression at any part of the cell cycle. TE induced a halving of the initial LI in all experimental groups, except the 24 hr group. This might be caused either by a decreased influx of cells in S or an inhibited DNA synthesis activity. The grain count values suggest that an inhibited DNA synthesis is the major cause. The microspectrophotometric results indicate that TE very rapidly blocks the entry of cells from GI to S. The pronounced inhibitory effect on influx in S and the decreased rate of cell progression through S and/or G2is in accordance with the effects of other tissue extracts
500
Lennart Olsson and Mogens H. Claesson
(Bichel, 1971; Elgjo, Laerum & Edgehill, 1972). Although the progression rate seemed normalized 24 hr after TE injection, the mitotic activity had not reached control levels at this time. The perturbations induced by SE were quantitatively similar to those of SE, but less pronounced. The difference between the effects of the two extracts are probably a result of different concentrations of the active substance(s). A plausible explanation, which is in agreement with both theoretical concepts (Weiss & Kavanau, 1957) and experimental data (Kiger et al., 1973), is that the T-cell population secretes the inhibitor. As the spleen contains about 45 % T-cells (Raff, 1970) it is to be expected that the SE would have a weaker effect compared to TE. In the basal cell layer of epidermis a water-soluble skin extract proved to inhibit mitotic activity, while physiological saline, TE, and SE had no effect. The results in the present study thus indicate a high degree of tissue specificity. The dye exclusion test showed an increase in cell death 24 hr after injection of TE. This late effect may reflect an increased cell decay among some of the arrested S and/or G, cells. These cells do not re-enter into the cell cycle. The present study has not only confirmed the existence of inhibitors of DNA synthesis, which have been demonstrated previously in various systems in vitro, but the study also suggests the existence of substances inhibiting the progression of cells through the G2phase and/or late S phase. This is a new observation in lymphoid cells, but it is comparable to the effects of skin chalones on the proliferation of epidermal cells (Elgjo et al., 1972). The chemical nature of the various water-soluble extracts with inhibitory effects on cellular proliferation are at present unknown. Cyclic-AMP has been suggested (Vorheers et al., 1973), but it can be excluded as the active component in extracts prepared the manner as those used in the present study. It is more likely that cyclic-AMP takes part in processes which trigger cells to start DNA synthesis, and where the first link is the removal of a tissue specific S phase inhibitor. While lymphocytopoietic inhibitors of the thymus have been studied to only a very limited extent, a thymocyte stimulating factor (thymosin) has been the subject of several investigations (Trainin, 1974). Essentially, thymosin stimulates T-cell differentiation and, perhaps, T-cell proliferation. It is surprising that no attempts have been made to relate thymosin t o inhibitors of lymphoid cell proliferation. It now seems reasonable to propose as a hypothesis that the production of thymic lymphoid cells is controlled in at least two ways. The thymosin stimulates the T cell differentiation and thymocyte emigration, while the extract investigated in our experiments (TE) inhibits the proliferation of thymoblasts, thereby controlling the size of the cell pool on which thymosin can act. Fig. 4 illustrates this hypothesis. The model suggests that proliferation of thymoblasts is regulated through a negative feedback mechanism. The inhibition affects both the initiation of DNA synthesis and mitosis and is carried out by some products released from differentiated small lymphocytes. The concentration of thymosin can probably be influenced by factors (e.g. antigens) in the microenvironment; factors which are known to influence the number of peripheral lymphocytes. The stimulation increases emigration of lymphocytes to the periphery, consequently a decrease in the number of cells that produce inhibitor substances, which results in an increase in the rate of proliferation of the blast cells. The model thus implicates both a regulation of proliferation and differentiation and is consistent with both stimdatory and
Lymphocyte production in murine thymus
501
BLOOD VESSEL
m
PROLIFERATION.0IFFERENTIATlON AND EMIGRATION
----_> INHIBITION
OF PROLIFERATION
FIG.4. A hypothesis for the production, differentiation and emigration of small lymphocytes in the thymus. I: Normal state. Thymosin (dotted area) secreted from medullary epithelial cells controls the differentiation of blast cells to small lymphocytes (dark cells) and regulates the emigration of cells. The differentiated cells produce a factor which partly inhibits blast cells proliferation. There is a sparse emigration of small lymphocytes. 11: Differentiative state. Stimulation (e.g. by antigens) causes an increased differentiation of small lymphocytes ready for emigration. Inhibitory substances from these cells inhibit further proliferation of blast cells. The emigration of lymphocytes is still sparse. 111:Proliferative state. Thymosin has disappeared. Differentiation rate is reduced, and small lymphocytes leave (or have left) the thymus resulting in lymphocytosis in the peripheral blood. The diminished number of differentiated cells in the organ causes an intense proliferation of thymic blast cells, because of the decreased levels of inhibitory substances.
inhibitory agents in the thymus. In various leukaemic diseases there is some proof that changed levels of humoral regulators of cell proliferation exist (Metcalf, Moore & Warner, 1969; Chan & Metcalf, 1973), but so far the role of tissue specific regulators in neoplastic tissues is still speculative (Bullough, 1969; Iversen, 1970). The present model-in spite of its limitations-can perhaps form the basis for studies on the production of lymphocytes under various physiological conditions including neoplasia in experimental animals and man. REFERENCES BICHEL, P.(1971)Autoregulation of ascites tumour growth by inhibition of the G-1 and G-2 phase. Europ. J. Cancer, 7,349.
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