rat ai~ ~ e ~ m t e lu c
Blur
Blut 39, 191-199 (1979)
9 Springer-Verlag 1979
Effect of T-Lymphocytes on Normal Haemopoiesis: Studies in Congenitally Athymic Nude Mice G. Harris 1 and S.N. Wickramasinghe 2 1 Division of Experimental Pathology, The Mathilda and Terence Kennedy Institute of Rheumatology, Bute Gardens, London, England Department of Haematology and MRC Experimental Haematology Unit, St. Mary's Hospital Medical School, University of London, London W2 1PG, England
Einflu6 yon T-Lymphozyten auf die normale H~irnopoese: Untersuchungen bei der kongenital athymischen Maus Zusalnmenfassung. Keimfreie nackte (nu/nu) M/iuse sind aufgrund des Fehlens eines funktionsffihigen Thymus kongenital ohne T-Lymphozyten. Sie sind deswegen zur Untersuchung der Interaktion zwischen T-Zellen und anderen biologischen Prozessen besonders geeignet. Vergleichende Untersuchungen zwischen nicht T-defizienten CBA-Miiusen zeigen, dal3 das Fehlen der T-Zellen zu einer St6rung der Hfimopoese in 2-6 Monate alten Tieren ffihrt. Diese zeigt sich in einer m~il3igen Makrozytose und einem stark reduzierten H 3TdR-Markierungsindex der Leukozyten nach Markierung in vivo. Trotz dieser St6rung k6nnen nu/nu-Miiuse so viele hiimatopoetische Zellen produzieren, dai3 normale Blutzellzahlen aufrechterhalten werden, allerdings nur beim Fehlen zusiitzlicher Belastung. Die Anfimie konventionell aufgezogener nu/nu-M~iuse ist wahrscheinlich dutch bakterielle Infektionen bedingt. Schliisselw/irter: T-Zelldefekt - Hfimopoese - Athymische nackte M[iuse Summary. The germ-free nude mouse represents a most useful animal for investigating the effects of a congenital deficiency of T-lymphocytes on various physiological processes, including haemopoiesis. Nude mice (nu/nu) of the CBA strain, nonmutant inbred CBA mice, and inbred C3H mice were reared in germ-free isolators and used to compare (1) the haematological parameters of nu/nu mice and CBA mice at different ages and (2) the labelling pattern of circulating leucocytes at various times after a single intraperitoneal injection of ~H-thymidine into 3-month-old nu/nu and C3H mice. The data suggest that the deficiency of T-lymphocytes in nu/nu mice may lead to a disturbance of haemopoiesis in 2 to 6-month-old animals which is characterised by a mild macrocytosis and a very marked reduction in the proportion of labelled leucocytes which can be seen in the blood after an injection of 3H-thymidine. Offprint requests to: Prof. S.N. Wickramasinghe, M.B., B.S., Ph.D., M.R.C. Path. (address see
above) 0006-5242/79/0039/0191/$ 1.80
192
G. Harris and S.N. Wickramasinghe Despite these perturbations, unstressed nu/nu mice were able to maintain adequate numbers of blood cells (other than lymphocytes) in their circulation. Nine-month-old nu/nu mice did not show a macrocytosis and the disappearance of the macrocytosis at this age was associated with an increase both in the blood lymphocyte count and in the mass of lymphoid tissue in the spleen and mesenteric lymph nodes. Key words: T-cell deficiency - Haemopoiesis - Nude mice
Several studies have indicated that thymic cells can influence the behaviour of spleen colony-forming cells (CFC-S)under certain experimental conditions. For example, Goodman and Grubbs [5] have demonstrated that the number of spleen colonies formed in irradiated F 1 hybrid mice following the injection of parental bone marrow cells is increased if parental thymocytes are given at the same time. Subsequent investigations have shown that this increase, which results from a more efficient seeding of parental bone marrow cells in the spleen, is associated with a shortening of the lag phase of the CFC-S growth curve, an unaltered slope of the exponential part of the CFC-S growth curve and the formation of larger spleen colonies [17]. In another study, Lord and Schofield [12] found that some CFC-S present in irradiated suspensions of marrow and spleen cells require the cooperation of live thymus cells before they could form spleen colonies when injected into irradiated mice. In yet another study, Hrsak [8] thymectomised mice of four different strains at the age of 2 months and found that 2-3 months later, such animals had (1) normal numbers of nucleated marrow cells in their femora and (2) increased numbers of bone marrow CFC-S when tested in lethally irradiated, non-thymectomised animals. However, bone marrow CFC-S from normal donors produced subnormal numbers of spleen colonies when injected into lethally irradiated thymectomised recipients suggesting that the T-lymphocyte-poor splenic microenvironment may be suboptimal for supporting the proliferation of CFC-S. All the above-mentioned animal studies reveal a "stimulatory" effect of Tlymphocytes on the CFC-S in experimental conditions which are unfavourable to the CFC-S or which require their maximal proliferation. By contrast, recent studies have revealed that the lymphoid cells of the blood of some patients with aplastic anaemia have a suppressive influence on the in vitro growth of erythroid colonies from normal human bone marrow cells [7] and that the lymphoid cells of the bone marrow of some patients with aplastic anaemia have a suppressive effect on the proliferation and differentiation of the patients own granulocytopoietic cells as well as a suppressive effect on the in vitro growth of granulocytopoietic colonies from normal human bone marrow [l, 10]. Although these in vitro results are of considerable interest, it is not yet certain whether lymphocyte-mediated suppressor effects operate in vivo either as a primary or secondary event in the pathogenesis of aplastic anaemia or in the regulation of normal haemopoiesis. However, the possibility that this sort of suppressive effect may operate in vivo at least in pathogical situations is suggested by the observation that some patients with aplastic anaemia who reject their bone marrow allografts subsequently
Effect of T-Lymphocytes on Normal Haemopoiesis
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recover from the aplasia by regenerating their own marrow, p r e s u m a b l y in response to the p r e - t r a n s p l a n t a t i o n therapy with i m m u n o s u p p r e s s i v e agents such as cyclophosphamide, total b o d y irradiation or horse a n t i - h u m a n thymocyte globulin [9, 18]. The investigations reported in this paper were u n d e r t a k e n to determine whether the presence of the t h y m u s was i m p o r t a n t for the p r o d u c t i o n of erythrocytes, granulocytes, a n d platelets u n d e r n o r m a l steady-state conditions. F o r this purpose mice reared in a germ-free isolator were used a n d a c o m p a r i s o n was m a d e (1) between the haematological parameters of the m o u s e m u t a n t n u d e ( n u / n u ) a n d C B A mice a n d (2) between the p a t t e r n of labelling of circulating leucocytes at various times after a single intraperitoneal injection of 3H-thymidine (3H-TdR) into n u d e mice a n d C 3 H mice. Wortis et al. [20] have shown that a p r i m a r y a b n o r m a l i t y in the n u d e m o u s e is a lack or severe developmental a b n o r m a l i t y of the epithelial element of the t h y m u s a n d the c o n s e q u e n t failure of b o n e marrow-derived precursor cells to develop into T-lymphocytes in the thymic m i c r o e n v i r o n m e n t ; the site n o r m a l l y occupied by the thymus contains very small structures whose histology bears no resemblance to that of n o r m a l thymus.
Material and Methods Animals
Inbred CBA mice and homozygous nude mice (nu/nu) were used for the determination of haematological parameters at various ages. Three-month-old inbred C3H mice and nu/nu mice were used for labelling with 3H-TdR in vivo. All these animals were reared in germ-free isolators and fed on sterile water and ordinary mouse pellets, sterilised by X-radiation. Although all the nu/nu mice studied were of the CBA strain, throughout this paper the phrase "CBA mice" is used to refer to the non-nude animals of this strain. Haematological Parameters
Groups of 3 CBA and 3 nu/nu mice were randomly selected at each of the ages of 1, 3, 6, and 9 months. Blood samples were withdrawn from ether-anaesthetised animals by cardiac puncture, collected into EDTA bottles, and used for haematological studies. The animals were then killed for various investigations including the determination of the total body weight, the weights of the spleen, thymus (in the CBA mice), and the mesenteric lymph nodes and the cellularity of the marrow in both femora. Haemoglobin levels were measured by the cyanmethaemoglobin method using a Coulter haemoglobinometer. The MCV and the standard deviation of the logarithms of the red cell volumes in each sample were determined using a Coulter Counter, Model F~ and Channelyzer as described by England and Down [3,4]. Total white cell counts were determined using a Coulter Counter, Model FN, and platelet counts were determined after diluting the blood in formolcitrate, using a Neubauer counting chamber. Differential leucocyte counts were performed on 200 consecutive cells in narrow longitudinal strips on May-Grtinwald-Giemsa-stained blood smears. Statistical Methods
The values for the reticulocyte counts, total white cell counts, neutrophil counts, lymphocyte counts, and platelet counts were subjected to a logarithmic transformation prior to any statistical analysis. The statistical significance of differences in the haematological parameters of nu/nu mice (at all ages) and CBA mice (at all ages) was determined by an analysis of variance. The occurrence of age-dependent differences in these parameters was also investigated separately in nu/nu and CBA mice by an analysis of variance.
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Labelling with ~H-TdR Each germ-free mouse (C3H or nu/nu) was given an intraperitoneal injection of 3 #Ci of (Me-3H) thymidine (specificactivity 20 Ci/mMol) per g body weight. Animals were killed in groups of 3 at various times (2 h-24 d) after the injection. Autoradiographs of methanol-fixed peripheral blood smears were prepared using AR10 stripping film (Kodak Ltd.) and exposed for 6 months at --20~ before developing and staining with the Giemsa stain, aH-TdR-labelling indices of lymphocytes and granuloeytes in the blood were determined from an examination of at least 100 cells of each cell type in each animal. Only cells with 4 or more grains over them were considered to be labelled: the grain count over an equal area of adjacent cell-free background was always less than 2, and usually 0.
Results Table 1 summarizes some of the features of the animals used for the c o m p a r i s o n between the haematological values of CBA a n d n u / n u mice at various ages. It is evident that the total n u m b e r s of nucleated m a r r o w cells in the two femora are similar in the CBA a n d n u / n u mice between the ages of 1 a n d 6 m o n t h s b u t are higher in the n u / n u mice at 9 months. I n addition, the n u / n u mice show a n abn o r m a l degree of increase in the mass of l y m p h o i d tissue with ageing, when compared with the C B A mice. The haematological data in the CBA a n d n u / n u mice are compared in Table 2. The m a i n features of the data are (1) no significant differences in the h a e m o g l o b i n levels (p = 0.74), reticulocyte counts (p = 0.10), neutrophil counts (p = 0.57), and platelet counts (p = 0.14) in the two types of mice, (2) higher MCVs in the 1-, 2- a n d 6 - m o n t h - o l d n u / n u mice, (3) higher total white cell counts in the CBA mice (p = 0.002), a n d (4) a l y m p h o p e n i a in the 1-, 2-, a n d 6-month-old n u / n u mice. A n analysis of variance using the data in all the age groups showed (1) highly
Table 1. Some characteristics~ of the germ-free animals used for the determination of haematological parameters Weight MLN b (mg)
Cellularity of marrow in both femora ( • 106)
Age (months)
Strain
Body (g)
Spleen (mg)
Thymus (mg)
1
CBA nu/nu
15.9 i 2.1 11.0 • 0.6
37.3 • 4.6 33.0 _+ 8.5
36.3 • 16.9 18.6 • 0.4 19.6 • 5.3 0 14.6 + 2.1 18.1 _+ 1.8
2
CBA nu/nu
27.1 • 0.3 17.0 i 0.9
46.0 i 1.7 51.6 • 6.5
36.3 +_ 5.5 39.0 i 13.0 23.7 +_ 2.6 0 24.6 i 8.7 23.7 • 1.2
6
CBA nu/nu
22.8 • 0.9 22.6 + 1.0
50.3 _+ 2.1 73.3 + 4.7
28.6 • 0
4.9 24.6 i 43.0 •
9
CBA nu/nu
26.2 i 0.8 24.1 • 0.5
52.3 • 1.5 78.6 • 7.0
20.6 • 0
0.6 26.0 • 11.0 19.4 • 0.4 46.6 _+ 8.1 29.5 _+ 5.2
Results are given as means • S.E. of means in groups of 3 animals b Mesenteric lymph nodes
9.5 28.3 _+ 4.0 8.6 25.9 • 3.0
Hb a (g/dl)
13.9 (12.9-14.9)
14.6 (14.3-14.9)
15.4 (15.1-15.6)
15.1 (13.9-15.8)
15.3 (13.1-16.2)
15.5 (15.3-15.9)
14.2 (12.0-15.5)
13.6 (12.5-15.2)
Strain
CBA
nu/nu
CBA
nu/nu
CBA
nu/nu
CBA
nu/nu
49.6 (47.9-51.3)
52.8 (48.2-55.5)
53.8 (51.3-55.8)
48.4 (45.4-51.2)
51.9 (50.3-54.1)
47.2 (46.6-47.6)
56.1 (51.0-59.9)
50.2 (49.9-50.5)
MCV a (fl)
0.166
0.166
0.160
0.165
0.157
0.149
0.164
0.158
(7 b
Mean of
5.94 (4.89-7.75)
4.15 (2.82-5.47)
4.52 (4.30-4.89)
6.02 (3.98-7.95)
3.34 (2.79-4.53)
6.99 (6.17-7.46)
3.32 (2.95-4.17)
5.74 (4.98-6.62)
WBC e ( • 109/1)
9~ Mean of results in groups of 3 animals. Observed ranges are given within brackets b cr = standard deviation of the natural logarithms of the red cell volumes ~ Median of results in groups of 3 animals. Observed ranges are given within brackets
Age (months)
Table 2. Haematological parameters of germ-free CBA and germ-free nu/nu mice
3.09 (1.56-5.04)
2.01 (1.00-3.04)
2.75 (2.63-2.84)
1.81 (1.53-2.28)
1.80 (1.03-2.92)
3.54 (2.73-4.51)
1.93 (1.32-2.38)
2.38 (1.94-2.64)
Neutrophils e ( • 10~/1)
2.66 (1.96-3.55)
2.01 (1.62-2.79)
1.75 (1.46-2.08)
3.94 (2.45-5.67)
1.38 (0.85-1.94)
3.39 (2.95-3.84)
1.48 (1.09-1.79)
3.47 (3.04-3.98)
(• lO~/1)
338 (264-433)
413 (182-756)
619 (482-741)
444 (224 650)
916 (510-1328)
731 (558-1106)
1145 (776-1770)
452 (396-516)
Lymphocytes Platelets e & monocytes c ( • 109/I)
2.0 (1.8-2.0)
2.2 (1.3-3.8)
4.1 (2.6-6.4)
1.6 (0.7-2.1)
3.6 (2.5-4.6)
-
5.8 (3.2-8.8)
3.9 (2.9-4.8)
(Vo)
Reticulocytes ~
9 ~3
~Z
Z
O
77" O
196
G. Harris and S.N. Wickramasinghe
Table 3. Labelling indices in peripheral blood white cells of 3-month-old germ-free C3H mice at various times after a single intraperitoneal injection of 3H-TdR
Timea
2h 1d
2d 3d 4d 9d 11 d 16 d 24 d
aH-TdR labelling indexb (~) lymphocytes
granulocytes
0 2.5 3.7 6.1 7.1 19.1 15.3 20.1 14.0
0 0.6 13.4 18.5 85.1 43.9 23.0 0 0
• • • • • • • •
0.5 2.0 0.9 3.1 1.6 9.6 6.7 9.8
• 1.4 + 4.7 • 14.8 • 7.1 • 19.9
Animals were killed in groups of 3 at each of the times shown b Results are given as means +_ S.E. of means Table 4. Labelling indices in peripheral blood white cells of 3-month-old germ-free nu/nu mice at
various times after a single intraperitoneal injection of aH-TdR Time
3H-TdR labelling index~ (~) lymphocytes
granulocytes
2h 6h
0 0
0 0
1d
O, O, 3.2
0
1.5, 1.4, 9.0 1.0, O, 1.7 0 0 0
8.0, O, 8.5 6.1, O, 15.3 0 0 0
2d 4d 7d 8d 14 d
a The data for each of the 3 animals studied at each time are given separately except when they were identical significant differences in the MCVs (p = 0.009) as well as in the lymphocyte plus m o n o c y t e counts (p = 0.001) of n u / n u a n d CBA mice a n d (2) statistically significant age-dependent differences in the MCVs (p = 0.043) a n d the total white cell counts (p = 0.035) in the n u / n u mice. Tables 3 a n d 4 show the labelling indices of the circulating leucocytes in the a n i m a l s which were given a single intraperitoneal injection of aH-TdR. It is evident that the pattern of labelling in the n u / n u mice (Table 4) is completely different from that in the C3H mice (Table 3). The very low labelling indices e n c o u n t e r e d in the blood leucocytes of the n u / n u mice were associated with abn o r m a l l y low grain counts over several of the labelled cells. These findings in the n u / n u mice appeared n o t to be the result of some technical fault as m a r r o w smears from the same n u / n u mice which were processed for a u t o r a d i o g r a p h y at the same time a n d in the same way as the blood smears showed several heavily labelled precursor cells b u t few or n o labelled granulocytes at all times after the injection of ZH-TdR.
Effect of T-Lymphocytes on Normal Haemopoiesis
197
Discussion
Rygaard and Povlsen [16] have demonstrated that 6 to 8-week-old germ-free nude mice show a leucopenia, a normal granulocyte count, and a lymphopenia. The lymphopenia is presumably due to a lack of T-lymphocytes as several studies have revealed that the lymph nodes and spleens of nude mice are virtually devoid of T-lymphocytes [11, 14, 15]. Our study has shown that the lymphopenia persists until the age of 6 months but is not evident at 9 months. The present investigation has revealed that 1 to 6-month-old nude mice have significantly higher MCVs when compared with CBA mice of the same age. It has also shown that the pattern of labelling of circulating lymphocytes and granulocytes at various times after a single intraperitoneal injection of 3H-TdR in germfree nude mice is strikingly different from that in germ-free C3H mice; the time taken for the first labelled cells to appear in the circulation was longer, the maximum labelling index achieved in the circulation and the average grain count over the labelled cells was much lower and the time taken for labelled cells to disappear completely from the circulation was much shorter in the nude mice (see Tables 3 and 4). It seems reasonable to postulate that both the mild macrocytosis and the peculiar pattern of labelling with aH-TdR referred to above are in some way related to the deficiency of T-lymphocytes, particularly in view of our findings that there was no macrocytosis in 9-month-old nude mice and that this disappearance of the macrocytosis was associated with an increase in the blood lymphocyte count as well as in the mass of lymphoid tissue in the spleen and mesenteric nodes. However, this hypothesis can only be proven by demonstrating that the transplantation of a normal thymus into neonatal nude mice causes the disapperance of the macrocytosis and the abnormal ~H-TdR labelling pattern. In any case, the mild macrocytosis must reflect a perturbation in the kinetics of erythropoiesis in the bone marrow of the nude mouse. In this connection it is of interest that in the human, macrocytosis is a feature of the blood picture of some patients suffering from a variety of congenital and acquired disorders associated with dyserythropoiesis, including acute leukaemia, aplastic anaemia and preleukaemic and pre-aplastic states. It is unlikely that the macrocytosis shown by the nu/nu mice was caused by vitamin B12 or folic acid deficiency as the deoxyuridinesuppressed values given by the bone marrow cells of these animals fell within the range of values given by the marrow cells of germ-free CBA mice, when determined by a modification of the method of Wickramasinghe and Longland [19] in which autologous serum was replaced by Hanks' solution. We have also electrophoresed lysates of saline-washed red cells from 2 to 9-month-old nu/nu and CBA mice on cellulose acetate paper (pH 8.6) for 0.5 h and have found that the macrocytosis encountered in the nu/nu mice is not associated with any abnormality in the types of haemoglobin synthesised: two major bands and three minor bands were visible in both nu/nu and CBA mice. It is noteworthy that despite the virtually complete deficiency of T-lymphocytes in nude mice, and the evidence referred to in the previous paragraph indicating the occurrence of some disturbances in haemopoiesis, such animals are capable of maintaining adequate numbers of circulating red cells, granulocytes and platelets for a period of at least 9 months when reared in a germ-free environment. We
198
G. Harris and S.N. Wickramasinghe
found no significant abnormality in the haemoglobin concentrations of 1 to 9month-old germ-free nude mice when compared with germ-free CBA mice of the same age. Our results confirm and extend an earlier report that 6 to 8-week-old nude mice have normal haemoglobin levels [16] and contrast with previous observations that an anaemia develops in neonatally thymectomised mice which were not reared in germ-free conditions [13]: the anaemia which develops in the latter situation is almost certainly caused by secondary infection. Although the bone marrow of the nude mouse can maintain a more or less normal rate of output of blood cells in the absence of T-lymphocytes, it may be unable to increase its output adequately in response to stresses such as haemorrhage or haemolysis. This possibility remains to be investigated. One explanation for the abnormal 3H-TdR-labelling data in nude mice may be that the T-lymphocytes, which are deficient in such mice, normally serve as a continuing source of radioactive molecules for incorporation into the D N A of the CFC-S and their differentiating progeny long after the end of the 10-40 min post-injection availability time [2] of 3H-TdR in the circulation. The radioactive material which is supplied by T-lymphocytes to haemopoietic cells may include macromolecular DNA, as recent studies of the transfer of radioactivity from aH-TdR-labelled mouse lymphocytes to V79 fibroblasts in vitro have shown that the transferred material consists initially of macromolecular D N A and subsequently of degradation pruducts of D N A [6]. If macromolecular D N A is transported from lymphocytes into haemopoietic cells in vivo, it is possible that other macromolecules, such as RNA and protein, are also transported. The functional consequences of the transfer of D N A from lymphocytes into other cells are still not known, but this type of transfer could be responsible for the stimulatory effect of thymic cells on the growth of spleen colonies from CFC-S [5, 12, 17]. Alternatively, the stimulatory effect of thymic ceils could be caused by other types of short-range cell to cell interactions in the marrow or spleen or by long-range stimulatory humoral substances elaborated by T-lymphocytes. Although the stimulatory effect of T-lymphocytes on the proliferation and differentiation of CFC-S could be based at least partly on a trephocytic function of lymphoid cells as discussed above, the suppressive effect of lymphoid cells from some patients with aplastic anaemia on the growth of haemopoietic cells in vitro [1,7, 10] cannot be explained on the basis of an inhibition of this trephocytic function as the data in the nude mice indicate that the lack of T-lymphocytes does not lead to the development of aplastic anaemia. It is possible that the suppressive effects, if relevant in vivo, result from (1) specific immunopathological mechanisms directed against bone marrow cells or (2) the excessive production of a hitherto unidentified physiological inhibiter of haemopoiesis by a subpopulation of lymphoid cells. Acknowledgement. We are grateful to Mrs. M. Chetty for determining the MCVs and to Dr. J.M. England for help with the mathematical analyses.
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References 1. Ascensao, J., Pahwa, R., Kagan, W., Hansen, J., Moore, M., Good, R. : Aplastic anaemia: evidence for an immunological mechanism. Lancet I, 669-671 (1976) 2. Cleaver, J.E. : Thymidine Metabolism and Cell Kinetics. P. 58. Amsterdam: North-Holland Publishing Company 1967 3. England, J.M., Down, M.C. : Red-cell-volume distribution curves and the measurement of anisocytosis. Lancet I, 701-703 (1974) 4. England, J.M., Down, M.C. : Measurement of the mean cell volume using electronic particle counters. Br. J. Haematol. 32, 403M09 (1976) 5. Goodman, J.W., Grubbs, C. G. : The relationship of the thymus to erythropoiesis. In: Hemopoietic cellular proliferation. Stohlman, F., Jr. (ed.), pp. 26-35. New York: Grune & Stratton 1970 6. Harris, G., Olsen, I., Furner, P. : Transport of D N A from lymphocytes to fibroblasts in culture. Differentiation 12, 1-13 (1978) 7. Hoffman, R., Zanjani, E.D., Lutton, J.D., Zalusky, R., Wasserman, L.R.: Suppression of erythroid-colony formation by lymphocytes from patients with aplastic anemia. N. Engl. J. Med. 296, 10-13 (1977) 8. Hrsak, I. : Influence of thymus on haemopoiesis in mice. Biomedicine 18, 213-219 (1973) 9. Jeannet, M., Speck, B., Rubenstein, A., Pelet, B., Wyss, M., Kummer, H. : Autologous marrow reconstitutions in severe aplastic anaemia after ALG pretreatment and HLA semiincompatible bone marrow cell transfusion. Acta Haematol. 55, 129-139 (1976) 10. Kagan, W.A., Ascensao, J.A., Pahwa, R.N., Hansen, J.A., Goldstein, G., Valera, E.B., Incefy, G.S., Moore, M.A.S., Good, R.A. : Aplastic anemia: presence in human bone marrow of cells that suppress myelopoiesis. Proc. Natl. Acid. Sci. USA 73, 2890-2894 (1976) 11. KSlsch, E., Davies, A. J.S., Leuchars, E. : The immune response to phage fd in normal and thymus-deprived animals of a low responding inbred strain and in genetically thymusless mice. Eur. J. Immunol. 2, 541-545 (1972) 12. Lord, B.I., Schofield, R. : The influence of thymus cells in hemopoiesis: stimulation of hemopoietic stem cells in a syngeneic, in vivo, situation. Blood 42, 395-404 (1973) 13. Metcalf, D. : The thymus, its role in immune responses, leukaemia, development and carcinogenesis. Recent results in cancer research, Vol. 5. Berlin, Heidelberg, New York: Springer 1966 14. Raft, M.C., Wortis, H. H. : Thymus dependence of 0-bearing cells in the peripheral lymphoid tissues of mice. Immunology 18, 931-942 (1970) 15. Raft, M.C. : Surface antigenic markers for distinguishing T and B lymphocytes in mice. Transplant. Rev. 6, 52-80 (1971) 16. Rygaard, J., Povlsen, C.O.: Effects of homozygosity of the nude (nu) gene in three inbred strains of mice. A detailed study of mice of three genetic backgrounds (BALB-c, C3H, C57BL-6) with congenital absence of the thymus (nude mice) at a stage in the gene transfer. Acta Pathol. Microbiol. Scand. [A] 82, 48-70 (1974) 17. Shinpock, S.G., Goodman, J.W. : Ability of thymic lymphocytes to alter C F U kinetics in radiation chimeras. Cell Tissue Kinet. 11, 111-117 (1978) 18. Thomas, E.D., Storb, E.R., Gilbert, B. : Recovery from aplastic anemia following attempted marrow transplantation. Exp. Hematol. 4, 97-102 (1976) 19. Wickramasinghe, S.N., Longland, J.E.: Assessment of deoxyuridine suppression test in diagnosis of vitamin BI~ or folate deficiency. Br. Med. J. 3, 148-150 (1974) 20. Wortis, H.H., Nehlsen, S., Owen, J.J.: Abnormal development of the thymus in "nude" mice. J. Exp. Med. 134, 681-692 (1971)
Received April 11, 1979/Accepted June 20, 1979
Blur
Blut 39, 191-199 (1979)
9 Springer-Verlag 1979
Effect of T-Lymphocytes on Normal Haemopoiesis: Studies in Congenitally Athymic Nude Mice G. Harris 1 and S.N. Wickramasinghe 2 1 Division of Experimental Pathology, The Mathilda and Terence Kennedy Institute of Rheumatology, Bute Gardens, London, England Department of Haematology and MRC Experimental Haematology Unit, St. Mary's Hospital Medical School, University of London, London W2 1PG, England
Einflu6 yon T-Lymphozyten auf die normale H~irnopoese: Untersuchungen bei der kongenital athymischen Maus Zusalnmenfassung. Keimfreie nackte (nu/nu) M/iuse sind aufgrund des Fehlens eines funktionsffihigen Thymus kongenital ohne T-Lymphozyten. Sie sind deswegen zur Untersuchung der Interaktion zwischen T-Zellen und anderen biologischen Prozessen besonders geeignet. Vergleichende Untersuchungen zwischen nicht T-defizienten CBA-Miiusen zeigen, dal3 das Fehlen der T-Zellen zu einer St6rung der Hfimopoese in 2-6 Monate alten Tieren ffihrt. Diese zeigt sich in einer m~il3igen Makrozytose und einem stark reduzierten H 3TdR-Markierungsindex der Leukozyten nach Markierung in vivo. Trotz dieser St6rung k6nnen nu/nu-Miiuse so viele hiimatopoetische Zellen produzieren, dai3 normale Blutzellzahlen aufrechterhalten werden, allerdings nur beim Fehlen zusiitzlicher Belastung. Die Anfimie konventionell aufgezogener nu/nu-M~iuse ist wahrscheinlich dutch bakterielle Infektionen bedingt. Schliisselw/irter: T-Zelldefekt - Hfimopoese - Athymische nackte M[iuse Summary. The germ-free nude mouse represents a most useful animal for investigating the effects of a congenital deficiency of T-lymphocytes on various physiological processes, including haemopoiesis. Nude mice (nu/nu) of the CBA strain, nonmutant inbred CBA mice, and inbred C3H mice were reared in germ-free isolators and used to compare (1) the haematological parameters of nu/nu mice and CBA mice at different ages and (2) the labelling pattern of circulating leucocytes at various times after a single intraperitoneal injection of ~H-thymidine into 3-month-old nu/nu and C3H mice. The data suggest that the deficiency of T-lymphocytes in nu/nu mice may lead to a disturbance of haemopoiesis in 2 to 6-month-old animals which is characterised by a mild macrocytosis and a very marked reduction in the proportion of labelled leucocytes which can be seen in the blood after an injection of 3H-thymidine. Offprint requests to: Prof. S.N. Wickramasinghe, M.B., B.S., Ph.D., M.R.C. Path. (address see
above) 0006-5242/79/0039/0191/$ 1.80
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G. Harris and S.N. Wickramasinghe Despite these perturbations, unstressed nu/nu mice were able to maintain adequate numbers of blood cells (other than lymphocytes) in their circulation. Nine-month-old nu/nu mice did not show a macrocytosis and the disappearance of the macrocytosis at this age was associated with an increase both in the blood lymphocyte count and in the mass of lymphoid tissue in the spleen and mesenteric lymph nodes. Key words: T-cell deficiency - Haemopoiesis - Nude mice
Several studies have indicated that thymic cells can influence the behaviour of spleen colony-forming cells (CFC-S)under certain experimental conditions. For example, Goodman and Grubbs [5] have demonstrated that the number of spleen colonies formed in irradiated F 1 hybrid mice following the injection of parental bone marrow cells is increased if parental thymocytes are given at the same time. Subsequent investigations have shown that this increase, which results from a more efficient seeding of parental bone marrow cells in the spleen, is associated with a shortening of the lag phase of the CFC-S growth curve, an unaltered slope of the exponential part of the CFC-S growth curve and the formation of larger spleen colonies [17]. In another study, Lord and Schofield [12] found that some CFC-S present in irradiated suspensions of marrow and spleen cells require the cooperation of live thymus cells before they could form spleen colonies when injected into irradiated mice. In yet another study, Hrsak [8] thymectomised mice of four different strains at the age of 2 months and found that 2-3 months later, such animals had (1) normal numbers of nucleated marrow cells in their femora and (2) increased numbers of bone marrow CFC-S when tested in lethally irradiated, non-thymectomised animals. However, bone marrow CFC-S from normal donors produced subnormal numbers of spleen colonies when injected into lethally irradiated thymectomised recipients suggesting that the T-lymphocyte-poor splenic microenvironment may be suboptimal for supporting the proliferation of CFC-S. All the above-mentioned animal studies reveal a "stimulatory" effect of Tlymphocytes on the CFC-S in experimental conditions which are unfavourable to the CFC-S or which require their maximal proliferation. By contrast, recent studies have revealed that the lymphoid cells of the blood of some patients with aplastic anaemia have a suppressive influence on the in vitro growth of erythroid colonies from normal human bone marrow cells [7] and that the lymphoid cells of the bone marrow of some patients with aplastic anaemia have a suppressive effect on the proliferation and differentiation of the patients own granulocytopoietic cells as well as a suppressive effect on the in vitro growth of granulocytopoietic colonies from normal human bone marrow [l, 10]. Although these in vitro results are of considerable interest, it is not yet certain whether lymphocyte-mediated suppressor effects operate in vivo either as a primary or secondary event in the pathogenesis of aplastic anaemia or in the regulation of normal haemopoiesis. However, the possibility that this sort of suppressive effect may operate in vivo at least in pathogical situations is suggested by the observation that some patients with aplastic anaemia who reject their bone marrow allografts subsequently
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recover from the aplasia by regenerating their own marrow, p r e s u m a b l y in response to the p r e - t r a n s p l a n t a t i o n therapy with i m m u n o s u p p r e s s i v e agents such as cyclophosphamide, total b o d y irradiation or horse a n t i - h u m a n thymocyte globulin [9, 18]. The investigations reported in this paper were u n d e r t a k e n to determine whether the presence of the t h y m u s was i m p o r t a n t for the p r o d u c t i o n of erythrocytes, granulocytes, a n d platelets u n d e r n o r m a l steady-state conditions. F o r this purpose mice reared in a germ-free isolator were used a n d a c o m p a r i s o n was m a d e (1) between the haematological parameters of the m o u s e m u t a n t n u d e ( n u / n u ) a n d C B A mice a n d (2) between the p a t t e r n of labelling of circulating leucocytes at various times after a single intraperitoneal injection of 3H-thymidine (3H-TdR) into n u d e mice a n d C 3 H mice. Wortis et al. [20] have shown that a p r i m a r y a b n o r m a l i t y in the n u d e m o u s e is a lack or severe developmental a b n o r m a l i t y of the epithelial element of the t h y m u s a n d the c o n s e q u e n t failure of b o n e marrow-derived precursor cells to develop into T-lymphocytes in the thymic m i c r o e n v i r o n m e n t ; the site n o r m a l l y occupied by the thymus contains very small structures whose histology bears no resemblance to that of n o r m a l thymus.
Material and Methods Animals
Inbred CBA mice and homozygous nude mice (nu/nu) were used for the determination of haematological parameters at various ages. Three-month-old inbred C3H mice and nu/nu mice were used for labelling with 3H-TdR in vivo. All these animals were reared in germ-free isolators and fed on sterile water and ordinary mouse pellets, sterilised by X-radiation. Although all the nu/nu mice studied were of the CBA strain, throughout this paper the phrase "CBA mice" is used to refer to the non-nude animals of this strain. Haematological Parameters
Groups of 3 CBA and 3 nu/nu mice were randomly selected at each of the ages of 1, 3, 6, and 9 months. Blood samples were withdrawn from ether-anaesthetised animals by cardiac puncture, collected into EDTA bottles, and used for haematological studies. The animals were then killed for various investigations including the determination of the total body weight, the weights of the spleen, thymus (in the CBA mice), and the mesenteric lymph nodes and the cellularity of the marrow in both femora. Haemoglobin levels were measured by the cyanmethaemoglobin method using a Coulter haemoglobinometer. The MCV and the standard deviation of the logarithms of the red cell volumes in each sample were determined using a Coulter Counter, Model F~ and Channelyzer as described by England and Down [3,4]. Total white cell counts were determined using a Coulter Counter, Model FN, and platelet counts were determined after diluting the blood in formolcitrate, using a Neubauer counting chamber. Differential leucocyte counts were performed on 200 consecutive cells in narrow longitudinal strips on May-Grtinwald-Giemsa-stained blood smears. Statistical Methods
The values for the reticulocyte counts, total white cell counts, neutrophil counts, lymphocyte counts, and platelet counts were subjected to a logarithmic transformation prior to any statistical analysis. The statistical significance of differences in the haematological parameters of nu/nu mice (at all ages) and CBA mice (at all ages) was determined by an analysis of variance. The occurrence of age-dependent differences in these parameters was also investigated separately in nu/nu and CBA mice by an analysis of variance.
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Labelling with ~H-TdR Each germ-free mouse (C3H or nu/nu) was given an intraperitoneal injection of 3 #Ci of (Me-3H) thymidine (specificactivity 20 Ci/mMol) per g body weight. Animals were killed in groups of 3 at various times (2 h-24 d) after the injection. Autoradiographs of methanol-fixed peripheral blood smears were prepared using AR10 stripping film (Kodak Ltd.) and exposed for 6 months at --20~ before developing and staining with the Giemsa stain, aH-TdR-labelling indices of lymphocytes and granuloeytes in the blood were determined from an examination of at least 100 cells of each cell type in each animal. Only cells with 4 or more grains over them were considered to be labelled: the grain count over an equal area of adjacent cell-free background was always less than 2, and usually 0.
Results Table 1 summarizes some of the features of the animals used for the c o m p a r i s o n between the haematological values of CBA a n d n u / n u mice at various ages. It is evident that the total n u m b e r s of nucleated m a r r o w cells in the two femora are similar in the CBA a n d n u / n u mice between the ages of 1 a n d 6 m o n t h s b u t are higher in the n u / n u mice at 9 months. I n addition, the n u / n u mice show a n abn o r m a l degree of increase in the mass of l y m p h o i d tissue with ageing, when compared with the C B A mice. The haematological data in the CBA a n d n u / n u mice are compared in Table 2. The m a i n features of the data are (1) no significant differences in the h a e m o g l o b i n levels (p = 0.74), reticulocyte counts (p = 0.10), neutrophil counts (p = 0.57), and platelet counts (p = 0.14) in the two types of mice, (2) higher MCVs in the 1-, 2- a n d 6 - m o n t h - o l d n u / n u mice, (3) higher total white cell counts in the CBA mice (p = 0.002), a n d (4) a l y m p h o p e n i a in the 1-, 2-, a n d 6-month-old n u / n u mice. A n analysis of variance using the data in all the age groups showed (1) highly
Table 1. Some characteristics~ of the germ-free animals used for the determination of haematological parameters Weight MLN b (mg)
Cellularity of marrow in both femora ( • 106)
Age (months)
Strain
Body (g)
Spleen (mg)
Thymus (mg)
1
CBA nu/nu
15.9 i 2.1 11.0 • 0.6
37.3 • 4.6 33.0 _+ 8.5
36.3 • 16.9 18.6 • 0.4 19.6 • 5.3 0 14.6 + 2.1 18.1 _+ 1.8
2
CBA nu/nu
27.1 • 0.3 17.0 i 0.9
46.0 i 1.7 51.6 • 6.5
36.3 +_ 5.5 39.0 i 13.0 23.7 +_ 2.6 0 24.6 i 8.7 23.7 • 1.2
6
CBA nu/nu
22.8 • 0.9 22.6 + 1.0
50.3 _+ 2.1 73.3 + 4.7
28.6 • 0
4.9 24.6 i 43.0 •
9
CBA nu/nu
26.2 i 0.8 24.1 • 0.5
52.3 • 1.5 78.6 • 7.0
20.6 • 0
0.6 26.0 • 11.0 19.4 • 0.4 46.6 _+ 8.1 29.5 _+ 5.2
Results are given as means • S.E. of means in groups of 3 animals b Mesenteric lymph nodes
9.5 28.3 _+ 4.0 8.6 25.9 • 3.0
Hb a (g/dl)
13.9 (12.9-14.9)
14.6 (14.3-14.9)
15.4 (15.1-15.6)
15.1 (13.9-15.8)
15.3 (13.1-16.2)
15.5 (15.3-15.9)
14.2 (12.0-15.5)
13.6 (12.5-15.2)
Strain
CBA
nu/nu
CBA
nu/nu
CBA
nu/nu
CBA
nu/nu
49.6 (47.9-51.3)
52.8 (48.2-55.5)
53.8 (51.3-55.8)
48.4 (45.4-51.2)
51.9 (50.3-54.1)
47.2 (46.6-47.6)
56.1 (51.0-59.9)
50.2 (49.9-50.5)
MCV a (fl)
0.166
0.166
0.160
0.165
0.157
0.149
0.164
0.158
(7 b
Mean of
5.94 (4.89-7.75)
4.15 (2.82-5.47)
4.52 (4.30-4.89)
6.02 (3.98-7.95)
3.34 (2.79-4.53)
6.99 (6.17-7.46)
3.32 (2.95-4.17)
5.74 (4.98-6.62)
WBC e ( • 109/1)
9~ Mean of results in groups of 3 animals. Observed ranges are given within brackets b cr = standard deviation of the natural logarithms of the red cell volumes ~ Median of results in groups of 3 animals. Observed ranges are given within brackets
Age (months)
Table 2. Haematological parameters of germ-free CBA and germ-free nu/nu mice
3.09 (1.56-5.04)
2.01 (1.00-3.04)
2.75 (2.63-2.84)
1.81 (1.53-2.28)
1.80 (1.03-2.92)
3.54 (2.73-4.51)
1.93 (1.32-2.38)
2.38 (1.94-2.64)
Neutrophils e ( • 10~/1)
2.66 (1.96-3.55)
2.01 (1.62-2.79)
1.75 (1.46-2.08)
3.94 (2.45-5.67)
1.38 (0.85-1.94)
3.39 (2.95-3.84)
1.48 (1.09-1.79)
3.47 (3.04-3.98)
(• lO~/1)
338 (264-433)
413 (182-756)
619 (482-741)
444 (224 650)
916 (510-1328)
731 (558-1106)
1145 (776-1770)
452 (396-516)
Lymphocytes Platelets e & monocytes c ( • 109/I)
2.0 (1.8-2.0)
2.2 (1.3-3.8)
4.1 (2.6-6.4)
1.6 (0.7-2.1)
3.6 (2.5-4.6)
-
5.8 (3.2-8.8)
3.9 (2.9-4.8)
(Vo)
Reticulocytes ~
9 ~3
~Z
Z
O
77" O
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G. Harris and S.N. Wickramasinghe
Table 3. Labelling indices in peripheral blood white cells of 3-month-old germ-free C3H mice at various times after a single intraperitoneal injection of 3H-TdR
Timea
2h 1d
2d 3d 4d 9d 11 d 16 d 24 d
aH-TdR labelling indexb (~) lymphocytes
granulocytes
0 2.5 3.7 6.1 7.1 19.1 15.3 20.1 14.0
0 0.6 13.4 18.5 85.1 43.9 23.0 0 0
• • • • • • • •
0.5 2.0 0.9 3.1 1.6 9.6 6.7 9.8
• 1.4 + 4.7 • 14.8 • 7.1 • 19.9
Animals were killed in groups of 3 at each of the times shown b Results are given as means +_ S.E. of means Table 4. Labelling indices in peripheral blood white cells of 3-month-old germ-free nu/nu mice at
various times after a single intraperitoneal injection of aH-TdR Time
3H-TdR labelling index~ (~) lymphocytes
granulocytes
2h 6h
0 0
0 0
1d
O, O, 3.2
0
1.5, 1.4, 9.0 1.0, O, 1.7 0 0 0
8.0, O, 8.5 6.1, O, 15.3 0 0 0
2d 4d 7d 8d 14 d
a The data for each of the 3 animals studied at each time are given separately except when they were identical significant differences in the MCVs (p = 0.009) as well as in the lymphocyte plus m o n o c y t e counts (p = 0.001) of n u / n u a n d CBA mice a n d (2) statistically significant age-dependent differences in the MCVs (p = 0.043) a n d the total white cell counts (p = 0.035) in the n u / n u mice. Tables 3 a n d 4 show the labelling indices of the circulating leucocytes in the a n i m a l s which were given a single intraperitoneal injection of aH-TdR. It is evident that the pattern of labelling in the n u / n u mice (Table 4) is completely different from that in the C3H mice (Table 3). The very low labelling indices e n c o u n t e r e d in the blood leucocytes of the n u / n u mice were associated with abn o r m a l l y low grain counts over several of the labelled cells. These findings in the n u / n u mice appeared n o t to be the result of some technical fault as m a r r o w smears from the same n u / n u mice which were processed for a u t o r a d i o g r a p h y at the same time a n d in the same way as the blood smears showed several heavily labelled precursor cells b u t few or n o labelled granulocytes at all times after the injection of ZH-TdR.
Effect of T-Lymphocytes on Normal Haemopoiesis
197
Discussion
Rygaard and Povlsen [16] have demonstrated that 6 to 8-week-old germ-free nude mice show a leucopenia, a normal granulocyte count, and a lymphopenia. The lymphopenia is presumably due to a lack of T-lymphocytes as several studies have revealed that the lymph nodes and spleens of nude mice are virtually devoid of T-lymphocytes [11, 14, 15]. Our study has shown that the lymphopenia persists until the age of 6 months but is not evident at 9 months. The present investigation has revealed that 1 to 6-month-old nude mice have significantly higher MCVs when compared with CBA mice of the same age. It has also shown that the pattern of labelling of circulating lymphocytes and granulocytes at various times after a single intraperitoneal injection of 3H-TdR in germfree nude mice is strikingly different from that in germ-free C3H mice; the time taken for the first labelled cells to appear in the circulation was longer, the maximum labelling index achieved in the circulation and the average grain count over the labelled cells was much lower and the time taken for labelled cells to disappear completely from the circulation was much shorter in the nude mice (see Tables 3 and 4). It seems reasonable to postulate that both the mild macrocytosis and the peculiar pattern of labelling with aH-TdR referred to above are in some way related to the deficiency of T-lymphocytes, particularly in view of our findings that there was no macrocytosis in 9-month-old nude mice and that this disappearance of the macrocytosis was associated with an increase in the blood lymphocyte count as well as in the mass of lymphoid tissue in the spleen and mesenteric nodes. However, this hypothesis can only be proven by demonstrating that the transplantation of a normal thymus into neonatal nude mice causes the disapperance of the macrocytosis and the abnormal ~H-TdR labelling pattern. In any case, the mild macrocytosis must reflect a perturbation in the kinetics of erythropoiesis in the bone marrow of the nude mouse. In this connection it is of interest that in the human, macrocytosis is a feature of the blood picture of some patients suffering from a variety of congenital and acquired disorders associated with dyserythropoiesis, including acute leukaemia, aplastic anaemia and preleukaemic and pre-aplastic states. It is unlikely that the macrocytosis shown by the nu/nu mice was caused by vitamin B12 or folic acid deficiency as the deoxyuridinesuppressed values given by the bone marrow cells of these animals fell within the range of values given by the marrow cells of germ-free CBA mice, when determined by a modification of the method of Wickramasinghe and Longland [19] in which autologous serum was replaced by Hanks' solution. We have also electrophoresed lysates of saline-washed red cells from 2 to 9-month-old nu/nu and CBA mice on cellulose acetate paper (pH 8.6) for 0.5 h and have found that the macrocytosis encountered in the nu/nu mice is not associated with any abnormality in the types of haemoglobin synthesised: two major bands and three minor bands were visible in both nu/nu and CBA mice. It is noteworthy that despite the virtually complete deficiency of T-lymphocytes in nude mice, and the evidence referred to in the previous paragraph indicating the occurrence of some disturbances in haemopoiesis, such animals are capable of maintaining adequate numbers of circulating red cells, granulocytes and platelets for a period of at least 9 months when reared in a germ-free environment. We
198
G. Harris and S.N. Wickramasinghe
found no significant abnormality in the haemoglobin concentrations of 1 to 9month-old germ-free nude mice when compared with germ-free CBA mice of the same age. Our results confirm and extend an earlier report that 6 to 8-week-old nude mice have normal haemoglobin levels [16] and contrast with previous observations that an anaemia develops in neonatally thymectomised mice which were not reared in germ-free conditions [13]: the anaemia which develops in the latter situation is almost certainly caused by secondary infection. Although the bone marrow of the nude mouse can maintain a more or less normal rate of output of blood cells in the absence of T-lymphocytes, it may be unable to increase its output adequately in response to stresses such as haemorrhage or haemolysis. This possibility remains to be investigated. One explanation for the abnormal 3H-TdR-labelling data in nude mice may be that the T-lymphocytes, which are deficient in such mice, normally serve as a continuing source of radioactive molecules for incorporation into the D N A of the CFC-S and their differentiating progeny long after the end of the 10-40 min post-injection availability time [2] of 3H-TdR in the circulation. The radioactive material which is supplied by T-lymphocytes to haemopoietic cells may include macromolecular DNA, as recent studies of the transfer of radioactivity from aH-TdR-labelled mouse lymphocytes to V79 fibroblasts in vitro have shown that the transferred material consists initially of macromolecular D N A and subsequently of degradation pruducts of D N A [6]. If macromolecular D N A is transported from lymphocytes into haemopoietic cells in vivo, it is possible that other macromolecules, such as RNA and protein, are also transported. The functional consequences of the transfer of D N A from lymphocytes into other cells are still not known, but this type of transfer could be responsible for the stimulatory effect of thymic cells on the growth of spleen colonies from CFC-S [5, 12, 17]. Alternatively, the stimulatory effect of thymic ceils could be caused by other types of short-range cell to cell interactions in the marrow or spleen or by long-range stimulatory humoral substances elaborated by T-lymphocytes. Although the stimulatory effect of T-lymphocytes on the proliferation and differentiation of CFC-S could be based at least partly on a trephocytic function of lymphoid cells as discussed above, the suppressive effect of lymphoid cells from some patients with aplastic anaemia on the growth of haemopoietic cells in vitro [1,7, 10] cannot be explained on the basis of an inhibition of this trephocytic function as the data in the nude mice indicate that the lack of T-lymphocytes does not lead to the development of aplastic anaemia. It is possible that the suppressive effects, if relevant in vivo, result from (1) specific immunopathological mechanisms directed against bone marrow cells or (2) the excessive production of a hitherto unidentified physiological inhibiter of haemopoiesis by a subpopulation of lymphoid cells. Acknowledgement. We are grateful to Mrs. M. Chetty for determining the MCVs and to Dr. J.M. England for help with the mathematical analyses.
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References 1. Ascensao, J., Pahwa, R., Kagan, W., Hansen, J., Moore, M., Good, R. : Aplastic anaemia: evidence for an immunological mechanism. Lancet I, 669-671 (1976) 2. Cleaver, J.E. : Thymidine Metabolism and Cell Kinetics. P. 58. Amsterdam: North-Holland Publishing Company 1967 3. England, J.M., Down, M.C. : Red-cell-volume distribution curves and the measurement of anisocytosis. Lancet I, 701-703 (1974) 4. England, J.M., Down, M.C. : Measurement of the mean cell volume using electronic particle counters. Br. J. Haematol. 32, 403M09 (1976) 5. Goodman, J.W., Grubbs, C. G. : The relationship of the thymus to erythropoiesis. In: Hemopoietic cellular proliferation. Stohlman, F., Jr. (ed.), pp. 26-35. New York: Grune & Stratton 1970 6. Harris, G., Olsen, I., Furner, P. : Transport of D N A from lymphocytes to fibroblasts in culture. Differentiation 12, 1-13 (1978) 7. Hoffman, R., Zanjani, E.D., Lutton, J.D., Zalusky, R., Wasserman, L.R.: Suppression of erythroid-colony formation by lymphocytes from patients with aplastic anemia. N. Engl. J. Med. 296, 10-13 (1977) 8. Hrsak, I. : Influence of thymus on haemopoiesis in mice. Biomedicine 18, 213-219 (1973) 9. Jeannet, M., Speck, B., Rubenstein, A., Pelet, B., Wyss, M., Kummer, H. : Autologous marrow reconstitutions in severe aplastic anaemia after ALG pretreatment and HLA semiincompatible bone marrow cell transfusion. Acta Haematol. 55, 129-139 (1976) 10. Kagan, W.A., Ascensao, J.A., Pahwa, R.N., Hansen, J.A., Goldstein, G., Valera, E.B., Incefy, G.S., Moore, M.A.S., Good, R.A. : Aplastic anemia: presence in human bone marrow of cells that suppress myelopoiesis. Proc. Natl. Acid. Sci. USA 73, 2890-2894 (1976) 11. KSlsch, E., Davies, A. J.S., Leuchars, E. : The immune response to phage fd in normal and thymus-deprived animals of a low responding inbred strain and in genetically thymusless mice. Eur. J. Immunol. 2, 541-545 (1972) 12. Lord, B.I., Schofield, R. : The influence of thymus cells in hemopoiesis: stimulation of hemopoietic stem cells in a syngeneic, in vivo, situation. Blood 42, 395-404 (1973) 13. Metcalf, D. : The thymus, its role in immune responses, leukaemia, development and carcinogenesis. Recent results in cancer research, Vol. 5. Berlin, Heidelberg, New York: Springer 1966 14. Raft, M.C., Wortis, H. H. : Thymus dependence of 0-bearing cells in the peripheral lymphoid tissues of mice. Immunology 18, 931-942 (1970) 15. Raft, M.C. : Surface antigenic markers for distinguishing T and B lymphocytes in mice. Transplant. Rev. 6, 52-80 (1971) 16. Rygaard, J., Povlsen, C.O.: Effects of homozygosity of the nude (nu) gene in three inbred strains of mice. A detailed study of mice of three genetic backgrounds (BALB-c, C3H, C57BL-6) with congenital absence of the thymus (nude mice) at a stage in the gene transfer. Acta Pathol. Microbiol. Scand. [A] 82, 48-70 (1974) 17. Shinpock, S.G., Goodman, J.W. : Ability of thymic lymphocytes to alter C F U kinetics in radiation chimeras. Cell Tissue Kinet. 11, 111-117 (1978) 18. Thomas, E.D., Storb, E.R., Gilbert, B. : Recovery from aplastic anemia following attempted marrow transplantation. Exp. Hematol. 4, 97-102 (1976) 19. Wickramasinghe, S.N., Longland, J.E.: Assessment of deoxyuridine suppression test in diagnosis of vitamin BI~ or folate deficiency. Br. Med. J. 3, 148-150 (1974) 20. Wortis, H.H., Nehlsen, S., Owen, J.J.: Abnormal development of the thymus in "nude" mice. J. Exp. Med. 134, 681-692 (1971)
Received April 11, 1979/Accepted June 20, 1979
