Quantitative studies of lymphocytes and other cell populations in the bone marrow of neonatally thymectomized C3H mice.код для вставкиСкачать
Quantitative Studies of Lymphocytes and Other Cell Populations in the Bone Marrow of NeonatallyThymectomized C3H Mice s. C . MILLER A N D D. G . OSMOND Department o f Anatomy, McGill University, P . 0. Box 6070,Station A, Montreal, Quebec, Canada H3C 3G1 ABSTRACT The effects of neonatal thymectomy on the development of the lymphoid, erythroid and granulocytic cell populations in mouse bone marrow have been assessed by quantitative techniques. The numbers per unit volume of bone marrow of 17 cell types were determined in neonatally thymectomized and sham thymectomized C3H mice a t two, four and eight weeks of age, and compared with those of normal C3H mice. After neonatal thymectomy the numbers of small lymphocytes, large and medium-sized lymphoid cells, and erythroid cells reached normal levels at two weeks but fell progressively to 1 8 % , 22% and 42% of normal, respectively, by eight weeks. In sham thymectomized mice these cell populations did not differ significantly from normal. Immature and mature granulocytes were elevated in numbers two weeks after either neonatal thymectomy or sham thymectomy, suggesting a transient non-specific stimulation of granulocytopoiesis. During continuous infusion of 3H-thymidine for ten days in neonatally thymectomized mice aged four weeks and eight weeks many bone marrow small lymphocytes remained unlabeled. The results demonstrate that early postnatal development of bone marrow lymphoid and erythroid cells proceeds normally in the absence of the thymus, in accord with the concept of the bone marrow as a primary site of lymphocyte production and differentiation. In addition, some slowly-renewing small lymphocytes in bone marrow appear to be thymus-independent cells. Large numbers of small lymphocytes are produced continuously in mammalian bone marrow (Osmond and Everett, '64; Miller and Osmond, '73; Osmond, '75) from which they migrate to peripheral lymphoid tissues, notably the spleen and lymph nodes (Brahim and Osmond, '70). Soon after their production, many bone marrow small lymphocytes show immunoglobulin molecules on their surface (Osmond and Nossal, '74), suggesting that this process represents a primary genesis and dissemination of B lymphocytes, i.e. cells with readily detectable surface immunoglobulin mediating humoral immune responses. Functional assays of antibody-forming cell precursors in bone marrow are consistent with this view (Stocker et al., '74). Of importance to such a concept of the bone marrow as a primary lymphoid organ is the question whether the development of intense lymphocytopoiesis is an intrinsic ANAT. REC., 184: 325-334. property of the marrow or is secondary to extramyeloid influences. The possibility that thymic factors may influence cell production in other lymphoid tissues has been suggested by the activity of thymic extracts in vitro (Stutman et al., '70) and by the effects of either thymic grafts or diffusion chamber implants on neonatally thymectomized animals in vivo (Miller and Osoba, '67). However, these studies have been concerned only with lymphocytes in the lymph nodes, spleen and blood. While the effects of neonatal thymectomy on peripheral lymphoid populations are well known (Miller and Osoba, '67), there appear to have been no quantitative studies of the effects on marrow lymphocytes. A relationship between the thymus and the production of erythrocytes in the marrow has been suggested by the observations that neonatal thymectomy is Received May 30, '75. Accepted Aug. 21, '75. 325 326 S. C. MILLER AND D. G . OSMOND (Coulter Electronics, Model B). The marrow was centrifuged, smeared in serum, and stained with MacNeal's tetrachrome. The total cellularity and absolute numbers of various marrow cell types per mm3 were determined by the method of Wller and Osmond ('74). Approximately 2,500 nucleated cells were examined along the middle third of each smear and classified as detaiIed elsewhere (Miller and Osmond, '74). RH-thymidine infusion and radioautography. Two further groups of ten neonatally thymectomized mice were infused subcutaneously at four weeks and eight weeks with 3H-thymidine (spec. act.: 6.7 Ci,/mM; New England Nuclear Corp., Boston, Mass.) at a rate of 2 clCi in 0.04 ml sterile water/gm body weight/day as described elsewhere (Miller and Osmond, '75). Animals were killed at 12 hours and at daily intervals from one to ten days MATERIALS AND METHODS after beginning isotope infusion. Smears Animals. Male C3H/HeJ mice (Jack- of femoral marrow were fixed in methanol son Laboratories, Bar Harbor, Me. ) were and prepared for radioautography by coating with Kodak NTB, liquid emulsion, exused. Operative procedures. Thymectomy and posing for 21 days, processing and staining sham thymectomy were performed under with MacNeal's tetrachrome. From each ether anesthesia within 16 hours of birth. animal, 2,000 nucleated bone marrow cells Through a midline incision the cervical were counted through the middle third of strap muscles were displaced laterally, the the smears and the proportion of small sternum was separated from the fmt two lymphocytes (< 8.0 p nuclear diameter), ribs and pulled forward. The thymus was identified as described elsewhere (Osmond ablated by gentle suction. The skin was and Everett, '64; Osmond, '67; Osmond sutured and sealed with aerosol plastic et al., '73; Miller and Osmond, '73), was dressing. Sham thymectomized mice were determined, From 1,000 small lymphocytes subjected to the same anesthesia and oper- in each animal the percentage of labeled ative exposure. All thymectomized animals cells was noted. In each experiment three used in the experiments showed an ab- or more radioautographic grains indicated sence of thymic tissue in the mediastinum positive labeling of small lymphocytes by when examined under a stereomicroscope comparison with counts of background grains over 500 erythrocytes. (X 18). Bone marrow sampling. Groups of ten RESULTS thymectomized and ten sham thymectomized mice were sampled at two, four and All neonatally thymectomized C3H mice eight weeks of age. A further group of showed a reduced growth rate from two ten normal, aged mice were sampled at weeks and the appearance of a progressive 78 weeks. To avoid diurnal variation in runting syndrome from three weeks oncell populations all animals were sampled ward. These features did not appear after at the same time of day (3 PM). Bone mar- neonatal sham thymectomy. row was gently flushed from femoral shaft Total cellularity o f the bone marrow. segments with fresh rat serum plus 3.8% In both neonatally thymectomized and sodium citrate (3: 1) and suspended by sham thymectomized mice the total numaspiration. Nucleated cells were enumer- ber of nucleated cells per unit volume of ated with an electronic particle counter femoral marrow was higher than normal followed by an apparent erythroid maturation arrest in opossums (Miller et al., '65) and anemia in mice (Metcalf, '66), and that thymus suspensions exert a beneficial effect on erythropoiesis when injected with bone marrow cells into lethally irradiated allogeneic mice (Goodman and Grubbs, '70; Lord and Schofield, '73). The present studies aimed to determine the effects of thymic ablation on the postnatal development of lymphoid, erythroid and myeloid (granulocytic) cell populations in mouse bone marrow. Quantitative techniques, developed previously to study murine bone marrow during normal growth and development (Miller and Osmond, '74), were used to enumerate cells at each morphologically distinguishable stage of differentiation at early intervals after either neonatal thymectomy or neonatal sham thymectomy. BONE MARROW OF NEONATALLY THYMECTOMIZED C3H MICE at two weeks of age ( p < 0.001 j but fell to normal by four weeks (fig. 1). Subsequently, in thymectomized mice the marrow cellularity continued to fall while sham thymectomized mice maintained normal values (fig, 1 ) . Bone m a r r w cell populations. Neonatal thymectomy resulted in selective changes in the major groups of nucleated cells in the bone marrow, as shown in figures 2 and 3. The increased marrow cellularity at two weeks was attributable entirely to significantly increased nunibers ( p < 0.001) of myeloid cells in both the neonatally thymectomized and sham thymectomized mice (fig. 2 ) . The myeloid cells fell to normal values by four to eight weeks in sham thymectomized mice. These values also fell in neonatally thymectomized mice at two to four weeks, but they failed to return completely to normal. The numbers of nucleated erythroid cells per unit volume of bone marrow were unaffected by neonatal thymectomy, except that they did not show the usual pubertal rise at eight weeks (fig. 2). The numbers of small lym- phocytes in the bone marrow of neonatally thymectomized mice attained normal high levels at two weeks (fig. 3 j . Thereafter, the sham thymectomized mice continued to follow normal levels but the values in neonatally thymectomized mice fell progressively to 18% of those in the sham operated mice by eight weeks. The large and medium-sized lymphoid cells fell simultaneously to 30% of sham operated values by eight weeks. Subpopulations of bone marrow cells are detailed in tables 1 and 2, as reported previously for normal C3H mice (Miller and Osmond, ’74). Fluctuations in the lymphoid, erythroid and myeloid cell lines affected cells at each stage of differentiation in a proportionate manner. In particular, the ratios of late, non-dividing forms in each cell line (small lymphocytes; late erythroblast s ; met amyelocytes plus band cells and polymorphonuclear leucocytes ) to the earlier, proliferative stages remained closely comparable in neonatally thymectomized, sham thymectomized and normal mice at all ages studied. Small numbers of plasma cells were 3.0 r, E E \ 2.5 “0 x -B 2 . 0 el 3 e 1.5 m 0 n v g 1.0 - m 0 z 0 0 I 2 3 4 5 6 7 8 Age (weeks) Fig. 1 Total numbers of nucleated cells per unit volume of femoral marrow in normal neonatally thymectomized (- -) and sham thymetomized ( - - - - - ) C3H mice of various ages. (-), 327 328 S . C. MILLER AND D. G . OSMOND 1600 I400 11. - MYELOID CELLS 1200 - - nE 1000 - E \ n -o aoo- X v) 600 - 0" 400 - 200 - 01 0 1 I 2 3 4 5 Age (weeks) 6 7 8 Total numbers of myeloid and erythroid cells per unit volume of femoral marrow (-), neonatally thymectomized (- -), and sham thymectomized ( - - - - - ) C3H mice of various ages. Fig. 2 in normal aoo SMALL LYMPHOCYTES T 700 600 E E 500 \ " -- 0 400 \ x v) 2 300 200 \ \ \ \\ \\ MEDIUM AND LARGE LYMPHOID CELLS 100 C I 2 3 4 5 A g e (weeks) 6 7 8 Total numbers of lymphoid cells per unit volume of femoral marrow in normal neonatally thymectomized (- -) and sham thymectomized (- - - --) C3H mice of various ages. Fig. 3 (-), BONE MARROW OF NEONATALLY THYMECTOMIZED C3H MICE 329 TABLE 1 Absolute numbers of cells in femoral marrow of neonatally thymectomized C 3 H mice of various ages ( x 103/mm3) Cell type Small lymphocyte Medium-sized lymphoid cell Large lymphoid cell Total lymphoid Proerythroblast Early erythroblast Intermediate erythroblast Late erythroblast Total erythroid Myeloblast Promyelocyte Myelocyte Metamyelocyte Band Polymorphonuclear Total myeloid Unclassified blast Unclassified mitotic figures Other Damaged forms 1 2wkl 4wkl 8wkl 656.62 36.4 90.6 2 7.2 17.0-C 0.9 764.2 2 38.7 4 . 0 i 0.9 15.5-C 4.1 5 7 . 9 i 15.2 244.7542.0 322.1 i60.7 24.3rl: 2.2 112.7rl: 3.9 823.8-C 53.9 298.6-C 18.2 224.62 19.8 11.92 0.8 1495.9 % 76.6 7.2% 1.2 28.92 4.3 9 . 6 i 0.7 234.7 2 30.9 452.9 rl: 45.2 64.8% 7.1 1 8 . 9 2 3.3 536.6251.3 4 . 0 2 1.0 10.12 1.5 50.0% 8.0 146.52 19.7 210.7226.4 23.0% 2.6 84.62 7.1 564.0 i26.8 224.4 2 17.3 181.3 1: 17.0 21.8-e 2.8 1099.0 i52.7 13.2-e 2.5 13.6f 1.9 11.22 1.5 174.62 17.6 87.2 2 17.0 18.42 5.1 8.1-C 2.3 113.8223.7 6.0C 1.06 18.12 3.2 38.2-C 5.4 94.9 2 12.8 157.2221.7 19.3C 1.8 94.42 8.2 627.2 2 36.4 208.72 17.2 181.22 15.6 3 4 . 7 r 3.4 1165.5265.5 2 . 4 2 0.4 13.72 1.1 4 . 9 2 0.9 132.2k 13.9 Mean 4 standard error; ten mice. TABLE 2 Absolute numbers of cells in femoral marrow of neonatally sham thymectomized C3H mice of various ages ( x 103/mm3) Cell type Small lymphocyte Medium-sized lymphoid cell Large lymphoid cell Total lymphoid Proerythroblast Early erythroblast Intermediate erythroblast Late erythroblast Total erythroid Myeloblast Promyelocyte Myelocyte Metainyelocyte Band Polymorphonuclear Total myeloid Unclassified blast Unclassified mitotic figures Other Damaged forms 1 Mean +- 2wkl 699.52 53.1 119.4" 11.8 29.6& 5.0 848.5 2 63.9 6 . 8 2 1.4 20.82 2.4 87.72 14.3 277.1 rl: 19.6 392.4 2 35.6 28.3C 3.6 102.42 9.4 876.1 C 87.3 266.5 C 26.5 263.8 i32.6 30.12 7.4 1567.1t 136.3 12.6C 2.9 26.0 2 3.3 11.12 1.1 225.4 C 19.3 4wkl 8wkl 661.7C34.8 101.12 9.6 20.22 2.5 783.0 C 40.2 4 . 6 2 1.0 14.3% 2.6 38.02 7.5 144.0 2 28.5 200.92 38.5 12.7C 0.9 51.5C 4.8 516.7 i37.2 193.5 2 19.5 136.12 19.3 18.52 2.8 929.1 & 56.1 6 . 6 2 0.9 15.7k 1.5 10.2, 1.3 177.0 C 19.6 471.1 227.2 72.4) 5.0 17.22 1.3 560.7 2 30.2 6 . 7 2 1.5 18.42 2.1 59.62 7.6 226.8 2 31.9 311.4538.2 1 8 . 0 i 1.4 59.6) 5.4 527.1 i26.4 226.22 10.8 207.9 2 18.6 31.62 3.1 1070.4 2 43.8 4 . 7 2 0.9 13.72 1.6 8 . 6 2 1.5 170.92 19.7 standard error; ten mice. found in normal bone marrow, increasing in incidence with age (table 3). In neonatally thymectomized mice the bone marrow plasma cells were greatly reduced in numbers but still appeared to increase with age (table 3). Labeling of bone marrow small Eymphocytes by "-thymidine. During continuous infusion of "-thymidine into neo- natally thymectomized mice many bone marrow small lymphocytes remained unlabeled, in contrast to the high labeling indices reached in normal animals (Miller and Osmond, '75). From four to ten days after beginning infusion the labeling of bone marrow small lymphocytes fluctuated from 8.0% to 32.0% in 4-week mice and from 24.0% to 46.0% in 8-week mice. 330 S. C. MILLER AND D. G. OSMOND TABLE 3 Incidence of plasma cells in femoral marrow of normal and neonatalLy thymectonzized C 3 H mice o f various ages ( p e r 1,000 nucleated cells) Normal mice Neonat ally thymectoniized mice 4wk 8wk 16wk 0.1 0.6 4.2 0.006 0.02 3 Approximately 5,000 nucleated cclls counted at each age (1,000 cells i n each of 5 mice). 2 Approximately 15,000 nucleated cells counted at each age (3,000 cells i n each of 5 mice). 3 No neonatally thymectomized mice survived to 16 weeks. 1 Bone marrow cell populations in old mice. Because the changes in bone marrow cell populations throughout the first eight weeks in neonatally thymectomized mice appeared to resemble normal age changes proceeding at an accelerated pace, a further group of aged (78-week old) normal C3H mice was examined. The number of nucleated cells in the femoral shaft (13.6 -t. 0 . 7 x 106,'femur) resembled closely the value in normal young adults (13.5 -C 0.7 X 1Oe/femur at 16 weeks. The relative incidence of the main marrow cell groups in aged mice were: small lymphocytes, 12.0 1 . 3 % ; erythroid cells, 7.0 e 1.0%; myeloid cells, 68.0 -r- 1 . 9 % , generally similar to the cell distribution in the 8-week neonatally thymectomized mice : small lymphocytes, 5.3 -t. 0.9%; erythroid cells, 10.4 5 1.7% and myeloid cells, 73.2 2 1.8%. DISCUSSION In normal C3H mice the bone marrow small lymphocytes reach maximal numbers at two to four weeks of age and are predominately rapidly renewed (Miller and Osmond, '74, '75). With increasing age the numbers and renewal rate of these cells decline (Miller and Osmond, '75). Recent radioautographic studies in mice confirm that, as in other rodents, the rapidly renewing bone marrow small lymphocytes are produced locally within the bone marrow by the proliferation of large and medium-sized lymphoid cells (M. Z. Kaiserman, S. C. Miller and D. G . Osmond, unpublished). The present quantitative studies in neonatally thymectomized C3H mice indicate that the bone marrow lymphocyte population can undergo a normal early postnatal development even in the absence of the thymus, previously noted only in percentage terms (Kalpaktsoglou et al., '69). Furthermore, bone marrow small lymphocyte renewal has been shown radioautographically to occur at a normal rapid rate in young congenitally athymic (nu/nu) mice (Osmond and Miller, '75; Ropke et al., '75). These results are in keeping with the concept that the production and maturation of lymphocytes in the bone marrow is thymus-independent. The cause of the delayed decline in marrow lymphocyte numbers after neonatal thymectomy is not clear. Since the large and medium-sized lymphoid cells fall in numbers as well as the small lymphocytes, and the labeling indices of the latter during 3H-thymidine infusion are lower than usual, a secondary reduction in marrow lymphocyte production seems to be the case. This might be due to a deficiency of a thymic lymphocytopoietic factor. If so, the post thymectomy delay in the effect wouId indicate either that such a thymic factor remains active for a prolonged period after release from the thymus or that it acts neonatally at a very primitive stage of marrow lymphocytopoiesis. Alternatively, the secondary decline in marrow lymphocyte numbers may well be an indirect effect of neonatal thymectomy secondary to the endocrine and other abnormalities of the runting syndrome. Neonatally thymectomized or congenitally athymic mice show adrenal cortical enlargement, reversible by implantation of a normal thymus (Pierpaoli and Sorkin, '72). Elevated circulatory levels of corticosteroids and hypersecretion in response to stress or infection could be responsible for a secondary decline in the cortisone-sensitive marrow lymphocytes (Fruhman and Gordon, '55; Harris, '61; Morrison and Toepfer, '67; Esteban, '68; Bennett, '70). Other contributory factors may be reduced endocrine secretion by the anterior pituitary, thyroid gland and gonads (Law et al., '64; Bianchi et al., '71; Fabris et al., '71a,b; Sakakura and Nishizuka, '72). Regardless of its cause, the decline in numbers of marrow lymphocytes implies that neonatally thymectomized mice suffer indirectly from a reduction in production of virgin B lymphocytes as well as an ab- BONE MARROW OF NEONATALLY THYMECTOMIZED C3H MICE sence of thymus-derived ( T ) lymphocytes. Small lymphocytes in normal mouse bone marrow develop readily detectable surf ace immunoglobulin as they mature (Osmond and Nossal, '74) and migrate to peripheral lymphoid tissues (Brahim and Osmond, '73) where they appear to form progenitors of antibody producing cells in the primary immune responses (Stocker et al., '74; Osmond, '75). The immunological defects of neonatally thymectomized mice during the runting syndrome may thus be due to deficiencies in both T and B lymphocytes. The reduction in numbers of bone marrow plasma cells noted in the present study may be due to both the lack of virgin antibody producing cell precursors themselves as well as of the T helper cells necessary for the activation of such precursor cells by many antigens. Bone marrow of normal mice contains a small population of long-lived small lymphocytes (Ropke and Everett, '73; Miller and Osmond, '75) which increases in numbers with age. Many are recirculating cells entering the marrow from the blood stream (Ropke and Everett, '74). While some of these cells normally are recirculating thymus-derived (T) lymphocytes (Howard and Scott, '72) the substantial proportions of small lymphocytes which remained unlabeled during 3H-thymidine infusion in the bone marrow of neonatalIy thymectomized mice in the present study suggests that some long-lived small lymphocytes in bone marrow are independent of the thymus in origin. They may be either indigenous, cy tokinetic ally resting marrow cells, as described in the rat (Haas et al., '71), or recirculating marrow-derived (B) cells. The maintenance of normal bone marrow erythroid cell populations for four weeks after neonatal thymectomy argues strongly against a direct dependence on thymic erythropoietic factors. The secondary disturbance in erythropoiesis seen in pubertal animals after neonatal thymectomy may be attributed to a generalized endocrine deficiency. Several hormones including growth hormone (Peschle et al., '72), thyroid hormone (Shalet et al., '66; Peschle et al., '71b), testosterone (Gurney and Fried, '65), cortisol and ACTH (Peschle et al., '71a), stimulate erythropoiesis in 331 normal and endocrine-deficient animals, while a marked atrophy of all the major endocrine glands occurs during severe runting in athymic animals (Law et al., '64; Bianchi et al., '71; Fabris et al., '71a,b; Sakakura and Nishizuka, '72). Granulocytic cells in the bone marrow are increased in numbers at two weeks after either neonatal thymectomy or sham thymectomy, in each case maintaining a normal ratio of proliferating to non-dividing forms. This is therefore a non-specific stimulation of granulocytopoiesis, probably occurring in response to tissue damage at operation. The slight elevation in numbers of bone marrow granulocytic cells persisting at four and eight weeks after neonatal thymectomy accords with previous observations in the blood and histological sections of bone marrow (Parrott, '62; Law et al., '64), and is probably secondary to infection and abnormal levels of hormones such as corticosteroids (Morrison and Toepfer, '67). The sequence of cellular changes in the bone marrow after neonatal thymectomy resembles normal ageing, greatly accelerated. By eight weeks after neonatal thymectomy the bone marrow resembles that of 78-week old normal mice. These features complement hematologic (Good et al., '62), immunologic (Metcalf et al., '67) and endocrine (Pierpaoli and Sorkin, '72) similarities between runting athymic and aged animals. ACKNOWLEDGMENTS The authors thank Mrs. E. D. Watson for technical assistance. This work was supported by the Medical Research CounciI of Canada. LITERATURE CITED Bennett, M. 1970 Hemopoietic environment necessary for an early stage in differentiation of antibody forming cells: effect of cortisol. J. Cell Physiol., 76: 197-205. Bianchi, E., W. Pierpaoli and E. Sorkin 1971 Cytological changes in the mouse anterior pituitary after neonatal thymectomy: a light and electron microscope sudy. J. Endoc., 51: 1-6. Brahim, F.,and D. G. Osmond 1970 Migration of bone marrow lymphocytes demonstrated by selective bone marrow labelling with thymidine-H3. Anat. Rec., 168: 139-160. 1873 The migration of lymphocytes from bone marrow to popliteal lymph nodes demonstrated by selective bone marrow label- 332 S. C. 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