197150a0Nature1974863196301121501520028-0836196310.1038/197150a0ukNatureNatureNATUREnatureNature is a weekly international journal publishing the finest peer-reviewed research in all fields of science and technology on the basis of its originality, importance, interdisciplinary interest, timeliness, accessibility, elegance and surprising conclusions. Nature also provides rapid, authoritative, insightful and arresting news and interpretation of topical and coming trends affecting science, scientists and the wider public./nature/journal/v197/n4863issueJournal homeArchiveCurrent issueAdvance online publicationPrivacy policySubscribeNature Publishing GroupCurrent issue197150a0Myelocyte-Metamyelocyte Transition in the Bone Marrow of the Dog
AU  - MALONEY, MARY A.
AU  - WEBER, CHARLOTTE L.
AU  - PATT, HARVEY M.Division of Biological and Medical Research, Argonne National Laboratory, Argonne, IllinoisTHE granulocytes constitute a steady-state renewal system, cells being born in the bone marrow and lost in the periphery. Although the general chronology of granulocyte development is known12, the dimension of time alone reveals only a part of the over-all picture. It is necessary to determine the flow of cells to understand the population relationships. This article is concerned with the flow of cells from the proliferating to the non-proliferating compartments in the bone marrow of the dog as revealed by autoradiographic analysis after labelling with tritiated thymidine. There are several transitions during granulocytopoiesis, that between myelocyte and metamyelocyte being particularly important, since the former is the last of the dividing granulocytes and the latter the first of the non-dividing granulocytes in the developmental sequence.
Two eight-month-old beagles were injected intravenously with tritiated thymidine (0-3 me./kg body-weight; specific activity 360 me./m.mole). The experimental procedures of bone marrow sampling and autoradiography have been described elsewhere3. It is generally agreed that tritiated thymidine is incorporated initially only in those cells that are synthesizing deoxyribonucleic acid (DNA) in preparation for mitosis (S period). Since granulocytes eventually reach a stage of maturation in which mitosis does not occur, some cell types, for example, metamyelocyte, will become labelled only with the passage of time. Labelled metamyelocytes appear within 3-4 h after injection of tritiated thymidine; the percentage of labelled cells then increases linearly for a few hours to a plateau followed by a second rise after 14 h. The results are given in Fig. 1.
Fig. 1. Initial pattern of labelled metamyelocytes after injection of tritiated thymidine. (Each point is derived from enumeration of 1,000 metamvelocytes; triangles, dog 4340; circles, dog "[ast]-m
It will be noted that the initial rate of increase of labelled metamyelocytes seems to be the same whether the autoradiographie analysis includes cells with 2 grains or more (2 +) or only cells with 4 grains or more (4 +). The linear increase extends over about 4 h with 2 + cells and 2 h with 4 + cells; it begins earlier and persists longer in the former instance. These results suggest that the more lightly labelled cells may be derived mainly from myelo-cytes which were near the beginning or end of their DNA synthetic period, while the more heavily labelled cells may be derived from a cohort near the middle of the S period. If this is so, the rate of uptake of thymidine must increase to a maximum around the middle of the S period and then decrease. The period of linear increase of 2 + metamyelocytes approximates 4-5-5-h duration of the myelocyte S period4; the agreement would perhaps be even closer if the progeny of myelocytes with less than 4 grains, which would appear as metamyelocyte false negatives, were included. The close correspondence of the duration of the S period and of the initial increase in labelled metamyelocytes indicates that there is little variation in the transitions from myelocyte DNA synthesis to myelocyte mitosis to metamyelocyte. A second rise in labelled metamyelocytes occurs some 10 h after the first increase. The rate of increase is less at this time, possibly because of the variation in the period preceding DNA synthesis, and also because of further dilution of the label by mitosis with resulting increase in false negatives. The 10-h interval may be a reflexion of the minimum duration of the myelocyte generation cycle.
Since the initial rate of entry of metamyelocytes is about 5 per cent per hour (Fig. 1), the turn-over or transit time would be 20 h. Labelled band cells were not seen even at 18 h after injection of tritiated thymidine, which means that the minimal transit time must exceed 15 h. It is noteworthy that a 20-h transit time was also obtained in earlier work by comparison of the time of appearance of label in metamyelocyte and band cell1. The initial rate of appearance of labelled metamyelocytes was considerably less in the latter instance, probably because insufficient thymidine was given to label all the myelocytes in synthesis of DNA. Since in the earlier investigation only about half the myelocyte mitoses were labelled during the first several hours after injection of tritiated thymidine, labelled and unlabelled cells must have entered the metamyelocyte compartment at the same time.
The magnitude of the metamyelocyte influx should correspond to the myelocybe efflux, assuming that few cells die or otherwise escape from the proliferating compartments. The initial labelling index of cells capable of mitosis and the duration of the S period provide a rather precise estimate of the birth or proliferation rate. This estimate will not be influenced by the possibility that: (1) all cells of a given type may not divide even though all may be capable of division; (2) the morphological boundaries of a given cell type may not include a complete generation cycle, that is, from the end of one mitosis to another. Some 37 per cent of myelocytes are labelled (2+ grains) a half-hour after injection of tritiated thymidine which, with an S period of 5 h, corresponds to a birth rate of about 7-5 per cent per hour. Since the metamyelocyte turnover is 5 per cent per hour, and the relative distribution of myelocytes to metamyelocytes in the marrow smear is 1 to 0-75, this means that for every 7-5 myelocytes that are born, only 3-75 myelocytes are transformed to metamyelocytes.
Cells are also entering the myelocyte compartment, and hence the myelocyte efflux must be greater than the myelocyte proliferation rate in the steady state with constant population. If we assume that all myeloblasts and promyelocytes eventually become myelocytes, the myelocyte efflux will be given by the sum of the proliferation rates of myeloblast, promyelocyte and myelocyte plus the presumed stem cell efflux (myeloblast influx). The latter is an indeterminate quantity at present, but it is probably very small relative to the succeeding fluxes. 40 per cent of myeloblasts, promyelocytes and myelocytes as a group are labelled at a half-hour after injection of tritiated thymidine; thus, the combined birth-rate with a 5-h S period is 8 per cent per hour. The proportional distribution of this group of cells to metamyelocytes is 1 to 0-6 and on this basis the relative efflux from the proliferating compartments would be 8 and the metamyelocyte influx 3. Whether we consider only the myelocyte proliferation rate or the approximate total myelocyte efflux, the results indicate a substantial 'over-production' or 'ineffective' granulocytopoiesis. A similar conclusion can be reached independently by comparing the expected yield after mitotic doubling of myelocytes labelled initially with 4 grains or more to the number of metamyelocytes with 2 grains or more. We can infer from the S period and the metamyelocyte appearance curve that all labelled myelocytes will have completed mitosis by 8 h after thymidine injection.
If only a part of the granulocyte production flows into the metamyelocyte compartment, it should be possible to account for the excess cells. Apropos of this, there is an increase in the percentage of labelled myelocytes during the first several hours after injection of tritiated thymidine, It is difficult to estimate the increase as long as some labelled cells are still undergoing mitosis. However, when myelocytes with 8 + grains at 0-5 h (21 per cent positive) are related to myelocytes with 4 + grains at 8 h after injection of tritiated thymidine (30 per cent positive), there is a 40 per cent increase. A similar increase does not occur in myeloblasts and promyelocytes. Thus, there is an appreciable 'sink' in the myelocyte compartment for accumulation of labelled cells. It follows that there must be a substantial number of myelocytes that do not participate in mitosis, but rather that are in a transitional phase prior to their loss in some way other than maturation to metamyelocyte. The transition from myelocyte to metamyelocyte requires about 1 h (3-h appearance time less 2 h for post-A period and mitosis); however, this could not be a factor in the accumulation of labelled myelocytes 8 h after thymidine administration. A prolonged myelocyte-metamyelocyte transition, postulated in our earlier experiments1, would be inconsistent with the present finding that the initial rate of entry of labelled cells into the metamyelocyte compartment is sufficient to account for its turn-over. It is of interest that there is no evidence of metamyelocyte attrition; labelled metamyelocytes can be accounted for afterwards as labelled band cells1.
Others have alluded to the possibility of some 'ineffective' erythrocytopoiesis5-6 and the fact that 'ineffective production' occurs in the case of the granulocytes is perhaps not too surprising. The myelocyte overproduction cannot be attributed to possible toxicity from incorporated tritium2 or to possible stimulation by thymidine7. If either of these factors were of consequence, this should be apparent in the rate of appearance of labelled metamyelocytes; the metamyelocyte turn-over is consistent with data obtained in other ways and after other doses of tritiated thymidine. It should be pointed out that this work \vas performed on young dogs arid, conceivably, the degree of overproduction could vary with age. We arc inclined to think that the so-called 'ineffective[ast] granulo -cytopoiesis represents an important facet of granulocyte regulation. Flexibility of the granulocyte system could be brought about by adjustment of the balance of 'effective' and 'ineffective' production. The population relationships and their regulation will be discussed in greater-detail elsewhere8.
This work was carried out under the auspices of the U.S. Atomic Energy Commission.Patt, , H. M., and Maloney, , M. A., in The Kinetics of Cellular Proliferation, 201 (Grune and Stratton, Inc., New York, 1959).Cronkite, , E. P., Bond, , V. P., Fliedner, , T. M., and Killman, , S., in Haemopoiesis, 70 (Churchill, Ltd., London, 1960).Maloney, , M. A., and Patt, , H. M., Proc. Soc. Exp. Biol. Med., 98, 801 (1958).PubMedISIChemPortMaloney, , M. A., Patt, , H. M., and Weber, , C. L., Nature, 193, 134 (1962).ArticlePubMedISIChemPortStohlman, , F., , jun., in The Kinetics of Cellular Proliferation, 318 (Grune and Stratton, Inc., New York, 1959).Lajtha, , L. G., and Oliver, , R., in Haemopoiesis, 289 (Churchill, Ltd. London, 1960).Greulich, , R. C., Cameron, , I. L., and Throsher, , J. D., Proc. U.S. Nat. Acad. Sci., 47, 743 (1961).ChemPortPatt, , H. M., and Maloney, , M. A., in Proc. Guinness Symp. Cell Proliferation, (Blackwell Sci. Publ.; in the press).