The cellular basis of long-term marrow injury after irradiation

Radiotherapyand Oncology, 3 (1985) 331-338 Elsevier

331

RTO 00110 Review Article

The cellular basis of long-term marrow injury after irradiation J. H. H e n d r y Radiobiology Section, Paterson Laboratories. Christie Hospital and Holt Radium Institute, Withington, Manchester M20 9BX, U.K.

(Received 18 May 1984, revisionreceived 13 November I984, accepted20 November 1984)

Key words': Bonemarrow injury;Long-termeffectsafter irradiation;Haemopoieticstemcells

Summary Haemopoietic recovery from radiation injury can appear complete when measured by blood cell counts, but this can hide deficiencies in the precursor cell populations because of compensatory mechanisms of increased numbers of divisions in the maturing cell populations and increased cycling of the stem cells. These mechanisms can operate for quite long but finite periods, before they fail which then leads to hypoplasia. Also, while these mechanisms are operating, small further injuries could precipitate marrow failure. Persistent injury in the stem cell population can be induced by quite small doses, and in mice the threshold total dose is probably in the region of 1.5 Gy using fractionated whole-body irradiations. The sensitivity of the environment varies enormously, depending largely on the proliferative stress applied to the cell populations involved in the particular assay technique used. When similar tests of reproductive integrity are applied, stromal progenitor cells are more radioresistant than haemopoietic stem cells. The contribution of environmental injuries to haemopoietic defects is uncertain and difficult to assess.

Introduction Cytotoxic treatment using irradiation or drugs can result in overt long-term haemopoietic injury. Knowledge of threshold doses and the mechanisms involved in such injury clearly is important for improving treatment strategies. Various animal species have been used to investigate different aspects of these questions. This review will concentrate on those qualitative aspects of human responses where the mechanisms can be elucidated in the mouse. These comprise the response of bone marrow stem cells to acute and protracted doses, the sensitivity of the environment, and dose-response relation-

ships measured for the production of residual haemopoietic injury.

The sensitivity of haemopoietic and stromai progenitor cells among species The long lineage of cell types in the differentiating haemopoietic cell hierarchy, in contrast to, for example, many epithelial hierarchies, means that there is the potential for assessing the radiosensitivity of many different types of progenitor cell using colony techniques. A simplified schematic diagram of the haemopoietic hierarchy is shown in Fig. 1,

0167-8140/85/$03.30 9 1985ElsevierSciencePublishers B.V. (BiomedicalDivision)

332

Maturecells Committed

S

t

e

m

~

~Pre-GM-CFC~GM-CFC~ W B C

Fig. 1. Schematic representation of the haemopoietic hierarchy, restricted to those cell types considered in this review. CFU-S, colony-forming units in the mouse spleen (stem cells); ERC, erythropoietin-responsive cells; BFU-E, erythroid burst-forming units; CFU-E, erythroid colony-forming units; RBC, red blood cells; pre-GM-CFC, assayed in diffusion chambers in vivo; GM-CFC, granulocyte/macrophage colony-forming cells; WBC, white blood cells; CFU-M, megakaryocytic colony-forming units; CFU-F, stromal (fibroblastoid) colony-forming units.

which includes the progenitor cells assayed for survival and reviewed in this paper. CFU-S are assayed in vivo by their ability to produce colonies in the mouse spleen, and ERC are also assessed in vivo. The other progenitor cells are detected by their ability to produce colonies in vitro, using different growth media and colony-stimulating factors. Many assessments of survival of progenitor cells have been made, using various species. The survival curves are summarised in Fig. 1, where cell survival S = n 9 exp(-D/Do). Mean values are given in cases where various estimates exist in the literature. The majority of the curves show very little "shoulder" to the near-exponential survival curves, i.e. n ~ 1, but it is probably most marked for the fibroblastic progenitor ceils (CFU-F). The only case where an attempt has been made to describe the curving shape in terms of the linear-quadratic parameters ~ and/~, which are now commonly used in the literature, is for the stem cells (CFU-S). In this description cell survival is described by S = exp - ( ~ D + /~D2), and this contrasts with the description above. Hence, when there is no shoulder, = 1~Do, and when there is a shoulder, ~ is the

inverse i n i t i a l slope to the survival curve. The ratio (~//~) is a measure of the curvature of the survival curve, which is important regarding the response to low dose-rate or multifractionated treatments. The value of ~ is about 1 Gy-1 and the value of (~//~) is about 21 Gy, when calculated from the parameters given in reference [13]. The latter value is within the range of values deduced using LDso/3o* and multifractionated treatments, of 7-35 Gy [8]. Also, it is consistent with data using low dose-rate exposures, where the LDso/3o of 6-8 Gy using high dose-rates ( - 1 Gy/min) can be increased by a factor of 1.3 to 1.4 by using 1-3 cGy/min (e.g. 30; 34). With cell types other than CFU-F and CFU-S, the survival data are virtually exponential (with no shoulder) and span only two decades in depopulation so that the survival curve can be described adequately by Do = (1/~ in these cases). These values are shown in Table I. In the mouse, the granulocyte/macrophage precursors (GM-CFC) are more resistant than the erythroid precursors (BFU-E, CFU-E) and the stem cells (CFU-S). The only stromal progenitor cell which has yet been cloned (CFU-F) and which produces TABLE I Values of sensitivity to low LET radiation. Cell

Species

Do (Gy)

Reference

CFU-S

Mouse

0.8-1.2

Pre-GM-CFC Pre-GM-CFC GM-CFC

Mouse Man Mouse

1.4 0.9 1.6-2.4

GM-CFC

Dog

0.7

GM-CFC CFU-M CFU-E BFU-E CFU-F

Man Mouse Mouse Mouse Mouse

1.4-1.6 1.3 0.7 0.7 1.54.0

CFU-F

Dog

4

Range reviewed in reference [15] [12] [12] Range reviewed in reference [15] Range reviewed in reference [15] Range reviewed in [29] [29] [45] Range reviewed in reference [15] [47]

* LDs0/3o: dose for death of 50% of mice by day 30.

333 colonies predominantly of fibroblasts (but in the mouse also endothelial cells and macrophages [49] has a sensitivity less than any of the haemopoietic progenitors (Fig. 1). GM-CFC in man have a sensitivity similar to that in the mouse, but in the dog their sensitivity inexplicably is much higher. The latter contrasts with the apparent lesser sensitivity of CFU-F in the dog than in the mouse.

The sensitivity of the haemopoietic environment Various assays have been developed for measuring the response of the haemopoietic environment to irradiation. Apart from histological methods in situ for assessing the sinusoidal component of the environment [21], the other assays involve the stimulated regeneration of environmental components in order to reveal latent radiation injury. These assays comprise (a) the production from ectopic marrow fragments of an environment supporting haemopoiesis [10,40]; (b) the recovery of the environment in femurs transplanted subcutaneously [1]; (c) the formation of fibroblastoid colonies in vitro from marrow cells [9] and (d) the formation in vitro from marrow fragments of an adherent layer which supports the maintenance of bone marrow stem ceils [6]. Each assay probably assesses a specific component of the environment, and the relationship between them is not fully understood. The relative sensitivities of the environment measured by different assays are shown in Fig. 3. As expected, greater sensitivity is expressed when the proliferative component of the response is high, e.g. in the production of colonies from CFU-F (curve C). It is noteworthy that the sensitivity of the femur measured in situ in terms of its content of CFU-F, was similar to that obtained when the content of CFU-F was assessed at 6 weeks after transplanting the femur ([32], curve B). Also, the latter sensitivity was similar to that obtained when the contents of CFU-S and GM-CFC were assessed (curve B also), indicating that the content of CFU-F could predict the ability of the femur to maintain its content of haemopoietic progenitors. Greater sensitivity is seen in terms of the ability of the marrow plug to

produce an environment under the kidney capsule (dashed curves, labelled D), capable of sustaining CFU-S and assayed 8 weeks after transplantation (Schofield, unpublished). The sensitivity is similar to that seen for CFU-F (curve C). Also, recovery in situ of the environment-producing cells was observed up to at least 9 weeks before the cells were removed and transplanted for assay under the kidney capsule (dashed curves, labelled D). The sensitivity of the adherent layers in vitro has been reported to be very high (curve E, Fig. 3) and the reason for this is unknown [7]. No morphological changes have been detected in the irradiated layer. A dose of 0.5 Gy was sufficient to abolish the ability of the layer to maintain the growth of normal stern cells, and it was affected by only 0.25 Gy [7]. In contrast, other studies indicate that radiation can stimulate the layer to maintain CFU-S [26]. Doses higher than 5 Gy are required to reduce the ability of the bone marrow to produce an adherent layer in vitro [7,39]. These studies indicate that long-term environmental injury can be induced by quite small doses, 1

F

01

CFU-F

~GM

-CFC CFU-E

0'0

"~ CFU-M ~'GM-CFC Pre-GM-CFC

0

i

i

i

T~

2

4

6

8

D o s e Gy Fig. 2. Survival o f h a e m o p o i e t i c a n d s t r o m a l p r o g e n i t o r cells to acute doses o f low L E T r a d i a t i o n (usually 7-rays). - - , mouse; ..... , h u m a n ; ........ , dog. Do values given in T a b l e I.

334 ~'~..\ ~\~\'~',

Loss of Sinusoids-"~ at 1 yr.

" ~

0"5 '\';~ \17 w k s ~ CFU-S,GM-CFC, CFU-F / ~ \ ~, \ in transplantedfemur ~\ ' \ \ at 6wks. cFuLs in\ , \ \ Kidney ~ '\~ ~ ~ CFU-F/femurin situ 0-1imnlant~ \~ ~ \ \ (+106 BM cells) ~\ ~\ \ C at 2-10 months Acute"\ \,,D ~\7 wks. \ 0"05~dherent ~ CFU-F layers ~ (Acute) in vitro Assay CFU-S at 7 wks. 0.01-

4

8 Dose Gy

12

16

Fig. 3. The sensitivity of the haemopoietic environment, using different assays. Curves taken from: A: Knospe et al. [21]; B: Piersma et al. [32]; C: mean values quoted in Hendry and Lord [15]; D and dashed curves: Schofield (unpublished): E: Dexter et al. [7].

down to a few Gy (Fig. 3). The injury may be latent, until it is revealed either in the long-term or by proliferation induced by subsequent injury.

Threshold whole-body doses for long-term injury in mice After large sublethal acute doses ( ~ 5 Gy) there is quite good recovery regarding long-term marrow function. The doubling time of the CFU-S population is about 24 h in mice, similar to that for unirradiated CFU-S repopulating lethally-irradiated mice, and this represents a cell cycle time as short as 6 h with 40% of CFU-S differentiating at each division [22]. By 3 weeks in mice (longer in man) most recovery has occurred, and some residual defects in haemopoiesis have been reported, for example, in the recovered level of CFU-S per femur (about 80%, reviewed in reference [15]), in the ability of grafts of recovered marrow to rescue lethally-irradiated recipients [33], and in the proliferative capacity of maturing cells [19]. However, these defects rarely are expressed in terms of blood cell out-

put, because of the ability of the hierarchy to expand the already large number of amplifying cell divisions between the stem cells and the mature cells, and also to increase the rate of cycling and differentiation of the stem cell population. An example of this after repeated irradiation, when the changes are quite marked, is shown in Fig. 4. Following the initial compensation for haemopoietic deficiencies by increased cycling and increased numbers of amplification divisions, there is a further compensation which develops in the long term. This is achieved in the small rodent by extramedullary haemopoiesis, e.g. in liver [41]. In larger species there is a slow extension of the bone marrow organ into inactive medullary regions, e.g. the long bones [36]. Few measurements in mice have been made after acute doses much lower than 5 Gy, but even after 0.5 Gy some defects have been reported, e.g. in CFU-S per femur which remains at 50% of control for up to 55 days before the final stage of recovery to near 100% [38], in the ability of adherent layers to maintain haemopoiesis (Fig. 2), and in the proliferation rate of maturing femoral marrow cells grown in the spleen [20]. More important for radiotherapy is the response to low dose-rate or multifractionated irradiation, and in these cases information is less extensive than for acute exposures. Repeated large doses of 4.5 Gy have been used to produce maximum tolerable injury which has been analysed in detail [14-16,43] and recently the doses 100 8O

~ 60 "5

RBC

t

WBC

J

ERC

GM-CFC

40

BFU-E~

~

/

20' 0 3-6 months after 4x4.5 Gy X-rays

Fig. 4. New steady-state achieved in the haemopoietic hierarchy at 6 months after 4 x 4.5 Gy X-rays (redrawn from ref. [18]).

335 190- ~

~_

~

~

v ~-rays

o8 g

t \ \\

04

0"2

~

9

/B

~ 0

...:s_~ ......

,

,

6

, 12

-,

, 18

Total dose Gy

Fig. 5. Levels per femur of nucleated cells (curve A), G M - C F C (B), C F U - F (C), and CFU-S (D), averaged between 3 and 9 months after four repeated doses of X-rays (redrawn from ref. 48). Total doses quoted on abscissa.

0-001 C

5

10 Dose

per treatment have been reduced to study dose-response relationships [48]. CFU-F are spared more than are haemopoietic progenitors by the use of low-dose fractions (Fig. 5). The threshold dose for the long-term depletion of CFU-F (e.g. below 0.9 of control) is about 4 x 1.5 Gy (doses delivered at 3-week intervals) in contrast to a lack of any significant threshold for CFU-S [48]. This difference also is seen with irradiations at low dose-rate [17]. Irradiations at 0.05 Gy/min (as used in human whole-body irradiations), spares CFU-F more than CFU-S when assayed immediately after irradiation, and this difference remains at 2 months (Fig. 6) when recovery has reached a plateau. With this doserate, threshold doses for levels at 2 months of CFU-S and CFU-F per femur are at most 2.5 Gy. A fundamental property of CFU-S is their selfrenewal ability, which must be considered in addition to their numbers. The self-renewal ability will determine the long-term ability of stem cells to maintain their population and to produce mature haemopoietic cells. Immediately after acute doses of up to 5 Gy [15] or after 7.7 Gy given continuously over 11 days [37], the ability of surviving stem cells to produce colonies containing numbers of new stem cells (measured as CFU-S per colony) is similar to that in controls. However, the ability is reduced after more prolonged treatments, e.g. at

15

Gy

Fig. 6. Survival of CFU-S (triangles and diamonds) or C F U - F (circles and squares) per femur, measured immediately after irradiation (open symbols) or at 2 months (closed symbols), using 7-rays at 4,2 Gy/min (diamonds and circles) or at 0.05 Gy/min (triangles and squares).

days 21-81 after 31 Gy given continuously over 45 days [37], and persistently reduced up to at least one year after four repeated doses of 4.5 Gy [18]. In general, defects of this nature are found when the stem cell population remains persistently subnormal. Whether this is due mainly to radiationinduced defects in the surviving stem cell population, to persistent induced cycling in the normallyquiescent stem cell population, or partly to environmental factors, has not yet been elucidated. Recovered levels of CFU-S per femur have been measured also after 15 daily whole-body exposures of between 0.05 and 0.70 Gy (unpublished data). The threshold dose for the long-term depletion of CFU-S femur (below 0.9 of control, between 2 and 6 months) was about 1.5 Gy total dose using 0.1 Gy per fraction, i.e. the same as the dose used in treatments of CLL [35]. With 0.3 Gy per fraction (total dose 4.5 Gy) the level of CFU-S per femur remained at 50% of normal between 2 and 6 months after irradiation. The data obtained using repeated doses, low dose-rates, or multifractionated

336 doses indicate that the threshold total dose for longterm haemopoietic injury is probably not more than about 1.5 Gy. The contribution of environmental defects to long-term marrow injury is unclear and difficult to assess. Clearly, defects in both haemopoiesis and in the haemopoietic environment separately can be measured, but generally this involves the use of proliferative stimuli to reveal latent injury. This will appear in tissue components at various times after irradiation, which depends largely on their normal rates of turnover. Also, when the proliferative capacity of stem cells is assessed by serial transplantation, the recipients are already irradiated and any influence of this on the graft may confound the interpretation of declines in haemopoiesis [37]. Residual injury is also produced by drug treatments, particularly alkylating agents [2,27,28], but there is very little information concerning the interaction of drugs and radiation for the production of residual injury (reviewed in reference [42]).

Partial body irradiation

Irradiation of sections of the body is followed by haemopoietic recovery with mechanisms dependent on the dose and the volume irradiated. There is evidence for local control of CFU-S turnover, and systemic control of CFU-differentiation ([11]; and see below). When the proportion of marrow which is shielded is small, say one leg of a mouse or 5% of the total, there is an initial decrease in numbers of CFU-S in that femur, within 15 min, which is dosedependent, to about 35% after 15 Gy to the rest of the body [4]. This is generally considered to represent induced differentiation, with possibly also some migration. It is followed by a marked release of CFU-S into the circulation more rapidly during the next 3 h than subsequently [5] when cycling of CFU-S increases markedly before reaching control levels by 48-96 h [3,11]. Hence after the initial perturbations, the numbers of CFU-S in the shielded region are restored and cycling decreases, even though the irradiated marrow still is markedly deficient in numbers of CFU-S which continue to cy-

cle rapidly. In contrast, when only one tibia is irradiated (which would affect mature cell production insignificantly), with the rest of the animal shielded, there is increased cycling in the tibia [11], which implicates a local control in the regulation and restoration of CFU-S numbers after depletion. Circulating CFU-S are known to have a low ability to renew themselves [25]. This suggests that CFU-S, migrating into a depleted area from a shielded region, may contribute initially to the production of blood cells but would have a low ability in the long term to colonise irradiated marrow. However, if 10% of the total marrow was shielded, this alone could provide continuously the normal complement of mature cells, based on studies with whole-body irradiation where 10% of CFU-S remaining in the whole (irradiated) animal support virtually full haemopoiesis during continuous irradiation (reviewed in reference [23]) or after repeated irradiation [18]. It has been shown that stromal progenitors are transplantable [31] so that endogenous levels can be increased persistently by marrow grafts [48]. Also, migration of stromal progenitors from unirradiated to irradiated regions of marrow in the same animal has been reported in some studies [46], but not in others [48].

Retreatments

There are few studies of the radiosensitivity of the stem cell population which has recovered following depletion by radiation, but changes have been reported to be small [14]. However, although stem cells have an extensive proliferative capacity, sufficient to provide haemopoiesis over several mouse life-spans [24], the recovered levels are reduced by subsequent radiation doses (see above). As the recovered level cannot be increased to control levels by large grafts of unirradiated marrow [18,44] and as the recovered level tends to decline with reference to controls at long times after irradiation [18], this suggests the presence of slowly developing environmental injury. This is in addition to injury in the stem cell population itself, which, however, corn-

337 pensates adequately for some time for persistent reductions in its size by up to a decade (see above). Nonetheless, it is possible that retreatments could easily tip the balance to below a level of recovery critical for survival of the animal. This could reveal latent injury and produce a severe response in a system appearing superficially from the complements of mature cells to be normal, and hence this is a cause for concern and for further investigations in the use of repeated cytotoxic therapy.

Acknowledgements I am grateful to my colleagues Dr. Nydia Testa and Dr. Ray Schofield for many discussions on the work reviewed here, and to the Cancer Research Campaign (U.K.) for support.

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