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IMPAIRMENT OF THE HEMO-LYMPHOPOIETIC CELL SYSTEM AND ITS MICRO-ENVIRONMENT BY IONIZING RADIATION. PATHOGENIS OF NON- STOCHASTIC AND NEO PLASTIC EFFECTS AND CONDITIONS FOR A LONG

Objective

The contracts represent a joint effort of four Community laboratories to improve the scientific basis and the practice for the diagnosis and treatment of radiation accident victims. The participating laboratories have a long standing expertise in this area and, for many years, have cooperated closely in research and treatment. They now aim to develop a European network for radiation accident mangement to serve as a centre of expertise for the development of strategies to manage radiation accidents should they ever occur in the Community. Moreover, such a network will have an important role in the training of doctors and nurses in the particular handling of accident victims and, thereby, transmit the unique personnal experience of the participating groups to other institutions.
The survival of persons exposed to irradiation in the lower range of possibly fatal doses depends mainly on the functional status of the hemopoietic tissue and its regenerative capacity. This has been clearly show during the recent accidents in nuclear power stations and industry. However, the anatomical distribution over the different bones of the hemopoietic stem cells and progenitor cells surviving an exposure may be quite different depending on the conditions of the exposure, ie whether it was total body irradiation with a homogeneous bone marrow dose distribution or a more or less inhomogeneous dose distribution. Under extreme conditions partial body irradiation may arise with certain fractions of the whole bone marrow organ being completely or nearly absolutely protected against the incident beam. Under the former conditions the regeneration events may be mainly local originating from resident surviving stem cells. However, it has to be asked whether after an inhomogeneous total body irradiation the local regeneration of the different bone marrow sites (receiving different degrees of damage) is associated with systemic equilibration processes due to exchanges of hemopoietic cell migration via the circulation. Clearly, after partial body irradiation where large doses in the range of 10 Gy had been received by a certain fraction of the marrow seeding of stem cells from the protected marrow could be the most essential prerequisite for any early repopulation. On the other hand in the early phase of regeneration the protected marrow may compensate in an essential way for the damage in the early phase of regeneration.
The research work was addressed to all these aforementioned aspects of hemopoietic regeneration and tolerance due to compensatory mechanisms.

Several conclusions of theoretical as well as of practical interest were drawn from data obtained using the canine model of partial body irradiation. First of all it could be demonstrated that the exposure of 30% of the total bone marrow mass resulted in a severe lymphocyte depression within 24 hours after the exposure, whereas the blood granulocyte and platelet values showed only minor changes within the first 3 weeks following the exposure. Therefore, an initial lymphocyte depression after irradiation may well be a monitor per se that in fact certain damage has been caused; however, the lymphocyte changes cannot be applied as a prognostic indicator of hemopoietic recovery. The reason for this is that in the case of partial body irradiation the protected bone marrow, whether 70% or 30% of the total mass, is able to compensate effectively for the damage in the exposed sites by rapid cell proliferation and differention within 24 hours after irradiation. On the other hand, it could be shown that in the highly irradiated bone marrow sites there was a significant degree of repopulation by hemopoietic elements already at day 7 after the exposure. This fact is clearly due to the seeding of stem cells from the protected marrow sites since in dogs receiving homogeneous total body irradiation with a much lower dose of 3.9 Gy progenitor cells could be detected at much lower numbers if any at day 7 or 9 after exposure.

The structural integrity of the bone marrow stroma and its functional status is the essential prerequisite for the maintenance of hemopoiesis and its regenerative and compensatory capacity under disturbed conditions. Thus, it is of importance to study the biology of the stroma and its elements under normal circumstances and the effects of different types of exposures to ionising radiation on the stroma itself.
One approach in the present programme was to study the fibroblastoid colony forming unit (CFU-F). These cells are considered to be representative of the progenitor cell population of the hemopoietic microenvironment. Consequently, they might be of importance in the longterm for the replacement of the long lived differentiated elements of the stroma, such as fibrocytes, reticulum cells etc.

The results obtained indicate that the concentration of the CFU-F in different bones is closely related to the hemopoietic activity in the respective bone marrow sites as measured by the incidence of hemopoietic progenitor cells, eg granulocyte-macrophage progenitor cell (GM-CFC) and bone-marrow erythroid burst-forming units (BFU-E). With respect to their radiobiological properties it is of interest that the clonogenic CFU-F are relatively radioresistant when compared to hemopoietic progenitor cells. The dose (D) values of 2.4 Gy of the canine bone marrow CFU-F are a factor of 4 higher than those for the GM-CFC, and they are obviously a factor of approximately 2 higher than the D values reported for human bone marrow CFIU-F. The presence of a shoulder, though quite small, on the survival curve is indicative of some capacity for accumulation of sublethal damage. Therefore, some capacity of sublethal damage repair could also be expected. However, when split dose irradiations were performed a significant loss of CFU-F after the first dose could be noted. Perhaps this loss was due to the fact that the CFU-F were irradiated in suspension and not in an adherent state. Neverthe less, when the survival data were corrected for the loss a clear split dose recovery became evident if the two doses were separated by an interval f 2 hours.

In the dogs which had received inhomogeneous total body irradiation (TBI) by a unilateral exposure the differences that could be detected between the CFU-F numbers in the ribs at least 1 year after the exposure were rather small. These findings show that the initial cell loss and the differences in the CFU-F numbers must have been nearly compensated within the 1 year period. On the other hand, there was clearly some late damage present in he hemopoietic tissue in the bone marrow that had received a dose of 2.7 Gy. This was obviously not directly related to the CFU-F. The data obtained from the partial body irradiated dogs clearly indicate that certain types of damage to the stroma are responsible for the delayed regeneration of hemopoiesis as reflected in the subnormal GM-CFC values in bone marrow sites that had received a single dose of 11.7 Gy. It could be shown that the CFU-F numbers in the irradiated sites also remained clearly subnormal for several months after the exposure. On the other hand, in this model microscopical examination revealed structural alterations in the stroma evident as an increased number of connective tissue fibres. Clearly, in areas in which fibrosis was marked there was little or no hemopoietic activity. However, despite such local defects the hemopoietic function in a given site was nearly normal after 1 year as became evident from the GM-CFC determinations in aspirates.

Sequential half body irradiation was employed to test whether a pre-irradiated stroma is able to allow a compensatory response of the hemopoietic tissue similar to that in an unirradiated state. It could be demonstrated that bone marrow sites that had partially recovered from a radiation dose of 11.7 Gy were able to compensate effectively for the damage that was produced in other parts of the skeleton by a sec ond irradiation with the same dose, if this second irradiation was performed 56 days after the first one. The compensation was achieved in the same way as observed in a previously unirradiated bone marrow, namely by a strongly increased proliferation within the following 3 weeks. Thus it can be concluded that the stroma that had received a dose of 11.7 Gy is able to supply the microenvironmental conditions required for such an extraordinary response of hemopoiesis.

Hemopoietic failure (aplasia) and malignancy as a consequence of radiation exposure are difficult to analyse in their stages of development. Even in mice no clear indication for the most critical decision between the two endpoints aplasia or neoplasia could be found. Stem cell depression may and may not be involved, the natural killer cell system had no influence. In a given leukemogenic protocol a synergism of a chemical agent and external radiation was seen, when each agent along was in a suboptimal dose. the enhancement of leukemogenesis in such protocols by preceeding radiation is thought to be a target cell phenomenon. During the recovery from a median dose of radiation more target cells seem to be present than under steady state conditions. If mutations in the N-ras gene are a critical event, the more recently established methods of molecular biology should be able to detect mutated genes in various hemopoietic organs during the latency period. This will contribute to the question of the target cell system.

The purpose of this study is to extend the knowledge base necessary to improve the understanding of the type of response of humans to radiation exposure and to establish therapeutic strategies that may improve the survival of persons accidentally exposed to ionizing radiation.

A radiation accident clinical database is being established. Considerable progress has been made through intensive efforts to establish an internationally acceptable computer compatible case history form in collaboration with experts in Moscow. It is now possible to extend the database to include several hundred radiation accident victims in order to correlate blood cell changes with clinical course and outcome. It is the first time in radiation accident research that such an effort is being made which is indispensible for establishing a knowledge based expert system for radiation accident maangement.

Significant progress has been possible in the further development of biomathematical computer assisted blood cell simulation models. In collaboration with the Department of Systems Engineering, it became possible to improve the granulocyte response model and to pave the way for an entirely new model for the megakaryocyte platelet system in order to understand platelet changes and to use them as biological indicators for radiation exposure.

Cytogenetic studies were performed in leukaemic patients after total body irradiation and bone marrow transfusion to study the type of chromosomal aberrations seen in host lymphocyte metaphases as a function of time after radiation exposure. These studies will serve to analyze the radiation dose to the lymphocyte systems and the extent and severity of graft versus host disease and relapse rate of leukaemia. This information is of importance to the improvement of the management of radiation accident victims.

Experimental studies were carried out in dogs using partial body irradiation to improve the understanding of the role of migratory stem cells an d the possibilities of the use of recombinant regulation factors to enhance haematopoietic regeneration after extensive radiation exposure.
The management of radiation accidents is typically hampered by the lack of information available on the extent of damage inflicted on the organism and on the amount of tissue spared from radiation which might bring about the recovery of the organism. It should now be possible to develop an expert system based on the experience of past accidents and to use a spectrum of biological indicators to determine the extent of damage, the likelihood of recovery and the risk of permanent damage with emphasis on effects on the haemopoietic and immune systems.

New treatment modalities have recently become feasible thanks to the development of bioengineered haemopoietic growth factors. It can be envisaged that a judicious treatment with these factors could allow an optimal stimulation to promote a more speedy and complete recovery of the haemopoietic and immune system.

The development of new techniques for the isolation of haemopoietic stem cells has enabled bone marrow transplantation to become a more reliable and safer procedure in cases of very severe radiation accidents. Moreover, new methods of supportive care have become available.

Finally, it may now be possible to reduce the risks to persons who have to participate in recovery operations by prophylactic measures. Haemopoietic injury could be mitigated after a planned radiation exposure during a rescue operation by reinfusing autologous stem cells taken previously from the patient. Such cells could be obtained most readily from blood. Factors,such as dextran sulphate, which mobilize stem cells from bone marrow into human blood so that greater numbers of stem cells can be collected, will be investigated. The logistics needed for the establishment of such banks will also be studied. (Ulm. Univ.).

Diagnosis and treatment of radiation accident victims must be based on a sound understanding of the underlying pathophysiological mechanisms of damage and recovery after total and partial body irradiation, including the definition of the conditions which might lead permanent damage.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Universität Ulm
Address
Oberer Eselsberg M 24
89070 Ulm
Germany