RADIATION DAMAGE AND RECOVERY OF THE IMMUNE SYSTEM

Cel

NON-STOCHASTIC EFFECTS FOLLOWING RADIATION OVER-EXPOSURE. MECHANISMS AND TREATMENT OF DOMAGE TO THE IMMUNE SYSTEM.

Our studies in BDF1 mice on the radiosensitivity and recovery of unprimed T-helper cells have indicated that these immunoregulatory cells are very radiosensitive and recover slowly, as some functional defects are still detachable 3 months after 200 cGy and complete recovery is only reached 6 months after 400 cGy. Recovery of T-helper cell activity from radiation damage is accelerated by injecting mice with synthetic thymosin alpha-1, a peptide of 28 amino acid residues identified in bovine thymus extracts and very effective in the induction of T-cell markers and functions.

We found that a synthetic peptide corresponding to residues 46-62 of mouse lysozyme, although not immunogenic itself, effectively inhibits the priming for T-cell responses when injected into mice together with immunogenic protein or peptide antigens. The inhibition observed strictly correlates with the capacity of the competitor to bind to the particular major histocompatibility complex (MHC) molecule presenting the foreign antigen, and its extent depends on the molar ratio between antigen and competitor.

This programme is part of the European Network of Experimental and Clinical Research and is focussed on radiation damage and recovery of the immune system, with the main objective of designing appropriate strategies for medical intervention in radiation accidents and radiotherapy. This study has investigated whether murine recombinant interleukin (IL)-3 injected into mice exposed to sublethal irradiation (100 to 200 cGy) can accelerate the recovery of thymocytes and peripheral T and B lymphocytes. It has previously reported by this laboratory that the synthetic nonapeptide valine, glutamine, glycine, glutamic acid, glutamic acid, serine asparagine, aspartate, lysine (position 163 to 171 of human IL-1 beta), when injected into mice immunodepressed by sublethal irradiation, induces restoration of T-helper cell activity and IL-2 production. The immunorestorative activity of the nonapeptide is similar to that of the human recombinant (hu r) IL-1 beta, but with no IL-1 like inflammatory effects which hamper the use of the whole protein as immunomodulator. Based on these findings the protective and restorative activities were compared of the 163 to 171 nonapeptide and hu r IL-1 beta on the 30 day survival of lethally irradiated mice. When mice were given a single injection of different doses of the nonapeptide or hu r IL-1 beta 20 hours before total body irradiation (TBI), both molecules were found to be able to increase the percent survival of mice exposed to 750 or 850 cGy, but not to 950 cGy. The nonapeptide, however, was less effective than hu r IL-1 beta and displayed a different dose response relationship, suggesting that the 2 molecules act through different radioprotective pathways. When mice were injected with the nonapeptide or hu r IL-1 beta immediately after exposure to 850 cGy, the percent survival was also increased but restoration was lower than protection in both cases. The nonapeptide was less effective than hu r IL-1 beta also in restoration, but the 2 m olecules displayed a comparable dose response relationship as if they shared similar mechanisms. These findings together indicate that the 163 to 171 nonapeptideis able to protect from lethal radiation injury and to restore viability. The nonapeptide is less effective than hu r IL-1 beta but, as it does not exhibit the IL-1 like side effects of the whole molecule, it appears an interesting compound for medical intervention in radiation accidents and radiotherapy.

IL-3 is a colony stimulating factor that regulates hemopoiesis, and is involved in the differentiation of pluripotent stem cells to mature cells of several lineages (neutrophils, macrophages, erythrocytes, eosinophils, magakaryocytes and mast cells). IL-3 also promotes limited self renewal of the multipotent stem cells which give rise to splenic colonies. Yet, there is no convincing evidence that IL-3 regulates the generation and growth of mature T and B lymphocytes. Experiments were performed to investigate whether murine recombinant (mu r) IL-3 injected into mice exposed to sublethal irradiation could accelerate the recovery of thymocytes and splenic T and B cells. In a first series of experiments, mice were given 300 cGy and daily injections of mu r IL-3 starting immediately after irradiation for 5 consecutive days. 7 days after irradiation, thymuses and spleens were assayed for cellularity, thymocyte mitotic response to concanavalin (Con)A, splenocyte mitotic responses to ConA and lipopolysaccharide (LPS), splenocyte antibody response to the hapten trinitrophenyl (TNP) and helper activity to the carrier horse red blood cells (HRBC) in cultures with the conjugate TNP-HRBC. Irradiated mice of other groups were similarly treated with mu r IL-3 for 10, 15, or 20 days and sacrificed 14, 21, or 28 days after irradiation, respectively. Under these conditions, IL-3 treatment did not accelerate the recovery from radiation damage in the thymus and spleen. In a second series of experiments, mice were given 100, 200, 300, or 400 cGy and daily injections of mu r IL-3 starting immediately after irradiation for 5 consecutive days. Mice were sacrificed 7 days after irradiation, and thymuses and spleens were assayed as indicated above. Results indicate that daily injections of 5 ug mu r IL-3 induced complete recovery of thymocyte cellularity and mitotic responsiveness to ConA in mice exposed to 200 but not to 300 or 400 cGy. Lower doses than 5 ug were not efficient in mice exposed to 200 cGy, whereas 1 ug was very effective after 100 cGy. Similar results were obtained for splenocyte count and mitotic responses. Fluorimetric analysis performed on thymocytes indicate that 5 ug mu r IL-3 completely reversed to normal values the CD4 and CD8 cell distribution altered by 200 cGy. Also the antibody response and helper activity of spleen cells were depressed by 200 cGy but could be recovered up to the level of unirradiated controls if mice were treated that 5 ug, but not 0.5 ug, mu r IL-3. In conclusion these findings indicate that injection of mu r IL-3 may induce complete recovery of T and B cellularity, and responsiveness to mitogens and antigens in sublethally irradiated mice provided larger amounts are injected after higher radiation doses. Thus, IL-3 regulates the generation and growth of mature T and B lymphocytes and appears as a powerful molecule that can be successfully used in radiation accidents and radiotherapy.

Treatment of irradiated persons by bone marrow transplantation may be successful if host and donor are human leucocyte antigen (HLA) compatible. Cell typing techniques must be rapid and very precise for the fine recognition of antigenic specificities. Professor G B Ferrara, Head of the Immunogenetics Laboratory at the Istituto Nazionale per la Ricerca sul Cancro in Genova, is collaborating with this laboraotry in the application of human monoclonal antibodies (mAbs) and recombinant deoxyribonucleic acid (DNA) techniques to the identification of HLA allelic speci ficities. 14 cell lines (MP1 to 14) secreting cytotoxic human immunoglobuline (Ig)M alloantibodies of restircted HLA specificities have been established. By use of mAb MP8 the HLA-DP polymorphism was analyzed and a supertypic determinant encoded by 7 DPB genes was detected. Furthermore, the HLA-DP typing was also performed using dot blot analysis with 14 synthetic oligonucleotide probes. Each probe was tested against genomic DNA amplified by polymerase chain reaction (PCR) using DP beta-specific primers. A total of 45 HLA homozygous B cell lines of know DPw specificities was analyzed and different hybridization patterns were found for each DPw specificity. The ologonucleotide hybridization performed on DPw negative B cell lines exhibited a pattern distinct from those of known DPw specificities, indicating the presence of novel DP allelic sequences. Thus, the use of these technologies has allowed reliable typing of HLA-DP antigens, which may play an important role in allogeneic bone marrow transplantation and in susceptibility to autoimmune diseases.
The contribution of the Laboratoire de Radiobiologie Appliquee can be described under two headings: the pathogenesis of severe acute radiation induced lesions and the treatment of radiation induced lesions.

Pathogenesis

Acute irradiation, with highly localized doses to pig skin, is the main experimental model used: it involves studies of induced radiolesions of the skin and in the underlying tissues (especially muscle). Studies on muscle radiolesions involve treatment.

Treatment

Screening of pharmacological treatment agents is performed on rabbits locally irradiated with iridium192 rays. The results of this screening are to be applied to locally irradiated pigs either by general or topical application.

Dziedzina nauki

Program(-y)

FP1-RADPROT 6C - Multiannual research and training programme (Euratom) in the field of radiation protection, 1985-1989

Temat(-y)

Data not available

Zaproszenie do składania wniosków

Data not available

System finansowania

CSC - Cost-sharing contracts

Koordynator

Comitato Nazionale per la Ricerca e per lo Sviluppo dell'Energia Nucleare e delle Energie Alternative

Wkład UE

Brak danych

Adres

Viale Regina Margherita 125
00198 Roma
Włochy

Koszt całkowity