A study of the relationship of the dynamics of development and characteristics of chimerism with manifestations of graft-vs.-host disease in the organs of mice after allogeneic transplantation of whole bone marrow

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In the сlinical practice, allogeneiс bone marrow transplantation (BMT) is often cause of the graft-versus-host disease (GvHD). GvHD is explained by the fact that T-lymphocytes, which are administered simultaneously with hematopoietic cells during transplantation and after then formed and matured in the timus of the recipient from donor progenitor cells, recognize and attack the cells of the host. However, a complete explanation of the phenomenon of the GvHD does not exists, and the chimerization of the recipient’s organism as a possible cause of damage of its organs is not taken into account. Therefore, the aim of this work was the modeling of allogeneic transplantation of the whole bone marrow (BM, experiment) and comparing its results with syngeneic transplantation (control) basing on the investigation of engraftment of cells of donor origin in the main GvHD target organs. Bone marrow (BM) donors were Tg(ACTB-EGFP)1Osb/J mice carrying a green fluorescent protein gene (EGFP), recipients were the animal of CBA and C57BL/6 inbred strains with age 2–10 months. 1 day before BMT (1.5×107 cells per mouse) all recipients were irradiated at a dose of 6.5 Gy (LD 50/30). After 1, 3, 5, 7, 11, 14, 21, 28, 35 and 55 days the development of chimerism in the liver, skin and colon of animals was examined using a fluorescent microscope. Already in 1 day, single fibroblast-like donor cells were found in the colon, in 7 days – in the skin and liver. 14–28 days after BMT, with donor cells mainly stroma in the liver, in the skin fibroblasts and keratinocytes were formed, in the colon villous cells and also stromal and parenchymal cells of Peyer’s patches which were died off after irradiation were substituted. Unlike control, in the experimental groups GFP+ giant fibroblasts about 30 mkm in length were found in the stroma of the liver, in the skin and in the colon; in the liver there was a lot of GFP+-bulkheads and fibroblast-like Ito’s cells of a very intricate configuration. To 35–55 days after allogeneic BMT cells of the donor origin in the liver and in the villi of the colon began to destroy, the villi became overgrown with GFP+-connective tissue cells and warped, wall of the colon became thin and the skin was fully substituted with a new one (all these things were never observed in the conrol groups). We propose a hypothesis that beside with GvHD traits like thinning of the colon wall and plenty of roundish GFP+-cells on inner surface of the skin, other signs of the studied after allogeneic BMT organs suggest that the cells of the organs which are formed from mesenchymal stem cells of the whole bone marrow become target for the recipient’s T-cells, i.e. suggest existence of host-versus-graft (HVG) reaction. Obvious manifestation of immune reactions after BMT directly coincides with the term of massive engraftment of the studied organs with cells of donor origin and restoration of the host’s own immune system, i.e. the development of chimerism determines the development of organ damage. This explains the events of GvHD from medical practice – atrophy of the mucous membranes, excess production of collagen, sclerosis of the bile ducts, skin damage, colitis – and the timing of its manifestation.

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Sobre autores

E. Bogdanenko

Institute of General Pathology and Pathophysiology

Autor responsável pela correspondência
Email: lenabogdval@mail.ru
Rússia, Moscow, 125315

L. Sergievich

Institute of Cell Biophysics Russian Academy of Sciences

Email: lenabogdval@mail.ru
Rússia, Pushchino, Moscow reg., 142290

A. Karnaukhov

Institute of Cell Biophysics Russian Academy of Sciences

Email: lenabogdval@mail.ru
Rússia, Pushchino, Moscow reg., 142290

N. Karnaukhova

Institute of Cell Biophysics Russian Academy of Sciences

Email: lenabogdval@mail.ru
Rússia, Pushchino, Moscow reg., 142290

I. Lizunova

Institute of Cell Biophysics Russian Academy of Sciences

Email: lenabogdval@mail.ru
Rússia, Pushchino, Moscow reg., 142290

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2. Fig. 1. Chimerization of skin with differentiation of donor cells: a – keratinocytes with nuclei (parakeratosis) in the stratum corneum 21 days after syngeneic bone marrow transplantation (BMT); b – a single keratinocyte deep in the skin on the inner side (dyskeratosis) 28 days after allogeneic BMT (arrow); c – outer side of new skin with many fluorescent cells 35 days after allogeneic BMT; d – fluorescent collagen strands, acellular mass and keratinocytes with nuclei in exfoliated dead skin 35 days after allogeneic BMT from the inside. Arrow 1 indicates a keratinocyte, arrow 2 indicates collagen strands; e – many GFP+ fibroblasts and keratinocytes 55 days after syngeneic BMT from the outside; e – dozens of round GFP+ cells and single fibroblasts from the inside 35 days after syngeneic BMT. Arrows indicate fibroblasts. Scale bar: 50 µm. Uv. volume: 20×.

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3. Fig. 2. Chimerization of the liver and spleen with differentiation of donor cells: a – GFP+ cells of irregular shape and triangular in the liver 1 day after allogeneic BMT; b – GFP+ structures from Ito cells in the liver in the form of vortices, rings and rays 35 days after allogeneic BMT; c, d – GFP+ megakaryocyte in the liver and spleen, respectively, 55 days after allogeneic BMT; e – hepatocyte of donor origin 35 days after syngeneic BMT; f – GFP+ hepatocytes (1) next to a GFP+ Ito cell of donor origin (2) 28 days after allogeneic BMT. Scale bar: 50 µm. Uv. volume: 40× (a, c–f), 10× (b).

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4. Fig. 3. Chimerization of the rectum with differentiation of donor cells: a – GFP+ fibroblasts of normal size 14 days later on the outer side of the intestine (in the muscle layer) after syngeneic BMT; b – GFP+ fibroblasts of giant size with branches on the outer side of the intestine (in the muscle layer) 14 days after allogeneic BMT; c – GFP+ cells similar to goblet cells (arrows), 14 days after allogeneic BMT; d – bases of villi of regular 6-sided shape in cross section 35 days after syngeneic BMT; e – deformed villi of different sizes, surrounded by layers of connective tissue GFP+ cells 35 days after allogeneic BMT. Scale bar: 50 µm. Uv. volume: 20× (a, b, d, d), 10× (c).

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