Cell Technologies in Biology and Medicine, No. 4, February, 2014
590
Effect of Immobilized Hyaluronidase on Stem and Progenitor Cells in Pulmonary Fibrosis
A. M. Dygai, E. G. Skurikhin, E. S. Khmelevskaya, N. N. Ermakova, A. M. Reztsova, O. V. Pershina, V. A. Krupin, I. E. Stepanova, V. M. Reztsova, A. V. Artamonov*, A. A. Bekarev*, P. G. Madonov*, and D. N. Kinsht* Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 4, pp. 248-244, October, 2013 Original article submitted February 15, 2012 The effect of immobilized hyaluronidase on stem and progenitor cells of the lungs was studied on the model of partially reversible toxic bleomycin-induced pulmonary fibrosis in C57Bl/6 mice. During the inflammation phase, immobilized hyaluronidase reduced infiltration of alveolar interstitium with hemopoietic stem cells Sca-1+, c-Kit+, CD34–, (CD3, CD45R (B220), Ly6C, Ly6G (Gr1), CD11b (Mac1), TER-119)–. Improvement of histological parameters of bleomycin lungs during the phase of collagen fiber deposition after the treatment was accompanied by accumulation of mesenchymal multipotent stromal cells (CD31–, CD34–, CD45–, CD44+, CD73+, CD90+, CD106+), decrease in the population of pan-hemopoietic cells (CD45+), accelerated restoration of the content of endothelial cells, and inhibition of clonal activity of fibroblast precursors (CD45–). Key Words: pulmonary fibrosis; immobilized hyaluronidase; hemopoietic stem cells; mesenchymal multipotent stromal cells; fibroblast progenitor cells High antifibrotic activity of immobilized hyaluronidase (imHD) synthesized by conjugation of testicular hyaluronidase to polyethylene glycol molecules using electron-beam synthesis technology was demonstrated on the model of partially reversible toxic bleomycin-induced pulmonary fibrosis [2]. It seems that imHD can be a new effective tool in the therapy of such chronic disease as idiopathic pulmonary fibrosis. However, despite previous studies of imHD, the mechanisms of the pharmacological effects of pegylated enzyme remain unknown. For instance, our understanding of possible mechanisms of imHD effects in pulmonary fibrosis is confined to its modulating effects on the structural and functional organization of matrix hyaluronan [2]. Research Institute of Pharmacology, Siberian Division of the Russian Academy of Medical Sciences, Tomsk; *Scientific Future Management, Novosibirsk, Russia. Address for correspondence:
[email protected]. O. V. Pershina
It is known that not only inflammatory cells (macrophages, lymphocytes, neutrophils, and plasma cells), but also hemopoietic stem cells, fibroblast progenitor cells, and mesenchymopoietic stem cells are involved in the pathogenesis of toxic bleomycin-induced pulmonary fibrosis [1,3,10]. It seems quite possible that violation of pulmonary fibrogenesis under the effect of imHD is underlain by changes in functional activity of various stem and progenitor cells. Here we studied the effect of imHD on progenitor and stem cells under conditions of pulmonary fibrosis.
MATERIALS AND METHODS The experiments were carried out on 7-8-week-old C57Bl/6 mice (n=300, conventional mouse strain obtained from nursery of the Research Institute of Pharmacology, Siberian Division of Russian Academy of Medical Sciences).
0007-4888/14/15640590 © 2014 Springer Science+Business Media New York
A. M. Dygai, E. G. Skurikhin, et al.
Pulmonary fibrosis was modeled by single intratracheal administration of 80 μg bleomycin (Bleomycetin, Lensfarm) in 30 μl physiological saline [9]. Hyaluronidase immobilized on polyethylene oxide was used (Scientific Future Management) [2]. The day of bleomycin administration was considered as day 0. Group 1 animals received 30 μl 0.9% NaCl (bleomycin solvent) into the trachea; group 2 mice received bleomycin and 0.9% NaCl (18 μl/mouse, solvent for imHD) intranasally on days 1, 3, 7, and 10 after intratracheal instillation of bleomycin (bleomycin control); group 3 animals received intranasal instillation of imHD (8 U/18 μl/mouse) on days 1, 3, 7, and 10 after intratracheal instillation of bleomycin. The animals were sacrificed on days 7 and 21 by CO2 overdosage and the materials for the analysis (lungs, bone marrow, and blood) were sampled. The inflammatory reaction and fibrogenesis in the lungs were verified morphologically. In nonadherent fraction of the bone marrow and lungs, CD45+ panhemopoietic cells and cells phenotypically similar to hemopoietic stem cells (HSC): Sca-1+, c-Kit+, CD34–, (CD3, CD45R (B220), Ly6C, Ly6G (Gr1), CD11b (Mac1), TER-119)– [10] were assayed. In adherent mononuclears of the lungs, bone marrow, and blood, expression of CD45 and formation of fibroblast colonies by CD45– cells (CFU-F) were analyzed. Then, CD45– cells were analyzed for the expression of surface markers CD31 (Platelet Endothelial Cell Adhesion Molecule-1), CD34 (stem/progenitor hemopoietic cells), CD44 (Hyaluronate receptor), CD73 (Ecto-5’nucleotidase), CD90 (Thy-1 glycoprotein), and CD106 (Vascular cell adhesion molecule-1) [10]. The data were processed by standard methods of variation statistics. Significance of differences was evaluated using parametric Student’s t test and nonparametric Mann–Whitney U test.
RESULTS In all mouse groups, CD34–, (CD3, CD45R (B220), Ly6C, Ly6G (Gr1), CD11b, TER-119)–, Sca-1+, and c-Kit+ cells were detected among isolated bone marrow and pulmonary mononuclears [10]. This phenotype is typical of HSC. On day 7 of the experiment (inflammation phase), HSC constituted 0.02% of the total number of examined nonadherent mononuclears. In bleomycin lungs of group 2 mice, this parameter decreased to 0.013%. In animals treated with imHD, the subpopulation of HSC decreased to 0.007% (Fig. 1). In the bone marrow of healthy mice, HSC population did not exceed 0.30% of the total number of examined nonadherent mononuclears. Intratracheal instillation of bleomycin had practically no effect on the content of HSC (0.31%). In
591 group 3, these parameters also remained unchanged (0.29%) (Fig. 1). On day 21 (phase of collagen fiber deposition), the population of CD45+ cells in the fraction of labeled nonadherent mononuclears were similar in histologically normal (68.6%) and bleomycin lungs (72.9%). At the same time, the percentage of pan-hemopoietic cells in fibrotic lungs decreased under the effect of imHD and constituted 52.6% of all labeled nonadherent cells. Interestingly, bleomycin and imHD had little effect on the population of bone marrow CD45+ cells (Fig. 1). Thus, imHD prevented bleomycin-induced infiltration of the lungs with HSC (phase of inflammation) and pan-hemopoietic cells (phase of collagen deposition). The pool of bone marrow CD45+ cells, including HSC, remained unchanged under these conditions. The use of previously developed protocol [10] allowed detection of CD45– cells in the fraction of adherent mononuclears of mouse lungs and bone marrow. On day 21 of the experiment, CD45– cells in the lungs of healthy animals constituted 31.4% (group 1, Fig. 2). Our findings coincided with previous reports [4], where CD45– cells constituted 30-40% of all labeled mononuclears in the lungs of healthy mice. In bleomycin lungs (group 2), this parameter tended to decrease (27.1%). In mice with pulmonary fibrosis treated with imHD, the relative content of CD45– cells increased to 47.4%.We revealed no differenced between the groups by the content of CD45– cells: 32.2, 33.6, and 31.8% in groups 1, 2, and 3, respectively. According to current views, damage to the alveolar-capillary membrane and myofibroblasts in airways and penetration of the plasma and blood cells in the pulmonary interstitium are followed by settlement of fibrocytes in the inflammatory focus [6,14]. In our experiments, single intratracheal instillation of bleomycin induced successive generation of fibroblast colonies by CD45– cells of the bone marrow (days 3, 7, 14, and 21), blood (days 7 and 21), and lungs (days 7, 14, and 21). In healthy animals, the growth of CFU-F was seen in bone marrow specimens, solitary colonies were detected in the culture of peripheral blood cells; no colonies were formed by adherent mononuclears of the lungs. Treatment with imHD considerably reduced the intensity of CFU-F formation in the culture of CD45– cells in fibrotic lungs and had no effect on the colony formation in bone marrow and blood samples (day 21, Fig. 2). These findings suggest that imHD selectively increased the count of CD45– cells in bleomycin lungs and reduced their clonal activity. Immunophenotyping of mononuclears according to the standards of lung mesenchymal multipotent stromal cells (MMSC) [8] using the protocol developed by us [10] detected CD45– cells expressing surface CD44,
592
Cell Technologies in Biology and Medicine, No. 4, February, 2014
Fig. 1. Quantitative and qualitative fluorescent cytometry analysis (dot-plots) of (CD3, CD45R (B220), Ly6C, Ly6G (Gr1), CD11b, TER-119)–, Sca-1+, c-Kit+, and CD34– expression in nonadherent cells isolated from the bone marrow (a) and lungs (b) of C57Bl/6 mice on day 7 of the experiment and content of CD45+ cells (panhemopoietic cells) in the lungs and bone marrow of C57Bl/6 mice on day 21 of the experiment (c). Here and in Fig. 2: I: 0.9% NaCl; II: bleomycin+0.9% NaCl; III: bleomycin+imHD.
CD73, CD90, and CD106 and not expressing CD31 and CD34 in the lungs and bone marrow of all mouse groups. On day 21, 1.7% of all isolated CD45– cells of the lungs in healthy animals were phenotypically identified as MMSC (Fig. 2). In bleomycin lungs of group 2 mice, this subpopulation constituted 2% of CD45– cells. In animals treated with imHD, the number of MMSC increased to 4.6% (Fig. 2). Analysis of surface markers CD31, CD34, CD44, CD73, CD90, and CD106 in the fraction of bone marrow CD45– cells showed that mononuclears with MMSC
phenotype constituted 0.4% of all analyzed CD45– cells in the bone marrow of healthy mice (Fig. 2). Bleomycin increased this parameter to 1% (group 2). In mice with pulmonary fibrosis treated with imHD, the content of MMSC-like cells was minimum (0.2%). Thus, imHD reduced the population of cells with phenotype CD31–, CD34–, CD45–, CD44+, CD73+, CD90+, CD106+ in the bone marrow and considerably increased the content of MMSC-like cells in the lungs. Quantitative changes in the cell composition of bleomycin lungs induced by imHD therapy can be
A. M. Dygai, E. G. Skurikhin, et al.
593
Fig. 2. Quantitative and qualitative fluorescent cytometry analysis (dot-plots) of CD45, CD73, and CD106 expression on adherent cells isolated from the bone marrow (a) and lungs (b) and the content of CFU-F in lung cell culture against the background of intratracheal administration of bleomycin and imHD (c) to C57Bl/6 mice on days 21 of the experiment. p
