Bulletin of Experimental Biology and Medicine, Vol. 156, No. 1, November, 2013 GENERAL PATHOLOGY AND PATHOPHYSIOLOGY
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Role of Matrix Metalloproteinases and Their Inhibitor in the Development of Early Pulmonary Fibrosis in Mice Infected with Influenza A/H5N1 A/GOOSE/Krasnoozerskoye/627/05 Virus
A. G. Anikina*,***, O. V. Potapova*,***, V. A. Shkurupii*,***, and A. M. Shestopalov** Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 156, No. 7, pp. 17-20, July, 2013 Original article submitted March 18, 2012 High levels of macrophages and fibroblasts expressing MMP-2, MMP-9, and MMP-10 against the background of progressing early fibrosis of the lungs (manifesting in an increase in volume density of type I, III, IV, and VI collagens) were found in C57Bl/6 mice infected with influenza A/H5N1 A/goose/Krasnoozerskoye/627/05 virus. Progressing fibrosis of the lungs in infected mice was associated with imbalance of collagen synthesis and degradation processes conjugated with high levels of macrophages and fibroblasts expressing TIMP-2. Key Words: influenza A/H5N1 virus; lungs; fibrosis; matrix metalloproteinases; tissue inhibitor of matrix metalloproteinase Early interstitial fibrosis of the lungs is a complication developing in humans and animals infected with A/H5N1 influenza [3,5,13]. The phenomenon of early fibrosis developing during the acute period of infection in parallel with tissue destruction is poorly studied and its prevention, therapy, and pathology are important problems. Matrix metalloproteinases (MMP), representatives of the zinc protease family, are involved in the degradation of extracellular matrix (ECM) components (including collagens), thus realizing the regulatory function in the connective tissue system in health and disease [2]. Gelatinases (MMP-2, MMP-9) are involved in catabolism of collagens I and IV fibrils [10,15] and stromelysins (MMP-10) hydrolyze collagens III, IV, and VI [12,14]. MMP activities are regulated by their specific TIMP [7]. *Research Center of Clinical and Experimental Medicine, Siberian Division of the Russian Academy of Medical Sciences, Novosibirsk; **Vector State Research Center of Virology and Biotechnology, Novosibirsk Region, Koltsovo; ***Novosibirsk State Medical University, Ministry of Health of the Russian Federation, Russia. Address for correspondence:
[email protected]. O. V. Potapova
We studied the expression of MMP and TIMP and their involvement in the regulation of collagens I, III, IV, and VI in the lungs of C57Bl/6 mice infected with influenza A/H5N1 A/goose/Krasnoozerskoye/627/05 virus (called influenza A/H5N1 virus below).
MATERIALS AND METHODS The study was carried out on 2-month-old male C57Bl/6 mice (n=60) from Breeding Center of Vector State Research Center of Virology and Biotechnology. Forty mice were infected intranasally with influenza A/H5N1 virus in a dose of 5 MLD50. Control group consisted of 20 intact mice. Inoculation and collection of the material were carried out at Vector Center (Koltsovo). The animals were kept under standard vivarium conditions with free access to water and food. Biological specimens for studies were collected in accordance with the International Regulations presented in the Helsinki Declaration. Lung specimens for histological and immunocytochemical (ICC) studies were collected on days 1,
0007-4888/13/15610011 © 2013 Springer Science+Business Media New York
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Bulletin of Experimental Biology and Medicine, Vol. 156, No. 1, November, 2013 GENERAL PATHOLOGY AND PATHOPHYSIOLOGY
3, 6, 10, and 14 after inoculation. The animals were sacrificed by cervical dislocation under ether narcosis. The specimens were fixed in 10% formalin solution in water, dehydrated in ascending alcohols and xylenes, and embedded in HISTAMIX paraffin (BioVitrum). For ICC studies by the indirect peroxidase method, the sections (3-4 μ) sliced on a Microm HM 355S rotation microtome (Thermo Scientific) were stained with primary specific antibodies: Collagen I (Sigma), Collagen III (BioGenex), Collagen IV (BioGenex), Collagen VI (Sigma), MMP-2 (Spring Bioscience), MMP-9 (Spring Bioscience), MMP-10 (Spring Bioscience), and TIMP-2 (Spring Bioscience). Lung sections for ICC studies were deparaffinized, rehydrated, and antigen retrieval was carried out in a microwave oven at 700 W. The sections were exposed with the primary antibodies according to the instructions, incubated with HRP conjugate and second antibodies (Spring Bioscience Detection System), processed in DAB substrate, and poststained with Mayer’s hematoxylin. Visualization was carried out under an AxioImager A1 microscope with an AxioCam MRc camera (Carl Zeiss). Morphometry was carried out in a closed test system of 100 points (3.52104 μ2) using instruments of
AxioVision software (rel. 4.8.2).. Numerical density (Nai) of fibroblasts and macrophages expressing MMP and TIMP and volume density (Vv) of collagens I, III, IV, and VI were evaluated. The means were estimated using Microsoft Office Excel 2007 and Statistica 6.0 software. The differences in the means were compared using Student’s t test. The differences were considered significant at p
