Shiro Mori,*t* Masato Nose,*. Masaaki ... Address correspondence to Dr. Shiro Mori, Department of Oral ...... Platt JL, Sibley RK, Michael AF: Interstitial nephritis.
American Journal of Pathology, Vol. 144, No. 6, june 1994 Copyright © Americant Society for Investigative Pathology
Interstitial Nephritis in Aleutian Mink Disease Possible Role of Cell-Mediated Immunity Against Virus-Infected Tubular Epithelial Cells
Shiro Mori,*t* Masato Nose,* Masaaki Miyazawa,* Masahisa Kyogoku,* James B. Wolfinbarger,: and Marshall E. Bloom* From the Department of Pathology,* Tohoku University School of Medicine, Sendai, Japan; Department of Oral and Maxillofacial Surgery fI,t Tohoku University School of Dentistry, Sendai, Japan; and the Laboratory of Persistent Viral Diseases,* National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana
Aleutian mink disease (AD) has been characterized by immune complex glomerulonephritis associated with persistent infection of Aleutian mink disease parvovirus (ADV). Histopathological examination of kidneys from ADV-infected mink in this study revealed that interstitial nephritis characterized by prominent damage ofrenal tubuli and lymphocyte infiltration was also common in AD along with glomerulonephritis. By using strand-specific in situ molecular hybridization technique, replication of ADV was observed in tubular epithelial ceUs, in addition to epithelial ceUs of Bowman's capsules and some glomerular cells of the infected mink Analysis of tubular lesions by a combination of immunohistochemistry and in situ hybridization revealed that the renal tubulipositivefor virion DNA or replicativeform DNA/mRNA of ADV were also positive for an activation marker ofimmunocompetent ceUls, which is shared by B lymphocytes and thymic epithelial ceUs. Infiltration of a subpopulation of Tlymphocytes around infected renal tubuli were observed but deposition of immune complexes in these tubular lesions was not demonstrable. ADV replication in epithelial ceUls of renal tubuli and cellmediated immune responses to the infected epithelial ceUs mayplay a role in thepathogenesis of interstitial nephritis in Aleutian mink disease. (Am J Pathol 1994, 144:1326-1333)
1326
Aleutian mink disease (AD) is caused by persistent infection of a nondefective parvovirus, Aleutian mink disease parvovirus (ADV), and is characterized by chronic viremia, extremely high levels of antiviral antibodies, polyclonal hypergammaglobulinemia, and plasmacytosis.1-3 It has been widely accepted that glomerulonephritis observed in AD is caused by the deposition of immune complexes (IC).45 Thus, AD has been studied as an animal model of IC-mediated glomerulonephritis. During the course of studies to identify cells supporting replication of ADV in adult mink tissues,6'7 we noticed the presence of another type of renal lesions that was histopathologically characterized by degeneration of tubular epithelial cells and infiltration of mononuclear cells into the surrounding interstitium. Henson et ale have described histopathological findings of the interstitial lesions in AD. However, pathogenetic relationship between the tubular damage, IC-mediated glomerular lesions and ADV replication has not been analyzed and no explanation for the mechanisms of tubular damage and interstitial nephritis have been provided. In this study we investigated the pathogenesis of interstitial nephritis in ADV-infected mink. Taking advantage of the highly sensitive, strand-specific in situ hybridization technique, 6,7 we examined possible roles of ADV replication in the kidney in the development of interstitial nephritis. We propose here that interstitial nephritis in ADV-infected mink may be caused by virus replication in epithelial cells of renal tubuli followed by cellular immunological reactions against the infected epithelial cells.
SM was supported by a NIH Visiting Fellow Award. Accepted for publication February 15, 1994. Address correspondence to Dr. Shiro Mori, Department of Oral and Maxillofacial Surgery II, Tohoku University School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980, Japan. Address reprint requests to Dr. Marshall E. Bloom, Laboratory of Persistent Viral Diseases, NIH, NIAID, Rocky Mountain Laboratories, 903 South 4th Street, Hamilton, MT 59840.
Virus-induced Interstitial Nephritis
1327 AJPJune 1994, Vol. 144, No. 6
Antibodies
Materials and Methods Animals, Virus, and Tissue Preparations
Rabbit antisera against ADV virion structural proteins (VP: p85 and p75), ADV major nonstructural protein (NS-1: p71), and mink immunoglobulins (Ig) have been described.6'11'12 Mouse monoclonal antibodies specific for a subpopulation of mink T lymphocytes (MTS-9.3) and an activation marker of mink B lymphocytes, which is also expressed by mink thymic epithelial cells, macrophages, and dendritic cells (MTB-5.6), have been described (Miyazawa M, Mori S, Spandrude GJ, Wolfinbarger JB, Bloom ME: Production and characterization of new monoclonal antibodies that distinguish subpopulations of mink lymphoid cells. Hybridoma 1994, 13: in press).
Nineteen adult sapphire mink (Aleutian genotype, a/a) and 13 adult pastel mink (non-Aleutian genotype, A/A) that were age-matched females from an ADVfree herd were housed as described.9 The a/a mink are more susceptible to fatal AD than A/A mink.10 Five sapphire mink and three pastel mink comprised uninfected negative control groups. Fourteen sapphire mink and 10 pastel mink were inoculated intraperitoneally with 107 50% lethal dose (LD50) of a standard ADV-Utah 1 inoculum9 and killed on a post inoculation day (PID) 10 or 60 as shown in Tables 1 to 4. Fresh tissue slices prepared from the kidney were immersed in O.C.T. compound (Miles Laboratories, Inc., Elkhart, IN) and quick-frozen in an acetone-dry ice mixture. Frozen tissue blocks were stored at -80 C until cryomicrotomy was performed. The rest of tissues were fixed with 10% formalin, embedded in paraffin, and used for standard light microscopical observation.
Immunohistochemical Staining Four-micron frozen sections were mounted on polyD-lysine-coated glass slides,12 air-dried at room temperature for 30 minutes and then fixed in cold acetone (4 C) for 10 minutes before another air drying. Sections were stained by the avidin-biotinperoxidase complex (ABC) immunoperoxidase technique as described previously.6'13 Specimens were counterstained with methyl green, but if the samples
Analysis of Infected Mink Sera by Counterimmunoelectrophoresis (CIEP) To examine anti-ADV antibody titers in mink sera, CIEP was performed as described previously.2 Table 1. Glomerular Lesions of the ADV-Infected Sapphire Mink
Animal Number
S-1 S-2 S-3 S-4 S-5 S-6 S-7 S-8 S-9 S-10 S-11 S-12 S-13 S-14 S-15 S-16 S-17 S-18 S-19
Anti-ADV
Pathological Findings of
PID*
Titert
Glomerulit
10 10 10 10 10 10 10 60 60 60 60 60 60 60
256 64 256 256 16 256 64 1024 4096 4096 4096 1024 4096 1024
Ig Deposits in Glomeruli§
Virion DNA Deposits in Glomeruli§
Virion DNA+ Cells in
Glomerulill
RF DNA/mRNA+ Cells in
Glomerulill
+
M+, L+ Mp 2+ Mp2+
M+, L+ Mp+, E Collapse Mp2 ,E,C
Mp2+
+
2+ 2+
PID: days after inoculation. t Anti-ADV antibody titers defined by counterimmunoelectrophoresis. t Key to pathological findings: M, mesangial expansion; L, lobulation; E, exudation; C, crescent; Mp, membranoproliferative glomerulonephritis; -, no pathological lesions; -, weak lesions; +, moderate lesions; and 2+, strong lesions. § -, No significant deposition; +, weak deposition; +, moderate deposition; and 2+, strong deposition. I A median section of the kidney was made and the positive cells in one quadrant of the cross-section were counted: +, 1 to 10; 2+, 11 to 20; 3+, 21 to 50; 4+, 51 to 100; and 5+,
