Biology of Blood and Marrow Transplantation 6:513-522 (2000) © 2000 American Society for Blood and Marrow Transplantation ASBMT Prevention of Coronary Vascular Disease by Transplantation of T-Cell–Depleted Bone Marrow and Hematopoietic Stem Cell Preparation in Autoimmune-Prone W/BF1 Mice Rosemarie P. Kirzner,1 Robert W. Engelman,2 Hajime Mizutani,3 Steven Specter,1 Robert A. Good 2 1Department of Medical Microbiology and Immunology, 2Department of Pediatrics, All Children’s Hospital, University of South Florida, St. Petersburg, Florida; 3Second Department of Internal Medicine, Osaka University Medical School, Suita, Osaka, Japan Correspondence and reprint requests: Rosemarie P. Kirzner, PhD, Department of Pathology and Area Laboratory Services, Brooke Army Medical Center, 3851 Roger Brooke Drive, Fort Sam Houston, TX 78234-6200. (Received March 8, 2000; accepted May 9, 2000) ABSTRACT This project was designed to investigate the application of bone marrow transplantation to a progressive and ulti- ؋ mately fatal systemic autoimmune disease. Male (NZW BXSB)F1 (W/BF1) mice develop acute systemic autoim- mune disease characterized by degenerative coronary vascular disease (CVD) with myocardial infarctions, hyperten- sion, thrombocytopenia, glomerulonephritis, and persistently elevated levels of circulating immune complexes. After preliminary studies established the onset of disease between 10 and 12 weeks of age, 6- to 8-week-old male W/BF1 mice were targeted for transplantation with either T-cell–depleted bone marrow or purified hematopoietic stem cells from haploidentical B6C3/F1 mice. Posttransplantation flow cytometric analysis of splenocytes demon- strated donor phenotypes in W/BF1 recipient mice that had received T-cell–depleted marrow or hematopoietic stem cell preparations (lineage negative, CD71 negative) from B6C3/F1 donors. Survival of W/BF1 mice transplanted with bone marrow from normal B6C3/F1 donors was very high, and assessment at 100 days after transplantation revealed reduction in onset and severity of disease. Autoantibodies to cardiolipin and double-stranded DNA were markedly reduced to levels present in normal mice. Immunohistochemistry of heart and kidney tissue revealed significant amelioration of degenerative CVD and glomerulonephritis in the majority of W/BF1 recipients of marrow trans- plants from B6C3/F1 donors. All engrafted W/BF1 mice displayed normal immunologic competence 100 days posttransplantation. KEY WORDS Bone marrow transplantation • Autoimmune • Coronary vascular disease INTRODUCTION tered in other autoimmune mouse strains [1-4]. The pro- ϫ Male (NZW BXSB)F1 (W/BF1) mice develop acute duction of anti–double stranded DNA (anti-dsDNA), autoimmune disease similar to human systemic lupus ery- platelet-associated anticardiolipin (anti-CLP), and thematosus (SLE) and subsequently express degenerative antiplatelet autoantibodies accounts for persistently elevated coronary vascular disease (CVD) with myocardial infarc- levels of circulating immune complexes and deposition of tions (MIs) that occur at a 4-fold higher rate than is encoun- the complexes in the subendothelium of the coronary vascu- lature. The autoantibody titers increase with age and are directly correlated to the increased incidence of multiple This work was done during the tenure of a Graduate Student Fellowship of MIs and mortality in mice of this strain. the American Heart Association, Florida Affiliate, Inc. Funding for this pro- The pathology and progression of autoimmune disease ject was provided by the Suncoast Cardiovascular Research and Education have been well characterized in the W/BF1 mouse after Foundation, funded by Helen Harper Brown and the Aging Institute of the 12 weeks of age. Previous studies have examined the use of National Institutes of Health (R37 AGO5628-11). T-cell–depleted bone marrow (TCDM) to prevent CVD in BB&MT 513 R.P. Kirzner et al. this model [5]. Thus, the present study investigated whether W/BF1 mice were transplanted at 6 to 8 weeks of age, and stem cell–purified bone marrow (BM), in addition to TCDM, B6C3/F1 were transplanted at 12 to 14 weeks of age. could be used to fully reconstitute the immune system of male W/BF1 mice before the onset of progressive and ulti- Dimethyl Myleran Preparation mately fatal systemic autoimmune disease, and thereby also to Dimethyl myleran (DMM) (courtesy of C.D. Buckner, prevent CVD and the associated mortality of mice of this Seattle, WA), which provides myeloablation without strain. Haploidentical male B6C3/F1 mice served as normal, immunosuppression, was provided in 50-mg bottles. Five nonautoimmune donors. Preliminary studies established that milliliters of DMM was dissolved in 100% dimethyl sulfox- between the ages of 6 and 8 weeks, male W/BF1 mice are free ide (DMSO), divided into 1-mL aliquots, and stored at from autoimmune disease; onset of disease occurs between 10 –80°C. One-milliliter aliquots were dissolved in 9 mL warm and 12 weeks of age and progresses rapidly thereafter. (37°C) phosphate-buffered saline (PBS) for a final solution W/BF1 mice suffer from autoimmune-associated hyper- of 1 mg/mL DMM in PBS, 10% DMSO. The solution was tension that has been found to be unrelated to certain host divided into 3-mL aliquots and stored at –20°C until use. factors, such as hormones, diet, stress, or steroids [6,7]. Thrombocytopenia in these mice arises from the removal and Preparation of BMT Recipients destruction of antibody-bound platelets by the reticuloen- Transplant recipients were prepared 48 hours before dothelial system, particularly in the spleen and liver [8,9]. The transplantation by total body irradiation with 8.0 Gy from a production of autoantibodies to dsDNA, platelets, and CLP, 137Cs source. Twenty-four hours after total body irradiation, as well as other platelet-bound autoantibodies, accounts for mice were also given 0.2 mg DMM via the retroorbital persistently elevated levels of circulating immune complexes. plexus (approximately 10 mg/kg). All recipients were main- Deposition of these complexes in the subendothelium of the tained in a laminar containment facility and fed sterilized vasculature leads to occlusive vascular disease in the heart, food and acidified water. kidney, and other tissues [6,10,11]. In the kidneys, mesangial proliferation, as well as endothelial proliferation and some BM Harvesting vascular occlusion, leads to rapidly progressing renal malfunc- All BM samples were prepared on ice using sterile tech- tion. The autoantibody titers increase with age and are niques throughout. Donor mice were killed by CO2 inhala- directly correlated to the increased incidence of multiple MIs tion immediately before harvest. The tibias and femurs were and ultimately to early mortality [8,11-13]. Large major coro- placed into ice-cold RPMI 1640 medium supplemented nary arteries usually remain relatively undamaged, but death with 10% fetal bovine serum (FBS), 1% glutamine, 1% results from the cumulative effects of multiple small MIs. penicillin/streptomycin, and 1% HEPES buffer, and mar- Proliferative thickening of the intima and endothelium, in the row was flushed with ice-cold, supplemented RPMI. The absence of obvious inflammatory infiltrates, gives rise to BM suspension was repeatedly resuspended to obtain a fully occlusive thrombi in the intramyocardial arterioles and to the dispersed, single-cell suspension, strained through 70-µm subsequent necrosis [3,4,14]. nylon filters to remove debris, and kept on ice. Platelets from female W/BF1 or BALB/c donors are immediately cleared by circulating antiplatelet antibodies TCDM Preparation when these elements are transferred to male W/BF1 recipi- Whole bone marrow (WBM) was pelleted in a cen- ents. Allogeneic BM transplants from BALB/c donors to trifuge at 500g and 4°C for 5 minutes. The supernatant was adult W/BF1 mice ameliorated the progression of thrombo- removed, and aThy 1.2 antibody (PharMingen, San Diego, ϫ 6 cytopenia [15,16]. Likewise, transplantation of W/BF1 BM CA) was added to the WBM pellet at 1 µg per 5 10 cells into autoimmune-resistant mouse strains leads to the devel- and allowed to stand on ice for 30 minutes. Selected low- opment of thrombocytopenia and lupus nephritis. Immuno- toxicity rabbit complement (Cedarlane, Hornby, Ontario, suppression by treatment with cytoxan prevents SLE and Canada) was diluted to a final dilution of 1:20, and 1 mL per CVD. Multiple studies have provided evidence that the 5 ϫ 107 cells was added and allowed to incubate for 45 min- defect resulting in autoimmunity resides in the stem cell utes at 37°C to yield TCDM. After incubation, cells were compartment and that replacement of defective stem cells washed twice with supplemented RPMI and counted. via bone marrow transplantation (BMT) may be an effective means of preventing the development of autoimmune dis- Stem Cell Purification eases or even, in some instances, of curing these diseases Hematopoietic stem cells were purified on a discontinu- after they have been launched [6,9,17,18]. ous Percoll gradient (Pharmacia, Piscataway, NJ), as described below, at pH 7.0 and 2.300 mOsm/kg. Cell counts and viability were determined using a hemacytometer and MATERIALS AND METHODS trypan blue dye exclusion for WBM suspended in supple- Animals mented RPMI. The BM suspension was centrifuged for Four-week-old male W/BF1 and 10- to 12-week-old 5 minutes at 500g at 4°C in a 15-mL conical gradient tube. male B6C3/F1 or BALB/c or 20- to 24-week-old MRL/lpr The supernatant was discarded, and 3 mL of 100% lpr (MRL/L) mice were obtained from Jackson Laboratory (1.094 g/mL) Percoll per mouse was added to the pellet, (Bar Harbor, ME). All animals were maintained under spe- flushed to resuspend the cells, mixed using a vortex mixer, cific pathogen-free conditions in a clean, conventional envi- layered with the remaining Percoll, and placed on ice for ronment in an American Association for Accreditation of 10 minutes. Gradient tubes were then centrifuged at 2000g Laboratory Animal Care (AAALAC)-accredited facility. for 36 minutes at 4°C.