MCMV induces neointima in IFN-γR-/- mice: Intimal cell apoptosis and persistent proliferation of myofibroblasts

MCMV strain Smith stock was purchased from American Type Tissue Collection (Rockville, MD) This virus stock had been propagated in SC-1 cells (mouse embryo fibroblast cell line). To enhance pathogenicity, the virus was passed three times in adult immunocompetent CD-1 mice, which were infected with MCMV by i.p. injection with 5 x 10⁵ plaque forming units (pfu). Two weeks after infection mice were sacrificed, salivary glands were collected, and virus stock was prepared as a 10% weight/volume homogenate. Concentration of virus in these homogenates was determined by a standard plaque assay on an infected 3T-12 fibroblast cell line. Final MCMV stock contained 4.2 x 10⁶ PFUs/ml of salivary gland homogenate. Control animals were infected with the same concentration of salivary gland homogenate obtained from control uninfected CD-1 mice.

Two months old mice were injected i.p. with either MCMV or control salivary gland homogenate. Starting at day 15 after infection and for as long as 6 months of age, mice were sacrificed on a biweekly basis by i.p. injection of sodium nembutal. Hearts and abdominal aortas were collected and histopathological and immunohistochemical analyses were performed. To both accelerate the development of vascular pathology and to deepen the immunocompromised state, these mice were irradiated with a sublethal dose of gamma radiation (300 Rads) at 4 months of age. Tissue sections were coded and scoring was performed in a blinded fashion.

H&E, Movat trichrome, and Verhoeff van Gieson, staining was performed on 4 μm sections of aortas fixed for 16 hours in neutral buffered formalin (10%), and embedded in paraffin.

Polyclonal antibodies specific for CD4, CD8, γ/δ-TCR, TGF-β₁, PDGF-A, PDGF-B, MCP-1 and polyclonal antibody to PCNA were purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, CA). Polyclonal antibody to B-lymphocyte marker (B-220) was purchased from Caltag Laboratories (Burlingame, CA). Monoclonal antibody specific for α-smooth muscle actin (clone 1A4) was purchased from Sigma (St. Louis, MO).

Tissues were collected, fixed in formalin, and embedded in paraffin. After deparaffinizing and two 5-minute washes in PBS, the sections were blocked for 20 minutes with a solution of 2% unconjugated goat anti-mouse IgG in 1 x PBS. Indicated dilutions of the primary antibodies were added to the slides and incubated for 2 hours at room temperature followed by two 5-minute washes with PBS. Biotinylated secondary IgG diluted 1:100 was added to the slides and incubated for 30 minutes. After two additional 5-minute PBS washes, the tissue sections were treated with a 3% solution of hydrogen peroxide to quench endogenous peroxidase activity. After two additional 5-minute washes, the slides were incubated for 30 minutes in the solution containing the avidin-biotin-peroxidase complex. Diaminobenzidine/H₂O₂ substrate was allowed to react with the peroxidase labeled tissue sections for 20 minutes. After two 1-minute rinses in distilled water, the tissue sections were counterstained with hematoxilin, cleared and dehydrated by successive gradations through 70%, 95%, and 100% ethanol followed by a final passage in xylene. The slides were mounted and analyzed by bright field microscopy.

Both immunocompetent 129S and immunodeficient IFN-γR-/- mice were infected by i.p. injection at age 2 months with either 1 x 10⁵ or 5 x 10⁴ pfus of MCMV stock. At 1 x 10⁵ pfus/mouse, 90% of IFN-γR-/- mice died with necrotizing hepatitis between days 5-10. Reducing the dose of MCMV to 5 x 10⁴ pfu resulted in lower mortality rates (30%). In the immunocompetent group no deaths occurred at either dose of MCMV. Surviving mice that received either dose of virus developed perivascular mononuclear cellular infiltrates in a number of organs including lungs, kidneys, and salivary glands. Vascular disease in the IFN-γR-/- group of mice began with development of an inflammatory infiltrate in the adventitia of aortas 3-4 weeks after infection in 30% of animals (Table 1) (Fig. 1A). This infiltrate was comprised predominantly of lymphohistiocytic cells (Fig. 1A). The vascular disease progressed over a time course of 4 months and affected between 50 and 65 % of animals (Table 1). In the group of IFN-γR-/- mice receiving sublethal (300 Rads) whole body irradiation 2 months after infection all examined animals exhibited vascular pathology (Table 1). Animals appeared healthy and all survived whole body irradiation. In the immunocompetent group of animals these infiltrates were cleared by 8 weeks after infection and from that point these animals were histologically indistinguishable from the uninfected group. On the contrary, in the IFN-γR-/- mice infected with MCMV, vascular disease progressed to the formation of neointima in aortas by 4 months of age, (Fig. 1B,C,D). Verhoeff-van Gieson staining indicated the focal destruction of aortic elastic fibrils in addition to intimal thickening (Fig. 1D) Controls in experiments using IFN-γR-/- mice included: 1) uninfected animals, 2) mice that were exposed only to the whole body irradiation and 3) mice that received only MCMV. Histopathological analysis of abdominal aortas collected from transgenic, infected and irradiated animals revealed severe vasculitis characterized by significant inflammatory infiltrate in the adventitia and media (Table 2), (Fig. 2). At the same time hearts showed no signs of disease (data not shown). Verhoeff-van Gieson (Fig. 2C) and Movat pentachrome (Fig. 2D) staining indicated the focal destruction of elastic tissue, as well as potential trafficking of the infiltrating cells through these breaks in the elastica. The extent of vascular damage in all mice from this group was similar. The most striking feature of the MCMV induced vascular disease was the formation of neointima in aortas of MCMV infected immunodeficient mice after irradiation (Table 2), (Fig 2). At the same time there was no vascular pathology in any of the control groups (Table 2) (Fig 2A). Verhoeff-van Gieson staining of aortas collected from IFN-γR-/- mice 30 days after irradiation (not shown) and IFN-γR-/- mice 60 days after irradiation (Fig. 2) indicated that the intimal vascular lesion induced by MCMV infection was characterized by collagen deposition and smooth muscle proliferation. We utilized monoclonal antibody specific for α-SMA, a marker for myofibroblast cells, and found that neointima seen in the aortas of MCMV infected experimental animals contained myofibroblasts. (Fig. 3).

Immunohistochemical characterization of the inflammatory infiltrate seen in the adventitia and media of the affected aortas revealed both helper T-lymphocytes (CD4⁺) (Fig. 4B) and cytotoxic T-lymphocytes (CD8⁺) (Fig. 4C) throughout the lesion. Since one of the characteristics of autoimmune vasculopathies is the appearance of high numbers of γ/δ-TCR [16] bearing cells, we studied this cell type in our model of vascular disease. Results from these experiments revealed a large number of γ/δ-TCR bearing lymphocytes throughout the lesion (Fig. 4D). We also investigated the presence of B-lymphocytes in the vascular lesion of MCMV infected mice. While T-lymphocytes were identified throughout the lesion these cells were mostly found in small follicles (Fig. 4A).

Growth factors possibly responsible for the neointimal proliferation were studied based on their putative importance in autoimmune vasculopathies in humans. Immunohistochemical analysis indicated a significant overproduction of TGF-β₁, PDGF-A, and PDGF-B in the MCMV induced intimal lesion (Fig. 5). While TGF-β 1 and PDGF-A immunoreactivity in affected arteries is localized to all three layers (intima, media, adventitia) (Fig. 5B and 5D), PDGF-B staining is localized to the media and neointima (Fig. 5F). MCP-1, a chemokine that has been implicated in pathogenesis of several autoimmune vascular diseases including SSc, was noted in the adventitia and media of affected arteries (Fig. 6). We did not detect MCP-1 positive staining in neointima (data not shown).

Further, we investigated the role of apoptosis and proliferation in vascular pathology triggered by MCMV infection of immunosuppressed mice. In situ TUNEL revealed positive staining in the intimal layer of affected aortas (Fig 7A). In addition, a small portion of infiltrating inflammatory cells stained positive by TUNEL (Fig. 7B). Smooth muscle/myofibroblast cells forming the neointima did not reveal any cells positive by TUNEL (Fig. 7A). Immunohistochemical staining for PCNA was used to identify cell proliferation. The majority of cells in the neointima (Fig. 7C and Fig. 8), as well as infiltrating inflammatory cells (Fig. 7D and FIG. 8), exhibit immunoreactivity for PCNA, indicating active proliferation.