Expression of the costimulatory molecule BB-1, the ligands CTLA-4 and CD28, and their mRNA in inflammatory myopathies

Abstract

To examine if the muscle fibers in patients with inflammatory myopathies have the potential to behave as antigen presenting cells (APCs), we investigated the expression of costimulatory molecules BB-1, B7-1 (CD80), and B7-2 (CD86), and their counterreceptors, CD28 or CTLA-4 (CD152), in the muscle biopsies of patients with polymyositis (PM), PM associated with human immunodeficiency virus infection (HIV-PM), sporadic inclusion body myositis (s-IBM), dermatomyositis (DM), and normal or disease controls. The expression of the B7 family of molecules on the muscle fibers was limited to BB-1. In PM, HIV-PM, and s-IBM, but not the disease controls, the nonnecrotic, MHC-class I-expressing muscle fibers, invaded or not by CD8+ T cells, had prominent membrane expression of BB-1. Several of the BB-1-positive fibers bound strongly in a cell-to-cell contact with their CD28 or CTLA-4 ligands on the autoinvasive CD8+ T cells, as confirmed by confocal microscopy. By reverse transcription-polymerase chain reaction, the expression of CD28 and CTLA-4 was up-regulated in PM, HIV-PM, and s-IBM, but not the controls. Because the BB-1-positive fibers expressed MHC-class I antigen and bound to up-regulated counterreceptors CD28 and CTLA-4 on the autoinvasive CD8+ T cells only in PM, HIV-PM, and s-IBM, the BB-1 molecule in these diseases should have a functional role in antigen presentation and T cell differentiation. These findings complement recent studies and suggest that in PM, HIV-PM, and s-IBM the muscle fibers are not only targets of CD8+ cytotoxic T cells but may also behave as "professional" APC.

Figures

Figure 1.

Transverse serial frozen sections of a muscle biopsy specimen from a patient with HIV-PM, stained with monoclonal antibodies against BB-1 (A), CD74 (B), and B7-1 (C). Note that BB-1 is expressed only on the muscle fibers but not on the surrounding lymphoid cells. Strong CD74 (B) and very weak B7-1 (C) expression is detected on the surface of some autoinvasive lymphocytes. Original magnification, ×400; scale bar: 50 μm.

Figure 2.

Transverse serial frozen sections of a muscle biopsy specimen from a patient with s-IBM, stained with Gomori trichrome (A) and with monoclonal antibodies against BB-1 (B), MHC-class I antigen (C), CD8 (D), and CD28 (E). The nonnecrotic (*) fibers (A) invaded by CD8+ T cells (D) express MHC-class I antigen (C) and BB-1 (B). Non-necrotic fibers, even if not in contact with the T cells (**), express BB-1 only on the cell membrane. The necrotic, degenerating, and small fibers express BB-1 throughout the cell. Many of the autoinvasive cells express the CD28 antigen (E). Original magnification, ×400: scale bar, 25 μm.

Figure 3.

Transverse serial frozen sections of a muscle biopsy specimen from a patient with HIV-PM, stained with Gomori trichrome (A) and with monoclonal antibody against BB-1 (B), MHC-class I antigen (C), CD8 (D), and CTLA-4 (E). The nonnecrotic fiber (*) invaded by CD8+ T cells (A and D), as well as healthy fibers remote from the inflammatory reaction express both MHC-class I antigen (C) and BB-1 (B). The pattern of BB-1 expression is similar to MHC-class I. Several of the autoinvasive T cells also express CTLA-4 (E). Original magnification, ×400: scale bar: 50 μm.

Figure 4.

Confocal microscopic images of a muscle biopsy from a patient with HIV-PM, dually immunostained for BB-1 (red) and CTLA-4 (green) (A), or for CD8 (red) and CTLA-4 (green) (B). A and B are serial sections. Areas with double staining are shown in yellow-orange due to the superimposition of green-to-red fluorescence. A: The CTLA-4 expression is restricted to the surface of the lymphocytes (green) invading or surrounding the BB-1-positive muscle fibers (red). A cell-to-cell contact between BB-1 and CTLA-4 is noted. No BB-1 staining is noted on the lymphocytes or CTLA-4 staining on the muscle fibers. B: Most of the CD8-positive autoinvasive cells express the CTLA-4 antigen, shown in yellow. Original magnification, ×800; scale bar, 10 μm.

Figure 5.

Transverse serial frozen sections of a muscle biopsy specimen from a patient with DMD immunostained with monoclonal antibodies against CD8 (A), CTLA-4 (B), and CD28 (C). Note that the autoinvasive CD8+ T cells are negative for CTLA-4 (B) and CD28 (C). Original magnification, ×400; scale bar, 50 μm.

Figure 6.

A: RT-PCR analysis of mRNA for CTLA-4 (316 bp), CD28 (238 bp), and control enzyme GAPDH (398 bp) in the muscle biopsies of patients with PM (lanes 1–3), HIV-PM (lanes 4–6) , and s-IBM (lanes 7–9), and controls (lanes 10–12). M indicates the size marker. Strong GAPDH and CTLA-4 mRNA expression and weak CD28 mRNA expression is seen in PM, HIV-PM, and s-IBM. Neither CTLA-4 nor CD28 mRNA expression was amplified in the normal control specimens. In s-IBM, only a very faintly amplified amount of the CD28 was observed. B: Semiquantitative expression of mRNA for CTLA-4 and CD28 in the studied muscle biopsy specimens. No apparent difference in the mean amount of CTLA-4 mRNA is noted in PM, HIV-PM, and s-IBM. The amount of amplified mRNA for CD28 in the muscles of s-IBM patients was very small.