Induction of HLA-B27 heavy chain homodimer formation after activation in dendritic cells

Abstract

Introduction: Ankylosing spondylitis (AS) is a severe, chronic inflammatory arthritis, with a strong association to the human major histocompatibilty complex (MHC) class I allele human leucocyte antigen (HLA) B27. Disulfide-linked HLA-B27 heavy-chain homodimers have been implicated as novel structures involved in the aetiology of AS. We have studied the formation of HLA-B27 heavy-chain homodimers in human dendritic cells, which are key antigen-presenting cells and regulators of mammalian immune responses.

Method: Both an in vitro dendritic-like cell line and monocyte-derived dendritic cells from peripheral blood were studied. The KG-1 dendritic-like cell line was transfected with HLA-B27 cDNA constructs, and the cellular distribution, intracellular assembly and ability of HLA-B27 to form heavy-chain homodimers was compared with human monocyte-derived dendritic cells after stimulation with bacterial lipopolysaccharide (LPS).

Results: Immature KG-1 cells expressing HLA-B27 display an intracellular source of MHC class I heavy-chain homodimers partially overlapping with the Golgi bodies, but not the endoplasmic reticulum, which is lost at cell maturation with phorbyl-12-myristate-13-acetate (PMA) and ionomycin. Significantly, the formation of HLA-B27 homodimers in transfected KG-1 cells is induced by maturation, with a transient induction also seen in LPS-stimulated human monocyte-derived dendritic cells expressing HLA-B27. The weak association of wildtype HLA-B*2705 with the transporter associated with antigen processing could also be enhanced by mutation of residues at position 114 and 116 in the peptide-binding groove to those present in the HLA-B*2706 allele.

Conclusion: We have demonstrated that HLA-B27 heavy-chain homodimer formation can be induced by dendritic cell activation, implying that these novel structures may not be displayed to the immune system at all times. Our data suggests that the behaviour of HLA-B27 on dendritic cells may be important in the study of inflammatory arthritis.

Figure 1

Expression of HLA-B27 in KG-1 cells. Unstimulated KG-1 cells (solid line) and KG-1 cells stimulated with PMA and ionomycin for 24 hours (dashed line) or 72 hours (dotted line), were surface stained for (a) DC markers CD11c and (b) W6/32 for MHC class I. (c) KG-1 and (d) KG-1.B27sv5 cells, unstimulated (solid line) or stimulated with PMA and ionomycin for 24 hours (dashed line), were surface stained with ME1, recognising HLA-B alleles. The grey curves indicate second stage anti-mouse FITC alone. (e) KG-1 and KG-1.B27 cells were metabolically labelled with ³⁵S-Trans label, and cell lysates immunoprecipitated at the indicated time points with W6/32 or ME1 antibodies, and digested with endoH. (f) KG-1.B27 cells were differentiated with PMA and ionomycin for 24, 48 or 72 hours, and stained with ME1 (green) and the Golgi dye Bodipy Ceramide (red). The white bar represents 5 μm. A panel below the 24 hour image displays a red and green merged volume section, from the region indicated by the white line.

Figure 2

Association of HLA-B27 with TAP in KG-1 cells. KG-1.B27sv5 and KG-1.B27.H114D.D116Ysv5 cells were stimulated for 24 hours, lysed in digitonin-containing buffer and immunoprecipitated with anti-TAP1 antibodies conjugated to Protein G beads. Samples and cell lysate controls were resolved on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with the indicated antibodies. Molecular mass markers are shown in kDa.

Figure 3

Induction of HLA-B27 heavy-chain dimers in KG-1 cells and human dendritic cells. KG-1, KG-1.B27sv5 and KG-1.B27.H114D.D116Ysv5 cells were stimulated with PMA and ionomycin for the indicated times (a) before preparation of cell lysates or surface biotinylation and (b) pulldown with streptavidin-agarose beads. Immunoblots were probed with HC10 or anti-tag pK. (c) In 24-hour stimulated KG-1 and KG-1.B27sv5, cell lysates were resolved under non-reducing conditions by two-dimensional gel electrophoresis, followed by immunoblotting with HC10 or pK. MHC class I dimers and monomers are indicated. (d) In human monocyte-derived dendritic cells were generated from peripheral blood monocytes of two HLA-B27-positive individuals and two negative controls, before being stimulated for 0, 24, 48 or 72 hours with lipopolysaccharides. Cells lysates were resolved under non-reducing conditions and immunoblotted for MHC-class I heavy chain (HC-10). Class I dimers and monomers are indicated. Molecular weight markers are shown in kDa.