Immune Reaction to Type XVII Collagen Induces Intramolecular and Intermolecular Epitope Spreading in Experimental Bullous Pemphigoid Models

Abstract

Bullous pemphigoid (BP), the most common autoimmune blistering disease, is induced by autoantibodies to type XVII collagen (COL17). Previous studies demonstrated that COL17 harbors several epitopes targeted by autoreactive T and B cells and that the target epitopes change sequentially during the disease course. To elucidate the details of the humoral immune response to COL17, we used an active BP mouse model in which BP is induced by the adoptive transfer of spleen cells from wild-type mice immunized with human COL17-expressing skin grafting to immunodeficient COL17-humanized (Rag-2-/-, mouse Col17-/-, human COL17+) mice. By immunoblot analysis, antibodies to the NC16A domain and other extracellular domains (ECDs) of COL17 were detected earlier than antibodies to intracellular domains (ICDs) in the active BP model. Time course analysis by enzyme-linked immunosorbent assay demonstrated a delayed peak of antibodies to ICD epitopes in active BP model. The blockade of CD40-CD40 ligand interaction soon after the adoptive transfer suppressed the production of antibodies to the non-collagenous 16A (NC16A) domain but not to an ICD epitope, suggesting the sequential activation from T and B cells against the ECD epitopes including the NC16A domain to those against ICD epitopes in vivo. Both wild-type mice immunized with a fragment of the NC16A domain and the recipients of those spleen cells produced IgG antibodies to ICD and ECD epitopes, showing intramolecular epitope spreading from the NC16A domain to other epitopes of COL17. Furthermore, we found that a portion of the active BP model mice show intermolecular epitope spreading from human COL17 to murine BP230. The appearance of antibodies to ICD epitopes of COL17 or of antibodies to murine BP230 did not correlate with the skin changes in the mice, suggesting that those antibodies have low pathogenicity. These results suggest that the immune response to the ECD epitopes of COL17, especially to the NC16A domain, triggers intramolecular, and intermolecular epitope spreading to ICD epitopes of COL17 and to murine BP230. These novel findings provide insight into the mechanism of epitope spreading in organ-specific, antibody-mediated autoimmune disorders.

Keywords: BP180; BP230; CD40 ligand; COL17; NC16A domain; active mouse model; autoantibody; autoimmunity.

Figures

Figure 1

Antibodies to the NC16A domain of COL17 decrease more rapidly than those to the dermal-epidermal junction (DEJ) in an active BP mouse model. (A) Schematic of the generation of an active BP mouse model. (B) Representative clinical presentations of the active BP model at 2 and 5 weeks after the adoptive transfer of immunized spleen cells and hematoxylin and eosin staining of the ear at 5 weeks (original magnification × 20). (C) Time course of disease severities for the Rag−/−/COL17-humanized recipients (n = 4). (D) Time course of titers of circulating IgG antibodies to the dermal-epidermal junction (DEJ) of the skin as determined by indirect IF using sera of an active BP model and normal human skin (n = 4). (E) Time course of the titers of circulating IgG antibodies to the NC16A domain as determined by ELISA (n = 4). Results are shown as mean ± SEM.

Figure 2

Antibodies to ICD epitopes of COL17 become detectable later than those to ECD epitopes by immunoblotting in an active BP model. (A) Schematic of human COL17 and recombinant proteins. TM, transmembrane domain; NH2, N-terminus; COOH, C-terminus; AA, amino acid. (B) Reactivity of sera from active BP model mice (n = 4, days 8 and 56) to fragments of COL17 as measured by immunoblotting. PC; positive control (BP patient serum), 8, day 8; 56, day 56; BP-1-4, active BP model mice #1-4, respectively; N1, negative control-1 (wild-type serum); N2, negative control-2 (normal human serum). Arrowheads indicate positive levels. (C) Relative intensities of reactivity as measured by immunoblotting. –, negative; ±, faint; +, weakly positive; ++, positive; +++, strongly positive.

Figure 3

The peak of antibodies to ICD-149 lags that of antibodies to the NC16A domain and ECD epitopes by ELISA in an active BP model. (A) Schematic of human COL17 and synthesized peptides. TM, transmembrane domain; NH2, N-terminus; COOH, C-terminus; AA, amino acid. (B) ELISA index of skin-grafted wild-type sera (day 35, n = 16) and untreated wild-type sera (n = 8) measured by ELISA. Sera were diluted to 1:100 for NC16A-R7 and 1:25 for ICD-149 and ECD-1330. (C) Time course of titers of circulating IgG antibodies to NC16A-R7 as determined by ELISA (n = 6). (D) Time course of titers of circulating IgG antibodies to ICD-149, ICD-320, ECD-917, ECD-1084 and ECD-1330 as determined by ELISA (n = 6, respectively). Results are shown as mean ± SEM.

Figure 4

Blockade of CD40–CD40 ligand interaction preferentially decreases antibodies to the NC16A domain but not to other epitopes of COL17. (A) Schematic of treatment of the active BP model with antibodies to murine CD40 ligand (CD40L). (B–E) Time course of titers of circulating IgG antibodies to NC16A-R7 (B), ICD-149 (C), ECD-917 (D), and ECD-1330 (E) in an anti-CD40L antibody-treated active BP model (n = 4) and in an untreated active BP model (n = 9) as determined by ELISA. Results are shown as mean ± SEM.

Figure 5

The immune response to the NC16A domain spreads to ICD and ECD epitopes of COL17 in vivo. (A) Schematic of the immunization of wild-type mice with NC16A-R7 and the adoptive transfer of spleen cells to make the active BP model. (B) Time course of titers of circulating IgG antibodies to NC16A-R7, ICD-149, and ECD-1330 in NC16A-R7-immunized wild-type mice (n = 5) as determined by ELISA. (C) Representative images of IgG deposition in indirect IF study using normal human skin (NHS) and wild-type mouse skin as substrates and 1:20 diluted sera from NC16A-R7-immunized wild-type mice at day 35 as primary antibodies. Arrowheads indicate IgG deposition around the basal keratinocytes. (D,E) Time course of titers of circulating IgG antibodies to NC16A-R7, ICD-149, and ECD-1330 in Rag−/−/COL17-humanized mice that received spleen cells from NC16A-R7-immunized mice (n = 8) (D) and in those that received spleen cells from untreated wild-type mice (n = 8) (E) as determined by ELISA. Results are shown as mean ± SEM.

Figure 6

Antibodies to murine BP230 are detected in a portion of the active BP model mice. (A) Representative images of IgG deposition in indirect IF study using normal human skin (NHS) and murine COL17−/− mouse skin as substrates and 1:20 diluted sera from an active BP model at day 35 and from wild-type mice as primary antibodies. Arrowheads indicate IgG deposition around the basal keratinocytes. (B) Schematic of murine BP230 and recombinant proteins. NH2, N-terminus; COOH, C-terminus; AA, amino acid; mBP230, murine BP230. (C) The sizes of recombinant proteins were confirmed by immunoblotting using anti-His-tag antibody. (D) Reactivity of sera from the active BP model (n = 8, days 21 and 84) to fragments of murine BP230 was measured by immunoblotting. NC, negative control (wild-type serum); PC, positive control (anti-His-tag antibody). Arrowheads indicate a positive level. Arrows indicate a positive band.

Figure 7

Epitope spreading in the skin-grafted mouse model and in the active BP model. A schematic of COL17 and BP230. In the skin-grafted mouse model, antibodies mainly react to the NC16A domain and weakly react to ECD epitopes of COL17. After the adoptive transfer of immunized spleen cells, intramolecular epitope spreading to ECD epitopes and to the outer portion of the ICD as well as intermolecular epitope spreading to BP230 occur, followed by intramolecular epitope spreading to the inner portion of the ICD. ES: epitope spreading.