Anti-PR3 immune responses induce segmental and necrotizing glomerulonephritis

Abstract

Wegener's granulomatosis (WG) is a life-threatening autoimmune vasculitis that affects lungs, kidneys and other organs. A hallmark of WG is the presence of classic anti-neutrophil cytoplasmic antibodies (c-ANCA) against self-proteinase 3 (PR3). Little is known about the role of these antibodies and PR3-specific immune responses in disease development. In this study, we demonstrate that PR3-specific autoimmune responses are pathogenic in non-obese diabetic (NOD) mice with an impaired regulatory arm of the immune response. Immunization of autoimmunity prone NOD mice with rmPR3 (recombinant mouse PR3) in complete Freund's adjuvant (CFA) resulted in high levels of c-ANCA, without detectable disease development. However, when splenocytes from these immunized mice were transferred into immunodeficient NOD-severe combined immunodeficiency (SCID) mice, the recipient mice developed vasculitis and severe segmental and necrotizing glomerulonephritis. No disease developed in NOD-SCID mice that received splenocytes from the CFA-alone-immunized donors (controls), indicating that disease development depends upon PR3-specific immune responses. In contrast to the pathology observed in NOD-SCID mice, no disease was observed when splenocytes from rmPR3-immunized C57BL/6 mice were transferred into immunodeficient C57BL/6-RAG-1(-/-) mice, suggesting that complex and probably multi-genetic factors play a role in the regulation of disease development.

Fig. 1

Recombinant mouse proteinase 3 (rmPR3) expression using the baculovirus system. Insect cells were infected with PR3-baculovirus for 4 days and rmPR3 was purified by affinity chromatography from the media and cell lysate. (a) Silver staining of a 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) following electrophoresis of eluates. (b) Immunoblot staining with anti-FLAG antibodies (left), anti-D-20 antibodies (middle) and anti-P-20 antibodies (right). P-20 and D-20 antibodies did not cross-react with human PR3.

Fig. 2

Recombinant mouse proteinase 3 (rmPR3) has enzymatic activity. (a) Zymogram gel of purified rmPR3. Purified human neutrophil elastase (hLE) was used as a positive control. (b) Proteolytic activity expressed as optical density (OD) measured at 410 nm using a specific nitroanilide substrate at two concentrations of rmPR3. The proteolytic activity was inhibited by the addition of α-1-anti-trypsin at 0·06 mg/ml of rmPR3. (c) PR3 enzymatic activity, expressed as optical density (OD) in the presence of different concentrations of nitroanilide substrate. The results shown are representative of three independent experiments. Note that for kinetic purposes, the rates are calculated in seconds.

Fig. 3

Non-obese diabetic (NOD) mice immunized with recombinant mouse proteinase 3 (rmPR3) develop classic anti-neutrophil cytoplasmic antibodies (c-ANCA). (a) NOD mice were primed by immunization with rmPR3 in complete Freund's adjuvant (CFA) on day 0, followed by boosts on days 20 and 40 with rmPR3 in incomplete Freund's adjuvant (IFA) (). The titre of anti-mPR3 antibodies in sera was monitored using enzyme-linked immunosorbent assay (ELISA) at different time-points. Control mice received CFA and IFA without antigen (). Values shown are mean ± standard deviation (n = 4), P < 0·005. (b) Anti-PR3 ELISA was performed in the presence of increasing concentrations of FLAG-peptide (0–1 mg/ml). (c) Autoantibodies are present on peripheral neutrophils/monocytes in NOD mice immunized with rmPR3 (right panel) but not CFA-alone-immunized mice (left panel). Indirect immunofluorescence staining of neutrophils with serum from NOD mice immunized with rmPR3. (d) This staining resulted in the characteristic granular cytoplasmic staining pattern. (e) Indirect immunofluorescence staining of neutrophils with serum from CFA-alone-immunized NOD mice. Original magnification of both images (d,e) ×600.

Fig. 4

Non-obese diabetic–severe combined immunodeficiency (NOD–SCID) mice transferred adoptively with splenocytes from recombinant mouse proteinase 3 (rmPR3)-immunized mice develop anti-PR3 antibodies. The NOD–SCID mice transferred adoptively with splenocytes from rmPR3-immunized mice (n = 3) () and adjuvant-alone-immunized mice (n = 2) () were bled at different time-points and the anti-PR3 antibody titre was measured using enzyme-linked immunosorbent assay.

Fig. 5

Non-obese diabetic–severe combined immunodeficiency (NOD–SCID) mice transferred adoptively with splenocytes from recombinant mouse proteinase 3 (rmPR3)-immunized mice develop lethal kidney failure. (a) Survival curve of NOD–SCID mice after adoptive transfer of splenocytes from rmPR3-immunized or adjuvant-alone-immunized, control mice. (b) Blood urea nitrogen (BUN) and (c) creatinine serum levels in NOD–SCID mice 20 days after they received 8 × 10⁷ splenocytes from mice immunized with rmPR3 () or with adjuvant alone ().The normal range for BUN is 18–29 mg/dl and 0·2–0·8 mg/dl for creatinine in mouse serum.

Fig. 6

Kidney sections from adoptively transferred mice. All NOD–SCID mice that received splenocytes from recombinant mouse proteinase 3 (rmPR3)-immunized NOD mice developed kidney pathology: (a) small artery fibrinoid necrosis (arrow) and protein cast (arrow); (b) glomerular fibrinoid necrosis (arrow); (c) crescentic glomerulonephritis (arrow). (d) No pathology was observed in any NOD–SCID mice that received splenocytes from mice immunized with complete Freund's adjuvant (CFA) alone. Kidney sections from C57BL/6-RAG-1^–/– mice that received splenocytes from either (e) rmPR3-immunized mice or (f) adjuvant-alone-immunized mice reveal no pathology. Original magnification is ×400. (g) The anti-PR3 antibody titre was measured using enzyme-linked immunosorbent assay. The C57BL/6RAG1^–/– mice adoptively transferred with splenocytes from rmPR3-immunized mice () (n = 3) and adjuvant-alone-immunized mice () (n = 2) were bled at different time-points.