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. 2011 Feb;22(2):336-48.
doi: 10.1681/ASN.2010010034. Epub 2011 Jan 13.

Myeloperoxidase-specific plasma cell depletion by bortezomib protects from anti-neutrophil cytoplasmic autoantibodies-induced glomerulonephritis

Julia Bontscho  1 , Adrian Schreiber, Rudolf A Manz, Wolfgang Schneider, Friedrich C Luft, Ralph Kettritz
Affiliations

Myeloperoxidase-specific plasma cell depletion by bortezomib protects from anti-neutrophil cytoplasmic autoantibodies-induced glomerulonephritis

Julia Bontscho et al. J Am Soc Nephrol. 2011 Feb.

Abstract

Anti-neutrophil cytoplasmic autoantibodies (ANCA) cause vasculitis and necrotizing crescentic glomerulonephritis (NCGN). Steroids and cytotoxic drugs reduce mortality but can cause significant adverse events. The proteasome inhibitor bortezomib (BTZ) prevents glomerulonephritis in mouse models of lupus but its efficacy in ANCA-associated glomerulonephritis is unknown. We induced anti-MPO IgG-mediated NCGN by transplanting wild-type bone marrow (BM) into irradiated MPO-deficient mice immunized with MPO. Four weeks after BM transplantation, we treated mice with steroid/cyclophosphamide (S/CYC) or BTZ. Compared with untreated control mice, both S/CYC and BTZ significantly reduced urine abnormalities, NCGN, and infiltration of neutrophils and macrophages. Response to BTZ depended on timing of administration: BTZ abrogated NCGN if begun 3 weeks, but not 5 weeks, after BM transplantation. BTZ treatment significantly reduced total and MPO-specific plasma cells in both the spleen and bone marrow, resulting in significantly reduced anti-MPO titers. Furthermore, BTZ affected neither B cells nor total CD4 and CD8 T cells, including their naive and effector subsets. In contrast, S/CYC reduced the total number of cells in the spleen, including total and MPO-specific plasma cells and B cells. In contrast to BTZ, S/CYC did not affect total and MPO-specific plasma cells in the bone marrow. Three of 23 BTZ-treated mice died within 36 hours after BTZ administration. In summary, BTZ depletes MPO-specific plasma cells, reduces anti-MPO titers, and prevents NCGN in mice.

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Figures

Figure 1.
Figure 1.
BTZ and S/CYC treatment prevents ANCA-induced necrotizing crescentic glomerulonephritis. Urine and renal histology in controls (black columns, CTR) or in mice treated with S/CYC (gray) and BTZ (white), respectively. Mice were sacrificed after 4 weeks of treatment. Panel (A) shows dipstick analysis and albuminuria ELISA. Panel (B) depicts renal tissue analysis; glomerular crescents and necrosis were expressed as the mean percentage of glomeruli with crescents and necrosis. Typical examples for each treatment group are depicted with ×20 magnification in the upper row and ×40 magnification in the lower row in panel (C). *P < 0.05.
Figure 2.
Figure 2.
BTZ and S/CYC treatments diminish glomerular PMN and macrophage influx. Panel (A) shows the percentage of glomeruli with PMN or macrophage infiltration and panel (B) the absolute number of these cells per glomerulus. Representative cross sections with GR-1 staining for PMN and CD68 staining for macrophages are depicted in panel (C). The groups consist of controls (CTR, black columns), S/CYC-treated mice (gray), and BTZ-treated mice (white). *P < 0.05.
Figure 3.
Figure 3.
BTZ reduces anti-MPO antibody titer. Results are shown in arbitrary units (405) nm. The anti-MPO titer was measured at randomization, after 1 week of treatment, and at sacrifice after 3 to 4 weeks of treatment. * indicates a significant difference compared with the control group at the indicated time point. # indicates a significant difference compared with the titer at randomization. P < 0.05.
Figure 4.
Figure 4.
BTZ does not reduce the number of total splenic plasma cells. Panel (A) shows the effect of S/CYC (gray columns) and BTZ (white) in comparison to untreated control mice (CTR, black) on the absolute number of splenic plasma cells analyzed by flow cytometry. Panel (B) depicts a typical experiment in a control mouse. The gated plasma cells express CD138 and cytoplasmic Ig κ light chains (cIg-κ). Panel (C) gives the absolute number of IgG and IgM secreting splenic plasma cells by ELISPOT with a typical example in panel (D). *P < 0.05.
Figure 5.
Figure 5.
BTZ and S/CYC do not reduce the number of BM plasma cells. Panel (A) shows the effect of S/CYC (gray columns) and BTZ (white) in comparison to control mice (CTR, black) on the absolute number of BM plasma cells analyzed by flow cytometry. Panel (B) depicts a typical experiment in a control mouse. The gated plasma cells express CD138 and cytoplasmic Ig κ light chains (cIg-κ). Panel (C) gives the absolute number of IgG and IgM secreting BM plasma cells by ELISPOT with a typical example in panel (D). *P < 0.05.
Figure 6.
Figure 6.
BTZ reduces MPO-specific plasma cells in spleen and BM. Panel (A) shows the effect of S/CYC (gray columns) and BTZ (white) in comparison to control mice (CTR, black) on the absolute number of MPO-specific splenic plasma cells. Panel (C) shows MPO-specific BM plasma cells. Panel (B) (splenic) and panel (D) (BM) depict typical experiments. No SAP means that the substrate streptavidin-AP was omitted. *P < 0.05.
Figure 7.
Figure 7.
BTZ does not affect the number of B cells in spleen and bone marrow. Panel (A) shows the effect of S/CYC (gray columns) and BTZ (white) in comparison to control mice (CTR, black) on the absolute number of splenic B220-positive B lymphocytes analyzed by flow cytometry. Panel (B) depicts a typical experiment in a control mouse. Panel (C) shows the effects on the absolute number of BM B lymphocytes with a typical example in panel (D). *P < 0.05.
Figure 8.
Figure 8.
Early but not late BTZ treatment protects from ANCA-induced NCGN. Urine dipstick analysis and albuminuria were measured at sacrifice (A) and renal tissue was examined (B). Black columns depict controls (CTR), white columns early BTZ, and dark gray columns late BTZ treatment. Glomerular crescents and necrosis were expressed as the mean percentage of glomeruli with crescents and necrosis. The treatment effect on circulating anti-MPO titers was assessed by ELISA. Results are shown in arbitrary units at outside diameter 405 nm. The anti-MPO titer was measured at random, 1 week after early BTZ treatment had started, and at sacrifice. * indicates a significant difference compared with the control group at the indicated time point. # indicates a significant difference compared with the titer at randomization. P < 0.05.
Figure 9.
Figure 9.
Early BTZ treatment has no effect on splenic T cells and reduces total and MPO-specific plasma cells. Panel (A) shows the effect of early BTZ treatment on the absolute number of splenic CD4 and CD8 T cells, panel (B) the effect on the CD4 and CD8 effector and naive subtypes, and panel (C) the effect of early BTZ treatment on the absolute number of splenic and BM total and MPO-specific plasma cells analyzed by flow cytometry and ELISPOT. White columns depicts early BTZ treatment and black columns the control group. *P < 0.05.
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