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. 2004 Jun 29;101(26):9751-6.
doi: 10.1073/pnas.0403620101. Epub 2004 Jun 21.

Targeted disruption of Nrf2 causes regenerative immune-mediated hemolytic anemia

Jong-Min Lee  1 , Kaimin Chan, Yuet Wai Kan, Jeffrey A Johnson
Affiliations

Targeted disruption of Nrf2 causes regenerative immune-mediated hemolytic anemia

Jong-Min Lee et al. Proc Natl Acad Sci U S A. .

Abstract

A basic leucine zipper transcription factor, NF-E2-related factor 2 (Nrf2), plays a critical role in the cellular defense mechanism by mediating a coordinate up-regulation of antioxidant responsive element-driven detoxification and antioxidant genes. Here, we report that targeted disruption of Nrf2 causes regenerative immune-mediated hemolytic anemia due to increased sequestration of damaged erythrocytes. Splenomegaly and spleen toxicity in Nrf2(-/-) mice raised a possibility of hemolytic anemia and splenic extramedullary hematopoiesis in Nrf2(-/-) mice. In support of this, hematology analysis revealed that Nrf2(-/-) mice suffer from anemia with abnormal red cell morphologies (i.e., Howell-Jolly bodies, acantocytes, and schistocytes). In addition, Nrf2(-/-) erythrocytes were more sensitive to H(2)O(2)-induced hemolysis, and erythrocyte-bound IgG levels were markedly increased in Nrf2(-/-) mice compared with Nrf2(+/+) mice. Because IgG bound to erythrocytes in the presence of oxidative damage in erythrocytes (regardless of Nrf2 genotype), these data support that Nrf2(-/-) erythrocytes have higher levels of damage compared with Nrf2(+/+) cells. Finally, Nrf2(-/-) mice showed increased levels of erythrocyte-bound IgG compared with Nrf2(+/+) mice after H(2)O(2) injection in vivo, suggesting that the decreased glutathione and increased H(2)O(2) render the Nrf2(-/-) mice more susceptible to toxicity. Taken together, these observations indicate that a chronic increase in oxidative stress due to decreased antioxidant capacity sensitizes erythrocytes and causes hemolytic anemia in Nrf2(-/-) mice, suggesting a pivotal role of Nrf2-antioxidant responsive element pathway in the cellular antioxidant defense system.

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Figures

Fig. 1.
Fig. 1.
Morphological changes in Nrf2-/- mice. Two distinctive morphological changes, such as white teeth (A) and splenomegaly (B), were observed in Nrf2-/- mice. (C) The organ to body-weight ratio data showed a dramatically increased spleen weight (14 mo, male, n = 7). S, spleen; K, kidney; L, liver; b, body. (D) Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining and (E) cyclooxygenase-2 immunostaining showed that Nrf2-/- spleen has more cell death and inflammation compared with Nrf2+/+ counterparts. (F) Primary splenocytes were incubated with H2O2 (24 h), and cell viability was measured by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (Promega)] cytotoxicity assay (n = 6). Each data bar or point represents mean ± SE. *, P < 0.05 by Student's t test.
Fig. 2.
Fig. 2.
Decreased expression levels of Nrf2-dependent ARE-driven genes in Nrf2-/- mice. Whole cell extracts (spleens) were used for Western blot analysis of HO-1 (A) and GCLC (B) and for NQO1 activity measurement (C) (n = 3). (D) RT-PCR analysis showed decreased levels of many ARE-driven genes in Nrf2-/- splenocyte culture. GCLM, glutamate-cysteine ligase modulatory subunit; FTL, ferritin light chain; FTH, ferritin heavy chain; TXNRD1, thioredoxin reductase-1; PRDX1, peroxiredoxin 1. PCR cycles are listed to the right of the gels.
Fig. 3.
Fig. 3.
Sensitized Nrf2-/- erythrocytes. Peripheral blood smear (A, Nrf2+/+; B, Nrf2-/-) showed that Nrf2-/- have morphological abnormalities such as Howell-Jolly bodies (C), schistocytes (D), and acantocytes (E), suggesting sensitized erythrocytes and hemolytic anemia in Nrf2-/- mice. For in vitro hemolysis, erythrocytes were incubated with H2O2 (37°C, 12 h). After centrifugation (600 × g, 10 min), Hb contents (F) and lactate dehydrogenase activity (G) in supernatant were measured as indicators of hemolysis (n = 4). *, P < 0.05 by Student's t test.
Fig. 4.
Fig. 4.
Immune-mediated hemolytic anemia resulting from oxidative damages. (A) Erythrocytes (≈5 × 106 cells) from Nrf2+/+ and Nrf2-/- mice were incubated (4°C, overnight) with FITC-conjugated anti-mouse IgG antibody. (B and C) After washing, fluorescent intensity was measured by flow cytometry. Each data bar represents mean ± SE (n = 5). (D) Erythrocytes (≈5 × 106 cells) from Nrf2+/+ (E, purple in G, and H) or Nrf2-/- mice (F, green in G, and I) were incubated (37°C, 1 h) with plasma (50 μl) from either Nrf2+/+ (green in H and in I) or Nrf2-/- mice (red in H and in I). (J) Nrf2+/+ erythrocytes were incubated (15°C, 10 h) with PBS (K)orH2O2 (L) to induce oxidative damage (L, echinocyte-like erythrocytes). After washing, Nrf2+/+ erythrocytes were incubated with plasma (50 μl) from either Nrf2+/+ (M, and purple in O), or Nrf2-/- mice (N, and green in O), and labeled with FITC-conjugated anti-mouse IgG antibody for flow cytometry analysis.
Fig. 5.
Fig. 5.
Sensitive Nrf2-/- erythrocytes due to decreased antioxidant potential and increased oxidative stress. (A) Peripheral blood was drawn from Nrf2+/+ (purple in D) and Nrf2-/- mice (green in D) before and after H2O2 injection (0.25 μmol/g body weight, tail vein, 1 h). (B) Erythrocytes (≈5 × 106 cells) were incubated with FITC-conjugated anti-mouse IgG antibody for flow cytometry analysis. (C) Fluorescence intensity of each mouse was corrected by subtracting fluorescence intensity of control erythrocytes (before H2O2 injection) from fluorescence intensity of H2O2-treated erythrocytes (n = 4). Nrf2-/- mice showed decreased total GSH (D) and increased H2O2 levels (E). Each data bar represents mean ± SE (n = 4). *, P < 0.05 by Student's t test.
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