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. 2012 Nov 1;189(9):4405-16.
doi: 10.4049/jimmunol.1201433. Epub 2012 Sep 28.

Prolonged production of reactive oxygen species in response to B cell receptor stimulation promotes B cell activation and proliferation

Matthew L Wheeler  1 , Anthony L Defranco
Affiliations

Prolonged production of reactive oxygen species in response to B cell receptor stimulation promotes B cell activation and proliferation

Matthew L Wheeler et al. J Immunol. .

Abstract

We have investigated the intracellular sources and physiological function of reactive oxygen species (ROS) produced in primary B cells in response to BCR stimulation. BCR stimulation of primary resting murine B cells induced the rapid production of ROS that occurred within minutes and was maintained for at least 24 h after receptor stimulation. While the early production of ROS (0-2 h) was dependent on the Nox2 isoform of NADPH oxidase, at later stages of B cell activation (6-24 h) ROS were generated by a second pathway, which appeared to be dependent on mitochondrial respiration. B cells from mice deficient in the Nox2 NADPH oxidase complex lacked detectable early production of extracellular and intracellular ROS after BCR stimulation but had normal proximal BCR signaling and BCR-induced activation and proliferation in vitro and mounted normal or somewhat elevated Ab responses in vivo. In contrast, neutralizing both pathways of BCR-derived ROS with the scavenger N-acetylcysteine resulted in impaired in vitro BCR-induced activation and proliferation and attenuated BCR signaling through the PI3K pathway at later times. These results indicate that the production of ROS downstream of the BCR is derived from at least two distinct cellular sources and plays a critical role at the later stages of B cell activation by promoting sustained BCR signaling via the PI3K pathway, which is needed for effective B cell responses to Ag.

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Figures

Figure 1
Figure 1. BCR stimulation of primary mouse B cells induces the rapid production of reactive oxygen species
Purified mouse primary B cells (A-C) or total spleen cells (D), isolated from wild-type (A and C) or MD4/IgHEL (B) spleens, were stimulated for the indicated times with 0.5 μg/ml PMA, 25 μg/ml anti-IgM F(ab’)2, 1 μg/ml soluble HEL, or 20 μg/ml anti-IgM-DCFDA. A) O2− production was measured by cytochrome C reduction from WT B cells which were unstimulated (open circles), or stimulated with PMA (filled triangles) or with anti-IgM (filled squares). Relative O2− levels are shown as ΔOD 550nm-490nm at 2 min intervals over 1 h. B) O2− production by WT non-transgenic (filled squares) and WT/MD4 IgHEL B cells (filled triangles) stimulated with soluble HEL (solid symbols) or left unstimulated (open circles) was measured by cytochrome C reduction as in A. C) O2− production by WT B cells was detected using the chemiluminescent reagent Diogenes, and is shown as relative light units (RLU) following stimulation with PMA (triangles) or anti-IgM (squares) D) H2O2 production in the vicinity of the BCR was measured at the indicated time points by flow cytometry after activation with anti-IgM conjugated to DCFDA. H2O2 production was measured as fluorescence in the FITC channel at each time point, and comparing levels in control B220− (non-B cells) and B220+CD93− mature B cells. All data are representative of at least 2-3 independent experiments.
Figure 2
Figure 2. Early BCR-induced ROS production is dependent on B cell expression of the Nox2-containing NADPH oxidase
A.) Expression of NADPH oxidase catalytic subunit isoforms in purified splenic B cells was measured by semi-quantitative RT-PCR using primers specific for Nox1-4 and Duox1-2. Shown are relative amounts of each transcript compared to a positive control tissue for each isoform. Wedges indicate 5-fold serial dilutions of cDNA (starting with 50 ng cDNA). B.) (Top panel) O2− was measured as in Fig 1A by cytochrome C reduction from B cells isolated from spleens of WT (filled symbols) and Ncf1−/− (open symbols) mice stimulated with PMA (triangles) or anti-IgM (squares) for the indicated times. (Bottom panel) O2− was measured by cytochrome C reduction from B cells purified from WT/MD4 (filled triangles) or Ncf1−/−/MD4 (filled squares) spleens and stimulated with soluble HEL for the indicated times. Basal ROS levels (open circles; top and bottom panel) were measured from unstimulated WT non-transgenic (top), or WT/MD4 (bottom) B cells. C) H2O2 production within the vicinity of the BCR was measured by flow cytometry as in Fig 1C from WT and Ncf1−/− B cells stimulated with 20 μg/ml anti-IgM-DCFDA for the indicated times. Shown are relative H2O2 levels from mature B cells (B220+CD93−), represented as a histogram for anti-IgM-DCFDA fluorescence. D) Intracellular O2− production by WT and Ncf1−/− B cells was measured by flow cytometry from cells loaded with the O2− indicator dye Mitosox-Red and stimulated for the indicated times with 20 μg/ml anti-IgM. O2− production by mature splenic B cells (B220+CD93−) was measured as fluorescence in the PE channel for the indicated time points.
Figure 3
Figure 3. Ncf1-deficient mice have relatively normal B cell development and maturation
A) Representative flow cytometry plots showing gating strategy for bone marrow and spleen B cell sub-populations. Mature and immature B cells in the spleen and BM are distinguished by surface CD93, while follicular, T1, T2, T3, and Mz+B1 (spleen), or mature re-circulating, pro-pre-B, newly formed-T1, and newly formed-T2 (bone marrow) were distinguished by surface CD23 and IgM levels as indicated in representative plots. B-D) Frequencies of bone marrow (B) and spleen (C and D) B cell sub-populations are shown as percentages of B220+ cells from WT and Ncf1−/− mice with a diverse (B and C) or fixed (MD4) BCR repertoire (D). Data represent n = 6-12 mice per group and similar results were found in 2-3 independent experiments. (* p< 0.05, ** p< 0.01 Student’s T test)
Figure 4
Figure 4. Deficiency in Nox2 NADPH oxidase-dependent early ROS production does not alter proximal BCR signaling or downstream B cell activation and proliferation
A) Cytoplasmic free calcium was measured by indo-1 fluorescence emission ratio at 405 and 530 nm in WT (black lines) and Ncf1−/− (grey lines) follicular B cells (B220+, CD93−, CD23+, IgMint-lo) stimulated with 2 μg/ml (dashed) or 20 μg/ml (solid) anti-IgM. Data are representative of n= 3 mice each from 3 independent experiments. B) BCR-induced phosphorylation of proximal (Syk) and downstream (ERK and AKT) targets was measured by flow cytometry in WT (solid black) and Ncf1−/− (dashed black) follicular B cells (B220+, CD24−, CD23+, IgMint-lo) stimulated with 25 μg/ml anti-IgM, and stained intracellulary with phospho-specific antibodies against activating phosphorylation sites of Syk, ERK1/2, and AKT. Basal phosphorylation status is represented by intracellularly stained unstimulated WT B cells (filled grey). Data are representative of n = 2-3 mice each from 3 independent experiments C) Upregulation of activation markers CD69 and CD86 was measured by flow cytometry from WT (solid black) and Ncf1−/− (black dashed) purified splenic B cells stimulated for 6 h (CD69) or 18 h (CD86) with 10 μg/ml anti-IgM. Data are representative of n = 2-3 mice in 3 independent experiments. D) Proliferation of WT and Ncf1−/− B cells was measured by BrdU incorperation in vitro 48 h after stimulation with anti-IgM (10 μg/ml), CpG ODN (500 ng/ml) or anti-CD40 + IL-4 (10 μg/ml and 10 ng/ml). Proliferation was measured by intracellular staining with anti-BrdU and flow cytometry, following an 18 h pulse with BrdU. Left panel shows representative histograms of BrdU+ WT (top) and Ncf1−/− (bottom) unstimulated, and anti-IgM stimulated B cells. Data are quantified in right panel as the percentage of WT (white bars) and Ncf1−/− (black bars) that have incorperated BrdU. Data are representative of n = 3 mice/group from 2 independent experiments.
Figure 5
Figure 5. Deficiency in Nox2 NADPH oxidase-dependent ROS production does not alter T cell-dependent germinal center formation or B cell antigen presentation, but enhances T cell-dependent antibody production
A) NP-specific IgM and IgG1 serum antibody titers were measured by ELISA from day 7 and day 14 NP-CGG immunized WT (open circles) and Ncf1−/− (filled circles) mice. Each circle represents an individual mouse (n = 6) (* p< 0.05, ** p< 0.01). Similar results were found a second independent experiment. B) Germinal center formation in response to immunization with T cell-dependent antigen (50 μg NP-CGG in alum IP) in WT and Ncf1−/− mice. Total numbers of GC B cells (IgD−low, GL7+, FAS+) per spleen are shown from day 7 and day 14 NP-CGG immunized WT (white bars) and Ncf1−/− (black bars) mice. C) Numbers of anti-NP IgG producing plasma cells per spleen from day 14 NP-CGG immunized WT (white bars) and Ncf1−/− (black bars) mice. D) Antigen presentation to cognate T cells by WT and Ncf1−/− B cells was measured by co-culturing CFSE-labeled OT-II T cells with WT/MD4 (top panel) or Ncf1−/−/MD4 (bottom panel) B cells pulsed with increasing amounts of HEL-OVA. Presentation of Ova to cognate T cells was determined by measuring OT-II proliferation by CFSE dilution following 72 h of co-culture. Data are representative of n= 3 mice/group from 2 independent experiments.
Figure 6
Figure 6. BCR stimulation for 6 h or more leads to sustained production of ROS independently of Nox2 NADPH oxidase
A-B) Purified splenic B cells from WT and Ncf1−/− mice were stimulated with 10 μg/ml anti-IgM for the indicated times, and superoxide levels were measured with the ROS indicator dye Mitosox-Red loaded into cells 30 min prior to harvesting cells at each time point. Specificity of the dye for ROS levels was determined by comparing the median fluorescence intensity (MFI) of Mitosox-Red in WT and Ncf1−/− B cells stimulated in the presence or absence of 5 mM N-acetylcysteine. Histograms (A) represent mitochondrial ROS levels at 0 h (grey filled) and 6 h after anti-IgM stimulation of WT (left) or Ncf1−/− (right) B cells, in the presence (black dashed) or absence (black solid) of 5mM NAC. B) Quantification of Mitosox-Red fluorescence (represented as MFI) from WT (white and light grey bars) and Ncf1−/− (black and dark grey bars) B cells measured at the indicated times over 24 h of anti-IgM stimulation in the presence (light grey and dark grey bars) or absence (white and black bars) of NAC. Data are representative of n =3 mice/group from 3 independent experiments. C) mRNA Expression of NADPH oxidase isoforms was measured, as described in Fig 2A, from purified resting splenic B cells (0 h), or in purified B cells stimulated for 6 h or 12 h with 10 μg/ml anti-IgM. Data are representative of two independent experiments. D) Intracellular superoxide levels were measured as described above with Mitosox-Red from unstimulated (filled grey histogram) and anti-IgM-stimulated WT B cells (12 hrs). Cells were treated for the last 2 h with 20 μg/ml antimycin-A (dashed black) or vehicle control (solid black) to enhance mitochondrial ROS production. Data are representative of n = 2-3 mice/group and similar results were obtained in 2 independent experiments.
Figure 7
Figure 7. Prolonged ROS production is required for optimal B cell activation and proliferation and for sustained PI3K signaling in response to BCR stimulation
A) Upregulation of the early activation marker CD69 was measured by flow cytometry 7 h after stimulation of WT (left) or Ncf1−/− (right) B cells with 10 μg/ml anti-IgM in the presence (black dashed) or absence (solid black) of 5 mM NAC. Data are representative of n = 2-3 mice/ group. B) Proliferation of WT and Ncf1−/− B cells in the presence or absence of NAC was measured by BrdU incorperation as in Fig 3D. Data are represented as the total number of BrdU positive B cells 48 h after stimulation with anti-IgM (10 μg/ml), CpG ODN (500 ng/ml), or anti-CD40 (10 μg/ml) + IL-4 (10 ng/ml) in the presence or absence of 5 mM NAC (** p<0.01). Data from A and B are representative of B cells from n = 2-3 mice/ group, and similar results were found in 3 independent experiments. C) BCR-induced calcium mobilization was measured as in Fig 4A in WT (top) and Ncf1-/- (bottom) follicular B cells stimulated with 25 μg/ml anti-IgM in the presence (dashed black lines) or absence (solid black lines) of 5 mM NAC. D) BCR-induced phosphorylation of Syk and ERK were measured as in Fig 4B in WT (left panels) and Ncf1-/- (right panels) follicular B cells stimulated with 25 μg/ml anti-IgM in the presence (dashed black) or absence (solid black) of 5 mM NAC. Data are representative of 4 (C) and 3 (D) independent experiments E) Phospho-ERK and phospho-S6 levels were measured by intracellular staining and flow cytometry, as described in figure 4b, of purified WT B cells stimulated for 12 h with anti-IgM (10 μg/ml) in the presence (dashed black) or absence (solid black) of 5mM NAC. F) Quantification of phospho-S6 (represented as the percentage of p-S6+ cells) from purified WT B cells stimulated with anti-IgM, as in E, in the presence or absence of NAC for the entire stimulation (12 h), or incubated for the last 2 h with NAC or 1 μM of the class I PI3K inhibitor GDC-0941. Data represent n=2-3 mice/group, and are pooled from 2-4 independent experiments. (** p<0.01 Student’s T test)
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References

    1. Kurosaki T, Shinohara H, Baba Y. B Cell Signaling and Fate Decision. Annu. Rev. Immunol. 2010;28:21–55. - PubMed
    1. Kurosaki T, Hikida M. Tyrosine kinases and their substrates in B lymphocytes. Immunological reviews. 2009;228:132–148. - PubMed
    1. Saijo K, Schmedt C, Su I, Karasuyama H, Lowell CA, Reth M, Adachi T, Patke A, Santana A, Tarakhovsky A. Essential role of Src-family protein tyrosine kinases in NF-kappaB activation during B cell development. Nature Immunol. 2003;4:274–279. - PubMed
    1. Reth M, Brummer T. Feedback regulation of lymphocyte signalling. Nature reviews. Immunology. 2004;4:269–277. - PubMed
    1. Maeda A, Scharenberg AM, Tsukada S, Bolen JB, Kinet JP, Kurosaki T. Paired immunoglobulin-like receptor B (PIR-B) inhibits BCR-induced activation of Syk and Btk by SHP-1. Oncogene. 1999;18:2291–2297. - PubMed

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