Unique B-1 cells specific for both N-pyrrolated proteins and DNA evolve with apolipoprotein E deficiency

peer reviewedLysine N-pyrrolation, a posttranslational modification, which converts lysine residues to N ε -pyrrole-L-lysine, imparts electronegative properties to proteins, causing them to mimic DNA. Apolipoprotein E (apoE) has been identified as a soluble receptor for pyrrolated proteins (pyrP), and accelerated lysine N-pyrrolation has been observed in apoE-deficient (apoE−/−) hyperlipidemic mice. However, the impact of pyrP accumulation consequent to apoE deficiency on the innate immune response remains unclear. Here, we investigated B-1a cells known to produce germline-encoded immunoglobulin M (IgM) from mice deficient in apoE and identified a particular cell population that specifically produces IgM antibodies against pyrP and DNA. We demonstrated an expansion of B-1a cells involved in IgM production in the peritoneal cavity of apoE−/− mice compared with wild-type mice, consistent with a progressive increase of IgM response in the mouse sera. We found that pyrP exhibited preferential binding to B-1a cells and facilitated the production of IgM. B cell receptor analysis of pyrP-specific B-1a cells showed restricted usage of gene segments selected from the germline gene set; most sequences contained high levels of non-templated-nucleotide additions (N-additions) that could contribute to junctional diversity of B cell receptors. Finally, we report that a subset of monoclonal IgM antibodies against pyrP/DNA established from the apoE−/− mice also contained abundant N-additions. These results suggest that the accumulation of pyrP due to apoE deficiency may influence clonal diversity in the pyrP-specific B cell repertoire. The discovery of these unique B-1a cells for pyrP/DNA provides a key link connecting covalent protein modification, lipoprotein metabolism, and innate immunity

Oxidative Deamination Activity of EGCG

(-)-Epigallocatechin-3-O-gallate (EGCG), the most abundant polyphenol in green tea, mediates the oxidative modification of proteins, generating protein carbonyls. However, the underlying molecular mechanism remains unclear. Here we analyzed the EGCG-derived intermediates generated upon incubation with the human serum albumin (HSA) and established that EGCG selectively oxidized the lysine residues via its oxidative deamination activity. In addition, we characterized the EGCG-oxidized proteins and discovered that the EGCG could be an endogenous source of the electrically-transformed proteins that could be recognized by the natural antibodies. When HSA was incubated with EGCG in the phosphate-buffered saline (pH 7.4) at 37°C, the protein carbonylation was associated with the formation of EGCG-derived products, such as the protein-bound EGCG, oxidized EGCG, and aminated EGCG. The aminated EGCG was also detected in the sera from the mice treated with EGCG in vivo. EGCG selectively oxidized lysine residues at the EGCG-binding domains in HSA to generate an oxidatively deaminated product, aminoadipic semialdehyde. In addition, EGCG treatment results in the increased negative charge of the protein due to the oxidative deamination of the lysine residues. More strikingly, the formation of protein carbonyls by EGCG markedly increased its cross-reactivity with the natural IgM antibodies. These findings suggest that many of the beneficial effects of EGCG may be partly attributed to its oxidative deamination activity, generating the oxidized proteins as a target of natural antibodies

Functional interaction between cyclooxygenase-2 and p53 in response to an endogenous electrophile

Cyclooxygenase-2 (Cox-2) is rapidly expressed by various stimuli and plays a key role in conversion of free arachidonic acid to prostaglandins. We have previously identified 4-hydroxy-2-nonenal (HNE), a lipid peroxidation-derived electrophile, as the potent Cox-2 inducer in rat epithelial RL34 cells and revealed that the HNE-induced Cox-2 expression resulted from the stabilization of Cox-2 mRNA that is mediated by the p38 mitogen-activated protein kinase signaling pathway. In the present study, we investigated an alternative regulatory mechanism of Cox-2 expression mediated by a transcription factor p53. In addition, to characterize the causal role for Cox-2, we examined the effects of Cox-2 overexpression in RL34 cells. To examine whether the HNE-induced Cox-2 expression was mechanistically linked to the p53 expression, we analyzed changes in Cox-2 and p53 expression levels in response to HNE and observed that the Cox-2 levels were inversely correlated with the p53 levels. Down-regulation of p53 followed by the activation of a transcription factor Sp1 was suggested to be involved in the HNE-induced Cox-2 gene expression. To characterize the effect of Cox-2 expression in the cells, we established the Cox-2-overexpressing derivatives of RL34 cells by stable transfection with Cox-2 cDNA. An oligonucleotide microarray analysis revealed a dramatic down-regulation of the proteasome subunit RC1 in the Cox-2 overexpressed cells compared to the empty-vector transfected control cells. Consistent with the Cox-2-mediated down-regulation of proteasome, a moderate reduction of the proteasome activities was observed. This proteasome dysfunction mediated by the Cox-2 overproduction was associated with the enhanced accumulation of p53 and ubiquitinated proteins, leading to the enhanced sensitivity toward electrophiles. These results suggest the existence of a causal link between Cox-2 and p53, which may represent a toxic mechanism of electrophilic lipid peroxidation products

The binding of the ONE-specific IgM mAbs to the apoptosis-induced cells evaluated using Annexin-V/PI.

<p>(<b>A</b>) Apoptosis-induced Jurkat cells were incubated with monoclonal antibodies and gated into populations according to the Annexin V and PI intensity; early apoptotic cells (single positive in Annexin-V but not PI) and late apoptotic and necrotic cells (double positive in Annexin-V/PI staining). Necrosis in Jurkat cells was induced by a freeze-thawing. (<b>B</b>) Bar graph representing relative fluorescence intensity of antibody binding to living cells, early apoptotic cells, late apoptotic cells, and necrotic cells. The non-immune control murine IgM and the ONE-specific IgM mAbs, 1F3, 3A8, and 3D10, isolated from the MFG-E8^−/− mice, were used. The means were tested for statistical significance by using Tukey’s HSD test, assuming equal variances. Statistically significant differences between the relative fluorescence intensity of living cells are indicated by asterisks (*, P<0.05; **, P<0.01). (<b>C</b>) Inhibition of Annexin-V binding to apoptotic Jurkat cells by the ONE-specific IgM mAbs. The apoptosis-induced Jurkat cells were incubated with the ONE-specific IgM mAbs (0, 50, and 100 µg/ml) and Annexin-V and the binding of Annexin V to the cells was analyzed by flow cytometry.</p

Presence of ONE-specific IgM antibodies in the MFG-E8^−/− mice.

<p>(<b>A</b>) Formation of ONE via 4-hydroperoxy-2-nonenal during the peroxidation of ω6 polyunsaturated fatty acids. (<b>B</b>) Separation of MFG-E8^+/+mice sera by gel filtration. The serum was eluted with PBS at the flow rate of 0.5 ml/min at room temperature with monitoring of the absorbance at 280 nm. The column system was composed of Hi Prep 16/60 Sephacryl S-300. The total IgM was determined by a direct antigen ELISA using the anti-IgM antibodies. The anti-ONE IgM titers were determined by a direct antigen ELISA using the ONE-modified BSA as the absorbed antigens. (<b>C</b>) Glomerular immunoglobulin deposition in 60-week-old female MFG-E8^+/+ (WT) and MFG-E8^−/− (KO) mice. Kidney sections were stained with Alexa Fluor 488-conjugated antibody against mouse IgG or IgM. <i>HE</i>: Hematoxylin and Eosin. (<b>D</b>) Cross-reactivity of antibodies eluted from the kidneys of MFG-E8^+/+ (WT) and MFG-E8^−/− (KO) mice. The IgM titer of the antibodies was determined by a direct antigen ELISA using native BSA, ONE-treated BSA, and DNA as the absorbed antigens. In panels B and C, the results are representative of three separate experiments with similar results. In panel D, the results represent the means ± SD of three separate experiments performed in duplicate determinations. There were no statistically significant differences between WT and KO groups.</p

Enhanced production of IgM antibodies in the MFG-E8^−/− mice.

<p>(<b>A</b>) Immunoblot analysis of the MFG-E8^+/+ (WT) and MFG-E8^−/− (KO) mice sera using the anti-mouse whole IgG Ab that cross-reacts not only with IgG, but also with IgM. <i>Left panel</i>, SDS-PAGE analysis. <i>Right panel</i>, immunoblot analysis. CBB, Coomassie Brilliant Blue. (<b>B</b>) Identification of the immunoglobulin class. The MFG-E8^+/+ and MFG-E8^−/− mice sera were analyzed by SDS-PAGE followed by an immunoblot analysis using the antibodies against individual immunoglobulins. (<b>C</b>) ELISA analysis of serum IgG and IgM levels in the MFG-E8^−/− mice compared to those in the wild-type mice. In panels A and B, the results are representative of at least three experiments with similar results. In panel C, the results represent the means ± SD of three separate experiments performed in duplicate determinations. Asterisks indicate a significant difference between WT and KO groups: **, P<0.01; ***, P<0.001. Differences were analyzed by the unpaired two-tailed Student’s t test.</p

Characterization of ONE-specific IgM mAbs.

<p>(<b>A</b>) Cross-reactivity of the ONE-specific IgM mAbs established from the MFG-E8^−/− mice. The IgM titer of the mAbs was determined by a direct antigen ELISA using dsDNA and native and aldehyde-modified BSA as the absorbed antigens. The results represent the means ± SD of three separate experiments performed in duplicate determinations. Asterisks indicate a significant difference between BSA and ONE-modified BSA groups: *, P<0.05; **, P<0.01; ***, P<0.001. Differences were analyzed by the unpaired two-tailed Student’s t test. (<b>B</b>) Sequence alignment of the hypervariable regions of ONE-specific IgM mAbs prepared from female MFG-E8^−/− mice. CDR1, CDR2, and CDR3 represent the Kabat definition complementarity-determining regions (CDRs). Hyphens (−) indicate sequence gaps, and the dots (•) indicate the sequence not determined. Sequences were aligned using the program CLUSTALW and were manually modified. Accession numbers for the sequences are as follows. Gen Bank: 9E10, CAN87019. Protein data bank: 26-2f, 1H0D; anti-Hiv-1, 1MF2. (<b>C</b>) V region gene use of ONE-specific IgM mAbs. All sequencing analyses were performed at least three times.</p

The ONE-specific IgMs promote the uptake of apoptotic cells by THP-1 monocytes.

<p>CellTracker Green-labeled Jurkat cells were induced to undergo late apoptotsis, followed by incubation with CellTrace Far Red-lableled THP-1 cells. After washing and fixation, the THP-1 cells were harvested by scraping with a rubber policeman. The percentage of double-positive macrophages was analyzed by flow cytometry. (<b>A</b>) Flow cytometric analysis of THP-1 cells engulfing apoptotic Jurkat cells in the presence and absence of IgM. The results are representative of three separate experiments with similar results. (<b>B</b>) Percentages of phagocytosis-positive THP-1 cells engulfing apoptotic Jurkat cells in the presence and absence of IgM. Data represented the mean ± SD of triplicate experiments. The means were tested for statistical significance by using Tukey’s HSD test, assuming equal variances. Statistically significant differences between the control and normal IgM values are indicated by asterisks (*, P<0.05; **, P<0.01).</p

Engulfment of apoptotic cells via MFG-E8.

<p>MFG-E8, secreted by activated macrophages and immature dendritic cells (<i>first step</i>), binds to apoptotic cells by recognizing phosphatidylserine (PS) (<i>second step</i>) which enhances the engulfment of apoptotic cells by macrophages (<i>third step</i>).</p