Sera were obtained from B6 mice, as well as from NZB/W, NZM2410, B6.Sle1, and B6.Sle1.Sle3 mice at 6–8 months of age. Tissues were prepared from groups of matched B6 and B6.TC mice of 4–6 months of age. Splenocytes were isolated from 6 BALB/c mice of 4 months of age and 13 NZB/W F1 female mice that were divided into 3 age groups: 6–10 weeks of age, 20–21 weeks of age, and 25–30 weeks of age. The treatment and care of animals were in accordance with the guidelines of the Office of Research, UTHSC, the University of Florida and the Norwegian Ethical and Welfare Board, and the study overall was approved by UTHSC Institutional Animal Care and Use Committee under the protocol #11-164.

For binding assays, we treated purified calf-thymus histones with recombinant PAD4 in vitro, as described previously (2, 3). To assess the extent of deimination, we analyzed the progress of the reaction by colorimetry of citrullines and testing the resulting dH by Western blot with an antibody to citrullinated histone H3 (Abcam, ab#5103). The results of this analysis are shown in Figure 1.

Flat bottom, 96 well microtiter plates (Immulon 4HBX; Thermo Electron Corp.) were coated overnight with 5  μg/ml of nH, poly l-lusine, bovine serum albumin (BSA) (Sigma), ovalbumin (OVA) (Sigma), protamine sulfate (Sigma), or dH, as previously described (25). Plates were washed three times with 0.05% Tween-20 in PBS and blocked with 2.5% BSA in 0.02% NaN₃ and PBS for 2 h. A 1:100 initial dilution of primary sera along with threefold serial dilutions in 1.6% Tween-20 and 1% BSA in PBS were incubated for 1 h in the plates. Then, serum dilutions were removed, and wells were washed with 0.1% Tween-20 in PBS. Alkaline phosphate-conjugated goat anti-mouse kappa (Southern Biotech) was added at 1:1,000 dilution in 1% BSA with 0.05% Tween-20 in PBS for 1 h. Phosphatase substrate (Sigma) was used to develop the ELISA, and OD values were read at 405 nm on a Multiscan Plus plate reader (Labsystems).

Serum antibodies against dsDNA were detected by ELISA exactly as described (26, 27). In short, calf-thymus dsDNA (10 μg/ml in PBS) was coated on microtiter plates (MaxiSorb; Nunc, Copenhagen, Denmark). Sera from mice were diluted twofold from 1:100 to 1:3,200 in PBS containing 0.02% Tween-20 and incubated in wells. ELISA readings were obtained with peroxidase-conjugated rabbit anti-mouse Fc-γ antibodies at 405 nm.

Seven-month-old B6.TC autoimmune female mice and age-matched control B6 IgH^a were dissected to recover a portion of spleen, bone marrow, kidney, and liver. Thymi from 4- to 6-month-old mice were similarly obtained. Tissue was cut, minced with scissors, and crushed between two sterile frosted glass slides. Dissociated tissues were washed in PBS (without Ca⁺⁺) and centrifuged at 5,000 × g for 5 min to pellet cells. Cell pellets were mixed with lysis buffer (65mM Tris pH 7.2, 2%SDS, 10% glycerol), containing protease inhibitors. To test for dH in tissue lysates, equal amounts of total protein were analyzed by Western blotting, as described below.

For Western blot analysis, proteins were resolved on 15% SDS-PAGE and transferred to nitrocellulose. Membranes were blocked in 5% BSA in 0.1% Tween-20 in TBS (TBST) overnight at 4°C. Subsequently, the membranes were incubated with sera at 1:500 dilution in TBS containing 2.5% BSA, 1% NP-40, and 0.1% SDS. After 2 h of incubation, membranes were washed with 1% NP-40 in TBS. Anti-mouse IgG–HRP was used for detection at 1:20,000 dilution in TBST for 1 h, and blots were developed using chemiluminescence (PerkinElmer).

Peptide inhibition assays included a preceding step, in which 3 µg of 20-mer peptides, both matching the amino terminus of H3 but either containing arginines or citrullines at positions 2, 8, and 17, were incubated with 1:300 dilutions of mouse sera for 1 h prior to use in binding to dH and nH on the membrane. Results of these Western blots were quantitated by infrared emission of secondary anti-mouse IgG antibodies (Odyssey). Separately, autoimmune sera were treated with DNase1 prior to Western blotting. Briefly, 400 μl of a 1:200 dilution of sera were incubated with 20 units of DNase1 (New England Biolabs) for 1 h at room temperature to limit the possibility that DNA–anti-DNA complexes present in sera contribute to the observed histone binding. Following this incubation, the sera were diluted 1:500 in Western blot binding buffer.

To probe for deiminated histone H3 (dH3) in B6.TC and B6 mice, equal amounts of tissue lysates were resolved on 12% SDS-PAGE and transferred to nitrocellulose. Membranes were blocked with 5% BSA in TBST for 30 min and incubated with anti-dH3 anti-citrullinated histone H3 rabbit antibodies (Abcam, ab#5103) overnight at 4°C. Membranes were washed and incubated with HRP-conjugated goat-anti-rabbit secondary IgG antibody for 1 h at room temperature, washed three times in TBST, and twice in TBS alone. The HRP activity was detected as above.

BALB/c mice were boosted twice with 100 µg of total histones (dH or nH) or 100 µg OVA, 14 and 2 days prior to the splenocyte proliferation assay. Ninety-six well tissue culture plates (Corning Incorporated) were filled with 100 µl aliquots of 100 µg/ml (or threefold serial dilutions) of dH, nH, or OVA in RPMI 1640 (Mediatech Inc.) supplemented with 10% FBS. Splenocytes were isolated and resuspended in RPMI with 10% FBS at 1 × 10⁶ cells/ml. One hundred microliters of cell suspension was added to each well, and plates were incubated at 37°C in 5% CO₂ for 72 to 96 h. Tritiated thymidine (1 μCi/well) was added for the last 17 h of incubation. Plates were harvested onto glass fiber filters, and thymidine incorporation was assessed by scintillation counting. Splenocyte proliferation assays were also performed using female NZB/W mice purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Splenocytes were collected from NZB/W mice of different ages. Following red blood cell lysis, the splenocytes were resuspended in DMEM-10 media with 10% FCS and 10,000 U/ml penicillin and 10 mg/ml streptomycin. One hundred thousand cells were incubated with dH or nH (20 μg/ml of protein) in triplicate wells. Tritiated thymidine incorporation (1 μCi/well) was measured after 20 h, 3 or 6 days by liquid scintillation, as described (28, 29).

To assess the production of anti-nH/dH autoantibodies in mice that spontaneously develop antinuclear autoantibodies, we tested sera from NZB/W and their recombinant inbred derivative strain NZM2410 for binding to nH and dH. In addition, we tested the contribution of lupus-predisposing genetic intervals Sle1 and Sle3 that were back-crossed from the NZM2410 onto the B6 background (B6.Sle1 and B6.Sle1.Sle3). Sle1 is a locus that breaks tolerance to chromatin, whereas Sle3 affects functions of myeloid cells (30). The parental strains, NZB and NZW, have distinct MHC, H-2^d and H-2^z, respectively. The lupus-predisposing H-2^z was maintained in the NZM2410 congenics, whereas the Sle1 and Sle3 congenics have the H-2^b from B6. Antibody binding to dH and nH was assessed by ELISA (Figure 2) and Western blot (Figure 3). As controls, sera from age- and sex-matched B6 mice were used.

By ELISA, NZB/W (Figure 2A), B6.Sle1 (Figure 2B), B6.Sle1.Sle3 (Figure 2C), and NZM2410 (Figure 2D) sera showed preference for nH over dH. This preferential binding was statistically significant for NZB/W, NZM2410, and B6.Sle1.Sle3, as assessed by paired, one-tailed T-test. Binding differed for different mice and dilutions but, in general, binding to nH was stronger than the binding to dH. We also confirmed the additive effect of Sle1 and Sle3 loci, as the B6.Sle1.Sle3 combination resulted in greater absorbance values relative to the B6.Sle1 mice. In parallel assays, sera from B6 mice (Figure 2E) showed no reactivity to histones. Both dH and nH were present in equivalent concentrations on the plates, as shown by the nearly identical binding of the LG2.2 monoclonal antibody (Figure 2F) whose epitope, the first 13 amino acid residues of histone H2B, is identical between nH and dH (31).

To examine the possibility that antibodies to dH arise first but are replaced by antibodies to nH, we collected mouse sera over time to identify mice during the conversion to anti-histone autoimmunity. In Figure 2G, we show that binding preference to nH over dH was present at an early time when anti-histone reactivity first appeared. This result indicates the two reactivities arise jointly, rather than in succession, as might be predicted by epitope spreading.

To dissect the preferential binding to nH, we used Western blotting. The stringency of binding was increased by including both SDS (0.05%) and NP-40 (0.5%) in the binding buffer. Indeed, under these conditions, the binding of serum antibodies from NZB/W, NZM2410 B6.Sle1, and B6.Sle1.Sle3 mice to dH was weaker relative to the binding to nH, such that many of the sera bound exclusively to nH (Figure 3A). A variety of binding patterns were observed, including exclusive binding to one or two core histones. Binding to dH3 was observed most often, whereas binding to deiminated H4 or H2A was rare. In addition, binding to a band with the mobility of the deiminated linker histone H1 was observed in several blots. Overall, binding was more biased toward nH over dH, although some IgG dH was also observed in individual NZM2410 and B6.Sle1.Sle3 mice. In no instance did binding to nH exceed binding to dH.

To examine the possibility that the binding to dH represented a truly separate population of antibodies, we conducted inhibition experiments using 20-mer peptides that matched the amino terminus of histone H3 and either contained arginines or citrullines at positions 2, 8, and 17 (Figure 3B). We observed that the arginine-containing peptide (orange bars) was a more effective inhibitor of binding to both nH and dH, relative to the citrulline-containing peptide (blue bars), suggesting that the antibody binding to either antigen reflects antibody specificity for nH and that the binding to dH likely represents cross-reactivity due to shared epitope structure.

We asked whether T cells from autoimmune mice also recognize dH by using NZB/W mice that spontaneously develop an autoimmune response against nuclear Ags, including DNA and histones (32). Splenocytes from NZB/W mice of different ages were tested for proliferation in the presence of dH or nH. At 6–10 weeks of age, prior to any measurable anti-DNA reactivity, the splenocytes did not proliferate in response to either form of histone (Figures 4A,B). At 20–21 weeks of age, anti-DNA autoantibodies could be detected in the sera of some NZB/W mice (indicated at the top of each panel), and splenocytes from these mice showed low levels of proliferation in response to nH and dH (Figures 4C,D). Thus, splenocyte responses to histones arose in parallel with, or slightly prior to, humoral responses to DNA.

Twenty-five-week-old NZB/W mice with established autoimmunity showed splenocyte proliferation in response to dH and nH (Figures 4E,F), suggesting the presence of histone-reactive T cells in the spleens of autoimmune mice. Although some mice showed a tendency to preferentially respond to dH, others preferred nH, as shown by data from two of the analyzed mice. Notably, preference could switch, depending on the length of stimulation (Figures 4E,F), suggesting the presence of a limited number of T cell clones with distinct specificities and growth characteristics. Because proliferation generally showed a bias for dH or nH rather than being equal, we infer that epitopes containing arginines or citrullines were both presented by the MHC and recognized by T cells in splenocytes.

To examine the ability of dH to drive a T cell response, we examined T cell proliferation in vitro. Splenocytes from BALB/c mice immunized with dH proliferated during incubation with dH (Figure 4G) to comparable extent as splenocytes from mice immunized with OVA and incubated with OVA (Figure 4I). By contrast, splenocytes from BALB/c mice immunized with nH showed no enhanced proliferation regardless of whether they were incubated with dH, nH, or media alone (Figure 4H).

To test for the presence of dH in vivo, we prepared tissue lysates of bone marrow, spleen, liver, kidney, and thymus from B6.TC mice and probed them with anti-dH by Western blot. The bone marrow lysates of autoimmune B6.TC mice and control mice at 7 months of age did not appreciably react with antibodies to dH3 (Figure 5A). By contrast, spleens of B6.TC mice had clearly increased levels of dH3 as compared to B6 controls or lysates of the Jurkat lymphoma cells (Figure 5B). These results provide a qualitative estimate rather than a precise measure of deimination. This is by necessity, as the cellular composition and disease process may affect histone deimination in a complex manner in a tissue such as the spleen. The overall amounts of histone H3 were similar in all samples, as indicated by the equivalent immunoreactivity of an anti-H3 antibody. Therefore, the spleens of autoimmune B6.TC mice contained increased quantities of dH3. In addition, the kidneys and the liver from individual autoimmune mice exhibited increased levels of dH3 (Figures 5C,D). Importantly, thymic extracts from 4- to 6-month-old B6 and B6.TC mice clearly showed constitutively elevated levels of dH (Figure 5E).