IgG autoantibodies against L1 p40 are elevated in pSLE, compared to healthy children, JIA, and JDM
As most adult SLE patients have IgG autoantibodies that recognize ORF1p/p40 encoded by L1 that can be detected both by immunoblotting and ELISA [24], we used the latter assay to quantitate anti-p40 reactive IgG in pSLE patients. At the time of diagnosis and with active disease, the 30 pSLE patients had much higher anti-p40 reactivity than healthy controls, JIA, and JDM patients (p < 0.0001) (Fig. 1A). Using the 95th percentile of the healthy controls as a cutoff for a “normal” range, 26 (87%) of the pSLE patients were above this cutoff, compared to only 2 of the healthy children. JIA patients showed a slight increase with 12 (38%) of patients having anti-p40 titers above this cutoff, but the difference compared to healthy controls was statistically significant at p = 0.0145. Children with JDM had only slightly higher titers than those of healthy children.
Anti-p40 autoantibody levels are higher in patients with active disease
Anti-p40 reactivity was significantly (p = 0.0003 by Wilcoxon matched-pair signed rank test) reduced in the second plasma sample from the pSLE patients taken either after several months of therapy, to which many of them had responded well and lowered their SLEDAI score to ≤ 4 (Fig. 1A), or at a time of inactive disease before a subsequent flare. In 22 (73%) of the patients, the titers were reduced, in 4 they were essentially unchanged, and in 4 they were somewhat increased (Fig. 1B). Nevertheless, 19 (63%) of the patients still had anti-p40 titers above the 95th percentile of the healthy controls, a statistically significant difference (p < 0.0001) to this control group.
Anti-p40 autoantibodies of IgA class are also elevated, but not IgM or IgE
To quantitate autoantibodies of other classes than IgG, we used different Ig class-specific secondary antibodies in the ELISA and found that the pSLE patients with active disease had elevated IgA reactivity (p < 0.0001), as did the patients with inactive diseases (p = 0.0032; Fig. 1C) compared to healthy children. In 25 patients, the titers were lower when their disease was inactive, while it was unchanged in 2 and somewhat increased in 3 (Fig. 1D). These differences were statistically significant (p = 0.0002 by Wilcoxon matched-pair signed rank test). In contrast, recognition of p40 by IgM antibodies was similar between the groups and IgE were generally very low (data not shown).
Anti-p40 autoantibodies correlate with disease activity and complement consumption
The IgG autoantibody titers also correlated with the SLE disease activity index (SLEDAI; r = 0.65, p = 0.0001) (Fig. 2A), erythrocyte sedimentation rate (ESR; r = 0.43, p = 0.02) (Fig. 2B), complement C3 and C4 consumption (r = − 0.55, p = 0.002 and r = − 0.51, p = 0.006, respectively) (Fig. 2C, D), and anti-dsDNA antibodies (r = 0.49, p = 0.03) (Fig. 2E). Collectively, these data indicate that higher anti-p40 IgG levels tend to accompany active disease. Anti-p40 IgA levels correlated positively with ESR (r = 0.445, p = 0.026), but did not correlate in a statistically significant manner with other measures of disease activity.
Detection of p40 in patient immune cells
We first searched for detectable p40 in leukocyte linages in the PBMC from 5 patients with pSLE and found that all of them had detectable p40 in a subset that varied from 2.4 to 44.2% (mean 16.1 ± 17.6%; standard deviation, SD) of their CD66b+ granulocytes, while much smaller fractions of their CD19+ B cells (0–5.2%) or CD14+ monocytes (0–19%) were positive compared to control antibody-stained cells analyzed by flow cytometry (Fig. 3A; gating strategy and representative results are shown in supplemental Fig. S1). To better explore this finding, we recruited 10 adult SLE patients and 5 healthy adult controls and analyzed their PMN and PBMC fractions by flow cytometry using more lineage markers. These experiments revealed that p40 was detectable in 12.2 ± 16.6% (range 0–56 %) of CD66b + cells in the PMN fractions (Fig. 3B). These values are statistically different from those in T cells (p = 0.007) and B cells (p = 0.015). In the lower-density PBMC fraction, a similar portion (14.1 ± 13.3%, range 0–40.9%) of the CD66b + cells were also positive for p40 (Fig. 3B), a statistically significant difference from the T cells (p = 0.016). Only two patients had detectable p40 in their B cells and none in their T cells, while the CD14+ cells in the PBMC fraction, which include monocytes and granulocytes, were positive in two patients (43.4% and 16.7%, respectively). In healthy controls, all leukocyte lineages were negative for p40 (Fig. 3C). These data indicate that neutrophils, particularly considering that they constitute approximately half of all immune cells in circulation, are the predominant cells expressing p40 protein in SLE patients, but that the number of positive cells varied broadly. Patients with the highest SLEDAI scores at the time of blood draw correlated with the highest portion of p40-positive neutrophils in the PMN (r = 0.669, p = 0.021) (Fig. 3D). A trend towards a similar correlation was seen between SLEDAI and the portion of p40-positive CD66b+ cells in the PBMC fractions (Fig. 3E), but this trends did not meet statistical significance.
The median fluorescence intensity of CD66b staining of neutrophils with detectable p40 was somewhat higher (p = 0.02) than of neutrophils without p40 in 7 of the p40-expressing patients (Fig. 3F), suggesting that the presence of p40 may be associated with a more activated neutrophil phenotype. As an independent validation, immunoblotting of cell lysates showed that the PMN fraction from SLE patients had more p40 than the PBMC fraction, while healthy controls were negative (Fig. 3G).
Markers of neutrophil activation and NETosis are elevated in pSLE patients
Because p40 appears to be expressed predominantly in neutrophils in both the pediatric and adult SLE patients, and perhaps more so in activated neutrophils, we measured markers for neutrophil activation and NETosis, which have been developed in our lab [26,27,28,29]. Myeloperoxidase (MPO) and/or neutrophil elastase (NE) in complex with free DNA in plasma reflect death of neutrophils, including (and perhaps mostly) by NETosis [25]. A second marker, levels of the S100A8/A9 proteins (also known as calprotectin), which reflects neutrophil activation was also measured. As shown in Fig. 4A–C, these markers were all elevated in the plasma of the 30 pSLE patients with active disease, as well as to a lower extent in samples taken from the patients during inactive disease. The levels of MPO-DNA and NE-DNA complexes correlated with each other both during active disease (r = 0.47, p = 0.018) (Fig. 4D) and during inactive disease (r = 0.52, p = 0.008) when the values were lower (not shown).
Anti-p40 autoantibodies correlate with markers of neutrophil death and activation
In pSLE patients with active disease, the titers of anti-p40 autoantibodies of IgG class correlated with MPO-DNA complexes (r = 0.412, p = 0.041) (Fig. 5A) and S100A8/A9 (r = 0.489, p = 0.013) (Fig. 5B). However, in inactive disease, only MPO-DNA complexes, but not S100A8/A9, correlated with anti-p40 IgG autoantibodies (Fig. 5C, D).
Anti-p40 IgA autoantibodies only correlated statistically significantly with MPO-DNA complexes (r = 0.15, p = 0.022) in active SLE (Fig. 6A). In healthy controls, all neutrophil markers and autoantibodies were low and did not correlate with each other in a statistically significant manner (Fig. 5E, F; Fig. 6E, F).