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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. REFERENCE
  9. Supporting Information

Objective

High mobility group box 1 (HMGB-1), a kind of proinflammatory mediator, is associated with inflammatory conditions and tissue damage. Previous studies have reported that circulating HMGB-1 levels in patients with active antineutrophil cytoplasmic autoantibody (ANCA)–associated vasculitis (AAV) were associated with renal manifestations and burdens of granulomatous inflammation. The current study aimed to investigate whether circulating HMGB-1 levels were associated with disease activity in AAV.

Methods

Plasma samples from 74 patients with AAV in active stage and 65 patients with AAV in remission were collected. The plasma levels of HMGB-1 were determined by enzyme-linked immunosorbent assay. Associations between plasma levels of HMGB-1 with clinical and pathologic parameters were analyzed.

Results

Plasma levels of HMGB-1 in active AAV patients were significantly higher than those in normal controls and AAV patients in remission (median 6.11 [interquartile range (IQR) 3.25–12.79] ng/ml versus median 1.12 [IQR 0.53–1.39] ng/ml, P< 0.001; median 6.11 [IQR 3.25–12.79] ng/ml versus median 3.04 [IQR 1.97–4.63] ng/ml, P < 0.001, respectively). Correlation analysis showed that plasma levels of HMGB-1 correlated with initial serum creatinine (r = 0.275, P = 0.018), estimated glomerular filtration rate (r = −0.277, P = 0.017), the Birmingham Vasculitis Activity Score (r = 0.308, P = 0.008), and C-reactive protein level (r = 0.309, P = 0.008). Among the patients with myeloperoxidase (MPO)–ANCA, those within the first quartile of plasma HMGB-1 levels had a significantly lower level of MPO-ANCA than those within the other 3 quartiles.

Conclusion

Circulating HMGB-1 levels might reflect the disease activity and renal involvement of AAV vasculitis.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. REFERENCE
  9. Supporting Information

Antineutrophil cytoplasmic autoantibody (ANCA)–associated vasculitis (AAV) is characterized by pauci-immune necrotizing inflammation of the small blood vessels. AAV comprises granulomatosis with polyangiitis (Wegener's) (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (Churg-Strauss). ANCAs are the serologic markers of the above small-vessel vasculitides. Proteinase 3 (PR3) and myeloperoxidase (MPO) are the 2 most important target antigens of ANCA in AAV ([1, 2]).

As an ancient nuclear protein, high mobility group box 1 (HMGB-1) exists within the nucleus ubiquitously, playing its nuclear role by stabilizing the structure of nucleosomes and inducing DNA bending ([3]). Later studies have demonstrated a novel role of HMGB-1 as a kind of proinflammatory mediator when placed extracellularly ([4]). Increased extracellular HMGB-1 has been confirmed to be associated with inflammatory conditions and tissue damage.

Besides the well investigated diseases such as sepsis and cancer, HMGB-1 might take part in the pathogenesis of some autoimmune diseases, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) ([5, 6]). The role of HMGB-1 in AAV is far from clear yet. Bruchfeld et al reported that HMGB-1 levels significantly increased in AAV with renal manifestations, irrespective of the underlying type of vasculitis ([7]), while Wibisono et al found that circulating levels of HMGB-1 were significantly higher in patients with GPA than MPA ([8]). Henes et al found the association between serum level of HMGB-1 and the burden of granulomatous inflammation in GPA ([9]). However, whether HMGB-1 as a kind of proinflammatory mediator is associated with disease activity of AAV is not known. In the current study, we measured circulating levels of HMGB-1 in AAV patients in both active stage and remission, and correlated circulating levels of HMGB-1 with clinical and pathologic parameters.

Box 1. Significance & Innovations

  • Circulating high mobility group box-1 (HMGB-1) level might reflect disease activity and renal involvement of antineutrophil cytoplasmic autoantibody (ANCA)–associated vasculitis (AAV).
  • The relatively large sample size of patients with followup data made it possible to investigate the association between HMGB-1 and clinicopathologic parameters.
  • This is the first report on HMGB-1 in AAV from China, where a majority of patients have a diagnosis of microscopic polyangiitis and most are myeloperoxidase-ANCA positive, which is different from white patients.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. REFERENCE
  9. Supporting Information

Patients and blood samples

Seventy-four patients with active AAV diagnosed at Peking University First Hospital from 2003 to 2011 were enrolled in this study. Plasma samples from these patients were collected on the day of renal biopsy and before the initiation of immunosuppressive treatment. Sixty-nine of the 74 patients received renal biopsy at diagnosis and before the initiation of immunosuppressive treatment. Plasma samples of 65 patients with AAV, who achieved complete remission after immunosuppressive therapy, were also collected at their regular ambulatory visits. “Remission” was defined as “absence of disease activity attributable to active disease qualified by the need for ongoing stable maintenance immunosuppressive therapy” (complete remission) or “50% reduction of disease activity score and absence of new manifestations” (partial remission) as described previously ([10]). The majority of these patients achieved remission 6 months after the initiation of immunosuppressive therapy. All these patients met the Chapel Hill Consensus Conference definition of AAV ([11]). Patients with secondary vasculitis or with any other coexisting renal disease, such as antiglomerular basement membrane nephritis, IgA nephropathy, diabetic nephropathy, lupus nephritis, or membranous nephropathy, were excluded. Disease activity was assessed in accordance with the Birmingham Vasculitis Activity Score (BVAS) ([12]). Among the AAV patients mentioned above, there were 49 patients who had plasma samples both in active stage and remission.

Plasma samples of 14 patients with SLE were collected as the disease control. All these patients fulfilled the 1997 American College of Rheumatology revised criteria for systemic lupus erythematosus ([13]). The mean ± SD Systemic Lupus Erythematosus Disease Activity Index ([14]) score was 15.5 ± 5.1 (range 9–25). Plasma samples of 14 age- and sex-matched healthy blood donors were collected as the normal control.

The blood samples of all the participants were drawn into EDTA tubes. The plasma was collected immediately after centrifugation at 2,000g for 15 minutes at 4°C. Then the plasma was stored in aliquots at −80°C until use. Repeated freeze/thaw cycles were avoided. The research was in compliance with the Declaration of Helsinki and was approved by the ethics committee of our hospital. Written informed consent was obtained from each participant.

Detection of ANCA

All the sera were tested for ANCAs at the time of presentation before immunosuppressive treatment was instituted. ANCA tests were performed by both indirect immunofluorescence (IIF) assay and antigen-specific enzyme-linked immunosorbent assay (ELISA). Standard IIF assays were performed according to the manufacturer (EUROIMMUN). In antigen-specific ELISAs, 2 highly purified known ANCA antigens, PR3 and MPO, purified as previously reported ([15]), were used as solid-phase ligands.

Measurement of plasma HMGB-1.

Plasma levels of HMGB-1 were tested using commercially available ELISA kits (Shino-TEST). The assay was conducted according to the manufacturer's instructions. In order to testify the validity of the ELISA method, i.e., whether ELISA could yield similar results to Western blot in AAV patients, plasma levels of HMGB-1 were also tested using Western blot as described previously ([16]). Furthermore, in order to exclude the potential interference of anti–HMGB-1 antibodies on the detection of HMGB-1 in AAV, circulating anti–HMGB-1 antibodies were tested (see Supplementary Appendix A, available in the online version of this article at http://onlinelibrary.wiley.com/doi/10.1002/acr.22187/abstract).

Renal histology

Renal histology of patients with AAV was evaluated according to the previous standardized protocol ([17-19]). The presence of glomerular lesions, including fibrinoid necrosis, crescents, and glomerulosclerosis, was calculated as the percentage of the total number of glomeruli in biopsy findings. Interstitial and tubular lesions were scored semiquantitatively on the basis of the percentage of the tubulointerstitial compartment that was affected per the following: interstitial infiltrate (“−” for 0%, “+” for 0–20%, “++” for 20–50%, and “+++” for >50%), interstitial fibrosis (“−” for 0%, “+” for 0–50%, and “++” for >50%), and tubular atrophy (“−” for 0%, “+” for 0–50%, and “++” for >50%).

Statistical analysis

Data were expressed as mean ± SD (for data that were normally distributed) or median and interquartile range (IQR; for data that were not normally distributed). Differences of quantitative parameters between groups were assessed using the t-test (for data that were normally distributed) or the nonparametric test (for data that were not normally distributed). Differences of nonindependent data were tested using the generalized estimating equations. Differences of not normally distributed paired samples were tested using Wilcoxon's signed rank test. Differences of semiquantitative results were tested using the Mann-Whitney U test. Differences of qualitative results were compared using the chi-square test. Pearson's test or Spearman's test was used for correlation analysis as appropriate. An analysis of covariance model was used for adjustment; the model regarded the concentration of HMGB-1 as a dependent variable, group as a factor, and serum creatinine as a covariate. Given that the results of circulating HMGB-1 were not normally distributed, by doing logarithmic transformation we converted the original data to its normal distribution representation for such adjustment. A P value less than 0.05 was considered to be statistically significant. All the statistics were analyzed using SPSS statistical software (version 17.0).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. REFERENCE
  9. Supporting Information

Demographic and general data

Among the 74 patients with AAV in active stage, 33 were male and 41 were female, with a mean ± SD age of 58.2 ± 14.2 years (range 14–82 years) at diagnosis. Sixty-seven of 74 patients were MPO-ANCA positive and the other 7 patients were PR3-ANCA positive. Fifty-two patients were diagnosed as MPA while the other 22 were diagnosed as GPA. The mean ± SD level of initial serum creatinine was 292.8 ± 170.7 μmoles/liter (range 69–915). The mean ± SD BVAS score was 19.3 ± 4.5 (range 11–30). In the renal specimens of the 69 patients, all had little or no staining for IgG, IgA, or IgM (≤1+ on a scale of 0–4+) by immunofluorescence microscopy. No electron dense deposit was detected by electron microscopy. The clinical and histopathologic data are listed in Table 1.

Table 1. Clinical and histopathologic data of patients with active AAV*
 Value
  1. Values are the number (percentage) unless indicated otherwise. Data were collected at presentation. AAV = antineutrophil cytoplasmic antibody–associated vasculitis; GFR = glomerular filtration rate; ENT = ear, nose, and throat; BVAS = Birmingham Vasculitis Activity Score.

General clinical data 
Subjects, no.74
Male/female, no.33/41
Age at disease onset, mean ± SD years58.2 ± 14.2
Serum creatinine, μmoles/liter 
Mean ± SD292.84 ± 170.71
Range69–915
Estimated GFR (ml/minute/1.73 m2) 
Mean ± SD27.80 ± 21.62
Range4.6–88.9
Renal insufficiency at diagnosis63 (85.1)
Urinary protein (gm/24 hours) 
Mean ± SD2.14 ± 1.60
Range0–7.35
Skin rash9 (12.2)
Arthralgia23 (31.1)
Muscle pain18 (24.3)
Pulmonary38 (51.4)
ENT29 (39.2)
Ophthalmic13 (17.6)
Gastrointestinal8 (10.8)
Nervous system7 (9.5)
BVAS, mean ± SD19.3 ± 4.5
Pathologic data 
Subjects, no.69
Glomerular lesions, mean ± SD 
Total crescents58.71 ± 28.44
Cellular crescents46.57 ± 26.97
Fibrous crescents11.31 ± 16.46
Tubulointerstitial lesions 
Interstitial infiltration (−/+/++/+++)2/15/34/18
Interstitial fibrosis (−/+/++)13/12/44
Tubular atrophy (−/+/++)8/16/45

Among the 65 patients in AAV remission, 30 were male and 35 were female. The mean ± SD level of serum creatinine at sampling was 187.8 ± 127.8 μmoles/liter. The BVAS scores were 0, 1, and 2 in 56, 6, and 3 patients at remission, respectively.

Plasma levels of HMGB-1.

HMGB-1 levels from the ELISA and Western blot correlated very well (r = 0.751, P < 0.001). Furthermore, circulating anti–HMGB-1 antibody levels (expressed by the optical density value) in active AAV patients were comparable to healthy controls and were significantly lower in active AAV patients than in active SLE patients (median 0.15 [IQR 0.06–0.57] versus median 0.20 [IQR 0.07–0.42]; P = 0.875 and median 0.15 [IQR 0.06–0.57] versus median 0.44 [IQR 0.15–1.17]; P < 0.001, respectively) (see Supplementary Figure 1, available in the online version of this article at http://onlinelibrary.wiley.com/doi/10.1002/acr.22187/abstract).

Plasma levels of HMGB-1 in active AAV patients were significantly higher than those in normal controls (median 6.11 [IQR 3.25–12.79] ng/ml versus median 1.12 [IQR 0.53–1.39] ng/ml; P < 0.001). There was no significant difference of plasma levels of HMGB-1 between active AAV patients and active SLE patients (median 6.11 [IQR 3.25–12.79] ng/ml versus median 7.01 [IQR 2.65–13.88] ng/ml; P = 0.973) (Figure 1). Among the patients with active AAV, plasma levels of HMGB-1 in PR3-ANCA–positive patients were significantly higher than those in MPO-ANCA–positive patients (median 16.36 [IQR 4.86–27.36] ng/ml versus median 5.96 [IQR 3.13–11.83] ng/ml; P = 0.042). In order to figure out whether lower HMGB-1 levels in MPO-ANCA–positive patients, as compared with those in PR3-ANCA–positive patients, were influenced by renal dysfunction, we used a covariance model for adjustment. After adjusting for serum creatinine values, the levels of plasma HMGB-1 in PR3-ANCA–positive patients were still significantly higher than those in MPO-ANCA patients (P = 0.003). There was no significant difference of plasma levels of HMGB-1 between patients with GPA and patients with MPA.

image

Figure 1. Plasma levels of high mobility group box 1 (HMGB1) in antineutrophil cytoplasmic autoantibody–associated vasculitis (AAV) patients in active stage and remission. SLE = systemic lupus erythematosus; HC = healthy controls.

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Compared with AAV patients in active stage, patients in remission had significantly lower levels of plasma HMGB-1 (median 3.04 [IQR 1.97–4.63] ng/ml versus median 6.11 [IQR 3.25–12.79] ng/ml; P < 0.001). After adjusting for serum creatinine values, the levels of plasma HMGB-1 in patients in active stage were still significantly higher than those at remission (P < 0.001) (Figure 1). The decrease in circulating HMGB-1 levels (from the active stage to remission) in patients receiving monthly intravenous cyclophosphamide and daily oral cyclophosphamide was comparable (median 0.43 [IQR 0.22–0.61] versus median 0.57 [IQR 0.4–0.78]; P = 0.685).

We then compared plasma levels of HMGB-1 in 49 AAV patients with plasma samples of both active stage and remission. The plasma levels of HMGB-1 were significantly lower in remission than those in active stage (median 6.38 [IQR 4.25–6.38] ng/ml versus median 3.27 [IQR 1.98–4.82] ng/ml; P < 0.001 by Wilcoxon's signed rank test), which was consistent with the above mentioned results. Forty-four out of these 49 patients had a decrease in plasma level of HMGB-1 in remission compared with those in active stage, whereas only 5 patients had a slight increase in plasma level of HMGB-1 in remission compared with that in active stage (Figure 2).

image

Figure 2. Changes of plasma high mobility group box 1 (HMGB1) levels in 49 antineutrophil cytoplasmic autoantibody–associated vasculitis patients with sequential plasma samples.

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In order to investigate circulating levels of HMGB-1 in those who did not achieve remission (before clinical remission was achieved) after the initiation of immunosuppressive therapy, we randomly selected 13 patients who received corticosteroids and monthly intravenous cyclophosphamide therapy, and measured circulating HMGB-1 levels in their sequential samples at followup (during the course of immunosuppressant therapy at the time points of the 2nd and 4th month after the initiation of immunosuppressive therapy). As shown in Figure 3, we can clearly see that the levels of circulating HMGB-1 decreased as immunosuppressive therapy progressed. Furthermore, among these samples, circulating HMGB-1 levels correlated with the BVAS during followup (r = 0.363, P = 0.011).

image

Figure 3. Changes of plasma high mobility group box 1 (HMGB1) levels in 13 antineutrophil cytoplasmic autoantibody–associated patients with sequential plasma samples at followup.

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Association between plasma HMGB-1 levels and clinicopathologic parameters of patients with active AAV

Among the 74 patients with AAV in active stage, correlation analysis showed that plasma levels of HMGB-1 correlated with initial serum creatinine (r = 0.275, P = 0.018), estimated glomerular filtration rate (GFR; r = −0.277, P = 0.017), BVAS (r = 0.308, P = 0.008), and C-reactive protein (CRP) level (r = 0.309, P = 0.008) (Figure 4). Since the majority of these patients were MPO-ANCA positive, we only investigated the association between HMGB-1 levels and ANCA levels in patients with MPO-ANCA. In this subgroup of patients, those within the first quartile of plasma HMGB-1 levels had significantly lower levels of MPO-ANCA than those within the other 3 quartiles (mean ± SD 98.75 ± 57.00 RU/ml versus 144.78 ± 53.83 RU/ml, P = 0.019; mean ± SD 98.75 ± 57.00 RU/ml versus 136.83 ± 52.60 RU/ml, P = 0.05; and mean ± SD 98.75 ± 57.00 RU/ml versus 142.64 ± 59.37 RU/ml, P = 0.034, respectively). No significant association between HMGB-1 levels and the pathology parameters of active nephritis in renal biopsies was found.

image

Figure 4. Plasma levels of high mobility group box 1 (HMGB1) correlated with A, serum creatinine (Scr), B, estimated glomerular filtration rate (eGFR), C, Birmingham Vasculitis Activity Score (BVAS), and D, C-reactive protein (CRP) level.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. REFERENCE
  9. Supporting Information

HMGB-1 is a 30 kd nonhistone, DNA-binding nuclear protein that functions in transcription when inside the nucleus and takes on proinflammatory properties when released extracellularly. The signal pathways of HMGB-1 involve a lot of dependent signaling molecules. Studies demonstrated that receptors for advanced glycation end products, Toll-like receptors 2, 4, and 9 may participate in HMGB-1 signaling ([20-22]). HMGB-1 was implicated in the pathogenesis of a broad spectrum of acute and chronic inflammatory conditions with elevated levels in sepsis, cancer, inflammatory bowel disease, RA, and in particular, SLE ([6, 23-27]). Abdulahad et al reported increased urinary levels of HMGB-1 in SLE patients, which might reflect both local renal inflammation as well as systemic inflammation ([28]). Urbonaviciute and Voll suggested that HMGB-1 could be a valuable biomarker for SLE disease activity as its probable involvement in the pathogenesis ([5]). The current study investigated the association between HMGB-1 levels and disease activity of AAV.

In this study, we found that plasma levels of HMGB-1 in active AAV patients were significantly higher than those in AAV patients in remission. The difference was still significant even after adjusting for the renal function. Further analysis showed that plasma levels of HMGB-1 correlated with CRP level, BVAS, and, to some extent, MPO-ANCA levels. These results indicated that HMGB-1 levels could reflect the disease activity of AAV. The underlying mechanism is not fully clear yet. HMGB-1 is an important proinflammatory mediator. Generally, stimulated neutrophils, macrophages, and monocytes could release HMGB-1, which acts as an inducer of macrophage activation including production of tumor necrosis factor α, interleukin 1 (IL-1), and other proinflammatory mediators, thus in turn regulates cytokine expression and promotes inflammatory cell recruitment ([29-31]). Recently, Th17 cells have been described as effector cells in AAV ([32]). Interestingly, increased Th17 activity and HMGB-1 may have a certain relationship. Shi et al found that in vitro stimulation of CD4+ T cells from patients with RA with increasing concentrations of recombinant HMGB-1 induced the production of IL-17 ([33]). Whether there are similar mechanisms in AAV requires further confirmation.

We found that HMGB-1 remained elevated in remission when compared with normal controls, which may be explained by the low-grade inflammatory activity or tissue damage despite clinical remission ([7]).

Bruchfeld et al demonstrated that HMGB-1 levels increased in AAV with renal involvement ([7]). A previous study from the same group found that in patients with chronic kidney disease, HMGB-1 levels correlated with GFR ([16]). In the current study, we demonstrated that in AAV patients, plasma levels of HMGB-1 correlated with serum creatinine, and estimated GFR, which was in line with and further extended the previous findings.

In conclusion, circulating levels of HMGB-1 might reflect disease activity and renal involvement of AAV. It might indicate a pathogenic role of HMGB-1 in AAV, which needs further study to confirm.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. REFERENCE
  9. Supporting Information

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be submitted for publication. Dr. Chen had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Wang, Gou, Chang, Yu, Zhao, Chen.

Acquisition of data. Wang, Gou, Chang, Yu, Zhao, Chen.

Analysis and interpretation of data. Wang, Gou, Chang, Yu, Zhao, Chen.

REFERENCE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. REFERENCE
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. REFERENCE
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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ACR_22187_sm_SupplAppA.doc45KSupplementary Data
ACR_22187_sm_SupplFig.tif7224KSupplementary Figure

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