High mobility group box 1 (HMGB1) and anti-HMGB1 antibodies and their relation to disease characteristics in systemic lupus erythematosus

Introduction: High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein. HMGB1, which is secreted by inflammatory cells and passively released from apoptotic and necrotic cells, may act as a pro-inflammatory mediator. As apoptotic cells accumulate in systemic lupus erythematosus (SLE), HMGB1 levels might be increased in SLE. HMGB1 may also serve as an autoantigen, leading to the production of anti-HMGB1 antibodies. In this study we determined levels of HMGB1 and anti-HMGB1 in SLE patients in comparison to healthy controls (HC) and analysed their relation with disease activity. Methods: The study population consisted of 70 SLE patients and 35 age-and sex-matched HC. Thirty-three SLE patients had quiescent disease, the other 37 patients were selected for having active disease. Nineteen of these had lupus nephritis. HMGB1 levels were measured with both Western blot and ELISA. Anti-HMGB1 levels were measured by ELISA. Clinical and serological parameters were assessed according to routine procedures. Results: HMGB1 levels in SLE patients could be measured reliably by Western blotting only, and were significantly increased compared to HC. During active disease HMGB1 levels increased, in particular in patients with renal involvement. Serum HMGB1 levels correlated with SLEDAI, proteinuria, and anti-dsDNA levels, and showed a negative correlation with complement C3. Anti-HMGB1 levels were significantly increased in SLE patients compared to HC, and positively correlated with HMGB1 levels. Conclusions: Levels of HMGB1 in the sera of SLE patients, in particular in those with active renal disease, are increased. Serum HMGB1 levels are related to SLEDAI scores and proteinuria, as well as to levels of anti-HMGB1 antibodies. These findings suggest that besides HMGB1, HMGB1-anti-HMGB1 immune complexes play a role in the pathogenesis of SLE, in particular in patients with renal involvement

Fluorescent Cell Barcoding as a Tool to Assess the Age-Related Development of Intracellular Cytokine Production in Small Amounts of Blood from Infants

Fluorescent Cell Barcoding (FCB) is a flow cytometric technique which has been used for assessing signaling proteins. This FCB technique has the potential to be applied in other multiparameter analyses. Since data on antigen (Ag)-specific T-cell immune responses, like intracellular cytokine production, are still lacking in infants because limited blood volumes can be obtained for analysis, the FCB technique could be very useful for this purpose. The objectives of this study were to modify the FCB method to be able to measure multiple Ag-specific cytokine reponses in T-cells upon simultaneous stimulation by various antigens and mitogens in small amounts of blood and to investigate the cytokine pattern of T-cell subsets in healthy infants aged six and twelve months. Blood samples, collected from 20 healthy infants aged six and twelve months, were stimulated in vitro with the antigens: phorbol-myristate-acetate (PMA), purified-protein-derivative (PPD), Tetanus-toxoid (TT), Staphylococcal-enterotoxin-B (SEB), and phytohemagglutinin (PHA). Each stimulus was barcoded by labelling with different intensities of fluorescent cell barcoding (FCB) markers. Intracellular production of interleukin-2, interferon-gamma, and tumor necrosis factor-alpha was measured simultaneously in just one blood sample of 600 µl whole blood. Significant age-related differences in cytokine production were shown for PMA, PHA, and TT in CD4+ T-cells, and for PMA, PHA, SEB, and TT in CD8+ T-cells. The intracellular cytokine production by CD4+ and CD8+ T-cells was higher at twelve months compared to six months of age for all antigens, except for PMA, which was lower at the age of twelve months. Based on the consistency in both T-cell subsets, we conclude that the new FCB method is a promising tool to investigate the age-related development of intracellular cytokine production in infants

Is disturbed clearance of apoptotic keratinocytes responsible for UVB-induced inflammatory skin lesions in systemic lupus erythematosus?

Apoptotic cells are thought to play an essential role in the pathogenesis of systemic lupus erythematosus (SLE). We hypothesise that delayed or altered clearance of apoptotic cells after UV irradiation will lead to inflammation in the skin of SLE patients. Fifteen SLE patients and 13 controls were irradiated with two minimal erythemal doses (MEDs) of ultraviolet B light (UVB). Subsequently, skin biopsies were analysed (immuno)histologically, over 10 days, for numbers of apoptotic cells, T cells, macrophages, and deposition of immunoglobulin and complement. Additionally, to compare results with cutaneous lesions of SLE patients, 20 biopsies of lupus erythematosus (LE) skin lesions were analysed morphologically for apoptotic cells and infiltrate. Clearance rate of apoptotic cells after irradiation did not differ between patients and controls. Influx of macrophages in dermal and epidermal layers was significantly increased in patients compared with controls. Five out of 15 patients developed a dermal infiltrate that was associated with increased epidermal influx of T cells and macrophages but not with numbers of apoptotic cells or epidermal deposition of immunoglobulins. Macrophages were ingesting multiple apoptotic bodies. Inflammatory lesions in these patients were localised near accumulations of apoptotic keratinocytes similar as was seen in the majority of LE skin lesions. In vivo clearance rate of apoptotic cells is comparable between SLE patients and controls. However, the presence of inflammatory lesions in the vicinity of apoptotic cells, as observed both in UVB-induced and in LE skin lesions in SLE patients, suggests that these lesions result from an inflammatory clearance of apoptotic cells

Urine levels of HMGB1 in Systemic Lupus Erythematosus patients with and without renal manifestations

INTRODUCTION: Lupus nephritis (LN) is a severe and frequent manifestation of systemic lupus erythematosus (SLE). Its pathogenesis has not been fully elucidated but immune complexes are considered to contribute to the inflammatory pathology in LN. High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein which is secreted from different types of cells during activation and/or cell death and may act as a pro-inflammatory mediator, alone or as part of DNA-containing immune complexes in SLE. Urinary excretion of HMGB1 might reflect renal inflammatory injury. To assess whether urinary HMGB1 reflects renal inflammation we determined serum levels of HMGB1 simultaneously with its urinary levels in SLE patients with and without LN in comparison to healthy controls (HC). We also analyzed urinary HMGB1 levels in relation with clinical and serological disease activity. METHODS: The study population consisted of 69 SLE patients and 17 HC. Twenty-one patients had biopsy proven active LN, 15 patients had a history of LN without current activity, and 33 patients had non-renal SLE. Serum and urine levels of HMGB1 were both measured by western blotting. Clinical and serological parameters were assessed according to routine procedures. In 17 patients with active LN a parallel analysis was performed on the expression of HMGB1 in renal biopsies. RESULTS: Serum and urinary levels of HMGB1 were significantly increased in patients with active LN compared to patients without active LN and HC. Similarly, renal tissue of active LN patients showed strong expression of HMGB1 at cytoplasmic and extracellular sites suggesting active release of HMGB1. Serum and urinary levels in patients without active LN were also significantly higher compared to HC. Urinary HMGB1 levels correlated with SLEDAI, and showed a negative correlation with complement C3 and C4. CONCLUSION: Levels of HMGB1 in urine of SLE patients, in particular in those with active LN, are increased and correlate with SLEDAI scores. Renal tissue of LN patients shows increased release of nuclear HMGB1 compared to control renal tissue. HMGB1, although at lower levels, is, however, also present in the urine of patients without active LN. These data suggest that urinary HMGB1 might reflect both local renal inflammation as well as systemic inflammation

Is serum HMGB1 a biomarker in ANCA-associated vasculitis?

Background: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are systemic inflammatory disorders that include granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), Churg-Strauss syndrome and renal limited vasculitis (RLV). Extracellular high-mobility group box 1 (HMGB1) acts as an alarmin and has been shown to be a biomarker of disease activity as well as an autoantigen in systemic lupus erythematosus (SLE) and, possibly, in AAV. This study aims to assess antibodies against HMGB1 and HMGB1 levels as biomarkers for AAV disease activity and predictors of relapsing disease.Methods: AAV patients with active disease and healthy controls (HC) were evaluated for anti-HMGB1 antibodies while serum HMGB1 levels were measured longitudinally in AAV patients at presentation, during remission, prior to and at relapses.Results: HMGB1 levels were similar between AAV patients at presentation (n = 52) and HC (n = 35) (2.64 +/- 1.80 ng/ml vs. 2.39 +/- 1.09 ng/ml; P = 0.422) and no difference regarding HMGB1 levels could be found among AAV disease subsets (GPA: 2.66 +/- 1.83 ng/ml vs. MPA: 3.11 +/- 1.91 ng/ml vs. RLV: 1.92 +/- 1.48 ng/ml; P = 0.369). AAV patients with renal involvement had lower HMGB1 levels than patients without renal involvement at presentation (2.35 +/- 1.48 ng/ml vs. 3.52 +/- 2.41 ng/ml; P = 0.042). A negative correlation was observed between HMGB1 levels and 24-hour proteinuria (rho = -0.361, P = 0.028). Forty-nine AAV patients were evaluated for HMGB1 levels during follow-up and no differences were observed between relapsing and nonrelapsing patients (P = 0.350). No significant increase in HMGB1 levels was observed prior to a relapse compared with the remission period and changes in HMGB1 levels were not associated with an increased risk for relapse in AAV. Positivity for anti-HMGB1 antibodies was low in patients with active AAV (three out of 24 patients).Conclusions: Serum HMGB1 levels at presentation are not increased and are lower in patients with renal involvement. Relapses are not preceded or accompanied by significant rises in HMGB1 levels and changes in HMGB1 levels are not related to ensuing relapses. Anti-HMGB1 antibodies are present in only a few patients in AAV. in contrast to SLE, HMGB1 is not a useful biomarker in AAV.Univ Groningen, Univ Med Ctr Groningen, Dept Rheumatol & Clin Immunol, NL-9713 GZ Groningen, NetherlandsUniversidade Federal de São Paulo, Escola Paulista Med Unifesp EPM, Div Rheumatol, BR-04023900 São Paulo, BrazilUniv Groningen, Univ Med Ctr Groningen, Dept Lab Med, NL-9713 GZ Groningen, NetherlandsUniv Groningen, Univ Med Ctr Groningen, Div Nephrol, Dept Internal Med, NL-9713 GZ Groningen, NetherlandsMartini Hosp, Dept Rheumatol & Internal Med, NL-9728 NT Groningen, NetherlandsUniversidade Federal de São Paulo, Escola Paulista Med Unifesp EPM, Div Rheumatol, BR-04023900 São Paulo, BrazilWeb of Scienc

Strong inhibition of TNF-α production and inhibition of IL-8 and COX-2 mRNA expression in monocyte-derived macrophages by RWJ 67657, a p38 mitogen-activated protein kinase (MAPK) inhibitor

In inflammatory processes, the p38 mitogen-activated protein kinase (MAPK) signal transduction route regulates production and expression of cytokines and other inflammatory mediators. Tumor necrosis factor α (TNF-α) is a pivotal cytokine in rheumatoid arthritis and its production in macrophages is under control of the p38 MAPK route. Inhibition of the p38 MAPK route may inhibit production not only of TNF-α, but also of other inflammatory mediators produced by macrophages, and indirectly of inflammatory mediators by other cells induced by TNF-α stimulation. Here we investigate the effects of RWJ 67657, a p38 MAPK inhibitor, on mRNA expression and protein production of TNF-α and other inflammatory mediators, in monocyte-derived macrophages. A strong inhibition of TNF-α was seen at pharmacologically relevant concentrations of RWJ 67657, but also inhibition of mRNA expression of IL-1β, IL-8, and cyclooxygenase-2 was shown. Furthermore, it was shown that monocyte-derived macrophages have a high constitutive production of matrix metalloproteinase 9, which is not affected by p38 MAPK inhibition. The results presented here may have important implications for the treatment of rheumatoid arthritis

Increase in IL-21 producing T-cells in patients with systemic lupus erythematosus

INTRODUCTION: Systemic lupus erythematosus (SLE) is an autoimmune disease accompanied by a disturbed T-cell balance skewed towards effector T-cells, in particular Th17-cells. The novel cytokine interleukin-21 (IL-21) is suggested to be crucial for triggering T-cell responses towards IL-17 producing cells. Thus, we aimed to investigate the ability of T-cells to produce IL-21 and IL-17 in SLE patients. METHODS: Peripheral blood of 34 SLE patients and 18 healthy controls (HC) was stimulated with phorbol myristate acetate (PMA) and calcium ionophore (Ca-Io). Percentages of IL-21- and IL-17A expressing T-cells were analysed by flow cytometry. The expression levels of the transcription factors B-cell lymphoma-6 (BCL-6) and factors retinoid-related orphan receptor (ROR-γt) were assessed in T-cells by real-time RT-PCR and flow cytometry. Additionally, IL-21 receptor (IL-21R) expression on B- and T-cells of patients and HC was analyzed. RESULTS: Significantly increased percentages of IL-21 expressing CD4(+ )T-cells and CD8(+ )T-cells were found in SLE patients as compared to HC. The percentages of IL-21(+ )CD4(+ )T-cells and CD8(+ )T-cells correlated significantly with the percentages of IL-17A(+ )CD4(+ )T-cells and CD8(+ )T-cells, respectively. The relative expression of BCL-6 and ROR-γt did not differ between SLE patients and HC. IL-21R expression occurred mainly on B-cells and was not different comparing SLE patients and HC. CONCLUSIONS: This study demonstrates an increased proportion of IL-21(+ )T-cells in SLE patients correlating with the proportion of IL-17(+ )T-cells. This suggests a pivotal role of IL-21 in the pathogenesis of SLE