J. Bijzet

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

IntroductionHigh 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.MethodsThe 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.ResultsHMGB1 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.ConclusionsLevels 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.

Time course of angiopoietin-2 release during experimental human endotoxemia and sepsis

IntroductionEndothelial activation leading to vascular barrier breakdown denotes a devastating event in sepsis. Angiopoietin (Ang)-2, a circulating antagonistic ligand of the endothelial specific Tie2 receptor, is rapidly released from Weibel-Palade and has been identified as a non-redundant gatekeeper of endothelial activation. We aimed to study: the time course of Ang-2 release during human experimental endotoxemia; the association of Ang-2 with soluble adhesion molecules and inflammatory cytokines; and the early time course of Ang-2 release during sepsis in critically ill patients.MethodsIn 22 healthy volunteers during a 24-hour period after a single intravenous injection of lipopolysaccharide (LPS; 4 ng/kg) the following measurement were taken by immuno luminometric assay (ILMA), ELISA, and bead-based multiplex technology: circulating Ang-1, Ang-2, soluble Tie2 receptor, the inflammatory molecules TNF-alpha, IL-6, IL-8 and C-reactive protein, and the soluble endothelial adhesion molecules inter-cellular adhesion molecule-1 (ICAM-1), E-selectin, and P-selectin. A single oral dose of placebo or the p38 mitogen activated protein (MAP) kinase inhibitor drug, RWJ-67657, was administered 30 minutes before the endotoxin infusion. In addition, the course of circulating Ang-2 was analyzed in 21 septic patients at intensive care unit (ICU) admission and after 24 and 72 hours, respectively.ResultsDuring endotoxemia, circulating Ang-2 levels were significantly elevated, reaching peak levels 4.5 hours after LPS infusion. Ang-2 exhibited a kinetic profile similar to early pro-inflammatory cytokines TNF-alpha, IL-6, and IL-8. Ang-2 levels peaked prior to soluble endothelial-specific adhesion molecules. Finally, Ang-2 correlated with TNF-alpha levels (r = 0.61, P = 0.003), soluble E-selectin levels (r = 0.64, P < 0.002), and the heart rate/mean arterial pressure index (r = 0.75, P < 0.0001). In septic patients, Ang-2 increased in non-survivors only, and was significantly higher compared with survivors at baseline, 24 hours, and 72 hours.ConclusionsLPS is a triggering factor for Ang-2 release in men. Circulating Ang-2 appears in the systemic circulation during experimental human endotoxemia in a distinctive temporal sequence and correlates with TNF-alpha and E-selectin levels. In addition, not only higher baseline Ang-2 concentrations, but also a persistent increase in Ang-2 during the early course identifies septic patients with unfavorable outcome.

A quantitative method for detecting deposits of amyloid A protein in aspirated fat tissue of patients with arthritis

OBJECTIVE To describe a new, quantitative, and reproducible method for detecting deposits of amyloid A protein in aspirated fat tissue and to compare it with smears stained with Congo red. METHODS After extraction of at least 30 mg of abdominal fat tissue in guanidine, the amyloid A protein concentration was measured by a monoclonal antibody-based sandwich ELISA. RESULTS The concentrations in 24 patients with arthritis and AA amyloidosis (median 236, range 1.1–8530 ng/mg tissue) were higher (p<0.001) than in non-arthritic controls, uncomplicated rheumatoid arthritis, and other types of systemic amyloidosis (median 1.1, range 1.1–11.6 ng/mg tissue). Patients with extensive deposits, according to Congo red staining, had higher concentrations than patients with minute deposits. CONCLUSION This is a new, quantitative, and reproducible method for detecting deposits of amyloid A protein in aspirated fat tissue of patients with arthritis, even when minute deposits are present as detected in smears stained with Congo red.

Higher levels of interleukin‐6 in cystic fluids from patients with malignant versus benign ovarian tumors correlate with decreased hemoglobin levels and increased platelet counts

Background. Recently, high pretreatment platelet counts and low pretreatment hemoglobin levels were found to be negative prognostic factors in patients with ovarian cancer. Interleukin‐6 (IL‐6) is a multifunctional cytokine with a diversity of functions leading to the induction of C‐reactive protein (CRP), increased platelet counts, and low hemoglobin levels. Different epithelial ovarian cancer cell lines are found to produce varying amounts of IL‐6. In this study, a possible relationship between IL‐6 levels in cystic fluids of benign and malignant ovarian tumors and pretreatment serum CRP, platelet counts, and hemoglobin levels was evaluated.

Skin autofluorescence is increased in systemic lupus erythematosus but is not reflected by elevated plasma levels of advanced glycation endproducts

OBJECTIVES To examine whether skin advanced glycation endproducts (AGEs) accumulation, plasma levels of AGEs-N(epsilon)-carboxymethyllysine (CML) and N(epsilon)-carboxyethyllysine (CEL)-and serum levels of soluble receptor for AGEs (sRAGE) are elevated in SLE patients compared with controls, and whether these parameters are related to disease activity and endothelial cell (EC) activation. METHODS Ten SLE patients (9 women, age 34 +/- 13 yrs, mean +/- s.d.) and 10 age- and sex-matched controls were included. Patients were analysed during inactive as well as active disease. Skin AGE accumulation was estimated using ultraviolet-A (UV-A) light for measurement of autofluorescence obtained by Excitation-Emission matrix Scanner (AF-EEMS). Levels of CML and CEL were determined by tandem mass spectrometry. Levels of sRAGE and of soluble vascular cell adhesion molecule-1 (sVCAM-1) were determined by ELISAs. RESULTS Skin AF-EEMS was increased in SLE patients compared with controls (P < 0.05). Levels of CML and CEL were comparable between patients and controls and were not influenced by disease activity. sRAGE and sVCAM-1 levels were higher in quiescent SLE patients compared with controls (P < 0.05) and increased further during active disease (P < 0.05). In patients with quiescent disease and controls, sRAGE levels correlated to sVCAM-1 levels (r = 0.579, P = 0.007). CONCLUSIONS Skin AGEs and levels of sRAGE and sVCAM-1 were elevated in SLE patients, whereas levels of CML and CEL were comparable with controls. As sRAGE even further increased during endothelial activation, it might be hypothesized that sRAGE acts as a decoy receptor. Why this proposed mechanism is insufficient to prevent increased AGE accumulation in the skin of SLE patients has to be established.

Screening for amyloid in subcutaneous fat tissue of Egyptian patients with rheumatoid arthritis: clinical and laboratory characteristics

Objective: To screen for amyloid and to assess associated clinical and laboratory characteristics in Egyptian patients with rheumatoid arthritis (RA). Methods: Abdominal subcutaneous fat aspirates were consecutively collected from 112 patients (103 women, nine men) having RA for five years or more. To detect amyloid, fat smears were stained with Congo red and the concentration of amyloid A protein in fat tissue was measured. Clinical, radiological, and laboratory characteristics of the patients were assessed. Results: Amyloid was detected in eight (7%) of the fat smears stained with Congo red. Compared with the Congo red stain, the sensitivity for detecting amyloid by measurement of amyloid A protein in fat tissue was 75% and the specificity was 100%. The amount of amyloid found was small for both methods. The median disease duration of the eight amyloid patients was significantly longer (17 years) than that of the non-amyloid patients (10 years). Bronchopulmonary disease and constipation were more common, whereas proteinuria and chronic renal insufficiency were not. The number of swollen joints and the number of red blood cells were significantly lower in the amyloid group. Conclusions: Quantification of amyloid A protein and staining with Congo red are strongly concordant methods of screening for amyloid in fat tissue. The prevalence of amyloid in Egyptian patients with RA is 7%. Proteinuria is not a discriminating feature, whereas long disease duration, constipation, bronchopulmonary symptoms, and a moderate to low number of red blood cells may help to identify the arthritic patients with amyloid.

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

IntroductionLupus 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.MethodsThe 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.ResultsSerum 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.ConclusionLevels 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.

High mobility group box1 (HMGB1) in relation to cutaneous inflammation in systemic lupus erythematosus (SLE)

Summary Photosensitivity is characteristic of systemic lupus erythematosus (SLE). Upon ultraviolet B (UVB) exposure, patients develop inflammatory skin lesions in the vicinity of sunburn cells (SBCs). High mobility group box 1 (HMGB1) is released from apoptotic and activated cells and exerts inflammatory actions through ligation to its receptors. Methods Eleven SLE patients and 10 healthy controls (HCs) were exposed to UVB. Skin biopsies were taken before and at one, three and 10 days after irradiation. Sections were stained for SBC, HMGB1, CD3, CD68, interferon-induced protein MxA and cleaved caspase 3. In vitro experiments with UVB-irradiated keratinocytes were also performed. Higher numbers of cells that had released HMGB1 were seen in the skin of SLE patients compared to HCs before and after irradiation. HMGB1-negative nuclei correlated with the presence of SBCs, and with the number of cleaved caspase 3 positive cells in lupus skin. Results HMGB1 release is increased in the skin of SLE patients compared to HCs. Upon UVB exposure, HMGB1 release further increases in SLE patients and is related to the number of apoptotic cells. Our data suggest that HMGB1, probably released from apoptotic keratinocytes, contributes to the development of inflammatory lesions in the skin of SLE patients upon UVB exposure.

Circulating monocytes in patients with acute coronary syndromes lack sufficient interleukin-10 production after lipopolysaccharide stimulation

Acute coronary syndromes (ACS) are associated with inflammation resulting from monocyte activation. We sought for differences in the production of pro‐ and anti‐inflammatory cytokines by monocytes from patients with ACS. C‐reactive protein (CRP) and neopterin were measured in 22 patients with acute coronary syndromes, 50 patients with stable vascular disease and 22 healthy controls. Production of tumour necrosis factor (TNF)‐α and interleukin (IL)‐10 was determined after, respectively, 6 and 24 h of incubation of full blood with lipopolysaccharide (LPS). Levels of CRP [median, interquartile range (IQR)][1·5 mg/l (0·8–4·5) ACS patient versus 2·1 (0·9–3·6) stable disease versus 0·4 (0·3–1·2) healthy controls] (P < 0·001) and neopterin [7·4 nmol/l (6·0–8·7) ACS patient versus 7·1(6·0–8·9) stable disease versus 6·4 (5·6–7·3) healthy controls] (P = 0·07) were higher in both the patient groups. IL‐10 production after LPS stimulation was greatly reduced in patients with acute coronary syndromes (16 175 pg/ml, 7559–28 470 pg/ml) as opposed to patients with stable disease (28 379 pg/ml, 12 601–73 968 pg/ml) and healthy controls (63 830 pg/ml, 22 040–168 000 pg/ml) (P = 0·003). TNF‐α production was not significantly different between the groups [7313 pg/ml (4740–12 615) ACS patient versus 11 002 (5913–14 190) stable disease versus 8229 (5225–11 364) healthy controls] (P = 0·24). Circulating monocytes in unstable coronary syndromes produce equal amounts of TNF‐α but less IL‐10 after stimulation with LPS in vitro as compared with healthy controls. We hypothesize that, in acute coronary syndromes, the production proinflammatory cytokines is not counterbalanced by anti‐inflammatory cytokines such as IL‐10.

Are urinary levels of high mobility group box 1 markers of active nephritis in anti-neutrophil cytoplasmic antibody-associated vasculitis?

The objective of this study is to evaluate urinary high mobility group box 1 (HMGB1) levels as markers for active nephritis in patients with anti‐neutrophil cytoplasmic antibody (ANCA)‐associated vasculitis (AAV) in comparison with urinary CD4+ effector memory T cells and urinary monocyte chemoattractant protein‐1 (MCP‐1). Twenty‐four AAV patients with active nephritis and 12 healthy controls (HC) were evaluated. In nine patients, samples were also obtained during remission. Urinary levels of HMGB1 were measured by Western blot. CD4+ T cells and CD4+ effector memory T cells (CD4+CD45RO+CCR7‐) were determined in urine and whole blood by flow cytometry. Measurement of urinary levels of MCP‐1 and serum HMGB1 levels were performed by enzyme‐linked immunosorbent assay (ELISA). AAV patients with active nephritis had higher median intensity of HMGB1 in urine than HC [10·3 (7·05–18·50) versus 5·8 (4·48–7·01); P = 0·004]. Both urinary HMGB1 and MCP‐1 levels decreased significantly from active nephritis to remission. The urinary MCP‐1/creatinine ratio correlated with Birmingham Vasculitis Activity Score (BVAS) (P = 0·042). No correlation was found between the HMGB1/creatinine ratio and 24‐h proteinuria, estimated glomerular filtration rate (eGFR), MCP‐1/creatinine ratio, BVAS and serum HMGB1. A positive correlation was found between urinary HMGB1/creatinine ratio and CD4+ T cells/creatinine ratio (P = 0·028) and effector memory T cells/creatinine ratio (P = 0·039) in urine. Urinary HMGB1 levels are increased in AAV patients with active nephritis when compared with HC and patients in remission, and urinary HMGB1 levels are associated with CD4+ T cells and CD4+ effector memory T cells in urine. Measurement of urinary HMGB1 may be of additional value in identifying active glomerulonephritis in AAV patients.