L.-S. Tam

Raised plasma concentration and ex vivo production of inflammatory chemokines in patients with systemic lupus erythematosus

Background: Chemokines are involved in leucocyte chemotaxis. Infiltrating leucocytes play an important role of tissue injury in systemic lupus erythematosus (SLE). Objective: To investigate the role of inflammatory chemokines and their association with interleukin 18 (IL18) in SLE pathogenesis and disease activity. Methods: Plasma concentrations and ex vivo peripheral blood mononuclear cell production of inflammatory chemokines IP-10, RANTES, MIG, MCP-1, TARC, IL8, and GROα, and proinflammatory cytokines IL18, IFNγ, IL2, IL4, and IL10 were assayed in 80 SLE patients with or without renal disease and 40 healthy controls by immunofluorescence flow cytometry and enzyme linked immunosorbent assay. Results: Plasma IP10, RANTES, MIG, MCP-1, GROα, and IL18 concentrations in all SLE patients were higher than in controls, and correlated significantly with SLEDAI score (all p<0.05). In SLE patients without renal disease, IP10, RANTES, MIG, MCP-1, IL8, and IL18 correlated positively with SLEDAI score, while in those with renal derangement, IP10, IL8, IL10, and IL18 correlated with disease activity (all p<0.05). Plasma IL18 concentration correlated positively with IP10, MIG, GROα, and IL8 in all SLE patients (all p<0.005). Mitogen induced increases in ex vivo production of IP10, MCP-1, TARC, IFNγ, IL4, and IL10 were higher in all SLE patients regardless of their difference in disease activity (all p<0.05). Patients with renal disease had an augmented ex vivo release of RANTES. Conclusions: The correlation of raised plasma concentration and ex vivo production of inflammatory chemokines with disease activity, and their association with IL18, supports the view that chemotaxis of Th1/Th2 lymphocytes and neutrophils is important in SLE pathogenesis.

Serum and urinary free microRNA level in patients with systemic lupus erythematosus

MicroRNAs circulating in body fluid have been suggested as biomarkers of various diseases. We studied the serum and urinary level of several miRNA species (miR-200 family, miR-205 and miR-192) in patients with systemic lupus erythematosus (SLE). We studied 40 SLE patients. Serum and urinary miRNA levels were determined and compared with that of healthy controls. The serum levels of miR-200a, miR-200b, miR-200c, miR-429, miR-205 and miR-192, and urinary miR-200a, miR-200c, miR-141, miR-429 and miR-192 of SLE patients were lower than those of controls. Glomerular filtration rate (GFR) correlated with serum miR-200b (r = 0.411, p = 0.008), miR-200c (r = 0.343, p = 0.030), miR-429 (r = 0.347, p = 0.028), miR-205 (r = 0.429, p = 0.006) and miR-192 (r = 0.479, p = 0.002); proteinuria inversely correlated with serum miR-200a (r = −0.375, p = 0.017) and miR-200c (r = −0.347, p = 0.029). SLE disease activity index (SLEDAI) inversely correlated with serum miR-200a (r = −0.376, p = 0.017). Serum miR-200b (r = 0.455, p = 0.003) and miR-192 (r = 0.589, p < 0.001) correlated with platelet count, while serum miR-205 correlated with red cell count (r = 0.432, p = 0.005) and hematocrit (r = 0.370, p = 0.019). These pilot results suggested that miRNA may take part in the pathogenesis of SLE. Further studies are needed to validate the role of serum miRNA as a biomarker of SLE.

Elevated Production of B Cell Chemokine CXCL13 is Correlated with Systemic Lupus Erythematosus Disease Activity

IntroductionB lymphocyte chemoattractant (BLC/CXCL13), a CXC chemokine, is involved in B1 and B2 cell trafficking for the activation of autoreactive T helper (Th) cells and autoantibody production in target organs during the development of lupus. CXCL13 can induce the trafficking of CXCR5+ T lymphocyte subset designated as follicular helper T lymphocytes (TFH) which are specifically involved in autoantibody production.Materials and MethodsWe herein measured the plasma concentrations of CXCL13, B-cell-activating factor of the TNF family (BAFF), and TFH-cells-related cytokine IL-21 and cell surface expression of TFH-related receptor CXCR5 and IL-21R on CD4+Th and CD19+B cells in 35 systemic lupus erythematosus (SLE) patients and 23 sex- and age-matched control subjects (NC) using enzyme-linked immunosorbent assay and flow cytometry, respectively.Results and DiscussionPlasma CXCL13, BAFF, and IL-21 concentrations were significantly higher in SLE patients than NC group (all p < 0.0001). Increase in CXCL13 concentration correlated positively and significantly with SLEDAI score in SLE patients (r = 0.399, p = 0.032). Cell surface expression of CXCR5 on Th and B cells and IL-21R on B cells was however significantly lower in SLE patients than controls (both p < 0.01). It may indicate that most differentiated TFH cells migrate out from circulation into lymphoid organ upon activation during the disease development of SLE.ConclusionsThe above results suggest that the elevated production of CXCL13, BAFF, and IL-21 may be associated with the function of TFH for the immunopathogenesis in SLE, and CXCL13 may serve as a potential disease marker of SLE.

Double-blind, randomized, placebo-controlled pilot study of leflunomide in systemic lupus erythematosus

Twelve systemic lupus erythematosus (SLE) patients with mild to moderate disease activity (SLEDAI of ≥6 and on prednisolone, 0.5 mg/kg/day) were included in a prospective, randomized, double-blind, placebo-controlled pilot study for 24 weeks. Six were randomized to receive oral leflunomide and six received placebo. Primary outcome of this study included the mean change of SLEDAI at 24 weeks. Secondary outcomes included the changes in proteinuria, complement levels, anti ds-DNA binding, and prednisolone dosage. The mean age of the 12 patients was 41 + 9 years, and the mean disease duration was 8.5 + 5.8 years. All were female except one patient. The disease activity of both groups of patients decreased significantly after six months of treatment (14.7 + 6.0 to 3.7 + 2.3 in leflunomide group, P 1/4 0.028, and 9.7 + 3.4 to 5.2 + 4.1 in placebo group, P 1/4 0.027). Reduction in the SLEDAI from baseline to 24 weeks was significantly greater in the leflunomide group than the placebo group (11.0 + 6.1 in the leflunomide group and 4.5 + 2.4 in the placebo group respectively, P 1/4 0.026). Minor adverse events included transient elevation in ALT, hypertension and transient leucopenia. In summary, leflunomide was more effective than placebo in treating SLE patients with mild to moderate disease activity and was safe and well-tolerated.

Elevated production of interleukin-18 is associated with renal disease in patients with systemic lupus erythematosus

Summary To investigate the production mechanism and proinflammatory role of the cytokine interleukin (IL‐18) in lupus nephritis, we investigated the plasma concentrations of IL‐18 and nitric oxide (NO) and the release of IL‐18 and NO from mitogen‐activated peripheral blood monomuclear cells (PBMC), in 35 SLE patients with renal disease (RSLE), 37 patients without renal disease (SLE) and 28 sex‐ and age‐matched healthy control subjects (NC). IL‐18 and NO concentrations were measured by ELISA and colourimetric non‐enzymatic assay, respectively. Gene expressions of IL‐18 and IL‐18 receptor were analysed by RT‐PCR. Plasma IL‐18 and NO concentrations were significantly higher in RSLE than NC (both P < 0·01). Elevation of plasma IL‐18 in RSLE correlated positively and significantly with SLE disease activity index and plasma NO concentration (r = 0·623, P < 0·0001 and r = 0·455, P = 0·017, respectively), and the latter also showed a positive and significant correlation with plasma creatinine (r = 0·410, P = 0·034) and urea (r = 0·685, P < 0·0001). There was no significant difference in gene expressions of IL‐18 and IL‐18 receptor in PBMC among RSLE, SLE and NC. Percentage increase in culture supernatant IL‐18 concentration was significantly higher in RSLE than SLE and NC (both P < 0·05). The basal NO release was significantly higher in RSLE than that in SLE and NC (both P < 0·005). IL‐18 is therefore suggested to play a crucial role in the inflammatory processes of renal disease in SLE.

Predictors of maternal and fetal outcomes in pregnancies of patients with systemic lupus erythematosus.

Disease activity 6 months before pregnancy of patients with systemic lupus erythematosus (SLE) associated with adverse maternal and fetal outcomes is not well studied. The aim of the study was to identify predictors of adverse maternal and fetal outcomes in pregnant SLE patients, based on patients’ background characteristics, clinical and laboratory data 6 months before pregnancy. Of 103 pregnancies, 55 pregnancies in 39 SLE patients were investigated. Clinical and laboratory data were recorded at regular intervals from 6 months before conception to 1 year after delivery. Primary outcomes included the predictors of combined adverse maternal and fetal outcomes. Potential explanatory variables included demographic, clinical and laboratory data 6 months before conception. Using logistic regression, history of nephritis (p = 0.001, odds ratio [OR] 13.3, 95% confidence interval [CI] 2.7–65.1) and a high SLE Disease Activity Index (SLEDAI) score 6 months before pregnancy (p = 0.015, OR 1.7, 95% CI 1.1–2.7) were associated with combined adverse maternal outcome, whereas flare during pregnancy (p = 0.003, OR 29.3, 95% CI 3.1–273.1) predicted combined adverse fetal outcome. The area under the curve for SLEDAI score of combined maternal outcome was 0.73 (95% CI 0.58–0.87). The optimal cut-off point according to the receiver operating characteristic curve was 4, with a sensitivity of 64% and a specificity of 75%. In conclusion, a history of nephritis or a SLEDAI score of 4 or more in SLE patients 6 months before conception predicts adverse maternal outcomes, while disease flare during pregnancy predicts adverse fetal outcomes. Pregnancies should be delayed until the disease has been in remission for 6 months.

Activation profile of Toll-like receptors of peripheral blood lymphocytes in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease associated with aberrant activation of T and B lymphocytes for the production of inflammatory cytokines and autoreactive antibodies. Animal studies of SLE have indicated that Toll‐like receptors (TLR) are important in the pathogenesis of murine lupus. In the present clinical study, differential protein expressions of TLR‐1–9 of monocytes and different lymphocyte subsets from patients with SLE and normal control subjects were determined by flow cytometry. Results showed that the expression of intracellular TLRs (TLR‐3, ‐8, ‐9) and extracellular TLRs (TLR‐1, ‐2, ‐4, ‐5, ‐6) were elevated in monocytes, CD4+ T lymphocytes, CD8+ T lymphocytes and B lymphocytes of SLE patients compared to control subjects (all P < 0·001). Moreover, cell surface expression of TLR‐4 on CD4+ T lymphocytes and CD8+ T lymphocytes, and TLR‐6 on B lymphocytes, were correlated positively with SLE disease activity index (SLEDAI) (TLR‐4 on CD4+ T lymphocytes and CD8+ T lymphocytes: r = 0·536, P = 0·04; r = 0·713, P = 0·003; TLR‐6 in B lymphocytes: r = 0·572, P = 0·026). In concordance with the above results, there is an observable increased relative induction (%) of inflammatory cytokine interleukin (IL)‐1β, IL‐6, IL‐10 and IL‐12, chemokines CCL2, CXCL8, CCL5 and CXCL10 from peripheral blood mononuclear cells (PBMC) upon differential stimulation by PolyIC (TLR‐3 ligand), lipopolysaccharide (TLR‐4 ligand), peptidoglycan (TLR‐2 ligand), flagellin (TLR‐5 ligand), R837 (TLR‐7 ligand) and CpG DNA (TLR‐9 ligand) in SLE patients compared to controls. These results suggest that the innate immune response for extracellular pathogens and self‐originated DNA plays immunopathological roles via TLR activation in SLE.

Increased arterial stiffness correlated with disease activity in systemic lupus erythematosus

To evaluate the relationships between arterial stiffness, disease activity and end-organ damage in systemic lupus erythematosus (SLE), non-invasive vascular assessments were made on 32 female SLE patients and 32 female normal controls. The patients had significantly increased brachial-ankle pulse wave velocity (PWV) (13.06 ± 1.79 vs. 11.50 ± 1.00 m/s; P < 0.001), heart-ankle PWV (8.98 ± 1.16 vs. 7.88 ± 0.73 m/s; P < 0.001), carotid augmentation index (AI) (21.6 ± 17.2% vs. 5.4 ± 14.0%; P = 0.001) and carotid intima-medial thickness (IMT) (0.753 ± 0.132 vs. 0.644 ± 0.092 mm; P = 0.002) when compared with controls. The disease activity and organ damage were evaluated by SLE disease activity index (SLEDAI) and systemic lupus international collaborating clinics (SLICC) damage index. Patients with active disease (SLEDAI ≥ 3) had significantly higher carotid AI (34.4 ± 9.7% vs. 17.8 ± 17.3%, P < 0.05) than stable ones (SLEDAI < 3) and those with organ damage (SLICC ≥ 1) had significantly higher heart-ankle PWV (9.69 ± 1.13 vs. 8.61 ± 1.02 m/s, P < 0.05) than those with SLICC = 0. After making adjustments for age, body mass index (BMI) and blood pressure, carotid AI was found to show correlation with SLEDAI and haPWV with SLICC. A carotid AI value of 33.3% identified SLEDAI ≥ 3 with a sensitivity of 83% and a specificity of 80%, whereas a heart-ankle PWV value of 9.0 m/s identified SLICC ≥ 1 with a sensitivity of 91% and a specificity of 67%. In conclusion, SLE was an independent risk factor of sub-clinical atherosclerosis and arterial stiffness may identify the presence of active disease.

Activation profile of intracellular mitogen-activated protein kinases in peripheral lymphocytes of patients with systemic lupus erythematosus.

IntroductionSystemic lupus erythematosus (SLE) is a systemic autoimmune disease associated with aberrant activation of T and B lymphocytes. Abnormal activation of intracellular signaling molecules in lymphocytes by inflammatory cytokines can instigate the inflammation in SLE.Materials and MethodsThe activation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) in inflammatory cytokine IL-18-activated monocytes, CD4+ T helper (Th) lymphocytes, CD8+ T lymphocytes, and CD19+B lymphocytes in 22 SLE patients and 20 sex- and age-matched control subjects were measured by flow cytometry.Results and DiscussionThe basal expressions of phospho-p38 MAPK in CD4+ T lymphocytes, CD8+ T lymphocytes, and B lymphocytes were significantly higher in SLE patients than controls (all p < 0.05). The expression of phospho-p38 MAPK in CD4+ T lymphocytes, CD8+ T lymphocytes and B lymphocytes, and phospho-JNK in CD8+ T lymphocytes and B lymphocytes was also significantly elevated in SLE patients upon the activation by IL-18, exhibiting significant correlation with the plasma concentrations of Th1 chemokine CXCL10 (all p < 0.05). The expression of phospho-JNK in IL-18 activated CD8+ T lymphocytes and the relative % fold increase of the expression of phospho-JNK upon IL-18 activation in B lymphocytes were significantly correlated with SLE disease activity index (both p < 0.05).ConclusionThe inflammation-mediated activation of JNK and p38 MAPK signaling pathways in T and B lymphocytes can be the underlying intracellular mechanisms causing lymphocyte hyperactivity in SLE.

SLE disease per se contributes to deterioration in bone mineral density, microstructure and bone strength

Objective The objective of this report is to assess the effect of systemic lupus erythematosus (SLE) disease itself on deterioration of bone mineral density (BMD), microstructure and bone strength. Method Thirty age-matched SLE patients on long-term glucocorticoids (GC) (SLE/GC), 30 SLE patients without GC (SLE/non-GC) and 60 healthy controls were examined. Areal BMD (aBMD) was measured by dual-energy X-ray absorptiometry. Bone geometry, volumetric BMD (vBMD), and architectural parameters at the nondominant distal radius were assessed by high-resolution peripheral quantitative computed tomography (HR-pQCT). Bone strength was estimated by HR-pQCT-based micro-finite element analysis. Results Adjusted for menopausal status and adjusted calcium level, when compared with controls, SLE/non-GC patients had significantly lower aBMD at femoral neck and total hip, and diminished radial total vBMD, cortical area, vBMD and thickness, respectively, by 8.3%, 8%, 2.7% and 9.2%, as well as significant compromised bone strength (stiffness, failure load and apparent modulus) by 8.3%, 9.1% and 9.5%, respectively. Similar alterations were also found in SLE/GC patients when compared to controls. In the premenopausal subgroup analysis, when compared with controls, total hip aBMD and radial cortical area were significantly lower in SLE/non-GC patients, and cortical area and thickness were significantly deficit in SLE/GC patients. However, no significant difference in any bone variables was present between SLE/GC and SLE/non-GC patients in the entire cohort or in the premenopausal subgroup. Conclusion SLE disease per se contributes to the deterioration in bone density, cortical microstructure and bone strength. This might help to explain the considerably higher fracture risk seen in SLE patients.