Ming-Chi Lu

Anti–citrullinated protein antibodies bind surface-expressed citrullinated Grp78 on monocyte/macrophages and stimulate tumor necrosis factor α production

OBJECTIVEnAnti-citrullinated protein antibodies (ACPAs), which are the most specific autoantibody marker in patients with rheumatoid arthritis (RA), correlate with disease activity; however, the role of ACPAs in RA pathogenesis has not been elucidated. We hypothesized that ACPAs may directly stimulate mononuclear cells to produce inflammatory cytokines. Thus, we identified cognate antigens of ACPAs on monocyte/macrophages and examined their immunopathologic roles in the pathogenesis of RA.nnnMETHODSnACPAs were purified from pooled ACPA-positive RA sera by cyclic citrullinated peptide-conjugated affinity column. After coculture of U937 cells with ACPAs, the tumor necrosis factor alpha (TNFalpha) production and NF-kappaB DNA binding activity of the cells were measured by enzyme-linked immunosorbent assay. The cognate antigens of ACPAs on the U937 cell surface were probed by ACPAs, and the reactive bands were examined via proteomic analysis.nnnRESULTSnACPAs specifically enhanced TNFalpha production and increased the DNA-binding activity of NF-kappaB in U937 cells. Proteomic analysis revealed that Grp78 protein (72 kd) was one of the cognate antigens of ACPAs. The truncated form of cell surface-expressed Grp78 (55 kd) on U937 cells contained citrulline capable of binding with ACPAs. After citrullination, glutathione S-transferase-tagged recombinant Grp78 (97.52 kd) became a 72-kd fragment and bound with ACPAs. ACPAs also bound to human monocytes and lymphocytes to promote TNFalpha production.nnnCONCLUSIONnWe clearly demonstrated that ACPAs enhance NF-kappaB activity and TNFalpha production in monocyte/macrophages via binding to surface-expressed citrullinated Grp78.

Pilocarpine Hydrochloride for the Treatment of Xerostomia in Patients with Sjögren's Syndrome in Taiwan—A Double-blind, Placebo-controlled Trial

n n Background/purposen Sjögrens syndrome (SS) is characterized by diminished exocrine secretions with the resultant symptoms of dry mouth and dry eye. As genetic predisposition and ethnicity may alter the effectiveness of drug treatment, evaluation of the efficacy and safety of the secretagogue pilocarpine hy-drochloride in the treatment of xerostomia in patients with SS in different populations is needed.n n n Methodsn Forty-four patients with SS were enrolled in this double-blind, placebo-controlled trial. Patients were randomized to receive 5 mg pilocarpine (Salagen) or placebo tablet four times daily for 12 weeks. Global evaluation and subjective responses of patients were assessed by questionnaires with visual analog scales and categorical checkboxes. Saliva production was also measured by modified Saxons test.n n n Resultsn Pilocarpine treatment significantly improved global assessment of dry mouth, symptoms associated with dry mouth (mouth comfort, ability to sleep and ability to speak), and saliva production compared to placebo. The drug was well tolerated and the most common adverse effect was sweating (5/23, 21.7%) resulting from the muscarinic agonist action of the drug. No serious drug-related adverse effect was found in this study.n n n Conclusionn The results of this study suggest that therapy with 5 mg pilocarpine four times daily is effective, safe and well tolerated for the relief of oral symptoms in patients with SS in Taiwan.n n

Release of surface-expressed lactoferrin from polymorphonuclear neutrophils after contact with CD4+T cells and its modulation on Th1/Th2 cytokine production

It is conceivable that a membrane component(s) is transferred from antigen‐presenting cells to T cells after antigenic stimulation. However, it is not clear whether a certain membrane component(s) is transferred from polymorphonuclear neturophils (PMN) to T cells for immunomodulation. In the presence study, we cocultured two of the three autologous cells—PMN, CD4+T, and red blood cells (RBC)—homotypically or heterotypically for 1 h. Spontaneous membrane exchange between autologous PMN‐PMN and PMN‐CD4+T but not between CD4+T‐CD4+T or RBC‐CD4+T was observed with a confocal microscope. Loss of membrane exchange between two paraformaldehyde‐fixed cells suggests that mutual membrane exchange is via cell–cell contact. Different combinations of cellular enzyme‐linked immunosorbent assay for measuring the binding between fixed cells and biotinylated cell lysates showed the same tendency. To identify the molecule(s) mediating PMN‐CD4+T binding, we compared the banding of biotinylated PMN lysates and the banding of plain PMN lysate probed by biotinylated CD4+T lysate in 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. We found that a 75‐ to 80‐kDa surface‐expressed molecule on PMN exists constantly to mediate PMN‐CD4+T binding. Peptide analysis disclosed that the molecule had 99.8% identity with lactoferrin (LF). The expression of LF on system lupus erythematosis (SLE)‐PMN is less than normal PMN. PMN‐CD4+T coculture increased LF expression on CD4+T. Normal PMN and human milk‐derived LF suppressed interferon‐γ (IFN‐γ) but enhanced interleukin (IL)‐10 production of anti‐CD3+anti‐CD28‐activated, normal CD4+T. In contrast, coculture of SLE‐PMN and autologous CD4+T suppressed IFN‐γ and IL‐10 production. These results suggest that the surface‐expressed LF released from PMN after contact with autologous CD4+T modulated its T helper cell type 1 (Th1)/Th2 cytokine production. Decreased LF expression on SLE‐PMN abnormally modulates Th1/Th2 production by CD4+T cells.

Anti-myeloperoxidase antibodies enhance phagocytosis, IL-8 production, and glucose uptake of polymorphonuclear neutrophils rather than anti-proteinase 3 antibodies leading to activation-induced cell death of the neutrophils

Anti-neutrophil cytoplasmic antibodies (ANCA) not only are triggered by target protein myeloperoxidase (MPO) and proteinase 3 (PR3) of polymorphonuclear neutrophil (PMN) but also react with primed PMN to exert the inflammatory process in vasculitis syndrome. To clarify the crucial role of PMN in ANCA-associated vasculitis and the related mechanism, PMN was cultured with monoclonal antibody MPO–ANCA and PR3–ANCA to determine the function of phagocytosis, Interleukin- 8 (IL-8) production, glucose uptake, and TNF-related apoptosis induced ligand (TRAIL) production. The spontaneous membrane expression of MPO and PR3 on PMN could be significantly increased by lipopolysaccharide (LPS) and TNF-α, but not by IL-8 or GRO-α. The PMN-stimulating activity of ANCA was demonstrated by enhancing phagocytosis, IL-8 production, and glucose uptake that was more prominent by MPO–ANCA. The PMN stimulation by ANCA was not through protein kinase, H2O2, or superoxide anion radicals as their inhibitors exerted no effect on ANCA-mediated activation. On the other hand, ANCA also accelerated PMN apoptosis and increased TRAIL production. These results demonstrate that activation-induced cell death (AICD) mechanism could be initiated in PMN with existence of ANCA. In conclusion, MPO–ANCA is more potent in stimulating PMN than PR3–ANCA. ANCA-activated PMN is not only responsible for the amplified inflammatory process in blood vessel but also initiates immune circuit via triggered macrophage/monocyte by apoptotic PMN through the mechanism of AICD elicited by ANCA.

Deranged Bioenergetics and Defective Redox Capacity in T Lymphocytes and Neutrophils Are Related to Cellular Dysfunction and Increased Oxidative Stress in Patients with Active Systemic Lupus Erythematosus

Urinary excretion of N-benzoyl-glycyl-Nε-(hexanonyl)lysine, a biomarker of oxidative stress, was higher in 26 patients with active systemic lupus erythematosus (SLE) than in 11 non-SLE patients with connective tissue diseases and in 14 healthy volunteers. We hypothesized that increased oxidative stress in active SLE might be attributable to deranged bioenergetics, defective reduction-oxidation (redox) capacity, or other factors. We demonstrated that, compared to normal cells, T lymphocytes (T) and polymorphonuclear neutrophils (PMN) of active SLE showed defective expression of facilitative glucose transporters GLUT-3 and GLUT-6, which led to increased intracellular basal lactate and decreased ATP production. In addition, the redox capacity, including intracellular GSH levels and the enzyme activity of glutathione peroxidase (GSH-Px) and γ-glutamyl-transpeptidase (GGT), was decreased in SLE-T. Compared to normal cells, SLE-PMN showed decreased intracellular GSH levels, and GGT enzyme activity was found in SLE-PMN and enhanced expression of CD53, a coprecipitating molecule for GGT. We conclude that deranged cellular bioenergetics and defective redox capacity in T and PMN are responsible for cellular immune dysfunction and are related to increased oxidative stress in active SLE patients.

Urinary Neutrophil Gelatinase-Associated Lipocalin Is a Potential Biomarker for Renal Damage in Patients with Systemic Lupus Erythematosus

Neutrophil gelatinase-associated lipocalin (NGAL) has been demonstrated to be a novel biomarker in acute and chronic kidney disease. We hypothesized that 24-hour urinary NGAL excretion may be a predictor for renal damage in patients with systemic lupus erythematosus (SLE). Thirty-four SLE patients with renal involvement (SLE-renal group), 8 SLE patients without renal involvement (SLE-nonrenal group), 14 patients with non-SLE autoimmune diseases (disease control or DC group), and 12 healthy volunteers (normal control or NC group) were compared for 24-hour urinary excretion of NGAL and different cytokines. We found that the 24-hour urinary NGAL excretion in the SLE-renal group was higher than that in the SLE-non-renal, DC, and NC groups. However, the excretion of interleukin-10, transforming growth factor-β1, and tumor necrosis factor-α was not different between the SLE-renal and SLE-non-renal groups. Furthermore, NGAL excretion in the SLE-renal group was correlated with serum creatinine levels and creatinine clearance, but not with the SLE Disease Activity Index score. Multivariate logistic regression analysis and receiver operating characteristic curve analysis revealed that 24-hour urinary NGAL excretion is a potential biomarker for renal damage in SLE patients, with higher sensitivity and specificity than anti-dsDNA antibody titers.

Tamm-Horsfall Glycoprotein Enhances PMN Phagocytosis by Binding to Cell Surface-Expressed Lactoferrin and Cathepsin G That Activates MAP Kinase Pathway

The molecular basis of polymorphonuclear neutrophil (PMN) phagocytosis-enhancing activity (PEA) by human purified urinary Tamm-Horsfall glyco- protein (THP) has not been elucidated. In this study, we found human THP bound to lactoferrin (LF) and cathepsin G (CG) expressed on the surface of PMN, identified by a proteomic study with MALDI-TOF- LC/LC/mass spectrometric analysis. Pre-incubation of 10% SDS-PAGE electrophoresed PMN lysates with monoclonal anti-LF or anti-CG antibody reduced the binding with THP. To elucidate the signaling pathway of THP on PMN activation, we found THP enhanced ERK1/2 phosphorylation, reduced p38 MAP kinase phosphorylation, but had no effect on DNA binding of the five NF-κB family members in PMN. To further clarify whether the carbohydrate-side chains or protein-core structure in THP molecule is responsible for THP-PEA, THP was cleaved by different degrading enzymes with carbohydrate specificity (neuraminidase and β-galactosidase), protein specificity (V8 protease and proteinase K) or glycoconjugate specificity (carboxylpeptidase Y and O-sialoglycoprotein endopeptidase). We clearly demonstrated that the intact protein-core structure in THP molecule was more important for THP-PEA than carbohydrate-side chains. Putting these results together, we conclude that THP adheres to surface-expressed LF and CG on PMN and transduces signaling via the MAP kinase pathway to enhance PMN phagocytosis.

Nifedipine suppresses Th1/Th2 cytokine production and increased apoptosis of anti-CD3 + anti-CD28-activated mononuclear cells from patients with systemic lupus erythematosus via calcineurin pathway

Increased Ca(2+) influx is found in mononuclear cells (MNC) of patients with systemic lupus erythematosus (SLE). The role of calcineurin and potential implication of calcium channel blocker to suppress the abnormal Ca(2+) influx in SLE remain to be determined. In the present study, we found that the expression and phosphatase activity of calcineurin, but not calcineurin inhibitor in SLE-MNC were greater than normal MNC. Functionally, 1 microM nifedipine could suppress SLE-MNC IFN-gamma secretion but 10 microM nifedipine was required for suppressing that of normal MNC. IL-10 secretion by both SLE-MNC and normal MNC was suppressed by 1 microM nifedipine. However, high dose of nifedipine (50 microM) suppressed NFATc1 activation in SLE-MNC and enhanced apoptosis of anti-CD3 + anti-CD28-activated SLE-MNC irrelevant to expression of Fas ligand. These data suggest that SLE-MNC overexpressed calcineurin and hyper-responded to L-type Ca(2+) channel blocker-mediated apoptosis and cytokine suppression. We proposed that L-type Ca(2+) channel blocker maybe a potential medication for controlling SLE.

Anti-agalactosyl IgG antibody in ankylosing spondylitis and psoriatic arthritis

Anti-agalactosyl IgG antibody (anti-Gal(0) IgG) has been regarded as a useful serological marker for rheumatoid arthritis (RA). It is unknown whether it is also elevated in serum and implicated in the pathogenesis of joint inflammation in seronegative spondyloarthropathy (SpA) such as ankylosing spondylitis (AS) and psoriatic arthritis (PsA). Sera were collected from 43 patients with AS or PsA with axial joint involvement, 22 patients with RA, and 25 healthy normal individuals for the detection of anti-Gal(0) IgG with a cup-type lectin enzyme immunoassay (Eitest CA.RF). The disease activity of the AS/PsA was evaluated by Bath Ankylosing Spondylitis Disease Activity Score (BASDAI), the serum C-reactive protein (CRP) and IgA were measured by nephelometry, and erythrocyte sedimentation rate (ESR) was measured by Westergren’s method. The median titers of anti-Gal(0) IgG were significantly elevated in patients with RA (167.85, 15.73∼797.58xa0AU/mL) and AS/PsA (186.15, 34.71∼651.19xa0AU/mL), compared to those of the normal controls (13.04, 12.00∼202.43xa0AU/mL). The titers of the anti-Gal(0) IgG in patients with AS/PsA were correlated to the BASDAI scores (r2u2009=u20090.422, SEEu2009=u20091.443, pu2009<u20090.001) and serum CRP (r2u2009=u20090.345, SEEu2009=u20092.434, pu2009<u20090.001) but not to IgA (r2u2009=u20090.0259, SEEu2009=u2009126.30, pu2009<u20090.001) or ESR (r2u2009=u20090.171, SEEu2009=u200931.053, pu2009=u20090.0059). Collectively, the anti-Gal(0) IgG is elevated and vaguely correlated with the disease activity of AS/PsA although its titers in these patients were erratic. The result of the present investigation has suggested that anti-Gal(0) IgG may be more ubiquitously present in inflammatory arthritides including RA or SpA.

Anti-CD45 antibody enhances lipoxygenase pathway of human naïve mononuclear cells and cyclooxygenase pathway of neutrophils.

Abstract.Objective and Design: Anti-CD45 antibody exhibits multiple biological effects on human mononuclear cells (MNC) and polymorphonuclear neutrophils (PMN). We intended to determine whether anti-CD45 antibody could affect arachidonic acid metabolism and thereby, the interactions between human naïve MNC and PMN.Materials and Methods: Human naïve MNC and PMN were incubated with monoclonal anti-human CD45 IgG F(ab’)2 antibody or non-specific IgG F(ab’)2 for 30xa0min. The mRNA expression of cyclooxygenase type 1 (COX-1), type 2 (COX-2), 5-lipoxygenase (5-LOX) and leukotriene A4 hydrolase (LTA4 hydrolase) in both cells was detected by RT-PCR and quantified by densitometric determination. The presence of COX-1 and COX-2 molecules in the cells was detected by Western blot. The concentration of PGE2 and LTB4 in cultured supernatants was measured by EIA kits.Results: Anti-CD45 IgG F(ab’)2 up-regulated LTA4 hydrolase mRNA expression and LTB4 production, but down-regulated COX-1 and COX-2 mRNA expression and PGE2 production, of naïve MNC compared to non-specific IgG F(ab’)2. In contrast, a reverse modulation by the specific antibody on PMN was observed including up-regulation of cyclooxygenase pathway and down-regulation of lipoxygenase pathway.Conclusions: A novel activity of anti-CD45 with reverse modulation on cyclooxygenase/lipoxygenase pathways was found such that the expression of COX-1 and COX-2 in PMN, and 5-LOX and LTA4 hydrolase in MNC were enhanced.