Gouty arthritis involves impairment of autophagic degradation via cathepsin D inactivation-mediated lysosomal dysfunction that promotes apoptosis in macrophages.

This study examined autophagy-lysosome pathway (ALP) perturbations in synovial monocytes/macrophages from patients with gouty arthritis (GA) and the associations of ALP perturbations with cell death. Synovial fluid mononuclear cells (SFMCs) and synovial tissues (STs) from patients with GA, as well as monosodium urate (MSU) crystal-exposed macrophages, underwent immunoblotting, quantitative polymerase chain reaction, and immunofluorescence analyses of markers linked to the ALP (microtubule-associated protein 1 light chain 3B [LC3B], p62, cathepsin D [CTSD], and lysosome-associated membrane protein 2 [LAMP2]) and cell death (caspase-3). GA STs underwent immunohistochemistry and immunofluorescence analyses to determine the distributions of LC3B-positive autophagosomes and macrophages. GA SFMCs and STs exhibited impaired autophagic degradation, indicated by elevated levels of LC3B and p62, along with CTSD upregulation and caspase-3 activation. Macrophages from GA STs exhibited significant accumulation of LC3B-positive autophagosomes. The temporal effects of MSU crystals on the ALP and the associations of these effects with cell death were investigated using a macrophage model of GA. MSU crystal-exposed macrophages exhibited early (2 h) autophagosome formation but later (6-24 h) autophagic flux impairment, demonstrated by p62 accumulation, lysosomal inhibitor failure to increase LC3B accumulation, and LC3B colocalization with p62. These macrophages exhibited autophagic flux impairment because of CTSD inactivation-mediated lysosomal dysfunction, which caused immature CTSD to accumulate within damaged LAMP2-positive lysosomes. This accumulation coincided with caspase-3-dependent cell death (24 h) that was unaffected by CTSD inhibition. These findings indicate that GA involves MSU crystal-induced impairment of autophagic degradation via CTSD inactivation-mediated lysosomal dysfunction, which promotes apoptosis in macrophages.

Molecular Basis for Paradoxical Activities of Polymorphonuclear Neutrophils in Inflammation/Anti-Inflammation, Bactericide/Autoimmunity, Pro-Cancer/Anticancer, and Antiviral Infection/SARS-CoV-II-Induced Immunothrombotic Dysregulation.

Polymorphonuclear neutrophils (PMNs) are the most abundant white blood cells in the circulation. These cells act as the fast and powerful defenders against environmental pathogenic microbes to protect the body. In addition, these innate inflammatory cells can produce a number of cytokines/chemokines/growth factors for actively participating in the immune network and immune homeostasis. Many novel biological functions including mitogen-induced cell-mediated cytotoxicity (MICC) and antibody-dependent cell-mediated cytotoxicity (ADCC), exocytosis of microvesicles (ectosomes and exosomes), trogocytosis (plasma membrane exchange) and release of neutrophil extracellular traps (NETs) have been successively discovered. Furthermore, recent investigations unveiled that PMNs act as a double-edged sword to exhibit paradoxical activities on pro-inflammation/anti-inflammation, antibacteria/autoimmunity, pro-cancer/anticancer, antiviral infection/COVID-19-induced immunothrombotic dysregulation. The NETs released from PMNs are believed to play a pivotal role in these paradoxical activities, especially in the cytokine storm and immunothrombotic dysregulation in the recent SARS-CoV-2 pandemic. In this review, we would like to discuss in detail the molecular basis for these strange activities of PMNs.

Molecular Basis of Accelerated Aging with Immune Dysfunction-Mediated Inflammation (Inflamm-Aging) in Patients with Systemic Sclerosis.

Systemic sclerosis (SSc) is a chronic connective tissue disorder characterized by immune dysregulation, chronic inflammation, vascular endothelial cell dysfunction, and progressive tissue fibrosis of the skin and internal organs. Moreover, increased cancer incidence and accelerated aging are also found. The increased cancer incidence is believed to be a result of chromosome instability. Accelerated cellular senescence has been confirmed by the shortening of telomere length due to increased DNA breakage, abnormal DNA repair response, and telomerase deficiency mediated by enhanced oxidative/nitrative stresses. The immune dysfunctions of SSc patients are manifested by excessive production of proinflammatory cytokines IL-1, IL-6, IL-17, IFN-α, and TNF-α, which can elicit potent tissue inflammation followed by tissue fibrosis. Furthermore, a number of autoantibodies including anti-topoisomerase 1 (anti-TOPO-1), anti-centromere (ACA or anti-CENP-B), anti-RNA polymerase enzyme (anti-RNAP III), anti-ribonuclear proteins (anti-U1, U2, and U11/U12 RNP), anti-nucleolar antigens (anti-Th/T0, anti-NOR90, anti-Ku, anti-RuvBL1/2, and anti-PM/Scl), and anti-telomere-associated proteins were also found. Based on these data, inflamm-aging caused by immune dysfunction-mediated inflammation exists in patients with SSc. Hence, increased cellular senescence is elicited by the interactions among excessive oxidative stress, pro-inflammatory cytokines, and autoantibodies. In the present review, we will discuss in detail the molecular basis of chromosome instability, increased oxidative stress, and functional adaptation by deranged immunome, which are related to inflamm-aging in patients with SSc.

Cross-Talk among Polymorphonuclear Neutrophils, Immune, and Non-Immune Cells via Released Cytokines, Granule Proteins, Microvesicles, and Neutrophil Extracellular Trap Formation: A Novel Concept of Biology and Pathobiology for Neutrophils.

Polymorphonuclear neutrophils (PMNs) are traditionally regarded as professional phagocytic and acute inflammatory cells that engulf the microbial pathogens. However, accumulating data have suggested that PMNs are multi-potential cells exhibiting many important biological functions in addition to phagocytosis. These newly found novel activities of PMN include production of different kinds of cytokines/chemokines/growth factors, release of neutrophil extracellular traps (NET)/ectosomes/exosomes and trogocytosis (membrane exchange) with neighboring cells for modulating innate, and adaptive immune responses. Besides, PMNs exhibit potential heterogeneity and plasticity in involving antibody-dependent cellular cytotoxicity (ADCC), cancer immunity, autoimmunity, inflammatory rheumatic diseases, and cardiovascular diseases. Interestingly, PMNs may also play a role in ameliorating inflammatory reaction and wound healing by a subset of PMN myeloid-derived suppressor cells (PMN-MDSC). Furthermore, PMNs can interact with other non-immune cells including platelets, epithelial and endothelial cells to link hemostasis, mucosal inflammation, and atherogenesis. The release of low-density granulocytes (LDG) from bone marrow initiates systemic autoimmune reaction in systemic lupus erythematosus (SLE). In clinical application, identification of certain PMN phenotypes may become prognostic factors for severe traumatic patients. In the present review, we will discuss these newly discovered biological and pathobiological functions of the PMNs.

Trogocytosis between Non-Immune Cells for Cell Clearance, and among Immune-Related Cells for Modulating Immune Responses and Autoimmunity.

The term trogocytosis refers to a rapid bidirectional and active transfer of surface membrane fragment and associated proteins between cells. The trogocytosis requires cell-cell contact, and exhibits fast kinetics and the limited lifetime of the transferred molecules on the surface of the acceptor cells. The biological actions of trogocytosis include information exchange, cell clearance of unwanted tissues in embryonic development, immunoregulation, cancer surveillance/evasion, allogeneic cell survival and infectious pathogen killing or intercellular transmission. In the present review, we will extensively review all these aspects. In addition to its biological significance, aberrant trogocytosis in the immune system leading to autoimmunity and immune-mediated inflammatory diseases will also be discussed. Finally, the prospective investigations for further understanding the molecular basis of trogocytosis and its clinical applications will also be proposed.

The Potential Role of Genetics, Environmental Factors, and Gut Dysbiosis in the Aberrant Non-Coding RNA Expression to Mediate Inflammation and Osteoclastogenic/Osteogenic Differentiation in Ankylosing Spondylitis.

Ankylosing spondylitis (AS) or radiographic axial spondyloarthritis is a chronic immune-mediated rheumatic disorder characterized by the inflammation in the axial skeleton, peripheral joints, and soft tissues (enthesis, fascia, and ligament). In addition, the extra-skeletal complications including anterior uveitis, interstitial lung diseases and aortitis are found. The pathogenesis of AS implicates an intricate interaction among HLA (HLA-B27) and non-HLA loci [endoplasmic reticulum aminopeptidase 1 (ERAP1), and interleukin-23 receptor (IL23R), gut dysbiosis, immune plasticity, and numerous environmental factors (infections, heavy metals, stress, cigarette smoking, etc.) The latter multiple non-genetic factors may exert a powerful stress on epigenetic regulations. These epigenetic regulations of gene expression contain DNA methylation/demethylation, histone modifications and aberrant non-coding RNAs (ncRNAs) expression, leading to inflammation and immune dysfunctions. In the present review, we shall discuss these contributory factors that are involved in AS pathogenesis, especially the aberrant ncRNA expression and its effects on the proinflammatory cytokine productions (TNF-α, IL-17 and IL-23), T cell skewing to Th1/Th17, and osteoclastogenic/osteogenic differentiation. Finally, some potential investigatory approaches are raised for solving the puzzles in AS pathogenesis.

The Cellular and Molecular Bases of Allergy, Inflammation and Tissue Fibrosis in Patients with IgG4-related Disease.

IgG4-related disease (IgG4-RD) is a spectrum of complex fibroinflammatory disorder with protean manifestations mimicking malignant neoplasms, infectious or non-infectious inflammatory process. The histopathologic features of IgG4-RD include lymphoplasmacytic infiltration, storiform fibrosis and obliterative phlebitis together with increased in situ infiltration of IgG4 bearing-plasma cells which account for more than 40% of all IgG-producing B cells. IgG4-RD can also be diagnosed based on an elevated serum IgG4 level of more than 110 mg/dL (normal < 86.5 mg/mL in adult) in conjunction with protean clinical manifestations in various organs such as pancreato-hepatobiliary inflammation with/without salivary/lacrimal gland enlargement. In the present review, we briefly discuss the role of genetic predisposition, environmental factors and candidate autoantibodies in the pathogenesis of IgG4-RD. Then, we discuss in detail the immunological paradox of IgG4 antibody, the mechanism of modified Th2 response for IgG4 rather than IgE antibody production and the controversial issues in the allergic reactions of IgG4-RD. Finally, we extensively review the implications of different immune-related cells, cytokines/chemokines/growth factors and Toll-like as well as NOD-like receptors in the pathogenesis of tissue fibro-inflammatory reactions. Our proposals for the future investigations and prospective therapeutic strategies for IgG4-RD are shown in the last part.

Pathogenic Roles of Autoantibodies and Aberrant Epigenetic Regulation of Immune and Connective Tissue Cells in the Tissue Fibrosis of Patients with Systemic Sclerosis.

Systemic sclerosis (SSc) is a multi-system autoimmune disease with tissue fibrosis prominent in the skin and lung. In this review, we briefly describe the autoimmune features (mainly autoantibody production and cytokine profiles) and the potential pathogenic contributors including genetic/epigenetic predisposition, and environmental factors. We look in detail at the cellular and molecular bases underlying tissue-fibrosis which include trans-differentiation of fibroblasts (FBs) to myofibroblasts (MFBs). We also state comprehensively the pro-inflammatory and pro-fibrotic cytokines relevant to MFB trans-differentiation, vasculopathy-associated autoantibodies, and fibrosis-regulating microRNAs in SSc. It is conceivable that tissue fibrosis is mainly mediated by an excessive production of TGF-ß, the master regulator, from the skewed Th2 cells, macrophages, fibroblasts, myofibroblasts, and keratinocytes. After binding with TGF-ß receptors on MFB, the downstream Wnt/ß-catenin triggers canonical Smad 2/3 and non-canonical Smad 4 signaling pathways to transcribe collagen genes. Subsequently, excessive collagen fiber synthesis and accumulation as well as tissue fibrosis ensue. In the later part of this review, we discuss limited data relevant to the role of long non-coding RNAs (lncRNAs) in tissue-fibrosis in SSc. It is expected that these lncRNAs may become the useful biomarkers and therapeutic targets for SSc in the future. The prospective investigations in the development of novel epigenetic modifiers are also suggested.

Efficacy and safety of rituximab in autoimmune and microangiopathic hemolytic anemia: a systematic review and meta-analysis.

BACKGROUND: The efficacy and safety of rituximab (RTX) on hemolytic anemia (HA) is unknown. Therefore we retrospectively analyze the efficacy and safety of RTX in autoimmune hemolytic anemia (AIHA) and microangiopathic hemolytic anemia (MAHA) from the previous literature. METHODS: Data in clinical trials and observational studies were collected from PubMed, Cochrane, Embase, and Google Scholar until Oct 15, 2018. The efficacy and safety of RTX in patients with AIHA or MAHA were assessed and overall response rates (ORRs), complete response rates (CRRs), adverse events (AEs) and relapse rates (RRs) were extracted if available. A meta-analysis was performed with a random-effects model, estimating mean proportions in all studies, and relative rates in comparative studies. RESULTS: After quality assessment, a total of 37 investigations encompassing 1057 patients eligible for meta-analysis were included. Pooled mean proportion of ORR was 0.84 (95% confidence interval [CI] 0.80-0.88), and that of CRR was 0.61 (95% CI 0.49-0.73). Mean AE rate was 0.14 (95% CI 0.10-0.17), and mean RR was 0.21 (95% CI 0.15-0.26). Relative ORR was 1.18 (95% CI 1.02-1.36), and relative CRR was 1.17 (95% CI 0.98-1.39) fold more than the respective non-RTX counter parts. Relative AE rate was 0.77 (95% CI 0.36-1.63), and relative RR was 0.93 (95% CI 0.56-1.55) fold less than the respective non-RTX counter parts. CONCLUSION: RTX is more effective than the treatments without RTX for AIHA and MAHA and is well-tolerated.

Advanced Glycation End Products of Bovine Serum Albumin Suppressed Th1/Th2 Cytokine but Enhanced Monocyte IL-6 Gene Expression via MAPK-ERK and MyD88 Transduced NF-κB p50 Signaling Pathways.

Advanced glycation end products (AGE), the most known aging biomarker, may cause "inflamm-aging" (i.e., chronic low-grade inflammation that develops with aging) in both aged and diabetes groups. However, the molecular bases of inflamm-aging remain obscure. We prepared AGE by incubating BSA (0.0746 mmol/L) + glucose (0.5 mol/L) at 37 °C in 5% CO2-95% air for 1-180 days. The lysine glycation in BSA-AGE reached 77% on day 30 and 100% after day 130, whereas the glycation of arginine and cysteine was minimal. The Nε-(carboxymethyl)-lysine content in BSA-AGE was also increased with increasing number of incubation days. The lectin-binding assay revealed that the glycation of BSA not only altered the conformational structure, but lost binding capacity with various lectins. An immunological functional assay showed that BSA-AGE > 8 µg/mL significantly suppressed normal human Th1 (IL-2 and IFN-γ) and Th2 (IL-10) mRNA expression, whereas AGE > 0.5 µg/mL enhanced monocyte IL-6 production irrelevant to cell apoptosis. The AGE-enhanced monocyte IL-6 production was via MAPK-ERK and MyD88-transduced NF-κBp50 signaling pathways. To elucidate the structure-function relationship of BSA-AGE-enhanced IL-6 production, we pre-preincubated BSA-AGE with different carbohydrate-degrading, protein-degrading, and glycoprotein-degrading enzymes. We found that trypsin and carboxypeptidase Y suppressed whereas ß-galactosidase enhanced monocyte IL-6 production. In conclusion, BSA-AGE exerted both immunosuppressive and pro-inflammatory effects that are the molecular basis of inflamm-aging in aged and diabetes groups.

Immunological dysfunction in chronic arsenic exposure: From subclinical condition to skin cancer.

Exposure to arsenic is a global health issue. Long-term arsenic exposure may associate with various cancers and many other pathological effects. Over 100 million people worldwide are exposed to arsenic particularly in countries such as Bangladesh, Chile, China, India, Mexico, Taiwan and the USA. Drinking of water contaminated with arsenic is the major route of human exposure. Skin lesions are considered to be the most common adverse effects associated with chronic arsenic exposure. Skin lesions usually develop with the latency period spanning more than 20 years from first exposure. Arsenic-induced Bowen's disease, the most frequently encountered carcinoma in situ resulting from chronic arsenic exposure, is characterized by multiple and recrudescent lesions. Long-term arsenic exposure results in impaired immunity in susceptible individuals. In the prenatal stage, enhanced placental inflammatory responses and reduced placental T cells by arsenic may result in decreased thymic size and functions in newborns. In childhood, arsenic exposure may reduce peripheral CD4+ cells and interleukin-2 secretion which leads to susceptibility to opportunistic infections. There was an impairment of macrophage function and oxidative DNA damage of peripheral polymorphonuclear leukocytes in adults with skin lesions. In arsenic-induced Bowen's disease lesions, a decrease in the number and functions of Langerhans cells and, in parallel, a selective CD4+ cell apoptosis was noticed. These findings provide scientific evidence for understanding the phenomenon of arsenic-induced immune escape in the early stage of carcinogenesis and a reasonable explanation for multiple and recrudescent arsenic cancers in the skin.

Tamm-Horsfall Protein is a Potent Immunomodulatory Molecule and a Disease Biomarker in the Urinary System.

Tamm-Horsfall protein (THP), or uromodulin (UMOD), is an 80-90-kDa phosphatidylinositol-anchored glycoprotein produced exclusively by the renal tubular cells in the thick ascending limb of the loop of Henle. Physiologically, THP is implicated in renal countercurrent gradient formation, sodium homeostasis, blood pressure regulation, and a defense molecule against infections in the urinary system. Investigations have also revealed that THP is an effective binding ligand for serum albumin, immunoglobulin G light chains, complement components C1 and C1q, interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor (TNF)-α, and interferon-γ through its carbohydrate side chains for maintaining circulatory and renal immune homeostasis. Thus, THP can be regarded as part of the innate immune system. UMOD mutations play crucial roles in congenital urolithiasis, hereditary hyperuricemia/gout, and medullary cystic kidney diseases. Recent investigations have focused on the immunomodulatory effects of THP on immune cells and on THP as a disease biomarker of acute and chronic kidney diseases. Our studies have suggested that normal urinary THP, through its epidermal growth factor (EGF)-like domains, binds to the surface-expressed EGF-like receptors, cathepsin G, or lactoferrin to enhance polymorphonuclear leukocyte phagocytosis, proinflammatory cytokine production by monocytes/macrophages, and lymphocyte proliferation by activating the Rho family and mitogen-activated protein kinase signaling pathways. Furthermore, our data support both an intact protein core structure and carbohydrate side chains are important for the different protein-binding capacities of THP. Prospectively, parts of the whole THP molecule may be used for anti-TNF-α therapy in inflammatory diseases, autoantibody-depleting therapy in autoimmune disorders, and immune intensification in immunocompromised hosts.

Anti-citrullinated protein antibodies promote apoptosis of mature human Saos-2 osteoblasts via cell-surface binding to citrullinated heat shock protein 60.

We hypothesized that anti-citrullinated protein antibodies (ACPAs) react with osteoblast surface citrullinated proteins and affect cell function, leading to joint damage in patients with rheumatoid arthritis (RA). First, we purified ACPAs by cyclic citrullinated peptide (CCP)-conjugated affinity column chromatography. The cognate antigens of ACPAs on Saos-2 cells, a sarcoma osteogenic cell line generated from human osteoblasts, were probed by ACPAs, and the reactive bands were analyzed using proteomic analyses. We found that ACPAs bind to Saos-2 cell membrane, and several protein candidates, including HSP60, were identified. We then cloned and purified recombinant heat shock protein 60 (HSP60) and citrullinated HSP60 (citHSP60) and investigated the effect of ACPAs on Saos-2 cell. We confirmed that HSP60 obtained from Saos-2 cell membrane were citrullinated and reacted with ACPAs, which induces Saos-2 cells apoptosis via binding to surface-expressed citHSP60 through Toll-like receptor 4 signaling. ACPAs promoted interleukin (IL)-6 and IL-8 expression in Saos-2 cells. Finally, sera from patients with RA and healthy controls were examined for their titers of anti-HSP60 and anti-citHSP60 antibodies using an enzyme-linked immunosorbent assay. The radiographic change in patients with RA was evaluated using the Genant-modified Sharp scoring system. Patients with RA showed higher sera titers of anti-citHSP60, but not anti-HSP60, antibodies when compared with controls. In addition, the anti-citHSP60 level was positively associated with increased joint damage in patients with RA. In conclusion, Saos-2 cell apoptosis was mediated by ACPAs via binding to cell surface-expressed citHSP60 and the titer of anti-citHSP60 in patients with RA positively associated with joint damage.

Use of HLA-B*58:01 genotyping to prevent allopurinol induced severe cutaneous adverse reactions in Taiwan: national prospective cohort study.

OBJECTIVE: To evaluate the use of prospective screening for the HLA-B*58:01 allele to identify Taiwanese individuals at risk of severe cutaneous adverse reactions (SCARs) induced by allopurinol treatment. DESIGN: National prospective cohort study. SETTING: 15 medical centres in different regions of Taiwan, from July 2009 to August 2014. PARTICIPANTS: 2926 people who had an indication for allopurinol treatment but had not taken allopurinol previously. Participants were excluded if they had undergone a bone marrow transplant, were not of Han Chinese descent, and had a history of allopurinol induced hypersensitivity. DNA purified from 2910 participants' peripheral blood was used to assess the presence of HLA-B*58:01. MAIN OUTCOME MEASURES: Incidence of allopurinol induced SCARs with and without screening. RESULTS: Participants who tested positive for HLA-B*58:01 (19.6%, n=571) were advised to avoid allopurinol, and were referred to an alternate drug treatment or advised to continue with their prestudy treatment. Participants who tested negative (80.4%, n=2339) were given allopurinol. Participants were interviewed once a week for two months to monitor symptoms. The historical incidence of allopurinol induced SCARs, estimated by the National Health Insurance research database of Taiwan, was used for comparison. Mild, transient rash without blisters developed in 97 (3%) participants during follow-up. None of the participants was admitted to hospital owing to adverse drug reactions. SCARs did not develop in any of the participants receiving allopurinol who screened negative for HLA-B*58:01. By contrast, seven cases of SCARs were expected, based on the estimated historical incidence of allopurinol induced SCARs nationwide (0.30% per year, 95% confidence interval 0.28% to 0.31%; P=0.0026; two side one sample binomial test). CONCLUSIONS: Prospective screening of the HLA-B*58:01 allele, coupled with an alternative drug treatment for carriers, significantly decreased the incidence of allopurinol induced SCARs in Taiwanese medical centres.

Anti-citrullinated protein antibodies suppress let-7a expression in monocytes from patients with rheumatoid arthritis and facilitate the inflammatory responses in rheumatoid arthritis.

We hypothesized that anti-citrullinated protein antibodies (ACPAs) could affect the expression of miRNAs in monocytes and contribute to the inflammatory responses in rheumatoid arthritis (RA). The expression profiles of 270 human miRNAs, co-cultured with ACPAs or human immunoglobulin G (IgG), were analyzed using real-time polymerase chain reaction. Ten miRNAs exhibited differential expression in U937 cells after co-cultured with ACPAs compared with human IgG. The expression levels of these miRNAs were investigated in monocytes from 21 ACPA-positive RA patients and 13 controls. Among these miRNAs, the expression levels of let-7a was decreased in monocytes from ACPA-positive RA patients. The expression levels of let-7a showed a negative correlation with positivity of rheumatoid factor in patients sampled. We found that transfection of U937 cells with let-7a mimic suppressed K-Ras protein expression. In the ACPA-mediated signaling pathway, transfection of U937 cells with let-7a mimic suppressed the ACPA-enhanced phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), and the expression and secretion of interleukin (IL)-1ß. In conclusion, ACPA-mediated decreased let-7a expression in monocytes from ACPA-positive RA patients. Decreased let-7a expression was associated with the positivity of RF in ACPA-positive RA patients. The decreased expression of let-7a could facilitate the inflammatory pathway via enhanced ACPA-mediated phosphorylation of ERK1/2 and JNK and increased expression of IL-1ß through an increase in the expression of Ras proteins.

Sjögren's Syndrome Antigen B Acts as an Endogenous Danger Molecule to Induce Interleukin-8 Gene Expression in Polymorphonuclear Neutrophils.

BACKGROUND: Sjögren's syndrome antigen B is expressed in the nucleus and surface membrane of human polymorphonuclear neutrophils and is released after cell death. However, its biological role is not clear. This study is aimed to investigate the effect of Sjögren's syndrome antigen B on human polymorphonuclear neutrophils. METHODS: Human recombinant Sjögren's syndrome antigen B (rSSB) purified from E. coli was incubated with human polymorphonuclear neutrophils as well as retinoid acid-induced granulocytic differentiated HL-60 cells, HL-60 (RA). Interleukin (IL)-8 protein production and mRNA expressions were measured by enzyme-linked immunosorbent assay and quantitative-polymerase chain reaction, respectively. Uptake of fluorescein isothiocyanate (FITC)-rSSB was assessed by flow cytometry and fluorescence microscopy. Moreover, mitogen-activated protein kinase (MAPK) pathways and nuclear factor-kappaB activation were investigated. RESULTS: Human rSSB stimulated IL-8 production from normal human neutrophils and HL-60 (RA) cells in a time- and dose-dependent manner. This IL-8-stimulated activity was blocked by chloroquine and NH4Cl, indicating that endosomal acidification is important for this effect. We found rSSB activated both MAPK pathway and nuclear factor-kappaB signaling to transcribe the IL-8 gene expression of cells. Furthermore, tumor necrosis factor-α exerted an additive effect and rSSB-anti-SSB immune complex exhibited a synergistic effect on rSSB-induced IL-8 production. CONCLUSIONS: Sjögren's syndrome antigen B might act as an endogenous danger molecule to enhance IL-8 gene expression in human polymorphonuclear neutrophils.

EGF receptor-dependent mechanism may be involved in the Tamm-Horsfall glycoprotein-enhanced PMN phagocytosis via activating Rho family and MAPK signaling pathway.

Our previous studies showed that urinary Tamm-Horsfall glycoprotein (THP) potently enhanced polymorphonuclear neutrophil (PMN) phagocytosis. However, the domain structure(s), signaling pathway and the intracellular events responsible for THP-enhanced PMN phagocytosis remain to be elucidated. THP was purified from normal human urine. The human promyelocytic leukemia cell line HL-60 was induced to differentiate into PMNs by all-trans retinoid acid. Pretreatment with different MAPK and PI3K inhibitors was used to delineate signaling pathways in THP-enhanced PMN phagocytosis. Phosphorylation of molecules responsible for PMN phagocytosis induced by bacterial lipopolysaccharide (LPS), THP, or human recombinant epidermal growth factor (EGF) was evaluated by western blot. A p38 MAPK inhibitor, SB203580, effectively inhibited both spontaneous and LPS- and THP-induced PMN phagocytosis. Both THP and LPS enhanced the expression of the Rho family proteins Cdc42 and Rac that may lead to F-actin re-arrangement. Further studies suggested that THP and EGF enhance PMN and differentiated HL-60 cell phagocytosis in a similar pattern. Furthermore, the EGF receptor inhibitor GW2974 significantly suppressed THP- and EGF-enhanced PMN phagocytosis and p38 and ERK1/2 phosphorylation in differentiated HL-60 cells. We conclude that EGF receptor-dependent signaling may be involved in THP-enhanced PMN phagocytosis by activating Rho family and MAP kinase.

Anti-citrullinated protein antibodies activated ERK1/2 and JNK mitogen-activated protein kinases via binding to surface-expressed citrullinated GRP78 on mononuclear cells.

In a previous study, we found that anti-citrullinated protein antibodies (ACPAs) enhance nuclear factor (NF)-κB activity and tumor necrosis factor (TNF)-α production by normal human peripheral blood mononuclear cells (PBMCs) and U937 cells via binding to surface-expressed citrullinated glucose-regulated protein 78 (cit-GRP78). However, the downstream signaling pathways remain unclear after binding. In the present study, we firstly measured the effects of different kinase inhibitors on ACPA-mediated TNF-α production from normal PBMCs and monocytes. Then, the native and phosphorylated mitogen-activated protein kinases (MAPKs) were detected in ACPA-activated U937 cells by Western blotting. We also explored the role of the phosphoinositide 3-kinase (PI3K)-Akt pathway in activating IκB kinase alpha (IKK-α) in ACPA-stimulated U937 cells. Finally, we measured the amount of cit-GRP78 from PBMC membrane extracts in RA patients and controls. We found that MAPK and Akt inhibitors, but not PI3K inhibitor, remarkably suppressed ACPA-mediated TNF-α production. Interestingly, ACPAs selectively activated extracellular signal-regulated kinase 1/2 (ERK1/2) and c-jun N-terminal kinase (JNK), but not p38 MAPK, in U937 cells. This activation was suppressed by cit-GRP78, but not GRP78. The JNK activation further enhanced the phosphorylation of Akt and IKK-α. The expression of cit-GRP78 on cell membrane was higher in RA than normal PBMCs. Taken together; these results suggest that through binding to surface, over-expressed cit-GRP78 on RA PBMCs, ACPAs selectively activate ERK1/2 and JNK signaling pathways to enhance IKK-α phosphorylation, which leads to the activation of NF-κB and the production of TNF-α .

The binding affinity and molecular basis of the structure-binding relationship between urinary Tamm-Horsfall glycoprotein and tumor necrosis factor-α.

In a previous study we noted significant THP binding to TNF-α, but did not explore the molecular basis of the structure-binding relationship. In this study, we used lectin-binding ELISA to assess the carbohydrate compositions of THP, BSA, IgG, TNF-α, and IFN-g. We identified ß(1,4)-N-acetylglucosamine oligomers (GlcNAc) and GlcNAc/branched mannose in BSA, IgG, TNF-α, and THP, but not in IFN-g. These carbohydrate moieties mediated binding with THP. Small amounts of Siaα(2,3)Gal/ GalNAc, Sia(2,6)Gal/GalNAc, and mannose residues were also present in THP and TNF-α. Binding affinity (K(d)) between THP and TNF-α by Scatchard plot analysis was 1.4-1.7 × 10⁻6 M, lower than antigen-antibody or ligand-receptor binding affinities. To elucidate the structure-binding relationship of THP-TNF-α, THP was digested with neuraminidase, ß-galactosidase, O-sialoglycoprotein endopeptidase, carboxypeptidase Y, or proteinase K. ß-galactosidase increased binding capacity of THP for TNF-α. Monosaccharide inhibition suggested that α-methyl-D-mannoside, GlcNAc, and GalNAc, but not sialic acid, suppress THP-TNF-α binding as detected by ELISA. We conclude that sugar-lectin and sugar-protein interactions between cognate sites in THP and TNF-α mediate their binding.

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-ß1, 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.