Gasdermin E-mediated keratinocyte pyroptosis participates in the pathogenesis of psoriasis by promoting skin inflammation.

BACKGROUND: Psoriasis is a common chronic inflammatory disease with an unclear aetiology. Keratinocytes in psoriasis are susceptible to exogenous triggers that induce inflammatory cell death. OBJECTIVES: To investigate whether gasdermin E (GSDME)-mediated pyroptosis in keratinocytes contributes to the pathogenesis of psoriasis. METHODS: Skin samples from patients with psoriasis and from healthy controls were collected to evaluate the expression of GSDME, cleaved caspase-3 and inflammatory factors. We then analysed the data series GSE41662 to further compare the expression of GSDME between lesional and nonlesional skin samples in those with psoriasis. In vivo, a caspase-3 inhibitor and GSDME-deficient mice (Gsdme-/-) were used to block caspase-3/GSDME activation in an imiquimod-induced psoriasis model. Skin inflammation, disease severity and pyroptosis-related proteins were analysed. In vitro, tumour necrosis factor (TNF)-α-induced caspase-3/GSDME-mediated pyroptosis in the HACAT cell line was explored. RESULTS: Our analysis of the GSE41662 data series found that GSDME was upregulated in psoriasis lesions vs. normal skin. High levels of inflammatory cytokines such as interleukin (IL)-1ß, IL-6 and TNF-α were also found in psoriasis lesions. In mice in the Gsdme-/- and caspase-3 inhibitor groups, the severity of skin inflammation was attenuated and GSDME and cleaved caspase-3 levels decreased after imiquimod treatment. Similarly, IL-1ß, IL-6 and TNF-α expression was decreased in the Gsdme-/- and caspase-3 inhibitor groups. In vitro, TNF-α induced HACAT cell pyroptosis through caspase-3/GSDME pathway activation, which was suppressed by blocking caspase-3 or silencing Gsdme. CONCLUSIONS: Our study provides a novel explanation of TNF-α/caspase-3/GSDME-mediated keratinocyte pyroptosis in the initiation and -acceleration of skin inflammation and the progression of psoriasis.

Psoriasis is chronic and autoinflammatory common skin disease that affects 2­3% of the world's population. The disease is characterized by persistent inflammation in various body systems, including the skin and joints. However, the exact cause of the disease is unclear. In this study from China, we found that in people with psoriasis a protein called 'gasdermin E' (or 'GSDME') is increased in a type of skin cell called keratinocytes. In psoriasis, these keratinocytes are susceptible to a type of cell death called 'pyroptosis'. We aimed to find out whether pyroptosis caused by GSDME in keratinocytes contributes to the development of psoriasis. To do this, we looked at samples of skin from people with psoriasis and compared these to samples from healthy controls (those without psoriasis). Firstly, we investigated the levels of GSDME, another protein called caspase-3 and other inflammatory factors in the skin lesions from patients with psoriasis. Secondly, we analysed previously published data from 24 patients with psoriasis. Finally, we carried out a range of experiments to confirm our findings. We found that keratinocyte pyroptosis was mediated by the messenger proteins TNF-α/caspase-3, and that GSDME played a key role in the initiation and acceleration of skin inflammation and the progression of psoriasis. Targeting the GSDME pathway may be a novel strategy in treating psoriasis.

Polydatin protects against gouty nephropathy by inhibiting renal tubular cell pyroptosis.

OBJECTIVE: To investigate the protective effect and mechanism of polydatin (PD) against gouty nephropathy (GN) in mice. METHODS: Twenty-four mice were randomly divided into three groups: the control group (no treatment), the GN group (300 mg/kg hypoxanthine + 150 mg/kg potassium oxonate), and the GN + PD group (300 mg/kg hypoxanthine + 150 mg/kg potassium oxonate + 50 mg/kg PD). Histological changes in the kidneys and the levels of uric acid (UA), blood urea nitrogen (BUN), and serum creatinine (SCr) in the sera were measured. In addition, the expression of gasdermin D (GSDMD) protein in renal tubular epithelial cells, and the expression of NOD-like receptor protein 3 (NLRP3), GSDMD, and caspase-1 proteins in the kidney tissues were determined by immunohistochemistry, immunofluorescence, and Western blot. RESULTS: In vitro, PD inhibited the expression of NLRP3, caspase-1, and GSDMD and protected the renal tubular epithelial cells from pyroptosis. In vivo, PD treatment significantly ameliorated the pathological changes in kidney tissue, and reversed the decrease of serum UA and BUN in GN model mice. The expression of NLRP3, GSDMD, and caspase-1 proteins was also decreased in the PD-treated GN mice. CONCLUSION: The results suggest that PD has a protective effect on mice with GN, which may be related to the downregulation of NLRP3, GSDMD, and caspase-1 proteins and the inhibition of renal tubular epithelial cells pyroptosis.

Blocking GSDME-mediated pyroptosis in renal tubular epithelial cells alleviates disease activity in lupus mice.

An increase in apoptosis and/or defects in the clearance of apoptotic cells resulting in massive secondary necrosis have been recognized as the main causes of systemic lupus erythematosus (SLE). Recent findings have revealed that gasdermin E (GSDME)-mediated pyroptosis is a mechanism associated with secondary necrosis. We aimed to investigate the effects of GSDME-mediated pyroptosis on disease activity in lupus mice. In vivo, high levels of GSDME expression were observed in the renal tubules of pristane-induced lupus (PIL) mice and SLE patients. In lupus mice, GSDME knockout or SP600125 administration effectively ameliorated lupus-like features by inhibiting GSDME-mediated renal tubular epithelial cell pyroptosis. In vitro, treatment with tumour necrosis factor-α (TNF-α) plus cycloheximide (CHX) or SLE sera induced HK2 cells to undergo pyroptosis in a caspase-3- and GSDME-dependent manner. Likewise, SP600125 significantly reduced GSDME expression and decreased pyroptosis in HK2 cells. GSDME-mediated pyroptosis may be associated with SLE pathogenesis, and targeting GSDME may be a potential strategy for treating SLE.

Attenuation of Rheumatoid Arthritis Through the Inhibition of Tumor Necrosis Factor-Induced Caspase 3/Gasdermin E-Mediated Pyroptosis.

OBJECTIVE: To determine the role of gasdermin E (GSDME)-mediated pyroptosis in the pathogenesis and progression of rheumatoid arthritis (RA), and to explore the potential of GSDME as a therapeutic target in RA. METHODS: The expression and activation of caspase 3 and GSDME in the synovium, macrophages, and monocytes of RA patients were determined by immunohistochemistry, immunofluorescence, and Western blot analysis. The correlation of activated GSDME with RA disease activity was evaluated. The pyroptotic ability of monocytes from RA patients was tested, and the effect of tumor necrosis factor (TNF) on caspase 3/GSDME-mediated pyroptosis of monocytes and macrophages was investigated. In addition, collagen-induced arthritis (CIA) was induced in mice lacking Gsdme, and the incidence and severity of arthritis were assessed. RESULTS: Compared to cells from healthy controls, monocytes and synovial macrophages from RA patients showed increased expression of activated caspase 3, GSDME, and the N-terminal fragment of GSDME (GSDME-N). The expression of GSDME-N in monocytes from RA patients correlated positively with disease activity. Monocytes from RA patients with higher GSDME levels were more susceptible to pyroptosis. Furthermore, TNF induced pyroptosis in monocytes and macrophages by activating the caspase 3/GSDME pathway. The use of a caspase 3 inhibitor and silencing of GSDME significantly blocked TNF-induced pyroptosis. Gsdme deficiency effectively alleviated arthritis in a mouse model of CIA. CONCLUSION: These results support the notion of a pathogenic role of GSDME in RA and provide an alternative mechanism for RA pathogenesis involving TNF, which activates GSDME-mediated pyroptosis of monocytes and macrophages in RA. In addition, targeting GSDME might be a potential therapeutic approach for RA.

Is hydroxychloroquine beneficial for COVID-19 patients?

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in December 2019. As similar cases rapidly emerged around the world1-3, the World Health Organization (WHO) declared a public health emergency of international concern on January 30, 2020 and pronounced the rapidly spreading coronavirus outbreak as a pandemic on March 11, 20204. The virus has reached almost all countries of the globe. As of June 3, 2020, the accumulated confirmed cases reached 6,479,405 with more than 383,013 deaths worldwide. The urgent and emergency care of COVID-19 patients calls for effective drugs, in addition to the beneficial effects of remdesivir5, to control the disease and halt the pandemic.

[Hydroxychloroquine alleviates 5-fluorouracil-induced enteritis in mice and its mechanism].

Objective To investigate the effect of hydroxychloroquine (HCQ) on 5-fluorouracil (5-FU)-induced enteritis in mice and its mechanism. Methods Thirty C57B6/J mice were randomly divided into 0-, 1-, 3-, 5- and 7-day groups and sacrificed separately at day 0, 1, 3, 5, and 7 after intraperitoneal administration of 5-FU 200 µL/d (50 mg/kg) at day 1-3. The double-stranded DNA (dsDNA) levels in serum and small intestinal fluid were detected by dsDNA quantification kit. Severity of enteritis was evaluated by diarrhea score. HCT-116 cells were cultured in vitro and treated with 5-FU at different concentrations (0, 0.01, 0.1, 0.5, 1, 10 µmol/L) for 72 hours or 5-FU at 1 µmol/L for different time (24, 48, 72 hours). The dsDNA concentration in the cell culture supernatant of each group was detected by dsDNA quantification kit. Twenty-four C57B6/J mice were randomly divided into 4 groups: control group (intraperitoneal injection and intragastric administration of 200 µL/d saline), model group (intraperitoneal injection of 50 mg/kg 5-FU of 200 µL/d, intragastric administration of saline 200 µL/d), HCQ treatment group (intragastric administration of 60 mg/kg HCQ of 200 µL/d, starting at 1 day before the first intraperitoneal injection of 50 mg/kg 5-FU of 200 µL/d) and HCQ group (intragastric administration of 60 mg/kg HCQ solution of 200 µL/d). And they were sacrificed after 6 days. Small intestine lesions were observed by HE staining. Apoptotic cells in the small intestine were detected by TUNEL staining. The expression levels of CD11c, Toll-like receptor 9 (TLR9) and nuclear factor κB (NF-κB) in the small intestine were assessed by immunofluorescence staining. Interleukin-1ß (IL-1ß) levels in the serum were detected by ELISA. Mouse bone marrow-derived dendritic cells (BMDCs) were cultured in vitro and stimulated by dsDNA using LipofectamineTM 3000. The expression of TLR9 and NF-κB in BMDCs were detected by Western blotting and immunofluorescent staining, respectively. IL-1ß level in the cell supernatant was detected by ELISA. Results Large amounts of apoptotic cells were observed in the small intestine of 5-FU-treated mice and the dynamic changes of dsDNA levels in the serum and small intestinal lavage fluid were consistent with that of diarrhea score. 5-FU triggered dsDNA release from HCT-116 cells in a dose- and time-dependent manner. HCQ alleviated the destruction in small intestinal epithelium and inhibited the expression levels of TLR9, NF-κB and IL-1ß in the serum. The infiltration of a large number of dendritic cells in the small intestine of model mice was observed. After BMDCs were stimulated with dsDNA, the expression of TLR9, NF-κB, and IL-1ß all significantly increased and HCQ significantly decreased. Conclusion HCQ alleviates 5-FU-induced enteritis in mice and inhibit TLR9 and NF-κB-dependent DNA sensing pathways and the secretion of IL-1ß in dendritic cells.

Bronchial epithelial pyroptosis promotes airway inflammation in a murine model of toluene diisocyanate-induced asthma.

Airway epithelial injury in response to allergens such as toluene diisocyanate (TDI) leads to persistent airway inflammation. Pyroptosis is recognized as a strong proinflammatory cell death process. However, the role of pyroptosis in bronchial epithelial injury and airway inflammation in TDI-induced asthma remains unknown. In this study, cytotoxic effect of TDI on 16HBE cells (a human bronchial epithelial cell line) was detected. Then a TDI-induced experimental asthma mouse model was established for in vivo study. Here we found that TDI induced pyroptosis in 16HBE cells, as evidenced by enhanced expressions of caspase-1 and elevated levels of LDH, IL-1ß and HMGB1. As expected, TDI-induced inflammatory cell death was significantly blocked by a specific NLRP3 inflammasome inhibitor. Intriguingly, in asthmatic mice, the increased cleavages of caspase-1 and pyroptotic executioner gasdermin D (GSDMD) in bronchial epithelial cells were decreased by NLRP3 inflammasome inhibitor. Furthermore, inhibition of NLRP3 inflammasome attenuated airway hyper-responsiveness and airway inflammation, accompanied by lower levels of IL-1ß, IgE and Th2-related cytokines. Our data suggest that bronchial epithelial pyroptosis exacerbates airway inflammation and hyper-responsiveness in TDI-induced asthma via NLRP3 inflammasome activation and GSDND cleavage. Therefore, NLRP3 inflammasome-mediated pyroptosis may be a potential treatment target for TDI-induced asthma.

Not only anti-inflammation, etanercept abrogates collagen-induced arthritis by inhibiting dendritic cell migration and maturation.

The application of tumor necrosis factor inhibitors (TNFi) is a major breakthrough in the treatment of rheumatoid arthritis (RA). While the anti-inflammatory nature of TNFi is thought to contribute to the therapeutic effects, recent data show that the pharmacology of TNF-α blockade is probably more complex than previously thought. This study investigates whether etanercept (ETN), one of the TNF antagonists, suppresses arthritis development through modulation of dendritic cell (DC) functions. Bone marrow-derived DCs (BMDCs) were stimulated with lipopolysaccharide (LPS) and treated with ETN for 24 hrs. DC functions, including maturation and migration, were determined. DCs from the lymph nodes (LNs) of ETN-treated collagen-induced arthritis (CIA) mice were analyzed for phenotypes and subsets. ETN efficiently inhibited the phenotypic maturation both in vitro and in vivo. ETN treatment delayed the onset and reduced the severity of arthritis in CIA mice. Moreover, ETN treatment strongly down regulated the number of both myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in LNs, possibly due to the depressive effect on the expression of CXCR4 on DCs in peripheral blood. The impaired DC migration to local LNs by ETN down regulated the number of T cells and B cells, and changed the LN cellular composition. The data show that TNF-α blockade has profound effects on DC maturation and migration, which may contribute to its immune regulatory effects in RA patients.

[MRP8, MRP14 and MRP8/14 promote phenotypic maturation of mouse bone marrow-derived dendritic cells in vitro].

Objective To investigate the effects of myeloid-related protein 8 (MRP8), MRP14 and MRP8/14 heterodimer on the phenotypic maturation of mice bone marrow-derived dendritic cells (BMDCs). Methods BMDCs were cultured and purified in vitro and divided into control group (equal volume of PBS), MRP14 (1 µg/mL) treatment group, MRP8 (1 µg/mL) treatment group and MRP8/14 (1 µg/mL) treatment group. Flow cytometry was used to detect the expression of costimulatory molecules, such as CD40, CD80, CD86 and major histocompatibility complex II (MHC II ) on the surface of BMDCs after stimulation. Results MRP14, MRP8 and MRP8/14 promoted the expression of CD40, CD80 and CD86, while MRP14 and MRP8 promoted the expression of MHC II on the surface of BMDCs. Moreover, the ability to promote the expression of CD80 and CD86 is stronger in MRP14 and MRP8/14 than MRP8. Conclusion MRP14, MRP8 and MRP8/14 promote the phenotypic maturation of BMDCs by increasing the expression of costimulatory molecules, and MRP14, MRP8 and MRP8/14 also differ in their ability to promote BMDCs to expresse various costimulatory molecules.

Mycophenolic Acid Synergizing with Lipopolysaccharide to Induce Interleukin-1ß Release via Activation of Caspase-1.

BACKGROUND: The previous study showed that mycophenolic acid (MPA) synergizing with lipopolysaccharide (LPS) promoted interleukin (IL)-1ß release, but the mechanism is unclear. This study aimed to investigate the mechanism of MPA synergizing with LPS to induce IL-1ß release. METHODS: Undiluted human blood cells, THP-1 human myeloid leukemia mononuclear cells (THP-1) cells, or monocytes were stimulated with LPS and treated with or without MPA, and the supernatant IL-1ß was detected by enzyme-linked immunosorbent assay. The mRNA levels of IL-1ß were detected by real-time quantitative polymerase chain reaction. The intracellular protein levels of nuclear factor kappa B (NF-κB) phospho-p65 (p-p65), precursor interleukin-1ß (pro-IL-1ß), NOD-like receptor pyrin domain containing-3 (NLRP3), and cysteine aspartic acid-specific protease-1 (caspase-1) p20 in THP-1 cell were measured by Western blot. RESULTS: The MPA alone failed to induce IL-1ß, whereas MPA synergized with LPS to increase IL-1ß in a dose-dependent manner (685.00 ± 20.00 pg/ml in LPS + 5 µmol/L MPA group, P = 0.035; 742.00 ± 31.58 pg/ml in LPS + 25 µmol/L MPA group, P = 0.017; 1000.00 ± 65.59 pg/ml in LPS + 75 µmol/L MPA group, P = 0.024; versus 408.00 ± 35.50 pg/ml in LPS group). MPA alone has no effect on the IL-1ß mRNA expression, LPS induced the expression of IL-1ß mRNA 2761 fold, and LPS + MPA increased the IL-1ß expression 3018 fold, which had the same effect with LPS group (P = 0.834). MPA did not affect the intracellular NF-κB p-p65 and pro-IL-1ß protein levels but activated NLRP3 inflammasome. Ac-YVAD-cmk blocked the activation of caspase-1 and subsequently attenuated IL-1ß secretion (181.00 ± 45.24 pg/ml in LPS + MPA + YVAD group vs. 588.00 ± 41.99 pg/ml in LPS + MPA group, P = 0.014). CONCLUSIONS: Taken together, MPA synergized with LPS to induce IL-1ß release via the activation of caspase-1, rather than the enhanced production of pro-IL-1ß. These findings suggested that patients immunosuppressed with mycophenolate mofetil may have overly activated caspase-1 during infection, which might contribute to a more sensitive host defense response to invading germs.

Isorhamnetin, the active constituent of a Chinese herb Hippophae rhamnoides L, is a potent suppressor of dendritic-cell maturation and trafficking.

Dendritic cells (DCs) have been recognized as major targets of immunosuppressive therapies for their significant roles in connecting innate and adaptive immunity. Isorhamnetin (Iso), one of the most common flavonoid compounds extracted from the Chinese herb Hippophae rhamnoides L, has been proved to have anti-inflammatory, anticarcinogenic, and antioxidant activities in many chronic inflammatory conditions, but the effects of Iso on DCs have rarely been reported before. Here we investigated the functions and the mechanisms of Iso on bone marrow-derived DCs (BMDCs) including maturation, phagocytosis, and trafficking. Our data showed that Iso effectively inhibited the maturation of lipopolysaccharide (LPS)-treated BMDCs by down regulation of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1ß and IL-12p70, up regulation of IL-10, and depression of costimulatory molecules CD40, CD80, and CD86, while had no effects on phagocytosis. Furthermore, Iso inhibited the migration of LPS-treated BMDCs, which may be due to its inhibition on chemokine receptor 7 (CCR7) expression. These findings strongly suggest that Iso is a potent immunosuppressive agent by inhibiting DC activation and trafficking, and may be used to prevent or treat chronic inflammation, autoimmune diseases, and graft rejections.

Sniping the scout: Targeting the key molecules in dendritic cell functions for treatment of autoimmune diseases.

Dendritic cells (DCs) are a power tool for manipulating immune system. They play important roles in the induction of immunity as well as inducing intrathymic and peripheral tolerance. After generated from stem cells in the bone marrow, DCs traffic to the peripheral tissues, where they capture and process antigens, express lymphocyte co-stimulators, migrate to the secondary lymph organs and present the processed antigen to naive T cells to either activate or tolerize them. These processes are modulated subtly and influenced by various factors. Aberrant regulation of the processes may cause autoimmunity. Investigation into the biology of DCs and the molecules and mechanisms that regulate them helps us understanding the pathogenesis of autoimmune diseases and reveals numerous steps for pharmacological manipulation. In this review, we made a sketch line of the critical events of DC biology that are potential pharmacologic targets for the treatment of autoimmune diseases.

The early and late stages of crowned dens syndrome: two case reports.

BACKGROUND CONTEXT: Crowned dens syndrome (CDS) is a rare form of calcium phosphate crystal depositions and often presents with recurrent neck pain, stiffness of neck, increased erythrocyte sedimentation rate, and episodes of fever. PURPOSE: The goal of this report is to identify the early and late stages of CDS and its consequences as the result of repeated attacks of CDS at cervical spine in its late stage. STUDY DESIGN: This is a case report. METHODS: We reported one case of early-stage CDS and one late-stage CDS. RESULTS: The two patients shared some common clinical features of acute attack of CDS, such as increased erythrocyte sedimentation rate, C-reactive protein, episode of fever, and increased white blood cells along with high blood glucose levels. The first case showed early phase of CDS with computed tomography (CT) scan that only showed mild calcification around the dens. The second case had appearance of late stage of CDS with more severe chronic degenerative changes of cervical spine. CONCLUSIONS: Early stage of CDS can be difficult to identify because of mild clinical symptoms, but CT scan is a preferable method to demonstrate densities surrounding the top and sides of the odontoid process. In the late stage of CDS, radiolographic features often include diffuse periodontoid calcifications, diffuse destructive discopathies, and apophyseal joint destruction, and patient might have severe neurological symptoms.

Cytokine signatures of human whole blood for monitoring immunosuppression.

How to evaluate status of the immune system is extremely critical for clinical immunosuppressive treatment. In this study, we tested the secretion of cytokines in undiluted whole blood samples stimulated with Phorbol 12-myristate 13-acetate (PMA) and ionomycin (IONO), and compared the effects of dexamethasone (DEX), cyclosporine A (CsA) or mycophenolic acid (MPA), either alone or in combination, on cytokine profiles. The results showed that both DEX and CsA dose-dependently inhibited the production of eleven cytokines: interleukin (IL)-2, IL-4, IL-5, IL-6, IL-13, IL-17, interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF). Unexpectedly, MPA showed no obvious influences except for the mild inhibition on GM-CSF production. In combination treatment, cytokine profiles reflect not only the synergistic effects among drugs, but also the specific effect of the individual drug. Thus, the effects of different immunosuppressants could be reflected through their specific cytokine signatures, which can be applied to maximize immunosuppressive effects, while to minimize risk of infections and help physicians to reasonably apply immunosuppressants.

[Effects of decitabine on proliferation and apoptosis of NB4 and K562 cells].

This study was aimed to investigate the effects of decitabine (DAC) on proliferation and apoptosis of leukemia NB4 and K562 cells. The proliferation inhibition of DAC on NB4 and K562 cells was detected by Trypan blue staining. After treatment of DAC at different concentrations, the changes of cell cycle and CD11b expression was determined by flow cytometry. The cell morphological changes were observed by Wright's staining. The DNA ladder was used to detect cell apoptosis. The results indicated that DAC significantly inhibited the proliferation of NB4 and K562 cells in dose-and time-dependent manner. The median inhibitory concentration (IC50) of DAC-treated NB4 and K562 cells for 72 h was 0.113 µmol/L and 0.138 µmol/L, respectively. After treating these two cell lines with DAC at different concentration for 72 h, the cell ratio in G0/G1 phase significantly increased, while the cell ratio in S phase obviously decreased in 0.15 µmol/L DAC group (P < 0.05). The expression levels of myeloid differentiation antigen CD11b of both cell lines significantly increased in contrast to the control group (P < 0.05). The cell morphology detected by Wright's staining displayed partial differentiation and apoptosis after treating NB4 and K562 cells with DAC for 48 h. Typical apoptotic DNA ladder was observed in 0.15 µmol/L DAC group at 48 h. It is concluded that DAC can inhibit NB4 and K562 cell proliferation, induce cell differentiation and apoptosis, but more obviously for NB4 cells.

[Effect of FK506 on cytokine secretion in whole blood].

OBJECTIVE: To investigate the effect of FK506 on cytokine secretions in whole blood from healthy individuals. METHODS: Blood samples collected from healthy volunteers were co-cultured with different concentrations of FK506 and stimulated with PMA and IONO. The concentrations of 8 cytokines including IL-2, IL-6, IL-12, IL-17, IFN-gamma, TNF-alpha, GM-CSF and G-CSF were detected by Bio-Plex suspension system. RESULTS: Compared with the control group, high-concentration FK506 (20 ng/ml) significantly inhibited the secretions of IL-2, IL-6, IL-12, IL-17, IFN-gamma, TNF-alpha, GM-CSF and G-CSF. At a moderate concentration (5 ng/ml), FK506 inhibited the secretion of GM-CSF significantly. CONCLUSION: FK506 effectively inhibits the secretion of proinflammatory cytokines including IL-6, IFN-gamma and TNF-alpha and also the secretion of IL-2, IL-12, IL-17, GM-CSF and G-CSF. FK506 might play the role of immunosuppression by inhibiting the production of these cytokines by the immune cells. Monitoring the levels of these cytokines might be a potential method for evaluating the adequacy of FK506 doses administered.

[Effects of apoptotic lymphocytes on proinflammatory cytokine secretion by hepatic sinusoidal endothelial cells].

OBJECTIVE: To investigate the effects of apoptotic lymphocytes on the secretion of cytokines by hepatic sinusoidal endothelial cells (HSEC). METHODS: Human HSEC cells were co-cultured for 16 h with allogenetic apoptotic lymphocytes induced by UVB irradiation. The supernatants were collected and the levels of interleukin-2, interferon-gamma, and tumor necrosis factor-alpha were detected by Luminex technique. RESULTS: All the cytokines were down-regulated by about 50% in HSECs after co-culture with the apoptotic lymphocytes as compared with those in the control group (P<0.05). CONCLUSIONS: Co-culture with apoptotic lymphocytes can down-regulate the secretion of pro-inflammatory cytokines in HSECs, which may contribute to tolerogenic microenvironment in the liver.

The "Miracle" fetus: an excellent model for apoptotic cell-induced immune tolerance?

For several decades, people have wondered why pregnant mothers do not reject fetuses bearing allogeneic paternal antigens. Several hypotheses have been proposed, including a physical barrier between fetus and mother, immaturity of fetal antigens and temporary dormancy of the maternal immune system. Based on the "cell death immune recognition model," the author proposes a hypothesis that pregnancy tolerance is an active immune response of the maternal immune system, which is induced by apoptotic cells bearing paternal antigens. The primary steps in the induction of pregnancy tolerance are apoptosis of fetal cells and spermatozoa, phagocytosis of those dying/dead cells by maternal antigen presenting cells (APC), migration of APC to local lymph nodes, and antigen presentation and induction of regulatory T cells in local lymph nodes. The hypothesis outlined below will not only help us to understand how pregnancy tolerance is induced, but also provide novel strategies to develop clinical measures for patients with infertility or pregnancy-related disorders.

[Necrotic cells induce and promote inflammatory responses in vitro].

OBJECTIVE: To investigate the effect of necrotic cells on the secretion of inflammatory cytokines. METHODS: RAW264.7 macrophages and necrotic mouse thymocytes induced by heating were incubated for 18 h at a ratio of 5:1 in the absence or presence of lipopolysaccharide (LPS, 100 ng/ml). The supernatant of the cell culture was collected and the expression and secretion of the pro-inflammatory cytokines were measured using Bio-Plex suspension system. RESULTS: The secretions of tumor necrosis factor-alpha (TNF-alpha) and interlukine-6 (IL-6) by macrophages co-cultured with the necrotic cells were significantly enhanced as compared with the control cells. The necrotic cells also significantly augmented the secretion of the pro-inflammatory cytokines induced by LPS. CONCLUSION: Necrotic cells not only induces pro-inflammatory cytokine expression by themselves but also work synergistically with LPS to enhance the cytokine production, suggesting the important roles of necrotic cells to initiate and maintain the inflammatory responses.

Cell death recognition model for the immune system.

It is essential for the immune system to recognize markers or understand rules required for discriminating antigens that should be actively responded to from those be tolerated. Although the classic self-nonself theory over the past five decades has been challenged by "danger" model and "infectious nonself" model, etc., no theories could fit for all. Cell death is important not only for its role in homeostasis, but also for its decisive effects on the immune responses. Different ways of cell death, apoptosis or necrosis, transmit fundamentally opposite driving forces for the immune system, inducing tolerance or initiating adaptive immune responses. The progress in understanding phagocytosis and process of apoptotic and necrotic cells leads the author to propose "cell death" recognition model for the immune system. Four principles are important in this model. First, only antigens shedding from apoptotic or necrotic cells rather than those from healthy cells, can be presented to naïve T cells. Second, either apoptotic cells or necrotic cells, but not healthy cells, can attract phagocytes, namely dendritic cells (DC) or macrophages that are also antigen presenting cells (APC), to scavenge dead cells. Third, macrophages or DC residing in non-lymphoid tissues phagocytose dying/dead cells, migrate to lymphoid tissues and present antigens to naïve T cells there. Fourth, tolerance or adaptive responses are not dependent on whether the antigens are self or nonself, but on the ways of cell death during antigen presentation. Importantly, tolerance and adaptive immunity are all dominant responses and the impact of cell death on immune responses is a dynamic balance between them. "Cell death" recognition model could more easily explain various immune phenomena, including infection, self tolerance and autoimmunity, tumor immunity as well as transplant rejection. Investigation into the roles and mechanisms of cell death mediated immune responses and finding out key modulators will prompt better understanding the ways of immune recognition and provide novel strategies for the management of autoimmunity, tumors, infections as well as transplantation.