Acidic sphingomyelinase induced by electrophiles promotes proinflammatory cytokine production in human bladder carcinoma ECV-304 cells

Electrophiles in environmental pollutants or cigarette smoke are high risk factors for various diseases caused by cell injuries such as apoptosis and inflammation. Here we show that electrophilic compounds such as diethyl malate (DEM), methyl mercury and cigarette smoke extracts significantly enhanced the expression of acidic sphingomyelinase (ASMase). ASMase activity and the amount of ceramide of DEM-treated cells were approximately 6 times and 4 times higher than these of non-treated cells, respectively. Moreover, we found that DEM pretreatment enhanced the production of IL-6 induced by TNF-α. Knockdown of ASMase attenuated the enhancement of TNF-α-dependent IL-6 production. On the other hand, enhancement of TNF-α-induced IL-6 production was observed in ASMase-overexpressing cells without DEM. Fractionation of the lipid raft revealed that the TNF receptor 1 (TNFR1) was migrated into the lipid raft in DEM-treated cells or ASMase-overexpressing cells. The TNF-α-induced IL-6 expression required the clustering of TNFR1 since IL-6 expression were decreased by the destruction of the lipid raft with filipin. These results demonstrated a new role for ASMase in the acceleration of the production of TNF-induced IL-6 as a pro-inflammatory cytokine and indicated that electrophiles could potentiate inflammation response by up-regulating of ASMase expression following formation of lipid rafts.     

Role of epiregulin in peptidoglycan-induced proinflammatory cytokine production by antigen presenting cells

We have previously found that epiregulin, a member of epidermal growth factor superfamily, is involved in proinflammatory cytokine production in bone marrow-derived macrophages. In this report, to further assess the role of epiregulin in innate immunity, we measured IL-6 production levels upon lipopolysaccharide and peptidoglycan stimulation in antigen presenting cells including macrophages and dendritic cells. Our analyses using epiregulin-deficient mice with mixed and inbred genetic backgrounds revealed that epiregulin deficiency results in the reduction of IL-6 production levels in both cell types upon peptidoglycan stimulation, and that the extent of this reduction is more evident under the BALB/c background compared with the C57BL/6J background. These results indicated that epiregulin may have a critical role in the regulation of peptidoglycan-mediated proinflammatory cytokine production in antigen presenting cells and innate immunity.     

Diacyltrehalose of Mycobacterium tuberculosis inhibits lipopolysaccharide- and mycobacteria-induced proinflammatory cytokine production in human monocytic cells

The lipids located in the outer layer of Mycobacterium tuberculosis, which include sulfolipid, phthiocerol dimycocerosate (PDIM), diacyltrehalose, and polyacyltrehalose, may play a role in host-pathogen interactions. These lipids were purified using thin-layer chromatography, and their ability to induce proinflammatory cytokines in human monocytes and in a human acute monocytic leukemia cell line (THP-1) was examined. None of the lipids tested induced significant interleukin (IL)-12p40 or tumor necrosis factor (TNF)-alpha production in monocytic cells. Diacyltrehalose significantly inhibited lipopolysaccharide- and M. tuberculosis-induced IL-12p40, TNF-alpha, and IL-6 productions in human monocytes, whereas other lipids had no effect. However, diacyltrehalose was unable to inhibit peptidoglycan-induced IL-12p40 production. These results suggest that diacyltrehalose is a mycobacterial factor capable of modulating host immune responses.     

The apoptotic process of human bladder carcinoma T24 cells induced by retinoid

Breakdown of the cytoskeletal network and redistribution of cytoplasmic organelles are early events of programmed cell death. Previous studies showed that retinoic acid induces programmed cell death in many tumor cell lines and that cytokeratins, particularly cytokeratin 18, are affected in the early events of apoptosis. In this study, patterns of cytoplasmic intermediate filaments (cytokeratin 18), actin filaments, and microtubules, as well as Bax and Bcl-2 proteins in human bladder carcinoma T24 cells were examined before and after retinoic acid treatment by immunocytochemistry and conventional electron microscopy. Our results demonstrate that the redistribution of Bax and Bcl-2 proteins in the subcellular compartment of T24 cells is correlated with reorganization of the cytoplasmic intermediate filament network and that cytokeratins are cleaved by caspases, as revealed by the M30 antibody which recognizes a specific caspase cleavage site within cytokeratin 18. The cytoskeletal architectures of microtubules are not significantly affected in the early apoptotic process, from our observations. We suggest that the breakdown in the intermediate filament network associated with the aggregation of mitochondria and lysosome may be a crucial event in the apoptotic process and that aggregation of cytoplasmic Bax may accelerate apoptotic death.     

IL-18 promotes type 1 cytokine production from NK cells and T cells in human intracellular infection

We investigated the role of IL-18 in leprosy, a disease characterized by polar cytokine responses that correlate with clinical disease. In vivo, IL-18 mRNA expression was higher in lesions from resistant tuberculoid as compared with susceptible lepromatous patients, and, in vitro, monocytes produced IL-18 in response to Mycobacterium leprae. rIL-18 augmented M. leprae-induced IFN-gamma in tuberculoid patients, but not lepromatous patients, while IL-4 production was not induced by IL-18. Anti-IL-12 partially inhibited M. leprae-induced release of IFN-gamma in the presence of IL-18, suggesting a combined effect of IL-12 and IL-18 in promoting M. leprae-specific type 1 responses. IL-18 enhanced M. leprae-induced IFN-gamma production rapidly (24 h) by NK cells and in a more sustained manner (5 days) by T cells. Finally, IL-18 directly induced IFN-gamma production from mycobacteria-reactive T cell clones. These results suggest that IL-18 induces type 1 cytokine responses in the host defense against intracellular infection.     

Elevated proinflammatory cytokine production by a skewed T cell compartment requires monocytes and promotes inflammation in type 2 diabetes

An appropriate balance between proinflammatory (Th17 and Th1) and anti-inflammatory (regulatory T cells [Tregs] and Th2) subsets of T cells is critical to maintain homeostasis and avoid inflammatory disease. Type 2 diabetes (T2D) is a chronic inflammatory disease promoted by changes in immune cell function. Recent work indicates T cells are important mediators of inflammation in a mouse model of T2D. These studies identified an elevation in the Th17 and Th1 subsets with a decrease in the Treg subset, which culminates in inflammation and insulin resistance. Based on these data, we tested the hypothesis that T cells in T2D patients are skewed toward proinflammatory subsets. Our data show that blood from T2D patients has increased circulating Th17 cells and elevated activation of Th17 signature genes. Importantly, T cells required culture with monocytes to maintain Th17 signatures, and fresh ex vivo T cells from T2D patients appeared to be poised for IL-17 production. T cells from T2D patients also have increased production of IFN-γ, but produce healthy levels of IL-4. In contrast, T2D patients had decreased percentages of CD4(+) Tregs. These data indicate that T cells in T2D patients are naturally skewed toward proinflammatory subsets that likely promote chronic inflammation in T2D through elevated cytokine production. Potential therapies targeted toward resetting this balance need to be approached with caution due to the reciprocal relationship between Th17 cells and Tregs. Understanding the unique aspects of T2D T cells is essential to predict outcomes of such treatments.     

Role of NF-kappa B in cytokine production induced from human airway epithelial cells by rhinovirus infection

Infection of human epithelial cells with human rhinovirus (HRV)-16 induces rapid production of several proinflammatory cytokines, including IL-8, IL-6, and GM-CSF. We evaluated the role of NF-kappaB in HRV-16-induced IL-8 and IL-6 production by EMSA using oligonucleotides corresponding to the binding sites for NF-kappaB in the IL-6 and IL-8 gene promoters. Consistent with the rapid induction of mRNA for IL-8 and IL-6, maximal NF-kappaB binding to both oligonucleotides was detected at 30 min after infection. NF-kappaB complexes contained p65 and p50, but not c-Rel. The IL-8 oligonucleotide bound recombinant p50 with only about one-tenth the efficiency of the IL-6 oligonucleotide, even though epithelial cells produced more IL-8 protein than IL-6. Neither the potent glucocorticoid, budesonide (10-7 M), nor a NO donor inhibited NF-kappaB binding to either cytokine promoter or induction of mRNA for either IL-8 or IL-6. Sulfasalazine and calpain inhibitor I, inhibitors of NF-kappaB activation, blocked HRV-16-induced formation of NF-kappaB complexes with oligonucleotides from both cytokines, but did not inhibit mRNA induction for either cytokine. By contrast, sulfasalazine clearly inhibited HRV-16 induction of mRNA for GM-CSF in the same cells. Thus, HRV-16 induces epithelial expression of IL-8 and IL-6 by an NF-kappaB-independent pathway, whereas induction of GM-CSF is at least partially dependent upon NF-kappaB activation.     

Porphyromonas gingivalis antagonises Campylobacter rectus induced cytokine production by human monocytes

Porphyromonas gingivalis and Campylobacter rectus are two major bacterial species implicated in the pathogenesis of periodontitis. P. gingivalis can antagonise the inflammatory response to other periodontal pathogens, a property commonly attributed to its lipopolysaccharide (LPS). The aim of this study was to investigate the capacity of P. gingivalis to antagonise C. rectus induced cytokine stimulation from human monocytes, and to investigate the involvement of its LPS. Primary human monocytes and Monomac-6 cells were challenged with culture supernatants from P. gingivalis and C. rectus, and levels of IL-1beta, IL-6 and IL-8 produced were measured by ELISA after 6h incubation. Purified P. gingivalis LPS was also added alone or in combination with C. rectus culture supernatant. Both species significantly stimulated the production of all three cytokines from the two cell lines, but P. gingivalis was considerably weaker inducer. Co-stimulation of the cells with P. gingivalis and C. rectus suppressed the cytokine-stimulatory capacity of the latter. P. gingivalis LPS alone was sufficient to antagonise IL-6 and IL-8, but not IL-1beta stimulation by C. rectus. In conclusion, mixed infections may impair host immune responses by reducing pro-inflammatory cytokine levels, which may be of relevance to the pathogenesis of periodontitis.     

Trichomonas vaginalis inhibits proinflammatory cytokine production in macrophages by suppressing NF-kappaB activation

Activation of NF-kappaB leads to the production of proinflammatory cytokines such as IL-12 and TNF-alpha that are involved in innate and adaptive immunity. We determined whether T. vaginalis-induced inflammatory response in macrophages associated with NF-kappaB. T. vaginalis adhesion led to transient NF-kappaB activation at 6 h but activation declined dramatically by 8 h. Super-shift assays showed that the gel-shifted complexes consisted of p65 (Rel A) and p50 (NF-kappaB1). NF-kappaB activation was accompanied by IkappaB-alpha degradation, and was inhibited by blocking T. vaginalis adhesion, indicating that the early NF-kappaB activation by T. vaginalis depends on IkappaB-alpha degradation. Quantitative real-time RT-PCR analyses revealed that the expression of TNF-alpha and IL-12 mRNA in T. vaginalis-adhesive cells was rapidly suppressed in comparison with LPS stimulation. We also observed that the parasite inhibited the nuclear translocation of NF-kappaB at 8 h, and diminished IL-12 and TNF-alpha production in response to LPS. In addition, inhibition of IkappaB-alpha degradation by MG-132 resulted in apoptosis. These results demonstrate that effects of T. vaginalis on NF-kappaB regulation are critical for cytokine production and the survival of macrophages. We suggest that there exist inhibitory mechanisms induced by T. vaginalis to evade host immunity.     

Secretory products from human adipocytes stimulate proinflammatory cytokine secretion from human endothelial cells

Hyperplasia and hypertrophy of fat cells can be found in obesity and increased adiposity is associated with endothelial dysfunction as an early event of atherosclerosis. However, it is unclear whether human adipocytes directly influence endothelial protein secretion. To study the crosstalk between fat and endothelial cells, human umbilical venous endothelial cells (HUVECs) were cultured in infranatants (Adipo) of primary differentiated human adipocytes. Interestingly, significantly increased secretion of 23 cytokines and chemokines from HUVECs was detected in four independent experiments after Adipo stimulation by protein array analysis detecting a total of 174 different proteins. Among those, time‐dependent Adipo‐induced upregulation of cytokine secretion in HUVECs was confirmed by ELISA for interleukin (IL)‐8, monokine induced by gamma interferon, macrophage inflammatory protein (MIP)‐1β, MIP‐3α, monocyte chemoattractant protein‐1, and IL‐6. Factors besides adiponectin, leptin, resistin, and tumor necrosis factor α appear to mediate these stimulatory effects. Our findings suggest that endothelial cell secretion is significantly influenced towards a proinflammatory pattern by adipocyte‐secreted factors. J. Cell. Biochem. 106: 729–737, 2009. © 2009 Wiley‐Liss, Inc.     

Piperine inhibits cytokine production by human peripheral blood mononuclear cells

Piperine, an amide isolated from Piper species (Piperaceae), has been reported to exhibit central nervous system depression, anti-pyretic and anti-inflammatory activity. Immunomodulatory and anti-tumor activity of piperine has been demonstrated in mouse carcinomas. However, there is little information available concerning the effect of piperine on humans. We evaluated the immunopharmacological activity of this compound in human immune cells. Human peripheral blood mononuclear cells (PBMCs) were exposed to piperine, and cell proliferation was determined by the MTS assay. Piperine significantly inhibited phytohemagglutinin-stimulated human PBMC proliferation after exposure for 72 h. This compound inhibited PBMC activity, with an IC(50) of 100.73 ± 11.16 μg/mL. Production of interleukin-2 (IL-2) and interferon-γ (IFN-γ) was measured using an ELISA assay and RT-PCR. Piperine inhibited IL-2 and IFN-γ production in the PBMCs. RT-PCR data indicated that IL-2 and IFN-γ mRNA expression in PBMCs is suppressed by piperine. This compound significantly inhibited the production of these two cytokines by activated PBMCs in a dose-dependent manner. In conclusion, piperine appears to have potential as an immunomodulatory agent for immune system suppression.     

Leukotriene E4 activates human Th2 cells for exaggerated proinflammatory cytokine production in response to prostaglandin D2

PGD(2) exerts a number of proinflammatory responses through a high-affinity interaction with chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) and has been detected at high concentrations at sites of allergic inflammation. Because cysteinyl leukotrienes (cysLTs) are also produced during the allergic response, we investigated the possibility that cysLTs may modulate the response of human Th2 cells to PGD(2). PGD(2) induced concentration-dependent Th2 cytokine production in the absence of TCR stimulation. Leukotrienes D(4) and E(4) (LTE(4)) also stimulated the cytokine production but were much less active than PGD(2). However, when combined with PGD(2), cysLTs caused a greater than additive enhancement of the response, with LTE(4) being most effective in activating Th2 cells. LTE(4) enhanced calcium mobilization in response to PGD(2) in Th2 cells without affecting endogenous PGD(2) production or CRTH2 receptor expression. The effect of LTE(4) was inhibited by montelukast but not by the P2Y(12) antagonist methylthioadenosine 5'-monophosphate. The enhancing effect was also evident with endogenous cysLTs produced from immunologically activated mast cells because inhibition of cysLT action by montelukast or cysLT synthesis by MK886, an inhibitor of 5-lipoxygenase-activating protein, reduced the response of Th2 cells to the levels produced by PGD(2) alone. These findings reveal that cysLTs, in particular LTE(4), have a significant proinflammatory impact on T cells and demonstrate their effects on Th2 cells are mediated by a montelukast-sensitive receptor.     

BAG-1 promotes apoptosis induced by N-(4-hydroxyphenyl)retinamide in human cervical carcinoma cells

N-(4-hydroxyphenyl)retinamide (4-HPR) is a synthetic apoptosis-inducing retinoid with cancer chemopreventive properties and lower toxicity than all-trans retinoic acid. BAG-1 is an antiapoptotic gene that is overexpressed in cervical and other cancers. In this study, we examined whether BAG-1 can inhibit 4-HPR-induced apoptosis in the C33A cervical carcinoma cell line. Surprisingly, although it inhibited apoptosis induced by five different apoptotic stimuli, overexpression of BAG-1 enhanced apoptosis induced by 4-HPR, producing a 2.5-fold lower IC(50) of 4-HPR. The effects of BAG-1 on 4-HPR-induced apoptosis were mediated by enhancing the caspase-3 activation pathway. Deletion mutation experiments showed that the central ubiquitin homology domain of BAG-1 protein was necessary for its promotion of 4-HPR-induced apoptosis, whereas its C-terminal Hsp70/Hsc70-interacting domain was required for its inhibition of staurosporine-induced apoptosis. These in vitro results suggest that the effectiveness of 4-HPR against the development of malignancy may be due to the overexpression of BAG-1 in cancer cells.     

Single-stranded oligonucleotides can inhibit cytokine production induced by human toll-like receptor 3

Toll-like receptor 3 (TLR3) can signal the production of a suite of cytokines and chemokines in response to double-stranded RNA (dsRNA) ligands or the dsRNA mimic poly(I-C). Using a human embryonic kidney 293T cell line to express human TLR3, we determined that poly(I-C)-induced signal could be significantly inhibited by single-stranded DNAs (ssDNAs), but not ssRNA or dsDNA. The ssDNA molecules that down-modulated TLR3 signaling did not affect TLR4 and do not require the hypomethylated CpG motif found in TLR9 ligands. The degree of modulation can be altered by the length, base sequence, and modification state of the ssDNAs. An inhibitory ssDNA was found to colocalize with TLR3 in transfected cells and in a cell line that naturally expresses TLR3. The inhibitory ssDNAs can compete efficiently with dsRNA for binding purified TLR3 ectodomains in vitro, while noninhibitory nucleic acids do not. The ssDNAs also decrease the levels of several cytokines produced by the human bronchial epithelial cell line BEAS-2B and by human peripheral blood mononuclear cells in response to poly(I-C) stimulation of native TLR3. These activities indicate that ssDNAs could be used to regulate the inflammatory response through TLR3.     

Taurine chloramine modulates cytokine production by human peripheral blood mononuclear cells

Summary.  The effect of taurine (Tau) and taurine chloramine (Tau-Cl) on the production of TNF-α, IL-1β, and IL-6 by peripheral blood mononuclear cells of healthy volunteers was examined. Cells were stimulated with bacterial lipopolysaccharide (LPS) in the presence of either Tau or Tau-Cl. After 24 h culture the cytokine concentrations were measured in both culture supernatants (secreted) and cell lysates (cell-associated) using ELISA. In LPS-stimulated cells Tau-Cl inhibited both the secreted and cell-associated IL-1β and IL-6, while exerted dual effect on TNF-α production: raising it slightly at low and reducing at higher concentration. By contrast, Tau had no significant effect on the cytokine production. These results indicate that Tau-Cl modulates synthesis of pro-inflammatory cytokines, and therefore it may play a role in the initiation and propagation of immune response. Received November 29, 2001 Accepted January 18, 2002 Published online August 30, 2002 Acknowledgments This research was supported by grants from the State Committee for Scientific Research of Poland (No 4 P05B 01018) and the Institute of Rheumatology (No I/14). The Institute of Rheumatology is supported by a core grant from the State Committee for Scientific Research of Poland. Authors' address: Ewa Kontny, Ph.D., Department of Pathophysiology and Immunology, Institute of Rheumatology, Spartanska 1, 02-637 Warsaw, Poland, E-mail: zpatiir@warman.com.pl Abbreviations: Tau, taurine; Tau-Cl, taurine chloramine; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; IL-1β, interleukin 1β; IL-6, interleukin 6; PBMC, peripheral blood mononuclear cells     

CMRF-35-like molecule 3 preferentially promotes TLR9-triggered proinflammatory cytokine production in macrophages by enhancing TNF receptor-associated factor 6 ubiquitination

TLRs are critical innate immune sensors in the induction of proinflammatory cytokines to eliminate invading pathogens. However, the mechanisms for the full activation of TLR-triggered innate immune response need to be fully understood. The murine CMRF-35-like molecule (CLM)-3 is a representative of CLM family belonging to the Ig superfamily. Considering that CLM-3 is selectively expressed in macrophages and the roles of CLM members in innate immune response remain unclear, in this study we investigated the role of CLM-3 in the regulation of TLR-triggered innate response. We found that CLM-3 was an endosome/lysosome-localized molecule, and was downregulated in macrophages by stimulation with TLR9 ligand, but not TLR4 and TLR3 ligands. Interestingly, CLM-3 selectively promoted production of TNF-α and IL-6 in macrophages triggered by TLR9, but not TLR4 or TLR3. CLM-3 enhanced activation of MAPKs and NF-κB pathways in TLR9-triggered macrophages. Furthermore, CLM-3-transgenic mice were generated, and CLM-3 expression was confirmed by mAb against CLM-3 that we prepared. Accordingly, the macrophages derived from CLM-3-transgenic mice were more sensitive to TLR9 ligand stimulation, with more pronounced production of TNF-α, IL-6, and increased activation of MAPKs and NF-κB pathways. Moreover, ubiquitination of TNFR-associated factor 6, a crucial signaling transducer of TLR-triggered MAPKs and NF-κB activation, was found to be significantly promoted by CLM-3 in macrophages. Collectively, the endosome/lysosome-localized CLM-3 can promote full activation of TLR9-triggered innate responses by enhancing TNFR-associated factor 6 ubiquitination and subsequently activating MAPKs and NF-κB.     

Hyperosmolarity induced by high glucose promotes senescence in human glomerular mesangial cells

Hyperglycemia is involved in the diabetic complication of different organs and can elevate serum osmolarity. Here, we tested whether hyperosmolarity promoted by high glucose levels induces cellular senescence in renal cells. We treated Wistar rats with streptozotocin to induce diabetes or with consecutive daily injections of mannitol to increase serum osmolarity and analyzed p53 and p16 genes in renal cortex by immunohistochemistry. Both diabetic and mannitol treated rats showed a significant increase in serum osmolarity, without significant signs of renal dysfunction, but associated with increased staining for p53 and p16 in the renal cortex. An increase in p53 and p16 expression was also found in renal cortex slices and glomeruli isolated from healthy rats, which were later treated with 30 mM glucose or mannitol. Intracellular mechanisms involved were analyzed in cultured human glomerular mesangial cells treated with 30 mM glucose or mannitol. After treatments, cells showed increased p53, p21 and p16 expression and elevated senescence-associated β-galactosidase activity. Senescence was prevented when myo-inositol was added before treatment. High glucose or mannitol induced constitutive activation of Ras and ERK pathways which, in turn, were activated by oxidative stress. In summary, hyperosmolarity induced renal senescence, particularly in glomerular mesangial cells, increasing oxidative stress, which constitutively activated Ras-ERK 1/2 pathway. Cellular senescence could contribute to the organ dysfunction associated with diabetes.