Human Peripheral Blood Monocytic Cells Research Articles

PdpC, a Type Six Secretion System Substrate, is Required for Erythrocyte Invasion in Francisella tularensis LVS

Francisella tularensis is an intracellular pathogen and the causative agent of tularemia. The F. tularensis type six secretion system (T6SS) is required for phagolysosomal escape and invasion of erythrocytes. An effector of the T6SS, PdpC, is required for phagosomal escape and we wanted to test if PdpC was also required for erythrocyte invasion. We constructed a pdpC-null mutant in the live vaccine strain, F. tularensis LVS. The pdpC-null strain is required for invasion of both human and sheep erythrocytes and reintroduction of a copy of pdpC, in trans, rescues this phenotype. Differential Immuno-Fluorescence Microscopy (DIFM) showed that the pdpC-null strain is affected in attachment as well as invasion. Further, a fluorescently labelled pdpC-null strain of F. tularensis LVS was unable to proliferate in THP-1 human peripheral blood monocyte cells. Finally, we constructed a fluorescent fusion of pdpC to emgfp and the resulting PdpC-EmGFP fusion protein localizes as discrete foci in a subset of broth cultured F. tularensis LVS cells. Our results confirm previous observations that PdpC is required for infection and virulence in phagocytic host cells and are the first description of an effector of the Type Six Secretion System that is required for erythrocyte invasion. The pdpC-emgfp strain will be a useful tool to further characterize the role of PdpC in both macrophage infection and red blood cell invasion. (This research was made possible by NASA West Virginia Space Grant Consortium Training Grant #NNX15A101H and by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence).

In-vitro performance of reinforced hydroxyapatite coatings deposited using vacuum plasma spray technique on Ti-6Al-4V

In this investigation the basic in-vitro assessment of substrate (Ti-6Al-4V), as-deposited and heat treated reinforced HA coatings has been performed to evaluate the biocompatible conduct of these bio-ceramic coatings. Briefly adherent fibroblast cell lines NIN3T3 and non-adherent human peripheral blood monocyte cells (hPBMCs) were utilized to survey the surface biocompatibility utilizing standard MTT, trypan blue tests while their reactive oxygen species profiling was done to comprehend the potential pressure that may hamper the human cell working. The outcomes demonstrated that the as-deposited and heat treated coatings displayed incredible cell reasonability for hPBMCs and NIN3T3 cells in contrast with bare substrates. Comparative results were observed for trypan blue dye exclusion assay. Besides, oxygen free radical levels demonstrated that there is no change in reactive oxygen species (ROS) levels in cells cultured on heat treated coatings. This suggests the materials utilized in coatings are not cytotoxic in nature and the phenomenon was true both for cell lines just as human primary cells. By and large natural assessment showed that coatings are protected to human cells in essential conditions.

C-Reactive Protein Stimulates Nicotinic Acetylcholine Receptors to Control ATP-Mediated Monocytic Inflammasome Activation.

Blood levels of the acute phase reactant C-reactive protein (CRP) are frequently measured as a clinical marker for inflammation, but the biological functions of CRP are still controversial. CRP is a phosphocholine (PC)-binding pentraxin, mainly produced in the liver in response to elevated levels of interleukin-1β (IL-1β) and of the IL-1β-dependent cytokine IL-6. While both cytokines play important roles in host defense, excessive systemic IL-1β levels can cause life-threatening diseases such as trauma-associated systemic inflammation. We hypothesized that CRP acts as a negative feedback regulator of monocytic IL-1β maturation and secretion. Here, we demonstrate that CRP, in association with PC, efficiently reduces ATP-induced inflammasome activation and IL-1β release from human peripheral blood mononuclear leukocytes and monocytic U937 cells. Effective concentrations are in the range of marginally pathologic CRP levels (IC50 = 4.9 µg/ml). CRP elicits metabotropic functions at nicotinic acetylcholine (ACh) receptors (nAChRs) containing subunits α7, α9, and α10 and suppresses the function of ATP-sensitive P2X7 receptors in monocytic cells. Of note, CRP does not induce ion currents at conventional nAChRs, suggesting that CRP is a potent nicotinic agonist controlling innate immunity without entailing the risk of adverse effects in the nervous system. In a prospective study on multiple trauma patients, IL-1β plasma concentrations negatively correlated with preceding CRP levels, whereas inflammasome-independent cytokines IL-6, IL-18, and TNF-α positively correlated. In conclusion, PC-laden CRP is an unconventional nicotinic agonist that potently inhibits ATP-induced inflammasome activation and might protect against trauma-associated sterile inflammation.

Peptidoglycan Suppresses Phagocytic Activities and Apoptosis of Macrophages in Colonic Mucosa Tissues of Crohn's Disease Patients and In Vitro.

BackgroundRac1 signaling plays a crucial role in controlling macrophage functions in CD. Peptidoglycan triggers several intracellular signaling pathways, including activation of Rac1, to regulate the function of macrophage. Suppressed Rac1 signaling in non-inflamed colonic mucosa of Crohn’s disease patients has been shown to correlate with increased innate immunity.Material/MethodsWe examined the effect of peptidoglycan on Rac1 signaling in macrophages and mucosal tissue samples collected from 10 patients with active Crohn’s disease and further investigated the effects of peptidoglycan on apoptosis and phagocytic activities of macrophages in vitro.ResultsMacrophage infiltration and Rac1 signaling was increased in inflamed mucosal tissues of Crohn’s disease patients. Immunoblotting assays revealed that peptidoglycan dose- and time-dependently increased the expression of Rac1-GTP, phosphorylated VAV1, and phosphorylated PAK1in RAW264.7 macrophages, which, however, was attenuated by 6-thioguanine. Peptidoglycan also dose-dependently inhibited phagocytic activities of human peripheral blood monocytic cells (PBMCs), which were partially abated by 6-thioguanine or NSC23766. Flow cytometry showed that peptidoglycan (3 μg/mL) decreased the proportion of apoptotic human PBMCs versus controls. The addition of 6-thioguanine or NSC3766 to peptidoglycan led to a sharper rise in the proportion of apoptotic human PBMCs than 6-thioguanine or NSC3766 alone.ConclusionsOur findings suggest that Rac1 signaling is a common molecular target shared by peptidoglycan and immunosuppressive treatment in intestinal macrophages. Inhibiting Rac1 activation may be crucial for optimizing macrophage immunity for treatment of Crohn’s disease.

Genetic variants may play an important role in mRNA-miRNA interaction: evidence for haplotype-dependent downregulation of ABCC2 (MRP2) by miRNA-379.

The functional influence of single-nucleotide polymorphisms (SNPs) of the ATP-binding cassette (ABC) transporter ABCC2 (MRP2) has been characterized in numerous studies. The aim of this study was to address the question of whether distinct ABCC2 haplotypes, which differ in their mRNA secondary structures, show an influence on the degree of mRNA and protein downregulation through miRNA interaction. A model using human peripheral blood monocytic cells (PBMCs) isolated from healthy Caucasian volunteers, with three defined ABCC2 haplotypes comprising the 5'-UTR SNP -24C>T, the 1249G>A SNP (V417I), and the silent 3972C>T SNP, was outlined. Cells were transiently transfected with miRNA-379, already known to target ABCC2 in HepG2 cells. ABCC2 was downregulated through miR-379 in a haplotype-dependent manner: the wild-type CGC/CGC was modestly affected (mRNA: -12.7±4.2%, protein: -9.9±0.1%), whereas variant haplotypes were more strongly suppressed: CGT/CGT (mRNA: -36.7±2.4%, protein: -21.6±0.4%) and TGT/TGT (mRNA: -55.7±1.2%, protein: -46.3±4.0%). In addition, glutathione-methylfluorescein efflux was significantly reduced in miR-379-transfected peripheral blood monocytic cells corresponding to ABCC2 protein expression. This observation may suggest a differential suppression of ABCC2 by miR-379 caused by haplotype-dependent differences in mRNA secondary structures, resulting in changes in mRNA target accessibility or mRNA stability.

Human IP10-scFv and DC-induced CTL synergistically inhibit the growth of glioma in a xenograft model.

The epidermal growth factor receptor (EGFR) mutant of EGFRvIII is highly expressed in glioma cells, and the EGFRvIII-specific dendritic cell (DC)-induced tumor antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) may hold promise in cancer immunotherapy. Interferon (IFN)-γ-inducible protein (IP)-10 (IP-10) is a potent inhibitor of angiogenesis and can recruit CXCR3+ T cells, including CD8+ T cells, which are important for the control of tumor growth. In this study, we assessed if the combination of IP10-EGFRvIIIscFv with DC-induced CTLs would improve the therapeutic antitumor efficacy. IP10-scFv was generated by linking the human IP-10 gene with the DNA fragment for anti-EGFRvIIIscFv with a (Gly4Ser)3 flexible linker, purified by affinity chromatography, and characterized for its anti-EGFRvIII immunoreactivity and chemotactic activity. DCs were isolated from human peripheral blood monocyte cells and pulsed with EGFRvIII-peptide, then co-cultured with autologous CD8+ T cells. BALB/c-nu mice were inoculated with human glioma U87-EGFRvIII cells in the brain and treated intracranially with IP10-scFv and/or intravenously with DC-induced CTLs for evaluating the therapeutic effect. Treatment with both IP10-scFv and EGFRvIII peptide-pulsed, DC-induced CTL synergistically inhibited the growth of glioma and prolonged the survival of tumor-bearing mice, which was accompanied by the inhibition of tumor angiogenesis and enhancement of cytotoxicity, thereby increasing the numbers of brain-infiltrating lymphocytes (BILs) and prolonging the residence time of CTLs in the tumor.

Recombinant Mammaglobin A Adenovirus-Infected Dendritic Cells Induce Mammaglobin A-Specific CD8+ Cytotoxic T Lymphocytes against Breast Cancer Cells In Vitro

Mammaglobin A (MGBA) is a novel breast cancer-associated antigen almost exclusively over-expressed in primary and metastatic human breast cancers, making it a potential therapeutic target for breast cancer. The development of dendritic cell (DC)-induced tumor antigen specific CD8+ cytotoxic T lymphocytes (CTLs) may hold promise in cancer immunotherapy. In this study we constructed recombinant replication-defective adenoviral (Ad) vectors encoding MGBA and evaluated their ability to trigger anti-tumor immunity in vitro. DCs were isolated from the human peripheral blood monocyte cells (PBMCs) of two HLA-A33+ healthy female volunteers, and infected with adenovirus carrying MGBA cDNA (Ad-MGBA). After that, the Ad-MGBA-infected DCs were used to stimulate CD8+ CTLs in vitro and the latter was used for co-culture with breast cancer cell lines. The data revealed that infection with Ad-MGBA improved DC maturation and up-regulated the expression of co-stimulatory molecules and the secretion of interleukin-12 (IL-12), but down-regulated interleukin-10 (IL-10) secretion from DCs. Ad-MGBA-infected DC-stimulated CD8+CTLs displayed the highest cytotoxicity towards HLA-A33+/MGBA+ breast cancer MDA-MB-415 cells compared with other CD8+CTL populations, and compared with the cytotoxicity towards HLA-A33−/MGBA+ breast cancer HBL-100 cells and HLA-A33−/MGBA− breast cancer MDA-MB 231 cells. In addition, Ad-MGBA-infected DC-stimulated CD8+ CTLs showed a high level of IFNγ secretion when stimulated with HLA-A33+/MGBA+ breast cancer MDA-MB-415 cells, but not when stimulated with HLA-A33−/MGBA+ HBL-100 and HLA-A33−/MGBA−MDA-MB-231 cells. In addition, killing of CD8+CTLs against breast cancer was in a major histocompability complex (MHC)-limited pattern. Finally, the data also determined the importance of TNF-α in activating DCs and T cells. These data together suggest that MGBA recombinant adenovirus-infected DCs could induce specific anti-tumor immunity against MGBA+ breast cancers, which could provide a novel strategy in the immunotherapy of breast cancer.

Human Monoclonal Antibodies against Clostridium difficile Toxins A and B Inhibit Inflammatory and Histologic Responses to the Toxins in Human Colon and Peripheral Blood Monocytes

Clostridium difficile infection (CDI) is a common and debilitating nosocomial infection with high morbidity and mortality. C. difficile mediates diarrhea and colitis by releasing two toxins, toxin A and toxin B. Since both toxins stimulate proinflammatory signaling pathways in human colonocytes and both are involved in the pathophysiology of CDI, neutralization of toxin A and B activities may represent an important therapeutic approach against CDI. Recent studies indicated that human monoclonal antibodies (MAbs) against toxins A and B reduce their cytotoxic and secretory activities and prevent CDI in hamsters. Moreover, anti-toxin A and anti-toxin B MAbs together with antibiotics also effectively reduced recurrent CDI in humans. However, whether these MAbs neutralize toxin A- and toxin B-associated immune responses in human colonic mucosa or human peripheral blood monocyte cells (PBMCs) has never been examined. We used fresh human colonic biopsy specimens and peripheral blood monocytes to evaluate the effects of these antibodies against toxin A- and B-associated cytokine release, proinflammatory signaling, and histologic damage. Incubation of anti-toxin A (MK3415) or anti-toxin B (MK6072) MAbs with human PBMCs significantly inhibited toxin A- and toxin B-mediated tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) expression. MK3415 and MK6072 also diminished toxin A- and toxin B-mediated NF-κB p65 phosphorylation in human monocytes, respectively, and significantly reduced toxin A- and B-induced TNF-α and IL-1β expression as well as histologic damage in human colonic explants. Our results underline the effectiveness of MK3415 and MK6072 in blocking C. difficile toxin A- and toxin B-mediated inflammatory responses and histologic damage.

Rab13 Is Upregulated During Osteoclast Differentiation and Associates with Small Vesicles Revealing Polarized Distribution in Resorbing Cells

Osteoclasts are bone-resorbing multinucleated cells that undergo drastic changes in their polarization due to heavy vesicular trafficking during the resorption cycle. These events require the precise orchestration of membrane traffic in order to maintain the unique characteristics of the different membrane domains in osteoclasts. Rab proteins are small GTPases involved in regulation of most, if not all, steps of vesicle trafficking. The investigators studied RAB genes in human osteoclasts and found that at least 26 RABs were expressed in osteoclasts. Out of these, RAB13 gene expression was highly upregulated during differentiation of human peripheral blood monocytic cells into osteoclasts. To study its possible function in osteoclasts, the investigators performed immunolocalization studies for Rab13 and various known markers of osteoclast vesicular trafficking. Rab13 localized to small vesicular structures at the superior parts of the osteoclast between the trans-Golgi network and basolateral membrane domain. Rab13 localization suggests that it is not involved in endocytosis or transcytosis of bone degradation products. In addition, Rab13 did not associate with early endosomes or recycling endosomes labeled with EEA1 or TRITC-conjugated transferrin, respectively. Its involvement in glucose transporter traffic was excluded as well. It is suggested that Rab13 is associated with a putative secretory function in osteoclasts.

Impact of oxLDL and LPS on C-type Natriuretic Peptide System is Different between THP-1 Cells and Human Peripheral Blood Monocytic Cells

Background: The C-type natriuretic peptide (CNP) has anti-inflammatory, anti-proliferative, and anti-migratory properties. The purpose of this study was to investigate the occurrence of CNP and its receptors (NPR2 and NPR3) in a human monocytic cell line (THP-1 cells) as well as in peripheral blood monocytic cells (PBMC). Impact of both, LPS and human oxLDL on expression pattern of CNP and its receptors shall be studied. Methods: Cells were cultured in standard medium with or without LPS or oxLDL. Expression levels of CNP, NPR2, NPR3, TNF-α, IL-1β, IL-6, CD14 and CD68 were measured at baseline, 24h, and 48h. Results: Baseline expression of all analysed genes was significantly higher in PBMC compared to THP-1 cells (all p<0.05). Expression levels of CNP, IL-1β, IL-6, and CD14 were significantly increased in PBMC following stimulation with LPS. In contrast, in THP-1 cells stimulated by LPS, significant increase in expression was found only for IL-6 (p=0.007). In THP-1 cells, oxLDL increased the expression levels of NPR3, TNF-α, IL-1β, IL-6, CD14, and CD68 significantly. In contrast, expression level of NPR2 was diminished by oxLDL (p=0.007). In PBMC NPR3 was significantly down-regulated (p=0.002). Treatment with oxLDL for 48h increased NPR2/3-ratio significantly in PBMC (22.5 vs. 4.8, p=0.010). In contrast, in THP-1 cells, NPR2/3-ratio was lowered significantly by oxLDL (0.31 vs. 17.0, p=0.008). Conclusion: Treatment with LPS or oxLDL leads to diverging changes in gene expression PBMC and THP-1 cells. With respect to CNP and its receptors, data gained from THP-1 cells should be further validated using naive human peripheral blood monocytes. However, THP-1 cells can serve as a negative control for e.g. future signalling pathway studies related to oxLDL effect on CNP system in monocytes/macrophages.

Targeting Migration inducting gene-7 inhibits carcinoma cell invasion, early primary tumor growth, and stimulates monocyte oncolytic activity

Expression of Migration inducting gene-7 (Mig-7) is limited to tumor cells and to date not found in normal tissues. Multiple tumor microenvironment factors, such as epidermal and hepatocyte growth factors, in concert with alphavbeta5 integrin ligation, induce Mig-7 mRNA expression. Gain or loss of Mig-7 protein studies shows that Mig-7 promotes invasion of colon and endometrial carcinoma cells. These data led us to hypothesize that targeting Mig-7 through various methods could decrease invasion, enhance monocyte cell killing of tumor cells, and inhibit disease progression. To begin testing this hypothesis, an in vitro chemoinvasion assay of endometrial carcinoma cells treated with Mig-7-specific or control antibodies was used. Mig-7 antibody significantly reduced invasion by >60% compared with controls. In another approach to test this hypothesis, an in vitro analysis of peptide-stimulated human peripheral blood monocyte cells and their killing of MCF-7 breast carcinoma cells was used. Mig-7 peptide treatment increased monocyte cell tumor necrosis factor expression and killing of MCF-7 cells 30-fold over no peptide stimulation and 3-fold over MUC-1 or control peptide treatments. Furthermore, stably expressing Mig-7-specific short hairpin RNA resulted in significantly reduced Mig-7 protein levels and early primary tumor growth in a xenograft nude mouse model. Reduced phosphorylation of ERK1/2, Akt, and S6 kinase as well as decreased membrane-type 1 matrix metalloproteinase activity were mechanisms through which Mig-7 protein caused these effects. Based on these collective data, Mig-7 expression could be a potential candidate for future targeted cancer therapies.

천초근 dichloromethane 추출물의 Jurkat T 세포에서 세포사멸 효과

천초근은 전통적으로 동양의학에서 항암제로 사용되어왔는데 인간 급성 백혈병 세포주인 Jurkat T 세포를 사용하여 천초근의 세포독성기작을 알아보았다. 천초근 뿌리(3 kg)를 메탄올로 추출, 증류한 후 물에 녹여 다시 dichloromethane으로 추출분획 하였다. 세포독성 활성을 보이는 dichloromethane 추출물을 연속적으로 HPLC를 통해 분리하였고 그 활성물질(65 mg)을 CCH1이라 명명하였다. CCH1을 0.5 <TEX>${\mu}g$</TEX>/ml에서 2.0 <TEX>${\mu}g$</TEX>/ml의 농도로 처리하고 세포사멸 과정을 보았다. 즉, mitochondria cytochrome c 방출, casapase-8, -9 및 caspase-3의 활성화, PARP 분해, DNA 단편화 현상들이 일어나는 것을 관찰하였다. 하지만, mitochondria cytochrome c 방출 억제자인 Bcl-xL이 과발현되는 Jurkat T 세포에서는 세포사멸현상이 일어나지 않았다. 이러한 결과는 CCH1이 mitochondria 의존적인 신호전달 과정을 통해서 세포사멸을 유도 한다고 할 수 있다. 그리고 CCH1에 의한 세포독성은 혈액에서 분리한 단핵구 세포보다 Jurkat T 세포에서 보다 강한 활성을 보였다. To understand cytotoxic activity of Rubia cordifolia L. (Rubiaceae), which has been used as a traditional oriental medicine, the mechanism underlying cytotoxic effect of its extract on human acute Jurkat T cells was investigated. The methanol extract of roots (3 kg) of R. codifolia was evaporated, dissolved in water, and then extracted by dichloromethane. The substances in the chloroform extract showing the most cytotoxic activity were further purified by a series of preparative HPLC. The extracted active substance (65 mg) was designated as CCH1. When Jurkat T cells were treated with CCH1 at concentration ranging from 0.5 to 2.0 <TEX>${\mu}g$</TEX>/ml, apoptotic phenomena of cells companying several subsequent biochemical reactions such as mitochondria cytochrome c release, activation of casapase-8, -9, and caspase- 3, degradation of PARP and DNA fragmentation occurred via mitochondria-dependent pathway. However, abrogation of apoptosis was observed in an ectopic expression of Bcl-xL, which is a suppressor for mitochondrial cytochrome c release. These results demonstrate that the cytotoxicity of CCH1 against Jurkat T cells is attributable to apoptosis mediated by mitochodria-dependent death-signaling regulated by Bcl-xL. In addition, the CCH1 is more potent to leukemia Jurkat T cell than to human peripheral blood monocyte cells (PBMC).

Mechanisms of LPS-induced CD40 expression in human peripheral blood monocytic cells

Mechanisms of LPS-induced CD40 expression in human peripheral blood monocytic cells

Mechanisms of LPS-induced CD40 expression in human peripheral blood monocytic cells

CD40 plays important roles in cell-mediated and humoral immune responses. In this study, we explored mechanisms underlying lipopolysaccharide (LPS)-induced CD40 expression in purified human peripheral blood monocytic cells (PBMCs) from healthy volunteers. Exposure to LPS induced increases in CD40 mRNA and protein expression on PBMCs. LPS stimulation caused IκBα degradation. Inhibition of NFκB activation abrogated LPS-induced CD40 expression. LPS stimulation also resulted in phosphorylation of mitogen-activated protein kinases, however, only Jun N-terminal kinase (JNK) was partially involved in LPS-induced CD40 expression. In addition, LPS exposure resulted in elevated interferon γ (IFNγ) levels in the medium of PBMCs. Neutralization of IFNγ and IFNγ receptor using specific antibodies blocked LPS-induced CD40 expression by 44% and 37%, respectively. In summary, LPS-induced CD40 expression on human PBMCs through activation of NFκB and JNK, and partially through the induction of IFNγ production.

Bromelain Inhibits Lipopolysaccharide-Induced Cytokine Production in Human THP-1 Monocytes via the Removal of CD14

Bromelain has been reported to have anti-inflammatory and immunomodulatory effects. However, the anti-inflammatory mechanism of bromelain is unclear. Therefore, we investigated the effect of bromelain on cytokine production from lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC) and monocytic leukemia THP-1 cells. The result showed that bromelain (50–100 μg/ml) significantly and reversibly reduced tumor necrosis factor (TNF)-α interleukin- (IL)-1β and IL-6 from LPS-induced PBMC and THP-1 cells. This effect was correlated with reduced LPS-induced TNF-α mRNA and NF-κB activity in THP-1 cells. In addition, bromelain dose-dependently inhibited LPS-induced prostaglandin E2, thromboxane B2 and COX-2 mRNA but not COX-1 mRNA. Importantly, bromelain degraded TNF-α and IL-1β molecules, reduced the expression of surface marker CD14 but not Toll-like receptor 4 from THP-1 cells. Taken together, the results suggest that the suppression of signaling pathways by bromelain's proteolytic activity may contribute to the anti-inflammatory activity of bromelain.

High extracellular inorganic phosphate concentration inhibits RANK–RANKL signaling in osteoclast‐like cells

In this work, we investigated the effect of inorganic phosphate (Pi) on the differentiation of monocyte/macrophage precursors into an "osteoclastic" phenotype, and we delineated the molecular mechanisms which could be involved in this phenomenon. This was achieved by stimulating human peripheral blood monocytic cells and RAW 264.7 monocyte-macrophage precursor cells to differentiate into osteoclast-like cells in the presence of receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). RANKL has been previously reported to stimulate the signaling kinases ERK 1/2, p38, Akt, JNK, and the DNA-binding activity of the transcription factors AP-1 and NF-kappaB. Increase in extracellular Pi concentration (1.5-4.5 mM) dose-dependently inhibits both osteoclastic differentiation and bone resorption activity induced by RANKL and M-CSF. Pi was found to specifically inhibit the RANKL-induced JNK and Akt activation, while RANKL-induced p38 and ERK 1/2 phosphorylation were not significantly affected. Moreover, we found that Pi significantly reduced the RANKL-stimulated DNA-binding activity of NF-kappaB. The effects of Pi on osteoclast differentiation and DNA-binding activity of NF-kappaB were prevented by Foscarnet, a sodium-phosphate cotransport inhibitor, suggesting that the effects of Pi occur subsequently to its intake. These results demonstrate that Pi downregulates the differentiation of osteoclasts via a negative feedback exerted on RANK-RANKL signaling.

Francisella tularensis LVS initially activates but subsequently down-regulates intracellular signaling and cytokine secretion in mouse monocytic and human peripheral blood mononuclear cells

Monocytic cells constitute an important defense mechanism against invading pathogens by recognizing conserved pathogens components. The recognition leads to activation of intracellular pathways involving nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPK), such as the c-Jun NH 2-terminal kinase (JNK), and p38. We show that in vitro infection with Francisella tularensis results in activation of NF-κB, phosphorylation of p38 and c-Jun, and secretion of TNF-α in adherent mouse peritoneal cells, in the mouse macrophage-like cell line J774A.1, in the human macrophage cell line THP-1, and in human peripheral blood monocytic cells. This occurred after infection with the human live vaccine strain, F. tularensis LVS or a mutant strain denoted Δ iglC, which lacks expression of a 23-kDa protein, or after addition of killed F. tularensis LVS. Addition of purified F. tularensis LPS resulted in no discernible effects on the cells. When the effects were followed up to 5 h, activation persisted in cultures with killed bacteria or infected with the Δ iglC strain. In contrast, the signal transduction activation and secretion of TNF-α were down-regulated within the 5 h period in mouse peritoneal cells, J774 cells or human peripheral blood mononuclear cells infected with F. tularensis LVS. Together, the results suggest that infection with live F. tularensis LVS bacteria leads to a rapid induction of a proinflammatory response in mouse and human cells but after internalization of bacteria, this response is completely or partly down-regulated in most cell types. This down-regulation does not occur when cells are infected with the mutant Δ iglC.

Oxysterol Activators of Liver X Receptor and 9-cis-Retinoic Acid Promote Sequential Steps in the Synthesis and Secretion of Tumor Necrosis Factor-α from Human Monocytes

Liver X receptor alpha (LXRalpha), is a nuclear hormone receptor that is activated by oxysterols and plays a crucial role in regulating cholesterol and lipid metabolism in liver and cholesterol efflux from lipid-loaded macrophages. Here we show that treatment of human peripheral blood monocytes or monocytic THP-1 cells with the LXR ligand 22(R)-hydroxycholesterol (22(R)-HC), in combination with 9-cis-retinoic acid (9cRA), a ligand for the LXR heterodimerization partner retinoid X receptor (RXR), results in the specific induction of the potent pro-apoptotic and pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). Promoter analysis, inhibitor studies, and order-of-addition experiments demonstrated that TNF-alpha induction by 22(R)-HC and 9cRA occurs by a novel two-step process. The initial step involves 22(R)-HC-dependent induction of TNF-alpha mRNA, and intracellular accumulation of TNF-alpha protein, mediated by binding of LXRalpha/RXRalpha to an LXR response element at position -879 of the TNF-alpha promoter. Subsequent cell release of TNF-alpha protein occurs via a separable 9cRA-dependent, LXRalpha-independent step that requires de novo transcription and protein synthesis. Our findings reveal a potentially new dimension of the physiological role of LXRalpha and identify a unique multistep pathway of TNF-alpha production that may be of consequence to the normal function of LXR in monocyte/macrophages and in disease conditions such as atherosclerosis.

Expression of interleukin-4 in apoptotic cells: stimulation of the type-2 cytokine by different toxins in human peripheral blood mononuclear and tumor cells.

Immunological reactivity is regulated by T-cell populations (type-1 and type-2 cells) via cytokine secretion, but their influence on apoptosis remains unclear. Intracellular expression of type-1 (interferon [IFN]-gamma) and type-2 (interleukin [IL]-4) cytokines and apoptosis-related molecules (Apo2. 7, Bcl-2 protein) was studied by flow cytometry in human peripheral blood mononuclear cells (PBMC), myeloma (U-266), monocytic (THP-1), and T-leukemia cells (MOLT-4) in response to toxins, which act on different intracellular targets (actinomycin D, cycloheximide, the mistletoe lectins [ML]-1 and ML-3, brefeldin A, staurosporine). The apoptosis-inducing toxins stimulated intracellular IL-4 expression mainly in PBMC with high expression of the mitochondrial apoptosis marker, Apo2.7, but with decreased level of the anti-apoptotic Bcl-2 protein. Up-regulation of IL-4 coincided with a significant down-regulation of IFN-gamma in CD4(+) and CD8(+) cells. The inhibitor of oxidative phosphorylation, oligomycin, and the caspase inhibitor, z-VAD-fmk, abolished IL-4 expression and DNA fragmentation in the PBMC. Also in the myeloma, monocytic, and T-leukemia cells, IL-4 was mainly observed in the Apo2.7(+) apoptotic cells in response to the toxins. We suggest that the different apoptotic toxins activate a common pathway in which IL-4 production plays a yet unknown intracellular role further downstream during apoptosis.

INTERLEUKIN–13 AND INTERLEUKIN–4 ACT AS INTERLEUKIN–6 INDUCERS IN HUMAN MICROGLIAL CELLS

The effect of interleukin 4 (IL–4) and IL–13 on IL–6 and IL–1β production by human embry-onic microglial cells and human peripheral blood monocyte cells (PBMC) was compared. IL–4 or IL–13 increase intra-cellular IL–6 mRNA and IL–6 titres in supernatants of microglial cells whereas they decreased IL–6 production by PBMC tested in the same conditions. IL–4 and IL–13 increased also IL–1β production by human microglial cells. Stimulations associating IL–4 or IL–13 and IL–1b had an additive effect on IL–6 production by microglial cells, whereas, in the same conditions, an inhibitory effect was observed in PBMC. In contrast, dexamethasone downregulated IL–6 production by microglial cells and PBMC. Finally, IL–1 receptor (IL–1r) type 1 and IL–1r type 2 were detected on human microglial cells but it was demonstrated that IL–13 and IL–4 acted as IL–6 inducers in human microglial cells independently of the IL–1/IL–1r pathway.