IL-12p40 mRNAs Research Articles

Unfolded Protein Response Differentially Regulates TLR4-Induced Cytokine Expression in Distinct Macrophage Populations.

Cellular stress responses are often engaged at sites of inflammation and can alter macrophage cytokine production. We now report that macrophages in distinct states of differentiation or in different temporal stages of inflammatory response exhibit differential sensitivity to cell stress mediated alterations in M1-like polarized inflammatory cytokine production. Tunicamycin (Tm) treatment of bone marrow derived macrophages (BMDM) cultured with M-CSF cultured bone marrow derived macrophages (M-BMDM) had markedly amplified M1-like responses to LPS, exhibiting higher levels of IL12p40 and IL12p35 mRNAs while BMDM cultured with GM-CSF, which normally express high IL12 subunit production in response to LPS, were relatively unaltered. Anti-inflammatory IL10 mRNA production in LPS-stimulated M-BMDM was greatly reduced by cell stress. These changes in cytokine mRNA levels resulted from altered rates of transcription and mRNA decay. Stress also altered cytokine protein production. Resident liver macrophages isolated from mice treated with Tm showed elevated levels of IL12 subunit mRNA production following LPS stimulation. Furthermore, macrophages infiltrating the liver during the early phase of acetaminophen injury (24 h) had little stress-mediated change in cytokine mRNA production while cells isolated in the later phase (48–72 h) exhibited higher sensitivity for stress elevated cytokine production. Hence cultured macrophages developed using different growth/differentiation factors and macrophages from different temporal stages of injury in vivo show markedly different sensitivity to cell stress for altered inflammatory cytokine production. These findings suggest that cellular stress can be an important modulator of the magnitude and character of myeloid inflammatory activity.

Kinetics of cytokine mRNA and protein expression by plastic adherent cells in the thymus after split-dose irradiation

Whole body irradiation causes significant apoptosis in various tissues such as the thymus. If apoptotic cells outnumber the phagocytic capacity of macrophages, apoptosis becomes secondary necrosis, inducing inflammatory cytokine expression in macrophages. Radiation also induces thymic lymphomas in C57BL/6 mice after four consecutive irradiations with 1.6 Gy X-rays with nearly 100% incidence. Since cancer development is modulated by a microenvironment involving macrophages, we examined the kinetics of thymocyte number and plastic adherent cell number in the thymus as well as cytokine mRNA expression by plastic adherent cells in the thymus after split-dose irradiation. Upon split-dose irradiation, thymocyte number changed dramatically, whereas plastic adherent cell number did not. Among cytokine mRNAs tested, IL-1β, IL-11 and IL-12p40 mRNAs were up regulated 2 days after the 1st and 2nd, 3rd and 4th, and 2nd and 3rd irradiations, respectively. On the other hand, TNF-α mRNA was up regulated 2 days after the 3rd irradiation and 2 weeks after the 4th irradiation. The level of IL-11 protein was also increased 2 days after 3rd and 4th irradiations. These results suggest that, upon split-dose irradiation, macrophages in the thymus produce various cytokines in a time-dependent manner, thereby contributing to induction of thymic lymphomas.

Characterization of the Localized Immune Response in the Respiratory Tract of Ferrets following Infection with Influenza A and B Viruses.

The burden of infection with seasonal influenza viruses is significant. Each year is typically characterized by the dominance of one (sub)type or lineage of influenza A or B virus, respectively. The incidence of disease varies annually, and while this may be attributed to a particular virus strain or subtype, the impacts of prior immunity, population differences, and variations in clinical assessment are also important. To improve our understanding of the impacts of seasonal influenza viruses, we directly compared clinical symptoms, virus shedding, and expression of cytokines, chemokines, and immune mediators in the upper respiratory tract (URT) of ferrets infected with contemporary A(H1N1)pdm09, A(H3N2), or influenza B virus. Gene expression in the lower respiratory tract (LRT) was also assessed. Clinical symptoms were minimal. Overall cytokine/chemokine profiles in the URT were consistent in pattern and magnitude between animals infected with influenza A and B viruses, and peak expression levels of interleukin-1α (IL-1α), IL-1β, IL-6, IL-12p40, alpha interferon (IFN-α), IFN-β, and tumor necrosis factor alpha (TNF-α) mRNAs correlated with peak levels of viral shedding. MCP1 and IFN-γ were expressed after the virus peak. Granzymes A and B and IL-10 reached peak expression as the virus was cleared and seroconversion was detected. Cytokine/chemokine gene expression in the LRT following A(H1N1)pdm09 virus infection reflected the observations seen for the URT but was delayed 2 or 3 days, as was virus replication. These data indicate that disease severities and localized immune responses following infection with seasonal influenza A and B viruses are similar, suggesting that other factors are likely to modulate the incidence and impact of seasonal influenza. Both influenza A and B viruses cocirculate in the human population, and annual influenza seasons are typically dominated by an influenza A virus subtype or an influenza B virus lineage. Surveillance data indicate that the burden of disease is higher in some seasons, yet it is unclear whether this is due to specific virus strains or to other factors, such as cross-reactive immunity or clinical definitions of influenza. We directly compared disease severities and localized inflammatory responses to different seasonal influenza virus strains, including the 2009 pandemic strain, in healthy naive ferrets. We found that the disease severities and the cytokine and chemokine responses were similar irrespective of the seasonal strain or the location of the infection in the respiratory tract. This suggests that factors other than the immune response to a particular virus (sub)type contribute to the variable impact of influenza virus infection in a population.

Ectosomes from neutrophil-like cells down-regulate nickel-induced dendritic cell maturation and promote Th2 polarization.

DCs are the first immune cells to be exposed to allergens, including chemical sensitizers, such as nickel, a human TLR4 agonist that induces DC maturation. In ACD, DCs can interact with PMNs that are recruited and activated, leading, in particular, to ectosome release. The objective of this work was to characterize the effects of PMN-Ect on DC functions in an ACD context. We first developed a standardized protocol to produce, characterize, and quantify ectosomes by use of human PLB-985 cells, differentiated into mature PMN (PLB-Ect). We then studied the in vitro effects of these purified ectosomes on human moDC functions in response to NiSO4 and to LPS, another TLR4 agonist. Confocal fluorescence microscopy showed that PLB-Ect was internalized by moDCs and localized in the lysosomal compartment. We then showed that PLB-Ect down-regulated NiSO4-induced moDC maturation, as witnessed by decreased expression of CD40, CD80, CD83, CD86, PDL-1, and HLA-DR and by decreased levels of IL-1β, IL-6, TNF-α, and IL-12p40 mRNAs. These effects were related to p38MAPK and NF-κB down-regulation. However, no increase in pan-regulatory DC marker genes (GILZ, CATC, C1QA) was observed; rather, levels of effector DC markers (Mx1, NMES1) were increased. Finally, when these PLB-Ect + NiSO4-treated moDCs were cocultured with CD4(+) T cells, a Th2 cytokine profile seemed to be induced, as shown, in particular, by enhanced IL-13 production. Together, these results suggest that the PMN-Ect can modulate DC maturation in response to nickel, a common chemical sensitizer responsible for ADC.

CCAAT/Enhancer-Binding Protein δ Is a Critical Mediator of Lipopolysaccharide-Induced Acute Lung Injury

Although inflammation plays a central role in the pathogenesis of acute lung injury, the molecular mechanisms underlying inflammatory responses in acute lung injury are poorly understood, and therapeutic options remain limited. CCAAT/enhancer-binding proteins, C/EBPβ and C/EBPδ, are expressed in the lung and have been implicated in the regulation of inflammatory mediators. However, their functions in lung pathobiological characteristics are not well characterized. Herein, we show that C/EBPβ and C/EBPδ are activated in mouse lung after intrapulmonary deposition of lipopolysaccharide (LPS). Mice carrying a targeted deletion of the C/EBPδ gene displayed significant attenuation of the lung permeability index (lung vascular leak of albumin), lung neutrophil accumulation (myeloperoxidase activity), and neutrophils in bronchial alveolar lavage fluids compared with wild-type mice. These phenotypes were consistent with morphological evaluation of lung, which showed reduced inflammatory cell influx and minimal intra-alveolar hemorrhage. Moreover, mutant mice expressed considerably less tumor necrosis factor-α, IL-6, and macrophage inflammatory protein-2 in bronchial alveolar lavage fluids in LPS-injured lung compared with wild-type mice. In contrast, C/EBPβ deficiency had no effect on LPS-induced lung injury. By using small-interfering RNA-mediated knockdown for C/EBPδ, we demonstrate, for the first time to our knowledge, that C/EBPδ plays a critical role for the tumor necrosis factor-α, IL-6, and macrophage inflammatory protein-2 production in LPS-stimulated alveolar macrophages. These findings demonstrate that C/EBPδ, but not C/EBPβ, plays an important role in LPS-induced lung inflammatory responses and injury.

MicroRNA profiling in duodenal ulcer disease caused by Helicobacter pylori infection in a Western population

Although the connection of microRNAs (miRNAs) to some diseases is well established, their involvement in chronic infections such as Helicobacter pylori has received less attention. The aim was to compare miRNA expression profiling in patients with duodenal ulcer (DU) due to H. pylori infection with that in infected patients without DU and in uninfected patients. The miRNA expression profile was determined by microarrays in antral mucosal samples from well-characterized dyspeptic patients (n = 46). The most significant set of miRNAs was subsequently analysed in an independent validation group of patients (n = 42). Transcripts for IL8, IL12p40, IL12p35 and IL23p19, the signalling molecules MYD88, GATA6, SOCS2 and STAT6 and H. pylori virulence factors cagA and VacA were analysed. Microarray experiments showed that 17 miRNAs were deregulated in the mucosa of H. pylori-infected patients. No significant differences were observed between normal and DU patients. PCR confirmed the up-regulation of miR-9, miR- 146a, miR-155 and miR-650 and the down-regulation of miR-96 and miR-204 in the independent validation set of patients. Importantly, miR-9, miR-96, miR-146a and miR-650 expression was specific to chronic-active gastritis. H. pylori-infected patients showed higher levels of IL8 and IL12p40 mRNAs and lower levels of GATA6 and SOCS2 mRNAs. The antral mucosa of patients with non-active or chronic-active gastritis showed significantly lower levels of GATA6, MYD88, SOCS2 and STAT6 mRNAs compared with patients without gastritis. The down-regulation of these factors was not correlated with the expression of any of the validated miRNAs. The exact role of the miRNA changes observed will require further study.

Influence of allitridin on transcription, expression and function of IL-12 genes in mice infected by murine cytomegalovirus

To investigate whether allitridin could interfere with the effects of murine cytomegalovirus (MCMV) infection on the transcription, expression and function of IL-12 genes in order to further explore the mechanism of allitridin against MCMV. Sixty mice were randomly divided into allitridin treated group, placebo and blank controls. Allitridin was intra-peritoneal injected to mice in treated group once a day with general dosage (25 mg x kg(-1)) at 24 hours after MCMV infection, and the same dosage of physiological saline were given to placebo and blank groups. Four experimental mice were sacrificed at 3, 5, 7, 10, 14 days after treatment (n = 4 per time point), respectively. The expression of IL-12 p70 and IFN-gamma in supernatant of spleen cell cultures were measured by double-antibody sandwich ELISA, and IL-12 p35 and p40 mRNAs in spleen cells were analyzed by RT-PCR. In systemic infection mice, the expression of both IL-12 p70 protein and p35 mRNA significantly increased on day 3 post-infection (pi); then rapidly and markedly decreased on day 5 pi and later. The level of IFN-gamma reached the peak on day 3 pi, then gradually dropped and returned to normal levels during the period of day 10 to 14 pi, and IL-12 p40 mRNA level was persistently and significantly higher after infection. In allitridin treated mice, the levels of IL-12 p70 protein, IL-12 p35 and p40 mRNAs reached the peak on day 3 after treatment (P < 0.05), and then rapidly dropped to the normal levels during the period of 5-14 days. Level of IFN-gamma was also reached the peak on day 3 after treatment; however, it dropped a little on day 5 and then gradually increased and was much higher than those of both placebo and bland controls during the period of day 7 to 14 after treatment (P < 0.01). Allitridin could completely correct the disturbance of expression of IL-12 gene caused by MCMV and persistently promote IFN-gamma expression, which was useful for enhancing the specific cellular immune reactions against CMV and clearance of CMV viruses from host. The result suggests another mechanism of allitridin against CMV.

UVB-induced suppression of resistance to Mycobacterium tuberculosisin guinea pigs after BCG vaccination: cellular and molecular changes in the skin (99.19)

Abstract Previously, we reported that ultraviolet-B radiation (UVR) suppressed BCG vaccine-induced resistance to Mycobacterium tuberculosis in guinea pigs as it induced immuno-suppression and increased pathogenesis. In this study, we investigated the cellular and molecular changes within the guinea pig epidermis.The dorsal skin of guinea pigs was exposed to 5.6 kJ/m2 UVR and the epidermal cells were harvested 24 h later. Although UVR did not induce any gross morphological changes in the epidermis, a significant increase in the angular nucleated keratinocytes and a reduction in the number of cells with high nuclear to cytoplasmic ratio and binucleated cells were observed. Flow cytometry studies indicated that the proportions of CD3+ and CD4+ T cells increased in the epidermis while spleen showed a decrease in the CD3+, CD4+, CD8+, CD25+, MHC class II+ and CD14+ cells. Upregulation of IL-10, IL-4, IL-12p40, TGF-β, IL-1β and INOS mRNA expression was observed in the UV-irradiated epidermal cells. In addition, IL-4, IL-12p40, IFN-γ, and IL-1β mRNAs were increased in the spleen cells after UVR. Furthermore, culture supernatants from in vitro UV-irradiated keratinocytes reduced the MHC class II expression in the peritoneal cells and reduced the spleen cell proliferation to ConA. Future studies will address the identity of the suppressive factors induced by UVR and the systemic effect of culture supernatants from UV-irradiated epidermal cells on M. bovis BCG or M. tuberculosis infection.

Periductal interleukin-17 production in association with biliary innate immunity contributes to the pathogenesis of cholangiopathy in primary biliary cirrhosis

An innate immune response to bacterial components is speculated to be involved in the pathogenesis of primary biliary cirrhosis (PBC). Recently, CD4-positive T helper type 17 (Th17) cells, characterized by the secretion of interleukin (IL)-17, have been implicated in the pathogenesis of autoimmune diseases. Human Th17 cells are generated from Th0 cells by IL-6 and IL-1 beta and maintained by IL-23. In this study, the role of IL-17 in PBC and its association with biliary innate immunity were examined. Using cultured human biliary epithelial cells (BECs), the expression of Th17-related cytokines and chemokines and changes therein on treatment with pathogen-associated molecular patterns (PAMPs) and IL-17 were examined. Immunohistochemistry for IL-17 and Th17-related cytokines was performed using tissue samples of human liver. Consequently, the expression of IL-6, IL-1 beta, IL-23p19 and IL-23/IL-12p40 mRNAs, and their up-regulation by PAMPs, were found in BECs. Moreover, BECs possessed IL-17-receptors and stimulation with IL-17 induced production of IL-6, IL-1 beta, IL-23p19 and chemokines. Several IL-17-positive cells had infiltrated damaged bile ducts and the expression of IL-6 and IL-1 beta was enhanced in the bile ducts of PBC patients. In conclusion, IL-17-positive cells are associated with the chronic inflammation of bile ducts in PBC which is associated causally with the biliary innate immune responses to PAMPs.

Foal Monocyte-Derived Dendritic Cells Become Activated uponRhodococcus equiInfection

Susceptibility of foals to Rhodococcus equi pneumonia is exclusive to the first few months of life. The objective of this study was to investigate the immediate immunologic response of foal and adult horse antigen-presenting cells (APCs) upon infection with R. equi. We measured the activation of the antigen-presenting major histocompatibility complex (MHC) class II molecule, costimulatory molecules CD40 and CD86, the cytokine interleukin-12 (IL-12), and the transcriptional factor interferon regulatory factor 1 (IRF-1) in monocyte-derived macrophages (mMOs) and dendritic cells (mDCs) of adult horses and foals of different ages (from birth to 3 months of age) infected with virulent R. equi or its avirulent, plasmid-cured derivative. Infection with virulent or avirulent R. equi induced (P <or= 0.01) the expression of IL-12p35 and IL-12p40 mRNAs in foal mMOs and mDCs at different ages. This response was likely mediated by the higher (P=0.008) expression of IRF-1 in foal mDCs at birth than in adult horse mDCs. R. equi infection promoted comparable expression of costimulatory molecules CD86 and CD40 in foal and adult horse cells. The cytokine and costimulatory response by foal mDCs was not accompanied by robust MHC class II molecule expression. These data suggest that foal APCs detect the presence of R. equi and respond with the expression of the Th1-inducing cytokine IL-12. Nevertheless, there seems to be a limitation to MHC class II molecule expression which we hypothesize may compromise the efficient priming of naïve effector cells in early life.

Expression of proinflammatory cytokines in osteoarthritis of the temporomandibular joint

Objective This study reports the expression of proinflammatory cytokines in temporomandibular joint (TMJ) of patients affected with temporomandibular osteoarthritis (OA). Design In twelve OA of the TMJ (OA–TMJ) affected patients and in six healthy volunteer subjects studied as control, the expression of IL1β (interleukin-1β), IL2, IL4, IL5, IL6, IL10, IL12p35, IL12p40, IL17, IFNγ (interferon-gamma), TNFα (tumor necrosis factor-alpha), and TNFβ mRNAs was evaluated. Using quantitative real-time RT-PCR technique, the cytokine levels, reported as Ct (cycle threshold), ΔCt (Ct cytokine − Ct 18S rRNA) and RQ (relative quantification), in patient and control groups were compared. Results Expression of IL1β, IL2, IL12p35, IL12p40, IL17, TNFα, TNFβ, and IFNγ mRNAs was significantly higher in patients as compared with controls. In particular, IL12 was the predominant cytokine expressed in patients (IL12p35 RQ = 30.2 and IL12p40 RQ = 29.0). Conversely, IL10 mRNA levels were higher in controls (RQ = 1.8). Conclusions These data suggest that not only IL1β, IFNγ, and TNFα but also IL10, IL12, and IL17 are involved in the OA–TMJ pathogenesis. Furthermore, an inflammatory response characterised by the predominant expression of IL12 mRNA and down-regulated expression of IL10 mRNA is associated with the degenerative changes observed in OA–TMJ.

Differences in the inducible gene expression and protein production of IL-12p40, IL-12p70 and IL-23: Involvement of p38 and JNK kinase pathways

The proper balance between IL-12p40-related cytokines controls the appearance of normal and pathological Th1 immune responses. In this study, we examined the inducible IL-12p40, IL-12p35 and IL-23p19 mRNA expression and protein production in human peripheral blood mononuclear cells (PBMC) and purified monocytes, isolated from healthy donors. We investigated how JNK and p38 MAPKs inhibitors influenced IL-12p40, IL-12p70 and IL-23 production. The cytokines’ quantity determination was performed by ELISA. qRT-PCR was performed for mRNA transcripts detection. All stimuli tested induced higher level of IL-12p40 and IL-12p19 mRNAs. LPS was the strongest inducer of IL-12p40 mRNA, whereas C3bgp stimulated the highest expression of IL-23p19 mRNA in human monocytes. IL-12p40 and IL-23 protein production observed was increased in the highest level after C3bgp stimulation. The inhibition of both JNK and p38 augmented IL-12p40 production. The inhibition of p38 MAPK downregulated IL-23 production and upregulated IL-12p40 production in stimulated monocytes and PBMC. These results provide evidence that in human monocytes and PBMC p38 MAP kinase activation has an opposite effect on the IL-12p40 and IL-23 expression.

Differential Effects of CpG DNA on IFN-β Induction and STAT1 Activation in Murine Macrophages versus Dendritic Cells: Alternatively Activated STAT1 Negatively Regulates TLR Signaling in Macrophages

Classical STAT1 activation in response to TLR agonists occurs by phosphorylation of the Y701 and S727 residues through autocrine type I IFN signaling and p38 MAPK signaling, respectively. In this study, we report that the TLR9 agonist CpG DNA induced Ifn-beta mRNA, as well as downstream type I IFN-dependent genes, in a MyD88-dependent manner in mouse myeloid dendritic cells. This pathway was required for maximal TNF and IL-6 secretion, as well as expression of cell surface costimulatory molecules. By contrast, neither A- nor B-type CpG-containing oligonucleotides induced Ifn-beta in mouse bone marrow-derived macrophages (BMM) and a CpG-B oligonucleotide did not induce IFn-beta in the macrophage-like cell line, J774. In BMM, STAT1 was alternatively activated (phosphorylated on S727, but not Y701), and was retained in the cytoplasm in response to CpG DNA. CpG DNA responses were altered in BMM from STAT1(S727A) mice; Il-12p40 and Cox-2 mRNAs were more highly induced, whereas Tlr4 and Tlr9 mRNAs were more repressed. The data suggest a novel inhibitory function for cytoplasmic STAT1 in response to TLR agonists that activate p38 MAPK but do not elicit type I IFN production. Indeed, the TLR7 agonist, R837, failed to induce Ifn-beta mRNA and consequently triggered STAT1 phosphorylation on S727, but not Y701, in human monocyte-derived macrophages. The differential activation of Ifn-beta and STAT1 by CpG DNA in mouse macrophages vs dendritic cells provides a likely mechanism for their divergent roles in priming the adaptive immune response.

THE EFFECTS OF INHALATION OF DIESEL EXHAUST ON MURINE MYCOBACTERIAL INFECTION

The authors investigated the effects of inhalation of diesel exhaust (DE) on murine mycobacterial infection in vivo. Eight-week-old female BALB/c mice were exposed to DE (3 mg/m3 of diesel exhaust particles [DEPs]) for 1 month, 2 months, or 6 months (for 7 hours a day, 5 days a week). Control mice were housed in a clean room for the same periods. On the day following the last DE exposure, control mice and DE-exposed mice were aerially infected with Mycobacterium tuberculosis (1 × 106 colony-forming units (CFU), Kurono strain). At 7 weeks after mycobacterial infection, the authors examined the lung tissues for histopathological changes and performed reverse transcriptase–polymerase chain reaction (RT-PCR) to measure the messenger RNA (mRNA) expression of several proinflammatory cytokines and inducible nitric oxide synthase (iNOS). Then, the homogenates of lungs and spleens were cultured on 1% (v/v) Ogawa's egg slant medium, and after a 4-week incubation period at 37°C, colonies on the medium were counted. After 1 month of DE exposure, the mycobacterial infection had slightly ameliorated. After 2 months of DE exposure, the expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-12p40, interferon (IFN)-γ, and iNOS mRNAs were slightly increased. However, after 6 months of DE exposure, the expression levels of IL-1β, IL-12p40, IFN-γ, and iNOS mRNAs were decreased, and the infection as measured by increased lung burden (CFU) actually increased. These results indicate that long-term DE exposure may increase pulmonary mycobacterial burden.

Expression of cytokine mRNAs in mice cutaneously exposed to formaldehyde

In this study, we have investigated the expression of cytokine mRNAs in mice cutaneously exposed to formaldehyde using semiquantitative RT-PCR. We show that formaldehyde induced the long-lasting expression of IL-4 and IFN-γ mRNAs and the transient expression of IL-13 mRNA in mouse spleen and draining lymph nodes. The transient increases in IL-2, IL-15, IL-12p40, IL-15 and IL-18 mRNAs, but long-lasting IL-15 mRNA were only seen in the formaldehyde-exposed mouse spleen. Moreover, a weak contact hypersensitivity (CH) and the significant increases in IL-4 and IFN-γ mRNAs were detected in the ear skin of formaldehyde-cutaneously exposed mice when rechallenged mouse ears. Furthermore, CH as measured by mouse ear swelling response was positively correlated with IL-4 and IFN-γ mRNA levels in the challenged ears. This study thus suggests that the induction of Th1 and Th2 cytokine mRNAs, particularly IL-4 and IFN-γ, are a common immunological feature caused by contact allergens irrespective of strong or weak contact allergens. The analysis of IL-4 and IFN-γ mRNAs may be useful markers in establishing the novel test for predicting chemical sensitizing potentials.

C/EBP beta gene inactivation causes both impaired and enhanced gene expression and inverse regulation of IL-12 p40 and p35 mRNAs in macrophages.

The transcription factor C/EBPbeta is believed to play a fundamental role in regulating activated macrophage functions. However, the molecular mechanisms and the target genes involved have been, so far, poorly characterized, partly due to the difficulty of reproducibly obtaining homogeneous and abundant primary macrophage populations. In this study, we describe the generation and characterization of immortalized macrophage-like cell lines from C/EBPbeta-deficient and wild-type mice. Using these cells, we were able to identify a number of genes involved in activated macrophage functions whose induction was affected in the C/EBPbeta(-/-) cells. IFN-gamma/LPS-dependent induction of IL-6, IL-1beta, TNF-alpha, inducible NO synthase, and plasminogen activator inhibitor-1 mRNAs was variably impaired, while IL-12 p40, RANTES and macrophage inflammatory protein-1beta mRNAs were up-regulated in the absence of C/EBPbeta. The differential mRNA expression correlated with differential transcription levels of the corresponding genes, and was in most cases confirmed in primary macrophage populations. Moreover, in sharp contrast to the enhanced induction of IL-12 p40 mRNA, C/EBPbeta(-/-) primary macrophages derived from both the bone marrow and the peritoneal cavity displayed totally defective expression of IL-12 p35 mRNA. Therefore, the IL-12 p35 gene represents a novel obligatory target for C/EBPbeta in macrophages and this may explain the defective production of bioactive IL-12 and the impaired Th1 responses of C/EBPbeta-deficient mice to Candida albicans infection observed in previous work.

Cytokine gene expression in peripheral blood mononuclear cells (PBMC) from patients with pharmacologically treated cystic echinococcosis.

The influence of pharmacological treatment on the immune response of patients with Echinococcus granulosus infection was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) to determine mRNA expression for IL-12 p35, IL-12 p40, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and IL-4 in PBMC from 12 patients before and after chemotherapy and from seven uninfected controls. Most patients' PBMC showed measurable amounts of IL-12 p35, IL-4, IFN-gamma and TNF-alpha mRNA in parasite antigen-stimulated and unstimulated cultures. Conversely, IL-12 p40 mRNA was detected almost exclusively in successfully treated patients (86%) after therapy. In these patients semiquantitative analysis of RT-PCR products showed a significant difference between IL-12 p40 mRNA mean levels before and after therapy (P = 0.03 in parasite antigen-stimulated cultures; P = 0.001 in unstimulated cultures). IL-4 mRNA was weakly expressed before therapy and more highly so after treatment in both groups of patients and under both culture conditions; IL-4 mRNA reached its highest level in post-therapy PBMC from patients in whom therapy failed (stimulated cultures). IFN-gamma and TNF-alpha mRNA expression increased in patients who responded to therapy and decreased in patients who did not. In contrast to IL-12 p35, IFN-gamma and TNF-alpha mRNAs, IL-12 p40 and IL-4 mRNAs were detected exclusively in patients, suggesting a close relationship between these two cytokines and cystic echinococcosis. Our findings indicate that chemotherapy influences the immune response, thus determining changes in Th1/Th2 cytokine mRNA patterns, predominantly in IL-12 p40 and IL-4 mRNA expression.

Cytokine and chemokine mRNA expression in neutrophils from CBA/NSlc mice infected with Plasmodium berghei ANKA that induces experimental cerebral malaria

To investigate the role of neutrophils in experimental cerebral malaria (ECM), in a previous study we found that early neutrophil depletion prevented the development of ECM and down regulated the expression of Th1 cytokines in the brain. To further clarify the mechanisms responsible for these findings, in the present study, using RT-PCR, we examined the expression of cytokine and chemokine mRNAs in neutrophils and macrophages after PbA infection. We found that, after infection, neutrophils not only expressed cytokines IL-2, IL-12p40, IL-18, IFN-γ and TNF-α mRNAs, but also mRNAs for Th1 chemoattractive chemokines, monokine-induced by IFN-γ (MIG), macrophage-inflammatory protein-1α (MIP-1α) and IFN-γ inducible protein-10 (IP-10). Neutrophil depletion down regulated the expression of IL-18 and MIG mRNAs in macrophages, but did not affect the expression of IFN-γ, TNF-α, MIP-1α and IP-10 mRNAs. Therefore, this study confirms our hypothesis that neutrophils may play a role in the pathogenesis of ECM via their expression of cytokines or chemokines.

Recombinant adenovirus induces maturation of dendritic cells via an NF-kappaB-dependent pathway.

Recombinant adenovirus (rAd) infection is one of the most effective and frequently employed methods to transduce dendritic cells (DC). Contradictory results have been reported recently concerning the influence of rAd on the differentiation and activation of DC. In this report, we show that, as a result of rAd infection, mouse bone marrow-derived immature DC upregulate expression of major histocompatibility complex class I and II antigens, costimulatory molecules (CD40, CD80, and CD86), and the adhesion molecule CD54 (ICAM-1). rAd-transduced DC exhibited increased allostimulatory capacity and levels of interleukin-6 (IL-6), IL-12p40, IL-15, gamma interferon, and tumor necrosis factor alpha mRNAs, without effects on other immunoregulatory cytokine transcripts such as IL-10 or IL-12p35. These effects were not related to specific transgenic sequences or to rAd genome transcription. The rAd effect correlated with a rapid increase (1 h) in the NF-kappaB-DNA binding activity detected by electrophoretic mobility shift assays. rAd-induced DC maturation was blocked by the proteasome inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) or by infection with rAd-IkappaB, an rAd-encoding the dominant-negative form of IkappaB. In vivo studies showed that after intravenous administration, rAds were rapidly entrapped in the spleen by marginal zone DC that mobilized to T-cell areas, a phenomenon suggesting that rAd also induced DC differentiation in vivo. These findings may explain the immunogenicity of rAd and the difficulties in inducing long-term antigen-specific T-cell hyporesponsiveness with rAd-transduced DC.

Induction of Cytokine Gene Expression in Mice after Repeated and Subchronic Oral Exposure to Vomitoxin (Deoxynivalenol): Differential Toxin-Induced Hyporesponsiveness and Recovery

A single oral exposure to vomitoxin (VT) in mice has been previously shown to induce in lymphoid tissues the rapid expression of cytokine mRNAs that are produced by both macrophages and T cells. To determine whether prior VT exposures positively or negatively modulate the cytokine response to the toxin in this model, we evaluated the effects of short-term oral (two to seven consecutive daily doses) and subchronic dietary (4 weeks) exposure to VT on expression of a panel of cytokine mRNAs. Effects of a single oral exposure to 0, 5, and 25 mg/kg body wt of VT or of two such daily consecutive doses on splenic cytokine mRNA abundance were compared 2 h after the last toxin administration using RT–PCR in combination with hybridization analysis. While robust cytokine mRNA responses occurred after a single VT exposure, attenuated but significant induction of interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β, and IL-12p40 mRNA was observed after a second VT dose. Similar but insignificant trends occurred with interferon (IFN)-γ, IL-2, IL-4, and IL-10 mRNAs. Serum TNF-α and IL-6 proteins mimicked cytokine mRNA responses although attenuation responses were less marked. Mice were also dosed with VT at 0, 0.5, 2, or 5 mg/kg body wt consecutively for 2, 4, or 7 days and cytokine mRNAs were assessed 2 h after the last treatment in spleen and Peyer's patches. Upon exposure to 2 and 5 mg/kg body wt VT, the relative abundance of IL-1β, IL-6, TNF-α, IL-12 p35, IL-12p40, IL-2, and IL-10 mRNAs increased with dose frequency whereas IFN-γ and IL-4 mRNAs were unaffected. When mice were fed 0, 10, and 25 ppm VT for 4 weeks, increased expression of mRNAs for TNF-α, IL-2, IFN-γ, and IL-10 was most prominent. However, when VT-fed mice were also challenged with an oral dose of VT equivalent to daily intake at 2 h prior to RNA isolation, vigorous mRNA responses were observed for IL-1β, IL-6, TNF-α, IL-12p40, IL-12p35, IL-2, IFN-γ, IL-4, and IL-10. In general, spleens were more responsive to the above effects than Peyer's patches. The results indicate that, following a single prior VT exposure, a significant but attenuated cytokine mRNA response occurred upon a second VT treatment. This hyporesponsiveness was overcome upon repeated exposures to the toxin. These data further support the contention that elevated cytokine expression may play a contributory role in the pathophysiologic and immunologic effects of VT and other trichothecene mycotoxins.