Trogocytosis of peptide-MHC class II complexes from dendritic cells confers antigen-presenting ability on basophils.

Th2 immunity plays important roles in both protective and allergic responses. Nevertheless, the nature of antigen-presenting cells responsible for Th2 cell differentiation remains ill-defined compared with the nature of the cells responsible for Th1 and Th17 cell differentiation. Basophils have attracted attention as a producer of Th2-inducing cytokine IL-4, whereas their MHC class II (MHC-II) expression and function as antigen-presenting cells are matters of considerable controversy. Here we revisited the MHC-II expression on basophils and explored its functional relevance in Th2 cell differentiation. Basophils generated in vitro from bone marrow cells in culture with IL-3 plus GM-CSF displayed MHC-II on the cell surface, whereas those generated in culture with IL-3 alone did not. Of note, these MHC-II-expressing basophils showed little or no transcription of the corresponding MHC-II gene. The GM-CSF addition to culture expanded dendritic cells (DCs) other than basophils. Coculture of basophils and DCs revealed that basophils acquired peptide-MHC-II complexes from DCs via cell contact-dependent trogocytosis. The acquired complexes, together with CD86, enabled basophils to stimulate peptide-specific T cells, leading to their proliferation and IL-4 production, indicating that basophils can function as antigen-presenting cells for Th2 cell differentiation. Transfer of MHC-II from DCs to basophils was also detected in draining lymph nodes of mice with atopic dermatitis-like skin inflammation. Thus, the present study defined the mechanism by which basophils display MHC-II on the cell surface and appears to reconcile some discrepancies observed in previous studies.

VEGF Requires the Receptor NRP-1 To Inhibit Lipopolysaccharide-Dependent Dendritic Cell Maturation.

To stimulate a productive T cell response, dendritic cells (DC) must undergo maturation characterized by heightened cell surface expression of MHC and costimulatory molecules as well as cytokine production. Conversely, the inhibition of DC maturation is a central mechanism of immune tolerance. The control of the DC maturation process relies on the integration of several cellular stimulatory or inhibitory signals. The soluble factors and their receptors controlling this central aspect of DC biology are incompletely characterized. We show that murine bone marrow-derived DC (BMDC) maturation induced by LPS, as opposed to polyinosinic:polycytidylic acid or cytosine-phosphate-guanine, is robustly inhibited by vascular endothelial growth factor (VEGF), a previously identified immunosuppressive cytokine. Using BMDC from wild type and conditional knockout mice, we show that neuropilin-1 (NRP-1), a known receptor of VEGF, is necessary to suppress LPS-dependent BMDC maturation. The absence of NRP-1 had no ostensible effects on the biology of BMDC in the absence of VEGF. However, NRP-1-deficient BMDC remained completely insensitive to the VEGF-dependent inhibition of BMDC maturation in culture. In the presence of VEGF, NRP-1 directly interacted with the LPS receptor TLR4 and suppressed downstream signaling through ERK and NF-κß, resulting in a sharp inhibition of MHC class II and costimulatory molecules (CD40, CD86) expression as well as proinflammatory cytokine production. Consequently, we identify NRP-1 as a target to optimize DC maturation within environments that are rich in VEGF, such as tumors.

Histone deacetylase inhibitor MS-275 augments expression of a subset of IFN-γ-regulated genes in Toxoplasma gondii-infected macrophages but does not improve parasite control.

Toxoplasma gondii is a ubiquitous apicomplexan parasite of mammals and birds and an important pathogen of humans. IFN-γ is the major mediator of host resistance against T. gondii but intriguingly, parasite-infected host cells including macrophages are severely impaired to respond to IFN-γ due to defective transcriptional activation of target genes. Here, we tested the possibility that the impaired responsiveness of T. gondii-infected macrophages to IFN-γ can be restored by inhibiting histone deacetylases (HDACs) using the class I-specific inhibitor MS-275. Treatment of RAW264.7 cells with MS-275 indeed increased MHC class II surface expression in infected and non-infected cells and largely abolished the inhibition of IFN-γ-regulated MHC class II expression exerted by T. gondii. Genome-wide transcriptome profiling revealed that MS-275 increased mean mRNA levels of IFN-γ-regulated genes particularly in non-infected macrophages. Transcript levels of 33% of IFN-γ secondary response genes but only those of a few primary response genes were also increased by MS-275 in T. gondii-infected cells. Importantly, the unresponsiveness of parasite-infected cells to IFN-γ was however not abolished by MS-275. Furthermore, MS-275 also up-regulated several anti-inflammatory cytokines or signaling molecules in T. gondii-infected macrophages. It additionally regulated expression of more than 2500 genes in non-infected macrophages expression of which was surprisingly counteracted by prior infection with T. gondii. FACS analysis and immunofluorescence microscopy revealed that MS-275 did not considerably diminish the number of parasite-positive cells or the intracellular replication in macrophages stimulated or not with IFN-γ. Thus, a supportive therapy using MS-275 appears inappropriate for treatment of toxoplasmosis.

Intestinal microbiota and immune related genes in sea cucumber (Apostichopus japonicus) response to dietary ß-glucan supplementation.

ß-glucan is a prebiotic well known for its beneficial outcomes on sea cucumber health through modifying the host intestinal microbiota. High-throughput sequencing techniques provide an opportunity for the identification and characterization of microbes. In this study, we investigated the intestinal microbial community composition, interaction among species, and intestinal immune genes in sea cucumber fed with diet supplemented with or without ß-glucan supplementation. The results show that the intestinal dominant classes in the control group are Flavobacteriia, Gammaproteobacteria, and Alphaproteobacteria, whereas Alphaproteobacteria, Flavobacteriia, and Verrucomicrobiae are enriched in the ß-glucan group. Dietary ß-glucan supplementation promoted the proliferation of the family Rhodobacteraceae of the Alphaproteobacteria class and the family Verrucomicrobiaceae of the Verrucomicrobiae class and reduced the relative abundance of the family Flavobacteriaceae of Flavobacteria class. The ecological network analysis suggests that dietary ß-glucan supplementation can alter the network interactions among different microbial functional groups by changing the microbial community composition and topological roles of the OTUs in the ecological network. Dietary ß-glucan supplementation has a positive impact on immune responses of the intestine of sea cucumber by activating NF-κB signaling pathway, probably through modulating the balance of intestinal microbiota.

Construction of novel immune-related signature for prediction of pathological complete response to neoadjuvant chemotherapy in human breast cancer.

BACKGROUND: The aim of this study was to construct a novel prediction model for the pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) using immune-related gene expression data. PATIENTS AND METHODS: DNA microarray data were used to perform a gene expression analysis of tumor samples obtained before NAC from 117 primary breast cancer patients. The samples were randomly divided into the training (n = 58) and the internal validation (n = 59) sets that were used to construct the prediction model for pCR. The model was further validated using an external validation set consisting of 901 patients treated with NAC from six public datasets. RESULTS: The training set was used to construct an immune-related 23-gene signature for NAC (IRSN-23) that is capable of classifying the patients as either genomically predicted responders (Gp-R) or non-responders (Gp-NR). IRSN-23 was first validated using an internal validation set, and the results showed that the pCR rate for Gp-R was significantly higher than that obtained for Gp-NR (38 versus 0%, P = 1.04E-04). The model was then tested using an external validation set, and this analysis showed that the pCR rate for Gp-R was also significantly higher (40 versus 11%, P = 4.98E-23). IRSN-23 predicted pCR regardless of the intrinsic subtypes (PAM50) and chemotherapeutic regimens, and a multivariate analysis showed that IRSN-23 was the most important predictor of pCR (odds ratio = 4.6; 95% confidence interval = 2.7-7.7; P = 8.25E-09). CONCLUSION: The novel prediction model (IRSN-23) constructed with immune-related genes can predict pCR independently of the intrinsic subtypes and chemotherapeutic regimens.

Research on the epigenetic modification of pancreatic cancer vaccine.

Pancreatic cancer is characterized as a type of gastrointestinal tumor with a poor prognosis and high degree of malignancy. CIITA gene was found highly methylated in pancreatic carcinoma cell line PANC-1 and responsible for the low expression of MHC-II that may lead to immune evasion. Here, we tried to prepare pancreatic cancer vaccine with PANC-1 cells via epigenetic modification to enhance the MHC-II expression. Then the vaccine was injected into C57BL/6J mice and the effect was examined. Our study found that the vaccine could promote the proliferation of antigen-specific T cells, enhance the killing activity of cytotoxic lymphocytes (CTL), promote Th1-type cells mediated secretion of cytokines IFN-gamma and IL-2 while inhibiting Th2-type cells mediated secretion of IL-4, and inhibit the secretion of TGF-beta. Generally, the epigenetically modified vaccine could enhance the body's anti-tumor immune response, providing feasibility research on cancer vaccine for therapy of pancreatic cancer.

Phycocyanobilin promotes PC12 cell survival and modulates immune and inflammatory genes and oxidative stress markers in acute cerebral hypoperfusion in rats.

Since the inflammatory response and oxidative stress are involved in the stroke cascade, we evaluated here the effects of Phycocyanobilin (PCB, the C-Phycocyanin linked tetrapyrrole) on PC12 cell survival, the gene expression and the oxidative status of hypoperfused rat brain. After the permanent bilateral common carotid arteries occlusion (BCCAo), the animals were treated with saline or PCB, taking samples 24h post-surgery. Global gene expression was analyzed with GeneChip Rat Gene ST 1.1 from Affymetrix; the expression of particular genes was assessed by the Fast SYBR Green RT-PCR Master Mix and Bioplex methods; and redox markers (MDA, PP, CAT, SOD) were evaluated spectrophotometrically. The PCB treatment prevented the H2O2 and glutamate induced PC12 cell injury assessed by the MTT assay, and modulated 190 genes (93 up- and 97 down-regulated) associated to several immunological and inflammatory processes in BCCAo rats. Furthermore, PCB positively modulated 19 genes mostly related to a detrimental pro-inflammatory environment and counteracted the oxidative imbalance in the treated BCCAo animals. Our results support the view of an effective influence of PCB on major inflammatory mediators in acute cerebral hypoperfusion. These results suggest that PCB has a potential to be a treatment for ischemic stroke for which further studies are needed.

Molecular changes of mesenchymal stromal cells in response to dexamethasone treatment.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types. The MSCs can be activated and mobilized if needed. AIM: This study aimed to investigate the response mechanism of MSCs under Dexamethasone (Dex) treatment by combining MSCs microarray and bioinformatics methods. MATERIALS AND METHODS: We downloaded the gene expression profile of rat's MSCs challenge with or without Dex (GSE3339) from Gene Expression Omnibus database, including 2 Dex treated samples and 3 untreated samples. The differentially expressed genes (DEGs) were identified by packages in R language. Then, Gestalt (Genomic Sequence Total Analysis and Lookup Tool) and EASE (Expression Analysis Systematic Explorer) to were employed to obtain the molecular events of MSCs under Dex treatment. RESULTS: A total of 17 genes were identified as DEGs between untreated and treated samples, and they were significant enriched in immune response and cell differentiation. The C3 gene was the common candidate gene selected from two different algorithms, and 24 conserved sites were identified in the 3'UTR of C3 gene. CONCLUSIONS: Genes associated with immune response and cell differentiation were dysregulated in MSCs under Dex.

Induction on differentiation and modulation of bone marrow progenitor of dendritic cell by methionine enkephalin (MENK).

Methionine enkephalin (MENK), the endogenous neuropeptide, is known to exert direct effects on the neuroendocrine and the immune systems and participates in regulation of various functions of cells related to both the innate and adaptive immune systems. Dendritic cells (DCs) play important role in initiating and regulating T cell responses. The aim of this work is to investigate the effects of MENK on differentiation, maturation, and function of DCs derived from murine bone marrow progenitors (BM-derived DCs). Our result showed that MENK could induce BM-derived DCs to polarize predominantly to mDC subtype, rather than pDC both in vivo and in vitro, and this was in favor of Th1 response. BM-derived DCs, after treatment with MENK, up-regulated the expressions of MHC class II and key costimulatory molecules. Result by RT-PCR showed MENK could increase expressions of delta and kappa receptors on BM-derived DCs. Also MENK promoted BM-derived DCs to secret higher levels of proinflammatory cytokines of IL-12p70, TNF-α. Furthermore, differentiated BM-derived DCs treated with MENK displayed higher activity to induce allogeneic T cell proliferation and MENK also inhibited tumor growth in vivo and induced apoptosis of tumor cells in vitro. Thus, it is concluded that MENK could be an effective inducer of BM-derived DCs and might be a new therapeutic agent for cancer, as well as other immune handicapped disease. Also we may consider MENK as a potential adjuvant in vaccine preparation.

Neonatal phytoestrogen exposure alters oviduct mucosal immune response to pregnancy and affects preimplantation embryo development in the mouse.

Treatment of neonatal mice with the phytoestrogen genistein (50 mg/kg/day) results in complete female infertility caused in part by preimplantation embryo loss in the oviduct between Days 2 and 3 of pregnancy. We previously demonstrated that oviducts of genistein-treated mice are "posteriorized" as compared to control mouse oviducts because they express numerous genes normally restricted to posterior regions of the female reproductive tract (FRT), the cervix and vagina. We report here that neonatal genistein treatment resulted in substantial changes in oviduct expression of genes important for the FRT mucosal immune response, including immunoglobulins, antimicrobials, and chemokines. Some of the altered immune response genes were chronically altered beginning at the time of neonatal genistein treatment, indicating that these alterations were a result of the posteriorization phenotype. Other alterations in oviduct gene expression were observed only in early pregnancy, immediately after the FRT was exposed to inflammatory or antigenic stimuli from ovulation and mating. The oviduct changes affected development of the surviving embryos by increasing the rate of cleavage and decreasing the trophectoderm-to-inner cell mass cell ratio at the blastocyst stage. We conclude that both altered immune responses to pregnancy and deficits in oviduct support for preimplantation embryo development in the neonatal genistein model are likely to contribute to infertility phenotype.

Both MHC and non-MHC genes regulate inflammation and T-cell response after traumatic brain injury.

Genetic regulation of autoimmune neuroinflammation is a well known phenomenon, but genetic influences on inflammation following traumatic nerve injuries have received little attention. In this study we examined the inflammatory response in a rat traumatic brain injury (TBI) model, with a particular focus on major histocompatibility class II (MHC II) presentation, in two inbred rat strains that have been extensively characterized in experimental autoimmune encephalomyelitis (EAE); DA and PVG. In addition, MHC and Vra4 congenic strains on these backgrounds were studied to give information on MHC and non-MHC gene contribution. Thus, allelic differences in Vra4, harboring the Ciita gene, was found to regulate expression of the invariant chain at the mRNA level, with a much smaller effect exerted by the MHC locus itself. Notably, however, at the protein level the MHC congenic PVG-RT1(av1) strain displayed much stronger MHCII(+) presentation, as shown both by immunolabeling and flow cytometry, than the PVG strain, dwarfing the effect of Ciita. The PVG-RT1(av1) strain had significantly more T-cell influx than both DA and PVG, suggesting regulation both by MHC and non-MHC genes. Finally, in terms of outcome, the EAE susceptible DA strain displayed a significantly smaller resulting lesion volume than the resistant PVG-RT1(av1) strain. These results provide additional support for a role of adaptive immune response after neurotrauma and demonstrate that outcome is significantly affected by host genetic factors.

Flexibility of the T cell repertoire. Self tolerance causes a shift of T cell receptor gene usage in response to insulin.

Bovine insulin(BI)-specific I-Ab-restricted T cell clones have been characterized for fine specificity and TCR gene usage. We have demonstrated that mouse strains carrying H-2b on three different genetic backgrounds (C57BL, BALB, and 129) rearrange and express the V beta 6 gene in a large proportion (36%) of insulin-specific clones. In these strains, the non-MHC background did not seem to influence TCR gene usage in response to BI. The V beta 6+ clones appeared to be selected by the antigen. In contrast, no V beta 6+ clones could be isolated from (B6 x DBA/2)F1 mice, where V beta 6+ (and V beta 8.1+) T cells are deleted by self tolerance to Mls-1a. Thus, although a small proportion of residual V beta 6+ cells had been demonstrated in Mls-1a mice, these cells could not be retrieved in a response that uses V beta 6 predominantly. In functional terms, therefore, the deletion of V beta 6 by self tolerance appears to be complete. Instead of V beta 6, the majority (up to 60%) of I-Ab- as well as I-Ad-restricted insulin-specific clones from the (B6 x DBA/2)F1 mice expressed V beta 8.2 and V beta 8.3. This shift of gene usage was not accompanied by any detectable change in the fine specificity pattern of response. Thus, in the insulin-specific response, the flexibility of T cell repertoire fully compensates for deletions caused by self tolerance.

Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression.

Interleukin 10 (IL-10) and viral IL-10 (v-IL-10) strongly reduced antigen-specific proliferation of human T cells and CD4+ T cell clones when monocytes were used as antigen-presenting cells. In contrast, IL-10 and v-IL-10 did not affect the proliferative responses to antigens presented by autologous Epstein-Barr virus-lymphoblastoid cell line (EBV-LCL). Inhibition of antigen-specific T cell responses was associated with downregulation of constitutive, as well as interferon gamma- or IL-4-induced, class II MHC expression on monocytes by IL-10 and v-IL-10, resulting in the reduction in antigen-presenting capacity of these cells. In contrast, IL-10 and v-IL-10 had no effect on class II major histocompatibility complex (MHC) expression on EBV-LCL. The reduced antigen-presenting capacity of monocytes correlated with a decreased capacity to mobilize intracellular Ca2+ in the responder T cell clones. The diminished antigen-presenting capacities of monocytes were not due to inhibitory effects of IL-10 and v-IL-10 on antigen processing, since the proliferative T cell responses to antigenic peptides, which did not require processing, were equally well inhibited. Furthermore, the inhibitory effects of IL-10 and v-IL-10 on antigen-specific proliferative T cell responses could not be neutralized by exogenous IL-2 or IL-4. Although IL-10 and v-IL-10 suppressed IL-1 alpha, IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 production by monocytes, it was excluded that these cytokines played a role in antigen-specific T cell proliferation, since normal antigen-specific responses were observed in the presence of neutralizing anti-IL-1, -IL-6, and -TNF-alpha mAbs. Furthermore, addition of saturating concentrations of IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha to the cultures had no effect on the reduced proliferative T cell responses in the presence of IL-10, or v-IL-10. Collectively, our data indicate that IL-10 and v-IL-10 can completely prevent antigen-specific T cell proliferation by inhibition of the antigen-presenting capacity of monocytes through downregulation of class II MHC antigens on monocytes.

Use of human bronchial epithelial cells (BEAS-2B) to study immunological markers resulting from exposure to PM(2.5) organic extract from Puerto Rico.

Fine particulate air pollutants, mainly their organic fraction, have been demonstrated to be associated with cardiovascular and respiratory health problems. Puerto Rico has been reported to have the highest prevalence of pulmonary diseases (e.g., asthma) in the United States. The aim of this study was to assess, for the first time, the immunological response of human bronchial epithelial cells (BEAS-2B) to organic extracts isolated from airborne particulate matter (PM(2.5)) in Puerto Rico. Organic extracts from PM(2.5) collected throughout an 8-month period (2000-2001) were pooled (composite) in order to perform chemical analysis and biological activity testing. BEAS-2B cells were exposed to PM(2.5) organic extract to assess cytotoxicity, levels of cytokines and relative gene expression of MHC-II, hPXR and CYP3A5. Our findings show that organic PM(2.5) consist of toxic as well as bioactive components that can regulate the secretion of cytokines in BEAS-2B, which could modulate inflammatory response in the lung. Trace element analyses confirmed the presence of metals in organic extracts highlighting the relative high abundance of Cu and Zn in polar organic extracts. Polar organic extracts exhibited dose-dependant toxicity and were found to significantly induce the release of interleukin 6 (IL-6), IL-1beta and IL-7 while significantly inhibiting the secretion of IL-8, G-CSF and MCP-1. Moreover, MHC-II transcriptional activity was up-regulated after 24 h of exposure, whereas PXR and CYP3A5 were down-regulated. This research provides a new insight into the effects of PM(2.5) organic fractions on specific effectors and their possible role in the development of respiratory inflammatory diseases in Puerto Rico.

(5R)-5-hydroxytriptolide inhibits the immune response of human peripheral blood mononuclear cells.

AIM: (5R)-5-hydroxytriptolide (LLDT-8) displayed immunosuppressive activities both in vitro and in autoimmune disease models. Here, we aim to further clarify the effect of LLDT-8 on the immune responses of human peripheral blood mononuclear cells (PBMC). METHOD: Cell proliferation of human PBMC from healthy donors was evaluated by [3H]-thymidine uptake. NK cell cytotoxicity was assayed using K562 cells in a [3H] lysis assay. Cytokine production was determined by enzyme-linked immunosorbent assay. The expression of cell surface molecules was detected with flow cytometry. The mRNA expression and the protein phosphorylation levels were detected by RT-PCR and Western immunoblot assay. RESULTS: LLDT-8 at 25 and 50 nM significantly inhibited the PHA- and recall antigens-induced T cell proliferation, and suppressed mixed lymphocyte reaction. LLDT-8 reduced cytokines production (IFN-gamma, IL-2, TNF-alpha) in PHA- and Sac-activated PBMC. LLDT-8 did not alter the increased expression of MHC class I/II and B7.1, but reduced B7.2 by approximately 30%. No effect of LLDT-8 was observed for the expression of T cell activation markers (CD69, CD154). However, LLDT-8 significantly reduced IFN-gamma-expressing T cell percentages and IFN-gamma mRNA transcription in PHA-activated T cells. It also inhibited the phosphorylation levels of JNK and p38. LLDT-8 did not affect NK cytotoxic activity against K562 cells. CONCLUSION: LLDT-8 was a promising immunosuppressant for human immune-related diseases.

Down-regulation of myelin-associated glycoprotein on Schwann cells by interferon-gamma and tumor necrosis factor-alpha affects neurite outgrowth.

To investigate the influence of inflammatory cytokines on the potential of peripheral nerves to regenerate, we analyzed the effect of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on the ability of immortalized Schwann cells to mediate outgrowth of neurites from primary DRG neurons. We found that IFN-gamma and TNF-alpha synergistically inhibited the neurite outgrowth-promoting properties of the Schwann cells by specifically down-regulating myelin-associated glycoprotein (MAG) at the levels of mRNA and cell surface protein by approximately 60%. Antibodies to MAg inhibited the outgrowth of neurites on Schwann cells to the same extent as treatment with the two cytokines. Since MAG appears to be involved in both neurite outgrowth and myelination, our findings may provide evidence for a mechanism, by which inflammatory cytokines interfere with Schwann cell-neuron interactions.

Aryl hydrocarbon receptor activation by benzo(a)pyrene inhibits proliferation of myeloid precursor cells and alters the differentiation state as well as the functional phenotype of murine bone marrow-derived macrophages.

Intensive research during the past decade has highlighted the impact of the regulatory function of the aryl hydrocarbon receptor (AhR) in immunity. In this study, we focused on the influence of AhR activation on the differentiation of murine bone marrow-derived myeloid precursor cells into mature macrophages. Our results show that the activation of AhR by subtoxic doses of the AhR ligand benzo(a)pyrene (BaP) impaired the proliferation of bone marrow cells (BMCs) whereas the proportion of resulting adherent cells was not affected. Flow cytometric analysis revealed that the number of mature bone marrow-derived macrophages (BMMs) was significantly decreased by AhR activation. However, expression of the murine macrophage marker F4/80, the major histocompatibility complex class II (MHC-II) and the Fcγ receptor I (FcγRI/CD64) were upregulated on BaP-exposed BMMs in an AhR-dependent manner. Analysis of cytokine secretion after BMM activation with heat-killed (hk) salmonellae showed that BaP exposure resulted in suppressed secretion of interleukin (IL)-1ß, IL-6 and the chemokine CXC motif ligand 1 (CXCL1). In contrast, the release of tumor necrosis factor (TNF)-α and IL-10 was increased following BaP exposure. In addition, the production of antimicrobial nitric oxide (NO) was increased AhR-dependently. Bacterial stimulation of BaP exposed BMMs also induced the expression of MHC-II and CD64, while the expression of F4/80 was dramatically decreased. In summary, this study demonstrates for the first time that sustained exposure over 6 days of bone marrow-derived myeloid precursors to subtoxic doses of BaP critically interferes with differentiation and activation of BMMs. We could convincingly show that AhR-induced gene regulation is crucial for homeostasis of pro- and anti-inflammatory cytokines during macrophage activation.

Sequence elements required for activity of a murine major histocompatibility complex class II promoter bind common and cell-type-specific nuclear factors.

We have examined the sequence elements and corresponding DNA-binding factors required for transient expression of the A alpha d promoter fused to the bacterial chloramphenicol acetyltransferase reporter gene in a variety of cultured cell lines. Deletion analysis demonstrated that only about 110 nucleotides of sequence 5' of the transcription start site are required for constitutive expression in the murine B-lymphoma cell line A20 or for gamma interferon-induced expression in the murine monocytic cell line WEHI-3. Linker-scanner mutation of this region indicated that at least three sequence elements are required for promoter activity. These elements correspond to the conserved sequence elements found in other human and mouse class II genes, the X box, the Y box, and the H box. Analysis of DNA-binding activity showed that the three most predominant factors present in extracts from WEHI-3, A20, or L cells (which do not express the class II genes) are actually a family of factors that bind to a fourth sequence element, overlapping the 3' end of the X-box sequence, that is homologous to the cyclic AMP-responsive enhancer element. A single common factor that binds to the Y box was detected in extracts from all cells tested, as has been seen with the Y-box elements of other class II genes. Another common factor was found that binds to the more conserved 5' region of the X-box element, although A20 extracts contained a second, distinct binding activity for this region. A common binding factor for the H-box element was detected in extracts from WEHI-3 and L cells. However, this activity was absent in A20 cell extracts. Instead, two different H-box-binding activities were detected, suggesting that different components are involved in class II gene expression in B cells and macrophages. Finally, gamma interferon treatment did not significantly alter the DNA-binding activity in WEHI-3 cells for any of the sequence elements shown to be required for induced chloramphenicol acetyltransferase expression.

Mutational analysis of the DRA promoter: cis-acting sequences and trans-acting factors.

Class II major histocompatibility genes are expressed at high levels in B lymphocytes and are gamma interferon (IFN-gamma) inducible in many other cells. Previously, we observed that DRA promoter sequences from positions -150 to +31 determine the tissue specificity of this class II gene. Moreover, Z and X boxes located between positions -145 and -87 conferred B-cell specificity and IFN-gamma inducibility upon a heterologous promoter. In this study, sequences from positions -145 to -35 in the DRA promoter were systematically mutated by using oligonucleotide cassettes. Z (-131 to -125), pyrimidine (-116 to -109), X (-108 to -95), Y (-73 to -61), and octamer (-52 to -45) boxes were required for B-cell specificity and, with the exception of the octamer box, for IFN-gamma inducibility. Z box and sequences flanking Z and X boxes helped to determine low levels of expression in T and uninduced cells. In phenotypically distinct cells, shared and distinct proteins bound to these conserved upstream sequences. However, few correlations between expression and DNA-binding proteins could be made. Similar proteins bound to Z and X boxes, and the Z box most likely represents a duplication of the X box.

Tumor necrosis factor alpha regulates expression of the major histocompatibility complex class II-associated invariant chain by binding of an NF-kappa B-like factor to a promoter element.

Expression of the major histocompatibility complex (MHC) class I and class II antigens and the class II-associated invariant chain (Ii) is strongly increased by treatment of cells with tumor necrosis factor alpha (TNF-alpha) and gamma interferon. We investigated elevation of expression of the invariant chain gene by TNF-alpha. Rat fibroblast cells transfected with the mouse Ii gene containing 802 base pairs of 5' sequences could be stimulated for Ii expression by treatment with TNF-alpha. Analysis of 5'-deleted Ii gene promoter-CAT constructs provided evidence for the presence of a TNF-alpha response box (TRB). Cloning of TRB in front of a non-TNF-alpha-responsive promoter could transfer the TNF-alpha stimulatory effect. We demonstrate binding of a TNF-alpha-induced factor to a kappa B-like motif within TRB. Mutations introduced into the kappa B element of the Ii promoter-CAT plasmid abolished the TNF-alpha-mediated stimulatory effect. Comparison of the TNF-alpha-induced factor and lipopolysaccharide-induced NF-kappa B in gel mobility shift assays upon partial protease digestion suggests similar DNA-binding protein cores. Further support for the NF-kappa B-like nature of the TNF-alpha-induced factor was obtained in methylation interference assays. The TNF-alpha-induced nuclear factor comprises DNA contact sites that are identical to those described for NF-kappa B. This TNF-alpha-induced factor also interacts with kappa B-like sequences of the MHC Kb, Ek alpha, and beta 2-microglobulin promoter, suggesting a common TNF-alpha-mediated regulatory signal for expression of MHC antigens and Ii.