CD11c Promoter Research Articles

Microglia can be induced by IFN-γ or IL-4 to express neural or dendritic-like markers

Microglia are resident cells in the central nervous system (CNS), of hematopoietic origin with a high plasticity. In this study, we examined whether adaptive immune system, involving in CNS maintenance and repair, can induce microglia to express markers of neural cells. We show that long exposure (above 10 days) of microglia to low doses (10 ng/ml) of the ‘proinflammatory’ T-cell derived cytokine, IFN-γ, induced them to express neuronal markers including γ-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD-67). In contrast, exposure of microglia to low doses (10 ng/ml) of the ‘anti-inflammatory’ T-cell derived cytokine, IL-4, induced the expression of oligodendrocyte markers and dendritic cell (DC) marker, CD11c. The microglial origin of the neural-like cells was confirmed using microglia from transgenic mice expressing GFP under promoter of the chemokine fractalkine receptor CX 3CR1, and diphtheria toxin receptor, under CD11c promoter. This study emphasizes that microglial plasticity includes their ability to give rise to neural-like cells and shows that cytokines produced by the adaptive immune system are involved in these processes.

351: Early recovery of host T cells predicts primary graft rejection following non-myeloablative conditioning allogeneic hematopoietic stem cell transplantation

The incidence of graft rejection is increasing in association with an increased use of non-myeloablative conditioning regimen, alternative donors, and cord blood transplantation. We investigated a role of host T cells in graft rejection following non-myeloablative conditioning bone marrow transplantation (BMT) in an MHC mismatched B6D2F1 (H-2b/d) into B6 (H-2b) mouse model of BMT, where host T cells can be activated to kill donor cells. BMT following 6 Gy, 7Gy, and 10Gy total body irradiation (TBI) resulted in a primary graft rejection, secondary graft rejection, and engraftment of donor cells, respectively. Interestingly, expansion of host T cells is observed in the bone marrow and spleen 2 weeks after BMT, followed by a brisk increase in number of host T cells in peripheral blood one week later. CD8+ T cells isolated from bone marrow produced interferon-gamma and lysed donor cells. We then examined whether donor-derived dendritic cells (DCs) play a role in this rejection, using transgenic mice carrying diphtheria toxin receptor (DTR) under the control of the CD11c promoter, which allows selective ablation of CD11c+ DCs by injection of diphtheria toxin (DTx), as donors. In the recipients conditioned with 6Gy TBI, depletion of donor-derived DCs with DTx injection reduced the host T cell-expansion and leads to transient mixed chimerism in contrast to primary graft rejection in controls. We then tested whether such an early recovery of host T cells could be predictive of the graft rejection in 7 adult recipients of non-myeloablative cord blood transplantation. Three week post-transplant, an overshoot of host T cells was observed in 2 patients who experienced rejection, but in none of 5 patients with engraftment. These results suggest that residual host T cells are associated with graft rejection and an early recovery of host T cells may be predictive of graft rejection after non-myloablative allogeneic hematopoietic stem cell transplantation. The incidence of graft rejection is increasing in association with an increased use of non-myeloablative conditioning regimen, alternative donors, and cord blood transplantation. We investigated a role of host T cells in graft rejection following non-myeloablative conditioning bone marrow transplantation (BMT) in an MHC mismatched B6D2F1 (H-2b/d) into B6 (H-2b) mouse model of BMT, where host T cells can be activated to kill donor cells. BMT following 6 Gy, 7Gy, and 10Gy total body irradiation (TBI) resulted in a primary graft rejection, secondary graft rejection, and engraftment of donor cells, respectively. Interestingly, expansion of host T cells is observed in the bone marrow and spleen 2 weeks after BMT, followed by a brisk increase in number of host T cells in peripheral blood one week later. CD8+ T cells isolated from bone marrow produced interferon-gamma and lysed donor cells. We then examined whether donor-derived dendritic cells (DCs) play a role in this rejection, using transgenic mice carrying diphtheria toxin receptor (DTR) under the control of the CD11c promoter, which allows selective ablation of CD11c+ DCs by injection of diphtheria toxin (DTx), as donors. In the recipients conditioned with 6Gy TBI, depletion of donor-derived DCs with DTx injection reduced the host T cell-expansion and leads to transient mixed chimerism in contrast to primary graft rejection in controls. We then tested whether such an early recovery of host T cells could be predictive of the graft rejection in 7 adult recipients of non-myeloablative cord blood transplantation. Three week post-transplant, an overshoot of host T cells was observed in 2 patients who experienced rejection, but in none of 5 patients with engraftment. These results suggest that residual host T cells are associated with graft rejection and an early recovery of host T cells may be predictive of graft rejection after non-myloablative allogeneic hematopoietic stem cell transplantation.

Cutting Edge: Dendritic Cells Are Essential for In Vivo IL-12 Production and Development of Resistance againstToxoplasma gondiiInfection in Mice

A powerful IFN-gamma response is triggered upon infection with the protozoan parasite, Toxoplasma gondii. Several cell populations, including dendritic cells (DCs), macrophages, and neutrophils, produce IL-12, a key cytokine for IFN-gamma induction. However, it is still unclear which of the above cell populations is its main source. Diphtheria toxin (DT) injection causes transient DC depletion in a transgenic mouse expressing Simian DT receptors under the control of the CD11c promoter, allowing us to investigate the role of DCs in IL-12 production. T. gondii-inoculated DT-treated and control groups were monitored for IL-12 levels and survival. We show in this study that DC depletion abolished IL-12 production and led to mortality. Furthermore, replenishment with wild-type, but not MyD88- or IL-12p35-deficient, DCs rescued IL-12 production, IFN-gamma-induction, and resistance to infection in DC-depleted mice. Taken together, the results presented in this study indicate that DCs constitute the major IL-12-producing cell population in vivo during T. gondii infection.

Se-methylselenocysteine enhances PMA-mediated CD11c expression via phospholipase D1 activation in U937 cells

CD11c/CD18 is expressed primarily on myeloid cells, where its expression is regulated both during differentiation and during monocyte maturation into tissue macrophages, and is also a receptor for fibrinogen and lipopolysaccharide (LPS). We focused on the molecular mechanisms leading to the activation of CD11c expression in differentiating U937 cells. During phorbol myristate acetate (PMA)-induced differentiation of U937 cells, we found that the mRNA expression of CD11c was increased. Se-methylselenocysteine (Se-MSC) potentiated up-regulation of CD11c expression and its promoter activity and increased PLD1 activity without affecting the level of PLD1 protein in PMA-treated cells. To examine the regulation mechanism of PMA and Se-MSC on CD11c gene expression through the activation of PLD1, we analyzed changes in the CD11c mRNA level and the promoter activity following treatment of a selective PLD inhibitor n-butanol. The combinatory effect of PMA and Se-MSC on CD11c gene expression was abolished by n-butanol in a dose-dependent manner. Further, introduction of PLD1 gene into U937 cells increased CD11c mRNA expression and activated CD11c promoter activity in a dose-dependent manner. These results showed that Se-MSC increased PMA-induced CD11c expression through the activation of PLD1 signaling pathway. To our knowledge, this is the first report that expression of the CD11c gene is regulated by PLD1 and is enhanced by Se-MSC during PMA-induced U937 differentiation.

The Shiga toxin B‐subunit targets antigen in vivo to dendritic cells and elicits anti‐tumor immunity

The non-toxic B-subunit of Shiga toxin (STxB) interacts with the glycolipid Gb3, which is preferentially expressed on dendritic cells (DC) and B cells. After administration of STxB chemically coupled to OVA (STxB-OVA) in mice, we showed that the immunodominant OVA(257-264) peptide restricted by K(b) molecules is specifically presented by CD11c+ CD8alpha- DC, some of them displaying a mature phenotype. Using mice carrying a transgene encoding a diphtheria toxin receptor (DTR) under the control of the murine CD11c promoter, which allows inducible ablation of DC, we showed that DC are required for efficient priming of CTL after STxB-OVA vaccination. Immunization of mice with STxB-OVA induced OVA-specific CD8+ T cells detected ex vivo; these cells were long lasting, since they could be detected even 91 days after the last immunization and were composed of both central and memory T cells. Vaccination of mice with STxB-OVA and STxB coupled to E7, a protein derived from HPV16, inhibited tumor growth in prophylactic and therapeutic experiments. This effect was mainly mediated by CD8+ T cells. STxB therefore appears to be a powerful carrier directly targeting DC in vivo, resulting in a strong and durable CTL response associated with tumor protection.

Insufficient APC Capacities of Dendritic Cells in Gene Gun-Mediated DNA Vaccination

Gene gun-mediated DNA immunization is a powerful mode of vaccination against infectious diseases and tumors. Many studies have identified dendritic cells (DC) as the central players in inducing immunity upon biolistic DNA vaccination; however, none of these studies directly quantify DC-mediated responses in comparison with immunity triggered by all Ag- and MHC-expressing cells. In this study we use two different approaches to decipher the relative role of DC vs other cell types in gene gun-induced immunity. First, we directly compared the immunization efficacy of different DNA constructs, which allow Ag expression ubiquitously (CMV promoter) or specifically in DC (CD11c promoter) and would encode either for soluble or membrane bound forms of Ag. Second, we immunized transgenic mice in which only DC can present MHC-restricted Ag, and directly compared the magnitudes of CTL activation with those obtained in wild-type mice. Surprisingly, our combined data suggest that, although DC-specific Ag expression is sufficient to induce humoral responses, DC alone cannot trigger optimal CD4 and CD8 T cell responses upon gene gun vaccination. Therefore, we conclude that DC alone are insufficient to mediate optimal induction of T cell immunity upon gene gun DNA vaccination and that broad Ag expression rather than DC-restricted approaches are necessary for induction of complete immune responses.

CD11c+ Dendritic Cells Are Required for Survival in Murine Polymicrobial Sepsis

CD11c+ dendritic cells (DCs) are APCs that link innate and adaptive immunity. Although DCs are lost from spleen and lymph nodes in sepsis, their role in outcome remains unclear. Transgenic mice (B6.FVB-Tg(.Itgax-DTR/EGFP.57)Lan/J) expressing the diphtheria toxin (DT) receptor on the CD11c promoter (DCKO mice) received 4 ng/kg DT, which resulted in depletion of 88-95% of mature myeloid and lymphoid DCs, with less depletion (75%) of plasmacytoid DCs. Pretreatment of DCKO mice with DT resulted in reduced survival in sepsis compared with saline-pretreated DCKO mice (0 vs 54%; p < 0.05) or DT-treated wild-type littermates (0 vs 54%; p < 0.05). This increased mortality was not associated with either increased bacteremia or plasma cytokine concentrations. Intravenous injection of 10(7) wild-type DCs improved survival in DCKO mice (42 vs 0%; p = 0.05). These data confirm that DCs are essential in the septic response and suggest that strategies to maintain DC numbers or function may improve outcome.

B Lymphocytes Participate in Cross-Presentation of Antigen following Gene Gun Vaccination

Although endocytosed proteins are commonly presented via the class II MHC pathway to stimulate CD4(+) T cells, professional APCs can also cross-present Ags, whereby these exogenous peptides can be complexed with class I MHC for cross-priming of CD8(+) T cells. Whereas the ability of dendritic cells (DCs) to cross-present Ags is well documented, it is not known whether other APCs may also play a role, or what is the relative contribution of cross-priming to the induction of acquired immunity after DNA immunization. In this study, we compared immune responses generated after gene gun vaccination of mice with DNA vaccine plasmids driven by the conventional CMV promoter, the DC-specific CD11c promoter, or the keratinocyte-specific K14 promoter. The CD11c promoter achieved equivalent expression in CD11c(+) DCs in draining lymph nodes over time, as did a conventional CMV-driven plasmid. However, immunization with DC-restricted DNA vaccines failed to generate protective humoral or cellular immunity to model Ags influenza hemagglutinin and OVA, despite the ability of CD11c(+) cells isolated from lymph nodes to stimulate proliferation of Ag-specific T cells directly ex vivo. In contrast, keratinocyte-restricted vaccines elicited comparable T and B cell activity as conventional CMV promoter-driven vaccines, indicating that cross-priming plays a major role in the generation of immune responses after gene gun immunization. Furthermore, parallel studies in B cell-deficient mu-MT mice demonstrated that B lymphocytes, in addition to DCs, mediate cross-priming of Ag-specific T cells. Collectively, these data indicate that broad expression of the immunogen is required for optimal induction of protective acquired immunity.

CD11c gene expression in hairy cell leukemia is dependent upon activation of the proto-oncogenes ras andjunD

AbstractHairy cell leukemia (HCL) is a chronic lymphoproliferative disease, the cause of which is unknown. Diagnostic of HCL is abnormal expression of the gene that encodes the β2 integrin CD11c. In order to determine the cause of CD11cgene expression in HCL theCD11c gene promoter was characterized. Transfection of theCD11c promoter linked to a luciferase reporter gene indicated that it is sufficient to direct expression in hairy cells. Mutation analysis demonstrated that of predominant importance to the activity of the CD11c promoter is its interaction with the activator protein-1 (AP-1) family of transcription factors. Comparison of nuclear extracts prepared from hairy cells with those prepared from other cell types indicated that hairy cells exhibit abnormal constitutive expression of an AP-1 complex containing JunD. Functional inhibition of AP-1 expressed by hairy cells reducedCD11c promoter activity by 80%. Inhibition of Ras, which represents an upstream activator of AP-1, also significantly inhibited the CD11c promoter. Furthermore, in the hairy cell line EH, inhibition of Ras signaling through mitogen-activated protein kinase/extracellular signal–regulated kinase kinases 1 and 2 (MEK1/2) reduced not only CD11c promoter activity but also reduced both CD11c surface expression and proliferation. Expression in nonhairy cells of a dominant-positive Ras mutant activated the CD11cpromoter to levels equivalent to those in hairy cells. Together, these data indicate that the abnormal expression of the CD11cgene characteristic of HCL is dependent upon activation of the proto-oncogenes ras and junD.

During differentiation of the monocytic cell line U937, Pur alpha mediates induction of the CD11c beta 2 integrin gene promoter.

CD11c is a member of the beta(2) integrin family of adhesion molecules that, together with CD18, forms a heterodimeric receptor on the surface of myeloid, NK, dendritic, and certain leukemic, lymphoma, and activated lymphoid cells. Monocytic differentiation is associated with an induction of both CD11c and CD18 gene expression. The resulting CD11c/CD18 receptor mediates firm adhesion to the vascular endothelium, transendothelial migration, chemotaxis, and phagocytosis. Monocytic differentiation can be mimicked in vitro by treatment of the promonocytic cell line U937 with PMA. Recently, we reported that in U937 cells, expression of the CD11c gene is controlled by an unidentified transcription factor that binds ssDNA. This finding suggested that DNA secondary structure plays an important role in controlling the CD11c gene and prompted us to search for additional ssDNA-binding activities with which this gene interacts. In this study, we report that in U937 cells, expression of the CD11c gene is mediated by the ssDNA-binding protein Puralpha. During PMA-induced differentiation, the ability of Puralpha to activate the CD11c promoter in U937 cells increases, as does that of Sp1. Together, these increases in the functional activity of both Puralpha and Sp1 combine to induce CD11c expression.

The leukocyte integrins are regulated by transcriptional and post-transcriptional mechanisms in a leukemic cell that overexpresses protein kinase C-ζ

Overexpression of protein kinase C-zeta (PKC-zeta) in the leukemic myeloid cell line U937 (U937-PKC-zeta cells), previously shown to induce leukemic cell differentiation, resulted in nearly complete downregulation of leukocyte integrins CD11a, CD11b, CD11d, and CD18, but not CD11c from the cell surface. The steady-state level of mRNAs for the downregulated leukocyte integrins was not detectable by Northern analysis. Nuclear run-on analysis revealed that transcription of all the leukocyte integrin genes except CD11c was reduced 70-90% as compared to control U937-Vector cells [U937 cells transfected with the empty vector pSV2M(2)6]. Transfection analysis of CD11-promoter-luciferase constructs confirmed that transcription of the leukocyte integrin genes was drastically downregulated in U937-PKC-zeta cells. The two c-jun binding sites in the CD11c promoter were essential for continued expression of CD11c in U937-PKC-zeta cells. Additionally, the 3' untranslated region (3' UTR) from CD11b, when fused to the luciferase gene, lead to the destabilization of this chimeric mRNA in U937-PKC-zeta cells. This indicates that downregulation of CD11b expression in U937-PKC-zeta cells is also the result of reduced stability of CD11b mRNA. Thus, overexpression of PKC-zeta in U937 cells leads not only to leukemic cell differentiation, but also to differential regulation of the leukocyte integrins.

C-Myc inhibits CD11a and CD11c leukocyte integrin promoters

The c-Myc transcription factor is an important regulator of cell growth and differentiation, and its gene repression ability seems to play a key role in Myc-mediated cellular transformation. Since Myc overexpression has been associated with reduced expression of β1 and β2 integrins, we have investigated the role of c-Myc on CD11a and CD11c transcription. c-Myc inhibited CD11a and CD11c integrin promoter activity in co-transfection experiments, and similar repression was obtained in cells where c-Myc expression (KmycB) or activity (Rat-1 c-MycERTM) is inducible. The c-Myc repression on the CD11c promoter was independent of the USF-binding site (USF-150), other putative Myc-binding elements, or the integrity of the initiator (Inr)-like sequence present at the major transcriptional start site. Analysis of deletion and mutant promoter constructs revealed that, in the absense of additional upstream cis-acting elements, an AP-1-binding site at -60 (AP1-60) is required for c-Myc repressor activity. The c-Myc repressor activity on both integrin promoters was abrogated by deletion of c-Myc residues 106 – 143, a domain involved in Inr-dependent transcriptional repression. These results demonstrate a direct effect of c-Myc on integrin gene transcription and suggest the existence of a c-Myc-dependent mechanism for coupling leukocyte integrin expression to the cell proliferative state.

C-Myc inhibits CD11a and CD11c leukocyte integrin promoters.

The c-Myc transcription factor is an important regulator of cell growth and differentiation, and its gene repression ability seems to play a key role in Myc-mediated cellular transformation. Since Myc overexpression has been associated with reduced expression of beta1 and beta2 integrins, we have investigated the role of c-Myc on CD11a and CD11c transcription. c-Myc inhibited CD11a and CD11c integrin promoter activity in co-transfection experiments, and similar repression was obtained in cells where c-Myc expression (KmycB) or activity (Rat-1 c-MycER) is inducible. The c-Myc repression on the CD11c promoter was independent of the USF-binding site (USF-150), other putative Myc-binding elements, or the integrity of the initiator (Inr)-like sequence present at the major transcriptional start site. Analysis of deletion and mutant promoter constructs revealed that, in the absence of additional upstream cisacting elements, an AP-1-binding site at -60 (AP1-60) is required for c-Myc repressor activity. The c-Myc repressor activity on both integrin promoters was abrogated by deletion of c-Myc residues 106-143, a domain involved in Inr-dependent transcriptional repression. These results demonstrate a direct effect of c-Myc on integrin gene transcription and suggest the existence of a c-Myc-dependent mechanism for coupling leukocyte integrin expression to the cell proliferative state.

The role of dendritic cells in T cell selection and survival.

Dendritic cells (DC) not only play an important role in the induction of immunity, but also have major functions in thymus and periphery in the absence of nominal antigen. In order to study these and other DC-functions in vivo, we expressed MHC class II I-E molecules under the control of the DC-specific CD11c promoter in C57B1/6 mice. With this targeted expression we were able to investigate DC functions in vivo without further manipulation of DC. We describe our findings with respect to the two major thymic events of negative and positive selection and demonstrate that DC are able and sufficient to induce negative selection of autoreactive CD4+ thymocytes by clonal deletion but do not induce positive selection. We further demonstrate in thymus-transplant experiments that DC play a major role in inducing survival of peripheral CD4+ mature T cells. This transgenic model therefore seems to be a valuable tool for the investigation of DC functions in vivo.

CCAAT-Enhancer-binding Proteins (C/EBP) Regulate the Tissue Specific Activity of the CD11c Integrin Gene Promoter Through Functional Interactions with Sp1 Proteins

The CD11c/CD18 integrin binds lipopolysaccharide, fibrinogen, and heparin, and mediates leukocyte adhesion, spreading, and migration. CD11c/CD18 is primarily found on myeloid cells and its expression is regulated during myeloid differentiation by transcriptional mechanisms acting on the CD11c gene promoter. We now describe that CCAAT/enhancer-binding proteins (C/EBP) contribute to the basal, tissue-specific and developmentally regulated activity of the CD11c promoter. A C/EBP-binding site within the CD11c promoter (CEBP-80) is bound by CEBPalpha in undifferentiated U937 cells and by C/EBPalpha- and C/EBPbeta-containing dimers in phorbol 12-myristate 13-acetate-differentiating cells, and its disruption decreased the CD11c promoter activity in a cell type-dependent manner. C/EBPalpha transactivated the CD11c promoter through the CEBP-80 element, and C/EBPalpha transactivation was also dependent on the Sp1-70- and Sp1-120 Sp1-binding sites. The -90/-50 fragment from the CD11c promoter, containing the adjacent CEBP-80, Sp1-70, and AP1-60 sites, differentially enhanced the activity of the minimal prolactin promoter in hematopoietic and epithelial cells. Altogether, these results demonstrate that C/EBP factors participate in the tissue-restricted and regulated expression of the CD11c/CD18 integrin through functional interactions with Sp1, suggest that Sp1-related factors modulate C/EBPalpha transcriptional activity on the CD11c promoter, and demonstrate the existence of a composite regulatory element recognized by C/EBP, Sp1, and AP-1 factors and whose enhancing effects are cell-type dependent.

Sp3 Mediates Transcriptional Activation of the Leukocyte Integrin Genes CD11C and CD11B and Cooperates with c-Jun to Activate CD11C

The leukocyte integrin genes CD11c and CD11b are expressed predominately in myelomonocytic cells. In previous experiments, the -70 to -65 and -121 to -103 regions of the CD11c promoter and the -66 to -59 region of the CD11b promoter were shown to be essential for Sp1-mediated activation of these genes. In vivo genomic footprinting had also revealed cell-specific binding of protein, presumably Sp1, to these regions. In this study, electrophoretic mobility shift analysis showed that the Sp1-related factor, Sp3, also binds at or near these same regions. Cotransfection of Sp3 along with CD11c promoter-luciferase constructs into Sp-deficient Drosophila Schneider 2 cells showed that Sp3 could activate the CD11c promoter. Deletion of both the -70 to -65 and -121 to -103 regions of the CD11c promoter resulted in the loss of activation by Sp3. Both sites showed activation by Sp3; however, the -70 to -65 region was more responsive to Sp3 than to Sp1. Similar transfection analysis of the -66 to -59 region of the CD11b promoter showed Sp3-dependent expression. Further, cotransfection analysis in Drosophila cells showed that Sp3, as was previously shown for Sp1, also synergizes with c-Jun to activate CD11c. Antisense experiments that knocked out endogenous Sp3 expression in the myelomocytic cell line, HL60, revealed that Sp3 participates in activation of the CD11c and CD11b promoters in vivo.

PU.1 negatively regulates the CD11c integrin gene promoter through recognition of the major transcriptional start site.

CD11c integrin expression is restricted to myeloid cells and activated B lymphocytes, mainly through the collaborative action of Sp1 and members of the AP-1 and C/EBP transcription factor families on the proximal region of the CD11c gene promoter. While analyzing the role of an initiator-like sequence at the major transcriptional start site, an inverted consensus GGAA Ets binding site was identified as a negative regulatory element whose disruption increases the activity of the CD11c promoter. The GGAA element was specifically recognized by PU.1 in THP-1 monocytic cells and by PU.1 and GABP-related proteins in U937 promonocytic cells. Mutational analysis indicated that PU.1 recognition depends not only on the GGAA consensus element but also on flanking sequences. The functional relevance of PU.1 binding was assayed in transactivation experiments in HeLa cells, where PU.1 co-expression led to a significant decrease in the activity of the CD11c promoter, demonstrating that PU.1 inhibits the activity of the CD11c promoter through a PU.1 binding site located at the major transcriptional start site (PU1-5). The inhibitory action of PU.1 on CD11c is in contrast with its positive regulatory effect on the CD11b and CD18 integrin gene promoters, which might contribute to the differentially regulated expression of CD11b/CD18 and CD11c/CD18 during monocyte extravasation and terminal maturation. In addition, since PU.1 transcriptional activity correlates with macrophage proliferation, PU.1 might modulate CD11c gene transcription according to the proliferative state of the cell.

An octamer element functions as a regulatory element in the differentiation-responsive CD11c integrin gene promoter: OCT-2 inducibility during myelomonocytic differentiation.

The integrin CD11c/CD18 mediates leukocyte adhesion to endothelium and other cell types and is a receptor for LPS, iC3b, and fibrinogen. CD11c expression is restricted to myeloid and activated B cells, is regulated during leukocyte differentiation, and constitutes a diagnostic tool for hairy cell leukemia. Mapping of in vivo DNA-protein interactions in the CD11c proximal promoter revealed three adjacent myeloid-specific interactions, one of which lies on an octamer consensus sequence, ATTT GCAT (Oct185). Oct185 disruption increased the CD11c promoter activity while decreasing its myeloid differentiation responsiveness, indicating that Oct185 contributes to the activity of the CD11c promoter and suggesting that Oct185 is a negative regulatory element whose function changes during myeloid differentiation. Oct185 is recognized by the ubiquitous Oct-1 factor in all cell lineages and by Oct-2 in B lymphoid lineage cells. Unexpectedly, Oct-2 binding to Oct185 was induced de novo upon monocytic differentiation of U937 and HL-60 cells but not during HL-60 granulocytic differentiation, as determined by electrophoretic mobility shift assays and immunochemical studies, and Oct-2 complexes were also observed in cultured adherent monocytes. Western blotting showed that the pattern of Oct-2 isoforms in myeloid cells is similar to that seen in B cells. The Oct-2 up-regulated expression in differentiating myeloid cells and its binding to the Oct185 negative regulatory element suggests its involvement in the differentiation-regulated activity of the CD11c promoter and might represent an important parameter for the myeloid- and B cell-restricted expression of the CD11c/CD18 integrin and other molecules with similar patterns of expression.

Targeted expression of major histocompatibility complex (MHC) class II molecules demonstrates that dendritic cells can induce negative but not positive selection of thymocytes in vivo.

It is well established that lymphoid dendritic cells (DC) play an important role in the immune system. Beside their role as potent inducers of primary T cell responses, DC seem to play a crucial part as major histocompatibility complex (MHC) class II+ "interdigitating cells" in the thymus during thymocyte development. Thymic DC have been implicated in tolerance induction and also by some authors in inducing major histocompatibility complex restriction of thymocytes. Most of our knowledge about thymic DC was obtained using highly invasive and manipulatory experimental protocols such as thymus reaggregation cultures, suspension cultures, thymus grafting, and bone marrow reconstitution experiments. The DC used in those studies had to go through extensive isolation procedures or were cultured with recombinant growth factors. Since the functions of DC after these in vitro manipulations have been reported to be not identical to those of DC in vivo, we intended to establish a system that would allow us to investigate DC function avoiding artificial interferences due to handling. Here we present a transgenic mouse model in which we targeted gene expression specifically to DC. Using the CD 11c promoter we expressed MHC class II I-E molecules specifically on DC of all tissues, but not on other cell types. We report that I-E expression on thymic DC is sufficient to negatively select I-E reactive CD4+ T cells, and to a less complete extent, CD8+ T cells. In contrast, it only DC expressed I-E in a class II-deficient background, positive selection of CD4+ T cells could not be observed. Thus negative, but not positive, selection events can be induced by DC in vivo.

CD11c Integrin Gene Promoter Activity During Myeloid Differentiation

The integrin CD11c/CD18 functions as a cell surface receptor for numerous soluble factors and proteins (LPS, fibrinogen, iC3b), mediates leukocyte interactions with other cell types and is a signal transducing receptor. CD11c/CD18 is found primarily on myeloid cells, where its expression is regulated both during differentiation and during monocyte maturation into tissue macrophages. To determine the transcription factors and cis-acting elements driving the developmentally-regulated expression of CD11c/CD18 the proximal regulatory region of the CD11c gene has been structurally and functionally characterized using the U937 and HL-60 cell lines as myeloid differentiation models. The tissue-specific activity of the CD11c promoter is conferred by two Spl-binding sites and an adjacent C/EBP-binding element, with a likely contribution from other transcription factors with a more limited tissue distribution (PU. 1. Oct-2, Myb). The participation of Sp1 in the transcription of the CD11c gene strongly suggests that CD11c/CD18 expression is dependent on the proliferative state of the cell, thus establishing a first level of control for the regulated expression of CD11c/CD18 during myeloid differentiation. The differentiation responsiveness of the CD11c promoter has been mapped to an AP-1-binding site whose mutation greatly decreases the inducibility of the promoter during the PMA-triggered differentiation of U937 cells. Although AP-1 mediates the responsiveness to several other differentiating agents including GM-CSF, additional elements are required for induction of the CD11c promoter activity upon Sodium Butyrate-triggered differentiation. In fact, the Sodium Butyrate-responsiveness and the presence of both AP-I- and C/EBP-binding sites suggests that the proximal regulatory region of the CD11c promoter might include an extracellular matrix-response element. As a whole, the transcription of the CD11c gene appears to be controlled by the proliferative state of the cell and is tightly coupled to progression along the myeloid differentiation pathway. The differentiation inducibility of the CD11c promoter has been further demonstrated after stable transfection into U937 cells, where the -361/+43 fragment retains the capacity to drive luciferase expression upon PMA-, GM-CSF- or Sodium Butyrate-triggered myeloid differentiation. Thus, while the characterization of the transcription factors regulating CD11c expression is still in progress, the CD11c promoter has been shown to constitute a very useful tool for the identification of myeloid-differenting agents which might be of potential therapeutical interest.