Dale Edelbaum

Generation and Function of Reactive Oxygen Species in Dendritic Cells During Antigen Presentation

Although reactive oxygen species (ROS) have long been considered to play pathogenic roles in various disorders, this classic view is now being challenged by the recent discovery of their physiological roles in cellular signaling. To determine the immunological consequence of pharmacological disruption of endogenous redox regulation, we used a selenium-containing antioxidant compound ebselen known to modulate both thioredoxin and glutaredoxin pathways. Ebselen at 5–20 μM inhibited Con A-induced proliferation and cytokine production by the HDK-1 T cell line as well as the LPS-triggered cytokine production by XS52 dendritic cell (DC) line. Working with the in vitro-reconstituted Ag presentation system composed of bone marrow-derived DC, CD4+ T cells purified from DO11.10 TCR-transgenic mice and OVA peptide (serving as Ag), we observed that 1) both T cells and DC elevate intracellular oxidation states upon Ag-specific interaction; 2) ebselen significantly inhibits ROS production in both populations; and 3) ebselen at 5–20 μM inhibits DC-induced proliferation and cytokine production by T cells as well as T cell-induced cytokine production by DC. Thus, Ag-specific, bidirectional DC-T cell communication can be blocked by interfering with the redox regulation pathways. Allergic contact hypersensitivity responses in BALB/c mice to oxazolone, but not irritant contact hypersensitivity responses to croton oil, were suppressed significantly by postchallenge treatment with oral administrations of ebselen (100 mg/kg per day). These results provide both conceptual and technical frameworks for studying ROS-dependent regulation of DC-T cell communication during Ag presentation and for testing the potential utility of antioxidants for the treatment of immunological disease.

A role for NF-κB–dependent gene transactivation in sunburn

Exposure of skin to ultraviolet (UV) radiation is known to induce NF-κB activation, but the functional role for this pathway in UV-induced cutaneous inflammation remains uncertain. In this study, we examined whether experimentally induced sunburn reactions in mice could be prevented by blocking UV-induced, NF-κB-dependent gene transactivation with oligodeoxynucleotides (ODNs) containing the NF-κB cis element (NF-κB decoy ODNs). UV-induced secretion of IL-1, IL-6, TNF-α, and VEGF by skin-derived cell lines was inhibited by the decoy ODNs, but not by the scrambled control ODNs. Systemic or local injection of NF-κB decoy ODNs also inhibited cutaneous swelling responses to UV irradiation. Moreover, local UV-induced inflammatory changes (swelling, leukocyte infiltration, epidermal hyperplasia, and accumulation of proinflammatory cytokines) were all inhibited specifically by topically applied decoy ODNs. Importantly, these ODNs had no effect on alternative types of cutaneous inflammation caused by irritant or allergic chemicals. These results indicate that sunburn reactions culminate from inflammatory events that are triggered by UV-activated transcription of NF-κB target genes, rather than from nonspecific changes associated with tissue damage.

Contrasting impacts of immunosuppressive agents (rapamycin, FK506, cyclosporin A, and dexamethasone) on bidirectional dendritic cell-T cell interaction during antigen presentation.

Rapamycin (RAP), tacrolimus (FK506), cyclosporin A, and glucocorticoids represent modern and classic immunosuppressive agents being used clinically. Although these agents have distinct molecular mechanisms of action and exhibit different immunoregulatory profiles, their direct influences on Ag presentation processes remain relatively unknown. Here we report quantitative and qualitative differences among the above four immunosuppressants in their impact on Ag-specific, bidirectional interaction between dendritic cells (DC) and CD4+ T cells. In the presence of relevant Ag, bone marrow-derived DC delivered activation signals to CD4+ T cells isolated from the DO11.10 TCR transgenic mice, leading to clonal expansion; secretion of IFN-γ, IL-2, and IL-4; and surface expression of CD69. Conversely, DO11.10 T cells delivered maturation signals to DC, leading to IL-6 and IL-12 production and CD40 up-regulation. FK506 (10−10–10−8 M) and cyclosporin A (10−9–10−7 M) each blocked efficiently and uniformly all the changes resulting from intercellular signaling in both DC→T cell and T cell→DC directions. Dexamethasone (10−9–10−6 M) suppressed all changes, except for CD69 up-regulation, rather incompletely. Remarkably, RAP (10−10–10−8 M) efficiently inhibited DC-induced T cell proliferation and T cell-mediated CD40 up-regulation by DC without abrogating other changes. Interestingly, T cell-independent DC maturation triggered by LPS stimulation was inhibited by dexamethasone, but not by other agents. Our results demonstrate contrasting pharmacological effects of RAP vs calcineurin inhibitors on Ag presentation, thus forming a conceptual framework for rationale-based selection (and combination) of immunosuppressive agents for clinical application.

Synthesis and Surface Expression of Hyaluronan by Dendritic Cells and Its Potential Role in Antigen Presentation

Hyaluronan (HA) is a large glycosaminoglycan consisting of repeating disaccharide units of glucuronic acid and N-acetylglucosamine. HA is known to act as a filling material of extracellular matrices and as an adhesive substrate for cellular migration. Here we report that dendritic cells (DC) express mRNAs for HA synthases and hyaluronidases, actively synthesize HA, and display HA on their surfaces. Interestingly, HA expression levels on DC were not significantly altered by their maturation states. With respect to physiological function, three specific HA inhibitors, i.e., bovine proteoglycan, a 12-mer HA-binding peptide (GAHWQFNALTVR) termed Pep-1, and an oligomeric Pep-1 formulation, all interfered with DC-induced activation of CD4+ T cells isolated from DO11.10 TCR transgenic mice. For example, Pep-1 oligomer efficiently inhibited DC-dependent cluster formation, IL-2 and IFN-γ production, and proliferation by DO11.10 T cells in vitro without affecting the viabilities of DC or T cells, DC function to uptake exogenous proteins, or DC-T cell conjugate formation at earlier time points. These observations suggest a paracrine mechanism by which DC-associated HA facilitates some of the late changes in T cell activation. Although T cells constitutively expressed mRNAs for HA synthases and hyaluronidases, their surface HA expression became detectable only after activation. Oligomeric Pep-1 and bovine proteoglycan both inhibited mitogen-triggered T cell activation in the absence of DC, suggesting an autocrine mechanism by which HA expressed by T cells assists their own activation processes. Finally, adoptively transferred DO11.10 T cells showed progressive mitosis when stimulated with Ag-pulsed DC in living animals, and this clonal expansion was inhibited significantly by administration of Pep-1 oligomer. Our findings may introduce a new concept that relatively simple carbohydrate moieties expressed on DC and perhaps T cells play an important immunomodulatory role during Ag presentation.

Effects of Inflammatory Cytokines and Phorbol Esters on the Adhesion of U937 Cells, a Human Monocyte‐Like Cell Line, to Endothelial Cell Monolayers and Extracellular Matrix Proteins

The accumulation of mononuclear phagocytes at sites of chronic inflammation is dependent on an increase in the rate of extravasation of blood‐borne monocytes through the vascular endothelium into the connective tissue. Once the monocytes have emigrated into the connective tissue, they may differentiate into tissue macrophages, presumably following interactions with extracellular matrix proteins. To study these processes, we tested the effects of cytokines and phorbol esters on the adhesion of U937 cells, a human monocyte‐like cell line, to cultured endothelial cells (EC) and to matrix proteins. In the absence of cytokines, very few of the U937 cells adhered to EC (5% or less in most experiments). When EC were pretreated for optimal periods of time (4–8 hr) with recombinant interleukin‐1α (IL‐1α), IL‐1β, tumor necrosis factor‐α (TNFα), or lymphotoxin (LT; also known as TNF‐β), 35–85% of the U937 cells were able to bind. Interferon‐γ (IFN‐γ) and interleukin–2 (IL‐2) did not stimulate U937‐EC binding, even though IFN‐γ was shown to increase EC adhesiveness for T lymphocytes. Phorbol esters also greatly stimulated U937‐EC adhesion but, in this case, the increase was due to an action on the U937 cells. A monoclonal antibody (MAb), 60.3, against the CD11/CD18 family of leukocyte adhesion molecules partially inhibited the adhesion of untreated and phorbol ester‐treated U937 cells to noncytokine‐treated EC. However, that MAb had no effect on U937 cell binding to TNF‐α‐treated EC. Thus U937 cells use both CD11/CD18‐dependent and ‐independent mechanisms to adhere to EC. In the absence of stimulating agents, only a small proportion of the U937 cells (2–20%) adhered to fibronectin (FN), and almost none bound to either laminin (LN) or gelatin (denatured type I collagen). In the presence of phorbol esters, a much larger proportion of the U937 cells adhered to FN, with only slight increases in the proportion of cells which bound to LN or gelatin. Additional adhesion assays performed in the presence of a pentapeptide containing the amino acid sequence arg‐gly‐asp (RGD), which is part of one of the cell‐binding domains of FN, demonstrated that the RGD‐containing peptide almost totally blocked the phorbol ester‐induced adhesion of U937 cells to FN. In contrast, the peptide had no inhibitory effect on the phorbol ester‐induced binding of U937 cells to EC.

New strategy for efficient selection of dendritic cell-tumor hybrids and clonal heterogeneity of resulting hybrids.

Heterotypic hybrids created between dendritic cells (DC) and tumor cells represent an efficient approach for loading DC with tumor-associated antigens (TAA), and DC-tumor hybrid vaccines have shown promising outcomes in various preclinical and clinical studies. Conventional DC-tumor hybrid preparations, however, are unavoidably contaminated by DCtumor aggregates and DC loaded with tumor cell debris. Here we describe a new strategy for selecting genuine DC-tumor hybrids. A HAT-sensitive/zeocin-resistant DC clone (XS106-7 Zeo) was fused with a GFP-transduced fibrosarcoma clone (S1509a-GFP) by polyethylene glycol and heterotypic hybrid clones were established by limiting dilution in the presence of HAT and zeocin. CD45 (DC origin) and GFP (tumor origin) were both expressed in 91% (51/56 clones) of the resulting clones, indicating high efficiency of our strategy. Marked heterogeneity was observed among the hybrid clones, and only one clone exhibited characteristic features of DC (CD86 and I-A expression, dendritic morphology, T cellstimulatory capacity, and IL-1b, IL-6, and TNFa production), suggesting that only small fractions of DC-tumor hybrids acquire and maintain the properties of parental DC. Finally, vaccination with this hybrid clone protected mice from subsequent growth of S1509a tumor cells, documenting the in vivo activity of DC-tumor hybrids in the complete absence of exogenous TAA.

ICAM-1 and LFA-1 on mouse epidermal Langerhans cells and spleen dendritic cells identify disparate requirements for activation of KLH-specific CD4 Th1 and Th2 clones+*

Abstract Expression of the adhesion molecules ICAM‐1 and LFA‐1 (CD11a/CD18) on mouse epidermal Lungerhans cells (LC) and on spleen dendritic cells (DC) from BALB/c mice was examined by staining with specific mAb and was evaluated by flow cytometry. LC were shown to express both ICAM‐1 and LFA‐1, whereas spleen DC expressed only LFA‐1. The contribution of these adhesion molecules to LC‐ or DC‐induced activation of keyhole limpet hemocyanin (KLH)‐specific, lad‐restricted, Th1 or Th2 clones was investigated in mAb blocking studies. At optimal doses, anti‐CD1la or anti‐CD18 mAb completely inhibited Th1 proliferation induced by either LC or DC. Anti‐ICAM‐1 also abrogated Th1 proliferation induced by LC, but only moderately reduced Th1 proliferation induced by DC. Inhibition in these experiments was specific, since isotype‐matched control Ab against other Ag constitutively expressed on LC (NLDC 145) or DC (33D1) had no effect on Th1 proliferation. In marked contradistinction, the capacity of LC to present KLH to our Th2 clones was resistant to treatment with the same mAb against ICAM‐1, CD11a or CD18. We conclude that interactions between ICAM‐1 and LFA‐1 on epidermal LC and LFA‐1 on spleen DC with their respective ligands on our Th1 clones are required for optimal presentation of protein Ag to Th1. Our results also indicate that neither ICAM‐1 nor LFA‐1 is required for the analogous activation of our Th2 clones by LC.