Ponciano D. Cruz

Dectin-2 is a pattern recognition receptor for fungi that couples with the Fc receptor γ chain to induce innate immune responses

Antigen presenting cells recognize pathogens via pattern recognition receptors (PRR), which upon ligation transduce intracellular signals that can induce innate immune responses. Because some C-type lectin-like receptors (e.g. dectin-1 and DCSIGN) were shown to act as PRR for particular microbes, we considered a similar role for dectin-2. Binding assays using soluble dectin-2 receptors showed the extracellular domain to bind preferentially to hyphal (rather than yeast/conidial) components of Candida albicans, Microsporum audouinii, and Trichophyton rubrum. Selective binding for hyphae was also observed using RAW macrophages expressing dectin-2, the ligation of which by hyphae or cross-linking with dectin-2-specific antibody led to protein tyrosine phosphorylation. Because dectin-2 lacks an intracellular signaling motif, we searched for a signal adaptor that permits it to transduce intracellular signals. First, we found that the Fc receptor γ (FcRγ) chain can bind to dectin-2. Second, ligation of dectin-2 on RAW cells induced tyrosine phosphorylation of FcRγ, activation of NF-κB, internalization of a surrogate ligand, and up-regulated secretion of tumor necrosis factor α and interleukin-1 receptor antagonist. Finally, these dectin-2-induced events were blocked by PP2, an inhibitor of Src kinases that are mediators for FcRγ chain-dependent signaling. We conclude that dectin-2 is a PRR for fungi that employs signaling through FcRγ to induce innate immune responses.

Dermal, subcutaneous, and tendon xanthomas: Diagnostic markers for specific lipoprotein disorders

Many patients with lipoprotein disorders are at increased risk for the development of premature atherosclerosis and, less commonly, other disorders that cause systemic morbidity. In some of these patients, xanthomas also develop and provide cutaneous markers for the lipoprotein disorder. As advances in molecular biology refine our understanding of lipoprotein metabolism, it has become increasingly clear that several types of xanthomas are associated with specific disease states. This article presents a differential diagnosis of xanthomas that incorporates contemporary thinking about lipoprotein disorders and focuses on the relationship between abnormalities in lipoprotein metabolism, content, or structure and the development of specific xanthomas.

Syndecan-4 Mediates the Coinhibitory Function of DC-HIL on T Cell Activation

Receptor-ligand interactions between APCs and T cells determine whether stimulation of the latter leads to activation or inhibition. Previously, we showed that dendritic cell-associated heparin sulfate proteoglycan-dependent integrin ligand (DC-HIL) on APC can inhibit T cell activation by binding an unknown ligand expressed on activated T cells. Because DC-HIL binds heparin/heparan sulfate and heparin blocks the inhibitory function of DC-HIL, we hypothesized that a heparin/heparan sulfate proteoglycan on activated T cells is the relevant ligand. Screening assays revealed that syndecan-4 (SD-4) is the sole heparan sulfate proteoglycan immunoprecipitated by DC-HIL from extracts of activated T cells and that blocking SD-4 abrogates binding of DC-HIL to activated T cells. Moreover, cell-bound SD-4 ligated by DC-HIL or cross-linked by anti-SD-4 Ab attenuated anti-CD3 responses, whereas knocked-down SD-4 expression led to enhanced T cell response to APC. Blockade of endogenous SD-4 using specific Ab or soluble SD-4 receptor led to augmented T cell reactions to syngeneic and allogeneic stimulation in vitro and exacerbated contact hypersensitivity responses in vivo. We conclude that SD-4 is the T cell ligand through which DC-HIL mediates its negative coregulatory function.

Expression of NF-κB in epidermis and the relationship between NF-κB activation and inhibition of keratinocyte growth

Summary Background Nuclear factor‐κB (NF‐κB) is a transcription factor involved in a number of signalling pathways in many cell types. NF‐κB in mice has been implicated as an important regulator of keratinocyte proliferation and differentiation.

Genomic scale analysis of the human keratinocyte response to broad-band ultraviolet-B irradiation

Ultraviolet B (UVB) radiation is an important inducer of many biologic changes in skin, of which keratinocytes are a key target. To gain better insight into changes in gene expression generated in the early phase after UVB exposure, we used complementary RNA (cRNA) microarray hybridization to compare differences in mRNA expression of UVB‐irradiated (single dose of 100 J/m2 broad‐band UVB) and sham‐irradiated primary cultured human keratinocytes. Six hours after irradiation, total RNA was isolated from keratinocytes, and cRNA was synthesized and hybridized to a GeneChip expression array (Affymetrix) consisting of 6800 genes. Based on a threshold of > twofold change, 187 genes (2.8%) were designated to be the most UVB‐responsive. Surprisingly, none of these genes had been shown previously to be modulated by UVB. Conversely, several genes in the microarray that had been reported previously to be UVB‐ responsive by other methods showed less (< twofold) or no change. Northern blotting of seven differentially modulated genes produced results similar to those derived from microarray technology, thereby validating the accuracy of screening. Clustering based on known or likely functions indicated that among 88 upregulated genes, nine encode for cytochrome c subunits, six for ribosomal proteins, and two for regulators of apoptosis. By contrast, many of the 99 downregulated genes are involved in transcription, differentiation and transport. These findings indicate that keratinocytes respond to a single low dose of broad‐band UVB irradiation by enhancing processes involved in energy production and translation, while suppressing those related to transcription, differentiation and transport.

Gpnmb is a melanosome-associated glycoprotein that contributes to melanocyte/keratinocyte adhesion in a RGD-dependent fashion.

Abstract:  Gpnmb is a glycosylated transmembrane protein implicated in the development of glaucoma in mice and melanoma in humans. It shares significant amino acid sequence homology with the melanosome protein Pmel‐17. Its extracellular domain contains a RGD motif for binding to integrin and its intracellular domain has a putative endosomal and/or melanosomal‐sorting motif. These features led us to posit that Gpnmb is associated with melanosomes and involved in cell adhesion. We showed that human Gpnmb is expressed constitutively by melanoma cell lines, primary‐cultured melanocytes and epidermal melanocytes in situ, with most of it found intracellularly within melanosomes and to a lesser degree in lysosomes. Our newly developed monoclonal antibody revealed surface expression of Gpnmb on these pigment cells, albeit to a lesser degree than the intracellular fraction. Gpnmb expression was upregulated by UVA (but not UVB) irradiation and by α‐melanocyte‐stimulating hormone (MSH) (but not β‐MSH); its cell surface expression on melanocytes (but not on melanoma cells) was increased markedly by IFN‐γ and TNF‐α. PAM212 keratinocytes adhered to immobilized Gpnmb in a RGD‐dependent manner. These results indicate that Gpnmb is a melanosome‐associated glycoprotein that contributes to the adhesion of melanocytes with keratinocytes.

The DC-HIL/syndecan-4 pathway inhibits human allogeneic T cell responses

T‐cell activation is regulated by binding of ligands on APC to corresponding receptors on T cells. In mice, we discovered that binding of DC‐HIL on APC to syndecan‐4 (SD‐4) on activated T cells potently inhibits T‐cell activation. In humans, we now show that DC‐HIL also binds to SD‐4 on activated T cells through recognition of its heparinase‐sensitive saccharide moiety. DC‐HIL blocks anti‐CD3‐induced T‐cell responses, reducing secretion of pro‐inflammatory cytokines and blocking entry into the S phase of the cell cycle. Binding of DC‐HIL phosphorylates SD‐4s intracellular tyrosine and serine residues. Anti‐SD‐4 Ab mimics the ability of DC‐HIL to attenuate anti‐CD3 response more potently than Ab directed against other inhibitory receptors (CTLA‐4 or programmed cell death‐1). Among leukocytes, DC‐HIL is expressed highest by CD14+ monocytes and this expression can be upregulated markedly by TGF‐β. Among APC, DC‐HIL is expressed highest by epidermal Langerhans cells, an immature type of dendritic cells. Finally, the level of DC‐HIL expression on CD14+ monocytes correlates inversely with allostimulatory capacity, such that treatment with TGF‐β reduced this capacity, whereas knocking down the DC‐HIL gene augmented it. Our findings indicate that the DC‐HIL/SD‐4 pathway can be manipulated to treat T‐cell‐driven disorders in humans.

DC-HIL/glycoprotein Nmb promotes growth of melanoma in mice by inhibiting the activation of tumor-reactive T cells.

DC-HIL/glycoprotein nmb (Gpnmb) expressed on antigen-presenting cells attenuates T-cell activation by binding to syndecan-4 (SD-4) on activated T cells. Because DC-HIL/Gpnmb is expressed abundantly by mouse and human melanoma lines, we posited that melanoma-associated DC-HIL/Gpnmb exerts similar inhibitory function on melanoma-reactive T cells. We generated small interfering RNA-transfected B16F10 melanoma cells to completely knock down DC-HIL/Gpnmb expression, with no alteration in cell morphology, melanin synthesis, or MHC class I expression. This knockdown had no effect on B16F10 proliferation in vitro or entry into the cell cycle following growth stimulation, but it markedly reduced the growth of these cells in vivo following their s.c. injection into syngeneic immunocompetent (but not immunodeficient) mice. This reduction in tumor growth was due most likely to an augmented capacity of DC-HIL-knocked down B16F10 cells (compared with controls) to activate melanoma-reactive T cells as documented in vitro and in mice. Whereas DC-HIL knockdown had no effect on susceptibility of melanoma to killing by cytotoxic T cells, blocking SD-4 function enhanced the reactivity of CD8(+) T cells to melanoma-associated antigens on parental B16F10 cells. Using an assay examining the spread to the lung following i.v. injection, DC-HIL-knocked down cells produced lung foci at similar numbers compared with that produced by control cells, but the size of the former foci was significantly smaller than the latter. We conclude that DC-HIL/Gpnmb confers upon melanoma the ability to downregulate the activation of melanoma-reactive T cells, thereby allowing melanoma to evade immunologic recognition and destruction. As such, the DC-HIL/SD-4 pathway is a potentially useful target for antimelanoma immunotherapy.

Epidermal Langerhans cell-targeted gene expression by a dectin-2 promoter

Despite their critical function as APCs for primary immune responses, dendritic cells (DC) and Langerhans cells (LC) have been rarely used as targets of gene-based manipulation because well-defined regulatory elements controlling LC/DC-specific expression have not been identified. Previously, we identified dectin-2, a C-type lectin receptor expressed selectively by LC-like XS cell lines and by LC within mouse epidermis. Because these characteristics raised the possibility that dectin-2 promoter may direct LC/DC-specific gene expression, we isolated a 3.2-kb nucleotide fragment from the 5′-flanking region of the dectin-2 gene (Dec2FR) and characterized its regulatory elements and the transcriptional activity using a luciferase (Luc) reporter system. The Dec2FR contains a putative TATA box and cis-acting elements, such as the IFN-stimulated response element, that drive gene expression specifically in XS cells. Dec2FR comprises repressor, enhancer, and promoter regions, and the latter two regions coregulate XS cell-specific gene expression. In transgenic mice bearing a Dec2FR-regulated Luc gene, the skin was the predominant site of Luc activity and LC were the exclusive source of such activity within epidermis. By contrast, other APCs (DC, macrophages, and B cells) and T cells expressed Luc activity close to background levels. We conclude that epidermal LC are targeted selectively for high-level constitutive gene expression by Dec2FR in vitro and in vivo. Our findings lay the foundation for use of the dectin-2 promoter in LC-targeted gene expression systems that may enhance vaccination efficacy and regulate immune responses.

Identification and expression profiling of a human C-type lectin, structurally homologous to mouse dectin-2.

Abstract:  A number of C‐type lectins on antigen‐presenting cells play an important role in regulating innate immunity. Previously, we identified the mouse C‐type lectins (dectin‐1, and dectin‐2) and human DECTIN‐1. To identify human DECTIN‐2, we employed degenerative polymerase chain reaction‐based cDNA cloning using RNA from human Langerhans cell (LC)‐like dendritic cells (DCs). This process yielded a cDNA encoding a C‐type lectin with 66.5% amino acid sequence homology to mouse dectin‐2, the same gene reported by Kanazawa et al. (J Invest Dermatol 2004: 122: 1522–1524) using the disparate approach of analyzing coding sequences in chromosome 12. Similar to their findings, we found gene expression in lung, spleen, and lymph node. Among resting leukocytes, it was expressed at highest levels by CD14+ monocytes, at lower levels by CD19+ B cells, and not at all by CD4+ T cells. Activation of CD19+ B cells with pokeweed mitogen down‐regulated gene expression, whereas expression in CD4+ T cells was induced by Con A. Among our novel findings are an alternatively spliced transcript lacking exon 2, expression in bone marrow and tonsil, expression in CD8+ T cells that is abrogated following activation with phytohemagglutinin, restricted expression to CD1a+ LC within epidermis, and preferential expression by plasmacytoid (rather than myeloid) DC. Finally, we found that treatment with interleukin‐4 (IL‐4), IL‐10, or UVB down regulated gene expression in CD14+ monocytes, whereas granulocyte‐macrophage colony‐stimulating factor, transforming growth factor‐β1, or tumor necrosis factor‐α treatment up‐regulated it. Our findings may form the basis for understanding the function of human DECTIN‐2 in innate immunity.