We isolated a novel molecule (DC-HIL) expressed abundantly by the XS52 dendritic cell (DC) line and epidermal Langerhans cells, but minimally by other cell lines. DC-HIL is a type I transmembrane protein that contains a heparin-binding motif and an integrin-recognition motif, RGD, in its extracellular domain (ECD). A soluble fusion protein (DC-HIL-Fc) of the ECD and animmunoglobulin Fc bound to the surface of an endothelial cell line (SVEC). This binding induced adhesion of SVEC to its immobilized form. Sulfated polysaccharides (e.g. heparin and fucoidan) inhibited binding of soluble DC-HIL-Fc and adhesion of SVEC. By contrast, an integrin inhibitor (RGDS tetramer) had no effect on binding to SVEC, but prevented adhesion of SVEC. This differential RGD requirement was confirmed by the finding that DC-HIL-Fc mutant lacking the RGD motif can bind to SVEC but is unable to induce adhesion of SVEC. Furthermore, DC-HIL appears to recognize directly these sulfated polysaccharides. These results suggest that DC-HIL binds to SVEC by recognizing heparan sulfate proteoglycans on endothelial cells, thereby inducing adhesion of SVEC in an RGD-dependent manner. We propose that DC-HIL serves as a DC-associated, heparan sulfate proteoglycan-dependent integrin ligand, which may be involved in transendothelial migration of DC.Dendritic cells (DCs 1 ) are a member of antigen-presenting cells (APC) family, which are characterized morphologically by the extension of long, lamellar dendrites (1). DC are distinguished from other APC (e.g. macrophages and B cells) by an unsurpassed potency in presenting antigens to naive T cells, thereby most efficiently initiating primary T cell-mediated immune responses (1). DC are widely distributed but comprise only a minuscule fraction (typically Ͻ5%) of the total cell population of a given peripheral tissue. After capturing antigens, tissue DC undergo maturation, losing endocytic capacity but acquiring increased immunostimulatory capacity (2). During maturation, DC migrate from peripheral tissues to T cell areas of secondary lymphoid organs where they activate T cells (2-4). Thus, DC migration is a critical step in initiating antigenspecific immune responses.Several adhesion molecules (e.g. cutaneous lymphocyte-associated antigen (5), lymphocyte function-associated antigen-1/ CD11a (6), intercellular adhesion molecule-1 (ICAM-1)/CD54 (6), CD44 (7), E-cadherin (8), and ␣ 6 integrins (9)) regulate the migration of DC through the entire life cycle. For example, blood-circulating precursors of Langerhans cells (LC), which are DC that reside in epidermis, attach to the blood vessel wall prior to initiating transendothelial migration into the epidermis where they develop into APC with phenotypes distinct from other members of the DC family. The LC homing and anchoring in the epidermis are probably controlled by lymphocyte-associated antigen (5) and E-cadherin (8), respectively. Conversely, after antigenic stimulation, LC dissociate from surrounding keratinocytes by down-regulating E-cadherin expression ...

Using a subtractive cDNA cloning strategy, we isolated previously five novel genes that were expressed abundantly by the murine dendritic cell (DC) line XS52, but not by the J774 macrophage line. One of these genes encoded a unique, DC-associated C-type lectin, termed "dectin-1." Here we report the characterization of a second novel gene that was also expressed in a DC-specific manner. Clone 1B12 encoded a type II membrane-integrated polypeptide of 209 amino acids containing a single carbohydrate recognition domain motif in the COOH terminus. The expression pattern of this molecule, termed "dectin-2," was almost indistinguishable from that for dectin-1; that is, both were expressed abundantly at mRNA and protein levels by the XS52 DC line, but not by non-DC lines, and both were detected in spleen and thymus, as well as in skin resident DC (i.e. Langerhans cells). Interestingly, reverse transcriptasepolymerase chain reaction and immunoblotting revealed multiple bands of dectin-2 transcripts and proteins suggesting molecular heterogeneity. In fact, we isolated additional cDNA clones encoding two distinct, truncated dectin-2 isoforms. Genomic analyses indicated that a full-length dectin-2 (␣ isoform) is encoded by 6 exons, whereas truncated isoforms (␤ and ␥) are produced by alternative splicing. We propose that dectin-2 and its isoforms, together with dectin-1, represent a unique subfamily of DC-associated C-type lectins. Dendritic cells (DC)1 are far more potent than other antigenpresenting cells (e.g. macrophages and B cells) in their capacity to activate immunologically naive T cells, and DC are, indeed, responsible for initiating T cell-mediated immune responses to a variety of antigens (1, 2). Members of the DC family are distributed to virtually all the organs (except the brain), where they serve as tissue resident antigen-presenting cells, playing critical roles in presenting environmental, microbial, and tumor-associated antigens to the immune system.Several years ago, we developed stable DC lines from the mouse epidermis (3). These lines, termed as the XS series, maintain many important features of skin resident DC, i.e. Langerhans cells, and they have provided a useful tool for the application of modern technologies for studying DC biology (3-13). Most recently, we employed the subtractive cDNA cloning strategy to identify genes that were expressed preferentially by DC. 2 Briefly, we constructed a DC-specific cDNA library by subtracting cDNAs prepared from the XS52 DC line with excess amounts of mRNAs isolated from the J774 macrophage line. Following three rounds of screening of this library, we identified five novel genes that were expressed selectively by XS52 DC, but not by J774 macrophages. One of these genes encoded a type II membrane-associated polypeptide of 244 amino acids (aa) containing a single carbohydrate recognition domain (CRD) motif at the COOH-terminal end. This polypeptide was, therefore, designated as DC-associated C-type (Ca 2ϩ -dependent) lectin-1 or dectin-1. Dectin-1 mRNA was expre...

In vivo brain microdialysis was used to determine the effects of the standard tricyclic antidepressant imipramine and the two selective serotonin reuptake inhibitors (SSRIs) antidepressants, fluoxetine and fluvoxamine, on extracellular levels of norepinephrine (NE), dopamine (DA), and serotonin (5-HT) in rat medial prefrontal cortex. When given intraperitoneally (IP), imipramine increased NE in the microdialysis perfusate, and elevated DA and 5-HT to a lesser extent. Similar dose-dependent increases in DA and 5-HT were detected after IP fluoxetine, although NE was less affected. In contrast, IP fluvoxamine produced no change in basal NE nor DA, although a large increase in 5-HT occurred at an intermediate dose. When administered directly into cortex, all three antidepressants increased 5-HT by the same amount in a dose-dependent fashion. Intracortical imipramine and fluoxetine increased NE, and fluoxetine and fluvoxamine both increased DA, with fluoxetine doing so at a lower concentration. These data suggest that the SSRIs are not entirely selective for serotonin in vivo.

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.

In vivo microdialysis was used to determine biogenic amines in medial prefrontal cortex of rats exposed to eight minutes of swim stress on two consecutive days. On the first day of stress, norepinephrine (NE) efflux increased by 183% over baseline after stress, while dopamine (DA) and serotonin (5-HT) remained stable throughout. On the second day of stress, a robust increase was observed in all 3 neurotransmitters measured, with (NE), (DA), and (5-HT) increasing by 310%, 441% and 496% respectively, and remaining elevated for an hour or more after stress. This suggests that the first exposure to swim stress, while not causing dramatic changes in biogenic amine release, may sensitize biogenic amines in medial prefrontal cortex to subsequent swim stress. Our results also serve as preliminary data concerning the neurochemical changes which might underlie the forced swimming model of "behavioral despair".

In vivo microdialysis was used to determine the effect of diazepam, flumazenil and FG-7142 upon the biogenic amine response to acute and repeated swim stress in the medial prefrontal cortex of the rat. Acute swim stress increased norepinephrine levels, although dopamine and serotonin levels remained stable. Upon re-exposure to swim stress twenty-four hours later, sustained increases (200-300% of baseline) in all three biogenic amines were detected. This enhanced response to re-stress was not seen in rats pretreated with either a benzodiazepine: agonist (diazepam, 2 mg/kg), an antagonist (flumazenil, 10 mg/kg), or an inverse agonist (FG-7142, 10 mg/kg) given prior to the first swim stress. Therefore, the sensitization of biogenic amine response to re-stress may be prevented by compounds which differ in their activity at the benzodiazepine receptor.

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