IntroductionDendritic cells (DCs) are extremely potent antigen-presenting cells (APCs), capable of initiating primary T-cell responses by presenting antigenic peptides in association with major histocompatibility complex (MHC) class I and II molecules on the cell surface (reviewed in Banchereau and Steinman 1 ). Circulating DCs are rare, necessitating the use of in vitro cultivation protocols to generate sufficient DCs from bone marrow or monocytes for in vitro studies or therapeutic use. [2][3][4][5] One such protocol, culturing peripheral blood (PB) derived CD14 ϩ monocytes in the presence of exogenous granulocyte macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 7-10 days, 2 has become the standard for production of human monocyte-derived DCs (moDCs). These immature moDC can be further matured by exposing the cells to toll-like receptor agonists or molecules such as tumor necrosis factor ␣ (TNF␣), interferon-␥ (IFN␥), and CD40 ligand (CD154). 1,6 Although these in vitro methods have been shown to generate moDCs that can induce immune responses in vitro and in vivo, it is unknown whether such cells correspond to any DC subset present in vivo.DCs are believed to contribute to the pathophysiology of inflammatory diseases such as rheumatoid arthritis (RA), which is characterized by chronically inflamed joints and other characteristic findings. 7-9 A variety of leukocytes accumulate in the RA synovium and synovial fluid, including large numbers of monocytes derived from the circulation. 10 DCs are also present in these tissues, although the physiologic processes that guide their differentiation from monocytes are not known.Like monocytes, circulating natural killer (NK) cells are recruited to inflamed tissues, including the RA joint, [11][12][13][14][15] and they coexist in various lymphoid organs with monocytes and DCs. [16][17][18][19][20][21] NK cells were initially defined on the basis of their cytotoxic activity 22 ; this activity, together with their production of both proinflammatory and anti-inflammatory cytokines, is believed to be important in the defense against infectious agents, especially viruses (reviewed in Lanier 23 ). Other studies have suggested that NK cells can regulate myelopoiesis [24][25][26] and induce maturation of GM-CSF and IL-4 derived moDCs in vitro, 27-29 but whether NK cells affect early stages of monocyte differentiation into moDCs is not known. On this basis, and given the colocalization of NK cells with monocytes and DCs in the inflamed RA joint, we reasoned that NK cells might play a role in the differentiation of monocytes into moDCs. Materials and methods Cell isolation and culturePeripheral blood mononuclear cells (PBMCs) from healthy volunteer blood donations at the Stanford Blood Center and synovial fluid mononuclear cells (SFMCs) from patients with immune-mediated arthritis were isolated by Ficoll density gradient centrifugation. CD14 ϩ monocytes and CD56 ϩ NK cells were purified from PBMCs or SFMCs using anti-CD14 and anti-CD56-coated microbeads (...

Monocytes and T helper (T H ) cells rapidly infiltrate inflamed tissues where monocytes differentiate into inflammatory dendritic cells (DCs IntroductionDCs are phenotypically and functionally divergent antigen presenting cells that originate from bone marrow precursors such as monocytes. [1][2][3][4][5][6] Encompassing a broad anatomical distribution, DCs continuously sample the microenvironment in search of danger signals recognized by a wide spectrum of pattern recognition receptors (PRRs). 1,2,6 Depending on the cytokine milieu and the pathogen encountered, DCs differentially up-regulate costimulatory molecules and secrete a variety of cytokines that dictate the nature of the T cell response. 7-10 DC exposure to particular intracellular pathogens or their products, including lipopolysaccharide (LPS) and double-stranded RNA, leads to DC-driven T H 1 differentiation primarily through IL-12 secretion. 11 Conversely, DCs mediate T H 2 differentiation in response to extracellular parasites, such as S mansoni, which likely results from diminished IL-12 secretion and increased OX40L expression. 7,8 Several reports have implicated DC-derived IL-1␤, IL-6, IL-23, and TGF-␤ in the polarization of T H 17 cells, 9,12-17 which mediate defense against extracellular bacteria and various fungal infections. 15 Psoriasis and atopic dermatitis are common inflammatory skin diseases characterized by the rapid accumulation of T H cells and DCs. [18][19][20][21][22] While both diseases share several pathologic features, 18,22 the T H cells implicated in the pathogenesis of each disease are distinct. 19,20,23 Infiltration of T H 1 and T H 17 cells is commonly observed in psoriatic lesions, 19 which is accompanied by elevated levels of the T H 1 cytokine, IFN-␥, and the T H 17 cytokines, Conversely, acute atopic dermatitis lesions contain elevated levels of T H 2 cells and their associated effector cytokines, 23 Not surprisingly, DCs that accumulate in each of these diseases exhibit markedly different phenotypes. 21,24,25 DCs present in psoriatic lesions express IL-12p40, IL-23, and TNF-␣ 25-27 while DCs present in atopic dermatitis lesions express low or undetectable levels of these cytokines. 25 DCs from each of these diseases also differentially express cell surface molecules such as DC-SIGN. 21 However, the cell types and/or factors that influence the generation of these DC subsets remain elusive.Given that memory T H cells and monocytes rapidly infiltrate inflamed tissues 2 and that activated T H cells secrete high levels of GM-CSF, 28 a cytokine that mediates DC differentiation from monocytes, 29,30 we hypothesized that T H cells instruct monocytes to differentiate into DCs. Our data demonstrate that not only do T H cells direct monocytes to differentiate into DCs, but that each T H cell subset drives the formation of phenotypically and functionally distinct DC subsets that resemble DCs previously identified in atopic dermatitis and psoriatic lesions. 21,24,25 These data support the hypothesis that memory T H cells influence t...

Dendritic cells (DC) trigger activation and IFN-γ release by NK cells in lymphoid tissues, a process important for the polarization of Th1 responses. Little is known about the molecular signals that regulate DC-induced NK cell IFN-γ synthesis. In this study, we analyzed whether the interaction between Qa-1b expressed on DC and its CD94/NKG2A receptor on NK cells affects this process. Activation of DC using CpG-oligodeoxynucleotides in Qa-1b-deficient mice, or transfer of CpG-oligodeoxynucleotide-activated Qa-1b-deficient DC into wild-type mice, resulted in dramatically increased IFN-γ production by NK cells, as compared with that induced by Qa-1b-expressing DC. Masking the CD94/NKG2A inhibitory receptor on NK cells in wild-type mice similarly enhanced the IFN-γ response of these cells to Qa-1b-expressing DC. Furthermore, NK cells from CD94/NKG2A-deficient mice displayed higher IFN-γ production upon DC stimulation. These results demonstrate that Qa-1b is critically involved in regulating IFN-γ synthesis by NK cells in vivo through its interaction with CD94/NKG2A inhibitory receptors. This receptor-ligand interaction may be essential to prevent unabated cytokine production by NK cells during an inflammatory response.

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