Gm–Csf Expands Dendritic Cells and Their Progenitors in Mouse Liver

Abstract

Dendritic cells (DCs) are rare but ubiquitous antigen-presenting cells situated in lymphoid and nonlymphoid organs throughout the body. The study of DCs located in the liver has been restricted by their relative scarcity and the difficulty of their isolation. Because granulocyte-macrophage colony-stimulating factor (GM-CSF) is a critical growth factor for DCs in vitro, we postulated that it would expand hepatic DCs in vivo. We found that adenoviralmediated GM-CSF overexpression in normal mice increased the number of liver DCs 400-fold to more than 100 million cells. GM-CSF-recruited DCs were CD11c ؉ DEC205 ؊ and had high expression of major histocompatibility complex (MHC) class II, CD54, and CD80 but low CD40 and CD86 staining. Further maturation occurred after overnight culture. In addition to CD11c ؉ DEC205 ؊ DCs, a population of CD11c ؊ DEC205 low/؊ cells resembling DC progenitors described previously in normal mice was expanded as serum GM-CSF levels increased. GM-CSF-recruited CD11c D endritic cells (DCs) are rare, heterogeneous leukocytes that are primarily responsible for the capture of antigens in the periphery and subsequent presentation to immune effector cells. 1-3 Although there is a large and rapidly expanding body of data regarding the phenotype and function of murine bone marrowderived DCs and splenic DCs, our understanding of hepatic DCs is rudimentary. [4][5][6] This is due to the scarcity of hepatic DCs and the difficulty of isolating them from normal mice. Most of our knowledge emanates from using granulocyte-macrophage colony-stimulating factor (GM-CSF) to propagate liver DCs in vitro from DC progenitors contained within hepatic nonparenchymal cells (NPCs). 7,8 This approach is analogous to the method commonly used to produce DCs from bone marrow cells. 4 Hepatic DC progenitors have low expression of major histocompatibility complex (MHC) class II and the DC-restricted markers DEC205, CD11c, and 33D1 and produce only low levels of allostimulation in a mixed leukocyte reaction. 7 However, they can differentiate (i.e., gain CD11c and MHC class II expression) into immature DCs in vitro in the presence of GM-CSF. After additional culture on type-1 collagen, the immature liver DCs then become mature with increased surface costimulatory molecule expression and T-cell stimulatory capacity. 7 More recently, the phenotype of freshly isolated CD11c ϩ liver DCs has been described. Consistent with studies of hepatic DCs propagated from DC progenitors in vitro, it was shown that freshly isolated liver DCs were immature, with low MHC class II and costimulatory molecule (CD40, CD80, and CD86) expression. 9 The ability of freshly isolated liver DCs to stimulate T cells has not been well defined.

Biliary Obstruction Selectively Expands and Activates Liver Myeloid Dendritic Cells

“…Although other investigators have demonstrated examples of DC recruitment in the setting of inflammatory responses both in vitro (24) and in vivo (25,26), selective expansion of myeloid DCs has not been demonstrated other than in the setting of exogenous cytokine administration (27). We have shown previously that overexpression of GM-CSF can expand liver myeloid DCs in vivo (28). However, we did not find expansion of DC precursors following BDL as we had after GM-CSF treatment.…”

Liver Dendritic Cells Are Less Immunogenic Than Spleen Dendritic Cells because of Differences in Subtype Composition

“…However, other cell types may express CD11c; therefore, its use is a limitation of this as well as other investigations. In the early studies, MHC class II-positive cells in the liver were found to have minimal expression of costimulatory molecules; however, we have recently reported that, using anti-CD11c immunohistochemistry after blocking nonspecific staining due to Fc␥ receptors, there is an exceedingly small number of DC present in normal murine liver (4,8). In the current study, we show that liver DC actually express low to moderate levels of costimulatory molecules.…”

“…Other indirect information regarding liver DC has been extrapolated from cultures of NPC grown in GM-CSF (3). These DC-like cells were immature, and their phenotype was skewed by the presence of GM-CSF, which we have shown to expand a uniformly myeloid population in vivo (4). Despite the belief that liver DC have little immunostimulatory capacity, we previously found that CD11c ϩ liver DC isolated from normal mice using a laborious process relying on density centrifugation as well as immunomagnetic bead sorting could generate significant T cell allostimulation (4).…”

“…To maximize the yield of DC for flow cytometry experiments, we then cut the liver into small pieces before incubation in 20 ml of collagenase solution for 40 min at 37°C. For experiments testing liver DC function, we omitted the in vitro collagenase incubation step because we previously found it to impair DC viability (4). Performance of the additional collagenase incubation in vitro did not affect the distribution of DC subtypes released or their expression of surface markers, merely the magnitude of the yield (not shown).…”

Natural Killer Dendritic Cells Have Both Antigen Presenting and Lytic Function and in Response to CpG Produce IFN-γ via Autocrine IL-12

“…Liver nonparenchymal cells were isolated as previously described, with minor modifications (23,24). Briefly, animals were euthanized by CO 2 inhalation before laparotomy, then the portal vein was cannulated in situ with a 26-gauge needle (BD Biosciences) and perfused with 2 ml of 1% (w/v) collagenase D (Sigma-Aldrich) in PBS.…”