G. Gordon MacPherson

Nomenclature of monocytes and dendritic cells in blood

Monocytes and cells of the dendritic cell lineage circulate in blood and eventually migrate into tissue where they further mature and serve various functions, most notably in immune defense. Over recent years these cells have been characterized in detail with the use of cell surface markers and flow cytometry, and subpopulations have been described. The present document proposes a nomenclature for these cells and defines 3 types of monocytes (classical, intermediate, and nonclassical monocytes) and 3 types of dendritic cells (plasmacytoid and 2 types of myeloid dendritic cells) in human and in mouse blood. This classification has been approved by the Nomenclature Committee of the International Union of Immunological Societies, and we are convinced that it will facilitate communication among experts and in the wider scientific community.

Relationships between Distinct Blood Monocyte Subsets and Migrating Intestinal Lymph Dendritic Cells In Vivo under Steady-State Conditions

The origins of dendritic cells (DCs) are poorly understood. In inflammation, DCs can arise from blood monocytes (MOs), but their steady-state origin may differ, as shown for Langerhans cells. Two main subsets of MOs, defined by expression of different chemokine receptors, CCR2 and CX3CR1, have been described in mice and humans. Recent studies have identified the inflammatory function of CCR2highCX3CR1low MOs but have not defined unambiguously the origin and fate of CCR2lowCX3CR1high cells. In this study, we show that rat MOs can also be divided into CCR2highCX3CR1low(CD43low) and CCR2lowCX3CR1high(CD43high) subsets with distinct migratory properties in vivo. Using whole body perfusion to obtain MOs, including the marginating pool, we show by adoptive transfer that CD43low MOs can differentiate into CD43high MOs in blood without cell division. By adoptive transfer of blood MOs followed by collection of pseudoafferent lymph, we show for the first time that a small proportion of intestinal lymph DCs are derived from CCR2lowCX3CR1high(CD43high) blood MOs in vivo under steady-state conditions. This study confirms one of the possible origins of CCR2lowCX3CR1high blood MOs and indicate that they may contribute to migratory intestinal DCs in vivo in the absence of inflammatory stimuli.

Intestinal Dendritic Cell Subsets: Differential Effects of Systemic TLR4 Stimulation on Migratory Fate and Activation In Vivo

Dendritic cells (DC) present peripheral Ags to T cells in lymph nodes, but also influence their differentiation (tolerance/immunity, Th1/Th2). To investigate how peripheral conditions affect DC properties and might subsequently regulate T cell differentiation, we examined the effects of a potent DC-activating, TLR-4-mediated stimulus, LPS, on rat intestinal and hepatic DC in vivo. Steady-state rat intestinal and hepatic lymph DC are αE2 integrinhigh (CD103) and include two subsets, signal regulatory protein α (SIRPα)hi/low, probably representing murine CD8αα−/+ DC. Steady-state lamina propria DC are immature; surface MHC class IIlow, but steady-state lymph DC are semimature, MHC class IIhigh, but CD80/86low. Intravenous LPS induced rapid lamina propria DC emigration and increased lymph DC traffic without altering SIRPαhigh/SIRPαlow proportions. CD80/86 expression on lymph or mesenteric node DC was not up-regulated after i.v. LPS. In contrast, i.v. LPS stimulated marked CD80/86 up-regulation on splenic DC. CD80/86 expression on intestinal lymph DC, however, was increased after in vitro culture with TNF-α or GM-CSF, but not with up to 5 μg/ml LPS. Steady-state SIRPαlow DC localized to T cell areas of mesenteric nodes, spleen, and Peyer’s patch, whereas SIRPαhigh DC were excluded from these areas. Intravenous LPS stimulated rapid and abundant SIRPαhigh DC accumulation in T cell areas of mesenteric nodes and spleen. In striking contrast, i.v. LPS had no effect on DC numbers or distribution in Peyer’s patches. Our results suggest that any explanation of switching between tolerance and immunity as well as involving changes in DC activation status must also take into account differential migration of DC subsets.

Subsets of migrating intestinal dendritic cells

Dendritic cells (DCs) in the intestine are heterogeneous. Phenotypically different populations of conventional DCs have been identified in the intestinal lamina propria, Peyer’s patches, and in the draining mesenteric lymph nodes, to which these DCs constitutively migrate. Markers used to identify these populations include major histocompatibility complex class II, CD11c, CD8α, CD11b, and CD103. Extensive studies in rats, summarized here, which involved collection of migrating DCs by thoracic duct cannulation after mesenteric lymphadenectomy, have clearly demonstrated that the subsets of migrating intestinal lymph DCs have different functional properties. The subsets might play different roles in the induction of oral tolerance and in driving systemic immune responses after vaccination or intestinal stimulation with Toll‐like receptor ligands. The use of these surgical techniques allows investigation of the functions of purified subsets of migrating DCs. However, in the rat, these studies are limited by the range of available reagents and are difficult to compare with data from other species in this fast‐moving field. Recent refinements have enabled the collection of migrating intestinal DCs from mice; our initial results are described here. We believe that these studies will generate exciting data and have the potential to resolve important questions about the functions of migrating intestinal DC subsets.

Dietary fish oil diminishes the antigen presentation activity of rat dendritic cells.

Rats were fed for 6 weeks on a low fat (LF) diet or on high fat diets containing safflower oil [SO; rich in n‐6 polyunsaturated fatty acids (PUFAs)] or fish oil (FO; rich in n‐3 PUFAs). Lymph‐borne dendritic cells (L‐DC) were isolated after cannulation of the thoracic duct and were used as antigen [keyhole limpet hemocyanin (KLH)] ‐presenting cells in an ex vivo assay that used KLH‐sensitized spleen lymphocytes as the responder cells. FO feeding significantly diminished the antigen presentation activity of L‐DC compared with L‐DC from rats fed each of the other diets. The antigen presentation activity of L‐DC from rats fed the SO diet was greater than that of L‐DC from rats fed the LF diet. Feeding the FO diet significantly reduced both the proportion of CD2‐positive L‐DC and the level of CD2 expression on L‐DC compared with feeding each of the other diets; the proportions of L‐DC staining positive for CD40, CD18, CD54, CD11a, and MHC II were unaffected by diet. However, FO feeding reduced the level of expression of CD18, CD11a, MHC II, and CD54 on L‐DC compared with feeding the other two diets; the level of expression of CD40 was unaffected by diet. This is the first study to report effects of dietary fatty acids on dendritic cells. The suppressive effect of FO feeding may account for some of the beneficial effects of n‐3 polyunsaturated fatty acids observed in clinical settings, such as prolonged survival of grafts and diminished chronic inflammatory responses. However, such an effect may also be detrimental because host defense toward bacterial and other antigens could be compromised. J. Leukoc. Biol. 62: 771–777; 1997.

Regulation of Intestinal Dendritic Cell Migration and Activation by Plasmacytoid Dendritic Cells, TNF-α and Type 1 IFNs after Feeding a TLR7/8 Ligand

Dendritic cells (DCs) migrating via lymph are the primary influence regulating naive T cell differentiation, be it active immunity or tolerance. How DCs achieve this regulation in vivo is poorly understood. Intestinal DCs are in direct contact with harmless or pathogenic luminal contents, but may also be influenced by signals from epithelial cells, macrophages, or other resident or immigrant cells. To understand the role of TLR7 and TLR8 in regulating intestinal DC function, we fed a TLR7/8 ligand (resiquimod (R-848)) to rats and mice and examined DC in pseudoafferent lymph (rat) and mesenteric lymph nodes (MLNs). Oral R-848 induced a 20- to 30-fold increase in DC output from the intestine within 10 h due to a virtually total release of lamina propria DCs. This resulted in an accumulation of DCs in the MLNs that in mice was completely TNF-α dependent. Surprisingly, intestinal lymph DCs (iL-DCs) released by R-848 did not up-regulate CD86, but did up-regulate CD25. In contrast, MLN-DCs from R-848-stimulated rats and mice expressed high levels of CD86. This DC activation in MLNs was dependent on type 1 IFNs. The major source of these rapidly released cytokines is plasmacytoid DCs (pDCs) and not classical DCs, because depletion of pDCs significantly reduces the R-848-stimulated increase in serum cytokine levels as well as the accumulation and activation of DCs in MLNs. These experiments show that TLR-mediated regulation of iL-DC functions in vivo is complex and does not depend only on direct iL-DC stimulation, but can be regulated by pDCs.

Dendritic cell–B‐cell interaction: dendritic cells provide B cells with CD40‐independent proliferation signals and CD40‐dependent survival signals

Dendritic cells (DC) have recently been shown to play an important role in B‐cell function. We have previously shown that DC can capture and retain unprocessed antigen in vitro and in vivo, and can transfer this antigen to naive B cells to initiate antigen‐specific antibody responses. We also demonstrated that DC were providing B cells with isotype‐switch signals independent of T cells but that T‐cell help was essential for antibody production. In this study, using B cells and DC from wild type (WT) and CD40 knockout (CD40KO) mice we show that DC initiate proliferation of B cells independently of CD40, because WT or CD40KO DC could induce proliferation of WT or CD40KO B cells, but proliferation was greater in the absence of CD40. DC also provide B cells with survival signals as WT DC improved viability of B cells after a 5‐day culture but survival was reduced in the absence of CD40 expression.

Dendritic cells, B cells and the regulation of antibody synthesis.

Summary: Dendritic cells (DC) are usually thought of as antigen‐presenting cells for T cells. However, recent studies from our laboratory and those of others have shown that they have important roles in B‐cell activation and regulation of antibody synthesis. Rat DC make short term interactions with resting B cells and these interactions can be stimulated by cross‐linking molecules on either cell surface. These DC can retain antigen in native form for at least 36 h in vivo and in vitro and can subsequently release it for recognition by B cells. In vivo antibody responses induced by antigen‐pulsed DC are skewed towards IgG. In vitro, naive B cells incubated with antigen‐pulsed DC subsequently secrete IgM and IgG when cultured with an antigen‐specific CD4+ T‐cell line, whereas if B cells are incubated with antigen without DC, only IgM is produced. These observations show that DC play an important role in the initiation of and regulation of antibody synthesis.

Systemic autoimmune disease induced by dendritic cells that have captured necrotic but not apoptotic cells in susceptible mouse strains

Systemic lupus erythematosus (SLE) is an autoimmune disorder of a largely unknown etiology. Anti‐double‐stranded (ds) DNA antibodies are a classic hallmark of the disease, although the mechanism underlying their induction remains unclear. We demonstrate here that, in both lupus‐prone and normal mouse strains, strong anti‐dsDNA antibody responses can be induced by dendritic cells (DC) that have ingested syngeneic necrotic (DC/nec), but not apoptotic (DC/apo), cells. Clinical manifestations of lupus were evident, however, only in susceptible mouse strains, which correlate with the ability of DC/nec to release IFN‐γ and to induce the pathogenic IgG2a anti‐dsDNA antibodies. Injection of DC/nec not only accelerated disease progression in the MRL/MpJ‐lpr/lpr lupus‐prone mice but also induced a lupus‐like disease in the MRL/MpJ‐+/+ wild‐type control strain. Immune complex deposition was readily detectable in the kidneys, and the mice developed proteinuria. Strikingly, female MRL/MpJ‐+/+ mice that had received DC/nec, but not DC/apo, developed a ‘butterfly’ facial lesion resembling a cardinal feature of human SLE. Our study therefore demonstrates that DC/nec inducing a Th1 type of responses, which are otherwise tightly regulated in a normal immune system, may play a pivotal role in SLE pathogenesis.

CD11c(high )dendritic cells are essential for activation of CD4+ T cells and generation of specific antibodies following mucosal immunization.

To generate vaccines that protect mucosal surfaces, a better understanding of the cells required in vivo for activation of the adaptive immune response following mucosal immunization is required. CD11chigh conventional dendritic cells (cDCs) have been shown to be necessary for activation of naive CD8+ T cells in vivo, but the role of cDCs in CD4+ T cell activation is still unclear, especially at mucosal surfaces. The activation of naive Ag-specific CD4+ T cells and the generation of Abs following mucosal administration of Ag with or without the potent mucosal adjuvant cholera toxin were therefore analyzed in mice depleted of CD11chigh cDCs. Our results show that cDCs are absolutely required for activation of CD4+ T cells after oral and nasal immunization. Ag-specific IgG titers in serum, as well as Ag-specific intestinal IgA, were completely abrogated after feeding mice OVA and cholera toxin. However, giving a very high dose of Ag, 30-fold more than required to detect T cell proliferation, to cDC-ablated mice resulted in proliferation of Ag-specific CD4+ T cells. This proliferation was not inhibited by additional depletion of plasmacytoid DCs or in cDC-depleted mice whose B cells were MHC-II deficient. This study therefore demonstrates that cDCs are required for successful mucosal immunization, unless a very high dose of Ag is administered.