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Dendritic cells (DC) are the most potent antigen-presenting cells and are therefore useful to induce immune responses against tumor cells in patients. DC can be generated in vitro from monocytes using GM-CSF and IL-4, the so-called monocyte-derived DC (MoDC). To achieve antitumor responses, MoDC must be able to migrate to the draining lymph nodes after injection to induce cytotoxic T cells. Therefore, we studied migration of MoDC in a rat model. Functional rat MoDC were generated from PVG-RT7B rats and injected subcutaneously into PVG rats. These rat strains differ only at one epitope of the leukocyte-common antigen, which can be recognized by the antibody His 41. The advantage is that migrated cells can be detected in the draining lymph nodes by staining sections with His 41+; thus, migration is not influenced by labeling procedures. Rat MoDC migrated to the T-cell areas of the draining lymph nodes, just as isolated Langerhans cells or spleen DC do. In contrast, monocytes also migrated to the B-cell areas and the medulla.

Dendritic cells (DC) are professional antigen-presenting cells, capable of priming naive T cell responses. Glucocorticoids (GC) are frequently used in asthmatic patients. In this study we describe the effects of GC on the development and function of monocyte-derived DC (MoDC) in vitro and in vivo. Monocytes from healthy individuals were isolated and incubated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 6 days, to induce maturation into MoDC. To study the role of GC on DC differentiation in vitro cells were incubated with dexamethasone at different stages of MoDC development. At day 6 cells were characterized phenotypically by flow cytometry and functionally in an allogeneic mixed leucocyte reaction. To study the effect of GC in vivo patients with mild/moderate atopic asthma were selected. In one group no GC were used, whereas the other group used inhalation GC. MoDC from these patients were generated as described above and tested functionally. Incubation of MoDC or its peripheral blood precursors with dexamethasone decreased the accessory potency dose-dependently. The functional differences could not be explained by the changes in the expression of MHC II and the costimulatory molecules CD40 and CD86. The relevance of this mechanism was confirmed for the in vivo situation as well. MoDC from patients using inhalation GC showed a decreased accessory potency. These data suggest a modulatory effect of GC therapy at the level of the peripheral blood monocyte. The results indicate that GC influence DC development and function in vitro as well as in vivo.

Background: Several studies suggest a role for dendritic cell in the pathogenesis of allergic disease., Objective: The purpose of this study was to compare function and phenotype of monocyte-derived dendritic cells (MoDC) from allergic asthmatic patients and healthy control subjects., Methods: MoDCs were developed by incubating adherent monocytes for 5 days with IL-4 and granulocyte-macrophage colony-stimulating factor. Phenotype was assessed with flow cytometry, and the antigen-presenting function was assessed with the allogeneic mixed leukocyte reaction and an autologous specific antigen presentation., Results: The morphology of the MoDCs was characteristic for immature dendritic cells. MoDCs from allergic asthmatic patients showed phenotypic differences in the expression of HLA-DR, CD11b, and the high-affinity receptor for IgE. A clearly enhanced accessory potential of MoDCs from atopic asthmatic patients in the mixed leukocyte reaction was also shown. Moreover, house dust mite-specific T-cell proliferation was increased., Conclusion: This study suggests the involvement of dendritic cells in the pathogenesis of atopic asthma by an increased immunostimulatory capacity of MoDCs.

In the present study about 0.3% to 1.6% of human bronchoalveolar lavage (BAL) cells were identified as typical dendritic cells (DC), having an irregular outline, lobulated nucleus, and clear distinguishable acid phosphatase activity or EBM11 (anti-CD68) reactivity in a spot near the nucleus. After DC enrichment, using transient adherence to plastic, FcR-panning, and a density metrizamide gradient, a population containing 7-8% typical DC was obtained. This DC-enriched low density fraction, containing the highest percentages of DC, very strongly induced T cell proliferation in an allogeneic mixed leucocyte reaction (MLR), which was significantly higher than that induced by other partly (un)fractionated BAL cells. These data indicate that DC seem to be the major accessory cells in the BAL fluid, and therefore may be important in the regulation of T cell immune responses in the lung.

In this study the antigen-presenting capacity of human peritoneal cells and the influence of continuous ambulant peritoneal dialysis (CAPD) were studied. On average 6% of the peritoneal cells were dendritic cells (DC), with no difference between CAPD and control peritoneal cells. DC were enriched by selecting for non-adherent, Fc receptor-negative, low density cells. A typical spot-like CD68 positivity was seen in DC, in contrast to the pancytoplasmic staining pattern in macrophages. Peritoneal DC morphologically and functionally showed features of cells belonging to the DC lineage. Peritoneal DC were superior antigen-presenting cells for both allo-antigen, and Candida albicans antigen or purified protein derivative. CAPD peritoneal macrophages were two- to three-fold better stimulator cells for allogeneic T cells compared with control macrophages. The level of integrins/adhesins or MHC class I or II, as measured semi-quantitatively on the FACS, could not account for this phenomenon. In addition, a double chamber system showed that dialysate-activated macrophages produced soluble factors that could enhance DC-induced allogeneic T cell proliferation. In conclusion, human peritoneal cells contain a relatively high percentage of classical DC. CAPD treatment does not impair the antigen-presenting capacity of peritoneal cells, but instead upregulates the allo-antigen-presenting capacity of peritoneal macrophages.

The migration of dendritic cell (DC)-enriched populations and alveolar macrophage (AM) populations isolated from PVG RT7.2 rats was studied after local administration to recipient PVG RT7.1 rats. The monoclonal antibody His41, which is directed against the common leukocyte antigen of the RT7.2 rat, was used to detect migrated cells. Injection of the splenic DC and AM subcutaneously into the footpads resulted in migration of both cell types to the popliteal lymph nodes after 24 h. DC located predominantly in the T cell-dependent areas, whereas AM located more in the medulla and medullary cords and spread throughout the outer cortex area. After intratracheal instillation of splenic DC, these cells were found predominantly in T cell-dependent areas of the draining lymph nodes of the lung after 24 h. In contrast, AM did not migrate to the draining lymph nodes after intratracheal instillation. Combined with those from earlier studies, these data show that DC present in the alveolar lumen may pick up airborne antigen and migrate to the draining lymph nodes of the lung, where they can induce primary T cell responses.

In a previous study we demonstrated that an increase of monocytes and dendritic cells (MDC) was In the current study, the bright autofluorescence of alveolar macrophages (AMs) was used to separate them efficiently from the MDC. Sorting of freshly isolated BAL cells resulted in a high-autofluorescent fraction, consisting predominantly of AMs, and a low-autofluorescent fraction containing the MDC, lymphocytes, and granulocytes. Thus, a clear separation between suppressive (AM) and stimulating (MDC) activity was obtained as shown in antigen-specific T cell responses. Flow cytometric parameters, density fractionation, and a series of ED monoclonal antibodies raised against rat macrophage antigens showed that both AMs and MDC were diverse populations. After overnight culture, more than 80% of an MDC population with a density range of 1.065-1.079 changed to a lower density (< 1.056) and morphologically developed into DCs with many processes. Concomitantly, monoclonal antibody ED1 expression changed from a granular pattern to a discrete juxtanuclear spot localization.

In the present study, splenic dendritic cells (DC) and alveolar macrophages (AM) were pulsed with antigen in vitro and subsequently intratracheally instilled to test whether these cells have the capacity to sensitize T cells in the draining lymph nodes of the lung. The data demonstrate that antigen-pulsed DC, instilled in the bronchoalveolar lumen, induce antigen-specific T cell priming in vivo in the draining lymph nodes. T cell priming is only seen with viable but not with killed antigen-pulsed DC. Amounts as low as 5 x 10(3) to 10 x 10(3) cells can still induce some responsiveness. In addition, it was found that instillation of viable as well as killed pulsed Ia-negative AM also leads to T cell priming, although about 10 times higher numbers of cells had to be used in comparison with DC. The results suggest that DC instilled in the bronchoalveolar lumen present antigen directly to naive T cells, whereas for AM other mechanisms are involved.

Recently we described the presence of a small number of DC among the peritoneal cells of steady state rats. These DC had the same morphological characteristics and a similar antigen-presenting capacity as DC isolated from the spleen. This study shows that in the peritoneal cavity, which is a non-lymphoid microenvironment, the number of DC increases after i.p. administration of BCG. Next to this relatively small influx of DC, the approximately three-fold increase of the total number of cells is predominantly caused by an enormous influx of neutrophilic granulocytes, and to a lesser extent by an influx of macrophages. The phenotype and the antigen-presenting capacity of peritoneal DC has not changed, while the number of Ia-positive M phi has increased. Nevertheless, due to a suppressive effect of the peritoneal M phi, the total peritoneal cell suspension is no longer capable of presenting antigen.

Dendritic cells (DC) are present in lymphoid organs and also in many non-lymphoid tissues. In this study, DC in the steady state peritoneal cavity of rats were identified morphologically and functionally. Approximately 1% of the peritoneal cells are DC. On cytocentrifuge preparations these cells had the same characteristics as lymph node and spleen DC: they had an irregular outline, all were strongly MHC class II positive and had acid phosphatase activity in a spot in a juxtanuclear position. Also ultrastructurally, peritoneal DC were similar to DC isolated from lymph node and spleen. Enrichment of peritoneal DC, using overnight culture and a Nycodenz gradient, resulted in a highly purified DC fraction. Functionally, peritoneal DC appeared to be very potent antigen-presenting cells, far more potent than peritoneal macrophages, which had an inhibitory rather than an accessory function.

Phenotypic heterogeneity between macrophages in situ has been reported, using different macrophage-specific monoclonal antibodies. Microenvironmental factors, which may play an important role in inducing this heterogeneity were studied in a local inflammatory response in vivo. Our results show that peritoneal exudate macrophages, formed during Thioglycollate induced chronic inflammation, acquire expression of the dendritic cell markers NLDC-145 and 6D2. In contrast to these phenotypic characteristics, NLDC-145+/6D2+ exudate cells do not function like dendritic cells in an allogeneic-MLR, but are suppressive when compared with steady state peritoneal cells. The expression of these membrane markers on exudate cells is discussed.

Identification of dendritic cells (DC) is usually done on the basis of their strong MHC class II expression, their typical dendritic morphology and their capacity to induce a strong proliferation of allogeneic T cells. However using these criteria DC can easily be confused with MHC class II positive macrophages (M phi). In addition, the lack of an antibody directed to a specific DC marker greatly hampers the discrimination between DC and M phi. In the present study it is shown that EBM11 (anti-CD68) is a marker specific for both human M phi and DC but in a distinctive way. Human DC locate the EBM11 reactivity in a discrete juxtanuclear spot in contrast to M phi which show EBM11 reactivity throughout the cytoplasm. This greatly improves identification of DC. Light and electron microscopy showed that the CD68 epitope is associated with (phago-)lysosomes. Remarkably the EBM11 spot was only seen in DC after short-term culture, which is an essential step in all classical DC enrichment procedures. Before culture, M phi and DC were indistinguishable. These results show the close relationship between M phi and DC and suggest an important role for the structure of the lysosomal apparatus in these antigen-presenting cells.

On light microscopical (LM) level dendritic cells (DC) isolated from lymphoid organs can be discriminated from macrophages (M phi) by the presence of acid phosphatase (APh) activity in a spot near the nucleus and constitutional expression of class II antigens. The aim of our study was to investigate whether DC and monocytes (Mo) enriched from human peripheral blood could be discriminated on the electron microscopical (EM) level. Therefore we developed a triple method by which we compared the presence of myeloperoxidase (MPO) containing vesicles, the localization of APh containing vesicles and expression of MHC class II and RFD1 (a DC-associated class II-like antigen) plasma-membrane antigens. DC, functionally characterized as potent stimulators in a MLR, are MPO-negative, whereas Mo show MPO in cytoplasmic granules. Although both DC and Mo show little APh activity at LM level, both types of cells show APh activity at the EM level but at different locations. In DC APh containing vesicles are present in a distinct juxtanuclear area, in contrast to Mo, which show APh activity in lysosomes scattered throughout the whole cytoplasm. Moreover, on both LM and EM level, DC are strongly class II positive, whereas Mo show variable labelling intensity for class II, while RFD1 was only found on DC.