Accessory and stimulating properties of dendritic cells and macrophages isolated from various rat tissues

Abstract

Single cell suspensions of rat lymphoid and nonlymphoid tissues were fractionated on discontinuous gradients of bovine serum albumin into high density and low density subfractions. In general, accessory activity required for responses of periodate-treated T lymphocytes was recovered only in a low density population containing a small percent of the total fractionated cells from lymph nodes, spleen, liver, skin, and peritoneal exudates. Further purification always led to an increase of both accessory activity and number of dendritic cells present in nonrosetting and nonadherent populations. After purification, a high recovery of the total accessory activity was found in fractions that contained a high percentage of dendritic cells resulting in a more than 1,000-fold enrichment in accessory activity per cell. No other fraction obtained during the purification contained significant accessory activity. In all cases, macrophage-enriched populations lacked accessory cell activity. With the exception of peritoneal exudate cell preparations, which contained an inhibitory cell, the level of accessory activity in a given population was always found to be a function of the number of dendritic cells present. Dendritic cells from all sources were nonadherent, nonphagocytic, radio- resistant, and nonspecific esterase negative. They expressed Ia antigens and lacked Fc receptors. Both epidermal and lymph node dendritic cells contain Birbeck granules, subcellular structures previously described only for Langerhans cells. Accessory activity requires viable dendritic cells but is unaffected by 1,000 rad of γ-irradiation. However, ultraviolet irradiation abolished the activity of accessory cells. The cells that responded to periodate were IgG-negative T cells, whereas IgG-positive B cells could not be stimulated under the same conditions. Only periodate-treated T cells and dendritic cells were needed for responses to occur; removal of virtually all macrophages from these purified preparations had no effect. Dendritic cells were also required as stimulators in mixed leukocyte cultures, whereas macrophages, even though Ia positive, were inert.

A new protocol for the propagation of dendritic cells from rat bone marrow using recombinant GM-CSF, and their quantification using the mAb OX-62

“…The allostimulatory ability of non-adherent cells obtained from GM-CSF-supplemented day 8 cultures of unfractionated rat BM was significantly improved by retaining the floating cells that would have been removed during medium exchanges using the 'murine' technique, at stimulator to responder ratios of 1:27 the MLR against PVG were 21747 ± 1484 and 6800 ± 1024, respectively (p < 0.01). These results are entirely consistent with previous reports that mature rat DC do not adhere to culture flasks (Klinkert et al, 1980(Klinkert et al, , 1982. Thus the removal of non-adherent cells during culture discards many of the mature DC and lowers the MLR activity obtained.…”

“…Apart from reducing DC purity, monocytes can also express class II Ag as a surface marker. Hence, simple determination of the number of class II + cells in culture, as has been performed in previous reports (Klinkert et al, 1982;MacPherson, 1989), may not adequately differentiate between monocytes and DC.…”

“…Although short-term culture (1-3 days) to aid in the isolation of DC from various rat tissues, including lymph nodes, skin, thoracic duct lymph and peritoneal exudate cells, has been described previously (Bowers and Berkowitz, 1986;Klinkert et al, 1980Klinkert et al, , 1982MacPherson, 1989;MacPherson et al, 1989), only two reports describe the culture of DC from rat bone marrow. In these papers, Bowers and Berkowitz (1986) used serum-free medium while Klinkert (1984) used medium supplemented with ConA-stimulated spleen cell supernatant to promote DC growth.…”

“…For TEM, day 6 BM cultures, depleted of monocytes by repanning, were further subjected to BSA gradient fractionation as previously described (Klinkert et al, 1980(Klinkert et al, , 1982. The low density fraction (ρ = 1.048) consisted of greater than 90% DC, and representative cells are shown in Fig.…”

“…Phenotypic analysis of these cells was enhanced by the use of OX-62, the new murine monoclonal antibody directed against an integrin-like rat DC surface marker (Brenan and Puklavec, 1992). This method utilises several of the previously described properties of rat DC precursors to maximise the yield of these cells in culture, name1y their lack of adherence to plastic flasks (Klinkert et al, 1980(Klinkert et al, , 1982, FcR status (Klinkert et al, 1980(Klinkert et al, , 1982 and their partial dependence on murine rGM-CSF (MacPherson, 1989;MacPherson et al, 1989). In addition to these measures, tissue culture flasks were coated with gelatin to augment DC growth (in a fashion analogous to the use of type 1 collagen to promote the maturation of liver-derived DC (Lu et al, 1994)).…”