Rat dendritic cells function as accessory cells and control the production of a soluble factor required for mitogenic responses of T lymphocytes.

Abstract

Transformation of T lymphocytes, induced by treatment with periodate or with neuraminidase plus galactose oxidase, requires the participation of accessory cells. Procedures were developed for the fractionation of rat lymph node cells, by which most of the lymphocytes can be recovered as a major population of cells that do not respond to mitogenic stimulation unless accessory cells from a separated minor population are added. Further purification led to a 1000-fold overall increase in accessory activity per cell, with a 50-70% yield. The purest preparations were virtually free of macrophages and contained more than 90% typical dendritic cells. Maximum responses occurred at a ratio of only one dendritic cell per 200 periodate-treated lymphocytes. This evidence thus indicates strongly that in rats, dendritic cells--not macrophages--function as accessory cells. Further, the number of dendritic cells in a preparation governed the magnitude of the mitogenic response and was limiting in the case of unfractionated lymph node cells. In addition, when oxidized with periodate or with neuraminidase plus galactose oxidase, the dendritic cell served as a very potent indirect stimulator of untreated responder lymphocytes. Both functions of the dendritic cell appeared to lack species specificity, since mouse dendritic cells were very active when tested with rat responder lymphocytes. A soluble factor (accessory cell-replacing factor), produced by cultures of lymph node or spleen cells subjected to oxidative mitogenesis, enabled otherwise unresponsive mitogen-treated lymphocytes to respond. Dendritic cells were required for the production of this factor but may not be solely responsible for its production.

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.…”

“…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.…”

“…This eliminates the option of decanting off granulocytes and other contaminating cells at an early stage (as performed in mouse DC cultures). Previous studies have shown, however, that DC precursors in rat BM are Fc − and do not adhere to plastic dishes and that these cells can be enriched by removing the plastic-adherent cells and depleting Fc + cells by using sheep red blood cell rosetting (Klinkert et al, 1982;Klinkert et al, 1980). To accomodate both properties of the DC precursors, we panned BM cell suspensions on normal serum-coated petri dishes prior to culture of rat BM in rGM-CSF supplemented medium and repanned the non-adherent cultured cells prior to analysis.…”

“…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)).…”