Identification of a novel cell type in peripheral lymphoid organs of mice. V. Purification of spleen dendritic cells, new surface markers, and maintenance in vitro.

Abstract

Dendritic cells (DCs; 1) have been purified from mouse spleen in good yield. Spleen cell suspensions were floated on dense bovine plasma albumin (BPA) columns, and the low density fraction was adhered to glass (2). The adherent cells consisted of DCs and immature macrophages most of which eluted in a viable state from the culture dish after overnight incubation. The macrophages were then removed by selective rosetting with opsonized erythrocytes and recentrifugation on dense BPA. This protocol resulted in a purified DC fraction, containing 1--3 X 10(5) DCs/spleen, which was homogeneous and distinctive in its properties. All cells exhibited the phase contrast and transmission electron microscopy (EM) cytologic features that were previously described for freshly isolated adherent DCs. By scanning EM, most purified DCs exhibited a remarkable array of bulbous protrusions of varying length and shape, unlike any other lymphoid cell. All DCs expressed surface Ia and other major histocompatibility complex (MHC)-linked alloantigens. DCs, however, lacked surface Ig and T-cell antigens, and did not bind or interiorize opsonized erythrocytes. Purified DCs have been maintined in vitro for 3 days. Recovery of cultured purified cells was 70% or more of starting cell numbers. When [3H]uridine-tagged DCs were mixed with nonlabeled heterogeneous spleen cells, 70--80% of the labeled DCs were recovered as viable cells 2--3 days later. Purified DCs did not readhere to tissue culture surfaces and did not proliferate, even when cultured with mitogenic doses of concanavalin A and lipopolysaccharide. Finally, DCs did not change their cytologic or surface properties after 3 days of culture. These observations extend the evidence that DCs are a novel cell type and provide useful properties and techniques for their further study.

Electron microscopic study of HLA‐DR and monocyte/macrophage staining cells in the rheumatoid synovial membrane

“…In Figure 8B, an enlargement of this type of cell within the infiltrate shows characteristic features of the IDC. Such cells showed irregularly shaped nuclei with finely dispersed chromatin, translucent cytoplasm which contained small vesicles and narrow tubules, whorlshaped endoplasmic reticulum, and few lysosomes, as has been described previously (6,21,22,(32)(33)(34). It is significant that such IDC were present mainly in those transitional areas which contained increased numbers of lymphocytes.…”

A rapid isolation procedure for dendritic cells from mouse spleen by centrifugal elutriation

“…The laborious standard isola tion procedure of D C (Steinman et al, 1979) takes 2 days and may lead to the activation of these cells through adhesion to tissue culture plates. Non-activated monocytes can be isolated by centrifugal elutriation (CE) (Figdor et al, 1986;Te Velde et al, 1990).…”

“…Spleen cells were incubated for 30 min with i saturating concentrations of monoclonal antibody or antiserum (Table I) Isolation of dendritic cells (DC) by the standard procedure DC were isolated according to the method described by Steinman et al (1979), with slight modifications. Spleens of B6 mice were injected with 1 ml collagenase (100 U /m l) (Clostridium histolyticum, type IV.…”

“…For many years the isolation procedure has been a major limiting step in the investigation of the functional and phenotypic properties of DC. The standard isolation proce dure of DC takes 2 days and includes an 18 h culture period involving cell adhesion to tissue culture plates (Steinman et aL, 1979). Adherence may induce biological and biochemical changes in DC which may* in turn, lead to the activation of these cells as has been described for monocytes (Figdor et al, 1986: Kelley et al, 1987Te Velde et al, 1990).…”

Dendritic cells: Understanding immunogenicity

“…A helpful choice, which took years to materialize, was to first enrich DC as a distinct cell type prior to studying their functional properties and comparing these to other cell types [15]. Others were using the expression of McDevitt's immune response gene-associated (Ia) antigens as a marker for functional accessory cells, but Ia antigens did not mark a specific cell type and later proved to be the MHC II products needed to present peptides.…”