L. J. Simar

Isolation of follicular dendritic cells from human tonsils and adenoids. I. Procedure and morphological characterization

Follicular dendritic cells have been isolated from human tonsils and adenoids and characterized at the ultrastructural level. Follicles were dissected and digested with different hydrolytic enzymes. The cells were separated by sedimentation at unit gravity. By this procedure we obtained follicular dendritic cells enveloping lymphocytes with their cytoplasmic extensions in a way analogous to that described for isolated thymic nurse cells. The ultrastructural features of isolated follicular dendritic cells are similar to those observed in situ. Prolonged enzymatic action caused loss of the enveloped lymphocytes.

Human follicular dendritic cells in vitro and follicular dendritic-cell-like cells.

Abstract.Human follicular dendritic cell (FDC)-like cells (FLC) have been utilized for the in vitro analysis of germinal center reactions. However, there is no consensus whether FLC represent FDC in vitro. The purpose of the present study has therefore been to determine distinguishing features of FDC and FLC in vitro. The expression of CD40, CD54, CD49d, cytokine (γ-IFN and IL-4)-dependent MHC-class II, and CD106 was observed to be specific for the determination of FDC in long-term culture. The cytokine-dependent emperipolesis of germinal center B cells was establised as another discriminating property for FDC in vitro. In 2 out of 72 long-term cultures of FDC, we encountered dividing cells among the non-dividing population of FDC. The dividing cells expressed accessory molecules similar to those of FDC but showed emperipolesis only for the initial few days of their growth. FDC did not enhance the CD40-dependent proliferation of germinal center B cells; in contrast, FLC augumented it. Both types of cells produced a significant amount of cytokine-dependent IL-6. Further studies are needed to determine whether FLC originate from FDC in vitro.

Quantitative study of left bundle branch fibrosis in left anterior hemiblock: A stereologic approach

Serial sectioning of the interventricular septum was carried out in 16 hearts, 8 from elderly subjects with no conduction disturbance and 8 from patients with chronic left anterior hemiblock. The histologic slides were studied stereologically, and the relative density of fibrosis was quantitatively assessed by the point counting technique at various levels of the main subdivisions of the left bundle branch system. Statistical analysis revealed the following: (1) Although some fibrosis was found in the control hearts, the density of fibrosis was consistently and significantly greater throughout the conduction system in patients with left anterior hemiblock. (2) In the group with hemiblock, the relative density of fibrosis tended to increase significantly from the posterior ramification to the midseptal fibers and, finally, to the anterior fascicle. (3) Among the eight patients with hemiblock, fibrosis appeared to be evenly distributed throughout the conduction system in four. It was predominantly located in the anterior and midseptal fibers in one patient and showed an increasing severity from the posterior to the midseptal and anterior fibers in the remaining three patients. From this quantitative study, it is concluded that left anterior hemiblock is a reliable sign of left bundle branch disease but that the underlying lesions are more widely distributed than would be from the expected electrocardiographic terminology since they were found predominantly in the anterior ramifications in only half of the studied cases.

Retention of immune complexes by Fc receptors on mouse follicular dendritic cells.

Follicular dendritic cells (FDC) are located inside lymph follicles and are mainly characterized by their capacity to retain antigens. We investigated this aspect in mice lymph nodes by using bovine serum albumin (BSA) labelled with 5‐nm colloidal gold particles and homologous anti‐BSA antibodies bound to 20‐nm gold particles. Gold‐labelled BSA injected alone in non‐immunized mice was only rarely found in FDC cytoplasmic interdigitations. Injected in the form of immune complexes, it was retained by FDC. Antigen‐free anti‐BSA antibodies injected under similar conditions as immune complexes were always found in draining lymph nodes in the same locations as BSA‐anti‐BSA immune complexes. F(ab′)2 from mouse immunoglobulins linked to colloidal gold particles were very rarely found between the FDC extensions, whereas it was intensely phagocytosed by macrophages. Our study permitted precise ultrastructural localization between FDC cytoplasmic extensions or inside macrophages and other cells of the lymph nodes, but it also pointed out that homologous antibodies linked to colloidal gold particles might be retained by FDC in the absence of antigens. These observations, carried out with colloidal gold, were checked by using 125I‐labelled anti‐BSA antibodies. Complement activation determinations of gold‐labelled antibodies or immune complexes showed that antibodies or immune complexes fixed on colloidal gold particles do not activate the complement. This observation enabled us to conclude that Fc receptors play a significant part in the retention of gold‐labelled antibodies or immune complexes by FDC of lymph nodes.

Precise localization of antigens on follicular dendritic cells.

SummaryHorse-spleen ferritin or bovine serum albumin conjugated to colloidal gold (BSA-gold) were injected subcutaneously in preimmunized mice. In draining lymph nodes both antigens were located in macrophages or between the cytoplasmic processes of follicular dendritic cells (FDC). Some of the antigens remained trapped on FDC until day 31 after injection. Simultaneous injection of both antigens showed that they were located between the infoldings of the same FDC. These cells are thus able to retain at least two different antigens on their surface. The peculiar arrangement of ferritin between the cytoplasmic infoldings suggests that this antigen is fixed on both cell membranes by specific antibodies. The trapped immune complexes could thus stabilize the FDC membrane system.The antigen retention requires the presence of specific antibodies since BSA-gold or ferritin injected without preimmunization were not found between FDC processes. Nonantigenic materials, such as colloidal gold or carbon particles, are not trapped by FDC, except when injected in large amounts.The antigens were trapped on the surface of FDC, however unfrequently in close contact with lymphocytes. FDC might protect lymphocytes against an excess of immune complexes and act as regulators of contacts between lymphocytes and immune complexes.

Moabs MAS516 and 5B5, two fibroblast markers, recognize human follicular dendritic cells

Follicular dendritic cells (FDC) are only located within follicles of secondary lymphoid tissues. The origin of this peculiar cell type is not clearly defined. To contribute to this study, we applied two monoclonal antibodies (MAS516 and 5B5) considered as specific for fibroblasts to tonsil cryosections and to isolated follicular dendritic cells. On the basis of an enzyme cocktail digestion of human tonsils and a fractionation procedure on albumin gradients, FDC can be prepared in the form of cell aggregates with associated lymphoid cells. MAS516 reacts with surface membrane molecules expressed by human fibroblasts, tissue macrophages and peripheral blood monocytes. With immunoperoxidase assays on tonsil cryosections connective tissue cells and macrophages are stained. Inside germinal centres, heavy labelling of the light zone was found. The MAS516 staining pattern is very similar to that of specific FDC markers DRC-1 or BU10. All isolated FDC reacted with MAS516 antibody. 5B5, considered as a typical fibroblast marker, reacts with human prolyl-4-hydroxylase which is an intracellular enzyme related to collagen biosynthesis. In cryosections, interfollicular and capsular areas showed 5B5 positive connective tissue fibroblasts. In germinal centres, some cells presenting features of FDC were 5B5 positive. After cell separation, 25%-50% of the isolated FDC were labelled with this antibody. This positivity of some FDC for 5B5 antibody may support the idea of their fibroblastic origin. The combination of observations realized in situ and after cell purification ensured an unequivocal recognition and identification of FDC.(ABSTRACT TRUNCATED AT 250 WORDS)

Human germinal center CD4^+CD57^+ T cells act differently on B cells than do classical T-helper cells

We have isolated two subtypes of helper T cells from human tonsils: CD4+ CD57+ cells, mostly located in the germinal center (GC), and CD4+ CD57- cells, distributed through the interfollicular areas but also present in the GC. In a functional study, we have compared the capacities of these T-cell subtypes to stimulate B cells in cocultures. In order to block T-cell proliferation while maintaining their activation level, we pretreated isolated T cells with mitomycin C prior to culture in the presence of B cells and added polyclonal activators such as PHA and Con A, combined or not with IL-2. Contrary to CD4+ CD57- cells, CD4+ CD57+ cells did not markedly enhance B-cell proliferation. Even when sIgD-B cells typical of germinal center cells were tested, the CD4 CD57 cells had no significant effect. This is in accordance with the location of these cells: They mainly occupy the light zones of the GC where few B cells divide. Even when added to preactivated, actively proliferating cells, CD4+ CD57+ cells failed to modulate B-cell multiplication. On the supernatants of B-cell-T-cell cocultures, we examined by the ELISA technique the effect of T cells on Ig synthesis. Contrary to CD57- T cells, whose effect was strong, CD57+ T cells weakly stimulated Ig synthesis. More IgM than IgG was generally found. Because CD57 antigen is a typical marker of natural killer cells, we tested the cytolytic activity of tonsillar CD4+ CD57+ cells on K562 target cells. Unlike NK cells, neither CD4+CD57+ nor CD4+ CD57- cells exhibit any cytotoxicity. Thus, germinal center CD4+ CD57+ cells are not cytolytic and do not strongly stimulate either B-cell proliferation or Ig secretion. CD4+ CD57- cells, however, enhance B-cell proliferation and differentiation, thus acting like the classical helper cells of the T-dependent areas.

Follicular Dendritic Cells in Lymph Nodes after X-irradiation

Follicular dendritic cells (FDC), non lymphoid cells present in lymph follicles, are characterized by numerous cytoplasmic processes retaining antigen-antibody complexes. Their origin, nature and function are unknown. Mice inguinal lymph nodes after 4.5 or 7.5 Gy X-irradiation were depleted of lymphoid cells. Ultrastructural observations during the first few days post-irradiation show that FDC are unaltered and possess dendritic processes enveloping dense material. Furthermore, they show intense metabolic activity. A lamina densa, never observed so well-developed in other lymph node cells, was detected around the nuclear envelope. The localization of junctions between FDC was analysed. FDC preserve their typical cytoplasmic processes even if lymphoid cells are rare. The latter thus seem not to be responsible for the maintenance of FDC integrity or their development. The possible role of this for antibody production is discussed. Irradiated lymph nodes of lymphoid cells are highly convenient for studying FDC. Isolation of FDC from irradiated lymph organs would seem to be possible.

Mouse lymph node follicular dendritic cells: quantitative analysis and isolation.

Follicular dendritic cells (FDC) are peculiar non-lymphoid cells only present in lymph follicles especially in germinal centers. They retain antigens in form of immune complexes on the surface of their membranes (1, 2, 3). Few data are available concerning their nature, origin and function. Their study is difficult as they represent only a low percentage of the cell population inside the germinal centers. Recently we achieved the isolation of FDC from human tonsils and adenoids (4). After isolation human FDC form spherical structures where they envelope lymphoid cells (mainly B cells, some T helper cells) with their cytoplasmic extensions. Isolated tonsillar FDC bear IgM, IgA, IgG and IGE but no IgD on their surface. They share also some antigens with macrophages (HLA-DR and Leu-M3) and express, at high density, C3b and Fc receptors (5).

A cetylpyridinium chloride (CPC) and ferric thiocyanate (Fe Th) method for polyanions demonstration on thin sections for electron microscopy

SummaryA technique for demonstration of glycosaminoglycans and other biological polyanions has been applied on floating thin sections for electron microscopy. The method is based on the formation of insoluble polyanions-quaternary ammonium salt complexes and the subsequent formation of electron dense ion association complexes following a ferric thiocyanate treatment. Mast cell granules and eosinophilic granule crystalloids were selectively stained. Nucleic acid contrast was selective to some extends.