Thymic nurse cells. Lymphoepithelial cell complexes in murine thymuses: morphological and serological characterization

Abstract

We describe a new cellular component of normal mouse thymuses, which is isolated by fractionated trypsin dissociation of minced thymus tissue followed by repeated unit gravity sedimentation. These cells are of unusually large size, with diameters of 30 μm and more. They represent cellular complexes of single large cells filled with high numbers of lymphoid cells. The majority of the engulfed lymphoid cells is not only fully intact, as judged by morphological criteria, but, moreover, includes a high proportion of mitotic figures. Electron microscopic investigations reveal the epithelial character of the large thymic nurse cells (TNC). The peripherally situated cytoplasmic tonofilament streams, and characteristic vacuoles filled with coarse, unidentified material, closely resemble cytoplasmic organelles found in the cortical reticuloepithelial cells described in situ. The internalized lymphocytes are located within caveolae lined by plasma membranes. These TNC caveolae are completely sequestered, and have lost any communication with the extracellular space, as demonstrated by the inability of an electrondense marker, cationized ferritin, to diffuse into the perilymphocytic clefts. The structural interactions between the membranes of the engulfed thymocytes with the surrounding TNC caveolar membranes were investigated both in ultrathin sections and in freeze-etch preparates. Two distinct contact types between both membranes were discerned: (a) complete, close contact along the entire lymphocyte circumference, and (b) more frequently, contact restricted to discrete, localized areas. Judging from their size and distribution, the localized contacts could correspond particle aggregates of freeze-etch preparates, which morphologically resemble certain stages of gap junction. Furthermore, we regularly found square arrays of particles of uniform size, which so far have been thought to be typical for cell membranes actively engaged in ion exchange. Tight junction-like particle arrays, which were present on TNC outer membranes, and probably represented disrupted contacts between adjacent TNC in the intact tissue, could not be found on caveolar or lymphocyte membranes. Finally, one of the most conspicuous specializations of the TNC caveolar membrane were membrane invaginations, which were arranged mainly in groups, and which probably reflect endo- or exocytotoxic events. We investigated the surface antigen phenotype of TNC by indirect immunofluorescence, with monoclonal antibodies against determinants of H-2- complex subregions as well as against lymphocyte differentiation markers. Semiquantification was reached with flow cytofluorimetry, followed by morphological control by fluorescence microscopy. The surface antigen formula of TNC is: Ig(-), Thy-l(-), H-2K(++), I-A (++), I-E/C(+), H-D(++), Ly-1(-), Ly-2(-), Qat-4(-), Qat-5(-), and peanut agglutinin (PNA)(-). Thymic macrophages, which were identified by double fluorescence, with rhodamine- coupled zymosan as a phagocytosis marker, were serologically identical with TNC. Free thymocytes, in contrast, had the following antigen formula: Ig(-), Thy-1(++), H-2K(+/-), I-A(-), I-E/C(-), H-2D(+/-), Ly-1(+/-), Ly-2(+), Qat- 4(-), Qat-5(-), and PNA(+). The unprecedented finding of high numbers of dividing thymocytes sojourning within thymic epithelial cells, and the particular specializations of the TNC caveolar membranes surrounding these engulfed thymocytes is the basis of a hypothesis that postulates that an intraepithelial differentiation cycle is one essential step in, intrathymic T lymphocyte generation.

Murine Thymic Nurse Cells Express ICAM‐1 on Caveolar and Vacuolar Membranes

“…After each round the suspended cells were removed and the remaining tissue fragments were incubated with fresh enzyme. The collected fractions were then examined by light microscopy, and the TNC-containing fractions (mainly from the D/C digestions) were pooled and enriched for TNCs by sequential sedimentation at unit gravity in an FCS gradient [2]. The TNC enriched media were finally centrifuged at 500 g.…”

CXCR4 chemokine receptors (CD184) and α4β1 integrins mediate spontaneous migration of human CD34+ progenitors and acute myeloid leukaemia cells beneath marrow stromal cells (pseudoemperipolesis)

“…By migrating into the same focal plane as the adherent stromal cells, the migrated cells acquire a dark appearance, whereas non-migrated cells remain refractile upon examination by phase contrast microscopy (see Fig 1). The term pseudoemperipolesis was defined to distinguish this type of cell interaction from 'true' emperipolesis, which involves the penetration of living cells into other cells, as described in situ for thymocytes that migrated into the cytoplasm of thymic nurse cells (Wekerle et al, 1980).…”

Murine Thymic Nurse Cells and Rosettes: Analysis of Adhesion Molecule Expression Using Confocal Microscopy and a Simplified Enrichment Method

“…TNC are cortical multicellular complexes composed of intra-TNC thymocytes (TNC-T) enclosed within epithelial cell vacuoles (Wekerle et al 1980;Wick et al 1991). ROS are characterized by thymocytes bound to a central stromal cell (Kyewski et al 1982).…”