When 2 inbred rat strains, the Brown-Norway rat and the Lewis rat were exposed to the same amount of CdCl2 for 15 days, a completely different immunological reaction pattern could be demonstrated. Despite the same amount of intrathymic cadmium in both strains, the Brown-Norway rat showed a significant decrease in thymocytes in the S-phase and a significant increase of thymocytes in the G2 phase and mitosis, in contrast with findings in the Lewis rats. A new method for estimating subtle forms of thymus atrophy showed a slight decrease in the number of the smallest thymocytes in the Brown-Norway rat after exposure to cadmium, in contrast with that in the Lewis rat. Evidence is presented that the approximately 1.7 times larger number of thymocytes/mg thymus in the Lewis rat, compared to the Brown-Norway rat, as well as the approximately 2.5 times lower proliferation rate of the thymocytes, and an approximately 1.5 times higher metallothionein content of the thymus medulla epithelial cells in the Lewis rat, might be responsible for the observed difference in toxicity. The zinc content of the thymus was not significantly decreased by exposure to CdCl2, and did not differ significantly between both strains.

In situ pre-existing complexes of epithelial cells and thymocytes having thymic nurse cell characteristics were visualized in the murine thymus cortex using dexamethasone as a potent killer of cortisone-sensitive thymocytes. The degradation and subsequent depletion of cortisone-sensitive thymocytes enclosed within cortical epithelial cells appeared to be paralleled by thymocyte degradation and depletion in thymic nurse cells isolated from thymic tissue fragments from dexamethasone-treated animals. This suggests that thymic nurse cells are derived from pre-existing sealed complexes of cortical epithelial cells and thymocytes. Not all thymocytes situated within in situ epithelial or thymic nurse cells complexes appear to be cortisone-sensitive: a minority of 1–2 thymocytes per complex survives the dexamethasone-treatment, thus constituting a minor subset of cortical cortisone-resistant thymocytes predominantly localized within cortical epithelial cells in situ and within thymic nurse cells derived from such structures. Cortisone resistance in thymocytes thus seems to be acquired within the cortical epithelial cell microenvironment. Cortisone-resistant thymocytes in thymic nurse cells express the phenotype of mature precursors of the T helper lineage, indicating that the in situ correlates of thymic nurse cells may play an important role in T cell maturation and selection.

Cadmium was administered subcutaneously to pregnant Wistar rats: 0.49 mg/kg as CdCl2 in saline daily, starting at the day of conception. Placentas and fetal livers were collected on day 14, 16, 18, 19 and 20 of gestation. Livers and thymuses from the newborns were collected 5 hours after delivery (day 22) and 1, 2 and 5 weeks after delivery. In these tissues concentrations of cadmium and zinc were determined by solid sampling ETA-AAS. Furthermore, the effect of cadmium administration on the glycogen content of the trophoblastic labyrinth and the fetal liver was studied. The concentration of cadmium in the placenta increased with time of exposure, indicating accumulation of cadmium in this organ. In the fetal liver, cadmium was present in a very low concentration, which slightly increased with longer exposure. The concentration of zinc in the placenta tends to decrease between day 14 and day 20. This decrease was observed both in control and in cadmium-exposed animals. Zinc levels increased in fetal livers from control dams, whereas this rise was markedly reduced in fetuses from cadmium-exposed animals. Placentas from cadmium-exposed animals had a changed glycogen pattern as compared to the controls, namely higher glycogen contents of the labyrinth at the end of pregnancy. However, notwithstanding lower zinc levels in the fetus and changed glycogen deposition in the placenta, it is not quite clear whether cadmium affects fetal development. No changes were observed in fetal weights or birthweights, nor in glycogen deposition of the fetal liver. Indications were obtained for reduced neonatal thymic weights.

Thymic nurse cells (TNC), defined as in vitro isolation products of thymic tissue, are epithelial cells harboring in their cytoplasm up to 200 intact, actively dividing thymocytes which are completely surrounded by vacuolar membranes. The TNC plasma membrane expresses major histocompatibility complex class I (H-2 K/D) and class II (I-A) antigens. The expression of MHC class I and class II antigens on the TNC vacuolar membranes was investigated with an improved in situ labeling technique. The major histocompatibility complex phenotype of the vacuolar membranes is H-2 K/D+, I-A2+ and thus identical to the TNC plasma membrane phenotype. By using the labeling technique, the TNC thymocyte population was examined for expression of the T cell differentiation antigens Thy-1, peanut agglutinin, Lyt-1, and Lyt-2, and the antigen expression was related to resistance of this population to cortisone. The majority of TNC thymocytes in individual TNC were cortisone-sensitive and expressed the immature phenotype of cortical thymocytes (Thy-1hi, PNAhi, Lyt-1lo, Lyt-2). A minority of the TNC thymocytes were cortisone-resistant and expressed a mature phenotype (Thy-1lo, peanut agglutininlo, Lyt-1hi). The existence of this minor mature population was confirmed in vivo: cortisone-resistant thymocytes were associated with cortical epithelial cells scattered throughout the thymic cortex of mice treated with dexamethasone. The major histocompatibility complex positive microenvironment of TNC and the heterogeneity in phenotype and resistance to cortisone of the TNC thymocytes, which is related to the state of maturation, indicate that TNC play an important role in the selection and differentiation of T cells.

Dendritic cells have been isolated from rabbit lymph nodes. Morphologically and phenotypically, they resemble dendritic cells from the mouse and rat. A comparison was made of the accessory cell function of dendritic cells and peritoneal macrophages in T-cell proliferation induced by phytohaemagglutinin (PHA) or Con A, or by a simultaneous treatment with the enzymes neuraminidase and galactose oxidase (NaGo). Dendritic cells seemed to be more effective than macrophages as accessory cells in these assays. However, macrophages suppress lymphocyte proliferation through the release of oxidating agents and production of prostaglandins. Elimination of this suppressive effect of the macrophages by addition of a combination of 2-mercaptoethanol (2-ME) and indomethacin in PHA-induced cell proliferation resulted in a much higher support by macrophages, giving results that were comparable to those obtained with dendritic cells, but in NaGo-induced proliferation, macrophages were still not as effective as dendritic cells in the presence of the drugs. Experiments in diffusion culture vessels and with interleukin-1-containing macrophage supernatants showed that support by accessory cells can be mediated by soluble factors in PHA-induced proliferation. In contrast, in NaGo-induced proliferation, lymphocytes and accessory cells have to interact directly.

Mitogen-induced proliferation of rabbit lymphocytes from the thymus (Thy), spleen (Spl) and lymph node (LN) was measured by [3H]thymidine incorporation. The various cell suspensions taken from normal animals showed wide differences in their response to Con A and PHA. Cell suspensions taken from steroid-treated animals did not show differences in response. The disappearance of these differences in mitogen reactivity after steroid treatment is caused by a decreased Con A response of LN cells and an increased Con A and PHA response of Thy cells. The results suggest the existence of a steroid-sensitive (Ss). Con A-responsive cell population in LN cells. The mitogen response of untreated and steroid-resistant (SR) Thy cells was further investigated in cell suspensions separated on density gradients and in cell suspensions enriched in, or depleted of cells bearing receptors for Fc (T gamma and T non-gamma, respectively). It is concluded that Ss thymocytes of high density are mitogen non-reactive, and that Ss cells of low and medium density consist of subpopulations of cells reactive to Con A and/or to PHA. Sr thymocytes still displayed heterogeneity in buoyant density and mitogen responsiveness. Heavy cells and T gamma cells (10% of the Sr cells were T gamma) showed a lowered mitogen response. The results are discussed in relation to data describing the localization of the cell types which differ in mitogen reactivity. The results support the idea of two differentiation pathways, one for Ss and the other for Sr thymocytes.

The specificity of a rat anti-rabbit thymocyte antiserum (ATS), as analysed with immunofluorescence techniques, is described. The antiserum was used in cytofluorometric studies to quantify the density of the corresponding T-cell antigen present on the various lymphocyte populations. On the basis of fluorescence intensity, the positive cells could be divided into four classes. Most of the thymus lymphocytes stained bright while minor subclasses were found with dull, medium and very bright staining. Steroid-resistant thymus cells stained medium and bright. Further analysis on buoyant density and characterization of ultrastructural nuclear morphology, combined with ultrastructural studies of control and steroid-resistant thymus lymphocytes in situ showed that the medium and bright fluorescent cells represent medullary and cortical cells respectively. Moreover, two types of large cells were observed. It is suggested that these two types represent different pools of cycling cells. One located in the outer cortical zone with light density and a medium fluorescence and the other located in the deep cortex with a heavy density and a very bright fluorescence for ATS; peripheral T cells express mainly dull fluorescence. A model for T-cell differentiation is proposed and discussed in respect to the density of T-cell antigen present on the thymocyte membrane.

We have investigated the possible role of intramembraneous particles as revealed by freeze-fracture electron microscopy in the plasma membrane of B lymphocytes from rabbits and mice as reflections of transmembrane structures of surface immunoglobulin receptor molecules. This was achieved by aggregation of the surface receptors using fluorochrome-conjugated antibodies, fixation and freezing of the cells in 35% glycerol. This procedure resulted in replicas of lymphocytes with well-preserved morphology (no ice-crystals), enabling the study of both protoplasmic and external fracture face in combination with surface receptor markers. It appeared that very small intramembraneous particles (3-6 nm diameter) were selectively clustered under patches of surface receptor label. This phenomenon was found on the external fracture face exclusively and not on the protoplasmic fracture face. 'Classical' intramembraneous particles (6-12 nm diameter) were not involved. We suggest that these small, clustered particles should be interpreted as transmembrane structures of surface immunoglobulin molecules.

Density separation of rabbit thymus lymphoid cells on a continuous and linear Ficoll-Metrizoate gradient resulted in the appearance of three subpopulations of lymphoid cells with peak fractions at densities of approx. 1.067, 1.077 and 1.084 g/ml. The subpopulation of the lowest density appeared to consist of cortisone-resistant cells of various size classes, all characterized by a relative low nucleo-cytoplasmic ratio. Glycerol-induced redistribution of particles within the plasma membrane (IMP) of the lymphoid cells, as revealed with the freeze-fracture technique, was present in the subpopulation of lowest density as well as in thymus lymphoid cells of cortisone-treated rabbits in a high percentage of the cells, and present in a very small percentage of the cells of highest density. It is concluded that an increase in the mobility of plasma membrane components may be a significant feature of T-cell education in the rabbit thymus.

Tannic acid as a prefixative for EM purposes was introduced by Futaesaku et al. (1972). The fixative creates conditions for enhancing electron density of different protein materials. By using a mixture of tannic acid and glutaraldehyde as prefixative, followed by a routine procedure of postfixation (OsO4) and poststaining (uranylacetate and lead citrate), membrane bound antibodies not conjugated with electron dense markers are made visible under the electron microscope.

Using absorption cytophotometry and flow cytofluorometrical DNA and protein estimation of single thymus lymphocytes we were able to establish that after injection of a large dose of antigen (ovalbumin) a subpopulation of lymphocytes arises in the thymus with high protein contents above that of those lymphocytes normally present, however, in small quantities in the thymus. By morphometrical analysis it was established that these lymphocytes are situated in the outermost cortex.