Corrado Ferrari

Ultrastructural characterization of human large granular lymphocyte subsets defined by the expression of HNK-1 (Leu-7), Leu-11, or both HNK-1 and Leu-11 antigens.

A peroxidase-colloidal gold double labeling system in immunoelectron microscopy was used to investigate the ultrastructural features of human large granular lymphocytes (LGL) subpopulations. Three subsets of LGL, Leu-7+-Leu-11-, Leu-7+-Leu-11+, Leu-7- -Leu-11+, were characterized using combinations of the monoclonal antibodies anti-Leu-7 and anti-Leu-11. They showed different ultrastructural patterns. In fact, Leu-7+-Leu-11- cells showed a high nuclear/cytoplasmic ratio (N/C), a round nucleus, a cytoplasm with few organelles, and a rather even surface. Moreover, most of them lacked electron-dense granules. On the other hand, Leu-11+ cells displayed a low N/C, an irregular-shaped nucleus, and a cytoplasm containing a well-developed Golgi apparatus, many mitochondria, vacuoles, vesicles, and numerous electron-dense granules. Moreover, they exhibited an irregular cell surface. Thus, Leu-7+-Leu-11- cells seemed to represent an immature form of LGL, while cells expressing the Leu-11 antigen showed a fine structure specific for functional NK cells. Our findings suggest that the expression of HNK-1 (Leu-7) and Leu-11 antigens respectively represents subsequent stages in NK cell differentiation.

Human normal‐resting epidermal Langerhans cells do express the type 3 complement receptor

The expression of CR3 by murine‐resting epidermal Langerhans cells (LC) is well established, but CR3 expression by human normal‐resting epidermal LC has not yet been demonstrated. In this study, highly sensitive immunostaining techniques, such as immunogold labelling in transmission‐ and scanning‐electron microscopy, were used on freshly isolated, LC‐enriched, normal human epidermal cells. Human normal resting epidermal LC were found to be CR3+, since a low but significant number of gold granules labelled the plasma membrane of all the LC observed under transmission‐electron microscopy, and all the epidermal cells showing LC morphology as observed by scanning‐electron microscopy.

Hairy cell leukemia cells express CD1A antigen

Five patients with hairy cell leukemia (HCL) were studied. Peripheral blood leukocytes, rosette‐forming cells (T) and non‐T‐cells were stained in immunofluorescence by a panel of monoclonal antibodies to investigate the phenotype of HCL cells (HCLC). In all patients HCLC showed B‐lymphocyte phenotype, although they were not stained by antibodies reactive for monocytes, natural killer cells, or T‐cells. However, in all instances the large majority of HCLC were unexpectedly stained with an antibody (anti‐CD1a) usually detectable only in early thymocytes and on Langerhans cells. This finding was further confirmed by immunoelectron microscopy. This type of ambiguity in the lineage of HCL could imply that HCLC might arise from cells differentiated towards the B‐cell lineage, still sustaining an early antigen of a different (T) lineage. These results, moreover, extend the range of the known distribution of the CD1a antigen, which could be useful in diagnosing HCL.

The S-100β protein in normal human peripheral blood is uniquely present within a discrete suppressor-T-cell compartment

The S-100-positive T lymphocytes, and, particularly, the S-100 beta subunit, are restricted, as demonstrated by quantitative subset analysis and double-labeling (gold-peroxidase) immunoelectron microscopy of T-cell subpopulations, to an unique T8-positive cell subset which interestingly was 9.3-negative and CD11b-positive. Since both the T8-positive, 9.3-negative and the T8-positive, CD11b-positive subpopulations have been demonstrated to show suppressive activities, the S-100-positive T cells seem to be closely restricted to a small T-suppressor-cell compartment. Although functional studies on viable isolated S-100 beta-positive cells are impossible to achieve, due to the lack of this protein on the cell membrane, its presence in a discrete T-suppressor compartment might suggest a possible role for the S-100 beta-positive T cells in the regulation of the immune system.

S-100-positive T cells are largely restricted to a CD8-positive, 9.3-negative subset

SummaryThe present report concerns the demonstration of the exclusive detection among peripheral blood T-lymphocytes of the S-100 protein within the CD8-positive subpopulation wich lacks the antigen recognized by the 9.3 monoclonal antibody. Highly purified human peripheral blood T-cell subsets, obtained by means of panning techniques, were first stained, by an immunofluorescence method, with purified anti-S-100 protein antibodies. The vast majority of S-100 protein- (and, specifically, itsβ subunit) positive cells were detected in the CD8-positive, 9.3-negative subset. This subset had previously been shown to comprise all the alloantigen-specific and histamine-inducible suppressor T-cells. Other T-subsets, even those showing either CD8-positivity (but 9.3-positivity) or 9.3-negativity (but CD8-negativity), were, as a rule, S-100 negative. Immunoelectronmicroscopy confirmed that the S-100-positive cells, showing peroxidase activity within the cytoplasm, were found exclusively within the CD8-positive, 9.3-negative subset. This finding of S-100 protein in cells of a specific T8 suppressor subset extends the range of the known distribution of this protein and may have important implications concerning its role in the modulation of immune responses.

Immunoperoxidase-immunogold double labelling in immunoelectronmicroscopy of large granular lymphocytes

A method for better characterization of mononuclear cell subpopulations using detection of 2 surface antigens simultaneously in electron microscopy was developed with immunoperoxidase-immunogold double labelling. Monoclonal antibodies of IgG and IgM classes were used in the first step, and colloidal gold-labelled anti-mouse IgG antibody and peroxidase-labelled anti-mouse IgM antibody (mu chain-specific) in the second step. Immunoelectronmicroscopy with such double labelling improves ultrastructural analysis of mononuclear cell subpopulations.

Immunoelectron microscopic characterization of a subpopulation of freshly isolated epidermal Langerhans cells that reacts with anti-CD23 monoclonal antibody

Large subsets of leucocytes were recently shown to express the low affinity receptor for the Fc portion of IgE. Because Langerhans cells (LC) are epidermal leucocytes, we investigated whether LC of normal human subjects might express this receptor. Whereas conventional immunofluorescence on epidermal sheets gave negative results, highly sensitive immunoelectron microscopy revealed that a subset (about one‐third) of freshly isolated LC express the CD23 molecule.

The immunogold-silver staining approach in the study of lymphocyte subpopulations in transmission electron microscopy

The potential of immunogold-silver staining has been evaluated in immunoelectron microscopic studies of human normal peripheral blood lymphocyte subpopulations. The cells were labeled, before being embedded in resin, using 5 nm colloidal gold particles and this was followed by silver enhancement. The use of colloidal gold particles permits detection of small amounts of antigen; the silver intensification forms a sphere of heavy metal around the gold granule giving rise to an ultrastructural marker which can be easily seen even at low magnification. The ultrastructural details of the cells were well preserved and there was no significant background staining. The major advantage of the present IGS technique is that it permits a rapid and simultaneous evaluation of both the immunophenotype and the ultrastructural characteristics of cells.

The fine structure of HNK-1 (Leu7) positive cells

SummaryThe ultrastructural characteristics of HNK-1 (Leu7) positive cells, visualized with a peroxidase labelled anti-mouse IgM serum, were analysed. Our investigation demonstrates: 1) the majority of Leu7 positive cells has a low nuclear/cytoplasmic ratio (N/C), an irregular outline, a well developed smooth endoplasmic reticulum, parallel tubular arrays (PTA) and electron dense granules; 2) the minority of Leu7 positive cells has a high N/C, regular profiles and lacks electron dense granules. The presence of two distinct ultrastructural patterns within Leu7 positive cells may represent: 1) the expression of subsequent stages of cell differentiation; 2) two distinct Leu7 positive cell subpopulations.

Langerhans cells are not damaged in contact allergic reactions in humans.

Fifteen biopsies from contact allergic reactions in sensitized patients were taken at different time intervals after application of simple chemicals. Two different procedures for electron microscopy were done. No signs of damage were ever seen in Langerhans cells. The authors are therefore of the opinion that there is still no proof for a target role of Langerhans cells in contact allergic reactions in humans.