Human epidermal Langerhans cells express the ICAM-3 molecule. Immunohistochemical and immunoelectron microscopical demonstration.

Three ligands have been described for the leucocyte integrin LFA-1, namely intercellular adhesion molecule (ICAM)-1, ICAM-2, and ICAM-3. ICAMs show differences in tissue distribution and inducibility. The recently described ICAM-3 is highly expressed on resting lymphocytes, monocytes and neutrophils. Here, we demonstrate that the whole human epidermal Langerhans cell (LC) population expresses this molecule. Immunohistochemical staining of skin sections with an anti-ICAM-3 monoclonal antibody displayed reactivity with dendritic epidermal cells regularly distributed along the epidermis. Highly-sensitive immunoelectron microscopy procedures, performed on freshly suspended epidermal cells both at transmission and scanning electron microscopic levels, enabled demonstration that the whole LC population expresses cell surface ICAM-3. In contrast, keratinocytes and melanocytes were consistently negative. The prominent expression of ICAM-3 by resident LC could imply a crucial part for this molecule in leucocyte-intercellular interactions in the skin.

New insights suggesting a possible role of a heat shock protein 70-kD family-related protein in antigen processing/presentation phenomenon in humans.

A possible role of the peptide binding protein (PBP) 72/74 in antigen processing and presentation has been recently suggested in mice. In order to evaluate a possible analogous role of a PBP72/74-related protein in humans, immunoelectron microscope investigations, functional studies, and immunofluorescence analyses were performed on normal human peripheral antigen-presenting cells. We demonstrated that the determinant recognized by antiheat shock protein (HSP) 72/73 monoclonal antibody (MoAb) is constitutively expressed on the cell surface of monocytes as well as of B cells. Moreover, the capability of monocytes to present a recall antigen to T cells was significantly decreased when preincubated with an anti-HSP72/73 MoAb. These data add further strength to a potential role of a protein related to human PBP72/74 homologue in antigen processing and/or presentation. Finally, the capability of anti-HSP72/73 MoAb to impair the ability of fixed monocytes to present a synthetic peptide demonstrates that cell surface-localized PBP72/74-related protein could play a role in antigen presentation.

A diffusion immunogold procedure for accurate ultrastructural investigation of renal cell membrane epitopes.

A role for renal antigenic targets has been supposed and sometimes convincingly demonstrated in the development of various types of experimental glomerulonephritides. In this report we describe a reliable protocol for accurate ultrastructural investigation of antigens on the renal cell surface by means of a pre-embedding technique associated with colloidal gold staining. Sprague-Dawley rats were injected with a monoclonal antibody specific for a 90-kD cell membrane glycoprotein and killed 12 or 48 h later; after prefixation, renal fragments were cryoprotected and snap-frozen. Cryostat sections were incubated with a 5-nm colloidal gold-goat antimouse antibody, postfixed in osmium tetroxide reduced with potassium ferrocyanide and embedded in Durcupan ACM. At the glomerular level, gold granules were localized on the endothelial cell surface. In the proximal tubules uniform labelling was noticed on the brush border microvilli, followed by later marking of the basolateral membranes. By this pre-embedding immunogold method we obtained suitable histological preservation and fine resolution of the cell membrane immunoreactive sites. This procedure represents a useful tool for ultrastructural studies on the interaction of circulating antibodies with renal cell surface antigens.

A new polychrome stain and simultaneous methods of histological, histochemical and immunohistochemical stainings performed on semithin sections of Bioacryl-embedded human tissues.

We describe a new polychrome stain and simultaneous methods of histological, histochemical and immunocytochemical staining performed on sections from human tissues embedded in the new hydrophilic resin Bioacryl. The polychrome stain involves the sequential use of Harris' Haematoxylin, silver methenamine, Light Green and Eosin or Safranin dyes and provides a highly specific visualization of the overall cytological tissue architecture. When histochemical, immunocytochemical, and polychrome stains are performed together on the same section, crisp images are obtained, yielding simultaneous data of histochemical and immunological reactivities with clear tissue architecture.

Melanocytes freshly isolated from normal human skin express the cell membrane receptor for the adhesive glycoprotein thrombospondin.

Thrombospondin (TSP) is an adhesive protein with multiple binding sites, which is able to mediate several cell-to-cell and cell-to-matrix interactions, particularly through its cell membrane receptor (TSP-R). Because human keratinocytes are able to synthesize and express TSP, and as TSP is also localized at the dermal-epidermal junction in normal human skin, we questioned whether epidermal cells are able to bind available TSP, that is, to express TSP-R. To investigate this, we employed gold immunoelectron microscopy on epidermal cells freshly isolated from normal human skin; the TSP-R was detected by OKM5 monoclonal antibody. Epidermal cells showing ultrastructural characteristics of melanocytes were gold-stained on their plasma membrane, whereas keratinocytes, Langerhans cells and lymphocytes were unstained. Although functional studies are clearly necessary to clarify the role(s) played by the TSP-R on the cell surface of melanocytes, it is tempting to speculate that the TSP-R may be important for melanocyte adhesion to the dermal-epidermal junction and to keratinocytes. Such adhesion may not only subserve the steric localization of melanocytes, but also have important implications for those functional activities of melanocytes which have been shown to require close contact between these cells and adjacent keratinocytes and/or basement membrane components.

A new insight into the suggestion of a possible antigenic role of a member of the 70 kD heat shock proteins.

In the present study the cellular distribution of the inducible (hsp72) and constitutive (hsc73) forms of human 70 kD heat shock protein was evaluated. A weak reactivity of the anti-hsp72/hsc73 MoAb was found in the cytoplasm of the unstressed human epidermal cells, while stressed cells showed an enhanced reactivity at the cytoplasmic level and the expression of the molecules on the cell surface. Moreover, the antigenic properties of the two proteins were investigated by sequence analysis. Our findings provided evidence for at least three regions in the hsp72 which can be considered good candidates to represent T-immunogenic antigens. This data as well as the cell surface localization of the hsp72, could suggest an antigenic role of the hsp72.

A new acrylic resin formulation: a useful tool for histological, ultrastructural, and immunocytochemical investigations.

We describe a new formulation for a hydrophilic resin, mostly composed of glycol methacrylate and hydroxypropyl methacrylate and here referred to as bioacryl, that allows the performance of morphological and immunohistochemical investigations at both light and electron microscopic levels. Immunolocalizations performed on bioacryl-embedded tissues are characterized by high specificity with virtually absent background staining. Finally, the new resin yields satisfactory fine-structural preservation, resulting in ultrastructural images of better quality than those obtained with Lowicryl K4M.

Adhesion molecules on the plasma membrane of epidermal cells. IV. Immunolocalization of the intercellular adhesion molecule-1 (ICAM-1, CD54) on the cell surface of a small subpopulation of keratinocytes freshly isolated from normal human epidermis.

The intercellular adhesion molecule-1 (ICAM-1) is a cell membrane glycoprotein displaying a pivvtal role in cell-cell interactions in the immune system, and is a ligand for LFA-1, which is expressed on leukocytes. ICAM-1 is expressed in different cell types, including epithelial cells in a number of organs; the universal feature on all these cells is ICAM-1 induction from very low ICAM-1 constitutive levels on unstimulated resting cells to very high ICAM-1 levels triggered by mediators released at sites of inflammation. Therefore, since a strong expression of ICAM-1 on keratinocyte (KC) surface was recently demonstrated in various inflammatory skin lesions, in this investigation we asked whether very low ICAM-1 levels might be present on the plasma membrane of unstimulated KC in normal skin. Crude epidermal cell suspensions, freshly isolated from normal human skin, were immunolabeled by anti-ICAM-1 monoclonal antibody and stained by two highly sensitive ultrastructural detection systems, namely, the immunogold (5-nm-sized particles) method and the immunogold-silver-enhancement method. The quantitative analysis of 1000 KC scrutinized under the electron microscope revealed that 17.2% KC were ICAM-1-positive, although a density per KC section (midplane) of merely 18.92 +/- 13.02 5 nm-sized particles was scored (n = 100), indicating that the amounts of ICAM-1 moieties on this KC subset are presumably low. The ICAM-1 expression on a subset of KC in normal skin might account for the trafficking to and from normal epidermis of LFA-1-positive cells, including migrating Langerhans cells and occasional leukocytes.

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.

Adhesion molecules on the plasma membrane of epidermal cells. III. Keratinocytes and Langerhans cells constitutively express the lymphocyte function-associated antigen 3.

It is now becoming clear that a collection of adhesion molecules is required on the surface of epidermal cells (EC) to establish the cell interactions that are necessary for skin immunologic reactions. In previous studies, we showed that human resting Langerhans cells (LC) express at least two members of the "integrins" family of adhesive molecules, as well as the intercellular adhesion molecule-1, which is a member of the immunoglobulin-related superfamily of molecules. This latter family includes another adhesive moiety, namely, the lymphocyte function-associated antigen-3 (LFA-3), which is the ligand for the T-lymphocyte-associated CD2 molecule, and has a broad tissue and organ distribution. In the present investigation the colloidal gold-immunoelectronmicroscopy immunostaining system and a quantitative analysis of the labeling provided decisive evidence for the weak but clear LFA-3 expression on virtually all keratinocytes (KC) and LC freshly isolated from normal human skin. Such constitutive expression of LFA-3 molecule on EC may be relevant for a number of functional interactions between LFA-3-positive EC and CD2-positive T lymphocytes within the cutaneous environment.

The immunogold-silver staining procedure in the study of freshly suspended Langerhans cells at the transmission electron microscopic level.

The potential of an immunogold-silver staining for the study of human suspended Langerhans cells at the transmission electron microscopic level was evaluated. Cells were labeled, by using a preembedding technique, with 5-nm colloidal gold particles followed by silver enhancement. The use of small colloidal gold particles permits a detection of small quantities of antigen; the metallic silver deposition around gold granules gives rise to a large electron-dense marker which can be easily detected even at low magnification. Ultrastructural details were well preserved, and the background was not significant. The major advantage of the present immunogold-silver staining is that it enables to detect labeled cells easily, even when limited amounts of antigenic moieties are present on a low percentage of cells. Therefore, a rapid and simultaneous evaluation of both immunophenotype and ultrastructural details of investigated cells is allowed.

Lymphocytes of a patient with lymphoproliferative disease of large granular lymphocytes express high natural killer, ADCC, and LAK activity.

The clinical, morphological, immunological, and molecular features of a case of expansion of large granular lymphocytes (LGL) are reported. Surface marker analysis of peripheral blood and spleen mononuclear cells showed that the majority of these cells were CD3-, CD2+, CD16+, and Leu 7-. Ultrastructural characteristics of CD16+ cells revealed a low nuclear/cytoplasmatic ratio, irregularly shaped nucleus, and numerous cytoplasmatic granules. Functional studies showed reduced proliferative responses to mitogens (PHA, Con A, PWM) and high levels of natural killer (NK) activity as well as antibody-dependent cell cytotoxicity (ADCC) and lymphokine-activated killer (LAK) activities. Molecular analysis of the T cell receptor genes revealed a germline configuration of the beta, gamma, and delta genes; however, as for normal NK cells, delta-related mRNA transcripts were found. Three months from diagnosis, the patient developed profound thrombocytopenia and splenectomy was carried out with complete normalization of the platelet counts and of hematological values while LGL lymphocytosis persisted. Although no tools are available for studying the monoclonality of CD3- lymphoproliferative disease, the clinical course, the absence of chromosomal abnormalities, and a liver histology indicative of chronic active hepatitis suggest that LGL expansion in this patient could be part of a benign, possibly reactive, process.

Morphometric evaluation of CD16-positive cells with respect to CD2 antigen coexpression.

We examined morphometric as well as functional characteristics of CD16-positive human peripheral blood lymphocytes on the basis of the coexpression of the CD2 antigen. For morphometric analyses, nuclear area and cellular area were determined by counting line cross-points of a superimposed quadratic lattice test system overlying nuclei and the whole cell, respectively. Moreover, to evaluate the cellular villousity degree, the maximum inscrible circle and an irregular polygon were inscribed within cell profiles. The cytoplasm fraction included between the plasmalemma and the traced irregular polygon was considered as the villous portion of the cell. Finally, the NK capability was measured in a 6-hr 51Cr-release assay with human K-562 myeloid cells as targets. Within the CD16-positive cell population, the CD16-positive/CD2-negative cells seem to represent the most efficient NK cell subset. To the higher NK capability correspond a higher villousity degree and a lower nuclear area/cellular area ratio of the CD2-negative/CD16-positive subset, when compared with CD2-positive/CD16-positive cells.

Subsets of keratinocytes and Langerhans' cells express epitopes associated with suppressor-inducer capabilities in resting normal human epidermis.

In recent years two cell populations with down-regulatory immune capabilities have been identified in murine epidermis. The present report demonstrates that even in human epidermis at least two populations of cells expressing suppressor-inducer phenotypes (i.e. CD45R-positive) exist, namely small subsets of keratinocytes and Langerhans' cells, respectively. Highly specific and sensitive 5-nm colloidal gold-immunoelectronmicroscopic techniques were carried out using anti-CD45R monoclonal antibodies, on freshly isolated crude epidermal cell suspensions, and 4000 cells were scrutinized in the electron microscope. Over 2% of the total epidermal cell population was CD45R+. Subpopulation analysis revealed that approximately 2% of keratinocytes and about 5% of the total Langerhans' cell population showed strong gold-plasma membrane staining, whilst the remaining epidermal cells were absolutely negative. Heterogeneity of staining together with this somehow surprising distribution of CD45R positivity on non-lymphoid epidermal cells was confirmed by the negative controls. These CD45R+ Langerhans' cells and keratinocytes are clearly candidates for the cells which have been functionally demonstrated as being capable of inducing down-regulation responsiveness in the human epidermis. However, functional investigations are needed to clarify the roles of the CD45R+ keratinocyte and Langerhans' cell subsets in the modulation of cutaneous immune responses.

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.