Ultrastructural localisation of sialoadhesin (siglec-1) on macrophages in rodent lymphoid tissues.

In previous studies it has been demonstrated that sialoadhesin is a macrophage-restricted adhesion receptor for lymphocytes and myeloid cells. It is under normal circumstances expressed by subpopulations of macrophages in lymphoid and haemopoietic tissues. In this study different immunoelectronmicroscopical techniques are used to investigate the ultrastructural localisation of sialoadhesin within the lymph node and spleen of rodents. The results show that sialoadhesin is selectively expressed by a subset of macrophages in peripheral lymphoid tissues. Sialoadhesin was localised predominantly on the plasma membrane and in particular in areas of intimate contact with lymphocytes, thereby visualizing putative local interaction between these cells. Interestingly, sialoadhesin was also detected in intracellular vesicles that were apparently taken up by macrophages. These findings are consistent with the putative role of sialoadhesin in local cell-cell interactions in lymphoid tissues. Surprisingly, sialoadhesin was also found at contact points of macrophages with other macrophages, sinus-lining cells and reticulum cells, suggesting that sialoadhesin also mediates interactions with these cell types.

The degree of branching of the glycans of alpha(1)-acid glycoprotein in asthma. A correlation with lung function and inflammatory parameters.

Alpha(1)-acid glycoprotein (AGP) is a plasma protein belonging to the group of acute-phase proteins. It contains five N-linked glycans which, depending on pathophysiologic state, differ in their degree of branching (i.e., in the relative proportions of di-, tri-, and tetraantennary glycans). Changes in the degree of branching of these glycans have been shown to affect various immunomodulatory properties of AGP. We wanted to investigate whether changes occur in the branching of AGP glycans in plasma and in bronchoalveolar lavage fluid (BALF) in asthma. For this purpose, we selected three groups of patients for study: patients with atopic asthma (AA), atopic nonasthmatic patients, and a group of patients with various interstitial lung diseases (ILDs). The plasma AGP concentration was normal in both atopic study groups, but was increased in ILD patients. In contrast, the branching of glycans of AGP was altered in subjects with AA, whereas it was normal in the other study groups. The presence of asthma symptoms correlated with the increased glycan branching of AGP in both plasma and BALF. Additionally, the degree of branching of AGP in BALF was related to FEV(1), to the provocative dose of histamine causing a 20% decrease in FEV (PD(20)), and to the number of eosinophils. In conclusion, asthma is accompanied by changes in the branching of AGP glycans that indicate an inflammatory reaction that differs markedly from a normal acute-phase response, in which decreased branching of AGP occurs.

Glucocorticoids modulate the development of dendritic cells from blood precursors.

Dendritic cells (DC) are professional antigen-presenting cells, capable of priming naive T cell responses. Glucocorticoids (GC) are frequently used in asthmatic patients. In this study we describe the effects of GC on the development and function of monocyte-derived DC (MoDC) in vitro and in vivo. Monocytes from healthy individuals were isolated and incubated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 6 days, to induce maturation into MoDC. To study the role of GC on DC differentiation in vitro cells were incubated with dexamethasone at different stages of MoDC development. At day 6 cells were characterized phenotypically by flow cytometry and functionally in an allogeneic mixed leucocyte reaction. To study the effect of GC in vivo patients with mild/moderate atopic asthma were selected. In one group no GC were used, whereas the other group used inhalation GC. MoDC from these patients were generated as described above and tested functionally. Incubation of MoDC or its peripheral blood precursors with dexamethasone decreased the accessory potency dose-dependently. The functional differences could not be explained by the changes in the expression of MHC II and the costimulatory molecules CD40 and CD86. The relevance of this mechanism was confirmed for the in vivo situation as well. MoDC from patients using inhalation GC showed a decreased accessory potency. These data suggest a modulatory effect of GC therapy at the level of the peripheral blood monocyte. The results indicate that GC influence DC development and function in vitro as well as in vivo.

MHC class II and invariant chain biosynthesis and transport during maturation of human precursor dendritic cells.

Dendritic cells (DC) are highly potent activators of the immune response. The precise mechanisms that give rise to the DC phenotype are not known. To investigate the mechanisms that contribute to the generation of the DC phenotype, precursor DC were freshly isolated from human blood and allowed to mature in vitro. These matured DC showed the phenotypical and functional characteristics of DC. Analysis of the MHC class II and invariant chain (li) biosynthesis revealed that upon maturation, class II synthesis was induced whereas li synthesis was significantly up-regulated. In mature DC, despite the presence of large amounts of li, export of MHC class II molecules from the endoplasmic reticulum was incomplete, up to 4 h after biosynthesis. Thus, MHC class II-li synthesis and transport in DC is highly regulated during maturation of DC. Analysis of the regulatory mechanisms may contribute to a better understanding of antigen-presenting capacities during the differentiation of DC.

Functional and phenotypic differences of monocyte-derived dendritic cells from allergic and nonallergic patients.

BACKGROUND: Several studies suggest a role for dendritic cell in the pathogenesis of allergic disease. OBJECTIVE: The purpose of this study was to compare function and phenotype of monocyte-derived dendritic cells (MoDC) from allergic asthmatic patients and healthy control subjects. METHODS: MoDCs were developed by incubating adherent monocytes for 5 days with IL-4 and granulocyte-macrophage colony-stimulating factor. Phenotype was assessed with flow cytometry, and the antigen-presenting function was assessed with the allogeneic mixed leukocyte reaction and an autologous specific antigen presentation. RESULTS: The morphology of the MoDCs was characteristic for immature dendritic cells. MoDCs from allergic asthmatic patients showed phenotypic differences in the expression of HLA-DR, CD11b, and the high-affinity receptor for IgE. A clearly enhanced accessory potential of MoDCs from atopic asthmatic patients in the mixed leukocyte reaction was also shown. Moreover, house dust mite-specific T-cell proliferation was increased. CONCLUSION: This study suggests the involvement of dendritic cells in the pathogenesis of atopic asthma by an increased immunostimulatory capacity of MoDCs.

The mannose receptor functions as a high capacity and broad specificity antigen receptor in human dendritic cells.

Dendritic cells, in contrast to B lymphocytes, must be able to efficiently internalize a diverse array of antigens for processing and loading onto major histocompatibility complex (MHC) class II molecules. Here we characterize the mannose receptor pathway in dendritic cells and show that mannose receptor-mediated uptake of antigens results in a approximately 100-fold more efficient presentation to T cells, as compared to antigens internalized via fluid phase. Immunocytochemistry as well as subcellular fractionation revealed the localization of the mannose receptor and MHC class II molecules in distinct subcellular compartments. The mannose receptor thus functions in rapid internalization and concentration of a variety of glycosylated antigens that become available for processing and presentation. This may contribute to the unique capacity of dendritic cells to generate primary T cell responses against infectious agents.

Mannose receptor mediated antigen uptake and presentation in human dendritic cells.

In an immature state, dendritic cells (DC) can capture antigen via at least two mechanisms. First, DC use macropinocytosis for continuous uptake of large amounts of soluble antigens. Second, they express high levels of mannose receptor that can mediate internalization of glycosylated ligands. We found that dendritic cells can present mannosylated antigen 100-1000 fold more efficiently than non-mannosylated antigen. Immunocytochemistry as well as subcellular fractionation demonstrated that the mannose receptor and MHC class II molecules were located in distinct subcellular compartments. These results demonstrate that the mannose receptor endows DC with a high capacity to present glycosylated antigens at very low concentrations.

Dynamics of nasal eosinophils in response to a nonnatural allergen challenge in patients with allergic rhinitis and control subjects: a biopsy and brush study.

BACKGROUND: Eosinophils are thought to play an important role in the symptomatology and pathophysiology of allergic rhinitis. Most quantitative studies on eosinophils in nasal mucosa have focused on the dynamics of eosinophils in the acute and late phases of the allergic reaction by using different cell sampling techniques. Little is known about the dynamics of eosinophils during a more prolonged period of allergen exposure and the activation of eosinophils induced by allergen challenge. OBJECTIVE: The aim of this study was to investigate the dynamics and activation of the eosinophils in the nasal mucosa of patients with an isolated grass pollen allergy during an out-of-season 2-week allergen exposure, mimicking the natural grass pollen season. METHODS: Seventeen patients with isolated grass pollen allergy and four control subjects were challenged daily with the allergen during a 2-week period in the winter. Nasal brush specimens were obtained before provocation and each day during the provocation period. Biopsy specimens were obtained once before, six times during, and once after the provocation period. Preparations made of nasal brush and nasal biopsy specimens were stained with the monoclonal antibody BMK 13 and Giemsa stain as paneosinophil markers and with the monoclonal antibody EG2 to identify activated eosinophils. RESULTS: We found significant increases in the total number of eosinophils and the number of activated eosinophils in the epithelium and lamina propria. These increases were most explicit in the second week. BMK 13 was found to be a paneosinophil marker superior to Giemsa staining. CONCLUSION: Eosinophils are not only involved in the acute and late phases of the allergic reaction but are probably even more involved in the chronic phase.

Immunohistochemical, morphological and ultrastructural resemblance between dendritic cells and folliculo-stellate cells in normal human and rat anterior pituitaries.

Immunolabeling of cryo-sections of human anterior pituitaries obtained at autopsy, and of cryo-sections of freshly prepared rat anterior pituitaries, with a panel of monoclonal antibodies against markers of the monocyte/dendritic cell/macrophage lineage, reveals in both species a characteristic pattern of immunopositive cells, among which many cells with dendritic phenotype are found. Cells characterized by marker expression of MHC-class II determinants and a dendritic morphology are present in both human and rat anterior pituitary. Markers characteristic of dendritic cells such as the L25 antigen and the OX62 antigen were present in anterior pituitaries from human and rat respectively. The population of MHC-class II expressing dendritic cells of the rat anterior pituitary is compared at the ultrastructural level with the folliculo-stellate cell population, which cell type has been previously characterized by its distinctive ultrastructure and immunopositivity for the S100 protein. Using immuno-electron microscopy of rat anterior pituitaries fixed with periodate-lysine-paraformaldehyde, we were able to distinguish non-granulated cells expressing MHC-class II determinants, whereas no MHC-class II expression was found in the granulated endocrine cells. Using double immunolabeling of cryo-sections of these rat AP with 25 nm and 15 nm gold labels, we demonstrated an overlap between the populations of MHC-class II-expressing and S100 protein-expressing cells. Furthermore, MHC-class II-expressing and S100-positive cells showed ultrastructural characteristics that have been previously ascribed to folliculo-stellate cells. At the light microscopical level in the rat AP, a proportion of 10 to 20% of the S100-positive cells was found immunopositive for the MHC-class II marker OX6. In the human AP, S100-positive folliculo-stellate cells and cells expressing the leukocyte common antigen CD45 were found to occupy predominantly different tissue compartments in the human anterior pituitary, namely the epithelial parenchyme cords and perivascular compartments respectively. A proportion of CD45+ cells was found in the parenchyme compartment and, vice versa, indicating an overlap of the tissue compartments in which both cell types occur. However, at the light microscopical level we could not find cells expressing both the S100 and CD45 marker. The present finding of a proportion of S100-positive pituitary cells with ultrastructural and immunohistochemical characteristics of both dendritic cells and folliculo-stellate cells, confirms the suggested heterogeneity of the latter cell group with respect to their ultrastructural phenotype and putative function. The possibility of a myeloid origin of part of the folliculo-stellate cell group in the AP, is discussed and might elucidate some of the discrepancies in the literature concerning the embryological origin of this cell group.

Analysis of dendritic-cell-induced primary T-cell responses between HLA genotypically identical individuals.

DCs are known for their superior antigen-processing and antigen-presenting capacities. They are capable of processing intact protein: either endocytosed exogenous proteins or newly synthesized endogenous viral and bacterial proteins. They are potent inducers of primary T-cell immune responses such as in allogeneic MLRs. It is also known that DCs can provide a strong stimulus for autologous T-cell proliferation. So far no information exists on the capacity of DCs to induce primary mH antigen-specific T-cell responses. Therefore, we investigated whether human DCs, isolated from peripheral blood, were able to generate specific T-cell responses between MLR-negative HLA genotypically identical individuals in vitro. To this end, unfractionated cells, monocytes, and B cells were assayed in parallel with DCs to compare their capacity to activate unprimed T cells in a primary MLR. DCs indeed induced significant proliferation between HLA genotypically identical siblings, whereas the other APCs were unable to evoke any T-cell response at all. As expected, besides these allogeneic T-cell responses, autologous T-cell responses were initiated by the DCs as well. Nonetheless, despite further detailed analyses of the responding T cells, neither proliferative nor cytotoxic mH antigen-specific reactivities could yet be detected using the stimulation protocols described herein.

Migratory properties and functional capacities of human skin dendritic cells.

The different cell types which migrated 'spontaneously' out of human skin explants during different periods of culture were characterized. Before culture, CD1a+ dendritic cells were observed not only in the epidermis but also in the dermis, whereas CD1b+ dendritic cells were present exclusively in the dermis. The populations of migrating cells were harvested and phenotyped on 3 successive days of culture. They always contained high percentages of CD1a+ cells. The other cells that migrated were T cells and macrophages. A relatively high proportion of the CD1a+ cells that migrated during the first 24 h culture period was also CD1b+. The number of cells which were positive for both CD1a and CD1b decreased in the following 2 days of culture. However, the purified CD1a+ cell populations isolated on the 3 consecutive days did not show any difference in their capacity to stimulate allogeneic T cells. The CD1a+ cells possess potent allo-activating capacities that are independent of whether or not they are positive for CD1b+. Three days after culture about half of the CD1a+ cells were still present in the epidermis and dermis, but no CD1b+ cells could be detected in the dermis. This suggests that the CD1b+ cells represent a population of active migrating cells.

Interleukin-8 production by human mesothelial cells after direct stimulation with staphylococci.

Mesothelial cells (MC) are able to produce interleukin-8 (IL-8) after stimulation with IL-1 beta or tumor necrosis factor alpha. The aim of our study was to investigate whether MC are able to produce IL-8 after direct stimulation with clinically relevant bacteria. We observed a significant IL-8 response by the MC which were directly stimulated with viable staphylococci.

Cellular composition of milky spots in the human greater omentum: an immunochemical and ultrastructural study.

BACKGROUND: Milky spots in the greater omentum of some animals are well organized perivascular infiltrates of leucocytes, and are considered to have characteristics of secondary lymphoid tissue. To determine whether milky spots in the human greater omentum can also be regarded as secondary lymphoid tissue, we studied milky spots in an unstimulated state. METHODS: Patients were selected on the basis of absence of disease in the peritoneal cavity that might influence the state of the milky spots. Using monoclonal antibodies against macrophages, B-lymphocytes and T-lymphocytes, and immunoperoxidase labeling, the number of these cells and their location in milky spots were studied by light microscopy. However, the stromal components of the greater omentum, especially those within the milky spots, were studied by electron microscopy. RESULTS: Milky spots in the human greater omentum are relatively uniform vascularized accumulations of mononuclear cells comprising macrophages (67.9% +/- 9.4, mean +/- standard deviation), B-cells (10.1% +/- 3.4), T-cells (10.2% +/- 3.7), and mast cells. However, no special B-cell and T-cell areas could be distinguished. On the ultrastructural level it was demonstrated that macrophages are present in different stages of maturation and can enter or leave the milky spots. Furthermore, no cells characteristic of secondary lymphoid organs, such as interdigitating cells or follicular dendritic cells, were seen. CONCLUSIONS: These data indicate that unstimulated milky spots in the human greater omentum are to a great extent just a preformed specific accumulation of primarily macrophages within the stroma of the greater omentum, and therefore, cannot be regarded as true secondary lymphoid tissue. Milky spots could serve as a gateway for, as well as a provider of peritoneal macrophages when the intra-abdominal status so requires. Finally, the data from this study are compared with the data of other studies of human milky spots and those in animals.

Localization and morphology of antigen-presenting cells in the adenoid of children with otitis media with effusion.

This study describes the localization of antigen-presenting cells (APC) in the different compartments in adenoids of children with otitis media with effusion (OME) and "healthy" children and adults. It is shown that the adenoid of children with OME contains a relatively high number of OKT6 and RFD1 positive cells. Moreover, accumulations of these cells are present in the extrafollicular areas of these adenoids. Very occasionally dendritic cells in the epithelial microenvironment contain Birbeck granules, indicating characteristic Langerhans cells. These OKT6 positive cells are not seen in the adenoids of the control group. Our results clearly show a relation between the presence of dendritic cells and the occurrence of OME. No differences are seen in localization and morphology of the APC in the studied adenoids.

Isolation and characterization of migratory human skin dendritic cells.

A method is described to isolate and characterize human skin dendritic cells (DC). This method is based on the migratory capacities of these cells. The cells migrated 'spontaneously' out of split-skin explants into the medium during a 24-h culture period and contained up to 75% CD1a+ cells. After removal of co-migrated T cells and macrophages, the highly enriched (> 95% CD1a+) DC showed potent allo-antigen-presenting capacities. About 25% of the CD1a+ cells were also positive for the dermal DC marker CD1b, whereas only 15-20% of the cells contained Birbeck granules, the characteristic cell organelle of the epidermal Langerhans cell. Before culture, CD1a+ DC were observed on cryostat sections not only in the epidermis but also in the dermis. After culture, the number of CD1a+ cells in both epidermis and dermis had decreased. Not all the cells had migrated during the culture period; some CD1a+ cells could still be detected in the epidermis and dermis after culture. Thus, using this method, potent allo-stimulating CD1a+ cells, migrating from both epidermis and dermis, can be obtained without the use of enzymes.

Enrichment and characterization of dendritic cells from human bronchoalveolar lavages.

In the present study about 0.3% to 1.6% of human bronchoalveolar lavage (BAL) cells were identified as typical dendritic cells (DC), having an irregular outline, lobulated nucleus, and clear distinguishable acid phosphatase activity or EBM11 (anti-CD68) reactivity in a spot near the nucleus. After DC enrichment, using transient adherence to plastic, FcR-panning, and a density metrizamide gradient, a population containing 7-8% typical DC was obtained. This DC-enriched low density fraction, containing the highest percentages of DC, very strongly induced T cell proliferation in an allogeneic mixed leucocyte reaction (MLR), which was significantly higher than that induced by other partly (un)fractionated BAL cells. These data indicate that DC seem to be the major accessory cells in the BAL fluid, and therefore may be important in the regulation of T cell immune responses in the lung.

Double labelling of major histocompatibility complex molecules and lysosomal protein lamp-1 on human dendritic cells.

In this study, double labelling for major histocompatibility complex (MHC) class I and class II molecules and for MHC molecules and the lysosomal membrane protein lamp-1 on ultrathin cryosections of dendritic cells isolated from human peripheral blood was performed. The plasma membrane proved to be positive for both MHC class I and MHC class II molecules and was labelled for only a very few lamp-1 molecules. MHC class I and MHC class II molecules did not co-localize intracellularly except in some peripherally located vesicles. However, many MHC class II-labelled vesicles were present in a juxtanuclear position but only some of them were co-labelled for lamp-1. These results indicate the presence of a separate, non-lysosomal compartment for class II molecules in dendritic cells.

Migration of dendritic cells into the draining lymph nodes of the lung after intratracheal instillation.

The migration of dendritic cell (DC)-enriched populations and alveolar macrophage (AM) populations isolated from PVG RT7.2 rats was studied after local administration to recipient PVG RT7.1 rats. The monoclonal antibody His41, which is directed against the common leukocyte antigen of the RT7.2 rat, was used to detect migrated cells. Injection of the splenic DC and AM subcutaneously into the footpads resulted in migration of both cell types to the popliteal lymph nodes after 24 h. DC located predominantly in the T cell-dependent areas, whereas AM located more in the medulla and medullary cords and spread throughout the outer cortex area. After intratracheal instillation of splenic DC, these cells were found predominantly in T cell-dependent areas of the draining lymph nodes of the lung after 24 h. In contrast, AM did not migrate to the draining lymph nodes after intratracheal instillation. Combined with those from earlier studies, these data show that DC present in the alveolar lumen may pick up airborne antigen and migrate to the draining lymph nodes of the lung, where they can induce primary T cell responses.

Ultrastructural study of macrophages of the rat thymus after cyclosporin treatment.

Young adult male Wistar rats were given 30 mg per kg of cyclosporin (CS) for 21 consecutive days. After CS treatment thymic medulla virtually disappears and the thymus is almost entirely composed of cortical tissue. Macrophages are scattered throughout the thymic cortex. These cells are very large, rounded, with inconspicuous prolongations and euchromatic nucleus with prominent nucleoli. These cells are loaded with lipid bodies and vacuolar cytoplasmic inclusions of different size and diverse content, but very rarely contain phagocytosed lymphocyte remnants. The cytoplasm between inclusions has very active aspect with abundance of polyribosomes, granular endoplasmic reticulum and vesicles. Mitoses of lymphocytes in the vicinity of macrophages are frequently seen. We discuss the morphological similarity between cortical macrophages of CS-treated thymus and macrophages of cortico-medullary zone (CMZ) of the normal rat thymus, as well as functional significance of described morphological characteristics of this type of thymic macrophages, which probably reflect the metabolism of arachidonic acid.