Distinct subpopulations of elicited human macrophages in peritoneal dialysis patients and women undergoing laparoscopy: a study on peroxidatic activity.

The endogenous peroxidatic activity (PA) pattern of peritoneal macrophages from 24 continuous ambulatory peritoneal dialysis (CAPD) patients and from five healthy women undergoing laparoscopy was studied. In general, the macrophages showed two different PA patterns in vivo: exudate and negative macrophages. However, two of 24 CAPD patients showed resident macrophages, the first described in vivo in man. Since, in general, the examined human peritoneal macrophages are exudate and PA-negative, this suggests, in accordance with the animal model system, that a chronic sterile inflammation exists in the peritoneal cavity of CAPD patients and healthy women undergoing laparoscopy. After 2 hr culture, blood monocytes and peritoneal macrophages transformed into cells with the characteristics of exudate-resident and resident macrophages, so isolation procedures that include short periods of culture can change the developmental stage of human monocytes and macrophages.

Separation of alveolar macrophages and dendritic cells via autofluorescence: phenotypical and functional characterization.

In a previous study we demonstrated that an increase of monocytes and dendritic cells (MDC) was In the current study, the bright autofluorescence of alveolar macrophages (AMs) was used to separate them efficiently from the MDC. Sorting of freshly isolated BAL cells resulted in a high-autofluorescent fraction, consisting predominantly of AMs, and a low-autofluorescent fraction containing the MDC, lymphocytes, and granulocytes. Thus, a clear separation between suppressive (AM) and stimulating (MDC) activity was obtained as shown in antigen-specific T cell responses. Flow cytometric parameters, density fractionation, and a series of ED monoclonal antibodies raised against rat macrophage antigens showed that both AMs and MDC were diverse populations. After overnight culture, more than 80% of an MDC population with a density range of 1.065-1.079 changed to a lower density (< 1.056) and morphologically developed into DCs with many processes. Concomitantly, monoclonal antibody ED1 expression changed from a granular pattern to a discrete juxtanuclear spot localization.

Localization of pepsinogen (A and C) and cellular differentiation of pepsinogen-synthesizing cells in the human gastric mucosa.

The localization of pepsinogens (PG A and PG C) was studied intracellularly in human gastric biopsies embedded in Lowicryl K4M, using affinity-purified antibodies and protein A-gold. The homogeneous secretory granules of the chief cells contained both PG A and PG C, as was proved by serial sections. Identical reaction was also seen in the core of the biphasic mucous neck cell granules, whereas the mantle did not label. The rough endoplasmic reticulum (RER) and Golgi complex of the chief cells and mucous neck cells contained ample label. Transitional cells identified by the presence of granules of both chief cells and mucous neck cells were recognized. This type of mucous neck cell is thought to transform into a chief cell. However, an increase of RER that could explain an increase of the pepsinogen production was not observed. A mixture of these granules was also found in cells morphologically characterized as young parietal cells, suggesting a common precursor for these three cell types. These observations make the transformation from mucous neck to chief cells questionable. Antral gland cells contained only PG C, as was shown in serial section, too.

The development of the resident pattern of endogenous peroxidatic activity in mouse peritoneal macrophages coincides with the expression of the differentiation antigen F4/80. A combined method for immunoperoxidase labeling and endogenous peroxidase cytochemistry.

In the mouse the maturation of mononuclear phagocytes was followed by comparing the ultrastructural pattern of endogenous peroxidatic activity (PA) at different time points during an acute peritonitis induced with newborn calf serum (NCS). Exudate macrophages demonstrate PA only in lysosomes, whereas resident macrophages have reaction product in the nuclear envelope (NE) and rough endoplasmic reticulum (RER). Transitional cells called "exudate-resident" macrophages have PA in the NE, RER, and some virginal lysosomes. In addition, peroxidase-negative macrophages were also present. A monoclonal antibody, F4/80, that specifically recognizes a mouse macrophage differentiation antigen (Austyn and Gordon, 1981) was used in this study. To compare the indirect immunoperoxidase labeling of this antigen and the endogenous peroxidase cytochemistry on the cellular level, a combined method was developed. Finally, the method was applied to the peritoneal cells at different time points after intraperitoneal injection of NCS in mice. The relative numbers of cells demonstrating the different patterns of endogenous PA and the proportions of each subpopulation expressing F4/80 antigen were estimated. It appeared that the expression of the antigen F4/80 coincides with the development of the resident pattern of PA. It is therefore concluded that the macrophages with the resident pattern of endogenous peroxidase are derived from monocyte-like exudate macrophages. In addition, the results indicate that both exudate-resident macrophages and at least a part of the peroxidase-negative macrophages are transitional forms.

Ontogeny of the rat thymus micro-environment: development of the interdigitating cell and macrophage populations.

The ontogeny of the rat thymus micro-environment and in particular the development of the interdigitating cell (IDC) and macrophage (M phi) populations has been studied. At day 15 of fetal life the thymus consisted of an epithelial primordium in which some Thy-1 positive thymocytes were present around local capillaries in a central area. At day 16 some Ia positive cells, which could not be further identified, and some monocyte-like M phi were observed in the central area. From day 17 the thymus became lobulated by ingrowth of small blood vessels with perivascular connective tissue from the surrounding capsule. An Ia positive epithelial reticulum developed which became populated by increasing numbers of thymocytes. Some strongly acid phosphatase positive M phi were present from this stage of development. From day 19 cortical and medullary areas could be distinguished in the thymus. The cortex consisted of an Ia positive epithelial reticulum in which closely packed thymocytes and scattered M phi were present. The medulla demonstrated a confluent Ia staining and consisted of an epithelial reticulum in which thymocytes, strongly non-specific esterase positive IDC and an occasional M phi were present. Also highly phagocytic IDC-like cells were observed in the medulla, most likely they comprise the population of differentiating IDC. Thymocyte proliferation areas, which were strongly pyroninophilic, were observed from day 21 in the cortex, just beneath the surrounding connective tissue capsule. A distinct cortico-medullary region with many M phi was present one week after birth. From this stage the IDC and M phi distribution was comparable with older thymi.

Ontogeny of the rat thymus micro-environment: development of the interdigitating cell and macrophage populations.

The ontogeny of the rat thymus micro-environment and in particular the development of the interdigitating cell (IDC) and macrophage (M phi) populations has been studied. At day 15 of fetal life the thymus consisted of an epithelial primordium in which some Thy-1 positive thymocytes were present around local capillaries in a central area. At day 16 some Ia positive cells, which could not be further identified, and some monocyte-like M phi were observed in the central area. From day 17 the thymus became lobulated by ingrowth of small blood vessels with perivascular connective tissue from the surrounding capsule. An Ia positive epithelial reticulum developed which became populated by increasing numbers of thymocytes. Some strongly acid phosphatase positive M phi were present from this stage of development. From day 19 cortical and medullary areas could be distinguished in the thymus. The cortex consisted of an Ia positive epithelial reticulum in which closely packed thymocytes and scattered M phi were present. The medulla demonstrated a confluent Ia staining and consisted of an epithelial reticulum in which thymocytes, strongly non-specific esterase positive IDC and an occasional M phi were present. Also highly phagocytic IDC-like cells were observed in the medulla, most likely they comprise the population of differentiating IDC. Thymocyte proliferation areas, which were strongly pyroninophilic, were observed from day 21 in the cortex, just beneath the surrounding connective tissue capsule. A distinct cortico-medullary region with many M phi was present one week after birth. From this stage the IDC and M phi distribution was comparable with older thymi.

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.

Effect of Bacillus Calmette-Guérin inoculation on numbers of dendritic cells in bronchoalveolar lavages of rats.

Dendritic cells (DC) are present in all lymphoid tissues and are widely distributed in the airway epithelium and lung parenchyma. In this study DC were morphologically and cytochemically identified in normal rat bronchoalveolar lavage (BAL), although in very low percentages. Furthermore, the total population as well as different Percoll density fractions demonstrated poor antigen-presenting capacity and even suppressed antigen-specific stimulation by rat splenic DC. In contrast, when an inflammatory response was induced by intratracheal inoculation of Bacillus Calmette-Guérin (BCG), an increase of Ia-positive cells, containing high percentages of monocytes and DC (MDC) was found. In BAL, DC increased about 25 times within 48 h after BCG inoculation. These BCG-induced BAL cells as well as the different density fractions showed a high antigen-presenting capacity at low concentrations. However, at higher concentrations they were suppressive, except for the highest density fraction which lacked alveolar macrophages (AM). These results indicate that the increased numbers of Ia-positive MDC during an inflammatory reaction are very likely responsible for antigen presentation in vitro. In contrast, AM suppress the antigen-specific T cell proliferation in a concentration dependent manner.

Characterization of non-lymphoid cells in Peyer's patches of the rat.

The aim of the present study was to correlate in vitro and in situ observations on nonlymphoid cells in Peyer's patches (PP) of the rat. By carrying out enzyme cytochemical reactions (acid phosphatase, APh, and non-specific esterase, NSE) and immunocytochemistry (Ia antigen staining) on cell suspensions and cryostat sections of PP, two classes of nonlymphoid cells could be distinguished. These were (1) strongly APh- and NSE-positive cells without or with a slight Ia membrane staining and (2) strongly Ia-positive cells with a weak APh and NSE activity. The first cell class comprised the classical macrophages which, except for tingible body macrophages, were glass-adherent. The second cell class was non-adherent and comprised dendritic (interdigitating) cells. The role of this Ia-positive non-adherent cell population was discussed, and a hypothesis was presented on the relation between mononuclear blood cells, veiled cells, and interdigitating cells in PP.

Relation between langerhans cells, veiled cells, and interdigitating cells.

Tissue macrophages are bone marrow derived mononuclear cells which play an important role in the immune response, especially as antigen presenting cells. They comprise a heterogeneous population of cells with phagocytic activity. On morphological functional and cytochemical criteria it is likely that the Langerhans cell (LC) in the epidermis, the veiled cell (VC) in the afferent lymph and the interdigitating cell (IDC) in the thymus dependent area of peripheral lymphoid organs and the thymus medulla belong to a subpopulation of the macrophages. They are low phagocytic, Ia positive and are highly immunogenic. VC and IDC may contain Birbeck granules, the characteristic organelles of the LC, suggesting a relationship between these cell types. An epithelial micro-environment as present in the skin epidermis and the thymus is necessary for the induction of these granules, which appear to have no immunological significance. In a scheme the development from monocyte into LC or into VC and subsequently IDC is postulated. Probably VC transport antigen from the skin area via the afferent lymphatics into the draining lymph node. In the thymus dependent area of this organ they present this antigen to T cells and mature into IDC. IDC in the medullary area of the thymus may also be involved in antigen presentation to immunocompetent T cells. However, in this central lymphoid organ a function in instruction of helper T cells may not be excluded.

Human peripheral blood dendritic cells concentrate in contrast to monocytes intracellular HLA class II molecules in a juxtanuclear position.

The localization of histocompatibility antigens of the HLA-D locus in dendritic cells (DC) and monocytes (Mo) isolated from human peripheral blood was investigated. Functionally DC were characterized by their capacity to act as strong stimulators in an allogeneic mixed leucocyte reaction. In cytospins, DC were differentiated from Mo by dendritic morphology, strong HLA class II and moderate RFD1 expression on the plasma membrane and acid phosphatase activity in a juxtanuclear spot. Ultrathin cryosections showed that DC had a heavily labelled plasma membrane for HLA-D. In addition, these antigens were found in intracellular vesicles predominantly located in the juxtanuclear zone. This pattern of labelling was not seen in Mo. Obviously, DC concentrate intracellular class II antigens in the same area as lysosomal activity. These results may indicate that this juxtanuclear area is a center of antigen processing in DC.

Langerhans cells in nasal mucosa of patients with grass pollen allergy.

Langerhans cells (LC) are known to be present in squamous epithelia of the human body. They are dendritic cells (DC) and characterized by the presence of Birbeck granules (BG). In previous studies, DC positive for CD1a and HLA-DR were found in the cylindrical epithelium and the lamina propria of the nasal mucosa. In our study, more CD1a cells occurred in the allergic patients than in the non-allergic controls. In a combined light microscopy (LM) and electron microscopy (EM) study, biopsies of nasal mucosa in allergic patients were studied. We used monoclonal antibodies against CD1a and HLA-DR, to identify DC in LM cryostat sections. The presence of BG identified most of the intra-epithelial DC as LC on the EM level, whereas a minority of DC in the lamina propria also contained BG. The ultrastructure of LC and DC in the ciliated cylindrical epithelium and the lamina propria is compared.

Localization of class II molecules in storage vesicles, endosomes and lysosomes in human dendritic cells.

Class II molecules are a prerequisite for antigen presentation. We studied whether class II molecules can be found in the endocytic and/or lysosomal route of dendritic cells (DC), which are very potent antigen-presenting cells. Therefore first immunolabelling for HLA-DR alpha chain was applied on ultrathin cryosections of cells of which plasma membrane HLA-DR/DQ molecules were labelled in suspension, followed by incubation with the endocytic marker BSA-gold. Second, immunolabelling for HLA-DR alpha chains was applied on ultrathin cryosections of cells on which enzyme cytochemistry for acid phosphatase (APh) was performed to see whether the class II positive vesicles belong to the lysosomal compartment. Third, this immunolabelling was applied on cryosections of cells pretreated with the protein synthesis inhibitor cycloheximide (CHX) to see whether the class II positive vesicles are derived from biosynthesis. We found limited uptake of BSA-gold into endosomes and lysosomes, some of which also contained endocytozed HLA-DR/DQ. APh and HLA-DR were observed in the same vesicles but also vesicles containing either HLA-DR or APh were found. However, many class II positive vesicles were found, which were apparently not accessible to exogenous molecules. Moreover, the amount of class II positive vesicles decreased after treatment of the cells with CHX, suggesting that these vesicles form part of the biosynthetic route. These results imply that there is a cluster of class II positive vesicles, probably a storage compartment, that has connections with the lysosomal system. The concentration of lysosomes and class II positive vesicles in the juxtanuclear area of DC is probably of crucial importance in the processing of antigens.

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.

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.

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.

Differential effect of cyclosporin application on epithelial cells of the rat thymus. Immunohistochemical study.

Young adult male Wistar rats were given 30 mg per kg of cyclosporin (CS) for 21 consecutive days. A panel of monoclonal antibodies was used to study the phenotype of thymic epithelial cells. After treatment with CS, subcapsular epithelial cells, although phenotypically similar to medullary epithelial cells, were changed in a similar manner to phenotypically distinct epithelial cells of the deep cortex. These cells became enlarged, stockier and their cytoplasmic prolongations were thicker and coarser compared with control cells and their number was not decreased. In contrast, the number of medullary epithelial cells was markedly reduced, whereby the cells with the most mature phenotype (CK8+10-19- and CK8+10+19-) were the most prominently depleted. No proliferation of thymic epithelial cells was detected as monitored by incorporation of 5-bromodeoxyuridine.