Macrophage-associated Antigens Research Articles

Immunohistochemical identification of osteoclasts and multinucleated macrophages

Osteoclasts are bone-resorbing multinuclear cells derived from hematopoietic stem cells which are specialised to carry out lacunar bone resorption. The immunophenotype of giant cell-containing bone lesions in a wide range of osteoclast-like giant cells was similarly assessed. Both multinucleated macrophages and osteoclasts were found to express CD68. Multinucleated macrophages, but not osteoclasts, expressed GrB and Ki67. CD13+/CD14+/CD68+/GrB−/Ki67−/CD56− all giant-cell lesions noted in giant cells of bone. Giant cells have an osteoclast phenotype in most giant cell-rich lesions of bone, which do not express the macrophage-associated antigens GrB and Ki67. Our results indicate that they are formed from osteoclast precursors of mononuclear phagocyte.

High intratumoral macrophage content is an adverse prognostic feature in anaplastic large cell lymphoma

Macrophage infiltration has been associated with prognosis in several cancers, including lymphoma, but has not been assessed systematically in anaplastic large cell lymphoma (ALCL). The aim of the study was to correlate expression of the macrophage-associated antigens CD68 and CD163 with pre-therapeutic parameters and outcome in a cohort of treatment-naive ALCL patients. Pre-therapeutic tumour specimens from 52 patients with ALCL were included in a tissue microarray. The intratumoral macrophage content was assessed by immunohistochemical staining for CD68 and CD163, and quantified using digital image analysis. Anaplastic lymphoma kinase (ALK)-positive patients were significantly younger and had a favourable outcome compared with ALK-negative ALCL patients (median age: 42 versus 59years; P=0.008). However, ALK expression was not a significant predictor when adjusting for age. Although classical risk factors were distributed evenly between the compared groups, high intratumoral content of CD68 and/or CD163 correlated with poor outcome, in both univariate and multivariate analyses. High intratumoral CD163 content showed the strongest adverse association with both overall and progression-free survival in ALK-negative patients (P<0.001). A high content of intratumoral CD68- and/or CD163-positive macrophages correlates with an adverse outcome in ALK-negative ALCL.

Immunophenotypic Identification and Characterization of Tumor Cells and Infiltrating Cell Populations in Meningiomas

Meningiomas are primary tumors of the central nervous system composed of both neoplastic and other infiltrating cells. We determined the cellular composition of 51 meningioma samples by multiparameter flow cytometric (MFC) immunophenotyping and investigated the potential relationship between mRNA and protein expression levels of neoplastic cells. For immunophenotypic, morphologic, and cytogenetic characterization of individual cell populations, a large panel of markers was used together with phagocytic/endocytic functional assays and MFC sorting. Overall, our results revealed coexistence of CD45(-) neoplastic cells and CD45(+) immune infiltrating cells in all meningiomas. Infiltrating cells included tissue macrophages, with an HLA-DR(+)CD14(+)CD45(+)CD68(+)CD16(-/+)CD33(-/+) phenotype and high phagocytic/endocytic activity, and a small proportion of cytotoxic lymphocytes (mostly T CD8(+) and natural killer cells). Tumor cells expressed multiple cell adhesion proteins, tetraspanins, HLA-I/HLA-DR molecules, complement regulatory proteins, cell surface ectoenzymes, and growth factor receptors. Noteworthy, the relationship between mRNA and protein levels was variable, depending on the proteins evaluated and the level of infiltration by immune cells. In summary, our results indicate that MFC immunophenotyping provides a reliable tool for the characterization of the patterns of protein expression of different cell populations coexisting in meningioma samples, with a more accurate measure of gene expression profiles of tumor cells at the functional/protein level than conventional mRNA microarray, independently of the degree of infiltration of the tumor by immune cells.

Clonal relationship between langerhans cell histiocytosis and myeloid sarcoma

Langerhans cells (LCs) represent the main antigen capturing, processing and presenting dentritic cells (DCs) resident in epithelial surfaces including the skin or the oral mucosa, thus leading to either the induction of immune responses or tolerance. They are classically considered as myeloid-derived DCs sharing initial steps of their differentiation with monocytes. LC histiocytosis (LCH) and the more aggressive variant LC sarcoma (LCS) are considered as the neoplastic counterparts of this DC cell type. The immunoreactivity for macrophage-associated antigens in conjunction with CD1a, S-100 protein and langerin (CD207) represents a useful tool to establish the diagnosis of LCH in paraffin-embedded material.1 Rare cases of neoplasms with LC differentiation features developing in patients previously diagnosed with follicular lymphoma or acute lymphoblastic lymphoma/leukaemia (ALL) of T- and B-cell lineage have been reported. In these cases, identical clonal IgH- or TCRγ-gene rearrangements were detected in the H/DC neoplasms and the lymphomas consistent with a common clonal origin.2, 3, 4, 5 The development of the histiocytic dentritic/cell (H/DC) neoplasm was associated with progressive disease in most of these cases. Thus, it has been suggested that these tumours may represent a part of the clonal evolution of the underlying lymphomas.4 The development of a LCS in a patient with an acute B-ALL in complete remission for 9 years has been attributed to a transcription factor-induced transdifferentiation.5 Both neoplasms showed an identical IgH rearrangement, but different expressions of PAX5 and ID2.5

The immunophenotype of osteoclasts and macrophage polykaryons

AimOsteoclasts are multinucleated cells which are specialised to carry out lacunar bone resorption. Osteoclasts form part of the mononuclear phagocyte system, and immunophenotypic criteria for distinction from macrophage polykaryons include...

PII: S8756-3282(10)01858-2

The expression of some candidate osteoclast markers, tartrate-resistant acid phosphatase (TRAP), macrophage associated antigens (MΦAg), and vitronectin receptor (VNR) on foreign body giant cells (FBGCs) was investigated in peri-implant tissues of loosened total joint arthroplasties. Osteoclasts showed distinct staining characteristics. They were strongly TRAP-positive at tartrate concentrations of 50–200 mM and expressed VNR and a restricted range of MΦAg. In contrast, FBGCs were shown to be significantly heterogeneous. Significant numbers of FBGCs were TRAP-positive at a 100 mM tartrate concentration and some were more intense than osteoclasts. A population of FBGCs did not express MΦAg such as CD11b, but expressed VNR. It was demonstrated that these candidate osteoclast markers were also positive on FBGCs. These results have highlighted the difficulty in distinguishing these two cell lineages and suggested that there might be some uncertainty in defining osteoclast-like cells in culture studies.

PII: S8756-3282(10)01850-8

The expression of some candidate osteoclast markers, tartrate-resistant acid phosphatase (TRAP), macrophage associated antigens (MΦAg), and vitronectin receptor (VNR) on foreign body giant cells (FBGCs) was investigated in peri-implant tissues of loosened total joint arthroplasties. Osteoclasts showed distinct staining characteristics. They were strongly TRAP-positive at tartrate concentrations of 50–200 mM and expressed VNR and a restricted range of MΦAg. In contrast, FBGCs were shown to be significantly heterogeneous. Significant numbers of FBGCs were TRAP-positive at a 100 mM tartrate concentration and some were more intense than osteoclasts. A population of FBGCs did not express MΦAg such as CD11b, but expressed VNR. It was demonstrated that these candidate osteoclast markers were also positive on FBGCs. These results have highlighted the difficulty in distinguishing these two cell lineages and suggested that there might be some uncertainty in defining osteoclast-like cells in culture studies.

Sinushistiozytose mit massiver Lymphadenopathie (Rosai-Dorfman-Erkrankung)

Sinus histiocytosis with massive lymphadenopathy (SHML) is a rare, painless lymphoproliferative disorder of unknown origin with a usually benign course. About 40% of the patients show an extranodal involvement with skin being the most common site in 27% of these patients. We describe a patient with widespread disease involving the respiratory tract, kidneys and skin. Histopathology revealed the characteristic features of SHML with emperipolesis and immunohistochemical positivity of histiocytes for S100 and macrophage-associated antigens.

Histiocytic sarcoma with secondary involvement of the skin and expression of CD1a: evidence of indeterminate cell differentiation?

Histiocytic sarcoma is an exceedingly rare malignant neoplasm composed of cells with a monocyte/macrophage phenotype. In the current nosology of histiocytic neoplasms, histiocytic sarcoma is separate from indeterminate cell histiocytosis, a generally benign disorder characterized by proliferation of a CD1a+ and S-100+ population of cells lacking Birbeck granules usually limited to the skin. We present a case of histiocytic sarcoma in a 64-year-old man presenting as a peritonsillar mass and secondarily involving the skin. The malignant cells in the extracutaneous foci of disease expressed macrophage-associated antigens including S-100 but were CD1a-. The malignant cells in the skin coexpressed CD1a and S-100 but lacked ultrastructural features of Langerhans cells, findings indicative of indeterminate cells. We discuss the clinical and histopathologic differential diagnosis in association with prior reported cases of histiocytic sarcoma, particularly in cases involving the skin and cases expressing the Langerhans cell-associated antigen CD1a.

Expression of β2 integrins and macrophage-associated antigens in meningeal tumours

This study assessed the expression of leukocyte integrins and macrophage-associated antigens in meningiomas. Fourteen benign meningiomas, ten atypical/anaplastic meningiomas, two hemangiopericytomas and one solitary fibrous tumour (SFT) were included. Frozen sections were immunostained using antibodies directed against leukocyte integrins, CD68, CD14, CD2, CD1a, DRC1 and CD34. Their expression was evaluated semi-quantitatively. Ki67 positive cells were counted. Arachnoid membranes served as controls. Arachnoid cells expressed the beta2-integrin subunit and KP1. Beta2 was detected in the tumour cells of 14 meningiomas. In nine cases, this was associated with an alpha-integrin subunit. There was no statistical difference in the expression of beta2 between benign and atypical/anaplastic meningiomas. KP1 was constantly expressed by the tumour cells of meningiomas. It was not expressed by other meningeal tumours. CD34 was detected in the fibrous meningiomas, hemangiopericytomas and the SFT. In each tumour, macrophages were more numerous than T lymphocytes. There was no statistical difference in the density of macrophages and T lymphocytes between the benign and atypical/anaplastic meningiomas. There was no correlation between the Ki67 proliferation index and macrophage infiltration. Meningiomas, through the expression of leukocyte antigens, have a very particular phenotype. The expression of beta2 integrins could play a role in the attraction of immunocompetent cells in the stroma of meningiomas.

Sinus histiocytosis with massive lymphadenopathy: evidence for its relationship to macrophages and for a cytokine‐related disorder

Sinus histiocytosis with massive lymphadenopathy (SHML) or Rosai-Dorfman disease is a rare histiocytic disorder of unknown origin. Immunophenotypically the histiocytes of SHML express intensively the S100 protein and in addition a panel of macrophage-associated antigens. Their exact relationship to either monocytes/macrophages or immune accessory dendritic cells is, however, still controversial. In this report recurrent nodal and extranodal manifestations of SHML of a 70-year-old patient were analysed by differential phenotyping using a panel of monoclonal and polyclonal antibodies to macrophage and immune accessory dendritic cell related antigens and by applying nonradioactive in-situ hybridization. We conclude that stimulation of monocytes/macrophages via macrophage colony stimulating factor (M-CSF) leading to immune suppressive macrophages represents a main mechanism for the pathogenesis of SHML. The study further provides evidence for the monocyte/macrophage but not dendritic cell differentiation of SHML histiocytes.

Responses of macrophage-associated antigen-expressing cells in the dental pulp of rat molars to experimental tooth replantation

Bacterial infection of the dental pulp is a major hindrance to successful pulp regeneration after tooth replantation. This study examined how macrophages and class II molecule-expressing cells of the pulp respond to tooth replantation, on the hypothesis that they contribute to the defence and repair of the traumatized pulp. Upper right first molars of 5-week-old male Wistar rats were replanted immediately after extraction; contralateral untreated teeth served as controls. Pulpal cells expressing macrophage-associated antigens were immunohistochemically demonstrated at 0 h (immediately after the replantation) to 84 days postoperatively using antirat monoclonal antibodies OX6 (anti-class II molecules), ED1 (pan-macrophage antibody, reactive also with dendritic cells) and ED2 (anti-resident macrophages). Between 3 and 7 days postoperatively, ED1+ and OX6+ cells, but not ED2+ cells, were concentrated in areas of degeneration formed in the coronal pulp, and frequently showed a marked accumulation along the pulp–dentine border of the cuspal area. Confocal laser scanning microscopy revealed that some of the OX6+ cells with a dendritic profile extended several cytoplasmic processes into the dentinal tubules communicating with the enamel-free area at the tip of the cusp. From 14–84 days, approx. two-thirds of specimens exhibited pulp-tissue regeneration with increasing formation of reparative dentine. Following the formation of sound reparative dentine, cells positive to each antibody were distributed more centrally in the pulp than in the controls, and thus did not show any accumulation along the pulp–dentine border. However, in the other specimens where a bone-like hard tissue had formed in the pulp chamber, many ED1+ and OX6+ cells were still concentrated in the remaining pulp tissue and showed a marked accumulation along the pulp–dentine border. Few ED2+ cells were observed in these specimens. These findings suggest that, following tooth replantation, exudative macrophages are actively engaged in eliminating dentinal tubule-derived infectious stimuli and that class II molecule-expressing cells, most probably containing dendritic cells, are positioned strategically at the outermost portion of the injured pulp to monitor incoming antigens. The intensity of the pulpal defence reaction may be dependent on the status of hard-tissue formation, which influences the amount of incoming antigens.

Podocytes undergo phenotypic changes and express macrophagic-associated markers in idiopathic collapsing glomerulopathy

Podocytes undergo phenotypic changes and express macrophagic-associated markers in idiopathic collapsing glomerulopathy

Podocytes undergo phenotypic changes and express macrophagic-associated markers in idiopathic collapsing glomerulopathy

Collapsing glomerulopathy (CG), a severe form of focal segmental glomerulosclerosis (FSG), is characterized by tuft retraction and consolidation in numerous glomeruli and changes in podocyte morphology and topography. Other glomeruli are less affected. Collapsing glomerulopathy is also characterized by tubulointerstitial atrophy and fibrosis. The pathophysiology of the glomerular and tubulointerstitial lesions is poorly understood. We studied renal tissue of five Black and three White patients, all human immuno-deficiency virus (HIV) negative, with nephrotic syndrome, renal failure, and histological evidence of CG. Immunohistochemistry identified normal podocyte phenotypes by podocalyxin, vimentin and complement receptor 1 (CR1) labeling. Three monoclonal antibodies were used to further characterize podocyte epitopes: anti-CD68 clone KP1, anti-CD68 clone PG-M1 and anti-M130 clone M18 (Ber-MAC3). Light microscopy of collapsed glomeruli showed podocyte swelling, vacuolization, multinucleation, "cobblestone-like" alignment around the glomerular tuft, and pseudo-crescent formation in Bowman's space. In collapsed glomeruli, podocalyxin, vimentin and CR1 labeling tagged both normal and vacuolated podocytes still attached to the GBM, but labeling was not found in cobblestone-like podocytes or in podocytes detached from the GBM. Conversely, numerous podocytes undergoing detachment and shedding into Bowman's space expressed macrophagic-associated epitopes. Cells with macrophagic-associated epitopes clumped in cystically dilated tubules and were aligned in tubules of smaller caliber. Their appearance was that of viable cells. There was no morphologic indication that these cells expressing macrophage-associated antigens originated from outside the glomeruli or outside the tubules. We conclude that in CG podocytes detach from the GBM, lose their normal podocytic phenotype and acquire macrophage differentiation antigens. The presence of cells with such antigens in tubular lumens suggests that detached metaplastic podocytes progress along the tubule or, alternatively, that CG tubular cells also undergo metaplastic changes into macrophage-like cells.

Hepatic overexpression of MHC Class II antigens and macrophage-associated antigens (CD68) in patients with biliary atresia of poor prognosis

The pathogenesis of biliary atresia (BA) is still unknown. Progression to cirrhosis despite restoration of bile flow by successful portoenterostomy suggests that it is a progressive disease of the liver and biliary tree. Whether immunologic factors play any role in the development of this disease remains uncertain. Aberrant expression of major histocompatibility complex (MHC) Class II antigens of HLA-DR on hepatocytes and biliary epithelium is regarded as important in the progression of hepatocellular and biliary damage mediated by cytotoxic T cells. This study was undertaken to evaluate expression of MHC Class II antigen and macrophage-associated antigens (CD68) in liver of patients with biliary atresia to determine their prognostic usefulness and possible role in the pathogenesis of the disease. Liver biopsy specimens from infants with BA (n = 15), neonatal hepatitis (n = 3), and normal livers (n = 6) were studied using an indirect immunoperoxidase staining using antibodies against MHC Class II antigen and macrophage-associated antigens (CD68) as well as routine H&E and Masson's trichome stain. In patients with biliary atresia, the liver biopsy specimen was obtained at the time of Kasai portoenterostomy. Expression of HLA-DR antigens and CD68 antigens was either absent or minimal in normal liver biopsy specimens. There were a few HLA-DR antigens and a few CD68-positive cells around portal tracts in all patients with neonatal hepatitis and five of the seven biliary atresia patients with successful Kasai portoenterostomy. In contrast, there was strong expression of HLA-DR antigen in bile ductules, inflammatory cells, and adjacent damaged hepatocytes and marked CD68-positive macrophage infiltrate in the portal tracts as well as hepatic lobules in two patients with good prognosis and in all eight patients with bad prognosis. Hepatic expression of MHC Class II antigen and CD68 antigens correlated well with the severity of clinical course in patients with BA and may act as a prognostic factor in these patients.

Heterogeneity of Expression of CD68 and Other Macrophage-associated Antigens in Human Long-term Bone Marrow Culture

The authors have performed an immunohistochemical study of intact human long-term bone marrow cultures (hLTBMC) grown directly onto glass slides. Between 4 and 12 weeks of growth, such cultures consist of a complex stromal layer supporting foci of haemopoietic cells which undergo granulocytic and monocytic differentiation. As part of a large panel of antibodies employed to characterize monocytes and macrophages within hLTBMC, we included six different anti-CD68 reagents and three antibodies representing a putative new CD group recognizing a macrophage-associated antigen of 130 kDa molecular weight. These gave heterogeneous immunostaining patterns with macrophages and stromal myofibroblastic cells.

Age-related changes in the immunoreactivity of the monocyte/macrophage system in rat molar pulp

Defence reactions of the dental pulp potentially involve a variety of immunocompetent cells, particularly class II major histocampatibility complex (MHC)-expressing cells and macrophages. In order to examine how the immunodefence potential of the pulp changes as a function of age, phenotypic distribution of pulpal cells expressing immunoreactivity to monoclonal antibody ED1 (reactive with nearly all macrophages and dendritic cells) was examined immunohistochemically in the lower first molars of developing (new-born to 10-week-old), adult (14–24-week-old) and aged (1–1.5-yr-old) Wistar rats. During tooth development, increasing numbers of ED1 + cells in the pulp also expressed immunoreactivity to ED2 (reactive with a differentiation-related antigen present on resident macrophages). ED1 + and ED2 + cells were distributed throughput the pulp before the tooth formation was completed. OX6+ (class II MHC-expressing) cells started to increase in number shortly after the eruption of the tooth and the increase continued even after the tooth formation had been completed. In aged rats, the density of the pulpal ED1 + cells was maintained at a relatively high level, whereas a significant decrease in the density of OX6+ cells was observed. These results indicate that the density and composition of pulpal cells expressing macrophage-associated antigens vary with increasing age, which most probably is related to changes in the immunological defence potential of the pulp against infection.

MHC expression in nonlymphoid tissues of the developing embryo: Strongest class I or class II expression in separate populations of potential antigen-presenting cells in the skin, lung, gut, and inter-organ connective tissue

We define expression of major histocompatibility complex (MHC) antigens in the nonlymphoid tissues of the developing rat. Antibodies to class I heavy and light chains (b2-m), and to class II MHC proteins were used. Strongest MHC expression was by individual cells in the skin, lung, gut, and inter-organ connective tissue. The class I + and class II + cells were distinct populations, differing in morphology, distribution, and expression of macrophage-associated antigens. A nonimmunologic role for MHC proteins in development has been proposed. Yet the distributions and antigenic profiles lead us to emphasize immunologic functions that may be served by the early presence of MHC + cells outside the forming lymphoid organs. Potential contributions to establishment of extrathymic or maternal/fetal tolerance are discussed. Localization of strongest MHC expression to individual connective tissue cells of the developing organs, rather than parenchymal cells, is of clinical relevance to transplantation of fetal tissue.

Increased numbers of cytokeratin-positive interstitial reticulum cells (CIRC) in reactive, inflammatory and neoplastic lymphadenopathies: hyperplasia or induced expression?

A total of 291 enlarged lymph nodes showing a range of reactive-inflammatory processes, primary and metastatic neoplasms were studied to determine the distribution and immunoprofile of their cytokeratin-positive interstitial reticulum cells (CIRC) in comparison with normal nodes. In 258/291 nodes (89%), CIRC numbers were distinctly increased in the subcapsular, paracortical and, occasionally, in the medullary zones; often, these increased CIRC formed networks around follicles, sinuses and vessels. CIRC had comparatively small, irregularly shaped bodies and dendritic processes; occasionally, giant forms were noted. CIRC contained cytokeratins (CK) 8 and 18 but not 19, as shown by immunohistochemistry, and by gel electrophoresis with subsequent immunoblotting. They co-expressed vimentin consistently, alpha-smooth-muscle actin frequently, and desmin less frequently. They did not contain desmoplakins, Factor VIII, S-100, LCA, B and T lymphocyte- and macrophage-associated antigens, chromogranin A, synaptophysin or the A-80 glycoprotein. We found no clear correlation between the increased CIRC and given nodal disease processes. However, CIRC were most abundant in nodes free of but draining malignant tumours; bizarre CIRC assemblies were noted in HIV lymphadenopathy. CIRC appear to represent a subset of the so-called "fibroblastic reticulum cells" of lymph nodes. Their function remains undetermined; their increase in diverse lymphadenopathies suggests that they partake in nodal reactions to injury. It remains unclear whether the increase in CIRC relative number is due to proliferation or to CK gene induction processes but their presence and potential capability to undergo hyperplasia with dysplastic forms should alert pathologists to possible diagnostic pitfalls. In addition, we discuss that CIRC may undergo transformation and represent the "cell of origin" of certain CK-positive tumours restricted to lymph nodes.

The expression of osteoclast markers on foreign body giant cells

The expression of some candidate osteoclast markers, tartrate-resistant acid phosphatase (TRAP), macrophage associated antigens (MΦAg), and vitronectin receptor (VNR) on foreign body giant cells (FBGCs) was investigated in peri-implant tissues of loosened total joint arthroplasties. Osteoclasts showed distinct staining characteristics. They were strongly TRAP-positive at tartrate concentrations of 50–200 mM and expressed VNR and a restricted range of MΦAg. In contrast, FBGCs were shown to be significantly heterogeneous. Significant numbers of FBGCs were TRAP-positive at a 100 mM tartrate concentration and some were more intense than osteoclasts. A population of FBGCs did not express MΦAg such as CD11b, but expressed VNR. It was demonstrated that these candidate osteoclast markers were also positive on FBGCs. These results have highlighted the difficulty in distinguishing these two cell lineages and suggested that there might be some uncertainty in defining osteoclast-like cells in culture studies.