[History and perspectives of the monocyte-macrophage system]. / Historie und Perspektive des Monozyten-/Makrophagensystems.

The macrophage cell system was identified by Metchnikoff on the basis of its phagocytic ability. Later on, the reticulohistiocytic system was defined as being composed of antigen-presenting reticulum cells and macrophages. Van Furth proposed that the mononuclear phagocyte system includes all tissue macrophages as well as antigen-presenting cells and blood monocytes as their precursors. Recent findings have shown that blood monocytes are not just transient forms involved in the recruitment of macrophages but that different dendritic and monocytic subpopulations can be observed in blood. In tissue, self-renewing macrophages derived from the yolk sac as well as monocyte-derived dendritic cells and monocyte-derived macrophages can be distinguished. Due to their plasticity and polarization, under inflammatory conditions monocyte-derived macrophages may be beneficial for the reestablishment of homeostasis or may contribute to mostly chronic diseases. Because of their ubiquitous distribution, monocytes and macrophages are increasingly considered to be possible therapeutic targets.

[Sex cord gonadal stromal tumors]. / Tumoren des Gonadenstromas.

According to the World Health Organization (WHO) classification from 2004, sex cord gonadal stromal tumors are divided into Leydig cell tumors, Sertoli cell tumors, granulosa cell tumors, tumors of the thecoma-fibroma group, incompletely differentiated sex cord gonadal stromal tumors, mixed forms of sex cord gonadal stromal tumors and tumors containing both germ cell and sex cord gonadal stromal elements. These tumors can appear sporadically or in combination with hereditary syndromes. To diagnose these rare tumors the combination of characteristic morphological aspects and various immunohistochemical markers is useful. Latest investigations demonstrate the potential role of mutation analyses in the diagnosis of this heterogeneous group of tumors.

Myoglobin expression in renal cell carcinoma is regulated by hypoxia.

Myoglobin is a member of the hemoprotein superfamily, which additionally includes hemoglobin, neuroglobin and cytoglobin. Cytoplasmic localized myoglobin functions as a radical scavenger and prevents hypoxia. Besides muscle tissue MB expression could also be observed in other tissues as well as in different types of cancer. For the correlation between the expression of myoglobin, hypoxia-inducible-factor-1α, and capillary density tissue of 86 different renal cell carcinomas were immunohistochemically stained with myoglobin-specific and hypoxia-inducible-factor-1α-specific antibodies as well as with CD31 antibody. Four different renal carcinoma cell lines were cultivated under hypoxic conditions and the expression of myoglobin and hypoxia-inducible-factor-1α was evaluated by real-time PCR and Western blot. Renal cell carcinoma including clear cell, papillary, and chromophobe subtypes expressed myoglobin with an inverse relationship to capillary density being highly significant for clear cell renal cell carcinoma. For hypoxia-inducible-factor-1α a significant correlation with capillary density could also be observed in clear cell RCC. In renal cell carcinoma cell lines hypoxia induced a significant increase of myoglobin expression up to 62 fold, whereas hypoxia-inducible-factor-1α only increased up to 5 fold. The PCR results of myoglobin expression could be confirmed by Western blot. Myoglobin seems to be a sensitive marker for hypovascularized tumor entities especially during the early phase of hypoxia. Such neoplasias may benefit from an antiangiogenic therapy.

The role of beta-catenin mutation and SOX9 expression in sex cord-stromal tumours of the testis.

The WHO classification of testis tumours includes the group of sex cord-stromal tumours. They are divided into several histological types, i.e. Leydig cell (LCT) and Sertoli cell tumours (SCT). Based on the physiological expression of ß-catenin in normal testis/Sertoli cells, it was previously shown that SCT can carry a ß-catenin mutation, causing a nuclear positivity for ß-catenin and cyclin D1. Furthermore, it could be shown that the stabilization of ß-catenin in Sertoli cells causes the loss of the Sertoli cell marker SOX9. We wanted to know whether the stabilization of ß-catenin in sex cord-stromal tumours influences SOX-9 expression and thus could be used in the diagnosis of sex cord-stromal tumours. Therefore, 53 cases of sex cord-stromal tumours and tumour-like lesions were investigated for their immunohistochemical expressions of ß-catenin, cyclin D1 and SOX9. In addition, mutation analyses of the ß-catenin gene (exon 3; CTNNB1) were performed. ß-catenin mutation in SCT results in nuclear ß-catenin and cyclin-D1 expressions on immunohistochemical analysis. The nuclear expression/stabilization of ß-catenin causes the loss of SOX9 in these tumours. In contrast, SOX9 is considerably expressed in non-mutated SCT as well as in Sertoli cells of non-neoplastic testes. In summary, immunohistochemical analyses of ß-catenin and SOX9 are useful to distinguish SCT from other sex cord-stromal tumours of the testis. Furthermore, the presence of SOX9 indicates that the cells of origin may be Sertoli cells.

Isozymes of acid esterase and acid phosphatase in permanent human hematopoietic cell lines.

Net enzyme activities of normal human blood cells were measured, and isoelectric focusing patterns of acid esterase (AcE) (EC 3.1.1.6) and acid phosphatase (AcP) (EC 3.1.3.2) were compared with corresponding data obtained for two acute T-lymphoblastic leukemia cell lines (JM, Molt-4), one acute B-lymphoblastic leukemia cell line (Ball-1), one acute non-B-non-T-lymphoblastic leukemia cell line (KM 3), and one promyelocytic leukemia cell line (HL-60). The AcE isozymes, found in the individual blood cell types, were regularly expressed by the corresponding cell lines. AcP was regularly expressed by lines HL-60 and KM 3, but lines JM, Molt-4, and Ball-1 showed additional isozymes and/or reduction of the intensity of the typical isozymes found in the presumed normal counterparts. This phenomenon resulted partly in an obscuration of the typical isozyme patterns. Our study documents the applicability of isozyme mapping to the characterization of permanent hematopoietic cell lines. The results suggest that long-term culture conditions can repress phenotypic properties and/or derepress gene activities.

Induction of hypoxanthine phosphoribosyltransferase deficiency in human U-937 cells.

The aim of the study was to establish enzyme-deficient mutants of the human permanent cell line U-937. Following chemical mutagenesis with the use of ethyl methanesulfonate, this cell line was chronically exposed to increasing concentrations of the toxic hypoxanthine analogue 6-thioguanine. Cells surviving hypoxanthine-aminopterin-thymidine selective media were separated by glass adherence with the use of 12-O-tetradecanoyl-phorbol-13-acetate. Three mutant clones were established, which have remained hypoxanthine phosphoribosyltransferase (HPRT) deficient for a period of 7 months, as shown by indirect measurements with the use of autoradiography and scintillation counting of cells exposed to [3H]hypoxanthine. Since the phenotypic properties and growth behavior of U-937 cells have remained unaltered after the induced mutation, a highly restricted chromosomal segment coding for HPRT seems to have been mutated.

Bimodal differentiation prospectives for promyelocytes.

In accordance with the long-range goal to demonstrate the direct derivation of blood monocytes from promyelocytes in human bone marrow, the promyelocytic cell line HL-60 in the present study was differently stimulated with various inducers for the purpose of documenting its capability to evolve into granulocytes or monocytes-macrophages. Each differentiation line was monitored with the use of marker enzymes, antigens, and cell-specific monoclonal antibodies. In addition, studies were done on normal human bone marrow and leukemias. The results provided strong evidence for the close cytogeneic relationship of granulocytes and monocytes and for a common progenitor at the maturation stage of promyelocytes-myelocytes for both cell lineages.

Expression of the monocyte-specific esterase gene in leukemia-lymphoma cell lines.

The expression of the monocyte esterase was examined in a panel of 77 continuous human leukemia-lymphoma cell lines representing all hematopoietic cell lineages and in 16 other cell lines. Accumulation of mRNA, determined by Northern blotting with the cDNA probe HMSE-1, and production of the protein, shown by isoelectric focusing on polyacrylamide gels, correlated with differentiation of the cells along the monocytic lineage. None of the lymphoid, erythroid, megakaryoblastic or Hodgkin's disease derived cell lines or the non-hematopoietic human tumor cell lines expressed the full-length mRNA of 2.0 kb. These results support the notion that this enzyme, a serine hydrolase with still unknown physiological functions, is specifically expressed in cells committed to the monocyte/macrophage cell lineage.

Lineage-specific methylation of the c-fms gene in blood cells and macrophages.

DNA methylation belongs to the multilevel genetic control system regulating differentiation processes and gene expression. The extent to which DNA methylation contributes to the differentiation of hematopoietic cells is elusive. In the present study we investigated the methylation state of the c-fms/M-CSF receptor gene in normal human blood cells and tissue macrophages. The methylation pattern of the c-fms gene as detected by isoschizomeric restriction analysis with MspI/HpaII showed only slight interindividual variations in normal donors, whereas constant differences were found between granulocytes and monocytes from the same donor. The second intron of the c-fms gene contains several CpG loci which were found to be hypomethylated on both alleles in monocytes and tissue macrophages. By contrast, these positions were methylated in granulocytes and lymphocytes that did not express the c-fms gene. In comparison to monocytes alveolar and peritoneal macrophages revealed an enhanced demethylation. There were constant differences in c-fms gene methylation between alveolar and peritoneal macrophages with a higher degree of demethylation in alveolar macrophages. We conclude that c-fms gene demethylation is involved in the differentiation of monocytes and macrophages from immature precursors and that the demethylation of lineage-specific growth factor receptor genes might provide an important step in lineage commitment of hematopoietic cells.

Modulation of c-fms proto-oncogene expression in human blood monocytes and macrophages.

The gene product of the c-fms proto-oncogene is a transmembrane protein with tyrosine-kinase activity that is obviously related to the receptor for the colony-stimulating-factor CSF-1. By Northern blot analysis, we investigated the expression of the cellular counterpart of v-fms in purified normal human blood mononuclear cells and different macrophage populations. The proto-oncogene c-fms expression was demonstrable in blood monocytes but not in blood lymphocytes. Short-term cultivated blood monocytes exhibited an increased expression of c-fms in comparison to freshly isolated blood monocytes, possibly due to a temporary down regulation of c-fms during the separation procedure of blood monocytes. A comparably high rate of fms-RNA expression was found in most of the analyzed samples of resident peritoneal macrophages, while resident alveolar macrophages showed a considerably lower level of c-fms expression. In this, alveolar macrophages resembled long-term cultivated adherent blood monocytes, which showed a down regulation of c-fms expression. By correlating these data obtained by Northern blot analysis with phenotypic properties of the analyzed monocyte/macrophage populations, it is concluded that different levels of c-fms expression in monocytes/macrophages correspond to their stage of differentiation and maturity.

Selective recognition of rat follicular dendritic cells (dendritic reticulum cells) by a new monoclonal antibody Ki-M4R in vitro and in vivo.

Using unstimulated rat peritoneal cells as immunogen a new monoclonal antibody Ki-M4R was produced. Ki-M4R recognizes follicular dendritic cells (dendritic reticulum cells) in germinal centers of lymphoid follicles in lymphatic tissue. In addition, sinus lining cells, endothelia of postcapillary venules, as well as mesangial cells of the renal glomerula immunoreact with Ki-M4R in vitro as well as in vivo. This antibody might be useful for studying the interaction of follicular dendritic cells and B-cell immune response.

Ki-M2R, a new specific monoclonal antibody, discriminates tissue macrophages from reticulum cells and monocytes in vivo and in vitro.

Utilizing rat peritoneal macrophages as the immunogen, a new monoclonal antibody enabling differential monitoring of the mononuclear phagocyte system (MPS) by immunohistochemistry has been raised. Designated Ki-M2R, this antigen could be detected with the immune alkaline phosphatase reaction on all macrophages including those of bone marrow, lymphatic sinuses, lymphoid follicles, splenic red pulp, and von Kupffer cells of the liver, as well as on macrophages of connective tissue, renal interstitial tissue, serous cavities, and gastrointestinal tract. Langerhans cells--the MPS-derived reticulum cells of the epidermis--interdigitating reticulum cells, and dendritic reticulum cells of lymphoid follicles were invariably negative. Blood monocytes were rendered positive only after evolving into macrophages upon appropriate stimulation. Thus, Ki-M2R selectively labels monocytes after transformation into macrophages.

Diversity of the human monocyte/macrophage system as detected by monoclonal antibodies.

Four monoclonal antibodies against the human monocyte/macrophage system, termed Ki-M1, Ki-M6, Ki-M7, and Ki-M8, are described with regard to their immunohistochemical tissue distribution pattern and their subcellular reactive sites. The differences found applying these analyses are also reflected by the various molecular weights of the recognized antigens. Based on these data it is proposed that the monocyte/macrophage system can be divided into the phagocytosing compartment on one hand and the immune accessory compartment on the other hand; the latter constitutes the interdigitating reticulum cells, the indeterminate dendritic cells, and the Langerhans cells, as well as the follicular dendritic cells (dendritic reticulum cells) as the accessory cells for T- and B-cell immune response, respectively.

Isolation of monocytic serine esterase and evaluation of its proteolytic activity.

Monocytes are characterized by high activity of alpha-naphthyl acetate esterase (ANAE), distinguishing them from all other blood cells. The physiological function of this monocyte marker enzyme has not yet been elucidated. In this study ANAE's potential proteolytic activity was analyzed because serine esterases/proteases can function as effector molecules in cell-mediated cytotoxicity and because monocytes-macrophages are known to exert cytotoxic effects on tumor cells. This enzyme was purified from the monocytic cell line U-937 by preparative isoelectric focusing and a three-step high-performance liquid chromatography that conserved its catalytic activity. It has a molecular mass of 60 kd, and partial amino acid sequence revealed that the enzyme is not identical to known serine esterases/proteases. The purified enzyme failed to digest a couple of peptides, indicating lack of protease activity. In addition, the esterolytic activity of ANAE was not inhibited by protease inhibitors. The isolation and purification of ANAE enable further studies concerning its function in monocytes-macrophages and its relation to monocytic cytotoxicity.

Isoelectric focusing pattern of acid phosphatase and acid esterase in human blood cells, including thymocytes, T lymphocytes, and B lymphocytes.

Lysosomal acid phosphatase and acid esterase from highly purified, viable human thymocytes, T lymphocytes, B lymphocytes, granulocytes, monocytes, platelets, and erythrocytes were subjected to direct measurement of activity and to isoelectric focusing in polyacrylamide thin-layer slabs. The typical patterns for each cell line were found to be distinctly different from the results recently presented in this journal. The study described here opens a new possibility for the biochemical identification of cells and their functional derivatives and neoplastic variants.

An improved method for elimination of mycoplasmas from cell cultures.

Cell lines infected by different species of mycoplasma (Mycoplasma orale, Mycoplasma hominis) were decontaminated by co-culture with human blood monocyte (BM)-derived macrophages and pooled human immunoglobulin preparations. Co-cultures with BM-derived macrophages or murine peritoneal macrophages (PM) alone were not successful. The phenotype of infected cell lines did not differ from that of uninfected cell lines as revealed by morphological, enzymecytochemical, and immunocytochemical analysis.

Apoptosis in the small intestinal allograft of the rat.

BACKGROUND: Necrosis and apoptosis are different cell death mechanisms. Necrosis is a pathological process that occurs after destruction of the cell membrane. Apoptosis is a DNA-dependent cell death mechanism, which occurs under physiological and pathological conditions. Although necrosis is a well-defined phenomenon in an acute graft rejection, the occurrence and relevance of apoptosis during this process is largely unknown. METHODS: The enterocyte apoptosis rates in allografted (n=24) and isografted (n=24) small intestines of the rat were compared using the in situ end-labeling technique. RESULTS: In situ end-labeling showed a dramatically increased number of apoptotic enterocytes in allografted small intestines, whereas increased labeling could not be observed in isogeneic small intestinal grafts. CONCLUSIONS: We suggest that graft rejection-associated apoptosis, in addition to necrosis, plays an important role in the course of organ failure, and that the degree of apoptosis represents another reliable indicator for the diagnosis and prognosis of transplant rejection.

Monocytic origin of human alveolar macrophages.

The monocytic lysosomal acid esterase (AcE; EC 3.1.1.6) comprises five isoenzymes, each having specific isoelectric points (pI) as well as antigenicity. In the present study attempts were made to retrace the monocytic origin of human alveolar macrophages (AM) by comparison of their isoenzyme patterns with those of blood monocytes. Resident AM obtained from bronchial lavages lacking any neutrophils and unstimulated monocyte admixture showed in addition to the five monocytic isoenzymes nine additional isoenzyme loci. In vitro stimulation of blood monocytes (BM) using lymphokine-conditioned media led to a gradual transition of the typical monocytic isoenzyme pattern into that of AM. It is concluded that AM originates from blood monocytes by tissue-specific stimulation. This cellular transformation can be modeled in vitro as far as morphology, cytochemistry, and isoenzyme pattern are concerned.

Human neutrophilic and eosinophilic granulocytes display different levels of c-fos proto-oncogene expression: an in situ hybridization study.

The cellular homologue of the retroviral oncogene v-fos has been shown to be involved in cell differentiation of hematopoietic cells. By use of the human promyelocyte cell line HL-60, several in vitro differentiation studies suggested a selective activation of c-fos during monocytic differentiation of myeloid precursor cells. In contrast to these observations, we found high levels of c-fos mRNA in purified normal human granulocytes, whereas c-fos was only faintly expressed in blood monocytes. In situ hybridization revealed that the high level of c-fos expression is restricted to neutrophilic granulocytes, whereas c-fos transcription is not detectable in eosinophilic granulocytes. These results indicate that in vitro differentiation systems can be misleading and may not reflect the in vivo situation. The high level of c-fos expression in neutrophilic granulocytes may be caused by superinduction due to the reduced capacity for protein synthesis in these cells.

Ki-M7 monoclonal antibody specific for myelomonocytic cell lineage and macrophages in human.

We describe a new monoclonal antibody, termed Ki-M7, which is specific to human myelomonocytic cell lineage and macrophages, as tested by immunohistochemical methods. Ki-M7 recognizes an intracytoplasmic antigen of molecular weight 29,000. Ultrastructurally, the antigen is localized in the lysosome and phagosome compartments and seems to be involved in generation of oxygen radicals during the respiratory burst. Dendritic cells, such as dendritic reticulum cells of lymphoid follicles and interdigitating reticulum cells of lymphoid T-zones, considered as accessory cells of the B- and T-cell immune response, respectively, do not show any reactivity with monoclonal antibody Ki-M7. Ki-M7 seems to be an appropriate reagent to clearly differentiate between the phagocytosing and the immune accessory population of the human monocyte/macrophage system.