Hans Kreipe

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.

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.

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.

Phenotypic differentiation patterns of the human monocyte/macrophage system

SummaryIn the present study phenotypic properties of non-stimulated and stimulated blood monocytes and of their normal macrophage derivatives were studied applying enzyme cytochemistry, isoenzyme analysis of acid esterase (EC 3.1.1.6), and immunohistochemical staining using a panel of newly established monoclonal antibodies specific for the monocyte/macrophage lineage. Certain marker profiles could be established for the various normal subpopulations within the monocyte/macrophage system, which were also observable in epithelioid cells and U-937 cell line considered as reactive and neoplastic differentiation variants of monocytes, respectively. Alveolar macrophages, in contrast to the other analysed monocyte/macrophage populations, showed a highly activated phenotype comparable to lymphokine stimulated blood monocytes and epithelioid cells. The results underline the concept that the adaptation of monocytes/macrophages to their particular microenvironment is of decisive importance for their definitive differentiation.

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.

Lectin binding and surface glycoprotein pattern of human macrophage populations

SummaryIn the present study unstimulated and stimulated human blood monocytes, untreated and phorbol ester treated U-937 cells, as well as human peritoneal and alveolar macrophages were studied with respect to their surface membrane properties. Binding of different lectins and electrophoretic patterns of tritium labeled surface glycoproteins were compared. The analysis of surface glycoproteins could be interpreted as evidence for a common origin of the analysed cell populations. Furthermore, banding patterns of glycoproteins might be useful to define certain activation states within monocyte/macrophage differentiation. In contrast, lectin binding pattern did not clearly discriminate macrophage subpopulations.

Heterogenous expression and putative structure of human monocyte/macrophage serine esterase l

Human monocyte serine esterase 1 (HMSE1) was purified from U937 cell extract. Since the N terminus of the enzyme was blocked, cleavage with trypsin was used to obtain several peptides accessible to amino acid sequencing. Based on partial amino acid sequence information, an oligonucleotide probe was synthesized and used to screen a U937 cDNA library. One clone was isolated and sequenced by us which contains an open reading frame of 503 amino acids that lacks about 50 amino acids at the N terminus relative to the protein. Computer analysis revealed an active site characteristic of known carboxylesterases with a catalytic active serine. Northern blot hybridization analysis revealed that the expression of HMSE1 is restricted to cells of the monocyte/macrophage system. In contrast to the moderate expression of HMSE1 in monocytes, alveolar macrophages showed very high amounts of the transcript. With the sequence features detected by computer analysis a structure model of HMSE1 as a dimeric, membrane-bound ectoenzyme was developed.

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.

Clonal Analysis of Agnogenic Myeloid Metaplasia

Agnogenic myeloid metaplasia (AMM) is a chronic myeloproliferative disorder that leads to a sustained proliferation of megakaryocytes and an increase of reticulin fibers within the bone marrow. Blood and bone marrow samples from patients with advanced AMM with fully developed myelofibrosis as well as cases in the cellular phase of the disease were investigated for clonality. Clonality was studied by X-linked restriction length polymorphism in conjunction with DNA methylation patterns. Granulocytes and total bone marrow cells proved to be monoclonal in origin whereas at least a minor portion of the peripheral lymphocytes were not clonally derived. Our findings indicate that the cellular phase of AMM as well as the fully developed disease progressed to myelofibrosis represent a monoclonal proliferation of pluripotent hematopoietic stem cells.

Human macrophage hybrid forming spontaneous giant cells

SummaryThymidine kinase-deficient clones of the human monocyte/ macrophage cell line U-937 were established and used for fusion experiments with separated normal human blood monocytes. A hybrid (H 29) was generated during HAT-selection procedure, about 50% of which formed spontaneous giant cells, as shown by morphological, immunocytochemical, and chromosomal analyses. It is concluded that giant cells originate from monocytes and display mitotic activity. Macrophage hybrids are the basic requirement for the elucidation of monocyte/macrophage heterogeneity and immortalization of their functional properties.