Abnormal expression of CD antigens in mastocytosis

Human mast cells are myeloid cells derived from human pluripotential CD34+ stem cells. Normal mast cells exhibit a myeloid immunophenotype characterized by the expression of CD117, CD33 and Fc epsilon RI in the absence of reactivity for CD14, CD15 and lymphoid-lineage-associated antigens. Multiparametric flow-cytometric studies have shown that mast cells from mastocytosis display unique immunophenotypic characteristics, including coexpression of CD2 and CD25 antigens together with abnormally high levels of the activation-related antigens CD35, CD63 and CD69 among others. Such aberrant immunophenotypic features are of great relevance in the diagnosis and differential diagnosis of the disease, flow-cytometric immunophenotyping of mast cells representing the most sensitive method for the diagnosis of tissue involvement in mastocytosis. From the pathogenetic point of view, the immunophenotypical patterns described suggest the existence of profound changes regarding the adhesion and activation status of mast cells in mastocytosis and may represent a useful tool for a better understanding of some pathophysiological aspects of the disease.     

Intra-Embryonic Haemopoietic Cells and Early MHC Expression

In the avian embryo the haemopoietic stem cells originate from the intra-embryonic area near dorsal aorta. The surface-marker expression of haemopoietic stern cells and their potential to produce different haemopoietic cells are still largely unknown. The surface antigen expression and particularly the MHC antigen expression on intra-embryonic haemopoietic cells was studied. Expression of B-F antigens, homologous to mammalian MHC class-I antigens, was found already on embryonic day (ED) 5. The first B-L antigens, analogous to mammalian MHC class-II antigens, were detected also from ED5 onwards. The appearance of surface antigens defined by MoAbs T10A6 and 3–298 during embryogenesis also was studied. The antigen defined with T10A6 was detected from ED4 onwards on endothelial cells but not on haemopoietic cells in the para-aortic region. The first 3–298⁺ haemopoietic cells were found on ED6, whereas endothelial cells were negative. These findings imply that some surface markers are shared with haemopoietic and endothelial cells indicating either a common embryonic origin or the importance of these molecules in embryonic stem-cell homing.     

Cell surface characterization of the human osteoclast: phenotypic relationship to other bone marrow-derived cell types

Among the differentiated progeny of stem cells transplantable by bone marrow are osteoclasts, the multinucleate cells which are the major agents of bone resorption. Although the osteoclast is well characterized from a structural and functional standpoint, its development and origin are still far from clear. We have used monoclonal antibodies to investigate the interrelationship between osteoclasts and other haemopoietic cells in man. We have analysed the distribution of 19 granulocyte-monocyte antigens in eight reactivity clusters on the non-neoplastic osteoclasts present within nine osteoclastomas (syn. giant cell tumours of bone) and a single example of aneurysmal bone cyst. We found that osteoclasts are antigenically effete, failing to express granulocyte-monocyte, common leucocyte or other haemopoietic determinants; the only monocyte antigens detected on osteoclasts are My-7 and two closely related specificities, MCS.2 and DüHL60.4, which are also expressed by tissues outside the haemopoietic system. Our findings, taken together with recent transplantation studies, cast further doubt on the view that osteoclasts are specialized bone-resorbing macrophage-derived giant cells, and support a hypothesis that they are the end product of fusion of a hitherto unidentified circulating mononuclear cell type, the preosteoclast, which constitutes a cell lineage separate from those originating from the conventional multipotential haemopoietic stem cell, although still of bone marrow origin.     

Mouse and rat IgE Cross-sensitization of mast cells and antigenic relationships.

Mouse and rat IgE fix firmly to the peritoneal mast cells from the other species, sensitizing them for anaphylactic reaction. Sensitization with IgE can be demonstrated by inducing degranulation either with specific antigens or with corresponding anti-IgE. Sensitization of rat mast cells by mouse IgE antibodies is more easily obtained than that of mouse mast cells by rat IgE antibodies. In this case, anti-IgE-induced degranulation is higher than antigen-induced degranulation. Heterologous sensitization by IgE is time requiring and temperature-dependent. Its kinetics depend upon IgE concentration. Cross-reactions between IgE from one species and anti-IgE from another species have been observed: anti-IgE for one species is able to neutralize PCA reaginic activity of sera from the other species; anti-rat IgE induces degranulation of mouse actively sensitized mast cells. The results suggest strongly that there exists a structural and functional similarity between the IgE molecules from the two species.     

Role of mast cells in intestinal mucosal function: studies in models of hypersensitivity and stress

Summary: A single layer of epithelial cells lines the gastrointestinal tract, forming a critical barrier between the luminal contents, which includes antigens and other noxious substances, and the body proper. It has become clear in recent years that the role of mast cells in the gastrointestinal mucosa is not only to react to antigens, but also to actively regulate the barrier and transport properties of the intestinal epithelium. Mucosal mast cells respond to both IgE/antigen-dependent and non-IgE-dependent stimulation, releasing bioactive mediators into adjacent tissues where they induce physiological responses. Studies in models of hypersensitivity and stress have provided evidence that changes in mucosal function are due to either direct action of mast cell mediators on epithelial receptors and/or indirect action via nerves/neurotransmitters.
Studies conducted in the authors' laboratories were funded by grants from the Canadian Institutes of Health Research and the Crohn's and Colitis Foundation of Canada.     

Human mast cells detected by monoclonal antibodies.

We report the establishment of seven mouse-mouse hybridoma cell lines secreting monoclonal antibodies with specificity for granule components of all human mast cells. Reactivity is directed against a molecule which is also found intracytoplasmically in human mature small intestinal enterocytes, liver parenchymal cells, and kidney proximal tubule epithelial cells. No reactivity of these antibodies was found with any other human or animal cell type examined. In particular, the antibodies did not react with basophils or other haemopoietic cell types. This study shows the potential of specific monoclonal antibodies as a tool for identifying and enumerating infiltrating mast cells in tissues. Such antibodies should be of value in investigations into the role of the mast cell in immunological reactions and hypersensitivity diseases.     

Mast cell membrane antigens and Fc receptors in anaphylaxis. I. Products of the major histocompatibility complex involved in alloantibody-induced mast cell activation.

Mast cell membrane antigens, coded by the K, I and D regions of the major histocompatibility complex of the mouse, were investigated for their presence at the cell surface and their participation in alloantibody-induced anaphylactic degranulation (DAAD). Anti-H-2 K, as well as anti-H-2 D antibodies were found to elicit DAAD. Recognition, on the mast cell membrane, of any product of the K or the D regions, either as the whole molecule, or as public or private antigens only, or even as a single private specificity, enabled alloimmune sera to trigger mast cell degranulation. By contrast, anti-Ia antibodies failed to elicit DAAD. By the autoradiographic technique, peritoneal mast cells were found to constitute a single homogeneous population, bearing H-2 D-coded antigens, although in smaller amounts than other peritoneal cells, but no Ia antigens or, if any, in much smaller amounts than other peritoneal cell type. These findings bring new evidence that mast cell alloantigens do participate in anaphylactic alloantibody-induced mast cell degranulation, by allowing bridging of (one?) Fc receptor with H-2 molecules.     

Mucosal mast cells in vivo and in vitro

There has recently been a flurry on the mast cell front caused, not least, by the publication of articles about the IL3-induced growth of mast cells in vitro from haemopoietic tissue. This has left bystanders, and one suspects not a few participants, confused about some of the issues involved. The reason for the confusion lies in the participation of disparate specialities - such as experimental haematology and immunoparasitology - among which communication has not been traditional: however, the revelations raise specific questions which demand an interdisciplinary approach. Thus the evidence is strong that the mast cells derived from cultured haemopoietic tissues are of a special type hitherto called atypical, intestinal or mucosal mast cells (MMC) which are known to occur in profusion in mucous membranes of helminth-infected animals. In this article Ellen Jarrett and David Haig link information about these cells obtained from both in-vivo and in-vitro experiments.     

Enrichment of c-kit+ Lin- haemopoietic progenitor cells that commit themselves to extrathymic T cells in in vitro culture of appendix mononuclear cells

The appendix as well as the small intestine have recently been found to carry c-kit+ stem cells which give rise to extrathymic T cells. In this study, the properties of c-kit+ stem cells in the appendix of mice were further characterized. When appendix mononuclear cells (MNC) were cultured in the presence of stem cell factor, interleukin-3, interleukin-6 and erythropoietin on a methylcellulose culture plate, the population of c-kitdull Lin- and that of c-kithi Lin- cells expanded. Morphological study revealed that these c-kithi Lin- cells were basophilic granular cells (possibly mast cells). Both populations of cultured appendix MNC were then injected into severe combined immunodeficient mice or cultured with Tst-4 thymic stroma cells. These in vivo and in vitro studies demonstrated that c-kitdull Lin- cells were oligopotent haemopoietic progenitor cells which gave rise to extrathymic T cells, while c-kithi Lin- cells lacked haemopoietic progenitor cell activity. In contrast to c-kit+ stem cells in the bone marrow, those in the appendix did not give rise to myeloid cells and conventional thymic T cells under any of the conditions tested. The present results suggest that the appendix primarily comprises c-kit+ cells which give rise to basophilic granular cells and extrathymic T cells and that such c-kit+ cells have the ability to replicate themselves in culture in vitro.     

The tryptase positive compact round cell infiltrate of the bone marrow (TROCI-BM): a novel histopathological finding requiring the application of lineage specific markers

AIMS: Compact tryptase-positive round cell infiltrates of the bone marrow (TROCI-BM) are very rare histopathological findings and may pose challenging problems with regard to the cell type involved (either mast cells or basophilic granulocytes) and the exact diagnosis. METHODS: A selected panel of immunohistochemical markers against mast cell and basophil related antigens, including CD25, CD34, CD117/Kit, and the 2D7 antigen (which is found only in basophilic granulocytes) on a total of 410 routinely processed bone marrow biopsy specimens (including 88 cases of systemic mastocytosis (SM), 20 cases of chronic myeloid leukaemia (CML), 92 cases of myeloid neoplasms other than CML, and 210 controls with normal/reactive bone marrows). RESULTS: In total, 17 cases with TROCI-BM could be identified: 11 SM (including two cases of well-differentiated SM and two mast cell leukaemias; MCL), 2 myelomastocytic leukaemia (MML), 2 CML with excess of basophils (secondary basophilic leukaemia (CMLba)), and 2 tryptase positive acute myeloid leukaemia (AML). Regarding the cell types involved, TROCI-BM cells were found to express CD117/Kit in all cases of SM and MCL. In MML and tryptase postitive AML, TROCI-BM cells were found to coexpress CD34 and Kit. The basophil specific antigen 2D7 was only detected in CD34/Kit negative TROCI-BM cells in two patients with CMLba. The activating point mutation D816V was detected in 8/11 patients with SM but not in any of the other haematological malignancies. CONCLUSIONS: In summary, a total of six rare myeloid neoplasms may present with a novel immunohistochemical phenomenon tentatively termed TROCI-BM.     

Monoclonal antibodies defining markers with apparent selectivity for particular haemopoietic cell types may also detect antigens on cells of neural crest origin

Monoclonal antibodies described as reacting with particular subsets of haemopoietic cells were screened against a variety of neuronal cell lines to further investigate their true specificity. While some reagents, e.g., monoclonal anti-cALL (J5), were found only reactive with haemopoietic cells, other monoclonals, e.g., BA1, BA2, NA134, OKT6, and OKT9, also bound to neuronal cells. Further investigation into the cross-reactivity of these antibodies to a variety of neural crest derived cell lines indicated that, with the exception of OKT9, differential binding patterns to different lines were obtained. This suggests that haemopoietic cell subsets and neural crest derived tissues can share similar differentiation antigens. For monoclonals OKT9 and BA2 this observation was confirmed biochemically, showing that it is not just antigenic determinants but similar molecular weight cell surface antigens that are shared between subsets of the two major cell types. A similar analysis using monoclonal antibodies raised against human neuronal cells or cell lines again indicates that some antigens appear unique to neural tissue while others are shared by haemopoietic cell subsets.     

Systemic mastocytosis with associated clonal hematologic nonmast cell lineage disease: a clinicopathologic review

Systemic mastocytosis (SM) is a heterogeneous disease with 6 subtypes, including systemic mastocytosis with associated clonal hematologic nonmast cell lineage disease (SM-AHNMD). Bone marrow biopsy specimens show multifocal aggregates of mast cells with predominantly spindle-shaped morphology associated with a myeloid or, less frequently, a lymphoproliferative neoplasm defined by World Health Organization criteria. Neoplastic mast cells abnormally express CD2 and/or CD25, which may be detected by flow cytometry or immunohistochemistry. The pathogenesis of SM-AHNMD is not well understood; however, combined KIT tyrosine kinase receptor mutations and additional genetic events in myeloid stem cells may have a pathogenic role. Reactive mast cell hyperplasia, monocytic/histiocytic proliferations, SM without sufficient criteria for a diagnosis of AHNMD, atypical mast cells associated with PDGFRA rearrangements, and other tryptase-positive myeloid proliferations should be excluded. Overall, the prognosis is poor and largely related to the AHNMD. Cytoreductive therapies, splenectomy, allogeneic bone marrow transplant, and tyrosine kinase inhibitors, excluding imatinib, may have potential efficacy in the treatment of these diseases.     

Evidence for the in vivo production and release into the serum of a T-cell lymphokine, persisting-cell stimulating factor (PSF), during graft-versus-host reactions.

The T-cell lymphokine, persisting-cell stimulating factor (PSF or interleukin-3), was detected in the serum of mice undergoing graft-versus-host reactions (GVHR). Gel filtration under non-dissociating conditions indicated that the PSF in the serum had an apparent molecular weight of 34,000, a figure identical with that of PSF generated from activated T cells in vitro, indicating that PSF was not bound by serum proteins. The GVHR was accompanied by increases in the numbers in the bone marrow and spleen of precursors of PSF-dependent mast cells, and increases in the numbers of mast cells, megakaryocytes and immature and mature neutrophils in the spleen. These effects of GVHR on haemopoietic cells paralleled those seen when haemopoietic tissues were stimulated with pure PSF in vitro and closely resembled those induced in previous studies by the presence of a tumour that secreted PSF alone. These studies are the first to show that PSF can enter the circulation during immune reactions in vivo and suggest that much of the stimulation of haemopoietic cells seen in GVHR, can be accounted for by the release of PSF from activated T cells.     

Immunohistochemical characterization of reactive and neoplastic mast cells

Mast cells are connective tissue elements likened to unicellular endocrine organs because of the wide diversity of physiologic and pathologic events associated with the secretion of biologically active compounds. Using an immunoperoxidase method (PAP), we studied tissue from patients with benign and malignant systemic mastocytosis and with a variety of reactive conditions. The following immunoreactive antigens were identified in mast cells: a heparinlike compound or compounds (HLC), prostaglandin, serotonin, and fibronectin. HLC is constantly present, staining mast cells in a granular fashion from most lesions. Serotonin and prostaglandin stain in a diffuse cytoplasmic manner in occasional lesions. Fibronectin is found in a surface location in selected cases. We found no clear association between the immunoreactivity of one compound in mast cells and one clinical symptom, e.g., HCL with bleeding, prostaglandin, or serotonin with systemic vasomotor activity or fibronectin with increased tissue fibrosis. However, patients with localized and systemic disease had symptoms that might have been attributed to more than one compound. Only occasional patients with reactive conditions showed such symptoms. The presence of these compounds, either alone or in combination, did not separate benign from malignant conditions. Other cells within selected tissues also stained with the antibodies tested. Despite the lack of exclusivity, these antibodies are useful in identifying mast cells within tissue sections and may have a role in the study of mast cell constituents.     

Human fetal osteoclasts fail to express macrophage antigens

A method for the isolation of osteoclasts from human fetal long bones in sufficient numbers for phenotypic studies has been devised. Using this technique we have studied the expression of cell surface antigens characteristic of mononuclear phagocytes and other haemopoietic cell types on fetal osteoclasts and compared their phenotype with mononuclear cells in the same preparations. We found that osteoclasts failed to express DrW(Ia) and 24 of 26 antigens (in 6 of 7 antigenic clusters) found on mononuclear phagocytes of the same developmental stage. This implies that osteoclasts represent the maturational end-stage of a cell lineage separate from that of conventional blood cells, and of mononuclear phagocytes in particular.     

What can We Learn from Leukemia as for the Process of Lineage Commitment in Hematopoiesis?

Most contemporary models of hematopoiesis assume lineage fidelity of early progenitor cells. Along with this concept normal hematopoietic cells and the majority of leukemias express exclusively myeloid or lymphoid specific antigens. On the other hand, growing evidence exists challenging the lineage fidelity model. Chronic myeloid leukemia (CML) in the blast crisis may switch to acute lymphoblastic leukemia (ALL) and as a result of the chemotherapy ALL may converse to acute myeloid leukemia (AML). Furthermore, a substantial portion of leukemia cases, named acute mixed-lineage leukemia (AMLL), show simultaneous expression of both myeloid and lymphoid antigens. Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements, correlating with myeloidlymphoid immunophenotype in AMLL, support the hypothesis of lineage infidelity of early progenitor cells, rather than the aberrant antigen expression. Based on a detailed characterization of AMLL we present a modified model of a “common myeloid/lymphoid progenitor cell”. This hypothetical very early hematopoietic progenitor cell shows a transient expression of myeloid and B-or T-lymphoid antigen and may also have rearranged its Ig and/or TCR genes. Subsequently, myeloid or lymphoid markers are downregulated and the hematopoietic cell enters either myeloid, T-lymphoid or B-lymphoid differentiation pathway.     

Human fetal osteoclasts fail to express macrophage antigens.

A method for the isolation of osteoclasts from human fetal long bones in sufficient numbers for phenotypic studies has been devised. Using this technique we have studied the expression of cell surface antigens characteristic of mononuclear phagocytes and other haemopoietic cell types on fetal osteoclasts and compared their phenotype with mononuclear cells in the same preparations. We found that osteoclasts failed to express DrW(Ia) and 24 of 26 antigens (in 6 of 7 antigenic clusters) found on mononuclear phagocytes of the same developmental stage. This implies that osteoclasts represent the maturational end-stage of a cell lineage separate from that of conventional blood cells, and of mononuclear phagocytes in particular.     

What can we learn from leukemia as for the process of lineage commitment in hematopoiesis?

Most contemporary models of hematopoiesis assume lineage fidelity of early progenitor cells. Along with this concept normal hematopoietic cells and the majority of leukemias express exclusively myeloid or lymphoid specific antigens. On the other hand, growing evidence exists challenging the lineage fidelity model. Chronic myeloid leukemia (CML) in the blast crisis may switch to acute lymphoblastic leukemia (ALL) and as a result of the chemotherapy ALL may converse to acute myeloid leukemia (AML). Furthermore, a substantial portion of leukemia cases, named acute mixed-lineage leukemia (AMLL), show simultaneous expression of both myeloid and lymphoid antigens. Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements, correlating with myeloid-lymphoid immunophenotype in AMLL, support the hypothesis of lineage infidelity of early progenitor cells, rather than the aberrant antigen expression. Based on a detailed characterization of AMLL we present a modified model of a "common myeloid/lymphoid progenitor cell". This hypothetical very early hematopoietic progenitor cell shows a transient expression of myeloid and B- or T-lymphoid antigen and may also have rearranged its Ig and/or TCR genes. Subsequently, myeloid or lymphoid markers are downregulated and the hematopoietic cell enters either myeloid, T-lymphoid or B-lymphoid differentiation pathway.     

Lipoteichoic acids selectively stimulate rat mast cells to cysteinyl leukotriene generation and affect mast cell migration after tumor necrosis factor (TNF)-priming

It is well established that mast cells play a critical role in the host defense against bacteria. Upon stimulation with bacteria and their antigens, mast cells release various mediators and cytokines that promote the development of inflammation at the site of infection. In the present study, we examined the ability of lipoteichoic acids (LTAs), some of the major components of cell walls of most gram-positive bacteria, to stimulate mast cell degranulation and histamine release as well as to generate of cysteinyl leukotrienes (LTs). We also studied the influence of LTAs on mast cell migration. Experiments were done on rat peritoneal mast cells and LTA from Staphyloccocus aureus and LTA from Bacillus subtilis were used. We have stated that neither S. aureus LTA nor B. subtilis LTA used at a wide range of concentrations (from 10(-4) to 10(5)ng/mL) induced mast cell degranulation and histamine release. However, stimulation of mast cells with both LTAs resulted in generation and release of significant levels of LTs. We have also documented that none of the LTAs stimulated rat mast cell migration, even in the presence of laminin. IL-6 priming did not influence mast cell migration towards LTAs, whereas, pretreatment of mast cells with TNF caused time-dependent mast cell migration in response to LTAs stimulation. Pretreatment of mast cells with anti-TNFR1 antibodies completely inhibited LTA-induced migratory response of TNF-primed mast cells. Our results showed that LTAs might be among important bacterial antigens involved in mast cell activation during bacterial infections.     

Exposure to Extremely Low Frequency Magnetic Fields Has No Effect on Growth Rate or Clonogenic Potential of Multipotential Haemopoietic Progenitor Cells

Abstract

Recent reports indicate an increased risk of acute myeloid leukaemia in children exposed to extremely low frequency magnetic fields (ELFMFs) emitted by high voltage power lines, suggesting that ELFMFs may act as weak tumour promoters. We have investigated possible interactions of weak ELFMFs with primitive haemopoietic cells in vitro using the multipotential progenitor cell line FDCP-mix(A4). We have determined the proliferative activity and clonogenic potential of cells under both optimal and sub-optimal growth conditions and exposed to either ambient laboratory ELFMFs or three other ELFMF regimes representative of those produced by high voltage power lines: nulled fields, Ca²⁺ion cyclotron resonance conditions at 50 Hz, and vertical 50 Hz fields of 6 μT_RMS. Using exposures of 1, 4, 7 and 21 days, we found no significant alteration of growth rate, cell-cycle state or clonogenic efficiency indicating that neither the proliferation nor self-renewal of multipotential FDCP-mix(A4) cells was perturbed.