Hermine Agis

Comparative immunophenotypic analysis of human mast cells, blood basophils and monocytes

Mast cells (MC), blood basophils (Ba) and moncoytes (Mo) are of haemopoietic origin. Lineage‐relationships and transdifferentiation between MC and Mo, or MC and Ba, have been considered, based on common expression of antigens. In this study, comparative phenotypic analyses on MC, Ba and Mo and on respective cell lines were performed using monoclonal antibodies (mAb) to previously defined and novel CD antigens (CD1–130). By cluster analysis, the overall (all 130 CD) phenotypic relationships (given as similarity indices, SI), between primary cells (MC, Ba and Mo) and corresponding cell lines (HMC‐1, KU‐812, U937) were 0.716, 0.779 and 0.757, respectively. When primary cells were compared, lower SI values were found (MC versus Ba, 0.509; MC versus Mo, 0.625; Mo versus Ba, 0.698). More distant relationships were found between MC versus Ba and MC versus Mo, compared with Ba versus Mo, for adhesion receptor (R)‐, complement R‐ and cytokine R profiles. Analysis of cytokine R revealed most significant dissimilarities between MC versus Ba and MC versus Mo (SIu2003<u20030.2). Moreover, in contrast to other CD subgroups and other lineages, MC and HMC‐1 differed from each other in cytokine R expression (SIu2003=u20030.286). Cytokine R detectable on HMC‐1 but not MC were granulocyte–macrophage colony‐stimulating factor (GM‐CSFR)α(CD116), CD40, Apo‐1/FAS(CD95) and gp130(CD130). Cytokine R detectable on Ba but not MC, were interleukin‐3 (IL‐3)Rα(CD123), IL‐1RII(CD121b), IL‐2Rα(CD25) and CD40. In summary, MC, Ba and Mo display a unique CD profile with MC being the most distantly related cell. The most significant mismatch within a given lineage is the loss of cytokine R on mature MC as compared with normal myeloid progenitors and HMC‐1 cells.

Indolent Systemic Mastocytosis with Elevated Serum Tryptase, Absence of Skin Lesions, and Recurrent Severe Anaphylactoid Episodes

Background: In contrast to aggressive mastocytosis, patients with indolent systemic mastocytosis (ISM) usually present with urticaria pigmentosa-like skin lesions. In those who lack skin lesions, mastocytosis is often overlooked or confused with endocrinologic, allergic, or other internal disorders. Case Report and Results: We report on a 33-year-old male patient in whom severe hypotensive episodes occurred after contact with ants or yellow jackets. Since no specific IgE was detected, the serum tryptase concentration was measured and found to be clearly elevated (70 ng/ml). Consecutive staging and examination of the bone marrow revealed ISM. The patient was advised to circumvent insect contact, to take antihistamines on demand, and to carry an epinephrine self-injector for emergency events. In a retrospective analysis of 40 patients seen between 1988 and 2003, only 2 had a life-threatening mediator-related episode before ISM was diagnosed. Conclusions: Our report confirms the diagnostic value of tryptase in patients with suspected mastocytosis. In addition, the report suggests that the lack of typical skin lesions does not exclude an indolent form of mastocytosis even if the serum tryptase is clearly elevated. Finally, our case further shows that mastocytosis can be an important differential diagnosis to be considered in patients with unexplained anaphylactoid or other mediator-related symptoms.

Effects of cyclosporin A and FK-506 on stem cell factor–induced histamine secretion and growth of human mast cells ☆ ☆☆ ★ ★★

Stem cell factor (SCF) is a key regulator of human mast cells (MCs) and a potential mediator of allergy. In this study the effects of cyclosporin A (CSA) and FK-506, two potent immunosuppressive drugs, on SCF-dependent histamine release and growth of human MCs were analyzed. Preincubation of tissue MCs with CSA (3 micrograms/ml) resulted in inhibition of histamine release provoked by either recombinant human (rh) SCF (70.3% +/- 20.6% inhibition, p < 0.001) or anti-IgE (76.7% +/- 21.9%, p < 0.001) or by rhSCF+ anti-IgE (77.4% +/- 13.9%, p < 0.001). Almost the same inhibition was produced by FK-506 (rhSCF: 82.0% +/- 18.9% inhibition, p < 0.001; anti-IgE: 71.5% +/- 16.7%, p < 0.001; rhSCF+ anti-IgE: 70.0% +/- 7.3%, p < 0.001). The effects of CSA and FK-506 on SCF-dependent release of histamine were dose-dependent (IC50: CSA, 1 to 10 ng/ml; FK-506, 0.3 to 3 ng/ml). IC50 values about three to 10 times higher were found for MCs preincubated with rhSCF before anti-IgE activation, compared with anti-IgE or SCF alone. SCF-dependent differentiation of human MCs was analyzed in a long-term suspension culture system (n = 6). Unexpectedly, CSA and FK-506 were unable to suppress, but even enhanced SCF-dependent growth of MCs and formation of MC tryptase in long-term culture. Together, CSA and FK-506 inhibit SCF-dependent release of histamine from human MCs and even augment SCF-dependent growth of human MCs in long-term culture.

Detection of differentiation- and activation-linked cell surface antigens on cultured mast cell progenitors.

Background:u2002 Mast cells (MC) are multifunctional effector cells of the immune system. They derive from uncommitted CD34+ hemopoietic progenitor cells (HPC). Depending on the stage of maturation and the environment, MC variably express differentiation‐ and activation‐linked antigens. Little is known, however, about the regulation of expression of such antigens in immature human MC.

Elevated tryptase levels selectively cluster in myeloid neoplasms: a novel diagnostic approach and screen marker in clinical haematology

Backgroundu2002 Recent data suggest that tryptase, a mast cell enzyme, is expressed in neoplastic cells in myeloid leukaemias. In several of these patients, increased serum tryptase levels are detectable.

Identification of basogranulin (BB1) as a novel immunohistochemical marker of basophils in normal bone marrow and patients with myeloproliferative disorders.

In myeloproliferative disorders (MPDs), basophils typically increase in number in the bone marrow (BM) and blood. In chronic myeloid leukemia (CML), basophilia is a diagnostic and prognostic variable. However, no reliable approach for routine detection and enumeration of basophils in BM sections is available. We applied the antibasogranulin antibody BB1 on paraffin-embedded BM sections in 21 control samples (normal BM), 45 patients with CML, 9 with chronic idiopathic myelofibrosis, 11 with polycythemia vera, 19 with essential thrombocythemia, and 7 with indolent systemic mastocytosis. As assessed by immunostaining of serial BM sections, BB1+ cells coexpressed myeloperoxidase, histidine decarboxylase, and leukosialin but did not express B- or T-cell-restricted antigens. BB1+ BM cells were found to be highly elevated in patients with CML compared with normal BM or other MPDs, with maximum counts found in accelerated phase CML (median, 160 cells/mm(2)). In summary, BB1 (basogranulin) is a new immunohistochemical basophil marker that should allow quantification of basophils in CML at diagnosis and during therapy.

Immunohistochemical detection of vascular endothelial growth factor (VEGF) in the bone marrow in patients with myelodysplastic syndromes: correlation between VEGF expression and the FAB category.

Recent data suggest that vascular endothelial growth factor (VEGF) is produced in neoplastic cells in various myeloid neoplasms and plays a key role as an autocrine regulator and mediator of angiogenesis. We examined the expression of VEGF in paraffin-embedded bone marrow sections obtained from normal donors (n = 5) and 46 patients with myelodysplastic syndromes [MDS, French–American–British (FAB)-type refractory anemia (RA), n = 10; refractory anemia with ringed sideroblasts (RARS), n = 10; refractory anemia with excess blasts (RAEB), n = 10; RAEB in transformation (RAEB-T), n = 8; chronic myelomonocytic leukemia (CMML), n = 8] by immunohistochemistry using an anti-VEGF antibody. In normal bone marrow, the anti-VEGF antibody was found to react with myeloid progenitor cells, immature monocytic cells, plasma cells and megakaryocytes, but not with erythroid cells or mature granulocytic cells. Higher levels of VEGF were found in patients with MDS, subtypes RAEB, RAEB-T and CMML, compared to patients with RA or RARS, or the normal bone marrow. These differences were found to result from expression of VEGF in immature myeloid cells in RAEB, RAEB-T and CMML. The microvessel density was also higher in patients with RAEB-T and CMML compared to RA and RARS or the normal bone marrow. Expression of VEGF mRNA was demonstrable in isolated neoplastic cells by reverse transcriptase-polymerase chain reaction in all patients examined. In aggregate, these data show that VEGF is expressed in bone marrow cells in patients with MDS. The amount of expressed VEGF is related to the percentage of immature myeloid cells (blasts and monocytic progenitors) and correlates with the FAB category.

Activation of human mast cells through stem cell factor receptor (KIT) is associated with expression of bcl-2

Background: Mast cells (MCs) are multifunctional effector cells of the immune system. These cells originate from pluripotent hemopoietic progenitors. In contrast to basophils and other leukocytes, MCs exhibit a remarkably long life span (years) in vivo. Although a role for stem cell factor (SCF) and SCF receptor (KIT) in long-term survival of MCs has been proposed, the underlying biochemical mechanisms remain unknown. Materials and Methods:We have examined expression of ‘survival-related’ molecules of the bcl-2 family including bcl-2 and bcl-xL, in primary human MCs and the human MC line HMC-1. Primary MCs were isolated from dispersed lung tissue by cell sorting using an antibody against KIT. mRNA expression was analyzed by RT-PCR and Northern blotting. Results: As assessed by RT-PCR, purified unstimulated lung MCs (>98% pure) exhibited KIT- and bcl-xL mRNA, but did not express bcl-2 mRNA. However, exposure of lung MCS to SCF (100 ng/ml) for 8 h resulted in expression of bcl-2 mRNA. Corresponding results were obtained by immunocytochemistry. In fact, exposure of MC to SCF resulted in expression of the bcl-2 protein whereas unstimulated MCs displayed only the bcl-xL protein without expressing the bcl-2 protein. The human MC leukemia cell line HMC-1, which contains a mutated and intrinsically activated SCF receptor, showed constitutive expression of both bcl-2 and bcl-xL at the mRNA and protein level. Conclusion: Our data show that human MCs can express members of the bcl-2 family. It is hypothesized that bcl-xL plays a role in KIT-independent growth of MCs, whereas bcl-2 may be involved in KIT-dependent functions of MCs.

Liposomal cytarabine for treatment of myeloid central nervous system relapse in chronic myeloid leukaemia occurring during imatinib therapy

Backgroundu2002 Central nervous system (CNS) relapse in chronic myeloid leukaemia (CML) is rare and if recorded is usually found to occur in patients with lymphoblastic transformation. The BCR/ABL tyrosine kinase inhibitor imatinib is highly effective in patients with CML, but hardly crosses the blood–brain barrier.

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.