Bone metabolism in patients more than five years after bone marrow transplantation.

We investigated the bone metabolism of 22 patients (median age 38 years) over 6 years after allogeneic bone marrow transplantation (BMT). Biplanar roentgenograms of the thoracic and lumbar spine were used to diagnose vertebral deformities caused by fractures. The actual bone mineral density (BMD) of the lumbar spine and the femoral neck were measured. Laboratory tests included calcium, phosphate, parathyroid hormone, a marker of bone resorption (beta-crosslaps, CTX), markers of bone formation (osteocalcin, bone-specific alkaline phosphatase), osteoprotegerin (OPG)--antagonist of the osteoclast differentiation factor RANKL, and sex hormone status. One patient had a vertebral fracture. Seven patients (28%) had osteopenia in the lumbar spine while 12 patients (48%) had osteopenia in the femoral neck. Bone resorption was increased in nine patients (43%) and bone formation was increased in four patients (20%). BMT recipients had significantly increased serum levels of OPG (P=0.029). Three women (75%) and four men (25%) were hypogonadal. The data showed that BMD is reduced and bone metabolism is still disturbed more than 6 years after BMT. The RANKL/osteoprotegerin system appears to play an important role in the pathophysiology of late post transplantation osteoporosis.

Serum thrombopoietin levels and anti-thrombopoietin antibodies in systemic lupus erythematosus.

Thrombocytopenia is a common phenomenon in patients suffering from systemic lupus erythematosus (SLE). The cause of thrombocytopenia in SLE, however, is poorly understood. In this study, 100 patients with SLE were evaluated for serum thrombopoietin levels, anti-thrombopoietin antibodies and routine laboratory parameters such as peripheral blood counts, parameters of blood chemistry and immunologic parameters of SLE. The median platelet count of SLE patients was 230 g/l and 19 were thrombocytopenic (range 8-148 g/l). Thrombopoietin levels in SLE patients were found to be significantly higher than in healthy controls (n = 96; median, 117 pg/ml vs 64 pg/ml, P < 0.01). When excluding thrombocytopenic SLE patients, thrombopoietin levels in SLE were still above controls (111 pg/ml, P < 0.01). The thrombopoietin levels were correlated to erythrocyte sedimentation rate and ECLAM score of disease activity, and inversely correlated to complement factor C4, but not to the platelet count. Anti-thrombopoietin antibody reactivity was found in 23% of SLE patients. Interestingly, these patients had lower platelet counts than SLE patients without anti-thrombopoietin antibodies (median 174 g/l and 253 g/l, respectively, P < 0.01), but thrombopoietin levels were not significantly different. Taken together, thrombopoietin levels are significantly higher in the sera of SLE patients than in healthy controls and anti-thrombopoietin antibodies are frequently found.

Quantitative, phenotypic, and functional evaluation of basophils in myelodysplastic syndromes.

BACKGROUND: The myelodysplastic syndromes (MDS) are a group of clonal haematological disorders characterized by cytopenia(s), reduced differentiation-capacity of myeloid cells, and impaired leukocyte function. However, little is known so far about basophil granulocytes in MDS. DESIGN: We have compared the numbers, phenotype and function of basophils in MDS patients with those in healthy subjects. A total numer of 23 patients with MDS (refractory anaemia, n = 8; refractory anaemia with ringsideroblasts, n = 7; refractory anaemia with excess of blasts/refractory anaemia with excess of blasts in transformation, n = 8) and 20 healthy donors were included. RESULTS: The numbers of blood basophils in MDS patients (34.6 +/- 62.9 microL-1) was lower compared to healthy controls (58.6 +/- 64.9 microL-1). Correspondingly, whole blood histamine levels were lower in MDS patients (MDS 34.1 +/- 29.1 ng mL-1 vs. normal donors 72.0 +/- 36.9 ng mL-1). Like "normal" basophils, basophils in MDS expressed interleukin-3 receptor alpha (CD123), E-NPP3 (CD203c), CR1 (CD35), CR3 (CD11b), CR4 (CD11c), membrane co-factor protein (CD46), decay-accelerating factor (CD55) and membrane attack complex inhibitory factor (CD59), as well as receptors for C3a, C5a (CD88), and IgE. Recombinant human (rh) C5a and anti-IgE induced significant release of histamine from basophils in both groups of donors without significant differences between MDS and healthy controls. CONCLUSIONS: The absolute numbers of basophils in MDS patients are lower than in normal donors. However, basophils in MDS do not differ from their "normal counterparts" in terms of complement receptor expression, IgE-receptor expression, or functional responses to respective ligands.

Decreased serum erythropoietin and its relation to anti-erythropoietin antibodies in anaemia of systemic lupus erythematosus.

OBJECTIVE: This study was performed to assess erythropoietin levels and anti-erythropoietin antibodies in patients with systemic lupus erythematosus (SLE). METHODS: The sera of 100 patients with SLE were investigated for serum erythropoietin levels and the presence of anti-erythropoietin antibodies by ELISA. Routine laboratory parameters such as peripheral blood count, relevant parameters of blood chemistry, and immunological parameters of SLE were recorded. RESULTS: Erythropoietin levels were significantly decreased in SLE patients when related to individual haemoglobin and haematocrit values (P<0.001), suggesting an inadequate erythropoietin response in SLE. Anti-erythropoietin antibodies were found in 46% of SLE patients, and erythropoietin levels (but not haemoglobin or haematocrit values) were significantly decreased in these patients compared with patients without anti-erythropoietin antibodies. Serum erythropoietin concentration as determined by ELISA was reduced in the presence of anti-erythropoietin antibodies. Furthermore, anti-erythropoietin antibodies also correlated with younger age, decreased serum levels of complement factors C3 and C4 and elevated anti-double-stranded DNA antibodies. CONCLUSIONS: We conclude that the anaemia of SLE is characterized by an inadequate erythropoietin response. Anti-erythropoietin antibodies are frequently present in SLE and interfere with the measurement of serum erythropoietin level. However, these antibodies are not associated with increased severity of SLE-associated anaemia.

Reciprocal translocation (3;5)(q26;q22) and possible BCHE gene involvement in an unusual myelogenous disorder with both myeloproliferative and dysplastic features.

We report on a 77-year-old male patient who presented with an unusual myelogenous disorder exhibiting both myeloproliferative and dysplastic features. The patient suffered from leukocytosis, eosinophilia, basophilia, transfusion dependent anemia, and rapidly progressing thrombocytopenia. Classical chromosome analysis and fluorescence in situ hybridization (FISH) revealed a reciprocal t(3;5)(q26;q22). Using yeast artificial chromosome (YAC) probes, the breakpoint on chromosome 3 was localized to the butyrylcholinesterase (BCHE) gene (3q26.1-q26.2). This gene has recently been implicated in the regulation of myeloid cells. Whether the BCHE gene was also involved in the deregulation of myelopoiesis, causing the unusual clinical picture in this case, remains unknown.

The mast cell as site of tissue-type plasminogen activator expression and fibrinolysis.

Recent data suggest that mast cells (MC) and their products (heparin, proteases) are involved in the regulation of coagulation and fibrino(geno)lysis. The key enzyme of fibrinolysis, plasmin, derives from its inactive progenitor, plasminogen, through catalytic action of plasminogen activators (PAs). In most cell systems, however, PAs are neutralized by plasminogen activator inhibitors (PAIs). We report that human tissue MC as well as the MC line HMC-1 constitutively produce, express, and release tissue-type plasminogen activator (tPA) without producing inhibitory PAIs. As assessed by Northern blotting, highly enriched lung MC (>98% pure) as well as HMC-1 expressed tPA mRNA, but did not express mRNA for PAI-1, PAI-2, or PAI-3. The tPA protein was detectable in MC-conditioned medium by Western blotting and immunoassay, and the MC agonist stem cell factor (c-Kit ligand) was found to promote the release of tPA from MC. In addition, MC-conditioned medium induced fibrin-independent plasmin generation as well as clot lysis in vitro. These observations raise the possibility that MC play an important role in endogenous fibrinolysis.

Treatment of hairy cell leukemia with cladribine (2-chlorodeoxyadenosine).

Hairy cell leukemia is a rare lymphoproliferative disorder resistant to conventional chemotherapeutic agents. Recently, the purine analogue cladribine (2-chlorodeoxyadenosine, 2-CdA) was introduced for the treatment of this disease. We report on 14 patients with hairy cell leukemia who were treated with 2-CdA at our department between 1993 and 1997. The patients received a single cycle of 2-CdA at a dose of 0.07 or 0.09 mg/kg/day by continuous infusion, over a seven-day period. Five patients were previously untreated, while the others had received prior treatment with interferon-alpha (seven patients), interferon-alpha and splenectomy (one patient) or interferon-alpha, splenectomy and pentostatin (one patient). Six patients achieved complete remission, three a good partial response and three partial remission. Two patients did not respond to treatment and one of them died from septicemia in aplasia. Relapse of the disease occurred in two patients. Side effects such as fever (WHO grade 2) and/or neutropenia (WHO grade 4) were noted in eight patients. Thus, 2-CdA is an effective treatment of hairy cell leukemia that can induce long lasting remissions in both, previously treated and untreated patients.

Low-molecular-weight heparin-induced skin necrosis.

We report a case of low-molecular-weight heparin (LMWH)-induced skin necrosis in a patient with chronic lymphatic leukemia. The patient had heparin-PF4 antibodies and the heparin-induced platelet activation (HIPA) test was positive, but platelet counts remained normal. Analysis of seven cases of LMWH-induced skin necrosis revealed that this complication occurred mostly in patients previously exposed to heparin, and that severe problems such as thrombocytopenia or thromboembolic complications were rare. This is in contrast to skin necrosis induced by unfractionated heparin (UFH), where a substantially higher number of patients suffered from thrombocytopenia and thromboembolism. In addition, most patients with UFH-induced skin necrosis were not pretreated with heparin. Therefore, it is possible that LMWH is less immunogenic than UFH and requires repeated exposure for induction of skin necrosis.

Metal ion-induced toxic histamine release from human basophils and mast cells.

Recent data suggest that distinct metal ions can be released from dental alloys or other biomaterials, and may cause toxic effects on various cells. In this study, the effects of 14 metal ions on histamine release from human blood basophils (n = 4), isolated tissue mast cells (lung n = 8, uterus n = 2, skin n = 1, gingiva n = 1), the basophil cell line KU-812, and the mast cell line HMC-1 were analyzed. Of the 14 metal ions, Ag+ (0.33 mM) and Hg2+ (0.33 mM) were found to induce release of histamine in blood basophils, KU-812, mast cells, and HMC-1. The effects of Ag+ and Hg2+ were dose dependent and were observed within 60 min of incubation. In primary mast cells and basophils, AU3+ (0.33 mM) also induced histamine release, whereas no effects of Au3+ on HMC-1 or KU-812 cells were seen. The other metal ions showed no effects on primary or immortal cells within 60 min. However, Pt4+ (0.33 mM) induced histamine liberation in HMC-1 and lung mast cells after 12 h. The Ag+- and Hg2+-induced rapid release of histamine from HMC-1 was associated with ultrastructural signs of necrosis, but not apoptosis. In contrast, prolonged exposure to Pt4+ (0.33 mM, 14 h) induced apoptotic cell death in HMC-1 cells, as assessed by electron microscopy and DNA analysis. Together, certain metal ions induce distinct cytopathogenic effects in mast cells and basophils. Whereas Ag+, Hg2+, and Au3+ cause direct toxicity, Pt4 causes cell death through induction of apoptosis. Whether such effects contribute to local adverse reactions to metal-containing biomaterials in vivo remains to be determined.

Phenotypic and functional characterization of mast cells derived from renal tumor tissues.

Mast cells (MCs) originate from multipotent hematopoietic progenitor cells. However, MCs in various organs are heterogenous in terms of mediator or receptor expression and response to diverse stimuli. We characterized the phenotype and functional properties of human renal mast cells (HRMCs). Tissue was obtained from 17 patients suffering from renal tumors (transitional cell carcinoma, n = 4; renal cell carcinoma, n = 13). HRMCs were isolated by collagenase digestion. Double staining with toluidine blue and immunofluorescence using monoclonal antibodies (mAbs) revealed expression of stem cell factor (SCF)-receptor (c-kit/CD117), CD9, CD29, CD33, CD43, CD44, CD54, and CD63 on HRMCs. In contrast, HRMCs were not recognized by mAbs to CD2, CD3, CD4, CD11b, CD14, CD15, CD16, CDw17, CD19, or CD23. HRMCs were also negative for CD116 (granulocyte-macrophage colony-stimulating factor [GM-CSF] receptor alpha), CD123 (interleukin [IL]-3Ralpha), CD121a (IL-1R type I), CD122 (IL-2Rbeta), and CD127 (IL-7R) and were also found to lack C5aR (CD88). Ligand-induced activation of HRMCs through immunoglobulin (Ig)E-R or SCF-R (c-kit) resulted in histamine secretion (control: <10%; alphaIgE, 1 microg/mL: 50.12 +/-5.18%; rhSCF, 100 ng/mL: 29.24 +/- 22.39), whereas recombinant C5a, erythropoietin (EPO), IL-1 through 10, and GM-CSF exerted no effects. As determined by in situ staining, HRMCs contained tryptase, but only low or undetectable amounts of chymase. Electron microscopy confirmed the presence of MCs in renal tissues and revealed a scroll-rich granule population in HRMCs. Together, HRMCs are tryptase+, C5aR- mast cells exhibiting phenotypic and functional properties similar to those of lung MCs.

The c-kit ligand stem cell factor and anti-IgE promote expression of monocyte chemoattractant protein-1 in human lung mast cells.

Recent data suggest that mast cells (MC) are involved in the regulation of leukocyte accumulation in inflammatory reactions. In this study, expression of leukocyte-chemotactic peptides (chemokines) in purified human lung MC (n = 16) and a human mast cell line, HMC-1, was analyzed. Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) showed baseline expression of monocyte chemoattractant protein (MCP)-1 mRNA in unstimulated MC. Exposure of MC to recombinant stem cell factor (rhSCF, 100 ng/mL) or anti-IgE (10 microgram/mL) was followed by a substantial increase in expression of MCP-1 mRNA. Neither unstimulated nor stem cell factor (SCF )-stimulated lung MC expressed transcripts for interleukin-8 (IL-8), macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, or RANTES by Northern blotting. The mast cell line HMC-1, which contains a mutated and intrinsically activated SCF-receptor, was found to express high levels of MCP-1 mRNA in a constitutive manner. Exposure of HMC-1 cells to rhSCF resulted in upregulation of MCP-1 mRNA expression, and de novo expression of MIP-1beta mRNA. The SCF-induced upregulation of MCP-1 mRNA in lung MC and HMC-1 was accompanied by an increase in immunologically detectable MCP-1 in cell supernatants (sup) (lung MC [<98%], control medium, 1 hour: 159 +/- 27 v SCF, 100 ng/mL, 1 hour: 398 +/- 46 pg/mL/10(6) cells; HMC-1: control, 1 hour: 894 +/- 116 v SCF, 1 hour: 1,536 +/- 265 pg/mL/10(6)). IgE-dependent activation was also followed by MCP-1 release from MC. MC-sup and HMC-1-sup induced chemotaxis in blood monocytes (Mo) (control: 100% +/- 12% v 2-hour-MC-sup: 463% +/- 38% v HMC-1-sup: 532% +/- 12%), and a monoclonal antibody (MoAb) to MCP-1 (but not MoAb to IL-8) inhibited Mo-chemotaxis induced by MC-sup or HMC-1-sup (39% to 55% inhibition, P < .05). In summary, our study identifies MCP-1 as the predominant CC-chemokine produced and released in human lung MC. MCP-1 may be a crucial mediator in inflammatory reactions associated with MC activation and accumulation of MCP-1-responsive leukocytes.

Immunophenotypic and functional characterization of human tonsillar mast cells.

Mast cells (MC) are proinflammatory immune cells residing in various organs. Tissue-specific heterogeneity of MC has been described. The aim of this study was to establish the phenotype and functional profile of human tonsillar mast cells (ToMC) and to compare ToMC with lung-, skin-, and uterus MC. Tonsillar tissue was obtained from 23 patients suffering from hyperplastic tonsils and dispersed by enzymatic digestion. With the use of a combined toluidine blue/immunofluorescence staining technique, isolated ToMC were found to react with monoclonal antibodies (mAb) to immunoglobulin E, CD9, CD43, CD44, CD46, CD54, CD55, and CD59, as well as mAb to stem cell factor (SCF) receptor (CD117/c-kit). ToMC were not recognized by mAb to other cytokine receptors or mAb to CD3, CD11b, CD14, CDw17, the skin MC marker CD88 (C5aR) or CD89 (Fc alphaR). Activation of ToMC by recombinant human (rh) SCF or anti-IgE resulted in histamine secretion, whereas no effects were seen with rhC5a, rh granulocyte-macrophage colony-stimulating factor, or rh interleukin-1 through -10. In summary, ToMC exhibit functional and phenotypic properties similar to lung- or uterus MC. Unlike skin MC, ToMC lack C5aR and are unresponsive to rhC5a.

Molecular and functional characterization of the urokinase receptor on human mast cells.

The urokinase receptor system is involved in several biological processes including extracellular proteolysis, cell invasion, and chemotaxis. Mast cells are multifunctional perivascular cells that play an important role in the regulation of microenvironmental events. We report that primary human mast cells and the human mast cell line HMC-1 express the receptor for urokinase. As assessed by Northern blotting and reverse transcription polymerase chain reaction technique, purified human lung mast cells and HMC-1 cells expressed urokinase receptor mRNA in a constitutive manner. Using a toluidine blue/immunofluorescence double staining technique and monoclonal antibodies, surface expression of urokinase receptor was demonstrable in lung, skin, uterus, heart, and tonsil mast cells, whereas the low density lipoprotein receptor-related protein was not detectable. Binding of monoclonal antibody VIM5 (recognizing the urokinase binding domain of urokinase receptor) to HMC-1 could be blocked by high molecular weight but not low molecular weight urokinase. Binding analyses performed with 123I-urokinase revealed expression of 271,000 +/- 55,000 high affinity urokinase binding sites per HMC-1 cell, with a calculated dissociation constant of 1. 29 +/- 0.3 nM. Purified urokinase induced dose-dependent migration of primary mast cells and HMC-1 in a chemotaxis assay without inducing release of histamine. The mast cell agonist stem cell factor also induced migration of HMC-1 and caused up-regulation of expression of urokinase receptor mRNA. Together, our data show that human mast cells express functional receptors for urokinase. Expression of urokinase receptors on mast cells may have implications for mast cell-dependent microvascular processes associated with fibrinolysis, migration, or local tissue repair.

Stem cell factor-induced downregulation of c-kit in human lung mast cells and HMC-1 mast cells.

Recent data suggest that local overexpression of the tissue-hormone c-kit ligand (stem cell factor [SCF]) is associated with accumulation of mast cells (MCs) and a decrease in expression of c-kit in the accumulated MCs [28]. In the present study, the effects of recombinant human (rh) SCF on expression of c-kit mRNA and c-kit protein in isolated human MCs and a human mast cell line, HMC-1, were analyzed. Incubation of isolated lung MC with rhSCF (100 ng/mL) for 120 minutes resulted in decreased expression of c-kit mRNA (optical density [OD], control: 100% vs. rhSCF: 37%). Almost identical results were obtained with HMC-1 cells (OD, control: 100% vs. rhSCF: 40 to 45%). As assessed by flow cytometry and monoclonal antibodies (mAbs) to c-kit, the SCF-induced decrease of c-kit mRNA in HMC-1 was associated with a substantial decrease in surface expression of c-kit (MFI, control: 100 +/- 21%, vs. MFI in cells incubated with rhSCF [100 ng/mL at 37 degrees C for 12 hours]: 8 +/- 2%, vs. MFI in cells incubated with rhSCF, 100 ng/mL, at 4 degrees C: 34 +/- 3%). The effects of rhSCF on c-kit expression in HMC-1 cells were dose- and time-dependent with maximum effects observed with 10-100 ng/mL of rhSCF after 4 to 12 hours. The SCF-dependent loss of c-kit was also accompanied by a decreased chemotactic response to rhSCF (control: 100%; rhSCF: 71 +/- 2%). This study shows that exposure of human lung MC and HMC-1 cells to recombinant SCF results in downregulation of c-kit mRNA and surface c-kit expression. These data may explain the partial loss of c-kit on MCs in areas of SCF overexpression.

Mast cell-lineage versus basophil lineage involvement in myeloproliferative and myelodysplastic syndromes: diagnostic role of cell-immunophenotyping.

Mast cells and blood basophils are distinct hemopoietic cells. They can be distinguished from each other and from all other lymphohemopoietic cells using antibodies against surface receptors or stored cytoplasmic molecules. In patients with myelodysplastic syndromes (MDS) or myeloproliferative syndromes (MPS), an elevation of metachromatically granulated cells (MCS) is frequently seen. These cells can be classified as basophils or mast cells using monoclonal antibodies (mAbs) against leukocyte antigens, including mast cell tryptase, c-kit (= mast cell growth factor [MGF] receptor), interleukin-3 receptor alpha chain (IL-3R alpha = CD123), and CD11b (C3biR). In a stable phase of MDS or MPS, the circulating MCS usually are basophils (histamine+, tryptase-, c-kit-, IL-3R alpha +, CD11b+). In an accelerated or terminal phase of disease, however, mast cell lineage involvement and circulating mast cell precursors (histamine+, tryptase+, c-kit+, IL-3R alpha-, CD11b-) are found in a subset of patients. The use of mAbs against mast cell antigens and granulocyte antigens is diagnostic in these patients.

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

Mast cells (MC), blood basophils (Ba) and monocytes (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 (SI < 0.2). Moreover, in contrast to other CD subgroups and other lineages, MC and HMC-1 differed from each other in cytokine R expression (SI = 0.286). Cytokine R detectable on HMC-1 but not MC were granulocyte-macrophage colony-stimulating factor (GM-CSFR)alpha(CD116), CD40, Apo-1/FAS(CD95) and gp130(CD130). Cytokine R detectable on Ba but not MC, were interleukin-3 (IL-3)R alpha(CD123), IL-1RII(CD121b), IL-2R alpha(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.

A case of malignant mastocytosis with circulating mast cell precursors: biologic and phenotypic characterization of the malignant clone.

The phenotypic and biologic properties of malignant cells in a case of aggressive mastocytosis with multi-organ involvement, circulating mast cell precursors and absence of skin infiltrates were analyzed. Circulating mast cell precursors were detected by immunostaining using antibodies against mast cell tryptase as well as by electron microscopy. These progenitors were tryptase+/chymase- (MCT) and accounted for 10 to 20% of nucleated mononuclear blood cells (MNC). A subset of them contained metachromatic granules. As assessed by combined toluidine blue/immunofluorescence staining, the granulated mast cell precursors were found to express CD9 (P24), CD33 (gp67) and CD44 (Pgp-1), but not basophil-related markers (CD11b (C3biR), CDw17 (lactosylceramide), CD123 (il-3R alpha))or monocyte-related antigens (CD14, CD15). Expression of the mast cell growth factor (MGF) receptor, c-kit(CD117), was also demonstrable, whereas the skin mast cell marker C5aR (CD88) could not be detected on mast cell precursors. The ligand of c-kit, recombinant human (rh) stem cell factor (SCF = MGF), induced histamine release from circulating mast cell progenitors, whereas rhC5a, a potent skin mast cell-/basophil-agonist, was ineffective over the dose-range (10(-9) to 10(-7(M)) tested. Analysis of mast cell antigens in malignant mastocytosis or mast cell leukemias may be helpful to establish a diagnosis and to determine the phenotype of the clone.

Differential expression of complement receptors on human basophils and mast cells. Evidence for mast cell heterogeneity and CD88/C5aR expression on skin mast cells.

Complement-dependent activation of immune cells is regulated by cell surface membrane receptors. In this study, expression of complement receptors (CR) on human blood basophils (n = 11), tissue mast cells (lung, n = 7; skin, n = 10; uterus, n = 4; tonsil, n = 3; heart, n = 10), and on respective human cell lines (basophil line KU-812, mast cell line HMC-1) was analyzed by the use of mAbs and indirect immunofluorescence. Normal blood basophils and KU-812 cells were found to express C5aR (CD88), membrane cofactor protein (CD46), decay-accelerating factor (CD55), and membrane attack complex inhibitory factor (CD59), as well as the previously recognized CR1 (CD35), CR3 alpha (CD11b), CR4 alpha (CD11c), and CR3/4 beta (CD18). Mast cells from all organs as well as HMC-1 cells expressed CD46, CD55, and CD59, but not CD11b, CD21, or CD35. The C5aR (CD88) was detectable on skin mast cells, a subset (5 to 15%) of cardiac mast cells, and on HMC-1 cells, but not on lung, uterus, or tonsillar mast cells (< 5%). Moreover, double immunoperoxidase staining (tryptase vs C5aR/CD88) revealed in situ expression of C5aR on skin, but not lung mast cells. Recombinant human (rh) C5a, at 10(-10) to 10(-7) M, induced secretion of histamine from basophils (rhC5a, 10(-8) M: 53.4 +/- 3.1% vs control < 5%) and from skin mast cells (rhC5a, 10(-8) M: 25.8 +/- 16.1% vs control < 10% histamine release), but not from other mast cells (rhC5a or control: < 10%, p > 0.05). The rhC5a-induced secretion of histamine from basophils and skin mast cells was inhibited by S5/1, a blocking Ab against CD88 (basophils: 37.2% to 75.1%; skin mast cells: 39.2% to 83.9% inhibition, p < 0.05). Together, this study shows that a) basophils and mast cells express a different profile of complement receptors, b) C5a-dependent mediator release in skin mast cells and basophils is mediated via CD88, and c) mast cells constitute a heterogeneous lineage in terms of expression of the C5a binding site CD88.

Response of L-929 fibroblasts, human gingival fibroblasts, and human tissue mast cells to various metal cations.

Recent data suggest that under certain conditions, various metal cations are released from dental alloys. These ions may produce adverse effects in various cell types in vivo. In this study, the cytopathogenic effects of 13 metal cations on murine L-929 fibroblasts, human gingival fibroblasts, and human tissue mast cells were analyzed in vitro. Several metal cations (dose range, from 0.0033 to 1.0 mmol/L) were found to induce dose-dependent inhibition of 3H-thymidine incorporation into cultured fibroblasts. The rank order of potency (lowest observed effect level, LOEL) for L-929 fibroblasts was: Ag+ > Pt4+ > Co2+ > In3+ > Ga3+ > Au3+ > Cu2+ > Ni2+ > Zn2+ > Pd2+ > Mo5+ > Sn2+ > Cr2+. A similar rank order of potency was obtained for primary human gingival fibroblasts: Pt4+ > Ag+ > Au3+ > In3+ > Ga3+ > Ni2+ > Co2+ > Zn2+ > Cu2+ > Cr2+ > Pd2+ > Mo5+ > Sn2+. In primary human mast cells, Ag+ and Au3+ caused dose-dependent toxic histamine release, whereas the other metal cations were ineffective over the dose range tested. To investigate the mechanism of metal cation-induced effects, we performed DNA as well as electron microscopic analyses on cultured fibroblasts. Both the DNA pattern and the ultrastructure of L-929 cells and gingival fibroblasts after exposure to cytopathogenic metal cations revealed signs of necrosis but no signs of apoptosis. Together, our data provide evidence that various metal cations produce dose-dependent cytopathogenic effects in distinct cell types, including human gingival fibroblasts and human tissue mast cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of human basophils and mast cells by multistep separation technique and mAb to CDw17 and CD117/c-kit.

Basophils and mast cells represent distinct cell lineages within the hemopoietic system. Based on the unique cell surface antigen profile of both cells, we have established methods which allow the reproducible purification to homogeneity (> 99%) of normal human basophil granulocytes from the peripheral blood and of mast cells from human dispersed tissues. Basophils (n = 9) were purified by current counterflow elutriation followed by depletion of monocytes with CD14 mAb conjugated to magnetic beads, and subsequent cell sorting for CD217+ cells. Basophil purity was 99.5 +/- 0.4% (range 98.7-99.9%). Mast cells were obtained from lung (n = 6), uterus (n = 1), mastocytosis bone marrow (n = 2), and human foreskin (n = 2). Mast cells were purified by collagenase digestion followed by current counterflow elutriation and sorting with CD117/c-kit mAb. Mast cell purity was 99.4 +/- 0.7% (range: 97.5-99.9%). Purified cells were more than 90% viable and were able to release histamine on induction with IgE plus anti-IgE. Furthermore, the PCR technique could be applied on pure cells and confirmed expression of high affinity IgE receptor (Fc epsilon R1) alpha chain mRNA. Thus, by combining isolation techniques including elutriation, magnetic cell depletion and cell sorting with mAb, functionally intact normal human basophils and mast cells can be enriched to homogeneity.