Philadelphia chromosome-negative acute hematopoietic malignancy: ultrastructural, cytochemical and immunocytochemical evidence of mast cell and basophil differentiation.

We describe a patient with fever and multiple osteolytic bone lesions accompanied by hypercalcemia, a duodenal ulcer, anemia, and thrombocytopenia. Bone marrow showed a dense infiltration by abnormal cells characterized by small basophil granula, erythrophagocytosis and nuclear atypia. These cells were positive for toluidine blue and partly for myeloperoxidase and chloroacetate esterase, expressed myeloid differentiation markers, and exhibited multiple numerical and structural chromosome aberrations. Molecular genetic analysis showed no breakpoint cluster region rearrangement. Electron microscopy demonstrated granula both of basophil and mast cell type. Concluding, in this patient an acute hematopoietic malignancy with many features of malignant mastocytosis but also with signs of a basophil differentiation. This is further support for a hematopoietic stem cell origin of human mast cells.

Interleukin-2 receptor expression in human mast cells and basophils.

Surgical human thymus, upper respiratory tract, lung and small and large bowel specimens were analyzed for the presence of interleukin 2 receptor (IL2-R)-positive cells. Histochemical (toluidine) and immunologic (anti-IL2-R monoclonal antibody) staining procedures revealed a distinct anti-IL2-R positivity in most of metachromatically staining cells. These positive cells were observed not only in tissues showing strong inflammatory reaction and mast cell hyperplasia, as in Crohn's disease, but also in those not histologically affected by pathologic conditions. This finding suggested that human mast cells, like T blast cells, express the p55 chain of IL2-R on their surface. To see whether IL2-R was being actively synthesized, a cell preparation rich in peripheral blood basophils (PBB), which are cells closely related to mast cells, was obtained. Ultrastructural analysis of PBB after indirect immunogold procedure revealed that the vast majority expressed the IL2-R. Moreover, the presence of intracellular reaction products in the cytoplasm of most membrane-positive PBB was indicative of active antigen synthesis. Furthermore, Northern blot analysis evidenced specific IL2-R mRNA in PBB, while its expression was augmented several times when PBB were cultured in the presence of stimulated T cell supernatant.

Human basophils selectively express the Fc gamma RII (CDw32) subtype of IgG receptor.

The role of IgG antibody in the sensitization of human basophils and mast cells to antigen is uncertain. To help resolve this uncertainty, we characterized by two-color fluorometric analysis the Fc receptors for IgG (Fc gamma R) on human basophils. Basophil-containing mononuclear cell fractions of atopic and nonatopic adult volunteers were incubated sequentially with fluorescein isothiocyanate-conjugated murine monoclonal IgE and biotinylated monoclonal antibodies (MAb) that bind specifically to the different Fc gamma R subtypes. Binding of biotinylated MAbs was visualized after subsequent incubation with phycoerythrin-strepavidin conjugate. Basophils did not react with a murine monomeric IgG2a, which binds specifically through its Fc to the high-affinity Fc gamma RI (CD64), or with MAbs specific for the low-affinity Fc gamma RIII (CD16). However, basophils reacted with a MAb specific for the low-affinity Fc gamma RII (CDw32). The profile of basophil Fc gamma R expression was not altered after brief IgE-mediated activation. In addition, pretreatment with gamma interferon, which induced expression of Fc gamma RI (CD64) on neutrophils, did not induce Fc gamma RI expression on basophils. These results indicate that the Fc gamma R present on basophils is exclusively of the Fc gamma RII (CDw32) subtype. The absence of the high-affinity Fc gamma RI (CD64) suggests that antigenic sensitization of basophils by monomeric IgG does not occur.

Pharmacokinetics and pharmacodynamic modeling of direct suppression effects of methylprednisolone on serum cortisol and blood histamine in human subjects.

Pharmacodynamic models for "directly suppressive" effects of methylprednisolone are based on the premise that receptor interactions of steroids are followed by immediate suppression of either the circadian secretion of cortisol or the constant rate recirculation of histamine-containing basophils that persists until inhibitory concentrations of methylprednisolone disappear. Methylprednisolone doses of 0, 10, 20, and 40 mg were given as the 21-succinate sodium salt in a balanced crossover study to six normal men. Plasma steroid concentrations and blood histamine were measured simultaneously. Both forms of methylprednisolone exhibited linear kinetic parameters. One dynamic model quantitates the baseline circadian pattern and the decline and return of cortisol with similar parameter estimates for all three dose levels. A similar model describes the monoexponential decline and the log-linear return to steady-state baseline of blood histamine. Similar inhibitory concentration values for both effects approximated the equilibrium dissociation constant of in vitro steroid receptor binding. The new models are more physiologically appropriate for these steroid effects than three other models that are commonly employed in pharmacodynamics. Steroid effects generally appear to be receptor mediated with either nongene immediate responses or gene-mediated delayed effects. These models allow quantitation of the rapid effects of steroids with simple equations and common fitted parameters for all steroid dose levels.

Spontaneous histamine release and histamine content in normal subjects and subjects with asthma.

Spontaneous histamine release (SHR) from basophils by simple incubation at 37 degrees C for 60 minutes and histamine content of basophils were assessed in normal subjects, patients with asthma, and methacholine-sensitive subjects without asthma (NAMS). SHR from basophils of normal subjects did not exceed 10% of the total histamine. A significantly higher SHR was observed in basophils from subjects with asthma than from normal subjects (p less than 0.002). Basophil SHR in patients with asthma not receiving medication was significantly greater than that in patients with asthma receiving medication (p less than 0.05). SHR from basophils in NAMS subjects was similar to that in normal subjects. SHR was highly dependent on temperature and Ca++ and Mg++ ions and appeared to be a slower event than anaphylactic release. The mean histamine content per basophil from normal subjects was 1.48 +/- 0.13 pg (mean +/- SEM). Basophils from subjects with asthma contained significantly less histamine than basophils from normal subjects (p less than 0.002). Histamine content per basophil from NAMS subjects was slightly lower than histamine content per normal basophil. No apparent relationship was found between the magnitude of SHR and the histamine content per basophil in the patients with asthma not receiving medication. Hypersensitivity to food or exercise does not appear to be essential for high SHR. High SHR appears to bear little, if any, relationship to cell damage or cell death. High SHR may be a factor that could serve as a marker for bronchial asthma. Further studies are needed to define the clinical relationship and pathophysiologic mechanism of SHR.

Medium pH determines the differentiation of human promyelocytic leukemia cells to basophils or eosinophils by culturing in a protein- and serum-free medium.

Cells containing large basophilic granules in the cytoplasm appeared after culturing human promyelocytic leukemia cells (HL-60) in a protein- and serum-free alkaline medium. These granules were stained with toluidine blue and alcian blue. Cells were morphologically and cytochemically similar to basophils. The existence of histamine in the cell lysates was detected in both uninduced and alkaline medium-induced HL-60 cells. After culturing HL-60 cells in a protein- and serum-free acidic medium, cells containing eosin-stained small granules (eosinophils) appeared. The differentiation of HL-60 cells to basophils or eosinophils may therefore depend on medium pH by culturing in a protein- and serum-free medium.

Modulation of human basophil growth in vitro by xiao-qing-long-tang (syo-seiryu-to), chai-pu-tang (saiboku-to), qing-fei-tang (seihai-to), baicalein and ketotifen.

Our previous study showed the inhibitory effect of Qing-Fei-Tang (Q.F.T.) and baicalein on the leukotriene (LT)B4 synthesis of human alveolar macrophages. It has recently been demonstrated that LTs support various cell growth, and basophil and its precursor numbers increase in atopic patients. Therefore, we examined the effect of anti-allergic drugs, including Q.F.T., Xiao-Qing-Long-Tang (X.Q.L.T.), Chai-Pu-Tang (C.P.T.), baicalein and ketotifen which have been used for treatment of bronchial asthma, on human basophil growth in vitro using cord blood mononuclear cells as a basophil precursor source and conditioned medium of T cell leukemia cell line Mo as a growth factor. Two-week cultured basophil numbers identified by alcian blue-safranin staining and those histamine contents assayed fluorometrically were inhibited by Q.F.T. (1.0 mg/ml), X.Q.L.T. (0.01-1.0 mg/ml), C.P.T. (0.01-1.0 mg/ml), baicalein (1-100 microM) or ketotifen (1-100 microM) in a dose-dependent manner while low dose (0.01-0.1 mg/ml) of Q.F.T. showed an enhancing effect on the basophil growth and the histamine content. However, LTB4 or LTC4 failed in restoring the basophil growth reduced by 1 mg/ml of C.P.T. or 100 microM of ketotifen. These results suggest that anti-allergic drugs may modulate basophil growth and differentiation in vitro and/or in vivo and therefore be useful and reasonable for controlling allergic diseases including bronchial asthma.

Structurally distinctive vasoactive intestinal peptides from rat basophilic leukemia cells.

Peptides recognized by rabbit antibodies to vasoactive intestinal peptide (VIP) were extracted from diisopropyl fluorophosphate-treated rat basophilic leukemia (RBL) cells and resolved by filtration on Sephadex G-25 in 50 mM acetic acid. The immunoreactive VIPs of RBL cells eluted from Sephadex G-25 at 35-41%, 53-60%, and 69-73% bed volume, but not at 63-68% as for the neuropeptide VIP1-28. The two forms of immunoreactive VIP larger than VIP1-28 reacted with antibodies to both VIP1-9 and VIP10-28, but the smallest was bound only by antibodies to VIP10-28. The smallest immunoreactive VIP was purified by ion-exchange and reverse-phase high-performance liquid chromatography, and the amino acid sequence was determined to be that of VIP10-28 with asparagine-free acid at the carboxyl terminus rather than the amide of VIP neuropeptide. Challenge of RBL cells with 1 microM ionophore A23187 at 37 degrees C released VIP10-28 rapidly to a mean of 75% at 5 min and 77% at 30 min. The VIP generated and released by mast cells thus consists of a mixture of peptides that all differ structurally from the neuropeptide VIP.

Basophils exhibit rearrangement of the bcr gene in Philadelphia chromosome-positive chronic myeloid leukemia.

Basophils were isolated from the peripheral blood of two patients with Philadelphia positive-chronic myeloid leukemia using monoclonal antibody Bsp-1 and fluorescence activated cell sorting. DNA blot analyses demonstrated rearrangement of the breakpoint cluster region gene in the isolated basophils, which suggests their leukemic origin. Isolated T cells from these patients that were cultured for 14 days in the presence of interleukin-2 lacked rearrangement of the breakpoint cluster region gene and are therefore unlikely to be derived from the chronic myeloid leukemia clone.

Sodium butyrate and a T lymphocyte cell line-derived differentiation factor induce basophilic differentiation of the human promyelocytic leukemia cell line HL-60.

Sodium butyrate induces basophilic differentiation of HL-60 promyelocytic leukemia cells that have been previously passaged in alkaline medium. A factor present in Mo conditioned medium (Mo-CM) acts synergistically with sodium butyrate to promote basophilic maturation in a dose-dependent fashion. The induced HL-60 cells exhibit nuclei at various stages of maturity and cytoplasmic granules staining azurophilic with May-Grünwald-Giemsa and metachromatically with toluidine blue. The histamine content of induced HL-60 cells is 50 ng/10(6) cells with sodium butyrate alone or 190 ng/10(6) cells with butyrate in combination with Mo-CM. Induced cells release histamine in response to anti-IgE and have receptors for the Fc portion of human IgE. The basophilic cell-differentiating activity present in Mo-CM appears to be distinct from several other cytokines including recombinant human interleukin-1 alpha, interleukin-2, interferon-gamma, interferon-alpha, murine interleukin-3, erythroid-potentiating activity, and purified human granulocyte/macrophage colony-stimulating factor. This is the first demonstration of a cell line that is capable of differentiation along the basophil lineage and could provide a useful model for examining biochemical and molecular events associated with basophil differentiation.

Cytochemical characterization of leukemic cells with numerous cytoplasmic granules.

Ten cases in which leukemic cells contained numerous cytoplasmic granules were examined by using a panel of cytochemical reactions. The diagnoses in the 10 cases were mast cell leukemia, chronic basophilic leukemia, and acute myeloid leukemia with basophilic differentiation in one case each, acute promyelocytic leukemia in two cases, acute megakaryoblastic leukemia in two cases, and blastic hairy cell leukemia in three cases. The cytochemical panel consisted of peroxidase, toluidine blue, chloroacetate esterase, aminocaproate esterase, tartrate-resistant acid phosphatase, and immunoalkaline phosphatase for platelet/megakaryocyte-specific antigen. The unusual cytologic features of leukemic cells in cases similar to our 10 cases have caused considerable diagnostic difficulties. In our 10 cases, however, the effective use of cytochemical studies helped to achieve accurate identification of the various types of leukemic cells. We conclude that the intelligent application of cytochemical techniques continues to be useful for the accurate cytodiagnosis of hematopoietic neoplasms.

Biochemical and morphological characterization of basophilic leukocytes from two patients with myelogenous leukemia.

Basophilic leukocytes from two patients with myelogenous leukemia were enriched to a purity of 10 to 45% by density gradient centrifugation. Ultrastructurally, these basophilic leukocytes contained segmented nuclei and granules with reticular patterns resembling those of normal basophils, and other granules with scroll and grating patterns resembling those of normal connective tissue mast cells. The 35S-labeled macromolecules isolated from these cells were approximately 140,000 m.w. Pronase-resistant proteoglycans bearing approximately 15,000 m.w. glycosaminoglycans. On incubation with chondroitinase ABC, nitrous acid, and heparinase, the 35S-labeled proteoglycans were degraded 50 to 84%, 16 to 43%, and 8 to 37%, respectively, indicating the presence of both chondroitin sulfate and heparin. As assessed by high performance liquid chromatography, the 35S-labeled chondroitin sulfate disaccharides liberated by chondroitinase ABC treatment were approximately 95% monosulfated chondroitin sulfate A and approximately 5% disulfated chondroitin sulfate E. The presence of heparin was confirmed by two-dimensional cellulose acetate electrophoresis of the 35S-labeled glycosaminoglycans. Cell preparations, enriched to 75% basophilic leukocytes by sorting for IgE+ cells, also synthesized 35S-labeled proteoglycans containing chondroitin sulfate and heparin. In one experiment, treatment of the cells with 1 microM calcium ionophore A23187 resulted in a 12% net release of both chondroitin sulfate and heparin containing 35S-labeled proteoglycans, a 57% net release of histamine, and the de novo generation of 8, 8, and 0.16 ng of immunoreactive equivalents of prostaglandin D2, leukotriene C4, and leukotriene B4, respectively, per 10(6) cells. Because only mast cells have been found to contain Pronase-resistant heparin proteoglycans, to generate PGD2 on cell activation, and to contain granules with scroll and grating patterns, these findings indicate that in some patients with myelogenous leukemia there are basophilic cells that possess properties of tissue mast cells.

[Determination of the heparin content in basophilic granulocytes by cytophotometry]. / Opredeleneie soderzhaniia geparina v bazofil'nykh granulotsitakh s pomoshch'iu tsitofotometrii.

A method for estimating heparin content in basophilic leucocytes by means of cytophotometry of alcian blue--heparin complex is proposed. Its application for measuring heparin amounts in basophils of healthy donors and of patients with allergies is demonstrated.

Stimulation of maturation of large immature histamine-containing basophilic cells from human peripheral blood, cord blood and bone marrow.

The development of FcR epsilon-bearing histamine-containing basophilic cells was studied in cultures of peripheral blood leucocytes and bone marrow leucocytes from normal individuals. The determination of basophilic cells was performed blindly. Before cultivation there were fairly similar numbers of basophilic cells in the samples from the three different sources (1.6 +/- 1.6 n = 13; 1.4 +/- 2.0 n = 21; 4.3 +/- 4.7 n = 8, respectively). During cultivation spontaneously appearing large blast-like basophilic cells were seen in good correspondence with a formation of histamine in the cultures (Spearman rank correlation coefficient = 0.9135, n = 13, P less than 0.001). This was more accentuated with bone marrow cells than with peripheral blood and cord blood cells. Conditioned medium (CM) was prepared from cells isolated from various tissues and stimulated by different means, e.g. peripheral blood from atopic individuals stimulated with allergen and unstimulated tonsil cells. Addition of the CM resulted in increased development of histamine-containing basophilic cells. Optimum stimulation was achieved with 10% CM. The basophilic stimulation by CM, as assessed as indices vs. unstimulated cultures, was more accentuated in cultures of peripheral blood cells than of bone marrow and cord blood cells (2.8 +/- 1.2; 1.8 +/- 0.5; 2.0 +/- 0.4, respectively). In contrast, the histamine formation was particularly evident in stimulated cultures of bone marrow cells, where more than four-fold increases of histamine were found. In bone marrow cells the histamine levels per basophilic cell also increased, whereas this was not the case in cord blood cells. A pronounced development of basophilic cells was achieved when using leukoagglutinin, provided the mitogen in the CM was eliminated. The formation of basophilic cells was prevented with mitomycin c and cycloheximide. In conclusion, the system described may provide important information on the development of histamine-containing basophilic cells at various maturation stages from different compartments, and mechanisms in a developing atopic disease.

Cell membrane glycoproteins of human mast cells: a biochemical comparison with basophils.

The relationship of mast cells (MCs) to other blood leucocytes, and to basophils in particular, is unclear. The relative distribution and abundance of cell surface glycoproteins of normal and neoplastic blood cells has been established as providing a "fingerprint" allowing assignment to a particular hemopoietic cell lineage. We have examined the major labeled glycoproteins of mast cells, basophils, HL-60 cells (granulocytic), and U937 cells (monocytic), after cell surface tritiation by the periodate-borohydride technique. Mast cells have a characteristic glycoprotein profile, different from that of basophils and other leukocytes; a major feature is the lack of the gp105-115 molecular weight band common to all other white blood cells. The data suggest that the tissue mast cell is more distantly related to other hemopoietic cells than has been previously recognized.

Some cytological aspects of bronchial asthma.

We report the correlation between blood eosinophilia obtained by chamber count, percent in peripheral blood and the resulting value of this percent by the total number of leukocytes, together with the nasal and sputum eosinophilia and basophils in blood, in 57 asthmatic patients. Seventeen of them were on treatment with systemic steroids. We found blood eosinophilia in 24 patients (42%), being the chamber count method the most reliable. There was a significant difference in blood eosinophils between symptomatic and asymptomatic patients (p less than 0.05) without systemic steroid treatment. We did not find correlation between eosinophilia and the type of asthma (intrinsic-extrinsic). Only 5 out of 40 patients had eosinophilia on nasal smear, all with rhinitic symptoms. We studied sputum eosinophils in 40 out of 57 patients. Thirty (75%) had eosinophilia while only 45% of them had blood eosinophilia. Neither did we find correlation between blood and sputum eosinophilia (p greater than 0.05). We also encountered basophilia in 11 patients (19.2%). The number of basophils was inferior in asymptomatic patients treated with systemic steroids than in asymptomatic without systemic steroid treatment. Any relation between number of basophils, kind of asthma or clinical status was not observed. We did not find correlation between number of basophils and eosinophils in blood (r = 0.24; p greater than 0.05).

Peripheral blood basophils, basophil progenitors, and nasal metachromatic cells in allergic rhinitis.

Relationships among mature blood basophils, blood basophil colony-forming units in culture (CFU-c), and nasal metachromatic cells (NMC) were investigated using hemopoietic and histochemical techniques in 29 patients with allergic rhinitis and in 7 nonatopic control subjects. Total blood granulocyte and basophil CFU-c were significantly elevated in atopy; the highest levels of basophil CFU-c were found in patients with low total NMC counts in nasal scrapings (Group I), compared with those with intermediate (Group II) or high (Group III) counts: 10 +/- 3 basophil CFU-c per 10(6) cells in 10 Group I patients, 4 +/- 2 in 10 Group II patients, 3 +/- 1 in 9 Group III patients, and 0.1 +/- 0.1 in nonatopic control subjects (p less than 0.05). Mean histamine content and frequency of histamine-positive granulocyte colonies correlated with counts of basophils in colonies (r = 0.864, p less than 0.001). Peripheral blood basophils, which stained metachromatically with toluidine blue at pH 0.5 after Mota's lead acetate but not after formalin fixation, were highest in atopic Group III and lowest in Group I; a similar relationship was observed only for NMC, which also failed to stain after formalin fixation. Metachromatic cells in colonies were similar to formalin-sensitive NMC and to peripheral blood basophils in their sensitivity to different fixatives. Nasal symptoms correlated inversely with the number of basophil CFU and directly with the number of either formalin-sensitive NMC or peripheral blood basophils. These findings confirm and extend evidence for increased nasal metachromatic cells, basophilia, and alterations in basophilopoiesis in atopy.

Action of ketotifen on histamine release and intracellular cAMP levels in basophil cultures from pollinic subjects.

Basophils from peripheral blood of 22 pollinic patients were isolated and were cultured in the presence of different concentrations of ketotifen. We studied the effect of this drug on the specific histamine release (Technicon autoanalyzer) and the levels of intracellular cAMP during days 1, 6 and 12 of culture period. We observed that ketotifen induced an inhibition of specific histamine release throughout the culture days, but was greater on the 12th day. The cAMP levels likewise increased during the period of culture in the presence of ketotifen.