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A slow reacting substance produced by rat basophilic leukemia cells, treated with ionophore A23187, was characterized by spectroscopic methods, enzymatic conversions, and chemical degradations as 5-hydroxy-6-S-cysteinylglycyl 7,9,11,14-eicosatetraenoic acid (leukotriene D). gamma-Glutamyltranspeptidase [gamma-glutamyltransferase; (5-glutamyl)-peptide: amino-acid 5-glutamyltransferase, EC 2.3.2.2] converted leukotriene C to a product identical to leukotriene D. This suggests that the stereochemistry of the arachidonyl moiety of leukotrienes C and D is the same [5(S)-6(R)-7,9-trans-11,14-cis]. Leukotriene D induces a faster contraction and, on a molar basis, is more potent than leukotriene C in the isolated guinea pig ileum bioassay.

In experiments using an immunofluorescent method to localize human eosinophil granule major basic protein (MBP) in cells and tissues, a small number of cells stained for MBP that subsequently could not be identified as eosinophils. Because the Charcot-Leyden crystal protein, another eosinophil protein, was recently identified in basophils, we tested whether MBP might also be a constituent of blood basophils. Highly purified, eosinophil-free basophil suspensions were prepared using the fluorescence-activated cell sorter (FACS) to sort basophil-containing mononuclear cell preparations stained with fluorescein-conjugated sheep IgG anti-human IgE antibody. Using these FACS-purified basophils, we found that: (a) enrichment for surface IgE-positive cells (greater than 95% basophils) by FACS also enriched for cells staining for MBP by immunofluorescence; (b) MBP appeared to be localized in the histamine-, heparin-containing granules; (c) significant quantities of MBP were measurable by radioimmunoassay (RIA) in freeze-thaw detergent extracts of purified basophils; and (d) RIA dose-response curves for extracts of purified eosinophils and basophils had identical slopes. The MBP content of basophils from normal individuals averaged 140 ng/10(6) cells, whereas purified eosinophils from normal donors and patients with the hyper-eosinophilic syndrome averaged 4,979 and 824 ng/10(6) cells, respectively. MBP was also detected by immunofluorescence and RIA in cells obtained from a patient with basophil leukemia, although the MBP content for basophil leukemia cells was lower than that for normal basophils. We conclude that basophils contain a protein that is immunochemically indistinguishable from eosinophil granule MBP.

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.

As reported previously, and confirmed here in 26 donors, the serum IgE level (2.6-5,500 ng/ml) correlates well (rs = 0.95, P less than 0.001) with the in vivo number of IgE molecules/basophil (6,000-600,000). The total number of IgE receptors/basophil was monitored by incubating them with an IgE-rich serum (15 microgram/ml), quantitatively stripping IgE from the cells at pH 3.7, and measuring eluted IgE by a direct radioimmunosorbent test. Saturation of receptors for each donor was achieved with 15 nM IgE (3 microgram/ml). The proportion of receptors occupied in vivo correlated with the serum IgE (rs = 0.84, P less than 0.001) whereas the average association constant of the receptors was independent of serum IgE and ranged from 7.1 X 10(8)/M to 2.8 X 10(10)/M, averaging 7.7 X 10(9)/M. Unexpectedly, the total number of IgE receptors/basophil was closely related to the serum IgE level. (rs = 0.92, P less than 0.001). Thus, either there is genetic association between serum IgE and the number of basophil IgE receptors, or, more likely, the receptor number is modulated by the serum IgE concentration.

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.

The platelet binding characteristics of platelet-activating factor (PAF), a basophil-derived lipid that causes aggregation and secretion in rabbit platelets, were studied. In an assay in which binding was quantitated by loss of PAF activity, suspensions of washed rabbit platelets rapidly removed PAF from solution. Rabbit erythrocytes, lymphocytes, and neutrophils also bound PAF, but their binding capacity was less than that of the platelets. PAF binding to rabbit platelets was saturable and dependent on temperature and concentration of PAF and platelets but not on the presence of extracellular Ca2+. Initial rates of PAF binding and platelet secretion were identical, although temperature dependence studies suggested that binding was not the rate-controlling step in PAF-induced platelet secretion. Analysis of binding kinetics suggested that a PAF concentrations saturable for platelet secretion, only some of the available platelet binding sites were occupied.

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.

Mononuclear cells from the peripheral blood of patients with chronic myelocytic leukaemia were grown and expanded in liquid culture in the presence of Con A-conditioned medium. An accelerated development of cells of the basophilic lineage was observed and resulted in the appearance of 85% mature basophils after 14 days of incubation. Transmission electron microscopy of developing basophils showed changes in the nucleus and active granule formation in the cytoplasm. By scanning electron microscopy, the immature cells were relatively smooth in comparison with the mature basophils which showed membranous microvilli. The chemical content of the cells at different days of culture was detected by X-ray microanalysis. Immature cells were characterized by a high level of phosphorus with a low level of sulphur. As maturation progressed, the amount of phosphorus decreased, while the level of the sulphur increased, reaching its highest peak in the mature basophils. The different amount of sulphur found in the cells during the maturational process most probably represents the amount of heparin located in the cell granules. This finding may be useful for studying the influence of growth factors on the development and differentiation of human basophils.

During IgE-mediated events, basophils secrete small molecular weight mediators (histamine, slow reacting substance, Eosinophil chemotactic factor) which are thought to participate in inflammatory processes. We here describe the IgE-mediated secretion of large molecular weight mediators which have the potential for the activation of the Hageman factor dependent pathways, and the generation of biologically active peptides. These large molecular weight basophil derived mediators may, through the activation of the Hageman factor dependent pathways, influence mechanisms which participate in both acute and chronic cell-mediated inflammatory processes. We suggest that these proteases may participate not only as mediators of the immediate hypersensitivity reaction, but may also function in important aspects of the entire inflammatory response.

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.

Charcot-Leyden crystals (CLC) are currently believed to be unique to the eosinophil and a hallmark of active eosinophilic inflammation or proliferation. The distinctiveness of the CLC to the eosinophil was questioned in 1965 by Archer and Blackwood (9), but their demonstration of CLC formation in basophils was ignored and later dismissed (1) as being the result of eosinophil contamination of basophil-enriched cell suspensions. We reexamined this question and showed that basophils obtained from the peripheral blood of normal individuals form CLC and that basophils contain a protein that is immunochemically indistinguishable from eosinophil CLC protein. These conclusions are based upon the findings that (a) crystal formation in basophils was demonstrated by specific histochemical staining of crystal-containing cells in highly enriched basophil suspensions prepared by fluorescence-activated cell sorter (FACS) purification of surface IgE-positive cells, (b) that enrichment for surface IgE-positive cells (primarily basophils) by the FACS also enriched for cells staining positively by immunofluorescence for eosinophil CLC protein, and (c) that CLC protein was measured by radioimmunoassay in cell extracts prepared from purified basophil suspensions containing 97-99% basophils and absolutely no contaminating eosinophils. These basophil extracts contained a protein immunochemically indistinguishable from eosinophil CLC protein. Based upon these findings, the CLC or the protein comprising the crystal (lysophospholipase) can no longer be considered as distinctive to the eosinophil. We must now consider the possibility that the presence of CLC in tissues, sputum, or stool may also represent basophil involvement in disease processes.

Proteoglycans of 300 000 mol.wt. were isolated from dispersed rat basophil tumour cells after labelling of the sulphated mucopolysaccharides with 35S in vitro:90% of the 35S-labelled mucopolysaccharides were extracted at high salt concentration. Alkali degradation of the 35S-labelled proteoglycans yielded glycosaminoglycan chains of 40 000 mol.wt. The composition of the salt-extracted 35S-labelled mucopolysaccharides, as defined by parallel or sequential degradation with chondroitinase AC, chondroitinase ABC and heparinase and resolution of the disaccharide-digestion products obtained with chondroitinase AC, was 48--61% chondroitin 4-sulphate, 20--30% dermatan sulphate, 10--15% heparin and 7--9% chondroitin 6-sulphate. Most of the salt-extracted 35S-labelled mucopolysaccharides were highly charged, with heparin and chondroitin 6-sulphate being relatively uniform in this regard, whereas chondroitin 4-sulphate and dematan sulphate exhibited a range of charge characteristics. The diversity of sulphated mucopolysaccharides present in the rat leukaemic basophil is in contrast with the predominance of heparin in the rat mast cell.

Platelet activating factor obtained from antigen-stimulated rabbit buffy coat leukocytes containing 10% IgE-sensitized basophils was shown by gas-liquid chromatographic and mass spectrometric analysis of products after acetolysis and by base and acid hydrolysis to be acetyl-alkylglyceryl phosphorylcholine. The alkyl side chains were composed of octadecyl (greater than 90%) and hexadecyl (less than 10%) residues.

The product of the postformalin ammoniacal silver reaction, which has been claimed to distinguish lysine-rich from arginine-rich histones with the light microscope on the basis of a color difference, was examined in developing erythroid cells of chick bone marrow with the electron microscope. Stem cells and early erythroblasts exhibit no, or little, ammoniacal silver reaction product, while small basophilic erythroblasts, polychromatophilic erythrocytes, and reticulocytes exhibit an increasing amount of reaction product as maturation proceeds. The reaction product is in the form of discrete electron-opaque particles associated with heterochromatin. The ammoniacal silver reaction in the erythroid cell series is interpreted as reflecting either the accumulation of newly synthesized arginine-rich histones or changes in the availability of reactive sites in preformed histones.

The central 95 percentile estimates of the normal white cell types (as determined by a standard differential count) were calculated from 777 normal individuals. The results were divided into groups by age and sex and expressed both as percentages and as absolute numbers of cells.A similar survey was made using the prototype of a new automated differential counting method (Hemalog-D(TM)). The two sets of laboratory values showed no statistically significant differences regarding age or sex and were strikingly similar in most cell types with the exception of band and juvenile forms of granulocytes. As expected, the normal range using the manual technique was somewhat broader than that found with the automated method. In addition to being the first comparison of machine counts with visual differentials this study provides additional insight into the age distributions of normal cells.

Intermediate lymph (efferent from the prefemoral lymph node) was collected for 600 hr from both flanks of each of four sheep that had an autograft of skin on the left flank and a homograft of skin on the right flank. 8 days after the grafts had been applied considerable numbers of large basophilic cells, apparently identical with those that appear during immune responses to conventional antigens, appeared in the lymph draining from the homografts. No such cells appeared in the lymph draining from the autografts. At this time the homografts were already showing signs of rejection and were apparently dead well before the cellular response in the lymph reached a peak, about 350 hr (14-15 days) after the homografts had been applied. During the peak of the response up to 40% of the cells in the lymph were basophilic cells and in one experiment such cells were leaving the lymph node at a rate of 200 million per hr. Peripheral lymph (afferent to the popliteal lymph node) draining from the sites of homografts of skin was collected from five sheep. This lymph contained few white cells (<1000 per mm(3)) and showed only an insignificant lymphoid cell reaction. Although the percentage of macrophage-like cells was increased significantly there were few signs of a lymphoid cell reaction; the lymph also contained much amorphous debris. Experiments in which the basophilic cells from the efferent lymph were labeled in vitro with thymidine-(3)H and returned to the sheep by intravenous injections were carried out in six sheep. The presence of the labeled cells in the grafts, blood, and other tissue was detected by liquid scintillation counting of nucleic acid extracts of biopsy and postmortem material and by radioautography. 2-3 labeled cells out of every 1000 injected entered the homografts but hardly any entered the autografts. However, labeled basophilic cells that had originated in response to bacterial antigens entered the homografts with equal facility. It is thus hard to believe that the immunological specificity of a lymphoid cell endows it with a specific "homing" capability. Furthermore, in all the experiments the specific radioactivities of the nucleic acids extracted from the blood mononuclear cells were approximately of the same order as those of the nucleic acids extracted from the homografts. It was concluded that most of the mononuclear cells that infiltrate homografts represent a random selection from the mononuclear cell population of the blood.