Human uterine mast cells. Isolation, purification, characterization, ultrastructure, and pharmacology.

As part of an ongoing investigation of human mast cell heterogeneity, we have isolated, partially purified, and characterized the uterine mast cell and compared it with mast cells isolated from other organs. The average histamine content of myometrium and leiomyofibroma obtained from hysterectomies was 2.1 +/- 0.3 (mean +/- SEM) microgram/g of tissue (n = 10), and the histamine content of the two tissues did not differ significantly. A mild collagenase, hyaluronidase, and DNase digestion was used to disperse the uterine mast cells, with an average yield of 9.5% (range, 0 to 21%). The average histamine/uterine mast cell was 2.1 +/- 0.2 pg (n = 3), and 61 +/- 7% (n= 3) of the uterine mast cells survived overnight culture. Early purification efforts with Percoll gradients have yielded up to 80% pure uterine mast cells, with an average of 27 +/- 10% (n = 5). Uterine mast cells released histamine in response to the secretogogues anti-IgE and A23187 but did not respond to substance P or to the basophil secretogogues FMLP, C5a, and 12-O-tetradecanoylphorbol-13-acetate. After 1 microgram/ml anti-IgE stimulation, the uterine mast cell appeared to make significant quantities of PGD2 (89 +/- 26 ng/10(6) cells, n = 6) (p less than 0.05), as assayed by RIA. Simultaneously, leukotriene C4 release was 45 +/- 15 ng/10(6) cells, (n = 6) (p less than 0.05), as assayed by RIA. Combined gas-chromatography mass spectroscopy analysis of anti-IgE-stimulated cell supernatants confirmed the production of PGD2. In pharmacologic studies, isobutyl-methylxanthine and isoproterenol blocked anti-IgE-induced histamine release. The uterine mast cell is similar to the lung mast cell in terms of response to secretogogues and release of arachidonic acid metabolites. Ultrastructurally, the uterine mast cell contains scroll granules, crystal granules, combined granules, homogeneously dense granules, and large lipid bodies, many with focal lucencies within them. Particle granules, most frequently present in gut mast cells of mucosal origin, were absent from uterine mast cells. Although certain features are analogous to the ultrastructure of skin or lung mast cells, the combination of structures is distinctive for uterine mast cells.     

Arachidonic acid metabolism in purified human lung mast cells

Arachidonic acid metabolism has been explored in preparations of purified human lung mast cells prelabeled with arachidonic acid (AA). Cells were of 83 to greater than 96% purity, and each experiment was performed with four to six different preparations of mast cells. After overnight culture of the purified cells in the presence of 3H-AA, followed by extensive washing in buffer, mast cell uptake of labeled AA was 61.4 +/- 14.8 pmol/10(6) cells with 21 +/- 2.4% of the label in phospholipids, 73 +/- 2.1% in neutral lipids, and 3.6 +/- 0.8% as free AA. Analysis of the distribution of radioactivity in phospholipid classes revealed 51.4 +/- 5.5% of the label in phosphatidylcholine, 14.5 +/- 1.6% in phosphatidylinositol, 12.0 +/- 3.0% in phosphatidylethanolamine, and 9.1 +/- 2.4% in sphingomyelin, with the rest in other phospholipid classes. Challenge of these cells with an optimal concentration of anti-IgE led to the release of 20 +/- 4.0% of cellular histamine and to a reduction in labeled phosphatidylcholine and phosphatidylinositol to 75.5 +/- 8.8% and 84.2 +/- 4.5% of the control levels, respectively, (p less than 0.05); anti-IgE challenge produced no statistically significant change in the quantities of other labeled phospholipids. Activation of human lung mast cells with anti-IgE led to the release of 3.4 +/- 1.3% of the cellular 3H as AA and AA metabolites (1.5 +/- 0.6% as unmetabolized AA) in conjunction with 16 +/- 4.3% of the cellular histamine. Although activation of human lung mast cells with ionophore A23187 caused 70 +/- 1.1% histamine release, a similar quantity of AA and AA metabolites was released (a total of 4.0 +/- 0.8% with 2.3 +/- 1.5% as unmetabolized AA). Analysis of the released metabolites by liquid scintillation spectrometry after high performance liquid chromatography separation showed that approximately equal amounts of metabolites were produced after mast cell activation with anti-IgE and ionophore A23187. In this series of experiments approximately equal amounts of cyclooxygenase and lipoxygenase products were generated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the anti-inflammatory effect of FK-506 on human mast cells.

We have examined the effects of FK-506 and of the struturally related macrolide rapamycin, which bind with high affinity to a specific binding protein (FKBP), to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized inflammatory mediators (sulfidopeptide leukotriene C4 and prostaglandin D2) from mast cells isolated from human lung parenchyma. FK-506 (0.1 to 300 nM) concentration dependently inhibited histamine release from lung parenchymal mast cells activated by anti-IgE. FK-506 was more potent in lung mast cells than in basophils (IC50 = 1.13 +/- 0.46 nM vs 5.28 +/- 0.88 nM; p less than 0.001), whereas the maximal inhibitory effect was higher in basophils than in lung mast cells (88.4 +/- 2.5% vs 76.4 +/- 3.8%; p less than 0.01). FK-506 had little or no inhibitory effect on histamine release from lung mast cells challenged with compound A23187, whereas it completely suppressed A23187-induced histamine release from basophils. FK-506 also inhibited the de novo synthesis of 5-lipoxygenase (sulfidopeptide leukotriene C4) and cyclo-oxygenase (prostaglandin D2) metabolites of arachidonic acid from mast cells challenged with anti-IgE. Unlike in basophils, Il-3 (3 to 30 ng/ml) did not modify anti-IgE- or A23187-induced histamine release from lung mast cells nor did it reverse the inhibitory effect of FK-506. Rapamycin (3 to 300 nM) had little or no effect on the release of histamine from lung mast cells, but it was a competitive antagonist of the inhibitory effect of FK-506 on anti-IgE-induced histamine release from human mast cells with a dissociation constant of about 12 nM. These data indicate that FK-506 is a potent anti-inflammatory agent that acts on human lung mast cells presumably by binding to a receptor site (i.e., FKBP).     

Distribution, density and heterogeneity of canine mast cells and influence of fixation techniques

 The present study was carried out to determine the physiological distribution of mast cell numbers and types in the dog according to tissue location, staining and fixation methods. Tissue samples from stomach, duodenum, lung, lymph node, skin and uterus were evaluated. Samples were fixed in formalin as well as in Carnoy’s fluid. The average number of mast cells was determined using a metachromatic staining method. Protease content of mast cells was examined with a double enzyme-immunohistochemical staining technique, using a histochemical reaction for chloroacetate esterase to detect chymase activity and an immunohistochemical staining method for the detection of tryptase. Canine mast cells can be subdivided into formalin-sensitive and -resistant mast cells. Three subtypes were identified according to their content of the mast cell-specific proteases tryptase (T) and chymase (C): T-, TC- and C-mast cells. Significant differences regarding the distribution of mast cell subtypes as well as the influence of the fixation method can be observed. This underlines the fact that data regarding mast cell heterogeneity from other species, obtained by different fixation methods, are not comparable. This fact has to be taken into consideration when evaluating mast cell subtypes under pathological conditions. Accepted: 29 January 1998     

Release of chemical mediators from partially purified human lung mast cells.

Human lung mast cells dispersed by enzymatic digestion of human lung fragments were concentrated to greater than 50% purity by sedimentation in isopycnic and velocity gradients. The dispersed lung mast cells had a characteristic ultrasturctural appearance including granules with a scroll or reticular structural appearance including granules with a scroll or reticular structure surrounded by perigranular membranes. Histamine and preformed eosinophilotactic activity sedimented with mast cells on isopycnic gradients, and mast cells and these mediators were separated from the bulk of the other lung cells after velocity gradient sedimentation. The histamine content of isolated lung mast cells was calculated to range from 1.0 to 5.5 pg/cell. The quantity of SRS-A generated with anti-IgE or specific antigen was relatively limited but confined to the mast cell-rich fractions and associated with release of histamine and eosinophilotactic activity.     

Lipopolysaccharides and lipoteichoic acids stimulate rat mast cells to cysteinyl leukotriene synthesis

We have investigated the ability of lipopolysaccharides (LPS) and lipoteichoic acids (LTA) to induce rat peritoneal mast cells to degranulation and histamine release, and to cysteinyl leukotriene (LT) generation. We have stated that LPS Salmonella Enteritidis, LPS Escherichia coli O111:B4 and LPS E. coli O55:B5 did not activate rat mast cells to degranulation and histamine release. However, LPSs induced LT synthesis and secretion; the strongest stimulant to generation of LT was LPS E. coli O55:B5 (concentration of LT in supernatant was 830.5 +/-15.2 pg/ml). We have also observed that LTA Staphylococcus aureus and LTA Bacillus subtilis stimulated rat mast cells to degranulation and histamine secretion, even though the percentage of the releases histamine was relatively low (10.0 +/- 1.4 and 10.4 +/- 5.4 at antigen concentration, respectively). At the same time, LTA of both of the bacterial species strongly activate LT generation by mast cells (concentrations of LT in supernatants were 777.9 +/- 11.2 pg/ml and 734.0 +/- 38.3 pg/ml, respectively, at the antigen concentration 50 ng/ml). Our results have shown that LPS oraz LTA activate rat mast cells to secretion of proinflammatory mediators.     

Co-culture of human lung-derived mast cells with mouse 3T3 fibroblasts: morphology and IgE-mediated release of histamine, prostaglandin D2, and leukotrienes

Human mast cells, dispersed from lung tissue by proteolytic treatment and enriched to a purity of 23 to 68% by density-gradient centrifugation, were maintained ex vivo for up to 13 days when co-cultured with mouse skin-derived 3T3 fibroblasts in RPMI 1640 containing 10% fetal calf serum. The human mast cells were adherent to the fibroblast cultures within 2 to 4 hr after seeding, and after 7 days of co-culture were localized between the layers of fibroblasts. The cell surfaces of the mast cells and the fibroblasts did not form tight junctions, but rather approached within 20 nm of each other. The co-cultured mast cells did not divide; they maintained their cellular content of histamine and TAMe esterase and resembled in vivo mast cells in that their secretory granules exhibited scroll patterns and their nuclei were oval. Both the freshly isolated and the co-cultured mast cells responded to activation with anti-human IgE by exocytosing histamine and generating and releasing arachidonic acid metabolites. When freshly isolated mast cells were activated immunologically, they exocytosed 38 +/- 8% of their total histamine content and released 28 +/- 1.9 ng (mean +/- range, n = 2) of immunoreactive equivalents of prostaglandin D2 (PGD2) per microgram of total cellular histamine, but did not generate significant amounts of either leukotriene C4 (LTC4) or leukotriene B4 (LTB4). The 1-wk co-cultured mast cells, on the other hand, exocytosed 43 +/- 2.4% of their total histamine content, and released 86 +/- 10, 43 +/- 20, and 5.2 +/- 5.2 ng (mean +/- SD, n = 4) of immunoreactive equivalents of PGD2, LTC4, and LTB4, respectively, per microgram of histamine. Thus, human lung-derived mast cells can be maintained ex vivo when co-cultured with fibroblasts, and, when treated with anti-IgE, they metabolize arachidonic acid via both the cyclooxygenase and the 5-lipoxygenase pathways.     

Potent histamine-releasing activity of atrahagin, a novel snake venom metalloproteinase

Poisonous snakebite wound is a popular disease worldwide. However, the pathogenesis remains unclear. In the present study, a novel metalloproteinase atrahagin in Chinese cobra (Naja atra) snake venom was purified, using heparin-sepharose followed by Superdex 75 gel filtration chromatography. Apart from its alpha-fibrinogenase activity, atrahagin potently activated human colon, lung and tonsil mast cells with the net histamine release being 25.9+/-4.4, 17.0+/-1.9, 13.2+/-3.6%, respectively. Time course studies revealed that the peak histamine release induced by atrahagin occurred at 12, 12 and 8 min following incubation of the enzyme with colon, lung and tonsil mast cells, respectively. The response of mast cells to atrahagin was abolished by preincubation of the cells with metabolic inhibitors or pertussis toxin, and by removal of Ca2+ and Mg2+ from the challenge buffer. In conclusion, activation of human mast cells by atrahagin indicated that the enzyme might contribute to the pathogenesis of snakebite wound.     

The human recombinant c-kit receptor ligand, rhSCF, induces mediator release from human cutaneous mast cells and enhances IgE-dependent mediator release from both skin mast cells and peripheral blood basophils.

The gene product of the steel locus of the mouse represents a growth factor for murine mast cells and a ligand for the c-kit proto-oncogene receptor, a member of the tyrosine kinase receptor class of oncogenes (for review, see O. N. Witte. 1990. Cell 63:5). We have studied the effect of the human recombinant c-kit receptor ligand stem cell factor (rhSCF) on the release of inflammatory mediators from human skin mast cells and peripheral blood basophils and compared its activity to that of rhIL-3, rhSCF (1 ng/ml to 1 microgram/ml) activated the release of histamine and PGD2 from mast cells isolated from human skin. Analysis by digital video microscopy indicated that purified human skin mast cells (84 +/- 5% pure) responded to rhSCF (0.1 to 1 microgram/ml) challenge with a rapid, sustained rise in intracellular Ca2+ levels that was accompanied by secretion of histamine. A brief preincubation (10 min) of mast cells with rhSCF (0.1 pg/ml to 1 ng/ml) significantly enhanced (100 +/- 35%) the release of histamine induced by anti-IgE (3 micrograms/ml), but was much less effective on IgE-mediated release of PGD2. In contrast, a short term incubation with rhSCF did not potentiate the secretion of histamine activated by substance P (5 microM). A 24-h incubation of mast cells with rhSCF did not affect the release of mediators induced by anti-IgE (3 micrograms/ml), probably due to receptor desensitization, rhSCF (1 ng/ml to 3 micrograms/ml) neither caused release of histamine or leukotriene C4 (LTC4) release from leukocytes of 14 donors, nor induced a rise in intracellular Ca2+ levels in purified (greater than 70%) basophils. Brief preincubation (10 min) of leukocytes with rhSCF (1 ng/ml to 3 micrograms/ml) caused an enhancement (69 +/- 11%) of anti-IgE-induced release of histamine that was significant at concentrations as low as 3 ng/ml (p less than 0.05), whereas it appeared less effective in potentiating IgE-mediated LTC4 release. In contrast, a prolonged incubation (24 h) with rhSCF (0.1 pg/ml to 100 ng/ml) did not enhance the release of histamine or LTC4 induced by anti-IgE (0.1 microgram/ml), whereas rhIL-3 (3 ng/ml) significantly potentiated the release of both mediators.(ABSTRACT TRUNCATED AT 400 WORDS)     

Histamine-containing cells obtained from the nose hours after antigen challenge have functional and phenotypic characteristics of basophils.

The pattern of mediators and appearance of cells that stain with alcian blue during human experimental early and late phase allergic reactions suggest that basophils accumulate in nasal secretions within hours of local Ag stimulation. To further explore whether the histamine containing cells that enter the nose after Ag challenge are mast cells or basophils, we studied their functional and phenotypic characteristics. Approximately 24 h after intranasal Ag provocation of subjects with allergic rhinitis, nasal lavage was performed, and the cells were isolated for degranulation studies, analysis of surface Ag, and viability. The average histamine content per alcian blue staining cell was 0.78 +/- 0.2 pg (n = 7), similar to that reported for peripheral blood basophils. Nasal cells were challenged in vitro with anti-IgE, ragweed Amb a I, and FMLP and their responses were compared to those of peripheral blood basophils isolated simultaneously from the same donors. Nasal leukocytes released histamine maximally at 0.1 micrograms/ml of anti-IgE (35.8 +/- 7.8%, n = 7) and responded to FMLP (25.4 +/- 9.9%, n = 7). The response of the cells to ragweed Amb a I and anti-IgE was attenuated compared to peripheral blood basophils. Anti-IgE-induced histamine release was calcium and temperature dependent. Dual color immunofluorescence and flow cytometric analysis of the recovered nasal cells coexpressed CD18, a leukocyte marker not expressed by mast cells. The nasal cells consistently had high levels of spontaneous histamine release (19.5 +/- 2.0%, n = 22). The viability of all cells, assessed by erythrosin B dye exclusion, was 70 +/- 2% (n = 15). However, the viability of IgE-bearing cells was only 28.3 +/- 5.7% (n = 4). The characteristics of histamine release and the nature of the cellular surface markers provide functional proof that the histamine-containing cells accumulating after nasal Ag challenge are basophils and not mast cells.     

Human skin mast cells: their dispersion, purification, and secretory characterization

Digestion of human foreskin with collagenase and hyaluronidase disperses approximately 3.4 X 10(7) nucleated cells per gram of tissue, of which mast cells constitute 4.7%. These may be purified to 80% by use of density gradient centrifugation. The majority of mast cells (79%) measured between 9 and 13 micron in diameter, and the mean histamine content was 4.6 pg/cell. Viability was demonstrated by trypan blue exclusion by 93% of the cells and the low spontaneous histamine secretion of less than 7% in functional studies. Anti-IgE released up to 17.5% of cell-associated histamine within 5 to 7 min. Calcium ionophore-induced release was optimal with 0.3 microM A23187 when 28.6% histamine was released. Unlike human lung mast cells, skin mast cells released histamine in response to compound 48/80 and poly-L-lysine. This release, which was complete within 20 sec, was totally dependent on intact glycolysis and oxidative phosphorylation and partially dependent on extracellular calcium. The same characteristics were observed with secretion induced by substance P and morphine. The weak activity of eledoisin and physalaemin suggests that the substance P receptor, like that of the rat mast cell, is not of the classical types described for smooth muscle. Morphine-induced secretion was partially blocked by naloxone in a manner not compatible with competitive antagonism at a classical opioid receptor. The sensitivity of skin mast cells to nonimmunologic stimulation clearly distinguishes them from mast cells of the lung and lymphoid tissues and provides evidence of functional heterogeneity within human mast cells.     

Pulmonary intravascular macrophages and hemodynamic effects of liposomes in sheep

We studied the effects of liposomes on the pulmonary circulation of sheep and found a close correlation between liposome retention in the lung and the intravascular macrophages. A test dose of liposomes (5.5 mumol of total lipids) injected intravenously transiently increased pulmonary arterial pressure from 24 +/- 2 to 55 +/- 16 (SD) cmH2O. The pulmonary arterial pressure responses were dose dependent and reproducible. The rise in pulmonary arterial pressure was blocked completely by indomethacin and 75% by a thromboxane synthase inhibitor. Systemic arterial thromboxane B2 concentration increased from a base-line level of less than 50 pg/ml to 250 +/- 130 pg/ml at the peak of the pressor response. Larger doses of liposomes (220 mumol of total lipids) infused intravenously over 1 h increased pulmonary arterial pressure maximally within the first 15 min. Lymph flow increased and lymph protein concentration decreased, suggesting venoconstriction. Over half (62.4 +/- 15.7%) of 111In-labeled liposomes remained in the lung after 2 h. Fluorescence and transmission electron microscopy showed that greater than 90% of the liposomes were associated with mononuclear cells in the lumen of the alveolar wall microvessels. We conclude that liposomes affect pulmonary arterial pressure transiently by a mechanism involving the arachidonate cascade, principally thromboxane. Our observations suggest that a population of pulmonary intravascular macrophages is likely to be the source of the thromboxane and the pulmonary hemodynamic and lymph dynamic changes that occur in a dose-dependent fashion, although interactions between liposomes, leukocytes, or endothelial cells, in addition to the macrophages, have not been completely ruled out. We believe this is the first demonstration that pulmonary intravascular macrophages may be the source of the arachidonate metabolites rather than endothelial cells, neutrophils, or perivascular interstitial cells.     

Mucosal mast cells. II. Effects of anti-allergic compounds on histamine secretion by isolated intestinal mast cells

Functional mast cells have been isolated from the lamina propria of the small intestine of rats infected with the nematode Nippostrongylus brasiliensis. The cells released histamine on challenge with specific antigen, anti-rat IgE, concanavalin A, and calcium ionophores but were less responsive than peritoneal mast cells (MMC) from the same animals. Intestinal mucosa mast cells (PMC) were refractory to the action of the basic secretagogues peptide 401 from bee venom and compound 48/80. The anti-allergic compounds disodium cromoglycate (less than or equal to 10(-3) M), AH 9679 (less than or equal to 10(-4) M), and theophylline (less than or equal to 10(-2)) did not inhibit antigen-induced histamine secretion by MMC, although these compounds were effective against PMC. In contrast, doxantrazole (10(-5) to 10(-3) M) inhibited the secretion of histamine from both MMC and PMC in a comparable dose-dependent fashion. Thus, we have established that mast cells from different sites are functionally heterogeneous not only in their response to various stimuli for histamine secretion, but also in their responses to different pharmacologic modulators of secretion. It cannot be assumed that anti-allergic compounds effective against mast cells in one tissue site or organ will be equally efficacious against mast cells in other sites. The extent of this functional heterogeneity must be established, and its investigation may provide new insights into the biochemical events involved in mast cell secretion.     

Mast cells from the human intestinal lamina propria. Isolation, histochemical subtypes, and functional characterization

With the use of a collagenase dispersion technique, cells were isolated from the lamina propria of the human small and large intestine. The cell suspensions contained 8% mast cells, which on average contained 1 to 2 pg of histamine/cell. With the use of histochemical procedures based upon fixative sensitivity and dye binding, which identify functionally distinct mast cell subtypes in the rat, dispersed human intestinal mast cells contained approximately equal proportions of two histochemical subtypes analogous to those in the rat. Whether these are functionally distinct as in the rat remains to be determined. The histochemically mixed mast cell populations from the human intestinal mucosa secreted histamine in a dose- and energy-dependent manner in response to anti-IgE and A23187, but not 48/80. Theophylline, doxantrazole, quercetin, and salbutamol all significantly inhibited anti-IgE-induced histamine secretion by human intestinal mast cells, but cromolyn sodium and the experimental antisecretory drugs, nedocromil sodium and FPL 52694, did not inhibit histamine secretion by the mast cell mixture to a statistically significant extent. Cromolyn sodium inhibited histamine secretion by 15 to 30%, and whether this reflected inhibition of one of the two histochemical mast cell subtypes to a greater extent than the other or all the cells to a minimal degree remains to be established. Control investigations of the intestinal cell isolation procedure indicated that these qualities did not reflect effects of the cell dispersal procedure. Further characterization and analysis of intestinal mast cells is essential to determine if functionally distinct mast cell subtypes exist in human tissues.     

Studies on Bovine Mast Cells: I. The Effect of Formalin Fixation

Studies are reported on the effect of aqueous formalin fixation on the bovine mast cell. Paraffin sections of bovine skin were prepared from tissues fixed for 7 to 72 hr in 10% neutral formalin in saline, and from tissues which were treated with water for various times both before and after formalin fixation. Metachromatic halos, scattering of the granules, and fraying of the cell outline, were seen in the mast cells of tissues washed in water before fixation. Exposure to water after fixation did not produce these artifacts. The tendency towards orthochromatic staining, and the occurrence of perinuclear clear zones are probably effects of the formalin and not of overstaining or of exposure of the tissue to water. The majority of the metachromatic material in the bovine mast cell is water soluble, and may be removed by washing in water before fixation, whereas the granules are relatively resistant to water. The optimum time of formalin fixation of bovine skin to permit studies on the metachromatic material of the mast cells was 24 to 36 hr.     

Mast cells and pulmonary fibrosis. Identification of a histamine releasing factor in bronchoalveolar lavage fluid.

As elevated bronchoalveolar lavage (BAL) fluid histamine levels are noted in patients with pulmonary fibrosis (PF), we assayed BAL fluid from 16 patients with PF for the presence of a histamine releasing factor (HRF). HRF activity was assayed by measuring release of the preformed mast cell-derived mediators, histamine, or beta-hexosaminidase (beta-hex) from a purified population of IL-3 dependent mouse bone marrow derived mast cells (MBMMC) or human blood basophils. Mean BAL cell free histamine levels in the patients with PF was 1226 +/- 1349 pg/ml, whereas BAL histamine levels in a comparison group of six non-PF patients was 118 +/- 60 pg/ml. HRF was significantly elevated in BAL fluid of patients with PF (mean beta-hex release 24.5 +/- 12.9%; range 6.8 to 52.4%) compared to the non-PF group of patients (mean beta-hex release 7.9 +/- 7.7%; range 1.8 to 20.7%). The PF HRF not only degranulated MBMMC, but also induced the generation of the arachidonic acid metabolite leukotriene C4 from MBMMC (24.6 +/- 4.2 ng leukotriene C4/10(6) MBMMC). The PF HRF did not appear to be a cytokine previously identified in BAL fluid of patients with PF (i.e., platelet derived growth factor or insulin growth factor-1) or a human cytokine able to degranulate human basophils (i.e., IL-1, or granulocyte-macrophage-CSF) as these recombinant human cytokines did not induce MBMMC beta-hex release. Physicochemical characterization of the HRF revealed that it was relatively heat stable, pronase sensitive and on Sephadex G-75 and G-200 column chromatography had an apparent molecular mass of 30 to 50 kDa. The ability of PF BAL to induce beta-hex release from MBMMC was not dependent on IgE as unsensitized or lactic acid treated MBMMC release similar amounts of beta-hex compared to MBMMC sensitized with IgE. Thus, BAL fluid of patients with PF contains an HRF that induces beta-hex release from MBMMC via an IgE-independent mechanism. The presence of the HRF could explain elevated BAL histamine levels in patients with PF.     

Human monocytes generate basophil histamine-releasing activities

Human peripheral blood monocytes generated activities during 24-h culture that were capable of triggering histamine release from 17 of 18 human basophil donors. Monocytes and their in vitro transformed macrophages continued to elaborate these basophil histamine-releasing activities for at least 3 wk in culture. In the 18 basophil donors tested, maximum histamine release induced by monocyte supernatants was 33.8 +/- 5.9% (mean +/- SEM) of total basophil histamine content; optimum anti-IgE-induced release was 38.8 +/- 6.2%. Basophil histamine release in response to monocyte activities was optimal at 37 degrees C and at calcium concentrations of 2 to 5 mM. Release was greater than 90% complete 1 min after challenge and was inhibited by anti-allergic drugs. The mechanism of release appeared to be independent of IgE binding. Gel filtration of supernatants derived from both day 1 (monocyte stage) and day 14 (macrophage stage) cultures demonstrated activity peaks with approximate m.w. of 12,000 and 30,000. In contrast to the marked responsiveness of basophils, only 2 of 10 human lung mast cell preparations responded; release in those preparations was low: 3% and 13% histamine release, respectively. Thus, monocytes produce potent histamine-releasing activities with differential actions on basophils and mast cells.     

Human lung mast cells: purification and characterization

Detailed studies of the biochemistry and pharmacology of mast cell-mediated inflammatory disorders have been hampered by the inability to purify human mast cells. We now report techniques to purify human lung mast cells to apparent homogeneity. The major purification steps are: 1) dispersion of lung fragments into a single-cell suspension with enzyme combinations (pronase-chymopapain, collagenase-elastase); 2) partial purification by countercurrent centrifugation elutriation (CCE); and 3) affinity column chromatography. Enzymatic dispersion yielded suspensions with congruent to 10(6) mast cells per gram of lung parenchyma in purities of 1.2 to 9.7%. Dispersed mast cells responded comparably to those in parent lung fragments to challenge with anti-human IgG and pharmacologic agonists. Elutriation of lung cell suspensions yielded mast cell-enriched fractions with purities up to 70%. High purity mast cell fractions were combined, passively sensitized with purified human penicillin (BPO)-specific IgE, and purified by a BPO-affinity column chromatography procedure. Post elutriation mast cell purities of 29 +/- 3.5% were increased to 84 +/- 3% (range 65 to 98%) by the affinity column. Short-term (24 hr) culture of column-purified mast cells allowed adherence of non-mast cell contaminants to tissue culture plates, further increasing purity (up to 100%). Purified mast cells were intact and functional as assessed by dye exclusion, survival in short-term culture, IgE-mediated histamine release, and modulation of release by the pharmacologic agonists adenosine, IBMX, prostaglandin E2, and fenoterol.     

Low density lipoprotein (LDL) inhibits histamine release from human mast cells

We examined the effect of low density lipoprotein (LDL) on histamine release from purified human lung mast cells. LDL inhibited anti-IgE- induced histamine release in a dose-dependent manner, with 100 micrograms/ml LDL-protein inhibiting histamine release by 53 +/- 8% (mean +/- SEM); half-maximal inhibition occurred at 40-80 micrograms/ml. LDL also inhibited calcium ionophore A23187-induced histamine release in a dose-dependent manner, with 1 mg/ml of LDL inhibiting histamine release by 83 +/- 9%; half maximal inhibition occurred at 220-280 micrograms/ml. Inhibition by LDL was time-dependent: half-maximal inhibition of anti-IgE- induced histamine release by LDL occurred at 30-50 minutes of incubation. The inhibitory effect of LDL was independent of buffer calcium concentrations (0-5 mM) or temperature (0-37 degrees C). These data are consistent with a newly defined immunoregulatory role for LDL.