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L M Lichtenstein

Publications and source records attributed to L M Lichtenstein.

At least 19 recordsLinked to original sources
PubMed1992

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)

Antibodies, Monoclonal↗
PubMed1992

Anti-human IgG causes basophil histamine release by acting on IgG-IgE complexes bound to IgE receptors.

We have reexamined the ability of anti-human IgG antibodies to induce histamine release from human basophils. A panel of purified murine mAbs with International Union of Immunological Societies-documented specificity for each of the four subclasses of human IgG was used. Of the 24 allergic subjects studied, the basophils of 75% (18/24) released greater than 10% histamine to one or more anti-IgG1-4 mAb, whereas none of the 13 nonatopic donor's basophils released histamine after stimulation with optimal amounts of anti-IgG mAb. The basophils of 85% (11/13) of the nonatopic donors did respond to anti-IgE challenge, as did 92% (22/24) of the atopic donor cells. Histamine release was induced most frequently by anti-IgG3, and 10/18 anti-IgG responder cells released histamine with mAb specific for two or more different subclass specificities. The rank order for induction of histamine release was anti-IgG3 greater than anti-IgG2 greater than IgG1 greater than anti-IgG4. As in our previous study using polyclonal anti-IgG, 100- to 300-micrograms/ml quantities of the anti-IgG mAb were required for maximal histamine release, about 1000-fold higher than those for comparable release with anti-human IgE. Specificity studies using both immunoassays and inhibition studies with IgE myeloma protein indicated that anti-IgG induced histamine release was not caused by cross-reactivity with IgE. Ig receptors were opened by lactic acid treatment so that the cells could be passively sensitized. Neither IgE myeloma nor IgG myeloma (up to 15 mg/ml) proteins could restore the response to anti-IgG mAb. However, sera from individuals with leukocytes that released histamine upon challenge with anti-IgG mAb could passively sensitize acid-treated leukocytes from both anti-IgG responder and nonresponder donors for an anti-IgG response. The only anti-IgG mAb that induced release from these passively sensitized cells were those to which the serum donor was responsive. Sera from non-IgG responders could not restore an anti-IgG response. These data led to the hypothesis that the IgG specific mAb were binding to IgG-IgE complexes that were attached to the basophil through IgE bound to the IgE receptor. This was shown to be correct because passive sensitization to anti-IgG could be blocked by previous exposure of the basophils to IgE. We conclude that anti-IgG-induced release occurs as a result of binding to IgG anti-IgE antibodies and cross-linking of the IgE receptors on basophils.

Antibodies, Anti-Idiotypic↗
PubMed1992

Preliminary identification and role of phosphodiesterase isozymes in human basophils.

We attempted to identify and establish the role of cyclic nucleotide phosphodiesterase (PDE) isozymes in human basophils by using standard biochemical techniques as well as describing the effects of isozyme-selective and nonselective inhibitors of PDE. The nonselective PDE inhibitors, theophylline and 3-isobutyl-1-methylxanthine, inhibited anti-IgE-induced release of histamine and leukotriene C4 (LTC4) from basophils. This inhibition was accompanied by elevations in cAMP levels. Rolipram, an inhibitor of the low Km cAMP-specific PDE (PDE IV), inhibited the release of both histamine and LTC4 from activated basophils and increased cAMP levels in these cells. In contrast, mediator release from basophils was not inhibited by either siguazodan or SK&F 95654, inhibitors of the cGMP-inhibited PDE (PDE III) or zaprinast, an inhibitor of the cGMP-specific PDE (PDE V). SK&F 95654 failed to elevate basophil cAMP in these experiments whereas zaprinast induced significant increases in cAMP content. The inhibitory effect of rolipram on mediator release was potentiated by siguazodan or SK&F 95654, but not by zaprinast. SK&F 95654 also enhanced the ability of rolipram to increase cAMP content. Forskolin, a direct activator of adenylate cyclase, inhibited IgE-dependent release of mediators from basophils and increased cAMP levels in these cells. These effects were enhanced by rolipram, but not by SK&F 95654 or zaprinast. The cell permeant analog of cAMP, dibutyryl cAMP, inhibited mediator release from these cells, a property not shared by either dibutyryl-cGMP or sodium nitroprusside, an activator of soluble guanylate cyclase. The presence of both PDE III and PDE IV was confirmed by partially purifying and characterizing PDE activity in broken cell preparations. Overall, these data lend support to the hypothesis that cAMP inhibits mediator release from basophils and suggest that the major PDE isozyme responsible for regulating cyclic AMP content in these cells is PDE IV, with a minor contribution from PDE III. However, the finding that zaprinast caused increases in cAMP without inhibiting mediator release indicates that cAMP accumulation is not invariably linked to an inhibition of basophil activation.

Basophils↗
PubMed1992

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.

Adult↗
PubMed1992

Immunophenotyping and functional analysis of purified human uterine mast cells.

Human mast cells have been purified from uterine tissues, and their surface marker profile and function have been evaluated as part of ongoing studies of mast cell heterogeneity. Using a panel of antibodies, purified uterine mast cells (UMC; 81% +/- 7% purity, n = 10) were analyzed by immunofluorescence and flow cytometry for surface expression of various antigens. Consistent with previous analyses of mast cells from other tissues, UMC expressed HLA class I, IgE, c-kit receptor, CD9, CD33, CD43, CD45, and CD54, while CD11a, CD11b, CD14, CD16, CD23, and CD64 were not detected. Unlike other mast cells, UMC expressed CD11c/CD18 (p150,95) and CD32 (Fc gamma RII). Additional antigens not previously studied on mast cells included the selectin LECAM-1 (Leu-8) and several beta 1 and beta 3 integrins; expression of very late activation antigen-4 (VLA-4) (CD49d/CD29), VLA-5 (CD49e/CD29), and the vitronectin receptor (CD51/CD61) was seen. Functional studies showed that treatment of human umbilical vein endothelial cells with interleukin-1 (5 ng/mL for 4 hours) resulted in a twofold to threefold increase in adhesiveness for UMC. Purification procedures did not alter histamine release responses to anti-IgE or the calcium ionophore A23187, and treatment of UMC with an anti-CD32 monoclonal antibody (IV.3) did not induce histamine release or alter anti-IgE-induced release. These data suggest that UMC may possess unique phenotypic characteristics, and support the concept of mast cell heterogeneity.

Antibodies, Monoclonal↗
PubMed1992

Tryptase and histamine as markers to evaluate mast cell activation during the responses to nasal challenge with allergen, cold, dry air, and hyperosmolar solutions.

We have used assays for histamine and for the specific mast cell enzyme, tryptase, to examine the response of the nasal mucosa to provocation with several different stimuli and to evaluate the reliability of histamine as a marker of mast cell activation. High levels of histamine detected in baseline lavages of some subjects are not associated with any detectable tryptase, suggesting they are not mast cell derived. During pronounced immediate allergic responses, however, mast cell degranulation clearly occurs, and a striking correlation between histamine and tryptase is observed. This correlation is weaker when a more modest allergic response is induced, possibly reflecting differential diffusion of the two mediators across the epithelium. Provocation of susceptible individuals with cold, dry air leads to increased recoveries of both histamine and tryptase, confirming that mast cell degranulation occurs during this reaction. Although hyperosmolarity of the nasal mucosa may contribute to mast cell degranulation induced by cold, dry air, a brief exposure of the nasal cavity to hyperosmolar mannitol was not associated with measurable production of tryptase. The combined use of histamine and tryptase measurements can therefore provide useful evidence regarding the role of mast cell activation in the pathogenesis of inflammatory responses.

Adult↗
PubMed1992

Intranasal beclomethasone inhibits antigen-induced nasal hyperresponsiveness to histamine.

To determine whether intranasal steroid treatment inhibits the increased sensitivity to histamine that occurs 24 hours after nasal antigen challenge, 12 allergic volunteers were entered into a double-blind study comparing placebo or 164 micrograms of beclomethasone dipropionate twice a day for 7 days. Beclomethasone dipropionate partially reduced the early mediator response to antigen and the influx of eosinophils 24 hours later. Comparing the initial histamine challenge with that done 24 hours after antigen with the subjects on placebo, there was a significant increase in sneezes, TAME-esterase activity, and albumin. Pretreatment with intranasal beclomethasone dipropionate resulted in a reduced response to histamine 24 hours after antigen challenge. A positive correlation occurred between the number of eosinophils in the lavage before histamine challenge and the level of TAME-esterase activity (rs = 0.67, p = 0.03) during the histamine challenge that followed antigen with the subjects on placebo. We thus confirmed the increase in nonspecific nasal airway responsiveness 24 hours after antigen challenge, with a concomitant increase in eosinophils, and demonstrate its inhibition by pretreatment with intranasal steroids.

Administration, Intranasal↗
PubMed1992

Clinical correlation of the venom-specific IgG antibody level during maintenance venom immunotherapy.

Allergen immunotherapy is associated with a significant increase of specific IgG antibodies that have been suggested as a mechanism of action and as a marker of efficacy for immunotherapy. The value of venom-specific IgG antibody determinations as a measure of clinical protection against sting anaphylaxis has been difficult to prove in individual patients. We performed 211 insect sting challenges in 109 patients over a 4-year period to determine the significance of venom IgG levels 3 micrograms/ml or lower. Systemic symptoms occurred in only 1.6% of those with venom IgG more than 3 micrograms/ml, but in 16% of those with less than 3 micrograms/ml IgG, and notably in 26% of patients with low venom IgG who had received less than 4 years of treatment. The venom IgG level had no predictive value in patients who had received more than 4 years of therapy. Honeybee sting data were inconclusive because of the small number of subjects. We conclude that low venom-specific IgG levels are associated with an elevated risk of treatment failure during the first 4 years of immunotherapy with yellow jacket or mixed vespid venoms.

Adult↗
PubMed1992

The effect of azelastine on the early allergic response.

To study the effect of azelastine on the immediate reaction to nasal allergen challenge, we performed a double blind, placebo-controlled cross-over clinical trial. Thirteen subjects with seasonal allergic rhinitis underwent nasal challenge with antigen 4 hr after a single oral 2 mg dose of azelastine. The response was monitored by counting the number of sneezes and by measuring the levels of histamine, prostaglandin D2, immunoreactive sulphidopeptide leukotrienes, kinis and TAME-esterase activity in recovered nasal lavages. After a single dose of azelastine, there was a significant reduction in sneezing (10 vs 2, P = 0.01) and in the median levels of recovered TAME-esterase activity (63.1 vs 17.5 c.p.m. x 10(-3), P = 0.01), immunoreactive sulphidopeptide leukotrienes (7.5 vs 2.1 ng/ml, P = 0.03) and kinins (1370 vs 251 pg/ml, P = 0.03), with no significant reduction in the median levels of histamine (3.7 vs 1.2 ng/ml, P = 0.2) and prostaglandin D2 (70 vs 70 pg/ml, P = 0.2) compared to placebo (numbers represent total increase over diluent challenge). These results suggest that azelastine does not inhibit mast cell activation but affects the consequences of released histamine, namely sneezing, increased vascular permeability and the generation of kinins. The results further suggest that other cells, in addition to mast cells, might be responsible for the generation of leukotrienes during the early allergic response, and that azelastine reduces their ability to generate this mediator or that inhibition of leukotriene release from mast cells occurs at lower drug concentrations.

Adult↗
PubMed1992

Immunologic mast cell-mediated responses and histamine release are attenuated by heparin.

Heparin has been shown to act as a competitive inhibitor of inositol 1,4,5-triphosphate (InsP3) receptors in various cell types. Because InsP3 is one of the second messengers involved in stimulus-secretion coupling in mast cells, it is possible that heparin may inhibit mast cell-mediated reactions. Therefore, in allergic sheep, we tested this hypothesis in two mast cell-mediated reactions induced by immunologic and nonimmunologic stimuli: immediate cutaneous reaction (ICR) and acute bronchoconstrictor response (ABR). In 12 sheep allergic to Ascaris suum antigen, the surface area of the skin wheal was determined 20 min after intradermal injection (0.05 ml) of increasing concentrations of specific antigen, compound 48/80, and histamine, without and after pretreatment with heparin (100, 300, or 1,000 U/kg i.v.). Antigen, compound 48/80, and histamine produced concentration-dependent increases in ICR. Heparin "partially" inhibited the ICR to antigen and compound 48/80 in a dose-dependent manner without modifying the ICR to histamine. The heparin preservative benzyl alcohol was ineffective. In 11 additional sheep, specific lung resistance was measured before and after inhalation challenges with antigen, compound 48/80, and histamine without and with aerosol heparin pretreatment (1,000 U/kg). Heparin blocked the antigen- and compound 48/80-induced bronchoconstriction without modifying the airway effects of histamine. In isolated human uterine mast cells, heparin inhibited the anti-immunoglobulin E- but not the calcium ionophore- (A23187) induced histamine release. These data suggest that heparin inhibits the ICR and ABR induced by stimuli that produce immunologic and nonimmunologic mast cell degranulation without attenuating the effects of histamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Local application of atropine attenuates the upper airway reaction to cold, dry air.

In some individuals, inhalation of cold, dry air (CDA) provokes symptoms of rhinitis, accompanied by an increase in the levels of inflammatory mediators and markers of plasma leakage of recovered nasal lavages. Because rhinorrhea is a major component of this reaction and because nasal glands are heavily innervated by the parasympathetic system, we assessed the effect of atropine on the nasal reaction to CDA. Using a double-blind, randomized, crossover design, we administered a total dose of 0.5 mg of atropine or placebo intranasally to 18 volunteers before provocation with CDA. The reaction was monitored with symptom scores and by measuring the concentrations of histamine, N-alpha-p-tosyl-L-arginine methyl ester (TAME)-esterase activity and albumin, as well as the osmolality of lavage fluids before and after the provocation. Atropine significantly reduced rhinorrhea, the levels of histamine, and TAME-esterase activity as well as the osmolality of recovered lavage fluids, but had no effect on nasal congestion or albumin. Even with atropine, however, rhinorrhea and TAME-esterase activity were still significantly increased over the prechallenge baseline. Our results demonstrate that atropine-sensitive parasympathetic efferent pathways contribute to the CDA-induced rhinitis. We speculate that (1) the glandular and the vascular events of the upper airway reaction to dry air have different pathophysiologic mechanisms; (2) a significant component of TAME-esterase activity in lavage fluids may be of glandular origin; and (3) in addition to parasympathetic nerve activation, other mechanisms are involved in the upper airway reaction to dry air. The mechanism(s) leading to the reduction of histamine is unknown.

Administration, Intranasal↗
PubMed1992

Ultrastructural immunogold localization of prostaglandin endoperoxide synthase (cyclooxygenase) to non-membrane-bound cytoplasmic lipid bodies in human lung mast cells, alveolar macrophages, type II pneumocytes, and neutrophils.

Lipid bodies are non-membrane-bound, lipid-rich cytoplasmic inclusions that occur in many mammalian cell types. Because lipid bodies are more prominent in cells associated with inflammation and are repositories of arachidonyl-phospholipids, a role for lipid bodies in the oxidative metabolism of arachidonic acid to form eicosanoids has been suggested. To evaluate further whether lipid bodies, in addition to serving as non-membranous sources of substrate arachidonate, are involved in eicosanoid formation, we used cells isolated from human lung to investigate the intracellular localization of prostaglandin endoperoxide (PGH) synthase (cyclooxygenase), the key initial, rate-limiting enzyme in the formation of prostaglandins and thromboxanes. Isolated lung cells containing a mixture of mast cells, alveolar macrophages, Type II alveolar pneumocytes, and neutrophils from short-term cultures were fixed in suspension in a dilute aldehyde mixture, post-fixed in osmium tetroxide, stained en bloc with uranyl acetate, dehydrated in a graded series of alcohols, and embedded in Epon. A post-embedding immunogold procedure was used with a primary PGH synthase monoclonal antibody and 20-nm gold-conjugated secondary antibody to demonstrate enzyme locations. Specificity controls were also done. We found PGH synthase in lipid bodies of human lung mast cells, alveolar macrophages, Type II alveolar pneumocytes, and neutrophils. Specific secretory and lysosomal granules and plasma membranes did not express PGH synthase. Specificity controls, including omission of the primary antibody or substitution with an irrelevant antibody, were negative. Absorption of the specific PGH synthase antibody with purified solid-phase PGH synthase resulted in a marked reduction of label in lipid bodies of all four cell types. These findings establish the presence of PGH synthase in lipid bodies of human lung mast cells, alveolar macrophages, Type II alveolar pneumocytes, and neutrophils and, in concert with previous studies, suggest that these cytoplasmic lipid-rich organelles may be non-membrane sites of eicosanoid formation.

Cells, Cultured↗
PubMed1992

Evaluation of a bedtime dose of a combination antihistamine/analgesic/decongestant product on antigen challenge the next morning.

The effects of CAAD (diphenhydramine hydrochloride, 50 mg; pseudoephedrine hydrochloride, 60 mg; acetaminophen, 1 g in 10% ethanol) were evaluated in a double-blind, three-way, placebo-controlled, cross-over study on 18 volunteers with allergic rhinitis. The number of sneezes following nasal challenge with antigen was significantly reduced after a bed-time dose of CAAD (P less than .005) and a single dose of diphenhydramine (P less than .001) given 2 hours before the challenge. The levels of N-alpha-tosyl-L-arginine methyl ester (TAME) activity decreased after diphenhydramine treatment, while histamine levels following challenge were not different. The drowsiness reported after CAAD was equal to placebo, but significantly less than diphenhydramine (P less than .002 for both). The active treatments reduced the actions of histamine without suppressing its release from mast cells. The effect of CAAD persists 10 hours after administration without inducing drowsiness.

Acetaminophen↗
PubMed1992

Effect of H1 receptor blockade on the early and late response to cutaneous allergen challenge.

We investigated whether cutaneous antigen-induced inflammatory cell infiltration and mediator release were modified by H1 receptor antagonists. Three chemically unrelated antihistamines (cetirizine, promethazine and chlorpheniramine) were tested in three groups of allergic subjects in a double-blind, crossover design. Chamber fluids were collected for 12 hr and histamine release, prostaglandin D2 production and cellular infiltration were quantified. Cetirizine significantly decreased late leukocyte migration into antigen-challenged chambers: eosinophils by 68% (P less than .04), basophils by 64% (P less than .04) and neutrophils by 72% (P less than .04), whereas mononuclear cells were not significantly affected. No alteration in the numbers of peripheral blood leukocytes or eosinophils occurred while on cetirizine treatment, suggesting that the decrease in inflammatory cells during the late phase reaction in the skin is not secondary to alterations in the peripheral leukocyte pool. In contrast, neither promethazine nor chlorpheniramine induced any significant alteration in inflammatory cell infiltration. All three antihistamines caused significant inhibition of the immediate reaction to antigen without any significant alteration in late phase reaction cutaneous reactivity. None of the three antihistamines caused any significant alteration in histamine or prostaglandin D2 levels. Thus, cetirizine may be an antihistamine uniquely capable of downregulating the late phase reaction inflammatory cell milieu without altering either early or late mediator production. The mechanisms involved and the clinical relevance of these findings remain to be explored.

Allergens↗
PubMed1992

An ultrastructural analysis of tumor-promoting phorbol diester-induced degranulation of human basophils.

Release reactions stimulated in human basophils by a variety of secretagogues show biochemical and morphologic differences as well as similarities. Biochemical differences include those of rate, amount, and order of mediator release, as well as mediator type released or generated. Morphologic diversity of release reactions includes prototypic anaphylactic degranulation (AND), or piecemeal degranulation (PMD), and a continuum of anatomic release comprised of PMD followed by AND that is seen when human basophils are stimulated by the bacterial peptide, formyl methionyl leucyl phenylalanine (FMLP). AND is characterized by extrusion of membrane-free granules through multiple plasma membrane pores; PMD is characterized by partially to completely empty, nonfused granule containers in the cytoplasm of basophils. AND is further characterized by diminished-to-absent granules and reduced cytoplasmic vesicles at peak histamine release intervals; PMD does not show decreases in numbers of granules, and cytoplasmic vesicles are plentiful. Smooth membrane-bound vesicles with granule particles and vesicles that appear empty comprise this organelle population. PMD is the single most evident activation change present in basophils that traffic into tissues in multiple diseases in vivo. In this study, we examined the ultrastructural kinetic morphology associated with stimulation of human basophils with tetradecanoyl phorbol acetate (TPA)--a tumor-promoting phorbol diester known to elicit histamine (but not LTC4) release. Partially purified human basophils were prepared for electron microscopy and examined either after control incubations in buffer alone or at 0 time, 1, 2, 5, 10, 30, and 45 minutes after TPA stimulation. Standard morphology and ultrastructural quantitation of vesicles and granules and contents of vesicles or alteration of granules was done and compared with previous ultrastructural kinetic analyses of human basophil release reactions stimulated by different triggers. Like biochemical studies that have determined that TPA is a unique secretogogue for human basophils, the morphology stimulated by TPA and associated with histamine release was also unique. For example, very minor images of AND were evident. Far greater amounts of PMD were imaged. PMD was associated with approximately 50% alteration of cytoplasmic granules by 45 minutes after TPA stimulation. This evidence of empty granules was associated with, and preceded by, a rapid, extensive, and sustained increase in particle-containing cytoplasmic vesicles, as compared with buffer controls (P < 0.001 for each TPA stimulation time compared with unstimulated basophils). In addition, previously undescribed interactions of releasing granules and their overlying plasma membranes characterized TPA-stimulated cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Basophils↗
PubMed1992

Production of granulocyte/macrophage colony-stimulating factor in human airways during allergen-induced late-phase reactions in atopic subjects.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) are hematopoietic growth factors that have been shown to induce proliferation and activation of inflammatory cells, and may play a role in allergic reactions. Since little is known about the involvement of cytokines in allergic inflammation in the lung, the levels of GM-CSF and IL-3 were measured in bronchoalveolar lavage (BAL) fluids obtained in the late phase after segmental lung antigen (Ag) challenge in 14 allergic rhinitis subjects with or without bronchial asthma. BAL fluids either after Ag (ragweed, dust mite, or timothy) or saline control challenge were recovered 19 h later. In 6 of the 14 patients, BAL fluids were concentration-dialyzed (20x) and assayed for cytokine activity. Cytokine assays were performed using the human megakaryocytic leukemic cell line M-07e, which is responsive to either GM-CSF or IL-3. The level of GM-CSF-equivalents was approximately 25 times higher in Ag-challenged sites (49.9 +/- 12.7 pg/ml; mean +/- SEM), compared to saline challenge sites (2.2 +/- 1.0, p < 0.01, n = 9). Neutralization experiments using a polyclonal specific antibody (Ab) against GM-CSF and IL-3 revealed that the bulk of the activity was GM-CSF. BAL fluids from Ag- and saline-challenged sites in one nonatopic subject contained no significant GM-CSF activity. Furthermore, the level of GM-CSF in Ag-challenged BAL fluid and the percentage of eosinophils in BAL from each subject correlated significantly (r = 0.73, p < 0.005, n = 14).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

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.

Cell Separation↗