Search PubMed⌕ Search

Biomedical subjects

L M Lichtenstein

Publications and source records attributed to L M Lichtenstein.

At least 91 records · Page 5Linked to original sources
PubMed1994

Modulation of antigen- and mitogen-induced proliferative responses of peripheral blood mononuclear cells by nonselective and isozyme selective cyclic nucleotide phosphodiesterase inhibitors.

Cyclic nucleotide phosphodiesterase (PDE) enzymes are felt to play a role in the regulation of inflammatory responses through their effects on cAMP. In this study, we investigated the effects of nonselective and isozyme selective PDE inhibitors on the proliferative responses of peripheral blood mononuclear cells (PBMCs) to ragweed (RW, a Th2 stimulus), tetanus toxoid (TT, a Th1 stimulus), and phytohemagglutinin in ragweed-allergic patients. The nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) produced nearly identical concentration-dependent inhibition for PBMCs cultured with either Ag (RW and TT) or mitogen (phytohemagglutinin). Neither the type V inhibitor, zaprinast, nor the type III inhibitor, siguazodan, was effective at inhibiting antigen- or mitogen-driven proliferative responses of human PBMCs. The type IV inhibitor, rolipram, was unique in its ability to inhibit the RW-driven proliferative response and, to a lesser extent, the TT-driven proliferative response. However, rolipram was ineffective at inhibiting the mitogen-driven proliferative response. Only with a combination of type III and type IV inhibitors could the efficacy on the TT response be made to approximate that of the type IV inhibitor alone in the RW-driven system. Although the efficacies of IBMX and rolipram were identical in the RW-driven system, the IC50 value of the latter was 10-fold lower, similar to the difference noted in the TT-driven system between IBMX alone and the combination of rolipram with siguazodan. Dose-response curves generated by using the D- or L-isomers of rolipram were not appreciably different from each other or from the curve generated with racemic rolipram. The addition of supraphysiologic doses of human rIL-2 and rIL-4 was unable to counteract the inhibitory effect of IBMX or rolipram. These data support the hypothesis that modulation of proliferation of PBMCs by selective PDE inhibitors varies in sensitivity with the type of stimulus used, and that the type IV PDE exerts the predominant cAMP-associated regulatory effect on allergen-driven proliferation. Finally, the inhibitory effect induced by rolipram is independent of its stereochemistry and cannot be exclusively attributed to deficits in IL-2 or IL-4.

Allergens↗
PubMed1994

IgE-dependent IL-4 secretion by human basophils. The relationship between cytokine production and histamine release in mixed leukocyte cultures.

IL-4 protein and mRNA have recently been detected in pure basophil cultures after stimulation. It has also been established in mixed leukocyte cultures obtained by elutriation and challenged with anti-IgE that the basophil is the only cell that makes detectable IL-4. We have used cells prepared using Percoll gradients (5 to 30% basophils) to study the relationship between histamine release and IL-4 synthesis. In cultures challenged with anti-IgE after a 15-min pretreatment with IL-3, detectable IL-4 secretion ranged from 40 to 630 pg/10(6) basophils in 18 of 22 donors, with no significant correlation (r = 0.21, p = 0.34) with basophil purity. IL-4 synthesis was dissociated from histamine release, with optimal production consistently occurring at 10 ng/ml anti-IgE, whereas histamine levels peaked at 50 to 100 ng/ml of stimulus. Stimulation with anti-IgE alone was sufficient for IL-4 secretion but protein levels were enhanced two- to threefold in low responders by increasing the calcium concentration to 5 mM with no significant changes in histamine release. The IgE-independent secretagogues, F-met peptide (1 microM) and C5a (25 ng/ml), demonstrated limited ability to generate detectable IL-4, despite promoting vigorous histamine release. Additional studies showed no significant difference between IL-4 secretion in atopic and nonatopic donors. Finally, IL-4 levels generated with specific Ags were comparable to those levels produced in response to anti-IgE. We predict that basophil IL-4 generation will have a proinflammatory effect, but it appears that the signal transduction mechanisms for its synthesis and release differ from those for histamine release and thus may require different methods of pharmacologic control.

Basophils↗
PubMed1994

Studies of the intracellular Ca2+ levels in human adult skin mast cells activated by the ligand for the human c-kit receptor and anti-IgE.

The human c-kit receptor ligand, rhSCF, is the only cytokine known to be active on human mast cells, but its intracellular signal transduction pathway is still unknown. We compared the effect of rhSCF on intracellular Ca2+ levels in purified (> 70% pure) adult skin mast cells with two other immunologic stimuli, namely, anti-IgE and substance P. Both rhSCF (1 microgram/mL) and anti-IgE (3 micrograms/mL) induced a rapid (< 20 sec) and sustained (T1/2 for decay > 10 min) increase in free cytosolic Ca2+ concentration. In contrast, substance P (5 microM) elicited a very rapid (< 1 sec) and transient (T1/2 for decay congruent to 5 sec) rise in intracellular Ca2+ levels. Intracellular cAMP levels were then increased by pharmacologic means to examine the role of the cyclic nucleotide in controlling the Ca2+ response in skin mast cells. A combination of the general phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX) (200 microM) and the adenylate cyclase activator, forskolin (30 microM) was effective in inhibiting the Ca2+ response induced by rhSCF or anti-IgE (82 and 68% inhibition, respectively), while IBMX and forskolin alone were much less effective. The phosphodiesterase isozyme IV inhibitor, rolipram (10 microM), variably affected the increase in Ca2+ levels induced by anti-IgE, but it exerted a significant inhibitory activity on anti-IgE- or rhSCF-induced response in the presence of forskolin (30 micrograms/mL) (33 and 67%, respectively). Two different protein kinase C (PKC) activators TPA (200 nM) and bryostatin 1 (200 nM) similarly inhibited rhSCF- (22 and 32%, respectively) and anti-IgE-induced (24 and 32%) Ca2+ response. Finally, the kinase inhibitor genistein (30 micrograms/mL) was a somewhat more effective inhibitor of the rise in intracellular Ca2+ induced by rhSCF (100%) than that activated by anti-IgE (54%) (P < 0.05). These data indicate that rhSCF and anti-IgE may act on human mast cells through a common pathway to increase free cytosolic Ca2+ levels and this effect is similarly modulated by various drugs.

Antibodies, Anti-Idiotypic↗
PubMed1994

Eosinophil transendothelial migration induced by cytokines. II. Potentiation of eosinophil transendothelial migration by eosinophil-active cytokines.

Activation of HUVEC monolayers by IL-1 beta or TNF-alpha induces migration of eosinophils across the endothelial monolayer (i.e., transendothelial migration) in vitro. In the present study, we demonstrate that culture of freshly isolated eosinophils in the presence of IL-5 for 24 to 48 h before use in the assay dramatically potentiated CD18-dependent eosinophil transendothelial migration through unstimulated endothelial monolayers. Granulocyte macrophage (GM)-CSF induced eosinophil transendothelial migration but did not induce neutrophil transendothelial migration. When IL-1 beta-activated endothelial cells were used, GM-CSF, IL-3, or IL-5 caused only modest potentiation of eosinophil transendothelial migration. Since activated endothelial cells are known to produce GM-CSF, we hypothesized that endothelial-derived GM-CSF might play a role in the process of IL-1 beta-induced eosinophil transendothelial migration. IL-1 beta-activated endothelial monolayers grown on the permeable supports produced 0.3 +/- 0.1 ng/ml of GM-CSF during a 4-h incubation and neutralizing Ab against GM-CSF inhibited eosinophil transendothelial migration by 48%, suggesting that endothelial-derived GM-CSF may participate in the response. Eosinophils purified from bronchoalveolar lavage 18 to 20 h after experimental Ag challenge in the lungs of allergic volunteers showed enhanced transendothelial migration, indicating that the cells may have undergone cytokine activation in vivo. Eosinophil-active cytokines may contribute to the preferential accumulation of eosinophils in vivo in part via potentiation of eosinophil transendothelial migration.

Adult↗
PubMed1994

Secretion of IL-4 from human basophils. The relationship between IL-4 mRNA and protein in resting and stimulated basophils.

Purified human basophils have been examined for secretion of IL-4 protein and expression of IL-4 mRNA after stimulation with several secretagogues. In general, these studies used a 15-min preincubation with IL-3, before challenge with secretagogues. Under these conditions, IL-4 release averaged 30 pg/10(6) basophils (range 4-70) after challenge with anti-IgE Ab. FMLP and C5a led to somewhat lower levels of secretion. A direct comparison of basophils at 88 to 99% purity with basophils from the same preparations, but at lower purities, showed that the amount of IL-4 secretion was proportional to the purity of the basophils. The presence of mRNA for IL-4 (as determined by reverse transcriptase-PCR, competitive reverse transcriptase-PCR, or Northern blots) was also strictly related to the purity of the basophils. IL-4 mRNA was also found to be constitutively present and was increased after stimulation. The concentration of polyclonal anti-IgE Ab required for optimal IL-4 release was somewhat less than that required for optimal histamine release. IL-4 secretion was slower (t1/2 of 1.5 h) than histamine release and was inhibited by cycloheximide. In a final series of studies, we found that IL-3 was not required for IL-4 secretion; a short 15-min preincubation with IL-3 resulted in the same or slightly less IL-4 release than no treatment with IL-3. In contrast, an 18-h pretreatment with IL-3 resulted in a nearly tenfold increase in IL-4 secretion. We conclude that human basophils secrete IL-4 in response to several secretagogues and that IL-3 priming is not necessary to observe IL-4 secretion.

Base Sequence↗
PubMed1994

Detection of allergen- and mitogen-induced human cytokine transcripts using a competitive polymerase chain reaction.

Human cytokines, IL-4, IL-5, and IFN-gamma play an important role in the regulation of IgE synthesis and atopic diseases. In this communication, we describe the development of a quantitative assay of steady-state cytokine mRNAs (IL-4, IL-5, and IFN-gamma) from a variety of cell sources, including peripheral blood mononuclear cells (PBMCs) stimulated with either a mitogen (PHA) or ragweed pollen allergen extract, and cells from allergen-challenged inflammatory sites. Quantitative analysis of IL-5, IL-4 and IFN-gamma transcripts was achieved by a competitive reverse transcription-polymerase chain reaction (RT-PCR) technique using internal standard (IS) cRNAs in the presence of specific oligonucleotide primers. Each IS was generated from a plasmid vector containing the respective cytokine cDNA modified by insertion with an SV40-DNA fragment. Both test RNA and IS were reverse-transcribed and subjected to the 'competitive' PCR in the same tube. We first demonstrate the linearity and reproducibility of this technique; second, we apply this competitive PCR assay to analyze quantitatively the expression of IL-4, IL-5, and IFN-gamma transcripts in PBMCs before and after stimulation with PHA or crude ragweed allergen. Finally, we analyzed cells isolated from the lung lavage fluids of an atopic subject following allergen challenge, and showed a significant increase of IL-4 and IL-5 transcripts, but not IFN-gamma, in the allergen-challenged site when compared to the control. This technique of PCR quantitation provides an easy and efficient tool to study the expression of cytokine genes in allergic inflammatory diseases.

Allergens↗
PubMed1994

Blood and bronchoalveolar eosinophils in allergic subjects after segmental antigen challenge: surface phenotype, density heterogeneity, and prostanoid production.

Eosinophil infiltration into the airways has been implicated in the pathophysiology of asthma. To improve our understanding of the function of eosinophils in asthma, we have compared the phenotype and function of eosinophils obtained simultaneously from blood and bronchoalveolar lavage (BAL) of allergic subjects 19 hours after segmental lung allergen challenge. Eosinophils were purified by discontinuous density gradient centrifugation, and their distribution at various layers was quantitated. Eosinophils at the 1.080 to 1.085 gm/ml interfaces from blood and BAL (purity > 70%) were analyzed by immunofluorescence and flow cytometry for several surface markers including adhesion-activation antigens. Eosinophils in BAL from antigen-challenged sites were markedly increased compared with control diluent-challenged BAL sites (0.3% +/- 1% vs 28.1% +/- 9.7%, n = 12, p < 0.002), and a greater percentage were hypodense (specific gravity < 1.080 gm/ml) than in peripheral blood (51.3 +/- 5.3 vs 19.0 +/- 4.4, n = 15, p < 0.01). In vitro, resting and activated BAL eosinophils biosynthesized less thromboxane B2 than blood eosinophils. Although both BAL and blood eosinophils expressed similar levels of Fc gamma RII (CD32), CD11a, and CD45, resting levels of Mo-1 (CD11b) were upregulated on BAL eosinophils (mean fluorescence intensity, 316% +/- 48% of blood eosinophils, n = 5, p < 0.05). Blood eosinophils stimulated in vitro with 1 mumol/L platelet activating factor or N-formyl-methionyl-leucyl-phenylalanine achieved levels of CD11b expression similar to those of BAL eosinophils. In contrast, CD11b expression on BAL eosinophils could not be further increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

The effect of terfenadine on unilateral nasal challenge with allergen.

To investigate the role of H1 receptor-mediated effects in allergic rhinitis, we challenged 12 allergic volunteers with allergen 2 hours after administration of either placebo or 60 mg of terfenadine. Filter paper discs were used for the unilateral administration of allergen and the collection of nasal secretions. Secretion weights, levels of histamine in recovered nasal secretions, and nasal airway resistance (NAR) were measured for each nostril separately, and the number of sneezes was counted. After placebo treatment, allergen challenge led to significant increases in ipsilateral and contralateral secretion weights, ipsilateral histamine levels, ipsilateral NAR, and sneezing. Contralateral histamine levels were not elevated. H1 antagonism with terfenadine markedly reduced the number of sneezes and partially decreased ipsilateral and contralateral secretion weights, without affecting the increase in NAR. Terfenadine premedication also lowered the amount of histamine in ipsilateral secretions after allergen challenge. Performing identical nasal challenges with a 10-fold lower dose of antigen produced similar results. Previous studies showed that terfenadine had no effect on methacholine provocation and completely abolished ipsilateral and contralateral secretion weights after histamine challenge. We conclude that sneezing after allergen challenge is caused almost exclusively by a reflex initiated through H1 receptors and that H1 antagonism has no influence on allergen-induced increases in NAR. Unilateral allergen challenge leads to bilateral increases in secretion weights, which are only partially inhibited by terfenadine, suggesting the involvement of mediators other than histamine in the nasonasal reflex. As reported earlier, terfenadine also decreases allergen-induced histamine release after challenge with the highest dose of antigen.

Adult↗
PubMed1994

Immunological aspects of allergic asthma.

Asthma is a chronic obstructive disease of the lower airways. It is characterized by episodic exacerbations of at least partially reversible airflow limitation, along with bronchial hyperreactivity and airway inflammation. This latter characteristic, airway inflammation, is now a major focus of investigation, in large part because of a growing body of evidence to support its critical role in the pathogenesis of this disorder. Investigations of the histopathologic, cytologic, and chemical events accompanying fatal and nonfatal asthma, as well as experimental allergen-induced allergic reactions of the lower airways, have confirmed that a wide variety of immunologic phenomena occur, including local release of preformed mediators, newly synthesized metabolites of arachidonic acid, and soluble pro-inflammatory proteins including kinins and cytokines; alterations in vascular permeability and the development of airways edema; leukocyte activation and accumulation; and immune-mediated neurogenic responses that affect airway smooth muscle and other local responses. This information has led to new pharmacologic approaches designed to antagonize each of these elements of the asthmatic diathesis, with varying degrees of success. Nevertheless, the development of novel antagonists of mediators, pro-inflammatory proteins, cells, adhesion molecules, neuropeptides, and their respective receptors have begun to yield additional insight into their relative importance in the immunologic basis of asthma.

Asthma↗
PubMed1994

Activated human basophils contain histamine in cytoplasmic vesicles.

Human basophils in contact allergy empty granules slowly--a secretory process termed piecemeal degranulation in contrast to the explosive extrusion of granules seen in anaphylactic degranulation. An ultrastructural enzyme-affinity method to detect histamine, employing diamine oxidase coupled to gold, was used to localize this proinflammatory mediator in human basophils stimulated to release histamine by FMLP. Cytoplasmic vesicles and granules of human basophils so stimulated for 20 s were gold-labeled, indicating the presence of histamine. Cytoplasmic vesicles may play a key role in the secretion of histamine from basophils in those diseases characterized by the ultrastructural morphology of piecemeal degranulation.

Basophils↗
PubMed1994

Physiologic responses and histamine release after nasal antigen challenge. Effect of atropine.

We enrolled nine allergic subjects in a double blind, placebo-controlled study to examine the effect of premedication with 0.6 mg of atropine on nasal antigen challenge. The challenge consisted of unilaterally stimulating the nasal septum with diluent followed by three increasing doses of antigen and recording responses bilaterally. Sneezes, symptoms, and nasal airway resistance (NAR) were recorded. Secretions were collected using preweighed filter paper discs and histamine was measured. Antigen challenge with the subjects on placebo led to significant dose-dependent increases in sneezes, symptom scores, ipsilateral and contralateral secretion weights, ipsilateral NAR, and total amount of ipsilateral histamine (p < 0.05 versus diluent). Bilaterally applied atropine led to significant inhibition of ipsilateral and contralateral nasal secretions as well as rhinorrhea scores (p < 0.05 versus placebo) but had no significant effect on other parameters. Challenge after atropine premedication led to higher increases in histamine concentration than placebo (p < 0.01). These results suggest that parasympathetically stimulated fluids did not contain histamine and diluted the histamine released by mast cells. To support this hypothesis, we challenged the same subjects with methacholine. The concentration of histamine decreased and was significantly lower than after challenge with antigen (p < 0.01). The data suggest that: (1) histamine is released locally at the site of antigen challenge, (2) the volume of glandular secretions is primarily controlled by parasympathetic stimulation, and (3) the total amount of a mediator recovered in a fixed time interval best reflects the underlying pathophysiologic events.

Adult↗
PubMed1994

Identification of IgE-bearing cells in the late-phase response to antigen in the lung as basophils.

We have carried out studies to ascertain whether the histamine-containing, IgE-bearing cells found in the bronchoalveolar lavage (BAL) fluid obtained during the late-phase response following subsegmental antigen challenge of human airways are predominantly basophils or mast cells. Four lines of evidence suggest that most are basophils: (1) The cells fulfill morphologic criteria for light microscopy. (2) Cell surface markers determined by immunofluorescence and flow cytometry revealed that the IgE-bearing cells express the leukocyte antigens Fc gamma RII and the beta 2 integrins, LFA-1 and Mac-1, but do not express the mast cell-associated c-kit receptor for stem cell factor. (3) The late-phase histamine-containing cells in late-phase BAL fluids have the functional characteristics of basophils in their secretory responses to anti-IgE, the f-met peptide, and phorbol ester TPA. (4) The cells have a functional histamine type 2 receptor, a characteristic of basophils, not mast cells. We conclude that basophils infiltrate the lower airways hours after antigen exposure. These cells may be responsible for the mediator release observed at that time.

Alcian Blue↗
PubMed1994

Recombinant stem cell factor-induced mast cell activation and smooth muscle contraction in human bronchi.

The effect of human recombinant stem cell factor (SCF) on inflammatory mediator release and smooth muscle contraction was evaluated in human isolated intralobar bronchi. Bronchi from 21 of 26 donors contracted in response to SCF. The threshold concentration was approximately 0.01 micrograms/ml. At 1 micrograms/ml, the tissues contracted to about 60% of the carbamylcholine-induced maximum contraction. The responses to SCF mimicked those obtained with anti-IgE. Thus, the contractions to SCF and anti-IgE were inhibited to a similar extent by a combination of a cysteinyl-leukotriene receptor antagonist and a histamine H1 receptor antagonist. SCF also mimicked the effect of anti-IgE in releasing histamine, i-LTD4, and PGD2 from the bronchi. At a threshold concentration for contraction (0.01 micrograms/ml), SCF had no effect on subsequent responses to anti-IgE in the bronchi. The data suggest that human recombinant SCF contracts airway smooth muscle by stimulating the release of contractile mediators from bronchial mast cells. The data fail to support the hypothesis that SCF primes bronchial mast cells to subsequent immunologic stimuli.

Antibodies, Anti-Idiotypic↗