Methacholine induces wheal-and-flare reactions in human skin but does not release histamine in vivo as assessed by the skin microdialysis technique

A number of investigations have indicated that cholinergic agonists release histamine from isolated mast cells and suggested that cholinergic stimulation releases histamine in vivo. The purpose of this study was to investigate whether the cutaneous wheal-and-flare reaction induced by methacholine challenge in human skin involves histamine release as measured by the skin microdialysis technique. Five hollow dialysis fibers were inserted intradermally in forearm skin in eight healthy subjects. Each fiber was perfused with Kreb's-Ringer bicarbonate at a rate of 3 μl/min. Dialysates were collected in 2-min fractions before skin challenge and for 20 min after intradermal injection of methacholine 10–^-3 -10^-1 M, the vehicle, and a positive control, codeine phosphate 0.3 mg/ml. Histamine was assayed spectrofluorometrically. Methacholine caused a statistically significant dose-related wheal-and-flare reaction, the flare reaction to methacholine 10^-1 M being comparable with that seen with codeine 0.3 mg/ml. No significant histamine release was observed with methacholine, cumulative histamine release of 16 ± 8nM by methacholine 10^-1 M being similar to vehicle responses of 15 ± 9 nM. Histamine release by codeine was 2524 ± 435 nM. In conclusion, methacholine-induced wheal-and-flare reactions in human skin appeared not to involve histamine release from skin mast cells.     

Quantitative measurement of extracellular histamine concentrations in intact human skin in vivo by the microdialysis technique: methodological aspects

Petersen LJ. Quantitative measurement of extracellular histamine concentrations in intact human skin in vivo by the microdialysis technique: methodological aspects. Calculation of recovery is needed in microdialysis studies to calculate absolute concentrations of compounds in the extracellular water space. The purposes of this study were to determine the extracellular concentration of histamine in intact human skin in vivo and to study the validity of absolute histamine measurements during allergic skin reactions. A skin microdialysis technique and two calibration techniques, the no net flux method and the flow rate method, were used to quantify histamine concentrations in resting skin. To validate these techniques, skin glucose concentrations were analysed as well. In addition, the influence of vasodilation and plasma extravasation on recovery was followed after intradermal injection of codeine, a mast-cell secretagogue. As expected, both calibration methods estimated skin glucose concentrations to be identical with venous blood glucose concentrations. However, skin histamine levels could not be calculated by the no net method, because the data did not meet the theoretic assumptions of this method. In contrast, histamine data fitted theoretically with the flow rate method, and skin histamine concentrations of 18.8±2.8 nM were found to be significantly greater than plasma histamine concentrations of 4.3±0.7 nM. Within minutes after intradermal injection of codeine, recovery increased significantly in a dose-dependent fashion. Vasodilation per se did not influence recovery. In conclusion, absolute assessment of skin histamine concentrations can be made by microdialysis by the flow rate method. The validity of such an estimate and the theoretic prerequisites for the calculations are discussed. Quantitative measurement of skin histamine levels during allergic reactions cannot be performed since recovery is altered by plasma extravasation after skin challenge.     

No release of histamine and substance P in capsaicin-induced neurogenic inflammation in intact human skin in vivo: a microdialysis study

Background Studies in rodents ‘skin have indicated substance P to be the main inflammatory mediator involved in neurogenic inflammation, acting partly by release of histamine from skin mast cells. The mediators released in neurogenic inflammation in human skin remain to be determined. Objectives To determine the effects of intradermally injected and topically applied capsaicin on the release of histamine and substance P and skin responses in intact human skin in vivo. Methods Extracellular skin levels of histamine and substance P were measured by microdialysis technique and assayed by enzyme and radio immunoassays. Two kinds of dialysis fibres (210μm, 2 kDa, and 500 μm, 20 kDa) were inserted intradermally into forearm skin for studies of histamine release to topically administered capsaicin and intradermally injected capsaicin and substance P. Results Baseline histamine skin levels were 8.0 ± 0.7 nM. Intradermally injected capsaicin (0.3–30μM, 7.5–750 pmol) caused significantly and dose-related flare and pain reactions, but no significant histamine release or weals. Intradermally injected substance P (1 and 3 μM, 25 and 75 pmol) released significant amounts of histamine (peak levels being 90 and 475 nM), evoked weal-and-flare reactions, but did not cause pain. Capsaicin 2% ointment, applied on the skin for 2.5 h, increased skin blood flow by 300–400% as measured by laser Doppler flowmetry, elicited a longstanding burning sensation, but did not release histamine. Substance P-like immunoreactivity (SP-LI) was below the 1.8 pM detection limit following insertion of 20 kDa dialysis fibre and after intradermal injection of capsaicin 3μM. Intradermal injection of injection of 1 μM of substance P increased SP-LI levels to values greater than 4500 pM, confirming the ability of the dialysis fibre to recover this peptide. Conclusions Capsaicin-induced neurogenic activation does not involve the release of histamine from mast cells or detectable amounts of substance P release from sensory nerves in normal human skin in vivo.     

Histamine release in immediate-type hypersensitivity reactions in intact human skin measured by microdialysis

The purpose of this study was to evaluate the application of a microdialysis technique for measurement of interstitial histamine levels in intact human skin. Three allergic subjects were investigated. Single dialysis fibers were glued to nylon tubings and inserted in forearm skin by means of a fine cannula. Dialysis fibers were inserted in triplicate and perfused with isotonic saline at a rate of 3 microliters/min. After a period of 2 h a 60-microliters base-line period was established. Then the patients were skin prick tested (SPT) with allergen in duplicate and a single saline control. Dialysate was collected in consecutive 30 microliters fractions. Histamine concentration in the dialysate was analyzed with a glass fiber fluorescence assay. Median base-line histamine level was 4 (range 4-7) ng/ml. Following allergen SPT, dialysate histamine concentration increased to 81 ng/ml (74-128), with maximum values 10-20 min after SPT. Intraindividual coefficient of variation on peak histamine levels was 18.9%. No histamine increase was seen following saline SPT. We consider microdialysis to be a valuable method for assessment of allergic mechanisms in intact human skin.     

The effect of cetirizine and loratadine on codeine-induced histamine release in human skin in vivo assessed by cutaneous microdialysis

Objective and Design To determine whether or not cetirizine and loratadine inhibit codeine- induced histamine release in human skin in vivo, we conducted a placebo-controlled double-blind trial in which histamine release was assessed by dermal microdiaysis.Subjects A group of ten normal volunteers were studied, each subject visiting the laboratory on three occasions with intervals of at least 2 weeks between visits.Treatment Cetirizine, loratadine (both 10 mg) or placebo was given orally 4h before provocation of weal and flare responses in the skin by intradermal injection of 25 l of 3 or 10 mg/ml codeine 1 mm from the centre of individual 216 m diameter microdialysis fibres inserted in the dermis.Methods Dialysate was collected at 2 min intervals for 4 min before and 20 min after codeine injection and histamine assayed spectrofluorometrically. Weal and flare responses to codeine were assessed in the opposite arm.Results Histamine concentrations in the microdialysis fibre outflow with 3 and 10 mg/ml codeine were maximal at 2–4 min when 910±156 and 1194±304 nM respectively were found in the placebo group. Cetirizine and loratadine did not modify either the kinetics or total histamine release while significantly (p<0.01) inhibiting weal and flare responses.Conclusions Neither cetirizine nor loratadine inhibited codeine-induced histamine release or modified the time course of its release in human skin in vivo when given in clinically used doses which are sufficient to significantly reduce weal and flare responses.accepted by M. J. Parnham     

Histamine is released in the wheal but not the flare following challenge of human skin in vivo: a microdialysis study

Background The mediator mechanisms of the cutaneous wheal and flare response, which underlies allergic skin and urticarial conditions, are controversial. The wheal results primarily from a direct effect of histamine on the local vascular bed, but to what extent does histamine diffuse within the wheal? The flare is neurogenic in origin, being disseminated within the dermis by axon reflexes, but do the neuropeptides released from the nerve endings cause the vasodilatation directly or do they induce the further release of histamine which then transduces the fiare? Objective We have addressed these questions by inserting 216 μm diameter microdialysis fibres into the dermis within the different areas of the wheal and flare to monitor changes in histamine levels provoked by intradermal injections of histamine, allergen, codeine and substance P. Twenty-one subjects participated in the investigations. Results The histamine concentration in unprovoked skin was 10.5 ± 0.6 nM. As the dialysis efficacy was 50%, this equates to tissue concentrations of 20 nM. All provicants released large amounts of histamine at the injection site, maximum histamine levels being 337–1293 nM. Diffusion of histamine within the wheai was poor, levels at 2.3 mm and 3.7 mm from the site of injection being 4–22% and 0.2–3.7% respectively of those 1 mm from the injection site. No increased histamine levels were detected in the flare with any provicant. Atraumatic delivery to the skin of histamine in infusion concentrations of 30–10000 nM caused concentration-related effects, at least 100 nM being necessary to induce a significant increase in skin blood flow, a threshold of 300–1000 being required to stimulate a visible flare and a measurable erythema, and 3000–10000 nM being the minimum for induction of a wheal. Thus the skin blood vessels and nerves are responsive to histamine, but at relatively high concentrations Conclusions These data support the theory that the flare reaction to local histamine injection or release is a neurogenic reflex not involving histamine release at its effector end.     

Measurement of interstitial cetirizine concentrations in human skin: correlation of drug levels with inhibition of histamine‐induced skin responses

BACKGROUND: The purpose of the present study was to measure the concentrations of cetirizine in the extracellular water compartment in intact human skin and assess simultaneously inhibition of histamine-induced wheal and flare reactions. METHODS: Skin cetirizine levels were collected by the microdialysis technique and analyzed by high-pressure liquid chromatography with mass spectrometry detection. Skin levels in 20 subjects were compared to plasma levels for 4 h after a single oral dose of 10 or 20 mg of cetirizine. Skin prick tests were performed with histamine 100 mg/ml. RESULTS: Plasma cetirizine levels increased within 30 min to reach peak values of 315+/-10 and 786+/-45 ng/ml 90-120 min after administration of 10 and 20 mg of cetirizine. This was followed by a slow decline. In the skin, dialysate cetirizine levels (non-protein-bound fraction only) peaked at 1.6+/-0.1 and 2.4+/-0.3 ng/ml at 120-180 min. In vivo recovery of cetirizine was 14.4+/-4.3%. It was estimated that the non-protein-bound concentration of cetirizine in the skin was 50-70% of corresponding plasma values. Both 10- and 20-mg doses of cetirizine inhibited wheal and flare reactions over 240 min. The time vs concentration profile of cetirizine in skin dialysate paralleled the inhibition of skin reactions, but no significant correlations were found between individual cetirizine levels in skin or plasma with wheal and flare reactions. CONCLUSIONS: Cetirizine concentrations in the skin could be monitored by the microdialysis technique. The results indicate no simple linear correlation between cetirizine skin levels and inhibition of skin reactions.     

The release of leukotriene B4 from human skin in response to substance P: evidence for the functional heterogeneity of human skin mast cells among individuals

Substance P is located in cutaneous nerve fibres and induces wheal and flare responses, accompanied by granulocyte infiltration, upon intradermal injection. Studies with animal skin and rat peritoneal mast cells have suggested that substance P induces the release of histamine and leukotriene B4 (LTB4), a potent chemoattractant for granulocytes, from skin mast cells. However, the release of LTB4 has not been detected from mast cells enzymatically isolated from human skin. In order to investigate the mechanism of granulocyte infiltration induced by substance P in human skin, we studied the release of LTB4 and histamine in response to substance P, and the effect of dexamethasone using human skin obtained from 22 nonallergic individuals. Histamine was released from all skin tissue samples in a dose-dependent manner. However, the amount of LTB4 release, both constitutive and inducible, was variable among skin preparations. Substance P induced a large release of LTB4 from the skin of eight donors (twice to six times that of the spontaneous release), but no or only negligible release from the skin of 14 donors. The amount of constitutive release of LTB4 correlated with the amount of tissue histamine. Dexamethasone selectively abolished the inducible release of LTB4, without an effect on histamine release and the constitutive release of LTB4. These results suggest that substance P induces the release of LTB4 in a certain population of human individuals by a glucocorticosteroid-dependent mechanism, and plays an important role in neurogenic inflammation with granulocyte infiltration.     

Responsiveness of human skin mast cells to repeated activation: an in vitro study

To assess human mast-cell (MC) behavior after repetitive activation, we cocultured human foreskin MC (SMC) with human foreskin fibroblasts (F). Under these conditions, we have previously demonstrated that SMC keep their viability and functional activity for up to 8 days. SMC were presensitized with atopic serum and repeatedly activated by consecutively increasing concentrations of anti-IgE antibodies (α-IgE, 0.0002–0.1). This treatment, which mimics the "rush desensitization" procedure, led to complete SMC unresponsiveness to activation by a-IgE at optimal concentrations, as evaluated by histamine release. However, presensitization of SMC with IgE antibodies before exposure to α-IgE restored their sensitivity to this stimulus. These data indicate that desensitization was probably due to lack of membrane-bound IgE rather than to downregulation of intracellular mechanisms. In fact, SMC challenged by an optimal concentration of α-lgE could release histamine upon a second activation by 2 h after the first activation, if the cells had been presensitized before the second challenge. SMC incubation with increasing concentrations of compound 48/80 (0.2–10μg/ml) led to MC unresponsiveness to an optimal concentration of this stimulus. Furthermore, SMC activated by an optimal concentration of compound 48/80 and rechallenged with the same agent were insensitive to the second activation for at least 24 h. In summary, we have shown that it is possible to induce "desensitization" in SMC to both IgEdependent and IgE-independent stimuli by incubating the cultures with consecutively increasing concentrations of the activator. SMC can release histamine when reactivated with a-IgE antibodies after presensitization by 2 h after the first challenge, while they reacquire their susceptibility to reactivation with compound 48/80 in only 2–3 days.     

Enhancement of basophil histamine release by interleukin-3: reduced effect in atopic subjects

The inducing and enhancing effects of interleukin-3 (IL-3) on basophil histamine release in patients with respiratory allergy ( n = 28) and in normal subjects ( n = 22) were compared. Leukocyte suspensions, prepared by dextran sedimentation, were stimulated with anti-IgE (1/5000), N-formyl-methionyl-leucyl-phenylalanine (FMLP, 1 μM), and IL-3 (0.1–10 ng/ml), and histamine concentration was measured by an automated fluorometric method. A trend toward higher histamine release after challenge with anti-IgE, FMLP, and IL-3 was found in atopic subjects. Preincubation of basophils with IL-3 resulted in a dose-dependent increase of anti-IgE- and FMLP-induced histamine release, with a more marked effect in nonatopic than in atopic subjects. Mean net enhancement of anti-IgE-induced histamine release by 10 ng/ml IL-3 was 2.5±5% in atopic subjects and 29.6±4.2% in nonatopic subjects ( P < 0.001). The enhancement of FMLP-induced histamine release by IL-3 was 10.3 ±3.9% in atopic patients and 29±2.4% in nonatopic subjects ( P < 0.01). In atopic subjects, a negative correlation was found between anti-IgE- or FMLP-induced histamine release and net enhancement by IL-3 ( r = -0.45, P < 0.02; r = 0.48, P < 0.01, respectively). The results of this study indicate that in atopic subjects IgE-mediated histamine release can scarcely be enhanced by a basophil response modifier such as IL-3. It is conceivable that the frequent basophil stimulation in atopic patients leads to a reduced sensitivity to the enhancing effect of IL-3.     

The release of histamine is associated with the inactivation of mast cell chymase during immediate allergic wheal reaction in the skin

BACKGROUND: Chymase released by mast cells can participate in the immediate allergic wheal. However, chymase may be susceptible to inactivation by protease inhibitors during degranulation. OBJECTIVE: To study the inactivation of chymase and the release of histamine in the immediate allergic wheal reaction. METHODS: Ten sensitive atopic subjects were prick-tested with the cow dander allergen, and skin biopsies were taken from the control skin and from the challenge site at 30 and 120 min. Tryptase (Tact) and chymase (Cact) activities in mast cells were measured enzyme-histochemically. Sequential double-staining was used to demonstrate the activity and immunoreactivity (Cprot) of chymase in the same mast cell as well as alpha1-proteinase inhibitor (alpha1-PI) and alpha1-antichymotrypsin (alpha1-AC) in Tact+ cells. Skin microdialysis was used to monitor histamine release after the allergen challenge for up to 120 min RESULTS: The numbers of Tact+ and Cact+ cells were already maximally decreased at 30 min by 37 +/- 17% and 61 +/- 31%, respectively (mean +/- SD, P < 0.0001). At the same time the Cact+/Cprot+ ratio decreased from 82 +/- 15% to 43 +/- 16% (P < 0.0001). The cumulative histamine release at 30 min correlated negatively with the Cact+/Tact+ (P = 0.047) and Cact+/Cprot+ (P = 0.024) ratios, but positively with the decrease in the number of Cact+ cells (P = 0.024). These data indicate that the higher the histamine release the lower the chymase activity. Also the number of Tact+ cells in the control skin correlated positively with the cumulative histamine release at 120 min (P = 0.043). In the control skin, 95 +/- 6% and 76 +/- 8% of the Tact+ cells displayed alpha1-AC and alpha1-PI, respectively. CONCLUSION: In addition to extensive degranulation of mast cells, chymase is also rapidly inactivated after the allergen challenge, possibly by pre-existing chymase inhibitors in the mast cells. This inactivation is associated with the release of histamine.     

Diagnostic value of scratch-chamber test,skin prick test,histamine release and specific IgE in birch-allergic patients with oral allergy syndrome to apple

BACKGROUND: The aim of the study was to examine the diagnostic value of skin prick test (SPT), scratch-chamber test (SCT), histamine release (HR) and specific immunoglobulin E (IgE) in birch-allergic patients with oral allergy syndrome to apple. METHODS: Ten birch-allergic patients with oral allergy syndrome to apple and 10 control subjects were included. All were tested with SPT, SCT, HR and specific IgE [CAP, Pharmacia, Sweden and Magic Lite (ML), ALK-ABELLO, Denmark]. RESULTS: The SPT with apple, acetone extract of apple (A72) and commercial apple extract showed sensitivities of 0.80, 0.90 and 0.10, respectively. The SCT with the same extracts showed sensitivities of 0.30, 0.50 and 0.20, respectively. The sensitivity of specific IgE to apple were 0.90 (CAP) and 0.10 (ML). The sensitivity of the HR test was 90% (A72), and 25% using the commercial extract. CONCLUSION: The SPT and HR test with apple and A72 showed a good diagnostic value with a sensitivity of more than 70% and a specificity of 100%. The SCT showed a poor sensitivity to apple, A72 and commercial apple extract. The ML test was not suitable in detecting specific IgE to apple compared with the CAP test. In daily practice a detailed case history about symptoms of oral allergy syndrome combined with a SPT with fresh apple peel or A72 will be useful.     

Poor association between allergen-specific serum immunoglobulin E levels, skin sensitivity and basophil degranulation: a study with recombinant birch pollen allergen Bet v 1 and an immunoglobulin E detection system measuring immunoglobulin E capable of binding to FcɛRI

BACKGROUND: Results from several studies indicate that the magnitude of immediate symptoms of type I allergy caused by allergen-induced cross-linking of high-affinity Fc epsilon receptors on effector cells (mast cells and basophils) is not always associated with allergen-specific IgE levels. OBJECTIVE: To investigate the association of results from intradermal skin testing, basophil histamine release and allergen-specific IgE, IgG1-4, IgA and IgM antibody levels in a clinical study performed in birch pollen-allergic patients (n = 18). METHODS: rBet v 1-specific IgEs were measured by quantitative CAP measurements and by using purified Fc epsilon RI-derived alpha-chain to quantify IgE capable of binding to effector cells. Bet v 1-specific IgG subclasses, IgA and IgM levels were measured by ELISA, and basophil histamine release was determined in whole blood samples. Intradermal skin testing was performed with the end-point titration method. RESULTS: Our study demonstrates on the molecular level that the concentrations of allergen-specific IgE antibodies capable of binding to Fc epsilon RI and biological sensitivities are not necessarily associated. A moderate association was found between cutaneous and basophil sensitivity. CONCLUSION: Our results highlight the quantitative discrepancies and limitations of the present diagnostic tools in allergy, even when using a single allergenic molecule. The quantity of allergen-specific serum IgE is only one component of far more complex cellular systems (i.e. basophil-based tests, skin tests) used as indirect diagnostic tests for IgE-mediated allergic sensitivity.     

Relevance of Ara h1, Ara h2 and Ara h3 in peanut-allergic patients, as determined by immunoglobulin E Western blotting, basophil–histamine release and intracutaneous testing: Ara h2 is the most important peanut allergen

BACKGROUND: A number of allergenic proteins in peanut has been described and the relative importance of these allergens is yet to be determined. OBJECTIVES: We have investigated the relevance of previously identified peanut allergens in well-characterized peanut-allergic patients by in vitro, ex vivo and in vivo assays. METHODS: Thirty-two adult peanut-allergic patients were included based on careful and standardized patient history and the presence of peanut-specific IgE. The diagnosis peanut allergy was confirmed using double-blind placebo-controlled food challenges in 23 patients. Major peanut allergens Ara h1, Ara h2 and Ara h3 were purified from peanuts using ion-exchange chromatography. IgE immunoblotting was performed and IgE-cross-linking capacity was examined by measuring histamine release (HR) after incubating patient basophils as well as passively sensitized basophils with several dilutions of the allergens. Intracutaneous tests (ICTs) using 10-fold dilution steps of the purified allergens and crude peanut extract were performed. RESULTS: Ara h2 was recognized most frequently (26 out of 32) in all tests and induced both positive skin tests and basophil degranulation at low concentrations, whereas Ara h1 and Ara h3 were recognized less frequently and reacted only at 100-fold higher concentrations as analysed with HR and intracutaneous testing (ICT). Next to the three tested allergens, proteins with molecular weights of somewhat smaller than 15 kDa were identified as a IgE-binding proteins on immunoblot in the majority of the patients (20 out of 32). CONCLUSION: We conclude that Ara h2 is, for our patient group, the most important peanut allergen, and that previously unidentified peanut proteins with molecular weights of somewhat smaller than 15 kDa may be important allergens as well. ICT in combination with basophil-HR and IgE immunoblotting provides insight in the patient specificity towards the individual peanut allergens.