Histamine-releasing Activity Research Articles

Response by authors: Assessment of histamine‐releasing activity of sera from patients with chronic urticaria showing positive autologous skin test on human basophils and mast cells. R. Asero and A. Tedeschi

Clinical & Experimental AllergyVolume 34, Issue 11 p. 1804-1805 Response by authors: Assessment of histamine-releasing activity of sera from patients with chronic urticaria showing positive autologous skin test on human basophils and mast cells. R. Asero and A. Tedeschi R. Asero, R. Asero Ambulatorio di Allergologia, Clinica San Carlo, Paderno DugnanoSearch for more papers by this authorA. Tedeschi, A. Tedeschi Unità Operativa di Medicina Interna I, IRCCS Ospedale Maggiore Policlinico, Milan, Italy, E-mail: [email protected]Search for more papers by this author R. Asero, R. Asero Ambulatorio di Allergologia, Clinica San Carlo, Paderno DugnanoSearch for more papers by this authorA. Tedeschi, A. Tedeschi Unità Operativa di Medicina Interna I, IRCCS Ospedale Maggiore Policlinico, Milan, Italy, E-mail: [email protected]Search for more papers by this author First published: 12 November 2004 https://doi.org/10.1111/j.1365-2222.2004.2116_3.xRead the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Volume34, Issue11November 2004Pages 1804-1805 RelatedInformation

Chronic urticaria: Expanding the autoimmune kaleidoscope

Chronic urticaria: Expanding the autoimmune kaleidoscope

Assessment of histamine‐releasing activity of sera from patients with chronic urticaria showing positive autologous skin test on human basophils and mast cells

All previous studies agree that only a proportion of sera from patients with chronic urticaria (CU) positive on the autologous serum skin test (ASST) are able to induce histamine release in vitro. A non-specific release of bradykinins during clotting of blood samples has been suggested; however, ASST seems rather specific and some data point to the existence of a mast cell-specific histamine-releasing factor. To assess whether, and to what extent, the use of both human basophils and mast cells increases the sensitivity of in vitro histamine release assays (HRAs) in ASST-positive patients with CU. The histamine-releasing activity of sera from 93 patients with CU selected on the basis of strong skin reactivity on ASST was assessed in vitro on basophils from 1 (n=86), 2 (n=31), or 3 (n=20) normal donors, and on mast cells from 1 (n=3), 2 (n=3), or 3 (n=87) normal donors. Sera from 88/93 (95%) patients induced significant histamine release from mast cells or basophils on at least one HRA. 76/93 (82%), 45/90 (50%), 22/80 (28%), and 6/12 (50%) sera were able to induce significant histamine release from cells of 2/5, 3/5, 4/5 and 5/5 donors, respectively. Sera from nearly all ASST-positive patients with CU are able to induce histamine release in vitro. However, the serum from each single patient seems to show its maximal activity on autologous mast cells in vivo, and functional in vitro tests show much variability and seem less sensitive than ASST in the detection of patients with histamine-releasing factors in their blood.

Evidence of in vivo basophil activation in chronic idiopathic urticaria

Rationale Approximately 40% of chronic idiopathic urticaria (CIU) subjects have autoantibodies to FceRIa or to IgE. However, the effect of such autoantibodies on circulating basophil activation status is unclear. We compared the basophils from CIU and allergic subjects for expression of cell surface activation markers. We also examined the serum of donors for histamine releasing activity (HRA) and for immunoreactivity to FceRIa protein. Methods Peripheral blood was obtained from CIU and allergic donors and fractionated by density gradient centrifugation. Enriched basophils (1–8%)were analyzed by flow cytometry for expression of activation markers including CD 69, CD 63, and CD203. Dilutions of serum (5, 25, 50%) were analyzed for HRA on basophils from a single donor. Serum was also tested for immunoreactivity by Western blotting to a standard cell lysate prepared from RBL-SX38 transfected with the human FceRIa. Results CIU subjects (n=5) demonstrated enhanced expression of all three surface markers as compared to allergic donors (n=5): CD 63, 220 vs. 13.9; CD69, 108 vs. 4.6 and CD203, 4.17 vs. 1.4 (net mean fluorescence intensity units). Three CIU subjects had evidence of significant serum HRA (>20%) on donor basophils. Two CIU donors had evidence of immunoreactivity for FceRIa as compared to none of the allergic donors. The two CIU subjects with both serum HRA and immunoreactivity also had altered basophil function as measured by anti-IgE mediated stimulation. Conclusions Basophils from CIU subjects express increased levels of activation markers as compared to allergic subjects. Heightened basophil activation in CIU may be due to circulating serum factors.

Partial alanine scan of mast cell degranulating peptide (MCD): importance of the histidine- and arginine residues.

The influence of the two histidine and two arginine residues of mast cell degranulating peptide (MCD) in activity and binding was studied by replacing these amino acids in the MCD sequence with L-alanine. Their histamine releasing activity was determined on rat peritoneal mast cells. Their binding affinity to the FcepsilonRIalpha binding subunit of the human mast cell receptor protein, was carried out using fluorescence polarization. The histamine assay showed that replacement of His13 by Ala o ccurred without loss of activity compared with the activity of MCD. Alanine substitutions for Arg7 and His8 resulted in an approximately 40 fold increase, and for Arg16 in a 14-fold increase in histamine-releasing activity of MCD. The binding affinities of the analogs were tested by competitive displacement of bound fluorescent MCD peptide from the FcepsilonRIalpha binding protein of the mast cell receptor by the Ala analogs using fluorescence polarization. The analogs Ala8 (for His) and Ala16 (for Arg) showed the same binding affinities as MCD, whereas analog Ala7 (for Arg) and analog Ala13 (for His) showed slightly better binding affinity than the parent compound. This study showed that the introduction of alanine residues in these positions resulted in MCD agonists of diverse potency. These findings will be useful in further MCD structure-activity studies.

Correlation between T-Cell and Mast Cell Activity in Patients with Chronic Urticaria

Background: The presence of autoantibodies reacting with the high affinity IgE receptor (FcΕRIα) usually indicates a more severe form of chronic urticaria (CU). Previously, we showed an increased lymphocyte reactivity in CU patients; however, the relation between enhanced cellular immunity and the presence of anti-FcΕRIα-specific autoantibodies has not been investigated. Methods: Cellular and humoral immune reactivity of 50 CU patients and 28 healthy controls was studied.Serum sIL-2R, neopterin, and tryptase levels were measured to assess T-cell, monocyte/macrophage and mast cell activity, respectively. Helicobacter pylori (HP)-specific IgG antibody, and IgE levels were also tested. Anti-FcΕRIα-specific autoantibody was determined by Western blotting. In vivo histamine-releasing activity of patients’ sera was assessed by the autologous serum skin test (AST). Results: 17/50 CU patients, who both had IgG-type anti-FcΕRIα-antibodies by Western blotting and a positive AST response, were classified as autoimmune CU. All patients with CU had significantly higher serum sIL-2R and tryptase levels than healthy controls (p = 0.000257, p = 0.000166, respectively), indicating T-cell and mast cell activation. Patients with higher sIL-2R levels also had higher tryptase levels; the strongest correlation was shown in the autoimmune subgroup of patients (ρ = 0.688, p = 0.002). There was a tendency towards higher tryptase levels in the autoimmune subgroup, as compared to the nonautoimmune CU patients. While the serum IgE was significantly lower in autoimmune than in nonautoimmune CU (p = 0.000836), there was no significant difference in their sIL-2R, neopterin and HP-specific IgG antibody levels. CU patients with a positive AST response (38/50) had significantly higher tryptase levels (p = 0.0107) when compared to the negative skin test group. Conclusions: The significant correlation between sIL-2R and tryptase levels in patients with CU indicates that T cell activation is proportional to mast cell degranulation in these patients. The increased level of tryptase in autoimmune CU may suggest more severe disease.

Histamine-releasing activity and binding to the FcepsilonRI alpha human mast cell receptor subunit of mast cell degranulating peptide analogues with alanine substitutions.

We have investigated the effects on mast cell binding and the histamine-releasing activity of l-alanine substitutions for the five lysine residues and the proline residue in the MCD peptide (1) sequence. All synthesized analogues Ala(2) (2), Ala(6) (3), Ala(11) (4), Ala(12) (5), Ala(17) (6), and Ala(21) (7) showed a loss of histamine release compared to the parent MCD peptide 1. The order of decreased potency was 1 > 6 > 7 > 4 > 2 > 3 > 5. The alanine-substituted analogues showed a 5- to 6-fold decrease in histamine release for analogues 6, 7, and 4 and a 10-fold decrease for analogue 2. A more significant loss was observed in analogue 3 with a 75-fold loss of activity. The greatest loss of activity was observed with alanine substituting for proline in position 12. This analogue 5 showed a 130-fold loss of histamine release compared to the parent peptide 1. The ability of each analogue to interact with the FcepsilonRIalpha subunit of the human mast cell receptor was analyzed by competitive binding of the fluorescent peptide 1 and the alanine analogues using fluorescence polarization. The binding affinities of analogues 4, 6, and 7 for the mast cell receptor were less than the affinity of the native peptide 1. Analogues 2, 3, and 5 showed an increase in binding affinity, with analogue 5 showing the highest increase compared to the native peptide 1. The order of increased affinity was 5 > 3 > 2 > 1 > 4, 6, 7. On the basis of these results, the possibility that analogue 5 inhibits peptide 1-stimulated histamine release was examined. We found that peptide 5 did not inhibit histamine release by peptide 1. The analogues 2, 3, and especially analogue 5 may be useful leads toward study of agents that prevent binding of IgE to mast cell receptors.

Non-IgE–dependent activation of human lung– and cord blood–derived mast cells is induced by eosinophil major basic protein and modulated by the membrane form of stem cell factor

The allergic reaction begins with the antigen-induced aggregation of occupied high-affinity IgE receptors expressed on mast cell surface, their activation, and the release of proinflammatory mediators that cause the "early phase" of this process. In addition, mast cell activation induces the onset of a "late phase" reaction characterized by the tissue infiltration of inflammatory cells, mainly eosinophils. We have hypothesized that during the late phase mast cells interact with and are activated by eosinophils. Here we report that highly purified human lung mast cells became responsive to eosinophil major basic protein (MBP) when in coculture with human lung fibroblasts. In addition, cord blood-derived mast cells maintained in coculture with 3T3 fibroblasts released more histamine and prostaglandin D(2) (PGD(2)) compared with cells maintained in suspension. The fibroblast-derived membrane form of stem cell factor (SCF) was found to be involved in the mast cell increased responsiveness to MBP. In fact, cord blood-derived mast cells cocultured with 3T3 in the presence of antisense for SCF or cocultured with fibroblasts that do not express the membrane form of SCF were inhibited in their histamine-releasing activity toward MBP. In addition, this form of SCF induced the expression of a pertussis toxin-sensitive G(i) protein, G(i3) that interacts with MBP to trigger mast cell non-IgE-dependent activation in a manner similar to other cationic compounds such as compound 48/80. Mast cell responsiveness to eosinophil mediators is a potentially novel evidence for an alternative pathway of allergen-independent activation able to contribute to the perpetuation of allergy.

Blood basophil numbers in chronic ordinary urticaria and healthy controls: diurnal variation, influence of loratadine and prednisolone and relationship to disease activity.

The basopenia of chronic urticaria relates to histamine releasing autoantibodies in the serum of patients with autoimmune urticaria. This reduction in circulating basophils may be due to active recruitment into weals. If so, it might be expected that numbers in blood would be reduced when urticaria is active and increased after treatment. The primary aim of this study was to look at diurnal variation of basophil numbers in patients with chronic ordinary urticaria (not physical or vasculitic) in relation to disease activity and the effect of treatment with antihistamines and corticosteroids, and to compare the results with healthy controls. A secondary aim was to compare a standard manual counting method with automated basophil counts and to look at numbers of other circulating leucocytes that might be relevant to urticaria pathogenesis. Manual basophil counts using a toluidine blue stain and automated 5-part differentials (Coulter Gen. S) were performed at 4-hourly intervals from 08.00 to 20.00 in 10 healthy controls (six women, age 24 to 63 years) and seven chronic urticaria patients (five women, 24 to 50 years). All chronic urticaria patients had severe daily or almost daily urticaria. Only one of six chronic urticaria sera showed in vitro basophil histamine releasing activity. Counts were performed without treatment, after a week of taking loratadine 10 mg daily and after 3 days of adding prednisolone at 0.6 mg/kg/day (maximum 40 mg). Daily urticarial activity scores (UAS) were derived from weal numbers and itch, maximum 7. There was no significant overall diurnal variation of basophil numbers in healthy controls or chronic urticaria patients. Mean (SE) manually counted basophil were higher in healthy controls than chronic urticaria (43.4/ microL (2.1) vs. 4.4 (0.8), P < 0.001). Basophil counts were reduced in healthy controls on steroids (19.2 (1.9), P < 0.001) but increased in chronic urticaria (8.9 (1.9), P < 0.001). Loratadine did not influence them. UAS fell on treatment (3.3 (0.4) baseline, 1.4 (0.5) on loratadine and 0.5 (0.2) on prednisolone with loratadine, P < 0.001). There was a negative linear correlation between basophil numbers and UAS in untreated chronic urticaria patients (P = 0.001, Spearman rank correlation). Manual and automated basophil counts showed poor agreement. Lymphocyte numbers were lower in chronic urticaria than healthy controls. Neutrophils increased whereas lymphocytes and eosinophils decreased in all subjects on prednisolone. They were unaffected by loratadine. The results are consistent with the hypothesis that circulating basophils may be recruited from blood into urticarial weals during disease activity. Automated counts are not suitable for assessing basophil numbers in chronic urticaria. The relevance of reduced lymphocyte numbers in chronic urticaria needs to be explored.

Histamine-releasing reaction induced by the N-terminal domain of Vibrio vulnificus metalloprotease

A zinc metalloprotease secreted by Vibrio vulnificus, an opportunistic human pathogen causing septicemia and wound infection, stimulates exocytotic histamine release from rat mast cells. This protease consists of two functional domains: the N-terminal domain that catalyzes proteolytic reaction and the C-terminal domain that promotes the association with a protein substrate or cell membrane. Like the intact protease, the N-terminal domain alone also induced histamine release from rat peritoneal mast cells in a dose- and time-dependent manner. However, the reaction induced was apparently weak and went on more slowly. The nickel-substituted protease or its N-terminal domain, each of which has the reduced proteolytic activity due to decreased affinity to a substrate, showed much less histamine-releasing activity. When injected into the rat dorsal skin, the N-terminal domain also evoked enhancement of the hypodermic vascular permeability, while the activity was comparable to that of the protease. Taken together, the protease may stimulate histamine release through the action of the catalytic center of the N-terminal domain on the target substance(s) on the mast cell membrane. The C-terminal domain may support the in vitro action of the N-terminal domain by coordination of the association of the protease with the membrane, but it may not modulate the in vivo action.

Studies on the association between immunoglobulin E autoreactivity and immunoglobulin E-dependent histamine-releasing factors.

It has been reported that serum immunoglobulin E (IgE) from certain atopic patients can sensitize basophils to release histamine in response to IgE-dependent histamine-releasing factors (HRFs). It has also been shown that patients suffering from severe forms of atopy may contain IgE autoantibodies. It was investigated whether HRF-responsive sera contained IgE autoantibodies and if there was an association between IgE autoreactivity and IgE-dependent responsiveness to HRF. The presence of HRF-responsive IgE (IgE+) in serum of patients with respiratory atopy was determined by stimulating stripped human basophils sensitized by serum with peripheral blood mononuclear cell (PBMC)-derived HRF, and measuring the release of histamine. In parallel, these sera were screened for the presence of IgE autoantibodies to nitrocellulose-blotted human cellular extracts. The capacity of IgE autoantigen-containing preparations to induce histamine release was tested in the stripped basophil assay. Eleven out of 52 sera contained IgE autoantibodies to blotted cellular extracts of human PBMCs or of the human epithelial cell line A431. No significant association was found between IgE autoreactivity and IgE-dependent responsiveness to HRF: 7/26 IgE+ sera contained IgE to human cellular extracts, and 4/26 of the sera without IgE+ did also. IgE autoantigen-containing extracts did not induce histamine release of appropriately sensitized basophils. By size-exclusion chromatography it was shown that a 32 000 MW autoantigen eluted in the >55 000 MW fraction, which indicates that this protein forms polymers or complexes with other macromolecules. This might explain the discrepancy between binding and histamine-releasing activity. A 20 000 MW IgE-defined autoantigen cross-reacted with a shrimp allergen. Our results indicate that IgE-reactivity to immunoblotted human protein and IgE-dependent HRF activity are distinct entities that may co-occur in atopic patients.

Classification of anti-FcepsilonRI and anti-IgE autoantibodies in chronic idiopathic urticaria and correlation with disease severity.

Circulating autoantibodies against FcepsilonRI, IgE, or both occur in approximately one third of patients with chronic idiopathic urticaria (CIU), but not all autoantibodies initiate histamine release. We sought to classify patients with CIU into subsets on the basis of serum bioactivity and immunoreactivity and to examine the relationship between newly defined subtype and disease severity. Sera from patients with CIU (n = 78), dermog-raphism (n = 15), and cholinergic urticaria (n = 10) and sera from healthy subjects (n = 39) were analyzed by means of Western blot analysis for anti-FcepsilonRI autoantibodies and for histamine release from basophils and dermal mast cells. In vivo reactivity of autologous serum was tested by means of intradermal injection, and CIU severity was determined on the basis of clinical interview. We classified sera from patients with CIU into 5 subsets: immunoreactive histamine-releasing anti-FcepsilonRI autoantibodies (n = 20 [26%]); immunoreactive anti-FcepsilonRI autoantibodies without histamine-releasing activity (n = 12 [15%]); anti-IgE-like autoantibodies (n = 7 [9%]); serum containing a mast cell-specific histamine-releasing factor (n = 7 [9%]); and sera with no identifiable factor (n = 32 [41%]). Patients with serum histamine-releasing activity had more severe urticaria than patients without such activity. Positive skin test responses to autologous sera were associated with histamine-releasing anti-FcepsilonRI autoantibodies but not with non-histamine-releasing anti-FcepsilonRI autoantibodies. Neither healthy subjects nor patients with dermographism or cholinergic urticaria had his-tamine-releasing anti-FcepsilonRI autoantibodies. These data support the specificity of functional anti-FcepsilonRI autoantibodies to CIU. The identification of distinctive subsets of patients suggests that other pathogenic mechanisms occur in CIU in addition to direct ligation of FcepsilonRI by autoantibodies causing dermal mast cell degranulation. Elucidating these mechanisms might lead to new treatments for CIU.

High-level expression of recombinant house dust mite allergen Der p 1 in Pichia pastoris.

The major house dust mite Der p 1 allergen is associated with allergic disease. Heterologous over-expression of biologically active Der p 1 was previously attempted but with limited success. The aim of this study was to establish an efficient system for the production of recombinant Der p 1. The proform of Der p 1 was expressed in Pichia pastoris as a fusion with the alpha mating factor signal sequence. The recombinant product was purified from culture medium and compared to Der p 1 isolated from mite culture, in terms of enzymatic activity as well as IgE binding capacity. ProDer p 1 was efficiently secreted into culture medium as a hyperglycosylated protein of 40-60 kDa. Postpurification dialysis in acidic buffer was required for the autocatalytic processing of Der p 1. During this treatment, the prosequence was efficiently removed to give highly glycosylated recombinant mature Der p 1. Competition ELISA experiments as well as cysteine proteinase activity assays indicated that recombinant processed Der p 1 was similar to natural Der p 1 isolated from mite cultures in terms of IgE binding and enzymatic activities. However, the histamine releasing activity of recombinant Der p 1 was slightly weaker than that of natural Der p 1. This efficient system for recombinant Der p 1 expression leads the way for the design of new diagnostics for house dust mite allergy, epitope mapping, allergen engineering, structural and immunological studies and new immunotherapeutic treatments.

Management of urticaria

Urticaria is predominantly due to release of mast cell mediators, mainly histamine. Chronic idiopathic urticaria, in which disease activity continues on most days for more than 6 weeks and there is no evidence of a physical urticaria or urticarial vasculitis, is common. An external cause is rarely identified even if a thorough history and appropriate investigation based on this are undertaken. However, approximately 50% of patients with chronic idiopathic urticaria (CIU) have a serum histamine releasing factor present. In one-third of patients with CIU this is an IgG autoantibody directed against the high affinity IgE receptor (FceRI) or less frequently against IgE. Patients with autoinimune urticaria, are clinically more severe. Urticaria can affect the quality of life markedly, being comparable to that of patients awaiting triple coronary by-pass surgery. The rational management of urticaria takes account of likely causes and mediators involved. However explanation and attention to general measures such as minimizing stress, overheating and alcohol are important. Aspirin, nonsteroidal anti-inflammatory drugs and opiates should be avoided if possible. Exclusion diets such as of food colourings and additives may be of some value to a limited number of patients. Second- and third-generation low sedation H1 antagonists are the treatment of choice and improve many patients. Addition of an H2 antagonist or a mast cell stabilizing drug may provide additional benefit for a few. There are reports that leukotriene receptor antagonists improve some patients. Oral steroids are reserved if possible for severe exacerbation of chronic urticaria, and disabling pressure urticaria. Third-line therapies involving immunosuppressive agents are only appropriate for patients with chronic urticaria refractory to other measures, and usually autoimmune. The encouraging results using short courses of oral cyclosporin, high dose intravenous immunoglobulin and plasmapheresis need to be confirmed in placebo-controlled trials, in patients with or without demonstrable serum histamine releasing activity.

Cytokines regulate development of human mast cells from hematopoietic progenitors.

Combination of stem cell factor (SCF) and interleukin-6 (IL-6) significantly promoted proliferation of human mast cells from cord blood CD34+ cells. Most of the cells, cultured in the presence of SCF and IL-6 for 10 weeks, expressed c-kit and contained a significant amount of histamine and tryptase and a low amount of chymase. Both tryptase-positive chymase-negative mast cells (MC(T)) and tryptase-positive chymase-positive mast cells (MC(TC)) were found in the same colony derived from a single cord blood CD34+ cell, suggesting that MC(T) and MC(TC) develop from common precursor cells. Single-cell culture of CD34+ cells revealed that committed mast cell progenitors are included in CD34+CD38+HLA-DR- cells. IL-4 significantly enhanced high-affinity immunoglobulin E (IgE) receptor (FcepsilonRI) alpha-chain messenger RNA expression and induced FcepsilonRI on SCF-dependent cord blood-derived human mast cells, resulting in high histamine-releasing activity upon cross-linking of FcepsilonRI. Another factor that up-regulated FcepsilonRI was IgE, and a combination of IL-4 and IgE markedly augmented FcepsilonRI expression on the mast cells. IL-4 and IgE may enhance FcepsilonRI expression by distinct mechanisms; IL-4 promotes FcepsilonRI alpha-chain gene transcription and thus increases alpha-chain protein synthesis in the cells, whereas the binding of IgE may anchor the FcepsilonRI on the cell surface, resulting in suppression of internalization of FcepsilonRI. Mast cells are progeny of hematopoietic stem cells. Recent discovery of a xenotransplantation model revealed that human hematopoietic stem cells can proliferate and differentiate into mature mast cells in the mouse skin 3 months after transplantation of human cord blood CD34+ cells, suggesting that this model may pave the way to clarification of the functions of human mast cells in vivo.

Further studies on the structural requirements for mast cell degranulating (MCD) peptide-mediated histamine release

Mast cell degranulating (MCD) peptide was modified in its two disulfide bridges and in the two arginine residues in order to measure the ability of these analogs to induce histamine release from mast cells in vitro. Analogs prepared were [Ala 3,15]MCD, [Ala 5,19]MCD, [Orn 16]MCD, and [Orn 7,16]MCD. Their histamine-releasing activity was determined spectrofluorometrically with peritoneal mast cells. The monocyclic analogs in which the cysteine residues were replaced pairwise with alanine residues showed three-to ten-fold diminished histamine-releasing activity respectively, compared with the parent MCD peptide. Substantial increases in activity were observed where arginine residues were replaced by ornithines. The ornithine-mono substituted analog showed an almost six-fold increase and the ornithine-doubly substituted analog three-fold increase in histamine-releasing activity compared with the parent MCD peptide. The structural changes associated with these activities were followed by circular dichroism (CD) spectroscopy. Changes in the shape and ellipticity of the CD spectra reflected a role for the disulfide bonds and the two arginine residues in the overall conformation and biological activity of the molecule.

Mast cell degranulating peptide binds to RBL-2H3 mast cell receptors and inhibits IgE binding

Fluorescent and biotinylated analogs of mast cell degranulating (MCD) peptide were synthesized and the labels fluoresceinisothiocyanate and N-hydroxysuccinimidobiotin were conjugated at position 1 in the MCD peptide sequence. The analogs with these moieties retained histamine-releasing activity as high as that of the parent MCD peptide in rat peritoneal mast cell assays. These labeled analogs were used in rat basophilic leukemia cells (RBL-2H3) to demonstrate by confocal microscopy and flow cytometry the specific binding of MCD peptide to mast cell receptors. Consequently MCD peptide was found to compete with and inhibit the binding of fluorescent IgE on RBL cells as monitored by flow cytometry. Thus MCD peptide may prove to be useful in the study of IgE receptor-bearing cells.

Chronic urticaria: novel clinical and serological aspects.

Recently, distinct studies have shown that: (a) chronic idiopathic urticaria (CIU) is autoimmune in 30-50% of cases; (b) in patients with CIU the autologous serum skin test is inhibited by heparin; and (c) basophil histamine release induced in vitro by CIU sera maybe complement-dependent. To carry out a comprehensive clinical and serological study on CIU based upon these observations. Three hundred and six adults with CIU underwent intradermal (ID) test with autologous serum; 57 of them with autologous heparinized plasma as well. Sera from 121 patients (plasmas from 17) were employed to induce in vitro histamine release from basophils of normal donors. The effects of heating (56 degrees C, 60 min), filtration through membrane, and preincubation with heparin were evaluated as well. Autologous serum and plasma induced a weal and flare reaction in 205 out of 306 (205/306; 67%) and in 8/57 (14%) patients, respectively. Positive plasma skin tests were observed only in patients showing strongly positive serum skin tests. Plasma did not elicit any skin reaction in 3/3 patients with dermatographism who showed a positive intradermal test with saline. Sera from 20/121 (16.5%) patients induced significant histamine release from basophils of normal donors. 19/20 sera were from patients with a positive intradermal test; thus, basophil histamine release assay was positive in 19/87 (21.8%) patients with a positive serum skin test. Heating at 56 degrees C x 1 h markedly reduced the histamine-releasing activity of both serum and plasma from in vitro reactors. Ultrafiltered fractions > 100 kDa of both sera tested retained the histamine-releasing activity, whereas fractions < 100 kDa were not able to induce any histamine release. Heparin dose-dependently inhibited histamine release induced by sera and plasma, and by basophil agonists such as anti-IgE, formyl-methionyl-leucyl-phenilalanine, and interleukin (IL)-3. 67% of our patients with CIU showed a positive autologous serum skin test. Sera from about 20% of those positive on autologous serum skin test induced histamine release from normal basophils in vitro probably as a consequence of the presence of functional autoantibodies. The marked difference between in vivo and in vitro findings could reflect the existence of a mast cell-specific histamine-releasing factor which does not release histamine from basophils of healthy blood donors. However, it might be also the result of in vivo priming of patients' cutaneous mast cells or of heterogeneity of basophil donors. At least in some cases complement seems essential for histamine-releasing activity of serum from patients with CIU. Heparin inhibits histamine release from both basophils (in vitro) and mast cells (in vivo), probably acting directly at a cellular level.

Leukotriene receptor antagonists in chronic urticaria.

. SOME patients with chronic idiopathic urticaria (CIU) do not respond to antihistamines and require long-term steroid administration or even cyclosporin to control their disease. Recently, several studies reported good results with antileukotriene drugs in CIU (1–4); however, a prospective investigation aiming to detect which patients are more likely to benefit from antileukotriene treatment has not been carried out yet. Twelve consecutive patients with unremitting, steroid-dependent urticaria were studied. Clinical features and results of the main laboratory investigations are shown in Table 1. Montelukast 10 mg once a day (n=5) or zafirlukast 20 mg twice a day (n=7) was started. The usual therapies were not changed at the start of the scheduled 3-week treatment period, but patients were left free to reduce or withdraw antihistamine treatment in the case of a remission of the disease. Patients recorded both the severity of urticaria and the drugs used, and were followed up weekly. The primary end point of the study was an adequate control of the disease, defined as absence or marked reduction of pruritus and urticaria without the need for systemic steroid therapy. One patient was excluded because of intolerance of both montelukast and zafirlukast (severe headache). Six of the remaining 11 patients (55%) responded to zafirlukast (n=3) or montelukast (n=3) (Table 1). In these subjects, leukotriene receptor antagonists (LRA) induced a nearly total remission of the disease: four could easily control their urticaria with cetirizine 10 mg every other day, and two could discontinue antihistamine treatment altogether. The effect of LRA was already appreciable after some days of treatment. Responders have now been followed up for 6–24 months; they are still taking LRA and are all still in remission. No clinical feature or specific ‘‘marker’’ (sex, age, duration of the disease, NSAID intolerance, autologous serum skin test [ASST] result, thyroid autoimmunity, or histamine-releasing activity of serum) was associated with the response to LRA (Table 1). Leukotrienes might well play a central pathogenic role in some patients with CIU. Their injection into human skin produces a wheal and flare reaction (5), and, on a molar basis, they are 100 times more potent than histamine; moreover, serum from some patients with CIU, particularly those positive on ASST, induces both histamine release and de novo production of sulfidoleukotrienes in human basophils (6). Interestingly, in one study (3), one nonresponder to LRA showed a complete response to the lipoxygenase inhibitor, zileuton, suggesting a possible pathogenic role for LTB4, a substance that enhances cutaneous microvascular permeability by a not yet identified neutrophil-derived mediator (7). Because zileuton is not available in Italy, we could not confirm these observations. Good result in 50% of cases of therapy resistance.

Serum histamine-releasing activity in a patient with idiopathic pulmonary haemosiderosis

idiopathic pulmonary haemosiderosis (IPH) is a rare disorder characterized by intermittent, diffuse alveolar bleeding. The pathogenesis of the diseases is unclear, although an association with milk or gluten hypersensitivity has been described, and an immune-mediated damage of alveolar capillaries has been suggested. A previous report showed the release of histamine after cow's milk intake in a newborn with cow's milk intolerance and IPH. here, we report the detection of serum histamine-releasing activity (HRA) in a 30-year-old woman with IPH. The serum taken during an active phase of the disease induced histamine release from basophils of two normal donors; conversely, when the patient was receiving prednisone and azathioprine, and the disease was in remission, the serum HRA was reduced. Serum fractions with a MW lower than 100 kDa displayed an enhanced HRA; in contrast, serum fractions with MW above 100 kDa were not able to induce histamine release, suggesting that the activity was due to a cytokine and not to an immunoglobulin. the detection of serum HRA provides further evidence that the immune system is activated in the course of IPH and supports an immunologic basis for the alveolar capillary damage, which is responsible for alveolar bleeding.