Neutrophils from allergic asthmatic patients produce and release metalloproteinase-9 upon direct exposure to allergens.

BACKGROUND: Asthma is characterized by airway inflammation and remodelling in which matrix metalloproteinases (MMPs) play an important role. MMP-9 is the major MMP found in the bronchoalveolar lavage fluids and bronchial biopsies from patients with allergic asthma after allergen challenge, where it correlates with the count of neutrophils and macrophages. However, the cellular sources of MMP-9 in this inflammatory condition have not yet been clearly identified. This work was undertaken to analyse whether neutrophils may be a source of MMP-9 in the allergic asthma condition upon allergen challenge. METHODS: Neutrophils from allergic asthmatic patients were in vitro stimulated, and the levels of MMP-9 release were measured in the cell culture supernatants using enzyme-linked immunosorbent assay (ELISA) and zymography. RESULTS: We show that MMP-9 is released by neutrophils, but not by eosinophils from allergic asthmatic patients in response to allergens to which the patients were sensitized. Neutrophils also released MMP-9 in response to anti-IgE Abs, and agonist Abs against FcεRI, FcεRII/CD23 and galectin-3. Inhibitors of transcription and translation, actinomycin D and cycloheximide, partially cancelled this process, suggesting that MMP-9 is also de novo synthesized in response to stimuli. We also show evidence that the MAPKs, p38 and extracellular signal-regulated kinase, as well as the transcription factor NF-κB, are involved, as specific chemical inhibitors of these cell-signalling pathways abolished the anti-IgE/allergen-dependent MMP-9 release. CONCLUSIONS: These data demonstrate that the exposure of neutrophils to allergens leads to generation of MMP-9, which may then lead to remodelling in asthma.

Neutrophil defensins: their possible role in allergic asthma.

BACKGROUND: Neutrophil defensins, originally identified as broad-spectrum antimicrobial peptides, have been implicated in the regulation of inflammatory and immunological processes. OBJECTIVES: To investigate whether the in vitro challenge of neutrophils from patients with bronchial asthma with allergens stimulated the release of alpha-defensins and whether levels released were dependent on lung infections. METHOD: The neutrophils were cultivated with different agonists and the concentration of alpha-defensin in cell-free supernatant was measured with enzyme-linked immunosorbent assay (ELISA). RESULTS: Neutrophils from allergic patients released alpha-defensins via an allergen-dependent mechanism. Our results indicate that the in vitro activation of neutrophils is highly allergen-specific. In this context, allergens other than those which produced clinical symptoms did not elicit alpha-defensin release, and allergens had no effect on neutrophils from healthy donors. However, neutrophils from both allergic patients and healthy controls were able to release alpha-defensins upon treatment with PMA. In the allergen-stimulated neutrophils, cells from asthmatic patients stimulated with a sensitizing allergen showed a significantly higher production of alpha-defensin under respiratory tract infection than cells from the same patients without such an infection. CONCLUSION: Neutrophils from allergic patients release alpha-defensins via an allergen-dependent mechanism.

Enhanced diagnosis of pollen allergy using specific immunoglobulin E determination to detect major allergens and panallergens.

BACKGROUND: Pollen is one of the main causes of allergic sensitization. It is not easy to make an etiological diagnosis of pollen-allergic patients because of the wide variety of sensitizing pollens, association with food allergy, and increasing incidence of polysensitization, which may result from the presence of allergens that are common to different species, as is the case of panallergens. OBJECTIVE: To compare the results of skin prick tests (SPT) using whole pollen extract with specific immunoglobulin (Ig) E determination for several allergens (purified panallergens included) in the diagnosis of polysensitized pollen-allergic patients. METHODS: The study sample comprised 179 pollen-sensitized patients who underwent SPT with pollen extract and allergen-specific IgE determination against different allergens. RESULTS: The level of concordance between the traditional diagnostic test (SPT) and IgE determination was low, especially in patients sensitized to the panallergens profilin and polcalcin. In the case of SPT, the results demonstrated that patients who are sensitized to either of these panallergens present a significantly higher number of positive results than patients who are not. However, IgE determination revealed that while patients sensitized to polcalcins are sensitized to allergens from a higher number of pollens than the rest of the sample, this is not the case in patients sensitized to profilins. On the other hand, sensitization to profilin or lipid transfer proteins was clearly associated with food allergy. CONCLUSIONS: Sensitization to panallergens could be a confounding factor in the diagnosis of polysensitized pollen-allergic patients as well as a marker for food allergy. However, more studies are required to further investigate the role of these molecules.

Enhancement of Antigen-specific functional responses by neutrophils from allergic patients.

It has been demonstrated that neutrophils from healthy donors or from patients with inflammatory disorders can bind immunoglobulin (Ig) E proteins through binding to Mac-2/epsilon bp. Functional responses to allergens were assessed by measuring the respiratory burst and intracellular Ca2+ levels, and binding of allergens to neutrophils was assessed by flow cytometry analysis and fluorescence microscopy. In this article, we demonstrate that neutrophils sensitized to specific allergens (from allergic patients), but not from healthy donors, are sensitive to allergens of the same type as those that produce clinical allergic symptoms. The activation of neutrophils was analyzed by the induction of a respiratory burst that was detected with luminol-dependent chemiluminescence. Intracellular Ca2+ levels increased parallel to those of the inducing allergens. In addition, the specific binding of allergens on the cell surface was revealed by flow cytometry and allergen-FITC-labeled staining analyses. The present data suggest a restricted recognition of allergen by sensitive neutrophils, probably associated with the specific binding of the allergen to its corresponding IgE molecule, which is bound to the Mac-2/epsilon bp structure. These findings demonstrate a functional role of allergen-associated neutrophils during the allergic state.

Neutrophils and asthma.

Although eosinophilic airway inflammation is recognized as an important feature of some patients with chronic, stable asthma, evidence supports an important role for neutrophils in asthma. Neutrophils are the first cells recruited to the site of the allergic reaction. Their presence may influence clinical presentation and has been linked to the development of severe chronic asthma and sudden severe attacks. Neutrophils are eliminated by apoptosis during the resolution of the allergic response.

An antibody-based ELISA for quantification of Ani s 1, a major allergen from Anisakis simplex.

Anisakis simplex is a nematode parasite that can infect humans who have eaten raw or undercooked seafood. Larvae invading the gastrointestinal mucosa excrete/secrete proteins that are implicated in the pathogenesis of anisakiasis and can induce IgE-mediated symptoms. Since Ani s 1 is a potent secreted allergen with important clinical relevance, its measurement could assess the quality of allergenic products used in diagnosis/immunotherapy of Anisakis allergy and track the presence of A. simplex parasites in fish foodstuffs. An antibody-based ELISA for quantification of Ani s 1 has been developed based on monoclonal antibody 4F2 as capture antibody and biotin-labelled polyclonal antibodies against Ani s 1 as detection reagent. The dose-response standard curves, obtained with natural and recombinant antigens, ranged from 4 to 2000 ng/ml and were identical and parallel to that of the A. simplex extract. The linear portion of the dose-response curve with nAni s 1 was between 15 and 250 ng/ml with inter-assay and intra-assays coefficients of variation less than 20% and 10%, respectively. The assay was specific since there was no cross-reaction with other extracts (except Ascaris extracts) and was highly sensitive (detection limit of 1.8 ng/ml), being able to detect Ani s 1 in fish extracts from codfish and monkfish.

Protection from nicotinamide inhibition of interleukin-1 beta-induced RIN cell nitric oxide formation is associated with induction of MnSOD enzyme activity.

We have studied the long-term effects of nicotinamide (NIC) on the synthesis of NO by insulin producing cells. NIC delays the formation of nitrite by interleukin (IL)-1 beta-(IL-1, 25 U/ml)-stimulated RINm5F cells, and previous exposure of cells to IL-1 for 15 h prevents this effect. The delay is associated with a lack of cytokine-induced inducible nitric oxide synthase (iNOS) enzyme activity in cell extracts. NIC (20 mM) inhibits NO synthase (NOS) activity in extracts from cells incubated with IL-1 for 6 h and 24 h, and oxyhemoglobin counteracts this inhibition. Hence, NIC could scavenge O2- and allow NO to inhibit the enzyme. The NO donor SIN-1 inhibits in a concentration-dependent manner iNOS activity, and the effect is potentiated by NIC. In intact cells, protection from NIC is associated with IL-1-induced expression of MnSOD activity, and reversible blockade of iNOS expression with pyrrolidine dithiocarbamate counteracts the NIC effect. We conclude that O2- plays a role in preventing NO inhibition of iNOS. The loss of this action coincides with the induction of MnSOD enzyme activity. In addition, the stimulation by NIC of IL-1-induced nitrite production in pyrrolidine dithiocarbamate-treated cells is a novel action that should be considered when the drug is proposed as potential agent for the prevention of insulin-dependent diabetes mellitus.

Plasma kallikrein amidolytic activity in patients with urticaria.

We have studied the plasma kallikrein amidolytic activity in healthy control subjects (inactive), patients with chronic urticaria (active) and patients with acute urticaria (active) from their admission to the emergency room (active) to the time after which their clinical symptomatology had disappeared (inactive). We found statistically significant differences (p < 0.01) in the active groups of urticaria patients. This leads us to believe that kallikrein participates in the development of symptomatology in these patients.

A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay to quantify Parietaria judaica major allergens, Par j 1 and Par j 2.

BACKGROUND: Parietaria pollen is one of the most important causes of pollinosis in Mediterranean countries. Parietaria judaica pollen extract presents two major allergens, Par j 1 and Par j 2, that belong to the lipid transfer protein family. OBJECTIVE: To develop an ELISA for quantification of both major allergens of P. judaica pollen extracts, and to assert correlation of these allergens content with the allergenic activity of extracts. METHODS: Natural Par j 1-Par j 2 allergens were purified by gel filtration, ion exchange, and affinity chromatography and identified by mass spectrometry. Rabbit antisera were obtained using this protein preparation as antigen and used for immunoaffinity purification of nPar j 1-Par j 2. BALB/c mice were immunized with the immunopurified nPar j 1-Par j 2 and after fusion and screening by direct ELISA, 5D4 monoclonal antibody was selected as capture antibody to develop a quantitative two-site ELISA. Bound proteins were detected by a biotinylated Par j 1-Par j 2-specific polyclonal antibody. RESULTS: The optimized ELISA was developed from 25 to 8000 pg/mL of purified Par j 1-Par j 2, and a linear portion of 200-1000 pg/mL. The intraassay and interassay coefficients of variation were lower than 7% and 14% respectively. The assay was very sensitive and specific as it had a detection limit of 25 pg/mL and did not detect reactivity with the same family plants, as Urtica. Par j 1-Par j 2 allergens content was measured in 14 P. judaica and two P. officinalis pollen extracts showing a significant correlation with their allergenic activity measured by enzyme allergosorbent test inhibition. CONCLUSIONS: The results proved the usefulness of the two-sandwich ELISA for the standardization of Parietaria pollen extracts intended for clinical use, because of its good correlation with allergenic potency.

Development of a sandwich-type ELISA for measuring Pla a 1, the major allergen of Platanus acerifolia pollen.

BACKGROUND: Platanus acerifolia is an important cause of pollinosis in Western European cities. Pla a 1, a nonglycosylated 18-kDa protein with a prevalence of 80%, is a major allergen in P. acerifolia pollen extracts. Our aim was to develop a Pla a 1-specific ELISA to quantify this protein in allergenic extracts and preparations for clinical use. METHODS: Pla a 1 was purified by cation exchange at pH 7.0, gel filtration, and anion exchange chromatography at pH 10.0. Monoclonal (mAb) and polyclonal antibodies were obtained by immunizing mice and rabbits with nPla a 1. One (5C1) of the 13 mAb obtained was used as capture antibody at 5 mug/ml and biotin-labeled specific polyclonal antiserum at 0.63 microg/ml served for detection. RESULTS: The prevalence of Pla a 1-specific IgE to purified Pla a 1 among 47 P. acerifolia-allergic patients was 79%. The Pla a 1-ELISA developed has a linear range of 3-25 ng/ml, high sensitivity with a detection limit of 0.5 ng/ml and is highly specific as none of the 24 pollen, mite, mold, and plant food extracts tested gave positive results. The assay could quantify Pla a 1-like proteins in other planetree pollen extracts. A good correlation was obtained between Pla a 1 content of 11 P. acerifolia pollen extracts (average content 0.69% of the total protein) and their IgE-binding activity. CONCLUSIONS: The described two-site sandwich ELISA to measure Pla a 1 is useful for standardization of planetree pollen extracts intended for clinical use.

L-selectin expression on neutrophils from allergic patients.

BACKGROUND: L-selectin (CD62L) is an adhesion molecule involved in leucocyte attachment to endothelium at sites of inflammation, and it has been demonstrated that L-selectin is rapidly shed after neutrophil activation. Recently, it has been reported that there is increasing evidence of neutrophil participation in asthma and the allergic process. OBJECTIVE: The present study was designed to determine whether an IgE-dependent mechanism can modulate L-selectin expression on the surface of neutrophils. Moreover, we analyse the potential implication of intracellular signal-transduction pathways and whether specific immunotherapy (IT), glucocorticoids and antihistamines might regulate this process. METHODS: Peripheral blood neutrophils from three groups of donors (asthmatic group without IT treatment, IT-treated asthmatic group and healthy group) were used. Cells were challenged in vitro with the specific allergen that produced clinical symptoms in asthmatic patients and also with the allergen to which the patients were not sensitive. Neutrophils from healthy donors were also challenged with allergens. Expression of CD62L on the neutrophil surface was analysed by flow cytometry, and soluble CD62L (sCD62L) in culture supernatant by ELISA. In an attempt to discover which IgE receptor is involved, we also challenged the neutrophils with monoclonal antibody to FcepsilonRI, FcepsilonRII (CD23) and galectin-3 receptors. RESULTS: When neutrophils from allergic patients were challenged with specific allergens that produce clinical allergy symptoms, L-selectin was down-regulated from the surface of those cells, accompanied by a concomitant up-regulation of soluble L-selectin in the supernatant. The challenge with antibodies against FCepsilonRI, FCepsilonRII (CD23) and galectin-3, induces down-modulation of L-selectin on the surface of the neutrophils in all three cases. Calphostin C, wortmannin and manoalide attenuated CD62L down-regulation, suggesting the potential implication of protein kinase C, phosphatidylinositol 3-kinase and phospholipase A(2) in the process. IT and glucocorticoids modulated allergen-dependent CD62L down-regulation, whereas antihistamines (terfenadine, loratadine and cetirizine) or nedocromil sodium did not affect the shedding of L-selectin. CONCLUSIONS: We present evidence that the neutrophil surface expression of CD62L can be modulated by an allergen-dependent mechanism. The modulation of CD62L expression can be induced through the three receptors of IgE. This process can be affected by IT.

Peripheral blood T lymphocytes in seasonal bronchial asthma.

Variations in T lymphocytes in asthmatic patients are related to disease severity. However, the effects of natural exposure to pollens on peripheral blood T lymphocytes have not been clarified. In this paper, the effects on peripheral blood CD4 and CD8 lymphocytes from pollen-sensitive subjects and from nonatopic donors were studied during and outside the pollen season. In patients who suffer from seasonal asthma, we found an increase in the CD4/CD8 bright ratio and a decrease in the mean number of CD4 receptors per cell during the pollen season. No variation was observed in healthy subjects. These results suggest that CD4 lymphocytes may be causally linked to the pathogenesis of seasonal bronchial asthma.

Plasma kallikrein amidolytic activity in bronchial asthma.

We have investigated plasma kallikrein amidolytic activity in the following groups of patients: 1. Normal control group of blood donors. 2. Extrinsic pollen-activated bronchial asthma patients, during periods of symptomatology and at a later time after the spring. 3. Subjects with atopic bronchial asthma in acute phase when admitted to our hospital's emergency room and later when clinically recovered. 4. Subjects with extrinsic bronchial asthma, sensitive to Dermatophagoides pteronyssinus and Dermatophagoides farinae with FEV1 < 80%. 5. Subjects with extrinsic bronchial asthma, sensitive to Dermatophagoides pteronissinus and Dermatophagoides farinae in a state of clinical rest. After 9 minutes of activation, the following results were found, with a significance of p < 0.01: There are significant differences between the normal group and those that we consider the active groups, asthma FEV1 < 80%, pollen-sensitive asthma in springtime and acute asthma. No significant differences exist between the normal group and inactive groups, inactive asthma, pollen-sensitive asthma out of springtime and acute asthma inactive. Significant differences exist in active groups (acute asthma and pollen-sensitive asthma in springtime) when they become inactive (acute asthma inactive and pollen-sensitive asthma out of springtime). The active groups have a higher plasma kallikrein amidolytic activity than both the inactive and control groups.

The study cellular subpopulations in peripheral blood from a normal reference group population (blood donors).

The spectacular development of monoclonal antibodies against cellular antigens an technology such as flow cytometry allow the investigation of cellular subpopulations that until now have been unknown. At the same time, the functional study of these subpopulations becomes of maximal interest, as this information could have future applications for pathological processes. Due ti this basic need for information, we have studied diverse lymphocytic subpopulations in a normal population that serves as a reference group, using the following antigens: CD3, CD4, CD8, CD20, CD45RA, CD25, LAM1, CD29, CD11b and CD23. Some of these subpopulations had not been previously studied in a normal reference group.

IgE-dependent release of myeloperoxidase by neutrophils from allergic patients.

BACKGROUND: The three forms of IgE receptor: the heterotrimeric high-affinity receptor for IgE (FcepsilonRI), the low-affinity receptor for IgE (FcepsilonRII/CD23) and the Mac-2/IgE-binding protein (epsilonBP), have previously been found on human neutrophils. We have previously shown that specific allergens are able to activate functional responses by neutrophils from allergic patients sensitized to those allergens. Neutrophils are present in the sites of allergic inflammation. The primary (azurophilic) granules of neutrophils contain a variety of enzymes that might potentiate inflammation, such as myeloperoxidase (MPO). It is not known whether specific allergens are able to elicit MPO release by neutrophils from allergic patients. METHODS: Neutrophils were challenged in vitro with the specific allergen that produced clinical symptoms in asthmatic patients. Also, the cells were challenged with allergens that the patients were not sensitive to. Neutrophils from normal subjects were also challenged with allergens. RESULTS: The in vitro challenge of neutrophils with allergens that the patients were sensitive to elicited a release of MPO by these cells. The in vitro activation of neutrophils was highly allergen-specific, in such a way that allergens other than those accounting for clinical symptoms did not evoke MPO release, and allergens were ineffective on neutrophils from healthy donors. CONCLUSION: An IgE-dependent mechanism might promote MPO release by neutrophils at allergic sites.

Production of soluble CD23 from peripheral blood lymphocytes of asthmatic patients.

The low-affinity receptor IgE (Fc epsilon RII) as shown to be identical to CD23 several years ago. The presence of the CD23 was demonstrated on a variety of human cells, such as T cells, monocytes, eosinophils, platelets, lymphocytes B, etc., and it can be cleaved in soluble fragments. We studied the in vitro production of soluble CD23 (sCD23) in PBMC of patients with bronchial asthma and in healthy donor cells. If we stimulate production with a polyclonal activator [Phytohemagglutinin (PHA) at a concentration of 10 micrograms/ml], it can be seen that the greatest amounts of sCD23 are produced on the fourth day and that production is greater in asthmatic patients than in control group (p < 0.01). When stimulated additionally by an antigen, the production of sCD23: Is greater when stimulated with PHA. Is released in quantities greater than in healthy group cells, when the PBMC are stimulated with the antigen to which the patients are sensitive (olive pollen). The quantity released depends on the concentration of the antigen added to the culture. Is not released in greater quantities by healthy groups cells in the presence of the antigen than in healthy group cells without the antigen. Is not released in greater quantities by cells of asthmatics in the presence of an antigen to which they are not sensitive (D. pteronyssinus). This leads us to conclude that the release of sCD23 in these patients could play a role in the physiopathology of extrinsic bronchial asthma.

Relationship of blood EG2+ eosinophils in patients with bronchial asthma.

The presence of eosinophils in peripheral blood has been previously associated with different degrees of activity in the evolution of disease in asthmatics. The eosinophil cationic protein is a mediator released by the eosinophils when they are activated due to various stimuli. The monoclonal antibody EG2 binds to human ECP, the epitopes which EG2 recognizes is only present in the secreted form of ECP, hence it only stains eosinophils which are undergoing activation and/or secretion. Flow cytometry was used to measure the level of eosinophils stained by the monoclonal antibody EG2 (eosinophil EG2+) in the peripheral blood of subjects with unstable extrinsic bronchial asthma, stable extrinsic bronchial asthma and healthy control group. A statistically significant higher level of eosinophil EG2+ was found in the group with unstable asthma than in either the group with stable asthma or the group of healthy subjects (p < 0.0002). No difference was found between the patients with stable asthma and the control group. The level of eosinophil EG2+ in peripheral blood, measured by flow cytometry, could be used as an indicator of inflammatory activity in patients suffering from bronchial asthma.

Variations of the cellular antigens CD4 and CD8 in pollinic patients during the spring.

In order to study the possible variations of CD4 and CD8 antigens in pollinic patients, we have studied 25 individuals (11 rhinoconjunctivitis and 14 rhinoconjunctivitis-asthma) before (T0), in the middle (T1) and after the spring (T2). The number of receptors per cell of CD4 start from values in T0, decrease in T1 and increase at the end of the spring (p < 0.00001), which could represent a mechanism to limit the clonal response to an antigen. An increase in the CD4/CD8 bright ratio could be indicate a higher helper mechanism in T1 with respect to T0 and T2 (p < 0.01). The opposite meaning is to given to the increase of CD8bright receptors in the asthmatic patients, and not only in those who suffer only from a rhinopathy (the only difference between the two groups of patients) during T2 over the T1 (p < 0.00001). The greater number of lymphocytes CD8 dim + during T1 with respect to T2 (p < 0.009) and the increase in the number of receptors of such cells during T1 with respect to T2 (p < 0.00001) suggests a possible intervention of these cells in the regulation of the response of the B and T lymphocytes. Of the two soluble factors CD4 (sCD4) and CD8 (sCD8), only the sCD4 increases during T1 (p < 0.0001) in an inverse manner as occurs with CD4 cell receptors, while the sCD8 remains unchanged during the three periods.(ABSTRACT TRUNCATED AT 250 WORDS)