Synergic production of neutrophil chemotactic activity by colonic epithelial cells and eosinophils

The presence of eosinophils in the lumen and mucosa of the intestine is characteristic of both ulcerative colitis (UC) and Crohn's disease (CD). There is evidence of eosinophil activation in the intestine during acute inflammatory episodes of these diseases; these episodes are also characterized by an influx of neutrophils, which have the potential to cause extensive tissue damage. We undertook a study to determine whether eosinophils in contact with colonic epithelial cells produce factors that may attract neutrophils in response to immunological stimulation. Neutrophil chemotactic activity (NCA) and concentrations of three neutrophil-attracting CXC chemokines – CXCL1 (Groα), CXCL5 (Ena78) and CXCL8 (IL8) – were measured in supernatants of T84 colonic epithelial cells and blood eosinophils or eosinophil-like myeloid leukaemia cells (AML14.3D10), alone or in combination. Cells were stimulated with serum-opsonized zymosan (OZ) particles. NCA (P < 0.005) and CXCL5 levels (P < 0.05) in the supernatants of OZ-stimulated epithelial/eosinophil co-cultures were significantly higher than in the supernatants of either cell type alone. Release of CXCL1 (P < 0.05) and CXCL8 (P < 0.01) from OZ-stimulated co-culture supernatants was significantly higher than from OZ-stimulated eosinophils but not higher than from OZ-stimulated epithelial cells. Eosinophils and colonic epithelial cells exhibit synergy in production of neutrophil chemoattractants in response to immunological stimulation. This may represent a mechanism for exaggerated recruitment of neutrophils to the intestine in response to acute infection in conditions that are characterized by the presence of eosinophils in the bowel.     

Enhanced basophil histamine release and neutrophil chemotactic activity predispose grain dust-induced airway obstruction.

BACKGROUND: The pathogenic mechanism of grain dust (GD)-induced occupational asthma (OA) remains unclear. OBJECTIVE: To understand further the mechanism of GD-induced OA. METHODS: Fifteen employees working in a same GD industry, complaining of work-related respiratory symptoms, were enrolled and were divided into two groups according to the GD-bronchoprovocation test (BPT) result: six positive responders were grouped as group III, nine negative responders as group II and five healthy controls as group I. Serum GD-specific immunoglobulin (Ig)E (sIgE), specific IgG (sIgG) and specific IgG4 (sIgG4) antibodies were detected by enzyme-linked immunosorbent assay. Basophil histamine release was measured by the autofluorometric method, and changes of serum neutrophil chemotactic activity were observed by the Boyden chamber method. RESULTS: For clinical parameters such as degree of airway hyperresponsiveness to methacholine, duration of respiratory symptoms, exposure duration, and prevalences of serum sIgE, sIgG and sIgG4 antibodies, there were no significant differences between group II and III (P > 0.05, respectively). Serum neutrophil chemotactic activity increased significantly at 30 min and decreased at 240 min after the GD-BPT in group III subjects (P < 0.05, respectively), while no significant changes were noted in group II subjects (P > 0.05). Basophil histamine release induced by GD was significantly higher in group III than those of group I or group II (P < 0.05, respectively), while minimal release of anti-IgG4 antibodies was noted in all three groups. CONCLUSIONS: These results suggest that enhanced basophil histamine release and serum neutrophil chemotactic activity might contribute to the development of GD-induced occupational asthma.     

Neutrophil chemotactic activity in milk-induced asthma

Four subjects with clinical histories of milk-induced asthma were studied (three allergic to cow's milk; one to soya milk). In each instance, skin prick tests, RAST (IgE and IgG₄), the basophil histamine release, and serum precipitins, using appropriate milk extracts, were negative. After the ingestion of milk all the subjects developed a reproducible and dose-dependent increase in airflow limitation. Three patients (two allergic to cow's milk; one to soya milk) gave a characteristic immediate-type reaction, which was maximal at 30 min after challenge. The fourth individual developed an isolated late-phase response, with maximal airways obstruction 3 hr after ingesting milk. In the three subjects who gave an early reaction, wheezing was accompanied by an elevation in circulating neutrophil chemotactic activity (NCA). This was not observed in the individual with the isolated late reaction. By Sephacryl S-400 gel-filtration chromatography it was shown that NCA of the early reactions eluted with proteins having an estimated molecular weight of 600,000 daltons. The immediate asthmatic response in peak expiratory flow rate and the elevation in NCA were inhibited by the prior oral administration of either disodium cromoglycate (DSCG) or oral beclomethasone dipropionate (BDP). In contrast, DSCG had no effect on airways obstruction in the subject with the isolated late asthmatic response, although inhibition was achieved by BDP.     

Gammadelta T-lymphocyte cytotoxic activity against Mycobacterium bovis analyzed by flow cytometry

Human exposure to bovine serum albumin (BSA) is very common and occurs through dietary and medicinal routes. Although great effort has been made to reduce exposure to BSA in pharmaceuticals to eliminate the threat of bovine spongiform encephalopathy, less attention has been given to assessing the human immune response after exposure to BSA. A sensitive quantitative radioimmunoassay was therefore developed to measure anti-BSA IgG antibodies in healthy subjects and in cancer patients participating in a randomized, placebo controlled clinical trial where they were exposed to BSA as an intrathoracic surgical sealant during pneumonectomy. Anti-BSA antibodies were detected in 55% of 60 healthy blood donors and 51% of 83 patients before lung cancer resection. The median antibody levels were the same in both cohorts; 0.086 μg/mL (range 0.016–19.5 μg/mL) for health blood donors and 0.062 μg/mL (range 0.009–44 μg/mL) for cancer patients. Six months after exposure of the cancer patients to BSA, the percentage of patients with anti-BSA antibody rose to 96% and the median antibody level rose to 19 μg/mL (range 0.009–258 μg/mL). Placebo-treated cancer patients showed no significant increase in the percentage of patients with anti-BSA antibody (41%) or the median antibody level (0.047 μg/mL; range 0.008–1.58) over 6 months. Western blot analysis confirmed the presence of anti-BSA antibody. Elevated levels of anti-BSA antibody were not associated with any detectable clinical events in either the healthy blood donors or the cancer patients.     

Immunologic release of histamine from dog lung: comparison of in vivo and in vitro responses in the same animal

The purpose of this study was to compare, for the first time, antigen-induced histamine release from the lung in the same natively allergic dogs both in vitro and in vivo. In six dogs, maximal antigen-induced histamine release from the lung correlated closely in vitro and in vivo (r = 0.94), although it varied widely between dogs (0% to 75.5% of total tissue histamine content); similarly, the antigen concentration to produce 50% of maximal histamine release varied sixfold between dogs (40 micrograms/ml to 250 micrograms/ml). In each of five other dogs, terbutaline sulfate administered intravenously caused a dose-dependent inhibition of antigen-induced histamine release from lung fragments in vitro: the maximal inhibition produced by 1 mg/kg was 60 +/- 4.5% (mean +/- SEM). In these same dogs, 10(-5)M terbutaline incubated with lung fragments in vitro caused inhibition of antigen-induced histamine release comparable to 1 mg/kg terbutaline in vivo. Increasing the dose of terbutaline in vitro produced maximal inhibition at 10(-4)M with no greater effect of the drug at 10(-3)M (71.4 +/- 3.8% inhibition). In both experimental situations propranolol caused a dose-dependent inhibition of beta-adrenergic modulation of Ascaris-induced release of histamine. This result supports the conclusion that terbutaline produced its effects by actions mediated by beta-adrenergic receptors on pulmonary mast cells. This experimental approach provides a suitable preparation in which to estimate the effective dose of agonists that modulate antigen-induced mast cell function in vivo.     

Secretion of a chemotactic factor for neutrophils and eosinophils by alveolar macrophages from asthmatic patients

The studies presented in this article demonstrate the release of an IgE-dependent chemotactic factor for polymorphonuclear neutrophils (PMN) and eosinophils by alveolar macrophages (AMs) from normal subjects (n = 15) and allergic asthmatic patients (n = 15). A 60-minute incubation of normal AMs previously sensitized by 20% nonheated allergic sera with anti-human IgE antibody or the related allergen induced the release of a chemotactic activity (CA) for PMN and eosinophils in culture supernatants. When AMs were obtained from asthmatic patients, direct incubation with anti-IgE or the related allergen induced the same CA, whereas incubation with an unrelated allergen failed to produce CA (neutrophil CA after addition of anti-IgE, 22.5 +/- 3.5 cells per high power field; with related allergen, 15.8 +/- 3.6; with unrelated allergen, 0.7 +/- 1.8; p less than 0.0001). A partial characterization of the neutrophil chemotactic factor was carried out. Enzymatic treatment by trypsin or carboxypeptidase or by heating (56 degrees C for 3 hr) failed to abolish the neutrophil CA. After gel filtration the greater part of the neutrophil CA (80%) was recovered among low-molecular-weight components (300 to 1300 daltons). A preliminary deactivation of PMN by leukotriene B4 suppressed the CA of AM supernatants. These results indicate that IgE-dependent stimulation of AMs produces a neutrophil and eosinophil CA, present in a low-molecular-weight fraction possibly related to leukotrienes, and emphasizes the role of AMs in inflammatory lung processes during allergic asthma.     

Immunologic release of neutrophil chemotactic activity from human lung tissue

The release of heat-stable neutrophil chemotactic activity (NCA) has been detected after challenge of isolated human lung tissue with anti-IgE. The major NCA released (NCAL) had similar physicochemical properties to the NCA detected in the circulation of asthmatic subjects after bronchial challenge with specific antigen (NCAAG). NCAL and NCAAG (1) had molecular weights of approximately 600,000 daltons as estimated by Sephacryl S-300 gel-filtration chromatography; (2) both eluted from DEAE-Sephacel (pH 7.8) between 0.1 and 0.2 molar NaCl; (3) had isoelectric points of between 6.5 and 6.8 as determined by chromatofocusing on Polybuffer Exchanger 94. In contrast to NCAAG, lung-derived neutrophil chemotactic activity appeared to be more heterogeneous after gel-filtration and anion-exchange chromatography. The release of NCA was complete by 15 min and there was no evidence of further release up to 12 hr. These observations indicate that high-molecular-weight NCA released from human lung tissue has similar properties to NCAAG and would support the view that NCAAG originates from lung tissue after antigen bronchial challenge in asthmatic subjects.     

Neutrophil chemotactic activity (NCA) in nasal secretions from atopic and nonatopic subjects

In order to elucidate the mechanism responsible for infiltration of nasal mucosa by granulocytes, we tested neutrophil chemotactic activity (NCA) in nasal lavages, by the modified Boyden chamber method, in 16 patients with perennial allergic rhinitis (AR), six ASA-sensitive patients with chronic rhinosinusitis (CRS), and seven normal, nonatopic control subjects (NC). Nasal secretions from all three groups showed significant NCA (mean 157.1±54.0, 62.2±20.7, and 39.4 ± 11.4% of FMLP chemotactic activity for AR, CRS, and NC subjects, respectively). Nasal secretions from patients with AR expressed significantly higher NCA ( P <0.02) than did secretions from NA patients.
NCA was unchanged by heating at 56°C for 60 min and was not susceptible to degradation by trypsin. Nasal challenge with Dermatophagoides pteronyssinus antigen induced clinical symptoms and resulted in significant increases in total protein and albumin concentrations in nasal lavages in AR patients, but failed to change the mean NCA activity for up to 40 min after the challenge. These results indicate that nasal secretions from both atopic and nonatopic patients express NCA, but its relation to allergic inflammation remains to be established.     

Heterogeneity of specific-IgE responses in workers sensitized to acid anhydride compounds

Acid anhydride compounds cause IgE-mediated respiratory sensitization in the workplace. In this study reaginic responses in four workers sensitized to phthalic anhydride (PA), hexahydrophthalic (HHPA), or himic anhydride (HA) were determined by direct RAST by use of PA-human serum albumin (HSA), HPPA-HSA, and HA-HSA methylcellulose disc substrates. RAST inhibition of binding to anhydride-HSA substrates was done with various concentrations of sodium salts, lysine, and HSA conjugates of PA, HHPA, HA, and trimellitic anhydride (TMA) in order to characterize cross-reactivity and specificity of humoral responses in these workers. Molar concentrations of anhydride bound to lysine and HSA carriers were assayed by protein hydrolysis followed by gas chromatographic analysis. Significant direct RAST binding against PA-HSA, HHPA-HSA, and HA-HSA was found in all four workers. PA-HSA-IgE binding in one PA-sensitive worker (no. 1) was inhibited significantly by PA-HSA alone and not by sodium PA or PA lysine. In contrast, another PA-sensitized worker (no. 2) exhibited 50% inhibition of PA-HSA binding by PA-HSA (1 X 10(-9) M), PA-lysine (7 X 10(-8) M), and NaPA (1 X 10(-7) M); no inhibition of PA-HSA by heterologous HHPA-, HA-, or TMA-inhibitory reagents was found in either of the PA workers. RAST binding to HHPA-HSA in the HHPA-sensitive worker was inhibited by HHPA-HSA alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of filter-bound IgA in inhibiting polymorphonuclear leukocyte chemotaxis

The effects of human serum IgA on human polymorphonuclear leukocyte (PMN) function were studied. In Boyden chambers, chemotactic migration against semipurified C5a as well as the formylated tripeptide FMLP was inhibited in the presence of IgA. Under such conditions, the release of superoxide anion was not changed. In addition, chemotaxis "under agarose" using the leading front evaluation remained unaltered in the presence of IgA despite IgA-induced aggregation of PMN and reduced density of migrating cells. Chemotaxis filters incubated with radiolabelled IgA demonstrated strong binding of IgA, and further, these IgA-pretreated filters impeded PMN migration. Incubation of PMN with IgA followed by subsequent washings did not affect chemotactic migration. These data show that filter binding of IgA is responsible for the inhibition of chemotactic PMN-migration in the Boyden chambers, most probably mediated by increased cell-substrate adhesion. Experiments using casein showed displacement of IgA by casein which enables PMN to fully respond to casein.     

Enhancement of Chemotactic Migration by the Local Anesthetic Tetracaine

The local amine anesthetic tetracaine added to a suspension of guinea pig or human neutrophilic granulocytes inhibited their random migration in Boyden chambers, but increased their chemotactic migration towards the chemotactic tripeptide f-Met-Leu-Phe, complement-activated normal guinea pig serum, and the eosinophil chemotactic factor ECF. Tetracaine not only increased the distance migrated by the leading cells, it also caused more cells to leave the upper filter surface and to migrate into the filter. The effect required the presence of the drug; cells preincubated with tetracaine and washed did not differ from control cells. It is suggested that tetracaine specifically enhanced a mechanism operative in a cell's response to a concentration gradient of a chemotactic factor.     

Generation of the eosinophil chemotactic factor (ECF) from various cell types by melittin

The peptide melittin, the main constituent of bee venom is a potent stimulus for the generation of an eosinophil chemotactic factor (ECF) from human polymorphonuclear neutrophils, rat mast cells and rat peritoneal cells depleted in mast cells. Optimal EFC induction required a sublytic activation of the cells. With each cell type the kinetics of ECF generation were similar in that after an early rise in activity a steep fall off occurred at later times of incubation suggesting a mechanism of inactivation. The induction of ECF by melittin is increased in the presence of calcium. The polar portion of the melittin molecule (aminoacids 20–26) is responsible for the generation of the chemotactic activity. Other peptides of honey bee venom such as the mast cell degranulating peptide (MCD) or apamine do not initiate ECF release. It appears that melittin leads to ECF induction via the phospholipase A₂-arachidonic acid dependent pathway of cell activation. Our data suggests that the lipid mediator ECF can be obtained from phagocytes and mast cells thus indicating the interdependence of inflammatory reactions.     

Allergens in Hymenoptera venom XIII: Isolation and purification of protein components from three species of vespid venoms

Pure venoms were collected from individual insects of the species Dolichovespula maculata, white-faced hornet, Vespula squamosa, southern yellow jacket, and Polistes exclamans, paper wasp (one species). The venoms were first fractionated by high-resolution gel filtration on a 1.6 m column of Sephadex G-75 superfine, and the components were then purified by high-performance, ion-exchange chromatography on a Mono-S cation exchange column followed by a further gel filtration step. The isolated components were evaluated for purity by sodium dodecyl sulfate polyacrylamide gel electrophoresis by use of two different types of silver stains, by assays for enzyme activities, and by immunodiffusion with the use of rabbit antisera. The protein components were isolated in highly purified states by these techniques. Only three significant proteins were found in V. squamous venom: phospholipase (PL) A and B, hyaluronidase (HYAL), and antigen 5 (Ag 5). D. maculata venom contained HYAL, Ag 5, two isozymes of PL A and B, a high-molecular-weight protein, and several trace proteins. No significant amounts of proteases were found in D. maculata venom. P. exclamans venom contained HYAL, PL A and B, Ag 5, a high-molecular-weight protein, and several minor proteins. In all three venoms the PL A and B activities were found to be in the same molecule and did not separate. Trace components with apparent PL A activity were observed in the venoms. The venoms were screened for a variety of esterases, proteases, peptidases, glucosidases, and phosphatases, and none were detected in more than trace amounts. Vespid venoms do not appear to contain significant amounts of acid phosphatases as bee venoms do.     

Purification and partial characterization of two major allergens from the house dust mite Dermatophagoides pteronyssinus

Two major allergens of the house dust mite, Dermatophagoides pteronyssinus (Dp), were purified, and their molecular weight and isoelectric points (pIs) were determined. Dp 42 was purified from an acetone-precipitated mite-excrement extract by a combination of hydrophobic interaction chromatography on phenyl Sepharose and copper-chelate chromatography. The molecular weight was determined to be 18,000 and 25,000 to 30,000 by gel filtration (G-75) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively, and pI values of 4.6, 5.6, and 6.6 were obtained by sucrose gradient isoelectric focusing (IEF). These values correspond well with those described for the identical allergen, P1. The pI 6.6 variant was considerably enriched in the purified material. Dp 42 constituted 6.4% of the dry weight of a reference whole mite-culture extract. Dp X was obtained partially purified by gel filtration (G-75), ammonium sulphate precipitation, and hydrophobic interaction chromatography. The molecular weight was 18,000 to 20,000 by gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Multiple pIs in the range 5 to 7 were found by sucrose gradient IEF and crossed IEF. The two purified allergens carried clearly distinct activities toward human IgE and appeared as potent allergens in crossed radioimmunoelectrophoresis, RAST, and RAST inhibition.     

Effects of dialyzable leukocyte extracts (DLEs) with transfer factor activity on leukocyte migration in vitro. III. Characterization of the antigen-independent migration inhibition factor in DLEs as a neutrophil immobilizing factor.

In earlier evaluations of the agarose LMI assay as an in vitro test for studying the nature and mechanism of action of TF, we reported the existence of a component in human DLEs which caused noncytotoxic inhibition of the random migration of human PMNs. The LMI was not dependent on the stimulation of viable mononuclear leukocytes by antigen or mitogen to effect the release of mediators of cellular immunity such as LIF; rather, the LMI was promoted by the direct action of a preexisting component in DLEs on PMNs. We now present evidence that this "antigen-independent" LMI activity in DLE'S is similar to a NIF shown previously by Goetzl and co-workers to be present in acid extracts of leukocytes and to be released by phagocytosing PMNs. The comparison is drawn from several parameters: (1) cellular origin, (2) molecular weight, (3) target cell, (4) susceptibility to inactivation by heating or by incubation with pronase, trypsin, or chymotrypsin, and (5) ability to cause noncytotoxic inhibition of random migration or chemotaxis of PMNs.     

Particle counting immunoassay of immunoglobulin E antibodies after their elution from allergosorbents by pepsin: an alternative to the radioallergosorbent test

The absorption of specific antibodies on allergen-coated cellulose discs, followed by pepsin digestion to release the IgE Fc″ fragment, which is then assayed by particle counting immunoassay (PACIA), is a new practical and reliable method to quantitate IgE antibodies and to standardize allergens. The coefficient of variation of repeated assays does not exceed 12.8% and the correlation coefficient with RAST was r = 0.96. Some discrepancies could be explained by differences in the antiserum specificities and the presence of anti-IgE autoantibodies. The determination of IgE antibodies by PACIA offers the following advantages: (I) no radioisotopes; (2) stability of reagents, since antibody-coated latex particles and the standards keep their activity for more than 1 yr; (3) low cost of reagents due to ease of preparation—PACIA does not require purified antibodies to measure IgE but only F(ab′)₂ fragments of the IgG fraction of the anti-IgE antiserum to coat latex; (4) avoidance of nonspecific absorption of the labeled anti-IgE on the solid phase; (5) prevention by elution of the inaccuracies observed in RAST at very high titers of IgE antibodies; (6) expression of the results in international units per milliliter to calculate the ratio of specific IgE Ab vs total IgE, with a high ratio suggesting that the patient is sensitive to a few allergens; (7) standardization of allergenic extracts; and (8) the particular specificity of the anti-IgE antiserum directed against a heat- and protease-resistant fragment of IgE.     

An inhibitory factor of DNA synthesis derived from the medium of short-time incubated human lymphocytes

The medium of human tonsillar lymphocytes incubated for 4 h without any in vitro activation was found to contain a non-dialyzable, soluble inhibitor of DNA synthesis. The spontaneous- as well as the PHA-stimulated DNA synthesis of lymphocytes were both affected by the inhibitor, which was not cytotoxic for its target cells. The factor was isolated by ammonium sulphate precipitation and was purified on a DEAE-cellulose column. It is assumed that the factor is continuously produced by lymphocytes in vivo and plays a role in the homeostatic regulation of lymphocytes.     

Macrophage handling of soluble immune complexes: Evaluation of mechanisms involved in the selective clearance of complexes from the circulation

The binding and release of soluble guinea pig IgG2-containing DNPBSA-anti-DNP complexes and antigen-free, covalently-linked anti-DNP IgG2 oligomers of similar size, by guinea pig peritoneal macrophages, has been examined in the absence and presence of monomeric IgG2, of unrelated antibody specificity, or the monovalent hapten, DNP lysine. Complex binding was found to differ from the binding of the oligomers in that it was about twice as efficient and was essentially irreversible even in the presence of an inhibitor of ingestion, cytochalasin B. On the other hand, quantitative complex release could be achieved, in the presence of the ingestion inhibitor, by including 1.5mM DNP lysine in the medium. Complex handling by macrophages at 37°C was also examined in the presence of monomeric IgG2, at its serum concn, and in the absence and presence of cytochalasin B. Inhibiting ingestion did not impair the capacity of the macrophages to take up complexes under these conditions. On the basis of these findings and previous reports that complexes bound to a receptor-bearing membrane undergo additional antibody-antigen bond formation [Dower et al, Biochemistry20, 6326–6334 (1981a) and Leslie, Protides biol. Fluids29, 431–434 (1982)] it is proposed that complex aggregation at the phagocyte surface may constitute the critical irreversible event required for the selective clearance of complexes in vivo. Other biological implications of receptor-mediated complex aggregation are also discussed.     

The biologic activity of mast cell granules. V. The effects of antihistamine treatment on rat cutaneous early- and late-phase allergic reactions

Mast cell-dependent late-phase allergic reactions (LPR) as sequelae of immediate hypersensitivity responses (IR) occur in both human and rat skin; thus the rat has served as a useful model to investigate the pathogenesis of cutaneous LPR. To analyze the roles that histamine might play in the generation of rat LPR, the effects of H1 and/or H2 antihistamines on both LPR and antecedent blueing responses (IR) were investigated. Systemically administered diphenhydramine and cimetidine, alone or in combination, reduced blueing reactions to histamine. However, blueing responses to anti-IgE were only partially abrogated by antihistamine treatment with diphenhydramine alone or the combination of antihistamines. Diphenhydramine treatment alone partially inhibited the histologic intensity of LPR in a dose-dependent manner. Although cimetidine treatment alone had no inhibitory effect, it potentiated the diphenhydramine-induced inhibition of LPR. The inhibitory action of antihistamine treatment was apparent only in reactions elicited by anti-IgE or mast cell granules containing histamine, since LPR caused by histamine-free mast cell granules were not affected by antihistamines. This observation suggests that the inhibitory effect of antihistamines on LPR was the result of a specific blockade of histamine receptors rather than the result of a nonspecific suppressive effect. Our findings demonstrate that cutaneous inflammation generated as a result of mast cell degranulation can be significantly reduced by treatment with H1 and H2 histamine receptor antagonists.