Monoclonal antibodies to immunoglobulin G4 induce histamine release from human basophils in vitro

Monoclonal murine antibodies to human IgE, IgG1, IgG2, IgG3, IgG4, IgM, and IgA1 were prepared and their abilities to stimulate histamine release from human peripheral blood leukocytes were investigated. As expected, anti-IgE caused 8% to 76% histamine release from the leukocytes of each of 14 nonallergic donors. In one subject anti-IgE alone had no effect, but a 15% histamine release did occur after the subsequent addition of a goat anti-mouse IgG antiserum to crosslink cell-bound anti-IgE molecules. Anti-IgG4 alone caused 5% to 59% histamine release from the cells of four of 14 donors. Subsequent challenge with goat anti-mouse IgG caused an additional release of up to 15% of the basophil histamine, but only from leukocytes of donors responsive to anti-IgG4 alone. No histamine release was detected after stimulation by monoclonal antibodies to IgG1, IgG2, IgG3, IgM, or IgA1. The results of this study indicate that monoclonal antibodies to IgG4 as well as to IgE can stimulate human peripheral blood leukocytes to release histamine. These observations support the theory that antigen-IgG4 interactions can stimulate histamine release from human basophils.     

Histamine Release from Human Leucocytes by IgG4 Subclass in the Sera of Allergic Children

Our studies on histamine release from normal washed leucocytes sensitised with sera of children allergic to various inhaled and ingested allergens show that besides IgE, IgG4 present in these sera was capable of sensitising leucocytes of normal donors, and these leucocytes released histamine on challenge with anti-IgG4. By contrast, antisera of other subclasses of IgG released very small amounts of histamine from sensitised leucocytes. A discordant correlation between skin tests and RAST IgE was observed in some children. Significantly greater amounts of histamine were released from normal leucocytes sensitised with serum from these children when challenged with anti-IgG4 compared to anti-IgE. These studies indicate that IgG4 merits further study as a reaginic marker in atopic diseases of the children with possible prognostic significance.     

In vitro basophil histamine-releasing activity of circulating IgG1 and IgG4 autoanti-IgE antibodies from asthma patients and the demonstration that anti-IgE modulates allergen-induced basophil activation

In this study we have examined the relationship between the in vitro basophil histamine-releasing activity of human IgG anti-IgE, isolated as euglobulin fractions from sera of asthmatic patients, and its IgG1/IgG4 subclass distribution. In particular, we have investigated whether IgG anti-IgE modulates allergen-induced basophil activation. The study has revealed that only a small proportion of IgG anti-IgE samples triggered histamine release from basophils of an asthmatic individual (4/21; 19%), a hay fever sufferer (4/10; 40%) and a healthy person (7/21; 33%). The basophil histamine-releasing activity of IgG anti-IgE did not seem to be determined by the IgG1/IgG4 subclass composition of the IgG anti-IgE preparation used. Furthermore, we have demonstrated that autoanti-IgE antibodies modulate allergen-induced basophil histamine release. The three modulatory effects exerted by IgG anti-IgE antibodies on allergen-triggered basophil activation (i.e. additive, synergistic and blocking) were not dependent on the subclass nature of IgG anti-IgE or the use of histamine-releasing anti-IgE preparations. Our data suggest that IgG anti-IgE antibodies in asthma patients may consist of two functionally distinct subpopulations: those which up-regulate (pro-allergic) and those which down-regulate (anti-allergic) the allergic release of mediators from mast cells and basophils.     

IgG4 and passive sensitization of basophil leukocytes

In contrast to IgE, myeloma IgG4 did not block passive sensitization of basophil leukocytes by IgE antibody. Atopic sera, selected for a high level of mite-specific IgG4, lost their sensitizing activity after they had been heated at 56 degrees C. After incubation of leukocytes with myeloma IgG4 or with sera with high levels of specific IgG4, the cells did not become responsive towards anti-IgG4 antiserum. After incubation of leukocytes with fluorescein-labelled myeloma IgE, these cells released histamine with antifluorescein antiserum. In contrast, after incubation with fluorescein-labelled myeloma IgG4, a subsequent incubation with antifluorescein antiserum did not result in histamine release. Therefore, we cannot confirm the hypothesis of Vijay and Perelmutter that IgG4 can sensitize basophil leukocytes in vitro.     

Circulating levels of IgG1 and IgG4 Anti-IgE antibodies and asthma severity

In this paper, we have determined the levels of IgG1 and IgG4 anti-IgE in the sera of 66 asthma patients suffering from mild ( n = 24), moderate ( n = 23), or severe ( n = 19) symptoms, and 20 nonatopic, healthy subjects. The study has revealed that although asthma patients have significantly elevated levels of IgG1 and IgG4 anti-IgE antibodies, the concentration of these autoantibodies is not related to the severity of asthma. This conclusion may be related to the known heterogeneity of autoanti-IgE antibodies in terms of their ability to trigger basophil histamine release.     

Mechanism of activation of human basophils by Staphylococcus aureus Cowan 1

We investigated the capacity of Staphylococcus aureus Cowan 1 and S. aureus Wood 46 to induce histamine release from human basophils in vitro. S. aureus Cowan 1 (10(5) to 10(7)/ml), which synthesizes protein A (Staph A), stimulated the release of histamine from basophils, whereas S. aureus Wood 46 (10(5) to 2 X 10(7)/ml), which does not synthesize Staph A, did not induce histamine secretion. Soluble Staph A (10(-3) to 10 micrograms/ml), but not staphylococcal enterotoxin A, induced histamine secretion from human basophils. Staph A binds through its classical site to the Fc region of human immunoglobulin G (IgG) and through its alternative site to the Fab portion of the different human immunoglobulins. Hyperiodination of Staph A, which destroys over 90% of the original Fc reactivity without altering the Fab-binding site, did not alter the ability of the protein to induce histamine release. The stimulating effect of Staph A was dose dependently inhibited by preincubation with human polyclonal IgG (0.3 to 100 micrograms/ml) and a human monoclonal IgM (0.3 to 100 micrograms/ml) which have F(ab')-Staph A reactivity. In contrast, rabbit IgG, which possesses only Fc-Staph A reactivity, and a Staph A-unreactive human monoclonal IgM did not inhibit Staph A activity. Similar results were obtained with intact S. aureus Cowan 1. Preincubation with either Staph A or anti-IgE (rabbit anti-Fc epsilon) resulted in complete desensitization to a subsequent challenge with the homologous stimulus. Staph A and anti-IgE induced partial cross-densensitization to the heterologous stimulus. Cells preincubated with anti-IgG (rabbit anti-Fc gamma) lost a small but significant part of their ability to release with Staph A but did not lose their response to anti-IgE. Basophils from which IgE had been dissociated by brief exposure to lactic acid no longer released histamine in response to anti-IgE and Staph A. When basophils from which IgE had been dissociated were incubated with human polyclonal IgE, they regained their ability to induce histamine in response to Staph A and anti-IgE. In contrast, two monoclonal IgEs which do not bind to Staph A did not restore the basophil responsiveness to Staph A. Furthermore, there was complete cross-desensitization between soluble Staph A and S. aureus Cowan 1, while cells desensitized to S. aureus Wood 46 released normally with Staph A and S. aureus Cowan 1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of IgG4 in histamine release from human basophil leucocytes. I. Sensitization of cells from normal donors

Several conflicting reports on the ability of IgG4 to mediate type I allergic reactions have appeared lately. We have developed a model system for testing this possibility, using passive sensitization of basophil leucocytes from normal individuals. At first, the system was optimized with regard to donor cells and anti-IgG4 antibody: among 3 normal individuals with cells showing high, medium and low responses to anti-IgE, only the high responder showed a reproducible, but low response to anti-IgG4. This response was consistent when testing anti-IgG4 from different sources, and could be potentiated by a phorbol ester TPA, although not up to the level of anti-IgE. The cells from the high responding donor and a monoclonal anti-IgG4 were selected for further studies. Serum pools from patients allergic to house dust mite (Dermatophagoides pteronyssinus) were used for passive sensitization. The pools contained allergen-specific IgE with or without concomitant IgG4 of the same specificity. The sensitized basophils reacted to anti-IgE and allergen, but to anti-IgG4 only to a small extent. However, when these serum pools were fractionated by affinity chromatography, only the IgE fractions and not the IgG4 fractions made the cells reactive towards specific allergen. It was still possible to elicit a reaction by triggering IgE with anti-IgE, whereas only a small reaction was seen, when IgG4-sensitized basophils were provoked with anti-IgG4. We conclude that IgG4 does not sensitize normal basophils.     

Basophil FcepsilonRI histamine release parallels expression of Src-homology 2-containing inositol phosphatases in chronic idiopathic urticaria

BACKGROUND: Basophils are implicated in the pathogenesis of chronic idiopathic urticaria (CIU). Autoantibodies to the IgE receptor (FcepsilonRI) and serum histamine releasing activity have been detected in some subjects with CIU, although their role in vivo is unclear. Basophils of patients with CIU have altered FcepsilonRI-mediated histamine release (HR); however, the mechanism is unknown. In the basophil FcepsilonRI signaling pathway, protein levels of Src-homology 2-containing-5'-inositol phosphatase (SHIP)-1 are inversely correlated with the release of mediators or releasability. A related phosphatase, SHIP-2, is a negative regulator of monocyte IgG receptor (FcgammaR) signaling . We hypothesized that SHIP levels are altered in CIU basophils. METHODS: Blood basophils were isolated from cold urticaria, CIU, or normal donors, and FcepsilonRI-dependent and independent HR were quantified. Protein levels of SHIP-1, SHIP-2, spleen tyrosine kinase, and phosphorylated Akt were determined by Western blotting. Subjects' serum was tested for serum histamine releasing activity and anti-FcepsilonRIalpha antibodies. RESULTS: CIU basophils displayed a bimodal response to anti-IgE activation. One half of CIU subjects' basophils had reductions in anti-IgE-induced HR and were designated nonresponders (CIU NR). CIU NR basophil HR remained diminished at 10-fold to 30-fold higher doses of anti-IgE. CIU anti-IgE responder basophils had HR similar to normal subjects. SHIP-1 and SHIP-2 proteins were increased in CIU NR basophils and were linked to reduced phosphoAkt after anti-IgE stimulation. CIU basophil anti-IgE response was not related to the presence of serologic factors. CONCLUSION: In CIU basophils, the observed changes in FcepsilonRI signaling pathway molecule expression may underlie changes in releasability. CLINICAL IMPLICATIONS: Patients with CIU can be segregated on the basis of basophil functional phenotype.     

IgG4 and release of histamine from human peripheral blood leukocytes

Allergen-specific IgG4 was found in the sera from many normal, nonallergic individuals. Basophil leukocytes from donors with IgG4 antibodies to an allergen, but without IgE antibodies to the same allergen, did not release histamine when challenged with this allergen. In some cases, anti-IgG4 antiserum induced a release of histamine from the leukocytes. However, these cells also release histamine after incubation with normal rabbit serum or normal sheep serum. It is concluded that the IgG4-RAST cannot be used for the detection of IgG short-term sensitizing antibodies.     

The effect of bacterial lipopolysaccharide (LPS) on histamine release from human basophils. I. Enhancement of immunologic release by LPS

Preincubation of human basophils with bacterial lipopolysaccharide (LPS) purified from the heptose-deficient mutant Salmonella minnesota R595 enhanced by an average of sixfold the response of peripheral blood basophils obtained from allergic donors to several allergens in vitro as judged by release of histamine. Enhancement occurred at suboptimal, optimal, and supraoptimal concentrations of antigen. No effect was seen if basophils were from a nonallergic donor, and LPS by itself rarely caused histamine release from any preparation of basophils. However, histamine release in basophils from nonallergic donors induced by antibody directed against IgE (anti-IgE) also was enhanced by LPS. Potentiation of histamine release occurred if basophils were pretreated with LPS before addition of anti-IgE for as little as 5 min; there was no increase in release if anti-IgE and LPS were added simultaneously to cells. LPS enhanced the rate of release without altering duration of the release response. LPS potentiation of release of histamine by F(ab')2 fragments of anti-IgE was equivalent to its effect on release triggered by the intact antibody molecule, confirming that the effect of LPS is not due solely to its interaction with the Fc component of the anti-IgE. These data thus provide evidence for modulation of basophil response to IgE-mediated stimuli by LPS, resulting in a significant enhancement of response. Enhancement by LPS appears to be independent of the stimulus which triggers the IgE receptor. The contribution of this mechanism to allergic disease or asthma remains to be determined.     

IgG receptors on the mast cell

Antisera to the IgG subclasses, 1, 2a, 2b, and 2c, induced histamine release from mast cells obtained from the peritoneal washings of Lister hooded rats. The maximum responses obtained with anti-IgG₁ and anti-IgG_2a were as great as that for anti-IgE (more than 60% histamine release). Cells from unresponsive Wistar rats which did not secrete appreciable amounts of histamine in response to any of the antisera, produced on active sensitization with ovalbumin a small but significant response on challenge with anti-IgG₁ and anti-IgG_2b as well as with anti-IgE. Passive sensitization with rat myeloma serum of mast cells from the unresponsive rats produced a large response on challenge with anti-IgE but no release to the anti-IgG group 2 subclasses. IgE myeloma serum (11000) neutralized the histamine-releasing activity of anti-IgE serum (87% inhibition) and the antisera to all subclasses of IgG. When the IgE in the myeloma serum was inactivated by heating, the response to the IgG antisera remained completely inhibited except for anti-IgG_2a where some reversal was observed. When purified myeloma IgE (30 g/ml) was used in place of whole serum, marked inhibition (86%) of the response to anti-IgE was obtained leaving the responses to the IgE subclasses unaffected (except for IgG_2a, which was 65% inhibited).     

Histamine release from rat peritoneal mast cells after passive sensitization with human reaginic serum or E myeloma protein

Significant histamine release (HR) (averaging 15 per cent) was attained from rat peritoneal cells upon incubation with human reaginic sera and a specific antigen (grass pollen) or a monospecific anti-IgE antiserum. Similar histamine values were obtained from rat mast cells (RMC) incubated with E myeloma protein and challenged with anti-IgE. Washing the cells after incubation with reaginic serum or E myeloma protein did not result in a decrease in HR upon challenge with grass pollen antigen or anti-IgE. No significant HR was observed in control situations. There was also no significant antigen-induced HR from RMC by nonreaginic (newborn) serum or by reaginic serum that was heated for 4 hours at 56 °C. Furthermore, anti-IgG, -IgA, and -IgM failed to induce HR from RMC incubated with reaginic serum. These observations support the role of human IgE in passive sensitization of heterologous cells (RMC) for the release of a chemical mediator (histamine).     

Antigen-, anti-F(ab')2- and anti-IgE-induced histamine release from rat mast cells.

The existence of IgE antibody molecules on peritoneal mast cells from rats immunized with dinitrophenylated ascaris extract was demonstrated by double antibody immunofluorescence staining and by histamine release induced by anti-IgE antibodies. Cross-linking of the IgE molecules, which are fixed to mast cells, by interaction with either the homologous antigen or with antibodies to the Fab or Fc regions of IgE, triggered in the presence of calcium ions a chain of intracellular reactions which involve cyclic nucleotide modulation and energy-requiring processes leading ultimately to the release of histamine from the granules of these cells. Although there was no apparent difference in the mechanism(s) underlying the reactions triggered by any of these three agents, the extent of histamine release caused by antibodies to the Fc region of the IgE was significantly lower than that induced by cross-linking of IgE molecules through their Fab regions by reactions with either anti-Fab antibodies or antigens.     

Immune responses to nematode exoantigens: sensitizing antibodies and basophil histamine release

Windelborg Nielsen B, Lind P, Hansen B, Reimert CM, Nansen P, Schiotz PO. Immune responses to nematode exoantigens: sensitizing antibodies and basophil histamine release.
High levels of IgE and eosinophilia are found in both allergy and helminth infections, but allergic symptoms are rare in naturally acquired helminth infections. The interrelation of specific IgE antibodies and in vitro basophil histamine release (HR) induced by exoantigens from the larval stages (L-₂/L₃) of the nematodes Toxocara canis and Ascaris suum was examined in 148 patients visiting an outpatient clinic for parasitic diseases. The antigen sensitivity of the basophils was found to be dependent not only on the absolute amount of antigen-specific IgE present in patient plasma, but also on the ratio between specific and total IgE. Thus, large HR was observed in some patients in response to helminth antigens despite low levels of both specific and total IgE content in plasma. Patients with eosinophilia showed greater IgE-mediated HR than the other patients examined. In contrast, only five patients showed HR after challenge with anti-IgG4, despite the presence of high levels of antigen-specific IgG4 and IgGl in all patients showing specific IgE antibodies.     

Failure of covalently cross-linked human IgG myeloma subclass protein to release histamine from human leukocytes

We have examined the ability of IgG subclass antibodies to release histamine from human leukocytes using covalently cross-linked oligomers of human myeloma proteins. Purified IgG1, G2, G3, G4, (or IgE) was incubated with dimethyl suberimidate to induce cross-linking. The resulting dimers, trimers, and higher molecular weight oligomers were isolated using gel filtration columns (Sephadex G200 and Ultrogel AcA 22) connected in tandem. None of the oligomers of IgG1, G2, G3, or G4 released histamine from leukocytes of donors whose basophils released histamine when challenged with IgE dimer. Furthermore, preincubation with subclass specific oligomers did not desensitize cells to challenge with IgE dimer or to anti-IgE. We conclude that, under our experimental conditions, oligomers of human IgG myeloma subclass antibodies do not trigger histamine release nor modulate IgE-mediated reactions.     

Induction of Histamine Release and Desensitization in Human Leukocytes

Protein A from Staphylococcus aureus has been found to react with all human leukocyte preparations tested. In 70 percent of the experiments the reaction leads to histamine release. Furthermore, protein A treatment of cells at 37 degrees C, both in complete and Ca-2+-free medium, results in the inhibition of anti-IgE-induced histamine release in all cell preparations, indicating that protein A and anti-IgE antibodies release histamine from the same cells. This inhibition seems to be due to the blocking or exhaustion of a step in the biochemical pathway, leading to histamine release activated by both protein A and anti-IgE. In some cell preparations desensitization but no histamine liberation is induced by protein A. No inhibition occurs if the protein A treatment is performed at 4 degrees C. It is concluded that protein A elicits histamine liberation and desensitization by acting on IgG present on the surface of the basophil granulocytes. Treatment of leukocytes at 37 degrees C with anti-IgE antibodies, or F(ab)2 fragments from such antibodies, also results in an inhibition of a subsequent anti-IgE-induced histamine release. Desensitization with low doses of anti-IgE results in an inhibition of the same type as that obtained with protein A. Supraoptimum amounts of anti-IgE or high amounts of monovalent Fab fragments from anti-IgE immunoglobulin G give an inhibition that could be due to a competition between the sensitizing and the challenging agents for combining with cell fixed IgE molecules. This inhibition is independent of temperature and calcium concentration.     

The release of basogranulin in response to IgE-dependent and IgE-independent stimuli: validity of basogranulin measurement as an indicator of basophil activation

BACKGROUND: Basogranulin, the novel basophil granule protein recognized by the monoclonal antibody BB1, can be released by stimulation with anti-IgE antibody or calcium ionophore. However, the kinetics and regulation of its secretion are unknown. OBJECTIVE: We quantified basogranulin and histamine release in response to a range of stimuli to assess whether basogranulin secretion is a reliable marker of basophil activation. METHODS: Isolated peripheral blood basophils were stimulated with anti-IgE antibody, calcium ionophore, N -formyl-Met-Leu-Phe, and complement C5a. The released basogranulin and histamine were quantified by dot blotting with BB1 and a fluorometric method, respectively. Basogranulin localization was confirmed by flow cytometry. RESULTS: Both basogranulin and histamine displayed a bell-shaped response curve when basophils were challenged with anti-IgE. Half-maximal release occurred within 30 seconds. Basogranulin levels were maximal by 15 minutes, whereas those for histamine continued increasing to 30 minutes. Wortmannin, a PI3-K inhibitor, suppressed the release of both mediators. Basophils from donors with the "nonreleaser" phenotype secreted neither mediator in response to anti-IgE. Non-IgE-dependent stimuli released both mediators in parallel in a concentration-dependent manner. The correlation between the relative amounts of each mediator released was highly significant (r =.901, P <.0001, n = 87). Flow cytometry revealed that some of the secreted basogranulin adhered to the cell surface. CONCLUSIONS: Basogranulin is secreted along with histamine in response to both FcepsilonR I-related and unrelated stimuli. It is therefore a valid marker of basophil activation and could provide the basis for an immunoassay that distinguishes between basophil and mast cell activation.     

IgG-, IgA-, and IgE-induced release of leukotriene C4 by monocytes isolated from patients with atopic dermatitis

Purified peripheral blood monocytes isolated from patients with atopic dermatitis (AD) and from nonallergic normal donors were compared for their abilities to release leukotriene C4 (LTC4), leukotriene B4 (LTB4) and beta-glucuronidase in response to challenge with aggregated immunoglobulins or anti-immunoglobulins. The relationship between mediator release and the number of monocytes that formed rosettes with immunoglobulin-coated indicator cells was examined. Patients with AD had twice as many IgA- and three times as many IgE-rosetting monocytes as normal donors (48 +/- 12% versus 27 +/- 10% and 40 +/- 15% versus 14 +/- 3%, respectively), and yet the amounts of IgA- and IgE-induced LTC4 released were similar for both groups. This apparent discrepancy did not result from a decreased capacity for arachidonate metabolism via the C5-lipoxygenase pathway, since stimulation of monocytes from patients and normal donors with the calcium ionophore A23187 induced similar amounts of LTC4 and LTB4 release (LTC4, 3.0 +/- 1.7 versus 3.0 +/- 1.0 ng/10(6) cells; LTB4, 5.3 +/- 0.7 versus 5.2 +/- 0.5 ng/10(6) cells, respectively). In addition, aggregated IgG-induced LTC4 release by monocytes of both groups was similar, concomitant with an equivalent number of IgG-rosetting cells. Determination of cytophilically bound IgG and IgE by flow cytometry demonstrated that monocytes from atopic patients had more IgG bound than monocytes from normal donors. Similar amounts of IgE were detected on most monocytes from both groups, despite the higher serum IgE levels of patients. However, approximately 3% to 8% of monocytes from atopic but not normal donors stained brightly for IgE, suggesting that relatively large amounts of cytophilic IgE were bound to a small percentage of the patients' monocytes. Challenge of monocytes with anti-IgE or anti-IgG induced release of similar amounts of LTC4 for both groups, despite the presence of more cytophilic IgG on monocytes from atopic donors. These data indicate that monocytes from patients with AD release LTC4 and LTB4 in response to challenge with aggregated IgE or anti-IgE, as well as aggregated IgG, IgA, and anti-IgG. However, under our in vitro conditions, stimulation of patients' monocytes with aggregated IgA or IgE was not associated with increased mediator release, despite higher percentages of IgA- and IgE-rosetting cells compared to normal donors.     

Redistribution of immunoglobulin receptors on human neutrophils and its relationship to the release of lysosomal enzymes;.

Release of the lysosomal enzyme beta-glucuronidase from human neutrophils was induced by IgG or its Fc fragment, aggregated by immune precipitation or by coating on latex particles. Such release was inhibited when the cells were preincubated with free IgG or Fc fragments; F(ab')2 fragments were ineffective in both inducing and inhibiting beta-glucuronidase release. Neutrophils incubated with IgG or Fc fragments, when challenged with anti-IgG antibody, released lysosomal enzymes without the release of the cytoplasmic marker lactic dehydrogenase; These studies indicate that human neutrophils have surface receptors for the Fc portion of IgG. Neutrophils treated with IgG or its Fc fragment and subsequently with fluorescein- or ferritin-labeled anti-IgG showed binding of Fc or IgG to the cell membrane. Under suitable conditions, polar capping of labeled antibody was seen by fluorescence or electron microscopy. These studies suggest that the immunoglobulin receptors on neutrophils are redistributed when they are cross-linked with antibody. Fluidity of the membrane receptors appeared to be time and temperature dependent. Compounds such as 2-deoxyglucose, colchicine, and cyclic AMP, which inhibit the release of lysosomal enzymes, also inhibited the redistribution of the surface receptors. Cytochalasin B, an agent which increases the release, was found to increase the receptor redistribution; The relationship between the release of lysosomal enzymes and receptor mobility is discussed;