Extracellular release of rat eosinophil peroxidase (EPO) I. Role of anaphylactic immunoglobulins

The release of intracellular peroxidase (EPO) was investigated in order to evaluate rat eosinophil activation by various immunoglobulin (Ig) isotypes. After successive incubations with purified rat IgG1, IgG2a, IgG2b, IgG2c, IgE, or IgM and their respective anti-Ig antisera, eosinophils released significant amounts of EPO (up to 26% of the intracellular content) only in the case of Ig with anaphylactic activities (IgG2a and IgE). Other classes and subclasses were unable to induce EPO exocytosis. Selective depletion and reconstitution experiments suggested that mast cells were not required in this process. Similar levels of EPO could be released after interaction of eosinophils with antigen-antibody complexes (IgG2a monoclonal antibody and Schistosoma mansoni antigen) immobilized on nonphagocytosable surfaces. These results indicate that EPO exocytosis can be obtained after cell activation with specific antibodies, and that this mechanism is independent of phagocytosis. A kinetic study of eosinophils from S. mansoni-infected rats revealed that IgG2a and IgE cytophilic antibodies induced EPO release after incubation with either specific antisera or specific antigen, which suggests the in vivo relevance of such findings. The present work underlines the parallelism of interaction of anaphylactic-type Ig with eosinophils and with mast cells. Moreover, EPO release seems to represent an interesting marker of eosinophil activation, because close relationships were established between the present findings and previous work on the effector function of rat eosinophils.     

Does IgE bind to and activate eosinophils from patients with allergy?

Human eosinophils have been reported to express both the mRNA and protein for the high affinity IgE receptor (FcepsilonRI); it is speculated that this receptor plays a role in eosinophil mediator release in allergic diseases. However, questions still remain. How much of the FcepsilonRI protein is actually expressed on the cell surface of the eosinophil? If they are present, are these IgE receptors associated with effector functions of eosinophils? To address these issues, we studied blood eosinophils from patients with ragweed hay fever. A high level of low affinity IgG receptor (FcgammaRII, CD32), but no expression of FcepsilonRI, was detectable on the eosinophil surface by standard FACS analysis. However, after in vitro sensitization with biotinylated chimeric IgE (cIgE), cell-bound cIgE was detected by PE-conjugated streptavidin. This cIgE binding was partially inhibited by anti-FcepsilonRI mAb, suggesting that eosinophils do express minimal amounts of FcepsilonRI detectable only by a sensitive method. Indeed, FACS analysis of whole blood showed that eosinophils express approximately 0.5% of the FcepsilonRI that basophils express. When stimulated with human IgE or anti-human IgE, these eosinophils did not exert effector functions; there was neither production of leukotriene C4 or superoxide anion nor any detectable degranulation response. In contrast, eosinophils possessed membrane-bound human IgG and showed functional responses when stimulated with human IgG or anti-human IgG. Thus, IgG and/or cytokines, such as IL-5, appear to be more important for eosinophil activation in allergic diseases than IgE.     

Cytophilic IgE on human blood and tissue eosinophils: detection by flow microfluorometry

Flow microfluorometry (FMF) was used to investigate the presence of cytophilic Ig (IgE or IgG) and the proportion of Fc receptor (Fc epsilon R or Fc gamma R)-bearing eosinophils among eosinophils from 21 hypereosinophilic patients. In a large majority of the cases, it was possible to detect cytophilic IgE significantly associated with serum IgE levels. Moreover, when lung and blood eosinophils were compared, the proportion of occupied Fc epsilon R was significantly increased on lung eosinophils, whereas very few cells had cytophilic IgG. This work provides further evidence that cytophilic IgE is not restricted to cells with high affinity Fc epsilon R, but can also be detected on the cell populations with low affinity IgE receptors. These findings support the view that eosinophils can act as effector cells in immediate hypersensitivity reactions and in diseases associated with increased IgE production and hypereosinophilia.     

Anti-human IgG causes basophil histamine release by acting on IgG-IgE complexes bound to IgE receptors.

We have reexamined the ability of anti-human IgG antibodies to induce histamine release from human basophils. A panel of purified murine mAbs with International Union of Immunological Societies-documented specificity for each of the four subclasses of human IgG was used. Of the 24 allergic subjects studied, the basophils of 75% (18/24) released greater than 10% histamine to one or more anti-IgG1-4 mAb, whereas none of the 13 nonatopic donor's basophils released histamine after stimulation with optimal amounts of anti-IgG mAb. The basophils of 85% (11/13) of the nonatopic donors did respond to anti-IgE challenge, as did 92% (22/24) of the atopic donor cells. Histamine release was induced most frequently by anti-IgG3, and 10/18 anti-IgG responder cells released histamine with mAb specific for two or more different subclass specificities. The rank order for induction of histamine release was anti-IgG3 greater than anti-IgG2 greater than IgG1 greater than anti-IgG4. As in our previous study using polyclonal anti-IgG, 100- to 300-micrograms/ml quantities of the anti-IgG mAb were required for maximal histamine release, about 1000-fold higher than those for comparable release with anti-human IgE. Specificity studies using both immunoassays and inhibition studies with IgE myeloma protein indicated that anti-IgG induced histamine release was not caused by cross-reactivity with IgE. Ig receptors were opened by lactic acid treatment so that the cells could be passively sensitized. Neither IgE myeloma nor IgG myeloma (up to 15 mg/ml) proteins could restore the response to anti-IgG mAb. However, sera from individuals with leukocytes that released histamine upon challenge with anti-IgG mAb could passively sensitize acid-treated leukocytes from both anti-IgG responder and nonresponder donors for an anti-IgG response. The only anti-IgG mAb that induced release from these passively sensitized cells were those to which the serum donor was responsive. Sera from non-IgG responders could not restore an anti-IgG response. These data led to the hypothesis that the IgG specific mAb were binding to IgG-IgE complexes that were attached to the basophil through IgE bound to the IgE receptor. This was shown to be correct because passive sensitization to anti-IgG could be blocked by previous exposure of the basophils to IgE. We conclude that anti-IgG-induced release occurs as a result of binding to IgG anti-IgE antibodies and cross-linking of the IgE receptors on basophils.     

Functional role of the alpha-chain of complement receptor type 3 in human eosinophil-dependent antibody-mediated cytotoxicity against schistosomes

The participation of complement receptor type 3 (CR3) in antibody-dependent effector function of human eosinophils against parasites was studied by using monoclonal antibodies directed against various surface molecules. Both adherence and cytotoxicity of hypodense eosinophils to IgE-coated schistosomula of Schistosoma mansoni were strongly inhibited by anti-CR3 antibodies (OKM1 or Mo1). The specificity of the inhibitory effect for the alpha-chain of CR3 was shown by the lack of inhibition of anti-beta-chain or anti-LFA1 alpha-chain monoclonal antibodies, although these antigens were expressed on human eosinophils. These results associated to previous works on IgE receptors demonstrate that both receptor for Fc fragments of IgE and CR3 are essential in IgE-dependent cytotoxicity of human eosinophils. Flow microfluorometry analysis revealed that hypodense eosinophils were more intensively stained by OKM1 antibodies than the normodense populations. In the case of IgG-mediated cytotoxicity by normodense eosinophils, only the enhancement of cytotoxicity due to monokine activation was inhibited by anti-CR3 alpha-chain antibodies. These findings suggest an increased expression of CR3 on eosinophils after activation either in vivo or in vitro. The participation of CR3 in IgE-mediated cytotoxicity against schistosomes was also required in the case of blood monocytes but not for platelet-mediated killing, which does not require prior adherence. The biologic role of CR3 is therefore extended to effector mechanisms involving eosinophils and two different isotypes of antibodies and possibly implied in immunity against schistosomes.     

Fc gamma receptors on rat eosinophils: isotype-dependent cell activation

Fc receptors for rat IgG subclasses (IgG2a, IgG2c, and IgG1) were studied on rat eosinophils by rosette formation with erythrocytes coated with monoclonal immunoglobulin (Ig) or anti-Ig antisera in a reverse assay. Inhibition experiments revealed that IgG2a and IgG2c bind to the same receptor (IgG2a/IgG2c Fc receptor), distinct from the receptor for IgG1. In addition to the recent demonstration of the blocking effect of IgG2c antibodies in immunity to schistosomes, the present results show that the existence of this common receptor led to the specific inhibition by IgG2c of IgG2a-mediated eosinophil peroxidase release. Kinetic experiments on Schistosoma mansoni-infected rat eosinophils indicate that the IgG2a/IgG2c Fc receptors were occupied by cytophilic antibodies of the IgG2a isotype during the early phase of infection and by IgG2c thereafter. By rosette experiments it was possible to displace both in vivo and in vitro cytophilically bound IgG2a from its receptor. These results confirm, therefore, the major role played by antibodies in the modulation of eosinophil effector function during schistosomiasis. They underline, moreover, the possible isotypic regulation of cell activation.     

Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry

BACKGROUND: Naturally induced antibodies binding to surface antigens of Plasmodium falciparum-infected erythrocytes can be detected by direct agglutination of infected erythrocytes or by indirect immunofluorescence on intact, unfixed, infected erythrocytes. Agglutinating antibodies have previously been shown to recognise Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). This protein is inserted by the parasite into the host cell membrane and mediates the adhesion to the venular endothelium of the host organism in vivo. METHODS: Erythrocytes infected at high parasitaemias with ethidium-bromide-labelled mature forms of P. falciparum parasites were sequentially exposed to immune plasma, goat anti-human immunoglobulin (Ig) G, and fluorescein-isothiocyanate-conjugated rabbit anti-goat Ig. Plasma antibodies recognising antigens exposed on the surface of parasitised erythrocytes were subsequently detected by two-colour flow cytometry. RESULTS: Binding of human antibodies to the surface of erythrocytes infected with adhesive strains of Plasmodium falciparum can be measured by the two-colour flow cytometry (FCM) assay described. In addition, we demonstrate that the adhesive capacity of a parasite isolate correlates with the capacity of human immune plasmas to label the isolate as detected by FCM. We also show that the antigens recognised by the labelling antibodies are strain specific and that their molecular weights are in the range previously described for PfEMP1 antigens. CONCLUSIONS: Our FCM assay predominantly detects antibodies that recognise PfEMP1 and thus constitutes a convenient assay for the analysis of acquisition, maintenance, and diversity of anti-PfEMP1-specific antibodies and for the examination of class and subclass characteristics.     

Requirements for allergen-induced airway hyperreactivity in T and B cell-deficient mice.

BACKGROUND: The pathogenesis of asthma is believed to reflect antigen-induced airway inflammation leading to the recruitment of eosinophils and activation of mast cells through cell-associated IgE. Controversies persist however, regarding the relative importance of different pathogenic cells and effector molecules. MATERIALS AND METHODS: A variety of gene-targeted mice were examined for the induction of cholinergic airway hyperresponsiveness (AH), allergic airway inflammation, mucus production, and serum IgE reactivity following intratracheal challenge with a potent allergen. AH was determined using whole-body plethysmography following acetylcholine challenge. Where possible, results were confirmed using neutralizing antibodies and cell-specific reconstitution of immune deficient mice. RESULTS: T and B cell-deficient, recombinase-activating-gene-deficient mice (RAG -/-) failed to develop significant allergic inflammation and AH following allergen challenge. Reconstitution of RAG -/- mice with CD4+ T cells alone was sufficient to restore allergen-induced AH, allergic inflammation, and goblet cell hyperplasia, but not IgE reactivity. Sensitized B cell-deficient mice also developed airway hyperreactivity and lung inflammation comparable to that of wild-type animals, confirming that antibodies were dispensable. Treatment with neutralizing anti-IL-4 antibody or sensitization of IL-4-deficient mice resulted in loss of airway hyperreactivity, whereas treatment with anti-IL-5 antibody or sensitization of IL-5-deficient mice had no effect. CONCLUSIONS: In mice, CD4+ T cells are alone sufficient to mediate many of the pathognomonic changes that occur in human asthma by a mechanism dependent upon IL-4, but independent of IL-5, IgE, or both. Clarification of the role played by CD4+ T cells is likely to stimulate important therapeutic advances in treatment of asthma.     

Recombinant IL-3 induces histamine release from human basophils

Human rIL-3 induces histamine release from some human basophils, with cells from atopics responding to a greater extent than non-atopic donors. The dose response curves were highly variable. IL-3 was active on purified basophils and the release process was slower and required more calcium than anti-IgE. Removal of surface IgE from basophils rendered them unresponsive to IL-3. The response could be restored by passive sensitization of basophils with IgE+, IgE known to bind histamine-releasing factors, and not IgE-, IgE unreactive with histamine-releasing factors. Thus, IL-3 uncovers IgE heterogeneity. IL-3 does not, however, directly interact with IgE+. Rather, passive sensitization with IgE+ or stimulation of basophils with low concentrations of several secretagogues renders the cells sensitive to IL-3. IL-3 may well play a pro-inflammatory role by potentiating the effects of IgE+ or various secretagogues.     

Evaluation of a biosensor immunoassay for simultaneous characterization of isotype and binding region of human anti-tocilizumab antibodies with control by surrogate standards

This article describes the simultaneous Biacore analysis of human anti-human antibodies (HAHAs) with respect to the binding region and the isotype by a combination of 11 single measurements per sample. The multiplexing single assay setup made efficient use of the four parallel flow cells on one biosensor chip by immobilization of full-length antibody and its constant (Fc) and antigen binding (Fab) fragments for differential binding analysis of anti-drug antibodies (ADAs). Thereby, a complete time-specific immunogenicity profile (intensity, isotype, specificity, and kinetics) of a patient could be obtained by assessing the response patterns of serially collected samples analyzed in a single measurement run. The use of functionally active standard conjugates allowed control of the assay performance throughout the whole procedure. The positive control standard conjugates mimicking polyclonal human ADAs of different isotypes were obtained by conjugating polyclonal rabbit antibodies against the therapeutic antibody to human immunoglobulin (Ig) M, IgG, or IgE. In this article, the qualification of the assay is demonstrated and the application of the methodology to six representative rheumatoid arthritis patients treated with the therapeutic humanized IgG1 antibody tocilizumab (anti-IL-6R) is shown to illustrate the versatility of the assay. The presented method allows one to differentiate specific ADAs from drug-unspecific responses (e.g., rheumatoid factors). In addition, the method can be used to discriminate between isotype responses of the IgG, IgM, and IgE types and, thereby, allows one to describe the time course of specific ADA formation and its disappearance on the single patient level.     

Costimulation blockade inhibits allergic sensitization but does not affect established allergy in a murine model of grass pollen allergy

Type I allergy is characterized by the development of an initial Th2-dependent allergen-specific IgE response, which is boosted upon a subsequent allergen encounter. Although the immediate symptoms of allergy are mainly IgE-mediated, allergen-specific T cell responses contribute to the late phase as well as to the chronic manifestations of allergy. This study investigates the potential of costimulation blockade with CTLA4Ig and an anti-CD154 mAb for modifying the allergic immune response to the major timothy grass pollen allergen Phl p 5 in a mouse model. BALB/c mice were treated with the costimulation blockers at the time of primary sensitization to the Phl p 5 allergen or at the time of a secondary allergen challenge. Costimulation blockade (CTLA4Ig plus anti-CD154 or anti-CD154 alone) at the time of sensitization prevented the development of allergen-specific IgE, IgM, IgG, and IgA responses compared with untreated but sensitized mice. However, costimulation blockade had no influence on established IgE responses in sensitized mice. Immediate-type reactions as analyzed by a rat basophil leukemia cell mediator release assay were only suppressed by early treatment but not by a costimulation blockade after sensitization. CTLA4Ig given alone failed to suppress both the primary and the secondary allergen-specific Ab responses. Allergen-specific T cell activation was suppressed in mice by early as well as by a late costimulation blockade, suggesting that IgE responses in sensitized mice are independent of T cell help. Our results indicate that T cell suppression alone without active immune regulation or a shifting of the Th2/Th1 balance is not sufficient for the treatment of established IgE responses in an allergy.     

Modulation of ongoing human immunoglobulin synthesis by natural killer cells

Freshly separated human NK cells (NKH-1+) inhibited IgE synthesis from IgE myeloma U266/AF-10 as much as 70% whereas they enhanced IgG and IgA synthesis 200 and 500% from the lymphoblastoid cell lines GM-1500 and GM-1056, respectively. The inhibition of IgE synthesis by NK cells was due to a direct cytolytic effect on AF-10. This could be reversed using K562 cells in a cold target competition assay. NK cells also inhibited spontaneous IgE as well as IgG and IgA synthesis from B cells of highly atopic donors. On the other hand the enhancement of Ig secretion by NKH-1+ cells was shown to be mediated by soluble factors released from NK cells. Furthermore when NK cells were preincubated with immune complexes (IgE-IC) constructed of human IgE and mouse IgG1 monoclonal anti-human IgE, inhibition of IgE synthesis was reversed, and in some cases actual enhancement of IgE synthesis was observed, while enhancement of IgG and IgA synthesis was not affected. In contrast to NK cells, T cells depleted of NK cells (T-NK), when activated by IgE-IC, suppressed IgE synthesis in an isotype specific fashion. Thus, NK and T-cell modulation of ongoing Ig synthesis involve distinct mechanisms.     

Role of IgE receptors in effector function of human eosinophils

After analysis of the technical parameters of the rosette assay with human IgE-coated erythrocytes, Fc epsilon receptors for IgE (Fc epsilon R) on human peripheral blood eosinophils were compared to Fc epsilon R on lymphocytes and monocytes. Antibodies directed against Fc epsilon R on lymphocytes and monocytes inhibited the IgE rosettes formed by eosinophils from hypereosinophilic patients, which suggests that Fc epsilon R on eosinophils were antigenically related to Fc epsilon R on lymphocytes and monocytes. Fc epsilon R on human eosinophils were shown to participate in the killing effect of Schistosoma mansoni schistosomula in vitro in the presence of purified eosinophils from highly hypereosinophilic patients (blood counts greater than 3000/mm3) and anti-schistosomula IgE antibodies present in S. mansoni-infected patient sera. Similar levels of inhibition of cytotoxicity were obtained after preincubation of eosinophils with aggregated human IgE or with anti-Fc epsilon R antibodies, whereas preincubation with aggregated IgG or with anti-C3b receptor antibodies did not decrease the killing effect for schistosomula targets. This IgE-dependent cytotoxic capacity seemed restricted to eosinophils with an abnormally low density ("hypodense" cells) present only in highly hypereosinophilic patients. These observations might be related to nonparasitic situations in which increased levels of IgE and tissue or blood eosinophils are observed.     

Interactions between eosinophils and antibodies: In vivo protective role against rat schistosomiasis

An original protocol of cell transfer from Schistosoma mansoni-infected rats to normal recipient rats is used to investigate the protective role of phagocytic cell populations, described as effector cells in vitro, against a challenge infection with S. mansoni. Nonadherent, eosinophil-enriched and -adherent, macrophage-rich cell preparations, injected via intradermal and subcutaneous routes at the precise site of exposure to cercariae, were able to significantly protect the recipient rats. The time-course study of this protective effect according to the time after infection of donor rats revealed that eosinophils were the major cell population involved in the early phase of infection (4 to 5 weeks), whereas macrophages could also be incriminated thereafter. A rosette assay using anti-immunoglobulin-coated erythrocytes indicated a sequence of the various antibody isotypes under study (IgG₁, IgG_2a, IgE) on the eosinophil surface, during the course of infection. As previously shown in vitro, cytophilic antibodies seemed to participate in the protective effect of eosinophils, since eosinophil-enriched cells from normal rats, sensitized in vitro with immune complexes present in infected rat serum, could also confer significant protection. These observations establish therefore the relevance between our previous in vitro studies and rat resistance to a challenge infection with S. mansoni, underlining the major role played by the interaction between antibodies and phagocytic cells (eosinophils and macrophages).     

Existence and function of a receptor for immunoglobulin A on human eosinophils

The existence of receptors for immunoglobulin A on human eosinophils is demonstrated by flow cytofluorometry. Between 5 and 60% of eosinophils purified from peripheral blood of hypereosinophilic patients are able to bind monomeric serum IgA. The addition of antihuman IgA antibodies to surface IgA-bearing eosinophils induces the exocytosis of peroxidase contained in the granules suggesting a cell activation due to IgA receptors. The inhibition of antiparasitic cytotoxicity by eosinophils preincubated with IgA under a polymeric form only, indicates the low affinity of IgA receptors as well as their participation in the effector function of eosinophils.     

IL-4-dependent IgE switch in membrane IgA-positive human B cells

IgE responses by human B cells, separated according to membrane Ig classes, were analyzed in a clonal assay using EL-4 thymoma cells as helper cells, T cell supernatant, and rIL-4. In cultures seeded by means of the autoclone apparatus of the FACS, IgE responses were generated frequently by either IgM (mu+/gamma-alpha-) or IgA (alpha +/mu-)-positive B cells (16 and 14% of the Ig producing wells, respectively), but rarely by IgG (gamma +/mu-)-positive B cells (1.3% of Ig producing wells). The total amounts of Ig secreted by IgM-, IgG-, or IgA-positive cells and the total proportions of responding autoclone wells (23-27%) were comparable. All IgE secretion was IL-4 dependent. When the Ig secretion patterns from alpha +/mu- vs alpha +/mu-epsilon- B cells were compared, most autoclone wells from both types of cells produced IgA only, and similar proportions of IgA producing wells (6.2 and 6.0%) also secreted IgE. In addition, IgE restricted responses occurred 6 times more frequently with alpha +/mu- than with alpha +/mu-epsilon- cells, which suggests that membrane IgA+E double-positive, IgE committed B cells occur in vivo. The isotype pattern generated by alpha +/mu-epsilon- B cells cannot be explained by a chance assortment of separate IgA and IgE precursors or by cytophilic antibody. Thus, IL-4 dependent switch to IgE occurred frequently in IgM- or IgA-positive, but rarely among total IgG-positive, B cells. This could be relevant to IgE production in mucosal tissues rich in IgA expressing B cells.     

Tyrosine phosphorylation is required for mast cell activation by Fc epsilon RI cross-linking.

We investigated the possible role of tyrosine phosphorylation in the activation process of mast cells by cross-linking of cell-bound IgE antibodies. Bone marrow-derived mouse mast cells (BMMC) were sensitized with mouse IgE antiDNP mAb and then challenged with multivalent Ag DNP conjugates of human serum albumin. Analysis of phosphotyrosine-containing proteins in their lysates by SDS-PAGE and immunoblotting revealed that cross-linking of cell-bound IgE antibodies induced a marked increase in tyrosine phosphorylation of several proteins. To obtain direct evidence for activation of protein-tyrosine kinases (PTK), phosphotyrosine-containing proteins in lysates of mast cells were affinity purified, and kinase activity of the immunoprecipitates was assessed by an in vitro kinase assay. The results clearly showed activation of PTK upon cross-linking of Fc epsilon RI. Activation of PTK was not detected by the same assay when the sensitized BMMC were challenged with monovalent DNP-lysine. Treatment of sensitized BMMC with either Ca2+ ionophore or PMA failed to induce the activation of PTK. A representative IgE-independent secretagogue, thrombin, induced histamine release from BMMC but failed to induce activation of PTK. The results excluded the possibility that PTK activation is the consequence of an increase in intracellular Ca2+ or activation of protein kinase C. Addition of genistein, a PTK inhibitor, to sensitized BMMC before Ag challenge inhibited not only Ag-induced PTK activation, but also inositol 1,4,5-trisphosphate production, and histamine release in a similar dose-response relationship. Other PTK inhibitors, such as lavendustin A and tyrphostin RG50864, also inhibited the Ag-induced activation of PTK and histamine release. The results collectively suggest that activation of PTK is an early event upstream of the activation of phospholipase C, and is involved in transduction of IgE-dependent triggering signals to mediator release.     

A role for IgE in intestinal immunity. Expression of rapid expulsion of Trichinella spiralis in rats transfused with IgE and thoracic duct lymphocytes

In these experiments we characterize the protective antibodies in immune serum that interact synergistically with immune thoracic duct lymphocytes (TDL) to induce rapid expulsion (RE) of Trichinella spiralis in adult rats. Antibodies with both reaginic and nonreaginic activity mediated RE upon passive transfer to adult rats that had been adoptively transfused with immune TDL 7 days earlier. In serum collected 28 days after a primary infection, the most important antibody was homocytotropic IgE. Native IgE produced by active infection was isolated from 28-day immune serum by salt precipitation and/or by sequential affinity chromatography. The murine mAb A2 and B5 (anti-rat IgE) were conjugated separately to Sepharose 4B affinity columns for affinity separations. IgE was shown to be pure by gel electrophoresis and Western blots and its m.w. was estimated at approximately 190,000. As little as 183 micrograms of purified IgE could induce RE after passive transfer to adult rats. The IgE was shown to be functional by PCA activity, Ag-binding on Western blots, and skin sensitization; the latter could be blocked by pretreatment with 1R162, a rat myeloma IgE. Monoclonal IgG of any isotype transferred in amounts up to 35 mg/rat could not transfer RE to rats previously transfused with TDL cells. Immune serum collected 3 mo after the primary infection contained insufficient IgE to transfer RE, but complex non-IgE fractions were protective. The data thus demonstrate that IgE is a functional Ig in the rat capable of mediating the rejection of challenge nematode infections of the gut in the absence of other specific Ig. Secondly, other Ig may also play a role, in particular, several weeks after the primary infection when specific IgE levels in serum have declined.     

Eosinophils Are Important for Protection,Immunoregulation and Pathology during Infection with Nematode Microfilariae

Eosinophil responses typify both allergic and parasitic helminth disease. In helminthic disease, the role of eosinophils can be both protective in immune responses and destructive in pathological responses. To investigate whether eosinophils are involved in both protection and pathology during filarial nematode infection, we explored the role of eosinophils and their granule proteins, eosinophil peroxidase (EPO) and major basic protein-1 (MBP-1), during infection with Brugia malayi microfilariae. Using eosinophil-deficient mice (PHIL), we further clarify the role of eosinophils in clearance of microfilariae during primary, but not challenge infection in vivo. Deletion of EPO or MBP-1 alone was insufficient to abrogate parasite clearance suggesting that either these molecules are redundant or eosinophils act indirectly in parasite clearance via augmentation of other protective responses. Absence of eosinophils increased mast cell recruitment, but not other cell types, into the broncho-alveolar lavage fluid during challenge infection. In addition absence of eosinophils or EPO alone, augmented parasite-induced IgE responses, as measured by ELISA, demonstrating that eosinophils are involved in regulation of IgE. Whole body plethysmography indicated that nematode-induced changes in airway physiology were reduced in challenge infection in the absence of eosinophils and also during primary infection in the absence of EPO alone. However lack of eosinophils or MBP-1 actually increased goblet cell mucus production. We did not find any major differences in cytokine responses in the absence of eosinophils, EPO or MBP-1. These results reveal that eosinophils actively participate in regulation of IgE and goblet cell mucus production via granule secretion during nematode-induced pathology and highlight their importance both as effector cells, as damage-inducing cells and as supervisory cells that shape both innate and adaptive immunity.     

Regulation of eosinophil function by phosphatidylinositol-specific PLC and cytosolic PLA(2)

We examined the regulatory role of cytosolic phospholipase A(2) (cPLA(2)) and phosphatidylinositol (PI)-specific phospholipase C (PLC) in the degranulation of human eosinophils and leukotriene (LT) C(4) synthesis. Activation with formyl-Met-Leu-Phe + cytochalasin B (fMLP/B) caused a time-dependent release of eosinophil peroxidase (EPO) and LTC(4), which was inhibited by pertussis toxin. By immunoblotting, eosinophil PLC-beta2 and -gamma2 isoforms were identified, and PLC activation was measured as a function of inositol 1,4,5-trisphosphate concentration. Stimulated release of EPO and intracellular Ca(2+) concentration was inhibited by ET-18-OCH(3), a PI-PLC inhibitor, whereas trifluoromethylketone (TFMK), a cPLA(2) blocker, had no inhibitory effect. Both TFMK and ET-18-OCH(3) attenuated stimulated arachidonate release and LTC(4) secretion, suggesting that activation of both PLC and cPLA(2) is essential for LTC(4) synthesis caused by fMLP/B. The structurally unrelated protein kinase C inhibitors bisindolylmaleimide, Ro-31-8220, and Go-6976 all blocked fMLP/B-induced EPO release but not LTC(4) secretion. 1,2-bis(2-Aminophenoxy)ethane-N,N,N',N'- tetraacetic acid acetoxymethyl ester, an intracellular Ca(2+) chelator, suppressed both EPO release and LTC(4) secretion. We found that fMLP/B-induced LTC(4) secretion from human eosinophils is regulated by PI-PLC through calcium-mediated activation of cPLA(2). However, cPLA(2) does not regulate eosinophil degranulation.