Nonatopic Donors Research Articles

IgE+ cells in the peripheral blood of atopic, nonatopic, and bee venom–hypersensitive individuals exhibit the phenotype of highly differentiated B cells

We have analyzed IgE+ cells in peripheral blood of atopic donors, donors hypersensitive to bee venom, and nonatopic control donors with two- and three-color flow cytometry. Although the percentage of IgE + cells varied among these groups, the overall phenotypic patterns were similar. Most IgE+ cells do not display typical B-cell markers, such as CD19, CD20, and CD21. A significant proportion of these cells stain for CD38, indicating that they are more differentiated. IgE+ cells express FcγRII and CD45RO, an isoform associated with an advanced level of differentiation. The majority of IgE+ cells do not coexpress other surface immunoglobulin isotypes. In the case of bee venom–hypersensitive donors, we have been able to identify a small population of IgE+ cells with a specificity for phospholipase A2, a major immunogenic component of bee venom. The phospholipase A2 + cells display a phenotype similar to that of the IgE+ cells. (J ALLERGY C LIN IMMUNOL 1995;95:587-96.)

High affinity IgE receptor-mediated prostaglandin E2 production by monocytes in atopic dermatitis.

High affinity IgE receptor (Fc epsilon R I) expression on monocytes and its upregulation on monocytes from patients with atopic dermatitis (AD) have been recently reported. In this study, we investigated whether prostaglandin E2 (PGE2) release from AD monocytes was Fc epsilon R I-dependent or not. The monocytes were stimulated with anti-Fc epsilon R I monoclonal antibody (mAb) and anti-Fc epsilon R II mAb. Cross-linking of Fc epsilon R I, but not that of Fc epsilon R II induced PGE2 release from monocytes. In order to confirm that the PGE2 release is IgE-dependent, stimulation with IgE+anti-IgE, IgG+anti-IgG and immune complexes made by incubation with AD patients' serum and recombinant Der fII (rDer fII-IC) were carried out in the culture. Significant PGE2 release by all the stimulants was observed, as compared with spontaneous release. PGE2 release by immune complex made by incubation with IgE-absorbed AD serum and rDer fII was much lower than that by rDer fII-IC. In monocytes from nonatopic donors, significant PGE2 release was observed when stimulated with IgE+anti-IgE, IgG+anti-IgG, rDer fII-IC, but not with anti-FC epsilon R I mAb and anti-Fc epsilon R II mAb. However, the release were significantly lower than respective PGE2 releases from AD monocytes. Taken together, cross-linking of Fc epsilon R I and IgE induced a PGE2 release from monocytes. This is a first report demonstrating an Fc epsilon R I-dependent mediator release from monocytes of AD patients.

Regulation of integrin-dependent release in human lung mast cells and basophils.

The interaction of cells with surfaces or components of the extracellular matrix alters cell responses and is regulated by integrins on the cell surface. We have used monoclonal antibodies to CD29 and CD49d followed by an F(ab)2 fragment of rabbit anti-mouse IgG1 to cross-link the integrins on the surface of human lung mast cells and basophils. We found that cross-linking either CD29 or CD49d failed to initiate mediator release from the basophils of non-atopic and atopic donors [histamine release (HR) = 1 +/- 0.5% for CD29 and 1 +/- 0.5% for CD49d, n = 10, NS]. In contrast we found that clustering CD29 caused significant HR from the basophils of asthmatic donors (HR = 21 +/- 5%, n = 10, p < 0.005). Clustering of CD49d also caused significant degranulation in the same donors (HR = 9 +/- 3%, n = 10, p < 0.11). Incubating the basophils of these asthmatic donors with a synthetic RGD peptide significantly reduced CD29- and CD49d-induced histamine release. CS-1 peptide was also found to inhibit CD29-induced histamine release but had no significant effect on CD49d-induced histamine release. The tyrosine kinase inhibitors, genistein and piceatannol, completely ablated CD29- and CD49d-induced degranulation. In summary, we have shown that cross-linking integrins can initiate mediator release from the basophils of asthmatic patients and that this appears to involve recognition of RGD and activation of tyrosine kinase.

House Dust Mite-Responsive Human T Cells Require both Interleukin 2 (IL2) and Interleukin 4 for Optimal Proliferation, Whereas IL2 Alone Is Sufficient for Proliferation of Tetanus Toxoid-Responsive T Cells

Production of interleukin 4 (IL4) and response to IL4 may be a key determinant of the atopic immune response. Proliferation requirements of allergen-specific T cells derived from atopics and nonatopics were determined. Peripheral blood lymphocytes were cultured with either house dust mite (HDM) or tetanus toxoid under limiting dilution conditions with interleukin 2 (IL2) alone or IL2 + IL4. The relative proportion of antigen-specific T cells responding under each of these conditions was determined by proliferation. The combination of IL2 + IL4 resulted in the highest proportion of responding HDM-specific T cells. Similar results were obtained with HDM-specific T cells from both atopic and nonatopic donors. These HDM and Der pI responsive cells were found to be CD4 +CD8 -. In contrast, tetanus toxoid-responsive T cells from both atopic and nonatopic donors grew preferentially in response to IL2 alone.

IgE-dependent IL-4 secretion by human basophils. The relationship between cytokine production and histamine release in mixed leukocyte cultures.

IL-4 protein and mRNA have recently been detected in pure basophil cultures after stimulation. It has also been established in mixed leukocyte cultures obtained by elutriation and challenged with anti-IgE that the basophil is the only cell that makes detectable IL-4. We have used cells prepared using Percoll gradients (5 to 30% basophils) to study the relationship between histamine release and IL-4 synthesis. In cultures challenged with anti-IgE after a 15-min pretreatment with IL-3, detectable IL-4 secretion ranged from 40 to 630 pg/10(6) basophils in 18 of 22 donors, with no significant correlation (r = 0.21, p = 0.34) with basophil purity. IL-4 synthesis was dissociated from histamine release, with optimal production consistently occurring at 10 ng/ml anti-IgE, whereas histamine levels peaked at 50 to 100 ng/ml of stimulus. Stimulation with anti-IgE alone was sufficient for IL-4 secretion but protein levels were enhanced two- to threefold in low responders by increasing the calcium concentration to 5 mM with no significant changes in histamine release. The IgE-independent secretagogues, F-met peptide (1 microM) and C5a (25 ng/ml), demonstrated limited ability to generate detectable IL-4, despite promoting vigorous histamine release. Additional studies showed no significant difference between IL-4 secretion in atopic and nonatopic donors. Finally, IL-4 levels generated with specific Ags were comparable to those levels produced in response to anti-IgE. We predict that basophil IL-4 generation will have a proinflammatory effect, but it appears that the signal transduction mechanisms for its synthesis and release differ from those for histamine release and thus may require different methods of pharmacologic control.

The Chemokine RANTES Is More than a Chemoattractant: Characterization of Its Effect on Human Eosinophil Oxidative Metabolism and Morphology in Comparison with IL-5 and GM-CSF

Eosinophils were shown to play a major role in the allergic inflammatory process leading to the clinical symptoms of atopic dermatitis. Only selected cytokines are capable of inducing a chemotactic response in eosinophils. In particular, the chemokine RANTES was recently shown to be a potent eosinophil chemotaxin. To examine the role of RANTES in eosinophil activation, we investigated the effect of RANTES and other chemokines on morphology and oxidative metabolism of highly purified eosinophils of normal nonatopic blood donors by assessment of functional as well as morphologic criteria. RANTES, and, to a lesser extent, MIP-1 alpha significantly induced the production of reactive oxygen species by human eosinophils, whereas MCP-1, MIP-1 beta, and interleukin (IL)-8/NAP-1 had no significant effects. RANTES stimulated only a subpopulation of the normal eosinophils. With the exception of IL-8, none of the cytokines tested had any significant effect on polymorphonuclear neutrophilic granulocytes. By scanning electron microscopy, RANTES induced characteristic changes that were completely abrogated in the presence of cytochalasin B. Based on functional and ultrastructural assays significant extracellular but not intracellular H2O2 production was detected and completely inhibited by cytochalasin B. Separation of eosinophils by discontinuous density gradients revealed the existence of two hypodense eosinophil populations, one which showed significantly reduced responses upon stimulation with RANTES. RANTES-induced production of reactive oxygen species was almost completely inhibited by staurosporine, wortmannin, or pertussis toxin. Based on these data it is evident that RANTES represents a potent eosinophil-specific activator of oxidative metabolism. Besides its chemotactic activity on T cells and eosinophils, therefore, RANTES may be involved in the functional activation of eosinophils in the skin of patients with atopic dermatitis.

Enhancement of IgE production by anti-CD40 antibody in atopic dermatitis

It has been recently recognized that the obligate requirement for T cells in the development of IgE responses can be substituted for by anti-CD40 antibody. In this study of patients with atopic dermatitis and high IgE levels, we have analyzed the role of the CD40 molecule in IgE production. Costimulation of peripheral blood mononuclear cells (PBMCs) from normal donors with interleukin-4 (IL-4) and anti-CD40 monoclonal antibody resulted in a selective increase in IgE production; either reagent alone, however, was ineffective. In contrast, addition of anti-CD40 monoclonal antibody alone to PBMCs or B cells from patients with atopic dermatitis markedly increased IgE production, even in the absence of exogenous IL-4. With the use of an ELISA spot assay, this increase in IgE production was attributed to an expansion of IgE-secreting B cells. In anti-IgM–stimulated lymphocyte cultures from patients with atopic dermatitis the costimulation with anti-CD40 induced strong lymphocyte proliferation. Similar results were observed with anti-IgM plus IL-4. The augmentation induced by anti-CD40 was inhibited by addition of anti-IL4 to anti-CD40–treated atopic dermatitis cells. In normal subjects the effects of anti-CD40 alone on IgE production could be observed after pretreatment of normal PBMCs with IL-4 for 3 days. The effects of anti-CD40 in atopic dermatitis may be explained in part by differences in CD40 expression. In freshly isolated PBMCs from patients with atopic dermatitis, the mean fluorescence intensity of CD40 expression on B cells was increased when compared with PBMCs from nonatopic donors, and stimulation of normal or atopic dermatitis PBMCs with IL-4 increased the intensity of CD40 staining of cells. (J A LLERGY C LIN I MMUNOL 1994;93:658-68.)

Granulocyte-macrophage colony-stimulating factor stimulates macrophages to respond to IgE via the low affinity IgE receptor (CD23)

We have found increased concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the bronchoalveolar lavage fluid of 11 patients with nocturnal asthma (15.3 ± 4.6 pg/ml) compared with normal subjects (2.3 ± 6.1 pg/ml) ( p = 0.03). In contrast to patients with asthma, low affinity IgE receptors (FcϵRII or CD23) are not expressed on monocytes obtained from healthy, nonatopic donors. FcϵRII expression was induced by the cytokines GM-CSF and interleukin (IL)-4 either alone or in combination. As assessed by flow cytometry, the combination of IL-4 and GM-CSF was found to be synergistic, inducing up to 54.8% ± 4.6% FcϵRII-positive monocytes compared with a maximum of 27.4% ± 5.0% and 30.0% ± 4.0% with IL-4 and GM-CSF alone, respectively ( p < 0.05 compared with either cytokine alone). Human monocytes from the peripheral blood of seven normal subjects were cultured for 24 hours with and without IL-4 or GM-CSF. With IL-4, addition of IgE/anti-IgE complexes failed to induce IL-1 secretion and inhibited IL-1 secretion induced by lipopolysaccharides. The addition of GM-CSF or IgE immune complexes alone resulted in no additional IL-1 secretion in supernatants of the untreated monocytes, whereas the IgE complexes did stimulate IL-1 secretion by monocytes cultured in GM-CSF, as measured by ELISA (from 0.7 ± 0.2 ng/ml to 2.3 ± 0.5 ng/ml; p < 0.01). This could be confirmed to represent an FcϵRII-dependent process insofar as blocking of the FcϵRII receptors abrogated the capacity of the IgE complexes to induce IL-1β (1.0 ± 0.4 ng/ml IL-1; p = NS in comparison with untreated monocytes). We conclude that GM-CSF is increased in the bronchoalveolar lavage fluid of patients with asthma and may be important in macrophage activation via induction of the low affinity IgE receptor, thereby making cells susceptible to IgE-dependent activation. (J A LLERGY C LIN I MMUNOL 1994;93:650-7.)

Role of interleukin-4 in human immunoglobulin E formation in hu-PBL-SCID mice.

We studied the role of IL-4 in human IgE formation in severe combined immunodeficient mice engrafted with peripheral blood mononuclear leukocytes (hu-PBL-SCID). PBL from four nonatopic donors produced only small (< 20 ng/ml) or undetectable amounts of IgE in SCID mice whereas engrafted PBL from seven atopic donors secreted IgE with IgE serum levels reaching a mean +/- SE of 184 +/- 37 ng/ml (n = 20). Serum IgE levels peaked 2-3 wk after PBL transfer and declined thereafter with a half-life of 1-2 wk. In contrast, IgG of all subclasses reached maximum serum levels 5-7 wk after PBL transfer and declined little thereafter. Injection of a neutralizing monoclonal antibody to the human IL-4 receptor (IL-4R) on day 0 inhibited completely the IgE formation and caused an approximate twofold reduction of IgG production of all subclasses. The anti-IL-4 R antibody had no effect on IgE secretion when administered 4 wk after PBL engraftment. Incubation of PBL with IL-4 before engraftment resulted in a 10-fold increase in IgE production and could be further enhanced by 100 fold if, in addition to preincubation with IL-4, IL-4 was injected daily for 5 d after PBL transfer. This treatment with IL-4 also induced two- to threefold increase in IgG levels. IFN-gamma had no effect on either IgE or IgG subclass production. In approximately 50% of the mice, one or more IgG subclasses increased disproportionally 5 wk after PBL injection as a result of monoclonal IgG formation. These data demonstrate that PBL from atopic donors secrete IgE in SCID mice in an IL-4-dependent manner, and that IgE production can be enhanced 10- to 100-fold with exogenous human IL-4 in these mice. This mouse model is amenable for the in vivo study of immunomodulators on human IgE formation.

Activation of human blood lymphocytes by house dust mite protein and Mycobacterium tuberculosis purified protein derivative: effects of interferon-gamma, interleukin-4, dexamethasone and cyclosporin A.

Peripheral blood mononuclear cells (PBMC) from nonatopic donors sensitive to Mycobacterium tuberculosis purified protein derivative (PPD) and from atopic donors sensitive to PPD and house dust mite antigen (HDM), were stimulated in vitro to proliferate in response to exogenous IL-2. In the presence of exogenous IFN-gamma, the response of cells from atopic donors to PPD was either unaffected or slightly enhanced, whereas the response to HDM was inhibited in a dose-dependent manner. The response of cells from nonatopic donors to PPD remained unchanged or was only slightly inhibited and this was not dose-dependent. Exogenous IL-4 did not reverse the inhibitory effects of IFN-gamma and a neutralizing antibody to IL-4 did not inhibit the proliferative response of cells to HDM and IL-2. Dexamethasone inhibited the IL-2-mediated response of cells from atopic donors stimulated with HDM, whereas the IL-2-mediated response of cells induced by PPD was either unchanged or stimulated by dexamethasone. Cyclosporin A inhibited the response of cells to both HDM and PPD. These results suggest that IFN-gamma can exert a selective effect on the response of T lymphocytes to different antigens and that it is possible to identify compounds able to regulate this activity.

Nedocromil sodium selectively inhibits IgE and IgG4 production in human B cells stimulated with IL-4.

We studied the effect of nedocromil sodium (NES) on human IgE and IgG subclass production. NES inhibited IgE and IgG4 production, without affecting IgM, IgA, IgG1, IgG2, or IgG3 production, by IL-4-stimulated mononuclear cells (MNC) obtained from non-atopic donors. Inhibition of IgE and IgG4 production by NES required the compound to be added on day 0 and to be present throughout the entire period of culture. Inhibition of IgE and IgG4 production was not mediated by known inhibitors, including IFN-alpha, IFN-gamma, transforming growth factor-beta, PGE2, or platelet-activating factor. Inhibition by NES required both T cells and monocytes because NES inhibited IgE production by B cells stimulated with IL-4 and anti-CD40 mAb in the presence, but not the absence, of T cells and monocytes. NES also inhibited IgE and IgG4 production in cultures stimulated with anti-CD3-treated T cells. However, in this case other isotypes were also inhibited by NES. In contrast to non-atopic donors' MNC, NES failed to inhibit spontaneous IgE and IgG4 production by MNC from atopic patients. However, NES was effective in inhibiting IL-4-induced IgE and IgG4 production in atopic patients' MNC without affecting IgM, IgA, IgG1, IgG2, or IgG3 production. NES inhibited IgE and IgG4 production by sIgE- and sIgG4-B cells, but not by sIgE+ and sIgG4+ B cells, respectively, stimulated with IL-4 or IL-4 plus anti-CD40 mAb in the presence of T cells and monocytes. These results suggest that NES may inhibit IL-4-induced switching of IgE and IgG4 production by sIgE- B cells and sIgG4- B cells, respectively, by affecting the interaction of B cells, T cells, and monocytes.

IL-10 and viral IL-10 prevent IL-4-induced IgE synthesis by inhibiting the accessory cell function of monocytes.

In the present study it is demonstrated that IL-10 and viral (v)-IL-10 inhibit IL-4-induced IgG4 and IgE synthesis in cultures of PBMC from healthy nonatopic donors. The inhibition occurred at the mRNA level. IL-10 strongly reduced IL-4-induced expression of both germline and productive epsilon transcripts in PBMC. The inhibitory effects were completely neutralized, or IgG4 and IgE synthesis was even enhanced, by anti-IL-10 mAb, demonstrating the specificity of the reaction. IL-4-induced IgG4 and IgE synthesis by PBMC was monocyte dependent. IL-4 failed to induce IgG4 or IgE synthesis by monocyte-depleted PBMC, but the production of these isotypes was completely restored by reconstitution with autologous monocytes. However, monocytes preincubated with IL-10 for 24 h failed to provide the accessory signals required for IL-4-induced IgG4 and IgE synthesis, indicating that the inhibitory effects of IL-10 are indirectly mediated via monocytes. This notion was further supported by the finding that IL-10 and v-IL-10 failed to inhibit IL-4-induced IgE synthesis in the absence of monocytes, i.e., when highly purified B cells were cultured in the presence of anti-CD40 mAb or cloned activated CD4+ T cells. Moreover, IL-10 failed to inhibit IL-4-induced germline epsilon mRNA synthesis in highly purified B cells. The inhibitory effects of IL-10 could not be restored by exogenous TNF-alpha or IL-6, indicating that the inhibitory effects were not mediated through inhibition of production of these cytokines. This is compatible with the observation that monocytes preincubated with IL-10 did not inhibit IgG4 or IgE synthesis in a monocyte- and T cell-independent culture system, in which purified B cells were cultured in the presence of anti-CD40 mAb and IL-4. Collectively, these data indicate that IL-10 mediates a potent, monocyte-dependent, inhibitory effect on IL-4-induced IgG4 and IgE production by human PBMC.

Seasonal enhancement of IL‐4 induced IgE synthesis by peripheral blood mononuclear cells of atopic patients

The role of IL-4 on IgE synthesis has been well established. IL-4 has been shown to promote IgE production by B cells from atopic and non-atopic donors. In this study, the effects of natural exposure to pollens on IL-4-induced IgE synthesis by peripheral blood mononuclear cells of atopic and non-atopic donors were examined. The results confirm production of IgE in an IL-4 dose-dependent manner by PBMC cultures of these two groups. When cultures were performed out of the pollen season, following stimulation by IL-4, no significant differences was observed between the levels of IgE produced by PBMC of atopic and non-atopic donors. In contrast, upon natural exposure to pollens, significant higher levels of IgE were measured in the atopic group than in the non-atopic one. These results show that the pollen season influences the IL-4-induced IgE synthesis by PBMC of allergic patients and are in keeping with seasonal rise of specific IgE antibodies.

Pokeweed mitogen induces IgE synthesis in the presence of a blocking antibody to the interferon-γ receptor

Background: Although pokeweed mitogen (PWEM) can induce peripheral blood mononuclear cells (PBMCs) to synthesize IgG, IgA, and IgM, such cultures fail to induce IgE synthesis. The present study examined the possibility that the stimulation of interferon-γ (IFN-γ) production plays a role in the failure of PWM to induce IgE synthesis. Methods: We examined PBMCs from eight normal control subjects for IFN-γ and IL-4 production. Since IFN-γ synthesis is known to inhibit IgE synthesis, we also examined the effect of a neutralizing anti-IFN-γ antibody and of two anti-IFN-γ receptor antibodies, monoclonal antibody (mAb) GIR208, which blocks cellular binding of IFN-γ, and mAB GIR94.5, which binds to the IFN-γ receptor but does not block the binding of IFN-γ to its receptor on PWM-stimulated PBMCs. Results: After stimulation with PWM, culture supernatants contained significantly more IFN-γ ( p = 0.001) and IL-4 ( P = 0.001) compared with supernatants from nonstimulated cultures. PWM-stimulated PBMCs also expressed higher levels of IFN-γ and IL-4 gene transcripts than unstimulated cells. When cultured in the presence of anti-IFN-γ, superantants from PWM-stimulated cultures also induced CD23 on Ramos B cells in an IL-4-dependent manner. In the presence of mAB GIR208 and a neutralizing anti-IFN-γ antibody, but not mAb GIR94.5, PWM stimulated PBMCs from eight normal control subjects and six patients with atopic dermatitis to produce IgE. Monoclonal antibody GIR208, however, did not enhance IgG synthesis. Furthermore, the exogenous addition of IFN-γ inhibited the IgE-stimulatory effect of mAb GIR208. Monoclonal antibody GIR208 was unable to induce purified B cells to synthesize IgE in the presence of IL-4. Conclusions: Thus unlike anti-CD40, mAb GIR208 does not act as a second signal for the induction of IgE synthesis. These results demonstrate that the induction of IFN-γ production contributes to the failure of PWM to stimulate synthesis of IgE in PBMCs from atopic and nonatopic donors.

Lol p I-induced IL-4 and IFN-γ production by peripheral blood mononuclear cells of atopic and nonatopic subjects during and out of the pollen season

The reciprocal effects of IL-4 and IFN-γ on IgE synthesis have been well established. It has also been shown that these two lymphokines are secreted by different subsets of CD4 + T cells (T H1 and T H2), and that T H2 helper T lymphocytes could be involved in the pathophysiology of allergic diseases. But little is known about the effects of an allergen on the profile of lymphokine synthesis by human peripheral blood mononuclear cells (PBMCs) of allergic and nonallergic subjects. We studied the production of IL-4 and IFN-γ by PBMCs of atopic and nonatopic donors after in vitro stimulation by the group 1 allergen from Lolium perenne pollen ( Lol p I), during and out of the grass pollen season. On natural exposure to pollen, Lol p I-induced IL-4 production was observed only with atopic donors (6 of 8), whereas the synthesis of IFN-γ was observed for all nonatopic donors (7 of 7) and most allergic patients (5 of 7). At the time of the study, higher amounts of IFN-γ were produced by PBMCs of nonatopic donors than by PBMCs of atopic patients. Out of the pollen season the production of IL-4 was not observed either by atopic ( n = 11) or by nonatopic subjects ( n = 5). On the other hand, IFN-γ was produced by PBMCs of most subjects (atopic, 10 of 11; nonatopic, 5 of 5), but at the time of the study no difference was observed between the two groups. These results show that Lol p I induces different profiles of IL-4 and IFN-γ production by PBMCs of atopic and nonatopic subjects. In atopic subjects this profile of lymphokine synthesis is influenced by the natural exposure to pollen, which is in keeping with the seasonal rise of IgE antibodies.

Differential effect of vasoactive intestinal peptide, somatostatin, and substance P on human IgE and IgG subclass production

We studied the effect of vasoactive intestinal peptide (VIP), somatostatin (SOM), and substance P (SP) on IL-4-stimulated human IgE and IgG subclass production. VIP and SOM, but not SP, inhibited IgE production without affecting IgM or IgA production by mononuclear cells (MNC) from nonatopic donors from 10 pM to 10 n M. These neuropeptides also differentially modulated IgG subclass production. While IgG1 production was not affected by VIP, SOM, or SP, all of the neuropeptides enhanced IgG2 production. By contrast, SOM and SP, but not VIP, inhibited IgG3 production, whereas VIP and SP, but not SOM, enhanced IgG4 production. The effect by neuropeptides was specific since each peptide effect was specifically blocked by each antagonist. To achieve this effect, neuropeptides must be added at the start of the culture and be present throughout the entire culture period. The inhibition of IgE production was not mediated by known inhibitors of IgE production, IFN-γ or PGE 2, because the addition of anti-IFN-γ mAb (10 μg/ml) or indomethacin (0.1 μ M) did not overcome the inhibition of IgE production. In contrast to MNC, neuropeptides did not affect IgG subclass production in purified B cells. IgE production was not induced by IL-4 in purified B cells. Neuropeptides also failed to modulate IgG subclass production in cultures of B cells with either T cells or monocytes. However, they modulated IgE production and IgG subclass production in B cells in the presence of T cells and monocytes. In purified B cells, IL-4 plus anti-CD40 mAb induced IgE production which was not inhibited by VIP or SOM. However, VIP or SOM, but not SP, inhibited IgE production in B cells cultured with both T cells and monocytes. Finally, the mechanism of modulation of IgE and IgG4 production was dependent on IL-4-induced switching, since neuropeptides modulated IgG4 and IgE production in surface IgG4-negative (sIgG4 −) and sIgE − B cells, respectively. In contrast, modulation of IgG2 and IgG3 production was not due to switching, since neuropeptides did not affect either IgG2 or IgG3 production in sIgG2 − or sIgG3 − B cells, respectively.

Decreased interferon gamma and increased interleukin-4 production in atopic dermatitis promotes IgE synthesis

The mechanism(s) responsible for increased IgE synthesis in atopic dermatitis (AD) are unknown, but they may be related to either decreased interferon gamma (IFN-gamma) and/or increased interleukin (IL)-4 production. In this study we examined peripheral blood mononuclear cells (PBMCs) from 21 patients with AD, six patients with psoriasis, and 22 nonatopic healthy controls for IFN-gamma and IL-4 production after stimulation with concanavalin A (Con A). The Con A-induced proliferative response of AD PBMCs was similar to the response of healthy controls (p = 0.9). After mitogen stimulation, however, AD culture supernatants contained significantly less IFN-gamma (p = 0.001) but increased IL-4 (p = 0.001) compared with supernatants from nonatopic controls. In contrast, PBMCs from patients with psoriasis produced normal levels of IFN-gamma and IL-4 in vitro. Since IL-4 is known to decrease IFN-gamma synthesis, we examined the effect of neutralizing anti-IL-4 on IFN-gamma production. Anti-IL-4 significantly increased IFN-gamma production in patients with AD (p = 0.008) and nonatopic controls (p = 0.02) but did not normalize IFN-gamma production by AD PBMCs. Supernatants from AD PBMCs, but not supernatants from nonatopic PBMCs, induced IgE synthesis in PBMCs from nonatopic donors (p = 0.02). When an anti-IFN-gamma receptor antibody, which blocks cellular binding of IFN-gamma, was added to supernatants from nonatopic controls their capacity to induce IgE synthesis was significantly greater (p = 0.03). These results demonstrate an imbalance of IL-4 and IFN-gamma production, which may contribute to increased IgE synthesis in AD.

Skin-derived aeroallergen-specific T-cell clones of Th2 phenotype in patients with atopic dermatitis

T-cell lines have been derived from aeroallergen-induced eczematous patch test sites of patients with atopic dermatitis (AD). Biopsy specimens were obtained 24 hours after allergen application to the skin, and only in vivo-activated T cells were propagated. From one patient, the T-cell lines were subcloned at 0.3 cells per well. With allergen-induced interleukin (IL) production, all clones tested (n = 13) were found to be specific for the allergen, producing IL-4 and granulocyte-macrophage-colony-stimulating factor. No IL-2 or interferon-γ was found. The allergen-specific proliferative response of these clones, although the response was dependent on exogenous IL-2 or IL-4, also proved that all clones were allergen specific. Under optimal stimulation (aCD3 plus phorbol myristate acetate), 15% of the clones appeared to be of Th0 phenotype and 70% of Th2 phenotype. In 15% of the clones, IL-4 was produced in the absence of IL-2, IL-5, or interferon-γ. Supernatants of all clones tested induced IgE production by B cells from normal non-atopic donors. The T-cell lines of the other patient demonstrated similar results; allergen-specific proliferation was dependent on exogenous IL-2 or IL-4 and stimulation with aCD3 plus phorbol myristate acetate demonstrated that the T cells in these lines were of the Th2 phenotype. In conclusion, our data reveal that, in AD, percutaneous sensitization to aeroallergens may occur and indicate that allergen-specific Th2 type T cells may be responsible for the high levels of (specific) IgE found in 80% of patients with AD.

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.

Aberrant interleukin (IL)-4 and IL-5 production in vitro by CD4+ helper T cells from atopic subjects.

The cytokine secretion profiles of T cell lines (TCL) specific for purified protein derivative (PPD) or streptokinase (SK), contemporarily derived from nine atopic and nine nonatopic individuals, were compared. Upon stimulation with phorbol myristate acetate (PMA) plus anti-CD3 monoclonal antibody (mAb), all TCL from both atopics and nonatopics produced interleukin (IL)-2 and interferon (IFN)-gamma. The mean IL-2 production by PPD- or SK-specific TCL from both atopics and nonatopics was similar, whereas the mean IFN-gamma production by TCL derived from atopics was significantly lower. In addition, both PPD- and SK-specific TCL from atopics produced detectable amounts of IL-4 and IL-5, whereas the corresponding TCL derived from nonatopics did not. A total number of 107 and 99 PPD-specific CD4+ T cell clones (TCC) were then derived from TCL of 4 atopic and 4 nonatopic donors and assessed for their profile of cytokine production in response to stimulation with either PMA plus anti-CD3 mAb or the specific antigen. Under both these experimental conditions, virtually all PPD-specific TCC from both atopic and nonatopic individuals produced IL-2 and IFN-gamma. In contrast, the great majority of PPD-specific TCC derived from nonatopic individuals did not produce IL-4 and IL-5, whereas high proportions of PPD-specific TCC derived from atopic donors displayed the ability to produce noticeable amounts of IL-4 and IL-5 besides IL-2 and IFN-gamma. These data indicate that CD4+ T cells from atopic individuals are able to produce IL-4 and IL-5 in response to bacterial antigens, such as PPD and SK, that usually evoke responses with a restricted type-1 T helper (Th1)-like cytokine profile in nonatopic individuals. Aberrant IL-4 production by Th cells may represent one of the immune alterations responsible for enhanced IgE antibody production in atopic people.