Suppression of IgE synthesis in vitro by allogeneic T cells from atopic and non-atopic subjects.

The role of T cells in the regulation of IgE synthesis by human PBMC was studied. PBMC or separated and recombined populations of T and B cells from both normal and atopic donors were cultured for 10 days with and without cycloheximide. IgE and IgG synthesis were determined by specific RIA. IgE synthesis was detected in 0/30 non-atopic, 6/34 mildly atopic and 25/31 severely atopic subjects. Autologous T cells from 10/26 atopic donors, whose B cells synthesised IgE, significantly suppressed this IgE synthesis. The addition of allogeneic T cells from atopic or non-atopic subjects to atopic B cells resulted in greater suppression of IgE synthesis than the addition of autologous T cells. These data support the notion that atopic subjects have naturally occurring IgE isotype-specific suppressor T cells as well as suppressor T cells which can be activated during incubation with alloantigen.     

Hydrocortisone enhances total IgE levels--but not the synthesis of allergen-specific IgE--in a monocyte-dependent manner.

Recently, hydrocortisone (HC), when combined with human IL-4, has been reported to increase IgE levels in supernatants (SN) of in vitro cultured leucocytes. In this study we investigated the influence of HC on allergen-specific IgE synthesis. Moreover, we examined the relevance of different cell types in this respect. Peripheral blood mononuclear cells (PBMC), T-cell depleted PBMC, CD14-depleted PBMC and highly purified B cells from 10 allergic (birch pollen and/or grass pollen) patients and five non-allergic individuals were investigated. The cells were incubated with HC and/or recombinant human IL-4 (rIL-4) for 8 days. A considerable increase of total IgE was observed in HC/rIL-4-stimulated cultures compared with rIL-4 alone, HC alone or non-stimulated cultures. We demonstrate that this effect depends on the presence of monocytes in in vitro cultures. These results were seen in every experiment, irrespective of healthy or atopic state of the blood donor. The increase of IgE could not be attributed to a rise of birch pollen-and/or grass pollen-specific IgE in patients allergic to these allergens, as shown by IgE-immunoblot. Radio-allergosorbent test (RAST) investigations of HC/rIL-4-stimulated cells cultures from allergic and non-allergic patients confirmed that HC/rIL-4-induced elevated IgE production was also not due to increased production of IgE, specific for important aero-allergens (pollens, house dust mite or animal dander). Therefore we conclude that newly synthesized IgE is not specific for allergens, but that sequential isotype switching in human B cells leads to increased polyclonal IgE production.     

Staphylococcus aureus modifies the cytokine-induced immunoglobulin synthesis and CD23 expression in patients with atopic dermatitis.

The influence of Staphylococcus aureus on peripheral blood lymphocytes (PBL) of patients with atopic dermatitis (AD) was analysed. The parameters studied were spontaneous and interleukin-inducible immunoglobulin (IgA, IgE, IgG) synthesis, as well as CD23 expression. Various heat-killed, clinical isolates of S. aureus were analysed. PBL from non-atopic donors served as controls. The time-course of co-cultured PBL with S. aureus showed a dose-dependent increase in immunoglobulin (Ig) synthesis from PBL of normal donors, whereas the Ig synthesis of atopic cells was significantly depressed. Additional stimulation with interleukin-4 (IL-4) also led to a pronounced suppression of the IgE and IgA synthesis in normal donor cells, while the effect of S. aureus on PBL of atopic donors was not markedly affected by IL-4. Transwell cultures of bacteria separated from PBL by a semi-permeable membrane induced stimulation of IgA and IgE synthesis in patients with AD. The Ig synthesis in the control group was not altered. Co-stimulation of S. aureus and IL-4 in this system led to a suppression of IgA with cells of both atopic and normal donors. IgE synthesis from atopic PBL was significantly stimulated. The CD23 expression of atopic PBL was increased by S. aureus and IL-4. Our data indicate that S. aureus may modulate the cytokine-dependent humoral immunity in patients with AD and that chronic colonization of the skin may be responsible for allergic skin reactions in AD.     

In Vitro Production of IgE and IgG Protein by Blood Mononuclear Cells from Non-Atopic and Atopic Donors

The production of IgE and IgG protein by human peripheral blood mononuclear cells in culture has been examined. Cells obtained from 18 grass-sensitive donors during the grass pollen season and from 11 atopic dermatitis patients (total serum IgE levels greater than 960 ng/ml), spontaneously produced significant amounts of IgE but not IgG with time. Similar results were obtained using B-cell enriched preparations from both groups. Cells from 16 non-atopic donors had mean levels of pre-formed IgE similar to those of grass pollen-sensitive donors, but there was no increase in culture IgE with time. Treatment of mononuclear cells with pokeweed mitogen did not influence the production of IgE but markedly increased the amount of IgG synthesized by non-atopic and atopic donor cells. Slight, but significant increases in culture IgE, but not IgG, were seen following a 7-day mixed lymphocyte reaction involving both unrelated non-atopic donor cells and the lymphoblastoid B-cell line, Raji. Treatment of cells with 50 HA units of influenza A/Hong Kong/1/68 (H3N2) virus or with 0.02% v/v (240 IU/ml) of a purified beta-interferon preparation did not alter IgE or IgG produced.     

IgE production in atopic patients is not related to IL-4 production.

To analyse whether there is a general defect in T or B cell function in atopic individuals we have measured cytokine and IgE production by peripheral blood lymphocytes, isolated from 19 atopic donors (17 asthma/rhinitis and two dermatitis patients) in comparison with 19 non-atopic controls. After stimulation of lymphocytes with anti-CD2 and anti-CD28, we found no significant difference in IL-2, IL-4 and interferon-gamma (IFN-gamma) production. To examine the correlation between the production of IgE and IL-4, we stimulated lymphocytes with anti-CD2 and rIL-2. Under this condition both T cell IL-4 and B cell IgE production can be measured. No significant difference was found for the amount of IgE and IL-4 produced between the two groups (P > 0.05). The non-atopic donors showed a good correlation between IL-4 and IgE production (r = 0.70). Surprisingly, within the atopic group there was no correlation between IgE and IL-4 production at all (r = -0.04). The ratio of IgE to IL-4 was higher (although not significantly) in the atopic group. Our data suggest that in atopic donors IgE production is less dependent on IL-4, and that other cytokines are involved.     

Distinct modulation by interferon-gamma (IFN-γ) of CD23 expression on B and T lymphocytes of atopic subjects

The low-affinity IgE receptor (FcεRII/CD23) plays a role in IgE production. Cytokines participating in IgE synthesis also modulate CD23 expression on lymphocytes, but whether this modulation is different in atopic subjects remains unclear. We studied CD23 expression on B and T lymphocytes in 10 asthmatic patients with Dermatophagoides pteronyssinus hypersensitivity and 10 healthy non-atopic subjects. Studies were performed by flow cytometry, in phytohaemagglutinin (PHA) or IL-4-stimulated mononuclear cell cultures, alone or in the presence of IFN-γ. Soluble CD23 (sCD23) released in the culture supernatants was measured by enzyme-linked immunoassay. Both PHA and IL-4 induced the expression of CD23 on lymphocytes of atopic and non-atopic subjects. Whereas PHA increased both the percentage and mean fluorescence intensity of CD23⁺ B and T cells, IL-4 alone did not increase the percentage of CD23⁺ T cells. The effects of IFN-γ were different in both groups, since it was able to reduce the percentage of PHA-stimulated CD23⁺ T cells only in non-atopic individuals. In non-atopic subjects more than atopic, levels of sCD23 were increased in the supernatants of PHA and IL-4 cultures. These results show that the modulation of CD23 expression is different on B and T cells, and that IFN-γ acts differently in atopic and non-atopic individuals.     

Oligodeoxynucleotides containing CpG motifs induce IL-12, IL-18 and IFN-gamma production in cells from allergic individuals and inhibit IgE synthesis in vitro.

The effects of phosphorothioate oligonucleotides containing CpG motifs (CpG-ODN) on cultured cells from allergic patients and non-atopic individuals were investigated. In peripheral blood mononuclear cells (PBMC) CpG-ODN led to a significant increase of IFN-gamma. By intracellular cytokine staining, IFN-gamma production could be attributed to NK cells and inhibition experiments indicated an IL-12-dependent mechanism. Moreover, CpG-ODN increased mRNA expression of IL-12 and IL-18 in PBMC. In this respect, no significant difference between allergic and non-atopic individuals was observed. Monocyte-derived dendritic cells were identified as one IL-12- and IL-18-producing source. In addition, stimulation of PBMC derived from atopic patients with CpG-ODN led to a considerable increase of polyclonal IgG and IgM synthesis. In contrast, the production of total IgE was suppressed. CpG-ODN induced a significant rise of IgG and IgM specific for allergens to which the patients were sensitized, whereas allergen-specific IgE levels remained unchanged. Our data suggest that CpG-ODN display a strong influence on the ongoing immune response and might represent potential adjuvants for specific immunotherapy of type I allergy.     

Interleukin-2 inhibits the interleukin-4-induced human IgE and IgG4 secretion in vivo.

The effect of interleukin-2 (IL-2) on IL-4-induced IgE and IgG4 secretion by B cells in peripheral blood mononuclear cell (PBMC) preparations from non-atopic healthy humans and atopic dermatitis patients was investigated. PBMC were cultured at an optimal concentration of recombinant IL-4 with or without addition of IL-2 for 10 days. Native and recombinant IL-2 inhibited the IL-4-induced IgE and IgG4 secretion in a dose-dependent manner by cells from both normal and atopic donors. Rabbit antibodies to IL-2 or to the monoclonal anti-IL-2 receptor antibody anti-TAC reversed the IL-2 effect. Culturing cells with IL-4 and IL-2 for 1 or 2 days only slightly suppressed the IgE and IgG4 secretion whereas addition of IL-2 to IL-4 containing cultures on day 4 or 5 inhibited the IgE and IgG4 secretion more effectively. This is in contrast to interferon-gamma (IFN-gamma) which inhibited the IL-4 induced IgE and IgG4 secretion when added for the first 24 or 48 h but had no effect when added on days 4 or 5. The data demonstrate that both IL-2 and IFN-gamma act as antagonists in the IL-4-induced IgE and IgG4 secretion by human B cells; while IL-2 appears to inhibit relatively late in culture, IFN-gamma has an early inhibitory effect, suggesting that the two lymphokines inhibit the IL-4 effect by different mechanisms.     

American cockroach Cr-PI allergen induces lymphocyte proliferation and cytokine production in atopic patients

Background Previous studies have shown cockroach-induced antigen-specific IgF-mediated asthma. In cockroach-Infested areas, more then 50% of asthmatic subjects may have positive skin reactions to this allergen. Partial purified Cr-PI allergen from American cockroaches contains allergens with molecular weights of 72 and 78k Da; however, little is known about its eifect on the lymphoeyte proliferation and cytokine production. Objective IgE synthesis is known to be regulated by interleukin-4 (IL-4) and interferon gamma (IFN-γ). Therefore, we studied Cr-PI allergen-induced cytokine production in atopic patients and healthy normal controls to understand each factors’ role in the disease. Methods Peripheral blood mononuclear cells (PBMC) from cockroach skin-sensitive patients and controls were stimulated with mitogen and Cr-PI for proliferative response and cytokine production. Cr-PI antigen-specific T-cell cultures of atopic patients and healthy normal controls were used to test C r-PI-lnduced proliferation and cytokine mKNA expression. Results PMBC ot atopic subjects showed a significantly (P < 0.01) higher stimulation index for Cr-PI induced proliferation (SI = l l.8.3.7) when compared with that of non-atopic subjects (SI =4.1 ± 0.8) and cord bloods (SI = 2.1 ± 0.4). Cr-Pl-induced IL-4 was observed only in the PBMC of atopic patients, whereas Cr-PI-induced IFNγ was detected in both atopic patients and normal controls. Likewise, Cr-PI-induced IL-4 mRNA expression in T-cell cultures was detected in all atopies but only one of nine controls. Conclusion IL-4 mKNA expression and IL-4 production in PBMC and T-cell cultures of atopic patients showed good correlation with clinical symptoms, skin-reactivity, specific IgE and proliferative response to Cr-PL These results suggests that cockroach allergen may be a hidden cause of asthma and other atopie diseases.     

Suppression by relevant allergen of the spontaneous in vitro production of rye group I specific IgE antibodies by human peripheral blood lymphocytes

The production in vitro of rye Group I specific IgE antibodies (rye I specific IgE Ab) by human peripheral blood lymphocytes (PBL) was examined. A sensitive enzyme-linked immunosorbent assay was developed which allowed the detection of rye I specific IgE Ab without interference from other immunoglobulin classes. A significant net synthesis of rye I specific IgE Ab by PBL from atopic subjects was detected only during the pollen season, which could not be observed with PBL from non-atopic donors whenever tested. This active synthesis in vitro of IgE antibodies by PBL from atopic patients in season was suppressed when PBL were pre-incubated with the relevant antigen. Furthermore, we are reporting data suggesting an inhibition of the passive release of preformed rye I specific IgE Ab from cells by the specific allergen.     

In vitro production of IgE in humans: kinetic studies and analysis of serum enhancing and inhibitory factors

By using a sensitive and reliable method for the measurement of IgE produced in vitro by peripheral blood lymphomonocytes, the following observations may be made. Serum obtained from atopic donors shows a significant capacity to enhance the in vitro synthesis of IgE of PBL from atopic as well as of non-atopic donors. On the other hand, serum obtained from non-atopic donors demonstrates a clear-cut inhibitory effect on IgE synthesis of cells from both atopic and non-atopic individuals. The kinetics of the IgE production was studied. As early as 30 min after initiation of culture, PBL of atopic individuals showed the capacity to synthesize large amounts of IgE. The IgE synthesis continued, with variations from case to case, for 7 days. This capacity of the cells is completely inhibited following incubation of the PBL for a short period of time with a protein synthesis inhibitor, cycloheximide. This in vitro procedure represents a valuable tool for the study and identification of the enhancing and suppressor factors of IgE synthesis present in the serum.     

Modulation of cell-bound and soluble CD23, spontaneous and ongoing IgE synthesis of human peripheral blood mononuclear cells by soluble IL-4 receptors and the partial antagonistic IL-4 mutant protein IL-4 (Y124D).

We studied the influence of human recombinant soluble interleukin-4 receptors (sIL-4R) and a partial antagonistic mutant IL-4 protein, IL-4(Y124D), on the in vitro CD23 expression, soluble (s)CD23 release and IgE synthesis of human peripheral blood mononuclear cells (PBMC). The data show that sIL-4R suppressed the IL-4-induced IgE synthesis of PBMC. sIL-4R fusion protein stabilized with human Fc gamma fragments showed a more pronounced effect than unconjugated sIL-4R. IL-4(Y124D) also suppressed the IL-4-induced IgE synthesis. The IL-4-induced antigen CD23 and its soluble fragments were suppressed by sIL-4R and IL-4(Y124D). PBMC of atopic donors with a spontaneous in vitro IgE synthesis showed a partial suppression of the IgE production after sIL-4R or IL-4(Y124D) application. The IgE synthesis and sCD23 release of normal donor PBMC were suppressed when the substances were applied 0-4 days after IL-4 treatment. After 4 days of IL-4 stimulation, sIL-4R and IL-4(Y124D) enhanced the IgE synthesis. These data demonstrate that sIL-4R and IL-4(Y124D) are suppressive for the primary IgE synthesis induced by IL-4. In contrast, the ongoing IgE synthesis was only partially modulated by sIL-4R and IL-4(Y124D), and in some conditions even an enhancement of the IgE production was observed. These data suggest a differential function for IL-4 in the early and late phase of PBMC IgE production.     

T cell clones providing helper function for IgE synthesis release soluble factor(s) that induce IgE production in human B cells: possible role for interleukin 4 (IL-4).

We have previously demonstrated that a proportion of human T cell clones (TCC) derived from tonsil or peripheral blood (PB) of non-allergic donors, upon triggering with phytohaemagglutinin (PHA) or anti-CD3 monoclonal antibody (MoAb), were able to provide help for IgE synthesis in B cells from both allergic and non-allergic individuals. In this study we show that, upon PHA stimulation, culture supernatants from 10 selected TCC active on IgE synthesis also provided helper activity for IgE, whereas supernatants from unstimulated cultures of the same TCC were ineffective. In contrast, culture supernatants derived from five PHA-stimulated TCC, unable to provide helper function for IgE synthesis, consistently failed to elicit production of IgE. While the induction of IgE synthesis by TCC occurred in B cells from virtually all allergic and non-allergic donors, their soluble factor(s) were found to be able to provide substantial help for IgE production only in B cells from a proportion of donors tested. In addition, B cells from non-atopic donors usually appeared to be less responsive than atopic B cells to the activity of such factor(s). In contrast, synthesis of both IgG and IgM was induced in every B cell donor by both TCC and their supernatants. Partial characterization of the factor(s) providing helper function for IgE synthesis in B cells showed that it apparently had a mol. wt between 10 and 50 kD and did not bind to immobilized IgE. Such an activity appeared to be associated with the presence of interleukin 4 (IL-4) in supernatants and it was inhibited by adding both gamma-interferon and anti-human IL-4 antibody in culture.     

Functional heterogeneity of human T cell clones from atopic and non-atopic donors.

Several distinct T helper (TH) subsets have been identified, based on the cytokines secreted. Recently, it has been demonstrated that these subsets can regulate the isotype of humoral responses. To investigate the possible differences in TH subsets between atopic donors which have elevated serum IgE and donors with normal serum IgE, we examined series of human TH cell clones. A total of 31 and 22 CD4+ T cell clones from the atopic and non-atopic donors, respectively, were characterized for the ability to help for IgE synthesis in vitro. T cell clones generated with allergen AgE from the atopic donor were autoreactive and all induced IgE synthesis. Tetanus toxoid-specific (TT) and phytohaemagglutinin clones were generated from both donors. There was significant heterogeneity between the T cells isolated with different stimuli from the same atopic donor. Also, there was a significant difference in the number of T cells generated from the atopic versus the non-atopic donor which helped for IgE, although there was no significant difference between the total number of T cells able to help for immunoglobulin synthesis of other isotypes. Most importantly, there was a higher frequency of clones able to support IgE synthesis between TT-specific T cell clones generated from the atopic versus the non-atopic donor. These results suggest that there are changes in subsets of TH cells specific for microbial antigens as well as allergens in atopics, which may have important implications for the aetiology of atopic disease.     

Interferon (IFN)-α, IFN-γ, interleukin (IL)-2, and arachidonic acid metabolites modulate IL-4-induced IgE synthesis similarly in healthy persons and in atopic dermatitis patients

The role of cytokines and arachidonic acid metabolites in the regulation of IgE production in healthy persons and in atopic dermatitis patients with elevated IgE levels was studied. Interleukin-4 (IL-4) induced IgE production in peripheral blood mononuclear cells (PBMCs) of all donors, and no significant difference was found between the amounts of IgE produced by healthy persons and atopic dermatitis patients. Similarly, recombinant interferon (IFN)-α and IFN-γ, as well as IL-2, inhibited IL-4-induced IgE production to a similar extent in both study groups. To evaluate the role of arachidonic acid (AA) metabolites in the regulation of IgE production, we added indomethacin, an inhibitor of the cyclooxygenase pathway, or nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase pathway, to IL-4-treated cultures. Both indomethacin and NDGA strongly inhibited IL-4-induced IgE production. They also inhibited IL-4-induced IgG4 synthesis. No significant difference in the amount of inhibition was found between the two study groups. We were unable to restore the NDGA-induced inhibition of IgE-production by adding leukotrienes B₄, C₄, D₄, or 5-HETE to the NDGA-treated cultures. PGE₂ also failed to restore the indomethacin-mediated inhibitory effect. Consequently, NDGA- and indomethacin-mediated inhibitory effects do not appear to be mediated by any single factor studied. Collectively, our results show IFNs and IL-2 to be similar in effect in the modulation of IL-4-induced IgE synthesis in healthy and atopic persons. In addition, our results show the importance of AA metabolites in the regulation of IgE and IgG4 synthesis in normal persons as well as in atopic dermatitis patients.     

Depressed lymphocyte transformation and the role of prostaglandins in atopic dermatitis.

We have shown that peripheral blood mononuclear cells (PBMC) from patients with atopic dermatitis have a reduced in vitro proliferative responsiveness to concanavalin A when compared with non-atopic controls. Addition of the cyclo-oxygenase inhibitor indomethacin caused a significant enhancement of the mitogen response in the patients, indicating a suppressive effect of cyclooxygenase products. We have further demonstrated increased levels of prostaglandin E2 in the supernatants of the PBMC cultures and increased levels of IgE immune complexes in the sera of the atopic dermatitis patients and therefore hypothesize that IgE immune complexes may cause increased monocyte production of prostaglandins which in turn appears to be responsible for a reduced lymphocyte proliferation.     

Allergen-induced interleukin-9 production in vitro: correlation with atopy in human adults and comparison with interleukin-5 and interleukin-13

BACKGROUND: The contribution of IL-9 to human atopy is supported by genetic studies. However, IL-9 production in response to allergen in vitro has been reported only in children. OBJECTIVE: Study IL-9 induction by allergen in adults, compare it with IL-5 and IL-13 and evaluate its association with atopy. METHODS: Peripheral blood mononuclear cell (PBMC) from control adults and from atopic patients were cultured with various allergens or phytohaemagglutinin (PHA) and secreted IL-5, IL-9 and IL-13 were measured by ELISA. RESULTS: IL-9 was produced in response to Dermatophagoides pteronyssinus (Der p) by PBMC from Der p-hypersensitive adults at levels equivalent to those induced by PHA but with slower kinetics. The induction of IL-9 was allergen specific, reflecting donor RAST profile. In Der p-triggered reactions of non-atopic and atopic subjects, IL-9 showed the highest selectivity for atopics, IL-5 and IL-13 being produced more frequently in non-atopic donors. Significant correlations with specific IgE titres were found for IL-9 with all allergens tested (Der p and two peptides of Bet v 1 birch allergen). For IL-5 and IL-13, they were in the same range for Der p but more variable for birch allergens. Patterns of cytokine production by individual patients in response to allergen reflected these differences: for Der p, IL-5, IL-9 and IL-13 productions were strongly correlated but for birch IL-5 differed from the latter two. The in vitro production of IL-9 reflected clinical hypersensitivity profiles and was higher in individuals with asthma than in those with disease limited to rhinitis and/or conjunctivitis. CONCLUSIONS: Allergen-triggered IL-9 production in vitro is an excellent marker for atopy in adults given its virtual absence in allergen-stimulated PBMC from non-atopic individuals and its correlation with allergen-specific IgE and asthma.     

Interleukin-4 and interferon-gamma production in atopic and non-atopic children with ashma

Previous studies have demonstrated increased production of interleukin-4 (IL-4) and reduced production of interferon (IFN)-γ in stimulated peripheral blood mononuclear cell cultures from children and adults with atopic dermatitis, however, it is unclear whether such an imbalance of cytokine production relates to other childhood atopic diseases such as asthma, and in particular to the presence of the atopic state per se. The production of IL-4 and IFNγ in phytohaemagglutin- (PHA)-stimulated peripheral blood mononuclear cell (PBMC) cultures from atopic and non-atopic children with moderately severe chronic persistent asthma, and a group of age-matched non-atopic controls who did not have asthma was examined. Atopic children with asthma produced significantly more IL-4 and less IFNγ than non-atopic children with asthma and non-atopic controls who did not have asthma. There was no significant difference in IL-4 or IFNγ production between non-atopic children with asthma and controls. These findir. 3 demonstrate that an imbalance of IL-4 and IFNγ production is present in atopic asthma as previously documented in atopic dermatitis, therefore suggesting that it is a feature of the atopic state per se.