The effect of the colostral cells on gene expression of cytokines in cord blood cells

Beneficial effect of maternal milk is acknowledged, but there is still question whether maternal milk from allergic mother is as good as from healthy one. In our study, we have assayed the effect of cells from colostrum of healthy and allergic mothers on gene expression of cytokines in cord blood cells of newborns of healthy and allergic mothers. Cytokines typical for Th1 (IL-2, IFN-gamma), Th2 (IL-4, IL-13), Tregs (IL-10, TGF-beta), and IL-8 were followed. We were not able to detect significant influence of colostral cells on gene expression of cytokines in cord blood after 2-day coculture using Transwell system. There was no difference in gene expression of cytokines in nonstimulated cord blood cells of newborns of healthy and allergic mothers, but generally increased gene expression of cytokines except IL-10 and TGF-beta after polyclonal stimulation was detected in cord blood cells of children of allergic mothers. There was no difference in IL-10 expression in stimulated cord blood cells of children of healthy and allergic mothers. Gene expression of TGF-beta was even decreased in stimulated cord blood cells of children of allergic mothers in comparison to healthy ones. We have not observed difference in the capacity of colostral cells of healthy and allergic mothers to influence gene expression of cytokines in cord blood cells, but we have described difference in the reactivity of cord blood cells between children of allergic and healthy mothers.     

Cytokine expression in cord blood cells of children of healthy and allergic mothers

To determine some early signs connected with the increased risk of future allergy development, gene expression and production of selected cytokines were tested in children of allergic mothers and compared with newborns of healthy mothers. Expression of IL-1β, IL-2, IL-4, IL-8, IL-10, IL-13, IFN-γ, TNF-α, TGF-β and EGF was tested in cord blood cells using real-time PCR and production of these cytokines was evaluated in cord sera by ELISA. Gene expression of IL-2, IL-4, IL-8, IFN-γ, IL-1β, TNF-α and TGF-β was decreased and that of IL-10, IL-13 and EGF increased in children of allergic mothers in comparison with those of healthy mothers. Significant differences in sera of healthy and allergic groups were only in IL-10 and EGF. Different relationship among serum cytokine levels reflects the fact that the cytokines are not produced only by blood cells. Significantly decreased production of EGF in newborns of allergic mothers could negatively influence maturation of mucosal membranes of these children and support thus their easier allergization. Allergic phenotype pointing to the bias to T_H2 response and to possibly impaired intestine maturation was apparent already on the level of cord blood and could serve as a predictive sign of increased allergy risk.     

Perinatal period cytokines related to increased risk of future allergy development

Testing of cytokine levels in colostrum, cord blood and amniotic fluid of healthy and allergic mothers and their newborns (using protein microarrays and quantitative analysis by ELISA) revealed differences in the levels of IL-5, IL-10, TGF-beta, TNF-alpha, EGF and eotaxin between healthy and allergic groups. Significantly higher concentration of IL-5 and IL-10 in the colostrum of allergic mothers and cord blood of their children and also tendency to a higher level of IL-4 found at allergic mothers and their children (but without statistical significance) indicate a bias to T(H)2 response in this group. The higher level of TGF-beta in the colostrum of healthy mothers should be involved in beneficial immunological tuning of their children including enhanced IgA formation and better intestine maturation. In amniotic fluid, concentration of TGF-beta was higher in children of allergic mothers. A significantly higher level of EGF was proved in the colostrum of healthy mothers and in cord blood of their children in comparison with allergic group. EGF deficiency in the allergic group could impair or delay intestine maturation and support thus allergy development.     

IgE against food and respiratory allergens in healthy and allergic mothers and their children

IgE against mixtures of common food or respiratory allergens were determined by ELISA in healthy (n = 38) and allergic (n = 62) mothers and their children. Significantly higher level of IgE against respiratory allergens was found in sera of allergic mothers and in cord blood of their children. No correlation between antibody level in maternal and newborn's sera was found; this argues against the transfer of IgE from mother to fetus and points rather to offspring's intrauterine sensitization. Specific IgE level in cord blood was higher in children who developed later allergy than in children who did not. Specific IgE level in colostrum was low both in healthy and allergic mothers; there was no correlation between high concentration of IgE against respiratory allergens in sera of allergic mothers and their colostrum, which does not support the idea of IgE transport from blood to mammary gland. Only slightly increased colostral IgE was detected in allergic mothers whose children manifested allergy later. Allergy of the mother and high level of anti-allergen IgE in her serum and in cord blood are the main predictive factors of future occurrence of allergy in the offspring. A combination of several predictive factors could have higher prognostic value.     

In vivo IL-10 gene delivery suppresses airway eosinophilia and hyperreactivity by down-regulating APC functions and migration without impairing the antigen-specific systemic immune response in a mouse model of allergic airway inflammation

IL-10 is an immunosuppressive cytokine. Although previous studies have reported that exogenous delivery of IL-10 reduced airway inflammation in experimental allergic airway inflammation, the mechanism of action has not been fully clarified. In this report, we elucidated a mechanism of action of IL-10 in vivo. BALB/c mice were immunized and aerosol challenged with OVA-Ag. We delivered the IL-10 gene to the mice before systemic sensitization or during aerosol Ag challenge by administering an IL-10-producing plasmid vector. Not only presensitization delivery of IL-10, as reported, but also delivery during inflammation strongly suppressed the development of airway eosinophilia and hyperreactivity. Presensitization delivery suppressed the Ag-specific Th2-type immune response in both the lung and spleen. In contrast, delivery in the effector phase suppressed the Th2 response only in the lung, whereas that in the spleen was not affected. IL-10 gene delivery did not induce the development of a regulatory phenotype of T cells or dendritic cells; rather, it suppressed the overall functions of CD11c(+) APCs of the lung such as Ag-presenting capacity, cytokine production, and transportation of OVA-Ag to lymph nodes, thus attenuating Th2-mediated allergic airway inflammation. Further, IL-10 revealed a distinct immunosuppressive effect in the presence of Ag and APCs. These results suggest that suppression of APC function in the lung, the site of immune response, played a critical role in the IL-10-mediated suppression of Ag-induced airway inflammation and hyperreactivity. Therefore, if delivered selectively, IL-10 could site specifically suppress the Ag-specific immune response without affecting systemic immune responses.     

High expression of IL-22 suppresses antigen-induced immune responses and eosinophilic airway inflammation via an IL-10-associated mechanism

Allergic inflammation in the airway is generally considered a Th2-type immune response. However, Th17-type immune responses also play important roles in this process, especially in the pathogenesis of severe asthma. IL-22 is a Th17-type cytokine and thus might play roles in the development of allergic airway inflammation. There is increasing evidence that IL-22 can act as a proinflammatory or anti-inflammatory cytokine depending on the inflammatory context. However, its role in Ag-induced immune responses is not well understood. This study examined whether IL-22 could suppress allergic airway inflammation and its mechanism of action. BALB/c mice were sensitized and challenged with OVA-Ag to induce airway inflammation. An IL-22-producing plasmid vector was delivered before the systemic sensitization or immediately before the airway challenge. Delivery of the IL-22 gene before sensitization, but not immediately before challenge, suppressed eosinophilic airway inflammation. IL-22 gene delivery suppressed Ag-induced proliferation and overall cytokine production in CD4(+) T cells, indicating that it could suppress Ag-induced T cell priming. Antagonism of IL-22 by IL-22-binding protein abolished IL-22-induced immune suppression, suggesting that IL-22 protein itself played an essential role. IL-22 gene delivery neither increased regulatory T cells nor suppressed dendritic cell functions. The suppression by IL-22 was abolished by deletion of the IL-10 gene or neutralization of the IL-10 protein. Finally, IL-22 gene delivery increased IL-10 production in draining lymph nodes. These findings suggested that IL-22 could have an immunosuppressive effect during the early stage of an immune response. Furthermore, IL-10 plays an important role in the immune suppression by IL-22.     

Th type 1-stimulating activity of lung macrophages inhibits Th2-mediated allergic airway inflammation by an IFN-gamma-dependent mechanism

In the mucosal immune system, resident dendritic cells are specialized for priming Th2-polarized immunity, whereas the Ag-presenting activity of macrophages has been linked with the development of Th1 phenotype. As an immune switch toward Th1 can protect against Th2-mediated allergic response, this study investigated the capacity of lung macrophages to stimulate Th1 responses during the secondary exposure to inhaled allergen, thereby suppressing Th2-mediated allergic airway inflammation in a murine model of allergic asthma. Following airway macrophage depletion in OVA-sensitized mice, lung T cells defaulted to a phenotype that produced less Th1 (IFN-gamma) and more Th2 (IL-4 and IL-5) cytokines, leading to more severe airway hyperreactivity and inflammation after intranasal Ag challenge. After OVA pulsing and adoptive transfer, lung macrophages selectively promoted a Th1 response in Ag-sensitized recipients and did not induce pulmonary eosinophilia. By contrast, OVA pulsing and adoptive transfer of a lung cell preparation, consisting of dendritic cells, B cells, and macrophages, promoted a Th2 response with an associated inflammatory response that was suppressed when macrophages were present and pretreated with IFN-gamma, but exacerbated when macrophages were depleted before IFN-gamma treatment. In addition, Th1-promoting activity of lung macrophages was not related to the autocrine production of IL-12p40. These results suggest that the Th1-promoting APC activity may be an inherent property of the lung macrophage population, and may play an important role, upon stimulation by IFN-gamma, in antagonizing an ongoing Th2 immunity and Th2-dependent allergic responses.     

Retinoic acid promotes the development of Th2-like human myelin basic protein-reactive T cells

To determine if retinoids might be beneficial in the treatment of multiple sclerosis (MS), all-trans-retinoic acid (tRA) was tested for its effects on proliferation and cytokine expression in human autoreactive T cells. tRA decreased human lymphocyte proliferation in vitro in a dose-dependent manner. In addition, tRA induced IL-4 gene expression in myelin basic protein (MBP)-specific T cell lines which had previously expressed a Th1-like phenotype. MBP-specific T cell lines generated in the presence of tRA had a Th2-like phenotype. Retinoids have previously been shown to have a similar effect on encephalitogenic T cells in experimental allergic encephalomyelitis (EAE; an animal model for MS) and treatment of EAE with retinoids stabilizes the disease. Since several oral retinoids have been shown to be safe in humans, retinoids may be beneficial in the treatment of MS.     

Elevated Antigen-Driven IL-9 Responses Are Prominent in Peanut Allergic Humans

Food allergies, and peanut allergy in particular, are leading causes of anaphylactic fatalities worldwide. The immune mechanisms that underlie food allergy remain ill-defined and controversial, in part because studies in humans typically focus on analysis of a limited number of prototypical Th1/Th2 cytokines. Here we determine the kinetics and prevalence of a broad panel of peanut-driven cytokine and chemokine responses in humans with current peanut allergy vs those with stable, naturally occurring clinical tolerance to peanut. Our primary focus is identification of novel indicators of immune dysregulation. Antigen-specific cytokine mRNA and protein responses were elicited in primary culture via peanut or irrelevant antigen (Leishmania extract, milk antigens) mediated stimulation of fresh peripheral blood cells from 40 individuals. Peanut extract exposure in vitro induced a broad panel of responses associated with Th2/Th9-like, Th1-like and Th17-like immunity. Peanut-dependent Type 2 cytokine responses were frequently found in both peanut allergic individuals and those who exhibit clinical tolerance to peanut ingestion. Among Th2/Th9-associated cytokines, IL-9 responses discriminated between allergic and clinically tolerant populations better than did commonly used IL-4, IL-5 or IL-13 responses. Comparison with responses evoked by unrelated control antigen-mediated stimulation showed that these differences are antigen-dependent and allergen-specific. Conversely, the intensity of IL-12, IL-17, IL-23 and IFN-γ production was indistinguishable in peanut allergic and peanut tolerant populations. In summary, the ability to generate and maintain cytokine responses to peanut is not inherently distinct between allergic and peanut tolerant humans. Quantitative differences in the intensity of cytokine production better reflects clinical phenotype, with optimally useful indicators being IL-9, IL-5, IL-13 and IL-4. Equivalent, and minimal, Ag-dependent pro-inflammatory cytokine levels in both healthy and peanut allergic volunteers argues against a key role for such cytokines in maintenance of clinical tolerance to food antigens in humans.     

The allergen-induced airway hyperresponsiveness in a human-mouse chimera model of asthma is T cell and IL-4 and IL-5 dependent

The cellular and molecular mechanisms involved in the airway hyperresponsiveness (AHR) of patients with allergic asthma remain unclear. A role for Th2 inflammatory cells was suggested based on murine asthma models. No direct evidence exists on the role of these cells in human asthma. The development of a mouse-human chimera might be useful, allowing the in vivo study of the components of the human immune system relevant to asthma. We investigated the role of allergen-reactive T lymphocytes in a human-mouse SCID model. SCID mice were reconstituted intratracheally with human PBMC from healthy, nonallergic, nonasthmatic donors and exposed to an aerosol of house dust mite allergen after i.p. injection with Dermatophagoides pteronyssinus I Ag and alum. The donor T lymphocytes had a Th1 cytokine phenotype. The reconstituted and allergen-challenged mice developed AHR to carbachol. The mouse airways and lungs were infiltrated with human T lymphocytes. No eosinophils or increases in human IgE were observed. The intrapulmonary human T lymphocytes demonstrated an increase in intracytoplasmic IL-4 and IL-5 and a decrease in IFN-gamma after exposure to allergen adjuvant. Antagonizing human IL-4/IL-13 or IL-5 resulted in a normalization of the airway responsiveness, despite a sustained intracellular Th2 cytokine production. These results provide evidence that the activated human allergen-reactive Th2 cells producing IL-4 or IL-5 are pivotal in the induction of AHR, whereas no critical role for eosinophils or IgE could be demonstrated. They also demonstrate that human allergen-specific Th1 lymphocytes can be driven to a Th2 phenotype.     

Segregation analysis indicates a major gene in the control of interleukine-5 production in humans infected with Schistosoma mansoni.

The interleukine-5 (IL-5) is a hormone of the immune system that is the main regulator of eosinopoiesis, eosinophil maturation and activation, and immunoglobulin A production. Thus, IL-5 contributes in several ways to human immune defenses against various pathogens, including helminths and infectious agents of the digestive and respiratory tracts. On the other hand, the increase in eosinophil number and the activation of these cells, which both have been related to elevated IL-5 production, are the cause of severe pathological disorders, as in asthma or hypereosinophilic syndromes. Although the immunological pathways leading to IL-5 synthesis have been identified, the reasons for the large variability observed in IL-5 production among subjects exposed to comparable antigenic stimulation are unknown. To investigate the role of genetic factors in this variability, we conducted a segregation analysis in a Brazilian population infected by the helminth parasite Schistosoma mansoni. The analysis was performed on IL-5 levels produced by blood mononuclear cells of these subjects after in vitro restimulation with either parasite extracts (IL-5/schistosomula sonicates [SS] phenotype) or a T-lymphocyte mitogen (IL-5/phytohemagglutin [PHA]). The results provide clear evidence for the segregation of a codominant major gene controlling IL-5/SS and IL-5/PHA production and accounting for 70% and 73% of the phenotypic variance, respectively; the frequency of the allele predisposing to low IL-5 production was approximately .22 for both phenotypes. No significant relationship was found between these genes and the gene controlling infection intensities by S. mansoni detected in a previous study. Linkage studies are ongoing to locate those genes that would help to characterize the genetic factors involved in pathological conditions such as severe helminth infections and allergic diseases.     

Thrombospondin-1 (TSP1)-producing B Cells Restore Antigen (Ag)-specific Immune Tolerance in an Allergic Environment

Restoration of the antigen (Ag)-specific immune tolerance in an allergic environment is refractory. B cells are involved in immune regulation. Whether B cells facilitate the generation of Ag-specific immune tolerance in an allergic environment requires further investigation. This paper aims to elucidate the mechanism by which B cells restore the Ag-specific immune tolerance in an allergic environment. In this study, a B cell-deficient mouse model was created by injecting an anti-CD20 antibody. The frequency of tolerogenic dendritic cell (TolDC) was assessed by flow cytometry. The levels of cytokines were determined by enzyme-linked immunosorbent assay. The expression of thrombospondin-1 (TSP1) was assessed by quantitative real-time RT-PCR, Western blotting, and methylation-specific PCR. The results showed that B cells were required in the generation of the TGF-β-producing TolDCs in mice. B cell-derived TSP1 converted the latent TGF-β to the active TGF-β in DCs, which generated TGF-β-producing TolDCs. Exposure to IL-13 inhibited the expression of TSP1 in B cells by enhancing the TSP1 gene DNA methylation. Treating food allergy mice with Ag-specific immunotherapy and IL-13 antagonists restored the generation of TolDCs and enhanced the effect of specific immunotherapy. In conclusion, B cells play a critical role in the restoration of specific immune tolerance in an allergic environment. Blocking IL-13 in an allergic environment facilitated the generation of TolDCs and enhanced the therapeutic effect of immunotherapy.     

Docetaxel promotes the generation of anti-tumorigenic human macrophages

The taxanes Docetaxel and Paclitaxel are two of the standard chemotherapies for patients with metastatic breast cancer. The functional effect of Docetaxel and Paclitaxel on human innate immune cells of the myeloid lineage is not well established, nor is the effects these agents have on differentiation of monocytes into macrophages and dendritic cells. Therefore, the aim with this project was to determine the effects of Docetaxel and Paclitaxel on primary human monocyte differentiation, activation and function. For this purpose, primary human monocytes were isolated from healthy donors and cultured with or without Docetaxel and Paclitaxel. We found that Docetaxel promoted the differentiation of primary human monocytes into pro-inflammatory macrophages with an M1 phenotype and an ability to present antigens to T cells. Monocytes treated with Docetaxel also displayed an elevated secretion of IL-8 and IL-1β, but did not promote generation of monocytic myeloid-derived suppressor cells. In conclusion, Docetaxel appears to have an immune stimulatory effect that would be beneficial for an anti-tumorigenic type of immune response, whereas Paclitaxel seems to have less effect on myeloid cells.     

A nematode immunomodulator suppresses grass pollen-specific allergic responses by controlling excessive Th2 inflammation

Helminth parasites modulate the immune system by complex mechanisms to ensure persistence in the host. Released immunomodulatory parasite components lead to a beneficial environment for the parasite by targeting different host cells and in parallel to a modulation of unrelated inflammatory responses in the host, such as allergy. The aim of this study was to investigate the effect of the potent helminth immunomodulator, filarial cystatin, in a murine model of airway inflammation and hyperreactivity induced by a clinically relevant aeroallergen (timothy grass (Phleum pratense) pollen) and on the function of peripheral blood mononuclear cells (PBMCs) from timothy grass pollen allergic patients. BALB/c mice were systemically sensitised with a recombinant major allergen of timothy grass pollen (rPhl p 5b) and then challenged with timothy grass pollen extract (GPE) via the airways. Filarial cystatin was applied i.p. during the sensitisation phase. Airway hyperresponsiveness to methacholine challenges, inflammation of airways, inflammatory cell recruitment, cytokine production and lung histopathology were investigated. In a translational approach, PBMCs from allergic subjects and healthy controls were treated in vitro with cystatin prior to stimulation with GPE. Administration of filarial cystatin suppressed rPhl p 5b-induced allergen-specific Th2-responses and airway inflammation, inhibited local recruitment of eosinophils, reduced levels of allergen-specific IgE and down-regulated IL-5 and IL-13 in the bronchoalveolar lavage (BAL). Ex vivo restimulation with cystatin of spleen cells from cystatin-treated mice induced the production of IL-10, while cystatin inhibited allergen-specific IL-5 and IL-13 levels. Human PBMCs from timothy grass pollen allergic patients displayed a shift towards a Th1 response after treatment with cystatin. These results show that filarial cystatin ameliorates allergic inflammation and disease in a clinically relevant model of allergy. This data indicate that filarial cystatin has a modulatory effect on grass pollen-specific responses warranting further investigation of potential preventive and therapeutic options in the treatment of allergies.     

Mutations in the gene for the common gamma chain (γc) in X-linked severe combined immunodeficiency

X-linked severe combined immunodeficiency (XSCID) consitutes a disorder of the immune system caused by mutations in the gene encoding the common gamma chain (γ_c), a subunit of the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors, which are necessary for lymphocyte development and function. In this study the IL2RG gene of 31 patients with severe combined immunodeficiency (SCID) was examined by nonradioactive single-strand conformation polymorphism and sequence analysis. Among the 11 patients with XSCID, ten different mutations were identified in the IL2RG gene, including eight novel mutations. Ninety percent of the mothers of the XSCID patients are carriers of the mutated allele. One patient showed low numbers of B-cells, a striking deviation from the classical B-cell-positive and T-cell-negative phenotype. Received: 23 June 1998 / Accepted: 6 September 1998     

Maternal transmission of resistance to development of allergic airway disease

Parental phenotype is known to influence the inheritance of atopic diseases, such as allergic asthma, with a maternal history being a more significant risk factor for progeny than paternal history. We hypothesized that recall Th1- or Th2-type immune responses during pregnancy would result in transfer of maternal factors that would differentially impact development of immune responsiveness in offspring. Following weaning, susceptibility and severity of allergic airway disease (a murine model of human asthma) was evaluated in progeny, disease being elicited by immunization with OVA-Al(OH)(3) and challenge with aerosolized OVA. We found that progeny of mothers with Th1-biased immunity to OVA subjected to recall aerosol challenge during pregnancy had reduced levels of Ag-specific IgE and airway eosinophilia compared with progeny of mothers with Th2-biased immunity to OVA or naive mothers. Interestingly, progeny of mothers with Th1-type immunity to a heterologous albumin, BSA, were not protected from developing OVA-induced allergic airway disease. These findings demonstrated that maternal transfer of protection from development of allergic airway disease to offspring in this model of maternal Th1-type immunity was Ag specific.     

IL-13 regulates the immune response to inhaled antigens

The large inhibitory effect of IL-13 blockers on the asthma phenotype prompted us to ask whether IL-13 would play a role in regulating the allergic immune response in addition to its documented effects on structural pulmonary cells. Because IL-13 does not interact with murine T or B cells, but with monocytes, macrophages, and dendritic cells (DCs), we examined the role of IL-13 in the activation of pulmonary macrophages and DCs and in the priming of an immune response to a harmless, inhaled Ag. We found that a majority of cells called "alveolar or interstitial macrophages" express CD11c at high levels (CD11c(high)) and are a mixture of at least two cell types as follows: 1) cells of a mixed phenotype expressing DC and macrophage markers (CD11c, CD205, and F4/80) but little MHC class II (MHC II); and 2) DC-like cells expressing CD11c, CD205, MHC II, and costimulatory molecules. Endogenous IL-13 was necessary to induce and sustain the increase in MHC II and CD40 expression by pulmonary CD11c(high) cells, demonstrated by giving an IL-13 inhibitor as a measure of prevention or reversal to allergen-primed and -challenged mice. Conversely, IL-13 given by inhalation to naive mice increased the expression of MHC II and costimulatory molecules by CD11c(high) cells in an IL-4Ralpha-dependent manner. We found that exogenous IL-13 exaggerated the immune and inflammatory responses to an inhaled, harmless Ag, whereas endogenous IL-13 was necessary for the priming of naive mice with an inhaled, harmless Ag. These data indicate that blockade of IL-13 may have therapeutic potential for controlling the immune response to inhaled Ags.     

Helminth infection protects mice from anaphylaxis via IL-10-producing B cells

Modulation of the immune system by infection with helminth parasites, including schistosomes, is proposed to reduce the levels of allergic responses in infected individuals. In this study we investigated whether experimental infection with Schistosoma mansoni could alter the susceptibility of mice to an extreme allergic response, anaphylaxis. We formally demonstrate that S. mansoni infection protects mice from an experimental model of systemic fatal anaphylaxis. The worm stage of infection is shown to mediate this protective effect. In vivo depletion studies demonstrated an imperative role for B cells and IL-10 in worm-mediated protection. Furthermore, worm infection of mice increases the frequency of IL-10-producing B cells compared with that in uninfected mice. However, transfer of B cells from worm-infected mice or in vitro worm-modulated B cells to sensitized recipients exacerbated anaphylaxis, which was attributed to the presence of elevated levels of IL-4-producing B cells. Worm-modulated, IL-10-producing B cells from IL-4-deficient, but not IL-5-, IL-9- or IL-13-deficient, mice conferred complete resistance to anaphylaxis when transferred to naive mice. Therefore, we have dissected a novel immunomodulatory mechanism induced by S. mansoni worms that is dependent on an IL-10-producing B cell population that can protect against allergic hypersensitivity. These data support a role for helminth immune modulation in the hygiene hypothesis and further illustrate the delicate balance between parasite induction of protective regulatory (IL-10) responses and detrimental (IL-4) allergic responses.