The interleukin 4 receptor gene and its role in recurrent airway obstruction in Swiss Warmblood horses

Recurrent airway obstruction (RAO) in horses is the result of an interaction of genetic and environmental factors and shares many characteristics with human asthma. Many studies have suggested that the interleukin-4 receptor gene (IL4R) is associated with this disease, and a QTL region on chromosome 13 containing IL4R was previously detected in one of the two Swiss Warmblood families. We sequenced the entire IL4R gene in this family and detected 93 variants including five non-synonymous protein-coding variants. The allele distribution at these SNPs supported the previously detected QTL signal. Subsequently, we investigated IL4R mRNA expression in bronchoalveolar lavage fluid cells. During exacerbation, IL4R expression was increased in RAO-affected offspring in the implicated family, but not in the other family. These findings support that IL4R plays a role in some cases of RAO.

Distinct reactivities of interleukin-4-specific antibodies with recombinant and native canine interleukin-4 in various assays

Interleukin 4 (IL-4) plays a central role in immune responses to parasites and allergens. IL-4 drives the differentiation of naive T cells into Th2 cells and regulates immunoglobulin class switching to IgE.Little is known about the role of IL-4 in canine allergies and parasite infections. Most of the information derives from measurement of IL-4 mRNA expression in dog tissues, but detection of IL-4 protein has been difficult so far, probably due to low sensitivity of available methods. Antibodies (Ab) specific for canine IL-4 are available from various sources, but these Ab have been produced against recombinant Escherichia coli-expressed canine IL-4 and there is only limited information on their reactivities with native canine IL-4. Therefore, in the present study, we tested six available canine IL-4-specific Ab for their reactivities with recombinant canine IL-4 expressed in E. coli (rec.IL-4) or in mammalian cells (mam.IL-4), and with supernatants from stimulated canine peripheral blood mononuclear cells (PBMCs) using several detection methods, including Western blotting, ELISA, cytokine bead assay, and intracellular IL-4 staining. Additionally, we tested a bovine IL-4-specific antibody that has been previously shown to cross-react with canine IL-4. All tested Ab except anti-bovine IL-4 reacted with rec.IL-4, and most of them reacted with mam.IL-4. However, only the cytokine bead assay was sensitive enough to allow the detection of IL-4 in supernatants of canine PBMCs.

Reduced incidence of insect-bite hypersensitivity in Icelandic horses is associated with a down-regulation of interleukin-4 by interleukin-10 and transforming growth factor-beta1

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by IgE-mediated reactions to bites of insects of the genus Culicoides. IBH does not occur in Iceland due to the absence of Culicoides. However, Icelandic horses exported to mainland Europe as adults (1st generation) have a >/=50% incidence of developing IBH. In contrast, their progeny (2nd generation) has a <10% incidence of IBH. Here we show that peripheral blood mononuclear cells (PBMC) from Icelandic horses born in mainland Europe and belonging either to the IBH or healthy subgroup produce less interleukin (IL)-4 after polyclonal or allergen-specific stimulation when compared with counterparts from horses born in Iceland. We examined a role of IL-10 and transforming growth factor (TGF)-beta1 in down-regulation of IL-4 in healthy 2nd generation Icelandic horses. Supernatants of PBMC from 2nd generation healthy horses down-regulated the proportion of IL-4-producing cells and IL-4 production in stimulated cultures of PBMC from 1st generation IBH. This inhibition was mimicked by a combination of IL-10 and TGF-beta1 but not by the single cytokines. Cultures of stimulated PBMC of healthy 2nd generation horses produced a low level of IL-4, but IL-4 production was increased by anti-equine IL-10 and anti-human TGF-beta1. This shows for the first time that in horses, IL-10 and TGF-beta1 combined regulate IL-4 production in vitro. It is suggested that in this naturally occurring IgE-mediated allergy, IL-10 and TGF-beta1 have a role in the down-regulation of IL-4-induced allergen-specific Th2 cells, thereby reducing the incidence of IBH.

Cloning of a full-length cDNA encoding bovine interleukin 4 by the polymerase chain reaction

A human interleukin 4 (hIL-4)-encoding cDNA (hIL4) probe was used to screen a bovine genomic library, and three clones containing sequences with homology to the human and mouse IL4 cDNAs were isolated. Sequence information obtained from one of these genomic clones was used to design an oligodeoxyribonucleotide primer corresponding to the transcription start point region for use in the polymerase chain reaction (PCR). The PCR-RACE protocol, designed for the rapid amplification of cDNA ends, was successfully used to generate a full-length bovine IL4 (bIL4) cDNA clone from polyadenylated RNA isolated from concanavalin A-stimulated bovine lymph node cells. The bIL4 cDNA is 570 bp in length and contains an open reading frame of 405 nucleotides (nt), coding for a 15.1-kDa precursor of 135 amino acids (aa), which should be reduced to 12.6 kDa for unglycosylated bIL4 after cleavage of a putative hydrophobic leader sequence of 24 aa. The aa sequence contains one possible Asn-linked glycosylation site. Bovine IL4 is shorter than mouse (mIL4) and hIL4, because of a 51-nt deletion in the coding region. Comparison of the overall nt and deduced aa sequences shows a greater homology of bIL4 with hIL4 than with mIL4. This homology is not evenly distributed, however, with the nt sequences 5' and 3' of the coding region showing a much greater homology between all three species than the coding sequence.

Expression and biological activities of bovine interleukin 4: effects of recombinant bovine interleukin 4 on T cell proliferation and B cell differentiation and proliferation in vitro

Interleukin 4 (IL-4) is a pleotropic cytokine affecting a wide range of cell types in both the mouse and the human. These activities include regulation of the growth and differentiation of both T and B lymphocytes. The activities of IL-4 in nonprimate, nonmurine systems are not well established. Herein, we demonstrate in the bovine system that IL-4 upregulates production of IgM, IgG1, and IgE in the presence of a variety of costimulators including anti-IgM, Staphylococcus aureus cowan strain I, and pokeweed mitogen. IgE responses are potentiated by the addition of IL-2 to IL-4. Culture of bovine B lymphocytes with IL-4 in the absence of additional costimulators resulted in the increased surface expression of CD23 (low-affinity Fc epsilon RII), IgM, IL-2R, and MHC class II in a dose-dependent manner. IL-4 alone increased basal levels of proliferation of bulk peripheral blood mononuclear cells but in the presence of Con A inhibited proliferation. In contrast to the activities of IL-4 in the murine system, proliferation of TH1- and TH2-like clones was inhibited in a dose-dependent manner as assessed by antigen-or IL-2-driven in vitro proliferative responses. These observations are consistent with the role of IL-4 as a key player in regulation of both T and B cell responses.

Expression of interleukin 4, interleukin 4 splice variants and interferon gamma mRNA in calves experimentally infected with Fasciola hepatica

Interleukin 4 (IL-4) is expected to play a dominant role in the development of T helper (Th) 2 cells. Th2 immune responses with expression of relatively large amounts of interleukin 4 (IL-4) but little interferon gamma (IFN-gamma) are characteristic for chronic helminth infections. But no information is available about IL4 expression during early Fasciola hepatica (F. hepatica) infections in cattle. Therefore, we investigated F. hepatica specific IL-4 and IFN-gamma mRNA expression in peripheral blood mononuclear cells (PBMCs) from calves experimentally infected with F. hepatica. Cells were collected prior to infection and on post-inoculation days (PIDs) 10, 28 and 70. Interestingly, PBMCs responded to stimulation with F. hepatica secretory-excretory products (FhSEP) already on PID 10 and expressed high amounts of IL-4 but not of IFN-gamma mRNA suggesting that F. hepatica induced a Th2 biased early immune response which was not restricted to the site of infection. Later in infection IL-4 mRNA expression decreased whereas IFN-gamma mRNA expression increased slightly. Isolated lymph node cells (LNCs) stimulated with FhSEP and, even more importantly, non-stimulated LN tissue samples indicated highly polarized Th2 type immune responses in the draining (hepatic) lymph node, but not in the retropharyngeal lymph node. During preliminary experiments, two splice variants of bovine IL-4 mRNA, boIL-4delta2 and boIL-4delta3, were detected. Since a human IL-4delta2 was assumed to act as competitive inhibitor of IL-4, it was important to know whether expression of these splice variants of bovine IL-4 have a regulatory function during an immune response to infection with F. hepatica. Indeed, IL-4 splice variants could be detected in a number of samples, but quantitative analysis did not yield any clue to their function. Therefore, the significance of bovine IL-4 splice variants remains to be determined.

Production and characterisation of monoclonal antibodies specific for bovine interleukin-4

Genetic immunisation is a simple method for producing polyclonal antibodies in mice. By this method, we produced antibodies against bovine interleukin-4 (BoIL-4). After a final injection with a recombinant BoIL-4 protein, nine stable hybridoma cell lines were established which secreted monoclonal antibodies (MAbs) against this cytokine. Specific binding of each of the MAbs to recombinant BoIL-4 produced by Escherichia coli, baculovirus, and Trypanosoma brucei was demonstrated in an indirect ELISA and/or in Western blotting. These MAbs recognise the same antigenic region localised in the first 47 amino acids of the mature protein. None of them was able to neutralise the biological activity of the BoIL-4 under the conditions tested but one allowed the detection of BoIL-4 by flow cytometry.

Interferon-gamma and interleukin-4 mRNA expression by peripheral blood mononuclear cells from pregnant and non-pregnant cattle seropositive for bovine viral diarrhea virus

The acceptance of the fetal allograft by pregnant women and mice seems to be associated with a shift from a Th 1 dominated to a Th 2 dominated immune response to certain infectious agents. The goal of this study was to examine cytokine expression in peripheral blood mononuclear cells (PBMCs) from cattle immune to bovine viral diarrhea virus (BVDV) to determine whether pregnancy also has an influence on the type of immune response in this species. Forty-six heifers and cows between 14 months and 13 years of age were included in this study. Twenty-four were seropositive and 22 seronegative for BVDV. Eleven of the seropositive animals and 11 of the seronegative animals were in the eighth month of gestation, the remaining animals were virgin heifers. PBMC from these animals were analyzed for Interferon (IFN)-gamma and Interleukin (IL)-4 mRNA expression by real-time RT-PCR after stimulation with a non-cytopathic strain of BVDV. Additionally, an ELISA was performed to measure IFN-gamma in the supernatants of stimulated cell cultures. In BVDV seropositive animals, IFN-gamma mRNA levels were significantly higher than in BVDV seronegative animals and there was a significant positive correlation between the changes in IFN-gamma and IL-4 mRNA expression. There was, however, no significant difference in IFN-gamma and IL-4 mRNA levels between pregnant and non-pregnant animals. These results are inconsistent with BVDV inducing a Th1 or Th2 biased immune response. Furthermore, a shift in the cytokine pattern during bovine pregnancy was not evident.

Mice deficient in interleukin-4 (IL-4) or IL-4 receptor alpha have higher resistance to sporozoite infection with Plasmodium berghei (ANKA) than do naive wild-type mice

BALB/c interleukin-4 (IL-4(-/-)) or IL-4 receptor-alpha (IL-4ralpha(-/-)) knockout (KO) mice were used to assess the roles of the IL-4 and IL-13 pathways during infections with the blood or liver stages of plasmodium in murine malaria. Intraperitoneal infection with the blood-stage erythrocytes of Plasmodium berghei (ANKA) resulted in 100% mortality within 24 days in BALB/c mice, as well as in the mutant mouse strains. However, when infected intravenously with the sporozoite liver stage, 60 to 80% of IL-4(-/-) and IL-4ralpha(-/-) mice survived, whereas all BALB/c mice succumbed with high parasitemia. Compared to infected BALB/c controls, the surviving KO mice showed increased NK cell numbers and expression of inducible nitric oxide synthase (iNOS) in the liver and were able to eliminate parasites early during infection. In vivo blockade of NO resulted in 100% mortality of sporozoite-infected KO mice. In vivo depletion of NK cells also resulted in 80 to 100% mortality, with a significant reduction in gamma interferon (IFN-gamma) production in the liver. These results suggest that IFN-gamma-producing NK cells are critical in host resistance against the sporozoite liver stage by inducing NO production, an effective killing effector molecule against Plasmodium. The absence of IL-4-mediated functions increases the protective innate immune mechanism identified above, which results in immunity against P. berghei infection in these mice, with no major role for IL-13.

Modulation of allergy incidence in icelandic horses is associated with a change in IL-4-producing T cells

BACKGROUND: Equine insect bite hypersensitivity (IBH) is an immediate-type hypersensitivity reaction provoked by insect-derived allergens. Icelandic horses living in Iceland do not have IBH due to absence of relevant insects, but acquire it at high frequency after being imported to mainland Europe. In contrast, their offspring born in mainland Europe has reduced IBH incidence. T helper 1 (Th1) and Th2 cells and cytokines were determined in Icelandic horses born in Iceland and on the continent and which either have IBH or are healthy. METHODS: Peripheral blood mononuclear cells (PBMC) from these horses were stimulated for 18 h during summer and winter with polyclonal T cell stimuli, IBH allergen(s) or irrelevant allergen(s). Cells were analysed by flow cytometry for interferon-gamma (IFN-gamma) and interleukin-4 (IL-4); RNA was analysed for IFN-gamma, IL-4, IL-5 and IL-13 mRNA. RESULTS: During summer, but not during winter, IBH PBMC stimulated polyclonally showed reduced IFN-gamma mRNA and IFN-gamma-producing cells when compared with those of healthy horses, regardless of origin. PBMC stimulated polyclonally or with IBH allergen showed increased IL-4 mRNA levels and higher numbers of IL-4-producing cells when born in Iceland or showing IBH symptoms. IL-5 and IL-13 mRNA were modulated neither by disease nor by origin. Abrogation of IL-4 production in healthy horses born in mainland Europe may be due, at least in part, to IL-10. There was an increased level of IL-10 in supernatants from PBMC of healthy horses born in mainland Europe and stimulated polyclonally or with IBH allergen. CONCLUSIONS: Modulation of IBH incidence is governed by altered Th1/Th2 ratio, which might be influenced by IL-10.

Comparison of genomic and proteomic data in recurrent airway obstruction affected horses using Ingenuity Pathway Analysis(R)

BACKGROUND: Recurrent airway obstruction (RAO) is a severe chronic respiratory disease affecting horses worldwide, though mostly in the Northern hemisphere. Environmental as well as genetic factors strongly influence the course and prognosis of the disease. Research has been focused on characterization of immunologic factors contributing to inflammatory responses, on genetic linkage analysis, and, more recently, on proteomic analysis of airway secretions from affected horses. The goal of this study was to investigate the interactions between eight candidate genes previously identified in a genetic linkage study and proteins expressed in bronchoalveolar lavage fluid (BALF) collected from healthy and RAO-affected horses. The analysis was carried out with Ingenuity Pathway Analysis(R) bioinformatics software. RESULTS: The gene with the greatest number of indirect interactions with the set of proteins identified is Interleukin 4 Receptor (IL-4R), whose protein has also been detected in BALF. Interleukin 21 receptor and chemokine (C-C motif) ligand 24 also showed a large number of interactions with the group of detected proteins. Protein products of other genes like that of SOCS5, revealed direct interactions with the IL-4R protein. The interacting proteins NOD2, RPS6KA5 and FOXP3 found in several pathways are reported regulators of the NFkappaB pathway. CONCLUSIONS: The pathways generated with IL-4R highlight possible important intracellular signaling cascades implicating, for instance, NFkappaB. Furthermore, the proposed interaction between SOCS5 and IL-4R could explain how different genes can lead to identical clinical RAO phenotypes, as observed in two Swiss Warmblood half sibling families because these proteins interact upstream of an important cascade where they may act as a functional unit.

Vaccination with a T-cell-priming Gag peptide of caprine arthritis encephalitis virus enhances virus replication transiently in vivo

CD4+ T cells are involved in several immune response pathways used to control viral infections. In this study, a group of genetically defined goats was immunized with a synthetic peptide known to encompass an immunodominant helper T-cell epitope of caprine arthritis encephalitis virus (CAEV). Fifty-five days after challenge with the molecularly cloned CAEV strain CO, the vaccinated animals had a higher proviral load than the controls. The measurement of gamma interferon and interleukin-4 gene expression showed that these cytokines were reliable markers of an ongoing immune response but their balance did not account for more or less efficient control of CAEV replication. In contrast, granulocyte-macrophage colony-stimulating factor appeared to be a key cytokine that might support virus replication in the early phase of infection. The observation of a potential T-cell-mediated enhancement of virus replication supports other recent findings showing that lentivirus-specific T cells can be detrimental to the host, suggesting caution in designing vaccine candidates.

A region on equine chromosome 13 is linked to recurrent airway obstruction in horses

REASONS FOR STUDY: Equine recurrent airway obstruction (RAO) is probably dependent on a complex interaction of genetic and environmental factors and shares many characteristic features with human asthma. Interleukin 4 receptor a chain (IL4RA) is a candidate gene because of its role in the development of human asthma, confirmation of this association is therefore required. METHODS: The equine BAC clone containing the IL4RA gene was localised to ECA13q13 by the FISH method. Microsatellite markers in this region were investigated for possible association and linkage with RAO in 2 large Warmblood halfsib families. Based on a history of clinical signs (coughing, nasal discharge, abnormal breathing and poor performance), horses were classified in a horse owner assessed respiratory signs index (HOARSI 1-4: from healthy, mild, moderate to severe signs). Four microsatellite markers (AHT133, LEX041, VHL47, ASB037) were analysed in the offspring of Sire 1 (48 unaffected HOARSI 1 vs. 59 affected HOARSI 2-4) and Sire 2 (35 HOARSI 1 vs. 50 HOARSI 2-4), age 07 years. RESULTS: For both sires haplotypes could be established in the order AHT133-LEXO47-VHL47-ASB37. The distances in this order were estimated to be 2.9, 0.9 and 2.3 centiMorgans, respectively. Haplotype association with mild to severe clinical signs of chronic lower airway disease (HOARSI 2-4) was significant in the offspring of Sire 1 (P = 0.026) but not significant for the offspring of Sire 2 (P = 0.32). Linkage analysis showed the ECA13q13 region containing IL4RA to be linked to equine chronic lower airway disease in one family (P<0.01), but not in the second family. CONCLUSIONS: This supports a genetic background for equine RAO and indicates that IL4RA is a candidate gene with possible locus heterogeneity for this disease. POTENTIAL RELEVANCE: Identification of major genes for RAO may provide a basis for breeding and individual prevention for this important disease.

Characterization of three human sec14p-like proteins: alpha-tocopherol transport activity and expression pattern in tissues

Three closely related human sec14p-like proteins (hTAP1, 2, and 3, or SEC14L2, 3, and 4, respectively) have been described. These proteins may participate in intracellular lipid transport (phospholipids, squalene, tocopherol analogues and derivatives) or influence regulatory lipid-dependent events. Here, we show that the three recombinant hTAP proteins associate with the Golgi apparatus and mitochondria, and enhance the in vitro transport of radioactively labeled alpha-tocopherol to mitochondria in the same order of magnitude as the human alpha-tocopherol transfer protein (alpha-TTP). hTAP1 and hTAP2 are expressed in several cell lines, whereas the expression level of hTAP3 is low. Expression of hTAP1 is induced in human umbilical cord blood-derived mast cells upon differentiation by interleukin 4. In tissues, the three hTAPs are detectable ubiquitously at low level; pronounced and localized expression is found for hTAP2 and hTAP3 in the perinuclear region in cerebellum, lung, liver and adrenal gland. hTAP3 is well expressed in the epithelial duct cells of several glands, in ovary in endothelial cells of small arteries as well as in granulosa and thecal cells, and in testis in Leydig cells. Thus, the three hTAPs may mediate lipid uptake, secretion, presentation, and sub-cellular localization in a tissue-specific manner, possibly using organelle- and enzyme-specific docking sites.

Natural killer T cell dysfunction in CD39-null mice protects against concanavalin A-induced hepatitis

Concanavalin A (Con A)-induced injury is an established natural killer T (NKT) cell-mediated model of inflammation that has been used in studies of immune liver disease. Extracellular nucleotides, such as adenosine triphosphate, are released by Con A-stimulated cells and bind to specific purinergic type 2 receptors to modulate immune activation responses. Levels of extracellular nucleotides are in turn closely regulated by ectonucleotidases, such as CD39/NTPDase1. Effects of extracellular nucleotides and CD39 on NKT cell activation and upon hepatic inflammation have been largely unexplored to date. Here, we show that NKT cells express both CD39 and CD73/ecto-5'-nucleotidase and can therefore generate adenosine from extracellular nucleotides, whereas natural killer cells do not express CD73. In vivo, mice null for CD39 are protected from Con A-induced liver injury and show substantively lower serum levels of interleukin-4 and interferon-gamma when compared with matched wild-type mice. Numbers of hepatic NKT cells are significantly decreased in CD39 null mice after Con A administration. Hepatic NKT cells express most P2X and P2Y receptors; exceptions include P2X3 and P2Y11. Heightened levels of apoptosis of CD39 null NKT cells in vivo and in vitro appear to be driven by unimpeded activation of the P2X7 receptor. CONCLUSION: CD39 and CD73 are novel phenotypic markers of NKT cells. Deletion of CD39 modulates nucleotide-mediated cytokine production by, and limits apoptosis of, hepatic NKT cells providing protection against Con A-induced hepatitis. This study illustrates a further role for purinergic signaling in NKT-mediated mechanisms that result in liver immune injury.