Characterization of monoclonal antibodies to equine interleukin-10 and detection of T regulatory 1 cells in horses

Interleukin-10 (IL-10) terminates inflammatory immune responses and inhibits activation and effector functions of T-cells, monocytes, macrophages and dendritic cells. IL-10 has also been found to be a key cytokine expressed by subpopulations of regulatory T-cells. In this report, we describe the generation and characterization of three monoclonal antibodies (mAbs) to equine IL-10. The antibodies were found to be specific for equine IL-10 using different recombinant equine cytokine/IgG fusion proteins. Two of the anti-equine IL-10 mAbs were selected for ELISA to detect secreted IL-10 in supernatants of mitogen stimulated equine peripheral blood mononuclear cells (PBMC). The sensitivity of the ELISA for detecting secreted IL-10 was found to be around 200pg/ml. The production of intracellular IL-10 was measured in equine PBMC by flow cytometry. PBMC were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of the secretion blocker Brefeldin A. All three anti-IL-10 mAbs detected a positive population in PMA stimulated lymphocytes which was absent in the medium controls. Around 80% of the IL-10(+) cells were CD4(+). Another 15% were CD8(+) cells. Double staining with IL-4 or interferon-gamma (IFN-gamma) indicated that PMA and ionomycin stimulation induced 80% IL-10(+)/IFN-gamma(+) lymphocytes, while only 5% IL-10(+)/IL-4(+) cells were observed. By calculation, at least 60% of the IL-10(+)/IFN-gamma(+) cells were CD4(+) lymphocytes. This expression profile corresponds to the recently described T regulatory 1 (T(R)1) cell phenotype. In summary, the new mAbs to equine IL-10 detected native equine IL-10 by ELISA and flow cytometry and can be used for further characterization of this important regulatory cytokine in horses.     

Monoclonal anti-equine IgE antibodies with specificity for different epitopes on the immunoglobulin heavy chain of native IgE

In this study we describe the generation of monoclonal antibodies (mAbs), which recognize different epitopes of the equine IgE constant heavy chain. Equi-murine recombinant IgE (rIgE), composed of the murine V(H)186.2 heavy chain variable region, linked to the equine IgE constant heavy chain and expressed together with the murine lambda(1) chain in J558L cells was used to immunize BALB/C mice. A total of 17 different mAbs were obtained, which recognized the rIgE heavy chain constant region. None of the mAbs reacted with monoclonal equine isotypes IgM, IgG1 (IgGa), IgG3 (IgG(T)), IgG4 (IgGb) or isolated equine light chains, IgGc and IgA from horse serum, or the native mAb B1-8delta, expressing the same heavy chain variable regions and light chains. One of the mAbs (alphaIgE-132) recognized the recombinant equine IgE, but did not recognize any protein in equine serum, i.e. native IgE. A total of 16 mAbs detected a serum protein of approximately 210,000Da on Western blots, corresponding to the expected MW of native IgE. In addition, one of the mAbs (alphaIgE-176) detected a protein of 76,000Da under reducing conditions, most likely the equine IgE heavy chain. According to binding inhibition studies, the equine IgE specific mAbs recognize at least two different epitopes of the equine IgE. In an ELISA using two anti-IgE mAbs which recognized different epitopes, no significant differences in the concentration of total serum IgE could be detected between adult Icelandic horses with IgE-mediated type I allergy (summer eczema) and healthy control animals. In Icelandic horse foals, no serum IgE could be measured 6 months post partum. All anti-IgE mAbs recognized a small population (1.3+/-0.5%) of leukocytes from adult Icelandic horses by surface immunofluorescence, but no cells could be detected in foal blood. The stained leukocytes from adult horses could be enriched by magnetic cell sorting and contained 32% basophils, 53% monocytes and/or large lymphocytes, 13% small lymphocytes and 2% eosinophils.     

Effect of age and pregnancy on the antibody and cell-mediated immune responses of horses to equine herpesvirus 1.

The cell-mediated immune response and antibody response of horses of varying ages and of pregnant horses to equine herpesvirus 1 antigen were examined. Six to eight month old horses showed either no increase or slight increases in anti-equine herpesvirus 1 serum neutralizing antibody following vaccination and revaccination with a modified live equine herpesvirus 1 vaccine. However, these same horses showed a marked increase in the cell-mediated immune response to equine herpesvirus 1 as measured by the lymphocyte transformation test. Eighteen to 21 month old horses showed four to 64-fold increases in anti-equine herpesvirus 1 serum neutralizing antibody titer following vaccination, but the cell-mediated immune response to equine herpesvirus 1 was low or absent. Only after revaccination did they show an increased cell-mediated immune response to equine herpesvirus 1. The cell-mediated immune response of mares in the latter stages of pregnancy to equine herpesivurs 1 was suppressed although antibody titers increased as much as 16-fold following exposure to virulent equine herpesvirus 1.     

Comprehensive network map of transcriptional activation of chicken type I IFNs and IFN-stimulated genes

Chicken type I interferons (type I IFNs) are key antiviral players of the chicken immune system and mediate the first line of defense against viral pathogens infecting the avian species. Recognition of viral pathogens by specific pattern recognition receptors (PRRs) induce chicken type I IFNs expression followed by their subsequent interaction to IFN receptors and induction of a variety of IFN stimulated antiviral proteins. These antiviral effectors establish the antiviral state in neighboring cells and thus protect the host from infection. Three subtypes of chicken type I IFNs; chIFN-α, chIFN-β, and a recently discovered chIFN-κ have been identified and characterized in chicken. Chicken type I IFNs are activated by various host cell pathways and constitute a major antiviral innate defense in chicken. This review will help to understand the chicken type 1 IFNs, host cellular pathways that are involved in activation of chicken type I IFNs and IFN stimulated antiviral effectors along with the gaps in knowledge which will be important for future investigation. These findings will help us to comprehend the role of chicken type I IFNs and to develop different strategies for controlling viral infection in poultry.     

Induction and sequencing of Rousette bat interferon alpha and beta genes

Bats are considered to be natural reservoirs for several viruses of clinical importance, including rabies virus, Nipah virus, and Hendra virus. Type I interferons (IFNs) is an important part of the immune system in the defense against viral infection. To investigate the function of type I IFNs upon viral infection in bats, the nucleic acid, and amino acid sequences of Egyptian Rousette (Rousettus aegyptiacus) IFN-alpha and -beta were characterized. Sequence data indicated that bat IFN-alpha consists of 562-bp encoded 187-aa, and IFN-beta consisted of 558-bp encoded 186-aa. Phylogenetic analysis of the overall identity of IFN-beta shared the highest sequence homology with pig IFN-beta in both nucleotide and amino acid level. Stimulation of bat primary kidney cells (BPKCs) and bat lung cell lines, Tb-1 Lu, with polyinosinic-polycytidylic acid (poly(I:C)) or exogenous bat type I IFNs resulted in increased type I IFNs mRNA expression in BPKCs, but not in Tb-1 Lu. Characterization of the bat IFN-alpha and -beta genes allows understanding of the immune responses upon stimulation in different tissues, thus providing practical strategies for control and treatment of clinically important diseases. These results are important especially for the virus infection, and suggest that future molecular studies on virus infection experiment of bats in vitro will require careful consideration of the differences of type I IFN expression patterns in different cell types.     

The involvement of mast cells and mast cell proteinases in the intestinal response to equine cyathostomin infection

Cyathostomins (Cyathostominae) are regarded as the most pathogenic equine nematode worldwide. These nematodes are difficult to control in equine populations due to emerging anthelmintic resistance and evasion of encysted larval cyathostomins to regular modern anthelmintics. Mast cells and their proteinases have been shown to play a role in the mammalian immune response to nematode infections. Involvement of mast cells and mast cell proteinases in the equine immune response to cyathostomin infection is proposed. A technique was established to perform immunohistochemical staining using polyclonal rabbit anti-equine mast cell proteinase-1 (eqMCP-1) and anti-equine tryptase on formalin-fixed large intestinal sections, from horses classified as cyathostomin positive and negative at the time of death based upon larval enumeration. Quantitative analysis of antibody labelled mast cells was used to detect mast cell proteinases in equine large intestinal sections positive and negative for cyathostomin larvae. This demonstrated an increase in equine tryptase labelled mucosal and submucosal mast cells in cyathostomin positive horses. This study has established an immunohistochemical technique to demonstrate mast cell proteinases in formalin-fixed large intestinal sections. This technique may be used to determine possible involvement of mast cells and their proteinases in the equine immune response to cyathostomin larvae. Further studies are required to define a specific role.     

Generation and characterization of monoclonal antibodies to equine CD16

The low-affinity Fc receptor CD16 plays a central role in the inflammatory and innate immune responses of many species, but has not yet been investigated in the horse. Using the predicted extracellular region of equine CD16 expressed as a recombinant fusion protein with equine IL-4 (rIL-4/CD16), we generated a panel of mouse monoclonal antibodies (mAbs) that recognize equine CD16. Nine mAbs were chosen for characterization based upon recognition of CD16, but not IL-4, in ELISA. All nine mAbs recognized full-length, cell-surface CD16 expressed as a GFP fusion protein by CHO cells, but not the closely related Fc receptor CD32 expressed in the same system. In flow cytometric analysis with equine peripheral leukocytes, the mAbs labeled cells in the granulocyte, monocyte, and lymphocyte populations in a pattern consistent with other species. Monocytes that were strongly labeled with CD16 mAb 9G5 were also positive for the LPS receptor CD14. Cytospins made with peripheral leukocytes were immunohistochemically labeled and showed mAb recognition of primarily mononuclear cells. ELISA revealed that the nine mAbs can be grouped into three patterns of epitope recognition. These new antibodies will serve as useful tools in the investigation of equine immune responses and inflammatory processes.     

A sensitive immunoassay for porcine interferon-α

Two murine monoclonal antibodies (mAbs) directed against different epitopes on recombinant porcine interferon-alpha (IFN-alpha) were selected and used to construct a two-site ELISA. This ELISA, when performed in a one-step version, detected about 0.5 units ml-1 of IFN-alpha and showed similar sensitivity but better precision than a cytopathic effect inhibition bioassay. Estimates of IFN-alpha in tissue culture medium by the two assays correlated well. In contrast, one or several factors in porcine serum reduced the sensitivity of the ELISA. Measurements of IFN-alpha in porcine serum was, however, possible in a two-step version of the ELISA, with a sensitivity of about 1 unit IFN-alpha ml-1. Results of ELISA and bioassay agreed, except that the ELISA possibly produced false positive results in two out of a total of 91 sera negative in the bioassay. In addition, one of 23 sera positive in the bioassay was negative in the ELISA.     

Generation and characterization of monoclonal antibodies to equine NKp46

The immunoreceptor NKp46 is considered to be the most consistent marker of NK cells across mammalian species. Here, we use a recombinant NKp46 protein to generate a panel of monoclonal antibodies that recognize equine NKp46. The extracellular region of equine NKp46 was expressed with equine IL-4 as a recombinant fusion protein (rIL-4/NKp46) and used as an immunogen to generate mouse monoclonal antibodies (mAbs). MAbs were first screened by ELISA for an ability to recognize NKp46, but not IL-4, or the structurally related immunoreceptor CD16. Nine mAbs were selected and were shown to recognize full-length NKp46 expressed on the surface of transfected CHO cells as a GFP fusion protein. The mAbs recognized a population of lymphocytes by flow cytometric analysis that was morphologically similar to NKp46+ cells in humans and cattle. In a study using nine horses, representative mAb 4F2 labeled 0.8-2.1% PBL with a mean fluorescence intensity consistent with gene expression data. MAb 4F2+ PBL were enriched by magnetic cell sorting and were found to express higher levels of NKP46 mRNA than 4F2- cells by quantitative RT-PCR. CD3-depleted PBL from five horses contained a higher percentage of 4F2+ cells than unsorted PBL. Using ELISA, we determined that the nine mAbs recognize three different epitopes. These mAbs will be useful tools in better understanding the largely uncharacterized equine NK cell population.     

Isotype-specific antibodies in horses and dogs with immune-mediated hemolytic anemia

Classes of antibody bound to erythrocytes were determined using direct immunofluorescence (DIF) flow cytometry in 3 horses and 12 dogs with immune-mediated hemolytic anemia (IMHA). Background levels of antibody binding were determined in samples from 12 horses and 12 dogs that were free of clinical disease. The range of nonspecific binding of a fluorescein isothiocyanate (FITC)-conjugated goat anti-equine immunoglobulin G (IgG) was 19.9–36.7%, but was eliminated by the use of the F(ab)₂ fragment of FITC-conjugated goat anti-equine IgG. Background binding by other class-specific antibodies to equine and canine erythrocytes was negligible. The DIF results were compared to the direct antiglobulin (Coombs) test in 5 horses and 20 dogs with anemia. The former assay was more sensitive in dogs with IMHA than was the Coombs' test (100% versus 58%). In contrast, the Coombs' test had better specificity than the DIF assay (100% versus 87.5%, respectively). Using clinical parameters or response to therapy as the comparison, the positive and negative predictive values for the DIF test were 92% and 100% compared to the values of the Coombs' test of 100% and 62%. The DIF assay detected low levels of cells bound with antibody (<30%) in 5 dogs that were Coombs' test-negative. For both species, performance of the DIF test was independent of the prozone effect. Five dogs with IMHA had IgG and IgM on erythrocytes, 5 had IgG, and 2 had IgM. Three horses had surface-bound IgG, including a horse with suspected penicillin-induced IMHA, a foal with neonatal isoerythrolysis, and a foal with clostridial septicemia. The DIF method was valuable in monitoring the response to therapy in the foal with neonatal isoerythrolysis.     

The development of equine immunity: Current knowledge on immunology in the young horse

The development of equine immunity from the fetus to adulthood is complex. The foal's immune response and the immune mechanisms that they are equipped with, along with changes over the first months of life until the immune system becomes adult‐like, are only partially understood. While several innate immune responses seem to be fully functional from birth, the onset of adaptive immune response is delayed. For some adaptive immune parameters, such as immunoglobin (Ig)G1, IgG3, IgG5 and IgA antibodies, the immune response starts before or at birth and matures within 3 months of life. Other antibody responses, such as IgG4, IgG7 and IgE production, slowly develop within the first year of life until they reach adult levels. Similar differences have been observed for adaptive T cell responses. Interferon‐gamma (IFN‐γ) production by T helper 1 (Th1)‐cells and cytotoxic T cells starts shortly after birth with low level production that gradually increases during the first year of life. In contrast, interleukin‐4 (IL‐4) produced by Th2‐cells is almost undetectable in the first 3 months of life. These findings offer some explanation for the increased susceptibility of foals to certain pathogens such as Rhodococcus equi. The delay in Th‐cell development and in particular Th2 immunity during the first months of life also provides an explanation for the reduced responsiveness of young horses to most traditional vaccines. In summary, all immune components of adult horses seem to exist in foals but the orchestrating and regulation of the immune response in immature horses is strikingly different. Young foals are fully competent and can perform certain immune responses but many mechanisms have yet to mature. Additional work is needed to improve our understanding of immunity and immune regulation in young horses, to identify the preferred immune pathways that they are using and ultimately provide new preventive strategies to protect against infectious disease.     

Muscovy duck reovirus infection rapidly activates host innate immune signaling and induces an effective antiviral immune response involving critical interferons

Muscovy duck reovirus (MDRV) is a highly pathogenic virus in waterfowl and causes significant economic loss in the poultry industry worldwide. Because the host innate immunity plays a key role in defending against virus invasion, more and more attentions have been paid to the immune response triggered by viral infection. Here we found that the genomic RNA of MDRV was able to rapidly induce the production of interferons (IFNs) in host. Mechanistically, MDRV infection induced robust expression of IFNs in host mainly through RIG-I, MDA5 and TLR3-dependent signaling pathways. In addition, we observed that silencing VISA expression in 293T cells could significantly inhibit the secretion of IFNs. Remarkably, the production of IFNs was reduced by inhibiting the activation of NF-κB or knocking down the expression of IRF-7. Furthermore, our study showed that treatment of 293T cells and Muscovy duck embryo fibroblasts with IFNs markedly impaired MDRV replication, suggesting that these IFNs play an important role in antiviral response during the MDRV infection. Importantly, we also detected the induced expression of RIG-I, MDA5, TLR3 and type I IFN in Muscovy ducks infected with MDRV at different time points post infection. The results from in vivo studies were consistent with those in 293T cells infected with MDRV. Taken together, our findings reveal that the host can resist MDRV invasion by activating innate immune response involving RIG-I, MDA5 and TLR3-dependent signaling pathways that govern IFN production.     

Molecular cloning and characterization of equine Toll-like receptor 9

Innate immunity relies on a series of germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to detect conserved microbial components. TLR9 is typically expressed intracellularly in immune cells such as dendritic cells and recognizes unmethylated bacterial or viral cytosine-phosphate-guanine DNA (CpG-DNA). To investigate innate immune responses through TLR9 signaling pathway in horses, we cloned and characterized equine TLR9. Protein sequence analysis shows that equine TLR9 has a typically conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, three leucine-rich repeat (LRR) motifs, with greater than 82% identity to human, monkey, bovine, canine, feline, porcine and ovine orthologs. Equine TLR9 mRNA expression was characterized for spleen, lymph node, and peripheral blood leukocyte samples. Flow cytometric analysis of equine TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in PMNs, CD4(+) and CD8(+) T-lymphocytes as well as other leukocytes; similar to human TLR9 expression. The conservation of equine TLR9 and high expression profile in leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity.     

Use of oromucosally administered interferon-alpha in the prevention and treatment of animal diseases

Interferon-alpha (IFN-alpha) is well known as a clinically effective antiviral and antineoplastic therapeutic agent. It has also been shown to have immunoregulatory properties. IFN-alpha stimulates a cell-mediated innate immune response and then participates in the transition of the initial host innate response to an effective adaptive immune response. IFN-alpha is produced in small quantities in nasal secretions during viral infections, prompting many authors to suggest that low-dose oromucosal administration of IFN-alpha effectively mimics nature. Moreover, the injectable high-dose interferon therapy currently approved for various human disorders causes numerous side effects. By contrast, oromucosal administration of IFN-alpha is not associated with toxic effects. Another distinct advantage is ease of administration: the IFN can be dissolved in drinking water or administered by nebulization to the oral or nasal cavity. This review describes the current state of knowledge concerning orally administered IFN-alpha, of both human and animal origin, as a prophylactic or therapeutic agent in veterinary medicine. We present the effects of IFN-alpha in such animals as cattle, pigs, horses, cats, dogs and chickens, and attempt to explain its mechanism of action following oromucosal administration. It is hoped that this review of the medical literature on the use of IFN-alpha in animals will give practitioners a better understanding of the challenges and benefits of using this interesting cytokine in clinical practice.     

Induction of a Th-1-biased IgG subclass response against equine herpesvirus type 1 in horses previously infected with type 4 virus

An immunoglobulin G (IgG) subclass response against equine herpesvirus type 1 (EHV-1) infection was investigated in horses that were na?ve to EHV-1/4 and those that had previously been exposed to EHV-4. The IgG subclass response was determined by an ELISA using EHV-1-specific recombinant gG protein as an antigen. In most horses na?ve to EHV-1/4, IgGa, IgGb, and IgG(T) were induced after experimental infection with EHV-1. In contrast, a subclass response dominated by IgGa and IgGb, with no apparent increase in IgG(T), was observed after EHV-1 infection in horses previously infected with EHV-4. Horses naturally infected with EHV-1 in the field showed similar responses. These results indicated that pre-infection with EHV-4 induced a Th-1-biased IgG subclass response against subsequent EHV-1 infection.     

Sandwich ELISA system for cartilage oligomeric matrix protein in equine synovial fluid and serum

Reasons for performing study: Measurement of cartilage oligomeric matrix protein (COMP) in serum has potential for diagnosis of equine osteoarthritis (OA), but clinical use is currently limited by the lack of specificity of an inhibition ELISA as well as by baseline increases due to exercise. Improved methods for ELISA with increased antigen specificity and sensitivity are therefore required for reliable measurement. Hypothesis: Measurement of the serum level of COMP by sandwich ELISA allows identification of horses with OA. Methods: New monoclonal antibodies (mAbs) were elicited against equine cartilage COMP, their epitopes were determined and a sandwich ELISA was developed. The concentrations of COMP in synovial fluid (SF; n = 100) and sera (n = 100) from OA cases were measured by sandwich ELISA as well as by inhibition ELISA and compared with concentrations in normal joints (n = 95) and horses (n = 50). Results: Immunoblots of enzymatically cleaved COMP showed that the new mAbs recognised different epitopes located on a 20 kDa fragment between K⁶³ and K²³⁸ of the EGF‐like repeats. Inhibition ELISA with any mAb detected significantly increased levels of COMP in OA SF compared with normal SF, whereas no significant difference was detected between serum levels of COMP in OA and normal horses. Conversely, sandwich ELISA with the combination of unlabelled 2A11 × biotinylated 11F10 mAbs detected a significant increase in COMP levels in both serum and SF from OA cases compared with levels in normal animals. Conclusions and potential relevance: Measurement of serum COMP with sandwich ELISA may be useful in identifying horses with OA.     

Expression of a 4-(hydroxy-3-nitro-phenyl) acetyl (NP) specific equi-murine IgE antibody that mediates histamine release in vitro and a type I skin reaction in vivo

Due to characteristic clinical signs, immunoglobulins of isotype E (IgE) are believed to be involved in several allergic diseases of the horse. To date, closer investigations have been hampered by the fact that neither purified equine IgE nor anti-equine IgE monoclonal antibodies were available for IgE isotype determination. As an approach to solve this problem, we constructed a stable cell line (EqE6) that expresses recombinant equi-murine IgE specific for 4-(hydroxy-3-nitro-phenyl) acetyl (NP). Biochemical analysis of the purified protein revealed a highly glycosilated IgE monomer of approximately 230,000 Da. The biological ability of the NP-IgE to mediate histamine release after crosslinking with antigen was demonstrated in vitro using equine blood leucocytes. In vivo, the intradermal application of NP-IgE followed by antigen crosslinking induced a type I hypersensitivity skin reaction in horses. Both results indicate that the recombinant NP-IgE contains an intact and functional Fc(epsilon) RI binding site and mediates effector functions in equine basophils and cutaneous mast cells. This equi-murine IgE can be used for the production of IgE-specific polyclonal and monoclonal antibodies. In addition, the NP specificity allows the antigen-specific activation of equine Fc(epsilon)-receptor-expressing cells, such as mast cells and basophils. This property could be used to investigate IgE-mediated mechanisms for a better understanding of equine type I allergic diseases.