Cloning and expression of recombinant equine interleukin-3 and its effect on sulfidoleukotriene and cytokine production by equine peripheral blood leukocytes

Interleukin-3 is a growth and differentiation factor for various hematopoietic cells. IL-3 also enhances stimulus-dependent release of mediators and cytokine production by mature basophils. Function of IL-3 has not been studied in horses because of lack of horse-specific reagents. Our aim was to produce recombinant equine IL-3 and test its effect on sulfidoleukotriene and cytokine production by equine peripheral blood leukocytes (PBL).Equine IL-3 was cloned, expressed in E. coli and purified. PBL of 19 healthy and 20 insect bite hypersensitivity (IBH)-affected horses were stimulated with Culicoides nubeculosus extract with or without IL-3. Sulfidoleukotriene (sLT) production was measured in supernatants by ELISA and mRNA expression of IL-4, IL-13 and thymic stromal lymphopoietin (TSLP) assessed in cell lysate by quantitative real-time PCR.Recombinant equine IL-3 (req-IL-3) had a dose dependent effect on sLT production by stimulated equine PBL and significantly increased IL-4, IL-13 and TSLP expression compared to non-primed cells.IL-3 priming significantly increased Culicoides-induced sLT production in IBH-affected but not in non-affected horses and was particularly effective in young IBH-affected horses (≤3 years).A functionally active recombinant equine IL-3 has been produced which will be useful for future immunological studies in horses. It will also allow improving the sensitivity of cellular in vitro tests for allergy diagnosis in horses.     

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.     

Production of monoclonal antibodies specific for native equine IgE and their application to monitor total serum IgE responses in Icelandic and non-Icelandic horses with insect bite dermal hypersensitivity

Immunoglobulin E forms a minor component of serum antibody in mammals. In tissues IgE is bound by FcvarepsilonRI receptors on the surface of mast cells and mediates their release of inflammatory substances in response to antigen. IgE and mast cells have a central role in immunity to parasites and the pathogenesis of allergic diseases in horses and other mammals. This paper describes the production of several novel monoclonal antibodies that detect native equine IgE in immunohistology, ELISA and Western blotting. An antigen capture ELISA to quantify equine IgE in serum has been developed using two of these antibodies. The mean serum IgE concentration of a group of 122 adult horses was 23,523ng/ml with a range of 425-82,610ng/ml. Total serum IgE of healthy horses was compared with that of horses with insect bite dermal hypersensitivity (IBDH) an allergic reaction to the bites of blood feeding insects of Culicoides or Simulium spp. IBDH does not occur in Iceland where Culicoides spp. are absent, but following importation into mainland Europe native Icelandic horses have an exceptionally high incidence of this condition. In the present study Icelandic horses with IBDH had significantly higher total IgE than healthy Icelandic horse controls (P<0.05). By contrast in horses of other breeds the difference in total serum IgE between those affected with IBDH and healthy controls was not statistically significant. Total serum IgE was also monitored in a cohort of Icelandic horses prior to import into Switzerland and for a period of 3 years thereafter. High levels of serum IgE were present in all horses at the start of the study but dropped in the first year after import. Thereafter the total serum IgE remained low in Icelandic horses that remained healthy but rose significantly (P<0.05) in those that developed IBDH. These results support the conclusion that IBDH is a type I hypersensitivity response to insect allergens but indicate that IBDH in Icelandic horses may have a different pathogenesis from the same condition in other breeds.     

Characterisation and quantification of equine interferon gamma

Interferon-gamma (IFN-gamma) is a key cytokine in cell-mediated immunity. To measure IFN-gamma production of equine lymphocytes (eqIFN-gamma), we developed a quantitative ELISA. Monoclonal antibodies (mAb) were produced against bacterially derived eqIFN-gamma. The mAbs recognised recombinant and lymphocyte-derived eqIFN-gamma in ELISA, Western blotting, as well as flow cytometric and microscopic analysis. In contrast to bacterially derived material, mammalian and insect cell-derived eqIFN-gamma was biologically active but could be neutralised by one of the monoclonal antibodies. Unexpectedly, glycosylation seemed to be required for antiviral activity of eqIFN-gamma.     

Monoclonal antibodies to equine CD23 identify the low-affinity receptor for IgE on subpopulations of IgM+ and IgG1+ B-cells in horses

CD23, also called FcεRII, is the low-affinity receptor for IgE and has first been described as a major receptor regulating IgE responses. In addition, CD23 also binds to CD21, integrins and MHC class II molecules and thus has a much wider functional role in immune regulation ranging from involvement in antigen-presentation to multiple cytokine-like functions of soluble CD23. The role of CD23 during immune responses of the horse is less well understood. Here, we expressed equine CD23 in mammalian cells using a novel IL-4 expression system. Expression resulted in high yield of recombinant IL-4/CD23 fusion protein which was purified and used for the generation of monoclonal antibodies (mAbs) to equine CD23. Seven anti-CD23 mAbs were further characterized. The expression of the low-affinity IgE receptor on equine peripheral blood mononuclear cells was analyzed by flow cytometric analysis. Cell surface staining showed that CD23 is mainly expressed by a subpopulation of equine B-cells. Only a very few equine T-cells or monocytes expressed CD23. CD23(+) B-cells were either IgM(+) or IgG1(+) cells. All CD23(+) cells were also positive for cell surface IgE staining suggesting in vivo IgE binding by the receptor. Two of the CD23 mAbs detected either the complete extracellular region of CD23 or a 22kDa cleavage product of CD23 by Western blotting. The new anti-CD23 mAbs provide valuable reagents to further analyze the roles of CD23 during immune responses of the horse in health and disease.     

A monoclonal antibody to equine interleukin-4

Interleukin-4 (IL-4) is secreted by T helper type 2 cells, mast cells, basophils and eosinophils. Detection of IL-4 can contribute the evaluation of cellular immune responses during infectious diseases, immunological disorders or vaccination. We used recombinant equine IL-4 to generate a monoclonal antibody (mAb) to equine IL-4. The mAb detected recombinant IL-4 in mammalian cells transfected with different plasmids containing IL-4 cDNA. After mitogen stimulation of equine peripheral blood mononuclear cells, an intracellular protein was recognized by the new mAb in 1–2% of lymphocytes using flow cytometric analysis. In the presence of the secretion blocker Brefeldin A, the protein accumulated and was detected in 4–8% of lymphocytes stimulated with phorbol 12-myristate 13-acetate and ionomycin. Double staining with the new mAb and T-cell or B-cell markers identified a subpopulation of CD4⁺ T-cells expressing the protein recognized by the mAb. In addition, the protein was detectable in cell culture supernatants of mitogen stimulated cells by ELISA when using the new mAb for coating of the plates and a polyclonal antiserum to equine IL-4 for detection. In conclusion, the new mAb detects equine IL-4 and can be used for intracellular staining and ELISA to measure this important cytokine.     

Monoclonal antibodies to equine interferon-alpha (IFN-alpha): new tools to neutralize IFN-activity and to detect secreted IFN-alpha

Interferon-alpha (IFN-alpha) is a type I interferon that is secreted during the early stages of the innate immune response and is often induced upon infection with viral pathogens. IFN-alpha production affects multiple downstream events influencing both innate and adaptive immune responses. Here, we describe the expression of an equine rIFN-alpha/IgG4 fusion protein in mammalian cells. The anti-viral activity of rIFN-alpha/IgG4 was found to be 70-fold higher than that of a previously described IFN-gamma/IgG1 as tested by bioassay. The purified rIFN-alpha was subsequently used for the generation of six monoclonal antibodies (mAbs) to equine IFN-alpha. Four of these mAbs inhibited the protective anti-viral effect of equine leukocyte IFN in bioassays. One mAb (clone 240-2) showed a high-neutralizing capacity. An ELISA was established using two anti-equine IFN-alpha mAbs (clones 29B and 240-2) and its analytical sensitivity for was found to be around 800 pg/ml and 3 U/ml for rIFN-alpha and equine leukocyte IFN, respectively. When analyzing samples with a likely dominance of IFN-alpha among type I IFNs, such as supernatants from equine peripheral blood mononuclear cells stimulated with CpG-oligodeoxyribonucleotides, the results obtained by ELISA and IFN bioassay showed a high agreement (r(2)(sp)=0.98). When analyzing samples likely containing a mixture of type I IFNs, such as serum and nasal secretions from virally infected horses, the ELISA only detected some of the IFN-activity recorded in the bioassay. Overall, the data showed that the new anti-equine IFN-alpha mAbs are valuable tools to detect native IFN-alpha for further characterization of the early innate immune response and anti-viral immunity in horses.     

Generation and characterization of murine monoclonal antibodies specific for bovine immunoglobulin E

Monoclonal antibodies were produced against serum-derived bovine immunoglobulin E (IgE). Culture supernatants of hybridomas were initially screened by enzyme-linked immunosorbent assay (ELISA). Supernatant-derived antibodies were concentrated and further characterized using ELISA, reverse cutaneous anaphylaxis, immunohistochemical staining, and immunoblotting of IgE-containing samples separated by SDS-polyacrylamide gel electrophoresis (PAGE). Eight monoclonal antibodies showed specificity for bovine epsilon immunoglobulin heavy chain. Two antibodies (E2 and E32) reacted in immunoblots of SDS-PAGE of serum IgE under reducing conditions. Additionally, E2, E5, and E32 detected epsilon chain in serum separated by SDS-PAGE and then renatured. Antigen-specific IgE was detected in Western blots by E5 and E32. Immunoperoxidase staining of IgE-containing cells in mesenteric lymph node sections was detected with E5, E21 and E32. All eight antibodies produced positive reverse cutaneous anaphylaxis reactions in calf skin. All functioned well in ELISA as a plate-sensitizing reagent for quantitation of total IgE; E5 and E32 worked well as a primary antibody in antigen-specific IgE assays. These antibodies will be useful in research applications and in diagnostic assays.     

Expression and characterisation of equine interleukin 2 and interleukin 4

In the present study, we describe the expression of equine IL2 and IL4. The cDNA of equine IL2 or IL4 was cloned in a mammalian expression vector, containing c-terminal myc- and six histidines His(6)-epitopes for recognition and purification of equine cytokines. The vector constructs were used for transfection of chinese hamster ovary (CHO) cells. Purified equine cytokines were characterised by western blotting. Equine IL2 was secreted with a molecular weight of approximately 17.1kDa, whereas IL4 was expressed in three different sizes of 17.1, 19.6 and 22.1kDa, probably due to different glycosylation modifications. The biological activities of both cytokines were tested by proliferation assays using leukoagglutinin (LAG) prestimulated equine PBMC. Both, equine IL2 and IL4 induced dose-dependent lymphocyte proliferation. In contrast to IL4, IL2 supported the proliferation of B cells.     

The effects of vaccination with tissue culture-derived viral vaccines on detection of antibodies to equine arteritis virus by enzyme-linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of serum antibodies to equine arteritis virus (EAV). Results from this assay produced a good correlation with results from virus neutralisation tests in horses which had not been regularly vaccinated with commercially available mammalian tissue culture-derived viral vaccines. Vaccination of some horses with tissue culture-derived vaccines induced the formation of antibodies to bovine serum. These antibodies reacted with the bovine protein contaminants in the EAV ELISA antigen, producing false-positive results. Non-viral protein contaminants were found to be closely associated with EAV in that they co-purified with the virus during gradient centrifugation.     

Evaluation of a polyvalent enzyme-linked immunosorbent assay incorporating a recombinant p44 antigen for diagnosis of granulocytic ehrlichiosis in dogs and horses

OBJECTIVE: To develop and evaluate a polyvalent ELISA incorporating a highly specific recombinant antigen (p44) for diagnosis of granulocytic ehrlichiosis in dogs and horses. ANIMALS: 32 dogs and 43 horses. PROCEDURE: Results of the ELISA were compared with results of indirect fluorescent antibody (IFA) staining and western immunoblotting incorporating whole-cell antigen. RESULTS: For the canine and equine samples, percentages of samples with positive IFA staining, western immunoblotting, and ELISA results were similar. For 29 (91 %) canine samples and 30 (70%) equine samples, results of IFA staining, western immunoblotting, and the ELISA were in complete agreement. Results of the ELISA for 3 canine serum samples known to contain antibodies to Ehrlichia canis and 12 equine serum samples known to contain antibodies to E risticii were negative. CONCLUSIONS AND CLINICAL RELEVANCE: Results of the present study suggest that a polyvalent ELISA incorporating a recombinant p44 antigen is suitable for detecting antibodies to E equi in dogs and horses.     

Asia 1型口蹄疫病毒VP1基因的表达鉴定及其多抗制备

本研究旨在表达口蹄疫病毒(FMDV)的VP1全基因并制备特异性的多克隆抗体。利用PCR方法扩增Asia 1 IND 49197株VP1全基因,将其克隆至原核表达载体pET-30a(+)中,在大肠杆菌BL21中进行表达。SDS-PAGE结果显示表达产物分子量约为31.6ku,以包涵体的形式存在。通过Ni-NTA Purification System纯化后进行western blot和间接ELISA分析,结果显示重组蛋白能够被FMD阳性血清识别,具有良好的反应性。将纯化的重组蛋白免疫新西兰白兔制备多克隆抗体,ELISA测定抗体效价为1∶20480,病毒中和试验测定抗体效价为1∶64。本研究所表达的VP1蛋白可用于开发检测Asia1口蹄疫抗体的诊断试剂,所制备的多克隆抗体为进一步研究VP1的结构、功能以及抗原表位的鉴定提供了条件。     

Cloning and expression of the extra-cellular part of the alpha chain of the equine high-affinity IgE receptor and its use in the detection of IgE

The high-affinity receptor for IgE (FcepsilonRI) plays a central role in IgE-mediated allergic reactions. Cross-linking of FcepsilonRI by IgE-antigen complexes results in the activation of mast cells and basophils and is thought to contribute to the immunopathology of Heaves, a chronic obstructive pulmonary disease of horses. Recombinant protein corresponding to the extra-cellular portion of the FcepsilonRI alpha subunit, cloned and sequenced previously, was expressed using both mammalian cells and insect cells. The yield of expressed protein was considerably greater using insect cells and the baculovirus expression system. The recombinant proteins differed in size between the two systems, presumably due to differences in the extent of glycosylation. However, recombinant protein from both cell systems bound equine IgE present in bronchoalveolar lavage fluid from horses with Heaves. These results suggest that the recombinant extra-cellular part of FcepsilonRI should be a useful tool with which to study equine IgE responses.     

Interleukin-1 stimulation of equine articular cells

Prostaglandin E2 (PGE2) and stromelysin are produced by equine chondrocytes and synovial cells in vitro in response to recombinant human (rh) interleukin-1 (IL-1) alpha and beta, and equine mononuclear cell supernatants (MCS) containing IL-1. However, culture conditions are important. PGE2 concentrations increase in proportion to the concentration of fetal calf serum (FCS) in the culture medium, whereas stromelysin concentrations are inversely proportional to the concentration of FCS. Equine MCS, containing a lower concentration of IL-1 than the concentration of rhIL-1 used in these experiments, stimulated production of much higher levels of PGE2 than rhIL-1. In addition, equine MCS induced the production of broadly similar levels of PGE2 by both chondrocytes and synovial cells, whereas rhIL-1 was more active on equine synovial cells than equine chondrocytes. Although equine MCS induced both stromelysin and PGE2 production by equine articular cells, on the whole rhIL-1 failed to induce stromelysin production. This supports previous observations of species restrictions in the activity of human IL-1 on equine cells. Therefore, experiments using mammalian cells and heterologous IL-1 should be interpreted with caution.     

抗东方马脑炎病毒结构蛋白E2单克隆抗体的制备及其抗原表位的鉴定

为制备抗东方马脑炎病毒(EEEV)结构蛋白E2的单克隆抗体(MAb)并鉴定其抗原表位,本研究以Bac-to-Bac真核表达系统表达EEEV E2蛋白,纯化后作为免疫原免疫BALB/c小鼠,取其脾淋巴细胞与小鼠骨髓瘤细胞SP2/0进行融合.以原核表达载体pET-30a表达并纯化的EEEV E2蛋白作为包被抗原建立间接ELISA方法筛选杂交瘤细胞,获得4株稳定分泌抗EEEV E2蛋白MAbs的杂交瘤细胞株,分别命名为6F3、6F11、7C11、8B11.Western blot与间接免疫荧光试验结果表明,获得的4株MAbs均与EEEV呈阳性反应,而与西方马脑炎病毒、乙型脑炎病毒以及登革热病毒1型～4型呈阴性反应.利用部分重叠的原核表达的短肽对E2蛋白抗原表位进行鉴定,初步确定MAb 6F11、7C11和8B11识别的抗原表位均为E-33 (321EGLEYTWGNHPPKRVW336),而MAb 6F3无短肽与其反应,推测可能为构象表位.本研究结果为建立EEEV型特异性检测方法、研究E2蛋白结构功能及该病的进一步防制奠定了基础.     

Detection of IgG and IgE serum antibodies to Culicoides salivary gland antigens in horses with insect dermal hypersensitivity (sweet itch).

We postulated that all horses exposed to the bites of Culcoides (midges) would have an antibody response to the antigen secreted in Culcoides saliva, but that IgE antibody would be restricted to allergic individuals. Using immunohistology on sections of fixed Culicoides, we have demonstrated the presence of antibodies in horse serum which recognise Culicoides salivary glands. Antibodies were detected in the serum of horses with insect dermal hypersensitivity and in the serum of normal horses exposed to Culicoides bites. In contrast, no antibodies were detected in serum from native Icelandic ponies which had not been exposed to Culicoides. Anti-salivary gland IgG antibodies were detected in serum from both allergic and healthy horses exposed to Culicoides. IgE antibodies were only detected in horses with signs of insect dermal hypersensitivity, they were not found in serum of healthy controls nor in the serum of horses with a history of hypersensitivity but in remission at the time of sampling. Using western blotting we confirmed the presence of antibodies to Culicoides antigens and demonstrated that individual horses react to different numbers of antigens. This paper demonstrates the ability of serum from allergic horses to detect Culcoides antigens and will enable further studies to isolate and characterise the allergens.     

Chronic equine laminitis is characterised by loss of GLUT1, GLUT4 and ENaC positive laminar keratinocytes

REASONS FOR PERFORMING STUDY: Equine laminitis is a multifactorial connective tissue disorder with major implications for the welfare of horses. There are few published studies on phenotypic markers for identification of equine laminar keratinocytes using immunohistochemical techniques. OBJECTIVES: To establish whether the epithelial sodium channel (ENaC) and the GLUT1 and GLUT4 facilitative glucose transporters may be used as phenotypic markers for identification of equine laminar keratinocytes using immunohistochemical techniques to monitor changes in the keratinocyte population in laminitis. METHODS: Histology and immunohistochemistry using polyclonal antibodies to the alpha subunit of ENaC (alphaENaC), GLUT1 and GLUT4 were used to compare the distribution of these proteins in normal and laminitic equine laminae. RESULTS: Immunohistochemistry with antibodies to alphaENaC, GLUT1 and GLUT4 confirmed the abundant expression of all 3 membrane proteins in healthy laminar keratinocytes. However, in laminitis, the Haematoxylin Van Gieson (HVG) technique revealed disordered laminar arrays and replacement with fibrous scar tissue. Immunostaining of laminitic samples confirmed the loss of alphaENaC, GLUT1 and GLUT4 positive keratinocytes. Other connective tissue cells did not stain positive for these proteins. CONCLUSIONS: This is the first report of alphaENaC and GLUT1/GLUT4 protein expression in equine laminar keratinocytes, which also confirms that the loss of laminar structure and function in chronic laminitis is accompanied by the loss of laminar keratinocytes. POTENTIAL RELEVANCE: alphaENaC, GLUT1 and GLUT4 may be used as phenotypic markers of metabolically active, differentiated equine laminar keratinocytes. Further in vitro studies are necessary to determine the effects of hypoxia, bacterial endotoxins, vasoactive amines, lactic acid and prostaglandins on the expression and activity of these plasma membrane keratinocyte markers.     

马传染性贫血病毒p26蛋白单克隆抗体的制备

为制备马传染性贫血病毒(EIAV)的单克隆抗体(MAb),本研究以原核表达并纯化的EIAV p26蛋白作为免疫原免疫8周龄的BALB/c小鼠,利用淋巴细胞杂交瘤技术,建立抗EIAV衣壳蛋白p26抗体的杂交瘤细胞.应用重组p26蛋白为抗原建立ELISA筛选方法,获得两株能够稳定分泌特异性MAb的杂交瘤细胞株,分别命名为E11和F8.经试验证明,这些抗p26MAb均能够与感染驴胎皮肤细胞的EIAV和经SDS-PAGE分离的EIAV总蛋白中的p26蛋白结合.这两株p26蛋白MAb的获得将为EIAV的检测及鉴别诊断奠定基础.     

Domain mapping and comparative binding features of eight dog IgE-specific reagents in ELISA, immunoblots, and immunohistochemistry.

Eight dog IgE-specific reagents including monoclonal and polyclonal antibodies (Ab) and a cross-reactive alpha chain of the human high affinity IgE receptor were mapped to recombinant fragments of the second (IgEf2) and third/fourth (IgEf3/4) domains of the dog IgE heavy chain. In ELISA, five out of eight reagents reacted to solid-phase bound IgEf2, of which two polyclonal Ab bound in addition to IgEf3/4. All Ab which recognized at least one recombinant IgE fragment, also bound to IgE in ELISA, immunoblots, and immunohistochemistry. In contrast, only one monoclonal Ab, that did not bind to the recombinant IgE fragments, reacted with immunoblots of serum and immunohistochemistry. The alpha chain could only be applied to ELISA with serum IgE. Furthermore, there was a wide range of heat-lability of binding reactions. Comparative analysis of available dog IgE-specific reagents enables more in-depth functional studies on IgE-mediated phenomena in dogs, and helps to further establish the dog as an animal model for allergy research.     

奶牛早孕因子重组蛋白的原核表达、纯化及多克隆抗体的制备

本研究旨在表达、纯化奶牛早孕因子重组蛋白及制备多克隆抗体.本试验构建奶牛早孕因子重组蛋白原核表达载体pET32a-EPF,转化至大肠杆菌BL21(DE3)内进行诱导表达,SDS-PAGE切胶纯化的早孕因子重组蛋白免疫BALB/c小鼠制备多克隆抗体,并用Western blotting和ELISA对重组蛋白及抗体进行检测.结果显示,成功表达并纯化了奶牛早孕因子重组蛋白,重组蛋白分子质量约37 ku,表达量约占菌体总蛋白的48.6%,纯化后目的蛋白纯度可达93%,重组蛋白可与所制备的多克隆抗体结合,ELISA测定的抗体效价为1:12 800.结果表明,本研究成功表达并纯化了奶牛早孕因子重组蛋白,为研究奶牛早孕诊断奠定基础.