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Characterization of monoclonal antibodies to equine interleukin-10 and detection of T regulatory 1 cells in horses.
Abstract: 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.
Publication Date: 2007-10-24 PubMed ID: 18061278DOI: 10.1016/j.vetimm.2007.10.012Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- N.I.H.
- Extramural
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- Non-P.H.S.
Summary
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This research paper details the creation and analysis of three antibodies that target Equine Interleukin-10 (IL-10), an important regulator in horse immune responses. The results confirmed the specificity of the antibodies for equine IL-10 and their potential for detecting IL-10 in controlled laboratory conditions.
Creation and Analysis of Monoclonal Antibodies
- The study produced three monoclonal antibodies (mAbs), specifically against equine IL-10. IL-10 is a critical immune system protein that suppresses inflammatory responses and impedes the activation of certain immune cells.
- The specificity of these antibodies was then analyzed using diverse recombinant equine cytokine/IgG fusion proteins, confirming that the antibodies were unique to equine IL-10.
Antibody Use in ELISA
- Two of the three mAbs were selected for use in an Enzyme-Linked Immunosorbent Assay (ELISA). This illustrated their capacity to detect IL-10 secretions in horse peripheral blood mononuclear cells (PBMC) in certain conditions.
- The ELISA demonstrated a sensitive detection level for secreted IL-10 at around 200pg/ml.
Flow Cytometry Analysis
- The research also evaluated the intracellular IL-10 production in horse PBMCs using flow cytometry, a technique used to analyze characteristics of cells in a sample.
- For stimulation, PBMCs were exposed to a variety of compounds including Phorbol 12-myristate 13-acetate (PMA) and ionomycin, which were both combined with the secretion blocker Brefeldin A.
- PMA-stimulated lymphocytes had a distinguishable presence of IL-10, while it was absent in the control samples.
Identification of Immune Cell Types
- Approximately 80% of detected IL-10 cells were CD4(+) lymphocytes while around 15% were CD8(+) cells.
- With the addition of other proteins for double staining, 80% of IL-10 positive cells were also shown to be interferon-gamma positive. Around 5% of the cells were found to be positive for both IL-10 and IL-4.
- The experiment figured at least 60% of IL-10 and interferon-gamma positive cells were CD4(+) lymphocytes. This profile aligns with the regulatory T1 cell phenotype.
Summary of Findings
- In conclusion, the newly produced antibodies could effectively denote native equine IL-10 using ELISA and flow cytometry. This finding paved the way for the further exploration of IL-10’s significant regulatory function in horse immune systems.
Cite This Article
APA
Wagner B, Hillegas JM, Brinker DR, Horohov DW, Antczak DF.
(2007).
Characterization of monoclonal antibodies to equine interleukin-10 and detection of T regulatory 1 cells in horses.
Vet Immunol Immunopathol, 122(1-2), 57-64.
https://doi.org/10.1016/j.vetimm.2007.10.012 Publication
Researcher Affiliations
- Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. bw73@cornell.edu
MeSH Terms
- Animals
- Antibodies, Monoclonal / immunology
- Enzyme-Linked Immunosorbent Assay
- Horses / immunology
- Interferon-gamma / biosynthesis
- Interleukin-10 / blood
- Interleukin-10 / immunology
- Interleukin-4 / biosynthesis
- T-Lymphocytes, Regulatory / classification
- T-Lymphocytes, Regulatory / immunology
- Tetradecanoylphorbol Acetate / pharmacology
Grant Funding
- 1 R01 HD049545 / NICHD NIH HHS
Citations
This article has been cited 17 times.- Chaimbeul SF, Rodrigues NNP, Thurston DD, Scoggin KE, Janes J, Jacobs CA, MacLeod JN, Stone AV, Menarim BC. PPARγ Agonism Modulates Synovial Macrophage and Cartilage Responses in an Equine Model of Synovial Inflammation-Implications for Joint Therapy. Biomolecules 2025 Sep 1;15(9).
- Harman RM, Sipka A, Oxford KA, Oliveira L, Huntimer L, Nydam DV, Van de Walle GR. The mammosphere-derived epithelial cell secretome modulates neutrophil functions in the bovine model. Front Immunol 2024;15:1367432.
- Giessler KS, Goehring LS, Jacob SI, Davis A, Esser MM, Lee Y, Zarski LM, Weber PSD, Hussey GS. Impact of the host immune response on the development of equine herpesvirus myeloencephalopathy in horses. J Gen Virol 2024 May;105(5).
- Holmes CM, Babasyan S, Wagner B. Neonatal and maternal upregulation of antileukoproteinase in horses. Front Immunol 2024;15:1395030.
- Keller LE, Tait Wojno ED, Begum L, Fortier LA. T Helper 17-Like Regulatory T Cells in Equine Synovial Fluid Are Associated With Disease Severity of Naturally Occurring Posttraumatic Osteoarthritis. Am J Sports Med 2023 Mar;51(4):1047-1058.
- Sipka A, Mann S, Babasyan S, Freer H, Wagner B. Development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ concentrations in plasma and cell culture supernatant. JDS Commun 2022 May;3(3):207-211.
- Keller LE, Tait Wojno ED, Begum L, Fortier LA. Interleukin-6 neutralization and regulatory T cells are additive in chondroprotection from IL-1β-induced inflammation. J Orthop Res 2023 May;41(5):942-950.
- Gressler AE, Lübke S, Wagner B, Arnold C, Lohmann KL, Schnabel CL. Comprehensive Flow Cytometric Characterization of Bronchoalveolar Lavage Cells Indicates Comparable Phenotypes Between Asthmatic and Healthy Horses But Functional Lymphocyte Differences. Front Immunol 2022;13:896255.
- Menarim BC, Gillis KH, Oliver A, Ngo Y, Werre SR, Barrett SH, Rodgerson DH, Dahlgren LA. Macrophage Activation in the Synovium of Healthy and Osteoarthritic Equine Joints. Front Vet Sci 2020;7:568756.
- Larson EM, Babasyan S, Wagner B. Phenotype and function of IgE-binding monocytes in equine Culicoides hypersensitivity. PLoS One 2020;15(5):e0233537.
- Saldinger LK, Nelson SG, Bellone RR, Lassaline M, Mack M, Walker NJ, Borjesson DL. Horses with equine recurrent uveitis have an activated CD4+ T-cell phenotype that can be modulated by mesenchymal stem cells in vitro. Vet Ophthalmol 2020 Jan;23(1):160-170.
- Schnabel CL, Steinig P, Koy M, Schuberth HJ, Juhls C, Oswald D, Wittig B, Willenbrock S, Murua Escobar H, Pfarrer C, Wagner B, Jaehnig P, Moritz A, Feige K, Cavalleri JM. Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent. BMC Vet Res 2015 Jun 23;11:140.
- Ayyildiz-Tamis D, Avcı K, Deliloglu-Gurhan SI. Comparative investigation of the use of various commercial microcarriers as a substrate for culturing mammalian cells. In Vitro Cell Dev Biol Anim 2014 Mar;50(3):221-31.
- Lewis DH, Chan DL, Pinheiro D, Armitage-Chan E, Garden OA. The immunopathology of sepsis: pathogen recognition, systemic inflammation, the compensatory anti-inflammatory response, and regulatory T cells. J Vet Intern Med 2012 May-Jun;26(3):457-82.
- Pinheiro D, Singh Y, Grant CR, Appleton RC, Sacchini F, Walker KR, Chadbourne AH, Palmer CA, Armitage-Chan E, Thompson I, Williamson L, Cunningham F, Garden OA. Phenotypic and functional characterization of a CD4(+) CD25(high) FOXP3(high) regulatory T-cell population in the dog. Immunology 2011 Jan;132(1):111-22.
- Wagner B, Burton A, Ainsworth D. Interferon-gamma, interleukin-4 and interleukin-10 production by T helper cells reveals intact Th1 and regulatory TR1 cell activation and a delay of the Th2 cell response in equine neonates and foals. Vet Res 2010 Jul-Aug;41(4):47.
- de Mestre A, Noronha L, Wagner B, Antczak DF. Split immunological tolerance to trophoblast. Int J Dev Biol 2010;54(2-3):445-55.
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