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Topic:Cytokines
Cytokines are small proteins that are important in cell signaling and are produced by various cell types in horses, particularly during immune responses. These molecules facilitate communication between cells and play a role in regulating inflammation, immune function, and hematopoiesis. In horses, cytokines are involved in numerous physiological and pathological processes, including infection, inflammation, and wound healing. Examples of cytokines studied in equine research include interleukins, interferons, and tumor necrosis factors. Their expression levels can change in response to disease states, making them potential biomarkers for assessing equine health and disease progression. This page compiles peer-reviewed research studies and scholarly articles that explore the role, regulation, and clinical implications of cytokines in equine health.
Immunologic testing of xeno-derived osteochondral grafts using peripheral blood mononuclear cells from healthy human donors. One means of treating osteoarthritis is with autologous or allogeneic osteochondral grafts. The purpose of this study was to evaluate the innate immunological response in humans toward xeno-derived osteochondral grafts that have been partially or entirely treated by the photooxidation process. Methods: The antigens tested included bovine, porcine, ovine and equine osteochondral samples that have been treated in successive steps of photooxidation. ELISPOT assays were used to evaluate the production of IL-1, IL-4, IL-6, IL-10, IL-12 and TNF-alpha by human monocytes in response to the antigens. R...
Early detection of dominant Env-specific and subdominant Gag-specific CD8+ lymphocytes in equine infectious anemia virus-infected horses using major histocompatibility complex class I/peptide tetrameric complexes. Cytotoxic T lymphocytes (CTL) are critical for control of lentiviruses, including equine infectious anemia virus (EIAV). Measurement of equine CTL responses has relied on chromium-release assays, which do not allow accurate quantitation. Recently, the equine MHC class I molecule 7-6, associated with the ELA-A1 haplotype, was shown to present both the Gag-GW12 and Env-RW12 EIAV CTL epitopes. In this study, 7-6/Gag-GW12 and 7-6/Env-RW12 MHC class I/peptide tetrameric complexes were constructed and used to analyze Gag-GW12- and Env-RW12-specific CTL responses in two EIAV-infected horses (A2164 an...
Immunostimulatory DNA activates production of type I interferons and interleukin-6 in equine peripheral blood mononuclear cells in vitro. This study aimed to evaluate different nucleic acid preparations as cytokine inducers in equine cells. To induce cytokine production, bacterial plasmid DNA or short synthetic oligodeoxyribonucleotides (ODN), with or without the transfection reagent lipofectin, were added to cultures of purified equine peripheral blood mononuclear cells (PBMC). Cytokine activity was detected with bioassays in cell culture supernatants after 24h of induction and cytokine mRNA expression was detected using RT-PCR at 6h post induction. For IFN-alpha/beta it was found that both plasmid DNA and phosphodiester ODN, c...
Equine interferon gamma synthesis in lymphocytes after in vivo infection and in vitro stimulation with EHV-1. Equine cytotoxic T lymphocyte (CTL) responses to equine herpesvirus-1 (EHV-1) are well characterised but little is known about the cytokine response after infection or vaccination. EHV-1 is common in horses and infects lymphocytes in vivo. This virus was used as a model to measure the synthesis of interferon gamma (IFN-gamma) by equine peripheral blood mononuclear cells (PBMC) after in vivo infection and/or in vitro stimulation with EHV-1. Both flow cytometry and ELISPOT assays were used to quantify equine IFN-gamma using a mouse anti-bovine IFN-gamma monoclonal antibody (clone CC302; shown to...
In vivo pretreatment with PGG-glucan fails to alter cytokine mRNA expression of equine peripheral blood mononuclear cells exposed to endotoxin ex vivo. Twelve healthy horses were assigned to treatment or control groups. Treated horses received PGG-Glucan[ED-1] (1 mg/kg, IV) 24 hours prior to peripheral blood mononuclear cell (PBMC) isolation. PBMCs were isolated and incubated in the presence of lipopolysachharide (LPS). At 0, 6, 12, 24 and 48 hours messenger RNA (mRNA) was extracted. Reverse transcription polymerase chain reaction (PCR) was performed and cytokine mRNA expression for tumor necrosis factor alpha (TNFalpha), interleukin 1beta (IL-1beta), interleukin 10 (IL-10) and interferon gamma (IFN-gamma) determined using real time PCR. A si...
Cytokine gene expression in response to SnSAG1 in horses with equine protozoal myeloencephalitis. Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome seen in horses from the Americas and is mainly caused by Sarcocystis neurona. Recently, a 29-kDa surface antigen from S. neurona merozoites was identified as being highly immunodominant on a Western blot. This antigen has been sequenced and cloned, and the expressed protein has been named SnSAG1. In a previous study, cell-mediated immune responses to SnSAG1 were shown to be statistically significantly reduced in horses with EPM in comparison to EPM-negative control horses. It therefore appears as though the parasite is able to i...
Horse cytokine/IgG fusion proteins–mammalian expression of biologically active cytokines and a system to verify antibody specificity to equine cytokines. Recombinant cytokines are valuable tools for functional studies and candidates for vaccine additives or therapeutic use in various diseases. They can also be used to generate specific antibodies to analyze the roles of different cytokines during immune responses. We generated a mammalian expression system for recombinant cytokines using the equine IgG1 heavy chain constant region as a tag for detection and purification of the expressed cytokine, demonstrated here using equine interferon-gamma (IFN-gamma), interleukin-2 (IL-2), interleukin-4 (IL4) and transforming growth factor-beta1 (TGF-beta1...
Chronic exacerbation of equine heaves is associated with an increased expression of interleukin-17 mRNA in bronchoalveolar lavage cells. Recent finding suggests that T-cells may be involved in the pathogenesis of heaves in horses. However, little is known concerning their possible contribution to pulmonary neutrophilia, a characteristic finding in heaves. Interleukin (IL)-17 is a cytokine secreted by activated T-cells that indirectly promotes the maturation, chemotaxis and activation of neutrophils. We therefore hypothesized that IL-17 may be involved in the recruitment of neutrophils into the airways and that its mRNA expression would be increased in bronchoalveolar lavage (BAL) cells of horses with heaves. Heaves susceptible ...
Differential regulation of the GLUT1 and GLUT3 glucose transporters by growth factors and pro-inflammatory cytokines in equine articular chondrocytes. Glucose serves as the major energy substrate for articular chondrocytes and as the main precursor for the synthesis of extracellular matrix glycosaminoglycans in cartilage. Chondrocytes have been shown to express several glucose transporter (GLUT) isoforms including GLUT1 and GLUT3. The aim of this investigation was to determine the effects of endocrine and cytokine factors on the capacity of equine articular chondrocytes for transporting 2-deoxy-d-[2,6-3H] glucose and on the expression levels of GLUT1 and GLUT3. Chondrocytes maintained in monolayer culture were stimulated for 24 h with TNF-al...
Ketamine inhibits LPS-induced tumour necrosis factor-alpha and interleukin-6 in an equine macrophage cell line. Ketamine is widely used in equine anaesthesia. Beside its anaesthetic and analgesic properties, ketamine possesses a cytokine-modulating activity. However, to date, no data are available regarding the inhibitory effect of ketamine on the cytokine response in horses. In horses, cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) play a pivotal role in the pathogenesis of equine endotoxaemia following gastrointestinal disorders. Hence, the objective of this study was to assess the influence of ketamine on LPS-induced TNF-alpha and IL-6 formation in an equine macro...
Inhibition of lymphocyte proliferation and activation: a mechanism used by equine invasive trophoblast to escape the maternal immune response. At days 36-38 of gestation, the equine invasive trophoblast cells migrate into the endometrium of the pregnant mare to form the endometrial cups. During their migration, they become surrounded by maternal CD4+ and CD8+ T lymphocytes, and stimulate a cytotoxic antibody response to the paternal major histocompatibility complex class I antigens that they express. Nevertheless, endometrial cup cells remain viable at the site of uterine invasion up to days 80-100 of gestation, suggesting the participation of immunomodulatory mechanisms to the maternal cellular immune response. To determine the effe...
Cold weather exercise and airway cytokine expression. Athletes who perform repeated exercise while breathing cold air have a high prevalence of asthmalike chronic airway disease, but the mechanism linking such activity to airway inflammation is unknown. We used a novel animal model (exercising horses) to test the hypothesis that exercise-induced chronic airway disease is caused by exposure of intrapulmonary airways to unconditioned air, resulting in the upregulation of cytokine expression. Bronchoalveolar lavage fluid (BALF) was obtained from eight horses 5 h after submaximal exercise while they breathed room temperature or subfreezing air in a r...
Cytokine induced metalloproteinase expression and activity does not correlate with focal susceptibility of articular cartilage to degeneration. To determine whether the focal susceptibility to cartilage degeneration in joints is related to a differential response to cytokine stimulation. Methods: Compare aggrecan and collagen catabolism in in-vitro models of cartilage degradation induced by retinoic acid (RA), interleukin-1 (IL-1), tumor necrosis factor alpha (TNF) and IL-1 plus oncostatin M (OSM). Glycosaminoglycan (GAG) and hydroxyproline (HyPro) quantification and Western immunoblot analyses of aggrecan and collagen degradation products were undertaken in explant cultures of normal cartilage from regions of equine joints with a kno...
Topical treatments in equine wound management. Wound repair is a complex series of coordinated events regulated by a delicately orchestrated cascade of cytokines and growth factors that restore the structural integrity of damaged tissue. Manipulation of the growth factor profile or wound environment through topical application of therapeutic agents could positively influence the rate and quality of wound repair. Transforming growth factor-beta,platelet-rich plasma, activated macrophage supernatant, and growth hormone are sources of mediators that may facilitate wound healing. Solcoseryl, ketanserin, tripeptide- and tetrapeptide-copper comp...
Modulation of articular chondrocyte activity by pirfenidone. Pirfenidone is under investigation as an anti-inflammatory and anti-fibrotic agent in several organs including lung. Since important features of arthritic conditions include inflammation and long-term damage to articular cartilage, we have investigated whether PD can suppress chondrocyte responses to bacterial lipopolysaccharide (LPS) and interleukin 1 (IL-1); modulators that induce a cascade of inflammatory responses that lead to articular joint tissue damage. PD (0 - 5microM) showed no effect on cell number or viability when incubated with high density primary equine chondrocyte cultures for...
Cytokine profiles of peripheral blood and airway CD4 and CD8 T lymphocytes in horses with recurrent airway obstruction. Equine recurrent airway obstruction (RAO) is thought to result from an aberrant immune response to inhaled antigens, modulated by T lymphocytes via the secretion of pro-inflammatory cytokines. However data relating to the phenotypes of the T lymphocytes present in peripheral blood and bronchoalveolar lavage fluid of RAO horses and their cytokine profiles are contradictory. The aim of this study was to further investigate the cytokine (IL-4, IL-5, IL-13 and INF-gamma) mRNA expression profile in peripheral blood lymphocytes and bronchoalveolar lavage lymphocytes from RAO and control horses, befo...
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, ...
Differential effects of virulent and avirulent equine infectious anemia virus on macrophage cytokine expression. Equine infectious anemia virus (EIAV) causes rapid development of acute disease followed by recurring episodes of fever, thrombocytopenia, and viremia. Most infected equid eventually bring the virus under immunological control. We recently reported the development of an equine-specific ribonuclease protection assay (RPA) to quantitate mRNA levels of 10 cytokines. Using this newly developed RPA, we now show significant differences in cytokine induction in equine monocyte-derived macrophages (EMDM) exposed to virulent and avirulent EIAV. Virulent EIAV17 induced significant increases in interleuk...
Distribution of TNF receptors and TNF receptor-associated intracellular signaling factors on equine tendinocytes in vitro. Although tumor necrosis factor (TNF) alpha is an important key factor in degeneration of equine superficial digital flexor tendon (SDFT), the dynamism of TNF receptors and associated factors on tendinocytes has not been elucidated. To reveal signaling events mediated by TNF-receptors (TNF-Rs) in tendinocytes, we focused on four signaling factors, TNF-R1, TNF-R2, TNF-R-associated factor 2 (TRAF2) and nuclear factor-kappa B (NF-kappaB), and investigated the distribution and production of these factors. Cultured tendinocytes were obtained from SDFTs of thoroughbred horses. The tendinocytes were t...
Equine herpesvirus-1 infection induces IFN-gamma production by equine T lymphocyte subsets. A commercial bovine IFN-gamma-specific monoclonal antibody was used to measure antigen-specific IFN-gamma production by equine lymphocytes. Paired PBMC samples were collected from six ponies prior to and 10 days after challenge infection with equine herpesvirus-1 (EHV-1). Each sample was stimulated in vitro with EHV-1, virus-free medium, or PMA and ionomycin, and labelled with monoclonal antibodies specific for various equine lymphocyte subset markers. Following fixation, intracellular IFN-gamma was detected using a FITC-conjugated bovine IFN-gamma-specific monoclonal antibody. In vitro restim...
Dual transduction of insulin-like growth factor-I and interleukin-1 receptor antagonist protein controls cartilage degradation in an osteoarthritic culture model. This study evaluated the potential of gene induced synoviocyte expression of a combination of insulin-like growth factor-I (AdIGF-I) and interleukin-1 receptor antagonist protein (AdIL-1Ra) to control articular cartilage degradation in vitro. Cartilage explants and synovial membrane were harvested from young mature horses. Synovial monolayers were established and either (1) maintained as untransduced controls; (2) transduced with AdIGF-I at 200 MOI in 500 microl serum-free medium; (3) transduced with AdIL-1Ra at 100 MOI; or (4) transduced with a combination of AdIGF-I (200 MOI) and AdIL-1Ra (1...
Does the cytotoxic effect of transient amyloid oligomers from common equine lysozyme in vitro imply innate amyloid toxicity? In amyloid diseases, it is not evident which protein aggregates induce cell death via specific molecular mechanisms and which cause damage because of their mass accumulation and mechanical properties. We showed that equine lysozyme assembles into soluble amyloid oligomers and protofilaments at pH 2.0 and 4.5, 57 degrees C. They bind thioflavin-T and Congo red similar to common amyloid structures, and their morphology was monitored by atomic force microscopy. Molecular volume evaluation from microscopic measurements allowed us to identify distinct types of oligomers, ranging from tetramer to oc...
Cytokine responses to Cyathostominae larvae in the equine large intestinal wall. To investigate cytokine responses in cyathostomin infection, we quantified mucosal interleukin-4 (IL-4), interleukin-10 (IL-10), tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma by reverse transcriptase-competitive polymerase chain reaction. The analysis was performed on large intestinal wall samples obtained from six anatomical sites spanning the caecum and colon of 17 naturally exposed horses. The numbers of developing larvae (DL) and early third stage larvae (EL3) were ascertained using transmural illumination and pepsin digestion techniques, respectively. Levels of each cytoki...
Rhodococcus equi-infected macrophages are recognized and killed by CD8+ T lymphocytes in a major histocompatibility complex class I-unrestricted fashion. The goal of this research was to examine the role of cytotoxic T lymphocytes (CTL) in the control of Rhodococcus equi and specifically to determine if R. equi-specific CD8+ CTL occurred in the blood of immune horses. Equine peripheral blood mononuclear cells stimulated with antigen-presenting cells either infected with R. equi or exposed to soluble R. equi antigen lysed R. equi-infected target cells. Lysis was decreased to background by depletion of either CD2+ or CD3+ cells, indicating that the effector cell had a T-lymphocyte, but not NK cell, phenotype. Stimulation induced an increased perc...
Bioactivity and secretion of interleukin-18 (IL-18) generated by equine and feline IL-18 expression constructs. Interleukin 18 (IL-18) is a cytokine capable of induction of IFNgamma, granulocyte monocyte-colony stimulating factor (GM-CSF), TNFalpha and IL-1 in immunocompetent cells. Equine and feline plasmid vectors expressing pro-IL-18, mature IL-18 and IL-18 fused to a synthetic signal sequence from human IL-1beta receptor antagonist protein (ILRAP), ILRAP-IL-18, have been generated. In vitro protein expression of these constructs was compared by Western blot analysis. These data demonstrated that ILRAP-IL-18 protein was secreted readily from transfected chinese hamster ovary (CHO) cells. A simple bio...
Evidence that use of an inactivated equine herpesvirus vaccine induces serum cytotoxicity affecting the equine arteritis virus neutralisation test. Several laboratories worldwide have recently experienced problems related to serum cytotoxicity with the equine arteritis virus (EAV) neutralisation test (VN) when using Office International des Epizooties (OIE) reference laboratory prescribed rabbit kidney (RK-13) indicator cells. Cytotoxicity can be mistaken for viral cytopathic effect and has led to increasing difficulties in test interpretation, consequently causing disruption to both equine breeding and disease surveillance. Results from experimental and field-derived data suggest that this serum cytotoxicity is associated with use of a t...
Depletion of natural killer cells does not result in neurologic disease due to Sarcocystis neurona in mice with severe combined immunodeficiency. Sarcocystis neurona is an apicomplexan parasite that is the primary etiologic agent of equine protozoal myeloencephalitis in horses. Protective immune responses in horses have not been determined, but interferon-gamma (IFN-gamma) is considered critical for protection from neurologic disease in mice. The role of adaptive and innate immune responses in control of parasites was explored by infecting BALB/c, IFN-gamma knockout (GKO), and severe combined immune deficient (SCID) mice with S. neurona (10(4) sporocysts/mouse). Immune competent BALB/c mice eliminated parasites within 30 days, with no s...
Clinical signs and hematologic, cytokine, and plasma nitric oxide alterations in response to Strongylus vulgaris infection in helminth-naïve ponies. The objective of this study was to determine the effect of infection with Strongylus vulgaris on serum cytokines and plasma nitric oxide (NO) concentrations in helminth-naive ponies. Group 1 (n = 21) was given 500 S. vulgaris L3 larvae and group 2 (n = 7) received a saline control. Ponies were monitored daily for clinical signs, and blood was collected for complete blood cell counts and serum cytokines (TNF, IL-1, IL-6) quantification. Group 1 ponies were depressed, anorexic, and febrile for variable periods of time. Plasma NO was increased on day 21 in group 1 and on days 9 and 21 in group 2....
IL-4 induced CD23 (FcepsilonRII) up-regulation in equine peripheral blood mononuclear cells and pulmonary alveolar macrophages. The objectives of this study were to quantify the induction of equine CD23 transcripts in equine peripheral blood mononuclear cells (PBMCs) and pulmonary alveolar macrophages cultured with recombinant equine IL-4 (rEq IL-4). PBMCs were isolated from blood drawn from four healthy horses. Bronchoalveolar lavage (BAL) fluid was collected from three healthy horses and alveolar macrophages were purified using adherence to plastic for 120 min. PBMCs and alveolar macrophages were cultured using four different conditions: rEq IL-4 and LPS, LPS alone, rEq IL-4 alone and a media control. Total RNA was i...
Comparison of TGF-beta 1 concentrations in bronchoalveolar fluid of horses affected with heaves and of normal controls. Airway remodeling may play an important role in heaves pathophysiology. Transforming growth factor-beta 1 (TGF-beta1) is a potent profibrotic cytokine, which might contribute to airway wall thickening and fibrosis of bronchiolar and alveolar submucosa. An ELISA designed for the measurement of human TGF-beta1 was used to measured total TGF-beta1 released in bronchoalveolar lavage fluid (BALF) of normal horses and of those affected with heaves in remission. The specificity of the assay for TGF-beta1 of the horse was confirmed using recombinant equine TGF-beta1. The influence of hay exposure on T...
