Effect of type of dietary polyunsaturated fatty acid supplement (corn oil or fish oil) on immune responses in healthy horses

The objective of this study was to compare effects of dietary polyunsaturated fatty acid supplementation (corn oil or fish oil) on selected immune responses in normal horses. Two groups of horses (n = 5) were randomly assigned a dietary supplement with either 3.0% corn oil or fish oil for a period of 14 weeks. Plasma fatty acid profiles were monitored to ensure uptake of dietary fatty acids. Cell-mediated immunity was assessed by a delayed-type hypersensitivity (DTH) skin test to keyhole limpet hemocyanin (KLH), and humoral immunity was assessed by measuring antibody titers to KLH. Production of prostaglandin E2 (PGE2), expression of tumor necrosis factor-alpha (TNF-alpha), and phagocytosis of latex beads by bronchoalveolar lavage fluid (BALF) cells were also assessed. Lipopolysaccharide (LPS)-stimulated BALF cells from horses fed corn oil showed a higher production of PGE2 compared with those from horses fed fish oil at 6 and 12 weeks. Production of TNF-alpha by LPS-stimulated BALF cells was higher in both groups of horses at 6, 8, and 12 weeks compared with pretrial values, and phagocytic activity of BALF cells was higher at 8 and 12 weeks, however, there were no differences between the 2 groups of horses. The DTH skin test and antibody titers to KLH revealed no differences between horses fed corn or fish oil. Based on these studies, dietary polyunsaturated fatty acids modulate the inflammatory response of horses. Both fatty acid supplements increased production of the proinflammatory cytokine TNF-alpha, whereas only corn oil increased production of the proinflammatory eicosanoid PGE2 by LPS-stimulated BALF cells. It is possible that fish oil, because it did not increase production of PGE2, could have value in the treatment of equine recurrent airway obstruction or other equine inflammatory diseases.     

Inhibition of interleukin-1 activity by equine synovial fluid.

The presence, in equine synovial fluid, of inhibitors of interleukin-1 (IL-1) activity has been investigated by means of an assay involving IL-1-mediated production of PGE2 by synovial cells. Inhibitors of IL-1 alpha and IL-1 beta were identified in normal synovial fluid and synovial fluid from two horses with early joint disease. Inhibitors of IL-1 alpha were also present in synovial fluid from two horses with long-standing joint disease. However, IL-1 beta inhibitory activity was not present in fluid from the horses with more chronic joint disease. The effect appeared to be specific for IL-1, and not a direct action on PGE2 production, as synovial fluid had no effect on lipopolysaccharide-mediated PGE2 production. It is suggested that the inhibitory activity may be involved physiologically in the control of IL-1 activity in the joint, and the loss of IL-1 inhibition may be at least as important biologically as increased production of IL-1.     

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.     

Adverse conditions in vitro stimulate chondrocytes to produce prostaglandin E2 and stromelysin

Chondrocytes subjected to adverse culture conditions in vitro are stimulated to produce the eicosanoid prostaglandin E2 (PGE2) and the neutral metalloproteinase stromelysin (proteoglycanase). This indicates the potential role of the chondrocyte in cartilage degeneration in equine clinical joint disease and suggests a mechanism which may be involved in the potentiation of the effects of other inflammatory mediators. Therefore, adverse conditions within the joint, such as decreased pH in an inflammatory focus and decreased access of nutrients to deeper layers of cartilage, might contribute to the activation of chondrocytes which leads to cartilage degradation.     

The role of p38 mitogen-activated kinase (MAPK) in the mechanism regulating cyclooxygenase gene expression in equine leukocytes

The goal of this study was to define the role for p38 mitogen-activated kinase (MAPK) in the signaling mechanism regulating pro-inflammatory cyclooxygenase (COX) gene expression in lipopolysaccharide (LPS)-activated equine leukocytes for the purposes of identifying novel targets for anti-inflammatory therapy in endotoxemic horses. The p38 MAPK has been shown to positively regulate inflammatory gene expression in human leukocytes and can be activated by a variety of stimuli including LPS, TNF-alpha, and IL-1. Activation-associated phosphorylated p38 MAPK has been implicated in the up-regulation of several inflammatory genes, including COX-2 which ultimately results in the production of prostanoids that are responsible for the pathophysiology associated with endotoxemia. Our hypothesis is that activation of p38 MAPK is essential for LPS-induced COX-2 expression in equine peripheral blood leukocytes. We tested our hypothesis by investigating the effects of the specific p38 MAPK inhibitors SB203580 and SB202190 on LPS-induced COX-2 protein expression and PGE(2) production in equine leukocytes. LPS stimulation activated p38 MAPK and increased COX-2 expression in a dose-dependent manner with maximal activation observed after 30min and 4h, respectively, at a concentration of 10 ng/ml LPS. In contrast, LPS stimulation did not affect COX-1 protein expression. Pretreatment with SB203580 or SB202190 significantly inhibited LPS-induced activation-associated p38 MAPK phosphorylation, COX-2 mRNA and protein levels, and PGE(2) production in equine leukocytes. Maximal inhibition of LPS-induced COX-2 protein expression was achieved at a concentration of 10 microM SB203580. We concluded that p38 MAPK is essential for LPS-induced COX-2 expression suggesting that p38 MAPK is a potential target for anti-inflammatory therapy during equine endotoxemia.     

Bradykinin stimulates prostaglandin E2 production and cyclooxygenase activity in equine nonglandular and glandular gastric mucosa in vitro

REASONS FOR PERFORMING STUDY: There are few data available regarding regulation of prostaglandin (PG) generation by equine gastric mucosae and the role of the cyclooxygenase (COX) isoforms in their production. OBJECTIVES: To: 1) characterise and quantify PGE2 output in vitro; 2) examine the sensitivity of PGE2 production to exogenous bradykinin (BK) exposure; 3) determine the contribution of the COX-1 and COX-2 pathways to basal and BK-stimulated PGE2 production; and 4) measure if BK influences electrogenic ion transport in equine gastric mucosae in vitro. METHODS: Full thickness gastric sheets were obtained from horses at post mortem, stripped of muscle layers and mounted in Ussing chambers. Tissues were exposed to bradykinin (BK, 0.1 micromol/l) either alone, or following pretreatment with a selective COX-2 inhibitor (NS-398, 1 micromol/l) or a nonselective COX inhibitor (piroxicam, 1 micromol/l), or were untreated. RESULTS: BK administration increased PGE2 output from the basolateral but not the apical faces of both tissue types. Piroxicam, but not NS-398, reduced basolateral PGE2 release below control levels in both tissue types. Both piroxicam and NS-398 pretreatment inhibited BK-stimulated PGE2 release. In separate experiments, BK was without effect upon electrophysiological parameters of tissues mounted in Ussing chambers. CONCLUSIONS: PGE2 is produced by the nonglandular and glandular equine gastric mucosae in vitro. Significantly more PGE2 is released basolaterally than apically. BK stimulated the production of PGE2 from the basolateral side of both tissue types. These findings suggest that COX-1 is a significant pathway for basal PGE2 production from the basolateral faces of both nonglandular and glandular equine gastric mucosae in vitro.     

Evaluation of the influence of prostaglandin E2 on recombinant equine interleukin-1beta-stimulated matrix metalloproteinases 1, 3, and 13 and tissue inhibitor of matrix metalloproteinase 1 expression in equine chondrocyte cultures

OBJECTIVE: To determine the effects of prostaglandin E2 (PGE2) on recombinant equine interleukin (IL)-1beta-stimulated expression of matrix metalloproteinases (MMP 1, MMP 3, MMP 13) and tissue inhibitor of matrix metalloproteinase 1 (TIMP 1) in vitro. SAMPLE POPULATION: Cultured equine chondrocytes. PROCEDURE: Stationary monolayers of first-passage chondrocytes were exposed to graduated concentrations of PGE2 with or without a subsaturating dose (50 pg/ml) of recombinant equine IL-1beta (reIL-1beta) to induce expression of MMP 1, MMP 3, MMP 13, and TIMP 1, followed by RNA isolation and northern blotting. In subsequent experiments, gene expression was similarly quantified from mRNA isolated from cultures pretreated with phenylbutazone to quench endogenous PGE2 synthesis, followed by exposure to reIL-1beta and exogenous PGE2 (5 mg/ml) with appropriate controls. RESULTS: Exogenous PGE2 (10 mg/ml) significantly reduced reIL-1beta-induced expression of MMP 1, MMP 3, MMP 13, and TIMP 1. Abrogation of cytokine induction with this dose of PGE2 was comparable to that for dexamethasone (10(-5) M) control. Similarly, pretreatment with phenylbutazone, followed by exposure to relL-1beta and PGE2 (5 mg/ml), was associated with a reduced expression of the genes of interest, an effect that was significant for MMP 1, MMP 13, and TIMP 1. CONCLUSIONS AND CLINICAL RELEVANCE: The MMP and TIMP 1 are important mediators in the pathophysiologic events in osteoarthritis. The potential for physiologically relevant regulation of expression of these genes by PGE2 is a consideration in the use of drugs that inhibit prostanoid synthesis in the treatment of equine arthropathies.     

Cellular aspects of inflammation. The Ciba-Geigy Prize for Research in Animal Health

The migration of leucocytes to sites of acute and chronic inflammation is an event of central importance to the maintenance of inflammatory processes; extravascular leucocytes are responsible for generating chemical mediators of inflammation and the phagocytosis of particulate matter. They may also be involved in the conversion of acute to chronic inflammatory lesions. Leucocytes are attracted to sites of tissue injury by a range of chemoattractants. This paper describes the development of a method for separating on Percoll gradients purified populations of equine polymorphonuclear and mononuclear leucocytes and use of the isolated cells in vitro studies. Two independent assay methods, the agarose microdroplet and the Boyden chamber microfilter techniques, were used. The assays were utilised in three ways: (a) to investigate the sensitivity of polymorphonuclear and mononuclear leucocytes to two standard chemoattractants, zymosan activated plasma and n-formyl-methionyl-leucyl phenylalanine; (b) to study the chemoattractant properties of leukotriene B4 and prostaglandin E2 for equine leucocytes; and (c) to investigate the inhibitory actions of several nonsteroidal anti-inflammatory drugs (NSAIDs) on equine leucocyte movement.     

Effects of the omega-3 fatty acid, alpha-linolenic acid, on lipopolysaccharide-challenged synovial explants from horses

OBJECTIVE: To determine the effects of pretreatment with alpha-linolenic acid, an omega-3 polyunsaturated fatty acid, on equine synovial explants challenged with lipopolysaccharide (LPS). ANIMALS: 8 mature mixed-breed horses (4 mares and 4 geldings). PROCEDURE: Synovial explants were assigned to receive 1 of 7 concentrations of alpha-linolenic acid, ranging from 0 to 300 microg/mL. At each concentration, half of the explants were controls and half were challenged with 0.003 microg of LPS as a model of synovial inflammation. Cell inflammatory response was evaluated by measurement of prostaglandin E2 production via an ELISA. Synovial cell viability, function, histomorphologic characteristics, and cell membrane composition were evaluated by use of trypan blue dye exclusion, hexuronic acid assay for hyaluronic acid, objective microscopic scoring, and high-performance liquid chromatography, respectively. RESULTS: Challenge with LPS significantly increased production of prostaglandin E2 and decreased production of hyaluronic acid. Treatment with alpha-linolenic acid at the highest dose inhibited prostaglandin E2 production. Cell viability and histomorphologic characteristics were not altered by treatment with alpha-linolenic acid or LPS challenge. Treatment with alpha-linolenic acid increased the percentage of this fatty acid in the explant cell membranes. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that investigation of alpha-linolenic acid as an anti-inflammatory medication for equine synovitis is warranted.     

The acute inflammatory process,arachidonic acid metabolism and the mode of action of anti-inflammatory drugs

Arachidonic acid is a polyunsaturated fatty acid covalently bound in esterified form in the cell membranes of most body cells. Following irritation or injury, arachidonic acid is released and oxygenated by enzyme systems leading to the formation of an important group of inflammatory mediators, the eicosanoids. It is now recognised that eicosanoid release is fundamental to the inflammatory process. For example, the prostaglandins and other prostanoids, products of the cyclo-oxygenase enzyme pathway, have potent inflammatory properties and prostaglandin E₂ is readily detectable in equine acute inflammatory exudates. The administration of nonsteroidal anti-inflammatory drugs results in inhibition of prostaglandin synthesis and this explains the mode of action of agents such as phenylbutazone and flunixin. Lipoxygenase enzymes metabolise arachidonic acid to a group of non-cyclised eicosanoids, the leukotrienes, some of which are also important inflammatory mediators. They are probably of particular importance in leucocyte-mediated aspects of chronic inflammation. Currently available non-steroidal antiinflammatory drugs, however, do not inhibit lipoxygenase activity. In the light of recent evidence, the inflammatory process is re-examined and the important emerging roles of both cyclo-oxygenase and lipoxygenase derived eicosanoids are explored. The mode of action of current and future antiinflammatory drugs offered to the equine clinician can be explained by their interference with arachidonic acid metabolism.     

Pig endometrial cells in primary culture: morphology, secretion of prostaglandins and proteins, and effects of pregnancy

Luminal epithelial, glandular epithelial, and stromal cells were isolated from pig endometrium by enzymatic dispersion and sieve filtration. The three cell types, maintained in primary culture, showed distinctly different morphologies when viewed by light and scanning electron microscopy. Immunocytochemical staining indicated that luminal and glandular epithelial cells were positive for both cytokeratin and vimentin. However, stromal cells were positive only for vimentin. Acid phosphatase activity was detected in the culture medium of glandular cells and increased (P less than .05) when progesterone (.1 microM) was included in the culture medium. The secretion of uteroferrin by glandular cells was also indicated by one-dimensional PAGE and Western blot analysis. Stromal cells produced more (P less than .01) prostaglandin E (PGE) than prostaglandin F2 alpha (PGF2 alpha), whereas glandular cells secreted more (P less than .01) PGF2 alpha than PGE. Pregnancy status affected prostaglandin secretion in that stromal cells secreted less (P less than .01) PGE and PGF2 alpha and glandular cells secreted less (P less than .05) PGF2 alpha when they were harvested from pregnant vs cyclic pigs. Furthermore, the PGE:PGF2 alpha ratio in medium from stromal cells was greater (P less than .01) for cells collected from pregnant pigs. This culture system provides an in vitro model for studying the hormonal regulation of the endometrium and potentially may be useful for studying interactions between endometrial cells and embryos in the pig.     

Effects of intramuscular meloxicam administration on prostaglandin E2 synthesis in the North American bullfrog (Rana catesbeiana)

Meloxicam is a commonly used nonsteroidal anti-inflammatory drug (NSAID) in veterinary medicine, but its use in amphibians has not been reported in the literature. NSAIDs are known to act by providing anti-inflammatory and analgesic actions by inhibiting the synthesis of prostaglandin E2 (PGE2). The objective of this study was to evaluate whether the intramuscular administration of meloxicam would decrease the circulating serum PGE2 levels in the North American bullfrog (Rana catesbeiana) following tissue trauma induced by a punch biopsy. Eighteen adult North American bullfrogs were randomly assigned to two treatment groups: meloxicam (0.1 mg/kg i.m.) and control (0.9% saline i.m.). Blood was obtained via cardiocentesis immediately prior to administration of the two treatment regimes and serum was frozen. A 4-mm punch biopsy was taken from the right triceps femoris muscle to induce an inflammatory response. Twenty-four hours later, a second blood sample was collected and serum was harvested and frozen. Serum PGE2 concentrations were measured using a commercial PGE2 enzyme assay (EIA) kit. Twenty-four hours following the biopsy, the mean circulating PGE2 levels of animals treated with meloxicam was 57.79 +/- 12.35 pg/ml, which did not differ significantly from animals that were treated with saline (85.63 +/- 17.55 pg/ml, P > or = 0.05). The calculated means of the absolute change between the circulating baseline PGE2 levels and the postinjury circulating PGE2 levels were significantly lower in animals treated with meloxicam (13.11 +/- 17.31 pg/ml) than in control animals treated with saline (46.14 +/- 38.02 pg/ml) (P < or = 0.05). These results suggest that the systemic administration of meloxicam at a dosage of 0.1 mg/kg once daily suppresses circulating serum PGE2 levels postinjury in the North American bullfrog.     

Influence of arachidonic acid metabolites in vitro and in uterine washings on migration of equine neutrophils under agarose

The influence of arachidonic acid metabolites on migration of equine neutrophils under agarose was investigated. Leukotriene B4 (LTB4) was chemotactic at concentrations between 0.1 and 1000 ng ml-1 and prostaglandin E2 (PGE2) at 1 and 10 ng ml-1 but not at higher or lower concentrations. Prostaglandin F2 alpha (PGF2 alpha) was not chemotactic for equine neutrophils at any concentration. Random migration was significantly inhibited (P less than 0.05) by suspension of neutrophils in LTB4 (0.1 to 1000 ng ml-1) and PGF2 alpha (0.1 ng ml-1) but not at high concentrations. There was a significant positive correlation between random migration of neutrophils suspended in uterine washings from persistently endometritic mares and concentrations of endogenous PGF (P less than 0.002) and PGE2 (P less than 0.05) in washings. Thus certain metabolites of arachidonic acid affect migration of equine neutrophils and may play a significant role in recruitment of neutrophils to sites of inflammation in the horse.     

Release of immunoreactive arachidonate metabolites by equine endometrium in vitro

The ability of equine endometrium to release prostaglandin (PG) F, PGE2, and leukotriene (LT) B4 was studied in vitro, using endometrial tissue from diestrous mares. Because of the high cross-reactivity of the PGF antiserum with PGF1 alpha and with PGF2 alpha, results were quoted as total immunoreactive PGF. Significant concentrations of these arachidonate metabolites were released into tissue culture medium between 1 and 24 hours of incubation. Significantly higher concentrations of PGE, but not of PGE2 or LTB4, were released from endometria of mares with chronic endometritis than from genitally normal mares. Prostaglandin F was released only in low concentrations from the endometrium of a mare with pyometra, but concentrations of PGE2 and LTB4 were similar to those of genitally normal mares.     

In vitro investigation of the interaction between nitric oxide and cyclo-oxygenase activity in equine ventral colon smooth muscle

The objective of this study was to determine if a correlation exists between the presence of nitric oxide and prostaglandin release in the equine ventral colon smooth muscle, since this relationship may accentuate the inflammatory process during intestinal injury. Tissue was collected from the ventral colon, cut into muscle strips oriented along the circular, longitudinal and taenial layers, and mounted in a tissue bath system. Samples of the bath fluid were collected before, following electrical field stimulation (EFS), and following EFS in the presence of L-NAME, a nitric oxide synthase inhibitor. Muscle strips were also obtained following systemic administration of a cyclo-oxygnease inhibitor and samples were collected using the previously described protocol. Concentrations of prostaglandins were determined in the fluid samples using an ELISA. Electrical field stimulated release of nitric oxide produced a significant increase in prostaglandin production which did not occur in the presence of L-NAME. Systemic administration of flunixin meglumine reduced prostaglandin levels at all sampling periods, although a small increase was present following EFS. The results of this study support the hypothesis that there is a correlation between the release of nitric oxide and the production of prostaglandins in the smooth muscle of the large colon. This association between nitric oxide and prostaglandin may act as an important regulatory mechanism for various physiological mechanisms, such as vascular smooth muscle tone, and may contribute to amplified tissue injury when the induced forms of both enzymes are activated during an inflammatory insult. This suggests that the use and development of COX2 and iNOS inhibitors may help attenuate the inflammatory response following intestinal injury.     

Inflammatory effects of platelet activating factor (PAF) in equine skin.

Intradermal administration of PAF (0.001-1 micrograms/site), but not lyso-PAF (10 micrograms/site), in the horse caused an increase in cutaneous vascular permeability which was maximal by 32 min. Responses to PAF and histamine were reduced by coadministration of the histamine 1 receptor antagonist chlorpheniramine, although only the inhibition of histamine-induced responses was dose-related and statistically significant. The cyclo-oxygenase inhibitor indomethacin was without effect on PAF-induced increases in vascular permeability. These findings suggest that the actions of PAF on equine skin microvasculature may be partly due to histamine release but not to prostanoid formation. Coadministration of prostaglandin (PG) E2 enhanced the oedematous responses to both PAF and histamine, although PGE2 failed to exert direct permeability-increasing activity. In addition, and in contrast to PAF and histamine, PGE2 increased cutaneous blood flow and skin surface temperature. PAF, but not lyso-PAF, also caused neutrophil infiltration into the skin which was maximal at 2 h. No significant effects on eosinophil or mononuclear cell numbers were apparent up to 24 h after injection of PAF. These results are consistent with the concept that PAF may be a mediator of inflammatory disorders of the skin in the horse.