Effects of interleukin-1beta and tumor necrosis factor-alpha on expression of matrix-related genes by cultured equine articular chondrocytes

OBJECTIVE: To determine the effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on expression and regulation of several matrix-related genes by equine articular chondrocytes. SAMPLE POPULATION: Articular cartilage harvested from grossly normal joints of 8 foals, 6 yearling horses, and 8 adult horses. PROCEDURE: Chondrocytes maintained in suspension cultures were treated with various doses of human recombinant IL-1beta or TNF-alpha. Northern blots of total RNA from untreated and treated chondrocytes were probed with equine complementary DNA (cDNA) probes for cartilage matrix-related genes. Incorporation of 35S-sulfate, fluorography of 14C-proline labeled medium, zymography, and western blotting were used to confirm effects on protein synthesis. RESULTS: IL-1beta and TNF-alpha increased steady-state amounts of mRNA of matrix metalloproteinases 1, 3, and 13 by up to 100-fold. Amount of mRNA of tissue inhibitor of metalloproteinase-1 also increased but to a lesser extent (1.5- to 2-fold). Amounts of mRNA of type-II collagen and link protein were consistently decreased in a dose-dependent manner. Amount of aggrecan mRNA was decreased slightly; amounts of biglycan and decorin mRNA were minimally affected. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment of cultured equine chondrocytes with IL-1beta or TNF-alpha resulted in marked alterations in expression of various matrix and matrix-related genes consistent with the implicated involvement of these genes in arthritis. Expression of matrix metalloproteinases was increased far more than expression of their putative endogenous inhibitor. Results support the suggestion that IL-1beta and TNF-alpha play a role in the degradation of articular cartilage in arthritis.     

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-alpha (100 ng mL(-1)), IL-1beta (100 ng mL(-1)), IGF-I (20 ng mL(-1)), TGF-beta (20 ng mL(-1)) and insulin (12.5 microg mL(-1)) before measuring uptake of non-metabolizable 2-deoxyglucose in the presence and absence of the glucose transport inhibitor cytochalasin B. Polyclonal antibodies to GLUT1 and GLUT were used to compare GLUT1 and GLUT3 expression in stimulated and un-stimulated alginate encapsulated chondrocytes by Western blotting. Results indicated that 2-deoxyglucose uptake was inhibited by up to 95% in the presence of cytochalasin B suggesting that glucose uptake into equine chondrocytes is GLUT-mediated. Insulin had no effect on glucose uptake, but treatment with IGF-I, TGF-beta, IL-1beta and TNF-alpha resulted in a significant increase (>65%) in 2-deoxyglucose uptake compared to control values. GLUT1 was found to be increased in chondrocytes stimulated with all the growth factors and cytokines but GLUT 3 was only upregulated by IGF-I. The data presented support a critical role for glucose in the responses of equine articular chondrocytes to pro-inflammatory cytokines and anabolic endocrine factors.     

Effects of the second-generation synthetic lipid A analogue E5564 on responses to endotoxin in [corrected] equine whole blood and monocytes

OBJECTIVE: To evaluate proinflammatory effects of the second-generation synthetic lipid A analogue E5564 on equine whole blood and isolated monocytes and to determine the ability of E5564 to prevent LPS (lipopolysaccharide)-induced procoagulant activity (PCA); tumor necrosis factor (TNF)-alpha production; and mRNA expression of TNF-alpha, interleukin (IL)-1beta, IL-6, and IL-10 by equine monocytes. SAMPLE POPULATION: Venous blood samples obtained from 19 healthy horses. PROCEDURES: Whole blood and monocytes were incubated with Escherichia coli O111:B4 LPS, E5564, or E5564 plus E coli O111:B4 LPS. Whole blood and cell supernatants were assayed for TNF-alpha, and cell lysates were assayed to determine PCA. Expression of mRNA for TNF-alpha, IL-1beta, IL-6, and IL-10 by monocytes was determined by use of real-time quantitative PCR assay. RESULTS: Minimal proinflammatory effects were detected in whole blood and monocytes. In addition, E5564 inhibited LPS-induced PCA and TNF-alpha production in a concentration-dependent manner. Furthermore, E5564 significantly inhibited LPS-induced mRNA expression of TNF-alpha, IL-1beta, and IL-10 and decreased LPS-induced expression of IL-6. CONCLUSIONS AND CLINICAL RELEVANCE: The second-generation synthetic lipid A analogue E5564 lacked agonist activity in equine whole blood and monocytes and was a potent antagonist of enteric LPS. Therefore, E5564 appeared to be the first lipid A analogue that has potential as an effective therapeutic agent in horses with endotoxemia.     

Cytokine expression by peripheral blood neutrophils from heaves-affected horses before and after allergen challenge

Heaves, also known as recurrent airway obstruction, is a common condition of horses characterised by pulmonary neutrophilia and reversible airway obstruction. This study evaluated the role of neutrophils in producing cytokines and chemokines that might be involved in the recruitment and activation of inflammatory cells in horses with heaves. Peripheral neutrophils were isolated from heaves-affected (n = 9) and control (n = 4) horses before and after 5 h of natural inhalation challenge. Expression of mRNA of two pro-inflammatory cytokines, tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta, along with two chemokines, IL-8 and macrophage inflammatory protein (MIP)-2, was evaluated. After exposure to mouldy hay, horses with heaves had significant airway obstruction and increased numbers of neutrophils in bronchoalveolar lavage samples, compared to control horses. However, there were no differences in the expression of mRNAs of TNF-alpha, IL-1beta, IL-8 and MIP-2 between the two groups, suggesting that the release of cytokines and chemokines by peripheral blood neutrophils is not necessary for the development of heaves.     

Effects of proinflammatory cytokines on canine articular chondrocytes in a three-dimensional culture

OBJECTIVE: To determine the effects of interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha on canine chondrocytes cultured in an agarose-based 3-dimensional (3-D) system. SAMPLE POPULATION: Humeral head articular cartilage chondrocytes obtained from 6 adult dogs. PROCEDURE: Chondrocytes were cultured in a 3-D system for < or = 12 days in serum-free medium with IL 1alpha, IL-1beta, or TNF-alpha at concentrations of 20, 50, or 100 ng/mL. After 1, 3, 6, and 12 days, glycosaminoglycan (GAG) concentrations in 3-D constructs; nitric oxide and prostaglandin E2 (PGE2) concentrations in media samples; and relative expressions of selected genes, including metalloproteinase (MMP)-13 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, were evaluated. Control specimens were comprised of chondrocytes cultured without proinflammatory cytokines. RESULTS: In control 3-D constructs, GAG content was significantly higher than for all other constructs. Compared with control values, relative expressions of MMP-13, TIMP-1, and TIMP-2 genes in the IL-1beta (50 ng/mL) group were significantly higher at day 1; at all evaluations, media concentrations of nitric oxide were significantly higher in all TNF-alpha-treated cultures; and concentrations of PGE2 in media samples were significantly higher in the IL-1beta (50 ng/mL) and IL-1beta (100 ng/mL) groups at days 1 and 3, in the IL-1beta (100 ng/mL) group at day 6, and in all TNF-alpha groups at days 1, 3, and 6. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that TNF-alpha more readily induces production of nitric oxide and PGE2 by canine chondrocytes, compared with IL-1beta. In vitro, IL-1alpha appeared to have a minimal effect on canine chondrocytes.     

Investigation of mRNA expression of tumor necrosis factor-alpha, interleukin-1beta, and cyclooxygenase-2 in cultured equine digital artery smooth muscle cells after exposure to endotoxin.

OBJECTIVE: To determine messenger RNA expression of cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-alpha, and interleukin- (IL)-1beta from cultured equine smooth muscle cells (SMC). SAMPLE POPULATION: Segments of palmar digital artery harvested from 6 clinically normal adult horses. PROCEDURE: Explants were collected from the tunica media of arteries for primary culture of SMC. Equine mononuclear cells were used as control cells. Subcultured vascular SMC and control cells were exposed to lipopolysaccharide (20 microg/ml and 100 ng/ml, respectively). Northern blot analysis with equine-specific probes for COX-2, TNF-alpha, and IL-1beta was performed, using isolated total cellular RNA. RESULTS: Although no message was detected for IL-1beta or TNF-alpha in control or endotoxin-exposed equine vascular SMC from all horses, COX-2 underwent a distinct substantial up-regulation after endotoxin exposure. Endotoxin-exposed equine mononuclear cells had up-regulation of IL-1beta and TNF-alpha mRNA. CONCLUSIONS AND CLINICAL RELEVANCE: Increased expression of COX-2 mRNA by equine vascular SMC may be an important early pathophysiologic event in the onset of endotoxemia in horses. Potentiated local vascular production of various prostanoids after increased expression of mRNA for COX-2 may result in vasoactive events observed with laminitis.     

Cytokine induction in pulmonary airways of horses with heaves and effect of therapy with inhaled fluticasone propionate

Work in humans and laboratory animals has identified a central role for cytokines and chemokines in development and persistence of lower airway inflammation. The objectives of this study were to determine interleukin (IL)-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha induction in bronchoalveolar lavage (BAL) of control horses and horses with heaves both during remission and exacerbation of the disease, and to determine the effect of therapy with inhaled fluticasone propionate on the cytokine profile of horses with heaves. IL-1 beta and TNF-alpha mRNA expression was significantly higher in horses with heaves after exposure to moldy hay compared to either values obtained during clinical remission or to healthy controls. IL-8 mRNA expression and protein concentrations were significantly higher in horses with heaves than in controls. Both IL-4 and IFN-gamma mRNA expression was increased at various times in heaves-susceptible horses compared to controls. IL-2, IL-5 and IL-10 mRNA expression was not detected in BAL cells of either group. Therapy with inhaled fluticasone propionate after induction of a severe heaves exacerbation resulted in complete resolution of clinical signs, normalization of pulmonary function tests, and significant decrease in BAL neutrophilia. This was associated with a significant decrease in IL-4 mRNA expression and increase in IFN-gamma/IL-4 ratio in horses with heaves. These results demonstrate the clinical efficacy of inhaled fluticasone propionate for the treatment of heaves and suggest a role for cytokines in the development of lower airway inflammation in heaves-susceptible horses.     

Direct stimulation of the oxidative activity of isolated equine neutrophils by TNF-alpha and IL-1beta

The capacity of the two cytokines TNF-alpha and IL-1beta to directly stimulate the oxidative activity of polymorphonuclear neutrophils remains debated. The purpose of this study was to verify if a direct stimulation of equine neutrophils by TNF-alpha and IL-1beta was possible. Equine neutrophils were isolated from blood by discontinuous density gradient centrifugation. The cell viability after isolation was >98%. The neutrophils were used at 1.25 x 10(6) cells by assay, immediately after isolation. The oxidative activity of neutrophils was measured by luminol- or lucigenin-enhanced chemiluminescence (CL), and the CL was recorded for 60 min. TNF-alpha and IL-1beta were used at concentrations ranging from 0.001 to 100 ng (0.0017-167 ng ml(-1)) for 1.25 x 10(6) neutrophils, and added to the cells just before the CL measurement. Both cytokines highly stimulated the lucigenin-enhanced CL of equine neutrophils in a dose-dependent manner. TNF-alpha was already active at 0.001 ng and IL-1beta at 0.01 ng. The CL response obtained with TNF-alpha was maximal after 5 min and more pronounced with luminol than with lucigenin. With IL-1beta, the luminol-enhanced CL response of neutrophils was short-lived and inversely proportional to the cytokine concentration: the CL response returned to baseline after 12 min, and became even lower than the baseline value for 10 and 100 ng IL-1beta. As luminol (but not lucigenin) enters the cell, we hypothesized that a rapid intracellular consumption of the luminol molecules occurred, explaining the rapid and intense CL response. The choice of the CL enhancer used in previous CL studies of neutrophils stimulation by cytokines could perhaps explain that controversial results were reported. In conclusion, we demonstrated a direct activation of the oxidative activity of equine neutrophils by TNF-alpha and IL-1beta, which was dose-dependent and obtained with very low doses equivalent to the plasma concentrations measured for both cytokines in equine septic shock. TNF-alpha and IL-1beta can thus aggravate neutrophils oxidative activity during septic shock in horses.     

Effect of transforming growth factor beta1 on chondrogenic differentiation of cultured equine mesenchymal stem cells

OBJECTIVE: To determine the morphologic and phenotypic effects of transforming growth factor beta1 (TGFbeta1) on cultured equine mesenchymal stem cells (MSC) and articular chondrocytes. SAMPLE POPULATION: Bone marrow aspirates and articular cartilage samples from a 2-year-old and two 8-month-old horses. PROCEDURE: After initial isolation and culture, MSC and chondrocytes were cultured in Ham's F-12 medium supplemented with TGF-beta1 at a concentration of 0, 1, 5, or 10 ng/ml. Medium was exchanged on day 2, and cells were harvested on day 4. Medium was assayed for proteoglycan (PG) content. Total RNA was isolated from cell cultures, and expression of aggrecan, decrin, collagen type-I, and collagen type-II mRNA was assessed by means of Northern blot analyses. Cell cultures were stained with H&E or toluidine blue and examined histologically. Additional cultures were examined after immunohistochemical staining for type-I and -II collagen. RESULTS: MSC cultures exposed to TGF-beta1 had an increased cellular density with cell layering and nodule formation that was most pronounced in cultures treated with 5 ng of TGF-beta1/ml. Expression of collagen type-II mRNA in MSC cultures exposed to 5 ng of TGF-beta1/ml was 1.7 times expression in control cultures, and expression of collagen type-I mRNA was 2.8 times expression in control cultures. Treatment of MSC with TGF-beta1 led to dose-related increases in area and intensity of type-II collagen immunoreaction. CONCLUSION: Results suggest that TGF-beta1 enhances chondrogenic differentiation of bone marrow-derived MSC in a dose-dependent manner.     

Effects of equine recombinant interleukin-1alpha and interleukin-1beta on proteoglycan metabolism and prostaglandin E2 synthesis in equine articular cartilage explants

OBJECTIVES: To evaluate the effects of equine recombinant interleukin-1alpha (rEqIL-1alpha) and recombinant interleukin-1beta (rEqIL-1beta) on proteoglycan metabolism and prostaglandin E2 (PGE2) synthesis by equine articular chondrocytes in explant culture. SAMPLE POPULATION: Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse. PROCEDURE: Expression constructs containing cDNA sequences encoding EqIL-1alpha and EqIL-1beta were generated, prokaryotically expressed, and the recombinant protein purified. Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse were separately randomized to receive rEqIL-1alpha or rEqIL-1beta treatments 10 to 500 ng/ml). Proteoglycan release was evaluated by 1,9-dimethylmethylene blue spectrophotometric analysis of explant media glycosaminoglycan (GAG) concentration and release of 35S-sulfate-labeled GAG to explant media. Proteoglycan synthesis was assessed by quantification of 35S-sulfate incorporation into proteoglycan. Explant media PGE2 concentrations were evaluated using a PGE2-specific enzyme-linked immunoassay. Data were collected at 48-hour intervals and normalized by DNA content. RESULTS: Proteoglycan release was induced by rEqIL-1alpha and rEqIL-1beta at concentrations > or =0.1 ng/ml, with 38 to 76% and 88 to 98% of total GAG released by 4 and 6 days, respectively. Inhibition of proteoglycan synthesis (42 to 64%) was observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days. Increased PGE2 concentrations were observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days. CONCLUSIONS AND CLINICAL RELEVANCE: The rEqIL-1 induced potent concentration-dependent derangement of equine chondrocyte metabolism in vitro. These findings suggest this model may be suitable for the in vitro study of the pathogenesis and treatment of joint disease in horses.     

Effects of enrofloxacin and magnesium deficiency on matrix metabolism in equine articular cartilage

OBJECTIVE: To investigate the effects of enrofloxacin and magnesium deficiency on explants of equine articular cartilage. SAMPLE POPULATION: Articular cartilage explants and cultured chondrocytes obtained from adult and neonatal horses. PROCEDURE: Full-thickness explants and cultured chondrocytes were incubated in complete or magnesium-deficient media containing enrofloxacin at concentrations of 0, 1, 5, 25, 100, and 500 microg/ml. Incorporation and release of sulfate 35S over 24 hours were used to assess glycosaminoglycan (GAG) synthesis and degradation. An assay that measured binding of dimethylmethylene blue dye was used to compare total GAG content between groups. Northern blots of RNA from cultured chondrocytes were probed with equine cDNA of aggrecan, type-II collagen, biglycan, decorin, link protein, matrix metalloproteinases 1, 3, and 13, and tissue inhibitor of metalloproteinase 1. RESULTS: A dose-dependent suppression of 35S incorporation was observed. In cartilage of neonates, 35S incorporation was substantially decreased at enrofloxacin concentrations of 25 mg/ml. In cartilage of adult horses, 35S incorporation was decreased only at enrofloxacin concentrations of > or =100 microg/ml. Magnesium deficiency caused suppression of 35S incorporation. Enrofloxacin or magnesium deficiency did not affect GAG degradation or endogenous GAG content. Specific effects of enrofloxacin on steady-state mRNA for the various genes were not observed. CONCLUSION AND CLINICAL RELEVANCE: Enrofloxacin may have a detrimental effect on cartilage metabolism in horses, especially in neonates.     

Expression of bone morphogenetic protein-6 and -2 and a bone morphogenetic protein antagonist in horses with naturally acquired osteochondrosis

OBJECTIVE: To determine the mRNA expression of bone morphogenetic protein (BMP)-6 and -2 and a BMP antagonist (Noggin) in horses with osteochondrosis. SAMPLE POPULATION: Samples of articular cartilage from affected stifle or shoulder joints of 10 immature horses with naturally acquired osteochondrosis and corresponding joints of 9 clinically normal horses of similar age; additionally, samples of distal femoral growth plate cartilage and distal femoral articular cartilage were obtained from a normal equine fetus. PROCEDURE: Cartilage specimens were snap-frozen in liquid nitrogen, and total RNA was isolated. Adjacent specimens were fixed in 4% paraformaldehyde for histologic examination. Expression of BMP-6, BMP-2, and Noggin mRNA was evaluated by real-time quantitative polymerase chain reaction (PCR) assays. Spatial tissue mRNA expression of BMP-6 was determined by in situ hybridization. RESULTS: Nucleotide sequences were obtained for portions of the BMP-6 propeptide and mature peptide region, as well as the signal and mature peptide region of Noggin. Expression of BMP-6, BMP-2, and Noggin mRNA was found to be similar in cartilage from normal and osteochondrosis-affected horses. Spatial expression of BMP-6 correlated with the middle and deep layers of articular cartilage; no differences were observed in overall expression between cartilage specimens from the 2 groups of horses. No expression of BMP-6 was found in the superficial layer, subchondral bone, or osteochondrosis-affected cleft fibrous tissue. CONCLUSIONS AND CLINICAL RELEVANCE: Although these signaling peptides may play important roles in cartilage differentiation, results did not provide evidence to suggest that they are involved in the disease process of osteochondrosis.     

The role of alveolar macrophages in the pathogenesis of recurrent airway obstruction in horses

When challenged with allergens and pro-inflammatory agents, such as Aspergillus fumigatus (AF), hay dust solution (HDS) and lipopolysaccharide (LPS), the innate immune response will not only activate the immune system but also increase the amount of pro-inflammatory cytokines in the bronchoalveolar space. The aim of this study was to assess the response of equine alveolar macrophages to different aerosolized challenges and to investigate the differences in this response between horses susceptible or nonsusceptible to recurrent airway obstruction (RAO). Seven susceptible and 5 nonsusceptible horses were challenged with saline, LPS, HDS, or AF, and bronchoalveolar lavage (BAL) cytology, total cell counts, and lung function were assessed. In addition, alveolar macrophages were isolated 6 and 24 hours after challenge, and macrophage mRNA expression of tumor necrosis factor (TNF)-alpha and interleukins (IL) IL-1beta, IL-6, IL-8, and IL-10 were measured by means of real-time (RT) polymerase chain reaction (PCR). There was a significant difference in lung function, neutrophil ratios, and total cell counts in the bronchoalveolar lavage fluid between RAO-susceptible and nonsusceptible horses. In addition, the expression of TNF-alpha, IL-1beta, and IL-8 by alveolar macrophages after challenges were higher in susceptible horses, than in nonsusceptible horses. In contrast, I1-6, considered an anti-inflammatory cytokine, showed a higher expression in nonsusceptible horses 6 hours after inhalation challenge with allergens and pro-inflammatory antigens. These data suggest that the differences between susceptible and nonsusceptible horses to RAO are not only dependent on adaptive immunity but also start with an innate immune response.