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Abstract

Objective—To determine whether adenosine influences the in vitro release of nitric oxide (NO) from differentiated primary equine articular chondrocytes.

Sample Population—Articular cartilage harvested from the metacarpophalangeal and metatarsophalangeal joints of 11 horses (3 to 11 years old) without history or clinical signs of joint disease.

Procedure—Chondrocytes were isolated, plated at a high density (105 cells/well), and treated with adenosine, the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), bradykinin, or other agents that modify secondary messenger pathways alone or in combination with bacterial lipopolysaccharide (LPS) or recombinant human interleukin-1α (rhIL-1α). Nitric oxide release was measured indirectly by use of the Griess reaction and was expressed as µmol of nitrite in the supernatant/µg of protein in the cell layer. Inducible nitric oxide synthase (iNOS) activity was determined by measuring the conversion of radiolabeled arginine to radiolabeled citrulline.

Results—Treatment of chondrocytes with adenosine alone had no significant effect on NO release. However, adenosine and NECA inhibited LPS- and rhIL-1α-induced NO release. This response was mimicked by forskolin, which acts to increase adenylate cyclase activity, but not by the calcium ionophore A23187. Treatment of chondrocytes with phorbol myristate acetate, which acts to increase protein kinase C activity, potentiated LPS-induced NO release. Adenosine treatment also significantly inhibited the LPS-induced increase in iNOS activity.

Conclusions and Clinical Relevance—Adenosine and the nonspecific adenosine receptor agonist NECA inhibited inflammatory mediator-induced release of NO from equine articular chondrocytes. Modulation of adenosine receptor-mediated pathways may offer novel methods for treatment of inflammation in horses with joint disease. (Am J Vet Res 2002;63:204–210)

Full access
in American Journal of Veterinary Research

Abstract

Objective

To investigate effects of intramammary infusion of β-1,3-glucan or recombinant ovine interleukin-2 (rOvIL-2) on blood and mammary leukocyte subpopulations and their expression of various surface antigens in sheep.

Animals

12 healthy multiparous, nonpregnant, non-lactating fine-wool Merino ewes.

Procedure

β-1,3-Glucan in pyrogen-free saline solution (PFSS; n = 6), rOvIL-2 in PFSS (3), or PFSS (3) was infused on days 0 and 7 in 1 udder half of each ewe. Jugular vein blood and mammary secretion samples were taken before infusion and on days 2, 7, and 21 after infusion. Total and differential leukocyte counts were obtained, and blood and mammary cells were labeled for flow cytometry.

Results

Slight swelling of the mammary glands was observed on day 2 after rOvIL-2 but not after β-1,3-glucan infusion. Both substances induced significant increase in mammary secretion leukocyte numbers, compared with controls. β-1,3-Glucan induced an influx of monocyte/macrophages, whereas neutrophils were the predominating cell population after rOvIL-2 infusion. β-1,3-Glucan induced selection for CD4+, B-cell+, WC1+, and L-selectin+ lymphocytes on day 14 after infusion. By comparison, rOvIL-2 induced selection for B-cell+ and L-selectin+ lymphocytes on days 14 and 21 and depletion of CD8+ and, to some degree, of IL-2R+ lymphocytes. β-1,3-Glucan induced an increase in the proportion of CD14+ leukocytes, indicating selective migration of monocyte/macrophages to the nonlactating udder.

Conclusion

β-1,3-Glucan and rOvIL-2 can modulate nonspecific immunity in the udder of sheep but may exert their effects by differing mechanisms.

Clinical Relevance

Stimulation of the nonspecific defense against udder infections may improve control of mastitis. (Am J Vet Res 1999;60:703–707)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To evaluate potential stimulatory or matrixsparing effects of insulin-like growth factor 1 (IGF-1 ), alone or in combination with a corticosteroid, in an interleukin 1 (IL-1)-induced model of cartilage degradation.

Samples

Cartilage from the weightbearing surfaces of trochlea and condyles of clinically normal 2-year-old male horses.

Procedure

Triamcinolone acetonide and IGF-1 effects were evaluated by assessing: matrix responses by sulfated glycosaminoglycan (GAG) assay and [35S]sulfated GAG synthesis; collagen content by hydroxyproline assay; and mitogenic response by [3H]thymidine incorporation into DNA and fluorometric assay of total DNA concentration.

Results

Conditioning of cartilage expiants with 10 ng of human recombinant IL-1α increased degradation and decreased synthesis of matrix proteoglycans (PG), without affecting matrix collagen content. Human recombinant IGF-1 decreased PG loss and reversed the reduction of PG synthesis in cartilage expiants conditioned with IL-1. Given alone, steroids decreased PG concentration and synthetic rate in normal cartilage. However, the previously diminished PG content, attributable to IL-1 conditioning, was not further exacerbated by steroid administration in IL-1-conditioned expiants. Combined treatment of normal cartilage expiants with IGF-1 and steroids resulted in PG preservation and increase in collagen content. Similar PG and collagen effects were not evident when treating IL-1-conditioned cartilage with IGF-1/steroid combinations. Decrease in chondrocyte proliferation was associated with steroid administration. Exposure to IGF and steroids prevented the decrease in mitogenesis that could lead to cellular loss, particularly in IL-1-conditioned expiants.

Conclusion

Combination IGF-1 and steroid treatment of normal cartilage cultures indicated substantial ability to override the anabolic suppression associated with steroids alone. Potentially, administration of corticosteroids, followed by IGF-1, may act to decrease propagation of detrimental mediator release while allowing appreciation of the chondroenhancing effects of IGF-1. These beneficial effects were considerably reduced in IL-1-induced cartilage damage. (Am J Vet Res 1997;58:524–530)

Free access
in American Journal of Veterinary Research

SUMMARY

Objective

To determine whether recombinant ovine interleukin (oIL)-1 or oIL-2 alters basal or hypothalamic peptide-induced secretion of ACTH from cultured sheep pituitary cells.

Animals

The pituitary gland was collected from castrated male sheep ranging from 0.5 to 1 year old.

Procedure

Cells were cultured for 3 to 5 days, then were treated with oIL for variable periods. Cells were washed and treated with the hypothalamic peptides corticotropin-releasing hormone (CRH) or arginine vasopressin (AVP) or both. Medium bathing the cells was collected and assayed for ACTH concentration.

Results

Ovine IL-1α and oIL-1β, but not oIL-2, increased the amount of ACTH released in response to CRH, AVP, and CRH and AVP combined. Both oIL were effective after 3, but not 18 or 24 hours of exposure. Treatment with oIL-1 did not affect basal release of ACTH. Exposure of cells to phorbol 12-myristate 13-acetate or calphostin C before treatment with oIL-1β inhibited the ability of the cytokine to augment ACTH release, suggesting a role for protein kinase C in the process.

Conclusions

Local concentration of oIL-1 in the sheep pituitary gland may have an important role in determining secretion of ACTH in response to CRH or AVP or both from the hypothalamus.

Clinical Relevance

The hypothalamic-pituitary-adrenocortical axis may be activated after immune challenge. The cytokine oIL-1 has been implicated as an important mediator in this process. The pituitary gland may be an important target for this effect. (Am J Vet Res 1998;59:107–110)

Free access
in American Journal of Veterinary Research

SUMMARY

Recombinant human interleukin-2 (rhil-2) was evaluated for its influence on total and differential wbc counts, lymphocyte blastogenic responsiveness to mitogens, and several measurements of neutrophil function in clinically normal and in dexamethasone-treated cattle. A single dose of rhil-2 (2.5 × 107 U) given sc had no influence on the total or differential wbc count; however, it did cause an inhibition of neutrophil random migration. The other measurements of neutrophil function (Staphylococcus aureus ingestion, cytochrome C reduction, iodination, and antibody-dependent and antibody-independent cell-mediated cytotoxicity) evaluated were not significantly altered. The rhil-2 treatment was associated with a significant (P < 0.01) decrease in uptake of [3H]thymidine in unstimulated lymphocytes and a tendency toward enhanced blastogenesis of lymphocytes stimulated with phytohemagglutinin. This enhancement was significant (P < 0.05) only when the results were expressed as a stimulation index. Lymphocyte responsiveness to concanavalin A and pokeweed mitogen was not significantly influenced by rhil-2 administration. Dexamethasone (0.04 mg/kg) administered every 24 hours for 3 consecutive days altered the wbc count and several measurements of lymphocyte and neutrophil function. The administration of a single dose of rhil-2 (2.5 × 107 U) 8 hours after the first dose of dexamethasone did not alter the influence of dexamethasone on any of the measurements. These results indicated that rhil-2 has some biologic activity in cattle, but when used as administered here, did not overcome the influence of dexamethasone on the in vitro measurements of lymphocyte and neutrophil function that were evaluated.

Free access
in American Journal of Veterinary Research

Summary

The effect of interleukin 1 (il-1) on equine articular cartilage was investigated, using a cartilage explant culture system. Measurement of [35S]O4 incorporation revealed synthesis of matrix proteoglycan by cartilage to be decreased 45, 59.7, and 37.5% after 1, 3, and 5 days, respectively, in culture in the presence of 5 U of il-1/ml. There was no change in proteoglycan degradation as determined by measurement of [35S]O4 release into the culture medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cartilage-conditioned medium indicated that exposure of cartilage to il-1 caused a decrease in total protein synthesis by 45, 68, and 87% after 1, 3, and 5 days, respectively, in culture while selectively inducing synthesis of the 57-kd neutral metalloproteinase stromelysin (matrix metallo-proteinase-3) in young and adult horses. Identification of stromelysin was confirmed by functional characterization and immunoprecipitation. Baseline total protein synthesis, as well as specific synthesis of stromelysin in cartilage from adult and aged horses, was markedly less than that of young horses. The il-1- induced reduction in total protein synthesis may not be a characteristic of equine articular cartilage from affected joints of horses with naturally acquired osteoarthritis as indicated by an overall increase in protein synthesis by osteoarthritic explants.

Introduction of il-1 into an equine articular cartilage explant culture system resulted in decrease of matrix component synthesis and increase in specific degradative enzyme synthesis and activity. Articular cartilage from aged horses had markedly less overall metabolic activity, compared with cartilage from young horses. Articular cartilage from affected joints of horses with naturally acquired osteoarthritis did not have metabolic alterations identical to those of il-1-stimulated normal articular cartilage from the same individual, necessitating reevaluation of the validity of the il-1-induced model of osteoarthritis. Osteoar thritis is a common, naturally acquired disease of horses, and tissue from animals of all ages and stages of osteoarthritis is available. The equine model of osteoarthritis may afford an important means of studying the alterations in articular cartilage metabolism as a function of age and disease severity.

Free access
in American Journal of Veterinary Research

SUMMARY

Direct effects of endotoxin (lipopolysaccharide [lps]) on equine wbc are known to stimulate the release of a variety of mediators including thromboxane, prostacyclin, and leukotrienes. In this study, 0.1 μg of lps/ml stimulated an early increase in tumor necrosis factor, succeeded by an increase in interleukin-1, but concentrations of lps up to 5.0 μg/ml caused no significant increase in superoxide anion release. The concentration of lps (0.1 μg/ml) used in this experiment was in the range of concentrations measured in plasma of some horses with gastrointestinal problems.

These results indicate that mediators released in response to low concentrations of lps may be responsible for many of the lps-induced pathophysiologic effects. This is indicated because concentrations of lps detected in plasma of some horses with severe gastrointestinal problems are approximately 0.1 μg/ml, a concentration that will stimulate cells to produce tumor necrosis factor, but will not stimulate any other measurable cytotoxic effect.

Free access
in American Journal of Veterinary Research

Abstract

Objective—To study the local immune response of calves to bovine respiratory syncytial virus (BRSV) infection with emphasis on IgE production and cytokine gene expression in pulmonary lymph.

Animals—Twelve 6- to 8-week-old Holstein bull calves. Six similar control calves were mock infected to obtain control data.

Procedure—Lymphatic cannulation surgery was performed on 12 calves to create a long-term thoracic lymph fistula draining to the exterior. Cannulated calves were exposed to virulent BRSV by aerosol. Lymph fluid collected daily was assayed for BRSV and isotype-specific IgE antibody, total IgG, IgA, IgM, and protein concentrations. Interleukin-4 (IL-4), interleukin- 2 (IL-2), and interferon-γ were semi-quantitated by reverse transcription-polymerase chain reaction (RT-PCR). Cell counts and fluorescence-activated cell scanner (FACSCAN) analysis of T-cell subsets were performed on lymph cells.

Results—Calves had clinical signs of respiratory tract disease during days 5 to 10 after infection and shed virus. Bovine respiratory syncytial virus-specific IgE in infected calves was significantly increased over baseline on day 9 after infection. Mean virus-specific IgE concentrations strongly correlated with increases in severity of clinical disease (r = 0.903). Expression of IL-2, IL-4, and interferon-γ was variably present in infected and control calves, with IL-4 expression most consistent during early infection.

Conclusions and Clinical Relevance—Infection with BRSV was associated with production of BRSV-specific IgE, and IL-4 message was commonly found in lymph cells of infected calves. This finding supports the concept that BRSV-induced pathophysiology involves a T helper cell type-2 response. Effective therapeutic and prophylactic strategies could, therefore, be developed using immunomodulation to shift the immune response more toward a T helper cell type-1 response. (Am J Vet Res 2000;61:291–298)

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in American Journal of Veterinary Research

Abstract

Objective—To determine messenger RNA expression of cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, and interleukin- (IL)-1β 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 µg/ml and 100 ng/ml, respectively). Northern blot analysis with equine-specific probes for COX-2, TNF-α, and IL-1β was performed, using isolated total cellular RNA.

Results—Although no message was detected for IL-1β or TNF-α 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-1β and TNF-α 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. (Am J Vet Res 2001;62:1957–1963)

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in American Journal of Veterinary Research

Abstract

Objective—To analyze effects of hay dust exposure on interleukin-8 (IL-8) concentration, percentage of neutrophils, and neutrophil chemotactic activity in bronchoalveolar lavage fluid (BALF) of horses with chronic obstructive pulmonary disease (COPD).

Animals—16 healthy horses and 29 horses with COPD.

Procedure—IL-8 concentration, percentage of neutrophils, and neutrophil chemotactic activity in BALF were measured. Values were analyzed with respect to hay dust exposure. These variables were also measured in 5 asymptomatic horses with COPD after the induction of clinical signs by changing feed from silage to hay.

Results—IL-8 concentrations and chemotactic activity in BALF were greater in horses with COPD, compared with healthy horses, and greater in horses with COPD exposed to hay dust, compared with nonexposed affected horses. An increase in IL-8 concentration accompanied by an increase in percentage of neutrophils in BALF and development of clinical signs of COPD were induced in asymptomatic horses with COPD by changing feed from silage to hay.

Conclusions and Clinical Relevance—Exposure of horses with COPD to hay dust components resulted in an increase in IL-8 secretion at the bronchoalveolar surface. This chemokine may play a role in the pathogenesis of COPD, because it causes neutrophil accumulation in the bronchoalveolar space. Our results underscore the importance of eliminating dust sources for the treatment and prevention of COPD in horses. (Am J Vet Res 2000;61:1369–1374)

Full access
in American Journal of Veterinary Research