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Abstract

Objective—To determine the effects of interleukin (IL)-1β on matrix synthesis and degradation by chondrocytes cultured in a 3-dimensional (3-D) gel medium.

Sample Population—Chondrocytes from 7 dogs.

Procedure—Articular chondrocytes were harvested and cultured in 3-D gel medium alone or with 10 or 20 ng IL-1βml that was added beginning on day 0, 3, 6, or 9. On days 3, 6, 12, and 20 of 3-D culture, samples of the liquid medium were evaluated for glycosaminoglycan (GAG), prostaglandin E2 (PGE2), and matrix metalloprotease (MMP)-3 content. The 3-D plug in each well was evaluated for histologic characteristics of viability, cell morphology, and proteoglycan staining, immunohistochemically stained for collagen type II, and spectrophotometrically analyzed for GAG content.

Results—Significant differences for all variables were detected between controls and each IL-1β group, among groups with different IL-1β concentrations, and among groups with IL-1β added at various time points. Chondrocytes exposed to IL-1β had loss of GAG, increased PGE2 and MMP-3 concentrations, and lack of collagen type-II synthesis. These IL-1β effects appeared to be time and concentration dependent.

Conclusions—Addition of IL-1β to chondrocytes in 3- D gel medium results in time- and concentrationdependent effects on matrix synthesis and degradation and provides an appropriate in vitro model for many of the pathophysiologic events associated with osteoarthritis. (Am J Vet Res 2000;61:766–770)

Full access
in American Journal of Veterinary Research

Abstract

Objective

To determine whether an established bovine mammary epithelial cell line expresses interleukin 8 (IL-8) mRNA and synthesizes antigenic IL-8 in response to lipopolysaccharide (LPS) stimulation.

Sample Population

A bovine mammary epithelial cell line (MAC-T).

Procedure

mRNA was isolated from cells stimulated with graded concentrations of LPS. The first strand of IL-8 cDNA was synthesized, using a reverse transcriptase (RT) reaction with a specific oligonucleotide. Amplification of IL-8 cDNA was obtained by use of polymerase chain reaction (PCR). The MAC-T-derived antigenic IL-8 was quantified by use of a commercial anti-human IL-8 kit in a sandwich ELISA.

Results

RT-PCR revealed expression of MAC-T-derived mRNA within the first hour after stimulation with LPS. Expression of IL-8 mRNA was correlated to production of IL-8 protein detected in medium by use of the sandwich ELISA. Amounts of antigenic IL-8 increased in a dose- and time-dependent manner, and were maximal (57 pg/ml) at 48 hours after stimulation with 20 μg of LPS/ml.

Conclusions

MAC-T cells secrete IL-8 in response to stimulation with LPS in a dose- and time-dependent manner. The results were consistent with our hypothesis that mammary gland epithelial cells can be a source of IL-8 during the early stage of mastitis. Therefore, IL-8 may have a pivotal role in resolving bacterial infections. (Am J Vet Res 1998;59:1563-1567)

Free access
in American Journal of Veterinary Research

SUMMARY

Objective

To determine whether clinical progression of paratuberculosis in cattle was associated with alterations in cytokine gene expression in affected tissues.

Animals

5 uninfected adult Holstein cows, 7 adult Holstein cows naturally infected with Mycobacterium paratuberculosis that did not have clinical signs of disease, and 4 adult Holstein cows naturally infected with M paratuberculosis that had progressive clinical signs of infection.

Procedure

Samples of ileum and cecal lymph nodes were obtained from each animal at the time of slaughter. A reverse transcriptase-competitive polymerase chain reaction assay was used to determine mRNA expression of interferon-γ (IFN-γ) and interleukin 4 in each sample.

Results

Interferon-γ gene expression was significantly higher in ileum and cecal lymph node samples from subclinically infected cows than from clinically infected cows.

Conclusions and Clinical Relevance

Progression of paratuberculosis to clinical stages is associated with reduced expression of IFN-γ at site of infection. If immune response to M paratuberculosis can be manipulated so that IFN-γ expression is increased, resistance to infection in cattle might be enhanced. (Am J Vet Res 1998;59:842–847)

Free access
in American Journal of Veterinary Research

SUMMARY

The role of interleukin 1 (il-1) as an inflammatory mediator during mastitis and the therapeutic effect of recombinant human il-1 receptor antagonist (il-1ra) for bovine mastitis was studied. Cows were intramammarily infused with lipopolysaccharide (25 μg) in 1 mammary gland. Half the cows also received infusions of 5 mg of il-1ra into the same mammary gland just prior to endotoxin infusion and 4, 8, and 12 hours later. After endotoxin infusion, tumor necrosis factor and high il-1 bioactivity were detected in whey from infused glands. Vascular permeability changes and neutrophil accumulation in milk paralleled the appearance of cytokines. A systemic reaction, characterized by pyrexia and an increase in blood cortisol concentration, also were observed. Milk yield was inhibited and milk composition was altered in infused and noninfused glands. The increase in il-1 bioactivity in milk after endotoxin infusion was almost completely prevented in glands receiving il-lra. However, il-lra had no effect on local inflammation, systemic reaction, or impairment in productive performance. These results indicate that il-1 does not mediate its effect within the milk compartment, and suggest either that il-1 is not critical to the mastitic response or that intramammary infusion of il-1ra does not place the antagonist where il-1 interacts with its receptor.

Free access
in American Journal of Veterinary Research

Summary

A study was conducted to determine whether serum interleukin-6 (il-6) activity increased in horses during experimentally induced endotoxemia and whether serum il-6 activity correlated to changes in clinical or laboratory data. Six clinically normal horses were given endotoxin iv (30 ng/kg of body weight) in 0.9% NaCl solution over 1 hour. Five of these and 1 additional horse served as controls and were given only 0.9% NaCl solution. Venous blood, for determination of serum il-6 activity and wbc count, was collected before and at various times through 8 hours after the start of endotoxin or NaCl infusion. Rectal temperature and heart and respiratory rates were recorded throughout the study period. Serum il-6 activity was determined by bioassay of proliferation of the B13.29 clone B.9 hybridoma cell line. From 1.5 through 5 hours after start of the infusion, serum il-6 activity was significantly (P < 0.05) increased in horses given endotoxin. Mean peak serum il-6 activity was observed between 3 and 4 hours. In response to endotoxin infusion, horses became lethargic, tachycardic, and febrile. Leukopenia developed by 1 hour, followed by leukocytosis at 8 hours. Significant (P < 0.05) positive association and linear correlation were apparent between mean serum il-6 activity and mean rectal temperature in the group of horses that were given endotoxin. Changes from baseline were not evident in any of the clinical or laboratory values in horses given only NaCl solution.

Free access
in American Journal of Veterinary Research

SUMMARY

In these studies, the effects of recombinant human interleukin 2 (rHuil-2) were examined on in vitro tumor cytotoxicity by canine blood lymphocytes obtained from peripheral vessels through use of a chromium release microcytotoxicity assay. Cytotoxic activity by lymphokine-activated killer cells was significantly increased, compared with that by untreated lymphocytes in a dose-dependent manner. The maximal effect was attained with 300,000 IU of rHuil-2/ml. Lymphokine-activated killing also was dependent on the duration of incubation with rHuil-2. After 1 day of rHuil-2 incubation, cytotoxicity was significantly increased, compared with that of untreated lymphocytes. Of the 3 times examined, cytotoxicity peaked after 3 days of rHuil-2 incubation. High levels of cytotoxic activity were still present at 7 days of incubation. Numbers of granular lymphocytes increased over the times examined. These results demonstrate functional and morphologic changes in canine peripheral blood lymphocytes obtained from peripheral vessels after incubation with rHuil-2 in a dose- and time-dependent manner.

Free access
in American Journal of Veterinary Research

Abstract

Objective—To determine the effect of caprine arthritis- encephalitis virus (CAEV) infection on expression of interleukin-16 (IL-16).

Animals—-6 goats experimentally infected with CAEV and 6 age-matched healthy uninfected control goats.

Procedure—-Peripheral blood mononuclear cells (PBMCs) and synovial membrane cells from infected and control goats cultured with or without phytohemagglutinin were analyzed for IL-16 mRNA by use of a reverse transcriptase-polymerase chain reaction assay with goat-specific primers, after cloning and sequencing of a 384-bp fragment of the goat IL-16 gene. Synovial fluid, serum, and culture supernatants of PBMCs and synovial cells of control and CAEVinfected goats were analyzed for IL-16 by use of an ELISA.

Results—-The 384-bp product was 86% homologous to the corresponding human IL-16 nucleotide sequence. Higher expression of IL-16 mRNA in PBMCs (unstimulated or stimulated with phytohemagglutinin) was detected in samples from CAEVinfected goats, compared with control goats, but the difference was not significant. Synovial membrane cells infected in vitro had higher expression than uninfected control cells. Higher IL-16 concentration was detected in synovial fluid, serum, and culture supernatants of PBMCs of infected goats than in samples from control goats.

Conclusions and Clinical Relevance—Infection with CAEV increases expression of IL-16, a proinflammatory and chemotactic cytokine. This cytokine appears to be constitutively expressed at low concentrations in normal uninfected PBMCs and synovial membrane cells. Increased production of IL-16 in CAEV infection may partly be responsible for increased lymphoid cell infiltrations observed in arthritic joints and other tissues of CAEV-infected goats. (Am J Vet Res 2002;63:1418–1422)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine response of interleukin-1α (IL-1α)-conditioned equine articular cartilage explants to insulin-like growth factor-1 (IGF-1).

Sample Population—Cartilage from the trochlea and condyles of the femur of a clinically normal 4-year-old horse.

Procedure—Effects of IGF-1 (0 to 500 ng/ml) after addition of IL-1α were evaluated by assessing matrix responses, using a sulfated glycosaminoglycan (GAG) assay, matrix 35SO4 GAG incorporation, and release of GAG. Mitogenic response was assessed by 3H-thymidine incorporation into DNA and fluorometric assay of total DNA concentration.

Results—Human recombinant IL-1α (40 ng/ml) increased the amount of labeled GAG released and decreased labeled and total GAG remaining in explants, and IL-1α decreased mitogenic response. Addition of IGF-1 counteracted effects seen with IL-1α alone. In general, IGF-1 decreased total and labeled GAG released into the medium, compared with IL-1α- treated explants (positive-control sample). Values for these variables did not differ significantly from those for negative-control explants. A significant increase in total and newly synthesized GAG in the explants at termination of the experiment was observed with 500 ng of IGF-1/ml. Labeled GAG remaining in explants was greater with treatment at 50 ng of IGF-1/ml, compared with treatment with IL-1α alone. Concentrations of 200 ng of IGF-1/ml abolished actions of IL-1α and restored DNA synthesis to values similar to those of negative-control explants.

Conclusions and Clinical Relevance—IGF-1 at 500 ng/ml was best at overcoming detrimental effects associated with IL-1α in in vitro explants. These beneficial effects may be useful in horses with osteoarthritis. (Am J Vet Res 2000;61:436–441)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine the complementary DNA (cDNA) sequence of interleukin-1 receptor antagonist (IL-1ra) in horses and compare messenger RNA (mRNA) expression of IL-1ra among horses of various breeds.

Sample Population—Blood samples from neonatal and adult horses examined for a variety of diseases.

Procedure—A polymerase chain reaction procedure was used to amplify a 220 base pair (bp) portion of the genomic DNA. The upstream and downstream regions of the cDNA sequence were determined by means of 5' and 3' rapid amplification of cDNA ends (RACE) procedures. Northern blot hybridization was used to examine steady-state mRNA expression of IL- 1ra.

Results—The consensus sequence of the cDNA obtained with the 5'-RACE procedure and the sequence for the 220 bp portion of the genomic DNA represented the putative sequence for secreted IL- 1ra. The predicted secreted IL-1ra amino acid sequence contained 176 residues with an in-frame stop codon; the N-terminal 25 amino acid residues resembled the signal peptide reported for human secreted IL-1ra. An approximately 1.3 kilobase pair (kb) band that represented a portion of the 3' end of the coding region and the 3' untranslated region was obtained by use of the 3' -RACE procedure. Northern blot hybridization detected a 1.6 kb transcript in blood RNA from adult Arabian, Belgian, Thoroughbred, and Standardbred horses.

Conclusions—Results suggest that the DNA for equine secreted IL-1ra has a short (29 bp) 5' untranslated region, a 534 bp coding region, and a long (approximately 1,080 bp) untranslated region. (Am J Vet Res 2000;61:920–924)

Full access
in American Journal of Veterinary Research

Summary

A study was performed to determine whether equine peritoneal macrophages produce interleukin 6 (il-6) in vitro in response to endotoxin. Peritoneal fluid was collected from 14 clinically normal adult horses and was used as the source of peritoneal macrophages. Macrophages from each horse were isolated and cultured separately in vitro in the absence or presence of various concentrations (0.5, 5, or 500 ng/ml) of endotoxin (lipopolysaccharide from Escherichia coli 055:B5). Culture medium supernatants were collected after 3, 6, 12, and 24 hours’ incubation and were frozen at −70 C until assayed for il-6 activity. Supernatant il-6 activity was determined by use of a modified colorimetric assay and the murine hybridoma cell line B 13.29 clone B.9, which is dependent on il-6 for survival.

Results indicated that equine peritoneal macrophages produce il-6 in vitro and that supernatant medium il-6 activity was significantly (P < 0.05) increased by exposure to endotoxin. Significant (P < 0.05) time and treatment effects on macrophage il-6 production were apparent. The il-6 activity peaked at 6 or 12 hours’ incubation, then remained high through 24 hours’ incubation, regardless of endotoxin exposure. Medium il-6 activity during 3 and 6 hours’ incubation was significantly (P < 0.05) greater in macrophages exposed to 5 or 500 ng of endotoxin/ml than in those exposed to 0.5 ng of endotoxin/ml; however peak il-6 activity was similar among all endotoxin concentrations. Endotoxin concentration did not have an effect on medium il-6 activity from macrophages exposed to endotoxin for 12 or 24 hours.

Free access
in American Journal of Veterinary Research