Search Results

You are looking at 1 - 10 of 32 items for :

  • "interleukins" x
  • Refine by Access: All Content x
Clear All

Abstract

Objective

To determine whether cytokines of homologous species might mediate the stimulatory effects of endotoxin on release of luteinizing hormone (LH) from pituitary cells.

Sample Population

Cells from pituitary glands collected from 8- to 14-month-old wethers.

Procedure

Cells from the anterior pituitary gland were cultured in the presence of recombinant ovine or bovine cytokines (interleukin [IL]-1α, IL-1β, and IL-2), tumor necrosis factor-α (TNF), and interferon-γ (IFN-γ). Luteinizing hormone that was released into the medium was measured. Cells were also cultured with modulators of signal transduction pathways to evaluate the second messenger system used by IL-1α and IL-1β.

Results

Similar to effects of endotoxin, IL-1α and IL-1β stimulated release of LH. Interleukin 2, TNF, and IFN-γ did not have a detectable effect on release of LH. Stimulation of LH release by IL-1αand IL-1β required activation of voltage-dependent Ca2+ channels and appeared to involve protein kinase C.

Conclusions

IL-1αand IL-1β may mediate the direct stimulatory effect of endotoxin on release of LH in vitro. Interleukin 2, TNF, and IFN-γ do not have a direct effect on release of LH; therefore, they do not mediate this effect of endotoxin.

Clinical Relevance

Stressors, including infection, are often associated with reduced fertility. Infection resulting in endotoxin release, production of interleukins, or both, can lead to direct stimulation of LH release from the pituitary gland. Inopportune release of LH via cytokines may interfere with normal pulsatile release of LH, thereby suppressing gonadal function. (Am J Vet Res 1998;59:1488–1493)

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

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

Summary

Because hepatocyte-stimulating factor/interleukin 6 (il-6) is the principal inducer of acute-phase protein synthesis in the liver, quantification of its activity in blood provides an early and sensitive assessment of the acute-phase response. Circulating il-6 activity was monitored in 4 adult horses for 72 hours after iv administration of endotoxin. In 4 experiments performed at weekly intervals and in randomized order, each horse was given endotoxin—1,000, 30, 1, and 0 ng/kg of body weight. Plasma il-6 activity was quantified as the ability to promote growth of the il-6-dependent B-cell hybridoma, B13.29 clone B9. Interleukin-6 activity (171 ± 10.2 U/ml) was found in all pretreatment plasma samples and was significantly (P < 0.05) increased above baseline from 2 to 12 hours after 1,000 ng of endotoxin/kg was given and at 3 hours after 30 ng of endotoxin/kg was given. After 1,000- or 30-ng/kg dosage of endotoxin, peak plasma il-6 activity (10,128 ± 4,096 and 1,555 ± 1,326 U/ml, respectively) was observed for 3 hours. The il-6 response of endotoxin-treated horses began about 1 hour after tumor necrosis factor appeared in the circulation, and its course closely approximated the endotoxin-induced febrile reaction. Significant increase in plasma il-6 activity was not detected in horses given 1 ng of endotoxin/kg or control buffer.

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine whether matrix metalloprotease 13 (MMP-13; collagenase 3) is produced by equine chondrocytes and to investigate modulation of its expression by recombinant human interleukin β (rhIL-1β) and corticosteroids.

Procedure

Equine chondrocytes in monolayer culture were stimulated with rhIL-1β. Total RNA was extracted, purified, and reverse transcribed into DNA. Using appropriate primers, a putative MMP-13 fragment was amplified by polymerase chain reaction, and cloned into a bacterial vector. The resultant fragment was purified and sequenced, then was used to prepare a digoxigenin-labeled cRNA probe. Monolayer cultures of first-passage chondrocytes were treated with rhIL-1β in the presence or absence of dexamethasone (10-6 M) or methylprednisolone acetate (10-9 M to 10-5 M), in addition to positive and negative controls. Cellular RNA was extracted and resolved on agarose gels and subjected to northern blot analysis, using the equine MMP-13 probe.

Results

Reverse transcriptase-polymerase chain reaction enabled isolation of a 0.6-kb fragment of equine MMP-13 cDNA that had 93% homology with the human MMP-13 cDNA sequence. rhIL-1 significantly stimulated MMP-13 expression in the chondrocytes. Methylprednisolone acetate inhibited the stimulatory effects of rhIL-1 in dose-dependent manner that was statistically significant at 10-5 M.

Conclusions

Novel information was gained on the existence of MMP-13 and its expression in equine chondrocytes, which suggests a possible role for this enzyme in matrix degradation in horses with arthritis. (Am J Vet Res 1996;57:1631–1634)

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

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

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

SUMMARY

Twenty-four horses were randomly allocated to 3 groups. Horses were anesthetized, subjected to a ventral midline celiotomy, and the large colon was exteriorized and instrumented. Group-1 horses served as sham-operated controls. Group-2 horses were subjected to 6 hours of low-flow colonic arterial ischemia, and group-3 horses were subjected to 3 hours of ischemia and 3 hours of reperfusion. Baseline (bl) samples were collected, then low-flow ischemia was induced by reducing ventral colonic arterial blood flow to 20% of bl. All horses were monitored for 6 hours after bl data were collected. blood samples were collected from the colonic vein and main pulmonary artery (systemic venous [sv]) for measurement of plasma endotoxin, 6-keto prostaglandin F (6-kPG), thromboxane B2 (txb 2), and prostaglandin E2 (pge 2) concentrations. Tumor necrosis factor and interleukin-6 activities were measured in colonic venous (cv) serum samples. Data were analyzed, using two-way anova, and post-hoc comparisons were made, using Dunnett's and Tu- key's tests. Statistical significance was set at P < 0.05. Endotoxin was not detected in CV or sv plasma at any time. There was no detectable tumor necrosis factor or interleukin-6 activity in CV samples at any time. There were no differences at bl among groups for CV or sv 6-kPG, pge 2, or txb 2 concentrations, nor were there any changes across time in group-1 horses. Colonic venous 6-kPG concentration increased during ischemia in horses of groups 2 and 3; CV 6-kPG concentration peaked at 3 hours in group-3 horses, then decreased during reperfusion, but remained increased through 6 hours in group-2 horses. Systemic venous 6-kPG concentration increased during reperfusion in group-3 horses, but there were no changes in group- 2 horses. Colonic venous pge 2 concentration increased during ischemia in horses of groups 2 and 3, and remained increased for the first hour of reperfusion in group-3 horses and for the 6-hour duration of ischemia in group-2 horses. There were no temporal alterations in sv pge 2 concentration. There was no difference in CV or sv ixb2 concentration among or within groups across time; however, there was a trend (P = 0.075) toward greater CV txb 2 concentration at 3.25 hours, compared with bl, in group-3 horses. Eicosanoid concentrations were significantly lower in sv, compared with CV plasma. Prostaglandin E2 and 6-kPG concentrations were approximately 3 to 8 and 5 to 10 times greater, respectively, in CV than in sv plasma. The increased concentrations of 6-kPG and pge 2 in CV plasma were likely attributable to their accumulation secondary to colonic ischemia. The increased values of these vasodilator eicosanoids may have a role in the reactive hyperemia observed during reperfusion. The increased 6-kPG concentration in sv plasma may represent spillover from the colonic vasculature, but more likely reflects systemic production.

Free access
in American Journal of Veterinary Research