To determine the in vitro effects of epinephrine, norepinephrine, and dobutamine on lipopolysaccharide (LPS)-stimulated production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) in blood from healthy dogs.
Blood samples from 9 healthy dogs.
Blood samples were incubated with LPS from Escherichia coli O127:B8 or PBSS (control) for 1 hour. Afterward, the samples were incubated with 10μM epinephrine, norepinephrine, or dobutamine or with saline (0.9% NaCl) solution (control) for 23 hours. Leukocyte viability was assessed by use of trypan-blue exclusion in blood from 2 dogs to ensure cell viability was not altered by the catecholamines. Tumor necrosis factor-α, IL-6, and IL-10 concentrations were measured in the supernatant in duplicate with a canine-specific multiplex bead-based assay. Blood samples from 2 dogs were used to create dose-response curves to evaluate whether the observed cytokine modulation was dependent on catecholamine concentration.
Incubation of blood with epinephrine and norepinephrine significantly increased LPS-stimulated production of IL-10, compared with the control. Epinephrine and norepinephrine significantly decreased LPS-stimulated production of TNF-α, compared with the control. Epinephrine and norepinephrine did not significantly alter LPS-stimulated production of IL-6. Dobutamine did not alter catecholamine production.
CONCLUSIONS AND CLINICAL RELEVANCE
Epinephrine and norepinephrine, but not dobutamine, had immunomodulatory effects on LPS-stimulated TNF-α and IL-10 production in blood from healthy dogs in this in vitro model of sepsis. Data suggested that dobutamine may have immune system-sparing effects in dogs with sepsis.
Objective—To determine the effects of interleukin
(IL)-1 and tumor necrosis factor (TNF)-α on canine
chondrocytes cultured in an agarose-based 3-dimensional
Sample Population—Humeral head articular cartilage
chondrocytes obtained from 6 adult dogs.
Procedure—Chondrocytes were cultured in a 3-D
system for ≤ 12 days in serum-free medium with IL-1α, IL-1β,
or TNF-α 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
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-1β
(50 ng/mL) group were significantly higher at day 1; at
all evaluations, media concentrations of nitric oxide
were significantly higher in all TNF-α–treated cultures;
and concentrations of PGE2 in media samples were
significantly higher in the IL-1β (50 ng/mL) and IL-1β
(100 ng/mL) groups at days 1 and 3, in the IL-1β
(100 ng/mL) group at day 6, and in all TNF-α groups at
days 1, 3, and 6.
Conclusions and Clinical Relevance—Results suggested
that TNF-α more readily induces production of
nitric oxide and PGE2 by canine chondrocytes, compared
with IL-1β. In vitro, IL-1α appeared to have a
minimal effect on canine chondrocytes. (Am J Vet Res
Objective—To determine the in vitro effects of selected growth factors on fibrochondrogenesis by synovial membrane cells from nonosteoarthritic (normal) and osteoarthritic joints of dogs.
Animals—5 dogs with secondary osteoarthritis of shoulder or stifle joints and 6 dogs with normal joints.
Procedures—Synovial membrane cells were harvested from normal and osteoarthritic joints and cultured in monolayer with or without (control) basic fibroblast growth factor, transforming growth factor-β1, and insulin-like growth factor-1. In the cultured cells, fibrochondrogenesis was measured by use of a real-time reverse transcriptase PCR assay to determine relative expressions of collagen I, collagen II, and aggrecan genes and of 3 genes involved in embryonic chondrogenesis: Sry-type homeobox protein-9 (SOX-9), frizzled-motif associated with bone development (Frzb), and regulator of G-protein signaling-10 (RGS-10). Tissue collagen content was measured via a hydroxyproline assay, and sulfated glycosaminoglycan content was measured via a 1,9-dimethylmethylene blue assay. Cellularity was determined via a double-stranded DNA assay. Immunohistochemical analysis for collagens I and II was also performed.
Results—In vitro collagen synthesis was enhanced by growth factor stimulation. Although osteoarthritic-joint synoviocytes could undergo a fibrocartilage-like phenotypic shift, their production of collagenous extracellular matrix was less than that of normal-joint synoviocytes. Gene expressions of SOX-9 and RGS-10 were highest in the osteoarthritic-joint cells; Frzb expression was highest in growth factor treated cells.
Conclusions and Clinical Relevance—Autogenous synovium may be a viable cell source for meniscal tissue engineering. Gene expressions of SOX-9 and RGS-10 may be potential future targets for in vitro enhancement of chondrogenesis.
Objective—To evaluate the effects of peroxisome proliferator-activated receptor gamma (PPARγ) agonists on tissue metabolism in cartilage and synovial explants from dogs.
Sample Population—Cartilage-synovial membrane expiants from 12 dogs.
Procedures—Expiants were cultured for 21 days without (negative control) or with interleukin (IL)-1β (positive control) or with IL-1β and 2 concentrations of 2 PPARγ agonists (15-deoxy-Δ 12,14-prostaglandin J2 [PGJ2] and pioglitazone). Media were collected on days 3, 7, 9, 12, 15, 18, and 21 and assessed for glycosaminoglycan (GAG), nitric oxide (NO), and prostaglandin E2 (PGE2) concentrations. Tissue GAG and hydroxyproline concentrations were determined in cartilage expiants collected on day 21.
Results—The GAG concentrations of cartilage expiants cultured in IL-1β (100 ng/mL) with 2 concentrations of PGJ2 were significantly higher than those in all other groups, whereas media GAG concentrations were significantly lower in the high-concentration PGJ2-treated groups, compared with all other groups. The PGE2 concentrations were significantly lower in the PGJ2 treatment groups, compared with the positive control and the pioglitazone treatment groups on days 3 to 21. The NO concentrations were significantly lower in PGJ2 treatment groups, compared with the other groups on days 3 and 12 to 21.
Conclusions and Clinical Relevance—PGJ2, an endogenous PPARγ agonist, may have anti-inflammatory and chondroprotective effects in an osteosteoarthritic joint environment (Am J Vet Res 2010;71:1142-1147)
Objective—To investigate the presence or absence of Toll-like receptor (TLR)-2 and TLR-4 in synovial tissues collected from stifle joints (SJs) of dogs with or without osteoarthritis.
Animals—21 purpose-bred research dogs, 3 client-owned dogs with SJ osteoarthritis, and 3 dogs without SJ osteoarthritis.
Procedures—Research dogs underwent arthroscopic surgery in 1 SJ to induce osteoarthritis via cranial cruciate ligament transection (CrCLt; n = 5), femoral condylar articular cartilage groove creation (6), or release of the caudal horn of the medial meniscus (5); 5 dogs underwent sham surgery. Synovial tissue specimens were obtained from both stifle joints of each dog 12 weeks after surgery, and TLR-2 and TLR-4 gene expression were determined via real-time reverse transcription PCR assays. Expression of TLR-4 protein was determined via an immunofluorescence technique in additional specimens obtained from osteoarthritic SJs of dogs with cranial cruciate ligament insufficiency and from dogs with nonosteoarthritic SJs.
Results—Synovial tissues from CrCLt-treated joints had significantly higher TLR-4 gene expression, compared with the contralateral control SJs or any other joint group. TLR-2 gene expression did not differ significantly among groups. Toll-like receptor-4 protein was detected in synovial tissues of osteoarthritic SJs but was rarely evident in nonosteoarthritic SJs.
Conclusions and Clinical Relevance—Increased TLR-4 gene expression in the synovial tissue of SJs with osteoarthritis secondary to CrCLt suggests that activation of innate immunity may play a role in the pathophysiology of SJ osteoarthritis in at least a subset of dogs.
Objective—To determine the quantity (concentration) and quality (molecular weight) of synovial fluid hyaluronan with respect to presence and severity of osteoarthritis in stifle joints of dogs.
Animals—21 purpose-bred dogs and 6 clinically affected large-breed dogs (cranial cruciate ligament [CrCL] disease with secondary osteoarthritis).
Procedures—Research dogs underwent arthroscopic surgery in 1 stifle joint to induce osteoarthritis via CrCL transection (CrCLt; n = 5 stifle joints), femoral condylar articular cartilage groove creation (GR; 6), or meniscal release (MR; 5); 5 had sham surgery (SH) performed. Contralateral stifle joints (n = 21) were used as unoperated control joints. Synovial fluid was obtained from research dogs at time 0 and 12 weeks after surgery and from clinically affected dogs prior to surgery. All dogs were assessed for lameness, radiographic signs of osteoarthritis, and pathologic findings on arthroscopy as well as for quantity and quality of hyaluronan.
Results—Clinically affected dogs had significantly greater degrees of pathologic findings, compared with dogs with surgically induced osteoarthritis (ie, those with CrCLt, GR, and MR stifle joints), and with respect to lameness scores, radiographic signs of osteoarthritis, pathologic findings on arthroscopy, and synovial fluid hyaluronan concentration. Synovial fluid from stifle joints of dogs with surgically induced osteoarthritis had hyaluronan bands at 35 kd on western blots that synovial fluid from SH and clinically affected stifle joints did not.
Conclusions and Clinical Relevance—Synovial fluid hyaluronan quantity and quality were altered in stifle joints of dogs with osteoarthritis, compared with control stifle joints. A specific hyaluronan protein fragment may be associated with early pathologic changes in affected joints.
Objective—To assess effects of in vitro meloxicam exposure on metabolism in articular chondrocytes from dogs with naturally occurring osteoarthritis
Sample—Femoral head cartilage from 16 dogs undergoing total hip replacement
Procedures—Articular cartilage samples were obtained. Tissue sulfated glycosaminoglycan (SGAG), collagen, and DNA concentrations were measured. Collagen, SGAG, chondroitin sulfate 846, NO, prostaglandin E2 (PGE2), and matrix metalloproteinase (MMP)-2, MMP-3, MMP-9, and MMP-13 concentrations in culture medium were analyzed. Aggrecan, collagen II, MMP-2, MMP-3, MMP-9, MMP-13, ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS)-4, ADAMTS-5, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, TIMP-3, interleukin-1β, tumor necrosis factor-α, cyclooxygenase-1, cyclooxygenase-2, and nducible nitric oxide synthase gene expression were evaluated. Comparisons between tissues cultured without (control) and with meloxicam at concentrations of 0.3, 3.0, and 30.0 μg/mL for up to 30 days were performed by means of repeated-measures analysis.
Results—Meloxicam had no effect on chondrocyte SGAG, collagen, or DNA concentrations. Expression of ADAMTS-5 was significantly decreased in all groups on all days, compared with the day 0 value. On day 3, culture medium PGE2 concentrations were significantly lower in all meloxicam-treated groups, compared with values for controls, and values remained low. Culture medium MMP-3 concentrations were significantly lower on day 30 than on day 3 in all meloxicam-treated groups.
Conclusions and Clinical Relevance—Results suggested that in vitro meloxicam treatment of osteoarthritic canine cartilage for up to 30 days did not induce matrix degradation or stimulate MMP production. Meloxicam lowered PGE2 release from this tissue, and effects on tissue chondrocyte content and matrix composition were neutral.
Objective—To identify proteins with differential expression between healthy dogs and dogs with stifle joint osteoarthritis secondary to cranial cruciate ligament (CCL) disease.
Sample—Serum and synovial fluid samples obtained from dogs with stifle joint osteoarthritis before (n = 10) and after (8) surgery and control dogs without osteoarthritis (9) and archived synovial membrane and articular cartilage samples obtained from dogs with stifle joint osteoarthritis (5) and dogs without arthritis (5).
Procedures—Serum and synovial fluid samples were analyzed via liquid chromatography–tandem mass spectrometry; results were compared against a nonredundant protein database. Expression of complement component 3 in archived tissue samples was determined via immunohistochemical methods.
Results—No proteins had significantly different expression between serum samples of control dogs versus those of dogs with stifle joint osteoarthritis. Eleven proteins (complement component 3 precursor, complement factor I precursor, apolipoprotein B-100 precursor, serum paraoxonase and arylesterase 1, zinc-alpha-2-glycoprotein precursor, serum amyloid A, transthyretin precursor, retinol-binding protein 4 precursor, alpha-2-macroglobulin precursor, angiotensinogen precursor, and fibronectin 1 isoform 1 preproprotein) had significantly different expression (> 2.0-fold) between synovial fluid samples obtained before surgery from dogs with stifle joint osteoarthritis versus those obtained from control dogs. Complement component 3 was strongly expressed in all (5/5) synovial membrane samples of dogs with stifle joint osteoarthritis and weakly expressed in 3 of 5 synovial membrane samples of dogs without stifle joint arthritis.
Conclusions and Clinical Relevance—Findings suggested that the complement system and proteins involved in lipid and cholesterol metabolism may have a role in stifle joint osteoarthritis, CCL disease, or both.