Steroid hormones strongly support bovine articular cartilage integration in the absence of interleukin-1beta

OBJECTIVE: Posttraumatic integration of articular cartilage at fracture sites is essential for mechanical stability of cartilage, and ruptured cartilage is a prerequisite for early osteoarthritis. This study was undertaken to investigate effects on articular cartilage integration mediated by steroid hormones, interleukin-1beta (IL-1beta), and combinations thereof. METHODS: Articular cartilage blocks were cultured in partial apposition for 2 weeks with ascorbic acid, testosterone, 17beta-estradiol, and dehydroepiandrosterone (DHEA), with or without IL-1beta. Mechanical integration was measured as adhesive strength, i.e., the maximum force at rupture of integrated cartilage blocks divided by the overlap area. Glycosaminoglycan content was used to study synthesized extracellular matrix. RESULTS: Culture in medium without supplements did not lead to integration (adhesive strength 0 kPa). With administration of ascorbic acid (100 microg/ml), the median adhesive strength was 49 kPa. In comparison with ascorbic acid alone, all steroid hormones induced a strong, concentration-dependent stimulation of integration (with maximum values observed with DHEA at 3 x 10(-5)M, testosterone at 10(-8)M, and 17beta-estradiol at 10(-11)M). For testosterone and 17beta-estradiol, this was also reflected by an increase of glycosaminoglycan content. Adhesive strength was increased with IL-1beta at 10 pg/ml, but not at 1 pg/ml or 100 pg/ml. In the presence of both IL-1beta and sex hormones, integration of articular cartilage was reduced. CONCLUSION: This is the first study to demonstrate that steroid hormones such as 17beta-estradiol, DHEA, and testosterone stimulate articular cartilage integration. This effect is abrogated by low concentrations of IL-1beta. In the absence of IL-1beta or after neutralization of IL-1beta, steroid hormones might be favorable adjuvant compounds to optimize cartilage integration.     

Alteration of articular cartilage frictional properties by transforming growth factor β, interleukin‐1β, and oncostatin M

Objective

To evaluate the functional effects of transforming growth factor β1 (TGFβ1), interleukin‐1β (IL‐1β), and oncostatin M (OSM) on the frictional properties of articular cartilage and to determine the role of cytokine‐mediated changes in cartilage frictional properties by extracting and redepositing lubricin on the surface of cartilage explants.

Methods

Neonatal bovine cartilage explants were cultured in the presence or absence of 10 ng/ml of TGFβ1, IL‐1β, or OSM over 48 hours. Boundary lubrication tests were conducted to determine the effects of endogenously produced surface localized lubricin and of exogenous lubricin at the tissue surface and in the lubricant solution. The initial friction coefficient (μ₀), equilibrium friction coefficient (μ_eq), and Young's modulus (E_Y) were determined from the temporal load data.

Results

IL‐1β and OSM decreased tissue glycosaminoglycan (GAG) content by ∼20% over 48 hours and decreased E_Y to a similar extent (11–17%), but TGFβ did not alter GAG content or E_Y. Alterations in proteoglycan content corresponded to changes in μ₀, but endogenous lubricin decreased boundary mode μ_eq. The addition of exogenous lubricin, either localized at the tissue surface or in the lubricating solution, did not modulate μ₀, but it did lower μ_eq in cytokine‐treated cartilage.

Conclusion

This study provides new insight into the functional consequences of cytokine‐mediated changes in friction coefficient. In combination with established pathways of cytokine‐mediated lubricin metabolism, these data provide evidence of distinct biochemical origins of boundary and biphasic pressure‐mediated lubrication mechanisms in cartilage, with boundary lubrication regulated by surface accumulation of lubricants and biphasic lubrication controlled by factors such as GAG content that affect water movement through the tissue.

     

Inhibition of interleukin‐1β‐stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage

Objective

To investigate the mechanism of the inhibitory action of hyaluronan (HA) on interleukin‐1β (IL‐1β)‐stimulated production of matrix metalloproteinases (MMPs) in human articular cartilage.

Methods

IL‐1β was added to normal and osteoarthritic (OA) human articular cartilage in explant culture to stimulate MMP production. Articular cartilage was incubated or preincubated with a clinically used form of 800‐kd HA to assess its effect on IL‐1β‐induced MMPs. Levels of secreted MMPs 1, 3, and 13 in conditioned media were detected by immunoblotting; intracellular MMP synthesis in chondrocytes was evaluated by immunofluorescence microscopy. Penetration of HA into cartilage tissue and its binding to CD44 were analyzed by fluorescence microscopy using fluoresceinated HA. Blocking experiments with anti‐CD44 antibody were performed to investigate the mechanism of action of HA.

Results

Treatment and pretreatment with 800‐kd HA at 1 mg/ml resulted in significant suppression of IL‐1β‐stimulated production of MMPs 1, 3, and 13 in normal and OA cartilage explant culture. Fluorescence histocytochemistry revealed that HA penetrated cartilage tissue and localized in the pericellular matrix around chondrocytes. HA‐binding blocking experiments using anti‐CD44 antibody demonstrated that the association of HA with chondrocytes was mediated by CD44. Preincubation with anti‐CD44 antibody, which suppressed IL‐1β‐stimulated MMPs, reversed the inhibitory effect of HA on MMP production that was induced by IL‐1β in normal and OA cartilage.

Conclusion

This study demonstrates that HA effectively inhibits IL‐1β‐stimulated production of MMP‐1, MMP‐3, and MMP‐13, which supports the clinical use of HA in the treatment of OA. The action of HA on IL‐1β may involve direct interaction between HA and CD44 on chondrocytes.

     

Interleukin‐1α induction of tensile weakening associated with collagen degradation in bovine articular cartilage

Objective

To determine whether interleukin‐1α (IL‐1α) induces tensile weakening of articular cartilage that is concomitant with the loss of glycosaminoglycans (GAGs) or the subsequent degradation of the collagen network.

Methods

Explants of young adult bovine cartilage obtained from the superficial (including the articular surface), middle, and deep layers were cultured with or without IL‐1α for 1 week or 3 weeks. Then, portions of the explants were analyzed for their tensile properties (ramp modulus, strength, and failure strain); other portions of explants and spent culture medium were analyzed for the amount of GAG and the amount of cleaved, denatured, and total collagen.

Results

The effect of IL‐1α treatment on cartilage tensile properties and content was dependent on the duration of culture and the depth of the explant from the articular surface. The tensile strength and failure strain of IL‐1α–treated samples from the superficial and middle layers were lower after 3 weeks of culture, but not after 1 week of culture. However, by 1 week of culture, IL‐1α had already induced release of the majority of tissue GAGs into the medium, without detectable loss or degradation of collagen. In contrast, after 3 weeks of culture, IL‐1α induced significant collagen degradation, as indicated by the amount of total, cleaved, or denatured collagen in the medium or in explants from the superficial and middle layers.

Conclusion

IL‐1α–induced degradation of cartilage results in tensile weakening that occurs subsequent to the depletion of GAG and concomitant with the degradation of the collagen network.

     

Mechanical injury potentiates proteoglycan catabolism induced by interleukin‐6 with soluble interleukin‐6 receptor and tumor necrosis factor α in immature bovine and adult human articular cartilage

Objective

Traumatic joint injury can damage cartilage and release inflammatory cytokines from adjacent joint tissue. The present study was undertaken to study the combined effects of compression injury, tumor necrosis factor α (TNFα), and interleukin‐6 (IL‐6) and its soluble receptor (sIL‐6R) on immature bovine and adult human knee and ankle cartilage, using an in vitro model, and to test the hypothesis that endogenous IL‐6 plays a role in proteoglycan loss caused by a combination of injury and TNFα.

Methods

Injured or uninjured cartilage disks were incubated with or without TNFα and/or IL‐6/sIL‐6R. Additional samples were preincubated with an IL‐6–blocking antibody Fab fragment and subjected to injury and TNFα treatment. Treatment effects were assessed by histologic analysis, measurement of glycosaminoglycan (GAG) loss, Western blot to determine proteoglycan degradation, zymography, radiolabeling to determine chondrocyte biosynthesis, and Western blot and enzyme‐linked immunosorbent assay to determine chondrocyte production of IL‐6.

Results

In bovine cartilage samples, injury combined with TNFα and IL‐6/sIL‐6R exposure caused the most severe GAG loss. Findings in human knee and ankle cartilage were strikingly similar to those in bovine samples, although in human ankle tissue, the GAG loss was less severe than that observed in human knee tissue. Without exogenous IL‐6/sIL‐6R, injury plus TNFα exposure up‐regulated chondrocyte production of IL‐6, but incubation with the IL‐6–blocking Fab significantly reduced proteoglycan degradation.

Conclusion

Our findings indicate that mechanical injury potentiates the catabolic effects of TNFα and IL‐6/sIL‐6R in causing proteoglycan degradation in human and bovine cartilage. The temporal and spatial evolution of degradation suggests the importance of transport of biomolecules, which may be altered by overload injury. The catabolic effects of injury plus TNFα appeared partly due to endogenous IL‐6, since GAG loss was partially abrogated by an IL‐6–blocking Fab.

     

Correlation of polymorphic variation in the promoter region of the interleukin‐1β gene with secretion of interleukin‐1β protein

Objective

Significant variation in interleukin‐1β (IL‐1β) protein secretion between subjects has been observed when using a lipopolysaccharide (LPS)/ATP–mediated ex vivo blood stimulation assay. To explore the potential relationships between genetic polymorphisms in the IL1B cytokine gene and cellular responses to inflammatory stimuli such as LPS, we investigated the hypothesis that polymorphisms within the promoter and exon 5 of the IL1B gene contribute to the observed differences in IL‐1β protein secretion.

Methods

The IL1B gene polymorphisms C−511T, T−31C, and C3954T were tested for association with LPS‐induced secretion of IL‐1β protein as measured by an ex vivo blood stimulation assay. Samples from 2 independent study populations (n = 31 and n = 25) were available for use in the ex vivo assay after consent was obtained to analyze the DNA.

Results

A specific haplotype, composed of the T allele at −511 and the C allele at −31, was significantly associated with a 2–3‐fold increase in LPS‐induced IL‐1β protein secretion. This association was observed in both of the independent study populations (P = 0.0084 and P = 0.0017).

Conclusion

These data suggest that polymorphisms within the promoter region of the IL1B gene contribute to observed differences in LPS‐induced IL‐1β protein secretion.

     

Enhanced interleukin‐1β and interleukin‐18 release in a patient with chronic infantile neurologic,cutaneous, articular syndrome

Chronic infantile neurologic, cutaneous, articular (CINCA) syndrome is a severe inflammatory disease that recently was associated with mutations in CIAS1. It was hypothesized that these mutations may lead to enhanced inflammatory responses. Herein, we provide evidence that inflammation in the CINCA syndrome is characterized by enhanced interleukin‐1β (IL‐1β) and IL‐18 release upon stimulation of blood cells and show that this release is caspase 1 dependent.     

A role for geranylgeranylation in interleukin‐1β secretion

Objective

Mevalonate kinase deficiency (MKD) is an autosomal‐recessive disorder characterized by recurring episodes of inflammation. MK catalyzes the phosphorylation of mevalonic acid, which is an early step in isoprenoid biosynthesis. The goal of our study was to determine whether a temporary shortage of certain isoprenoid end products and/or the accumulation of mevalonic acid is the cause of interleukin‐1β (IL‐1β) secretion in MKD.

Methods

We studied the effect of the addition of intermediate metabolites and inhibitors of the isoprenoid biosynthesis pathway on IL‐1β secretion by peripheral blood mononuclear cells (PBMCs) of patients with MKD and healthy controls.

Results

Inhibition of enzymes involved in geranylgeranyl pyrophosphate (GGPP) synthesis or geranylgeranylation of proteins led to a marked increase of lipopolysaccharide‐stimulated IL‐1β secretion in PBMCs of control subjects. Furthermore, the increased IL‐1β secretion by PBMCs of patients with MKD was reversed by supplementation with GGPP as well as with mevalonic acid. IL‐1β secretion was increased only when control PBMCs were incubated with excessive amounts of mevalonic acid. Finally, a reduction in IL‐1β secretion by MKD PBMCs was also observed when sterol biosynthesis was inhibited, favoring nonsterol isoprenoid biosynthesis.

Conclusion

Our results indicate that a shortage of geranylgeranylated proteins, rather than an excess of mevalonate, is likely to cause increased IL‐1β secretion by PBMCs of patients with MKD.

     

Mechanotransduction of bovine articular cartilage superficial zone protein by transforming growth factor β signaling

Objective

Mechanical signals are key determinants in tissue morphogenesis, maintenance, and restoration strategies in regenerative medicine, although molecular mechanisms of mechanotransduction remain to be elucidated. This study was undertaken to investigate the mechanotransduction process of expression of superficial zone protein (SZP), a critical joint lubricant.

Methods

Regional expression of SZP was first quantified in cartilage obtained from the femoral condyles of immature bovines, using immunoblotting, and visualized by immunohistochemistry. Contact pressure mapping in whole joints was accomplished using pressure‐sensitive film and a load application system for joint testing. Friction measurements on cartilage plugs were acquired under boundary lubrication conditions using a pin‐on‐disk tribometer modified for reciprocating sliding. Direct mechanical stimulation by shear loading of articular cartilage explants was performed with and without inhibition of transforming growth factor β (TGFβ) signaling, and SZP content in media was quantified by enzyme‐linked immunosorbent assay.

Results

An unexpected pattern of SZP localization in knee cartilage was initially identified, with anterior regions exhibiting high levels of SZP expression. Regional SZP patterns were regulated by mechanical signals and correlated with tribological behavior. Direct relationships were demonstrated between high levels of SZP expression, maximum contact pressures, and low friction coefficients. Levels of SZP expression and accumulation were increased by applying shear stress, depending on location within the knee, and were decreased to control levels with the use of a specific inhibitor of TGFβ receptor type I kinase and subsequent phospho‐Smad2/3 activity.

Conclusion

These findings indicate a new role for TGFβ signaling in the mechanism of cellular mechanotransduction that is especially significant for joint lubrication.

     

Rosiglitazone induces interleukin‐1 receptor antagonist in interleukin‐1β–stimulated rat synovial fibroblasts via a peroxisome proliferator–activated receptor β/δ–dependent mechanism

Objective

To study the potency of 2 peroxisome proliferator–activated receptor γ (PPARγ) agonists, 15‐deoxy‐Δ^12,14‐prostaglandin J₂ (15‐deoxy‐PGJ₂) and rosiglitazone, to modulate the expression of interleukin‐1 receptor antagonist (IL‐1Ra) in rat synovial fibroblasts.

Methods

Levels of messenger RNA for IL‐1Ra and PPAR isotypes (α, β/δ, γ) were assessed by real‐time polymerase chain reaction in rat synovial fibroblasts exposed to 10 ng/ml of IL‐1β. PPAR levels were assessed by Western blotting and secreted IL‐1Ra levels by immunoassay. The potency of PPARγ agonists and the PPARβ/δ agonist GW‐501516 on IL‐1Ra levels was tested in the range of 1–10 μM and at 100 pM, respectively. The contribution of PPARγ to the effects of rosiglitazone on IL‐1Ra secretion was examined either by its overexpression or by inhibition using wild‐type or dominant‐negative constructs and the antagonist GW‐9662 (10 μM), respectively. The dominant‐negative strategy was also performed to investigate the possible contribution of PPARβ/δ and NF‐κB activation.

Results

IL‐1β–induced IL‐1Ra production was increased by 10 μM rosiglitazone but was reduced dose‐dependently by 15‐deoxy‐PGJ₂. Both agonists lowered IL‐1β secretion, but rosiglitazone alone reduced the imbalance of IL‐1β/IL‐1Ra toward basal levels. Enhancement of IL‐1β–induced IL‐1Ra production by rosiglitazone was not affected by PPARγ overexpression or by its inhibition with dominant‐negative PPARγ or GW‐9662. Inhibition of NF‐κB was also ineffective against rosiglitazone but abolished the stimulating effect of IL‐1β on IL‐1Ra. All PPAR isotypes were expressed constitutively in rat synoviocytes, but PPARγ decreased dramatically upon IL‐1β exposure, whereas PPARβ/δ remained stable. Dominant‐negative PPARβ/δ abolished the enhancement of IL‐1Ra by rosiglitazone, whereas GW‐501516 reproduced the effect of rosiglitazone on IL‐1Ra secretion.

Conclusion

Rosiglitazone stimulates IL‐1Ra production by a PPARβ/δ mechanism in activated rat synovial fibroblasts, further contributing to its potential antiarthritic properties and opening new perspectives for the modulation of inflammatory genes by specific PPAR agonists in articular cells.

     

Role of interleukin‐1β in NLRP12‐associated autoinflammatory disorders and resistance to anti–interleukin‐1 therapy

Objective

A new class of autoinflammatory syndromes called NLRP12‐associated disorders (NLRP12AD) has been associated with mutations in NLRP12. Conflicting data on the putative role of NLRP12 in interleukin‐1β (IL‐1β) signaling have been found in in vitro analyses. This prospective study was undertaken to assess the secretion of IL‐1β and 3 IL‐1β–induced cytokines (IL‐1 receptor antagonist [IL‐1Ra], IL‐6, and tumor necrosis factor α [TNFα]) in patients' peripheral blood mononuclear cells (PBMCs) cultured ex vivo and to evaluate the patients' response to IL‐1Ra (anakinra), a major drug used in the treatment of autoinflammatory disorders.

Methods

Patients' disease manifestations and cytokine measurements were recorded before anakinra treatment was started, during 14 months of therapy, and after discontinuation of anakinra treatment.

Results

Spontaneous secretion of IL‐1β by patients' PBMCs was found to be dramatically increased (80–175 fold) compared to healthy controls. Consistent with these findings, anakinra initially led to a marked clinical improvement and to a rapid near‐normalization of IL‐1β secretion. However, a progressive clinical relapse occurred secondarily, associated with an increase in TNFα secretion, persistent elevated levels of IL‐1Ra and IL‐6, and a reactivation of IL‐1β secretion. Anakinra was discontinued after 14 months of therapy.

Conclusion

Our findings provide in vivo evidence of the crucial role of IL‐1β in the pathophysiology of NLRP12AD. This is the first time anakinra has been used to treat this disorder. This study provides new insights into the mechanisms underlying resistance to anti–IL‐1 therapy observed in a few patients with autoinflammatory syndromes. Our data also point to the potential of ex vivo cytokine measurements as predictors of response to treatment.