Selective decrease of donor-reactive Tregs after liver transplantation limits Treg therapy for promoting allograft tolerance in humans.

Promoting immune tolerance to transplanted organs can minimize the amount of immunosuppressive drugs that patients need to take, reducing lifetime risks of mortality and morbidity. Regulatory T cells (Tregs) are essential for immune tolerance, and preclinical studies have shown their therapeutic efficacy in inducing transplantation tolerance. Here, we report the results of a phase 1/2 trial (ARTEMIS, NCT02474199) of autologous donor alloantigen-reactive Treg (darTreg) therapy in individuals 2 to 6 years after receiving a living donor liver transplant. The primary efficacy endpoint was calcineurin inhibitor dose reduction by 75% with stable liver function tests for at least 12 weeks. Among 10 individuals who initiated immunosuppression withdrawal, 1 experienced rejection before planned darTreg infusion, 5 received darTregs, and 4 were not infused because of failure to manufacture the minimal infusible dose of 100 × 106 cells. darTreg infusion was not associated with adverse events. Two darTreg-infused participants reached the primary endpoint, but an insufficient number of recipients were treated for assessing the efficacy of darTregs. Mechanistic studies revealed generalized Treg activation, senescence, and selective reduction of donor reactivity after liver transplantation. Overall, the ARTEMIS trial features a design concept for evaluating the efficacy of Treg therapy in transplantation. The mechanistic insight gained from the study may help guide the design of future trials.

Elevated de novo lipogenesis, slow liver triglyceride turnover, and clinical correlations in nonalcoholic steatohepatitis patients.

De novo lipogenesis (DNL) converts carbon substrates to lipids. Increased hepatic DNL could contribute to pathogenic liver triglyceride accumulation in nonalcoholic steatohepatitis (NASH) and therefore may be a potential target for pharmacological intervention. Here, we measured hepatic DNL using heavy water in 123 patients with NASH with fibrosis or cirrhosis, calculated the turnover of hepatic triglycerides to allow repeat labeling studies, and determined the associations of hepatic DNL with metabolic, fibrotic, and imaging markers. We found that hepatic DNL was higher in patients with fibrotic NASH [median (IQR), 40.7% contribution to palmitate (32.1, 47.5), n=103] than has been previously reported in healthy volunteers and remained elevated [median (IQR), 36.8% (31.0, 44.5), n=20] in patients with cirrhosis, despite lower liver fat content. We also showed that turnover of intrahepatic triglyceride pools was slow (t½ >10 days). Furthermore, DNL contribution was determined to be independent of liver stiffness by magnetic resonance imaging but was positively associated with the number of large very low density lipoprotein (VLDL) particles, the size of VLDL, the lipoprotein insulin resistance score, and levels of ApoB100, and trended toward negative associations with the fibrosis markers FIB-4, FibroSure, and APRI. Finally, we found treatment with the acetyl-CoA carboxylase inhibitor firsocostat reduced hepatic DNL at 4 and 12 weeks, using a correction model for residual label that accounts for hepatic triglyceride turnover. Taken together, these data support an important pathophysiological role for elevated hepatic DNL in NASH and demonstrate that response to pharmacological agents targeting DNL can be correlated with pretreatment DNL.

Dietary sugar restriction reduces hepatic de novo lipogenesis in adolescent boys with fatty liver disease.

BACKGROUNDHepatic de novo lipogenesis (DNL) is elevated in nonalcoholic fatty liver disease (NAFLD). Improvements in hepatic fat by dietary sugar reduction may be mediated by reduced DNL, but data are limited, especially in children. We examined the effects of 8 weeks of dietary sugar restriction on hepatic DNL in adolescents with NAFLD and correlations between DNL and other metabolic outcomes.METHODSAdolescent boys with NAFLD (n = 29) participated in an 8-week, randomized controlled trial comparing a diet low in free sugars versus their usual diet. Hepatic DNL was measured as percentage contribution to plasma triglyceride palmitate using a 7-day metabolic labeling protocol with heavy water. Hepatic fat was measured by magnetic resonance imaging-proton density fat fraction.RESULTSHepatic DNL was significantly decreased in the treatment group (from 34.6% to 24.1%) versus the control group (33.9% to 34.6%) (adjusted week 8 mean difference: -10.6% [95% CI: -19.1%, -2.0%]), which was paralleled by greater decreases in hepatic fat (25.5% to 17.9% vs. 19.5% to 18.8%) and fasting insulin (44.3 to 34.7 vs. 35.5 to 37.0 µIU/mL). Percentage change in DNL during the intervention correlated significantly with changes in free-sugar intake (r = 0.48, P = 0.011), insulin (r = 0.40, P = 0.047), and alanine aminotransferase (ALT) (r = 0.39, P = 0.049), but not hepatic fat (r = 0.13, P = 0.532).CONCLUSIONOur results suggest that dietary sugar restriction reduces hepatic DNL and fasting insulin, in addition to reductions in hepatic fat and ALT, among adolescents with NAFLD. These results are consistent with the hypothesis that hepatic DNL is a critical metabolic abnormality linking dietary sugar and NAFLD.TRIAL REGISTRYClinicalTrials.gov NCT02513121.FUNDINGThe Nutrition Science Initiative (made possible by gifts from the Laura and John Arnold Foundation, Ambrose Monell Foundation, and individual donors), the UCSD Altman Clinical and Translational Research Institute, the NIH, Children's Healthcare of Atlanta and Emory University's Children's Clinical and Translational Discovery Core, Children's Healthcare of Atlanta and Emory University Pediatric Biostatistical Core, the Georgia Clinical and Translational Science Alliance, and the NIH National Institute of Diabetes, Digestive, and Kidney Disease.

Dynamics and turnover of memory CD8 T cell responses following yellow fever vaccination.

Understanding how immunological memory lasts a lifetime requires quantifying changes in the number of memory cells as well as how their division and death rates change over time. We address these questions by using a statistically powerful mixed-effects differential equations framework to analyze data from two human studies that follow CD8 T cell responses to the yellow fever vaccine (YFV-17D). Models were first fit to the frequency of YFV-specific memory CD8 T cells and deuterium enrichment in those cells 42 days to 1 year post-vaccination. A different dataset, on the loss of YFV-specific CD8 T cells over three decades, was used to assess out of sample predictions of our models. The commonly used exponential and bi-exponential decline models performed relatively poorly. Models with the cell loss following a power law (exactly or approximately) were most predictive. Notably, using only the first year of data, these models accurately predicted T cell frequencies up to 30 years post-vaccination. Our analyses suggest that division rates of these cells drop and plateau at a low level (0.1% per day, ∼ double the estimated values for naive T cells) within one year following vaccination, whereas death rates continue to decline for much longer. Our results show that power laws can be predictive for T cell memory, a finding that may be useful for vaccine evaluation and epidemiological modeling. Moreover, since power laws asymptotically decline more slowly than any exponential decline, our results help explain the longevity of immune memory phenomenologically.

De novo lipogenesis is essential for platelet production in humans.

Acetyl-CoA carboxylase (ACC) catalyses the first step of de novo lipogenesis (DNL). Pharmacologic inhibition of ACC has been of interest for therapeutic intervention in a wide range of diseases. We demonstrate here that ACC and DNL are essential for platelet production in humans and monkeys, but in not rodents or dogs. During clinical evaluation of a systemically distributed ACC inhibitor, unexpected dose-dependent reductions in platelet count were observed. While platelet count reductions were not observed in rat and dog toxicology studies, subsequent studies in cynomolgus monkeys recapitulated these platelet count reductions with a similar concentration response to that in humans. These studies, along with ex vivo human megakaryocyte maturation studies, demonstrate that platelet lowering is a consequence of DNL inhibition likely to result in impaired megakaryocyte demarcation membrane formation. These observations demonstrate that while DNL is a minor quantitative contributor to global lipid balance in humans, DNL is essential to specific lipid pools of physiological importance.

Discovery of Half-life of Circulating Hepatitis B Surface Antigen in Patients With Chronic Hepatitis B Infection Using Heavy Water Labeling.

In a pilot study, heavy water labeling was used to determine hepatitis B surface antigen (HBsAg) turnover rates in chronic hepatitis B (CHB) patients. The mean (standard deviation) half-life of HBsAg in blood was 6.7 (5.5) days, which reflects recent production in the liver and supports strategies aimed at reducing HBsAg production in CHB patients.

Origin and differentiation of human memory CD8 T cells after vaccination.

The differentiation of human memory CD8 T cells is not well understood. Here we address this issue using the live yellow fever virus (YFV) vaccine, which induces long-term immunity in humans. We used in vivo deuterium labelling to mark CD8 T cells that proliferated in response to the virus and then assessed cellular turnover and longevity by quantifying deuterium dilution kinetics in YFV-specific CD8 T cells using mass spectrometry. This longitudinal analysis showed that the memory pool originates from CD8 T cells that divided extensively during the first two weeks after infection and is maintained by quiescent cells that divide less than once every year (doubling time of over 450 days). Although these long-lived YFV-specific memory CD8 T cells did not express effector molecules, their epigenetic landscape resembled that of effector CD8 T cells. This open chromatin profile at effector genes was maintained in memory CD8 T cells isolated even a decade after vaccination, indicating that these cells retain an epigenetic fingerprint of their effector history and remain poised to respond rapidly upon re-exposure to the pathogen.

Imaging regiospecific lipid turnover in mouse brain with desorption electrospray ionization mass spectrometry.

Compartmentalization of metabolism into specific regions of the cell, tissue, and organ is critical to life for all organisms. Mass spectrometric imaging techniques have been valuable in identifying and quantifying concentrations of metabolites in specific locations of cells and tissues, but a true understanding of metabolism requires measurement of metabolite flux on a spatially resolved basis. Here, we utilize desorption ESI-MS (DESI-MS) to measure lipid turnover in the brains of mice. We show that anatomically distinct regions of the brain have distinct lipid turnover rates. These turnover measurements, in conjunction with relative concentration, will enable calculation of regiospecific synthesis rates for individual lipid species in vivo. Monitoring spatially dependent changes in metabolism has the potential to significantly facilitate research in many areas, such as brain development, cancer, and neurodegeneration.

Leukemia cell proliferation and death in chronic lymphocytic leukemia patients on therapy with the BTK inhibitor ibrutinib.

BACKGROUND. Ibrutinib is an effective targeted therapy for patients with chronic lymphocytic leukemia (CLL) that inhibits Bruton's tyrosine kinase (BTK), a kinase involved in B cell receptor signaling. METHODS. We used stable isotopic labeling with deuterated water (2H2O) to measure directly the effects of ibrutinib on leukemia cell proliferation and death in 30 patients with CLL. RESULTS. The measured average CLL cell proliferation ("birth") rate before ibrutinib therapy was 0.39% of the clone per day (range 0.17%-1.04%); this decreased to 0.05% per day (range 0%-0.36%) with treatment. Death rates of blood CLL cells increased from 0.18% per day (average, range 0%-0.7%) prior to treatment to 1.5% per day (range 0%-3.0%) during ibrutinib therapy, and they were even higher in tissue compartments. CONCLUSIONS. This study provides the first direct in vivo measurements to our knowledge of ibrutinib's antileukemia actions, demonstrating profound and immediate inhibition of CLL cell proliferation and promotion of high rates of CLL cell death. TRIAL REGISTRATION. This trial was registered at clinicaltrials.gov (NCT01752426). FUNDING. This study was supported by a Cancer Center Support Grant (National Cancer Institute grant P30 CA016672), an NIH grant (CA081554) from the National Cancer Institute, MD Anderson's Moon Shots Program in CLL, and Pharmacyclics, an AbbVie company.

Identifying nonalcoholic fatty liver disease patients with active fibrosis by measuring extracellular matrix remodeling rates in tissue and blood.

Excess collagen synthesis (fibrogenesis) in the liver plays a causal role in the progression of nonalcoholic fatty liver disease (NAFLD). Methods are needed to identify patients with more rapidly progressing disease and to demonstrate early response to treatment. We describe here a novel method to quantify hepatic fibrogenesis flux rates both directly in liver tissue and noninvasively in blood. Twenty-one patients with suspected NAFLD ingested heavy water (2 H2 O, 50-mL aliquots) two to three times daily for 3-5 weeks prior to a clinically indicated liver biopsy. Liver collagen fractional synthesis rate (FSR) and plasma lumican FSR were measured based on 2 H labeling using tandem mass spectrometry. Patients were classified by histology for fibrosis stage (F0-F4) and as having nonalcoholic fatty liver or nonalcoholic steatohepatitis (NASH). Magnetic resonance elastography measurements of liver stiffness were also performed. Hepatic collagen FSR in NAFLD increased with advancing disease stage (e.g., higher in NASH than nonalcoholic fatty liver, positive correlation with fibrosis score and liver stiffness) and correlated with hemoglobin A1C. In addition, plasma lumican FSR demonstrated a significant correlation with hepatic collagen FSR. CONCLUSION: Using a well-characterized cohort of patients with biopsy-proven NAFLD, this study demonstrates that hepatic scar in NASH is actively remodeled even in advanced fibrosis, a disease that is generally regarded as static and slowly progressive. Moreover, hepatic collagen FSR correlates with established risks for fibrotic disease progression in NASH, and plasma lumican FSR correlates with hepatic collagen FSR, suggesting applications as direct or surrogate markers, respectively, of hepatic fibrogenesis in humans. (Hepatology 2017;65:78-88).

Circulating protein synthesis rates reveal skeletal muscle proteome dynamics.

Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders.

A pilot study demonstrating a non-invasive method for the measurement of protein turnover in skin disorders: application to psoriasis.

BACKGROUND: Previous studies of epidermal kinetics in psoriasis have relied on invasive biopsy procedures or the use of radioactive labels. We previously developed a non-invasive method for measuring keratin synthesis in human skin using deuterated water labeling, serial collection of tape strips and measurement of deuterium enrichment in protein by mass spectrometry. This powerful method can be applied to measure other skin proteins and lipids collected by tape stripping. Here, for the first time, we apply this technique to investigate the epidermal kinetics of psoriasis, the first step in defining a kinetic profile for normal skin versus activated or quiescent psoriatic skin. METHODS: Psoriatic subjects were given (2)H2O orally as twice-daily doses for 16-38 days. Affected and unaffected skin was sampled by tape stripping and washing (modified Pachtman method). Proteins were isolated from the tape strips by a method that enriches for keratin. Turnover times were determined by gas chromatography/mass spectrometry. Kinetic data were compared to transepidermal water loss (TEWL). RESULTS: Deuterium-labeled protein from lesional psoriatic skin appeared at the skin surface within 3-8 days of label administration, whereas labeled protein from non-lesional skin requires 10-20 days to appear. Psoriatic skin had similar rate of growth despite varying anatomic location. Proteins recovered from tape strips were identified by nanoscale liquid chromatography/tandem mass spectrometry. Isolated peptides were >98% from keratin in uninvolved skin and >72% keratin in psoriatic skin. Revealing that one-quarter of all newly synthesized proteins in psoriatic skin are antimicrobial defense and other immune-related proteins. TEWL values were greater in lesional than non-lesional skin, suggesting barrier compromise in psoriatic skin despite increased clinical thickness. CONCLUSIONS: This simple, elegant, and non-invasive method for measuring epidermal protein synthesis, which can also be adapted to measure epidermal lipids, provides a metric that may reveal new insights into the mechanisms and dynamic processes underlying psoriasis and may also provide an objective scale for determining response to therapeutic agents in pre-clinical and clinical trials. This opens a pathway to the non-invasive study of kinetics of protein formation in psoriasis or other skin diseases.

The effect of long term calorie restriction on in vivo hepatic proteostatis: a novel combination of dynamic and quantitative proteomics.

Calorie restriction (CR) promotes longevity. A prevalent mechanistic hypothesis explaining this effect suggests that protein degradation, including mitochondrial autophagy, is increased with CR, removing damaged proteins and improving cellular fitness. At steady state, increased catabolism must be balanced by increasing mitochondrial biogenesis and protein synthesis, resulting in faster protein replacement rates. To test this hypothesis, we measured replacement kinetics and relative concentrations of hundreds of proteins in vivo in long-term CR and ad libitum-fed mice using metabolic (2)H(2)O-labeling combined with the Stable Isotope Labeling in Mammals protocol and LC-MS/MS analysis of mass isotopomer abundances in tryptic peptides. CR reduced absolute synthesis and breakdown rates of almost all measured hepatic proteins and prolonged the half-lives of most (≈ 80%), particularly mitochondrial proteins (but not ribosomal subunits). Proteins with related functions exhibited coordinated changes in relative concentration and replacement rates. In silico expression pathway interrogation allowed the testing of potential regulators of altered network dynamics (e.g. peroxisome proliferator-activated receptor gamma coactivator 1-alpha). In summary, our combination of dynamic and quantitative proteomics suggests that long-term CR reduces mitochondrial biogenesis and mitophagy. Our findings contradict the theory that CR increases mitochondrial protein turnover and provide compelling evidence that cellular fitness is accompanied by reduced global protein synthetic burden.

Measurement of human plasma proteome dynamics with (2)H(2)O and liquid chromatography tandem mass spectrometry.

Dysfunction of protein turnover is a feature of many human diseases, and proteins are substrates in important biological processes. Currently, no method exists for the measurement of global protein turnover (i.e., proteome dynamics) that can be applied in humans. Here we describe the use of metabolic labeling with deuterium ((2)H) from (2)H(2)O and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of mass isotopomer patterns to measure protein turnover. We show that the positions available for (2)H label incorporation in vivo can be calculated using peptide sequence. The isotopic incorporation values calculated by combinatorial analysis of mass isotopomer patterns in peptides correlate very closely with values established for individual amino acids. Inpatient and outpatient heavy water labeling protocols resulted in (2)H label incorporation sufficient for reproducible quantitation in humans. Replacement rates were similar for peptides deriving from the same protein. Using a kinetic model to account for the time course of each individual's (2)H(2)O enrichment curves, dynamics of approximately 100 proteins with half-lives ranging from 0.4 to 40 days were measured using 8 µl of plasma. The measured rates were consistent with literature values. This method can be used to measure in vivo proteome homeostasis in humans in disease and during therapeutic interventions.

Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium.

This study demonstrated the chondrogenic effect of hydrostatic pressure on human bone marrow stromal cells (MSCs) cultured in a mixed medium containing osteogenic and chondrogenic factors. MSCs seeded in type I collagen sponges were exposed to 1 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for 4 h per day for 10 days, or remained in identical culture conditions but without exposure to pressure. Afterwards, we compared the proteoglycan content of loaded and control cell/scaffold constructs with Alcian blue staining. We also used real-time PCR to evaluate the change in mRNA expression of selected genes associated with chondrogenic and osteogenic differentiation (aggrecan, type I collagen, type II collagen, Runx2 (Cbfa-1), Sox9, and TGF-beta1). With the hydrostatic pressure loading regime, proteoglycan staining increased markedly. Correspondingly, the mRNA expression of chondrogenic genes such as aggrecan, type II collagen, and Sox9 increased significantly. We also saw a significant increase in the mRNA expression of type I collagen, but no change in the expression of Runx2 or TGF-beta1 mRNA. This study demonstrated that hydrostatic pressure enhanced differentiation of MSCs in the presence of multipotent differentiation factors in vitro, and suggests the critical role that this loading regime may play during cartilage development and regeneration in vivo.

Gene regulation ex vivo within a wrap-around tendon.

This study tested the hypothesis that physiologic tendon loading modulates the fibrous connective tissue phenotype in undifferentiated skeletal cells. Type I collagen sponges containing human bone marrow stromal cells (MSCs) were implanted into the midsubstance of excised sheep patellar tendons. An ex vivo loading system was designed to cyclically stretch each tendon from 0 to 5% at 1.0 Hz. The MSC-sponge constructs were implanted into 2 tendon sites: the first site subjected to tension only and a second site located at an artificially created wrap-around region in which an additional compressive stress was generated transverse to the longitudinal axis of the tendon. The induced contact pressure at the wraparound site was 0.55 +/- 0.12 MPa, as quantified by pressure-sensitive film. An MSC-sponge construct was maintained free swelling in the same bath as an unloaded control. After 2 h of tendon stretching, the MSC-sponge constructs were harvested and real-time PCR was used to quantify Fos, Sox9, Cbfa1 (Runx2), and scleraxis mRNA expression as markers of skeletal differentiation. Two hours of mechanical loading distinctly altered MSC differentiation in the wrap-around region and the tensile-only region, as evidenced by differences in Fos and Sox9 mRNA expression. Expression of Fos mRNA was 13 and 52 times higher in the tensile-only and wrap-around regions, respectively, compared to the free-swelling controls. Expression of Sox9 mRNA was significantly higher (2.5-3 times) in MSCs from the wraparound region compared to those from the tensile-only region or in free-swelling controls. In contrast, expression levels for Cbfa1 did not differ among constructs. Scleraxis mRNA was not detected in any construct. This study demonstrates that the physiologic mechanical environment in the wrap-around regions of tendons provides stimuli for upregulating early response genes and transcription factors associated with chondrogenic differentiation. These differentiation responses begin within as little as 2 h after the onset of mechanical stimulation and may be the basis for the formation of fibrocartilage that is typically found in the wrap-around region of mature tendons in vivo.

Depth-varying density and organization of chondrocytes in immature and mature bovine articular cartilage assessed by 3d imaging and analysis.

Articular cartilage is a heterogeneous tissue, with cell density and organization varying with depth from the surface. The objectives of the present study were to establish a method for localizing individual cells in three-dimensional (3D) images of cartilage and quantifying depth-associated variation in cellularity and cell organization at different stages of growth. Accuracy of nucleus localization was high, with 99% sensitivity relative to manual localization. Cellularity (million cells per cm3) decreased from 290, 310, and 150 near the articular surface in fetal, calf, and adult samples, respectively, to 120, 110, and 50 at a depth of 1.0 mm. The distance/angle to the nearest neighboring cell was 7.9 microm/31 degrees , 7.1 microm/31 degrees , and 9.1 microm/31 degrees for cells at the articular surface of fetal, calf, and adult samples, respectively, and increased/decreased to 11.6 microm/31 degrees , 12.0 microm/30 degrees , and 19.2 microm/25 degrees at a depth of 0.7 mm. The methodologies described here may be useful for analyzing the 3D cellular organization of cartilage during growth, maturation, aging, degeneration, and regeneration.

In vitro physical stimulation of tissue-engineered and native cartilage.

Because of the limited availability of donor cartilage for resurfacing defects in articular surfaces, there is tremendous interest in the in vitro bioengineering of cartilage replacements for clinical applications. However, attaining mechanical properties in engineered cartilaginous constructs that approach those of native cartilage has not been previously achieved when constructs are cultured under free-swelling conditions. One approach toward stimulating the development of constructs that are mechanically more robust is to expose them to physical environments that are similar, in certain ways, to those encountered by native cartilage. This is a strategy motivated by observations in numerous short-term experiments that certain mechanical signals are potent stimulators of cartilage metabolism. On the other hand, excess mechanical loading can have a deleterious effect on cartilage. Culture conditions that include a physical stimulation component are made possible by the use of specialized bioreactors. This chapter addresses some of the issues involved in using bioreactors as integral components of cartilage tissue engineering and in studying the physical regulation of cartilage. We first consider the generation of cartilaginous constructs in vitro. Next we describe the rationale and design of bioreactors that can impart either mechanical deformation or fluid-induced mechanical signals.

Microenvironment regulation of extracellular signal-regulated kinase activity in chondrocytes: effects of culture configuration, interleukin-1, and compressive stress.

OBJECTIVE: To compare extracellular signal-regulated kinase (ERK) activity in response to interleukin-1 (IL-1) in chondrocytes under various culture configurations designed for the study of cartilage biology and repair, and also in response to dynamic load for chondrocytes in cartilage. METHODS: Isolated bovine articular chondrocytes were maintained in serum-supplemented medium under 4 culture configurations: high-density monolayer, attached to a cut surface of cartilage, within tissue-engineered constructs, or within intact cartilage explants. Samples were subjected to a change of medium with or without IL-1. Cartilage explants were also subjected to dynamic compression. RESULTS: In chondrocyte monolayers, both basal and IL-1-stimulated ERK activities were similarly elevated at 0.5 hours after medium change, diminishing by 74% after 16 hours. In contrast, chondrocytes in other culture configurations exhibited lower basal levels of ERK activity and a moderate activation of ERK in response to IL-1 that was sustained over the 16-hour treatment time. The dynamic component of loading of cartilage explants led to a 5-fold activation of ERK, compared with free-swelling controls, that was indistinguishable from the effects of IL-1. CONCLUSION: ERK signaling in response to IL-1 in chondrocyte monolayers exhibited a pattern that was distinct from that in other culture systems, suggesting that the extracellular matrix plays an important regulatory role in modulating the response to extracellular stimuli. Since IL-1 and dynamic loading have distinct effects on chondrocyte biosynthesis, signaling pathways other than ERK participate in the chondrocyte responses to these stimuli.