lactoferrin and Osteoarthritis

Bovine lactoferricin (LfcinB), a multifunctional peptide, was recently demonstrated to be anti-catabolic and anti-inflammatory in human articular cartilage. LfcinB blocks IL-1-mediated proteoglycan depletion, matrix-degrading enzyme expression, and pro-inflammatory mediator induction. LfcinB selectively activates ERK1/2, p38 (but not JNK), and Akt signaling. However, the relationship between these pathways and LfcinB target genes has never been explored. In this study, we uncovered the remarkable ability of LfcinB in the induction of an anti-inflammatory cytokine, IL-11. LfcinB binds to cell surface heparan sulfate to initiate ERK1/2 signaling and activate AP-1 complexes composed of c-Fos and JunD, which transactivate the IL-11 gene. The induced IL-11 functions as an anti-inflammatory and chondroprotective cytokine in articular chondrocytes. Our data show that IL-11 directly attenuates IL-1-mediated catabolic and inflammatory processes ex vivo and in vitro. Moreover, IL-11 activates STAT3 signaling pathway to critically up-regulate TIMP-1 expression, as a consecutive secondary cellular response after IL-11 induction by LfcinB-ERK-AP-1 axis in human adult articular chondrocytes. The pathological relevance of IL-11 signaling to osteoarthritis is evidenced by significant down-regulation of its cognate receptor expression in osteoarthritic chondrocytes. Together, our results suggest a two-step mechanism, whereby LfcinB induces TIMP-1 through an IL-11-dependent pathway involving transcription factor AP-1 and STAT3.

Topics: Adult; Aged; Animals; Anti-Inflammatory Agents; Cartilage, Articular; Cattle; Chondrocytes; Female; Humans; Interleukin-11; Lactoferrin; Male; MAP Kinase Signaling System; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Osteoarthritis; STAT3 Transcription Factor; Tissue Inhibitor of Metalloproteinase-1; Transcription Factor AP-1; Up-Regulation

This study aims to evaluate the efficacy of high-molecular-weight hyaluronic acid (HMWHA) and lactoferrin (LF) injections on temporomandibular joint (TMJ) cartilage and subchondral bone in mono-iodoacetate (MIA)-induced temporomandibular joint osteoarthritis model in rats.. In this in vivo study, a total of 24 rats were divided into three groups as follows: saline group (Group 1), HMWHA group (Group 2), and LF group (Group 3) including eight rats in each group. The intra-articular injections were administered once a week for three weeks after osteoarthritis was induced. All animals were euthanized 28 days after induction of osteoarthritis, and TMJs were harvested for histomorphometric, immunohistochemical, and micro-computed tomography (CT) analysis.. There was no significant difference between the HMWHA and LF groups in terms of the histomorphometric and immunohistochemical analysis results (p>0.05). According to the micro-CT analysis, the LF group had the highest mean bone volume fraction (74.9±0.5) and trabecular thickness (0.122±0.002), while the saline group had the lowest mean values (55.0±0.3 and 0.071±0.002, respectively) (p<0.001). There was no significant difference between the HMWHA and LF groups according to the micro-CT analysis (p>0.05). Both groups had better healing effects than the saline group in all analyses.. Lactoferrin has a healing effect at least as much as HMWHA in MIA-induced TMJ osteoarthritis. We suggest that LF may be evaluated in future clinical studies as a promising agent in the treatment of osteoarthritis.

Topics: Animals; Hyaluronic Acid; Immunohistochemistry; Injections, Intra-Articular; Lactoferrin; Osteoarthritis; Rats; Temporomandibular Joint; X-Ray Microtomography

Topics: Animals; Apoptosis; Cell Proliferation; Chondrocytes; Lactoferrin; Osteoarthritis; Rats; Signal Transduction

Progressive skeletal and connective tissue disease represents a significant clinical burden in all of the mucopolysaccharidoses. Despite the introduction of enzyme replacement strategies for many of the mucopolysaccharidoses, symptomatology related to bone and joint disease appears to be recalcitrant to current therapies. In order to address these unmet medical needs a clearer understanding of skeletal and connective tissue disease pathogenesis is required. Historically the pathogenesis of the mucopolysaccharidoses has been assumed to directly relate to progressive storage of glycosaminoglycans. It is now apparent for many lysosomal storage disorders that more complex pathogenic mechanisms underlie patients' clinical symptoms. We have used proteomic and genome wide expression studies in the murine mucopolysaccharidosis I model to identify early pathogenic events occurring in micro-dissected growth plate tissue. Studies were conducted using 3 and 5-week-old mice thus representing a time at which no obvious morphological changes of bone or joints have taken place. An unbiased iTRAQ differential proteomic approach was used to identify candidates followed by validation with multiple reaction monitoring mass spectrometry and immunohistochemistry. These studies reveal significant decreases in six key structural and signaling extracellular matrix proteins; biglycan, fibromodulin, PRELP, type I collagen, lactotransferrin, and SERPINF1. Genome-wide expression studies in embryonic day 13.5 limb cartilage and 5 week growth plate cartilage followed by specific gene candidate qPCR studies in the 5week growth plate identified fourteen significantly deregulated mRNAs (Adamts12, Aspn, Chad, Col2a1, Col9a1, Hapln4, Lum, Matn1, Mmp3, Ogn, Omd, P4ha2, Prelp, and Rab32). The involvement of biglycan, PRELP and fibromodulin; all members of the small leucine repeat proteoglycan family is intriguing, as this protein family is implicated in the pathogenesis of late onset osteoarthritis. Taken as a whole, our data indicates that alteration of the extracellular matrix represents a very early event in the pathogenesis of the mucopolysaccharidoses and implies that biomechanical failure of chondro-osseous tissue may underlie progressive bone and joint disease symptoms. These findings have important therapeutic implications.

Topics: Animals; Biglycan; Bone Diseases; Collagen Type I; Disease Models, Animal; Extracellular Matrix; Extracellular Matrix Proteins; Eye Proteins; Fibromodulin; Gene Expression Profiling; Glycoproteins; Glycosaminoglycans; Immunohistochemistry; Lactoferrin; Mass Spectrometry; Mice; Mice, Knockout; Mucopolysaccharidosis I; Nerve Growth Factors; Osteoarthritis; Proteoglycans; Proteomics; Serpins

Chondrocyte apoptosis is largely responsible for cartilage degeneration in osteoarthritis (OA). Interleukin-1 beta (IL-1β) is widely used as a chondrocyte apoptosis-inducing agent, while lactoferrin (LF) is an anabolic reagent which has the potential to inhibit chondrocyte apoptosis. We assessed the effects of LF on cartilage degeneration in IL-1β-induced chondrocytes and in a rat model of OA, and explored the potential molecular mechanisms involved.. Human articular chondrocytes (HACs) were treated with IL-1β alone or in combination with LF. MTT and flow cytometric assays were used to detect changes after treatment with LF. Western blotting was used to examine the relevant molecules regulating apoptosis.. We found that IL-1β reduced the viability of HACs, whereas 200 μg/mL of LF significantly counteracted the inhibitory effect of IL-1β. LF significantly inhibited IL-1β-induced HAC apoptosis. The protein expression of the apoptotic markers Caspase-3 and PARP was also significantly reduced in the LF treatment group when analyzed by western blotting. Furthermore, we found that LF triggered CREB1 phosphorylation in IL-1β-induced HAC apoptosis through AKT1 signaling. In addition, LF promoted the repair of articular cartilage damage in a rat OA model with elevated p-CREB levels.. These studies suggest that LF has an anti-apoptotic effect on IL-1β-induced chondrocytes, and thus may be a promising novel therapeutic agent for OA.

Topics: Animals; Apoptosis; Cartilage, Articular; Cell Survival; Chondrocytes; Cyclic AMP Response Element-Binding Protein; Disease Models, Animal; Humans; Interleukin-1beta; Lactoferrin; Male; Middle Aged; Osteoarthritis; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Wound Healing

Osteoarthritis (OA) treatments have major limitations which include systemic toxicity, reduced joint retention and inability to inhibit disease progression. In this study, the therapeutic potentials of 100% iron saturated-bovine lactoferrin encapsulated in alginate-chitosan polymeric nanocarriers (AEC-CP-Fe-bLf-NCs) were examined in in vitro inflammatory OA model and in collagen-induced arthritis (CIA) mice. By diminishing IL-1β induced apoptotic and oxidative stress, chondrocyte protection and proliferation was up-regulated with C-CP-Fe-bLf-NCs as compared to void and C-CP-Apo(metal free)-bLf-NCs. Oral administration of nanocarriers in mice was non-toxic and it significantly induced disease modifying activity by reducing joint inflammation and significantly downregulating the expression of catabolic genes, IL-1β, NO, JNK and MAPK. In addition, up-regulation of type II collagen, aggrecan and inflammation depleted iron and calcium metabolisms via inhibition of miRNA of iron transporting receptors was shown in AEC-CP-Fe-bLf-NCs treated mice. In addition, AEC-CP-Fe-bLf-NCs dissoluted calcium pyrophosphate crystals found in mice joints indicating the significantly important therapeutic ability of nanoformulated Fe-bLf to be utilized in the treatment of chronic inflammatory rheumatic diseases such as OA.

Topics: Administration, Oral; Alginates; Animals; Anti-Infective Agents; Anti-Inflammatory Agents; Cattle; Chitosan; Drug Carriers; Gene Expression Regulation; Glucuronic Acid; Hexuronic Acids; Interleukin-1beta; Iron; Joints; Lactoferrin; Male; Mice; MicroRNAs; Nanostructures; Osteoarthritis; Oxidative Stress

Dexamethasone (Dex) is commonly used for osteoarthritis (OA) with excellent anti-inflammatory and analgesic effect. However, Dex also has many side effects following repeated use over prolonged periods mainly through increasing apoptosis and inhibiting proliferation. Lactoferrin (LF) exerts significantly anabolic effect on many cells and little is known about its effect on OA chondrocytes. Therefore, the aim of this study is to investigate whether LF can inhibit Dex-induced OA chondrocytes apoptosis and explore its possible molecular mechanism involved in. MTT assay was used to determine the optimal concentration of Dex and recombinant human LF (rhLF) on chondrocytes at different time and dose points. Chondrocytes were then stimulated with Dex in the absence or presence of optimal concentration of rhLF. Cell proliferation and viability were evaluated using MTT and LIVE/DEAD assay, respectively. Cell apoptosis was evaluated by multi-parameter apoptosis assay kit using both confocal microscopy and flow cytometry, respectively. The expression of extracellular signal-regulated kinase (ERK), FAS, FASL, and Caspase-3 (CASP3) at the mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and immunocytochemistry, respectively. The optimal concentration of Dex (25 μg/ml) and rhLF (200 μg/ml) were chosen for the following experiments. rhLF significantly reversed the detrimental effect of Dex on chondrocytes proliferation, viability, and apoptosis. In addition, rhLF significantly prevented Dex-induced down-regulation of ERK and up-regulation of FAS, FASL, and CASP3. These findings demonstrated that rhLF acts as an anabolic effect on chondrocytes through significantly reversing Dex-induced chondrocytes apoptosis. This study may contribute to further investigating the clinical application of LF on OA.

Topics: Apoptosis; Cartilage, Articular; Caspase 3; Cell Proliferation; Cell Survival; Chondrocytes; Dexamethasone; Extracellular Signal-Regulated MAP Kinases; Fas Ligand Protein; fas Receptor; Gene Expression Regulation; Humans; Lactoferrin; Middle Aged; Osteoarthritis; RNA, Messenger; Up-Regulation

Bovine lactoferricin (LfcinB) is a heparan sulfate-binding peptide with multiple bioactivities. In human articular cartilage, LfcinB antagonizes interleukin-1 β (IL-1β) and fibroblast growth factor 2 (FGF-2) in proteoglycan metabolism, catabolic protease expression, and induction of pro-inflammatory mediators. LfcinB specifically activates ERK1/2, p38 and Akt, but whether these signaling pathways control the expression of LfcinB target genes remained unknown. In this report, we characterized a novel aspect of LfcinB-mediated genetic response in human articular chondrocytes, tissue inhibitor of metalloproteinase 3 (TIMP-3) induction. Inhibition of individual signaling pathways revealed that ERK1/2 functions as the major pathway in TIMP-3 expression, whereas Akt plays a minor role. Further investigation identified Sp1 as a critical transcriptional activator in TIMP-3 regulation, and Sp1 activity is modulated by ERK1/2, not Akt. Comparative quantification indicates that significant downregulation of TIMP-3 occurs in OA chondrocytes, suggesting a beneficial role of LfcinB in OA pathogenesis. Our results collectively provide new insights into the mechanism of action of LfcinB, and support the candidacy of LfcinB as a chondroprotective agent.

Topics: Adult; Aged; Animals; Anti-Bacterial Agents; Blotting, Western; Cartilage, Articular; Case-Control Studies; Cattle; Cells, Cultured; Chondrocytes; Electrophoretic Mobility Shift Assay; Humans; Immunoenzyme Techniques; Lactoferrin; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Osteoarthritis; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sp1 Transcription Factor; Tissue Inhibitor of Metalloproteinase-3

At the whole organ level, degenerative mechanisms in bone and cartilage are primarily attributed to modifications in loading pattern. Either a change in magnitude or location can initiate a degenerative path. At the micro scale we often see changes in structure such as porosity increase in bone and fibrillation in cartilage. These changes contribute to a reduced structural integrity that weakens the bulk strength of tissue. Finally, at the cell level we have modeling and remodeling pathways that may be disrupted through disease, drugs and altered stimulus from the micro and macro scales. In order to understand this entire process and the roles each level plays a multiscale modeling framework is necessary. This framework can take whole body loadings and pass information through finer spatial scales in order to understand how everyday dynamic movements influence micro and cellular response. In a similar manner, cellular and microstructural processes regulate whole bulk properties and modify whole organ strength. In this study we highlight the multiscale links developed as part of the open-source ontologies for the Physiome Project using the lower limb as an example. We consider the influence of remodeling in (i) anabolic treatments in cortical bone; and (ii) subchondral bone and cartilage degeneration.

Topics: Aged; Anterior Cruciate Ligament; Anthropometry; Bone and Bones; Bone Remodeling; Bone Resorption; Cartilage, Articular; Computer Simulation; Gait; Humans; Imaging, Three-Dimensional; Inflammation; Lactoferrin; Magnetic Resonance Imaging; Models, Anatomic; Models, Biological; Osteoarthritis; Osteoporosis; Porosity

To analyze lactoferrin expression on synovial fluid (SF) and peripheral blood neutrophils of patients with rheumatoid arthritis (RA) and to compare it with the lactoferrin expression on neutrophils from patients with osteoarthritis (OA).. Paired samples of peripheral blood and SF were obtained from 14 patients with RA and 9 patients with OA. Lactoferrin expression was evaluated on cell surfaces by cytofluorimetric analysis utilizing both polyclonal antibodies and the monoclonal anti-lactoferrin antibody AGM 2.29. Data are presented as mean fluorescence intensity.. In patients with RA, the expression of membrane lactoferrin was significantly increased on SF neutrophils in comparison with those in peripheral blood. This increase was found using both polyclonal antibodies and AGM 2.29 (p = 0.0001, p = 0.0017, respectively). In patients with OA, the difference was not significant. In addition, lactoferrin expression on SF neutrophils of patients with RA was significantly increased compared with that found on SF neutrophils of patients with OA (polyclonal antibodies, p = 0.0015; AGM 2.29, p = 0.005). In patients with RA, no correlation was found between lactoferrin expression and disease activity.. Our results provide evidence for an activation of neutrophil granulocytes at site of inflammation in RA and indicate that lactoferrin surface expression represents a reliable neutrophil activation marker.

Topics: Adult; Aged; Arthritis, Rheumatoid; Female; Humans; Lactoferrin; Male; Middle Aged; Neutrophil Activation; Neutrophils; Osteoarthritis; Severity of Illness Index; Synovial Fluid

Lactoferrin is a multifunctional immunoregulatory protein, stored in specific granules of neutrophil granulocytes, from which it is released following cell activation. As activated neutrophils play a crucial role in the destruction of synovial joints in rheumatoid arthritis, we evaluated lactoferrin concentration in synovial fluid and sera from 21 patients with rheumatoid arthritis and 11 patients with osteoarthritis. We also measured lactoferrin levels in sera from 12 healthy controls. Lactoferrin was measured by a solid-phase inhibition immunoassay. Median lactoferrin levels were significantly higher in synovial fluid from rheumatoid arthritis than from osteoarthritis patients (P = 0.0002). In contrast, no significant difference was found between serum lactoferrin from patients with rheumatoid arthritis or osteoarthritis compared with normal controls. In patients with rheumatoid arthritis, lactoferrin concentrations were higher in synovial fluid than in sera (P = 0.036). In both rheumatoid arthritis and osteoarthritis no correlation was found between serum and synovial fluid lactoferrin (P = 0.51 and P = 0.5, respectively). In synovial fluid from patients with rheumatoid arthritis, lactoferrin concentrations correlated with neutrophil granulocyte count (P < 0.0001), but neither serum nor synovial lactoferrin levels correlated with disease activity (P = 0.32 and P = 0.25, respectively). In conclusion, lactoferrin is a reliable marker of neutrophil activation at sites of inflammation in rheumatoid synovitis, but does not represent a marker of disease activity.

Topics: Adult; Aged; Aged, 80 and over; Anti-Inflammatory Agents; Anti-Inflammatory Agents, Non-Steroidal; Antirheumatic Agents; Arthritis, Rheumatoid; Autoantibodies; Female; Humans; Immunoassay; Lactoferrin; Leukocyte Count; Male; Middle Aged; Neutrophils; Osteoarthritis; Prednisolone; Synovial Fluid

Topics: Adult; Aged; Aged, 80 and over; Arthritis, Juvenile; Arthritis, Rheumatoid; Female; Ferritins; Humans; Iron; Iron-Regulatory Proteins; Iron-Sulfur Proteins; Lactoferrin; Male; Middle Aged; Osteoarthritis; Psoriasis; RNA-Binding Proteins; Synovial Fluid; Transferrin

We studied the state of alpha 2-macroglobulin (alpha 2M), an important inhibitor of cartilage-degrading proteinases, in relation to activation of neutrophils in 82 patients with several types of arthritis, including 52 with rheumatoid arthritis and 11 with osteoarthritis. Levels of total inactive alpha 2M (i alpha 2M), which comprises alpha 2M complexed to proteinases and alpha 2M inactivated by oxidation or hydrolysis, were measured with a monoclonal antibody specific for i alpha 2M. In addition, levels of alpha 2M complexed to proteinases were quantitated with specific assays. Neutrophil activation was assessed by measuring elastase-alpha 1-antitrypsin complexes and lactoferrin. In 83% of the 82 patients tested, the synovial fluid (SF) to plasma ratio of i alpha 2M exceeded 1, indicating an intraarticular generation. Levels of i alpha 2M significantly correlated with neutrophil numbers (P less than 0.0005) and with levels of elastase-alpha 1-antitrypsin complexes and of lactoferrin (P less than 0.00001 for both). Moreover, part of i alpha 2M consisted of alpha 2M complexed to elastase-like and chymotrypsin-like proteinases, presumably, neutrophil elastase and cathepsin G, respectively. However, the amount of i alpha 2M was approximately 10-fold larger than the amount complexed to these proteinases. In vitro inactivation of alpha 2M by activated neutrophils was only partly inhibitable by eglin C, a specific inhibitor of both elastase and cathepsin G. Release of reactive oxygen species was presumably responsible for the additional inactivation of alpha 2M, because eglin C completely abolished the inactivation of alpha 2M by cell-free supernatant of activated neutrophils. Thus, our results suggest a predominant role of neutrophils in the inactivation of alpha 2M in the SF of patients with inflammatory joint diseases. However, this inactivation could be explained only in part by the release of neutrophilic proteinases. We propose that the inactivation of alpha 2M in SF was due to the concerted action of both reactive oxygen species and lysosomal proteinases.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; alpha 1-Antitrypsin; alpha-Macroglobulins; Antibodies, Monoclonal; Arthritis; Arthritis, Rheumatoid; Female; Humans; Joints; Lactoferrin; Male; Middle Aged; Neutrophils; Osteoarthritis; Pancreatic Elastase; Synovial Fluid

Iron-binding proteins (lactoferrin, transferrin and ferritin) and free iron were measured in synovial fluid (SF) from 30 patients with rheumatoid arthritis (RA) and 20 osteoarthritis (OA) patients. The iron-binding proteins except transferrin were significantly increased in RA SF as compared with OA SF. Similarly, free iron was also significantly higher in RA SF than in OA SF, whereas the ferritin saturation index, transferrin saturation index and bound iron were more significantly decreased in RA SF than in OA SF. These results suggest that RA SF contains sufficient micromolar amounts of free iron to allow hydroxyl radical formation. Also the capacity of iron-binding proteins to bind free iron is inadequate in the presence of a large amount of iron-binding proteins which are present in RA SF.

Topics: Antigen-Antibody Complex; Arthritis, Rheumatoid; Carrier Proteins; Complement System Proteins; Ferritins; Humans; Iron; Iron-Binding Proteins; Lactoferrin; Osteoarthritis; Rheumatoid Factor; Synovial Fluid; Transferrin; Transferrin-Binding Proteins

Topics: Arthritis, Rheumatoid; Humans; Immunoglobulins; Iron; Lactoferrin; Lactoglobulins; Osteoarthritis; Synovial Fluid