Mari Ainola

MMPs, IL-1, and TNF are Regulated by IL-17 in Periodontitis

Periodontitis is characterized by periodontal tissue destruction. Since interleukin-17 (IL-17) has been reported to up-regulate IL-1β and tumor necrosis factor-alpha (TNF-α), it was hypothesized that it is increased in periodontitis and up-regulates these cytokines and tissue-destructive matrix metalloproteinases (MMP) in local migrant and resident cells. Immunocytochemistry disclosed elevated IL-1β, TNF-α, and IL-17 levels in periodontitis. These cytokines induced proMMP-1 and especially MMP-3 in gingival fibroblasts, whereas MMP-8 and MMP-9 were not induced. IL-17 was less potent as a direct MMP inducer than IL-1β and TNF-α, but it induced IL-1β and TNF-α production from macrophages, and IL-6 and IL-8 from gingival fibroblasts. In accordance with these findings, immunocytochemistry disclosed that MMP-1 and MMP-3 were increased in periodontitis. Gingival fibroblasts may play an important role in tissue destruction in periodontitis via cytokine-inducible MMP-1 and MMP-3 production, in which IL-17 plays a role as a key regulatory cytokine.

Macrophages – Key Cells in the Response to Wear Debris from Joint Replacements

The generation of wear debris is an inevitable result of normal usage of joint replacements. Wear debris particles stimulate local and systemic biological reactions resulting in chronic inflammation, periprosthetic bone destruction, and eventually, implant loosening, and revision surgery. The latter may be indicated in up to 15% patients in the decade following the arthroplasty using conventional polyethylene. Macrophages play multiple roles in both inflammation and in maintaining tissue homeostasis. As sentinels of the innate immune system, they are central to the initiation of this inflammatory cascade, characterized by the release of proinflammatory and pro-osteoclastic factors. Similar to the response to pathogens, wear particles elicit a macrophage response, based on the unique properties of the cells belonging to this lineage, including sensing, chemotaxis, phagocytosis, and adaptive stimulation. The biological processes involved are complex, redundant, both local and systemic, and highly adaptive. Cells of the monocyte/macrophage lineage are implicated in this phenomenon, ultimately resulting in differentiation and activation of bone resorbing osteoclasts. Simultaneously, other distinct macrophage populations inhibit inflammation and protect the bone-implant interface from osteolysis. Here, the current knowledge about the physiology of monocyte/macrophage lineage cells is reviewed. In addition, the pattern and consequences of their interaction with wear debris and the recent developments in this field are presented.

Acid attack and cathepsin K in bone resorption around total hip replacement prosthesis.

Normal bone remodeling and pathological bone destruction have been considered to be osteoclast‐driven. Osteoclasts are able to attach to bare bone surface and produce an acidic subcellular space. This leads to acid dissolution of hydroxyapatite, allowing cathepsin K to degrade the organic type I collagen‐rich osteoid matrix under the acidic condition prevailing in Howship lacunae. Using a sting pH electrode, the interface membrane around a loosened total hip replacement prosthesis was found to be acidic. Confocal laser scanning disclosed irregular demineralization of the bone surface in contact with the acidic interface. Cathepsin K, an acidic collagenolytic enzyme, was found in interface tissue macrophages/giant cells and pseudosynovial fluid. Tissue extracts contained high levels of cathepsin K messenger RNA (mRNA) and protein. These observations suggest the presence of an acid‐ and cathepsin K‐driven pathological mechanism of bone resorption, mediated not by osteoclasts in subosteoclastic space, but rather by the uncontrolled activity of macrophages in extracellular space.

Messenger ribonucleic acid expression of 16 matrix metalloproteinases in bone–implant interface tissues of loose artificial hip joints

Matrix metalloproteinases (MMPs) have been reported to be the major factors responsible for aseptic loosening of artificial hip joints. So far, messenger ribonucleic acid (mRNA) expression patterns of seven MMPs have been reported, but that of many other MMPs which have been newly discovered or recently considered to be responsible for prosthetic loosening is still unknown. In this study, mRNA expression pattern of 16 different types of MMPs were analyzed to evaluate which MMPs were locally produced and contributed to prosthetic loosening. Synovium-like interface tissues between bone and prosthesis were collected from 18 cases of aseptic loose artificial hip joint at revision surgery. Six cases of normal synovium were used as controls. Total RNA was extracted by single-step acid guanidinium-thiocyanate-phenol-chloroform procedure. mRNA expression of MMPs was analyzed by semiquantitative reverse transcription-polymerase chain reaction. Based on local expression pattern of MMPs at the mRNA level, aseptic loose artificial hip joint was characterized by elevated expression of MMP-1, MMP-9, MMP-10, MMP-12, and MMP-13; moderate expression of MMP-2, MMP-7, MMP-8, MMP-11, membrane type (MT)1-MMP (MMP-14), MT2-MMP (MMP-15), MT3-MMP (MMP-16), MT4-MMP (MMP-17), and MMP-19; lower expression of MMP-3; and little significance of MMP-20. The MMPs detected in this study can potentially degrade almost all components of the periprosthetic extracellular matrix. Thus, many MMP type enzymes possibly contribute to prosthetic loosening and osteolysis through pathologic extracellular matrix degradation and connective tissue/bone remodeling around prostheses.

Osteoarthritis as an autoinflammatory disease caused by chondrocyte-mediated inflammatory responses.

Osteoarthritis (OA) is considered to be primarily a disease of the hyaline articular cartilage, which secondarily affects subchondral bone and synovial membrane. The exact nature and mechanisms of OA, particularly during the early phases of the disease, are unknown. OA per se might in part relate to the poor inherent repair capacity of the articular cartilage, which during the lifetime of modern (long-lived) human beings, is gradually subjected to progressive and accumulative wear and tear. However, the idea of OA as a simple wear-and-tear disease has been widely rejected because various biologic processes, such as inflammation and enzymatic cartilage degradation, are apparently involved in its pathogenesis. Recent findings provide possible new explanatory pathogenic models that intimately link the two phenomena—biomechanical wear and tear of the cartilage (osteoarthrosis) and inflammation (osteoarthritis)—to each other. Although mesenchymal progenitor cells have been found in the cartilage matrix (1), cartilage cannot recruit circulating mesenchymal stem cells and is unable to organize the repair according to the developmental programs that, during the embryonic and fetal stages, created the delicate molecular collagen and architectural tissue structure of true synovial diarthrodial joints. Structural degeneration, cartilage thinning, and sclerosis of the subchondral bone plate are indeed common in the elderly population, but clinically manifest OA with pain is, fortunately, much rarer. This depends in part on the avascular nature of the cartilage; cartilage contains neither blood nor lymphatic vessels. This makes it impossible for the cartilage to recruit leukocytes and, according to the conventional view, makes it unable to mount an inflammatory response that is recognizable by the classic signs of redness, local inflammatory swelling, pain, increased local temperature, and impaired function. The question is whether cartilage can mount an inflammatory cellular (chondrocyte) response, which then via transfer of the locally produced messenger molecules to synovium causes synovitis, with classical signs of inflammation. Cartilage is also aneural, which might be useful from the functional point of view, considering the high peak loads and trauma that the cartilage covering the ends of the bone is naturally subjected to during cyclic activities and traumatic “high-energy” activities in particular. Due to the lack of primary afferent nociceptive nerves, cartilage also cannot be a source of nociceptive or inflammatory pain. Pain in OA is therefore considered to be caused by secondary involvement of the synovium, that is, osteoarthritic synovitis, which has an unclear pathogenesis. In any case, it has been emphasized that the lymphocyte-rich mononuclear cell infiltrates seen in OA are relatively similar to those seen in rheumatoid arthritis (RA) and that, at the superficial level at least, the differences between secondary OA synovitis and primary RA synovitis are quantitative rather than qualitative. The results reported by Opolka and coworkers (2) elsewhere in this issue of Arthritis & Rheumatism can be used to question this line of speculation. The investigators show that both in micromass and monolayer culture, primary costal chondrocytes from newborn mice produce neuronal transmitters, substance P (SP), and some of them probably also norepinephrine (NE) (because they contained tyrosine hydroxylase, the ratelimiting enzyme of its biosynthesis). Furthermore, chondrocytes contain neurokinin type 1 (NK-1) receptor and -adrenergic (possibly all subtypes, although monolayer cultures did not contain 1a or 2c) and Y. T. Konttinen, MD, PhD, D. C. E. Nordström, MD, PhD: Helsinki University Central Hospital, Helsinki, Finland; T. Sillat, MD: Coxa Hospital for Joint Replacement, Tampere, Finland; G. Barreto, BSc: Orton Orthopaedic Hospital of the Orton Foundation, Helsinki, Finland; M. Ainola, PhD: University of Helsinki, Helsinki, Finland. Address correspondence to Y. T. Konttinen, MD, PhD, Institute of Clinical Medicine, Department of Medicine, Biomedicum 1, PO Box 700, Helsinki University Central Hospital, Helsinki FIN00029, Finland. E-mail: yrjo.konttinen@helsinki.fi. Submitted for publication October 3, 2011; accepted in revised form October 25, 2011.

Toll-like receptors in human chondrocytes and osteoarthritic cartilage.

Background and purpose Degenerating cartilage releases potential danger signals that react with Toll-like receptor (TLR) type danger receptors. We investigated the presence and regulation of TLR1, TLR2, and TLR9 in human chondrocytes. Methods We studied TLR1, TLR2, TLR4, and TLR9 mRNA (qRT-PCR) and receptor proteins (by immunostaining) in primary mature healthy chondrocytes, developing chondrocytes, and degenerated chondrocytes in osteoarthritis (OA) tissue sections of different OARSI grades. Effects of a danger signal and of a pro-inflammatory cytokine on TLRs were also studied. Results In primary 2D-chondrocytes, TLR1 and TLR2 were strongly expressed. Stimulation of 2D and 3D chondrocytes with a TLR1/2-specific danger signal increased expression of TLR1 mRNA 1.3- to 1.8-fold, TLR2 mRNA 2.6- to 2.8-fold, and TNF-α mRNA 4.5- to 9-fold. On the other hand, TNF-α increased TLR1 mRNA] expression 16-fold, TLR2 mRNA expression 143- to 201-fold, and TNF-α mRNA expression 131- to 265-fold. TLR4 and TLR9 mRNA expression was not upregulated. There was a correlation between worsening of OA and increased TLR immunostaining in the superficial and middle cartilage zones, while chondrocytes assumed a CD166× progenitor phenotype. Correspondingly, TLR expression was high soon after differentiation of mesenchymal stem cells to chondrocytes. With maturation, it declined (TLR2, TLR9). Interpretation Mature chondrocytes express TLR1 and TLR2 and may react to cartilage matrix/chondrocyte-derived danger signals or degradation products. This leads to synthesis of pro-inflammatory cytokines, which stimulate further TLR and cytokine expression, establishing a vicious circle. This suggests that OA can act as an autoinflammatory disease and links the old mechanical wear-and-tear concept with modern biochemical views of OA. These findings suggest that the chondrocyte itself is the earliest and most important inflammatory cell in OA.

Epithelial-mesenchymal transition downregulates laminin α5 chain and upregulates laminin α4 chain in oral squamous carcinoma cells

Basement membranes maintain the epithelial phenotype and prevent invasion and metastasis. We hypothesized that expression of basement membrane laminins might be regulated by epithelial-mesenchymal transition (EMT), hallmark of cancer progression. As EMT is mediated by transcription factor Snail, we used oral squamous carcinoma cells obtained from a primary tumor (43A), from its EMT-experienced recurrence (43B) and Snail-transfected 43A cells (43A-SNA) displaying full EMT, as a model to study laminins and their receptors. Northern blotting, immunofluorescence, and immunoprecipitation showed a gradual loss of laminin-511 and its receptor Lutheran from 43A to 43B and 43A-SNA cells. In contrast, neoexpression of laminin α4 mRNA was found congruent with synthesis of laminin-411. Chromatin immunoprecipitation disclosed direct binding of Snail to regions upstream of laminin α5 and α4 genes. Immunofluorescence and immunoprecipitation showed a switch from hemidesmosomal integrin α6β4 to α6β1 and neoexpression of α1β1 in 43A-SNA cells, and upregulation of integrin-linked kinase in both 43B and 43A-SNA cells. The cells adhered potently to laminin-511 and fibronectin, whereas adhesion to laminin-411 was minimal. In contrast, laminin-411 inhibited cell adhesion to other extracellular matrix proteins. In conclusion, EMT induces a switch from laminin-511 to laminin-411 expression, which may be directly controlled by Snail. Concomitant changes take place in laminin- and collagen-binding receptors. Laminin-411 reduces adhesion to laminin-511 and fibronectin, suggesting that tumor cells could utilize laminin-411 in their invasive behavior.

Chemical and physical properties of regenerative medicine materials controlling stem cell fate

Abstract Regenerative medicine is a multidisciplinary field utilizing the potential of stem cells and the regenerative capability of the body to restore, maintain, or enhance tissue and organ functions. Stem cells are unspecialized cells that can self-renew but also differentiate into several somatic cells when subjected the appropriate environmental cues. The ability to reliably direct stem cell fate would provide tremendous potential for basic research and clinical therapies. Proper tissue function and regeneration rely on the spatial and temporal control of biophysical and biochemical cues, including soluble molecules, cell–cell contacts, cell–extracellular matrix contacts, and physical forces. The mechanisms involved remain poorly understood. This review focuses on the stem cell–extracellular matrix interactions by summarizing the observations of the effects of material variables (such as overall architecture, surface topography, charge, ζ-potential, surface energy, and elastic modulus) on the stem cell fate. It also deals with the mechanisms underlying the effects of these extrinsic, material variables. Insight in the environmental interactions of the stem cells is crucial for the development of new material-based approaches for cell culture experiments and future experimental and clinical regenerative medicine applications.

Involvement of a disintegrin and a metalloproteinase 8 (ADAM8) in osteoclastogenesis and pathological bone destruction

Objectives: The eventual role of a disintegrin and a metalloproteinase 8 (ADAM8) in osteoclastogenesis was studied in erosive rheumatoid arthritis (RA) and in vitro. Methods: ADAM8 protein and mRNA expression was measured in RA pannus and synovitis and compared to osteoarthritic (OA) synovial membrane. Human monocytes were isolated and stimulated with proinflammatory cytokines and their ADAM8 expression and surface ADAM8 were measured. Human peripheral blood monocytes and RAW 264.7 mouse monocyte/macrophage cells were stimulated to osteclast like-cells, and their expression of ADAM8 and osteoclastic markers (calcitonin receptor, integrin β 3, cathepsin K, TRAP) were analysed. Transfection and small interfering RNA (siRNA) were used to assess the role of ADAM8 in formation of polykaryons. Results: Increased numbers of ADAM8 positive cells were shown particularly in the pannus-cartilage/bone junction close or adjoining to TRAP positive multinucleate cells under formation (60 (2)% in pannus, 47 (2)% in synovitis vs 10 (1)% in OA, p<0.001). Human pannus contained high ADAM8 mRNA copy numbers (23 (7) in pannus, 14 (4) in synovitis vs 1.7 (0.3) in OA, p<0.001). Functional studies in vitro disclosed ADAM8 mRNA and protein, which was first converted to a proteolytically active and then to fusion-active form. Gene transfection and siRNA experiments enhanced and inhibited, respectively, expression of osteoclast markers and maturation of multinuclear cells. Conclusions: ADAM8 may be involved in bone destruction in RA because it is upregulated in RA pannus adjacent to developing erosions and enhances maturation of osteoclast-like cells.

Trypsin-2 degrades human type II collagen and is expressed and activated in mesenchymally transformed rheumatoid arthritis synovitis tissue

It has traditionally been believed that only the human collagenases (matrix metalloproteinase-1, -8, and -13) are capable of initiating the degradation of collagens. Here, we show that human trypsin-2 is also capable of cleaving the triple helix of human cartilage collagen type II. We purified human trypsin-2 and tumor-associated trypsin inhibitor by affinity chromatography whereas collagen type II was purified from cartilage extracts using pepsin digestion and salt precipitation. Degradation of type II collagen and gelatin by trypsin-2 was demonstrated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, zymography, and mass spectrometry, and tumor-associated trypsin inhibitor specifically inhibited this degradation. Although human trypsin-2 efficiently digested type II collagen, bovine trypsin did not. Furthermore, immunohistochemical staining detected trypsin-2 in the fibroblast-like synovial lining and in stromal cells of human rheumatoid arthritis synovial membrane. These findings were confirmed by reverse transcriptase-polymerase chain reaction and nucleotide sequencing. Trypsin-2 alone and complexed with alpha(1)-proteinase inhibitor were also detected in the synovial fluid of affected joints by time-resolved immunofluorometric assay, suggesting that trypsin-2 is activated locally. These results are the first to assess the ability of human trypsin to cleave human type II collagen. Thus, trypsin-2 and its regulators should be further studied for use as markers of prognosis and disease activity in rheumatoid arthritis.