Tarvo Sillat

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.

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.

Basement membrane collagen type IV expression by human mesenchymal stem cells during adipogenic differentiation

During adipogenic differentiation human mesenchymal stem cells (hMSC) produce collagen type IV. In immunofluorescence staining differentiating hMSCs started to express collagen type IV when Oil Red O‐positive fat droplets appeared intracellularly. Quantitative real time‐polymerase chain reaction confirmed progressive increase of collagen type IV α1 and α2 mRNA levels over time, 18.6‐ and 12.2‐fold by day 28, respectively, whereas the copy numbers of α3–α6 mRNAs remained rather stable and low. Type IV collagen was in confocal laser scanning microscopy seen around adipocytes, where also laminins and nidogen were found, suggesting pericellular deposition of all key components of the fully developed basement membrane. Immunofluorescence staining of matrix metalloproteinase‐2 (MMP‐2, 72 kD type IV collagenase, gelatinase A) and MMP‐9 (92 kD type IV collagenase, gelatinase B) disclosed only faint staining of MSCs, but MMP‐9 was strongly induced during adipogenesis, whereas MSC supernatants disclosed in zymography pro‐MMP‐2 and faint pro‐MMP‐9 bands, which increased over time, with partial conversion of pro‐MMP‐2 to its active 62 kD form. Differentiation was associated with increasing membrane type 1‐MMP/MMP‐14 and tissue inhibitor of metalloproteinase‐2 (TIMP‐2) staining, which may enable participation of type IV collagenases in basement membrane remodelling via ternary MT1‐MMP/TIMP‐2/MMP‐2 or −9 complexes, focalizing the fully active enzyme to the cell surface. MMP‐9, which increased more in immunofluorescence staining, was perhaps preferentially bound to cell surface and/or remodelling adipocyte basement membrane. These results suggest that upon MSC‐adipocyte differentiation collagen type IV synthesis and remodelling become necessary when intracellular accumulation of fat necessitates a dynamically supporting and instructive, partly denatured adipogenic pericellular type IV collagen scaffold.

Laminin Production and Basement Membrane Deposition by Mesenchymal Stem Cells upon Adipogenic Differentiation

The aim was to study laminin (LM) synthesis, integration, and deposition into the basement membrane (BM) during adipogenesis. Human bone marrow-derived mesenchymal stromal cells (MSCs) were induced along the adipogenic lineage. LM chain mRNA and protein levels were followed using quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF) staining, transmission electron microscopy (TEM), and immunoprecipitation. MSCs produced low levels of LM mRNAs but were not surrounded by BM in IF and TEM imaging. LM-α4, LM-β1, and LM-γ1 mRNAs increased during adipogenesis 3.9-, 5.8-, and 2.8-fold by day 28. LM-411 was immunoprecipitated from the ECM of the differentiated cells. Immunostaining suggested deposition of LM-411 and some LM-421. BM build-up was probably organized in part by integrin (Int) α6β1. At day 28, TEM images revealed BM-like structures around fat droplet-containing cells. The first signs of BM formation and Int α6β1 were seen using IF imaging at day 14. Laminin-411 and Int α6β1 were expressed in vivo in mature human subcutaneous fat tissue. Undifferentiated human MSCs did not organize LM subunits into BM, whereas LM-411 and some LM-421 are precipitated in the BM around adipocytes. This is the first demonstration of LM-411 precipitation during hMSC adipogenesis around adipocytes as a structural scaffold and Int-regulated signaling element.

Brief Report: First identification of H4 histamine receptor in healthy salivary glands and in focal sialadenitis in Sjögren's syndrome

OBJECTIVE The conventional H(1) and H(2) histamine receptors have >10,000-fold lower avidity for histamine than H(4) histamine receptor, which has been implicated in autoimmune diseases. This study was undertaken to compare H(4) histamine receptor levels in the salivary glands (SGs) of healthy controls with those in the SGs of patients with primary Sjögrens syndrome (SS). METHODS H(4) histamine receptor messenger RNA (mRNA) was analyzed using real-time quantitative polymerase chain reaction, and the receptor protein was examined using immunostaining. Effects of the H(4) histamine receptor agonist ST-1006 on cytokine synthesis by human SG (HSG) cells were analyzed using xMAP technology and enzyme-linked immunosorbent assay. RESULTS Healthy SGs contained H(4) histamine receptor mRNA. The receptor protein was localized to the acinar and ductal epithelial cells. H(4) histamine receptor agonist stimulated HSG cells to produce the cytokines interleukin-8 and vascular endothelial growth factor. SS patients had low H(4) histamine receptor levels. CONCLUSION H(1) and H(2) histamine receptor antagonists are not effective in the treatment of autoimmune diseases. However, such antagonists do not affect the newly discovered H(4) histamine receptor. Dendritic cells and lymphocytes are nonprofessional histamine-producing cells, which produce histamine at 100-1,000-fold lower rates than mast cells do. Saliva contains only 0.31-12.4 ng/ml histamine, which is too low to stimulate H(1) or H(2) histamine receptor, but stimulates H(4) histamine receptor half maximally. Our findings show that H(4) histamine receptor is strongly expressed in tubuloacinar SG cells, which emphasizes the role of these cells in the pathogenesis of SS.

Osteogenic differentiation of mesenchymal stromal cells in two-dimensional and three-dimensional cultures without animal serum

IntroductionBone marrow-derived mesenchymal stromal cells (MSCs) have been intensely studied for the purpose of developing solutions for clinical tissue engineering. Autologous MSCs can potentially be used to replace tissue defects, but the procedure also carries risks such as immunization and xenogeneic infection. Replacement of the commonly used fetal calf serum (FCS) with human platelet lysate and plasma (PLP) to support cell growth may reduce some of these risks. Altered media could, however, influence stem cell differentiation and we address this experimentally.MethodsWe examined human MSC differentiation into the osteoblast lineage using in vitro two- and three-dimensional cultures with PLP or FCS as cell culture medium supplements. Differentiation was followed by quantitative polymerase chain reaction, and alkaline phosphatase activity, matrix formation and matrix calcium content were quantified.ResultsThree-dimensional culture, where human MSCs were grown on collagen sponges, markedly stimulated osteoblast differentiation; a fourfold increase in calcium deposition could be observed in both PLP and FCS groups. PLP-grown cells showed robust osteogenic differentiation both in two- and three-dimensional MSC cultures. The calcium content of the matrix in the two-dimensional PLP group at day 14 was 2.2-fold higher in comparison to the FCS group (p < 0.0001), and at day 21 it was still 1.3-fold higher (p < 0.001), suggesting earlier calcium accumulation to the matrix in the PLP group. This was supported by stronger Alizarin Red staining in the PLP group at day 14. In two-dimesional PLP cultures, cellular proliferation appeared to decrease during later stages of differentiation, while in the FCS group the number of cells increased throughout the experiment. In three-dimensional experiments, the PLP and FCS groups behaved more congruently, except for the alkaline phosphatase activity and mRNA levels which were markedly increased by PLP.ConclusionsHuman PLP was at least equal to FCS in supporting osteogenic differentiation of human MSCs in two- and three-dimensional conditions; however, proliferation was inferior. As PLP is free of animal components, and thus represents reduced risk for xenogeneic infection, its use for human MSC-induced bone repair in the clinic by the three-dimensional live implants presented here appears a promising therapy option.

Role and regulation of VEGF and its receptors 1 and 2 in the aseptic loosening of total hip implants

It was hypothesized that vascular endothelial growth factor (VEGF) in fibroblasts participates in aseptic loosening of total hip replacement (THR) implants. Therefore, osteoarthritic (OA) samples (n = 11) were compared with synovial membrane‐like interface tissues from revision THR (n = 10). VEGF‐A and its receptors were stained using streptavidin‐immunoperoxidase method. Their regulation by hypoxia and cytokines were studied in cultured fibroblasts using quantitative real‐time polymerase chain reaction (qRT‐PCR). VEGFR1+ lining cells (p < 0.01), stromal fibroblast‐like cells (p = 0.001) and stromal macrophage‐like cells (p < 0.05) were more numerous in rTHR than in OA. As to VEGFR2+, only stromal fibroblast‐like cells in rTHR outnumbered those found in OA (p < 0.05). VEGFRs in synovial fibroblasts were not affected by hypoxia, but VEGF increased 2.4‐fold (p < 0.05). Interleukin‐4 up‐regulated VEGFR1 expression 23‐fold. This is the first study to describe a difference between rTHR and OA in VEGF receptors, particularly VEGFR1. Hypoxia increased VEGF, but the VEGFR1 increase in the lining and stroma is probably IL‐4 driven, in accordance with the M2‐type macrophage dominance in interface tissues. VEGF/VEGFR system is also affected by hypoxia and may play a role in angiogenesis and bone pathology in aseptic loosening of total hip implants.

Histamine transport and metabolism are deranged in salivary glands in Sjögren’s syndrome

OBJECTIVE To study histamine transport and metabolism of salivary gland (SG) epithelial cells in healthy controls and SS patients. METHODS Enzymes and transporters involved in histamine metabolism were analysed in cultured human submandibular salivary gland (HSG) epithelial cells and tissue sections using quantitative real-time PCR and immunostaining. HSG cells were used to study [(3)H]histamine uptake [(±1-methyl-4-phenylpyridinium (MPP)] and efflux by liquid scintillation counting. RESULTS mRNA levels of l-histidine decarboxylase (HDC) and histamine-N-methyltransferase (HNMT) were similar in the control and SS glands, but diamine oxidase was not expressed at all. Organic cation transporter 3 (OCT3) in healthy SG was localized in the acinar and ductal cells, whereas OCT2 was restricted to the myoepithelial cells. Both transporters were significantly decreased in SS at mRNA and protein levels. OCT3-mRNA levels in HSG cells were significantly higher than those of the other studied transporters. Uptake of [(3)H]histamine was inhibited by MPP in a time-dependent manner, whereas [(3)H]histamine-preloaded HSG cells released it. CONCLUSION Ductal epithelial cells are non-professional histamine-producing cells able to release histamine via OCTs at the resting state up to ∼100 nM, enough to excite H3R/H4R(+) epithelial cells, but not H1R, which requires burst release from mast cells. At the stimulated phase, 50-60 μM histamine passes from the interstitial fluid through the acinar cells to saliva, whereas uptake by ductal cells leads to intracellular degradation by HNMT. OCT3/histamine/H4R-mediated cell maintenance and down-regulation of high histamine levels fail in SS SGs.

Do changing toll-like receptor profiles in different layers and grades of osteoarthritis cartilage reflect disease severity?

Objective. Cartilage degeneration in osteoarthritis (OA) leads to release of potential danger signals. The aim of our study was to profile OA cartilage for the Toll-like receptor (TLR) danger signal receptors. Methods. Osteochondral cylinders from total knee replacements were graded using OA Research Society International score and stained for proteoglycans, collagenase-cleaved type II collagen, and TLR 1–10, which were analyzed histomorphometrically. Results. Grade 1 OA lesions contained 22%–55% TLR 1–9-positive cells in the surface zone, depending on the TLR type. In Grade 2 TLR, immunoreactivity was 60%–100% (p < 0.01) and it was even higher in Grades 3 and 4 (p < 0.01 vs Grade 1). TLR-positive cells in Grade 1 middle zone were low, 0–19.9%, but were 5.1%–32.7% in Grade 2 (p < 0.01) and 34%–83% in Grades 3–4 samples (p < 0.001). TLR values in Grade 5 were low (14.3%–28.7%; p < 0.001). In Grades 3–4 OA, cartilage matrix stained strongly for TLR. In Grade 1, COL2-3/4M was restricted to chondrocytes, but was increasingly seen in matrix upon progress of OA to Grade 4, and then declined. Conclusion. Cells in the gliding surface zone are fully equipped with TLR in mild OA. Their proportion increases and extends to the middle or even the deep zone, reflecting OA progression. COL2A-3/4M staining suggests Endo180-mediated intake for intralysosomal degradation by cathepsins in Grade 1, but in higher grades this chondrocyte-mediated clearance fails and the matrix demonstrates extensive collagenase-induced damage. Detached and/or partially degraded matrix components can then act as endogenous danger signals (damage-associated molecular patterns or DAMP) and stimulate increasingly TLR-equipped chondrocytes to inflammation. At the peak inflammatory response, soluble TLR may exert negative feedback, explaining in part the low TLR levels in Grade 5 OA.