Konttinen Yt

Specificity of the anticollagenase action of tetracyclines: relevance to their anti-inflammatory potential.

The concentrations of doxycycline and 4-de-dimethylaminotetracycline required to inhibit 50% of collagenase activity were found to be 15 to 30 microM for human neutrophil and gingival crevicular fluid collagenases. Fibroblast collagenase was relatively resistant to inhibition by tetracyclines; the 50% inhibitory concentrations of doxycycline and 4-de-dimethylaminotetracycline were 280 and 510 microM, respectively.

Human Neutrophil Collagenase MMP-8 in Peri-implant Sulcus Fluid and its Inhibition by Clodronate

The exact molecular mechanisms of the loosening of a dental implant are not well-known. The characteristics of implant sulci are similar to those of periodontal sulci regarding gingival crevicular fluid (GCF) and peri-implant sulcular fluid (PISF). Proteolytic enzymes, matrix metalloproteinases (MMPs), participate in peri-implant tissue remodeling. Clodronate is a well-tolerated bisphosphonate-group drug currently used in bone-resorption-related diseases in humans. The mechanisms of bisphosphonate action are not clarified. Collagenase activity in diseased PISF was significantly higher than in the clinically healthy group. Immunoblotting disclosed that diseased PISF contained increased immunoreactives MMP-8 compared with the healthy PISF. The residual latent collagenase activity in the diseased PISF was activated by gold thioglucose and inhibited completely by 100 pM of doxycycline closely resembling pure neutrophil collagenase (MMP-8). The presence of MMP-8 in diseased but not in clinically healthy PISF may prove to be a useful biochemical indicator to monitor peri-implant health and disease. Pure human neutrophil collagenase (MMP-8) and the MMP-8 present in PISF and in the GCF of both loosening implants and periodontitis-affected teeth were efficiently inhibited in vitro by clodronate (50% inhibition [IC50] was achieved by 150 uM of clodronate), an osteoactive, antiresorptive bisphosphonate. Furthermore, the new finding suggests an extended and hitherto-undescribed potential for clodronate in preventing the loosening of both implants and teeth, based on a dual beneficial effect: prevention of both bone resorption/osteolysis and of soft tissue/dental ligament destruction. Potential new therapeutic indications based on the collagenase-inhibiting effect of clodronate provide potential new therapeutic indications for a variety of diseases involving connective tissue breakdown, such as periodontal disease, arthritides, and tumor invasion.

Neuropeptides in Experimental and Degenerative Arthritis

Abstract: Classical symptoms of both inflammatory and degenerative arthritides may contribute to neurogenic responses like wheal, flare, edema, and pain. Rheumatoid arthritis (RA) is an autoimmune disease with an immunogenetic background. Neurogenic inflammation has been considered to play an essential role in RA, in part because of the symmetrical involvement (cross‐spinal reflexes) and the predominant involvement of the most heavily innervated small joints of the hands and the feet (highly represented in the hominiculus). In contrast, osteoarthritis (OA) is considered to arise as a result of degeneration of the hyaline articular cartilage, which secondarily results in local inflammation and pain. However, it is possible that the age‐related and predominant (compared to nociceptive nerves) degeneration of the proprioceptive, kinesthetic and vasoregulatory nerves can represent the primary pathogenic events. This leads to progressive damage of tissue with extremely poor capacity for self‐regeneration. Inflammation, be it primary/autoimmune or secondary/degenerative, leads to peripheral sensitization and stimulation, which may further lead to central sensitization, neurogenic amplification of the inflammatory responses and activation of the neuro‐endocrine axis. Neuropeptides serve as messengers, which modulate and mediate the actions in these cascades. Accordingly, many neuropeptides have been used successfully as experimental treatments, most recently VIP, which effectively controlled collagen‐induced arthritis in mice. Therefore, it can safely be concluded that better treatment/control of disease activity and pain can be achieved by blocking the cascade leading to initiation and/or amplification of inflammatory process combined with effects on central nociceptive and neuroendocrine responses.

Macrophage activation results in bone resorption.

Monocytes or macrophages from important accessory cells in the regulation of bone metabolism and destruction. Cells of the mononuclear phagocyte lineage form the precursor cells of the osteoclasts. Soluble products produced by activated macrophages regulate progenitor cell proliferation, recruitment, differentiation, and activity of osteoblasts and osteoclasts. After osteoclasts are removed from the resorption site, macrophages process bone surfaces and create a cement line before osteoblasts enter to form new bone. Although osteolysis associated with normal bone remodeling is seen as an osteoclast driven process, it may be that in chronic inflammation macrophage activation and vascular derangements lead to low pH, local bone demineralization (acid attack), and H+ mediated stimulation of the primary afferent nociceptive nerve fibers (bone pain). Osteoclasts are not able to attach to demineralized bone or to osteoid surfaces. However, if macrophages degrade the demineralized organic bone matrix, chemotactic factors and attachment sites for osteoclasts are produced. In such a scenario, the osteoclast-osteoblast mediated activation, resorption, and formation cycle would be secondarily activated. Such events may play a role in the most common orthopaedic problem related to macrophage activation, aseptic loosening of orthopaedic joint implants, which is secondary to a chronic foreign body reaction and to micromovement.

Membrane Components of Treponema denticola Trigger Proteinase Release from Human Polymorphonuclear Leukocytes

Tissue destruction during periodontitis is believed to be primarily brought about by leukocyte proteinases. We postulate that oral spirochetes cause discharge of polymorphonuclear leukocyte (PMN) lysosomal enzymes. Effects of Treponema denticola 53-kDa outer membrane protein, lipopolysaccharide (LPS), and peptidoglycan on degranulation of matrix metalloproteinases (MMP)-8 (collagenase) and -9 (gelatinase), cathepsin G, and elastase by human peripheral blood PMNs were studied by specific enzyme assays and Western blot analysis. T. denticola 53-kDa outer membrane protein was found to be a particularly efficient inducer of MMP-8 release. The induction was comparable with that of phorbol myristate acetate, a known inducer of PMN specific granule discharge. All of the treponemal substances, most notably the 53-kDa protein and LPS, induced release of MMP-9, a component of C-type granules. Both collagenase and gelatinase released from PMNs were mostly in active forms. Release of cathepsin G and elastase was also observed with the 53-kDa protein treatment. The other T. denticola substances did not induce release of these serine proteinases. Lactate dehydrogenase was not released from PMNs by the treatments, indicating that the degranulation was specific and not caused by toxic effects of the substances. This was confirmed by transmission electron microscopy of PMNs treated with the 53-kDa protein that showed rapid vacuole formation and cell shape changes but no disintegration of the cells. Thus, T. denticola may participate in the PMN-dependent extracellular matrix degradation during the course of periodontal inflammation by triggering the secretion and activation of matrix metalloproteinases.

In vivo inhibition of human neutrophil collagenase (MMP-8) activity during long-term combination therapy of doxycycline and non-steroidal anti-inflammatory drugs (NSAID) in acute reactive arthritis.

We studied the in vivo effect of long‐term doxycycline treatment combined with NSAID on human interstitial collagenases, other matrix melalloproteinases, serine proteinases, tissue inhibitor of matrix metalloproteinase‐1 (TIMP‐1) and lactoferrin from saliva and serum during the course of acute reactive arthritis (ReA). Collagenase activity and serine proteases (elastase‐like, cathepsin G‐like and trypsin‐like activities) of saliva (n= 10) and gelatinase, lactoferrin and TIMP‐1 of saliva (n=10) and serum (n= 10) samples before and after 2 months doxycycline treatment, combined with NSAID, were studied by quantitative SDS‐PAGE assay, ELISA assay and by spectrophotometric assay. The cellular source and molecular forms of salivary collagenase were characterized by immunoblotting using specific antisera. We found that activities of total and endogenously active interstitial collagenase reduced significantly. The salivary collagenase was found to originate from neutrophils. No fragmentation of either pro 75‐kD and active 65‐kD MMP‐8 was detected after 2 months doxycycline treatment. However, during 2 months doxycycline and NSAID treatment no reduction of salivary and serum gelatinase, lactoferrin and TIMP–1‐levels and salivary serine protease activities were detected. The in vivo inhibition of collagenase (MMP‐8) activity during long‐term doxycycline therapy in human saliva containing inflammatory exudate of ReA patients may contribute to the reduced tissue destruction observed in recent clinical and animal model studies in arthritides during long‐term doxycycline/tetracycline treatment.

Complement in acute and chronic arthritides: assessment of C3c, C9, and protectin (CD59) in synovial membrane.

OBJECTIVES: To investigate the role of complement cascade induced damage and protection against it in acute arthritides compared to rheumatoid arthritis and other chronic joint derangements. METHODS: C3c, C9, and protectin (CD59) were examined by avidin-biotin-peroxidase complex staining. RESULTS: Marked deposits of C3c and C9 were found in synovial vasculature and intercellular matrix of the lining in rheumatoid arthritis and in acute arthritides (including bacterial, reactive, and osteoarthritis flare up). Furthermore, protectin was not visible in synovial lining cells and was relatively weakly expressed in stromal and endothelial cells in rheumatoid arthritis; also in acute arthritides protectin expression was weak. In contrast, C3c and C9 deposits were not found in chronic conditions associated with degenerative diseases (osteoarthritis and osteochondritis dissecans) or mechanical causes (patellar luxation and a ruptured meniscus), in which also the protectin expression was prominent in synovial lining, endothelial and some stromal cells. CONCLUSIONS: Activation of the complement in rheumatoid arthritis and in acute arthritides seems to be associated with a decreased protection of synovial cells against cellular effects and lysis mediated by membrane attack complex.

Tumor Necrosis Factor-a and its Receptors, p55 and p75, in Gingiva of Adult Periodontitis

Tumor necrosis factor-a. (TNF-a), a pro-inflammatory cytokine, can stimulate matrix metalloproteinase synthesis and osteoclastic bone resorption. We hypothesized that elevated expression of TNF-a and its p55 and p75 receptors (TNF-R) in gingival tissue might associate with periodontitis. Immunohistochemistry was used for the study of the localization of TNF-a and its p55 and p75 TNF-R in adult periodontitis (AP) gingival tissue, in comparison with that in healthy control specimens. TNF-a and p55 TNF-R were detected in sulcular epithelial basal cells and in monocyte/macrophages, fibroblasts, and endothelial cells in the AP gingival tissue specimens, but mainly in fibroblasts and endothelial cells in control specimens. P75 TNF-R was occasionally found in monocyte/macrophage-like cells in gingival tissue specimens. The percentage of TNF-a-containing cells was not increased in AP compared with controls (13.2% ± 6.1% vs. 12.8% ± 7.6%), but, due to the increased cellularity of AP samples, the number of TNF-a positive cells/mm2 was clearly increased (1621 ± 663 vs. 664 ±191, p > 0.001). Thus, AP gingival tissue has an elevated expression of TNF-a and especially its p55 receptor, suggesting that TNF-a may contribute to tissue degradation in periodontitis.

Doxycycline in the protection of serum alpha-1-antitrypsin from human neutrophil collagenase and gelatinase.

The concentration of doxycycline required to inhibit 50% (50% inhibitory concentration for serpinase activity) of alpha-1-antitrypsin degradation by purified neutrophil collagenase was found to be approximately 20 microM, a value similar to the 50% inhibitory concentration of doxycycline required to inhibit collagen degradation by neutrophil collagenase. Doxycycline also efficiently inhibited phorbol myristate acetate-triggered neutrophil-mediated degradation of alpha-1-antitrypsin. This suggests that doxycycline can protect alpha-1-antitrypsin from collagenase and gelatinase in the presence of other proteases and biologically active molecules that are released by triggered neutrophils. The protection of a bodys alpha-1-antitrypsin shield from serpinolytic activity of collagenase and matrix metallproteinases can result in inhibition of serine proteases such as neutrophil elastase. Tetracyclines may thus protect matrix constituents from a wider spectrum of neutral proteases than previously recognized, not just from the matrix metalloproteinases collagenase and gelatinase.

Activation of periprosthetic connective tissue in aseptic loosening of total hip replacements.

In aseptic loosening of initially well inserted total hip prostheses, implant wear debris and cyclic mechanical loading lead to a foreign body type of chronic inflammatory reaction, then to osteolysis, and finally to loosening of the implant. In the present work the reactive and adaptive changes of the periprosthetic tissues and pseudojoint were characterized by analysis of the local cell proliferation. Immunohistochemical demonstration of proliferating cells was performed by application of affinity purified rabbit antihuman Ki-67 antibodies to periprosthetic tissues obtained from revision operations for loose total hip prostheses. The fibrous areas and, in particular, the cell rich, vascular areas of the interface tissue (between implant and bone) and the pseudocapsule around aseptically loosened implants contained higher numbers of proliferating cells than the tissues around well fixed implants. In addition, the pseudosynovial lining occasionally contained some Ki-67 positive proliferating cells. Somewhat surprisingly, proliferating vascular endothelial cells were relatively rare. These findings suggest that reactive (interface tissues) and adaptive (pseudojoint and capsule formed around the artificial joint) tissue changes in loosening total hip prostheses comprise proliferation of local fibroblastlike cells. It is concluded that periprosthetic tissues of the loosened total hip prosthesis represent activated mesenchymal tissue.