Nick Amos

The role of synovial macrophages and macrophage-produced cytokines in driving aggrecanases, matrix metalloproteinases, and other destructive and inflammatory responses in osteoarthritis

There is an increasing body of evidence that synovitis plays a role in the progression of osteoarthritis and that overproduction of cytokines and growth factors from the inflamed synovium can influence the production of degradative enzymes and the destruction of cartilage. In this study, we investigate the role of synovial macrophages and their main proinflammatory cytokines, interleukin (IL)-1 and tumour necrosis factor-alpha (TNF-α), in driving osteoarthritis synovitis and influencing the production of other pro- and anti-inflammatory cytokines, production of matrix metalloproteinases, and expression of aggrecanases in the osteoarthritis synovium. We established a model of cultures of synovial cells from digested osteoarthritis synovium derived from patients undergoing knee or hip arthroplasties. By means of anti-CD14-conjugated magnetic beads, specific depletion of osteoarthritis synovial macrophages from these cultures could be achieved. The CD14+-depleted cultures no longer produced significant amounts of macrophage-derived cytokines like IL-1 and TNF-α. Interestingly, there was also significant downregulation of several cytokines, such as IL-6 and IL-8 (p < 0.001) and matrix metalloproteinases 1 and 3 (p < 0.01), produced chiefly by synovial fibroblasts. To investigate the mechanisms involved, we went on to use specific downregulation of IL-1 and/or TNF-α in these osteoarthritis cultures of synovial cells. The results indicated that neutralisation of both IL-1 and TNF-α was needed to achieve a degree of cytokine (IL-6, IL-8, and monocyte chemoattractant protein-1) and matrix metalloproteinase (1, 3, 9, and 13) inhibition, as assessed by enzyme-linked immunosorbent assay and by reverse transcription-polymerase chain reaction (RT-PCR), similar to that observed in CD14+-depleted cultures. Another interesting observation was that in these osteoarthritis cultures of synovial cells, IL-1β production was independent of TNF-α, in contrast to the situation in rheumatoid arthritis. Using RT-PCR, we also demonstrated that whereas the ADAMTS4 (a disintegrin and metalloprotease with thrombospondin motifs 4) aggrecanase was driven mainly by TNF-α, ADAMTS5 was not affected by neutralisation of IL-1 and/or TNF-α. These results suggest that, in the osteoarthritis synovium, both inflammatory and destructive responses are dependent largely on macrophages and that these effects are cytokine-driven through a combination of IL-1 and TNF-α.

Interferon-γ inhibits interleukin-1β-induced matrix metalloproteinase production by synovial fibroblasts and protects articular cartilage in early arthritis

IntroductionThe first few months after symptom onset represents a pathologically distinct phase in rheumatoid arthritis (RA). We used relevant experimental models to define the pathological role of interferon-γ (IFN-γ) during early inflammatory arthritis.MethodsWe studied IFN-γs capacity to modulate interleukin-1β (IL-1β) induced degenerative responses using RA fibroblast-like synoviocytes (FLS), a bovine articular cartilage explant (BACE)/RA-FLS co-culture model and an experimental inflammatory arthritis model (murine antigen-induced arthritis (AIA)).ResultsIFN-γ modulated IL-1β driven matrix metalloproteinases (MMP) synthesis resulting in the down-regulation of MMP-1 and MMP-3 production in vitro. IFN-γ did not affect IL-1β induced tissue inhibitor of metalloproteinase-1 (TIMP-1) production by RA FLS but skewed the MMP/TIMP-1 balance sufficiently to attenuate glycosaminoglycan-depletion in our BACE model. IFN-γ reduced IL-1β expression in the arthritic joint and prevented cartilage degeneration on Day 3 of AIA.ConclusionsEarly therapeutic intervention with IFN-γ may be critical to orchestrate tissue-protective responses during inflammatory arthritis.

The suppression of rat collagen-induced arthritis and inhibition of macrophage derived mediator release by liposomal methotrexate formulations

Abstract.Objective and Design: This study was designed to determine whether liposomes are suitable vehicles for the delivery of methotrexate (MTX-γ-DMPE) for arthritis therapy.¶Material or Subjects: Liposomal formulations containing either egg lecithin (EPC), cholesterol (CHOL) and phosphatidic acid (PA) (MTX-EPC) or distearoylphosphatidylcholine (DSPC), CHOL and distearoylphosphatidylethanolamine conjugated to polyethyleneglycol (PEG) (MTX-PEG) were employed. Rat peritoneal macrophages (rPMφ) were used to test the mechanism of action of these liposomes in vitro, whilst, the rat collagen-induced arthritis (CIA) model was used to evaluate the in vivo efficacy of MTX-EPC and MTX-PEG.¶Treatment: In vitro, rPMφ were incubated with liposomal MTX concentrations ranging from 0 to 15 μg/well. In vivo, rats were given 4 daily intravenous injections of liposomal MTX (2.5 mg/Kg).¶Methods: IL-1β and prostaglandin-E2 (PGE2) release from rPMφ were quantified by immunoradiometric assay. Arthritis progression, in vivo, was measured by serial clinical score and hind paw diameter measurements.¶Results: MTX-EPC and MTX-PEG respectively (15 μg of MTX and 0.15 mg of lipid) were powerful inhibitors of both IL-1β (77 ± 2.3%; 79 ± 4.0%) and PGE2 (75.5 ± 4.9%; 68.5 ± 2.3%) release (mean ± SEM % inhibition) from lipopolysaccaride stimulated rPMφ. In vivo, only MTX-EPC exerted an anti-inflammatory effect, clinical score (p < 0.001) and paw diameter (p < 0.001) measurements being significantly lower than in control rats, after 2 days treatment.¶Conclusions: MTX-EPC and MTX-PEG are potent inhibitors of pro-inflammatory mediators in vitro, but liposomes with long circulation times do not appear to have therapeutic potential for treating arthritis in vivo.

Differential effects of methotrexate and liposomally conjugated methotrexate in rat adjuvant-induced arthritis

In this study we evaluated the comparative efficacy of free and liposomally conjugated methotrexate on both disease indtiction and suppression of acute inflammation in rat adjuvant‐induced arthritis. Rats were given either empty liposomes (E‐LIPO), free methotrexate (MTX) or the liposomally conjugated methotrexate (MTX‐LIPO) at a dose of 100/μg/day for 7 consecutive days by the intravenous route. When MTX treatment was initiated on the day of arthritis induction the drug suppressed bul did not abolish the development of joint inflammation. Free MTX had no significant anti‐inliammatory effect upon an established arthritis when dosing was commenced on day 11 post‐adjuvant induction. Conversely, MTX‐LIPO did not affect the progression of the arthritis when dosing was started on day 0, but exerted a significant anti‐inflammatory effect on an established arthritis. MTX‐LIPO treatment was significantly less haematotoxic than free MTX.

Interleukin‐1β (IL‐1β) inhibition: a possible mechanism for the anti‐inflammatory potency of liposomally conjugated methotrexate formulations in arthritis

Liposomes with conventional and long‐circulation times were employed as carriers for the methotrexate derivative MTX‐γ‐DMPE (MTX‐EPC and MTX‐PEG respectively), their mechanism of action was investigated in vitro and in vivo and their therapeutic efficacy assessed using the rat collagen‐induced arthritis (CIA) model. At non‐toxic dose, both MTX‐EPC and MTX‐PEG inhibited the lipopolysaccharide (LPS) induced release of IL‐1β from activated rat peritoneal macrophages (rPMΦ) in a dose and time dependent manner. Free methotrexate (MTX) was not active in this respect. After a single intravenous injection (i.v.), and at equivalent doses, both free MTX (500 μg) and MTX‐EPC inhibited the LPS induced rise in plasma IL‐1β levels observed in MTX‐PEG and saline treated rats. When used to treat established CIA, MTX‐EPC resulted in significantly lower clinical score (CS) (1.0±0.42 (P<0.001)) and hind paw diameter (HPD) (6.5±0.34 mm (P<0.001)) measurements than controls (3.0±0.26; 7.33±0.41 mm), after only two i.v. doses, and remained significantly lower for the entire experimental period. By day 24 both CS (2±0.61 (P<0.001)) and HPD (6.97±0.25 mm (P<0.002)) measurements had also become significantly lower in MTX‐PEG treated rats than in saline treated controls (3.62±0.17, 7.92±0.38 mm) and remained lower until day 30. Joint inflammation in MTX treated rats was completely ameliorated by day 20 but the health and well being of the animals was compromised and the experiment terminated at this time‐point. Our results clearly demonstrate that both MTX‐EPC and MTX‐PEG liposomes have potential for development into therapeutic modalities for the treatment of inflammatory joint disease in man.

Effect of Three Lipophilic Methotrexate Derivatives Upon Mediator Release by Lipopolysaccharide-stimulated Rat Peritoneal Macrophages

Abstract— The ability of methotrexate and three lipophilic derivatives (methotrexate‐γ‐dimyristoylphos‐phatidylethanolamine (MγD), methotrexate‐α‐dimyristoylphosphatidylethanolamine (MαD) and metho‐trexate‐α‐γ‐di‐dimyristoylphosphatidylethanolamine (MαγD) to modulate mediator release by lipopolysaccharide‐stimulated rat peritoneal macrophages was investigated. At nontoxic concentrations, approximately 10 nmol/105 cells, MαD and MγD produced 11·06±1·0 and 75·6 ± 5·2%, respectively, inhibition of tumour necrosis factor (TNF) release (mean ± s.e.m., n = 4). At this same dose MαγD resulted in 68·8 ± 2·1 % inhibition of TNF but cellular ATP levels were reduced by 80%. The inhibitory activity of all three derivatives was dose‐dependent. Non‐derivatized methotrexate at a concentration of 25 nmol/105 cells had no inhibitory effect upon TNF release (14·7 ±0·8%, n = 3). Determination of prostaglandin E2 (PGE2) levels in the same samples demonstrated that all three conjugates were powerful inhibitors of prostaglandin release. At a quarter of the conjugate concentrations described above the monoamides MαD (31 nmol/105 cells) and MγD (2·5 nmol/105 cells) maintained their effects on PGE2 production with 73±2·3 and 71±2·0% (n=4) inhibition, respectively. At this lower concentration, however, the diamide MαγD (3·1 nmol/105 cells) was less effective in reducing the amount of PGE2 released from the macrophages (29 ± 18%, n = 4). Maximal PGE2 inhibition by each of the conjugates was attained at approximately 5 nmol/105 cells. Unconjugated methotrexate (range of 2·5–20 nmol/105 cells) did not inhibit the release of PGE2 from lipopolysaccharide‐stimulated macrophages.

Contractile, but not endothelial, dysfunction in early inflammatory arthritis: a possible role for matrix metalloproteinase‐9

BACKGROUND AND PURPOSE Excess morbidity/mortality in rheumatoid arthritis (RA) is associated with increased incidence of cardiovascular disease. In this ‘proof‐of‐concept’ study, vascular function was characterized in the murine collagen‐induced arthritis (mCIA) model, the benchmark choice for evaluation of the pathological processes and assessment of new therapies.