Tetracyclines (doxycycline and minocycline) augmented (one- to twofold) the PGE2 production in human osteoarthritis-affected cartilage (in the presence or absence of cytokines and endotoxin) in ex vivo conditions. Similarly, bovine chondrocytes stimulated with LPS showed (one- to fivefold) an increase in PGE2 accumulation in the presence of doxycycline. This effect was observed at drug concentrations that did not affect nitric oxide (NO) production. In murine macrophages (RAW 264.7) stimulated with LPS, tetracyclines inhibited NO release and increased PGE2 production. Tetracycline(s) and l-N-monomethylarginine (l-NMMA) (NO synthase inhibitor) showed an additive effect on inhibition of NO and PGE2 accumulation, thereby uncoupling the effects of tetracyclines on NO and PGE2 production. The enhancement of PGE2 production in RAW 264.7 cells by tetracyclines was accompanied by the accumulation of both cyclooxygenase (COX)-2 mRNA and cytosolic COX-2 protein. In contrast to tetracyclines, l-NMMA at low concentrations (≤100 μM) inhibited the spontaneous release of NO in osteoarthritis-affected explants and LPS-stimulated macrophages but had no significant effect on the PGE2 production. At higher concentrations, l-NMMA (500 μM) inhibited NO release but augmented PGE2 production. This study indicates a novel mechanism of action of tetracyclines to augment the expression of COX-2 and PGE2 production, an effect that is independent of endogenous concentration of NO.

Doxycycline and minocycline are members of the tetracycline family of broad spectrum antibiotics. During recent years, it has been established that tetracyclines, which are rapidly absorbed and have a prolonged half-life, exert biological effects independent of their antimicrobial activity 1 . Such effects include inhibition of MMPs3, 2, 3 , NOS expression 4 , tumor progression, bone resorption 5, 6 , angiogenesis 7 , and inflammation 8 . Among these, the role of MMPs, NO, and inflammation have been implicated in the pathophysiology of rheumatoid arthritis (RA) and osteoarthritis (OA). Yu et al. 9 have shown that prophylactic administration of doxycycline markedly reduced the severity of OA in dog models. In human subjects, the safety and efficacy of minocycline was assessed in the treatment of RA, where a double-blind, randomized, multicenter trial indicated that the drug was safe and effective for patients with mild and moderate disease 10 .

PGs are produced at elevated levels in inflamed tissues like rheumatoid synovium 11, 12 . PGE1 and PGE2 contribute to synovial inflammation by increasing local blood flow and potentiating the effects of mediators, such as bradykinin, that induce vasopermeability 13 . PGE2 has been shown to inhibit chondrocyte growth, to trigger osteoclastic bone resorption 14 , and to up-regulate IL-1β production 15 , suggesting that this molecule may also contribute to the pathophysiology of joint erosion in arthritis.

NO, another multifunctional mediator produced by and acting on various cells, participates in cartilage destruction in arthritis. The most compelling evidence for NO as a mediator of tissue injury has been in arthritis, based on studies conducted in animal models 16, 17 , human OA 18 , and RA 19 .

We have recently observed that human OA-affected cartilage (but not normal cartilage) spontaneously releases NO and PGE2 in ex vivo conditions in quantities sufficient to cause cartilage damage 18, 20 . This is primarily because human OA-affected cartilage shows up-regulation of OA-affected NOS (OA-NOS) and COX-2.

Due to the culpatory role of NO and PGE2 in arthritis, their destructive effect in joints, including activation of metalloproteases 1 , we evaluated the action of tetracyclines on the release of PGE2 from OA-affected human cartilage in ex vivo conditions 18 and COX-2 expression in LPS-stimulated murine macrophages (RAW 264.7).

In the present study we report that a) low concentrations of doxycycline and minocycline (5–10 μg/ml) augment the accumulation of PGE2 in OA-affected cartilage in ex vivo conditions independent from effects of intracellular NO; b) low concentrations of l-NMMA (≤100 μM) and tetracyclines (20–40 μg/ml) inhibit NO production equivalently, but at such concentrations only tetracyclines augment PGE2 production in LPS-stimulated murine macrophages; c) tetracycline (40 μg/ml) and l-NMMA (500 μM) additively inhibit NO production and augment PGE2 production in LPS-stimulated RAW 264.7 cells; and d) doxycycline and minocycline up-regulate COX-2 expression at the level of COX-2 mRNA and protein accumulation in murine macrophages stimulated with LPS. Taken together, the data indicate that tetracyclines exert independent and opposing effects on the regulation of NO and PGE2 production. These observations may have significance in the clinical application of tetracyclines for the treatment of arthritis.

Murine macrophage (RAW 264.7) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). An anti-murine iNOS and COX-2 Ab were obtained from Transduction Laboratories (Lexington, KY). OA-affected cartilage was obtained from OA patients who underwent knee replacement surgery and were free of steroidal/nonsteroidal antiinflammatory drugs for at least 2 wk before surgery. Doxycycline, minocycline, hydrocortisone, and LPS were obtained from Sigma (St. Louis, MO).

The bovine cartilage was obtained from young calves. Isolation of bovine chondrocytes from hoofs was conducted as described previously 21 . Briefly, normal bovine cartilage was washed in RPMI 1640 and cut into small pieces and digested with enzymes. Cartilage pieces were incubated with trypsin (0.25%) in RPMI 1640 (Life Technologies, Gaithersburg, MD) for 30 min at 37°C before they were washed and reincubated in hyaluronidase (0.2%) and collagenase (0.2%), dissolved in RPMI 1640 containing 5% FBS for 16 h at 37°C with continuous agitation (100 rpm). The cells were passed through 75-μ nylon mesh and washed twice with PBS to remove cell debris. The released cells were suspended in RPMI 1640 plus 10% FBS plus antibiotics and plated in a 24-well plate (Becton Dickinson, Lincoln Park, NJ) at a density of 5 × 105 cells/2.0 cm2. After 48 h, the medium was changed and the cells were preincubated in fresh media with various modulators before stimulating them with LPS (100 μg/ml) and analyzed for NO and PGE2 levels at 72 h poststimulation.

This assay was basically conducted as described previously 18 . Briefly, OA-affected cartilage was obtained from tibial plateau and femoral condyle of OA patients undergoing knee replacement surgery. OA-affected cartilage was cut into 3-mm discs; 4–6 discs (∼100–200 mg) were placed in organ cultures in 2 ml medium (F-12 with 0.1% human albumin) for 24–72 h in the incubator. The medium was analyzed for nitrite and PGE2 accumulation by modified Griess reaction 22 and radioimmunoassay, respectively 23 .

RAW 264.7 cells were induced with LPS (100 ng/ml) in the presence or absence of tetracyclines or hydrocortisone for 14–20 h. Following induction, the cells were pelleted at 4°C and resuspended in Tris buffer 10 mM (pH 7.4) containing 10 μg/ml each of chymostatin, antipain, leupeptin, and pepstatin, 1 mM DTT, and 1 mM PMSF. Cells were lysed in a Polytron PA 1200 homogenizer (Kinematica, Switzerland) after 3 cycles of rapid freeze-thawing. The lysate was centrifuged at 18,000 rpm for 60 min at 4°C in an Eppendorf centrifuge (Eppendorf, Madison, WI) and the soluble supernatant was used as cell-free extracts. The protein was measured by bicinchoninic acid (BCA) assay reagent (Pierce, Rockford, IL) using BSA as standard 24 .

Equal amounts of protein (25–50 μg) estimated by bicinchoninic acid (BCA) reagent (Pierce) were loaded onto SDS-PAGE gels and stained to verify the concentrations of various protein fractions by examining the intensities of the protein bands on the gel. The Western blot was probed with a specific anti-iNOS or anti-COX-2 mAb. The blots were developed using the enhanced chemiluminescence (ECL) Western blot system (Amersham, Arlington Heights, IL). Quantitation of the bands was performed using a densitometer (Molecular Dynamics, Sunnyville, CA).

Total RNA was isolated using TRI Reagent (MRC, Cincinnati, OH). Northern blot analysis was conducted as described by Church and Gilbert 25 . Briefly, 20 μg of RNA was subjected to electrophoresis in 1% agarose formaldehyde gel. The gel was then transferred by capillary action onto a nylon membrane (zeta Probe, Bio-Rad Laboratories, Melville, NY). The membrane was hybridized with [32P]dCTP-labeled COX-2 cDNA (a kind gift from Dr. Paul Worley, Johns Hopkins University) and GAPDH. After hybridization, the blot was exposed to Kodak x-ray film (Kodak, Rochester, NY) for 24–48 h with intensifying screens at −70°C. Quantitation of the intensity of the COX-2/GAPDH bands was performed using a densitometer (Molecular Dynamics).

Data are expressed as mean ± SD, and statistical analysis was performed using GraphPad Software (V1.14). The t test or nonparametric (Mann-Whitney or Wilcoxon test) was performed for experiments as described in the figure legends.

Human OA-affected cartilage exhibits up-regulated OA-NOS and COX-2 that spontaneously release NO and PGE2, respectively, in ex vivo conditions 18, 20 . In the present study, we examined whether doxycycline or minocycline could modulate human COX-2-mediated spontaneous release of PGE2 in these ex vivo conditions. Generally accepted pharmacologically relevant concentrations of tetracyclines were selected for this study, based on previous reports 2, 26, 27, 28 . OA-affected cartilage slices were incubated in endotoxin-free medium containing 0.1% human albumin with and without 5–80 μg/ml of doxycycline in ex vivo conditions for 72 h (Table I). The spontaneously released PGE2 and nitric oxide (monitored by estimating the stable end product, nitrite), were examined in the medium, as described previously 20 .

Relatively low concentrations of doxycycline (10–20 μg/ml) had no significant effect on the spontaneous accumulation of nitrite but induced a significant increase in the accumulation of PGE2. However, addition of 40 μg/ml of doxycycline significantly blocked nitrite accumulation in OA-affected cartilage in ex vivo conditions and also caused increased accumulation of PGE2, as observed with 10–20 μg/ml of doxycycline. Doxycycline at 80 μg/ml resulted in significant inhibition of nitrite accumulation, with no effect on the spontaneous release of PGE2. These experiments suggest that the augmentation of PGE2 in OA-affected cartilage by doxycycline lies within a narrow window, depending upon the concentration of doxycycline. Furthermore, this effect of doxycycline is independent of NO concentration since a) low concentrations of tetracycline (10–20 μg/ml) augment PGE2 production but have no significant effect on NO production and b) a high concentration of tetracycline (80 μg/ml), which inhibits NO production, has no significant effect on PGE2 production.

In view of the above observation, we tested the effect of tetracyclines on PGE2 and NO production in the presence of cytokines and endotoxin (Table II). OA-affected cartilage, when incubated with cytokines and endotoxin, showed a significant increase in the accumulation of NO and PGE2.

As seen in the previous experiments, cytokine- and endotoxin-induced PGE2 could also be augmented by low concentrations of doxycycline (5–10 μg/ml), which had no significant effect on the levels of NO in the medium. Similarly, 20- to 40-μg/ml concentrations of doxycycline had no significant effect on PGE2 production but inhibited NO production. Furthermore, higher concentrations of doxycycline (80 μg/ml) not only inhibited NO production significantly but also inhibited PGE2 production, when compared with the cytokine- and endotoxin-induced PGE2 production.

We also tested the effect of another tetracycline derivative, minocycline, on the release of PGE2 in the presence of cytokines and endotoxin in OA-affected cartilage. Minocycline (5 μg/ml) had no significant effect on NO production and (unlike doxycycline) had no effect on PGE2 production. Minocycline (10 μg/ml) caused significant augmentation of PGE2 production and (like doxycycline) had no significant effect on NO production. Increasing concentrations of minocycline (20 to 40 μg/ml) significantly inhibited NO production, with no effect on the PGE2 production. Minocycline (80 μg/ml) (like doxycycline in this experiment) inhibited both NO and PGE2 production. Thus, these two tetracyclines exert biphasic dose-dependent effects on PGE2 that are independent from their effects on NO.

To further examine the role of NO in the regulation of PGE2 in OA-affected cartilage, l-NMMA (a competitive substrate inhibitor of NOS) was added at different concentrations (25–500 μM) to OA-affected cartilage explants, and the levels of PGE2 and NO were estimated in the medium. l-NMMA (50–100 μM), which inhibited NO accumulation by ∼70% or less, had no significant effect on the levels of PGE2 production (Table III). However, a higher concentration of l-NMMA (500 μM), which inhibited NO production by ≥85%, caused a significant augmentation of PGE2 production, as previously described 48 . These experiments indicate that a threshold inhibition (≥85% of that induced by LPS) of NO production by l-NMMA needs to be achieved before the augmentation of PGE2 is be observed (Table III). This suggests that low concentration of NO is sufficient to inhibit COX-2-mediated PGE2 production. The nonspecific effects of NOS inhibitor l-NMMA can be ruled out since other NOS inhibitors, such as l-N5-(1-iminoethyl)-ornithine · HCL (l-NIO) and l-nitrocitrulline, also inhibit NO production and augment PGE2 accumulation in OA-affected cartilage (data not shown). The effects of l-NMMA are distinct from those observed with tetracyclines, since augmentation of PGE2 production can be achieved at concentrations of tetracycline that do not significantly inhibit the NO accumulation. These experiments suggest that tetracyclines exert differential and independent effects on PGE2 and NO production.

We tested the effect of doxycycline in bovine chondrocytes that, upon stimulation with LPS, show up-regulation of NO and PGE2 within 72 h 21 . Primary bovine chondrocytes stimulated with LPS in the presence of 1 to 2.5 μg/ml showed no effect on accumulation of NO, but there was a significant increase in the production of PGE2 (Table IV). Doxycycline at 5 μg/ml inhibited NO production and augmented PGE2 accumulation, whereas 10 μg/ml of doxycycline inhibited both NO and PGE2 production in LPS-stimulated bovine chondrocytes. These experiments show a biphasic effect of doxycycline on NO and PGE2 production, as observed in the OA-affected human cartilage cultures.

Based on the above studies, we sought to evaluate the mechanism of action of tetracyclines on COX-2 expression in a murine macrophage model for the following reasons: a) the biochemistry, enzymology, and molecular biology of iNOS and COX-2 is well characterized in these cells 29, 30 ; b) our inability, after several attempts, to precisely and reproducibly quantitate the expression of COX-2 directly from the OA-affected cartilage without disturbing the architecture of the cartilage, which plays a significant role in PGE2 production; and c) the regulation of NO and PGE2 in OA-affected cartilage and LPS-stimulated murine macrophages seems to be similar based on our and other previous observations 4, 20, 31, 32, 48 .

RAW 264.7 cells were activated with LPS (100 ng/ml) to induce iNOS and COX-2 33 with and without 20–80 μg/ml of doxycycline and minocycline. Table V shows concentration-dependent inhibition of nitrite accumulation in cells stimulated with LPS in the presence of 20–80 μg/ml of doxycycline or minocycline at 16 h of incubation as previously reported 4 . Doxycycline (20 μg/ml) inhibited LPS-stimulated NO production by murine macrophages, while having no effect on PGE2 production. In contrast, higher concentrations of doxycycline or minocycline (40 μg/ml) both augmented PGE2 and inhibited NO production, results comparable to those observed in OA explant tissue. The inhibition of NO production by l-NMMA to levels comparable to those achieved by the tetracyclines at 40 μg/ml had no effect on PGE2 production.

We further tested the effects of l-NMMA on NO and PGE2 production by murine macrophages. Inhibition of iNOS with competitive inhibitors of NOS (l-NMMA) at low concentrations (≤100 μM) significantly inhibited NO accumulation (by ∼60%) and had no effect on the levels of PGE2, whereas hydrocortisone blocked both PGE2 and NO production (Fig. 1). l-NMMA (500 μM) inhibited >90% nitrite accumulation and significantly augmented PGE2 production, consistent with various reports in the literature 20, 31, 34 . These data are consistent with those obtained using OA explants and again indicate that low concentrations of NO are sufficient to inhibit COX-2-mediated PGE2 production.

To further examine the effects of NOS inhibition, we monitored the NO and PGE2 levels in RAW 264.7 cells stimulated with LPS in the presence of l-NMMA (25–500 μM) plus tetracyclines (40 μg/ml) (Fig. 2). Doxycycline or minocycline alone (40 μg/ml) augmented PGE2 production with inhibition of NO production similar to that seen in Fig. 1. Less than 100 μM of l-NMMA blocked NO production, with no significant effects on PGE2 production, and ≥200 μM l-NMMA inhibited NO production and augmented PGE2 production. Combination of 25–100 μM of l-NMMA (which did not significantly augment PGE2 production) with 40 μg/ml of tetracyclines additively augmented PGE2 production. Similarly, the decrease in nitrite accumulation was also additive in the presence of l-NMMA and tetracyclines. It should be noted that 200–500 μM of l-NMMA, concentrations that augment PGE2 production, additively increased PGE2 production in the presence of tetracyclines. These experiments show that the mechanism of inhibition of NOS by l-NMMA 35 and tetracyclines 4, 36 are distinct, as is the mechanism by which l-NMMA and tetracyclines augment PGE2 production.

We have previously reported that tetracyclines inhibit iNOS protein expression, as examined by Western blot analysis, but had no significant effect on the total COX-2 protein expression 4 . We 48 and others 37 have shown that ∼90% of COX-2 is localized in the perinuclear membrane fraction. In our studies, using subcellular fractionation, we have also noted that ∼10% or a lesser amount of COX-2 protein is localized to a soluble cytosolic fraction, which we have designated as cytosolic COX-2 (Cy-COX-2) 48 . Since there was no effect of tetracyclines on the expression of nuclear COX-2 but there was an increase in the accumulation of PGE2, we examined the expression of Cy-COX-2 protein in murine macrophages stimulated with LPS in the presence and absence of various concentrations of doxycycline and minocycline. Cytosolic cell-free extracts were prepared as described in Materials and Methods. The cytosolic fraction was analyzed for 133-kDa iNOS and 72-kDa Cy-COX-2 by Western blotting, using specific Abs. Fig. 3 shows a dose-dependent inhibition of iNOS protein expression in the presence of both doxycycline (10–65% inhibition) and minocycline (13–82% inhibition), as previously observed 4 , and a simultaneous dose-dependent augmentation of Cy-COX-2 expression by doxycycline or minocycline. Hydrocortisone as expected inhibited both iNOS and COX-2 expression. There was no significant effect on the levels of β-actin synthesis in the same samples (data not shown). The experiments suggest that the effects of doxycycline or minocycline on iNOS and Cy-COX-2 are specific, but the mechanism of inhibiting iNOS and augmenting Cy-COX-2 seems to be distinct.

We have previously reported that tetracyclines suppress iNOS mRNA accumulation leading to inhibition of iNOS protein expression, specific activity, and accumulation of nitrite 4, 36 . In the current studies, Northern blot analysis of COX-2 mRNA (at 4 and 16 h) was also conducted to evaluate the effect of these agents on COX-2 expression. As shown in Fig. 4 a, doxycycline and minocycline augmented the accumulation of COX-2 mRNA in RAW 264.7 cells stimulated with LPS for 4 h. Similarly, cycloheximide, as previously described, also augmented the accumulation of COX-2 mRNA in the presence of LPS 38 . Doxycycline/minocycline and cycloheximide had no additive effect on the accumulation of COX-2 mRNA, thus indicating that de novo protein synthesis is not a prerequisite for COX-2 mRNA accumulation in the presence of LPS and tetracyclines. Hydrocortisone, as expected, inhibited the accumulation of COX-2 mRNA.

We examined the effect of tetracyclines on the accumulation of COX-2 mRNA 16 h poststimulation. These experiments also showed that 40 μg/ml of doxycycline and minocycline significantly augmented COX-2 mRNA accumulation by >150%, as compared with LPS-stimulated cells, whereas hydrocortisone (10 μM), as expected, inhibited COX-2 mRNA accumulation (Fig. 4 b). l-NMMA, at concentrations (75–150 μM) that inhibited NO production by 60–80%, had no significant effect on the accumulation of COX-2 mRNA. l-NMMA at 500 μM, which inhibits NO production by >90% and augments PGE2 production by one- to twofold, also had no significant effect on the accumulation of COX-2 mRNA 48 . These experiments indicate that (unlike l-NMMA) the action of doxycycline and minocycline on COX-2 is at the level of COX-2 mRNA accumulation. This may be due to increased transcription or posttranscriptional modification (or both) of the COX-2 mRNA.

Our studies indicate that tetracycline, doxycycline, and minocycline share the property to a) inhibit iNOS expression 4, 36 and b) augment soluble COX-2 and PGE2 production independent of the effects of intracellular NO. This conclusion is supported by the observation that tetracyclines stimulate COX-2 mRNA, cytosolic COX-2 protein expression, and PGE2 production at concentrations that have no significant effect on NO production. Furthermore, the additive effect of tetracyclines and l-NMMA (with respect to inhibition of NO and augmentation of PGE2) is consistent with independent mechanisms of actions of tetracyclines.

Previous studies have shown that inhibition of NO by competitive inhibitors of NOS augment PGE2 in a NO-dependent manner 31, 32, 34, 39 . One of the important observations was the requirement of a threshold level of inhibition of NO by l-NMMA before an augmentation of PGE2 could be observed in OA-affected cartilage and murine macrophages. These observations indicate that low concentrations of intracellular NO are sufficient to inhibit COX-2 mediated PGE2 production. Furthermore, the nonspecific effects of l-NMMA at high concentration could be ruled out because other NOS inhibitors, such as l-N6-(1-iminoethyl)-lysine · HCl (l-NIL), l-N5-(1-iminoethyl)-ornithine · HCL (l-NIO), and nitrocitrulline not only inhibit accumulation of nitrite in OA-affected cartilage and LPS stimulated murine macrophages but also augment significantly the accumulation of PGE2. The mechanism by which tetracyclines modulate iNOS or COX-2 seems to be at the level of their respective mRNA accumulation. Our recent experiments indicate that the decrease in iNOS mRNA by tetracyclines is primarily due to an increase in iNOS mRNA degradation and not transcription of iNOS gene 36 . The mechanism by which COX-2 mRNA is up-regulated needs to be elucidated. However, it can be speculated that one (or both) of the mechanisms described below may be operational: a) COX-2 is an immediate early gene that does not require de novo protein synthesis for transcription 40 . Therefore, like cycloheximide, tetracycline treatment may preferentially lead to accumulation of early de novo insensitive mRNA; or b) the accumulation of the COX-2 mRNA by tetracyclines at 16 h may be due to stabilization of the COX-2 mRNA.

Our data, taken together with previous findings by other investigators, indicate that tetracyclines exert pleiotropic functions independent of their antimicrobial activities, which include inhibition of MMPs 2 , NOS expression 4 , tumor progression, bone resorption 5, 6 , angiogenesis 7 , and inflammation 8 . We speculate that the pleiotropic properties of tetracyclines may be partially attributed to their ability to target other multifunctional signaling molecules, such as PGE2 and NO. PGE2, when overproduced in cells, is known to exert diverse effects on cellular functions, which include activation of MMPs 41 , induction or protection of apoptosis depending on the cell type 42, 43 , promotion of metastatic growth of tumor 44, 45 , inhibition of chondrocyte proliferation 46 , and up-regulation of IL-1 transcription and cAMP levels in various cell types 15, 47 . Our studies suggest that the nonmicrobial actions of tetracyclines are complex, underscoring their potential role as a double-edged sword that differentially regulates two pleiotropic mediators: NO and PGE2.

In view of the present use of doxycycline and minocycline in clinical trials for RA and scleroderma, the results suggest caution in the use of tetracyclines as antiinflammatory drugs in ongoing clinical trials.

We thank Dr. Paul Worley for generously providing COX-2 cDNA and Una Yearwood for preparation of the manuscript.

3

Abbreviations used in this paper: MMP, matrix metalloproteinase; NO, nitric oxide; NOS, NO synthase; iNOS, inducible NOS; ecNOS, endothelial NOS; OA-NOS, osteoarthritis-affected NOS; l-NMMA, l-N-monomethylarginine; COX, cyclooxygenase; Cy-COX-2, cytosolic COX-2; RA, rheumatoid arthritis; GAPDH, glyceraldehyde phosphate dehydrogenase.

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