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,
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Department of Rheumatology, Hospital for Joint Diseases, New York, NY 10003; and Departments of
Pathology and
Medicine, New York University Medical Center, New York, NY 10016
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-1ß 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
Procurement of bovine chondrocytes
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 x 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.
Assay of OA-NOS in organ cultures
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 .
Preparation of cell-free extracts
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 .
Western blot analysis
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).
Northern blot analysis
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).
Statistical analysis
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 .
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Effect of doxycycline and minocycline on PGE2 production in OA-affected cartilage in the presence of cytokines and endotoxin
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.
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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.
Effect of NOS inhibitor (L-NMMA) on PGE2 production in OA-affected cartilage
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.
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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.
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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.
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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.
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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.
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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 ß-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.
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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. 4a,
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.
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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. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ashok R. Amin, Department of Rheumatology, Room 1600, Hospital for Joint Diseases, 301 East 27th Street, New York, NY 10003. E-mail address:
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.
Received for publication July 20, 1998. Accepted for publication December 9, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß in murine peritoneal macrophages. J. Immunol. 145:3333.[Abstract]
B and JNK activation. FEBS Lett. 418:135.[Medline]
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