Doxycycline Modulates Nitric Oxide Production in Murine Lung Epithelial Cells Jeffrey C

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Doxycycline Modulates Nitric Oxide Production in Murine Lung Epithelial Cells Jeffrey C. Hoyt, Janelle Ballering, Hiroki Numanami, John M. Hayden and Richard A. Robbins This information is current as of September 24, 2021. J Immunol 2006; 176:567-572; ; doi: 10.4049/jimmunol.176.1.567 http://www.jimmunol.org/content/176/1/567 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Doxycycline Modulates Nitric Oxide Production in Murine Lung Epithelial Cells1

Jeffrey C. Hoyt,2*† Janelle Ballering,* Hiroki Numanami,* John M. Hayden,* and Richard A. Robbins*†

Many effective therapeutic agents exhibit effects that are different from their intended primary mode of action. Antibiotics such as doxycycline and erythromycin A are no exception. They also display anti-inﬂammatory activity. Using LA4 murine lung alveolar epithelial cells, effects of doxycycline and erythromycin A on inducible NO synthase (iNOS) NO production as well as iNOS protein and mRNA production were investigated. Induction of iNOS was accomplished by treatment with cytomix (TNF-␣, IL-1␤, and IFN-␥ each at 5 ng/ml). Production of NO or iNOS was not detected in controls with or without erythromycin A. In the presence of cytomix, erythromycin A did not decrease NO, nitrite, iNOS protein, or mRNA production. In contrast, doxy-

cycline caused a dose-dependent decrease in NO, nitrite, iNOS protein, and mRNA production in cytomix-treated cells. Doxycy- Downloaded from cline at 30 ␮g/ml produced a 90% decrease in nitrite and NO production and a 52% decrease in iNOS mRNA transcription compared with cytomix treatment alone. Actinomycin D treatment suggests that doxycycline decreases stability of iNOS mRNA in cytomix-treated cells. To determine a mechanism for the decrease in iNOS expression, NF-␬B and AP-1 transcription regulatory systems and p38 MAPK were examined. Doxycycline treatment gave no statistically signiﬁcant change in NF-␬B activation but did decrease p38 MAPK protein in cytomix-treated cells by 50%, suggesting that p38 MAPK may be responsible for stabilization of iNOS mRNA. These results demonstrate that doxycycline decreases NO production from iNOS by destabilization of iNOS mRNA http://www.jimmunol.org/ via decreased expression of p38 MAPK. The Journal of Immunology, 2006, 176: 567–572.

he common obstructive airway disorders such as asthma, NO produced by inducible NO synthase (iNOS)3 and that this inhi- chronic bronchitis, diffuse panbronchiolitis, and bronchi- bition is due, at least in part, to decreased stabilization and expression T ectasis are all invariably associated with chronic airway of iNOS mRNA and its translation to form active iNOS enzyme. The inflammation (1–3). Despite treatment with anti-inflammatory effect of doxycycline on the NF-␬B transcriptional regulatory system therapies, these disorders are often progressive, suggesting that the was examined as well as the effects of p38 MAPK on iNOS mRNA current anti-inflammatory therapies are inadequate. stability. The results confirm this hypothesis in vitro and suggest that by guest on September 24, 2021 Macrolides and tetracyclines are bacterial and fungal secondary doxycycline treatment may be an effective airway anti-inflammatory metabolites that possess antibacterial action but also exhibit dis- therapy in diseases such as asthma, chronic bronchitis, diffuse pan- tinct and separate anti-inflammatory actions. These antibacterial bronchiolitis, and bronchiectasis. compounds have been known to reduce inflammation associated with a variety of inflammatory diseases since the 1970s (4, 5). Materials and Methods Among these compounds are erythromycin A and many of its de- Culture of LA4 cells rivatives such as azithromycin, clarithromycin, and roxithromycin, The clonal murine lung alveolar epithelial cell line LA4 was purchased as well as tetracycline and several related compounds such as from the American Type Culture Collection (7). LA4 cells were grown in doxycycline. These compounds have exhibited anti-inflammatory 25-cm2 tissue culture flasks (Corning Costar) in Ham’s F-12 containing activity against a variety of inflammatory airway disorders (5). 10% FCS, 2 mM L-glutamine, and penicillin-streptomycin (100 U/ml, 100 However, concern has been raised about development of microbial mg/ml) until confluent. After washing with serum-free medium doxycycline, dissolved in distilled water, or erythromycin A, dissolved in DMSO, antibiotic resistance limiting macrolide usefulness and suggesting was added to confluent LA4 cultures for 3 h, and the cells were cultured for a need for alternative anti-inflammatory therapies. 18 h in serum-free Ham’s F-12 with or without cytomix (CM). CM is a One marker of inflammation has been measurement of NO, a combination of recombinant human TNF-␣, human IL-1␤, and murine ␥ gas produced by lung epithelial cells, which has been shown to be IFN- , each at 5 ng/ml concentration (all cytokines from R&D Systems), that is used to stimulate the expression of iNOS (8, 9). Concentrations of elevated in several inflammatory airway conditions (6). We hy- test chemicals ranged from 0.3 to 30 ␮g/ml for doxycycline and 0.3 to 60 pothesized that doxycycline possesses potent anti-inflammatory ␮g/ml for erythromycin A based on reports of clinically relevant serum and properties in murine lung epithelial cells, leading to a reduction of tissue levels (10, 11). Serum levels of doxycycline are reported to be up to 79 ␮g/ml (depending on the individual) (10) and for erythromycin in the range of 1.9 to 6.5 ␮g/ml (11). Ampicillin (50 ␮g/ml) and sparsomycin (0.5 ␮ *Research Service, Carl T. Hayden Veterans Affairs Medical Center, Phoenix, AZ g/ml) were used as control compounds. All control cultures contained the 85012; and †Respiratory Sciences, University of Arizona, Tucson, AZ 85721 appropriate amount of carrier solvent alone. Received for publication May 17, 2005. Accepted for publication October 13, 2005. Cell viability assays The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Cell viability was determined by assay for release of lactate dehydrogenase with 18 U.S.C. Section 1734 solely to indicate this fact. (LDH) into cell culture supernatants using a commercially available LDH assay kit (Sigma-Aldrich). 1 This research is funded by a Veterans Affairs Merit Review Grant. 2 Address correspondence and reprint requests to Dr. Jeffrey C. Hoyt, Carl T. Hayden Veterans Affairs Medical Center, Research (RS/151), Building 27, 650 East Indian 3 Abbreviations used in this paper: iNOS, inducible or type II nitric oxide synthase; School Road, Phoenix, AZ 85012, E-mail address: [email protected] CM, cytomix; LDH, lactic dehydrogenase.

Determination of NO concentration in culture flask head space Table II. Effect on NO productiona and nitrite in culture flask supernatants After 18 h of stimulation with CM, culture flasks were tightly sealed and Treatment Doxycycline (SD) Erythromycin A (SD) o incubated at 37 C. After 3 h head space gas was withdrawn using a needle Control 18 ppb NO (1) 21 ppb NO (5) and syringe. NO concentrations were measured using a NO chemilumi- Ͻ CM alone 2680 (237) 1980 (76) nescence analyzer (Sievers) with a detection sensitivity of 1 parts per 30 ␮g/ml alone 10 (6) 8 (2) billion (ppb). The analyzer was calibrated daily according to the manufac- 30 ␮g/ml ϩ CM 8 (2) 1800 (172) turer’s directions using a gas of known NO concentration. Nitrite concen- 10 ␮g/ml alone 8 (0.8) — — trations of the cell culture supernatant were determined spectrophotometri- 10 ␮g/ml ϩ CM 1094 (277) — — cally using the Griess reaction (12). 3 ␮g/ml alone 7 (1) 8 (2) ␮ ϩ Protein determination 3 g/ml CM 1200 (78) 1900 (643) 0.3 ␮g/ml alone 9 (2) 5 (1) Protein concentrations were determined spectrophotometrically using the 0.3 ␮g/ml ϩ CM 2400 (144) 1870 (333) Bradford assay (13). a A549 cells were stimulated in the presence of CM with and without doxycycline or erythromycin A. NO was measured as described in Materials and Methods. Data Cell lysis, SDS-PAGE, and Western blotting shown represent the mean Ϯ SD of at least two sets of data assayed in triplicate. Cell extracts were prepared for Western blotting using a modification of previously described methods (14). Cells were lysed using 10 mM Tris- HCl (pH 7.4) containing 1% SDS and 1 mM EDTA and a premade mixture transcription. RNA was isolated from cultures at various times. RT-PCR of protease inhibitors (Sigma-Aldrich). Cell lysates were clarified by cen- was used to determine the amount of iNOS mRNA present at each time. trifugation before electrophoresis. SDS-PAGE was performed using a 4% ␬ Downloaded from T acrylamide stacking gel and a 7.5% T acrylamide separating gel for Effect of doxycycline on NF- B iNOS or a 10% T separating gel of p38 MAPK (15). After electrophoresis The effect of doxycycline on the NF-␬B transcription regulatory system was complete, the separated proteins were transferred to a polyvinylidene was determined using a commercially available ELISA kit (Active Motif). difluoride membrane for further analysis (16). Transfer was performed us- The primary Ab used in this kit detects an epitope of p50/p65 that is ing a Tris-HCl (25 mM), glycine (192 mM), and methanol (20% v/v) buffer ␬ o accessible only when NF- B is activated and bound to its consensus DNA- system. The transfer was performed at 4 C for 16 h at 30 V and then for binding sequence. Cell extracts were prepared according to manufacturer’s 30 min at 60 V. Abs for iNOS (Transduction Laboratories) or p38 MAPK instructions. LA4 cells were treated with 30 ␮g/ml doxycycline for 3 h http://www.jimmunol.org/ (Santa Cruz Biotechnology) were used to immunochemically detect iNOS followed by addition of CM for 1 h before preparation of cell extracts for or p38 MAPK protein on the transfer membranes using goat anti-rabbit IgG NF-␬B analysis. For each analysis each well contained 5 ␮g protein. alkaline phosphatase as the secondary Ab-enzyme conjugate and NBT/5- bromo-4-chloro-3-indolyl phosphate phosphatase substrate tablets (Sigma- Statistical analysis Aldrich) in 0.1 M Tris-HCl (pH 9.1) as the substrate system. Five micrograms of Ab were used at each step during the development procedures. Data were analyzed by one-way ANOVA with Fisher’s protected least The transfer membranes were blocked with BLOTTO (20 mM Tris base, significant difference (Fisher’s PLSD). In all cases, a p Ͻ 0.05 was con- Ϯ 180 mM NaCl, 4% nonfat dry milk, 0.02% NaN3) to minimize nonspecific sidered to be significant. The data are expressed as mean SD. binding. Results

RNA isolation and RT-PCR by guest on September 24, 2021 LA4 cell culture supernatants from all treatment types were as- iNOS mRNA was analyzed by RT-PCR. Total cellular RNA was extracted sayed for LDH release as a measure of cell viability. There were from adherent cells using a modification of the methods of Chomczynski no significant differences in the LDH activity in the culture super- and Sacchi (17). The RNA was reverse transcribed using a commercially available RT-PCR kit (Promega) using the sense and antisense primers natants from all treatment types (data not shown). (Table I) added at 0.2 ␮M final concentration. Using a PerkinElmer model Doxycycline, but not erythromycin A, decreased NO production 480 thermal cycler, reverse transcription was performed on total cellular in CM (5 ng/ml each of IL-1␤, TNF-␣, and IFN-␥)-stimulated RNA at 48oC for 45 min. PCR for iNOS was performed in the same sample LA4 cells (Table II). Doxycycline markedly inhibited NO produc- tubes at 94°C for 2 min followed by 28 cycles consisting of 94°C for 45 s, tion at the highest concentration used. NO production was also 60°C for 45 s, 72°C for 2 min, followed by 72°C for an additional 7 min. ␤-Actin (primers shown in Table I) was used as a “housekeeping gene” and inhibited at the lower doxycycline concentrations, in a concentra- RT-PCR was performed in a similar manner as for iNOS except for an tion-dependent manner, but the decrease was not as dramatic. The annealing temperature of 50°C. The DNA fragments were separated by differences in NO production between CM-treated and CM plus 3 electrophoresis on a 2% agarose gel in TAE buffer (40 mM Tris base, 20 ␮g/ml, 10 ␮g/ml, or 30 ␮g/ml doxycycline was statistically sig- mM acetic acid, 2 mM Na2 EDTA, pH 8.5) and then analyzed and quan- Ͻ ␮ titated by densitometry. Increasing PCR cycles gave increasing amounts of nificant ( p 0.05). The difference with CM plus 0.3 g/ml doxy- DNA through a fairly broad range with linearity obtained from about cy- cycline was not statistically significant ( p Ͼ 0.05). In contrast, cles 24–32, beginning with 1 ␮g of DNA (r ϭ 0.99 for iNOS and ␤-actin).

Stability of iNOS mRNA Table III. Effect of doxycycline and erythromycin on nitrite productiona LA4 cells were treated with CM or CM plus doxycycline for 18 h as described above and then treated with actinomycin D (10 ␮g/ml) to stop Treatment Doxycycline (SD) Erythromycin A (SD) Control 0.2 ␮M nitrite (0.2) 0.1 ␮M nitrite (0.3) CM alone 5.9 (0.7) 8.9 (1.7) Table I. Oligonucleotides used for PCR ampliﬁcationa 30 ␮g/ml alone 0.3 (0.2) 0.1 (0.2) 30 ␮g/ml ϩ CM 0.4 (0.1) 9 (0.9) Primers 10 ␮g/ml alone 0.4 (0.1) — — 10 ␮g/ml ϩ CM 5.5 (0.7) — — iNOS 3 ␮g/ml alone 0.2 (0.4) 0.1 (0.07) iNOS sense 5Ј-CCCTTCCGAAGTTTCTGGCAGCAGC-3Ј 3 ␮g/ml ϩ CM 6.2 (1.6) 8.5 (2.4) iNOS antisense 5Ј-GGCTGTCAGAGCCTCGTGGCTTTGG-3Ј 0.3 ␮g/ml alone 1.4 (0.2) 0.1 (0.4) ␤-actin 0.3 ␮g/ml ϩ CM 5.8 (0.2) 8.8 (0.9) ␤-actin sense 5Ј-TGACCCAGATCATGTTTGAG-3Ј ␤-actin antisense 5Ј-TCATGAGGTAGTCAGTCAGG-3Ј a A549 cells were stimulated in the presence of CM with and without doxycycline or erythromycin A. Nitrite was measured as described in Materials and Methods. a Primer pairs used for RT-PCR experiments. Experimental procedures were per- section. Data shown represent the mean Ϯ SD of at least two sets of data assayed in formed as described in Materials and Methods. triplicate. The Journal of Immunology 569

FIGURE 3. Effect of doxycycline on iNOS mRNA. Lane 1, bp markers; FIGURE 1. Effect of doxycycline on iNOS protein. Lane 1, Molecular lanes 2–4, control (no treatment); lanes 5–7, CM alone; lanes 8–10, 30 mass markers (kDa); lane 2, control (no treatment); lane 3, CM alone; lane ␮g/ml doxycycline; lanes 11 and 12, 30 ␮g/ml doxycycline plus CM; lane 4, 30 ␮g/ml doxycycline; lane 5, 30 ␮g/ml doxycycline plus CM; lane 6, 13, bp markers; lanes 14–16, 3 ␮g/ml doxycycline; lanes 17–19, 3 ␮g/ml 10 ␮g/ml doxycycline; lane 7, 10 ␮g/ml doxycycline plus CM; lane 8, 3 doxycycline plus CM; lanes 20–22, 0.3 ␮g/ml doxycycline; and lanes ␮g/ml doxycycline; and lane 9, 3 ␮g/ml doxycycline plus CM. 23–25, 0.3 ␮g/ml doxycycline plus CM. erythromycin A exhibited little inhibition of NO production at all concentrations tested. Assays for NO and nitrite were performed production in CM-stimulated cells compared with CM alone was on at least two sets of samples in triplicate. observed (Fig. 3). Lower concentrations of doxycycline did not In similar experiments, cell culture supernatants were examined significantly alter iNOS mRNA production. Although there was for nitrite content (Table III). In the absence of superoxide, LA4 some variability in iNOS mRNA detected by RT-PCR, CM-in- Downloaded from cells produce mostly nitrite (9, 18, 19). Furthermore, nitrite and duced iNOS mRNA was clearly lowered by doxycycline at 30 nitrate concentrations correlate in LA4 cells even in the presence ␮g/ml (Fig. 3, p Ͻ 0.05 when compared by scanning of superoxide (9, 18, 19). Doxycycline, at a concentration of 30 densitometry). ␮g/ml, decreased production of nitrite in CM-stimulated LA4 cells Erythromycin A did not reduce production of iNOS mRNA in compared with cells only treated with CM ( p Ͻ 0.01). Lower CM-stimulated cells (Fig. 4). These findings are consistent with concentrations of doxycycline did not produce statistically signif- the insignificant change in NO and nitrite production seen after http://www.jimmunol.org/ icant differences. In contrast, erythromycin A, at all concentrations erythromycin treatment. Expression of the ␤-actin gene and pro- tested, did not alter nitrite production. duction of its mRNA was not altered during any of the treatments The effect of doxycycline and erythromycin A on production of discussed (data not shown). Erythromycin A at a concentration of iNOS protein was examined (Figs. 1 and 2). Analysis was per- 60 ␮g/ml did not alter iNOS mRNA expression or production of formed by Western blot transfer of SDS electrophoresis gels to nitrite (data not shown). polyvinylidene difluoride membranes followed by immunochemi- In addition, ampicillin (50 ␮g/ml) and sparsomycin (0.5 ␮g/ml), cal development with anti-iNOS Ab. At the highest concentration both used as control compounds, did not alter iNOS mRNA ex- of doxycycline (30 ␮g/ml), iNOS protein was not detected even pression or the production of nitrite (data not shown). Concentra- by guest on September 24, 2021 though the CM control and samples from lower doxycycline con- tions of sparsomycin greater than 0.5 ␮g/ml were cytotoxic. centrations do contain iNOS protein (Fig. 1). At a concentration of Doxycycline treatment does not result in a statistically signifi- 10 ␮g/ml doxycycline, iNOS protein is present in ϳ50% of the cant decrease in NF-␬B in CM-stimulated cells with respect to CM amount in the CM control. At 3 mg/ml doxycycline, the iNOS treatment alone (Fig. 5). In a companion experiment, treatment of protein is roughly the same as in the CM control. No further de- LA4 cells with erythromycin A results in a statistically significant crease in iNOS protein is seen when the doxycycline concentration decrease in NF-␬B after CM treatment (data not shown), but there is decreased to 0.3 ␮g/ml (data not shown). In contrast, erythro- is no effect on iNOS mRNA transcription. mycin A at the concentrations tested did not decrease iNOS protein Expression of p38 MAPK protein was affected by doxycycline production (Fig. 2). in CM-treated LA4 cells (Fig. 6). The presence of doxycycline in The effect of doxycycline and erythromycin A on iNOS mRNA CM-treated cells decreased the amount of p38 MAPK protein by expression was evaluated by use of semiquantitative RT-PCR 50% of that produced relative to either treatment alone. (Figs. 3 and 4). ␤-Actin was used as a control. Expression of the The presence of 10 ␮g/ml actinomycin D in CM-treated LA4 ␤-actin gene and production of its mRNA was not altered during cell cultures results in a decrease in iNOS mRNA decay in the any of the treatments discussed (data not shown). At a concentra- presence of doxycycline over a 6-h time course (Fig. 7). The ab- tion of 30 ␮g/ml doxycycline, a 50% decrease of iNOS mRNA sence of doxycycline results in no significant decrease in iNOS mRNA production.

FIGURE 4. Effect of erythromycin A on iNOS mRNA. Lane 1, bp markers; lanes 2 and 3, control (no treatment); lanes 4 and 5, CM alone; lanes 6 and 7, 30 ␮g/ml erythromycin A; lanes 8 and 9, 30 ␮g/ml eryth- FIGURE 2. Effect of erythromycin A on iNOS protein. Lane 1, Molec- romycin A plus CM; lane 10, bp markers; lanes 11 and 12, 3 ␮g/ml eryth- ular mass markers (kDa); lanes 2 and 3, control (no treatment); lanes 4 and romycin A; lanes 13 and 14, 3 ␮g/ml erythromycin A plus CM; lanes 15 5, CM alone; lanes 6 and 7, 30 ␮g/ml erythromycin A; and lanes 8 and 9, and 16, 0.3 ␮g/ml erythromycin A; and lanes 17 and 18, 0.3 ␮g/ml eryth- 30 ␮g/ml erythromycin A plus CM. romycin A plus CM. 570 DOXYCYCLINE MODULATES NO PRODUCTION

FIGURE 5. Effect of doxycycline on NF-␬B activation in CM-stimulated LA4 cells. Left column, CM treatment alone; right column, CM plus 30 ␮g/ml doxycycline. Total cell extracts, 5 ␮g of protein per assay well, FIGURE 7. ␮ were used in each case. Assay for NK-␬B was performed with at least two Stability of iNOS mRNA in the presence of 10 g/ml ac- samples each assayed in triplicate. SDs are shown. tinomycin D.

Discussion iNOS (28). The p38 MAPK pathway influences stability of various Downloaded from Doxycycline effects NO production catalyzed by iNOS in CM- mRNAs by promoting translation via AU-rich elements in the 3Ј- stimulated LA4 murine lung epithelial cells. Both production of Ϫ untranslated region of these molecules (24, 29). Doxycycline de- NO itself and its oxidation end product, nitrite (NO ) are sig- 2 creases the amount of p38 MAPK protein produced in CM-treated nificantly decreased by doxycycline (Tables II and III). In ad- cells (Fig. 6). In addition, doxycycline appears to decrease the dition, doxycycline decreases the amount of iNOS mRNA (Fig. stability of iNOS mRNA in CM-treated cells (Fig. 7). These data 3) and its translation to form iNOS protein (Fig. 1). Erythro- mycin A had no effect of either NO or nitrite production (Tables suggest that the doxycycline-induced decrease in p38 MAPK pro- http://www.jimmunol.org/ II and III) or on expression of iNOS mRNA (Fig. 4) or its tein may be responsible for the observed decrease in iNOS mRNA translation to form iNOS protein (Fig. 2). The unrelated anti- stability. biotics, ampicillin and sparsomycin used as controls, did not Regulation of inflammatory gene transcription represents a com- effect NO production or expression of iNOS mRNA. This sug- plex interplay between transcription factors and other factors me- ␬ gests that the observed effects are specifically related to doxy- diating stability of mRNA. Previous studies suggest that NF- B cycline exposure and are not general, nonspecific effects of ex- plays an important role in regulating iNOS transcription (30). posure to antibiotic compounds. However, our results suggest that NF-␬B plays at best a minor role ␬ in the doxycycline-modulated decrease in iNOS-catalyzed NO

Analysis of the NF- B transcription regulatory signaling path- by guest on September 24, 2021 way suggests that it may not play a significant role in attenuating production in murine LA4 cells. It seems likely that the decrease expression of iNOS mRNA after exposure to doxycycline (Fig. 5). in iNOS mRNA and protein that is observed upon doxycycline The primary Ab in the NF-␬B ELISA kit (Active Motif) that was treatment is due to decreased stability of iNOS mRNA that is a used detects an epitope of p50/p65 that is accessible only when function of a concomitant decrease in p38 MAPK. This concept is NF-␬B is activated and bound to its consensus DNA-binding se- supported by the recent work of Fechir et al. (31) that demonstrates quence. This would provide a reliable measure of the degree of that inhibition of p38 MAPK decreases stability of iNOS mRNA, NF-␬B activation in the presence or absence of doxycycline in ultimately leading to decreased iNOS expression. The effects of CM-treated cells. erythromycin on p38 MAPK have been previously reported else- The p38 MAPK pathway plays an important role in the inflam- where (32, 33). matory response (20). A primary function of p38 MAPK is to Tetracycline and similar antibiotics such as doxycycline have regulate the stability of inflammatory protein mRNAs such as long been known to effectively decrease inflammation in rheuma- MCP-1 (21), GM-CSF and vascular endothelial growth factor (22, toid arthritis (34, 35). The present results support the possibility 23), TNF-␣, IL-1, cyclooxygenase 2, IL-6, IL-8 (24–27), and that doxycycline may have potential as an anti-inflammatory treatment for inflammatory lung diseases such as asthma, diffuse panbronchiolitis, chronic bronchitis, and bronchiectasis. Antibiotics such as the macrolides (erythromycin A and derivatives) (36, 37) and tetracycline derivatives such as doxycycline (38–40) have been successfully used to treatment inflammation in a variety of clinical settings. However, successful treatment of inflammation requires long-term drug therapy which includes the possibility of the induction of resistance to antibiotics, such as erythromycin A, in potentially pathogenic bacteria. The possible increase in antibiotic-resistant Mycobacterium spp. (41, 42)., No- cardia spp. (43, 44), and other bacterial pathogens is a serious problem that is often encountered when treating inflammatory lung diseases with erythromycin-type antibiotics. Therefore, other po- FIGURE 6. Effect of doxycycline of p38 MAPK. Lane 1, molecular tential anti-inflammatory therapies which might not induce resis- mass markers (kDa); lane 2, Control; lane 3, doxycycline (30 ␮g/ml) tance, such as doxycycline, would be beneficial for treatment of alone; lane 4, CM (5 ng/ml for each component) alone; lane 5, CM plus chronic inflammatory lung diseases. The data presented in this doxycycline. Twenty micrograms of protein was loaded per lane. article suggest that doxycycline may be useful as an anti- The Journal of Immunology 571 inflammatory therapy in inflammatory lung diseases where emer- 17. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by gence of resistance to doxycycline may not be a serious clinical acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156–159. problem. 18. Robbins, R. A., J. H. Sisson, D. R. Springall, K. J. Nelson, J. A. Taylor, In addition to its prominent role in tissue damage during chronic N. A. Mason, J. M. Polak, and R. G. Townley. 1997. Human lung mononuclear inflammation, NO is also believed to have roles in which its re- cells induce nitric oxide synthase in murine airway epithelial cells in vitro: role of TNF␣ and IL-1␤. Am. J. Respir. Crit. Care Med. 155: 268–273. duction would be detrimental, such as in the defense against mi- 19. Robbins, R. A., P. A. Jinkins, T. W. Bryan, S. C. Prado, and S. A. Milligan. 1998. crobial pathogens (45). iNOS has been demonstrated to be an im- Methotrexate inhibition of inducible nitric oxide synthase in murine lung epithe- portant antimicrobial defense mechanism (46). The NO produced lial cells in vitro. Am. J. Respir. Cell Mol. Biol. 18: 853–859. can react with superoxide produced by macrophages or neutro- 20. Saklatvala, J. 2004. The p38 MAP kinase pathway as a therapeutic target in inflammatory disease. Curr. Opin. Pharmacol. 4: 372–377. phils, forming peroxynitrite (45), which can damage a variety of 21. Goebeler, M., K. Kilian, R. Gillitzer, M. Kunz, T. Yoshimura, E. B. Brocker, microbial targets including lipids, heme clusters, aromatic amino U. R. Rapp, and S. Ludwig. 1999. The MKK6/p38 stress kinase cascade is critical acids, thiols, and DNA (47). for tumor necrosis factor-␣-induced expression of monocyte-chemoattractant Blood A reduction in iNOS expression and NO production is also seen protein-1 in endothelial cells. 93: 857–865. 22. Kracht, M., and J. Saklatvala. 2002. Transcriptional and post-transcriptional con- in cystic fibrosis (48). This observed decrease in iNOS expression trol of gene expression in inflammation. Cytokine 20: 91–106. and NO production may be, in part, responsible for the continued 23. Clark, A. R., J. L. Dean, and J. Saklatvala. 2003. Post-transcriptional regulation infection and colonization of the cystic fibrosis lung by Pseudo- of gene expression by mitogen-activated protein kinase p38. FEBS Lett. 546: monas aeruginosa (49). 37–44. 24. Winzen, R., M. Kracht, B. Ritter, A. Wilhelm, C. Y. Chen, A. B. 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