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Cutting Edge: CD40 Engagement Up-Regulates Cyclooxygenase-2 Expression and Prostaglandin E₂ Production in Human Lung Fibroblasts¹

Ying Zhang^*, H. James Cao^,§, Beth Graf^*, Heather Meekins, Terry J. Smith^,§ and Richard P. Phipps^2,*,

^* University of Rochester Cancer Center and Departments of Microbiology and Immunology, Pediatrics, and Environmental Medicine, Rochester, NY 14642; and the Division of Molecular and Cellular Medicine, Department of Medicine, and the ^§ Department of Biochemistry and Molecular Biology, Albany Medical College and Samuel S. Stratton Veterans Affairs Medical Center, Albany, NY 12208

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
A newly emerging view of fibroblasts is that they are vital for initiating inflammation and respond to and direct the activities of leukocytes. Human fibroblasts can express CD40, an activation Ag the ligand of which is displayed by activated leukocytes. We demonstrate here that CD40 engagement on human lung fibroblasts dramatically increases proinflammatory PGE₂ synthesis. This up-regulation is mediated through an induction of cyclooxygenase-2 (Cox-2) since Cox-2-selective inhibitors block the up-regulation. Western and Northern blot analyses demonstrated that Cox-2 protein and mRNA are dramatically increased in fibroblasts following CD40 engagement. We conclude that CD40 is a major pathway in human fibroblasts for the induction of Cox-2. There is intense interest in devising strategies for disruption of the CD40-CD40 ligand system to blunt inflammation. Such an intervention would be expected to attenuate the up-regulation of fibroblast Cox-2 and PGE₂ production at the site of tissue injury.

	Introduction

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Current therapy for chronic inflammation and established fibrotic conditions is inadequate. Advancement in the amelioration of fibrotic lung disease, rheumatoid synovial destruction, and other tissue derangement will require additional insights into the mechanisms of inflammation and repair (1). In nearly all body tissues, fibroblasts are the final effector cells mediating both repair and fibrosis (2). Substantial evidence suggests that fibroblasts from distinct anatomic regions differ with regard to their phenotype (3, 4). They are not only interactive regulatory cells capable of response to signals emanating from infiltrating hemopoietic cells but also are sites for diverse molecular biosynthesis (5, 6, 7). For example, it has recently become appreciated that fibroblasts are key sentinel cells that first recruit migratory white blood cells to the site of tissue injury through the synthesis of an array of chemokines (8). Another recent finding concerning fibroblasts relates to their expression of CD40, a 50-kDa member of the TNF- receptor superfamily previously thought to be expressed only on cells of bone marrow origin (9, 10). Engagement of CD40 on fibroblasts from human lung, orbit, thyroid, and gingiva activates the transcription factor NF-B and induces the fibroblasts to synthesize high levels of IL-6 and the chemotactic and angiogenic factor IL-8 (6, 11). During inflammation and in fibrotic conditions, activated T lymphocytes, eosinophils, and mast cells displaying CD40 ligand (CD40L,³ also known as gp39, CD154) are translocated to sites adjacent to fibroblasts (12, 13). Following this recruitment, interaction with resident fibroblasts through the CD40-CD40L bridge enhances the inflammatory process by inducing synthesis of cytokine mediators and adhesion molecules (6, 10, 14). Thus, the CD40-CD40L system appears to be a critical pathway for fibroblast/immune system cross-talk.

Prostanoids, metabolites of arachidonic acid, are produced at high levels in chronic inflammation and are the focus of intensive examination as potential therapeutic targets. PGE₂ is one of the most important mediators in chronic inflammatory disorders such as interstitial lung disease and rheumatoid arthritis. The key and rate-limiting steps in the biosynthesis of prostanoids are catalyzed by cyclooxygenases (Cox, also known as prostaglandin-endoperoxide H synthase, EC 1.14.99.1), membrane-bound, bifunctional enzymes. Two forms of Cox (Cox-1 and Cox-2) have been cloned and characterized (15). In spite of similar catalytic activity, the two isozymes are encoded by separate genes, and their patterns of expression are very different. Typically, Cox-1 is constitutively expressed and considered a "housekeeping" enzyme, important for physiologic regulation. In contrast, Cox-2 is encoded by an early response gene and is ordinarily not expressed or only at very low levels. Cox-2 expression can be induced following stimulation with certain cytokines, hormones, and lipid mediators (7, 16). PGE₂ has many functions including vasodilatation, enhancement of pain, and alteration of immune responses (17). However, the pathways crucial for induction of Cox-2 expression are not well understood.

Fibroblasts from certain anatomic regions are capable of dramatic Cox-2 expression (7). Moreover, as a prominent cell type in most body tissues, they may be viewed as potentially important sites of PGE₂ production. The genes activated by CD40 engagement in fibroblasts that are directly relevant to inflammation encouraged us to consider that CD40-CD40L interaction may represent a novel pathway for induction of prostanoid production. In this report, we test this hypothesis and demonstrate that human lung fibroblasts respond through the CD40-CD40L bridge by massively inducing Cox-2 expression and PGE₂ synthesis. These findings support the concept that stimulation of fibroblast CD40 is a powerful signaling pathway for the induction of Cox-2 expression and for mediating inflammation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Abs and reagents

Human CD40L-containing membranes were provided by Dr. Marilyn R. Kehry (18). SC58125 was a gift of Dr. Peter Isakson (Searle, Skokie, IL). Recombinant human IFN- was purchased from Genzyme (Cambridge, MA), indomethacin was from Sigma Chemical Co. (St. Louis, MO), NS-398 and mAbs against human Cox-1 (CX-102) and Cox-2 (CX-229) for Western blot analysis were from Cayman Chemical Co. (Ann Arbor, MI), monoclonal anti-Cox-2 Ab was from Transduction Laboratories (Lexington, KY), polyclonal anti-Cox-1 Ab was from Santa Cruz Biotech, Inc. (Santa Cruz, CA), and mouse IgG1 and goat IgG1 were from Biosource International (Camarillo, CA).

Cell culture

L828 is a normal, nontransformed human lung fibroblast strain previously isolated by our laboratory (9). The fibroblasts were maintained in MEM (Life Technologies, Gaithersburg, MD), containing 10% FBS (HyClone, Logan, UT) and gentamicin (50 µg/ml) and were used between the 5th and 15th passages.

RNA isolation and Northern blot analysis

L828 fibroblasts were harvested and lysed in Tri-Reagent (1 ml/10⁷ cells; Molecular Research Center, Inc., Cincinnati, OH). Total cellular RNA was isolated from each sample as per the manufacturer’s protocol. Twenty micrograms of RNA per sample were electrophoresed in a 1% formaldehyde agarose gel. For Northern blot analysis, the method used was previously described by Wang et al. (7). The hybridization probes specific for Cox-1 and Cox-2 were generated from 1.6- and 1.4-kb human cDNAs and labeled with [³²P]dCTP by the random-primed technique.

Western blot analysis of Cox-1 and Cox-2 protein levels

Fibroblast monolayers were harvested in an ice-cold lysis buffer (15 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, 1 mM EDTA, 20 mM Tris-HCl (pH 7.5) containing 10 µg/ml soybean trypsin inhibitor and 10 µM PMSF). The protein concentrations of lysates were quantified using the bicinchoninic acid method (Pierce, Rockford, IL). Ten micrograms of cellular protein from each sample were run through a 12% SDS-PAGE, and the membranes were immunoblotted with CX-102 and CX-229 as previously described (7). The density of the resulting bands was analyzed with a BioImage scanner (Milligen, Bedford, MA).

Immunocytochemistry

L828 fibroblasts were seeded in eight-well chamber slides and cultured as indicated in the figure legends. Cells were fixed with 2% paraformaldehyde, treated with 3% H₂O₂, blocked with 2% horse serum, and incubated overnight at 4°C with either 10 µg/ml of an anti-Cox-2 mAb, anti-Cox-1 polyclonal Ab, or isotype control Abs to assess nonspecific staining. Biotinylated horse anti-mouse or anti-goat IgG (heavy + light chain) (1:200; Vector Labs, Inc., Burlingame, CA) was used as secondary Ab and streptavidin-horseradish peroxidase (1:1000; Jackson ImmunoResearch Labs, Inc., West Grove, PA) as substrate. Cells were visualized with 3-amino-9-ethylcarbazole staining (Zymed, San Francisco, CA).

Assay of PGE₂ production

Cells were seeded into 48-well culture plates (1 x 10⁵ cells/well) and cultured as indicated in the figure legends. At selected time points, conditioned medium was collected and assessed for PGE₂ content by enzyme immunoassay (Cayman Chemical Co.).

Data analysis

The statistical significance between treatment groups was analyzed using a paired Student t test. Results were considered significant at p < 0.05.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
CD40 engagement stimulates massive PGE₂production by human lung fibroblasts

Human fibroblasts in culture express CD40 surface protein and respond to CD40L (9, 10, 11). The expression of CD40 can be up-regulated by IFN- (9). In the current studies, recombinant CD40L was used as a cross-linking reagent for CD40 activation on L828, a strain of human lung fibroblasts. As shown in Figure 1A, the level of PGE₂ released by L828 cells into the medium, stimulated with IFN- and CD40L, is considerably higher than that of untreated cultures or fibroblasts treated solely with IFN- or CD40L. CD40L augmented the production of PGE₂ in a time-dependent manner. The increase in PGE₂ levels was observed as early as 5 h and persisted up to 120 h (the duration of the study). We next assessed whether Cox-1 and/or Cox-2 participated in the enhanced PGE₂ production. As shown in Figure 1B, indomethacin (10 µM), a nonselective inhibitor of Cox, completely abolished the stimulatory effect of CD40L on PGE₂ production (p < 0.05 compared with control). To determine the relative contributions of Cox-1 and Cox-2 to the enhanced PGE₂ production, NS-398 and SC58125, selective Cox-2 inhibitors, were utilized (Fig. 1B). Both NS-398 (10 µM) and SC58125 (5 µM) significantly inhibited (p < 0.05) PGE₂ production in response to a 24-h treatment with IFN- plus CD40L, whereas the basal untreated level of PGE₂ produced by L828 was relatively unaffected. The remarkable inhibitory effects of indomethacin, NS-398, and SC58125 were also observed at 48 and 72 h (data not shown). The drugs did not affect cell viability as determined by trypan blue exclusion.

View larger version (21K): [in this window] [in a new window]   FIGURE 1. CD40 engagement of lung fibroblasts results in a time-dependent increase in PGE2 production. L828 lung fibroblasts were seeded in 48-well culture plates (1 x 105 cells/well). Untreated control groups were cultured for 72 h in serum-containing (0.5%) medium followed by replacement with fresh serum-free medium (A). IFN--treated fibroblasts were cultured for 72 h with IFN- (500 U/ml), and the medium was then replaced with fresh serum-free medium containing IFN-. CD40L-treated cells were cultured for 72 h in serum-containing medium, followed by replacement with serum-free medium with CD40L. Cultures receiving IFN- plus CD40L were pretreated for 72 h with IFN- and then incubated with IFN- plus CD40L. At intervals indicated along the abscissa, culture medium was collected and subjected to enzyme immunoassay for PGE2. B, Increased PGE2 production following CD40 activation can be attenuated by indomethacin, NS-398, and SC58125. Fibroblasts were treated as described above. Cultures received indomethacin (10 µM), NS-398 (10 µM), or SC58125 (5 µM) treatment during the incubations. Medium from each well was collected after 24 h and assessed for PGE2 content. Data are presented as mean ± SD of four experiments (A) and mean of triplicate cultures ± SD from a representative experiment (B).

L828 cells were cultured with nothing, IFN-, CD40L, or CD40L plus IFN-, and the cells were lysed and analyzed for Cox-2 protein by Western blot using a specific mAb (Fig. 2). Both IFN- and CD40L enhanced slightly the level of Cox-2 protein when added separately, compared with unstimulated cells. However, pretreatment with IFN- followed by CD40L stimulation dramatically increased Cox-2 protein expression at least 12-fold (Fig. 2). Cox-2 appeared as a single band with a molecular mass of 72 kDa. One explanation for this enhancement of the induction is that IFN- up-regulates the expression of CD40 on fibroblasts (9). The increased display of surface CD40 molecules, once engaged by CD40L, resulted in a dramatic Cox-2 induction. CD40-mediated induction of Cox-2 protein was observed as early as 5 h (data not shown) and preceded the increase in PGE₂ production (Fig. 1A). Cox-1 protein was also detected in L828 fibroblasts as a single 68-kDa band. In contrast to Cox-2, Cox-1 was not affected by any of the treatments (Fig. 2).

View larger version (18K): [in this window] [in a new window]   FIGURE 2. Engagement of CD40 with CD40L increases the expression of Cox-2 protein in lung fibroblasts. L828 fibroblasts were cultured as described in Figure 1 for 72 h, and then the medium was replaced with serum-free medium containing nothing, IFN-, CD40L, or IFN- plus CD40L for 20 h. Monolayers were then harvested, and 10 µg of cellular protein were subjected to SDS-PAGE, transferred to membranes, and immunoblotted with anti-human Cox-2 or anti-human Cox-1 mAbs (top). Bottom, relative densities of Cox-2 protein bands.

To investigate whether the induction of Cox-2 mediated through CD40 engagement uniformly affected all the fibroblasts in the monolayers, we performed immunocytochemistry using specific Abs. Shown in Figure 3 are typical results with lung fibroblasts untreated (Fig. 3A) or treated with IFN- (Fig. 3B), CD40L (Fig. 3C), or a combination of IFN- and CD40L (Fig. 3D) for 20 h followed by staining with Cox-2 (Fig. 3, top) or Cox-1 specific Abs (Fig. 3, bottom). Positive staining for Cox-2 was observed in cultures treated with CD40L or IFN- alone, while untreated cells appear essentially unstained. Far more intense staining, particularly around the nuclear envelope and in the cytoplasm, was observed in the IFN--primed, CD40L-triggered cells (Fig. 3D, top). We noted that not all of the fibroblasts stimulated by IFN- pretreatment followed by CD40L engagement stained for Cox-2. We speculate that this reflects the fibroblast heterogeneity observed previously (1, 19). While the majority of lung fibroblasts in these experiments (>93%) express CD40 (6), 70% express Cox-2. In contrast to the heterogeneous Cox-2 staining, a Cox-1 signal was observed in essentially all fibroblasts regardless of treatment (Fig. 3, bottom, A–D). Thus, our results from Western blotting and immunocytochemical analysis demonstrate that the expression of Cox-2 protein, but not Cox-1 protein, can be induced greatly in lung fibroblasts following CD40 engagement.

View larger version (78K): [in this window] [in a new window]   FIGURE 3. Immunostaining of Cox-1 and Cox-2 in fibroblasts. L828 fibroblasts were cultured as described in Figure 1 for 72 h, and then the medium was replaced with serum-free medium containing nothing, IFN-, CD40L, or IFN- plus CD40L for 20 h. Cells were then fixed and stained for Cox-2 (top) or Cox-1 (bottom) protein with specific Abs. Original magnification, x 400.

The next experiments focused on whether engagement of CD40 by CD40L up-regulated Cox-2 mRNA. To quantitate steady-state Cox-1 and Cox-2 mRNA levels, Northern hybridization with specific Cox-2 and Cox-1 cDNA probes was performed (Fig. 4). No Cox-2 mRNA expression was detectable in untreated cultures, and only minimal expression was seen in cells treated solely with IFN- or CD40L. However, the Cox-2 probe strongly hybridized to a 4.8-kb transcript and to a smaller transcript (7) expressed in CD40L-treated lung fibroblasts primed by IFN-. In these cells, Cox-2 mRNA levels were dramatically up-regulated, at least 10- to 25-fold above control samples after 4 h of engagement. In contrast to the massive induction of Cox-2 mRNA, Cox-1 mRNA levels were uninfluenced by any of these treatments, indicating Cox-1 mRNA is constitutively expressed in human fibroblasts and most likely contributes to the basal prostaglandin production. This Cox-1 mRNA appeared as a 5-kb species, consistent with our previous studies in human orbital and dermal fibroblasts (7). These results support the data showing the constitutive expression of Cox-1 protein observed in Figures 2 and 3.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Steady-state Cox-2 mRNA levels are increased in human lung fibroblasts activated through CD40. L828 fibroblasts were cultured in 100-mm culture dishes for 72 h (as in Fig. 1), and then the medium was replaced with serum-free medium containing nothing, IFN-, CD40L, or IFN- plus CD40L for 4 h. Twenty micrograms of total cellular RNA per sample were electrophoresed, transferred, and hybridized with a Cox-2 or Cox-1 cDNA probe as described in Materials and Methods.After radioautography, the blot was stripped and rehybridized with a probe complimentary to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative intensities of the Cox-2 signals were analyzed by densitometry, normalized, and plotted.

	Acknowledgments

 
We thank Dr. Marilyn R. Kehry for providing human CD40-containing membranes and Dr. Peter Isakson for SC58125. We also thank Dr. Josue Padilla for technical assistance.

	Footnotes

 
¹ This research was supported by the Rochester Area Pepper Center and United States Public Health Service Grants CA11198, DE11390, HL56002, EY11708, EY08976, ES01247, and HL36543 and by a Merit Review Award from the Department of Veterans Affairs.

² Address correspondence and reprint requests to Dr. Richard P. Phipps, Box 704, Cancer Center, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642. E-mail address:

³ Abbreviations used in this paper: Cox, cyclooxygenase; CD40L, CD40 ligand.

Received for publication November 5, 1997. Accepted for publication December 3, 1997.

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