Cyclic AMP stimulates platelet-derived growth factor B chain mRNA expression in murine macrophage cell lines

Prostaglandin E2 plays a role in cytokine production presumably by altering intracellular levels of cAMP. In this paper, we report on the differential expression of cytokine genes in murine macrophages in response to stimulation with activators of cAMP. Macrophages were cultured with or without cAMP activators in the presence or absence of LPS. Prior to treatment, macrophages do not express interleukin-1β, but do express low levels of tumour necrosis factor α and platelet-derived growth factor B chain mRNAs. After culture with cAMP-inducers, including PGE2, dibutyryl cAMP and forskolin, PDGF B chain mRNA is induced. Forskolin, for example, induced expression PDGF B chain mRNA to a level ranging from 25% to 200% of the level induced by LPS in 6 h. In contrast, cAMP-inducers enhance the expression of IL-1β and TNF-α mRNAs, but only in the presence of LPS. The combination of forskolin and LPS does not appear to act synergistically on PDGF B chain mRNA levels, suggesting that LPS-stimulated effects are not mediated through a cAMP-dependent pathway. Furthermore, macrophages differentially express cytokine genes in response to treatment with inducers of intracellular cAMP.


Introduction
A variety of diseases characterized by fibrosis share common elements. The pathogenesis of these disorders includes the proliferation of fibroblasts and the deposition of extracellular matrix (for a review see Reference 1). This often begins as an inflammatory reaction with leukocyte infiltration followed by the elaboration of cytokines. In the absence of inhibitory signals, the aberrant production of these mediators sustains the connective tissue accumulation which results in permanent alteration in tissue structure and function.
Macrophages play a central role in the tissue response to injury by releasing cytokines and mediate immune-mesenchymal cell interactions and trigger the proliferation fibroblasts and the deposition of connective tissue. 2 Although the mechanisms responsible for the activation of macrophages are not fully understood, evidence suggests that soluble factors including cytokines and arachidonic acid metabolites, including prostaglandin .E2 (PGE2), play a critical role in the process. 3 '4 We have previously reported on the induction of expression of PDGF genes in rat peritoneal macrophage in response to treatment with cytokines, including interleukin-2, 5 and herein examine the role of PGE2 and agents which stimulate the accumulation of intracellular cJkMP on the expression of the PDGF B chain gene in the murine macrophage cell line ANA-1.

Materials and Methods
Reagents: Forskolin, dibutyryl-cAMP (dBcAMP), 8- To determine cytokine protein levels, immunoblot analysis was performed as previously described s with the following minor modifications. Supernatants (10011) from cultured cells were blotted on nitrocellulose filters. The filters were blocked with gelatin and sequentially incubated with anti-human PDGF BB antibody (Genzyme, Cambridge, MA) followed by biotin-conjugated anti-rabbit IgG. The filters were then incubated with avidin-conjugated alkaline phosphatase followed by a substrate for colour development.
Data presented herein are representative examples of experiments performed a minimum of three times unless otherwise specified.

Results
PGE2 and cAMP inducers elevation of PDGF B chain mRNA expression: Treatment of ANA-1 macrophages with PGE2 stimulates the expression of PDGF B chain mRNA (Fig. 1). PDGF B chain mRNA is expressed in ANA-1 cells following culture with as little as 2 ng/ml of PGE2 and peak levels were induced at 20ng/ml of PGE2. Similar results were obtained after incubation of ANA-1 cells with forskolin, which is known to stimulate adenylate cyclase activity. 9  determine whether the induction of cytokine mRNA expression was specific for PDGF B chain, we monitored the expression of IL-lfl and TNF<z in ANA-1 cells cultured for 4h with either LPS alone, forskolin alone or both LPS and forskolin (Fig. 2). Northern blots revealed that only PDGF B chain mRNA expression was stimulated by forskolin alone. In contrast to PDGF B chain mRNA, the expression of IL-I[ and TNF<z mRNAs were greatly induced by stimulation with LPS alone. Furthermore, treatment of ANA-1 cells with LPS and forskolin only yielded slightly higher levels of IL-I and TNF<z mRNAs than LPS alone, further suggesting a stimulatory role for a cAMP-dependent pathway in the control of PDGF B chain gene expression.
A quantitative analysis of the .expression of PDGF B chain and TNF<z mRNAs is shown in Fig. 3. Blots were prepared from ANA-1 cells treated with either LPS alone, forskolin alone or both LPS and forskolin for 4 h. Scanning densitometric analysis (Fig. 3) revealed that PDGF B chain mRNA was induced over 40-fold following treatment with forskolin, and that this level was not further enhanced by LPS. The induction of TNF<z mRNA expression by LPS was over 19-fold and was enhanced to 17-fold by the addition of forskolin to LPS. LPS alone marginally induced PDGF B chain mRNA expression, as did forskolin alone for TNF-z mRNA levels. analysis. PDGF B chain mRNA is marginally expressed in untreated cells and is rapidly induced within I h of treatment with forskolin. Maximal expression is achieved between 4 and 18 h of culture, after which the PDGF B chain mRNA level gradually declines.
Modulation of PDGF B chain .mRNA expression by agents which elevate intracellular cAMP levels: To confirm that the stimulatory effects, of forskolin on the expression of PDGF B chain mRNA expression were due to the enhancement of cAMP levels, ANA-1 cells were cultured in the presence or absence of several agents which elevate intracellular cAMP. These include PGE2 forskolin, dBcAMP, 8-bromo-cAMP, IBMX (an inhibitor phosphodiesterase and, thus, indirectly enhancing intracellular levels of cAMP by blocking its degradation), 1 and cholera toxin (which triggers continuous activation of adenylate cyclase by altering the a subunit of the stimulatory GTP-binding protein). 1 All of the agents which triggered the elevation of intracellular cAMP stimulated the enhanced expression of PDGF B chain mRNA (Fig. 4). In contrast, the addition of dBcGMP to ANA-1 cells failed to stimulate levels of PDGF B chain mRNA above background (not shown).
Effects of cAMP-inducing agents on PDGF B chain protein production: Immunoblot analysis was performed to determine whether incubation of ANA-1 with cAMP-inducing agents triggered the production and secretion of PDGF B chain protein. Fig. 5 shows that PDGF B chain protein is released by macrophages following culture with a variety of agents which trigger the accumulation of intracellular cAMP. In contrast, treatment with a cGMP-inducer fails to induce ANA-1 cells to secrete detectable levels of PDGF B chain protein.

Discussion
The expression of PDGF-like molecules by macrophages in response to tissue injury has been well documented (for a review, see Refer- chain protein is produced and secreted by macrophages following treatment with cAMP-inducing agents. ence 12). Alveolar macrophages from normal lungs do not spontaneously release PDGF-like mediators, but can be induced to secrete these cytokines followinR in vitro treatment with a variety of agents. -In contrast, constitutive production of high levels of PDGF is observed in macrophages isolated from the lungs of animals undergoing the repair process following pulmonary injury induced by exposure to cytotoxic The involvement of macrophage-derived fibrogenic cytokines, such as PDGF, in the development of fibrosis is not restricted to the lung. PDGF has been found in the peritoneal fluid of patients with endometriosis 2 as well as in patients undergoing peritoneal dialysis 27 or intraperitoneal immunotherapy for the treatment of abdominal tumours. 2u In addition, hyperplasia of smooth muscle cell and accumulation of extracellular matrix along with a lipid component are associated with the development of atherosclerosis. 29 This is believed to result from the release of a PDGF-like mediator from infiltrating macrophages. Finally, human wound fluid contains PDGF-like molecules. At present, it is not clear whether the immunoreactive PDGF described herein is identical to the platelet-derived molecules or similar to the smaller molecular weight peptides recently described. 2' While these disorders all share the common denominator of local PDGF release by macrophages, it is unclear what triggers the local production. Since the release of prostaglandins, including PGE2, occurs during inflammatory/prefibroti responses, we hypothesized that PGEa could act in an autocrine/paracrine feed-forward manner to trigger the production of PDGF B chain by macrophages.
Our data are in concurrence with other studies demonstrating a stimulatory role for PGEa and cAMP-inducers in the control of a subset of macrophage functions, including cholesterol ester 32 33 34 clearance and IL-6 production. The role of cAMP in the regulation of IL-1 and TNF-t genes has remained somewhat controversial, with some laboratories reporting that cAMP inducers inhibit IL-1 and TNF-a gene expression, while others state that they stimulate, expression (for a review, see Reference 35).
With regard to the transcription of IL-1 genes, studies performed in murine peritoneal macrophages demonstrate an inhibitory role for cAMP 36 37 and cAMP-inducers.' However, in human monocytes, it acts primarily as a stimulant. -4 Our data reveal that the cAMP inducer, forskolin, has a marginal stimulatory effect on LPS-induced expression of IL-1 mRNA in the fetal liver-derived ANA-1 murine macrophage cell line (Fig. 2). Thus, the action of cAMP-inducing agents on the production of IL-1 seems to be species, as well as cell type, specific.
In conclusion, these data demonstrate that agents which trigger the accumulation of intracellular cAMP specifically stimulate the expression of the PDGF B chain gene, without altering the endogenous or LPS-induced expression of TNF-cz or IL-113 mRNA. Hence, the expression of cytokine genes may be divided into two classes, ones (such as PDGF B chain) which are activated by inducers of cAMP and others (including TNF<z and IL-113) which are not.