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We previously described that recombinant interleukin-1β (IL-1β) induced the significant release of substance P (SP) via a cyclooxygenase (COX) pathway in primary cultured rat dorsal root ganglion (DRG) cells. In the present study, we examined the involvement of two types of phospholipase A2 (PLA2) enzymes, which lie upstream of COX in the prostanoid-generating pathway, in the IL-1β-induced release of SP from DRG cells. The expression of type ΙΙΑ secretory PLA2 (sPLA2-IIA) mRNA was undetectable by ribonuclease protection assay in non-treated DRG cells, while in DRG cells incubated with 1 ng/mL of IL-1β, the expression was induced in a time-dependent manner. On the other hand, type IV cytosolic PLA2 (cPLA2) mRNA was constitutively expressed in the non-treated DRG cells, and treatment with 1 ng/mL of IL-1β for 3 h significantly increased the levels of cPLA2 mRNA. The IL-1β-induced SP release was significantly inhibited by the sPLA2 inhibitor, thioetheramide phosphorylcholine (TEA-PC), and the cPLA2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3). Furthermore AACOCF3 suppressed the induction of sPLA2-IIA mRNA expression induced by IL-1β. These observations suggested that two types of PLA2, sPLA2-IIA and cPLA2, were involved in the IL-1β-induced release of SP from DRG cells, and that the functional cross-talk between the two enzymes might help to control their activity in the prostanoid-generating system in DRG cells. These events might be key steps in the inflammation-induced hyperactivity in primary afferent neurons of spinal cord.
Substance P (SP) is a neuropeptide which has been shown to have various physiological functions. In the soma of primary afferent neurons in the spinal dorsal root ganglion (DRG), SP is biosynthesized via transcription of three types of mRNAs encoding α-, β-, and γ-precursor preprotachykinins derived from one gene (Krause et al. 1987), and released from central and peripheral nerve terminals of primary afferent neurons in response to several stimuli. Substance P released at the central site plays a crucial role in the transmission of nociceptive information to the CNS (Randic and Miletic 1977; Hirota␣et al. 1985). The peptide released at the peripheral site induces vasodilation, increases vascular permeability (Lembeck and Holzer 1979; Saria 1984), activates adjacent cells such as mast cells and macrophages and evokes inflammatory responses, including hyperalgesia (Walsh et al. 1995).
Many recent studies have demonstrated that the release of SP from primary afferent neurons is modulated by several mediators including prostaglandins (PGs) (Hingtgen et al. 1995), 5-hydroxytryptamine (Inoue et al. 1997), nitric oxide (Kamisaki et al. 1995) and N-methyl-d-aspartate (Liu et al. 1997). Among the many types of PGs, it has been shown that PGE2 and PGI2 are involved in the development of inflammatory hyperalgesia. The production of these PGs is remarkably increased at local inflammatory sites (Hay et al. 1997; Murata et al. 1997; Safieh-Garabedian et al. 1997), suggesting that these molecules are closely related to the change in nociceptive perception. The production of each PG is regulated in three steps mediated by distinct enzymes: first arachidonic acid is released from membrane phospholipids by phospholipase A2 (PLA2), then it is converted sequentially to the common prostanoid precursor (PGH2) by cyclooxygenase (COX), and finally it is metabolized to bioactive PGs by terminal PG synthases.
It has recently been indicated that PLA2 belongs to a family of heterogeneous enzymes that can be divided into two main classes based on molecular mass and function. Type IIA secretory phospholipase A2 (sPLA2-IIA) has a molecular mass of 14 kDa, requires millimolar concentrations of Ca2+ for its catalytic activity, and displays no selectivity for the fatty acid. The expression of this enzyme is markedly induced upon exposure to proinflammatory stimuli in many cell types including vascular smooth muscle␣cells (Nakano et al. 1990), astrocytes (Oka and Arita 1991),␣macrophages (Arbibe et al. 1997), mesangial cells␣(Vervoordeldonk et al. 1996) and fibroblasts (Kuwata et al. 1998). Moreover, sPLA2-IIA is also found in a soluble form at inflammatory sites such as in human synovial fluid␣from patients with rheumatoid arthritis (Kramer et al.␣1989). In addition, it has been demonstrated that carrageenin-induced inflammatory responses were suppressed by a selective inhibitor of type II PLA2 (Miyake et al. 1993; Garcia-Pastor et al. 1999). Therefore, it appears that sPLA2-IIA plays an important role in several responses at inflammatory sites.
The high molecular mass PLA2 (85 kDa), referred to as cytosolic PLA2 (cPLA2; type IV), selectively liberates arachidonic acid, requires micromolar concentrations of Ca2+ for its activity, and is activated via phosphorylation by protein kinase C and by mitogen-activated protein kinase (Lin et al. 1993; Dennis 1994; Glover et al. 1995). cPLA2 is constitutively expressed in most mammalian cells. Recent reports indicated that proinflammatory cytokines including interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) increased the expression of cPLA2 in several cell types (Lin et al. 1992; Schalkwijk et al. 1993; Murakami et al. 1995), suggesting that cPLA2 contributes not only to physiological functions, but also to the various responses observed under inflammatory conditions.
Several studies have demonstrated interaction between neurons and the immune system, for example, between SP and IL-1β. We previously reported that IL-1β directly evoked the release of SP from cultured DRG cells, the mechanism of which might involve the cyclooxygenase systems (Inoue et al. 1999). Thus, in the present study, to confirm the involvement of PLA2s in the IL-1β-induced release of SP from primary afferent neurons, we examined the effects of IL-1β on the PLA2 subtypes in rat DRG cells. Both sPLA2-IIA and cPLA2 were found to be required for IL-1β-induced release of SP from cultured DRG cells. Moreover, we suggested the possibility of significant cross-talk between these two enzymes in IL-1β-stimulated DRG cells.
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- Materials and methods
Prior studies indicated that sPLA2 and cPLA2 have major roles in the synthesis of inflammatory eicosanoids (Balsinde and Dennis 1997; Dennis 1997). Thus in this study, as a candidate for the rate-limiting enzyme in the PG biosynthesis pathway, we focused on PLA2, and investigated whether PLA2 is involved in the IL-1β-induced release of SP from cultured DRG cells.
We observed that the concentration of SP in the medium of cultured DRG cells treated with rat recombinant IL-1β increased in a dose- and time- dependent manner. IL-1β is a polypeptide cytokine with a wide variety of immunogenic and inflammatory activities that it is thought to play an important role in host response under pathological conditions. This cytokine is released from a variety of cells including macrophages, fibroblasts, mesangial cells, glial cells and neurons (Bianchi et al. 1998). Safieh-Garabedian et al. (1997) demonstrated that intraplanter injection of an endotoxin such as lipopolysaccharide into rat hindpaw evoked a localized inflammatory hyperalgesia, and significantly elevated the level of IL-1β in the skin of the injected paw compared with the non-treated paw. Moreover, it has been reported that the hyperalgesia induced with complete Freund's adjuvant was significantly ameliorated by IL-1 receptor antagonist. IL-1β also has been observed to induce hyperalgesia after injection into rat hindpaw (Ferreira et al. 1988), and after intracerebroventricular or intraperitoneal administration (Watkins et al. 1994). Several studies have suggested that hyperalgesia is induced by IL-1β through a change in primary afferent neuronal activity or sensitivity in mice and rats (Fukuoka et al. 1994; Malcangio et al. 1996). However, it still remains to be elucidated whether IL-1β acts directly by sensitizing primary afferent nociceptors, or indirectly through the generation of another mediator that could affect other cells or stimulate primary afferent nociceptors. As illustrated in Fig. 1(a), it took hours to detect a significant increase in SP release from DRG cells, suggesting that other functional proteins participated in the IL-1β-induced events.
In our study, the expression of sPLA2-IIA mRNA was not detected in non-treated DRG cells. Treatment of cultured DRG cells with 1 ng/mL of IL-1β for 3 h significantly induced the expression of sPLA2-IIA transcript. This persisted until 24 h after treatment before the level of expression gradually declined. We examined the effect of the sPLA2 inhibitor TEA-PC on the IL-1β-induced release of SP from cultured DRG cells. Previous study has indicated that TEA-PC is a preferential inhibitor of sPLA2 with little effect on cPLA2 at the concentration used in this study. (Magolda et al. 1985). TEA-PC at the dose used here completely suppressed the IL-1β-induced release of SP from DRG cells. We concluded therefore that IL-1β exerts these effects through the induction of sPLA2-IIA at the transcriptional level. This paper is a first report that up-regulation of sPLA2-IIA is necessary for the release of SP from primary afferent neurons induced by IL-1β. The increase in SP release from the terminals of these neurons may imply an enhancement in nociceptive perception. Thus, the results presented here indicate the possibility that the sPLA2-IIA enzyme is involved in the induction of hypersensitivity of primary afferent neurons in inflammation.
Furthermore, we examined the possible role of cPLA2 in this pathway. In contrast to sPLA2-IIA, cPLA2 mRNA was expressed even in non-treated DRG cells. There have been several reports that cPLA2 mRNA is expressed under normal conditions, suggesting that this enzyme has an important role in not a pathological, but physiological state. In cultured DRG cells, however, the expression of cPLA2 mRNA was significantly up-regulated in response to 1 ng/mL of IL-1β already at 3 h. Up-regulation of cPLA2 activity by pro-inflammatory cytokines was demonstrated in a number of cell systems including mouse mast cells (Murakami et al. 1995), rat mesangial cells (Schalkwijk et al. 1993) and human vascular smooth muscle cells (Beasley 1999). The involvement of cPLA2 in inflammatory responses induced by various stimuli has been shown using selective inhibitors and antisense oligonucleotides (Amandi-Burgermeister et al. 1997; Wu et al. 1997). Moreover, studies using cPLA2-deficient mice have demonstrated that the production of PGE2 by peritoneal macrophages stimulated with lipopolysaccharide was markedly decreased compared with that in wild type mice (Bonventre et al. 1997; Uozumi et al. 1997). The cPLA2 inhibitor AACOCF3, which has 500-fold greater potency against cPLA2 than sPLA2 (Street et al. 1993), significantly inhibited the IL-1β-induced release of SP from DRG cells at concentrations comparable to those generally used to assess the function of cPLA2 in cells (Riendeau et al. 1994; Kuwata et al. 1998). Thus, the facilitation of cPLA2, not only sPLA2-IIA, expression is also involved in the IL-1β-induced SP release from DRG cells. These observations indicate that the activity of cPLA2 contributes to the␣inflammation-induced hyperactivity of primary sensory afferents.
In this study, the degree to which the mRNA expression was induced by IL-1β was less for cPLA2 than sPLA2-IIA in our models. Therefore, we speculate that cPLA2, compared with sPLA2-IIA, has a distinct role in the IL-1β-induced release of SP in DRG cells. For example, cPLA2 may support sPLA2-IIA in these responses. So, to investigate this hypothesis, we examined the possibility that the activity of cPLA2 enzyme is required for the IL-1β-induced expression of sPLA2-IIA mRNA. Interestingly, 10 µm AACOCF3, which is similar to the dose that exerts an inhibitory effect on SP release as shown in Fig. 5, significantly reduced the induction of sPLA2-IIA mRNA expression by IL-1β. These results implied that certain metabolites produced by the cPLA2-dependent pathway are crucial for the expression of sPLA2-IIA mRNA and that the inhibitory effect of AACOCF3 on the IL-1β-induced SP release may be, at least in part, due to suppression of sPLA2-IIA mRNA expression in DRG cells. In contrast, TEA-PC had no effect on the up-regulation of cPLA2 mRNA expression by IL-1β, suggesting that the induction of cPLA2 mRNA may be not unaffected by the activity of sPLA2-IIA enzyme. Our results are consistent with the recently proposed hypothesis that prior up-regulation of cPLA2 is necessary for sPLA2 to exert its own activity in several cell types (Balsinde and Dennis 1996; Balsinde et al. 1998; Kuwata et al. 1998). Although the molecules which contribute to this event remain to be identified, several interesting reports have surfaced. Kuwata et al. (1998) suggested that some lipoxygenase-derived products are involved in the cytokine-induced expression of sPLA2-IIA. Moreover, Rao et al. (1996) have shown that 12- and 15-hydroperoxyeicosatetraenoic acids, which are metabolites of 12-/15-lipoxygenase, induced the expression of c-fos and c-jun which form the transcription factor AP-1 in vascular smooth muscle cells.
Furthermore, we examined the possibility that the activities of PLA2 enzymes are required for the induction of COX-2 by IL-1β in cultured DRG cells. But neither TEA-PC nor AACOCF3 had an effect on the level of COX-2 mRNA expression induced by IL-1β in these cells, suggesting that the mechanism of COX-2 mRNA induction following treatment with IL-1β is independent of the activities of PLA2 enzymes.
The cultured DRG cells used in this study form a mixed-culture system which comprises primary afferent neurons and growing non-neuronal cells such as Schwann cells and fibroblasts. Whether both types of PLA2 enzymes are located in neurons or non-neuronal cells in this DRG preparation is unknown, although several forms of these enzymes have been identified in various peripheral nerve tissues, where they could play a role in the maintenance and repair of the nerve membrane (Quik 1987; Edstrom et al. 1996; Hornfelt et al. 1999). Paul and Gregson (1992) detected immunoreactivity for sPLA2-II in the Schwann cells. Interestingly, Hornfelt et al. (1999) have reported that in mouse DRG cells, immunoreactivity for PLA2-170, a new type of sPLA2, was detected in neuronal cell bodies and axons and was up-regulated after sciatic nerve injury for 24 h. It is thought that the cytokines play an important role in the repair of nerve injury. Thus we believe that these reports are in line with the results presented, i.e. that IL-1β-induced the up-regulation of both types of PLA2.
In conclusion, the present study showed that both sPLA2-IIA and cPLA2 are essential for the IL-1β-induced release of SP from cultured DRG cells. Furthermore, the results suggested functional cross-talk between the two types of PLA2 enzymes. Although the molecular mechanism of this cross-talk is not known, this finding may help to improve our understanding of the IL-1β-induced responses in DRG cells. It is thought that the actions of IL-1β observed in this model actually occur under inflammatory conditions in the primary afferent nerve system. Based on the results therefore we speculate that these enzymes are candidates for targets in inflammation-induced hyperalgesia.