GPCRs regulate multiple aspects of responses to injury in the lung, including inflammation, vascular permeability, pulmonary edema, airway smooth muscle tone, cell survival, proliferation, and repair. Stimulation of some GPCRs is known to upregulate the expression of proinflammatory genes, such as cytokines and chemokines [14
]. Acute lung injury triggers an inflammatory response driven by cytokines and chemokines that results in a rapid influx of neutrophils into lung tissue. Activation of neutrophils within lung tissue causes the release of toxic mediators that can worsen and perpetuate lung injury. Signaling pathways by which GPCRs stimulate cytokine production represent potential therapeutic targets for treating lung inflammatory diseases, but these signaling mechanisms are not completely understood.
GPCRs transduce intracellular signals by activating heterotrimeric G proteins, which have been classified into families based on structural and functional properties of the Gα subunit. GPCRs coupled to the Gq
family of Gα proteins have the capability in some cell types to stimulate cytokine gene expression through the activation of the proinflammatory transcription factor NF-κB. Examples of this include Tacr1 [8
], PAF receptor [15
], PAR-2 [16
], and the BK B2 receptor [17
]. Because most such studies were performed in transformed cells lines and/or with overexpressed receptors, we examined in the current experiments the ability of these GPCRs to induce chemokine expression in primary lung cells.
Our results indicated that IL-8 levels were significantly increased in human primary alveolar epithelial cells in response to SP, BK, PAR-2A, and PAF, four ligands known to stimulate Gq
-coupled GPCRs. In contrast, human lung endothelial cells responded with increased cytokine production only to PAF, and rat lung endothelial cells responded to PAF and SP but not BK or PAR-2A. Thus, compared with epithelial cells, endothelial cells showed a more restricted response in the capacity to secrete IL-8 or CINC-1 in response to the panel of GPCR ligands we used. The magnitude of chemokine expression was also more pronounced in epithelial cells as compared with endothelial cells. These results are consistent with the concept that epithelial cells are a major source of cytokine production in vivo following lung injury, with high concentrations of chemokines secreted by epithelial cells serving to attract inflammatory cells to sites of injury. The failure of endothelial cells to respond to some of the GPCR ligands tested may occur because they do not express the appropriate receptors or because they lack the intracellular signaling pathways necessary for the response. For example, we have observed that in A549 cells transfected with the tachykinin 1 receptor, receptor stimulation with SP results in Gq
-coupled calcium release ([8
], our unpublished observations). In contrast, in 293 cells transfected with Tacr1, SP treatment does not elicit calcium release [18
]. The mechanisms underlying differential responses such as these to GPCR stimulation are unknown and represent an interesting avenue of future investigation.
PAF has been well established as a mediator of lung injury in studies using in vivo models [19
]. Because PAF is a potent inducer of pulmonary edema, much attention has been focused on this aspect of its function. Our results demonstrate that PAF also induces a robust increase in proinflammatory gene expression in lung epithelial cells. PAF has been shown previously to activate NF-κB, which is a key regulator of chemokines such as IL-8 [15
]. PAF has been demonstrated to upregulate cytokine expression in lung cells, including lung fibroblasts [22
], airway smooth muscle cells [23
], and transformed bronchial epithelial cells [24
]. The results in the present study are to our knowledge the first to demonstrate IL-8 induction by PAF in primary lung epithelial cells.
Endothelial cells participate in inflammatory responses by regulating vascular permeability and the egress of inflammatory cells from the bloodstream. These processes are controlled by inflammatory mediators that induce acute changes in endothelial cell gene expression. Although adhesion molecules are considered to be the most important proteins in this regard, previous studies have shown that endothelial cells also respond to inflammatory mediators, including those acting through GPCRs, by upregulating cytokine gene expression. For example, PAF induced the upregulation of tumor necrosis factor-α in human umbilical vein endothelial cells [26
]. PAR-2 agonists caused increases in IL-8 in human umbilical vein endothelial cells [27
] and dermal microvascular endothelial cells [28
]. The present study is the first to report increased CINC-1 or IL-8 expression by SP treatment in endothelial cells, although there is precedence for SP inducing upregulation of COX-2, an enzyme responsible for the production of inflammatory lipid mediators [29
], and for SP causing the activation of NF-κB, an important factor mediating chemokine gene transcription [30
We previously characterized a signal transduction pathway by which Tacr1 receptor stimulation results in NF-κB activation and IL-8 production [8
]. The results documented in the present work extend these studies in several important ways. The previous study was performed in A549 cells, a transformed lung cancer cell line. Here we demonstrate that an equivalent pathway is operative in primary cells, which increases the potential relevance to normal physiological processes. In our previous study, we studied intracellular signaling transduced by receptors that were overexpressed by transfection of Tacr1 cDNA; treatment of untransfected A549 cells with SP did not result in NF-κB activation. In the experiments described here, signaling triggered by endogenous tachykinin and PAF receptors was investigated. These responses by endogenous receptors on primary lung cells are likely to more closely reflect signaling mechanisms in the intact lung. Whereas we previously examined only one GPCR ligand in epithelial cells, here we investigated multiple ligands in both epithelial and endothelial cells. As we obtained similar responses in the different model systems, our results provide evidence for a general signal transduction pathway responsible for promoting proinflammatory gene expression by GPCRs in multiple cell types.
The overall effects of signaling inhibitors were generally similar between human SAECs and RLMVECs, but some minor differences were observed that deserve comment. First, the phospholipase C inhibitor U73122 inhibited PAF-induced IL-8 production in SAECs at 10 μM but not at 1 μM. In contrast, SP-induced CINC-1 production was significantly inhibited by 1 μM U73122 (). Although the reason for this difference is not understood, it is consistent with previous results suggesting that higher concentrations of U73122 are necessary to inhibit phospholipase C mediated effects in lung epithelial cells compared with endothelial cells. For example, we previously observed similar effects in A549 cells [8
], and other studies in lung epithelial cells have typically employed U73122 concentrations of at least 10 μM [31
]. In endothelial cells, a variety of events mediated by phospholipase C have been shown to be inhibited by 1 μM U73122 [35
]. A second difference in the response of the different cell types was the increase in CINC-1 expression following treatment of endothelial cells with 1 μM manumycin A that was not observed in SAECs (). This differential response to different doses of manumycin appears to be unusual but not unprecedented. Low doses of ras inhibitors, including manumycin, stimulated the growth of ras-transformed cells at low concentrations and inhibited growth at higher doses [39
]. No information is available regarding the mechanisms underlying these effects. A third difference was the lack of response to the Raf-1 inhibitor GW5074 in the endothelial cells. One possibility to explain this result is that in epithelial cells Raf-1 is required, but in endothelial cells this pathway depends on other forms of Raf, such as B-Raf, which are not as readily inhibited by GW5074 [40
Human IL-8 and rat CINC-1 are both acutely upregulated in response to a variety of injurious or proinflammatory stimuli and share common features by which gene expression is controlled. The IL-8 promoter contains an NF-κB binding site, and this factor is the primary regulator of IL-8 at the transcriptional level. In addition, the proximal promoter contains binding sites for NF-IL6 (C/EBP) and AP-1, which in some circumstances are required for maximal increases in gene expression. IL-8 expression may also be controlled at the level of message stability mediated by sequences in the 3′ untranslated region of the mRNA [41
]. Likewise, NF-κB binding to a site in the proximal promoter region of CINC-1 is the most important mechanism controlling its upregulation [42
]. Similar to IL-8, the CINC-1 gene contains consensus sequences for NF-IL6 binding motif and message instability, but the functional activity of these elements has not been demonstrated. The presence of an AP-1 site in the CINC-1 gene has not been reported. The results of the present study are consistent with a major role for NF-κB in mediating the upregulation of IL-8 and CINC-1 in response to GPCR ligands, although additional roles for these other factors cannot be excluded.
depicts a proposed signal transduction pathway activated by ligands for Gq
-coupled GPCRs based on the results shown here for SAECs and RLMVECs and in our earlier study in A549 cells [8
]. In the present study, signaling pathways in primary cells were investigated using chemical inhibitors. In the cells used here, transfection methods that were effective in introducing DNA constructs into the primary cells also induced significant cell death (not shown). As we are examining signaling pathways activated during injury, this problem precluded the use of potentially more specific genetic inhibitors such as dominant-negative mutants. However, because we have examined the effects of inhibiting signaling molecules that are often closely linked (e.g. Ras/Raf/Erk), the overall evidence for involvement of these molecules is enhanced over the use of single signaling inhibitors in isolation. Although minor differences in the responses to some inhibitors were observed, the overall results implicating the signaling molecules shown in the model were consistent among the different cell types. The activation of phospholipase C by GPCRs through Gq
leading to calcium release and PKC activation has been well characterized in many cell types. This proximal phospholipase C/calcium/PKC cascade is commonly linked to downstream activation of the Ras/Raf/Erk pathway. In some cells these pathways are linked by the calcium-activated tyrosine kinase Pyk2 [11
]. Our results with the AG 17 inhibitor are consistent with the involvement of Pyk2 in the primary cells we used, but the limited specificity of this tyrosine kinase inhibitor does not allow us to definitively rule out the potential requirement for other kinases. Erk activation is well known to stimulate the activation of NF-κB in response to a variety of external stimuli, but the exact mechanism by which this occurs and the nature of any potential intermediates remains unclear. One potential mechanism involves ribosomal S6 kinase 1, an Erk substrate which has been implicated in activating NF-κB through both IκB-dependent and -independent mechanisms [43
Signaling model for activation of cytokine gene expression by ligands for Gq-coupled GPCRs
The signaling mechanisms we have identified for PAF- and SP-induced upregulation of chemokine expression are generally consistent with previous experiments with Gq
-coupled GPCRs. For example, treatment of A549 cells with BK led to NF-κB activation mediated by the BK B2 receptor through a Ras/Raf/Erk-dependent pathway [17
]. PAR-2-induced upregulation of IL-8 production in A549 cells was dependent on PKC, MEK, and tyrosine kinase activity [16
]. SP-induced upregulation of NF-κB in human endothelial cells was dependent on intracellular calcium release [30
In summary, we have demonstrated increased cytokine production in primary lung epithelial and endothelial cells in response to multiple GPCR ligands. The intracellular signaling mechanisms responsible for the upregulation of IL-8 by PAF in primary epithelial cells and the increased expression of CINC-1 by SP in primary endothelial cells appeared similar based on responses to signaling inhibitors. The inhibitor studies were consistent with a model involving phospholipase C/calcium/PKC and Ras/Raf/Erk pathways. These results provide potential molecular targets for inhibition of GPCR-induced proinflammatory events. Furthermore, these findings suggest that the signaling pathway we have identified is a general mechanism for activating proinflammatory gene expression by GPCRs.