Impact of VIP and cAMP on the regulation of TNF-α and IL-10 production: implications for rheumatoid arthritis
- 7k Downloads
Vasoactive intestinal peptide (VIP) is an anti-inflammatory immunomodulatory neuropeptide with therapeutic potential demonstrated for collagen-induced arthritis. The aim of this study was to characterise its potential anti-arthritic effect on human monocytes, macrophages, T cells, and rheumatoid arthritis synovial membrane cells. Monocytes, macrophages, and T cells derived from human peripheral blood were treated with VIP and compared with other cAMP-elevating drugs for a range of activating stimuli. Cytokine production was assessed for cell cultures and, in addition, the ability of VIPs to activate cAMP response element binding protein. VIP partially suppressed monocyte- and macrophage-derived tumour necrosis factor α (TNF-α) with no effect on IL-10, whereas VIP fails to regulate IL-10 and TNF-α production by T lymphocytes. No such modulation of cytokine profile was observed for rheumatoid arthritis synovial membrane cells. Elevation of intracellular cAMP, on the other hand, potently suppressed macrophage TNF-α production and modulated T-cell response by inhibiting TNF-α and IFN-γ. VIP's lack of effect on IL-10 and its slight effect on TNF-α results from cAMP being rapidly degraded as the phosphodiesterase IV inhibitor, rolipram, rescues cAMP-dependent activation of cAMP response element binding protein. Interestingly, macrophages stimulated with phorbol 12-myristate 13-acetate/ionomycin displayed an augmented IL-10 response upon addition of dibutyryl cAMP, with corresponding downregulation in TNF-α, suggesting a complex interaction between protein kinase C and protein kinase A in cytokine regulation. In conclusion, VIP may represent an efficaceous anti-arthritic treatment modulating macrophage and T-cell cytokine profiles when used alongside a phosphodiesterase inhibitor.
KeywordsIL-10 macrophage T cells TNF-α VIP
activating transcription factor-1
cAMP response element binding protein
enzyme-linked immunosorbent assay
fetal calf serum
median inhibitory concentration
macrophage-colony stimulating factor
nuclear factor κB
peripheral blood mononuclear cells
protein kinase A
protein kinase C
phorbol 12-myristate 13-acetate
rheumatoid arthritis synovial membrane cell
Roswell Park Memorial Institute [medium]
T helper cell type 1/2
tumour necrosis factor α
vasoactive intestinal peptide.
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterised by the dysregulated expression of many proinflammatory cytokines including tumour necrosis factor α (TNF-α), with increased yet insufficient production of anti-inflammatory cytokines including IL-10 . The validation of TNF-α as a therapeutic target in RA has encouraged the investigation of signalling pathways regulating its production by cells relevant to the pathophysiology of this disease. One pathway known to downregulate proinflammatory TNF-α production and, consequently, upregulate the anti-inflammatory cytokine IL-10 is that elicited by the second messenger cAMP [2, 3]. This pathway may therefore represent a good therapeutic target due to its opposing effects on TNF-α and IL-10. Previously, we and others demonstrated that rolipram, a phosphodiesterase (PDE) IV inhibitor, reduced the clinical and histological severity of collagen-induced arthritis (CIA) [4, 5]. These studies demonstrated the potential for the cAMP/protein kinase A (PKA) pathway in treatment of autoimmune diseases such as RA.
Another stimulator of the cAMP/PKA pathway whose principle immunomodulatory functions are anti-inflammatory is the vasoactive intestinal peptide (VIP). VIP is a 28-aminoacid neuropeptide belonging to the glucagon/secretin family, found in the nervous system and in the immune system, where it is detected in a variety of cell types including mast cells, neutrophils, and mononuclear cells. The effects of VIP are transduced via three known receptors, VPAC1, VPAC2, and PAC1, all of which are coupled to adenylate cyclase via heterotrimeric G proteins. In vivo, VIP has a therapeutic effect in the CIA mouse model [6, 7] and protects from lipopolysaccharide (LPS) shock by suppression of TNF-α [8, 9] and nuclear factor κB (NF-κB) activation . Furthermore, in vitro studies showed that VIP inhibits the production of proinflammatory factors TNF-α, IL-6, IL-12 [11, 12], chemokines [13, 14], and nitric oxide (NO)  and stimulates the production of the anti-inflammatory cytokine IL-10 , most of these effects being apparently mediated through the VPAC1 receptor. In addition, neuropeptides such as VIP have been shown to inhibit activities both of stimulated T cells (VIP being described as a Th2 cytokine), effectively suppressing T helper cell type 1 (Th1) differentiation  and of macrophages  and to antagonise inflammatory mediators such as histamine, prostaglandin E2, leukotrienes, and neurokinins . The mechanism by which VIP antagonises LPS-induced production of proinflammatory TNF-α and abrogates production of anti-inflammatory IL-10 is suggested to result from a fine balance between cAMP response element DNA binding factors where VIP increases the phosphorylation of PKA-dependent cAMP response element binding protein (CREB) and decreases the phosphorylation of c-Jun N-terminal kinase-dependent c-Jun phosphorylation, without affecting the amount of CRE binding: changes of CRE binding complexes from high c-Jun/low CREB (LPS treated) to low c-Jun/high CREB (VIP treated) leads to an inhibition of TNF-α mRNA expression, whereas the corresponding stimulation in IL-10 gene expression is due to an increase in CRE binding by VIP [; reviewed in ].
It would appear from these studies that VIP has therapeutic potential based on its ability to ameliorate CIA in mice [6, 7], this effect possibly mediated by cAMP. However, the effect of VIP on human cells and particularly on RA synovial cells is unknown. Thus the aim of this study was to examine the potential of VIP as a therapeutic agent in chronic inflammatory diseases such as RA by investigating its effects on human macrophages, T cells, and synovial cells – all of which play an important role in the pathology of RA – and compare the findings with murine VIP data already published.
Materials and methods
Capture and detection antibodies for human TNF-α, IL-10, and IFN-γ ELISAs were purchased from Pharmingen International, Oxford, UK. Direct colorimetric immunoassay kit for detection of cAMP was purchased from Merck Biosciences, Nottingham, UK. Macrophage-colony stimulating factor (M-CSF) was obtained from Genetics Institute, Boston, MA, USA. Rolipram, a PDE IV inhibitor, was a gift from Dr Peter Scholz (Schering, Berlin, Germany). PDE-resistant dibutyryl cAMP and forskolin, an activator of adenylate cyclase, were purchased from Sigma, Poole, Dorset, UK. VIP was synthesised at the Advanced Biotechnology Centre, Imperial College School of Medicine at Charing Cross Hospital, London, UK. Antibodies to CREB and phospho-CREB were purchased from New England Biolabs, Beverly, MA, USA. All reagents used in these tissue-culture experiments were tested for the presence of LPS/endotoxin contamination and were found to be below the lower level of detection of the limulus amoebocyte assay (Cambrex BioScience, Wokingham, Berkshire, UK). In addition, rolipram, dibutyryl cAMP, forskolin, and VIP were tested for cytotoxicity and displayed no toxicity at the concentrations being used in this study as determined by (3-[4,5-dimethylhiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and trypan blue exclusion.
Purification of T lymphocytes and monocytes
Human peripheral blood mononuclear cells (PBMCs) were obtained by density centrifugation of human venous blood buffy coats (purchased from the North London Blood Transfusion Service, Colindale, UK) through Ficoll/ Hypaque (specific density 1.077 g/ml; Nycomed Pharma AS, Oslo, Norway). The resulting PBMCs were centrifugally elutriated in 1% fetal calf serum (FCS) RPMI 1640 medium in a Beckman JE6 elutriator. Lymphocyte and monocyte purity was assessed by flow cytometric analysis of binding of fluorochrome-conjugated anti-CD3, anti-CD19, anti-CD14, and anti-CD45 antibodies (Becton Dickinson, Oxford, UK). T cells obtained were routinely of >90% purity and monocytes of >85% purity.
Differentiation of monocytes to macrophages
Peripheral blood monocytes obtained by centrifugal elutriation were seeded at 1 × 106 ml-1 in assay medium in T-75 medium tissue-culture flasks. M-CSF was added to a final concentration of 100 ng/ml. Cells were cultured for 7 days at 37°C in a 5% CO2 humidified atmosphere. Adherent cells were then washed twice in FCS-free RPMI 1640 and removed from the plastic with cell-dissociation medium (Sigma). The resulting cells were washed twice more and resuspended in RPMI 1640/10% FCS ready for use.
Isolation of RA synovial membrane mononuclear cells
RA synovial membrane cells (RA-SMCs) were obtained from synovial membrane tissue samples provided by the Rheumatology Clinic and the Department of Musculoskeletal Surgery, Charing Cross Hospital, London, UK. All patients gave their signed consent, and ethical approval was obtained from the Riverside Research Ethics Committee, London. Patients met the American College of Rheumatology (ACR) 1987 revised criteria for RA. Synovial membranes obtained were from patients who failed to respond to antirheumatic regimens currently available in the rheumatology clinic and will be discussed later in this manuscript. In brief, tissue was cut into small pieces and digested in medium containing 0.15 mg/ml DNAse type I (Sigma) and 5 mg/ml collagenase A (Roche, Lewes, Sussex, UK) for 1 to 1.5 hours at 37°C. Cell debris was excluded by passing cells through a nylon mesh, and cells were then washed and collected in RPMI-10% FCS at 1 × 106 cells/ml and used immediately for spontaneous cytokine production by RA-SMCs.
Detection of intracellular cAMP
A number of signals are known to stimulate the production of cAMP through the action of adenylate cyclase converting ATP to cAMP. Intracellular cAMP was measured using a colorimetric direct immunoassay in accordance with the manufacturer's instructions. Briefly, 5 × 105 monocytes/macrophages were incubated with test reagents, and then cells were lysed in 0.1 N HCl at room temperature for approximately 10 min. In the capture microtitre plate provided, 100 μl lysate and controls were added per well along with 50 μl conjugate and 50 μl antibody solution and incubated for 2 hours at room temperature on a plate shaker. The plate was then emptied and washed three times in wash buffer provided. Colour development was detected using 200 μl pNpp substrate solution and incubated for 1 hour at room temperature and stopped by the addition of 50 μl stop solution. These assays were read and quantified on a Labsystems Multiscan Bichromatic plate reader at 405 nm and analysed with a Deltasoft II programme (BioMetallics Inc, Princeton, NJ, USA). The minimal sensitivity of the assay was 0.078 pmol/ml cAMP. All results are expressed as the mean concentration of cAMP obtained per condition.
Cytokine determination by ELISA
Sandwich ELISAs were used to measure human IL-10, TNF-α, and IFN-γ. In the IL-10 assay, the anti-IL-10 monoclonal antibody 9D7 was used as the capture antibody, and biotinylated 12G8 was used as the detection antibody. The ELISA was performed as previously described, with a standard curve of recombinant human IL-10 from 10,000 to 13 pg/ml . TNF-α ELISA was carried out as described using 61E71 as the coating antibody and a rabbit polyclonal anti-TNF-α antibody as the detection antibody. This polyclonal anti-TNF-α antibody was in turn detected by a horseradish-peroxidase-conjugated goat anti-rabbit IgG(H+L) (Jackson ImmunoResearch, West Grove, PA, USA). The standard curve of recombinant human TNF-α covered the range of 20,000 to 8 pg/ml . In addition, human IFN-γ ELISA was carried out in accordance with the manufacturer's specifications (PharMingen International, Oxford, UK). These ELISAs were quantified by tetramethylbenzide dichloride activity in response to the horseradish peroxidase conjugate and read on a Labsystems Multiscan Bichromatic plate reader at 450 nm and analysed with the Deltasoft II programme (BioMetallics). The minimal sensitivity of the ELISAs were 8 pg/ml for human TNF-α and 13–40 pg/ml for the human IL-10 and IFN-γ ELISAs. All results are expressed as the mean concentration of cytokine ± SD obtained per condition.
Western blot analysis of phospho-CREB
Macrophages were seeded at 5 × 106 cells/ml in 24-well plates in RPMI 1640/10% FCS. To inhibit phosphodiesterase activity prior to VIP treatment, macrophages were pretreated for 1 hour with 10 μM rolipram and then stimulated for 30 min with 10 μM VIP before harvesting of cell lysates. The stimulation time was previously defined in our laboratory as optimal for activation of CREB. After stimulation, cells were lysed on ice for 15 min in lysis buffer (1% NP-40, 200 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 1 mM Na3VO4, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 10 μg/ml aprotinin). Lysed samples (10 μg) were separated by electrophoresis on a 10% SDS–polyacrylamide gel and transferred to a nitrocellulose membrane. Phosphorylated proteins were detected using antibodies raised against phospho-CREB followed by anti-rabbit horseradish peroxidase conjugate and enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech UK Ltd, Little Chalfont, Buckinghamshire, UK). Protein bands were visualised by autoradiography using Hyperfilm (Amersham Pharmacia Biotech UK).
Comparison of data was assessed using GraphPad Prism version 3.0 (GraphPad Software Inc, San Diego, CA, USA). Statistical differences were determined with Student's t-test. Differences were regarded as significant when *P < 0.05, **P < 0.01, or ***P < 0.001.
VIP, rolipram, and dibutyryl cAMP suppress LPS-induced monocyte TNF-α production without upregulating IL-10
VIP, rolipram, and dibutyryl cAMP suppress LPS-induced macrophage TNF-α production without upregulating IL-10
TNF-α fails to modulate VIP regulation of LPS-induced macrophage IL-10
2182 ± 317.7
1904 ± 255.7 (12.7%)
1123 ± 23.92
922.7 ± 49.93 (17.8%)
1481 ± 494.6
1606 ± 906.3 (-8.4%)
IL-10 fails to modulate VIP regulation of LPS-induced macrophage TNF-α
1400 ± 62.02
630.3 ± 61.55 (55%)
3041 ± 624.9
2195 ± 224.9 (28%)
119.5 ± 6.944
88.26 ± 14.48 (26%)
The lack of any great effect of VIP on monocyte-derived macrophages and monocytes themselves led us to postulate that there was an endogenous phosphodiesterase activity intrinsic to these human cells. This was confirmed by the inability of VIP to activate/phosphorylate CREB, a downstream effector molecule to the cAMP-dependent PKA pathway, in M-CSF-treated macrophages (Fig. 2g, lane 2). Of particular interest is the fact that simultaneous addition of VIP and the PDE IV inhibitor rolipram restored activation of CREB (Fig. 2g, lane 3), suggesting that in the absence of a PDE inhibitor these cells quickly and efficiently break down cAMP produced in response to VIP. Rolipram on its own inhibits PDE IV activity (cAMP breakdown) but does not stimulate cAMP production and as such was not included in this phospho-western result. This control, however, failed to activate CREB by phosphorylation of residue Ser 133 on all blots tested. Of particular interest, however, is the observation that activating transcription factor-1 (ATF-1) is activated by LPS (ATF-1 is also recognised by the CREB antibody used) (lane 1), an effect that is abrogated in the presence of VIP (lane 2), and that the combination of VIP and rolipram activates both CREB and ATF-1 upon LPS stimulation (lane 3).
VIP fails to modulate T-cell production of IL-10, TNF-α, and IFN-γ
In addition, VIP has been shown to modulate T-cell function in the murine system of CIA, shifting a Th1 cytokine response to a Th2-like response . We investigated this modulation in the context of the human T cells stimulated by PMA/ionomycin. Unlike its effect in the murine system, VIP failed to modulate human T-cell activity. PMA/ionomycin-induced T-cell IFN-γ production was not significantly affected by VIP (Fig. 3c). Unlike VIP however, elevation of intracellular cAMP did modulate IFN-γ production. The addition of cell-permeable dibutyryl cAMP suppressed IFN-γ production from PMA/ionomycin-stimulated T cells (IC50 = 9 μM) (Fig. 3f). In a separate set of experiments, IFN-γ was also suppressed by the adenylate cyclase activator forskolin (data not represented graphically) from PMA/ionomycin-stimulated control levels of 6963 ± 230 pg/ml to 5691 ± 265 pg/ml (P = 0.027) and 4968 ± 372 pg/ml (P = 0.025) at concentrations of 10 μM and 20 μM respectively (IC50 = 6 μM), and by rolipram, the PDE IV inhibitor, from 6963 ± 230 pg/ml to 2685 ± 204 (P = 0.002) and 2262 ± 94 (P = 0.002) at 1 μM and 10 μM respectively (IC50 = 2 μM) (data not represented graphically). In addition, these data were reproducible for concanavalin-A-stimulated T cells from human peripheral blood.
cAMP modulates PMA/ionomycin stimulated macrophage cytokine profile
Macrophages have a high endogenous PDE IV activity: VIP induction of cAMP is augmented by rolipram
Rolipram and VIP augment IL-10 production in a stimulus- and cell-specific manner
Elevation of intracellular cAMP by the phosphodiesterase-resistant dibutyryl cAMP augments production of IL-10 by PMA/ionomycin-stimulated macrophages. The lack of augmentation of IL-10 production by VIP is suggested by a short-lived elevation in cAMP as a result of high endogenous PDE activity. Here, we have investigated VIP modulation of cytokine production in the presence of rolipram, an inhibitor of PDE IV activity. Our results demonstrate both stimulus- and cell-type-specific responses to VIP in the presence of rolipram. The addition of VIP and rolipram on their own or in combination in the absence of an activating stimulus failed to induce cytokine production. VIP augmented macrophage IL-10 production when stimulated by PMA/ionomycin in the presence of rolipram (Fig. 6a). This was not the case, however, when macrophages were stimulated with LPS; LPS-induced IL-10 production was unaffected by rolipram alone or rolipram+VIP (Fig. 6b). In addition, stimulated T cells also failed to show an augmentation of IL-10 production upon treatment by VIP and rolipram (Fig. 6c). On the other hand, rolipram augmented VIP suppression of TNF-α production in a cell-nonspecific and stimulation-nonspecific manner, as observed for PMA/ionomycin- and LPS-stimulated macrophages and concanavalin-A-stimulated T cells (data not shown).
VIP fails to modulate spontaneous IL-10 and TNF-α production by RA-SMCs
In a murine model of arthritis (CIA), VIP has been described as a potent anti-inflammatory mediator effectively reducing paw swelling, clinical score, and histological severity of disease [6, 7]. This neuropeptide downregulates macrophage and T-cell function as well as modulating T-cell phenotype by altering Th1/Th2 balance in favour of Th2-like cells. There are no such compelling data for the efficacy of VIP in human tissues. The data presented in this paper would argue against VIP alone being a useful therapeutic agent in the treatment of human chronic inflammatory disorders such as RA, because the peptide failed to significantly modulate in vitro TNF-α IL-10 expression by human cells. The lack of effect of VIP in monocyte/macrophage cultures stimulated with LPS may have been due to a high level of endogenous intrinsic phosphodiesterase activity, resulting in a short-lived cAMP in these cell types. This question was addressed by the use of rolipram to inhibit PDE IV and dibutyryl cAMP, which is resistant to PDEs. The treatment of macrophages with VIP in the presence of rolipram facilitated activation of CREB but did not augment IL-10 cytokine production. In addition, this lack of sensitivity of cells to VIP is not as a result of a window of opportunity of action. VIP was added 1 hour prior to stimulation. However, some reports have described VIP to be a more effective anti-inflammatory agent if it is administered at the same time as or after stimulation; addition of VIP to cultures 1 hour before, simultaneously with, or 2 hours after stimulation showed no significant differences to TNF-α/IL-10 ratios in this study.
The effect of VIP in modulating T-cell function was observed by Delgado and colleagues and by Williams and colleagues, in studies in which murine lymph node cells from CIA mice demonstrated a shift in ratio of IFN-γ/IL-5, from Th1 in favour of a Th2 profile [6, 7]. We wished to investigate this modulation of a Th1-driven response (IFN-γ production) in the context of stimulated human T cells. Indeed, VIP failed to modulate IFN-γ. However, elevation of intracellular cAMP by rolipram, dibutyryl cAMP, and forskolin dose-dependently suppressed IFN-γ and TNF-α production. This would suggest that VIP activation of the cAMP pathway is not involved in T-cell IFN-γ production or that the cAMP is rapidly degraded by an active phosphodiesterase present in the cell. This T-cell unresponsiveness to VIP with respect to production of TNF-α, IL-10, and IFN-γ is not a consequence of PMA/ionomycin stimulation, as PHA and concanavalin A also failed to exhibit VIP responsiveness. Alternatively, reports thus far describing modulation of T-cell activity have used PBMCs and lymph node cells, whereas our present studies used purified T cells, which appear relatively insensitive to VIP. We suggest that the modulatory effect of VIP on T-cell cytokine production is indirect, through the regulation of effector functions of antigen-presenting cells (APCs). The role of Th differentiation is likely to play a role where VIP has been described to bias the Th1/Th2 balance in favour of Th2, thus indirectly modulating T-cell cytokine production . The data presented in this paper focus on mature human T-cell modulation by VIP, which has no direct effect on cytokine production; the confirmation of an effect of VIP on T-cell differentiation warrants further investigation in the human system using naïve T cells from cord blood.
Unlike its effect in the murine system, VIP has little effect in modulating IL-10 production by human peripheral blood derived monocytes, macrophages, and T cells. It suppresses monocyte TNF-α production upon stimulation with LPS and is less potent in M-CSF differentiated macrophages. Results obtained with the PDE IV inhibitor rolipram and the PDE-resistant dibutyryl cAMP suggest that the cAMP generated is a potent inhibitor of LPS-induced TNF-α, whereas IL-10 is relatively unaffected. The slight inhibition of IL-10 by elevation of cAMP is thought to be a consequence of the potent inhibition of TNF-α. The failure of VIP to augment macrophage IL-10 production, unlike the murine system, is likely to result from the lack of activation/phosphorylation of CREB, a transcription factor that is readily activated in the murine system by VIP . This is likely to be due to instability of cAMP that results from PDE activation. The combined treatment with VIP and rolipram both activated CREB and augmented IL-10 production. In addition, VIP failed to modulate spontaneous IL-10 or TNF-α production by RA-SMCs. However, spontaneous TNF-α production was suppressed by the PDE-resistant dibutyryl cAMP. This would again suggest that cAMP is failing to activate CREB by a mechanism which involves high PDE activity.
Thus, VIP inhibition of TNF-α was less effective in macrophages than in monocytes and was completely ineffective in RA-SMCs, which suggests that there is an increase in PDE activity during differentiation. One point of note regarding the responsiveness of macrophages and RA-SMCs to VIP is that the effective doses are higher than in earlier reports. In addition to the PDE IV activity, this might be explained by modulation of expression of the VIP receptors (VPAC1 and VPAC2) on these cells. Indeed, in the case of RA-SMCs, it is possible that the method of isolation from synovial membrane tissue might downregulate VIP-receptor expression. Additionally, this could be accounted for by the drug regimen encountered by the patient, where patient tissue obtained by surgery results from the failure to respond to treatments given, decreasing sensitivity to VIP through downmodulation of the receptors. Monocytes, however, were more sensitive to VIP than macrophages, and as such would also suggest that maturation might influence VIP responsiveness through modulation of receptor expression. The relative expression of these receptors is currently under investigation.
Alternatively, this slight discrepancy between effective doses of VIP (IC50) in our data and data already published may result from both different methods of isolation and different cell populations. Our studies use highly purified cells obtained by the centrifugal elutriation of PBMCs, resulting in >90–95% monocytes and T cells that are not prestimulated in any way as a consequence of the purification protocol. Reports in the literature on human cells describe VIP to potently suppress TNF-α (IC50 approximately 20 nM) in whole blood cultures and purified monocytes, where monocytes were separated by clumping and adherence, activating stimuli which may prime VIP responses . In addition, VIP suppresses LPS-induced monocyte IL-8 production (IC50 approximately 0.1 M)  and LPS-induced peripheral blood mononuclear cell TNF-α production, its potency and overall effect being dependent on the age of the subject where VIP inhibited LPS-induced TNF-α in young patients but stimulated TNF-α in older subjects . Thus, responsiveness to VIP can be regulated by many factors, including cell type and differentiation status, method of purification, activation stimulus encountered, age of subject, and drug regimens encountered by donors.
Although VIP activity has been documented to be regulated in a cAMP-dependent manner, there are additional cAMP-independent mechanisms capable of transducing VIP function. One such mechanism involves the inhibition of NF-κB, a crucial factor for the expression of inflammatory mediators such as TNF-α . Thus, the effects of VIP can be explained not only through the cAMP/PKA pathway. This dichotomy in mechanisms of VIP action may explain differential regulation of proinflammatory and anti-inflammatory cytokines: inhibition of NF-κB suppresses TNF-α production, whereas activation of the cAMP/PKA/ CREB pathway in the presence of low endogenous PDE activity not only suppresses TNF-α but also positively regulates IL-10 production. Further investigation into the cAMP-dependent mechanism led to an interesting observation which described ATF-1 activation by LPS-stimulated macrophages whereas CREB was not activated; the addition of VIP abrogated this activation. The combined treatment of VIP+rolipram, however, resulted in activation of both CREB and ATF-1 upon LPS stimulation. It is possible that, in addition to NF-κB, the suppressive effect of VIP on TNF-α production is achieved through inhibition of ATF-1 activation, whereas activation of CREB by VIP upon PDE inhibition augments IL-10 production in a cell- and stimulus-specific manner.
Treatment of RA by neuropeptides such as VIP alone may not be efficacious but may be useful in combination with a PDE inhibitor. This, however, cannot be explained wholly by a failure to elevate cAMP, as treatment with dibutyryl cAMP and other cAMP-elevating drugs alone failed to induce monocyte, macrophage, and T-cell IL-10 but could, however, downregulate TNF-α upon stimulation and spontaneous TNF-α production by RA-SMCs. In the presence of an exogenous stimulus such as LPS for monocytes and macrophages and PMA/ionomycin for T cells, TNF-α production was downregulated whereas IL-10 was relatively unaffected. Of particular interest is the observation that elevation of cAMP augments macrophage IL-10 production with a concomitant potent inhibition of TNF-α upon PKC activation by PMA/ionomycin. This augmentation was not observed for VIP alone; however, treatment with VIP and simultaneous PDE inhibition by rolipram did augment IL-10 production by PMA/ionomycin-stimulated macrophages. This augmentation response is specific, as it was not reproducible in LPS-stimulated macrophages and stimulated T cells. This suggests that there is a complex relationship between the PKC and cAMP-dependent PKA pathways regulating macrophage IL-10 production and suggests that IL-10 augmentation is specific not only to cell type but also to the stimulus.
Comparing these data with those found in the mouse, the augmentation of IL-10 and downregulation of TNF-α and IFN-γ results in a Th2 cytokine profile that is dependent on cell types present, differentiation status, and the activation stimulus encountered. The effectiveness of VIP as an anti-inflammatory agent in vivo for CIA in the mouse contrasts starkly with in vitro human data. This may be due to differences between murine and human cells but it is also possible that VIP activates regulatory circuits in vivo that are not seen in vitro. The differences in VIP sensitivity between these two systems requires further work and is currently being investigated in our laboratory. Cyclic-AMP levels can be raised by increasing the activity of adenylate cyclase or by decreasing the activity of phosphodiesterases. In the light of the observation that a physiological activator of adenylate cyclase, like VIP, failed to modulate cytokine production by RA synovial cells, probably due to high PDE activity in these cells, our findings suggest that a PDE inhibitor is more likely to be effective in human RA than an activator of adenylate cyclase. However, the possibility of a combined approach using VIP together with a PDE inhibitor merits further investigation.
This report has demonstrated that, in humans, VIP is relatively ineffective as an anti-inflammatory mediator capable of augmenting IL-10 while concurrently inhibiting TNF-α production. This, however, is specific to cell type and to the stimulus and is dependent on endogenous PDE activity. Macrophages stimulated by phorbol ester in the presence of the PDE-resistant dibutyryl cAMP augment IL-10 and strongly suppress TNF-α. No such modulation of anti-inflammatory IL-10 was observed in T cells or macrophages stimulated by LPS. These data suggest efficacy for a combination of VIP and a phosphodiesterase inhibitor.
This work has been funded by a Wellcome Trust project grant and the Kennedy Institute of Rheumatology is supported by a core grant from the Arthritis and Rheumatism Council of Great Britain.
- 9.Delgado M, Martinez C, Pozo D, Calvo JR, Leceta J, Ganea D, Gomariz RP: Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) protect mice from lethal endotoxemia through the inhibition of TNF-a and IL-6. J Immunol. 1999, 162: 1200-1205.PubMedGoogle Scholar
- 10.Delgado M, Munoz-Elias EJ, Kan Y, Gozes I, Fridkin M, Brenneman DE, Gomariz RP, Ganea D: Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit tumour necrosis factor alpha transcriptional activation by regulating nuclear factor-kB and cAMP response element-binding protein/c-Jun. J Biol Chem. 1998, 273: 31427-31436. 10.1074/jbc.273.47.31427.CrossRefPubMedGoogle Scholar
- 15.Delgado M, Ganea D: Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide prevent inducible nitric oxide synthase transcription in macrophages by inhibiting NF-kB and IFN regulatory factor 1 activation. J Immunol. 1991, 162: 4685-4696.Google Scholar
- 25.Hernanz A, Tato E, De la Fuente M, de Miguel E, Arnalich F: Differential effects of gastrin-releasing peptide, neuropeptide Y, somatostatin and vasoactive intestinal peptide on interleukin-1 beta, interleukin-6 and tumour necrosis factor-alpha production by whole blood cells from healthy young and old subjects. J Neuroimmunol. 1996, 71: 25-30. 10.1016/S0165-5728(96)00118-X.CrossRefPubMedGoogle Scholar
- 26.Foxwell B, Brown K, Bondeson J, Clarke C, de Martin R, Brennan F, Feldmann M: Efficient adenoviral infection with IkappaB alpha reveals that macrophage tumour necrosis factor alpha production in rheumatoid arthritis is NF-kappaB dependent. Proc Natl Acad Sci USA. 1998, 95: 8211-8215. 10.1073/pnas.95.14.8211.PubMedCentralCrossRefPubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.