Adrenomedullin Is Both Proinflammatory and Antiinflammatory: Its Effects on Gene Expression and Secretion of Cytokines and Macrophage Migrat
http://www.100md.com
内分泌学杂志 2005年第3期
Departments of Medicine (L.Y.F.W., B.M.Y.C.) and Physiology (Y.-Y.L., F.T.), The University of Hong Kong, Hong Kong, China
Address all correspondence and requests for reprints to: Dr. Louisa Y. F. Wong, Department of Medicine, The University of Hong Kong, Pokfulam, Hong Kong. E-mail: lyfwong@hkucc.hku.hk.
Abstract
Adrenomedullin (ADM) is a potent vasorelaxant peptide that plays important roles in cardiovascular homeostasis and inflammatory response. ADM derived from macrophages is one of the major sources of ADM that is produced in the inflammatory process. To assess the functions of ADM in inflammation, we studied the temporal changes in ADM production and its effect on secretion of macrophage migration inhibitory factor (MIF) and cytokine response of NR8383 rat macrophages activated by lipopolysaccharide (LPS). NR8383 cells were stimulated by LPS in the absence and presence of exogenous ADM, and the concentrations of ADM, MIF, and proinflammatory cytokines (IL-6, TNF-, and IL-1?) in the culture media and gene expressions of the cells were measured. We confirmed that the secretion and mRNA expression of ADM in the macrophages were markedly increased by LPS. ADM increased initial secretion of MIF and IL-1? from both nonstimulated and LPS-stimulated cells, and it also increased basal and LPS-induced IL-6 secretion of the cells by 2- to 15-fold. However, it reduced secretion of TNF- from LPS-stimulated cells by 34–56%. Our results suggest that ADM modulates MIF secretion and cytokine production and plays important roles in both the initiation and propagation of the inflammatory response.
Introduction
ADRENOMEDULLIN (ADM) IS a potent vasorelaxant peptide originally isolated from extracts of human pheochromocytoma (1). ADM has multiple regulatory functions, the most distinctive of which arises from its vasodilatory and hypotensive effect. ADM relaxes vascular smooth muscle cells (VSMCs) through the elevation of intracellular cAMP (2, 3). It also acts on endothelial cells (ECs) by activating adenylate cyclase and nitric oxide synthase, resulting in dilation of blood vessels (4, 5). ADM circulates in plasma in picomolar concentrations, and its plasma level is increased in a variety of diseases, including hypertension (6), congestive heart failure (7), acute myocardial infarction (8, 9), and septic shock (10).
ADM secretion is raised in various experimental and clinical infections (11, 12) as well as in inflammatory conditions (13, 14, 15). Lipopolysaccharide (LPS) is a main component of bacterial endotoxin. Release of LPS from dying bacteria initiates the production of a cascade of proinflammatory mediators and a series of systemic inflammatory response to infection, resulting in septic shock (16). In rats, iv injection of LPS increased ADM expression in various tissues including lung, heart, liver, and kidney (17). Transgenic mice overexpressing ADM are resistant to septicemic shock (18). These findings suggest that ADM may have important roles in the pathophysiology of inflammatory response and endotoxic shock in addition to those for the regulation of vascular tone.
ADM is widely synthesized and secreted from most of the cells in the body including VSMCs, ECs, and fibroblasts, which generally secrete ADM at high rates (19). IL-1?, TNF-, and LPS potently stimulate ADM production in VSMCs, ECs, and fibroblasts (20, 21, 22). The macrophage is a major source of the cytokines that are induced in response to LPS administration (23). Production of ADM in macrophages and related cells is markedly induced by LPS and interferon- and, to a lesser extent, by differentiation and activation factors (phorbol ester and retinoic acid) (24, 25, 26) and accounts for a significant proportion of ADM produced during inflammatory processes.
Although ADM is up-regulated in conditions of inflammation and sepsis, it also exerts a regulatory effect on the production of the inflammatory cytokines, at least in macrophages (24) and fibroblasts (22). In this study, we aimed to study in cultured macrophages the time course of production of ADM and its effect on the production of the inflammatory cytokines, including IL-6, TNF-, and IL-1?. We also studied the effect of ADM on secretion of macrophage migration inhibitory factor (MIF), which is an important regulator of macrophage function and a critical mediator of inflammatory disease and septic shock (27, 28, 29). This may allow a better understanding of the role of ADM and interplay between ADM, MIF, and the inflammatory cytokines during inflammatory responses.
Materials and Methods
Materials
Eschericheria coli LPS (serotype 026:B6) and BSA were obtained from Sigma Chemical Co. (St. Louis, MO). RPMI 1640 medium and fetal bovine serum (FBS) were obtained from Gibco BRL (Gaithersburg, MD). Rat ADM, [125I]ADM, and ADM antiserum were obtained from Peninsula (Belmont, CA). Rabbit -globulin and goat antirabbit serum were obtained from Antibodies Inc. (Davis, CA).
Cell culture
NR8383 rat macrophages were obtained from American Type Culture Collection (Rockville, MD). The cells were cultured in RPMI 1640 medium containing 2 mM glutamine and 10% heat-inactivated FBS (RPMI/10% FBS) at 37 C in a humidified atmosphere containing 5% CO2. The cells were grown to confluence and passed weekly.
Preparation of conditioned medium
NR8383 cells at passages 11–20 were used and grown at 2 x 106 cells in each 25-cm2 culture flask. The cells were serum deprived for 24 h in the control medium containing 1% FBS in RPMI 1640 medium before they were stimulated by LPS or ADM.
Experiment 1: ADM production in macrophages
NR8383 cells were stimulated with LPS at 1–1000 ng/ml. For time-course experiments, parallel cultures were set up and conditioned media were removed at 1, 3, 6, and 24 h after stimulation and subjected to RIA for ADM. Cell pellets were harvested by centrifugation and subjected to quantification of ADM mRNA expression. To determine the effect of exogenous ADM on the expression level of ADM mRNA in macrophages, the cells were stimulated with ADM at 100 nM in the presence or absence of LPS at 1–1000 ng/ml, and the cells were harvested for quantification of ADM mRNA level.
Experiment 2: effect of ADM on production of inflammatory cytokines and MIF
NR8383 cells were stimulated with LPS (1–1000 ng/ml) in the presence or absence of ADM at 100 nM. The concentrations of IL-6, TNF-, and IL-1? in culture media were measured at 1, 3, 6, and 24 h of incubation using sandwich ELISAs specific for rat IL-6, TNF-, and IL-1? (R&D Systems Inc., Minneapolis, MN). The mRNA levels of the cytokines were determined by semiquantitative RT-PCR for cells stimulated by 100 ng/ml LPS with or without ADM at 3, 6, and 24 h.
To study the effect of ADM on MIF secretion, NR8383 cells were incubated with ADM at 0.1–100 nM in the presence or absence of LPS for 1–24 h. The MIF concentration in the culture medium was measured by a sandwich enzyme immunoassay specific for rat MIF (ChemiKine, Chemicon, Temecula, CA), and MIF mRNA expression was determined by RT-PCR.
RIA for ADM
Duplicate 100-μl samples were diluted with RIA buffer and incubated with 100 μl of [125I]ADM for the determination of immunoreactive (ir)-ADM concentration as described previously (30). Rat ADM, ADM antiserum, and 125I-labeled ADM were purchased from Peninsula. The detection limit of the assay was 5 pg ir-ADM per tube. The intra- and interassay coefficients of variation for ADM were 7 and 10%, respectively.
Quantification of mRNA expression using RT-PCR
Total RNA (2.5 μg) was extracted from the cells using the NucleoSpin RNA II kit (Clontech Laboratory, Inc., Palo Alto, CA) and transcribed into cDNA with the SuperScript II reverse transcriptase (Life Technologies, Inc., Carlsbad, CA) according to the manufacturer’s instructions. After RT, the mRNA levels of ADM, MIF, or the cytokines (IL-6, TNF-, and IL-1?) were measured by semiquantitative PCR with the corresponding forward and reverse primers as listed in Table 1. TATA-box binding protein (TBP) was used as an internal control to normalize the input cDNA level. The PCR profile for ADM and TBP consisted of initial activation for 15 min at 94 C followed by 27 cycles of PCR using denaturation at 94 C for 1 min, annealing at 56 C for 1 min, and extension at 72 C for 1 min, whereas that of TNF-, IL-6, IL-1?, and MIF consisted of 22 cycles of PCR with annealing temperature at 58 C instead of 56 C. The gene-specific PCR products were analyzed on 6% polyacrylamide gels together with the TBP PCR product using a gel documentation and analysis system (GeneGenius, Syngene Co., Cambridge, UK). The mRNA expression level of the test gene was expressed in arbitrary units after normalizing the band intensity with that of the corresponding TBP internal control.
TABLE 1. PCR primers for rat ADM, MIF, and inflammatory cytokines
Statistical analysis
All data were expressed as mean ± SEM. The data were analyzed using ANOVA, followed by a multiple comparison test (Scheffé test) with a statistical software program (SPSS for Windows, version 7.5, SPSS, Chicago, IL). A P value of <0.05 was used as the level of significance for all analyses.
Results
ADM production in macrophages
Ir-ADM was secreted by NR8383 cells without any stimulation and accumulated in the medium at a concentration of 17.5 ± 1.7 fmol/106 cells after 24 h of incubation, and the secretions of ir-ADM were increased by LPS stimulation (Fig. 1). Two-way ANOVA of the ir-ADM levels indicated significant effects of concentration (P < 0.001) and time (P < 0.001) of LPS treatment. LPS significantly increased ADM secretion by 4.8- to 20.8-fold of basal level at 10–1000 ng/ml, respectively (P < 0.01), but showed no effect at 1 ng/ml (Fig. 1). The ir-ADM levels at 100 and 1000 ng/ml LPS were significantly higher than those at 1 and 10 ng/ml LPS (P < 0.001), but there was no significant difference between that of 100 and 1000 ng/ml LPS. With the addition of 1000 ng/ml LPS, the level of ir-ADM at 24 h was significantly higher than those at 3 and 6 h (P < 0.001), but there was no significant difference between that of 3 and 6 h. ADM mRNA expression was estimated by semiquantitative RT-PCR after scanning densitometry and normalization to TBP expression, and the results are shown in Fig. 2, A and B. ADM mRNA was expressed constitutively in NR8383 cells at a low level (Fig. 2B, lane 1), but the expression was increased by LPS stimulation (Fig. 2B, lanes 3–10). Two-way ANOVA of the ADM mRNA levels also indicated significant effects of concentration (P < 0.001) and time (P < 0.001) of LPS treatment. The ADM mRNA levels at 10–1000 ng/ml LPS were significantly higher than that at 1 ng/ml LPS (P < 0.001), but there was no significant difference among those of 10, 100, and 1000 ng/ml LPS. ADM mRNA levels at 24 h of incubation were significantly higher than those at 3 and 6 h (P < 0.001) for LPS at 100 and 1000 ng/ml. As shown in Fig. 2A, the level of ADM mRNA was not changed at 3 h after LPS stimulation except for LPS at 1000 ng/ml in which it increased by 2-fold from basal level (P < 0.05). At 6 h, ADM mRNA expression was increased by 1.9- to 3.5-fold (P < 0.05) for LPS at 1–1000 ng/ml. At the end of 24 h, ADM mRNA level was increased by 7.5- to 11.5-fold for LPS at 10–1000 ng/ml LPS (P < 0.01), whereas it decreased back to near basal level in the 1 ng/ml LPS-stimulated cells. The mRNA levels paralleled the ADM peptide levels. Addition of exogenous ADM (at 100 nM) significantly increased ADM mRNA levels in both non-LPS-stimulated and 1 ng/ml LPS-stimulated cells at 6 h of stimulation (Fig. 2C). However, it did not affect ADM expression at other time intervals or for LPS at higher concentrations.
FIG. 1. ADM secretion of NR8383 cells stimulated with LPS. NR8383 cells were stimulated with LPS at 1–1000 ng/ml for 24 h. ADM concentrations in the culture media were measured at 1, 3, 6, and 24 h by RIA specific for ADM. Each value represents the mean ± SEM of four separate sets of experiments. *, P < 0.05; #, P < 0.01 vs. no LPS added.
FIG. 2. RT-PCR analysis of ADM gene expression in NR8383 cells. ADM mRNA level in NR8383 cells was estimated at 3, 6, or 24 h after stimulation with LPS with or without exogenous ADM (at 100 nM) by RT-PCR after normalizing with TBP mRNA level. Each value represents mean ± SEM of four separate sets of experiments. A, ADM mRNA of LPS-stimulated cells; *, P < 0.05; #, P < 0.01 vs. control with no LPS. B, Representative results of gene-specific PCR products of ADM and TBP mRNA at 6 h of incubation analyzed by gel electrophoresis. C, ADM mRNA at 6 h of incubation in the absence (closed bar) or presence (open bar) of exogenous ADM. *, P < 0.05 vs. LPS only.
Effect of ADM on cytokine productions
LPS induced cytokine production of IL-6, TNF-, and IL-1? in NR8383 cells in a dose-dependent manner, but the temporal changes in the rate of secretion were different for the three cytokines. The rate of IL-6 secretion started to increase at 3 h after LPS stimulation and was markedly elevated in the subsequent incubation period (Fig. 3A). ADM (at 100 nM) augmented IL-6 production 2.7-fold in non-LPS-stimulated cells and by 5.7-, 14.6-, 2.9-, and 1.8-fold in NR8383 cells stimulated by LPS at 1, 10, 100, and 1000 ng/ml, respectively (Fig. 3B). Expression levels of the mRNA of the cytokines were determined by RT-PCR after normalization to TBP expression. Figure 3C shows the representative results of gene-specific PCR products of IL-6, IL-1?, TNF-, and TBP analyzed by gel electrophoresis. IL-6 mRNA level was undetectable in the nonstimulated cells (Fig. 3C, lanes 1, 4, and 7) but was markedly induced by LPS at 3, 6, and 24 h of stimulation (Fig. 3C, lanes 2, 5, and 8). Coadministration of ADM and LPS further increased IL-6 mRNA level by 92 and 56% at 3 and 6 h of stimulation, respectively (P < 0.01), compared with LPS alone (Fig. 3D).
FIG. 3. Effect of ADM on IL-6 production in NR8383 cells. NR8383 cells were stimulated with LPS in the absence (closed bar) or presence (open bar) of exogenous ADM at 100 nM. IL-6 concentrations of the culture media were measured at 1, 3, 6, and 24 h by ELISA. A, Time course of secretion of IL-6. B, IL-6 concentrations at 6 h of incubation of non-LPS-stimulated cells (left) and LPS-stimulated cells (right); the cross-hatched bar represents the basal level of the control with no LPS or AM added. C, Representative results of gene-specific PCR products of IL-6, IL-1?, TNF-, and TBP analyzed by gel electrophoresis. D, IL-6 mRNA level was estimated by RT-PCR after normalizing with TBP mRNA level. Each value represents the mean ± SEM of four separate sets of experiments. *, P < 0.05; #, P < 0.01 vs. LPS only.
The basal secretion level of TNF- from NR8383 cells was below the detection limit (15 pg/ml). No measurable change in TNF- concentration in the culture medium was obtained when the cells were incubated with ADM alone. LPS induced production of TNF- in NR8383 cells, and TNF- concentration was rapidly increased upon LPS stimulation and continued to increase for 6 h before it started to level off (Fig. 4A). ADM (at 100 nM) significantly suppressed TNF- production from LPS-stimulated cells, by 34–56% compared with LPS alone (Fig. 4B). The inhibitory effect was observed even at high concentrations of LPS. Expression of TNF- mRNA was highly induced at 3 and 6 h of LPS stimulation but was reduced to near basal level at 24 h (Fig. 4C). Addition of exogenous ADM did not affect TNF- expression of LPS-stimulated NR8383 cells at 3 and 6 h but significantly reduced it at 24 h (Fig. 4C).
FIG. 4. Effect of ADM on TNF- production in NR8383 cells. NR8383 cells were stimulated with LPS in the absence (closed bar) or presence (open bar) of exogenous ADM at 100 nM. TNF- concentrations of the culture media were measured at 1, 3, 6, and 24 h by ELISA. A, Time course of secretion of TNF-; B, TNF- concentrations at 3 h of incubation; C, TNF- mRNA level was measured by RT-PCR after normalizing with TBP mRNA level; the cross-hatched bar represents the basal level of the control with no LPS or AM added. Each value represents the mean ± SEM of four separate sets of experiments. *, P < 0.05; #, P < 0.01 vs. LPS only.
LPS increased IL-1? secretion from NR8383 cells rapidly upon stimulation, but the rate of secretion gradually decreased after 6 h of incubation (Fig. 5A). ADM (at 100 nM) enhanced initial secretion of IL-1? for both non-LPS-stimulated and LPS-stimulated cells (P < 0.05) at 1 h of incubation (Fig. 5B) but slightly reduced IL-1? concentration of 1 ng/ml LPS-stimulated cells at 24 h of incubation (Fig. 5C). IL-1? mRNA expression was elevated by LPS, but no significant effect of exogenous ADM on IL-1? mRNA expression was observed in the 100 ng/ml LPS-stimulated cells (Fig. 5D).
FIG. 5. Effect of ADM on IL-1? production in NR8383 cells. NR8383 cells were stimulated with LPS in the absence (closed bar) or presence (open bar) of exogenous ADM at 100 nM. IL-1? concentrations of the culture media were measured at 1, 3, 6, and 24 h by ELISA. A, Time course of secretion of IL-1?; B, IL-1? concentrations at 1 h of incubation; C, IL-1? concentrations at 24 h of incubation; D, IL-1? mRNA level was measured by RT-PCR after normalizing with TBP mRNA level; the cross-hatched bar represents the basal level of the control with no LPS or AM added. Each value represents the mean ± SEM of four separate sets of experiments. *, P < 0.05; #, P < 0.01 vs. LPS only.
Effect of ADM on secretion and expression of MIF
MIF was secreted constitutively from NR8383 cells even without LPS stimulation, and LPS significantly increased MIF content in the media at the concentrations of 10–1000 ng/ml (Fig. 6A). As shown in the figure, MIF concentrations increased rapidly in the first hour of stimulation, but the rate of secretion decreased gradually in the subsequent incubation period. At the end of 24 h incubation, MIF concentrations were increased by 44–56% by LPS at 10–1000 ng/ml (P < 0.05). ADM (at 0.1–100 nM) significantly increased initial MIF secretion of NR8383 cells by 2.2- to 3.2-fold (P < 0.05) after the first hour of incubation (Fig. 6B) but did not affect the MIF concentration at 24 h (Fig. 6C). However, when ADM was administered simultaneously with LPS (at 100 ng/ml), there was no significant effect of ADM on MIF secretion of the LPS-stimulated cells except at a high concentration of ADM (at 100 nM) that increased initial MIF concentration by 41% (P < 0.05) compared with LPS alone (Fig. 6B). Figure 7 shows the results of MIF mRNA expression in nonstimulated cells and the cells stimulated by LPS (100 ng/ml) alone or LPS with ADM at 100 nM. MIF mRNA was expressed at a significant level in NR8383 cells even without any stimulation and was significantly increased by LPS at 6 and 24 h of stimulation. However, there was no significant effect of exogenous ADM (at 100 nM) on MIF mRNA expression in the LPS-stimulated cells.
FIG. 6. MIF secretion from NR8383 cells stimulated with ADM and LPS. NR8383 cells were stimulated with LPS and/or ADM at 0.1–100 nM. MIF concentration of the culture medium was measured at 1, 3, 6, and 24 h by ELISA specific for MIF. A, Time course of secretion of MIF from LPS-stimulated cells; B and C, MIF concentrations at 1 h (B) and at 24 h (C) of the cells stimulated by ADM alone (closed bar) or ADM and 100 ng/ml LPS (open bar). Each value represents the mean ± SEM of three to four separate sets of experiments. *, P < 0.05 vs. control with no ADM or LPS; #, P < 0.05 vs. LPS only.
FIG. 7. Semiquantitative RT-PCR analysis of MIF gene expression in NR8383 cells. NR8383 cells were stimulated with 100 ng/ml LPS in the absence (closed bar) or presence (open bar) of ADM at 100 nM. Expression levels of MIF mRNA were measured at 1, 3, 6, and 24 h by RT-PCR after normalizing with TBP mRNA level. Each value represents the mean ± SEM of three to four separate sets of experiments. *, P < 0.05; #, P < 0.01 vs. control with no LPS or ADM added.
Discussion
In this study, it was shown that ADM production and gene expression were dose-dependently and time-dependently stimulated by LPS in cultured NR8383 macrophages. This is consistent with the observations of other investigators that ADM gene expression is maintained in a suppressed state in resting macrophages and up-regulated by LPS (24, 25, 26). LPS induced a significant elevation of ADM production in NR8383 cells at a threshold dose of 10 ng/ml. This was similar to the findings of Isumi et al. (21) on ADM secretion from rat ECs. The temporal change of LPS-induced ADM production in NR8383 cells was similar to that obtained in RAW264.7 macrophages (24). The production level of ADM in NR8383 cells under 100 ng/ml LPS stimulation was approximately 1.29 fmol/105 cells·h as an average over a 24-h incubation. This is approximately three times higher than that of cultured RAW264.7 macrophages (24) but comparable to that of cultured VSMCs and ECs (20, 21). Our results confirm that the macrophage is another important source of ADM when activated by inflammatory stimulus such as LPS. The difference in the secretion rates is probably because of the difference in the macrophage cell lines that were used.
In NR8383 cells, LPS stimulated production of inflammatory cytokines, including TNF-, IL-1?, and IL-6, at concentrations as low as 1 ng/ml, but there was a distinctive difference in the time of induction of cytokine secretion among the different cytokines. There were rapid increases in secretion of IL-1? as early as 1 h and of TNF- at 3 h, but they started to reduce at 6 h after LPS stimulation. On the other hand, similar to the secretion of ADM, secretion of IL-6 in the LPS-stimulated cells was elevated much more significantly at 6–24 h of incubation. Strong induction of IL-1? and TNF- mRNA occurred well before that of ADM. Although it has been reported that TNF- and IL-1? show weak or no direct effect in ADM secretion in monocytes and macrophages (24, 25), they have been shown to increase ADM secretion additively with LPS in ECs when coadministered with LPS (21). The results of our time-course study demonstrate that the induction of IL-1? and TNF- precedes that of ADM and may suggest a possible contribution of these two cytokines to the induction of ADM gene expression in the macrophages when provided with a second triggering signal such as LPS.
MIF is an important mediator of the inflammatory response and is present at a high level in resting macrophages (31). It was shown in our results that MIF was constitutively expressed in NR8383 cells, and both its gene expression and secretion were elevated by LPS stimulation. Gene expression of MIF was increased by LPS at 6–24 h after stimulation, probably to replenish the storage pool of MIF in the cells. ADM significantly increased the initial release of MIF in both nonstimulated and LPS-stimulated cells. It also increased initial IL-1? secretion in resting and LPS-stimulated NR8383 cells. Because MIF and IL-1? are potent mediators of the inflammatory response, our results suggest that ADM may play a role as a proinflammatory factor in the initiation of the inflammatory response through the stimulation of MIF and IL-1? secretion.
On the other hand, ADM was found to continually suppress TNF- production in LPS-stimulated NR8383 cells up to 24 h, and TNF- gene expression was reduced by ADM at 24 h of incubation. Our results agree with the previous findings of an inhibitory effect of ADM on TNF- production in the macrophages (24). However, it was shown in our study that ADM markedly increased production of IL-6 in both nonstimulated and LPS-stimulated NR8383 cells. Our findings contrast with the results of Kubo et al. (24) where a reduction of IL-6 production was observed. The discrepancy may be because of the difference in the concentration of ADM that was used for stimulation and the different properties of the cells used. The stimulatory effect of ADM on IL-6 production has previously been reported in fibroblasts (22) but not in macrophages or other cell types. Based on our findings, LPS, in addition to directly increasing IL-6 production, may have an indirect effect on IL-6 production through ADM synthesis and secretion, as supported by a simultaneous increase in IL-6 and ADM in the LPS-stimulated cells. The increase in IL-6 then may have an inhibitory effect on the synthesis and secretion of TNF- and IL-1? because IL-6 has been shown to inhibit LPS-induced TNF- production in cultured macrophages (32) and suppress IL-1? and TNF- production in peripheral blood mononuclear cells (33). TNF- and IL-1? are proinflammatory, whereas IL-6 is antiinflammatory (34). Thus, our results indicate that ADM may also function as a peptidergic regulator of the inflammatory reactions through the inhibition of TNF- and stimulation of IL-6 and strongly support the notion that ADM is an antiinflammatory factor that suppresses the progression of inflammation. The promoter region of the ADM gene contains a consensus sequence for the nuclear factor NF-IL6 (35) that is induced by the stimulation with LPS, TNF-, IL-1, and IL-6 (36, 37). Therefore, although ADM can be used as a feedback regulator for the production of the inflammatory cytokines, it may also self-regulate its expression by either increasing secretion of its inducer, IL-1?, or suppressing its own inducer, TNF- .
The present study shows that ADM gene expression and secretion were elevated in NR8383 rat macrophages after LPS stimulation. The results suggest that ADM is both proinflammatory and antiinflammatory and that it plays a functional role in the initiation as well as progression of the inflammatory response by enhancing initial release of MIF and IL-1? and subsequently by inhibiting TNF- and stimulating IL-6 production. When overproduced in extreme inflammatory conditions, ADM may lead to feedback inhibition of TNF- and IL-1? production directly or through stimulating IL-6 production. Because TNF- and IL-1? are mediators of endotoxic shock, overexpression of ADM may therefore protect the host against the toxicity of endotoxin administration. The data shown here indicate that ADM stimulates secretion of MIF and modulates production of inflammatory cytokines in rat macrophages, and its role in the inflammatory process changes with time after the onset of inflammatory challenge.
References
Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, Eto T 1993 Adrenomedullin: a novel hypotensive peptide isolated from human phaeochromocytoma. Biochem Biophys Res Commun 192:553–560
Eguchi S, Hirata Y, Kano H, Sato K, Watanabe Y, Watanabe TX, Nakajima K, Sakakibara S, Marumo F 1994 Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells. FEBS Lett 340:226–230
Ishizaka Y, Ishizaka Y, Tanaka M, Kitamura K, Kangawa K, Minamino N, Matsuo H, Eto T 1994 Adrenomedullin stimulates cyclic AMP formation in rat smooth muscle cells. Biochem Biophys Res Commun 200:642–646
Kato J, Kitamura K, Kangawa K, Eto T 1995 Receptors for adrenomedullin in human vascular endothelial cells. Eur J Pharmacol 289:383–385
Shimekake Y, Nagata K, Ohta S, Kambayashi Y, Teraoka H, Kitamura K, Eto T, Kangawa K, Matsuo H 1995 Adrenomedullin stimulates two signal transduction pathways, cAMP accumulation and Ca2+ mobilization, in bovine aortic endothelial cells. J Biol Chem 270:4412–4417
Ishimitsu T, Nishikimi T, Saito Y, Kitamura K, Eto T, Kangawa K, Matsuo H, Omae T, Matsuoka H 1994 Plasma levels of adrenomedullin, a newly identified hypotensive peptide, in patients with hypertension and renal failure. J Clin Invest 94:2158–2161
Jougasaki M, Wei CM, McKinley LJ, Burnett Jr JC 1995 Elevation of circulating and ventricular adrenomedullin in human congestive heart failure. Circulation 92:286–289
Kobayashi K, Kitamura K, Hirayama N, Date H, Kashiwagi T, Ikushima I, Hanada Y, Nagatomo Y, Takenaga M, Ishikawa T, Imamura T, Koiwaya Y, Eto T 1996 Increased plasma adrenomedullin in acute myocardial infarction. Am Heart J 131:676–680
Yoshitomi Y, Nishikimi T, Kojima S, Kuramochi M, Takishita S, Matsuoka H, Miyata A, Matsuo H, Kangawa K 1998 Plasma levels of adrenomedullin in patients with acute myocardial infarction. Clin Sci 94:135–139
Nishio K, Akai Y, Murao Y, Doi N, Ueda S, Tabuse H, Miyamoto S, Dohi K, Minamino N, Shoji H, Kitamura K, Kangawa K, Matsuo H 1997 Increased plasma concentrations of adrenomedullin correlate with relaxation of vascular tone in patients with septic shock. Crit Care Med 25:953–957
Shoji H, Minamino N, Kangawa K, Matsuo H 1995 Endotoxin markedly elevates plasma concentration and gene transcription of adrenomedullin in rat. Biochem Biophys Res Commun 215:531–537
Elsasser TH, Sartin JL, Martinez A, Kahl S, Montuenga L, Pio R, Fayer R, Miller M, Cuttitta F 1999 Underlying disease stress augments plasma and tissue adrenomedullin (AM) responses to endotoxin: colocalized increases in AM and inducible nitric oxide synthase within pancreatic islets. Endocrinology 140:5402–5411
Hirata Y, Mitaka C, Sato K, Nagura T, Tsunoda Y, Amaha K, Marumo F 1996 Increased circulating adrenomedullin, a novel vasodilatory peptide, in sepsis. J Clin Endorinol Metab 81:1449–1453
Ueda S, Nishio K, Minamino N, Kubo A, Akai Y, Kangawa K, Matsuo H, Fugimura Y, Yoshikoa A, Masui K, Doi N, Murao Y, Miyamoto S 1999 Increased plasma levels of adrenomedullin in patients with systemic inflammatory response syndrome. Am J Respir Crit Care Med 160:132–136
Evereklioglu C, Yurekli M, Er H, Ozbec E, Hazneci E, Cekmen M, Inaloz HS 2000 Increased plasma adrenomedullin levels in patients with Behcet’s disease. Dermatology 201:312–315
Manthey CL, Vogel SN 1992 The role of cytokines in host response to endotoxin. Rev Med Microbiol 3:72–79
Shindo T, Kurihara H, Kurihara Y, Morita H, Yazaki Y 1998 Upregulation of endothelin-1 and adrenomedullin gene expression in the mouse endotoxin shock model. J Cardiovasc Pharmacol 31:S541–S544
Shindo T, Kurihara H, Maemura K, Kurihara Y, Kuwaki T, Izumida T, Minamino N, Ju KH, Morita H, Oh-hashi Y, Kumada M, Kangawa K, Nagai R, Yazaki Y 2000 Hypotension and resistance to lipopolysaccharide-induced shock in transgenic mice overexpressing adrenomedullin in their vasculature. Circulation 101:2309–2316
Tomoda Y, Isumi Y, Katafuchi T, Minamino Naoto 2001 Regulation of adrenomedullin secretion from cultured cells. Peptides 22:1783–1794
Sugo S, Minamino N, Shoji H, Kangawa K, Kitamura K, Eto T, Matsuo H 1995 Interleukin-1, tumor necrosis factor and lipopolysaccharide additively stimulate production of adrenomedullin in vascular smooth muscle cells. Biochem Biophys Res Commun 207:25–32
Isumi Y, Shoji H, Sugo S, Tochimoto T, Yoshioka M, Kangawa K, Matsuo H, Minamino N 1998 Regulation of adrenomedullin production in rat endothelial cells. Endocrinology 139:838–846
Isumi Y, Minamino N, Kubo A, Nishimoto N, Yoshizaki K, Yoshioka M, Kangawa K, Matsuo H 1998 Adrenomedullin stimulates interleukin-6 production in Swiss 3T3 cells. Biochem Biophys Res Commun 244:325–331
Nathan CF 1987 Secretory products of macrophages. J Clin Invest 79:319–326
Kubo A, Minamino N, Isumi Y, Katafuchi T, Kangawa K, Dohi K, Matsuo H 1998 Production of adrenomedullin in macrophage cell line and peritoneal macrophage. J Biol Chem 273:16730–16738
Kubo A, Minamino N, Isumi Y, Kangawa K, Dohi K, Matsuo H 1998 Adrenomedullin production is correlated with differentiation in human leukemia cell lines and peripheral blood monocytes. FEBS Lett. 426:233–237
Zaks-Zilberman M, Salkowski CA, Elsasser T, Cuttitta F, Vogel S 1998 Induction of adrenomedullin mRNA and protein by lipopolysaccharide and paclitaxel (Taxol) in murine macrophages. Infect Immunol 66:4669–4675
Bernhagen J, Calandra T, Mitchell RA, Martin SB, Tracey KJ, Voelter W, Manogue KR, Cerami A, Bucala R 1993 MIF is a pituitary-derived cytokine that potentiates lethal endotoxaemia. Nature 365:756–759
Bernhagen J, Calandra T, Bucala R 1998 Regulation of the immune response by macrophage migration inhibitory factor: biological and structural features. J Mol Med 76:151–161
Donnelly SC, Bucala R 1998 Macrophage migration inhibitory factor: a regulator of glucocorticoid activity with a critical role in inflammatory disease. Mol Med Today 3:502–507
Hwang ISS, Tang F 1999 The distribution and gene expression of adrenomedullin in the rat brain: peptide/mRNA and precursor/active peptide relationships. Neurosci Lett 267:85–88
Calandra T, Bernhagen J, Mitchell RA, Bucala R 1994 The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor. J Exp Med 179:1895–1902
Di Santo E, Alonzi T, Poli V, Fattori E, Toniatti C, Sironi M, Ricciardi-Castagnoli P, Ghezzi P 1997 Differential effects of IL-6 on systemic and central production of TNF: a study with IL-6-deficient mice. Cytokine 9:300–306
Schindler R, Mancilla J, Endres S, Ghorbani R, Clark SC, Dinarello CA 1990 Correlations and interactions in the production of interleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. Blood 75:40–47
Tilg H, Dinarello CA, Mier JW 1997 IL-6 and APPs: anti-inflammatory and immunosuppressive mediators. Immunol Today 18:428–432
Ishimitsu T, Miyata A, Matsuoka H, Kangawa K 1998 Transcriptional regulation of human adrenomedullin gene in vascular endothelial cells. Biochem Biophys Res Commun 243:463–470
Akira S, Isshiki H, Sugita T, Tanabe O, Kinoshita S, Nishio Y, Nakajima T, Hirano T, Kishimoto T 1990 A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J 9:1897–1906
Kiehntpof M, Herrmann F, Brach MA 1995 Functional NF-IL-6/CCAAT enhancer-binding protein is required for tumor necrosis factor -inducible expression of the granulocyte colony-stimulating factor (CSF), but not the granulocyte/macrophage CSF or interleukin 6 gene in human fibroblasts. J Exp Med 181:793–798(Louisa Y. F. Wong, Bernar)
Address all correspondence and requests for reprints to: Dr. Louisa Y. F. Wong, Department of Medicine, The University of Hong Kong, Pokfulam, Hong Kong. E-mail: lyfwong@hkucc.hku.hk.
Abstract
Adrenomedullin (ADM) is a potent vasorelaxant peptide that plays important roles in cardiovascular homeostasis and inflammatory response. ADM derived from macrophages is one of the major sources of ADM that is produced in the inflammatory process. To assess the functions of ADM in inflammation, we studied the temporal changes in ADM production and its effect on secretion of macrophage migration inhibitory factor (MIF) and cytokine response of NR8383 rat macrophages activated by lipopolysaccharide (LPS). NR8383 cells were stimulated by LPS in the absence and presence of exogenous ADM, and the concentrations of ADM, MIF, and proinflammatory cytokines (IL-6, TNF-, and IL-1?) in the culture media and gene expressions of the cells were measured. We confirmed that the secretion and mRNA expression of ADM in the macrophages were markedly increased by LPS. ADM increased initial secretion of MIF and IL-1? from both nonstimulated and LPS-stimulated cells, and it also increased basal and LPS-induced IL-6 secretion of the cells by 2- to 15-fold. However, it reduced secretion of TNF- from LPS-stimulated cells by 34–56%. Our results suggest that ADM modulates MIF secretion and cytokine production and plays important roles in both the initiation and propagation of the inflammatory response.
Introduction
ADRENOMEDULLIN (ADM) IS a potent vasorelaxant peptide originally isolated from extracts of human pheochromocytoma (1). ADM has multiple regulatory functions, the most distinctive of which arises from its vasodilatory and hypotensive effect. ADM relaxes vascular smooth muscle cells (VSMCs) through the elevation of intracellular cAMP (2, 3). It also acts on endothelial cells (ECs) by activating adenylate cyclase and nitric oxide synthase, resulting in dilation of blood vessels (4, 5). ADM circulates in plasma in picomolar concentrations, and its plasma level is increased in a variety of diseases, including hypertension (6), congestive heart failure (7), acute myocardial infarction (8, 9), and septic shock (10).
ADM secretion is raised in various experimental and clinical infections (11, 12) as well as in inflammatory conditions (13, 14, 15). Lipopolysaccharide (LPS) is a main component of bacterial endotoxin. Release of LPS from dying bacteria initiates the production of a cascade of proinflammatory mediators and a series of systemic inflammatory response to infection, resulting in septic shock (16). In rats, iv injection of LPS increased ADM expression in various tissues including lung, heart, liver, and kidney (17). Transgenic mice overexpressing ADM are resistant to septicemic shock (18). These findings suggest that ADM may have important roles in the pathophysiology of inflammatory response and endotoxic shock in addition to those for the regulation of vascular tone.
ADM is widely synthesized and secreted from most of the cells in the body including VSMCs, ECs, and fibroblasts, which generally secrete ADM at high rates (19). IL-1?, TNF-, and LPS potently stimulate ADM production in VSMCs, ECs, and fibroblasts (20, 21, 22). The macrophage is a major source of the cytokines that are induced in response to LPS administration (23). Production of ADM in macrophages and related cells is markedly induced by LPS and interferon- and, to a lesser extent, by differentiation and activation factors (phorbol ester and retinoic acid) (24, 25, 26) and accounts for a significant proportion of ADM produced during inflammatory processes.
Although ADM is up-regulated in conditions of inflammation and sepsis, it also exerts a regulatory effect on the production of the inflammatory cytokines, at least in macrophages (24) and fibroblasts (22). In this study, we aimed to study in cultured macrophages the time course of production of ADM and its effect on the production of the inflammatory cytokines, including IL-6, TNF-, and IL-1?. We also studied the effect of ADM on secretion of macrophage migration inhibitory factor (MIF), which is an important regulator of macrophage function and a critical mediator of inflammatory disease and septic shock (27, 28, 29). This may allow a better understanding of the role of ADM and interplay between ADM, MIF, and the inflammatory cytokines during inflammatory responses.
Materials and Methods
Materials
Eschericheria coli LPS (serotype 026:B6) and BSA were obtained from Sigma Chemical Co. (St. Louis, MO). RPMI 1640 medium and fetal bovine serum (FBS) were obtained from Gibco BRL (Gaithersburg, MD). Rat ADM, [125I]ADM, and ADM antiserum were obtained from Peninsula (Belmont, CA). Rabbit -globulin and goat antirabbit serum were obtained from Antibodies Inc. (Davis, CA).
Cell culture
NR8383 rat macrophages were obtained from American Type Culture Collection (Rockville, MD). The cells were cultured in RPMI 1640 medium containing 2 mM glutamine and 10% heat-inactivated FBS (RPMI/10% FBS) at 37 C in a humidified atmosphere containing 5% CO2. The cells were grown to confluence and passed weekly.
Preparation of conditioned medium
NR8383 cells at passages 11–20 were used and grown at 2 x 106 cells in each 25-cm2 culture flask. The cells were serum deprived for 24 h in the control medium containing 1% FBS in RPMI 1640 medium before they were stimulated by LPS or ADM.
Experiment 1: ADM production in macrophages
NR8383 cells were stimulated with LPS at 1–1000 ng/ml. For time-course experiments, parallel cultures were set up and conditioned media were removed at 1, 3, 6, and 24 h after stimulation and subjected to RIA for ADM. Cell pellets were harvested by centrifugation and subjected to quantification of ADM mRNA expression. To determine the effect of exogenous ADM on the expression level of ADM mRNA in macrophages, the cells were stimulated with ADM at 100 nM in the presence or absence of LPS at 1–1000 ng/ml, and the cells were harvested for quantification of ADM mRNA level.
Experiment 2: effect of ADM on production of inflammatory cytokines and MIF
NR8383 cells were stimulated with LPS (1–1000 ng/ml) in the presence or absence of ADM at 100 nM. The concentrations of IL-6, TNF-, and IL-1? in culture media were measured at 1, 3, 6, and 24 h of incubation using sandwich ELISAs specific for rat IL-6, TNF-, and IL-1? (R&D Systems Inc., Minneapolis, MN). The mRNA levels of the cytokines were determined by semiquantitative RT-PCR for cells stimulated by 100 ng/ml LPS with or without ADM at 3, 6, and 24 h.
To study the effect of ADM on MIF secretion, NR8383 cells were incubated with ADM at 0.1–100 nM in the presence or absence of LPS for 1–24 h. The MIF concentration in the culture medium was measured by a sandwich enzyme immunoassay specific for rat MIF (ChemiKine, Chemicon, Temecula, CA), and MIF mRNA expression was determined by RT-PCR.
RIA for ADM
Duplicate 100-μl samples were diluted with RIA buffer and incubated with 100 μl of [125I]ADM for the determination of immunoreactive (ir)-ADM concentration as described previously (30). Rat ADM, ADM antiserum, and 125I-labeled ADM were purchased from Peninsula. The detection limit of the assay was 5 pg ir-ADM per tube. The intra- and interassay coefficients of variation for ADM were 7 and 10%, respectively.
Quantification of mRNA expression using RT-PCR
Total RNA (2.5 μg) was extracted from the cells using the NucleoSpin RNA II kit (Clontech Laboratory, Inc., Palo Alto, CA) and transcribed into cDNA with the SuperScript II reverse transcriptase (Life Technologies, Inc., Carlsbad, CA) according to the manufacturer’s instructions. After RT, the mRNA levels of ADM, MIF, or the cytokines (IL-6, TNF-, and IL-1?) were measured by semiquantitative PCR with the corresponding forward and reverse primers as listed in Table 1. TATA-box binding protein (TBP) was used as an internal control to normalize the input cDNA level. The PCR profile for ADM and TBP consisted of initial activation for 15 min at 94 C followed by 27 cycles of PCR using denaturation at 94 C for 1 min, annealing at 56 C for 1 min, and extension at 72 C for 1 min, whereas that of TNF-, IL-6, IL-1?, and MIF consisted of 22 cycles of PCR with annealing temperature at 58 C instead of 56 C. The gene-specific PCR products were analyzed on 6% polyacrylamide gels together with the TBP PCR product using a gel documentation and analysis system (GeneGenius, Syngene Co., Cambridge, UK). The mRNA expression level of the test gene was expressed in arbitrary units after normalizing the band intensity with that of the corresponding TBP internal control.
TABLE 1. PCR primers for rat ADM, MIF, and inflammatory cytokines
Statistical analysis
All data were expressed as mean ± SEM. The data were analyzed using ANOVA, followed by a multiple comparison test (Scheffé test) with a statistical software program (SPSS for Windows, version 7.5, SPSS, Chicago, IL). A P value of <0.05 was used as the level of significance for all analyses.
Results
ADM production in macrophages
Ir-ADM was secreted by NR8383 cells without any stimulation and accumulated in the medium at a concentration of 17.5 ± 1.7 fmol/106 cells after 24 h of incubation, and the secretions of ir-ADM were increased by LPS stimulation (Fig. 1). Two-way ANOVA of the ir-ADM levels indicated significant effects of concentration (P < 0.001) and time (P < 0.001) of LPS treatment. LPS significantly increased ADM secretion by 4.8- to 20.8-fold of basal level at 10–1000 ng/ml, respectively (P < 0.01), but showed no effect at 1 ng/ml (Fig. 1). The ir-ADM levels at 100 and 1000 ng/ml LPS were significantly higher than those at 1 and 10 ng/ml LPS (P < 0.001), but there was no significant difference between that of 100 and 1000 ng/ml LPS. With the addition of 1000 ng/ml LPS, the level of ir-ADM at 24 h was significantly higher than those at 3 and 6 h (P < 0.001), but there was no significant difference between that of 3 and 6 h. ADM mRNA expression was estimated by semiquantitative RT-PCR after scanning densitometry and normalization to TBP expression, and the results are shown in Fig. 2, A and B. ADM mRNA was expressed constitutively in NR8383 cells at a low level (Fig. 2B, lane 1), but the expression was increased by LPS stimulation (Fig. 2B, lanes 3–10). Two-way ANOVA of the ADM mRNA levels also indicated significant effects of concentration (P < 0.001) and time (P < 0.001) of LPS treatment. The ADM mRNA levels at 10–1000 ng/ml LPS were significantly higher than that at 1 ng/ml LPS (P < 0.001), but there was no significant difference among those of 10, 100, and 1000 ng/ml LPS. ADM mRNA levels at 24 h of incubation were significantly higher than those at 3 and 6 h (P < 0.001) for LPS at 100 and 1000 ng/ml. As shown in Fig. 2A, the level of ADM mRNA was not changed at 3 h after LPS stimulation except for LPS at 1000 ng/ml in which it increased by 2-fold from basal level (P < 0.05). At 6 h, ADM mRNA expression was increased by 1.9- to 3.5-fold (P < 0.05) for LPS at 1–1000 ng/ml. At the end of 24 h, ADM mRNA level was increased by 7.5- to 11.5-fold for LPS at 10–1000 ng/ml LPS (P < 0.01), whereas it decreased back to near basal level in the 1 ng/ml LPS-stimulated cells. The mRNA levels paralleled the ADM peptide levels. Addition of exogenous ADM (at 100 nM) significantly increased ADM mRNA levels in both non-LPS-stimulated and 1 ng/ml LPS-stimulated cells at 6 h of stimulation (Fig. 2C). However, it did not affect ADM expression at other time intervals or for LPS at higher concentrations.
FIG. 1. ADM secretion of NR8383 cells stimulated with LPS. NR8383 cells were stimulated with LPS at 1–1000 ng/ml for 24 h. ADM concentrations in the culture media were measured at 1, 3, 6, and 24 h by RIA specific for ADM. Each value represents the mean ± SEM of four separate sets of experiments. *, P < 0.05; #, P < 0.01 vs. no LPS added.
FIG. 2. RT-PCR analysis of ADM gene expression in NR8383 cells. ADM mRNA level in NR8383 cells was estimated at 3, 6, or 24 h after stimulation with LPS with or without exogenous ADM (at 100 nM) by RT-PCR after normalizing with TBP mRNA level. Each value represents mean ± SEM of four separate sets of experiments. A, ADM mRNA of LPS-stimulated cells; *, P < 0.05; #, P < 0.01 vs. control with no LPS. B, Representative results of gene-specific PCR products of ADM and TBP mRNA at 6 h of incubation analyzed by gel electrophoresis. C, ADM mRNA at 6 h of incubation in the absence (closed bar) or presence (open bar) of exogenous ADM. *, P < 0.05 vs. LPS only.
Effect of ADM on cytokine productions
LPS induced cytokine production of IL-6, TNF-, and IL-1? in NR8383 cells in a dose-dependent manner, but the temporal changes in the rate of secretion were different for the three cytokines. The rate of IL-6 secretion started to increase at 3 h after LPS stimulation and was markedly elevated in the subsequent incubation period (Fig. 3A). ADM (at 100 nM) augmented IL-6 production 2.7-fold in non-LPS-stimulated cells and by 5.7-, 14.6-, 2.9-, and 1.8-fold in NR8383 cells stimulated by LPS at 1, 10, 100, and 1000 ng/ml, respectively (Fig. 3B). Expression levels of the mRNA of the cytokines were determined by RT-PCR after normalization to TBP expression. Figure 3C shows the representative results of gene-specific PCR products of IL-6, IL-1?, TNF-, and TBP analyzed by gel electrophoresis. IL-6 mRNA level was undetectable in the nonstimulated cells (Fig. 3C, lanes 1, 4, and 7) but was markedly induced by LPS at 3, 6, and 24 h of stimulation (Fig. 3C, lanes 2, 5, and 8). Coadministration of ADM and LPS further increased IL-6 mRNA level by 92 and 56% at 3 and 6 h of stimulation, respectively (P < 0.01), compared with LPS alone (Fig. 3D).
FIG. 3. Effect of ADM on IL-6 production in NR8383 cells. NR8383 cells were stimulated with LPS in the absence (closed bar) or presence (open bar) of exogenous ADM at 100 nM. IL-6 concentrations of the culture media were measured at 1, 3, 6, and 24 h by ELISA. A, Time course of secretion of IL-6. B, IL-6 concentrations at 6 h of incubation of non-LPS-stimulated cells (left) and LPS-stimulated cells (right); the cross-hatched bar represents the basal level of the control with no LPS or AM added. C, Representative results of gene-specific PCR products of IL-6, IL-1?, TNF-, and TBP analyzed by gel electrophoresis. D, IL-6 mRNA level was estimated by RT-PCR after normalizing with TBP mRNA level. Each value represents the mean ± SEM of four separate sets of experiments. *, P < 0.05; #, P < 0.01 vs. LPS only.
The basal secretion level of TNF- from NR8383 cells was below the detection limit (15 pg/ml). No measurable change in TNF- concentration in the culture medium was obtained when the cells were incubated with ADM alone. LPS induced production of TNF- in NR8383 cells, and TNF- concentration was rapidly increased upon LPS stimulation and continued to increase for 6 h before it started to level off (Fig. 4A). ADM (at 100 nM) significantly suppressed TNF- production from LPS-stimulated cells, by 34–56% compared with LPS alone (Fig. 4B). The inhibitory effect was observed even at high concentrations of LPS. Expression of TNF- mRNA was highly induced at 3 and 6 h of LPS stimulation but was reduced to near basal level at 24 h (Fig. 4C). Addition of exogenous ADM did not affect TNF- expression of LPS-stimulated NR8383 cells at 3 and 6 h but significantly reduced it at 24 h (Fig. 4C).
FIG. 4. Effect of ADM on TNF- production in NR8383 cells. NR8383 cells were stimulated with LPS in the absence (closed bar) or presence (open bar) of exogenous ADM at 100 nM. TNF- concentrations of the culture media were measured at 1, 3, 6, and 24 h by ELISA. A, Time course of secretion of TNF-; B, TNF- concentrations at 3 h of incubation; C, TNF- mRNA level was measured by RT-PCR after normalizing with TBP mRNA level; the cross-hatched bar represents the basal level of the control with no LPS or AM added. Each value represents the mean ± SEM of four separate sets of experiments. *, P < 0.05; #, P < 0.01 vs. LPS only.
LPS increased IL-1? secretion from NR8383 cells rapidly upon stimulation, but the rate of secretion gradually decreased after 6 h of incubation (Fig. 5A). ADM (at 100 nM) enhanced initial secretion of IL-1? for both non-LPS-stimulated and LPS-stimulated cells (P < 0.05) at 1 h of incubation (Fig. 5B) but slightly reduced IL-1? concentration of 1 ng/ml LPS-stimulated cells at 24 h of incubation (Fig. 5C). IL-1? mRNA expression was elevated by LPS, but no significant effect of exogenous ADM on IL-1? mRNA expression was observed in the 100 ng/ml LPS-stimulated cells (Fig. 5D).
FIG. 5. Effect of ADM on IL-1? production in NR8383 cells. NR8383 cells were stimulated with LPS in the absence (closed bar) or presence (open bar) of exogenous ADM at 100 nM. IL-1? concentrations of the culture media were measured at 1, 3, 6, and 24 h by ELISA. A, Time course of secretion of IL-1?; B, IL-1? concentrations at 1 h of incubation; C, IL-1? concentrations at 24 h of incubation; D, IL-1? mRNA level was measured by RT-PCR after normalizing with TBP mRNA level; the cross-hatched bar represents the basal level of the control with no LPS or AM added. Each value represents the mean ± SEM of four separate sets of experiments. *, P < 0.05; #, P < 0.01 vs. LPS only.
Effect of ADM on secretion and expression of MIF
MIF was secreted constitutively from NR8383 cells even without LPS stimulation, and LPS significantly increased MIF content in the media at the concentrations of 10–1000 ng/ml (Fig. 6A). As shown in the figure, MIF concentrations increased rapidly in the first hour of stimulation, but the rate of secretion decreased gradually in the subsequent incubation period. At the end of 24 h incubation, MIF concentrations were increased by 44–56% by LPS at 10–1000 ng/ml (P < 0.05). ADM (at 0.1–100 nM) significantly increased initial MIF secretion of NR8383 cells by 2.2- to 3.2-fold (P < 0.05) after the first hour of incubation (Fig. 6B) but did not affect the MIF concentration at 24 h (Fig. 6C). However, when ADM was administered simultaneously with LPS (at 100 ng/ml), there was no significant effect of ADM on MIF secretion of the LPS-stimulated cells except at a high concentration of ADM (at 100 nM) that increased initial MIF concentration by 41% (P < 0.05) compared with LPS alone (Fig. 6B). Figure 7 shows the results of MIF mRNA expression in nonstimulated cells and the cells stimulated by LPS (100 ng/ml) alone or LPS with ADM at 100 nM. MIF mRNA was expressed at a significant level in NR8383 cells even without any stimulation and was significantly increased by LPS at 6 and 24 h of stimulation. However, there was no significant effect of exogenous ADM (at 100 nM) on MIF mRNA expression in the LPS-stimulated cells.
FIG. 6. MIF secretion from NR8383 cells stimulated with ADM and LPS. NR8383 cells were stimulated with LPS and/or ADM at 0.1–100 nM. MIF concentration of the culture medium was measured at 1, 3, 6, and 24 h by ELISA specific for MIF. A, Time course of secretion of MIF from LPS-stimulated cells; B and C, MIF concentrations at 1 h (B) and at 24 h (C) of the cells stimulated by ADM alone (closed bar) or ADM and 100 ng/ml LPS (open bar). Each value represents the mean ± SEM of three to four separate sets of experiments. *, P < 0.05 vs. control with no ADM or LPS; #, P < 0.05 vs. LPS only.
FIG. 7. Semiquantitative RT-PCR analysis of MIF gene expression in NR8383 cells. NR8383 cells were stimulated with 100 ng/ml LPS in the absence (closed bar) or presence (open bar) of ADM at 100 nM. Expression levels of MIF mRNA were measured at 1, 3, 6, and 24 h by RT-PCR after normalizing with TBP mRNA level. Each value represents the mean ± SEM of three to four separate sets of experiments. *, P < 0.05; #, P < 0.01 vs. control with no LPS or ADM added.
Discussion
In this study, it was shown that ADM production and gene expression were dose-dependently and time-dependently stimulated by LPS in cultured NR8383 macrophages. This is consistent with the observations of other investigators that ADM gene expression is maintained in a suppressed state in resting macrophages and up-regulated by LPS (24, 25, 26). LPS induced a significant elevation of ADM production in NR8383 cells at a threshold dose of 10 ng/ml. This was similar to the findings of Isumi et al. (21) on ADM secretion from rat ECs. The temporal change of LPS-induced ADM production in NR8383 cells was similar to that obtained in RAW264.7 macrophages (24). The production level of ADM in NR8383 cells under 100 ng/ml LPS stimulation was approximately 1.29 fmol/105 cells·h as an average over a 24-h incubation. This is approximately three times higher than that of cultured RAW264.7 macrophages (24) but comparable to that of cultured VSMCs and ECs (20, 21). Our results confirm that the macrophage is another important source of ADM when activated by inflammatory stimulus such as LPS. The difference in the secretion rates is probably because of the difference in the macrophage cell lines that were used.
In NR8383 cells, LPS stimulated production of inflammatory cytokines, including TNF-, IL-1?, and IL-6, at concentrations as low as 1 ng/ml, but there was a distinctive difference in the time of induction of cytokine secretion among the different cytokines. There were rapid increases in secretion of IL-1? as early as 1 h and of TNF- at 3 h, but they started to reduce at 6 h after LPS stimulation. On the other hand, similar to the secretion of ADM, secretion of IL-6 in the LPS-stimulated cells was elevated much more significantly at 6–24 h of incubation. Strong induction of IL-1? and TNF- mRNA occurred well before that of ADM. Although it has been reported that TNF- and IL-1? show weak or no direct effect in ADM secretion in monocytes and macrophages (24, 25), they have been shown to increase ADM secretion additively with LPS in ECs when coadministered with LPS (21). The results of our time-course study demonstrate that the induction of IL-1? and TNF- precedes that of ADM and may suggest a possible contribution of these two cytokines to the induction of ADM gene expression in the macrophages when provided with a second triggering signal such as LPS.
MIF is an important mediator of the inflammatory response and is present at a high level in resting macrophages (31). It was shown in our results that MIF was constitutively expressed in NR8383 cells, and both its gene expression and secretion were elevated by LPS stimulation. Gene expression of MIF was increased by LPS at 6–24 h after stimulation, probably to replenish the storage pool of MIF in the cells. ADM significantly increased the initial release of MIF in both nonstimulated and LPS-stimulated cells. It also increased initial IL-1? secretion in resting and LPS-stimulated NR8383 cells. Because MIF and IL-1? are potent mediators of the inflammatory response, our results suggest that ADM may play a role as a proinflammatory factor in the initiation of the inflammatory response through the stimulation of MIF and IL-1? secretion.
On the other hand, ADM was found to continually suppress TNF- production in LPS-stimulated NR8383 cells up to 24 h, and TNF- gene expression was reduced by ADM at 24 h of incubation. Our results agree with the previous findings of an inhibitory effect of ADM on TNF- production in the macrophages (24). However, it was shown in our study that ADM markedly increased production of IL-6 in both nonstimulated and LPS-stimulated NR8383 cells. Our findings contrast with the results of Kubo et al. (24) where a reduction of IL-6 production was observed. The discrepancy may be because of the difference in the concentration of ADM that was used for stimulation and the different properties of the cells used. The stimulatory effect of ADM on IL-6 production has previously been reported in fibroblasts (22) but not in macrophages or other cell types. Based on our findings, LPS, in addition to directly increasing IL-6 production, may have an indirect effect on IL-6 production through ADM synthesis and secretion, as supported by a simultaneous increase in IL-6 and ADM in the LPS-stimulated cells. The increase in IL-6 then may have an inhibitory effect on the synthesis and secretion of TNF- and IL-1? because IL-6 has been shown to inhibit LPS-induced TNF- production in cultured macrophages (32) and suppress IL-1? and TNF- production in peripheral blood mononuclear cells (33). TNF- and IL-1? are proinflammatory, whereas IL-6 is antiinflammatory (34). Thus, our results indicate that ADM may also function as a peptidergic regulator of the inflammatory reactions through the inhibition of TNF- and stimulation of IL-6 and strongly support the notion that ADM is an antiinflammatory factor that suppresses the progression of inflammation. The promoter region of the ADM gene contains a consensus sequence for the nuclear factor NF-IL6 (35) that is induced by the stimulation with LPS, TNF-, IL-1, and IL-6 (36, 37). Therefore, although ADM can be used as a feedback regulator for the production of the inflammatory cytokines, it may also self-regulate its expression by either increasing secretion of its inducer, IL-1?, or suppressing its own inducer, TNF- .
The present study shows that ADM gene expression and secretion were elevated in NR8383 rat macrophages after LPS stimulation. The results suggest that ADM is both proinflammatory and antiinflammatory and that it plays a functional role in the initiation as well as progression of the inflammatory response by enhancing initial release of MIF and IL-1? and subsequently by inhibiting TNF- and stimulating IL-6 production. When overproduced in extreme inflammatory conditions, ADM may lead to feedback inhibition of TNF- and IL-1? production directly or through stimulating IL-6 production. Because TNF- and IL-1? are mediators of endotoxic shock, overexpression of ADM may therefore protect the host against the toxicity of endotoxin administration. The data shown here indicate that ADM stimulates secretion of MIF and modulates production of inflammatory cytokines in rat macrophages, and its role in the inflammatory process changes with time after the onset of inflammatory challenge.
References
Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, Eto T 1993 Adrenomedullin: a novel hypotensive peptide isolated from human phaeochromocytoma. Biochem Biophys Res Commun 192:553–560
Eguchi S, Hirata Y, Kano H, Sato K, Watanabe Y, Watanabe TX, Nakajima K, Sakakibara S, Marumo F 1994 Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells. FEBS Lett 340:226–230
Ishizaka Y, Ishizaka Y, Tanaka M, Kitamura K, Kangawa K, Minamino N, Matsuo H, Eto T 1994 Adrenomedullin stimulates cyclic AMP formation in rat smooth muscle cells. Biochem Biophys Res Commun 200:642–646
Kato J, Kitamura K, Kangawa K, Eto T 1995 Receptors for adrenomedullin in human vascular endothelial cells. Eur J Pharmacol 289:383–385
Shimekake Y, Nagata K, Ohta S, Kambayashi Y, Teraoka H, Kitamura K, Eto T, Kangawa K, Matsuo H 1995 Adrenomedullin stimulates two signal transduction pathways, cAMP accumulation and Ca2+ mobilization, in bovine aortic endothelial cells. J Biol Chem 270:4412–4417
Ishimitsu T, Nishikimi T, Saito Y, Kitamura K, Eto T, Kangawa K, Matsuo H, Omae T, Matsuoka H 1994 Plasma levels of adrenomedullin, a newly identified hypotensive peptide, in patients with hypertension and renal failure. J Clin Invest 94:2158–2161
Jougasaki M, Wei CM, McKinley LJ, Burnett Jr JC 1995 Elevation of circulating and ventricular adrenomedullin in human congestive heart failure. Circulation 92:286–289
Kobayashi K, Kitamura K, Hirayama N, Date H, Kashiwagi T, Ikushima I, Hanada Y, Nagatomo Y, Takenaga M, Ishikawa T, Imamura T, Koiwaya Y, Eto T 1996 Increased plasma adrenomedullin in acute myocardial infarction. Am Heart J 131:676–680
Yoshitomi Y, Nishikimi T, Kojima S, Kuramochi M, Takishita S, Matsuoka H, Miyata A, Matsuo H, Kangawa K 1998 Plasma levels of adrenomedullin in patients with acute myocardial infarction. Clin Sci 94:135–139
Nishio K, Akai Y, Murao Y, Doi N, Ueda S, Tabuse H, Miyamoto S, Dohi K, Minamino N, Shoji H, Kitamura K, Kangawa K, Matsuo H 1997 Increased plasma concentrations of adrenomedullin correlate with relaxation of vascular tone in patients with septic shock. Crit Care Med 25:953–957
Shoji H, Minamino N, Kangawa K, Matsuo H 1995 Endotoxin markedly elevates plasma concentration and gene transcription of adrenomedullin in rat. Biochem Biophys Res Commun 215:531–537
Elsasser TH, Sartin JL, Martinez A, Kahl S, Montuenga L, Pio R, Fayer R, Miller M, Cuttitta F 1999 Underlying disease stress augments plasma and tissue adrenomedullin (AM) responses to endotoxin: colocalized increases in AM and inducible nitric oxide synthase within pancreatic islets. Endocrinology 140:5402–5411
Hirata Y, Mitaka C, Sato K, Nagura T, Tsunoda Y, Amaha K, Marumo F 1996 Increased circulating adrenomedullin, a novel vasodilatory peptide, in sepsis. J Clin Endorinol Metab 81:1449–1453
Ueda S, Nishio K, Minamino N, Kubo A, Akai Y, Kangawa K, Matsuo H, Fugimura Y, Yoshikoa A, Masui K, Doi N, Murao Y, Miyamoto S 1999 Increased plasma levels of adrenomedullin in patients with systemic inflammatory response syndrome. Am J Respir Crit Care Med 160:132–136
Evereklioglu C, Yurekli M, Er H, Ozbec E, Hazneci E, Cekmen M, Inaloz HS 2000 Increased plasma adrenomedullin levels in patients with Behcet’s disease. Dermatology 201:312–315
Manthey CL, Vogel SN 1992 The role of cytokines in host response to endotoxin. Rev Med Microbiol 3:72–79
Shindo T, Kurihara H, Kurihara Y, Morita H, Yazaki Y 1998 Upregulation of endothelin-1 and adrenomedullin gene expression in the mouse endotoxin shock model. J Cardiovasc Pharmacol 31:S541–S544
Shindo T, Kurihara H, Maemura K, Kurihara Y, Kuwaki T, Izumida T, Minamino N, Ju KH, Morita H, Oh-hashi Y, Kumada M, Kangawa K, Nagai R, Yazaki Y 2000 Hypotension and resistance to lipopolysaccharide-induced shock in transgenic mice overexpressing adrenomedullin in their vasculature. Circulation 101:2309–2316
Tomoda Y, Isumi Y, Katafuchi T, Minamino Naoto 2001 Regulation of adrenomedullin secretion from cultured cells. Peptides 22:1783–1794
Sugo S, Minamino N, Shoji H, Kangawa K, Kitamura K, Eto T, Matsuo H 1995 Interleukin-1, tumor necrosis factor and lipopolysaccharide additively stimulate production of adrenomedullin in vascular smooth muscle cells. Biochem Biophys Res Commun 207:25–32
Isumi Y, Shoji H, Sugo S, Tochimoto T, Yoshioka M, Kangawa K, Matsuo H, Minamino N 1998 Regulation of adrenomedullin production in rat endothelial cells. Endocrinology 139:838–846
Isumi Y, Minamino N, Kubo A, Nishimoto N, Yoshizaki K, Yoshioka M, Kangawa K, Matsuo H 1998 Adrenomedullin stimulates interleukin-6 production in Swiss 3T3 cells. Biochem Biophys Res Commun 244:325–331
Nathan CF 1987 Secretory products of macrophages. J Clin Invest 79:319–326
Kubo A, Minamino N, Isumi Y, Katafuchi T, Kangawa K, Dohi K, Matsuo H 1998 Production of adrenomedullin in macrophage cell line and peritoneal macrophage. J Biol Chem 273:16730–16738
Kubo A, Minamino N, Isumi Y, Kangawa K, Dohi K, Matsuo H 1998 Adrenomedullin production is correlated with differentiation in human leukemia cell lines and peripheral blood monocytes. FEBS Lett. 426:233–237
Zaks-Zilberman M, Salkowski CA, Elsasser T, Cuttitta F, Vogel S 1998 Induction of adrenomedullin mRNA and protein by lipopolysaccharide and paclitaxel (Taxol) in murine macrophages. Infect Immunol 66:4669–4675
Bernhagen J, Calandra T, Mitchell RA, Martin SB, Tracey KJ, Voelter W, Manogue KR, Cerami A, Bucala R 1993 MIF is a pituitary-derived cytokine that potentiates lethal endotoxaemia. Nature 365:756–759
Bernhagen J, Calandra T, Bucala R 1998 Regulation of the immune response by macrophage migration inhibitory factor: biological and structural features. J Mol Med 76:151–161
Donnelly SC, Bucala R 1998 Macrophage migration inhibitory factor: a regulator of glucocorticoid activity with a critical role in inflammatory disease. Mol Med Today 3:502–507
Hwang ISS, Tang F 1999 The distribution and gene expression of adrenomedullin in the rat brain: peptide/mRNA and precursor/active peptide relationships. Neurosci Lett 267:85–88
Calandra T, Bernhagen J, Mitchell RA, Bucala R 1994 The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor. J Exp Med 179:1895–1902
Di Santo E, Alonzi T, Poli V, Fattori E, Toniatti C, Sironi M, Ricciardi-Castagnoli P, Ghezzi P 1997 Differential effects of IL-6 on systemic and central production of TNF: a study with IL-6-deficient mice. Cytokine 9:300–306
Schindler R, Mancilla J, Endres S, Ghorbani R, Clark SC, Dinarello CA 1990 Correlations and interactions in the production of interleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. Blood 75:40–47
Tilg H, Dinarello CA, Mier JW 1997 IL-6 and APPs: anti-inflammatory and immunosuppressive mediators. Immunol Today 18:428–432
Ishimitsu T, Miyata A, Matsuoka H, Kangawa K 1998 Transcriptional regulation of human adrenomedullin gene in vascular endothelial cells. Biochem Biophys Res Commun 243:463–470
Akira S, Isshiki H, Sugita T, Tanabe O, Kinoshita S, Nishio Y, Nakajima T, Hirano T, Kishimoto T 1990 A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J 9:1897–1906
Kiehntpof M, Herrmann F, Brach MA 1995 Functional NF-IL-6/CCAAT enhancer-binding protein is required for tumor necrosis factor -inducible expression of the granulocyte colony-stimulating factor (CSF), but not the granulocyte/macrophage CSF or interleukin 6 gene in human fibroblasts. J Exp Med 181:793–798(Louisa Y. F. Wong, Bernar)