ß2-Adrenoceptor agonist suppresses renal tumour necrosis factor and enhances interleukin-6 gene expression induced by endotoxin

Akio Nakamura,1, Edward James Johns2, Akira Imaizumi1, Yukishige Yanagawa1 and Takao Kohsaka3

1 Department of Paediatrics, Teikyo University School of Medicine, Tokyo, 3 Department of Immunology, National Children's Medical Centre, Tokyo, Japan and 2 Department of Physiology, University of Birmingham, Birmingham, UK



   Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. ß2-Adrenoceptor activation regulates tumour necrosis factor (TNF)-{alpha} and interleukin-6 (IL-6) production in cultured renal cells. However, it remains uncertain whether, in vivo, the administration of ß2-adrenoceptor agonists regulate renal TNF-{alpha} and IL-6 mRNA following lipopolysaccharide (LPS) stimulation to cause endotoxaemia. This study was performed in order to evaluate the effect of ß2-adrenoceptor agonist on renal TNF-{alpha} and IL-6 production.

Methods. Four-week-old Wistar rats pre-treated with the ß2-adrenoceptor agonist terbutaline or formoterol, and/or the ß- and ß2-adrenoceptor antagonists (propanolol, ICI118,551), were injected with LPS (1 mg i.p.), and then 2, 4 or 6 h later, kidneys (cortex, medulla), spleen, thymus and plasma were collected to assay TNF-{alpha} and IL-6 mRNA levels and their respective protein release.

Results. Administration of ß2-adrenoceptor agonists suppressed TNF-{alpha} mRNA expression in the whole kidney, by 61% (P<0.05), as well as plasma, spleen and thymus TNF-{alpha} protein and mRNA expression 2 hours after injection of LPS. On the other hand, although IL-6 levels in plasma, spleen and thymus mRNA expression were suppressed significantly by administration of ß2-adrenoceptor agonists, the basal- and LPS-induced IL-6 mRNA levels in the whole kidney were increased 1.6- and 1.2-fold (P<0.05), respectively, by treatment with ß2-adrenoceptor agonists. ß2-Adrenoceptor agonist suppressed LPS-induced TNF-{alpha} mRNA expression by 35% (P<0.05) and stimulated LPS-induced IL-6 mRNA expression by 1.5-fold (P<0.05) in the medullary region of kidney.

Conclusions. ß2-Adrenoceptor agonists down-regulate renal TNF-{alpha} mRNA expression following LPS-induced endotoxaemia. This effect was particularly apparent in the renal medulla. IL-6 mRNA expression in the renal medulla was up-regulated by the agonists whereas plasma, spleen and thymus IL-6 levels were completely inhibited by the agonist, which suggests the existence of tissue specific regulation of IL-6 production in the kidney by ß2-adrenoceptor activation.

Keywords: ß2-Adrenoceptor; IL-6; kidney; rat; TNF-{alpha}



   Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Endotoxaemia caused by Gram-negative bacteria can result in sepsis and organ dysfunction, including kidney damage and renal failure [1,2]. The pathological mechanisms responsible for this renal dysfunction involves several mediators, and an important class is the inflammatory cytokines [3,4]. Fouqueray et al. [4] demonstrated that tumour necrosis factor (TNF)-{alpha} and interleukin-6 (IL-6) were produced promptly by isolated glomeruli after in vivo or in vitro exposure to lipopolysaccharide (LPS), suggesting an important role of these cytokines in the development of endotoxin-induced renal dysfunction.

There is evidence that ß2-adrenoceptor activation can modulate the production of TNF-{alpha} and IL-6 in some tissues and organs. In liver cells, Liao et al. [5] documented that ß2-adrenoceptor-mediated processes were able to regulate TNF-{alpha} and IL-6 production. Severn et al. [6] demonstrated suppression of TNF-{alpha} production by isoproterenol in cultured human blood cells, which was mediated by increased intracellular cAMP levels. Hetier et al. [7] investigated the regulation of TNF-{alpha} gene expression in the microglia cells, and observed that isoproterenol, via an action at ß2-adrenoceptors, was able to influence the regulatory processes of TNF gene expression. In the kidney, we have reported that TNF-{alpha} and IL-6 gene transcription, mRNA accumulation and protein levels were suppressed by ß2-adrenoceptor activation with terbutaline using cultured renal resident macrophage cells [8,9] and mesangial cells [10]. Although ß2-adrenoceptor activation regulated TNF-{alpha} and IL-6 production in cultured renal cells, it remains uncertain whether, in vivo, the administration of ß2-adrenoceptor agonist would be able to modulate TNF-{alpha} and IL-6 production in the kidney.

The objective of the present study was to clarify whether ß2-adrenoceptor stimulation could modulate TNF-{alpha} and IL-6 production in the kidney. The findings provide evidence that administration of the ß2-adrenoceptor agonists down-regulated LPS-induced TNF mRNA levels and up-regulated IL-6 mRNA levels in the medullary regions of kidney.



   Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reagents
ISOGEN was obtained from Nippon Gene (Tokyo, Japan) and ICI 118,551 was obtained from Funakoshi (Tokyo, Japan). The Multiprime DNA labelling system and [{alpha}-33P]dCTP were purchased from Amersham International PLC (Little Chalfont, Buckinghamshire, UK). Biodyne A membranes were obtained from Pall Ultrafine Filtration (Glen Cove, NY, USA). Rat TNF-{alpha} and IL-6 ELISA kits were obtained from Biosource International Inc. (Camarillo, CA, USA). RT RNase H-reverse Transcriptase kit was obtained from Super Script, Bethesda Research laboratories (Gaithersberg, MD, USA). Unless stated, reagents were obtained from Sigma Chemical Co. (St Louis, MO, USA).

Rat preparation and protocols
The animal experimentation was conducted in accordance with the Teikyo University Guide for the Care and Use of Laboratory Animals. To investigate the time-course changes of TNF-{alpha} and IL-6 mRNA, 4-week-old Wistar rats were divided into six groups and subjected to the protocol as shown in Figure 1Go. Rats in groups I–VI were injected intraperitoneally with LPS (1 mg), terbutaline (1 mg), ICI 118,551 (1 mg) and/or saline at 20 min intervals. Eight rats were used 2 h after treatment in groups I–IV and six rats were used 2 h after treatment in group V or VI. Four rats were used 4 or 6 h after treatment in each group. Two, 4 or 6 h later, rats were euthanized under ether anaesthesia. Kidneys, spleen and thymus were collected in order to measure TNF-{alpha} and IL-6 mRNA levels and plasma was taken to measure TNF-{alpha} and IL-6 protein content in the circulation. To evaluate the dose-response effect of terbutaline and the effect of other ß2-agonists and antagonists on TNF-{alpha} and IL-6 in the cortex and medulla of kidney, LPS (1 mg), terbutaline (0.25 mg, 0.5 mg, 1 mg) and formoterol (1 mg) as a ß2-adrenoceptor agonist, or propanolol (1 mg) as non-selective ß-adrenoceptor antagonist were injected intraperitoneally (n=4) at 20 min intervals. Two hours later, rats were euthanized under ether anaesthesia, the kidney was separated into cortex and medulla, and mRNA levels were measured in these regions using semi-quantitative reverse transcription (RT)-PCR.



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Fig. 1. Experimental protocols used in this study. Group I rats were injected with saline (1 ml, i.p.) three times at 20 min intervals. Group II rats were injected with LPS (1 mg, i.p.) and saline at 20 min intervals. Group III rats were injected with the ß2-adrenoceptor agonist, terbutaline, (1 mg, i.p.) and saline 20 min apart. Group IV rats were injected with terbutaline (1 mg, i.p.), LPS (1 mg, i.p.) and saline at 20 min intervals. Group V rats were injected with the ß2-adrenoceptor antagonist, ICI 118,551 (1 mg), terbutaline (1 mg) and LPS (1 mg) intraperitoneally at 20 min intervals. Group VI rats were injected with ß2-adrenoceptor antagonist, ICI 118,551 (1 mg), terbutaline (1 mg) and saline intraperitoneally every 20 min.

 

Analysis of TNF-{alpha} and IL-6 production
Plasma concentrations of TNF-{alpha} and IL-6 were estimated using an ELISA kit according to the manufacturer's instructions, and tissue mRNA levels in the whole kidney, spleen and thymus were measured using northern blot hybridization analysis as described in our previous study [11]. Briefly, total RNA was extracted from the kidney, spleen and thymus using ISOGEN as per the manufacturer's instruction. For northern blot hybridization, the 600 bp EcoRI/HindIII fragment derived from the cDNA insert of clone pGEM [12] for murine IL-6 or the 420 bp HinfI fragment for human ß-actin (National Children's Research Centre, Japan, Tokyo) was used as a cDNA probe for IL-6 or ß-actin, respectively. The cDNA for TNF-{alpha} was constructed with RT-PCR product (546 bp) using RNA isolated from rat kidney. These cDNA probes were labelled using the oligolabelling method in the presence of [{alpha}-33P]dCTP which was utilised as a hybridization probe. RNA samples (10 or 20 µg) were applied to a Biodyne A membranes, hybridized simultaneously and exposed for the same time. The ß-actin cDNA probe was used as a loading control after the TNF-{alpha} or IL-6 probes had been stripped from the membrane (Figure 2Go). The membranes were exposed on an imaging plate (BAS III, Fuji Photo Film Co Ltd, Tokyo, Japan) and the incorporated radioactivity was measured with a Bioimage analyzer (BAS 2000, Fuji Photo Film Co Ltd). The radioactivity was expressed as photostimulated luminescence (PSL) units and was used as an index of mRNA level. The data were expressed as amount of cytokine product over the amount of ß-actin product (cytokines : ß-actin ratio).



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Fig. 2. Representative northern blots for ß-actin mRNA in the kidney, spleen and thymus. The ß-actin cDNA probe was labelled using the oligolabelling method in the presence of [{alpha}-33P]dCTP, which was utilised as a hybridization probe. RNA samples (10 µg, n=4–8) used 2 h after the injection or 20 µg RNA (n=4) taken at 4 or 6 h were applied to a Biodyne A membrane. Lane 1: control, lane 2: LPS, lane 3: terbutaline, lane 4: LPS plus terbutaline, lane 5: LPS plus terbutaline plus ICI 118,551, lane 6: terbutaline plus ICI 118,551.

 
TNF-{alpha} and IL-6 mRNA levels in the cortex and medullary regions of the kidney were measured using the semi-quantitative RT-PCR analysis. Total RNA (2 µg) of kidney was carried out with RT RNase H-reverse Transcriptase kit, which was used for the synthesis of the first strand cDNA and subsequently subjected to amplification with the PCR for IL-6, TNF-{alpha} and ß-actin primers. The following primers have been published previously [17,18] and were used for cytokine gene amplification:

IL-6 (sense) 5'-GACTGATGTTGTTGACAGCCAGTGC-3', IL-6 (antisense) 5'-TAGCCACTCCTTCTGTGACTCTAACT-3', TNF-{alpha} (sense) 5'-CACCACGCTCTTCTGTCTACTGAAC-3', TNF-{alpha} (antisense) 5'-CCGGACTGCGTGATGTCTAAGTACT-3', ß-actin (sense) 5'-TGGAATCCTGTGGCATCCATGAAAC-3', ß-actin (antisense) 5'-TAAAACGCAGCTCAGTAACAGTCCG-3.

To carry out the PCR, tubes containing 50 µl of the reaction mixture were placed in the Programmed Tempcontrol system set up as follows: (i) denaturing for 1 min at 95°C; (ii) annealing primers for 1 min at 60°C; (iii) extending the primers for 1 min at 72°C. For the purpose of semi-quantification of the RT-PCR, it was necessary to correct the amplification process for tube-to-tube variability in amplification efficiency. In our study, ß-actin mRNA was used as an internal standard for the semi-quantification of the RT-PCR. To confirm the reliability of the semi-quantification, we extracted total RNA from rat kidney, diluted it to different concentrations and performed RT-PCR at 30 cycles for IL-6 and TNF-{alpha}, and at 25 cycles for ß-actin [13]. There was a significant linear correlation between starting total RNA and the PCR product for IL-6, TNF-{alpha} and ß-actin [13]. A portion (20 µl) of the PCR solution was electrophoresed in 2% agarose gel, stained with ethidium bromide, and visualized with an ultraviolet trans-illuminator. Imaging and analysis of the PCR products separated by electrophoresis were performed using a fluorescence imaging analyser (FluorImager SI, Molecular Dynamics-Japan, Tokyo, Japan). The images of ethidium-bromide stained DNA fragments on the gels were scanned into the analyser and quantified using ImageQuaNT (Microsoft) software in the computer, and were expressed as relative fluorescent units (r.f.u.). RT-PCR of ß-actin served as an internal control and the inter-assay coefficient of variation for ß-actin was 0.075. The cytokines/ß-actin ratio was used as an index of mRNA level.

Statistics
Statistical analysis was undertaken using ANOVA followed by the Bonferroni and Dunnet tests for multiple comparisons of plasma and mRNA levels between groups. P values of <0.05 were accepted as statistically significant. Results were expressed as mean±standard error (SE of the mean).



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 Materials and methods
 Results
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 References
 
Plasma IL-6 and TNF-{alpha} levels
Table 1Go shows the responses in plasma IL-6 and TNF-{alpha} levels 2 h after injection of LPS, terbutaline and/or ICI 118,551. LPS (1 mg, i.p.) injection stimulated plasma IL-6 and TNF-{alpha} levels by 14- and 10-fold, respectively. Administration of terbutaline (1 mg, i.p.) together with the LPS decreased the responses in plasma IL-6 and TNF-{alpha} significantly (P<0.05) to control levels, but following the co-administration of the ß2-adrenoceptor antagonist, ICI118,551, the increases in IL-6 and TNF-{alpha} were restored. Terbutaline alone (Group III) or terbutaline plus ICI 118,551 (Group VI) had no significant effect on plasma levels of either IL-6 or TNF-{alpha}. These findings indicated that ß2-adrenoceptor activation suppressed the LPS-induced increases in plasma levels of IL-6 and TNF-{alpha}.


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Table 1. Modulation of plasma interleukin-6 (IL-6) and tumour necrosis factor (TNF)-{alpha} protein levels by the ß2-adrenoceptor agonist, terbutaline

 

TNF-{alpha} mRNA
The effects of ß2-adrenoceptor activation with terbutaline on TNF-{alpha} mRNA in the whole kidney, spleen and thymus are shown in Figure 3Go. TNF-{alpha} mRNA levels in the whole kidney increased significantly (P<0.05) by 1.9-fold 2 h after injection of LPS (1 mg), and 4 and 6 h after injection were reduced to control levels. Although terbutaline alone had no effect on renal TNF-{alpha} mRNA, it prevented the LPS-induced increase in TNF-{alpha} mRNA in the whole kidney by 61% compared with the group treated with LPS alone (P<0.05). This terbutaline-mediated suppression was overcome by co-administration of ICI 118,551, which suggested that ß2-adrenoceptor activation suppressed renal TNF-{alpha} production. Similarly, spleen and thymus TNF-{alpha} mRNA levels (Figure 3Go) were stimulated 2 h after LPS (1 mg) injection and these responses were also blocked in the presence of the ß2-adrenoceptor agonist, terbutaline. Four and 6 h after LPS injection, there was no evidence of regulation of renal TNF mRNA by ß2-adrenoceptor activation. The ß2-adrenoceptor mediated actions on renal cortex and medullary TNF-{alpha} mRNA are shown in Figure 4Go. ß2-Adrenoceptor activation with terbutaline and formoterol did not change the TNF-{alpha} mRNA level in the cortex region of the kidney (Figure 4AGo). However, terbutaline inhibited LPS-induced TNF-{alpha} mRNA significantly (P<0.05) in the renal medulla in a dose-dependent manner (Figure 4BGo). Formoterol, a ß2-adrenoceptor agonist, also significantly inhibited LPS-induced TNF-{alpha} mRNA in the medullary region (P<0.05) (Figure 4BGo).



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Fig. 3. This shows representative northern blots for TNF-{alpha} mRNA at 2, 4 and 6 h after treatment in the kidney, spleen and thymus, and bar graphs for renal TNF-{alpha} mRNA (n=4–8) 2 h after treatment. The TNF-{alpha} cDNA probe was labelled using the oligolabelling method in the presence of [{alpha}-33P]dCTP, which was utilised as a hybridization probe. RNA samples (10 µg, n=4–8) 2 h after treatment or 20 µg RNA (n=4) at 4 or 6 h were applied to a Biodyne A membrane. Lane 1: control, lane 2: LPS, lane 3: terbutaline, lane 4: LPS plus terbutaline, lane 5: LPS plus terbutaline plus ICI 118,551. mRNA units in each group are represented as the mean±SE and are expressed as amount of TNF mRNA over the amount of ß-actin. *P<0.05 vs control group. {dagger}P<0.05 vs LPS alone treated group.

 


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Fig. 4. TNF-{alpha} mRNA levels in the cortex (A) and medullary (B) region of the kidney using RT-PCR. The data for mRNA are expressed as amount of TNF-{alpha} mRNA over the amount of ß-actin mRNA (cytokines : ß-actin ratio) and are the mean±SE from four rats. *P<0.05 vs control group. {dagger}P<0.05 vs LPS alone treated group.

 

IL-6 mRNA
The effects of ß2-adrenoceptor activation with terbutaline on IL-6 mRNA in the whole kidney, spleen and thymus are shown in Figure 5Go. IL-6 mRNA levels in the whole kidney were increased significantly (P<0.05) by ~1.8-fold 2 h after injection of LPS (1 mg). Four hours after LPS injection, IL-6 mRNA was still enhanced, but had diminished 6 h after the injection. The basal level of renal IL-6 mRNA was increased 1.6-fold in the presence of terbutaline alone (P<0.05) and the response to LPS was also enhanced when terbutaline was co-administered (P<0.05). This action of terbutaline on IL-6 mRNA levels in the whole kidney was inhibited by the addition of ICI 118,551, which suggested that it was ß2-adrenoceptor activation that stimulated renal IL-6 production. However, increases in spleen and thymus IL-6 mRNA levels stimulated 2 h after LPS (1 mg) injection were prevented by ß2-adrenoceptor stimulation with terbutaline. These responses indicated that a different mechanism was involved whereby ß2-adrenoceptor activation modulated IL-6 production in kidney, spleen and thymus. Four and 6 h after the treatments, renal IL-6 mRNA levels were unaffected by ß2-adrenoceptor activation. The way in which renal IL-6 mRNA levels were suppressed by ß2-adrenoceptor activation is shown in Figure 6Go. ß2-Adrenoceptor activation with terbutaline and formoterol had no effect either on basal levels of IL-6 mRNA in the cortex, and was unable to modulate the LPS-induced increases in these mRNA levels (Figure 6AGo). In contrast, terbutaline stimulated LPS-induced IL-6 mRNA significantly (P<0.05) in renal medulla and in a dose-dependent manner (Figure 6BGo). Furthermore, renal medullary IL-6 mRNA was increased in the presence of terbutaline alone (P<0.05). Formoterol, a ß2-adrenoceptor agonist, also stimulated IL-6 mRNA in the medulla significantly (P<0.05).



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Fig. 5. Representative northern blots for IL-6 mRNA at 2, 4 and 6 h after treatment in the kidney, spleen and thymus, and bar graphs for renal IL-6 mRNA levels (n=4–8) 2 h after treatment. IL-6 cDNA probe was labelled using the oligolabelling method in the presence of [{alpha}-33P]dCTP, which was utilised as a hybridization probe. RNA samples (10 µg, n=4–8) 2 h after treatment or 20 µg RNA samples (n=4) at 4 or 6 h were applied to a Biodyne A membrane. Lane 1: control, lane 2: LPS, lane 3: terbutaline, lane 4: LPS plus terbutaline, lane 5: terbutaline plus ICI 118,551. mRNA units in each group are represented as the mean±SE and are expressed as amount of IL-6 mRNA over the amount of ß-actin. *P<0.05 vs control group. {dagger}P<0.05 vs LPS alone treated group. **P<0.05 vs terbutaline alone treated group.

 


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Fig. 6. IL-6 mRNA levels in the cortex (A) and medullary (B) region of the kidney using RT-PCR. mRNA units are expressed as amount of IL-6 mRNA over the amount of ß-actin mRNA (cytokines : ß-actin ratio) and were the mean±SE from four rats. *P<0.05 vs control group. {dagger}P<0.05 vs LPS alone treated group.

 



   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
TNF is a primary inducer in the genesis of endotoxaemia and is regarded as a central mediator of the pathophysiological changes associated with LPS [3,14]. The present study demonstrated that LPS-induced TNF-{alpha} production in the kidney was markedly suppressed in the presence of the ß2-adrenoceptor agonists. The inhibitory effect of the ß2-adrenoceptor agonist on renal TNF production may be one mechanism whereby renal dysfunction and injury caused by overproduction of TNF in the kidney may be prevented. However, the inhibitory mechanism of the ß2-adrenoceptor agonists remains unclear. Previous investigators [57,15] have indicated that the inhibition of TNF-{alpha} production by ß2-adrenoceptor activation was dependent upon an increase in intracellular cAMP levels. Olivier et al. [16] reported that the increase in intracellular cAMP concentration decreased the NF-{kappa}B-mediated function of TNF-{alpha} gene transcription. However, the inhibitory mechanism of ß2-adrenoceptor agonists on TNF production may involve several other factors besides the cAMP–protein kinase A (PKA) pathway. Seldon et al. [17] reported that the inhibitory actions of ß2-adrenoceptor agonists on TNF-{alpha} transcription and/or translation of TNF-{alpha} gene were regulated by a cAMP/cAMP-dependent PKA cascade and cAMP-independent mechanisms. In our previous studies we reported that ß2-adrenoceptor activation suppressed mitogen-activated protein kinase (p42/p44), which in turn decreased TNF production in renal resident macrophage cells [8].

IL-6 is also involved in mediating various components of the immune and inflammatory response. For example, IL-6 can induce the adhesion of circulating cells to the glomerular capillary wall [18]. Moreover, the metabolic alterations observed in sepsis are associated with changes in the production of IL-6 [19]. We demonstrated in the present study that the ß2-adrenoceptor agonist up-regulated IL-6 production in the kidney some 2 h after the LPS challenge whereas plasma IL-6, spleen and thymus IL-6 mRNA were down-regulated by the ß2-adrenoceptor agonist. Liao et al. [5] observed that ß2-adrenoceptor-mediated processes increased LPS-induced IL-6 production in liver cells, while Maimone et al. [20] reported that exposure of astrocytes to norepinephrine elevated IL-6, which was mediated predominantly by ß2-adrenoceptors and the activation of adenylate cyclase. It is recognised that intracellular cAMP plays an important role in the stimulation of IL-6 gene expression [21] and it has been suggested that up-regulation of IL-6 production due to ß2-adrenoceptor activation is mediated through the cAMP–PKA pathway. On the other hand, Straub et al. [22] demonstrated that isoproterenol inhibited IL-6 secretion in the spleen, while our own studies also indicated an inhibitory effect of ß2-adrenoceptor activation following LPS-induced IL-6 gene transcription in rat astrocytes [23]. Furthermore, in an in vivo study, epinephrine infusion into human subjects did not affect IL-6 production following an LPS challenge [24]. Therefore, the action of ß2-adrenoceptor stimulation on IL-6 production is quite complex and may depend on tissue and cell-specific mechanisms.

Activation of the cAMP–PKA pathway has been reported to induce a decrease in TNF-{alpha} mRNA levels and an increase in IL-6 mRNA [16,21]. The findings of the present study suggested that formation of cAMP through ß2-adrenoceptor activation plays an important role in the regulation of the production of these cytokines in the kidney, especially in the medullary region. On the other hand, in the plasma, spleen and thymus, the decrease in both TNF-{alpha} and IL-6 mRNA by the ß2-adrenoceptor agonist suggested that other factors, such as the MAPK pathway [8,17], may be involved in the regulation of the production of these cytokine production. In the kidney, the effect of the ß2-adrenoceptor agonist on TNF-{alpha} and IL-6 mRNA was observed to be significant in the medullary region. This is supported by a previous report that ß2-adrenoceptors are distributed predominantly to the outer and inner stripe of the outer medulla [25]. The changes in medullary TNF-{alpha} and IL-6 mRNA levels due to ß2-adrenoceptor activation reflect the levels of these cytokines in the whole kidney. The present study demonstrated that IL-6 mRNA was enhanced by terbutaline alone in the absence of LPS. IL-6 mRNA was not expressed constitutionally and was not increased by the injection of ß-adrenoceptor agonist alone [26]. However, in the present study, kidneys in the control group expressed IL-6 mRNA, which suggested the possibility that treatment in the control group might induce the activation of the IL-6 gene expression. Therefore, although normally terbutaline itself does not have the ability to increase IL-6 gene expression, because IL-6 gene expression in the control group was already activated, terbutaline induced an enhancement of IL-6 mRNA. This is compatible with the enhancement of LPS-induced IL-6 mRNA by the ß2-adrenoceptor agonist.

ß2-Adrenoceptor activation has been found to suppress the inflammatory responses induced by TNF-{alpha}, and the concomitant enhancement of IL-6 production caused by ß2-adrenoceptor activation may play a crucial role in the acute inflammatory responses by controlling the level of pro-inflammatory cytokine (such as TNF-{alpha}) production [27]. Furthermore, ß2-adrenoceptor activation potentiates the production of the anti-inflammatory cytokine IL-10 in human blood cells [24] while it has been reported that ß2-adrenoceptor agonists inhibit IL-12 production [28]. This implies that ß2-adrenoceptors could play an important role in the treatment of T helper 1-mediated autoimmune diseases. Thus, ß2-adrenoceptor agonists may be useful in the treatment of some patients with serious infections and could potentially offer protection from immune-mediated damage to the kidney. The potential role of ß2-adrenoceptor stimulation as a therapeutic tool for cytokine inhibition must be tested, particularly in relation to glomerular diseases, which are associated with TNF-{alpha} overproduction.



   Acknowledgments
 
This study was supported by grants from the Human Science Foundation of Japan.



   Notes
 
Correspondence and offprint requests to: Dr Akio Nakamura, Department of Paediatrics, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173, Japan. Back



   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 12. 8.99
Revision received 18. 6.00.