Noradrenaline reduces the stimulatory effect of interleukin-1α on reactive oxygen species production by oyster immunocytes

Invertebrate Biology I20(4): 358-364. 0 200 1 American Microscopical Society, Inc.

Noradrenaline reduces the stimulatory effect of interleukin- la on reactive oxygen species production by oyster immunocytes

Arnaud Lacoste," Shelagh K. Malham, Anne Cueff, and Serge A. Poulet

Station Biologique de Roscoff, CNRS-UniversitC Paris VI-INSU Place Georges Teissier, B.P. 74, F-29682 Roscoff cedex, France

Abstract. A growing body of evidence suggests that interleukin-la (IL-la) is present in invertebrates. Both invertebrate and human IL- l a can bind to invertebrate receptors and stimulate invertebrate immune functions. The present study shows that TL- la increases reactive oxygen species (ROS) production by oyster immunocytes. However, physiological doses of noradrenaline (NA) exert a suppressive effect on IL- la stimulation in vitro. The P-adrenoceptor agonist isoproterenol mimicked the effects of NA and the P-adrenoceptor antagonist propanolol blocked the NA-induced suppression of hemocyte responsiveness to IL- 1 a. The type IV phosphodiesterase inhibitor rolipram acted in synergy with isoproterenol to reduce hemocyte response to 1L- 1 a and the protein kinase A inhibitor H-89 suppressed the effects of isoproterenol. These results suggest that circulating NA impairs IL-la-stimulation of oyster hemocyte via a p-adrenoceptor/cyclic AMP/protein kinase-A signaling pathway. Considering that mollusc immunocytes secrete NA, an autocrine regulatory loop may also modulate the ability of these cells to respond to 1L- I a.

Additional key words: catecholamines, cytokines, chemiluminescence, Crussostreu gigus

Close hi-directional interactions are known to occur between the immune and neuroendocrine systems in vertebrates. These interactions, which play a central role in homeostasis, result from the presence of common ligands and receptors in both systems. Indeed, the immune system signals neuroendocrine tissues at multiple levels via cytokines and, reciprocally, the neuroendocrine system regulates immune functions via hormones such as catecholamines (CA), glucocorti- coids, and neuropeptides (Blalock 1994; Sternberg 2000).

A growing body of evidence shows that the basic mechanisms and molecules involved in the vertebrate immune-neuroendocrine axis are also present in invertebrates. Indeed, the presence of various cytokines such as TL-la, IL-2, IL-6, and TNF-a has been dem- onstrated in several invertebrate phyla (Hughes et al. 1990, 1992; Beck & Habicht 1991, 1996; Raftos et al. 1991; Stefano et al. 1991; Granath et al. 1994; Owve- Missi-Oukem-Boyer et al. 1994; Cooper et al. 1995; Ottaviani & Franceschi I996), and the invertebrate neuroendocrine system involves central neurotransmit- ters, neuropeptides, and hormones such as acetylcho- line, pro-opiomelanocortin-derived peptides, and cat-

" Author for correspondence. E-mail: lacoste@ sb-roscoff.fr

echolamines (Rosza 1984; Stefano et al. 1993; Stefano & Salzet 1999; Lacoste et al. 200 I ). Furthermore, data suggest that an immune-neuroendocrine axis exists in invertebrates. Indeed, cytokines are known to induce the release of noradrenaline (NA) produced by hemocytes, and ACTH has been shown to stimulate phagocytosis in the gastropod Planorbarius comeus (Ottaviani & Franceschi 1996).

Numerous studies have suggested that catecholamines play essential roles in several physiological processes in molluscs including feeding (Teyke et al. 1993), locomotion (Sakharov & SalAnski 1982), res- piration (Syed & Winlow 199 l), reproduction (Marti- nez & Rivera 1994), and larval settling and metamorphosis in free-living marine species (Pires et al. 1997). However, little is known about how these hormones participate in neuro-immune interactions in molluscs. In the present study, we have investigated how NA interferes with IL- l a modulation of oyster hemocyte reactive oxygen species (ROS) production.

Methods Drugs

Noradrenaline, the a-adrenoceptor agonist phenylephrine, the P-adrenoceptor agonist isoproterenol, the a-adrenoceptor antagonist prazosin, the P-adrenocep-

Noradrenaline and 1L- 1 a stimulation in molluscan hemocytes 359

tor antagonist propanolol, the phosphodiesterase type IV specific inhibitor rolipram, the protein kinase-C specific inhibitor calphostiii C, the protein kinase-A specific inhibitor H-89, and human recombinant TL- I a were all obtained from Sigma (France).

Hemolymph collection and hemocyte treatments

Oysters of Crussostrea gigus (THUNBERG 1793), weighing 60-70 g, were maintained in polyethylene tanks (60-70 oysters per tank) containing 110 1 of aer- ated and continuously flowing (SO l/h) natural seawater at 14-15°C. Animals were left undisturbed for a 10- day acclimation period. Hemolymph (0.5- 1 ml/oyster) was collected from the pericardial cavity using 2-ml syringes and 26 GX%" needles. Hemolymph was pooled to obtain 30-35-ml samples and cells were pel- leted by centrifugation (200 g, 5 min, 15°C). Hemo- cytes were then resuspended in modified Hank's bal- anced salt solution (MHBSS) consisting of HBSS adjusted to ambient seawater salinity (31 ppm) and containing 2 g/l of D-glucose (Anderson et al. 1994). Hemocyte concentration was then determined using a hemacytometer and adjusted to lo6 cell/ml. Cell suspensions were divided into 1-ml aliquots and incubated for 20 min at 15°C in MHBSS containing either NA, phenylephrine, or isoproterenol at concentrations of 0.01, 0 . 1 , and 1 .O FM indicated in the text. In some experiments, a 10 min pre-incubation in the presence of antagonists or inhibitors preceded the incubation with adrenergic drugs. Interleukin-la (0.01-10 ng/ml) was then added to the cells and samples were pro- cessed for chemiluminescence assays 10 rnin later. Pre- liminary experiments showed that, at the concentrations used, none of the drugs had any significant effect on chemiluminescence (CL).

Chemiluminescence assay

Zymosan (Sigma, France) particles were used as a stimulant for the oyster hemocytes. Zymosan particles were suspended in filtered seawater at a concentration of 10 mg/ml, heated for 30 min at IOO"C, washed twice in filtered seawater and resuspended in MHBSS at a concentration of 1 mg/ml. At the time of use, the number of zymosan particles was estimated using a hemacytometer. The chemilumigenic probe used was luminol (5-amino-2, 3-dihydro- I , 4-phthalazinedione, Sigma). Stock solutions were prepared according to Scott & Klesius (1981) and stored at -20°C for 1 week maximum. A working solution ( lo- ' M in sterile de- ionized water) was prepared < 1 h before use. Luminol

M final concentration) was added to 1-ml cell suspensions containing lo6 cell/ml, and baseline CL was recorded for 20 min. Zymosan was then added

(80 zymosan particledcell) and CL response was recorded using a Lumat LB 9507 luminorneter (EG&G Berthold) every 4 min for 100 min.

CL counts were plotted against time and the total CL response was expressed as the area under the curve minus the background level. The resulting counts are expressed as a percentage of control.

Statistical analyses

Data are presented as mean and standard error of at least three experiments. Significant differences between various group means were determined by one- way ANOVA. Significant effect of particular drug concentrations was shown via Dunnett's multiple comparisons test. For all statistical analyses, P<.O I was considered significant.

Results

The CL-response curves shown in Fig. l a were obtained from aliquots (1 O6 cell/ml each) originating from the same sample of pooled hemolymph. These responses are representative of results obtained from all other pools examined and they show typical CL- responses of untreated samples (controls). Moreover, they show that exposure of hemocytex to TL- 1 Q! (0.0 1 - 10 ng/ml) produced a dose dependent increase of the CL-response of hemocytes in the oyster Crusso\treu gigas. Both resting and zymosan-induced raw RLU/s values from individual cell pools usually show consid- erable variation in molluscs (Anderson et al. 1994), although the effects of IL- la and other drug concentrations on hemocyte CL-re5ponse were rather repro- ducible. Therefore, CL data from each treated aliquot originating from a given pool were expressed as per- cent of control to permit intrapool comparisons. Re- sults in Fig. l b show that the IL-la-induced CL-response increase was significant (Pc.0 I ) at 1L- 1 a concentrations of 1 ng/ml (150.74 2 lS.O3% of control) and 10 ng/ml (181.62 -+ 16.64% of control). In oyster hemocytes pretreated with 0.1 or 1.0 yM NA (Fig. 2), the stimulating effect of 1 ng/ml of TL-la was suppressed (Pc.01) and higher IL- la concentrations were needed to stimulate the CL-response.

To determine whether the effect of NA was mediated by a- or P-adrenoceptors, the effects of specific agonists and antagonists were tested. Pretreatment of the hemocytes with the a-adrenoceptor agonist phenylephrine (0.01-1.0 yM) did not significantly alter their capacity to respond to IL- la (Fig. 3a), however, the P-adrenoceptor agonist isoproterenol caused a dose-dependent inhibition of hemocyte stimulation by 1L-la (Fig. 3b). Indeed, in the presence of 1.0 or 10.0 ng/ml 1L- 1 a, the CL-response of hemocytes pretreated

360

150 -

Interleukin l a (nglml) :

*

-0 (Con) 2o 4 a h +O.OI

15

10

5

0 0 20 40 60 80 100

Time (min)

* T

0.01 0.1 1.0 10.0 lnterleu kin-I a (nglm I)

Fig. 1. Concentration-dependent stimulation of the luminol- dependent CL-response of oyster hemocytes by IL- 1 a. (a) CL-response from individual cell aliquots (1 O6 cell/ml) from a common pool of hemocytes. Cells were incubated for 20 min in MHBSS alone as a control (Con) or in MHBSS containing 0.01-10.0 ng/ml 1L-la. Basal hemocyte CL was recorded for 20 min, the cells were then stimulated by the addition of zymosan particles (ZY) and the CL-response was evaluated. (b) Stimulation of the luminol-dependent CL of oyster hemocytes by IL-la at concentrations of 0.01-10.0 nghnl. Data are mean CL values and standard errors expressed as a percentage of control (cell samples unexposed to IL- la). *=significantly different from controls (P<.Ol).

with 0.1 or 1.0 kM of isoproterenol was not significantly increased. Incubating the hemocytes in the presence of the a-adrenoceptor antagonist prazosin (10 kM) before the addition of NA (1 kM) did not significantly modify the suppressive effect of NA on the response to IL- la (Fig. 4a), however, in the presence of the P-adrenoceptor antagonist propanolol (10 kM), the suppressive effect of NA (1 pM) was significantly (P<.01) reduced (Fig. 4b).

To determine whether the P-agonist effect may be mediated by CAMP, rolipram, a selective type IV phos-

Lacoste, Malham, Cueff, 8r Poulet

Noradrenaline (yM) : mo.01

0.1 01 .0

* T

0.01 0.1 1.0 10.0

lnterleu kin-I a (nglml)

Fig. 2. The stimulatory effect of 1L- 1 a on the hemocyte CL- response is reduced in the presence of 0.01-1.0 FM NA. Data are mean CL values and standard errors expresscd as a percentage of control (cell samples incubated with the various noradrenaline concentrations and unexposed to IL- 1 a). *=significantly different from controls (P<.Ol).

phodiesterase inhibitor, was used. Separately, suboptimal concentrations of rolipram (10 nM) or isoproterenol (0.01 kM) did not significantly inhibit the stimulation of hemocyte ROS production by 1L- 1 a (Fig. 5). However, experiments where hemocytes were pretreated with both rolipram and isoproterenol show that rolipram significantly (P<.OI) enhanced the inhibitory effect of a suboptimal concentration of isoproterenol on the response of hemocytes to IL-la. The synergic effect of the combination was apparent when hemocytes were stimulated with 1 or 10 ng/ml TL-la.

Finally, to determine whether the P-agonist inhibition of the hemocyte response to 1L-la may be mediated by protein kinase C or protein kinase A, the effects of calphostin C, a protein kinase-C inhibitor and H-89, a protein kinase-A inhibitor, were tested. Calphostin C (80 nM) had no significant effect on the P-agonist inhibition of hemocyte response to 1L- la (Fig. 6a) whereas, in the presence of the protein kinase A inhibitor H-89 (25 kM), the suppressive effect of isoproterenol (1 pM) was significantly (P<.01) reduced (Fig. 6b).

Discussion

Although invertebrates do not possess a complex immune system compared to vertebrates, they are ca- pable of effective cellular and humoral responses (Sminia & Van der Knaap 1987). Indeed, invertebrate immunocytes (also called hemocytes) are responsible for defense reactions such as wound repair (Fisher I 988), encapsulation, phagocytosis (Chu 1 98S), and

Noradrenaline and TL- 1 a stimulation in molluscan hemocytes 36 1

Phenylephrine (pM) : a * *

0.01 0.1 1.0 10.0

Interleukin4a (nglml)

0.01 0.1

*

1 .o

I

*

10.0

Interleukin-l a (nglml)

Fig. 3. Effects of adrenergic receptor agonists on the hemocyte CL-response stimulation by IL-la. (a) Effects of the u-adrenoceptor agonist phenylephrine and (b) effects of the P-adrenoceptor agonist isoproterenol. Data are mean CL values and standard errors expressed as a percentage of control (cell samples incubated with the various agonisl concentrations and unexposed to IL-1 a). *=significantly different from controls (P<.O I ) .

reactive oxygen species production (Bachirre et al. 1991; Pipe 1992; Noel et al. 1993; Greger et al. 1995; Bramble & Anderson 1998). These immune functions are thought to be mediated by diverse cytokines (Hughes et al. 1990, 1992; Beck & Habicht 1991, 1996; Raftos et al. 1991; Stefano et al. 1991; Granath et al. 1994; Owve-Missi-Oukem-Boyer et al. 1994; Cooper et al. 1995; Ottaviani & Franceschi 1996). IL- I a- and IL-6-like molecules have been purified and characterized in invertebrates (Hughes et al. 1990; Raftos et al. 1991; Ottaviani & Franceschi 1996). The echinoderm IL-1 is known to bind specifically to a

0 NA + Prazo Q) 0 C 150 gz T

0.01 0.1 1.0 10.0

In terleu kin-I a (nglm I)

b 2001.NA * a uNA+Propa a T

0.01 0.1 1.0 10.0

lnterleu kin-I a (nglm I)

Fig. 4. Effects of adrenoceptor antagonists and NA on the hemocyte response to IL-la. (a) Effects of the ai-adrenoceptor antagonist prazosin and (b) effects of the P-adrenoccptor antagonist propanolol. Data are mean CL values and standard errors expressed as a percentage of control (cell samples incubated with NA or NA+antagonists and unexposed to IL-la). *=significantly different from controls (P<.OI). Matching letters above bars indicate values that arc signi li- cantly different from each other (P<.Ol).

membrane receptor at the surface of the hemocyte (Beck et al. 2000) and to stimulate hernocyte immune functions such as phagocytosis (Beck et al. 1993). Hu- man IL-1 can bind to the invertebrate 11,-1 receptor (Beck et al. 2000) and previous studies have shown that human IL-1 can also stimulate phagocytosis and cell migration in bivalves at concentrations ranging from 0.1-100 ng/ml (Hughes et al. 1991). In the present study, we show that human IL-la can also stimulate ROS production in hemocytes of the oyster, C. gigas. However, results also indicate that NA, the prin- cipal circulating catecholamine in oysters, causes a

362 Lacoste, Malham, Cueff, & Poulet

0.01 0.1 1.0 10.0

Interleukin-la (nglml)

Fig. 5. Synergistic effect of the type IV phosphodiesterase inhibitor roliprani (Ro) and a suboptimal concentration (0.0 1 FM) of isoproterenol (ISO) on the IL- 1 a-induced increase of hernocyte CL-response. Data are mean CL values and standard errors expressed as a percentage of control (cell samples incubated with [SO, Ro or ISO+Ro and unexposed to IL- I a). :!:=significantly different from controls (P<.Ol). Matching letters above bars indicate values that are significantly different from each other (P<.Ol).

\ignificant (P<.0 1) dose-dependent inhibitory effect on hemocyte response to IL-la .

NA concentrations of 0.1-1 p,M fall within the rang- es reported in oyster or scallop tissues (Osada & No- mura 1989; Pani & Croll 1995) and they are known to exert physiological effects in bivalves (Muneoka & Kamura 1982). Thus, the inhibitory effect of NA on the hemocyte response to IL- la is likely to occur in vivo, especially during stress or when NA concentrations increase in tissues (Osada & Nomura 1989).

To determine whether a - or P-adrenoceptors mediate the effect of NA on hemocyte response to IL-la , specific agonists where used. Phenylephrine, which is known to be active as an a-adrenoceptor agonist in Cmssostrea gigas (Coon & Bonar 1987), had no effect on hemocyte responsiveness to IL- 1 a, whereas the P-adrenoceptor agonist isoproterenol inhibited the IL- la-stimulated increase of hemocyte CL-response. Moreover, in the presence of the P-adrenoceptor antagonist propanolol the effect of NA was reduced. Tak- en together, these results indicate that P-adrenergic receptors are involved in the suppressive effect of NA on hernocyte responsiveness to IL- la. Previous studies have shown that a P-adrenergic receptor-cyclic AMP- dependent signaling pathway exists in mollusc foot- muscle sarcolemma and can be modulated in vivo and in vitro by isoproterenol and propanolol at micromolar concentrations (Pertseva et al. 1992).

The present results also show that the P-adrenergic

IS0 IS0 + CalC

1

0.01 0.1 1.0 10.0

Interleukin-I a (nglml)

b * a T

0.01 0.1 1.0 10.0

Interleukin-l a (nglml)

Fig. 6. Effects of protein kinase inhibitors on the isoproterenol-induced reduction of heniocyte response to IL- 1 a. (a) Effects of the protein kinase-C inhibitor calphostin C (CalC, 80 nM) and (b) effects of the protein kinase-A inhibitor H- 89 (25 p,M). Data are mean CL values and standard errors expressed as a percentage of control (cell samples incubated with isoproterenol (ISO) ( 1 pM) or ISO+inhibitors and unexposed to IL- 1 a). *=significantly different from controls (P<.01 ). Matching letters above bars indicate values that are significantly different from each other (P<.O I ) .

inhibition of oyster hemocyte responsiveness to IL- 101

involves CAMP and protein kinase A. Several in vitro studies have shown that adrenergic neuroendocrine hormones and drugs inhibit immune cell functions in vertebrates (Koff & Dunegan 1985; Nielson 1987; Tosk et al. 1993) and NA has been shown to inhibit the capacity of mammalian immune cells to respond to y-interferon or lipopolysaccharide stimulation (Koff et al. 1986) and to suppress the in vitro reactivity of rat T lymphocytes to mitogens (Felsner et al. 1995). However, few studies have examined the effect of NA on the invertebrate immune system and the present re-

Noradrenaline and IL- 1 a stimulation in molluscan hemocytes 363

sults demonstrate for the first time the existence of a P-adrenoceptor-CAMP-protein kinase A signaling 173-180. pathway in mollusc immune cells.

~~~~~~~~i~~,~, the neuroendocrine system is not the only source of catecholamines in molluscs; hemocytes are also known to secrete N A upon stimulation by cor- ticotropin-releasing factor, adrenocorticotropic hor- =,

Ostrea edulis and Crassostrea gigas. Dis Aquat Org I I :

Beck G & Habicht GS 199 1 . Purification and biochemical characterization of an invertebrate interleukin- I . Mol Im- muno'. 28: 577-584.

1996. Characterization of an IL-6-like molecule ~

from an echinoderm (Asterias jbrbesi). Cytokine 8: 507-

mone, or even IL- la (Ottaviani & Franceschi 1996). These data and the present results suggest that the se- cretion of endogenous N A may act in an autocrine or paracrine fashion to modulate hemocyte responsive- nesc to TL-la.

The mechanisms by which N A causes reduced hemocyte responsitivity to 1L-la remain to be determined. Noradrenaline-induced down-regulation of key elements required for ROS production may prevent the stimulation of hemocyte CL-response by normal doses of IL- 1 a. Alternatively, expression of IL- l a receptors may be affected by this hormone. Similar regulation is known to exist in mammals where activation of the P-adrenergiclcAMP1protein kinase A signaling pathway, by local and systemic release of catecholamines, leads to destabilization of messenger RNA encoding cell surface molecules involved in T-cell activation (Wajeman-Chao et al. 1998).

The present study represents a further step in the characterization of immune-neuroendocrine interactions in invertebrates. The existence of a P-adrenoceptor mediated second messenger system in molluscan immune cells suggests that immune-neuroendocrine interactions in these animals are more complex than previously thought. Further work is in progress to determine whether catecholamines can also modulate molluscan immunocyte responses to other cytokines. Results from such studies and comparison between invertebrate and vertebrate systems are expected to help in understanding the basic principles that have led to the construction and the evolution of immune-neuroendocrine relationships in animals.

Acknowledgments: This work was supported by grants from the Conscil Regional de Bretagne, Departement du Fin- istkre, CBtes d' Armor et Ille-et-Vilaine, and the Section RC- gionale Conchylicole de Bretagne Nord. We would like to thank Dr B. Kloareg and members of his group for advice concerning luminometry.

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Noradrenaline reduces the stimulatory effect of interleukin-1α on reactive oxygen species production by oyster immunocytes