Synergism between interleukins 1β and 6 on noradrenergic nerves in rat myenteric plexus

Synergism Between lnterleukins Nerves in Rat Myenteric Plexus ANNE RijHL, Intestinal Diseases

SUZANNE

HURST,

and STEPHEN M. COLLINS

Research Program, McMaster University Medical Centre, Hamilton, Ontario, Canada

Bac&?round/Aims: Because levels of interleukins lb and 6 (IL-Q and 11-6) are elevated during intestinal Trichinella spirahs infection, they may mediate the changes in enteric neural function in that model. IL-lb suppresses norepinephrine release from the myenteric plexus, but the effect of IL-6 is unknown. Therefore, we investigated the effects of IL-6 alone and in combination with IL-Q on norepinephrine release. Methods: Longitudinal muscle myenteric plexus or myenteric nerve varicosity preparations from jejunum of noninfected rats were loaded with [3H]norepinephrine, and 3H release was measured after a preincubation with or without human recombinant IL-6, alone or in combination with human recombinant IL-Q. Results: 1 ng/mL of IL-6 augmented 3H release, 100 ng/mL suppressed 3H release, whereas 10 ng/mL had no effect. However, IL-6 (10 ng/mL) plus a subthreshold concentration of human recombinant IL-Q significantly suppressed 3H release, and this was abolished by adding anti-IL-6 antibody or an IL-1 receptor antagonist. Conclusions: Because 3H release reflects [3H]norepinephrine release, our results show that IL-6 exerts a dual effect on norepinephrine release. Furthermore, there is synergism between IL-lb and IL-6 resulting in suppression of norepinephrine release. Therefore, both cytokines may contribute to the suppression of norepinephrine release observed in the inflamed intestine.

I

ntestinal

in

inflammation

motility

lp and 6 on Noradrenergic

in

humans

by changes inflammatory bowel

is accompanied

with

disease’-” and in small mammals after nematode infe’ction.“-’ Studies in the latter have suggested that the inflammation-associated motility changes reflect, at least

The ability of inflammation

of exogenous

teric plexus has been established as for norepinephrine.‘““’ gest that IL-l substance

to mimic

P contentI

the effects

release in the myenfor acetylcholine

Recent

preliminary

may also be involved

data sug-

in the increase

and in the suppression

lease in the rat infected

as well in

of NE re-

with T. spiralis.” However,

the

role of IL-6 has yet to be determined. The present the ability

study was conducted,

of IL-6 to influence

in part, to evaluate

NE release from the rat

myenteric plexus. Furthermore, because our preliminary data support a role for IL- 1 p as a mediator of the suppression of NE release in the inflamed intestine’> and because both

cytokines

we investigated

are present

in the inflamed

the interactions

between

6 on NE release. This was prompted

intestine,”

IL-lp

and IL-

by the knowledge

of synergistic interactions between IL-1p and IL-6 in other biological systems.‘“.” Finally, we used a recently described

nerve varicosity

plexus to determine

preparation

whether

of the myenteric

interactions

and IL-6 occur at the level of the adrenergic or whether

an intermediary

between

IL-lp

nerve ending

cell type is necessary.‘”

In all experimental protocols, human recombinant (hr) cytokines were used. Approximately 75%-780/o of the amino acid sequence

of IL-10

is conserved

across a wide

range of species,‘” and previous studies have shown that hrIL-lb is biologically active in murine and rat preparations (for review see Dinarello”). Although human and rat IL-6 share only a 58% homology in amino acid sequence,“’ hrIL-6 already has been shown to be highly active in both murine and rat systems.20m2i

in part, alterations in smooth muscle and enteric nerve function. These include the demonstrations of an increase

Materials

in substance P content’ and a suppression of acetylcholine and norepinephrine (NE) release’,‘O from the myenteric plexus. The changes occur as a result of the inflammatory response of the host.‘-‘” Increases in levels of the inflammatory cytokines interleukins lb (IL-lp) and 6 (IL6) have been documented in the myenteric plexus and longitudinal muscle in the rat intestine infected with Tricbinella spiralis’’ and are therefore putative mediators

Animals

of these changes.

IL-1p

on neurotransmitter

and Methods

Male Sprague-Dawley rats (180 - 200 g) were supplied by Charles River Breeding farms (Montreal, Quebec, Canada). Abbreviations used in this paper: anti-IL-6ab, anti-human IL-6 antibody; EFS, electrical field stimulation; hr, human recombinant; LMMP, longitudinal muscle myenteric plexus; NE, norepinephrine; NV, myenteric nerve varicosities; ra, receptor antagonist. 0 1994 by the American Gastroenterological Association 00185085/94/$3.00

994

Rats

RiiHL

were

kept in filtered cages on a 12-hour

and under allowed

GASTROENTEROLOGY

ET AL.

conditions

of controlled

light-dark

temperature.

cycle

They were

neutralized

food and water ad libitum.

compounds

NaH2P04,

acid from Mallinkrodt

fluid and

(Oakville, act,

KY); pargyline,

acid,

and

(St. Louis, MO); [‘H]NE

from New England lation

were used: NaCI,

NCS-II

tissue

hrIL-1P

1 X 10” U/pg).

ment of Pathology,

(sp act,

acid,

from

Sigma

15.0 Ci/mmol)

solubilizer

from

and the polyclonal

rabbit

were a gift from Dr. J. Gauldie McMaster

was a gift

from

University).

of Synergen

Rats were killed by decapitation,

chromatography

at the ligament

jejunum

superfusate

A previously nerve varicosities the method

described

by cervical dislocation,

1.2 MgCI,;

were obtained previously

was

for 10 minutes.

(in mmol/L)

glucose; 0.11 ascorbic acid (as antioxidant); bubbled

oxidase inhibitor).

with 95% oxygen/S%

The method described.

[‘H)NE

of 0.5 pmol/L

The supernatant

cordingly, cytokines

in experiments for periods

added during

than 40 minutes,

The

a pH of 7.4. was previously

the last 40 minutes

Ac-

to the

[‘H)NE

The pellet

was

HEPES;

20 minutes.

desipramine

reuptake

with I’HINE the LMMP preparations were mounted between two parallel silver electrodes in a water-jacketed superfusion chamber and maintained at 37°C. The preparations were superfused with Krebs’ buffer and superfusate samples were collected in 2-mL fractions equilibration

every 2 minutes

period of 40 minutes,

for 60 minutes.

After an

‘H release was evoked by

electrical field stimulation (EFS) at 30 V for 0.5 milliseconds with a frequency of 10 Hz for 1 minute, which are optimal conditions for measurements of evoked NE release from rat myenteric plexus. “’ At the end of the superfusion period, 4 mL of ACS scintillation fluid were added to each fraction, and the ‘H content was measured by liquid scintillation spectrome-

maximal

and

was again

effect of cytokines is obtained

of 10 Fmoli

and centrifuged pellet

at

was resus-

concentration

of 1

allowed

to sit at room

before

it was used in

In experimental preincubation

to

at 37°C for

twice with Locke’s

The obtained

for at least 20 minutes SO-minute

was added

at a concentration

of noradrenaline,

The suspension

The superfusion

been described.“’ Briefly, after the preincubation

[‘H]NE

of 0.1 PCiimL

in Locke’s buffer at a final protein

preparation

has previously

preparation,

2O,OOOg, 4°C for 20 minutes. mg/mL.

10

was oxygenated

The NV were then washed

L to prevent

cytokines,

release

140

10 glucose;

acid; 0.11 ascorbic

The suspension

at a concentration

buffer, containing

Measurement of [3H]NE Release From LMMP used to assess {‘H]NE

(in mmol/L)

1 MgCl,;

at 24°C for 30 minutes.

To label the varicosity the suspension

pended

was

containing

2.5 CaCl,;

acid, and 0.03 pargyline. allowed to rehydrate

All supernatants

the varicosities

0.004 ethylenediaminetetraacetic

temperature

of the preincubation.

in Locke’s buffer

‘H release experiments.

method

on ice, and the

containing

and 0.03 pargyline

where tissues were exposed

longer

was spun at lOOOg, 4°C

was placed

gauze, and spun at 2O,OOOg, 4°C

11.1

at 37°C for 40 minutes.“’

with scissors and homogenized

The homogenate

for 50 minutes.

was added to the tissue at a concentration

(15.0 Cilmmol)

and

described

were pooled, filtered through

The buffer was continuously CO* to maintain

was removed, technique

from

120.0 NaCI; 5.9 KCI;

for loading the LMMP preparations

from

mus-

described.”

1.2 NaH*PO,;

was adapted

et aLL4 Rats were killed

pellet was washed with the sucrose solution.

were tied at each end and placed

15.5 NaHCO,;

to NE.”

LMMP was placed in 0.32 mol/L sucrose

resuspended

in Krebs’ buffer containing

to

thin layer

was used to prepare

the small intestine

NaCl; 5 KCl; 5 NaHCO?;

(a monoamine

technique

This technique

by Hammond

peeled from the underlying

2.5 CaCl,;

is still bound

described

(NV).i8

on the serosal surface.

LMMP preparations

study,

Preparation of Nerve Varicosities

Briefly, the LMMP was cautiously the jejunum

in

fractions and the

was used to show that more than 80%~ of the

‘H in the collected

for 3 X 5 seconds.

of Treitz. Longitudinal

using a method

‘H

‘H content

in the tissue. ‘H release was considered

on ice. The tissue was minced

and the jejunum

plexus (LMMP) preparations

to deter-

The evoked

of the total

release because in a previous

tissue after scoring obtained

in the tissue.

as a fraction

residual radioactivity reflect [‘HlNE

above. The isolated

Preparation of Longitudinal Muscle Myenteric Plexus Specimen

the proximal

acetic acid, 4 mL ofscintilla-

the LMMP was isolated using the peeling

removed starting

and solubilized

These samples were then

the tissue which was the sum of all superfusate

anti-

(Depart-

The IL-1 -receptor

Dr. R. Thompson

‘H content

No. 4

Amersham

CO).

cle myenteric

with 100 PL ofglacial

release was expressed

from UBI (Lake Placid, NY; sp

hrIL-6

human IL-6 antibody antagonist

ascorbic

HEPES

Nuclear (Boston, MA); ACS liquid scintil-

Ontario);

(Denver,

CaCI,,

KCI, glucose, sucrose, and acetic

(Paris,

ethylenediaminetetraacetic Chemicals

dry, weighed,

tissue solubilizer.

mine the residual

The following NaHCOi,

were blotted

tion fluid were added, and the samples were counted

Materials MgCI,,

try. The tissues

with 1 mL NCS-II

Vol. 107,

protocols

periods

involving

were chosen as a

on the release of NE from the NV

after 50 minutes

NE was added for the last 20 minutes

of preincubation.”

of the incubation,

the aliquots were allowed to sit at room temperature further 20-minute period before stimulation.

and for a

Measurement of [3H]NE Release From NV Scorpion venom in a concentration of 10 PglmL was used to stimulate 3H release from NV. Five hundred microliters of the nerve varicosity suspension was transferred into plastic tubes, and 10 PL of the scorpion venom were added. The aliquots were incubated at room temperature for 20 minutes. After this stimulation period, duplicate 2OO+L aliquots were centrifuged at 11,500g for 5 minutes. Two milliliters of ACS scintillation fluid was added to lSO+L aliquots of the

October 1994

supernatant,

IL-P AND IL-6 IN MYENTERIC PLEXUS

and the ‘H content

by liquid scintillation rations,

of the samples

spectrometry.

‘H release was considered

was measured

was added

[‘H]NE

As with the LMMP prepa-

of a subthreshold

to reflect [‘H]NE

previous

release.

for the last 20 minutes.

The determination

concentration

was based on our

of IL-lp

study, lx and a subthreshold

The release of [IH]NE was calculated as dpm/mg protein, and the evoked [‘H]NE release was expressed as percentage of

chosen on the basis of preliminary

baseline.

Locke’s buffer,

the preincubation 10 pmol/L.

Protocols Involving hr Cytokines First, experiments of IL-6

To examine

lease from LMMP,

100 ng/mL)

was added

To evaluate hrIL-6

to the superfusate

Control

and changes

at varying

liminary

experiments

while

in evoked ‘H release

(O.l-

100 ng/mL)

demonstrating

a dose-response

a maximal

tissues were preincubated hrIL-I p (0.1 ng/mL) combination threshold ies,”

used throughout

the synergistic

interactions

our present study. of IL-lb

with subthreshold

and IL-6 (10 ng/mL)

for 120 minutes. concentration

was established and IL-6,

concentrations

of

either

alone or in

The determination

of a sub-

of IL-lb

was based on previous

and the IL-6 concentration

stud-

was based on our dose-re-

To evaluate investigate reversed

the specificity

if the combined by blocking

were performed human

using

a neutralizing (anti-IL-bab)

In control

hrIL-1P mixture.

to the control

hrIL-6 was combined and the mixture then

added

In experiments

bated with the antagonist

before the mixture

experiments

with a 1:lO dilution

of the anti-IL-bab,

at 4°C for 30 minutes

to the NV suspension. with saline.

were per-

In some experiments,

In controls,

In another

was preincubated

hrIL-6

with the IL-lra

before the cytokines

were preincubated

and was

set of experiments,

the

(10 FglmL)

were added. Con-

with saline.

All studies involved at least four animals, means _’ SEM of at least four separate experiments

and data are each involv-

ing tissue from one animal. Because of interexperimental tion in the EFS-induced tokine exposure proportional

as a percentage

For statistical

data were stabilized

Comparisons

between

varia-

NE release, values obtained

were expressed

value for that tissue.

analysis,

after cy-

of the control variances

of the

using arcsine transformations.

two groups

were made using Student’s

t test, whereas one-way analysis of variance was used for comof more than two groups.

considered

A P value of ~0.05

was

significant.

Results Effect of Cytokines on [3H]NE Uptake

hrIL-6

was added

Preincubation

6 at different did

by the protocol,

with the hrIL-6-antibody the tissue was preincu-

(10 pg/mL)

at 37°C for 20 minutes

Control

tissues were preincu-

[jH]NE

preparations

or with

uptake

with

IL-lb

plus

by the tissue

ILIL-6,

(Table

1).

Immediate Effects of IL-6 on [3H]NE Release

at 4°C before it was

using IL-lra,

of the LMMP

concentrations,

not change

hrIL-6 was mixed with

If demanded

of the cytokines.

and

Data Expression and Statistical Analysis

anti-

(in a 1:lO ratio of antibody

for 30 minutes tissue.

polyclonal

in buffer,

venom.

the following

was preincubated

trol suspensions

could be

using anti-IL-bab,

was added simultaneously

before addition

rabbit

experiments,

saline and preincubated added

and to

of

at 20,0001:

or a specific IL-1 receptor

with the antibody

excess) at 4°C for 30 minutes to the tissue.

effects

effect of the cytokines

(ra).2’,26 In experiments

was preincubated

in the NV preparation,

either one of them, separate experiments

IL-6 antibody

antagonist

of the cytokine

were resuspended

formed as with the LMMP preparations.

parisons

sponse curve of the effects of IL-6.

with

To determine the specificity of the cytokine effects and to support the contention of synergism between IL-lp and IL-6

effect of IL-6

after 120 minutes.

curve for hrIL-lb

for the hrIL- 1 p preparation To study

for 120

twice

at a concentration

with scorpion

at 37°C for 15 minutes

containing

At the end of

were then centrifuged

the pellets

NV suspension

preparations

of IL-6 was

experiments.

the NV were washed

The suspensions

preincubated

with saline.

concentration

desipramine

the NV were stimulated

time was chosen on the basis of pre-

on ‘H release from the LMMP preparation Similarly,

(1 or

the tissues

Krebs’ buffer

concentrations

The incubation

hrIL-6

effect of IL-6, LMMP

in oxygenated

minutes.

onto the tissue,

tissues were treated

a delayed

preincubated

of 1, 10, or 100

superfused

set of experiments,

stimulated

were monitored.

affects ‘H re-

Changes of the basal ‘H outflow

In another

were electrically

were

and directly

by a washout period.

were monitored.

to study the effects

IL-6 immediately

hrIL-6 at a concentration

ng/mL was repeatedly followed

were performed

whether

period, containing

for 20 minutes,

995

There evoked IL-6

release was

was

no

change

of ‘H from

added

directly

in

the

the LMMP to the

basal

outflow

preparations

superfusate

or

when

(data

not

shown).

bated with saline at 37°C for 20 minutes. To exclude effects because of possible tion, hrIL-6 at different kines were boiled

concentrations

for 20 minutes

endotoxin

contamina-

and the combined

before being

added

Concentration Dependence of IL-6 Effect

cytoAs shown

to the

tissues

effects

To examine interactions of the cytokines at the level of the neural membranes, NV were preincubated for 50 minutes with

preparations.

hrIL- 1p (0.0 1 ng/mL) and hrIL-6 (10 ng/mL) either alone or in combination; controls were preincubated with saline, and

mL)

in Figure

on electrically

and biphasic. caused

concentrations

These Lower

effects

‘H release

from

were concentration

concentrations

an increase (50-

1, IL-6 had profound

evoked

in

of hrIL-6

‘H

release,

100 ng/mL)

caused

delayed

the LMMP dependent (O.l-

whereas

1 ngi higher

a suppression

of

996

RUHL ET AL.

GASTROENTEROLOGY Vol. 107, No. 4

Table 1. [3H]NE Uptake Cytokine (WmL)

by the LMMP

IL-lo,

31

Mean r SEM (dw/mg)

27,964

t

simultaneously.

1522

20,991

t

for 120 minutes

3407

With hrll-6

and hrll-1P

Either Alone or in Combination

slgnlhcant

‘H release. A concentration ‘H release by 49% of IL-6 suppressed

IL-6. 0.1

11.6. 1

IL-6, 10

11.6. 50

11.6. 100

0.1 + 10

6

7

5

3

11

10

20,798

wth either hrll-6

13H]NE uptake was calculated

There were no statlstlcally

as compared

0.1 5

NOTE. Tissues were premcubated (0.1 ngfmL)

After Preincubation

IL-10 + ILK 0

”

tssue.

Preparations

differences

t

1995

at dlfferent

from total 3H outfkw between treatment

24.600

and the residual

(0.1-100

27,665

2 1110

ng/mL),

hrll-10

radvxxtlvlty

m the tissue: calculated

? 4% (P < O.Ol), and 100 ng/mL A concentration

potentiating

experiments

interactions

designed

between

As illustrated LMMP

of 10 ng/mL did not alter ‘H release, and this concentration was used in the subsequent

f 6533

IL-lp

and IL-6.

Concentration Dependence of IL-Q Effect to our

previously

reported

in Figure

preparation

threshold

with

findings, ’’

concentrations

changes.

bated with hrIL-1P

2 6% of control,

that had been preincubated + 10.4% of control. were combined,

was significantly

suppressed

(50.4%

with

of ‘H release (100%

both

that

anti-IL-6ab

or saline. abolished

was observed

cytokines

in

the

+ 3.9% of control group;

hrIL-6 was The pretreat-

the suppression

in the tissues absence

P < 0.001)

(Figure

treated

of anti-IL-6ab

in the antibody-treated

kine group vs. 65% + 3.9% of control treated

How-

‘H release

+ 22.3% of control;

In a separate set of experiments,

preincubated

used, we could only show a maximal

of

significant

ever, when the two cytokines

with

was used at a concentration

cause

with hrIL-6 alone was 99.3%

ever, in contrast to our previous study in which a hrILlb preparation with higher specific activity had been 30% suppression

not

of the

or IL-6 at sub-

alone was 93.5%

ment of IL-6 with the antibody

2). How-

IL-lp

did

a preincubation

of ‘H release when IL-lp 100 ng/mL.

+ 4362

and hrlL-10

‘H release from tissue that had been preincu-

P < 0.001).

(Table

32,625

2, preincubation

either

hrIL-lb showed a concentration-dependent suppression of evoked ‘H release from the LMMP preparations after period of 120 minutes

% 1834

or hrlL-6 (10 ng/mL)

results were corrected for the weight of the respectwe

and ‘H release from tissue

Similar

27,753

of 0.1 ng/mL.

Interactions of IL-Q and IL-6:Findings in the LMMP Preparation

‘H release by 31% + 10% (P < 0.05) controls.

25,337

at a concentrabon

groups.

of 1 ng/mL of IL-6 increased

with the respective

to investigate

-’ 4448

concentrabons

cyto-

in the cytokine-

3). Similarly,

in an-

other set of experiments

it was shown that pretreatment

of the tissues with IL-lra

abolished

suppression

of ‘H release (112%

the cytokine-induced t

7.5% of control

67% + 6.6% of control; P < 0.01) (Figure 4). As shown in Figure 5, boiling of the hrIL-6

vs.

for 20

minutes abolished both the stimulatory effect and the inhibitory effects of the cytokine. Boiling both hrIL-1P and hrIL-6 for 20 minutes abolished the synergistic effect of the cytokines.

Interactions of IL-l/3 and IL-6:Findings in the Nerve Varicosity Preparation

2

.

..I

1

CONCENTRATION

1.

5.

1..

OFbrIL-L(ng/ml)

Flgure 1. Concentration dependence of the delayed effect of IL-6 on evoked 3H release from LMMP preparations. Tissues were preincubated with hrlL6 at the indicated concentrations for 120 minutes. 3H release was evoked by EFS. Each bar represents at least six separate experiments at the respective concentration. Results have been calculated as means ? SEM. Evoked 3H release from IL-6-treated tissues is expressed as a percentage of the evoked release from control tissues. The dotted line represents 3H release from the respective control groups (100%). Controls were preincubated with saline. *Significant difference from control group (P < 0.01). **Significant difference from control group (P < 0.05).

As shown in Figure 6, preincubation of the nerve varicosity preparation with either IL-lp (0.01 ng/mL) or IL-6 (10 ng/mL) alone did not cause significant changes: ‘H release from control varicosities was evoked 15.9% -+ 2.6% of basal release; evoked ‘H release from nerve varicosities that had been preincubated with hrILlp alone was 14.2% 5 2.2%; and evoked ‘H release from NV that had been preincubated with hrIL-6 alone was 15.5% + 2.4%. In contrast, evoked ‘H release from NV that had been preincubated with both cytokines was only 7.0% t 1.5%, and this was significantly different

IL-P AND IL-6 IN MYENTERIC PLEXUS

October 1994

Table 2. Dose-Response

Relationship

for IL-lp 0

0.1

1

10

0

6.5 ? 1.8

14.5 t 7.4

25.0 f 4.6”

IL-lfl (ng/mL)

Inhibition of [?i]NE release (%)

997

100 32.0 + 3.3”

NOTE. Given are mean 2 SEM from four separate experiments. “Significant difference from control (P < 0.05).

from control when

hrIL-6

(P < 0.05). This suppression had been preincubated

was abolished

with

Evoked “H release from these preparations 3.0%

(Figure

varicosity

7). Similarly,

preparations

kine-induced

suppression

IL-lra

jH

preparations

more, the anti-IL-6ab

plexus

to NE,”

‘H

release is considered

to reflect the release of l’H]NE

in

release.

The group

had any intrin-

3, 4, 7, and 8). Furtherwith hrIL-lb

(data not shown).

of cytokines it focuses

that

than

study. In addition,

pretreatment

with

evoked by electrical

study further

on neural

function

on the effects

characterizes in the gut.

of IL-6 on NE

sympathetic

neurons

in the myenteric

interactions

between

IL-6 and IL-lp.

Specifically, release

plexus,

To measure NE release from sympathetic release was quantitated

the effects

after the myenteric

from

abolished

as well as

ng/mL).

of an adrenergic

from our experiments

central, neurons,

‘H

trast,

effect at higher

autonomic,

neural origin indicate

of

that IL-6

exposure

of the tissue

and consisted

of a stimu-

(0.1 - 1 ng/mL)

concentrations

there are no previous

or enteric shown

release

and this pro-

of IL-6 on neurotransmitter

it has been

desipra-

release from the myenteric

for 120 minutes,

To our knowledge,

of the action

studies show

l’H]NE

field stimulation,‘“.‘”

latory effect at lower concentrations an inhibitory

bound

our previous

plexus. The effect of IL-6 required to the cytokine

of the ‘H released

6-hydroxydopamine,

virtually

vides indirect evidence the released ‘H. The results

80%

is still

exerts a dual effect on l’H]NE

Discussion The present

the present

more

mine, or tetrodotoxin

did not cross-react

was

from the myenteric

the cyto-

cytokine

nor the IL-lra

On the basis of a

study in which thin layer chromatography

of the nerve

8).

(Figures

had been loaded with l’H]NE.

used to show that

sic effect on the evoked 3H release from LMMP or nerve varicosity

ration

previous ?

abolished

of evoked

was .19.6% 5 3.1% (Figure the anti-IL-6ab

was 15.3%

pretreatment

with

evoked 3H release in the IL-lra-treated Neither

anti-IL-6ab.

nervous

previously

and

(50-100 reports

release in the systems.

that

In con-

IL-1B affects

plexus prepa-

120 120 100 100 80 80 60 60 40 40 20 20 0 0

Figure 2. Effects of IL-lb and IL-6 alone or in combination on evoked 3H release from LMMP preparations. Tissues were preincubated with ), hrlL-6 (10 ng/mL; LR),or a combination thereof (m) for 120 minutes. 3H release was evoked by EFS. Each bar repre sents at least six separate experiments. Results have been calculated as means ? SEM. Evoked 3H release from cytokine-treated tissues is expressed as a percentage of the evoked release from control tissues. Controls were preincubated with saline (0). *Significant difference from control group (P < 0.001).

Pigure 3. Effect of neutralizing IL-6 antibody on IL-lband IL-6induced suppression of evoked 3H release from LMMP preparations. Tissues were preincubated with saline (0). hrll-10 (0.1 ng/mL) and ), the cytokines plus antibody (RI), or the antibody alone (W) for 120 minutes. ‘H release was evoked by EFS. Each bar represents at least four separate experiments. Results have been calculated as means 2 SEM. Evoked 3H release is expressed as a percentage of the evoked release from control tissues. *Significant difference from control group (P < 0.02). **Significant difference from cytokine group (P < 0.001); not different from control group.

998

RiiHL ET AL.

GASTROENTEROLOGY Vol. 107, No. 4

2

IL-6,

cr Figure 4. Effect of IL-1 receptor antagonist on IL-lb- and IL-6- induced suppression of evoked 3H release from LMMP preparations. Tissues were preincubated with saline (O), hrll-lb (0.1 ng/mL) and ). the cytokines plus IL-lra (LB), or IL-lra alone (0) for 120 minutes. ‘l-l release was evoked by EFS. Each bar represents at least four separate experiments. Results have been calculated as means + SEM. Evoked 3H release is expressed as a percentage of the evoked release from control tissues. *Significant difference from control group (P < 0.02). **Significant difference from cytokine not different from control group. group (P -c 0.01);

release

neurotransmitter

tem,27,‘X the autonomic

in

the

central

nervous

sys-

lng/ml

IL-6, lOOng/ml

IL-lD+IL-6

Figure 5. Effect of heat treatment of IL-lb and IL-6 on cytokine-induced suppression of evoked 3H release from LMMP preparations. HrlL-6 at a concentration of 1 ng/mL or 100 ng/mL and hrll-1)3 (0.1 ng/mL) plus hrlL-6 (10 ng/mL) were added to the LMMP preparations without pretreatment (LR)or after boiling for 20 minutes (W). Tissues were preincubated for 120 minutes. 3H release was evoked by EFS. Results have been calculated as means + SEM from four separate experiments at each concentration. Evoked 3H release is expressed as a percentage of the evoked release from control tissues. Controls were preincubated with saline (0). Boiling of the cytokines abolished the respective effects of the untreated cytokines. *Significant differ**Significant difference from ence from control group (P < 0.01). control group (P < 0.05). “Significant difference from control group (P < 0.001).

nervous system,‘” and the enteric

nervous system. “,” In the enteric

nervous system,

IL-lb

suppresses neurotransmitter release from the myenteric plexus with a delayed onset of action,‘“,” similar to the

myenteric

pattern

fects occur at the level of the neural membrane.

displayed

by IL-6 at higher concentrations

in the

ties of sympathetic plexus

nerve suggest

terminals

prepared

from the

that the cytokine-induced

ef-

However,

present study. In summary, the observed effects of IL-6 in the enteric nervous system seem to be unique in that

because the nerve varicosity preparation is an impure one, we cannot exclude the possibility of cytokine interactions

IL-6 exerts opposite tion involved.

with

When

IL-lp

concentrations,

effects depending

and IL-6 were combined they

caused

on the concentra-

membranes

from other cell types with

the subse-

in subthreshold

a significant

50%

sup-

pression of NE release from the LMMP. A similar phenomenon was also observed using the nerve varicosity preparation. The suppression of [‘HINE release by IL-6 and IL-lp does not reflect changes in neurotransmitter uptake

because

preincubation

of the myenteric

plexus,

in the presence of cytokines at different concentrations and in different combinations, did not affect the uptake of radiolabeled NE. In addition, the heat sensitivity of the recombinant cytokine effects proves that the actions of hrIL-1P and hrIL-6 are not because of endotoxin or other contaminants. The specificity of the cytokine responses is also supported by the experiments using antiIL-6 antibody and the IL-lra. With regard to the mechanisms underlying the combined action of IL-6 and IL-lp in the myenteric plexus, there are several interpretations. The abilities of IL-lb and IL-6 to suppress ‘I-e release of NE from the varicosi-

Figure 6. Effects of IL-lb and IL-6 alone or in combination on evoked 3H release from nerve varicosity preparations. NV suspensions were preincubated with saline (0) hrlL-1S (0.01 ng/mL; mL; l!R), or a combination thereof(M) for 50 minutes. 3H release was stimulated with scorpion venom. Results have been calculated as means ? SEM, and each barrepresents seven separate experiments. Evoked 3H release is expressed as a percentage of basal 3H outflow. *Significant difference from all other groups (P < 0.05).

IL-P AND IL-6 IN MYENTERIC PLEXUS

October 1994

quent

of membrane-associated

production

Although

mediators,

Our results show that the combination pression

of subthreshold

of IL-1 p and IL-6 causes a significant

sup-

of NE

release,

and we interpret

reflect synergism

between

IL-1p and IL-6. The possibility

of an additive unlikely

effect of the two cytokines

when

suppression

our data

the dose-response

of NE

are examined.“’ concentrations

release

appears

relationships

The combination

of IL-lp

of 0.1 ng/mL and 10 ng/mL,

to

to be for the

based on radioligand proaches.

binding

Receptors

quenced,“.” complex

been cloned

and IL-6 at

able literature

respectively,

IL-6 activate e.g., protein

protein,

and sequenced.“,” suggests different

30%

suppression of NE release, and this required a concentration of 100 ng/mL for each cytokine. Therefore, it is extremely unlikely that the combination IL-6 in a substantially lower concentration activate

enough

magnitude

receptors

to produce a suppression

of 50% if the actions

were mediated

we consider

subthreshold concentrations synergism. “’ or antagonistic

in the

of the two cytokines

via the same receptors in an additive

ner. In conclusion,

Additive

of IL-lp and range could

the response

of IL-lb

man-

to the two

and IL-6 to reflect

interactions

between

IL- 1 p

implies

a synergistic

interaction

between

that IL-1p and IL-6 are acting

receptor-transduction

pathways

that

these cytokines through

finally

different

converge. “I

flgure 7. Effect of neutralizing IL-6 antibody on IL-@- and IL-6induced suppression of evoked 3H release from netve varicosity preparations. NV suspensions were preincubated with saline (II), hrll-1P (0.01 ng/mL) and hrll-6 (10 ng/mL) ( ). the cytokines plus antibody (a). or the antibody alone (m) for 50 minutes. 3H release was stimulated with scorpion venom. Results have been calculated as means 2 SEM, and each bar represents at least four separate experiments. Evoked 3H release is expressed as a percentage of basal 3H outflow. *Significant difference from all other groups (P < 0.05).

protein

Furthermore,

the availIL-lb

pathways,

signaling

signal,‘?

of

whereas orhers have shown a 5’-cyclic

adenosine

induced

responses.”

ki-

In contrast,

binding

of IL-6 to its receptor complex activates transduction

pathway

mono-

protein

cellular

the

an intra-

that results in rapid phos-

of tyrosine.”

The existence showing

and

as a mediator

(CAMP) and the CAMP-dependent

phorylation

and have

that, in other systems,

kinase C has been invoked

nase A in IL-lp

of cytokine

receptors

on nerves in the

plexus is supported

indirectly

by recent studies

IL-1 receptors on neurons

system. ‘+“) identified

and IL-6 could occur if the two cytokines were acting through a common receptor-signal transduction system because of cross competition for binding sites. In contrast,

phosphate

myenteric

ap-

intracellular

role for the second messenger

a maximal

is

and se-

gp 130; both subunits

the IL-1 -induced

caused

and molecular

of a specific IL-6 binding

a signal-transducing

myenteric

preparation,

studies

for IL-1 have been cloned

caused a 50% suppression of NE release. In contrast, each of these cytokines, when incubated alone with the plexus

there

and it is known that IL-6 binds to a receptor

consisting

cytokines

by each of these

this directly,

considerable evidence in the literature that identifies two distinct receptors for IL-lb and IL-6. These data are

such as prostaglandins. concentrations

we have not examined

999

In addition,

several

in the central previous

studies

effects of IL-1 on the function

invertebrates,

nervous have

of neurons

as well as in the mammalian

brain,

of and

direct interactions between IL-lp and neurons have been postulated.““~“’ Although the effects on IL-6 on immune cells have been well described, considerably less is known about the action of this cytokine on other cell types, including identify

nerves. However, a neuroprotective

there are several studies role of IL-6

that

in the central

Figure 8. Effect of IL-1 receptor antagonist on IL-lp- and IL-6- induced suppression of evoked 3H release from nerve varicosity preparations. NV suspensions were preincubated with saline (O), hrlL-10 (0.01 ng/mL) and hrll-6 (10 ng/mL) ( ), the cytokines plus IL-lra (8), or IL-lra alone (W) for 50 minutes. 3H release was stimulated with scorpion venom. Results have been calculated as means + SEM, and each bar represents at least four separate experiments. Evoked 3H release is expressed as a percentage of basal 3H outflow. *Significant difference from all other groups (P i 0.05).

1000

GASTROENTEROLOGY Vol. 107, No. 4

Ri.iHL ET AL.

nervous system, “.“‘,‘J~ and IL-6 has been shown to induce neuronal that

differentiation.”

neuroglial

that neurons

6.‘” Direct

interactions

also been implied

IL-lp

tors on sympathetic to suppress

The finding

infected infection,

synergistically

studies

not detectable

are expressed

between

in view of the fact intestine

of rats

the enteric phase of the

an increase in IL-1p protein externa.”

Although

in the muscle layer of noninfected

Thus, both IL-6 and IL-lp changes in myenteric neural

rats, IL”

could contribute to the function that have been

intestine

following

fection. c)V’O Based on our findings a modulatory

IL-6 is

tissue after infection.

IL-lp

and

6. Castro GA, Badial-Aceves F, Smith JW, Dudrick SJ, Weisbrodt NW: Altered small bowel propulsion associated with parasitism. Gastroenterology 1976; 71:620-625. 7. Sukhdeo MVK, Croll NA: Gut propulsion in mice infected with Trichinellaspiralis. J Parasitol 1981;67:906-910. 8. Swain MG, Agro A, Blennerhassett P, Stanisz A, Collins SM. Increased levels of substance P in the myenteric plexus of TrichC nellainfected rats. Gastroenterology 1992;102:1913-1919. 9. Collins SM, Blennerhassett PA, Blennerhassett MG, Vermillion DL. Impaired acetylcholine release from the myenteric plexus of Trichinellrtinfected rats. Am J Physiol 1989; 257:G898-G903. 10. Swain MG, Blennerhassett PA, Collins SM. Impaired sympathetic nerve function in the inflamed rat intestine. Gastroenterology 1991; 100:675-682. 11

Khan I, Collins SM. Expression of pro-inflammatory cytokines in the muscularis externa from the inflamed intestine of rat. Gastroenterology 1994; 107:691-700.

12.

Main C, Blennerhassett B, Collins SM. Human recombinant interleukin-1P suppresses acetylcholine release from rat myenteric plexus. Gastroenterology 1993; 104:1648-1654.

13.

Hurst S, Collins SM. Interleukin-lb modulation of norepinephrine release from rat myenteric nerves. Am J Physiol 1993;264:G30G35.

role for IL-6 in neuro-

transmitter release during intestinal inflammation. Depending on its concentration, IL-6 may either potentiate the effects of the proinflammatory

5. Russell DA, Castro GA. Physiology of the gastrointestinal tract in the parasitized host. In: Johnson LR, ed. Physiology of the gastrointestinal tract. New York: Raven, 1987:1749-1780.

T. spiralis in-

between

peptide

IL-1 or coun-

teract the effects of IL- 1.

Farthing MJG, Lennard-Jones JE. Sensitivity of the rectum to distension and the anorectal distension reflex in ulcerative colitis. Gut 1978; 19:64-69.

4. Rao SSC, Read NW, Brown C, Bruce C, Holdsworth CD. Studies on the mechanism of bowel disturbance in ulcerative colitis. Gastroenterology 1987; 93:934-940.

of a dual effect of IL-

6 on NE release and an interaction IL-6, we propose

IL-6

in the muscle and my-

levels rise to 9 11 pg/mg

shown in the inflamed

myentetic

NE release.

layer from the inflamed

we have estimated

that

with recep-

in the

relevance

with T. spiralis. During

have

in the cen-

we postulate

interactions

by 140% in the muscularis 6 protein

IL-6 and neurons

endings

of synergistic

gains functional

plexus

and it has been

and IL-6 interact

nerve

that both cytokines enteric

between

system.“‘.“” In summary,

tral nervous

and IL-lb

IL-6,

may be direct target cells for IL-

in other functional

in our experiments, plexus

In the brain, there is evidence

cells synthesize

speculated

3

14. Hurst SM, Stepien H, Stanisz A, Blennerhassett P, Sharkey K, Bunnett N, Collins SM. The relationship between the pro-inflammatory peptides interleukin-1P and substance P in the inflamed rat intestine (abstr). Gastroenterology 1992;102:A640. 15.

Collins SM, Blennerhassett P. Hurst S, Khan I, Thompson RC. The role of endogenous interleukin-lfi in enteric nerve and muscle changes in the inflamed nematode infected rat intestine (abstr). Gastroenterology 1992; 102:A608.

16.

Mizutani H, May LT, Sehgal PB, Kupper TS. Synergistic interactions of IL-1 and IL-6 in T cell activation. J lmmunol 1989; 143:896-901.

17.

neural membranes. Our results raise the possibility that the dual effect of IL-6 and the interactions between ILlp and IL-6 are important factors in the modulation of

4lmawi WY, Lipman ML, Stevens AC, Zanker B, Hadro ET, Strom TB. Abrogation of glucosteroid-mediated inhibition of T cell proliforation by the synergistic action of IL-l, 11-6, and IFN-y. J lmmunol 1991; 146:3523-3527.

18.

Hurst S, Collins SM. Interleukin-la inhibits the release of ‘Hioradrenaline from rat myenteric plexus varicosities (abstr). FA3EB J 1991; 5:A1273.

neural function

19.

Iinarello CA. Interleukin-1 L991; 77:1627-1652.

20.

Uorthemann W, Braciak TA, Hattori M, Lee F, Fey GH. Structure If the rat interleukin-6 gene and its expression in macrophagederived cells. J Biol Chem 1989;264:16072-16082.

21.

tndus T, Geiger T, Hirano T, Kishimoto T, Tran-Thi TA, Decker K, ieinrich PC. Regulation of synthesis and secretion of major rat acute-phase proteins by recombinant human interleukin-6 (BSFZ/11-6) in hepatocyte primary cultures. Eur J Biochem L988; 173:287-293.

22.

iama T, Miyamoto M, Tsukui H, Nishio C, Hatanaka H. Interleutin-6 as a neurotrophic factor for promoting the survival of culured basal forebrain cholinergic neurons from postnatal rats. Jeurosci Lett 1989;104:340-344.

23.

jatoh T. Nakamura S, Taga T, Matsuda T. Hirano T, Kishimoto _, Kaziro Y. Induction of neuronal differentiation in PC12 cells

In summary, we have shown that exogenously applied IL-6 affects NE release from the rat jejunal myenteric plexus in a dose-dependent dual manner: lower concentrations of IL-6 increase NE release, whereas higher concentrations suppress this release. Furthermore, we have shown synergistic interactions between IL-1p and IL-6 which involve direct interactions of the cytokines with

during

intestinal

inflammation.

Ongoing

studies in our laboratory are investigating the putative sources of endogenous cytokines in the myenteric plexus, and preliminary results indicate that enteroglial cells cultured from rat myenteric plexus express messenger RNA for IL-6 and IL- 1 p.49

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IL-8 AND IL-6 IN MYENTERIC PLEXUS

October 1994

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Accepted June 27, 1994. Received November 2,1993. Address requests for reprints to: Stephen M. Collins, M.D., Intestinal Diseases Research Program, McMaster University, HSC-BNSC, 1200 Main Street West, Hamilton, Ontario L8N 325, Canada. Fax: (905) 522-3454. Supported by the Medical Research Council of Canada and by grant (to A.R.) from the Deutsche Forschungsgemeinschaft (Ru 528/ l-l).