(Received for publication, October 3, 1994; and in revised form, December 9, 1994)
From the
Interleukin-13 (IL-13), a novel cytokine produced by activated
lymphocytes modulates some monocyte functions, but no data is available
concerning the signal transduction pathway. We show here, the
inhibitory effect of IL-13 on
12-O-tetradecanoylphorbol-13-acetate (TPA)-triggered reactive
oxygen intermediate production in human monocytes and the signals
involved in this response. Our results show that IL-13 produces rapid
and transient phosphoinositide hydrolysis and intracellular
Ca mobilization. Furthermore, IL-13 induces
intracellular cAMP accumulation through inositol
1,4,5-trisphosphate-dependent Ca
mobilization.
Metabolic inhibitors were used to relate the first steps in signaling
pathways to the inhibitory effect of IL-13 on TPA-triggered reactive
oxygen intermediate production. Indeed, inhibitors of phospholipase C
(neomycin), intracellular Ca
mobilization
(8-[N,N-diethylamino]-octyl
3,4,5-trimethoxybenzoate hydrochloride), adenylate cyclase
(
-tetrahydrocannabinol), and protein kinase A (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide)
impair the IL-13 inhibitory response. Altogether these observations
indicate that modulatory effect of IL-13 on the TPA-induced oxidative
burst is the result of the intracellular cAMP accumulation through an
inositol 1,4,5-trisphosphate-induced Ca
mobilization-dependent pathway.
Interleukin 13 (IL-13) ()is a novel lymphokine of 112
amino acids with a molecular mass of 12.5 kDa produced by activated
Th-2 cells(1) . This cytokine induces cell surface phenotype
changes and displays immunomodulatory effects on B cells and human
monocytes ( (2) and for reviewed, see (3) ). In B
cells, IL-13 induces proliferation and differentiation(4) ,
promotes CD23 expression and the production of certain immunoglobulin
isotypes such as IgG4 and IgE(5) . IL-13 displays many effects
on monocytes. It induces morphological changes (2) and enhances
the expression of several members of the integrin superfamily and class
II major histocompatibility complex antigens, whereas it down-regulates
the expression of CD14 and Fc
R receptors (CD64, CD32, and
CD16)(6) . IL-13 inhibits immunodeficiency virus type 1
production, but the precise mechanism remains to be
elucidated(7, 8) . It reduces pyrogen-induced
expression of procoagulant activity(9) . In
lipopolysaccharide-stimulated monocytes(1, 6) , IL-13
inhibits the production of chemokines (IL-8, macrophage inflammatory
proteins 1
), hematopoietic growth factors (granulocyte/macrophage
colony-stimulating factor, granulocyte colony-stimulating factor), and
proinflammatory cytokines (e.g. tumor necrosis factor
,
IL-1, IL-6). Thus, IL-13 is considered as an anti-inflammatory
cytokine.
However, the effect of IL-13 on the reactive oxygen
intermediates (ROI) production, an important function of monocytes that
are involved in infectious and inflammatory processes, has not yet been
investigated. The NADPH-oxidase system is the key step in the
regulation of ROI generation. The oxidase is normally dormant in
resting phagocytes, but it can be rapidly activated by a number of
stimuli such as protein kinase C agonists like phorbol esters,
12-O-tetradecanoylphorbol-13-acetate (TPA). It is known that
the oxidase requires interaction at the plasma membrane level between
membrane and cytosolic components, these include flavocytochrome b, the cytosolic proteins p47 and p67, and the
small GTP-binding proteins Rap1A, Rac-1, and Rac-2 (for review, see (10, 11, 12) ). Classically, protein kinase C
phosphorylates specific proteins (p47, p67) that constitute the
predominant regulatory mechanism governing the activation of the
oxidase(13) .
While several studies have been oriented
toward phosphorylation and activation of respiratory burst, the
reversal of the event by dephosphorylation and inactivation of the
active phagocyte to the normal resting state have not been fully
elucidated. Agents that increase the intracellular concentration of
cAMP have been shown to be negative modulators of TPA-triggered
respiratory burst in polymorphonuclear leukocytes (14) and in
Ehrlich ascites tumor cells (15) . The regulation of
intracellular cAMP level involves at least two independent systems, a G
protein pathway coupled with adenylate cyclase and the direct
activation of catalytic subunits of adenylate cyclase. In particular,
the Ca-calmodulin complex can activate specific I and
III adenylate cyclase isozymes(16) . A coupling of
phospholipase C and adenylate cyclase effector systems through
Ca
has also been reported in
leukocytes(17, 18) .
In this study, we investigate
the capacity of IL-13 to modulate respiratory burst in human monocytes
and the signaling pathways used by the cytokine to modify this
function. Our findings indicate that IL-13 inhibits TPA-triggered ROI
production by a mechanism involving a rapid and transient phospholipase
C-dependent Ca mobilization and the consequent
protein kinase A activation.
Figure 1: Effect of IL-13 pretreatment on TPA-triggered oxidative burst in human monocytes. The generation of CL was monitored after TPA (100 nM) (a), IL-13 (100 ng/ml) (d), Hanks' balanced solution (control) (e), or IL-13/anti-IL-13 antiserum complex (f) addition at time 0, as described under ``Experimental Procedures.'' In parallel experiments, monocytes were incubated for 5 min before TPA with IL-13 (100 ng/ml) (c) or IL-13/anti-IL-13 antiserum complex (b). The curves are representative of three separate experiments, each performed in triplicate.
Figure 2:
Effect of IL-13 pretreatment on
TPA-triggered chemiluminescence production in human monocytes in the
absence (0Ca) or in the presence
of extracellular calcium and metabolic inhibitors. Total CL emission
(area under curve) was observed for 10 min after TPA (100 nM)
(&cjs2108;) or IL-13 (100 ng/ml)+TPA (&cjs2113;) stimulation with
or without (CONTROL) inhibitors as described under
``Experimental Procedures.'' When inhibitors were used,
monocytes were preincubated (10 min) with 1 µM
-THC, 10 µMH89,
10 µM neomycin sulfate (NEO), 10 µMTMB8, 0.5 µM indomethacin (IND).
Data are means ± S.E. of three separate experiments, each
performed in triplicate.**, p <
0.01.
To investigate the possible
involvement of the cAMP-dependent protein kinase pathway in the
inhibitory effect of IL-13 on TPA-triggered ROI production, cells were
preincubated with -THC, an inhibitor of adenylate
cyclase, or H89, an inhibitor of protein kinase A. The results obtained
show that these inhibitors completely block the effect of IL-13 (Fig. 2). Moreover, forskolin (10 µM), a direct
activator of adenylate cyclase, or dibutyryl cAMP (5 mM), a
cell permeable analogue of cAMP, caused 50% inhibition of ROI
production induced by TPA. These effects were reversed in the presence
of H89 (10 µM).
We also investigated Ca mobilization as another candidate pathway in IL-13 modulation of
the oxidative burst. In the absence of extracellular
Ca
, the effect of IL-13 on TPA-induced ROI production
was maintained. In the presence of neomycin sulfate (inhibitor of
phospholipase C) or TMB8 (inhibitor of InsP
-induced
intracellular Ca
release from intracysternal stores),
the IL-13 inhibitory effect was impaired, suggesting that phospholipase
C and subsequent production of InsP
-induced intracellular
Ca
release can be related to the inhibitory response (Fig. 2). The involvement of prostaglandin in this mechanism is
not suggested. Indeed, in the presence of indomethacin (inhibitor of
the cyclooxygenase pathway), the effect of IL-13 on TPA-triggered ROI
production was maintained.
Figure 3:
Time
course of cAMP accumulation induced by IL-13 in human monocytes.
Monocytes were incubated with () or without (
) IL-13 (100
ng/ml). In parallel experiments, cells were preincubated for 10 min
with (
) neomycin sulfate (0.5 mM) or (
) TMB8 (10
µM) before IL-13 addition. cAMP was quantified as
described under ``Experimental Procedures.'' Data are means
± S.E. of three separate experiments, each performed in
triplicate. *, p < 0.05;**, p <
0.01.
Figure 4:
Time
course of IL-13-stimulated inositol phosphate formation in human
monocytes. Monocytes were labeled with myo-[2-H]inositol, washed, and incubated
with (
) or without (
) IL-13 (100 ng/ml).
[
H]inositol phosphates were measured as described
under ``Experimental Procedures.'' Data are means ±
S.E. of three separate experiments, each performed in triplicate. *, p < 0.05;**, p <
0.01.
Figure 5:
Kinetics of
[Ca] response in single cells after
stimulation with IL-13. Monocytes were loaded with Fluo 3-AM, and, at
the time indicated by arrows, IL-13 (100 ng/ml) was added to cells in
Hanks' balanced solution with (A) or without (B) Ca
. In parallel experiments, cells were
preincubated for 10 min in the presence of 10 µM TMB8 (C) before IL-13 addition. Determination of
[Ca
] was measured as described under
``Experimental Procedures.'' Records of cell responses are
representative of three separate experiments each performed in
triplicate.
In the present study we determine the initial events induced by IL-13, which can regulate the protein kinase C-triggered respiratory burst in human monocytes. Classically, the activation of protein kinase C by TPA results in the activation of NADPH-oxidase, inducing the so-called respiratory burst(26, 27, 28) . Probably, phosphorylation of cytosolic oxidase proteins, p47 and p67 by activated protein kinase C is a key step in this response(11, 13) .
ROI and proinflammatory cytokines are likely to be involved in phagocyte antimicrobial activity and inflammatory responses. IL-13 produced by activated Th-2 cells is described to suppress the production of proinflammatory monokines(1, 6) . However, no information is available on the action of IL-13 on human ROI production.
Our results
demonstrate that IL-13 inhibits the TPA-induced oxidative burst in
human monocytes when added 5 min before TPA and also for longer periods
up to 18 h (data not shown). The nonspecific inhibition of ROI
production is excluded since in the presence of anti-IL-13 antiserum
the effect of IL-13 is cancelled. The IL-13 down-modulatory effect on
human monocyte oxidative burst supports that described with another
anti-inflammatory cytokine, IL-4. Indeed, preexposure of human
monocytes to IL-4 for 24 h decreases the respiratory burst induced by
TPA(29) . IL-4 also inhibits this response when other stimuli
such as zymosan, platelet-activating factor, chemotactic peptide and
IFN- are used(29, 30) . An explanation for the
similar biological activities displayed by IL-13 and IL-4 is that their
receptors share a common subunit critical for cellular signal
transduction(31) .
In contrast to the finding with
human monocytes, the respiratory burst induced by TPA in murine
resident peritoneal macrophages is enhanced by murine IL-4 used under
similar experimental conditions (32) . Recent data show that
P600 (murine IL-13) and murine IL-4 are unable to modify ROI response
triggered by TPA in murine biogel-elicited macrophages(33) .
These results suggest the necessity to be cautious when extrapolating
data obtained with murine cells to the human system. As suggested for
IL-4, the role of IL-13 in host defense and inflammation can be
significant. ROI may damage various cells at high concentrations;
however, their role is important in antimicrobial activity. The partial
inhibition by IL-13 of TPA-triggered ROI production (35%) could reduce
inflammatory reactions without completely abolishing the antimicrobial
process.
Having established that IL-13 induces an inhibitory effect
on ROI production, we investigated the signal transduction pathways
used by this cytokine to induce the deactivation of the ROI response.
In leukocytes, agents which increase intracellular cAMP concentrations
(prostaglandins E and E
, dibutyryl cAMP,
forskolin, and 1-isobutyl-3-methylxanthine) have often been shown to
inhibit various cellular functions such as respiratory
burst(34, 35, 36) . Some authors have
recently proposed a mechanism of protein kinase A-dependent
phosphorylation and subsequent activation of a specific phosphatase.
This event results in dephosphorylation of NADPH-oxidase subunits and
thus inactivation of the enzyme(14, 15) . Our data
suggest the involvement of the cAMP-dependent protein kinase pathway in
IL-13-induced inhibition of respiratory burst. Indeed, the direct
involvement of cAMP in the IL-13 signaling pathway is demonstrated by a
sustained increase of cAMP levels after cytokine stimulation.
Furthermore, in the presence of adenylate cyclase
(
-THC) and protein kinase A (H89) inhibitors, the
effect of IL-13 on ROI production is not observed. IL-13 seems to mimic
the forskolin or dibutyryl cAMP inhibitory effect on TPA-triggered ROI
production.
Different studies show that the accumulation of cAMP can
be subsequent to intracellular Ca mobilization(17, 37, 38) . Coupling of
phospholipase C and cAMP pathways through Ca
was
reported in leukocytes (18, 39) and human
neuroblastoma cells(40) . A direct activation of type I and III
adenylate cyclase by Ca
and calmodulin previously
described (41, 42) could be a possible mechanism. Our
experiments show that cAMP production is markedly reduced by prior
treatment of human monocytes with an inhibitor of phospholipase C
(neomycin sulfate) or an inhibitor of InsP
-induced
Ca
release (TMB8). These observations suggest that
IL-13 causes intracellular cAMP accumulation by
Ca
-dependent processes.
It was of interest to
verify the capacity of IL-13 to promote phospholipase C activation and
intracellular Ca rise. We demonstrate here that IL-13
induces immediate and transient hydrolysis of inositol phospholipids
and a rapid increase in intracellular Ca
levels
inhibited by TMB8. These results suggest that IL-13 causes
phospholipase C activation and a release of Ca
from
InsP
-sensitive Ca
stores. The regulation
of phospholipase C is distinct for two of the known phospholipase
families, the phospholipase C-
and phospholipase C-
. The
phospholipase C-
family is regulated by G proteins, while
phospholipase C-
1 and -
2 are regulated by protein-tyrosine
kinase (for review, see (43) ). The involvement of a
protein-tyrosine kinase in the IL-13 signal transduction pathway was
strongly suggested in a recent report. Indeed, genistein (one inhibitor
of tyrosine kinase) completely blocked the IL-13-activated
IL-4-dependent nuclear transcription factor in monocytic U937
cells(44) . A study concerning the suggested activation of
phospholipase C-
through tyrosine phosphorylation induced by IL-13
has already been undertaken in our laboratory.
Otherwise, we relate
these initial events induced by IL-13 to the inhibition of the human
monocyte biological response, the oxidative burst. Indeed, in the
presence of TMB8 and neomycin sulfate, this inhibitory effect of IL-13
is impaired, whereas, in the absence of extracellular Ca or in the presence of indomethacin (inhibitor of cyclooxygenase
pathway), IL-13 still induces a significant inhibition of ROI
production. This last observation strongly suggests that
prostaglandins, classically described to increase intracellular cAMP
levels and inhibit ROI production in activated
phagocytes(35, 37) , are not involved in IL-13
down-regulation of the biological response studied. Our results support
the involvement of InsP
-dependent Ca
mobilization and subsequent cAMP accumulation in the IL-13
inhibitory effect on ROI production.
In conclusion, we describe here
for the first time the initial steps in the signaling pathway of IL-13
related to respiratory burst decrease. Indeed, direct measurement of
second messengers and the use of metabolic inhibitors allowed us to
establish a temporal sequence and a link between
InsP-dependent Ca
mobilization, the cAMP
protein-kinase pathway and inhibition of the protein kinase C-triggered
respiratory burst in human monocytes pretreated with IL-13. The
modulation of this important human monocyte function strengthens the
notion that IL-13 could play a central role in the inflammatory process
without totally inhibiting the monocyte oxygen-dependent
anti-infectious capacity. Furthermore, work will be required to
evaluate if the anti-inflammatory properties of IL-13 displayed in in vitro models, could be of clinical significance in the
future for the treatment of inflammatory human diseases.