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J Virol, March 1998, p. 2496-2499, Vol. 72, No. 3
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Autocrine Regulation and Experimental Modulation of
Interleukin-6 Expression by Human Pulmonary Epithelial Cells Infected
with Respiratory Syncytial Virus
Zili
Jiang,
Masaru
Kunimoto, and
Janak A.
Patel*
Division of Pediatric Infectious Diseases,
Department of Pediatrics, Children's Hospital at University of
Texas Medical Branch, Galveston, Texas 77555
Received 26 June 1997/Accepted 24 November 1997
 |
ABSTRACT |
The mechanisms of regulation of interleukin-6 (IL-6) production in
respiratory syncytial virus (RSV)-infected respiratory epithelial cells
were evaluated in A549 cell cultures. Incubation with purified RSV
resulted in significant production of IL-1
, IL-1
, IL-6, and tumor
necrosis factor alpha (TNF-). Addition of saturating concentrations
of neutralizing antibodies against IL-1, IL-1, or TNF- into
purified RSV-infected cell cultures resulted in a significant
inhibition of IL-6 production, although anti-IL-1 antibody had the
most predominant effect (80% inhibition). Anti-IL-1 antibody also
almost completely blocked the expression of mRNA for IL-6. Addition of
therapeutic concentrations of dexamethasone (1 µM) or ribavirin (90 µg/ml), an antiviral agent, also significantly inhibited the
synthesis of IL-6. Hence, in clinical settings, pharmacological agents
such as the specific antagonists of IL-6-inducing cytokines, as well as
dexamethasone and ribavirin, could be used to modulate IL-6 production.
| |
TEXT |
Respiratory syncytial virus (RSV) is
the most common causative agent of bronchiolitis and pneumonia in
infants and young children (7). Mounting evidence suggests
that the respiratory epithelium has the potential to initiate and
modulate immune responses (11). The immunologic role of the
respiratory epithelial cells in infections due to respiratory viruses
such as RSV may be even more important, because it is also the primary
target cell for RSV infection.
Interleukin-6 (IL-6) is detected in the respiratory secretions during
RSV infection in vivo (6, 14, 17, 18), as well as in
RSV-infected respiratory epithelial cell cultures in vitro (1, 4,
16). During RSV infection, IL-6 may play an immunoregulatory role: IL-6 has been shown to contribute to recovery from infection by
promoting humoral and cellular defense mechanisms, such as facilitation
of the terminal differentiation of B lymphocytes into immunoglobulin
(Ig)-secreting cells and activation and subsequent proliferation of
primary antigen-receptor-dependent T lymphocytes (13, 24).
On the other hand, IL-6 may also play a proinflammatory role: IL-6 may
contribute to the symptoms and signs of acute clinical illnesses
characterized by fever, leukocytosis, increased vascular permeability,
and increased synthesis of hepatic acute-phase proteins (22,
24).
Since we have previously shown that RSV-infected epithelial cells
produce IL-1, IL-1, and TNF- (19), which by
themselves are known to induce the synthesis of IL-6, we hypothesized
that IL-6 production could be regulated by these IL-6-inducing
cytokines in an autocrine manner in the epithelial cells. Furthermore,
because corticosteroids, as anti-inflammatory agents, and ribavirin, as an antiviral agent, have been used clinically in the management of RSV
infection, we hypothesized that dexamethasone (a corticosteroid) and
ribavirin would inhibit the synthesis of IL-6 induced by RSV in the
epithelial cells. These hypotheses were examined with A549, a type II
pulmonary epithelial carcinoma cell line.
Long strain (A1) RSV was grown in Hep-2 (American Type Culture
Collection, Rockville, Md.) cell cultures and purified by polyethylene glycol precipitation and a sucrose gradient as previously described (19). A549 (American Type Culture Collection) cell cultures were grown as 100% confluent monolayers in Eagle's minimal essential medium supplemented with 5% (vol/vol) heat-inactivated fetal calf serum, 100 U of penicillin per ml, 100 mg of streptomycin per ml, and 2 mM glutamine (Sigma, St. Louis, Mo.), and incubated at 37°C in 5%
CO2 for 48 h. Quantitative detection of IL-1,
IL-1, IL-6, and tumor necrosis factor alpha (TNF-) in the cell
culture supernatants was performed with commercial cytokine-specific
enzyme-linked immunosorbent assay (ELISA) kits (Quantitine kits; R & D
Systems, Minneapolis, Minn.). Recombinant human IL-1, IL-1, and
TNF- and their respective neutralizing polyclonal antibodies were
purchased from R & D Systems.
Expression of mRNA for IL-6 in A549 cells was analyzed by reverse
transcription-PCR as previously described (19). Sense and
antisense primers for human IL-6 were purchased from Clontech (Palo
Alto, Calif.). To ensure the conditions of the PCR assay, an IL-6 cDNA
fragment was used as a positive control. As an internal control, human
G3PDH primers and the respective cDNA fragment (Clontech) were used
under the same conditions. The expected sizes of the amplified DNA
products for IL-6 and G3PDH were 628 and 838 bp, respectively.
Induction of IL-6 synthesis by RSV.
After incubation of A549
cells with live purified RSV at a multiplicity of infection (MOI) of 1, IL-6 was detected in the cell culture supernatants in significant
amounts at 24 h (mean, 366.5 pg/ml), with a further rise at
48 h (1,748.6 pg/ml), while the control cells produced negligible
quantities of IL-6 (<10 pg/ml) at all time points. After 48 h,
due to further cell detachment and destruction caused by RSV infection,
the assay could not be continued. The rate of production of IL-6
induced by purified RSV was exponential, while that induced by
exogenous recombinant TNF- was linear (Fig.
1). RSV infection and replication were indeed essential for the initial production of IL-6 or IL-6-inducing mediator(s), since extracellular inactivation of infectivity of purified RSV by UV irradiation did not result in any enhancement of
IL-6 production (Fig. 1).
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FIG. 1.
Kinetics of IL-6 accumulation, as determined by ELISA,
in supernatants of A549 cell cultures stimulated by sham media,
purified RSV (pRSV; MOI = 1), UV-irradiated pRSV (UV-pRSV), or
1,200 pg of TNF- per ml. The values expressed are means ± standard deviations of triplicate observations. *, P < 0.05 (stimuli versus sham [Mann-Whitney rank sum test]).
|
|
Blocking of RSV-mediated production of IL-6 by neutralization of
IL-1, IL-1, and TNF-.
The potential role for IL-1,
IL-1, and TNF- as the soluble mediators that induce IL-6
production was evaluated, since we have previously detected enhanced
expression of the genomic transcripts and secreted proteins of these
cytokines in RSV-infected A549 cell cultures (19). At
48 h after incubation with purified RSV, the mean concentrations
of IL-1, IL-1, and TNF- were approximately 120, 20, and 75 pg/ml, respectively. Compared with production of IL-6 stimulated by
purified RSV alone (expressed as 100%), addition of saturating
concentrations of neutralizing antibodies against IL-1 (150 µg/ml), IL-1 (150 µg/ml), TNF- (75 µg/ml), or all of them
together significantly blocked IL-6 production by approximately 80, 20, 66, and 80%, respectively (Fig. 2). As controls, these antibodies also blocked the production of IL-6 induced
by the respective exogenous cytokines by more than 95% (data not
shown). Control goat IgG (Sigma) or mouse IgG (R & D Systems) did not
block IL-6 induced by purified RSV. Since these experiments suggested
that IL-1 was the primary inducer of IL-6 protein, we next evaluated
whether IL-1 regulated the gene transcription of IL-6. Indeed,
addition of anti-IL-1 antibody also significantly inhibited the
expression of mRNA (Fig. 3).
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FIG. 2.
Demonstration of effect of cytokine-neutralizing
antibodies (Ab) on IL-6 accumulation, as determined by ELISA, in
supernatants of A549 cell cultures stimulated for 24 h with sham
media, purified RSV (pRSV; MOI = 1), or pRSV coincubated with
control antibodies (goat IgG) or neutralizing antibodies against
IL-1, IL-1, and TNF-. The values expressed are means ± standard deviations of triplicate observations. *, P < 0.05 (stimuli versus sham [Mann-Whitney rank sum test]). The
relative amounts of IL-6 induced by the various stimuli are expressed
as a percentage on top of each bar. (pRSV was assigned 100%.)
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FIG. 3.
Demonstration of effect of neutralizing antibody (Ab)
against IL-1 on mRNA expression of IL-6 and G3PDH, as determined by
reverse transcription-PCR in 105 A549 cells stimulated with
purified RSV (pRSV; MOI = 1) for various time intervals. The graph
shows laser densitometer analysis of IL-6 DNA bands (normalized to
G3PDH bands). The results are from representative experiments performed
in duplicate.
|
|
Effects of dexamethasone and ribavirin on IL-6 production.
Simultaneous incubation of 1 µM dexamethasone and purified RSV with
A549 resulted in a significant inhibition of IL-6 production (60%
inhibition). Dexamethasone also suppressed IL-6 induced by exogenous
recombinant IL-1 (73% inhibition [Fig.
4]). This inhibition of IL-6 production
occurred despite the lack of cytotoxicity of dexamethasone, as
determined by the trypan blue dye exclusion test, and the lack of
effect on RSV replication (data not shown). The production was further
reduced by addition of increasing concentrations of dexamethasone (10 to 1,000 µM), but with increasingly higher cytotoxicity (data not
shown).
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FIG. 4.
Demonstration of effect of 1 µM dexamethasone (Dex)
and 90 pg of ribavirin per ml on IL-6 accumulation, as determined by
ELISA, in A549 cells stimulated for 24 with sham media, 250 pg of
recombinant IL-1 per ml, or purified RSV (pRSV). The values
expressed are means ± standard deviations of six observations.
The relative amounts of IL-6 induced by the various stimuli are
expressed as a percentage on top of each bar. (pRSV was assigned
100%.) *, P < 0.05 (Mann-Whitney rank sum test).
|
|
Simultaneous incubation of ribavirin (90 µg/ml) and purified RSV also
resulted in inhibition of IL-6 production (98% inhibition
[Fig.
4]).
However, unlike dexamethasone, ribavirin did not inhibit
the production
of IL-6 induced by exogenous recombinant IL-1
(Fig.
4). Ribavirin
also completely blocked the replication of
RSV, as determined by lack
of cytopathicity due to RSV for 7 days
after infection. The inhibitory
effect of ribavirin on virus replication
and IL-6 production occurred
at concentrations as low as 11 µg/ml.
Overall, our results offer direct evidence that RSV replication is
essential for initial induction of IL-6 synthesis, albeit
largely
through the autocrine activity of IL-6-inducing soluble
mediators
released by RSV-infected cells. With neutralizing antibodies
against
IL-1
, IL-1
, or TNF-
, it was shown that the cytokines
IL-1
and TNF-
are the predominant IL-6-inducing soluble mediators.
Furthermore, it was shown that ribavirin and dexamethasone blocked
the
production of IL-6 by RSV-infected epithelial cells, suggesting
the
need for clinical evaluation of the effects of these pharmacological
agents on the regulation of IL-6-related host defense mechanisms
during
RSV infection.
In the steady state, IL-6 is usually not produced by normal cells, but
its expression is readily induced by viral infections,
and a variety of
cytokines, such as IL-1, TNF-
, platelet-derived
growth factor, IL-3,
and granulocyte-macrophage colony-stimulating
factor induce IL-6
production (
2,
15,
16,
22,
24).
However, not all types of
cells respond similarly to all of these
factors. IL-1, for example, is
probably the most potent inducer
of IL-6 in fibroblasts but induces
little IL-6 in bone marrow
cells, which, in contrast, respond very well
to IL-3 or granulocyte-macrophage
colony-stimulating factor
(
24). Cromwell et al. observed that
the bronchial epithelial
cells secrete IL-6, which can be enhanced
by cytokines such as IL-1
and TNF-
(
4).
The mechanism of regulation of IL-6 during RSV infection has not been
previously explored. In this respect, the present study
shows that
IL-1
, IL-1
, and TNF-
are all involved in the regulation
of
IL-6 production by RSV-infected A549 cells in an autocrine
manner.
Furthermore, it was shown that IL-1
, which was the most
predominant
inducer of IL-6, regulated IL-6 synthesis at the transcription
level;
however, posttranscriptional and translational mechanisms
cannot be
excluded. Because the combined sum of the individual
IL-6-inducing
activities of IL-1
, IL-1
, and TNF-
exceeds 100%,
it is likely
that these cytokines enhance the activities of each
other. For example,
IL-1 is known to increase the expression of
TNF-
receptor
(
23). Nonetheless, the IL-6-enhancing abilities
of IL-1
,
IL-1
, and TNF-
correlate with the relative amounts
of these
cytokines in the supernatants of RSV-infected epithelial
cells.
Clinical modulation of disease severity due to RSV infection has been
attempted with two pharmacological agents
ribavirin
and dexamethasone.
The present study shows that at concentrations
comparable to
therapeutically achievable levels in the airway
secretions when
delivered topically by aerosols (
3,
5,
8),
both of these
pharmacologic agents can inhibit IL-6 production
by RSV-infected
epithelial cells. Ribavirin, a synthetic nucleoside
similar to
guanosine and inosine, possesses broad antiviral properties
(
3,
8). Our studies suggest that ribavirin inhibits IL-6
production,
which is probably related to its property as an antiviral
agent, and
not related to direct regulation of cytokine synthesis
pathways,
because ribavirin did not inhibit IL-6 production by
a nonviral agonist
such as IL-1
. On the other hand, the suppressive
effect of
dexamethasone was unrelated to viral replication. Levine
and colleagues
have shown that in vitro dexamethasone preconditioning
significantly
inhibits both the secretion of immunoreactive IL-6
and the accumulation
of IL-6 mRNA induced by TNF-
in BEAS-2B,
a human bronchial
epithelial cell line (
12). The mechanisms
of
dexamethasone-mediated repression of IL-6 gene expression and
secretion
in virus-infected epithelial cells have not been explored;
however,
evidence suggests that both transcription and posttranscription
regulatory mechanisms may be involved in uninfected epithelial
cells
(
10,
20).
While corticosteroids have been shown to provide a beneficial effect in
the management of acute lower airway inflammation
associated with acute
viral laryngotracheobronchitis and asthma
(
9,
25), evidence
for clinical benefit in acute RSV bronchiolitis
has been contradictory
(reviewed in reference
21). The benefit
of ribavirin
in the management of acute RSV bronchiolitis has
been shown in a number
of studies; however, its use remains controversial
(reviewed in
reference
21). There are additional concerns for
the
early use of these pharmacological agents during acute infection
that
relate to the possible impairment of the host defenses involved
in
recovery from acute illness and subsequent long-lasting immunity
against RSV infection.
In conclusion, the present study provides direct evidence for an
autocrine mechanism of enhanced IL-6 expression in RSV-infected
respiratory epithelial cells which is primarily mediated by IL-1
and
TNF-
, and, to a lesser extent, by IL-1
. Use of pharmacological
agents such as specific antagonists of IL-6 or IL-6-inducing cytokines
dexamethasone and ribavirin may inhibit the acute-phase response
as
well as long-lasting cellular and humoral immunity induced
by IL-6
during RSV infection; however, this requires further clinical
studies.
| |
ACKNOWLEDGMENTS |
This work was supported by NIDCD grant R29-DC-02129.
We thank Natsuki Nakajima, Edgar Molina, and Todd Elliott for technical
help.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Pediatric Infectious Diseases, Department of Pediatrics, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0371. Phone: (409) 772-2798. Fax: (409) 747-1753. E-mail:
janak.patel{at}utmb.edu.
| |
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J Virol, March 1998, p. 2496-2499, Vol. 72, No. 3
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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