Interaction between Porcine Reproductive-Respiratory Syndrome Virus and Bacterial Endotoxin in the Lungs of Pigs: Potentiation of Cytokine Production and Respiratory Disease

Abstract

Porcine reproductive-respiratory syndrome virus (PRRSV) is a key agent in multifactorial respiratory disease of swine. Intratracheal administration of bacterial lipopolysaccharides (LPSs) to PRRSV-infected pigs results in markedly enhanced respiratory disease, whereas the inoculation of each component alone results in largely subclinical disease. This study examines whether PRRSV-LPS-induced respiratory disease is associated with the excessive production of proinflammatory cytokines in the lungs. Gnotobiotic pigs were inoculated intratracheally with PRRSV and then with LPS at 3, 5, 7, 10, or 14 days of infection and euthanatized 6 h after LPS inoculation. Controls were inoculated with PRRSV or LPS only or with phosphate-buffered saline. Virus titers, (histo)pathological changes in the lungs, numbers of inflammatory cells, and bioactive tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6 levels in bronchoalveolar lavage fluids were examined. All pigs inoculated with PRRSV-LPS developed severe respiratory disease, whereas the controls that were inoculated with PRRSV or LPS alone did not. PRRSV infection significantly enhanced cytokine production in response to LPS. Peak TNF-alpha, IL-1, and IL-6 titers were 10 to 100 times higher in the PRRSV-LPS-inoculated pigs than in the pigs inoculated with PRRSV or LPS alone; and the titers correlated with the respiratory signs. The levels of neutrophil infiltration and the pathological changes detected in the lungs of PRRSV-LPS-inoculated pigs resembled those detected when the effects of PRRSV and LPS inoculated alone are combined, but with no synergistic effects between PRRSV and LPS. These data demonstrate a synergism between PRRSV and LPS in the induction of proinflammatory cytokines and an association between induction of these cytokines and disease.

Full text

JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2003, p. 960–966 Vol. 41, No. 3
0095-1137/03/$08.00⫹0 DOI: 10.1128/JCM.41.3.960–966.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Interaction between Porcine Reproductive-Respiratory Syndrome
Virus and Bacterial Endotoxin in the Lungs of Pigs: Potentiation
of Cytokine Production and Respiratory Disease
Steven Van Gucht, Kristien Van Reeth,* and Maurice Pensaert
Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium
Received 1 August 2002/Returned for modification 22 October 2002/Accepted 15 December 2002
Porcine reproductive-respiratory syndrome virus (PRRSV) is a key agent in multifactorial respiratory
disease of swine. Intratracheal administration of bacterial lipopolysaccharides (LPSs) to PRRSV-infected pigs
results in markedly enhanced respiratory disease, whereas the inoculation of each component alone results in
largely subclinical disease. This study examines whether PRRSV-LPS-induced respiratory disease is associated
with the excessive production of proinflammatory cytokines in the lungs. Gnotobiotic pigs were inoculated
intratracheally with PRRSV and then with LPS at 3, 5, 7, 10, or 14 days of infection and euthanatized 6 h after
LPS inoculation. Controls were inoculated with PRRSV or LPS only or with phosphate-buffered saline. Virus
titers, (histo)pathological changes in the lungs, numbers of inflammatory cells, and bioactive tumor necrosis
factor alpha (TNF-␣), interleukin-1 (IL-1), and IL-6 levels in bronchoalveolar lavage fluids were examined. All
pigs inoculated with PRRSV-LPS developed severe respiratory disease, whereas the controls that were inoc-
ulated with PRRSV or LPS alone did not. PRRSV infection significantly enhanced cytokine production in
response to LPS. Peak TNF-␣, IL-1, and IL-6 titers were 10 to 100 times higher in the PRRSV-LPS-inoculated
pigs than in the pigs inoculated with PRRSV or LPS alone; and the titers correlated with the respiratory signs.
The levels of neutrophil infiltration and the pathological changes detected in the lungs of PRRSV-LPS-
inoculated pigs resembled those detected when the effects of PRRSV and LPS inoculated alone are combined,
but with no synergistic effects between PRRSV and LPS. These data demonstrate a synergism between PRRSV
and LPS in the induction of proinflammatory cytokines and an association between induction of these
cytokines and disease.
European strains of porcine reproductive-respiratory syn-
drome virus (PRRSV) fail to cause respiratory disease as such.
Nevertheless, PRRSV is considered one of the most important
etiological agents in multifactorial respiratory disease of swine
both in Europe and in the United States (18). Few studies,
however, have been able to reproduce clinical respiratory dis-
ease by experimental inoculation of PRRSV followed by inoc-
ulation of a secondary virus or bacterium (4, 6, 17, 21). Vari-
ations in the severities of clinical signs and a lack of
reproducibility are the main problems with these types of stud-
ies. Even a single experimental infection with respiratory vi-
ruses results in intrinsic variations in virological, inflammatory,
and clinical parameters. Therefore, a second infection may
enhance this variation, as the outcome of the second infection
is in part dependent on that of the first infection.
We have previously developed an alternative dual-inocula-
tion model consisting of a primary inoculation with PRRSV
followed by inoculation of a nonreplicating agent, namely, li-
popolysaccharide (LPS) from Escherichia coli (8). LPS is a
major component of the outer membrane and the main endo-
toxin of gram-negative bacteria. Intratracheal administration
of LPS (20 ␮g/kg of body weight) to PRRSV-infected pigs
resulted in severe respiratory disease, characterized by tachy-
pnea, abdominal breathing, dyspnea, and high fever. In con-
trast, inoculation of PRRSV or LPS alone resulted in subclin-
ical or mild disease. This model proved to be reproducible, in
contrast to the classic dual-infection models consisting of in-
oculation of PRRSV followed by inoculation of a second rep-
licating agent. In addition, we believe that the combination of
PRRSV and LPS has practical relevance. Most pigs become
infected with PRRSV between 4 and 16 weeks of age, and the
virus persists in the lungs for up to 40 days after inoculation (9;
B. Mateusen, D. Maes, H. Nauwynck, B. Balis, M. Verdonck,
and A. de Kruif, Proc. 17th IPVS Congr., vol. 2, p. 240, 2002).
Also, most pigs are exposed to LPS under farm conditions, as
LPS is present in stable dust at concentrations ranging up to
4.9 ␮g/m
3
. Furthermore, LPS is released at high concentrations
in the lungs during pulmonary infections with gram-negative
bacteria (16, 27).
The proinflammatory cytokines interleukin-1 (IL-1), tumor
necrosis factor-␣(TNF-␣), and IL-6 are important mediators
of several respiratory diseases. IL-1 and TNF-␣are among the
first cytokines that are produced in the lungs during an infec-
tion. They cause infiltration and activation of leukocytes in the
lungs, increased microvascular permeability, and pulmonary
dysfunctions (3, 20, 25). IL-1 and TNF-␣also induce a cascade
of secondary cytokines, such as IL-6. IL-6 is a potent inducer of
acute-phase proteins in the liver (12). Although IL-6 is gener-
ally considered a proinflammatory cytokine, it also has some
anti-inflammatory properties (19). IL-6 can down-regulate the
production of IL-1 and TNF-␣and suppress their activities by
inducing the production of IL-1 receptor antagonists and sol-
uble TNF-␣receptors. Furthermore, the production of each of
* Corresponding author. Mailing address: Laboratory of Virology,
Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133,
B-9820 Merelbeke, Belgium. Phone: 00 32 9 264 73 66. Fax: 00 32 9 264
74 95. E-mail: kristien.vanreeth@rug.ac.be.
960

the three cytokines in the lungs has been associated with gen-
eral signs of disease such as fever, depression, and anorexia.
The present study was undertaken to test the hypothesis that
PRRSV-LPS-induced respiratory disease is associated with the
excessive production of proinflammatory cytokines in the
lungs. Therefore, we compared the levels of production of
IL-1, TNF-␣, and IL-6 in the lungs of pigs after dual inocula-
tion of PRRSV and LPS with those after the inoculation of
each agent alone. Correlations between cytokine levels and
respiratory signs, macroscopic and microscopic lung patholo-
gies, and the infiltration of inflammatory cells in the bronchoal-
veolar spaces were examined.
MATERIALS AND METHODS
Virus and LPS preparations. PRRSV (Lelystad virus strain) (26) was used in
the present study. The virus used for inoculation was at the fifth passage in
alveolar macrophages, which had been obtained from 4- to 6-week-old gnotobi-
otic pigs. The inoculation dose was 10
6
50% tissue culture infective doses per pig.
E. coli O111:B4 LPS was obtained from Difco Laboratories (Detroit, Mich.) and
was used at a dose of 20 ␮g/kg of body weight. This dose was based on data from
earlier experiments and was selected because it caused no clinical disease and
minimal IL-1 and TNF-␣secretion into the lungs (24). Virus and LPS were
diluted in sterile pyrogen-free phosphate-buffered saline (PBS; Gibco, Merel-
beke, Belgium) to obtain a 3-ml inoculum.
Pigs, experimental design, and sampling. Thirty-eight colostrum-deprived pigs
(age, 4 weeks) delivered by cesarean section were used in the study. They were
housed in individual Horsefall-type isolation units with positive-pressure venti-
lation and were fed commercial ultrahigh-temperature-treated cow’s milk. All
inoculations were performed intratracheally with an 18-gauge needle that was
inserted through the skin cranial to the sternum.
The pigs were allocated to four groups (see Table 1). Fourteen pigs were
inoculated with PRRSV; and 3 (n⫽2),5(n⫽3),7(n⫽6), 10 (n⫽2), or 14
(n⫽1) days later they were inoculated with LPS (PRRSV-LPS group). These
pigs were euthanatized 6 h after the LPS inoculation. This time point was chosen
because previous virus-LPS experiments showed that cytokine production peaks
at 3 to 8 h after the LPS inoculation and declines afterwards (24). Fourteen pigs
were inoculated exclusively with PRRSV and euthanatized at 3 (n⫽3),5(n⫽
3),7(n⫽3), 10 (n⫽4), and 14 (n⫽1) days after inoculation (PRRSV control
group). Five pigs were inoculated exclusively with LPS and euthanatized 6 h later
(LPS control group). Five pigs were mock inoculated with PBS and euthanatized
6 h later (PBS control group). All pigs were clinically monitored until euthanasia.
Samples from the left lung were collected for virological, histopathological,
and standard bacteriological examinations. The right lung was used for lung
lavage by a method described earlier (22). The brochoalveolar lavage (BAL)
fluids that were recovered were separated into cells and cell-free fluids by
centrifugation (400 ⫻g, 10 min, 4°C). For four of the six pigs that were inocu-
lated with PRRSV and then with LPS 7 days later, both the left and right lungs
were used for lung lavage.
Clinical and pathological examinations. Pigs were monitored for clinical signs
daily throughout the experiment and every hour after the LPS inoculation. A
respiratory disease score was attributed to each pig at the time of euthanasia.
Scores ranged from 0 to 4 (0, normal; 1, tachypnea when stressed; 2, tachypnea
at rest; 3, tachypnea and dyspnea at rest; 4, severe tachypnea and dyspnea with
labored, jerky breathing).
Macroscopic lung lesions were evaluated by visual inspection. For histopatho-
logical examination, samples of the cardiac and diaphragmatic lung lobes were
fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and
stained with hematoxylin-eosin.
BAL fluid cells were counted in a Tu¨rk chamber, and cytocentrifuge prepa-
rations were stained with Diff-Quik (Baxter, Du¨dingen, Switzerland) to deter-
mine the percentage of neutrophils and mononuclear cells.
Cytokine bioassays. Cell-free BAL fluids were concentrated 20 times by dial-
ysis against a 20% (wt/vol) solution of polyethylene glycol (molecular weight,
20,000) and cleared of residual virus by centrifugation at 100,000 ⫻gbefore
analysis in bioassays for cytokines. Bioassays for IL-1, IL-6, and TNF-␣have
been described in detail elsewhere (7, 23).
IL-1 was assayed by determination of its capacity to stimulate proliferation of
D10(N4)M cells in the presence of concanavalin A (grade IV; Sigma, Bornem,
Belgium) and recombinant human IL-2 (Genzyme, Cambridge, Mass.). The
percentage of proliferation was determined by the thiazolyl blue [3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] conversion procedure, and op-
tical densities were measured. The number of biological units per milliliter of
BAL fluid was determined as the dilution that produced 50% maximal prolifer-
ation. To confirm the specificity of the bioassay, D10 cells were incubated with
monoclonal rat anti-mouse IL-1 receptor type 1 antibodies (Genzyme).
TNF-␣activity was measured in a cytotoxicity assay with porcine kidney (PK)
(15) subclone 15 cells (a gift from G. Bertoni, Bern, Switzerland) in the presence
of actinomycin D. The plates were stained with crystal violet and read spectro-
photometrically. The number of biological units per milliliter was defined as the
dilution that produced 50% cytotoxicity. Specificity was demonstrated by neu-
tralization of samples with rabbit anti-human TNF-␣antibodies (Innogenetics,
Zwijnaarde, Belgium).
IL-6 was assayed by determination of its capacity to stimulate proliferation of
B9 cells (a gift from L. A. Aarden, Amsterdam, The Netherlands). Percent
proliferation was determined by the thiazolyl blue [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide] conversion procedure, and optical densities
were measured. The number of biological units per milliliter of BAL fluid was
determined as the dilution that produced 50% maximal proliferation. To confirm
TABLE 1. Respiratory scores, virus titers, and numbers of inflammatory cells in BAL fluids
Inoculum No. of
pigs
Time of euthanasia after
inoculation with: Mean respiratory
score
a
Mean virus titer
(log
10
TCID
50b
/g)
Mean no. (10
6
) of BAL cells
PRRSV (days) LPS (h) Neutrophils Mononuclear cells
PBS 5 —
c
—0 Negative 2 ⫾2 115 ⫾52
PRRSV 3 3 —0 4.4 ⫾1.1 5 ⫾8 154 ⫾115
35—0 5.4 ⫾1.3 4 ⫾3 117 ⫾42
37—0 6.0 ⫾0.6 7 ⫾2 216 ⫾71
410—0 5.7 ⫾0.9 38 ⫾38 409 ⫾219
114—0 6.0 3 337
LPS 5 —6 0 Negative 303 ⫾105 233 ⫾60
PRRSV-LPS 2 3 6 2 ⫾1.4 6.0 ⫾018⫾23 121 ⫾22
356 3⫾0 5.1 ⫾2.2 296 ⫾163 208 ⫾14
6 7 6 3.2 ⫾0.8 5.5 ⫾0.3 320 ⫾263 275 ⫾144
2 10 6 2.5 ⫾0.7 5.9 ⫾1.3 380 ⫾474 486 ⫾142
1 14 6 3 5.7 576 483
a
Respiratory scores range from 0 to 4 (see text for the definition of each score).
b
TCID
50
, 50% tissue culture infective dose.
c
—, not applicable.
VOL. 41, 2003 PRRSV POTENTIATES ENDOTOXIN RESPIRATORY EFFECTS 961

the specificity of the bioassay, samples were neutralized with goat anti-porcine
IL-6 antibodies (R&D Systems, Abingdon, United Kingdom).
Bioassays were done with twofold dilutions of samples in 96-well microtitra-
tion plates. Laboratory standards were run in each bioassay. Samples were tested
in two or three individual bioassays, and the geometric means were calculated.
Virological and bacteriological examinations. Tissue samples from the dia-
phragmatic lobe of the left lung were used for virological and bacteriological
examinations. PRRSV titrations were performed on alveolar macrophages by
standard methods (26). For bacteriology, samples of lung tissue were plated on
bovine blood agar and cultured aerobically. A nurse colony of coagulase-positive
Staphylococcus species was streaked diagonally on each plate. The plates were
inspected for bacterial growth after 48 and 72 h. The colonies were then iden-
tified by standard techniques.
Statistical analysis. Standard two-sample Mann-Whitney tests were used to
compare respiratory disease scores and cytokine titers. Correlation coefficients
were calculated by the Spearman rank correlation test. Pvalues ⬍0.05 were
considered significant. Statistical analyses were performed by using SPSS (ver-
sion 6.1) software.
RESULTS
The lungs of all pigs were free of bacteria by culture.
PRRSV titers are presented in Table 1. PRRSV was isolated
from the lungs of all virus-inoculated pigs but not from pigs
inoculated with LPS or PBS only. There were no differences in
virus titers between the pigs inoculated with PRRSV and LPS
combined and the pigs inoculated with PRRSV alone or at the
different time points after inoculation of PRRSV.
Clinical signs. Mean respiratory scores are presented in
Table 1. Pigs that received PBS or LPS only remained asymp-
tomatic. Pigs inoculated with PRRSV only showed no respira-
tory signs at any day after inoculation. They showed mild
anorexia and dullness between 3 and 5 days after inoculation.
In contrast, all pigs inoculated with PRRSV-LPS developed
marked respiratory signs. All pigs were clinically normal before
the LPS inoculation but developed tachypnea, dyspnea with
labored, abdominal breathing, and depression within 1 to 2 h
after LPS. These signs were still present at the time of eutha-
nasia. There were no differences in disease severity among the
pigs inoculated with LPS at 3, 5, 7, 10, or 14 days after inoc-
ulation of PRRSV. Respiratory disease scores were signifi-
cantly (P⬍0.05) higher for the group inoculated with PRRSV-
LPS than for any other group.
Macroscopic and microscopic lung pathologies. PBS-treated
control pigs did not have macroscopic or microscopic lung
pathologies (Fig. 1). The lungs of PRRSV-inoculated pigs had
a mottled appearance with multifocal red and tan areas. Mul-
tifocal interstitial pneumonia was found microscopically. Inter-
FIG. 1. Hematoxylin-eosin staining of the lungs of pigs inoculated with PBS only (a), LPS only (b), PRRSV only (10 days after inoculation) (c),
and PRRSV and then LPS over a 10-day interval (d). Interalveolar septal thickening was comparable in pigs inoculated with PRRSV-LPS and pigs
inoculated with PRRSV alone. Magnifications, ⫻100.
962 VAN GUCHT ET AL. J. CLIN.MICROBIOL.

alveolar septal thickening with infiltration of mononuclear cells
was the major feature and increased from 3 to 14 days after
PRRSV inoculation. Inoculation with LPS only resulted in
milder pneumonic lesions. Macroscopic lesions were charac-
terized by focal areas of atelectasis and interlobular edema.
The characteristic histopathological features were thickening
of the interalveolar septa, although it was less pronounced than
that after inoculation with PRRSV, and bronchiolar infiltra-
tion with neutrophils and macrophages. Intra-alveolar edema
and focal transudation of erythrocytes were occasionally seen.
The macroscopic and microscopic lung lesions after
PRRSV-LPS inoculation resembled the combination of the
lesions seen after inoculation of PRRSV or LPS alone. The
lungs were mottled with small red and tan areas and interlob-
ular edema. Microscopically, there was thickening of the inter-
alveolar septa due to an infiltration of mononuclear cells and
neutrophils. The degree of septal thickening was comparable
to that seen after inoculation of PRRSV only.
Infiltration of inflammatory cells. The BAL cells of the
PBS-treated control pigs consisted mainly of mononuclear cells
(mean, 115 ⫻10
6
cells) and a few neutrophils (mean, 2 ⫻10
6
cells) (Table 1). PRRSV-inoculated pigs showed an influx of
mononuclear cells in the bronchoalveolar spaces, and this in-
flux increased from 3 to 14 days after inoculation. Starting at 7
days after inoculation with PRRSV, the mean number of
mononuclear cells was at least two times higher in PRRSV-
inoculated pigs than in PBS-treated control pigs. The number
of neutrophils in all except one of the PRRSV-inoculated pigs
was comparable to that in PBS-treated control pigs; one
PRRSV-inoculated pig had 91 ⫻10
6
neutrophils. The LPS
inoculation induced infiltration of both neutrophils (mean, 303
⫻10
6
cells) and mononuclear cells (mean, 233 ⫻10
6
cells).
Pigs inoculated with PRRSV-LPS showed an influx of both
mononuclear cells and neutrophils. The amount and kinetics of
mononuclear cell infiltration in the pigs inoculated with
PRRSV-LPS were comparable to those in the PRRSV-inocu-
lated control pigs. The neutrophil numbers in the pigs inocu-
lated with PRRSV-LPS, on the other hand, were generally
comparable to those in the LPS-inoculated control pigs. Only
3 of the 14 pigs inoculated with PRRSV-LPS had larger num-
bers of neutrophils (567 ⫻10
6
to 786 ⫻10
6
) than the LPS-
inoculated control pigs. One pig inoculated with PRRSV-LPS
showed no neutrophil infiltration at all (1 ⫻10
6
), and three
pigs inoculated with PRRSV-LPS showed only minor neutro-
phil infiltration (19 ⫻10
6
to 44 ⫻10
6
) compared to that
detected in the LPS-inoculated control pigs. Two of these pigs
were inoculated with LPS 3 days after PRRSV inoculation,
which explains the low mean number of neutrophils in this
group.
Biologically active IL-1, TNF-␣, and IL-6 in BAL fluids.
Figure 2 shows the IL-1, TNF-␣, and IL-6 titers in the BAL
fluids of individual pigs after inoculation of PRRSV-LPS,
PRRSV only, and LPS only. PBS-treated control pigs had no
detectable IL-1, TNF-␣, or IL-6. Ten of 14 PRRSV-inoculated
pigs had elevated IL-1 titers, with the highest titers (183 to 339
U/ml) detected 10 days after inoculation. Only 3 of these 14
pigs (which were euthanatized 7, 10, and 14 days after inocu-
lation, respectively) had detectable TNF-␣titers (28 to 61
U/ml). Ten pigs had detectable IL-6 titers (61 to 343 U/ml).
LPS inoculation induced the production of all three cytokines
in the lungs. IL-1 (titers, 28 to 1,022 U/ml) and IL-6 (titers,
1,276 to 2,659 U/ml) were detected in all five pigs, and TNF-␣
(titers, 28 to 133 U/ml) was detected in three pigs.
Compared to the pigs inoculated with PRRSV and LPS
alone, 10 of 14 pigs inoculated with PRRSV-LPS showed sig-
nificantly (P⬍0.05) increased titers of at least one of the three
cytokines. In nine pigs the titers of IL-1 (titers, 2,172 to 20,480
U/ml), TNF-␣(titers, 164 to 6,047 U/ml), and IL-6 (titers,
2,511 to 378,724 U/ml) were strongly increased; and in one pig
only the titer of IL-1 (2,840 U/ml) was increased. The highest
cytokine titers were detected in pigs inoculated with LPS 5 to
14 days after the PRRSV inoculation, and they were 10 to 100
times higher than the cytokine titers of the control pigs inoc-
ulated with PRRSV or LPS only. Four pigs inoculated with
FIG. 2. Titers of proinflammatory cytokines in BAL fluids of
PRRSV-LPS-inoculated pigs and pigs inoculated with PRRSV only or
LPS only. Each dot corresponds to one pig: F, pigs inoculated with
LPS at the indicated day after PRRSV inoculation; E, pigs inoculated
with PRRSV only; ‚, pigs inoculated with LPS only. The dotted line
represents the detection limit.
VOL. 41, 2003 PRRSV POTENTIATES ENDOTOXIN RESPIRATORY EFFECTS 963

PRRSV-LPS, on the other hand, did not show enhanced levels
of cytokine production. These pigs had negligible levels of
TNF-␣(titers, ⬍20 to 31 U/ml), and the levels of IL-1 (titers,
191 to 1,571 U/ml) and IL-6 (titers, 266 to 2425 U/ml) were
comparable to those for the LPS-treated control pigs.
The left and right lungs of pigs that were inoculated with
PRRSV-LPS and whose lungs were lavaged showed no differ-
ence in cytokine titers or cell counts (P⬎0.05) (data not
shown).
Table 2 presents the correlation between respiratory scores,
cytokine levels, and numbers of inflammatory cells in BAL
fluids. The levels of all three cytokines were tightly correlated
with each other and with the respiratory scores and the neu-
trophil numbers. There was, however, little correlation be-
tween neutrophil numbers and respiratory scores. The number
of infiltrated mononuclear cells did not correlate with cytokine
levels or respiratory scores. The four pigs inoculated with
PRRSV-LPS that did not have increases in cytokine levels also
had lower neutrophil numbers (1 ⫻10
6
to 129 ⫻10
6
). The
cytokine titers and BAL cell numbers did not correlate with the
virus titers (data not shown).
DISCUSSION
This study demonstrates that a PRRSV infection sensitizes
the lungs for production of proinflammatory cytokines upon
exposure to LPS. Moreover, the cytokine titers were tightly
correlated with the appearance of respiratory signs. We have
previously documented a similar phenomenon for another re-
spiratory virus of swine that causes subclinical disease, porcine
respiratory coronavirus (PRCV) (24). Like PRRSV, PRCV
infection enhanced the levels of production of TNF-␣and IL-1
in response to LPS, and the levels of both cytokines correlated
with the severity of disease. The pathogenesis of PRRSV-LPS-
induced disease appears to be similar to the pathogenesis of
PRCV-LPS-induced disease. As IL-1 and TNF-␣have over-
lapping effects and potentiate the effects of each other, we
consider them both to be central mediators in virus-LPS-in-
duced disease. IL-6 levels in the lungs of pigs inoculated with
both virus and LPS were assessed for the first time in the
present study, and they were also found to be markedly en-
hanced. IL-6 is probably induced as a secondary cytokine in
response to IL-1 and TNF-␣, which may explain the tight
correlation between IL-6 levels and IL-1 and TNF-␣levels.
Because IL-6 has both pro- and anti-inflammatory activities, it
may either contribute to disease or counteract the activities of
IL-1 and TNF-␣.
We have indications that the tachypnea and dyspnea result-
ing from PRRSV-LPS or PRCV-LPS inoculations are due to a
functional process, such as bronchoconstriction, rather than to
structural lung damage. First, the onset of respiratory signs is
hyperacute. In another study of PRRSV-LPS inoculation, it
was shown that respiratory signs started within 1 h after LPS
inoculation, reached a climax 2 to 4 h later, and were clearly
diminished 12 h later (8). Second, the microscopic lesions in
the lungs of pigs inoculated with PRRSV-LPS and with
PRRSV only did not differ much. Pigs in both groups had
interstitial pneumonia typical of PRRSV infection, and LPS
inoculation had little extra effect. The inoculation with LPS as
such caused a marked increase in the numbers of neutrophils
in BAL fluids, but there were no differences in neutrophil
numbers between pigs inoculated with PRRSV-LPS and those
inoculated with LPS alone. Third, it is well known that IL-1
and TNF-␣can cause bronchial hyperreactivity (2, 15) and
bronchoconstriction (10), leading to asthma-like symptoms.
Moreover, TNF-␣and IL-1 were shown to act synergistically in
the induction of bronchoconstriction in the rat lung (10).
Therefore, simultaneous overproduction of these cytokines af-
ter PRRSV-LPS inoculation may cause increased and sus-
tained contraction of bronchi, which may explain the acute
respiratory signs.
We cannot explain why four pigs inoculated with PRRSV-
LPS, which showed clear respiratory signs, had only low cyto-
kine titers and negligible neutrophil infiltration. There were no
consistent differences in PRRSV titers or the numbers of
mononuclear cells in BAL fluids between these and the other
pigs. Because LPS exerts its effect locally, our initial hypothesis
was that the LPS inoculum probably did not reach the right
lung in those pigs and that cytokine production and neutrophil
infiltration might have been restricted to the left lung. To test
this hypothesis we lavaged both the left and the right lungs of
four pigs inoculated with PRRSV-LPS. There were no differ-
ences in the levels of cytokine production or neutrophil infil-
tration between the two lung halves. Therefore, it can be as-
sumed that the LPS inoculum is distributed equally between
both lung halves in most pigs. The true reason for the variabil-
ity in cytokine production and neutrophil infiltration among
pigs inoculated with PRRSV-LPS is unclear.
There have been few studies on the interactions between
viruses and LPS in vivo. To our knowledge, PRRSV and
PRCV are the first respiratory viruses shown to act synergis-
tically with LPS in the induction of respiratory disease and
cytokines. Recently, it has been described that systemic infec-
TABLE 2. Correlation coefficients between respiratory scores, cytokine titers, and numbers of inflammatory cells in BAL fluids
Parameter Correlation with:
Respiratory score IL-1 titer TNF-␣titer IL-6 titer Neutrophils no. Mononuclear cell no.
Respiratory score 11 0.81 0.70 0.71 0.59 NS
a
IL-1 titer —
b
1 0.75 0.85 0.80 0.43
TNF-␣titer ——1 0.84 0.74 0.40
IL-6 titer ———1 0.84 NS
Neutrophil no. — ——— 1 0.61
Mononuclear cell no. — ——— — 1
a
NS, no significant correlation (P⬎0.05).
b
—, not applicable.
964 VAN GUCHT ET AL. J. CLIN.MICROBIOL.

tion of mice with lymphocytic choriomeningitis virus or vesic-
ular stomatitis virus leads to fatal shock upon intraperitoneal
inoculation of a sublethal dose of LPS (13, 14). It appeared
that the shock syndrome was caused by the overproduction of
TNF-␣. Mice inoculated with virus-LPS had 3- to 50-fold
higher serum TNF-␣levels compared to those in the sera of
mice inoculated with LPS alone. By use of knockout mice, it
was demonstrated that virus-induced interferon was responsi-
ble for the increased sensitivity to LPS (5, 13). Both alpha/beta
interferon and gamma interferon were able to sensitize cells to
LPS. It is unlikely, however, that alpha interferon is involved in
the sensitization of PRRSV-infected pigs to LPS, because al-
pha interferon production is minimal during infection with
PRRSV (1, 23).
It remains to be seen whether the PRRSV-induced infiltra-
tion of the lungs with mononuclear cells contributes to the
increased responsiveness to LPS. PRRSV induces infiltration
of monocytes in the lungs, reaching a peak at 25 days after
inoculation (9). In mice, it was shown that monocytes infiltrat-
ing the lungs in response to monocyte chemoattractant protein
type 1 (MCP-1) have increased levels of expression of CD14,
the LPS receptor, and become primed for enhanced TNF-␣
production in response to LPS (11). It is possible that mono-
cytes attracted to PRRSV are an important source of cytokines
upon LPS exposure and that they are responsible for the en-
hanced cytokine response compared to the response of unin-
fected lungs. In this study, the number of mononuclear cells in
the bronchoalveolar spaces did not correlate with the respira-
tory signs. There are two important considerations in this re-
gard. First, the profiles of the cells in the BAL fluid of pigs
inoculated with PRRSV-LPS were partly the result of the LPS
inoculation and, as such, did not reflect the situation before the
LPS inoculation. Second, we counted the mononuclear cells in
the BAL fluids and not in the interstitium, while interstitial
monocytes may be important targets for LPS.
In conclusion, respiratory viruses like PRRSV, which do not
cause respiratory signs on their own, can sensitize the lungs for
the production of proinflammatory cytokines and respiratory
signs upon exposure to bacterial endotoxins. This interaction
may be important in the development of multifactorial respi-
ratory disease, as is often seen in the field.
ACKNOWLEDGMENTS
This work was supported by grant 5772A from the Belgian Ministry
of Agriculture. S.V.G. and K.V.R. are fellows of the Fund for Scientific
Research Flanders (FWO-Vlaanderen).
We thank Lieve Sys, Fernand De Backer, and Chantal Vanmaercke
for excellent technical assistance. We also thank G. Bertoni for pro-
viding recombinant porcine TNF-␣and PK(15) subclone 15 cells and
L. A. Aarden for providing B9 cells.
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