Vol.2, No.1, 40-48 (2012) Open Journal of Immunology
Cytokine responses in infants infected with
respiratory syncytial virus
Morten Breindahl1,2*, Klaus Rieneck2, Claus Nielsen2, Tage Justesen3, Klaus Bendtzen2,
Klaus Müller2,4
1Department of Pediatrics, Hillerod Hospital, Hillerod, Denmark; *Corresponding Author: breindahl@dadlnet.dk
2Institute for Inflammation Research (IIR), Department of Rheumatology, Rigshospitalet University Hospital, Copenhagen, Denmark
3Department of Clinical Microbiology, Hillerod Hospital, Hillerod, Denmark
4Pediatric Clinic II, Rigshospitalet University Hospital, Copenhagen, Denmark
Received 19 December 2011; revised 20 January 2012; accepted 2 February 2012
Introduction: Variability in severity of Respira-
tory Syncytial Virus (RSV) infection is reportedly
due to differences in inflammatory response.
Objective: To characterize the c ytokine response
in RSV+ infants aged 0 - 36 months and to relate
their responses to disease severity. Methods:
Nasopharyngeal aspirations (NPAs) were ana-
lyzed for RSV and IL-1
, IL-2, IL-4, IL-5, IL-6,
IL-10, IL-12, IL-1RA, IL-4R, IFN-
, sTNFR1,
sTNFR2, and TNF-α. Clinical data were collected
from the medical records. Results: We included
331 infants of whom 214 were RSV+. In com-
parison to RSV- infants, they had significantly
higher levels of TNF-
, IL-6, IL-1
, and IFN-
(p <
0.05). This also applied to anti-inflammatory cy-
tokine IL-10 levels, but these levels were re-
markably lower than levels of TNF-
, IL-6, and
. sTNFR1/2 were significantly increased in
RSV+ infants. Hospitalized patients had signifi-
cantly higher levels of TNF-
, sTNFR2, and IL-10
(p < 0.05) than non-hospitalized patients. The
cytokine response could not be related to dis-
ease severity. We found no evidence of a skewed
Th1/Th2 immune profile. Conclusion: In acute
RSV disease, infected infants’ NPAs contain a
significant amount of pro-inflammatory cyto-
kines. Whether this response is beneficial or
deleterious remains unanswered. Interpersonal
variations in cytokine responses might be linked
to an inherited tendency to variations in disease
Keywords: Respiratory Syncytial Virus; Bronchiolitis;
Inflammation; Cytokines; Infants Aged 0 - 3 Years
A number of findings suggest that the variability in the
severity of respiratory syncytial virus (RSV) infections
may be rooted in individual differences in inflammatory
response [1,2] and not to RSV polymorphisms
Ex vivo studies have focused on cytokine production
by cord blood cells [3-6] or peripheral blood mononu-
clear cells [7-9], or they have measured cytokines in se-
rum [10]. The direct clinical applicability of these results,
however, remains, questionable since RSV has never
been reported to cause a viremic phase.
In vivo human studies of cytokine levels in airway spe-
cimens from RSV+ infants indicate a pronounced pro-
duction of inflammatory and immunoregulating cytokines
such as interleukin (IL-)-2, IL-5, IL-6, IL-8, IL-9, IL-10,
IL-11, tumor necrosis factor (TNF), Regulated on Acti-
vation Normal T-cell Expressed and Secreted (RANTES),
and Eosinophil Cationic Protein (ECP) [11-13]. However,
the clinical significance has been difficult to ascertain
since the majority of these studies report unsystematic
measurements of cytokine candidates in small groups of
patients. Moreover, only a few studies have included an
appropriate control group.
We studied a large cohort of 0 - 36-month-old infants
with acute upper and/or lower respiratory tract infection
with the purpose of characterizing the magnitude of their
disease symptoms during winter seasons. We evaluated
the cytokine response in nasal pharyngeal aspirations
(NPAs) to characterize the cytokine responses in RSV+
infants compared to RSV– infants, and to relate cytokine
levels to disease severity.
2.1. Participants
2.1.1. Inclusion Criteria
Eligible subjects were infants aged 0 - 36 months re-
ferred to the Pediatric Emergency Room (PER) at Hillerød
Hospital during three consecutive winter seasons (Octo-
Copyright © 2012 SciRes. OPEN AC CESS
M. Breindahl et al. / Open Journal of Immunology 2 (2012) 40-48 41
ber-March) from 2000-2003. The children were admitted
due to symptoms of acute upper and/or lower respiratory
tract infection (URTI/LRTI), i.e. wheezing, increased res-
piratory rate (RR), asthmatic breathing, rales, crackles,
dyspnea, apnea, cyanosis.
2.1.2. Exclusion Criteria
We excluded patients undergoing treatment with cor-
ticosteroids or agents with possible symptomatic effects
on respiratory symptoms, i.e. β2-agonists and antibiotics,
and patients with recurrent episodes of bacterial pneu-
monia, chronic inflammatory diseases, immune deficien-
cies, chromosome aberrations, and congenital cardiopul-
monary diseases, e.g. congenital heart disease (CHD) and
bronchopulmonary dysplasia (BPD).
2.1.3. Study Design
This study was designed as an open, one-center cohort
3.1. Inclusion
The principal investigator examined all eligible pa-
tients within 24 hours after referral to the PER. The fol-
lowing baseline information was collected: Gestational
age at time of birth (GA), birth weight (BW), known
presence of chronic lung disease (BPD, cystic fibrosis,
asthma, etc.), asthmatic bronchitis, atopic disease, dispo-
sition to allergy and/or asthma, and duration of respira-
tory symptoms before referral to the PER.
The physical examination focused on weight (W),
height (H), rectal body temperature (Tp), transcutaneous
oxygen saturation (SAT) (without supplemental oxygen
being provided), skin color, RR, retractions of the chest
wall, flaring of alae nasi, and use of accessory muscles
during inspiration. C-reactive protein (CRP), pH, partial
pressure of oxygen, and carbon dioxide were measured.
Decisions on treatment of the included infants, time of
discharge, and the need for clinical outpatient follow-up
were left at the discretion of the house officer on duty.
After discharge, the following information was ex-
tracted from the medical records: date of discharge,
length of stay (LOS) in hospital, RSV test result, con-
firmed positive culture of a representative tracheo-
bronchial aspiration (TBA) or blood, antibiotic treatment,
bronchodilators, steroids, supplemental oxygen, nasal con-
tinuous positive airway pressure (n-CPAP), mechanical
ventilation, and death.
3.2. Sampling Technique
NPAs were sampled by introducing a catheter (char-
riére 08, Unoplast A/S, Denmark) attached to a sterile
trachea suction unit (Maersk Medical A/S, Denmark)
into the nostrils to the nasopharynx and subsequently
applying vacuum (25 cm H2O) during a 5-second retrac-
tion of the catheter. The catheter was flushed into the
sterile mucus trap with 2 ml sterile, isotonic sodium chlo-
ride, which was immediately placed on ice and trans-
ported to our laboratories.
The samples were weighed using a precision scale
weight (Mettler Toledo, Dual Range, Precision 0.01g)
and centrifuged at 440 g for 10 minutes at 4˚C. The su-
pernatants were separated from cell pellets by decanta-
tion in toto into another sterile tube and briefly homoge-
nized. Aliquots of 250 l of the supernatant were trans-
ferred to 5 - 6 sterile cryo tubes (NUNC A/S, Denmark)
containing 20 l 10% azide, gently turned several times,
and immediately frozen at –80˚C until final batch-wise
cytokine analysis. The pellet was resuspended in 0.5 ml
Phosphate-Buffered Saline (PBS) and kept at 4˚C until
RSV examination within less than 12 hours. The analysis
was performed by direct immunofluorescence (DIF)
staining (Merifluor RSV, Meridian Bioscience Inc.,
Cincinnati, Ohio, USA) at the Department of Clinical
Microbiology according to the manufacturer’s instruc-
3.3. Analyses of Inflammatory Mediators
IL-4 and TNF-α (PeliKine Compact kits, CLB, Am-
sterdam, the Netherlands) and soluble TNF-receptor 1
(sTNFR1) and 2 (sTNFR2) (Quantikine kits, R&D Sys-
tems, Abingdon, UK) were quantified in accordance with
the manufacturers’ manuals. The inter- and intra-assay
coefficients of variability of all assays were <10%. All
cytokine analyses were carried out in duplicate. The de-
tection limit of the enzyme-linked immunosorbent assays
(ELISAs) used ranged from 0.4 - 3.0 pg/ml.
IL-1 receptor antagonist (IL-1Ra) and sIL-4R were
measured in duplicate by an in-house double-sandwich
ELISA using monospecific polyclonal antibodies to puri-
fied recombinant cytokines. Briefly, immuno-Maxisorp
plates (Nunc, Roskilde, Denmark) were coated with pro-
tein-A affinity-purified IgG. The plates were blocked
with 1% human serum albumin in PBS. The assays were
calibrated with international standards of the respective
cytokines (National Institute for Biological Standards
and Controls, Potters Bar, Herfordshire, UK). Bioti-
nylated rabbit antibodies were used as detecting antibod-
ies along with streptavidin-peroxidase (Kirkegaard and
Perry La., Gaithersburg, MD, USA). Development was
carried out with 1,2-phenylenediamine dihydrochloride
and measured at 492 nm. The inter- and intra-assay coef-
ficients of variability for the concentration range [15.0
pg/mL - 1.0 ng/mL] were <15%.
IL-2, IL-4, IL-5, IL-10, TNF-α and interferon-γ (IFN-γ)
were quantified in duplicate by use of the Human
Copyright © 2012 SciRes. OPEN AC CESS
M. Breindahl et al. / Open Journal of Immunology 2 (2012) 40-48
T-helper 1 and 2 (Th1/Th2) Cytokine Cytometric Bead
Array kit (Becton Dickinson (BD) Biosciences Pharm-
ingen, San Diego, California, USA) in accordance with
the manufacturer’s manual.
3.4. Ethics
The local ethics committee (1999-1-1) and the Danish
Data Management (2000-41-0313) approved the study,
and written, informed consent was obtained from the
parents of all patients before participation.
3.5. Statistical Analyses
Data were computed with SPSS Version 11.5 (SPSS
Inc, Chicago, Illinois). The distribution was significantly
different from a normal distribution (Kolmogorov-Smir-
nov with Lilliefors Significance Correction and Shapiro-
Wilk had p-values < 0.001), and consequently non-pa-
rametric statistical methods were applied.
The Mann-Whitney U-test was used for two-indepen-
dent-samples tests of cytokine levels in the following
groups: 1) RSV+/RSV–; 2) female/male; and 3) hospi-
talized/non-hospitalized. Contingency tables were ana-
lyzed using Fisher’s exact test for calculations involving
groups < 5; otherwise Yates’ corrected chi-square was
applied. For numeric and non-parametric continuous data,
Spearman’s rho was calculated.
4.1. Description of Cohort
Figure 1 provides an overview of the 361 screened
study patients. We recruited 331 infants of whom 158
(48%) were girls. Table 1 shows baseline characteristics
of the cohort. Episodes of asthmatic bronchitis treated
with bronchodilators and/or steroids had been present in
17% of the infants. Atopic diseases, diagnosed by a phy-
sician, were present in 20%, and 49% were predisposed
for such disease, since asthma and/or allergy defined as >
1 positive skin provocation test had been diagnosed in
first-degree siblings and/or parents.
Physical examination revealed that 49 (15%) of the
infants had normal skin color, 83 (25%) had flushing,
192 (58%) had paleness, and 5 (1.5%) had cyanosis of
the extremities. Significant subcostal chest wall retrac-
tions were present in 258 (78%), 54 (16%) had none,
whereas 17 (5%) had minor localized suprasternal, su-
praclavicular, or intercostal retractions without involve-
ment of the subcostal tissue.
Only 59 (18%) were immediately discharged as out-
patients, whereas 272 (83%) were hospitalized. The hos-
pitalized infants were significantly younger (p < 0.001),
were generally more premature at birth (p = 0.030), and
had a lower SAT (p < 0.007) and a higher RR (p = 0.034)
than out-patients (Table 2).
Figure 1. Patient flow chart.
Table 1. Baseline characteristics.
Variable Median Range
Age (weeks) 17.3 1 - 156
GA (weeks) 40 28 - 43
BW (g) 3460 835 - 5500
DUR (days) 5 1 - 30
Tp (˚C) 37.6 35.9 - 40.6
SAT (%) 94 85 - 100
RR (n/min) 52 20 - 100
Baseline characteristics of the total cohort showing median and range.
Please refer to list of abbreviations.
Copyright © 2012 SciRes. OPEN AC CESS
M. Breindahl et al. / Open Journal of Immunology 2 (2012) 40-48
Copyright © 2012 SciRes. OPEN AC CESS
Almost 2/3 (65%, n = 214) of the included infants (n = 331)
were RSV+, while the remaining 117 (35%) were RSV–.
RSV+ infants were significantly younger than RSV–
infants, had increased RR upon admission, and were
hospitalized longer (Table 3). However, we found no
significant differences between RSV+ and RSV– infants
in terms of clinical or paraclinical parameters (i.e., GA,
BW, duration of symptoms before referral, Tp, SAT, C-
reactive protein [CRP], pH, or partial pressure of either
oxygen [pO2] or carbon dioxide [pCO2]). Males and fe-
males did not differ from each other (data not shown).
Nearly all of the hospitalized infants (97%, 263/272)
received inhalations with bronchodilators, 17% (45/272)
were treated with corticosteroids, and 11% (31/272) had
additional fluid administered either intravenously or
orally through a nasogastric tube. Hospitalized infants
treated with n-CPAP (18%, 50/272) had significantly lo-
wer age, longer LOS, and higher RR compared to non-
n-CPAP-treated infants (not shown). They also had sig-
nificantly lower GA and BW. At the time of n-CPAP
treatment, they had respiratory acidosis with lower pH
and higher pCO2 values than non-hospitalized patients
(data not shown). None of the patients died during hos-
Table 2. Characteristics of hospitalized and non-hospitalized.
Hospitalized (n = 272) Non-hospitalized (n = 59)
Median Range Median Range
p value
Age (weeks) 15 1 - 149 38 4 - 156 <0.001
GA (weeks) 40 28 - 43 40 36 - 42 0.030
BW (g) 3460 835 - 5500 3453 2340 - 4770 0.428
DUR (days) 4 1 - 30 4 1 - 30 0.509
Tp (˚C) 37.6 35.9 - 40.6 37.6 36.1 - 40.1 0.840
SAT (%) 94 85 - 100 97 88 - 100 0.007
RR (n/min) 52 28 - 100 48 20 - 90 0.34
Non-parametric analyses (Mann-Whitney U-test) of the two independent variables (hospitalized and non-hospitalized), showing me-
dian, range, and subsequent p-values. Significant differences highlighted with bold typing. Please refer to list of abbreviations.
Table 3. Characteristics of RSV+ and RSV–.
RSV+ (n = 214) RSV– (n = 117)
Variable Median Range Median Range p values
Age (weeks) 15 1 - 149 25 1 - 156 0.002
GA (weeks) 40 28 - 42 40 32 - 43 0.159
BW (g) 3420 835 - 5500 3560 1580 - 5200 0.091
DUR (days) 5 1 - 14 5 1 - 30 0.573
LOS (days) 3 0 - 27 1 0 - 12 <0.001
Tp (˚C) 37.6 35.9 - 40.6 37.5 36.1 - 40.4 0.283
SAT (%) 94 86 - 100 95 85 - 100 0.550
RR (n/min) 52 28 - 100 48 20 - 90 0.007
CRP (ng/ml) 129 1 - 2388 112 1 - 1392 0.929
pH 7.37 7.25 - 7.45 7.36 7.12 - 7.45 0.314
pO2 6.40 3.50 - 12.30 7.35 4.30 - 10.50 0.224
pCO2 5.90 4.40 - 9.30 5.90 4.10 - 13.40 0.793
Non-parametric analyses (Mann-Whitney U-test) of the two independent variables (RSV+ and RSV– infants) showing median, range,
and subsequent p-values. Significant differences highlighted with bold typing. Please refer to list of abbreviations.
M. Breindahl et al. / Open Journal of Immunology 2 (2012) 40-48
4.2. Analyses of Cytokines in
Nasopharyngeal Aspirations
The nasopharyngeal aspirates from RSV+ infants con-
tained significantly higher levels of the pro-inflammatory
mediators TNF-α, IL-6, IL-1β, and IFNγ (Figures 2(a)-
(d)) than those of RSV– infants. Though the same ap-
plied to the levels of the anti-inflammatory cytokine
IL-10 (Figure 2(e)), the IL-10 levels were remarkably
lower than those of TNF-α, IL-6 and IL-1β.
We also measured the levels of sTNFR1 and sTNFR2,
which correlated significantly in 35 randomly selected
patients (Spearman’s rho 0.950, p < 0.001, not shown).
We therefore proceeded with in-depth analyses of
sTNFR2 only. The levels of this receptor were also in-
creased in RSV+ infants (Figur e 2(f)).
The levels of TNF-α, sTNFR2, and IL-10 (Figures
3(a)-(c)) were higher in hospitalized infants than in
out-patients. This was not the case for the other cytokines
tested (data not shown).
No significant differences in cytokine levels were
found upon subgroup analysis of the cohort stratified into
1) age groups: young infants: 0 - 2 months, infants: 3 - 11
months, and young children: 12 - 36 months; 2) n-CPAP
treatment: n-CPAP+, n-CPAP–; 3) bacterial contamina-
tion in TBA; 4) degree of retractions: suprasternal, su-
praclavicular, intercostal, subcostal; or 5) scoring of skin
color: normal, flushing, paleness, cyanosis (not shown).
4.3. Th1/Th2-Related Cytokines
To further characterize the Th1/Th2 profile of the cy-
-tokines in NPAs, we made a computerized random se-
lection of approximately 60 samples to be tested for IL-2,
IL-4, IL-5, IL-12, and sIL-4R.
The measured levels were either very low (maximum
15280N =
TNF (pg/ml) ELISA
P = 0.024
11949N =
RSV vs IL-6
IL-6 (pg/ml) FACS
P = 0.019
3016N =
RSV vs IL-1B (TBA)
IL-1B (pg/ml) FACS
P = 0.028
(a) (b) (c)
4716N =
IFNg (pg/ml) FACS
P = 0.001
4716N =
RSV vs IL-10
IL-10 (pg/ml) FACS
P = 0.008
17094N =
RSV vs sTNF-R2
sTNF-R2 (pg/ml) ELISA
P = 0.002
(d) (e) (f)
Figure 2. Boxplots showing RSV+ and RSV– infants versus the levels of (a) TNF-α; (b) IL-6; (c) IL-1β; (D) IFN-γ; (e) IL-10 and (f)
sTNFR2. Non-parametric analyses by Mann-Whitney U-test show significant differences between RSV+ and RSV– infants (p <
Copyright © 2012 SciRes. OPEN AC CESS
M. Breindahl et al. / Open Journal of Immunology 2 (2012) 40-48 45
14325N =
Hospitalisation vs TNF
TNF (pg/ml) FACS
P < 0.001
22440N =
Hospitalisation vs sTNF-R2
sTNF-R2 (pg/ml) ELISA
P = 0.031
14225N =
Hospitalisation vs IL-10
IL-10 (pg/ml) FACS
P = 0.027
(a) (b) (c)
Figure 3. Boxplots showing hospitalized and non-hospitalized infants versus the levels of (a) TNF-α; (b) sTNFR2 and (c) IL-10.
Non-parametric analyses by Mann-Whitney U-test show significant differences between hospitalized and non-hospitalized (p < 0.05).
concentrations of IL-12 < 70 pg/ml) or below detectable
thresholds (IL-2, IL-4, IL-5, and sIL-4R).
4.4. Cytokine Profile versus Severity of Disease
We performed statistical analysis of subgroups of the
study cohort divided into 1) eligible RSV+ patients, aged
0 - 6 months and 2) eligible RSV+ patients, aged 0 - 3
months, in order to spot possible deviations in cytokine
profile as reflected by their disease severity. We found no
statistically significant differences in either subgroup
when correlating with the measured cytokines between
infants with 1) very short (1 day) vs long (2 days) hos-
pitalization; 2) short (2 days) vs long (3 days) hospi-
talization; 3) normocapnia vs hypercapnia; or 4) with no
need for respiratory support (–n-CPAP) vs need for res-
piratory support (+n-CPAP) (data not shown).
We examined a large group of infants with acute res-
piratory distress referred to the PER for immediate pedi-
atric evaluation during three winter seasons in Denmark.
Most infants had been ill for approximately one week
upon referral. Hospitalized infants constituted almost
83%. Compared to the non-hospitalized group, they were
younger and had clinical symptoms indicating more se-
vere disease, e.g. lower SAT and higher RR. The major-
ity of these infants were RSV+ and they were signifi-
cantly younger than the RSV– infants, which is in line
with the notion that RSV infection results in more seri-
ous illness at younger age [14].
Apart from younger age, other well-known risk factors
include prematurity, congenital heart disease, lung dis-
ease, and immunodeficiency. However, it has been diffi-
cult to identify reliable prognostic factors for develop-
ment of severe disease in previously healthy infants [15].
A number of cytokine gene polymorphisms may be
associated with severe RSV disease, and high levels of
inflammatory cytokines in RSV-infected infants have
previously been demonstrated in both acute URTI [16]
and RSV-related bronchiolitis [17]. However, it has not
been thoroughly investigated whether the degree of in-
flammation, as measured by the levels of these mediators,
predicts the clinical course of the disease. Specific cyto-
kine profiles associated with RSV-infection have not
been reported. In general, it has not been shown whether
the inflammatory response seen upon RSV infection dif-
fers from the response to other viruses, and whether this
reaction is beneficial or deleterious [18]. In fact, the im-
maturity of the infant immune system may alter the out-
come of this viral infection, but at the same time, the
viral infection possibly shapes the development of the
infant’s immune system and its future responses [19].
In our cohort, we found a predominantly pro-inflame-
matory cytokine response in NPAs from RSV+ infants
that was significantly different from that found in RSV-
infants. In particular, TNF-α, IL-6, and IL-1β were found
at concentrations likely to be of pathogenic importance.
These findings are consistent with the previous observa-
tion that RSV binding to Toll-like receptor (TLR)-4 in
airway epithelium may result in release of pro-inflame-
matory cytokines through activation of nuclear factor-
kappa B [20], which controls the expression of numerous
RSV-inducible inflammatory and immunomodulatory
genes. However, multiple TLRs activate the innate im-
mune system upon RSV infection, and though TLR acti-
vation may cause a specific cytokine production, viral
factors may well counteract these pathways, thereby
contributing to RSV proliferation [21].
Despite the finding of elevated concentrations of in-
flammatory cytokines in RSV+ infants along with the
observation that these infants needed longer hospitalize-
Copyright © 2012 SciRes. OPEN AC CESS
M. Breindahl et al. / Open Journal of Immunology 2 (2012) 40-48
tion than RSV– infants, we were unable to demonstrate
any direct correlation between the level of inflammatory
cytokines and the severity of pulmonary symptom scores.
Due to the association between the development of
asthma and severe RSV infection, researchers have specu-
lated whether the immune response elicited upon RSV is
a Th2-predominant phenotype. Though animal models
have indicated a skewed Th1/Th2 cytokine balance in
RSV-related disease, this observation has not yet been
systematically confirmed in humans [22,23]. An excess
type 2 immune response during RSV bronchiolitis has
been suggested based on ratios of Th1- and Th2-related
cytokines from nasal lavage fluid and stimulated PBMC
[22]. Similarly, Th2 responses have been detected in NPA
from children during viral lower respiratory tract infec-
tion, but these responses were not unique for RSV infec-
tion [24].
We found very low levels of Th1- and Th2-related cy-
tokines. Even though the concentrations of IFN-γ and
IL-10 were significantly higher in RSV+ than in RSV-
infants, they were so low that a biological effect must be
doubtful. IL-10 is known to down-regulate pro-inflame-
matory and to upregulate anti-inflammatory mediators -
including the shedding of sTNFR2. This soluble receptor
has a dual action, since it serves as an effective inhibitor
of ligand activity (here: TNF-α) at high concentrations,
and acts as a facilitator of ligand-binding at low concen-
trations. To our knowledge, the present study is the first
to show sTNFR2 in infants with RSV infection. Al-
though the levels were significantly higher in RSV+ in-
fants, they were within the lower range of that seen in
sera of healthy children and in children with inflamma-
tory conditions [25,26]. Along with the fact that TNF
concentrations were highly elevated in the airway secre-
tion, this suggests that at the levels found here sTNFR2
is unlikely to effectively suppress TNF-α activity in the
airway secretion of these patients. On the contrary, one
may speculate that the sTNFR2 levels may facilitate
TNF-α activity in airway secretion. We were unable to
further substantiate this, since the ratio of TNF-α to
sTNFR2 did not correlate with the clinical severity of
During the acute phase, RSV infection seems to be
characterized by a predominantly pro-inflammatory im-
mune response. We demonstrated no association between
the level of pro-inflammatory cytokines and the clinical
severity of the infection. However, the cytokines ob-
served in airway secretions from these patients may be
involved in the pathogenesis of RSV-associated bron-
chiolitis—or they may represent an appropriate defense
mechanism against the viral infection. Thus, it remains
unanswered whether the cytokines measured are to be
considered “friend” or “foe”.
In conclusion, this large-scale prospective study of pa-
tients aged 0 - 36 months with symptoms of URTI/LRTI
provides evidence of a predominantly pro-inflammatory
cytokine response in RSV+ infants significantly different
from that encountered in RSV– infants. RSV+ infants
also had significantly higher concentrations of sTNFR2,
a finding not previously reported. However, the data do
not suggest that cytokine profiling of airway secretions
can be used as a prognostic tool for determining the risk
of developing severe disease manifestations in a clinical
M. Breindahl and K. Müller wrote the study protocol.
M. Breindahl was responsible for the planning of the
study, collected all data and processed all samples, did
the statistical analyses and wrote the first draft of this
manuscript. He supervised and held responsibility for all
laboratory analyses, of which he conducted a significant
part himself. K. Müller supervised the study, and he, K.
Bendtzen, K. Rieneck, C. H. Nielsen, and T. Justesen
revised and approved all drafts of this manuscript. Lone
Bredahl and Marianne Thomsen performed the majority
of cytokine analyses at the Institute for Inflammation
Research. T. Justesen and the technical staff at the De-
partment of Clinical Microbiology, Hillerød Hospital,
conducted the RSV tests.
Funding has been granted from the Medical Research Foundation in
Region III, East-Danish Medical Research Forum (ØSFF), Frederiks-
borg County Research Foundation, Rosalie Petersen’s Foundation,
Captain Harald Jensen and Wife’s Foundation, Mrs Olga Bryde Niel-
sen’s Foundation, Tvergaard Foundation, the Danish Medical Associa-
tion’s Research Foundation, the Danish Pediatric Society (DPS). K.
Bendtzen provided additional funding through applications to the
Lundbeck Foundation and the Danish Biotechnology Program. We
would also like to thank the parents of the included infants for their
willingness to let their acutely ill children participate in this study.
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BPD Bronchopulmonary Dysplasia PBS Phosphate-Buttered Saline
BW Birth Weight PER Pediatric Emergency Room
CF Cystic Fibrosis RR Respiratory Rate
CHD Congenital Heart Disease RSV Respiratory Syncytial Virus
DUR Duration of illness before referral to
PER RSV– Respiratory Syncytial Virus negative
GA Gestational Age RSV+ Respiratory Syncytial Virus positive (patients)
H Height SAT Transcutaneous Oxygen Saturation
IFN-γ Interferon-gamma sIL4-R Soluble Interleukin 4 Receptor
IIR Institute for Inflammation Research sTNFR1/2 Soluble Tumor Necrosis Factor Receptor
1 and 2
IL Interleukin TBA Tracheo-Bronchial Aspiration
IL-1-Ra Interleukin 1 Receptor Antagonist Th1/Th2 T-Helper cell type 1 and 2
LOS Length of Stay TLR Toll-Like Receptor
LRTI Lower Respiratory Tract Infection TNF-α Tumor Necrosis Factor Alfa
n-CPAP Nasal Continuous Positive Airway
Pressure Tp Temperature
NPA Nasopharyngeal Aspiration URTI Upper Respiratory Tract Infection
PBMC Peripheral Blood Mononuclear Cells W Weig ht