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MAJOR ARTICLE
Differences in Participation of Innate and Adaptive
Immunity to Respiratory Syncytial Virus in Adults
and Neonates
Subramaniam Krishnan,1,4 Mary Craven,4 Robert C. Welliver,5 Nafees Ahmad,3 and Marilyn Halonen2,3,4
1
Graduate Program in Microbiology and Immunology, Departments of 2Pharmacology and 3Microbiology and Immunology, and 4Arizona Respiratory
Center, University of Arizona Health Sciences Center, Tucson; 5Department of Pediatrics, State University of New York at Buffalo
Innate and adaptive immune responses to respiratory syncytial virus (RSV) in neonates were assessed by cord
blood mononuclear cell (MC) cytokine expression and proliferation and these responses were compared with
those from adult peripheral blood MCs. In adult cells, inactivated and live virus invoked cytokines reflecting
both innate and adaptive immunity (interleukin [IL]–6, interferon [IFN]–g, IL-2, tumor necrosis factor [TNF]–
a, and IL-10). Low levels of IL-4 were detected, although only with inactivated virus. In contrast, in neonatal
cells, inactivated virus invoked large levels of the innate immune cytokines IL-6, TNF-a, and IL-10 and reduced
levels of IFN-g and IL-12 but no adaptive cytokines. Live virus induced fewer innate (IL-6, IL-10, and IFNg) and no adaptive immune cytokines. RSV-induced proliferation was absent in neonatal MCs, although positive
in adult MCs. Thus, exposure to RSV does not appear to occur before birth, and adaptive immune insufficiency
or greater innate responses may account for early life RSV-induced illnesses.
Respiratory syncytial virus (RSV) is the principal cause
of severe lower respiratory tract infection in infants and
young children [1]. Infection rates are ∼70% in the first
year of life, and all children are infected by age 3 [2].
Disease manifestations vary markedly among individuals and may include pneumonia, bronchiolitis, or mild
upper airway congestion alone. Morbidity is highest
during the first 6 months of life [3]. In older children
and adults, symptoms are mild and are usually restricted to the upper respiratory tract.
Variability in the development of a protective immune response to this virus may account for the very
large interindividual differences in disease phenotype
that accompany exposure in infants. Information re-
Received 18 September 2002; accepted 3 March 2003; electronically published
10 July 2003.
Presented in part: American Academy of Allergy, Asthma and Immunology
(AAAAI), New Orleans, 16–21 March 2001 (abstract 311); AAAAI, San Diego, 3–
8 March 2000 (abstract 887).
Financial support: National Institutes of Health (grants AI-44697, P50 HL 67672,
and AI-42268).
Reprints or correspondence: Dr. Marilyn Halonen, Dept. of Pharmacology and
Microbiology and Immunology, PO Box 245030, Rm. 2342A, University of Arizona
HSC, Tucson, AZ 85724 ([email protected]).
The Journal of Infectious Diseases 2003; 188:433–9
2003 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2003/18803-0013$15.00
garding the dynamics of the development of an immune
response to RSV in early life is sketchy. The development of detectable antibody responses to RSV is evident
in many children within the first year of life and in
most children before age 3 [4]. Maternal antibody provided passively to the fetus is present through the first
few months of life when prevalence of the more-severe
forms of RSV illness is greatest, thus casting doubt on
a major protective role for antibody-mediated immunity [5]. Innate immunity to RSV also has been reported, as demonstrated by increased levels of tumor
necrosis factor (TNF)–a, interleukin (IL)–1b, IL-6, and
IL-8 in nasal washings of infants during active infection
[6]. Perhaps the greatest lack of clarity concerns the
presence, temporal nature, and degree of variability of
cell-mediated immunity, with possibilities of an overzealous immune response [7, 8], immune deviation [9,
10], or transient early life immune deficiency [11], all
of which have been offered as suggestions to account
for the variability in disease severity.
To clarify the role of adaptive immune development
to RSV, it is necessary to determine the timing of the
initial exposure to RSV and address especially the possibility of in utero exposure. Monocyte-derived macrophages obtained from cord blood have been reported
Adult and Neonate Responses to RSV
• JID 2003:188 (1 August) • 433
to produce IL-6 and TNF-a after exposure to live but not
inactivated virus [12], thus suggesting that the capacity to produce an innate immune response is present at birth. Recently,
proliferative responses and interferon (IFN)–g expression were
detected to RSV-infected, UV light–inactivated cells in onethird of infants exposed to an RSV season at the appropriate
time of gestation [13]. However, whether this response is specific for RSV was not clearly shown.
As an initial phase of studies establishing the course of development of the immune response to RSV in early life, we
report here a comparison of the responses to RSV stimulation
of mononuclear cells (MCs) obtained from the umbilical cord
blood of neonates and the peripheral blood of healthy adult
volunteers, both randomly selected. We reasoned that the immune response is likely to be different depending on the state
of the virus (inactivated or live) that initially induces immunity
and that information as to which is the more prominent form
for antigen presentation in vivo is not known. We thus examined responses to in vitro challenge of MCs with both inactivated and live virus. We asked whether MCs at birth exhibited adaptive and/or innate immunity to RSV by assessing
cytokine expression and proliferation. We sought to determine
how the neonate response (if it occurred) differed from the
response in adults (i.e., after many exposures to this antigen),
to identify any evidence for adaptive immunity to RSV developing before birth.
SUBJECTS, MATERIALS, AND METHODS
Cord and adult blood MC isolation. Cord blood was obtained from 26 randomly selected healthy neonates after scheduled caesarian section births by umbilical venipuncture in heparinized syringes after delivery of the placenta. Whole blood
was obtained from 20 randomly selected healthy human volunteers (ages 20–50) in heparinized Vacutainer tubes (Becton
Dickinson). For adult blood, 7 mL was layered onto 4 mL of
lymphocyte separation medium (LSM; ICN Biomedicals) and
centrifuged at 1000 g for 20 min to obtain the MCs. The band
of MCs was removed, washed in Hank balanced salt solution
with phenol red (Life Technologies, Gibco-BRL), and centrifuged; the pellet was resuspended in RPMI 1640 medium supplemented with l-glutamine, Pen/Strep, HEPES, and 5% fetal
calf serum (all from Life Technologies). The protocol for cord
blood MCs isolation was similar but included a modification
of the procedure of Landesburg et al. [14] to remove the contaminating nucleated red blood cells (nRBCs). After LSM separation, the interface layer containing cord blood MCs and
nRBCs was carefully aspirated, mixed with dextran (6% Dextran 500 in 0.9% NaCl; Sigma Chemical) in a ratio of 4 parts
interface layer to 1 part dextran, and incubated at 37C for 10
min. The mixture was then layered over LSM and centrifuged
434 • JID 2003:188 (1 August) • Krishnan et al.
as described above. If the interface layer had nRBC contamination, the dextran sedimentation step and LSM density centrifugation were repeated up to 2 times until no contamination
was evident. This procedure has been shown to be without
effect on the MC proportions [14]. Monocytes comprised 20%–
26% of the MC population for both adult and cord samples.
Informed consent was obtained from patients or their parents or guardians, and human experimentation guidelines of
the US Department of Health and Human Services were followed in the conduct of clinical research.
Viral cultures. RSV A2 strain (ATCC-VR 1302) was grown
in HEp-2 cells (ATCC) in Eagle minimal essential medium
supplemented with l-glutamine, Pen/Strep, and 5% fetal calf
serum (all from Life Technologies), as recommended by ATCC.
After 36 h of infection (indicative of 80%–90% cytopathic effect), HEp-2 cells were disrupted with sterile beads, and the
culture fluid was centrifuged at 500 g for 10 min and preserved
at ⫺70C. The RSV titer was determined in a standard plaqueforming unit assay [15] for the live and (subsequently) inactivated frozen aliquots. The titers of virus for the 2 aliquots
were 1 ⫻ 107 pfu/mL and 0.7 ⫻ 107 pfu/mL. The 0.7 ⫻ 107 pfu/
mL aliquot was inactivated by UV light irradiation (253.7 nm)
for 20 min at a distance of 10 cm from the UV light source.
Complete inactivation of virus was confirmed by the absence
of plaque formation of UV-treated RSV preparation (data not
shown). Uninfected HEp-2 cell culture fluid was processed similarly for use as controls.
MC culture and stimulation. MCs were counted and resuspended to a concentration of 2 ⫻ 10 6 cells/mL in RPMI 1640,
and 2-mL cultures were stimulated with either live RSV A2
strain 5 ⫻ 10 5 pfu or UV-inactivated RSV A2 strain 3.5 ⫻ 10 5
pfu (final concentrations). Initial dose-response data showed
that inactivated RSV induced the same cytokine responses over
the range 105–106 pfu. Unstimulated cells and cells stimulated
with 100 mL of uninfected HEp-2 cell supernatant cultures
served as negative controls, and cells stimulated with the mitogens concanavalin A (ConA; 10 mg/mL) and PMA (10 ng/
mL) (final concentration) served as positive controls.
Infectivity assessment. To verify that MCs were infected
with live RSV (as has been reported elsewhere [16]), adult and
cord blood MCs stimulated with live RSV were assessed at 48
h for viral proteins on the cell surface by flow cytometry; we
used anti-RSV protein antibody provided by Chemicon International. Each sample had appropriate isotype subtracted. Similar to the report of Midulla et al. [16], live virus yielded a
mean SD of 17.0% 11.4%, which was significantly greater
than the inactivated virus control (5.6% 5.1%; n p 6; P p
.048). Assessment of lymphocyte staining revealed no difference
between live RSV (7.0% 5.5%) and inactivated RSV (3.5%
3.6%) (data not shown).
Cytokine assays. Supernatants from the stimulated and
1 IL-4 (figure 1). Cytokines that were not detectable included
Figure 1. Adult mononuclear cell (MC) cytokine responses to inactivated respiratory syncytial virus (RSV), by day of incubation. Adult peripheral blood MCs (4 ⫻ 106 cells in 2-mL cultures) from 10 subjects were
stimulated in vitro with 3.5 ⫻ 105 pfu of UV-inactivated RSV (final concentration). Supernatants were collected on days 3 and 5 and assayed
for cytokines by ELISA. *P ! .05, vs. control (t test). IFN, interferon; IL,
interleukin; TNF, tumor necrosis factor.
control cell cultures were collected on days 1, 3, and 5. The
supernatants were assayed for these 9 cytokines: IFN-g, TNFa, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, and IL-13. They were
assessed by ELISA by means of commercially available kits
(R&D Systems) according to the manufacturer’s instructions.
The IL-12 kit measured the p70 subunit of IL-12. The sensitivities of the cytokine ELISAs are as follows: IL-2, !31.2 pg/
mL; IFN-g, !15.6 pg/mL; IL-4, !0.25 pg/mL; IL-13, !62.5 pg/
mL; IL-5, !7.8 pg/mL; IL-10, !7.8 pg/mL; IL-12, !0.31 pg/mL;
IL-6, !31.2 pg/mL; and TNF-a, !4.4 pg/mL.
Proliferation assay. Cord blood and peripheral blood MCs
(2–4 ⫻ 10 5 cells per well) were cultured in 96-well microtiter
plates. MCs were cultured with ConA (10 mg/mL) and PMA
(10 ng/mL), UV-inactivated RSV (1.75 and 3.5 ⫻ 10 5 pfu), and
uninfected HEp-2 supernatant controls for 5 days. Proliferation
assays were performed by means of commercially available kits
(Cell Proliferation ELISA Biotrak System, version 2, Amersham
Biosciences) according to manufacturer’s instructions.
Statistical analysis. Cytokine levels in supernatants of stimulated adult MCs have been previously found to be log normally
distributed [17]; thus, values here have been analyzed in this
manner. Differences from control were assessed by Student’s
paired t test, and differences between 2 groups were assessed by
unpaired t test. P ! .05 was considered to be significant.
RESULTS
Adult MC responses to RSV. Cytokine levels in supernatants
from adult blood MCs (n p 10) stimulated with inactivated
RSV on days 3 and 5 are shown in figure 1. The response on
day 5 included detectable levels of the following cytokines in
decreasing rank order: IL-6 1 IFN-g ∼ IL-2 ∼ TNF-a 1 IL-10
IL-5, IL-12, and IL-13. The variation among individual subjects
in the quantity of any one cytokine released (as indicated by
error bars) was much more limited than the variation in expression among the different cytokines, for which mean values
were distributed over 4 logs. Varying the time of incubation
revealed that significant maximal cytokine levels were reached
already at day 2 (data not shown) for all cytokines except for
IL-2 (reached at day 3) and IL-10 (reached only at day 5). RSV
concentrations of 1.75 and 3.5 ⫻ 10 5 pfu as the stimulant gave
comparable results, whereas stimulation with 3.5 ⫻ 10 4 pfu resulted in lower levels of cytokines (data not shown). Stability
of the responses over time within individuals was examined in
cells obtained from blood samples drawn 3 weeks apart, and
these second samples yielded no significant differences in mean
level in the stimulated cells for any cytokine (data not shown).
The response in adult blood MCs (n p 10) to live RSV provided a similar pattern of cytokine secretion on days 3 and 5
(figure 2). Compared with inactivated virus, no IL-4, less IL2, and more IFN-g were expressed, and IL-10 levels reached
significance at an earlier time point after stimulation (by day
3). The relative order of cytokine expression was still maintained at IL-6 1 IFN-g 1 IL-2 ∼ TNF-a 1 IL-10. Cytokines
that were not detectable included IL-4, IL-5, IL-12, and IL-13.
Neonate MC responses to RSV. Cytokine levels in supernatants from cord blood MCs (n p 14) stimulated with inactivated RSV on days 3 and 5 are shown in figure 3. The
responses on day 5 included detectable levels of the following
cytokines in decreasing rank order: IL-6 1 TNF-a 1 IL-10 1
IFN-g 1 IL-12 (figure 3). The responses were evident on day
1 (data not shown) and were essentially maintained on days 3
Figure 2. Adult mononuclear cell (MC) cytokine responses to live
respiratory syncytial virus (RSV), by day of incubation. Adult peripheral
blood MCs (4 ⫻ 106 cells in 2-mL cultures) from 10 subjects were stimulated in vitro with 5 ⫻ 105 pfu of live RSV (final concentration). Supernatants were collected on days 3 and 5 and assayed for cytokines by
ELISA. *P ! .05, vs. control (t test). IFN, interferon; IL, interleukin; TNF,
tumor necrosis factor.
Adult and Neonate Responses to RSV • JID 2003:188 (1 August) • 435
Figure 3. Neonate mononuclear cell (MC) cytokine responses to inactivated respiratory syncytial virus (RSV), by day of incubation. Cord
blood MCs (4 ⫻ 106 cells in 2-mL cultures) from 14 neonates were stimulated in vitro with 3.5 ⫻ 105 pfu of UV-inactivated RSV (final concentration). Supernatants were collected on days 3 and 5 and assayed for
cytokines by ELISA. *P ! .05, vs. control (t test). IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.
and 5 for all cytokines except IFN-g, which was undetectable
on day 1. No IL-2, IL-4, IL-5, or IL-13 was detectable at any
of the 3 time points after stimulation. The innate immune
cytokines IL-12, IL-6, and TNF-a were significantly greater in
neonates than in adults. The adaptive immune cytokines IL-2
and IL-4 that were expressed by adult cells were undetectable
in neonatal cells.
The response in cord blood MCs (n p 12) to live RSV
yielded several differences from that to inactivated RSV (figure
4). First, the response on days 1 (data not shown) and 3 consisted only of IL-6. On day 5, detectable levels of IL-6, IL-10,
and IFN-g were produced, although IL-6 and IL-10 levels were
significantly reduced, compared with the inactivated virus response. No IL-2, IL-4, IL-5, IL-13, TNF-a, or IL-12 was detectable at any of the 3 time points after stimulation. Compared
with inactivated virus, the overall cytokine expression was
greatly decreased in cord blood MCs in response to live RSV.
Adult and neonate MC responses to mitogen. Cytokine
levels in supernatants from the same adult (n p 10 ) and cord
(n p 14) blood MCs stimulated with mitogen ConA/PMA for
24 h are shown in figure 5. The cytokine responses in adult
cells to mitogen were greater than those induced by RSV stimulation for all cytokines and were expressed in a different rank
order: IL-2 ∼ IFN-g 1 TNF-a 1 IL-6 1 IL-10 1 IL-13 1 IL-5
1 IL-4. No IL-12 was detectable. Cord blood cell responses to
mitogen were also different from those to the virus. Cord blood
MCs produced greater amounts of IL-2, IFN-g, IL-4, IL-13,
and TNF-a to mitogen than to viral stimulation (either live or
inactivated). IL-6, IL-10, and IL-12 were produced in lesser
amounts in cord blood MCs stimulated with mitogen, com436 • JID 2003:188 (1 August) • Krishnan et al.
pared with inactivated virus, but were essentially equivalent
between mitogen and live viral stimulations. The rank order
for mitogen-stimulated cord blood MCs was IL-2 1 TNF-a 1
IL-6 1 IFN-g 1 IL-13 1 IL-10 1 IL-4. Levels of IL-5 and IL12 were undetectable. When comparing the cytokine response
in adult versus cord blood MCs to mitogen, the following observations were made: levels of IFN-g, IL-4, IL-5, IL-10, and
IL-13 were significantly greater in adults than in neonates,
whereas IL-2, IL-6, and TNF-a levels were comparable.
Adult and neonate proliferative responses to RSV. Proliferation to inactivated RSV was assessed in neonatal MCs (n p
5) and compared with peripheral blood MCs (n p 5) from
healthy adults (figure 6). Mitogen and HEp-2 supernatants were
used as stimulants for positive and negative controls, respectively.
No detectable proliferation to RSV was observed in neonatal cells,
although positive proliferative responses to ConA/PMA were observed in neonatal cells (∼3-fold increase over unstimulated controls). Fetal age was 122 weeks at the time of the RSV season
that occurred during the gestational period for each of these 5
infants. Positive proliferative responses to both RSV and ConA/
PMA were seen in adult cells (4–5- and 9–12-fold increase over
unstimulated controls, respectively).
DISCUSSION
Given that the respiratory illnesses to RSV are most devastating
in the first 6 months of life [3], this study sought to establish
the nature of the immune response to RSV at birth as an
indicator of the in vivo status of the immune cells before direct
exposure to RSV from the environment. Subsequent immune
responses of the infants at first encounter with RSV were likely
to contribute to the severity of illness through 3 possibilities:
Figure 4. Neonate mononuclear cell (MC) cytokine responses to live
respiratory syncytial virus (RSV), by day of incubation. Cord blood MCs
(4 ⫻ 106 cells in 2-mL cultures) from 12 neonates were stimulated in vitro
with 5 ⫻ 105 pfu of live RSV (final concentration). Supernatants were
collected on days 3 and 5 and assayed for cytokines by ELISA. *P !
.05, vs. control (t test). IFN, interferon; IL, interleukin; TNF, tumor necrosis
factor.
Figure 5. Comparison of adult and neonate cytokine responses to
mitogen. Adult (n p 10) and cord (n p 14) blood mononuclear cells (MCs)
(4 ⫻ 106 cells in 2-mL cultures) were stimulated in vitro with concanavalin
A (ConA; 10 mg/mL) and PMA (10 ng/mL) (final concentration). Supernatants were collected on day 1 and assayed for cytokines by ELISA.
*P ! .05, vs. control (t test); #P ! .05, adult response vs. neonate response
(t test). IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.
an overzealous innate immune response, an immune insufficient adaptive response, or an adaptive response polarized in
a Th2 direction. We also assessed the response to RSV in adult
peripheral blood cells to provide an indication of the nature
of a fully developed immune response to RSV in multiply exposed humans.
The present study was designed to (1) identify the developed
immune status to RSV in adults, (2) identify any indication of
previous exposure in infants, and (3) predict the likely immune
contribution to the severity of illness in newborns. Thus, cytokine response patterns were assigned to 3 categories: innate
(IL-6, TNF-a, IL-12, IFN-g, and IL-10), T helper type 1/T
cytotoxic type 1 (Th1/Tc1) (IL-2 and IFN-g), and T helper
type 2/T cytotoxic type 2 (Th2/Tc2) (IL-4, IL-5, IL-10, and IL13) immune responses. The immune response to inactivated
RSV in adult cells included production of both innate and
adaptive immune cytokines. The extremely low levels of IL-4,
the lack of IL-5 and IL-13, and the high levels of IFN-g and
IL-2 support categorizing the adaptive portion of the response
as predominantly a Th1/Tc1-type immune response. The significant, although low, expression of IL-4 suggests the possibility of a small Th2 or Tc2 response but also includes the
possibility of natural killer cell participation.
Live RSV stimulation in adults yielded responses that were
very similar to those observed with inactivated virus, as shown
by a similar rank order of cytokine expression. One difference
was the complete lack of detectable IL-4 with live RSV, essentially excluding even a small role for Tc2 or Th2 cells. Overall,
these data support a scenario in which both innate and adaptive
immunity are part of the immune response to RSV in adults,
with the latter being primarily a Th1/Tc1 type of response.
An emerging concept purports that pathogens that induce
Th1 responses are those that invoke large production of IL-12,
whereas production of IL-10 and/or IL-4 is inhibitory to Th1
and facilitatory for Th2 responses [18–20]. Studies with some
viruses, however, have demonstrated Th1 responses that are
independent of IL-12 [21]. As seen here, RSV can be placed
among the latter group of viruses. Future studies will determine
whether IFN-a/b and/or IL-18 [22] might be involved.
Stimulation of cord blood MCs resulted in several differences
from the adult response. The cord blood cells responded vigorously to inactivated RSV with a response that appears to be
only innate in that no IL-2, IL-4, IL-5, or IL-13 could be detected, whereas large amounts of IL-6, TNF-a, and IL-10 and
moderate amounts of IFN-g were produced. Although IFN-g
and IL-10 are not uniquely produced from innate immune cells,
the simultaneous lack of IL-2 production strongly suggests that
the IFN-g and IL-10 from the cord blood cells are part of the
innate immune response and likely have come from non–T cell
sources. Of interest, in the cord blood cells, a small but significant amount of IL-12 was induced, and thus we cannot
clearly conclude that IFN-g production is independent of IL12 in the cord blood responses. Except for IFN-g, cord blood
innate cytokines in response to inactivated RSV were produced
in amounts substantially greater than those from adult cells,
including IL-10, which was increased ∼3-fold, and TNF-a and
IL-6, which were increased 14–30 times.
The immune response to live RSV in the cord blood cells
Figure 6. Adult and neonate proliferative responses to respiratory
syncytial virus (RSV) and mitogen. Adult (n p 5) and neonate (n p 5)
blood mononuclear cells (MCs) (2–4 ⫻ 105 cells per well) were cultured
in vitro with concanavalin A (ConA; 10 mg/mL) and PMA (10 ng/mL), UVinactivated RSV (1.75 ⫻ 105 and 3.5 ⫻ 105 pfu) and uninfected HEp-2
supernatant controls for 5 days, and proliferation assays were performed.
*P ! .05, vs. unstimulated control (t test).
Adult and Neonate Responses to RSV • JID 2003:188 (1 August) • 437
was similar to inactivated RSV with respect to only being innate
in nature. However, several differences from the inactivated
virus response were noted. With live virus, TNF-a did not reach
detectability. The IFN-g and IL-10 responses were much slower
in developing, reaching significance (and levels equivalent to
those with inactivated virus) only at day 5. IL-6 was greatly
reduced (by 10-fold) in level, compared with inactivated virus.
As a result of these differences, the innate immune response
of neonates to live virus was not greater than that of the adult
MCs. Thus, the possibility of an overly vigorous innate response
is not supported, nor is a Th2-deviated response. On the basis
of these data, if the in vivo response is driven primarily by live
virus, an adaptive immune insufficiency remains the most likely
setting for severe responses to RSV seen in early life RSV exposure. If inactivated virus plays the predominant role, an
overly vigorous innate response may also participate. We cannot, of course, completely exclude any possibility of development of transient immune deviation toward Th2 at the time
of a subsequent RSV exposure. However, neither the newborn
nor the adult responses assessed provide any support for such
a response. Longitudinal follow-up studies in infants through
their early exposures to RSV are needed.
The greatly decreased response to live virus in cord blood
MCs suggests that live virus invokes the innate response via
less efficient pathways or actively suppresses innate cytokine
stimulation induced by inactivated virus. No similar suppressive effect on the innate immune response was significant in
adult cells. Although our results differ from those of Matsuda
et al. [12], who reported IL-6 and TNF-a from cord blood
monocyte–derived macrophages were produced by live but not
inactivated RSV, other studies have shown that live RSV can
induce suppressive factors, including IL-1 inhibitor activity [23]
and IFN-g [24], that decrease the overall immune response.
Although the possibility that RSV enhances host cell death
might be suggested for smaller responses, this notion is not in
keeping with the findings of Krilov et al. [25], who have shown
that RSV actually decreased cell death in adult and cord blood
MCs, compared with controls. We do not have an explanation
for the discrepancy with Matsuda et al. [12], other than the
possibility that macrophages alone respond differently than
monocytes in the context of cord blood.
To determine whether the lack of an adaptive response to
RSV in cord blood cells was indicative of a general incapacity
to produce T cell cytokines, mitogen stimulation was performed
and was found to induce cytokine production for all cytokines
except IL-12 (also absent in adult cells) and IL-5. Such a capacity of cord blood cells to produce many adaptive immune
cytokines thus strengthens our conclusion that the response to
RSV in cord blood cells is solely innate and signifies that exposure before birth appears not to have occurred.
Studies have shown that fetal MCs are capable of proliferating
438 • JID 2003:188 (1 August) • Krishnan et al.
in response to mitogenic and allergenic stimuli from ∼22 weeks’
gestation [26], and when we were doing these studies, Legg et
al. [13] reported that antenatal sensitization is associated with
proliferation and IFN-g production in those infants 122 weeks’
gestation during the RSV season. At that point, we determined
that most of the neonates in our study had not been older than
22 weeks during the RSV season that occurred during their
gestation. We then tested a group of cord blood MCs from
infants whose gestation had been 122 weeks during the RSV
season. Proliferative responses to RSV were not detected in any
of these neonatal MCs, whereas adult peripheral blood MCs
showed significant proliferative responses. Thus, we found no
evidence of adaptive immunity to RSV before birth. This difference from the results of Legg et al. [13] may be due to the
stimulus they used, which was not RSV but whole cells that
had been infected with RSV and then UV inactivated. Thus,
the response in that study may be due to one or more cellsurface antigens, rather than to RSV per se.
Establishing the basic nature of the infant immune system
status in response to RSV is also important in future development of vaccines against RSV disease. Attempts to enhance
human immunization against RSV have not met with success.
In the 1960s, a formalin-inactivated vaccine tested in infants
led to disease enhancement during subsequent RSV infection
[27], which may have been Th2 mediated. Thus, the need to
clarify the nature of immune response to RSV in the infant in
relation to host contribution to illness manifestations is further
emphasized.
In summary, although cord blood MCs were shown to be
capable of producing cytokines indicative of adaptive immunity
by mitogen stimulation, it is clear that stimulation with both
live and inactivated RSV leads to only innate responses, and,
thus, our results provide no evidence for in utero sensitization.
Adaptive, as well as innate, immune responses were evident
among the adult cells for both inactivated and live virus. Our
study provides the working hypothesis that humans are born
with a capacity to mount an innate immune response to RSV
and supports the concept that development of adaptive immunity occurs after birth and appears to be biased toward Th1/
Tc1 polarization.
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