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Regular Articles |
From the Departments of Pediatrics
* and Pathology,
University of Michigan Medical School, and the Department of Epidemiology,
University of Michigan
School of Public Health, Ann Arbor, Michigan; and the Department of
Immunology and the Walther Oncology
Center,
Indiana University School
of Medicine, Indianapolis, Indiana
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present studies examined the role of IL-12 in the immune response to RSV infection in a mouse model using C57BL/6 mice. IL-12 is important in the initial phase of bacterial, parasitic, and viral infections, and for the development of the T helper type 1 (Th1) response.13-17 Production of IL-12 is thought to favor differentiation and function of (Th1) T cells while inhibiting the differentiation of Th2 cells. In many viral infections, IL-12 promotes viral clearance and host recovery from infection.18-21 The role of IL-12 in RSV infection in relation to airway hyperreactivity and mucus production has not been specifically addressed, but other studies have speculated on its importance in acute bronchiolitis in humans.22 This study focused on the physiological and immune response to RSV infection in the C57BL/6 mouse strain, a strain that appears to have a favorable response to RSV.
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Materials and Methods |
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Specific pathogen-free C57BL/6 mice (H-2b) and DBA/J mice (H-2d) were purchased from Jackson Laboratories (Bar Harbor, ME) and housed in University of Michigan animal facilities under pathogen-free conditions. Stat-4 deficient mice were grown and maintained by Dr. Mark Kaplan at Indiana University.
Virus
RSV A2 was grown and harvested in Dr. Maassab’s lab at the University of Michigan School of Public Health. The virus had been through 48 passages in Hep-2 cells (human epidermoid carcinoma cells from the larynx). It was subsequently passed multiple times in Vero cells (green monkey kidney cells), MRC-5 cells (human lung cells), and Hep-2 cells for growth and amplification of the virus.
Infection
Pathogen-free mice were infected intratracheally with 3 x 105 plaque- forming units (PFU) in 30 µl media. Control mice received 30 µl of vehicle intratracheally. Mice were anesthetized with sodium pentobarbital (50 mg/kg) and ketamine (40 mg/kg) injected intraperitoneally. A tracheostomy was performed and RSV was injected directly into the trachea with a Hamilton syringe (Reno, NV). Following infection, the incision was closed with surgical staples and mice were allowed to recover. No mice died from this level of viral infection or from anesthesia.
Measurement of Airway Hyperreactivity
Airway hyperreactivity was measured using a Buxco mouse
plethysmograph (Buxco, Troy, NY) which is specifically designed for low
tidal volumes as previously described.23,24
Briefly, the
mouse to be tested was anesthetized as described above, and intubated
via cannulation of the trachea with an 18-gauge metal tube. The mouse
was placed on a Harvard pump ventilator (Harvard, Holliston, MA) (tidal
volume 0.4 ml, frequency 120 breaths/min, positive end-expiratory
pressure 2.5–3.0 cm H2O) and was ventilated for
5 minutes before the methacholine challenge. The plethysmograph was
sealed and readings monitored by computer. Since the box is a closed
system, a change in lung volume was represented by a change in box
pressure (Pbox) that was measured by a
differential transducer. The system was calibrated with a syringe that
delivered a known volume of 2 ml. A second transducer was used to
measure the pressure swings at the opening of the trachea tube
(Paw), referenced to the body box (ie, pleural
pressure), and to provide a measure of transpulmonary pressure
(Ptp) (Ptp =
Paw - Pbox). The tracheal
transducer was calibrated at a constant pressure of 20 cm
H2O. Resistance is calculated by the Buxco
software by dividing the change in pressure (Ptp)
by the change in flow (F) (
Ptp/F;
units = cmH2O/ml/s) at two time points from
the volume curve based on a percentage of the inspiratory volume. Once
baseline levels were stabilized and initial readings were taken, a
methacholine challenge was given intravenously via cannulation of one
of the tail veins with a 27-gauge needle. A dose-response curve (0.01
to 0.5 mg/kg) was performed and an optimal dose of 0.1 mg/kg of
methacholine was obtained. This dose was used throughout the rest of
the experiments in this study. After the methacholine challenge, the
response was monitored and the peak airway resistance was recorded as a
measure of airway hyperreactivity.
Lung Homogenates
The right upper lobe from each mouse was flash-frozen in liquid nitrogen and kept frozen at -80°C. Just before running the ELISA assays, the samples were weighed and homogenized in 1 ml of homogenization buffer containing 1 Complete tablet (Boehringer Mannheim, Germany) and 0.1% Triton-X in 50 ml of phosphate-buffered saline (PBS).
ELISAs
Cytokines were quantitated from homogenized PBS lung aqueous extracts using a double ligand ELISA system. The murine ELISAs have been developed in our laboratories using a previously described method.25 ELISAs were conducted as follows: flat-bottomed 96-well microtiter plates (Nunc Immuno-Plate I 96-F, Lincolnshire, IL) were coated with capture antibody diluted to 3.2 µg/ml in coating buffer (borate-buffered saline, pH 8.6) and incubated overnight at 4°C. Nonspecific binding sites were blocked with 2% bovine serum albumin (BSA) in PBS and incubated for 1 hour at 37°C. Plates were washed and specimens added in triplicate followed by incubation at 37°C and washing. Biotinylated detection antibody was added and the plates incubated at 37°C for 1 hour. Plates were washed and conjugated streptavidin-peroxidase was added, followed by washing and the addition of chromogen substrate (OPD). Finally plates were incubated at room temperature, the reaction terminated with 3 mol/L H2SO4 and read at 490 nm in an ELISA reader. The individual polypeptides were standardized to total protein (ng/µg total protein). Our ELISAs routinely detect protein at concentrations above 50 pg/ml. These ELISAs are specific and do not cross-react to any other chemokine or cytokine.
Production of Anti-IL-12 Antibodies
Rabbit anti-murine IL-12 antibodies were prepared by multiple-site
immunization of New Zealand White rabbits with recombinant murine IL-12
(R&D, Rochester, MN) in Complete Freund’s Adjuvant. Polyclonal
antibodies were titered by direct ELISA and specifically verified by
the failure to cross-react to mIL-3, mIL-1
, mTNF, mIL-4, hIL-12,
mIL-10, mMIP-1, IL-6, mJE, mMIP-1ß, hMCP-1, hIL-8, hRANTES,
hMIP-1, hTNF, and hMIP-1. The IgG portion of the serum was
purified over a protein A column and used in a sandwich ELISA. Whole
serum (0.5 ml) was used in vivo to block IL-12 during the
RSV infection.
In Vivo Neutralization of IL-12
Neutralization of IL-12 was carried out using a polyclonal rabbit
anti-murine IL-12 antibody developed in our laboratory as above. The
anti-IL-12 or control antibody was administered intraperitoneally 1
hour before infection and every 2 days postinfection until day 14. The
in vivo half-life of the antibody was 
30 hours.
BAL Microscopy
Bronchoaveolar lavage samples (BAL) samples were collected from each mouse just after airway hyperreactivity data were collected and after cervical dislocation. 1 ml of sterile 0.9 N saline was instilled intratracheally and was suctioned out after a few seconds. Samples were placed in Eppendorf tubes and centrifuged for 1500 rpm for 5 minutes. Supernatant was removed and cells were resuspended in PBS and cytospin-fixed and stained with Diff-Quick (Dade Behring Ag., Dudingen, Switzerland).
Morphometric Analysis of Peribronchial Eosinophil Accumulation
Mouse lungs from each time point were preserved in 1 ml of 4% paraformaldehyde. The fixed lungs were embedded in paraffin, and multiple 50-µm sections were differentially stained with hematoxylin-eosin for the identification of eosinophils. Eosinophils were quantified by counting 100 hpf/lung using multiple step sections of the lung. The eosinophils counted were only in the peribronchial region to assure enumeration of only those eosinophils within or immediately adjacent to the airway.
Goblet Cell Hypertrophy
Periodic acid-Schiff (PAS)-stained lung sections (prepared as above) were used to assess goblet cells in the small-to-moderate sized airways. Goblet cell hypertrophy was assessed qualitatively and quantitatively (counting numbers per airway).
Statistics
Numerical results were expressed as means ± SEM. Analysis of variance was used to determine the level of difference between groups. Pairs of groups were compared by unpaired two-tailed Student’s t-test. Analysis of variance was used to compare changes between different strains with same treatment and significance was determined with P values <0.05.
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Results |
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The hypothesis that host genetic difference affects response to
RSV was tested by comparing airway hyperreactivity (AHR) responses and
BALs in C57BL/6 (H-2b) mice to those in DBA/2
mice (H-2d) during primary RSV infection.
Pathophysiological response to RSV was determined by measuring change
in airway resistance after a methacholine challenge. Comparison of the
strains showed that the BALB/c and DBA/2 mice had significantly higher
airway hyperreactivity over almost the entire time course after RSV
exposure when compared to the C57BL/6 mice (Figure 1)
. Increased hyperreactivity was
apparent by day 8 in DBA/2 mice, continued to increase to day 12, and
then declined to baseline by later time points postinfection. The
C57BL/6 mice had minimal change in hyperreactivity over the same
postinfection time course. Interestingly, C57BL/6 and DBA-2 mice had
similar levels of virus on day 4 of infection with no evidence of
productive virus at the time of peak airway hyperreactivity at day 12
(data not shown).
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Immune Cytokine Profile in Mice with Mild Response to Primary RSV Infection
The pulmonary physiological response observed in the different
mouse strains may be due to the cytokine environment established at the
site of infection. In these studies, we were interested in examining
the basis for the mild pathophysiological response to RSV seen in the
C57Bl/6 mouse strain. Cytokine profiles were determined by running
ELISA assays on whole lung homogenates. Cytokines examined included
interferon-
, IL-4, IL-12, and IL-13. The production of IFN-
showed an increase early in the response while IL-12 production was
up-regulated at later time points; specifically, at day 8 through day
14 of infection (Figure 2)
. IL-13 and
IL-4 showed slight decreases over the same time period (data not
shown). Increased IL-12 levels appeared to correlate with mild response
to RSV infection suggesting that it may be an important cytokine
involved in the immune response to RSV in the C57BL/6 mice.
Accordingly, IL-12 production in lungs of infected DBA/2 mice did not
increase over the same period of infection over background levels,
ranging from 0.65 to 0.85 ng/lung on the various days (Figure 2)
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IL-12 is known to be an important immunoregulatory cytokine
favoring differentiation and function of Th1 cells while inhibiting
differentiation of Th2 cells.14,26
Although there is not a
great deal of information available on the importance of IL-12 in RSV
infection, several investigators have suggested that it may have an
immunomodulatory role.10,22,27
To confirm that IL-12 was
in fact the cytokine responsible for the mild response to RSV,
antibodies specific for IL-12 were used to neutralize the cytokine
in vivo. Neutralizing anti-IL-12 or control antibodies were
given intraperitoneally 1 hour before intratracheal RSV (3 x
105
PFU) and every other day thereafter until day
12 of infection. The mice were then examined for changes in airway
hyperreactivity at specific time points after infection (Figure 3)
. The mice treated with neutralizing
antibodies against IL-12 had a significant increase in airway
hyperreactivity, whereas untreated mice and mice treated with control
antibodies had minimal airway hyperreactivity, comparable to previous
experiments. IL-12 levels were reduced to baseline values in
neutralized animals (data not shown) while IL-13 levels showed a slow
increase with peak levels observed on day 12 (Figure 4)
. In control, uninfected mice,
anti-IL-12 had no effect on airway hyperreactivity (data not shown).
Microscopic examination of bronchoalveolar lavage fluid demonstrated
minimal mucus production in control serum-treated mice and untreated
mice, with a large amount of mucus production seen in the
anti-IL-12-treated animals at days 8 and 12 (Figure 5)
. Histologically, the
anti-IL-12-treated mice had increased inflammation (Figure 6)
with a significantly greater number of
pulmonary eosinophils (Figure 6)
. PAS staining revealed increased
numbers of mucus-producing goblet cells in anti-IL-12 treated mice
compared to RSV/control mice (Figure 7)
.
Thus, production of IL-12 during RSV infection appears to be an
important component in protecting the host from detrimental
pathophysiologic responses.
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Th1 and Th2 lymphocytes are thought to be activated by specific
cytokines resulting in a differentiated immune response. Binding of the
cytokine to its receptor results in activation of its associated signal
transduction pathways resulting in a unique response. The signal
transducer and activator of transcription (Stat) family of proteins are
involved in one of these intracellular signal transduction pathways.
IL-12 activates T helper cells via a Stat-4-induced signaling pathway.
To confirm the importance of IL-12 in abrogating severe airway
hyperreactivity responses in RSV infection, we performed similar RSV
infection experiments in Stat-4-deficient mice. Figure 8
shows that Stat-4-/- mice on a
C57BL/6 background had significantly higher airway hyperreactivity
responses compared to Stat 4+/+ mice. This pronounced effect was
present as soon as day 4 after infection and persisted at day 12 which
is similar to the effects observed in anti-IL-12-treated mice.
Histologically, Stat 4 -/- mice had increased numbers of eosinophils
and goblet cell hypertrophy when compared to RSV controls but not to
the extent observed in anti-IL-12-treated C57BL/6 mice (data not
shown).
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Discussion |
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.31,34,35
IL-12 has been shown to be
inversely related to length of respiratory failure in children with
RSV,36
and decreased levels of IL-12 have been shown in
humans with asthma and RSV bronchiolitis further supporting its role in
preventing severe symptoms in airway disease.22,37
Additional studies have demonstrated that exogenous IL-12 can attenuate
allergen-induced airway hyperreactivity and decreases pulmonary
eosinphilia in murine models.38,39
Altogether, IL-12
appears to be a protective factor against the development of airway
hyperreactivity and eosinophilia. The intensity and nature of the response to RSV may be determined by the local cellular and cytokine response. The specific cytokines produced at the site of infection may then direct the immune response with the recruitment and activation of specific immune cells.40 An alternative hypothesis might suggest that primary RSV infection drives an immature immune system toward a specific immune response that persists and acts in subsequent viral infections and on development of allergen sensitivity.41-43 These responses can be broadly categorized into Th1 or Th2-like immune responses. RSV infection is thought to induce a Th2-like response in infected individuals who exhibit severe forms of the disease.28,30 This immune response has also been observed in individuals with asthma and development of this Th2 type response may be the link between early RSV bronchiolitis and development of severe asthma. Prominent cytokines involved in Th2 responses are IL-4, -5, and -13. Mouse models of RSV infection in BALB/c (H-2d) mice after sensitization with viral antigens, have demonstrated increased mucus production and increased airway hyperreactivity with a preponderance of Th2 cytokines.44-46 IL-13 is a key cytokine involved in asthmatic responses and has been associated with increased mucus production and hyperreactivity.47,48 In contrast, the Th-1 cytokine IL-12 observed in the infected mice in these studies is associated with pathophysiologically milder response to the infection. The current study has suggested that IL-13 production is inversely related to IL-12 production, possibly indicating an IL-12/IL-13 balance during RSV responses. We are presently addressing these issues with additional in vitro and in vivo studies. In support of this concept, recent studies in our laboratory have shown that IL-13 is elevated in DBA/2 and BALB/c (H-2d) mice during RSV infection.49 These latter strains of mice have pronounced airway hyperreactivity and mucus production after RSV infection and low IL-12 levels. Neutralization of IL-13 in the DBA/J and BALB/c mice resulted in abrogation of airway hyperreactivity, increase in pulmonary IL-12 levels, and decreased evidence of mucus production suggesting that IL-13 is a key mediator of pulmonary pathology leading to morbidity in RSV infection. This correlates well with the rise in IL-13 before and during the most severe pathophysiologic responses, suggesting that IL-13 is inducing other mediators, such as mucus and eosinophil products, for the pulmonary dysfunction. The relationship between IL-13 and mucus production in models of RSV infection and asthma has been strongly established.48,49 The reduction in IL-13 levels before the reduction in airway physiology may indicate the prolonged detrimental effect of the IL-13-induced responses similar to those found in the previous study in RSV-infected DBA-2 mice.49
The mechanism of IL-12 function in diminishing severe pathology in RSV
and other viral diseases may be contentious. IL-12 binds to its
receptor and through a Stat-4 signal transduction pathway, acts to
increase IFN-.50-52
The over-expression of IFN in the
airway greatly diminishes the adverse affects observed in severe RSV
infections.53
Other studies have demonstrated that
monocyte-derived IL-12 production is inversely related to duration of
respiratory failure54
and IL-12-induced NK cells can
reduce lung eosinophilia and do not enhance disease
severity.55
Interestingly, administration of IL-12 during
RSV antigen vaccination reduced subsequent Th2 responses, but did not
improve vaccine-enhanced RSV lung illness.56
There are a
number of pathways in addition to IFN production that IL-12 and
Stat-4 may be affecting during the pulmonary responses. IL-12 may be
acting directly on recruited leukocytes to enhance viral clearance by
activating effector cell populations.31,34,35,41
This
could be occurring via the recruitment and activation of NK cells
and/or CD8+ cytotoxic T cells. Another pathway may function via the
regulation of other cytokines by IL-12, such as IL-13, and maintaining
an effective anti-viral response in the lungs of infected animals.
Whatever the specific mechanism, IL-12 appears to modulate adverse
responses, including mucus production, eosinophil accumulation and
airway hyperreactivity. Thus, administration of exogenous IL-12 may
have therapeutic applications, especially in those individuals who have
low endogenous levels.
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Footnotes |
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Supported by National Institutes of Health grants HL59178, AI36302, HL31963.
Accepted for publication April 26, 2001.
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