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Release in the Lung But Not for the Differentiation of Viral-Reactive Th1-Type Lymphocytes1
Department of Pathology and Molecular Medicine and Division of Infectious Diseases, Centre for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in the lung was
similar in wt and IL-12-/- mice during pulmonary viral
infection. Upon Ag stimulation in vitro, lymphocytes from draining
lymph nodes or spleen of infected IL-12-/- mice released
large amounts of IFN-, but not IL-4, which were comparable to those
released by wt lymphocytes. Furthermore, a predominantly IgG2a response
to adenoviral infection was unimpaired in IL-12-/- mice.
These significant anti-adenoviral Th1-type responses in
IL-12-/- mice led to an efficient clearance of
virus-infected cells in the lung. Whether IL-18 was involved in
IL-12-independent anti-adenoviral immune responses was
investigated. Abrogation of endogenous IL-18 by an Ab resulted in
diminished IFN- release and lymphocytic infiltrate in the lung
during adenoviral infection. Nevertheless, the development of
lymphocytes of the Th1 phenotype was unimpaired in the absence of both
IL-12 and IL-18. In contrast to their intact ability to mount
Th1-type responses to viral infection,
IL-12-/- mice suffered impaired Th1-type immune responses
to pulmonary mycobacterial infection. Our findings suggest that IL-12,
although induced, is not required for Th1-type responses to respiratory
viral infection, in contrast to mycobacterial infection. IL-18 is
required for the optimal release of IFN- in the lung during viral
infection, but is not required for the generation of virus-reactive
Th1-type lymphocytes. Th1 differentiation during respiratory adenoviral
infection may involve molecules different from IL-12 or
IL-18. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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release
from Th1 and NK cells (1, 2). In comparison, IL-18, a
recently identified IFN--inducing factor, may compensate for at
least some of the functional activities of IL-12 in certain models of
intracellular infections (3, 4, 5). However, we have recently
provided experimental evidence that the function of IL-12 cannot be
compensated for by any other cytokine, including IL-18, during host Th1
immune responses against pulmonary mycobacterial infection
(6). We have further demonstrated that in addition to its
indispensable effect on T cells, IL-12, but not IL-18, is critically
required for IFN- release by macrophages during pulmonary
mycobacterial infection (7). These findings have thus
established a unique role for IL-12 in host defense against
intracellular bacterial infection in the respiratory system.
In addition to intracellular bacterial infection, the respiratory tract
is the most common mucosal site for viral infections. Unfortunately,
relatively little is known about the role of endogenous IL-12 in host
resistance to viral infections, particularly those occurring in the
respiratory tract, despite the fact that exogenously administered
rIL-12 was shown to enhance immune protection to a number of viral
infections (2). While it is scientifically plausible to
speculate about the importance of endogenous IL-12 in antiviral immune
responses in the lung, recent evidence has suggested a far more
complicated picture. Indeed, IL-12 seems induced during most, if not
all, viral infections (2, 8) and was shown to be required
for IFN- release and type 1 immune protection during viral
infections by murine CMV (9) or herpes simplex virus 1
(10). However, IL-12 has also been shown to be required
only for early, but not later, IFN- responses upon primary influenza
virus infection (11). Furthermore, following i.v. or i.p.
infection with lymphocytic choriomeningitis virus (12) or
mouse hepatitis virus (13), a Th1-type immune response,
characterized by both T cell IFN- release and IgG2a production,
remained intact in the absence of IL-12. The mechanisms underlying such
IL-12-independent Th1 immune responses have remained unclear.
Apparently, given some unique aspects of antiviral immune responses,
there is a need to further understand the role of IL-12 and to identify
additional modulatory molecules in antiviral Th1 immune responses.
Our current study aimed to investigate the role of IL-12 in host immune responses to both primary and secondary respiratory adenoviral infections in wild-type (wt)3 and IL-12-deficient mice. Both in vivo and ex vivo studies were designed to examine 1) cellular and cytokine responses in the lung postadenoviral infection, 2) Th phenotypes of lymphocytes isolated from lung draining lymph nodes and spleen, 3) antiviral humoral immune responses, 4) the differences in immune responses to respiratory adenoviral and mycobacterial infections, and 5) if IL-12 was not required, the role of IL-18 in anti-adenoviral Th1-type immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both male and female mice at the age of 10–14 wk were used. The generation of IL-12p40-/- mice (C57BL/6 background) has previously been described, and these mice demonstrate a normal development of immune systems (14). These mice were bred in our central animal facility. C57BL/6 mice (Harlan, Indianapolis, IN) were used as wild-type controls for IL-12 p40-/- mice. All mice were housed in autoclaved cages with autoclaved bedding, food, water, and microfilter lids in a pathogen-free level B facility. All experiments were performed in accordance with the guidelines of the animal research ethics board of McMaster University.
Infectious agents
The wt type 5 adenovirus was amplified, purified, and titrated in our laboratory as previously described (15). In some experiments a replication-deficient adenovirus was also used. This virus has its E1 and E3 genomic regions partially deleted but remains fully infectious. For transgene expression experiments a recombinant replication-deficient adenoviral vector expressing murine eotaxin was used. Live Mycobacterium bovis Calmette-Guérin bacillus (BCG) was obtained from Connaught Laboratories (North York, Canada). It was grown in Middlebrook 7H9 broth (Difco, Detroit, MI) supplemented with Middlebrook OADC enrichment (Life Technologies, Gaithersburg, MD), 0.002% glycerol, and 0.05% Tween-80.
Establishment of pulmonary adenoviral or mycobacterial infection
Pulmonary adenoviral infection was established by intranasal (i.n.) administration of 0.5 x 109 PFU of wt adenovirus in a total volume of 30 µl/mouse following a procedure previously described (6). Pulmonary mycobacterial infection was established by intratracheal instillation of live BCG at a dose of 0.5 x 106 CFU in a total volume of 40 µl/mouse as previously described (6).
Abrogation of endogenous IL-18 in IL-12-/- mice
In some experiments, 10 and 20 µg/mouse of a rat anti-murine IL-18 Ab (R&D Systems, Minneapolis, MN) were administered, i.n. and i.p., respectively, to IL-12-/- mice at the time of adenoviral infection. These mice were further injected i.p. with 20 µg/mouse of anti-IL-18 on days 3 and 5 postinfection and were then sacrificed, and samples were collected on day 7. As control, a group of IL-12-/- mice was treated in the same way with purified normal rat IgG (Sigma, St. Louis, MO); 0.3–1 µg of this anti-murine IL-18 Ab has a capacity to neutralize 15 ng of IL-18.
Lymphocyte isolation from the mediastinal lymph nodes (MLN)/spleens and Ag stimulation assay
Anaesthetized mice were bled retro-orbitally, and peripheral blood samples were processed for serum collection. The spleen was removed and saved in PBS on ice. The thoracic cavity was opened, and MLN were removed before removing the lung for lavage. MLN pooled from several mice of the same group were ground between two microscopic slides, and cells were released into culture medium containing 10% FCS and 1% P/S. The resultant MLN-derived cell suspension was filtered through two layers of nylon membrane (55 µm). Spleens pooled from several mice of the same group were meshed through a metal screen, and cells were collected in culture medium containing 10% FCS and 1% penicillin/streptomycin. RBC were lysed with sterile water. This method of RBC lysis does not affect assay results. Cells were allowed to settle on ice for 15 min, and the top cell suspension was removed from cellular debris. Both MLN and spleen cell suspensions were centrifuged and resuspended in culture medium.
One half million of MLN cells or splenocytes were plated into each well of 96-well plates in a final volume of 300 µl. Each condition was set up in triplicate wells. Cells were cultured for 72 h in the absence or the presence of UV-inactivated wt adenovirus (25 PFU/cell) or mycobacterial purified protein derivative (PPD) Ags (M. tuberculosis-derived; 10 µg/ml). Culture supernatants were cleared and stored at -70°C until cytokine assay.
Bronchoalveolar lavage (BAL), cytologic analysis, and macrophage purification
BAL was conducted by following a well-described standard
procedure previously described (6, 7, 16). Briefly, after
collecting the MLN and spleens, the lung was removed from the thoracic
cavity with the heart and a portion of the trachea intact. To collect
BAL fluid, a polyethylene tube (Becton Dickinson, Sparks, MD) was used
to cannulate the trachea. Lungs were lavaged twice with PBS (0.25 and
0.20 ml), and 
0.4 ml of BAL fluid was consistently recovered. Such
techniques ensure gentle noninvasive handling and even lavage of both
lungs (6, 7, 16). BAL samples were spun at 4000 rpm for 2
min at 4°C, and supernatants were removed and stored at -20°C for
cytokine or Ig assays. Cell pellets were resuspended in 500–800 µl
of PBS, and total cells were determined on a hemocytometer. Cytospins
were made in a cytospin machine (Shandon, Pittsburgh, PA) and stained
using Diff-Quick stain (Baxter, McGraw Park, IL) for differential cell
counting. Routinely, 300–500 cells/cytospin were differentiated in a
random fashion.
For experiments involving macrophage cultures, macrophages were isolated from BAL fluids of naive or virus-infected mice according to a procedure previously described (7). Briefly, cells purified from BAL were counted and cultured in 96-well plates at a density of 0.1 x 106 cells/well in 300 µl of culture medium for 3 days under different conditions. Supernatants were stored at -20°C until cytokine measurement.
Measurement of anti-adenoviral Abs by ELISA
ELISA for measuring anti-adenoviral total IgG, IgG2a, and IgA levels in the serum or BAL fluids was conducted as previously described (17). Briefly, 96-well plates were precoated with 5 µg/well of protein extract from wt adenovirus-infected HeLa cells overnight at 4°C. After washing, each well was treated with 50 µl of Tris-Tween reagent diluent (0.24% Tris-HCl, 0.876% NaCl, 0.037% KCl, 0.05% Tween-20, 0.05% BSA, 0.02% NaN3, and 0.01% bromocresol purple, pH 7.4) for 30 min at 37°C. Diluted serum or BAL samples were then incubated in Ag-coated wells for 60 min at 37°C. After washing, the wells were incubated with 50 µl/well of a rat-anti murine IgG, IgG2a, or IgA biotinylated Ab (Sigma) 1/10,000 diluted with Tris-Tween buffer for 30 min at 37°C, and then incubated with 50 µl/well of 1/2000 diluted extravidin-peroxidase conjugate (Sigma) for 15 min at 37°C. The color reaction was developed in the presence of substrate tetramethylbenzidine (Sigma) for 20 min, and the OD was measured at 450 nm. The content of specific anti-adenovirus total IgG, IgA, or IgG2a was expressed as titer determined by using a formula:1/dilution factor ÷ optical density x 0.05. This formula represents an improved way to determine end-point titers, which minimizes the variation between assays compared with the conventional way of taking the highest reciprocal dilution as the end-point titer (17). The OD reading that was immediately >2-fold the average background reading was used for calculation.
Measurement of cytokines in BAL and lymphocyte culture supernatants
Cytokines were measured in BAL and lymphocyte culture
supernatants by specific ELISA. All ELISA kits were purchased from
either R&D Systems (IFN- and IL-4) or BioSource (Montreal, Canada;
IL-12). The sensitivity of detection for all of these ELISA kits was
5 pg/ml.
Examination of adenovirus-mediated transgene expression in the lung by RT-PCR
A dose of 0.5 x 109 PFU of a replication-deficient adenovirus that has been engineered to express the murine eotaxin transgene was i.n. delivered into the lung of C57BL/6 or IL-12-/- mice. On days 3, 7, 12, and 21 postviral infection, lungs were snap-frozen in liquid nitrogen and homogenized for total RNA extraction. RT-PCR was then performed with total RNA samples to evaluate transgene mRNA expression by using primers specific for murine eotaxin, following the protocol previously described (18). The sense and antisense primers were 5'-GTCTCTAACGAGTTCTCCTTCAAG-3' and 5'-TTCAGAGGGCTATACTGCCTTCCA-3', respectively. Amplified RT-PCR products were then electrophoresed and verified for size in a 1% agarose gel. The correct size of the transgene-derived PCR product should be 813 bp. The same amounts of reverse transcribed products were also subjected to PCR amplification using primers specific for rodent GAPDH as a housekeeping control to verify amounts of total RNA used for RT-PCR (18). The correct size of GAPDH PCR product was expected to be 555 bp.
Processing and histologic assessment of lung tissues
Lungs were fixed in 10% formalin by perfusion. Both left and right lungs were sectioned from top to bottom, resulting in four or five cross-sectional pieces of tissue from each side. Tissues were then embedded in paraffin, cut into 4- to 5-µm sections, and stained with hematoxylin and eosin.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine whether adenoviral infection in the lung will induce
IL-12 production, we measured IL-12 content in BAL fluids from the
lungs of C57BL/6 mice infected with wt adenovirus. We focussed on the
examination of samples collected on day 7 postprimary infection and day
3 postsecondary infection. We have previously shown that the primary
immune response to lung adenoviral infection peaks around day 7
(15). There were significant amounts of IL-12
released into the lung by day 7 postprimary viral infection and by day
3 (day 13 postprimary infection) postsecondary infection (Fig. 1
). Of interest, little IL-12 was
released in the lung of C57BL/6 mice infected with a
replication-deficient adenovirus (Fig. 1), suggesting that viral
replication and/or sufficient viral Ags are required for the optimal
release of IL-12 in the lung. As expected, there was no measurable
IL-12 in samples from the lungs of IL-12-/-
mice (not shown).
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Because IL-12 was induced by wt adenoviral infection in the lung,
we examined whether IL-12 was required for primary or secondary
anti-adenoviral immune-inflammatory responses in the tissue. Day 7
postprimary infection was chosen since we have previously demonstrated
that cellular, particularly lymphocytic, responses, peak around this
time point and markedly decline between days 10–12 following infection
with replicable adenovirus in the lung (15). The number of
leukocytes, particularly macrophages and lymphocytes, was markedly
increased in the lungs of C57BL/6 mice (a normal PBS-treated mouse lung
contains only 1 x 105 of macrophages
(16); Fig. 2a).
In comparison, the number of these leukocyte subsets was similarly
increased in the lungs of IL-12-/- mice and
slightly smaller numbers were not statistically significantly different
from those in wt control mice. Such similar cellular responses in the
lungs of both C57BL/6 and IL-12-/- mice during
primary adenoviral infection were also observed earlier (day 4;
macrophages, 18.6 x 104 vs 19.9 x
104; lymphocytes, 1.3 x
104 vs 2.9 x 104 in
C57BL/6 and IL-12-/- mice, respectively).
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b). Of interest, the neutrophilic
response in the lungs of IL-12-/- mice was
significantly smaller than that in C57BL/6 mice during the secondary
immune-inflammatory response (Fig. 2b).
The similarity in cellular responses to wt adenoviral infection between
C57BL/6 and IL-12-/- mice was also
histopathologically observed on day 7 postprimary infection (Fig. 3, a and b) or day
3 postsecondary infection (not shown). An intense peribronchial and
perivascular inflammatory infiltrate was observed in the lungs of both
C57BL/6 and IL-12-/- mice and was composed
primarily of mononuclear cells, with some neutrophils. Patches of such
responses were also seen in lung parenchyma. Importantly, we observed a
similar extent of bronchial epithelial injury in the lungs of both
C57BL/6 and IL-12-/- mice (not shown).
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responses in the lungs of C57BL/6 and
IL-12-/- mice postadenoviral infection
IL-12 is critically required for the optimal release of type 1
cytokine IFN- in vivo in a number of models of intracellular
infection (4, 6, 9, 10, 11, 14, 19, 20, 21). We investigated
whether IL-12 was also critically required for IFN- production
during pulmonary adenoviral infection. To our surprise, similarly
increased amounts of IFN- protein were detected in BAL from both
C57BL/6 and IL-12-/- mice 7 days postprimary
infection (Fig. 4). The level of IFN-
in the lung induced by reinfection with adenovirus (day 10 postprimary
infection) was also significant, but lower in
IL-12-/- mice than in their counterpart
controls (Fig. 4).
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Th phenotype of lymphocytes from MLN and spleens of C57BL/6 and IL-12-/- mice postadenoviral infection
Having examined the level of IFN- release in the lung during
adenoviral infection, we set out to examine the nature of lymphocytes
isolated from both local and peripheral lymphoid tissues by analyzing
cytokine profiles stimulated by adenoviral Ags. To this end, both
C57BL/6 and IL-12-/- mice were infected via the
airway with wt adenovirus and sacrificed on day 7. Some mice were
subjected to a secondary exposure to the virus on day 10 and sacrificed
on day 13. Both MLN and spleen were removed, total mononuclear cells
were isolated and stimulated in vitro with UV-inactivated adenovirus,
and the levels of Ag-specific Th1-type IFN- or Th2-type IL-4 recall
responses were compared. On day 7 postprimary adenoviral infection,
lymph node-derived lymphocytes from both C57BL/6 and
IL-12-/- mice responded significantly to
adenoviral stimulation by releasing IFN-, although cells from
IL-12-/- mice released less IFN- (Fig. 5a). Such low level IFN-
release by lymphocytes from IL-12-/- mice may
provide a potential mechanism for the trend for a lower level of
IFN- in the lungs of these mice (Fig. 4). Splenocytes from both
C57BL/6 and IL-12-/- mice released similar
amounts of IFN- upon challenge with adenoviral Ags (Fig. 5b). The specificity of such Ag recall responses was shown
by the fact that these cells responded very little to challenge with an
irrelevant mycobacterial Ag PPD (not shown). In contrast, only a
minimum of IL-4 was released by cells from both C57BL/6 and
IL-12-/- mice (Fig. 5c), suggesting
that the phenotype of lymphocytes in IL-12-/-
mice remained Th1.
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responses of lymphocytes from lymphoid
tissues of mice after two repeated pulmonary adenoviral infections. Of
note, lymph node- or spleen-derived lymphocytes from both C57BL/6 and
IL-12-/- released remarkable amounts of IFN-
upon recall challenge with adenoviral Ags (Fig. 6, a and b), and
the differences between C57BL/6 and IL-12-/-
mice were not significantly different. In contrast, little IL-4 was
released under these conditions (Fig. 6c). Overall, the
results from these experiments suggest that IL-12 is not critically
required for the development and maintenance of the Th1 phenotype of
immune cells in lymphoid tissues.
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responses in macrophages
We have observed that lymphocytes from both infected C57BL/6 and
IL-12-/- mice released comparable amounts of
IFN- upon adenoviral Ag recall challenge in vitro, and yet the level
of this cytokine in the lungs of IL-12-/- mice
was significantly lower than that in C57BL/6 mice postsecondary
adenoviral infection. Because adenovirus infects not only airway
epithelial cells but also alveolar macrophages in the lung
(15), and we have recently reported IL-12-dependent
IFN- release by lung macrophages during mycobacterial infection
(7), we investigated whether lung macrophages could
release IFN- in response to adenoviral infection and whether this
IFN- response was IL-12 dependent. By using alveolar macrophages
from naive IL-12-/- mice, we found that
adenoviral infection per se could not release IFN-, whereas
exogenously added IL-12 alone released only a very small amount of
IFN- (Fig. 7a). However, significant amounts of IFN-
were released by macrophages stimulated with both adenovirus and IL-12
(Fig. 7a). Furthermore, we compared macrophages from the
lungs of C57BL/6 and IL-12-/- mice adenovirally
infected for 10 days and reinfected for 3 days. We found that
macrophages from infected C57BL/6 mice spontaneously released more
IFN- than those from IL-12-/- mice (Fig. 7b). Similarly, upon
stimulation with a macrophage agonist (LPS), cells from infected
C57BL/6 mice released at least 10 times more IFN-. These findings
suggest that some of the IFN- we measured in the lung may have
derived from activated macrophages and that this IFN- response in
macrophages, unlike that in lymphocytes, is critically dependent on
IL-12.
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Having demonstrated that the Th1-type cellular immune response to
pulmonary adenoviral infection is in large part independent of IL-12,
we investigated whether there was an impairment in humoral immune
responses. IL-12 has been shown to be capable of enhancing Ig secretion
by B cells (22, 23). We have recently shown that
anti-adenoviral IgG arose as a major isotype of Igs most
significantly in the peripheral blood in rodents postpulmonary
adenoviral infection (17). We thus examined the titer of
total anti-adenoviral IgG in the peripheral blood. Very little
anti-adenoviral IgG was measured on day 7 postprimary adenoviral
infection, but a markedly increased level was observed in both C57BL/6
and IL-12-/- mice by day 21 (Fig. 8a). The level of such IgG was
very similar between C57BL/6 and IL-12-/- mice.
Postsecondary adenoviral infection, the level of anti-adenoviral
IgG was small by day 13, but it was markedly increased by day 21 (11
days postsecondary infection), which doubled the amount measured at the
same time postprimary adenoviral infection. Of importance, there was no
significant difference between C57BL/6 and
IL-12-/- mice (Fig. 8b). To examine
whether such humoral immune responses to adenoviral infection in
IL-12-/- mice remained a Th1 phenotype, we
further compared the level of IgG2a isotype in sera between C57BL/6 and
IL-12-/- mice after secondary adenoviral
infection (days 3 and 11). We found that the levels of
anti-adenoviral IgG2a were similar to those of total IgG in both
C57BL/6 and IL-12-/- mice, suggesting that the
majority of total anti-adenoviral IgG were IgG2a in nature (Table I). Of importance, the levels of
anti-adenoviral IgG2a in IL-12-/- mice were
not decreased compared with those in C57BL/6 counterparts. We also
measured the level of anti-adenoviral IgA in BAL fluids collected
11 days postsecondary adenoviral infection (we previously found that
anti-adenoviral IgA was compartmentalized largely to the lung
(17)). The levels of IgA in the lungs of both C57BL/6 and
IL-12-/- were similarly low (average IgA
titers, 110 and 72 in the lungs of C57BL/6 and
IL-12-/- mice, respectively). These findings
suggest that IL-12 was not required for the generation of
anti-adenoviral Igs during the primary or secondary adenoviral
infection.
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It is known that the elimination of adenoviral-infected cells in
the lung is attributed to cytotoxic activities of immune cells. We
postulated that the rate of clearance of adenoviral infected cells in
the lung would not be markedly affected by the lack of IL-12, since we
have demonstrated similar levels of Th1-type cellular and
cytokine responses in both C57BL/6 and IL-12-/-
mice. To this end, we administered to C57BL/6 and
IL-12-/- mice a recombinant adenoviral vector
expressing the transgene coding for murine eotaxin, a chemokine with
very restricted effects on eosinophils (24), and
investigated the extent of elimination of adenovirus-infected cells in
the lung by examining the kinetic expression of adenovirus-mediated
eotaxin transgene mRNA by PCR. We found that eotaxin mRNA was similarly
expressed in the lungs of C57BL/6 and IL-12-/-
mice (Fig. 9). The level of eotaxin
transgene expression started to decline by day 12 and similarly
markedly decreased by day 21, suggesting that the host immune response
in both C57BL/6 and IL-12-/- was similarly
effective in controlling adenoviral infection.
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Because IL-18 shares many functional similarities with IL-12
(3), we investigated the role of this cytokine in
IL-12-independent Th1-type responses to pulmonary adenoviral
infection. To this end, an anti-IL-18 Ab was administered three
times from the time of pulmonary infection to
IL-12-/- mice, and the level of IFN- in the
lung and the phenotypic response of lymphocytes were analyzed 7 days
postprimary adenoviral infection. The level of IFN- in the lungs of
IL-12-/- mice receiving the control Ab was
similar to that in IL-12-/- mice without Ab
treatment (Figs. 4 and 10a).
To our surprise, however, abrogation of IL-18 resulted in a dramatic
reduction in the level of IFN- in the lung, which was 10 times lower
than that in the control Ab-treated group (Fig. 10a). Upon
examination of cellular responses in the lung, while the number of
macrophages remained similarly elevated in both control and
anti-IL-18 Ab-treated mice, the number of lymphocytes decreased by
50% in anti-IL-18-treated mice (Table II). We next examined whether there was
an impairment in the generation of virus-reactive Th1 lymphocytes in
local and systemic lymphoid tissues in anti-IL-18-treated
IL-12-/- mice. Of interest, lymphocytes
isolated from MLN or spleen of control or anti-IL-18-treated mice
released similar amounts of Th1-type cytokine IFN- in response to
adenoviral recall stimulation (Fig. 10b). In a separate
experiment, even when cultured in the presence of anti-IL-18 Abs,
the ability of these cells to release IFN- in response to adenoviral
Ag stimulation was not weakened (not shown). These findings suggest
that Th1 differentiation was not impaired in mice lacking both IL-12
and IL-18.
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Because the mycobacterium represents a classic intracellular
bacterial pathogen, we were interested to compare the role of IL-12 in
the development of Th1 immune responses against pulmonary mycobacterial
infection with its role in anti-adenoviral Th1 responses. To this
end, mice were infected via the airway with mycobacterial BCG. In this
model of pulmonary mycobacterial infection that we have previously
established (6), cellular responses were not markedly
induced until 3 wk postinfection. Indeed, on day 27 postinfection, the
numbers of neutrophils, lymphocytes, and macrophages increased
minimally in the lungs of IL-12-/- mice
compared with those in control counterparts (not shown)
(6). These IL-12-dependent cellular responses to
mycobacterial infection contrast with IL-12-independent responses to
adenoviral infection (Fig. 2). We then measured the levels of
IFN- in BAL fluids collected from days 14, 27, and 37. The level of
IFN- markedly increased by day 14, peaked by day 27, and
significantly decreased by day 37 in the lungs of C57BL/6 mice
postmycobacterial infection (Fig. 11)
in contrast to the small amount of IFN- detected in the lungs of
IL-12-/- mice. This again contrasts sharply
with a marked IFN- response in the lungs of
IL-12-/- mice during adenoviral infection
(Fig. 4).
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response in vitro. Lymphocytes from MLN or
spleens of infected C57BL/6 mice released large quantities of IFN-
upon Ag recall challenge (Fig. 12,
a and b). However, inconsistent with the lack of
IFN- protein in the lung, there was an apparently impaired IFN-
response to mycobacterial Ag challenge by lymphocytes from either local
draining lymph nodes or spleen of infected
IL-12-/- mice (Fig. 12, a and
b). These findings indicate that the generation of
mycobacterial reactive Th1 lymphocytes is critically dependent upon the
presence of IL-12. This is in stark contrast with the unimpaired
ability to generate adenovirus-reactive Th1 cells of
IL-12-/- mice (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and the optimal lymphocytic infiltration in the
lung. However, IL-18 is not required for the differentiation of
adenoviral-reactive Th1-type lymphocytes. The IL-12-independent
Th1-type immune response to respiratory adenoviral infection sharply
contrasts with the critical requirement of IL-12 for Th1-type responses
against respiratory mycobacterial infection. Our findings provide the
rationale for using IL-18, rather than IL-12, as an immune adjuvant in
vaccines against mucosal viral infections and warrant further studies
to identify key cytokines involved in Th1 differentiation during
respiratory viral infection.
IL-12 has been well recognized for its critical role in the
differentiation of Th1 phenotype, the release of type 1 cytokine
IFN- from NK and T cells (1, 2, 3) as well as macrophages
(7, 25), and the activation of T cytotoxic activities
(1, 2, 3) during host responses to a number of infectious
diseases caused by intracellular bacteria and parasites. IL-12 was also
shown to be capable of activating functional activities of B
lymphocytes (22, 23). However, whether IL-12 is as
important in host antiviral immune responses has remained controversial
(8), and little is known about its role or the mechanisms
alternative to IL-12 in host Th1 responses against viral infection at
the respiratory tract, the commonest site of viral infection. It has
apparently become difficult to make extrapolation from limited
published studies primarily involving the models of systemic viral
infection or at tissue sites other than the respiratory tract, because
the emerging idea is that the relative contribution of IL-12 to host
antiviral responses may depend on the site of infection and the type of
virus (8, 9, 10, 11, 12, 13).
Our data demonstrate that in the absence of IL-12, the host is able to
mount an intense tissue immune-inflammatory response to respiratory
viral infection both qualitatively and quantitatively similar to that
in wt mice. The only significant difference at the cellular level we
observed between wt and
IL-12-/- mice is in the
number of neutrophils. The smaller number of neutrophils in the lungs
of IL-12-/- suggests that IL-12 is involved in
the optimal generation of signals for neutrophilic accumulation in the
lung during viral infection. We have also provided evidence that the
phenotype of lymphocytes from either the local draining lymph nodes or
the peripheral lymphoid tissue in IL-12-/-
remains Th1, because they released large amounts of IFN-, but little
IL-4, upon specific viral Ag recall stimulation. This was found to be
true of lymphocytes isolated from IL-12-/- mice
either postprimary or postsecondary adenoviral infection. This
near-normal IFN- response by lymphocytes in
IL-12-/- mice does not, however, explain why
there was a significantly lower level of IFN- detected in BAL fluids
of IL-12-/- mice postsecondary adenoviral
infection. Because we have recently reported that in addition to
lymphocytes, pulmonary macrophages may be a prominent source of IFN-
during respiratory intracellular mycobacterial infection
(7), it is likely that the total IFN- measured in the
lung was partially accounted for by macrophage-derived IFN-. This
led us to investigate whether the IFN- response in lung macrophages
elicited by adenoviral infection was IL-12 dependent. Indeed, we found
that IFN- release by lung macrophages required both adenovirus and
IL-12. In addition to macrophages, it is possible that activation of NK
cells by IL-12 during secondary viral infection also contributes to
IFN- release in the lung. We have also examined the humoral immune
response in IL-12-/- mice. B cells have been
shown to bear IL-12R, and IL-12 could enhance B cell Ab secretion
(23, 24). We found that IL-12-/-
mice could mount as vigorous anti-adenoviral IgG responses of the
Th1 type as in their normal counterparts. The integrity of Th1-type
immune responses seen in IL-12-/- mice is
further supported by our finding that the duration of
adenoviral-mediated transgene expression in the lung was similar
between IL-12-/- and C57BL/6 mice. We have
previously shown that adenovirus infects not only airway epithelial
cells but also macrophages in the lung (15, 26), and it is
well known that anti-adenoviral cytotoxic responses by CD8 T cells
are the major mechanisms underlying the clearance of virus-infected
cells and limited virus-mediated transgene expression in these cells
(15, 27, 28). Although we have not conducted a CTL assay
to directly address the role of CD8 T cells in our current study, our
findings suggest that the clearance of virus-infected cells was not
impaired in IL-12-/- mice. Indeed, we observed
a similar extent of bronchial epithelial damage in the lungs of both wt
control and IL-12-/- mice during primary or
secondary adenoviral infection.
The role of endogenous IL-12 in host defense against primary influenza
infection has previously been studied using anti-IL-12 Abs
(11). In this study IL-12 was found involved in the early,
but not later, immune responses, including IFN- release in the lung.
However, the phenotype of lymphocytes, antiviral humoral responses, and
the compensating molecular mechanisms remain to be determined in this
study. In comparison, our observations argue against the critical role
of IL-12 in host defense against both primary and secondary pulmonary
adenoviral infections. This conclusion is supported by observations
made through examination of tissue cellular responses, IFN- level in
the lung, type1/2 profile of virus-reactive lymphocytes from both
regional lymph nodes and peripheral lymphoid tissues, and humoral
responses. In support of our findings, Th1 immune protection against
murine hepatitis viral infection has very recently been found to be
independent of IL-12 (13). Immune control of systemic
viral infection with lymphocytic choriomeningitis virus has also been
found to be independent of IL-12 (12). However,
different from the adenovirus used in our study, lymphocytic
choriomeningitis virus is noncytopathic and does not induce IL-12
release in vivo (12). It appears that whether a virus is
cytopathic or lytic is important in dictating IL-12 responses by the
host, because we also found that compared with wt adenovirus,
replication-deficient adenovirus released little IL-12 in the
lung.
Of particular importance, we have investigated the potential role of
IL-18 in IL-12-independent Th1 immune responses to pulmonary adenoviral
infection. We reveal that in contrast to IL-12, IL-18 is required for
the release of IFN- and for optimal lymphocytic accumulation in the
lung during IL-12-independent anti-adenoviral Th1-type responses.
However, IL-18, like IL-12, is not required for the development of
virus-reactive lymphocytes of the Th1 phenotype in both local and
systemic lymphoid tissues during respiratory viral infection. These
important findings suggest that IL-18 plays a much more critical role
than IL-12 in IFN- responses to viral infection by stimulating not
only lymphocytes but also other cell types, such as NK cells. Immune
protection from viral infection by IL-18 via IFN- induction has also
recently been suggested in a model of herpes simplex virus 1 infection
by using rIL-18 to treat mice before infection (29). Of
interest, very recently, Cousens et al. (30) identified
IFN-
/ß as IFN--inducing factors alternative to IL-12 in a
systemic model of lymphocytic choriomeningitis virus infection. Many
differences can be noted between our model and theirs. First, LCV is
noncytopathic virus that does not induce IL-12 release in vivo
(12), and therefore, although still unknown, it is
plausible to speculate that it cannot induce IL-18 in vivo and that the
requirement of IFN-/ß for IFN- release could be associated
specifically with LCV infection. Secondly, different from our
respiratory viral infection model, they analyzed responses in a
systemic viral infection model. Thirdly, we found that the lack of both
IL-12 and IL-18 did not hinder the development of virus-reactive
Th1-type lymphocytes in lymphoid tissues. This last finding from our
studies is in agreement with the finding that IL-18 does not have a
direct Th1-differentiating effect (3) but, on the other
hand, does not support the current understanding that IL-12 is the only
critical Th1 driver (1). Thus, the findings reported by us
and Cousens et al. still leave an issue of fundamental importance
unsolved: the nature of the cytokine(s) driving a Th1 differentiation
during viral infection.
Contrasting with IL-12-independent Th1 immune responses to respiratory
adenoviral infection, our evidence indicates that IL-12 is critically
required not only for IFN- release but also for the development of
Th1 lymphocytes during respiratory mycobacterial infection. Apparently,
other cytokines, including IL-18 and IFN-/ß, cannot compensate for
the function of IL-12 in the model. Collectively, our findings indicate
that IL-12 is differentially required for Th1 immune responses in the
lung depending on the nature of intracellular pathogens. It is critical
for host Th1 responses to intracellular bacterial infections but not to
intracellular viral infections.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Zhou Xing, Health Sciences Centre, Room 4H19, Department of Pathology and Molecular Medicine, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5. E-mail address:
3 Abbreviations used in this paper: wt, wild type; BCG, Calmette-Guérin bacillus; i.n., intranasal; MLN, mediastinal lymph nodes; BAL, bronchoalveolar lavage; PPD, purified protein derivative.
Received for publication June 30, 1999. Accepted for publication December 10, 1999.
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