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* British Columbia Center for Disease Control and
Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada; and
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Host defense to C. trachomatis infection involves humoral and cell-mediated immune responses, and both appear to be required for the full expression of immunity. Dendritic cells (DCs)3 are potent APCs that function as the principal in vivo activators of naive T cells for initiating adaptive immune responses, including T cell- and B cell-mediated immunity (13). DCs predominantly reside in blood, bone marrow, lymphoid organs, and subepithelial tissues frequently exposed to Ags. The ability of DCs to potently activate naive T cell responses lies in a well-orchestrated developmental program. Immature DCs in the peripheral tissue are highly active in phagocytosing and processing microbial Ags and undergo a maturation process as they migrate to draining lymphoid tissues (14). In T cell-dependent areas of lymphoid tissue, DCs carrying Ags interact with naive T cells to initiate primary Ag-specific immune responses (15). DCs have been studied in chlamydia immunobiology. They are known to secrete IL-12 when pulsed ex vivo with inactivated chlamydia organisms and to elicit protective immunity to challenge infection when organism-pulsed DCs are adoptively transferred (6, 16).
C. trachomatis mouse pneumonitis (MoPn) infection of the
murine genital tract and lung induces cellular as well as humoral
immune responses, with almost sterile protection against homologous
reinfection. Strikingly, immunization with inactivated chlamydia is
unable to elicit protective immunity (6, 16, 17, 18, 19). As
mentioned, immunization with DCs pulsed ex vivo with inactivated
chlamydia organisms is able to induce solid protection against
challenge infection and protection correlated with Ag-specific
Th1-biased immunity and was similar in magnitude to that observed
following infection (6, 16). The results indicate that the
underlying mechanism in the initiation and maintenance of protective
immunity induced by viable chlamydia may at least partly depend on the
mobilization and maturation of DCs. Presumably immunization with
inactivated organisms does not directly result in the mobilization of
mature DCs. Indeed, several studies have provided evidence for the
accumulation of MHC class II+ cells or
dendritic-like cells at local sites following C. trachomatis
infection of the genital tract (20) or peritoneal cavity
(19). Studies have also shown that in vitro infection of
epithelial cells with C. trachomatis results in the
secretion of a variety of proinflammatory cytokines and chemokines,
such as IL-1
, IL-1
, IL-6, IL-8, IL-18, GRO-, and GM-CSF, that
could result in the mobilization and maturation of DCs. Secretion was
induced only by viable, but not by inactivated, organisms and was
inhibited by antibiotics (21, 22, 23). These observations
suggest that chlamydia-induced secretion of cytokines and chemokines
from host cells may play a critical role in the activation of
DC-mediated immunity. Inactivated organisms fail to do so and thereby
may fail to induce protective immune responses.
This study was directed toward evaluating the usefulness of adenoviral GM-CSF transfection in enhancing protective immunity following the intrapulmonary delivery of inactivated chlamydia organisms. GM-CSF is a potent proinflammatory cytokine known to mobilize and mature DCs (15, 24, 25). The adenovirus vector carrying the murine GM-CSF gene is known to increase the numbers of DCs at local sites and enhance the development of Th1-mediated immunity (24). In the present study we found that adenoviral GM-CSF gene transfer significantly enhanced protective immunity elicited by inactivated chlamydial organisms, while similarly administrated inactivated organisms without prior GM-CSF transfection was unable to induce significant immune protection. Protection was correlated with systemic chlamydia-specific CD4+ Th1 and mucosal IgA responses. Our findings have implications for the development of a safe and effective vaccine against chlamydial infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The C. trachomatis MoPn strain Nigg was grown in HeLa 229 cells. Chlamydia elementary bodies (EBs) were purified on discontinuous density gradients of Renografin-76 (Squibb Canada, Quebec, Canada) as described previously (23). Purified EBs were aliquoted and stored in a sucrose-phosphate-glutamic acid buffer at -80°C. The same stock of EBs was used for all experiments. The infectivity of purified EBs was titrated by infection of HeLa cell monolayers for 24–36 h, followed by the fixation of cells with methanol and enumeration of inclusions. Cells containing chlamydial inclusions were detected by staining with anti-chlamydial LPS mAb (ViroStat, Portland, ME) as described previously (23). Portions of purified EBs were inactivated by UV light (G15T8 UV lamp; D. William Fuller, Inc., Chicago, IL) at a distance of 5 cm for 1 h at room temperature. No chlamydial growth was detected when UV-inactivated EBs (UVEBs) were inoculated onto HeLa monolayers at a dose equivalent to 108 inclusion-forming units (IFU)/106 HeLa cells. For convenience, the number of UVEBs used in all experiments was calculated based on the number of IFU of the corresponding viable EBs preparations before UV light exposure.
Female BALB/c mice, 6–8 wk old, were purchased from Charles River Canada (St. Constant, Canada). Mice were housed under specific pathogen-free conditions. All animal procedures used in this study were approved by the animal care committee of University of British Columbia.
Gene transfer and immunization
A replication-deficient human type 5 adenoviral construct (adenovector) carrying murine GM-CSF cDNA in the E1 region of the viral genome (AdGM) was constructed as described previously (26). An adenoviral vector add170-3 without the GM-CSF transgene (Ad) was used as a control.
Mice were anesthetized, and AdGM or Ad was intranasally (i.n.) delivered to lungs as described previously (25). Briefly, a dose of 3 x107 PFU of viral vector in 50 µl PBS was delivered to mouse lungs. The same amount of PBS was delivered i.n. to control mice. On day 7 after gene transfer, some of the mice were sacrificed for measurement of GM-CSF levels in bronchoalveolar lavage (BAL); other groups of mice were immunized i.n. with 1 x 105 IFU of UVEBs twice on consecutive days on two occasions at 3-wk intervals.
BAL analysis
BAL was performed at the indicated time points. Mice were usually sacrificed on day 7 postinstillation of adenovector or at indicated times. A total of 450 µl PBS was used to lavage the lung on two occasions. BAL fluids were centrifuged in a microcentrifuge at 2000 rpm for 5 min, and supernatants were stored at -80°C until cytokine assays.
Challenge infection and quantification of MoPn
For a comparison of immune responses between groups immunized with viable or UVEBs without GM-CSF gene transfer, mice were immunized i.n. with 5000 IFU viable EBs or 1 x 105 IFU UVEBs, respectively. On days 3, 7, 9, 10, 14, and 21, mice were sacrificed for assays of sera Abs and Ag-driven cytokine production by spleen cells.
On day 21 after immunization with UVEBs post-GM-CSF gene transfection, mice were i.n. challenged with 5000 IFU of viable MoPn and monitored daily for body weight changes. Mice were sacrificed at 10 days following inoculation, during the time of peak chlamydial growth in immunologically naive mice (27). Lungs were aseptically removed and homogenized with a cell grinder in 3 ml cold sucrose-phosphate-glutamic acid buffer. Tissue suspensions were centrifuged at 3000 x g for 15 min at 4°C to remove coarse tissue debris and were frozen at -80°C until tested. For quantification of MoPn in lung homogenates, HeLa 229 monolayers were inoculated with serially diluted tissue supernatants for 2 h, followed by culture for 30 h. Cells with chlamydial inclusions were detected using an anti-LPS mAb as previously described (28).
Determination of MoPn-specific Ab levels
Sera were collected at the indicated time points for the determination of MoPn-specific Ab responses using an EB ELISA as previously described (28). A 96-well ELISA plate (Corning Glass Works, Corning, NY) was coated with 105 IFU of MoPn EBs in 100 µl of 0.1 M NaHCO3 buffer at 4°C overnight. Abs bound to the coated Ags were detected using biotin-conjugated, isotype-specific anti-mouse IgG2a and IgG1 (Caltag, Burlingame, CA) and IgA (BD PharMingen, San Diego, CA), followed by the addition of streptavidin and substrate (Sigma, St. Louis, MO). ELISA Ab titers in sera were expressed as the geometric mean titer ± SEM and represented the inverse dilution of sera using the end point (cutoff at OD 405, 0.5) of the titration curves. Chlamydial MoPn-specific IgA in the supernatants of lung homogenates or BALs was also measured by ELISA. The lungs of mice were homogenized as described for the quantitation of MoPn EBs, but were centrifuged at 10,000 rpm. The clarified supernatant was diluted in 1/10 PBS for anti-MoPn IgA ELISA. The lung MoPn-specific IgA titer was expressed as an OD value at 405 nm.
Spleen cell culture and cytokine determination
Spleens were collected at the same time points as sera for Ab
measurement. Spleen cells were cultured at 5 x
106 cells/ml in the presence or the absence of
UVEBs (1 x 105 IFU/ml) for 3 days
(28). Culture supernatants were harvested for IFN-
,
TNF-, IL-10, and IL-4 analysis by sandwich ELISA. Cytokines in the
lung homogenate supernatants or BAL were also tested using ELISA. Ab
pairs for determination of mouse IFN-, TNF-, IL-10, IL-4, and
GM-CSF, and matched standard murine cytokines were purchased from BD
PharMingen. To determine whether CD4 T cells were responsible for
IFN- production, a CD4 mAb or isotype control Ig (BD PharMingen) was
added to spleen cell culture.
Histopathological analysis
The lungs from mice were isolated at indicated time points for fixation in 10% buffered formalin and embedded in paraffin as described previously (28). Tissue sections (7 µm) were stained with H&E and examined under light microscopy by a person blinded to the identification of mouse groups.
Immunohistochemical staining
Lung tissues were lyophilized with OCT embedding compound (Sakura Finetek, Torrance, CA) in liquid nitrogen and stored at -80°C. Five-micrometer sections were fixed with cold acetone and incubated with 0.5% H2O2 in methanol to block endogenous peroxidase activity. Slides were stained with rat anti-mouse DC mAb (TIB-227; American Type Culture Collection, Manassas, VA) or control Ig (BD PharMingen) in 2% goat serum/PBS, respectively. Following incubation with a 1/250 dilution of biotin-strepavidin-conjugated F(ab')2 mouse anti-rat IgG and a 1/500 dilution of peroxidase-conjugated strepavidin (Jackson ImmunnoResearch Laboratories, West Grove, PA), the slides were visualized through the addition of diaminobenzidine substrate (Roche, Laval, Canada) and counterstained with hematoxylin. Images were acquired and processed with a computer equipped with a digital camera (CoolSNAP; Media Cybernetics, Silver Spring, MD).
Statistical analysis
Data are shown as the mean ± SEM unless otherwise indicated. Results were analyzed by Student’s t test and were considered statistically significant at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous data and other studies have demonstrated that
infection immunity is necessary and sufficient for eliciting protective
immunity against C. trachomatis MoPn challenge infection
(6, 16, 17, 18, 19). Understanding the mechanism for this is
important for the development of an effective chlamydial vaccine. We
initially compared differences in immune responses between
infection-elicited immunity and i.n. immunization with UVEBs by
characterizing Ag-specific Ab and cytokine responses. Striking
differences in systemic and pulmonary immunities were observed between
two groups as shown in Fig. 1
. By day
10–14 after primary infection, mice produced significant systemic
MoPn-specific IgG2a and IgG1 Abs (Fig. 1, a and
b), spleen cell IFN- response (Fig. 1c), and
lung IgA Ab response (Fig. 1e). After primary infection the
lung IFN- level peaked around day 10 and declined to minimal levels
at day 21 postinfection (Fig. 1d). In contrast, i.n.
immunization with UVEBs did not induce significant immune responses.
Mice who recovered from primary infection exhibited almost sterile
protection against homologous reinfection, whereas mice who received
UVEBs were fully susceptible to challenge infection (Fig. 1f). These data confirm the inability of mice to generate
anti-MoPn immunity following i.n. immunization with UVEBs.
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, g–i;
infection group, 7.1 ± 3.1 DCs/field; UVEB group, 2.1 ± 0.7
DCs/field; naive mice, 1.5 ± 0.6 DCs/field; mean ± SEM
number of DCs; three to five mice per group; p <
0.05). Thus, infection with viable EBs preferentially increased the
number of DCs at the infection site compared with immunization with
inactivated EBs. Accumulation of DCs preceded Ag-specific cellular and
humoral immune responses (Fig. 1). The results suggest the immunization
with UVEBs fails to induce protective immune responses at least partly
because of its inability to increase the number of DCs at the local
immunization site. Intranasal delivery of a transgene for GM-CSF via an adenovector increased the numbers of DCs at local tissue sites and induced Ag-specific cellular and humoral immunities following UVEB mucosal immunization
We next explored the importance of DCs in the initiation of Ag-specific immune responses using a transgene strategy. Similar to the report by Stampfli et al. (25), mice that received an i.n. administration of AdGM had significantly higher levels of GM-CSF in the BAL on day 7 after GM-CSF gene transfer than mice instilled with Ad (AdGM group, 12 ± 1.9 pg/ml; Ad group, 0.5 ± 0.1 pg/ml of GM-CSF in BAL; mean ± SEM of four mice; p < 0.01). The levels of GM-CSF in the lungs decreased to background levels by day 28 after GM-CSF transfer (data not shown). As previously reported using the same strategy to study allergy induction (24, 25), increased GM-CSF expression was correlated with the appearance of large numbers of DCs in the lungs of mice who received AdGM, as demonstrated by immunohistochemistry with anti-DC Ab (TIB-229; American Type Culture Collection) staining. In contrast, few DCs were found in the lungs of mice who received Ad, indicating that the appearance of DCs was not due to the nonspecific effect of adenoviral vector effect. These findings confirm that i.n. delivery of the GM-CSF transgene causes transient local GM-CSF expression and increased the number of DCs in the lung. We next tested our hypothesis that UVEBs should be able to induce protective immunity when sufficient DCs are present at the local immunization site using the GM-CSF gene transfer strategy.
We initially determined whether UVEB immunization post-GM-CSF gene
transfer selectively induces a Th1-like, IFN- producing, T
cell-dominant response. As shown in Fig. 2a, spleen cells from
intranasal UVEB-immunized mice post-GM-CSF gene transfer had
significantly elevated levels of IFN-, TNF-, and IL-10 upon in
vitro stimulation with MoPn EBs (p < 0.05
compared with mice immunized with UVEB after administration of
adenovector). Naive spleen cells or spleen cells from control mice
(Ad/UVEB) failed to produce significant amounts of IFN-, TNF-,
and IL-10 regardless of Ag stimulation, indicating that immunization
with UVEBs following GM-CSF gene transfer primed chlamydia Ag-specific
T cells in vivo. There was only marginal IL-4 production, with no
significant differences among the groups of mice (Fig. 2a).
Increased production of IFN- from spleen cells was dependent on CD4
T cells, since anti-CD4 Ab abolished IFN- production (data not
shown). We measured serum Abs against chlamydia, which provides further
information on the biological activity of Th responses in vivo. Mice
immunized with UVEB post-GM-CSF gene transfer had significantly higher
titers of Ag-specific serum IgG2a and IgG1, but no detectable
Ag-specific serum IgA (Fig. 2b and data not shown).
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b, IgA levels in the
lungs of the mice immunized with UVEB after GM-CSF transfection were
significantly higher than those in control groups and similar to the
levels observed in mice infected with viable EB (vEB). Thus, the data
indicate that the immunization of UVEB post-GM-CSF gene transfer was
able to induce not only systemic Ag-specific immune responses, but also
mucosal Ag-specific IgA responses. Immunity induced by UVEBs following transgene GM-CSF provides protection against MoPn infection
To address the potential role of enhanced mucosal and systemic
immune responses in protecting against infection, we next assessed
protective immunity by measuring chlamydia growth and body weight
changes following intranasal challenge with 5000 IFU of MoPn. Mice
immunized with UVEB following GM-CSF gene transfer displayed milder
systemic illness and significantly less body weight reduction and began
to regain body weight earlier compared with control mice (Fig. 3a). We further evaluated the
effect of UVEB immunization after GM-CSF gene transfer by quantitating
infectious chlamydia recovery from mouse lung tissues. As shown in Fig. 3b mice immunized with UVEB post-GM-CSF gene transfer had
>1000-fold less chlamydia recovered from lung tissue compared with
other control groups (p < 0.05). Mice
immunized with UVEB following Ad transfer were not protected.
Collectively, these observations show that the transfer of GM-CSF gene
indeed enhances protective immunity following UVEB immunization. The
degree of the protective immunity was similar to that observed among
previously infected mice (Fig. 3b). Protection was unlikely
to be due to the direct effect of GM-CSF alone, since mice that
similarly received AdGM without UVEBs were not protected (Fig. 3, a and b).
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To investigate the potential role of enhanced immune responses in
protecting against the tissue-damaging effects of chlamydial infection,
we next characterized the pathological features following challenge
infection. We processed and examined lung tissues on day 10
postinfection (Fig. 4, e–h)
using the morphology of lung before challenge among each group of mice
as control (Fig. 4, a–d). There was much less severe
peribronchial and perivascular infiltration of mononuclear cells in the
lungs of mice immunized with UVEB post-GM-CSF gene transfer (Fig. 4g). The inflammatory infiltrate was comparable to that
observed in challenged mice who had recovered from previous infection
(vEB; Fig. 4h). In contrast, diffuse mononuclear cell
infiltration involving between 60 and 90% of the lung parenchyma was
present in infected control mice (Fig. 4e) and in mice
immunized with UVEB after delivery of adenovector (Ad/UVEB; Fig. 4f).
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To determine whether local GM-CSF expression is required for the
induction of protection, we compared the effects of alternate routes of
GM-CSF gene delivery on cellular and humoral immune responses and on
protective immunity. As expected, mice i.p. immunized with UVEB after
i.n. delivery of AdGM (i.n. AdGM/i.p. UVEB) did not have significant Ab
responses or spleen cell cytokine production upon Ag stimulation in
vitro (Fig. 5, a–c). Mice
immunized by i.p. injection of UVEB after i.p. delivery of AdGM or i.n.
administration of UVEB after i.n. delivery of AdGM had strong Ab (IgG2a
and IgG1) responses in sera and spleen cell Ag-driven cytokine (IFN-
and TNF-) responses (Fig. 5, a–c). Thus, the effect of
AdGM as an adjuvant appears to be limited to the local site where the
immunogen is delivered. Despite developing systemic immune responses,
however, the i.p. immunized mice did not demonstrate protective
immunity against challenge infection (Fig. 5d). Since both
IFN- and mucosal IgA appear to be critical components of protective
immunity against C. trachomatis infection, we measured lung
IFN- and IgA levels among groups of mice. The results showed that
i.p. immunized mice (i.p. AdGM/i.p. UVEB) had little local IgA
response, whereas they exhibited high levels of IFN- in lung
homogenates (Fig. 6, a and
b). The high IFN- in lung homogenates correlated with the
extensive mononuclear cell inflammation seen histopathologically (Fig. 6, c, e, and f) and the higher growth
of chlamydia in lung tissue (Fig. 5d). Intranasally
immunized mice (i.n. AdGM/i.n. UVEB) had higher lung IgA responses,
lower lung IFN- levels (Fig. 6, a and b), much less
mononuclear cell inflammation (Fig. 6d), and lower growth of
chlamydia in the lung (Fig. 5d) compared with infected
control mice (Fig. 6, c, e, and f).
The results indicate that local IgA may neutralize chlamydial growth,
thereby limiting lung inflammation and local IFN- production in
response to challenge infection. The absence of local IgA responses
correlated at least in part with the failure to induce protective
immunity following i.p. immunization of UVEBs and AdGM.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently Shaw et al. (29) reported that DCs pulsed with chlamydial major outer membrane protein or UVEB induced Ag-specific CD4 T cell proliferation in vitro, but that only immunization with DCs pulsed with UVEB induced Th1-mediated protective immunity in vivo. Immunization with DCs pulsed with major outer membrane protein elicited Th2 immune responses without significant protection against challenge infection. The authors suggested that the nature of the Ag used to pulse DCs may influence the maturation of DCs and thereby skew the Th1-Th2 balance of the immune response in vivo (29). In the absence of maturation, Ag-loaded DCs may induce Ag-specific tolerance or polarize the immune response to a type 2 pattern (30). Whole EBs, as used in the current study, not only contain protective epitopes, but also appear to promote the maturation of DCs, perhaps through interaction with Toll-like receptors.
Epithelial cells maintain an effective antimicrobial barrier to mucosal
infection in part through the direct induction of innate antimicrobial
factors in response to infection, which results in the initiation of an
inflammatory response followed by recruitment of immune cells to the
local infection site. The initial site of C. trachomatis
infection is at mucosal surfaces such as the genital, respiratory, or
ocular surfaces. In vitro infection of epithelial cells by C.
trachomatis causes the secretion of many chemokines,
proinflammatory cytokines, and other mediators through mechanisms
dependent on chlamydia-derived protein synthesis. Inactivated chlamydia
are unable to induce chlamydia-dependent cytokine secretion by
epithelial cells (22, 23), suggesting that cytokine
secretion by epithelial cells requires in vivo metabolism by the
organism. For the present study we hypothesized that mediators secreted
by epithelial cells following chlamydial infection recruit inflammatory
and immune cells to the local site, which are critical to the induction
of protective immunity. During this process, DCs may be mobilized and
matured by molecules, such as GM-CSF, IL-1, TNF-, macrophage
inflammatory protein-1 (MIP-1), MIP-1, MIP-3, RANTES, and
macrophage-derived chemokine. The relative in vivo importance of each
of the molecules in DC mobilization and maturation has not yet been
determined (31, 32, 33, 34, 35, 36, 37). Gene transfer technology provides a
novel way to evaluate the importance of local cytokine expression in
this process. Previous studies have demonstrated that adenovirus
vectors are in many ways well suited for gene transfer to epithelial
cells and have been informatively used for experimental study in tumor,
allergy, and infection models (24, 26, 38, 39). The
strategy has advantages over the use of recombinant cytokines, in that
the transgene can be expressed in a dose-dependent manner in a specific
tissue for a limited period of time. In the present study we used a low
dose of the adenoviral construct (26) delivered via the
airway to target epithelial cells and to certain extent alveolar
macrophages (40), since these cell types are probably
major targets for MoPn infection in vivo. We observed that the potent
effect of GM-CSF was closely associated with the appearance of
increased numbers of DCs, enhanced Ag-specific CD4-Th1 immune
responses, and increased lung Ag-specific IgA levels and resulted in
protection against challenge infection. In the absence of
adenovirus-mediated GM-CSF transfection, UVEB immunization did not
induce significant immune responses or protection (Figs. 2 and 3).
Local production of GM-CSF at the site of immunization appeared to be
particularly important, since mice delivered GM-CSF and UVEB by
separate routes had undetectable Ag-specific immune responses and no
protection against challenge infection. Protection induced by
immunization with UVEB post-GM-CSF transfection was unlikely to be due
to the direct effect of GM-CSF, since mice similarly immunized with
adenovector encoding GM-CSF alone were as susceptible to challenge
infection as mock control mice (Fig. 3b).
Interestingly, mice i.p. immunized with UVEB following GM-CSF gene
transfer generated a strong chlamydia-specific Th1 immune response and
systemic Ab response. However, mice were not protected against lung
challenge infection. This observation is similar to a previous report
by Su et al. (6), who reported that i.p. immunization with
heat-killed EB plus exogenous IL-12 elicited strong Th1-dominated
immune responses, but no protective immunity against chlamydia genital
tract infection. The authors suggested that Ag-specific T cell
populations may not be appropriately sensitized after systemic
immunization with inactivated EBs plus IL-12 to enable their homing to
the genital mucosa (6). In our system we observed that
mice i.p. immunized with UVEB after delivery of AdGM lacked local lung
IgA responses despite having high serum Abs (IgG2a and IgG1) responses
and spleen cell cytokine responses, including IFN- production upon
Ag restimulation in vitro (Figs. 5 and 6). Lung homogenate IFN- on
day 10 postchallenge did not correlate with protection, since mice
recovered from previous infection or immunized locally with UVEB
post-GM-CSF gene transfer showed significant protective immunity and
yet exhibited very low levels of IFN- in the lung, whereas infected
naive mice or i.p. immunized mice had poor protection, extensive
pulmonary inflammation, and high lung levels of IFN- (Fig. 6).
Previous studies have consistently shown that IFN- is central to
immunity against C. trachomatis genital tract or lung
infection (41, 42, 43, 44). The experiments reported here
demonstrate that local IgA is required for optimal protective immunity
to chlamydia, and it may be that the protective effect of mucosal IgA
is most apparent in the presence of Th1-related cytokines, such as
IFN-. Mucosal immunity can be most efficiently induced by the
mucosal immunization (45, 46). Mucosal IgA may neutralize
chlamydia organisms when encountering the pathogen in the mucosal tract
and thereby limit the need to mount a vigorous inflammatory response to
cope with a large infection load, a concept that is probably important
for vaccine development.
In conclusion, the differing efficacies of in vivo immunization with dead vs live chlamydia are probably due to the inability of dead chlamydia to induce the secretion of proinflammatory mediators such as GM-CSF and thereby mobilize DCs at the site of immunization. Information from our studies indicates that the design of a vaccine to prevent chlamydia infection should not only target the activation of the CD4+ Th1 cells and IgA-producing B cells with mucosal homing properties, but also enhance the mobilization and maturation of APCs such as DCs for Ag processing and presentation through the use of transient transgene expression such as GM-CSF or other key cytokine adjuvants.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert C. Brunham, British Columbia Center for Disease Control, 2077-655 West 12th Avenue, Vancouver, British Columbia, Canada V5Z 4R4. E-mail address: robert.brunham{at}bccdc.ca
3 Abbreviations used in this paper: DC, dendritic cell; BAL, bronchoalveolar lavage; EB, elementary body; IFU, inclusion-forming unit; i.n., intranasally; MIP, macrophage inflammatory protein; MoPn, mouse pneumonitis; UVEB, UV-inactivated EB; vEB, viable EB.
Received for publication February 27, 2002. Accepted for publication October 1, 2002.
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References |
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transgene attracts dendritic cells to established murine tumors and suppresses tumor growth. J. Clin. Invest. 105:1383.[Medline]
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J. Rey-Ladino, K. M. Koochesfahani, M. L. Zaharik, C. Shen, and R. C. Brunham A Live and Inactivated Chlamydia trachomatis Mouse Pneumonitis Strain Induces the Maturation of Dendritic Cells That Are Phenotypically and Immunologically Distinct Infect. Immun., March 1, 2005; 73(3): 1568 - 1577. [Abstract] [Full Text] [PDF]
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) or 105 IFU of UVEB (
). On days 3, 7, 10, 14,
and 21 postinstillation, sera, spleen, and lungs (six mice per group
per time point) were collected for Ab and cytokine determinations by
ELISA, respectively. MoPn-specific IgG2a (a) and IgG1
(b) responses in sera were expressed as the geometric
mean titer ± SEM of the inverse dilution of the samples. Spleen
cells were cultured at 5 x 106 cells/ml in the
presence of UVEB (1 x 105 IFU/ml) for 3 days. Culture
supernatants were harvested for IFN-
