| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY 12208;
Laboratory of Immunohistochemistry and Immunopathology, Institute of Pathology, University of Oslo, Rikshospitalet, Oslo, Norway; and
Department of Medicine, University of California, San Diego, CA 92093
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
5-fold more systemic IgA and 6-fold less nasal IgA Ab than wild-type mice due to defective transport into mucosal tissues. Wild-type, but not pIgR–/– mice were protected against infection with serotype 14 S. pneumoniae, which causes mucosal colonization but does not induce systemic inflammatory responses in mice. The relative importance of secretory IgA in host defense was further shown by the finding that intranasally vaccinated IgA gene-deficient mice were not protected from colonization. Although secretory IgA was found to be important for protection against nasal carriage, it does not appear to have a crucial role in immunity to systemic pneumococcus infection, because both vaccinated wild-type and pIgR–/– mice were fully protected from lethal systemic infection by serotype 3 pneumococci. The results demonstrate the critical role of secretory IgA in protection against pneumococcal nasal colonization and suggest that directed targeting to mucosal tissues will be needed for effective vaccination in humans. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Dimeric IgA, with an incorporated J chain, is the predominant Ab isotype expressed by B cells in the mucosal lamina propria. It is actively transported by the epithelial polymeric Ig receptor (pIgR) and released into mucosal secretions as secretory IgA (SIgA), a complex of dimeric IgA with a bound secretory component (the extracellular domain of the pIgR; Ref. 4). Luminal SIgA is believed to interfere with pathogen adherence to mucosal epithelial cells, a process called immune exclusion (9). Human serum IgA has also been reported to induce complement-mediated killing of S. pneumoniae and to enhance phagocytosis (8). Mice with a disruption in the pIgR gene (pIgR–/– mice) cannot transport IgA or IgM into the mucosal lumen, and thus offer an opportunity to determine the importance of respiratory SIgA in host defense against pneumococcal colonization. Previous studies have shown that pIgR–/– mice have reduced protection against influenza virus infection in the upper respiratory tract, which cannot be substituted by serum IgG (2). Nevertheless, it has been reported that IgA is not necessary for vaccine-induced protection against bacterial and viral mucosal pathogens, including the influenza virus (10), rotavirus (11), Shigella flexneri (12), and Helicobacter pylori (13).
We have now evaluated protection by mucosal and systemic Abs in pIgR–/– and wild-type mice after intranasal (i.n.) and parenteral immunization with polysaccharide conjugate vaccine, using IL-12 as a mucosal adjuvant as previously described (14). The role of SIgA in the defense against pneumococcal respiratory infection was also investigated using IgA gene-deficient (IgA–/–) mice. The results show that while serum Ab can protect against systemic infection (15), secretory IgA is critical for control of nasal carriage.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
pIgR–/– mice on a B6 x 129 background were originally developed as described (16). Due to the defective transport of IgA into the mucosal lumen, there are significantly higher levels of IgA in sera of these pIgR–/– mice compared with wild-type mice. IgA gene-deficient (IgA–/–) mice (17) were obtained from Baylor College of Medicine (Houston, TX). Wild-type C57BL/6 x 129 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and used as controls for both strains. All mice were bred and maintained at the Albany Medical College Animal Facility following Institutional Animal Care and Use Committee guidelines (Albany, NY).
Immunizations
To induce mucosal Ab responses, mice were immunized i.n. using IL-12 as an adjuvant essentially as described previously (14). In brief, mice were anesthetized and immunized i.n. on days 0 and 21 with 2.5 µg of serotype 14 PS conjugate vaccine (PS14-CRM197,; kindly provided by Wyeth Vaccines, Pearl River, NY) mixed with 1 µg of murine IL-12 (Wyeth Vaccines), followed by i.n. administration of 1 µg of murine IL-12 alone on days 1, 2, and 3. The total volume of vaccine and IL-12 per inoculation was 25 µl in PBS containing 1% normal mouse serum (NMS). PS3-CRM197 serotype 3 vaccine (1 µg in 200 µl saline; Wyeth Vaccines) was given i.p on days 0 and 21 together with 2 mg/ml alum (Rehydrogel Low Viscosity Gel; Reheis, Berkeley Heights, NJ). Control animals received either no pretreatment before infection or were treated with IL-12 only. No immune responses were noted in the absence of IL-12 coadministration.
Sera and nasal washes
Serum samples were obtained by tail vein bleeding or by retro orbital puncture under anesthesia. In addition, 3 days after infection, anesthetized mice were bled by cardiac exsanguination. To collect nasal washes, Ringer’s solution was inoculated into the tracheas and 250 µl of outflow were collected from the nose of each mouse, followed by assays for detecting nasal carriage as well as Ab expression. Levels of Ab in nasal washes were normalized by volume.
ELISA
Concentrations of Igs and PS-specific Abs in serum and nasal wash samples were measured by ELISA. Briefly, Maxisorp or Polysorp ELISA plates (Nalge Nunc International, Rochester, N.Y.) were coated overnight at 4°C in PBS containing 1 µg/ml goat anti-mouse Ig (H+L) to capture total Ig or 15 µg/ml serotype 3 or 14 PS (American Type Culture Collection, Manassas, VA) to capture PS-specific Ab. After washing, 2-fold dilutions of serum or nasal wash samples were incubated in the plates at 37°C for 2 h. Detection was performed using alkaline phosphatase-labeled goat anti-mouse Ig(H+L), IgG and IgA Abs (Southern Biotechnology Associates, Birmingham, AL) followed by the addition of p-nitrophenylphosphate substrate. Absorbance was measured in a microplate reader at 405 nm.
Bacterial infection
The S. pneumoniae strain A66.1, which expresses PS3, and strain TJO983, which expresses PS14, (provided by Dr. D. E. Briles, University of Alabama Birmingham, AL) were cultured at 37°C in Todd-Hewitt broth until mid-log phase, washed repeatedly, resuspended in fresh broth containing 15% glycerol, and stored at –70°C until use. To establish pneumococcus carriage, mice were anesthetized 10 days after the last immunization and inoculated i.n. with 10 µl of serotype 14 pneumococcal bacteria containing 2 x 108 CFU/ml in Ringer’s solution. The mice were sacrificed 3 days later to determine nasal colonization by plating nasal washes on blood agar (14). The lower limit of bacterial detection in nasal washes was 10 CFU per mouse. To analyze systemic infection, mice were infected i.p. with 100 µl of serotype 3 pneumococcal bacteria containing 2 x 107 CFU/ml in PBS and were monitored daily for survival.
Passive protection
Serum samples from normal or immune pIgR–/– mice were pooled and incubated at 55°C for 1 h to inactivate complement. Serotype 14 pneumococci were incubated for 1 h on ice with 10% serum diluted in Ringer’s solution. After centrifugation, the opsonized bacteria were resuspended in Ringer’s solution at a concentration of 5 x 107 CFU/ml and mice were infected i.n. as described above. The mice were sacrificed 24 h later to determine nasal colonization.
Histology
Mice were sacrificed 3 days after i.n. infection and the lungs were removed for histological analyses. Paraffin-embedded tissues were sectioned to a thickness of 5 µm and stained with H&E by standard methods.
Statistical analysis
Statistical comparisons of Ab levels were performed on log-transformed data by the Student’s t test. Mann-Whitney U test analysis was used for comparison of nasal carriage between experimental groups.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Serotype 14 PS conjugate vaccine was delivered i.n. to wild-type and pIgR–/– mice using IL-12 as an adjuvant to induce mucosal immunity. The immunized mice were then bled 4 wk later (7 days after secondary boosting) and serum samples were examined before bacterial challenge by ELISA to determine PS14-specific systemic Ab levels (Fig. 1A). Although there were no significant differences in total and IgG serum Ab levels between the two groups of mice, IgA was found to accumulate in the sera of pIgR–/– mice. Lung infection with serotype 14 S. pneumoniae induced similar increases in systemic Ag-specific Ab production in the two groups of mice (Fig. 1B). Naive mice had no detectable PS14-specific Abs in sera, and i.n. infection of these mice induced a small but detectable serum Ab response (data not shown).
|
Mucosally immunized pIgR–/– mice are not protected against nasal carriage by S. pneumoniae
Ten days after boosting with vaccine, the mice were challenged i.n. with serotype 14 pneumococci and 72 h later, the number of pneumococci in the nose was quantified (Fig. 2A). The effect of immunization on protection against bacterial carriage was found to be significant only in wild-type mice (p < 0.05 compared with unimmunized mice treated only with IL-12). Immunization resulted in more than a 10-fold decrease in bacterial load in these mice but no significant decrease was observed in pIgR–/– mice. Nasal vaccination did not induce the same level of Ab responses in all mice (Fig. 1) and the numbers of nasal-associated bacteria tended to have a negative correlation with Ab levels in nasal washes, i.e., the five wild-type mice with total nasal PS14-specific Ab levels >0.1 OD were also the mice that had nearly complete clearance of pneumococci from their noses (Fig. 2B). There was also a slight negative correlation between the numbers of nasal-associated bacteria and serum levels of PS14-specific Ab. In contrast to the results in wild-type mice, immunization had no discernable effect on nasopharyngeal carriage in pIgR–/– mice (no significant difference from unimmunized mice). Although significant levels of serum (but not nasal) Abs were detected in these mice, there was no correlation between numbers of nasal-associated pneumococci and levels of serum Ab in individual pIgR–/– mice. These results indicate that SIgA is necessary for protection against pneumococcal colonization.
|
To further address the role of SIgA in protection against pneumococcal nasal carriage, studies were performed using IgA–/– mice. Vaccination i.n. with PS14 conjugate vaccine in the presence of IL-12 tended to induce lower overall serum Ab responses in IgA–/– mice compared with wild-type mice but these differences were not statistically significant (Fig. 3A). However, the IgA–/– mice produced little respiratory Ab (Fig. 3B) and immunization of IgA–/– mice did not induce protection against bacterial colonization (Fig. 3C). There was no correlation between serum Ab titers and bacterial carriage in individual IgA–/– mice. As above, the vaccination protected wild-type mice from colonization, which was correlated with significant mucosal Ab titers.
|
The results indicated that mucosal Ab plays a necessary role in protection against nasal colonization, a role that cannot be replaced by Ab in serum. However, it was not clear whether the mucosal Abs possessed unique protective functions or if serum Abs could also mediate protection if they were able to reach the bacteria in the upper respiratory tract. To investigate the protective capability of serum PS14-specific Abs present in pIgR–/– mice, bacteria were opsonized in vitro with immune serum collected from pIgR–/– mice vaccinated i.n. with IL-12 as above and then used to challenge wild-type and IgA–/– mice. The observed passive protection provided by pIgR–/– immune serum in both strains (Fig. 4) indicates that the susceptibility of vaccinated pIgR–/– mice to pneumococcal carriage simply reflects the inability of Abs to be transported to the respiratory tract mucosal surface.
|
Because nasal colonization of humans by S. pneumoniae occurs asymptomatically and in the absence of inflammation, it was important to ensure the lack of lung inflammation in our mouse model of colonization. Mice were sacrificed 3 days after i.n. infection and lungs were prepared for histological analysis. No significant tissue alternations were observed after serotype 14 pneumococcal infection (Fig. 5), suggesting that nasal infection with this strain does not lead to lung inflammation, which in turn, could initiate systemic infection. Three days after i.n. challenge, bacterial burden in the upper respiratory tract, lung, and bloodstream were also examined by plating nasal washes, lung homogenates, and whole blood on blood agar plates. All mice were nasally colonized after i.n. infection (1000 CFU per mouse); however, there were few bacteria in the lower respiratory tract (10 CFU per mouse) in either pIgR–/– or wild-type mice. There were no detectable bacteria in the blood of infected pIgR–/– or wild-type mice. Thus, i.n. inoculation of serotype 14 bacteria did not lead to systemic infection.
|
In a final set of experiments, the role of Abs in protection against systemic infection was determined. For this purpose, mice were immunized i.p. and then challenged i.p with pneumococcal serotype 3, a serotype that, unlike serotype 14, leads to invasive disease. It was found that immunization of both wild-type and pIgR–/– mice induced high levels of serum Abs and that pIgR–/– mice had much higher IgA Ab titers compared with wild-type mice (Fig. 6A). In addition, both groups of mice were fully protected from a lethal dose of bacteria (Fig. 6B). Thus, although pIgR–/– mice do not express secretory Ab and are not protected against nasal carriage, they are fully protected against systemic infection after parenteral vaccination.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The importance of respiratory Abs in the host defense against pneumococcal colonization was established using pIgR–/– mice that lack the ability to actively secrete dimeric IgA and pentameric IgM into the mucosal lumen (2). In addition, we exploited an i.n. vaccination protocol that used IL-12 as a mucosal adjuvant and that was previously shown by our laboratory and others to yield high levels of protective Abs in the lungs of mice (18, 19). Vaccination i.n. with pneumococcal PS14 conjugate vaccine induced similar patterns of specific serum Ab isotype responses in wild-type and pIgR–/– mice, and almost the same levels of serum Abs after infection. However, nasal IgA Ab was not detected in pIgR–/– mice and protection against nasal carriage was not observed. Although mucosal leakiness in pIgR–/– mice has been described (16), we did not observe leakage of serum IgA into the upper respiratory tracts of these mice. In individual wild-type mice, levels of nasal bacteria were negatively correlated with Ab levels in nasal washes; in contrast, there was no correlation between numbers of pneumococci in the nose and serum Ab levels in individual pIgR–/– mice. There was also no defect in the ability of serum Abs from pIgR–/– mice to opsonize pneumococci, but in the absence of pIgR, the Abs could not be actively secreted into the mucosal lumen. Passive protection from nasal carriage that was provided by pIgR–/– immune serum demonstrated the functional activity of these Abs and the necessity of secreting them into the mucosal lumen to decrease nasal colonization. It has been reported that IgA is not necessary for vaccine-induced protection against bacterial and viral mucosal pathogens, including the influenza virus (10), rotavirus (11), S. flexneri (12), and H. pylori (13). Nevertheless, our results agree with previous studies demonstrating the susceptibility of pIgR–/– mice to respiratory virus infections (2).
Histological analysis of lungs from mice infected with the serotype 14 pneumococcus strain showed lack of inflammatory cell infiltration. Therefore, the mice cleared colonization through a noninflammatory mechanism rather than as an indirect effect of protection against an inflammatory systemic infection.
IgA–/– mice were previously shown by our laboratory to have a general defect in specific antiviral Ab production after i.n. immunization, a defect that could be overcome through the use of IL-12 as a vaccine adjuvant (19). In this study, we found that incorporating IL-12 into the vaccination protocol led to essentially comparable amounts of total serum Ab levels in wild-type and IgA–/– mice, but the latter mice lacked protection against nasal carriage. This finding clearly demonstrates that SIgA is the most important Ab isotype in mediating protection against pneumococcal carriage. We do not at this point know whether this induced SIgA may mediate protection through interaction with other immune factors induced by IL-12, although IL-12 given by itself before i.n. challenge conferred no protection (Fig. 2A). However, SIgA does not appear to play a crucial role in defense against systemic infection once the pathogen has breached the mucosal barrier.
The protective activity of IgA against S. pneumoniae and other pathogens has previously been studied (8, 20), but it has been difficult to distinguish the activity of serum IgA vs SIgA, especially when both are produced in the same host at the same time. The use of pIgR–/– mice in this study allowed us to distinguish between the protective role of SIgA against nasal carriage, which may lead to epithelial penetration, and systemic immunity against blood-borne S. pneumoniae. In this regard, it is interesting to note that human pIgR interacts directly with a surface protein of S. pneumoniae, SpsA (CbpA; Refs. 21, 22, 23), through Ig-like domains 3 and 4 of the receptor (24, 25). This interaction may supportS. pneumoniae adherence and epithelial penetration (23, 25), but excess secretory component, either free or bound to SIgA in nasal secretions, will favor blocking of pneumococci. However, it should also be noted that SpsA (CbpA) does not bind to murine pIgR (24) and pIgR–/– mice are not differently susceptible to pneumococcal invasion compared with pIgR+/+ mice (Fig. 6).
A full understanding of SIgA function is not only of theoretical interest, but also of practical significance in designing appropriate vaccination strategies. Although nasal colonization of humans by S. pneumoniae occurs asymptomatically, it is likely that bacteria can be spread to susceptible populations or mutate into antibiotic-resistant strains. Investigations into the protective function of SIgA against nasal colonization with S. pneumoniae suggest that vaccination efforts against respiratory pathogens should focus primarily on inducing effective mucosal immune responses (26). The present work has not only confirmed some of the properties of IgA previously documented (27), but has also definitively demonstrated the crucial role for SIgA in local defense against pneumococcal colonization under noninflammatory situations.
|
Footnotes |
|---|
1 This study was supported by National Institutes of Health Grant AI 41715 and a grant from Philip Morris, Inc. 
2 Address correspondence and reprint requests to Dr. Dennis W. Metzger, Center for Immunology and Microbial Disease, MC-151, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208. E-mail address: Metzged{at}mail.amc.edu
3 Abbreviations used in this paper: PS, polysaccharide; pIgR, polymeric Ig receptor; SIgA, secretory IgA; i.n., intranasal(ly); NMS, normal mouse serum.
Received for publication May 3, 2004. Accepted for publication July 16, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
4
7 integrin expression but does not require IgA antibody production. J. Immunol. 166:1894.RI (CD89). J. Infect. Dis. 182:1139.[Medline]
This article has been cited by other articles:
|
|
 |
|
  D. M. Ferreira, M. Darrieux, D. A. Silva, L. C. C. Leite, J. M. C. Ferreira Jr., P. L. Ho, E. N. Miyaji, and M. L. S. Oliveira Characterization of Protective Mucosal and Systemic Immune Responses Elicited by Pneumococcal Surface Protein PspA and PspC Nasal Vaccines against a Respiratory Pneumococcal Challenge in Mice Clin. Vaccine Immunol., May 1, 2009; 16(5): 636 - 645. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. N. Wolfe, G. S. Kirimanjeswara, E. M. Goebel, and E. T. Harvill Comparative Role of Immunoglobulin A in Protective Immunity against the Bordetellae Infect. Immun., September 1, 2007; 75(9): 4416 - 4422. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. D. Baron, R. Singh, and D. W. Metzger Inactivated Francisella tularensis Live Vaccine Strain Protects against Respiratory Tularemia by Intranasal Vaccination in an Immunoglobulin A-Dependent Fashion Infect. Immun., May 1, 2007; 75(5): 2152 - 2162. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. E. Fasching, T. Grossman, B. Corthesy, A. G. Plaut, J. N. Weiser, and E. N. Janoff Impact of the Molecular Form of Immunoglobulin A on Functional Activity in Defense against Streptococcus pneumoniae Infect. Immun., April 1, 2007; 75(4): 1801 - 1810. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. J. Davids, J. E. D. Palm, M. P. Housley, J. R. Smith, Y. S. Andersen, M. G. Martin, B. A. Hendrickson, F.-E. Johansen, S. G. Svard, F. D. Gillin, et al. Polymeric Immunoglobulin Receptor in Intestinal Immune Defense against the Lumen-Dwelling Protozoan Parasite Giardia J. Immunol., November 1, 2006; 177(9): 6281 - 6290. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Rice, T. Peng, J. S. Spence, H.-Q. Wang, R. M. Goldblum, B. Corthesy, and B. J. Nowicki Pyelonephritic Escherichia coli Expressing P Fimbriae Decrease Immune Response of the Mouse Kidney J. Am. Soc. Nephrol., December 1, 2005; 16(12): 3583 - 3591. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F.-E. Johansen, E. S. Baekkevold, H. S. Carlsen, I. N. Farstad, D. Soler, and P. Brandtzaeg Regional induction of adhesion molecules and chemokine receptors explains disparate homing of human B cells to systemic and mucosal effector sites: dispersion from tonsils Blood, July 15, 2005; 106(2): 593 - 600. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, nonimmunized pIgR–/– mice; 
, immunized wild-type mice; 
, immunized pIgR–/– mice.