The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2008, 181, 1787 -1797
Copyright © 2008 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vasilevsky, S.
Right arrow Articles by Snapper, C. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vasilevsky, S.
Right arrow Articles by Snapper, C. M.

Macrophages Pulsed with Streptococcus pneumoniae Elicit a T Cell-Dependent Antibody Response upon Transfer into Naive Mice1

Sam Vasilevsky*, Jesus Colino*, Roman Puliaev*, David H. Canaday{dagger} and Clifford M. Snapper2,*

* Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814; and {dagger} Division of Infectious Disease, Case Western Reserve University, Cleveland, OH 44106


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Macrophages are less effective than DC at priming naive CD4+ T cells, suggesting that DC are unique in initiating T cell-dependent Ab responses. We compared the ability of DC and macrophages, pulsed in vitro with Streptococcus pneumoniae, to elicit protein- and polysaccharide-specific Ig isotype production upon adoptive transfer into naive mice. S. pneumoniae-activated DC secreted more proinflammatory and anti-inflammatory cytokines, expressed higher levels of surface MHC class II and CD40, and presented S. pneumoniae or recombinant pneumococcal surface protein A (PspA) to a PspA-specific T hybridoma more efficiently than macrophages. However, upon adoptive transfer into naive mice, S. pneumoniae-pulsed macrophages elicited an IgM or IgG anti-PspA and anti-polysaccharide response comparable in serum titers and IgG isotype distribution to that induced by DC. The IgG anti-PspA response, in contrast to the IgG anti-polysaccharide, to S. pneumoniae-pulsed macrophages was T cell-dependent. S. pneumoniae-pulsed macrophages that were paraformaldehyde-fixed before transfer or lacking expression of MHC class II or CD40 were highly defective in eliciting an anti-PspA response, although the anti-polysaccharide response was largely unaffected. To our knowledge, these data are the first to indicate that macrophages can play an active role in the induction of a T cell-dependent humoral immune response in a naive host.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Numerous studies have provided compelling evidence that dendritic cells (DC)3 are unique in their capacity to prime naive T cells (1, 2). The role of other APC, such as macrophages and B cells, is thought instead to be primarily the promotion of various effector functions of T cells previously primed by DC. Thus, early studies demonstrated that DC were at least 100-fold more potent at stimulating a primary MLR than macrophages or B cells (3, 4, 5). In addition splenic DC, but not peritoneal macrophages (PerM) or unfractionated (B cell-rich) spleen cells, pulsed in vitro with a number of different soluble proteins, and injected into the footpads of mice, induced significant priming of CD4+ T cells from the draining lymph node (LN), as evidenced by DNA synthesis in an in vitro restimulation assay (6). Similarly, in the presence of peptide Ag, splenic DC were significantly more efficient than splenic B cells, or PerM, splenic macrophages, or bone marrow-derived macrophages (BMM) in promoting DNA synthesis or IL-2 production in naive, peptide-specific transgenic CD4+ T cells in vitro (7, 8). However, it has also been demonstrated that macrophages are heterogeneous in their ability to present Ag to naive CD4+ T cells (9). Thus, ~20% of murine splenic macrophage precursors were found to present native or peptide Ag to, and activate, naive TCR transgenic CD4+ T cells. This presentation correlated with the ability of presenting macrophages to release IL-12. DC have also been found to be the main APC for stimulating primed T cells in vitro, when cultured ex vivo from mice immunized with different soluble proteins; macrophages from immunized mice were nonstimulatory nor were they able to transfer Ag to DC (10). Purified splenic DC, but not splenic or PerM, are also potent APC for restoring the in vitro T cell-dependent primary anti-SRBC response or trinitrophenyl (TNP)-keyhole limpet hemocyanin-induced anti-TNP response in mixtures of B and T cells (11, 12).

Consistent with the studies mentioned, numerous studies have demonstrated the ability of Ag- or pathogen-pulsed DC to elicit immune responses upon adoptive transfer into naive mice (13). However, little is known regarding a similar function for adoptively transferred, Ag-pulsed macrophages. A series of studies by Moser and colleagues (14, 15, 16, 17) indeed demonstrated the ability of nonelicited PerM, as well as splenic DC, pulsed with soluble protein Ag and adoptively transferred into naive mice, to both elicit specific Ab responses and T cell priming in vivo. However, it is unclear from these studies whether the Ag-pulsed macrophage itself was playing an active and direct role in initiating immunity or perhaps was serving to transfer Ag to other, endogenous APC. This latter issue, however, was addressed by Pozzi et al. (18) who investigated the ability of adoptively transferred peptide-pulsed BMM and DC to elicit CTL responses in transgenic CD8+ T cells specific for the peptide bound to MHC class I. Using the approach of adoptive transfer into bone marrow chimeras, both type of APC were shown to directly induce, without requiring peptide transfer to host APC, CD8+ T cell proliferation, cytokine secretion, and differentiation into CTL and memory cells. Although macrophages that were s.c. injected required pulsing with 10-fold more peptide than DC to elicit a comparable response, this function was shown to be a result of lower migration of macrophages to LN and not APC potency per se. Equivalent migration of APC to the spleen and subsequent CD8+ T cell responses were observed, however, using the i.v. route for APC injection.

Several in vivo studies support the notion that endogenous DC are indeed critical, nonredundant APC for initiating immunity. Conventional DC (CD11chigh) can be depleted by injection of diphtheria toxin into transgenic mice that express the diphtheria toxin receptor under the control of the CD11c promoter. DC depletion in these mice results in the abrogation of CTL priming in response to cross-presentation of cell-associated (19) or exogenous soluble (20) Ag. Further CTL generation in response to Listeria monocytogenes and Plasmodium yoelii was also inhibited following DC depletion (19). Another study demonstrated that DC depletion in mice infected with lymphocytic choriomeningitis virus inhibited priming of virus-specific CD8+ T cells, despite the presence of lymphocytic choriomeningitis virus in all major APC (DC, macrophages, and B cells) (21). Priming of CD4+ T cells in response to i.v. injection of soluble or cell-associated protein is also inhibited by splenic conventional DC depletion, whereas LN plasmacytoid DC can substitute for conventional DC for CD4+ T cell priming following s.c. immunization (20). Additionally, following DC depletion, mice immunized i.v. with Mycobacterium tuberculosis, exhibit a substantial delay in specific CD4+ T cell priming (22). Of interest, no studies have been reported to date on the effects of DC depletion on the induction of a T cell-dependent Ab response. Furthermore, although both static and live imaging microscopy has clearly demonstrated the ability of Ag-expressing DC to form stable interactions with, and activate, naive CD4+ T cells (23), the ability of macrophages to mediate a similar function has not been resolved.

To our knowledge, it remains to be determined whether macrophages can play an active and direct role in initiating a CD4+ T cell-dependent humoral response in vivo. We previously demonstrated that mice immunized with intact Streptococcus pneumoniae capsular type 14 (Pn14) elicit an IgG response specific for pneumococcal surface protein A (PspA) and pneumococcal capsular polysaccharide type 14 (PPS14) that is dependent on CD4+ T cells and CD40-CD40L interactions (24). In contrast, the IgM anti-PPS14 response is T cell-independent. Furthermore, we reported that bone marrow-derived DC (BMDC) pulsed with Pn14 in vitro and transferred i.v. into naive, wild-type (WT) mice elicit IgG anti-PPS14 and IgG anti-PPS14 responses that are dependent on viable BMDC before transfer, their ability to secrete IL-6, and the presence of endogenous CD4+ T cells in the recipient mice (25). Whereas the IgG anti-PspA response required BMDC expression of MHC class II, CD40, and B7-1/B7-2, the IgG anti-PPS14 did not. Using this approach, we now wished to directly compare Pn14-pulsed BMDC with two distinct populations of macrophage, BMM and PerM, for their ability to induce anti-S. pneumoniae humoral immunity upon adoptive transfer, and determine the mechanism by which this ability might occur. We show for the first time that macrophages pulsed with Pn14 can play an active, direct role in eliciting a specific humoral response in a naive mouse, comparable to that observed using BMDC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6, BALB/c, and athymic nude mice (BALB/c background) were obtained from the National Cancer Institute (Frederick, MD). CD40–/– mice (BALB/c background; strain name CNCr.129P2-Cd40tm1Kik/J) and MHC class I–/– mice (C57BL/6 background, strain name B6.129-H2dlab1-E{alpha}/J) were obtained from The Jackson Laboratory. CD40–/– and MHC class II–/– mice were bred and maintained at Uniformed Services University of the Health Sciences in a pathogen-free environment, and were used between 7 and 12 wk of age.

Reagents

Recombinant PspA (family 1, seroclade 2) was expressed in Saccharomyces cerevisiae BJ3505 and purified as previously described (26). Purified PPS14 was purchased from the American Type Culture Collection. Lipopeptide Pam3Cys-Ser-Lys4 (Pam3Cys) and purified LPS from Escherichia coli K12 strain (LPS) were purchased from InvivoGen. Phosphorothiorated 30-mer CpG oligodeoxynucleotide (27) was synthesized in the Biomedical Instrumentation Center (Uniformed Services University of the Health Sciences).

Preparation of S. pneumoniae Pn14

A frozen stock of Pn14 was thawed and subcultured on BBL pre-made blood agar plates (VWR International). Isolated colonies in blood agar were grown in Todd Hewitt broth (BD Biosciences) to mid-log phase, collected, and heat killed by incubation at 60°C for 1 h. Sterility was confirmed by subculture on blood agar plates. After extensive washings, the bacterial suspension was adjusted with PBS to give an absorbance reading at 650 nm of 0.6, which corresponded to 109 CFU/ml. Bacteria were then aliquoted at 1010 CFU/ml and frozen at –80°C until their use as Ag for mouse immunizations.

Pneumococcal strain R6–14 (R6–14)

The S. pneumoniae strain R6J (R6cps::Janus), a variant of the strain R6 in which the capsule locus is substituted by a Janus cassette, was provided by Dr. K. Trzcinski and M. Lipsitch (Harvard School of Public Health, Boston, MA). An IgG1{kappa} mouse mAb specific for the capsular polysaccharide of S. pneumoniae type 14 (44.1) was provided by Dr. A. Lucas (Children’s Hospital Oakland Research Institute, Oakland, CA). Two IgG2a mouse mAbs specific for the family 1, seroclade 2 of PspA (DC10-IA5 and CF9IIB7) were provided by Dr. R. Schuman (Biosynexus, Inc., Gaithersburg, MD). An isogenic variant of S. pneumoniae strain R6 expressing capsular polysaccharide type 14 (R6–14) was constructed by transformation with chromosomal DNA, essentially as described (28). The expression of serologically intact Cps14 on the selected smooth colonies was confirmed by colony-blot using the mouse mAb 44.1 specific for Cps14 as detection Ab. DNA from one isolate was purified and used to retransform R6J into a Cps14-expressing strain again. This process of backcross transformation was repeated three times to minimize uncontrolled recombinational replacements in loci other than cps. R6–14 expresses quantitatively equivalent levels of Cps14 relative to the donor strain (Pn14) as determined by quantitative sandwich ELISA. The PspA is serologically identical with the PspA of the recipient R6 strain.

PspA-specific T hybridomas

CD4+ T cell hybridomas specific for PspA peptide in association with MHC class IId were produced and screened according to a previously published protocol (29). Briefly, BALB/c mice were injected in the footpad with 50 µg of recombinant PspA emulsified in CFA (Sigma-Aldrich). On day 7, popliteal LN cells were cultured with PspA at 5 µg/ml in the presence of IL-2. Cells were then harvested on day 6 and fused, using polyethylene glycol (Boehringer Mannheim), with the fusion partner BW1100. Clones that grew in hypoxanthine/aminopterin/thymidine (Sigma-Aldrich) selection medium were screened for PspA specificity and MHC restriction. One CD4+CD8– T hybridoma, BALD4, was selected for use in these studies.

Preparation of BMDC

BMDC were prepared as previously described (25). Briefly, bone marrow cells were cultured at 1.25 x 106 cells/ml (24-well plates) in RPMI 1640 plus 5% FBS, 10,000 IU penicillin and 10 µg/ml streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM nonessential amino acids, and 25 mM HEPES (culture medium), supplemented with 10 ng/ml murine rGM-CSF (Sigma-Aldrich). After 7 days of culture, nonadherent cells were harvested.

Preparation of BMM

BMM were obtained using a similar approach to that for BMDCs, with slight modifications. Bone marrow cells were cultured at 1 x 106 cells/ml in cell culture medium supplemented with 10 ng/ml murine M-CSF (Sigma-Aldrich). Cells were plated in 6-well plates in a volume of 4–5 ml/well. On days 3 and 5, three-fourths culture medium was removed and fresh culture medium was added. On day 7, BMM were harvested by washing plates with sterile PBS to remove nonadherent cells. Cells were detached from the plate by adding 2 ml of detachment buffer (4 mg/ml lidocaine, 5 mM EDTA, PBS) for 3–5 min with pipetting.

Isolation of PerM

Culture medium (10 ml) was injected into the peritoneal cavity using a 23-gauge needle. After 3 min, lavage fluid containing the peritoneal cells was withdrawn using an 18-gauge needle. Cells were pelleted by centrifugation at 400 x g for 10 min. RBC were removed by lysis with ACK lysing buffer. Cells were resuspended in culture medium, and cultured in 6-well plates (5 ml/plate) at 1 x 106 cells/ml overnight, followed by washing of plates with sterile PBS to remove nonadherent cells. Adherent cells were then detached by adding 2 ml of detachment buffer (4 mg/ml lidocaine, 5 mM EDTA, PBS) for 3–5 min with pipetting.

In vitro culture of APC

APC were plated in culture medium without added GM-CSF or M-CSF, at 1 x 106 cells/ml in 24-well cell culture plates (Costar). After 30 min, to allow cells to settle and to completely reverse the transient metabolic effects of the lidocaine on the macrophage population, varying concentrations of Pn14 or TLR ligands were added to the cultures for 24 h.

APC stimulation of IL-2 production by PspA-specific T cell hybridoma

APC were plated at 2 x 104 APC/well in 96-well flat-bottom tissue culture plates (Costar), containing serial dilutions of PspA or bacteria. After 2 h of pulse with the Ag at 37°C, the T cell hybridoma was added to the cultures at 1 x 105 cells/well and cocultured with the APC for 24 h in the presence of free Ag. APC derived from C57BL/6 bone marrow cells were used in every experiment as a control of MHC-restricted Ag presentation. Negative controls included cultures of the T cell hybridoma and Ag in the absence of APC, and APC cultured with Ag in the absence of T cell hybridoma.

Adoptive transfer of Pn14-pulsed APC

APC were cultured at 1 x 106 cells/ml for 4 h in the presence of 2 x 109 CFU of Pn14 in vitro. Free bacteria were then removed from the cell cultures by washing about six times in cold PBS, each time followed by centrifugation for 10 min at 350 x g at 4°C. A control tube containing a mixture of thymocytes and CM-DiI (chloromethylbenzamido derivative DiI; Molecular Probes) fluorescent-labeled Pn14 at the same initial bacterial density was used to monitor the progress of the washings. If greater than 100 free bacteria were found in pelleted cells, using an inverted fluorescent microscope, the washings were continued. Pn14-pulsed cells were then resuspended in fresh medium at 1 x 106 cells/200 µl, and 200 µl were injected i.v. into each mouse.

Measurement of cytokine concentrations in culture supernatant by ELISA

IL-6, IL-10, IL-12, and TNF-{alpha} concentrations released into the culture medium by activated APC or T hybridoma cells were measured by ELISA according to the manufacturer’s instructions (BD Pharmingen). TGF-β was measured by ELISA (TGF-β1 mouse/rat/porcine/canine Quantikine ELISA kit, catalog no. MB100B; R&D Systems). The ELISA measured only active TGF-β. To measure total TGF-β (active and latent), the TGF-β in culture supernatant was first purposefully activated using hydrochloric acid followed by neutralization using sodium hydroxide/HEPES according to the manufacturer’s instructions, then assayed immediately by ELISA. Standards were included in every plate, and the samples were tested in duplicate.

Flow cytometric analysis

All steps were performed on ice. Fc{gamma}R were blocked with 10 µg/ml rat IgG2b,{kappa} anti-mouse Fc{gamma}RI/II/III (clone 2.4G2). Cells were stained for 30 min with either PE-Armenian hamster IgG1,{lambda}2 anti-mouse CD11c (clone HL3), FITC-mouse IgG2b,{kappa} anti-mouse MHC class IId (clone AMS-32.1), FITC-rat IgG2a,{kappa} anti-mouse CD40 (clone 3/23), PE-rat IgG2a,{kappa} anti-mouse CD86 (clone GL1), Armenian hamster IgG2a,{kappa} anti-mouse CD80 (clone 16-10A1), or PE-rat IgG1,{kappa} anti-mouse CD14 (clone rmc5-3). All mAbs were purchased from BD Pharmingen. FITC rat anti-mouse F4/80 (clone C1:A3-1) was purchased from Accurate Chemical and Scientific. Irrelevant isotype- and species-matched mAbs were used as staining controls. Cells were analyzed on an EPICS XL-MCL flow cytometer (Beckman Coulter). Dead cells and debris were eliminated from analysis by excluding cells positive for propidium iodide and gating on the appropriate forward and side scatter profile.

Measurement of serum Ig isotype titers

Serum titers of PspA- and PPS14-specific Ig isotypes were measured by ELISA as previously described (30).

Fluorescent labeling of Pn14 with CM-DiI

Pn14 (1 x 109 CFU) were incubated at 37°C for 10 min, in 5 mM CM-DiI in PBS, followed by additional 30 min of incubation at 4°C, and washed before use.

Phagocytosis assay

APC were pulsed with CM-DiI-labeled Pn14. At different times, APC were washed in PBS, and viable cells were analyzed by flow cytometry with gating, according to size, to exclude free bacteria. To control for cell surface binding of Pn14, cytochalasin D (5 µg/ml; Calbiochem) was added to APC cultures (31). The treatment markedly inhibited the internalization of CM-DiI-labeled bacteria (<4% of the mean fluorescence intensity uptake by untreated cells).

Fixation of BMM

BMM were fixed by incubation for 30 min in 1% paraformaldehyde in PBS at room temperature and washed three times in PBS before use.

Statistics

Data were expressed as geometric mean ± SEM of the individual results. Student’s t test was used to determine statistical significance between groups. A value for p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Phenotypic characterization of unstimulated and Pn14-activated BMDC, BMM, and PerM populations in vitro

We previously demonstrated that BMDC pulsed with intact heat-killed Pn14 elicited, via distinct mechanisms, both protein- and polysaccaride-specific Ig isotype responses upon adoptive transfer into naive mice (25). Using this approach, we now wished to compare the ability of Pn14-pulsed macrophages and DC to elicit Ag-specific Ig isotype responses in vivo. Heat-killed Pn14 was used in all experiments. Two distinct macrophage populations were used: freshly harvested PerM obtained in the absence of thioglycolate elicitation, and macrophages obtained after culturing bone marrow cells for 6 days in M-CSF (BMM). A similar approach, in which bone marrow cells were instead cultured in GM-CSF, was used to produce BMDC. BMDC were only lightly adherent to the tissue culture plastic, and were easily removed by pipetting (32). In contrast, BMM and PerM were firmly adherent. We effected detachment by using lidocaine as previously described (33), instead of the more traditional method of cell scraping, because we found the latter caused significant cell death (data not shown). Although reports have shown that lidocaine can inhibit macrophage O2 consumption, digestion of immune complexes, or metabolic activity, all of these inhibitory effects could be completely reversed within 30 min following washing of the macrophages in medium (34, 35, 36). In this regard, we cultured lidocaine-detached macrophages in medium alone for at least 30 min before adding bacteria or other activators.

The three cell populations cultured in medium alone each exhibited distinct patterns of expression of a number of key cell surface phenotypic markers (Fig. 1). As expected (37, 38), BMM and PerM both expressed, in a comparable fashion, significantly higher levels of F4/80, CD11b, and CD14 than BMDC. Whereas all three populations expressed CD11c, BMM exhibited the highest expression. Of interest, whereas BMDC broadly expressed a high mean level of MHC class II, compared with the uniformly low expression by BMM, a major subpopulation of PerM constitutively exhibited high levels of MHC class II. In contrast, all three populations expressed low levels of CD40, although PerM exhibited somewhat higher levels than either BMDC or BMM. CD86 expression on unstimulated BMDC was low, whereas BMM and PerM exhibited moderately higher and comparable levels. In contrast, significant CD80 expression was observed on both BMDC and BMM, but found at much lower levels on PerM. These data confirm two distinct populations of macrophages that each contrast with BMDC and suggest <3% cross-contamination between the macrophage and DC populations.


Figure 1
View larger version (35K):
[in this window]
[in a new window]

  		FIGURE 1. Cell surface phenotype of unstimulated and Pn14-activated BMDC, BMM, and PerM. APC were stained with fluorochrome-labeled mAbs specific for the indicated cell surface proteins after a 24-h culture period (1 x 106 cells/ml) in medium alone or in the presence of Pn14 (2 x 109 CFU/ml), and analyzed by flow cytometry. Staining with species- or isotype-matched control mAbs are below each set of tracings. Results representative of two independent experiments.

 
Following 24 h of culture with intact Pn14, expression of MHC class II, CD80, CD86, CD40, CD14, CD11b, and CD11c each decreased significantly on viable PerM relative to cells cultured in medium alone (Fig. 1). In contrast, CD40 expression was markedly up-regulated on both BMDC and BMM following Pn14-mediated activation, whereas each moderately increased CD86. No significant modulation of either MHC class II or CD80 was observed in BMM, whereas BMDC exhibited a moderate increase in CD80, although no significant change in mean MHC class II expression was shown. BMDC also up-regulated CD11c. Collectively, the phenotypic profiles of both unstimulated PerM and Pn14-activated BMDC suggest that these cells may, during some time interval, be potentially effective APC for CD4+ T cells. However, low expression of MHC class II on BMM, in the absence or presence of Pn14, despite relatively high expression of costimulatory molecules following activation, suggests that these cells are likely to function poorly as APCs.

BMDC produce higher levels of proinflammatory and anti-inflammatory cytokines than BMM or PerM in response to Pn14

Secretion of cytokines by activated APC can significantly impact on CD4+ T cell activation, and differentiation into distinct subsets, with subsequent differing effects on humoral immunity (39, 40, 41). In particular we previously demonstrated that endogenous proinflammatory cytokines stimulate, and the anti-inflammatory cytokine IL-10, inhibits humoral immunity in response to Pn14 (40). In this regard, we wished to compare the ability of BMDC, BMM, and PerM to secrete key proinflammatory (IL-6, TNF-{alpha}, and IL-12), and (IL-10) anti-inflammatory cytokines in vitro in response to graded doses of Pn14. As illustrated in Fig. 2A, BMDC secreted significantly (p < 0.05) higher levels of all four cytokines than BMM or PerM, at the two highest doses of Pn14, and higher levels of IL-6 at all doses. BMM made significantly higher levels of IL-12 than PerM at all doses of Pn14, whereas the latter was more sensitive to low dose Pn14 for TNF-{alpha} secretion. We previously demonstrated that Pn14 elicits cytokine production by splenic macrophages and DC through the combined action of TLR2, TLR4, and TLR9 (42). In this regard, a similar pattern of IL-12 secretion as that observed for intact Pn14 was also observed using optimal amounts of individual TLR ligands (i.e., LPS (TLR4), CpG oligodeoxynucleotide (TLR9), and Pam3Cys (TLR2)) (43) (Fig. 2B). Likewise, BMDC secreted significantly higher amounts of TNF-{alpha} than BMM or PerM in response to all three TLR ligands, and PerM made significantly more TNF-{alpha} than BMM at least in response to LPS. In a separate experiment to measure release of TGF-β, an anti-inflammatory cytokine, BMDC, BMM, and PerM were cultured for 24 h in the presence of either medium alone or Pn14. No detectable (<1 pg/ml) active TGF-β was observed in either medium alone or in the presence of Pn14 from any of the three cell types (Fig. 2C). Detectable amounts of total (active plus latent) TGF-β were released constitutively by all three cell populations with only modest, though significant differences between the cell types (PerM>BMDC>BMM). In the presence of Pn14, BMDC showed a only modest, though significant, increase in total TGF-β release, whereas BMM and PerM showed modest, though significant, decreases (Fig. 2C). Collectively, although these data indicate that BMDC elicit a more robust in vitro, proinflammatory cytokine response to Pn14 than either BMM or PerM, it is not clear whether the higher levels of IL-10 secreted by BMDC would counteract the potential in vivo Ig-stimulatory effects of the enhanced IL-6, IL-12, and TNF-{alpha} response (40). Alternatively, as we reported previously, autocrine IL-10 might enhance APC-mediated induction of Ig secretion by delaying APC apoptosis (41).


Figure 2
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 2. Cytokine secretion by BMDC, BMM, or PerM in the absence or presence of Pn14 or TLR ligands. APC were cultured for 24 h in medium alone or in the presence of varying concentrations of Pn14 (A), LPS (2 µg/ml), CpG-ODN (2 µg/ml), or Pam3CSK4 (150 ng/ml) (B), or 2 x 109 CFU/ml of Pn14 (C). Concentrations of cytokines in the culture supernatant were measured by ELISA. Values represent the geometric mean ± SEM. p < 0.05. Results are representative of two independent experiments.

 
BMM>BMDC>PerM for phagocytosis of Pn14

Although both macrophages and DC can phagocytose intact bacteria, the rate by which this occurs could impact on their ability to act as APC. In particular, we were interested in establishing conditions to obtain roughly comparable bacterial uptake by BMDC, BMM, and PerM in vitro, to better compare their ability to induce humoral responses upon adoptive transfer into naive mice. In a first set of experiments, Pn14 was labeled with the fluorescent dye CM-Dil and added to cultures of APC at a ratio of 800 CFU Pn14 to APC for varying periods of time. The mean fluorescence intensity of the three APC populations was measured to compare the relative uptake of bacteria at each time point. Because fluorescence could reflect cell surface binding rather than actual uptake, we included control groups consisting of the various APC cultured with Pn14 in the presence of cytochalasin D, an inhibitor of microfilament assembly, and hence phagocytosis, for 300 min (the latest time point). We previously demonstrated that cytochalasin D blocks Pn14 uptake into BMDC (31). As illustrated in Fig. 3A the efficiency of uptake of Pn14 was BMM>BMDC>PerM. With the addition of concentrations of Pn14 higher than 800 CFU/APC, these differences could be somewhat narrowed over time (Fig. 3B). In a separate experiment we determined that BMDC, BMM, and PerM internalized 27, 58, and 14 CFU of Pn14 per cell, respectively, following 4 h of culture with 2000 CFU Pn14/APC. We used these conditions for our subsequent adoptive transfer experiments.


Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 3. Uptake of Pn14 by BMDC, BMM, and PerM. APC were cultured in the presence of CM-Dil-labeled Pn14. A, APC were cultured for the indicated times with 8 x 108 CFU of labeled Pn14 (800 CFU/APC). B, APC were cultured for 240 min with the indicated concentrations of labeled Pn14. Cytochalasin D ("cyto") was used to control for surface binding of labeled Pn14. APC were analyzed by flow cytometry. Results are representative of two independent experiments.

 
Pn14-pulsed BMM and PerM elicit humoral immune responses comparable to BMDC upon adoptive transfer into naive mice

BMDC, BMM, and PerM were pulsed with Pn14 in vitro under the conditions cited above. After thorough washing to remove free bacteria, 1 x 106 Pn14-pulsed APC were injected i.v. per mouse. As controls, free Pn14 and unpulsed APC were also i.v. injected into separate groups of mice. The percentages of Pn14-pulsed BMDC and BMM found in the spleen, 24 h after i.v. injection were 0.01% and 0.03% of the total spleen cell population, respectively (significant, p < 0.05 difference for one representative experiment of two independent experiments). Fourteen days postimmunization with either Pn14-pulsed APC or free Pn14, all groups were boosted i.p. with free Pn14 to asses the potential generation of memory during the primary immunization. Sera were collected on days 0 (pre-bleed), 7, 14 (following primary immunization with Pn14-pulsed APC or free Pn14), and 21 (following secondary immunization with free Pn14) for determination of titers of IgG specific for PspA, and IgM and IgG specific for the PPS14. As illustrated in Fig. 4A, Pn14-pulsed BMDC, BMM, and PerM each effectively primed naive mice, in a comparable manner, for a secondary IgG anti-PspA response following boosting, on day 14, with free Pn14. Thus, day 21 serum titers of IgG anti-PspA were significantly (p < 0.05) higher in all groups primed with Pn14-pulsed APC relative to mice primed with unpulsed APC, and relative to naive mice, 7 days after immunization with free Pn14. Additionally, a small, but significant, primary IgG anti-PspA response was also elicited by all Pn14-pulsed APC, in contrast to undetectable titers in mice primed with unpulsed APC (Fig. 4A). In contrast, none of the Pn14-pulsed APC populations primed mice for a secondary IgM or IgG anti-PPS14 response following boosting with free Pn14 (Fig. 4A). However, all Pn14-pulsed APC induced substantial primary IgM and IgG anti-PPS14 responses, in contrast to the undetectable titers observed in mice injected with unpulsed APC. Additionally, no significant differences in day 21 serum titers of PspA-specific IgG3, IgG1, IgG2b, or IgG2a were observed between any of the groups primed with Pn14-pulsed APC and boosted with free Pn14 (Fig. 4B). With the exception of significantly higher day 14 serum titers of PPS14-specific IgG3 and IgG1 in mice injected with Pn14-pulsed BMM relative to BMDC, PPS14-specific IgG isotypes were otherwise comparable among the three groups (Fig. 4B).


Figure 4
View larger version (37K):
[in this window]
[in a new window]

  		FIGURE 4. Adoptive transfer of Pn14-pulsed BMDC, BMM, and PerM into naive, WT mice. APC were cultured in medium alone or pulsed with Pn14, and injected i.v. into naive BALB/c mice (n = 6 mice per group). Mice were boosted i.p. with free Pn14 (2 x 108 CFU/mouse) 14 days following injection of APC. Another group of BALB/c mice (n = 6) were immunized and boosted on day 14 with free Pn14 alone. A, Sera were collected on the indicated days for measurement of serum Ig titers. B, Titers of secondary (day 21) PspA-specific and primary (day 14) PPS14-specific IgG isotypes were measured from sera analyzed in A. The data show the geometric mean ± SEM of the individual titers. *, p < 0.05. Results are representative of two independent experiments.

 
Pn14-pulsed BMM play an active role as APC for initiating the PspA-specific IgG response

The ability of Pn14-pulsed macrophages to initiate a humoral immune response upon adoptive transfer into naive mice could reflect an active process mediated by the transferred macrophages or, alternatively, the passive transfer by macrophages of intact Pn14 or Pn14-derived Ags to other immune cell types. Furthermore, Pn14-pulsed macrophages could be playing a direct and critical role in presenting Pn14-derived proteins to CD4+ T cells in vivo or, alternatively, might be actively transferring Pn14 proteins to endogenous APC. In the following series of studies to test these possibilities, we focused solely upon BMM, as opposed to PerM, because of our ability to obtain substantially larger numbers of BMM for our experiments. BMM were pulsed with Pn14 as described for adoptive transfer (see Fig. 4), then either fixed with paraformaldehyde or left untreated, immediately before i.v. injection. The percentages of Pn14-pulsed viable and fixed BMM found in the spleen 24 h after i.v. injection were 0.02% and 0.06% of the total spleen cell population, respectively (significant difference, p < 0.05, for one representative experiment of two independent experiments). As illustrated in Fig. 5, viable, but not fixed, Pn14-pulsed BMM elicited a primary PspA-specific IgG response, and primed mice for a secondary IgG response following boosting with free Pn14, similar to that observed in Fig. 4. In distinct contrast, fixed Pn14-pulsed BMM elicited an IgG anti-PPS14 response that was not significantly different from that for viable BMM, whereas the IgM anti-PPS14 was only modestly, though, significantly reduced (Fig. 5). In a separate experiment, serving as a control, BALB/c mice (seven per group) were immunized and boosted with free heat-killed Pn14 with or without further paraformaldehyde fixation. No significant differences in the serum titers of IgG anti-PspA, as well as IgM or IgG anti-PPS14 were observed (data not shown) strongly suggesting that paraformaldehyde did not destroy key antigenic epitopes in the bacteria. Thus, viable BMM are required in vivo for induction of the PspA-, though not PPS14-specific, Ig response.


Figure 5
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 5. Adoptive transfer of viable and paraformaldehyde-fixed Pn14-pulsed BMM. BMM obtained from BALB/c mice were pulsed in vitro with Pn14. BMM were then either left untreated or fixed in paraformaldehyde, and immediately injected i.v. into naive BALB/c mice (n = 5 mice per group). Mice were immunized i.p. 14 days later with free Pn14 (2 x 108 CFU/mouse). A separate group of mice (n = 5) were immunized and i.p. boosted on day 14 with free Pn14 alone (2 x 108 CFU/mouse). Sera were collected on the indicated days for measurement of serum Ig titers. Data show the geometric mean ± SEM of the individual titers. *, p < 0.05, comparing viable vs fixed BMM. Results are representative of two independent experiments.

 
To assess whether transferred Pn14-pulsed BMM were playing an active role as APC in vivo for induction of the PspA-specific IgG response, we first wished to determine whether this response required endogenous T cells. We previously demonstrated that both the IgG anti-PspA and IgG anti-PPS14 response to free Pn14 was dependent on CD4+, but not CD8+ T cells, whereas the IgM anti-PPS14 response was T cell-independent (24, 44). Pn14-pulsed BMM or free Pn14 were injected into either naive WT or athymic nude mice, the latter being markedly deficient in T cells. As illustrated in Fig. 6, both Pn14-pulsed BMM and free Pn14 significantly (p < 0.05) primed WT mice, but not athymic nude mice, for a subsequent secondary IgG anti-PspA response following boosting with free Pn14. In distinct contrast Pn14-pulsed BMM elicited an equivalent primary IgG anti-PPS14 response in WT and athymic nude mice (Fig. 6). Boosting of WT mice, initially injected with Pn14-pulsed BMM, with free Pn14 increased the IgG anti-PPS14 response only to those levels seen in WT mice immunized with free Pn14 alone, confirming that there was no significant priming by Pn14-pulsed BMM for induction of PPS14-specific IgG. These data thus demonstrate that the IgG anti-PspA response to Pn14-pulsed BMM is dependent on endogenous CD4+ T cells.


Figure 6
View larger version (25K):
[in this window]
[in a new window]

  		FIGURE 6. Adoptive transfer of Pn14-pulsed BMM into athymic nude mice. BMM obtained from BALB/c mice were pulsed in vitro with Pn14. BMM (i.v.) or free Pn14 (2 x 108 CFU/mouse, i.p.) were then injected into either naive BALB/c or athymic nude (BALB/c background) mice (n = 5 mice per group). All groups of mice were i.p. immunized 14 days later with free Pn14 (2 x 108 CFU/mouse). Sera were collected on the indicated days for measurement of serum Ig titers. Data show the geometric mean ± SEM of the individual titers. p < 0.05. Results are representative of two independent experiments.

 
We next wished to address whether the IgG anti-PspA response to Pn14-pulsed BMM required cognate interactions between the transferred BMM and the endogenous CD4+ T cells. Thus, BMM were obtained from mice genetically deficient in MHC class II. WT and MHC class II–/– BMM were pulsed with Pn14 in vitro and injected i.v. into naive WT mice followed by boosting i.p. with free Pn14 on day 14. A separate group of WT mice were immunized and boosted with free Pn14 alone as a control. In contrast to WT Pn14-pulsed BMM, MHC class II–/– BMM exhibited a complete failure to prime for a secondary IgG anti-PspA response (Fig. 7A). In contrast, both WT and MHC class II–/– Pn14-pulsed BMM elicited equivalent IgM and IgG anti-PPS14 responses. In a second, analogous set of studies, we compared WT and CD40–/– Pn14-pulsed BMM for their ability to elicit Ig responses upon adoptive transfer into WT mice. During cognate interactions, CD40L on activated T cells binds CD40 expressed by APC, resulting in delivery of a potent costimulus for cytokine secretion and phenotypic maturation of the APC (45). In this regard, we observed that the primary IgG anti-PspA response elicited by Pn14-pulsed CD40–/– BMM was markedly reduced relative to WT BMM, whereas priming by CD40–/– BMM for the secondary response was partially, though significantly, reduced as well (Fig. 7B). In contrast, CD40–/– and WT BMM elicited essentially equivalent IgM and IgG anti-PPS14 responses (Fig. 7B).


Figure 7
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 7. Adoptive transfer of Pn14-pulsed BMM from WT vs MHC class II–/– or CD40–/– mice into naive WT mice. BMM obtained from C57BL/6 (WT) or MHC class II–/– mice (C567BL/6 background) (A) or BALB/c (WT) or CD40–/– mice (BALB/c background) (B) were pulsed in vitro with Pn14. BMM (i.v.) or free Pn14 (2 x 108 CFU/mouse i.p.) were then injected into naive C57BL/6 (A) or BALB/c (B) mice (n = 5 mice per group). All groups of mice were immunized i.p. 14 days later with free Pn14 (2 x 108 CFU/mouse). Sera were collected on the indicated days for measurement of serum Ig titers. Data show the geometric mean ± SEM of the individual titers. *, p < 0.05, in comparing BMM obtained from WT vs knockout mice. Each experiment is representative of two independent experiments.

 
BMDC pulsed with either recombinant PspA or intact Pn14 are more potent stimulators of IL-2 secretion by a PspA-specific T hybridoma, than similarly treated BMM

Collectively, the data so far strongly suggest that Pn14-pulsed BMM, in addition to BMDC, can play an active role as APC for CD4+ T cells when adoptively transferred into naive mice. In a final set of experiments we therefore wished to assess the relative ability of BMDC and BMM to act as APC for CD4+ T cells upon uptake of Pn14. We thus created an MHC class IId-restricted CD4+ T hybridoma (BALD4) specific for the Pn14 cell wall protein, PspA (see Materials and Methods). In preliminary studies, we observed that BMDC cultured with Pn14 failed to stimulate BALD4 for IL-2 secretion (data not shown). This observation was in distinct contrast with either recombinant PspA, intact S. pneumoniae capsular type 2 (strain D39) or its unencapsulated isogenic mutant (strain R36A). In light of previous reports that different strains of S. pneumoniae can express antigenically distinct families/seroclades of PspA, we concluded that BALD4 recognized an epitope of PspA not expressed by our Pn14 strain. We thus genetically engineered a new strain of S. pneumoniae capsular type 14 (R6–14) that produced the same family 1/seroclade 2 of PspA as strain R36A (both recognized by the mAbs DC10-IA5 and CF9IIB7, and able to stimulate BALD4), but expressed the same serotype 14 capsular polysaccaride as Pn14 (both recognized by the mAb 44.1) (see Materials and Methods).

BMDC and BMM were cocultured with BALD4 for 24 h in the presence of varying concentrations of PspA (0.001–25 µg/ml) or R6-Pn14 or its unencapsulated isogenic mutant R6 (0.03–67 x 106 CFU/ml). At the end of the culture period, supernatant was collected for determination of IL-2 concentrations by ELISA. In three independent experiments, BMDC were found to be 12 ± 2-fold (arithmetic mean ± SEM) more efficient at presenting PspA than BMM (p < 0.05) (Fig. 8). This efficiency reflects the amount of additional PspA required for BMM to give an IL-2 response equivalent to that induced by BMDC. The difference in efficiency between BMDC and BMM cocultured with either R6–14 or R6, for induction of IL-2 by BALD4 was even more marked than that observed for PspA: 443 ± 34 for R6–14 (two independent experiments) and 418 ± 117 for R6 (three independent experiments) (Fig. 8). Of interest, although it has been suggested that the capsular polysaccaride might interfere with, or otherwise modulate, the presentation of bacterial protein to T cells (46, 47, 48, 49), the IL-2 induction profiles using R6–14 and R6 were essentially identical. The results in Fig. 8 were aligned so that the amount of PspA, calculated by an inhibition ELISA, contained in a given dose of R6–14 or R6 is directly (vertically) beneath the concentration given for PspA in the uppermost display. This indicates that BMDC, though not BMM, present PspA ~100-fold more efficiently to BALD4 when expressed by the intact bacteria as opposed to being present in an isolated, soluble form (Fig. 8). Collectively, these data strongly suggest that Pn14-pulsed BMM can play an active role as APC for eliciting a robust TD humoral immune response in vivo, despite a markedly lower APC efficiency in vitro relative to BMDC.


Figure 8
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 8. APC capacity of BMDC vs BMM pulsed with either free PspA or unencapsulated or encapsulated Pn for a PspA-specific T hybridoma. BMDC and BMM obtained from BALB/c mice were cultured in the presence of the indicated concentrations of PspA (A), R6–14 (B), or R6 (C). BALD4 (PspA-specific T hybridoma) cells were then added to these cultures, in the continued presence of the added Ag/bacteria, and cultured for 24 h. Concentrations of IL-2 released by BALD4 cells in the culture supernatant were measured by ELISA. Results are representative of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Although many studies have documented the ability of macrophages to internalize pathogen-associated Ags, and present them to preprimed T cells (50, 51, 52, 53, 54), it is widely assumed that the induction of CD4+ T cell-dependent Ab responses in naive mice is uniquely dependent upon initial Ag presentation by DC (1, 2). Following this event, Ag-specific B cells engage in cognate interactions with DC-primed CD4+ T cells to elicit effector functions critical for induction of Ig secretion and class switching (55). Although a role for macrophages as APC in this latter process is largely obscure, macrophages may regulate humoral immunity through other mechanisms, including transfer of intact Ag to B cells (56, 57), secretion of BAFF, a B cell survival factor and inducer of Ig class switching (58), or local release of C3 (59). Although the indirect evidence in support of a central role for DC as APC in naive mice is extensive, there is to our knowledge, no published data directly demonstrating that endogenous DC are indeed indispensable for eliciting in vivo humoral immune responses. Thus, although several studies, using CD11c diphtheria toxin receptor transgenic mice, treated with diphtheria toxin to deplete DC, demonstrated a critical role for endogenous DC in eliciting a CD8+ CTL response (19, 20, 21) or CD4+ T cell priming (22), in the model systems studied, no comparable data on humoral immunity have as yet been reported. Furthermore, although a number of imaging studies have documented early DC-CD4+ T cell interactions in situ, following immunization with protein Ag (23), there is little indication that potential macrophage CD4+ T cells were extensively investigated, especially in the spleen, leaving open their possible role as initiating APC for naive T cells.

Two groups studied the ability of Ag-pulsed macrophages to elicit immunity upon adoptive transfer into naive mice, and surprisingly both reported that they were indeed capable of acting in a manner comparable to that of Ag-pulsed DC. In the series of studies by Moser and colleagues (15, 16, 17) macrophages pulsed in vitro with protein Ag were shown to elicit specific Ab production upon injection into naive mice. However, the possibility that the injected macrophages were acting largely via passive transfer of Ag to endogenous DC was not ruled out. However, a study by Pozzi et al. (18), demonstrated the ability of peptide-pulsed macrophages to elicit CD8+ CTL responses upon injection into naive mice, in which a role for endogenous APC in the recipients was clearly ruled out.

In our current study we demonstrate that Pn14-pulsed macrophages play an active role in eliciting the IgG anti-PspA response, upon their transfer into naive mice. This response was abrogated in T cell-deficient recipients, consistent with our earlier studies using free Pn14, in which an absolute dependence on CD4+ T cells was demonstrated (24). We further show that the induction of the IgG anti-PspA response by Pn14-pulsed macrophages was not due exclusively to passive transfer of PspA to endogenous APC. Thus, macrophages from mice genetically deficient in MHC class II or CD40, or WT macrophages made nonviable before transfer, were defective in eliciting the IgG anti-PspA response in WT mice, following in vitro pulsing with intact Pn14. This requirement for macrophage expression of MHC class II and CD40 further supports the notion that these cells were acting as APC for CD4+ T cells. We believe that it is highly unlikely that the observed Ig-inducing effects of the Pn14-pulsed macrophage populations used in this study were due to contaminating DC. Firstly, we used cells that were firmly adherent to the tissue culture plastic following 7 days of culture in the presence of M-CSF-1, a feature not traditionally observed for DC. Secondly, phenotypic analysis of the macrophage populations revealed an essentially unimodal population of cells with a relatively high expression of F4/80, CD14, and CD11b, characteristic of macrophages (37, 38). Finally, the ability of the Pn-pulsed BMM population to present the bacterial PspA to a PspA-specific T hybridoma was ~400-fold less efficient than Pn-pulsed BMDC.

In contrast to the in vivo IgG anti-PspA response induced by Pn14-pulsed BMM, the IgG anti-PPS14 response was not dependent on BMM expression of MHC class II or CD40, was largely independent of BMM viability immediately before transfer, and was T cell-independent. However, as previously described (24), the IgG anti-PPS14 response to free Pn14 was T cell-dependent. The IgG anti-PPS14 response elicited by Pn14-pulsed BMM also differed in several respects with what we previously observed using Pn14-pulsed BMDC (25). Using BMDC, the IgG anti-PPS14 response did dependent on BMDC viability and was T cell-dependent, although with the exception of the IgG1 isotype, not dependent on BMDC expression of MHC class II or CD40. The mechanism by which Pn14-pulsed BMM elicited the anti-PPS14 response is not clear, although macrophages have been previously implicated in stimulating T cell-independent Ab responses. Thus, it was demonstrated that macrophages can directly enhance proliferation of B cells in vitro (58). Furthermore, in vitro T cell-independent B cell responses to TNP-Ficoll, TNP-LPS and TNP-Brucella abortus are dependent on the presence of macrophages (60, 61, 62, 63). B cells that are stimulated by TNP-Ficoll-pulsed macrophages appear to be a distinct B cell subset that is uniquely responsive to macrophage signals (64). Hence, direct activation of B cells by macrophages, even perhaps macrophages in a nonviable state, might account for their inductive effect.

We demonstrate that Pn14-pulsed macrophages can stimulate a PspA-specific T hybridoma in an MHC class II-restricted manner following uptake of intact Pn14 or free PspA, although with much lower efficiency than DC. Others, using naive or primed CD4+ T cells have also demonstrated a similar difference in APC efficiency (1, 2). We directly compared presentation of a protein expressed by an intact extracellular bacterium with the same protein, produced in an isolated, soluble form. In this regard, we show that DC exhibit a much greater APC efficiency than macrophages when using intact S. pneumoniae, as opposed to soluble PspA, where the differences are less marked. The reason for this difference is unclear, although the requirements for APC presentation of soluble and particulate Ags are distinct (65, 66, 67). Our data appear to contrast with earlier work reporting that macrophages, but not DC, are capable of presenting particulate OVA to primed T cells (68). The poor efficiency of macrophages as APC, relative to DC, may relate both to their lower surface expression of MHC class II and their tendency to be highly degradative (69). Of note, although it has been suggested that polysaccharides might interfere with, or otherwise regulate, APC presentation of associated proteins (46, 47, 48, 49), we find no difference in APC efficiency when using an encapsulated S. pneumoniae vs its unencapsulated, isogenic variant.

Our data notwithstanding, studies documenting the ability of DC to act as superior APC for naive CD4+ T cells, would appear to argue against a role for endogenous macrophages as initiators of humoral immunity. However, this conclusion could be an oversimplification, especially in regard to Ig responses induced by intact bacteria. Thus, the differences observed in APC efficiency between macrophages and DC are relative, not absolute. The threshold level for initial priming of naive CD4+ T cells required to initiate an in vivo humoral immune response is not clear. Furthermore, macrophages are uniquely positioned in secondary lymphoid organs, and functionally specialized, to efficiently internalize incoming bacteria (38, 70). In this regard, we observed that BMM were more efficient than BMDC in uptake of Pn14. Large numbers of macrophages may also be recruited into secondary lymphoid organs such as the spleen, especially in response to a systemic bacterial infection. Macrophages present in an inflammatory milieu will likely up-regulate cell surface costimulatory molecules and release cytokines that may enhance naive CD4+ T cell priming. We demonstrate that in response to Pn14, which contain multiple TLR ligands, macrophages up-regulate CD40 and CD86 and release proinflammatory cytokines, including the Th1-inducing cytokine IL-12 (71). Of interest, it was previously demonstrated that macrophages pulsed in vitro with soluble protein in the absence of a TLR ligand elicit a predominant Th2 Ab response upon adoptive transfer (14, 15), whereas additional stimulation with a TLR ligand resulted in the induction of both Th1 and Th2 IgG isotypes, similar to DC pulsed with Ag in the absence of TLR stimulation (16). In this regard, we demonstrate that Pn14-pulsed DC and macrophages also elicited a similar PspA-specific IgG isotype profile upon transfer in vivo, and subsequent boosting with free Pn14. Macrophages are also functionally heterogeneous (70, 72, 73), even in regard to their ability to act as APC. In particular, one study demonstrated that 20% of splenic macrophage precursors could process and present a soluble protein Ag to naive CD4+ T cells, that was positively correlated with their ability to secrete IL-12 (9). Thus, a particular macrophage subset, used in studies of APC function, may not accurately reflect the overall APC potential of the mixed macrophage population in vivo, especially within an inflammatory milieu.


   Acknowledgments
 
We thank Dr. Alex Lucas (Children’s Hospital Oakland Research Institute, Oakland, CA) for the IgG1{kappa} mouse mAb specific for the capsular polysaccharide of S. pneumoniae type 14 (44.1) and Dr. Rick Schuman (Biosynexus, Inc., Gaithersburg, MD) for the two IgG2a mouse mAbs specific for PspA (DC10-IA5 and CF9IIB7). We also thank Drs. Kryzysztof Trzcinski and Marc Lipsitch (Harvard School of Public Health, Boston, MA) for the R6J strain.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant 2R01 AI49192 from the National Institutes of Health and the Uniformed Services University of the Health Sciences Dean’s Research and Education Endowment Fund. Back

Opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences.

2 Address correspondence and reprint requests to Dr. Clifford M. Snapper, Department of Pathology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814. E-mail address: csnapper{at}usuhs.mil Back

3 Abbreviations used in this paper: DC, dendritic cell; LN, lymph node; Pn14, intact Streptococcus pneumoniae capsular type 14; PspA, pneumococcal surface protein A; PPS14, capsular polysaccharide, serotype 14; BMM, bone marrow-derived macrophage; PerM, peritoneal macrophage; BMDC, bone marrow-derived DC; TNP, trinitrophenyl; WT, wild type. Back

Received for publication March 5, 2008. Accepted for publication May 23, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392: 245-252. [Medline]
  2. Steinman, R. M.. 1991. The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9: 271-296. [Medline]
  3. Steinman, R. M., N. Nogueira, M. D. Witmer, J. D. Tydings, I. S. Mellman. 1980. Lymphokine enhances the expression and synthesis of Ia antigens on cultured mouse peritoneal macrophages. J. Exp. Med. 152: 1248-1261. [Abstract/Free Full Text]
  4. Steinman, R. M., B. Gutchinov, M. D. Witmer, M. C. Nussenzweig. 1983. Dendritic cells are the principal stimulators of the primary mixed leukocyte reaction in mice. J. Exp. Med. 157: 613-627. [Abstract/Free Full Text]
  5. Steinman, R. M., M. D. Witmer. 1978. Lymphoid dendritic cells are potent stimulators of the primary mixed leukocyte reaction in mice. Proc. Natl. Acad. Sci. USA 75: 5132-5136. [Abstract/Free Full Text]
  6. Inaba, K., J. P. Metlay, M. T. Crowley, R. M. Steinman. 1990. Dendritic cells pulsed with protein antigens in vitro can prime antigen-specific, MHC-restricted T cells in situ. J. Exp. Med. 172: 631-640. [Abstract/Free Full Text]
  7. Levin, D., S. Constant, T. Pasqualini, R. Flavell, K. Bottomly. 1993. Role of dendritic cells in the priming of CD4+ T lymphocytes to peptide antigen in vivo. J. Immunol. 151: 6742-6750. [Abstract]
  8. Croft, M., D. D. Duncan, S. L. Swain. 1992. Response of naive antigen-specific CD4+ T cells in vitro: characteristics and antigen-presenting cell requirements. J. Exp. Med. 176: 1431-1437. [Abstract/Free Full Text]
  9. Askew, D., J. Gatewood, E. Olivas, K. Havenith, W. S. Walker. 1995. A subset of splenic macrophages process and present native antigen to naive antigen-specific CD4+ T-cells from mice transgenic for an {alpha}β T-cell receptor. Cell Immunol. 166: 62-70. [Medline]
  10. Crowley, M., K. Inaba, R. M. Steinman. 1990. Dendritic cells are the principal cells in mouse spleen bearing immunogenic fragments of foreign proteins. J. Exp. Med. 172: 383-386. [Abstract/Free Full Text]
  11. Inaba, K., R. M. Steinman, W. C. Van Voorhis, S. Muramatsu. 1983. Dendritic cells are critical accessory cells for thymus-dependent antibody responses in mouse and in man. Proc. Natl. Acad. Sci. USA 80: 6041-6045. [Abstract/Free Full Text]
  12. Inaba, K., R. M. Steinman. 1985. Protein-specific helper T-lymphocyte formation initiated by dendritic cells. Science 229: 475-479. [Abstract/Free Full Text]
  13. Colino, J., C. M. Snapper. 2003. Dendritic cells, new tools for vaccination. Microbes Infect. 5: 311-319. [Medline]
  14. De Becker, G., T. Sornasse, N. Nabavi, H. Bazin, F. Tielemans, J. Urbain, O. Leo, M. Moser. 1994. Immunoglobulin isotype regulation by antigen-presenting cells in vivo. Eur. J. Immunol. 24: 1523-1528. [Medline]
  15. De Becker, G., V. Moulin, F. Tielemans, F. De Mattia, J. Urbain, O. Leo, M. Moser. 1998. Regulation of T helper cell differentiation in vivo by soluble and membrane proteins provided by antigen-presenting cells. Eur. J. Immunol. 28: 3161-3171. [Medline]
  16. De Becker, G., V. Moulin, B. Pajak, C. Bruck, M. Francotte, C. Thiriart, J. Urbain, M. Moser. 2000. The adjuvant monophosphoryl lipid A increases the function of antigen-presenting cells. Int. Immunol. 12: 807-815. [Abstract/Free Full Text]
  17. Moser, M.. 2001. Regulation of Th1/Th2 development by antigen-presenting cells in vivo. Immunobiology 204: 551-557. [Medline]
  18. Pozzi, L. A., J. W. Maciaszek, K. L. Rock. 2005. Both dendritic cells and macrophages can stimulate naive CD8 T cells in vivo to proliferate, develop effector function, and differentiate into memory cells. J. Immunol. 175: 2071-2081. [Abstract/Free Full Text]
  19. Jung, S., D. Unutmaz, P. Wong, G. Sano, K. De los Santos, T. Sparwasser, S. Wu, S. Vuthoori, K. Ko, F. Zavala, et al 2002. In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens. Immunity 17: 211-220. [Medline]
  20. Sapoznikov, A., J. A. Fischer, T. Zaft, R. Krauthgamer, A. Dzionek, S. Jung. 2007. Organ-dependent in vivo priming of naive CD4+ but not CD8+ T cells by plasmacytoid dendritic cells. J. Exp. Med. 204: 1923-1933. [Abstract/Free Full Text]
  21. Probst, H. C., M. van den Broek. 2005. Priming of CTLs by lymphocytic choriomeningitis virus depends on dendritic cells. J. Immunol. 174: 3920-3924. [Abstract/Free Full Text]
  22. Tian, T., J. Woodworth, M. Skold, S. M. Behar. 2005. In vivo depletion of CD11c+ cells delays the CD4+ T cell response to Mycobacterium tuberculosis and exacerbates the outcome of infection. J. Immunol. 175: 3268-3272. [Abstract/Free Full Text]
  23. Cahalan, M. D.. 2008. Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs. Annu. Rev. Immunol. 26: 585-626. [Medline]
  24. Khan, A. Q., A. Lees, C. M. Snapper. 2004. Differential regulation of IgG anti-capsular polysaccharide and antiprotein responses to intact Streptococcus pneumoniae in the presence of cognate CD4+ T cell help. J. Immunol. 172: 532-539. [Abstract/Free Full Text]
  25. Colino, J., Y. Shen, C. M. Snapper. 2002. Dendritic cells pulsed with intact Streptococcus pneumoniae elicit both protein- and polysaccharide-specific immunoglobulin isotype responses in vivo through distinct mechanisms. J. Exp. Med. 195: 1-13. [Medline]
  26. Chen, Q., G. Sen, C. M. Snapper. 2006. Endogenous IL-1R1 signaling is critical for cognate CD4+ T cell help for induction of in vivo type 1 and type 2 antipolysaccharide and antiprotein Ig isotype responses to intact Streptococcus pneumoniae, but not to a soluble pneumococcal conjugate vaccine. J. Immunol. 177: 6044-6051. [Abstract/Free Full Text]
  27. Sen, G., Q. Chen, C. M. Snapper. 2006. Immunization of aged mice with a pneumococcal conjugate vaccine combined with an unmethylated CpG-containing oligodeoxynucleotide restores defective immunoglobulin G antipolysaccharide responses and specific CD4+-T-cell priming to young adult levels. Infect. Immun. 74: 2177-2186. [Abstract/Free Full Text]
  28. Trzcinski, K., C. M. Thompson, M. Lipsitch. 2003. Construction of otherwise isogenic serotype 6B, 7F, 14, and 19F capsular variants of Streptococcus pneumoniae strain TIGR4. Appl. Environ. Microbiol. 69: 7364-7370. [Abstract/Free Full Text]
  29. Canaday, D. H., A. Gehring, E. G. Leonard, B. Eilertson, J. R. Schreiber, C. V. Harding, W. H. Boom. 2003. T-cell hybridomas from HLA-transgenic mice as tools for analysis of human antigen processing. J. Immunol. Methods 281: 129-142. [Medline]
  30. Chattopadhyay, G., A. Q. Khan, G. Sen, J. Colino, W. Dubois, A. Rubtsov, R. M. Torres, M. Potter, C. M. Snapper. 2007. Transgenic expression of Bcl-xL or Bcl-2 by murine B cells enhances the in vivo antipolysaccharide, but not antiprotein, response to intact Streptococcus pneumoniae. J. Immunol. 179: 7523-7534. [Abstract/Free Full Text]
  31. Colino, J., C. M. Snapper. 2003. Two distinct mechanisms for induction of dendritic cell apoptosis in response to intact Streptococcus pneumoniae. J. Immunol. 171: 2354-2365. [Abstract/Free Full Text]
  32. Steinman, R. M., G. Kaplan, M. D. Witmer, Z. A. Cohn. 1979. Identification of a novel cell type in peripheral lymphoid organs of mice. V. Purification of spleen dendritic cells, new surface markers, and maintenance in vitro. J. Exp. Med. 149: 1-16. [Abstract/Free Full Text]
  33. Davies, J. Q., S. Gordon. 2005. Isolation and culture of murine macrophages. Methods Mol. Biol. 290: 91-103. [Medline]
  34. Duddridge, M., C. A. Kelly, C. Ward, D. J. Hendrick, E. H. Walters. 1990. The reversible effect of lignocaine on the stimulated metabolic activity of bronchoalveolar lavage cells. Eur. Respir. J. 3: 1166-1172. [Abstract]
  35. Hijlstra, A., W. Van Dorp, M. R. Daha, G. Q. Leslie. 1980. The effect of lidocaine on the processing of soluble immune aggregates and immune complexes by peritoneal macrophages. Immunology 41: 237-244. [Medline]
  36. Hoidal, J. R., J. G. White, J. E. Repine. 1979. Influence of cationic local anesthetics on the metabolism and ultrastructure of human alveolar macrophages. J. Lab. Clin. Med. 93: 857-866. [Medline]
  37. Hume, D. A.. 2006. The mononuclear phagocyte system. Curr. Opin. Immunol. 18: 49-53. [Medline]
  38. Gordon, S.. 1999. Macrophages and the immune response. W. E. Paul, ed. Fundamental Immunology 4th Edition.533 Lippincott Williams & Wilkins, Philadelphia, PA.
  39. Reinhardt, R. L., S. J. Kang, H. E. Liang, R. M. Locksley. 2006. T helper cell effector fates: who, how and where?. Curr. Opin. Immunol. 18: 271-277. [Medline]
  40. Khan, A. Q., Y. Shen, Z. Q. Wu, T. A. Wynn, C. M. Snapper. 2002. Endogenous pro- and anti-inflammatory cytokines differentially regulate an in vivo humoral response to Streptococcus pneumoniae. Infect. Immun. 70: 749-761. [Abstract/Free Full Text]
  41. Colino, J., C. M. Snapper. 2003. Opposing signals from pathogen-associated molecular patterns and IL-10 are critical for optimal dendritic cell induction of in vivo humoral immunity to Streptococcus pneumoniae. J. Immunol. 171: 3508-3519. [Abstract/Free Full Text]
  42. Lee, K. S., C. A. Scanga, E. M. Bachelder, Q. Chen, C. M. Snapper. 2007. TLR2 synergizes with both TLR4 and TLR9 for induction of the MyD88-dependent splenic cytokine and chemokine response to Streptococcus pneumoniae. Cell Immunol. 245: 103-110. [Medline]
  43. Beutler, B., Z. Jiang, P. Georgel, K. Crozat, B. Croker, S. Rutschmann, X. Du, K. Hoebe. 2006. Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large. Annu. Rev. Immunol. 24: 353-389. [Medline]
  44. Wu, Z. Q., Y. Shen, A. Q. Khan, C. L. Chu, R. Riese, H. A. Chapman, O. Kanagawa, C. M. Snapper. 2002. The mechanism underlying T cell help for induction of an antigen-specific in vivo humoral immune response to intact Streptococcus pneumoniae is dependent on the type of antigen. J. Immunol. 168: 5551-5557. [Abstract/Free Full Text]
  45. Clark, E. A., A. Craxton. 2003. A CD40 bridge between innate and adaptive immunity. Immunity 18: 724-725. [Medline]
  46. Leonard, E. G., D. H. Canaday, C. V. Harding, J. R. Schreiber. 2003. Antigen processing of the heptavalent pneumococcal conjugate vaccine carrier protein CRM(197) differs depending on the serotype of the attached polysaccharide. Infect. Immun. 71: 4186-4189. [Abstract/Free Full Text]
  47. Leyva-Cobian, F., E. R. Unanue. 1988. Intracellular interference with antigen presentation. J. Immunol. 141: 1445-1450. [Abstract]
  48. Ishioka, G. Y., A. G. Lamont, D. Thomson, N. Bulbow, F. C. Gaeta, A. Sette, H. M. Grey. 1992. MHC interaction and T cell recognition of carbohydrates and glycopeptides. J. Immunol. 148: 2446-2451. [Abstract]
  49. Mouritsen, S., M. Meldal, I. Christiansen-Brams, H. Elsner, O. Werdelin. 1994. Attachment of oligosaccharides to peptide antigen profoundly affects binding to major histocompatibility complex class II molecules and peptide immunogenicity. Eur. J. Immunol. 24: 1066-1072. [Medline]
  50. Hsieh, C.-S., S. E. Macatonia, A. O'Garra, K. M. Murphy. 1993. Pathogen-induced Th1 phenotype development in CD4+ {alpha}β-TCR transgenic T cells is macrophage dependent. Int. Immunol. 5: 371-382. [Abstract/Free Full Text]
  51. Hsieh, C. S., S. E. Macatonia, C. S. Tripp, S. F. Wolf, A. O'Garra, K. M. Murphy. 1993. Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science 260: 547-549. [Abstract/Free Full Text]
  52. Hamilton-Easton, A., M. Eichelberger. 1995. Virus-specific antigen presentation by different subsets of cells from lung and mediastinal lymph node tissues of influenza virus-infected mice. J. Virol. 69: 6359-6366. [Abstract]
  53. Usherwood, E. J., T. L. Hogg, D. L. Woodland. 1999. Enumeration of antigen-presenting cells in mice infected with Sendai virus. J. Immunol. 162: 3350-3355. [Abstract/Free Full Text]
  54. Ziegler, H. K.. 1984. The processing and presentation of Listeria monocytogenes antigens by macrophages. Clin. Invest. Med. 7: 269-272. [Medline]
  55. Allen, C. D., T. Okada, J. G. Cyster. 2007. Germinal-center organization and cellular dynamics. Immunity 27: 190-202. [Medline]
  56. Phan, T. G., I. Grigorova, T. Okada, J. G. Cyster. 2007. Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells. Nat. Immunol. 8: 992-1000. [Medline]
  57. Carrasco, Y. R., F. D. Batista. 2007. B cells acquire particulate antigen in a macrophage-rich area at the boundary between the follicle and the subcapsular sinus of the lymph node. Immunity 27: 160-171. [Medline]
  58. Craxton, A., D. Magaletti, E. J. Ryan, E. A. Clark. 2003. Macrophage- and dendritic cell–dependent regulation of human B-cell proliferation requires the TNF family ligand BAFF. Blood 101: 4464-4471.
  59. Kopf, M., S. Herren, M. V. Wiles, M. B. Pepys, M. H. Kosco-Vilbois. 1998. Interleukin 6 influences germinal center development and antibody production via a contribution of C3 complement component. J. Exp. Med. 188: 1895-1906. [Abstract/Free Full Text]
  60. Boswell, H. S., S. O. Sharrow, A. Singer. 1980. Role of accessory cells in B cell activation. I. Macrophage presentation of TNP-Ficoll: evidence for macrophage-B cell interaction. J. Immunol. 124: 989-996. [Abstract]
  61. Chused, T. M., S. S. Kassan, D. E. Mosier. 1976. Macrophage requirement for the in vitro response to TNP Ficoll: a thymic independent antigen. J. Immunol. 116: 1579-1581. [Abstract/Free Full Text]
  62. Corbel, C., F. Melchers. 1983. Requirement for macrophages or for macrophage- or T cell-derived factors in the mitogenic stimulation of murine B lymphocytes by lipopolysaccharides. Eur. J. Immunol. 13: 528-533. [Medline]
  63. Sinha, A. A., C. Guidos, K.-C. Lee, E. Diener. 1987. Functions of accessory cells in B cell responses to thymus-independent antigens. J. Immunol. 138: 4143-4149. [Abstract]
  64. Boswell, H. S., A. Ahmed, I. Scher, A. Singer. 1980. Role of accessory cells in B cell activation. II. The interaction of B cells with accessory cells results in the exclusive activation of an Lyb5+ B cell subpopulation. J. Immunol. 125: 1340-1348. [Medline]
  65. Kovacsovics-Bankowski, M., K. Clark, B. Benacerraf, K. L. Rock. 1993. Efficient major histocompatibility complex class I presentation of exogenous antigen upon phagocytosis by macrophages. Proc. Natl. Acad. Sci. USA 90: 4942-4946. [Abstract/Free Full Text]
  66. Guermonprez, P., J. Valladeau, L. Zitvogel, C. Thery, S. Amigorena. 2002. Antigen presentation and T cell stimulation by dendritic cells. Annu. Rev. Immunol. 20: 621-667. [Medline]
  67. Ziegler, H. K., C. A. Orlin, C. W. Cluff. 1987. Differential requirements for the processing and presentation of soluble and particulate bacterial antigens by macrophages. Eur. J. Immunol. 17: 1287-1296. [Medline]
  68. Kapsenberg, M. L., M. B. Teunissen, F. E. Stiekema, H. G. Keizer. 1986. Antigen-presenting cell function of dendritic cells and macrophages in proliferative T cell responses to soluble and particulate antigens. Eur. J. Immunol. 16: 345-350. [Medline]
  69. Savina, A., S. Amigorena. 2007. Phagocytosis and antigen presentation in dendritic cells. Immunol. Rev. 219: 143-156. [Medline]
  70. Gordon, S., I. Fraser, D. Nath, D. Hughes, S. Clarke. 1992. Macrophages in tissues and in vitro. Curr. Opin. Immunol. 4: 25-32. [Medline]
  71. Trinchieri, G.. 2003. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat. Rev. Immunol. 3: 133-146. [Medline]
  72. Rutherford, M. S., A. Witsell, L. B. Schook. 1993. Mechanisms generating functionally heterogeneous macrophages: chaos revisited. J. Leukocyte Biol. 53: 602-618. [Abstract]
  73. Celada, A., C. Nathan. 1994. Macrophage activation revisited. Immunol. Today 15: 100-102. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
J. Colino, G. Chattopadhyay, G. Sen, Q. Chen, A. Lees, D. H. Canaday, A. Rubtsov, R. Torres, and C. M. Snapper
Parameters Underlying Distinct T Cell-Dependent Polysaccharide-Specific IgG Responses to an Intact Gram-Positive Bacterium versus a Soluble Conjugate Vaccine
J. Immunol., August 1, 2009; 183(3): 1551 - 1559.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vasilevsky, S.
Right arrow Articles by Snapper, C. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vasilevsky, S.
Right arrow Articles by Snapper, C. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS