| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
From the Department of Pathology,*University of Michigan Medical School, Ann Arbor; and the Department of Pathology and Laboratory Medicine,Veterans Administration Ann Arbor Healthcare System, Ann Arbor, Michigan
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
mRNA but indicated biased interleukin (IL)-1, IL-6, and IL-12 expression with PPD challenge. All beads elicited comparable levels of CXCL9, CXL10, CCL2, CCL17, and CCL22 mRNA, but PPD beads caused biased CXCL2 CXCL5, CCL3, and CCL4 expression whereas both pathogen Ags induced CCL7. Immunohistochemical, electron microscopic, and flow cytometric analyses showed that Ag beads mobilized CD11c+ dendritic cells (DCs) of comparable maturation. Transfer of DCs from PPD Ag-challenged lungs conferred a Th1 anamnestic cytokine response in recipients. Surprisingly, transfer of DCs from the helminth SEA-challenged lungs did not confer the expected Th2 response, but instead rendered recipients incapable of Ag-elicited IL-4 production. These results provide in vivo evidence that lung DCs recruited under inflammatory conditions favor Th1 responses and alternative mechanisms are required for Th2 commitment.
DCs are critical participants in the innate response through their ability to link innate and acquired immunity.8 These Ag-presenting cells have been the subject of intense investigation and are known to recognize pathogen-associated molecular patterns by way of Toll-like receptors as well as provide co-stimulatory signals to Ag-specific T lymphocytes.9 Presently, there remains controversy regarding the mechanisms by which DCs are recruited to sites of infection and the means by which they shape the nature of acquired T-cell responses. In addition, because of their limited numbers, the study of DCs has depended heavily on DCs generated in culture, which likely do not fully reflect in vivo functions. A means to systematically study DC biology in vivo could significantly advance our understanding of innate responses and their relationship to acquired immunity. As potential sites of DC recruitment, granulomas might provide an ideal means to study DC biology.
We previously described models of polarized Th1 and Th2 cell-mediated anamnestic pulmonary granulomas in sensitized mice challenged with Ag-coated agarose beads.10-12 These highly synchronized models have allowed detailed analyses of cellular and molecular biological events involved in anamnestic T-cell-mediated granuloma formation. In the present study, we wished to determine whether a similar approach could be used to study critical events in the innate granulomatous response to mycobacterial and helminthic Ags represented by Mycobacteria bovis purified protein derivative (PPD) and Schistosoma mansoni soluble egg Ags (SEA). Respectively, these Ags induce Th1- and Th2-acquired immunity; therefore analysis of the innate phase of the response might help elucidate mechanisms directing polarized immune responses. In this report, we provide a comprehensive characterization of the innate Ag bead granuloma with special focus on DC recruitment and function. We demonstrate that Ag bead challenge invokes recruitment of MHCII+CD11c+ DCs. Furthermore different Ags invoked different patterns of cytokine and chemokine production with the potential to mobilize and activate DCs.
In draining lymph nodes, PPD bead challenge directly elicited a Th1 cytokine pattern, whereas SEA beads induced Th2 polarization in a two-stage manner with an initial Th0-like period as described for intact parasite eggs,13 therefore it was predicted that DCs from bead granulomas might similarly initiate polarized responses. Unexpectedly, DCs from PPD and SEA innate granulomas had similar phenotypes with regard to surface markers and cytokine expression. Transfer of PPD bead DCs conferred the predicted anamnestic Th1 response in naïve recipients, but surprisingly, SEA bead DCs failed to confer Th2 characteristics. Thus unlike that reported for resident lung DCs,14-16 granuloma DCs impaired Th2 commitment suggesting that SEA-induced Th2 commitment requires additional or alternative mechanisms. This study represents the first comprehensive analysis of innate pulmonary granuloma formation and the first in vivo analysis using directly transferred Ag-presenting DCs recruited during pathogen Ag-elicited inflammation in the lung.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Female, 8- to 10-week-old, CBA mice were obtained from Jackson Laboratories, Bar Harbor, ME, and maintained under specific pathogen-free conditions. The University of Michigan Committee on Use and Care of Animals approved all animal studies.
Antigen Bead Challenge and DC Transfer Protocols
Primary innate lung inflammatory responses were elicited in naïve mice by intravenous injection of 6000 Sepharose 4B beads coupled to M. bovis purified protein derivative (PPD) (Department of Agriculture, Veterinary Division, Ames, IA) or to S. mansoni soluble schistosome egg Ags (SEA) (the World Health Organization, Geneva, Switzerland), as described.10 Bovine serum albumin (BSA) (Sigma, St. Louis, MO) was coupled to beads as a control nonpathogenic Ag challenge. For secondary responses, mice were rechallenged with respective Ag beads 16 days after primary injection. In DC-priming studies, CD11c+ DCs were isolated as below 3 days after primary bead injection and 2 x 105 DCs were adoptively transferred to each naïve recipient intravenously. Ten days later recipient mice were challenged by tail vein injection with respective Ag beads. Replicates consisted of independent experiments each using three to five mice per group.
Morphometry and Transmission Electron Microscopy
Granulomas were measured blindly from formalin-inflated lungs that were paraffin-embedded, sectioned, and then stained with hematoxylin and eosin. Granuloma area was measured by computerized morphometry. A minimum of 20 lesions was measured per lung. For transmission electron microscopy, lungs were inflated with 2.5% glutaraldehyde and then submitted for standard osmium fixation and plastic embedding. Thin sections were placed on grids and examined using an LEO electron microscope (Carl Zeiss, LEO Electron Microscopy, Thornwood, NY).
Immunohistochemisty
Frozen tissue sections (5 to 7 µm thick) were mounted on poly-L-lysine-coated slides, fixed with acetone, and then rehydrated in phosphate-buffered saline (PBS). Sections were pretreated 10 minutes with 0.03% H2O2 and then avidin and biotin. The sections were covered with biotinylated 10 µg/ml of hamster anti-mouse CD11c monoclonal antibody (BD Pharmingen, San Diego, CA). Biotinylated nonimmune hamster IgG served as a control. Slides were incubated for 1 hour at room temperature, then rinsed and overlaid with a 1-µg/ml streptavidin peroxidase (Sigma), and incubated another 30 minutes at room temperature. The slides were rinsed again in PBS and overlaid with a 1:20 dilution of AEC staining solution (Sigma) for 20 minutes at room temperature. Finally, the sections were rinsed, counterstained with Gill’s hematoxylin, and mounted with coverslips.
CD11c+ DC Isolation from Lungs and Granulomas
After perfusion with cold RPMI 1640, lungs were excised, placed in cold RPMI 1640 medium, and then homogenized in a Waring blender. Intact granulomas were collected over a no. 100 stainless steel mesh and rinsed with cold RPMI 1640. The filtrate, containing CD11c+ DCs not associated with beads, was also collected and spun down. Naïve, unchallenged lungs were similarly homogenized. Single cell suspensions were obtained by collagenase digestion. Briefly, granulomas or naïve lung homogenates were incubated with digestion medium containing RPMI (JRH Biosciences, Lenexa, KS), 10% fetal bovine serum (Intergen, Purchase, NY), 10 mmol/L glutamine, and 1000 U/ml type IV collagenase (Sigma Chemical Co.). After 15 minutes of incubation with agitation at 37°C, the digest was passed through a no. 100 stainless steel mesh and washed four times by centrifugation in RPMI 1640. CD11c+ DCs were isolated from the dispersed cell preparations using an EasySep PE selection kit (StemCell Technologies, Vancouver, BC, Canada) combined with a phycoerythrin-labeled anti-CD11c antibody (BD PharMingen). The enrichment of the recovered CD11c+ cells was confirmed by flow cytometric analysis and was routinely greater than 95%.
Flow Cytometry
Monoclonal antibodies used to identify mouse DC populations included fluorescein isothiocyanate-, phycoerythrin-, and Cy-chrome-conjugated anti-CD11c (HL3), phycoerythrin-conjugated anti-I-Ak (11-5.2), anti-CD8a (53-6.5), anti-CD11b/Mac-1a (M1/70), anti-CD40 (3/23), anti-CD80 (16-10-A1), anti-CD86 (GL-1) (all from BD PharMingen), anti-DEC-205 (NLDC-145) and anti-F4/80 (A3-1) (both from Serotec). All isotype controls and anti-CD16/CD32 (2.4G2) were from BD Pharmingen. After blocking with anti-CD16/CD32 for 5 minutes, cells were stained with fluorescent-labeled antibodies or isotype control antibodies in 2% fetal bovine serum-PBS buffer. A FACScan flow cytometer with CellQuest software (B-D, San Jose, CA) was used for data acquisition and analysis. Depending on the number of cells available, 10,000 to 100,000 events per sample were analyzed.
Real-Time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Transcript Analysis
Real-time RT-PCR analyses were performed as described.17 Poly(A) pure mRNA was isolated from cells using mRNA isolation kits (Ambion, Austin, TX) and reverse-transcribed using Reverse Transcription System kits (Promega, Madison, WI). Real-time PCR was performed on samples before and after reverse transcription to assess genomic DNA contamination levels in a sample preparation. DNase treatment was applied whenever contamination was detected. mRNA was considered detectable only when the signal was 10-fold more abundant than any contamination level. The ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) was used for real-time PCR analysis with comparative CT method. TaqMan (Applied Biosystems) predeveloped reaction kits were used for most target genes. De novo designed primer probe sequences have been previously reported.18 In all cases, the thermal cycling condition was programmed according to the manufacturer’s instructions. Data are expressed as arbitrary units as described previously.17
Lymph Node Cell Culture and Cytokine Assays
Mediastinal lymph nodes were collected aseptically at the time of lung harvest and teased into a single cell suspension. After washing, the cells were cultured in RPMI 1640 medium (JRH Biosciences) containing 10% fetal bovine serum (Intergen), 10 mmol/L glutamine, and 100 µg/ml streptomycin and 100 U/ml penicillin (RPMI-fetal bovine serum) at 5 x 106 cells/ml in 100-mm dishes with 5 µg/ml of PPD or SEA for 48 hours at 37°C in a humidified 5% CO2 atmosphere. Supernates were collected by centrifugation and stored at -45°C before performing cytokine assays. All cytokines used in this study were obtained as purified carrier-free recombinant proteins from PeproTech Inc., Rocky Hill, NJ, and R&D Systems, Minneapolis, MN. Interleukin (IL)-2, IL-4, and interferon (IFN)-
were measured by standard enzyme-linked immunosorbent assay using commercially available reagents (R&D Systems and BD Pharmingen); sensitivities ranged from 15 to 50 pg/ml.
Statistics
Analysis of variance was used for intergroup comparisons with P < 0.05 considered to indicate significance. Pairwise tests were done with Dunnett error protection at 95% confidence interval.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
We previously demonstrated that pulmonary Ag bead challenge of Ag-sensitized mice offers an effective means to study sequential events in the development of anamnestic, Th1- and Th2-mediated hypersensitivity-type granulomas. In the present study, we wished to determine whether this approach could be used to study key events of the innate response by challenging naïve mice with beads bearing pathogen-associated Ags known to ultimately evoke Th1 and Th2 responses, specifically mycobacterial purified protein derivative (PPD) and soluble schistosomal egg Ags (SEA). In the first phase of the study we characterized the histology of Ag bead-challenged lungs throughout a 20-day period (Figure 1)
. Pathogen Ag-coated beads elicited focal reactions that were consistent with granulomas and approximately half of the area reported for the anamnestic Ag bead response.19
Interestingly, the reaction to mycobacterial PPD occurred more rapidly than the SEA response and more neutrophils were observed in these lesions on day 2. The SEA-elicited lesions tended to decline more slowly and were noted to leave more matrix scarring during the involutional period. Both pathogen Ags elicited more vigorous responses than a control nonpathogen Ag BSA.
|
, immunohistochemical staining showed a rim of CD11c+ cells around all bead types; this was most prominent on day 3 after Ag bead injection. Scattered resident interstitial CD11c+ were also present in naïve and noninflamed lungs. Electron microscopy identified mononuclear cells with complex dendritic processes in close proximity to beads either adherent to endothelial cell basement membrane or in direct contact with bead surfaces (Figure 2)
.
|
, shows the number of F4/80+, CD11c+, CD11c+MHCII+, Gr-1hi cells measured as a percentage of CD45+ leukocytes in dispersed PPD and SEA bead-challenged lungs. Antigen bead challenge increased proportions of F4/80+, CD11c+, and Gr1hi cells corresponding to the accumulation of inflammatory cells observed histologically. The number of CD11c+ cells reached maximum on day 3 after injection, respectively, comprising 43% and 25% of CD45% cells in the PPD and SEA models. (Figure 2, A and B)
. Unlike naïve lung CD11c+ cells, the majority of Ag-elicited CD11c+ cells expressed high levels of MHCII. As reported by others,16,20
a majority of CD11c+ cells in the lung displayed CD11b and weak F4/80 co-expression consistent with myeloid origin (data not shown). Although both CD11c+ and F4/80+ populations had weak Gr-1 expression, Gr-1bright cells were a distinct population that did not overlap with CD11c+ populations and were more dominant in PPD bead-challenged lungs, which corroborated histological findings. The CD11c+ population in the lung was heterogeneous with regard to DEC-205, CD8a, and CD11b expression, but was negative for B220 and CD4. Two distinct populations were identified, CD11cbright CD11bdim, and CD11cdimCD11bbright.16,20
Interestingly, the former did not increase with Ag challenge and retained an immature phenotype. In summary, Ag bead challenge mobilized CD11c+CD11b+MHCII+ cells consistent with DCs of myeloid origin.
|
To begin a mechanistic analysis of events associated with the innate granuloma, we measured cytokine mRNA expression in lungs using quantitative real-time RT-PCR assay of pathogen Ag beads and compared this to nonpathogen BSA bead challenge. Preliminary time course studies indicated that most transcript induction occurred during the first 72 hours after bead challenge. Figure 4
shows the induction of several known innate response-related cytokines, IL-1ß, tumor necrosis factor (TNF)-, GM-CSF, IL-6, IL-12p35, and IL12p40. The patterns revealed Ag-related differences in transcript induction. Specifically, PPD bead challenge was associated with the highest transcript levels of IL-1ß, GM-CSF, IL-6, and IL-12p35. In addition, although IL-12 p40 and TNF- transcripts were detected in all groups, PPD challenge tended to evoke higher levels. SEA bead challenge evoked minimal IL-12p35 mRNA that was comparable to BSA bead challenge. However, SEA beads did induce more IL-6 than BSA beads. The finding of greater IL-12 induction in the PPD response suggests innate Ag recognition and cytokine induction could potentially promote the Th1-biased immune response21
observed in the adaptive phase of the PPD response. Transcripts for cytokines that promote Th2 responses, IL-4 and IL-10, were not significantly increased in the lungs of any bead-challenged group during the 3-day study period (not shown).
|
shows the induction patterns of 12 chemokines, most of which displayed significant up-regulation over virtually nil baseline levels in naive lungs (time 0). Of these, CXCL2, CXCL5, CCL3, and CCL4 were more dominant in PPD bead-challenged lungs indicating a PPD-dependent effect. Interestingly, CXCL9 and CXCL10, both IFN--amplified chemokines, did not display biased expression with primary PPD bead challenge as observed in the anamnestic response,12
pointing to a requirement for adaptive immunity to fully establish chemokine polarization patterns. In addition, although most of chemokine transcripts achieved maximal levels by 24 hours, CXCL9 did not reach maximum until 48 to 72 hours.
|
The above described cytokine and chemokine transcript analyses indicated that innate granuloma formation is associated with rapid cytokine and chemokine induction. Moreover, there was evidence of innate pathogen Ag recognition resulting in different induction patterns for some of these mediators.
Induction of Differential Adaptive Immunity During Innate Ag Bead Granuloma Formation
Having defined key immunopathological events occurring during the innate granulomatous response we next analyzed draining lymph nodes (DLNs) for evidence of evolving adaptive immunity. Figure 6
shows the cytokine profiles of DLN cultures prepared 4 days after primary or secondary rechallenge after primary Ag bead exposure. The primary DLN response in PPD bead-challenged mice consisted of IL-2 and IFN-; this Th1 pattern was accentuated with rechallenge on day 16. The primary response to SEA beads was a Th0 profile with concomitant IL-2, IFN-, IL-4, IL-5, and substantial IL-10. By day 16 this had polarized toward a Th2 profile with decreased IL-2 and IFN- and increased IL-5 and IL-13. Thus, the PPD response showed early and direct commitment to a Th1 profile, whereas the SEA response evolved from a Th0 to a Th2 dominant profile similar to sensitization with intact parasite eggs.13
|
Because DCs were potentially responsible for initiating the observed DLN responses, we examined granuloma DC populations for phenotypic differences. We first compared the activation/maturation status of Ag bead-elicited lung DCs, by measuring surface expression of MHCII, CD40, CD80, and CD86 by flow cytometry. The CD11c+ cells shifted from low to high MHCII, CD40, CD80, and CD86 expression after Ag bead injection. Table 1
compares the mean fluorescent intensity (MFI) of the CD11c+ populations in naïve unchallenged and 3-day Ag bead-challenged lungs. MHCII and CD40 expression increased 5- and 10-fold, respectively, among 3-day Ag bead-challenged lung DCs. CD80 and CD86 expression increased approximately twofold. It should be noted that DCs from BSA bead granulomas showed similar activation profiles (not shown).
|
. Not all chemokines were affected because CCL3 (MIP1) did not change after challenge. Interestingly, IL-12p35, IL-12p40, and IL-10 transcripts were negligible and did not show up-regulation among CD11c+ cells from challenged lungs. This was also the case for IL-4, IFN-, IL-1ß, IL-6, and TNF- transcripts (data not shown). All CD11c populations expressed CCR1, CCR5, and CXCR4 transcripts with evidence of augmented CCR5 mRNA in challenged mice. Unlike DCs in lymphoid tissues, lung DCs did not express CCR7. In addition, CCR3, CCR6, CCR8, CCR9, CXCR1/2, CXCR3, and CXCR5 transcripts were negligible (not shown). Notably, CCR2 transcripts increased nearly 100-fold among CD11c+ cells from challenged lungs compared to those of naïve lungs. Unexpectedly, the phenotypes of SEA and PPD bead lung DCs were strikingly similar despite the disparate effects of the eliciting Ags. Taken together, the data indicated that inflammatory CD11c+ DCs from challenged lungs expressed genes consistent with activated DCs present in peripheral nonlymphoid tissues.22
Ag Bead DCs Promote Th1 but Not Th2 Characteristics on Transfer
Lung derived DCs are believed to be critical for CD4+ T-cell priming; yet only lymphoid tissue but not peripheral tissue DCs are considered to be capable of initiating T-cell activation.23
Ag bead-elicited lung DCs had some features of mature activated DCs (Table 1)
. Because SEA and PPD Ags induced divergent CD4+ T-cell responses, we wished to determine whether the Ag bead-elicited lung DCs could similarly induce polarized Th responses. Ag bead DCs were isolated and directly transferred to syngeneic recipients (2 x 105/recipient). Ten days later, recipients were challenged with respective Ag beads then DLNs were harvested and cultured on day 4 after challenge (day 14). Parallel challenged naïve mice were used for controls. Figure 7
shows cytokine profiles of DLN cultures after challenge. As noted above primary PPD bead challenge of naïve mice resulted in a detectable early Th1 profile, whereas SEA beads induced a Th0 pattern with concomitant IL-2, IFN-, and IL-4 production (Figure 7
, right column). Compared to naïve mice, priming with PPD bead DCs conferred an enhanced Th1 pattern with twofold and fourfold increases of IL-2 and IFN- (Figure 7
, left column). This effect was associated with an anamnestic inflammatory response to PPD beads and could not be transferred with DCs from non-Ag beads (data not shown). Surprisingly, priming with SEA bead lung DCs did not confer the expected Th2 cytokine profile, but rather elicited IL-2 and IFN- but not IL-4, resembling a more Th1-like profile. The experiment was repeated four times and in one, DC-primed mice were further boosted with soluble SEA before SEA bead challenge and still the IL-4 response was blocked by DC transfer. It was noted that the IFN- promoting ability of SEA bead DCs was weaker than their PPD counterparts, suggesting that SEA lung DCs could not invoke a vigorous Th1 response. Taken together, the data indicated that bead-elicited, activated lung DCs were capable of conferring a Th1 adaptive response, but were unable to establish a Th2 response.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Our histomorphological analysis revealed a transient innate inflammatory response to Ag-coated beads with subtle kinetic differences that suggested Ag-dependent effects. Mycobacterial PPD elicited rapid focal inflammation that reached a peak on day 3 that quickly resolved thereafter. The helminth Ag, SEA, reached maximum on day 4 and showed a slower resolution rate. Both pathogen Ag bead responses were greater than that for BSA beads suggesting an element of innate recognition of pathogen-associated molecular patterns. The response to BSA beads likely results from a nonspecific release of mediators from cells because of local tissue injury/irritation combined with activation of complement and coagulation pathway factors. DCs may process BSA Ags but no Toll-like receptor recognition has been described for bovine albumin.
Kinetic analyses of cytokine and chemokine transcript expression likewise provided evidence of innate Ag-recognition. TNF-, IL-1, and IL-6 transcripts were all induced by 24 hours and PPD beads elicited the greatest levels. BSA beads primarily induced only TNF- whereas SEA beads induced TNF- and low levels of IL-1 and IL-6. By 48 hours there was enhanced expression of IL-12 p35 and p40 transcripts in the PPD response but not in the other responses. The delayed IL-12 expression would be consistent with derivation from a recruited population or might reflect a maturation/activation interval. As discussed below, the selective induction of IL-12 mRNA by PPD beads is possibly relevant in linking the innate PPD response to subsequent acquired Th1 cell-mediated immunity.21
Sources of IL-12 could include mononuclear phagocytes, which bear Toll-like receptors (TLR). PPD contains lipoprotein-lipopolysaccharide complexes25
that could be recognized by such receptors.26
Chemokine transcripts were primarily induced by 24 hours with the exception of CXCL9, CCL3, and CCL8, which appeared by 48 to 72 hours. As reported for the secondary response, CXCL2, CXCL5, CCL3, and CCL4 were biased to the innate PPD granuloma,27 suggesting that innate recognition signals may help establish this bias. It is known that the neutrophil chemotactins, CXCL2 and CXCL5, are induced by endotoxin therefore it is conceivable that PPD lipopolysaccharide complexes could potentially mediate direct cellular activation and mediator production by way of the Toll-like LPS receptor, TLR-4.28,29 The enhanced expression of neutrophil chemotactins in the innate PPD granuloma corresponds to the increased neutrophil influx that we observed in these lesions. Transcripts for CCL3 showed delayed expression kinetics that paralleled IL-12. This would be fully consistent with our demonstration that CCL3 is dependent on endogenous IL-12 expression during PPD granuloma formation.18 CCL3 and CCL4 are potentially chemotactic for monocytes, DCs, and CCR5+ Th1 cells, providing coordinating recruitment signals. In addition, several other chemokines displayed rapid Ag nonspecific induction, these included CXCL9/10 and CCL17, ligands for known Th1 and Th2 cell-associated chemokine receptors. Thus, cells of the acquired immune system are adapted to respond to chemokines generated by the innate response, providing a means for rapid local mobilization of immune cells. CCL2 was also among the rapidly elicited Ag-nonspecific chemokines and transcripts or its receptor, CCR2, was notably enhanced among granuloma DCs. This would be fully consistent with reports implicating CCL2 in the trafficking of DCs.30,31
Some chemokines including CXCL11, CCL8, and CCL22, which show strong expression during secondary Ag bead granuloma formation,18 were weakly represented in the innate granuloma indicating that adaptive immune signals profoundly amplify their expression. Interestingly, the innate response to SEA beads did not display a distinctly biased chemokine pattern. Toll receptors are reportedly not required for the generation of Th2 responses32 and the relatively modest cytokine response elicited by SEA beads might support this notion. However, CCL7 transcripts were expressed at levels comparable to that elicited by PPD, which were threefold higher than for nonpathogen Ag bead challenge suggesting some form of innate recognition of SEA. Recently, TLR-2 has been implicated in recognition of lysophosphatidylserine residues derived from schistosome eggs,33 providing precedent for innate recognition of helminth Ags. Taken together, our findings suggest the existence of a reflexive innate chemokine response designed to recruit all potentially relevant immune-related cells; however that response can be tailored to a pathogen likely through recognition of pathogen-associated molecular patterns.
Our study extends the findings of Iyonaga and colleagues4
who reported PPD bead granulomas as a site of DC recruitment. We demonstrate that DCs undergo maturation at the site of recruitment displaying expression of class II major histocompatibility proteins and other co-stimulatory molecules when challenged with either pathogen or nonpathogen Ag. Furthermore we show that such DCs when directly transferred to naïve recipients have the capacity to transfer a Th1 anamnestic response. Under noninflamed conditions, the lung is thought to favor a Th2 default response.15,16
However, pathogens often breach the mucosa evoking inflammation and DC mobilization. Studies of rat lungs indicate that resident DCs internalize Ag efficiently but weakly express MHCII and co-stimulatory molecules and confer Th2 responses on adoptive transfer. However, after overnight culture with Ag and GM-CSF in combination with TNF- or CD40L those DCs became potent antigen-presenting cells (APC) that induced Th1 responses on adoptive transfer.15
Our models of Ag bead challenge elicit an environment rich in mediators that apparently similarly activated CD11c+ DCs.
In unprimed mice, SEA beads behaved like parasite eggs inducing an initial Th0 that progressed to a Th2 cytokine pattern;13 therefore it was surprising that SEA bead DCs were unable to transfer a Th2 response. The SEA bead DCs were clearly biologically active as they prevented Th2 differentiation even on Ag boosting. Others have reported successful transfer of Th2 responses using culture generated DCs loaded with SEA.34 However, these cells were not exposed to inflammatory mediators that occur under conditions of parasite egg deposition. Because SEA DCs could not initiate Th2 differentiation then SEA must invoke other pathways to establish a Th2 response. A number of potential mechanisms will need to be investigated such as Ag-presenting B cells, alternative DC subpopulations, and regulatory T cells.35,36
The less vigorous Th1 response induced by SEA DCs as compared to PPD DCs may reflect our observed differences in IL-12. Although isolated lung DCs expressed negligible IL-12 transcripts, PPD induces greater overall IL-12 production in lungs and DLNs compared to SEA (not shown). Although APC-derived IL-1237-39
is considered critical for Th1 differentiation,23
recent evidence suggests that the induction and maintenance of high levels of T-bet are necessary for initial Th1 commitment and IL-12/STAT4 signaling amplifies the amount of IFN- produced by individual cells.40
Thus, transferred SEA DCs may provide signals for T-bet induction but in a milieu deficient in IL-12, resulting in less IFN- production.
In conclusion, we describe models of pulmonary Ag bead-elicited innate granuloma formation that offer a potential in vivo approach to systematically analyze critical mechanisms of the DC response to pathogen Ags as well as determine their relationship to acquired immunity. We further provide the first demonstration of the in vivo Ag-presenting function of lung DCs recruited during pathogen Ag-elicited innate granuloma formation. We show that Ag bead-recruited DCs can confer Th1 responses on adoptive transfer and do not favor Th2 responses even when exposed to a potent Th2 Ag. These findings have important implications with regard to manipulating adaptive immunity in the lung.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported by the National Institutes of Health–National Institute of Allergy and Infectious Diseases (grant AI43460) and the Department of Veterans Affairs.
Schistosome life stages or materials for this work were supplied through National Institutes of Health–National Institute of Allergy and Infectious Diseases contract NO1-AI-55270.
Accepted for publication November 25, 2003.
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  B.-C. Chiu, V. R. Stolberg, and S. W. Chensue Mononuclear Phagocyte-Derived IL-10 Suppresses the Innate IL-12/IFN-{gamma} Axis in Lung-Challenged Aged Mice J. Immunol., September 1, 2008; 181(5): 3156 - 3166. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Lei, B. L. Plattner, and J. M. Hostetter Live Mycobacterium avium subsp. paratuberculosis and a Killed-Bacterium Vaccine Induce Distinct Subcutaneous Granulomas, with Unique Cellular and Cytokine Profiles Clin. Vaccine Immunol., May 1, 2008; 15(5): 783 - 793. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. R. Baskin, H. Bielefeldt-Ohmann, A. Garcia-Sastre, T. M. Tumpey, N. Van Hoeven, V. S. Carter, M. J. Thomas, S. Proll, A. Solorzano, R. Billharz, et al. Functional Genomic and Serological Analysis of the Protective Immune Response Resulting from Vaccination of Macaques with an NS1-Truncated Influenza Virus J. Virol., November 1, 2007; 81(21): 11817 - 11827. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B.-C. Chiu, V. R. Stolberg, C. M. Freeman, and S. W. Chensue Mononuclear Phagocyte-Derived Interleukin-10 Suppresses the Innate Pulmonary Granuloma Cytokine Response in Aged Mice Am. J. Pathol., September 1, 2007; 171(3): 829 - 837. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Freeman, V. R. Stolberg, B.-C. Chiu, N. W. Lukacs, S. L. Kunkel, and S. W. Chensue CCR4 Participation in Th Type 1 (Mycobacterial) and Th Type 2 (Schistosomal) Anamnestic Pulmonary Granulomatous Responses J. Immunol., September 15, 2006; 177(6): 4149 - 4158. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Wen, C. M. Hogaboam, J. Gauldie, and S. L. Kunkel Severe Sepsis Exacerbates Cell-Mediated Immunity in the Lung Due to an Altered Dendritic Cell Cytokine Profile Am. J. Pathol., June 1, 2006; 168(6): 1940 - 1950. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Vermi, F. Facchetti, E. Riboldi, H. Heine, S. Scutera, S. Stornello, D. Ravarino, P. Cappello, M. Giovarelli, R. Badolato, et al. Role of dendritic cell-derived CXCL13 in the pathogenesis of Bartonella henselae B-rich granuloma Blood, January 15, 2006; 107(2): 454 - 462. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Osterholzer, T. Ames, T. Polak, J. Sonstein, B. B. Moore, S. W. Chensue, G. B. Toews, and J. L. Curtis CCR2 and CCR6, but Not Endothelial Selectins, Mediate the Accumulation of Immature Dendritic Cells within the Lungs of Mice in Response to Particulate Antigen J. Immunol., July 15, 2005; 175(2): 874 - 883. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Aksoy, C. S. Zouain, F. Vanhoutte, J. Fontaine, N. Pavelka, N. Thieblemont, F. Willems, P. Ricciardi-Castagnoli, M. Goldman, M. Capron, et al. Double-stranded RNAs from the Helminth Parasite Schistosoma Activate TLR3 in Dendritic Cells J. Biol. Chem., January 7, 2005; 280(1): 277 - 283. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B.-C. Chiu, C. M. Freeman, V. R. Stolberg, J. S. Hu, K. Zeibecoglou, B. Lu, C. Gerard, I. F. Charo, S. A. Lira, and S. W. Chensue Impaired Lung Dendritic Cell Activation in CCR2 Knockout Mice Am. J. Pathol., October 1, 2004; 165(4): 1199 - 1209. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
