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Departments of
*
Biology and
Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and
Division of Immunology and Gnotobiology, Institute of Microbiology, Czech Academy of Science, Prague, Czech Republic
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the murine upper respiratory tract, nasal-associated lymphoid tissue (NALT) is believed to be the equivalent of the Waldeyer’s ring of humans (10). It consists of a paired lymphoid tissue located at the floor of the nasal cavity lined by ciliated respiratory epithelium, and has been postulated in different studies as a possible functional equivalent in the upper respiratory tract to PP in the gut (reviewed in Ref. 11). Several findings support this hypothesis. First, the cellular composition of NALT is similar to PP (12, 13, 14); both tissues contain a major population of naive B cells as well as naive (CD45RBhigh) T cells (12). Second, it has been shown in rats that both NALT and PP have overlaying epithelium containing M cells (15, 16) and follicle-associated epithelium (17) that may serve as entry-sites for different pathogens (18, 19). Third, upon stimulation with Ag, the major isotype of Ab produced by NALT B cells is IgA (12, 13, 20, 21). Salivary glands and tear glands were suggested as possible effector sites for IgA production (22, 23), which is reflected by the presence of IgA Abs in saliva and tear fluid. Despite these similarities, differences between PP and NALT, such as a markedly diverging expression and function of homing receptors (24), have been reported. Other hallmarks of mucosal inductive sites, like formation of germinal centers as well as isotype-switching and expansion of surface IgA+ B cells upon Ag-stimulation have not been directly demonstrated in NALT. Moreover, even though specific CTLs have been observed in cervical or mediastinal LN after intranasal (i.n.) immunization (25, 26), it is unknown whether CTLs are induced in NALT.
Using i.n. infection with reovirus serotype 1/Lang, an established mucosal pathogen that infects and elicits mucosal immune responses in both the gastrointestinal (4, 27, 28, 29) and respiratory tract (30, 31), we have structurally and functionally analyzed murine NALT. Germinal centers, as well as IgA+ and IgG2a+ B cells, were induced and expanded in NALT, and potent reovirus-specific IgA responses were detected in the upper respiratory tract (NALT, palatine salivary glands, and submandibular LN) and gastrointestinal tract (PP, small intestine, and mesenteric LN). Importantly, virus-specific CTLs were detected in NALT as well as in mediastinal, submandibular, and cervical LN. Limiting dilution analysis revealed a 5- to 6-fold higher precursor-CTL frequency in NALT compared with cervical LN. Thus, NALT is a mucosal inductive site in the upper respiratory tract for specific humoral and cellular immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male C3HeB/FeJ (referred to as C3H), BALBc/ByJ, and DBA/2 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). C.B-17 (C.B-Igh1b/IcrTac) mice were from Taconic Farms (Germantown, NY). All mice were used at the age of 8–14 wk. Stock mice were housed in the animal facility of the Department of Biology, University of Pennsylvania (Philadelphia, PA). After infection with reovirus, mice were kept physically separated from naive stock mice in a Trexler plastic isolator (Standard Safety, McHenry, IL).
L-929 fibroblasts were grown in M199 medium (Life Technologies, Grand Island, NY) containing 5% FCS (Life Technologies), 2 mM L-glutamine (Life Technologies), 1000 U/ml penicillin, and 0.1 mg/ml streptomycin (Life Technologies). CTLs were grown in RPMI-1640 (Life Technologies) containing 10% FCS, L-glutamine, penicillin, streptomycin, 50 µg/ml gentamicin (Life Technologies), and 50 µM 2-ME (Sigma Aldrich, St. Louis, MO). For organ fragment cultures, Kennett’s HY medium (Life Technologies) supplemented with 10% FBS, L-glutamine, penicillin, streptomycin, and gentamicin was used.
Histology, immunohistochemistry, and immunofluorescence
NALT was frozen in OCT compound (TissueTek; EMSCO, Philadelphia, PA), horizontal 5-µm cryosections were cut on a Cryocut 1800 cryotome (Leica; Dolbey-Jamison, Norristown, PA) and after air-drying fixed inice-cold acetone. Some slides were stained with hematoxylin (Mayer’s hematoxylin; Sigma Aldrich) and eosin (Sigma Aldrich). For immunohistochemical staining, slides were incubated in 0.3% H2O2 for 30 min, then blocked with Superblock (Pierce, Rockford, IL) for 30 min, and with biotin/avidin block (Vector Laboratories, Burlingame, CA) following the manufacturer’s instructions. Slides were incubated for 60 min with 50 µl of 2.5 µg/ml biotinylated anti-IgD (clone AMS9.1; BD PharMingen, San Diego, CA), 2.5 µg/ml biotinylated anti-CD4 (clone GK1.5; BD PharMingen), 2.5 µg/ml biotinylated anti-CD8 (clone 53.6-72; BD PharMingen), or 4 µg/ml biotinylated Ulex Europaeus agglutinin I (UEA; Vector Laboratories), followed by an incubation with HRP-conjugated streptavidin (1/1000; BD PharMingen). All reagents were diluted in a solution of 10% Superblock in PBS. 3,3-Diaminobenzidine (Sigma Aldrich) was used as substrate according to the manufacturer’s instructions, followed by counterstaining with hematoxylin (Gill’s hematoxylin; Fisher, Pittsburgh, PA), and overlaid with Permount (Fisher).
For immunofluorescence, slides were incubated for 30 min with 50 µl of the following FITC-conjugated reagents: PNA (Pierce, coupled to FITC in our laboratory as described in Ref. 32), 10 µg/ml anti-IgD (clone 11-26c.2a; BD PharMingen), 10 µg/ml anti-IgA (Southern Biotechnology Associates, Birmingham, AL), and 10 µg/ml anti-IgG2a (Southern Biotechnology Associates). Sections were overlaid with Vectashield containing 4',6'-diamidino-2-phenylindole (Vector Laboratories).
Flow cytometry
Single cell suspensions (2 x
105–106/sample) of NALT or
submandibular LN were stained for 20 min at 4°C with FITC-conjugated
anti-CD19 (clone 1D3; BD PharMingen), PE-conjugated anti-CD4
(GK1.5; BD PharMingen), FITC-CD8 (53-6.72; BD PharMingen), FITC-PNA,
PE-
L chain (Southern Biotechnology Associates), FITC-IgA (Southern
Biotechnology Associates), and FITC-IgG2a (Southern Biotechnology
Associates). Cells were washed and fixed in 1% paraformaldehyde in PBS
and analyzed on a FACScan flow cytometer (BD Biosciences, Mountain
View, CA). WinMDI2.8 (The Scripps Research Institute, La Jolla, CA)
software was used for evaluation.
Virus preparation and infection
Third passage stocks of reovirus type 1/Lang (33) were produced and purified as described (34). For i.n. infection, mice were lightly anesthetized with 0.1 mg/g body weight Avertin (2,2,2,-tribromoethanol; Aldrich, Milwaukee, WI) and 1–2.5 x 107 PFU reovirus was applied to both nostrils with a micropipette in a total volume of 25 µl saline/0.5% gelatin.
Virus titration
For determination of viral titers in various tissues, C3H mice (n = 3–4 per time point) were sacrificed by CO2 asphyxiation and cervical dislocation on days 2, 5, 7, and 14 after i.n. infection. After perfusion through the right ventricle with 20 ml PBS, the upper right lobe of the lungs, the entire trachea, one palatine salivary gland, and a 1-cm piece of terminal ileal small intestine were removed, washed, and weighed. The tissues were homogenized in 3 ml saline/0.5% gelatin and serial dilutions incubated on monolayers of L-929 fibroblasts in 6-well tissue culture plates (Costar, Cambridge, MA) for 45 min at 37°C, and thereafter overlaid with 3 ml of 1% Agar in complete M199 medium and cultured at 34°C. Cultures were overlaid with more Agar/M199 after 3 and 6 days. Plaques were counted after 7 days incubation.
Analysis of Ab production in organ fragment cultures
On days 0, 4, 7, 11, and 14 after i.n. infection, C3H mice (n = 3–4 per time point) were sacrificed and blood collected by heart puncture for serum isolation. After perfusion with 20 ml PBS, the entire small intestine and mesenteric LN were surgically removed. PP were visually detected and excised from the small intestine. After decapitation, submandibular LN were removed. NALT was isolated after removal of the mandible as described elsewhere (13). Palatine salivary glands were isolated after removal of the hard palate.
For organ fragment culture (35), tissues were sterilized by sequential washes as described in detail elsewhere (36). Individual PP or LN, 3 x 3 mm pieces of small intestine from jejunum, individual palatine salivary glands, and NALT from one mouse (a pair of NALT still attached to a small piece of nasal epithelium overlaying the palate) were incubated in wells of 24-well tissue culture plates (Costar) in 1 ml complete Kennett’s HY medium for 7 days under a 90% O2/10% CO2 atmosphere at 37°C.
Reovirus-specific IgM, IgA, and IgG2a Abs were measured by RIA. For this purpose, flexible polyvinyl plates (Serocluster; Costar) were coated with 2.5 x 109 particles of reovirus per well in 50 µl PBS overnight at 4°C. Plates were blocked with 1% BSA in PBS and incubated with organ fragment culture supernatant fluid overnight at 4°C. Thereafter, plates were incubated for 6 h at room temperature with 125I-labeled anti-IgA, anti-IgG2a, or anti-IgM Abs (all from Southern Biotechnology Associates). Radioactivity of individual wells was measured using a 1272 Clinigamma gamma counter (Wallac, Gaithersburg, MD). Total IgA Abs were measured by RIA as described earlier (35), and a standard curve of purified, monoclonal IgA was used to convert cpm to nanograms per milliliter.
In vitro restimulation and analysis of cytotoxic lymphocytes
Seven days after i.n. infection of C3H mice with reovirus,
single cell suspensions of NALT, mediastinal, cervical, and
submandibular LN pooled from 16–20 mice were restimulated in vitro by
incubation in 96-well round-bottom microtiter plates (2 x
105/well; Costar) in the presence of 5 x
104 virus-pulsed, irradiated,
thioglycolate-elicited peritoneal exudate cells (4). After
24 h, ConA-conditioned medium (5% (v/v)) mixed with
methyl-a-D-mannopyranosid (100 µM; Sigma Aldrich) was
added. Effector cells were restimulated the same way after 7 days of
culture, and after a total of 13 days of culture, cellular cytotoxicity
was assessed in a standard 51Cr-release
cytotoxicity assay (37). L-929 fibroblasts infected with
reovirus (or uninfected for control) were labeled with 100 µCi
51Cr (NEN, Boston, MA) and then incubated with
effector cells at different E:T ratios (3000 target cells/well). After
5 h of incubation in V-bottom microtiter plates (Costar), 100 µl
supernatant fluids were collected, mixed with 1 ml scintillation fluid
(Cytoscint; ICN Pharmaceuticals, Costa Mesa, CA), and 
-emission
measured on a LS6500 multipurpose scintillation counter (Beckman
Coulter, Fullerton, CA).
Limiting dilution of precursor cytotoxic lymphocytes
Replicate cultures (n = 17–24) containing varying numbers of either NALT or cervical LN cells (4.7 x 102–1.2 x 105 for NALT, 3.9 x 103–5 x 105 for cervical LN) obtained from C3H mice 7 days after i.n. infection were restimulated in vitro for 7 days as described above. Thereafter, the contents of individual microtiter wells were harvested and equally divided into two wells of V-bottom microtiter plates containing either reovirus-infected or -uninfected L-929 cells (3000 target cells/well, labeled with 51Cr) for a 5 h standard 51Cr-release cytotoxicity assay as described above. Cultures were considered to demonstrate cytotoxicity if the resulting 51Cr release was at least three times more than background of spontaneous release. Cytotoxicity was considered virus-specific if the release from infected targets was at least 20% higher from infected than from uninfected targets. The 95% confidence intervals of the linear regression curves were calculated using the Sigmaplot 2.00 software (Jandel Corporation, SSPS, Chicago, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We analyzed the structure of NALT by immunohistochemistry.
As shown in Fig. 1
, NALT in the naive C3H
mouse is organized in distinct B and T cell areas (Fig. 1). As in PP, B
cells reside in follicular areas, whereas T cells, particularly
CD8+ T cells, predominantly occupy the
parafollicular spaces between the B cell follicles. NALT is lined with
an epithelium that binds the lectin UEA (Fig. 1). UEA is specific for
-L-fucose residues which are typically present on M cells and
follicle-associated epithelium (16, 38). Staining with
labeled UEA was restricted to the epithelium on the luminal side of
NALT (Fig. 1). A detailed view of the luminal epithelium revealed
staining of individual cells within the epithelium. These data
demonstrate that NALT displays the typical structure and organization
of a secondary lymphoid tissue and contains epithelial cells that
potentially enable it to efficiently take up Ag from the nasal
cavity.
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, B cells were
the most abundant cell population in NALT comprising 47–78% of
lymphocytes (Fig. 2, upper panel) depending on the strain of
mice. The ratio of B:T cells varied from 3.8:1 in C.B17 to 0.9:1 in C3H
mice, whereas the CD4:CD8 ratio varied from 3:1 in C.B17 to 1.6:1 in
C3H mice (Fig. 2, lower panel). These data reveal that the
cellular composition of NALT is similar to PP. As most studies using
reovirus have been done in C3H mice, this strain was used for all
further analyses.
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To address the function of NALT, C3H mice were infected i.n. with
1–2.5 x 107 PFU of reovirus serotype
1/Lang in a volume of 25 µl. Replicating virus was detected in the
respiratory tract (NALT, palatine salivary gland, lung, and trachea),
and to a lesser extent in the small intestine, and was cleared within
7–14 days after infection (Fig. 3).
These data demonstrate that the respiratory tract is the major site of
viral infection after i.n. inoculation, but that coincidental
swallowing of virus leads to infection and possible cross-priming of
the gastrointestinal tract.
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L chainlow, Ref.
3) was observed in NALT on day 7 and increased through day
14, in parallel with expansion of both surface
IgAlow and IgG2alow
germinal center B cells, as well as surface
IgAhigh and IgG2ahigh
memory type B cells (Ref. 3 ; Fig. 4). Staining with anti-IgG2b Ab gave
similar results (data not shown). A similar expansion of
PNA+, IgA+, and
IgG2a+ B cells was observed in submandibular LNs,
although with slightly delayed kinetics compared with NALT (Fig. 4).
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). No PNA-binding,
IgA+, or IgG2a+ cells were
detected before infection. Seven days after inoculation with reovirus,
germinal centers characterized by the presence of PNA-binding cells
were observed preferentially at the periphery of NALT. These
PNA-positive follicles colocalized with IgA-stained and IgG2a-stained
areas, whereas IgD+ cells predominantly occupied
the PNA-negative regions. By day 14 of infection, PNA-binding cells
were concentrated at the luminal side of NALT and strongly stained
individual IgA+ and IgG2a+
cells were scattered throughout NALT. These findings demonstrate that
germinal center reactions with generation and expansion of IgA- and
IgG2a-switched B cells are induced in NALT after i.n. infection with
reovirus.
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Organ fragment cultures (35) of mucosal and lymphoid
tissues of the respiratory and gastrointestinal tract were performed to
address production of total and virus-specific Abs after i.n. reovirus
infection. Marginal amounts of total IgA were produced by NALT and
palatine salivary glands of naive mice, whereas the components of GALT
produced substantial quantities of IgA before stimulation with reovirus
(Fig. 6A). After infection,
total IgA production was stimulated in NALT and palatine salivary
glands, and simultaneously a slight increase was observed in PP and
small intestine. However, the total output of IgA was 4- to 150-fold
higher (on day 7 or 0, respectively) from PP compared with NALT.
Similarly, production of total IgA was generally higher in small
intestine and mesenteric LN compared with palatine salivary glands and
submandibular LN, respectively.
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B, top row). Whereas production
rapidly declined from NALT, secretion of virus-specific IgA by palatine
salivary glands was sustained for at least 2 wk. Only marginal amounts
of virus-specific IgA were detected in other mucosal tissues of the
oral cavity such as lip or tongue (data not shown). Large amounts of
virus-specific IgA were detected in supernatant fluid of PP and small
intestine fragment cultures indicating cross-priming during i.n.
inoculation. Virus-specific IgA was also produced by mesenteric and to
a lesser extent by submandibular LN which drain the gastrointestinal
and upper respiratory tract, respectively. Compared with IgA, the
production of virus-specific IgG2a was delayed in NALT, but by day 14
postinfection, NALT was producing considerable amounts of
virus-specific IgG2a (Fig. 6B, middle row). Similarly,
virus-specific IgG2a responses were induced in submandibular and
mesenteric LN. In contrast, no appreciable production of virus-specific
IgG2a occurred in PP, palatine salivary glands, or the small intestine.
Virus-specific IgM was detected in PP, submandibular, and mesenteric LN
as early as 4 days postinfection, whereas no virus-specific IgM Abs
were detected in supernatant fluids of NALT, palatine salivary gland,
and small intestine fragment cultures (Fig. 6B, bottom row).
The major isotype of virus-specific Ab induced in serum was IgG2a
concomitant with production of virus-specific IgM, but only trace
amounts of virus-specific IgA (data not shown). Generation of reovirus-specific CTL in NALT
It is not known to date whether cytotoxic cellular immune
responses can be induced in NALT. To address this, 7 days after i.n.
infection with reovirus, cells from NALT, mediastinal, submandibular,
and cervical LN from 16–20 mice were isolated and pooled. After in
vitro restimulation for 13 days, the presence of virus-specific CTL was
assessed in a standard 51Cr-release cytotoxicity
assay. Potent virus-specific CTL were obtained from NALT, as well as
from the LN draining the respiratory tract (Fig. 7). Identical restimulation of NALT and
LN cells isolated from naive mice did not result in the outgrowth of
virus-specific CTL (Fig. 7).
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). This ratio is similar when the
frequencies are adjusted to total numbers of CD8+
cells in the initial NALT or cervical LN populations (NALT: 23.5%
CD8+ cells, frequency =
962/106; cervical LN: 27.8%
CD8+ cells, frequency =
165/106; ratio = 5.8:1).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is well accepted that mesenteric LN serve to amplify immune
responses initiated in PP. Unlike this, it remains elusive which, and
whether at all, LN of the respiratory tract fulfill a similar function
for NALT. However, some of our findings may implicate such a role for
the submandibular LNs. Whereas only modest expansion of cells was
observed in NALT after i.n. infection (<2-fold within first 7 days
postinfection), a more pronounced expansion of cells occurred in
submandibular LN (11-fold) over the same time period. Furthermore,
increased numbers of cells were still found in submandibular LN 14 days
postinfection, although by that time cell numbers in NALT were not
significantly different from naive mice. In contrast, our analysis of
precursor CTL frequencies in NALT vs cervical LN (and presuming similar
numbers for submandibular LNs) revealed a higher frequency of
virus-specific CTL in NALT (Fig. 7). Moreover, kinetic analysis of
generation of PNA+, IgA+,
and IgG2a+ B cells showed a delay in the
generation of these cells in submandibular LN compared with NALT (Fig. 4), i.e., whereas percentages of PNA+,
IgA+, and IgG2a+ cells
peaked or reached a plateau after 
7 days in NALT, the respective
cells appeared later in submandibular LNs. Taken together, these
findings may suggest that submandibular LN can indeed amplify the
specific responses generated in NALT, and that these structures are
functionally equivalent to PP and mesenteric LN, respectively.
Despite these similarities, the essentially different physiological
functions of the respiratory tract (gas exchange) vs the
gastrointestinal tract (uptake of food) result in distinct requirements
for the immune components present at these sites. GALT largely ignores
food Ags to allow uptake of nutrients, and mounts only minimal immune
responses to the components of the resident bacterial flora, but at the
same time serves to exclude potential pathogens. In contrast, the
immune components of the respiratory tract, in particular the lower
respiratory tract, are not permissive and attempt to keep the
noncolonized lungs sterile. These functional differences among a number
of subtle differences might be best reflected in the predominance of
different isotypes at these sites. Clearly, IgA is the major isotype
secreted in the gut, and its limited inflammatory effector potential is
the ideal "low-key" isotype for this site. In contrast, there is
increasing evidence that the lower respiratory tract is mainly
controlled and protected by IgG Abs that have broader effector
potential than IgA. Studies in influenza-infected SCID mice after
transfer of neutralizing mAbs of different isotypes (39),
and more recently in influenza-infected IgA knockout mice
(40) have provided elegant evidence for this notion. IgG
Abs may originate from the systemic circulation and enter the lung by
transudation (41), or be produced locally in
bronchus-associated lymphoid tissue (BALT). Indeed, BALT in rodents has
been shown to mount specific immune responses to Ags present in the
lung lumen (reviewed in Ref. 42), as opposed to a more
discrete function in humans (43). It may be postulated
that NALT may serve an "intermediate" function between BALT and
GALT; although it is in physical connection with the alimentary tract
and not sterile, NALT also serves as an important "gate-keeper" for
the well-protected lower respiratory tract. This intermediate status
may be reflected in the mixed IgA/IgG2a isotype-pattern prevalent at
this site. As shown in Fig. 6, NALT not only produces IgA, but also
considerable amounts of IgG2a Abs (Fig. 6B, middle row).
Similarly, IgG2a was produced in larger quantities than IgA in
submandibular LN, whereas IgA predominated over IgG2a in mesenteric LN.
A more detailed comparison of the functions of BALT vs NALT in
comparison to GALT will be particularly interesting. We are currently
investigating this in a model of reovirus infection of rats.
Several other studies have described humoral immune responses in murine
NALT (12, 13, 20, 21). In these studies, Ab production was
assessed using the ELISPOT technique. In general, the results correlate
well with our findings using the organ fragment culture technique. Both
IgA- and IgG-producing cells were observed in NALT after i.n. infection
with live influenza virus (21, 21, 44). Our results show
local production of both IgA and IgG2a Abs in NALT after i.n. infection
with reovirus. This is in line with the reported production of a mixed
Th1/Th2-type cytokine profile after respiratory infection with reovirus
(45). Others described IgA as the clearly predominating
local isotype in NALT (12, 13). In these studies, cholera
toxin subunit B was used either as Ag or as an adjuvant in combination
with bacterial Ag, which may somehow mediate this skewing of the B cell
response toward IgA. However, the use of the fragment culture technique
may allow for a more quantitative analysis of Ab production than
ELISPOT, as it integrates the number of specific cells with the actual
secretion of Ab, yielding information about total Ab production
capacity of an entire organ or tissue. For example, we show that 7 days
postinfection, NALT produces 4 times less total IgA than PP (Fig. 6A), which is probably due to the smaller number of total
cells present in NALT. Nevertheless, equal amounts of virus-specific
IgA are produced, which likely indicates a higher frequency of
Ag-specific IgA-producing B cells in NALT than in PP.
In this study, we report the first direct demonstration of CTL
generation in NALT. NALT CTL appear to kill infected targets more
efficiently at lower E:T ratios than CTL from the draining LNs of the
same animals (Fig. 7). More strikingly, the 5- to 6-fold higher
precursor CTL frequency in NALT compared with cervical LN (Fig. 8)
clearly shows that NALT is a potent inductive site for specific CTL
responses upon i.n. infection. A similarly increased precursor CTL
frequency in PP over peripheral LN was observed after local
gastrointestinal reovirus infection (4). It will be
interesting to pinpoint effector sites of these CTL to establish
whether they specifically emigrate to mucosal effector sites of the
respiratory tract through a homing-receptor mediated process, or
whether a more random distribution to different nonlymphoid tissues
occurs, as shown recently by Masopust et al. (46) in
systemically and orally primed mice. Furthermore, we demonstrate the
generation of germinal centers in NALT after i.n. infection along with
expansion of IgA+ and
IgG2a+ B cells, and local production of
reovirus-specific IgA and IgG2a Abs. Altogether, these findings provide
direct evidence that NALT is an inductive site for humoral mucosal
immune responses. Thus, tracking patterns of distribution of CTL and
specific B lymphoblasts after induction in local mucosal inductive
sites (e.g., in NALT vs PP) may represent a promising approach to
address cross-priming and communication between distant mucosal sites,
and a way to experimentally assess questions concerning the regulation
and functional characteristics of the common mucosal immune system.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to: Dr. John J. Cebra, Department of Biology, University of Pennsylvania, 415 South University Avenue, Philadelphia, PA, 19104-6018. E-mail address: jcebra{at}sas.upenn.edu
3 Abbreviations used in this paper: GALT, gut-associated lymphoid tissue; NALT, nasal-associated lymphoid tissue; i.n., intranasal(ly); PP, Peyer’s patch; LN, lymph node; UEA, Ulex Europaeus agglutinin I; BALT, bronchus-associated lymphoid tissue.
Received for publication June 27, 2001. Accepted for publication December 4, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
T cell receptor-positive intraepithelial lymphocytes and Peyer’s patch lymphocytes. Eur. J. Immunol. 23:1333.[Medline]
2-microglobulin-deficient mice. J. Virol. 71:5782.[Abstract]
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P. Gueirard, P. Ave, A.-M. Balazuc, S. Thiberge, M. Huerre, G. Milon, and N. Guiso Bordetella bronchiseptica Persists in the Nasal Cavities of Mice and Triggers Early Delivery of Dendritic Cells in the Lymph Nodes Draining the Lower and Upper Respiratory Tract Infect. Immun., July 1, 2003; 71(7): 4137 - 4143. [Abstract] [Full Text] [PDF]
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A. W. Zuercher, H.-Q. Jiang, M. C. Thurnheer, C. F. Cuff, and J. J. Cebra Distinct Mechanisms for Cross-Protection of the Upper Versus Lower Respiratory Tract Through Intestinal Priming J. Immunol., October 1, 2002; 169(7): 3920 - 3925. [Abstract] [Full Text] [PDF]
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). Lightly anesthetized naive C3H mice were infected i.n. with
1–2.5 x 107 PFU reovirus serotype 1/Lang in a volume
of 25 µl. After 7 days, mice (n = 20) were
sacrificed and perfused with PBS. Various dilutions of single cell
suspensions were cultured for 7 days as described above. Contents of
individual wells were assessed for cytotoxicity against
51Cr-labeled reovirus-infected or uninfected L-929
fibroblasts in a 51Cr-release assay. Results were evaluated
as described in Materials and Methods. Frequencies (f)
of precursor CTL among total cells are shown.