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Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4
7-MAdCAM-1 interactions, and that
MAdCAM-1 and VCAM-1 expressed by NALT follicular dendritic cells may
play an important role in lymphocyte recruitment and
retention. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intranasal (i.n.) immunization is an effective route for stimulating mucosal immunity to a variety of pathogens (7, 8, 9, 10, 11), soluble proteins including cholera toxin (12, 13), and microparticle-delivered Ags (14). This route of immunization induces strong Ag-specific mucosal and systemic IgA and IgG Ab responses (6, 7, 9, 10, 11, 12, 13) and stimulates elevated CTL responses to viral and OVA peptides (15, 16, 17). Thus, the elevations in humoral and cell-mediated immunity coupled with the ease of administering Ags make this a favorable route of immunization not only for local respiratory immunity, but also for providing immunity at distal mucosal sites (8, 9, 12, 13, 18, 19). In particular, i.n. immunization provides immunity in the genito-urinary tract (11, 13, 20, 21, 22) where direct immunization is often hampered by epithelial cell turnover and hormonal influences. For instance, mice immunized i.n. with the HIV gp160 protein produced HIV-1-neutralizing IgA and IgG Abs in serum, lung, and vagina (11). Elevations in Ag-specific IgG and IgA were reported in vaginal secretions of mice following i.n. immunization with plasmid DNA as well as viral and bacterial vectors (8, 22, 23). Although i.n. immunization may prove to be a highly effective method of inducing immunity in the genito-urinary tract, it is unknown how immunization of the NALT leads to immunity at this distal mucosal site.
The ability of NALT to induce an immune response at a distal mucosal site may lie in part within the specialized ligands expressed by its high-walled endothelial venules (HEV). These ligands, or addressins, interact with specific homing receptors expressed by B and T lymphocytes and allow for trafficking of the lymphocytes from blood into lymph tissue. However, the addressin profiles displayed by these HEV have yet to be identified, although some functional attributes of preferential homing by NALT lymphocytes have been observed. Lymphocytes isolated from rat NALT preferentially homed back to NALT, to cervical lymph nodes, and to mesenteric lymph nodes (MLN) rather than to the PP (24). This evidence suggests that the NALT addressin profile may differ from the PP profile.
Naive lymphocyte homing to the PP has been well characterized. It
has been shown that the mucosal vascular addressin, MAdCAM-1, plays an
important role in the trafficking of naive B and T cells into PP
(25, 26, 27). MAdCAM-1 is expressed in the PP, MLN, and gut
lamina propria (28, 29). Both B and T cells interact with
MAdCAM-1 through the cellular ligand, 47
(30). In some tissues, MAdCAM-1 can also bind cells
expressing L-selectin through the expression of the peripheral node
addressin (PNAd) carbohydrate on the MAdCAM-1 glycoprotein backbone
(26, 31). In contrast, naive lymphocyte trafficking into
peripheral lymph nodes (PLN) is mediated by L-selectin binding to PNAd
expressed on glycoproteins other than MAdCAM-1 (32, 33).
Thus, there appears to be a definite separation between mucosal and
peripheral type lymphocyte recirculation pathways, and it has yet to be
determined whether NALT HEV express an exclusive mucosal or peripheral
phenotype, or a combination of both.
In this study we show that NALT HEV express a unique addressin profile that resembles neither a strict mucosal nor a peripheral phenotype. All NALT HEV express PNAd, either alone or in conjunction with MAdCAM-1. This profile differs greatly from the PP, the inductive site for the gut-associated lymphoreticular tissue (GALT). The functionality of this PNAd expression was explored through an ex vivo binding assay, where lymphocytes bound to NALT HEV primarily through PNAd-L-selectin interactions. NALT lymphocytes also displayed unique expression of L-selectin profiles. In addition, NALT displayed both MAdCAM-1 and VCAM-1 on the follicular dendritic cells (FDC) within its B cell areas. Finally, the relative location of HEV within the NALT differed greatly from that in the PP. This study suggests that NALT is a unique mucosal tissue, where lymphocyte binding is primarily mediated by PNAd and where the location of MAdCAM-1 and VCAM-1 expressed by FDC may play an important role in lymphocyte recruitment and retention.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free BALB/c mice were purchased from the National Cancer Institute at 5–6 wk of age, or BALB/cBY mice were bred and maintained in the Animal Resources Center at Montana State University (Bozeman, MT). All mice were kept under pathogen-free conditions in horizontal laminar flow cabinets and were fed sterile food and water ad libitum. The mice were free of bacterial and viral pathogens as determined by Ab screening and by histopathologic analysis of major organs and tissues. The mice were used at 6–8 wk of age in these experiments.
Monoclonal Abs
The rat anti-mouse mAbs used were anti-PNAd mAb MECA 79
(32), anti-MAdCAM-1 mAb MECA 367 (28),
anti-L-selectin mAb MEL-14 (34), anti-VCAM-1 mAb
426 (MVCAM.A; PharMingen, San Diego, CA), PE-conjugated MEL-14
(PharMingen), anti-7 mAb FIB 30
(35), anti-CD45R/B220 mAb RA3-6B2 (PharMingen),
anti-CD4 mAb RM4-5 (PharMingen), and CD8 mAb 53-6.7
(PharMingen). Also used was a hamster anti-mouse CD3 mAb 500-A2
(PharMingen).
Tissue isolation and collection
MLN, PLN, and PP were isolated from normal BALB/c mice. Each set of lymphoid tissue was pooled from five different mice, washed in RPMI 1640 medium (BioWhittaker, Walkersville, MD), and frozen using Tissue-Tek O.C.T. compound embedding medium (Miles, Elkhart, IN) in a 15- by 15-mm Tissue Tek Cryomold. Samples were stored at -80°C until use. NALT tissues were collected by removing the soft palates as previously described (5, 14). Briefly, euthanized mice were decapitated, their heads were immobilized, and the lower jaws, including tongue, were removed. Palates were scored along the outer edge and removed gently. Palates were washed in RPMI 1640 medium, blotted dry, and arranged in the cryomolds with their ventral faces (containing the NALT) oriented at the bottom of the mold and frozen in O.C.T. as described above. For double immunofluorescent and immunoperoxidase staining, 5-µm frozen sections were cut, air-dried, fixed in acetone at 4°C, and air-dried before rehydration.
Double immunofluorescent staining for tissue addressins
Lymphoid tissue sections were incubated with MECA 367 supernatant (rat anti-mouse MAdCAM-1 mAb) or normal rat serum for 30 min at room temperature. Specific detection of MAdCAM-1 was obtained upon incubation of a 1/150 dilution of TRITC conjugate goat anti-rat IgG (H+L) Ab (Southern Biotechnology Associates, Birmingham, AL) in the dark for 30 min at room temperature. Sections were then stained for PNAd using a 1/50 dilution of MECA 79 (FITC-conjugated rat IgM anti-mouse PNAd) mAb in the dark for 30 min at room temperature. To prevent nonspecific binding of FITC-conjugated MECA 79 mAb to free arms of the TRITC-goat anti-rat IgG Ab, sections were blocked with 1% rat serum for 30 min before addition of the FITC-conjugated MECA 79 mAb. After rinsing, slides were coverslipped using Vecta-Shield mounting medium (Vector Laboratories, Burlingame, CA).
Immunohistochemical staining of MAdCAM-1 and VCAM-1 location within NALT
Serially cut frozen sections were rehydrated in Dulbecco’s PBS
(DPBS) containing 0.2% normal goat serum (NGS), and endogenous
peroxidase was blocked with Dako peroxidase blocking reagent (Dako,
Carpinteria, CA). Nonspecific binding was blocked using 10% NGS in
DPBS for anti-MAdCAM-1 and VCAM-1 mAb staining or 10% NGS with
2.5% mouse serum in DPBS for anti-B220 mAb staining for 30 min at
room temperature. Endogenous avidin and biotin were blocked with
avidin/biotin blocking solution (Vector Laboratories). Sections were
incubated with a 1/200 dilution of biotinylated rat anti-mouse
VCAM-1 mAb, a 1/500 dilution of biotinylated rat anti-mouse mouse
B220 mAb, a MECA 367 mAb supernatant, or an isotype-matched rat IgG
control for 30 min at room temperature. Sections incubated with MECA
367 mAb were then treated with a 1/250 dilution of biotinylated goat
F(ab')2 anti-rat Ig (
and light chain,
adsorbed against mouse Ig; BioSource International, Camarillo, CA) for
30 min at room temperature. All sections were then incubated with a
1/500 dilution of streptavidin-HRP (SA-HRP; BioSource International),
and HRP was visualized with a precipitate of 3-amino-9-ethylcarbazole
(AEC; Sigma, St. Louis, MO). After AEC development for 3–5 min, the
sections were counterstained with hematoxylin (Richard-Allan
Scientific, Kalamazoo, MI) and counterslipped using Immuno-Mount
mounting medium (Shandon-Lipshaw, Pittsburgh, PA).
Double immunohistochemical staining of NALT lymphocytes and addressins
Frozen sections were rehydrated and blocked as described above. A 10% NGS and 2.5% mouse serum block was used for the anti-B220 mAb staining, whereas a 10% NGS block was used for the anti-CD3 mAb staining. Sections were incubated with 100 µg/ml of MECA 79, MECA 367 supernatant, or mAb isotype control for 30 min at room temperature, followed by a 1/250 dilution of biotinylated goat anti-rat Ig (BioSource International) for 30 min at room temperature, then treated with a 1/500 dilution of SA-HRP (BioSource International) for 20 min. Location of the HRP was visualized with unenhanced diaminobenzidine (Vector Laboratories).
After diaminobenzidine development, sections were reblocked for endogenous peroxidase, and 0.2% BSA was added to the PBS wash to prevent nonspecific Ab binding. Sections were blocked with 10% NGS and 1% rat serum. Sections were then stained with a 1/100 dilution of biotinylated hamster anti-mouse CD3 mAb (PharMingen) for 1 h, a 1/750 dilution of biotinylated rat anti-mouse B220 mAb (PharMingen), or isotype-matched mAb control for 30 min, followed by a 1/500 dilution of SA-HRP (BioSource International). SA-HRP was visualized with VIP substrate kit (Vector Laboratories). Sections were counterstained with hematoxylin (Richard-Allan Scientific). Slides were coverslipped using Crystal/Mount aqueous mounting medium (Biomeda, Foster City, CA).
Ab blocking of naive lymphocyte adhesion to HEV
A modification of the Stamper-Woodruff protocol (36) was performed. Murine MLN were isolated, suspended in RPMI 1640 medium, and subjected to Dounce homogenization. The resulting cell suspension was filtered through NITEX fabric (Fairmont Fabrics, Mountain View, CA). Following centrifugation, the MLN lymphocytes were resuspended in DMEM (Sigma) and 2.5% BSA at a concentration of 1 x 107 cells/ml.
Frozen tissue samples were cut to a thickness of 7 µm and allowed to air-dry for 30 min. A hydrophobic circle was drawn around each section, and the sections were then placed on an orbital shaker (GeneMate, Intermountain Scientific, Bountiful, UT) at 4°C at 80 rpm. MECA 79 or MECA 367 mAb supernatant was added, and the Ab was rotated on the sections for 45–60 min to allow binding to MAdCAM-1 or PNAd and then decanted. One hundred microliters of lymphocytes (1 x 106) were added and rotated over the sections for 30 min to allow binding to HEV. Lymphocytes bound to HEV were fixed by placing sections in a cold 1.5% glutaraldehyde solution.
To block lymphocyte binding to L-selectin or
47, cells were
treated with 50 µg/ml MEL-14 (rat anti-mouse L-selectin) mAb or
FIB 30 (rat anti-mouse
47) mAb,
respectively, for 30 min. Pretreated cells were then rotated over the
sections as described above.
Comparison of L-selectin expressed on NALT, PP, MLN, and PLN lymphocytes
Lymphocytes were isolated from NALT using procedures previously described (4, 6). Briefly, dissected NALT was disrupted by Dounce homogenization, and released lymphocytes were collected in serum-free HBSS (Sigma). PP and PLN lymphocytes were obtained by Dounce homogenization of the tissues as described above, and the resulting cell suspensions were filtered through NITEX. Lymphocytes were stained with FITC-anti-B220, FITC-anti-CD4, or FITC-anti-CD8 mAbs and PE-MEL-14 rat anti-mouse L-selectin mAb. FL1 and FL2 gains and compensation were set by the analysis of single-color FITC or PE. Two-color analyses were performed using a FACSCalibur (Becton Dickinson, Mountain View, CA). Ten thousand events per sample were collected.
Statistical analysis
Results were analyzed using paired Student’s t test. Significant p values are indicated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lymphocyte trafficking into the PP requires MAdCAM-1 expression by
its HEV (25, 26). If the NALT is indeed analogous to PP
and behaves as a mucosal inductive site, it would be expected that its
HEV would also mediate lymphocyte binding primarily through MAdCAM-1.
To assess the NALT addressin phenotype, its HEV were examined for
expression of both mucosal and peripheral addressins. NALT HEV were
stained simultaneously with mAb for MAdCAM-1 (MECA 367 mAb and
TRITC-labeled secondary) and PNAd (FITC-conjugated MECA 79 mAb). The
number of HEV expressing MAdCAM-1, PNAd, or both addressins in each
NALT, PP, or MLN section was determined. As expected in a mucosal
tissue, the NALT HEV expressed MAdCAM-1; however, the total percentage
of HEV expressing mucosal addressin was significantly less than that
observed in the PP (Fig. 1
,
A–F, and Table I). In fact,
all the MAdCAM-1-positive HEV in the NALT also coexpressed PNAd. This
coexpression was observed in 62.8% of the NALT HEV. No NALT HEV
expressed MAdCAM-1 only. In contrast, 52.4% of PP HEV expressed
MAdCAM-1 only, whereas the remaining 47.6% of PP HEV expressed
MAdCAM-1 and PNAd (p < 0.0001). In addition,
there was a significant difference in the number of HEV expressing PNAd
alone in the NALT (37.2%) compared with that in the MLN (2.5%;
p < 0.01). NALT also differs from the PLN, where
nearly 100% of HEV expressed PNAd alone. Thus, NALT HEV express a
unique addressin profile, one that differs from the strictly peripheral
phenotype of the PLN as well as from the intestinal PP and MLN HEV
addressin profiles.
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, I and M). Staining for VCAM-1 showed localization
within the B cell areas, but not on HEV within the NALT (Fig. 1Q). However, staining for MAdCAM-1 in the NALT consistently
appeared diffuse, whereas staining for VCAM-1 appeared intense. This
staining profile differed greatly from that in the PP, which showed
dark staining with the anti-MAdCAM-1 mAb in the B cell areas (Fig. 1, G and K). In addition, NALT FDC displayed
addressin profiles different from those of MLN, which showed very
little VCAM-1 staining and a higher incidence of MAdCAM-1 (Fig. 1, H, L, and P). In contrast, although PLN displays
VCAM-1 on its FDC, very little MAdCAM-1 staining is observed in the
follicles (Fig. 1, J, N, and R). Taken together,
these Ab stains provide further evidence for the expression of a
distinct addressin profile in the NALT. Initial naive lymphocyte binding to NALT HEV is mediated primarily by PNAd –L-selectin interaction
To determine whether the PNAd expressed by NALT HEV was
functional, we used a Stamper-Woodruff ex vivo binding assay. Frozen
tissue sections were treated with mAbs specific for MAdCAM-1 or PNAd or
with irrelevant isotype-matched Abs. For blocking of cellular
homing receptors, naive MLN lymphocytes were pretreated with mAbs
specific for 47 or
L-selectin or with a rat IgG2a isotype control Ab. The number of cells
bound per HEV per node in the presence of addressin-specific Ab was
compared with control binding, defined as lymphocytes bound to HEV in
the presence of isotype-matched control Ab. Blocking of the PNAd
expressed on the HEV of NALT with the MECA 79 mAb resulted in a 60%
reduction of binding compared with that in controls (Fig. 2). This reduction of lymphocyte binding
was similar to that observed in PLN sections treated with MECA 79 mAb.
In contrast, treatment of PP with MECA 79 mAb resulted in no
significant reduction in naive lymphocyte binding
(p < 0.01). Treatment of NALT HEV with MECA
367 mAb had little effect (<20%) on the number of lymphocytes bound
per HEV, indicating that the expressed MAdCAM-1 has no apparent role in
naive lymphocyte binding. These results were in direct contrast with
those for PP, where treatment of sections with MECA 367 mAb resulted in
>90% reduction in binding. In addition, NALT HEV displayed no
similarities in binding to MLN HEV, as 50% of naive lymphocyte binding
to MLN was blocked by MECA 367 mAb. Thus, initial naive lymphocyte
binding to NALT HEV was most similar to naive lymphocyte binding to
peripheral lymph node HEV and dissimilar to the binding observed in the
PP, again providing evidence that NALT acts in a manner unique from
other characterized mucosal tissues.
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). Blocking of L-selectin using MEL-14
mAb resulted in >90% reduction in binding of naive lymphocytes to
NALT HEV. Binding of lymphocytes to NALT HEV was reduced to a lesser
extent by blocking of
47 with FIB 30 mAb,
which reduced binding by about 60% compared with the control value.
MEL-14 mAb blocking of naive lymphocyte binding to NALT was similar to
that observed in the PLN, where this mAb blocked nearly all naive
lymphocyte binding. However, as expected, treatment with FIB 30 mAb had
no effect on binding in the PLN. In the PP, MEL-14 mAb reduced binding
of naive lymphocytes to HEV by 
30% compared with the control value
(p < 0.01), whereas FIB 30 mAb showed a >90%
reduction in naive lymphocyte binding (p <
0.0001). In the MLN, naive lymphocytes exhibit characteristics of
binding to both mucosal and peripheral addressin, evidenced by an
80% reduction in binding by both FIB 30 and MEL-14 mAbs
(p < 0.01). These results suggest that the
NALT HEV express an addressin phenotype distinct from those of other
characterized mucosal sites, and that naive lymphocyte binding to NALT
HEV is primarily mediated by L-selectin- PNAd interactions. However,
47-MAdCAM-1
interactions may also play a role, as evidenced by the reduction in
binding observed upon treatment of naive lymphocytes with FIB 30 mAb.
These results were consistent with the NALT phenotype, in which all HEV
coexpress PNAd, unlike PP HEV. NALT HEV mediated initial naive
lymphocyte binding through different addressin-receptor pairs than did
PP HEV. Thus, NALT HEV expressed more of a peripheral phenotype in both
form and function. This preferential lymphocyte binding to the NALT HEV
via PNAd rather than MAdCAM-1 suggested that the L-selectin expression
on NALT B and T cells might be different from that on lymphocytes in
the PP.
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Based upon the observation that naive lymphocyte binding to NALT
HEV was primarily mediated by L-selectin-PNAd interactions, it would be
expected that NALT lymphocytes express profiles of L-selectin
expression more similar to those of PLN than to those of PP
lymphocytes. Two-color flow cytometric analyses of lymphocytes isolated
from NALT, PP, and PLN were performed to determine the expression of
L-selectin on B cells, CD4+ T cells, and
CD8+ T cells (Fig. 4). As expected for naive lymphocytes,
nearly all the NALT B and T cells expressed L-selectin, similar to the
L-selectin+ populations observed in the PLN. This
result differed from that in the PP, where a significant portion of the
B220+ and CD4+ lymphocytes
were L-selectin negative (Fig. 4 and Table II). The NALT L-selectin-positive
lymphocyte homing receptor profile appeared to differ greatly from that
in the PP, and these data substantiated the results of the ex vivo
binding assays, where initial binding was mediated primarily through
L-selectin interactions with PNAd.
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Although van der Ven and Sminia had observed HEV within the T
cell areas of murine NALT (2), the expressed mucosal and
peripheral addressins had not yet been fully characterized. It
was therefore unknown whether the various HEV phenotypes unique to
the NALT segregated to specific B cell zones or T cell areas within the
tissue. To determine whether the location of HEV within the NALT
affected the type of addressin expressed, a sequential double
immunoperoxidase staining was performed. Staining of murine NALT with
anti-B220 and anti-CD3 mAbs showed clearly defined B and T cell
regions. HEV were localized to both the T and B cell regions (Table III); 39.7% of HEV that expressed PNAd
were located in the B cell areas, whereas the remaining
PNAd+ HEV (58.6%) were located within the T cell
areas. No significant difference was observed for the distribution of
NALT HEV coexpressing PNAd and MAdCAM-1 in the B and T cell areas. In
addition, our evidence suggests that these double-positive HEV were
more likely to be located within the B cell areas of the NALT, than
were the HEV expressing PNAd alone (p = 0.05).
However, compared with the PP, in which only 16.2% of double-positive
HEV were located in the B cell areas, a significantly greater number of
NALT double-positive HEV were located within the B cell area
(p < 0.001; Table III).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results show that naive lymphocytes from nonimmunized mice bind to
the NALT HEV using a peripheral addressin homing receptor pair. The
mucosal ligand-addressin
47-MAdCAM-1
interactions have been shown to mediate both naive and memory
lymphocyte homing to the PP and extralymphoid mucosal sites such as the
gut lamina propria (27, 37). L-selectin can also play a
role in naive lymphocyte trafficking into the PP (26), and
this can be observed in the results of the ex vivo binding assay, where
treatment of lymphocytes with MEL-14 mAb reduces binding to PP HEV
(Fig. 3). Almost all naive lymphocytes display L-selectin on their cell
surface and thus are able to traffic throughout many different lymphoid
sites in the body through interactions with PNAd. NALT HEV may express
PNAd to facilitate naive lymphocyte trafficking through the nasal
passages. Thus, naive lymphocytes can readily circulate through the
lymphoid tissue that first encounters nasally introduced Ag.
However, this does not readily explain why all NALT HEV express PNAd.
In this respect, NALT more resembles a PLN than a PP. Several
experiments have shown that lymphocyte homing to the upper airways of
humans and sheep does not appear to be mediated primarily by
47-MAdCAM-1
interaction, (38, 39, 40). Also, MECA 79 mAb has been shown to
block naive lymphocyte binding to HEV of human tonsils
(41). I.n. immunization induces B cells that have both
L-selectin and 47 on
their cell surface, in contrast to enteric immunization, which induces
mainly 47-expressing
B cells (42). In addition, i.n. immunization has been more
effective than intragastric immunization at producing protective immune
responses in the lower respiratory tract (18, 19). This
may reflect a specific homing pathway of effector cells induced in the
NALT rather than the PP. It appears as though L-selectin-PNAd
interactions may be most important for lymphocyte trafficking into the
respiratory tract, and this conclusion is reflective of the addressin
profile of the NALT HEV. Because the respiratory tract contains both
mucosal (upper airways and bronchi) and systemic (alveolar spaces)
compartments, the coexpression of a peripheral phenotype with the
mucosal addressin may enable trafficking of lymphocytes that facilitate
both systemic and mucosal immune responses.
The presence of MAdCAM-1 as well as VCAM-1 and PNAd within the NALT
presents interesting implications for the theory of the common mucosal
immune system. Recent studies have shown that
47–MAdCAM-1
interactions do not play a major role in lymphocyte trafficking into
the lung or pulmonary tissues (39, 40, 41, 42), but do provide a
mechanism for protective immunity against gut pathogens
(43). Therefore, it has been suggested that this
addressin/homing receptor pair should be regarded as intestinal rather
than mucosal (44). However, our studies in the mouse have
shown that
47-MAdCAM-1
interactions may appear to play some role in the recruitment of
lymphocytes to the nasal tissues and may thus provide a method of
dissemination of Ab-producing cells to distal mucosal effector sites,
including the intestinal and reproductive tracts.
47-MAdCAM-1
interactions may also play a role in the initial binding of naive
lymphocytes to NALT HEV. This is evidenced by a 60% reduction in
binding in lymphocytes treated with the
anti-7 FIB 30 mAb (Fig. 3). These data may
suggest a mechanism for the entrance of intestinal lymphocytes, which
have been shown to preferentially express
47 and enter lymphoid
tissue primarily through
MAdCAM-1-47
interactions (26, 28). Alternatively, the
47–MAdCAM-1
interaction may play a significant role in tight adhesion of
lymphocytes to NALT HEV. In the three-step model of lymphocyte homing,
the initial binding of lymphocytes to NALT HEV may be primarily
mediated by L-selectin-PNAd interactions, whereas subsequent tight
binding might be mediated primarily by
MAdCAM-1-47
interactions (26, 27).
The 47-MAdCAM-1
interaction also may play a significant role in the trafficking of
memory lymphocytes to NALT. Some memory lymphocytes preferentially
express 47 and home
to MAdCAM-1 in the mucosal PP and lamina propria (45, 46).
In addition, MAdCAM-1 has been shown to be up-regulated in inflamed
mucosal tissue (29, 47, 48). It has yet to be determined,
however, whether MAdCAM-1 is up-regulated on the HEV of inflamed NALT
and if such an up-regulation would result in a higher percentage of
lymphocytes binding through
47 interaction.
Another method for naive lymphocyte recruitment and/or retention in
NALT is suggested by the expression of MAdCAM-1 and VCAM-1 by the FDC
in the NALT. MAdCAM-1 expression on PP FDC has been recently
characterized (49). MAdCAM-1 expression within the PP
follicles could mediate binding by
47+
memory lymphocytes, whereas binding to the follicles of PLN appeared to
be primarily mediated by
VCAM-1-41
interaction. It has also been shown (50) that VCAM-1
interactions with the 4 subunit can mediate
binding to the germinal centers of human tonsil. However, VCAM-1 also
binds both 47- as
well as 41-expressing
lymphocytes (51), suggesting that the
47+
lymphocytes might bind in the germinal centers through this route as
well. In addition, adhesion to VCAM-1 may prevent apoptosis and allow
positive selection of B cells within the follicle (52).
Although binding studies of lymphocytes to the NALT follicles have yet
to be undertaken, intense staining with anti-VCAM-1 mAb in the NALT
suggests that lymphocytes expressing both
47 and
41 integrins are able
to traffic into these germinal centers, where they then mature and
differentiate.
Finally, our results indicated that compared with PP, a greater
percentage of the NALT HEV were located within B cell areas. It
appeared as though the double-positive HEV would more likely be located
within the B cell areas of the NALT. The reason for this apparent
localization of the MAdCAM-1+ HEV is unclear, but
this localization of HEV that can support binding of
47+
in the B cell areas might provide a new mechanism for B cell
localization. Rather than entering the lymphoid tissue in the
paracortex and migrating to the B cell areas in response to chemotactic
factors, as has been recently reported (53), these
lymphocytes might simply enter the B cell areas directly, where
MAdCAM-1 and VCAM-1 expression on dendritic cells might also enable B
cell survival and differentiation. However, if T lymphocytes enter
these B cell areas as well, some type of migration would still
occur.
In summary, characterization of addressin expression revealed that NALT HEV were phenotypically and functionally distinct. This addressin profile was demonstrated to be important for initial naive lymphocyte binding mediated by PNAd. Lymphocytes isolated from the NALT also displayed a homing receptor profile that more resembled lymphocytes derived from peripheral tissues. Finally, the location of the HEV within the NALT differed dramatically from that observed in the PP. Collectively, these results suggest that NALT is a truly unique inductive mucosal tissue that cannot be equated with the intestinal PP. In addition, these data may provide evidence for a unique lymphocyte homing pathway, where T and B cells activated in the NALT are able to preferentially circulate to alternate mucosal tissues.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David W. Pascual, Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717-3610. E-mail address:
3 Abbreviations used in this paper: NALT, nasal-associated lymphoid tissue; FDC, follicular dendritic cell; GALT, gut-associated lymphoreticular tissue; HEV, high-walled endothelial venule; i.n., intranasal; MAdCAM-1, mucosal cell adhesion molecule-1; MLN, mesenteric lymph node; PP, Peyer’s patches; PLN, peripheral lymph node; PNAd, peripheral node addressin; DPBS, Dulbecco’s PBS; NGS, normal goat serum: SA, HRP, streptavidin-HRP; AEC, 3-amino-9-ethylcarbazole.
Received for publication March 10, 1999. Accepted for publication May 13, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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47/4P in lymphocyte adherence to fibronectin and VCAM-1 and in homotypic cell clustering. J. Cell Biol. 117:179.This article has been cited by other articles:
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