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Cutting Edge |
E Versus L-Selectin1
Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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4
7 interactions, as is the case for
gut-associated lymphoreticular tissue. To assess the role of L-selectin
in effector B cell immunity, L-selectin-deficient mice were
intranasally immunized with cholera toxin (CT), and mucosal immune
responses were compared with C57BL/6 mice. The absence of L-selectin
correlated with a reduction in CT-specific secretory-IgA responses in
nasal passages and reproductive tract, but not intestinal lamina
propria. Cell sorting experiments showed that an L-selectin-dependent
subset was responsible for CT-specific responses in nasal passages and
reproductive tract, whereas an
E7+ B cell subset was
responsible for L-selectin-independent intestinal immunity. This study
provides evidence for compartmentalization of the common mucosal immune
system into "intestinal" vs "nonintestinal" effector
sites. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Memory T and B lymphocyte homing to the gut subsequent to oral
immunization is mediated through
47 interaction with
mucosal addressin cell adhesion molecule-1 (MAdCAM-1)
(7, 8, 9, 10). Memory T cells appear to be retained in the
lamina propria and intraepithelial compartment of the intestine via the
expression of E7
integrin and its interaction with E-cadherin, although similar
expression has not been observed on B lymphocytes (11).
However, in the nasal-associated lymphoid tissue and the related
lymphoid tissues of the head and neck, naive lymphocyte homing is
mediated primarily through peripheral node addressin (PNAd) and its
interaction with L-selectin (12, 13). In addition,
lymphocyte homing to the upper airways in sheep is mediated through
peripheral homing receptor addressin interactions (14),
and lymphocyte homing to the RT appears to be mediated by homing
receptors other than
47 (15, 16). These results combined suggest that there is
"compartmentalization" of the CMIS.
L-selectin is expressed by nearly all naive T and B lymphocytes,
and it is required for trafficking of lymphocytes to lymphoid tissues
throughout the body. L-selectin interactions with PNAd carbohydrate
expressed by MAdCAM-1 on high endothelial venules of Peyer’s patches
allows for the trafficking of naive lymphocytes to this tissue.
However, in studies conducted in L-selectin-deficient
(L-Sel-/-) mice (17, 18),
cellularity of Peyer’s patches was unaffected by the loss of
L-selectin (17). This suggests a compensatory homing
mechanism via 47
expression, while peripheral lymph nodes were severely reduced in size
and cell number. More importantly, immunization studies revealed that
L-Sel-/- mice have reduced peripheral
delayed-type hypersensitivity responses, as well as delayed graft
rejection in the skin (19, 20, 21). Although immune responses
of L-Sel-/- mice to peripheral
immunization have been well defined, it remains to be determined the
loss of L-selectin will have on mucosal immune responses.
Because the nasal-associated lymphoid tissue appears to rely on
L-selectin-PNAd homing interactions rather than on
47-MAdCAM-1
interactions, we hypothesized that the loss of L-selectin might lead to
a reduction in the mucosal immune response after i.n. immunization. We
determined the effect of L-selectin deficiency on mucosal effector
immune responses in NP, RT, and intestinal lamina propria (iLP)
following i.n. immunization with cholera toxin (CT), a potent mucosal
adjuvant for producing mostly Th2-dependant Ab responses (22, 23). Comparison of CT B subunit (CT-B)-specific Ab titers from
L-Sel+/+ and L-Sel-/-
mice revealed that Ag-specific immune responses are abated in NP and
RT, but not in iLP. Further investigation shows that compensation for
the loss of L-selectin in the iLP can be contributed to a subset of
mucosal effector cells unique to the intestine, and provides additional
evidence for the compartmentalization of the CMIS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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A breeding colony of L-Sel-/- mice on a B6 background (Refs. 17 and 18 ; The Jackson Laboratory, Bar Harbor, ME) was maintained. C57BL/6 (L-Sel+/+) mice were purchased from B&K Universal (Kent, WA). Mice were immunized via nasal drip on day 0 with 5 µg CT in 10 µl sterile PBS (List Biological Laboratories, Campbell, CA) and were boosted on days 7 and 14 postprimary immunization with 2.5 µg CT. Mice were sacrificed 16 days after primary immunization. Serum, fecal, and vaginal wash samples were collected on days 0, 7, and 14 postprimary immunization.
ELISA detection of CT-B-specific IgA and IgG
A CT-B (List Biological Laboratories)-specific ELISA was performed as previously described (23).
Lymphocyte isolation and purification
NP, iLP, and RT lymphocytes were isolated as previously
described (15, 23, 24) using collagenase Type IV (Sigma,
St. Louis, MO; Ref. 25). Tissues from 5–10 mice per group
were used in each experiment. One to three million viable lymphocytes
per tissue per mouse from NP and iLP and 
50,000 viable lymphocytes
per mouse from RT were recovered.
B cell ELISPOT for detection of IgG and IgA Ab-forming cells (AFC)
Standard B cell ELISPOT methods were used as previously described (23).
Flow cytometry and lymphocyte sorting
NP, iLP, and RT lymphocytes combined from 15 mice 16 days
postimmunization were stained for three- or four-color flow cytometry
analysis as follows: cells were stained with
anti-7 FIB 504 mAb supernatant, followed by a
biotinylated goat anti-rat IgG (BioSource International, Camarillo,
CA), then by addition of 1% rat serum, followed by streptavidin-APC.
For three-color analysis, anti-L-selectin mAb MEL-14-PE, and
anti-B220-CyChrome mAb were used, while
anti-47
heterodimer DATK-32-PE mAb, anti-E M290-FITC mAb,
and anti-B220-CyChrome mAb (BD PharMingen, San Diego, CA) were used
for four-color analysis. B220+ lymphocytes were
sorted on a FACSVantage (BD Biosciences, Mountain View, CA) according
to 7 and L-selectin staining.
7high and
7low populations were sorted from
L-Sel-/- and L-Sel+/+
iLP, whereas
7low/L-selectinhigh
and 7low
L-selectinlow populations were sorted from
L-Sel+/+ NP and RT. All cells were sorted at
97% purity, counted, and the respective populations used in a B cell
ELISPOT.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The comparison of CT-B-specific mucosal and serum responses in
L-Sel+/+ and L-Sel-/-
mice subsequent to i.n. immunization with CT revealed that loss of
L-selectin has a significant impact on immune responses in the RT,
resulting in a nearly 32-fold reduction in vaginal IgA titers in
L-Sel-/- mice and complete abatement of IgG
response when compared with L-Sel+/+ mice (Fig. 1
; p <0.001). In contrast,
fecal and serum CT-B-specific IgA titers were not significantly
different between L-Sel+/+ mice and
L-Sel-/- mice. Surprisingly, serum
CT-B-specific IgG titer was significantly lower in
L-Sel-/- mice than in
L-Sel+/+ mice by 16-fold (p
<0.001), suggesting that the lack of L-selectin resulted in a weaker
systemic IgG response. IgG titers in fecal samples were not detectable
in L-Sel+/+ or
L-Sel-/- mice.
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, C and
D). Our ELISPOT data also corroborated the results observed
by ELISA, as there was no significant difference in the number of
CT-B-specific AFC between L-Sel+/+ and
L-Sel-/- iLP, but the Ag-specific response in
RT was completely abated in L-Sel-/- mice.
Collectively, these results suggest the importance of L-selectin for
the development of effector immunity for some mucosal tissues as
evident in the RT but not the iLP. However, what remains unclear is how
the loss of L-selectin selectively diminishes CT-B-specific mucosal
responses in nonintestinal mucosal effector tissues and how intestinal
mucosal effector responses remain intact. Therefore, we analyzed the
specific mucosal effector lymphocytes that populate the NP, RT,
and iLP. The iLP contains a unique subset of effector B lymphocytes
To determine homing receptor expression on effector B lymphocytes
from NP, RT, and iLP, three- and four-color FACS staining for B220,
L-selectin, and 7 was performed on the cells from 16-day
i.n. immunized L-Sel+/+ and
L-Sel-/- mice. Three distinct populations of B
lymphocytes were found in the mucosal effector tissues (Fig. 2A):
L-selectinhigh/7low
and
L-selectinlow/7low
were found in all effector sites, and an
L-selectinlow/7high
phenotype was displayed by 13–23% of iLP lymphocytes. Few lymphocytes
could be recovered from L-Sel-/- NP or RT (Fig. 2B), suggesting that L-selectin is required for lymphocyte
trafficking to these effector tissues.
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7high
and
L-selectinlow/7low
B220+ subsets with DATK 32 mAb, which recognizes
the 47 heterodimer, and with
anti-E (CD103) M290 mAb (Fig. 2, C and
D) revealed that in iLP, the
7high subset also expressed
Ehigh (Fig. 2C). The
7low populations found in the NP
and RT did not express E. However, the
E-negative populations in these tissues and in the iLP
were positive for the 47 heterodimer
(Fig. 2D), whereas the
7high/Ehigh
population in the iLP expressed lower levels of
47.
E7 does not bind to
MAdCAM-1 and does not appear essential for iLP homing
(26); therefore, expression of
E7 may contribute to
the retention of B cells at this site. Alternatively, the
E7+
population represents a resident iLP B cell population that may have
been stimulated by CT immunization. Recent studies also suggest that an
as yet unidentified endothelial cell ligand expressed in intestine may
mediate E7 binding
(27), suggesting that the
E7+
population may have migrated to the iLP through selective homing
interactions independent of L-selectin.
In contrast, staining in the RT and NP revealed that the
7low population expressed only
47low. This lymphocyte
population may be dependent on L-selectin for trafficking to effector
sites, where activation of the lymphocyte then causes rapid
down-regulation of L-selectin. It is well known that L-selectin is
expressed on nearly all naive lymphocytes, but it is rapidly
down-regulated following lymphocyte activation (28, 29).
L-selectin mediates memory lymphocyte trafficking to peripheral sites
such as inflamed skin (7). Further investigation of
L-selectin and 7 staining on lymphocytes isolated from
blood at 16 days postimmunization revealed that these cells are
L-selectinhigh/7low,
contributing to the notion that the
L-selectinlow/7low
effector subsets might have down-regulated L-selectin upon entry into
the mucosal tissues. It is also possible that the
L-selectinlow/7low
subset may have trafficked to the effector sites before 16 days
postimmunization. The absence of a
7high population in RT and NP
suggests that memory lymphocyte trafficking to these sites is dependent
on L-selectin for the initial rolling interaction along endothelial
cells in the effector sites. It is also possible that homing of the
7low subset might be mediated
through 47 binding to MAdCAM-1 that could
be up-regulated in response to immunization. Although FACS analyses
have clearly identified three distinct populations of lymphocytes in
effector sites, it is unclear which of these populations is responsible
for the majority of CT-B-specific and total IgG and IgA production in
the various effector sites.
The L-selectinlow/7low B
lymphocyte subset provides CT-B-specific response in NP and RT
Cell sorting experiments were performed to determine which subset
of lymphocytes provides CT-specific and total IgA and IgG AFC in
effector tissue. NP and RT B220+ lymphocytes were
sorted for
L-selectinhigh/7low
vs
L-selectinlow/7low
(see Fig. 2A for examples of sorted populations), and AFC
responses were enumerated by ELISPOT. For i.n. CT-immunized
L-Sel+/+ NP and RT (Fig. 3, A and B), the
L-selectinlow/7low
subset of B lymphocytes contained the majority of both IgG and IgA
CT-B-specific and total AFC. In the NP, the
L-selectinhigh population accounted for <10% of
specific and total AFC. However, this population did not provide AFC in
RT (Fig. 3B). Additional sorting experiments revealed that
the lymphocytes contained within these effector populations could be
classified as IgDlow memory cells (data not
shown). Because the lymphocyte yields from NP and RT in
L-Sel-/- mice were poor, similar cell sorting
experiments could not be conducted on these tissues.
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7high
(E7+) subset produces
CT-B-specific and total IgA AFC in iLP
In contrast to the NP and RT, the iLP contains a
7high
(E7+)
cell population and very few L-selectinhigh
lymphocytes. Results of NP and RT sorting indicated that the
contribution of the L-selectinhigh population to
AFC in iLP would be minimal at best. Therefore, sorting experiments
were conducted with the 7high and
7low populations (see Fig. 2A for examples of sorted populations). Because lymphocyte
trafficking to the iLP appeared independent of L-selectin, sorted
populations from both L-Sel+/+ and
L-Sel-/- mice were obtained. The
7high lymphocyte population
provided the majority of IgA CT-B-specific and total AFC in both
L-Sel-/- and L-Sel+/+
mice (Fig. 3C), confirming the hypothesis that this subset
does provide an L-selectin-independent mechanism for inducing immunity
in the intestine following i.n. immunization.
Unexpectedly, upon analysis of the contribution of the
7low lymphocyte subset to AFC
response, these studies revealed that the number of total IgA AFC in
the 7low lymphocyte population of
L-Sel-/- mice was reduced (300 AFC) when
compared with the number of AFC obtained from the
7low population in
L-Sel+/+ mice (2300 AFC). The number of total
IgA AFC produced in the 7low
population in L-Sel+/+ mice equaled more than
half the number of AFC found within the
7high lymphocyte population
(2300 vs 4000 AFC). Although there is a subset of
7low lymphocytes present in the
gut, it was found not to be specific for CT. This population may be
dependent upon L-selectin for trafficking and may have been induced via
the i.n. route of immunization, resulting in a reduced total IgA
response in the L-Sel-/- mice. It also
resembles the
L-selectinlow/7low
lymphocyte subset observed in NP and RT.
This study has identified important differences in intestinal vs
nonintestinal mucosal sites. Most importantly, we have identified a
subset of
E7+
B220+ effector lymphocytes in the iLP that are
independent of the expression of L-selectin to traffic to the iLP. In
addition, we have identified a subset of
L-selectinlow/7low/47+
lymphocytes that provides the majority of CT-B-specific and total
immune response in the NP and RT. Surprisingly, this subset provides a
significant contribution to total IgA response in the iLP as well. This
subset might represent lymphocytes that are Ag stimulated in the nasal
inductive site and subsequently traffic to distal mucosal sites,
whereas the
E7+
population may represent a resident intestinal lymphocyte population
stimulated by CT immunization, or a gut-specific population that has
homed from the NP.
However, it is important to determine whether the subsets of
lymphocytes induced in this experiment are strictly Th2-type, or
whether immunization with a Th1-type Ag might induce different
responses. Preliminary studies in our laboratory indicate that i.n.
immunization with attenuated adenovirus induces
L-selectinlow/7low/47+
populations in both NP and RT, whereas both
L-selectinlow/7low/47+
and
E7+
populations are induced in iLP, indicating that these populations may
be stimulated via i.n. immunization, regardless of the type of Ag. More
importantly, it remains to be determined whether a loss of L-selectin
will result in reduced effector NP and RT responses after oral
immunization. However, we have recently shown that oral immunization of
L-Sel-/- mice with a Salmonella
vaccine vector expressing colonization factor Ag I results in
attenuated mucosal IgA Abs while maintaining serum IgG responses
(30). In fact, a preferential bias in serum IgG2a Abs was
noted when compared with similarly immunized
L-Sel+/+ mice. Ongoing studies in our laboratory
are addressing whether the types of Th cells induced impact effector B
cell development and what effector B cells are induced subsequent to
oral immunization with CT. Collectively, the evidence suggests that
several pathways exist for B cell homing to effector tissues. The
question remains as to whether these alternative pathways can
compensate for deficiencies in any of one of these mechanisms and
whether they are dependent on the types of Th cells induced. In
conclusion, this study provides evidence for separation of the CMIS
into "intestinal" vs "nonintestinal" effector sites and
suggests a novel requirement for peripheral addressin-homing receptor
interactions in mucosal effector sites. This evidence provides further
support for the notion that the CMIS is, in fact,
compartmentalized.
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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: dpascual{at}montana.edu
3 Abbreviations used in this paper: i.n., intranasal; CMIS, common mucosal immune system; RT, reproductive tract; GALT, gut-associated lymphoreticular tissue; NP, nasal passages; MAdCAM-1, mucosal addressin cell adhesion molecule-1; PNAd, peripheral node addressin; iLP, intestinal lamina propria; CT, cholera toxin; CT-B, CT B subunit; AFC, Ab-forming cells; L-Sel-/-, L-selectin-deficient.
Received for publication May 8, 2001. Accepted for publication July 13, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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47 by circulating CD4+ cells with memory for intestinal rotavirus. J. Clin. Invest. 100:1204.[Medline]
47. J. Immunol. 161:4227.47 and LFA-1 in lymphocyte homing to Peyer’s patch-HEV in situ: the multistep model confirmed and refined. Immunity 3:99.[Medline]
47 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell 74:185.[Medline]
7(P) integrins on mouse lymphocytes: relevance to the mucosal immune system. Eur. J. Immunol. 21:2591.[Medline]
-galactosidase. Hum. Gene Ther. 6:895.[Medline]
1-induced expression of the mucosa-related integrin E on lymphocytes is not associated with mucosa-specific homing. Eur. J. Immunol. 25:1487.[Medline]
E(CD103)7 mediates adhesion to intestinal microvascular endothelial cell lines via an E-cadherin-independent interaction. J. Immunol. 166:3506.This article has been cited by other articles:
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  E. Tuaillon, D. Valea, P. Becquart, Y. Al Tabaa, N. Meda, K. Bollore, P. Van de Perre, and J.-P. Vendrell Human Milk-Derived B Cells: A Highly Activated Switched Memory Cell Population Primed to Secrete Antibodies J. Immunol., June 1, 2009; 182(11): 7155 - 7162. [Abstract] [Full Text] [PDF]
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D. W. Pascual, X. Wang, I. Kochetkova, G. Callis, and C. Riccardi The Absence of Lymphoid CD8+ Dendritic Cell Maturation in L-Selectin-/- Respiratory Compartment Attenuates Antiviral Immunity J. Immunol., July 15, 2008; 181(2): 1345 - 1356. [Abstract] [Full Text] [PDF]
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A. Rynda, M. Maddaloni, D. Mierzejewska, J. Ochoa-Reparaz, T. Maslanka, K. Crist, C. Riccardi, B. Barszczewska, K. Fujihashi, J. R. McGhee, et al. Low-Dose Tolerance Is Mediated by the Microfold Cell Ligand, Reovirus Protein {sigma}1 J. Immunol., April 15, 2008; 180(8): 5187 - 5200. [Abstract] [Full Text] [PDF]
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 T. Matsumoto, M. Noguchi, O. Hayashi, K. Makino, and H. Yamada Hochuekkito, a Kampo (traditional Japanese herbal) Medicine, Enhances Mucosal IgA Antibody Response in Mice Immunized with Antigen-entrapped Biodegradable Microparticles Evid. Based Complement. Altern. Med., October 29, 2007; (2007) nem166v1. [Abstract] [Full Text] [PDF]
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K. L. Csencsits and D. W. Pascual Absence of L-Selectin Delays Mucosal B Cell Responses in Nonintestinal Effector Tissues J. Immunol., November 15, 2002; 169(10): 5649 - 5659. [Abstract] [Full Text] [PDF]
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0.05.
