| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Rocky Mountain Laboratories, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Hamilton, MT 59840
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Lß2 heterodimer that binds vascular
ICAM-1, and most displayed enhanced levels of the
4ß1 integrin that interacts with VCAM-1.
E and ß7low integrin chains
were detected on approximately 15 and 30% of infiltrating T cells,
respectively. Lymphocytes derived from the spleen or draining lymph
nodes expressed this same integrin profile, suggesting that cells are
recruited to the genital mucosa from the systemic circulation without
significant selection pressure for these markers. Immunofluorescent
staining for the corresponding vascular addressins revealed intense
expression of VCAM-1 on small vessels within
Chlamydia-infected genital tracts and up-regulation of
ICAM-1 on endothelial, stromal, and epithelial cells. Mucosal addressin
cell adhesion molecule-1 was not detected within genital tissues. These
results indicate that T lymphocyte homing to the genital mucosa
requires the interaction of Lß2 and
4ß1 with endothelial ICAM-1 and VCAM-1,
respectively, which is the same pathway that directs lymphocytes to
systemic sites of inflammation. Homing pathways defined for the
intestinal mucosa and assumed to be relevant to all mucosal sites are
not well represented in the genital tract. The identification of T
lymphocyte trafficking pathways shared between systemic and mucosal
tissues should facilitate vaccine strategies aimed at maximizing immune
responses against Chlamydia and other pathogens of the
urogenital tract. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The molecular signals that govern recruitment of recirculating
lymphocytes to the genital mucosa are not known. Lymphocyte trafficking
to the intestinal mucosa, which has provided a model for homing to all
mucosal tissues, has been shown to depend upon receptor-ligand
interactions distinct from those dictating migration into systemic
tissues. Thus, lymphocytes emigrating into sites of cutaneous delayed
type hypersensitivity (9, 10) or systemic inflammation (11) express the
Lß2 and 4ß1
integrins that direct adherence to endothelial cells expressing ICAM-1
and VCAM-1, respectively. In contrast, homing to the Peyer’s patches
of the small intestine depends on expression of the
4ß7 integrin, which confers binding
specificity for the mucosal addressin cell adhesion molecule-1
(MAdCAM-1),2 which is the
predominant ligand on intestinal endothelium (12). Identification of
4ß7-MAdCAM-1 interactions as determinants
of lymphocyte homing to the intestinal mucosa suggested that regulated
expression of the ß7 chain may determine the capacity for
expression of immunity at all mucosal surfaces (12), providing a
mechanism for expression of a common mucosal immune response. However,
T cell homing programs for trafficking to mucosal sites outside of the
gastrointestinal tract have not been defined.
The genital tract is somewhat unique among mucosal surfaces in that it lacks organized lymphoid elements, possessing instead small numbers of mononuclear cells scattered throughout the subepithelial stroma (13, 14). In the absence of a rudimentary follicular structure, induction of immunity to genital pathogens must occur outside of the genital tract followed by recruitment of recirculating cells into infected sites. This contrasts sharply with the resident immune system of the intestinal mucosa that consists of the Peyer’s patches, submucosal lymphocytes, and a large population of CD8+ intraepithelial lymphocytes (IELs) poised between crypt epithelial cells to defend the integrity of the mucosal barrier (15, 16). Given that many sexually transmitted pathogens such as Chlamydia, gonococci, HIV, and herpesvirus can establish infection through either the rectal or genital routes, an understanding of pathways that direct lymphocyte homing to each of these tissues is essential to the development of efficacious vaccines that will provide protection at each of these sites.
In this report, we utilized a murine model of chlamydial infection of the female genital tract to define the receptor-ligand interactions that direct lymphocytes to the genital mucosa. The ability of Chlamydia to infect epithelial cells lining the genital as well as the gastrointestinal tract was utilized to compare directly the expression of homing markers on lymphocytes derived from each of these mucosal sites. Results revealed that T lymphocyte recruitment to the genital mucosa is directed by the same set of interactions that direct T cells to systemic sites of inflammation and distinct from those that dictate trafficking to the intestinal mucosa. The implications of these findings for the development of immunization strategies aimed at maximizing the expression of immunity at the genital mucosa are discussed.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Female C57BL/6J and BALB/cByJ mice, 8 to 12 wk of age, were obtained from The Jackson Laboratory (Bar Harbor, ME). Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility in filter top cages under standard environmental conditions and provided food and water ad libitum.
C. trachomatis
The C. trachomatis strain mouse pneumonitis (MoPn) was grown in HeLa 229 cells, and elementary bodies were purified by discontinuous density centrifugation as previously described (17).
Infection of mice
Mice pretreated with 2.5 mg of medroxy-progesterone acetate (Depo-Provera, The Upjohn Co., Kalamazoo, MI) on day -5 were infected vaginally by depositing 5 µl of 250 mM sucrose, 10 mM sodium phosphate, 5 mM L-glutamic acid (pH 7.2; SPG) containing 1500 inclusion-forming units (IFU) of MoPn into the vaginal vault. Mice were infected enterically by depositing 50 µl of SPG containing 108 IFU C. trachomatis MoPn into the esophagus using a Jorgensen feeding needle. The course of chlamydial infection was monitored by swabbing the vaginal vault or the rectum with Calgiswabs (Spectrum Medical Industries, Los Angeles, CA) at selected intervals postinfection followed by enumeration of recovered IFUs on HeLa cell monolayers using indirect immunofluorescence as described previously (3).
Isolation of mucosal lymphocytes
Lymphocytes were isolated from the genital tract (vagina to ovary) or small intestine using procedures developed for the isolation of intestinal IELs (18), unless stated otherwise. Briefly, tissues were bluntly dissected from adult mice and transferred to tissue culture dishes containing Ca2+/Mg2+-free HBSS. Fecal matter was flushed from the intestinal lumen and Peyer’s patches were bluntly dissected for separate analysis. Organs were bisected longitudinally and then cut into 5-mm sections. Minced fragments were washed at least three times by gravity sedimentation and then transferred to Ca2+/Mg2+-free HBSS containing 5% FCS, 1 mM DTT, and 1.3 mM EDTA in Teflon-coated flasks. Flasks were rotated at 150 rpm for 30 min at 37°C and cell-containing supernatants decanted and held on ice. This procedure was repeated three times for a total of four rotations, and recovered cells were pooled, centrifuged, and counted. Lymphocytes from intestinal samples were further purified on Percoll gradients (18). Because of the low recovery of cells from genital tissues, no further separation of lymphocytes from contaminating epithelial cells was attempted. Where specifically indicated, lymphocytes from similarly prepared tissues were isolated in the presence of RPMI 1640-5% FCS containing 1 mM CaCl2, 1 mM MgCl2, and 100 µg/ml collagenase (Boehringer Mannheim, Indianapolis, IN) (18) for two cycles of rotation. In each experiment, tissues were pooled from three to six mice to obtain sufficient numbers of cells for analysis.
Monoclonal Abs
Fluorochrome-conjugated, mouse-reactive mAbs used to stain
isolated lymphocytes were obtained from PharMingen (San Diego, CA) and
are as follows: ß TCR (clone H57-597), 
TCR (clone GL3),
CD3
(clone 145-2C11), CD4 (L3T4; clone RM4-5), CD8 (Ly-2; clone
53-6.7), CD8ß (Ly-3.2; clone 53-5.8), NK cells (clone 2B4), CD11a
(integrin L chain; clone 2D7), CD11c (integrin
x chain; clone HL3), CD18 (integrin ß2
chain; clone C71/16), CD29 (integrin ß1 chain; clone
Ha2-5), CD44 (clone IM7), CD45R (B220 Ag; clone RA3-6B2), CD45RB (clone
16A), CD49d (integrin 4 chain; clone R1-2), CD62L
(L-selectin; clone MEL-14), CD103 (integrin E chain),
integrin ß7 chain (clone M293), or LPAM-1
(4ß7 complex; clone DATK32). Binding of
biotinylated mAbs was detected with streptavidin-RED613 (Life
Technologies, Grand Island, NY). Interference between mAbs binding to
distinct integrin chains present on the same cell precluded attempts at
colocalization in most cases. Epithelial cells were identified with
polyclonal rabbit anti-keratin Abs (Organon Teknika, Durham, NC)
plus FITC-conjugated goat anti-rabbit IgG (Zymed, San Francisco,
CA). Vascular addressins were detected using fluorochrome-conjugated
Abs recognizing murine CD54 (ICAM-1-1, clone YN1/1.7.4; American Type
Culture Collection (ATCC), Rockville, MD), CD106 (VCAM-1, clone M/K2.7;
ATCC), or MAdCAM-1 (clone MECA-367 (PharMingen) or clone MECA-89
(ATCC)). A panel of fluorescein-conjugated mAbs specific for variable
region genes of the TCR ß-chain were also obtained from PharMingen
and are as follows: Vß2 (clone B20.6), Vß3 (clone KJ25), Vß4
(clone KT4), Vß5.1/5.2 (clone MR9-4), Vß6 (clone RR4-7), Vß7
(clone TR310), Vß8.1/8.2 (clone MR5-2), Vß9 (clone MR10-2), Vß10
(clone B21.5), Vß11 (clone RR3-15), Vß12 (clone MR11-1), Vß13
(clone MR12–3), Vß14 (clone 14-2), and Vß17a
(clone KJ23).
Flow cytometry
Isolated lymphocytes were plated in 96-well round-bottom plates (Corning, Corning, NY) at a concentration no greater than 106 lymphocytes/well. Pelleted cells were resuspended in specific combinations of the mAbs indicated above at final mAb concentrations of 20 µg/ml in HBSS-5% FCS and incubated on ice for 30 min. Samples stained with biotinylated or unlabeled mAbs were washed twice in HBSS containing 5% FCS and pellets resuspended in the appropriate concentration of secondary Ab or streptavidin-RED613 for an additional 30 min on ice. After the final incubation, cells were washed twice in HBSS and once in PBS (pH 7.2), fixed in 2% paraformaldehyde in PBS, and stored in the dark at 4°C for examination on a Becton Dickinson FACS within 24 h. During collection of data, lymphocytes were gated on the basis of forward and side scatter characteristics as verified by detection of CD3 but not keratin markers, and 2500 to 5000 cell events were collected. Additional gates for CD4+ or CD8+ cells were set during data analysis, which reduced the number of cell events accordingly (usually to 800–2200 cell events). Each experiment was repeated two to four times to assess variation in the staining profile of cells from different donors or different time points postinfection and to ensure reproducibility of the results obtained.
Immunofluorescent detection of vascular addressins
Tissues including uteri, cervix/vagina, and Peyer’s patches were removed from three normal and three MoPn-infected mice, snap frozen by immersion in liquid nitrogen, and stored at -80°C. Tissues were mounted in OCT medium (Miles Inc., Elkhart, IN), and 5-µm sections were cut on a cryostat microtome, transferred to microscope slides, air dried, and fixed with 3.7% formaldehyde in PBS at room temperature for 5 min. A representative section of each tissue was stained with toluidine blue to ensure proper orientation within each tissue, and remaining slides were washed once with PBS. Sections were incubated with primary mAbs recognizing the murine vascular addressin ICAM-1, VCAM-1, or MAdCAM-1, or with a polyclonal rabbit antiserum specific for the major outer membrane protein (MOMP) of MoPn, or with buffer alone (Tris-buffered saline, pH 7.3, containing 3% BSA) for 30 min at room temperature. Sections were then washed three times for 5 min each in PBS and incubated with appropriate concentrations of fluorochrome-conjugated secondary Abs for 30 min at room temperature. These included mouse Ig-absorbed tetramethyl rhodamine-conjugated goat anti-rat Ig (Southern Biotechnology Associates, Birmingham, AL) for detection of addressins or mouse Ig-absorbed FITC-conjugated goat anti-rabbit Ig (Southern Biotechnology Associates) for detection of MOMP. After incubation, sections were washed three times for 5 min each and viewed with a Nikon Microphot SA epifluorescence microscope and photomicrographs were taken with Fujichrome Provia 400 daylight film. Images were digitized using a Polaroid SprintScan 35-mm slide scanner, and figures were assembled in gray scale without further manipulation using Adobe Photoshop Version 4.0.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Lymphocytes were isolated from the genital tracts of normal female
C57BL/6 mice (18) and analyzed by flow cytometry for expression of T
cell subset markers. Approximately 30% of the CD3+
lymphocytes were double-negative cells expressing neither CD4 nor CD8,
whereas the remainder consisted of roughly equal proportions of
CD4+ and CD8ß+ T cells (Table I
). No CD8+ T cells
were detected. Most lymphocytes expressed the conventional ß TCR
with less than 5% of either subset displaying a -encoded
receptor in each of several experiments (data not shown). Vaginal
infection with C. trachomatis increased the recovery of
genital tract lymphocytes by up to 75-fold at 12 days postinfection
(from 0.2–1.1 x 105 lymphocytes/normal genital tract
to 0.8–1.5 x 106 lymphocytes/infected genital tract)
and resulted in an altered distribution of T cell subsets with an
increase in the proportions of CD4+ and CD8+ T
cells and an apparent loss of the double-negative subset (Table I).
|
),
in general agreement with published reports of IEL subsets (19, 20).
CD8+ cells could be further divided into
CD8ß+ and CD8+ subsets, with
expression of the instead of the ß TCR by 30–50% of the
CD8+ population (data not shown). Enteric infection
with C. trachomatis did not alter significantly the number
of intestinal lymphocytes recovered (0.9–1.7 x 106
lymphocytes/normal or infected small intestine) nor their phenotypic
profile (Table I). Collectively, these data indicate that cellular
infiltrates within the genital vs the intestinal mucosa can be
distinguished by the presence of double-negative T cells,
CD8+ T cells, + CD8+ T cells, and
the magnitude of the host inflammatory response to infection. Integrin profiles on mucosal lymphocytes
Lymphocyte homing to systemic tissues usually relies on binding of
the 4ß1 integrin to endothelial
VCAM-1 (21, 22) whereas homing to the intestinal mucosa depends on
4ß7 interactions with MAdCAM-1 (23, 24).
Once localized to the intestine, retention of lymphocytes at the
epithelial border is thought to depend on
Eß7 binding to E-cadherin (25). To
determine which of these receptor-ligand interactions may be important
to lymphocyte homing and retention within the reproductive mucosa,
genital tract lymphocytes were examined by flow cytometry for
expression of a panel of integrin - and ß-chains. Mice were
infected vaginally with C. trachomatis before cell harvest
to stimulate a mucosal inflammatory response and provide sufficient
numbers of lymphocytes for analysis. Intestinal IELs were analyzed in
parallel to provide a direct basis for comparison and a positive
control for marker expression.
CD4+ and CD8+ T cells recovered from the
mucosa of the genital tract were largely negative for the
E chain that is prominent on intestinal lymphocytes but
expressed uniformly the L chain that combines with
ß2 to form LFA-1 (Fig. 1).
Genital tract lymphocytes, but not intestinal lymphocytes, also
displayed enhanced levels of the ß1 integrin that
complexes with 4 to form the receptor for VCAM-1. In
contrast, ß7 expression was up-regulated on most
intestinal lymphocytes but was expressed by fewer than 30% of genital
tract lymphocytes (Fig. 1). All CD4+ intestinal IELs
expressed Lß2, but only two-thirds of
intestinal CD8+ cells displayed this marker because of the
absence of the L chain from some cells (Fig. 1).
Expression of other markers was not different on lymphocytes derived
from the genital vs intestinal mucosa, in that all cells expressed low
levels of 4 and only 0 to 5% of cells stained positive
for L-selectin or CD11c (x) (data not shown) (26, 27).
The trends exemplified by these data were also observed in two
experiments utilizing cells pooled from noninfected donors, although
the absolute proportion of integrin-positive cells varied by 10
to 15% for certain markers between experiments (primarily
ß7).
|
Lß2 (LFA-1) and
4ß1 (VLA-4) as primary integrins on T
lymphocytes homing to the genital mucosa. To ensure that this receptor
profile extended to submucosal lymphocytes, expression was also
analyzed on cells isolated from collagenase-digested tissues. Under
these conditions, the majority of CD4+ cells from the
female genital tract expressed ß1, and 25% also
expressed ß7 (Fig. 2A). Intestinal
lymphocytes displayed the reciprocal pattern, with most cells staining
positive for ß7 but low to negative for ß1.
The intensity of ß7 staining is nearly 1 log higher on
lamina propria lymphocytes isolated through collagenase digestion of
the small intestine (Fig. 2A) compared with
expression on the IEL subset recovered with reducing agents (see Fig. 1), distinguishing ß7high and
ß7low T cell subsets in the small intestine
(28). By comparison, the level of expression on the positive subset of
genital tract lymphocytes would be characterized as
ß7low. Differential representation of
ß1 vs ß7 integrins on lymphocytes
infiltrating the genital vs intestinal mucosae appears to be
independent of the method used to recover cells for analysis.
|
B
demonstrate the expression of ß1 vs ß7
integrins on CD4+ and CD8+ T cells isolated
from collagenase-digested genital tracts collected 5 days after a
secondary challenge with 1500 IFU MoPn. As found previously,
infiltrating T cells expressed predominantly the ß1
integrin chain, and only a small subset stained positive for
ß7low (Fig. 2B). Similarity of integrin and Ag receptor profiles on spleen, lymph node, and genital tract lymphocytes
The dominance of 4ß1 and
Lß2 expression by genital tract
lymphocytes suggested that these cells may be representative of the
recirculating pool of peripheral lymphocytes. A phenotypic comparison
of integrin profiles on lymphocytes derived from the spleen, iliac
lymph nodes, or genital tracts of BALB/c mice supported this
possibility (Fig. 3). Expression of
selected markers was similar at all three sites, in that
CD4+ T cells were largely negative for E,
positive for ß1, and negative for ß7. This
contrasts with the integrin profile of intestinal CD4+
lymphocytes, which is E+,
ß1-, and ß7+
(Fig. 3).
|
ß TCR with fewer than 5%
of cells staining positive for -encoded receptor molecules in
several separate experiments. As one approach toward defining selection
pressures that may be imposed on lymphocyte homing to the genital
mucosa, expression of selected Vß specificities was
analyzed using a panel of FITC-conjugated mAbs recognizing 13 distinct
Vß gene products (Vß2, -3, -5, -6, -7, -8,
-9, -10, -11, -12, -13, -14, and -17). Spleen and lymph node
lymphocytes from the same animals were stained in parallel to define
the panel of receptor expression on cells homing to systemic tissues.
Vß8 was identified as the predominant receptor
specificity on lymphocytes from all three locations, with expression
being detected on 18 to 25% of CD4+ and CD8+ T
cells from each site (data not shown). Expression of Vß3
ranked second, and low levels of the remaining Vß
specificities were randomly distributed. By this criterion, there was
no evidence for selection of specific TCR specificities on cells homing
to the genital mucosa. Lymphocytes infiltrating the genital tract are memory cells
It has been suggested that only memory cells carry the capacity
for mucosal homing, with naive cells being restricted to the peripheral
circulation and systemic tissues (21, 30, 31). To test this hypothesis
at the genital mucosa, lymphocytes isolated from
Chlamydia-infected genital tracts were typed for expression
of CD44 and CD45RB to define their status as naive or memory cells.
CD4+ lymphocytes uniformly displayed the
CD44high,CD45RBlow phenotype, consistent with
definition of a memory subset of T cells. CD44 was also up-regulated on
CD8+ lymphocytes, but expression of CD45RB on these cells
was inconsistent, with approximately half of the cells expressing the
CD45RBlow phenotype and half displaying the
CD45RBhigh phenotype (Fig. 4). The profile of intestinal IELs was
similar in that the majority of CD4+ cells clearly
displayed a memory phenotype whereas the CD8+ population
was heterogenous with regard to expression of CD45RB (Fig. 4). The
failure of CD8+ T cells to down-regulate CD45RB following
receptor-mediated activation has been reported previously (32),
however, suggesting that this marker may not be a reliable indicator of
memory status in the CD8+ subset.
|
Identification of Lß2 and
4ß1 as major integrins on genital tract
lymphocytes distinguished this site from the intestinal mucosa, where
4ß7-MAdCAM-1 interactions dictate
lymphocyte homing. If the expression of ligands on vascular endothelium
reflects the integrin profile of infiltrating cells, induction of
ICAM-1 and VCAM-1 rather than MAdCAM-1 would be predicted within the
genital tract. This prediction was tested by immunofluorescent
localization of ICAM-1, VCAM-1, and MAdCAM-1 on vascular endothelium of
normal and Chlamydia-infected genital tracts.
For ease of sectioning, the uterus, oviduct, and ovaries from each of
three mice were examined separately from the cervix and vagina. The
uteri of infected mice exhibited gross evidence of acute inflammation
manifested by edema and vascular congestion. Microscopically, the
endometrium was edematous, and the lumen was filled with a
polymorphonuclear leukocyte exudate. Staining of tissues from infected
mice with Abs specific for the MOMP of Chlamydia revealed an
extensive epithelial infection (Fig. 5A) with clear,
well-circumscribed inclusions restricted to epithelial cells in the
endometrium (Fig. 5B) and endocervix (not shown).
|
A, HEV) and in microvessels
within the lamina propria of mucosal villi (not shown). VCAM-1
expression was not detected in these sections, and ICAM-1 staining was
weak and restricted to small vascular structures within the lamina
propria (Fig. 6, B and C, and not shown). In
contrast, MAdCAM-1 was essentially undetectable in either the normal
(Fig. 6D) or Chlamydia-infected (Figs. 6G and 7B)
uterus using mAb against either of two distinct epitopes on the
MAdCAM-1 molecule (MECA 89 and MECA 367). Instead, VCAM-1 and ICAM-1
were the predominant addressins detected within the uterine mucosa
(Fig. 6). VCAM-1 was found only in rare focal small arteries in the
muscular wall of the normal uterus (Fig. 6E, arrow) but was
expressed in small vessels (Figs. 6H and
7A) and focal epithelial cells (Fig. 7A) scattered throughout the endometrium of infected
mice. ICAM-1 displayed a broader distribution in the normal uterus and
was detected on vascular and epithelial cells as well as on stromal
elements within the lamina propria (Fig. 6F).
Expression of this addressin was further up-regulated following
infection with Chlamydia (Fig. 6I) with
extension to all layers of the uterus including the epithelium, stroma,
and muscularis (Fig. 7, C and D). In fact,
expression of ICAM-1 by vascular structures within infected uteri was
difficult to appreciate because of the massive staining of stromal
cells. Examination of cervical/vaginal tissues from these same mice
revealed no notable differences in expression of ICAM-1 and MAdCAM-1,
with expression being consistently positive and negative, respectively.
However, it was our impression that VCAM-1 was expressed at higher
levels in the cervix than in the uterus of normal mice. In the normal
uterus, VCAM-1 expression was restricted to small arteries in the deep
muscularis, whereas in the cervix it was also frequently detected in
small vessels of the submucosa. Findings were similar in all three mice
examined. Cumulatively, these findings suggest that a dichotomy of
addressin expression exists between the genital and intestinal mucosae,
whereby ICAM-1 and VCAM-1 appear to be predominant ligands in the
genital tract vs MAdCAM-1 as the predominant ligand in the intestinal
tract.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß TCR molecules. Large subsets of CD8+ T cells
or receptor-bearing cells similar to those present in the
intestinal mucosa were not found in the genital tract. All T cells from
reproductive tissues expressed the broadly functional
Lß2 heterodimer that participates in the
final stage of lymphocyte diapedesis during emigration into cutaneous,
systemic, or mucosal sites of inflammation (21, 30). The majority of
cells also displayed the 4ß1 integrin that
has been implicated in trafficking to nonmucosal sites such as the
heart, liver, and central nervous system (21, 22, 33). Expression of
homing receptors traditionally associated with mucosal homing and
retention, the 4ß7 and
Eß7 integrins, respectively, was detected
on a small proportion of cells not only from the genital tract but also
from the spleen and lymph node, defining the population of cells
infiltrating genital tissues as being broadly similar to those found in
major lymphoid organs. This notion was supported by detection of
similar proportions of Vß8 receptor bearing T cells at
all three sites, although additional selection pressures for genital
homing markers or receptor specificities not evaluated in these studies
cannot be excluded. Expression of ß7 integrins by
lymphocytes infiltrating the genital mucosa distinguished these cells
from Peyer’s patch or lamina propria lymphocytes in that the former
were ß1high/ß7low
and the latter,
ß1low/ß7high. Only
cells exhibiting the
ß1low/ß7high
profile have been shown to efficiently bind the MAdCAM-1 ligand on
venule endothelial cells,
ß1high/ß7low cells
interacting preferentially with VCAM-1 (12). Considered collectively,
these data suggest that Lß2 and
4ß1 are primary integrins involved in
lymphocyte homing to the genital mucosa during chlamydial
infection.
The significance of this receptor profile was confirmed by
immunofluorescent detection of the corresponding vascular ligands on
genital tract endothelial cells. ICAM-1 and VCAM-1 were both
constitutively expressed in the normal genital tract, but expression
was dramatically induced following infection with C.
trachomatis. In contrast, MAdCAM-1 was not detected in the genital
tract either before or after chlamydial infection using mAbs
recognizing either of two distinct epitopes on the MAdCAM-1 molecule,
mAbs that readily stained MAdCAM-1 on gut-associated lymphoid tissue.
Therefore, it must be concluded that MAdCAM-1 is expressed at very low
levels, if at all, in the genital mucosa. It appears that T lymphocyte
trafficking to the genital tract depends instead on the interaction of
Lß2+,4ß1+
T cells with vascular ICAM-1 and/or VCAM-1. This feature, as well as
the magnitude of the inflammatory response and the phenotype of
recruited cells, distinguishes the expression of immunity within the
genital tract from that within the intestinal tract, even though
lymphocytes from both sites belong to the larger subset of memory T
cells.
The immunologic basis for utilization of distinct homing pathways at
different mucosal sites is of interest as it relates to the function
and microenvironment of tissues at each mucosal surface. For example,
the intestinal mucosa is host to a variety of bacteria, resident
commensals as well as potential pathogens. Elicitation of a vigorous
4ß1+ type 1 T cell-mediated
immune response at every encounter would be inappropriate, because
inflammation would compromise the absorptive function of the luminal
epithelium. To accommodate these environmental conditions, intestinal
4ß7+ lymphocytes appear to be
under strict negative control by regulatory cytokines. Disruption of
this delicate balance by deletion of the gene encoding IL-10 can result
in development of inflammatory bowel disease (34), whereas both IL-10
and TGF-ß contribute to the intestinal lymphocyte-mediated
suppression of peripheral blood T cell responses to Escherichia
coli Ags (35). The uterine mucosa more closely resembles the
liver, central nervous system, and other systemic tissues in that the
coexistence of even commensal bacteria cannot be tolerated, and
sterility is maintained through the vigilance of recirculating
4ß1+ lymphocytes. Constitutive
up-regulation of VCAM-1 in the uninfected cervical mucosae may reflect
a higher frequency of exposure to environmental pathogens within the
lower vs upper genital tract. The lung is another site intolerant to
bacterial pathogens that is poor in
4ß7+ lymphocytes and vascular
MAdCAM-1 expression (36), supporting the concept that
4ß7+ lymphocytes predominate
only where the host inflammatory response must be down-regulated to
preserve tissue function and/or to allow the coexistence of a resident
bacterial population.
Identification of a systemic homing pathway for T cell trafficking to
the genital mucosa also has implications for the functional expression
of a common mucosal immune system (15, 37), whereby cells primed in the
intestine migrate to distant mucosal sites (38) to mediate protection
against a subsequent challenge. Enteric immunization with live C.
trachomatis has been shown to provide protection against a
subsequent vaginal challenge (39), but whether this reflected
immigration of CD4+ lymphocytes from the intestinal mucosa
via a common Lß2-ICAM-1 pathway or the
concomitant but unintentional infection of genital tissues as a result
of grooming behavior in mice is not clear. In the present experiments,
chlamydial shedding was detected within the vaginal vault of
enterically infected animals (unpublished data), supporting the
possibility that genital immunity developed as a result of an
unintended primary chlamydial exposure at this site. However, the
potential for intestinally primed Lß2+,
4ß7+ T cells to migrate into genital
tissues following adhesive interactions with locally expressed ICAM-1
cannot be excluded.
Knowledge of the homing signals required to direct mononuclear cells to
mucosal tissues should facilitate the delivery of vaccines specific for
Chlamydia or other mucosal pathogens, because the site of
initial Ag exposure dictates the integrin profile of circulating,
Ag-specific cells (40, 41). In addition to the potential for enteric
immunization mentioned above, sharing of the
4ß1-VCAM-1 pathway between reproductive
and systemic tissues implicates parenteral immunization as an effective
route to induce protection against pathogens of the urogenital tract.
Indeed, the development of salpingitis in mice immunized parenterally
with the C. trachomatis MOMP was significantly reduced when
compared with mice immunized via the Peyer’s patches (42). A third
alternative, direct immunization of the vaginal mucosa, may actually
prove to be the least efficient route because of the hormonally
regulated expression of a cornified epithelium, low numbers of stromal
macrophages, and T cells (13, 14, and this paper), and the absence of
organized lymphoid tissue. Ultimately, definition of the homing
programs relevant to all available mucosal immunization sites may be
required before attempts to provide cross-protection through
vaccination can be implemented successfully.
It is likely that the collective program of interactions dictated by
hormonal influences, receptor usage, chemoattractant gradients, and
integrin activation determines the ultimate efficacy of the
inflammatory response at any site of inflammation. It is now apparent
that these programs differ for cells homing to the intestinal vs the
genital mucosa, at least with regard to the integrins and vascular
ligands required for lymphocyte extravasation. Identification of the
4ß1-VCAM-1 homing pathway as a major
system for directing lymphocytes to the genital mucosa suggests that
mucosal immunization strategies may not be required to induce T
cell-mediated immunity at this site. Instead, parenteral immunization
may prove efficacious in providing protection against
Chlamydia and other pathogens of the urogenital tract.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Abbreviations used in this paper: MAdCAM-1, mucosal addressin cell adhesion molecule-1; IEL, intraepithelial lymphocyte; MoPn, mouse pneumonitis; IFU, inclusion-forming unit; MOMP, major outer membrane protein.
Received for publication September 24, 1997. Accepted for publication November 20, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-dependent and -independent pathways. J. Immunol. 158:3344.[Abstract]
- T-cell receptor on intestinal CD8+ intraepithelial lymphocytes. Nature 333:855.[Medline]
4ß7 and LFA-1 in lymphocyte homing to Peyer’s patch-HEV in situ: the multistep model confirmed and refined. Immunity 3:99.[Medline]
Eß7 integrin. Nature 372:190.[Medline]
4ß7 and Eß7 integrins on thymocytes, intestinal epithelial lymphocytes and peripheral lymphocytes. Eur. J. Immunol. 26:897.[Medline]
This article has been cited by other articles:
|
|
 |
|
  P. Steven, J. Rupp, G. Huttmann, N. Koop, C. Lensing, H. Laqua, and A. Gebert Experimental Induction and Three-Dimensional Two-Photon Imaging of Conjunctiva-Associated Lymphoid Tissue Invest. Ophthalmol. Vis. Sci., April 1, 2008; 49(4): 1512 - 1517. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Li, A. K. Murthy, M. N. Guentzel, J. Seshu, T. G. Forsthuber, G. Zhong, and B. P. Arulanandam Antigen-Specific CD4+ T Cells Produce Sufficient IFN-{gamma} to Mediate Robust Protective Immunity against Genital Chlamydia muridarum Infection J. Immunol., March 1, 2008; 180(5): 3375 - 3382. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Li, M. N. Guentzel, J. Seshu, G. Zhong, A. K. Murthy, and B. P. Arulanandam Induction of Cross-Serovar Protection against Genital Chlamydial Infection by a Targeted Multisubunit Vaccination Approach Clin. Vaccine Immunol., December 1, 2007; 14(12): 1537 - 1544. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. R. Roan and M. N. Starnbach Antigen-Specific CD8+ T Cells Respond to Chlamydia trachomatis in the Genital Mucosa J. Immunol., December 1, 2006; 177(11): 7974 - 7979. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. R. Roan, T. M. Gierahn, D. E. Higgins, and M. N. Starnbach Monitoring the T cell response to genital tract infection PNAS, August 8, 2006; 103(32): 12069 - 12074. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Gupta, R. Janani, Q. Bin, P. Luciw, C. Greer, S. Perri, H. Legg, J. Donnelly, S. Barnett, D. O'Hagan, et al. Characterization of Human Immunodeficiency Virus Gag-Specific Gamma Interferon-Expressing Cells following Protective Mucosal Immunization with Alphavirus Replicon Particles J. Virol., June 1, 2005; 79(11): 7135 - 7145. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 R. P. Morrison and H. D. Caldwell Immunity to Murine Chlamydial Genital Infection Infect. Immun., June 1, 2002; 70(6): 2741 - 2751. [Full Text] [PDF]
|
|
|
|
|
|
U. Palendira, A. G. D. Bean, C. G. Feng, and W. J. Britton Lymphocyte Recruitment and Protective Efficacy against Pulmonary Mycobacterial Infection Are Independent of the Route of Prior Mycobacterium bovis BCG Immunization Infect. Immun., March 1, 2002; 70(3): 1410 - 1416. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Belay, F. O. Eko, G. A. Ananaba, S. Bowers, T. Moore, D. Lyn, and J. U. Igietseme Chemokine and Chemokine Receptor Dynamics during Genital Chlamydial Infection Infect. Immun., February 1, 2002; 70(2): 844 - 850. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E.-L. Johansson, L. Wassen, J. Holmgren, M. Jertborn, and A. Rudin Nasal and Vaginal Vaccinations Have Differential Effects on Antibody Responses in Vaginal and Cervical Secretions in Humans Infect. Immun., December 1, 2001; 69(12): 7481 - 7486. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. L. Csencsits, N. Walters, and D. W. Pascual Cutting Edge: Dichotomy of Homing Receptor Dependence by Mucosal Effector B Cells: {alpha}E Versus L-Selectin J. Immunol., September 1, 2001; 167(5): 2441 - 2445. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. L. Wormley Jr., J. Chaiban, and P. L. Fidel Jr. Cell Adhesion Molecule and Lymphocyte Activation Marker Expression during Experimental Vaginal Candidiasis Infect. Immun., August 1, 2001; 69(8): 5072 - 5079. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. U. Igietseme, J. L. Portis, and L. L. Perry Inflammation and Clearance of Chlamydia trachomatis in Enteric and Nonenteric Mucosae Infect. Immun., March 1, 2001; 69(3): 1832 - 1840. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-K. Kim, L. Devine, M. Angevine, R. DeMars, and P. B. Kavathas Direct Detection and Magnetic Isolation of Chlamydia trachomatis Major Outer Membrane Protein-Specific CD8+ CTLs with HLA Class I Tetramers J. Immunol., December 15, 2000; 165(12): 7285 - 7292. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. S. Rott, M. J. Briskin, and E. C. Butcher Expression of {alpha}4{beta}7 and E-selectin ligand by circulating memory B cells: implications for targeted trafficking to mucosal and systemic sites J. Leukoc. Biol., December 1, 2000; 68(6): 807 - 814. [Abstract] [Full Text]
|
|
|
|
|
|
R. A. Hawkins, R. G. Rank, and K. A. Kelly Expression of Mucosal Homing Receptor alpha 4beta 7 Is Associated with Enhanced Migration to the Chlamydia-Infected Murine Genital Mucosa In Vivo Infect. Immun., October 1, 2000; 68(10): 5587 - 5594. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. G. Morrison and R. P. Morrison In Situ Analysis of the Evolution of the Primary Immune Response in Murine Chlamydia trachomatis Genital Tract Infection Infect. Immun., May 1, 2000; 68(5): 2870 - 2879. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. G. Feng, W. J. Britton, U. Palendira, N. L. Groat, H. Briscoe, and A. G. D. Bean Up-Regulation of VCAM-1 and Differential Expansion of {beta} Integrin-Expressing T Lymphocytes Are Associated with Immunity to Pulmonary Mycobacterium tuberculosis Infection J. Immunol., May 1, 2000; 164(9): 4853 - 4860. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Kelly, J. C. Walker, S. H. Jameel, H. L. Gray, and R. G. Rank Differential Regulation of CD4 Lymphocyte Recruitment between the Upper and Lower Regions of the Genital Tract during Chlamydia trachomatis Infection Infect. Immun., March 1, 2000; 68(3): 1519 - 1528. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Dupuy, D. Buzoni-Gatel, A. Touze, D. Bout, and P. Coursaget Nasal Immunization of Mice with Human Papillomavirus Type 16 (HPV-16) Virus-Like Particles or with the HPV-16 L1 Gene Elicits Specific Cytotoxic T Lymphocytes in Vaginal Draining Lymph Nodes J. Virol., November 1, 1999; 73(11): 9063 - 9071. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. L. Perry and S. Hughes Chlamydial Colonization of Multiple Mucosae following Infection by Any Mucosal Route Infect. Immun., July 1, 1999; 67(7): 3686 - 3689. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-K. Kim, M. Angevine, K. Demick, L. Ortiz, R. Rudersdorf, D. Watkins, and R. DeMars Induction of HLA Class I-Restricted CD8+ CTLs Specific for the Major Outer Membrane Protein of Chlamydia trachomatis in Human Genital Tract Infections J. Immunol., June 1, 1999; 162(11): 6855 - 6866. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. L. Perry, K. Feilzer, S. Hughes, and H. D. Caldwell Clearance of Chlamydia trachomatis from the Murine Genital Mucosa Does Not Require Perforin-Mediated Cytolysis or Fas-Mediated Apoptosis Infect. Immun., March 1, 1999; 67(3): 1379 - 1385. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Eriksson, M. Quiding-Jarbrink, J. Osek, A. Moller, S. Bjork, J. Holmgren, and C. Czerkinsky Specific-Antibody-Secreting Cells in the Rectums and Genital Tracts of Nonhuman Primates following Vaccination Infect. Immun., December 1, 1998; 66(12): 5889 - 5896. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Quayle, W. M. P. Coston, A. K. Trocha, S. A. Kalams, K. H. Mayer, and D. J. Anderson Detection of HIV-1-Specific CTLs in the Semen of HIV-Infected Individuals J. Immunol., October 15, 1998; 161(8): 4406 - 4410. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
