Role of T-cell-associated lymphocyte
function-associated antigen-1 in the pathogenesis
of experimental colitis
Kevin P. Pavlick1*, Dmitry V. Ostanin1*, Kathryn L. Furr1, F. Stephen Laroux1, Carla M. Brown1,
Laura Gray1, Christopher G. Kevil2and Matthew B. Grisham1
1Department of Molecular and Cellular Physiology and2Department of Pathology, Louisiana State University Health Sciences
Center, 1501 Kings Highway, P.O. Box 33932, Shreveport, LA 71130-3932, USA
Keywords: adhesion molecules, CD11a, cytokines, inflammatory bowel disease, mesenteric lymph nodes
The b2integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) is important for
lymphocyte trafficking and activation as well as recruitment to sites of tissue inflammation. The
objective of this study was to assess the role of ‘T-cell-associated’ LFA-1 in the pathogenesis of
chronic colitis in vivo. Transfer of CD41CD25?T cells isolated from wild-type (wt) mice into
immunodeficient recipients [recombinase-activating gene-1-deficient (RAG-1?/?)] produced moderate
to severe colitis, whereas RAG-1?/?mice injected with CD11a-deficient (CD11a?/?; LFA-1?/?) donor
T cells displayed minimal macroscopic and histological evidence of colitis. Surface expression of
L-selectin, a4, a4b7and chemokine receptor-7 were similar for wt and CD11a?/?donor T cells.
Attenuated disease in the CD11a?/?! RAG-1?/?animals was associated with decreased numbers of
CD41T cells in the mesenteric lymph nodes (MLNs), spleen and intestinal lamina propria (LP). In
addition, significant reductions in Th1 cytokines were observed following ex vivo stimulation of
mononuclear cells obtained from the MLNs and colonic LP. Interestingly, mononuclear cells obtained
from the spleens of CD11a?/?! RAG-1?/?exhibited enhanced pro-inflammatory cytokine production
compared with splenocytes obtained from wt ! RAG-1?/?colitic mice. Taken together, our data
suggest that T-cell-associated CD11a (LFA-1) expression plays a dual role in the initiation of chronic
gut inflammation by facilitating naive T-cell priming/activation and expansion within MLNs and by
augmenting pro-inflammatory cytokine production following secondary stimulation by antigen-
presenting cells in the colonic interstitium.
Recent experimental and clinical studies suggest that Crohn’s
disease (CD) may result from a complex interaction among
genetic, environmental and immune factors. Indeed, a number
of different studies, using genetically engineered and immune-
manipulated mice, demonstrate that chronic colitis results
from a dysregulated immune response to components of the
normal gut flora (1, 2). It is well recognized that the intestinal
and/or colonic interstitium of patients with active CD contains
substantial numbers of CD4+T cells with a Th1 phenotype
characterized by the production of IL-2, IFN-c and tumor
necrosis factor-a (TNF-a). It is thought that the production of
these T-cell-derived cytokines perpetuates or ‘drives’ chronic
gut inflammation by activating tissue macrophages to release
pro-inflammatory cytokines and mediators, including TNF-a,
IL-1b, IL-12, nitric oxide and reactive oxygen species (3).
Together, Th1- and macrophage-derived cytokines and medi-
ators activate the microvascular endothelium within the gut
to enhance expression of adhesion molecules thereby pro-
moting the recruitment of potentially injurious phagocytic
leukocytes such as neutrophils and monocytes. Because
several different animal models of chronic colitis suggest
that CD4+T cells play a critical role in initiating and/or per-
petuating chronic gut inflammation, it is important to under-
stand the mechanisms involved in leukocyte trafficking
*These authors contributed equally to this work.
Correspondence to: M. B. Grisham; E-mail: firstname.lastname@example.orgReceived 14 October 2005, accepted 29 November 2005
Transmitting editor: C. TerhorstAdvance Access publication 13 January 2006
International Immunology, Vol. 18, No. 2, pp. 389–398
ª The Japanese Society for Immunology. 2006. All rights reserved.
For permissions, please e-mail: email@example.com
by guest on June 9, 2013
and activation in T-cell-dependent models of chronic gut
Although several different T-cell-associated adhesion mol-
ecules [e.g. CD62L, CD11a/CD18, chemokine receptor-7
(CCR7), a4b7] have been identified as important molecular
determinants for T-cell migration to and activation within
secondary lymphoid tissue as well as recruitment to target
tissue, little is known regarding their potential roles in the
initiation and perpetuation of chronic gut inflammation. One
such T-cell-associated adhesion molecule that is known to be
important in T cell trafficking and activation is the b2integrin
lymphocyte function-associated antigen-1 (LFA-1; CD11a/
CD18; aLb2). LFA-1 is a heterodimeric protein composed of
an alpha (CD11a, aL) and beta chain (CD18, b2), which is
expressed on T and B cells, granulocytes and macrophages
(4). Multiple LFA-1 ligands have been identified that include
the intracellular adhesion molecules (ICAM-1–5) (5–9) and the
junctional adhesion molecule-1 (CD166) (10). Interaction of
LFA-1 with its counter receptor ICAM-1 has been shown to
promote the recirculation of naive Tcells to certain secondary
lymphoid tissue such as peripheral lymph nodes (PLNs);
however, its role in mediating migration to mesenteric lymph
nodes (MLNs) is less well characterized. LFA-1 has also been
demonstrated to facilitate the interaction of T cells with
antigen-presenting cells (APCs) to promote T cell activation,
polarization and proliferation in vitro (11–14). In addition, this
T-cell-associated integrin is involved in the firm adhesion and
transendothelial cell migration of activated T cells to in-
flammatory foci (15–18). Anecdotal evidence for a role for
LFA-1 in intestinal inflammation comes from the observation
that CD4+T cells obtained from the intestines of CD patients
have increased CD11a expression compared with cells from
healthy controls (19–21).
A number of intervention studies targeting different adhe-
sion molecules have been performed in inflammatory bowel
disease (IBD) models with varying degrees of success. For
example, ICAM-1 blocking strategies have been successful in
down-regulating colonic inflammation in animal models of
erosive, self-limiting IBD (22, 23). Additionally, anti-ICAM
strategies have been successful in attenuating inflammation
observed in human ulcerative colitis (24, 25). In other studies,
delivery of anti-vascular cell adhesion molecule-1 and
MAdCAM-1 mAbs demonstrated limited efficacy in attenuat-
ing the severity of inflammation in both T-cell-independent and
-dependent models of colitis (26–30). Furthermore, while
transfer of T cells deficient in the b7integrin subunit delayed
the onset of colitis in the CD45RBhightransfer model of chronic
colitis (28), b7deficiency did not influence the development of
spontaneous colitis in IL-2?/?mice (31). Surprisingly, few, if
any, studies have addressed the role of LFA-1 in the
pathogenesis of chronic gut inflammation. This is surprising
since efalizumab, a humanized anti-CD11a mAb, has proven
effective in both experimental and clinical studies for the
treatment of certain T-cell-dependent diseases such as
Therefore, the objective of this study was to assess the role
of LFA-1 in the pathogenesis of chronic gut inflammation in a
T-cell-dependent model of chronic colitis. Recombinase-
activating gene-1-deficient (RAG-1?/?) mice were reconsti-
tuted with naive CD4+CD25?T cells obtained from either
wild-type (wt) or CD11a (LFA-1)-deficient donor animals. We
demonstrate that transfer of CD11a-deficient T cells fail to
induce chronic colitis in RAG-1?/?recipient mice, whereas
transfer of wt T cells induced severe colitis in the same
immunodeficient recipients. Failure to induce colitis appeared
to be dueto defects in Tcellpriming/activation within the MLNs
thereby limiting the numbers of Th1 disease-producing cells
that populate the colonic interstitium.
Wild-type (wt) and RAG-1?/?mice on a C57BL/6 background
were purchased from the Jackson Laboratories (Bar Harbor,
ME, USA). CD11a-deficient (LFA-1?/?) mice on a C57BL/6
background were obtained from Christie M. Ballantyne (Baylor
College of Medicine) and bred at the Louisiana State
University (LSU) Health Sciences Centers animal facility.
Animals were maintained on 12/12 h light/dark cycles in
free conditions. All animals were given standard laboratory
involving the use of animals were reviewed and approved by
the Institutional Animal Careand Use Committee ofLSU Health
Sciences Center and performed according to the criteria
outlined by the National Institute of Health.
All antibodies used in the experimental procedures were
purchased either at BD PharMingen, San Diego, CA, USA or
eBioscience, San Diego, CA, USA. The following antibodies
raised against mouse antigens were used for cell isolation and
flow cytometric analysis: biotin-conjugated anti-CD4 (GK1.5);
FITC-conjugated anti-B220, anti-CD8a, anti-CD45RB, anti-
CD44 and anti-Mac-1 (CD11b); PE-conjugated anti-CD25,
(LPAM-1) (integrin a4b7 complex), anti-CD49d (integrin a4
chain) and anti-CCR7; PE–Cy5-conjugated anti-CD3 and anti
CD62L; allophycocyanin-conjugated anti-CD4(clone GK1.5).
T-cell transfer model of chronic colitis
Chronic colitis was induced in RAG-1?/?mice using a minor
modification of a method previously described (35). Briefly,
donor spleens were surgically removed from either C57BL/6
wt or CD11a?/?female mice and teased into a single-cell
suspension in PBS containing 4% FBS (FACS buffer). CD4+
T cells were enriched using negative selection by first label-
ing with FITC-conjugated anti-B220, anti-CD8 and anti-Mac-1
andthen withanti-FITCmagnetic microbeads followed bysep-
aration on a type CS column using VarioMACS magnetic
separator according to manufacturer’s instructions (Miltenyi
Biotec, Auburn, CA, USA). Unlabeled flow-thru cells were
(GK1.5), streptavidin and PE-conjugated anti-CD25 mAb.
Cells were sorted for CD4+CD25?
(Becton Dickinson, San Jose, CA, USA) with >98% purity on
post-sort analysis. Male RAG-1?/?C57BL/6 mice (8- to 10-
weeks old) were injected (intra-peritoneally) with 7.5 3 105
390Role of LFA-1 in experimental colitis
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CD4+CD25?Tcells, from either wt or CD11a?/?, suspended in
500 ll of PBS. Clinical signs of disease (e.g. loss of body
weight and loose stool/diarrhea) were followed and recorded
weekly for 8 weeks from the time of injection.
Tissue lymphocyte isolation and flow cytometric analysis
Lymphocytes were obtained from the MLNs, spleen, intestine
and colon and analyzed by flow cytometry as previously
described (36). Briefly, MLNs and spleens were removed
aseptically and teased into a single-cell suspension using the
frosted ends of two glass slides in FACS buffer on ice. The
suspension was then passed through a 26-gauge syringe to
obtain a single-cell suspension. After pelleting, RBCs were
removed by hypotonic lysis and the resulting leukocytes were
re-suspended in FACS buffer containing anti-FcR (CD16/32)
antibody at 5 3 107cells ml?1. After incubation with anti-FcR
mAb, ~1 3 106cells were placed into individual wells of
a round bottom 96-well plate, pelleted and stained with
appropriate antibodies. After the staining, cells were fixed
for 15 min on ice in freshly prepared 2% ultrapure formalde-
hyde (Polysciences, Inc., Warrington, PA, USA) and analyzed
next day on the FACScalibur (BD Biosciences, San Diego, CA,
Analysis of intestinal intra-epithelial lymphocytes (IELs) was
performed using a modification of the method described
previously (31). Briefly, small and large intestines were
removed from mice flushed of luminal contents and trimmed
of excess fat and connective tissue. Small and large intestines
were opened longitudinally and cut into small (0.5–1.0 cm)
pieces in PBS on ice. Pieces were then incubated in pre-
warmed (37?C) PBS/4% FCS/0.2 mM EDTA/10 mM D-glucose
on a rotating shaker for 20 min at 250 r.p.m. at 37?C. After
incubation, intestinal pieces were vortexed for 3–5 s. Super-
natants from individual animals were collected in separate
50-ml conical tubes and kept on ice. Incubations were per-
formed at least three times to insure complete removal of
epithelium. Intestinal pieces from individual animals were pro-
natants were pelleted by centrifugation and re-suspended in
30 ml of 40% Percoll (Amersham Biosciences). IELs were
further enriched by centrifugation over a 40/70% Percoll
gradient for 25 min, 1000 g at room temperature. After the
centrifugation, the pellet of IELs was washed and then re-
suspended in FACS buffer containing anti-FcR mAb. Viable
cells were counted using 0.4% tryphan blue dye/PBS solution.
Lamina propria lymphocytes (LPLs) were prepared by
digestion of finely minced intestinal pieces remaining after
IEL isolation with RPMI-1640/4% FBS and containing collage-
nase type VIII (200 U ml?1) for 40 min at 250 r.p.m. in a 37?C
shaker (15). Lymphocytes were further enriched by centrifu-
gation over a 40/70% Percoll gradient. The LPL pellet was
washed, re-suspended in FACS buffer containing anti-FcR
mAb and counted. ~1 3 106cells were placed in individual
wells of a 96-well plate and stained. After the staining, cells
were fixed for 15 min on ice in freshly prepared 2% ultrapure
formaldehyde (Polysciences, Inc.) and analyzed the next day
on the FACScalibur (BD Biosciences). Data were analyzed
using FlowJo software (Tree Star, Inc., Ashland, OR, USA;
version 5.7.2 for PC). Percentages of specific subsets of
lymphocytes were compared between wt and CD11a?/?
Ex vivo stimulation and cytokine determinations
Mononuclear cells from spleen, MLN and colonic lamina
propria (LP) of individual animals were prepared as described
above with minor modification. Following RBC lysis, mono-
nuclear leukocytes from the spleen were prepared by density
centrifugation through Ficoll-PaqueTMPLUS (Amersham Bio-
sciences). Colonic lamina propria mononuclear cells (LPMCs)
were prepared as described above using a 40/70% Percoll
density gradient while a single-cell suspension of mono-
nuclear cells from MLNs was used without further enrichment.
All cells were washed in complete RPMI 1640 media con-
taining 4% FBS, L-glutamine and 100 U ml?1each penicillin
and streptomycin and counted. A total of 5 3 105mononuclear
cells were plated in either control (uncoated) or CD3e-coated
(BD Falcon, San Diego, CA, USA) 96-well flat bottom plates.
Anti-mouse FG-purified anti-CD28 (eBioscience, 1 lg ml?1
final concentration) or medium alone (for control uncoated
plate) was added and cells were incubated for 48 h at 37?C in
a humidified incubator with 10% CO2. At the end of incubation,
cells were removed by centrifugation and the supernatants
were collected, frozen and stored at ?70?C. Cytokine de-
termination in collected supernatants was determined using
the Inflammation and Th1/Th2 Cytometric Bead Array kits (BD
Biosciences) as described by the manufacturer.
Macroscopic and histological evaluation
At8 weeksfollowing Tcellreconstitution,micewere euthanized
and their colons removed. Colons were cleaned of fecal ma-
terial and scored for macroscopic evidence of inflammation
using a modification of the method described by Conner et al.
(37). Normal colons were assigned a score of 0; mild bowel
wall thickening in the absence of visible hyperemia was given
a score of 1; moderate bowel wall thickening and hyperemia
was given a score of 2; severe bowel wall thickening with
rigidity and marked hyperemia was assigned a score of 3 and
severe bowel thickening with rigidity, hyperemia and colonic
adhesions was given a score of 4. The colons were then
divided into proximal and distal sections and each segment
was measured and weighed for ratio comparisons as an index
of inflammation. A small piece of each section was placed in
10% buffer formalin and stored overnight at 4?C. The fixed
tissue was then rinsed of formalin with PBS, partially hydrated
in ethanol and embedded in JB-4 medium (Polysciences, Inc.)
or parafin. Samples were sectioned (5 lm) and stained with
hematoxylin and eosin to evaluate standard histopathology
changes. Histopathological analysis was performed in a
blinded manner and scored using a published method (35).
Briefly, eight parameters were used that include (i) the degree
of inflammatory infiltrate in the LP, range 1–3; (ii) Goblet cell
loss as a marker of mucin depletion, range 0–2; (iii) reactive
epithelial hyperplasia/atypia with nuclear changes, range 0–3;
(iv) the number of IELs in the epithelial crypts, range 0–3;
(v) abnormal crypt architecture(distortion, branching, atrophy,
crypt loss), range 0–3; (vi) number of crypt abscesses, range
0–2; (vii) mucosal erosion to frank ulcerations, range 0–2 and
Role of LFA-1 in experimental colitis391
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The severity of inflammation in both sections of the colon was
based on the sum of the scores in each parameter with a max-
imum score of 20.
CD11a-deficient T cells fail to induce chronic colitis in
To investigate the importance of T-cell-associated LFA-1 in the
development of chronic colitis, CD4+CD25?T cells isolated
from spleens of wt or CD11a?/?mice were transferred into
RAG-1?/?immunodeficient mice. Beginning 5 weeks following
T-cell transfer, RAG-1?/?mice that received wt cells (wt !
RAG) began to lose body weight, and developed loose stools/
diarrhea consistent with the development of chronic colitis
(Fig. 1A). In contrast, RAG-1?/?mice that received CD11a?/?
cells (CD11a?/?! RAG) continued to gain weight throughout
Macroscopic inspection of the colons obtained 8 weeks
following T-cell transfer revealed significant hyperemia, bowel
wall thickening and adhesions in the wt ! RAG mice, whereas
little or no macroscopic evidence of inflammation was
observed in the CD11a?/?! RAG group (Fig. 1B). In addition,
the CD11a?/?! RAG mice had significantly lower colon
weight–length ratios compared with the wt ! RAG group,
suggesting decreased colonic inflammation in these mice
(Fig. 1C). Histological inspection revealedthat wt ! RAG mice
had a profuse transmural leukocyte infiltrate accompanied by
goblet cell loss, crypt abscesses and abnormalities and bowel
wall thickening, whereas colons from CD11a?/?! RAG mice
exhibited little or no evidence of chronic colitis (Fig. 2A).
Reduced colonic inflammation in CD11a?/?! RAG mice was
confirmed using blinded histopathological scoring, which
demonstrated minimal inflammation in the proximal and distal
portions of the colon in the large majority of the CD11a?/?!
RAG mice compared with the almost 70% of wt ! RAG group
It should be noted that the CD11a?/?! RAG mice remained
healthy and had no signs of colitis after 18 weeks post-transfer
(data not shown). To insure that the failure to induce colitis
by transfer of CD11a?/?into immunodeficient recipients was
not due to alterations in surface expression of other adhesion
molecules and chemokine receptors necessary for naive T-cell
trafficking, we evaluated the surface expression of CD62L
(L-selectin), CCR7, LPAM-1(a4b7) and CD49d (a4). We found
that CD11a?/?CD4+CD25?Tcells expressed similar levels to
wt Tcells of all indicated surface markers despite having <1%
of the wt levels of LFA-1 (Fig. 3).
T-cell-associated CD11a is required for lymphocyte
expansion in the MLNs, spleen and colon
It has been proposed by different groups of investigators that
naive Tcells must first migrate to the MLNs where they interact
with APCs displaying their cognate/enteric antigens to
become activated to proliferate and polarize to Th1 colitogenic
T cells. Therefore, we wished to ascertain whether failure to
induce colitis by transfer of CD11a?/?was due to defects in
T-cell expansion within the MLNs as well as in the spleen and
intestine. We found that the number of CD3+CD4+T cells
present in the MLNs and spleen of the CD11a?/?! RAG mice
were reduced by ~70% compared with the wt ! RAG animals
(Fig. 4A) In addition, we found that while colons obtained from
the colitic wt ! RAG group possessed large numbers of
CD3+CD4+IELs and LPLs consistent with activation and
Weight-to-Length Ratio (mg/mm)
% Original Weight
Week 1Week 2
Week 5Week 6
Week 7 Week 8
Fig. 1. Development of colitis in RAG-1?/?mice reconstituted
with CD4+CD25?cells isolated from wt (wt ! RAG) or CD11a?/?
(CD11a?/?! RAG) donor mice. (A) The percent change in weight
and 67 for wt ! RAG and CD11a?/?! RAG groups, respectively.
(B) Macroscopic evidence of inflammation. Data are expressed as
the mean 6 SEM. Pooled data from at least three independent trans-
fers, n ¼ 56 and 51 for wt ! RAG and CD11a?/?! RAG, respectively.
Pooled data from at least three independent transfers, n ¼ 56 and 67
for wt ! RAG and CD11a?/?! RAG groups, respectively. Filled bars
represent wt ! RAG and open bars represent the CD11a?/?! RAG
mice. Significant differences (*) from the colitic wt ! RAG group was
determined by two-sided Student’s t-test (P < 0.05).
392Role of LFA-1 in experimental colitis
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expansion of naive T cells in immunodeficient recipients (38),
the absolute numbers of CD3+CD4+Tcells within IEL and LPL
compartments of the colon obtained from CD11a?/?! RAG
mice were significantly reduced (Fig. 4C). We also observed
a trend for reduced numbers of IELs and LPLs in the small
bowel of the CD11a?/?! RAG mice; however, these differ-
ences were not statistically significant (Fig. 4B).
CD11a deficiency does not affect surface expression of
T-cell-associated activation markers
It is well appreciated that transfer of naive CD4+T cells into
immunodeficient recipients results in the activation and
polarization of these cells into a memory/effector phenotype
(i.e. Th1), which is important in the pathogenesis of IBD (38,
39). In addition, LFA-1 has been proposed to play an important
role in the activation of T cells via its interaction with ICAM-1
located on APCs within the MLNs as well as the gut interstitium
(12, 40, 41). To determine whether CD11a deficiency alters
expression of T-cell-associated activation markers, we exam-
ined the surface expression of CD45RB, CD44, CD69 and
CD25 on wt versus CD11a?/?T cells obtained from MLNs,
spleen and colonic LP of reconstituted RAG-1?/?mice. As
expected, we observed a shift to an activated/memory
phenotype (CD44high, CD45RBlow) in the lymphocytes from
all three tissues analyzed. In addition, we observed equivalent
frequencies of expression of the four different activation
markers on CD3+CD4+cells obtained from MLNs, suggesting
that CD11a was not required for T-cell activation within this
lymphoid tissue (Fig. 5B). Surface expression of the different
activation markers confirmed that the CD3+CD4+cell pop-
ulations isolated from the spleens and the colonic LP of both
groups were similar as well (Fig. 5A and C). Taken together,
these data suggest that T-cell-associated CD11a (i.e. LFA-1)
is not required for the activation and conversion from naive to
an activated/memory phenotype within the MLNs, spleen or
Fig. 2. Histopathology of the colon. Colons were fixed, embedded
in parafin, sectioned (5 lm) and stained with hematoxylin and eosin.
(A) Representative micrographs are shown for wt ! RAG and
CD11a?/?! RAG. Micrographs represent 3100 magnification.
(B) Blinded histopathological scores of the distal colon. Data are
expressed as the mean 6 SEM (n ¼ 15 for wt ! RAG, n ¼ 40 for
group were determined by two-sided Student’s t-test (P < 0.05).
(C) Incidence and severity of colonic inflammation. Histopathological
scores of distal colon were grouped as follows: no colitis (score 0–2),
mild (3–9), moderate (10–14) and severe inflammation (15–20).
Fig. 3. Surface expression of indicated markers for T cells isolated
from wt (solid lines) and CD11a?/?(dotted lines) donors. Spleen cells
that were used for transfer into immunodeficient RAG?/?mice were
assessed for the frequency of adhesion molecule expression to
determine if the differential disease susceptibility could be due to the
alteration of adhesion molecules in CD11a?/?donors.
Role of LFA-1 in experimental colitis393
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It has been postulated that CD4+CD25+regulatory cells
(Treg) may arise from the CD25?T cells transferred into
recipient mice (42, 43). Interestingly, we observed an increase
in CD25+staining in the spleens of both reconstituted groups,
compared with naive wt spleen, which could be due to an
expanded regulatory cell population. This confirms recent
findings by Liang et.al. that show a conversion of sorted
CD4+CD25?cells into CD4+CD25+cells with regulatory
properties similar to the naturally arising Tregs (42). However,
the percentage as well as absolute numbers of CD3+CD4+
positive cells that were also CD25bright(>100 fluorescent units
expression) were similar in both groups and only slightly
higher than naive wt splenocytes: 8.3% for CD11a?/?! RAG
and 8.1% for wt ! RAG compared with 5.3% for naive wt
spleen (Fig. 5A–C). Taken together, these data suggest that
failure of CD11a?/?T cells to induce disease was not due to
increased generation of Treg cells.
Fig. 4. Total CD3+CD4+T-cell numbers isolated from wt ! RAG (filled
bars) and CD11a?/?! RAG (open bars) in the MLNs and spleen
(A), small intestine (B) and colon (C). Cells were analyzed by two-
color flow cytometry for surface expression of CD3 and CD4.
Absolute numbers of CD3CD4 double-positive cells in each tissue
were calculated by multiplying percentage of the total cells that are
CD3+CD4+by the total number of cells. Data presented are averages
of at least two separate experiments (n > 3) 6 SEM. Significant
differences (*) between the two groups were determined by two-sided
Student’s t-test (P < 0.05).
Fig. 5. Surface expression of T-cell activation markers isolated from
spleen (A), MLNs (B) and colonic LP (C) of wt ! RAG (solid lines) and
CD11a?/?! RAG (dotted lines). In (A), the filled gray area represents
surface staining of wt donor splenocytes before transfer into RAG?/?.
Cells were initially gated according to their forward and side scatter
properties characteristic for the mononuclear cells and then based on
simultaneous expression of both CD3 and CD4 markers using FlowJo
software as indicated in the Methods.
394 Role of LFA-1 in experimental colitis
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Mononuclear cells obtained from CD11a?/?! RAG mice
have impaired cytokine generation
Both experimental colitis and human IBD are associated with
the accumulation of T cells as well as monocytes, macro-
phages and PMNs in the colon that secrete pro-inflammatory
cytokines that are thought to be important in the pathogenesis
of colonic inflammation. It has also been demonstrated by dif-
ferent laboratories that T-cell-associated LFA-1 co-stimulatory
signaling is important in regulating immune properties of
Thcells (14, 44). Therefore, we wished to determine whether
T cells from CD11a?/?! RAG mice retain the potential for
mounting pro-inflammatory responses following ex vivo
stimulation. In order to more closely mimic the cellular
composition in the different tissues, mononuclear cells were
isolated from the MLNs, spleen and colonic LP of wt ! RAG
and CD11a?/?! RAG mice and T cells were activated with
plate-bound anti-CD3 and soluble anti-CD28 mAbs in vitro.
We found leukocytes isolated from the MLNs of colitic wt !
RAG produced 15-, 85- and 7-fold more TNF-a, IFN-c and IL-2
protein, respectively, compared with cells obtained from
CD11a?/?! RAG mice (Fig. 6B). In addition, these leukocytes
also produced significantly greater amounts of IL-5 and IL-4
(27- and 7-fold, respectively) although the absolute amounts
of the different cytokines were skewed much more toward the
Th1/pro-inflammatory cytokines (Fig. 6B). Surprisingly, mono-
nuclear cells obtained from spleens of CD11a?/?! RAG
mice, actually produced 4- and 5-fold more TNF-a and INF-c
protein, respectively, than did cells isolated from the colitic
wt ! RAG mice (Fig. 6A). It should be noted that no significant
cytokine production was observed in the absence of plate-
bound CD3 and soluble CD28 demonstrating that cytokine
production was dependent upon T-cell activation.
Despite the fact that adoptively transferred CD11a?/?Tcells
are impaired in their ability to expand in the MLN and spleen,
they still exhibit an activated/memory phenotype with respect
to expression of different surface activation markers. One
possible explanation for lack of disease in the CD11a?/?!
RAG mice may be that there are simply too few cytokine-
producing T cells present in the colon to initiate colitis.
Therefore, we sought to investigate the potential for LPMCs
obtained from diseased and non-diseased colons to produce
Th1/pro-inflammatory cytokines in vitro. As expected, we
found that LPMCs from wt ! RAG mice produced 10-, 41-
and 7-fold more TNF-a, IFN-c and IL-2 protein when stimulated
with plate-bound anti-CD3 and soluble anti-CD28 compared
with those produced by equivalent numbers of LPMCs
isolated from CD11a?/?! RAG mice (Fig. 6C). In addition,
the ability of LPMCs from CD11a?/?! RAG mice to produce
Th2/regulatory cytokines was also blunted. Again, no signifi-
cant cytokine production was observed when mononuclear
leukocytes were plated onto plastic wells devoid of CD3 and
It is becoming increasingly appreciated that chronic gut in-
flammation results from a dysregulated mucosal immune re-
sponse to normal enteric antigens in genetically susceptible
individuals (45). This concept is best exemplified experimen-
tally by the adoptive transfer model of naive T cells into
immunodeficient recipient SCID or RAG-deficient mice, which
induces moderate to severe colitis (1, 39). It has been
proposed that the chronic colitis induced by adoptive transfer
arises from enteric antigen-driven activation, polarization and
expansion of naive Tcells within the MLNs to produce effector
Th1 cells. These cells then leave the MLNs, enter the systemic
circulation and migrate to the intestinal interstitium where they
initiate chronic intestinal inflammation (14, 39, 46). Although
Fig. 6. Cytokineproductionbymononuclearcells isolatedfromspleen
(A), MLNs (B) and colonic LP (C) of wt ! RAG (filled bars) and
CD11a?/?! RAG (open bars). Mononuclear cells from indicated
tissues were prepared from three pooled animals per group as
described in the Methods. Samples were run in triplicate using
Cytometric Bead Array (CBA) mouse Th1/Th2 kit and BD CBA software
according to the manufacturer’s instructions. Data shown were
normalized to the cytokine levels in unstimulated control samples
(105total plated mononuclear cells without CD3 and CD28 stimulation)
and represent average values 6 SEM. from 105total plated mono-
nuclear cells. Significant differences between the groups, where
applicable, are indicated by (*) and were determined by two-sided
Student’s t-test (P < 0.05).
Role of LFA-1 in experimental colitis 395
by guest on June 9, 2013
recognition and interaction of the MHC Class II-bound antigen
by the TCR is critical for the initial immune response within the
MLN, a variety of different adhesion molecules localized on
T cells are also required for co-stimulatory signals as well as
cell–cell interactions. One adhesion molecule thought to play
a critical role in T-cell biology yet has received little attention in
immune-based models of IBD is the b2integrin LFA-1 (47). It
has been shown that LFA-1 plays an important role in several
aspects of cell-mediated immunity including migration (re-
circulation) to and activation/expansion within MLNs as well as
the recruitment of activated/memory Tcells to inflamed tissue
(48). Data obtained in the current study demonstrate that
transfer of CD11a?/?CD4+CD25?Tcells into immunodeficient
RAG-1?/?mice fails to induce chronic colitis, whereas transfer
of wt T cells induced moderate to severe gut inflammation.
of CD3+CD4+Tcells within the MLNs, colonic interstitium and
spleen. These data are consistent with the proposal that LFA-1
is critical for migration and/or priming within the MLN and the
subsequent recruitment of these T cells into the colonic
interstitium to initiate disease. Recent studies have shown
that expression of different adhesion molecules can influence
the inhibitory nature of anti-LFA treatment using in vitro
adhesion assays, supporting an accessory role for LFA-1 in
trafficking of naive Tcells to some secondary lymphoid tissues
(49). It is well documented for example that LFA-1 is important
for the migration (i.e. recirculation) of naive T cells from the
blood into PLNs (40, 50, 49). However, these studies also
demonstrated that LFA-1 had only ‘a limited role’ in the
homing of lymphocytes to MLNs and Peyer’s patches and no
effect on homing to the spleen. This accessory role of LFA-1 is
supported by the findings that the a4integrins (a4b1and a4b7)
are important for the migration of lymphocytes into certain
secondary lymphoid tissue (e.g. MLNs) and can compensate
for the loss of LFA-1 (51, 52). These data coupled to the fact
that CD62L, CCR7, a4and a4b7expression on CD11a?/?were
virtually identical to those observed on wt CD4+CD25?donor
Tcells suggest that CD11a?/?Tcells were able to migrate into
MLNs via T-cell-associated a4integrins, CCR7 and possibly
CD62L (i.e. L-selectin). Consequently, we would propose that
the paucity of lymphocytes in the MLNs and colonic tissue of
CD11a?/?! RAG mice versus wt ! RAG mice may be due to
defects in the initial T-cell priming/activation and consequent
expansion within the MLNs.
The extent of the primary response of naive CD4 T cells,
which involves activation, expansion and polarization, dictates
if a proper immune response is mounted to a specific antigen.
Activation of T cells requires two signals; the first signal is
antigen dependent, involving the interaction of the antigen–
MHC on an APC and the TCR, while the second signal is
antigen independent, involving multiple T-cell surface mol-
ecules and their ligands on APCs. In particular, LFA-1/ICAM
interactions are important for optimal T-cell activation by
mediating and stabilizing the T-cell-APC contact referred to as
the immunologic synapse (53). LFA-1 on T cells is thought to
lower the threshold level ofactivation andpromoteproliferation
of Tcells. The generalized lack of T-cell expansion in multiple
tissues, including the spleen, would appear to support the
idea that LFA-1 is more important for priming and expansion
than for trafficking to the MLNs (Fig. 4). These in vivo data
agree well with other investigators who have demonstrated
that LFA-1?/?lymphocytes have major defects in alloantigen
(MLR)- or Con A-stimulated proliferation in vitro (15, 54). In
addition to priming within the MLNs, our data would suggest
that T-cell-associated LFA-1 is important for Th1 cytokine
production as T cells obtained from MLNs and colonic LP of
CD11a?/?! RAG produced much smaller amounts of IFN-c
and/or TNF-a following ex vivo stimulation with plate-bound
CD3 and soluble CD28 compared with the colitic wt ! RAG
animals (Fig. 6). It could be argued that the cytokine profiles
observed in our ex vivo studies were due to non-specific
activation of the different cell types (Tcells, macrophages and
dendritic cells) found in our mononuclear cell preparations of
MLN, spleen and colon. However, as noted previously, no
cytokine generation could be observed by cells from any
tissue in the absence of CD3 and CD28, suggesting that T-cell
activation was required for cytokine synthesis. Although it is
possible that certain T-cell-derived cytokines (e.g. IFN-c)
could influence the production of macrophage and/or den-
dritic cell cytokines during the incubation period, the fact
remains that Th1 cytokines were depressed to a much greater
extent in cells derived from the CD11a?/?! RAG mice than
their colitic counterparts. These data are interesting for two
reasons. First, although we observed large and significant
reductions in IFN-c and/or TNF-a production by cells obtained
from CD11a?/?! RAG MLNs and colon, T-cell activation
marker expression was consistent with activated/antigen-
experienced cells and virtually identical to those obtained
from the colitic wt ! RAG animals, suggesting that expression
of activation markers may be dissociated fromcell proliferation
and cytokine expression. A second interesting observation
made in these studies is that colonic LPMCs isolated from
CD11a?/?! RAG animals appeared to be deactivated i.e.
they appeared to be refractory in their ability to produce both
Th1 and Th2 cytokines following TCR activation. These data
suggest the novel finding that LFA-1 is required not only for
priming within the MLNs but is also required for ‘secondary’
activation of T cells that have gained access to the gut
One possible mechanism to account for reduced cytokine
production by MLNs and LPMCs isolated from CD11a?/?!
RAG mice is that lack of LFA-1 on Tcells somehow skews the
polarization of T cells to a Th2 and/or regulatory phenotype.
Indeed, it is known that blocking the interaction of LFA-1 with
ICAM-1 expressed on APCs inhibits Th1 polarization and
cytokine production (12, 44, 55). In fact, blocking of both
ICAM-1 and ICAM-2 in the interaction of naive T cells with
APCs induces a >100-fold increase in the production of
Th2 cytokines, IL-4 and IL-5, which will down-regulate Th1-
dependent inflammation (44). Our data would suggest that
this is not the case as we found that although the production of
Th1 cytokines from MLNs and LPMCs of CD11a?/?! RAG
mice were significantly reduced compared with their colitic
counterparts, the production of Th2/regulatory cytokines was
almost undetectable. In addition, we observed that the
numbers of CD4+CD25+T cells isolated from the MLNs,
spleen and gut were very small and were not different from
those isolated from tissues of the colitic wt ! RAG mice.
Another unexpected finding in the current study was that
splenic mononuclear cells obtained from CD11a?/?! RAG
396Role of LFA-1 in experimental colitis
by guest on June 9, 2013
compared with cells obtained from wt ! RAG mice. The
mechanisms responsible for this rather surprising finding were
not delineated in the current study; however, it may be that
a larger percentage of the mononuclear cells in the spleen
preparation were IFN-c- and TNF-a-producing T cells and/or
NK cells. This exaggerated cytokine production profile raises
the interesting possibility that extra-intestinal tissue inflamma-
tion (i.e. lung, liver, spleen) may be present in the CD11a?/?!
RAG mice. Although possible, the animals appeared healthy
and gained weight throughout the 8- to 10-week experiment.
In addition to recirculation and activation, LFA-1 is known to
be important for the firm adhesion of activated lymphocytes to
the post-capillary microvascular endothelium (56–58). Ding
et al. (59) showed that T-cell adhesion and migration across
stimulated endothelium (e.g. TNF-a, INF-c and MIP-1a) is me-
diated by LFA-1 and not VLA-4. However, LFA-1-independent
pathways exist and it has also been shown that human
lymphocytes can migrate across stromal cell-derived factor-1
stimulated human umbilical vein endothelial cells monolayers
independent of both VLA-4 and LFA-1 (56, 59, 60). Therefore,
it is reasonable to propose that specific T-cell populations
utilize several adhesion pathways to migrate from the
peripheral circulation into the tissue after they have adhered
to the endothelium. Additional adhesion molecules may be
able to facilitate LFA-1-deficient T-cell migration across the
endothelium and thus explaining the small but significant
numbers of Tcells in the colon of CD11a?/?! RAG mice. The
observation of decreased numbers of T cells in the colons of
these mice is consistent with previous findings using a T-cell-
dependent delayed type sensitivity model, which showed that
infiltration of LFA-1?/?lymphocytes injected into wt mice was
reduced by 50% in ears sensitized with oxazolone (50). Since
the isolation of T cells from the colons of CD11a?/?! RAG
mice was performed at one specific point in time, it is possible
that these cells were recruited to the colonic interstitium in
numbers similar to those observed in colitic wt ! RAG mice at
an earlier time but were rendered either anergic or unable to
be retained within the interstitium. Data obtained in the current
study demonstrate that LPMCs isolated from the colons of
CD11a?/?! RAG mice may exist in a ‘deactivated’ or
refractory state, suggesting that LFA-1 is critical for secondary
activation within the tissue. Current studies are underway to
address these potential mechanisms.
In summary, we have shown that T-cell-associated LFA-1 is
necessary for the induction of chronic colonic inflammation
in a T-cell-dependent mouse model of Crohn’s colitis. Our
findings indicate that T-cell-associated LFA-1 is critical for the
priming/activation, expansion and polarization of naive Tcells
within the MLNs. In addition, our data suggest that LFA-1 is
also important for secondary activation of T cells gaining
access to the colonic interstitium. We propose that LFA-1 may
represent an important target for immune therapy for patients
This work was supported by DK64023, the Arthritis Center of
Excellence at LSU Health Sciences Center and the Yamanouchi
inflammatory bowel disease
intracellular adhesion molecule
lymphocyte function-associated antigen-1
lymphocyte Peyers patch adhesion molecule-1
lamina propria lymphocyte
lamina propria mononuclear cell
Louisiana State University
mesenteric lymph node
peripheral lymph node
recombinase-activating gene-1 deficient
tumor necrosis factor-a
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