Alpha L-integrin I domain cyclic peptide antagonist selectively inhibits T cell adhesion to pancreatic islet microvascular endothelium.

Abstract

Insulitis is a hallmark feature of autoimmune diabetes that ultimately results in islet beta-cell destruction. We examined integrin requirements and specific inhibition of integrin structure in T cell and monocyte adhesion to pancreatic islet endothelium. Examination of cell surface integrin expression on WEHI 7.1 T cells revealed prominent expression of beta-, beta(1)-, alpha(L)-integrins, and low expression of alpha(M)-integrins; whereas WEHI 274.1 monocytes showed significant staining for beta(2)-, beta(1)-, alpha(M)-molecules and no expression of alpha(L)-molecules. Unstimulated islet endothelium showed constitutive levels of ICAM-1 counter-ligand expression with minimal VCAM-1 expression; however, TNF-alpha stimulation increased cell surface density of both molecules. TNF-alpha increased T cell and monocyte rolling and adhesion under hydrodynamic flow conditions. Administration of a cyclic peptide competitor for the alpha(L)-integrin I domain binding sites (cyclo1,12-PenITDGEATDSGC) blocked T cell adhesion without inhibiting monocyte adhesion. Examination of T cell rolling revealed that cLAB.L treatment increased the average rolling velocity on activated endothelium and significantly decreased the fraction of T cells rolling at < or =50 microm/s, suggesting that cLAB.L treatment interferes with signal activation events required for the conversion of T cell rolling to firm adhesion. These data demonstrate for the first time that cyclic peptide antagonists against alpha(L)-integrin I domain attenuate T cell recruitment to islet endothelium.

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doi: 10.1152/ajpgi.00267.2004
288:G67-G73, 2005. First published 19 August 2004;
Am J Physiol Gastrointest Liver Physiol
Meng Huang, Kametra Matthews, Teruna J. Siahaan and Christopher G. Kevil
endothelium
inhibits T cell adhesion to pancreatic islet microvascular
L-Integrin I domain cyclic peptide antagonist selectively
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?L-Integrin I domain cyclic peptide antagonist selectively inhibits T cell
adhesion to pancreatic islet microvascular endothelium
Meng Huang,1Kametra Matthews,1Teruna J. Siahaan,2and Christopher G. Kevil1
1Department of Pathology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, Louisiana;
and2Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas
Submitted 22 June 2004; accepted in final form 14 August 2004
Huang, Meng, Kametra Matthews, Teruna J. Siahaan, and
Christopher G. Kevil. ?L-Integrin I domain cyclic peptide antagonist
selectively inhibits T cell adhesion to pancreatic islet microvascular
endothelium. Am J Physiol Gastrointest Liver Physiol 288: G67–G73,
2005. First published August 19, 2004; doi:10.1152/ajpgi.00267.
2004.—Insulitis is a hallmark feature of autoimmune diabetes that
ultimately results in islet ?-cell destruction. We examined integrin
requirements and specific inhibition of integrin structure in T cell and
monocyte adhesion to pancreatic islet endothelium. Examination of
cell surface integrin expression on WEHI 7.1 T cells revealed prom-
inent expression of ?-, ?1-, ?L-integrins, and low expression of
?M-integrins; whereas WEHI 274.1 monocytes showed significant
staining for ?2-, ?1-, ?M-molecules and no expression of ?L-mole-
cules. Unstimulated islet endothelium showed constitutive levels of
ICAM-1 counter-ligand expression with minimal VCAM-1 expres-
sion; however, TNF-? stimulation increased cell surface density of
both molecules. TNF-? increased T cell and monocyte rolling and
adhesion under hydrodynamic flow conditions. Administration of a
cyclic peptide competitor for the ?L-integrin I domain binding sites
(cyclo1,12-PenITDGEATDSGC) blocked T cell adhesion without
inhibiting monocyte adhesion. Examination of T cell rolling revealed
that cLAB.L treatment increased the average rolling velocity on
activated endothelium and significantly decreased the fraction of T
cells rolling at ?50 ?m/s, suggesting that cLAB.L treatment inter-
feres with signal activation events required for the conversion of T
cell rolling to firm adhesion. These data demonstrate for the first time
that cyclic peptide antagonists against ?L-integrin I domain attenuate
T cell recruitment to islet endothelium.
lymphocyte function-associated antigen-1; leukocyte; pancreas; diabetes
AUTOIMMUNE DIABETES (type 1 diabetes; T1D) is a multifaceted
metabolic disorder characterized by hyperglycemia, which re-
sults in the long-term development of cardiovascular and
neuropathic complications. The etiology of T1D is complex,
however. Autoimmunity and insulitis are believed to be key
aspects of this disease (6, 36). Islet infiltration of leukocytes,
such as CD4?and CD8?T cells, are believed to contribute to
?-cell destruction (4, 16, 43, 45, 47). Examination of leukocyte
adhesion molecules expressed during autoimmune diabetes
reveals that lymphocyte function-associated antigen-1 (LFA-1;
?L?2-antigen), Mac-1 (?M?2-integrin), ?4- and ?1-integrins,
and L-selectin may all be involved in development of disease
(7, 8, 15, 29, 31, 32). Consistent with these observations,
reports have shown increased ?L?2-integrin expression on
mononuclear cells from T1D patients, which correlated with
islet cell autoantibody titers and increased expression of
counter-ligand endothelial cell adhesion molecules, such as
ICAM-1 (2, 33). However, the specific molecular mechanisms
required for immune cell adhesion to pancreatic islet micro-
vascular endothelium remains unknown.
Recruitment of leukocytes into extravascular tissues occurs
through a concerted multistep process involving leukocyte
rolling, firm adhesion, and emigration across the vascular
endothelium. Leukocyte firm adhesion and transmigration are
governed, in part, by the ?2-integrin family, or CD18 integrins,
which consists of four different adhesion proteins: ?L?2-
(LFA-1), ?M?2- (Mac-1), ?X?2- (p150/95), and ?D?2-proteins.
Importantly, expression of the ?2-integrins is limited to leuko-
cytes, indicating their importance for immune responses. Mem-
bers of the immunoglobulin superfamily serve as the major
ligands for the ?2-integrins, including ICAM-1, -2, and -3, and
others (18, 19). Integrin-mediated cell adhesion is controlled
through multiple mechanisms that include alterations in surface
density of ligand and changes in receptor affinity and avidity
(24, 44). Several studies have demonstrated that the ?-chain I
domain is important for specific ligand binding and integrin
function (5, 42, 51). The I domain is highly homologous
among integrins that contain one, yet specific differences have
been noted between the metal ion-dependent adhesion site of
the ?L- and ?M-molecules (9, 27). These and other differences
have been suggested to be functionally important in terms of
specific integrin actions, yet the precise biological role of these
motifs and possible therapeutic intervention at these sites has
not been determined.
Here we examine the specific adhesion molecule require-
ments involved during leukocyte-islet microvascular endothe-
lial cell interactions using specific cyclic peptide antagonists
that are designed to compete with ?L-integrin I domain binding
to its ligand, combined with a high temporal and spatial
resolution in vitro flow chamber model. We report that T cell,
but not monocyte, adhesion to islet microvascular endothelium
is critically dependent on ?L-integrin I domain interaction with
ligand even when other adhesion molecules are present (e.g.,
VCAM-1/VLA-4). We also show that inhibition of ?L-integrin
I domain function increases the average rolling velocity of T
cells by decreasing the number of T cells rolling at velocities
?50 ?m/s. These data demonstrate that cyclic peptide compe-
tition with the ?L-integrin I domain for ligand may be useful in
attenuating T cell recruitment during autoimmune diabetes.
MATERIALS AND METHODS
Cells, antibodies, and materials. The WEHI 7.1 mouse T cell,
WEHI 274.1 mouse monocyte cell, and MS1 mouse pancreatic islet
Address for reprint requests and other correspondence: C. G. Kevil, Dept. of
Pathology, Louisiana State University Health Sciences Center-Shreveport,
1501 Kings Hwy., Shreveport, LA 71130 (E-mail: ckevil@lsuhsc.edu).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Am J Physiol Gastrointest Liver Physiol 288: G67–G73, 2005.
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microvascular endothelial cell lines were purchased from American
Type Culture Convention (ATCC; Manassas, VA). All cell culture
medium materials and recombinant mouse TNF-? were purchased
from Sigma (St. Louis, MO). Corning cell culture plasticware was
purchased from VWR (Suwanee, GA). Anti-CD54 (ICAM-1) FITC
(3E2), anti-CD18 (?2-integrin) phycoerythrin (PE) (C71/16), anti-
CD11a (?L-integrin) PE (2D7), anti-CD11b (?M-integrin) PE (M1/
70), anti-CD29 (?1-integrin) FITC (Ha2/5), anti-CD16/32 (2.4G2) Fc
block, isotype control rat IgG2a and IgG2b PE, and isotype control
hamster IgG1 and IgM FITC were all obtained from BD (Franklin
Lakes, NJ). Anti-CD106 (VCAM-1) FITC (429) and isotype control
rat IgG2a were obtained from Ebiosciences (San Diego, CA).
Peptide synthesis. Cyclic peptides cLAB.L (cyclo1,12-Pen-
ITDGEATDSGC) and cyclic CGFCG control peptide were synthe-
sized by using the solid phase method by Multiple Peptide Systems
(San Diego, CA) as previously reported (46, 50). The crude cLAB.L
peptide was purified by preparative HPLC using reversed-phase C-18
column. The pure product was analyzed by NMR and fast atom
bombardment mass spectrometry.
Cell culture. Cells were cultured by using ATCC recommended
guidelines along with recommended media. WEHI cell lines were
grown in suspension in T-75 flasks, whereas adherent MS1 endothe-
lial cell cultures were grown in T-25 flasks. For parallel plate flow
chamber studies, MS1 cells were seeded into 35-mm culture dishes
and grown to confluency.
Flow cytometry analysis. The working dilutions of the ?2-, ?1-,
?L-, and ?M-antibodies, and ICAM-1, and VCAM-1 antibodies and
their isotype controls were 1:320, 1:320, 1:80, 1:80, 1:80, and 1:80,
respectively. Cultured WEHI and MS1 cell lines were harvested and
washed in 10 ml of FACS buffer (PBS ? 1% FBS). An aliquot of 5 ?
105cells was used for adhesion molecule analysis. All cells were
preincubated on ice for 20 min with 50 ?l of 1:100 anti-FC-receptor
antibody to block nonspecific binding. The above diluted antibodies
were then added to the cells and incubated on ice for 20 min. The cells
were washed twice with 1 ml of FACS buffer and resuspended in 300
?l of FACS buffer. Immunofluorescence stained samples were ana-
lyzed on a FACS Calibur flow cytometer (Becton-Dickinson) made
available through the Research Core Facility at Louisiana State
University Health Sciences Center. Data analysis was performed by
using CELL Quest software (Becton-Dickinson). The cells were gated
on the basis of forward vs. side scatter, and 10,000 events were
collected.
In vitro parallel plate flow chamber assays. Parallel plate flow
chamber studies were performed as previously reported with slight
modifications (21, 34). Briefly, endothelial cell monolayers were
stimulated with TNF-? (10 ng/ml) or PBS vehicle for 6 h. Monocytes
or T cells were labeled with CMTPX CellTracker Red for 30 min at
room temperature (Molecular Probes, Eugene, OR). The 200,000
cells/ml of either monocytes or T cells were perfused over endothelial
monolayers using a parallel plate flow chamber maintained at 37°C
(GlycoTech, Rockville, MD). In some experiments, 100 ?M of
cLAB.L or control cyclic peptide was combined with labeled leuko-
cytes and, subsequently, used for parallel plate flow studies. Cells
were injected into the flow chamber in HBSS at a controlled physi-
ological shear rate of 1.2 dynes/cm2using a programmable digital
syringe pump (KD Scientific, New Hope, PA). Cells were viewed on
a Nikon model TE2000 inverted microscope equipped with a
Hamamatsu ORCA-ER digital charge-coupled device camera and
viewed under epifluorescent illumination. Digital video of fluorescent
cell signals were captured at a rate of 29 images per second over a
3-min period using Simple PCI software (Compix, Cranberry Town-
ship, PA). Digital images were analyzed to yield position measure-
ments for every object in each frame. Position measurements were
acquired and computed by the motion tracking analysis feature of
Simple PCI software to determine leukocyte rolling velocity. Counts
of rolling and firmly adherent cells were determined by computer
analysis and confirmed by manual visual review of image sequences.
Statistical analyses. Data were statistically compared by using
Prism 4.0 software (GraphPad). The number of firmly adherent and
rolling cell data was compared by using a standard ANOVA with a
Tukey’s posttest to determine statistical differences between experi-
mental groups. Numbers from firm adhesion and rolling cell data are
reported as means ? SD. Rolling velocity data from 1,200 cells per
treatment group were compared by using a Kruskal-Wallis nonpara-
metric ANOVA with a Dunnett’s post test to determine statistical
differences between experimental groups. Rolling velocity data are
reported as a vertical box and whisker plot with a mean line and a
relative frequency histogram distribution.
RESULTS
Identification of T cell and monocyte surface adhesion mol-
ecules. Members of the ?2- and ?1-integrin families have been
reported to be important for facilitating leukocyte-endothelial
cell adhesion interactions (11, 20). We determined surface
adhesion molecule expression on established monocyte and T
cell lines to identify possible integrins that could participate
during leukocyte adhesion. Figure 1 shows surface expression
of ?1-, ?2-, ?L-, and ?M-integrins between the two cell lines.
Fig. 1, A–D sequentially illustrates ?2-, ?L-, ?M-, and ?1-flow
cytometry profiles of T cells. Fig. 1, E–H shows the same flow
cytometry profiles for monocytes. Both cell lines displayed
high expression for ?2- and ?1-integrin, suggesting that mem-
bers of these adhesion molecules are available for cell-cell
interactions. The T cells showed high expression of ?L-integrin
and low expression of ?M-integrin. Conversely, the monocytes
displayed high expression of ?M-integrin and no expression of
?L-integrin. Together, these data clearly demonstrate that the T
cells are LFA-1highand Mac-1low, whereas the monocytes are
Mac-1highand LFA-1 negative.
Characterization of pancreatic islet microvascular endothe-
lial cell adhesion molecule expression. Leukocyte ?2- and
?1-integrin-dependent adhesion is largely attributed to binding
of the endothelial counter-ligands ICAM-1 and VCAM-1,
respectively (19). In both experimental autoimmune diabetes
models and clinical specimens, increased expression of
ICAM-1 and VCAM-1 can be observed on islet microvascular
endothelium (8, 10, 29). Several different proinflammatory
cytokines induce the expression of these adhesion molecules,
such as TNF-? and IFN-?, and have been reported in autoim-
mune diabetes models and from T1D patients (17, 25, 49).
Therefore, we examined induction of adhesion molecule ex-
pression in pancreatic islet microvascular endothelial cells in
response to TNF-?. Figure 2A shows that islet microvascular
endothelial cells display constitutive surface expression of
ICAM-1 that can be upregulated by stimulation with TNF-?
(10 ng/ml). Moreover, Fig. 2B reveals minimal constitutive
VCAM-1 surface expression that can also be induced by
TNF-? (10 ng/ml). These data demonstrate that the in vitro
islet microvascular endothelial cell adhesion molecule expres-
sion profile corresponds similarly to previous studies examin-
ing these molecules in clinical and animal model specimens.
Effect of cLAB.L on TNF-? mediated monocyte and T cell
firm adhesion. A recent study has shown that cyclic peptide
antagonists against the ?L-integrin I domain can attenuate
leukocyte-epithelial cell adhesive interactions under static con-
ditions (52). However, studies are lacking that examine
whether these cyclic peptide antagonists interfere with leuko-
cyte-microvascular endothelial cell interactions under hydro-
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dynamic flow conditions. Here we used the cLAB.L cyclic
peptide to interfere with leukocyte-islet microvascular endo-
thelial cell adhesion. This peptide is derived from the I-domain
sequence of the ?L-integrin and has been shown to specifically
bind to D1 of ICAM-1 (50). We quantified leukocyte firm
adhesion with or without cyclic peptide treatment under basal
or TNF-?-stimulated conditions. Figure 3A shows that TNF-?
stimulation increases monocyte adhesion that is not attenuated
by cLAB.L peptide treatment. Conversely, TNF-? treatment
increases T cell adhesion that is completely attenuated by
cLAB.L treatment (Fig. 3B). This inhibition is likely dependent
on conversion of the ?L?2-integrin to the “active” conforma-
tion, in that cLAB.L peptide treatment did not alter the number
of adherent T cells under nonstimulated conditions. Figure 3, C
and D shows that control peptides did not affect monocyte or
T cell adhesion in response to TNF-?. These data demonstrate
that the cLAB.L peptide specifically inhibits adhesion of T
cells containing an active ?L?2-integrin conformation.
Effect of cLAB.L on T cell rolling. Recent reports (12, 21)
suggest that adhesion molecules involved in leukocyte firm
adhesion can modulate various aspects of rolling. Moreover,
the ?L?2-integrin has been reported to facilitate leukocyte slow
rolling that may subsequently influence firm adhesion (3, 38).
Figure 4 shows the effect of cLAB.L on the numbers and
velocities of rolling T cells. Figure 4A demonstrates that
cLAB.L treatment does not affect the number of rolling cells in
response to TNF-?. However, treatment with cLAB.L signif-
icantly increased the average rolling velocity. The mean rolling
velocity for unstimulated conditions was 121.5 ?m/s, 88.7
?m/s for TNF-? treatment, 107.0 ?m/s for TNF-? plus
cLAB.L, and 82.3 ?m/s for TNF-? plus control peptide (Fig.
4B). Figure 4C shows that TNF-? activation of islet microvas-
cular endothelial cells increases the frequency of cells rolling at
slower velocities compared with unstimulated endothelium.
Interestingly, treatment with cLAB.L significantly decreased
the frequency of cells rolling between 0 and 50 ?m/s. To-
gether, these data clearly demonstrate that inhibition of ?L-
integrin I domain does not alter the ability of T cells to roll;
rather, it increases the overall average rolling velocity and
decreases the frequency of slow rolling cells.
DISCUSSION
T cell recruitment with subsequent islet ?-cell destruction is
a primary pathological feature of autoimmune diabetes. Recent
advances in islet autoantibody detection have enabled predic-
tion of T1D development in patients with susceptible haplo-
types and suggest that therapeutic intervention may be feasible
before the development of frank diabetes (35). Whereas it is
now possible to clinically diagnose autoimmune diabetes be-
fore symptoms occur, it is of little use if there are no interven-
tions to interrupt immune cell attack on pancreatic islets.
Fig. 1. Surface integrin expression of WEHI 7.1 T cells and WEHI 274.1
monocytes. A–D: illustrate ?2-, ?L-, ?M-, and ?1-surface-integrin staining of
WEHI 7.1 T cells, respectively. E–H: demonstrate ?2-, ?L-, ?M-, and ?1-
surface-integrin staining of WEHI 274.1 monocytes, respectively.
Fig. 2. Surface adhesion molecule expres-
sion of pancreatic islet microvascular endo-
thelium. A: constitutive and TNF-? (10 ng/
ml)-mediated surface CD54 (ICAM-1) ex-
pression. B: constitutive and TNF-? (10 ng/
ml)-mediated surface CD106 (VCAM-1)
expression. Dashed line curve indicates iso-
type control, solid line curve shows consti-
tutive staining, and filled curve indicates
expression after TNF-? (10 ng/ml) treat-
ment.
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Recent interventional efforts have focused on inhibiting T cell
receptor (TCR) engagement and subsequent T cell activation;
however, other avenues for blocking immune cell recruitment
and activation may provide additional benefit (13, 14).
Studies (26, 28, 33, 37) from both animal models and
clinical specimens implicate a role for cell adhesion molecules
during the development of autoimmune diabetes. Increased
leukocyte and endothelial cell adhesion molecule expression
has been observed at initial diagnosis of T1D. Inhibition of
some of these adhesion molecules can attenuate disease pro-
gression in the autoimmune diabetes-prone nonobese diabetic
(NOD) mouse model (2, 26, 29, 30, 33, 37). These observa-
tions clearly suggest that cell adhesion molecules participate in
the pathogenesis of autoimmune diabetes, yet the specific
molecular requirements and roles of these proteins are poorly
understood.
Recent studies (42, 44) have advanced our understanding of
how leukocyte integrins, such as ?L?2-integrins, facilitate cell
adhesion. Several molecular mechanisms have been identified
that influence the conversion of ?L?2-integrins from a resting
to activated state, including divalent cation binding and con-
formational changes in protein structure (9, 22, 40). Once
activated, ?L?2-integrins have been suggested to play a role in
cell adhesion that may participate in cell rolling and during the
transition to firm adhesion (23, 38). However, the direct effect
of competitive ligand antagonists with the ?L-integrin I domain
during leukocyte-islet microvascular endothelial cell interac-
tions has not been examined. In this study, we have examined
the importance and mechanism of the ?L-integrin I domain in
mediating T cell and monocyte adhesion to pancreatic islet
microvascular endothelial cells.
cLAB.L peptide (cyclo1,12-PenITDGEATDSGC) was de-
rived from 10 amino acid residues (237Ile-246Gly) of the ?L-
integrin I domain. The Pen1 and Cys12 residues were added to
the NH2- and COOH-terminals of the237Ile-246Gly sequence,
respectively, to give a linear peptide PenITDGEATDSGC. The
thiol groups in Pen1 and Cys12 were oxidized to make a
disulfide bond to produce a cyclic peptide cLAB.L. The
cLAB.L peptide has been shown to bind to domain 1 (D1) of
ICAM-1 and inhibit both homotypic and mixed lymphocyte
reactions, as well as heterotypic T-cell adhesion to epithelial
cell monolayers (46, 52, 53). The conformation of cLAB.L as
determined by NMR and molecular dynamics simulations
shows that cLAB.L peptide has a stable ?II?-turn at Asp4-
Gly5-Glu6-Ala7 and a ?I?-turn at Pen1-Ile2-Thr3-Asp4 (50). It
is interesting to find that the X-ray structure of the I domain at
239Asp-Gly-Glu-242Ala has a ?-turn conformation, suggesting
that the cyclic formation of cLAB.L maintains the conforma-
tion of these residues. In addition, docking studies (50) of
cLAB.L peptide onto D1 of ICAM-1 indicate that Ile2 to Thr8
residues of cLAB.L peptide interact with the F and C strands of
ICAM-1. These results suggest that cLAB.L peptide binds to F
and C stands of D1 of ICAM-1, thereby blocking the interac-
tion between ?L-integrin I domain and ICAM-1.
Our data clearly demonstrate that ?L-integrin I domain-
ligand interactions play a dominant role in mediating T cell
Fig. 3. Effect of cLAB.L peptide on monocyte and T cell firm adhesion. A: effect of 100 ?M cLAB.L peptide treatment on resting and TNF-?-mediated
monocyte adhesion to islet endothelium. B: effect of 100 ?M cLAB.L peptide on resting and TNF-? stimulated T cell adhesion to islet endothelium. C: effect
of 100 ?M control peptide on monocyte adhesion to islet endothelium. D: effect of 100 ?M control peptide on T cell adhesion to islet endothelium. *P ? 0.01
TNF-? (?) vs. TNF-? (-). #P ? 0.01 TNF-? (?) vs. TNF-? (?) 100 ?M cLAB.L (?).
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adhesion to islet microvascular endothelium as demonstrated
by cLAB.L peptide. Importantly, other cell adhesion molecules
were found on both cell types, specifically VCAM-1 and
VLA-4, which were not able to mediate firm adhesion after
inhibition of ?L-integrin with the cLAB.L peptide. Our data
also indicate that the cLAB.L peptide specifically blocks ?L?2-
antigen-mediated adhesion, because it did not affect adhesion
of monocytes that only express ?M?2-(Mac-1) and ?1-integrin
(VLA-4). Because cLAB.L is derived from the I-domain of ?L,
these data indicate that this region of the I domain is important
for ?L?2-integrin-mediated leukocyte-endothelial cell adhe-
sion, and further demonstrate that this segment of the ?L-
integrin I domain selectively differentiates ?L- vs. ?M-integrin
binding to ICAM-1. Taken together, these data demonstrate for
the first time that the ?L-integrin I domain is critical for T cell
adhesion to islet microvascular endothelium and that this
adhesion can be prevented by peptide antagonists that compete
for the ?L-integrin I domain ligand.
Recent studies (12, 21) have shown that ?L?2-integrin may
influence the nature of leukocyte rolling. Whereas the selectin
proteins are the primary mediators of immune cell capture and
rolling, studies have shown that ?2-integrins and their ligands,
such as ICAM-1, clearly modulate cell rolling. Sigal et al. (41)
has shown that ?L?2-integrins can support rolling under phys-
iological shear stress that is dependent on ICAM-1 surface
density. Another study, by Dunne et al. (3) examined leukocyte
rolling parameters in ?2-, ?L-, or ?M-integrin null mice and
found increased average rolling velocities in ?L- and ?M-
mutants, along with a higher increase in rolling velocities of
?2-null mutants. An important finding of this study was that
leukocyte adhesion efficiency was similarly decreased in ?L-
and ?2-mutants, but only slightly attenuated in ?M-mutants.
These results further indicate that ?L?2-antigen is important for
the conversion of leukocyte rolling to firm adhesion. Interest-
ingly, our data demonstrate that competitive inhibition for
?L-integrin I domain ligand significantly increases average
leukocyte rolling velocities. Inhibition of ?L-integrin I domain
binding primarily affected slow rolling velocities (0–50 ?m/s),
which may impede the transition of leukocyte rolling to firm
adhesion. Together with previous studies, our data strongly
suggest an important role for ?L-integrin I domain in facilitat-
ing T cell slow rolling and transition to firm adhesion on islet
microvascular endothelium.
Much effort is currently focused on identifying small mol-
ecule ?L-antagonists with the anticipation that these com-
pounds will provide new avenues for therapeutic intervention
in several chronic inflammatory diseases, including autoim-
mune diabetes (1, 39, 48). Here we have examined the utility
of cyclic peptide antagonists against ?L-integrin I domain in
preventing T cell-islet endothelial cell interactions. Our data
show that peptide antagonists competing for ?L-integrin I
domain ligand specifically inhibit T cell adhesion, enabling
other leukocyte adhesion. These data are the first to describe
the effect of ?L-integrin I domain antagonists in a physiolog-
ical leukocyte-islet microvascular endothelial cell hydrody-
namic flow model. Therapeutic interventions using this type of
approach may prove useful in attenuating insulitis and ?-cell
destruction observed in T1D.
GRANTS
This work was supported by the Louisiana State University Health Sciences
Center-Shreveport Center for Excellence in Rheumatology and Arthritis, and
National Institute of Diabetes and Digestive and Kidney Diseases Grant
DK-43785. Financial support was also received from Self Faculty Scholar
from The University of Kansas and National Institutes of Health Grant
EB-00226 (to T. J. Siahaan).
Fig. 4. Effect of cLAB.L peptide on T cell rolling
parameters. A: number of rolling T cell per 10 s for
various treatment conditions. *P ? 0.01 control vs.
treatment groups. B: mean rolling velocity of T cells for
various treatment conditions. *P ? 0.01 control vs.
TNF-? or TNF-? plus 100 ?M control peptide, #P ?
0.05 TNF-? vs. TNF-? plus 100 ?M cLAB.L peptide.
C: frequency of rolling T cell populations at different
average velocities. *P ? 0.01 control vs. TNF-? or
TNF-? plus 100 ?M control peptide. #P ? 0.05 TNF-?
vs. TNF-? plus 100 ?M cLAB.L.
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