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Am J Physiol Gastrointest Liver Physiol 288: G67-G73, 2005. First published August 19, 2004; doi:10.1152/ajpgi.00267.2004
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INFLAMMATION/IMMUNITY/MEDIATORS

{alpha}L-Integrin I domain cyclic peptide antagonist selectively inhibits T cell adhesion to pancreatic islet microvascular endothelium

Meng Huang,1 Kametra Matthews,1 Teruna J. Siahaan,2 and Christopher G. Kevil1

1Department of Pathology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, Louisiana; and 2Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas

Submitted 22 June 2004 ; accepted in final form 14 August 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 ≤50 µm/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.

lymphocyte function-associated antigen-1; leukocyte; pancreas; diabetes

AUTOIMMUNE DIABETES (type 1 diabetes; T1D) is a multifaceted metabolic disorder characterized by hyperglycemia, which results 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 {beta}-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; {alpha}L{beta}2-antigen), Mac-1 ({alpha}M{beta}2-integrin), {alpha}4- and {beta}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 {alpha}L{beta}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 microvascular 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 {beta}2-integrin family, or CD18 integrins, which consists of four different adhesion proteins: {alpha}L{beta}2- (LFA-1), {alpha}M{beta}2- (Mac-1), {alpha}X{beta}2- (p150/95), and {alpha}D{beta}2-proteins. Importantly, expression of the {beta}2-integrins is limited to leukocytes, indicating their importance for immune responses. Members of the immunoglobulin superfamily serve as the major ligands for the {beta}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 {alpha}-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 {alpha}L- and {alpha}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 requirements involved during leukocyte-islet microvascular endothelial cell interactions using specific cyclic peptide antagonists that are designed to compete with {alpha}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 {alpha}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 {alpha}L-integrin I domain function increases the average rolling velocity of T cells by decreasing the number of T cells rolling at velocities <50 µm/s. These data demonstrate that cyclic peptide competition with the {alpha}L-integrin I domain for ligand may be useful in attenuating T cell recruitment during autoimmune diabetes.


   MATERIALS AND METHODS
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 MATERIALS AND METHODS
RESULTS
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 GRANTS
 REFERENCES
 
Cells, antibodies, and materials. The WEHI 7.1 mouse T cell, WEHI 274.1 mouse monocyte cell, and MS1 mouse pancreatic islet microvascular endothelial cell lines were purchased from American Type Culture Convention (ATCC; Manassas, VA). All cell culture medium materials and recombinant mouse TNF-{alpha} 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 ({beta}2-integrin) phycoerythrin (PE) (C71/16), anti-CD11a ({alpha}L-integrin) PE (2D7), anti-CD11b ({alpha}M-integrin) PE (M1/70), anti-CD29 ({beta}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-PenITDGEATDSGC) and cyclic CGFCG control peptide were synthesized 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 endothelial 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 {beta}2-, {beta}1-, {alpha}L-, and {alpha}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 x 105 cells was used for adhesion molecule analysis. All cells were preincubated on ice for 20 min with 50 µl 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 µl of FACS buffer. Immunofluorescence stained samples were analyzed 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-{alpha} (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 µM of cLAB.L or control cyclic peptide was combined with labeled leukocytes and, subsequently, used for parallel plate flow studies. Cells were injected into the flow chamber in HBSS at a controlled physiological shear rate of 1.2 dynes/cm2 using 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 Township, PA). Digital images were analyzed to yield position measurements 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 experimental 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 nonparametric 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
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 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Identification of T cell and monocyte surface adhesion molecules. Members of the {beta}2- and {beta}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 {beta}1-, {beta}2-, {alpha}L-, and {alpha}M-integrins between the two cell lines. Fig. 1, A–D sequentially illustrates {beta}2-, {alpha}L-, {alpha}M-, and {beta}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 {beta}2- and {beta}1-integrin, suggesting that members of these adhesion molecules are available for cell-cell interactions. The T cells showed high expression of {alpha}L-integrin and low expression of {alpha}M-integrin. Conversely, the monocytes displayed high expression of {alpha}M-integrin and no expression of {alpha}L-integrin. Together, these data clearly demonstrate that the T cells are LFA-1high and Mac-1low, whereas the monocytes are Mac-1high and LFA-1 negative.



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  		Fig. 1. Surface integrin expression of WEHI 7.1 T cells and WEHI 274.1 monocytes. A–D: illustrate {beta}2-, {alpha}L-, {alpha}M-, and {beta}1-surface-integrin staining of WEHI 7.1 T cells, respectively. E–H: demonstrate {beta}2-, {alpha}L-, {alpha}M-, and {beta}1-surface-integrin staining of WEHI 274.1 monocytes, respectively.

 
Characterization of pancreatic islet microvascular endothelial cell adhesion molecule expression. Leukocyte {beta}2- and {beta}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-{alpha} and IFN-{gamma}, and have been reported in autoimmune diabetes models and from T1D patients (17, 25, 49). Therefore, we examined induction of adhesion molecule expression in pancreatic islet microvascular endothelial cells in response to TNF-{alpha}. Figure 2A shows that islet microvascular endothelial cells display constitutive surface expression of ICAM-1 that can be upregulated by stimulation with TNF-{alpha} (10 ng/ml). Moreover, Fig. 2B reveals minimal constitutive VCAM-1 surface expression that can also be induced by TNF-{alpha} (10 ng/ml). These data demonstrate that the in vitro islet microvascular endothelial cell adhesion molecule expression profile corresponds similarly to previous studies examining these molecules in clinical and animal model specimens.



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  		Fig. 2. Surface adhesion molecule expression of pancreatic islet microvascular endothelium. A: constitutive and TNF-{alpha} (10 ng/ml)-mediated surface CD54 (ICAM-1) expression. B: constitutive and TNF-{alpha} (10 ng/ml)-mediated surface CD106 (VCAM-1) expression. Dashed line curve indicates isotype control, solid line curve shows constitutive staining, and filled curve indicates expression after TNF-{alpha} (10 ng/ml) treatment.

 
Effect of cLAB.L on TNF-{alpha} mediated monocyte and T cell firm adhesion. A recent study has shown that cyclic peptide antagonists against the {alpha}L-integrin I domain can attenuate leukocyte-epithelial cell adhesive interactions under static conditions (52). However, studies are lacking that examine whether these cyclic peptide antagonists interfere with leukocyte-microvascular endothelial cell interactions under hydrodynamic flow conditions. Here we used the cLAB.L cyclic peptide to interfere with leukocyte-islet microvascular endothelial cell adhesion. This peptide is derived from the I-domain sequence of the {alpha}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-{alpha}-stimulated conditions. Figure 3A shows that TNF-{alpha} stimulation increases monocyte adhesion that is not attenuated by cLAB.L peptide treatment. Conversely, TNF-{alpha} treatment increases T cell adhesion that is completely attenuated by cLAB.L treatment (Fig. 3B). This inhibition is likely dependent on conversion of the {alpha}L{beta}2-integrin to the "active" conformation, 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-{alpha}. These data demonstrate that the cLAB.L peptide specifically inhibits adhesion of T cells containing an active {alpha}L{beta}2-integrin conformation.



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  		Fig. 3. Effect of cLAB.L peptide on monocyte and T cell firm adhesion. A: effect of 100 µM cLAB.L peptide treatment on resting and TNF-{alpha}-mediated monocyte adhesion to islet endothelium. B: effect of 100 µM cLAB.L peptide on resting and TNF-{alpha} stimulated T cell adhesion to islet endothelium. C: effect of 100 µM control peptide on monocyte adhesion to islet endothelium. D: effect of 100 µM control peptide on T cell adhesion to islet endothelium. *P < 0.01 TNF-{alpha} (+) vs. TNF-{alpha} (-). #P < 0.01 TNF-{alpha} (+) vs. TNF-{alpha} (+) 100 µM cLAB.L (+).

 
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 {alpha}L{beta}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-{alpha}. However, treatment with cLAB.L significantly increased the average rolling velocity. The mean rolling velocity for unstimulated conditions was 121.5 µm/s, 88.7 µm/s for TNF-{alpha} treatment, 107.0 µm/s for TNF-{alpha} plus cLAB.L, and 82.3 µm/s for TNF-{alpha} plus control peptide (Fig. 4B). Figure 4C shows that TNF-{alpha} activation of islet microvascular 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 µm/s. Together, these data clearly demonstrate that inhibition of {alpha}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.



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  		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-{alpha} or TNF-{alpha} plus 100 µM control peptide, #P < 0.05 TNF-{alpha} vs. TNF-{alpha} plus 100 µM cLAB.L peptide. C: frequency of rolling T cell populations at different average velocities. *P < 0.01 control vs. TNF-{alpha} or TNF-{alpha} plus 100 µM control peptide. #P < 0.05 TNF-{alpha} vs. TNF-{alpha} plus 100 µM cLAB.L.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
T cell recruitment with subsequent islet {beta}-cell destruction is a primary pathological feature of autoimmune diabetes. Recent advances in islet autoantibody detection have enabled prediction of T1D development in patients with susceptible haplotypes 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 before symptoms occur, it is of little use if there are no interventions to interrupt immune cell attack on pancreatic islets. 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 progression in the autoimmune diabetes-prone nonobese diabetic (NOD) mouse model (2, 26, 29, 30, 33, 37). These observations 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 {alpha}L{beta}2-integrins, facilitate cell adhesion. Several molecular mechanisms have been identified that influence the conversion of {alpha}L{beta}2-integrins from a resting to activated state, including divalent cation binding and conformational changes in protein structure (9, 22, 40). Once activated, {alpha}L{beta}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 {alpha}L-integrin I domain during leukocyte-islet microvascular endothelial cell interactions has not been examined. In this study, we have examined the importance and mechanism of the {alpha}L-integrin I domain in mediating T cell and monocyte adhesion to pancreatic islet microvascular endothelial cells.

cLAB.L peptide (cyclo1,12-PenITDGEATDSGC) was derived from 10 amino acid residues (237Ile-246Gly) of the {alpha}L-integrin I domain. The Pen1 and Cys12 residues were added to the NH2- and COOH-terminals of the 237Ile-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 {beta}II'-turn at Asp4-Gly5-Glu6-Ala7 and a {beta}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 {beta}-turn conformation, suggesting that the cyclic formation of cLAB.L maintains the conformation 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 interaction between {alpha}L-integrin I domain and ICAM-1.

Our data clearly demonstrate that {alpha}L-integrin I domain-ligand interactions play a dominant role in mediating T cell 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 {alpha}L-integrin with the cLAB.L peptide. Our data also indicate that the cLAB.L peptide specifically blocks {alpha}L{beta}2-antigen-mediated adhesion, because it did not affect adhesion of monocytes that only express {alpha}M{beta}2- (Mac-1) and {beta}1-integrin (VLA-4). Because cLAB.L is derived from the I-domain of {alpha}L, these data indicate that this region of the I domain is important for {alpha}L{beta}2-integrin-mediated leukocyte-endothelial cell adhesion, and further demonstrate that this segment of the {alpha}L-integrin I domain selectively differentiates {alpha}L- vs. {alpha}M-integrin binding to ICAM-1. Taken together, these data demonstrate for the first time that the {alpha}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 {alpha}L-integrin I domain ligand.

Recent studies (12, 21) have shown that {alpha}L{beta}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 {beta}2-integrins and their ligands, such as ICAM-1, clearly modulate cell rolling. Sigal et al. (41) has shown that {alpha}L{beta}2-integrins can support rolling under physiological shear stress that is dependent on ICAM-1 surface density. Another study, by Dunne et al. (3) examined leukocyte rolling parameters in {beta}2-, {alpha}L-, or {alpha}M-integrin null mice and found increased average rolling velocities in {alpha}L- and {alpha}M-mutants, along with a higher increase in rolling velocities of {beta}2-null mutants. An important finding of this study was that leukocyte adhesion efficiency was similarly decreased in {alpha}L- and {beta}2-mutants, but only slightly attenuated in {alpha}M-mutants. These results further indicate that {alpha}L{beta}2-antigen is important for the conversion of leukocyte rolling to firm adhesion. Interestingly, our data demonstrate that competitive inhibition for {alpha}L-integrin I domain ligand significantly increases average leukocyte rolling velocities. Inhibition of {alpha}L-integrin I domain binding primarily affected slow rolling velocities (0–50 µm/s), which may impede the transition of leukocyte rolling to firm adhesion. Together with previous studies, our data strongly suggest an important role for {alpha}L-integrin I domain in facilitating T cell slow rolling and transition to firm adhesion on islet microvascular endothelium.

Much effort is currently focused on identifying small molecule {alpha}L-antagonists with the anticipation that these compounds will provide new avenues for therapeutic intervention in several chronic inflammatory diseases, including autoimmune diabetes (1, 39, 48). Here we have examined the utility of cyclic peptide antagonists against {alpha}L-integrin I domain in preventing T cell-islet endothelial cell interactions. Our data show that peptide antagonists competing for {alpha}L-integrin I domain ligand specifically inhibit T cell adhesion, enabling other leukocyte adhesion. These data are the first to describe the effect of {alpha}L-integrin I domain antagonists in a physiological leukocyte-islet microvascular endothelial cell hydrodynamic flow model. Therapeutic interventions using this type of approach may prove useful in attenuating insulitis and {beta}-cell destruction observed in T1D.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
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).


   FOOTNOTES
 

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{at}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.


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