Skip Navigation


International Immunology Advance Access originally published online on January 13, 2006
International Immunology 2006 18(2):389-398; doi:10.1093/intimm/dxh378
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
18/2/389    most recent
dxh378v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (8)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Pavlick, K. P.
Right arrow Articles by Grisham, M. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pavlick, K. P.
Right arrow Articles by Grisham, M. B.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

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. Kevil2 and Matthew B. Grisham1

1 Department of Molecular and Cellular Physiology and 2 Department of Pathology, Louisiana State University Health Sciences Center, 1501 Kings Highway, P.O. Box 33932, Shreveport, LA 71130-3932, USA

Correspondence to: M. B. Grisham; E-mail: mgrish{at}lsuhsc.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The ß2 integrin 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 CD4+CD25– 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, {alpha}4, {alpha}4ß7 and 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 CD4+ T 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.

Keywords: adhesion molecules, CD11a, cytokines, inflammatory bowel disease, mesenteric lymph nodes


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
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-{gamma} and tumor necrosis factor-{alpha} (TNF-{alpha}). 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-{alpha}, IL-1ß, IL-12, nitric oxide and reactive oxygen species (3). Together, Th1- and macrophage-derived cytokines and mediators activate the microvascular endothelium within the gut to enhance expression of adhesion molecules thereby promoting 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 perpetuating chronic gut inflammation, it is important to understand the mechanisms involved in leukocyte trafficking and activation in T-cell-dependent models of chronic gut inflammation.

Although several different T-cell-associated adhesion molecules [e.g. CD62L, CD11a/CD18, chemokine receptor-7 (CCR7), {alpha}4ß7] 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 ß2 integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18; {alpha}Lß2). LFA-1 is a heterodimeric protein composed of an alpha (CD11a, {alpha}L) and beta chain (CD18, ß2), 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 T cells 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 inflammatory 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 adhesion 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 attenuating 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 ß7 integrin subunit delayed the onset of colitis in the CD45RBhigh transfer model of chronic colitis (28), ß7 deficiency 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 psoriasis (32–34).

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 reconstituted 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 due to defects in T cell priming/activation within the MLNs thereby limiting the numbers of Th1 disease-producing cells that populate the colonic interstitium.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Animals
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 standard animal cages with filter tops under specific pathogen-free conditions. All animals were given standard laboratory rodent chow and water ad libitum. All experimental procedures involving the use of animals were reviewed and approved by the Institutional Animal Care and Use Committee of LSU Health Sciences Center and performed according to the criteria outlined by the National Institute of Health.

Antibodies
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, anti-CD69, anti-lymphocyte Peyers patch adhesion molecule-1 (LPAM-1) (integrin {alpha}4ß7 complex), anti-CD49d (integrin {alpha}4 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 labeling with FITC-conjugated anti-B220, anti-CD8 and anti-Mac-1 and then with anti-FITC magnetic microbeads followed by separation on a type CS column using VarioMACS magnetic separator according to manufacturer's instructions (Miltenyi Biotec, Auburn, CA, USA). Unlabeled flow-thru cells were collected and labeled with biotin-conjugated anti-CD4 (GK1.5), streptavidin and PE-conjugated anti-CD25 mAb. Cells were sorted for CD4+CD25– using FACSVantage (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 x 105 CD4+CD25– T cells, from either wt or CD11a–/–, suspended in 500 µl 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 x 107 cells ml–1. After incubation with anti-FcR mAb, ~1 x 106 cells 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 formaldehyde (Polysciences, Inc., Warrington, PA, USA) and analyzed next day on the FACScalibur (BD Biosciences, San Diego, CA, USA).

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. Supernatants from individual animals were collected in separate 50-ml conical tubes and kept on ice. Incubations were performed at least three times to insure complete removal of epithelium. Intestinal pieces from individual animals were processed separately and never mixed. The resultant pooled supernatants 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 collagenase 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 centrifugation over a 40/70% Percoll gradient. The LPL pellet was washed, re-suspended in FACS buffer containing anti-FcR mAb and counted. ~1 x 106 cells 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–/– CD4+CD25– injected RAG-1–/– animals.

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, mononuclear leukocytes from the spleen were prepared by density centrifugation through Ficoll-PaqueTM PLUS (Amersham Biosciences). Colonic lamina propria mononuclear cells (LPMCs) were prepared as described above using a 40/70% Percoll density gradient while a single-cell suspension of mononuclear cells from MLNs was used without further enrichment. All cells were washed in complete RPMI 1640 media containing 4% FBS, L-glutamine and 100 U ml–1 each penicillin and streptomycin and counted. A total of 5 x 105 mononuclear cells were plated in either control (uncoated) or CD3{varepsilon}-coated (BD Falcon, San Diego, CA, USA) 96-well flat bottom plates. Anti-mouse FG-purified anti-CD28 (eBioscience, 1 µg 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 determination 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
At 8 weeks following T cell reconstitution, mice were euthanized and their colons removed. Colons were cleaned of fecal material 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 µm) 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 (viii) submucosal spread to transmural involvement, range 0–2. The severity of inflammation in both sections of the colon was based on the sum of the scores in each parameter with a maximum score of 20.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
CD11a-deficient T cells fail to induce chronic colitis in immunodeficient mice
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 the 8-week observation period and appeared healthy (Fig. 1A). 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 revealed that 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 which displayed moderate to severe pan-colitis (Fig. 2B and C). 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({alpha}4ß7) and CD49d ({alpha}4). We found that CD11a–/– CD4+CD25– T cells expressed similar levels to wt T cells of all indicated surface markers despite having <1% of the wt levels of LFA-1 (Fig. 3).


Figure 1
View larger version (15K):
[in this window]
[in a new window]
  		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 (±SEM.); pooled data from at least three independent transfers, n = 56 and 67 for wt -> RAG and CD11a–/– -> RAG groups, respectively. (B) Macroscopic evidence of inflammation. Data are expressed as the mean ± SEM. Pooled data from at least three independent transfers, n = 56 and 51 for wt -> RAG and CD11a–/– -> RAG, respectively. (C) Colon weight–length ratios. Data are expressed as the mean ± SEM. Pooled data from at least three independent transfers, n = 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).

 

Figure 2
View larger version (42K):
[in this window]
[in a new window]
  		Fig. 2. Histopathology of the colon. Colons were fixed, embedded in parafin, sectioned (5 µm) and stained with hematoxylin and eosin. (A) Representative micrographs are shown for wt -> RAG and CD11a–/– -> RAG. Micrographs represent x100 magnification. (B) Blinded histopathological scores of the distal colon. Data are expressed as the mean ± SEM (n = 15 for wt -> RAG, n = 40 for CD11a–/– -> RAG). Significant differences (*) from the colitic wt -> RAG 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).

 

Figure 3
View larger version (19K):
[in this window]
[in a new window]
  		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.

 
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 T cells 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 expansion of naive T cells in immunodeficient recipients (38), the absolute numbers of CD3+CD4+ T cells 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 differences were not statistically significant (Fig. 4B).


Figure 4
View larger version (16K):
[in this window]
[in a new window]
  		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) ± SEM. Significant differences (*) between the two groups were determined by two-sided Student's t-test (P < 0.05).

 
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 examined 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 populations 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 colon.


Figure 5
View larger version (22K):
[in this window]
[in a new window]
  		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.

 
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.

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, macrophages 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 different laboratories that T-cell-associated LFA-1 co-stimulatory signaling is important in regulating immune properties of Th cells (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-{alpha}, IFN-{gamma} 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, mononuclear cells obtained from spleens of CD11a–/– -> RAG mice, actually produced 4- and 5-fold more TNF-{alpha} and INF-{gamma} 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.


Figure 6
View larger version (16K):
[in this window]
[in a new window]
  		Fig. 6. Cytokine production by mononuclear cells isolated from spleen (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 (105 total plated mononuclear cells without CD3 and CD28 stimulation) and represent average values ± SEM. from 105 total plated mononuclear cells. Significant differences between the groups, where applicable, are indicated by (*) and were determined by two-sided Student's t-test (P < 0.05).

 
Despite the fact that adoptively transferred CD11a–/– T cells 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-{alpha}, IFN-{gamma} 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 significant cytokine production was observed when mononuclear leukocytes were plated onto plastic wells devoid of CD3 and CD28.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
It is becoming increasingly appreciated that chronic gut inflammation results from a dysregulated mucosal immune response to normal enteric antigens in genetically susceptible individuals (45). This concept is best exemplified experimentally 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 T cells 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 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 ß2 integrin LFA-1 (47). It has been shown that LFA-1 plays an important role in several aspects of cell-mediated immunity including migration (recirculation) to and activation/expansion within MLNs as well as the recruitment of activated/memory T cells to inflamed tissue (48). Data obtained in the current study demonstrate that transfer of CD11a–/– CD4+CD25– T cells into immunodeficient RAG-1–/– mice fails to induce chronic colitis, whereas transfer of wt T cells induced moderate to severe gut inflammation. Failure to induce colonic inflammation correlated with a paucity of CD3+CD4+ T cells 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 T cells 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 {alpha}4 integrins ({alpha}4ß1 and {alpha}4ß7) 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, {alpha}4 and {alpha}4ß7 expression on CD11a–/– were virtually identical to those observed on wt CD4+CD25– donor T cells suggest that CD11a–/– T cells were able to migrate into MLNs via T-cell-associated {alpha}4 integrins, 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 molecules 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 of activation and promote proliferation of T cells. 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-{gamma} and/or TNF-{alpha} 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 (T cells, 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-{gamma}) could influence the production of macrophage and/or dendritic 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-{gamma} and/or TNF-{alpha} 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 from cell 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 interstitium.

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 T cells 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 mice produced significantly larger amounts of IFN-{gamma} and TNF-{alpha} 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-{gamma}- and TNF-{alpha}-producing T cells and/or NK cells. This exaggerated cytokine production profile raises the interesting possibility that extra-intestinal tissue inflammation (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-{alpha}, INF-{gamma} and MIP-1{alpha}) is mediated 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 T cells 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 T cells 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 with IBD.


   Acknowledgements
 
This work was supported by DK64023, the Arthritis Center of Excellence at LSU Health Sciences Center and the Yamanouchi USA Foundation.


   Abbreviations
 
APC   antigen-presenting cell
CCR7   chemokine receptor-7
CD   Crohn's disease
IBD   inflammatory bowel disease
ICAM   intracellular adhesion molecule
IEL   intra-epithelial lymphocyte
LFA-1   lymphocyte function-associated antigen-1
LP   lamina propria
LPAM-1   lymphocyte Peyers patch adhesion molecule-1
LPL   lamina propria lymphocyte
LPMC   lamina propria mononuclear cell
LSU   Louisiana State University
MLN   mesenteric lymph node
PLN   peripheral lymph node
RAG-1–/–   recombinase-activating gene-1 deficient
TNF-{alpha}   tumor necrosis factor-{alpha}
wt   wild type

   Notes
 
Transmitting editor: C. Terhorst

* These authors contributed equally to this work. Back

Received 14 October 2005, accepted 29 November 2005.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Powrie, F. and Leach, M. W. 1995. Genetic and spontaneous models of inflammatory bowel disease in rodents: evidence for abnormalities in mucosal immune regulation. Ther. Immunol. 2:115.[Medline]
  2. Groux, H. and Powrie, F. 1999. Regulatory T cells and inflammatory bowel disease. Immunol. Today 20:442.[CrossRef][ISI][Medline]
  3. Laroux, F. S. and Grisham, M. B. 2001. Immunological basis of inflammatory bowel disease: role of the microcirculation. Microcirculation 8:283.[CrossRef][ISI][Medline]
  4. Springer, T. A., Dustin, M. L., Kishimoto, T. K. and Marlin, S. D. 1987. The lymphocyte function-associated LFA-1, CD2, and LFA-3 molecules: cell adhesion receptors of the immune system. Annu. Rev. Immunol. 5:223.[ISI][Medline]
  5. Butini, L., de Fougerolles, A. R., Vaccarezza, M. et al. 1994. Intercellular adhesion molecules (ICAM)-1 ICAM-2 and ICAM-3 function as counter-receptors for lymphocyte function-associated molecule 1 in human immunodeficiency virus-mediated syncytia formation. Eur. J. Immunol. 24:2191.[Medline]
  6. de Fougerolles, A. R. and Springer, T. A. 1992. Intercellular adhesion molecule 3, a third adhesion counter-receptor for lymphocyte function-associated molecule 1 on resting lymphocytes. J. Exp. Med. 175:185.[Abstract/Free Full Text]
  7. Klickstein, L. B., York, M. R., Fougerolles, A. R. and Springer, T. A. 1996. Localization of the binding site on intercellular adhesion molecule-3 (ICAM-3) for lymphocyte function-associated antigen 1 (LFA-1). J. Biol. Chem. 271:23920.[Abstract/Free Full Text]
  8. Bailly, P., Tontti, E., Hermand, P., Cartron, J. P. and Gahmberg, C. G. 1995. The red cell LW blood group protein is an intercellular adhesion molecule which binds to CD11/CD18 leukocyte integrins. Eur. J. Immunol. 25:3316.[Medline]
  9. Yoshihara, Y., Oka, S., Nemoto, Y. et al. 1994. An ICAM-related neuronal glycoprotein, telencephalin, with brain segment-specific expression. Neuron 12:541.[CrossRef][ISI][Medline]
  10. Ostermann, G., Weber, K. S., Zernecke, A., Schroder, A. and Weber, C. 2002. JAM-1 is a ligand of the beta(2) integrin LFA-1 involved in transendothelial migration of leukocytes. Nat. Immunol. 3:151.[CrossRef][ISI][Medline]
  11. Abraham, C., Griffith, J. and Miller, J. 1999. The dependence for leukocyte function-associated antigen-1/ICAM-1 interactions in T cell activation cannot be overcome by expression of high density TCR ligand. J. Immunol. 162:4399.[Abstract/Free Full Text]
  12. Perez, O. D., Mitchell, D., Jager, G. C. et al. 2003. Leukocyte functional antigen 1 lowers T cell activation thresholds and signaling through cytohesin-1 and Jun-activating binding protein 1. Nat. Immunol. 4:1083.[CrossRef][ISI][Medline]
  13. Ragazzo, J. L., Ozaki, M. E., Karlsson, L., Peterson, P. A. and Webb, S. R. 2001. Costimulation via lymphocyte function-associated antigen 1 in the absence of CD28 ligation promotes anergy of naive CD4+ T cells. Proc. Natl Acad. Sci.USA 98:241.[Abstract/Free Full Text]
  14. Bachmann, M. F., McKall-Faienza, K., Schmits, R. et al. 1997. Distinct roles for LFA-1 and CD28 during activation of naive T cells: adhesion versus costimulation. Immunity 7:549.[CrossRef][ISI][Medline]
  15. Schmits, R., Kundig, T. M., Baker, D. M. et al. 1996. LFA-1-deficient mice show normal CTL responses to virus but fail to reject immunogenic tumor. J. Exp. Med. 183:1415.[Abstract/Free Full Text]
  16. Springer, T. A. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76:301.[CrossRef][ISI][Medline]
  17. Sligh, J. E., Jr, Ballantyne, C. M., Rich, S. S. et al. 1993. Inflammatory and immune responses are impaired in mice deficient in intercellular adhesion molecule 1. Proc. Natl Acad. Sci. USA 90:8529.[Abstract/Free Full Text]
  18. Lehmann, J. C., Jablonski-Westrich, D., Haubold, U., Gutierrez-Ramos, J. C., Springer, T. and Hamann, A. 2003. Overlapping and selective roles of endothelial intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 in lymphocyte trafficking. J. Immunol. 171:2588.[Abstract/Free Full Text]
  19. Bernstein, C. N., Sargent, M. and Rector, E. 2002. Alteration in expression of beta 2 integrins on lamina propria lymphocytes in ulcerative colitis and Crohn's disease. Clin. Immunol. 104:67.[CrossRef][ISI][Medline]
  20. Kirman, I. and Nielsen, O. H. 1996. LFA-1 subunit expression in ulcerative colitis patients. Dig. Dis. Sci. 41:670.[Medline]
  21. Vainer, B., Nielsen, O. H. and Horn, T. 2000. Comparative studies of the colonic in situ expression of intercellular adhesion molecules (ICAM-1, -2, and -3), beta2 integrins (LFA-1, Mac-1, and p150,95), and PECAM-1 in ulcerative colitis and Crohn's disease. Am. J. Surg. Pathol. 24:1115.[CrossRef][ISI][Medline]
  22. Bendjelloul, F., Maly, P., Mandys, V. et al. 2000. Intercellular adhesion molecule-1 (ICAM-1) deficiency protects mice against severe forms of experimentally induced colitis. Clin. Exp. Immunol. 119:57.[CrossRef][ISI][Medline]
  23. Bennett, C. F., Kornbrust, D., Henry, S. et al. 1997. An ICAM-1 antisense oligonucleotide prevents and reverses dextran sulfate sodium-induced colitis in mice. J. Pharmacol. Exp. Ther. 280:988.[Abstract/Free Full Text]
  24. van Assche, G. and Rutgeerts, P. 2002. Antiadhesion molecule therapy in inflammatory bowel disease. Inflamm. Bowel Dis. 8:291[CrossRef][ISI][Medline]
  25. van Deventer, S. J., Tami, J. A. and Wedel, M. K. 2004. A randomised, controlled, double blind, escalating dose study of alicaforsen enema in active ulcerative colitis. Gut 53:1646.[Abstract/Free Full Text]
  26. Hesterberg, P. E., Winsor-Hines, D., Briskin, M. J. et al. 1996. Rapid resolution of chronic colitis in the cotton-top tamarin with an antibody to a gut-homing integrin alpha 4 beta 7. Gastroenterology 111:1373.[CrossRef][ISI][Medline]
  27. Podolsky, D. K., Lobb, R., King, N. et al. 1993. Attenuation of colitis in the cotton-top tamarin by anti-alpha 4 integrin monoclonal antibody. J. Clin. Invest. 92:372.[ISI][Medline]
  28. Picarella, D., Hurlbut, P., Rottman, J., Shi, X., Butcher, E. and Ringler, D. J. 1997. Monoclonal antibodies specific for beta 7 integrin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) reduce inflammation in the colon of scid mice reconstituted with CD45RBhigh CD4+ T cells. J. Immunol. 158:2099.[Abstract]
  29. Burns, R. C., Rivera-Nieves, J., Moskaluk, C. A., Matsumoto, S., Cominelli, F. and Ley, K. 2001. Antibody blockade of ICAM-1 and VCAM-1 ameliorates inflammation in the SAMP-1/Yit adoptive transfer model of Crohn's disease in mice. Gastroenterology 121:1428.[CrossRef][ISI][Medline]
  30. Soriano, A., Salas, A., Salas, A. et al. 2000. VCAM-1, but not ICAM-1 or MAdCAM-1, immunoblockade ameliorates DSS-induced colitis in mice. Lab. Invest. 80:1541.[ISI]
  31. Sydora, B. C., Wagner, N., Lohler, J. et al. 2002. beta7 Integrin expression is not required for the localization of T cells to the intestine and colitis pathogenesis. Clin. Exp. Immunol. 129:35.[CrossRef][ISI][Medline]
  32. Dedrick, R. L., Walicke, P. and Garovoy, M. 2002. Anti-adhesion antibodies—efalizumab, a humanized anti-CD11a monoclonal antibody. Transpl. Immunol. 9:181.[CrossRef][Medline]
  33. Gottlieb, A. B., Krueger, J. G., Wittkowski, K., Dedrick, R., Walicke, P. A. and Garovoy, M. 2002. Psoriasis as a model for T-cell-mediated disease—immunobiologic and clinical effects of treatment with multiple doses of efalizumab, an anti-CD11a antibody. Arch. Dermatol. 138:591.[Abstract/Free Full Text]
  34. Lebwohl, M., Tyring, S. K., Hamilton, T. K. et al. 2003. A novel targeted T-cell modulator, efalizumab, for plaque psoriasis. N. Engl. J. Med. 349:2004.[Abstract/Free Full Text]
  35. Laroux, F. S., Norris, H. H., Houghton, J. et al. 2004. Regulation of chronic colitis in athymic nu/nu (nude) mice. Int. Immunol. 16:77.[Abstract/Free Full Text]
  36. Ostanin, D. V., Pavlick, K. P., Bharwani, S. et al. 2006. T-cell-induced inflammation of the small and large intestine in immuno-deficient mice. Am. J .Physiol. Gastrointest. Liver Physiol., 289:G109.
  37. Conner, E. M., Brand, S., Davis, J. M. et al. 1997. Proteasome inhibition attenuates nitric oxide synthase expression, VCAM-1 transcription and the development of chronic colitis. J. Pharmacol. Exp. Ther. 282:1615.[Abstract/Free Full Text]
  38. Aranda, R., Sydora, B. C., McAllister, P. L. et al. 1997. Analysis of intestinal lymphocytes in mouse colitis mediated by transfer of CD4+, CD45RBhigh T cells to SCID recipients. J. Immunol. 158:3464.[Abstract]
  39. Powrie, F., Mauze, S. and Coffman, R. L. 1997. CD4+ T-cells in the regulation of inflammatory responses in the intestine. Res. Immunol. 148:576.[CrossRef][ISI][Medline]
  40. Warnock, R. A., Askari, S., Butcher, E. C. and von Andrian, U. H. 1998. Molecular mechanisms of lymphocyte homing to peripheral lymph nodes. J. Exp. Med. 187:205.[Abstract/Free Full Text]
  41. Shimizu, Y. 2003. LFA-1: more than just T cell velcro. Nat. Immunol. 4:1052.[Medline]
  42. Liang, S., Alard, P., Zhao, Y., Parnell, S., Clark, S. L. and Kosiewicz, M. M. 2005. Conversion of CD4+. J. Exp. Med. 201:127.[Abstract/Free Full Text]
  43. Curotto de Lafaille, M. A., Lino, A. C., Kutchukhidze, N. and Lafaille, J. J. 2004. J. Immunol. 173:7259.[Abstract/Free Full Text]
  44. Salomon, B. and Bluestone, J. A. 1998. LFA-1 interaction with ICAM-1 and ICAM-2 regulates Th2 cytokine production. J. Immunol. 161:5138.[Abstract/Free Full Text]
  45. Singh, B., Read, S., Asseman, C. et al. 2001. Control of intestinal inflammation by regulatory T cells. Immunol. Rev. 182:190.[CrossRef][ISI][Medline]
  46. Powrie, F. 1995. T cells in inflammatory bowel disease: protective and pathogenic roles. Immunity 3:171.[CrossRef][ISI][Medline]
  47. Anderson, D. C. and Springer, T. A. 1987. Leukocyte adhesion deficiency: an inherited defect in the Mac-1, LFA-1, and p150,95 glycoproteins. Annu. Rev. Med. 38:175.[CrossRef][ISI][Medline]
  48. Pribila, J. T., Quale, A. C., Mueller, K. L. and Shimizu, Y. 2004. Integrins and T cell-mediated immunity. Annu. Rev. Immunol. 22:157.[CrossRef][ISI][Medline]
  49. Hamann, A., Jablonski-Westrich, D., Duijvestijn, A. et al. 1988. Evidence for an accessory role of LFA-1 in lymphocyte-high endothelium interaction during homing. J. Immunol. 140:693.[Abstract]
  50. Andrew, D. P., Spellberg, J. P., Takimoto, H., Schmits, R., Mak, T. W. and Zukowski, M. M. 1998. Transendothelial migration and trafficking of leukocytes in LFA-1-deficient mice. Eur. J. Immunol. 28:1959.[CrossRef][ISI][Medline]
  51. Berlin-Rufenach, C., Otto, F., Mathies, M. et al. 1999. Lymphocyte migration in lymphocyte function-associated antigen (LFA)-1-deficient mice. J. Exp. Med. 189:1467.[Abstract/Free Full Text]
  52. Issekutz, T. B. 1991. Inhibition of in vivo lymphocyte migration to inflammation and homing to lymphoid tissues by the TA-2 monoclonal antibody. A likely role for VLA-4 in vivo. J. Immunol. 147:4178.[Abstract]
  53. Dustin, M. L. and Shaw, A. S. 1999. Costimulation: building an immunological synapse. Science 283:649.[Free Full Text]
  54. Shier, P., Otulakowski, G., Ngo, K. et al. 1996. Impaired immune responses toward alloantigens and tumor cells but normal thymic selection in mice deficient in the beta2 integrin leukocyte function-associated antigen-1. J. Immunol. 157:5375.[Abstract]
  55. Smits, H. H., de Jong, E. C., Schuitemaker, J. H. et al. 2002. Intercellular adhesion molecule-1/LFA-1 ligation favors human Th1 development. J. Immunol. 168:1710.[Abstract/Free Full Text]
  56. Kavanaugh, A. F., Lightfoot, E., Lipsky, P. E. and Oppenheimer-Marks, N. 1991. Role of CD11/CD18 in adhesion and transendothelial migration of T cells. Analysis utilizing CD18-deficient T cell clones. J. Immunol. 146:4149.[Abstract]
  57. Oppenheimer-Marks, N., Davis, L. S., Bogue, D. T., Ramberg, J. and Lipsky, P. E. 1991. Differential utilization of ICAM-1 and VCAM-1 during the adhesion and transendothelial migration of human T lymphocytes. J. Immunol. 147:2913.[Abstract]
  58. Van Epps, D. E., Potter, J., Vachula, M., Smith, C. W. and Anderson, D. C. 1989. Suppression of human lymphocyte chemotaxis and transendothelial migration by anti-LFA-1 antibody. J. Immunol. 143:3207.[Abstract]
  59. Ding, Z., Xiong, K. and Issekutz, T. B. 2001. Chemokines stimulate human T lymphocyte transendothelial migration to utilize VLA-4 in addition to LFA-1. J. Leukoc. Biol. 69:458.[Abstract/Free Full Text]
  60. Ding, Z. M., Babensee, J. E., Simon, S. I. et al. 1999. Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration. J. Immunol. 163:5029.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea