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Tissue Specificity of E- and P-Selectin Ligands in Th1-Mediated Chronic Inflammation

Protein Design Laboratories, Inc., Fremont, CA 94555

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The demonstrated role of E- and P-selectin ligands in the recruitment of Th1 cells raises the question of tissue specificity determination by pathogenic T cells. We took advantage of the fact that chronic Th1-mediated inflammation in the scid/scid CD4⁺CD45RB^high T cell transfer model can occur at multiple tissue sites, resembling inflammatory bowel disease in the colon and psoriasis in the skin. We show that the majority of infiltrating effector T cells from psoriatic skin expresses high levels of functional P-selectin ligand (87 ± 3%), detected by P-selectin-Ig (PIg), while a significantly smaller subset of T cells from colitic lesions expresses this ligand (24 ± 2%). Similarly, E-selectin ligand is preferentially expressed on CD4⁺ T cells infiltrating the skin (24 ± 2%), but only on very few CD4⁺ T cells infiltrating the colon (CIT; 1.3 ± 0.8%). In contrast, CD4⁺ T cells infiltrating the skin express ₄ß₇ at a significantly lower level than CIT (mean fluorescence intensity, 28 vs 61, respectively), although, interestingly, _Eß₇ was expressed at high levels on both populations. Analysis of the disease-inducing potential of PIg⁺ and PIg^- CD4⁺ CIT cells revealed that both populations not only express similar levels of the gut-homing molecule ₄ß₇ (mean fluorescence intensity, 50 vs 56, respectively), but do not differ in their capacity to express IFN-. Furthermore, CIT depleted of cells expressing functional P-selectin ligand were able to induce colitis upon transfer, suggesting that induction of colitis in this model may be independent of E- and P-selectin. These results indicate that adhesion molecule expression and the homing pattern of inflammatory T cells are regulated by the local environment independently of their inflammatory capacity.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Recent data about adhesion molecules that direct the recruitment and localization of lymphocytes have raised the question to what degree alterations in patterns of adhesion molecule expression are associated with specific functional phenotypes or with particular autoimmune or inflammatory conditions. Recently, activated or memory T cells (e.g., CD45RO⁺ in the human or CD45RB^low in the mouse) that have acquired the ability to migrate through nonlymphoid tissues are quite heterogeneous in their homing potential. These cells differentially express L-selectin and ₄ß₇, homing receptors for peripheral and mucosal lymphoid organs, respectively (1), and _Eß₇, which is preferentially found on T cells that localize to the intraepithelial spaces of the intestinal mucosa. T cells that migrate to the skin express the cutaneous lymphocyte-associated Ag (CLA),³ a ligand for the vascular E-selectin (2, 3). Memory T cells are also quite functionally heterogeneous in their capacity to produce effector cytokines and in their capacity to extravasate into particular sites. Despite many studies examining the correlation of adhesion receptor expression with functional phenotype, the question of how the expression of adhesion molecules is regulated on inflammatory T cells in chronic autoimmune conditions is not well understood (4, 5, 6, 7).

In a recent study Austrup et al. (8) demonstrated in mice that either in vitro or in vivo differentiated Th1 cells, but not Th2 cells, preferentially express ligands for E- and P-selectin and migrate selectively to acutely inflamed tissues via E- and P-selectin-dependent interactions, suggesting an association of the Th1 phenotype with the expression of ligands for E- and P-selectin. These data contrast with findings in humans, in whom E-selectin ligand expression (i.e., CLA) is found on both Th1 and Th2 subsets (9, 10, 11). To investigate adhesion molecule expression on effector T cells in a more chronic inflammatory disease setting in the mouse, we used a novel experimental animal model in which inflammatory T cells at different tissue sites arise from a common naive donor T cell pool (12). This model takes advantage of the fact that scid/scid mice receiving naive T cells in conjunction with LPS plus rmIL-12 (p70) or staphylococcus endotoxin B can develop inflammatory disease at multiple organ sites, namely inflammatory bowel disease in the colon and psoriasis in the skin (12). Both diseases result from a polarized Th1 inflammatory response (this manuscript and Refs. 12 and 13), allowing analysis of the expression of adhesion molecules, including functional E- and P-selectin ligands, L-selectin, ₄ß₇, and _Eß₇, on functionally similar T cells from different local microenvironments. Our data suggest that inflammatory T cells from psoriatic skin (SIT) preferentially express ligands for E- and P-selectin, while inflammatory T cells from colitic tissue (CIT) do not. Instead, CIT cells preferentially express ₄ß₇, and upon transfer the E- and P-selectin ligand-negative CIT cells are, in fact, able to induce chronic colitis. These results provide evidence that induction of Th1-induced inflammatory bowel disease in mice may occur through selectin-independent mechanisms, suggesting that therapeutic intervention of inflammatory bowel disease may be better addressed by targeting integrin-dependent mechanisms.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Mice

Female BALB/c mice (donor mice) were purchased from The Jackson Laboratory (Bar Harbor, ME), and C.B-17/Icr scid/scid (recipient mice) were purchased from Taconic Farms (Germantown, NY). All mice were housed in a specific pathogen-free environment at the Protein Design Labs, animal facility and were used between 4–12 wk of age. Sentinel mice were used to screen for the following pathogens: mouse hepatitis virus, Sendai virus, pneumonia virus of mice, reovirus serotype 3, Theiler’s murine encephalomyelitis virus, Mycoplasma pulmonis, and parvovirus. Random screens of mice for pinworms were also conducted. None of the pathogens listed above was detected at any time. Mice were housed two to five per microisolator. All scid/scid mice were handled with gloves under a class II hood, fed sterile food and water ad libitum, and maintained inside a laminar flow tent (Bioclean Maywood, NJ) in sterile microisolators that were changed weekly. Donor mice were housed in conventional cages that were changed weekly.

Induction of psoriasiform lesions and colitis

The scid/scid CD4⁺CD45RB^high T cell transfer model employed here has been previously described (12). Briefly, splenocytes were collected from 6- to 12-wk-old donor mice (BALB/c). CD4⁺ T cells were selected by positive selection. The resulting CD4⁺-enriched population was >90% pure by flow cytometric analysis. These cells were then labeled with anti-CD4-FITC (9004D, PharMingen, San Diego, CA), and anti-CD45RB-PE (01145A, PharMingen; both at 10 µg/ml) and sorted using a FACStar (Becton Dickinson, San Jose, CA) cell sorter. Double-positive cells (CD4⁺CD45RB⁺) were collected, selecting the cells that expressed high levels of CD45RB (brightest 45%). The collected cell population was >95% pure and viable and was injected i.v. (tail vein) or s.c. (near base of tail) into C.B-17/Icr scid/scid mice, aged 4–6 wk (3 x 10⁵ cells each in 200 µL). An i.p. injection of 20 µg of LPS (Sigma L-2012) and 10 ng of IL-12 p70 (19361V, PharMingen) was given on day 1 following T cell transfer, as this resulted in an increased disease penetrance and severity of psoriasis (12).

Skin-infiltrating and colon-infiltrating lymphocyte cell isolation

In animals that developed both psoriasis and colitis, lymphocytes from the skin (SIL) and the colon (CIL) were isolated simultaneously from the same animals via enzyme digestion. In short, colons, pinnae, and eyelids were minced separately with sterile scissors, and the pieces were washed with HBSS over a 100-µm pore size nylon cell strainer (Falcon, Becton Dickinson, Franklin Lake, NJ) to remove surface debris. Infiltrating cells were extracted by incubating the cut pieces in 25 ml of warm (37°C) HBSS medium without Ca²⁺/Mg²⁺ (10-543F, BioWhittaker, Walkersville, MD) supplemented with 25 mM HEPES buffer, pH 7.0 (17-737E, BioWhittaker), and 10% FBS (SH30071.03, HyClone, Logan, UT) for 20 min at 37°C. The remaining pieces were washed over nylon mesh; resuspended in RPMI 1640 medium (12-702F, BioWhittaker) augmented with 25 mM HEPES buffer, 10% FBS, 400 U/ml DNase (104159, Boehringer Mannheim, Indianapolis, IN), and 400 U/ml collagenase (1088874, Boehringer Mannheim); and incubated for 90 min at 37°C on a rocker. The resultant cell suspension was filtered sequentially through 100- and 40-µm pore size nylon mesh filters and then washed twice in RPMI 1640 supplemented with 25 mM HEPES and 10% FBS.

In vitro stimulation of SIL and CIL and detection of cytokines

SIL and CIL were separately resuspended at 10⁶/ml in complete RPMI 1640 medium supplemented with 10% FBS and 5 x 10^-5 2-ME, 2 mM L-glutamine (Life Technologies, Gaithersburg, MD), 10 U/ml penicillin, 100 µg/ml streptomycin (Life Technologies), and 10 mM HEPES. A total of 200 µl of these suspensions was then placed in 96-well tissue culture plates (3072, Falcon) and incubated for 48 h with anti-CD3 (clone 145-2C11, provided by J. Tso, Protein Design Laboratories) and anti-CD28 (PharMingen), each at 1 µg/ml. Supernatants from three separate culture wells were collected and tested by ELISA for IFN-, TNF-, IL-4, and IL-10 (all capture and detection Abs were purchased from PharMingen). The ELISA procedure involved coating a 96-well flat-bottom Immulon 4 plate (011-010-3850, Dynatech, Chantilly, VA) overnight at 4°C with 50 µl of a 2 µg/ml solution of capture Ab in carbonate buffer (pH 9.0). Plates were then washed with PBS with 0.05% Tween-20 and blocked with 200 µl of a sterile solution of PBS with 3% BSA (A7030, Sigma, St. Louis, MO) for 1 h at 37°C. In between all of the following steps, plates were washed with PBS/Tween. IFN-, IL-4, TNF-, and IL-10 standards (PharMingen) as well as sample supernatants were added to wells and incubated for 2 h at 37°C. Biotin-conjugated secondary Abs for IFN-, IL-4, TNF-, and IL-10 (PharMingen) were then added to the respective plates at 2 µg/ml in 3% BSA/PBS solution and incubated for 1 h at 37°C. HRP-labeled streptavidin (016-030-084, Jackson ImmunoResearch Laboratories, West Grove, PA) was then added at a concentration of 1 µg/ml and incubated for 25 min. O-Phenylenediamine (4664, Sigma) was used as substrate buffer according to the manufacturer’s protocol. Assay results were read on a Molecular Devices (Sunnyvale, CA) plate reader, and data was analyzed using SOFTmax software.

Cell surface FACS staining and analysis

One million viable SIL or CIL cells in 100 µl were treated with 0.5 µg/ml Fc block (anti-mouse CD32 (FcIII/II receptor), PharMingen, 01241A, clone 2.4 G2) to reduce nonspecific Fc receptor-dependent staining, then stained for 20 min with 0.5 µg of one or more of the following FITC- or PE-conjugated mAbs: anti-mouse L-selectin (PharMingen, 01265B), anti-mouse CD4 (PharMingen, L3T4), anti-mouse CD45RB (PharMingen, 16A), anti-mouse CD103 (integrin IEL chain, PharMingen, M290), anti-mouse LPAM-1 (integrin ₄ß₇ complex; PharMingen, DATK32), HECA-452 (anti-CLA Ab, FITC-conjugated HECA-452, kindly provided by Dr. Eugene C. Butcher, Stanford University, Palo Alto, CA), or 0.6 µg/ml purified mouse P-selectin-IgG fusion protein (human IgG1, PharMingen). The ability of HECA-452 to detect E-selectin ligands on CD4⁺ T cells was confirmed by the similarity of our results with those of Thoma et al. (21), who employed E-selectin-Ig and P-selectin-Ig chimeras to stain T cells in the scid/scid CD4⁺CD45RB^high scid/scid transfer model of colitis. After washing, secondary detection Abs, either FITC- or PE-conjugated anti-human Ig and light chains (Tago, Burlingame, CA; no. 4903 and 4200) were added in the presence of 10% normal mouse serum for an additional 20 min, then cells were fixed with 1% paraformaldehyde in PBS and analyzed according to standard procedures on a Becton Dickinson FACScan flow cytometer using CellQuest software (Becton Dickinson, San Jose CA). In some experiments, isolated CIL were stained with anti-CD4 and P-selectin-IgG and sorted on a FACStar cell sorter (Becton Dickinson). CD4⁺ cells gated for P-selectin-IgG-positive (top 20%) or P-selectin-IgG-negative were collected separately.

Intracellular staining

Intracellular staining for cytokines was performed on SIL and CIL according to the protocol of PharMingen with the following modifications. SIL and CIL cells were incubated at 10⁶/ml in complete medium overnight with 1 µg/ml each of anti-mouse CD3 and anti-mouse CD28 (PharMingen). The cells were then incubated for 4 h with 0.66 µl/ml GolgiStop (PharMingen). Cells were harvested, washed, treated with 1 µg/ml Fc Block (PharMingen), and stained for 30 min with 0.5 µg of anti-CD4 FL (PharMingen) for two-color analysis. In the case of three-color analysis, cells were stained with anti-CD4 APC (PharMingen) and 0.6 µg of purified mouse P-selectin-IgG fusion protein (human IgG1, PharMingen). PE-conjugated anti-human Ig and light (Tago 4903) was used to detect the P-selectin-IgG fusion protein. Cells were fixed and permeabilized with 250 µl of Cytofix/Cytoperm solution (PharMingen) for 20 min and washed twice with Perm/Wash buffer (PharMingen). For two-color analysis, cells were stained with anti-mouse IFN--PE (XMG 1.2, PharMingen) or with anti-mouse IL-4-PE (11B11, PharMingen) or rat IgG1-PE isotype control (R3-34, PharMingen). For three-color analysis, cells were stained for 30 min with anti-mouse IFN--FITC (XMG 1.2, PharMingen), anti-mouse IL-4-FITC (11B11, PharMingen), or rat IgG1-FITC isotype control (R3-34, PharMingen).

Histopathologic analysis and evaluation of disease incidence

Necropsies were performed on mice 16 wk after cell transfer. Tissue samples from pinnae, eyelid, tail, and colon were collected and fixed in paraformaldehyde solution and submitted to Comparative Bioscience (Sunnyvale, CA) for section preparation. To record disease severity, semiquantitative histological scores from 0–4 were given based on the severity of inflammation from three different sections of the tissue. The histological scores were then averaged from all three sections of one particular tissue, and the scores were reported as the average of all mice examined. Histologic evaluation was blindly conducted by three independent investigators. Histological scoring for the skin: 0 = no signs of inflammation; 1 = low focal areas of infiltration; 2 = low level of mononuclear cell infiltration, mild thickening of epidermis, mild to moderate acanthosis; 3 = high level of mononuclear cell infiltration, high vascular density, thickening of the epidermis, acanthosis, (rete pegs and hyperplasia of epidermis and keratinocytes), microabscesses, thinning of the granular cell layer; and 4 = very extensive infiltration in epidermis and dermis, very high vascular density, extreme thickening of epidermis, pustule formation, and destruction of granular cell layers. Histological scoring for the colon was: 0 = no signs of inflammation; 1 = low focal areas of infiltration; 2 = low level of mononuclear cell infiltration, mild thickening of the colon wall; 3 = high level of mononuclear cell infiltration, high vascular density, thickening of the colon wall; and 4 = very extensive transmural infiltration, loss of goblet cells with extreme thickening of the colon wall and high vascular density. Mice that had ear thickness of 25 mm or more and/or clinical signs of disease were considered diseased. Similarly, mice that showed signs of loose stool over an extended period of time (>2 wk) and/or showed signs of weight loss of >10% were considered to have colitis.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
SIT or CIT isolated from chronic lesions are CD45RB^low, L-selectin^low, and produce similar Th1-like cytokine profiles

Naive minor haplotype mismatched CD4⁺/CD45RB^high cells isolated from BALB/c mice transferred into naive scid/scid mice induce chronic mucosal inflammation that resembles Crohn’s disease in humans (13). We and others have recently demonstrated that the transfer of minor haplotype-mismatch naive T cells results in the development of psoriasiform lesions, especially in the presence of microbial products (12, 14). Because some animals develop a dual Th1-driven inflammatory condition (12, 14) in both the colon and the skin, this model provides an ideal system to compare the expression of adhesion molecules on functionally similar CD4⁺ T cells from different inflammatory sites derived from the same naive donor T cell pool and activated on the same haplotype-mismatched background. To establish that inflammatory T cells from both inflammatory sites, the colon and the skin, exhibit similar states of activation after they migrate to their respective tissue, T cells were isolated from both sites after disease had become established (6–10 wk) and analyzed for CD45RB and L-selectin expression together with their cytokine expression profile. Both SIT and CIT cells when analyzed by FACS showed a significant shift from CD45RB^high to CD45RB^low compared with naive donor cells before transfer (Fig. 1). The mean fluorescence intensities of CD45RB for SIT and CIT was more than 1 log lower than naive T cells (Fig. 1, bold lines), and both SIT and CIT showed a marked decrease in the percentage of CD45RB^high cells (3 ± 2% in SIT and 1 ± 1% in CIT cells) compared with the naive CD4⁺ donor cells (97–99% CD45RB^high; Table II). Moreover, both SIT cells and CIT cells expressed very low levels of L-selectin (Fig. 1) and were shifted significantly lower compared with the high expression of L-selectin on normal BALB/c splenocytes (Fig. 1, solid lines).

View larger version (27K): [in this window] [in a new window]   FIGURE 1. CD4+ CIT and SIT exhibit similar levels of activation. Gated CD4+ T cells from normal BALB/c spleen, CIT, or SIT were evaluated for expression of CD45RB or L-selectin by flow cytometry as described in Materials and Methods. Histograms show the staining profile of CIT or SIT (bold lines) and normal spleen (solid lines). Histogram profiles of CIT or SIT stained with isotype control Abs are shown by dotted lines. The markers shown delineate the CD4+CD45RBhigh donor T cell population from BALB/c spleen that is initially transferred to scid/scid mice. Similar results were obtained in at least 10 separate experiments.

SIT preferentially express ligands for E- and P-selectin, while CIT preferentially express ₄ß₇; _Eß₇ is up-regulated on both SIT and CIT

The similar state of activation of SIT and CIT cells, but localization in different tissues allowed us to address whether adhesion molecules associated with cutaneous or mucosal sites are differentially expressed on these inflammatory T cells. In humans, expression of E-selectin ligand activity on T cells is preferentially associated with cutaneous localization. E-selectin ligand activity on T cells in humans is known as the cutaneous lymphocyte Ag, CLA, and is detected with mAb HECA-452 (3). CLA is comprised of sialyl Lewis-x-like carbohydrates modifying PSGL-1, among other cell surface proteins (15). In human peripheral blood, CLA⁺ T cells comprise a small subset of activated/memory cells, but are highly enriched in cutaneous sites of chronic inflammation where E-selectin is also highly expressed on venular endothelium (2). P-selectin ligand activity is also found on a subset of memory/activated T cells in human blood (16, 17). Although several studies in mice and humans have characterized the expression of E- and P-selectin ligand activities on activated T cells in vitro as partially overlapping, the relationship between E- and P-selectin ligand expression on T cells in vivo has not been carefully examined. Furthermore, although E- and P-selectin ligand activities are preferentially found on in vitro derived Th1 but not Th2 cells in the mouse, and the Th1-inducing cytokine IL-12 induces expression of E- and P-selectin ligand activities on T cells in vitro in mouse and man (18, 19, 20), both Th1 and Th2 cytokines are produced by CLA⁺ T cells in vivo in humans (9). To test the role of tissue specificity of adhesion molecules on chronic inflammatory Th1 cells in the mouse, SIT and CIT were tested for their expression of E- and P-selectin ligand activities and compared with normal splenic CD4⁺ T cells from BALB/c mice (Fig. 2, A and B).

View larger version (21K): [in this window] [in a new window]   FIGURE 2. A and B, Expression of E- and P-selectin and 4ß7 on chronic inflammatory Th1 cells depends on the tissue of origin and differs from the T cell donor profiles. CD4+CD45RBhigh BALB/c donor mice (A), CD4+ CIT and CD4+ SIT (B) were evaluated for the expression of E-selectin, P-selectin ligand, 4ß7, and Eß7 by flow cytometry as described in Materials and Methods. Fluorescence histograms show staining of CIT or SIT (bold lines), and CD4+CD45RBhigh gated splenocytes (solid lines) for the indicated receptor. The dotted lines show staining of CIT or SIT with an isotype control Ab. Percentages shown indicate the fraction of gated CD4+CD45RBhigh BALB/c cells positive for the respective cell surface protein. The x-axis shows the fluorescence intensity, and the y-axis indicates the cell number.

The significant difference in E- and P-selectin ligand levels in SIT cells compared with CIT cells suggests that there are tissue-specific differences in the expression of E- and P-selectin ligands on chronic inflammatory T cells in mice. In recent studies, Thoma et al. (21) have also shown that while only 19% of P-selectin-ligand expressing T cells isolated from the inflamed lamina propria secrete IFN-, while the majority of P-selectin ligand-expressing T cells isolated from the peritoneal cavity of mice with colitis secrete IFN-. These results do not rule out a role for E- and/or P-selectin in initial recruitment of cells into the colon; however, they do indicate that cutaneous, but not mucosal, environments support high expression of both E- and P-selectin ligands. These results also raise the question of how the differential expression of E- and P-selectin ligands is regulated on T cells in different tissues under similar inflammatory conditions. The expression of E- and P-selectin ligands on inflammatory T cells can be regulated at the time of their initial encounter with Ag within secondary lymphoid organs, as has been suggested recently (9). However, given that expression of selectin ligands on long term activated T cells in culture is highly dynamic, it is also possible that further modulation of selectin ligand expression occurs once cells have accumulated at the inflammatory site. In humans, for example, E-selectin ligand expression is induced on cultured T cells in the presence of either IL-12 or TGF-ß (18, 22). Indeed, IL-12 has been shown to promote T cell adhesion to endothelial selectins through the induction of FucT-VII mRNA, a gene important for the formation of selectin ligands (19). However, high levels of IL-12 are found in both cutaneous and mucosal sites in the chronic inflammatory model employed here (12, 13), suggesting that additional factors, present in the mucosa and absent in skin, may counteract the ability of IL-12 to regulate the expression of E- and P-selectin ligands. Alternatively, perhaps in vivo, IL-12 alone may not be sufficient to maintain the expression of these ligands, and the additional factors that are required for maintaining expression are present in the skin, but are missing in the colon. Furthermore, these studies suggest that the high levels of active TGF-ß in the colon mucosa (23) may also not be sufficient for the in vivo expression of E- and P-selectin ligands. In summary, although both diseases are supported by a Th1 environment, other factors must play a role in these tissues to account for the differential expression of selectin ligands.

Expression of ₄ß₇ and _Eß₇ integrin was also examined on SIT and CIT cells. ₄ß₇ is the homing receptor for lymphocyte trafficking into Peyer’s patches and gut lamina propria. Its ligand, MAdCAM-1, is selectively expressed on postcapillary venules in these tissues and through interactions with ₄ß₇ mediates lymphocyte trafficking to normal and inflamed gastrointestinal mucosa (24, 25, 26, 27, 28, 29, 30, 31, 32). Abs either directed against ß₇ or ₄ have been demonstrated to attenuate established colitis in cotton-top tamarin monkeys and in mice (33, 34, 35). As shown in Fig. 2B and Table II, high levels of ₄ß₇ were preferentially expressed on CIT cells, as nearly all CIT cells express ₄ß₇ (MFI, 60), whereas ₄ß₇ was expressed only on 26% of SIT cells (MFI, 27). Interestingly, while most SIT do not express ₄ß₇, the donor cells initially transferred to recipient all express ₄ß₇, albeit at a lower level than CIT cells (MFI, 19; Fig. 2A).

Similar observations regarding low expression of ₄ß₇ in cutaneous inflammatory sites have been observed in humans. For instance, Picker et al. (36) found that the percentage of ₄ß₇ integrin-expressing memory/effector T cells was diminished in skin compared with that in peripheral blood. In summary, the down-regulation of ₄ß₇ in SIT and up-regulation in CIT is further evidence of the tissue-specific differences between mucosal and cutaneous environments.

_Eß₇, like ₄ß₇, is also associated with mucosal tissues, as it is commonly known as the integrin that is highly expressed on intraepithelial lymphocytes (IELs) (37, 38). However, unlike ₄ß₇, very few _Eß₇-expressing cells are found outside the gut. A small subset of activated/memory T cells in the peripheral blood in humans express _Eß₇, and in the mouse, very few splenic or lymph node T cells express _Eß₇. It has been postulated that _Eß₇ may serve as a retention molecule for the intraepithelial site of the gut mucosa as it binds to E-cadherin expressed on epithelial cells (37, 39, 40). Surprisingly, both SIT and CIT were found to express high levels of _Eß₇ (59 and 56%, respectively), despite the fact that most SIT and CIT do not reside in between epithelial cells but are either within the dermis or the lamina propria. This data suggest that a broader role for _Eß₇ in various chronic inflammatory diseases should be addressed and that perhaps _Eß₇ may serve as an important "velcro" molecule, ensuring the retention of potentially pathogenic T cells at the inflamed tissue site. The finding of _Eß₇ up-regulation on inflammatory T cells in different tissues is similar to findings in human patients, where increased expression of _Eß₇ has been found in inflammatory T cells from patients with inflammatory bowel disease and rheumatoid arthritis (41, 42, 43). In addition, Ludviksson et al. (44) have recently shown increased expression of _Eß₇ on T cells isolated from the lamina propria of IL-2^-/- mice with colitis. Interestingly, although a significant number of lamina propria cells and T cells in nonmucosal inflammatory sites express _Eß₇, no other ligand for _Eß₇ has yet been identified. In both mice and humans, in vitro T cell stimulation in the presence of TGF-ß results in the up-regulation of _Eß₇ (29, 45, 46, 47), suggesting that TGF-ß might play an important role in the expression of this integrin at inflammatory sites.

Both P-selectin ligand-positive and -negative CD4⁺ CIT cells belong to the Th1 phenotype

Although very few T cells in the colons of mice suffering from colitis in our model were found to express P-selectin ligand, it was possible that this minority T cell population is responsible for the induction of colitis. To determine whether the P-selectin ligand-positive (P-selectin-IgG⁺) minority population or the P-selectin ligand-negative (P-selectin-IgG^-) majority population in CIT represents the functional T cell effector pool in colitis, we first determined whether there is a correlation between P-selectin ligand expression and cytokine production in the colon.

T cells from the inflamed lesions of the colon were isolated, stained for CD4 and P-selectin-IgG, and sorted into P-selectin-IgG-positive and -negative populations. Both populations were cultured overnight in the presence of anti-CD3 and anti-CD28, and supernatants were tested by ELISA for IFN-, TNF-, IL-4, and IL-10. Freshly sorted cells were also permeabilized for intracellular cytokine staining to evaluate IFN- protein expression by individual cells. As shown in Table III, the cytokine data indicate a Th1 phenotypical bias for both P-selectin-IgG-positive and -negative cells, because we detected significantly higher levels of IFN- and TNF- in both cell populations compared with IL-10 and IL-4. The slightly higher amounts of IFN- and TNF- found in supernatants from P-selectin-IgG-positive cells and the slightly higher amount of IL-4 found in supernatants from P-selectin-IgG-negative cells are not significantly different and do not reflect significant functional differences. Indeed, the data from intracellular cytokine staining confirms that P-selectin ligand-high (MFI, 24.2), P-selectin ligand-medium (MFI, 22.4), and P-selectin ligand-low (MFI, 26.1) CD4⁺ T cells express similar amounts of IFN- (Fig. 3). Thus, P-selectin ligand expression in the colon does not seem to correlate with a particular Th phenotype. Although this model is Th1 driven, as it is crucially dependent on IL-12 and partially dependent on IFN- (12), the role of IL-4 remains to be addressed. In humans, psoriatic T cells as well as colitic T cells have often been shown to produce both IFN- and IL-4 (48, 49).

View larger version (31K): [in this window] [in a new window]   FIGURE 3. CIT cells with surface expression of high, medium, and low amounts of P-selectin ligand express similar amounts of intracellular IFN-. CIT cells were stained intracellularly for IFN- and IL-4 as described in Materials and Methods. A PE-conjugated rat IgG isotype control Ab was used to detect nonspecific staining. Cells were also stained with anti-mouse CD4 and P-selectin-IgG. CD4+ T cells were divided into high, medium, and low P-selectin expression populations via FACS analysis. The percentages of these populations expressing IFN- are depicted in histograms as well as the MFIs. Histograms of CD4+-gated, P-selectin-IgGhigh/med/low IFN-+ cells (bold line) are shown compared with the isotype control (dotted line). Staining of cells with P-selectin-Ig and anti-CD4 was unaffected by the permeabilization treatment (data not shown).

P-selectin ligand-negative CD4⁺ CIT express ₄ß₇ and are able to induce colitis upon transfer into naive scid/scid recipients

To directly test whether E- and P-selectin-ligand-negative CD4⁺ CIT are able to induce chronic colitis, we first compared CD4⁺ P-selectin-IgG-positive and CD4⁺ P-selectin-IgG-negative cells for ₄ß₇, a marker that has been shown to be important for the homing of inflammatory Th1 cells into mucosal sites (24, 33, 34, 35). As shown in Fig. 4, there was no significant difference in the expression of ₄ß₇ between P-selectin-IgG-negative CIT and P-selectin-IgG-positive CIT (MFI, 50 and 56, respectively). To further evaluate the disease-inducing potential of P-selectin-IgG-negative CIT, we transferred 2 x 10⁵ P-selectin-IgG-negative CIT to naive scid/scid mice and evaluated disease development 16 wk after T cell transfer to ensure full disease development. (Due to insufficient numbers, P-selectin-IgG-positive CITs could not be tested in transfer studies.) As shown in Table IV, all mice that received P-selectin-IgG-negative CIT developed colitis, although, interestingly, none of these mice showed signs of psoriasis, as determined by clinical observation, measurement of ear thickness (>25 µm), and histology. Although the outcomes of these transfer experiments are surprising, they are not totally unexpected, because one other group has recently demonstrated that only LN CD4⁺ T cells that express functional E- and P-selectin ligand, but not E- and P-selectin-negative LN CD4⁺ T cells, home to acutely inflamed skin (50). Furthermore, studies in P-selectin-deficient mice showed a significant reduction of CD4⁺ T cells into the skin in oxazolone-induced delayed-type contact hypersensitivity (51). Overall, these data indicate that E- and P-selectin ligand expression on inflammatory CD4⁺ T cells may be required for skin homing, but may not be required for the induction of mucosal inflammation; however, additional studies with blocking Abs to E- and P-selectin or with E- and P-selectin ligand-deficient mice are required to further investigate the role of E- and P-selectin in psoriasis and colitis.

View larger version (29K): [in this window] [in a new window]   FIGURE 4. 4ß7 integrin is expressed uniformly on both P-selectin ligand-positive and P-selectin ligand-negative CD4+ CIT cells. Freshly isolated CIT cells were stained with anti-mouse CD4 APC-conjugated Ab, anti-mouse 4ß7 FITC-conjugated Ab, and unconjugated mouse PIg. The P-selectin-IgG was detected by a PE-labeled anti-human IgG secondary Ab as described in Materials and Methods. Fluorescence histograms representing the 4ß7 staining of gated CD4+ P-selectin-Ig-positive or CD4+ P-selectin-Ig-negative cells are shown. The MFI of cells staining for 4ß7 are shown. The x-axis represents fluorescence intensity for 4ß7, and the y-axis indicates cell number.

	Acknowledgments

 
We thank Patricia Lekas for performing cell sorting.

	Footnotes

 
¹ A.C. and K.H. contributed equally to this manuscript.

² Address correspondence and reprint requests to Dr. Rolf O. Ehrhardt, PDL, Inc., 34801 Campus Drive, Fremont, CA 94555. E-mail address:

³ Abbreviations used in this paper: CIL, colon infiltrating lymphocytes; CIT, colon infiltrating T cells, SIT, skin-infiltrating T cells; SIL, skin infiltrating lymphocytes; CLA, cutaneous lymphocyte-associated Ag; IEL, intraepithelial lymphocytes; LN, lymph node; MFI, mean fluorescence intensity; PIg, P-selectin-Ig.

Received for publication April 8, 1999. Accepted for publication August 24, 1999.

	References

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