Regional differences in L -selectin expression in murine intestinal lymphocytes

GASTROENTEROLOGY 1998;114:965–974

Regional Differences in L-Selectin Expression in Murine Intestinal Lymphocytes FRANK SEIBOLD,* BEATRICE SEIBOLD–SCHMID,‡ YINGZI CONG,* FENG YU SHU,§ ROBERT P. MCCABE,* CASEY WEAVER,‡ and CHARLES O. ELSON* *Division of Gastroenterology and Hepatology, Department of Medicine, and Departments of §Microbiology and ‡Pathology, University of Alabama at Birmingham, Birmingham, Alabama

Background & Aims: The expression of the lymphocyte homing receptor and activation marker L-selectin is different in colon and small intestinal intraepithelial lymphocytes (IELs). In this study, the mechanism of this difference in L-selectin expression was investigated. Methods: L-selectin expression on lymphocytes was measured by flow cytometry. L-selectin messenger RNA (mRNA) was detected by reverse-transcription polymerase chain reaction. L-Selectin expression on peripheral lymphocytes was analyzed after incubation with cytokines, food and bacterial antigens, and homogenates of small and large bowel. Results: L-selectin was expressed by none of the small intestinal IELs but by 30% of those in the colon and by 60% of splenocytes. mRNA for L-selectin was detectable in isolated lymphocytes of all three sites. L-Selectin was downregulated in colon IELs during colitis and up-regulated in small intestinal IELs after in vitro culture for 48 hours. Incubation of splenocytes with small intestinal homogenates led to a rapid down-regulation of Lselectin (1% vs. 60% untreated). Preincubation with a metalloproteinase inhibitor prevented L-selectin loss. Conclusions: The mechanism of the differential expression of L-selectin in mouse small intestine and colon appears to be an increased functional activity of a metalloproteinase (sheddase) in the small intestine compared with the colon.

ntestinal lymphocytes have distinctive phenotypes in comparison to peripheral lymph node or splenic lymphocytes. Most studies in mucosal immunology have focused on small intestinal lymphocytes, but recent studies indicate that colon lymphocytes are different from their counterparts in the small intestine. For example, intraepithelial lymphocytes (IELs) of the murine colon are mostly CD41 cells, whereas IELs of the small intestine are predominantly CD81 cells.1 These differences may be related to the luminal environment of the bowel at these anatomic sites, which are distinct: small intestinal lymphocytes are mainly exposed to various food antigens, whereas colonic lymphocytes encounter anti-

I

gens of the bacterial flora. Further differences between lymphocytes of the colon and small intestine relate to their development, which is thymus independent in a fraction of small intestinal lymphocytes. In preliminary studies into the expression of different activation markers on lymphocytes of different regions of the intestine, we found that small intestinal IELs and lamina propria lymphocytes (LPLs) appear more activated than their colonic counterparts. One of the most striking differences found was the absence of L-selectin2 in the small intestine but not the colon. L-Selectin is a homing receptor that is involved in the trafficking of lymphocytes.3 It is also considered to be an activation marker because naive T cells shed L-selectin on antigen exposure and subsequent activation. L-Selectin, previously called Leu 8 in humans or Mel 14 in mice,4 is rapidly shed after stimulation of cells with calcium ionophores but is reexpressed after 24 hours.5 The mode of stimulation is important in that the mitogens anti-CD3 and concanavalin A have only minor effects on L-selectin expression.5 Because memory CD41 T cells are negative for Lselectin,6,7 the virtual absence of L-selectin in the small intestine but presence in the colon suggest that there is a higher proportion of memory cells or more activation in the small intestine compared with the colon. Thus, the present studies focused particularly on L-selectin expression to gain a better understanding of the mechanism of these regional differences among mucosal lymphocytes.

Materials and Methods Mice C3H/HeJ, C3H/HeJBir, BALB/c, and C57Bl/6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME). C3H/HeSnJ scid/scid were bred in our facility and maintained Abbreviations used in this paper: IEL, intraepithelial lymphocyte; IFN, interferon; IL, interleukin; LPL, lamina propria lymphocyte; MLN, mesenteric lymph node; PCR, polymerase chain reaction; PMA, phorbol myristate acetate; TCR, T-cell receptor. r 1998 by the American Gastroenterological Association 0016-5085/98/$3.00

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in horizontal laminar flow cabinets. Mice were used between 8 and 12 weeks of age.

Cell Isolations IELs were isolated as described previously.1 Briefly, small and large intestines were removed from groups of 5 mice and placed in separate Petri dishes containing chilled Hank’s balanced salt solution (Cell Growth Mediatech, Herndon, VA) medium. Peyer’s patches were removed from the small intestine, and lymphoid follicles were removed from the colon. Then each segment of the intestine was opened longitudinally, washed several times in Hank’s balanced salt solution containing 100 U/mL penicillin, 100 µg/mL streptomycin, and 25 mg/mL gentamycin. Finally, the intestine was cut into 1-cm segments that were transferred in 125-mL flasks containing 25 mL RPMI 1640 supplemented with 50 µmol/L with 2mercaptoethanol, 2 mmol/L sodium pyruvate, 2 mmol/L L-glutamine, 25 mg/L gentamicin, 100,000 U/L penicillin, 100 mg/L streptomycin, 25 mmol/L HEPES buffer, and 2% fetal calf serum. The flasks were incubated for 30 minutes at 37°C with gentle stirring. The intestinal tissue was transferred to a 50-mL centrifuge tube (Falcon; Becton Dickinson Labware, Lincoln Park, NJ) and was shaken vigorously for 30 seconds. Then the contents were filtered through a stainless steel sieve. The tissue fragments were returned to the 125-mL flasks, and the process was repeated three times. IELs from four incubations were pooled, washed, and passed through a loosely packed glass wool column. The column-passed cells were purified further by centrifugation over a discontinuous 40%/ 75% Percoll (Sigma Chemical Co., St. Louis, MO) gradient, recovering the cells at the interface. LPLs were recovered by incubating the residual intestinal tissue in RPMI medium supplemented with collagenase VIII (20 mg/100 mL medium; Sigma) for four 30-minute intervals. The cells were purified in a 40%/75% Percoll gradient. The viability of IELs and LPLs was .95%. Peyer’s patch cells and cecal follicle cells were also recovered by collagenase digestion. Lymphocytes from spleen, mesenteric lymph node, and caudal lymph node were teased apart, washed, and used in experiments. Splenic red cells were lysed using ammonium chloride lysing buffer (0.15 mol/L Tris-NH4Cl).

Analysis of Lymphocyte Phenotype by Flow Cytometry Cells were washed in phosphate-buffered saline containing 0.1% sodium azide and 2% fetal calf serum and were incubated for 30 minutes with either fluorescein-, phycoerythrin-, or biotin-derivatized antibodies, as listed below, for 30 minutes. After washing, streptavidin-tandem was used as secondary antibody for the biotin-derivatized antibody. After further washing, the cells were fixed in 1% paraformaldehyde in phosphate-buffered saline. Antibodies anti-CD2 (RM2-5), anti-CD8 (53-6.7), anti–L-selectin (Mel 14), anti-CD44 (IM7), anti–CD45 RB (23G2), anti-CD69 (H1.2F3), anti–b7integrin (M293), and anti–a4b7-integrin (LPAM1 complex)

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were from Pharmingen (San Diego, CA). Antibodies anti-CD3 (F 500), anti-CD4 (RM4–5), and anti-Thy1 (53-2.1) were from the University of Alabama at Birmingham Multipurpose Arthritis Center Core Facility.

Measurement of Messenger RNA by Reverse-Transcription Polymerase Chain Reaction RNA was extracted from small intestinal IELs that had been freshly isolated, incubated in medium only, or incubated with 500 U/mL interferon (IFN)-a. RNA was isolated from small intestinal IELs, colon IELs, spleen cells, liver, and the murine epithelial cell line Mode K; the latter two were used as negative controls. RNA extraction was performed according to the RNeasy protocol (Qiagen, Inc., Chatsworth, CA). Reverse transcription was performed by mixing 1 µg RNA, 1 µL oligo(dT) (Sigma), and 3.4 µL H2O and heating for 10 minutes at 70°C. Then 2 µL of 0.1 mol/L dithiothreitol (GIBCO BRL, Gaithersburg, MD), 1 µL deoxynucleoside triphosphates (10 mmol/L; Pharmacia, Uppsala, Sweden), 1 µL RNAsin (Promega, Madison, WI), and 4.1 µL of 53 first-strand buffer (GIBCO BRL) were added and incubated for 2 minutes at 42C. Finally, 1 µL of MMLV reverse-transcriptase (GIBCO BRL) was added, and the tube was incubated for 60 minutes at 37°C. The reaction was heated to 70°C to inactivate the reversetranscriptase. The primers for L-selectin were as follows: 58-AGAGAGACTTGCAGAGAGAC-38 and 58-CCTGCATCACAGATGLACGT38. A reaction tube contained 5 µL of 103 polymerase chain reaction (PCR) buffer (Perkin-Elmer Corp., Norwalk, CT), 3 mmol/L MgCl2, 1 µL of 10 mmol/L deoxynucleoside triphosphate (Pharmacia), 1 µL of each primer, 0.4 µL of AmpliTaq DNA polymerase (5 U/µL; Perkin-Elmer), and 2 µL of the complementary DNA (cDNA) sample. Samples were heated to 95°C for 2 minutes and then cycled at 94°C for 45 seconds, 60°C for 2 minutes, and 72°C for 3 minutes for 35 cycles. PCR amplification with b-actin primers (Clonetech Laboratories, Palo Alto, CA) was performed to assess variations in total RNA or cDNA loading between samples. Detection of PCR products was performed by electrophoresis in a 1% agarose gel and ethidium bromide staining.

Lymphocyte Cultures Lymphocyte stimulation with antigens. Lymphocytes from spleen, mesenteric lymph node (MLN), or intestine were stimulated in vitro for 0.5, 2, or 24 hours with a variety of antigens and mitogens followed by flow-cytometric analysis for L-selectin expression. Soy protein was used in a concentration of 50 mg/mL as a food antigen because soy is a major component of the mouse diet. Bacterial antigens were obtained by isolation of cecal contents that were mixed with deoxyribonuclease (100 µg/mL) and MgCl2 (100 mmol/L). One milliliter of this suspension was added to 1 mL of glass beads. The cells were disrupted at 5000 rpm for 3 minutes in a Mini-Bead Beater

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Results

apparatus (BioSpec Products, Bartlesville, OK). The supernatant was filtered through a 0.45-µm Millipore filter (Millipore Corp., Bedford, MA) before being used in cultures at a concentration of 200 µg/mL. Phorbol myristate acetate (PMA) was used at a concentration of 0.1 µmol/L. The metalloproteinase inhibitor KD-IX-73-5, N-R-(2-(hydroxyaminocarbonyl)methyl)-4-methylpentanoyl-Lalanine 2-aminoethyl amide (Peptides International, Louisville, KY) was added at a concentration of 50 µL/mL. Supernatant of the hybridoma cell line 145 2C11 (anti-CD3) was used at a concentration of 10% to stimulate T cells. Lipopolysaccharide from Escherichia coli 055285 (Sigma) was used at 20 µg/mL. Incubation of lymphocytes with cytokines. Lymphocytes from spleen or MLN were incubated for 0.5, 2, or 24 hours with cytokines, and then L-selectin expression was measured by flow-cytometric analysis. The following concentrations of cytokines or cytokine inhibitors were used: 0.1, 0.01, and 0.001 ng/mL tumor necrosis factor a; 500 U/mL IFN-a; 100 and 500 U/mL IFN-g; 0.25, 0.025, and 0.0025 µg/mL interleukin (IL)-10; 5 ng/mL transforming growth factor b; 0.1 µg/mL IL-4; 10 µg/mL anti–IL-4; and 10 µg/mL anti–IL-10. Lymphocyte-epithelial cocultures. Syngeneic spleen or MLN lymphocytes were cocultured with the following epithelial cell lines: Mode K cells (a gift of Dr. D. Kaiserlian, Lyon, France) and mouse colon adenocarcinoma line MCA38 (from Dr. B. Barna, Cleveland Clinic, Cleveland, Ohio). To test the influence of possible factors from epithelial cells, supernatants from the following cell lines were used: HT29 (ATCC), DLD 1 (ATCC), MCA38, Caco2 (ATCC), and Mode K. Furthermore, cell lysates (prepared by glass bead shaking, see above) of the above-mentioned cell lines and of normal small intestine, colon, liver, and spleen were used at a concentration of 0.5 mg/mL. Cocultures were performed with and without antigen-presenting cells present. Splenic adherent cells were used as antigen-presenting cells. Statistical methods. Data were converted to means and SD. The significance of differences between means was tested using Fisher’s Exact Test or Student’s t test.

L-Selectin

Expression of Lymphocytes

L-Selectin was virtually undetectable in small intestinal IELs, whereas 30% of CD31 colonic IELs expressed L-selectin (P , 0.001). Similarly, in the lamina propria (LPL) fraction, L-selectin was expressed by 10% of the CD31 small intestinal lymphocytes vs. 30% of the colonic LPLs (P , 0.01). Cecal follicle lymphocytes expressed L-selectin in 25% of the CD31 cells compared with 13% of CD31 small intestinal Peyer’s patch lymphocytes (P , 0.05) (Table 1). When L-selectin expression was determined in lymphocytes from the proximal vs. distal small intestine, a significant difference was found: lymphocytes of the most proximal 5 cm of small intestine expressed no L-selectin, whereas lymphocytes of the most distal 5 cm of small intestine expressed L-selectin in 25% of the CD31 cells (P , 0.01). It was shown previously that CD2 is not expressed on small intestinal IELs but is present in 80% of colon IELs1 and that a significant portion of small intestinal IELs lack CD5.8 Therefore, we compared CD2 and CD5 expression with that of L-selectin. The expression of CD2 and CD5 paralleled that of L-selectin, being much lower in the small intestinal IELs than in colonic IELs (CD2, P , 0.001; CD5, P , 0.001) (Table 1). In contrast to the small intestinal IELs, lymphocytes of the Peyer’s patches expressed high CD2 but low L-selectin. CD2 and CD5 expression was absent from CD81 T cells in the small intestinal IELs, whereas CD41 cells of the small intestine always expressed a high percentage of CD2 (95%) or CD5 (75%). In contrast, L-selectin expression was independent of whether T cells expressed CD41 or CD81. We were then interested in how homing receptors other than L-selectin were regulated in the intestine. The homing receptor aE/b7 was expressed by 80% of the

Table 1. Expression of Homing Receptors, CD2, and CD5 on CD31 T Cells of Different Anatomic Compartments Percent positive (%)

L-Selectin

aEb7 b7 CD2 CD5

Colon IELs

Small intestine IELs

Colon LPLs

Small intestine LPLs

Cecal follicle

Peyer’s patches

30 6 10 20 6 5 75 6 8 96 6 3 88 6 8

363 80 6 11 97 6 3 18 6 10 20 6 6

30 6 10 NT 72 6 10 96 6 3 NT

10 6 6 NT 83 6 8 34 6 10 NT

13 6 6 562 65 6 7 95 6 4 95 6 3

13 6 4 962 68 6 8 95 6 3 96 6 2

MLN

Caudal lymph node

Spleen

55 6 14 19 6 4 60 6 10 95 6 3 96 6 3

58 6 15 20 6 4 64 6 9 95 6 3 94 6 5

60 6 15 11 6 3 65 6 11 96 6 3 96 6 3

NOTE. Lymphocytes were isolated from the colon, small intestine, cecal follicle, Peyer’s patches, MLN, caudal node, and spleen; were stained with antibodies against L-selectin, aEb7, b7, CD2, and CD5; and were analyzed by flow cytometry. Results are expressed as means 6 SD of the percentage of positive cells of the CD31 population. Each data point represents the results of 9–15 mice. NT, not tested.

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small intestinal IELs, whereas this marker was found at a much lower proportion in colon IELs (P , 0.01), Peyer’s patches, or spleen (Table 1). Low L-Selectin Expression on Small Intestinal IELs Correlates With Activation Measurement of other activation markers such as CD44, CD45RB, and CD69 showed that small intestinal IELs and LPLs are more activated than their colonic counterparts (Table 2). Although less activated than small intestine, colonic IELs and LPLs had a more activated phenotype with an up-regulation of CD69 when compared with peripheral lymphocytes. Not surprisingly, lymphocytes of cecal follicle and Peyer’s patches were more similar to the intestinal lymphocytes than they were to lymphocytes of MLN or spleen (Table 2). Regulated Expression of L-Selectin in Mucosal Lymphocytes

nal IELs that had up-regulated L-selectin in culture were then stimulated with PMA for 30 minutes, the L-selectin expression was rapidly down-regulated again (Figure 2). Despite this, incubation of small intestinal IELs in medium for longer periods of time never led to L-selectin expression comparable to spleen cells (.80%). To investigate whether the low L-selectin expression in fresh small intestinal IEL isolates was caused by decreased transcription of messenger RNA (mRNA), we analyzed L-selectin mRNA levels of spleen cells, colon IELs, and small intestinal IELs. Lymphocytes of all three anatomic sites expressed mRNA for L-selectin (Figure 3). There was no difference whether mRNA of freshly isolated or cultured lymphocytes was used (data not shown). This result shows that the absence of L-selectin in small bowel IELs is not caused by an absence of transcription. L-Selectin

Expression and Subtype of T-Cell Receptor

Because decreased L-selectin expression correlates with increased expression of other activation markers, we asked whether local inflammation would alter, i.e., decrease, the L-selectin expression on colonic IELs. To investigate the regulation of L-selectin in the presence of inflammation, we looked at L-selectin expression of colon lymphocytes during colitis induced by transfer of CD41, CD45RBhi T cells into a scid/scid syngeneic recipient. During colitis, colonic IELs, which normally express L-selectin in about 30% of the CD31 cells, had no L-selectin expression at all (Figure 1). In an animal with a mild colitis (C3H/HeJBir mouse), L-selectin was downregulated to a lesser extent (Figure 1). Conversely, when small intestinal IELs, which normally do not express L-selectin, were incubated in vitro in medium only, L-selectin expression increased gradually over time (Figure 2). The addition of either IL-2 (30 U) or anti-CD3 to such cultures had no appreciable effect on the degree of L-selectin up-regulation (data not shown). If small intesti-

The low expression of L-selectin in small intestine was possibly related to the different subgroups of T-cell receptor (TCR) expressed there, e.g., gd TCR is found on many small intestinal IELs. Fifty-two percent of the spleen gd T cells expressed L-selectin, whereas 65% of the spleen ab T cells did. Thus, neither the subclass of the TCR nor the CD4 and CD8 status of the small intestinal IELs was likely responsible for the low expression of L-selectin. This is in contrast to CD2 and CD5 expression, which was found only on 2%–3% of the gd TCR cells, compared with 25% of ab TCR cells (data not shown). L-Selectin

Is Decreased by Exposure to Small Intestinal Homogenates

To determine what factors are involved in the low expression of the small intestine, T cells from the spleen and MLN were incubated with bacterial or food antigens. Incubations ranging between 30 minutes

L-selectin

Table 2. Expression of CD4 and CD8 and Activation Markers CD44, CD45RBhi, and CD69 on Lymphocytes of Different Anatomic Localizations Percent positive

CD4 CD8 CD41CD81 CD44 CD45RB CD69

Colon IELs

Small intestine IELs

Colon LPLs

Small intestine LPLs

Cecal follicle

Peyer’s patches

46 6 7 22 6 8 261 43 6 9 78 6 8 15 6 5

965 66 6 6 15 6 4 75 6 6 52 6 7 85 6 14

50 6 9 21 6 10 362 55 6 11 77 6 10 35 6 12

48 6 8 36 6 7 662 50 6 8 26 6 7 40 6 12

83 6 6 12 6 5 161 64 6 7 44 6 8 29 6 6

81 6 11 10 6 4 261 65 6 7 36 6 9 50 6 7

MLN

Caudal lymph node

Spleen

58 6 9 34 6 5 261 33 6 6 62 6 11 662

56 6 10 35 6 6 261 49 6 7 71 6 9 662

50 6 8 40 6 7 161 50 6 6 60 6 9 461

NOTE. Cells were isolated and stained with the above-mentioned antibodies and were analyzed by flow cytometry. Results are expressed as means 6 SD of the percentage of positive cells of the CD31 population. Each data point represents the results of 9–15 mice.

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Figure 1. Proportion of L-selectin–positive CD41 T cells in the colon in the presence or absence of colitis. Colon IELs were isolated from groups of mice with no, mild, or severe colitis as described in Materials and Methods. Cells were stained with both anti–L-selectin and anti-CD41 and then were analyzed by two-color flow cytometry, gating on the CD41 population. None, healthy C3H/HeJ mice; mild colitis, spontaneously colitic C3H/HeJBir mice; and severe colitis, C3H/SnJ scid/scid mice that had received C3H/HeJ CD41 CD45RBhi T cells 10 weeks before analysis. These data represent the means 6 SD of 4–5 animals in each group from 2 separate experiments.

and 24 hours with these antigens did not lead to a significant change in L-selectin expression. In addition, small intestinal IELs up-regulated L-selectin during 24 hours despite incubation with anti-CD3, IL-2, bacterial antigens, or food antigen. Moreover, incubation of splenocytes with sterile-filtered luminal content of small intestine or colon, with lysed bacteria, with soy protein, or

Figure 2. Proportion of L-selectin–positive small intestinal IELs cultured in vitro for various times. After culture, cells were stained with anti–L-selectin and analyzed by flow cytometry. An aliquot incubated for 48 hours was exposed to 0.1 mmol/L PMA for 30 minutes before staining and analysis (48 hours 1PMA). These data represent the means 6 SD of replicates from 1 of 3 experiments with similar results.

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Figure 3. Reverse-transcription PCR for L-selectin mRNA. Total RNA was isolated and reverse transcribed to cDNA, and L-selectin mRNA was amplified by PCR using specific primers. The products were electrophoresed on agarose gels and stained with ethidium bromide. Lane 1, small intestinal IELs; lane 2, colonic IELs; lane 3, spleen cells; lane 4, Mode K epithelial cell line; and lane 5, liver homogenate. Size markers are shown to the left of lane 1. This experiment was repeated three times with similar results.

with lipopolysaccharide did not change L-selectin expression. PMA, however, down-regulated L-selectin within 30 minutes, although it was gradually reexpressed within the following hours (Figure 4). Stimulation with antiCD3 caused only slight down-regulation of L-selectin from 96% to 80% 6 8% after 24 hours of incubation. Assuming that some regulatory factor leading to down-regulation of L-selectin may be present in the small intestine, we tested a variety of cytokines for their effects on L-selectin. Transforming growth factor b is secreted by intestinal epithelial cells; however, the incubation with transforming growth factor b or other cytokines, such as tumor necrosis factor a, IFN-a, IFN-g, IL-2, or IL-10, showed no effect on L-selectin expression at the time points tested (30 minutes, 2 hours, and 24 hours). Because a down-regulatory effect of epithelial cells or their products remained possible, we cocultured lymphocytes with different epithelial cell lines, but only minor changes could be found in L-selectin expression. Supernatant of these cell lines, which contain secreted products of the epithelial cells, were also not able to down-regulate L-selectin. Homogenates of the epithelial cell lines induced a modest degree of L-selectin down-regulation of spleen cells when used in high concentration. In contrast, homogenates of the small intestine induced strong shedding of L-selectin (Figure 5). Homogenates of the colon at similar concentrations down-regulated L-selectin but to a lesser extent (Figure 5). The percentage of loss of L-selectin was dependent on the dose of homogenized tissue added (Figure 6). If epithelial cells were depleted from the small intestine and if the remaining tissue was homogenized, the supernatant still was able to downregulate L-selectin, and homogenates of freshly isolated

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A

B

C

D

Figure 4. L-Selectin expression on CD41 MLN incubated in vitro with various stimulants. (A ) Untreated MLN; (B ) MLN incubated with 0.1 mmol/L PMA for 30 minutes; (C ) MLN incubated with homogenized small intestine for 2 hours; and (D ) MLN incubated with colon homogenate for 2 hours. The data are representative of the results of 3 independent experiments.

small intestinal epithelial cells had no significant effect on the L-selectin expression of peripheral lymphocytes (data not shown). Lastly, homogenates of colon tissue from C3H/HeSnJ scid/scid had effects similar to those of normal C3H/HeJ colon or inflamed colon (CD45RBhi model) homogenates, suggesting that gut lymphocytes are not the source of sheddase activity in the intestinal homogenates that is detected in the in vitro assay (data not shown). In contrast to L-selectin expression, CD2 expression was unchanged after stimulation of the cells either with PMA or with homogenates of small intestine. Effect of Metalloproteinase Inhibitor When cells were incubated with a specific metalloproteinase inhibitor (KD-IX-73-5) and then stimulated with PMA, L-selectin expression was not decreased (Figure 5). This effect was found whether using spleen cells or colonic and small intestinal IELs (the latter after culture for 12 hours to up-regulate L-selectin). Incubation with this metalloproteinase inhibitor also

prevented a shedding of L-selectin after exposing MLN lymphocytes to homogenates of small intestine (Figure 5). This inhibition of L-selectin shedding was dependent on the concentration of the metalloproteinase inhibitor added (Figure 7). As a control, small intestinal IELs and spleen cells were incubated with proteinase inhibitor for 24 hours, but the proteinase inhibitor itself had no up-regulatory functions.

Discussion This study extends previous reports on differences among mucosal lymphocytes in different regions of the murine intestine. For instance, small intestinal IELs are predominantly CD81 cells, whereas colon IELs are predominantly CD41. In addition, double-positive CD41CD81 are observed mainly in the small intestinal IELs and not in the colon IELs. Likewise, small intestinal IELs contain a major proportion of gd TCR-expressing cells, whereas colonic IELs are mainly ab TCRs. The

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A

B

C

D

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E

Figure 5. Inhibition of L-selectin down-regulation by a metalloproteinase inhibitor. L-Selectin expression was measured on spleen cells incubated in vitro under various conditions that were then stained with anti–L-selectin antibody and analyzed by flow cytometry. (A ) untreated control; (B ) cells incubated with 0.1 mmol/L PMA for 30 minutes; (C ) the same as in B but in the presence of metalloproteinase inhibitor KD-IX-73-5; (D ) cells incubated with small intestinal homogenate; and (E ) the same as in D but in the presence of metalloproteinase inhibitor KD-IX-73-5. The data are representative of results of 3 independent experiments.

expression of the latter appears to be dependent on bacterial colonization because germfree animals have very few ab TCR-expressing IELs. These differences may therefore relate to the degree or type of antigenic stimulation. Other markers, including CD2, CD5, and L-selectin, have much lower expression in small intestinal IELs than in the colon.1,8 In these studies, we have focused particularly on the

differences in L-selectin expression among mucosal lymphocytes from different parts of the bowel. As shown in the current study, small intestinal mucosal cells, both IELs and LPLs, express almost no L-selectin, whereas some 30% of their colonic counterparts do express this cell surface molecule. Several mechanisms might be envisioned to account for this difference, one of which is lymphocyte activation with subsequent shedding. How-

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Figure 6. Loss of L-selectin is proportional to the amount of intestinal homogenate added to the lymphocytes. Lymphocytes (1 3 106 ) from MLN were incubated for 90 minutes with either small bowel (—h—) or large bowel (··e··) homogenate at different concentrations (as shown). After incubation, lymphocytes were washed, stained with both anti– L-selectin and anti-CD4, and analyzed by flow cytometry. These data represent the means 6 SD of 3 experiments.

ever, this explanation is a bit difficult to reconcile with the huge antigenic load present in the colon and the preponderance of antigen-dependent ab TCR-expressing cells there. The colon is known to have a tighter, less permeable epithelium than the small intestine, and perhaps this limits actual antigenic exposure of the mucosal lymphocytes there. Indeed, L-selectin expression

Figure 7. Competitive inhibition of L-selectin shedding by a metalloproteinase inhibitor. Lymphocytes (1 3 106 ) from MLN were preincubated with the metalloproteinase inhibitor KD-IX-73-5 for 15 minutes at various concentrations as shown. Then 300 mg/mL of either colon homogenate (··e··) or small intestine (—h—) homogenate was added. After incubation for 90 minutes, lymphocytes were washed, stained with both anti–L-selectin and anti-CD4, and analyzed by flow cytometry. These data represent the means 6 SD of 3 experiments.

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was found to be down-regulated in inflamed colons proportionate to the severity of inflammation. Another explanation of this difference between colon and small intestine is the presence in the small intestine of a higher proportion of memory cells, which have been traditionally found to be L-selectin negative.6,7 However, recent reports show that naive cells can be L-selectin negative and memory cells L-selectin positive; therefore, the significance of L-selectin as a marker of memory cells is uncertain.9 Other activation markers, such as CD44, CD45RB, and CD69, are expressed to a higher degree in small intestinal IELs than in colon IELs, indicating that small intestinal IELs are indeed more activated than their colonic counterparts. However, the latter markers show that only a portion of small intestinal IELs are activated and not all of them as would be expected if the difference in L-selectin was simply caused by cell activation. Thus, other factors must be involved. Within the small intestine, the proximal IELs express less L-selectin than do distal IELs. This suggests that activation of lymphocytes by food or other antigens might be involved. Therefore, we incubated mucosal lymphocytes with bacterial and food antigens but could identify no differences in L-selectin expression under such conditions. In addition, incubation of splenic lymphocytes with IL-2, IL-10, IFN-g, or tumor necrosis factor a did not reduce L-selectin expression. L-Selectin expression on some cells lines has been found to be increased after incubation with IFN-a,10 but in this study, IFN-a had no influence on the L-selectin expression on the small intestinal IELs. It is now recognized that there are important interactions between lymphocytes and intestinal epithelial cells; however, culturing lymphocytes with epithelial cell lines or with homogenates of small intestinal epithelial cells had no effect on L-selectin expression, and coculture with transforming growth factor b, a cytokine produced by epithelial cells, also had no effect. However, we cannot exclude a contribution by epithelial cells in vivo, which are exposed to a variety of stimuli from the local environment that may not be reflected in these in vitro assays. Because CD2 and CD5 expression was greatly reduced in the small intestine as well, we analyzed the expression of L-selectin relative to either CD2 or CD5 expression. In contrast to L-selectin, CD2 and CD5 are not expressed on CD81 T cells. It has been reported previously that CD5 and CD2 are absent in the majority of gd TCR IELs.8,11–13 Thus, the subtype of TCR expressed in different locations may be related to the L-selectin expression.14 Indeed, gd TCR–positive T cells express L-selectin less frequently, but this does not explain the very low L-selectin expression in small intestinal IELs. In another report, all

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extrathymic T cells in the liver and intestine were found to be CD441 and L-selectin negative.15 However, because not all small intestinal IELs are extrathymic but almost all IELs lack L-selectin expression, the thymic or extrathymic origin of IEL T cells cannot explain the differences. A proportion of L-selectin–negative small intestinal IELs reexpress L-selectin after incubation in culture medium alone (with or without IL-2) for 24 hours. Similar results have been obtained with T cells obtained from nonintestinal sites.14,16 Using reverse-transcription PCR, mRNA for L-selectin was identified in extracts of small intestinal IELs, colonic IELs, and spleen cells, indicating that the lack of expression of L-selectin in small intestinal IELs is not related to a lack of gene transcription in these cells. In humans, very low levels of L-selectin mRNA were found in LPLs. The levels of mRNA encoding L-selectin increased after stimulation of human spleen cells with PMA but not in LPLs similarly stimulated.17 In this study, a partial methylation of an MSP-1 site proximal to the L-selectin gene was identified in LPLs, indicating that the gene had been transcriptionally active.17 The ability of murine small intestinal IELs to reexpress L-selectin and the detection of L-selectin mRNA in freshly isolated small intestinal IELs indicate that small intestinal IELs are not a separate lineage of cells that are unable to express this molecule but that factors present in the microenvironment of the small intestine reduce its expression. In an attempt to identify such a microenvironmental factor, we incubated spleen lymphocytes with a homogenate of small intestine. This led to a rapid down-regulation of L-selectin expression that was comparable to that observed after incubation with PMA, a known potent down-regulator of L-selectin expression. Recently it has been shown that L-selectin is shed from the cell surface by the action of a metalloproteinase and that hydroxy acid–based metalloproteinase inhibitors block L-selectin down-regulation.18 As shown in Figures 5–7, we found that a hydroxyamic-based metalloproteinase inhibitor not only inhibited the down-regulation of L-selectin of PMA-stimulated lymphocytes but also inhibited the L-selectin down-regulation induced by a homogenate of the small intestine. Homogenates of the colon contain significantly less of this proteinase activity, as was shown by dilution studies, as well as by the quantities of inhibitor needed to competitively block the enzymatic activity. The functional assay used in these experiments cannot distinguish whether this increased activity is caused by a greater amount of the enzyme being present or whether there are differences in activation or inhibitors in the small intestine vs. colon. Not much is known about this enzyme, which has

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been termed a ‘‘sheddase,’’ except that it appears ubiquitous rather than cell type specific because a broad array of L-selectin–negative cell types transfected with L-selectin cDNA are able to shed this receptor.19,20 The enzyme appears to be expressed in a latent form in many cells, including lymphocytes. This is shown by spleen and lymph node lymphocytes that have L-selectin on their surface normally in a latent form. However, they can activate the enzyme and shed L-selectin rapidly when stimulated with PMA. The enzyme remains latent in homogenates of spleen cells, which do not induce shedding of L-selectin from fresh peripheral lymphocytes (data not shown). This contrasts with homogenates of intestinal tissue that do contain either active enzyme or an activator of the enzyme as shown by the data presented in this report. Consistent with this difference, L-selectin expression is lower in intestinal than in peripheral lymphocytes. Within the intestine, this activity is greater per unit protein in the small intestine than in the colon (Figures 6 and 7), which again correlates well with the very low L-selectin expression in small intestinal lymphocytes and somewhat greater expression in colon lymphocytes. In preliminary studies, we did not detect a difference in enzyme activity in homogenates from inflamed, normal, or immunodeficient colons. We postulate that the differences in L-selectin expression observed during colitis are likely caused by differences in activation of the enzyme rather than in the amount of enzyme present in tissues. Although the factors that regulate the expression of this sheddase have not been defined in these studies, the differences in the expression of activation markers in small intestine vs. colon would suggest that the degree of lymphocyte activation in these mucosal sites is likely to be one factor regulating sheddase expression. The cleaving of L-selectin may represent an important mechanism for T-cell retention in the intestine. Cells leaving the intestine may reexpress L-selectin, and this would enhance their ability to migrate to lymph node and other lymphoid tissues. Indeed, L-selectin knockout mice have much smaller peripheral lymph nodes than wild-type animals, which is consistent with an important role of L-selectin in lymphocyte trafficking.21 This information plus the data presented in this report raise the interesting possibility that mucosal lymphocytes in the colon traffic more readily into mesenteric lymph node or other lymphoid tissues than do cells from the small intestine. Indeed, we have recently found that activation of lymphocytes in the spleen can be detected within hours after delivery of a specific antigen into the colon. The data presented in this report confirm some previous work showing regional differences within the mucosal immune system of the intestine and provide

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some new insights into the complex microenvironmental factors that influence these differences. These microenvironmental factors may lead to a greater expression of the metalloproteinase inhibitor in the small bowel than in the large bowel. Several cell types could be responsible for the production of this enzyme. Homogenates of the small intestine depleted of epithelial cells still were able to induce the down-regulation of L-selectin, indicating that epithelial cells are not the only source (data not shown). Another possibility would be that the down-regulation is caused by the SB IELs themselves. However, homogenates of lymphocytes that were stimulated with PMA for 30 minutes (and therefore not expressing L-selectin) before their homogenization were not able to downregulate L-selectin (data not shown). It is known that the metalloproteinase is ubiquitous, but whether there are differences in the concentration of the enzyme or there are factors leading to activation of the enzyme needs to be evaluated. Nevertheless, these data indicate that the different microenvironments in regions of the intestine can have an important influence on local mucosal lymphocytes.

References 1. Beagley KW, Fujihashi K, Lagoo AS, Black CA, Sharmanov AT, Yamamoto M, Kiyono H, McGhee JR, Elson CO. Differences in intraepithelial (IEL) T cell subsets isolated from murine small versus large intestine. J Immunol 1995;154:5611–5619. 2. Seibold F, McCabe RP, Weaver C, Elson CO. Colonic IEL appear to be less activated than small intestinal IEL (abstr). Gastroenterology 1996;110:A1012. 3. Bargatze RF, Jutila MA, Butcher EC. Distinct roles of L-selectin and integrins a4b7 and LFA1 in lymphocyte homing to Peers patch HEV in situ: the multistep model confirmed and refined. Immunity 1995;3:99–108. 4. Camerini V, Panwala C, Kronenberg M. Regional specialization of the mucosal immune system. J Immunol 1993;151:1765–1776. 5. Bu¨hrer C, Berlin C, Jablonski-Westrich D, Holzmann B, Thiele H-G, Hamann A. Lymphocyte activation and regulation of three adhesion molecules with supposed function in homing: LECAM-1, LPAM-1/2 and CD44. Scand J Immunol 1992;35:107–120. 6. Bradley LM, Atkins GG, Swain SL. Long-term memory T cells from the spleen lack Mel-14, the lymph node homing receptor. J Immunol 1992;148:324–331. 7. Ohgama J, Katoh M, Hirano M, Arase H, Arase-Fukushi N, Mishima M, Iwabuchi K, Ogasawara K, Onol K. Functional studies on Mel 141 and Mel 142 T cells in peripheral lymphoid tissues. Immunobiol 1994;190:225–242. 8. Croitoru K, Bienenstock J, Ernst PB. Phenotypic and functional assessment of intraepithelial lymphocytes bearing a forbidden alpha b TCR. Int Immunol 1994;10:1467–1473.

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9. Steeber DA, Green NE, Sato S, Tedder TF. Lymphocyte migration in L-selectin–deficient mice: altered subset migration and aging of the immune system. J Immunol 1996;157:1096–1106. 10. Appenheimer M, Scarozza A, Cheddy M, Evans S. L-Selectin gene expression is regulated by an interferon alpha responsive signal transduction pathway (abstr). FASEB J 1996;10:A1200. 11. Boll G, Rudolphi A, Spieß S, Reimann J. Regional specialization of intraepithelial T cells in the murine small and large intestine. Scand J Immunol 1995;41:103–113. 12. Lefrancois L. Phenotypic complexity of intraepithelial lymphocytes of the small intestine. J Immunol 1991;1747:1746–1751. 13. Budd RC, Russell JQ, van Houten N, Cooper SM, Agita H, Wolfe J. CD2 expression correlates with the proliferative capacity of ab1 or gd1CD42CD82 T cells in lpr mice. J Immunol 1992;148: 1055–1064. 14. Wallace DL, Beverley PCL. Characterization of a novel subset of T cells from human spleen that lacks L-selectin. Immunology 1993;78:623–628. 15. Ohtsuka K, Hasegawa K, Sato K, Arai K, Watanabe H, Asakura H, Abo T. A similar expression pattern of adhesion molecules between intermediate TCR cells in the liver and intraepithelial lymphocytes in the intestine. Microbiol Immunol 1994;38:677– 683. 16. Mobley JL, Rigby SM, Dailey MO. Regulation of adhesion molecule expression by CD 8 T cells in vivo. J Immunol 1994;153:5443– 5452. 17. Berg M, Murakawa Y, Camerini D, James SP. Lamina propria lymphocytes are derived from circulating cells that lack the Leu 8 lymph node homing receptor. Gastroenterology 1991;101: 90–99. 18. Walcheck B, Kahn J, Fisher JM, Wang BB, Fisk RS, Payan DG, Feehan C, Betageri R, Parlak K, Spatola AF, Kishimoto TK. Neutrophil rolling altered by inhibition of L-selectin shedding in vitro. Nature 1996;380:720–723. 19. Chen A, Engel P, Tedder TF. Structural requirements regulate endoproteolytic release of L-selectin adhesion receptor from cell surface of leukocytes. J Exp Med 1995;182:519–530. 20. Kahn J, Ingraham RH, Shirley F, Magaki GI, Kishimoto TK. Membrane proximal cleavage of L-selectin: identification of the cleavage site and a 6 kD transmembrane peptide fragment of L-selectin. J Cell Biol 1994;125:461–470. 21. Arbones ML, Ord DC, Ley K, Radich H, Maynard-Curry C, Capon DJ, Tedder TF. Lymphocyte homing and leukocyte rolling and migration are impaired in L-selectin (CD62L) deficient mice. Immunity 1994;1:247–252.

Received March 4, 1997. Accepted January 12, 1998. Address requests for reprints to: Charles O. Elson, M.D., Division of Gastroenterology and Hepatology, University of Alabama at Birmingham, UAB Station, Birmingham, Alabama 35294-0007. Fax: (205) 934-8493. Supported by grant Nr Se 739/2-1 from the Deutsche Forschungsgemeinschaft (to F.S.) and by grant PO1 DK44240 from the National Institutes of Health.