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Mucosal T Lymphocyte Numbers Are Selectively Reduced in Integrin _E (CD103)-Deficient Mice¹

Michael P. Schön^2,*, Anu Arya^*, Elizabeth A. Murphy^*, Cassandra M. Adams^*, Ulrike G. Strauch^*, William W. Agace^*, Jan Marsal^*, John P. Donohue^*, Helen Her, David R. Beier, Sara Olson^§, Leo Lefrancois^§, Michael B. Brenner^*, Michael J. Grusby^*, and Christina M. Parker^3,*

^* Division of Rheumatology, Immunology, and Allergy, and Division of Genetics, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; Department of Immunology and Infectious Disease, Harvard School of Public Health, Boston, MA 02115; and ^§ Department of Medicine, University of Connecticut Health Center, Farmington, CT 06030

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mucosal lymphocyte integrin _E(CD103)ß₇ is thought to be important for intraepithelial lymphocyte (IEL) localization or function. We cloned the murine integrin gene encoding _E, localized it to chromosome 11, and generated integrin _E-deficient mice. In _E^-/- mice, intestinal and vaginal IEL numbers were reduced, consistent with the known binding of _Eß₇ to E-cadherin expressed on epithelial cells. However, it was surprising that lamina propria T lymphocyte numbers were diminished, as E-cadherin is not expressed in the lamina propria. In contrast, peribronchial, intrapulmonary, Peyer’s patch, and splenic T lymphocyte numbers were not reduced in _E-deficient mice. Thus, _Eß₇ was important for generating or maintaining the gut and vaginal T lymphocytes located diffusely within the epithelium or lamina propria but not for generating the gut-associated organized lymphoid tissues. Finally, the impact of _E deficiency upon intestinal IEL numbers was greater at 3–4 wk of life than in younger animals, and affected the TCR ß⁺ CD8⁺ T cells more than the T cells or the TCR ß⁺ CD4⁺CD8^- population. These findings suggest that _Eß₇ is involved in the expansion/recruitment of TCR ß⁺ CD8⁺ IEL following microbial colonization. Integrin _E-deficient mice will provide an important tool for studying the role of _Eß₇ and of _Eß₇-expressing mucosal T lymphocytes in vivo.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mucosal T lymphocytes appear to be functionally distinct from those in peripheral blood. Indeed, intestinal intraepithelial lymphocytes (IEL)⁴ have been found to differ from PBL in their Ag-recognition specificity (1, 2, 3), accessory costimulatory molecule expression (4), TCR ß and type, and thymus-dependent vs -independent T cell development (reviewed in 5). There are estimated to be as many T lymphocytes in the intestinal immune system as in the spleen (6). Furthermore, many infectious agents invade via mucosal epithelia, emphasizing the importance of mucosal T lymphocytes for immune surveillance and immune responses to mucosal pathogens under normal conditions. In addition, intestinal T lymphocytes have been implicated in the pathogenesis of inflammatory bowel diseases, based upon the development of intestinal inflammation in animals with defects in T lymphocyte regulation (7). Thus, it will be important to understand the mechanisms whereby mucosal lymphocytes selectively localize and function.

Following primary stimulation in organized lymphoid tissues, such as mesenteric lymph nodes and Peyer’s patches, some activated intestine-derived lymphocytes recirculate and then preferentially return to the intestinal tract. The selective expression of chemokine receptors and adhesion molecules are thought to contribute to T cell homing (8). Once in the intestine, lymphocyte subpopulations localize to particular microenvironments. For example, the CD8⁺ T cells are found preferentially within the epithelium, where they comprise 90% of the resident population, whereas CD4⁺ T cells predominate in the lamina propria, where they constitute more than half of the T lymphocytes (9). Targeted migration along chemokine gradients and selective adhesion to extracellular matrix or to cellular counterreceptors may account for the localization and retention of T cell subsets within mucosal microenvironments.

One candidate to mediate the selective localization or retention of intraepithelial T lymphocytes is the integrin _E(CD103)ß₇. This integrin is expressed selectively on >90% of intestinal IEL and on 45–50% of lamina propria T lymphocytes (9, 10, 11) in both mice and humans. It is also found on T lymphocytes in some other mucosal epithelia, such as the genitourinary epithelium (12), on 40% of bronchioalveolar lavage T cells obtained from normal humans (13) and on some cells of dendritic morphology in rats (14). Furthermore, _Eß₇ expression can be induced on T lymphocytes and murine mast cells by culture in the presence of TGF-ß1 (15), a cytokine produced by intestinal epithelial cells (16) as well as other cell types. In contrast, _Eß₇ is expressed on <5% of PBL in humans (10), on only 15% of splenic T lymphocytes in mice (17), and has not been found on B lymphocytes, underscoring its selective expression on mucosal T cells.

The _Eß₇ integrin mediates T cell adhesion to epithelial cells (18) through its binding to E-cadherin (19, 20, 21), a member of the cadherin family of adhesion molecules that is expressed selectively on epithelial cells. Cadherins are characterized by their tissue-specific distribution and are known to mediate homophilic adhesion of cells within tissues (22). In addition, evidence has suggested that _Eß₇-dependent adhesion is regulated by inside-out signals, based upon the observation that _Eß₇ function is enhanced following stimulation through the TCR (21). Integrin _Eß₇ also appears to transmit an intracellular signal, as anti-human _E mAbs enhance T cell proliferation in response to suboptimal anti-CD3 stimulation (9, 23), and as an anti-murine _E mAb induces T cell-mediated lysis of FcR-bearing target cells in the absence of a signal from the TCR (17). Thus, the _Eß₇ integrin may be important in the localization or function of T cells, dendritic cells, and/or mast cells.

To determine the role of _Eß₇ in in vivo immune responses, the murine integrin _E-encoding gene (Itgae) was cloned and localized to chromosome 11. Then, integrin _E-deficient mice were generated. Study of these animals revealed an altered distribution of T lymphocytes within epithelia and in the intestinal lamina propria, in the absence of experimentally induced infection or inflammation. Thus, these studies demonstrate an important role for _Eß₇ in modulating the homeostasis of T lymphocyte numbers in selected tissue sites.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Integrin _E-encoding gene (Itgae) cloning

A murine 129/Sv-derived -Fix II library (Stratagene, La Jolla, CA) was screened by hybridization with an _E cDNA probe incorporating nucleotides 1088–1253 of the murine _E cDNA sequence (15). Inserts were subcloned and partial sequence determined by deletion cloning, followed by dideoxy chain termination analysis. The nucleotide sequence within identified exons was identical to nucleotides 14–2771 of the murine _E cDNA reported previously with the exception of only 4/2757 bases (nucleotide changes t411c, g668a, c1057g, and g1058c; amino acid changes I207E and R337A).

Localization of the Itgae gene

Primers were designed to amplify a region corresponding to intronic sequence of Itgae to test for single-strand conformation polymorphisms (SSCPs) between mouse strains. These were analyzed as previously described (24). Briefly, oligonucleotides were radiolabeled with [³²P]ATP using polynucleotide kinase, and genomic DNAs from a series of mouse strains were amplified using standard protocols (anneal at 55°C for 1 min, extend at 72°C for 2 min, and denature at 94°C for 1 min for 40 cycles, with a final extension at 72°C). A total of 2 µl of the amplified reaction was added to 8.5 ml USB (United States Biochemical) stop solution, denatured at 94°C for 5 min, and immediately placed onto ice. A total of 2 µl of each reaction is loaded on a 6% nondenaturing acrylamide sequencing gel and electrophoresed in 0.5x TBE buffer for 2–3 h at 40 W in a 4°-cold room. A primer pair based upon sequence in the intron between exons 2 and 3 (5-AAGGTCAGATGAGCAATATGT-3' (forward) and 5'-GCCAGCAGACTCAGCATTACT-3' (reverse)) identified a polymorphism between C57BL/6J and Mus spretus. This primer pair was used to analyze DNA prepared from the BSS ((C57BL/6JEi x SPRET/Ei)F₁ x SPRET/Ei) backcross (25). The strain distribution pattern was analyzed using the Map Manager Program (26).

Construction of the _E targeting vector and generation of _E^-/- mice

A targeting construct was generated (Fig. 1a) and transfected by electroporation into the 129/Sv-derived embryonic stem cell line ES-D3. Transfected cells were selected for G418 and gancyclovir resistance, as reported previously (27), and cloned. Clones that incorporated a single copy of the construct into the genome by homologous recombination were identified by Southern blot analysis of genomic DNA using a 5' probe (Fig. 1a), a 3' probe, and the neomycin resistance gene. Such clones were injected into BALB/c blastocysts to generate chimeric animals. The _E^+/- progeny of matings between chimeric animals and BALB/c mice were intercrossed to generate F₂ (129/Sv x BALB/cJ) offspring, which were utilized in these experiments except where otherwise indicated. Two independent clones were used to generate chimeric animals that expressed similar phenotypes. All animals were housed under specific pathogen-free (SPF) conditions.

View larger version (46K): [in this window] [in a new window]   FIGURE 1. Genomic organization and targeted disruption of the murine integrin E-encoding gene. a, Schematic representation of the E-encoding gene (top), the targeting construct (middle), and the disrupted allele (bottom). Exons (thick lines), introns (thin lines), and restriction endonuclease sites are shown (n, NcoI; B, BamHI; p, PstI; X, XhoI; H, HindIII; for PstI and NcoI, selected cleavage sites are indicated). b, Schematic representation of the E polypeptide indicating the location of the 5' untranslated region (crosshatched fill pattern), the extra region unique to E (speckled fill pattern), cleavage site (arrow), I domain (grey fill pattern), divalent cation binding domains (black fill pattern), transmembrane domain (diagonal line fill pattern), the regions of E encoded by each exon (below the schematic), and the location of the targeted exon (*). c, Southern blot analysis after NcoI digestion (top panel) and PCR analysis (bottom panel) of DNA isolated from offspring of E+/- mouse intercrosses. d, Intestinal IEL freshly isolated from E+/+ and E-/- mice were stained as indicated. e, Splenocytes derived from E+/-, E+/+ and E-/- mice were stimulated with Con A for 5 days, and then cultured for 5 days with TGF-ß1 to up-regulate E expression, surface radiolabeled, solubilized, and E expression analyzed by immunoprecipitation and SDS-PAGE.

Genomic DNA was isolated from tail biopsies by proteinase K digestion, followed by phenol extraction. Southern blot analysis was performed using 10 µg genomic DNA, Hybond-N (Amersham, Arlington Heights, IL) filters and digoxigenin-labeled probes (Nonradioactive DNA labeling and detection kit; Boehringer Mannheim, Indianapolis, IN) according to standard procedures.

PCR was performed on genomic DNA using primer sequences flanking the inserted neomycin resistance gene to yield products of 1100 bp from disrupted and 900 bp from wild-type alleles (primers used: 5'-GCA ACA ACG CAT CGT TCA TAT GGA-3' and 5'-GTG CTC TGT CTA TTG TTC CCC TCC TT-3'; conditions: anneal at 62°C for 1 min, extend at 72°C for 2 min, and denature at 94°C for 1 min for 40 cycles, with a final extension at 72°C).

Cells and culture conditions

A single cell suspension of splenic leukocytes was stimulated with 5 µg/ml Con A (Sigma, St. Louis, MO) and cultured for 5 days. A total of 5 ng/ml human recombinant TGF-ß1 was added to the medium, and the cells were grown for an additional 5 days to induce _E expression.

Purification of intestinal IEL, flow cytometry, and estimation of Peyer’s patch size

Intestinal IEL were isolated as described (28) with minor modifications. Briefly, small intestines were flushed with HBSS, trimmed to remove Peyer’s patches, opened longitudinally, and cut into 3- to 5-mm fragments. The fragments were incubated in a shaking water bath at a rate of 120 shakes per minute in medium (RPMI 1640 containing 10⁴ U/ml penicillin/streptomycin, 20 µg/ml gentamicin, 2% bovine calf serum, and 20 mM HEPES buffer) at 37°C for 20–30 min. The intestinal pieces were shaken manually for three cycles of 15 s each to release intestinal IEL. The resulting cell suspensions were collected, pooled, passed through a glass wool column, and the IEL separated from epithelial cells using 44/66% Percoll density gradient centrifugation. To estimate the size of Peyer’s patches, their length and width were multiplied to estimate their relative cross-sectional area in mm². To isolate Peyer’s patch lymphocytes, the Peyer’s patches were mechanically disrupted by rubbing the patches between two frosted glass slides.

For FACS analysis, 10⁵ cells were stained with saturating concentrations of mAb (buffer: 2% BSA, 0.05% NaN₃ in PBS; blocking reagents: 10% mouse serum (for rat Abs) or 10% goat serum and 20 µg/ml mAb 2.4G2 (for hamster Abs)). In some experiments, dead cells were excluded by propidium iodide staining. Samples were analyzed using a FACScan and FACScalibur for four-color analysis and a FACSort and the Cell Quest software (Becton Dickinson, San Jose, CA) for one color analysis.

Cell surface iodination with ¹²⁵I and immunoprecipitation

A total of 4 x 10⁷ TGF-ß1-stimulated splenocytes was surface iodinated and then solubilized in TBS/0.5% Triton X-100 (Sigma). Then, 1 x 10⁷ cell equivalents were precleared with protein G-Sepharose resin (Pharmacia, Piscataway, NJ), immunoprecipitated with 5 µg of purified mAb followed by protein G-Sepharose, washed, and the immunoprecipitated proteins were analyzed by SDS-PAGE using 7% gels under reducing conditions (29).

Abs

The following mAbs were used as controls: rat IgG1 (R59-40; PharMingen, San Diego, CA), rat IgG2A (R35-95; PharMingen), rat IgG2B (SFR3-DR5; American Type Culture Collection (ATCC), Manassas, VA), and hamster IgG (UC8-4B3; PharMingen). The following Ags were detected by mAbs: CD3 (500A2; PharMingen, and 145-2C11; ATCC), CD4 (RM4-5; PharMingen), CD8 (53-6.72; ATCC), CD8ß (53-5.8; ATCC), CD11b (_M integrin, Mac-1, M1/70; ATCC), CD18 (ß₂ integrin, 2E6; ATCC), CD25 (IL-2R -chain, 3C7; PharMingen), CD29 (ß₁ integrin, Ha2/5; PharMingen), CD45R/B220 (RA3-6B2; PharMingen), CD45RB (MB23G2; ATCC, and 16A; PharMingen), CD49a (₁ integrin, Ha31/8; PharMingen), CD49b (₂ integrin, HM2; PharMingen), CD49d (₄ integrin, P/S2, or R1-2; ATCC), CD49e (₅ integrin, HM5; PharMingen), CD49f (₆ integrin, GoH3; Dianova, Hamburg, Germany), CD49d/ß₇ (₄ß₇, DATK32 (30); LeukoSite, Cambridge, MA), CD51 (_v integrin, H9.2B8; PharMingen), CD54 (ICAM-1, YN1/1.7.4; ATCC), CD62L (L selectin, MEL-14; ATCC), CD90 (Thy-1, AT15.E; R. MacDonald Ludwig Institute for Cancer Research, Epalinges, Switzerland), CD103 (_E integrin, M290 (11); P. Kilshaw, Department of Immunology, AFRC Babraham Institute, Cambridge, U.K. or 2E7 (17); L. Lefrancois, Department of Medicine, University of Connecticut Health Center, Farmington, CT), ß₇ (M293; P. Kilshaw), CD106 (VCAM-1, M/K-2.7; ATCC), CD32/CD16 (Fc-II/IIIR, 2.4G2; ATCC), MHC class I (M1/42.3, rat IgG2A; ATCC), anti-TCR ß (H57-597; PharMingen), and anti-TCR (GL3; PharMingen). FITC-conjugated goat anti-hamster and mouse anti-rat secondary Abs used in FACS or direct immunofluorescent staining of intestinal sections were purchased from Jackson ImmunoResearch (West Grove, PA), and biotinylated goat-anti-hamster and mouse adsorbed rabbit-anti-rat serum were obtained from Vector Laboratories (Burlingame, CA).

Histochemistry

For histochemistry, tissue samples were embedded in JB-4 plastic resin (Polysciences, Warrington, PA) and 3-µm sections stained with hematoxylin-eosin. For immunohistochemistry, tissue samples were frozen in OCT and 5- to 10-µm cryostat-cut sections stained using the ABC (avidin/biotin complex)-immunoperoxidase kit according to the manufacturer’s instructions (Vector Laboratories). For analysis of lamina propria T cell numbers, a cell was counted as a lamina propria lymphocyte if it did not overlap the basement membrane and was contained within villi rather than crypts. This criteria may account for the unusually high ratio of CD4⁺/CD8⁺ T cells observed within the lamina propria in these studies, as some cells were counted as IEL that were largely within the lamina propria. For analysis of T cell numbers within vaginal tissue sections, the number of intraepithelial T lymphocytes within an entire tissue section derived from the middle third of the vagina was determined and expressed per mm basement membrane. For evaluation of T lymphocyte numbers within lung tissue, the left lung was frozen in OCT, the average number of anti-CD3 mAb stained cells in at least seven randomly selected high power (40x) fields was determined and used to calculate the number of T lymphocytes/mm². In addition, the average number of CD3⁺ cells per bronchus was determined evaluating all of the bronchi within these randomly located tissue sections.

Statistical analysis

Statistical analysis was performed using an unpaired, two-tailed Student’s t test when n = 3 and the Mann-Whitney nonparametric U test when n > 3, unless otherwise indicated. Analysis was performed using the Instat software (GraphPad Software, San Diego, CA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Itgae was cloned, and its chromosomal localization was determined

Genomic clones encoding murine _E were identified by hybridization with PCR products encoding fragments of the murine _E cDNA (GenBank accession nos. AF133070-AF133085). Based upon comparison with the murine _E cDNA sequence (15) and identification of the conserved consensus splice sites (Ref. 31, and data not shown), a partial map of the murine _E intron/exon structure was generated (Fig. 1a). To determine the chromosomal location of Itgae, SSCP analysis was used as previously described (24). Primers corresponding to intronic sequence between exons 2 and 3 of Itgae were analyzed and found to identify an SSCP between inbred mouse strains (see Materials and Methods). The BSS-interspecific backcross was genotyped and the allele distribution pattern analyzed using the Map Manager program. Itgae was found to map to chromosome 11 with a logarithm of odds likelihood score of 27.4. No recombinants were found in 91 progeny between Itgae and the marker D11Abb1. The position of Itgae with respect to flanking microsatellite markers was: D11 Mit4 - 5.5 ± 2.4 cm - Itgae, D11Abb1 - 1.1 ± 1.1 cM - D11 Mit7, D11 Mit32, D11 Mit34 (mapping data submitted to the Mouse Genome Database).

Integrin _E-deficient mice were generated

To generate integrin _E-deficient mice, exon 10 within the integrin _E-encoding gene was replaced with a neomycin resistance gene by homologous recombination (Fig. 1, a–c). FACS analysis confirmed that intestinal IEL isolated from _E^-/- mice lacked _E expression (Fig. 1d). In addition, splenocytes from _E^-/- mice were cultured in the presence of TGF-ß1, a cytokine that up-regulates _E expression on wild-type T cells. Integrin _E was not detected on the surface of TGF-ß1-treated splenocytes derived from _E^-/- mice by immunoprecipitation or FACS analysis. In contrast, it was readily detected on the surface of cells derived from _E^+/+ animals, and was detected at reduced levels on cells from _E^+/- animals (Fig. 1e, and data not shown). Thus, _E^-/- mice lacked expression of the _E polypeptide, while heterozygous animals expressed intermediate levels of _Eß₇ both in immunoprecipitation (Fig. 1e) and in FACS analysis (data not shown). Integrin _E deficiency did not alter fecundity, morphogenesis, or overall weight gain, and most animals survived for >18 mo in a SPF facility (data not shown).

Integrin _E-deficient mice had normal or increased numbers of splenic T lymphocytes

Because _E is selectively expressed on leukocytes, studies were performed to determine the impact of _E deficiency upon the histologic appearance of organized lymphoid tissues. In this initial evaluation, _E deficiency had no apparent effect on the size of the thymus or on the immunohistologic appearance of the thymus, peripheral lymph nodes, or spleen after staining with anti-CD3, anti-CD4, and anti-CD8 mAbs. In addition, there were no changes in the serum levels of IgM, IgG isoforms, or IgA in _E^-/- as compared with _E^+/+ mice (data not shown).

To further evaluate the impact of _Eß₇ expression upon the number and subset composition of splenic T lymphocytes, additional studies were performed. First, the overall number of leukocytes/spleen after RBC lysis was similar in _E^-/- and _E^+/+ mice three to four generations backcrossed toward the C57BL/6 strain and then intercrossed (N_3–4) (_E^-/- mice: 1.07 ± 0.15 x 10⁸ cells/spleen; _E^+/+ mice: 1.14 ± 0.16 x 10⁸ cells/spleen; n = 3; p = 0.55). In addition, flow cytometry was used to determine the total number of splenocytes expressing CD3, CD4, or CD8 using the formula: (the total number of splenocytes) x (the proportion of the total splenocyte population that expressed each marker). In this analysis, the number of CD3⁺, CD4⁺, and CD8⁺ splenocytes was not altered significantly by _E deficiency. In contrast, when mice N₁₀ to the BALB/c strain were evaluated, the total number of splenic leukocytes was increased by 24% in _E-deficient mice (_E^-/- mice: 1.07 x 10⁸ ± 0.11 cells/spleen; _E^+/+ mice: 0.86 x 10⁸ ± 0.04 cells/spleen; n = 3; p = 0.04). In addition, the numbers of CD3⁺, CD4⁺, and CD8⁺ splenocytes were increased by 14%, 28%, and 13%, respectively in _E^-/- mice (Fig. 2), while CD4⁺CD8⁺ double positive splenic T cells were not detected in either _E^+/+ or _E^-/- mice (n = 1, pooling cells isolated from three mice of each genotype, data not shown). Overall, peripheral lymphocyte compartments appeared similar in _E^-/- and _E^+/+ mice, with a trend toward increased numbers of CD4⁺ and CD8⁺ splenic T cell numbers in _E^-/- animals on the BALB/c genetic background.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. The proportion of splenocytes expressing CD3, CD4, and CD8 increased slightly by E deficiency. Single cell splenocyte populations were prepared from E+/+ or E-/- mice and stained with directly conjugated anti-CD3, CD4, or CD8 mAbs, as indicated. Flow cytometry was performed to determine the proportion of cells that expressed each marker, analyzing the total splenocyte population. After subtracting the background staining with isotype-matched noncell binding control mAbs, the percentage of of the total splenic leukocyte population that was stained with each mAb was determined. Then, the total number of stained cells/spleen was calculated by multiplying the total number of leukocytes/spleen x the percentage that expressed the analyzed marker, as determined by flow cytometry. The bars represent the average number of positive splenocytes/spleen for each marker (n = 3 for each group). *, p < 0.05; **, p < 0.01.

Integrin _E-deficient mice had fewer T cells diffusely distributed within the intestinal and vaginal epithelia

The number of intestinal IEL in adult _E^-/- and _E^+/+ littermates also was compared by immunohistology, with the expectation that intestinal IEL numbers would be reduced by _E deficiency due to the loss of the _Eß₇/E-cadherin-mediated adhesion. Indeed, the number of jejunal IEL was reduced by 54% in the _E^-/- progeny of F₂ (BALB/c x 129/Sv) mice as compared with _E^+/+ mice of similar genetic background (p < 0.002, Mann-Whitney U test; n = 5 _E^+/+ and 8 _E^-/- mice) (Figs. 3 and 4a). In these studies, it was apparent that the number of intestinal IEL was influenced by genetic background and/or environment, as there were more intestinal IEL in _E^+/+ mice on the (129/Sv x BALB/c) or the C57BL/c backgrounds than on the BALB/c background. However, _E^-/- mice had reduced intestinal IEL numbers in all genetic backgrounds examined, including groups of mice housed in two independent animal facilities. Thus, reduced intestinal IEL numbers represents a consistent feature of _E-deficient mice.

View larger version (123K): [in this window] [in a new window]   FIGURE 3. Diffusely distributed T cells are diminished in intestinal mucosa of E-deficient mice. Cryostat-cut sections of proximal jejunum from E+/+ and E-/- adult mice backcrossed two generations to the C57BL/6 strain were stained with anti-E (M290), anti-CD3 (500A2), anti-CD4 (RM4-5), and anti-CD8 (53-6.72), as indicated. Scale bar, 20 µm.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. The number of intestinal and vaginal IEL is reduced by E deficiency. a, Proximal jejunal tissue sections were stained with anti-CD3 (500A2 or 145-2C11), and the number of stained cells/1000 villus epithelial cells was determined by microscopic evaluation. For (BALB/c x 129/Sv)F2, n = 5 E+/+ and 8 E-/- mice, for BALB/c (N1–5), n = 9 mice. b, Vaginal tissue sections derived from the middle third of the vagina were stained with anti-CD3 (500A2), and the average number of stained IEL/mm epithelium was determined for E+/+ or E-/- mice (n = 3 animals for each genotype). c, Pulmonary sections from E+/+ or E-/- mice were stained with anti-CD3 (500A2), and the average number of stained cells per bronchiolus was determined. d, Pulmonary sections were stained with anti-CD3, and the number of stained cells/mm2 lung parenchyma was determined. Results from E-/- mice are indicated as -/- and from E+/+ mice are indicated as +/+ (n = 5 E+/+ and 6 E-/- mice). Bars indicate the average ± SD. Strains shown in bold were housed in a SPF facility at Harvard University, and strains shown in normal text were housed in the SPF facility at the Dana-Farber Cancer Institute. *, p 0.05; **, p 0.01.

Integrin _E-deficient mice have reduced numbers of lamina propria T cells but not of Peyer’s patch T cells

It appeared that the number of lamina propria T lymphocytes was reduced in immunohistochemical analysis of tissue sections from (129/Sv x BALB/c) mice. This was not an expected finding because E-cadherin, the known _Eß₇ counterreceptor, is not expressed in the lamina propria (32). To quantitate this difference, the number of T lymphocytes within the villus lamina propria that did not appear to be in contact with either the basement membrane or epithelium was determined per 0.5-mm villus length. In this analysis, the number of lamina propria lymphocytes was significantly diminished by _E deficiency in animals of the (129/Sv x BALB/c) background (CD3⁺ lamina propria lymphocytes/0.5-mm villus length in _E^+/+ mice: 76 ± 6 vs 39 ± 9 in _E^-/- mice; p < 0.01; n = 3). In a second group of mice partially backcrossed toward the BALB/c strain, integrin _E deficiency had a similar impact upon lamina propria T lymphocyte numbers (in _E^+/+ mice, 70 ± 7 T cells/0.5-mm villus length vs 30 ± 6 in _E^-/- mice; p = 0.002; n = 3). Thus, _E^-/- mice had significantly fewer lamina propria T lymphocyte numbers than _E^+/+ mice in these two groups. However, when additional groups of mice were evaluated, _E deficiency did not appear to have an impact upon lamina propria T lymphocyte numbers (Fig. 5a). In these two additional groups, it was notable that the _E^+/+ mice had fewer lamina propria T lymphocytes than those in the groups initially studied (Fig. 5a). This could be due to differences in genetic background, as these later groups of mice were backcrossed toward either the C57BL/6 or BALB/c strains. However, it seems more likely that environmental factors may have had an effect. Of note with respect to this possibility, the two more fully backcrossed groups of mice were housed in an animal facility where the cages were changed twice/wk, and the microisolator cages were vented with an air circulation system. These animals had relatively few lamina propria T lymphocytes and _E deficiency had no effect on lamina propria T cell numbers. In contrast, the other groups of mice were housed in a facility where the cages were changed once/wk, and the microisolator cages were not vented. In the setting of this less-stringent environment, there were more lamina propria T lymphocytes in _E^+/+ mice, and _E deficiency had a significant impact upon lamina propria T lymphocyte numbers.

View larger version (41K): [in this window] [in a new window]   FIGURE 5. The number of lamina propria lymphocytes is reduced by E deficiency, while there was not a consistent effect upon Peyer’s patch size or composition. a, Proximal jejunal tissue sections were stained with anti-CD3 (500A2 or 145-2C11), and the average number of stained cells wholly enclosed within the lamina propria lymphocytes per 0.5-mm villus length was determined by microscopic evaluation. b, The average small intestinal Peyer’s patch size was estimated based upon the calculation: estimated size = length x width in mm2. For the C57BL/c (N3–4) experimental group, n = 6. c, Peyer’s patch lymphocytes were stained with anti-CD3, anti-CD4, and anti-CD8, and the proportion of stained cells within the lymphocyte light scatter gate was determined by flow cytometry. Results from E-/- mice are indicated as -/- and from E+/+ mice are indicated as +/+. Bars indicate the average ± SD. Unless otherwise stated, n = 3 for each experimental group. Strains shown in bold were housed in a SPF facility at Harvard University and strains shown in normal text were housed at the Dana-Farber Cancer Institute. *, p 0.05; **, p 0.01.

Integrin _E disruption has a preferential effect upon ß T cell localization

The T cells populate the intestinal epithelium at an earlier age than ß T cells (33, 34) and the recruitment/expansion of TCR ß⁺ intestinal IEL depends upon microbial colonization of the intestinal tract (33). Thus, studies were performed to determine at what age reduced numbers of intestinal IEL were detected in _E-deficient animals. In immunohistochemical analyses, intestinal IEL were not observed until 5 days of age in either _E^+/+ or _E^-/- mice. By 3 wk of age, the intestinal IEL number increased to 40–50 intestinal IEL/1000 epithelial cells in _E^+/+ mice, with only a 20% reduced number of intestinal IEL in _E^-/- mice. Finally, the intestinal IEL number reached a plateau in animals of both genotypes at 4–5 wk of age. In mice older than 5 wk of age, the number of proximal jejunal IEL in _E^-/- mice was only 46% of that seen in _E^+/+ mice (Fig. 6). Overall, this kinetic analysis suggested that _E might be more important in the localization of ß than of T cells and that the impact of _E deficiency upon intestinal IEL numbers becomes apparent at a time when TCR ß cells are increasing in number, presumably due to bacterial colonization.

View larger version (16K): [in this window] [in a new window]   FIGURE 6. The impact of E deficiency upon IEL numbers is greater in mice >25 days of life than in neonatal animals. Proximal jejunum sections from E+/+ (squares) and E-/- (circles) mice of increasing age were stained with anti-CD3 (500A2). When more than one mouse was sacrificed at a given time point, the number of animals is indicated, and the average number of intestinal IEL is graphed. As the number of intestinal IEL determined in animals >35 days of age did not appear to change with increasing age, the mean intestinal IEL number in animals >40 days was graphed. Mice were derived from intercrosses of 129/Sv x BALB/c animals. Error bars indicate SD. Statistical analysis was performed using the Mann-Whitney U test.

View larger version (33K): [in this window] [in a new window]   FIGURE 7. Integrin E deficiency preferentially reduces the number of TCR ß-expressing intestinal IEL on the C57BL/6 background. Intestinal IEL isolated from mice backcrossed for two to three generations to the C57BL/6 strain were reacted with the indicated mAb and analyzed for expression by FACS, evaluating the total lymphocyte population (a), the TCR ß+CD8+ lymphocytes (b), or the TCR ß+ T cells (c). Profiles from one mouse of each genotype are shown, that are representative of results from four E-/- and six E+/+ or E+/- mice.

Integrin _E disruption reduced the numbers of CD8⁺ TCR ß⁺ IEL subsets but had a lesser impact upon the number of CD4⁺ IEL

Within the intestinal epithelium, _Eß₇ is expressed by >90% of the CD8⁺ T cells but by only half of the CD4⁺ T cells (9, 11). Thus, it seemed likely that _Eß₇ disruption might affect the number of CD8⁺ IEL more than the number of CD4⁺ IEL. Thus, three-color FACS analysis was performed to evaluate the subsets of TCR ß⁺ intestinal IEL in _E^-/- as compared with _E^+/- or _E^+/+ mice backcrossed toward the C57BL/6 strain, in which the TCR /TCR ß cell ratio was altered (Fig. 7a). In this analysis, the proportions of intestinal TCR ß⁺ IEL that expressed CD8, CD8ß, or both CD4 and CD8 were not consistently altered in _E^-/- as compared with _E^+/- or _E^+/+ mice (Fig. 7, b and c). However, the proportion of TCR ß⁺ IEL that were CD4⁺CD8^- was greater in _E^-/- than in _E^+/+ or _E^+/- mice (7.3 ± 0.9% of the IEL in _E^+/+ or _E^+/- mice vs 12.9 ± 1.0% of the IEL in _E^-/- mice; n = 4; p = 0.028). As the total number of IEL was reduced to 50% of normal, an apparent doubling of the CD4⁺CD8^- subset suggests that CD4⁺CD8^- T cell numbers were not increased but rather remained unchanged, while the other subsets were reduced in number in _E-deficient mice. In contrast, in the BALB/c strain, where _E deficiency had relatively little impact upon IEL numbers (Fig. 4a), the proportion of IEL that expressed CD4 was not altered in _E-deficient mice (Fig. 8). Finally, within the lamina propria of mice backcrossed toward the C57BL/6 strain, the CD4⁺ and CD8⁺ populations both were reduced in number with a slightly greater impact upon the number of CD4⁺ cells (Fig. 5a). Overall, _E deficiency resulted in diminished numbers of CD8⁺ IEL but not of CD4⁺ IEL in backgrounds where _E deficiency had a dramatic impact upon IEL numbers, but resulted in reduced numbers of both CD4⁺ and CD8⁺ lamina propria subpopulations.

View larger version (33K): [in this window] [in a new window]   FIGURE 8. Intestinal IEL isolated from E-/- mice N3–4 to the BALB/c strain express higher levels of CD44. Intestinal IEL isolated from either E+/+ (grey fill pattern) or E-/- (black line) mice were evaluated by FACS analysis for expression of T cell and adhesion molecules as indicated. * indicates Abs that reproducibly stained E-/- intestinal IEL with a different average MFI intensity than E+/+ intestinal IEL. Histograms shown are representative of three experiments.

While the expression of many adhesion molecules on IEL was not altered by _E deficiency, the expression of CD44 was increased

Given the expression of _Eß₇ on >80% of intestinal IEL, it was surprising that the intestinal IEL numbers were reduced by only 30% in mice partially backcrossed to the BALB/c strain. Thus, additional FACS analyses were performed to determine whether _E^-/- IEL derived from mice N_3–4 to BALB/c express higher levels of an adhesion molecule that might functionally compensate for _E deficiency in IEL localization, and thus reduce the impact of _E deficiency on intestinal IEL number. Importantly, the expression of ₄ß₇, the other ß₇ integrin, was not altered on intestinal IEL or Peyer’s patch lymphocytes (Fig. 8, and data not shown). While there were some interindividual variations in cell surface expression of other adhesion receptors, many adhesion molecules were expressed, on average, at similar levels on intestinal IEL isolated from _E^-/- or _E^+/+ mice. These included CD18 (LFA-1 ß₂-chain), CD54 (ICAM-1), CD62L (L-selectin), CD29 (ß₁ integrin subunit), CD49d (₄), and CD49a (₁ integrin subunit) (Fig. 8). Other adhesion molecules were not detected on intestinal IEL isolated from either _E^-/- or _E^+/+ mice, including CD49b (₂), CD49e (₅), CD49f (₆), and CD51 (_V) (data not shown). Overall, these studies emphasize the relatively limited expression of other adhesion molecules, such as the ß₁ integrins, on intestinal IEL. On IEL derived from _E^-/- mice, the only consistent shift in adhesion molecule expression was increased expression of CD44 (average mean fluorescence intensity (MFI) 118 on cells derived from _E^-/- mice vs 78 on cells derived from _E^+/+ mice; n = 4; p < 0.03), a hyaluronate receptor (35, 36). This molecule could participate in mucosal T cell adhesion within the intestinal mucosa, as hyaluronate is expressed within the lamina propria (37). In addition, CD45RB was expressed a slightly higher levels on IEL derived from _E^-/- than on IEL derived from _E^+/+ mice (average MFI = 890 vs 780; p = 0.0005; n = 3, not visible in the profile due to the logarithmic scale). This finding, while highly reproducible, was of unclear functional significance.

	Discussion

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Overall, the organization of the _E-encoding gene was strikingly similar to that of other I-domain containing integrins, _M and _X (38, 39), with a high degree of conservation in the intron/exon boundaries and exon spacing. In addition, an exon was identified between exons 5 and 6 that was not homologous to exons within other integrins, suggesting that it may have resulted from a gene-insertion event. This exon encoded 44 amino acids, corresponding to the X-(extra)-domain identified within the predicted human _E (40) and murine _E subunits (15) (Fig. 1, a and b). The exons were numbered based upon homology with the other I domain-containing integrins, and the exon unique to integrin _E was designated E (Fig. 1). SSCP analysis was used to localize Itgae to mouse chromosome 11, tightly linked to Abr, and was consistent with the localization of sequence-tagged sites derived from the human homologues of both Itgae (SHGC-12572, WI-22591) and Abr (WI-19743) to 17p13 on the human transcript map (http://www.ncbi.nlm.nih.gov/genemap98 (41)).

In the _E ^-/- mice, there were reduced numbers of intestinal IEL in three different genetic backgrounds, evaluating mice housed in two different animal facilities. Thus, reduced intestinal IEL numbers was a consistent feature of _E deficiency, although the magnitude of this effect varied with genetic background and/or environment. IEL numbers were also reduced in the vaginal epithelium, while the number of lymphocytes in lung parenchyma or adjacent to the bronchiolar epithelium was not affected. While lamina propria T lymphocyte numbers also were diminished by _E deficiency in some strains and/or environmental conditions, the number of T lymphocytes in Peyer’s patches and spleen was not diminished by disruption of the _E-encoding gene. Thus, _E deficiency appeared to function selectively in the generation/localization of IEL and lamina propria T lymphocytes but not in the generation of organized lymphoid tissues in the mucosa. While other explanations may exist, these data are most consistent with a role of _Eß₇ in modifying T lymphocyte localization to the intestinal mucosa, but suggest that other receptors, such as CD44 and possibly _Lß₂ (42), may partially compensate for the loss of _Eß₇.

It was notable that the proportion of intestinal IEL that expressed the TCR-ß was reduced in _E-deficient mice and that the impact of _E deficiency upon IEL number was greater in animals that were 5 wk of age or older than in younger mice. Thus, the impact of _E deficiency upon intestinal T lymphocyte numbers appeared during the time period when microbial colonization results in increased numbers of TCR ß-expressing intestinal IEL. Finally, the impact of _E deficiency upon IEL/lamina propria lymphocyte (LPL) numbers may have been influenced by environmental factors, as LPL numbers were reduced in some groups but not others. Overall, these findings suggested that integrin _Eß₇ may play a role in the recruitment/expansion of TCR ß⁺ IEL that is triggered by microbial colonization of the intestine. Based upon the flow cytometry analysis of IEL, it appeared that the ß T cell subset that was CD4⁺CD8^- was not affected by _E deficiency. In contrast, the CD8⁺, CD8ß⁺, and CD4⁺CD8⁺ subsets were diminished to a similar extent, consistent with the expression of _Eß₇ on a larger proportion of CD8⁺ than of CD4⁺ intestinal IEL.

While reduced IEL numbers were consistent with the known function of _Eß₇, we were surprised to find reduced numbers of CD3⁺ cells in the intestinal lamina propria of _E^-/- mice, as E-cadherin is not detected within the lamina propria by immunohistology (Ref. 32, and our unpublished observations). This finding suggested one of two major possibilities, either that intestinal IEL play a role in maintaining lamina propria lymphocyte numbers or that _Eß₇ mediates another adhesive interaction that results in the homing or retention of lymphocytes within the lamina propria. While integrin ß₇-deficient mice had markedly reduced numbers of lamina propria T lymphocytes, we found that intestinal IEL and lamina propria T lymphocytes were present in normal numbers in RAG-2-deficient mice that were reconstituted with bone marrow from ₄^-/- fetuses (our unpublished observations). Taken together with the reduced lamina propria lymphocyte number observed in _E^-/- mice, these studies suggest that _Eß₇ participates in the localization of lamina propria T lymphocytes. Additional studies will be required to define the cellular and molecular basis for this interaction.

By comparing the impact of targeted integrin gene disruption, insight is gained into the relative roles of the ₄ß₇ and _Eß₇ integrins. In integrin ß₇-deficient mice, there are markedly reduced numbers of T and B cells in Peyer’s patches, intestinal lamina propria, and in the intestinal epithelium (43). In addition, in mice whose T lymphocytes lack the ₄ subunit, there are reduced numbers of T and B lymphocytes in Peyer’s patches, while the numbers of IEL and of lamina propria T lymphocytes were not reduced (Ref. 44, and our unpublished observations). These findings suggest that ₄ß₇ is essential for B and T cell localization to Peyer’s patches, but not for T lymphocyte localization to the intestinal epithelium or lamina propria. Finally, in the _E^-/- mice described in this report, the number of IEL and lamina propria T lymphocytes was partially reduced, without an impact on the localization of B or T cells to Peyer’s patches. Thus, it appears that both _Eß₇ and ₄ß₇ can function in the localization of T lymphocytes to the intestinal lamina propria and epithelium, while _Eß₇ does not mediate T lymphocyte localization to Peyer’s patches. The integrin _E-deficient mice described herein will provide an important reagent with which to further define the in vivo functions of _Eß₇, and of the cells that express it, in health and disease.

	Acknowledgments

 
We thank T. J. Smith and J. H. Weis for murine _E cDNA probes; H. B. Warren for histopathologic analyses; P. Kilshaw and R. MacDonald for mAbs; C. Nagler-Anderson for technical advice; X. Hu, E. Meluleni, and J. Connolly for technical assistance; and M. Hemler, V. Hsu, J. Higgins, M. Carr, and G. J. Russell for helpful discussions.

	Footnotes

 
¹ This work was supported by a grant from the Deutsche Forschungsgemeinschaft (to M.P.S. and U.G.S.), grants from the National Institute of Health (NIH) and the Mathers Foundation (to M.J.G., who is a Scholar of the Leukemia Society of America), National Research Service institutional training grants from the NIH (to C.M.A. and J.P.D), a fellowship from the Swedish Foundation for International Cooperation in Research and Higher Education (STINT) (to W.W.A), grants from NIH (to M.B.B.), grants from NIH, the Crohn’s and Colitis Foundation, the Cancer Research Institute, and a Pilot and Feasibility grant from the Center for the Study of Inflammatory Bowel Diseases (to C.M.P), Grant RO1HD29028 from National Institute for Child Health and Human Development, and Grant ROHG00951 from the National Center for Human Genome Research (to D.R.B.).

² Current address: Department of Dermatology, Heinrich-Heine-University, Moorenstr. 5, D-40225 Düsseldorf, Germany

³ Address correspondence and reprint requests to Dr. Christina M. Parker, Brigham and Women’s Hospital, Smith-552B, 1 Jimmy Fund Way, Boston, MA 02115. E-mail address:

⁴ Abbreviations used in this paper: IEL, intraepithelial lymphocyte; Itgae, integrin gene encoding _E; SSCP, single strand conformation polymorphism; SPF, specific pathogen-free; MFI, mean fluorescence intensity.

Received for publication January 12, 1999. Accepted for publication March 17, 1999.

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