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The Journal of Immunology, 2003, 171: 745-753.
Copyright © 2003 by The American Association of Immunologists

CD49d Overexpression and T Cell Autoimmunity1

Ru-Ran Mo*, Julie K. Eisenbraun*, Joanne Sonstein{dagger}, Ronald A. Craig{ddagger}, Jeffrey L. Curtis{dagger}, Lloyd M. Stoolman{ddagger}, Jun Chen* and Raymond L. Yung2,*,§

Divisions of * Geriatric Medicine and Rheumatology and {dagger} Pulmonary and Critical Care Medicine, Department of Internal Medicine, and {ddagger} Department of Pathology, University of Michigan, and § Geriatrics Research, Education, and Clinical Center, Ann Arbor Veterans Affairs Health System, Ann Arbor, MI 48109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
D10.G4.1 (D10) cells, a murine conalbumin-reactive Th2 cell line, made to overexpress the {beta}2 integrin LFA-1 by pharmacological manipulation or by transfection become autoreactive and are capable of inducing in vivo autoimmunity. However, whether this is specific to LFA-1 and whether overexpression of other T cell integrin molecules has the same effect are unknown. We examined the functional consequences of T cell CD49d ({alpha}4 integrin) overexpression by transfecting murine CD49d cDNA into D10 cells. Similar to the LFA-1-transfected cells, the CD49d-overexpressing T cells are autoreactive and proliferate in response to APCs in an MHC class II-dependent manner in the absence of nominal Ag. Additionally, CD49d overexpression is associated with increased in vitro adhesion to endothelial cells and increased in vivo splenic homing. However, in contrast to LFA-1 overexpression, increased T cell CD49d expression is not associated with autoreactive cytotoxicity or the ability to induce in vivo autoimmunity. In addition to the novel observation that CD49d overexpression is sufficient to induce T cell autoreactivity, our results also support the hypothesis that the ability to induce in vivo autoimmunity is related to T cell cytotoxicity and not to T cell proliferation function in the D10 murine adoptive transfer model of autoimmunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The integrin family of adhesion molecules includes a series of noncovalently linked {alpha} and {beta} glycoprotein chains that are expressed on a variety of cell types (1, 2). These molecules have been shown to play an important role in leukocyte migration and adhesion to vascular endothelium. At least 16 {alpha}- and 8 {beta}-chains have been identified. A number of integrins are expressed on lymphocytes including LFA-1 (CD11a/CD18, {alpha}L{beta}2), very late activation Ag-4 (VLA-4)3 (CD49d/CD29, {alpha}4{beta}1), lymphocyte Peyer’s patch adhesion molecule-1 (CD49d/{beta}7, {alpha}4{beta}7), and {alpha}v{beta}3 (CD51/CD61). Of these, LFA-1 and VLA-4 have been studied in the greatest detail.

T cell integrins have been shown to play an important role in the pathogenesis of many autoimmune diseases. For example, autoreactive LFA-1-overexpressing T cells have been found in patients with systemic lupus erythematosus (3, 4). Interestingly, the subset of lupus patients with vasculitis also has T cells that overexpress the CD49d Ag (4). However, the pathogenic significance of these autoreactive T cells is unclear. We recently established a murine model to determine the role of T cell integrin overexpression in the induction of autoimmunity. In earlier studies, it was shown that human and mouse CD4+ T cells treated with DNA-hypomethylating agents overexpress LFA-1 and become autoreactive (5, 6, 7, 8, 9, 10). The drug-treated cells lose the requirement for nominal Ags and will proliferate in response to MHC class II molecules on APCs alone. Importantly, the LFA-overexpressing cells will induce apoptosis in syngeneic macrophages (M{phi}) without specific Ag (6, 8, 9, 10), thereby providing a potential source of autoantigens that may then take part in the autoimmune process. Adoptive transfer of the drug-treated autoreactive T cells into syngeneic murine hosts results in the development of in vivo autoimmunity characterized by the presence of autoantibodies, immune complex glomerulonephritis, and autoimmune liver and lung diseases (6, 8, 9, 10). This murine system appears to be independent of T cell cytokine profile, because both drug-treated Th1 and Th2 cells are autoreactive and can induce autoimmunity (11). Interestingly, the spleen appears to be critical in this model, because splenectomized mice receiving the autoreactive cells do not develop evidence of autoimmune disease (12). To directly show that the observed in vitro T cell autoreactivity is due to increased T cell LFA-1 expression, we transfected the human CD11a cDNA into a tetanus toxoid-reactive human CD4+ T cell line (7) and the murine CD18 cDNA into D10 cells (9). In both human and murine T cells, the resulting LFA-1 overexpression is associated with the induction of in vitro autoreactivity, similar to the drug-treated T cells. Finally, we showed that adoptive transfer of the CD18-transfected D10 cells resulted in the development of autoantibodies (anti-ssDNA, anti-dsDNA) and immune complex glomerulonephritis in syngeneic AKR mice. However, the underlying mechanisms by which the autoreactive D10 cells induce in vivo autoimmunity are unclear. It is also unknown whether the ability to induce T cell autoreactivity and in vivo autoimmunity is unique to LFA-1 and whether overexpression of other T cell integrin, such as the {alpha}4 integrins, will have similar effects. This is an important question because the {alpha}4 integrins have been implicated in a number of human (13, 14, 15) and animal (16, 17, 18) models of autoimmune diseases. In this report, we examined the role of {alpha}4 integrin overexpression in autoimmunity by stably transfecting a cloned murine T cell line with a CD49d cDNA construct. We then asked whether the transfected cells become autoreactive and whether adoptive transfer of the {alpha}4-overexpressing T cells will induce in vivo autoimmunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and peritoneal M{phi} isolation

Six- to 8-wk-old female AKR (Iak) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in a specific pathogen-free environment. Peritoneal M{phi} were obtained by i.p. thioglycolate (BD Biosciences, San Diego, CA) injection and harvested 3 days later as previously described (6, 8, 9).

T cell and endothelial cell culture

D10 cells (19) obtained from the American Type Culture Collection (Manassas, VA) were maintained by challenging them weekly with irradiated syngeneic AKR/J mouse splenocytes and conalbumin (8, 9). Because of a report that the D10 cell line may contain an autoreactive subset (20), the D10 cells were subcloned by limiting dilution at ≤0.2 cells/well, and a nonautoreactive subclone was selected. All cells used for functional or cytofluorographic analysis were studied 6 days after challenge. Dermal microvascular endothelial cells from AKR mice were isolated according to a previously published protocol (21). Identity of the isolated endothelial cells was confirmed by their typical cobblestone appearance and Von Willibrand factor (21) and CD34 (22) expression.

Generation of the pSub2-CD49d constructs and transfection into D10 cells

The expression vector pSub2 that we used previously to generate the D10/CD18 transfectants (9) was donated by Dr. M. Clarke (University of Michigan). The pSub2 polylinker site was cleaved with EcoRV, and EcoRI(NotI) adapters (Life Technologies, Gaithersburg, MD) ligated to the blunt ends. A full-length 4-kb cDNA-encoding murine CD49d in pBSK plasmid, kindly donated by Dr. I. Weissman (Stanford University, Stanford, CA) (23), was excised with NotI and cloned into the NotI sites generated by the adapters. The cDNA orientation in the subclones was determined by digestion with BglII, and a plasmid containing the complete cDNA in the sense and antisense (control) direction was selected. The construct was linearized by digestion with ScaI, and D10 cells were transfected by electroporation as before (7, 9). Stable transfectants were selected and maintained by culturing in medium containing 250 µg/ml Geneticin (G418) (Life Technologies). Finally, the transfectants were subcloned by limiting dilution at ≤0.2 cells/well.

DNA isolation and Southern analysis

Southern analysis was performed using previously published protocols (7, 9). Briefly, 20 µg of genomic DNA from control or transfected D10 cells were digested with NotI (Boehringer Mannheim, Mannheim, Germany), fractionated through agarose gels, and transferred to nylon filters (MagnaGraph; Micro Separations, Westborough, MA). The filters were probed using a 32P-labeled full-length CD49d cDNA, excised from pSub2 with NotI, and exposed to x-ray film.

Flow-cytometric analysis

Cultured T cells were stained with mAbs conjugated with FITC or PE including anti-CD3, -TCR, -CD18, -CD28, -CD29, -{beta}7, -CD49d, -CD62L, -CD49d/{beta}7 (DATK32) and goat anti-mouse Ig (Coulter, Hialeah, FL) as previously described (6, 7, 8, 9), and analyzed on a Coulter ELITE flow cytometer. All of the mAbs used in these studies were purchased from BD PharMingen (San Diego, CA) unless stated otherwise.

Proliferation assay

Proliferation assays were performed as described (6, 7, 8, 9), using irradiated AKR splenocytes as APCs. For the inhibition studies, the CD49d-transfected D10 cells were preincubated with mAbs against CD49d, CD29, {beta}7, CD18, Iak, Iab/d, or control Ig Abs for 30 min at 37°C, before coculturing with the APCs.

Cytotoxicity assay

For cytotoxic response, peritoneal M{phi} were labeled with 51Cr-labeled Na2Cr2O7 (New England Nuclear, Boston, MA) and used as targets as previously described (6, 9). Con A (2 µg/ml, final concentration) stimulation was used as positive control. All of the cytotoxicity experiments were performed in quadruplicate, and the results are expressed as cytotoxicity index and presented as the mean ± SEM.

Static adhesion assay

Second-passage microvascular endothelial cells were allowed to grow to 90% confluence in 1% gelatin-coated 24-well plates (Costar, Cambridge, MA). Untransfected and CD49d-transfected D10 cells were labeled with 51Cr by culturing 10 x 106 cells with 100 µCi of 51Cr-labeled Na2Cr2O7 in 1 ml at 37°C for 1 h. The 51Cr-labeled D10 cells were then allowed to adhere to the endothelial cells for 30 min at 37°C. The adhered cells were then lysed with Nonidet P-40, and radioactivity was counted by a scintillation spectrometer (6, 9). For the functional blockade experiments, the CD49d-transfected D10 cells were preincubated with anti-CD49d, -CD29, or -{beta}7 mAbs for 30 min at 37°C and washed three times with RPMI 1640, before adhesion to the endothelial cells.

Flow adhesion assay

The parallel-plate flow chamber used in the present study has been described previously in detail (24, 25). Microvascular endothelial monolayers on coverslips were stimulated with TNF-{alpha} (2.5 ng/ml) for 16 h at 37°C and mounted on an inverted microscope. A circular glass window in the top plate allows direct examination of the monolayer during the experiment. The monolayer was perfused for 5–10 min with perfusion medium. A total of 106 cells per milliliter of perfusion medium of the CD49d-transfected and untransfected D10 cells were drawn through the chamber at 2.0 dyne/cm2 for 5 min and then decreased to 0.2 dyne/cm2 for 5 min. Leukocyte adhesion was determined by counting the number of T cells in 20 randomly selected high-power microscope fields during the final minute at each level of flow.

Western blot

Tyrosine phosphorylation of anti-CD3-stimulated D10 and D10 transfectants was determined as before (26). Briefly, the cells were stimulated with anti-CD3 (10 mg/ml) for 10 min and cross-linked with goat anti-rat IgG. Proteins from the cells were resolved on 10% SDS-polyacrylamide gel and transferred to nitrocellulose-1 membrane. The membrane was then incubated with HRP-conjugated anti-phosphotyrosine Ab (Chemicon International, Temecula, CA) and anti-mouse IgG (Chemicon International). Detection was performed using the ECL system (Amersham, Arlington Heights, IL) as before (27). The membrane was then stripped and probed with anti-{beta}-actin Abs (Chemicon International).

T cell adoptive transfer

To induce autoimmunity, 5 x 106 CD49d-transfected and untransfected D10 cells were injected i.v. via the tail vein into 11 and 10 6-wk-old AKR mice, respectively, every 2 wk for a total of six injections. This is the same protocol we have used successfully to induce autoimmunity using LFA-1-overexpressing D10 cells (6, 8, 9, 10, 11) and is based on the murine graft-vs-host T cell adoptive transfer model (28). The mice were sacrificed 4 wk after the last adoptive transfer. A complete necropsy was done.

ELISA

Splenic B cells from AKR/J mice were isolated by MACS (Miltenyi Biotec, Auburn, CA) using CD19 microbeads (27) and cocultured with the D10 transfectants for 72 h as before (29). Anti-ssDNA Abs in the supernatant were measured by ELISA using previously published protocols (8, 9, 10, 11).

In vivo splenic homing

Untransfected and CD49d-transfected D10 cells were labeled with CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA), CFSE (both from Molecular Probes, Eugene, OR), or PKH26 green fluorochrome (Sigma-Aldrich, St. Louis, MO) using the protocols provided by the manufacturers (12). Viability of the cells was determined by trypan blue exclusion and was similar in both the labeled and unlabeled cells. The labeled transfected and untransfected cells were injected i.v. via the tail vein, and the mice were killed 24 h later. Labeled D10 cells were detected by staining splenocytes with FITC- or PE-conjugated anti-CD4 and then enumerating cells expressing both CD4 and CMFDA or CFSE as before (12).

Statistical analysis

The difference between means was tested using Student’s two-tailed t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of CD49d-transfected D10 cells

D10, which has previously been shown to induce autoimmunity after transfection with the CD18 cDNA (9), was first subcloned by limiting dilution, and an Ag-reactive subclone was selected. A full-length cDNA-encoding murine CD49d was cloned into the mammalian expression vector pSub2, linearized by digestion with ScaI, and transfected into the D10 subclone by electroporation, and stable transfectants were selected using G418. The transfected cells were subcloned by limiting dilution (0.2 cell/well). Incorporation of the full-length CD49d cDNA was confirmed by Southern analysis (Fig. 1A). Experiments were then performed to determine the effect of CD49d transfection on the cell surface expression of adhesion and costimulatory molecules by flow-cytometric analyses. The cultured T cells were stained with anti-CD3, -TCR, -CD18, -CD28, -CD29, -CD49d, -{beta}7, and -CD62L (Fig. 1B). The results show that D10 cells normally express a low level of CD49d, CD29, and {beta}7 that is only slightly above background staining. Transfection of CD49d cDNA caused a 10-fold increase in CD49d expression in D10 cells but no significant change in the expression of other adhesion or costimulatory Ags. Interestingly, the CD49d transfectants showed increased expression of the corresponding {beta}7 but not the CD29 chain, suggesting that the overexpressed {alpha}4 chains are preferentially forming a heterodimer with the endogenous {beta}7 chains. To confirm this, the transfected cells were stained with the DATK32 mAb that is specific for the {alpha}4{beta}7 heterodimer (Fig. 1C). The reason why the CD49d-transfected D10 cells overexpress the corresponding {beta}7 and not the CD29 chain is unclear. However, examination of 15 other CD49d-transfected clones all showed similar increased expression of {beta}7 and not the CD29 chain. This suggests that the selective increase in the {beta}7 chain in response to increased CD49d expression is not a random but a regulated process in D10 cells.



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  		FIGURE 1. A, Southern blot analysis of CD49d-transfected D10 cells. DNA from the pSub2-CD49d construct (a), untransfected D10 cells (b), and subcloned CD49d transfectants (c) were digested with NotI, fractionated by agarose gel electrophoresis, transferred to nylon filters, and hybridized with the full-length CD49d cDNA. The full-length CD49d cDNA in the transfectant and the pSub2-CD49d construct is identified by the 4.0-kb band. B, CD49d transfectants () and untransfected D10 cells (->) were stained with anti-CD3, -TCR, -CD18, -CD28, -CD29, -{beta}7, -CD49d, and -CD62L mAbs. The x-axis represents fluorescence intensity on an arbitrary scale, and the y-axis represents cell number. C, CD49d transfectants and D10 cells stained with anti-CD49d/{beta}7 Abs (DATK32) or isotype-matched Abs ({blacktriangleleft}).

 
T cell proliferation response

Because D10 cells overexpressing the integrin LFA-1 no longer require Ag and will proliferate in the presence of APCs alone (8, 9, 10, 11), we determined whether CD49d overexpression has the same effect in D10 cells. Three CD49d-transfected clones (clones 16, 29, and 39) were tested for autoreactive proliferation response against irradiated syngeneic splenocytes. All of the CD49d transfectants were shown to proliferate without conalbumin, similar to the CD18-transfected D10 cells (Fig. 2A). Because clone 29 most closely resembled the CD18 transfectants in its autoreactivity response, the line was chosen for further characterization and for the in vivo adaptive transfer experiment. Clone 29 was found to proliferate in the presence of APCs and suboptimal concentrations of conalbumin (Fig. 2B). In addition, the CD49d-transfected D10 cells became autoreactive and proliferated in response to autologous APCs without the addition of Ag (Fig. 2C), similar to D10 cells made to overexpress CD18 by transfection (9). The increased T cell Ag proliferation response in the transfectants is reversed by the addition of saturating amount (10 µg/ml, final concentration) of functionally blocking mAbs against CD49d, {beta}7, or the appropriate MHC class II Ag (Fig. 2D). Anti-CD18 treatment also partially reversed the increased Ag proliferation response, consistent with the known costimulatory role that LFA-1 plays in normal T cell Ag response. Similarly, the autoreactive proliferation response of the CD49d transfectants is found to be CD49d and MHC class II dependent (Fig. 2E).



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  		FIGURE 2. CD49d overexpression causes autoreactivity in D10 cells. A, Autoreactive proliferation of CD18 transfectants, three different CD49d clones (clones 16, 29, and 39), and untransfected D10 cells were compared. B, The Ag proliferation response of untransfected (D10) and CD49d transfected (clone 29) D10 cells showing that the CD49d transfectants will proliferate in the presence of suboptimal concentrations of Ag (conalbumin). C, Autoreactive proliferation response of untransfected and CD49d-transfected D10 cells in the presence of the indicated number of irradiated splenocytes. Effects of saturating amount (10 µg/ml, final concentration) of functional Ab blockade on the Ag (D) and autoreactive (E) proliferation responses of CD49d-transfected D10 cells. The results represent the mean ± SEM of quadruplicate determinations.

 
Tyrosine phosphorylation

We have previously shown increased signaling in LFA-1-overexpressing T cells. (26). We next determined whether T cells overexpressing CD49d also have increased signaling as measured by tyrosine phosphorylation. The results show that both the CD18- and CD49d-transfected cells have increased TCR/CD3-mediated tyrosine phosphorylation of cellular protein substrates (Fig. 3).



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  		FIGURE 3. CD49d transfectants display increased TCR/CD3-mediated tyrosine phosphorylation of cellular protein substrates. A, Western blotting using anti-phosphotyrosine Abs. Lanes 1, Untransfected D10; 2, D10/CD18 transfectants; 3, CD49d transfectant clone 16; 4, clone 29; and 5, clone 39. B, Densitometric analysis of tyrosine-phosphorylated proteins. Bar graph showing relative expression of the two most prominent bands (45 and 15 kDa) in D10 (equal to 1), CD18, and clone 16, 29, and 39 CD49 transfectants. The results are corrected for {beta}-actin controls.

 
T cell cytotoxicity

Earlier studies have established that untransfected D10 cells will lyse syngeneic M{phi}, but only in the presence of conalbumin (8). In contrast, CD18-overexpressing D10 cells that cause in vivo autoimmunity induce apoptosis in syngeneic M{phi} in vitro without Ag (8, 9) by a process that likely involves TNF-related apoptosis-inducing ligand (Apo2 ligand) and TNF-like weak inducer of apoptosis (Apo3 ligand) (30). This has led us to propose that such M{phi} killing in lymphoid organs, including the spleen, may provide a source for autoantigens that contribute to the production of autoantibodies and in vivo autoimmunity in our D10 model. Therefore, we determined whether CD49d overexpression will also induce D10 cells to kill syngeneic M{phi} in the absence of Ag. The results showed that, unlike CD18 overexpression, clone 29 CD49d transfectants do not cause increased M{phi} killing compared with untransfected D10 cells (cytotoxicity index: untransfected D10 cells vs CD49d transfectants = 100 vs 107 ± 9.9, mean ± SEM; p = NS; n = 5 independent experiments). Clones 16, 29, and 39 all had similar cytotoxicity response (mean + SD: 39 + 2, 33 + 2, and 35 + 3%, respectively). Positive controls for the experiments have included D10 and D10/CD49d Ag cytotoxicity response (cytotoxicity index: D10 vs CD49d transfectants = 126 ± 11 vs 152 ± 20, mean ± SEM; p = NS), with results similar to what was previously reported (6, 8). In addition, Con A stimulation caused a further increased (+27%) in M{phi} killing in the CD49d transfectants.

Adhesion to microvascular endothelial cells

Primary dermal microvascular endothelial cells were isolated from 3- to 4-wk-old female AKR mice. Vascular endothelial cells were identified according to morphological appearance, Von Willibrand factor, and CD34 expression (data not shown). In vitro adhesion function of the CD49d transfectants was assessed by adhesion assays, performed under static and flow conditions. Under static condition, four times more CD49d transfectants were found to adhere to TNF-stimulated endothelial cells compared with untransfected D10 cells (p < 0.01) (Fig. 4A). The increased adhesion of the CD49d transfectants was reversed with the addition of anti-CD49d, but not anti-{beta}7 or CD29 Abs (Fig. 4B). Binding of CD49d transfectant under high (physiological) flow rate (2.0 dyne/cm2) was similar to that of untransfected D10 cells (data not shown). However, a 4-fold increase in binding to endothelial cells was observed under low-flow condition (0.2 dyne/cm2) (p < 0.01) (Fig. 4C). The simulated flow adhesion data confirm the results of the static adhesion assays and are consistent with the notion that {alpha}4 integrin is involved in the firm adhesion of leukocytes to vascular endothelial cells, and less in the initial contact or rolling stage of the interaction.



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  		FIGURE 4. D10 CD49d overexpression is associated with increased adhesion to primary endothelial cells. A, Static adhesion assay showing CD49d transfectants adhere four times more to microvascular endothelial cells than untransfected D10 cells. The results represent the mean ± SD of four independent experiments and are expressed relative to the radioactivity of adhered untransfected D10 cells (equal to 100%). B, The increased adhesion of the D10/CD49d transfectants is secondary to increased CD49d expression. Preincubation of the CD49d transfectants with anti-CD49d but not anti-{beta}7 decreased T cell binding to levels comparable with that seen in untransfected D10 cells. The results represent the mean ± SD of quadruplicate determinations. C, D10/CD49d transfectants have increased binding to endothelial cells under simulated flow condition. The results represent mean ± SD of the number of untransfected D10 and CD49d transfectants that adhere to the microvascular endothelial monolayer under low-flow state (0.2 dyne/cm2) in 20 randomly selected areas under high-power magnification.

 
In vivo effects of D10 CD49d overexpression

Adoptive transfer of D10 cells made to overexpress LFA-1 by pharmacological treatment (8, 10, 11, 12) or by CD18 transfection (9) caused in vivo autoimmunity in syngeneic mice, with features that include the development of anti-ssDNA and -dsDNA Abs, immune complex glomerulonephritis, and interstitial pneumonitis. Because CD49d-overexpressing D10 cells are also autoreactive and because CD49d overexpressing T cells have been found in lupus patients with vasculitis (4), we next determined the ability of the CD49d transfectants to induce in vivo autoimmunity, using the same adoptive transfer protocol that we used previously (8, 9, 10, 11, 12). Interestingly, although clone 29 CD49d transfectants display similar in vitro autoreactive proliferation as the CD18 transfectants, the D10/CD49d transfectants failed to elicit an autoimmune response in syngeneic hosts. Mice receiving the CD49d-overexpressing T cells did not develop anti-ssDNA or -dsDNA Abs (Fig. 5). Total serum IgG (OD: D10 vs CD49d, mean ± SD = 0.432 ± 0.006 vs 0.436 ± 0.008; p = NS) and IgM (OD: D10 vs CD49d, mean ± SD = 0.045 ± 0.039 vs 0.015 ± 0.006; p = NS) levels were also determined by ELISA and were found to be comparable in both groups, excluding the possibility that the lack of difference in autoantibody levels is due to variability in Ig levels. Histologic analysis also failed to show evidence of autoimmune features or vasculitis in mice receiving the CD49d transfectants (Fig. 6).



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  		FIGURE 5. Measurement of anti-DNA Abs. Sera were obtained from 5-mo-old female MRL/lpr mice, mice receiving control D10 cells (10 mice), and mice receiving CD49d-transfected D10 cells (11 mice). Anti-ssDNA (A) and anti-dsDNA (B) Abs were measured by ELISA.

 


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  		FIGURE 6. H&E stain of a representative lung (A and B), kidney (C and D), and liver (E and F) sections from mice receiving control D10 cells (A, C, and E) or CD49d transfectants (B, D, and F) showing no evidence of in vivo autoimmunity in contrast to the pathology described in mice receiving the D10/CD18 transfectants (9 ).

 
ELISA

Anti-ssDNA measurements were done following in vitro cocultures of CD49d transfectants with B cells from AKR/J mice. Consistent with the in vivo data, the CD49d transfectants did not promote B cell anti-DNA response (OD, mean ± SD of quadruplicate determinations: B cell alone, 0.106 ± 0.012; B cells with D10, 0.126 ± 0.008; B cells with clone 16, 0.119 ± 0.01; B cells with clone 29, 0.117 ± 0.004; B cells with clone 39, 0.129 ± 0.08; and MRL/lpr, 3.668 ± 0.247).

In vivo splenic homing

We have previously shown that D10 homing to the spleen is critical for the development of in vivo autoimmunity in the D10 murine autoimmunity model, and splenectomy will protect the recipients receiving the LFA-1-overexpressing D10 cells from developing the disease (12). To exclude the possibility that T cell CD49d overexpression may cause decreased splenic homing by directing the CD49d transfectants to mucosal or other lymphoid organs and thus protect the recipients from developing autoimmunity, we examined the effect of CD49d expression on in vivo T cell homing. This was initially done by injecting 5 x 106 CMFDA-labeled D10 cells or CD49d transfectants into AKR mice. The number of CD4+CMFDA+ cells in the spleens of the recipient mice was then determined by flow-cytometric analysis 24 h later. CD49d transfectants were found to traffic to the spleen three to four times more than the control D10 cells (p < 0.05) (Fig. 7A). It is possible that the observed increased splenic homing is the result of unintended differences in the number of cells given to the individual animal in the adoptive transfer process. To exclude this possibility, AKR mice were given an i.v. mixture of either 5 x 106 CFSE (green)-labeled D10 cells and 5 x 106 PKH26 (red)-labeled CD49d transfectants, or 5 x 106 CFSE-labeled CD49d transfectants and 5 x 106 PKH26-labeled D10 cells. The number of D10 and CD49d transfectants in the spleen was then enumerated by flow cytometry 24 h later. Mice that received the CFSE-labeled D10 cells and PKH26-labeled CD49d transfectants were found to have significantly more CD49d transfectants in the spleen (Fig. 7B) compared with untransfected D10 cells (p < 0.001). Similarly mice receiving the PKH26-labeled D10 cells and CFSE-labeled CD49d transfectants were found to have significantly increased D10/CD49d splenic homing (D10 vs CD49d, mean ± SD, 87 ± 5 vs 132 ± 10 per 100,000 events; n = 8; p < 0.001).



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  		FIGURE 7. D10 CD49d overexpression causes increase in vivo splenic homing. A, A total of 5 x 106 CMFDA-labeled D10 or CD49d transfectants were injected i.v. into nine AKR mice. The spleen cells were harvested 24 h later, and the percentages of CMFDA-positive cells were enumerated by flow-cytometric analysis. The results showed that significantly more CD49d transfectants homed to the spleen (p < 0.05). The results are expressed as the percentage of spleen cells that are CMFDA positive. Four AKR mice were given 5 x 106 CFSE-labeled D10 cells and 5 x 106 PKH26-labeled CD49d transfectants, and the spleens were harvested 24 h later. A significantly greater number of CD49d transfectants was found to home to the spleen (B) (p < 0.001). The results are expressed as the number of CFSE-positive or PKH26-positive events per 100,000 events during the flow-cytometric studies.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In addition to their adhesion and T cell migration functions, the integrins are increasingly recognized for their role in Th cell activation (31) and in their ability to transduce bidirectional signals into and out of the cell (32, 33). T cell integrins have a major role in the initial Ag-independent adhesion to APCs. The initiation of the subsequent Ag-specific T cell proliferative response involves the engagement of the TCR/CD3 complex with antigenic peptide presented by a MHC molecule on the APC. However, this interaction is generally not sufficient to stimulate resting T cells. Additional costimulatory signals, such as those provided by CD7/CD28, LFA-1/ICAM-1 (34, 35, 36), or VLA-4/VCAM-1 (34) interactions, are necessary for optimal T cell effector functions. Adhesion through VLA-4 triggers tyrosine phosphorylation of a 105-kDa protein in lymphocytes (37) and may both promote (38) and inhibit (39) cell death. It appears that integrin- and CD28-mediated costimulations are distinct but may complement each other at different stages of the Th cell activation cascade (31). Compared with LFA-1 and VLA-4, the role of lymphocyte Peyer’s patch adhesion molecule-1 in Th cell activation and the resulting downstream functional consequence are less clear. The subpopulation of activated or memory T cells has been shown to express a high level of {alpha}4{beta}7 (40). Although in vitro stimulation of resting T cells is associated with a late up-regulation of integrin cell surface expression (41), early down-regulation of {alpha}4{beta}7 is also seen in activated T lymphocytes upon recognition of alloantigen or in the presence of superantigen (42).

In the current study, we determined the effects of CD49d overexpression on T cell autoimmunity by stably transfecting the {alpha}4 cDNA into D10 cells. In addition to CD49d overexpression, we found that transfecting the {alpha}4 cDNA into D10 cells results in increased cell surface expression of the corresponding {beta}7 but not the CD29 chain. The reason for the selective {beta}7 up-regulation in D10 cell is unknown but may be related to the observation that increased {beta}7 expression is a phenotype of memory T cells. Others have reported that transfecting the human CD49d cDNA into mouse L cells could induce de novo surface expression of host {beta}7 and {beta}1 chains, suggesting that the {alpha}4 subunit requires the {beta} subunit for surface expression (43). Our current results show that D10 cells overexpressing the {alpha}4 integrin also lose the requirement for nominal Ag and will proliferate in response to APCs alone, similar to the LFA-1-overexpressing T cells. The possibility that CD49d transfection caused overexpression of other adhesion or costimulatory molecules was excluded by flow-cytometric cell staining. The autoreactivity is MHC class II specific, as anti-Iak but not -Iad/b will reverse the D10 autoreactivity. Interestingly, the autoreactivity is also abolished by treatment with either anti-CD49d or anti-{beta}7 Abs, suggesting that both the heterodimer subunits are involved in this reaction.

We did not determine the basis by which overexpression of CD49d induced T cell autoreactivity in this report. Potential mechanisms include increased costimulatory signals and/or overstabilization of normally low-affinity TCR-Ia interactions. Recently it was shown that CD49d itself can also act as a cellular ligand for both {alpha}4{beta}1 and {alpha}4{beta}7, with functional consequences (44). Increased CD49d expression may therefore also provide an increased costimulatory signal to the adjacent CD49d transfectant cells. We have previously examined the mechanism in T cell autoreactivity caused by overexpression of CD18 and showed that CD18 overexpression permits TCR signal transmission in response to a normally subthreshold stimulus presented by M{phi}, consistent with overstabilization of TCR-Ia interaction. In addition, CD18 overexpression also causes increased tyrosine phosphorylation, although this alone is not sufficient to induce a proliferative response to a low level of stimuli (26). Therefore, it is reasonable to postulate that overstabilization of TCR-Ia also occurs in the CD49d-overexpressing D10 cells.

In contrast to the LFA-1-overexpressing D10 cells, the {alpha}4 transfectants do not kill syngeneic M{phi} and are incapable of inducing autoimmunity in vivo. Given the important role of the spleen in our T cell adoptive transfer model system, one potential explanation would be that the {alpha}4 overexpression may result in decreased trafficking of the autoreactive cells to the spleen, possibly through increased homing to other lymphoid organs, such as those in the gut mucosa. However, our data showed that {alpha}4 overexpression is associated with significantly increased T cell splenic homing. The idea that CD49d may participate in tissue- or organ-specific T cell trafficking is supported by existing literature. Naive-phenotype T cells recirculate through both peripheral and mucosal sites, whereas tissue-specific recirculation is a property of memory-phenotype (often {alpha}4{beta}7high) cells (45). Others have also shown that {alpha}4{beta}7high memory T lymphocytes home efficiently to the spleen (46). Mucosal addressin cell adhesion molecule-1 is expressed by the sinus-lining cells closest to the splenic lymphoid white pulp (47). The entrance and retention of T and B cells into the white pulp is a highly organized process (48, 49). The location of mucosal addressin cell adhesion molecule-1 in the spleen suggests that interaction with {alpha}4{beta}7 may be an important determinant in lymphocyte splenic homing. In addition, high {alpha}4{beta}7-expressing CD4+ T cells have also been described in the spleen (50).

The reason for the differential ability of the LFA-1- and CD49d-overexpressing D10 cells to induce in vivo autoimmunity is uncertain. The ability of the autoreactive LFA-1-overexpressing, but not the CD49d-overexpressing, D10 cells to cause M{phi} apoptosis may help explain the difference. D10 cells have been shown to be capable of inducing apoptosis in syngeneic M{phi} through Fas-independent pathways (30). Although how LFA-1 overexpression increases D10 cytotoxicity is unclear, many investigators have reported an important role of LFA-1 in modulating T cell cytotoxic functions (51, 52, 53, 54). In contrast, the {alpha}4 integrins have not been implicated in this process. Increased monocyte apoptosis has been reported in lupus patients, and it has been proposed that, if this occurs in the appropriate lymphoid organ such as the spleen, it may provide a source of antigenic nucleic acids that contributes to the autoimmune process in these patients (30, 55). Future experiments comparing the ability of autoreactive T cells to induce in vivo autoimmunity in control and apoptosis knockout mice may provide the definitive answer to this question. Another approach is to generate specific integrin transgenic mice. However, this approach may not be feasible, because integrin has been shown to contribute to the thymic deletion of potentially self-reactive T cells (56). Therefore, it is likely that the integrin-overexpressing autoreactive T cells will be deleted in the transgenic animals.

In summary we have shown that the ability to induce T cell autoreactivity is not limited to LFA-1, but that overexpression of other integrins such as CD49d is also sufficient to allow T cells to lose the requirement for Ag to proliferate, presumably through overstabilization of TCR-Ia interaction. However, in contrast to LFA-1, increased T cell CD49d expression is not sufficient to induce killing of APCs or in vivo autoimmunity. This will suggest that the ability of autoreactive T cells to cause autoimmune disease is dependent on their ability to induce apoptosis of APCs in lymphoid organs such as the spleen.


   Acknowledgments
 
We thank Dr. Bruce C. Richardson (University of Michigan) for his intellectual input and critical review of the manuscript.


   Footnotes
 
1 This work was supported by the Michigan Multipurpose Arthritis Center, Public Health Service Grants 1KO8AR01977-01A1, 1RO1 AI42753, 1RO1 HL61577, American Federation for Aging Research (Paul Beeson Physician Faculty Scholar Award), and the Geriatrics Research, Education, and Clinical Center of the Ann Arbor Veterans Affairs Medical Center. Back

2 Address correspondence and reprint requests to Dr. Raymond L. Yung, Room 5312, Cancer Center and Geriatrics Center Building, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0940. E-mail address: ryung{at}umich.edu Back

3 Abbreviations used in this paper: VLA-4, very late activation Ag-4; M{phi}, macrophage; CMFDA, 5-chloromethylfluorescein diacetate. Back

Received for publication January 29, 2003. Accepted for publication May 14, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Springer, T. A.. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76:301.[Medline]
  2. Kilger, G., B. Holzmann. 1995. Molecular analysis of the physiological and pathophysiological role of {alpha}4 integrins. J. Mol. Med. 73:347.[Medline]
  3. Richardson, B. C., J. R. Strahler, T. S. Pivirotto, J. Quddus, G. E. Bayliss, L. A. Gross, K. S. O’Rourke, D. Powers, S. M. Hanash, M. A. Johnson. 1992. Phenotypic and functional similarities between 5-azacytidine-treated T cells and a T cell subset in patients with active systemic lupus erythematosus. Arthritis Rheum. 35:647.[Medline]
  4. Takeuchi, T., K. Amano, H. Sekine, J. Koide, T. Abe. 1993. Upregulated expression and function of integrin adhesive receptors in systemic lupus erythematosus patients with vasculitis. J. Clin. Invest. 92:3008.
  5. Scheinbart, L. S., M. A. Johnson, L. A. Gross, S. R. Edelstein, B. C. Richardson. 1991. Procainamide inhibits DNA methyltransferase in a human T cell line. J. Rheumatol. 18:530.[Medline]
  6. Quddus, J., K. J. Johnson, J. Gavalchin, E. P. Amento, J. S. Warren, C. Chrisp, R. L. Yung, B. C. Richardson. 1993. Treating activated CD4+ T cells with either of two distinct DNA methyltransferase inhibitors, 5-azacytidine or procainamide, is sufficient to induce a lupus-like disease in syngeneic mice. J. Clin. Invest. 92:38.
  7. Richardson, B. C., D. Powers, F. Hooper, R. L. Yung, K. O’Rourke. 1994. Lymphocyte function associated antigen-1 overexpression and T cell autoreactivity. Arthritis Rheum. 37:1363.[Medline]
  8. Yung, R. L., J. Quddus J, C. E. Chrisp, K. J. Johnson, B. C. Richardson. 1995. Mechanisms of drug-induced lupus. I. Cloned Th2 cells modified with DNA methylation inhibitors in vitro cause autoimmunity in vivo. J. Immunol. 154:3025.[Abstract]
  9. Yung, R. L., D. Powers, K. Johnson, E. Amento, D. Carr, T. Laing, J. Yang. S. Chang, N. Hemati, B. C. Richardson. 1996. Mechanisms of drug-induced lupus. II. T Cells overexpressing LFA-1 cause a lupus-like disease in syngeneic mice. J. Clin. Invest. 97:2866.[Medline]
  10. Yung, R., S. Chang S, N. Hemati, K. Johnson, B. Richardson. 1997. Mechanisms of drug-induced lupus. IV. Comparison of procainamide and hydralazine with analogs in vitro and in vivo. Arthritis Rheum. 40:1436.[Medline]
  11. Yung, R., M. Kaplan, D. Ray, K. Schneider, R. R. Mo, K. Johnson, B. Richardson. 2001. Autoreactive murine Th1 and Th2 cells kill syngeneic macrophages and induce autoantibodies. Lupus 10:539.[Abstract/Free Full Text]
  12. Yung, R., R. Williams, K. Johnson, L. Stoolman, S. Chang, B. Richardson. 1997. Mechanisms of drug-induced lupus. III. Gender specific differences in splenic T cell homing explain increased disease severity in female mice. Arthritis Rheum. 40:1334.[Medline]
  13. Archelos, J. J., S. C. Previtali, H. P. Hartung. 1999. The role of integrins in immune-mediated diseases of the nervous system. Trends Neurosci. 22:30.[Medline]
  14. Takeuchi, T., T. Abe. 2002. Role of adhesion molecules in vasculitis syndrome. Intern. Med. 41:41.[Medline]
  15. Bernstein, C. N., M. Sargent, E. Rector. 2002. Alteration in expression of {beta}2 integrins on lamina propria lymphocytes in ulcerative colitis and Crohn’s disease. Clin. Immunol. 104:67.[Medline]
  16. Yednock, T. A., C. Cannon, L. C. Fritz, F. Sanchez-Madrid, L. Steinman, N. Karin. 1992. Prevention of experimental autoimmune encephalomyelitis by antibodies against {alpha}4{beta}1 integrin. Nature 356:63.[Medline]
  17. Baron, J. L., E. P. Reich, I. Visintin, C. A. Janeway, Jr. 1994. The pathogenesis of adoptive murine autoimmune diabetes requires an interaction between {alpha}4-integrins and vascular cell adhesion molecule-1. J. Clin. Invest. 93:1700.
  18. Issekutz, T. B.. 1991. Inhibition of in vivo lymphocyte migration to inflammation and homing to lymphoid tissues by the TA-2 monoclonal antibody: a likely role for VLA-4 in vivo. J. Immunol. 147:4178.[Abstract]
  19. Kaye, J., S. Porcelli, J. Tite, B. Jones, C. A. Janeway, Jr. 1983. Both a monoclonal antibody and antisera specific for determinants unique to individual cloned helper T cell lines can substitute for antigen and antigen-presenting cells in the activation of T cells. J. Exp. Med. 158:836.[Abstract/Free Full Text]
  20. Saizawa, M. K., E. Hug, S. Haque, P. Portoles, S. Suzuki, K. Eichmann. 1992. Autoreactivity of low but not of high CD4 variants of an antigen-specific, I-A-restricted mouse T cell clone. J. Immunol. 148:702.[Abstract]
  21. Murphy, H. S., N. Bakotoulos, M. K. Dame, J. Varani, P. A. Ward. 1998. Heterogeneity of vascular endothelial cell: differences in susceptibility to neutrophil mediated injury. Microvasc. Res. 56:203.[Medline]
  22. Kriehuber, E., S. Breiteneder-Geleff, M. Groeger, A. Soleiman, S. F. Schoppmann, G. Stingl, D. Kerjaschki, D. Maurer. 2001. Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages. J. Exp. Med. 194:797.[Abstract/Free Full Text]
  23. Neuhaus, H., M. C. Hu, M. E. Hemler, Y. Takada, B. Holzmann, I. L. Weissman. 1991. Cloning and expression of cDNAs for the {alpha} subunit of the murine lymphocyte-Peyer’s patch adhesion molecule. J. Cell Biol. 115:1149.[Abstract/Free Full Text]
  24. Gerszten, R. E., F. W. Luscinskas, H. T. Ding, D. A. Dichek, L. M. Stoolman, M. A. Gimbrone, Jr, A. Rosenzweig. 1996. Adhesion of memory lymphocytes to vascular cell adhesion molecule-1-transduced human vascular endothelial cells under simulated physiological flow conditions in vitro. Circ. Res. 79:1205.[Abstract/Free Full Text]
  25. Spertini, O., F. W. Luscinskas, G. S. Kansas, J. M. Munro, J. D. Griffin, M. A. Gimbrone, Jr, T. F. Tedder. 1991. Leukocyte adhesion molecule-1 (LAM-1, L-selectin) interacts with an inducible endothelial cell ligand to support leukocyte adhesion. J. Immunol. 147:2565.[Abstract/Free Full Text]
  26. Kaplan, M. J., L. Beretta-Hanash, R. Yung, B. C. Richardson. 2000. LFA-1 overexpression causes autoreactivity by overstabilizing low affinity TCR-Ia interactions. Immunol. Invest. 29:427.[Medline]
  27. Mo, R. R., J. Chen, Y. Han, C. Bueno-Cannizares, D. E. Misek, S. Hanash, R. L. Yung. 2003. T cell chemokine receptor expression in aging. J. Immunol. 170:895.[Abstract/Free Full Text]
  28. Tary-Lehmann, M., A. G. Rolink, P. V. Lehmann, Z. A. Nagy, U. Hurtenbach. 1990. Induction of graft-versus-host-associated immunodeficiency by CD4+ T cell clones. J. Immunol. 145:2092.[Abstract]
  29. Richardson, B. C., M. R. Liebling, J. L. Hudson. 1990. CD4+ cells treated with DNA methylation inhibitors induce autologous B cell differentiation. Clin. Immunol. Immunopathol. 55:368.[Medline]
  30. Kaplan, M. J., D. Ray, R. R. Mo, R. L. Yung, B. C. Richardson. 2000. TRAIL (Apo2 ligand) and TWEAK (Apo3 ligand) mediate CD4+ T cell killing of antigen-presenting macrophages. J. Immunol. 164:2897.[Abstract/Free Full Text]
  31. Van Seventer, G. A., R. T. Semnani, E. M. Palmer, B. L. McRae, J. M. van Seventer. 1998. Integrins and T helper cell activation. Transplant. Proc. 30:4270.[Medline]
  32. Coppolino, M. G., S. Dedhar. 2000. Bi-directional signal transduction by integrin receptors. Int. J. Biochem. Cell Biol. 32:171.[Medline]
  33. Hynes, R.. 2002. Integrins: bidirectional, allosteric signaling machines. Cell 110:673.[Medline]
  34. van Seventer, G. A., W. Newman, Y. Shimizu, T. B. Nutman, Y. Tanaka, K. J. Horgan, T. V. Gopal, E. Ennis, D. O’Sullivan, H. Grey, et al 1991. Analysis of T cell stimulation by superantigen plus major histocompatibility complex class II molecules or by CD3 monoclonal antibody: costimulation by purified adhesion ligands VCAM-1, ICAM-1, but not ELAM-1. J. Exp. Med. 174:901.[Abstract/Free Full Text]
  35. van Seventer, G. A., Y. Shimizu, K. J. Horgan, G. E. Luce, D. Webb, S. Shaw. 1991. Remote T cell co-stimulation via LFA-1/ICAM-1 and CD2/LFA-3: demonstration with immobilized ligand/mAb and implication in monocyte-mediated co-stimulation. Eur. J. Immunol. 21:1711.[Medline]
  36. Brunmark, A., A. M. O’Rourke. 1997. Augmentation of mature CD4+ T cell responses to isolated antigenic class II proteins by fibronectin and intercellular adhesion molecule-1. J. Immunol. 159:1676.[Abstract]
  37. Nojima, Y., D. M. Rothstein, K. Sugita, S. F. Schlossman, C. Morimoto. 1992. Ligation of VLA-4 on T cells stimulates tyrosine phosphorylation of a 105-kD protein. J. Exp. Med. 175:1045.[Abstract/Free Full Text]
  38. Damle, N. K., K. Klussman, G. Leytze, A. Aruffo, P. S. Linsley, J. A. Ledbetter. 1993. Costimulation with integrin ligands intercellular adhesion molecule-1 or vascular cell adhesion molecule-1 augments activation-induced death of antigen-specific CD4+ T lymphocytes. J. Immunol. 151:2368.[Abstract]
  39. Koopman, G., R. M. Keehnen, E. Lindhout, W. Newman, Y. Shimizu, G. A. van Seventer, C. de Groot, S. T. Pals. 1994. Adhesion through the LFA-1 (CD11a/CD18)-ICAM-1 (CD54) and the VLA-4 (CD49d)-VCAM-1 (CD106) pathways prevents apoptosis of germinal center B cells. J. Immunol. 152:3760.[Abstract]
  40. Schweighoffer, T., Y. Tanaka, M. Tidswell, D. J. Erle, K. J. Horgan, G. E. Luce, A. I. Lazarovits, D. Buck, S. Shaw. 1993. Selective expression of integrin {alpha}4{beta}7 on a subset of human CD4+ memory T cells with hallmarks of gut-trophism. J. Immunol. 151:717.[Abstract]
  41. Shimizu, Y., G. A. van Seventer, K. J. Horgan, S. Shaw. 1990. Regulated expression and binding of three VLA ({beta}1) integrin receptors on T cells. Nature 345:250.[Medline]
  42. Hernandez-Caselles, T., M. Martinez-Esparza, A. I. Lazarovits, P. Aparicio. 1996. Specific regulation of VLA-4 and {alpha}4{beta}7 integrin expression on human activated T lymphocytes. J. Immunol. 156:3668.[Abstract]
  43. Webb, D. L., P. J. Conrad, L. Ma, M. L. Blue. 1996. Induction of mouse {beta} integrin expression following transfection with human {alpha}4 chain. J. Cell. Biochem. 61:127.[Medline]
  44. Altevogt, P., M. Hubbe, M. Ruppert, J. Lohr, P. von Hoegen, M. Sammar, D. P. Andrew, L. McEvoy, M. J. Humphries, E. C. Butcher. 1995. The {alpha}4 integrin chain is a ligand for {alpha}4{beta}7 and {alpha}4{beta}1. J. Exp. Med. 182:345.[Abstract/Free Full Text]
  45. Mackay, C. R., W. L. Marston, L. Dudler. 1990. Naive and memory T cells show distinct pathways of lymphocyte recirculation. J. Exp. Med. 171:801.[Abstract/Free Full Text]
  46. Williams, M. B., E. C. Butcher. 1997. Homing of naive and memory T lymphocyte subsets to Peyer’s patches, lymph nodes, and spleen. J. Immunol. 159:1746.[Abstract]
  47. Kraal, G., K. Schornagel, P. R. Streeter, B. Holzmann, E. C. Butcher. 1995. Expression of the mucosal vascular addressin, MAdCAM-1, on sinus-lining cells in the spleen. Am. J. Pathol. 147:763.[Abstract]
  48. Stevens, S. K., I. L. Weissman, E. C. Butcher. 1982. Differences in the migration of B and T lymphocytes: organ selective localization in vivo and the role of lymphocyte-endothelial cell recognition. J. Immunol. 128:844.[Abstract]
  49. Kraal, G., I. L. Weissman, E. C. Butcher. 1983. Differences in in vivo distribution and homing of T cell subsets to mucosal versus non-mucosal lymphoid organs. J. Immunol. 130:1097.[Abstract]
  50. Andrew, D. P., L. S. Rott, P. J. Kilshaw, E. C. Butcher. 1996. Distribution of {alpha}4{beta}7 and {alpha}E{beta}7 integrins on thymocytes, intestinal epithelial lymphocytes and peripheral lymphocytes. Eur. J. Immunol. 26:897.[Medline]
  51. Matsumoto, G., Y. Omi, U. Lee, T. Nishimura, J. Shindo, J. M. Penninger. 2000. Adhesion mediated by LFA-1 is required for efficient IL-12-induced NK and NKT cell cytotoxicity. Eur. J. Immunol. 30:3723.[Medline]
  52. Poggi, A., R. Carosio, G. M. Spaggiari, C. Fortis, G. Tambussi, G. Dell’Antonio, E. Dal Cin, A. Rubartelli, M. R. Zocchi. 2002. NK cell activation by dendritic cells is dependent on LFA-1-mediated induction of calcium-calmodulin kinase. II. inhibition by HIV-1 Tat C-terminal domain. J. Immunol. 168:95.[Abstract/Free Full Text]
  53. Wang, P., M. Malkovsky. 2000. Different roles of the CD2 and LFA-1 T-cell co-receptors for regulating cytotoxic, proliferative, and cytokine responses of human V{gamma}9/V{delta}2 T cells. Mol. Med. 6:196.[Medline]
  54. Huang, G. T., X. Zhang, N. H. Park. 2000. Increased ICAM-1 expression in transformed human oral epithelial cells: molecular mechanism and functional role in peripheral blood mononuclear cell adhesion and lymphokine-activated-killer cell cytotoxicity. Int. J. Oncol. 17:479.[Medline]
  55. Richardson, B. C., T. Buckmaster, D. F. Keren, K. J. Johnson. 1993. Evidence that macrophages are programmed to die after activating autologous, cloned, antigen-specific, CD4+ T cells. Eur. J. Immunol. 23:1450.[Medline]
  56. Quddus, J., A. Kaplan, B. C. Richardson. 1994. Anti-CD11a prevents deletion of self-reactive T cells in neonatal C57BR mice. Immunology 82:301.[Medline]



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