HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Murakami, K. Articles by Pober, J. S. Search for Related Content PubMed PubMed Citation Articles by Murakami, K. Articles by Pober, J. S.

Human Endothelial Cells Augment Early CD40 Ligand Expression in Activated CD4⁺ T Cells Through LFA-3-Mediated Stabilization of mRNA¹

Kenji Murakami^2,*, Weilie Ma^*, Ramsay Fuleihan and Jordan S. Pober^3,*

^* Molecular Cardiobiology Program, Boyer Center for Molecular Medicine, and Department of Pediatrics, Yale University School of Medicine, New Haven, CT 06510

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human endothelial cells (EC) augment CD40 ligand (CD40L) expression on PHA-activated CD4⁺ T cells at early times (e.g., 4–6 h). Fixed EC, devoid of mRNA, are comparable to living EC in their capacity to augment early CD40L expression on CD4⁺ T cells. Fixed EC increase T cell mRNA expression of both IL-2 and CD40L compared with PHA alone at 6 h. EC are unable to increase the rate of transcription of CD40L compared with PHA alone as measured with a promoter-reporter gene, although they do increase transcription of an IL-2 promoter-reporter gene. Fixed EC prolong the half-life of CD40L mRNA >2-fold. Inclusion of anti-human LFA-3 (CD58) mAb or pretreatment of EC with an LFA-3 antisense oligonucleotide blocks EC-induced increases in CD40L expression, whereas mAb to ICAM-1 or pretreatment with ICAM-1 antisense oligonucleotide does not. Moreover, mAb to LFA-3 reverses the capacity of EC to prolong the half-life of CD40L mRNA, whereas mAb to ICAM-1, even in combination with mAb to ICAM-2, does not. We conclude that EC use LFA-3 to increase early CD40L protein expression on newly activated CD4⁺ T cells by stabilizing CD40L mRNA.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The CD40 ligand (commonly referred to as CD40L⁴ but officially designated CD154), is a 33-kDa type II membrane protein that is structurally homologous to TNF in its extracellular domain (1). CD40L is transiently expressed on activated but not resting CD4⁺ Th cells (1, 2, 3, 4). Interaction of CD40L on T cells with CD40 on B cells is critical for the development and function of the humoral immune system (5). For example, in vitro studies have shown that either mAb specific for CD40L (6) or soluble CD40Ig (1, 2) can block the ability of CD4⁺ T cells to activate B cells. Furthermore, cells transfected to express CD40L acquire the capacity to stimulate B cell proliferation and IgE secretion (7). These in vitro findings on the role of CD40L in humoral immunity have been supported by in vivo studies of the immune responses in mice deficient for either CD40 or CD40L (8, 9, 10, 11). In such mice, Ig class switching in response to T-dependent Ag is defective, resulting in accumulation of IgM, whereas responses to T-independent Ags are normal. CD40 or CD40L knockout mice also fail to form germinal centers in their lymphoid organs. Moreover, in humans, the X-linked hyperIgM (XHIM) syndrome has been linked to a defect in the CD40L molecule (12, 13, 14, 15, 16). XHIM patients show immune deficiencies similar to those found in CD40 or CD40L knockout mice.

CD40L has functions in vivo that extend beyond humoral immunity. This possibility was originally suggested by the observation that patients with the XHIM syndrome were more susceptible to infection by microbes such as Pneumocystis carinii, normally controlled by cell-mediated immunity. The role of CD40L in protective T cell responses may be mediated through CD40L-induced maturation of dendritic cell capacity to present Ag (17), CD40L-induced cytokine production by macrophages (18), or CD40L-induced adhesion molecule expression on endothelium (19, 20, 21), a key event in immune-mediated inflammation. Interruption of this signaling system in vivo by administration of anti-CD40L mAb limits experimental autoimmune diseases such as lupus nephritis and acute or chronic graft-vs-host disease (22, 23, 24, 25). Ab to CD40L also reduces T cell-mediated allograft rejection (24, 25).

Because CD40L is absent on resting T cells, inhibition of the induction of new CD40L expression on activated T cells presents a therapeutic target for immunosuppression that could be as effective as blocking CD40L engagement of CD40 by mAb. The induction of CD40L expression on resting human peripheral blood CD4⁺ T cells in response to PHA appears to be biphasic with early expression observable at 4–6 h and a second peak of expression observable at 24 h (26). Cotreatment of T cells with PMA plus PHA can increase the early expression of CD40L without affecting the magnitude of the second peak. Indeed, many of the original studies of CD40L regulation used PMA cotreatment to measure CD40L expression at early times (1, 3, 14, 27). Human endothelial cells (EC), like PMA, also can increase CD40L expression on PHA-activated CD4⁺ T cells during the early phase, i.e., at 4–6 h after activation (26). Neither blood monocytes nor B lymphoblastoid cells are able to replace EC as a signal for early CD40L expression. The purpose of the present study was to investigate the mechanism by which EC augment early expression of CD40L on activated CD4⁺ T cells. We find that EC do not modulate CD40L gene transcription but instead appear to stabilize CD40L mRNA. We also show that this action of EC is largely mediated through an LFA-3:CD2 interaction. These data provide the first evidence that EC, like professional APC, can provide costimulation via mRNA stabilization and that, in humans, LFA-3, like B7 molecules, is capable of mediating this effect.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Monoclonal Abs and antisense oligonucleotides (ASO)

Murine mAb used for FACS analysis of surface molecules used in this study were anti-CD40L PE-conjugated mAb and anti-CD25 PE-conjugated mAb (both from PharMingen, San Diego, CA). Isotype-matched FITC- and PE-conjugated irrelevant Ab (Coulter, Keenesaw, GA) were used as negative controls. Murine anti-human CD4, anti-CD8, anti-CD3, anti-CD16 (for NK cells), anti-CD56 (for NK cells), anti-CD19 (for B cells), and anti-CD14 (for monocytes) mAbs for cell purification were also purchased from Coulter. Inhibitory mouse mAb TS2/9 (anti-CD58/LFA-3) and 6E6 (anti-ICAM-1) were gifts from Dr. Paula Hochman (Biogen, Cambridge, MA) and Dr. Dario Altieri (Yale University, New Haven, CT), respectively. Anti-ICAM-2 mAb was obtained from Serotec (Oxford, England). K16/16 (nonbinding IgG1 control) was a gift from Dr. Donna Mendrick (Brigham and Women’s Hospital, Boston, MA).

Single-strand DNA ASO and scrambled control (SC) oligonucleotides were gifts from Dr. Frank Bennett (ISIS Pharmaceuticals, Carlsbad, CA). The sequence and composition of the oligonucleotides used in this study are reported in Table I. Oligonucleotides were either partially 2'-O-methoxyethyl modified or uniformly modified as indicated. The 2'-O-methoxyethyl modification has previously been reported to increase binding affinity for the target RNA and confer greater nuclease resistance (28, 29). The LFA-3 ASO inhibits LFA-3 expression by an RNase H-dependent mechanism, while the ICAM-1 ASO inhibits ICAM-1 expression by non-RNase H mechanism as previously reported (29). The effects of these reagents on cultured human EC have been recently described.⁵

Human EC were isolated from umbilical veins and cultured as previously described on human plasma fibronectin (FN)- or gelatin-coated tissue culture plastic (Falcon, Lincoln Park, NJ) in 20% FBS, medium 199, 2.5 mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 µg/ml) (all from Life Technologies, Grand Island, NY), and EC growth factor (Collaborative Biomedical Products, Bedford, MA) as previously described (26). Cultures were used at passage level 2 to 5, at which time the cells are 100% positive for von Willebrand factor and for CD31 but negative for CD45, indicating an absence of leukocyte contaminants.

PBMC were isolated by leukapheresis from healthy adult volunteer donors and further purified by centrifugation over lymphocyte separation medium (Oreganon Teknika, Durham, NC) according to the manufacturer’s instructions. The isolated PBMC were washed three times in HBSS (Mg²⁺, Ca²⁺ free) and either used immediately or suspended in 10% DMSO and 90% heat-inactivated FBS and cryopreserved in liquid nitrogen. No differences were seen in the responses of cells recovered from cryopreservation compared with freshly isolated cells.

CD4⁺ T cells were isolated from PBMC by negative selection. Cells were sequentially depleted of peripheral blood adherent cells (PBAC) by adsorption on FN-coated bacteriological plastic and then depleted of HLA-DR⁺, CD8⁺, and CD16⁺ cells by negative panning with mAb LB3.1 (IgG2b, anti-HLA-DR monomorphic determinant; a gift from Dr. Jack Strominger, Harvard University, Cambridge, MA), OKT8 (anti-CD8; CRL 8014, American Tissue Culture Collection, Manassas, VA) and 3G8 (IgG1, anti-CD16; a gift from Dr. Jay Unkeless, Mt. Sinai School of Medicine, New York, NY) at saturating conditions for 30 min at 4°C. The cells were then washed three times with RPMI 1640 (Life Technologies) and 5% FBS to remove excess Ab and enriched using goat anti-mouse IgG-bound Dynabeads (Dynal., Lake Success, NY) according to manufacturer’s instruction. The purity of the CD4⁺ T cell was determined by direct immunofluorescence labeling (all Abs from Coulter) with anti-CD4, anti-CD8, anti-CD3, anti-CD16 (for NK cells), anti-CD56 (for monocytes), anti-CD19 (for B cells), and anti-CD14 (for monocytes) using a FACSort flow cytometer (Becton Dickinson, San Jose, CA) and Lysis II software. The CD4⁺ enriched T cell populations used in these studies were 95% CD4⁺ cells and contained no detectable (<1%) CD14⁺, CD16⁺ CD56⁺, or CD19⁺ cells and a very small number (<2%) of CD8⁺ cells. Where indicated, PBAC were recovered from FN-coated dishes by treatment with EDTA. These populations, enriched for professional APCs, consist largely of CD14⁺ monocytes (80%) and CD19⁺ B cells (0–10%).

ASO treatment

EC in 100-mm tissue culture plates (Falcon) were transfected with either 25 nM SC or 25 nM ASO as indicated in the text using lipofectin (Life Technologies) according to methods described elsewhere (30). Forty-eight to 72 h after transfection, cells were harvested with trypsin. LFA-3 and ICAM-1 surface expression was quantified by direct immunofluorescence labeling with FITC-conjugated anti-LFA-3 and FITC-conjugated anti-ICAM-1 (both from Coulter), respectively, and FACS analysis.

Induction of CD40L and flow cytometry

CD4⁺ T cells were cultured in the presence or absence of optimal concentrations of PHA-L (3 µg/ml; Sigma, St. Louis, MO) or PHA plus PMA (10 ng/ml; Sigma) on human plasma FN-coated wells either alone or in the presence of accessory cells (EC or PBAC) at a ratio of 5:1 T cells to accessory cells. All cultures were maintained in RPMI 1640, supplemented with 10% FBS, 2.5 mM glutamine, and penicillin (100 U/ml)/streptomycin (100 µg/ml). At indicated times, T cells were recovered from the cultures in two steps. First, nonadherent cells were collected by washing with Dulbecco’s PBS (Mg²⁺, Ca²⁺ free). Second, the cultures were incubated for 30 min with PBS/5 mM EDTA at 37°C to recover the adherent T cells. Both populations were pooled and washed once with PBS/1% BSA before incubation with a directly conjugated mAb for 30 min at 4°C. After immunofluorescence labeling, cells were washed once in PBS/1% BSA, followed by two washes in PBS and then fixed with 1% paraformaldehyde before analysis by FACS. Corrected mean fluorescence intensities (MFI) were calculated by subtracting the MFI for the isotype-matched control Ab from the MFI for the specific Ab for each treatment condition. Percent inhibition of the corrected mean fluorescence of control were calculated as follows: {1 - [corrected MFI (in the presence EC with mAb) - corrected MFI (in the absence of EC)]/[corrected MFI (in the presence of EC with control mAb) - corrected MFI (in the absence of EC)]} x 100.

Inhibition of costimulator signals

Freshly isolated CD4⁺ T cells (2 x 10⁶/ml) were added to FN-coated 24-well tissue culture plate containing ASO or SC-pretreated EC plus PHA (3 µg/ml) or to empty wells in the presence of PHA (3 µg/ml). Alternatively, costimulator function was blocked by inclusion of 10 µg/ml of inhibitory mAb (TS 2/9, 6E6, or K16/16 control) in cocultures using untreated EC. After 6 h of cultivation, CD4⁺ T cells were collected and CD40L expression on CD4⁺ T cells was examined by FACS.

Transfection of promoter-reporter gene and transcriptional analysis

A human CD40L promoter-reporter gene that includes the transcriptional start site was constructed by cloning a 550-bp segment (-495 to +67) into the HindIII site of the pGL3 promoter vector (Promega, Madison, WI). This promoter includes both previously identified NF-AT binding motifs, located at –259 to –265 and at –62 to –69 relative to the transcription start site, and is sufficient to confer activation-dependent transcription of reporter gene constructs in either a transformed T cell line or normal peripheral T cells (27, 31). A human IL-2 promoter-reporter gene, previously described (32), was used as positive control. Adult human PBMC (3 x 10⁶/ml) were primed for transfection by culturing in RPMI 1640 medium containing 10% FBS, 2.5 mM glutamine, and penicillin (100 U/ml)/streptomycin (100 µg/ml) in the presence of a low concentration (1 µg/ml) of PHA for 19.5 h to induce transfection competence. After washing, cells were resuspended in fresh medium at 2 x 10⁷/ml. Aliquots of 0.25 ml were electroporated in a Bio-Rad Gene Pulser at 250 V and 960 µF at room temperature in the presence of 60 µg/ml reporter gene DNA. The gap width of the cuvettes was 0.4 cm. Transfected human CD4⁺ T cells were then isolated from the whole PBMC population by negative selection using mAbs. Purified CD4⁺ T cells (2 x 10⁶/ml) were stimulated in the presence of EC and PHA (3 µg/ml) or PHA in the absence EC or unstimulated for 3 or 6 h. Cells were harvested and lysed in 100 µl of reporter lysis buffer (Promega). Lysates (40 µl for each determination) were analyzed in triplicate for luciferase activity by using a luciferase assay kit following the manufacturer’s protocol (Promega) and a Lumat LB9501 luminometer (EG&G, Gaithersburg, MD). Data are reported as mean relative light units (RLU).

RNA preparation and Northern blot analysis for mRNA expression and stability

RNA preparation (using a guanidinium isothiocyanate lysis method, Trizol, Life Technologies), electrophoresis, transfer to nitrocellulose, hybridization, and washing were performed as previously described (32). EC may also contain mRNA for CD40L (33). To ensure that only T cell RNA was analyzed, EC were fixed in 1% paraformaldehyde/2% FBS in PBS at 37°C for 12 min, washed three times in medium 199 containing 5% FBS, and then treated with 10 µg/ml RNase A for 30 min at 37°C before initiation of coculture with CD4⁺ T cells as described above. This treatment results in a complete loss of detectable EC mRNA (data not shown and Ref. 34). Equivalence of loading was estimated by monitoring the 18S and 28S ribosomal RNA bands on the formaldehyde gel by ethidium bromide staining and UV illumination. Probes were labeled with [³²P]dCTP (New England Nuclear, Boston, MA) by random priming using a kit according to the manufacture’s instructions (Pharmacia Biotech, Piscataway, NJ). Probes used were as follows: the EcoRI fragment of the human CD40L clone (15), the PstI fragment of the human IL-2 clone pTCGF-11 (American Type Culture Collection no. 39673), and the HpaI and SalI fragment of a human HLA-A2 clone (35). The levels of CD40L and IL-2 mRNA were normalized to that of a stable mRNA species (HLA-A2) by densitometric analysis of autoradiographs using a computing densitometer and Imagequant program (Molecular Dynamics, Sunnyvale, CA).

Northern blotting was also used to measure mRNA decay. In this case, CD4⁺ T cells were stimulated with PHA (3 µg/ml) in the presence or absence of fixed EC. After 4 h, the cultures were treated with 200 nM of 5,6-dichloro-1-ß-D-ribobenzimidazole (DRB; Sigma) to stop further gene transcription. Total RNA was harvested for Northern blot analysis at the indicated time points (15, 30, and 60 min) following DRB addition. For quantitation, the intensity of the specific mRNA hybridization signal for CD40L was compared with that of HLA-A2 as described above. For decay measurements, the time point 0 min values of CD40L signals were taken as 100%. Where indicated, 10 µg/ml of blocking mAbs were included in the culture system to assess the role of costimulator molecules on mRNA stabilization.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Early expression of CD40L protein and mRNA in activated CD4⁺ T cells is augmented by EC

Although incubation of resting CD4⁺ T cells with EC does not result in detectable CD40L up-regulation (data not shown and Ref. 26), previous studies from our laboratory demonstrated that human EC provide contact-dependent signals that augment the level of CD40L expressed on PHA-activated CD4⁺ T cells at early times (e.g., 4–6 h) compared with T cells activated by PHA in the presence of conventional APC (e.g., PBAC or B lymphoblastoid cells) or by PHA alone (26). We confirm this result in Fig. 1 and further demonstrate that in this assay EC are as potent as PMA, a pharmacological T cell activator commonly used to boost CD40L expression (1, 3, 14, 27). Also as reported previously, the enhanced expression of CD40L induced by EC compared with PBAC is less striking at 24 h, when T cells activated in the presence of conventional APCs catch up (26). In other words EC augment early but not late CD40L expression. The superiority of EC as accessory cells is not seen for all T cell activation markers because EC are no better at increasing CD25 expression on PHA-activated CD4⁺ T cells than are PBAC or even PHA alone (Fig. 1).

View larger version (34K): [in this window] [in a new window]   FIGURE 1. Effect of EC or PMA on the time course of CD40L and CD25 expression on PHA-activated CD4+ T cells. CD4+ T cells were incubated with PHA (5 µg/ml) or PHA plus PMA (10 ng/ml) in the presence or absence of EC or PBAC as indicated. Expression of CD40L and CD25 on cell surface were examined by FACS. A, Fluorescence histograms of anti-CD40L staining (solid line) vs control mAb (filled curve). B, Quantitation of CD40L and CD25 expression (corrected MFI as a function of time of treatment). One of two experiments with similar results is shown.

View larger version (14K): [in this window] [in a new window]   FIGURE 2. Effect of fixed vs living EC on the time course of CD40L and CD25 expression on PHA-activated CD4+ T cells. CD4+ T cells were incubated with PHA (5 µg/ml) or PHA plus PMA (10 ng/ml) in the presence or absence of living or fixed EC as indicated. Expression of CD40L and CD25 on the cell surface were examined by FACS and plotted as corrected MFI as a function of time of treatment. One of two experiments with similar results is shown.

View larger version (44K): [in this window] [in a new window]   FIGURE 3. Effect of fixed EC on the level of CD40L and IL-2 mRNA in PHA-activated CD4+ T cells. CD4+ T cells were activated by PHA alone in the presence of fixed EC or in combination with PMA. RNA was extracted and analyzed by Northern blot using CD40L and IL-2 probes. Lanes 1 and 5, in the presence of EC with PHA (5 µg/ml); lanes 2 and 6, PHA; lanes 3 and 7, PHA and PMA (10 ng/ml); lanes 4 and 8, in the absence of both EC and mitogen. One of three experiments with similar results is shown.

Increased steady-state levels of CD40L mRNA could arise from an increased rate of transcription or from a decreased rate of mRNA degradation or both. Previous studies from our laboratory have shown that EC increase IL-2 synthesis by PHA-activated CD4⁺ T cells primarily by increasing the rate of gene transcription as measured by nuclear run off (34). However, nuclear run off assays in normal T cells are technically difficult to perform, and therefore we optimized conditions for introduction of promoter-reporter genes into normal T cells (32). The capacity of EC to increase the rate of IL-2 transcription is readily observed by this approach (32), and our method has been employed by others to study CD40L transcription in normal T cells (27). To investigate whether transcription of CD40L is also increased in CD4⁺ T cells by EC, we examined transcription of a CD40L promoter-reporter gene. This CD40L construct is functional in normal peripheral blood CD4⁺ T cells as demonstrated by an increase in luciferase activity in response to PHA (Fig. 4). As previously observed, EC increase the transcription of an IL-2 promoter-reporter gene in normal CD4⁺ T cells over that of the response to PHA alone at 3 and 6 h (32). However, EC fail to increase the response of the CD40L promoter-reporter gene in replicate cultures. In fact, EC may slightly inhibit transcriptional activity of the CD40L promoter-reporter gene compared with PHA alone (Fig. 4). These data suggest that the effect of EC on CD40L expression is not mediated by enhanced transcription, although we cannot exclude a transcriptional action on the endogenous CD40L gene through an element not contained in our promoter-reporter constructs.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Effect of EC on transcription of CD40L and IL-2 in PHA-activated CD4+ T cells. Promoter-reporter gene-transfected CD4+ T cells were incubated for 3 or 6 h with PHA (3 µg/ml) in the presence or absence of EC, and then analyzed by luciferase assay. One of three experiments with similar results is shown.

To determine whether the EC-mediated increase in CD40L mRNA could be attributed to mRNA stabilization, we stimulated CD4⁺ T cells with PHA alone or PHA in the presence of fixed EC for 4 h, and then treated the cultures with the RNA synthesis inhibitor DRB to follow mRNA decay. Replicate samples were harvested immediately at 15 min, 30 min, or 60 min of DRB treatment and CD40L mRNA was quantitated by Northern blotting. The results were normalized to MHC class I mRNA (HLA-A2) on the same blot, a long-lived mRNA whose level of expression is unaffected by short-term DRB treatment. As shown in Fig. 5, EC prolong the half-life of CD40L mRNA to >60 min, whereas the half-life of this transcripts in the absence of EC is <30 min. Taken in combination with the promoter-reporter gene studies, these data suggest that the primary effect of EC on early CD40L expression is mediated by mRNA stabilization.

View larger version (39K): [in this window] [in a new window]   FIGURE 5. Effect of EC on the stability of CD40L mRNA in PHA-activated CD4+ T cells. A, CD4+ T cells were stimulated with PHA (3 µg/ml) in the presence or absence of fixed EC, DRB was added at 4 h, and total RNA was analyzed by Northern blotting using CD40L and HLA-A2 probes at the indicated time points thereafter. B, The CD40L signals from the blot were quantitated, normalized to HLA-A2 levels, and plotted vs the time of DRB treatment taking the time 0 min value as 100%. One of three experiments with similar results is shown.

To identify the costimulator molecules involved in the interactions between EC and CD4⁺ T cells that augment CD40L expression, we used both ASOs and inhibitory mAbs for LFA-3 and ICAM-1. We targeted these molecules because they had each been shown to participate in T cell interaction with EC (26, 36)⁵ and because human EC lack B7 molecules (36, 37), previously shown to influence CD40L mRNA stabilization (38). As shown in Table II, mAb to LFA-3 block the EC effect on CD40L expression by 50% or more, whereas mAb to ICAM-1 do not block this effect and actually appear to enhance expression. In the same experiments, LFA-3 ASO pretreatment also blocks the effect of EC by about 40%, whereas ICAM-1 ASO, like ICAM-1 mAb, is actually stimulatory. The combination of anti-LFA-3 and anti-ICAM-1 mAb is no more effective than anti-LFA-3 mAb alone. These data suggest that the blockade of the EC LFA-3/T cell CD2 interaction can markedly reduce the degree of EC-mediated augmentation of early CD40L expression on CD4⁺ T cells, whereas EC ICAM-1 signals are not important for this effect.

View larger version (27K): [in this window] [in a new window]   FIGURE 6. Effect of inhibitory mAb on EC-mediated stabilization of CD40L mRNA. A, CD4+ T cells were stimulated with PHA (3 µg/ml) in the presence of fixed EC and the blocking mAb (10 µg/ml) for 4 h. DRB was added and total RNA was analyzed by Northern blotting using CD40L and HLA-A2 probes 30 min later. One of three similar experiments is shown. B, The CD40L signals from each of three experiments were quantitated, normalized to HLA-A2 levels, and plotted vs the time of DRB treatment taking the time 0 min values as 100%. Each bar depicts the mean and SD.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

LFA-3 appears to play a central role in a variety of T cell interactions with EC. For example, anti-LFA-3 mAb inhibits cytolytic T lymphocyte killing of allogeneic EC (39). LFA-3 may stimulate increased T cell adhesion to EC (40), an action that may recruit circulating T cells into an inflammatory site in vivo (41). Inhibition of LFA-3 also blocks EC augmentation of cytokine synthesis (36, 42) and IL-2 transcription (32). The regulation of early CD40L expression by this pathway is one more example of the importance of the LFA-3 molecule as a costimulator of T cell activation by human EC.

Increased mRNA stabilization is an important mechanism of costimulation provided by professional APCs. A variety of protooncogene, transcription factor, and lymphokine genes encode mRNAs that display extreme liability (half-life <30 min) (43). T cell surface effector molecules may also be regulated by mRNA stabilization (44). These effects are generally thought to be rendered through a CD28 signal, because agonistic anti-CD28 mAb stabilizes mRNAs of several cytokines in activated T cells, including the mRNA for IL-2, IFN-, TNF, and GM-CSF (45). Indeed, anti-CD28 mAb can also stabilize CD40L mRNA in the presence of anti-CD3 mAb (38). EC, unlike professional APCs, do not express CD80/CD86 (B7-1/B7-2) (36, 37). Therefore, EC must be able to stabilize CD40L mRNA in CD4⁺ T cells by some alternative to the B7/CD28 pathway. Recently, Wang et al. (44) reported that T cell LFA-1 engagement was able to stabilize mRNA for the urokinase plasminogen activator receptor. However, the EC augmentation of CD40L expression is not inhibited by blockade of the EC ligands for LFA-1, namely ICAM-1 and ICAM-2. Our data instead implicate the LFA-3/CD2 pathway. To the best of our knowledge, this is the first example of a CD2 signal causing mRNA stabilization.

The biochemical basis of mRNA stabilization is currently under intensive investigation. Many short-lived mRNAs contain reiterations of a specific sequence (AUUUA) in their 3'-untranslated region (UTR) called AU-rich elements (AREs) (43, 46). The first direct evidence that AREs can function as a potent mRNA destabilizing element came from a study in which a conserved region of 51 nt containing AUUUA motifs from the 3'-UTR of human GM-CSF mRNA was inserted into the 3'-UTR of ß-globin mRNA; the otherwise stable ß-globin mRNA was destabilized (46). Several experiments have shown that a number of AREs from the c-fos, c-myc, nur77, junB, IFN-, and IL-3 mRNAs also function as RNA destabilizing elements (46, 47, 48, 49, 50). Two types of motifs, coupled to or located in U-rich regions, have been characterized and may be differentially regulated. Type I motifs have multiple copies of AUUUA, whereas the sequence UUAUUUA(U/A)(U/A) defines type II motifs and facilitates binding of specific protein factors to AREs (51). The decameric sequence UUAUUUUAUU is also thought to be a functional degradation motif (52). CD40L mRNA includes four type I motifs and one type II motif in its 3'-UTR (53). Signals that influence mRNA lifetimes are thought to alter protein binding to these motifs; studies are in progress to determine whether CD2 signals can regulate the mRNA binding activity of such proteins.

What might be the biological significance of early CD40L expression in response to signals produced by EC? Recent studies point to a crucial role for CD40L on T cells either as an inducer of costimulator expression (e.g., B7 molecules) on professional APCs such as dendritic cells or monocytes or as a receptor for outside-in signals that directly costimulate T cells (17). Regardless of mechanism, CD40L-negative T cells are less effectively activated by APCs and may even be inactivated by Ag. Circulating memory T cells, newly recruited into an immune reaction, could encounter Ag before their expression of CD40L. Early induction of CD40L in response to signals received during attachment to and transmigration through the endothelial lining of the microvasclulature could reduce the time window during which T cells are susceptible to inactivation by Ag. This may be one more mechanism by which blockade of an LFA-3 signal through Ab or ASO prevents immune injury in vivo (54).

	Acknowledgments

 
We thank Ms. Louise Benson and Gwendolyn Davis for assistance in EC culture.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant HL51014. K.M. was supported by a fellowship from Japan Science and Technology Corporation.

² Current address: National Institute of Animal Health, 3-1-1 Kannon dai, Tsukuba, Ibaraki 305, Japan.

³ Address correspondence and reprint requests to Dr. Jordan S. Pober, Molecular Cardiobiology Program, Boyer Center for Molecular Medicine, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06536-0812. E-mail address:

⁴ Abbreviations used in this paper: CD40L, CD40 ligand; ARE, AU-rich element; ASO, antisense oligonucleotides; DRB, 5,6-dichloro-1-ß-D-ribobenzimidazole; EC, endothelial cells; FN, fibronectin; MFI, mean fluorescence intensity; PBAC, peripheral blood adherent cells; RLU, relative light units; SC, scrambled control oligonucleotides; UTR, untranslated region; XHIM, X-linked hyperIgM.

⁵ W. Ma, S. Flournoy, T. P. Condon, C. F. Bennett, and J. S. Pober. Antisense reduction of LFA-3 and ICAM-1 expression on cultured human endothelial cells inhibits allogeneic CD4⁺ T cell activation and sensitization without induction of anergy. Submitted for publication.

Received for publication April 16, 1999. Accepted for publication June 15, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Hollenbaugh, D., L. S. Grosmaire, C. D. Kullas, N. J. Chalupny, S. Braesch-Andersen, R. J. Noelle, I. Stamenkovic, J. A. Ledbetter, A. Aruffo. 1992. The human T cell antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor: expression of a soluble form of gp39 with B cell co-stimulatory activity. EMBO J. 11:4313.[Medline]
2. Armitage, R. J., W. C. Fanslow, L. Strockbine, T. A. Sato, K. N. Clifford, B. M. Macduff, D. M. Anderson, S. D. Gimpel, T. Davis-Smith, C. R. Maliszewski, E. A. Clark, C. A. Smith, K. H. Grabstein, D. Cosman, M. K. Spriggs. 1992. Molecular and biological characterization of a murine ligand for CD40. Nature 357:80.[Medline]
3. Lane, P., A. Traunecker, S. Hubele, S. Inui, A. Lanzavecchia, D. Gray. 1992. Activated human T cells express a ligand for the human B cell-associated antigen CD40 which participates in T cell-dependent activation of B lymphocytes. Eur. J. Immunol. 22:2573.[Medline]
4. Lederman, S., M. J. Yellin, A. Krichevsky, J. Belko, J. J. Lee, L. Chess. 1992. Identification of a novel surface protein on activated CD4⁺ T cells that induces contact-dependent B cell differentiation (help). J. Exp. Med. 175:1091.[Abstract/Free Full Text]
5. Kroczek, R. A., D. Graf, D. Brugnoni, S. Giliani, U. Korthäuer, A. Ugazio, G. Senger, H. W. Mages, A. Villa, L. D. Notarangelo. 1994. Defective expression of CD40 ligand on T cells causes "X-linked immunodeficiency with hyper-IgM (HIGM1)". Immunol. Rev. 138:39.[Medline]
6. Noelle, R. J., M. Roy, D. M. Shepherd, I. Stamenkovic, J. A. Ledbetter, A. Aruffo. 1992. A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells. Proc. Natl. Acad. Sci. USA 89:6550.[Abstract/Free Full Text]
7. Spriggs, M. K., R. J. Armitage, L. Strockbine, K.N. Clifford, B.M. Macduff, T. A. Sato, C. R. Maliszewski, W. C. Fanslow. 1992. Recombinant human CD40 ligand stimulates B cell proliferation and immunoglobulin E secretion. J. Exp. Med. 176:1543.[Abstract/Free Full Text]
8. Castigli, E., F. W. Alt, L. Davidson, A. Bottaro, E. Mizoguchi, A. K. Bhan, R. S. Geha. 1994. CD40-deficient mice generated by recombination-activating gene-2-deficient blastocyst complementation. Proc. Natl. Acad. Sci. USA 91:12135.[Abstract/Free Full Text]
9. Kawabe, T., T. Naka, K. Yoshida, T. Tanaka, H. Fujiwara, S. Suematsu, N. Yoshida, T. Kishimoto, H. Kikutani. 1994. The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation. Immunity 1:167.[Medline]
10. Renshaw, B. R., III W. C. Fanslow, R. J. Armitage, K. A. Campbell, D. Liggitt, B. Wright, B. L. Davison, C. R. Maliszewski. 1994. Humoral immune responses in CD40 ligand-deficient mice. J. Exp. Med. 180:1889.[Abstract/Free Full Text]
11. Xu, J., T. M. Foy, J. D. Laman, E. A. Elliott, J. J. Dunn, T. J. Waldschmidt, J. Elsemore, R. J. Noelle, R. A. Flavell. 1994. Mice deficient for the CD40 ligand. Immunity 1:423.[Medline]
12. Allen, R. C., R. J. Armitage, M. E. Conley, H. Rosenblatt, N. A. Jenkins, N. G. Copeland, M. A. Bedell, S. Edelhoff, C. M. Disteche, D. K. Simoneaux, W. C. Fanslow, J. Belmont, M. K. Spriggs. 1993. CD40 ligand gene defects responsible for X-linked hyper-IgM syndrome. Science 259:990.[Abstract]
13. Aruffo, A., M. Farrington, D. Hollenbaugh, X. Li, A. Milatovich, S. Nonoyama, J. Bajorath, L. S. Grosmaire, R. Stenkamp, M. Neubauer, R. L. Roberts, R. J. Noelle, J. A. Ledbetter, U. Francke, H. D. Ochs. 1993. The CD40 ligand, gp39, is defective in activated T cells from patients with X-linked hyper-IgM syndrome. Cell 72:291.[Medline]
14. DiSanto, J. P., J. Y. Bonnefoy, J. F. Gauchat, A. Fischer, G. de Saint Basile. 1993. CD40 ligand mutations in X-linked immunodeficiency with hyper-IgM. Nature 361:541.[Medline]
15. Fuleihan, R., N. Ramesh, R. Loh, H. Jabara, F. S. Rosen, T. Chatila, S. M. Fu, I. Stamenkovic, R. S. Geha. 1993. Defective expression of the CD40 ligand in X chromosome-linked immunoglobulin deficiency with normal or elevated IgM. Proc. Natl. Acad. Sci. USA 90:2170.[Abstract/Free Full Text]
16. Korthäuer, U., D. Graf, H. W. Mages, F. Brière, M. Padayachee, S. Malcolm, A. G. Ugazio, L. D. Notarangelo, R. J. Levinsky, R. A. Kroczek. 1993. Defective expression of T-cell CD40 ligand causes X-linked immunodeficiency with hyper-IgM. Nature 361:539.[Medline]
17. Durie, F. H., T. M. Foy, S. R. Masters, J. D. Laman, R. J. Noelle. 1994. The role of CD40 in the regulation of humoral and cell-mediated immunity. Immunol. Today 15:406.[Medline]
18. Alderson, M. R., R. J. Armitage, T. W. Tough, L. Strockbine, W. C. Fanslow, M. K. Spriggs. 1993. CD40 expression by human monocytes: regulation by cytokines and activation of monocytes by the ligand for CD40. J. Exp. Med. 178:669.[Abstract/Free Full Text]
19. Hollenbaugh, D., N. Mischel-Petty, C. P. Edwards, J. C. Simon, R. W. Denfeld, P. A. Kiener, A. Aruffo. 1995. Expression of functional CD40 by vascular endothelial cells. J. Exp. Med. 182:33.[Abstract/Free Full Text]
20. Karmann, K., C. C. W. Hughes, J. Schechner, W. C. Fanslow, J. S. Pober. 1995. CD40 on human endothelial cells: inducibility by cytokines and functional regulation of adhesion molecule expression. Proc. Natl. Acad. Sci. USA 92:4342.[Abstract/Free Full Text]
21. Yellin, M. J., J. Brett, D. Baum, A. Matsushima, M. Szabolcs, D. Stern, L. Chess. 1995. Functional interactions of T cells with endothelial cells: the role of CD40L-CD40-mediated signals. J. Exp. Med. 182:1857.[Abstract/Free Full Text]
22. Durie, F. H., A. Aruffo, J. Ledbetter, K. M. Crassi, W. R. Green, L. D. Fast, R. J. Noelle. 1994. Antibody to the ligand of CD40, gp39, blocks the occurrence of the acute and chronic forms of graft-vs-host disease. J. Clin. Invest. 94:1333.
23. Mohan, C., Y. Shi, J. D. Laman, S. K. Datta. 1995. Interaction between CD40 and its ligand gp39 in the development of murine lupus nephritis. J. Immunol. 154:1470.[Abstract]
24. Larsen, C. P., E. T. Elwood, D. Z. Alexander, S. C. Ritchie, R. Hendrix, C. Tucker-Burden, H. R. Cho, A. Aruffo, D. Hollenbaugh, P. S. Linsley, K. J. Winn, T. C. Pearson. 1996. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 381:434.[Medline]
25. Kirk, A. D., D. M. Harlan, N. N. Armstrong, T. A. Davis, Y. Dong, G. S. Gray, X. Hong, D. Thomas, Jr J. H. Fechner, S. J. Knechtle. 1997. CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates. Proc. Natl. Acad. Sci. USA 94:8789.[Abstract/Free Full Text]
26. Karmann, K., C. C. W. Hughes, W. C. Fanslow, J. S. Pober. 1996. Endothelial cells augment the expression of CD40 ligand on newly activated human CD4⁺ T cells through a CD2/LFA-3 signaling pathway. Eur. J. Immunol. 26:610.[Medline]
27. Schubert, L. A., G. King, R. Q. Cron, D. B. Lewis, A. Aruffo, D. Hollenbaugh. 1995. The human gp39 promoter: two distinct nuclear factors of activated T cell protein-binding elements contribute independently to transcriptional activation. J. Biol. Chem. 270:29624.[Abstract/Free Full Text]
28. Altmann, K. H., N. M. Dean, D. Fabbro, S. M. Freier, T. Geiger, R. Häner, D. Hüsken, P. Martin, B. P. Monia, M. Müller, F. Natt, P. Nicklin, J. Phillips, U. Pieles, H. Sasmor, H. E. Moser. 1996. Second generation antisense oligonucleotides: from nuclease resistance to biological efficacy in animals. Chimia 50:168.
29. Baker, B. F., S. S. Lot, T. P. Condon, S. Cheng-Flournoy, E. A. Lesnik, H. M. Sasmor, C. F. Bennett. 1997. 2'-O-(2-Methoxy)ethyl-modified anti-intercellular adhesion molecule 1 (ICAM-1) oligonucleotides selectively increase the ICAM-1 mRNA level and inhibit formation of the ICAM-1 translation initiation complex in human umbilical vein endothelial cells. J. Biol. Chem. 272:11994.[Abstract/Free Full Text]
30. Bennett, C. F., M. Y. Chiang, H. Chan, J. E. E. Shoemaker, C. K. Mirabelli. 1992. Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. Mol. Pharmacol. 41:1023.[Abstract]
31. Lobo, F. M, R. Zanjani, N. Ho, T. A. Chatila, R. L. Fuleihan. 1999. Calcium-dependent activation of TNF family gene expression by Ca²⁺/calmodulin kinase type IV/Gr and calcineurin. J. Immunol. 162:2057.[Abstract/Free Full Text]
32. Hughes, C. C. W., J. S. Pober. 1996. Transcriptional regulation of the interleukin-2 gene in normal human peripheral blood T cells. Convergence of costimulatory signals and differences from transformed T cells. J. Biol. Chem. 271:5369.[Abstract/Free Full Text]
33. Mach, F., U. Schönbeck, G. K. Sukhova, T. Bourcier, J. Y. Bonnefoy, J. S. Pober, P. Libby. 1997. Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for CD40-CD40 ligand signaling in atherosclerosis. Proc. Natl. Acad. Sci. USA 94:1931.[Abstract/Free Full Text]
34. Hughes, C. C. W., J. S. Pober. 1993. Costimulation of peripheral blood T cell activation by human endothelial cells: enhanced IL-2 transcription correlates with increased c-fos synthesis and increased Fos content of AP-1. J. Immunol. 150:3148.[Abstract]
35. Winter, C. C., B. M. Carreno, R. V. Turner, S. Koenig, W. E. Biddison. 1991. The 45 pocket of HLA-A2.1 plays a role in presentation of influenza virus matrix peptide and alloantigens. J. Immunol. 146:3508.[Abstract]
36. Hughes, C. C. W., C.O.S. Savage, J. S. Pober. 1990. Endothelial cells augment T cell interleukin 2 production by a contact-dependent mechanism involving CD2/LFA-3 interaction. J. Exp. Med. 171:1453.[Abstract/Free Full Text]
37. Karmann, K., J. S. Pober, C. C. W. Hughes. 1994. Endothelial cell-induced resistance to cyclosporin A in human peripheral blood T cells requires contact-dependent interactions involving CD2 but not CD28. J. Immunol. 153:3929.[Abstract]
38. Klaus, S. J., L. M. Pinchuk, H. D. Ochs, C. L. Law, W. C. Fanslow, R. J. Armitage, E. A. Clark. 1994. Costimulation through CD28 enhances T cell-dependent B cell activation via CD40-CD40L interaction. J. Immunol. 152:5643.[Abstract]
39. Collins, T., A. M. Krensky, C. Clayberger, W. Fiers, Jr M. A. Gimbrone, S. J. Burakoff, J. S. Pober. 1984. Human cytolytic T lymphocyte interactions with vascular endothelium and fibroblasts: role of effector and target cell molecules. J. Immunol. 133:1878.[Abstract]
40. Shaw, S., G. E. G. Luce, R. Quinones, R. E. Gress, T. A. Springer, M. E. Sanders. 1986. Two antigen-independent adhesion pathways used by human cytotoxic T-cell clones. Nature 323:262.[Medline]
41. van Kooyk, Y., P. van de Wiel-van Kemenade, P. Weder, T. W. Kuijpers, C. G. Figdor. 1989. Enhancement of LFA-1-mediated cell adhesion by triggering through CD2 or CD3 on T lymphocytes. Nature 342:811.[Medline]
42. Ma, W., J. S. Pober. 1998. Human endothelial cells effectively costimulate cytokine production by, but not differentiation of, naive CD4⁺ T cells. J. Immunol. 161:2158.[Abstract/Free Full Text]
43. Caput, D., B. Beutler, K. Hartog, R. Thayer, S. Brown-Shimer, A. Cerami. 1986. Identification of a common nucleotide sequence in the 3'-untranslated region of mRNA molecules specifying inflammatory mediators. Proc. Natl. Acad. Sci. USA 83:1670.[Abstract/Free Full Text]
44. Wang, G. J., M. Collinge, F. Blasi, R. Pardi, J. R. Bender. 1998. Posttranscriptional regulation of urokinase plasminogen activator receptor messenger RNA levels by leukocyte integrin engagement. Proc. Natl. Acad. Sci. USA 95:6296.[Abstract/Free Full Text]
45. Lindsten, T., C. H. June, J. A. Ledbetter, G. Stella, C. B. Thompson. 1989. Regulation of lymphokine messenger RNA stability by a surface-mediated T cell activation pathway. Science 244:339.[Abstract/Free Full Text]
46. Shaw, G., R. Kamen. 1986. A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46:659.[Medline]
47. Treisman, R.. 1985. Transient accumulation of c-fos RNA following serum stimulation requires a conserved 5' element and c-fos 3' sequences. Cell 42:889.[Medline]
48. Peppel, K., J. M. Vinci, C. Baglioni. 1991. The AU-rich sequences in the 3' untranslated region mediate the increased turnover of interferon mRNA induced by glucocorticoids. J. Exp. Med. 173:349.[Abstract/Free Full Text]
49. Stoecklin, G., S. Hahn, C. Moroni. 1994. Functional hierarchy of AUUUA motifs in mediating rapid interleukin-3 mRNA decay. J. Biol. Chem. 269:28591.[Abstract/Free Full Text]
50. Chen, C. Y. A., A. B. Shyu. 1994. Selective degradation of early-response-gene mRNAs: functional analyses of sequence features of the AU-rich elements. Mol. Cell. Biol. 14:8471.[Abstract/Free Full Text]
51. Chen, C. Y. A., A. B. Shyu. 1995. AU-rich elements: characterization and importance in mRNA degradation. Trends. Biochem. Sci. 20:465.[Medline]
52. Ma, W. J., S. Cheng, C. Campbell, A. Wright, H. Furneaux. 1996. Cloning and characterization of HuR, a ubiquitously expressed Elav-like protein. J. Biol. Chem. 271:8144.[Abstract/Free Full Text]
53. Gauchat, J. F., J. P. Aubry, G. Mazzei, P. Life, T. Jomotte, G. Elson, J. Y. Bonnefoy. 1993. Human CD40-ligand: molecular cloning, cellular distribution and regulation of expression by factors controlling IgE production. FEBS Lett. 315:259.[Medline]
54. Sultan, P., J.S. Schechner, J.M. McNiff, P.S. Hochman, C.C.W. Hughes, M.I. Lorber, P.W. Askenase, J.S. Pober. 1997. Blockade of CD2-LFA-3 interactions protects human skin allografts in immunodeficient mouse/human chimeras. Nat. Biotechnol. 15:759.[Medline]

This article has been cited by other articles:

				B. J. Hamilton, X.-W. Wang, J. Collins, D. Bloch, A. Bergeron, B. Henry, B. M. Terry, M. Zan, A. J. Mouland, and W. F. C. Rigby Separate cis-trans Pathways Post-transcriptionally Regulate Murine CD154 (CD40 Ligand) Expression: A NOVEL FUNCTION FOR CA REPEATS IN THE 3'-UNTRANSLATED REGION J. Biol. Chem., September 12, 2008; 283(37): 25606 - 25616. [Abstract] [Full Text] [PDF]

				D. Daoussis, I. Antonopoulos, A. P. Andonopoulos, and S.-N. C. Liossis Increased expression of CD154 (CD40L) on stimulated T-cells from patients with psoriatic arthritis Rheumatology, February 1, 2007; 46(2): 227 - 231. [Abstract] [Full Text] [PDF]

				J. Choi, J. Walker, S. Boichuk, N. Kirkiles-Smith, N. Torpey, J. S. Pober, and L. Alexander Human Endothelial Cells Enhance Human Immunodeficiency Virus Type 1 Replication in CD4+ T Cells in a Nef-Dependent Manner In Vitro and In Vivo J. Virol., January 1, 2005; 79(1): 264 - 276. [Abstract] [Full Text] [PDF]

				K. Singh, J. Laughlin, P. A. Kosinski, and L. R. Covey Nucleolin Is a Second Component of the CD154 mRNA Stability Complex That Regulates mRNA Turnover in Activated T Cells J. Immunol., July 15, 2004; 173(2): 976 - 985. [Abstract] [Full Text] [PDF]

				D. Daoussis, A. P. Andonopoulos, and S.-N. C. Liossis Targeting CD40L: a Promising Therapeutic Approach Clin. Vaccine Immunol., July 1, 2004; 11(4): 635 - 641. [Full Text]

				R. Zhang, C. J. Fichtenbaum, D. A. Hildeman, J. D. Lifson, and C. Chougnet CD40 Ligand Dysregulation in HIV Infection: HIV Glycoprotein 120 Inhibits Signaling Cascades Upstream of CD40 Ligand Transcription J. Immunol., February 15, 2004; 172(4): 2678 - 2686. [Abstract] [Full Text] [PDF]

				C. Chougnet Role of CD40 Ligand dysregulation in HIV-associated dysfunction of antigen-presenting cells J. Leukoc. Biol., November 1, 2003; 74(5): 702 - 709. [Abstract] [Full Text] [PDF]

				B. J. Hamilton, A. Genin, R. Q. Cron, and W. F. C. Rigby Delineation of a Novel Pathway That Regulates CD154 (CD40 Ligand) Expression Mol. Cell. Biol., January 15, 2003; 23(2): 510 - 525. [Abstract] [Full Text] [PDF]

				P. A. Kosinski, J. Laughlin, K. Singh, and L. R. Covey A Complex Containing Polypyrimidine Tract-Binding Protein Is Involved in Regulating the Stability of CD40 Ligand (CD154) mRNA J. Immunol., January 15, 2003; 170(2): 979 - 988. [Abstract] [Full Text] [PDF]

				L.-P. Berg, M. J. James, M. Alvarez-Iglesias, S. Glennie, R. I. Lechler, and F. M. Marelli-Berg Functional Consequences of Noncognate Interactions Between CD4+ Memory T Lymphocytes and the Endothelium J. Immunol., April 1, 2002; 168(7): 3227 - 3234. [Abstract] [Full Text] [PDF]

				U. Schonbeck and P. Libby CD40 Signaling and Plaque Instability Circ. Res., December 7, 2001; 89(12): 1092 - 1103. [Abstract] [Full Text] [PDF]

				B. Barnhart, P. A. Kosinski, Z. Wang, G. S. Ford, M. Kiledjian, and L. R. Covey Identification of a Complex that Binds to the CD154 3' Untranslated Region: Implications for a Role in Message Stability During T Cell Activation J. Immunol., October 15, 2000; 165(8): 4478 - 4486. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Murakami, K. Articles by Pober, J. S. Search for Related Content PubMed PubMed Citation Articles by Murakami, K. Articles by Pober, J. S.