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The Journal of Immunology, 2006, 176: 750-761.
Copyright © 2006 by The American Association of Immunologists

Human Monocytes as Intermediaries between Allogeneic Endothelial Cells and Allospecific T Cells: A Role for Direct Scavenger Receptor-Mediated Endothelial Membrane Uptake in the Initiation of Alloimmunity1

He Xu2, Kiran K. Dhanireddy and Allan D. Kirk

Transplantation Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Recipient monocytes, T cells, and donor endothelial cells (ECs) are recognized as critical components of allograft rejection. We have recently shown that human monocytes infiltrate vascularized allografts before clinical rejection and have thus hypothesized that monocytes, rather than costimulation-poor ECs, initiate an alloimmune response. However, the nature of the interactions between ECs, monocytes, and T cells has been incompletely defined. Specifically, it is not clear whether these cells interact in a hierarchical manner, nor is it apparent what constitutes an interaction. We therefore studied human ECs, monocytes, and T cells in various isolated in vitro combinations to define the salient features of their contact and to determine whether their interactions were sequential in nature. We find that T cells proliferate poorly to allogeneic ECs and autologous monocytes but well to autologous monocytes following allogeneic EC contact. We show that monocytes gain their stimulatory capacity by phagocytizing allogeneic but not autologous EC membranes in a process governed by scavenger receptors. This process facilitates the subsequent presentation of intact donor HLA molecules to T cells (semidirect presentation). Moreover, monocytes are receptive to T cell help only after exposure to ECs and require CD4+ T cells to optimally express costimulatory molecules and foster Ag presentation. Our results indicate that monocytes engage allogeneic ECs through scavenger receptors and are then primed to facilitate T cell activation in a codependent manner. This reciprocal codependence allows for monocytes to serve as a regulated bridge between the allograft and T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Initial acute allograft rejection is thought to be a T cell-mediated process driven predominantly by direct alloantigen recognition. However, the manner in which T cells recognize donor Ags and are activated in vivo remains incompletely defined, particularly in humans. Specifically, it has been shown in some animal models that allograft parenchymal and endothelial cells (ECs)3 are incapable of directly presenting alloantigen in vivo and are thus unable to initiate rejection even when T cells have access to the graft (1, 2, 3). Secondary lymphoid organs are thought to provide an environment where naive T cells receive the costimulatory signals required for activation, presumably by intermingling specialized APCs with a critical number of T cells (1). Donor-derived dendritic cells (DCs) are well known for carrying intact alloantigen to secondary lymphoid tissues for direct presentation (4). Although this fact is clearly applicable to organs rich in DCs like skin grafts, kidneys are less replete with these specialized APCs, and, even so, it has been shown that human allograft rejection can occur in the absence of donor-derived APCs (5). Thus, there are likely other intermediaries between the graft and naive T cells.

One cell with potential mobile APC capabilities is the host monocyte. Unlike peripheral tissues such as the graft endothelium, activated monocytes have the ability to express CD40 and B7 costimulatory molecules (6, 7, 8, 9, 10, 11, 12), and these molecules have been shown to be critical for optimal naive alloimmune responses (13, 14, 15). Recently, several human studies have implicated monocytes as playing a more immediate role in allograft rejection than previously suspected. For example, when human renal allograft recipients are profoundly T cell-depleted using mAb preparations, rejection still occurs despite severe lymphopenia and is characterized by intense infiltration of the allograft by monocytes (16). Indeed, rejection does not occur until monocytes are available in the peripheral circulation, and graft dysfunction occurs upon the arrival at the graft of activated monocytes even without a prominent T cell infiltrate. Monocytes have also been shown to be among the first cells infiltrating human renal allografts immediately upon reperfusion (17, 18). Thus, monocytes could be envisioned as interacting with graft cells such as the vascular endothelium and conveying alloantigen and costimulation to T cells.

Several questions exist when considering mobile APCs. For example, what specifically defines an "interaction" with allogeneic cells? How does the Ag get taken in by the cell and in what form is it conveyed? Acute rejection has been typically thought to be predominantly a process driven by direct alloantigen presentation (4), and recipient-derived monocytes would be expected to be capable only of indirect alloantigen presentation. Also, given the minimal injury to allografts using modern transplantation and preservation techniques (histologically, human live donor grafts have no detectable necrosis), is Ag limited to the cells liberated by cell death or are live cells capable of delivering Ag for presentation? Furthermore, if monocytes are able to imbibe Ag, how do they avoid promiscuous activation of cells that they encounter during transit?

To investigate human monocytes and their potential role in conveying alloantigen to T cells, we have initiated studies investigating the nature of monocytic interactions with the allogeneic endothelium and autologous T cells. We have specifically evaluated the effect of isolated interactions, because in vivo it is apparent that monocytes encounter graft cells remote from substantial numbers of T cells (16). We find that monocytes directly engulf live allogeneic but not autologous EC membranes using a scavenger receptor-dependent process. This process facilitates the representation of intact MHC molecules to T cells and subsequent T cell activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents and mAbs

Recombinant human IFN-{gamma} and TNF-{alpha} were purchased from R&D Systems. Human AB serum, gelatin, PKH-26-GL fluorescent cell linker compound kits, polyguanylic acid (poly(G)), and polycytidylic acid (poly(C)) were obtained from Sigma-Aldrich.

The mAbs and their FITC or PE conjugates used for flow cytometry included the mouse isotype control Abs IgG1, IgG2a, and IgG1 (BD Biosciences), human-specific mouse Abs against CD4, CD8, CD14, CD16, CD20, CD40, CD58, CD80, CD83, CD86, CD134L, CD154 (5c8), annexin V, and HLA-DR (BD Biosciences), and CD40, CD106, CD62E, and von Willebrand factor (Serotec). The biotin-conjugated, mouse anti-human HLA class I Ag A1 (HLA-A1) was purchased from United States Biological. Biotin-conjugated mouse IgM (BD Biosciences) was used as an isotype control Ab for the HLA-A1. PE-conjugated streptavidin was purchased from Caltag Laboratories. FITC-conjugated, monoclonal, active caspase-3 apoptosis kit 1 was purchased from BD Pharmingen. The agonist CD28-specific monoclonal IgG2a (clone 9.3) and Fab were gifts from Dr. D. Harlan (Bethesda, MD). Human EC growth factor was purchased from Sigma-Aldrich.

Mouse human-specific Abs against CD2 (clone 35.1, IgG2a), CD8 (clone 51.1, IgG2a), CD11a (clone OKM 1, IgG2b), CD14 (clone 63D3, IgG1), CD16 (clone 3G8, IgG1), CD20 (clone 1F5, IgG2a), and HLA-DR (clone 2.06, IgG1) were prepared from American Type Culture Collection hybridomas.

EC culture and activation

Human ECs were isolated for coculture studies from the cadaveric aorta by digestion with 0.01% collagenase (Crescent Chemical). Primary cultures and subcultures were conducted in EC culture medium (Invitrogen Life Technologies) supplemented with 10% FCS (HyClone Laboratories) and EC growth factor. The cells were verified to be endothelial by staining for von Willebrand factor by FACS analysis. For positive control activation, ECs were treated with TNF-{alpha} (250 U/ml, for 4 h) or IFN-{gamma} (10,000 U/ml, for 48–72 h), and activation was verified by expression and/or up-regulation of CD54, CD62E, CD106, HLA class I, and HLA-DR as determined by FACS using a FACScan (BD Biosciences). Both resting and cytokine-stimulated ECs were stained with anti-human CD40, CD80, and CD86 to determine the expression of these costimulatory molecules.

For assessment of functional CD40 on ECs, human D1.1 cells (American Type Culture Collection), CD4-negative derivatives of the Jurkat line capable of providing a contact-dependent helper function (19), were used as a stimulant. D1.1 cultures were maintained at 1 x 106 cells/ml in RPMI 1640 medium containing 10% FCS, penicillin, and streptomycin. Intact EC monolayers were coincubated with D1.1 cells (5 x 106) in the presence or absence of the anti-CD154 Ab 5c8 (100 µg/ml) or anti-CD80 h1f1 (100 µg/ml; Genetics Institute/Wyeth) at 37°C for 2 h. After removal of the D1.1 cells, EC monolayers were stained with mouse IgG1-FITC (isotype control) or anti-CD62E-FITC for 30 min, washed three times with FACS buffer, and incubated with PBS (Invitrogen Life Technologies) containing 20 mM HEPES (BioWhittaker) (pH 7.4), 10 mM EDTA, and 0.5% BSA (Sigma-Aldrich) at 4°C for 20 min and then at 37°C for 20 min. Detached ECs were analyzed by FACScan. Resting ECs and ECs activated by recombinant human TNF-{alpha} were used as negative and positive controls, respectively. Because monocytes were cocultured with ECs in several experiments, both resting and activated EC were confirmed to be CD14-negative.

Leukocyte purification

Human monocytes and lymphocytes were obtained by leukapheresis from normal volunteers enrolled in a National Institutes of Health Institutional Review Board blood product procurement protocol following informed consent. Human PBMCs were isolated by Ficoll (Sigma-Aldrich) density gradient centrifugation. Cells were washed three times with Ca2+- and Mg2+-free PBS. Human monocytes were either isolated from PBMCs by negative selection methods or obtained as elutriated monocytes from the Department of Transfusion, National Institutes of Health. Briefly, PBMCs were resuspended in RPMI 1640 supplemented with 5% FCS, incubated with anti-CD2, anti-CD16, and anti-CD20 Abs at 4°C for 60 min, and washed three times with Ca2+- and Mg2+-free PBS. Cells were incubated with goat anti-mouse IgG-coated Dynabeads (Dynal) at 4°C for 60 min. After magnetic removal of the beads, cells were washed twice in RPMI 1640 and diluted with tissue culture medium (10% FCS in RPMI 1640 medium) at 2 x 106 cells/ml. Cell phenotype was verified by FACS with CD14+CD86+ cells at >90% (mean, 91 ± 2.8%), CD3+CD4+ cells at 2.2 ± 1.2%, CD3+CD8+ cells at 1.0 ± 0.9%, CD3+CD20+ cells at 0.5 ± 0.6%, and CD3+CD16+ cells at 0.9 ± 0.5%.

Human CD4+ T cells were purified from PBMCs by negative selection. Briefly, PBMCs were incubated with anti-human CD8, CD11a, CD14, CD16, CD20, and HLA-DR at 4°C for 60 min and then washed three times with RPMI 1640 medium at 4°C. Cells were then incubated with goat anti-mouse IgG-coated Dynabeads at 4°C for 60 min. After magnetic removal of the beads, cells were washed twice with RPMI 1640 at 4°C and diluted with tissue culture medium at 2 x 106 cells/ml for the T cell-monocyte interaction studies. Cell phenotype was verified by FACS.

Human splenocytes were isolated from human cadaveric spleen and aorta discarded after clinical organ procurement for transplantation. Briefly, spleen was minced, and the resultant suspension was filtered by a fine nylon filter to retain connective tissue capsule fragments. Splenocytes were washed three times with cold PBS after lysing RBCs. Cell viability was determined by trypan blue (Invitrogen Life Technologies) exclusion and was >85%. Splenocytes were stored in freezing medium (10% DMSO in FCS) at –80°C for 24 h and then stored in liquid nitrogen until used. Cells were thawed rapidly before the experiments, and cell viability was confirmed by trypan blue exclusion.

Allogeneic mixed lymphocyte-EC reactions

All studies were performed using nontrypsinized, intact, confluent EC monolayers to mimic the in vivo conditions of initial PBMC-EC interactions. Human ECs were grown to confluence in 96-well, flat-bottom tissue culture plates and examined microscopically to ensure viability before each experiment. Monolayers were treated with mitomycin C (Sigma-Aldrich) at 50 µg/ml for 38 min and washed three times with RPMI 1640 medium. PBMCs (2 x 105) or purified CD4+ T cells were added to each well supplemented with RPMI 1640 containing 10% human AB serum. Mixed lymphocyte-EC reactions were conducted for 6 days, with cells pulsed (50 µCi/ml [3H]thymidine) during the final day of culture, harvested, and counted as described above. To verify the costimulation requirements of this system, mixed lymphocyte-EC reactions were in some cases supplemented by the addition of anti-CD28 (50 µg/ml) or Fab (50 µg/ml) for 6 days. These and all other experiments were repeated at least three times with equivalent results.

PBMC-EC or monocyte-EC cocultures and flow cytometry

To detect EC membrane uptake, coculture experiments were performed using intact EC monolayers labeled with PKH-26, a fluorescent compound that incorporates aliphatic reporter molecules into the cell membrane by selective partitioning (20). ECs were labeled with PKH-26 at room temperature for 5 min and then washed twice with RPMI 1640, seeded in gelatin-coated 12-well flat-bottom tissue culture plates, grown to confluence, and examined microscopically before each experiment. ECs were also verified to be viable before and after interactions with monocytes by trypan blue exclusion and negative staining with FITC-conjugated monoclonal anti-active caspase-3 and annexin V Ab to exclude apoptosis by FACS analysis. Labeled ECs were >95% positive for PKH-26 by FACS. Unlabeled ECs were also seeded in 12-well tissue culture plates and used after EC monolayers formed. Human PBMCs or purified monocytes were diluted with tissue culture medium and then added to 12-well, flat-bottom culture plates with EC monolayers at 4 x 106 cells/well. In some experiments, PBMC were added to a Transwell chamber (Corning Costar), and the chamber was placed in 12-well, flat-bottom plates containing unlabeled EC monolayers or PHK26-labeled EC monolayers.

To determine whether EC membrane uptake was allogeneic specific, frozen human splenocytes were isolated from a cadaveric donor spleen from which aortic ECs had been recovered and stored in standard freezing medium in liquid nitrogen. Splenocytes were thawed in a 37°C water bath and then transferred to culture medium followed by centrifugation at 4°C. Cellular debris and dead cells were removed by Ficoll centrifugation. Cells were washed three times with culture medium, and viability was confirmed using trypan blue exclusion. Cells were then coincubated with allogeneic ECs and autologous ECs labeled with or without PKH-26 for 16 h, and cells were collected followed by FACS analysis gating on monocytes by forward and side scatter and CD14 expression. To determine whether cell membrane uptake by monocytes was EC-specific, in an additional experiment a coculture of human monocytes or PBMCs with unlabeled or PKH-26-labeled allogeneic fibroblasts (a gift from Dr. K. Pechhold, Bethesda, MD) was performed for 16 h, followed by FACS analysis.

In additional experiments, PKH-26-labeled gelatin was coated on culture plates and incubated at 37°C for 2 h. Unlabeled, washed ECs were transferred into these wells. Cocultures were conducted at 37°C, and cells were collected at varying time points. Unstimulated PBMCs or monocytes were used as controls. Uptake inhibition studies were performed using PBMCs or monocytes that were preincubated with poly(G) (a scavenger receptor inhibitor) at 1.0, 0.5, 0.25, 0.125, or 0.0625 mg/ml for 60 min and then added to unlabeled or PKH-26-labeled EC monolayers, and cocultures were carried out for 16 h. Poly(C), a structural homolog of poly(G), was used as a specific negative control for poly(G) in inhibitory experiments.

PBMCs or monocytes collected from cocultures were washed once with 4°C FACS buffer and stained with FITC-labeled Abs specific for CD4, CD8, CD20, CD14, CD40, CD58, CD80, CD86, CD134L, and/or HLA-DR at 4°C for 30 min. After a final wash with cold FACS buffer, cells were analyzed by FACS with the electronic gate set on CD14+ or PKH26-stained CD14+ cells. All coculture experiments were repeated at least three times with equivalent results. Additionally, these cells were also stained for CD83 to determine whether the monocyte population changed over time to DC-like cells.

Confocal microscopic evaluation of monocyte from monocyte-EC coculture

Monocytes collected from PKH-26-labeled EC-monocyte cocultures were washed three times with PBS and then incubated with anti-CD14-FITC at 4°C for 30 min. Cells were washed three times with PBS and then fixed with 1.6% paraformaldehyde on ice for 20 min. Cells were washed twice with PBS and then diluted with FACS buffer, and cell slides were prepared by Cytospin (Shandon Southern Products) and examined by confocal microscopy (MRC600; Bio-Rad).

Tissue typing and verification of intact HLA class I transfer

Human ECs and leukocytes were HLA-typed by the clinical HLA typing laboratory at the Walter Reed Army Medical Center using a complement-dependent microcytotoxicity assay. To verify the expression of HLA-A1 before the experiments, EC monolayers were examined microscopically before each experiment and stained with a biotin-conjugated mAb specific for the HLA-A1 on ice for 30 min. EC monolayers were washed twice with PBS buffer and then incubated with PE-conjugated streptavidin for 30 min, followed by three washes. ECs were detached by incubation in PBS containing 20 mM HEPES (pH 7.4), 10 mM EDTA, and 0.5% BSA at 4°C for 20 min and then at 37°C for 20 min. Detached ECs were analyzed by FACScan, and these ECs were positive for the HLA-A1.

To determine the uptake of EC-derived HLA-A1 Ag by human monocytes, human PBMCs were evaluated by FACS analysis to confirm the absence of HLA-A1 Ag expression. An EC-PBMC coculture was carried for 18 h. Cells were collected after incubation and washed twice with FACS buffer. Cells were then incubated with mouse mAb specific for HLA-A1 and CD14 as well as mouse isotype control Ig at 4°C for 30 min. PE-conjugated streptavidin was added to cells after the final wash and incubated at 4°C for 30 min. Cells were analyzed by FACS gating on CD14+ after two washes with FACS buffer.

Coculture of purified CD4+ T cells and EC-conditioned monocytes

To generate EC-conditioned monocytes, purified monocytes were coincubated with intact EC monolayers for 48 h in tissue culture medium at 37°C and collected by repeated gentle washes without destroying the EC monolayers. Monolayers were confirmed to be intact by microscopic examination. Cellular debris was removed by Ficoll centrifugation. Cells were then washed three times with PBS at 4°C, and the viability of cells was confirmed by trypan blue exclusion.

Washed, EC-conditioned monocytes were diluted with tissue culture medium. Purified autologous CD4+ T cells were rested for 48 h then added to 12-well tissue culture plates and coincubated with EC-conditioned monocytes for 24 h (T cell/monocyte ratio was 5:1) at 2.5 x 106 cells/ml in tissue culture medium. Coincubation of resting monocytes with purified autologous CD4+ T cells was conducted as a negative control. Cells were collected, washed twice with FACS buffer, and stained with mAbs against human CD14, CD40, CD80, CD86, and HLA-DR at 4°C for 30 min. Cells were washed twice with FACS buffer and evaluated by FACScan analysis gating on the CD14+ monocyte population.

To determine whether EC-conditioned monocytes could induce purified CD4+ T cell proliferation, a 5-day EC-conditioned monocyte-T cell coculture was performed. Briefly, live cells were washed three times and diluted with RPMI 1640 medium containing 10% human AB serum. EC-conditioned monocytes (2 x 104) were incubated with 1 x 105 purified CD4+ T cells in 96-well round-bottom culture plates with or without blocking mAbs directed against CD80, CD86, and CD154 at 37°C for 5 days. After incubation with 50 µCi/ml [3H]thymidine on the final day, cells were harvested onto glass fiber filters, and proliferation was measured by incorporation of [3H]thymidine using a liquid scintillation counter (PerkinElmer Life Sciences). All coculture experiments were repeated at least three times with equivalent results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Human ECs remain costimulation-poor despite activation by cytokines or exposure to T cells

Effective APCs express the costimulatory molecules CD80 and CD86. However, human ECs typically do not express these molecules even when optimally activated. To verify this assumption, we evaluated both resting and activated ECs. Resting ECs expressed minimal CD40 and were devoid of CD80, CD86, and the adhesion molecules CD62E, CD54, and CD106 (Fig. 1, a and b). Following incubation with rIFN-{gamma}, ECs expressed CD40, CD54, CD62E, CD106, and HLA-DR but remained negative for surface CD80, CD86 (Fig. 1b), and CD134L (not shown). TNF-{alpha} stimulation improved CD40 expression but did not alter HLA-DR expression or induce CD80 or CD86 expression. Stimulation with TNF-{alpha} or rIFN-{gamma} did not alter CD58 expression (not shown). ECs simultaneously stimulated with both rIFN-{gamma} and TNF-{alpha} also remained CD80-, CD86-, and CD134L-negative (data not shown). The CD40 was functional in that D1.1 T cells augmented an EC adhesion molecule expression that was inhibitable by an anti-CD154 Ab but not by an isotype control Ab (data not shown). However, even after activation by CD154-expressing T cells, ECs remained CD80-, CD86-, and CD134L-negative. Consistent with these findings, activated human ECs (those expressing HLA-DR, CD40, CD54, CD62E, and CD106) had no ability in vitro to activate purified CD4+ T cells under the conditions of this study (Fig. 1c). These conditions were sufficient to induce robust resting CD4+ T cell proliferation when allogeneic monocytes served as stimulators (data not shown). Thus, the inability of ECs to induce proliferation was not the result of overly stringent in vitro conditions. Interestingly, T cells did proliferate when monocytes were present during the interaction (as did PBMCs) (Fig. 1c). Thus, human ECs were poor candidates for primary APCs and would be unlikely to activate naive CD4+ T cells under physiological conditions despite direct alloantigen expression.



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  		FIGURE 1. Human aortic ECs do not express CD80 or CD86 even after exogenous cytokine-mediated activation and fail to stimulate purified CD4+ T cells. This failure to activate CD4+ T cells is due to inadequate B7 costimulation. a, Flow cytometric data shows that human ECs derived from cadaveric aorta up-regulate the adhesion molecules CD62E, CD54, and CD106 (open histogram) when TNF-{alpha}-stimulated. b, Cytokine-stimulated human ECs up-regulate CD40 (open histogram). IFN-{gamma} but not TNF-{alpha} stimulation results in HLA-DR up-regulation on human EC (open histogram). In contrast, cytokine-stimulated human ECs fail to up-regulate costimulatory molecule CD80 and CD86 (open histogram). c, Purified human CD4+ T cells proliferate poorly in response to human ECs but well when cultured as bulk PBMCs. d, ECs can be induced to stimulate purified CD4+ T cells if CD28 is cross-linked by the addition of anti-CD28 but not CD28-specific Fab.

 
To confirm that the failure of ECs to serve as effective T cell stimulators was related to their lack of B7 expression, ECs were coincubated with purified CD4+ T cells in the presence of anti-CD28 Ab, an isotype control, or anti-CD28 Fab. Intact anti-CD28 (but not isotype control Ab or Fab) induced T cell proliferation in the presence of allogeneic ECs (but not autologous cells), indicating that the lack of B7 by the ECs was a fundamental defect in their ability to primarily stimulate isolated T cells (Fig. 1d). Thus, ECs require an external source of costimulation (for example, B7 molecules) to activate isolated T cells, and ECs are not capable of providing this source despite cytokine activation.

Human monocytes require T cell help after exposure to allogeneic ECs to optimally express costimulation molecules

Human monocytes are typically thought to behave as mobile APCs and are known to be able to costimulate T cells via B7 molecule expression (21, 22). Thus, these cells could serve as the required source of B7 in the periphery. Unstimulated human monocytes, evaluated as part of freshly obtained or incubated PBMCs, expressed CD58, CD86, and HLA-DR, but not CD40, CD80, or CD134L. Following coculture with ECs and T cells (as PBMCs), monocytes increased their surface expression of CD40, CD80, and HLA-DR (Fig. 2) but not of CD58 or CD134L (not shown). However, this up-regulation was not an independent effect of exposure to allogeneic ECs. Unlike the monocytes in PBMC-EC cocultures, purified monocytes cocultured with ECs in isolation did not express surface CD40 or CD80 (Fig. 2). HLA-DR expression was increased in isolated coculture, and there was no significant change in CD86 expression for either coculture condition. Thus, although monocytes could express B7 molecules, their exposure to ECs alone was insufficient to induce this expression. Rather, some other component of PBMCs was needed to facilitate monocyte APC function. Therefore, under peripheral lymphopenic conditions, monocytes, like ECs, could be expected to be suboptimal APCs.



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  		FIGURE 2. The up-regulation of CD40 and CD80 on human monocytes is dependent on the presence of CD4+ T cells. Shown are flow cytometric analyses of human monocyte populations gating on CD14+ as purified resting monocytes in resting PBMCs, undergoing allogeneic ECs stimulated in the presence or absence of purified human CD4+ T cells, or as PBMCs. Isotype control results are shown in the gray histograms, whereas the experimental results are shown in the open histograms. Resting purified monocytes and monocytes in resting PBMCs express CD86 and HLA-DR but not CD40 or CD80 expression following a 3-day culture. Purified monocytes, stimulated by allogeneic ECs for 3 days, similarly failed to express CD40 or CD80. In contrast, allogeneic EC-stimulated PBMCs markedly up-regulated the costimulatory molecules CD40 and CD80. Purified monocytes were able to express CD40 and CD80 following allogeneic EC stimulation in the presence of CD4+ T cells.

 
To determine whether CD4+ T cells were permissive for EC-induced monocyte activation, purified autologous CD4+ T cells were added back to the isolated monocyte-EC cocultures and then evaluated after 3 days by FACS. CD4+ T cells reconstituted the ability of monocytes in monocyte-EC cocultures to express CD40 and CD80 (Fig. 2). To determine whether this effect required temporal colocalization between T cells, EC-conditioned monocytes were collected from monocyte-EC cocultures after 48 h and separated from the ECs. These EC-conditioned monocytes were negative for CD40 and CD80; however, following coincubation with autologous CD4+ T cells in the absence of ECs for an additional 24 h, these monocytes expressed CD40 and CD80 (Fig. 3). Thus, monocyte activation did not require T cell exposure to be simultaneous with EC exposure. Monocytes stimulated with EC for 72 h did not express CD40 or CD80, and monocytes that were not exposed to ECs did not up-regulate CD40 and CD80 when incubated with T cells (Fig. 3). Thus, monocytes required help, in this case in the form of exposure to autologous T cells, some time after a priming exposure to allogeneic ECs for optimal costimulation molecule expression.



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  		FIGURE 3. Monocyte expression of CD40 and CD80 following EC exposure does not require simultaneous exposure to ECs and T cells. Shown is a flow cytometric analysis of human monocyte populations from purified resting monocytes, EC-conditioned monocytes, and EC-conditioned monocytes following their coculture with CD4+ autologous T cells with gating on CD14+ monocytes. Forty-eight- and 72-h EC-stimulated monocytes as well as unstimulated monocytes were negative for surface expression of CD40 and CD80. EC-conditioned monocytes, when removed from the EC and subsequently incubated with CD4+ autologous T cells, up-regulated CD40 and CD80 expression (row 4). Coincubation of resting monocytes with resting autologous T cells did not result in up-regulation of CD40 and CD80.

 
Monocytes are primed for interactions with T cells by internalizing allogeneic EC membranes via scavenger receptors

Given that monocytes were activated only after EC exposure and subsequent CD4+ T cell exposure, we next examined the nature of the priming interaction between monocytes and ECs. EC monolayers were labeled with PKH-26, a membrane dye, and were then incubated with PBMCs or purified monocytes for various lengths of time and analyzed by FACS to determine whether monocytes physically engulfed EC membranes. Monocytes derived from PBMC (with T cells present)-EC cocultures took up PKH-26 following incubation with labeled ECs (Fig. 4a) as early as 60 min following coculture and increased their PKH-26 content after 2, 4, and 24 h of coincubation (Fig. 4a). PKH-26-positive monocytes expressed surface CD40 and CD80, but PKH-26-negative monocytes remained surface CD40- and CD80-negative (Fig. 4b), indicating that EC priming for subsequent T cell help was associated with membrane uptake. The engulfment of EC membranes was monocyte-specific, because neither CD4+ nor CD8+ T cells became positive for PKH-26 (data not shown). In addition, purified CD4+ T cells from CD4+ T cell-EC cocultures also remained negative for PKH-26 (data not shown). To determine whether the engulfment of EC membranes was dependent on the presence of T cells (as activation was shown to be) or some other PBMC-derived factor, purified monocytes were cocultured with PKH-26-labeled ECs. Interestingly, PKH-26 transfer to purified monocytes was detected both by FACS (data not shown) and confocal microscopy (Fig. 4c). However, as shown above, despite membrane uptake the monocytes did not express costimulation molecules in the absence of T cells. Thus, monocytes, but not T cells, engulfed EC membranes during EC-monocyte contact independently of the presence of T cells or T cell-derived factors, but EC membrane uptake was not sufficient for monocyte activation and costimulation of molecule expression. Membrane uptake was a distinct event from activation, although activation was predicated on EC exposure and uptake.



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  		FIGURE 4. Direct allogeneic cell uptake by monocytes. Shown is flow cytometric analysis of human monocyte populations from PBMC-EC cocultures. a, Monocyte populations stain positive for PKH-26 as early as 2 h following their interaction with PKH-26-labeled ECs and increase their uptake of PKH-26 following 4- and 24-h coincubation with labeled ECs. b, PKH-26-positive monocytes have enhanced expression of CD40 and CD80 following a 3-day coincubation (open histograms) as compared with PKH-26-negative monocytes. c, Shown are the results of confocal microscopic evaluation of monocytes after interaction with PKH-26-labeled EC monolayers showing dual staining for CD14-FITC and PKH-26 (x60).

 
To determine whether the uptake of EC membranes was allospecific (perhaps driven by unique carbohydrate differences but not necessarily HLA specificities), we studied monocytes derived from the splenocytes of a deceased human donor in which we had established an aortic EC population. When monocytes were cocultured with autologous PKH-26-labeled ECs for 16 h, they remained PKH-26-negative (Fig. 5). In contrast, when incubated with two allogeneic sources of ECs, the monocytes became positive for PKH-26, suggesting that these monocytes were able to discriminate between self and foreign sources of endothelium. In an additional study, coincubation of monocytes or PBMCs with PKH-26-labeled allogeneic fibroblasts resulted in positive PKH-26 staining on monocytes, indicating that this effect was not EC-specific (data not shown).



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  		FIGURE 5. Monocytes do not engulf autologous EC membranes. Shown are flow cytometric analyses of human monocyte populations from splenocyte-EC cocultures. Monocytes derived from splenocytes became positive for PKH-26 following their interaction with two separate PKH-26-labeled allogeneic EC populations (EC-1 and EC-2, open histograms) but did not stain positive when incubated with stained autologous ECs. The gray histograms in each panel show background from cocultures of splenocytes with unlabeled ECs.

 
In addition to active monocyte phagocytosis of EC membranes, several trivial explanations could explain the observed PKH-26 uptake results. We therefore investigated a variety of passive mechanisms to determine whether membrane uptake was an active process. To determine whether labeled ECs released PKH-26 into the culture medium that could then be taken up via passive scavenging, human PBMCs were incubated with PKH-26-labeled ECs in a Transwell system that prohibited cell-to-cell contact. Monocytes separated from ECs did not become PKH-positive, indicating a requirement for direct cell-to-cell contact (data not shown). To determine whether the uptake required contact with live cells or merely contact with any PKH-26-labeled entity, PBMCs were cocultured with unlabeled ECs that were seeded on PKH-26-labeled, gelatin-coated culture plates. PBMCs collected from these cocultures similarly did not become PKH-26-positive (data not shown). Thus, monocytes engulfed membranes specifically from live, nonself cells after direct cell-to-cell contact and not passively as a result of adherence to cellular debris. Furthermore, membrane uptake did not result in EC disruption, lysis, apoptosis, or death. EC monolayers remained intact and viable after 24 h of coincubation with PBMCs or purified monocytes. Thus, uptake was not simply a clearing of apoptotic ECs. Additionally, although monocyte adhesion to EC monolayers increased when EC monolayers were preconditioned with activating cytokines or hypoxia/hypothermia, these maneuvers did not lead to demonstrably increased membrane uptake. Thus, membrane engulfment was not dependent upon EC activation, injury, or apoptosis.

One mechanism known to facilitate live cell engulfment by monocytes involves the function of scavenger receptors, a broad family of integral membrane proteins (SR-A, SR-B, SR-D, and SR-E class) that function as innate pattern recognition receptors and mediate the cellular binding and internalization of many negatively charged macromolecules (23). A scavenger receptor blockade with scavenger receptor ligands such as poly(G) has been shown to prevent phagocytosis of apoptotic cells and glycosylated pathogens by macrophages (24). To evaluate the role of scavenger receptors in this process, human PBMCs were pretreated with poly(G) or poly(C), a structural homolog of poly(G), and were then coincubated with PKH-26-labeled ECs. Poly(G) but not poly(C) inhibited the uptake of EC membranes by monocytes in a dose-dependent manner (Fig. 6). Thus, the monocyte uptake of EC membranes was a contact-dependent process mediated by scavenger receptors.



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  		FIGURE 6. EC membrane uptake by monocytes is blocked in a dose-dependent fashion by scavenger receptor blockade with poly(G). Monocytes were incubated for 16 h with PKH-26-labeled allogeneic ECs in the presence of the indicated concentration of poly(G) or poly(C), a structural homolog of poly(G) that does not inhibit scavenger receptor function, and then assessed by flow cytometry gating on CD14+ monocytes.

 
One potential consequence of membrane uptake that would have a bearing on alloimmunity is alloantigen internalization and representation. It has been demonstrated that monocytes pulsed with allogeneic cell membranes can process immunodominant peptides for indirect presentation, and it has been equally established that monocytes can directly present MHC Ag to allogeneic T cells (reviewed in Ref.25). Recently, the prospect of intact MHC molecule transfer has been proposed as a mechanism for direct MHC presentation by an autologous cell (26), so-called "semidirect presentation" (27). We therefore looked to see whether monocyte EC engulfment facilitated intact HLA molecule transfer and surface expression using an Ab that recognized intact HLA as opposed to the HLA peptide. We evaluated HLA-A1-positive ECs coincubated with PBMC that were HLA-A1-negative as determined by molecular HLA typing as well as by FACS. The cells were interrogated with FACS by gating on monocytes both by forward and side scatter and on CD14 expression to rule out the possibility of false-positive PKH-26 staining of contaminating allogeneic ECs (neither resting nor activated ECs express CD14). The coincubation of HLA-A1-positive ECs with HLA-A1-negative PBMC resulted in transfer of the surface expression of HLA-A1 on CD14+ monocytes (Fig. 7). Thus, EC uptake not only facilitated monocyte priming and monocyte-T cell interactions, it also provided a mechanism for alloantigen presentation and transport.



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  		FIGURE 7. HLA-A1-negative monocytes become surface A1-positive after incubation with A1-positive ECs. Shown are flow cytometric analyses of ECs that express HLA-A1 (left panel, open histograms). Monocytes not coincubated with EC monolayers lack HLA-A1 expression on their surface (middle panel). Monocytes incubated with HLA-A1-positive ECs express HLA-A1 on their surfaces (right panel, open histograms).

 
EC-conditioned monocytes costimulate, activate, and induce proliferation in CD4+ T cells

Because it was evident that monocytes were primed for interaction with T cells by exposure to the allogeneic endothelium, the functional relevance of monocyte-EC conditioning was then assessed. Purified resting CD4+ T cells proliferated poorly in response to EC monolayers and when incubated with autologous monocytes (Figs. 1C and 8). However, when EC-conditioned monocytes were generated from purified monocyte-EC cocultures, these monocytes stimulated robust T cell proliferation that could not be augmented by additional anti-CD28 stimulation. Although, EC-conditioned monocytes did not express surface CD40 and CD80 before coincubation with T cells, the monocyte-induced T cell proliferation was inhibited by mAbs specific for CD80, CD86, or CD154 (Fig. 8), and a combined blockade with these Abs completely blocked T cell proliferation. Thus, monocyte EC uptake facilitated a proliferative T cell response that was costimulation-sensitive, yet costimulation molecule expression was predicated on exposure to T cells. Monocytes, even though primed with foreign cell membranes, reserved their costimulation molecule expression until they were in direct contact with T cells, at which time they up-regulated CD40 and CD80 and facilitated reciprocal T cell activation. As such, peripheral monocytes engaged with an allogeneic endothelium would be expected to reserve their costimulatory capabilities until they are in a T cell-rich environment, most typically a secondary lymphoid organ. Accordingly, monocytes in transit likely remain suboptimal APCs until they are in an appropriate environment, with appropriateness defined by excess T cell presence.



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  		FIGURE 8. EC-conditioned monocytes induced autologous CD4+ T cell proliferation that was inhibited by costimulation blockade. Shown are the results of proliferation studies evaluating CD4+ T cell proliferation from T cells or monocytes alone or from T cells cocultured with monocytes, EC-conditioned monocytes, or EC-conditioned monocytes in the presence of anti-CD80 and/or CD86 and/or CD154 (or isotype control Abs). CD4+ T cell proliferation was induced by EC-conditioned monocytes and inhibited by costimulation-specific mAbs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Monocytes are recognized as participants in human allograft rejection, and their role has been made more apparent by clinical lymphocyte depletion protocols (16, 17, 28, 29). It is unclear whether monocytes assume more responsibility for rejection in a lymphopenic environment or whether lymphopenia simply makes them more readily observable. Nevertheless, monocytes are clearly present histologically before and during all functionally significant cellular rejections. The data reported herein specifically address the initial events occurring when monocytes infiltrate a vascularized allograft and demonstrate a mechanism by which they can initiate an alloimmune response. These data further show the specific temporal and stoichiometric relationships required for a coordinated response between ECs, T cells, and monocytes and reveal a novel mechanism (direct cell engulfment) for response initiation. These studies begin to address the choreography between cells involved in rejection and suggest that there is a clear hierarchy required for delivering activating signals and Ags among the participants in cellular alloimmunity.

It is known that APCs, specifically murine DCs, require priming by CD4+ T cells to gain the ability to activate a cytotoxic response (30, 31, 32, 33). We have now shown that monocytes require an interaction with foreign cells to become receptive to priming and that this prepriming can be achieved through scavenger receptor-mediated cell uptake outside of a secondary lymphoid organ. This observation is novel and is the first report of scavenger receptor involvement in human alloimmunity. The scavenger receptor system is increasingly recognized as facilitating the engulfment of foreign cells and lipoprotein bodies based on carbohydrate recognition and is now seen as an important part of innate immunity (23, 24, 34). Thus, we suspect that the ability to distinguish autologous from allogeneic cells is related to carbohydrate moiety recognition, which is not a function of MHC. However, we cannot exclude the role of the small number (<1%) of T cells facilitating this discrimination. Elucidating this role will require additional study. Because allogeneic fibroblast membranes are also subject to uptake, this may represent a more general incompatibility than previously recognized, one that facilitates the presentation of intact HLA Ags and provides a means for direct allorecognition in the absence of donor-derived APCs. However, the specific characterization of scavenger receptor usage and the signaling involved will require considerable additional study.

Interestingly, the macrophage type A scavenger receptor has been recently identified as a differentially overexpressed transcript during rat allograft rejection (35), and scavenger receptors have also been increasingly implicated in advanced atherosclerotic disease (36). This finding is consistent with the advanced vasculopathy typically seen in transplanted organs (37) and deserves more intense scrutiny. These data also provide one explanation for why alloimmune responses are delayed considerably by profound peripheral monocyte depletion but eventually proceed upon monocyte resurgence (16).

These data suggest that monocytes in vivo become partially primed and capable of direct alloantigen recognition in the periphery but do not become capable of mature reciprocal responses until they gain access to T cells. Because T cells are mobilized only after Ag presentation, this would limit monocyte priming to the resting spots of T cells (such as the nodes) and limit peripheral activation. Clearly, mechanisms are required to limit peripheral T cell activation during chance encounters of cells in transit from sites of inflammation to lymph nodes, and additional regulation is needed to avoid nonactivated monocytes from inappropriately stimulating T cells by chance encounters in nodal tissue. This ordered response satisfies both of these requirements.

This study is also consistent with others in showing that human ECs, unlike mouse ECs, are incapable of expressing CD80 and CD86 (38, 39), making them suboptimal costimulatory cells and incapable alone of fostering a naive immune response or of promoting naive T cell Th1 differentiation (40). These findings are also consistent with studies by Adams et al. (41) and, more recently, Vudattu et al. (42) in showing that the presence of monocytes markedly influences the proliferative capacity of naive T cells. The conditions associated with the experiment are important in interpreting our findings and have been chosen to specifically relate to initial immune interactions in lymphopenic hosts. ECs have been shown by many investigators to induce CD4+ T cell proliferation under certain in vitro conditions, specifically when the cells are preincubated with IFN-{gamma} 2 (43, 44, 45, 46). However, this condition is not consistent with the physiological absence of IFN-{gamma} at the time of initial monocyte contact with allogeneic EC posttransplant reperfusion (17) and, thus, may not reflect the most likely conditions present during transplantation. Although CD8+ T cells have been shown in vitro to provide IFN-{gamma} and to stimulate the proliferative capacity of ECs (40), we have shown in humans via serial protocol biopsies that CD8+ T cells are rare in the early infiltrates of renal allografts in depleted hosts (16). Additionally, most prior studies have used ECs that have been trypsinized and replated with responding T cells, often in dense aggregates (e.g., U-bottom plates). We have used intact EC monolayers for our studies to more precisely mimic the early conditions seen clinically, particularly in patients following depletional induction. Thus, although conditions can be created in vitro to foster EC-mediated T cell proliferation, we do not feel that these conditions reflect those present posttransplant or that ECs independently induce naive T cell activation in vivo.

It is also important to draw a distinction between naive T cell responses and those of committed memory T cells. Purified CD8+CD45RO+ T cells can proliferate in response to allogeneic ECs directly, and studies have shown that CD2, LFA-3, and CD59 play a major role in this response. Similarly, many of the non-B7 costimulatory molecules present on the human ECs in this study (CD58, OX40L, and the ICOS ligand) have been shown to support the proliferation and cytokine secretion of memory and/or polyclonally stimulated T cells independent of the effects of B7 costimulation (47, 48, 49, 50, 51). However, there is no effect of these molecules on naive T cells, and they cannot by themselves induce a de novo response. Because the addition of CD28 signaling markedly changes the proliferative capacity of naive cells in this study, we favor a primacy of B7 costimulation during initial alloimmune interactions.

Monocytes are drawn to an allograft to some extent in proportion to the degree of reperfusion injury (17). The critical requirement of this cell type for T cell activation described herein helps explain the marked augmentation of alloimmunity that occurs commensurate with aggressive reperfusion injury and monocyte graft infiltration after T cell depletion (16). It is interesting to note that one of the most striking effects of methylprednisolone treatment (a glucocorticosteroid widely used at the time of allograft reperfusion) is monocyte clearance from the circulation (K. K. Dhanireddy and A. Kirk, manuscript in preparation) that is independent of the use of depleting Abs. Although steroids have many effects that could mediate their antirejection effects, their influence on monocyte mobilization may be one that has been overlooked. Although our in vitro results suggest that membrane engulfment is independent of EC activation, injury, or apoptosis, we cannot discount the possibility that increased adhesion would facilitate membrane uptake. Berliner and coworkers (52) have investigated interactions between monocytes and allogeneic ECs and have shown that allospecific T cells do facilitate monocyte adhesion. Indeed, we favor a model in which EC activation brings monocytes into proximity and allows this membrane uptake to occur.

Our observation of patients treated with potent lymphocyte-depleting agents combined with these in vitro studies lead us to suggest a model for alloimmune activation in a naive lymphocyte-depleted host (Fig. 9). We propose that alloimmunity is initiated by monocyte attraction to a reperfused vascularized allograft (17, 18) and the resultant scavenger receptor-mediated EC membrane engulfment. Monocytes then traffic without costimulation molecule expression to areas replete with T cells, where they can present intact HLA Ags via a semidirect method (27) and receive reciprocal priming signals from CD4+ lymphocytes. This process appears to be at least partially mediated by the IFN-{gamma} receptor on monocytes (H. Xu and A. Kirk, manuscript in preparation). Once primed, monocytes express CD80 and CD40 and have the necessary surface molecules to mature a cytotoxic T cell response (31, 32). This reciprocal codependence of T cells and monocytes helps insure that immune activation occurs only when there are alloantigen-expressing cells (in this case, ECs), monocyte activation, and a critical number of T cells. This mechanism is not thought to be exclusive, because tissue-based DCs clearly have the capacity to mediate Ag transfer to lymph nodes. However, this mechanism can substantially augment the capacity of the local environment to deliver and present Ags in response to local tissue injury and factors increasing chemotaxis. In circumstances like allotransplantation, this augmented capacity may take on a more prominent role.



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  		FIGURE 9. A model for the role of monocytes in allograft rejection. Following reperfusion and the resultant increase in monocyte chemotactic/adhesion factors, monocytes are attracted to the allograft and initiate EC membrane engulfment driven by scavenger receptor recognition. Monocytes then traffic without costimulation molecule expression to the secondary lymphoid tissues. During transit, the monocytes are unable to support sporadic T cell activation. However, upon entering areas replete with T cells, they can present intact HLA Ags via a semidirect method and receive reciprocal priming signals from CD4+ lymphocytes. Once primed, monocytes express CD80 and CD40 and are thus able to mature a cytotoxic T cell response. This reciprocal codependence of T cells and monocytes helps insure that immune activation occurs only when there are alloantigen-expressing cells (in this case, ECs), monocyte activation, and a critical number of T cells.

 
We conclude that human monocytes are primed by a direct interaction with allogeneic ECs through a process involving the incorporation of allogeneic EC membranes and the representation of HLA. This process is mediated via monocyte scavenger receptors and influences the mobile APCs to become receptive to subsequent interactions with CD4+ T cells. Interactions with CD4+ T cells induce the up-regulation of costimulatory molecules, "licensing" (32) the monocytes to mature a cytotoxic response. Accordingly, we suggest that monocytes are an important bridge between the allograft and T cells in initial alloimmune interactions. This finding suggests additional routes for intervention following allograft transplantation.


   Acknowledgments
 
We thank Dr. Josh Farber for his critical reading of this manuscript and helpful suggestions and for the continuous support of members of the Transplantation Branch.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was funded by the Division of Intramural Research of the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health. Partial salary support for K.K.D. was generously supplied by the Georgetown University Department of Surgery. Back

2 Address correspondence and reprint requests to Dr. He Xu, Room 5-5832, Building 10 CRC, Center Drive, Bethesda, MD 20892. E-mail address: hex{at}intra.niddk.nih.gov Back

3 Abbreviations used in this paper: EC, endothelial cell; DC, dendritic cell; HLA-A1, anti-human HLA class I Ag A1; poly(G), polyguanylic acid; poly(C), polycytidylic acid. Back

Received for publication April 19, 2005. Accepted for publication October 25, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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