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The Journal of Immunology, 1999, 163: 1010-1018.
Copyright © 1999 by The American Association of Immunologists

Alloantibody-Mediated Class I Signal Transduction in Endothelial Cells and Smooth Muscle Cells: Enhancement by IFN-{gamma} and TNF-{alpha}1

Hong Bian and Elaine F. Reed2

Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, NY 10032


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chronic rejection is the major limiting factor to long term survival of solid organ allografts. The hallmark of chronic rejection is transplant atherosclerosis, which is characterized by the intimal proliferation of smooth muscle cells, endothelial cells, and fibroblasts, leading to vessel obstruction, fibrosis, and eventual graft loss. The mechanism of chronic rejection is poorly understood, but it is suspected that the associated vascular changes are a result of anti-HLA Ab-mediated injury to the endothelium and smooth muscle of the graft. In this study we have investigated whether anti-HLA Abs, developed by transplant recipients following transplantation, are capable of transducing signals via HLA class I molecules, which stimulate cell proliferation. In this report we show that ligation of class I molecules with Abs to distinct HLA-A locus and HLA-B locus molecules results in increased tyrosine phosphorylation of intracellular proteins and induction of fibroblast growth factor receptor expression on endothelial and smooth muscle cells. Treatment of cells with IFN-{gamma} and TNF-{alpha} up-regulated MHC class I expression and potentiated anti-HLA Ab-induced fibroblast growth factor receptor expression. Engagement of class I molecules also stimulated enhanced proliferative responses to basic fibroblast growth factor, which augmented endothelial cell proliferation. These findings support a role for anti-HLA Abs and cytokines in the transduction of proliferative signals, which stimulate the development of myointimal hyperplasia associated with chronic rejection of human allografts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Aform of chronic rejection, termed accelerated transplant atherosclerosis, is the leading cause of late heart, lung, and kidney graft loss and is estimated to affect more than 40% of recipients within the first 5 yr following transplantation (1, 2, 3). Chronic rejection manifests itself as a progressive vasculo-occlusive disease, resulting in ischemic injury and deterioration of organ function. The histologic appearance of transplant atherosclerosis shows marked proliferation and hyperplasia of vascular smooth muscle cells (SMC)3 and endothelial cells (EC), implying that augmented EC and SMC responsiveness to growth factors contributes to the pathogenesis of the disease. Indeed, immunohistochemical analyses support a role for growth factors and growth factor receptors in the formation of vascular lesions associated with chronic rejection. Increased localization of platelet-derived growth factor (PDGF) and its receptor have been identified in areas of intimal hyperplasia associated with cardiac and renal allograft vasculopathy (4, 5). In addition, both acidic fibroblast growth factor (FGF) and its receptors were found to be increased following cardiac transplantation (5, 6). The expression of basic FGF (bFGF) and FGFR1 isoforms has also been shown to be up-regulated in the vessels of cardiac and renal allografts undergoing chronic rejection (6, 7).

Although the etiology of transplant atherosclerosis is poorly understood, there is substantial evidence to suggest that anti-HLA Abs are involved in the process of chronic rejection. Numerous studies have shown that patients developing anti-donor HLA Abs following transplantation are at increased risk of transplant atherosclerosis and graft loss (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). Furthermore, passive transfer of sera containing anti-donor MHC Abs has been shown to accelerate the development of transplant atherosclerosis in experimental models of transplantation (25, 26, 27). Moreover, allografts transplanted to B cell- or Ig-deficient mice fail to develop fibrotic arteriopathic lesions, whereas prominent fibrotic lesions occur in recipients developing humoral immunity to the allograft (28, 29, 30). Recent experimental studies have also shown that the genesis of transplant-associated arteriosclerosis depends upon the interaction of anti-MHC Abs with helper T cells and macrophages (28). These results imply that the cytokines and growth factors produced by T helper cells and macrophages act in concert with anti-MHC Abs to the development of transplant atherosclerosis.

In addition to their classical role in Ag presentation, HLA class I molecules have been shown to serve as signal-transducing molecules regulating cell growth (31, 32, 33, 34, 35, 36, 37, 38, 39). In light of these observations, we have postulated that anti-HLA Abs trigger the development of transplant atherosclerosis by binding to HLA class I molecules on endothelium and smooth muscle of the graft and transducing signals that stimulate cell proliferation. To explore this hypothesis, we tested the effect of human anti-HLA class I alloantibodies on cultured human aortic EC and SMC. In addition, we studied the effect of anti-HLA Abs in the context of the macrophage/T lymphocyte-derived cytokine TNF-{alpha} and the T helper cell-derived cytokine IFN-{gamma} to determine whether the combined factors enhance anti-HLA Ab-mediated alterations on EC or SMC. The results reveal that binding of human anti-HLA Abs to class I molecules expressed on EC and SMC stimulates rapid protein tyrosine phosphorylation, increased FGFR expression, and augmented cell proliferation. Exposure to TNF-{alpha} or IFN-{gamma} had no direct effect on FGFR expression on EC or SMC but rather increased class I expression and augmented anti-HLA class I Ab-mediated induction of FGFR expression. These findings indicate that anti-HLA Abs synergize with inflammatory cytokines to induce increased FGFR expression, thereby rendering the cells responsive to FGFs and stimulating cell proliferation. The mechanism underlying transplant atherosclerosis may reside in the induction of EC and SMC proliferation by anti-HLA Abs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

Human aortic ECs and SMCs from single donors were obtained from Clonetics (San Diego, CA) and propagated at 37°C, 5% CO2, in EC growth medium (EGM) containing 10 ng/ml human epidermal growth factor (EGF), 1.0 mg/ml hydrocortisone, 50 mg/ml gentamicin, 50 µg/ml amphotericin B, 3 mg/ml bovine brain extract, and 5% FCS (Clonetics). Assays were performed on EC and SMC monolayers that were 70–90% confluent and used between passages 2 and 6.

HLA typing

Human aortic ECs and SMCs were HLA typed using the two color fluorescence microlymphocytotoxicity technique (40), using HLA class I typing reagents obtained from One Lambda (Los Angeles, CA). The HLA phenotypes of cells used in this study are as follows: EC No. 2709 (HLA-A2, A68, B51, and BX); EC No. 2337 (HLA-A3, A11, B7, and B37); and SMC No. 2634 (A1, AX, B8, and B56).

Human anti-HLA alloantibodies

Sera containing anti-HLA-A2, anti-HLA-A1, and anti-HLA-B51 Abs were obtained from renal allograft recipients transplanted with a kidney mismatched for HLA-A2, HLA-A1, or HLA-B51, respectively. All sera were tested for the presence of lymphocytotoxic anti-HLA Abs on an HLA reference panel representing all established HLA-A, -B, -C, -DR, and -DQ Ags using the standard microlymphocytotoxicity assay as previously described (40). Sera from two patients (SJ, DN) producing anti-HLA-A2 Abs, two patients (RR, DC) producing anti-HLA-A1 Abs, and one patient (NN) producing anti-HLA-B51 Abs were selected for use in the study. These sera did not show any reactivity to HLA class II molecules, as determined by screening on the HLA reference panel of B lymphocytes. Sera obtained from these patients before transplantation, without anti-HLA Abs, were used as negative controls in parallel experiments.

Murine Abs

The following murine Abs were used: W6/32 (IgG2b), a mAb that binds to a monomorphic epitope on HLA class I Ags obtained from the American Type Culture Collection (Manassas, VA); and mouse IgG, used as an isotype control supplied from Sigma (St. Louis, MO).

Preparation of human IgG and F(ab')2 fragments

The IgG fraction of the serum was prepared by affinity chromatography using protein A (41). Following IgG purification, the protein was extensively dialyzed in PBS using a molecular cutoff of 14,000 daltons and adjusted to 10 mg/ml. F(ab')2 fragments of human anti-HLA Abs were prepared by digesting 10 mg of IgG with pepsin (0.1 mg/ml) in acetate buffer (pH 4.0) for 24 h at 37°C as previously described (41). The digested IgG was dialyzed against PBS and passed over a protein A-Sepharose CL-4B column supplied by Sigma. The unbound fraction was collected, and the purity of the F(ab')2 fragment was assessed on a 10% SDS polyacrylamide gel. The F(ab')2 fragments were used at a concentration of 5 mg/ml.

Proliferation assays

Human aortic EC and SMC were seeded into 96-well flat-bottom plates at 5000 cells/well and left to attach overnight in EGM. After 18 h of incubation, EGM was removed and replaced with EGM containing 5% FCS. On day 2, 200 µl of 5 mg/ml anti-HLA IgG or control IgG was added to the cultures. Where indicated, recombinant human bFGF (rhbFGF) (0.6 ng/ml) or polyclonal neutralizing Abs to human bFGF, PDGF, or TGF-ß (obtained from R&D Systems, Minneapolis, MN) were added at concentrations ranging from 10 µg/ml to 0.01 µg/ml together with anti-HLA IgG. [3H]TdR incorporation was determined by detaching the cells with 0.125% trypsin/0.05% EDTA, harvesting, and scintillation counting after 24, 48, and 72 h on an LKB Beta Plate Cell Harvester (Turku, Finland). All data are expressed as the mean cpm of triplicate determinations (SD < 10%). The percentage inhibition of anti-HLA IgG-induced cell proliferation by neutralizing Abs was calculated from the formula: [(cpm of cells treated with IgG) - (cpm of cells treated with IgG and neutralizing Abs)]/(cpm of cells treated with IgG and neutralizing Abs).

Quantitation of bFGF

EC and SMC were seeded into 24-well plates at a concentration of 56,000 cells/well. After 18 h of incubation at 37°C, EGM was removed and replaced with EGM containing 5% FCS. On day three, 1 ml containing 5 mg/ml of anti-HLA class I IgG or control IgG was added to the cells and incubated for up to 48 h at 37°C. Supernatants were collected from the cultures, and the amount of bFGF was quantitated using the Quantikine human FGF basic immunoassay (R&D Systems) according to the manufacturer’s specifications.

Phosphotyrosine immunoblotting

Tyrosine phosphorylation was measured by immunoblotting EC lysates with the anti-phosphotyrosine mAb PY20 as previously described (42, 43). Briefly, EC monolayers (1 x 106) were incubated in T25 flasks at 37°C for 16 h in EGM without supplements and treated for 3 min with 5 ml of 5 mg/ml anti-HLA IgG or negative control IgG. The samples were lysed directly in SDS sample buffer, boiled for 5 min, and centrifuged for 10 min at 14,000 x g. The supernatant was electrophoresed on 5–20% gradient SDS-PAGE, blotted on polyvinylidene difluoride (PVDF) membrane from Pierce (Rockford, IL), and blocked with 5% BSA. The immunoblot was incubated with biotinylated PY20 obtained from Zymed Laboratories (San Francisco, CA) for 1 h at room temperature, washed, and incubated with avidin-alkaline phosphatase (Zymed) for 1 h at room temperature. The blot was washed, developed, and analyzed.

Flow cytometry

EC and SMC seeded in T25 flasks were incubated for up to 72 h in 5 ml EGM media containing 2.5 ml anti-HLA class I IgG, 2.5 ml negative control IgG, 10 µg/ml mAb W6/32, or 10 µg/ml murine isotype control IgG. Where indicated, EC and SMC were preincubated for 24 h in TNF-{alpha} (200 U/ml) or IFN-{gamma} (500 U/ml) obtained from R&D Systems before the addition of anti-HLA Abs. The cells were washed three times and detached with 0.125% trypsin/0.05% EDTA. Expression of FGFR was determined by indirect immunofluorescence on a FACScan flow cytometer as previously described (42). Expression of ICAM-1, HLA class I, and VCAM-1 was determined by direct immunofluorescence using mAbs from Coulter (Miami, FL) and R&D Systems (Minneapolis, MN). Cell fluorescence was analyzed on a FACScan flow cytometer using CellQuest Software obtained from Becton Dickinson (Mountain View, CA). Gates for forward and side scatter measurements were set on EC or SMC, and a minimum of 10,000 events were acquired. Instrument calibration was performed using CaliBRITE beads and FACScomp software (Becton Dickinson).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anti-HLA class I Abs stimulate tyrosine phosphorylation in ECs

One of the earliest events observed during signal transduction is the activation of tyrosine kinases. To determine whether ligation of HLA class I molecules on EC with anti-HLA-A2 Abs stimulates tyrosine phosphorylation of intracellular proteins, Western blot studies were performed. EC were treated with anti-HLA-A2 Abs from patients SJ and DN for various periods of time, and cell lysates were electrophoresed and immunoblotted with the anti-phosphotyrosine mAb PY20. Treatment of ECs with the IgG fraction of sera containing anti-HLA-A2 Abs for 10 min resulted in an increase in tyrosine phosphorylation of cellular proteins at molecular masses ranging from 35 to 150 kDa, with prominent bands at approximate molecular masses of 70, 120, and 140 kDa. (Fig. 1Go, lanes B and C). A similar pattern of tyrosine phosphorylation was observed when EC were treated with W6/32, a mAb directed against a monomorphic epitope on HLA class I (Fig. 1Go, lane D). In contrast, tyrosine phosphorylation was not observed when ECs were treated with IgG prepared from an anti-HLA Ab negative serum obtained from SJ before transplantation (Fig. 1Go, lane A). These results demonstrate that the binding of anti-HLA Abs to HLA-A2 Ags expressed by ECs transduces signals, resulting in increased protein tyrosine phosphorylation.



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  		FIGURE 1. Tyrosine phosphorylation studies of EC following stimulation with anti-HLA-A2 Abs. Lanes A, EC treated with 5 mg/ml pretransplant IgG from SJ for 10 min; B, EC treated with 5 mg/ml anti-HLA-A2 IgG from SJ for 10 min; C, EC treated with 5 mg/ml anti-HLA-A2 IgG from DN for 10 min; D, EC treated with 10 µg/ml W6/32 IgG for 10 min. The results of one of four representative experiments are presented.

 
Anti-HLA class I Abs stimulate EC proliferation

To determine whether anti-HLA alloantibodies stimulate cell proliferation, human aortic ECs (No. 2709) expressing the HLA-A2 Ag were cultured in the presence of the IgG fraction of sera containing anti-HLA-A2 Abs. Incubation of ECs with anti-HLA-A2 Abs, in the absence of EC growth supplements, led to a significant increase in cell proliferation during the 48-h period of study (Fig. 2Go). Addition of anti-HLA-A2 IgG from patient SJ induced a 3-fold increase in the amount of [3H]TdR incorporation at 24 h and a 3.5-fold increase in cell proliferation at 48 h. Similarly, treatment of EC with anti-HLA-A2 IgG from patient DN induced a 2.5-fold increase in cell proliferation at 24 h and a 3-fold increase at 48 h. Incubation of ECs with medium containing mAb W6/32 had a similar effect on cell growth. In contrast, IgG prepared from sera obtained from patients SJ and DN before transplantation, without anti-HLA Abs, did not stimulate cell proliferation. These results indicate that ligation of class I molecules with human anti-HLA Abs recognizing polymorphic epitopes on HLA class I molecules induces EC proliferation.



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  		FIGURE 2. Ligation of HLA class I molecules with anti-HLA-A2 Abs stimulates cell proliferation. Quiescent EC were cultured with 10 µg/ml of murine isotype control IgG, 10 µg/ml mAb W6/32, 5 mg/ml of pretransplant IgG from patient SJ, 5 mg/ml of anti-HLA-A2 IgG from patient SJ, 5 mg/ml of pretransplant IgG from patient DN, or 5 mg/ml of anti-HLA-A2 IgG from patient DN. [3H]TdR incorporation was determined when the Abs were added and after 24 and 48 h. The data are expressed as the mean cpm of triplicate determinations (SD < 10%). One of five representative experiments is presented.

 
Effect of anti-HLA class I Abs on FGFR expression by EC

To investigate the mechanism of anti-HLA class I Ab-induced cell proliferation, we determined to what extent cell proliferation was dependent on the binding of growth factors to EC. Since FGFs are known to be the primary growth factors regulating EC proliferation, we evaluated the ability of anti-HLA-A2 Abs to stimulate EC growth in the presence of neutralizing Abs to bFGF. As shown in Fig. 3Go, the addition of anti-bFGF-neutralizing Abs significantly inhibited anti-HLA class I-induced cell proliferation. The capacity of anti-HLA-A2 Abs from DN and SJ to induce cell proliferation was inhibited by 70% and 50%, respectively. In contrast, neutralizing Abs to other growth factors produced by EC, such as TGF-ß and PDGF showed no inhibition of anti-HLA Ab-induced cell growth. These results suggest that anti-HLA Abs stimulate EC proliferation through the activation of FGFR.



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  		FIGURE 3. Inhibition of anti-HLA-A2-induced EC proliferation by anti-bFGF neutralizing Abs. EC were treated with 5 mg/ml anti-HLA-A2 IgG from patient SJ or DN in the presence and absence of neutralizing Abs to bFGF (1 µg/ml), PDGF (10 µg/ml), and TGF-ß (10 µg/ml). [3H]TdR incorporation was determined after 24 and 48 h, and the results obtained at 48 h are presented. The data are expressed as the percentage inhibition of proliferation. One of four experiments is presented.

 
Class I signaling may stimulate proliferation, either by stimulating bFGF production or by increasing FGFR expression, thereby facilitating ligand binding. To determine whether class I ligation leads to an increased production of bFGF, ECs were deprived of growth factors for 24 h and stimulated with anti-HLA-A2 IgG, and the amount of bFGF present in the culture supernatant was measured after 48 h. There was no difference in the amount of bFGF present in supernatants from ECs incubated with IgG containing anti-HLA-A2 Abs or IgG without anti-HLA Abs (data not shown). We therefore determined whether Ab ligation of class I molecules on EC altered FGFR expression on the surface of the EC. The experimental design was to treat EC monolayers with anti-HLA-A2 Abs and determine the number of EC expressing FGFR by FACS analysis. In the presence of anti-HLA-A2 Abs from SJ, 55% of the cells expressed FGFR, whereas in untreated EC (not shown), or EC exposed to anti-HLA Ab negative IgG, less than 15% of cells were FGFR positive (Fig. 4GoA). Similar results were obtained when EC were exposed to anti-HLA Abs from DN (Fig. 4GoB). Ligation of class I molecules with anti-HLA-A2 IgG resulted in a significant increase in the number of cells expressing FGFRs (65%), compared with EC treated with IgG without anti-HLA Abs (15%), or untreated EC (16%, not shown). We also examined the response of EC to anti-HLA Abs reacting with HLA-B molecules. Treatment of EC No. 2709 with anti-HLA-B51 Abs (patient NN) stimulated FGFR expression (70%), whereas anti-HLA Ab negative sera had no effect (Fig. 4GoC). The highest level of FGFR expression (96%) was observed when EC No. 2709 were stimulated with the mAb W6/32, which recognizes a monomorphic epitope on all HLA-A, -B, and -C molecules (Fig. 4GoD). These results indicate that both HLA-A and HLA-B molecules are capable of traducing signals. However, it appears that simultaneous engagement of HLA-A, -B, and -C molecules transduces the maximal activation signal in EC.



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  		FIGURE 4. Ligation of class I molecules by anti-HLA-A and anti-HLA-B locus Abs induced FGFR expression on EC. EC were treated with anti-HLA-IgG for 24 h, and the expression of FGFRs was determined by indirect immunofluorescence on a FACScan flow cytometer. A, EC treated with 5 mg/ml pretransplant IgG from SJ (dashed line); EC treated with 5 mg/ml anti-HLA-A2 IgG from SJ (solid line). B, EC treated with 5 mg/ml pretransplant IgG from DN (dashed line); EC treated with 5 mg/ml anti-HLA-A2 IgG from DN (solid line). C, EC treated with 5 mg/ml pretransplant IgG from NN (dashed line); EC treated with 5 mg/ml anti-HLA-B51 IgG from NN (solid line). D, EC treated with 10 µg/ml murine isotype control (dashed line); EC treated with 10 µg/ml mAb W6/32 (solid line).

 
To determine whether increased FGFR expression following class I ligation is dependent on the release and/or uptake of FGF, ECs were stimulated with mAb W6/32 in the presence or absence of neutralizing Abs to bFGF and tested for FGFR expression by FACS. The addition of anti-bFGF-neutralizing Abs failed to block anti-HLA class I-mediated induction of FGFR expression on EC. Treatment of EC with mAb W6/32 (10 µg/ml) for 24 h stimulated FGFR expression on 53% of EC. Similarly, treatment of EC with mAb W6/32 (10 µg/ml) in the presence of anti-bFGF-neutralizing Abs (1 µg/ml) stimulated FGFR expression on 51% of the cells. These results indicate that class I-mediated induction of FGFR expression is not dependent on the release and/or uptake of bFGF.

To investigate the time course of FGFR expression following class I ligation, ECs were treated with anti-HLA Abs for various time intervals and tested for FGFR expression by FACS analysis (Table IGo). FGFR expression was up-regulated as early as 1 h after exposure of EC to anti-HLA-Abs and remained at high levels thereafter. This suggests that signal transduction via MHC class I leads to the rapid release of intracellular stores of FGFR.


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  		Table I. Time course of FGFR expression on EC stimulated with anti-HLA-A2 Abs

 
To exclude the possibility that signaling was mediated by Fc receptors, F(ab')2 fragments were prepared from the anti-HLA-A2-positive IgG (SJ) and tested for their capacity to induce FGFR expression on EC. As shown in Fig. 5Go, addition of F(ab')2 fragments of anti-HLA-A2 Abs to EC cultures stimulated an increase in FGFR expression that was similar to the level of expression on cells treated with the intact anti-HLA-A2 IgG. These results clearly indicate that signaling is mediated via HLA class I molecules and not by Fc receptors.



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  		FIGURE 5. Ligation of class I molecules with F(ab')2 fragments of anti-HLA-A2 IgG stimulates FGFR expression on EC. Figure shows EC treated with 5 mg/ml pretransplant IgG from SJ (solid line), EC treated with 5 mg/ml anti-HLA-A2 F(ab')2 from SJ (dark solid line), and EC treated with 5 mg/ml anti-HLA-A2 IgG (dashed line). EC were treated with anti-HLA Abs for 24 h, and the FGFR level was determined by indirect immunofluorescence on a FACScan flow cytometer. One of two representative experiments is shown.

 
To examine the specificity of class I-mediated induction of FGFR, we tested the ability of anti-HLA-A2 Abs to induce FGFR expression on EC that do not express the HLA-A2 Ag. As summarized in Table IIGo, anti-HLA-A2 Abs specifically up-regulated FGFR expression in HLA-A2-positive EC (No. 2709), yet had no effect on HLA-A2-negative EC (No. 2337). In contrast, treatment of HLA-A2-negative EC (No. 2337) with mAb W6/32 stimulated FGFR expression. To eliminate the possibility that FGFR induction was mediated by cytokines contaminating the IgG preparation, we tested the ability of the anti-HLA-A2 IgG for its capacity to alter adhesion molecule expression on EC. Since ICAM-1 and VCAM-1 expression are known to be increased by cytokines, such as IL-1ß and TNF-{alpha}, present in human sera (44), we determined whether the anti-HLA-A2 IgG could up-regulate expression of these molecules. The anti-HLA-A2 IgG was incubated with EC No. 2709 and EC No. 2337 for 24 h and stained for ICAM-1 and VCAM-1 expression. The anti-HLA-A2 IgG did not induce VCAM-1 expression or cause an increase in the expression of ICAM-1 above the baseline level (data not shown). These experiments demonstrate that induction of FGFRs on EC following exposure to anti-HLA-A2 Abs is due to specific binding of anti-HLA Abs to HLA-A2 molecules expressed by the EC and rules out the possibility that induction of FGFR is due to non-HLA Abs or to cytokines present in the Ig preparation.


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  		Table II. Anti-HLA-A2 Abs specifically induce FGFR expression on HLA-A2-positive EC1

 
The above data indicate that anti-HLA Ab binding to class I molecules on EC transduces signals that lead to increased expression of FGFR. To determine whether increased FGFR expression on EC is accompanied by augmented proliferative responses to bFGF, quiescent EC were stimulated with anti-HLA-A2 Abs in the presence and absence of recombinant human bFGF (rhbFGF) (Table IIIGo). The addition of rhbFGF to anti-HLA-A2 Ab-treated EC resulted in a proliferative response that was approximately 3 times greater than cultures treated with anti-HLA-A2 IgG alone and 2.5 times greater than EC cultures treated with bFGF alone. These results show that, following class I ligation, EC proliferative responses to bFGF are enhanced.


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  		Table III. Effect of bFGF on anti-HLA-A2 Ab-induced proliferation of EC

 
Ligation of HLA class I molecules on SMCs by anti-HLA Abs stimulates FGFR expression

Our findings that ligation of class I molecules with human anti-HLA Abs results in increased FGFR expression on EC suggested that anti-HLA Abs may have a similar effect on SMC. To explore this possibility, SMC were treated with anti-HLA-A1 Abs, and cell surface expression of FGFRs was quantitated by FACS analysis. The addition of anti-HLA-A1 IgG from patient RR (Fig. 6GoA) and DC (Fig. 6GoB) to HLA-A1-positive SMC induced a 3-fold increase in the amount of FGFR at 24 h. In contrast, IgG prepared from anti-HLA Ab negative sera from these same patients had no effect on FGFR expression. These results are consistent with the effect of anti-HLA class I Abs on EC and indicate that binding of anti-HLA Abs to class I Ags induces FGFR expression on SMC.



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  		FIGURE 6. Induction of FGFR expression on SMC treated with anti-HLA-A1 Abs. SMC were treated with anti-HLA Abs for 24 h, and the cell surface expression of FGFR was determined by indirect immunofluorescence on a FACScan flow cytometer. A, SMC treated with 5 mg/ml pretransplant IgG from patient RR (dashed line) and SMC treated with 5 mg/ml anti-HLA-A1 IgG for patient RR (solid line). B, SMC treated with 5 mg/ml pretransplant IgG from patient DC (dashed line) and SMC treated with 5 mg/ml anti-HLA-A1 IgG from patient DC (solid line). One of four representative experiments is presented.

 
TNF-{alpha} and IFN-{gamma} potentiate anti-HLA class I Ab-mediated induction of FGFR expression

Numerous experimental and clinical studies have indicated that cytokines are important in the processes underlying allograft rejection since they have been shown to regulate the differentiation and activation of immune effector cells and alter the expression of MHC and adhesion molecules on EC and SMC (45, 46, 47, 48, 49, 50, 51, 52). We therefore examined the effect of two inflammatory cytokines, TNF-{alpha} and IFN-{gamma}, on HLA class I-mediated induction of FGFR expression. For these experiments, EC and SMC were pretreated with TNF-{alpha} or IFN-{gamma} for 24 h, stimulated with mAb W6/32 in the presence of TNF-{alpha} or IFN-{gamma} for an additional 24 h, and cell surface expression of FGFR was quantitated by FACS. As shown in Fig. 7Go, addition of TNF-{alpha} alone had no effect on FGFR expression. In contrast, exposure of EC and SMC to the combination of anti-HLA Abs and TNF-{alpha} stimulated a 4-fold increase in FGFR above the level induced by anti-HLA Ab alone (Fig. 7Go, A and C). Similarly, exposure of EC and SMC to IFN-{gamma} alone had no effect on FGFR expression whereas cells pretreated with IFN-{gamma} followed by stimulation with anti-HLA Abs showed a 3-fold increase in FGFR, compared with cells treated with Abs alone (Fig. 7Go, B and D). The capacity of human anti-HLA-A2 Abs to up-regulate FGFR expression on EC No. 2709 was also augmented by IFN-{gamma} and TNF-{alpha} (Fig. 7Go, E and F). These results indicate that both TNF-{alpha} and IFN-{gamma} augment anti-HLA Ab-mediated class I signaling in EC and SMC, resulting in higher levels of FGFR expression.



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  		FIGURE 7. Effect of TNF-{alpha} and IFN-{gamma} on anti-HLA Ab-mediated induction of FGFR. EC and SMC were pretreated with cytokine for 24 h and stimulated with anti-HLA Abs and cytokine for an additional 24 h, and cell surface expression of FGFR was measured by FACS. A, EC treated with 200 U/ml TNF-{alpha} (dashed line); EC treated with 10 µg/ml mAb W6/32 (solid line); and EC treated with 200 U/ml TNF-{alpha} and 10 µg/ml mAb W6/32 (dark solid line). B, EC treated with 500 U/ml IFN-{gamma} (dashed line); EC treated with 10 µg/ml mAb W6/32 (solid line); and EC treated with 500 U/ml IFN-{gamma} and 10 µg/ml mAb W6/32 (dark solid line). C, SMC treated with 200 U/ml TNF-{alpha} (dashed line); SMC treated with 10 µg/ml mAb W6/32 (solid line); and SMC treated with 200 U/ml TNF-{alpha} and 10 µg/ml mAb W6/32 (dark solid line). D, SMC treated with 500 U/ml IFN-{gamma} (dashed line); SMC treated with 10 µg/ml mAb W6/32 (solid line); and SMC treated with 500 U/ml IFN-{gamma} and 10 µg/ml mAb W6/32 (dark solid line). E, EC treated with 200 U/ml TNF-{alpha} (dashed line); EC treated with 10 mg/ml anti-HLA-A2 IgG (SJ) (solid line); and EC treated with 200 U/ml TNF-{alpha} and 10 mg/ml anti-HLA-A2 IgG (SJ) (dark solid line). F, EC treated with 500 U/ml IFN-{gamma} (dashed line); EC treated with 10 mg/ml anti-HLA-A2 IgG (SJ) (solid line); and EC treated with 500 U/ml IFN-{gamma} and 10 mg/ml anti-HLA-A2 IgG (SJ) (dark solid line) .

 
It is well established that IFN-{gamma} and TNF-{alpha} can up-regulate the surface expression of HLA class I. Thus, TNF-{alpha} and IFN-{gamma} may potentiate class I signaling by up-regulating class I expression and increasing the binding of anti-HLA Abs to EC and SMC. To test this hypothesis, anti-HLA Abs and cytokines were added simultaneously to EC, and cell surface expression of class I and FGFR was measured at various time intervals. We then determined whether the capacity of anti-HLA Abs to induce maximal FGFR expression correlated with an increased density of class I on the EC. As shown in Fig. 8Go, there was a positive relationship between increased class I expression and the ability of anti-HLA Abs to stimulate FGFR expression. There was no significant increase in the density of HLA class I or FGFR on EC treated with Ab + IFN-{gamma} or Ab + TNF-{alpha} for 1 or 6 h, compared with cells stimulated with anti-HLA Ab alone. In contrast, there was a 6-fold increase in the density of class I Ag and a 2-fold increase in the density of FGFR on EC treated with Ab + TNF-{alpha} or Ab + IFN-{gamma} for 24 h, compared with cells treated with anti-HLA Ab alone. These results indicate that IFN-{gamma} and TNF-{alpha} enhance anti-HLA Ab-induced FGFR expression because they increase MHC class I gene expression.



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  		FIGURE 8. Relationship between the density of class I expression and capacity of anti-HLA Abs to induce FGFR expression on EC. EC were treated with anti-HLA Abs alone (10 µg/ml mAb W6/32), or together with IFN-{gamma} (500 U/ml) or TNF-{alpha} (200 U/ml), and cell surface expression of HLA class I (bars) and FGFR (lines) was measured on a FACScan flow cytometer at 1, 6, and 24 h. The results are expressed as the mean intensity of fluorescence on a log scale. One of four representative experiments is presented.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although anti-HLA Abs have been implicated in chronic rejection, their precise role in the disease process is not well understood. Chronic rejection is characterized by the proliferation of intimal smooth muscle cells and endothelial cells in the walls of the arteries, resulting in occlusion of the vessels and fibrosis of the graft. A consistent finding in graft atherosclerotic lesions is Ig deposits in affected vessel walls and within the media (2). In contrast to hyperacute rejection or acute humoral rejection, the endothelium remains intact, suggesting that anti-HLA Abs do not cause a severe destructive vasculitis or necrosis. Our studies are the first to show that ligation of class I molecules with human anti-HLA Abs recognizing polymorphic residues located on the class I heavy chain transduce activation signals in EC and SMC and initiate cell proliferation in a model relevant to the development of transplantation-associated vasculopathies. Thus, engagement of class I molecules by anti-HLA Abs stimulated tyrosine phosphorylation of intracellular proteins, increased FGFR expression, and enhanced proliferative responses to bFGF. Our findings that anti-HLA Abs specifically induce FGFR expression only in cells bearing the appropriate HLA Ag, and that this effect is retained in the IgG and F(ab')2 fraction of the serum, supports the hypothesis that signaling is mediated by anti-HLA Abs and not by non-HLA Abs or Fc receptors. Furthermore, the Ig fraction of the anti-HLA Abs failed to induce ICAM-1 and VCAM-1 expression on EC, ruling out the possibility that FGFR up-regulation was caused by cytokines or other low m.w. mediators.

Numerous studies using lymphocytes as target cells have shown that HLA class I molecules can transduce signals that can regulate various aspects of cell metabolism, including activation and cell growth or cell cycle arrest and apoptosis, depending upon the Ab specificity and degree of molecular aggregation. (31, 36, 37, 38, 53, 54, 55, 56, 57, 58, 59, 60, 61). Cross-linking of class I molecules on T cells can stimulate tyrosine phosphorylation of multiple proteins including PLC-{gamma}1 (56, 59, 62, 63) and increased intracellular Ca+2 levels (36, 64, 65), and may result in IL-2 production, IL-2R expression, and cellular proliferation (36, 38, 56). In contrast, mAbs against class I molecules have also been shown to inhibit lymphocyte activation in response to triggering through the TCR or by T and B cell mitogens (66, 67, 68). Genestier et al. have recently shown that mAbs that bind to specific epitopes on the {alpha}1 domain of HLA class I H chain can induce apoptotic cell death of activated, but not resting, peripheral T lymphocytes (32, 33). The current studies demonstrate that anti-HLA Abs to polymorphic epitopes on both HLA-A locus and HLA-B locus molecules effectively transduce activation signals in EC and SMC irrespective of the epitope they recognize. This suggests that the mechanism whereby anti-HLA Abs stimulate EC and SMC FGFR expression relates to their capacity to cross-link these molecules rather than to induce conformational changes in the class I molecule. Consistent with this interpretation is the observation that cross-linking class I molecules by bivalent murine IgG molecules is required for the generation and transduction of proliferative signals (42, 69).

It is well established that TNF-{alpha} and IFN-{gamma} play an important role in mediating allograft rejection since increased production of these inflammatory cytokines has been found in association with episodes of human liver, kidney, and heart allograft rejection (46, 47, 48, 51, 70). The current studies show that IFN-{gamma} and TNF-{alpha} synergize with anti-HLA Abs to transduce maximal activation signals to EC and SMC, resulting in augmented FGFR expression. The ability of TNF-{alpha} and IFN-{gamma} to amplify class I signaling was related to their capacity to up-regulate class I MHC Ag expression on the surface of EC and SMC and enhance the binding of anti-HLA Abs. These results indicate that the intensity of the signal generated following engagement of class I is dependent upon the number of class I molecules ligated by anti-HLA Abs. Consistent with this interpretation is the observation that treatment of EC with mAb W6/32 directed against total class I (HLA-A, -B, and -C) stimulated a higher level of FGFR expression than treatment with Abs to individual A locus or B locus molecules. These findings have important clinical implications since, during acute allograft rejection, IFN-{gamma} and TNF-{alpha} are released by alloreactive T cells and macrophages infiltrating the graft. Therefore, patients producing anti-HLA Abs concomitant with an acute rejection episode may be at increased risk of developing transplant atherosclerosis. In support of this conclusion are previous data from our laboratory showing that development of transplant atherosclerosis is strongly associated with the production of anti-donor HLA Abs and the occurrence of multiple rejection episodes (20). These findings also predict that patients developing Abs to more than one of the donor’s mismatched HLA Ags may have a greater potential to transduce activation signals to EC and SMC and therefore be at higher risk of developing transplant atherosclerosis.

The data demonstrate that FGFR expression is rapidly up-regulated on the surface of the EC and SMC following ligation of class I molecules by anti-HLA Abs. We also found that anti-HLA class I Ab-mediated proliferation could be inhibited by the addition of neutralizing Abs to bFGF. Together, these results indicate that the FGFR is a major costimulatory molecule for the generation of class I-mediated proliferative signals. FGFRs belong to the superfamily of tyrosine kinase growth factor receptors (71, 72). FGF are known to induce cell proliferation by binding to FGFRs and stimulating receptor dimer formation and receptor autophosphorylation (73). FGF binding subsequently triggers a series of downstream events, including activation of p21ras, phopholipase C-{gamma}, p90/FRS2, Shc, and mitogen-activated protein (MAP) kinases, and activation of nuclear transcription factors culminating in cell proliferation (73, 74, 75). This suggests that the signaling pathway triggered after HLA class I engagement by anti-HLA Abs induces the expression of FGFR, rendering the EC and SMC responsive to FGFs and stimulating cell proliferation. Consistent with this interpretation, we found that anti-HLA Ab induction of FGFR expression was accompanied by increased EC responsiveness to bFGF and augmented cell proliferation. These results are also in agreement with our recent studies, which showed that HLA class I-mediated induction of cell proliferation correlates with inactivation of the Rb protein in the Jurkat T cell line and in human aortic EC (76). HLA class I-mediated inactivation of Rb was specifically inhibited by neutralizing Abs to bFGF, confirming the role of FGFR in the signaling process. The molecular mechanisms involved in the activation of the FGFR by HLA class I remains to be elucidated, but it is possible that the signal transduction pathways activated following class I ligation share a common pathway regulating FGFR metabolism. For example, phospholipase C is activated following class I signaling, and it is a known substrate of the FGFR (59, 77).

In conclusion, our data indicate that transplant-associated atherosclerosis may be mediated by anti-HLA Abs that bind to class I molecules on the endothelium and smooth muscle of the allograft and transduce signals that stimulate FGFR expression and cell proliferation. Our data also show that the inflammatory cytokines TNF-{alpha} and IFN-{gamma} may play a key role in this process by up-regulating MHC class I expression on EC and SMC and, as a result, by enhancing the binding of anti-HLA Abs to the cell. Increased binding of anti-HLA Abs to class I molecules amplifies the intensity of the signals generated, resulting in augmented FGFR expression and maximal responsiveness to FGFs. Thus, prevention of transplant atherosclerosis will require the identification of agents that can block the autocrine and paracrine effects associated with the FGFR. Inhibition of IFN-{gamma} and TNF-{alpha} production and/or activity should also be considered as a therapeutic intervention for chronic rejection.


   Acknowledgments
 
We thank Dr. Paul Harris for his advice and critical review of the manuscript.


   Footnotes
 
1 This work was supported by National Institute of Allergy and Infectious Diseases Grant RO1 AI/HL 42819 and a Charles H. Leach II Foundation Grant-In-Aid from the American Heart Association, New York City affiliate. Back

2 Address correspondence and reprint requests to Dr. Elaine F. Reed, Department of Pathology, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, Room 14-401, New York, NY 10032. E-mail address: Back

3 Abbreviations used in this paper: SMC, smooth muscle cell; FGF, fibroblast growth factor; bFGF, basic FGF; rhbFGF, recombinant human bFGF; EC, endothelial cells; EGM, EC growth medium; PDGF, platelet-derived growth factor. Back

Received for publication February 18, 1999. Accepted for publication April 29, 1999.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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