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Abstract
Peptide growth factors have been reported to contribute to the atherogenic process, and they are known to mediate signals for vascular remodeling. Using syngeneic and allogeneic rat aorta transplant models, we analyzed the impact of cold ischemia time up to 24 hours and reperfusion injury on development of transplant arteriosclerosis during the first 2 months after transplantation. The expression of the transforming growth factor-β (TGF-β) family as well as the platelet-derived growth factor (PDGF) and its receptors was studied by use of immunohistochemistry, followed by semiquantitative evaluation and multivariate analysis. In the syngeneically transplanted aortas, the expression of TGF-β1, PDGF, and the two PDGF receptors in the neointima increased significantly with the extent of cold ischemia time. Furthermore, there was a significant induction of the latent TGF-β binding protein in the neointima as well as TGF-β2 in the media, both correlating with the observation time after transplantation. In the allogeneic grafts, all examined proteins were already induced strongly 2 weeks after transplantation, even at the shortest ischemic period studied (1 hour). However, no positive correlation between growth factor expression and cold ischemia or observation time could be found. Double immunohistochemistry revealed that macrophages express PDGF and its receptors as well as TGF-β1. Smooth muscle cells express both types of PDGF receptors, and a few T cells express TGF-β1 as well as PDGF receptors. In summary, TGF-β and PDGF are induced by allogeneic as well as ischemic stimuli in transplanted aortas, suggesting a role in the pathogenesis of transplant arteriosclerosis and representing a potential target for therapeutic intervention.
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Presented in part at the Joint XIIth World Congress of Cardiology and the XVIth Congress of the European Society of Cardiology, Berlin, Germany, September 10-14, 1994. Drs Waltenberger and Akyurek contributed equally to this article.
- Received November 14, 1995.
- Accepted March 28, 1996.
Atherogenesis has a great impact on both cardiovascular and total morbidity and mortality in Western societies. It is characterized as a slowly progressive and complex process involving several cell types and various mechanisms.1 The most prominent features are the migration and proliferation of SMCs and the deposition of extracellular matrix in the vessel wall, resulting in vascular remodeling.2
Several growth factors have been found to be induced during atherogenesis,3 4 5 inflammation, and repair processes. In fact, mitogenic activity has been shown to be present in the atherosclerotic lesion.6 7 8 Infusion of recombinant PDGF-BB9 as well as increasing the expression of PDGF-BB10 or TGF-β111 in the arterial wall using gene transfer technology has been shown to induce a rapid and pronounced neointima formation with distinct morphological differences induced by these two genes. Moreover, antagonizing either PDGF12 or TGF-β113 in vivo resulted in the reduction of intimal SMC accumulation after angioplasty.
Two major goals in atherosclerosis research are to analyze the mechanisms of atherogenesis and to test therapeutic approaches in a preclinical setting, the latter requiring the establishment of reliable and valid animal models. We and others have been developing and characterizing an in vivo model of transplantation arteriosclerosis that allows the isolated analysis of morphological and molecular changes in the arterial wall.14 15 16 17
In contrast to the conventional type of atherosclerosis, transplant arteriosclerosis occurs as a result of chronic vascular rejection18 and represents a distinct pathological entity that differs both in cellular composition, plaque distribution, and dynamics.19 20 Different variables include immunologic incompatibilities between donor and recipient, number and severity of major rejection episodes, and the duration of the cold ischemia time before implantation of the graft. Because of its rapid development within weeks after transplantation, this form of atherosclerosis can be studied in animal models in a standardized fashion, allowing the investigation of the expression of various molecules involved in this process.
However, when syngeneic transplantations are performed, this model is suitable for studying the mechanisms of arteriosclerosis in the absence of rejection, allowing us to study the impact of nonimmunologic parameters such as the role of an ischemia-reperfusion injury.
In the present study, we have assessed expression of the TGF-β family, including the LTBP, and of PDGF and its receptors in a rat aortic transplant model using immunohistochemical techniques.
Methods
Animals
Male PVG (RT1c) and DA (RT1a) rats (Møllegaard, Skensved, Denmark) aged 3 to 4 months and weighing 120 to 200 g were used. The rats were serologically negative for the most common viruses, such as the Hantaan, Kilham, REO3, Coronavirus, Sendai, and Theiler's mouse encephalomyelitis viruses, and for Mycoplasma pneumoniae. The rats were housed at our facility for 2 weeks before surgery and received food and water ad libitum. Beginning 1 week before transplantation, the rats were fed a 0.5% cholesterol pellet diet (AnalyCen). The allogeneic transplantations were performed from DA donors to PVG recipients, and the syngeneic transplantations were done among PVG rats. In each group, we assessed a total of 42 animals, covering the entire spectrum of variables tested with different ischemia time periods of 1, 4, or 24 hours and posttransplantation observation times of 2, 4, 6, or 8 weeks.
Aorta Transplantation
Recipient rats were anesthetized by an intraperitoneal injection of a mixture of chloral hydrate (195 mg/kg body weight) and pentobarbital (45 mg/kg; Apoteksbolaget) and donor rats with thiobarbital at 120 mg/kg (Inactin; Byk Gulden). The abdominal aorta of each recipient rat was dissected free from just below the renal arteries to the bifurcation, and a 10- to 15-mm-long portion from the donor rat was excised as described previously.15 17 Grafts were stored at 4°C for 1, 4, or 24 hours in a histidine-buffered preservation solution (Frödin solution).21 Afterward, each graft was implanted orthotopically and anastomosed. After up to 8 weeks, the rats were anesthetized and the grafts excised. From each recipient rat, 10- to 15-mm-long nontransplanted portions from the abdominal aorta were harvested to serve as negative controls for immunohistochemical analysis. Each transplanted and nontransplanted segment was divided into two parts, one of which was frozen in a mixture of cold isopentane and dry ice and kept at −70°C until processed for immunohistochemistry. The other part was fixed in 4% buffered formaldehyde at room temperature and embedded in paraffin. These sections were then stained with van Gieson and Mayer's hematoxylin and eosin for histological and comparative studies.
Antibodies
Antibodies against latency-associated peptides of the three TGF-β isoforms and the antibody against LTBP were used as previously described.22 23 In addition, we used antibodies against PDGF B-chain (PGF007, Mochida Co),5 the PDGF β-receptors (Ab1, Oncogene Science),24 and the PDGF α-receptors25 as previously described by others. For identification of various cell types, cell markers were identified on monocytes/macrophages (ED1, Serotec) and on macrophages (ED2, Serotec), α-actin was identified in SMCs (anti-SMC, Dakopatts), and CD4 and CD8 were detected on T lymphocytes (W3/25 and OX8, respectively, kindly provided by A.F. Williams, Oxford, UK) as described in our earlier study.15 Furthermore, the activation of cells was assessed by the use of the antibody OX6 (generously provided by A.F. Williams, Oxford, UK) directed against major histocompatibility complex (MHC) class II antigen.
Immunohistochemistry
Cryosections (5 μm) were kept at −70°C and fixed in ice-cold acetone for 10 minutes before the staining procedure. Endogenous peroxidase activity was quenched by 0.3% hydrogen peroxide in methanol. The ABC technique was performed as previously described22 25 with a Vectastain ABC kit (Vector Laboratories). The sections were counterstained with hematoxylin.
Assessment of growth factor expression was done semiquantitatively. A staining-intensity scale of 0 (no positive cell) to 3 (many positive cells) was applied to estimate the level of immunoreactivity and thus the expression levels of the various molecules. All sections were examined in duplicate and evaluated by two of the authors blindly and independently.
Double immunohistochemistry was performed as described before.26 Briefly, one of the antibodies against cell-marker proteins, ie, ED1, α-actin, CD4 (all mouse monoclonal), and von Willebrand's antigen (rabbit polyclonal), were combined with one of the antibodies against PDGF (monoclonal), PDGF α- or β-receptors, or TGF-β1 (polyclonal). First, frozen sections were stained with a polyclonal antibody, coupled with biotinylated anti-rabbit IgG and ABC complex, which was then visualized with 3,3′-diaminobenzidine. For the PDGF antibody, a biotinylated anti-mouse IgG was used as the secondary antibody. The sections were then rinsed with 0.1 mol/L glycine-HCl buffer (pH 2.2) for 30 minutes to remove immunocomplexes. Second, the sections were incubated with a mouse monoclonal antibody, which was then coupled with rabbit anti-mouse Ig and rinsed with Tris-buffered saline (pH 7.6), incubated with alkaline phosphatase anti-alkaline phosphatase (Dakopatts), and then visualized with naphthol AS-MX phosphate used as a substrate and fast blue BB salt as a chromogen. For control, we included sections in which only the first or second primary antibody was omitted. We also included sequential sections that were stained with each of the antibodies for comparison.
Statistical Analysis
Statistical calculations were performed with the use of StatView and SuperAnova programs. As expected, the distribution of ischemic time intervals was skewed on the left. Log10 transformation (after adding 1) yielded a nonskewed distribution of values >0, allowing parametric analyses. Factors contributing to the variance in the log (1 plus the number of cells) variable were analyzed by ANOVA. Independent variables were time after transplantation, ischemic time intervals (log10 transformed), and their interaction. This ANOVA model was tested separately in four different groups (syngeneic/allogeneic; intima/media), always predicting the expression of TGF-β isoforms, LTBP, or PDGF and its receptors.
The correlational pattern between the presence of various cell types (ED1- and ED2-positive macrophages, CD4- and CD8-positive T lymphocytes, and MHC class II–positive cells) and the TGF-β isoforms, LTBP, or PDGF and its receptors was analyzed separately in syngeneic and allogeneic transplantation models. Two nonparametric correlational analyses were used: Spearman's correlation between rank-ordered raw numbers and contingency analyses (Fisher's exact test) between dichotomized variables (0 or >0 expression). Significant results were similar on most occasions and were considered significant with a probability level at <.05.
Results
The transplanted aortas were compared with normal nontransplanted aortas regarding morphology as well as expression of cell markers and growth factors. Detailed description of the morphological changes and their correlations with ischemia time and length of observation after transplantation has been reported previously.15
Nontransplanted (Own) Aortas
In the normal nontransplanted aorta in PVG rats, no intimal thickening was found. No immunoreactivity for the examined growth factors was found other than for the PDGF α-receptor that was seen in the medial layer. In addition, a diffuse weak staining for LTBP was found in the same layer.
Syngeneic Transplantation Model
In the syngeneic model, a strong positive correlation between the duration of ischemia and the formation of the neointima was found.15 The intimal thickening seen with the longer ischemia time was almost comparable to that found in the allogeneic grafts (Fig 1⇓).
Immunohistochemical staining of transplanted rat aortas for the localization of PDGF and its receptors as well as TGF-β1. All the grafts shown were removed from the recipient rat 2 weeks after transplantation. The syngeneic transplants were subjected to either 1 hour or 24 hours of cold ischemia time and compared with the allogeneic transplants after 24 hours of cold ischemia time. Note the intimal thickening seen in most of the syngeneic grafts with 24 hours of ischemia time. A, PDGF-BB, syngeneic transplant, 1 hour; B, PDGF-BB, syngeneic transplant, 24 hours; C, PDGF-BB, allogeneic transplant, 24 hours; D, PDGF α-receptor, syngeneic transplant, 1 hour; E, PDGF α-receptor, syngeneic transplant, 24 hours; F, PDGF α-receptor, allogeneic transplant, 24 hours; G, PDGF β-receptor, syngeneic transplant, 1 hour; H, PDGF β-receptor, syngeneic transplant, 24 hours; I, PDGF β-receptor, allogeneic transplant, 24 hours; J, TGF-β1, syngeneic transplant, 1 hour; K, TGF-β1, syngeneic transplant, 24 hours; and L, TGF-β1, allogeneic transplant, 24 hours. The internal elastic lamina of the aortic wall is also shown. The sections were counterstained with hematoxylin. rec indicates receptor. Original magnification ×400.
A clear activation of the inflammatory cells as well as a gradual induction of growth factors could be demonstrated (Fig 1A, 1B, 1D, 1E, 1G, 1H, 1J, and 1K⇑⇑⇑⇑⇑⇑⇑⇑). Expression of growth factors was induced in the neointima and was significantly correlated with the extent of the ischemic stimulus. The results of these growth factor expressions after various ischemia times at 2 weeks of observation are plotted in Fig 2⇓. Ischemia time was the most important predictor of expression of the following proteins in the intima: TGF-β1 (F=6.40; P=.03), PDGF-BB (F=5.35; P=.043), and PDGF α-receptor (F=6.41; P=.03). The expression of the PDGF β-receptor in the aortic wall correlated significantly with ischemia time at certain observation time points (F=4.72; P=.035) (Fig 2⇓). This receptor was induced not only in the neointima but also, to a lesser extent, in the medial layer (Fig 1G, 1H, and 1I⇑⇑⇑). Moreover, the capillary endothelial cells in the adventitia of the grafted aorta also expressed this receptor.
Ischemia time-dependent PDGF B-chain (•), PDGF β-receptor (▪), α-receptor (▵), and TGF-β1 (×) in the intima (A), media (B), and adventitia (C) of the syngeneic grafts after 2 weeks of observation. All semiquantitative evaluations were scored blindly from 0 to 3, given as mean±SEM, and compared with 1 hour of ischemia time by ANOVA. *P<.05, **P<.01.
LTBP was also induced in the neointima of syngeneic aortic grafts (not shown). Its occurrence, however, correlated best with observation time after transplantation (F=5.09; P=.048) and was not significantly dependent on the ischemic stimulus. The same was true for the entire TGF-β family of molecules (TGF-β1, TGF-β2, TGF-β3, and LTBP) when assessed together (F=6.24; P=.016). TGF-β was also induced in the neointima by ischemia (Fig 1J and 1K⇑⇑). However, this was significant for only some of the observation times assessed (TGF-β3, F=6.11; P=.030).
Unlike most of the other molecules studied, the expression of TGF-β2 increased significantly in the media of the transplanted aorta (not shown). This increase was time dependent and correlated well with the observation period after transplantation (F=8.27; P=.021). For some but not all time points observed, induction of TGF-β2 was also correlated with the length of the ischemic stimulus (F=6.11; P=.033). The latter was also true for PDGF (F=5.64; P=.039).
Several cells that became positively stained with markers for T cells and macrophages accumulated within the transplanted aortic segments. This process of leukocyte trapping showed a strong temporal and spatial codistribution with the expression of various growth factors or growth factor–related molecules (Table⇓). LTBP and TGF-β3 expression were significantly correlated with the presence of ED2-positive macrophages, CD4-positive T lymphocytes, and MHC class II–expressing cells, and TGF-β3 correlated positively with the immunoreactivity of ED1-positive monocytes/macrophages. The presence of PDGF immunoreactivity showed an association with MHC class II–positive cells, and the presence of the PDGF β-receptor was associated with the presence of ED2-positive macrophages, CD4-positive T lymphocytes, and MHC class II–positive cells.
Growth Factors and Cell-Type Markers: Association Between Immunodetectable Growth Factors or Growth Factor–Related Molecules With the Expression of Cell Surface Markers
Allogeneic Transplantation Model
In the allogeneic combination, there were clear morphological changes in the transplanted aorta with different durations of ischemia and observation time. The thickness of the intima increased with time during the entire observation period15 as the numbers of macrophages, T cells, and SMCs increased in this layer. Medial thickness remained fairly constant; however, after a long observation time, there was a tendency for disappearance of the SMCs between the elastic fibers of the media.
Higher levels of overall immunoexpression of all TGF-β isoforms, LTBP, and PDGF and its receptors could be demonstrated in the allogeneic grafts than in the syngeneic grafts (Fig 1C, 1F, 1I, and 1L⇑⇑⇑⇑). The marked difference was noted especially in those grafts with short ischemia time. For example, the expression level of PDGF was significantly higher in the aortic segments of the allogeneic transplantation model than in those in the syngeneic model when all examined grafts were included (1.13±0.18 versus 0.72±0.10; P=.034; unpaired t test). Likewise, significant differences in the expression levels were seen for TGF-β1 (0.56±0.09 versus 0.27±0.06; P=.009), TGF-β2 (0.66±0.09 versus 0.22±0.06; P<.0001), TGF-β3 (0.57±0.10 versus 0.13±0.04; P<.0001), LTBP (0.86±0.12 versus 0.51±0.08; P=.015), and the PDGF β-receptor (1.13±0.18 versus 0.72±0.10; P<.034). All of the induced immunoreactivities were detected clearly in all three layers of the allogeneically transplanted aortas (Fig 1⇑), except for TGF-β3, which was expressed much more weakly than other TGF-β isoforms. The level of growth factor expression was already high in the aorta with the shortest ischemia (1 hour) and observation times (2 weeks), and it did not significantly change over time, nor did the duration of the ischemia affect growth factor protein level.
Intense immunoreactivity for all cell marker proteins was induced after transplantation of an allogeneic aortic section, indicating the presence and accumulation of monocytes/macrophages, CD4- and CD8-positive T cells, and MHC class II–expressing cells. The intensity of the expression of the various marker molecules did not differ significantly with regard to ischemia time periods or observation time. However, intensity did show significant correlations with the expression of LTBP, TGF-β3, and the PDGF-receptors (Table⇑).
Double Immunohistochemistry
Double immunohistochemistry was performed to identify the cell types expressing the growth factors examined. Some macrophages as detected by ED1 antibody expressed PDGF (Fig 3A⇓), PDGF β-receptor (Fig 3B⇓), and PDGF α-receptor (Fig 3C⇓) as well as TGF-β (not shown). Most of the SMCs in the intima and media expressed the PDGF β-receptor (Fig 3D⇓) and α-receptor (Fig 3E⇓) as well as the PDGF B-chain (not shown). A few T cells scattered in the intima and adventitia also expressed either one or both PDGF receptors (Fig 3F⇓). The vasa vasorum in the adventitia stained positive for both von Willebrand's factor and PDGF β-receptor (not shown).
Double immunohistochemical staining of allogeneically transplanted rat aortas for the identification of cell types expressing PDGF and its receptors. A, Many ED1-positive monocytes/macrophages (Mø) (blue) express PDGF (brown); B, PDGF β-receptor (β-rec) (brown); and C, PDGF α-receptor (α-rec) (brown). D, Most of the α-actin–containing SMCs (blue) express PDGF β-receptor (brown) as well as PDGF α-receptor (E) (brown). F, Some T cells labeled with CD4 (blue) express PDGF β-receptor (brown). Positively stained cells with both of the antibodies are indicated by small arrows, and the internal elastic laminae of the aortic wall are indicated by arrowheads. Original magnification ×400.
Discussion
In this study, we tested whether growth factors of the PDGF and TGF-β families are involved in the process of vascular remodeling in a rat aorta transplant model by analyzing the time course of their expression after different degrees of ischemia/reperfusion injury. In our previous study using the same model,15 we showed a significant increase in the numbers of inflammatory cells, such as macrophages, T cells, and MHC class II–positive cells, correlating with the duration of ischemic time in the syngeneic grafts. We studied the expression of various growth factors and their receptors in the different layers of the aortic graft compared with the recipient's own aorta. The time course of dynamic changes in the immunoreactivity of these molecules was assessed over a period of up to 8 weeks after transplantation.
There was a stronger degree of correlation between the extent of ischemia-reperfusion injury and growth factor expression in the syngeneic model. In this model, the growth factors were found to be induced in the later stage, when intimal thickening and morphological changes occur. This mainly reflects the fact that the process is slower in that situation, and illustrates that any change in growth factor expression can be registered during the observation period between 2 and 8 weeks after transplantation. In the allogeneic combination, on the other hand, the induction of growth factor expression was already strong after observation periods of 2 to 4 weeks, regardless of ischemia time, and no clear difference in expression levels was detectable. In the present study, we did not include observation periods <2 weeks, making it difficult to detect and quantify any changes that occurred within this early time period. In the allogeneic model, lymphocyte activation occurs rapidly after recognition of incompatible MHC antigens, leading to proliferation of lymphocytes as well as release of lymphokines. Both PDGF and TGF-β are known to be induced by allogeneic stimuli mediated through other lymphokines. Thus, PDGF was found to be induced by interleukin-127 and, conversely, was found to amplify the immune response by stimulating interleukin-2 production in T cells.28 Some T cells scattered in the intima were found to be positive for PDGF receptors as evidenced by double immunohistochemistry. TGF-β, on the other hand, has been thought to suppress and modify the immune response.29 30 31 32 33 34 The main producers of PDGF and TGF-β are monocytes/macrophages, as demonstrated by double immunohistochemistry and as demonstrated by several previous studies.5 29 Hence, macrophages, as identified by both the ED1, ED2, and OX6 antibodies in the present study, seem to play an important role in the allogeneic situation. This also seems to be true in the syngeneic situation, because their presence at a certain stage after transplantation highly correlates with the presence of PDGF or TGF-β protein. The activation of macrophages appears to be a shared phenomenon, triggered both by immunologic stimuli during allograft rejection as well as by inflammatory stimuli secondary to the ischemia-reperfusion injury. This also seems to hold true during conventional atherogenesis, in which macrophages and other cells of the immune system can be found in the vessel wall.35
Parallel changes were observed in the media and the intima. This can be explained in part by the fact that the cell populations in these two layers of the aortic wall partly overlap owing to the migration of medial SMCs into the intima.36 The disappearance of SMCs from the medial layer in the allogeneic grafts, which may represent medial necrosis secondary to cytotoxic effects of cellular mediators,37 may also explain the observation time–dependent reduction of expression of some of the factors. The PDGF α-receptor, for example, is expressed in normal vascular SMCs.38 Expression in the neointima as revealed by double immunostaining may reflect SMC migration/chemotaxis.39 Furthermore, we found significant induction of PDGF β-receptor expression in the aortic wall, especially in the neointima of the syngeneic model. The PDGF-mediated stimulation seems to occur in a paracrine manner, because the expression of PDGF ligand, found strongly in the neointimal macrophages, correlates well with the extent of ischemic stimulus. These results imply that PDGF might play a central role in vascular remodeling. PDGF produced by macrophages in the intima can stimulate proliferation of neointimal cells via the PDGF β-receptor and migration of the medial SMCs via the α-receptor. This is in agreement with previous studies by others.1 9 38
Another possible PDGF action might be a stimulation of local angiogenesis via the β-receptor, because a strong induction was also found in vasa vasorum. Considering previous reports on the endothelial expression of PDGF β-receptor40 and the angiogenic activity of PDGF-BB,41 this upregulation might indicate the presence of active neovascularization of microcirculation around the grafted aorta. Similar induction of the PDGF β-receptor has been found in rat heart transplantation.42 43 Basic fibroblast growth factor,44 which is also known to be of importance at the site of the vascular injury, has been shown to play a similar role.45
TGF-β expression is upregulated in our model in a similar manner as in the more widespread vascular repair model.46 However, it is induced at a later stage than the expression of PDGF. TGF-β induces PDGF,47 but the reverse has also been shown.48 TGF-β may exert different actions in this context: it may be induced as a late, endogenous antagonist and self-regulator of inflammation, as we had postulated earlier.29 Furthermore, TGF-β is a well-known inhibitor of vascular SMC proliferation.49 On the other hand, TGF-β may contribute to the development of fibrotic changes,22 29 50 51 which do occur during vascular remodeling. Thus, TGF-β seems to play a dual role in the development of atherosclerosis. However, to exert these actions, TGF-β needs to be activated in the vessel wall.52 A recent report53 on positive associations between a decreased ratio of active TGF-β and increased levels of apoprotein(a) as well as plasminogen activator inhibitor-1 in the serum of patients with coronary atherosclerosis suggests an interesting possibility that active TGF-β in the serum might antagonize the development of atherosclerosis through inhibition of SMC proliferation.
In the present study, we did not examine the activated fraction of TGF-β, but the elevated level of LTBP in the transplanted aortas may indicate an accumulation after being secreted,29 ie, continuous production of the latent TGF-β and subsequent activation may have occurred in the arteriosclerotic grafts.54 In fact, LTBP staining showed a strong positive correlation with the presence of inflammatory cells, suggesting that most of the TGF-β isoforms might be secreted as large latent complexes containing LTBP.23
In summary, we have characterized the time course and intensity of growth factor induction in ischemia-induced transplant arteriosclerosis. This may help us to understand the pathophysiology of vascular remodeling and may provide a molecular basis for the design of novel therapeutic approaches.
Selected Abbreviations and Acronyms
| Ig | = | immunoglobulin |
| LTBP | = | latent transforming growth factor-β binding protein |
| MHC | = | major histocompatibility complex |
| PDGF | = | platelet-derived growth factor |
| SMC | = | smooth muscle cell |
| TGF | = | transforming growth factor |
Acknowledgments
This work was supported in part by grant Wa734/1-1 from Deutsche Forschungsgemeinschaft (to Dr Waltenberger) and by grants from the Swedish Medical Research Council, the National Heart and Lung Foundation, and the Foundation of Njursjukasförening in CUWX-länen. The PGF007 monoclonal antibody was a gift from Mochida Co Pharmaceuticals, Japan, and the antibodies W3/25, OX8, and OX6 were kindly provided by A.F. Williams, Oxford, UK. We wish to thank Carl-Henrik Heldin for his continuous support and Ulla Svensson for expert technical assistance.
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