© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Inhibition of Arteriosclerosis by T-Cell Depletion in Normocholesterolemic Rabbits Immunized With Heat Shock Protein 65
From the Institute for Biomedical Aging Research, Austrian Academy of Sciences (B.M., M.M., Q.X., G.W.), and the Institute for General and Experimental Pathology, University of Innsbruck Medical School (B.M., M.M., H.D., G.W.), Innsbruck, Austria; GBF, Department of Genexpression (M.S.), Braunschweig, Germany; and Stressgen Biotechnologies Corp (E.W.), Victoria, BC, Canada. Dr Metzler is currently at the Division of Cardiology, Department of Internal Medicine, University Hospital of Innsbruck, Innsbruck, Austria.
Correspondence to Georg Wick, MD, Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria. E-mail IBA{at}oeaw.ac.at
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Abstract—Previous studies in our laboratory have shown that arteriosclerotic changes can be induced in normocholesterolemic rabbits by immunization with mycobacterial heat shock protein (hsp) 65. To further investigate the immunologic mechanisms underlying such vascular lesions, 39 male New Zealand White rabbits were treated by triple immunization with fortified Freund's complete adjuvant containing 5 mg/mL Mycobacterium tuberculosis as a source of hsp65 and simultaneous immunosuppressive therapy twice per week with either anti-CD3 monoclonal antibody (1 mg/kg) and prednisolone (1 mg/kg) or prednisolone (1 mg/kg) alone. Sixteen weeks after the first immunization the animals were killed, and as expected, severe arteriosclerotic lesions in the intima of the aortic arch were found in 9 of 10 immunized rabbits. However, only 1 of 10 rabbits immunized and immunosuppressed with the combined anti-CD3 monoclonal antibody and prednisolone treatment showed a single moderate lesion in the aorta, whereas 5 of 9 rabbits immunized and immunosuppressed by prednisolone treatment alone showed lesions, albeit mild. In conclusion, the early inflammatory stages of arteriosclerotic lesions induced by immunization with hsp65 can be inhibited by immunosuppressive therapy with anti-CD3 monoclonal antibody.
Key Words: arteriosclerosis • immunosuppression • heat shock proteins • stress proteins • lymphocytes
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Introduction |
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It has been established by various groups that atherosclerotic lesions in humans and rabbits contain large numbers of T lymphocytes,1 2 3 4 5 half of which express major histocompatibility class (MHC) class II antigens and some of which also express interleukin-2 receptors,6 indicating a state of activation. The presence of T cells in atherosclerotic lesions could be important, because these cells can secrete factors chemotactic for monocytes/macrophages and smooth muscle cells, determine the differentiation and function of B cells and monocytes/macrophages, and modulate lipoprotein uptake by the latter.7 8 However, the antigen(s) responsible for this immunoactivation has not been identified. Two major candidates have been proposed: (1) Oxidized LDL (oxLDL) has been shown to be a chemokine for T lymphocytes9 and to activate T lymphocytes derived from atherosclerotic lesions10 by an MHC class II–dependent mechanism.11 Interestingly, immunization of hypercholesterolemic rabbits with oxLDL has been shown to protect these animals from atherosclerosis development.12 13 (2) Heat shock protein 65 (hsp65), as we have shown, may be an (auto)antigen initiating the pathogenesis of atherosclerosis,14 15 16 17 and several lines of evidence support this hypothesis. First, T-cell proliferation to hsp65 was found not only in lesion-derived cells from hsp65-immunized rabbits but also in nonimmunized rabbits fed a cholesterol-rich diet exclusively.18 Second, hsp65 immunization of normocholesterolemic New Zealand White rabbits leads to formation of arteriosclerotic lesions demonstrating the typical features of their human counterparts, ie, inflammatory cell accumulation and smooth muscle cell proliferation, but without foam cells. In addition, a combination of immunization with hsp65-containing material and a cholesterol-rich diet led to the development of complicated atherosclerotic lesions, including foam cells, exactly paralleling the situation in classic human lesions.18
Therefore, the question arose as to whether T lymphocytes play a key role in the induction of arteriosclerosis in normocholesterolemic rabbits immunized with hsp65-containing material. In the present experiment, we addressed this issue by immunosuppression with monoclonal antibodies (mAbs) against CD3 lymphocytes, antibodies known to result in rapid lymphopenia and impaired T-cell immune response.19 20
We provide evidence herein that the formation of arteriosclerotic lesions by immunization with hsp65-containing material can be abolished by immunosuppression with an anti-CD3 mAb plus prednisolone.
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Methods |
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Animals, Reagents, and Immunization
Thirty-nine male New Zealand White rabbits, 10 to 11 weeks old and weighing between 1800 and 2200 g, were obtained from Savo/Charles River Co (Kisslegg im Allgäu, Germany). All animals were selected for serum cholesterol levels <2.6 mmol/L (100 mg/dL) with an average value of 1.34±0.44 mmol/L and were individually housed under conventional conditions in wire-bottom cages at 22°C with a relative humidity of 60%. They received water ad libitum and were fed a normal, standard diet (T775, Tagger & Co). The animals were separated into 4 groups. Ten rabbits were immunized with Freund's complete adjuvant (FCA) fortified with 5 mg/mL Mycobacterium tuberculosis. FCA (lot No. 3114-33-8 containing 0.5 mg/mL Mycobacterium butyricum) and heat-killed M tuberculosis H37Ra (lot No. 3114-33-) were purchased from Difco Laboratories. Recombinant Mycobacterium bovis hsp65 (BCG 65K, batch No. mA-11B) was a gift from Dr van Embden, National Institute of Public and Environmental Protection, Bilthoven, The Netherlands. (The production of hsp65 was supported by the United Nations Development Program/World Bank/World Health Organization special program for research and training in tropical diseases.) Immunization schedules were detailed previously.18 21 In brief, rabbits received 3 intracutaneous injections from the nape downward at 5-week intervals (Table 1
). Each milliliter of emulsion (1
mL per rabbit) consisted of 0.5 mL of PBS, pH 7.2, and 0.5 mL of FCA
(15 mg of mycobacteria per rabbit). The 1-mL emulsion was always
administered at 4 sites. Before the first intracutaneous immunization,
2 groups of rabbits were immunosuppressed with either an anti-CD3 mAb
(1 mg/kg) and prednisolone (1 mg/kg) or prednisolone (1 mg/kg) alone
for 5 subsequent days. These 2 groups were then treated twice per week
with the same dose as before immunization, determined to be appropriate
based on preliminary experiments.
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The group treated with prednisolone alone (1 mg/kg twice per week)
served as a control because the anti-CD3 mAb must be combined with
another immunosuppressive agent to prevent a possible anaphylactic
reaction due to formation of antibodies against the injected anti-CD3
mAb. Without additional application of prednisolone, the rapid
depletion of T lymphocytes by intravenous application of
anti-CD3 mAb would wane within 
2 weeks.22 Ten animals
served as untreated controls. All experimental animal procedures were
done in accordance with institutional guidelines.
Production and Purification of mAbs
The mAb-producing anti-CD3 hybridoma (clone L11/135; catalog No.
TIB 188) was obtained from the American Type Culture Collection (ATCC,
Manassas, Va). This mouse antibody is of the IgG1 subclass and is
specific for CD3 on all rabbit T cells.23
Antibody-containing ascites fluid was produced by injecting these cells
into the peritoneal cavities of pristane-primed BALB/c mice, and
precipitation of the immunoglobulin fraction was performed with
saturated ammonium sulfate.
Blood Cholesterol Determination
Blood samples (1 to 2 mL) were collected from the central ear
artery of rabbits after a 16-hour fast. Serum cholesterol
values were measured at 4-week intervals by using an enzymatic
procedure (catalog No. 352-100, Sigma). In brief, 10 µL of serum was
added to 1 mL of solution from a cholesterol test kit and
incubated for 18 minutes at room temperature, followed by photometric
measurement at a wavelength of 500 nm (Dynatech Laboratories Inc).
Serum cholesterol levels of all rabbits before treatment
were <100 mg/dL (2.6 mmol/L).
Blood Glucose Determination
Blood samples (1 to 2 mL) were collected from the central ear
artery of rabbits after a 16-hour fast. Serum glucose values were
measured at 4-week intervals by using an enzymatic procedure (catalog
No. 315-100, Sigma). In brief, 5 µL of serum was added to 1 mL of
solution from a glucose test kit and incubated for 18 minutes at room
temperature, followed by photometric measurement at a wavelength of 505
nm (Dynatech Laboratories Inc). Serum glucose levels of all rabbits
before treatment were <75 mg/dL (4.1 mmol/L).
Enzyme-Linked Immunosorbent Assay
Determination of anti-hsp65 antibodies was performed essentially
as described.24 In brief, recombinant hsp65 (1 µg/mL)
was coated onto flat-bottom ELISA plate wells (Petra-Plastic 1.1041E)
overnight at 4°C. After being washed with PBS supplemented with Tween
20 (0.05%, vol/vol) and blocked with 1% BSA (Sigma) in PBS, rabbit
serum was added in appropriate dilutions and incubated for 1 hour at
room temperature. A horseradish peroxidase–labeled swine anti-rabbit
immunoglobulin conjugate (catalog No. P217, Dako) was then added, and
the plates were incubated for 1 hour at room temperature followed by 4
washes with PBS/Tween. Finally, 100 µL of citrate phosphate buffer
(0.1 mol/L, pH 4.2) containing 0.53 mg/mL
2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid (Sigma) was added,
and absorbance was measured after 30 minutes with a Microelisa
Autoreader (Dynatech Laboratories Inc) at 410 nm.
Lymphocyte Culture
Before and 8 and 16 weeks after the first immunization,
heparinized blood (15 IU/mL of preservative-free heparin, Immuno AG)
was obtained from the central ear artery and diluted 1:2 in RPMI 1640
(Seromed). Blood mononuclear cells were isolated by density gradient
centrifugation over Lymphoprep (d=1.077
g/mL, Nycomed Pharma AS) as described
previously.25
Peripheral mononuclear cells (105 per well) were cultivated in duplicate in round-bottom microtiter plates (Falcon 3077) in 0.2 mL of RPMI 1640 supplemented with 5% fresh autologous serum, 5x10-5 mol/L 2-mercaptoethanol, streptomycin (100 µg/mL), penicillin (100 IU/mL), and 2 µg/mL concanavalin A (Pharmacia) or antigens in appropriate dilutions, including a purified protein derivative of mycobacteria (PPD, Statens Seruminstitut). The proliferative response of the cells was determined by measuring the incorporation of [3H]thymidine (1 µCi per well; specific activity 5 Ci/mmol, 185 GBq/mmol; Amersham) during the last 8 hours of a 48-hour culture at 37°C and 5% CO2.
Fluorescence-Activated Cell Sorting (FACS)
Analysis of Blood Mononuclear Cells
For flow cytometric analysis,
2x105 peripheral blood mononuclear
cells were incubated in PBS containing 1% BSA in a total volume of 100
µL with predetermined, appropriately diluted mAbs against a rabbit
pan–T cell surface marker (L11/135, ATCC), Ia antigen (2C4; ATCC
catalog No. CRL 1760), and CD4 (catalog No. MCA 799, Serotec, Oxford,
UK) for 60 minutes at 37°C. After 3 washes with PBS–1% BSA, the
cells were incubated with FITC-conjugated rabbit anti-mouse
immunoglobulin for 1 hour and washed again. Fluorescence
measurements were performed in an FACScan (Becton Dickinson and Co).
Details of FACS settings and methods of quantification of
fluorescence intensity for the labeled cells are described
elsewhere.25 26
Arteriosclerotic Lesion Measurement
Animals were killed by heart puncture under ketamine (25
mg/kg) and xylazine (5 to 10 mg/kg) anesthesia. Some serum
was used immediately for supplementation of cell culture medium, and
the rest was stored frozen at -70°C. The aortas were carefully
removed intact from the aortic arch to the iliac bifurcation and cut
longitudinally for documentation of macroscopically visible intimal
lesions on a plastic template.21 The total surface area of
the aortas covered by lesions was determined by computerized planimetry
(IBM PC-AT 486 image analyzer; Optimas 4.1 program, Bioscan)
after scanning the templates (GT8000 Epson) at a resolution of 400 dots
per inch in black-and-white mode. All drawings of the lesions and
planimetric evaluations were done by the same operator. These data were
used to calculate the intimal surface area (mm2)
affected by arteriosclerotic lesions.
Several portions of uninvolved and lesioned aortic intimas from each group were subdivided and processed for conventional histology. Tissue fragments were fixed in 4% phosphate-buffered (pH 7.2) formaldehyde, embedded in paraffin, and sectioned for hematoxylin-eosin (HE) staining.
Statistics
Statistical tests were carried out with the Mann-Whitney
U test for analysis of
arteriosclerotic lesions, antibody titers, and
proliferative cell responses and paired Student's t test
for comparison of blood cholesterol and glucose levels
(StatView SE+Graphics for the Macintosh computer, Abacus Concepts,
Inc).
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Results |
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Macroscopic and Microscopic Assessment of Arteriosclerotic Lesions
Sixteen weeks after the first immunization, 9 of 10 rabbits in the FCA-immunized group showed arteriosclerotic lesions in their aortic intimas similar to our previous observations.18 21 The number of lesions present in the individual rabbits varied from 1 to 7. Only 1 of 10 rabbits from the group receiving 2 intravenous injections per week of combined anti-CD3 mAb and prednisolone developed arteriosclerotic lesions. Moreover, 1 of 10 rabbits from the nonimmunized control group showed minor arteriosclerotic lesions. In the group treated with 2 injections of prednisolone (1 mg/kg) only per week, 5 of 9 rabbits developed arteriosclerotic lesions, mainly located in the aortic arch. Quantification of lesion areas in the aortic intima revealed a significant increase (P<0.01) in FCA-immunized animals (Table 1
). In fact, no significant quantitative
difference in lesion areas between control and immunosuppressed animals
(anti-CD3 and prednisolone, Table 1) was found. As shown
previously,21 the histological
characteristics of immunization-induced lesions included intimal
thickening with mononuclear cell infiltration, migration of smooth
muscle cells, and extracellular matrix deposition, but an absence of
foam cells (Figure 1B). The exact
immunohistochemical description of these
arteriosclerotic lesions has been detailed
previously.21 The arch portions of all aortas, even those
without macroscopically apparent lesions after 16 weeks of treatment,
were examined microscopically on serial cross sections. Infiltrating
mononuclear cells were occasionally found in the aortic intimas of
group 4 rabbits, with some extracellular matrix deposition (Figure 1C), whereas aortas of group 3 animals (Figure 1D)
appeared similar to those of normal rabbits (Figure 1A).
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It has been demonstrated that T lymphocytes are present in
arteriosclerotic lesions and are induced by either
immunization or a cholesterol-rich diet.6 21
In the present experiment, we also found a number of T lymphocytes
in the lesions of immunized rabbits, but not in the normal vessel
wall (Figure 2).
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Serum Antibodies to hsp65
To determine serum antibody levels, ELISA plates were coated with
recombinant mycobacterial hsp65, ovalbumin, PPD, or mouse
immunoglobulins. The data summarized in Tables 2 and 3
show significantly increased antibody titers against hsp65 and PPD in
FCA-immunized rabbits. The antibody titer against the control antigen
ovalbumin remained low (
1:10) in all groups (data not shown).
The anti-hsp65 and anti-PPD titers in the immunized group
immunosuppressed with anti-CD3 mAb and prednisolone remained
significantly lower than positive controls. The anti–mouse
immunoglobulin titer in the group immunosuppressed with anti-CD3 mAb
and prednisolone rose to 3830±2340 (not shown in these Tables) at the
end of the experiment, but without symptoms of serum sickness. In all
other groups, this titer remained low.
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Blood Cholesterol and Blood Glucose Levels
Blood cholesterol levels in rabbits immunized with FCA
remained below 2.6 mmol/L (100 mg/dL), ie, the same range as
untreated controls, confirming previous results indicating that
immunization alone does not elevate blood cholesterol
levels in rabbits receiving a normal diet.21
Owing to the glucose-liberating effect of glucocorticoids, blood
glucose levels in rabbits treated with prednisolone alone or with
combined anti-CD3 mAb and prednisolone after immunization with FCA rose
progressively from the beginning of the immunosuppressive therapy to
the end of the experiment (Figure 3).
However, the blood glucose level of the FCA-immunized group treated
with prednisolone alone was significantly higher than that in
FCA-immunized rabbits without immunosuppressive therapy (7.27±1.23
versus 4.65±0.91 mmol/L, P<0.01).
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) were found to be similar to
controls (
). Serum glucose levels of immunized rabbits
immunosuppressed by prednisolone (
) were significantly higher after
) were
not significantly elevated, *P<0.05.
Peripheral T-Cell Response
Figure 4 summarizes the results of
proliferation assays of blood mononuclear cells cultivated for 48 hours
in the presence or absence of various antigens or with the T-cell
mitogen concanavalin A as a positive control, which strongly stimulated
the cells of all rabbits. As expected, the cells of immunized animals
reacted specifically with hsp65 and PPD, ie, hsp65-containing material,
but not with ovalbumin. The peripheral lymphocyte
cell response of FCA (ie, hsp65)-immunized rabbits treated with
anti-CD3 mAb and prednisolone was significantly decreased
(P<0.02) compared with FCA-immunized animals. The cells of
FCA-immunized animals also showed a higher proliferative response to
hsp65 or PPD than those in medium without antigen or with
ovalbumin.
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) weeks after the first immunization. The
cells (105 per well) were cultivated in duplicate in
round-bottom microtiter plates in 0.2 mL of RPMI 1640 containing 5%
autologous serum and antigen supplement (ovalbumin 30 µg/mL,
PPD 30 µg/mL, or recombinant hsp65 5 µg/mL). The proliferative
response of cells was determined by measuring the incorporation of
[3H]thymidine (1 µCi per well) that was added for the
last 8 hours of the 48-hour culture period. Columns show
[3H]thymidine uptake ±SD and are means of 5 rabbits from
each group. *The [3H]thymidine uptake in the groups
immunized with FCA and intravenously treated with the
combination of anti-CD3 mAb and prednisolone or prednisolone alone are
significantly different from the group immunized with FCA only,
*P<0.02.The spleen cell response was determined in the same manner as the peripheral mononuclear cell response. Although the effect of immunosuppression with anti-CD3 mAb and prednisolone was also clearly evident in spleen cells, it was less distinct than that seen with peripheral mononuclear cells (data not shown).
FACS Analysis of Blood Mononuclear Cells
FACS analysis was used to test the effect of various forms
of immunosuppressive treatment on the composition of
peripheral blood mononuclear cell populations. The
viability of labeled peripheral blood mononuclear cells was
determined by FACS scatter analysis, and only living cells were
gated and subjected to immunofluorescence
measurements. Flow cytometric studies showed that treatment with
anti-CD3 mAb significantly reduced CD3+ and
CD4+ peripheral blood mononuclear
cells (P<0.02) as well as the Ia+
peripheral blood mononuclear cells, albeit at a lower level
(Table 4).
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Our clinical and experimental data provide ample evidence that atherosclerosis can start as an inflammatory, immunologic disease induced by an autoimmune reaction against hsp60. Arteriosclerosis is characterized by vascular areas containing mononuclear and proliferating smooth muscle cells as well as extracellular matrix components, resulting in hardening and thickening of the arterial wall. Atherosclerotic lesions are localized in the intima and additionally contain foam cells and deposits of cholesterol crystals, manifested as fatty streaks. Most humans possess hsp65-reactive humoral antibodies and T cells, which afford vital protection from potentially dangerous microorganisms expressing cross-reactive antigens of the hsp60 family of heat shock proteins. These polyclonal effector mechanisms allow for recognition of various hsp65 epitopes, several of which provide the basis for potential pathogenetic cross-reactions with human hsp60.
The role of T lymphocytes in atherogenesis remains controversial, with different data published by laboratories using various animal models.27 28 29 30 The opinion that T lymphocytes do not play a priming role in atherogenesis is supported by the fact that T cell–deficient mice develop atherosclerotic lesions to the same extent as do normal mice after receiving a cholesterol-rich diet.30 31 32 Hansson et al33 reported that rats in which T lymphocytes were eliminated by an mAb developed even larger proliferative arterial lesions after balloon-catheter injury. On the other hand, some data support the important role of T lymphocytes in atherogenesis, because T lymphocytes have been shown to be prominent components of early lesions, not only in human atherosclerotic plaques1 but also in apoE-deficient mice.34 Moreover, Emeson et al27 showed atherosclerosis inhibition in CD4 T cell–ablated and nude (nu/nu) C57BL/6 hyperlipidemic mice.
In contrast to our approach with normocholesterolemic rabbits, previous published experiments in this field involved feeding the animals a hypercholesterolemic diet,27 28 29 30 as is also the case for studies showing enhanced development of arteriosclerosis after immunosuppression with cyclosporin A28 29 or in immune-deficient mice.30 Because cyclosporin A can also increase arterial blood pressure,35 damage endothelial cells,36 induce elevated plasma total cholesterol levels,37 and increase endothelin production,38 this chemical seems to be unsuitable for investigations on the role of T cells in atherogenesis.
It is also becoming evident that immunity against modified LDL, in contrast to immune reactions against hsp65/60, may be protective. Thus, immunization of hypercholesterolemic rabbits with oxLDL protects them against cholesterol-induced arteriosclerosis,12 13 although the mechanism by which it exerts this effect remains unknown. One theory is the elimination of oxidatively modified circulating LDL by high-titer antibodies against oxLDL.
The presumed mechanism by which hsp65 immunization of rabbits leads to formation of arteriosclerotic lesions differs from the mechanism by which oxLDL immunization reduces arteriosclerosis, ie, an autoimmune cross-reaction between antigenically highly conserved heat shock protein. We speculate that these 2 phenomena are not contradictory, although the mechanisms and animal models are different. In humans, the 2 phenomena could coincide. The hsp60 or portions thereof are present on the surface of stressed endothelial cells and serve as targets for circulating lymphocytes and antibodies against hsp65/60 provoked by immunization with bacterial hsp65.39 40 Schett et al41 showed that these anti-hsp65/60 antibodies can mediate both complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity of stressed (eg, by heat, tumor necrosis factor, etc) but not unstressed endothelial cells. Furthermore, we have shown that a cholesterol-rich diet and simultaneous immunization with hsp65 synergistically leads to more severe atherosclerotic lesions than does a cholesterol-rich diet alone and that arteriosclerotic lesions provoked by hsp65 immunization are reversible, whereas those arising as a consequence of hypercholesterolemia are not.42 It has been shown that long-term treatment with glucocorticosteroids does not influence heat shock protein expression in the aortic wall of animals.43
To further scrutinize our hypothesis that T cells support the
development of arteriosclerotic lesions in this
model, we analyzed the effect of immunosuppression on the
formation of arteriosclerotic lesions by selective
depletion of CD3+ lymphocytes with mAbs. Although
immunosuppression with prednisolone alone led to decreased T
lymphocytes (Table 4) and moderately decreased antibody titers
against hsp65 and PPD, this treatment alone did not inhibit development
of arteriosclerotic lesions. The most likely
explanation, beyond the incomplete depletion of lymphocytes, is the
significant rise of blood glucose levels provoked by long-term
administration of prednisolone (Figure 3). Because diabetes is a
classic risk factor for arteriosclerosis, this
result is not surprising. Interestingly, blood glucose levels did not
increase significantly in the group treated with anti-CD3 mAb and
prednisolone, but so far, we have no explanation for this phenomenon.
Finally, we provide data herein that the inflammatory lesions provoked
in normocholesterolemic rabbits by immunization with
hsp65-containing material can be almost completely abolished by
intravenous administration of anti-CD3 mAb, which depletes
mature T lymphocytes.
We hypothesize that the first stage of atherosclerosis is inflammatory in nature, and with age, is aggravated by high cholesterol levels to form typical atherosclerotic plaques. The sequence of this hypothesis is supported by the fact that mononuclear cells preexist in the intima of unaffected arteries of children.17 Inhibition of the first step, by immunosuppression, is thus of particular importance and probably of potent clinical relevance. Recently, the prominent role of inflammation in the progression of arteriosclerosis in humans was described in a large study on the protective effect of aspirin.44 The present data suggest that the progression of arteriosclerosis development could be delayed or even prevented by immunosuppression. We are now investigating the effects of immunosuppression in rabbits in which atherosclerosis is induced by a high-cholesterol diet only.
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Acknowledgments |
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This study was supported by grants P12213-MED from the Austrian Science Fund, P5651 from the Jubiläumsfonds of the Austrian National Bank, the State of Vorarlberg and the State of Tyrol (to G. Wick). We thank A. Jennewein and G. Sturm for excellent technical assistance, E. Rainer for help in animal experimentation, and T. Öttl for the preparation of photographs.
Received June 1, 1998; accepted December 15, 1998.
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