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(Circulation Research. 2002;90:1064.)
© 2002 American Heart Association, Inc.

Molecular Medicine

Overexpression of Interleukin-10 by Activated T Lymphocytes Inhibits Atherosclerosis in LDL Receptor–Deficient Mice by Altering Lymphocyte and Macrophage Phenotypes

Laura J. Pinderski, Michael P. Fischbein, Ganesamoorthy Subbanagounder, Michael C. Fishbein, Nobuhiko Kubo, Hilde Cheroutre, Linda K. Curtiss, Judith A. Berliner, William A. Boisvert

From the Departments of Medicine (L.J.P, G.S., J.A.B.), Experimental Pathology (L.J.P., M.C.F., J.A.B.), Surgery (M.P.F.), and Microbiology and Immunology (M.P.F.), University Of California Los Angeles; Department of Immunology (N.K., L.K.C., W.A.B.), The Scripps Research Institute, La Jolla; and the Division of Developmental Immunology (H.C.), The La Jolla Institute For Allergy and Immunology, San Diego, Calif.

Correspondence to Laura J. Pinderski, University of Alabama, Birmingham, Division of Cardiology, THT 332, 1900 University Blvd, Birmingham, AL 35294-0006. E-mail lpinderski{at}cardio.com.uab.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous studies demonstrated that interleukin-10 (IL-10) overexpression decreases formation of early fatty-streak lesions in mice independent of lipoprotein levels. The present studies, using bone marrow transplantation, demonstrate that overexpression of IL-10 by T cells inhibits advanced atherosclerotic lesions in LDL receptor–null mice fed an atherogenic diet. In mice receiving bone marrow from the IL-10 transgenic mice compared with those receiving wild-type marrow, there was a 47% decrease in lesion size and a marked decrease in lesion complexity with an 80% reduction in the necrotic core. Accumulation of cholesterol and phospholipid oxidation products in the aorta was decreased by 50% to 80%, unrelated to plasma lipid or IL-10 levels. Our studies also provide insight into the mechanism of the IL-10–mediated decrease in lesion size. Although a strong influence toward a Th1 phenotype has previously been demonstrated in atherosclerotic models, T lymphocytes in the IL-10 transgenic (Tg) group revealed a marked shift to a Th2 phenotype, with decreased IFN- production and an increase in IL-10. Evaluation of specific immunoglobulin subclasses demonstrated a preponderance of IgG₁ isotype, characteristic of a Th2 influence on B cell immunoglobulin class-switching in the IL-10 Tg group. A major finding of these studies was altered monocyte/macrophage function in the IL-10 Tg group. Monocytes showed a decrease in activation resulting in decreased expression of IFN-. Furthermore, macrophage foam cells within lesions of the IL-10 Tg group exhibited markedly decreased apoptosis. These studies demonstrate that T lymphocyte IL-10 can influence the function of other immune cells to reduce the development of advanced atherosclerotic lesions in mice.

Key Words: cytokine • LDL receptor • B lymphocytes • antibodies • apoptosis

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Atherosclerosis has been demonstrated to be a chronic inflammatory process involving various immune cells, particularly macrophages and T lymphocytes. We and others have shown that interleukin (IL)-10 overexpression can inhibit fatty-streak formation, independent of lipoprotein levels, in C57BL/6J mice fed a cholate containing atherogenic diet.^1,2 However, these studies were restricted to examining the development of early lesions in C57BL/6J mice on a cholate-containing diet, which is known to alter lipid metabolism and induce inflammatory cytokines in the liver.³ Therefore, cholate may artificially predispose the atherosclerotic process in these animals to be highly inflammatory, partially exaggerating the antiinflammatory role of IL-10. Because of the therapeutic potential of IL-10 in human atherosclerosis, the present studies were directed at determining whether IL-10 can retard development of advanced atherosclerotic lesions in an animal model that did not require a cholate-containing diet and, more importantly, examining the mechanism of IL-10 inhibition of atherogenesis.

Evidence that IL-10 may play a role in advanced lesions was demonstrated by several in vivo studies. IL-10 has been demonstrated within human atherosclerotic plaques,^4,5 primarily in macrophages. Increased local levels of IL-10 has been associated with decreased apoptotic activity as measured by TUNEL staining, as well as decreased local iNOS expression within these diseased human arteries.⁴ Decreased levels of IL-10, contrasted with increased levels of IL-6, have been demonstrated in patients with unstable coronary syndromes.⁶ In vitro studies have demonstrated that oxidized lipids can alter IL-10 expression within human monocytes,⁵ and IL-10 can alter human aortic endothelial cell function to inhibit oxidized phospholipid-induced monocyte-endothelial interactions in vitro.² Both the in vivo and the in vitro studies suggested, but did not directly test, potential mechanisms by which IL-10 might inhibit atherosclerosis.

Our present studies used transplantation of bone marrow from either wild-type or IL-10 transgenic mice, in which the murine IL-10 gene is under the control of a human IL-2 promoter,⁷ into LDL receptor–deficient (LDL-R^-/-) mice. This model allows for localized overexpression of IL-10, because transgenic expression is restricted to T lymphocytes that have encountered antigen or otherwise had their T-cell receptor engaged. These studies tested the hypothesis that IL-10 overexpression by T cells could reduce advanced atherosclerotic lesion formation by shifting the T-cell phenotype from a T-helper 1 (Th1) phenotype, previously seen in atherosclerosis,^8,9 to a Th2 phenotype. Previous studies in inflammatory bowel disease suggested that such a switch might be possible. Through cell-cell interaction and cytokine secretion, T-helper lymphocytes can impact B lymphocyte and monocyte/macrophage function. The data presented here demonstrate that IL-10 overexpression in activated T lymphocytes inhibits development of advanced lesions and decreases accumulation of cholesterol and phospholipids in hyperlipidemic LDL-R^-/- mice. These data suggest that this inhibition is mediated by the specific shift toward a Th2 phenotype with subsequent alteration in monocyte and lymphocyte function.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animal Models
LDL-R^-/- mice were initially obtained from Jackson Laboratory (Bar Harbor, Maine). All mice were weaned at 4 weeks and initially fed using a chow diet (No. 5015; Harlan-Teklad). Mice were housed under a 12-hour light/dark cycle in specific pathogen-free conditions. Marrow cells used for repopulation of the irradiated mice were isolated from either 8-week-old C57BL/6J male mice (WT) or IL-10 transgenic male mice on a C57BL/6J background (IL-10 Tg).⁷

Nineteen 6-week-old male LDL-R^-/- mice were subjected to bone marrow transplantation (BMT), as previously described.¹⁰ The recipient mice received bone marrow from either WT (n=10) or IL-10 Tg (n=9) mice. After BMT, mice were fed chow diet (No. 5015) for 4 weeks, and blood was obtained for baseline lipid determination. Circulating white cells were counted to confirm reconstitution of the peripheral leukocyte population and marrow engraftment. For 20 weeks, the mice were then fed an atherogenic diet (15.8% fat; 1.25% cholesterol; no cholate; diet No. 94059 Harlan-Teklad) previously used by our group and others.¹⁰ All studies were carried out under institutional review board approval.

Polymerase Chain Reaction
Confirmation of appropriate bone marrow engraftment was performed using polymerase chain reaction (PCR). For details, see the expanded Materials and Methods section found in the online data supplement available at http://www.circresaha.org.

Lesion Morphology
At euthanasia, the hearts and aortas were prepared as previously described.¹¹ The heart and proximal aorta were immediately frozen in OCT medium and sectioned. Every third section was stained with Oil Red O, with total lesion area quantified using these sections as previously described.² For this analysis, 10 WT and 9 IL-10 Tg mice were used. The remainder of the frozen sections were utilized for immunostaining or staining with Masson’s trichrome (for details, please see the expanded Materials and Methods in the online data supplement).

Lipid Analysis
For details concerning liquid analysis, please see the expanded Materials and Methods in the online data supplement.

IL-10 ELISA
Total circulating IL-10 was measured in the plasma of the mice at 0, 10, and 20 weeks on the atherogenic diet utilizing a Mouse IL-10 Quantikine Immunoassay kit (R&D Systems).

Intracellular Cytokine Staining and Flow Cytometric Analysis
These studies were performed using methods similar to those described previously.¹² For details, please see the expanded Materials and Methods in the online data supplement.

Immunoglobulin Subclass Determination
Total IgG, IgG_2a, IgG₁, and malondialdehyde-specific¹³ IgG_2a and IgG₁ in the plasma of the mice was quantitated with a sandwich ELISA technique as described.⁸

Immunostaining
Immunohistochemistry was performed on 5-µm thick cryostat sections, as discussed in the expanded Materials and Methods in the online data supplement.

Statistical Analysis
All statistical analysis was performed using 1-way ANOVA with StatView (Abacus Concepts, Inc). Data presented are mean±SE.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Confirmation of BMT Engraftment
To confirm successful engraftment of donor bone marrow cells, circulating white peripheral blood cells (WPBCs) from all mice (WT, n=10; IL-10 Tg, n=9) were examined 4 weeks after BMT. Total cell number and mononuclear cell populations were comparable for mice from both groups (data not shown). PCR performed on DNA extracted from circulating WPBC indicated the expected IL-10 transgene-derived 507-bp product in all the IL-10 Tg marrow–recipient mice and complete absence of this product in the WT marrow–recipient mice (Figure 1).

View larger version (124K): [in this window] [in a new window]   Figure 1. PCR confirms bone marrow engraftment in each group. DNA was extracted from circulating white blood cells in each mouse in both groups 4 weeks after BMT. PCR was performed with primers specific for the IL-10 transgenic construct. PCR products were run on a 1.5% agarose gel, and the transgene-derived 507-bp product was visualized in all the IL-10 transgenic BMT recipients. Despite equal initial amounts of DNA, the WT recipients did not display the specific product. A representative gel is shown.

IL-10 Overexpression Does Not Alter Circulating Lipoprotein Profiles
Total cholesterol and triglyceride levels were similar at all time points between the two groups (Figure 2). HDL cholesterol was not significantly different at baseline between the two groups. There was a mildly lower HDL cholesterol in the IL-10 Tg group at the time of euthanasia.

View larger version (25K): [in this window] [in a new window]   Figure 2. Lipoprotein profiles do not differ between the WT and IL-10 Tg group. Plasma was obtained from all mice at 0 and 20 weeks on the atherogenic diet. Total cholesterol and triglycerides did not differ significantly between the two groups at any time point. HDL cholesterol was not significantly different at baseline, but was mildly decreased in the IL-10 Tg BM–recipient group at the time of euthanasia. *Significant change from baseline to time of euthanasia; significant difference between WT and IL-10 Tg group. Data presented as mean±SE; WT n=10, IL-10 Tg n=9. Solid bar indicates WT group; hatched bar, IL-10 Tg recipient group.

IL-10 Overexpression Decreases Atherosclerotic Lesion Development
Analysis of the lesion area in the proximal aortas (using Oil Red O staining) revealed a significant decrease in atherosclerotic lesion development in the LDL-R^-/- mice that had received bone marrow from IL-10 transgenic donor mice, compared with those recipient mice that had received bone marrow from wild-type mice (WT 144 955±81 µm² versus IL-10 Tg 77 129±56 µm²; P<0.0001; Figure 3).

View larger version (52K): [in this window] [in a new window]   Figure 3. Atherosclerotic lesions are significantly decreased in the IL-10 Tg BM–recipient group. Aortas were harvested at the time of euthanasia, sectioned, and stained with Oil Red O. Proximal aortic atherosclerotic lesion area was quantitated and found to be approximately 47% decreased in the IL-10 Tg group (P<0.001). Data presented as mean±SE; WT n=10, IL-10 Tg n=9.

IL-10 Overexpression Decreases Aortic Accumulation of Lipids in LDL-R^-/- Mice
Analysis of the distal aorta showed a significantly greater amount of total cholesterol in the WT-recipient mice (803.3±213.0 µg/mg wet weight tissue; n=10) compared with the IL-10 Tg–recipient mice (540.9±210.6 µg/mg wet weight tissue; P<0.01, n=9). We and others have previously shown that certain oxidized phospholipids, particularly POVPC and PGPC, increase monocyte-endothelial interactions in vitro and are present in significant amounts in atherosclerotic lesions in animals.¹⁴ We observed an increase in these particular oxidized phospholipids within the WT-recipient mice, compared with the IL-10 Tg BM–recipient mice. POVPC and PGPC were significantly reduced in the IL-10 transgenic group with respect to wet weight (POVPC 66±8.3%, P=0.04; PGPC 83.7±9.5%, P=0.009; n=6) and to a lesser extent with respect to total phosphorus in the lipid extract, an indicator of total phospholipid (POVPC 54±11.2%, P=0.09; PGPC 78±13%, P=0.017; n=6). These results suggest that differences in bioactive phospholipids probably arise from both a decrease in total phospholipid accumulation in the IL-10 transgenic mice (as reflected by decrease in bioactive lipids with respect to wet weight), as well as a lesser but significant IL-10–mediated decrease in oxidation in the IL-10 group (as measured with respect to phosphorus), where the PGPC decrease was significant and the POVPC decrease nearly significant.

Circulating IL-10 levels Are Not Different Between WT- and IL-10 Tg–Recipient Mice
Plasma IL-10 levels were assayed by ELISA in all mice at 0, 10, and 20 weeks on the atherogenic diet. There was no significant difference between the two groups at any time point (data not shown).

Intracellular Cytokine Staining/Flow Cytometry Reveals a Th2 Lymphocytic Shift in the IL-10 Tg Group
Because the IL-10 transgene in this murine construct is under the control of the IL-2 promoter, the expression of transgene-derived IL-10 is restricted to activated T lymphocytes. Expression of cell-surface CD69 and intracellular IL-2 were examined as markers of activation and were similar between the two groups of mice (74% versus 78% and 34% versus 33%, respectively). Despite comparable activation of CD4⁺ T lymphocytes in both groups, there was a marked shift from a Th1 to a Th2 phenotype in CD4⁺ T lymphocytes in the IL-10 Tg BM group, demonstrated by a 1.5-fold decrease in IFN- production and a 9-fold increase in IL-10 production compared with the CD4⁺ lymphocytes from the WT BM–recipient mice (Figure 4A). Importantly, not only was the proportion of T lymphocytes producing the cytokines altered, but the level of cytokine expression by the lymphocytes was also markedly affected. An even more prominent shift in cytokine expression was seen in CD4⁺ splenocytes between the two groups (Figure 4B).

View larger version (19K): [in this window] [in a new window]   Figure 4. Intracellular cytokine staining reveals a Th2 phenotype in the IL-10 Tg BM–recipient group. Peripheral blood lymphocytes (A) and splenocytes (B) were analyzed from mice in each group at the time of euthanasia, and representative plots are shown. T lymphocytes were identified by size, granularity, and positive staining for CD4 (quadrants 2 and 4). After partial cell membrane permeabilization, phycoerythrin-conjugated antibodies against either IL-10 or IFN- were introduced, and cells were then subjected to 2-color flow cytometry. Cells with positive staining for cytokines are in quadrants 1 and 2; cells with double positive staining cells are in quadrant 2. The WT group made negligible amounts of IL-10, but actively produced IFN-, whereas the IL-10 Tg group had a marked shift toward elaboration of IL-10, with decreased IFN.

Examination of Immunoglobulin Subclasses Reveals Opposite CD4⁺ T-Helper Influence on B Lymphocytes in the IL-10 Tg Group Compared With the WT Group
During atherogenesis, antibodies against malondialdehyde-LDL (anti–MDA-LDL) are formed¹⁵ and have been demonstrated to undergo a class shift from IgG_2a to IgG₁ during lesion progression.⁸ We measured anti–MDA-LDL antibodies to determine whether the T-lymphocyte phenotypic difference identified above impacted immunoglobulin subclass production. As in previous studies,⁸ the total levels of IgG did not differ between the 2 groups of mice at 10 or 20 weeks on the atherogenic diet (data not shown). The ratio of Th2 (IgG₁) to Th1 (IgG_2a) anti–MDA-LDL antibodies, however, was significantly higher at both time points in the recipient mice that had received IL-10 Tg BM compared with the WT BM–recipient mice (Figure 5). This B-lymphocyte immunoglobulin subclass shift is additional support for an enhanced Th2 state in the IL-10 Tg group of mice.

View larger version (44K): [in this window] [in a new window]   Figure 5. Immunoglobulin subclass analysis reveals a Th2 influence on B lymphocytes in the IL-10 Tg BM–recipient group. Sandwich ELISAs were performed on plasma from mice in each group to quantify immunoglobulin subclasses. The total IgG did not differ between the WT and IL-10 Tg groups (data not shown). The ratio of anti–MDA-LDL IgG1 to IgG2a, however, suggests a Th2 influence on the B lymphocytes in the IL-10 Tg BM–recipient group (n=7) at both time points measured, compared with the WT (n=8) group. Differences between WT and IL-10 Tg groups are statistically significant.

Microscopic Analysis of Lesion Components
Although the atherosclerotic lesions of both groups of mice contained lipid-laden cells, fibrous caps, and at least some necrotic cores, the lesions of WT BM–recipient mice were considerably more complex and advanced (Figure 6A). In contrast, lesions in the IL-10 Tg group were highly cellular, resembling large fatty streaks, with small to absent necrotic cores. MOMA-2 staining revealed these cellular areas to contain mainly macrophages (data not shown). Morphometric analysis of these sections confirmed significantly greater cellularity of the atherosclerotic lesions in the IL-10 Tg BM–recipient mice (Figure 6B). Conversely, necrotic cores made up a much higher percentage of lesional area in the WT BM–recipient mice (Figure 6B). Because the formation of the necrotic core precedes smooth muscle replication and matrix synthesis in mice, it is not surprising that the matrix area is reduced in the IL-10 transgenic group (Figure 6B). This difference in lesion composition suggested a possible alteration in macrophage fate within the lesions of the two groups of mice. Therefore, apoptotic activity was assessed using an antibody to the activated form of caspase-3. For this analysis, sections from fatty streaks of comparable lesion area were compared between the two groups (8 sections per group). There was a striking difference in caspase staining in the WT group, primarily within MOMA-2–positive cells (Figure 6C). All sections of the WT group showed heavy staining, whereas it was difficult to find caspase staining in the IL-10 group. Comparison of fatty-streak lesions in the two groups revealed that individual macrophages appeared larger in the lesions of the IL-10 Tg group. This qualitative size difference may reflect an ability of these macrophages to load greater amounts of lipid without undergoing apoptosis and could potentially account in part for the differences in oxidative lipid products seen within the aortas of the two groups. Immunostaining with anti-CD4, -CD8, and -CD3 antibodies revealed no lymphocytes within the lesions of either group of mice after 20 weeks on the atherogenic diet (data not shown). This finding is consistent with previous documentation that although T lymphocytes are present in significant numbers early in lesion development in LDL-R^-/- mice, the presence of intralesional T lymphocytes decreases as lesions progress over time.¹⁶ Thus, there appeared to be a protective effect of the T cell–derived IL-10 in this BMT mouse model of atherosclerosis, in terms of both macrophage apoptosis and ultimate lesion development, despite the absence of T lymphocytes within the advanced lesions.

View larger version (72K): [in this window] [in a new window]   Figure 6. Microscopic analysis of the lesions in the WT and IL-10 Tg groups. A, Trichrome staining of lesions in the proximal aorta revealed a higher cellularity in the lesions of the IL-10 Tg group, contrasted with more extensive lipid-filled necrotic cores within the WT lesions. Nearly all intralesional cells were MOMA-2 positive (data not shown). B, Morphometric analysis revealed compositional differences between the WT- and IL-10 Tg–recipient group’s lesions. A representative WT section is shown, with the necrotic core (NC) area bordered in yellow and the cellular area (C) bordered in green. L indicates lumen; A, adventitia. In these analyses, n indicates multiple representative cross-sections analyzed for each mouse in both groups. WT lesions (n=21 representative aortic cross-sections) had a significantly larger area of necrotic core (P<0.0001) vs the IL-10 Tg group (n=24). Furthermore, the WT lesions (n=30) had significantly less cellularity (P=0.004) compared with the IL-10 Tg lesions measured (n=24). Data presented as mean±SE. C, Immunostaining for activated caspase-3 revealed a marked increase in apoptotic cells within the WT lesions. A representative, typical fatty-streak type lesion is shown for both the WT and IL-10 Tg BM–recipient groups. Differences were also observed in more advanced lesions, although to a lesser degree.

Intracellular Cytokine Staining/Flow Cytometry Indicates Marked Activation of Circulating Monocytes Within the WT-Reconstituted Mice Compared With Minimal Activation in the IL-10 Tg BM–Reconstituted Group of Mice
Intracellular cytokine staining with flow cytometric analysis was used to characterize the monocyte population. Initially, the monocyte population was identified by size, granularity, NK-negative, and CD11b-positive selection. Gated monocytes were subsequently assessed for activation states via the measurement of IFN-, previously shown to be expressed in activated monocytes.^17,18 At the time of euthanasia, there was a 6-fold decrease in the amount of IFN- produced by the monocytes in the group of mice that had received IL-10 Tg bone marrow compared with monocytes from the WT group of mice. This dramatic difference was evident both in terms of the number monocytes producing IFN-, as well as the amount of IFN- expressed. Finally, this pattern was noted in both the circulating monocytes (Figure 7), as well as those residing within the spleen (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 7. Intracellular cytokine staining reveals a marked decrease in monocyte activation in the IL-10 Tg group. Peripheral blood lymphocytes were analyzed from mice in each group at the time of euthanasia, and representative plots are shown. Monocytes were identified by size, granularity, negative staining for NK1.1 (data not shown), and positive staining for CD11b. Intracellular IFN- was identified as described in Figure 4. Monocytes from the IL-10 Tg BM–recipient mice produced 9-fold less IFN- compared with WT controls.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
These studies demonstrate that IL-10 overexpression in lymphocytes leads to a significant decrease in the development of advanced atherosclerotic lesions. Previous in vivo work examining IL-10 in atherogenesis was restricted to studying the development of early fatty-streak lesions in C57BL/6J mice on a cholate-containing diet.^1,2 Our present studies utilized the LDL-R^-/- mouse, which develops complex lesions on a cholate-free atherogenic diet. Even after 20 weeks on this diet, the majority of lesions in the mice transplanted with bone marrow from IL-10 transgenic donors were arrested at the fatty-streak stage. In marked contrast, animals transplanted with wild-type bone marrow formed advanced atherosclerotic lesions with large necrotic cores, as has been observed in our previous studies.¹⁰ We have shown recently that LDL-R^-/- mice subjected to BMT and fed the atherogenic diet for 16 weeks had significantly larger aortic sinus lesion areas compared with mice that were not subjected to BMT.¹⁹ Whether this increase was due to the effect of BMT on initiation or progression is not known because only 1 time point was examined. However, because both groups of mice in our present study were subjected to BMT in an identical manner, the comparison of their lesion formation should be valid.

Whereas the previously documented effect of IL-10 in decreasing atherosclerotic initiation^1,2 may play a role in this observed decrease in advanced lesions, our present findings suggest that IL-10 also affects processes associated with lesion progression. A high degree of apoptosis was detected among the intralesional cells of the WT mice, primarily within the macrophages surrounding the lipid-laden cores. In contrast, little to no apoptosis was seen within lesions of similar cellularity in the IL-10 Tg BM–recipient mice. This difference in lesions of comparable size suggests a decrease in progression as well as initiation. Monocyte migration into arterial walls with subsequent transformation into macrophages and then foam cells characterizes the early fatty-streak stage in atherosclerosis. Apoptosis of foam cells, which is influenced by cytokine expression and macrophage activation state,²⁰ contributes to formation of the necrotic core and occurs during progression of atherosclerotic lesions. Reduction in macrophage apoptosis most likely accounts for the decreased formation of necrotic cores in the transgenic group. Interestingly, IL-10 has been shown in other systems to inhibit apoptosis.²¹ Moreover, IL-10 increases stimulation as well as production of reactive oxygen species and IL-6 while inhibiting the incidence of apoptosis in human monocytes.²² Previous studies of human atherosclerotic plaques revealed an inverse association between presence of IL-10 and TUNEL staining in atherosclerotic lesions,⁴ suggesting our murine results are applicable to the human disease process. Lastly, because necrotic cores participate in destabilization of plaques,²³ our murine results may have direct therapeutic implications.

Because plasma levels of IL-10 were not altered in the IL-10 Tg group, the observed effects on lesion progression cannot be attributed to systemic alterations in this cytokine, but are instead mediated by alterations in lymphocyte function. We demonstrate that IL-10 overexpression can promote development of the Th2 phenotype despite ongoing hyperlipidemia, previously shown to induce a Th1 phenotype.^8,24 We show this phenotypic switch results in decreased production of IFN- by lymphocytes and splenocytes in the IL-10 Tg group. Underscoring the relevance of this IFN- decrease are findings that IFN- receptor–deficient mice develop decreased atherosclerosis,²⁵ and IFN- administration increased atherosclerosis formation in murine models.²⁶ Recent work investigating the impact of pentoxifylline on atherosclerosis in mice also suggested decreased lesion progression associated with a Th2 phenotype; mice with decreased lymphocyte IFN- did not progress significantly beyond the fatty-streak stage compared with the advanced lesions seen in the control group.²⁷

Overexpression of lymphocyte IL-10 altered B cell IgG class in response to oxidized phospholipids. The Th2 phenotype in the IL-10 Tg group influenced B lymphocytes in these mice to produce a preponderance of IgG₁ antiphospholipid antibodies. Previous studies have not only documented specific antibodies in atherosclerosis, but correlated immunoglobulin subclass with extent of disease in both animal models and humans.^8,24 Immunoglobulins of the IgG_2a subclass specific for oxidized epitopes have been quantitated in atherosclerosis, and IgG subclass switching has been demonstrated during the progression of atherosclerosis.^8,28 These immunoglobulins most likely modulate atherogenesis. Mice inoculated with oxidized lipids develop specific antibodies, which may have a protective effect in vivo¹⁵; moreover, treatment of apolipoprotein E–deficient mice with intravenous immunoglobulins significantly decreased early and late lesion formation.²⁹ Thus, the T-cell effect on atherosclerosis in our studies may be executed partially through alterations in B-lymphocyte function.

The central cell type in atherosclerosis is the macrophage.³⁰ Importantly, we present evidence that macrophage function was affected by the Th2 phenotypic alterations we created. Compared with animals fed a chow diet, circulating monocytes in fat-fed animals are activated as manifested by expression of cytokines, adhesion molecules, and other surface-activation markers.³¹ In the present studies, we demonstrated a major difference in monocyte activation between the two groups as measured by IFN- expression. Overall, a 9-fold decrease in IFN- was observed in the monocytes of the IL-10 Tg group, with most monocytes producing no detectable IFN-. Previous reports regarding macrophage function in atherosclerosis^24,32,33 suggest that a dramatic decrease in monocyte activation would have significant impact on atherosclerotic development. Furthermore, the significant reduction in specific bioactive phospholipids within the aortas of the IL-10–overexpressing group suggests that modulation of macrophage function may lead to alterations in lipid processing and oxidation within the vessel wall.

T lymphocytes are present in significant numbers within atherosclerotic lesions of both humans and apolipoprotein E–deficient mice, as well as prominent in early lesions of LDL-R^-/- mice.¹⁶ Previous studies have shown a 2.5-fold increase in T-cell infiltration into early atherosclerotic lesions in IL-10–deficient mice, as well as an increase in IFN- in the more advanced lesions of these mice.¹ Although T cells were not detectable in the lesions presently studied after 20 weeks on the atherogenic diet, previous descriptions¹⁶ suggest they were most likely present within the early lesions, playing a pivotal role at that time. T lymphocyte–derived cytokines could also be acting outside of the lesion to exert critical effects on the monocytes. In arthritis, another chronic inflammatory disease, IL-10 overexpression localized to one joint area had profound effects on monocyte-mediated inflammation at distant joints, even without alterations in free-circulating IL-10.³⁴

In our disease model, the altered character of the lesions in the IL-10 Tg group may be even more important than the decreased lesion burden. Interruption of the CD40-CD40L system has limited impact on total atherosclerotic lesion area, but dramatically changes lesion composition.^35,36 Arguably lesion composition, even more than alteration in total lesion size, has implications for atherosclerotic complications in humans and may therefore be a more appropriate therapeutic target. Although IL-10 may exert some of its influences through CD40-CD40L, ³⁷ the changes in lesions that we observed in IL-10 Tg BM–recipient mice (predominantly a decrease in apoptosis and lipid cores, accompanied by increased cellularity) were not identical to those seen with direct CD40-CD40L interruption (largely represented by increases in fibrous changes and a decrease in monocyte content).^35,36 In summary, this study demonstrates 4 important novel findings regarding lymphocyte overproduction of IL-10 and atherosclerosis: (1) T-cell IL-10 decreased lesion development even at 20 weeks of cholesterol feeding in the LDL-R^-/- mouse, which develops advanced complex lesions; (2) overexpression of IL-10 by T lymphocytes created a Th2 phenotype as indicated by their cytokine expression profile even during the inflammatory stress of atherosclerosis; (3) B-lymphocyte phenotype, as manifested by immunoglobulin subclass, was also altered toward a Th2 phenotype; and (4) T-cell IL-10 inhibited macrophage activation and susceptibility to apoptosis. These IL-10–mediated alterations in lymphocytes and macrophages, as well as alterations in matrix deposition previously documented,¹ likely contribute to both initiation and progression of atherosclerosis, suggesting a potential regulatory role for IL-10 in all stages of atherogenesis. Because of the therapeutic potential of these observations to the treatment of atherosclerosis, it will be important in the future to determine whether lymphocyte IL-10 overexpression occurring exclusively during lesion progression or at late stages of lesion remodeling can also influence lesion development and character. Taken together, these and past studies suggest that lymphocyte-derived as well as circulating IL-10 may promise therapeutic strategies for the treatment of atherosclerosis.

	Acknowledgments

 
L.J.P. was supported by NIH University of California Los Angeles Cardiovascular Scientist Training Program grant T32-HL-07895. L.K.C. is supported by NIH grant HL35297; J.A.B. by NIH grant HL30568; and W.A.B. by NIH grant HL61731. The authors acknowledge technical assistance from Julia Ozbolt and Allis Ip. Antibodies against MDA-LDL were a kind gift from Dr Joseph Witztum, University of California San Diego.

Received May 16, 2001; revision received April 3, 2002; accepted April 9, 2002.

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