378 Journal of Atherosclerosis and Thrombosis　Vol.17, No.4

Original Article

379Resident Peritoneal Cells and Atherogenesis

Resident Peritoneal Inflammatory Cells are Pivotal in the Development of Experimental Atherosclerosis

Waldir G M Relvas1, Maria C O Izar1, Helena R C Segreto1, Adelmo J Giordani1, Silvia S M Ihara2, Mario Mariano3, Jose D Lopes3, Ana F Popi3, Tatiana Helfenstein1, Daniel Pomaro2, Rui M S Póvoa1, Antonio C C Carvalho1, and Francisco A H Fonseca1

1Department of Medicine, Federal University of Sao Paulo, Sao Paulo, Brazil2Department of Pathology, Federal University of Sao Paulo, Sao Paulo, Brazil3Department of Immunology, Federal University of Sao Paulo, Sao Paulo, Brazil

Aim: Based on evidence that ionizing radiation can ameliorate chronic and autoimmune diseases in patients and experimental animals, we investigated the effects of radiation on the induction and development of experimental atherogenesis.Methods: Male New Zealand rabbits were divided into 5 groups and given an atherogenic diet for 90 days. Peritoneal and thoracic areas (9 Gy) were irradiated on the 1st and 45th days for groups 1 and 2, the 45th day for groups 3 and 4, and not at all for group 5. Prior to irradiation, the peritoneal cavity of animals from groups 1 and 3 was washed with buffered saline. Cells collected by peritoneal wash-ing were reinfused into the peritoneal cavity of the same animal after irradiation. Animals from groups 2 and 4 were intraperitoneally injected with saline as a control.Results: Despite similar lipid profiles among the experimental groups, the percentage of aortas cov-ered by plaques was remarkably reduced (p＜0.001) among animals submitted to irradiation (groups 2 and 4). These differences were completely abolished in irradiated animals reconstituted with their own peritoneal cells.Conclusions: These findings point to an important role of resident inflammatory peritoneal cells in experimental atherogenesis.

J Atheroscler Thromb, 2010; 17:378-385.

Key words; Atherogenesis, Rabbits, Peritoneal cells, Ionizing radiation

ferent leukocyte subpopulations has been proposed in the pathophysiology of this disease6, 7). In this context, the contribution of these cells to early atherogenesis has emerged, and details regarding the origin and type of the inflammatory cells involved in this process pro-vide insights for new approaches to therapy6-8).

Several reports have demonstrated that periodic irradiation known as “total lymphoid irradiation” can improve the health and survival of mice in certain autoimmunity models (reviewed by Loor et al.)9) as well as humans10-12).

Recently, e Brito et al.13) investigated the possible role of B-1 cells in the evolution and fate of systemic lupus erythematosus (SLE) in female lupus-prone New Zealand Black/New Zealand White (NZB/NZW) F1 mice. The investigation was based on the fact that B1-b cells reside within the peritoneal cavity

Introduction

Atherosclerosis is a chronic inflammatory disease of multiple origins characterized by an accumulation of lipoproteins and mononuclear cells in the sub-endothelial space of blood vessels1-3). The initial stages of the disease involve the recruitment of blood leuko-cytes, while later stages involve many inflammatory mediators, regulated by cells of both the innate and adaptive immune systems4, 5). Recently, the role of dif-

Address for correspondence: Francisco A H Fonseca, Setor de Lípides, Aterosclerose e Biologia Vascular, Universidade Federal de São Paulo−UNIFESP, Rua Pedro de Toledo 276, São Paulo, SP, Brasil, CEP 04039-030E-mail: fahfonseca@terra.com.brReceived: July 27, 2009Accepted for publication: October 6, 2009

378 Journal of Atherosclerosis and Thrombosis　Vol.17, No.4

Original Article

379Resident Peritoneal Cells and Atherogenesis

in an extra medullar reservoir, being capable of migrat-ing to distant lesions and spontaneously proliferating in stationary cultures of adherent peritoneal cells14-16). Interestingly, these cells have a hybrid monocyte/macrophage pattern. An increased rate of differentia-tion into adherent phagocytic cells was triggered by components of the extracellular matrix, inducing the B-1 cells to produce high levels of several cytokines15). In addition, having been completely destroyed by ion-izing radiation, the cells repopulated the peritoneal cavity in 45 days in mice13, 14). Further, B-1 cells par-ticipate in the genesis of inflammatory giant cells17), are endowed with tolerogenic properties18) and facili-tate the growth and metastatic potential of experimen-tal melanoma19, 20). e Brito et al.13) demonstrated that, in this particular model of SLE (NZB/NZW) F1 mice, irradiation significantly halted the development of SLE, even though B-1 cells seemed not to be affected by radiation in (NZB/NZW) F1 mice. Inter-estingly, a dramatic decrease in mortality in irradiated animals was observed since no control mice survived over 10 months, whereas 80% of irradiated specimens were alive at 16 months of age without symptoms of lupus disease.

Here we investigated the possible role of resident inflammatory peritoneal cells in the induction and development of atherogenesis by diet in rabbits21-25). The results suggested that selective irradiation abro-gated atherogenesis in the model and induced signifi-cant remission of established lesions. Still, these results seem to be dependent on the presence of resident inflammatory peritoneal cells.

Methods

AnimalsMale New Zealand white rabbits were kept in

individual cages on a 12-hour dark/light cycle and fed a 0.5% cholesterol-enriched diet for 90 days plus water ad libitum. The protocol was in accordance with the institution’s guidelines for the care and use of labo-ratory animals and was approved by the local ethics committee. As shown in Table 1, animals were ran-domly assigned to one of five groups: goups 1 and 2 received irradiation of the peritoneal and pleural cavi-ties on the 1st and 45th days, groups 3 and 4 were similarly irradiated but only on the 45th day; and group 5 was not irradiated. Prior to irradiation, the peritoneal cavity of the animals from groups 1 and 3 was washed with a 0.9% saline solution in order to collect resident cells. The cell suspension was main-tained at 4℃. Following irradiation, the cell suspen-sion obtained from each rabbit was re-inoculated into

their peritoneal cavity.

Irradiation of Peritoneal and Pleural CavitiesAnimals were anesthetized with ketamine hydro-

chloride (Ketalar®, Pfizer) and xylazine (Rompum®, Bayer). Shortly after, they were fixed in a Styrofoam bed, in dorsal decubitus, for simulation (Acuity Simu-lator Varian) and transportation to the cobalt unit (60 Co source, CGR, ALCION Ⅱ, MEV, Varian). Each animal received a radiation dose of 9 Gy, collimated to the thorax and abdomen, at a dose rate of 1.3 Gy min－1 with parallel and opposed fields.

Peritoneal WashPrior to the irradiation procedure, the peritoneal

cavity was washed with saline (0.9%) and the sample collected was re-infused in the animals of groups 1 and 3 immediately after irradiation. Only saline replaced the wash solution collected from animals of groups 2 and 4. In some animals, histology (Giemsa) and flow cytometry (using RAM-11, and IgM) were performed to identify the presence of inflammatory cells.

Flow Cytometry, and Histomorphometrical AnalysisAnimals were sacrificed on the 90th day and aor-

tas were carefully removed from their origin in the heart to the iliac bifurcation. The aortas were cut lon-gitudinally, fixed in 10% formalin, and stained with the Sudam Red dye to identify lipid-enriched areas. A histomorphometrical analysis was conducted using the Image Tool® software to estimate the percentage of the aorta covered by plaques. To determine plaque composition, immunohistochemical analyses were done using RAM-11 (DAKO) for macrophages and 1A4 (DAKO) for smooth muscle cells. Collagen con-tent was examined by Picrosirius staining plus polar-ization microscopy. Flow cytometry was used to mea-sure the number of peritoneal cells before and after irradiation.

Table 1. Experimental protocol

GroupsDays of

treatmentn

Time ofirradiation

Peritoneal cellreinfusion

12345

9090909090

8888

10

1st and 45th days1st and 45th days

45th day45th day

Not irradiated

yesnoyesnono

380 Relvas et al. 381Resident Peritoneal Cells and Atherogenesis

Laboratory MeasurementsThe lipid profile of all experimental animals was

processed in the Central Laboratory of UNIFESP. Samples maintained at －80℃, were defrosted at room temperature for two hours and the lipid profile was obtained by enzymatic methods. The SERA-PAK reagent was used for determining total cholesterol and triglyceride (Bayer) levels and HDL-C IMMUNO FS (DiaSys) was used for determining HDL-C levels, in ADVIA 1650 (Bayer, Germany). The effects of irradi-ation on blood cells were examined at baseline and weekly up to four weeks after the procedure (Cell Dyn 3700, Abbott, USA). Concentrations of enzymes in liver were determined using an AU 640 (Olympus, USA).

Statistical AnalysisData are expressed as the mean and SEM. Results

were analyzed using a one-way ANOVA. Differences were considered statistically significant at a value of p＜0.05.

Results

AnimalsAt baseline, no differences were observed in the

weight or lipid profile of the animals from the differ-ent experimental groups (data not shown). After treat-ment, as shown in Table 2, the weight and lipid pro-file were similar between the five groups of rabbits.

Characterization of Peritoneal Cells and the Effects of Irradiation

Numbers of peritoneal cells with the monocyte/macrophage morphology (Fig.1) decreased markedly after irradiation (Fig.2). A decrease of 78.4% at the gate characterizing lymphocytes was observed. The

possible B-1 phenotype of these lymphocytes could not be determined with certainty when commercial monoclonal anti-rabbit IgM and anti-rabbit macro-phage antibodies were used.

Evolution of Atherosclerosis and Effects of RadiationAs shown in Table 3 and Fig.3 and 4, animals

subjected to irradiation exhibited a remarkable reduc-tion in the area of the aorta covered by plaques. Con-versely, irradiated animals reinfused with peritoneal cells had the same percentage of the aorta covered as control animals.

Animals irradiated on day 45 had smaller areas of plaques when compared with non-irradiated controls suggesting some controlling effect of irradiation on established lesions. This interpretation is based on results showing that reinfused animals irradiated on day 45 had more plaques than non-reconstituted ani-

Table 2. Weight and lipid profile at day 90

Groups Weight Total cholesterol LDL-C HDL-C Triglycerides

12345

3,159 (112)2,926 (47)2,943 (27)2,947 (39)3,095 (90)

1,159 (118)1,039 (119)1,005 (146)1,102 (157)1,233 (188)

1,093 (114)881 (79)835 (94)

1,056 (157)1,174 (183)

40 (6)39 (4)32 (3)28 (3)34 (4)

134 (29)170 (77)173 (93)

91 (35)128 (35)

Groups 1 and 2= irradiated twice (at days 1 and 45 day); group 3 and 4= irradiated only at day 45. Group 5=control (not irradiated). Peritoneal wash solution obtained prior to the irradiation procedure was replaced in the peritoneal cavity after irradiation for the animals of groups 1 and 3, and saline infused intraperitoneally was used as a control for the animals of groups 2 and 4. All the animals received an atherogenic diet (0.5% choles-terol). Data are expressed as the mean (SEM); groups 1 to 4 (n=8); group 5 (n=10). LDL= low density lipopro-tein; HDL=high density lipoprotein; weight is expressed in grams; lipids are in mg/dL. No significant differ-ences were observed for each variable between groups (ANOVA).

Fig.1. Lympho/mieloid cells from the peritoneal cavity of a normal rabbit.

380 Relvas et al. 381Resident Peritoneal Cells and Atherogenesis

mals. Immunohistochemical analyses to determine the composition of plaques showed high macrophage and low smooth muscle cell content (as shown in Fig.5 A, B, C, D), as well as low macrophage and high smooth muscle cell and collagen content (Fig.5 E, F, G, H), in all groups.

Effects of Irradiation on Hematologic ParametersAs shown in Table 4, there was no apparent

effect of irradiation on red blood cells. However, there was a progressive decrease in leukocyte numbers from baseline to four weeks, mainly neutrophils (－54.01%) and lymphocytes (－73.64%). Platelets were less affected by irradiation.

Discussion

Our findings point towards a surprising new aspect of the atherogenetic process, that an extra medullar reservoir of inflammatory cells in the perito-neal cavity is extensively involved in atherogenesis. This assumption is supported by the observation that irradiation of the thoracic-abdominal region of rabbits maintained on an atherogenic diet almost completely abolished plaque formation. This effect might be due to depletion of lymphoid reactive cells from lymph nodes and other organs. In fact, after irradiation, a progressive decrease in leukocyte numbers was noted. Nevertheless, this interpretation was ruled out based

Fig.2. Figures show the percentage of cells in the R1 (lymphocytes) and R2 (macrophages and granulocytes) gates. Selection is based on the size and complexity of the cells using the parameters SSC and FSC.

The total number of cells in non-irradiated animals was 4.2×106, the number of lymphocytes was 7.4×105, and the number of macrophages was 2.5×106. In irradiated animals, the total number of cells was 3.4×106, the number of lymphocytes was 1.6×105, and the number of macrophages was 2.7×106. The analyses of the absolute number of cells showed a 78.4% reduction of cells of the lymphocyte gate after irradiation.

Table 3. Areas of aortas, areas of plaques and percentage of aortas occupied by plaques according to group

VariablesGroup 1

n =8Group 2

n =8Group 3

n =8Group 4

n =8Group 5n =10

p

Area of aorta＊

Area of plaque†

Aorta covered by plaques, %‡

1,303 (92)842 (162)70.7 (7.3)

1,133 (52)287 (101)

23.5 (7.5)

1,105 (50)497 (123)43.8 (10.5)

968 (25)81 (11)

8.4 (1.3)

1,351 (92)924 (156)64.3 (8.8)

0.0030.0010.001

Aorta and plaque areas are in mm2; aorta covered by plaques is the percentage of aortas positively stained by Sudam Red dye (%). Data are the mean (SEM). ＊Group 4 ＜ group 1 (p=0.019); group 4 ＜ group 5 (p=0.003). †Group 1 ＞ group 2 (p=0.036); group 1 ＞ group 4 (p=0.002); group 2 ＜ group 5 (p=0.007); group 4 ＜ group 5 (p＜0.001). ‡Group 1 ＞ group 2 (p=0.002); group 1 ＞ group 4 (p＜0.001); group 2 ＜ group 5 (p=0.005); group 3 ＞ group 4 (p=0.029); group 4 ＜ group 5 (p＜0.001). Comparisons were made with ANOVA followed by the Tukey test.

382 Relvas et al. 383Resident Peritoneal Cells and Atherogenesis

on experiments in which resident peritoneal cells were washed out from the peritoneal cavity before irradia-tion with reinfusion of these cells after irradiation. Under these experimental conditions, the intensity of plaque formation was similar to that observed in con-trol animals, showing that the reduction in the num-ber of blood leukocytes did not prevent plaques for developing in irradiated animals, highlighting the con-tribution of inflammatory cells from other sources, such as the resident peritoneal cells. Interestingly, the irradiation exerted some anti-atherogenic effect even in animals on the atherogenic diet for 45 days, in which plaques had already developed. In other words, irradiation might not only block the formation of plaques but arrest/regress the development of lesions that have already formed. The mechanisms by which irradiation exerts its anti-atherogenic effects are not entirely clear. Irradiation is known to kill intestinal endothelial cells and induce chronic diarrhea, so the absorption of cholesterol-enriched food would be decreased in irradiated animals. However, the recon-stitution of irradiated animals with peritoneal cells reestablished the atherogenesis. In addition, irradia-tion neither changed the levels of liver enzymes in serum (Table 4), nor affected the microscopic analysis of the liver or spleen (data not shown).

Irradiation depletes approximately 78.4% of lymphoid-like cells in the peritoneal cavity of normal rabbits and in the mouse, the existence of a popula-tion of lympho/myeloid cells denominated B-1 cells is

Fig.3. Representative aortas from five experimental groups.

Group 5 was used as a control (non-irradiated animals) receiving the atherogenic diet. In groups 3 and 4, the animals were irradi-ated on day 45, but only those from group 3 received their own peritoneal cells after irradiation. In groups 1 and 2, the animals were irradiated twice (on the 1st and 45th days) but only those from group 1 were reconstituted with their own cells after irradia-tion. Note that irradiation attenuated the development of athero-mas while reinfusion of the peritoneal cavity with resident perito-neal cells allowed plaque formation similar to control animals.

Fig.4. Figure shows box-plots of the distribution of atheromas in the animals.

Fig. 4a indicates the total plaque area among groups. Groups 1 and 5 showed a greater area when compared with groups 2 and 4; ANOVA, p≤0.001. Fig. 4b indicates the total area of the aorta covered by plaques among the groups. Groups 1, 3 and 5 showed a greater percentage of area occupied by plaques than did group 4. Group 2 showed a smaller percentage when compared with groups 1 and 5; ANOVA, p≤0.001.

382 Relvas et al. 383Resident Peritoneal Cells and Atherogenesis

well established. It has also been demonstrated that these cells facilitate parasite infectivity26), promote tumor growth19, 20), have tolerogenic properties18), are radiosensitive13), migrate from the peritoneal cavity to distant inflammatory lesions and differentiate into a mononuclear phagocyte14, 16). Nevertheless, there are no reports in the literature considering the possible existence of B-1 cells in the peritoneal and pleural cav-ities of rabbits. Using commercial monoclonal anti-bodies and a marker for macrophages we did not suc-ceed in characterizing with certainty the B-1 cell sub-type in the peritoneal cavity of the rabbit. Therefore, the role of these cells in atherogenesis remains specula-

tive.The involvement of B-1 cells in the pathogenesis

of atherosclerosis has been reported by Silverman et al.27). The authors postulated that modified LDL par-ticles could be highly stimulatory for certain B-1 clones. In their opinion, during the evolution of the adaptive immune system, the neo-self antigenic milieu may have been exploited for the natural selection of primordial clonal specificity. We strongly agree with this important concept, and the extra-medular reser-voir of B1-b cells in the peritoneal cavity might repre-sent a new paradigm in the field of atherosclerosis, with this clone of inflammatory cells recognized as

Fig.5. Histological and immunohistochemical analyses in segments of aorta (×100).

A and B show Hematoxylin and Eosin staining; B and F show RAM-11-positive staining (brown) for macrophages; C and G show 1A4-posi-tive staining for smooth muscle cells; D and H show Picrosirius staining for collagen.

Table 4. Effects of irradiation on hematologic and liver parameters

Parameter Baseline1-wk afterirradiation

2-wk afterirradiation

3-wk afterirradiation

4-wk afterirradiation

HemoglobinHematocritLeukocytesNeutrophilsEosinophilsBasophilsLymphocytesMonocytesPlateletsASTALT

12.7 (0.4)38.2 (1.2)

3,923 (1,136)1,533 (559)

9 (9)64 (33)

1,324 (330)222 (93)

305,667 (32,539)29.2 (2.6)39.0 (3.4)

15.0 (0.9)43.0 (1.4)

3,910 (370)2,614 (951)

2 (2)73 (73)

891 (223)331 (285)

234,500 (35,500)28.0 (3.0)27.5 (3.5)

14.4 (0.6)43.0 (1.5)

3,510 (1,135)2,289 (995)

83 (41)26 (21)

817 (181)296 (157)

189,333 (75,393)33.3 (8.4)29.0 (4.3)

13.7 (0.1)39.8 (1.3)

2,990 (310)1,583 (21)

135 (113)57 (22)

787 (117)183 (59)

400,000 (51,000)27.5 (8.5)35.5 (17.5)

13.1 (1.0)39.2 (2.6)

1,467 (230)705 (111)

1 (0)0 (0)

349 (76)15 (6)

243,333 (49,640)52.0 (10.6)45.7 (9.9)

Hematologic parameters observed in rabbits at baseline and after irradiation (n=5). Values are expressed as the mean (SEM). Hemoglobin (g/dL); Hematocrit (%); Leukocytes (number/μL); AST=aspartate aminotransferase; ALT=alanine aminotransferase.

384 Relvas et al. 385Resident Peritoneal Cells and Atherogenesis

part of our evolution, initially as an important defense mechanism for maintenance of cellular homeostasis, but now as an important pathway for atherosclerotic plaque development.

How could the oxidized lipoprotein particles trigger immune responses involving the peritoneal inflammatory cells ? In fact, it was reported that oxi-dized epitopes from low-density lipoproteins were rec-ognized by a natural phosphorylcholine-specific germ-line-encoded B-1 cell antibody, T1527). These findings support the notion of interaction between atheroscle-rosis and these inflammatory cells28).

Summarizing, our study adds to the study of ath-erosclerosis in at least two ways. First, we developed a new promising experimental model to examine the role of peritoneal inflammatory cells in the athero-genic process, and second, we added the novelty of radiation to the conventional diet-induced atheroscle-rosis in the rabbit model. Furthermore, through the reinfusion of peritoneal inflammatory cells after irra-diation, we established the relevance of these cells to atherogenesis while concomitantly demonstrating an oustanding attenuation of plaque formation when these cells are not reinfused.

The characterization of these inflammatory cells and the pathways for both differentiation and migra-tion to the vessel wall will constitute the next step in the study of this complex disease.

Acknowledgments

This study was supported by FAPESP (The State of São Paulo Research Foundation). Dr. Relvas is also a recipient of a research fellowship from CNPq (National Counsel of Technological and Scientific Development).

References

1) Binder CJ, Silverman GJ: Natural antibodies and the autoimmunity of atherosclerosis. Springer Semin Immu-nopathol, 2005; 26: 385-404

2) Loppnow H, Werdan K, Buerke M: Vascular cells con-tribute to atherosclerosis by cytokine- and innate-immu-nity-related inflammatory mechanisms. Innate Immun, 2008; 14: 63-87

3) Galkina E, Ley K: Leukocyte influx in atherosclerosis. Curr Drug Targets, 2007; 8: 1239-1248

4) Staprans I, Pan XM, Rapp JH, Feingold KR: The role of dietary oxidized cholesterol and oxidized fatty acids in the development of atherosclerosis. Mol Nutr Food Res, 2005; 49: 1075-1082

5) Weber C, Zernecke A, Libby P: The multifaceted contri-butions of leukocyte subsets to atherosclerosis: lessons from mouse models. Nat Rev Immunol, 2008; 8: 802-815

6) Lamon BD, Hajjar DP: Inflammation at the molecular interface of atherogenesis: an anthropological journey. Am J Pathol, 2008; 173: 1253-1264

7) Libby P: Inflammatory mechanisms: the molecular basis of inflammation and disease. Nutr Rev, 2007; 65: S140- 146

8) Williams KJ, Feig JE, Fisher EA: Rapid regression of ath-erosclerosis: insights from the clinical and experimental literature. Nat Clin Pract Cardiovasc Med, 2008; 5: 91-102

9) Loor F, Jachez B, Montecino-Rodriguez E, Klein AS, Kuntz L, Pflumio F, Fonteneau P, Illinger D: Radiation therapy of spontaneous autoimmunity: a review of mouse models. Int J Radiat Biol Relat Stud Phys Chem Med, 1988; 53: 119-136

10) Fuks Z, Strober S, Bobrove AM, Sasazuki T, McMichael A, Kaplan HS: Long term effects of radiation of T and B lymphocytes in peripheral blood of patients with Hodg-kin’s disease. J Clin Invest, 1976; 58: 803-814

11) Meredith RF, Buchsbaum DJ: Pretargeted radioimmuno-therapy. Int J Radiat Oncol Biol Phys, 2006; 66: S57-59

12) Singh TP, Rabah R, Cooper LT, Walters HL: Total lym-phoid irradiation: new therapeutic option for refractory giant cell myocarditis. J Heart Lung Transplant, 2004; 23: 492-495

13) e Brito RR, De Lorenzo BH, Xander P, Godoy LC, Lopes JD, da Silva NP, Sampaio SC, Mariano M: Role of dis-tinct immune components in the radiation-induced abro-gation of systemic lupus erythematosus development in mice. Lupus, 2007; 16: 947-954

14) Almeida SR, Aroeira LS, Frymuller E, Dias MA, Bogsan CS, Lopes JD, Mariano M: Mouse B-1 cell-derived mononuclear phagocyte, a novel cellular component of acute non-specific inflammatory exudate. Int Immunol, 2001; 13: 1193-1201

15) Ferreira KS, Almeida SR, Ribeiro CH, Mariano M, Lopes JD: Modulation of proliferation, differentiation and cyto-kine secretion of murine B-1b cells by proteins of the extracellular matrix. Immunol Lett, 2003; 86: 15-21

16) Popi AF, Motta FL, Mortara RA, Schenkman S, Lopes JD, Mariano M: Co-ordinated expression of lymphoid and myeloid specific transcription factors during B-1b cell differentiation into mononuclear phagocytes in vitro. Immunology, 2009; 126: 114-122

17) Bogsan CS, Novaes e Brito RR, Palos Mda C, Mortara RA, Almeida SR, Lopes JD, Mariano M: B-1 cells are piv-otal for in vivo inflammatory giant cell formation. Int J Exp Pathol, 2005; 86: 257-265

18) De Lorenzo BH, Brito RR, Godoy LC, Lopes JD, Mari-ano M: Tolerogenic property of B-1b cells in a model of allergic reaction. Immunol Lett, 2007; 114: 110-118

19) Perez EC, Machado J Jr, Aliperti F, Freymuller E, Mariano M, Lopes JD: B-1 lymphocytes increase metastatic behav-ior of melanoma cells through the extracellular signal-reg-ulated kinase pathway. Cancer Sci, 2008; 99: 920-928

20) Staquicini FI, Tandle A, Libutti SK, Sun J, Zigler M, Bar-Eli M, Aliperti F, Perez EC, Gershenwald JE, Mariano M, Pasqualini R, Arap W, Lopes JD: A subset of host B lym-phocytes controls melanoma metastasis through a mela-noma cell adhesion molecule/MUC18-dependent interac-tion: evidence from mice and humans. Cancer Res, 2008;

384 Relvas et al. 385Resident Peritoneal Cells and Atherogenesis

68: 8419-842821) Silva EP, Fonseca FA, Ihara SS, Izar MC, Lopes IL, Pinto

LE, Badimon JJ, Tuffik S, Paiva TB, Kasinski N, de Paola AV, Carvalho AC: Early benefits of pravastatin to experi-mentally induced atherosclerosis. J Cardiovasc Pharmacol, 2002; 39: 389-395

22) Yanni AE: The laboratory rabbit: an animal model of ath-erosclerosis research. Lab Anim, 2004; 38: 246-256

23) Russell JC, Proctor SD: Small animal models of cardiovas-cular disease: tools for the study of the roles of metabolic syndrome, dyslipidemia, and atherosclerosis. Cardiovasc Pathol, 2006; 15: 318-330

24) Verbeuren TJ: Experimental models of thrombosis and atherosclerosis. Therapie, 2006; 61: 379-387

25) Soliman A, Kee P: Experimental models investigating the

inflammatory basis of atherosclerosis. Curr Atheroscler Rep, 2008; 10: 260-271

26) Popi AF, Godoy LC, Xander P, Lopes JD, Mariano M: B-1 cells facilitate Paracoccidioides brasiliensis infection in mice via IL-10 secretion. Microbes Infect, 2008; 10: 817-824

27) Silverman GJ, Shaw PX, Luo L, Dwyer D, Chang M, Horkko S, Palinski W, Stall A, Witztum JL: Neo-self anti-gens and the expansion of B-1 cells: lessons from athero-sclerosis-prone mice. Curr Top Microbiol Immunol, 2000; 252: 189-200

28) Shaw PX, Goodyear CS, Chang MK, Witztum JL, Silver-man GJ: The autoreactivity of anti-phosphorylcholine antibodies for atherosclerosis-associated neo-antigens and apoptotic cells. J Immunol, 2003; 170: 6151-6157