Defective B1 Cell Homing to the Peritoneal Cavity and Preferential Recruitment of B1 Cells in the Target Organs in a Murine Model for Systemic Lupus Erythematosus This information is current as of September 26, 2021. Toshihiro Ito, Sho Ishikawa, Taku Sato, Kenji Akadegawa, Hideaki Yurino, Masahiro Kitabatake, Shigeto Hontsu, Taichi Ezaki, Hiroshi Kimura and Kouji Matsushima J Immunol 2004; 172:3628-3634; ; doi: 10.4049/jimmunol.172.6.3628 Downloaded from http://www.jimmunol.org/content/172/6/3628

References This article cites 26 articles, 8 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/172/6/3628.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Defective B1 Cell Homing to the Peritoneal Cavity and Preferential Recruitment of B1 Cells in the Target Organs in a Murine Model for Systemic Lupus Erythematosus1

Toshihiro Ito,*† Sho Ishikawa,* Taku Sato,* Kenji Akadegawa,* Hideaki Yurino,* Masahiro Kitabatake,* Shigeto Hontsu,*† Taichi Ezaki,‡ Hiroshi Kimura,† and Kouji Matsushima2*

We previously reported that B chemoattractant (BLC; CXCL13) was highly and ectopically expressed in aged (NZB ؋

NZW)F1 (BWF1) mice developing lupus nephritis, and that B1 cells were preferentially chemoattracted toward BLC. We dem- onstrate in this study that B1 cells fail to home to the peritoneal cavity in aged BWF1 mice developing lupus nephritis, and that they are preferentially recruited to the target organs including the kidney, lung, and when injected i.v. In contrast, B1 cells Downloaded from homed to the peritoneal cavity in aged BALB/c mice as effectively as in young mice. Accumulation of B1 cells to the omentum milky high high spots was also impaired in aged BWF1 mice compared with young mice. CD11b F4/80 cells with morphology were confirmed to be a major cell source for BLC in the peritoneal cavity both in young and aged BWF1 mice. However, the number of BLC-producing peritoneal was markedly decreased in aged BWF1 mice. These results suggest that the decreased number of BLC-producing peritoneal macrophages together with ectopic high expression of BLC in aged BWF1 mice result in abnormal B1 cell trafficking during the development of murine lupus. The Journal of Immunology, 2004, 172: 3628–3634. http://www.jimmunol.org/

ϫ he (NZB NZW)F1 (BWF1) strain of mice spontane- 7). Involvement of B1 cells in IgM-mediated autoimmune diseases ously develop systemic autoimmune disorders resembling such as autoimmune hemolytic anemia was also demonstrated (8, T systemic lupus erythematosus (SLE)3 in humans (1, 2). It 9). However, it remains to be elucidated whether B1 cells class- is characterized by production of a variety of IgG autoantibodies switch from IgM to IgG in the development of autoimmune dis- and massive deposition of immune complexes in glomeruli in the eases. Accumulating data also suggest that B1 cells contribute to kidney. More than 95% of these mice die from renal failure before the innate immunity by producing natural IgM Ab in the circula- 12 mo of age. A marked mononuclear cell infiltration in the target tion and by secreting IgA Ab in the intestinal mucosa (10Ð13). It by guest on September 26, 2021 organs, including the kidney and lung, is also observed in aged has been recently reported that BLC is essential for B1 cell homing

BWF1 mice. We previously demonstrated that B lymphocyte che- to the peritoneal cavity and for immunity (14). In the moattractant (BLC; CXCL13) was highly and ectopically ex- present study, we investigated a role of BLC expression in aged pressed by myeloid dendritic cells in the target organs including BWF1 on aberrant B1 cell trafficking, including B1 cell homing to the thymus, kidney, and lung in aged BWF1 mice, but not in aged the peritoneal cavity. We found that B1 cell homing to the peri- NZB and NZW mice, and that B1 cells were preferentially che- toneal cavity was impaired and preferential B1 cell trafficking to moattracted toward BLC (3, 4). the target organs in aged BWF1 mice. We also demonstrated that B1 cells are a specialized cell population that are distinguished the number of BLC-producing peritoneal macrophages, which from conventional B cells (B2 cells) by their origin, cell surface were reported to be a major cell source for BLC in the peritoneal phenotype, unique tissue distribution, and capacity for self-re- cavity (14), was markedly decreased in aged BWF1 mice. Patho- newal, and have also been considered to be involved in autoanti- logical significance of abnormal B1 cell trafficking in the devel- body production in the development of autoimmune diseases (5Ð opment of murine lupus is discussed.

*Department of Molecular Preventive Medicine, School of Medicine, University of Materials and Methods Tokyo, Tokyo, Japan; †Second Department of Internal Medicine, Nara Medical Uni- versity, Nara, Japan; and ‡Department of Anatomy and Developmental Biology, Mice School of Medicine, Tokyo Women’s Medical University, Tokyo, Japan ϫ (NZB NZW)F1 (BWF1), NZB, NZW, and BALB/c mice, originally Received for publication July 9, 2003. Accepted for publication January 20, 2004. obtained from the Shizuoka Laboratory Animal Center (Shizuoka, Japan), The costs of publication of this article were defrayed in part by the payment of page were maintained in our animal facility at University of Tokyo. Female charges. This article must therefore be hereby marked advertisement in accordance BWF1 and BALB/c mice aged 8Ð10 wk were used as young mice, and with 18 U.S.C. Section 1734 solely to indicate this fact. BWF1 mice aged 8Ð12 mo with moderate-to-severe proteinuria and 1 This work was supported by Solution-Oriented Research for Science and Technol- BALB/c mice aged 8 mo were used as aged mice. ogy (Japan Science and Technology Corporation) and the Long-Range Research Ini- tiative (Japan). Antibodies 2 Address correspondence and reprint requests to Dr. Kouji Matsushima, Department of Molecular Preventive Medicine, School of Medicine, University of Tokyo, 7-3-1 Hongo, Rat mAbs specific for mouse CD11b (M1/70), CD11c (HL-3), B220/ Bunkyo-ku, Tokyo 113-0033, Japan. E-mail address: [email protected] CD45R (RA3-6B2), CD5 (53-7.3RRH), MHC class II (ER-TR3), and 3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; BLC, B lym- CD16/32 (2.4G2) were purchased from BD PharMingen (San Diego, CA). phocyte chemoattractant; pAb, polyclonal Ab; PBLk, peripheral blood leukocyte; Anti-F4/80 (CI: A3-1) mAb and goat anti-BLC polyclonal Ab (pAb) were CMTMR, 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine; purchased from Serotec (Raleigh, CA) and Genzyme Techne (Minneapolis, SDF-1, stromal cell-derived factor 1; SLC, secondary lymphoid tissue chemokine. MN), respectively.

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 3629 Downloaded from http://www.jimmunol.org/

FIGURE 1. Failure of B1 cell homing to the peritoneal cavity in aged BWF1 mice developing lupus nephritis. Four million CFSE-labeled peritoneal B1 ٗ Ⅵ ϫ 6 cells ( )or4 10 CMTMR-labeled splenic B2 cells ( ) suspended in 0.2 ml of PBS were injected i.v. into young BWF1 (A), aged BWF1 (B), young BALB/c (C), or aged BALB/c (D) mice. The recruitment to various lymphoid tissues was analyzed 24 h after injection. The results were presented as the Ϯ mean percentage SD in four independent experiments in BWF1 mice and in three in BALB/c mice. Note that B1 cells fail to home to the peritoneal cavity Ͻ ءءء Ͻ ءء Ͻ ء in aged BWF1 mice developing lupus nephritis. Statistical analysis was performed by Student’s t test. , p 0.03; , p 0.01; , p 0.001. PerC, Peritoneal cell; Thy, thymus; LN, ; mLN, mesenteric lymph node; PP, Peyer patches; Spl, .

Cell preparation aged BWF1 mice labeled with CFSE and B1 cells from young BWF1 mice by guest on September 26, 2021 labeled with CMTMR suspended in 0.2 ml of PBS was injected i.v. into Peritoneal lavage cells were isolated by flushing the peritoneal cavity with young BWF1 mice. Mice were sacrificed 24 h after i.v. injection and sub- 4Ð6 ml of RPMI 1640 supplemented with 5% FCS. Cell suspensions pre- jected to flow-cytometric and immunofluorescent analysis. The number of pared from mouse spleen and lymph nodes were passed through nylon cells localized in the various organs was calculated by the percentage of mesh. Peripheral blood leukocytes (PBLk) were obtained by centrifugation labeled cells in each organ, and the percentage of recruited cells to the total on Lymphorite-M (Cedarlane, Hornby, Ontario, Canada). RBC in the number of injected cells was presented using an Epics Elite cell sorter. In spleen and PBLk were lysed by ammonium chloride solution. nonlymphoid organs, 10 nonconsecutive microscopic fields of cryostat sec- tion were examined, and the numbers of CFSE-labeled B1 cells and Flow cytometry CMTMR-labeled B2 cells in each field were counted. The mean percentage Flow-cytometric analyses of peritoneal cells, spleen cells, lymphoid cells, of B1 cells among total number of fluorescent cells was presented. and PBLk were performed using an Epics Elite cell sorter (Coulter Elec- Immunofluorescent staining tronics, Hialeah, FL) as described previously (3). After blocking FcR with anti-CD16/32 for 10 min, peritoneal cells were stained with FITC-conju- For immunofluorescent analysis of target organs, spleen, lung, kidney, and gated anti-CD11b mAb and PE-conjugated anti-CD11c mAb, and biotin- thymus from aged BWF1 mice were fixed in paraformaldehyde, embedded conjugated anti-B220, F4/80, or class II mAb followed by allophycocya- in Tissue-Tek OCT compound (Miles, Elkhart, IN), and then frozen in ϩ nin-streptavidin (BD PharMingen). CD11bhighCD11clowF4/80 liquid nitrogen. Six-micron cryostat sections were then incubated with bi- macrophages were sorted to Ͼ95% purity using an Epics Elite cell sorter. otin-conjugated anti-B220 mAb followed by allophycocyanin-streptavidin. Sorted macrophages were spun down onto a slide glass and stained with Avidin/biotin blocking solution (Vector Laboratories, Burlingame, CA) Giemsa solution (Merck, Tokyo, Japan). In some experiments, peritoneal was used to decrease nonspecific staining. Finally, the sections were ana- cells were strained with FITC-conjugated anti-CD5 mAb, PE-conjugated lyzed by Olympus IX-70 confocal laser-scanning microscope system anti-B220 mAb, and biotin-conjugated anti-F4/80 mAb followed by allo- (Olympus Optical, Tokyo, Japan). phycocyanin-streptavidin. CD5ϩB220ϩF4/80Ϫ B1 cells in the peritoneal cavity and CD5ϪB220ϩF4/80Ϫ B2 cells in the spleen were sorted on an Histological and immunocytochemical examination Epics Elite cell sorter. More than 95% of B1 and B2 cells were CD19 Peritoneal macrophages obtained by cytospin centrifugation were stained positive. with goat anti-BLC pAb, followed by biotinylated rabbit anti-goat IgG Injection of CFSE- and 5-(and-6)-(((4-chloromethyl)benzoyl) (DAKO, Carpinteria, CA) and HRP-labeled streptavidin (DAKO). Avidin/ biotin blocking solution and normal rabbit IgG were used to decrease non- amino)tetramethylrhodamine (CMTMR)-labeled B cells specific staining. Cell labeling of B1 cells with CFSE (Molecular Probes, Eugene, OR) and Reverse transcription and real-time quantitative PCR analysis B2 cells with CMTMR (Molecular Probes) was performed according to the manufacturer’s instructions. These cells were all labeled in 10 ␮Mat37¡C Total RNA was isolated from peritoneal cells by using RNAzol B (Tel- for 15 min. The cell viability after CFSE and CMTMR staining and before Test, Friendswood, TX) according to manufacturer’s instructions. It was injection was Ͼ99% in dye exclusion tests using trypan blue. The mixture then reversely transcribed into cDNA using Superscript II preamplification of 4 ϫ 106 CFSE-labeled peritoneal B1 cells and/or 4 ϫ 106 CMTMR- kit (Invitrogen, Carlsbad, CA) and amplified with specific oligonucleotide labeled splenic B2 cells suspended in 0.2 ml of PBS were injected i.v. into primers. The sense and the antisense primers used were as follows: BLC Ј Ј Ј BWF1 or BALB/c mice. In some experiments, the mixture of B1 cells from primer, 5 -TCTCTCCAGGCCACGGTATTCT-3 and 5 -ACCATTTGGC 3630 ABERRANT B1 CELL TRAFFICKING IN MURINE LUPUS

ACGAGGATTCAC-3Ј, and GAPDH primer, 5Ј-AGTATGACTCCACT number of CFSE-labeled B1 cells accumulated in the omentum CACGGCAA-3Ј and 5Ј-TCTCGCTCCTGGAAGATGGT-3Ј. PCR was milky spots in aged BWF1 mice compared with young mice (Fig. performed by thermal cycler for 35 cycles of 94¡C for 30 s, 58¡C for 45 s, 2A). In contrast, B1 cell accumulation in the omentum milky spots and 72¡C for 45 s, and final extension was done at 72¡C for 10 min. The PCR products of GAPDH and BLC were examined by 2.5% agarose gel was intact in aged BALB/c mice (Fig. 2B). B1 cell homing to the electrophoresis. Real-time quantitative PCR analysis was performed by peritoneal cavity was also normal in aged BALB/c mice. A similar using ABI 7700 sequence detector system (PE Applied Biosystems, Foster number of B1 cells was detected in the peritoneal cavity and spleen City, CA). FAM-labeled primers were used as target hybridization probes in aged BALB/c mice compared with that in young BWF mice. for BLC and GAPDH (BLC, 5Ј-CATCATAGTTCGGATTCAAGTTA 1 CGCCCCC-3Ј; GAPDH, 5Ј-AAGGGACACAGTCAAGGCCGAGAAT-3Ј). Furthermore, there was no difference in homing ability to the peri- The thermal cycling condition included 50¡C for 2 min and 95¡C for 10 min, toneal cavity between B1 cells obtained from young BWF1 mice followed by 45 cycles of amplification at 95¡C for 15 s and 55¡C for 1.5 min and those from aged mice. A similar number of B1 cells from for denaturing and annealing, respectively. PCR were run in triplicate. BLC either origin was detected in the milky spots and peritoneal cavity quantity was normalized by the level of GAPDH. when injected together into young BWF1 mice (Fig. 2C). Statistical analysis Preferential recruitment of B1 cells in the target organs in aged Statistical analysis was performed using Student’s t test. The 95% confi- BWF mice dence limit was taken as significant. 1 When the mixture of CFSE-labeled B1 cells and CMTMR-labeled

Results B2 cells was injected i.v. into the same aged BWF1 mice, B1 cells Impaired B1 cell trafficking in aged BWF1 mice developing were preferentially recruited in the cellular infiltrates (B220 pos- lupus nephritis itive) in the target organs such as the kidney, lung, and thymus, Downloaded from We conducted B1 cell homing experiments where CFSE-labeled whereas they were similarly recruited to follicles in the B1 or CMTMR-labeled B2 cells were injected i.v. into young or spleen (Fig. 3A). The mean percentages of B1 cells among total Ϯ Ϯ aged BWF mice to examine the change in B1 cell trafficking number of fluorescent cells were 50.15 3.44, 62.33 10.60 1 Ͻ Ϯ Ͻ Ϯ Ͻ during the development of lupus nephritis in these mice. A signif- ( p 0.05), 88.81 6.88 ( p 0.001), and 83.00 3.91 ( p icant number of B1 cells homed to the peritoneal cavity in young 0.001) in the spleen, thymus, kidney, and lung, respectively (Fig. 3B). These data confirmed that the percentage of B1 cells recruited http://www.jimmunol.org/ BWF1 mice when injected i.v. with CFSE-labeled B1 cells (Fig. to the target organs was significantly higher than that of B2 cells 1A). However, in aged BWF1 mice, B1 cells failed to home to the peritoneal cavity (Fig. 1B). In contrast, B1 cells did home to the in aged BWF1 mice. peritoneal cavity in aged BALB/c mice as well as in young BALB/c mice (Fig. 1, C and D) We next investigated the accu- Identification of BLC-producing cells in the peritoneal cavity mulation of B1 cells in the milky spots, which are reported to be To elucidate the mechanism for defective B1 cell homing, RT- the entry sites for B1 cells into the (14). The decreased PCR and real-time quantitative PCR analysis on BLC expression by guest on September 26, 2021

FIGURE 2. Decreased B1 cell accumulation to the omentum milky spots in aged BWF1 mice. Immunofluorescent and flow-cytometric analyses of CFSE (green) and CMTMR (red) cells in the omentum milky spots, peritoneal cells, and spleen. Four million labeled peritoneal B1 cells were injected i.v. into young and aged BWF1 or BALB/c mice. Omentum milky spots were analyzed 24 h after injection under a confocal laser-scanning microscope system. A representative and histological and flow-cytometric analysis of three independent experiments was presented. Similar results were obtained in other experiments. A, Immunofluorescent analysis of the omentum milky spots in young and aged BWF1 mice. B, Immunofluorescent and flow-cytometric analyses of the omentum milky spots, peritoneal cells, and spleen in young and aged BALB/c mice. C, Immunofluorescent and flow-cytometric analyses of labeled B1 cells from young and aged BWF1 mice. The mixture of CFSE-labeled B1 cells from aged BWF1 mice and CMTMR-labeled B1 cells from young BWF1 mice were injected i.v. into young BWF1 mice. The Journal of Immunology 3631 Downloaded from http://www.jimmunol.org/

FIGURE 3. Preferential B1 cell recruitment to nonlymphoid organs including the kidney and lung. B1 and B2 cells were labeled with CFSE (green) and

CMTMR (red), respectively, and the mixture of B1 and B2 cells at a ratio of 1:1 was injected i.v. into the same aged BWF1 mice. Mice were sacrificed 24 h after injection. A, Cryosections of the spleen, lung, kidney, and thymus were photographed under a confocal laser-scanning microscope system (ϫ200).

Cellular infiltrates were highlighted using biotin-conjugated B220 mAbs (blue). A representative histology of three independent experiments was presented. by guest on September 26, 2021 Similar findings were observed in other experiments. B, Percentage of CFSE-labeled cells (B1 cells) in transferred cells in each organ. Ten nonconsecutive microscopic fields were examined, and the numbers of CFSE-labeled B1 cells and CMTMR-labeled B2 cells in each field were counted. The mean percentage of B1 cells among the total number of fluorescent cells was presented. The results are presented as the mean Ϯ SD. Statistical analysis was .p Ͻ 0.001 ,ءء ;p Ͻ 0.05 ,ء .performed by Student’s t test in comparison with the percentage in the spleen in the peritoneum were performed. BLC was expressed in the peri- Discussion toneal cells in both young and aged BWF1 mice, although BLC We demonstrated that B1 cells failed to home to the peritoneal gene expression was significantly lower in aged BWF1 mice than cavity in aged BWF1 mice, whereas a significant number of B1 in young mice (Fig. 4A, a and b). It contrasted with our previous cells did home in young BWF1 mice. This was also supported by study demonstrating that BLC gene expression was markedly in- the fact that markedly decreased number of B1 cells appeared in creased in the target organs including the kidney, lung, and thymus the milky spots in aged BWF mice when injected i.v. These re- in aged BWF mice (3). RT-PCR analysis on highly purified leu- 1 1 sults are not attributed to mere age-dependent phenomena, because kocyte subpopulations demonstrated that BLC was highly ex- B1 cell homing to the peritoneal cavity was intact in aged BALB/c pressed in peritoneal CD11bhighCD11clow cells (Fig. 4B). Sorted mice. There was also no difference in homing ability between B1 CD11bhighCD11clow cells showed a morphological appearance of cells from young mice and those from aged mice. Instead of hom- macrophages and expressed F4/80, a cell surface marker for mac- rophage while they were low in class II expression and negative ing to the peritoneal cavity, B1 cells were recruited to the target for B220 (Fig. 5A). These findings were consistent with previous organs such as the lung, kidney, and thymus in aged BWF1 mice. reports (14). Furthermore, immunocytochemical analysis con- This is probably due to ectopic and high expression of BLC in firmed BLC protein expression by peritoneal macrophages both in these organs as demonstrated in our previous study (3) and also due to reduced number of BLC-producing macrophages in the young and aged BWF1 mice (Fig. 5B). peritoneum in aged BWF1 mice. In agreement with previous find- Decreased cell number of BLC-producing peritoneal ings (14), the peritoneal macrophage was a major cell source for macrophages in aged BWF mice 1 BLC in the peritoneal cavity both in young and aged BWF1 mice. Although the number of total peritoneal cells and B1 cells in the We found that the number of macrophages in the peritoneal cavity peritoneal cavity was significantly increased in aged BWF1 mice was markedly decreased in aged BWF1 mice compared with those compared with those in young BWF1 mice (Fig. 6, A and B), the in young BWF1 mice, whereas the number of B1 cells in the peri- number of peritoneal macrophages in aged BWF1 mice was mark- toneal cavity was increased in aged BWF1 mice. Increased B1 cells edly reduced compared with those in young BWF1 mice (Fig. 6C). and decreased macrophages in the peritoneal cavity of aged BWF1 3632 ABERRANT B1 CELL TRAFFICKING IN MURINE LUPUS

FIGURE 4. Constitutive and high expression of BLC by peritoneal macrophages. A, RT-PCR (a) and quantitative real-time PCR (b) analysis on peritoneal cells (PerC) obtained from young or aged BWF1 mice. Total cellular RNAs ob- tained from young or aged BWF1 mice were used for RT-PCR and quantitative real-time PCR analysis as described in Materials and Methods. A representative result of three exper- iments is presented. B, Identification of BLC- expressing cells in the peritoneal cavity. Perito- neal cells from young BWF1 mice were stained with FITC-labeled anti-CD11b Ab and PE-la- Downloaded from beled anti-CD11c Ab and were sorted on Epics Elite cell sorter. RNA from each fraction was analyzed by RT-PCR for BLC expression. http://www.jimmunol.org/

mice are possibly due to enhanced IL-10 expression in the perito- growth factor for B1 cells and as a negative regulator for macro- neal cells in aged BWF1 mice (data not shown), because it is re- phages (15). Enhanced B1 cell generation by increased expression

ported that IL-10 produced by B1 cells plays a role as an autocrine of IL-10 in the peritoneal cavity may overwhelm decreased B1 cell by guest on September 26, 2021

FIGURE 5. Characterization of CD11bhighCD11clow cells in the peritoneal cavity. A, Sorted CD11bhighCD11clow cells were stained with Giemsa staining. Peritoneal cells from young BWF1 mice were stained with FITC-labeled anti-CD11b Ab, PE-labeled anti-CD11c Ab, and biotinylated Ab, as indicated on each histogram, followed by allophycocyanin-streptavidin. B, CD11bhighCD11clowF4/80ϩ cells were sorted and cytospun on a slide glass. Cells were stained with goat anti-BLC pAb followed by biotinylated rabbit anti-goat IgG and HRP-labeled streptavidin as described in Materials and Methods. The Journal of Immunology 3633

FIGURE 6. The number of peritoneal macro- phages was decreased in aged BWF1 mice. The absolute cell number of peritoneal cells (A), B1 cells (B), and CD11bhighCD11clowF4/80ϩ perito- neal macrophages (C) in young (n ϭ 11) and ϭ aged (n 4) BWF1 mice. Statistical analysis was ,ءء ;p Ͻ 0.01 ,ء .performed by Student’s t test p Ͻ 0.001.

homing to the peritoneal cavity. In contrast, this situation in the BWF1 mice (24). IgG anti-DNA Ab are a serologic hallmark for peritoneal cavity in aged BWF1 mice would facilitate B1 cell exit SLE and important mediators for kidney damage (25, 26). Fur- from the peritoneal cavity. thermore, accumulating data suggest that B1 cells play an impor-

Coinjection of B1 and B2 cells into aged BWF1 mice resulted in tant role in mucosal immunity in the gut (13). Macpherson et al. preferential recruitment of B1 cells in the target organs such as the (12) suggested that IgA produced by B1 cells in the gut contributed Downloaded from thymus, kidney, and lung, whereas a similar number of B1 and B2 to the defense against direct penetration of commensal bacteria cells was recruited to the spleen. These results are also well ex- into the systemic circulation. Therefore, it is tempting to speculate plained by our previous findings that B1 cells are more efficiently that aberrant B1 cell trafficking in aged BWF1 mice may result in chemoattracted toward BLC than B2 cells (3). Because BLC ex- penetration of pathogens into systemic circulation and induction of pression in is reported in chronically ac- vigorous IgG anti-DNA Ab production that cross-reacts with

tivated lymphoid tissues such as and Peyer’s patches (16, mammalian DNA. http://www.jimmunol.org/ 17), BLC expression in high endothelial venules in inflammatory It has been a matter of debate whether B1 cells are involved in cellular infiltrates is also possibly involved in preferential B1 cell IgG autoantibody production in aged BWF1 mice because of the recruitment to the target organs. Chemotaxis assay showed that B1 lack of firm evidence for IgG class switching in vivo. Recent study cells migrated toward stromal cell-derived factor 1 (SDF-1) and has suggested that CXCR5ϩCD4ϩ T cells designated as follicular secondary lymphoid tissue chemokine (SLC) in addition to BLC, Th cells enhanced IgG and IgA response in the absence of APCs and that only BLC showed preferential chemotactic activity to B1 (16, 27). Preliminary experiments showed that the number of cells (data not shown). It has been reported recently that SDF-1 CXCR5ϩCD4ϩ T cells with the phenotype of follicular Th cells ϩ expression is enhanced in the glomeruli in the kidney in aged was increased in aged BWF1 mice, and that CD4 T cells obtained by guest on September 26, 2021 BWF1 mice (18). However, RT-PCR analysis showed no increase from aged BWF1 mice enhanced IgG Ab production by B1 cells in the level of SDF-1 expression in the target organs between (data not shown). We are now extensively investigating the role of ϩ ϩ young and aged BWF1 mice (data not shown). SLC is another CXCR5 CD4 T cells in IgG autoantibody production by B1 chemokine that may possibly affect B1 cell homing to the perito- cells. neal cavity. However, little SLC expression was detected in peri- Collectively, we have demonstrated that B1 cells fail to home to toneal cells both in young and aged BWF1 mice (data not shown). the peritoneal cavity and are preferentially recruited to the target Constitutive SLC expression was observed in the target organs organs in aged BWF1 mice developing murine lupus. These find- such as the kidney as well as in lymphoid tissues in both young and ings would provide a new insight into the pathological significance aged BWF1 mice. These results suggest that preferential recruit- of B1 cells in the development of SLE and a novel way for the ment of B1 cells in the target organs such as the kidney and lung regulation of murine lupus by targeting B1 cell trafficking. compared with B2 cells is most likely attributed to high and ec- topic expression of BLC in these organs. It is unlikely that mi- Acknowledgments grated cells in the target organs were contaminated macrophages We are very grateful to Dr. Kaoru Hamada for helpful comments. or other cell type, because the purity of B1 cells (CD5ϩB220ϩF4/ 80Ϫ) was Ͼ95%. It was also confirmed that all CD5ϩB220ϩF4/ 80Ϫ B cells were CD19 positive. We, therefore, favor the idea that References ectopic high expression of BLC in the target organs and decreased 1. Theofilopoulos, A. N. 1985. Murine models of systemic lupus erythematosus. Adv. Immunol. 37:269. number of BLC-producing macrophages in the peritoneal cavity 2. Kotzin, B. L. 1996. Systemic lupus erythematosus. Cell 85:303. contribute to aberrant B1 cell trafficking in aged BWF1 mice. 3. Ishikawa, S., T. Sato, M. Abe, S. Nagai, N. Onai, H. Yoneyama, Y. Zhang, Although we do not know the physiological significance of B1 T. Suzuki, S. Hashimoto, T. Shirai, et al. 2001. 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