Redundant Role of CCL25/TECK and CCL28/MEC in IgA + Plasmablast Recruitment to the Intestinal Lamina Propria After Rotavirus Infection This information is current as of September 27, 2021. Ningguo Feng, María C. Jaimes, Nicole H. Lazarus, Denise Monak, Caiqui Zhang, Eugene C. Butcher and Harry B. Greenberg J Immunol 2006; 176:5749-5759; ; doi: 10.4049/jimmunol.176.10.5749 Downloaded from http://www.jimmunol.org/content/176/10/5749

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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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Redundant Role of Chemokines CCL25/TECK and CCL28/ MEC in IgA؉ Plasmablast Recruitment to the Intestinal Lamina Propria After Rotavirus Infection1

Ningguo Feng,2*† Marı´a C. Jaimes,2*† Nicole H. Lazarus,§ Denise Monak,‡ Caiqui Zhang,*† Eugene C. Butcher,§ and Harry B. Greenberg3*†‡

Rotaviruses (RV) are the most important cause of severe childhood diarrheal disease. In suckling mice, infection with RV results ,in an increase in total and virus-specific IgA؉ plasmablasts in the small intestinal lamina propria (LP) soon after infection providing a unique opportunity to study the mechanism of IgA؉ cell recruitment into the small intestine. In this study, we show that the increase in total and RV-specific IgA؉ plasmablasts in the LP after RV infection can be blocked by the combined administration of Abs against chemokines CCL25 and CCL28, but not by the administration of either Ab alone. RV infection in Downloaded from CCR9 knockout mice still induced a significant accumulation of IgA؉ plasmablasts in the LP, which was blocked by the addition of anti-CCL28 Ab, confirming the synergistic role of CCL25 and CCL28. The absence of IgA؉ plasmablast accumulation in LP following combined anti- treatment was not due to changes in proliferation or apoptosis in these cells. We also found ؉ ␣ that coadministration of anti-CCL25 and anti-CCL28 Abs with the addition of anti- 4 Ab did not further inhibit IgA cell ؉ ␤ ␣ accumulation in the LP and that the CCL25 receptor, CCR9, was coexpressed with the intestinal homing receptor 4 7 on IgA plasmablasts. Finally, we showed that RV infection was associated with an increase in both CCL25 and CCL28 in the small http://www.jimmunol.org/ ␣ ␤ intestine. Hence, our findings indicate that 4 7 along with either CCR9 or CCR10 are sufficient for mediating the intestinal .migration of IgA؉ plasmablasts during RV infection. The Journal of Immunology, 2006, 176: 5749–5759

ecretory IgA in the intestinal lumen constitutes the first patches (PP) and/or mesenteric lymph nodes (MLN), where affin- line of defense against a vast array of Ags (both food and ity maturation and isotype switching from IgM to IgA occurs (1). pathogens) that enter through the gut. IgA is produced by Most of these fully differentiated B cells, which are predominantly S 4 ϩ plasma cells (PC) in the intestinal lamina propria (LP) as a IgA plasmablasts, exit PP and MLN via the lymphatics and tho- dimeric molecule associated with J-chain. This form of IgA binds racic duct, enter the blood circulation, and then specifically traffic by guest on September 27, 2021 to the polymeric IgR expressed basolaterally on intestinal epithe- to the intestinal LP, where they can subsequently perform effector lial cells. The ligand-polymeric IgR undergoes transcytosis to the functions. apical cell surface for luminal release by cleavage of the extracel- The tissue-specific recruitment of lymphocytes is a tightly reg- lular receptor domain, which is incorporated into the quaternary ulated multistep process that involves the interaction between structure of secretory IgA as so-called bound secretory component homing (integrins and selectins) and chemokine receptors on the (1, 2). lymphocyte surface and cell adhesion molecules and chemokines B cells activated in the intestine undergo expansion and differ- expressed on the vascular endothelium of specific target tissues entiation predominantly in the germinal centers of the Peyer’s (3). The requirements for multiple protein-protein interactions dur- ing lymphocyte homing ensure a high degree of specificity in tar-

*Department of Medicine Stanford University School of Medicine, Stanford, CA geting cells. † ␣ ␤ 94305; Veterans Affairs (VA) Palo Alto Health Care System, Palo Alto, CA 94304; There is considerable evidence that the integrin 4 7 plays a ‡Department of Microbiology and Immunology, Stanford University School of Med- § critical role in lymphocyte homing to the gut. For instance, in both icine, Stanford, CA 94305; and Laboratory of Immunology and Vascular Biology, ϩ Department of Pathology, Stanford University School of Medicine, Stanford, CA humans and mice, gut lymphocytes including IgA PC, express 94305 ␣ ␤ high levels of 4 7 (4, 5) and interact with mucosal addressin Received for publication September 19, 2005. Accepted for publication February cell-adhesion molecule 1 (MADCAM-1), which is constitutively 20, 2006. expressed in intestinal mucosa (6, 7). In addition, both oral and The costs of publication of this article were defrayed in part by the payment of page rectal mucosal immunizations induced B cells expressing high lev- charges. This article must therefore be hereby marked advertisement in accordance ␣ ␤ with 18 U.S.C. Section 1734 solely to indicate this fact. els of 4 7 as compared with cells elicited after systemic immu- 1 ␤ This work was supported by National Institutes of Health Grant AI21362-20, a VA nization (8). Furthermore, 7 knockout (KO) mice have reduced merit review grant, Digestive Disease Center Grant DK56339, and National Institutes numbers of IgAϩ PC in the gut (9). of Health Grant AI47822. Among chemokines, CCL25/TECK (thymus-expressed chemo- 2 N.F. and M.C.J. contributed equally to this work. kine) and CCL28/MEC (mucosae-associated epithelial chemo- 3 Address correspondence and reprint requests to Dr. Harry B. Greenberg, VA Palo kine), and their respective receptors CCR9 and CCR10, are in an Alto Health Care System, 3801 Miranda Avenue, MC154C, Palo Alto, CA 94304. E-mail address: [email protected] anatomic position to play an important role in B cell homing to the 4 Abbreviations used in this paper used in this paper: PC, plasma cell; LP, lamina gut (10–13). CCL25 is almost exclusively expressed in the small propria; PP, Peyer’s patch; MLN, mesenteric lymph nodes; MADCAM-1, mucosal intestine (SI) and is expressed at high levels by crypt epithelial and addressin cell-adhesion molecule 1; KO, knockout; SI, small intestine; CT, cholera endothelial cells. CCL25 attracts B cells that express CCR9 and toxin; RV, rotavirus; WT, wild type; GFP-VLP, GFP-labeled RV-like particle; DPI, ϩ days postinfection; SP, ; 7-AAD, 7-aminoactinomycin D; int, intermediate. IgA on their surface. Mouse IgA cells from PP and MLN were

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 5750 IgAϩ PLASMABLAST HOMING TO LP AFTER RV INFECTION highly responsive to CCL25 in contrast to cells from the upper Mice and infections respiratory tract, indicating that the expression of CCR9 is induced ϩ BALB/c mice were originally purchased from The Jackson Laboratory and during the development of IgA plasmablasts/PC in the lymphoid propagated and maintained at the Palo Alto Veterans Affairs Health Care ϩ tissues of the intestinal mucosa (10, 13, 14). In addition, CCR9 System Veterinarian Medicine Unit (Palo Alto, CA). CCR9-deficient B cells are rare in the colon and absent in other mucosal tissues (CCR9 KO) mice on C57BL/6 and 129 hybrid background and the hybrid (10). In contrast, the expression of CCL28 is higher in the colon control mice were originally obtained from Dr. P. Love (National Institutes of Health, Bethesda, MD) and propagated and maintained in the same than in the SI (15, 16), and it has been suggested that the differ- facility. All studies were approved by the Stanford Institutional Animal ential expression of CCL25 and CCL28 along the intestine helps to Care Committee. ϫ 3 compartmentalize the intestinal immune response (10). CCL28 is Five-day-old suckling mice were orally inoculated with 3 10 ID50 of also found in a variety of mucosal tissues including the salivary ECw or, in some experiments, with 2000 CFU of the auxotrophic aroA mutant strain SL7207 (Salmonella typhimurium 2337-65 derivative hisG46 gland, mammary gland, small and large intestine, tonsil, appendix, DEL407[aroA::Tn1(Tcs)]) (B.A.D. Stocker, Stanford University, Stanford, and trachea, and it has been previously shown that CCR10 is CA). Bacterial strains were routinely grown at 37°C in Luria-Bertani broth ϩ highly expressed on IgA PC (16, 17). Because of the variety of or agar (Difco). At indicated DPI, mice were sacrificed, and SI, MLN, and tissues in which CCL28 is expressed, it was suggested that the spleen (SP) were collected. Lymphocytes from intestinal LP, MLN, and SP CCL28/CCR10 interaction may function as a unifying mucosal were isolated as described previously (26). Because PP in suckling mice ϩ intestines were difficult to isolate, whole SI were digested to isolate lym- homing mechanism for IgA plasmablasts/PC (17). However, it phocytes. Isolated lymphocytes were used for flow cytometry analysis (see has recently been shown that nasal-associated lymphoid tissue- below). Additional SI were collected for immune fluorescent staining (see derived human B cells, which also express CCR10, have limited below).

␣ ␤ Downloaded from access to the SI because they express low levels of CCR9 and 4 7 ␣ ␤ (18). Hence, the coexpression of homing and chemokine receptors In vivo treatment with Abs to CCL25, CCL28, and integrin 4 7 contributes greatly to the compartmentalization of the mucosal im- Five-day-old BALB/c, CCR9 KO, or WT control mice were infected with mune system, with important implications for the development of ECw and injected i.p. with rat mAbs against CCL25 (IgG2B, clone 89818) effective vaccines (19). and CCL28 (IgG2B, clone 134306) (R&D Systems) either individually or Pabst et al. (13) demonstrated that CCR9-deficient mice had in combination, or purified rat IgG2B as an isotype control Ab (R&D ϩ Systems) at days 0, 2, 4, and 6 postinfection. The first dose of Ab was 25 decreased numbers of IgA PC in the LP and an impaired mucosal ␮ ␮ ␣ http://www.jimmunol.org/ g, and the subsequent doses were 50 g. In some experiments, anti- 4 Ab immune response to orally administered OVA. In vivo treatment of (IgG2B, clone PS/2; American Type Culture Collection) (100 ␮g for the cholera toxin (CT)-immunized mice with anti-CCL25 or anti- first dose, and 200 ␮g for the subsequent doses) was injected i.p. either CCL28, resulted in diminished Ag-specific IgA response in the SI alone or in combination with anti-CCL25 and/or anti-CCL28 Abs. Mice (12). Because these previous studies were not performed within the were sacrificed at day 7 postinfection. Lymphocytes from SI, MLN, and SP were collected for FACS analysis, and additional SI were collected for context of a natural intestinal infection, we examined the roles of immunohistologic analysis. Treatment of CCR9 KO mice consisted of anti- ␣ ␤ ␣ CCL25 and CCL28, along with their interaction with 4 7,ingut CCL28 and/or anti- 4 following the same schedule and doses as for homing of IgAϩ plasmablasts/PC during murine rotavirus (RV) BALB/c mice. infection. RV causes severe diarrhea in young children (20) and many Immunofluorescent staining and confocal microscopy of by guest on September 27, 2021 mammalian and avian species including infant mice. The virus intestinal tissues replicates to high titers in the mature epithelial cells of the SI and At different DPI (as indicated in Fig. 2), 5-day-old suckling mice were induces a strong intestinal IgA response, which is important for sacrificed, and SI were collected. At each time point at least four mice were protection against reinfection (21). In mice, RV-specific B cells are examined. To avoid potential bias between litters, mice were collected responsive to CCL25 and CCL28 (10, 22), and B cells lacking ␤ from different litters at each time point. Collected intestinal specimens 7 were placed in a horizontal position and frozen in Tissue-Tek optimal are defective in mediating clearance of chronic infection in im- cutting temperature compound (Sakura Finetek). Sections (7 ␮m) were mune-deficient mice (9). Acute RV infection also induces signif- then fixed in methanol or acetone for 10 min and washed in PBS. Before icant polyclonal activation of B cells (23). We recently observed staining, sections were treated with 2 mM CuCl2 in 10 mM Tris buffer (pH that RV infection in suckling mice speeds up the accumulation of 7.5) for1htoreduce autofluorescence (27). After washing three times in ϩ PBS, sections were blocked with 5% normal goat serum and stained with IgA PC in the intestinal LP when compared with noninfected GFP-VLP (10 ␮g/test) for1hatroom temperature. Sections were then controls. Hence, this model system provides a useful opportunity washed and stained with Texas Red-labeled goat anti-mouse IgA (Southern to examine the role of several trafficking molecules (CCL25, Biotechnology Associates) for 45 min at room temperature. Finally, after ␣ ␤ washing, the slides were mounted using aqua-poly/mount mounting me- CCL28, and 4 7) in the seeding of LP of suckling mice with IgAϩ PC after an enteric viral infection. dium (Polysciences). Of note, we and others have previously demonstrated the specificity of the staining using GFP-VLPs to identify RV-specific B cells by showing that there is insignificant binding of GFP-VLPs to B cells in noninfected Materials and Methods mice and that GFP-VLP binding can be specifically blocked using unla- Virus and virus-like particles beled VLPs or purified virus (28, 29). It is also important to note that after RV infection, the predominant Ab response is directed against the two viral The virulent wild-type (WT) murine RV strain, ECw, was used to infect proteins (VP2 and VP6), which are the components of the GFP-VLPs (30). mice. This virus causes diarrhea in 100% of 5-day-old suckling mice, Confocal microscopic images were obtained using the software Laser- which lasts for ϳ7–9 days (24). The virus was propagated in suckling sharp 2.0 and a MRC-1024 laser scanning confocal imaging system (Bio- mice, used as a clarified intestinal homogenate, and its ID50 was deter- Rad) connected to a Nikon Eclipse TE300 inverted microscope (Nikon). mined as described previously (24). IgA-secreting cells appeared as large IgAϩ cells detectable in LP (Fig. 1, GFP-labeled RV-like-particles (GFP-VLP) were generated by coinfect- A, C, and D) and PP parafollicular region (data not shown) and were B220 ing Spodoptera frugiperda 9 (Sf9) cells with two recombinant baculovi- negative (data not shown). For each section, 5–10 images were collected ruses at a multiplicity of infection Ն5 PFU/cell, as described previously after scanning the entire section under the microscope. We first identified (25). One baculovirus expressed RF (bovine RV) VP6 and the other a areas with IgAϩ cells and adjusted fields to maximize the presence of IgAϩ fusion protein consisting of GFP fused to the N terminus of RF VP2 de- cells and acquired both red and green images using sequential acquisition. leted in the first 92 aas. Infected cultures were collected 5–7 days postin- If there were no IgAϩ cells detected in a section, 5–10 random images were fection (DPI) and purified by density gradient centrifugation in CsCl. The acquired. concentration of protein in the purified GFP-VLP suspension was esti- Total IgAϩ cells (in red) and GFP-VLPϩ/IgAϩ cells (in yellow) were mated by the Bradford method, using BSA as a standard. counted, and data were expressed as number of IgAϩ cells per field and The Journal of Immunology 5751

Quantification of IgA production in SI by ex vivo intestinal fragment culture Ex vivo intestinal fragment culture for measuring IgA secretion from SI was performed as described previously (31). Five-day-old CCR9 KO suck- ling mice were orally infected with ECw and treated with anti-CCL28 or isotype control as described above. At 7 DPI, mice were sacrificed, and SI sections of ϳ0.5 cm long were collected and individually weighted. The intestinal sections were opened longitudinally, washed, and cultured in

95% O2 and 5% CO2 chamber. After 3 days of culture, the media were collected, and total and RV-specific IgA concentration were determined by ELISA as described previously (32). IgA production by intestinal frag- ments was expressed as micrograms of IgA per 100 mg of intestine weight. Measurement of intestinal CCL25 and CCL28 by ELISA ELISA kits for the detection of CCL25 and CCL28 were purchased from R&D Systems. SI from RV-infected or uninfected suckling mice were collected at 1 and 3 DPI and immediately frozen at Ϫ70°C. Before assay, 250 ␮l of PBS with protease inhibitor mixture including 4-(2-aminoethyl) benzenesulfonyl fluoride HCl (1 mM), E-64 (10 ␮M), EST (40 ␮M), leu- peptin hemisulfate (10 ␮M), pepstatin A (1 ␮M), N␣-tosyl-lys chloro- methyl ketone, HCl (40 ␮M), and N␣-tosyl-phe chloromethyl ketone (40 ␮M) (Calbiochem) was added to each sample and homogenized. The ho- Downloaded from mogenates were centrifuged at 10,000 rpm for 10 min. Supernatants were serially diluted before addition to ELISA plates. Mouse CCL25 and CCL28 detection was performed according to the manufacturer’s instructions. Pro- tein concentration for each sample was determined with a BCA protein assay kit (Pierce). The amount of chemokine in each intestinal sample was expressed as picograms of CCL25 or CCL28 per milligrams of tissue

protein. http://www.jimmunol.org/ Statistical analysis

FIGURE 1. Confocal microscopy analysis of intestinal LP sections Statistical analyses were performed with StatView (SAS Institute). Non- from BALB/c-infected suckling mouse 7 DPI (A), noninfected age- parametric Mann-Whitney U test (two-tailed) was used to analyze immu- matched control (12 days old) (B), BALB/c-infected suckling mouse 21 nofluorescent staining data. Two-way ANOVA was used to evaluate the statistical significance of treatment factors such as RV infection, anti- DPI (C), and noninfected age-matched control (26 days old) (D). Tissues chemokine Ab treatment, Ab production by ex vivo intestinal fragment in A and B were stained with anti-IgA Texas Red and in C and D with culture, and quantification of chemokines by ELISA. Because it was nec- anti-IgA Texas Red and GFP-VLPs and examined under ϫ40 magnifica- ϩ ϩ ϩ essary to combine several independent experiments with variable baseline tion. The arrow shows IgA PC (A) and GFP-VLP /IgA cells (C). results, we also included an additional factor that identified individual ex- by guest on September 27, 2021 periment in the ANOVA model. Therefore, the p values for treatment ef- fects reported in Results were adjusted for interexperiment variations. In addition, the data were log transformed for the chemokine’s quantification data by ELISA to normalize the data distribution. percentage of GFP-VLPϩ cells per total IgAϩ cells, respectively. For anal- ysis, the average number of IgAϩ cells per field for each mouse was cal- Results culated, before determining the mean for each group at individual time RV infection speeds up the accumulation of total and RV- points. The data presented in graphs were obtained from sections of jeju- ϩ num. Of note, total and RV-specific IgA responses were similar in ileum specific IgA PC in intestinal LP in suckling mice (data not shown). Five-day-old BALB/c mice were orally infected with WT RV ECw. At various DPI, mice were sacrificed and the SI isolated and Flow cytometry analysis frozen in Tissue-Tek optimal cutting temperature compound. The frozen tissue sections were stained with Texas Red-labeled goat Isolated SI LP, MLN, and SP lymphocytes were stained with different anti-IgA and GFP-VLP to detect IgAϩ B cells and RV-specific B combinations of GFP-VLP and the following anti-mouse Abs (all obtained cells, respectively. IgAϩ and GFP-VLPϩ cells were observed by from BD Pharmingen unless specified): IgA FITC (Southern Biotechnol- ϩ ␣ ␤ confocal microscopy. The subset of IgA cells that we identified ogy Associates); B220 PerCP (RA3-6B2); 4 7 PE or allophycocyanin Ϫ (DATK32.2, allophycocyanin-labeled by Chromoprobe); CCR9 allophy- were large, B220 and primarily located in the LP and parafol- cocyanin (242503; R&D Systems); CD138 PE (281-2); and a dump PE or licular region of PP (data not shown), indicating these cells were biotin, which included Thy 1.2 (53-2.1), CD-11c (HL3), CD-11b (M1/7), IgAϩ plasmablasts and PC. Fig. 1 shows IgAϩ cells in the intes- and IgD (11–26). After staining, cells were washed once and fixed with 1% tinal LP of infected and noninfected mice at 12 days of age (equiv- paraformaldehyde (Electron Microscopy Sciences). At least 500,000 cells ϩ were acquired and four- or five-color flow cytometry was performed using alent to 7 DPI). IgA cells could be detected in the LP of RV- a FACSCalibur or a LSRII (BD Biosciences), and data analysis was per- infected suckling mice starting 5 DPI (10 days after birth; Fig. 1A), formed with CellQuest Pro (BD Biosciences). Dead cells were excluded by and the numbers increased rapidly during the course of infection forward and side scatter gating. (Fig. 2). In contrast, IgAϩ cells were virtually nondetectable in the To measure lymphocyte proliferation in vivo, infected suckling BALB/c ␮ LP of noninfected mice during the first 11 days after birth (equiv- mice were injected i.p. with 100 g of BrdU (BD Pharmingen) at 6 DPI. ϩ Mice were sacrificed 16–18 h after BrdU administration, and lymphocytes alent to 6 DPI; Fig. 1B), and the accumulation of IgA cells oc- from LP, MLN, and SP were isolated. BrdU staining was performed using curred slowly during the first 2 wk after birth (equivalent to 9 DPI) the BrdU Flow Kit (BD Pharmingen) following the manufacturer’s instruc- (Fig. 2). The speed of accumulation in uninfected mice increased tions. To compare the levels of lymphocyte apoptosis after anti-chemokine thereafter and reached the levels comparable to the infected mice Ab treatment, LP and MLN lymphocytes from anti-chemokine or isotype around 3 wk of age (equivalent to 16 DPI) (Fig. 2). The amounts control Ab-treated mice at 7 DPI were collected and stained using the ϩ annexin V-PE apoptosis detection kit I (BD Pharmingen) following the of IgA cells accumulating in the LP (expressed as number of cells manufacturer’s instructions. per field) were significantly greater in the infected group between 5752 IgAϩ PLASMABLAST HOMING TO LP AFTER RV INFECTION

RV-specific IgAϩ cells, identified by GFP-VLP and IgA dou- ble-positive staining, could be detected in infected mice starting at 6 DPI, and the highest numbers were detected at 21 DPI (Fig. 1C). However, the proportion of RV-specific IgAϩ cells of total IgAϩ cells in LP of infected mice was low, especially during the first 10 days of infection (6% or less; Fig. 2). Therefore, most of the IgAϩ cells in LP during RV infection were non-RV specific, and cells were presumably generated as the result of polyclonal B cell activation after infection, as previously shown in adult mice (23). The rapid accumulation of IgAϩ cells in the small intestinal LP of RV-infected suckling mice as detected by immunohisto- FIGURE 2. Number of IgAϩ cells and VLPϩ, RV-specific cells in the logic examination was confirmed by FACS analysis. Mice were LP of infected suckling mice as a function of time. Intestines from infected and noninfected suckling mice were collected at indicated time points and sacrificed at 7 DPI, and lymphocytes from LP, MLN, and SP analyzed for the presence of IgAϩ and IgAϩ/VLPϩ cells. For each section, were isolated. For analysis, cells were gated on large lympho- 5–10 images were collected. Total IgAϩ cells and GFP-VLPϩ/IgAϩ cells cytes, dump (Thy 1.2, CD11c, CD11b, IgD) negative, B220 ϩ were counted, and data were expressed as number of IgA cells per field intermediate or low and percentages of IgAϩ cells were deter- and percentage of GFP-VLPϩ cells per total IgAϩ cells. The average of the ϩ ϩ mined (Fig. 3A). The mean percentage of IgA plasmablasts in Downloaded from total IgA cells for each mouse was calculated and used to compute the the LP of RV-infected suckling mice at 7 DPI was ϳ30 times group mean and SEM, presented in the graph, for each time point. There were significant statistical differences in the number of total IgAϩ cells higher than in age-matched noninfected mice (Fig. 3, A and B) ϭ ϩ between infected and noninfected groups from days 5 to 11 postinfection ( p 0.006). The number of IgA plasmablasts was also sig- (p Ͻ 0.05, Mann-Whitney U). nificantly higher in the MLN (5.8 times) of infected mice as compared with the control group (Fig. 3B)(p ϭ 0.018). The ϩ difference in percentages of IgA plasmablasts between in- http://www.jimmunol.org/ day 5 and 11 postinfection (day 10–19 after birth) than in the fected and noninfected mice in LP and MLN remained signif- Ͻ icant at 10 DPI (data not shown). In contrast, the percentages of noninfected age-matched controls ( p 0.05, Mann-Whitney U ϩ test). IgA plasmablasts in the SP of both infected and noninfected by guest on September 27, 2021 FIGURE 3. Flow cytometry analysis of IgAϩ plasmablast accumulation in the intestinal LP after RV infection. A, Lym- phocytes were isolated from the intesti- nal LP of infected (7 DPI) and age- matched noninfected BALB/c and CCR9KO suckling mice and stained with mAbs against Thy1.2/IgD/CD11c/ ␣ ␤ CD11b (dump), B220, IgA, and 4 7. For analysis, cells were gated on large, dumpϪ, B220int/low lymphocytes. Based on this subpopulation, dot plots were cre- ated showing expression of IgA and ␣ ␤ Ϫ 4 7. The percentages of large dump IgAϩ cells expressing or not expressing ␣ ␤ 4 7 are indicated in the right quadrants. B, Percentage of large, dumpϪ, B220int/low IgAϩ plasmablasts in the intestinal LP and MLN from noninfected and infected BALB/c and CCR9 KO suckling mice (7 DPI). Frequencies of cells expressing IgA were obtained as described in A. Data are summarized from three inde- pendent experiments with two to four ,ء .mice per group for each experiment Significant differences between infected and noninfected mice (p ϭ 0.006 in the LP, and p ϭ 0.018 in MLN). C, Coex- ␣ ␤ pression of CCR9 and 4 7 in MLN and intestinal LP large, dumpϪ, B220int/low IgAϩ, and GFP-VLPϩ lymphocytes. The Journal of Immunology 5753 groups were very low (ϳ0.02–0.03%) and were not signifi- cantly different between the 2 groups (data not shown) ( p Ͼ 0.05). It is important to note that when we examined the LP by FACS analysis, we were unable to dissect out PP and isolated lymphoid follicles. However, the results of the FACS analysis are highly comparable to the results obtained by histologic examination (Figs. 2, and 3, A and B), where we were able to reliably discriminate between PP and the LP, supporting the proposition that the FACS analysis is primarily measuring cells in the LP. The striking dif- ferences in IgAϩ cell numbers in the intestinal LP between RV- infected and noninfected suckling mice at early time points postin- fection provided a unique opportunity to study the role of homing and chemokine receptors and their ligands in the migration of FIGURE 4. Effect of Salmonella infection on IgAϩ plasmablast/PC re- IgAϩ plasmablasts/PC into the LP during a mucosal viral cruitment into the intestinal LP and MLN. Lymphocytes were isolated from infection. the intestinal LP and MLN of Salmonella-infected (10 DPI, right side dot plots) and age-matched noninfected (left side dot plots) BALB/c mice. ϩ Phenotype of RV-specific and IgA B cells elicited during acute Cells were stained and analyzed as described in Fig. 3A. Representative dot plots showing the percentage of ␣ ␤ ϩ and ␣ ␤ Ϫ IgAϩ in the MLN (up- infection 4 7 4 7 Downloaded from per row) and intestinal LP (lower row) of two independent experiments. To better characterize the large lymphocytes, dump negative, B220 intermediate, GFP-VLPϩ,orIgAϩ cells elicited during acute RV infection in the MLN and the intestinal LP, five-color presence of IgAϩ plasmablasts in LP, MLN, and SP was examined staining for FACS analysis was performed. As shown in Fig. at 7 DPI by FACS (Fig. 5, A and B). When anti-CCL25 and CCL28 ϩ ␣ ␤ ϩ ϩ 3C, in the MLN, 94% of the IgA cells were 4 7 , and among were used together, the numbers of IgA PC in LP were reduced those 75% coexpressed CCR9. Interestingly, in the MLN, a by 62 Ϯ 4.2% (mean Ϯ SEM) as compared with control mice (Fig. http://www.jimmunol.org/ ϩ ␣ ␤ Ͻ lower percentage of the GFP-VLP cells expressed 4 7 5, A and B, and Table I) ( p 0.0001). Interestingly, when the Abs (50%), but similar to the total IgAϩ population, of those, 72% were used individually, there was a reduction trend in the number coexpressed CCR9. Similarly, in the LP, a high percentage of of IgAϩ PC in the LP compared with the isotype control-treated ϩ ␣ ␤ Ϯ Ϯ IgA cells expressed 4 7 (80%), but this percentage was only mice (11 4.4 and 19 7.1% for anti-CCL25 and anti-CCL28, 38% in the RV-specific cells. Moreover, the expression of respectively) that was not statistically significant (Fig. 5B)(p Ͼ CCR9 appeared to be down-regulated in the intestinal LP for 0.05). In addition, we observed that the reduction, when using the both IgAϩ and VLPϩ subsets, because only 22 and 36% of the combination of anti-CCL25 and anti-CCL28, was higher in the ϩ ␣ ␤ ϩ ϩ Ϯ cells expressed this receptor, respectively. The VLP popula- 4 7 subset of IgA plasmablasts (71.4 2.7%) than in the ␣ ␤ Ϫ Ϯ by guest on September 27, 2021 tion in both the LP and the MLN had a lower proportion of 4 7 subset (54 5.2%), and this difference was significant ␣ ␤ ϩ ϩ ϭ 4 7 cells than the total IgA population (Fig. 3C). We also ( p 0.01) (Fig. 5A and Table I). looked at the expression of CD138 on total IgAϩ cells (data not The reduction of IgAϩ plasmablasts in the LP after anti- shown) in the MLN and intestinal LP and found 73 and 62% to chemokine treatment was also confirmed by immunofluorescent be CD138ϩ, respectively, suggesting that the majority of the staining of intestines collected at 7 DPI (Fig. 5C). The reduction IgAϩ cells elicited after infection are plasmablasts or PC. between the anti-CCL25/CCL28 and the isotype-treated groups, as ϭ ϩ measured by immunohistology, was 61.4% ( p 0.04), similar to Effect of Salmonella infection in the accumulation of total IgA the reduction measured by FACS. In addition, no apparent differ- cells in the MLN and intestinal LP ences in the structure of germinal centers or number of cells in the To determine whether the increase in the total number of IgAϩ parafollicular region of the PP were observed by histologic exam- cells in the MLN and intestinal LP after RV infection in suckling ination between groups (data not shown), suggesting that the Ab mice was due to a specific effect of RV, we infected suckling mice treatment had little or no histologic impact on the intestinal induc- with an unrelated intestinal pathogen, S. typhimurium. Interest- tive site of the B cell response. ingly, up to 10 days after Salmonella infection, we could not detect The administration of anti-CCL25 and anti-CCL28 not only re- an increase in the percentage of IgAϩ cells in the MLN (Fig. 4). In duced the accumulation of total IgAϩ plasmablasts/PC in the LP the intestinal LP, at 7 DPI there was a slight increase in the number but also the numbers of RV-specific B cells (Fig. 5A and Table I). of IgAϩ cells (1%, data not shown). However, this increase was However, the reduction observed for VLPϩ plasmablasts was not not sustained over time, as it was during RV infection, and by 10 as pronounced as for total IgAϩ plasmablasts/PC (41.2 Ϯ 9vs DPI there was no difference in the percentage of IgAϩ plasma- 62 Ϯ 4.2%, respectively), although still significant ( p ϭ 0.0035). ␣ ␤ ϩ blasts/PC in the LP of Salmonella-infected suckling mice when The 4 7 subset of RV-specific B cells was more efficiently ␣ ␤ Ϫ Ϯ Ϯ ϭ compared with noninfected mice (Fig. 4). We concluded from blocked than the 4 7 subset (68.1 5.2 vs 29.4 10.6%) ( p these experiments that the increase in numbers of total IgAϩ plas- 0.007) (Table I). mablasts/PC seen during acute RV infection was not a nonspecific As a consequence of Ab treatment, the numbers of IgAϩ plas- consequence of all enteric infections in suckling mice. mablasts were increased in the MLN and SP. This effect was con-

ϩ sistent between experiments and considerably higher in the SP Effects of anti-CCL25 and CCL28 in IgA plasmablast than in the MLN, where the numbers of IgAϩ plasmablasts in- accumulation in the LP after RV infection creased by 133 Ϯ 50.9% compared with the isotype control-treated To study the role of chemokines CCL25 (TECK) and CCL28 group ( p ϭ 0.03) (Fig. 5D). Of note, most of the IgAϩ cells de- ϩ ␣ ␤ (MEC) in intestinal LP accumulation of IgA plasmablasts, we tected in the SP of treated mice expressed 4 7. The change in treated suckling mice with anti-CCL25 and anti- CCL28 Abs ei- number of IgAϩ plasmablasts in the MLN was small after Ab ther individually or in combination at 0, 2, 4, and 6 DPI, and the treatment (23 Ϯ 10.5%) and not significant (Fig. 5D)(p Ͼ 0.05). 5754 IgAϩ PLASMABLAST HOMING TO LP AFTER RV INFECTION Downloaded from http://www.jimmunol.org/

FIGURE 5. Effect of treatment with anti-CCL25 and/or anti-CCL28 in RV-infected suckling mice. A, Representative dot plots showing the percentage ␣ ␤ ϩ ␣ ␤ Ϫ ϩ ϩ of 4 7 and 4 7 IgA and GFP-VLP lymphocytes in the MLN and intestinal LP of infected BALB/c mice treated with anti-CCL25 and anti-CCL28 or an isotype control. Analysis was done with the basic gating strategy described in Fig. 3A. B, Percentage of reduction of large, dumpϪ, B220int/low IgAϩ lymphocytes in the intestinal LP of infected BALB/c mice treated with anti-CCL25 and anti-CCL28, anti-CCL25 only, and anti-CCL28 only as compared Significant ,ء .with infected isotype control-treated mice were calculated from three independent experiments with 2–4 mice per group for each experiment differences between the anti-CCL25 and anti-CCL28 treatment vs isotype control Ab treated (p Ͻ 0.05). C, Number of IgAϩ cells detected per field in intestinal sections from BALB/c suckling mice 7 DPI treated with anti-CCL25 and anti-CCL28 or isotype control were calculated from three mice per group ,Significant differences between the anti-CCL25 and anti-CCL28 treatment vs isotype control treatment (p Ͻ 0.05). D ,ء .(see Materials and Methods) by guest on September 27, 2021 Percentage increase of large, dumpϪ, B220int/low IgAϩ plasmablasts in the SP and MLN of infected BALB/c suckling mice treated with anti-CCL25 and anti-CCL28 as compared with isotype control (determined using the gating strategy described in Fig. 3A). The data from three independent experiments Significant difference between the anti-CCL25- and anti-CCL28-treated group vs isotype ,ء .with two to four mice per group for each experiment are shown control group (p ϭ 0.03).

The treatment with anti-chemokine Abs did not alter IgAϩ The anti-chemokine treatment did not modify the levels of repli- plasmablast replication or apoptosis in RV-infected suckling cation of IgAϩ plasmablasts in either LP or MLN ( p Ͼ 0.05) mice (Table II). In a separate set of experiments, we stained cells with To exclude the possibility that the reduction of IgAϩ cells in the annexin V and 7-aminoactinomycin D (7-AAD) to assess the ef- fects of Ab treatment on apoptosis of IgAϩ plasmablasts. The per- LP was caused by an effect of the anti-CCL25 and anti-CCL28 ϩ treatment on cell proliferation, mice were injected with BrdU at 6 centage of IgA cells stained with annexin V alone (measurement DPI to label replicating cells in vivo and sacrificed 16–18 h later.

Table II. Effect of combined anti-CCL25 and anti-CCL28 treatment on Table I. Differential effect of combined anti-CCL25 and anti-CCL28 cell division and apoptosis as measured by BrdU incorporation or treatment in BALB/c mice and anti-CCL28 treatment in CCR9 KO mice annexin staining of LP B cells 7 DPIa ␣ ␤ ϩ ␣ ␤ Ϫ ϩ on the accumulation of 4 7 and 4 7 subsets of IgA plasmablasts a in the LP BrdUϩ Annexinϩ Annexinϩ/7-AADϩ

BALB/c CCR9 KO LP Isotype control 37.7 Ϯ 4.5 26.1 Ϯ 1.1 3.8 Ϯ 0.8 Total IgAϩ VLPsϩ Total IgAϩ VLPsϩ Anti-CCL25 ϩ CCL28 35.6 Ϯ 3.7 27.2 Ϯ 3.4 3.6 Ϯ 0.3 MLN Total 62 Ϯ 4.2b 41.2 Ϯ 9b 71.1 Ϯ 4.6b 36.6 Ϯ 8.0b Isotype control 54.6 Ϯ 2.6 14.9 Ϯ 3.8 16.4 Ϯ 0.3 ␣ ␤ ϩ Ϯ b,c Ϯ b,c Ϯ b Ϯ b 4 7 71.4 2.7 68.1 5.2 75.7 4.2 57.9 17.5 Anti-CCL25 ϩ CCL28 50 Ϯ 11.5 19.9 Ϯ 1.0 11.8 Ϯ 0.5 ␣ ␤ Ϫ Ϯ b Ϯ b Ϯ b Ϯ b 4 7 54 5.2 29.4 10.6 63.8 5.9 34.8 7.0 a Five-day-old mice were infected with RV and treated with Abs against a Five-day-old mice were infected with RV and treated with Abs against CCL25 CCL25 and CCL28. IgAϩ plasmablasts/PC were labeled in vivo with BrdU or and CCL28. Total IgAϩ and VLPϩ cells in LP were detected at 7 DPI by FACS. Data stained with annexin and 7-ADD (see Materials and Methods). Data (mean Ϯ (mean Ϯ SEM) are expressed as percentage of reduction compared to the isotype SEM) are expressed as percentage of BrdUϩ, annexinϩ, or 7-AADϩ per total control-treated mice. Numbers derived from three independent experiments with 2–4 IgAϩ plasmablast/PC in MLN or LP at 7 DPI. There are no statistical differences mice per group for each experiment. between anti-chemokine Ab treatment and isotype control Ab treatment in any b Significant reduction compared to isotype control-treated mice ( p Ͻ 0.05). group. Percentages are derived from two independent experiments with 2–3 mice c ␣ ␤ ϩ ␣ ␤ Ϫ Ͻ Significant difference between 4 7 and 4 7 subsets ( p 0.05). per group. The Journal of Immunology 5755 of an early stage of apoptosis) or costained cells for annexin V and 7-AAD (measurement of dead cells or a late stage of apoptosis) in anti-CCL25/anti-CCL28-treated mice were not different from con- trol mice in either LP or MLN (Table II) ( p Ͼ 0.05). Effects of RV infection and anti-CCL28 treatment on IgAϩ plasmablast accumulation in intestinal LP of CCR9 KO To confirm and better dissect the roles of CCL25 and CCL28 in IgAϩ plasmablasts migration to the intestinal LP, we repeated the experiments previously described in BALB/c pups using CCR9 KO mice. RV infection significantly increased the IgAϩ plasma- blast population in both LP and MLN of infected CCR9 KO mice compared with noninfected mice (Fig. 3, A and B). Moreover, the increase was similar to that seen in infected WT controls and BALB/c suckling mice (data not shown and Fig. 3B). These results indicated that RV infection could effectively speed up the accu- mulation of IgAϩ plasmablasts in the intestinal LP in the absence of CCR9.

However, when CCR9 KO mice were infected and treated with Downloaded from anti-CCL28 Ab, the number of IgAϩ plasmablasts in the LP was reduced by 71.1 Ϯ 4.6% ( p ϭ 0.02) (Table I). The percentage ϩ ␣ ␤ ϩ ϩ ␣ ␤ Ϫ reductions of IgA 4 7 and IgA 4 7 lymphocytes were also significant ( p Ͻ 0.01), but the difference between these two subsets was not significant ( p Ͼ 0.05) (Table I). The blockade of ϩ

RV-specific (VLP ) cells in the LP of CCR9 KO mice by anti- http://www.jimmunol.org/ CCL28 was also significant when compared with isotype control Ab-treated mice ( p ϭ 0.02, 0.04, and 0.02 for VLPϩ, VLPϩ ␣ ␤ ϩ ϩ ␣ ␤ Ϫ 4 7 , and VLP 4 7 , respectively). As a control for the CCR9 KO mice, we treated WT mice with Abs to CCL25 and CCL28 alone or in combination. Only combined anti-CCL25 and anti-CCL28 treatment could effectively reduce the IgAϩ plasma- blasts accumulation in the LP, as was observed for WT BALB/c mice (data not shown). by guest on September 27, 2021 Effect of anti-chemokine treatment on intestinal IgA production To investigate whether, as a consequence of the reduced number of IgAϩ plasmablasts in the LP after anti-chemokine Ab treatment, there would be an impact in the intestinal IgA production, we isolated SI fragments from RV-infected and anti-CCL28-treated CCR9 KO mice at 7 DPI, cultured them in vitro for 3 days, and measured IgA by ELISA in the culture supernatant. The intestinal IgA production, expressed as micrograms of IgA per 100 mg of intestine, was significantly reduced in CCR9 KO mice treated with anti-CCL28 compared with the isotype control Ab-treated mice (71.7% reduction) ( p ϭ 0.01) (Fig. 6A). However, no reduction FIGURE 6. Effect on intestinal IgA of anti-CCL28 and anti-␣ treat- was observed in the amount of RV-specific IgA between groups 4 ment in CCR9 KO mice. A, Effect on intestinal IgA secretion. CCR9 KO (data not shown), which is probably due to the less efficient block- mice were infected and treated with anti-CCL28 or an isotype control, and ing of RV-specific plasmablasts accumulation in the LP after the SI were harvested at 7 DPI. SI fragments were cultured ex vivo for 3 days, anti-chemokine treatment (Table I). Intestinal IgA production was and total IgA was measured in the culture supernatant. Data from two also determined from intestinal fragments of RV-infected BALB/c independent experiments with three mice per group for each experiment, -Significant differ ,ء .mice treated with anti-CCL25 and anti-CCL28 Abs, and results expressed as mean IgA (␮g/100 g tissue) and SEM were comparable to the anti-chemokine-treated CCR9 KO mice ence between the anti-CCL28 treatment vs isotype control treatment (p ϭ ␣ ␤ ϩ (data not shown). 0.01). B, Representative dot plots showing the percentage of 4 7 and ␣ ␤ Ϫ ϩ 4 7 IgA plasmablasts in CCR9 KO mice, 7 DPI treated with isotype ␣ ␤ ␣ ϩ ␣ The interaction between the intestinal homing receptor 4 7 and control, anti-CCL28, anti- 4, or anti-CCL28 4. Analysis was done the chemokines CCL25 and CCL28 in IgAϩ plasmablasts using the same gating strategy described in Fig. 3A. C, Mean and SEM of Ϫ ϩ accumulation in LP the percentage of reduction of large, dump , B220int/low IgA plasmablasts in the LP of CCR9-infected mice treated with anti-CCL28 and anti-CCL28 ␣ ␤ It has been shown that 4 7 is crucial for lymphocyte homing to ϩ ␣ anti- 4 compared with isotype control-treated mice. Data from two in- the gut. We investigated whether combined anti-␣4␤7 and anti- dependent experiments with four mice per group for each experiment. chemokines CCL25 and CCL28 treatment would result in a more effective blockade of IgAϩ plasmablast accumulation in the LP after RV infection, compared with anti-chemokine treatment reductions of IgAϩ plasmablasts in the LP were similar among ␣ alone. For this purpose, CCR9 KO mice were infected and treated anti- 4 and anti-CCL28-treated mice (Fig. 6B), and, interestingly, ␣ ␣ ␣ with anti- 4, anti-CCL28, or anti- 4 plus CCL28. The percentage we observed that the concomitant administration of anti- 4 and 5756 IgAϩ PLASMABLAST HOMING TO LP AFTER RV INFECTION

␣ Ͻ anti-CCL28 did not enhance blockade over anti- 4 alone (Fig. 6, B the SI of RV-infected mice both at 3 and 5 DPI ( p 0.05). In and C). These results suggest that the specificity of IgAϩ plasma- addition, CCL28 was also significantly increased at 3 DPI ( p ϭ blasts for intestinal gut homing is determined by the coexpression 0.007). These results suggested that RV infection in suckling mice ␣ ␤ of the homing receptor 4 7 and either CCR9 or CCR10. Of note increases the quantity of CCL25 and CCL28 in the SI, hence fa- ␣ ␣ ␤ because PS/2 is an anti- 4 Ab, it will interact with both 4 7 and cilitating the recruitment into the LP of the cells expressing their ␣ ␤ 4 1 receptors. However, we conducted similar experiments using receptors. ␣ ␤ a specific anti- 4 7 Ab (DAKT32, IgG2a) and obtained similar results (data not shown). Discussion In this study, we document the impact of RV infection on the early Effect of RV infection on CCL25 and CCL28 expression in SI of accumulation of IgAϩ plasmablast and PC in the intestinal LP of suckling mice ␣ ␤ infant mice and the role that the integrin 4 7 and the chemokine To measure CCL25 and CCL28 expression after RV infection, we receptors CCR9 and CCR10, along with their ligands, play in this isolated the SI of BALB/c suckling mice at 1, 3, and 5 DPI. The process. quantities of chemokines in the intestine were determined by It has been previously shown that the number of plasma- ELISA, and results were expressed as picograms per milligrams of blasts/PC in the MLN, PP, and intestinal LP greatly depends on tissue protein (Fig. 7). At 1 DPI, there was no significant difference exogenous antigenic exposure (33). In fact, in these tissues hardly ϩ in CCL25 or CCL28 expression between infected and noninfected any IgA PC could be detected in the first weeks of life in unin- mice ( p Ͼ 0.05). However, CCL25 was significantly increased in fected suckling mice, but immediately after weaning the number of ϩ IgA PC increased considerably (Ref. 33 and Fig. 2). In suckling Downloaded from mice (5 days old), we were able to determine that RV infection not only induces a virus-specific immune response but also a massive bystander activation/proliferation of B cells in the MLN (Fig. 3B) and PP (data not shown). This effect appears to be specific to only some enteric pathogens, because we show that Salmonella infec-

tion does not lead to proliferation of B cells in the intestine (Fig. http://www.jimmunol.org/ 4). We selected Salmonella for comparison because it is, like RV, an enteric pathogen of both mice and humans. Previous reports suggest that reovirus infection of suckling mice might have a sim- ilar effect as RV on B cell activation (34). It has recently been proposed, in adult mice, that the mechanism by which RV induces the polyclonal activation of B cells is likely to be TLR4 mediated (35), and future experiments will determine whether this is also the case in suckling mice. Because Salmonella also activates TLR4, it is unclear why B cell proliferation was not seen during this infec- by guest on September 27, 2021 tion (36). Our data also show that this polyclonal activation resulted in an enhanced accumulation of total IgAϩ plasmablast/PC to the intes- tinal LP of suckling mice during the first 2 wk after infection (Figs. 1 and 2). Taking into account the substantial and easily quantified differences in the number of intestinal IgAϩ plasmablasts/PC be- tween RV-infected and noninfected suckling mice, RV infection provided an excellent system to study the mechanisms that control the specific B cell population of the intestinal LP after a natural viral infection, and perhaps, in the absence of infection as well. In this study, we were able to characterize the homing pheno- type of an important subset of both RV-specific and total IgAϩ ␣ ␤ plasmablast/PC in the MLN that coexpressed 4 7 and CCR9 on their surface (Fig. 3C). We believe that these cells are likely to also express CCR10, because in previous experiments we have shown that after RV infection a large subset of IgAϩ and RV-specific B cells from the MLN migrated to both CCL28 and CCL25 (22). Of note, this result is comparable with our previous findings in humans, where we showed that during acute infection ␣ ␤ in children, RV-specific plasmablasts coexpressed 4 7, CCR9, and CCR10 and that the overall circulating plasmablast population had up-regulated the expression of these receptors on their surface FIGURE 7. RV infection increases the quantity of CCL25 and CCL28 (22). We hypothesized, as has been suggested for T cells (37), that in the SI of suckling mice. Five-day-old BALB/c mice were infected with because the activation of the B cells occur in the inductive sites of RV. At 1, 3, and 5 DPI, mice were sacrificed, and SI were isolated. The the intestine (i.e., PP and MLN), dendritic cells in these tissues amount of CCL25 (A) and CCL28 (B) in small intestinal samples was ␣ ␤ determined by ELISA (see Materials and Methods). Data from three in- would induce expression of high levels of 4 7, CCR9, and dependent experiments with three to four mice per group for each exper- CCR10 (i.e., gut homing imprinting phenotype) in all the locally iment, expressed as the geometric mean of the amount of chemokine (in activated B cells, and not only the RV-specific ones. However, the ϩ -Signifi- intestinal LP RV-specific and total IgA plasmablasts/PC ex ,ء .picograms) per milligrams of total intestinal protein and SEM Ͻ ␣ ␤ cant difference between the infected and noninfected group (p 0.05). pressed lower levels of 4 7 (Fig. 3, A and C) compared with the The Journal of Immunology 5757

MLN, and we could not detect significant levels of expression of play their respective roles at different points in the multistep hom- ␣ ␤ CCR9 (Fig. 3C). The fact that only a small percentage of RV- ing model. For instance, the interaction of the integrin 4 7 and its ␣ ␤ specific plasmablast/PC express 4 7 in the intestinal LP of suck- receptor MADCAM-1 brings the lymphocytes in contact with the ling mice is in agreement with previous studies in adult mice (28) endothelium, where the tethered cells have an opportunity to detect and could reflect down-regulation of this receptor once the cells chemotactic gradients from the underlying tissue. In contrast, the ␤ reach their effector site. Although prior studies indicate that 7 KO CCL25/CCR9 and CCL28/CCR10 interactions lead to migration B cells fail to mediate anti-RV effects in RAG 2 KO mice (9), the across the endothelium and potentially to the cell localization and ␤ 7 KO experiments were done using cells derived from the SP of retention in the intestinal LP. Interestingly, both CCL25 and ␣ ϩ immune mice, and these cells may have a different effector phe- CCL28 enhance the 4-dependent adhesion of IgA plasmablasts notype than the cells studied here. Concerning the low expression to MAdCAM-1 (12). of CCR9 in the LP, our findings are in agreement with a previous An alternative, but less likely, explanation of our findings would study (13) and also suggests that upon entry into the intestinal LP, be that the accumulation of IgAϩ plasmablasts observed in the LP ϩ RV-specific and total IgA plasmablast/PC down-regulate CCR9 after RV infection is mainly due to sideways migration of these expression, perhaps because they have reached their effector site. cells from intestinal inductive sites. However, if this was the case, An alternative explanation could be that these cells are activated the combined anti-chemokine treatment would not be expected to by the ongoing RV infection and down-regulate CCR9 as a result significantly reduce the number of plasmablasts/PC detected by of this activation. It remains to be determined whether the expres- FACS in the SI (which includes the inductive sites) nor would sion of CCR10 is also down-regulated in the plasmablast/PC in the redistribution of cells to the SP and MLN be anticipated (Fig. 5D). intestinal LP during RV infection. Downloaded from ϩ In addition, it has been shown that many of the plasmablasts Our data suggest that the enhanced accumulation of IgA plas- found near to the gut-associated lymphoid tissue follicles ap- mablast/PC in the LP observed following RV infection is the result parently belong to exhausted B cell clones with decreased J- of recruitment of lymphocytes activated in the PP and MLN. Of chain expressing potential and increased class switching to IgG note, short-term homing experiments would be required to directly (reviewed in Ref. 19). test this hypothesis, but such experiments have been extremely diffi- Our experiments demonstrating the inhibition of recruitment cult to perform in suckling mice. Therefore, we provided indirect of plasmablasts/PC to the intestinal LP only by the combined http://www.jimmunol.org/ evidence to support the proposition that plasmablasts trafficking from effects of Abs to CCL25 and CCL28 provide, for the first time, inductive sites are responsible for the LP accumulation. evidence of the functionally redundant role of the chemokine First, we showed that this accumulation could be significantly receptors CCR9 and CCR10 and their ligands. Two different inhibited by Abs directed to the chemokines CCL25 and CCL28 sets of experiments support this conclusion. First (Fig. 3, A and and the integrin ␣ . Interestingly, previous studies of short-term 4 B), we showed that in CCR9 KO suckling mice, a normal re- homing in adult mice using the PS/2 Ab have demonstrated the sponse to RV infection was elicited because we found compa- inhibitory effect of this Ab in lymphocyte homing to the intes- ϩ rable numbers of RV-specific and total IgA plasmablasts/PC tine (38). Second, the chemokine Ab treatment did not have an

in the intestinal LP and MLN of these animals compared with by guest on September 27, 2021 effect on cell division or apoptosis of plasmablasts/PC in the controls. Furthermore, in these KO mice, the length and sever- LP. Third, after treating the mice with the anti-chemokines Abs, ity of diarrhea during RV infection was the same as controls we detected a redistribution of the plasmablasts/PC to systemic (data not shown). Second, as shown in Fig. 5, A and B, only sites in the SP (Fig. 5D), and these cells were predominantly ␣ ␤ ϩ. In addition, using histologic analysis, we observed that when WT BALB/c mice were treated with anti-CCL25 and 4 7 CCL28 simultaneously could we significantly reduce the num- PP in the anti-chemokine-treated mice appeared identical with ϩ those in untreated or isotype-treated control mice after RV in- ber of RV-specific and total IgA plasmablasts/PC in the LP, fection. This last result led us to believe that the reduced num- whereas treatment with each Ab alone had little or no effect. ber of plasmablast/PC in the LP after anti-chemokine adminis- This result might have been expected because, as discussed pre- tration was not the result of an effect of the Ab treatment on the viously, we have evidence to support the fact that these cells intestinal inductive site. coexpress the two chemokine receptors on their surface. Finally, we were also able to show that RV infection not only Of note, these results are inconsistent with two previous re- had an effect on the number and homing phenotype of B cells ports. Hieshima et al. (12) showed that in adult mice immunized with CT, individual treatment with either anti-CCL25 or CCL28 elicited during acute infection, but also lead to increased ϩ CCL25 and CCL28 expression in the intestine of suckling mice alone had a significant effect on the number of CT IgA PC in at early time points after infection (Fig. 7). The up-regulation of the intestinal LP. Also, Pabst et al. (13) showed an impaired these chemokines would facilitate the recruitment of the cells response to OVA plus CT in CCR9-deficient mice. The fact that bearing their respective receptors on their surface. Interestingly, these studies were conducted in adult mice, that the immuni- a previous report in humans showed that CCL28 was up-regu- zations were not exclusively enteric, and that the nature of the lated upon inflammation in the colon (39), and it has also been Ag studied differed from the one in our study, could account for shown in mice, during influenza infection, that there is up-reg- the differences observed. In addition, it is unclear what the ac- ulation of the expression of inflammatory chemokines in the tual homing and repertoire expressed on the lung (40). Whether the effect of RV infection on chemokine surface of the B cells was in the study by Hieshima et al. (12). expression is related to inflammation as in the studies men- If different from the one in our study, specifically if the Ag-specific tioned above or is mediated through other mechanisms remains cells do not coexpress CCR9 and CCR10, this could explain the dif- to be determined. ferences observed using individual anti-chemokine treatment. Taken together, all of these results suggest that after RV infec- The finding of functional redundancy for chemokine recep- tion there is active recruitment of RV-specific and total IgAϩ plas- tors and their ligands does not appear to be exclusive to the gut mablasts/PC into the intestinal LP in which the chemokines because in skin homing, CCR4 and CCR10 are simultaneously ␣ CCL25 and CCL28 and the integrin 4 play an active role. It is expressed in a subset of effector T cells, can both support hom- important to clarify here that these three molecules would likely ing of T cells to skin, and either one or the other is required for 5758 IgAϩ PLASMABLAST HOMING TO LP AFTER RV INFECTION lymphocyte recruitment in cutaneous delayed-type hypersensi- 8. Kantele, A., J. M. Kantele, E. Savilahti, M. Westerholm, H. Arvilommi, tivity (41, 42). A. Lazarovits, E. C. Butcher, and P. H. Makela. 1997. Homing potentials of ␣ circulating lymphocytes in humans depend on the site of activation: oral, but It is also important to point out that the addition of the anti- 4 not parenteral, typhoid vaccination induces circulating antibody-secreting Ab to the anti-chemokine treatment did not augment the reduction cells that all bear homing receptors directing them to the gut. J. Immunol. 158: ϩ 574–579. of virus-specific or total IgA plasmablast in the LP. Moreover, 9. Kuklin, N. A., L. Rott, N. Feng, M. E. Conner, N. Wagner, W. Muller, and we showed (Table I) that the population that is preferentially H. B. Greenberg. 2001. Protective intestinal anti-rotavirus B cell immunity is ϩ ␣ ␤ blocked by the anti-chemokine treatment is the ␣ ␤ one. These dependent on 4 7 integrin expression but does not require IgA antibody pro- 4 7 duction. J. Immunol. 166: 1894–1902. two findings are in agreement with the phenotype of the B cells 10. Bowman, E. P., N. A. Kuklin, K. R. Youngman, N. H. Lazarus, E. J. Kunkel, ␣ ␤ induced during infection, because these B cells coexpress 4 7, J. Pan, H. B. Greenberg, and E. C. Butcher. 2002. The intestinal chemokine CCR9, and, most likely, CCR10, and treatment with the anti-␣ thymus-expressed chemokine (CCL25) attracts IgA antibody-secreting cells. 4 J. Exp. Med. 195: 269–275. and the anti-chemokine Abs is apparently targeting the same cell 11. Campbell, D. J., and E. C. Butcher. 2002. Intestinal attraction: CCL25 functions population. in effector lymphocyte recruitment to the small intestine. J. Clin. Invest. 110: Of interest, even when using these three Abs at optimal con- 1079–1081. 12. Hieshima, K., Y. Kawasaki, H. Hanamoto, T. Nakayama, D. Nagakubo, centrations, we were only able to reduce by ϳ60% the number of A. Kanamaru, and O. Yoshie. 2004. CC chemokine ligands 25 and 28 play es- IgAϩ cells present in the intestinal LP, compared with isotype sential roles in intestinal extravasation of IgA antibody-secreting cells. J. Immu- nol. 173: 3668–3675. control-treated mice. This finding suggest that other molecules 13. Pabst, O., L. Ohl, M. Wendland, M. A. Wurbel, E. Kremmer, B. Malissen, and might also be involved in homing of lymphocytes to the gut and R. Forster. 2004. Chemokine receptor CCR9 contributes to the localization of that a subset of IgAϩ plasmablasts/PC primed in the PP and MLN plasma cells to the small intestine. J. Exp. Med. 199: 411–416. 14. Lazarus, N. H., E. J. Kunkel, B. Johnston, E. Wilson, K. R. Youngman, and could express a different combination of integrin/chemokine re- E. C. Butcher. 2003. A common mucosal chemokine (mucosae-associated epi- Downloaded from ceptors, allowing them to be recruited into the LP. This hypothesis thelial chemokine/CCL28) selectively attracts IgA plasmablasts. J. Immunol. is supported by preliminary experiments, where we have infected 170: 3799–3805. ␤ 15. Wang, W., H. Soto, E. R. Oldham, M. E. Buchanan, B. Homey, D. Catron, suckling 7 KO mice and have observed a delayed but significant N. Jenkins, N. G. Copeland, D. J. Gilbert, N. Nguyen, et al. 2000. Identification accumulation of RV-specific and IgAϩ PC in their LP. Additional of a novel chemokine (CCL28), which binds CCR10 (GPR2). J. Biol. Chem. 275: experiments in these KO mice, as well as the generation of dual 22313–22323. 16. Pan, J., E. J. Kunkel, U. Gosslar, N. Lazarus, P. Langdon, K. Broadwell,

CCR9/10 KO mice will allow us to better understand whether M. A. Vierra, M. C. Genovese, E. C. Butcher, and D. Soler. 2000. A novel http://www.jimmunol.org/ other molecules are involved in homing to the gut. Previous reports chemokine ligand for CCR10 and CCR3 expressed by epithelial cells in mucosal tissues. J. Immunol. 165: 2943–2949. suggest that the chemokine receptors CXCR4 and CXCR6 might 17. Kunkel, E. J., C. H. Kim, N. H. Lazarus, M. A. Vierra, D. Soler, E. P. Bowman, also play a role in human PC recruitment to the bone marrow and and E. C. Butcher. 2003. CCR10 expression is a common feature of circulating other tissues including the intestine where their respective ligands and mucosal epithelial tissue IgA Ab-secreting cells. J. Clin. Invest. 111: 1001–1010. (CXCL12 and CXCL16) are expressed (43). Alternatively, the 18. Johansen, F. E., E. S. Baekkevold, H. S. Carlsen, I. N. Farstad, D. Soler, and possibility remains that the plasmablast/PC population that we de- P. 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