The pathogenic connection of type I IFN and its role in regulating the migration response of Ag delivery by B cells into lymphoid follicles in an autoimmune condition has not been well-identified. Here, we show that there was a significantly larger population of marginal zone precursor (MZ-P) B cells, defined as being IgMhiCD1dhiCD21hiCD23hi in the spleens of autoimmune BXD2 mice compared with B6 mice. MZ-P B cells were highly proliferative compared with marginal zone (MZ) and follicular (FO) B cells. The intrafollicular accumulation of MZ-P B cells in proximity to germinal centers (GCs) in BXD2 mice facilitated rapid Ag delivery to the GC area, whereas Ag-carrying MZ B cells, residing predominantly in the periphery, had a lower ability to carry Ag into the GCs. IFN-α, generated by plasmacytoid dendritic cells, induced the expression of CD69 and suppressed the sphingosine-1-phosphate-induced chemotactic response, promoting FO-oriented Ag transport by MZ-P B cells. Knockout of type I IFN receptor in BXD2 (BXD2-Ifnαr−/− ) mice substantially diffused the intrafollicular MZ-P B cell conglomeration and shifted their location to the FO-MZ border near the marginal sinus, making Ag delivery to the FO interior less efficient. The development of spontaneous GCs was decreased in BXD2-Ifnαr−/− mice. Together, our results suggest that the MZ-P B cells are major Ag-delivery B cells and that the FO entry of these B cells is highly regulated by type I IFN–producing plasmacytoid dendritic cells in the marginal sinus in the spleens of autoimmune BXD2 mice.
The localization of B cells in appropriate microenvironments of secondary lymphoid organs plays an important role in determining the fate of B cell immunity. Specifically, the B cell migration pattern into the follicular (FO) regions of the spleen can alter the tempo and frequency of T–B cell contact and, thus, has been proposed as one mechanism to regulate the development of autoreactive B cells (1, 2). Early studies by Cyster et al. (3) showed that under conditions of clonal selection, anergic B cells failed to enter the FO areas, instead arresting at the T–B cell boundary in a process known as FO exclusion. FO exclusion has been proposed as one B cell checkpoint mechanism to prevent B cell tolerance loss (4). Although chemokines and chemokine receptors have been paramount in shaping B cell migration patterns, we and other investigators (5–7) showed that local cytokine production could provide additional signals to regulate the migration and retention of B cells in the FOs. In autoimmune murine models, B cells can act as APCs and/or Ag-delivery cells to promote the development of spontaneous autoreactive germinal centers (GCs) (8–12). Treatment of autoimmune disease with B cell–depletion therapy depletes B cells that mainly serve as APCs or Ag-delivery cells (9, 13–15).
The BXD2 mouse strain is one of ∼20 BXD recombinant inbred strains that we analyzed for the development of autoimmune disease, including the development of features resembling human systemic lupus erythematosus and rheumatoid arthritis (16). The development of highly mutated multireactive autoantibodies that can induce kidney and joint disease is one of the important pathogenic features of BXD2 mice (16, 17). We found that BXD2 mice spontaneously form GCs in the spleens and that the expression of the gene encoding activation-induced cytidine deaminase (Aicda) in the GC B cells can be stimulated by activated CD4+ T cells from BXD2 mice (18). There is an abundance of Th-17 cells in the FOs and GC area. The high levels of IL-17 produced by these cells is associated with the upregulation of regulator of G-protein signaling (Rgs)13 and Rgs16 and the migration arrest of B cells in response to FO- and GC-oriented chemokines, CXCL12 and CXCL13, enabling them to form stable T–B cell conjugates and facilitate GC formation in the spleens of BXD2 mice (6).
The importance of IFN-α in the spontaneous development of autoantibodies and immune complex deposition in the glomeruli was demonstrated using murine models of lupus (19–21). Clues to the possible role of IFN-α in the production of high-affinity autoantibodies can be derived from their normal physiologic mechanisms of action. IFN-α was shown to promote Ab class-switch recombination and plasma cell differentiation under nonautoimmune conditions (22, 23). To determine the reason for the coexistence of high levels of IFN-α and IL-17 in the spleens of BXD2 mice, we observed that there was a significantly increased IgMhiCD1dhiCD21hiCD23hi B cell population in the spleens of BXD2 mice. B cells with this phenotype are referred to as marginal zone precursor (MZ-P) B cells in mice (24, 25). We further identified that the entry of MZ-P B cells into the FO region is promoted by IFN-α, which is primarily produced by plasmacytoid dendritic cells (pDCs) that are localized in the marginal sinus (MS). Abrogation of IFN-αR in BXD2 mice attenuated the development of spontaneous GCs and autoimmune disease. Importantly, injection of BXD2 and BXD2-Ifnαr−/− mice with 2,4,6-trinitrophenyl-protein (TNP)-Ficoll demonstrated that MZ-P B cells serve as the major Ag-transporting B cells that directly deliver TNP into the GCs. Deficiency of IFN-α abrogated this response. Our results suggest that FO entry of Ag-delivery MZ-P B cells facilitated by IFN-α may be an important mechanism to promote the autoimmune response in BXD2 mice.
Materials and Methods
Female homozygous C57BL/6 and BXD2 recombinant inbred mice were obtained from The Jackson Laboratory (Bar Harbor, ME); B6-Ifnαr−/− mice were obtained from Dr. Jocelyn Demengeot (Instituto Gulbenkian de Ciência, Oeiras, Portugal). B6-Ifnαr−/− - mice were backcrossed with BXD2 mice for seven generations using a marker-assisted speed congenic approach with 146 markers. All mice were housed in the University of Alabama at Birmingham (UAB) Mouse Facility under specific pathogen-free conditions in a room equipped with an air-filtering system. The cages, bedding, water, and food were sterilized. All mouse procedures were approved by the UAB Institutional Animal Care and Use Committee.
Flow cytometry analysis and cell sorting
Flow cytometry was performed on fluorescently labeled single-cell suspensions derived from spleens using the method we described previously (6, 182 before obtaining single-cell suspensions for Ab labeling. For the intracellular detection of IFN-α, cells were permeabilized (Cytofix/cytoperm, BD Biosciences), washed (Perm/Wash, BD Biosciences), and then incubated with a 1:1 mixture of anti-IFN–α Abs (clones RMMA-1 and [18F]). Cells (100,000/sample) were washed twice with FACS buffer and fixed in 1% paraformaldehyde/FACS solution before analysis by flow cytometry using a BD LSR II flow cytometer (BD Biosciences). The analysis was performed using FlowJo Software (Tree Star, Ashland, OR). Forward-angle light scatter was used to exclude dead and aggregated cells. Some results are presented as fluorescence histograms with the relative number of cells on a linear scale versus the relative fluorescence intensity on a log scale. Acquisition and gating of FO, marginal zone (MZ), and MZ-P B cells were carried out based on the method described by Allman and Pillai (24). FO B cells were defined as CD23hisIgMlo+hisIgDhiCD21intCD1dloCD19+ cells; MZ B cells were defined as CD23null+losIgMhisIgDloCD21hiCD1dhiCD19+ cells; MZ-P B cells were defined as CD23hisIgMhisIgDhiCD21hiCD1dhiCD19+ cells. All gatings were set up to ensure that the percentage of each subpopulation of B cells obtained from normal B6 mice was equivalent to the previously reported results (24).
For cell sorting to isolate the different B cell populations, anti-CD19 microbeads (Miltenyi Biotec, Auburn, CA) were used to isolate whole B cells. Subsequently, the single-splenic B cell preparation was labeled with fluorescent conjugated Abs and sorted into FO, MZ, and MZ-P B cells based on the expression of CD21, CD23, and IgM, as described above. Sorting was carried out using a BD FACS Aria Cell Sorter (BD Biosciences). All sorting for pDCs was completed by positive selection using anti-PDCA1 microbeads (Miltenyi Biotec).
Immunofluorescent staining and confocal image analysis
6).+ GC response and TNP+ cells was carried out using Olympus DP2-BSW software (Olympus America, Center Valley, CA) according to the method we described previously (
Cells, including pDCs and B cell subpopulations, were cultured with RPMI 1640 medium (Invitrogen) supplemented with 2 mM l-glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, 5.5 × 10−5 M 2-mercaptoethanol, and 10% FCS for all in vitro experiments.
IFN-α stimulations and measurements
For CD69 expression on different B cell subpopulations in response to IFN-α under in vitro conditions, single spleen cells were cultured in 96-well (Costar, Cambridge, MA) tissue-culture plates at 37°C/5% CO2 in triplicate wells (1 × 104 cells per well) and stimulated with IFN-α (20 ng/ml) for 12 h. To measure IFN-α levels produced by pDCs, single-cell suspensions were prepared by collagenase D (2 mg/ml) digestion of spleen tissues for 30 min at 37°C/5%CO2 before sorting for pDCs. Enriched pDCs were subsequently cultured in 96-well (Costar) tissue-culture plates at 37°C/5% CO2 in triplicate wells (1 × 104 cells per well) and stimulated with medium only, CpG (3 μM), or CpG (3 μM)-N–[1-(2,3-Dioleoyloxy)propyl]-N,N,N trimethylammonium methylsulfate (DOTAP) complex (F. Hoffman-La Roche, Boulder, CO). After 24 h, culture supernatants were collected, and IFN-α levels were measured by ELISA (PBL Biomedical, Piscataway, NJ). CpG-DOTAP preparation for in vitro culture was formulated by mixing 19.2 μg (3 nmol) CpG in 120 μl PBS with 30 μl DOTAP, incubating for 15 min, and then diluting with 850 μl medium to make 3 μM CpG-DOTAP solution. For in vivo measurements of IFN-α, 5 μg CpG was administered i.v. into each mouse tail vein. Five micrograms of CpG was mixed with 50 μl PBS, whereas in another reaction tube, 30 μl DOTAP was mixed with 70 μl PBS. The CpG solution was then mixed with the DOTAP solution and incubated at room temperature for 15 min prior to administration. Serum levels of IFN-α in each mouse were measured 4 h later.
Cell migration assay
Single-cell suspensions of anti-CD19 MACS column (Miltenyi Biotec)-purified spleen B cells from B6, BXD2, or BXD2-Ifnαr −/−26). The stimulated cells were loaded (2 × 106) into the upper well insert (5-μm pore size) of a Transwell system (5 μm pore size, Costar), and sphingosine-1-phosphate (S1P) was added to the bottom chamber at a final concentration of 20 nM. After incubation for 2 h at 37°C in a 5% CO2 incubator, the migrated cells were harvested and resuspended in 300 μl FACS buffer. The cells that remained in the inserts or migrated to the lower chamber were counted using a flow cytometer, and the separation of the CD19+ B cells into migrated and nonmigrated cell subsets was determined. The chemotaxis index was calculated by dividing the number of cells that migrated in response to chemokine by the number of cells that migrated in the absence of chemokine.
Isolation and quality control of RNA
RNA was isolated from 0.2 to 10 × 106 cells using TRIzol reagent (Invitrogen) or from <0.2 × 106 cells using the PicoPure RNA Isolation Kit (Molecular Devices, Sunnyvale, CA). The quality of the isolated RNA was determined using an Experion Automated Electrophoresis System (Bio-Rad, Hercules, CA). RNAs that exhibited an 18S/28S ratio >1.8 were converted to cDNA using the First Strand cDNA Synthesis Kit (Fermentas, Glen Burnie, MD).
Quantitative real time-PCR
The expression of Aicda, S1p1, and S1p3 was analyzed using a quantitative real-time PCR (QRT-PCR) method. Briefly, the QRT-PCR mixtures contained SYBR Green PCR Master Mix (Bio-Rad) with the following primers: Aicda forward: GCCACCTTCGCAACAAGTCT; Aicda reverse: CCGGGCACAGTCATAGCAC (137 bp); S1p1 forward: TGCTGTAACTGAAGGCTCAC; S1p1 reverse: GGATGCTAGTGGACACCATAG (108 bp); S1p3 forward: CCGTAGTGATTGTGGTGAGTG; S1p3 reverse: GGACAGCCAGCATGATGAAC; Cdca3 forward: GAGTAGCAGACCCTCGTTCAC; Cdca3 reverse: TCTCTACCTGAATAGGAGTGCG; Cdca5 forward: GGACTTCACTTACTAAGCCTTC; Cdca5 reverse: GACATCTGGGACCTCTACTG; Cdca8 forward: ACAAGGAAGAGGCAGAAG; Cdca8 reverse: CCGTTGATGGAGATGTTG; Gapdh forward: AGGTCGGTGTGAACGGATTTG; Gapdh reverse: TGTAGACCATGTAGTTGAGGTCA (136 bp). A final volume of 25 μl was used for QRT-PCR in an IQ5 thermocycler (Bio-Rad). Amplification conditions were 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and annealing for 30 s. The annealing temperature used was 54°C for Cdca8; 56°C for Gapdh; 57°C for S1p3; 58°C for Aicda, Cdca3, and Cdca5; and 60°C for S1p1 and S1p3.
QRT-PCR products were normalized relative to that of Gapdh to correct for potential differences in template input to determine relative mRNA levels. Results are expressed as fold differences in the expression of the indicated gene relative to the expression of Gapdh. Standard curves were generated for every target using six 4-fold serial dilutions.
To specify cellular agents for Ag transport, 50 μg TNP-Ficoll (Biosearch Technologies, Novato, CA) was administered i.v. into each mouse, and mice were sacrificed at the indicated time points. A portion of the spleen was snap-frozen in 2-methylbutane for histological analysis, as described above. Another portion of the spleen was made into a single-cell suspension and labeled for detection of TNP, as described above.
All results are shown as mean ± SEM. A two-tailed t test was used when two groups were compared for statistical differences. The ANOVA test was used when more than two groups were compared for statistical differences. The distribution differences of MZ and MZ-P in the anatomical MZ area versus those in the anatomical follicular area between different strains of mice were tested using the χ2 test. p values <0.05 were considered significant.
Increased IFN-α production by pDCs localized to the marginal periphery of B cell FOs in BXD2 mice
The type I IFNs, including IFN-α, are highly associated with the presence of several human autoimmune diseases (20, 27, 28) and, as demonstrated by murine models of lupus, are a causative factor for the onset of disease (21, 29). A specialized subset of dendritic cells, the pDCs, were found to efficiently generate massive amounts of type I IFNs (30). Because of the unique lupus and arthritis features developed by BXD2 mice, we questioned whether high numbers of IFN-α–producing pDCs are present in these mice. Flow cytometry analysis indicated that there was a significantly higher percentage of IFN-α–producing pDCs in the spleens of BXD2 mice compared with the spleens of B6 mice (Fig. 1A ). On gating of the IFN-α–producing cells, we found that the pDCs are the primary IFN-α–producing cells in the spleens of BXD2 mice (Fig. 1B ), which is consistent with previous reports that pDCs are efficient producers of type I IFNs (30–34). Stimulation of pDCs by type A CpG, a Toll-like receptor 9 ligand, can elicit the expression and secretion of IFN-α (30, 33). We found that pDCs isolated from the spleens of BXD2 mice were more responsive to in vitro stimulation with type A CpG or type A CpG-DOTAP and produced significantly greater amounts of IFN-α in response to these agents than equivalent numbers of pDCs isolated from the spleens of B6 mice (Fig. 1C ). Immunofluorescent imaging of spleen sections of B6 mice showed small numbers of pDCs (green) that formed a single layer surrounding nonreactive FOs (Fig. 1D ). In the spleens of wild-type (WT) naive BXD2 mice, there were large FOs with active PNA+ GCs (blue). Surrounding these active GCs were increased numbers of pDCs (green) that were located away from the GCs but were primarily in the marginal periphery of the FOs in proximity to the MS (Fig. 1D ).
GC formation is partially diminished in IFN-αR–deleted BXD2 mice
To determine the role of type I IFN in GC formation, the presence of GCs in WT BXD2 and age-matched BXD2-Ifnαr−/− mice was compared. Confocal microscopy analysis of frozen spleen sections confirmed the presence of well-formed PNA+ GCs (PNA+; blue) located close to the central areas of FOs in 3-mo-old WT BXD2 mice. In age-matched BXD2-Ifnαr−/− mice, there were fewer and smaller PNA+ GCs (Fig. 2A ). Quantification of the number of PNA+ FOs confirmed that there was a smaller number of PNA+ GCs per section of area in the spleens of BXD2-Ifnαr−/− mice compared with WT BXD2 mice (Fig. 2B ), and flow cytometry indicated that BXD2 mice exhibited a higher frequency of PNA+Fas+ GC B cells than BXD2-Ifnαr−/− mice (Fig. 2C ). This decreased formation of GCs in BXD2-Ifnαr−/− mice was associated with significantly reduced levels of Aicda transcripts, which encode the activation-induced cytidine deaminase protein required for class-switch recombination and somatic hypermutation, in B cells enriched from the spleens of BXD2 mice (Fig. 2D ).
Increased IgMhiCD21hiCD23hi B cells in BXD2 mice
The presence of pDCs in the marginal periphery of FOs suggests that type I IFNs can signal B cells in the MZ and B cells that traffic near the border region between the MZ and FO. We showed previously that the percentage of MZ B cells, defined by their anatomic location in the MZ and exhibiting the CD21hi CD23lo phenotype, is lower in the spleens of BXD2 mice than age-matched B6 mice, whereas the percentage of FO B cells is higher (6). To determine whether this reduction in mature MZ B cells in BXD2 mice is due to a lack of precursor or altered B cell development under the influence of pDCs in the MS, we analyzed the phenotype of B cells in B6, BXD2, and BXD2-Ifnαr−/− mice. The relative expression of CD23 on the MZ B cells subdivides these cells into two functionally different populations (24, 25, 35, 36). The CD23loIgMhiCD21hi B cells are considered to be mature MZ B cells whereas the CD23hi IgMhiCD21hi B cells are thought to be MZ-precursor (MZ-P) (24, 25, 35, 36).
As defined by IgMhiCD21hi, the highest percentage of MZ B cells was seen in B6 mice, with lower percentages in BXD2 and in BXD2-Ifnαr−/− mice (Fig. 3A ). However, the majority of the IgMhiCD21hi MZ B cells from the spleens of BXD2 mice exhibited the CD23hi MZ-P phenotype (Fig. 3B , middle panel), which was significantly greater compared with B6 mice, in which the majority of IgMhiCD21hi MZ B cells exhibited the CD23lo MZ phenotype (Fig. 3B , upper panel). Although exhibiting a higher frequency of IgMhiCD21hiCD23hi MZ-P B cells than in B6 mice, BXD2-Ifnαr −/− mice displayed significantly decreased percentages than their WT counterparts for this particular cell population (Fig. 3B , lower panel).
Flow cytometry analysis for the FO, CD23lo MZ, and CD23hi MZ-P B cells also showed that MZ and MZ-P B cell populations express high levels of CD1d compared with the FO B cell population (Fig. 3C ). The percentages of MZ and MZ-P B cells expressing CD1d are comparable, whereas FO B cells express significantly less CD1d than either of the two populations (Fig. 3D ). There was no difference in the expression of IgD and CD93 (AA4) in IgMhiCD21hiCD23hi B cells from all three strains of mice (IgDhiCD93−/lo; data not shown). The results suggest that these abnormally expanded B cells in BXD2 mice exhibit the same characteristics as the previously described MZ-P B cells (24, 25, 35, 36).
The total counts of IgMhiCD21hi MZ B cells in the spleens were higher in WT BXD2 mice and BXD2-Ifnαr−/− mice than in the B6 mice as a result of the significantly enlarged spleen sizes of these BXD2 mice (6) (Fig. 3E ). There were fewer conventional CD23loIgMhiCD21hi MZ B cells but greatly elevated CD23hiIgMhiCD21hi MZ-P B cells in the spleens of BXD2 mice compared with the spleens of B6 mice (Fig. 3E ). Although the total spleen cell count was nearly equivalent between WT BXD2 and BXD2-Ifnαr−/− mice, there was a significantly lower percentage and lower total counts of MZ-P B cells in the BXD2-Ifnαr−/− mice compared with the BXD2 mice (Fig. 3E ).
To determine whether the percentage of MZ-P B cells correlated with the levels of IFN-α in BXD2 mice, the MZ-P B cells, as a percentage of IgMhiCD21hi B cells, were calculated in 1-, 3-, and 8-mo-old BXD2 mice (Supplemental Fig. 1A). The results indicated that the percentage of MZ-P B cells peaked in 3-mo-old BXD2 mice (Supplemental Fig. 1A). Consistent with the role of type I IFN in promoting the development of MZ-P B cells, 3-mo-old BXD2 mice exhibited the highest serum IFN-α levels in response to type A CpG compared with 1- and 8-mo-old mice (Supplemental Fig. 1B). The lower ability of pDCs from older BXD2 mice to produce type I IFN seems to be associated with their maturation status; increases in the expression of CD80 and CD86 were observed in pDCs from 8-mo-old BXD2 mice (Supplemental Fig. 1C).
Compared with FO and CD23lo MZ B cells, MZ-P B cells from BXD2 mice exhibited the highest proliferative response after anti-CD40 and anti-IgM stimulation (Supplemental Fig. 2A, 2B). In vivo, MZ-P B cells also exhibited the highest expression of genes involved in cell division, including Cdca3, Cdca5, and Cdca8, compared with MZ or FO B cells (Supplemental Fig. 2C). BXD2 MZ-P B cells exhibited higher message levels of these proliferation genes than B6 MZ-P B cells, demonstrating the unique hyperproliferation phenotype of this B cell subpopulation in BXD2 mice.
Increased counts and intrafollicular concentration of MZ-P B cells in proximity to GCs in type I IFN signaling-competent WT BXD2 mice
MZ B cells can be distinguished by their phenotype (CD21hiIgMhiCD23lo) and their location outside of the MS. This location enables them to encounter blood-borne IgM-immune complexes (37). In contrast, the location of MZ-P B cells is less well established. Compared with FO (CD1dloCD23hi) and MZ (CD1dhiCD23lo) cells, only MZ-P B cells exhibit the CD1dhiCD23hi phenotype; therefore, we used anti-CD1d and anti-CD23 staining to determine the location of the MZ-P B cells in the spleens of the B6, BXD2, and BXD2-Ifnαr−/− mice (Fig. 4A–F ). As expected, CD1dhiCD23lo MZ B cells (green) surrounded the B cell FO in proximity to the MS in the spleens of all three strains of mice (Fig. 4A–C ). In the spleens of BXD2 mice, there is an area of CD1dhi B cells that are also CD23hi near the GCs (yellow) (Fig. 4B , 4E ). These CD1dhiCD23hi B cells are situated adjacent to a GC and opposite from the CD4+ T cell area, suggesting that these CD1dhiCD23hi B cells are located near the light zone of a GC (38, 39). In contrast, significantly decreased numbers of MZ-P B cells were distributed diffusely within the B cell FOs of B6 mice (Fig. 4A , 4D ). Similarly, in the absence of type I IFN signaling in BXD2-Ifnαr−/− mice, small numbers of MZ-P B cells were distributed diffusely, many of which were near the FO–MZ border of the FO. There was an absence of concentrated clusters of MZ-P B cells in BXD2-Ifnαr−/− mice (Fig. 4C , 4F ), even in the presence of a developing GC in BXD2-Ifnαr−/− mice (Fig. 4F ).
Quantitative analysis (counting the number of CD1dhiCD23lo MZ B cells per visual field) showed that the majority of MZ B cells resided in the MZ (Fig. 4G , open area), whereas a minor fraction was located in the FO (Fig. 4G , solid area). In contrast, the majority of CD1dhiCD23hi MZ-P B cells resided in the FO in the spleens of BXD2 mice (Fig. 4H , solid area). The χ2 test indicated a significantly greater percentage of MZ-P B cells residing in the FO relative to the MZ in the spleens of WT BXD2 mice compared with the spleens of B6 or BXD2-Ifnαr−/− mice (Fig. 4H ), whereas no significant differences were observed for the residence of MZ B cells among the B6, BXD2, and BXD2-Ifnαr−/− mice (Fig. 4G ).
Type I IFN signaling induces and maintains higher CD69 surface expression and decreases S1p1 message levels by MZ-P B cells in BXD2 mice
S1P1 surface expression is known to antagonize the surface expression of CD69 (40), and its expression on B cells is known to promote their localization to the MZ (41). Because greater fractions of MZ-P B cells are localized within the FO in BXD2 mice than in B6 or BXD2-Ifnαr−/− mice (Fig. 4H ), we sought to determine the expression of CD69 and S1p1. The highest surface expression of CD69 was observed on freshly isolated MZ-P B cells compared with MZ and FO B cells (Fig. 5A ). CD69 surface expression on MZ and MZ-P B cells was decreased in BXD2-Ifnαr−/− mice compared with their WT counterparts (Fig. 5A ). The percentage of CD69+IgMhiCD21hiCD23hi MZ-P B cells was significantly greater compared with the other two CD69+ B cell populations in BXD2 mice (Fig. 5B ). In contrast, MZ-P B cells from BXD2-Ifnαr−/− mice exhibited significantly suppressed levels of CD69 (Fig. 5B ).
To further test whether type I IFNs can increase the surface expression of CD69 on the individual B cell populations, cell-sorted FO, MZ, and MZ-P B cells were stimulated with IFN-α in vitro. Culture with IFN-α led to significant induction of CD69 surface expression on all three B cell populations from BXD2 mice (Fig. 5C ). More importantly, IFN-α significantly increased CD69 surface expression on MZ-P B cells to a greater level than FO or MZ B cells in BXD2 mice, indicating that MZ-P B cells have a greater sensitivity to IFN-α (Fig. 5D ). In contrast, CD69 surface expression on MZ-P B cells from BXD2-Ifnαr−/− mice remained significantly suppressed (Fig. 5D ).
Consistent with the greater percentage of MZ-P B cells from BXD2 mice that expressed CD69, the cell-sorted MZ-P B cells expressed lower levels of S1p1 transcripts compared with the CD23lo MZ B cells from these mice (Fig. 5E ). In contrast, MZ B cells displayed comparable levels of S1p1 transcripts (Fig. 5E ), regardless of whether type I IFN signaling was present. Furthermore, S1p3 transcript levels were significantly suppressed in MZ-P B cells, independent of type I IFN signaling (Fig. 5E ). Analysis of the chemotactic response toward S1P in vitro indicated that the migration of the MZ B cell in response to S1P was equivalent between BXD2 and BXD2-Ifnαr−/− mice (Fig. 5F ). However, the S1P-induced chemoattractive effect on MZ-P B cells from the BXD2 mice was lower than that of MZ-P cells from BXD2-Ifnαr−/− mice (Fig. 5F ). Consistent with the elevated expression of CD69 on the BXD2 MZ-P cells, in vitro IFN-α stimulation of B cells from BXD2 mice led to further reduction of the chemotactic response toward S1P, especially in the CD23hi MZ-P B cell population; this decrease was not observed in B cells isolated from BXD2-Ifnαr−/− mice (Fig. 5F ). Together, these results suggest a mechanistic model in which the production of type I IFNs, including IFN-α, by pDCs localized to the marginal periphery of B cell FOs in BXD2 spleens can upregulate the surface expression of CD69 on MZ-P B cells to induce intrafollicular aggregations that may collectively facilitate GC induction.
Type I IFNs promote Ag delivery to GCs by MZ-P B cells in BXD2 mice
One critical function of B cell migration into the FO and GC region is to serve as Ag-delivery cells (42). To determine whether MZ-P B cells could transport Ag and be influenced by type I IFNs, BXD2 and BXD2-Ifnαr−/− mice were analyzed 1 h after TNP-Ficoll administration. TNP-Ficoll was shown to bind to MZ B cells via a complement receptor (CD21–CD35)-dependent manner. This facilitates the visualization of the shuffling of TNP capturing CD21+ B cells (43, 44). Because MZ and MZ-P B cells express high levels of CD21, we determined whether MZ and MZ-P B cells exhibit an equivalent ability to bind to and deliver TNP-Ficoll into the FOs. Flow cytometry analysis indicated that there was near equivalent binding of TNP-Ficoll to MZ and MZ-P B cells from BXD2 and BXD2-Ifnαr−/− mice, which was significantly higher than TNP-Ficoll binding to CD21lo FO B cells (Fig. 6A , 6B ). However, histological analysis revealed substantial accumulation of TNP-Ficoll carried by MZ-P B cells in proximity to the GCs in BXD2 mice. A representative GC is shown and is adjacent to concentrations of TNP+ MZ-P B cells (dark blue) (Fig. 6C ). Although MZ B cells could trap TNP-Ficoll, the majority of TNP+ MZ B cells (light pink) were located in the MZ and not adjacent to the developing GC (Fig. 6D , 6G ). Consistent with the earlier observation that MZ-P B cells have a diffuse distribution and a preference for locations near the FO–MZ border in the spleens from BXD2-Ifnαr−/− mice (Fig. 6E ), we did not observe significant aggregates of TNP+ MZ-P B cells inside the B cell FO. Rather, many of the TNP+ MZ-P B cells remained near the FO–MZ border in proximity to the MS (Fig. 6F ). The average number of MZ-P TNP+ B cells in the FO was 74 ± 6 in BXD2 mice, compared with only 16 ± 4 in BXD2-Ifnar−/− mice (Fig. 6G ). This result shows that MZ-P B cells are especially capable of carrying Ag into the FO in BXD2 mice that have intact IFN-αR signal.
We established previously that BXD2 mice spontaneously develop high levels of circulating high-affinity nephritogenic and arthrogenic pathogenic autoantibodies and that the spontaneous formation of GCs in the spleen is critical to the production of these high-affinity pathogenic autoantibodies (16–18). In the current study, we showed that there are increased counts of pDCs in the spleens of BXD2 mice. These pDCs exhibit significantly elevated expression of IFN-α and are the primary producers of this cytokine. We further showed that type I IFNs play a role in the development of lupus in the BXD2 mice by demonstrating that a deficiency of the IFN-αR in these mice leads to a reduction in the spontaneous formation of GCs. Strikingly, although the type I IFN signature and the expanded development of Th-17 cells were reported to be associated with lupus in humans (45–47), IFN-α by itself was found to suppress Th-17 development (48). Because IFN-α is mainly produced by pDCs that are located in the MZ, and we previously showed that Th-17 cells are preferentially located in the inner FO or inside a GC (6), our results suggest that spatial compartmentalization of IFN-α–producing pDCs and IL-17–producing CD4 T cells in the BXD2 spleens is one mechanism to enable the cooperative effects of these two cytokines.
In the spleens of naive mice, pDCs are scattered mainly in the T cell area and in the red pulp and are only rarely found in the MZ (49); however, upon infection with virus or after CpG stimulation, pDCs migrate to the MZ area and produce IFN-α within the first 36 h of exposure (49), suggesting that the relocation of pDCs into the MZ area and production of IFN-α in this area is part of an early host defense mechanism (49). Our observations suggest that the location of IFN-α–producing pDCs in the MZ region serves to act on cells in the immediate vicinity, including those in the MZ and the FO regions in near proximity, to affect a chain of events that lead to GC induction and maintenance.
BXD2 mice, unlike their B6 counterparts, display an elevated percentage and number of MZ-P B cells, of which the majority of IgMhiCD21hi B cells are CD23hi, rather than CD23lo. Compared with normal B6 MZ-P B cells, MZ-P B cells from BXD2 mice exhibited a dramatically elevated proliferative response by anti-IgM and anti-CD40 stimulation. Consistent with these in vitro observations, MZ-P B cells directly isolated from BXD2 mice demonstrated higher levels of cell cycle– and cell division cycle–associated genes than FO or MZ B cells and MZ-P B cells from B6 mice. Among these, proteins encoded by Cdca3 (Tome-1) and Cdca8 (Borea) are important regulators of mitosis (50, 51). Protein encoded by Cdca5 (Sororin) is required for sister chromatid cohesion (52). The hyperproliferative phenotype of MZ-P B cells from BXD2 mice may account for the larger percentage of these cells in the spleens of these mice.
Interestingly, BXD2-Ifnαr−/− mice display decreased numbers of MZ-P B cells compared with WT BXD2 mice. IFN-αR deletion shifts the migration pattern of MZ-P B cells toward the FO–MZ border and away from the FO interior and GCs, possibly facilitating the migration of MZ-P B cells to the MZ and their permanent transition to MZ B cells in response to the chemical milieu in the MZ (40, 53). Accordingly, BXD2-Ifnαr−/− mice exhibit decreased MZ-P B cells, with a concomitant increase in the number of MZ B cells compared with WT BXD2 mice. Examining BXD2 mice at different ages, we observed peak counts of MZ-P B cells at 3 mo of age. This trend correlates with the ability of pDCs to produce IFN-α, which is high at 3 mo of age and becomes suppressed at 8 mo of age. The downregulation of IFN-α in older BXD2 mice correlated with the increased expression of maturation markers (CD80 and CD86) on pDCs. This is consistent with the previous finding that mature pDCs express higher levels of CD80 and CD86 to make pDCs more efficient APCs but reduce their type I IFN production (33). Chronic exposure to and activation by Ags could induce the phenotypic shift in pDCs from primary IFN-producers to efficient APCs in BXD2 mice. An upward trend of serum levels of IFN-α from 1 to 3 mo of age in BXD2 mice can promote MZ-P B cell expansion and their inward FO movement to induce and maintain GCs. This is consistent with increased percentages of MZ-P B cells in BXD2 mice from 1 to 3 mo of age. The finding that these occur prior to significant development of autoantibody production, which occurs at 4 to 6 mo of age (17), indicates that MZ-P B cell expansion and its regulation by type I IFN is an early event prior to disease onset.
Importantly, our results suggest that type I IFNs exhibit an important effect to promote the FO-oriented migration of MZ-P B cells. Because CD69 surface expression is diminished in FO, MZ, and MZ-P B cells from BXD2-Ifnαr−/− mice, type I IFN can regulate S1p1 expression within each of these B cell populations. Despite IFN-α stimulation under in vitro conditions, CD69 surface expression in all three B cell populations in BXD2-Ifnαr−/− mice remained suppressed. Interestingly, MZ-P B cells in IFN-αR intact BXD2 mice expressed the highest levels of cell surface CD69. When stimulated with IFN-α, MZ-P B cells from BXD2 mice exhibited the highest surface expression of CD69 compared with FO or MZ B cells. Consistent with the antagonistic relationship between CD69 and S1P1 expression, when IFN-αR is deleted, S1p1 transcript levels become elevated. Furthermore, S1P3, known to be highly expressed in MZ B cells, had significantly depressed transcript levels in the MZ-P B cells, regardless of whether type I IFN signaling was present or absent. S1P3 stabilizes the positioning of B cells in the MZ (41). Its depressed levels in MZ-P B cells further suggest that, compared with MZ cells, MZ-P B cells exhibit a higher tendency to migrate into the FO. We also determined the chemotactic responses of MZ and MZ-P from BXD2 and BXD2-Ifnαr−/− mice to CXCL12, CXCL13, CCL19, and CCL21; CXCL13 was the only chemokine to provide differentially chemoattractive effects to MZ and MZ-P B cells from these two strains. MZ and MZ-P B cells from BXD2 mice exhibited a stronger migration response toward CXCL13 compared with the same cells from BXD2-Ifnαr−/− mice (data not shown). Therefore, the results suggest that the dominant effects of IFN-α to regulate the FO entry of MZ-P B cells were via its suppression of the S1P chemotactic response. Potentially, pDCs residing in the MZ can generate massive amounts of IFN-α to achieve this.
McHeyzer-Williams et al. (54) reported that during the initial phase of protein immunization, naive B cells specifically recognize the protein Ag and transport this Ag to initiate the T cell–dependent B cell response in checkpoint I. They further proposed that these B cells contact cell-associated Ags for priming during this early initiation phase of adaptive immunity. In parallel, naive Th cells expand, differentiate into effector Th cells, and migrate to the T–B borders to contact peptide–MHC II–expressing Ag-primed B cells in checkpoint II. Despite this importance of B cells as the initial Ag-transporting B cells in adaptive immunity, the identity and unique property of these initial Ag-transporting B cells and how these B cells can affect the development of autoimmune disease are not well known (55). Our present study suggests that MZ-P B cells are the dominant B cells that transport Ag directly into a GC in BXD2 mice. In the context of autoimmunity, this continuous supply of Ag to the GCs can potentially drive and maintain the generation of autoantibody-producing plasma cells. However, in BXD2-Ifnαr−/− mice, MZ-P B cells carrying Ag are now relegated to the periphery near the FO–MZ border in proximity to the MS and, thereby, prevent the influx of Ag-delivery B cells into the inner FOs. The present results suggest that production of IFN-α in the MS to drive the FO migration of Ag-transporting MZ-P B cells into the GC area exhibits the potential to break the closely monitored checkpoints I and II of the T-dependent humoral responses, leading to the development of spontaneous GCs and pathogenic autoantibodies in BXD2 mice.
We thank Marion L. Spell of the UAB AIDS FACS Core Facility and Enid Keyser of the Arthritis and Musculoskeletal Disease Center Analytic and Preparative Cytometry Facility for operating the FACS instrument. We thank Albert Tousson of the High Resolution Imaging Facility for assistance with operating the confocal imaging equipment. We thank Dr. Jocelyn Demengeot, Instituto Gulbenkian de Ciência, Oeiras, Portugal, for providing B6-Ifnαr−/− mice. We thank Dr. Fiona Hunter for expert review of the manuscript and Carol Humber for excellent secretarial assistance.
Disclosures The authors have no financial conflicts of interest.
This work was supported by a grant from the American College of Rheumatology Research and Education Foundation Within Our Reach: Finding a Cure for Rheumatoid Arthritis campaign, the Alliance for Lupus Research – Target Identification in Lupus program, a Department of Veterans Affairs Merit Review Grant (1I01BX000600-01), Daiichi-Sankyo, and National Institutes of Health Grants (1AI 071110-01A1 and ARRA 3RO1AI71110-02S1) (all to J.D.M.), The University of Alabama at Birmingham Skin Diseases Research Center (to L.T.), and the Arthritis Investigator Award supported by the Arthritis Foundation (to H.-C.H).
The online version of this article contains supplemental material.
Abbreviations used in this paper:
- N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N trimethylammonium methylsulfate
- follicle or follicular
- germinal center
- marginal sinus
- marginal zone
- marginal zone precursor
- plasmacytoid dendritic cell
- peanut agglutinin
- quantitative real-time PCR
- University of Alabama at Birmingham
- Received March 17, 2009.
- Accepted October 21, 2009.
- Copyright © 2010 by The American Association of Immunologists, Inc.