1 Department of Pathology and 2 Department of Medicine, University of Utah School of Medicine, 50 N Medical Drive, Salt Lake City, UT 84132, USA
Correspondence to: J. H. Weis; E-mail: john.weis{at}path.utah.edu
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Abstract |
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Keywords: B cells, microphthalmic, interferon, marrow, RANK/RANKL, mouse
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Introduction |
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Two contrasting models can be proposed to account for the depressed levels of B cell precursors in the marrow of osteopetrotic mice. The first suggests that the reduced bone cavity does not possess enough space to support B cell development: similar losses in B cell numbers are evident in a variety of osteopetrotic model systems (6). An alternative model suggests that the osteopetrotic marrow is inhospitable for B cell development due to the over/under-expression of products, such as cytokines or chemokines, required for B cell precursor development. To test this second model, we analyzed normal and mi total marrow samples for the aberrant expression of marrow products. The mi marrow demonstrated elevated expression of receptor activator of nuclear factor B (RANK) ligand (RANKL) (7), a gene product intimately involved in the regulation of bone morphology (8). In addition, we also observed the elevated expression of Fragilis 5, a gene whose expression is regulated, in part, by type I IFNs (9), and elevated expression of SDF-1
and BLC, two chemokines important in the development of B lineage cells in the marrow (1013).
The process of bone homeostasis is maintained by a dynamic interplay between osteoclasts (which resorb bone) and osteoblasts (which form bone). During bone development, RANKL, found on osteoblasts as well as bone marrow stromal cells, T cells and pro-B cells, binds to RANK to initiate osteoclastogenesis (1417). RANK is found on osteoclasts, dendritic cells, activated T cells and hematopoietic precursors (14, 18, 19). Binding of RANKL to RANK has also been shown to induce the expression in osteoclasts of c-fos, a transcription factor involved in osteoclast differentiation, which in turn induces the expression of IFN-ß. Mice deficient in a subunit of the type I IFNR (IFNAR1/) or IFN-ß (IFN-ß/) demonstrated an increase in the number of osteoclasts in the bone when compared with wild-type mice. It has been proposed that the induction of IFN-ß through RANKL signaling via the c-fos pathway serves as a negative regulator of osteoclastogenesis (20).
IFN-ß, along with the IFN- family, comprises the type I IFNs, a group of pleiotropic-acting cytokines that function as part of the innate immune system (21). It has been demonstrated that IL-7-responsive lymphoid progenitors (B cells) were susceptible to IFN-
/ß-induced apoptosis at certain stages of development in vitro (22). Further in vivo investigations showed a reversible block in B cell development with type I IFN treatment, beginning with the pro-B cell stage (23). Type I IFNs act through a receptor that includes a common
subunit; cells lacking this IFNAR1 subunit are insensitive to the effects of these cytokines (24).
The preceding information led us to propose a model to explain the deficiency of B cell precursors in the marrow of mi mice. We propose that the environment of the mi marrow leads to the over-production of RANKL by osteoblasts and/or stromal cells in an attempt to increase the functional activity of resident osteoclasts. These osteoclasts respond to the excess RANKL by over-activation of c-fos and the over-production of IFN-ß. The release of excess IFN-ß within the marrow leads to the expression of IFN response genes (such as Fragilis 5) as well as the induction of apoptosis of maturing B lymphocytes. In order to test this model, we generated mice that were homozygous for the mi mutation and which also lacked the IFNAR1 protein. These mice demonstrated a partial restoration of maturing B cells and precursors in the marrow, suggesting that IFN signaling in the mi marrow is detrimental to B cell development.
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Methods |
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A PCR-based genotyping assay was used to detect the 3-bp deletion in the mi gene and the presence of the neo-cassette and disruption of IFNAR1 in genomic DNA. Primers used were as follows: Mitf-F 5'-TGACGTCAGTACGCACATGC-3', Mitf-R 5'-GCAGATGAACATGCGATTGTAC; IFNAR1-F 5'-AAAAGACGAGGCGAAGTGG-3', IFNAR1-R 5'-CATTCCACGAAGATGTGCTG-3' and neo-R 5'- AATTCGCCAATGACAAGACGC-3'. PCR parameters were 95°C denaturation (1 s), 60°C annealing (1 s) and 72°C extension (5 s) for 25 cycles using an Air Thermo Cycler (Idaho Technologies) (25). Fragments were electrophoresed on an 8% bis-acrylamide (BioRad) gel for 2 h at 65 W for adequate separation. Gels were exposed to film for 1 h (32P label). Mitf forward and reverse primers yielded a 195-bp fragment for wild-type animals, 192-bp fragment for mi animals and both sizes for heterozygote animals. IFNAR1 forward and reverse primers produced a 149-bp fragment. IFNAR1 forward and neo reverse primers yielded a 249-bp fragment.
Matings yielded mi IFNAR1/ mice with similar physical characteristics as normal mi mice. Wild-type littermates from mi and mi IFNAR1 KO breeding pairs were used as control animals. The genotype for mice used in each experiment is indicated in the figures. Experiments used age-matched mice.
FACS analysis
Single-cell suspensions were obtained from the bone marrow and spleen of each animal. Femurs and tibias from wild-type littermates were flushed with PBS + 0.1% BSA. Bones from mi and mi IFNAR1-deficient animals were sliced with a razor blade to release the cells. Spleens from each animal were mechanically disrupted through a cell strainer. RBCs were lysed in ACK lysis buffer (0.15 M NH4 Cl, 10.0 mM KHCO3 and 0.1 mM Na2EDTA) for 5 min at room temperature. Cells were aliquoted into tubes for staining and re-suspended in PBS + 0.1% BSA containing 10% mouse serum, 10% rat serum, antibody and Fc blocker (CD16/CD32, BD PharMingen). Cells were incubated in the antibody mixes for 20 min on ice. The following antibodies were used: from BD PharMingen, B220 (clone RA3-6B2), CD19 (1D3), Ly-6G/Gr-1 (RB6-8C5), CD3 (145-2C11) and streptavidinPE; from Caltag Laboratories, F4/80 (C1:A3-1). Cells were stained with a secondary antibody and filtered through nylon mesh to obtain a single-cell suspension. Stained cells were analyzed on the BD FACScan and enumerated with CellQuest software. Isotype controls were used to detect non-specific binding. Figures 1 and 5 were based upon 10 000 live events while Figs 35 are representative of 5000 live events. Live cells were differentiated from dead cells by staining with the vital dye, 7-AAD (BD PharMingen).
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Colony-forming units assay
A total of 1 x 106 cells were isolated from the bone marrow of each animal as previously described. RBCs were lysed and live cells were counted via trypan blue exclusion. Cells were re-suspended in 0.3 ml volume of RPMI media (Invitrogen) supplemented with 10% FBS (Hyclone)/1% Penstrep (Invitrogen) and containing either 20 ng ml1 IL-7 or 100 ng ml1 SCF (R&D Systems). Cells and media mixtures were added to 3 ml of methylcellulose (M3231 -erythropoietin, -cytokines, Stem Cell Technologies) and vortexed. Duplicate wells of 1.1 ml were plated into separate wells of a 6-well dish and incubated for 7 days. After the incubation period, the total number of cell colonies was enumerated blindly. Data are representative of the average number of colonies between the duplicate wells for each experiment. Error bars were derived from averaging duplicate wells from each experiment.
Detection of type I IFN transcripts
RNA was isolated from the leg bones of +/+ and mi/mi mice as previously described and used as a template for cDNA synthesis (7). Relative transcript levels for IFN-, IFN-ß, IFN-
, IFN-
and limitin were evaluated for each sample via continuous fluorescence monitoring PCR using the LightCycler (Roche Diagnostics) (26). Copy numbers for each type I IFN were calculated using the LightCycler software and corrected by normalization to ß-actin transcript levels. Primers for type I IFNs were as follows: IFN-
forward, 5'-CTCTCCTGCCTGAAGGACAG and IFN-
reverse, 5'-CTGCTGATGGAGGTCATTGC; IFN-ß forward, 5'-CAAGAAAGGACGAACATTCG and IFN-ß reverse, 5'-AGACATTCTGGAGCATGTCT; IFN-
forward, TCCAGCAGTGTCTAGCACACAG and IFN-
reverse, TCCCATGTGTCTGGAGGAGC; IFN-
forward, TTCTGGGCAGTACCATGACCG and IFN-
reverse, ATTGAAGATAGTTAGTGCCAGAG; limitin forward, 5'-AAGTGCTGAAGAGCCCAAGAGAG-3' and limitin reverse, 5'-TAGCGGAAGAACCCTCGGAAGTAG-3'.
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Results |
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We previously demonstrated the elevated expression of IFN response genes in the marrow of mi mice, suggesting the presence of type I IFNs but the expression of such cytokines was not measured directly. To test this question, we screened multiple RNA preparations from wild-type and mi bone marrows for the expression of -IFNs (using a consensus sequence for the members of this gene family), IFN-ß and the other type I IFNs, limitin (also termed IFN-
), IFN-
and IFN-
. Real-time reverse transcription (RT)PCR analysis of these cytokines either could not detect expression levels over background (IFN-
, IFN-ß and IFN-
) despite robust detection in controls (LPS- or dIdC-treated macrophages) or there was no difference between the samples (IFN-
) (data not shown). However, the expression of limitin was elevated in the wild-type marrow compared with the mi sample (data not shown). Specifically, the wild-type sample demonstrated 3.05 limitin transcripts (±0.92 transcript) per 1000 ß-actin transcripts while the mi/mi marrow sample demonstrated 0.85 limitin transcripts (±0.25 transcripts) per 1000 ß-actin transcripts. The limitin transcript data were an average of four different samples examined from each genotype with a statistical significance of P < 0.05. Limitin has been described as a cytokine that specifically suppresses B cell lymphopoiesis by inducing the nuclear transduction of Daxx (3032). If the role of limitin in the marrow is, in part, to limit B cell expansion and the mi marrow lacks B cells and their precursors, then it is not surprising to see limited expression of this cytokine in the mi marrow compared with wild-type marrow. The same RTPCR assays were performed on total splenic RNA: only limitin, which was elevated in the mi compared with wild-type spleen, showed differences in expression (data not shown). Animals with the mi mutation have normal numbers and types of B cells in the spleen and lymph nodes due to the peripheralization of B cell development (such as in the spleen), thus limitin expression appears to coincide with sites of B cell lymphopoiesis.
The inability to demonstrate elevated levels of IFN-ß in the marrow of the mi animal (compared with wild type) did not eliminate the possibility that this cytokine may be instrumental in the RANKL-mediated exclusion of B cells in the developing animal since our analyses were done with animals 34 weeks of age instead of at birth when the marrow is seeded with precursors from the fetal liver. Additionally, localized concentrations of the cytokine in marrow microenvironments that could not be detected in a total RTPCR assay could still provide a potent enough signal to induce maturing B cells to undergo apoptosis. Thus, an alternative approach to this question would be to remove the ability of lymphocyte precursor cells to respond to IFN-ß in the marrow of the mi osteopetrotic marrow and determine if a partial or full restoration of lymphocyte precursors in the marrow would be observed. Accordingly, we created a colony of animals that were deficient in the type I IFNR (IFNAR1) and that were homozygous for the mutant mi allele. IFNAR1-deficient animals do not respond to IFN- or IFN-ß. The IFNAR1-deficient animals (wild type for Mitf) do have reduced trabecular bone mass (osteoporosis) due to an increase in osteoclast numbers (20); however, percentages of other bone marrow-derived cell types were normal (33). Analysis of cross-sections of femurs from the IFNAR1-deficient, mi/mi animal did show reduced bone density compared with that of the receptor-replete, mi/mi animal (data not shown). Percentages of offspring were similar to that of the receptor-positive, +/mi breeding (data not shown). The harvest of total cell numbers from the mi/mi IFNAR1 wild-type bone marrow was virtually identical to those obtained from the mi/mi IFNAR1 homozygous KO marrow (see below), indicating that total marrow space and cell occupancy was roughly equivalent.
Loss of type I IFN response restores B cells in the marrow of mi/mi mice
The mice described above possessing the mi mutation and lacking the IFNAR1 protein were compared with mice with the mi mutation but having a functional IFNAR1. Cells were harvested from the bone marrow and spleens of these mice and were stained with various cell-surface markers. In the bone marrow (Fig. 1A) the B220 antibody recognized a 1.6% positive cell population in the mi mouse while identifying a 7.7% positive population in the mi IFNAR1 KO, a 5-fold increase overall. This trend was duplicated with CD19 staining, illustrating a difference of 0.5% compared with 7.7% between these animals. Concomitant with the increased percentage of B cells in the marrow of the mi receptor-deficient animal was a decrease in cells committed to a granulocyte lineage (Gr-1 positive). Although the data shown in Fig. 1(A) are from single mi/mi and mi/mi IFNAR1 KO animals, a similar percentage increase of B cells was seen in identical experiments with additional animals. Specifically, three additional analyses demonstrated B220-positive cells from mi/mi, IFNAR1 wild-type marrow with an average of 3.1% (SD of 1.39), and from mi/mi IFNAR1-deficient marrow with an average of 11.2% (SD of 5.04; P < 0.05) (data not shown). Gr-1-positive cells from the same experiments were an average of 91.2% (SD of 0.77) for wild-type marrow, and from mi/mi IFNAR1-deficient marrow, an average of 72.5% (SD of 2.87; P < 0.001) (data not shown).
Interestingly, the number of macrophage lineage cells (F4/80 positive) also increased in these animals compared with those mi animals that possessed the IFNAR1 protein (7.3% compared with 1.5%; P < 0.04). The colonization of B cells in a peripheral lymphoid organ, such as the spleen, was not altered for mi animals in the presence or absence of the IFNAR1 protein (Fig. 1B). These data suggest that the presence of inhibitory doses of IFN-ß are localized in the marrow cavity and are not widespread in the animal.
The analysis of the IFNAR1-deficient mice was extended using the Mitf wild-type littermates. Analysis of bone marrow constituents between IFNAR1-replete and -deficient animals did not show any major alteration in cell types and percentages (Fig. 2). This was also the case of splenic constituents since there were no significant differences in B cell or T cell percentages between the IFNAR1+/+ and IFNAR1/ lineages (data not shown). Although the mi mutant protein can act as a dominant negative, no such effect was evident in these experiments (Fig. 2).
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A more accurate determination of potential precursor cells in the marrow is a colony-forming unit (CFU) assay in which lymphocyte precursors can be enumerated by growth in IL-7-supplemented methylcellulose, and granulocyte precursors can be similarly analyzed by growth in SCF-supplemented methylcellulose. Total marrow was obtained from wild-type animals (+/+), heterozygote animals (+/mi) or homozygous mi animals (mi/mi) with or without IFNAR1, plated in the presence of IL-7 or SCF, and CFUs were counted. The mi animals (regardless of the presence or absence of IFNAR1) routinely possessed 6 x 105 total marrow cells per two pooled femurs (data not shown). This quantity of cells was 10% of the quantity obtained from either mi/+ or +/+ animals regardless of the presence or absence of IFNAR1. Plates were blindly read by two individuals: duplicate wells were averaged. For IL-7-dependent CFU (Fig. 4A), the absence of IFNAR1 (black bar) resulted in a greater number of precursor cells compared with marrow cells that possess the receptor (white bar) for the three possible mi genotypes. We have never observed IL-7-dependent CFU present in mi/mi, IFNAR1+/+ samples (5) (Fig. 4A, far right panel). However, by removing the ability of these cells to respond to IFN-ß, IL-7 CFUs were now obtained from the marrow of the mi/mi animals. The marrows from such mice were also used to screen for SCF-dependent CFU. There was no difference in CFU obtained from the various mi genotypes with or without the IFNAR1 protein (Fig. 4B).
The enrichment for IL-7 CFU in the marrow of the Mitf wild-type and heterozygote animals lacking the IFNAR1 protein compared with those expressing the receptor (Fig. 4A, left and middle panels) (without the receptor, black bar) suggested that there is a pool of precursor cells in the marrow whose numbers were expanded in the absence of type I IFN signaling. We sought to identify these precursors by first depleting marrow samples of lineage-committed cells followed by FACS analysis of the remaining cells. For these analyses, C57BL/6 mice were compared with C57BL/6 mice lacking the IFNAR1 protein. Two different strategies were taken. First, cells expressing Ter119, CD19 and Gr-1 (thus already committed to B lymphocyte or granulocyte lineages) were removed by magnetic bead depletion. The remaining cells (which represent immature marrow cells with the potential to contribute to multiple lineages) were stained with antibodies against the c-kit receptor and B220. The IFNAR1-deficient marrow showed a significant increase in cells expressing c-kit only (8.5% wild type versus 36.2% for the KO) (Fig. 5A). In addition, the receptor-deficient marrow demonstrated the loss of a significant percentage of B220+ single-positive cells (18.56.2%).
The second strategy utilized marrow samples that were depleted of cells possessing Ter119 and Gr-1 (removing cells committed to a granulocyte lineages), and then counterstained for c-kit, B220 and the IL-7R (Fig. 5B). Depleted marrow obtained from the IFNAR1-deficient animal consistently included more immature precursor cells (double negatives) than did the wild-type animal. This difference is most evident in the B220/IL-7 analysis in which the wild-type animal possessed 19.7% double-positive B lineage precursors and 29.4% double-negative cells, while the receptor-deficient animal possessed 5.2% B lineage precursors and 48.3% double-negative cells. These data do not identify the end-stage capabilities of double-negative precursor cells, but do suggest that the lack of response to type I IFNs increases the percentage of uncommitted precursors in the marrow. It is not known which of the type I IFNs (including loss of response to limitin) would be responsible for the differences observed in the B cell precursor subsets.
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Discussion |
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Takayanagi et al. demonstrated that production of IFN-ß is induced in osteoclasts following RANK signaling (20). Their analyses considered this cytokine within an autocrine autoregulatory loop. However, an accentuation of this loop could lead to the release of excess IFN-ß. Type I IFNs including IFN-ß have been shown to inhibit the proliferation of IL-7-dependent bone marrow cells in short-term culture (22). These studies were expanded in vivo and demonstrated that mice treated with type I IFNs exhibited a block in marrow B cell differentiation (23, 37). Cooper and associates proposed that type I IFNs predispose maturing B cells for apoptosis (29). Mice lacking the type I IFNR do possess a skewed antibody repertoire (28). If differentiating B cells are indeed poised upon the razors edge of survival or apoptosis via type I IFN priming, then over-production of IFN-ß in the marrow would be expected to deplete the site of maturing B cells.
The generation of the mi mouse that lacked expression of the key receptor component for the type I IFNs allowed for us to directly test this hypothesis: these mice did show a return of B cell precursors (CD19+ and B220+) as well as a dramatic increase of marrow precursors in general (defined as Gr-1/B220 double-negative cells). It should be noted, however, that the level of B cell precursor reconstitution in the marrow of the mi IFNAR1-deficient animal did not equal that of the normal wild-type mouse, indicating that there are additional factors within the mi marrow that compromise B cell development.
The analysis of the control animals, those with one or two wild-type Mitf alleles, in the presence or absence of the IFNAR1, surprisingly demonstrated that the number of IL-7-dependent CFUs (per cell count) was elevated in the receptor-deficient animals compared with wild-type animals. The same phenomenon was not observed for SCF-dependent-CFUs, suggesting a direct inhibitory effect on IL-7-dependent lymphocyte precursor cells by the type I IFNs.
The key question raised by these data is why lymphocyte precursors returned to the mi marrow when type I IFN signaling was lost. The data derived from both the mi and Mitf wild-type animals lacking IFNAR1 demonstrated that normal type I IFN signaling suppressed the maintenance of lymphocyte progenitor cells (as defined by IL-7-dependent CFU) but that such signaling was required for full expansion of the maturing cells (as evidenced by the reduction of B220/IL-7R double-positive cells in wild-type, IFNAR1-deficient marrow). These data suggest that the type I IFNs are critical in the homeostatic maintenance of marrow lymphocyte precursors. Akin to the RANKL and RANK network for osteoblast and osteoclast functions, there must exist a type I IFN pathway that is functional in the normal marrow in the absence of any exogenous stimulation such as a viral infection. This pathway presumably places a restriction upon the key lineage checkpoints utilized by either (or both) uncommitted precursor cells or lymphocyte progenitors.
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Acknowledgements |
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Abbreviations |
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CFU | colony-forming unit |
KO | knockout |
mi | microphthalmic |
MITF | microphthalmia-associated transcription factor |
NIH | National Institutes of Health |
OPG | osteoprotegerin |
RANK | receptor activator of nuclear factor ![]() |
RANKL | receptor activator of nuclear factor ![]() |
RT | reverse transcription |
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Notes |
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Received 14 January 2005, accepted 26 August 2005.
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References |
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