1 Institute for Cellular Therapeutics, University of Louisville, Louisville, Kentucky
2 Department of Physiology and Biophysics, University of Louisville, Louisville, Kentucky
3 Department of Pathology, University of Louisville, Louisville, Kentucky
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
NOD mice have well-characterized abnormalities that affect antigen presentation (14), including defects in the protein kinase C activation pathway (3) and low expression levels of CD80 and CD86, which affect costimulation (5). The decreased expression of CD86 impairs T-cell activation and the upregulation of CD152, which is important in the induction of T regulatory cells (6). Langmuir et al. (7) demonstrated defective production of myeloid progeny in NOD bone marrow cells, most notably cells that coexpress Ly6C and heat-stable antigen (HSA), resulting in impaired responses to cytokines, including interleukin (IL)-3, granulocyte macrophagecolony stimulating factor (GM-CSF), and IL-5.
NOD mice do not produce normal myeloid dendritic cells in vivo (5,8,9). Until recently this was believed to be caused by a cell-intrinsic defect. However, when bone marrow cells from NOD mice are cultured in vitro, mature myeloid dendritic cells are produced (10). The significance of this paucity of myeloid dendritic cells in NOD mice was demonstrated when diabetes progression was delayed in naive NOD mice after the adoptive transfer of mature myeloid dendritic cells that were either incubated in vitro with islet cells or obtained from the pancreatic lymph nodes (11,12). In contrast, immature dendritic cells had no effect (12,13). Transfer of -interferontreated NOD dendritic cells also provided long-lived protection against autoimmunity (14). Taken together, these studies indicate that NOD mice have defective myeloid dendritic cells in vivo and that these cells are important in the maintenance of self-tolerance to the antigens that mediate autoimmunity and diabetes. The fact that normal levels of myeloid dendritic cells are generated in vitro indicates that the defect is extrinsic to the myeloid dendritic cells or their progenitors.
Mixed allogeneic chimerism eliminates autoimmunity in NOD mice (15). Prediabetic female NOD mice do not become diabetic after bone marrow transplantation (15). No insulitis is present after transplant, indicating control of the preexisting autoimmunity (15,16). We hypothesized that mixed chimerism supplies missing factors that correct the differentiation of myeloid cells. Notably, bone marrow cells from the chimeras have a distinct population of HSA+/Ly6C+ cells, whereas naive NOD bone marrow cells do not. Moreover, NOD cells contribute to the HSA+/Ly6C+ population, demonstrating that the defect is not an inability to produce myeloid precursor cells, but rather a lack of some factor or cellular interaction that instructs precursor cells to this lineage. In vitro culture of NOD bone marrow cells with Flt3-ligand resulted in production of myeloid cells similar to that observed in control bone marrow cells. Strikingly, Flt3-ligand treatment of NOD mice restored the production of HSA+/Ly6C+ myeloid progenitors and increased numbers of mature myeloid dendritic cells and plasmacytoid dendritic cells in the spleen and pancreatic lymph nodes. A significant increase in regulatory T-cells in pancreatic lymph nodes was also seen. Importantly, insulitis and diabetes progression were both significantly delayed in the Flt3-ligandtreated NOD mice. These data provide in vivo evidence that peripheral tolerance mediated by mature myeloid dendritic cells is critical to the control of autoimmune diabetes.
![]() |
RESEARCH DESIGN AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies.
All monoclonal antibodies (mAbs) used in this study were purchased from BD/Pharmingen (San Diego, CA) and included mAbs against HSA-phycoerythrin (PE), AA4.1fluorescein isothiocyanate (FITC), Ly6C-biotin or -FITC, H-2Kd-FITC, H-2Kk-PE, CD11c-allophycocyanin or -FITC, CD11b-APC or -FITC, B220-peridinin-chlorophyll-protein, CD80-FITC, and CD86-FITC.
Chimera preparation.
Bone marrow cells were harvested and resuspended to 100 x 106 cells/ml in chimera media (medium 199 and 50 µg/ml gentamicin; Gibco/BRL, Grand Island, NY). Fully allogeneic NOD chimeras were prepared by irradiating NOD mice (H2g7) with 1,000 cGy total body irradiation (TBI) (-cell 40; Nordion, Ottawa, ON, Canada) and administering 30 x 106 B10.BR bone marrow cells in chimera media as previously described (15). For mixed allogeneic chimeras, NOD mice were given either 750 cGy TBI 46 h before infusion with untreated 30 x 106 B10.BR bone marrow cells or 950 cGy TBI plus 40 x 106 T-celldepleted B10.BR bone marrow cells.
Assessment of chimerism.
Recipients were characterized for allogeneic engraftment using two-color flow cytometry 30 days posttransplantation as previously described (17). Briefly, whole blood was collected, and 100 µl aliquots were stained with antiH-2KdFITC and antiH-2KkPE for 30 min and either analyzed fresh or fixed in 1% formaldehyde (Polysciences, Warrington, PA) until analysis. Data were acquired on a FACSCalibur flow cytometer (Becton Dickinson, San Diego, CA) and analyzed using CellQuest software (Becton Dickinson).
In vitro culture of bone marrow cells.
Bone marrow cells from NOD, B10.BR, and C57BL/10 mice were harvested and placed in culture at 33°C at 5% C02. Cells were cultured at 1 x 106 cells/ml in long-term bone marrow cell media (Iscoves modified Dulbeccos medium [Gibco] supplemented with 20% horse serum [Gibco], 104 mol/l ß-mercaptoethanol [Sigma], 105 mol/l hydrocortisone [Sigma], 100 units/ml penicillin, 100 mg/ml streptomycin [Gibco], and 2 mmol/l L-glutamine [Gibco]) in the absence and presence of the following cytokines used singly or in combination: GM-CSF (103 units/ml; Genzyme, Cambridge, MA), stem cell factor (SCF) (1.2 units/ml; Genzyme), and Flt3-ligand (10 ng/ml; generously provided by Amgen, Thousand Oaks, CA). Cells were harvested at selected time points and stained for the coexpression of HSA and Ly6C as well as dendritic cell subsets: myeloid (CD11c+/CD11b+/B220) and plasmacytoid (CD11c+/CD11b/B220+) dendritic cells.
In vivo Flt3-ligand treatment of NOD mice.
Prediabetic (7- to 9-week-old) female NOD mice were treated daily for 10 days with 10 µg of Flt3-ligand subcutaneously. On day 11, peripheral blood and collagenase-digested spleen and pancreatic lymph nodes were analyzed for the presence of dendritic, T-, and natural killer cells and compared with untreated age-matched control NOD mice. Other groups were treated with the 10-day course of Flt3-ligand (10 µg/day) or saline and followed for diabetes progression or insulitis. Progression of diabetes was monitored by urine glucose testing using Chemstrip uGK test strips (Roche, Indianapolis, IN). From the fixed pancreatic samples, 0.7-µm sections were obtained from control and Flt3-ligandtreated mice, stained with hematoxylin and eosin, and assessed for insulitis as previously described (18). A minimum of 120 islets were scored per sample on a blinded basis.
Statistical analysis.
Diabetes progression curves of the Flt3-ligandtreated or saline controls were compared using SPSS for Windows 11.0.1 statistical software package. A Cox-regression analysis was performed with the age at diabetes set as the timed event. Each treatment group consisted of 16 mice followed until at least 40 weeks of age. Log-rank results indicate that the P value comparison between the two curves was 0.0084 or P < 0.01.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The bone marrow HSA+/Ly6C+ population in B10.BR and C57BL/10 mice is myeloid.
To determine the basis for the cellular deficiency in the HSA+/Ly6C+ population in NOD, bone marrow cells were harvested from NOD, B10.BR, and C57BL/10 mice and analyzed for other lineage markers (Fig. 2A). When the HSA+/Ly6C+ cells were gated and analyzed for the expression of CD11b and CD11c, nearly 100% of the HSA+/Ly6C+ population was CD11b+ (Fig. 2). Of these cells, 30% also expressed CD11c, a phenotype typical of myeloid dendritic cells (Fig. 2C vs. F and I).
Fully chimeric NOD mice express the HSA+/Ly6C+ cell population similar to donor strain levels.
To examine whether chimerism corrected the myeloid defect in the NOD marrow, B10.BRNOD chimeras were prepared, and 1 month after transplantation, the recipients were typed for donor chimerism. All mice (n = 9) exhibited >99% donor B10.BR-derived cells in peripheral blood. Bone marrow cells from chimeras were harvested and examined for the coexpression of HSA and Ly6C. The HSA+/Ly6C+ cell population in the chimeric NOD bone marrow (68.5 ± 19.0%) was significantly greater compared with naive NOD mice (2.0 ± 1.2%, P < 0.05) (Fig. 3A), and was similar to the HSA+/Ly6C+ cell population in naive B10.BR mice (64.9 ± 13.9%, P = 0.35).
|
The HSA+/Ly6C+ population was further analyzed for the relative contribution by NOD versus B10.BR bone marrow cells using flow cytometry. Figure 4A depicts a representative HSA/Ly6C stain of the total bone marrow cell population from a B10.BRNOD mixed chimera compared with controls. Both B10.BR-derived H2-Kk+ cells and NOD-derived H2-Kd+ cells from chimeric mice contributed to the HSA+/Ly6C+ population (Figs. 4BE). These data indicate that the lack of the HSA+/Ly6C+ population in naive NOD mice is not due to an intrinsic inability to produce myeloid precursors, but rather due to a lack of signal(s) that would instruct precursor cells to this lineage.
|
In vitro culture of NOD bone marrow cells results in the expression of an HSA+/Ly6C+ cell population.
We next determined whether NOD bone marrow cells could be induced to produce the HSA+/Ly6C+ population in vitro. Bone marrow cells from NOD, B10.BR, and C57BL/10 mice were harvested and placed in culture with the following hematopoietic growth factors used singly or in combination: GM-CSF, SCF, and Flt3-ligand. Interestingly, culture of NOD bone marrow cells in long-term bone marrow cell media alone resulted in a slight increase in the HSA+/Ly6C+ population, from 0.6% in fresh cells to 6.8% after 7 days in culture (Fig. 5A). The HSA+/Ly6C+ population decreased in the B10.BR and C57BL/10 cultures with time. The HSA+/Ly6C+ populations in the 7-day cultures were lower than in fresh bone marrow cells, ranging from 52.8 to 24% for B10.BR cells and from 44.7 to 32% for C57BL/10 cells. However, the percentage of cells in these cultures was always higher than that in the NOD cultures (Fig. 5A). Viable cells were not obtained on day 10, when the cells were cultured in media alone; therefore, no day 10 results are shown.
|
All of the HSA+/Ly6C+ cells are CD11b+, with many of the cells coexpressing CD11c. When NOD bone marrow cells were cultured with Flt3-ligand, the percentage of CD11b+/CD11c+ myeloid dendritic cells present within the HSA+/Ly6C+ cell population increased to a level similar to that in B10.BR and C57BL/10 cultures, with each culture reaching the highest percentage of myeloid dendritic cells on day 5 (Fig. 5C). In addition, the HSA+/Ly6C+/CD11c+ population also stained brightly for MHC class II (data not shown), indicative of mature myeloid dendritic cells. Taken together, these data indicated that culture of NOD bone marrow cells with Flt3-ligand was sufficient for differentiation of the myeloid precursor cells into mature myeloid dendritic cells, confirming the in vivo observation that precursor cells are present in NOD marrow but need the proper signal for differentiation to occur.
In vivo treatment of NOD mice with Flt3-ligand decreases insulitis and delays onset of diabetes.
We therefore hypothesized that treatment of NOD mice with Flt3-ligand in vivo would restore myeloid cell differentiation and lead to the generation of mature myeloid dendritic cells, which would help to control the peripheral autoimmune processes that lead to diabetes. To test this hypothesis, 7- to 9-week-old prediabetic NOD mice were treated with either Flt3-ligand (10 µg/day) or saline for 10 consecutive days and monitored for diabetes progression. The first of the untreated NOD mice became diabetic at 16 weeks of age (Fig. 6A). At 40 weeks, 75% of untreated mice had developed diabetes. In the Flt3-ligandtreated group disease progression was significantly delayed or prevented (P < 0.01). The first conversion to diabetes in the Flt3-ligandtreated group did not occur until 24 weeks of age, and 70% of the animals remained disease-free at 44 weeks.
|
Flt3-ligand treatment of NOD mice increases HSA+/Ly6C+ cells in bone marrow and mobilizes predominantly myeloid dendritic cells.
We evaluated Flt3-ligandtreated NOD mice for an increase in HSA+/Ly6C+ cells in the bone marrow and mobilization of myeloid dendritic cells and predendritic cells into peripheral blood. The HSA+/Ly6C+ population is indeed significantly increased in the NOD bone marrow after Flt3-ligand treatment (26.8 ± 2.2%) compared with untreated NOD bone marrow cells (3.7 ± 0.3%; P < 0.01). Although both dendritic cell subsets were detected in the peripheral blood, the majority were of myeloid dendritic cell phenotype (Fig. 6D), paralleling the in vitro data for culture of NOD bone marrow cells with Flt3-ligand. There was also a significant increase in both myeloid dendritic cells and predendritic cells in the spleen and pancreatic lymph nodes after treatment with Flt3-ligand as long as 5 weeks after treatment (Tables 1 and 2, respectively). Notably, the myeloid dendritic cells obtained from the pancreatic lymph nodes after Flt3-ligand treatment were significantly increased in number and had increased cell surface expression of both CD80 and CD86. However, CD80 and CD86 expression was not increased in the myeloid dendritic cells found in the spleen (Tables 1 and 2). Increased costimulatory molecule expression is indicative of mature myeloid dendritic cells, which have been shown to delay diabetes onset upon adoptive transfer into naive NOD mice (13). Concomitant with the increase in mature myeloid dendritic cells in the pancreatic lymph nodes is a sixfold increase in the numbers of CD4+/CD25+ T-cells in the Flt3-ligandtreated NOD mice compared with untreated controls (P < 0.05), indicative of the generation of T regulatory cells (Fig. 6E).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
We first reproduced the observation that bone marrow from NOD mice contained a significantly lower percentage of HSA+/Ly6C+ cells compared with BALB/c (7). Although a significant difference between NOD and B10.BR mice in the lymphocyte-gated HSA+/Ly6C+ population was detected, an even more striking difference was observed when myeloid cells were included in the analysis (Fig. 4A). Hence, total bone marrow cells were used to analyze all data reported. The HSA+/Ly6C+ population detected in naive B10.BR mice has the same mean fluorescence intensity as the HSA+/Ly6C population in the naive NOD bone marrow cells (Fig. 4), possibly representing a precursor stage where the myeloid cells accumulate due to the lack of instruction for further development.
The establishment of chimerism in NOD mice restored the production of the HSA+/Ly6C+ population to normal levels. Bone marrow cells from fully allogeneic B10.BRNOD chimeras have a distinct HSA+/Ly6C+ population, significantly increased over that seen in naive NOD mice and similar to that in B10.BR bone marrow cells. This result was expected because fully allogeneic NOD chimeras have, in essence, the hematopoietic compartment of a B10.BR mouse. Strikingly, NOD mixed chimeras also developed the HSA+/Ly6C+ cell population. The percentage of HSA+/Ly6C+ cells did not correlate with the percentage of donor chimerism, as would be expected if the cells were only of donor origin. When the HSA+/Ly6C+ population was stained for donor versus recipient, we indeed confirmed that NOD-derived cells contributed to the restored HSA+/Ly6C+ myeloid population.
To confirm that NOD bone marrow cells could produce myeloid precursor cells, NOD bone marrow cells were cultured in the presence of hematopoietic growth factors in vitro. Steptoe et al. (10) recently demonstrated that culturing NOD bone marrow cells in the presence of IL-4 and GM-CSF not only induced the production of myeloid dendritic cells, but the NOD cultures had a greater percentage of dendritic cells when compared with cultures obtained from other mouse strains. Here we show that when NOD bone marrow cells were cultured in vitro with long-term bone marrow cell media alone, a small increase in the HSA+/Ly6C+ cells was detected (1% of the cells to 6%). When NOD bone marrow cells were cultured with Flt3-ligand, the percentage of HSA+/Ly6C+ cells was significantly increased by day 5 in culture and approached control strain levels on days 7 and 10. Although the numbers of NOD HSA+/Ly6C+ cells were low on day 3 of culture, by day 5 the levels were near to that of the control strains, reflecting normal differentiation of these cells. The decrease in the HSA+/Ly6C+ population detected in the B10.BR and C57BL/10 bone marrow cell cultures in the presence of Flt3-ligand may represent a normal maturation that occurs in the HSA+/Ly6C+ precursor population in response to Flt3-ligand, whereas the 5- to 10-day expression in the cultures are indicative of newly developed precursor cells (Fig. 5). Moreover, HSA+/Ly6C+ expression in bone marrow cells from all three strains exhibited similar kinetics after 3 days in culture with Flt3-ligand.
Notably, culture of NOD bone marrow cells with Flt3-ligand resulted in the production of HSA+/Ly6C+ myeloid dendritic cells. Interestingly, the CD11b+/CD11c+ population exhibited a higher peak of expression than that of the other mouse strains on day 5 of culture. These results are reminiscent of previous reports in that the dendritic cell population absent in NOD bone marrow cells can be generated and in fact is increased over other strains once placed in culture with differentiating cytokines (10) or under competitive reconstitution conditions (19).
We next asked whether the restoration of the HSA+/Ly6C+ population in vivo would delay diabetes progression. Several lines of evidence indicate that myeloid dendritic cells are important in the maintenance of peripheral tolerance in NOD mice. The induction of low-level apoptosis that leads to increased peripheral tolerance through T regulatory cells was mediated by increased numbers of myeloid dendritic cells in the pancreatic lymph nodes (20). Moreover, the transfer of ex vivo differentiated mature NOD myeloid dendritic cells decreases the incidence and progression of diabetes (12,13,21). Therefore, we hypothesized that treatment of NOD mice in vivo with Flt3-ligand would induce endogenous differentiation of myeloid dendritic cells needed to maintain peripheral tolerance, thus delaying diabetes progression. When we treated prediabetic NOD mice with a single 10-day course of Flt3-ligand, the incidence of diabetes was significantly decreased and the time of onset also significantly delayed. In addition, there was a marked reduction of insulitis in the NOD mice treated with Flt3-ligand. The increase of myeloid dendritic cells, as well as the sixfold increase in CD4+/CD25+ T-cells in the pancreatic lymph nodes, was striking and supports the hypothesis that Flt3-ligand treatment of NOD mice enhances mechanisms of peripheral tolerance. Moreover, the observation that the myeloid dendritic cells generated in the spleen had a lower level of expression of CD80 and CD86 would further support this mechanism of disease progression because this phenotype induced a delay in disease onset after adoptive transfer in NOD mice (13).
CD4+/CD25+ T regulatory cells have been shown to play an important role in the maintenance of tolerance and prevention of autoimmunity (13). NOD mice exhibit impaired production of T regulatory cells (13). It is of note that Flt3-ligandtreated NOD mice contained significantly greater numbers of CD4+/CD25+ cells in their pancreatic lymph node compartment compared with untreated controls. Although the CD4+/CD25+ T-cell phenotype can be indicative of newly activated T-cells, and is not strictly demonstrative of T regulatory cells, the significant increase in CD4+/CD25+ T-cells in the pancreatic lymph nodes of Flt3-ligandtreated mice is suggestive of an increase in regulatory cells because it occurred in mice demonstrated to have reversal of insulitis and a decreased incidence in overall diabetes. In addition, the increase in cells of this phenotype is reminiscent of the increase of regulatory cells in NOD mice observed after adoptive transfer of myeloid dendritic cells (13) or after the induction of low-level ß-cell apoptosis (20), both of which were associated with diabetes prevention.
Together, the data presented here demonstrate that NOD mice are lacking a key component for the differentiation of myeloid cells, and that the induction of mixed chimerism provides a needed factor that results in the production of myeloid precursors in the bone marrow. Whether Flt3-ligand initiates a cascade of events or is the key cytokine restored by chimerism is under evaluation. Notably, Flt3-ligand culture in vitro restores the production of HSA+/Ly6C+ cells and myeloid dendritic cells by NOD bone marrow cells. The in vivo treatment of NOD mice with Flt3-ligand increases the endogenous production of both myeloid and plasmacytoid dendritic cells in the pancreatic lymph nodes and the spleen and significantly decreases the incidence and delays the onset of diabetes. Further treatment with Flt3-ligand may increase the efficacy in controlling autoimmune diabetes and provide a relatively benign therapeutic approach for inducing and maintaining peripheral tolerance in autoimmune diseaseprone patients.
![]() |
ACKNOWLEDGMENTS |
---|
The authors thank Drs. Hilary McKenna, H. Leighton Grimes, and Thomas Mitchell for helpful suggestions; Sherry Willer for technical assistance; Douglas J. Lorenz for advice on statistical analysis; Carolyn DeLautre for manuscript preparation; the staff of the animal facility for outstanding animal care; and AMGEN for providing the growth factors.
Address correspondence and reprint requests to Suzanne T. Ildstad, MD, Director, Institute for Cellular Therapeutics, the Jewish Hospital Distinguished Professor of Transplantation, University of Louisville, 570 S. Preston St., Suite 404, Louisville, KY 40202-1760. E-mail: suzanne.ildstad{at}louisville.edu
Received for publication January 28, 2004 and accepted in revised form April 29, 2004
FITC, fluorescein isothiocyanate; GM-CSF, granulocyte macrophage-colony stimulating factor; HSA, heat-stable antigen; IL, interleukin; mAb, monoclonal antibody; MHC, major histocompatibility complex; PE, phycoerythrin; SCF, stem cell factor; TBI, total body irradiation
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|