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* South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio, TX 78284; Departments of
Medicine and
Pathology, University of Texas Health Science Center, San Antonio, TX 78229;
Veterans Administration Center for Research on AIDS and HIV-1 Infection, San Antonio, TX 78229; and
¶ Department of Molecular Genetics and Microbiology and
|| Institute of Cellular and Molecular Biology, University of Texas, Austin, TX 78712
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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bm12 mice), we sought to elucidate the role of the donor non-T cell compartment and molecular determinants therein in the pathogenesis of GVHD. In this model the acute GVHD-inducing effects of purified B6 wild-type (wt) CD4+ T cells was inhibited by wt non-T cells in a dose-dependent manner. Paradoxically, unlike the chronic GVHD phenotype observed in bm12 mice transplanted with B6wt unfractionated splenocytes, bm12 recipients of B6ccr2-null unfractionated splenocytes developed acute GVHD and died of IFN-
-mediated bone marrow aplasia. This switch from chronic to acute GVHD was associated with increased target organ infiltration of activated CD4+ T cells as well as enhanced expression of Th1/Th2 cytokines, chemokines, and the antiapoptotic factor bfl1. In vitro, ccr2-/- CD4+ T cells in unfractionated splenocytes underwent significantly less activation-induced cell death than B6wt CD4+ T cells, providing another potential mechanistic basis along with enhanced expression of bfl1 for the increased numbers of activated T cells in target organs of B6ccr2-/- splenocytebm12 mice. Collectively, these findings have important clinical implications, as they implicate the donor non-T cell compartment as a critical regulator of GVHD and suggest that ccr2 expression in this cellular compartment may be an important molecular determinant of activation-induced cell death and GVHD pathogenesis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to these observations in humans, a careful analysis of the elegant work of Sprent et al. (8, 9) provides indirect evidence for an important role for the non-T cell compartment in murine GVHD. In the current study, using the well-established murine model of GVHD used by Sprent et al. (8, 9), we sought to provide direct evidence for a role for the non-T cell compartment in GVHD pathogenesis and to determine some of the molecular determinants in this cellular compartment that may impact on the course of GVHD.
In agreement with the findings of Sprent et al. (8, 9), we confirm that in a murine model system in which a single MHC class II-disparate Ag induces GVHD (B6bm12 mice), irradiated bm12 recipients of CD4+ T cells purified from the unfractionated splenocytes of B6 wild-type (wt) mice developed acute GVHD, whereas, paradoxically, those transplanted with wt unfractionated splenocytes developed chronic GVHD. Based on these differential GVHD phenotypes, we hypothesized that the non-T cell compartment in unfractionated splenocytes negatively influences the acute GVHD-inducing effects of CD4+ T cells. In support of this hypothesis, we found that non-T cells cotransplanted with CD4+ T cells inhibited the acute GVHD induced by T cells in a dose-dependent manner.
Based on these findings, we sought to determine potential molecular determinants in the non-T cell compartment that may confer protection against the acute GVHD mediated by purified CD4+ T cells. In recent years there has been a burgeoning literature documenting various members of the chemokine system as key mediators of immune responses, and they have been implicated in diverse disease states ranging from atherosclerosis to HIV-1 infection (10, 11, 12). Furthermore, because they serve as critical mediators of leukocyte recruitment and activation, various members of the chemokine system may potentially play a critical role in the migration of activated cells to target organs during GVHD. In support of a role for this class of molecules in GVHD, there are a handful of studies implicating macrophage inflammatory protein-1
(MIP-1) and its receptor, CCR5, in different model systems of murine GVHD (13, 14, 15).
For the aforementioned reasons, we surmised that chemokine receptors may serve as critical molecular determinants of GVHD pathogenesis. Since recent clinical studies have implicated monocytes as an important cellular determinant in inhibiting the acute GVHD induced by donor T cells during PBSCT (6, 7), we sought to determine whether CCR2 expression serves as a molecular determinant of GVHD, as it is highly expressed on monocytes and is a high affinity receptor for monocyte chemotactic protein-1 (11, 12, 16). Our findings indicate that this is indeed the case, and we elucidated some of the mechanisms by which the expression of this receptor on donor cells influences disease pathogenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Culture reagents RPMI 1640, DMEM, antibiotics, FCS, HEPES, PBS, and 2-ME were purchased from Invitrogen (Grand Island, NY). All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) and fluorescent-labeled Abs were purchased from BD PharMingen (San Diego, CA).
Mice
All mice were on a C57BL/6J (B6, H-2b) background. Eight- to 12-wk-old B6.C-H2bm12/KhEg (bm12), B10.BR (H-2k), and B6-IFN-R-/- mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Ccr2-/- and ccr5-/- mice (each were F10 generation on C57BL/6 background) and appropriate litter backcrossed wt controls were described previously (17, 18). All mice were maintained in microisolator cages and received neomycin-treated, acidified water. By FACS we found that the proportion of T cell subsets (CD4+; CD8+, NK1.1) and B cells (B220+) in the spleens of wt and ccr2-/- mice were similar (data not shown).
Model of murine chronic GVHD
Recipient bm12 mice received 600 cGy of irradiation from a 137Cs source 8–12 h before cell transfer. Donor mice were euthanized, and spleens were harvested. Single-cell suspensions of splenocytes were depleted of RBC using RBC lysis buffer and were resuspended in PBS. Recipient mice received 10 x 106 or 2.5 x 106 spleen cells in 200 µl of PBS via tail vein injection. All mice were followed until end-point analysis (see below).
T cell purification
CD4+ T cells were magnetically purified using the CD4 Multisort Kit according to the instructions of the manufacturer (Miltenyi Biotec, Auburn, CA), and 1 x 105 CD4+ T cells were adoptively transferred to recipient bm12 mice. The negative fraction isolated after magnetic sorting was designated the non-T cell compartment, and this population was cotransplanted with purified CD4+ T cells in varying ratios as depicted in Fig. 1a. The purity of the purified CD4+ T cells and non-T cell compartment was consistently >95%. For bone marrow transplantation experiments, 2 x 106 bone marrow cells derived from B6wt or B6IFNR-/- mice were cotransplanted with 10 x 106 B6ccr2-/- unfractionated splenocytes into sublethally irradiated bm12 mice.
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Mice were monitored every other day for the presence of skin changes, diarrhea, and weight loss. In addition to weight loss of >10% of initial body weight, clinical features such as hunched posture, ruffled fur, pallor, and inability to feed were taken into consideration, and such moribund animals were euthanized. In initial experiments we found that the majority of the bm12 recipients of CCR2-deficient splenocytes lost 8–10% of their body weight by 3–4 wk post-transplant and succumbed rapidly after this time point. For immunological studies equal numbers of bm12 recipients of wt or syngeneic unfractionated splenocytes were also sacrificed at this time point to provide controls for the mice transplanted with CCR2-null splenocytes.
Histopathology
The organs (liver, spleen, skin, lungs, and gastrointestinal tract) were harvested and fixed in 10% buffered formalin. The sections were stained with H&E and examined by light microscopy. Long bones were also harvested, fixed in 0.4% paraformaldehyde, and subsequently decalcified, sectioned, and stained with H&E. Individual sections were read by R. L. Reddick, a pathologist (blinded to the groups), for evidence of GVHD using a quantitative scale as described previously (19, 20).
Isolation of liver and bone marrow-infiltrating leukocytes
Liver-infiltrating leukocytes were prepared as described previously by Yoneyama et al. (21). In brief, livers harvested from recipient mice were minced, pressed through a stainless steel mesh, and resuspended in 10% FCS/DMEM. The cell suspensions were treated with 33% Percoll containing heparin (100 U/ml) and centrifuged at 2000 rpm for 10 min to remove liver parenchymal cells. The pellet was treated with RBC lysis solution, washed and resuspended in 10% FCS/DMEM, and used for FACS and cytokine production assays. In some experiments the bone marrow was harvested by flushing the long bones such as femur with 5–10 ml of cold PBS. The resulting cell pellet was depleted of RBC, and leukocyte subtypes were identified by FACS.
Cell labeling and culture
A red fluorescent membrane linker PKH26 (Sigma-Aldrich) was used to label the donor cells to visualize their migration in bm12 mice (22). To quantify and visualize the fractions of dividing T lymphocytes that were undergoing activation-induced cell death (AICD) in vitro we used the methods described by Wells et al. (23). Single-cell suspensions of splenocytes (1 x 107 cells/ml) resuspended in PBS were incubated with equal volumes of 5 µM CFSE. (Molecular Probes, Eugene, OR) for 5–10 min at room temperature. Unbound CFSE was quenched by addition of an equal volume of FCS. The labeling efficiency was 99%, and all cells remained labeled for the duration of the 72-h cell culture. The labeled cells were washed twice and resuspended in RPMI 1640 containing 10% FCS, 25 mM HEPES, and 5 µM 2-ME at a concentration of 2 x 106 cells/ml in 24-well plates. Soluble CD3 Ab (2 µg/ml) was added to stimulate the T cells. After 72 h in culture, the CFSE-labeled splenocytes were washed in cold PBS, 2% FCS, and 0.01% sodium azide and stained with allophycocyanin-conjugated mAb against CD4 and annexin V-PE in the presence of Fc-blocking Ab (blocks nonspecific binding to FcR). After staining, a vital dye, TOPRO-3 (Molecular Probes), was added to each sample before acquisition of FACS data to distinguish between live and dead cells. In some experiments cells were costimulated with soluble CD28 (5 µg/ml) and CD3 (2 µg/ml) Abs.
Flow cytometry and ELISA
Four-color flow cytometry was performed on a FACSCalibur scan (BD Biosciences, San Jose, CA) using CellQuest software, fluorescence compensation was achieved using appropriate single fluorochrome-labeled samples, and 100,000 events were collected. FACS analysis of the liver-infiltrating cells was performed as described previously (17). ELISA was performed using commercial kits (from BD PharMingen and R&D Systems (Minneapolis, MN)) as described previously (17, 22).
Donor lymphocyte chimerism was assessed by determining the number of B6 (H-2b) CD4+ T cells in the spleens of B10.BR (H-2k) mice 7 days after cell injection. Unfractionated splenocytes (10 x 106) derived from B6wt or B6ccr2-/- mice were injected i.v. into sublethally irradiated (600 cGy) MHC class I and class II disparate B10.BR mice. Splenocytes from B10.BR mice were labeled with allophycocyanin-conjugated CD4+, PE-conjugated H-2Kb, and FITC-conjugated H-2Kk murine Abs obtained from BD PharMingen. Control mAb (mouse IgG2a; BD PharMingen) values were subtracted from those obtained with the relevant mAbs. Ten thousand events were analyzed by four-color flow cytometry using a FACScan (BD Biosciences). Cells were gated for lymphocytes based on forward and side scatter settings.
RNase protection assay
RNase protection assay was performed to detect and quantify mRNA expression using the RiboQuant Multiprobe RNase protection Assay Kit (BD PharMingen) as described by the manufacturers.
Statistical analysis
Time curves for progression to death were prepared by the Kaplan-Meier method using SAS (version 6.12; SAS Institute, Cary, NC). Significance between animal groups was computed by t test or the Wilcoxon rank/median test depending on whether the data were normally distributed.
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Results |
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As described previously by Sprent et al. (8, 9), we found that sublethally irradiated bm12 mice transplanted with B6wt unfractionated splenocytes (B6wtbm12) had minimal lethality during the first 90 days post-transplant (Fig. 1a; Kaplan-Meier plot 1 and data not shown). In contrast, and as would be expected based on the well-described pathogenic effects of CD4+T cells in GVHD, inoculation of purified B6wt CD4+ T cells was associated with a rapid disease course (Fig. 1a; compare Kaplan-Meier plots 1 and 4). These findings indicated that the non-T cell compartment present in the B6wt unfractionated splenocytes may mediate the differential disease course observed in bm12 mice receiving B6wt CD4+ T cells vs those transplanted with B6wt splenocytes. Supporting this conclusion, transplantation of increasing concentrations of the B6wt non-T cells along with B6wt CD4+ T cells ameliorated the acute GVHD induced by CD4+ T cells (Fig. 1a). This protective effect mediated by the non-T cell compartment was not complete and may have been related to the partial loss and/or altered ratios of specific leukocyte subtypes during the purification step of the transplanted cells (data not shown).
Unfractionated CCR2-null splenocytes induce an acute GVHD phenotype
The clinical course of bm12 mice transplanted with B6ccr2-/- CD4+ T cells (Fig. 1b; Kaplan-Meier plot 5) was similar to that of B6wt CD4+ T cellsbm12 mice (Fig. 1a; Kaplan-Meier plot 4). Paradoxically, the clinical course of bm12 recipients of B6ccr2-/- splenocytes was not similar to that of mice transplanted with B6wt splenocytes (Fig. 1b; compare Kaplan-Meier plots 2 and 4). Indeed, B6ccr2-/- splenocytesbm12 mice had an aggressive clinical course that was more reminiscent of that induced by B6wt or B6ccr2-/- CD4+ T cells than the chronic GVHD induced by B6wt splenocytes (Fig. 1, a and b). Transplantation of lower doses of B6ccr2-/- splenocytes (2.5 x 106) into bm12 hosts induced a similar acute GVHD phenotype (n = 20 mice/group; Kaplan-Meier plots not shown). The specificity of a dominant role of CCR2 expression in donor cells in GVHD pathogenesis was further demonstrated by the observation that mice transplanted with splenocytes from mice lacking expression of another highly related chemokine receptor, namely CCR5, had a clinical course identical with that observed in B6wt splenocytesbm12 mice (Fig. 1b).
Acute GVHD in B6ccr2-/- unfractionated splenocytesbm12 mice: clinicopathological correlates
Two weeks after transplantation, B6ccr2-/- splenocytesbm12 mice were lethargic and had a hunched posture as well as ruffled fur; however diarrhea, a classical feature of acute GVHD, was not observed. The clinicopathological features in the livers of B6ccr2-/- splenocytesbm12 mice were also atypical. Although histopathological analysis revealed that the livers of B6ccr2-/- splenocytesbm12 mice had a diffuse lymphoid infiltrate predominantly in the portal and perisinusoidal areas, there was no evidence of hepatitis, endothelialitis, or nonsuppurative destructive cholangitis (Fig. 2, a–c).
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bm12 mice is due to bone marrow aplasiaOn clinical inspection we observed that mice transplanted with wt or CCR2-null CD4+ T cells had marked pallor. Sprent et al. (8, 9, 24) in a similar model of GVHD demonstrated that this lethality induced by CD4+ T cells was secondary to fulminant bone marrow aplasia. In agreement, we found that mice transplanted with wt CD4+ T cells induced anemia, leukopenia, and bone marrow aplasia (Table I), and the inoculation of CCR2-deficient CD4+ T cells induced a similar hematological profile.
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bm12 mice was due to bone marrow aplasia, a cause of death typically associated with transplantation of CD4+ T cells in this model system. Notably, the degree of pallor as well as the hematological profiles of mice transplanted with CCR2-null splenocytes were similar to or more severe than those found in recipients of wt or CCR2-null CD4+ T cells (Table I). Histopathologically, the bone marrows of B6ccr2-/- splenocytesbm12 mice were aplastic (data not shown), and there was a marked reduction in cell numbers in the bone marrows of these mice (Table I).
Compared with mice that were sublethally radiated but not transplanted, B6wt splenocytesbm12 mice also developed leukopenia, anemia, thrombocytopenia (Table I), and reduced bone marrow cellularity; however, the extent of bone marrow aplasia was not as prominent as that found in B6ccr2-/- splenocytesbm12 mice (Table I). The hematological profiles shown in Table I reflected changes detected in recipient mice 3 wk post-transplantation, and re-examination of the bone marrows at later time points revealed that the aplasia in B6wt splenocytesbm12 was transient, whereas it was persistent in B6ccr2-/- splenocytesbm12 mice (data not shown). Mirroring the temporary hematological crisis observed in B6wt splenocytesbm12 mice, Sprent et al. (8, 9, 24) also observed that bm12 recipients of wt B6 splenocytes underwent a crisis at 
2 wk post-transplantation (hunched posture and lethargy), but then achieved full recovery.
Accelerated GVHD in B6ccr2-/- unfractionated splenocytesbm12 mice is linked to increased infiltration of activated CD4+ T cells
We speculated that the shift from a chronic to an acute GVHD phenotype as well as bone marrow aplasia in mice transplanted with donor CCR2-null splenocytes were secondary to enhanced infiltration of activated CD4+ T cells and cytokine/chemokine expression in the target organs. Also, to indirectly elucidate the role of CCR2 expression in the non-T cell compartment in GVHD pathogenesis, we determined whether these effector mechanisms were similar in mice transplanted with B6ccr2-/- splenocytes or CD4+ T cells (B6wt or B6ccr2-/-), but distinct from those transplanted with wt splenocytes.
Three observations support the idea that the degree of infiltration of CD4+/CD44+ T cells in bm12 recipients of CD4+ T cells or splenocytes derived from wt, CCR2-null, or syngeneic bm12 mice correlated with the GVHD phenotype (acute vs chronic) in these animals. First, paralleling the nearly identical aggressive disease course in recipients of wt or CCR2-null CD4+ T cells, the extent of infiltration of CD4+/CD44+ T cells in the spleens and livers was similar in these two groups of mice (Fig. 2, d and e). The degree of CD4+/CD44+ T cell infiltration in these two groups of mice was significantly higher than that in mice transplanted with syngeneic CD4+ T cells that had a benign disease course (Fig. 2, d and e). Second, the chronic GVHD course in recipients of B6wt splenocytes was associated with a lesser degree of infiltration in the spleens and livers of CD4+/CD44+ T cells compared with animals transplanted with B6wt or CCR2-null CD4+ T cells (Fig. 2, d and e). Third, the percentage of CD4+/CD44+ T cells in spleen and liver is higher in bm12 recipients of B6ccr2-null splenocytes compared with bm12 recipients of B6wt splenocytes. (Fig. 2, d and e).
CCR2 expression influences AICD of CD4+ T cells and apoptotic pathways
Having determined that CCR2 expression influenced the degree of accumulation of activated CD4+ T cells in target organs, we sought to understand the potential mechanisms underlying this observation. The expansion of lymphoid cells is a tightly regulated process, and apoptosis is an important mechanism to offset uncontrolled proliferation and prevent damage of host tissues by an overly aggressive immune response (23, 25, 26, 27, 28, 29, 30). Activation of T lymphocytes can therefore promote either cell proliferation or apoptotic cell death. We hypothesized that a decrease in AICD and/or enhanced proliferation in the T cell compartment of splenocytes derived from B6ccr2-/- splenocytesbm12 mice might contribute in part to the increased numbers of activated T cells observed in these mice. However, the complex cascade of activation events induced by cell transplantation and GVHD can potentially confound analyses of the role of CCR2 expression in AICD; thus, we used an in vitro approach to test our hypothesis. An additional advantage of an in vitro approach is that we can synchronize the cell cycle of T cells (26).
Recent studies by several groups have shown that analysis of T cell proliferation and apoptosis can be assayed quantitatively after labeling cells with CFSE (31, 32). After excluding the dead cells using a vital dye, we used annexin V and CD4 staining in conjunction with CFSE to quantify the extent of apoptosis in each cell division. Following stimulation with CD3, CFSE-labeled splenocytes from nontransplanted B6wt or CCR2-null mice underwent similar numbers of cell divisions, suggesting that there were no gross defects in the proliferative capacity of CCR2-null splenocytes (Fig. 3, a–d). However, dual staining with annexin V and CD4 of the CFSE-labeled splenocytes revealed that the number of CD4+ annexin V+ T cells was significantly lower in the CD3-stimulated CCR2-null than in wt splenocytes (Fig. 3, e–i). Fig. 3j shows that CD3-stimulated CCR2-null cells had at least an 11–16% reduction in the percentage of CD4+ annexin V+ T cells in each cell division compared with CD3-stimulated wt splenocytes. This degree of inhibition in apoptosis is probably physiologically relevant, as we observed that after addition of Fas-Fc to anti-CD3-stimulated B6wt splenocytes there was a similar degree of inhibition of apoptosis (3–11%; data not shown), and these findings were in agreement those of Wasem et al. (33).
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bm12 mice may be secondary to reduced apoptosis following activation of CCR2-null splenocytes.
In light of the finding that B6ccr2-/- unfractionated splenocytesbm12 mice had increased numbers of activated CD4+ T cells that might be secondary to an impaired ability of CCR2-null CD4+ T cells in whole splenocytes to undergo AICD, we next determined whether we could link these findings to altered expression of various pro- and antiapoptotic factors in the splenocytes of B6ccr2-/- splenocytesbm12 and B6wt splenocytesbm12 mice (Fig. 4). We observed an up-regulation in the expression of the antiapoptotic gene bfl1 in the splenocytes of B6ccr2-/- splenocytesbm12 mice compared with B6wt splenocytesbm12 mice, whereas we detected no significant differences in these two groups of animals in the expression of other anti- and proapoptotic factors or caspases (Fig. 4, a and b; data not shown). One limitation of this analysis is that these in vivo data do not allow us to distinguish donor vs host cells and cell subtypes that express/up-regulate bfl1.
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bm12 mice is associated with a cytokine/chemokine storm with a highly skewed production of IFN-
We surmised that accumulation of activated CD4+ T cells would influence cytokine production in the target organs of B6ccr2-/- splenocytesbm12 mice. This was a relevant hypothesis to address because previous studies in humans and mice have demonstrated that dysregulated cytokine production, including the Th1 (IFN-) and Th2 (IL-4) profile in GVHD may determine the nature of the inflammatory response and the final outcome (34, 35, 36, 37, 38, 39, 40). We determined the 1) spontaneous, 2) Ag-induced (irradiated bm12 splenocytes were used as a source of APCs), and 3) Con A-induced production of cytokines IFN- and IL-4 in the spleens of bm12 hosts 3 wk post-transplant. Fig. 5, a–d, shows the findings obtained from splenocytes 48 h after stimulation with either Ag or mitogen. B6ccr2-/- unfractionated splenocytesbm12 mice produced significantly higher amounts of IFN- both spontaneously and in response to Ag (bm12 splenocytes; Fig. 5b). Con A-induced IFN- was not different among the different groups (Fig. 5c). The spontaneous and Con A-induced production of IL-4 was also enhanced significantly in B6ccr2-/- unfractionated splenocytesbm12 mice (Fig. 5, c and d). Enhanced production of IL-4 and IFN- was also observed at later time points (72 and 96 h) after stimulation (data not shown).
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bm12 and B6 splenocytebm12 mice. We found that the mean expression levels of several chemokines (lymphotactin, RANTES, eotaxin, MIP-1
, MIP-1, MIP-2, inducing protein-10, monocyte chemotactic protein-1, and T cell activating gene-3) was enhanced significantly in B6ccr2-/-bm12 mice compared with bm12 recipients of wt cells (Fig. 5e). Serum TNF- levels were similar in mice that were recipients of wt or CCR2-null splenocytes (Fig. 5f), suggesting that elevated levels of this cytokine were unlikely to be playing a major role in the GVHD pathogenesis in B6ccr2-/- splenocytesbm12 mice.
Accelerated GVHD in B6ccr2-/- unfractionated splenocytesbm12 mice is due to IFN--mediated bone marrow aplasia
The production of nanogram levels of spontaneous and Ag-induced IFN- in the spleens of B6ccr2-/-bm12 mice was striking (Fig. 5, a and b). There is extensive evidence documenting the myelosuppressive properties of IFN- in both humans and mice (41, 42, 43, 44). We therefore tested the hypothesis that the bone marrow microenvironment of mice transplanted with CCR2-null splenocytes also contains high levels of IFN- that secondarily induce profound bone marrow aplasia. In agreement with this hypothesis, Con A-stimulated bone marrow cells from B6ccr2-/-bm12 mice produced extremely high levels of IFN-, 100-fold higher than the amount of IL-4 produced (Fig. 5, g and h).
Using the identical murine model of GVHD studied herein, Welniak et al. (45) previously demonstrated a critical role for production of IFN- by donor CD4+ T cells in mediating acute GVHD. These authors showed that transplantation of IFN--null CD4+ T cells into bm12 hosts prevented the lethal bone marrow aplasia induced by IFN--intact CD4+ T cells (Fig. 6a). Based on this observation (Fig. 6a) and our experimental findings presented to date, the GVHD outcomes of transplanting CD4+ T cells or splenocytes based on the IFN- levels in the bone marrow microenvironment of these different donor groups are depicted in Fig. 6b. To provide a causal link between the high IFN- levels found in the bone marrow microenvironment of B6ccr2-/- splenocytesbm12 mice and accelerated GVHD in these mice, we transplanted bm12 mice with the donor cells shown in Fig. 6c. We envisaged the following scenario. The syngeneic B6 bone marrow cells cotransplanted with B6-CCR2-null splenocytes would repopulate the bone marrows of host bm12 mice. However, the high levels of IFN- found in the bone marrow microenvironment due to cotransplantation of CCR2-null splenocytes would be myelosuppressive and lead to acute GVHD (Fig. 6c). In contrast, cotransplantation of bone marrow cells null for the receptor for IFN- would protect these cells from the myelosuppressive effects of IFN- (Fig. 6c). In agreement with this conceptual scenario, we found that cotransplantation of IFN-R-null, but not IFN-R-intact, bone marrow cells protected mice from the detrimental effects of the cotransplanted CCR2-null splenocytes (Fig. 6d).
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The role of chemokines in leukocyte migration in different models of inflammation (e.g., asthma and experimental autoimmune encephalomyelitis) has been well documented (10, 11, 12), and they are also likely to be involved in the trafficking of the alloreactive donor T cells to target organs. We therefore excluded the possibility that the GVHD phenotype of B6ccr2-/- splenocytesbm12 mice was secondary to altered migratory properties of ccr2-null splenocytes compared with wt donor splenocytes. To examine this possibility, we labeled splenocytes derived from wt or ccr2-/- mice with a fluorescent membrane linker PKH26 and injected them into sublethally irradiated bm12 hosts. Previous studies have established that 24 h after i.v. injection, the sites of maximum accumulation of cells are the spleen, liver, and lung (46). We found that 24 h after inoculation of donor cells, both the percentage and the absolute number of PKH+ ccr2-/- and wt splenocytes in the spleens, livers, and lungs of the recipient bm12 mice were similar (Fig. 7 and data not shown), suggesting that gross differences in the migratory properties of the donor cells were unlikely to account for the contrasting GVHD phenotypes induced by unfractionated wt or ccr2-null splenocytes.
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We also excluded the possibility that the contrasting GVHD phenotypes induced by B6wt or B6ccr2-null splenocytes was secondary to the efficiency with which donor cells engrafted. Unfortunately, Abs are not available to distinguish between B6wt donor and bm12 recipient cells. An alternative approach to test this hypothesis was described recently by Welniak et al. (45). This approach involved inoculation of B6wt and B6ccr2-/- whole splenocytes (H-2b+) into sublethally irradiated MHC disparate B10.BR strain (H2k+; B10.BR). Seven days post-transplantation, the frequencies of B6ccr2-/- (0.31 ± 0.33) and B6wt (0.19 ± 0.16) cells in recipient B10.BR mice were similar (n = 5 mice/group; p = 0.3), suggesting comparable engraftment efficiencies by the donor cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several decades of studies in clinical and experimental transplantation have clearly demonstrated that donor T cells are the principal mediators of GVHD. However, with the increasing use of allogeneic PBSCT over the last decade several studies have begun to probe why the incidence and severity of acute GVHD after allogeneic PBSCT are not significantly different from those seen after allogeneic bone marrow transplantation despite the fact that the transplanted PBSCT allograft contains higher proportions of T cells. This lower incidence of acute GVHD after allogeneic PBSCT is thought to be due to quantitative differences in the leukocyte subsets in the transplanted PBSCs vs bone marrow cells. PBSCT grafts contain 50-fold more CD14 cells and only 10-fold more T cells than bone marrow cells (6, 7). These higher proportions of monocytes/monocyte progenitors as well as potentially other non-T cell leukocyte subsets are hypothesized to exert a suppressive effect on the alloantigen-induced T cell proliferation (6, 7). More recently, studies have also alluded to an important role for NK cell subsets in donor cells in GVHD pathogenesis (4, 50, 51).
In addition to elucidating that the non-T cell compartment is an important cellular determinant of GVHD pathogenesis, we identified CCR2 expression in this compartment as a potential molecular determinant of GVHD. Mice who received CCR2 wt unfractionated splenocytes developed chronic GVHD. In contrast, mice transplanted with CCR2-deficient splenocytes developed a switch from chronic to acute GVHD. We show that CD4+ T cells derived from CCR2 wt or null animals were equipotent in their ability to induce acute GVHD. This suggested that the shift from chronic to acute GVHD following transplantation of CCR2-null splenocytes was not due to an intrinsic defect in the CD4+ T cells that lack expression for CCR2. We integrated the GVHD phenotypes as well as the in vivo and in vitro cellular and immunological findings of this study (Table II) into a tentative model that might explain the mechanisms by which CCR2 expression influences GVHD pathogenesis (Fig. 8).
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bm12 mice, B6ccr2-/- splenocytebm12 mice had increased expression of bfl1, a member of the bcl family that has recently been shown to play an important role mediating antiapoptotic signals in T cells (52, 53). In vitro we did not observe any gross increase in the proliferation rate of CD4+ T cells in splenocytes lacking CCR2 expression, suggesting that this increase in activated T cell numbers may be secondary to a dysregulation in cell apoptosis. Supporting this, we found that CD3- as well as CD3/CD28-stimulated CD4+ T cells in whole CCR2-null splenocytes underwent reduced apoptosis. Whether this phenotype observed in our experiments is due to a defect solely in the T cell compartment and/or to a dysregulation in apoptosis-inducing signals in the non-T cell compartment is currently under investigation. Notably, there is growing appreciation of a role for the chemokine system, including CCR2, in apoptosis (54, 55, 56). Our findings might also shed light on the potential mechanisms underlying the bone marrow aplasia observed in some clinical situations following transplantation. For example, donor leukocyte therapy is used for treating patients with chronic myeloid leukemia who relapse following allogeneic bone marrow transplantation (57, 58). Although this form of therapy results in a remission rate >70%, the efficacy of donor leukocyte infusions and the ease of therapy are counterbalanced by the potential for significant toxicity. The reported treatment-related mortality rate of donor leukocyte therapy is almost 20%, and two of the major toxicities of this treatment are marrow aplasia and GVHD that occur in up to 50 and 90% of responders, respectively (57, 58).
In this context, a striking finding of this study was that transplantation of CCR2-null donor cells was associated with nearly complete bone marrow aplasia. Because transfer of syngeneic bone marrow cells along with CCR2-null splenocytes did not protect the recipient mice against bone marrow aplasia, it is conceivable that the depletion of the bone marrow progenitors occurred in a class II-independent manner. Full interpretation of our findings requires us to consider the findings of two prior studies. First, in classical work by Sprent et al. (24) using a similar model of GVHD, inoculation of B6 CD4+ T cells into lightly irradiated (B6xbm12) F1 hosts caused nearly 100% mortality by day 21, and the acute lethality was secondary to profound aplasia of host hemopoietic cells 2–3 wk after cell transfer. Sprent et al. (24) speculated that this destruction of hemopoietic cells by donor CD4+ T cells was limited to host class II+ cells and argued that the injected CD4+ cells destroyed host hemopoietic cells via direct class II-restricted CTL activity. However, the potential role of IFN- in mediating bone marrow aplasia was not determined by these authors. Second, Murphy’s group (45) have demonstrated that compared with inoculation of wt cells, transplantation of IFN- null CD4+ T cells has a limited ability to induce bone marrow aplasia.
Our findings suggest that the high level of IFN- in the bone marrow microenvironment may be a major determinant in the bone marrow aplasia observed in B6ccr2-/- splenocytesbm12 mice. These high levels of IFN- induced bone marrow aplasia in a class II-independent manner, as supported by the observation that cotransplantation of syngeneic B6 wt bone marrow cells with B6 CCR2-null splenocytes failed to ameliorate the bone marrow aplasia. However, cotransplantation of bone marrow cells from IFN-R-/- mice with CCR2-null splenocytes significantly protected bm12 recipients from bone marrow aplasia.
Thus, our findings suggest that recipients of CCR2-null whole splenocytes develop IFN--dependent bone marrow aplasia that may be secondary to two potential mechanisms: 1) a cytokine spillover from CD4+ T cells activated in a class II-dependent manner by local APCs (in this scenario the CD4+ T cells are not interacting directly with bone marrow progenitors), and 2) aplasia as a consequence of the production of IFN- by a non-CD4+ T cell type activated in a class II-independent fashion (in this scenario this non-CD4+ T cell type would undergo activation in the proinflammatory environment created by allogeneic T cells interacting with local APCs).
Several caveats need to taken into account when interpreting the findings presented in this paper. First, there are several murine models of GVHD, and the findings obtained using different models can be difficult to compare. Also, our model does not involve the use of lethal radiation before transplantation, a common characteristic of other models. However, the B6B6-bm12 model is based on the allogeneic response induced by a minimal (three-amino acid) antigenic mismatch between the host and the donor that may mimic the clinical transplantation setting, where it is preferable to avoid a complete mismatch (8, 9). Second, we do not directly show that the CCR2-deficient non-T cell compartment is responsible for the phenotype induced by transfer of CCR2-null splenocytes. However, because there was no difference in the phenotype induced by transfer of CD4+ T cells derived from the unfractionated splenocytes of CCR2-null or wt mice (regardless of the dose used; data not shown), we infer that CCR2-null CD4+ T cells do not have an intrinsically higher propensity to induce acute GVHD. Thus, the accelerated GVHD phenotype observed after injection of CCR2-null splenocytes, but not wt splenocytes, was probably due to the failure of the CCR2-deficient, non-T cell compartment to inhibit the acute GVHD-inducing effects of CCR2-null CD4+ T cells or to the combined defects of the T cell and non-T cell compartments in the CCR2-deficient splenocytes. Thus, we propose that the loss of CCR2 expression in the non-T cell compartment results in a dysregulation in the cross-talk that normally occurs between T cells and the non-T cell compartment (Fig. 8). Third, we did not identify the specific cell type in the non-T cell compartment that confers protection against GVHD. However, one good candidate is monocytes. This is based on the finding that CCR2 is highly expressed on monocytes, and previous clinical studies have implicated a potential role of monocytes in inhibiting alloantigen-induced T cell responses in GVHD (6, 7, 10, 12, 16).
Despite these caveats, our findings provide the impetus for a greater scrutiny of the role of leukocyte subsets in the non-T cell compartment and molecular determinants therein in modulating GVHD pathogenesis. Enrichment of this cell population before transplantation may be a valuable addition to transplantation strategies. Additionally, a further understanding of the mechanisms by which CCR2 expression in the non-T cell compartment may negatively influence allogeneic T cell responses may lead to novel therapeutic strategies to ameliorate GVHD.
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2 A.R.R. and M.P.Q. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Seema S. Ahuja, Department of Medicine (MC 7870), University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900. E-mail address: ahuja{at}uthscsa.edu
4 Abbreviations used in this paper: GVHD, graft-versus-host disease; AICD, activation-induced cell death; BMT, bone marrow transplant; MIP, macrophage inflammatory protein; PBSCT, peripheral blood stem cell transplant; wt, wild type.
Received for publication May 30, 2003. Accepted for publication August 14, 2003.
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is critical to the recruitment of CD8+ T cells to the liver, lung, and spleen during graft-versus-host disease. Blood 96:2973. cell outgrowth, and sustained neutrophilic inflammation. J. Exp. Med. 192:205. are overcome to control infection with Leishmania donovani in CC chemokine receptor (CCR) 5-, macrophage inflammatory protein-1-, or CCR2-deficient mice. J. Immunol. 163:5519. and IL-4 in acute graft-versus-host disease after allogeneic bone marrow transplantation in mice. J. Clin. Invest. 102:1742.[Medline]
constitutively expressed in the stromal microenvironment of human marrow cultures mediates potent hematopoietic inhibition. Blood 87:4149. by stromal cells inhibits murine long-term repopulating hematopoietic stem cell activity. Exp. Hematol. 27:895.[Medline]
and TNF-. Br. J. Haematol. 78:442.[Medline]
on CD4+ T cell-mediated graft-versus-host disease: effects of conditioning. Biol. Blood Marrow Transplant. 6:604.[Medline]
and CD40 signaling induces allospecific T cell anergy, preventing graft-versus-host disease. J. Clin. Invest. 109:549.[Medline]
B-dependent expression of antiapoptotic factors. J. Exp. Med. 194:1299.This article has been cited by other articles:
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  G. Socie and B. R. Blazar Acute graft-versus-host disease: from the bench to the bedside Blood, November 12, 2009; 114(20): 4327 - 4336. [Abstract] [Full Text] [PDF]
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|
|
|
C. M. Capitini, S. Herby, M. Milliron, M. R. Anver, C. L. Mackall, and T. J. Fry Bone marrow deficient in IFN-{gamma} signaling selectively reverses GVHD-associated immunosuppression and enhances a tumor-specific GVT effect Blood, May 14, 2009; 113(20): 5002 - 5009. [Abstract] [Full Text] [PDF]
|
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 |
|
 Z. Kaya, S. Goser, S. J. Buss, F. Leuschner, R. Ottl, J. Li, M. Volkers, S. Zittrich, G. Pfitzer, N. R. Rose, et al. Identification of Cardiac Troponin I Sequence Motifs Leading to Heart Failure by Induction of Myocardial Inflammation and Fibrosis Circulation, November 11, 2008; 118(20): 2063 - 2072. [Abstract] [Full Text] [PDF]
|
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S. Goser, M. Andrassy, S. J. Buss, F. Leuschner, C. H. Volz, R. Ottl;, S. Zittrich;, N. Blaudeck, S. E. Hardt, G. Pfitzer, et al. Cardiac Troponin I but Not Cardiac Troponin T Induces Severe Autoimmune Inflammation in the Myocardium Circulation, October 17, 2006; 114(16): 1693 - 1702. [Abstract] [Full Text] [PDF]
|
|
|
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|
|
S. Goser, R. Ottl;, A. Brodner, T. J. Dengler, J. Torzewski, K. Egashira, N. R. Rose, H. A. Katus, and Z. Kaya Critical Role for Monocyte Chemoattractant Protein-1 and Macrophage Inflammatory Protein-1{alpha} in Induction of Experimental Autoimmune Myocarditis and Effective Anti-Monocyte Chemoattractant Protein-1 Gene Therapy Circulation, November 29, 2005; 112(22): 3400 - 3407. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. H. Terwey, T. D. Kim, A. A. Kochman, V. M. Hubbard, S. Lu, J. L. Zakrzewski, T. Ramirez-Montagut, J. M. Eng, S. J. Muriglan, G. Heller, et al. CCR2 is required for CD8-induced graft-versus-host disease Blood, November 1, 2005; 106(9): 3322 - 3330. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. R Blazar and W. J Murphy Bone marrow transplantation and approaches to avoid graft-versus-host disease (GVHD) Phil Trans R Soc B, September 29, 2005; 360(1461): 1747 - 1767. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Wysocki, A. Panoskaltsis-Mortari, B. R. Blazar, and J. S. Serody Leukocyte migration and graft-versus-host disease Blood, June 1, 2005; 105(11): 4191 - 4199. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Wysocki, S. B. Burkett, A. Panoskaltsis-Mortari, S. L. Kirby, A. D. Luster, K. McKinnon, B. R. Blazar, and J. S. Serody Differential Roles for CCR5 Expression on Donor T Cells during Graft-versus-Host Disease Based on Pretransplant Conditioning J. Immunol., July 15, 2004; 173(2): 845 - 854. [Abstract] [Full Text] [PDF]
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0.01 for differences between B6wt
