Loss of RARγ Results in Perturbed Hematopoiesis, with Significantly Elevated Granulopoiesis
We have observed that RARα and RARγ, but not RARβ isoforms are widely expressed in immature and maturing hematopoietic cell types ((Purton et al., 2006
) and unpublished data). Furthermore, the natural ligand for RARs, ATRA, has potent effects on enhancing HSC self-renewal and promoting differentiation of more mature granulocyte/macrophage progenitors, which we have previously demonstrated are likely due to the different actions of RARα and RARγ on hematopoietic cells (Purton et al., 1999
; Purton et al., 2000
; Purton et al., 2006
mice do not have any observable hematopoietic defects in vivo (Kastner et al., 2001
; Purton et al., 2006
), whereas RARγ-/-
mice have significantly reduced numbers of HSCs accompanied by increased numbers of immature progenitor cells in their BM (Purton et al., 2006
). To determine if loss of RARγ also affected the production of mature hematopoietic cells, we examined the cellularity and hematopoietic composition of peripheral blood (PB), BM, spleen and thymus preparations obtained from RARγ null, heterozygous and wildtype mice.
Eight week old RARγ-/-
mice had significantly elevated PB and BM leukocytes compared to their wildtype littermates (). The elevated leukocyte levels were comparable to those achieved by oncoretroviral overexpression-induced models of MPD in C57Bl/6 background strains (Wernig et al., 2006
). Spleen leukocyte numbers were also elevated in RARγ-/-
mice, but this increase was not significant compared to the wildtype mice. Eight week old RARγ-/-
mice also presented with splenomegaly, with spleen weights significantly increased (1.5 to 3-fold) compared to their wildtype littermates. In contrast, the thymic cellularity was significantly reduced (25%) in RARγ-/-
mice compared to their wildtype littermates (). RARγ-/-
mice have a growth deficiency (Lohnes et al., 1993
), and at 8 weeks of age were approximately 20% smaller than their wildtype littermates (Table S1
). When normalized to body size the thymocyte cellularity was similar to that of wildtype mice, however, there were significant increases in BM and spleen cellularity in RARγ-/-
8 week old RARγ-/- Mice have Elevated Numbers of Granulocytes
We used immunophenotypical analysis to further explore the effects of loss of RARγ on the production of mature hematopoietic cell lineages. There were marked increases in the numbers of CD11b+ Gr-1+ granulocytes in the PB ( and Table S2
), BM ( and Table S2
) and spleen ( and Table S2
) of RARγ-/-
mice compared to their wildtype littermates. These increases occurred in both the immature and mature granulocyte compartments in the BM (Walkley et al., 2002
) ( and Table S2
There were also significant reductions in the numbers of B lymphocyte subsets and erythrocytes in the BM of the RARγ-/-
mice (Tables S1
), however the numbers of these cells were unaltered in the PB and spleen (Table S1
). In contrast to RARγ-/-
mice, RAR +/-
mice did not have altered numbers of mature hematopoietic cells ( and Table S1
The Increased Granulocyte Compartment in RARγ Null Mice Arises at an Immature Progenitor Level
We further focused on investigating the mechanisms behind the increased granulopoiesis in the RARγ-/-
mice. Both the immature (CD11b+ Gr-1 dim) and mature (CD11b+ Gr-1 bright) granulocyte subsets were significantly elevated in the BM, suggesting that the increase in granulocytes was arising from an immature progenitor population (Walkley et al., 2002
). Furthermore, we have recently shown that RARγ-/-
mice have increased numbers of immature colony-forming cells (CFU-GEMM), which likely arise from accelerated differentiation of HSCs into progenitor cells (Purton et al., 2006
). We therefore investigated the numbers of more committed granulocyte progenitors in the BM of RARγ-/-
The numbers of day 7 GM-CSF, SCF+G-CSF and G-CSF-responsive colony-forming cells (CFCs) were significantly increased in RARγ-/-
BM compared to that of wildtype littermates (). The numbers of more mature day 3 cluster-forming cells were also significantly increased in these mutants compared to wildtype mice (). The PB and spleen of RARγ-/-
mice also had increased numbers of mature myeloid CFCs (CFU-GM) (), but the numbers of immature CFCs (CFU-GEMM) and immunophenotypic HSC/progenitor cells in these organs were not significantly different to RARγ+/+
mice (Table S3
). These data demonstrated that there were increased numbers of committed myeloid progenitors in the BM, spleen and PB of RARγ-/-
mice. In contrast, the lack of CFU-GEMM and HSC/progenitor cells in spleen and PB suggested that RARγ-/-
mice were not presenting with an HSC mobilization phenotype.
8 Week Old RARγ-/- Mice Have Increased Numbers of Granulocyte Progenitors
To determine if the increased numbers of progenitors could be due to altered cytokine sensitivity, we investigated the numbers of BM CFCs formed in submaximal and supramaximal concentrations of G-CSF (Walkley et al., 2002
). Both RARγ-/-
BM cells showed a similar cytokine response, hence it did not appear that the progenitor cells had altered sensitivity to cytokines (). A similar result was also observed in response to IL-3 (unpublished data).
It was possible that the increased numbers of CFCs were due to increased survival of these progenitors. To assess this, we established CFC assays and delayed cytokine addition to 24 and 48 hours. The numbers of CFCs that formed were then assessed at 7 days post-initiation. There were no differences in the survival of progenitors stimulated with G-CSF (), suggesting that the myeloid expansion was not a secondary response due to altered cell death of granulocyte progenitors.
Finally, we assessed the frequencies of common myeloid progenitors (CMP), granulocyte/macrophage progenitors (GMP) and megakaryocyte/erythroid progenitors (MEP) in RARγ-/-
BM (Akashi et al., 2000
). The frequency of GMPs was significantly increased (1.8-fold) in RARγ-/-
BM compared to that of their wildtype littermates (). Given that RARγ-/-
mice also had significantly increased BM leukocyte cellularity compared to RARγ+/+
mice (), this resulted in an overall significant 2.1-fold increase in absolute numbers of GMPs in RARγ null BM compared to wildtype BM.
RARγ Null Granulocytes have Normal Functional Potential
The elevated granulopoiesis in RARγ-/-
mice could be compensatory if these cells had impaired functional capacity, hence we tested several of their functional properties. The oxidative bursts response was similar in BM samples of either genotype (Figure S1
). Furthermore, recruitment of cells after intradermal injection of Zymosan A (which elicits an acute inflammation response) was similar in RARγ-/-
mice compared to their wildtype littermates (Figure S1
), and there was no difference between the morphology of the cell types that were recruited to the ear in response to Zymosan A (data not shown). Finally, there was no difference in the percentage of BM granulocytes undergoing apoptosis when assessed by annexin V staining (Figure S1
). These data suggested that RARγ-/-
granulocytes had similar functional potential to those of RARγ wildtype granulocytes.
Ageing RARγ Null Mice Have a Profound Myeloproliferative-Like Disease with Excessive Extramedullary Hematopoiesis
We have recently reported that small numbers of RARγ-/-
mice survive to approximately 12 months of age (Purton et al., 2006
). To determine if the increased granulocyte phenotype persisted for the lifespan of RARγ-/-
mice we investigated the cellularity and hematopoietic composition of different organs obtained from 12 month old RARγ-/-
The average PB leukocyte counts of the older RARγ-/- mice were dramatically increased compared to their wildtype littermates (RARγ+/+= 10.54 ± 0.51; RARγ-/-= 29.6 ± 3.32 × 103/μl blood, P<0.005). This was accompanied by significantly increased numbers of granulocytes in the PB, spleen and BM (). Platelets were also significantly elevated in the PB of these older knockout mice (RARγ+/+= 1480 ± 46.7; RARγ-/-= 1888 ± 66.7 × 103/μl blood, P<0.005). In contrast, erythrocyte numbers were significantly reduced in the PB of these aged mutant mice (RARγ+/+= 9.77 ± 0.32; RARγ-/-= 8.87 ± 0.15 × 106/μl blood, P<0.05).
12 Month Old RARγ-/- Mice Have Profoundly Elevated Numbers of Mature and Immature Granulocytes and Progenitor Cells
Analysis of immature progenitors in these mice revealed that RARγ-/- mice had significantly increased numbers of CFU-GEMM and CFU-GM in their PB and spleen (), accompanied by strikingly elevated numbers of lineage-restricted granulocyte/macrophage CFCs in their bone marrow (). Significantly reduced numbers of mature B cells and erythrocytes were also observed in the bone marrow of the 12 month old RARγ-/- mice ().
The frequencies of the lineage-negative, c-kit-positive, Sca-1-negative (LKS-) progenitor cells were significantly increased in BM and spleens of 12 month old RARγ-/-
mice (Table S4
). The HSC-containing lineage-negative, c-kit-positive, Sca-1+ (LKS+) cells were also markedly elevated in the spleens of these mice (Table S4
Histological analysis of different organs of 9-12 month old mice revealed that the bone marrow of the RARγ-/-
mice was extremely hypercellular compared to their wildtype littermates (). The trabecular bones were virtually absent, and the cortical bones were dramatically thinner in these older RARγ-/-
mice (). Bone marrow cells of the RARγ-/-
mice were predominantly developing myeloid cells, with some megakaryocytes also obvious (). Reduced B lymphocyte foci were also evident in the spleens of the RARγ-/-
mice compared to their wildtype littermates (Figure S2
). Myeloperoxidase staining revealed that extramedullary hematopoiesis was occurring in the liver of the RARγ-/-
mice, however we did not observe significant numbers of hematopoietic cells in their kidneys compared to their wildtype littermates (Figure S2
). Strikingly, there were large foci of immature and maturing hematopoietic cells (including granulocytes, monocytes, megakaryocytes and erythrocytes) developing in adipose tissue in these older of the RARγ-/-
mice, but not in their wildtype littermates (). During their lifespan, however, none of the animals developed leukemia or lymphoma.
Ageing RARγ-/- Mice Exhibit a Profound Myeloproliferative-Like Disease. Representative sections of organs from 9-12 month old RARγ+/+ or RARγ-/- mice
Given the significantly increased myeloid compartment in the mice with tissue infiltration, together with lack of evidence of malignant transformation, the phenotype of the RARγ-/-
mice best corresponds with that of a myeloproliferative-like disease (MPD-like, or MPS) (Kogan et al., 2002
The Myeloproliferation in RARγ Null Mice Is Not Intrinsic to the Hematopoietic Compartment, but Is Induced by the RARγ Null Microenvironment
Myeloproliferative-like diseases are thought to arise from hematopoietic cells (Kogan et al., 2002
). In a previous study we investigated the HSC frequency in the RARγ-/-
BM (Purton et al., 2006
). During the 6 months of monitoring recipient mice post-transplant we did not observe a MPS in wildtype mice that were transplanted with BM from the RARγ-/-
mice. However, these transplants were performed with competing BM from wildtype congenic mice, which may have masked or prevented the occurrence of the myeloproliferation. Therefore we repeated the transplants using whole BM without competing cells and compared the hematopoietic phenotypes to that of 8 week old RARγ null mice ().
RARγ-/- Mice Have a Microenvironment-Induced Myeloproliferative Syndrome
In contrast to the MPS observed in RARγ-/- animals, at 8 weeks post-transplant all wildtype recipients of either RARγ+/+ or RARγ-/- BM had similar PB leukocyte and granulocyte counts (). The PB cellularity did not increase during the 6 months the mice were monitored post-transplant (at 6 months post-transplant, leukocytes: RARγ+/+= 13.72 ± 0.93; RARγ-/-= 11.5 ± 0.61 × 103/μl blood; granulocytes: RARγ+/+= 0.91 ± 0.17; RARγ-/-= 0.80 ± 0.12 × 103/μl blood). Bone marrow and spleen cellularity and lineage contribution were also comparable between the recipients of the two genotypes (data not shown). Complete reconstitution by RARγ+/+ or RARγ-/- cells was confirmed by immunophenotypical analysis at each time point of analysis, hence these results were not due to an inability of the RARγ-/- BM cells to engraft in the congenic recipients. These data therefore demonstrate that the myeloproliferation did not occur when RARγ-/- hematopoietic cells were supported by a wildtype microenvironment.
To determine if the microenvironment of the RARγ null mice was inducing the MPS we performed reciprocal transplants. Congenic wildtype cells were transplanted into lethally irradiated RARγ+/+
recipient mice. By 5 weeks post-transplant, RARγ-/-
mice transplanted with wildtype cells had significantly elevated PB leukocytes and granulocytes (, Table S5
). The increases in leukocytes and granulocytes were even more profound compared to 8 week old non-transplanted RARγ-/-
mice (, Table S2
). Immunophenotypical analysis confirmed the hematopoietic cells were of wildtype origin rather than endogenous recovery of RARγ null hematopoietic cells.
In addition to the markedly elevated granulocytes in these transplant recipients, BM B lymphopoiesis and erythropoiesis were also significantly suppressed when the wildtype congenic BM was transplanted into the RARγ-/-
mice (, Table S5
). This was also more profound compared to the B lymphocyte and erythrocyte phenotype observed in the BM of 8 week old RARγ-/-
mutants (Table S1
). Hence, the myeloproliferative-like disease observed in RARγ-/-
mice was not intrinsic to the hematopoietic cells, but was induced by the RARγ deficient microenvironment.
The RARγ Deficient Microenvironment Has Normal HSC Niche Potential
We have previously reported that 8 week old RARγ-/-
mice have three-fold reduced numbers of HSCs accompanied by increased numbers of progenitors, including day 12 colony-forming unit-spleen (CFU-S) (Purton et al., 2006
). Given that the trabecular bone has been described as being a key component of the HSC niche (Calvi et al., 2003
; Zhang et al., 2003
) and that trabecular bone was virtually absent in 12 month old RARγ-/-
mice (), we wished to determine whether the microenvironment of 8 week old RARγ-/-
mice was impaired in its ability to support HSCs.
Histological sections of undecalcified tibiae revealed significantly reduced trabecular bone in 8 week old RARγ-/- mice. Histomorphometric quantitation of the tibiae sections from RARγ-/- mice demonstrated significantly fewer and more dispersed trabeculae, decreased trabecular volume but normal trabecular thickness in tibiae obtained from RARγ-/- compared to wildtype mice (LEP and NA Sims, manuscript in preparation). This resulted in an overall 1.6-fold reduction in the number of trabeculae in these mice. Despite this, there were similar percentages of osteoblasts per bone surface in 8 week old RARγ-/- mice compared to wildtype mice. In contrast, the reduction in trabecular bone was accompanied by 1.6-fold increased numbers of osteoclasts per bone surface, indicating increased osteoclastogenesis was the predominant cause of the osteopenia in 8 week old RARγ-/- mice (LEP and NA Sims, manuscript in preparation).
To assess whether this reduction in trabecular bone impaired HSC self-renewal, we transplanted lethally irradiated RARγ+/+
recipient mice with wildtype congenic BM. At 8 weeks post-transplant (a similar time of exposure to the niche as that of the 8 week old mutants used in our previous studies (Purton et al., 2006
)) we assessed the numbers of CFCs, CFU-S and HSCs (using the limiting dilution assay) in the transplanted mice as previously described (Purton et al., 2006
; Szilvassy et al., 1990
; Walkley et al., 2005
The numbers of CFU-GM were significantly increased in PB and spleen, but not BM of RARγ-/-
recipient mice (Figure S3A-C
). In contrast, there were no differences in the numbers of CFU-GEMM, LKS
+, CFU-S or HSCs when wildtype BM was exposed to an RARγ-/-
microenvironment compared to an RARγ+/+
niche (Figure S3D-H
). In vitro co-culture assays revealed that RARγ-/-
stromal cells increased the production of maturing wildtype BM cells during 14 days of culture (Figure S3I
). Collectively, these data suggest that the RARγ-/-
microenvironment increases the proliferation and production of relatively mature hematopoietic cells, but does not affect the numbers of immature progenitors and HSCs.
Loss of RARγ Results in Markedly Increased Production of TNFα
In order to further elucidate the mechanisms behind the myeloproliferation in the RARγ-/-
mice, we examined the expression of different inflammatory mediators, myeloid cytokines and JunB, which is a putative target of retinoic acid (Balmer and Blomhoff, 2002
), and induces an MPD (Passegue et al., 2004
), in the hematopoietic organs of the RARγ mutants.
The expression of TNFα was significantly increased in all three hematopoietic organs in RARγ-/-
mice compared to their wildtype littermates (). In contrast, expression of other pro-inflammatory cytokines, IL-2 and IL-6 were not markedly altered in the organs (Table S6
). The expression of IL-4 was significantly reduced in the BM and slightly but significantly elevated in the spleen of RARγ-/-
mice (Table S6
The Microenvironment-Induced Myeloproliferative Syndrome Observed in RARγ-/- Mice is Partially Due to Increased TNFα Signaling
The expression of JunB was not altered in the BM (Table S6
). Neither of the two major myeloid cytokines, GM-CSF or G-CSF were elevated in any organ (Table S6
). In contrast, G-CSF expression was significantly reduced in the thymus of RARγ-/-
mice compared to their wildtype littermates (Table S6
Given that we had not observed altered numbers of T lymphocyte subsets in RARγ-/-
mice (Table S1
), we further investigated the expression of TNFα in purified populations of T lymphocytes. TNFα was predominantly expressed by thymocytes expressing CD4 and/or CD8 (Table S6
), consistent with a previous report investigating the synthesis of TNFα by T lymphocyte populations (Giroir et al., 1992
TNFα is a pro-inflammatory cytokine that is known to reduce the numbers of B lymphocytes in the BM and preferentially promote granulopoiesis via reductions in CXCL12 expression in BM (Ueda et al., 2005
; Ueda et al., 2004
). CXCL12 was unaltered in RARγ-/-
BM and spleen, and was elevated in the thymus (Table S6
). Hence, the reduced B lymphopoiesis observed in RARγ-/-
BM (Table S1
) did not appear to be a result of reduced CXCL12 expression that normally occurs in the BM during inflammation.
The Microenvironment-Induced Hematologic Defects are Partially Rescued When RARγ Null Mice are Transplanted with TNFα Null Hematopoietic Cells
To determine the contribution of deregulated TNFα signaling to the occurrence of the MPS we transplanted RARγ+/+ or RARγ-/- mice with TNFα-/- BM. PCR genotyping of BM obtained at the time of analysis confirmed full engraftment of the transplanted mice with TNFα-/- BM. Furthermore, Q-RT-PCR studies on BM, spleen and thymus harvested from these mice confirmed that the levels of expression of TNFα in RARγ-/- recipients were reduced to that of the wildtype hosts transplanted with TNFα-/- BM (data not shown).
Transplantation of TNFα-/-
BM significantly reduced the MPS in the RARα-/-
recipient mice ( and S4
). When compared to the myeloproliferation observed in RARγ-/-
mice transplanted with wildtype BM, in those transplanted with TNFα-/-
BM the fold-increases in PB leukocytes were reduced by approximately 50% (). The numbers of BM leukocytes were not altered (data not shown), whereas the spleen leukocyte counts were restored to the levels of the wildtype transplant recipients ().
While still elevated above that of wildtype recipients, the fold-increases in granulocytes in all organs were reduced by approximately 50% in BM and spleen, and were 3-fold reduced in PB of RARγ-/-
mice transplanted with TNFα-/-
BM compared to those transplanted with wildtype BM (, S4
The numbers of B220+/IgM+ cells remained significantly lower in the BM of RARγ-/- recipients transplanted with TNFα-/- BM, however, the numbers of immature B220+/IgM- cells in these recipients were restored to that of wildtype recipients (). Finally, BM erythropoiesis in RARγ-/- recipients transplanted with TNFα-/- BM was restored to the levels observed in wildtype recipients (). These data indicate that deregulated TNFα production significantly contributes to, but is not the sole cause of, the myeloproliferative-like disease that is induced by the microenvironment of RARγ null mice.
Absolute Requirement of an RARγ Null Microenvironment to Sustain the Myeloproliferation
Approximately 1/3rd of the RARγ-/- recipients had PB leukocyte counts above 48 × 106 cells/ml (twice that normally achieved by oncoretroviral MPD in a C57BL/6 background) at 8 weeks post-transplant, and had to be euthanized due to poor condition. These mice also had severe anemia, reflected by significantly reduced PB erythrocyte counts and hemoglobin content (data not shown).
PB smears of these recipient mice revealed highly elevated numbers of circulating immature myeloid cells and numerous abnormal erythrocytes in the RARγ-/- recipients compared to the wildtype recipients (). FACS analysis confirmed the cells in both recipient types were of wildtype donor origin (), and showed that the majority (74.9 ± 1.89%, n = 3) of the PB leukocytes in the RARγ-/- recipients were immature myeloid cells co-expressing intermediate levels of Gr-1 and CD11b (). In contrast the same donor cells transplanted into a wildtype microenvironment had PB content of approximately 15.1 ± 1.55% cells (n = 4) expressing higher levels of Gr-1 and CD11b ().
Absolute Requirement for an RARγ-/- Microenvironment to Sustain the Myeloproliferative-Like Disease
To determine if leukemic transformation of the cells had occurred, we transplanted whole spleen cells consisting of 2.5 × 106 leukocytes (containing 25% immature granulocytes by FACS) from one such primary RARγ-/- recipient () into lethally irradiated wildtype and RARγ-/- secondary recipient mice and monitored their recovery during 8 weeks post-transplant.
At 8 weeks post-transplant the average CD45.1+ donor reconstitution was similar between groups (approximately 80%, data not shown). Surprisingly, the MPD-like phenotype was highly dependent upon the secondary recipient genotype, demonstrating that the hematopoietic cells had not undergone leukemic transformation during the primary transplant. When injected into wildtype secondary recipients, the percentage of donor-derived granulocytes at 8 weeks post-transplant reverted to 26.6 ± 6.36% (n = 5), with many mature granulocytes evident (). In marked contrast, when injected into RARγ-/- recipients the splenic leukocytes reconstituted an average of 78.87 ± 9.31% immature granulocytes in the PB of these recipients (, n = 5, P<0.003 wildtype vs RARγ-/- recipients). These data therefore revealed that an RARγ null microenvironment was absolutely required to maintain the MPS.