Abstract
Extramedullary hematopoiesis (EMH) is a pathological process secondary to underlying bone marrow (BM) insufficiency in adults. It is characterized by the emergence of multipotent hematopoietic progenitors scattered around the affected tissue, most likely in the spleen, liver, and lymph node, etc. EMH in patients frequently receives less medical attention and is neglected unless a compressive or obstructive hematopoietic mass appears to endanger the patient's life. However, on a biological basis, EMH reflects the alteration of relationships among hematopoietic stem and progenitor cells (HSPCs) and their original and new microenvironments. The ability of hematopoietic stem cells (HSCs) to mobilize from the bone marrow and to accommodate and function in extramedullary tissues is rather complicated and far from our current understanding. Fortunately, many reports from the studies of drugs and genetics using animals have incidentally found EMH to be involved. Thereby, the molecular basis of EMH could further be elucidated from those animals after cross-comparison. A deeper understanding of the extramedullary hematopoietic niche could help expand stem cells in vitro and establish a better treatment in patients for stem cell transplantation.
Keywords
Introduction
Extramedullary hematopoiesis (EMH) is the production of mature blood cells outside of the medullary cavity of bones. This can be a physiological process, for example, during fetal development where hematopoiesis is initiated in the yolk sac (YS) followed by aorta–gonad–mesonephros (AGM), placenta, liver, and spleen before lodgment in the bone marrow (BM). However, it usually occurs postnatally as a pathological compensation due to a result of BM failure. EMH is often seen in patients with myelofibrosis, myeloproliferative disorders, and hemoglobinopathy, especially in thalassemia and sickle cell anemia (107). It is well known that cross talk between hematopoietic stem cells (HSCs) and their niches ensure steady-state hematopoiesis in the marrow. However, during pathological development, hematopoiesis occurs in extramedullary sites where microenvironments are still poorly defined. In this review, we have summarized the recent studies on EMH from clinical reports and publications in experimental hematology. In addition, we focus on the findings of EMH in laboratory animals that are experimentally manipulated. Embryos and fetuses are direct resources for the study of the interaction between HSCs and their niche outside the BM; however, animal models in adulthood with EMH created by drugs, microorganism infection, or a specific gene knockedin or deletion are not only informative about affected patients, but also provide a tool to resolve the molecular basis of hematopoiesis occurring in a variety of tissues (Table 1).
Extramedullary Hematopoiesis (EMH) in Manipulated Laboratory Animals
Patients with EMH in Atypical Sites
EMH can be microscopic or present as a mass lesion and shows features of so-called benign EMH with a mixed population of hematopoietic precursor cells including erythroid lineage, granulocytic lineage, and megakaryocyte (130). Four major theories involving changes in stem cells and/or their microenvironment can explain the development of most frequent occurrences of EMH: (i) severe BM failure; (ii) myelostimulation; (iii) tissue inflammation, injury, and repair; and (iv) systemically or locally abnormal production of chemokines (43). In most reported cases, the foci of EMH appear in the spleen and liver. Other sites, including the lymph nodes, retroperitoneum, spinal cord, kidneys, adrenal glands, gastrointestinal tract, lung, and breasts, are seen occasionally (20). Some clinical reports from 1994 to 2014 are briefly illustrated here for the emphasis of EMH that can present in many atypical locations (Table 2). Many of these cases have hallmarks in common, indicating that a new hematopoietic microenvironment could possibly be created in any of a patient's nonhematopoietic organs, while the demand of blood is increased during the progression of the diseases (cases 1–4, 7–10). However, EMH may also occur as an occasional event without anemia or specific underlying diseases (cases 5, 6).
Clinical Patients With EMH in Atypical Tissues
Case 1
A 50-year-old woman (92) with a history of myelofibrosis for 5 years was admitted to hospital after 48 h of diffuse abdominal pain, diarrhea, nausea, and vomiting. She received a splenectomy 8 months earlier for severe anemia due to marked splenomegaly. Histopathological examination revealed myeloid metaplasia and EMH in both ovaries and fallopian tubes with remarkable presence of all three myeloid hematopoietic cell lineages.
Case 2
A 55-year-old woman (20) with diseases advancing from essential thrombocytosis to idiopathic myelofibrosis developed progressive splenomegaly. The patient underwent therapeutic splenectomy and later showed conversion of idiopathic myelofibrosis to acute myeloid leukemia. The physical examination revealed two indurated erythematous violaceous nodules in her left mammary region and three nodules of similar characteristics on her scalp. A histological study of biopsies taken from a breast lesion and from the scalp showed a dense infiltrate in the reticular dermis composed of myeloid cells at different stages of maturation and megakaryocytes. Immunohistochemistry was positive for myeloperoxidase and CD68, with an insignificant number of CD34+ cells. These findings were consistent with cutaneous EMH.
Case 3
A 65-year-old lady (36) with a history of bronchial asthma developed a right lower lobe lung mass. Chest X-ray and CT studies showed an infiltrating mass resembling malignancy. Cytological examination of the mass by fine needle aspiration revealed pulmonary EMH. Hypercellular smears were composed of islands of erythroid precursors, granulocytic series, and some mature and immature mega-karyocytes. BM aspiration and biopsy were also performed and showed normocellular marrow without evidence of myelofibrosis in special stains. This was a rare case from a patient with respiratory disease.
Case 4
A 69-year-old white woman (3) who had a history of bradycardia required pacemaker placement. The patient was admitted to hospital with severe dyspnea. Her chest X-ray showed cardiomegaly and bilateral interstitial infiltrates interpreted as pulmonary edema. Physical examination revealed hepatomegaly as well. Transbronchial lung biopsy showed perivascular and peribronchiolar fibrosis and a heterogeneous cellular infiltrate of myeloid and erythroid precursors and megakaryocytes. These findings were indicative of pulmonary involvement by agnogenic myeloid metaplasia with an in situ EMH, rather than heart disease. This case was absent of the evidence for intravascular emboli of EMH.
Case 5
A 43-year-old Iranian woman (101) complained of back pain radiating to both of her lower extremities 2 months before admission. The pain was nonresponsive to conventional medical treatments, and mild splenomegaly was noted in her general physical examination. By MRI, the compressive spinal cord lesion and later an intracranial lesion were confirmed. A microscopic study of the lesion indicated erythroid, myeloid, and megakaryocytic proliferation. In a peripheral blood smear, the nucleated RBCs (red blood cells) and teardrop spherocytes were also present. This was a rare case report of sacral, lumbar, thoracic, and cranial involvement in the patient with EMH. The low incidence of EMH in neuroaxis indicates that cells with hematopoietic potential find little support in the CNS environment (97).
Case 6
A 38-year-old woman was diagnosed with moderately differentiated invasive ductal carcinoma in the upper and lower inner quadrants of her right breast. The patient's medical history included hypothyroidism with treatment of hormone replacement and a seizure disorder, but no hematologic disease was recorded. The patient was treated with neoadjuvant chemotherapy including doxorubicin and cyclophosphamide followed by paclitaxel. She received a bilateral mastectomy and right axillary lymph node dissection subsequently. EMH in mastectomy specimens was found to be primarily composed of myeloid cells at different stages of maturation. Given the presence of EMH in the breast in this patient with history of neoadjuvant chemotherapy, a possible role for chemotherapeutic agents in the pathogenesis of EMH was postulated (119).
Case 7
A 78-year-old man (98) with history of multiple myeloma and long-standing mild anemia developed a violaceous nodule on the tongue. By physical examination, a 1.5-cm rubbery violaceous nodule covered by nonulcerated mucosa was seen on the right lateral border of the tongue. A microscopic view of the specimen, a round mass of fibrin, showed an eccentric irregular lumen bordered by papillary endothelial hyperplasia. Conspicuous foci of EMH were present near the lumen consisting mostly of red cell precursors with an admixture of myeloid cells. Coexistence of papillary endothelial hyperplasia (Masson's pseudoangiosarcoma) and EMH demonstrated that the lesion could harbor cells with hemangioblast potential.
Case 8
A 29-year-old nulliparous female (21) was admitted to the hospital with heavy vaginal bleeding. The hematologic examination showed an abnormal paraclinical state. She was diagnosed by a transvaginal ultrasound confirming a 10.4 × 9.7 × 9.5-cm mass that occupied the fundus and body of the uterus. An exploratory laparotomy with uterine myomectomy was performed, and gross examination of the specimen revealed a single nodular mass with a white-tan swirling cut surface. By microscopic examination, benign leiomyoma with small cellular aggregates was confirmed. The cellular aggregates were further identified by immunohistochemistry staining as benign hematopoietic precursor cells for erythroid (CD44+ and CD71+), myeloid (CD33+, CD34+, CD45+, and myeloperoxidase) lineages, and megakaryocytes (CD61+). In addition, there were numerous intravascular thrombi containing hematopoietic precursor cells. EMH foci were not identified within normal myometrium. Occurrence of EMH in uterine leiomyoma and intravascular thrombi is very rare.
Case 9
A 40-year-old male (5) with δ-β thalassemia complained of nonspecific upper abdominal pain and anorexia for 6 months. Laboratory investigations showed an abnormal hematocrit of 30.6% and a reticulocyte count of 4.3%, but no other comorbid illnesses. Ultrasonographic and CT examination revealed an enlarged right adrenal gland and splenomegaly (18 cm). He underwent a laparoscopic adrenalectomy, and the excised specimen was 8 × 9 cm with a reddish brown surface, but no fat component. Histological examination confirmed the mass was composed of erythroid lineages and megakaryocytes.
Case 10
A 49-year-old male (40) with thalassemia intermedia underwent a splenectomy and maintained infrequent blood transfusion for 30 years. An increased density at the lower lobe of the right lung was incidentally detected on a chest radiograph. Physical examination revealed a pale conjunctiva, an icteric sclera, and marked hepatomegaly. A CT revealed in the thorax the lobulated and diffusely enhanced solid masses measuring 27 × 18 mm at maximum diameter located at the posterior mediastinum, bilaterally at the paraspinal area and parasacral region, which did not cause any erosion in the adjacent bone. Solid masses were observed alternatively in the MRI and revealed this mass to be T1 and T2 isointense to muscle with fat strands inside with heterogeneous enhancement. Although a biopsy was not conducted, CT/MRI image features combined with patient's history of thalassemia intermedia confirmed EMH (40).
Emh Exhibits in the Embryo and Fetus During Development
Embryonic hematopoiesis initiated spatially and temporally in different tissues sets the stages for developing the capacity of adult hematopoiesis. Those tracks of prenatal hematopoiesis become the most common sites for development of EMH in adults. Therefore, microenvironments in prenatal hematopoietic tissues are widely considered to have the potential to reactivate hematopoiesis (43). Both YS and placenta are extraembryonic tissues and should be excluded for the discussion of EMH in adulthood, but they are important places not only for de novo generation of primitive and definitive HSCs (86,94) but also for expansion of HSC pools. Hence, the explored microenvironment from five critical fetal hematopoietic tissues, including YS and the placenta, of animals should be addressed.
Yolk Sac
The YS is the initial site of blood cell production during murine and human ontogeny. Erythropoiesis is prevailed at embryonic (E) day 7.5 in mice (86). Dramatic expansion of the primitive erythroid (EryP) lineage is taken during E8.25–E8.5. Subsequently, the number of EryP colony-forming cells (CFCs) within the YS greatly declined to undetectable levels at E9.0. Low numbers of definitive erythroid (EryD) progenitors are present in the YS as early as at E8.25, prior to the establishment of circulation. These progenitors consist entirely of burst-forming unit-erythroids (BFU-Es). The definitive nature of cells was confirmed by the presence of βmajor, but no βH1 globin (85). Ncx1-/- mutant embryos fail to initiate a heartbeat on embryonic day E8.25 but continue to develop through E10. By using Ncx1-/- mutant embryos as a study tool, Liu et al. detected normal numbers of definitive progenitors in Ncx1-/- versus wild-type (WT) YS, confirming a de novo generation of definitive hematopoietic progenitor cells in the YS (64).
The differentiation of erythrocytes and endothelial cells from hemangioblasts occur within the YS during normal development of mouse embryos (4). Huang and Auerbach isolated nonadherent, density <1.077 g/cm3, AA4.1+ YS HSCs and cocultured them (1 × 104) with a YS-derived endothelial cell line (YS-EC, C166) for 7 days to confirm a suitable microenvironment of YSs for expansion of early precursor cells (T-cells, B-cells, and myeloid cells) (63). This conclusion is in parallel with the study from culture of the nonadherent cells with YS endoderm (YS-EC)- or mesoderm (YS-MC)-derived cell lines that resulted in significantly fewer neutrophils and immature myeloid cells but higher mature macrophages than culture of hematopoietic cells with adult BM stromal cell lines (127). Furthermore, in vitro growth of adult murine BM high proliferative potential CFCs in coculture with YS-EC- and YS-MC-derived cells gained a significant increase in total nucleated cells when compared with coculture with adult BM stromal cell lines (126).
Aorta–Gonad–Mesonephros
Medvinsky et al. separately explanted E9, 10, and 11 tissues (AGM, YS, liver, head/heart, and body remnants) from embryos. These tissues were cultured for 2–3 days and dissociated; sequentially, these cells were injected into lethally irradiated mice for colony-forming unit in the spleen day 11 (CFU-S11) and long-term repopulating (LTR)-HSC activity by transplantation assay. When E9 tissues were cultured and cell suspensions were injected, no CFU-S11 was found in the recipient mice. However, beginning at early- and mid-E10, significant CFU-S11 activity could be found in cultured AGM and YS, but not in liver, control head/heart, or body remnants. They observed at least a 15-fold enhancement in LTR-HSC activity from the cultured E10 AGM region when compared with uncultured AGM. This work provided a demonstration to show the capability of AGM for expansion of LTR-HSCs (71). Taoudi et al. had also characterized the CD45+ compartment in the E11.5 AGM region and identified two functionally distinct populations. The major population (VE-cadherin-/CD45+) predominantly contained mature blood cells and a low frequency of progenitor cells. The second, a rare VE-cadherin+/CD45+ population, were blast-like cells and contained a high frequency of hematopoietic progenitors (112). The VE-cadherin+/CD45+ fraction was highly efficient in producing definitive HSCs as transplanted mice were reconstituted (111).
Fetal Liver (FL)
FL is a suggested place for the expansion of HSC pools during E14–16. At day 12 of gestation, multilineage repopulating activity was first detected in the liver as 50 repopulating units (RU) per liver. The number of RU per liver increased 10-fold and 33-fold by E14 and E16 of gestation, respectively, and decreased thereafter (23). Cell cycle analysis confirmed that around 40% of purified Thylo/Sca-1+/Lin-/lo HSCs in FL had greater than 2n DNA content estimated by Hoechest 33342 staining (24). Chang et al. (12) have studied hematopoiesis in FL from E12.5 to E17.5 compared with that in adult BM. Although specific genes [Pax 5, CD19, CXCR4, interleukin (IL)-7 receptor a, and VLA-4] for B-lymphopoiesis were only marginally expressed in E12.5 FL, the B-cells' reconstitution capacity of hematopoietic stem and progenitor cells (HSPCs) from early FL was equal to that from late FL and adult BM (12). During pregnancy, the elevated plasma estrogen induces a decreased B-lymphopoiesis in adult mice (70) and also inhibits T-cell development in the thymus (96). This downregulation of B-cell production lies on adult HSPCs' sensitivity to suppression by estrogens (69) and protects the fetus from attack by the mother's adaptive immune system. Interestingly, during fetal development, an intensive B-lymphopoiesis occurs in the middle- and late-stage FL despite high estrogen levels (12). Hlobeňová et al. provided evidence that HSPCs are primed for estrogen sensitivity in FL at the E17–20 developmental stage, while the HSPCs from early E14 FL barely gain estrogen sensitivity after being transplanted into the adult BM microenvironment (34). Kinoshita et al. have reported that primary culture of fetal hepatic cells from E14.5 murine embryos supported expansion of embryonic Lin–/Sca-1 +/c-Kit+ (LSK) cells, giving rise to myeloid, lymphoid, and erythroid lineages (52). In contrast, a hematopoietic cell-derived paracrine factor, oncostatin M (OSM), acts on neighboring hepatocytes and induces maturation of hepatocytes (47). Besides, B-lymphopoiesis in FL was downregulated selectively by OSM in contrast to that regulated by estrogen in adult BM (52).
Placenta
The YS, AGM region, and FL are well recognized organs consecutively for hematopoiesis; however, the small number of HSCs generated in the YS and AGM region cannot completely account for the number of HSCs in FL at E12. In addition, there is a 2-day time lag between HSC generation in the AGM and initiation of HSC expansion in FL. These observations suggest the presence of another hematopoietic site for HSC generation to fill a time gap between the AGM region and FL (55). Sasaki et al. identified hematopoietic cell clusters expressing CD31, CD34, and c-Kit in mouse placenta. These clusters were attached to the endothelial wall of capillary vessels, the so-called vascular labyrinth region, from E10.5 to E12.5, and were morphologically similar to those seen in the AGM region at E10.5 (100). Rare hematopoietic cells (0.1 RUs/embryo equivalents) capable of contributing to blood chimerism of irradiated adult recipients 12 weeks posttransplantation were found in the placenta already at E10.5. Placental HSC pools expand until E12.5–E13.5 and contain >15-fold more HSCs than the AGM. At E13.5, the average competitive repopulation level of HSCs in the FL was similar to placental HSCs. The size of the placental HSC pool was maintained through E13.5, but it diminished >10-fold by E15.5, while the HSC pool in the FL continued to expand (25). Analysis of Ncx1-/- embryos by tracking Runx1+ and CD41+ HSCs verified that HSC development is de novo initiated in the placental vasculature independent of blood flow at E10.5–11.5 (94).
Fetal Spleen (FS)
Mice injected with embryonic spleen cells from E14.5 showed long-term multilineage reconstitution. The splenic rudiment is colonized at E12.5 and actively starts its hematopoietic activity around E14.5 of gestation. At this stage, LTR activity is readily detected, suggesting that the spleen is colonized by HSCs (26). FL-derived HSCs (c-Kit+/Thy-1.1lo/Lin-/Sca-1+/Mac-1lo) begin to seed the FS at E14.5–E15.5. LT-HSC activity is detectable in fetal blood at E12.5–E17.5 with a slight increase at E14.5 and a slight decrease at E15.5 measured by competitive reconstitution assay (16).
The development of macrophage populations during splenic hematopoiesis was analyzed from Rag2GFP embryos. In these animals, lymphoid but not myeloid precursors were marked with GFP (129). The presence of Mac1+/F4/80+ macrophages in the E15 FS was determined, and they represented 40% of the total splenocytes. These data were in parallel with an immunohistochemistry study performed on splenic sections from E15.5 Rag2GFP embryos that showed the majority of the nucleated cells expressing F4/80. The abundant macrophage population was disorganized and evenly distributed throughout the sections. GFP-marked cells interspersed in the section indicated the rare presence of committed lymphoid progenitors. This result shows that E15.5 FS is largely composed of monocytes/macrophages that appear homogeneously distributed throughout this organ. Meanwhile, macrophage differentiation is driven by a signal secreted by the FS stroma (7).
Emh in the Adult Laboratory Animals
The extramedullary site in adult animals may acquire hematopoietic potential upon a modification of its microenvironment enabling engraftment, support, and control of HSCs. This is of a high interest, since such a microenvironment significantly differs from that in the BM. The BM stroma supporting normal adult hematopoiesis is unique in several aspects. It is comprised of a vascular niche and an endosteal niche in which osteoblasts and osteoclasts are included and found nowhere outside the BM. The osteoclasts elevate the local and systemic calcium level, and recent studies have demonstrated that the lodgment of HSCs in the endosteal niche is mediated in part by the calcium-sensing receptor (113). The osteoblasts also play a crucial role as the HSC regulators (8) and express numerous signaling molecules including stromal cell-derived factor-1α (SDF-1α), osteopotin (79), and vascular cell adhesion molecule 1 (VCAM-1) that are supposed to be involved in retention of HSCs in the BM niche (35). Furthermore, the endosteal BM hematopoietic niche was shown to be hypoxic (52), and the low-oxygen conditions induced a transcription factor called hypoxiainducible factor-1α (HIF-1α). It has been suggested that elevated HIF-1α activity maintains HSCs (61). In the following reviewed literature, we briefly demonstrate that EMH can be readily induced in animals by various insults occurring independently or coincidently: (i) specific myelosuppressive regimen; (ii) mobilization of HSPCs from the BM; (iii) creation of a mesenchymal/stromal layer mimicking BM microenvironment in an extramedullary site; (iv) an increased production of a hematopoietic growth factor locally; (v) an increased production of erythropoietin activating stress erythropoiesis; and (vi) a decreased oxygen tension in an extramedullary microenvironment. Those insults, however, can be elicited through drug treatment, gene manipulation, and microorganism infection in the laboratory animals. It is worth noting that in mice the spleen is the organ that becomes hematopoietic as part of the normal response to erythroid stress. The stimulus of erythropoietin does not increase BM erythropoiesis, but rather induces stress erythropoiesis in the spleen. By contrast, in humans the spleen is not a normal hematopoietic organ (90).
Animal Models of Emh Induced by Drugs, Cytokines, and Growth Factors
Chemicals and hematopoietic stimulators that are cytotoxic to BM or mediated by inflammatory responses, respectively, were reported to result in EMH in treated animals. Therefore, those drugs, cytokines, and growth factors that are capable of inducing EMH consistently in the experimental animals could be used further for stem cell research.
Drugs
Cyclophosphamide is one of the most common drugs used in cancer chemotherapy and for patient conditioning prior to HSC transplantation (102). Treatment of cyclophosphamide induces mobilization of hematopoietic progenitors into the blood and causes myelosuppression and extramedullary hematopoiesis. In mice after cyclophosphamide (135 or 300 mg/kg) administration, the numbers of CFU-S reached a peak at 7 days in the spleen and then declined. Flow cytometry analysis also demonstrated the evidence in parallel that the percentage of CD34+ and CD117+ HSPCs in the spleen increased at 7 days. Therefore, these proofs indicated that the spleen became a major reservoir of HSCs after cyclophosphamide treatment on around day 7 (102,120). The most commonly used mobilization agent, G-CSF, induced HSPC mobilization through increasing the response of the osteocytic network to sympathetic suppressive signal and eliminating endosteal macrophages that lead to suppression of the osteoblastic niche function (2). The dual-negative regulation in the BM caused a remarkable reduction in endosteal osteoblasts, bone formation, CXCL12 expression, and F4/80+ osteomacs and inhibited expression of endosteal cytokines resulting in major impairment for HSC retention (122). Data showing (33) that combined treatment of cyclophosphamide/G-CSF (4 mg/5 μg per mouse) in mice also promoted mobilization of HSPCs from the BM to the adipose tissue. After the cyclophosphamide/ G-CSF treatment, the defined hematopoietic stem/progenitor cell (LSK) population in the stromal vascular fraction (SVF) of epididymal adipose tissue was increased compared with the untreated control. Moreover, the SVF treated with cyclophosphamide/G-CSF contained 3.5-fold more colony-forming cells compared with the SVF treated with control. Hence, adipose tissue is a novel extramedullary tissue possessing phenotypic and functional HSPCs induced by cyclophosphamide/G-CSF (33). It is known that adipose tissue surrounded by the complex vasculature and stromal cells constitutes a hypoxic environment and provides an appropriate microenvironment for HSPC expansion (33). In BM, expression of VCAM-1 was reduced in mice during cyclophosphamide/G-CSF treatment. In contrast, matrix metalloproteinase (MMP)-9 gradually accumulated in BM extracellular fluids on day 6 following administration of cyclophosphamide/G-CSF (59). MMP-9 plays a role to abolish the retention of HSCs in BM via SDF-1/CXCR4 axis (59).
The chemotherapeutic agent 5-fluorouracil (5-FU) induced hematotoxicity to BM that causes anemia and hemocytopenias, which is a basis of its use for clinical treatment. 5-FU also induced the phenomenon of EMH that in the 5-FU-treated mice at the end of the administration period, polyploidy nuclei of granulocytes within atrophy of the white pulp of the spleen were observed (74). In BM, levels of fibroblast growth factor 2 (FGF2) was elevated and associated with EMH after 5-FU treatment (41).
The toxicity of BMS-182248, an immunoglobulin (cBR96)-cytotoxic drug (doxorubicin) conjugate, was investigated in Sprague–Dawley male rats. The effects in BMS-182248-treated rats were noted with thymic atrophy, BM hypocellularity, splenic lymphoid, and red pulp depletion and spleen weight increase. Meanwhile, BMS-182248-treated female rats had increased EMH compared to placebo-treated controls (19).
A low dose of nicotine induced proliferation of human embryonic stem cell-derived endothelial cells (hESC-ECs) and increased their expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) genes (128). In contrast, sustained exposure to nicotine was associated with significant inhibition of rolling and migration of enriched HSPCs (Lin-/CD34+) across BM endothelial cells in response to SDF-1α in vitro. Decreased expression of β2-integrin, not CXCR4, on HSPCs was also found upon exposure to nicotine. In vivo exposure to nicotine induced the increased generation of BFU-E, CFU-E, CFU-GM, and CFU-GEMM in the spleen but not in the BM (87).
Cytokines
IL-5 transgenic mice with constitutive overexpression of IL-5 can potentially induce colonization of spleen with mesenchymal/stromal progenitor cells (CFU-Fs) from BM, which provides the necessary microenvironment for establishment of hematopoiesis in extramedullary sites (49). The size of spleens was significantly larger (two- to threefold) and a significant increase (10- to 40-fold) in numbers of CFU-mix, CFU-GM, CFU-M, and CFU-Eos in comparison to control WT mice. However, IL-5 does not affect transendothelial migration of HSPCs toward SDF-1 (49). Similarly, activators of the gp130 signal transducer like the IL-6–IL-6R complex may represent the most powerful stimulators for extramedullary hematopoietic progenitor cells (91). Increasing weight of liver and spleen in IL-6–sIL-6R mice was caused by a marked extramedullary proliferation of hematopoietic cells (CFU-GM and BFU-E) in both organs. In histomorphological analysis of livers and spleens of IL-6–sIL-6R mice, the liver tissue with several hematopoietic foci contained predominantly granulocyte precursors, blastic precursors, and terminally differentiated segmental granulocytes. Spleen tissue with activated EMH had shown enlarged granulopoietic and erythropoietic areas as well as an increased number of megakaryocytes in IL-6–sIL-6R mice (91). In the transgenic (Tg) mice expressing IL-27 under the control of a liver-specific human serum amyloid P component (SAP) promoter, an enhanced myelopoiesis and impaired B-cell development with splenomegaly was observed. IL-27 Tg mice had an increased number of CD34– LSK cells in BM, but they failed to show any HSC activity by no increase in the number of B-, T-, and myeloid cells. IL-27 Tg mice developed splenomegaly with an average twofold increase in spleen weight at 8 to 12 weeks of age. Histological analysis revealed that the number of megakaryocytes appears to be increased in spleen and in BM of IL-27 Tg compared with WT mice (103). EMH was also induced by recombinant human IL-3 at subcutaneous injection sites in cynomolgus monkeys (50). Histologically, the subcutaneous tissues at injection sites in the rhIL-3-treated monkeys were moderately expanded by EMH and mixed leukocytic infiltrates. The EMH was characterized by proliferation of immature hematopoietic cells from the myeloid, megakaryocytic, and erythroid series. The severity of EMH was dose dependent with rhIL-3 and occurred most prominently in the high-dose animals. IL-3 administration also resulted in expansion and mobilization of hematopoietic progenitor cells into the peripheral circulation. Presumably, these circulating progenitor cells then localize and proliferate in extramedullary tissues capable of supporting EMH, including skin (50).
Growth Factors
Systemic administration of FGF2 in mice disrupted normal BM hematopoiesis in part through reduced expression of SDF-1. At the end of the 21-day treatment period, the spleens from FGF2-treated mice were significantly larger than those from the control mice. Histologic examination of the spleens revealed that FGF2-treated mice had less white pulp and more red pulp than vehicle-treated mice, which suggests an increased EMH in the FGF2 treated mice. The livers of FGF2-treated mice displayed occasional clusters of hematopoietic cells, which were mainly absent from the livers of vehicle-treated mice. Peripheral blood smears from FGF2-treated mice stained by May–Giemsa contained significantly more immature myeloid cells (particularly myelocytes and metamyelocytes) than those from control mice (78).
In mice after the administration of FLT3 ligand for 10 consequent days, Michael et al. (106) have observed pronounced EMH in the mouse spleen with the presence of all hematopoietic lineages. Neutrophils at different stages of maturation appeared in the spleen of FLT3 ligand-treated mice, but not in control mice. Clusters of monocytes, characterized by the condensation of nuclear chromatin and folding of the nucleus, as well as mature megakaryocytes with highly folded nuclei and finely granular cytoplasm, could be clearly seen. Analysis of phenotype and morphology of splenocytes obtained from FLT3 ligand-treated mice suggested that dendritic cells (B7–2+) and granulocytes were the two main populations of cells to increase in the spleen following the treatment. Administration of FLT3 ligand also resulted in a significant increase in the liver weight. The authors had observed the appearance of large groups of nonparenchymal cells (NLDC-145+/B7–2+) that formed compartments associated with the portal triad and hepatic veins (106).
Tumor-derived PDGF-BB (platelet-derived growth factor-BB) can target PDGFR+ stromal cells in organs such as liver and spleen, where PDGF-BB induces erythropoietin (EPO) expression and activates EMH (125). Xue et al. observed a significant increase in Ter-119+/Ki-67+ erythroid progenitors in the spleen and liver of mice carrying murine T241 fibrosarcoma-derived PDGF-BB tumors. The authors also detected a significantly elevated number of BFU-Es, CFU-Es, and granulocyte-macrophage colonyforming units (CFU-GMs) in the liver and spleen of mice with T241-PDGF-BB tumors compared to those with T241-vector tumors. The expression levels of EPO mRNA were significantly elevated in both liver and spleen from mice with T241-PDGF-BB tumors compared to those with T241-vector tumor. Treatment with PDGFR-β-specific antibody significantly inhibited PDGF-BB tumor growth and prevented PDGF-BB-induced splenomegaly and hepatomegaly (125).
Tumor-derived VEGF acts as an endocrine-like hormone to induce EMH by targeting distal organs in the host (124). In tumor-bearing mice, circulating VEGF induced hepatomegaly and splenomegaly owing to vessel dilation, tortuosity, and activation of hematopoiesis. Tumor-derived VEGF modulates the hematopoietic system by suppression of BM hematopoiesis and then leads to activation of EMH in liver and spleen. Histological examination of liver tissues showed visible hematopoietic islets in liver sections from T241-VEGF tumor-bearing mice. The sinusoidal hepatic vasculature (CD31+) became highly dilated in livers of T241-VEGF tumor-bearing mice, but not in livers of T241-vector tumor-bearing mice. These findings demonstrated that tumor-derived VEGF significantly contributes to hepatic hematopoiesis, and the vascular architecture is significantly altered in this organ. Histological examination of the spleen showed that apparent borders between the white pulp (WP) and red pulp (RP) under physiological conditions vanished and were replaced by a mixture of WP and RP without any distinctive borders throughout the entire spleen of T241-VEGF tumor-bearing mice. The erythroid progenitor cells stained with Ter-119+/PI were observed in T241-VEGF tumor-bearing mice (124).
In summary, a myelosuppressive microenvironment disfavors the retention of HSCs in BM. Cyclophosphamide and G-CSF can suppress niche function in BM due to their cytotoxicity to osteoblasts and osteomacs that results in a decline in the expression of endosteal cytokines and VCAM-1 and impair bone formation. Cyclophosphamide and G-CSF also cause disruption of the CXCR4/CXCL12 chemotactic interaction between HSPCs and stromal cells in the BM via MMP9 protease activation. However, overexpression of cytokines IL-27, VEGF, and FGF2 can also prevent HSCs from differentiation or retention that result in BM dysfunction. Stem cell niches in EMH can be constructed by a hypoxic microenvironment mimicking BM or by activation of stromal cells in extramedullary site through IL-5 and PDGF induction. A presence of local stimulator for HSPC expansion like IL-3, IL-6, and FLT-3 can also stimulate EMH directly.
Animal Models of Emh Induced by Genetic Defects
Mutations That Result in Myeloproliferative Disorder
EMH in the spleen is a characteristic feature of the chronic myeloproliferative neoplasms (MPN) and various neoplastic or reactive myeloid conditions (45). A previous study indicated that the V617F mutation in the Janus kinase 2 gene (JAK2V617F) was frequently and preferentially identified in BM and peripheral blood cells of MPN patients (60). Moreover, the JAK2V617F mutation is frequently present in splenic EMH cells associated with MPN (37). Thus, the precursor cells that lead to extramedullary hematopoietic expansion most likely originate from the transformed BM clone. In addition, the overexpression of Bcl-xL in JAK2V617F-positive megakaryocytes suggests that the JAK2/STAT5/Bcl-xL pathway may be important to the myeloproliferative process in splenic EMH (37). A JAK2 small molecule inhibitor called G6 significantly reduced EMH in the liver and splenomegaly of JAK2V617F mice (53). Thus, the potency of JAK2/STAT5 pathway inhibitors as a pharmaceutical alternative to surgical intervention for JAK2V617F-positive MPN patients with significant splenomegaly is undergoing clinical trial.
Overexpression of high mobility group AT-hook 2 (HMGA2) gene is found in a number of benign and malignant tumors, including MPNs (80). HMGA2 transgenic mice showed splenomegaly with an expansion of red pulp, an increase in the proportion of Ter-119+ erythroid cells, and a perturbation of the normal spleen architecture, suggesting EMH in the spleen of HMGA2 Tg mice. However, proportions of B- and T-cells in enlarged spleens of HMGA2 Tg mice were similar to those in normal spleens of WT mice, suggesting that numbers of B- and T-cells were also increased in the spleen, with the potential of lymphoma development. Meanwhile, BFU-Es and CFU-GMs can be grown from spleen cells in the presence of human EPO and IL-6, and murine IL-3 and SCF (38).
Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas (89) and acute myeloid leukemias (AML) (66). Sasaki et al. reported the characterization of conditional knock-in (KI) mice in which the most common IDH1 mutation, IDH1R132H, was inserted into the endogenous murine Idh1 locus and was expressed in all hematopoietic cells (Vav-KI mice) or specifically in cells of the myeloid lineage (LysM-KI mice) (99). The IDH1R132H mutation in all hematopoietic cells (Vav-KI mice), and in the myeloid lineage in particular (LysM-KI mice), results in the accumulation of LSK and lineage-restricted progenitors (LRP), common lymphoid progenitor (CLP) cells first in the BM and later in the spleen. Macroscopic analysis of these mutants shows LysM-KI mice facilitating age-dependent reduction of BM cellularity and anemia, suggesting a myelosuppression. Histologically, architecture of enlarged spleen became increasingly disorganized, with increased numbers of CFU-GM and CFU-GEMM (99).
Children with neurofibromatosis type 1 (Nf1) are predisposed to juvenile myelomonocytic leukemia (JMML), an aggressive MPN that is refractory to conventional chemotherapy (62). Conditional inactivation of the Nf1 tumor suppressor in hematopoietic cells of mice causes a progressive MPN that formulates JMML and chronic myelomonocytic leukemia (CMML) (117). Chang et al. observed more dramatic effects of Nf1 inactivation in the spleen, with BFU-E and CFU-E colony numbers increased 30-fold in Nf1 mutant mice, while splenocytes were grown in methylcellulose medium containing erythropoietin (100 ng/ml). Nonetheless, Chang et al. also proposed that treatment with MEK inhibitor (PD0325901) not only greatly decreased splenomegaly but also reduced the frequency of splenic BFU-E and CFU-E colonies and of BM CFU-E to nearnormal levels and reverted splenic histology toward normal (13).
Somatic loss-of-function mutations in the 10–11-translocation-2 (TET2) gene occur in a significant proportion of patients with myeloid malignancies (22). TET2 loss confers increased self-renewal of HSPCs in vivo (32). In an animal model, Tet2-/- mice by week 20 of age developed significantly enlarged spleens (weight >250 mg) compared to littermate controls (75). FACS analysis of the spleen of Tet2-/- mice revealed a significant enlargement of both LSK and CD11b+/Gr1+ populations consistent with HSC, granulocytic, and monocytic expansion. Numbers of white blood cells (WBCs), neutrophils, and monocytes were enormous in peripheral blood showing monocytosis (75).
NF-κB-inducing kinase (NIK) plays critical roles in the development of lymph nodes and Peyer's patches and microarchitecture of the thymus and spleen via NF-κB activation (39). Alymphoplasia (aly/aly) mice have a point mutation in the NIKG855R gene that causes a defect in the activation of an NF-κB member RelB (68,81,104). BALB/cAaly/aly mice showed significant female-dependent splenomegaly with EMH, which was not significant in C57BL/6aly/aly mice (105). The ratio of spleen to body weight was significantly increased in 21-week-old BALB/cAaly/aly mice compared to the littermate aly/+ control mice. The splenomegaly appeared to be age dependent because it was milder in 8-week-old mice. Frequency of erythroid precursors (Ter-119+/CD45–) in total splenocytes from 21-week-old female BALB/cAaly/aly was increased (105). These data were in parallel supported by the finding that Relb-/- mice exhibit significant splenomegaly and splenic EMH with an increase in erythrocyte precursors (121).
Mutations That Result in Myelofibrosis
The identification of inactivating mutations in the PcG gene unveiled a tumor-suppressor function in myeloid malignancies, including primary myelofibrosis (PMF) (31). The study showed that loss of PcG gene, Bmi, causes pathological hematopoiesis similar to PMF (82). In a mouse model, transfer of Bmi1-/-/Ink4a-Arf -/- hematopoietic cells induced abnormal megakaryocytopoiesis in recipient BM accompanied by marked increase in HSCs (89) and multipotent progenitors (MPP) in the spleen as an evidence of EMH. Massive fibrosis of the spleen in recipient mice was also found. The recipient mice repopulated by Bmi1-/-/Ink4a-Arf-/- BM cells had marked hepatosplenomegaly and hypoplastic BM with severe fibrosis at their terminal stage. EMH was also evident in the liver. Increased expression of HMGA2 in Bmi1-/-/Ink4a-Arf-/- hematopoietic cells was found as well (83).
Gata1 is a member of the GATA family of transcription factors indispensable for appropriate maturation of hematopoietic cells of many lineages, including erythroid cells and megakaryocytes (MKs) (9). HSCs from the hypomorphic Gata1low mutant mice with deletion of regulatory sequences hypersensitive site 1 (HS1) gene in X chromosome lack the expression of CXCR4 (72). The mutation is lethal in the C57BL/6 strain but is viable in CD1 strains that efficiently recruit the spleen as extramedullary hematopoietic site in response to age. Gata1low mutant mice also show thrombocytopenia, fibrosis, and reduced hematopoiesis in the BM (67). All the hemizygous Gata1low/0 males with splenectomy died of severe anemia within 1 month after surgery, suggesting that only hemizygous females were rescued by the presence of the normal allele. In −2.7kbGata1GFPGata1low/0 males with GFP-Gata1 expression upon HS2 instead of HS1 regulation, Gata1 was expressed by a minority (10–18%) of the c-Kit+/CD34+ cells of the marrow and by the majority (>80%) of the c-Kit+ (both CD34+ and CD34-) of the spleen. In −2.7kbGata1GFPGata1low/0 mice, the c-Kit+ cells purified from the spleen had greater cloning efficiency (BFU-E) than −2.7kbGata1GFPGata1+/0 control (18–72% vs. 0–0.2%). These results indicated that the inability of Gata1low HSPCs to produce hematopoietic colonies is rescued in the spleen by a yet unelucidated mechanism that allows the cells to activate Gata1 expression via the HS2 enhancer (73).
Mutations That Result in Myelodysplastic Syndrome (MDS)
The TEL-Syk fusion protein was isolated from a patient with myelodysplasia with megakaryocyte blasts. In vivo, TEL-Syk expression in pre-B-cells (CD19+) blocks B-cell differentiation, leading to lymphoid leukemia (56,123). Mice transplanted with TEL-Syk transduced fetal liver hematopoietic cells developed hypocellular splenomegaly and EMH followed rapidly by BM failure and extreme splenic/hepatic fibrosis accompanied by extensive apoptosis (28). In fact, the TEL-Syk chimeric mice had approximately twofold fewer splenocytes in total, resulting in nearly a fivefold difference in cell number/weight ratio of the spleen. The cells in the spleens of the TEL-Syk chimeras were predominately Ly6G+/CD11b+ neutrophils or F4/80+/CD11b+ monocytes/macrophages, with lower percentages of T- and B-lymphocytes, compared to the spleens of vector control chimeras. TEL-Syk expression also caused temporal increases in circulating inflammatory cytokines like IL-6, -12, -13, G-CSF, IP-10, interferon-γ (IFN-γ), monocyte chemotactic protein-1 (MCP-1), monokine induced by γ interferon (MIG), macrophage inflammatory protein a (MIP-1α), T-cell activation gene 3 (TCA-3), TIMP metallopeptidase inhibitor 1 (TIMP-1), and triggering receptor expressed on myeloid cells 1 (TREM-1) (28).
Expression of the immediate early response gene X-1 (IEX-1) is diminished significantly in HSCs in a subgroup of patients with early stage myelodysplastic syndromes (109), but it is not clear whether the deregulation contributes to the disease. IEX-1-/- mice developed MDS after total body irradiation (93). The BM erythroid dysplasia occurred in parallel to increasing EMH, with a relatively higher level of CD71+/Ter-119+ erythroblasts and internuclear bridge and binucleated erythroid precursors in irradiated IEX-1-/- spleens than WT counterparts. Increased erythropoiesis within the spleen was in an attempt to compensate insufficient erythropoiesis of IEX-1 KO HSCs in irradiated mice. Insufficient production of IEX-1 KO megakaryocytes and increased cell cycling of KO HSCs cells (LSK) were also found in BM. IEX-1-/- HSCs from BM produced significantly fewer circulating platelets and red blood cells when transplanted into WT mice, despite their enhanced repopulation capability. These findings highlight a role for this early response gene in multiple differentiation steps within hematopoiesis, including erythropoiesis and in the regulation of HSC quiescence (93).
Miscellaneous
Genetic depletion of FoxP3 regulatory T-cells (Tregs) greatly increased the number of the myeloid progenitors in the spleen during immune responses with staphylococcal enterotoxin B (SEB, 2 μg/ml) challenge (57). The number of myeloid progenitors (CFU-GM) in the spleen of the FoxP3-deficient mice was increased compared to WT mice. In contrast, the numbers of CFU-GM in the BM were not significantly increased in the FoxP3-deficient mice. Myelopoietic activities and hematopoietic IL-3- and GM-CSF-producing effector T-cells in the spleen, but not in the BM of FoxP3-/- mice, were increased. These results demonstrated that Tregs function to restrain the myelopoietic cytokine producing T-cells and, therefore, play an active role in prevention of EMH induced upon antigenic stimulation in the spleen (57).
Mammalian HSCs are kept quiescent in the endosteal niche, a hypoxic zone of the bone marrow (BM) (54,88). HIF-1α is functionally induced in low-oxygen conditions, and it has been suggested that the hypoxic BM niche maintains HIF-1α activity, thereby maintaining HSCs (61). In HIF-1α-deficient mice, Ki-67+ G1 fraction from BM HSCs (CD34- LSK) was increased and the reconstitution capacity of HSCs was reduced after BM transplantation via a p16Ink4a/p19Arf-dependent manner (110). In the aged HIF-1α-/- mice, the authors observed an increased number of LSK cells in the extramedullary site in spleen. The RP and WP structures of the spleen were disorganized in the aged HIF-1α-/- mice because of EMH. Transplantation of 1 × 106 splenic MNCs rescued the lethally irradiated recipients, which suggests that splenic LSK cells from HIF-1α-/- mice support the hematopoiesis of recipient mice (110).
In summary, the stem cell niche in EMH can be induced by genetic manipulations in laboratory animals. For example, FoxP3 knockout mice produce IL-3 and GM-CSF in the spleen, and TEL-SyK chimeric mice increase production of several inflammatory cytokines in the circulation. Moreover, a malignant transformation and expansion of HSCs by a gene mutation can also result in EMH in animals. For instance, loss of tumor-suppressor gene Bmi and NF1 predispose to chronic myeloproliferative neoplasms. DNA hypermethylation caused by mutation of TET2, IDH1, and IDH2 increases leukemogenesis and leukemic stem cell maintenance. Loss of IEX-1 and HIF-1α also promotes a faster proliferation rate of HSCs. Leukemic proliferation in the BM turns the normal stromal microenvironment into malignant niches that outcompete native HSPC niches for CD34+ cell engraftment (18). Osteopetrosis in mice caused by NIK point mutation results in BM failure and EMH. An age-dependent onset of EMH by genetic aberration of NIK, IDH1 and IDH2, and HIF-1α is also of concern.
Animal Models of Emh Induced by Bacterial, Viral, or Parasitic Infection
Bacterial Infections Induce EMH
IFN-γ-deficient mice infected with Mycobacterium bovis strain (BCG) undergo a dramatic remodeling of the hematopoietic system. Spleens isolated from infected IFN-γ-/- mice 5 weeks after intraperitoneal administration of BCG showed change in size and color of the organs. Examination of cell types within the spleen confirmed that replacement of the normal lymphoid population with extramedullary hematopoietic elements, predominantly of the granulocyte at various stages of maturity, had occurred. Significant increase in CFCs in IFN-γ-/- mice 3 days after infection verified the large expansion of hematopoietic progenitors in the spleen following BCG infection. Besides, a tremendous expansion (~10×) of splenic CFC at day 7 postinfection in IFN-γ-/- mice compared with that in WT mice occurred. The BM counterpart of IFN-γ-/- mice had large foci of band-form granulocytes. In addition, systemic levels of cytokines, particularly IL-6 and G-CSF, were elevated (77).
Infection with Anaplasma phagocytophilum, a Gram-negative, lipopolysaccharide (LPS)-negative, obligate intracellular bacterium, results in marked decreases in the number of BFU-E and CFU-GM in the BM along with concurrent increases of those lineage-committed HPCs in the spleen. BM cells isolated from mice at day 6 after intraperitoneal infection produced significantly elevated levels of myelosuppressive cytokines, for example, monocyte chemoattractant protein 1 (JE), keratinocyte-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2), and proinflammatory cytokines TNF-α and IL-6 compared to BM cells isolated from uninfected mice. There was significant downregulation of CXCL12 in BM cells as well. In cytology study, there was loss of erythrocytes and B-lymphocytes from the BM along with increased granulopoiesis. These changes were accompanied by splenomegaly due to expansion of B-lymphocytes in lymphoid follicles and marked increase in Ter-119+ erythroid precursors in RP. Megakaryocytes were also characterized in the spleen at days 4 to 8 postinfection (44).
Infection with Ehrlichia muris, a pathogen closely related to E. chaffeensis, resulted in anemia, thrombocytopenia, and a marked reduction in BM cellularity in mice (65). CFU assays conducted on days 10 and 15 postinfection revealed a significant decrease in multipotential myeloid and erythroid progenitors in BM. Splenomegaly and disorganized architecture of the spleen from a mouse on day 11 postinfection were also noted. The spleen section throughout infection showed the lymphoid nodules were pale and poorly defined. The nodule has an enlarged germinal center and an expanded marginal zone. Meanwhile, egress of B-cells was accompanied by the decrease in SDF-1 expression in the BM. The RP exhibited increased cellularity due to the EMH, primarily erythropoiesis. The Ter-119+ cells in the E. muris-infected spleens were largely immature erythroid cells, as a majority of the Ter-119+ cells expressed high levels of CD71 and c-Kit. These data reveal that the spleen is a site of EMH during acute E. muris infection (65).
Systemic Salmonella infection commonly induces prolonged splenomegaly in murine or human hosts (95). A marked increase in the number of phagocytes (CD11b+), NK cells (NK1.1+ and Gr-1+), CD4, CD8 T-cells, and B-cells was detected in the spleens of infected mice. Infected mouse Ter-119+ cells represented 80–85% of spleen cells at the peak of splenomegaly compared with 10–30% of all spleen cells in uninfected mice. At the peak of infection, the majority of spleen cells were immature CD71+/ Ter-119+ reticulocytes, indicating that massive erythropoiesis occurs in response to Salmonella infection. Mice injected with anti-EPO had a much lower frequency of CD71+/Ter-119+ cells in the spleen after Salmonella infection, conclusively demonstrating that the expansion of these precursors was due to increased EPO expression (42).
S. aureus infection in skin is able to result in HSPCs trafficking to wounds where HSPCs contribute to the innate immune response by differentiating into mature polymorphonuclear leukocytes (PMN) (51). HSPC numbers, identified by flow cytometry as Lin-/c-Kit+ cells, increased significantly in the BM from days 3 to 5 in response to S. aureus infection. In contrast, HSPC numbers significantly increased in S. aureus-inoculated wounds compared with saline-treated wounds (29). The physiological significance of HSPCs to this response was examined by systemic depletion of HSPCs using the anti-c-kit antibody ACK2. After antibody depletion, a 60% decrease in BM HSPCs leading to a 30% reduction of BM PMN numbers and a decreased 42% to 50% of wound PMN numbers in S. aureus-infected mice were found at 7 days postinfection. Besides, not only PMNs but also CMPs (c-Kit+/Sca-1-/CD34+/CD16/32lo/Lin-/Ly6G-) on day 5 were significantly elevated in S. aureus-inoculated wounds compared with saline-treated wounds. These data indicate that S. aureus infection elicited EMH via SCF-c-Kit axis in the abscess (29).
CpG oligodeoxynucleotides (or CpG ODN) mimicked to bacteria DNA can be synthesized to induced an immune response (114,115). Challenging mice with immunostimulatory CpG-ODN sequences led to transient splenomegaly, with a maximum increase in spleen weight at day 6 after CpG ODN intraperitoneal injection and subsequently normalizes. The induction of splenomegaly by CpG-ODNs was sequence specific, dose dependent, and associated with an increase in splenocytes and in numbers of CFU-GM and BFU-E. Cell surface phenotyping of splenic cells by FACS analysis revealed a transient, but significant, increase in the B220-CD3 double-negative compartment at day 6. There was a slight increase in the number of CFU-GM in BM at day 4 that preceded the splenic increase at day 6, as if a mobilization of BM-derived progenitor cells to the spleen may have taken place (108).
Parasitic Infections Induce EMH
Trypanosoma vivax infection causes severe anemia reported for natural cases of bovine trypanosomosis (84). A transient fivefold increase in the number of reticulocytes in peripheral blood 14 days postinfection was evaluated. Histopathological investigations in various organs showed multifocal inflammatory infiltrates associated with EMH in the liver. A multifocal to coalescing lesion, primarily centered on the portal tracts, but also involving centrilobular veins, was observed in the liver. A very high density of EMH foci was noted in the liver sinusoids. The spleens were uniformly enlarged, thereby characterizing marked splenomegaly, but did not manifest any congestion through the white pulp. This showed disorganized lymphoid structure associated with marked infiltration of activated macrophages, lymphocytes, and plasma cells (11).
Plasmodium infection is associated with splenomegaly and disruption of the splenic microarchitecture in humans as well as in mouse malaria models (116). Myeloid hyperplasia is noted as associated with splenomegaly. The resolution of acute parasitemia in several malaria models is positively correlated with the degree of splenic myelopoiesis (118). In mice with P. chabaudi infection, mobilization of myeloid progenitors via CCL2/CCR2 (C-C chemokine receptor type 2) interaction resulted in the presence of CMPs (Lin-/c-Kithi/CD27+/CD16/32-) and GMPs (CD16/32+) in the spleen. The increased presence of myeloid progenitors in the spleen during infection equated with the significantly increased myeloid potential (CFU-GM) of this organ, suggesting establishment of EMH in the spleen (6).
Viral Infections Induce EMH
Murine cytomegalovirus (MCMV) causes splenomegaly and the expansion of hematopoietic islands in the enlarged spleen of acutely infected mice (58). A significant increase in splenic weight of mice was observed through both intraperitoneal and intravenous infection. MCMV infection that significantly increased the numbers of myeloid progenitors (CFU-GM) and erythroid progenitors (BFU-E) confirmed EMH in the spleen. All developmental stages from early BFU-E to Ter-119+ cells in the splenic EMH could also be observed. Notably, the proportion of Ter-119+ splenocytes correlated with the splenic weight postinfection, indicating that EMH substantially contributes to MCMV-induced splenomegaly (46). Jordan et al. also found that depletion of NK cells by using anti-asialo GM1 antibody completely abolished the expansion of Ter-119+ cells after MCMV infection (46). Moreover, Arase et al. had previously proposed that the Ly49H+ NK cells directly recognize MCMV-infected cells expressed viral m157 protein on the cell surface (1). Combination of these two data reveals a role of NK cells in the establishment of EMH following MCMV infection. NK cells secrete IFN-γ and TNF-α that can activate quiescent HSCs during infection (46). However, Ter-119+ cells could expand either in IFNGR-/- mice after MCMV infection or in C57BL/6 mice with TNF-α depletion achieved by using Etanercept, indicating that IFN-γ and TNF-α signaling is dispensable for induction of EMH (46). In contrast, Ter-119+ cells were completely abrogated in perforin-knockout mice (Prf1-/-) after MCMV infection. Hence, it was the cytotoxic function of NK cells rather than cytokine production that is required for the development of EMH (46). Furthermore, this conclusion is in parallel to Murray's finding (77).
Conclusion
Rigorously defined in vitro clonogenic assays and in vivo reconstitution assays developed in recent years have easily allowed recognition of HSPC activity in nonhematopoietic tissues (14). However, the recruitment of HSCs from the BM into peripheral blood and accommodation to extramedullary organs for expansion and differentiation is a complex process (10,15,27,48,110). From a biological point of view, EMH could serve as an alternative hematopoietic microenvironment for HSPCs to avoid struggling in a myelosuppressive condition in BM. HSCs stay in the spleen cycle twice as frequently as they do in BM (76), suggesting that hematopoietic recovery could employ EMH. Studies involving animal models of EMH demonstrated the link between MSCs and HSPCs, influencing their mobilization/retention and self-renewal/differentiation through cell–cell contacts and production of growth factors and chemokines. The understandings of HSPCs/niche interactions are expanding and promise to improve engraftment of transplanted BM, allow expansion of HSCs in vitro, and provide protection of hematopoiesis from the radiotherapy damage (17).
Footnotes
Acknowledgments
The authors thank Chun-Wei Chen, Jo-Ting Wang, Rong-Kai Liu, and Chia-Wei Kuo for assistance in organizing references. This study was supported by the Ministry of Science and Technology (NSC 99–2314-B-020–001-MY3, NSC 102–2320-B-020–001-MY2, NSC 102–2314-B-039–021-MY3, and MOST 103–2633-B-039–002) and Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW104-TDU-B-212–113002) in Taiwan. This study was also supported in part by China Medical University and Hospital (DMR-104–053) awarded to Dr. Shao-Chih Chiu.