Roles for common cytokine receptor gamma-chain-dependent cytokines in the generation, differentiation, and maturation of NK cell precursors and peripheral NK cells in vivo

NK cells differentiate in adult mice from bone marrow hemopoietic progenitors. Cytokines, including those that signal via receptors using the common cytokine receptor gamma-chain (gamma(c)), have been implicated at various stages of NK cell development. We have previously described committed NK cell precursors (NKPs), which have the capacity to generate NK cells, but not B, T, erythroid, or myeloid cells, after in vitro culture or transfer to a fetal thymic microenvironment. NKPs express the CD122 Ag (beta chain of the receptors for IL-2/IL-15), but lack other mature NK markers, including NK1.1, CD49b (DX5), or members of the Ly49 gene family. In this report, we have analyzed the roles for gamma(c)-dependent cytokines in the generation of bone marrow NKP and in their subsequent differentiation to mature NK cells in vivo. Normal numbers of NKPs are found in gamma(c)-deficient mice, suggesting that NK cell commitment is not dependent on IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21. Although IL-2, IL-4, and IL-7 have been reported to influence NK cell differentiation, we find that mice deficient in any or all of these cytokines have normal NK cell numbers, phenotype, and effector functions. In contrast, IL-15 plays a dominant role in early NK cell differentiation by maintaining normal numbers of immature and mature NK cells in the bone marrow and spleen. Surprisingly, the few residual NK cells generated in absence of IL-15 appear relatively mature, expressing a variety of Ly49 receptors and demonstrating lytic and cytokine production capacity.     

The host environment regulates the function of CD8+ graft-versus-host-reactive effector cells

We have examined how the host environment influences the graft-vs-leukemia (GVL) response following transfer of donor T cells to allogeneic chimeras. Donor T cells induce significant GVL when administered in large numbers to established mixed chimeras (MC). However, when using limiting numbers of T cells, we found that late transfer to MC induced less GVL than did early transfer to freshly irradiated allogeneic recipients. Late donor T cell transfer to MC was associated with marked accumulation of anti-host CD8 cells within the spleen, but delayed kinetics of differentiation, reduced expression of effector molecules including IFN-gamma, impaired cytotoxicity, and higher rates of sustained apoptosis. Furthermore, in contrast to the spleen, we observed a significant delay in donor CD8 cell recruitment to the bone marrow, a key location for hematopoietic tumors. Increasing the numbers of T cells transferred to MC led to the enhancement of CTL activity and detectable increases in absolute numbers of IFN-gamma(+) cells without inducing graft-vs-host disease (GVHD). TLR-induced systemic inflammation accelerated differentiation of functional CTL in MC but was associated with severe GVHD. In the absence of inflammation, both recipient T and non-T cell populations impeded the full development of GVHD-inducing effector function. We conclude that per-cell deficits in the function of donor CD8 cells activated in MC may be overcome by transferring larger numbers of T cells without inducing GVHD.     

Changes in the populations of null, NK1.1+, and Thy1lo lymphocytes in the bone marrow of tumor-bearing mice: effect of indomethacin treatment.

Mouse bone marrow produces many "null" lymphocytes which lack B and T lineage markers (B220-Thy1-). A subset of these cells expresses the natural killer (NK) cell marker, NK1.1. In addition, some rapidly renewed bone marrow lymphocytes express low intensities of Thy1 (Thy1lo). In view of their possible implication in tumor-host interactions these various cell populations have now been examined in mice injected with either the nonmetastatic Ehrlich ascites (EA) tumor or the Lewis lung carcinoma (LLc), a highly metastatic solid tumor. In each case, the number of null lymphocytes, as defined by a lack of radioautographic labeling of either B220 glycoprotein or Thy1, increased markedly in both the bone marrow and spleen. Treatment with the prostaglandin inhibitor, indomethacin, enhanced the increase in null cells in the bone marrow and spleen of LLc-bearing mice. The number of null small lymphocytes expressing NK1.1, as detected by combined radioautographic and immunoperoxidase techniques, increased almost 30-fold in LLc-bearing mice. The number of Thy1lo small lymphocytes increased in parallel with null cells during EA tumor growth. The findings accord with the hypothesis that the null lymphocyte population produced in mouse bone marrow includes newly formed NK lineage cells which sequentially express NK1.1 and Thy1lo. The present work demonstrates that the populations of null, NK1.1+, and Thy1lo lymphocytes in mouse bone marrow expand rapidly during the early growth of transplanted tumors, the initial increase in null lymphocytes apparently being curtailed by prostaglandin production. The results suggest that the production of null lymphocytes in mouse bone marrow is responsive to tumor development, possibly providing cells to be involved in tumor-host interactions.     

Synthesis of colony-stimulating factor (CSF) and differentiation-inducing factor (D-factor) by osteoblastic cells, clone MC3T3-E1

The role of osteoblasts in inducing the proliferation and differentiation of bone marrow cells was examined. Conditioned medium obtained from mouse osteoblastic cell (MC3T3-E1) cultures stimulated colony formation of mouse bone marrow cells (CSF) and differentiation of mouse myeloid leukemia cells (M1) into macrophage-like cells (D-factor). The CSF activity increased time dependently in parallel with the increase of alkaline phosphatase activity during the culturing of the MC3T3-E1 cells. The activity of the D-factor attained a maximum on days 12 - 15 and decreased thereafter. Both the CSF and the D-factor were eluted in a range of 25,000 to 67,000 daltons on gel filtration. The fraction containing both factors exhibited bone-resorbing activity. These results suggest that osteoblasts are involved in bone resorption at least in part by enhancing the proliferation and differentiation of osteoclast progenitors.     

Population dynamics of "null" and Thy1lo lymphocytes in mouse bone marrow: genesis of cells with natural killer cell lineage characteristics.

The population dynamics of "null" small lymphocytes lacking B and T lineage markers in mouse bone marrow have been examined using a combination of immunolabeling and hydroxyurea (HU) deletion techniques. The binding of the B lineage-associated mAb, 14.8, and anti-Thy1.2 to bone marrow cells has been detected radioautographically. Null cells lacking 14.8 and Thy1.2 determinants (14.8- Thy1-) formed a substantial subset (12-14%) of bone marrow small lymphocytes, representing 0.5 x 10(6) cells per femur (2-3% of nucleated cells). HU treatment revealed an exceptionally rapid turnover of the null small lymphocyte population (T1/2, 7.5 hr) compared with 14.8+ cells (T1/2, 20.5 hr) and Thy1+ cells (T1/2, 53 hr). Small lymphocytes bearing low intensities of Thy1 (Thy1lo) were also rapidly renewed (T1/2, 28 hr) whereas those with high intensities of Thy1 (Thy1hi) were renewed only slowly (T1/2, 123 hr). During ontogeny, null small lymphocytes first appeared in the fetal liver by Day 11 and the fetal spleen by Day 16, but increased rapidly in the bone marrow in early postnatal life. Double immunolabeling techniques demonstrated that 10% of null small lymphocytes in the bone marrow expressed NK1.1 antigen, while larger proportions bound to tumor (YAC.1) cells in vitro and displayed Fc receptors. The NK1.1-bearing fraction of null small lymphocytes in bone marrow was depleted by HU treatment only after an initial delay. NK1.1 was also expressed on subsets of Thy1lo cells and Thy1hi cells. The results have revealed the continuous production in mouse bone marrow of null and Thy1lo small lymphocytes, totaling 1-3 x 10(7) cells/day and 1.2 x 10(6) cells/day, respectively. The findings suggest that the large-scale production of null lymphocytes in mouse bone marrow includes the genesis of NK lineage cells which express NK1.1 and Thy1lo during a period of terminal maturation.     

Heterogeneity of NK1.1+ T cells in the bone marrow: divergence from the thymus.

NK1.1+ T cells in the mouse thymus and bone marrow were compared because some marrow NK1.1+ T cells have been reported to be extrathymically derived. Almost all NK1.1+ T cells in the thymus were depleted in the CD1-/-, beta2m-/-, and Jalpha281-/- mice as compared with wild-type mice. CD8+NK1.1+ T cells were not clearly detected, even in the wild-type mice. In bone marrow from the wild-type mice, CD8+NK1.1+ T cells were easily detected, about twice as numerous as CD4+NK1.1+ T cells, and were similar in number to CD4-CD8-NK1.1+ T cells. All three marrow NK1.1+ T cell subsets were reduced about 4-fold in CD1-/- mice. No reduction was observed in CD8+NK1.1+ T cells in the bone marrow of Jalpha281-/- mice, but marrow CD8+NK1.1+ T cells were markedly depleted in beta2m-/- mice. All NK1.1+ T cell subsets in the marrow of wild-type mice produced high levels of IFN-gamma, IL-4, and IL-10. Although the numbers of marrow CD4-CD8-NK1.1+ T cells in beta2m-/- and Jalpha281-/- mice were similar to those in wild-type mice, these cells had a Th1-like pattern (high IFN-gamma, and low IL-4 and IL-10). In conclusion, the large majority of NK1.1+ T cells in the bone marrow are CD1 dependent. Marrow NK1.1+ T cells include CD8+, Valpha14-Jalpha281-, and beta2m-independent subsets that are not clearly detected in the thymus.     

Cutting edge: Thymic NK cells develop independently from T cell precursors

Although NK cells in the mouse are thought to develop in the bone marrow, a small population of NK cells in the thymus has been shown to derive from a GATA3-dependent pathway. Characteristically, thymic NK cells express CD127 and few Ly49 molecules and lack CD11b. Because these NK cells develop in the thymus, the question of their relationship to the T cell lineage has been raised. Using several different mouse models, we find that unlike T cells, thymic NK cells are not the progeny of Rorc-expressing progenitors and do not express Rag2 or rearrange the TCRγ locus. We further demonstrate that thymic NK cells develop independently of the Notch signaling pathway, supporting the idea that thymic NK cells represent bona fide NK cells that can develop independently of all T cell precursors.     

Low calcium environment effects osteoprotegerin ligand/osteoclast differentiation factor

In coculture with osteoblastic cell line MC3T3-E1 (E1) and mouse bone marrow cells, we reported that numbers of osteoclasts rose significantly on exposure to a low-calcium environment. Here we examined how osteoblasts influence osteoclastogenesis under a low-calcium environment. Comparing low extracellular calcium with a regular calcium environment, osteoprotegerin ligand (OPGL)/osteoclast differentiation factor (ODF) mRNA expression show more increase in the culture of low-calcium environment than in that of a regular calcium environment. Calcium-sensing receptor (CaSR), which was supposed as one of the mechanisms of recognizing extracellular calcium, existedon the surface of E1 cells. When E1 cells stimulated with agonists of CaSR, gadolinium, and neomycin, OPGL/ODF mRNA expression decreased. Moreover, these agonists reduced osteoclast formation in coculture. Taken together, it is possible that osteoblasts may recognize extracellular calcium via CaSR and regulate osteoclastogenesis.     

High expression of the Thy-1 antigen on natural killer cells recently derived from bone marrow

The subset of murine natural killer (NK) cells that kills lymphoma targets contains about 50% cells expressing the Thy-1 antigen and this has been one of the reasons for assigning NK cells to the T-cell differentiation lineage. It has now been shown that the proportion of the Thy-1+ NK cells is not constant: ca. 90% of the NK cells appearing in the spleens of irradiated mice injected 10-14 days previously with bone marrow cells (anti-Thy-1 plus complement treated) express this antigen. The donor origin of these Thy-1+ NK cells was demonstrated by using semisyngeneic bone marrow cells in transfers but this same phenomenon could also be observed after entirely syngeneic transfers, excluding the possibilities that this Thy-1+ NK activity is due to activated T cells or to the effect of T-cell activation products on NK cells. Additionally, these early NK cells expressed the asialo-GM1 antigen, which is found on murine NK cells but not on cytotoxic T cells. These data suggest that the precursors for NK cells in the bone marrow are Thy-1-, and that the first splenic NK cells derived from these progenitors express this antigen.     

Origin and differentiation of natural killer cells. I. Characteristics of a transplantable NK cell precursor

To study the origin and differentiation of natural killer (NK) cells, we developed an assay for the transplantable precursor of NK(YAC-1) cells present in the bone marrow. Mice were depleted of endogenous NK(YAC-1) cells by injection of anti-asialo GM1 antibody, followed by lethal whole body irradiation. Normal syngeneic bone marrow cells were transplanted into such pretreated mice. Regeneration of NK(YAC-1) activity in the recipient mice was monitored by two different assays: the ability of spleen cells to lyse YAC-1 cells in vitro and the ability to clear i.v. injected, 125IUdR-labeled YAC-1 cells from the lungs. With both assays, a dose-response relationship between the number of bone marrow cells injected and the degree of NK(YAC-1) activity generated could be demonstrated. However, the lung clearance assay appeared superior because the NK regeneration could be detected earlier and with lower numbers of injected marrow cells. With this assay, several characteristics of the NK precursors and their differentiation could be defined. 1) The generation of mature, lytic NK cells from their transplantable precursor requires an intact "marrow microenvironment" in the recipient mice, because differentiation failed to occur in mice rendered osteopetrotic by estradiol treatment. 2) The NK(YAC-1) precursors lack the surface antigens (NK-2.1, asialo GM1, Qa-5, Thy-1) that are characteristically seen on mature NK cells. 3) The NK-precursors could be eliminated from the bone marrow with anti-Qa-2 or anti-H-2 antisera + complement, indicating that these two antigens are expressed on the precursors. The relationship between NK(YAC-1) precursors and multipotent myeloid stem cells (CFU-S) was investigated by utilizing W/Wv and Sl/Sld mutant mice. Bone marrow cells of W/Wv anemic mice, although markedly deficient in CFU-S, have a normal frequency of NK(YAC-1) precursors. Sl/Sld mice that lack a suitable microenvironment for the development of CFU-S allowed normal differentiation of NK(YAC-1) precursors when transplanted with normal bone marrow cells. Together, these data suggest that multipotent myeloid progenitor cells, as defined by the CFU-S assay, and the NK(YAC-1) precursors are not closely related.     

Regulatory mechanisms of mast cell differentiation

Mast cells are a progeny of the multipotential hematopoietic stem cell. Most of progenies of the stem cell complete their differentiation within the bone marrow, but precursors of mast cells leave the bone marrow, migrate in blood, and invade into tissues. After the invasion, precursors proliferate and differentiate into mast cells. An appreciable proportion of mast cells retain proliferative potential after differentiation, and even after degranulation, some mast cells can proliferate and recover the original morphology. Proliferation of mast cells are regulated by both T cell-derived factors (i.e., IL-3 and IL-4) and fibroblast-derived factor(s). Mice of either W/Wv or Sl/Sld genotype lack mast cells, but mast cells do develop when bone marrow cells of W/Wv or Sl/Sld mice were cultured in the presence of T cell-derived factors. Mast cells derived from W/Wv mice cannot respond fibroblast-derived factor(s) and fibroblasts derived from Sl/Sld mice cannot support mast cells of normal mouse origin. Phenotypes of mast cells are determined by the environment in which the mast cells differentiated. However, when mast cells are transplanted into a new environment which is different from the original one, the mast cells acquire the phenotype which are dependent on the second environment.     

Propranolol attenuates calorie restriction- and high calorie diet-induced bone marrow adiposity

We investigated the effects of β-adrenergic activation on bone marrow adiposity and on adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). C57BL/6 mice were subjected to a control (CON), high calorie (HIGH) or low calorie (LOW) diet for 12 weeks. In each group, mice were treated with vehicle (VEH) or propranolol. The number of adipocytes per area bone marrow was increased in LOWVEH and HIGHVEH mice compared with CONVEH mice, which was attenuated by propranolol. Isoproterenol increased lipid droplet accumulation and adipogenic marker gene expression in 3T3-L1 preadipocytes and mouse BMSCs, which were blocked by propranolol. Conditioned medium obtained from MC3T3-E1 osteoblasts suppressed adipogenic differentiation of 3T3-L1 cells, which was significantly attenuated by treatment of MC3T3-E1 cells with isoproterenol. These data suggest that β-adrenergic activation enhances bone marrow adipogenesis via direct stimulation of BMSCs adipogenesis and indirect inhibition of osteoblast anti-adipogenic potential. [BMB Reports 2014; 47(10): 587-592]     

Effect of extracellular calcium concentrations on osteoclast differentiation in vitro

We have been researching with the purpose of understanding the hard organization mineralization-suppression mechanism under a low calcium environment. So far, we have discerned clearly that the femur of an incubated newborn rat under a low calcium environment will develop bone formation dyscrasia such as hypertrophy of the caput. In this experiment, MC3T3-E1 (E1) cells, which are osteoblast-like cells, and mouse bone marrow were incubated in a coculture system under a low calcium environment and manifestation of osteoblast cells and their ability to resorb were examined. The results suggested that as the calcium concentration in the medium decreased manifestation of osteoclast cells increased, and as the degree of mineralization of E1 cells advanced, the number of manifestation of osteoblast cells decreased. We, therefore, report that there is a possibility that extracellular calcium concentrations involve the process of differentiation of bone marrow cells into osteoblast cells.     

Effect of the mi allele on mast cells, basophils, natural killer cells, and osteoclasts in C57Bl/6J mice

The osteopetrotic, microphthalmic (mi/mi) mouse lacks functional osteoclasts and has also been reported to be deficient in mast cells and natural-killer (NK) cells. The later deficiencies could be secondary to the osteopetrotic marrow, or a direct result of the mi allele. Therefore, heterozygotes were examined for these cell types, since these mice do not exhibit osteopetrosis. Adult +/mi animals have approximately 50%, and mi/mi animals examined by histologic techniques or tissue histamine levels have 0-10%, of the peritoneal, dermal, and intestinal mast cells compared with that of +/+ animals. Leukocyte histamine, indicative of the number of basophils, demonstrates the same pattern. Histamine content per mast cell in +/+ and +/mi animals is identical. The number of large granular lymphocytes (LGL) in splenic leukocyte preparations from +/mi animals is 50% that of +/+ animals, and these cells are undetectable in preparations from mi/mi mice. NK activity against YAC-1 cells paralleled the number of LGL present. The resorptive response of neonatal calvaria to parathyroid hormone was delayed in the case of cultured +/mi bone compared with that of +/+ bone, but the final rate of calcium release was identical. These data indicate that 1) the presence of one mi allele can affect the development of four distinct cell types, and 2) osteopetrosis alone does not account for the lack of mast cells, basophils, and NK cells in mi/mi mice.     

Lack of spontaneous and inducible natural killer cell activity in human bone marrow: presence of adherent suppressor cells

Human bone marrow cells collected from ribs of patients undergoing thoracotomy had low or no natural killer (NK) cell activity against K562 in a 4-hour chromium release assay. In vitro overnight treatment with interferon or interleukin 2 of bone marrow cells resulted in no induction or augmentation of NK cell activity. In the presence of adherent bone marrow cells interferon was unable to enhance NK cell activity of blood lymphocytes, although the baseline level of NK cell activity was not suppressed. These results suggest that adherent bone marrow cells regulate the development of active NK cells and that bone marrow components do not provide a favorable environment for the functional differentiation of NK cells.     

MYC and PIM2 co-expression in mouse bone marrow cells readily establishes permanent myeloid cell lines that can induce lethal myeloid sarcoma in vivo

The hematopoietic cell malignancy is one of the most prevalent type of cancer and the disease has multiple pathologic molecular signatures. Research on the origin of hematopoietic cancer stem cells and the mode of subsequent maintenance and differentiation needs robust animal models that can reproduce the transformation and differentiation event in vivo. Here, we show that co-transduction of MYC and PIM2 proto-oncogenes into mouse bone marrow cells readily establishes permanent cell lines that can induce lethal myeloid sarcoma in vivo. Unlike the previous doubly transgenic mouse model in which coexpression of MYC and PIM2 transgenes exclusively induced B cell lymphoma, we were able to show that the same combination of genes can also transform primary bone marrow myeloid cells in vitro resulting in permanent cell lines which induce myeloid sarcoma upon in vivo transplantation. By inducing cancerous transformation of fresh bone marrow cells in a controlled environment, the model we established will be useful for detailed study of the molecular events involved in initial transformation process of primary myeloid bone marrow cells and provides a model that can give insight to the molecular pathologic characteristics of human myeloid sarcoma, a rare presentation of solid tumors of undifferentiated myeloid blast cells associated with various types of myeloid leukemia.     

Impaired NK cell development in an IFN-gamma transgenic mouse: aberrantly expressed IFN-gamma enhances hematopoietic stem cell apoptosis and affects NK cell differentiation

Aberrant expression of IFN-gamma has been demonstrated to cause a wide variety of alterations in cell function and development. Previously we reported that constitutive expression of IFN-gamma in bone marrow (BM) and thymus results in a total absence of B cells and a substantial decrease in the number of hematopoietic progenitor cells. In this study, we demonstrate a severe deficiency of NK1.1(+)CD3(-) cells in this transgenic mouse model. Compared with normal control littermates, we found a pronounced reduction of NK cells in IFN-gamma transgenic mouse spleen and liver despite maintenance of normal function. In addition, we observed a reduced number of BM cells in the IFN-gamma transgenic mouse despite normal expression of hematopoietic growth factors in the BM. Interestingly, these cells were less responsive to stem cell factor (SCF) despite c-kit expression on hematopoietic stem cells (HSCs). We observed that addition of exogenous IFN-gamma inhibited proliferation of HSCs and differentiation of NK precursors from HSCs in normal mice in response to SCF, IL-7, fms-like tyrosine kinase 3 ligand, and IL-15. Furthermore, we found that HSCs express the IFN-gammaRalpha subunit and undergo apoptosis in response to exogenous IFN-gamma. Thus, we have demonstrated the occurrence of a severe deficiency of NK cells and lower numbers of BM cells in an IFN-gamma transgenic mouse model. Furthermore, because exogenous IFN-gamma affects the responsiveness to hematopoietic growth factors such as SCF in vitro, our results indicate that chronic expression of IFN-gamma in vivo leads to widespread immune system defects, including alterations in NK cell differentiation.