Kohji Morimoto

CD44 and Hyaluronan Binding by Human Myeloid Cells

The CD44 cell surface molecule has been shown to be the principal cell surface receptor for hyaluronan (or hyaluronic acid), a glycosaminoglycan component of marrow extracellular matrix. However, its affinity for hyaluronan is not constitutive, since it depends on the cell type, the stage of differentiation and on activation by external stimuli including certain anti-CD44 antibodies and phorbol esters. Except for a few lymphoid cell lines, hematopoietic cells do not spontaneously bind hyaluronan and initial studies reported that, contrary to lymphocytes, myeloid cells could not be activated to bind hyaluronan. Because CD44 plays an important role in the initial phases of hematopoiesis, as shown by experiments using blocking anti-CD44 monoclonal antibodies, its capacity to mediate adhesion of primitive myeloid cells has been investigated. It was found that CD44 could mediate spontaneous adhesion to hyaluronan of immature myeloid cell lines KG1, KG1a, and TF1, which serve as a model for hematopoietic progenitors. However, despite expressing high amounts of CD44, no more than 15% of bone marrow progenitors could adhere to hyaluronan. Recent experiments have shown that a very important feature of CD44 is its capacity to be rapidly activated by certain antibodies and cytokines (GM-CSF and KL) from a low affinity to a high affinity state for hyaluronan. These data shed light on striking similarities in the functional regulation of CD44 and of the two integrin receptors VLA-4 (a4b1), and VLA-5 (a5b1), which are also expressed on hematopoietic progenitors. The relevance of these data to the regulation of normal hematopoiesis and mobilization of CD34+ progenitors in the view of cell grafting is analyzed. In addition, we show that in idiopathic myelofibrosis, the amount of hyaluronan is markedly increased in the extracellular matrix from the myeloproliferative spleen. Considering that the production of cytokines is enhanced in this disease, we discuss whether CD44-hyaluronan interaction may have a role in the pathophysiology of this myeloproliferative syndrome.

Senescent B lymphopoiesis is balanced in suppressive homeostasis: Decrease in interleukin-7 and transforming growth factor-β levels in stromal cells of senescence-accelerated mice

The suppression of the B cell population during senescence has been considered to be due to the suppression of interleukin-7 (IL-7) production and responsiveness to IL-7; however, the upregulation of transforming growth factor-β (TGF-β) was found to contribute to B cell suppression. To investigate the mechanism of this suppression based on the interrelationship between IL-7 and TGF-β during senescence, senescence-accelerated mice (SAMs), the mouse model of aging, were used in this study to elucidate the mechanisms of B lymphopoietic suppression during aging. Similar to regular senescent mice, SAMs showed a decrease in the number of IL-7–responding B cell progenitors (i.e., colony-forming unit pre-B [CFU-pre-B] cells in the femoral bone marrow [BM]). A co-culture system of B lymphocytes and stromal cells that the authors established showed a significantly lower number of CFU-pre-B cells harvested when BM cells were co-cultured with senescent stromal cells than when they were co-cultured with young stromal cells. Interestingly, cells harvested from a senescent stroma and those from the control culture without stromal cells were higher in number than those harvested from a young stroma, thereby implying that an altered senescent stromal cell is unable to maintain self-renewal of the stem cell compartment. Because TGF-β is supposed to suppress the proliferative capacity of pro-B/pre-B cells, we added a neutralizing anti-TGF-β antibody to the co-culture system with a pro-B/pre-B cell-rich population to determine whether such suppression may be rescued. However, unexpectedly, any rescue was not observed and the number of CFU-pre-B cells remained unchanged when BM cells were co-cultured with senescent stromal cells compared with the co-culture with young stromal cells, which essentially showed an increase in the number of CFU-pre-B cells (P < 0.001 in 5 μg/ml). Furthermore, TGF-β protein level in the supernatant of cultured senescent stroma cells was evaluated by enzyme-linked immunoabsorbent assay, but surprisingly, it was found that TGF-β concentration was significantly lower than that of cultured young stromal cells. Thus, TGF-β activity was assumed to decline particularly in a senescent stroma, which means a distinct difference between the senescent suppression of B lymphopoiesis and secondary B lymphocytopenia. Concerning proliferative signaling, on the other hand, the level of IL-7 gene expression in cells from freshly isolated BM decreased significantly with age. Therefore, the acceleration of proliferative signaling and the deceleration of suppressive signaling may both be altered and weakened in a senescent stroma (i.e., homeosupression).

Comparison of erythropoietic response to androgen in young and old senescence accelerated mice.

In this study, to clarify whether the functional capacity of hemopoietic progenitor cells and the micro-environment of aged mice are identical with those of the young, we investigated the changes in the number of hemopoietic progenitor cells and the production of regulatory cytokines from splenic cells as well as changes in the serum levels of cytokine in senescence-accelerated mice (SAM) after administration of 19-nandrolone decanoate (19-ND), a synthetic androgenic anabolic steroid. 19-ND induced an increase in erythroid colony-forming units (CFU-E), erythroid burst-forming units (BFU-E), and granulocytic-macrophage committed progenitor cells (CFU-GM) in bone marrow and spleen; especially remarkable increases were observed in the splenic CFU-E in both young and old mice. Antigen expression analysis of hemopoietic organs revealed that total TER-119+ cells per spleen of young and old mice with androgen treatment rose 2.6- and 3.2-fold over their respective control values. The responsiveness of hemopoietic progenitor cells to androgen did not change with age. Injection of 19-ND into young and old mice markedly enhanced the erythropoietin levels but not IL3 and GM-CSF levels in the serum of both groups. Cytokine production assessed by pokeweed mitogen-stimulated spleen condition medium showed an age-related decline. Androgen treatment could not influence IL-3 and GM-CSF production of spleen. These findings suggest that the spleen of both old and young mice served as the major site of regenerative repopulation of hemopoietic progenitors, especially the late erythroid progenitors in 19-ND-treated mice. The proliferative reserve of erythropoiesis with androgen treatment in aged mice was not reduced more than that in treated-young mice.

Age-related changes in myelopoietic response to lipopolysaccharide in senescence-accelerated (SAM) mice.

The effects of in vivo lipopolysaccharide (LPS) administration on myelopoiesis were examined in senescence-accelerated (SAM) mice. Young mice injected with LPS exhibited: (a) increased femoral proliferative pool size; (b) transient reduction in femoral non-proliferative pool size and number of femoral colony forming unit-granulocyte macrophages (CFU-GMs); (c) marked increase in splenic CFU-GMs; and (d) transient increase in S-phase of femoral CFU-GMs. The responses of old mice after LPS administration differed from those of young mice in the following points: (a) no recovery of the femoral non-proliferative pool or femoral CFU-GMs, (b) less significant augmentation of the femoral proliferative pool and splenic CFU-GMs, and (c) prolonged reduction in S-phase of femoral CFU-GM. Injection of LPS into mice resulted in a hyperproduction of colony-stimulating activity (CSA) in bone followed by production of colony-inhibitory activity (CIA) in young mice and in contrast, an excessive CIA secretion from bone without an increase in CSA levels in old mice. These imbalances in the regulatory factors derived from non-hemopoietic cells in the bones may lead to an inappropriate response of myelopoiesis in aged SAM mice after LPS administration, which may play a key role in infections.

Age-related decreases in the reconstituting ability of hemopoietic cells and the ability of hemopoietic microenvironment to support hemopoietic reconstitution in senescence accelerated (SAM-P) mice

The effect of the aging process on the hemopoietic system in senescence-accelerated (SAM-P) mice with respect to the reconstituting ability of hemopoietic cells and the ability of the microenvironment to support hemopoietic reconstitution was investigated by bone marrow transplantation (reconstitution assay). When the bone marrow cells, obtained from young or old mice, were transplanted to lethally irradiated young SAM-P mice no difference in the reconstituting pattern of femoral spleen colony-forming units (CFU-S), splenic CFU-S and splenic granulocyte macrophage colony-forming units (CFU-GM) was observed between the mice transplanted with young and old donor cells. However, the reconstitution of femoral CFU-GM in mice transplanted with old donor cells was delayed compared to that in mice transplanted with young donor cells. Moreover, the recovery of WBC in mice transplanted with old donor cells was noticeably delayed. When the bone marrow cells obtained from young mice were transplanted to young or old recipient mice, no difference in the reconstituting pattern of femoral CFU-S and CFU-GM as well as splenic CFU-S and CPU-GM was observed between young and old recipient mice. However, the recovery of WBC in old recipient mice was noticeably delayed. These data indicate that the functions of both the hemopoietic cells and the hemopoietic microenvironment deteriorated with age in SAM-P mice.

Colony promoting activity: differences from interleukin-3 and similarities to interleukin-6

The supernatant of long-term bone marrow culture contains colony promoting activity (CPA) which does not have granulocyte-macrophage colony stimulating activity (CSF) but which enhances granulocyte-macrophage (GM) colony formation in the presence of CSF. CPA might consist of IL-3 or IL-3-like activity, since CPA stimulates proliferation and differentiation of more immature cells to CSF-responding granulocytes-macrophage progenitors (GM-CFC), and since IL-3 also stimulates immature hemopoietic cells to proliferate and differentiate to functional blood cells. IL-1 and IL-6 are also known to enhance GM colony formation. One or both of these molecules can accordingly be another candidate for CPA. In the present study, GM-CSF activity of IL-3 was dependent on the batch of serum: it was negative in the presence of fetal calf serum, but positive in the presence of horse serum. In contrast, GM-CSF and CPA showed no such dependence on the batch of serum. The addition of IL-3 to GM colony assay system did not enhance but rather suppressed GM colony formation. The supernatant of long-term bone marrow culture which contains substantial levels of CPA did not stimulate proliferation of IL-3 dependent DA-1 cells, but facilitated the proliferation of IL-6 dependent, MH60.BSF2 cells. No detectable level of IL-1 activity was found in the supernatant. These results indicate that CPA is different from IL-1, IL-3 or GM-CSF, but similar to or the same as IL-6.

In vivo effect of a large amount of allogeneic granulocytes on reconstitution of hemopoietic cells of irradiated mice

The in vivo effects of allogeneic granulocytes on the reconstitution of splenic and bone marrow CFUs and CFUc numbers were investigated using irradiated mice. When allogeneic granulocytes were intraperitoneally injected into irradiated BDF1 mice (260 rads), the reconstitution of CFUs in both spleen and bone marrow as well as the hematocrit were enhanced, while the reconstitution of splenic or bone marrow CFUc numbers was transiently suppressed and then enhanced. The magnitude of enhancement was dose-dependent. These results suggest that granulocytes injected into irradiated mice might act as enhancing effectors on the in vivo reconstitution of hemopoietic cells.