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Physiological parameters such as pH and oxygen tension probably play significant roles in the regulation of haemopoiesis in the bone marrow microenvironment, but these roles have yet to be characterized in detail. We have found that changes in culture pH (0.2 units) can cause significant changes in the culture composition of mature cells and colony-forming cells (CFCs), especially in the presence of erythropoietin (Epo). Peripheral blood (PB) CD34+ cells cultured at different pH values (7.15–7.6) were characterized using total cell counts, colony assays, morphological analysis, haemoglobin staining, flow cytometry, immunocytochemical staining, and Western blots. Cultures performed at high (7.6) pH contained greater numbers of haemoglobin-positive and band-3-positive cells, and acquired these erythroid differentiation markers sooner than standard (7.35) and low (7.1) pH cultures. Flow cytometry using CD71 and CD45RA antigens also indicated that erythroid differentiation proceeds faster at high pH and is blocked at an intermediate stage by low pH. Morphological data confirmed that high pH cultures had been shifted towards late-stage erythroid compartments as compared to low and standard pH cultures. These findings have important implications both in elucidating the regulatory role of pH in the bone marrow microenvironment and for the design of in vitro systems to study the development of erythroid cells.
It has been recognized since early this century that pH buffering is an important and complex control system in mammals. Cells subjected to different pH environments exhibit distinct responses in terms of differentiation and protein secretion patterns. In haemopoietic cells, changes in intracellular pH have been associated with activation of lymphocytes, neutrophils and platelets ( Ozaki, 1992). Also, the proliferation of, and erythropoietin (Epo) secretion by, murine macrophages have been shown to be pH-dependent, with optimal values at higher than physiological pH (7.6–8.0) ( Rich, 1988). Although the connection between pH and haemopoietic cells is more difficult to make in vivo because of interactions with CO2, O2 and bicarbonate, there are suggestions of a link between blood pH and the generation and function of erythroid cells. One such condition is end-stage renal failure, in which both acidosis (low blood pH) and anaemia (shortage of red blood cells) are primary symptoms ( Segal et al, 1988 ). In addition, Yang (1992) and Yang et al (1995 ) have demonstrated that some respiratory failure patients who exhibit acidosis also have impaired function of the primary ion channel found in erythroid cells.
In order to provide insight into these pathophysiological conditions and to further understand the effects of culture pH on the erythroid lineage, we have conducted experiments to examine in detail the effects of culture pH on the ex vivo differentiation of erythroid lineage cells. Since bone marrow pH in vivo is likely to be as low as 7.1, and dramatic effects on haemopoietic cell lines were noted at pH 7.6 ( Endo et al, 1994 ), we chose to examine three pH values spanning this range (7.1, 7.4, 7.6). In previous work we have demonstrated that culture pH has a significant effect on haemopoietic cells ( McAdams et al, 1997 ). These studies demonstrated important changes in cloning efficiency with pH and provided preliminary indications of changes in the differentiation programme of haemopoietic (particularly erythroid) progenitors cultured ex vivo. In the current studies we used the previously developed culture system to perform a detailed analysis of erythroid differentiation markers and draw new conclusions about the effects of culture pH on the kinetics of erythroid differentiation. These studies were performed with CD34+ progenitors, thus extending our investigation of pH effects to this clinically important cell population. Based both on our in vitro results and an examination of the bone marrow microvasculature, we propose that pH may be an important regulator of erythroid proliferation within the bone marrow microenvironment.
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- MATERIALS AND METHODS
Changes in culture pH have been shown to affect haemopoietic differentiation in several systems. For the myeloid lineage, Fischkoff et al (1984 ) and Fischkoff & Rossi (1990) demonstrated that HL-60 (a human myeloid leukaemia cell line) cells, which normally differentiate into monocytes when cultured at pH 7.2, will instead differentiate into eosinophils when cultured at pH 7.6. For the erythroid lineage, Endo et al (1994 ) investigated the effects of pH on the human erythroid cell lines KU-812 and K562. Their experiments indicated that a significantly greater proportion of cells acquire a mature erythroid phenotype (haemoglobin-positive) when cultured at pH 7.6 than when cultured at lower pH values. A progressive increase in differentiation was noted as pH increased, indicating gradual changes rather than a step-change phenomenon.
Our initial studies investigating the effects of culture pH on ex vivo haemopoietic cultures ( McAdams et al, 1997 ) led us to hypothesize that the rate and/or extent of differentiation of cells of the erythroid lineage accelerated with increasing extracellular pH, over the range 7.1–7.6. In this report we have provided data supporting this hypothesis by examining multiple markers of erythroid differentiation in the culture of CD34+ peripheral blood progenitor cells. The culture data in Table I show that culture at low pH resulted in fewer total cells without significantly affecting the number of BFU-E. Increasing the pH, however, resulted in a significant drop in BFU-E content, and an increase in the percentage of haemoglobin-containing cells. These data suggest that the generation of more mature erythroid cells from BFU-E progenitors is inhibited by low pH, whereas at high pH it is accelerated, resulting in a loss of BFU-E. An alternative explanation is that the low pH conditions promote greater destruction of mature erythroid cells rather than inhibiting their production. However, our previous study ( McAdams et al, 1997 ) utilizing both pH-adjusted methylcellulose cultures and liquid cultures (same culture conditions as in these studies) failed to document the presence of any (even non-viable) mature erythroid cells, or increased cellular debris that one would expect to accompany the destruction hypothesis. A further examination of the cellular phenotype in these cultures was performed using morphological (Fig 1) and flow cytometry (Fig 2) analyses. Both analyses indicated that the distribution of erythroid cell types was more immature at low pH, and more mature at high pH. The flow cytometry analysis, in particular, demonstrated a kinetic effect in that similar patterns of change occurred, but at different rates in different pH environments. The time course profile for benzidine-positive cells in Fig 3 provided further confirmation of this change in the rate of differentiation. The use of band 3 as an additional erythroid marker (Fig 4) supports the hypothesis, showing increased expression at high pH and essentially no expression at low pH. Together, these results indicate that a low pH-induces block or bottleneck in erythroid differentiation occurs at a post-BFU-E stage after the cell has up-regulated CD71 and begun to accumulate haemoglobin, but before it acquires late erythroid markers such as band 3 ( Table II). The morphological analysis suggests that this is most probably somewhere within the basophilic normoblast stage. The Western blots (Fig 5) were consistent with our assessment of the location of the low pH-induced block, showing that the low pH cultures contained a small amount of haemoglobin, but no band 3 protein.
Both the structure of the bone marrow ( Adler, 1984) and measurements of pH in various tissues strongly suggest that substantial pH gradients exist in the bone marrow microenvironment. In normal brain tissue, pH values range from 6.7 to 7.5 ( Gerweck & Seetharaman, 1996). Measurements of normal subcutaneous tissue using a sensitive fluorescence ratio imaging technique have found an average pH value of 7.23 ( Martin & Jain, 1994). In addition, the same study documented steep pH gradients as the distance from blood vessels (pH 7.4) was increased to 10 μm (pH 7.25) or 30 μm (pH 7.1). The latter two values correspond to about one or three cell diameters in distance from the blood vessel. Structural studies of bone marrow show that a significant amount of the bone marrow extravascular space is >50 μm from the nearest sinus ( Adler, 1984; Weiss, 1976).
We propose that pH plays a role in regulating the development of erythroid cells within the bone marrow. The later stages of erythroid differentiation are often accomplished within erythroblastic islands, in which a ‘nurse’ macrophage cell assists in the maturation and enucleation of the erythroid cells with which it maintains intimate contact in the bone marrow microenvironment ( Tavassoli & Yoffey, 1983). Since mature erythroid cells lack motility, these erythroblastic islands are located immediately adjacent to the sinus wall ( Jandl, 1987a), thus facilitating the egress of mature erythroid cells from the bone marrow. We suggest two possible physiological roles in vivo of our observations that reduced pH slows erythroid development. One possible role may be to regulate the rate at which erythroid development occurs within the erythroblastic island. Due to the large size (>50 μm) of the erythroblastic island ( Wickramasinghe, 1975), erythroid cells at the interior are likely to be exposed to a lower pH than are the cells at the periphery. This would lead to an increasing rate of differentiation of the erythroid cells radially outward from the macrophage, which may facilitate the release of the most mature erythroid cells at the periphery into the circulation.
Another role may be to allow the primitive erythroid progenitor, which may be generated away from the sinus wall, time to reach an appropriate macrophage near the sinus before beginning its differentiation programme. Maturation of erythroid progenitors far into the bone marrow interior may be disadvantageous because of the lack of motility of mature erythroid cells ( Jandl, 1987a). The lower pH further away from the sinuses would tend to slow down or inhibit the erythroid maturation process. The primitive erythroid cells would gradually migrate or be pushed towards the sinuses by the general outward flow ( Lord, 1990) of haemopoietic cells within the bone marrow environment. As the erythroid progenitors encounter increasing pH values near the sinus, they would accelerate their programme of differentiation at a more appropriate location in the bone marrow with the assistance of macrophages. This is in contrast to granulocytic development — in which granulocytic post-progenitors are formed deeper in the bone marrow and are actively motile, propelling themselves towards the sinuses as they differentiate ( Jandl, 1987a). This may account for the less profound effects of culture pH on the granulocytic lineage in comparison to the erythroid lineage ( McAdams et al, 1997 ).
The observation that the rate and/or extent of erythroid differentiation is sensitive to changes in pH has implications in several areas. First, in studies characterizing the effects of other agents and conditions on the erythroid lineage, it is very important to control and monitor pH throughout the course of an experiment. This may be particularly important in cultures utilizing stromal cells or high-density cultures in monolayers, since the metabolic output of such cultures may produce significant changes in local pH values even if the bulk medium pH remains unaltered ( Akatov et al, 1985 ). Second, this report demonstrates that pH gradients within bone marrow tissue may be an important mechanism in regulating erythroid development. Further studies on the effects of pH on haemopoietic cells may provide additional insight into these regulatory mechanisms.