Martin Rosendaal

Hypoxia is a Major Stimulator of Osteoclast Formation and Bone Resorption

Hypoxia is known to act as a general stimulator of cells derived from marrow precursors. We investigated the effect of oxygen tension on the formation and function of osteoclasts, the cells responsible for bore resorption, which are of promonocytic origin. Using 7‐ and 13‐day cultures of mouse marrow cells on ivory discs, we found that reducing oxygen tension from the ambient atmospheric level of 20% by increasing the proportion of nitrogen caused progressive increases in the formation of multinucleated osteoclasts and resorption pits. Peak effects occurred in 2% oxygen, where stimulations of resorption up to 21‐fold were measured. Significant stimulations of osteoclast formation and resorption were observed even in severely hypoxic cultures gassed with 0.2% oxygen. Short‐term cultures of cells disaggregated from rat bones indicated that hypoxia did not alter the resorptive activity of mature osteoclasts, but reduced their survival or adherence. In 3‐day organ cultures of mouse calvarial bones, exposure to 2% oxygen resulted in maximal, fivefold stimulation of osteoclast‐mediated calcium release, an effect equivalent to that of prostaglandin E2 (PGE2), a reference osteolytic agent. Hypoxia also caused a moderate acidosis in calvarial cultures, presumably as a result of increased anaerobic metabolism; this observation is significant because osteoclast activation is dependent on extracellular acidification. Our experiments reveal a previously‐overlooked mechanism of considerable potential importance for the regulation of bone destruction. These findings may help explain the bone loss associated with a wide range of pathological states involving local or systemic hypoxia, and emphasize the importance of the vasculature in bone.

Gap-Junction Communication Pathways in Germinal Center Reactions

Intercellular channels called gap junctions enable multicellular organisms to exchange information rapidly between cells. Though gap junctions are held to be ubiquitous in solid tissues, we have only recently found them in the lymphoid organs. Functional direct cell-cell communication has now been confirmed by us and other groups in bone marrow, thymus, and in secondary lymphoid tissues. What functions do they serve in the lymphoreticular system where, so far, only cytokines/growth factors and adhesion molecules have been considered as regulators? Here we show evidence for and refer to published work about functional direct cellcell communication through gap junctions in germinal center reactions and make proposals for their role in the immune response. We found a large amount of the connexin43 (Cx43) gap junctions in the germinal centers of secondary lymphoid follicles. Ultrastructurally and immunohistologically, most of the junctions were detected on the processes of follicular dendritic cells (FDC) enveloping nondividing centrocytes in the light zone of germinal centers where B-cell selection is thought to take place. Further support for this finding came by revealing the Cx43 mRNA in situ at the same location as the protein. On antigen challenge, gap junctions appeared on the FDC as they formed meshworks in germinal centers. In order to find out which germinal center cells communicate directly, we separated FDC-rich, low-density, B-cell fractions from human tonsil. In culture, we injected single FDC with the low-molecular-weight fluorescent dye, Lucifer Yellow (Mr 457 Da), which passed between adjacent FDC and sometimes from FDC to B cells. Based on these findings and their assigned functions in other tissues, gap junctions may contribute to germinal center reactions in the following ways: (1) they may regulate follicle pattern formation by controlling FDC growth, (2) they may be involved in FDC-B-cell signaling contributing to the final rescue of selected B cells from apoptosis, and (3) they may enable FDC to work as a functional syncytium providing a cellular internet for integrating germinal center events. Data supporting these interpretations are briefly discussed.

A cautionary tale: how to delete mouse haemopoietic stem cells with busulphan

In this study treating mice with the ‘correct’ dose of busulphan did not necessarily destroy all haematopoietic stem cells. In certain circumstances host stem cells survived undetected and subsequently resumed haemopoiesis. This may apply to the use of busulphan clinically. We found that the following conditions determined the deletion of mouse stem cells using busulphan: (1) graft size – grafting more than 106 marrow cells (∼0·3% of the animals stem cells) concealed the survival of stem cells; (2) dose of busulphan – insufficient busulphan did not kill all host stem cells; (3) old or improperly stored busulphan failed to delete all host stem cells; furthermore (4) the survival of host stem cells should be assessed by typing many kinds of circulating cells; and (5) tests should be carried out to determine if busulphan has killed all host stem cells by typing circulating blood cells at appropriate intervals.

Physical and kinetic properties of haemopoietic progenitor cell populations from mouse marrow detected in five different assay systems.

Properties of haemopoietic progenitor cells detected in several different assays have been compared in order to position them within the haemopoietic developmental lineage. The spleen colony-forming cell (CFUs), the high proliferation potential colony-forming cell (HPP-CFC) and two granulocyte-macrophage colony-forming cells (GM-CFC-1 and GM-CFC-2) have been studied. Two experimental techniques were used: separation of cells on the basis of their buoyant density and comparison of the survival of haemopoietic cells after donor mice had been injected with the cytotoxic drug 5-fluorouracil (5-FU). On linear BSA gradients the modal buoyant densities of CFUs, HPP-CFC and GM-CFC-1 were the same, 1.070 g cm-3; the density of GM-CFC-2 was higher, 1.075 g cm-3. GM-CFC-2 colonies were much smaller and contained far fewer cells than HPP-CFC or GM-CFC-1 colonies, even after prolonged culture, and this suggests that dense haemopoietic progenitors have less proliferation potential. This was confirmed by comparison of the size of colony formed, under identical culture conditions, by progenitors of different densities. Mean colony diameter was inversely related to the density of the progenitor cell. With the exception of GM-CFC-1, low density progenitors were more resistant to the cytotoxic effects of 5-FU than high density precursor cells (GM-CFC-2). Consequently, the GM-CFC-1 could be distinguished from GM-CFC-2 on the basis of buoyant density and from the other low density populations on the basis of post-FU kinetics. The reasons why the GM-CFC-1 should be more sensitive to 5-FU than other low density progenitors are discussed and the relation of these low density precursors to one another in terms of their position within the haemopoietic developmental lineage is elucidated.

Characterization of a Developmentally Early Macrophage Progenitor Found in Normal Mouse Marrow

Summary. In tissue culture high proliferation potential colony forming cells (HPP‐CFC, Bradley & Hodgson, 1979) formed large colonies in which macrophages predominated. Granulocyte/macrophage colony forming cells (GM‐CFC, Bradley & Metcalf, 1966) formed smaller colonies of granulocytes and macrophages. We compared HPP‐CFC from normal adult mouse bone marrow with GM‐CFC from the same source. We found that HPP‐CFC differed from GM‐CFC in the following ways: (1) HPP‐CFC formed larger colonies containing more than 5 × 104 cells. (2) They required spleen conditioned medium (Metcalf & Johnson, 1978) as well as pregnant mouse uterine extract (PMUE, Bradley et al, 1971) as stimulants. (3) Only 9% of HPP‐CFC were killed by the phase specific drug hydroxyurea. 36–55% of GM‐CFC were killed by the same treatment. (4) The modal density of HPP‐CFC was 1·070 g/cm3. That of GM‐CFC was 1·080 g/cm3.

Regulatory Pathways in Blood-forming Tissue with Particular Reference to Gap Junctional Communication

Blood formation by pluripotent stem cells and their progeny is thought to be regulated by receptorligand interactions between cellsubstrate, cell-cell and cell-matrix in the bone marrow. Primitive stem cells form progenitors and, in their turn, these give rise to haemopoietic progeny which are more specifically committed in that they can form progressively fewer types of blood cells. Recently we have established that direct cell-cell communication via gap junctions may be part of this regulatory system. Connexin43 gap junctions metabolically couple the three dimensional meshwork of bone marrow stromal cells to form a functional syncytium in which some blood-forming cells are also coupled. The expression of gap junctions in the bone marrow is markedly upregulated when there is an urgent and substantial demand for blood-formation; for example, following cytotoxic injury after 5-fluorouracil or irradiation; or during neonatal blood-formation and in the epiphysis of growing bones. Chemical blockade of gap junctions blocks blood-formation in long-term cultures but is reversible after the blockade has been relieved. This short review highlights briefly the known regulatory mechanisms of blood-formation with especial attention to gap junctional communication.

Patterns of haemopoietic recovery after stress—II. Treatment with fluorouracil

The mouse haemopoietic system is not permanently damaged by repeated injections of cytotoxic fluorouracil. It contains approximately normal numbers of nucleated femoral and spleen colony-forming cells (CFUs) after seven monthly injections of the drug and normal numbers of high proliferation potential colony-forming cells (HPP-CFC) after five serial injections. Furthermore, the mouse is fully fertile after seven injections of fluorouracil. The mouse recovers quickly after treatment because it regenerates from cells which were not killed by the drug. Within 14 days of treatment with fluorouracil there are almost twice the normal number of femoral macrophage and high proliferation potential colony-forming cells (M-CFC and HPP-CFC). These numbers then fall but are returning to normal 6 weeks after the drug was administered. In this quick recovery the response of the haemopoietic system differs from its response to the loss of blood cells caused by sub-lethal irradiation, or lethal irradiation, or treatment with busulphan. When mice are treated twice with fluorouracil, the second injection 14 days after the first, the number of femoral M-CFC two days after the second injection, is 16-fold the number in controls, but the number of femoral HPP-CFC is only twice the number in controls. When the interval between the two injections is 21 days, the number of femoral M-CFC is almost 8% of that of mice treated once, but the number of HPP-CFC is 67%. The characteristic response of each type of cell to repeated treatment with fluorouracil is probably due to the number of its precursors which are killed by the drug and to the interval between successive injections. A second injection of fluorouracil, 28 days after the first, speeds the growth rate of HPP-CFC. Their doubling time is 6 h shorter than that of mice treated once. Haemopoietic tissue from mice treated repeatedly with fluorouracil can only outgrow normal marrow under certain conditions. The nature of these conditions and the mechanisms involved are discussed in the light of contradictory findings.

Assessing cultured colonies automatically

The number of colonies formed by macrophage colony-forming cells and high proliferation potential colony-forming cells was assessed by an image processor. The processor counted and sized colonies accurately, reproducibly, rapidly (2 s/dish) and objectively. The processor also measured the amount of light (in grey levels) the colonies transmitted. The optical density of a colony (the sum of its grey levels) was related to its cellularity. Thus the image processor compared both the number of colonies in samples and their cellularity. Samples of marrow containing high proliferation potential colony-forming cells of different proliferative capacity were prepared by injecting fluorouracil into mice and collecting their marrow 2-10 days later (marrow samples called FU2-FU10). These samples were cultured with one of three sources of synergistic factor titrated over seven dilutions. Colonies contained approx. 5 X 10(4) cells after 11 days culture but the way that FU2-FU10 marrow grew depended on the interval between treating donors with fluorouracil and collecting their marrow. Samples collected 2-4 days after fluorouracil formed more colonies containing more cells with small increases of synergistic factor whereas samples collected after 8-10 days did neither. It was important to culture samples of marrow with the appropriate synergistic factor for the interval after fluorouracil. Factor(s) derived from the 5637 cell line acted optimally on high proliferation potential colony-forming cells in samples collected 2-8 days after fluorouracil, and factor(s) derived from Wehi 3B cells on high proliferation potential colony-forming cells in samples collected 6-10 days after fluorouracil. Factor(s) derived from placental conditioned medium acted well on samples collected between 2 and 10 days. The proliferative capacity of samples of marrow could also be compared by estimating growth curves for high proliferation potential colony-forming cells in samples collected at successive intervals after fluorouracil.

Patterns of recovery of high proliferation potential colony-forming cells after stressing the haemopoietic system-I

The rates at which the number of high proliferation potential colony-forming cells and other haemopoietic cells recovered after different first stresses and a standard second stress were studied. The following first stresses were compared; different doses of sublethal irradiation (2.57, 4.84 and 5.5 Gy) followed by endogenous repopulation; lethal irradiation followed by exogenous repopulation; lethal irradiation of radio-protected mice followed by endogenous repopulation; and treatment with busulphan. Six to 16 weeks after these first stresses a standard second stress was applied. This was i.v. injection of fluorouracil. Two, four and six days later the number of high proliferation potential colony-forming cells in femora was determined and recovery curves for these cells were calculated. Their number increased exponentially in this period in all mice studied except radio-protected, lethally irradiated ones. In these, the exponential increase occurred between four and eight days after fluorouracil. The rate of increase was faster than normal in sublethally irradiated and radio-protected, lethally irradiated animals whose haemopoietic systems repopulated endogenously; in lethally irradiated, exogenously reconstituted animals it was the same as normal and in busulphan-treated animals it was slower than normal. The marrow from these differently stressed mice was also cultured with seven doses of two synergistic factors to contrast the growth of high proliferation potential colony-forming cells in the mice whose first stresses had differed. The cultures were assessed automatically by the CLIP 4 image processor. The high proliferation potential colony-forming cells of sublethally irradiated and busulphan-treated mice required more synergistic factor than normals to form a given number of cells/femur.

Investigating the role of CONNEXIN43 in marrow

Abstract We have found connexin43 (Cx43) gap junctions in mouse haemopoietic tissue and proposed that they play a part in the division of primitive haemopoietic stem cells (PHSC, Rosendaal et al., 1997). Normal resting haemopoiesis, which produced the circulating blood cells, is carried out by progenitors, formed by the division of PHSC. They divide when their progeny are needed to repopulate a severely depleted blood-forming system, e.g. when the definitive haemopoietic system is founded in the embryo and neonate or after treating an adult with a cytotoxic drug which selectively deletes dividing haemopoietic cells (e.g. 5-fluorouracil). To divide, PHSC must receive signals and we believe that communication via Cx43 gap junctions is involved. We graft into toe-notched reporter mice, all of whose stem cells have been deleted by busulphan, a competing mixture of two kinds of congenic stem cells with different glucose-phosphate isomerase markers, 1 a and 1 b . The competing populations of stem cells have different degrees of expression of Cx43, +/+1 a versus +/+1 b , or +/+1 a versus +/− 1 b , or +/+1 a versus −/−1 b . At intervals thereafter we assay the proportions of Gpi-1 a and 1 b in five blood cells. This allows three levels of comparison, (1) of repopulation of donors by stem cells, (2) of repopulation of reporter mice by grafted stem cells and (3) of repopulation of reporter mice after subsequent challenge. (1) We treat donor 1 a and 1 b mice with 5-fluorouracil and then compete the same femoral fractions of their marrow in reporter mice. (2) We compete untreated 1 a and 1 b foetal liver stem cells from +/+, +/− or −/− in reporter mice. (3) We stress with different cytotoxic drugs the blood-forming systems of reporter mice after they have been grafted with these combinations of stem cells. Rosendaal et al. (1997) Leukemia 11, 1281–9.