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 E 2 (PGE 2 ), 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.

The effects of providing low oxygen tension in the gas phase of two different types of cell culture systems were investigated. The clonal growth of granulocyte-macrophage progenitor cells in an agar culture system was improved markedly by incubation within a low oxygen tension gas phase (48 mmHg--6.8%) instead of the conventional air (135 mmHg--19%), the effects being measured by increases in numbers of colony forming cells detected and in the colony sizes. The increased efficiency of colony formation was observed both with mouse and human marrow cells. A similar effect was observed in a liquid adherence culture system with primary cultures of foetal mouse fibroblasts both at clonal and higher cell densities.

Direct cell/cell communication occurs through gap junctions (GJ). We mapped GJ expression in secondary lymphoid organs and found, for the first time, a high density of connexin43 (Cx43) GJ in follicular dendritic cells (FDC) in close association with lymphocytes (Krenacs T. and Rosendaal M., J. Histochem. Cytochem. 1995. 43: 1125-1137). In this work, we used a combination of ultrastructural, immunocytochemical, molecular methods, and functional dye transfer experiments to study which germinal center cells are involved in direct cell/ cell communication and how GJ expression is regulated during antigen responses. One week after injecting the footpad of mice with 50 micrograms lysozyme, Cx43 GJ were detected on elongated cells in the paracortex of their popliteal lymph nodes. Repeated challenge led to the formation of secondary follicles with enlarged FDC meshwork full of Cx43 GJ. This positive correlation may reflect an importance for GJ in the pattern formation of FDC and lymphoid follicles. In human tonsil, the density of GJ and FDC was highest in the light zone of germinal centers where the fate of B cells is thought to be decided. Cx43 colocalized with CD21 and CD35 antigens in the vicinity of desmosomal junctions on FDC embracing lymphocytes. Freeze-fracture hallmarks of GJ of 200-400 nm were also found on FDC in the vicinity of desmosomal plaques. Furthermore, Northern blot analysis showed the consistent presence of Cx43 mRNA in human tonsil and spleen. Most Cx43 message was localized in situ to cells with FDC morphology and some to a few germinal center lymphocytes. To investigate functional cell coupling, we set up FDC/B cell cultures from the low density cell fractions of human tonsils. Cx43 plaques associated with lymphocytes were detected both on elongated FDC processes in early cultures (up to 4 h) and in established FDC/B cell clusters (between 4 and 24 h). In early cultures, we injected FDC with Lucifer Yellow, a fluorescent dye which passes through GJ: the dye spread into adjacent FDC and occasionally from FDC into CD19+ B cells. Based on these results, we propose that direct cell/cell communication through Cx43 GJ is involved in FDC/FDC and in FDC/B cell interactions. The functionally coupled FDC meshwork may serve as a communication channel synchronizing germinal center events. FDC may also deliver crucial direct signals through GJ involved in the rescue of high-affinity B cell clones from apoptotic cell death.

No abstract

We investigated the expression of gap junction connexins26, -32, and -43 in normal, reactive, and diseased human lymphoid tissue with single and double immunolabeling and confocal laser scanning microscopy. In all tissues, connexin43 positivity was detected in follicular dendritic cells positive for CD21 and CD35 antigens, around lymphoendothelial cells moderately positive for Factor VIII, CD31 and cathepsin-D antigens; and somewhat in vascular endothelia including high endothelial venules strongly positive for Factor VIII and CD31 antigens. The ultrastructural hallmark of gap junctions, pentalaminar structures with appropriate spacing, was found in follicular dendritic cell processes. Connexin43 was also detected between smooth muscle and stromal cells of the gut, in capsular fibroblasts, and in tonsil epithelium. Neither connexin32 nor -26 was revealed, except for connexin26 in the tonsil epithelium. In follicular dendritic cells, connexin43 co-localized closely with the desmosomal proteins desmoplakin and desmoglein, suggesting that cell adherence has a role in gap junction formation. Most connexin43 was observed in sinus lining cells of lymph nodes involved in malignancies and in follicular dendritic cells in the light zone of germinal centers where maturing but still proliferating lymphocytes are situated. In the light of their distribution, gap junctions may play a part in regulating the growth of germinal centers and in integrating activating or controlling signals in follicular dendritic and sinus lining cell networks. Because connexin43 is the connexin of stromal cells, finding it in follicular dendritic cells in consistent with the proposal that these cells originate from resident stromal cells.