Kodetthoor B. Udupa

In vitro culture of proerythroblasts: characterization of proliferative response to erythropoietin and steroids

Summary This study characterized variables affecting the in vitro liquid culture of proerythroblasts. When bone marrow from mice depleted of haemoglobin containing cells, was cultured in vitro in the presence of human urinary erythropoietin (Ep) a significant degree of erythroid cell proliferation and maturation occurred as measured directly by 3H‐thymidine (3H‐TdR) incorporation into DNA (autoradiographical measurement). Proliferation increased indirect proportion to the dose of Ep added to the culture. We also demonstrated a highly significant positive correlation between proliferation measured directly by 3H‐TdR incorporation into DNA or indirectly by 59Fe incorporation into haem. Ep was a potent stimulator of proerythroblast proliferation. We also examined the role of the androgenic and non‐androgenic steroids on in vitro proliferation. All the hormones tested were stimulatory but only in the presence of Ep. The androgenic steroids primarily affected the more mature erythroid precursors whereas the glucocorticoids were more general growth promoters. Their addition in physiologic concentration to liquid culture reduced Ep requirements. Thus when both testosterone and hydrocortisone were added to culture the Ep concentration that produced the same degree of proliferation as a culture containing Ep alone was decreased by 90%. This finding is important as it indicates that in vitro culture conditions can be created that more closely mimic in vivo erythropoiesis where Ep requirements are far less.

Age and the Hematopoietic System

The aging process is characterized by a decline in the function of many organ systems. Changes that occur in cardiovascular, endocrine, and immune function with aging have been described extensively. The bone marrow clearly plays an important role in normal physiology, being responsible for oxygen delivery to tissues as well as participating in normal hemostasis and host defense. There is evidence that anemia is common in older persons and that the increased morbidity and susceptibility to infection that occurs in the elderly is in part related to altered granulocyte function. This review will discuss normal bone marrow function and describe the most recent evidence implicating alterations in hematopoiesis with age.

Nicotine-induced proliferation of isolated rat pancreatic acinar cells: effect on cell signalling and function.

Abstract.  Objectives: The aim of the current study was to investigate whether nicotine treatment would induce the proliferation of isolated rat primary pancreatic acinar cells in culture by activating mitogen‐activated protein kinase (MAPK) signalling and exocrine secretion. Materials and Methods: A nicotine dose‐ and time‐response curve was initially developed to determine the optimal dose and time used for all subsequent studies. Proliferation studies were conducted by cell counting and confirmed further by bromodeoxyuridine (BrdU) incorporation and flow cytometry assays. MAPK signalling studies were conducted by Western blot analysis. Localization of ERK1/2 signals, with or without nicotine and the MAPK inhibitor, was visualized by immunofluorescence. Results: Nicotine treatment caused dose‐dependent activation of extracellular signal‐regulated kinases (ERK1/2), the maxima occurring at 100 µm and at 3 min after treatment; the response was suppressed by the ERK1/2 inhibitor. Maximal nicotine‐induced cell proliferation occurred at 24 h, and UO126‐treatment significantly reduced this response. Exposure of cells to 100 µm nicotine for 6 min significantly enhanced both baseline and cholecystokinin‐stimulated cell function, and these effects were not affected by treatment with the inhibitor of ERK1/2 but were suppressed by mecamylamine, a nicotinic receptor antagonist. Conclusions: Our results suggest that nicotine treatment induced cell proliferation of isolated pancreatic acinar cells and that this is coupled with the activation of MAPK signalling with no effect on its function. Hence, in primary cells, the mechanism of induction and regulation of these two processes, cell proliferation and cell function, by nicotine treatment are independent of each other.

Effect of age on marrow macrophage number and function

Employing flow cytometry and a monoclonal antibody against the murine macrophage antigen, Mac- 1, we found a significant increase in the number of marrow macrophages in aged mice. This was reflected as significant increase with age in the number of α- naphthyl acetate esterase positive cells, as well as in colony forming unit- macrophage (CFU- M) progenitor cells. Macrophages from the marrow of old mice generated significantly less tumor necrosis factor α (TNFα) than did macrophages from young mice, either spontaneously or when activated by granulocyte- macrophage colony stimulating factor (GM- CSF). Furthermore, conditioned medium (CM) derived from either marrow or peritoneal macrophages of old mice caused less suppression of burst forming unit- erythroid (BFU- E) colony growth than did CM obtained from young mice. Aging, therefore, is associated with an increase in the number of marrow macrophages that have an impaired ability to generate or release cytokines. The increase in macrophage number may reflect a compensation for their reduced function. Altered macrophage number and function may contribute to the age- related decline in hematopoietic reserve capacity.

Evidence suggesting a stimulatory role for interleukin-10 in erythropoiesis in vitro

Interleukin‐10 (IL‐10) has been shown to exert anti‐inflammatory effects by suppressing macrophage proliferation and inhibiting cytokine production. In this study we show that in the presence of erythropoietin (EPO), the addition of IL‐10 results in a significant dose‐dependent increase in both Burst Forming Unit‐Erythroid (BFU‐E) and Colony Forming Unit‐Erythroid (CFU‐E) colony growth in both serum‐containing and serum‐free murine cultures in vitro. IL‐10 acts at the later stages of erythroid cell proliferation and differentiation as the increase in colony number was greater in CFU‐E than in BFU‐E, and was similar when IL‐10 was added to BFU‐E cultures at the time of culture initiation as when its addition to culture was delayed for 7 days. Furthermore, no increase in BFU‐E colony number was noted when IL‐10, added at the time of culture initiation, was neutralized by the addition to culture of a monoclonal anti‐IL‐10 antibody up to 7 days later. The increases in BFU‐E by IL‐10 addition were not the result of prolongation of BFU‐E colony lifespan, which was not significantly different in IL‐10 treated and control cultures, respectively. Rather IL‐10 stimulated the proliferation of erythroid clusters that were now large enough to be recognized as colonies. IL‐10‐induced stimulation of erythropoiesis appeared to be independent of its inhibitory effects on macrophage function, as stimulation of erythroid colony growth was similar in macrophage‐containing and depleted cultures. Studies to determine if the IL‐10 effect was direct or indirect yielded equivocal results. A limiting dilution assay suggested a direct effect. However, a log/log dose response curve with IL‐10 did not pass through the origin suggesting an indirect effect. These studies indicate that IL‐10 acts synergistically with EPO to significantly increase stimulation of erythroid differentiation and proliferation in vitro and may be involved in the regulation of normal erythropoiesis in vivo.

Age-related alteration in hepatic acyl-CoA : cholesterol acyltransferase and its relation to LDL receptor and MAPK

The aim of this study was to evaluate changes in the regulation of lipid metabolism and mitogen-activated protein kinases (MAPK) in the liver of C57BL/6 mice as they age. This was done by assessing the status of total cholesterol content and its enzyme, acyl-CoA: cholesterol acyltransferase (ACAT), in liver microsomal preparations and the low-density lipoprotein receptor (LDLr) mRNA expression in the livers of 4-24-month-old C57B/6 mice, without exogenous cholesterol feeding. With aging, there was an increase in cholesterol content and ACAT activity in liver microsomes. Northern blot analysis and real-time quantitative polymerase chain reaction data showed that ACAT-2 mRNA increased with age as well. LDLr expression decreased significantly in an age-dependent manner. In addition, we studied the basal and activated forms of MAPK, e.g. extracellular regulatory kinase (ERK-1/2), c-jun NH2-terminal kinase (JNK-1/2) and p38 MAPK. During aging, there was a considerable decrease in phosphorylated ERK-1/2 level while JNK-1/2 and p38 MAPK levels increased with age. Our studies showed an altered LDLr expression and altered phosphorylated MAPK in the liver of C57BL/6 mice during aging. These alterations might contribute to the development of atherosclerosis, hypercholesterolemia and other cholesterol-related conditions.

Effect of nicotine on exocytotic pancreatic secretory response: role of calcium signaling

BackgroundNicotine is a risk factor for pancreatitis resulting in loss of pancreatic enzyme secretion. The aim of this study was to evaluate the mechanisms of nicotine-induced secretory response measured in primary pancreatic acinar cells isolated from Male Sprague Dawley rats. The study examines the role of calcium signaling in the mechanism of the enhanced secretory response observed with nicotine exposure.MethodsIsolated and purified pancreatic acinar cells were subjected to a nicotine exposure at a dose of 100 μM for 6 minutes and then stimulated with cholecystokinin (CCK) for 30 min. The cell’s secretory response was measured by the percent of amylase released from the cells in the incubation medium Calcium receptor antagonists, inositol trisphosphate (IP3) receptor blockers, mitogen activated protein kinase inhibitors and specific nicotinic receptor antagonists were used to confirm the involvement of calcium in this process.ResultsNicotine exposure induced enhanced secretory response in primary cells. These responses remained unaffected by mitogen activated protein kinases (MAPK’s) inhibitors. The effects, however, have been completely abolished by nicotinic receptor antagonist, calcium channel receptor antagonists and inositol trisphosphate (IP3) receptor blockers.ConclusionsThe data suggest that calcium activated events regulating the exocytotic secretion are affected by nicotine as shown by enhanced functional response which is inhibited by specific antagonists… The results implicate the role of nicotine in the mobilization of both intra- and extracellular calcium in the regulation of stimulus-secretory response of enzyme secretion in this cell system. We conclude that nicotine plays an important role in promoting enhanced calcium levels inside the acinar cell.

Erythropoietin enhancement of rat pancreatic tumor cell proliferation requires the activation of ERK and JNK signals

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells by binding to its specific transmembrane receptor EPOR. Recent studies, however, have shown that the EPOR is additionally present in various cancer cells and EPO induces the proliferation of these cells, suggesting a different function for EPO other than erythropoiesis. Therefore, the purpose of the present study was to examine EPOR expression and the role of EPO in the proliferation and signaling cascades involved in this process, using the rat pancreatic tumor cell line AR42J. Our results showed that AR42J cells expressed EPOR, and EPO significantly enhanced their proliferation. Cell cycle analysis of EPO-treated cells indicated an increased percentage of cells in the S phase, whereas cell numbers in G0/G1 phase were significantly reduced. Phosphorylation of extracellular regulatory kinase 1/2 (ERK1/2) and c-Jun NH(2) terminal kinase 1/2 (JNK1/2) was rapidly stimulated and sustained after EPO addition. Treatment of cells with mitogen-activated protein/ERK kinase (MEK) inhibitor PD98059 or JNK inhibitor SP600125 significantly inhibited EPO-enhanced proliferation and also increased the fraction of cells in G0/G1 phase. Furthermore, the inhibition of JNK using small interference RNA (siRNA) suppressed EPO-enhanced proliferation of AR42J cells. Taken together, our results indicate that AR42J cells express EPOR and that the activation of both ERK1/2 and JNK1/2 by EPO is essential in regulating proliferation and the cell cycle. Thus both appear to play a key role in EPO-enhanced proliferation and suggest that the presence of both is required for EPO-mediated proliferation of AR42J cells.

Effect of donor age on long-term culture of bone marrow in vitro.

Previous reports suggest that 33 degrees C is the optimum temperature for the long-term culture of murine bone marrow in vitro. We found that horse serum was an important variable determining our ability to culture marrow from immature (less than 10 weeks) mice at 37 degrees C. While some batches supported growth others did not. The ability of deficient horse serum to support growth could be corrected if the culture was fed at mid-week with additional medium. Marrow from aged mice (96 weeks and older) could invariably be cultured at 37 degrees C irrespective of the horse serum batch used for culture. Adult mice (20-24 weeks) gave intermediate results between immature and aged mice. All three groups of mice studied could be cultured at 33 degrees C irrespective of the batch of horse serum used or the age of the donor. These results indicate that horse serum provides a factor(s) which varies in concentration from batch to batch and is essential for normal marrow growth in vitro. The requirement for this factor is greater at higher temperature and in younger animals. Marrow from adult and aged mice was then simultaneously cultured at 33 degrees C and 37 degrees C. For adult mice the cell recovery at 33 degrees C was initially significantly higher than at 37 degrees C but at later times became less. As a result cumulative recovery at both temperatures was equal. Cumulative cell recovery and culture survival time was significantly less in the aged than the young mice cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Murine marrow stromal response to myelotoxic agents in vitro.

Previous studies have shown that the haemopoietically active murine long‐term bone marrow culture (LTBMC) is a useful model for studying drug‐induced suppression and recovery of myelopoiesis. We studied the effects on stromal morphology and stromal progenitors, assessed as colony forming unit‐fibroblasts (CFU‐F), of the addition of either the antimetabolite methotrexate (MTX) or the betalactam ceftazidime (CEF) to LTBMC. The examination of 500 μg/ml CEF‐treated cultures revealed a stroma that appeared empty, with modest reduction in total stromal counts, and significant decreases in fat‐containing and endothelial cells. In contrast, treatment with 1 μM MTX for 1 week initially caused minimal morphologic stromal changes, thereafter total stromal cell count as well as fibroblastoid, endothelial, fat containing and macrophage cells significantly increased. Haemopoiesis and the stroma recovered. Both CEF and MTX reversibly suppressed stromal progenitor cells in LTBMC. When added directly to CFU‐F cultures, the concentrations resulting in a 50% colony growth inhibition were 214 μg/ml for CEF and 375 nM for MTX. These results suggest that CEF, but not MTX, has direct toxic effects on the stroma of established LTBMC. Stromal cell increase following MTX treatment probably indicates a stromal response that may contribute to haemopoietic cell recovery.